Novel Benzimidazole Analogs as Inhibitors of EGFR Tyrosine Kinase

Article abstract of DOI:10.1111/j.1747-0285.2012.01407.x

A series of new benzimidazole congeners were synthesized, and their structures were elucidated on the basis of elemental analyses and spectral studies (¹H NMR, FT-IR and EI-MS). Preliminary pharmacokinetic studies showed a promising outlook for further in vivo evaluation. The newly synthesized compounds were tested in vitro on human breast carcinoma cell line (MCF-7) in which EGFR is highly expressed. Most of the tested compounds exhibited antitumor activity with IC₅₀ values in the micro to nano molar range.

Full text of DOI:10.1111/j.1747-0285.2012.01407.x

ª 2012 John Wiley & Sons A/S
doi: 10.1111/j.1747-0285.2012.01407.x
Chem Biol Drug Des 2012
Research Letter
Novel Benzimidazole Analogs as Inhibitors of
EGFR Tyrosine Kinase
Soni Yadav1,2, Deepa Sinha3, Sanjay K.
cancer therapy. Protein kinases (PTKs) catalyze the phosphorylation
Singh2 and Vinay K. Singh1,*
of tyrosine and serine ⁄ threonine residues in various proteins
involved in the regulation of all functions (21). They can be broadly
classified as receptor such as epidermal growth factor receptor
(EGFR), or non-receptor kinases. Inappropriate or uncontrolled acti-
vation of many of these kinases, by over-expression, constitutive
activation, or mutation, has been shown to result in uncontrolled
cell growth (22). The epidermal growth factor receptor belongs to
the family of transmembrane growth factor receptor PTKs. The
EGFR erbB1 and erbB2 PTKs have been identified as interesting tar-
gets for medicinal chemistry programs especially in cancer therapy.
Excellent descriptions of the involvement of erbB family proteins in
cell physiology and disease applications have been reported and
reviewed (23–26).
1Department of Chemistry, DrKNMIET, Modinagar, Meerut 201204,
India
2Department of Applied Sciences, Institute of Engineering and
Technology, Lucknow 226021, India
3Department of Applied Sciences, HMRITM (Affiliated to GGSIP
University), Delhi 110036, India
*Corresponding author: V. K. Singh, vinayk2003@rediffmail.com
A series of new benzimidazole congeners were
synthesized, and their structures were elucidated
on the basis of elemental analyses and spectral
studies (1H NMR, FT-IR and EI-MS). Preliminary
pharmacokinetic studies showed a promising out-
look for further in vivo evaluation. The newly syn-
thesized compounds were tested in vitro on
human breast carcinoma cell line (MCF-7) in which
EGFR is highly expressed. Most of the tested com-
pounds exhibited antitumor activity with IC50 val-
ues in the micro to nano molar range.
Overexpression of these receptors was found in a number of can-
cers (e.g., breast, ovarian, colon, and prostate); their expression lev-
els often correlate with vascularity and are associated with poor
prognosis in patients (27). Inhibitors of the EGFR PTK are therefore
expected to have great therapeutic potential in the treatment of
malignant and non-malignant epithelial diseases. Drug discovery
efforts have targeted this aberrant kinase activity in cancer, asthma,
psoriasis, and inflammation (28). Recent advances in the identifica-
tion of erbB family kinase inhibitors have created hope for the mod-
ulation of uncontrolled cell growth in cancer therapy for solid
tumors (29). This strongly suggests that these targets represent
drug intervention opportunities because of pivotal role in governing
cellular proliferation, survival, and metastasis. A great number of
different structural classes of tyrosine kinase inhibitors has been
reported and reviewed (23–25). Recent success in the clinical evalu-
ation of EGFR tyrosine kinase (TK) inhibitors such as gefitinib and
erlotinib strongly suggests small molecular EGFR-TK inhibitors to be
promising new anticancer drugs. The most promising small molecule
selective EGFR-TK inhibitors include pyrrolopyrimidines, aminoqui-
nazolines, and benzimidazole analogs.
