Molecular dynamics guided development of indole based dual inhibitors of EGFR (T790M) and c-MET

Article abstract of DOI:10.1016/j.bioorg.2018.04.001

Secondary acquired mutation in EGFR, i.e. EGFR T790M and amplification of c-MET form the two key components of resistant NSCLC. Thus, previously published pharmacophore models of EGFR T790M and c-MET were utilized to screen an in-house database. On the ba

Full text of DOI:10.1016/j.bioorg.2018.04.001

Accepted Manuscript
Molecular dynamics guided development of indole based dual inhibitors of
EGFR (T790M) and c-MET
Pankaj Kumar Singh, Om Silakari
PII:
S0045-2068(18)30198-6
DOI:
https://doi.org/10.1016/j.bioorg.2018.04.001
YBIOO 2324
Reference:
Bioorganic Chemistry
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2 March 2018
4 April 2018
6 April 2018
Please cite this article as: P. Kumar Singh, O. Silakari, Molecular dynamics guided development of indole based
dual inhibitors of EGFR (T790M) and c-MET, Bioorganic Chemistry (2018), doi: https://doi.org/10.1016/j.bioorg.
2018.04.001
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Molecular dynamics guided development of indole based dual inhibitors of EGFR
(T790M) and c-MET
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
Abstract
Secondary acquired mutation in EGFR, i.e. EGFR T790M and amplification of c-
MET form the two key components of resistant NSCLC. Thus, previously published
pharmacophore models of EGFR T790M and c-MET were utilized to screen an in-house
database. On the basis of fitness score, indole-pyrimidine scaffold was selected for further
evaluation. Derivatives of indole-pyrimidine scaffold with variedly substituted aryl
substitutions were sketched and then docked in both the targets. These docked complexes
were then subjected to molecular dynamic simulations, to study the stability of the complexes
and evaluate orientations of the designed molecules in the catalytic domain of the selected
kinases. Afterwards, the complexes were subjected to MM-GBSA calculation, to study the
effect of substitutions on binding affinity of double mutant EGFR towards these small
molecules. Finally, the designed molecules were synthesized and evaluated for their
inhibitory potential against both the kinases using in-vitro experiments. Additionally, the
compounds were also evaluated against EGFR (L858R) to determine their selectivity towards
double mutant, resistant kinase [EGFR (T790M)]. Compound 7a and 7c were found to be
possess nanomolar range inhibitory (IC50) potential against EGFR (T790M), 7h showed good
inhibitory potential against c-MET with IC50 value of 0.101 µM. Overall, this work is one of
the earliest report of compounds having significant dual inhibitory potential against
secondary acquired EGFR and cMET, with IC50 values in nanomolar range.
Keywords: Indole-pyrimidine; molecular dynamic simulations; EGFR (T790M); c-MET

1. Introduction
Kinases form a major class of molecular targets in lung adenocarcinomas harbouring
activating mutations. These kinases result in over-activation of downstream signalling
pathways that regulate the process of cell growth, proliferation, and survival. The
identification of these driver kinases has previously led to the clinical use of small molecule
kinase inhibitors such as erlotinib and gefinitib [1, 2]. These molecules act as competitive
inhibitors of ATP and have been proven efficacious over conventional chemotherapies in
patients harbouring epithelial growth factor receptor (EGFR) overexpressed adenocarcinomas
[3]. 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: (1) alteration in the driver oncogene such as acquired secondary mutation T790M in
EGFR (2) activation of signaling pathway(s) via parallel signalling, as in case of
amplification of wildtype c-MET (hepatocyte growth factor receptor) in the L858R mutant
EGFR overexpressed lung cancer and (3) reactivation of signalling pathways downstream of
a driver oncogene, Nuclear factor-κB (NFκB)-containing complex activation is one such
example. One more mechanism of resistance involves transformation of cell lineage such as
epithelial (Non-small cell lung cancer) to another i.e., mesenchymal (Small cell lung cancer)
[4].
Among all, secondary acquired gate keeper residue mutation, T790M in EGFR, is
observed in ~50% of EGFR-mutant patients who develop resistance to EGFR inhibition and
is a pivotal mechanism of resistance. Initially, researchers suggested that gatekeeper mutation
results in steric hindrance towards small molecule inhibitors thereby leading to development
of resistance [5]. Later, studies disclosed that secondary acquired kinase do not possess
resistance due to steric hindrance but rather the affinity of the kinase is altered back in favour
of ATP and hence, ATP competitively inhibits the inhibitors [6]. To counter this secondary
acquired mutation, researchers focussed on developing second generation kinase inhibitors,
covalent inhibitors. Several studies focused on developing covalent inhibitors for EGFR
T790M, via analysis of catalytic mechanism of binding with Cys797 [7] and utilization of
different cysteine-trapping fragments [8] such as isothiocyanates [9], have been conducted.
However, they suffer with lack of selectivity and therefore have poor safety profile. This
further led to the development of third generation kinase inhibitors, which are covalent but
are selective towards double mutant EGFR. Selectivity in these agents is claimed due to the
hydrophobic interactions between the inhibitors and mutated residue M790 in the catalytic

