Design, synthesis, biological evaluation, QSAR analysis and molecular modelling of new thiazol-benzimidazoles as EGFR inhibitors

Article abstract of DOI:10.1016/j.bmc.2020.115657

Heterocyclic rings such as thiazole and benzimidazole are considered as privileged structures, since they constitute several FDA-approved drugs for cancer treatment. In this work, a new set of 2-(2-(substituted) hydrazinyl)-4-(1-methyl-1H-benzo[d]imidazol-2-yl) thiazoles 4a-q were designed as epidermal growth factor receptor (EGFR) inhibitors and synthesized using concise synthetic methods. The new target compounds have been evaluated in vitro for their suppression activity against EGFR TK. Compounds 4n, 4h, 4i, 4a and 4d exhibited significant potency in comparison with erlotinib which served as a reference drug (IC50, 71.67–152.59 nM; IC50 erlotinib, 152.59 nM). Furthermore, MTT assay revealed that compounds 4j, 4a, 4f, 4h, 4n produced the most promising cytotoxic potency against the human breast cancer cell line (MCF-7) (IC50; 5.96–11.91 μM; IC50 erlotinib; 4.15 μM). Compound 4a showed promising activity as EGFR TK inhibitor as well as anti-breast cancer agent. In addition, 4a induced apoptotic effect and cell cycle arrest at G2/M phase preventing the mitotic cycle in MCF-7 cells. Moreover, 4a upregulated the oncogenic parameters; caspase-3, p53, Bax/Bcl-2 as well as it inhibited the level of PARP-1 enzyme. QSAR study was carried out for the new derivatives and it revealed the goodness of the models. Furthermore, molecular docking studies represented the binding modes of the promising compounds in the active pocket of EGFR.

Full text of DOI:10.1016/j.bmc.2020.115657

Journal Pre-proofs
Design, synthesis, biological evaluation, QSAR analysis and molecular mod‐
elling of new thiazol-benzimidazoles as EGFR inhibitors
Aladdin M. Srour, Nesreen S. Ahmed, Somaia S. Abd El-Karim, Manal M.
Anwar, Salwa M. El-Hallouty
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DOI:
Reference:
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https://doi.org/10.1016/j.bmc.2020.115657
BMC 115657
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Bioorganic & Medicinal Chemistry
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2 April 2020
26 June 2020
15 July 2020
Please cite this article as: Srour, A.M., Ahmed, N.S., Abd El-Karim, S.S., Anwar, M.M., El-Hallouty, S.M.,
Design, synthesis, biological evaluation, QSAR analysis and molecular modelling of new thiazol-benzimidazoles
as EGFR inhibitors, Bioorganic & Medicinal Chemistry (2020), doi: https://doi.org/10.1016/j.bmc.2020.115657
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Design, synthesis, biological evaluation, QSAR analysis and molecular
modelling of new thiazol-benzimidazoles as EGFR inhibitors
Aladdin M. Srour1, Nesreen S. Ahmed1, Somaia S. Abd El-Karim1, Manal M. Anwar1*, Salwa
M. El-Hallouty2
1Department of Therapeutic Chemistry, National Research Centre, Dokki, Cairo 12622, Egypt
2Drug Bioassay-Cell Culture Laboratory, Department of Pharmacognosy, National Research
Centre, Dokki, Giza, 12622, Egypt
*Corresponding authors:
E-mail addresses: manal.hasan52@live.com
somaia_elkarim@hotmail.com
Abstract
Heterocyclic rings such as thiazole and benzimidazole are considered as privileged
structures, since they constitute several FDA-approved drugs for cancer treatment. In this work, a
new set of 2-(2-(substituted) hydrazinyl)-4-(1-methyl-1H-benzo[d]imidazol-2-yl) thiazoles 4a-q
were designed as epidermal growth factor receptor (EGFR) inhibitors and synthesized using
concise synthetic methods. The new target compounds have been evaluated in vitro for their
suppression activity against EGFR TK. Compounds 4n, 4h, 4i, 4a and 4d exhibited significant
potency in comparison with erlotinib which served as a reference drug (IC50, 71.67 - 152.59 nM;
IC50 erlotinib, 152.59 nM). Furthermore, MTT assay revealed that compounds 4j, 4a, 4f, 4h, 4n
produced the most promising cytotoxic potency against the human breast cancer cell line (MCF-
7) (IC50; 5.96-11.91 µM; IC50 erlotinib; 4.15 µM). Compound 4a showed promising activity as
EGFR TK inhibitor as well as anti-breast cancer agent. In addition, 4a induced apoptotic effect
and cell cycle arrest at G2/M phase preventing the mitotic cycle in MCF-7 cells. Moreover, 4a
upregulated the oncogenic parameters; caspase-3, p53, Bax/Bcl-2 as well as it inhibited the level
of PARP-1 enzyme. QSAR study was carried out for the new derivatives and it revealed the
goodness of the models. Furthermore, molecular docking studies represented the binding modes
of the promising compounds in the active pocket of EGFR.
Keywords: Thiazole; benzimidazole; EGFR inhibitors; Breast cancer; Apoptosis; caspase-3;
p53; Bax/Bcl-2; QSAR; molecular docking
1