Key words: antitumor, benzimidazole, EGFR, nanomolar, spectros-
copy
Received 13 February 2012, revised 19 March 2012 and accepted for
publication 9 April 2012
Benzimidazoles and related derivatives are a class of fused hetero-
cycles that are of considerable interest because of the diverse
range of their biological properties, for example, anticancer, diuretic,
anti-inflammatory, anticonvulsant, and antihypertensive activities (1–
9). They also demonstrate several other useful and interesting prop-
erties such as hypertensive adrenergic blocker, selective phosphodi-
esterase inhibitor, against prostate disorders, and dihydrofoalate
reductase inhibitor (10–18).
In the same direction, and in continuing effort to find more potent
selective lead compound, herein, we describe a series of benzimid-
azole conjugated with quinazolone derivatives mimicking gefitinib
and erlotinib as possible antitumor agents that may act through
EGFR inhibition.
In addition, some derivatives are calcium antagonists and share the
common property of interfering with the influx of extracellular calcium
via the calcium L channel. Recently, quinozolone chemistry has found
new direction because of some resemblance with folic acid (19,20).
Protein tyrosine kinases are enzymes involved in many cellular pro-
cesses such as cell proliferation, metabolism, survival, and apopto-
sis. Several protein tyrosine kinases are known to be activated in
cancer cells and to drive tumor growth and progression. Blocking
tyrosine kinase activity therefore represents a rational approach to
Chemistry
The chemicals used in the synthesis are purchased from Aldrich
and Merck and are of analytical grades. Analytical thin layer chro-
1

Yadav et al.
matography (TLC) on silica gel plates containing UV indicator was
employed routinely to follow the course of reactions and to check
the purity of products. All reagents and solvents were purified
and dried by standard techniques. IR spectra were recorded on a
PerkinElmer FT-IR series using KBr cell (PerkinElmer, Cambridge,
MA, USA). NMR spectra were measured in CDCl3 by Bruker
400 MHz apparatus with TMS as an internal standard. EI-MS spec-
tra were recorded on a JEOL SX102 ⁄ DA (KV 10 mA) instrument.
Elemental analysis was performed on elemental analyzer Gmbh vari-
able system. Radio complexation and radio chemical purity was
checked by instant strip chromatography (silica gel impregnated
paper chromatography) with ITLC-SG (Gellman Sciences, Ann Arbor,
MI, USA). The gamma scintillation counting was carried out at ECA
(Electronic Corporation of India Ltd., Hyderabad, India) Gamma Ray
Spectrometer K 2700 B. All the reaction steps were monitored by
TLC [chloroform:methanol:hexane 4:3:1].
QSAR analysis
The QSAR investigations were carried out by the linear-free energy
relationship (LFER) model proposed by Hansch and Leo. The selec-
tion of parameters is the first step in any QSPR study. In this study,
parameters that were considered relevant to the Schiff base series
(electronic, hydrophobic, and steric) were selected and considered
as consistent, which included molar refractivity, Vander Waals vol-
ume (VDW), Connolly accessible area, Connolly molecular area, Con-
nolly solvent excluded area, dipole–dipole energy (DD), partition
coefficient (logP), HOMO, etc. The above-mentioned parameters
were calculated by MM2 studies using Chem 3D 6.0 software
(PerkinElmer Inc.). Geometries of all compounds were completely
optimized by the same software package.
Scheme 1: Chemical Scheme for synthesis of novel benzimida-
zoles.
left under refrigeration for 24 h. Subsequently, the acidified solution
was poured into ice-cold water (100 mL) slowly and carefully with
constant stirring. A solid that separated out was filtered off and
washed with water repeatedly to remove any sulphonated products
formed during the course of the reaction. The crude compound thus
obtained was treated with an aqueous solution of sodium bicarbon-
ate (10%). Evolution of carbon dioxide as effervescence was
observed. A little more sodium bicarbonate solution was added to
ensure the complete reaction with the carboxylic acid. When there
was no more effervescence, the remaining solid was filtered off,
and the filtrate was neutralized with diluted hydrochloric acid. The
solid thus obtained was filtered off and washed with water. It was
dried in vacuo and recrystallized from acetone as white crystalline
needles, m.p. 148–149 ꢀC [149 ꢀC]13, yield, 70%.
