Structure-based drug design and biological evaluation of 2-acetamidobenzothiazole derivative as EGFR kinase inhibitor

Article abstract of DOI:10.3109/14756366.2014.887707

EGFR tyrosine kinase has been reported mainly in 40-80% of non-small lung cancers, in addition to colon and breast cancers. In this study, we illustrate the synthesis of a highly potent antitumor agent. The synthesized compound 4 was screened at NCI, USA, for antitumor activity against non-small lung cancer, colon cancer and breast cancer cell lines. Results indicated that this compound is more potent antitumor agent compared to erlotinib against all tested cell lines except breast cancer (MDA-MB-468) cell line. In addition, it was tested initially at a single dose concentration of 100 μM over 11 different kinases. At this concentration, 94.45% inhibition of the enzymatic activity of EGFR kinase was observed, while the inhibition in activity was below 55% in all other kinases. Compound 4 was further tested in a 10-dose IC₅₀ mode and showed IC₅₀ value of 0.239 μM for EGFR kinase. In vivo acute toxicity of this compound was also tested.

Full text of DOI:10.3109/14756366.2014.887707

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ISSN: 1475-6366 (print), 1475-6374 (electronic)
J Enzyme Inhib Med Chem, Early Online: 1–6
2014 Informa UK Ltd. DOI: 10.3109/14756366.2014.887707
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SHORT COMMUNICATION
Structure-based drug design and biological evaluation of
2-acetamidobenzothiazole derivative as EGFR kinase inhibitor
Moustafa T. Gabr, Nadia S. El-Gohary, Eman R. El-Bendary, and Mohamed M. El-Kerdawy
Department of Medicinal Chemistry, Faculty of Pharmacy, Mansoura University, Mansoura, Egypt
Abstract
Keywords
EGFR tyrosine kinase has been reported mainly in 40–80% of non-small lung cancers, in
addition to colon and breast cancers. In this study, we illustrate the synthesis of a highly potent
antitumor agent. The synthesized compound 4 was screened at NCI, USA, for antitumor activity
against non-small lung cancer, colon cancer and breast cancer cell lines. Results indicated that
this compound is more potent antitumor agent compared to erlotinib against all tested cell
lines except breast cancer (MDA-MB-468) cell line. In addition, it was tested initially at a single
dose concentration of 100 mM over 11 different kinases. At this concentration, 94.45% inhibition
of the enzymatic activity of EGFR kinase was observed, while the inhibition in activity was
below 55% in all other kinases. Compound 4 was further tested in a 10-dose IC50 mode
and showed IC50 value of 0.239 mM for EGFR kinase. In vivo acute toxicity of this compound was
also tested.
Acetamidobenzothiazole, breast cancer,
colon cancer, non-small lung cancer,
EGFR tyrosine kinase inhibitor, in vivo acute
toxicity
History
Received 15 December 2013
Revised 22 January 2014
Accepted 23 January 2014
Published online 5 March 2014
Introduction
EGFR tyrosine kinase. With this aim, a 3D pharmacophore
model was created based on erlotinib binding pose to the active
site of EGFR tyrosine kinase comprising two hydrophobic
features, two hydrogen bond acceptors and one hydrogen bond
donor. Herein, we report the discovery of a potent and highly
selective EGFR tyrosine kinase inhibitor 4. The structure based
design strategy developed utilizing QSAR techniques, enabled
the understanding of the pharmacophoric requirements of
erlotinib as EGFR tyrosine kinase inhibitor (Supplementary
Figure S1).
The epidermal growth factor receptor (EGFR) is a transmembrane
glycoprotein belonging to the human epidermal receptor (HER)
family1. It plays a vital role in signal transduction pathways,
regulating key cellular functions such as cell proliferation,
survival, adhesion, migration and differentiation2. The binding
of a ligand to EGFR induces conformational changes within the
receptor which increase its intrinsic catalytic activity of a tyrosine
kinase and result in autophosphorylation, which is necessary for
biological activity3,4. Mutations that lead to EGFR overexpression
or over activity have been associated with a variety of human
cancers, including lung5, colon6 and breast7 cancers. Therefore,
Experimental
inhibitors of EGFR-inhibiting EGFR kinase activity by competing Molecular modeling and computational studies
with its cognate ligands-may potentially constitute a new class of
Protein structure preparation
effective drugs in clinical use or cancer therapy8,9
.