1. Introduction
Cancer disease represents one of the crucial public health problems worldwide. The
incidence and mortality statistics reported that it is growing in the economically developing and
developed countries1. Breast cancer (BC) is the most prevalent neoplasm in women reaching to
23% of the total cancer cases leading to about 14% of the cancer deaths among females2.
Numerous signaling cascades are deregulated resulting in increment of cell proliferation,
survival, in addition to the emergence of resistance against different anticancer drugs. In most
cases of BC, deregulation detected results in inactivation of tumor suppressor genes as well as
hyperactivation of proto-oncogenes2,3. BC is considered as a heterogeneous group of neoplasms
and represents a considerable challenge in chemotherapeutic and immunological fields4,5.
Epidermal growth factor receptor tyrosine kinase (EGFR TK) is a receptor tyrosine kinase
(RTK) of the ErbB family6. It produces key regulation of multiple bioactivities, including
motility of the cell, adhesion, regulation of the cell cycle, alongside with angiogenesis, apoptosis,
and metastasis7,8. Additionally, this enzyme is considered to be one of the most commonly
altered oncogenes in solid cancers including head and neck, breast, colorectal as well as non-
small cell lung cancers (NSCLC)6,9. The pathological alterations of EGFR in cancers occurs
either through kinase-activating mutation or EGFR over-expression, which is related to poor
clinical cancer outcomes including early recurrence, increased metastasis risk and reduced
survival11-13. Therefore, EGFR represents a characteristic rational target for BC drug discovery.
There are various small molecules acting as EGFR inhibitors that are now available in clinics;
including the reversible inhibitors gefitinib, erlotinib, and lapatinib and irreversible inhibitors
afatinib, dacomitinib and osimertinib14,15 (Fig. 1).
2

Fig. 1. Clinically available EGFR inhibitors
Thiazole motif is a fascinating building block in pharmaceutical chemistry for designing
and synthesizing multiple bioactive agents16-23. Different studies investigated that many thiazole
analogs exhibited very potent antitumor activity targeting specific pathways such as; suppressing
microtubule polymerization as well as inhibition of various molecular targets such as receptor
tyrosine kinases (RTKs), non-receptor tyrosine kinases (nRTKs), phosphatidylinositol-3- kinases
(PI3Ks), serine/threonine kinases (STKs), Bcl-2 family, histone deacetylases (HDACs) and
tumor necrosis factor-α (TNF-α) 24-26. In addition, various thiazole-based compounds were
determined as apoptosis inducers and antimetastatic agents so that they were transferred into
clinical trials as therapeutic anti-cancer candidates25,26. Dasatinib and Dabrafenib are
representative thiazole drugs of selective protein kinase inhibitory effects27,28 (Fig. 2). Molecular
docking studies showed that the nitrogen and sulphur atoms represent valuable binding sites for
H-interaction with various amino acid residues in the active pockets of different protein kinases
including EGFR26,29
.
Benzimidazole ring is the core structure of numerous compounds possessing a wide
spectrum of bioactivities30-39. Furthermore, benzimidazole scaffold is considered as an isostere of
purine nucleotides facilitating them to bind with the biopolymers in the human bodies so, it is an
important motif in various potent anticancer agents38,40. Figure 2 represents some potent
3

antitumor derivatives bearing benzimidazole - heterocyclic scaffolds, such as; selumetinib41,
Hoechst-3325842 and bendamustine43.
Fig. 2. Different thiazole and benzimidazole – based anticancer drugs inhibiting various
molecular targets
Despite the initial high response levels obtained by the first-generation reversible EGFR
inhibitors, gefitinib and erlotinib and the second-generation irreversible ErbB family blockers,
afatinib and dacomitinib, patients would eventually become resistant to the treatment. The third-
generation EGFR TKIs have been developed and optimized with a view to combat the acquired
resistance to the first- and second-generation EGFR TKIs44,45. Osimertinib is the only
third‐generation EGFR TKIs that was approved by Food and Drug Administration, while
nazartinib, olmutinib, avitinib and rociletinib are still under clinical development44.45. It could be
noted that benzimidazole nucleus constitutes the basic structural core of nazartinib, whereas
avitinib and osimertinib, bear indolopyrimidine and indole rings, respectively, which are
bioisosteres to the benzimidazole ring system. The rational design of new potent anticancer
agents of more selectivity towards various protein kinases takes place through the molecular
hybridization of two or more pharmacophoric units recognized and derived from known
bioactive molecules to reach a final compound that would progress to clinical trials and
development. 24,46. Based on this aspect, the strategy of the present study was directed towards
4

