Novel imidazo[2,1-b]thiazole-based anticancer agents as potential focal adhesion kinase inhibitors: Synthesis, in silico and in vitro evaluation

Article abstract of DOI:10.1111/cbdd.13896

The purpose of this study was to synthesize imidazo[2,1-b]thiazole derivatives, characterize them with spectroscopical techniques and investigate for cytotoxic and apoptotic effects on glioma C6 cancer cell line. The in vitro anticancer activities were al

Full text of DOI:10.1111/cbdd.13896

Received: 3 March 2021
DOI: 10.1111/cbdd.13896
Revised: 9 May 2021
Accepted: 15 May 2021
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R E S E A R C H A R T I C L E
Novel imidazo[2,1-b]thiazole-based anticancer agents as potential
focal adhesion kinase inhibitors: Synthesis, in silico and in vitro
evaluation
Faika Başoğlu1,2
Serap Çetinkaya4
Nuray Ulusoy-Güzeldemirci1
Gülşen Akalın-Çiftçi3
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Abdulilah Ece5
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1Department of Pharmaceutical Chemistry,
Faculty of Pharmacy, Istanbul University,
Istanbul, Turkey
2Department of Pharmaceutical Chemistry,
Faculty of Pharmacy, European University
of Lefke, Northern Cyprus, Turkey
3Department of Biochemistry, Faculty of
Pharmacy, Anadolu University, Eskisehir,
Turkey
4Department of Molecular Biology
and Genetics, Science Faculty, Sivas
Cumhuriyet University, Sivas, Turkey
Abstract
The purpose of this study was to synthesize imidazo[2,1-b]thiazole derivatives, char-
acterize them with spectroscopical techniques and investigate for cytotoxic and apop-
totic effects on glioma C6 cancer cell line. The in vitro anticancer activities were also
investigated against focal adhesion kinase. Most of the compounds, particularly the
derivatives carrying 3-oxo-1-tiya-4-azaspiro[4.5]decane moiety, exhibited higher or
comparable activities in comparison with the reference drug, cisplatin. Compounds
with methyl, propyl, phenyl moieties at the eighth and second position of the spiro-
thiazolidinone ring showed high FAK inhibitory activities. In addition, molecular
docking studies shed light on the binding modes of the synthesized compounds. The
critical interactions with amino acid residues located in the active site were revealed.
The results obtained from both biological assay data and computational results might
provide insight into developing new inhibitors against focal adhesion kinase.
5Department of Pharmaceutical Chemistry,
Faculty of Pharmacy, Biruni University,
Istanbul, Turkey
Correspondence
Faika Başoğlu, Department of
Pharmaceutical Chemistry, Faculty of
Pharmacy, Istanbul University, 34116,
Istanbul, Turkey.
K E Y W O R D S
focal adhesion kinase, Imidazo[2,1-b]thiazole, molecular docking
Email: fabasoglu@eul.edu.tr
Abdulilah Ece, Department of
Pharmaceutical Chemistry, Faculty of
Pharmacy, Biruni University, 34010,
Istanbul, Turkey.
Email: aece@biruni.edu.tr
Funding information
Istanbul Üniversitesi
1
INTRODUCTION
of problems in drug delivery and inherent radio, and chemo-
resistance (Lin et al., 2017; Patil et al., 2013). Therefore, the
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Cancer (carcinoma) is one of the serious diseases that are still
being studied extensively. Among the most aggressive human
cancers are gliomas, a type of primary malignant brain tumour.
Despite advances in regular therapy, patients with gliomas
have a low prognosis. At the present day, adjuvant chemo-
therapy and radiotherapy are administered to glioma patients
after surgery. The efficacy of chemotherapy is limited because
development of novel anticancer drugs that possess good bio-
logical activity and fewer side-effects are required.
In medicinal chemistry, imidazo[2,1-b]thiazoles have a
broad spectrum of pharmacological activities including an-
tiviral (Conti et al., 2019), antibacterial (Atta et al., 2011;
Gadad et al., 2000; Mohan & Kiran, 1991), antifungal (Çapan
et al., 1999; Juspin et al., 2010; Ulusoy et al., 2002) and
Chem Biol Drug Des. 2021;00:1–13.
wileyonlinelibrary.com/journal/cbdd
© 2021 John Wiley & Sons A/S.