Anilinoquinazolines are the most developed class of drugs The crystal structure of EGFR kinase domain (PDB ID: 1M17)
that inhibit EGFR tyrosine kinase intracellularly10,11
in complex with an irreversible inhibitor was obtained from
Anilinoquinazoline-containing compounds, erlotinib the protein data bank (PDB; http://www.rcsb.org/pdb/home/
.
(TarcevaÕ)12,13 and gefitinib (IressaÕ)14 have been approved for home.do). Refinement of crude PDB structure of receptor was
chemotherapeutic treatment of patients with advanced non-small performed. Polar hydrogens were added, Kollman charges were
cell lung cancer. Our strategy is directed toward designing ligands assigned and atomic solvation parameters were added, the internal
which are structurally similar to the basic skeleton, 4-anilinoqui- degrees of freedom and torsions were set for all the designed
nazoline of erlotinib through replacing quinazoline ring with small molecules. The optimized receptor was then saved as mol
benzothiazole since both are isosteric with adenine portion of file and used for docking simulation.
ATP and can mimic the ATP competitive binding regions of
Ligand structure preparation
The 2D structure of the compound 4 was built and then converted
into the 3D with the help of vLife MDS 3.0 software. The 3D
structure was then energetically minimized up to the rms gradient
Address for correspondence: Nadia S. El-Gohary, Department of
of 0.01 using CHARMM22 force field. All conformers were then
Medicinal Chemistry, Faculty of Pharmacy, Mansoura University,
energetically minimized up to the rms gradient of 0.01 and then
saved in separate folder.
Mansoura 35516, Egypt. Tel: +2 010 00326839. Fax: +2 050 2247496.
E-mail: dr.nadiaelgohary@yahoo.com

2
M. T. Gabr et al.
J Enzyme Inhib Med Chem, Early Online: 1–6
Docking protocol
Yield 58%, m.p. 162–163 ꢀC. IR spectrum (KBr, v, cmꢁ1):
3255, 3210 (NH2), 3115 (2NH), 1655 (C ¼ O). 1H NMR
spectrum: (Acetone-d6, ꢀ ppm): 2.96 (s, 2H, NH2), 4.45 (s, 2H,
CH2), 6.96 (s, 2H, 2NH), 7.24 (d, 1H, Ar–H), 7.37 (d, 1H, Ar–H),
7.71 (s, 1H, Ar–H). HRMS: m/z (ESI) Calcd for C9H8ClN4OSꢁ,
[M–H]ꢁ: 255.0186; found: 255.0189.
Docking simulation was done by SwissDock software15. All the
conformers were virtually docked at the defined cavity of the
receptor. The number of placements was fixed at 30 placements
and the rotation angle was fixed at 30ꢀ. By rotation angle, the
ligand gets rotated for different poses. By placements, the method
will check all the 30 possible placements into the active site
pocket and results out few best placements out of 30. For each
ligand, all the conformers with their best placements and their
Synthesis of ethyl 1-[2-((6-chlorobenzothiazol-2-yl)amino)-2-
oxoethyl]-5-hydroxy-1H-pyrazole-4-carboxylate (4)
dock score will be saved in output folder. The method also A mixture of compound 3 (0.257 g, 0.001 mol), diethyl ethox-
highlights the best placement for the best conformer of one ymethylenemalonate (0.216 g, 0.001 mol) and anhydrous potas-
particular ligand which is having best (minimum) dock score. sium carbonate (0.207 g, 0.0015 mol) in acetonitrile (15 mL) was
After docking simulation, the best docked conformer of com- heated at reflux temperature for 12 h. After cooling, the solution
pound 4 and receptor was merged and its complex was then was acidified with dilute hydrochloric acid and the precipitated
˚
energetically optimized by defining the radius of 10 A measured dark yellow solid was collected by filtration, dried and crystal-
from the docked ligand. Stepwise energy optimization was lized from ethanol.
done by first hydrogen, second side chains and finally the
backbone of receptor.