construction of new derivatives bearing thiazol- benzimidazole motif hybridized with different
alicylic, aromatic and / or heterocyclic rings via methylene-hydrazinyl linker at thiazole-C3
position, taking into account the impact of molecular orientation, the variation in the ring size as
well as the presence of heteroatoms which could provide hydrogen bond interactions with the
amino acid residues in the active pocket of EGFR TK (Fig. 3).
The designed compounds were synthesized, and their structural formulae were confirmed
by different elemental and spectral data. All the newly synthesized derivatives were evaluated as
EGFR inhibitors. The derivatives that exhibited promising EGFR inhibitory effects were further
assessed as anticancer agents against human breast cancer cells (MCF-7). The candidate which
revealed characteristic EGFR inhibition and showed potent anticancer activity against MCF-7
cells was selected as a representative example to find out further mechanistic anticancer effects
of the new derivatives against; cell cycle, apoptosis, caspase-3, p53, Bax/Bcl-2 as well as PARP-
1 inhibition. QSAR study and molecular modelling were also performed to elucidate the binding
modes of the active candidates with amino acid residues of EGFR and rationalize their
pharmacological effects.
5

Fig. 3. Proposed hypothetic model of new thiazol-benzimidazole compounds as EGFR TKIs
2. Results and discussion
2.1. Chemistry
The synthetic approaches of construction of the target thiazol-benzimidazole hybrids 4a-
q were illustrated in (Scheme 1). To synthesize a series of thiosemicarbazones 2a-q, the
appropriate carbonyl compounds 1a-q were allowed to react with thiosemicarbazide in refluxing
ethanol which includes a catalytic amount of acetic acid via Schiff base condensation46-60
.
Hantzsch condensation of the formed thiosemicarbazone derivatives 2a-q with 2-bromo-1-(1-
methyl-1H-benzo[d]imidazol-2-yl)ethan-1-one (3) in refluxing ethanol61,62 afforded the
corresponding
targeted
2-(2-(substituted)hydrazinyl)-4-(1-methyl-1H-benzo[d]imidazol-2-
yl)thiazole derivatives 4a-q. Characterization and the purity assessment of the newly synthesized
compounds were carried out by melting points determination, spectroscopic methods (IR, 1H and
13C NMR) and elemental analyses. The resultant data confirmed the formation of analytically
pure desired products.
6

IR spectra of the compounds 4a-q showed absorption stretching bands in the region 3417
1
- 3285 cm-1 representing NH groups of the hydrazono linker. Furthermore, H NMR chemical
shifts represented the protons of the new compounds at their expected regions which confirmed
their chemical structures. A singlet signal appeared at the range δ 4.16 – 4.30 ppm due to the
three protons of benzimidazole-CH3 group, the methine proton of the hydrazono CH=N group
was represented as a singlet signal in the aromatic region at δ 7.90 – 8.20 ppm, the aromatic
protons appeared as multiplet signals at the range δ 7.30- 8.01 ppm, while the NH proton
appeared as a broad D2O exchangeable singlet at the region δ 10.78 - 13.14 ppm. At the same
time, the eight protons of the cyclopentyl ring of 4a appeared as two multiplets at δ 1.70-1.74
and 1.79-1.82 ppm, while the ten protons of the cyclohexyl moiety of 4b were displayed as three
multiplets at δ 1.61-1.65, 2.25-2.31and 2.47-2.49 ppm. In addition, singlet signals appeared at
the region δ 2.28 - 2.34 ppm due to the methyl protons of p-phenyl-CH3, furan-CH3, p-phenyl-
N(CH3)2 and -N=C-CH3 of compounds 4d, 4m, 4i and 4p, respectively. With respect to the
compounds bearing methoxy groups as 4e, 4f, 4g exemplified singlet signals at the region δ 3.71
- 3.85 ppm.
13
Also, C NMR spectra of the compounds 4a-q exhibited the typical signals due to the
aliphatic or alicyclic carbons in the expected ranges. For example, a singlet signal appeared at
the range δ 31.16 - 35.24 ppm due to N-CH3 carbon, the signals referring to the cyclopentyl and
cyclohexyl carbons of compounds 4a, 4b appeared at the range δ 25.04 - 33.27 ppm and the
signals due to OCH3 carbon of compounds 4e,f,g presented at the region δ 55.85 – 60.60 ppm.
The additional CH3 carbon of compounds 4d, 4m appeared at δ 14.02, 21.48 ppm respectively,
while -N(CH3)2 and -N=C-CH3 carbons of compounds 4i and 4p presented as singlet signals at δ
33.0, 33.03 ppm. The resultant data of the elemental analyses strongly characterized the newly
reported derivatives 4a-q.
7