A classical Hansch multivariate regression analysis using the least-
square method was chosen to derive QSAR equations for the data
set. The level of significance of each coefficient was judged by sta-
tistical procedure such as F tests. Statistic analysis was carried out
by employing the method of least square with stepwise selection
and elimination procedure. For each equation, several indices of
best fit were considered: the regression coefficient 'r', the standard
deviation 's', and the measure of level of statistical significance 'F'.
Two models were found which are as follows:
QSAR Model for benzimidazole congeners (Model 1)
ꢀlog C ¼ 1:01432ðꢁ0:5208Þ ꢀ VDW 0:0104ðꢁ0:538Þ
2-Phenyl-3-[5-{2-(1H-benzimidazolyl)-2-
chlorophenyl} ethyl] 4(3H) quinazolone
n ¼ 15 jrj ¼ 0:910 s ¼ 0:198 F ¼ 99:125
The synthetic steps were used as shown in Scheme 1.
This step involves the cyclization of a carboxylic acid into a benz-
imidazole on reaction with o-phenylenediamine. Thus, 2-phenyl-3-[5-
(2-chloro-3-carboxylatophenyl) ethyl] 4(3H) quinazolone and o-pheny-
lenediamine in a molar ratio of (0.02 mol) and (0.025 mol), respec-
tively, in dry pyridine (50 mL) were heated under reflux for 6 h
under anhydrous reaction conditions. Subsequently, the contents
after cooling to room temperature were poured into ice-cold diluted
hydrochloric acid (100 mL). The solid that separated out was
allowed to settle down and filtered off. It was washed successively
with water and dried overnight under vacuum. Recrystallization from
Synthesis 2-Phenyl-3-[5-(2-chloro-3-carboxylato-
phenyl) ethyl] 4(3H)-quinazolone
A mixture of 2-phenyl-3-(hydroxy-ethyl) 4(3H) quinazolone (0.06 mol)
and o-chloro benzoic acid (0.06 mol) was dissolved in concentrated
sulfuric acid (50 mL) by stirring and cooling the reaction mixture
occasionally. When a clear solution was obtained, the resultant
solution was further stirred for 1 h at room temperature and was
2
Chem Biol Drug Des 2012

Inhibitors of EGFR Kinases
Biodistribution study in mice
Albino mice strain (A) (taken in triplicate set) was used for the tis-
sue distribution studies. Animal handling and experimentation were
carried out as per the guidelines of the Institutional Animal Ethics
Committee.
diluted ethanol afforded 2-phenyl-3-[4-{2-(1H-benzimidazolyl)-2-chlor-
ophenyl] 4(3H) quinazolone in analytically pure form. It melted at
184–185 ꢀC, yield, 65%.
A mixture of 2-phenyl-3-[4-{2-(1H-benzimidazolyl) 2-chlorophenyl}
ethyl] 4(3H) quinazolone (0.01 mol), an aromatic aldehyde (0.01 mol)
and an aromatic primary amine (0.01 mol), was heated under reflux
for 4 h under anhydrous reaction conditions. Subsequently, solvent
ethanol was distilled off, and the remaining pasty mass was triturated
with petroleum ether (b.p. 60–80 ꢀC). It was treated with diluted
hydrochloric acid (50 mL). Solidification occurred. The solid was fil-
tered off and washed with 1% NaOH solution initially and finally with
water. It was dried in vacuum and recrystallized from ethanol.
An equal dose of 10 lCi of labeled test compound was injected in
mice through tail vein of each animal. At different time intervals,
mice were killed, blood sample was collected, and different tissue
and organs were dissected and analyzed. The radioactivity was
measured in a gamma counter. The actual amount of radioactivity
administered to each animal was calculated by subtracting the
activity left in the tail from the activity injected. Radioactivity accu-
mulated in each organ was expressed as percentage administered
dose per gram of tissue. Total volume of the blood was calculated
as 7% of the body weight.