Yield 45%, m.p. 195–197 ꢀC. IR spectrum (KBr, v, cmꢁ1):
3260 (OH), 3150 (NH), 1710 (COOC2H5), 1685 (C ¼ O). 1H
NMR spectrum: (CDCl3, ꢀ ppm): 1.30 (t, 3H, CH2CH3), 3.55
(s, 2H, CH2), 4.05–4.35 (q, 2H, CH2CH3), 7.20–7.80 (m, 4H,
Ar–H, pyrazole–H), 10.40 (s, 1H, OH), 11.50 (s, 1H, NH). 13C
NMR: spectrum: (CDCl3, ꢀ ppm): 14.6, 56.9, 61.0, 95.8, 121.0,
125.8, 128.0, 132.0, 135.5, 145.8, 157.2, 161.4, 167.2. HRMS:
m/z (ESI) Calcd for C15H12ClN4O4Sꢁ, [M–H]ꢁ: 379.0346; found:
379.0352. CHN Analysis for C15H13ClN4O4S: Found
(Calculated): C: 47.56 (47.31), H: 3.25 (3.44), N: 14.47 (14.71).
Synthesis
Melting points (ꢀC) were recorded on Fisher–Johns melting point
apparatus and are uncorrected. The infrared spectra were recorded
using Nicolet Magna-IR Fourier-Transform 560 Spectrometer
(v in cmꢁ1) at the Department of Chemistry, Georgia State
University, USA. Nuclear magnetic resonance (1H and 13C NMR)
spectra were obtained on Bruker Avance 400 MHz spectrometer
using CDCl3, and Acetone-d6 as solvents at the Department of
Chemistry, Georgia State University, USA. The chemical shifts
are expressed in d ppm using tetramethylsilane (TMS) as internal
reference. Mass spectra were recorded on nano LC-Q-TOF micro
(Waters Micromass) spectrometer in negative ion mode as
necessary at the Department of Chemistry, Georgia State
University, USA. Reaction times were monitored using TLC
plates, Silica gel 60 F254 pre-coated (E. Merck) and the spots
were visualized by UV(366 nm). Chloroform:methanol (9:1) was
adopted as an elution solvent. 2-Amino-6-chlorobenzothiazole (1)
was purchased from Richest Co., China.
A general approach to synthesize compound 4 is outlined in
Scheme 1, started with the synthesis of 2-chloro-N-(6-chloroben-
zothiazol-2-yl)acetamide (2). Chloroacetyl chloride (1.13 g,
0.01 mol) was added slowly with stirring to a mixture of
2-amino-6-chlorobenzothiazole (1) (1.85 g, 0.01 mol) and triethy-
lamine (0.1 mL) in carbon tetrachloride (20 mL). The mixture
was heated at reflux temprature for 12 h, then the solvent was
evaporated under reduced pressure. The remaining solid was
crystallized from ethanol. Yield 61%, m.p. 140–142 ꢀC16.
Tyrosine kinase assay
Initial screening over 11 kinases
The tested compound 4 was dissolved in DMSO and tested at a
single concentration of 100 mM with a final DMSO concentration
of 2%. Compound 4 was then added to reaction plates containing
the particular kinase in assay buffer [20 mM 4-(2-hydroxyethyl)-
1-piperazineethanesulfonic acid (HEPES), pH 7.5, 10 mM MgCl2,
1 mM ethylene glycol tetra-acetic acid (EGTA), 0.02% Brij35,
0.02 mg/mL bovine serum albumin (BSA), 0.1 mM Na3VO4,
2 mM dithiothreitol (DTT), 1% DMSO]. Reactions were initiated
by the addition of a mixture of ATP (Sigma, St. Louis, MO) and
33P ATP (Perkin Elmer, Waltham, MA) to a final concentration of
10 mM. Reactions were carried out at room temperature for 2 h,
followed by spotting of the reactions onto P81 ion exchange filter
paper (Whatman Inc., Piscataway, NJ). Unbound phosphate was
removed by extensive washing of filters in 0.75% phosphoric
acid17. Kinase activity data was reported as the percentage
remaining enzyme activity after subtraction of enzyme inhibitory
activity of DMSO control reactions as background (Table 1).