Scheme 1: Synthetic route of the new thiazol-benzimidazole - based compounds
2.2. Biological evaluation
2.2.1. EGFR PK inhibition assay
The EGFR inhibitory profile of all the target thiazol-benzimidazole derivatives was
evaluated utilizing Erlotinib as a standard drug. The obtained data were expressed as IC50 values
(nM) as shown in Table 1. It has been noticed that all the tested compounds 4a-q, exhibited a
wide range of EGFR inhibitory activity in the nanomolar range with IC50 ranging from 71.67 to
1235.34 nM. Accordingly, a wide variety of substituents can be tolerated at the terminal position
of the parent thiazol-benzimidazole motif. It has been noted that the incorporation of 5-nitrofuran
to the parent thiazol-benzimidazole scaffold via the methylene-hydrazineyl linker as compound
4n exhibited two folds more potent inhibitory effect than that of the reference inhibitor erlotinib
(IC50, 71.67 nM; IC50 erlotinib, 152.59 nM). Replacing the 5-nitrofuran appendage with 4-
8

nitrophenyl ring as the derivative 4h led to a slight reduction in the potency but still 1.7 folds
higher than that of the reference drug (IC50, 85.03 nM). This significant activity might be due
the presence the nitro functionality which might produce an extra arene-cation binding leading to
more fixation in the active pocket of the enzyme. Despite the marked decrease in the potency
upon the replacement of the p-nitrophenyl functionality with p-dimethylamino-phenyl,
cyclopentyl or p-methylphenyl groups, the compounds 4i, 4a and 4d appeared more potent than
erlotinib of IC50 values; 103.71, 109.71 and 150.36 nM, It could be investigated that the
decrease in the polarity of the molecules decreases the effectiveness of the examined compounds.
Furthermore, the conjugation of 3,4-di-OCH3-phenyl, 5-methylfuran and cyclohexyl moieties
with the parent thiazol-benzimidazole methylene hydrazine motif as compounds 4f, 4m, 4b
resulted in a pronounced decrease in the inhibitory effect by approximately 1.3-1.8 folds less
than that of erlotinib of IC50 values; 208, 212, 278 nM. Further dramatic reduction in the activity
was also detected by the rest of the compounds bearing different substituted phenyl and
heterocyclic rings. This could be explained by the increase in the substituent bulkiness negatively
affects the potency of the molecule due to changing its the geometry with respect to the enzyme.
2.2.2. In vitro cytotoxicity
The newly synthesized thiazol-benzimidazole compounds which exhibited the most
promising EGFR inhibitory effect (4a, 4b, 4d, 4f, 4h, 4i, 4j, 4m, 4n) were selected as
representative examples to evaluate their anticancer activity. MCF-7 cell line has diverse
characters of differentiated mammary epithelium, including the capability of
processing estradiol by means of cytoplasmic estrogen receptors as well as expressing epidermal
growth factor receptors63,64. Accordingly, this type of breast cancer cells was selected to evaluate
the cytotoxic activity of the latter compounds in comparison with erlotinib as a reference
anticancer drug using MTT assay65. The obtained results were expressed as half maximal
inhibitory concentration IC50 (µM) and summarized in Table 1.
The gained data exhibited that the most effective cytotoxic agent was the p - fluorophenyl
derivative 4j which was approximately equipotent to erlotinib (IC50; 5.96, IC50 erlotinib; 4.15
µM). The electron-withdrawing power of fluorine atom, alongside with its increased carbon–
fluorine bond energy, significantly potentiates the metabolic stability of the host molecule in
addition to increasing its lipophilic character which facilitates cell membrane permeation
resulting in a pronounced anticancer effect66. Furthermore, the cyclopentyl 4a and the di-
9

methoxyphenyl 4f derivatives were less likely to inhibit the viability of the cancer cells than
erlotinib of IC50 values; 6.30, 7.56 µM, respectively. Although the derivative 4h, 4n, 4i and 4d
exhibited significant EGFR inhibition effects, they produced cytotoxic effects less than that of
erlotinib by approximately two-four folds (IC50; 9.87, 11.91, 17.31, 18.15 µM, respectively).
Such contradiction between the enzyme inhibition action and the cytotoxic effect is often
troublesome in the field of discovery of enzyme inhibitors. It has been reported that there are
many factors affecting this problem, such as improper physicochemical properties contributing to
lower cancer cells accumulation67,68. There is also no clear link between the anti-angiogenic
action of the compounds being tested and their cytotoxic effects toward the cancer cells, since
the in vitro environment does not imitate the in vivo one due to the absence of blood supply or
vascular endothelial cells69. Consequently, the in vitro EGFR suppression may not be completely
translated to cancer cells suppression, but primarily applies to other mode of actions. A marked
drop in the activity was also observed by incorporation of the cyclohexyl and 5-methylfuranyl
moieties with the parent benzimidazole-thiazole methylene hydrazine scaffold as compounds 4b,
4m of IC50 values 17.30 to 20.69 µM, respectively.
Table 1: In vitro EGFR inhibitory effects of the new thiazol-benzimidazole compounds and their
anticancer activity against MCF-7 cells
EGFR kinase
inhibition
IC50 (nM)
Anticancer
activity against
MCF-7 cells
IC50 (µM)
Compound No
R, R’ = C5H9
R, R’ = C6H11
109.71 ± 3.55
278.88 ± 9.33
1235.34 ± 48.2
150.36 ± 6.57
311.75 ± 11.03
208.09 ± 12.36
622.64 ± 26.4
85.03 ± 3.51
103.71 ± 2.91
253.74 ± 7.84
406.09 ± 16.1
513.63 ± 19.1
212.82 ± 8.12
71.67 ± 2.57
6.30 ± 0.37
17.30 ± 0.66
---
18.15 ± 0.84
---
7.56 ± 0.23
---
9.87 ± 0.76
17.31 ± 0.92
5.96 ± 0.29
---
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
R = C6H5; R’ = H
R = 4-CH3-C6H5; R’ = H
R = 4-OCH3-C6H4; R’ = H
R = 3,4-di-OCH3-C6H3; R’ = H
R = 3,4,5-tri-OCH3-C6H2; R’ = H
R = 4-NO2-C6H4; R’ = H
R = 4-N(CH3)2-C6H4; R’ = H
R = 4-F-C6H4; R’ = H
R = 4-Br-C6H4; R’ = H
R = 2,4-di-Cl-C6H3; R’ = H
R = 5-CH3-2-furanyl; R’ = H
R = 5-NO2 -2-furanyl; R’ = H
---
20.69 ± 1.57
11.91 ± 0.85
10