1
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BAŞOĞLU et AL.
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antitumor (Andreani et al., 1982; Andreani et al., 1992, 1993)
activities. Moreover, the clinical efficacies of tiazofurin, da-
satinib and bleomycins have indicated the importance of
incorporating the thiazole moiety in antitumor drug design
(Paunescu et al., 2015; Rouf & Tanyeli, 2015).
bovine serum (Gibco, Paisley, UK), NIH/3T3 mouse em-
bryonic fibroblast cells and Glioma C6 rat cancer cells
were brooded. All of the media were put in with 100 IU/ml
penicillin-streptomycin (Gibco, Paisley, UK), and the cells
were incubated at 37°C in a humidified atmosphere of 5%
CO2 and 95% air. Exponentially growing cells were plated
at 2 × 104 cells/mL into 96-well microtiter tissue culture
plates (Nunc, Roskilde, Denmark) and were incubated for
24 hr before the supplementation of the compounds. The
stock solutions of compounds were prepared with DMSO
(dimethyl sulfoxide), and extra dilutions were done using
the fresh culture medium. Finally, DMSO's concentra-
tion in the last culture medium was less than 0.1% (Ciftci
et al., 2015).
Focal adhesion kinase (FAK) is a tyrosine kinase that
plays a major role in cellular survival and movement path-
ways. FAK is a potential target for the prevention and treat-
ment of both primary cancers and tumour metastasis (Frisch
et al., 1996; Ilic et al., 1995; Mitra et al., 2005). Only in the
past decade, phase I and II clinical trials have been initiated
for small molecule inhibitors of FAK (Infante et al., 2012;
Shanthi et al., 2014; Yoon et al., 2015). Therefore, inhibition
of FAK activity may be a novel strategy for cancer therapy.
Previous studies reported that compounds carrying imid-
azole and thiazole rings display anticancer activity by inhib-
iting FAK (Dao et al., 2015; Jain et al., 2013; Lv et al., 2018).
The present study aims to investigate the synthesis of a se-
riesofnovelimidazo[2,1-b]thiazolederivatives,whichinclude
either N-cyclohexylidene or 3-oxo-1-thia-4-azaspiro[4.5]
decan-4-yl that could have potential anticancer activity. The
characterization of these compounds with spectroscopic tech-
niques was also carried out, and in vitro anticancer activities
were evaluated. Additionally, computational studies were
employed to gain new insights into interaction modes of the
compounds with the active site of FAK.
2.2.2
MTT assay for
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cytotoxicity of compounds
The level of cellular reduction of tetrazolium salt, 3-(4,5
-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide
(MTT) to formazan by mitochondrial succinate dehydroge-
nase was quantified as previously described in the litera-
ture with small modifications (Scheuber et al., 1983). After
24 hr of preincubation, the tested compounds and cisplatin
(positive control) were added to give final concentration
in the range 3.9–500 µg/ml, and the cells were incubated
for 24 hr. Then, MTT was added to a final concentration
of 0.5 mg/ml, and the cells were incubated for further
four hours at 37℃. After the medium was removed, the
formazan crystals were solubilized by the addition of 200
µl DMSO to each well and absorbance was read at 540 nm
with a microtitre plate spectrophotometer (Bio-Tek plate
reader, Winooski, VT, USA). Every concentration was re-
peated in three wells, and IC50 values were defined as the
drug concentrations that reduced absorbance to 50% of the
control values (Ciftci et al., 2015).
2
METHODS AND MATERIALS
Experimental
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2.1
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Details for general synthesis procedure for the compounds
are given in supplementary data. All synthesized compounds
were recrystallized from 96% ethanol. After the purification,
white solid powders were obtained with good yield.
Elemental analyses were carried out on a Thermo Finnigan
Flash EA 1,112 elemental analyser. Melting points were exam-
ined using a Büchi B-540 melting point apparatus in open capil-
lary tubes and are uncorrected. FT-IR spectra were recorded on
KBr discs, using a Shimadzu IR Affinity-1 FT-IR spectrophotom-
eter. 1H-NMR, 13C-NMR (APT), 13C-NMR (DEPT) and HSQC
(1H-13C) spectra were measured on a Varian UNITY INOVA
(500 MHz) spectrometer using DMSO-D6. Mass spectra were re-
corded on a Thermo Finnigan LCQ Advantage Max instrument.