Radiolabeling of the compounds with
Technetium
Radiolabeling of compounds was carried out by taking 100 lL of
0.03 nM solution of the compounds dissolved in DMSO and taken in
a shielded vial. Further 60 lL of 1 · 10)2 M SnCl2Æ2H2O (dissolved
in N2 Purged 1 mL 10% acetic acid) was added followed by freshly
eluted saline solution of sodium pertechnetate (NaTcO4) (74 MBq,
100 mL). The pH of the reaction mixture was adjusted to 6.5 with
0.1 M NaHCO3 solution and shaken to mix the contents well. The
vial was allowed to incubate for 20–30 min at room temp. Labeling
of the compound, radiochemical purity as well as Rf of the 99mTc-
based complex was determined by ITLC-SG strips using 0.9% NaCl
aqueous solution (saline) as developing solvent and simultaneously
in acetone and PAW (Pyridine, acetic acid and water in 3:5:1.5
ratio). Each ITLC was cut into 0.1-cm segments, and counts of each
segment were taken.
Measurement of potential cytotoxicity
The synthesized compounds were tested against (MCF-7) human
breast adenocarcinoma cell line (originally obtained in 1977, from
the Michigan Cancer Foundation). Routine culture maintenance and
experimental studies were carried out at 37 ꢀC in a cell incubator
with humid atmosphere at 5% CO2. Cell propagation was achieved
in DMEM (Dubecco's modified eagle minimal medium) with phenol
red, 10% fetal bovine serum, L-glutamine, penicillin, streptomycin,
and gentamycin as described in the previous literature. Before any
experiment, the cells were transferred for 4 days to a defined med-
ium, containing phenol red-free DMEM, supplemented with 10%
charcoal stripped. Estrogen (17 b-estrodiol) in the concentration up
to 100 lg was added to defined medium. Doxorubicin is taken as
standard. The MTT assay with 3-(4, 5-dimethylthiazole-2-yl)-2, 5-
phenyltetrazolium bromide was used to determine the number of
viable cells. For assay, MCF-7 cells (1 · 104 cells ⁄ well) were plat-
ted in a 96-well tissue culture plate and exposed to the compounds
under investigation. Cells were processed with the MTT assay for
24, 48, and 72 h of incubation. In brief, 10 lL of MTT (final con-
centration = 250 lg ⁄ mL) in PBS (phosphate buffer saline) was
added to every well containing 100-lL cell suspension in medium,
and the cultures were incubated at 37 ꢀC for 5 h. The reaction mix-
ture was carefully taken out, and 100 lL of DMSO was added to
each well and pipetted up and down several times unless it
became homogenic. After 10 min, the color was read at 540 nm
using spectrophotometer plate reader (Bio-Rad, Tokyo, Japan). The
inhibitory effect on cell proliferation was determined after 72 h of
treatment with various concentrations (0.1–300 nM) of the tested
compound.
In vitro serum stability assay
The fresh human serum was prepared by allowing blood collected
from healthy volunteers to clot for 1 h at 37 ꢀC in a humidified
incubator maintained at 5% carbon dioxide, 95% air. Then the sam-
ple was centrifuged at 400 rpm, and the serum was filtered
through 0.22 lm syringe filter into sterile plastic culture tubes. The
above freshly prepared technetium radio complexes were incubated
in fresh human serum at physiological conditions, that is, at 37 ꢀC
at a concentration of 100 nM ⁄ mL and then analyzed by ITLC-SG at
different time intervals to detect any dissociation of complex. Per-
centage of free pertechnetate at a particular time point that was
estimated using saline and acetone as mobile phase, represented
percentage dissociation of the complex at that particular time point
in serum.
Blood kinetic studies
Result and Discussion
The blood clearance study was performed in normal rabbit, weigh-
ing 2–2.5 kg. Of the 99mTc-labeled compounds (0.3 mL), 5 MBq
was administered intravenously through the dorsal ear vein. At
different time intervals about 0.5-mL blood samples were with-
drawn from the dorsal vein of other ear, and radioactivity was
measured in the gamma counter. The data from the experiment
were expressed as percentage of administered dose at each time
interval.
In the search for useful 99mTc probes for early detection and staging
of EGFR positive tumors, we have designed and synthesized the
new ligands by modifying a known tyrosine kinase inhibitor, Skelton
of quinazoline. In the scheme, the synthesis of final benzimidazole
[4A–4E] was described. The structures of all the newly synthesized
compounds were elucidated with different spectroscopic techniques
such as IR, NMR, mass spectroscopy, and by elemental analysis
Chem Biol Drug Des 2012
3

Yadav et al.