domain of the double mutant kinase. Osimertinib, recently reported EGFR (T790M) inhibitor,
was also based on the same concept that molecules with hydrophobic interaction with the
mutant methionine-790 gatekeeper residue could result in potent and selective inhibitors. It is
also potent inhibitor of EGFR (L858R) due to its increased affinity towards small molecule
inhibitors in place of ATP [10]. However, as covalent inhibitors are site specific in nature,
any alteration in target residues can limit their efficacy. In recent years another acquired
mutation, C797S in EGFR, has been disclosed and reported to make third generation
inhibitors ineffective. Additionally, another mutation in the P-loop residue (L718Q) has been
reported to result in resistance against third generation inhibitors such as osimertinib. One of
the mechanism suggests that mutant Gln718 affects the conformational space of the EGFR–
osimertinib complex, preventing its interaction with Cys797 [11].
Another key mechanism of resistance against kinase inhibitors in NSCLC (Non-small cell
lung cancer) is amplification of c-MET, upon administration of first generation inhibitors. It
has been one of the earliest mechanism of resistance which rendered EGFR inhibitors
ineffective [12]. Amplification of c-MET leads to the reactivation of PI3K/Akt signaling
pathway by forming a MET-ErbB-3 heterodimer, previously formed by EGFR. Basically, c-
MET behaves as a substitute for EGFR in the signalling pathway. Thus, despite continuous
suppression of EGFR by the inhibitor, a critical downstream signalling pathway continues,
and resistance to the inhibitor emerges [13]. In patients with resistance to first-generation
EGFR tyrosine kinase inhibitors generated by c-MET amplification, it is unlikely that a third
generation covalent inhibitor would be effective, but the combination of a double mutant
EGFR inhibitor and a c-MET/mTOR/ PI3K/Akt pathway inhibitor may prove to be an
effective strategy [14]. Clinical trials are underway to test the effect of dual inhibition of c-
MET and EGFR to overcome this mode of resistance [15].
Thus, in our study we focussed on targeting c-MET and secondary acquired mutant
EGFR (T790M) via non-covalent inhibitors. Focus was laid on 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 towards EGFR T790M. Thus, we attempted to design molecules with
higher affinity towards T790M EGFR along with c-MET using in-silico techniques and
further synthesis and biological evaluation.
2. Results and Discussion