In the present study, the degree of selectivity index (SI) of
the synthetic compounds is expressed as follows:
SI = IC50 of pure compound in a normal cell line / IC50
of the same pure compound in cancer cell line, where IC50 is
the concentration required to kill 50% of the cell population.
2.2.3
Flow cytometric analyses of apoptosis
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After cells were incubated with compounds 4g, 4k, 5c, 5d, 5e,
6a-6f and cisplatin at IC50 concentrations, phosphatidylserine
externalization which indicates early apoptosis was measured
by Annexin V-PI (BD, Pharmingen) on a flow cytometer
(BD FACS Aria) (Ciftci et al., 2015) for 24 hr. Annexin V
staining protocol was applied according to the manufacturer's
instructions (BD, Pharmingen). The cells were processed for
2.2
Biochemistry
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2.2.1
Cell culture method
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In Dulbecco's Modified Eagle's Medium (DMEM - Sigma,
Deisenhofen, Germany) supplemented with 10% foetal

BAŞOĞLU et AL.
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data acquisition and analysed on a Becton-Dickinson FACS
Aria, which uses FACSDiva version 6.1.1. Software (Ciftci
et al., 2015).
2.3.3
Calculation of the molecular properties
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Some selected physicochemical properties of the synthesized
compounds were calculated using the Qik-Prop module of
Maestro (Table 4) (Schrödinger, 2016c).
2.2.4
FAK (Phospho-Tyr397) activity
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detection by ELISA
3
RESULTS AND DISCUSSION
Chemistry
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Initially, C6 cancer glioma cells were administrated by IC50
concentrations of compounds 4c, 4h, 4l, 5d, 6b, 6d, 6e, 6f
and cisplatin for 24 hr. FAK (Phospho-Tyr397) effectiveness
of compounds was examined by Colorimetric Cell-Based
ELISA FAK (Phospho-Tyr397) activity kit (Aviva Systems
Biology, San Diego, USA) according to manufacturer's in-
structions (Altintop et al., 2018).
3.1
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The rationale for the design and synthesis of N2-(substituted
cyclohexylidene)-6-(phenyl/4-chlorophenyl)imidazo[2,1-b]
thiazol-3-acetohydrazides and 2-[6-(phenyl/4-chlorophenyl)
imidazo[2,1-b]thiazol-3-yl]-N-(2-nonsubstituted/methyl-
6,7,8,9-alkyl/aryl-3-oxo-1-thia-4-azaspiro[4.5]decan-4-yl)
acetamides derivatives are based on previous studies that
showed efficient anticancer potency. Both imidazo[2,1-b]
thiazoles and thiazolidinone scaffolds are known to possess
many various anticancer activities (Ali et al., 2014; Deep
et al., 2015; Deng et al., 2019; Jain et al., 2012; Manasa
et al., 2020; Romagnoli et al., 2015; Szychowski et al., 2017;
Szychowski et al., 2017; Tahmasvand et al., 2020).
Additionally, Levamisole containing imidazo[2,1-b]thiazole
nucleus was approved by FDA in 1990 and has been used in
cancer treatment since then (FDA, 2021; Moertel et al., 1995;
Vacchelli et al., 2012) Those imidazo[2,1-b]thiazole
2.3
Computational section
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2.3.1
Ligand and protein preparation
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Prior to computational studies, all compounds must be pre-
pared for calculations. Accordingly, all compounds were
prepared using the LigPrep (Ligand preparation) module of
the Schrödinger suite (Schrödinger, 2016a). OPSL2005 (op-
timized potential liquid simulations) force field was used for
minimizations (Jorgensen et al., 1996; Jorgensen & Tirado-
Rives, 1988; Shivakumar et al., 2010). All probable states of
the compounds at pH 7.0 2.0 were generated.
