Synthesis, characterization, and biological activity of a novel series of benzo[4,5]imidazo[2,1-b]thiazole derivatives as potential epidermal growth factor receptor inhibitors

Article abstract of DOI:10.3390/molecules24040682

Based on the analysis of epidermal growth factor receptor (EGFR) complexes with gefitinib with molecular docking, the scaffold-hopping strategy, combination of the active substructures, and structural optimization of EGFR inhibitors, a novel series of benzo[4,5]imidazo[2,1-b]thiazole derivatives was designed, synthesized, and evaluated for antitumor activity in human cancer cell lines and cellular toxicity against human normal cell lines using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay and EGFR inhibitory activities in vitro. Some target compounds such as 2-(benzo[4,5]imidazo[2,1-b]thiazol-3-yl)-N-(2-hydroxyphenyl)acetamide (D04) and 2-(benzo[4,5]imidazo[2,1-b]thiazol-3-yl)-N-(naphthalen-1-yl)acetamide (D08) have shown significant antitumor activity against the EGFR high-expressed human cell line HeLa. All the target compounds showed hardly any antitumor activity against the EGFR low-expressed human cell line HepG2, and nearly no cellular toxicity against the human normal cell lines HL7702 and human umbilical vein endothelial cell lines (HUVEC). The inhibitory activities against EGFR kinase in vitro of the three target compounds were greatly consistent with the anti-proliferative activities. The preliminary structure–activity relationships of the target compounds were summarized. Conclusively, the novel benzo[4,5]imidazo[2,1-b]thiazole derivatives as novel potential EGFR inhibitors may be used as the potential lead compounds for the development of antitumor agents.

Full text of DOI:10.3390/molecules24040682

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molecules
Article
Synthesis, Characterization, and Biological Activity of
a Novel Series of Benzo[4,5]imidazo[2,1-b]thiazole
Derivatives as Potential Epidermal Growth Factor
Receptor Inhibitors
Xinshan Deng 1, Xiaoyu Tan 1, Tiantian An 1, Qingqing Ma 1, Zhe Jin 1,*, Ce Wang 1,
Qingguo Meng 2 and Chun Hu 1,
*
1
Key Laboratory of Structure-Based Drug Design & Discovery, Ministry of Education,
Shenyang Pharmaceutical University, Shenyang 110016, China; xinshandeng@126.com (X.D.);
tanxiaoyuaikaoyan@163.com (X.T.); antiantiansyphu@163.com (T.A.); maqingqing01@163.com (Q.M.);
wangce0412@163.com (C.W.)
Department of Pharmacy, Yantai University, Yantai 264005, China; qinggmeng@163.com
Correspondence: jinzheln@163.com (Z.J.); chunhu@syphu.edu.cn (C.H.); Tel.: +86-24-43520246
2
*
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Academic Editor: Wolfgang Sippl
Received: 21 November 2018; Accepted: 4 February 2019; Published: 14 February 2019
Abstract: Based on the analysis of epidermal growth factor receptor (EGFR) complexes
with gefitinib with molecular docking, the scaffold-hopping strategy, combination of the
active substructures, and structural optimization of EGFR inhibitors, a novel series of
benzo[4,5]imidazo[2,1-b]thiazole derivatives was designed, synthesized, and evaluated
for antitumor activity in human cancer cell lines and cellular toxicity against human
normal cell lines using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) colorimetric assay and EGFR inhibitory activities in vitro
such as 2-(benzo[4,5]imidazo[2,1-b]thiazol-3-yl)-N-(2-hydroxyphenyl)acetamide
.
Some target compounds
D04 and
(
)
2-(benzo[4,5]imidazo[2,1-b]thiazol-3-yl)-N-(naphthalen-1-yl)acetamide (D08) have shown significant
antitumor activity against the EGFR high-expressed human cell line HeLa. All the target compounds
showed hardly any antitumor activity against the EGFR low-expressed human cell line HepG2,
and nearly no cellular toxicity against the human normal cell lines HL7702 and human umbilical
vein endothelial cell lines (HUVEC). The inhibitory activities against EGFR kinase in vitro of the
three target compounds were greatly consistent with the anti-proliferative activities. The preliminary
structure–activity relationships of the target compounds were summarized. Conclusively, the novel
benzo[4,5]imidazo[2,1-b]thiazole derivatives as novel potential EGFR inhibitors may be used as the
potential lead compounds for the development of antitumor agents.
Keywords: EGFR inhibitors; heterocycle; synthesis; molecular docking; antitumor activity
1. Introduction
Presently, many populations are affected by cancer. In 2012, 14.2 million new cancer cases and
8.2 million deaths occurred, and by 2030, this statistic is reported to increase to about 19 million [1].
To find an effective way to prevent cancer from developing and stop patients from deteriorating,
over the past decade, a novel series of selective chemotherapeutic drugs such as imatinib, gefitinib,
erlotinib, and afatinib has been launched worldwide for the treatment of cancer (Figure 1). The action
targets of these innovative drugs are mainly directed to impact the signal transduction pathway of the
epidermal growth factor receptor (EGFR, Figure 2) [2].
Molecules 2019, 24, 682; doi:10.3390/molecules24040682
www.mdpi.com/journal/molecules