Table 1: Chemical profile of quinazoline-benzimidazole analogs 4[A]–4[E]
O
C
CH2CH2
N
N
N
H5C6
N
Cl
R
CHNHR1
Analysis
Nitrogen %
Compound no.
R
R1
m.p. (ꢀC)
Yield
Colour
Molecular formula
Molecular weight
Calcd.
Found
1
2
3
4
5
Phenyl
Phenyl
Styryl
Styryl
4-methyl-phenyl
4-methoxy-phenyl
4-methyl-phenyl
7-methoxy-phenyl
4-methyl-phenyl
175–180
170
184
162–164
180–182
60
50
49
51
55
Yellow
White
Light brown
Brown
C43H34N5OCl
C43H34N5O2Cl
C45H36N5OCl
C45H36N5O2Cl
C44H36N5O2Cl
671.5
687.5
697.5
713.5
701.5
10.42
10.18
10.03
9.81
10.30
9.90
9.95
9.50
9.42
4-methoxy-phenyl
White
9.98
Table 2: Spectral analysis of quinazoline-benzimidazole analogs 4[A]–4[E]
O
C
CH2CH2
H5C6
N
N
N
N
Cl
CHNHR1
R
Compound
No.
R
R1
Spectral analysis
LC-MS (m ⁄ z)
1
2
3
Phenyl
4-methyl-phenyl
IR (kBr) (mmax in cm)1): 1695 (test amide C=O), 1735 (ester C=O), 1630 (C=N), 3345 (N-17). 671
1HNMR (CDCl3) (d ppm): 6.75–7.60 (m, 26H, ArH), 3.25 (s, 3H,
CH3), 3.75 (t, 2H, N-CH2), 2.80 (s, 1H, N-CH).
Phenyl
Styryl
4-methoxy-phenyl IR (kBr) (mmax in cm)1): 1690 (test amide C=O), 1705 (ester C=O), 1630 (C=N), 3360 (N-17). 687
1HNMR (CDCl3) (d ppm): 6.75–7.85 (m, 26H, ArH), 2.55 (s, 3H, OCH3), 3.75
(t, 2H, N-CH2), 2.80 (s, 1H, N-CH).
4-methyl-phenyl
IR (kBr) (mmax in cm)1): 1695 (test amide C=O), 1788 (ester C=O), 1632
(C=N), 3345 (N-17). 1130 (C=C)
697
713
1HNMR (CDCl3) (d ppm): 6.75–7.65 (m, 26H, ArH), 3.25 (s, 3H, CH3), 3.85
(t, 2H, N-CH2), 2.80 (s, 1H, N-CH).
4
5
Styryl
7-methoxy-phenyl IR (kBr) (mmax in cm)1): 1680 (test amide C=O), 1735 (ester C=O), 1638 (C=N), 3345
(N-17). 1130 (C=C)
1HNMR (CDCl3) (d ppm): 6.55–7.65 (m, 26H, ArH), 2.55 (s, 3H, OCH3), 3.72 (t, 2H,
N-CH2), 2.80 (s, 1H, N-CH).
4-methoxy-phenyl 4-methyl-phenyl
IR (kBr) (mmax in cm)1): 1690 (test amide C=O), 1735 (ester C=O), 1635 (C=N), 3340 (N-17). 701
1HNMR (CDCl3) (d ppm): 6.35–7.60 (m, 26H, ArH), 3.25 (s, 3H, CH3), 3.70 (t, 2H, N-CH2),
2.80 (s, 1H, N-CH). 2.50 (s, 3H, OCH3)
(Tables 1 and 2). Comparing the TLC with the starting materials,
which resulted a single spot different from the starting materials,
checked the synthesized ligand. The spectral evidence confirms the
presence of different functionalities (IR at 3351, 1640, 1467 cm)1).
Similarly, NMR multiplet in the range of (6.2–8) ppm of 5–15 hydro-
gen also confirms the presence of aromatic rings. It also confirms
the proposed stoichiometry and structure for the phenothiazinyl
quinazolones. Integral of NMR confirms the number of protons in
phenothiazinyl quinazolones as well as coupling constant confirms
the nature of double bonds.