The molecular docking studies were performed using
high resolution (2.3 Å) X-ray crystal structure of focal
adhesion kinase domain (PDB ID: 2ETM) in complex
with 7H-Pyrrolo[2,3-d]pyrimidine derivative (7PY, na-
tive ligand). The raw crystal structure was also prepared
using PrepWizard (Protein Preparation Wizard) (Sastry
et al., 2013) in Maestro of Schrödinger-2016 software pack-
age (Schrödinger, 2016b). As a first step, the PrepWizard
was used for adding missing hydrogen atoms and removing
all water molecules and heteroatoms. The native ligand was
kept for binding site detection. Then, the protein structure
was refined by assigning the bond orders and revising the
missing side-chains. In the final step, the steric clashes be-
tween the atoms were removed by minimizing whole struc-
ture using OPLS2005 force field.
2.3.2
Molecular docking
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The docking calculations were carried out using Glide SP
(standard precision) module (Friesner et al., 2004; Halgren
et al., 2004). A grid that illustrated the binding pocket was
generated. The native ligand, 7PY, was used as reference to
identify the size and centre of the receptor grid.
SCHEME 1 Rationally designed template for the targeted
compounds as potential anticancer agents

4
BAŞOĞLU et AL.
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O
SCHEME 2 General synthetic
O
O
S
O
pathway of the imidazo[2,1-b]thiazole
derivatives (4a-m, 5a-5f, 6a-6f). Reagents
and conditions: (i) acetone, 36h; (ii) 99%
ethanol, reflux, 20 min; (iii) 99% ethanol,
98% NH2NH2.H2O, reflux, 5h; (iv) 99%
ethanol, cyclic ketone derivatives, reflux,
6h; (v) toluene, mercaptoacetic acid or
2-mercaptopropionic acid, reflux, 7h
O
i
R
H N
2
+
O
Br-
R
N
N+
Br
S
H2N
1
ii
CH2COOC 2H5
HBr
CH2CONHNH 2
N
iii
R
N
R
N
S
N
S
2
3
R5
O
R
R1
iv
R2
R4
O
R3
N
S
R2
N
N
HS
NH
R4
COOH
v
N
R5
R
R1
R4
N
S
R1
NH
N
O
R5
O
4
S
R2
R3
5, 6
Comp.
4a
4b
4c
4d
4e
R
H
H
Cl
Cl
Cl
Cl
Cl
Cl
Cl
R1
H
Pr
H
H
H
H
H
Pr
H
R2
Me
H
Me
2-CNEt
R3
-
-
-
-
-
-
-
-
-
-
-
-
-
H
H
H
H
H
H
Me
Me
R4
H
H
H
H
H
R5
H
H
H
H
COOEt
H
Me
4f
H
H
H
diMe
diMe diMe
H
H
H
H
H
H
H
diMe
H
H
H
H
4g
4h
4i
4j
4k
4l
4m
5a
5b
5c
5d
5e
H
H
H
H
H
H
H
Me
H
H
H
H
H
H
H
H
H
H
ter-butyl
H
Cl ter-butyl
Cl
Cl
H
Cl
Ph
Ph
H
Me
H
H
H
H
H
H
Me
H
H
H
Cl 4-OHPh
Cl
Cl
Cl
H
H
Pr
Cl ter-butyl
Cl Ph
Cl 4-OHPh
H
Cl
Cl
Cl
5f
6a
6b
6c
6d
6e
Pr
H
H
H
H
Me diMe
Pr
Me
Me
Me
H
H
H
Cl ter-butyl
Cl Ph
6f
H

BAŞOĞLU et AL.
5
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TABLE 1 IC50 values of the synthesized compounds on C6 cancer cells and NIH/3T3 cell lines
IC50 (μM)
IC50 (μM)
NIH/3T3 cell
line
SI
NIH/3T3 cell
line
SI
Comp.
C6 cell line
value
Comp.
C6 cell line
values
4a
4b
4c
4d
4e
4f
4g
4h
4i
>500
>500
>500
275 35.35
>500
>500
8.33 2.08
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
–
–
–
5a
5b
5c
5d
5e
5f
68.33 12.58
>500
13.66 2.08
12.66 0.57
19.83 0.76
>500
12.66 0.57
28.33 2.88
18.00 2.00
10.83 1.25
19.66 3.21
16.33 1.52
36 8.48
>500
>500
>7.32
-
10.24
2.03
1.64
-
12.38
2.03
2.17
20.62
1.27
3.83
140.0 28.28
25.67 6.03
32.5 3.54
>500
156.67 40.41
57.5 3.54
39.0 1.41
223.33 25.17
25.0 7.07
62.5 3.24
ND
>1.82
–
–
>60.02 6a
–
6b
6c
6d
>500
–
4j
4k
4l
106.66 2.88
4.83 0.28
>500
>4.69
>103.5 6e
–
–
6f
4m
>500
Cisplatin
Abbreviations: ND, not determined; SI, selectivity index.