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The over-expression and/or mutation of EGFR plays an important role in the growth of cancer
including cell proliferation, anti-apoptosis, metastasis, and angiogenesis [ ]. The EGFR has been
reported as an attractive target for cancer therapy [ ]. Over recent years, many scientists have
discovered the mechanism of the EGFR that the binding of specific ligands such as epidermal growth
factor (EGF) and transforming growth factor (TGF ) to the extracellular domain of EGFR induces
3
4–6
α
α
the dimerization, activation of intrinsic kinase activity, and subsequent autophosphorylation of EGFR
at multiple residues in the cytoplasmic region, while the downstream signaling proteins including
phosphoinositide 3-kinase (PI3-K), Akt, mammalian target of rapamycin (mTOR), Ras, Raf, MEK, and
extracellular signal-regulated kinase (ERK) stimulate several signal transduction cascades, leading to
DNA synthesis and cell proliferation (Figure 2) [7–10]. As it can obviously influence the proliferation
of human cancer cells, and even result in the apoptosis of human cancer cells, the EGFR has become
a very luminous target for various antitumor candidates [11].
N
F
N
O
HN
N
Cl
HN
N
N
O
O
N
O
N
N
H
N
gefitinb
imatinib
F
HN
Cl
H
N
HN
N
N
O
O
O
O
N
O
N
O
N
O
erlotinib
afatinib
Figure 1. The chemical structure of the selective chemotherapeutic drugs.
Figure 2. The signal transduction pathway of EGFR.
Gefitinib, as a selective small-molecule EGFR tyrosine kinase inhibitor indicated for the treatment
of adults with locally advanced or metastatic non-small cell lung cancer (NSCLC) with activating
mutations of EGFR tyrosine kinase, was approved by the USA Food and Drug Administration
(FDA) in 2003 [11]. In this study, to discover a novel EGFR inhibitor, gefitinib was used as the
lead molecule. According to the scaffold-hopping strategy of drug design [12], the scaffold of the
lead molecule was changed to benzo[4,5]imidazo[2,1-b]thiazole, a novel heterocycle core. As the
two side chains of the quinazoline ring in gefitinib serve to improve physical properties and confer