4o
4p
4q
R = 2-thienyl; R’ = H
R = 2-pyrrolyl; R’ = CH3
R = 4-pyridinyl; R’ = H
773.24 ± 26.1
342.06 ± 13.7
569.41 ± 22.4
152.59 ± 6.69
---
---
---
4.15 ± 0.18
erlotinib
2.2.3. The effect of representative target compounds against the normal hTERT-RPE1 cells
One of the characteristics that distinguish various antiproliferative drugs from each other
is the frequency and the severity of side effects to the normal cells at the therapeutic doses. The
safety profile of compounds (4a, 4b, 4d, 4f, 4h, 4i, 4j, 4m, 4n) was examined against the normal
hTERT-RPE1 cell line. The suppression percentage of the normal cells by each tested compound
was determined via MTT assay65,70 (Table 2). It has been noted that most of the tested
derivatives exhibited promising safety profile of inhibition percentages ranging from 0-22%.
Unfortunately, compounds 4-methylphenyl 4d, 3,4-dimethoxyphenyl 4f and 5-nitro-2-furanyl 4d
represented inhibition percentages of normal cells 68%, 66% and 65%, respectively.
Table 2. Inhibition percentages of the cytotoxic compounds against
normal hTERT-RPE1 cells
Compound No.
% inhibition
11.9%
22%
4a
4b
4d
4f
68%
66%
4.6%
0%
4h
4i
6.9%
8.7%
65%
4j
4m
4n
2.2.4. Cell cycle analysis and detection of apoptosis
Cancer cells obviate apoptosis since mutant cell aggregation prevents apoptosis
pathways. Thus, induction of apoptosis is considered as one of the most important tools that
confirm the effectiveness of cancer treatment71. Cell cycle checkpoints are G1 (restriction or
11

start), S (metaphase), and G2/M72. The main function of the anticancer candidates is cessation
the cell division at these checkpoints72. Due to the well-balanced bioactivity i.e. promising EGFR
inhibition potency and anticancer activity, the cyclopentyl derivative 4a was chosen for further
elucidation of its actions towards cell cycle progression and apoptosis induction in MCF-7 cells.
Thus, MCF-7 cells were incubated with compound 4a at its IC50 value (6.30 µM) for 24 h to
analyze its effect on the normal cell cycle profile and apoptosis induction. The cells were stained
with Annexin V/PI and examined using flow cytometry procedure65. The obtained data
investigated that there was cell accumulation of percentages 24.2 % and 38.16 % at pre G1 and
G2/M phases in MCF-7 cells treated with 4a, comparing to 1.44 % and 12.39 % of the untreated
MCF-7 cells. This result represents that there was cell cycle arrest at G2/M phase and cessation
of mitotic cycle. Studies showed that erlotinib arrest the breast cancer cell G2/ M phase1,56 which
comes in agreement with that gained by compound 4a (Table 3) (Fig. 4).
Table 3: Results of cell cycle analysis of compound 4a
Compound
No
DNA content %
%G2-M %Pre G1
%G0-G1
%S
Comment
cell growth arrest at
G2/M
4a/
MCF-7
36.07
25.77
38.16
24.29
53.92
33.69
12.39
1.44
Untreated
MCF-7
12

Fig. 4. Cell Cycle analysis results of compound 4a
2.2.5. Apoptosis assay
To evaluate the apoptotic effect of compound 4a, flow cytometry technique was carried
out using propidium iodide (PI) and annexin-V-FITC in MCF-7 cells29,65,73. MCF-7 cells were
stained with the two dyes; annexin V/propidium iodide (PI) after treating them with 4a at its
IC50 concentration of 6.30 µM for a period of 24 h. Flow cytometry technique has detected the
corresponding red (PI) and green (FITC) fluorescence. There was an increase in the late
apoptosis produced by the examined compound from 0.31 % (DMSO control) to 14.53 % and an
increase in the early apoptosis from 0.47.87% (DMSO control) to 7.59 % with necrosis percent
2.17 % vs 0.66 % produced by DMSO control (Fig. 5,6). It has been observed that the late
apoptosis percentage caused by 4a was higher than that of the early phase which makes it more
challenging to recover the apoptotic cells to safe ones. The obtained results revealed that 4a
inhibits cancer cell proliferation through apoptosis (Fig. 5,6).
13