TABLE 3 FAK inhibitory effects of the compounds 4c, 4h, 4l,
5d, 6b, 6d, 6e, 6f and Cisplatin on C6 cell line
TABLE 2 Percentages of typical quadrant analysis of Annexin V
FITC/Propidium iodide flow cytometry of C6 cancer cells treated with
Cisplatin and most active compounds
Inhibition %
Early
Late
Compounds
FAK
apoptotic
cells%
apoptotic
cells%
Viable
cells%
Necrotic
cells%
Comp.a
4c (>500 μM)
4h (>500 μM)
4l (>500 μM)
5d (12.66 0.57 μM)
6b (28.33 2.88 μM)
6d (10.83 1.25 μM)
6e (19.66 3.21 μM)
6f (16.33 1.52 μM)
Cisplatin (36 8.48 μM)
20.58 3.31
21.74 2.87
5.93 0.04
37.29 2.85
68.71 0.73
47.78 3.93
46.64 8.59
72.55 3.63
32.03 0.21
Control
Cisplatin
4.8
2.2
8.4
12.9
2.5
7.1
0.2
4.7
3.5
4.0
5.7
5.6
2.1
3.7
10.6
7.8
23.4
24.5
6.3
1.6
14.9
3.9
87.4
75.1
63.6
29.0
11.6
77.9
33.4
22.0
85.5
79.3
14.2
84.3
77.0
4.1
12.1
20.3
34.7
61.9
13.5
64.8
58.5
7.2
4g
4k
5c
5d
5e
6a
6b
6c
6d
6e
6f
7.7
30.6
4.0
9.0
49.5
6.1
imidazo[2,1-b]thiazole derivative. The tail group was planned
as either N-cyclohexylidene or 3-oxo-1-thia-4-azaspiro[4.5]
decan-4-yl, and the linker between the head and tail groups
was designed as hydrazone and hydrazide, respectively.
The tail group that was expected to be key important for
the anticancer activity consists of 2-methylcyclohexyliden,
7.3
13.5
aC6 cancer cells were cultured for 24 hr in medium with active compounds (4g,
4k, 5c, 5d, 5e, 6a-f and cisplatin) at IC50 values. At least 10,000 cells were
examined for each sample, and quadrant analysis was achieved.
4-propylcyclohexyliden,
2-(2-cyanoethyl)cyclohexyliden,
2-ethoxycarbonylcyclohexyliden,3,3,5-trimethylcyclohexyliden,
2-ter-butylcyclohexyliden, 2-phenylcyclohexyliden,4-pheny
lcyclohexyliden, 4-(4-hydroxyphaneyl) cyclohexyliden and
their spirothiazolidinone derivatives.
Over and above that, substituents with different electro-
negativity and size were also considered. Therefore, some
small and bulky substitutions that have low electronegativity
derivatives were synthesized by extremely easy method with
a high yield (Guzeldemirci & Kucukbasmaci, 2010).
In the current study, the target novel compounds were
designed as the head group (region A), the linker chain (re-
gion B) and the tail group (region C) to shed light on future
research (Scheme 1). The head group was considered as an

6
BAŞOĞLU et AL.
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FIGURE 1 Flow cytometric analysis of C6 cancer cells treated with IC50 values of compounds 4g, 4k, 5c, 5d, 5e, 6a-6f and cisplatin
for 24 hr. At least 10,000 cells were examined for each sample, and quadrant analysis was carried out

BAŞOĞLU et AL.