Synthesis of N-(6-chlorobenzothiazol-2-yl)-2-hydrazinylacetamide
(3)
Testing against EGFR kinase
A mixture of compound 2 (2.6 g, 0.01 mol) and hydrazine hydrate
99% (5 g, 0.1 mol) in ethanol (30 mL) was heated at reflux
temprature for 14 h. The reaction mixture was cooled and the
precipitated solid was collected by filtration, washed with water,
dried and crystallized from ethanol.
Compound 4 was tested in a 10-dose IC50 mode with threefold
serial dilutions starting at 20 mM. Staurosporine was used as a
control compound in a 10-dose IC50 mode with fivefold serial
dilutions starting at 20 mM. Reaction was carried out at 10 mM
ATP concentration17. IC50 value of 4 was calculated.
COOC2H5
HO
O
O
O
N
N
S
N
S
N
NH NH2
N
Cl
iii
i
ii
NH2
N
N
H
N
H
N
H
S
S
Cl
Cl
Cl
Cl
1
2
3
4
Scheme 1. Reaction conditions and reagents: (i) chloroacetyl chloride, CCl4, 12 h, 61%; (ii) hydrazine hydrate, C2H5OH, reflux, 14h, 58%; (iii) diethyl
ethoxymethylenemalonate, anhydrous K2CO3, CH3CN, reflux, 12h, 45%.

DOI: 10.3109/14756366.2014.887707
2-Acetamidobenzothiazole derivative as EGFR-TK inhibitor
3
Table 1. Percentages of enzymatic inhibition exerted by compound 4 on 11 human protein and lipid kinases.
% Kinase inhibition
Tyrosine kinases contain
SH2 Domain
Tyrosine kinases contain
SH2 and SH3 Domains
Other human protein or lipid kinases
Compounds
TYK2
LCK
JAK3
ABL-1
BTK
LYN
Cyclin D1
MEK1
mTOR
AKT1
EGFR
4
9.2
99.6
nt
43.3
99.5
nt
9.1
99.8
nt
11.7
99.5
nt
8.1
99.7
nt
18.7
99.8
nt
23.7
100.1
nt
15.7
99.7
nt
54.3
nt
81.3
25.0
99.3
nt
94.4
97.8
nt
Staurosporine
LY294002
Bold underlined value represents the best result. % Activity in each enzyme is the mean of two different readings. Test compound was used in a single
dose concentration of 100 mM. Reference compounds were used in a single dose concentration of 20 mM. % Inhibition was calculated after subtraction
of the activity of DMSO. nt, Indicates compound not tested against enzyme.
Antitumor screening
Table 2. GI50 values (mM) of compound 4 and erlotinib over cell lines of
non-small lung cancer, colon cancer and breast cancer subpanels.
Full in vitro five-dose antitumor assay
The human tumor cell lines of the cancer screening panel were
GI50
grown in RPMI 1640 medium containing 5% fetal bovine serum
Subpanel tumor cell lines
Compound 4
Erlotinib
and 2 mM L-glutamine. For a typical screening experiment, cells
were inoculated into multiwell microtiter plates in 100 mL at
plating densities ranging from 5000 to 40 000 cells/well depend-
ing on the doubling time of individual cell lines. After cell
inoculation, the microtiter plates were incubated at 37 ꢀC, 5%
CO2, 95% air and 100% relative humidity for 24 h and then two
plates of each cell line were fixed in situ with trichloroacetic acid
(TCA) to represent a measurement of the cell population for each
cell line at the time of compound addition. The tested compound
was solubilized in DMSO at 400-fold the desired final maximum
test concentration and stored frozen prior to use. At the time of
compound addition, an aliquot of frozen concentrate was thawed
and diluted to twice the desired final maximum test concentration
with complete medium containing 50 mg/mL gentamicin.