2.1.In-silico analysis
.Previously reported ligand-based pharmacophore models for EGFR (T790M) and cMET,
generated using Discovery studio were employed to screen and cross screen an in-house
small molecule database containing around 200 molecules with diverse scaffolds such as
benzimidazole, oxindole, indole, flavones and thiazolidinones. Pharmacophore mapping tool
was employed for this purpose and molecules possessing significant fit score in mapping via
both pharmacophores were selected for further analysis. This screening yielded a total of 18
molecules with indole scaffold, similar compounds have been reported previously to possess
anti-microbial and cytotoxic potential [16-18], which were then subjected to 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). Molecules selected after docking analysis, in EGFR
T790M, showed that an amino group present at the second position of the pyrimidine
containing molecules acts as donor group and interacts via hydrogen bond with Gln791,
Met793 and Lys745 in many of the designed compounds while the ring of indole formed the
hydrophobic interactions with various other hinge region residues. Similarly, in cMET, free
NH2 in many of the designed compounds formed hydrogen bond with Asp1231 and the
hydrophobic region was occupied by benzyl group substituted at N1 of the indole. All the
compounds showed good docking scores (Glide XP G-score) in both the kinases; ranging
from -8.22 to -6.98 kcal/mol within the EGFR (T790M) protein and -7.42 to -4.59 kcal/mol
in cMET. Docking was followed by molecular dynamic simulations of the designed
molecules in complex with EGFR (T790M) as well as cMET, for a period of 30 ns.
Simulations studies 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.
Additionally, presence of bromine in the side ring was found to be vital as it occupied small
hydrophobic cavity in the catalytic domain of EGFR (T790M) enhancing the overall
hydrophobic and van der waal interactions, while in some derivatives it also formed halogen
bond with neighbouring residues. The RMSD values of the protein and ligands in the
complex with top compounds (7c and 7h) reflected the overall stability of the complex for the
given period of 30 ns and the graphs for the same are represented in Fig.1 and Fig.2.

<figure1>
<figure2>
The 3D interaction diagram of the designed molecules 7c in EGFR T790M and 7h in cMET
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 -75.706 to -49.003 in EGFR
(T790M) and -84.334 to -66.319 in cMET (Table 1).
<figure3>
<figure4>
<table1>
2.2.Chemistry
The designed compounds were synthesized according to the scheme 1. In the first step,
alkylation of indole-3-carbaldehyde was performed by reacting it with variedly substituted
benzyl chlorides in the presence potassium carbonate to yield different N-substituted indole-
3-carbaldehydes. Followed by claisen-schmidt condensation of the obtained N-substituted
indole-3-carbaldehydes with p-bromoacetophenone using piperidine as catalyst in methanol
to afford 1,3-diaryl/heteroaryl propenones. The obtained 1,3-diaryl propenones were purified
via recrystallization. Then, 1,3-diaryl/heteroaryl propanones were treated with guanidine
hydrochloride, using sodium hydroxide as catalyst in methanol to afford desired products.
Finally column chromatography was performed to obtain pure derivatives. All the
1
compounds were characterized by IR, Mass, H-NMR and 13C-NMR. In IR spectrum, the
indole-pyrimidine derivatives showed the presence of strong absorption bands of multiple
1
C=N from ~1680 to ~1580 cm-1. The synthesis of final compounds were confirmed in H-
NMR. Almost each spectrum showed singlet of only proton present in pyrimidine nucleus,
ranging from ~7.3 to ~7.8 ppm according to different derivatives. Rest of the aromatic
protons were observed in a similar pattern from ~7.0 to ~ 8.4 ppm. A singlet of two proton at
13
~5.5 ppm was also observed in each spectrum for -CH2 of the benzyl groups. C spectrum
exhibited characteristic peaks at ~163 ppm for pyrimidines. Rest of the aromatic carbons
were observed from 120 to 134 ppm with a characteristic peak at 55 ppm for benzylic carbon.
Similarly, mass spectrometry also showed quasi ion peaks at expected m/z values,