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TABLE 4 Binding energies and
selected molecular properties of the native
ligand and compounds 4a-4m, 5a-5f, 6a-6f
Compound
MWa
%HOAb
PSAc
QPlogP0/wd
DSe
MM/GBSAf
4a
4b
4c
4d
4e
4f
4g
4h
4i
366.4
394.5
400.9
439.9
548.9
428.9
443.0
428.9
443.0
443.0
462.9
462.9
478.9
475.0
503.0
503.0
517.1
537.0
553.0
482.6
489.0
517.1
517.1
531.1
551.1
377.4
100
100
100
94.2
100
100
100
100
100
100
100
100
100
100
100
100
100
100
89.07
100
100
95.76
100
100
100
100
69.06
71.11
68.13
93.93
94.07
69.60
68.83
71.17
64.45
71.01
68.20
71.04
94.75
73.19
73.27
76.98
76.99
76.97
99.51
73.62
68.23
69.62
74.95
74.86
74.94
65.14
5.39
6.13
5.52
5.16
5.52
6.46
6.78
6.56
6.84
6.66
7.17
7.10
6.46
5.57
6.06
6.36
6.53
6.95
6.26
6.24
5.88
5.98
6.75
6.92
7.64
3.90
−5.441
−5.606
−4.490
−4.651
−4.844
−5.382
−5.035
−4.801
−4.894
−5.151
−4.781
−4.637
−5.692
−6.060
−4.587
−6.617
−5.977
−5.459
−5.588
−4.443
−6.622
−5.432
−6.715
−5.762
−5.558
−7.740
−52.572
−62.942
−50.817
−40.530
−61.477
−59.494
−67.270
−61.831
−65.487
−53.405
−42.677
−61.928
−64.445
−78.772
−45.650
−76.899
−82.917
−57.943
−60.849
−55.226
−81.730
−50.252
−78.799
−69.911
−70.194
−71.812
4j
4k
4l
4m
5a
5b
5c
5d
5e
5f
6a
6b
6c
6d
6e
6f
NL
aMolecular weight (g/mol) (recommended value <500).
bPercentage oral absorption (<25% weak and >85% strong).
cPolar surface area (Å) (recommended value ≤140 Å).
dLogarithm of the octanol/water ratio coefficient of compound (recommended value <5).
eDocking Score (kcal/mol).
fMM/GBSA dG Bind Energy (kcal/mol).
such as methyl, propyl, tert-butyl and phenyl were chosen.
Additionally, to investigate the electronegativity effect on ac-
tivity, more electronegative substituents such as 2-cyanoethyl,
ethoxycarbonyl and 4-hydroxyphenyl were chosen.
Scheme 2. The structures of the compounds were character-
ized by several spectroscopic methods.
MTT assay was used to figure out antiproliferative ef-
fectiveness of the synthesized compounds on rat glioma C6
cancer cell lines (Table 1). To investigate whether the com-
pounds possess either non-toxic or toxic to healthy (normal)
cells, the cytotoxic effects of imidazo[2,1-b]thiazole deriva-
tives (4a-4m, 5a-5f, 6a-6f) on NIH/3T3 mouse embryonic
fibroblast cell line were evaluated as well by MTT assay.
Because compound 6d had critical inhibitory effects on
C6 cell line with 10.83 1.25 µg/ml, it was identified to be
the most encouraging anticancer agent among the series of
the synthesized compounds. This result showed that the pro-
pyl substituent on the cyclohexane ring at the 4th position
of the azaspiro[4.5]decane moiety considerably increased
The synthesis of the heretofore unlisted compounds
(4a-m, 5a-f, 6a-f) is outlined in Scheme 1. N2-(substituted
cyclohexylidene)-6-(4-chlorophenyl/phenyl)imidazo[2,1-b]
thiazol-3-acetohydrazides (4a-4m) was synthesized starting
from
2-[6-(4-chlorophenyl/phenyl)imidazo[2,1-b]thiazol-
3-yl]acetohydrazides and then 2-[6-(phenyl / 4-chlorophenyl)
imidazo[2,1-b]thiazol-3-yl]-N-(2-nonssbstited/methyl-
6,7,8,9-alkyl/aryl-3-oxo-1-thia-4-azaspiro[4.5]decan-4-yl)
acetamides (5a-5f, 6a-6f) were synthesized by the reaction
of compounds 4a-m with thioglycolic acid or thiolactic acid.
All novel imidazo[2,1-b]thiazole derivatives are reported in

8
BAŞOĞLU et AL.
|
FIGURE 2 2D ligand interaction diagram for NL (Top), active
compound 6d (middle) and inactive compound 4h (bottom). H-
bond is represented purple arrow
anticancer activity against the C6 cancer cell line by induc-
ing apoptosis. Also, this compound had selectivity by its low
toxic effect on towards the NIH/3T3 cell line.