Fig. 5. Apoptosis induction analysis caused by the derivative 4a
14

Fig. 6. Analysis of cell cycle and activation of apoptosis caused by
the derivative 4a against MCF-7 cells
15

2.2.6. The impact of the derivative 4a on Bax/Bcl-2 /p53 levels
Apoptotic process controls a cell via two main pathways; the extrinsic pathway (the death
receptor) and the intrinsic pathway (mitochondrial pathway)74. The Bcl-2 family protein displays
a major role in tumor growth or suppression the intrinsic apoptotic pathway75. On the other hand,
the pro-apoptotic Bax plays an essential role in cell apoptosis76. The anti-apoptotic Bcl-2
overexpresses in the cancer cell enhancing the cell survival by inhibition apoptosis. Accordingly,
the balance between these two proteins decides the cell fate76. Furthermore, p53 is a tumor
suppressor protein serving as a transcription factor. It is expressed in response to multiple
cellular stresses, including DNA disruption77. Commonly, it is mutated in different human
cancers78. Various studies exhibited the implication of the p53 pathway in the regulation of the
G2 to M transition preventing entry into mitosis, thus mediates either growth arrest or
apoptosis. p53 is considered as one of the most relevant tumor suppressor genes77,78. In addition,
the tumor suppressor gene p53 controls the expression of Bcl-2 and Bax genes78. Thus, this work
investigated also the impact of 4a on the regulation of the apoptotic markers p53, Bax and Bcl-2.
MCF-7 cancer cells were incubated with compound 4a at its IC50; 6.30 µM for 24 h. The results
exhibited that the examined compound 4a boosted the levels of p53 and Bax proteins by 9.85
and 4.95 folds, respectively in comparison to the untreated cells. Reversely, 4a reduced Bcl-2
level by 2 folds comparing to the control cells (Table 4).
2.2.7. Upregulation of caspase-3
Caspases constitute a family of cysteine proteases that are essential in launching the
programmed cell death (apoptosis). It has been investigated that some types of caspases initiate
intracellular event cascade, where others are known effectors which produce their effects further
downstream leading to cellular breakdown via intrinsic protein breakdown65,73. The events of
apoptosis including cell shrinkage, chromatin condensation, and DNA fragmentation all need the
involvement of caspase-373. MCF-7 cells were treated with 4a at its IC50 concentration of 6.30
µM for 24 h in order to detect the level of caspase-3. The tested compound 4a elevated the level
of caspase -3 by 12 folds comparing to the untreated cancer cells (Table 4).
16