additionally, an M+2 peak was observed in each case due to presence of -Br group in each
molecule.
<scheme1>
2.3.In-vitro biological evaluation
For the in vitro evaluation of kinase inhibitory potential of the synthesized compounds,
enzymatic assay against EGFR (T790M) and cMET were performed. Additionally evaluation
against EGFR (L858R) was also performed, which is involved in development of NSCLC in
around 50 % patients on the 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. Compound 7a with ethyl
substitution at para position of benzyl attached to indole was found to possess significant
selective EGFR (T790M) inhibitory activity over EGFR (L858R) with IC50 value 0.097 µM
in EGFR (T790M) and 0.913 µM in EGFR (L858R). It also showed significant cMET
inhibitory potential with IC50 value 0.518 µM. Another compound 7c having 4-isopropyl
substituted benzyl substitution showed excellent non-selective inhibition in both EGFR
(T790M) and EGFR (L858R) with IC50 values 0.094 and 0.099 µM, respectively.
Additionally it also showed modest cMET inhibitory activity with IC50 value 0.595. However,
upon substituting isopropyl with isobutyl the inhibitory activity in compound 7b fell to IC50
value 0.569 µM. Compound 7h with 3-chloro substitution was found to be most potent cMET
inhibitor with IC50 value 0.101 µM as summarized in Table 2. The inhibitory potential
against EGFR (T790M) can be correlated with hydrophobic interactions between mutated
residue M790 and bulk of the substituent group at N-benzyl group. Isopropyl substituent was
found to be most optimum for significant inhibitory potential.
<table2>
3. Conclusion
Acquired secondary mutation T790M in EGFR and amplification of cMET are two most
important resistance mechanisms hampering chemotherapy in NSCLC. Therefore, in this
study in-silico methodologies were employed to design some indole-pyrimidine scaffold
based reversible dual inhibitors of EGFR (T790M) and cMET. The designed compounds
were further synthesized and evaluated for their kinase inhibitory potential using enzymatic

assay. Compound 7a, 7c and 7h 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. The obtained results lay ground
for further efforts to develop small molecule dual inhibitors of EGFR T790M-cMET for
resistant NSCLC.
4. Material and methods
4.1.In-silico study
Previously published pharmacophore models for EGFR (T790M) and c-MET [19] were
utilized to screen an in-house database of synthetically feasible compounds via
pharmacophore mapping tool available with Discovery studio software [20]. 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 module of Schrödinger
[21]. The screened molecules were first docked into the grid of PDB ID: 2W2P for EGFR
(T790M) followed by docking into the grid of 3DKF for c-MET and vice-versa, 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 hinge region of the active site were selected.
To check the stability of the designed molecules in the catalytic domain of 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 [22]. 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 [23].
4.2.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 were recorded on a FT-IR
1
13
spectrophotometer, using KBr pellet. H NMR and C NMR spectra of the molecules were
generated on a Bruker spectrophotometer at 400MHz and 100MHz (TMS as internal
standard), respectively using CDCl3 or DMSO as solvent. 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.
4.2.1. Synthesis of variedly substituted benzyl-1H-indole-3-carbaldehyde (3a-i).
A mixture of indole-3-carbaldehyde (1) (0.35 g, 2.5 mmol) and potassium carbonate (5
mmol) in acetone (10 ml) was stirred for 5 minutes. Next, variedly substituted benzyl
chlorides (2a-i) (2.5 mmol) were added into the reaction mixture and stirred at 50°C.
Completion of reaction was monitored by TLC. After completion of reaction, the mixture