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a favorable pharmacokinetic profile in animals and humans [13], based on the efficacy of the target
compounds that should be preferentially considered, the side chains in the core scaffold were tentatively
ignored. The pharmacodynamic aniline group in gefitinib was retained in the target compounds with
a –CH2CO– linker (Figure 3). Moreover, to gain an understanding of the interactions between these
benzo[4,5]imidazo[2,1-b]thiazole derivatives and EGFR, hereinafter docking studies are also presented.
Herein, we report on the synthesis, characterization, and biological activity against cancer and normal
human cell lines of a novel series of benzo[4,5]imidazo[2,1-b]thiazole derivatives as EGFR inhibitors.
Figure 3. The design of the target compounds based on the structure of gefitinib.
2. Results and Discussion
2.1. Virtual Screening
In general, the molecular docking technique can help to understand the binding mode of the
target compounds with EGFR, rationalize the molecular level interactions between the bio-target and
ligand, and play an important role in new drug discovery.
In this study, gefitinib was chosen as the lead molecule, which was transformed to become the
target compounds. The crystallographic structure of the EGFR-gefitinib complex [14], which was
encoded as 4I22 in the Protein Data Bank (PDB) of Research Collaboratory for Structural Bioinformatics
(RCSB), was used for the docking model.
Molecular docking studies between the EGFR and the target compounds with gefitinib were
performed with Molegro Virtual Docker 2010 (v4.1) (Molegro ApS, Aarhus, Denmark). The docking
algorithm was set at 1500 maximum iterations with a simple evolution population size of 50 and
a minimum of 10 runs. Its original inhibitor (gefitinib) was deleted and all the water molecules
were removed, adding a further degree of freedom in terms of variability in the docking results.
Schematic diagrams of the interactions between the EGFR and the small molecules were analyzed
by the Discovery Studio 2016 client and the PyMOL. All parameters of the binding energy and the
H-bond energy of the target compounds were revealed to be as good as the lead one (Table 1).
Table 1. The results of the docking studies.
Compound
Binding Energy (kJ/mol)
H-Bond Energy (kJ/mol)
D01
D02
D03
D04
D05
D06
D07
D08
D09
D10
D11
D12
D13
D14
D15
D16
D17
D18
gefitinib
−108.06
−130.13
−122.44
−100.89
−125.13
−113.96
−97.81
−0.28
−3.93
−2.50
−0.83
−1.80
−2.82
−1.36
−2.46
0
−2.50
−2.44
−4.19
0
−0.84
−2.50
−1.47
−0.76
−1.09
−0.56
−121.82
−123.99
−96.51
−107.71
−133.45
−125.90
−110.38
−111.54
−110.86
−115.62
−102.07
−131.54

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Based on the docking results, a significant hydrogen bond was formed with MET793 for gefitinib,
and the other EGFR residues that interacted with gefitinib were LEU718, VAL726, ALA743, LYS745,
LEU788, MET790, GLN791, MET793, PRO794, LEU844, and THR854 (Figure 4). Surprisingly, there
was an equal hydrogen bond that formed with MET793, and the same other EGFR residues interacted
excluding GLN791 with EGFR for the target compounds D04 and D08 (Figure 5). Therefore, the docking
results indicate that the target compounds would become potent EGFR inhibitors.
Figure 4. The 3D and 2D representation of the EGFR crystal structure in complex with gefitinib (PDB
ID: 4I22). (left) The 3D representation of the EGFR crystal structure in complex with gefitinib; (right
)
The 2D representation of the EGFR crystal structure in complex with gefitinib.
Figure 5. The interactions of EGFR with D04 (orange), D08 (purple), and gefitinib (yellow) shown
by PyMOL.
2.2. Chemistry
As shown in Scheme 1, all the target compounds were rooted from benzene-1,2-diamine (
The 1H-benzo[d]imidazole-2-thiol ( ) was prepared by reacted with CS2 and Na2CO3, and then easily
converted to the intermediate with ethyl 4-chloro-3-oxobutanoate. Subsequently, the intermediate
was prepared by cyclization of the intermediate in the presence of concentrated sulfuric acid at
50 ◦C, and then the intermediate
was directly hydrolyzed and acidified to yield the intermediate 5.
1).
2
1
3
4
3
4
Finally, the reaction between the intermediate
5 and aniline or aniline derivative resulted in the target
compounds D01 to D18.