The cell proliferation was measured by MTT assay, and the results
were expressed as IC50 values. The activity data are given in
Table 3. The inhibition of the EGFR activity by 4[A]–4[E] was evalu-
4
Chem Biol Drug Des 2012

Inhibitors of EGFR Kinases
ated in human breast cancer cell line, MCF-7. These cells are also
known to over-express EGFR, which leads to continuous activation
of the EGFR pathway involved in cell proliferation. The inhibitory
effects on MCF-7 cell proliferation were determined after 72 h of
treatment with various concentrations (10)5 to 10)10 M) of the
tested compound, and the results were expressed as IC50 values
ranging from 0.030 to 0.190 lM.
Table 3: Antiproliferative activity of the compounds 4[A]–
4[E]against the MCF-7 cells and their corresponding EGFR-TK activ-
ity
Compound
IC50 (lM) EGFR-TK
IC50 (lM) MCF-7
4[A]
4[B]
4[C]
4[D]
4[E]
0.072 0.003
0.062 0.005
0.049 0.005
0.081 0.006
0.190 0.005
9.02 1.24
6.31 0.41
5.40 1.34
10.30 0.39
15.42 1.52
Preliminary complexation of novel synthesized compounds with
99mTc was found to give sufficiently stable complexes under physio-
logical conditions. The in vitro serum stability of the radio com-
plexes is necessary parameter meant to measure the effectiveness
of chelating moiety to coordinate the radio metal. Generally, there
is transchelation of radio metal to serum proteins particularly albu-
min. In vitro serum stability of the complexes clearly indicates that
initially there was fall in the stability of the complex but further
showing a constant stability. Initial fall in the labeling efficiency
after the addition of fresh serum could be attributed to the trans-
chelation that could have taken place in serum because of high
affinity of plasma proteins for metal ions.
The values are the mean € SD of independent experiments.
Concentration of compound resulting in 50% inhibition of EGFR-TK activity.
The retention of drug in the blood of the animal depends upon the
pharmacological and physical properties of the drugs. Nearly all the
phenothiazinyl quinazolones shows a very rapid clearance of radio-
activity from the blood. Approximately 50–65% of activity was
removed within 1 h and more than 90% in 3 h (Figure 1). It shows
rapid kinetics, which may be attributed to the hydrophilic nature of
the drug–radio-metal complexes.
Figure 1: Blood kinetic study of labeled benzimidazole analogue
in rabbit [4C].
Biodistribution of the radio complexes is an important phenomenon
to study because it gives an idea about its excretory metabolic
pathway and in vivo distribution of the radio complex drug. Accu-
mulation of low amount of radioactivity in the stomach precludes
the presence of free pertechnetate, which indicates in vivo stability
of preparation. The percentage distribution of drug in various
organs of mice is shown as percentage of injected dose per organ
or tissue at different time intervals (Figure 2). The drug localized in
the liver and kidneys; with the passage of time, the activity in kid-
ney amplified for most of the compounds. This shows that the
major route of excretion of activity is through kidneys. Accumulation
of drugs in liver may also be because of protein binding nature of
drugs. Very slight accumulation of activity was observed in lungs,
spleen, and stomach. Negligible accumulation occurs in heart and
brain. Biodistribution in tumor-bearing mice showed that the com-
pound is specific for tumor cells, so, further it can be used for diag-
nostic
purpose
through
SPECT ⁄scintigraphic
techniques.
Biodistribution of the 99mTc complex in healthy animals showed a
rather fast blood and soft tissue clearance between 0 and 2 h, fol-
lowed by a slower clearance between 2 and 6 h.
Conclusion
Because breast cells are known to overexpress EGFR, which leads to
continuous activation of the EGFR pathway involved in cell proliferation,
and therefore, we measured antitumor activity of the compounds in vi-
tro on human breast carcinoma cell line (MCF-7). Most of the tested
compounds exhibited potent inhibitory activity against MCF-7 cell line.
Figure 2: Biodistribution study of labeled benzimidazole ana-
logue in normal and tumor bearing mice [4C].
Chem Biol Drug Des 2012
5

Yadav et al.
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