was filtered to remove K2CO3 and concentrated under vacuum. Obtained crude product was
dried and recrystallized with diethyl ether to afford the pure product (3a-i).
4.2.2. Synthesis of variedly substituted (E)-3-(1-benzyl-1H-indol-3-yl)-1-(4-
bromophenyl)prop-2-en-1-one (5a-i)
To a solution of N-substituted indole-3-carbaldehyde, 3a-i (0.23 g, 10 mmol) and 4’-
bromoacetophenone (4) (0.2 g, 10 mmol) in methanol (10 ml), few drops of piperidine were
added. The reaction mixture was stirred for 2 h at 50°C and then left overnight in refrigerator.
The reaction mixture was neutralized with diluted hydrochloric acid (1:1) and the solid
formed was filtered off, washed with water, air dried and recrystallized from absolute
ethanol. (5a-i).
4.2.3. Synthesis of final compounds of Series I (7a-i).
A mixture of chalcones, 5a-i (0.1 g, 0.25 mmol) and guanidine hydrochloride, 6 (0.25 mmol)
solution in methanol (10 ml) containing sodium hydroxide (0.25 mmol) was stirred at 50°C
for 6-8 h. After cooling, the reaction mixture was poured onto ice-water (50 mL) and the
solid formed was filtered off, air dried to obtain the crude products (7a-i) which were purified
by column chromatography (Silica gel # 60-120; Petroleum Ether: Ethyl acetate::80: 20).
4-(4-Bromophenyl)-6-(1-(4-ethylbenzyl)-1H-indol-3-yl)pyrimidin-2-amine (7a) light yellow
solid, Yield: 33.5%, mp: 112-114oC, Rf 0.51, IR (ν cm-1) 3307 (-NH2), 1660 (C=N), 1632
1
(C=N), 1590 (C=N), 872 (C-Br); H NMR (400 MHz, CDCl3) δ (ppm): 8.23 (s, NH2), 8.03-
8.01 (d, J = 8 Hz, 1H), 7.99-7.96 (m, 1H), 7.92-7.89 (m, 1H), 7.72 (s, 1H), 7.66-7.64 (d, J = 8
Hz, 1H), 7.61-7.59 (m, 1H), 7.48-7.46 (m, 2H), 7.35-7.33 (m, 1H), 7.25 (s, 1H), 7.15-7.06
(m, 4H), 5.33 (s, 2H), 2.68 (m, 2H), 1.27 (m, 3H); 13C NMR (100 MHz, CDCl3) 163.55,
163.09, 158.82, 134.45, 134.97, 132.96, 131.94, 130.67, 129.38, 129.10, 128.80, 128.60,
128.50, 127.14, 126.16, 106.95, 55.53, 25.68, 15.55; MS (+ESI): m/z 483.10 (M+H)+, 485.10
(M+2+H)+. Anal. Calcd for (C27H23BrN4): C, 67.08; H, 4.80; N, 11.59; Found C, 67.21; H,
4.77; N, 11.61.

4-(4-Bromophenyl)-6-(1-(4-(tert-butyl)benzyl)-1H-indol-3-yl)pyrimidin-2-amine (7b) Light
Yellow solid, yield: 35.6%, mp: 110-112oC, Rf 0.45, IR (ν cm-1) 3399 (-NH2), 1637 (C=N),
1
1612 (C=N), 1572 (C=N), 872 (C-Br); H NMR (400 MHz, CDCl3) δ (ppm): 8.40-7.38 (m,
1H), 7.89-7.86 (m, 2H), 7.57-7.55 (d, J = 8Hz, 2H), 7.31-7.23 (m, 6H), 7.08-7.06 (m, 3H),
13
5.21 (s, 2H), 1.25 (s, 9H); C NMR (100 MHz, CDCl3) δ (ppm): 163.88, 163.56, 158.55,
137.56, 137.02, 133.51, 132.31, 131.89, 130.56, 128.72, 128.69, 127.13, 127.00, 126.95,
126.76, 125.99, 125.84, 124.66, 124.13, 118.63, 110.23, 53.54, 31.35; MS (+ESI): m/z
511.14 (M+H)+, 513.13 (M+2+H)+. Anal. Calcd for (C29H27BrN4): C, 68.10; H, 5.32; N,
10.95; Found C, 68.23; H, 5.29; N, 10.98.
4-(4-Bromophenyl)-6-(1-(4-isopropylbenzyl)-1H-indol-3-yl)pyrimidin-2-amine (7c) light
yellow solid, yield: 30.15%, mp: 125-127oC, Rf 0.48, IR (ν cm-1) 3349 (-NH2), 1644 (C=N),
1
1574 (C=N), 1511 (C=N), 871 (C-Br); H NMR (400 MHz, CDCl3) δ (ppm) 8.33-8.30 (m,
1H), 7.90-7.88 (m, 2H), 7.70 (s, 1H), 7.59-7.57 (d, J = 8 Hz, 2H), 7.36-7.34 (d, J = 8Hz, 2H),
7.32-7.29 (m, 2H), 7.20-7.14 (m, 2H), 7.10-7.08 (d, J = 8Hz, 2H) 5.23 (s, 2H), 2.89 (m, 1H),
1.25 (m, 6H); 13C NMR (100 MHz, CDCl3) δ (ppm) 163.85, 163.55, 163.48, 149.25, 148.77,
138.59, 137.54, 136.99, 133.80, 132.61, 131.88, 130.66, 127.40, 127.20, 126.26, 125.52,
124.66, 124.13, 121.77, 121.45, 116.96, 115.42, 110.82, 50.67, 33.84, 23.96; MS (+ESI):
m/z 497.12 (M+H)+, 499.12 (M+2+H)+. Anal. Calcd for (C28H25BrN4): C, 67.61; H, 5.07; N,
11.26; Found C, 67.74; H, 5.04; N, 11.29.
4-(4-Bromophenyl)-6-(1-(3-(trifluoromethyl)benzyl)-1H-indol-3-yl)pyrimidin-2-amine (7d)
light yellow solid, yield: 25.6%, mp: 117-119oC, Rf 0.48, IR (ν cm-1) 3309 (-NH2), 1644
1
(C=N), 1574 (C=N), 1564 (C=N), 812 (C-Br); H NMR (400 MHz, CDCl3) δ (ppm): 8.40-
8.38 (m, 1H), 7.91-7.89 (d, J = 8 Hz, 2H), 7.72 (s, 1H), 7.62-7.60 (d, J = 8 Hz, 2H), 7.54-7.50
13
(m, 2H), 7.42-7.38 (m, 2H), 7.32-7.23 (m, 4H), 5.40 (s, 2H); C NMR (100 MHz, CDCl3)
164.13, 163.41, 163.20, 138.25, 137.54, 136.79, 131.92, 131.81, 131.48, 130.09, 129.92,