Additional 4-, 10-fold or 1/2 log serial dilutions were made to
provide a total of five compound concentrations plus control.
Aliquots of 100 mL of these dilutions were added to the
appropriate microtiter wells already containing 100 mL of
medium, resulting in the required final compound concentrations.
Following compound addition, the plates were incubated for
additional 48 h at 37 ꢀC, 5% CO2, 95% air and 100% relative
humidity. For adherent cells, the assay was terminated by the
addition of cold TCA. Cells were fixed in situ by gentle addition
of 50 mL of cold 50% (w/v) TCA (final concentration, 10% TCA)
and incubated for 60 min at 4 ꢀC. The supernatant was discarded,
and the plates were washed five times with tap water and air dried.
Sulforhodamine B (SRB) solution (100 mL) at 0.4% (w/v) in 1%
acetic acid was added to each well, and plates were incubated for
10 min at room temperature. After staining, unbound dye was
removed by washing five times with 1% acetic acid and the plates
were air dried. Bound stain was subsequently solubilized with 10
mM trizma base and the absorbance was read on an automated
plate reader at a wavelength of 515 nm18–20. GI50 values (mM) of
compound 4 and erlotinib are shown in Table 2.
Non-small lung cancer
A549/ATCC
HOP-62
HOP-92
NCI-H226
NCI-H23
NCI-H460
NCI-H522a
Colon cancer
COLO 205
HCC-2998
HCT-116
HCT-15
HT29
SW-620
Breast cancer
MCF-7
MDA-MB-231
HS 578T
BT-549
MDA-MB-468
T-47D
1.31
10
0.353
0.196
1.43
1.09
1.72
15.8
3.98
39.8
31.6
6.3
0.0573
1.00
1.53
2.11
1.38
1.44
0.353
1.69
50.1
79.4
6.31
3.98
63.10
79.4
1.36
0.317
1.71
1.27
0.56
1.32
100
6.31
6.31
31.6
0.126
3.98
The cell lines overexpressing EGFR in each tumor subpanel and their
GI50 values are bold and underlined.
study using the tested compound in different concentrations and
mice were observed for toxic symptoms, signs of poisoning and
mortality continuously after dosing21. Finally, the number of
survivors in each group was noted after 24 h. The arithmetical
method of Karber was used for the determination of LD50 values
(mg/kg) of the selected compounds22.
Results and discussion
Molecular modeling and computational studies
With the software LigandScout23, a 3D interaction map was
generated from the ligand (erlotinib) co-crystallized in the protein
In vivo acute toxicity testing
Adult male Swiss albino mice (weighing 20–25 g) were obtained binding site (PDB ID: 1M17; http://www.rcsb.org/pdb/home/
from animal house, Department of Pharmacology, Faculty of home.do). The generated structure-based pharmacophore showed
Pharmacy, Mansoura University, Mansoura, Egypt. Animals were that two main hydrophobic features accommodating the hydro-
housed in microlon boxes in a controlled environment (tempera- phobic residues of the ligand in the binding pocket
ture 25 2 ꢀC) with a regular 12 h light/12 h dark and were (Supplementary Figure S2). Compound 4 forms impressive
allowed free access to standard laboratory food and water.
alignment with the 3D pharmacophore of erlotinib binding pose
Animals were divided into groups each consisting of six mice. which may indicate that both compounds possess similar
One of these groups served as control and received normal saline. arrangements of pharmacophoric characters required for activity
The other groups were subjected to acute intraperitoneal toxicity (Supplementary Figure S3).