Table 4: The effect of compound 4a on the levels of Bax/Bcl-2 /p53/ caspase-3 in MCF-7 cells
Compound
No
Bax
Conc.
Pg/mL
Bcl-2
Conc.
ng/mL
P53
Conc.
Casp-3
Conc.
FLD
FLD
FLD
FLD
Pg/mL
ng/mL
4a/
MCF-7
201.3±7.56 4.95 2.745±0.071 0.48 712.3±22.4 9.85
40.63±2.9 5.696±0.27 72.30±3.5
18.31±2.3
1.49±0.05
12.28
1
1
1
1
Untreated
MCF-7
2.2.8. PARP-1 Cleavage Assay
Poly (ADP-ribose) polymerase-1(PARP-1) inhibitors represent a new group of drugs
which target breast cancers. PARP-1 is a nuclear enzyme that is essential in repairing of single-
stranded DNA (ss DNA) breaks78,79. Interestingly, clinical studies investigate that the anticancer
activity of PARP-1-inhibitors is not restricted to BRCA-1 or BRCA-2 mutated cancers, but also
attacks the non-BRCA mutated breast and ovarian cancers and exhibit a significant impact in
combination therapy80,81. Thus, it was of interest to study the inhibitory activity of compound 4a
against PARP-1 enzyme in the MCF-7 cancer cells using Western blot analysis82. The obtained
results showed that 4a reduced the level of PARP-1enzyme by 1.5 folds when compared to the
control untreated MCF-7 cells (Table 5).
Table 5: The effect of compound 4a on the level of PARP-1 in MCF-7 cells
PARP-1
Compound No
pg/mL
257.06
370.7
FLD
0.693
1
4a/MCF-7
Untreated MCF-7
3. QSAR Analysis
Quantitative structure-activity relationship (QSAR) analysis is a technique that is used to
correlate between the physicochemical properties of a series of chemical compounds and their
bioactivities74. The 2D-QSAR study was introduced utilizing the CODESSA PRO package in
order to validate and correlate the detected EGFR inhibiting results with the structural features of
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the tested compounds83. Molecular descriptors of the 2D-QSAR correlating the chemical
structures of the new thiazol-benzimidazole derivatives with their biological activity were
expressed as 1/IC50 nM and are expressed in Table 6. They were arranged according their levels
of significance (t-criterion). The descriptors were acquired using the BMLR (Best Multiple
Linear Regression) process, initiated to find the strongest n-parameter regression equations and
focused on the maximum R2 (Squared Coefficient of Correlation) and F (Fisher Criteria)
values83,84
.
Table 6. Descriptor of the BMLR-QSAR model for the tested compounds as EGFR inhibitors
Entry ID
Coefficient
-0.278096
0.0542838
s
t
Descriptors
1
2
0
0.057
0.006
-4.841
9.617
Intercept
D1
FHACA Fractional HACA
(HACA/TMSA) (MOPAC PC)
Max. e-e repulsion for atom N
Max. bond order for atom H
3
4
D2 0.000674413
D3 0.195435
0.0001
0.055
5.263
3.549
N = 17, n = 3, R2 = 0.899, R2cvOO = 0.828, R2cvMO = 0.838, F = 38.712, s2 = 2.565
1(IC50, nM) = ‒0.278096 + (0.0542838 x D1) + (0.000674413 x D2) + (0.195435 x D3)
Internal validation methodology utilized by the CODESSA PRO using both Leave One
Out (LOO), which includes the creation a number of models with one example excluded at a
time, and leave several Out (LMO) that develops a number of models with several data points
excluded at a time (up to 20% of the total data points) is used to test the reliability and statistical
validity of the obtained BMLR-QSAR model, which is considered the deemed acceptable
methodology in the present study due to the minimal data points85–90. The association found for
the internal validation approaches are R2cvOO = 0.828, R2cvMO = 0.838. Both are strongly
associated with the R2 value of the attained QSAR model obtained (R2 = 0.899). The standard
regression deviation (s2 = 2.565) and the Fisher test value (F = 38.712) are also statistical
parameters which help in supporting the QSAR model. Table 6 presents the predicted/estimated
IC50 values for the chemical entities as reported by the achieved BMLR-QSAR model. The
findings showed that compound 4n is the most potent derivative in this series, showing error
value (difference between estimated and observed IC50 values) = −17. Furthermore, compounds
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4n,h,i are the most potent derivatives amongst the new chemical entities, compared with the
standard reference Erlotinib (IC50 = 152 nM), revealing an estimated bio-properties (IC50 = 211,
205 and 275 nM) so far matching their observed potencies (IC50 =195, 225 and 275 nM) with
error values (-17, 0 and 20) respectively. Whereas compounds 4a, d (IC50 = 352, 467 nM and
445, 433 nM for the observed and estimated IC50, respectively) came with reasonable error
values = -92, -34. It is interested to mention that the lowest bio-active compound in this study 4c
showed also the largest gab between the observed and estimated bio results (error values = 2194)
and can be considered as an outlier (Fig. 7). Based on the detected and estimated bio-
observations, it could be concluded that, the accomplished BMLR-QSAR model is statistically
significant and verified by the robust correlations. Successful accomplishment of this study is
correlated to the uniformity of scaffolds chemical structures. Furthermore, the accomplished
model could be assumed for improving new hits of better efficacies compared with the standard
drugs.
Table 7. Observed and estimated/predicted activity values for the compounds being tested as
EGFR inhibitors depending on the BMLR-QSAR model
Entry Compound No Observed IC50, nM Estimated IC50, nM
Errora
-110
-279
793
-691
-34
1
2
858
985
2278
722
433
352
857
631
225
1470
529
3705
1277
275
195
968
1264
1485
1413
467
445
824
665
205
843
920
1511
976
275
211
4e
4k
4o
4j
3
4
5
4d
4a
4b
4m
4h
4g
4f
6
-92
7
33
8
-34
9
20
10
11
12
13
14
15
627
-391
2194
301
0
4c
4l
4i
-17
4n
19