128.70, 126.27, 125.53, 125.20, 124.80, 124.47, 123.84, 122.49, 121.81, 121.74, 116.70,
115.56, 110.15, 50.13; MS (+ESI): m/z 523.06 (M+H)+, 525.06 (M+2+H)+. Anal. Calcd for
(C26H18BrF3N4): C, 59.67; H, 3.47; N, 10.71; Found C, 59.61; H, 3.46; N, 10.78.
4-(4-Bromophenyl)-6-(1-(4-methylbenzyl)-1H-indol-3-yl)pyrimidin-2-amine (7e)
light
yellow solid, yield: 25.8%, mp: 120-122oC, Rf 0.42, IR (ν cm-1) 3369 (-NH2), 1648 (C=N),
1590 (C=N), 1577 (C=N), 872 (C-Br); 1H NMR (400 MHz, CDCl3) δ (ppm): 8.43-8.41 (d, J
= 8 Hz, 1H), 7.91-7.89 (m, 2H), 7.64-7.60 (t, J = 8 Hz, 2H), 7.51 (s, 1H), 7.37-7.34 (m, 2H)
7.31-7.27 (m, 2H), 7.15-7.11 (m, 2H), 7.08-7.05 (m, 2H), 5.34 (s, 2H), 5.07 (s, NH2) 2.32 (s,
13
3H); C NMR (100 MHz, CDCl3) 163.82, 163.44, 139.23, 137.84, 137.51, 137.09, 134.07,
133.34, 132.78, 131.85, 130.44, 129.85, 129.60, 128.58, 127.10, 126.98, 124.58, 121.82,
116.38, 115.47, 110.41, 103.62, 50.40, 21.11; MS (+ESI): m/z 469.09 (M+H)+, 471.09
(M+2+H)+. Anal. Calcd for (C26H21BrN4): C, 66.53; H, 4.51; N, 11.94; Found C, 66.67; H,
4.48; N, 11.96.
4-(4-Bromophenyl)-6-(1-(4-fluorobenzyl)-1H-indol-3-yl)pyrimidin-2-amine (7f) yellow
solid, yield: 37.8%, mp: 115-117oC, Rf 0.48, IR (ν cm-1) 3369 (-NH2), 1648 (C=N), 1581
(C=N), 1511 (C=N), 812 (C-Br); 1H NMR (400 MHz, CDCl3) δ (ppm): 8.42-8.40 (dd, J13 =
6.8 Hz J12 = 1.6 Hz, 1H), 7.94-7.91 (m, 3H), 7.62-7.60 (d, J = 8 Hz, 2H), 7.38 (s, 1H), 7.32-
13
7.28 (m, 3H), 7.16-7.12 (m, 2H), 7.02-6.98 (m, 2H), 5.35 (s, 2H), 5.11 (s, NH2); C NMR
(100 MHz, CDCl3) δ (ppm) 163.97, 163.47, 163.42, 137.37, 137.00, 132.19, 132.15, 131.89,
130.27, 128.69, 128.61, 126.29, 124.67, 122.93, 121.83, 121.56, 116.03, 115.82, 114.97,
110.30, 103.67, 49.91; MS (+ESI): m/z 473.06 (M+H)+, 475.06 (M+2+H)+. Anal. Calcd for
(C25H18BrFN4): C, 63.44; H, 3.83; N, 11.84; Found C, 63.69; H, 3.82; N, 11.88.
4-(4-Bromophenyl)-6-(1-(4-chlorobenzyl)-1H-indol-3-yl)pyrimidin-2-amine (7g) yellow
solid, yield: 24.6%, mp: 113-115oC, Rf 0.41; IR (ν cm-1) 3354 (-NH2), 1654 (C=N), 1568