4
M. T. Gabr et al.
J Enzyme Inhib Med Chem, Early Online: 1–6
The SwissDock software15 was used to find plausible docking the benzothiazole ring interacted with the residues Lys-721, Met-
poses for compound 4 into the crystal structure of EGFR kinase 769, Leu-768, Leu-820, Pro-770, Leu-694 and Gly-772, while the
domain (PDB ID: 1M17; http://www.rcsb.org/pdb/home/ pyrazole moiety interacted with the residues Asp-831, Val-702
home.do). Evaluation was done using two scoring functions; and Phe-699 of the second enzyme hydrophobic subsite.
simple fitness and full fitness (Supplementary Table S1). Furthermore, it was involved in three hydrogen bonds with the
Compound 4 was deeply embedded into the ATP-binding cleft active site of EGFR tyrosine kinase (Figure 1).
of EGFR tyrosine kinase (Supplementary Figure S4). Analysis of
the docking pose of compound 4 using Lead IT software showed Chemistry
good complementarities between the docked ligand and the
The synthetic protocol for compound 4 is outlined in Scheme 1.
hydrophobic subsites of the enzymatic cavity. In the first subsite,
The starting compound, 2-amino-6-chlorobenzothiazole (1) was
allowed to react with chloroacetyl chloride in carbon tetrachloride
to produce the acetamide derivative 216. Reaction of 2 with
hydrazine hydrate in ethanol gave the hydrazinylacetamide analog
3. Condensation of 3 with diethyl ethoxymethylenemalonate in
acetonitrile and in the presence of anhydrous potassium carbonate
gave the targeted ethyl pyrazole-4-carboxylate derivative 4.
Tyrosine kinase assay
In vitro profiling of compound 4 was performed at Reaction
Biology Corporation to assess its inhibitory activity of EGFR
tyrosine kinase. Kinase activity was assessed using HotSpot
technology, a miniaturized radioisotope-based filter binding
assay17. Kinase activity data was reported as the percent
remaining enzyme activity after subtraction of enzyme inhibitory
activity of the negative control (DMSO). Results are presented as
percentage enzyme inhibition and compared to staurosporine as a
reference EGFR tyrosine kinase inhibitor. Compound 4 displayed
94.4% inhibition of the enzymatic activity of EGFR kinase at
100 mM (Figure 2 and Table 1).
Compound 4 was further tested over EGFR kinase in order to
determine its IC50 value, where a 10-dose IC50 mode with
threefold serial dilutions starting at 20 mM concentration was
applied against staurosporine24–26 as a reference kinase inhibitor.
Compound 4 showed IC50 value of 0.239 mM, while the IC50
value for the non-selective kinase inhibitor staurosporine was
0.0533 mM.
Figure 1. 2D Interaction of compound 4 with the binding site of EGFR-
TK. Dashed lines represent hydrogen bonds. Hydrophobic interactions are
shown by green solid lines.
Figure 2. Kinase profile assay of compound 4 against different human protein and lipid kinases.

DOI: 10.3109/14756366.2014.887707
2-Acetamidobenzothiazole derivative as EGFR-TK inhibitor
5
Compound 4 was also examined over other 10 different human
kinases containing SH2 or both SH2 and SH3 domains and other
human protein or lipid kinases adopting the kinase profile assay17
using staurosporine and LY294002 as control compounds. Results
showed that compound 4 exhibited weak to moderate inhibitory
activity in the range of 8.1–54.3% with the highest inhibitory
activity against mTOR kinase with percentage inhibition value of
54.3% (Figure 2 and Table 1).
The high selectivity of compound 4 toward EGFR kinase
might be attributed to difference in the geometry of the binding
pocket of this enzyme that enables the fitting and interaction of
compound 4. It is worth mentioning that the mechanism of its
unique inhibitory activity is still unclear.