16
17
1017
1703
537
480
637
4p
4q
1066
a Error is the difference between the observed and estimated property values.
Fig. 7. BMLR-QSAR model plot of correlations representing
the observed vs. predicted 1/(IC50), nM values
for the tested compounds as EGFR inhibitors
4. Molecular docking studies
In order to rationalize the accomplished results of EGFR enzymatic assay and to obtain
an insight into the relationship between the chemical structures of the target thiazol-
benzimidazole compounds and their potencies, molecular docking study was carried out for the
highly potent targets 4a, 4d, 4h, 4i and 4n within the active site of EGFR using Molecular
Operating Environment (MOE®) 2008.10 software91. Firstly, the original co-crystallized ligand,
erlotinib was re-docked in the active site of EGFR (PDB code: 1M17)92,93 (Fig. 8A, B). It
revealed score energy -12.85 kcal/mol at root mean square deviation (RMDS) value equal to 7.32
Å giving perfect superimposition of the docking pose and the co-crystallized of erlotinib (Fig.
8C).
As reported in the docking of erlotinib, the N1 atom of its quinazoline core formed a
hydrogen bond acceptor with the backbone of Met769 (distance: 2.70 Å), while the methoxy-
ethoxy group inserted into the hydrophobic region II and formed hydrophobic interaction with
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Leu694, Leu768 and Gly772 residues. Moreover, the ethynylphenyl moiety located in the
hydrophobic pocket I and displayed hydrophobic interactions with Ala719, Lys721, Lue764,
Thr766, Leu820, Thr830 and Asp83194. Then, the newly synthesized compounds 4a, 4d, 4h, 4i
and 4n were docked into the ATP-active site of EGFR and the docking scores are listed in table
8. By comparison of the docking energy scores of compounds 4a, 4d, 4h, 4i and 4n with that of
the native ligand erlotinib, it has been observed that all derivatives illustrated variable and
promising scores ranging from -11.32 to -14.20 kcal/mol.
Table 8. Docking scores of the compounds 4a, 4d, 4h, 4i, 4n and erlotinib within the active
pocket of EGFR using MOE software version 2008.10
Docking score
Compound No.
(Kcal/mol)
-12.85
-11.32
-12.83
-13.46
-13.72
-14.20
erlotinib
4a
4d
4h
4i
4n
By inspection of the recorded data in figures 9-13, it was noticed that all compounds 4a,
4d, 4h, 4i and 4n were embedded nicely within the active site of EGFR through two hydrogen
bond acceptors between the sidechain of Lys721 and the two nitrogen atoms of the
benzimidazole and the thiazole rings, respectively. Also, the thiazole ring shared the binding via
arene-arene interaction with the essential amino acid Phe699. Furthermore, the amino acid
Lys721 demonstrated extra arene-cation with the centroid of 4-substituted phenyl in case of 4d,
4h, 4i and 4n that gave the chance for more fixation within the pocket.
On the other hand, the superimposition model between the new targets 4a, 4d, 4h, 4i, 4n
and the original ligand erlotinib within EGFR approved that all of them settled in the same active
site and were surrounded by the same hydrophobic interactions. In case of erlotinib, the
hydrophilic interactions based on the interaction with Met769, while in case of our targets, the
they based on binding with Lys721 (Fig. 14).
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Based on the previous docking results, it can be deduced that the conjugation of
benzimidazole nucleus with thiazole moiety improved the binding strength within the EGFR
active pocket through hydrogen bond formation with the key amino acid Lys721 and
hydrophobic interaction with Phe699.
A
B
22

C
Fig. 8. 2D & 3D Views (A & B) of the original ligand, erlotinib re-docked in the active site of
EGFR (PDB ID: 1M17) using MOE software. 3D representation (C) of the superimposition of
the docking pose (yellow) and the co-crystallized (red) of erlotinib with an RMSD of 0.76 Å
23

A
B
24

Fig. 9. A & B diagrams illustrate the 2D & 3D binding pattern of compound 4a into ATP-active
pocket of EGFR (PDB code: 1M17)
A
B
25

Fig. 10. A & B diagrams illustrate the 2D & 3D binding pattern of compound 4d into ATP-
active pocket of EGFR (PDB code: 1M17)
A
B
26

Fig. 11. A & B diagrams illustrate the 2D & 3D binding pattern of compound 4h into ATP-
active pocket of EGFR (PDB code: 1M17)
A
B
27

Fig. 12. A & B diagrams illustrate the 2D & 3D binding pattern of compound 4i into ATP-active
pocket of EGFR (PDB code: 1M17)
A
B
Fig. 13. A & B diagrams illustrate the 2D & 3D binding pattern of compound 4n into ATP-
active pocket of EGFR (PDB code: 1M17)
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Fig. 14. 3D representation of the superimposition of erlotinib (red), 4a (yellow), 4d (green), 4h
(blue), 4i (violet) and 4n (brown) within the ATP-active pocket of EGFR (PDB code: 1M17)
5. Conclusion
In conclusion, a new class of thiazol-benzimidazole compounds has been designed and
synthesized as EGFR PK inhibitors targeting human breast cancer (MCF-7). All the new
derivatives were screened for their potential suppression activity against EGFR. The compounds
4n, 4h, 4i, 4a, 4d revealed more potent inhibiting effect than erlotinib, displaying IC50 values
ranging from 71.67-150.36 nM vs IC50 erlotinib, 152.59 nM. On the other hand, the compounds 4f,
4m, 4b showed 1.3-1.8 folds reduction in the potency in comparison with erlotinib revealing
IC50 values; 208, 212, 278 nM. The compounds 4a, 4b, 4d, 4f, 4h, 4i, 4j, 4m, 4n were examined
as cytotoxic agents using MTT assay utilizing erlotinib as a standard drug. The compounds 4j,
4a, 4f appeared as the most promising cytotoxic candidates of IC50 values 5.96, 6.30, 7.56 µM,
respectively, IC50 erlotinib; 4.15 µM. The safety profile of the most active compounds (4a, 4b, 4d,
4f, 4h, 4i, 4j, 4m, 4n) was evaluated against the normal hTERT-RPE1 cell line. Most of the
compounds appeared as safe agents on the normal cells.
The biological evidence revealed that the compound 4a caused marked apoptosis degree
with necrosis percentage 2.17%. It led to high cell aggregation and cell cycle disruption at G2/M
phase in MCF-7 cancer cells. Additionally, the increased expression of p53, caspase-3, Bax /
Bcl-2 ratio as well as the significant decrease in the level of PARP-1enzyme have verified the 4a
apoptotic pathway.
29