1
(C=N), 1531 (C=N), 852 (C-Br); H NMR (400 MHz, CDCl3) δ (ppm): 8.40-8.38 (d, J =
8Hz, 1H), 7.89-7.86 (m, 3H), 7.58-7.56 (d, J = 8Hz, 2H), 7.33 (s, 1H), 7.26-7.17 (m, 5H),
7.03-7.01 (d, J = 8Hz, 2H), 5.35 (s, NH2), 5.25 (s, 2H); 13C NMR (100 MHz, CDCl3) δ
(ppm): 163.88, 163.48, 163.28, 137.25, 136.88, 134.89, 133.76, 131.81, 130.34, 129.24,
129.05, 128.87, 128.69, 128.57, 128.17, 128.11, 126.22, 124.22, 121.79, 121.54, 114.91,
110.26, 103.56, 49.82; MS (+ESI): m/z 491.03 (M+H)+, 493.03 (M+2+H)+. Anal. Calcd for
(C25H18BrClN4): C, 61.30; H, 3.70; N, 11.44; Found C, 61.41; H, 3.68; N, 11.46.
4-(4-Bromophenyl)-6-(1-(3-chlorobenzyl)-1H-indol-3-yl)pyrimidin-2-amine (7h) Yellow
solid, yield: 35.2%, mp: 120-122oC, Rf 0.44, IR (ν cm-1) 3368 (-NH2), 1653 (C=N), 1562
1
(C=N), 952 (C-Cl); H NMR (400 MHz, CDCl3) δ (ppm): 8.68-8.66 (m, 1H), 8.46 (s, 1H),
7.65-7.63 (d, J = 8 Hz, 2H), 7.55 (s, 1H), 7.47-7.44 (m, 1H), 7.34-7.26 (m, 4H), 7.24-7.18
(m, 4H), 5.50 (s, 2H); 13C NMR (100 MHz, CDCl3) δ (ppm): 163.82, 162.44, 140.07, 137.37,
137.20, 134.06, 132.03, 131.71, 130.64, 128.95, 127.93, 127.18, 126.54, 125.80, 123.22,
122.77, 121.24, 114.54, 110.61, 101.77, 47.24; MS (+ESI): m/z 491.03 (M+H)+, 493.03
(M+2+H)+. Anal. Calcd for (C25H18BrClN4): C, 61.30; H, 3.70; N, 11.44; Found C, 61.45; H,
3.66; N, 11.44.
4-(4-Bromophenyl)-6-(1-(2-chlorobenzyl)-1H-indol-3-yl)pyrimidin-2-amine
(7i)
light
yellow solid, yield: 32.5%, mp: 116-118oC, Rf 0.48, IR (ν cm-1) 3342 (-NH2), 1655 (C=N),
1
1571 (C=N), 1514 (C=N), 952 (C-Cl); H NMR (400 MHz, CDCl3) δ (ppm): 8.70-8.68 (m,
1H), 8.42 (s, 1H), 7.65-7.63 (d, J = 8 Hz, 2H), 7.54 (s, 1H), 7.49-7.47 (m, 1H), 7.34-7.26 (m,
13
4H), 7.24-7.18 (m, 4H), 5.59 (s, 2H); C NMR (100 MHz, CDCl3) 163.78, 163.16, 162.10,
136.91, 136.88, 134.42, 131.71, 131.22, 129.32, 129.09, 128.47, 128.20, 127.28, 125.98,
123.52, 122.80, 120.78, 114.17, 110.03, 101.20, 47.74; MS (+ESI): m/z 491.03 (M+H)+,
493.03 (M+2+H)+. Anal. Calcd for (C25H18BrClN4): C, 61.30; H, 3.70; N, 11.44; Found C,
61.39; H, 3.73; N, 11.43.