Antitumor screening
Comparison of the antitumor activity of compound 4 with the
activity of erlotinib was performed against non-small lung cancer
(NCI-H522) cell line which harbors EGFR wild type27. Moreover,
colon cancer (HCT-116, HCT-15 and HT-29) cell lines were
selected for the comparison since they express high levels of
EGFR28–30, e.g. HCT-116 and HT-29 cell lines express approxi-
mately 100 000–150 000 EGFRs per cell29. Breast cancer (MDA-
MB-468 and MDA-MB-231) cell lines were also selected for the
same comparison, whereas, MDA-MB-468 cell line was proved
by Western blot to express the highest EGFR level compared to
colon cancer (HCT-116, HT-29, IEC-6 and Caco-2) and breast
cancer (SKBR-3) cell lines31. In addition, MDA-MB-231 cell line
expresses high levels of EGFR compared with MCF-10 A cells32.
Compound 4 was evaluated for its antitumor activity in
accordance with the current protocol of the National Cancer
Institute (NCI), USA18–20. The data was reported as mean-graph
of the percent growth of the treated cells, and presented as
percentage growth inhibition (GI%). It exhibited lethal effects
(4100% inhibition) at a single dose (10 mM) against non-small
lung cancer (NCI-H522), colon cancer (HCT-116, HCT-15 and
HT29) and breast cancer (MDA-MB-468 and MDA-MB-231) cell
lines in which EGFR is overexpressed in varying levels (Figure 3).
Therefore, it was carried over by the NCI for the five-dose
screening.
Figure 3. % Inhibition expressed by compound 4 at a single-dose
concentration of 10 mM over the selected cancer cell lines.
such as dogs. It provides an objective measure to compare and
rank the toxicity of substances. The LD50 is defined as the amount
of the substance required to kill 50% of a given test population,
it is usually expressed as the amount of substance per kg of body
weight. Compound 4 was screened for potential acute toxicity
in mice21 and the dose (mg/kg) required to kill 50% of animals
(LD50) within 24 h was calculated. Compound 4 showed LD50
value of 365 mg/kg, while the LD50 value for the reference
antitumor agent, 5-fluorouracil was 115 mg/kg.
Non-small lung cancer (NCI-H522), colon cancer (HCT-116,
HCT-15, and HT29) and breast cancer (MDA-MB-468 and MDA-
MB-231) cell lines were incubated with five concentrations
(0.01–100 mM) of compound 4 and were used to create log
concentration – % growth inhibition curves. The GI50 values of
compound 4 and erlotinib against the selected cancer cell lines
are listed in Table 2. Results indicated that compound 4 is
more potent than erlotinib against all selected cell lines except
MDA-MB-468. It showed the lowest GI50 value (57.3 nM) against
non-small lung (NCI-H522) cancer cell line.
Conclusion
In conclusion, a highly potent, safe and selective antitumor agent
as well as EGFR tyrosine kinase inhibitor has been synthesized
and can be used as a promising lead for new selective inhibitors
for EGFR tyrosine kinase. Also, it is worth mentioning that the
development of new selective inhibitors for such kinase might
open the way for new selective therapeutics for non-small lung,
colon and breast cancers.
Further investigation of antitumor activity of compound 4
against additional cancer cell lines belonging to non-small lung
cancer, colon cancer and breast cancer subpanels is presented in
Table 2. Compound 4 was proved to be highly selective against
non-small lung cancer (NCI-H522) cell line compared to the other
cell lines belonging to the same subpanel. Moreover, it showed
moderate selectivity against colon cancer (HT29) cell line
compared to the other cell lines belonging to the same subpanel.
With regard to the breast cancer subpanel, compound 4 showed
GI50 values in the submicromolar range only against breast cancer
(MDA-MB-468 and MDA-MB-231) cell lines.
Acknowledgements
The authors would like to express their sincere thanks to the staff
members of the Department of Health and Human Services, National
Cancer Institute (NCI), Bethesda, Maryland, USA for performing the
antitumor testing of the newly synthesized compound. Thanks to Reaction
Biology Corporation, USA for performing the kinase profile assay.
Declaration of interest
The authors report no conflicts of interest. The authors alone are
responsible for the content and writing of this article.
In vivo acute toxicity testing
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