4.3.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. 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 (PV3193) was utilized. The 2X EGFR (ErbB1) L858R / Tyr 04 mixture was prepared
in 50 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 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 (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 10 µL Kinase Reaction consists of 3.9 - 39.3 ng EGFR (ErbB1) T790M and
2 µM Tyr 04 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 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
(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,
0.01% BRIJ-35, 10 mM MgCl2, 1 mM EGTA. After the 1 hour Kinase Reaction 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 at 445 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).
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:
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
Conflict of interest
Authors declare no conflict of interest.
Acknowledgement

This work was supported by the DBT under grant no. BT/PR8275/BID/7/455/2013.
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Figure legends:
Fig. 1: RMSD plot of 7c in complex with EGFR (T790M) for the time period of 30 ns.
Fig. 2: RMSD plot of 7h in complex with cMET for the time period of 30 ns.
Fig. 3: 3D interaction diagram along with contact summary of 7c in complex with EGFR
(T790M) after MD simulations of 30 ns.
Fig. 4: 3D interaction diagram along with contact summary of 7h in complex with cMET
after MD simulations of 30 ns.
Scheme 1. Synthetic scheme of designed indole-pyrimidine analogues.

Table 1 Designed compounds with their binding energies and predicted activity.
S.No. Compound
MM-GBSA
binding energy
(EGFR(T790M)
-75.706
Predicted activity-
EGFR(T790M)
(µM)
MM-GBSA
binding energy
(cMET)
Predicted
activity-
cMET (µM)
0.124
1.
2.
3.
4.
5.
6.
7.
8.
9.
0.827
-79.450
7a
7b
7c
7d
7e
7f
-63.435
-71.350
-70.200
-49.003
-61.855
-73.181
-71.889
-60.450
1.627
0.158
0.941
1.610
1.581
1.610
0.982
1.490
-68.614
-66.319
-73.562
-81.705
-81.573
-68.174
-84.334
-82.288
0.128
0.115
0.123
0.135
0.126
0.137
0.124
0.125
7g
7h
7i
Table 2. In-vitro kinase inhibitory data of designed compounds against EGFR (T790M),
EGFR (L858R), cMET
S.NO. Compound
ID
R (subtituents)
EGFR
(T790M)
(µM)
EGFR
cMET
(µM)
(L858R) (µM)
0.913
0.452
0.099
0.998
0.866
0.336
0.587
0.666
0.518
0.571
0.595
0.293
0.588
0.421
0.183
0.101
1.
2.
3.
4.
5.
6.
7.
8.
7a
7b
7c
7d
7e
7f
4-ethyl
4-isobutyl
4-isopropyl
3-trifluoromethyl
4-methyl
0.097
0.569
0.094
0.494
0.757
0.304
0.287
0.195
4-fluoro
7g
7h
4-chloro
3-chloro

7i
2-chloro
---------
---------
0.713
0.907
--------
0.827
0.032
-------
0.507
------
0.030
9.
Erlotinib
XL184
10.
11.

Highlights:
-
-
-
-
In-silico tools were used to identify scaffold with dual kinase inhibitory potential
Focus was laid on hydrophobic interaction with mutant M790
Molecules, also, having good binding affinity with cMET were selected
In-vitro evaluation against EGFR (L858R), EGFR (T790M) and cMET was
performed

Molecular dynamics guided development of indole based dual inhibitors of EGFR
(T790M) and c-MET
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
Graphical Abstract