The ketone-hydrazone derivatives (except compound 4d, 4j,
4g and 4k) among the synthesized compounds did not exhibit
cytotoxic activity against C6 cancer cell line (IC50 > 500 µM).
Compounds 4d and 4j expressed cytotoxic activity with IC50
values of 275 35.35 and 106.66 2.88 µM, respectively.
However, compounds 4g and 4k exhibited considerable cyto-
toxic effect with IC50 values of 8.33 2.08 and 4.83 0.28 µM,
respectively. Also, the cytotoxicity of the compounds was low
against NIH/3T3 cell line (Table 1).
As the SI demonstrates the differential activity of
compounds, the greater the SI value is, the more selec-
tive it is. According to the calculated SI data shown in
Table 1, the selectivity index rankings are as follow;
4k>4g>6d>6a>5c>5a>4j>6f>6c>6b=5d. The rest of
the compounds displayed very low selectivity.
The apoptotic effects of the most effective anticancer
agents among the new series were evaluated for C6 can-
cer cell lines based on Annexin V-PI binding capacities in
flow cytometry. The early apoptotic effects of compound 6d
and cisplatin on the C6 cancer cell line were identified as
5.7 and 2.2%, respectively, while their late apoptotic effects
were observed as 30.6 and 10.6%, respectively (Table 2 and
Figure 1). The results showed that compound 6d possessed
more apoptotic activity against the C6 cancer cell line than
cisplatin. Moreover, the significance of the imidazo[2,1-b]
thiazole scaffold for apoptotic activity against the C6 cell line
was clearly observed.
We should note that FAK also known as PTK2, which
has been identified as a crucial regulator of growth factor
receptor, is either prompted or overexpressed in various can-
cers (Altintop et al., 2018). Hence, FAK has been known
to be a pharmacological target for preventing carcinoma,
and numerous compounds that have FAK inhibitory activity
have been designed and synthesized (Cao et al., 2016; Lee
et al., 2015; Roy-Luzarraga & Hodivala-Dilke, 2016; Yoon
et al., 2015; Zhao & Guan, 2011). Also, the significance of
imidazole and thiazole derivatives as FAK inhibitors (Dao
et al., 2015; Jain et al., 2013) prompted us to evaluate the
inhibitory effects of compounds 4c, 4h, 4l, 5d, 6b, 6d, 6e, 6f
on FAK (Phospho-Tyr397) activity (Table 3). According to
the assay, we administered IC50 values of these compounds
to C6 cell lines. Then, the OD450 values obtained for the
phosphorylated FAK were normalized using the OD450 val-
ues that were obtained for GAPDH after 24 hr. Mean per

BAŞOĞLU et AL.
9
|
FIGURE 3 Electrostatic potential at
the binding site mapped onto the molecular
surface of FAK in complexed with docked
6d (left) and 4h (right)
cent absorbance of untreated control cells were assumed to
be 0%. Then, % inhibition rates of compounds and cisplatin
administered C6 cells were calculated. Data points represent
means for three independent wells. According to the assay,
the most effective FAK inhibitor on C6 cancer line was de-
termined to be compound 6f (72.54925 3.63%) followed
by compounds 6b (68.71 0.73%), 6d (47.78 3.93%) and
6e (46.64 8.59%) (IC50 concentrations were 16.33 1.52,
28.33 2.88,10.83 1.25and19.66 3.21µM,respectively).
overlayed. The RMSD value between the docked pose and
the crystal conformation of the native ligand was found as
0.651 Å. Although the docking programmes perform well in
generating pharmacological active conformations of the li-
gands and detecting the binding modes to the receptor, the
current scoring functions are not predicted to distinguish be-
tween inactive and active compounds with a high success rate
(Ece & Fatma, 2013; Mascarenhas & Ghoshal, 2008; Tahtaci
et al., 2018). However, we have obtained good results with
Glide software previously (Er et al., 2017; Hou et al., 2011;
Tahtaci et al., 2018). The docking scores of the synthesized
compounds (4a-4m, 5a-5f, 6a-6f) are given together with
7PY in Table 4.
Glide docking performed well in classifying active com-
pounds, for example 6d, which had a higher docking score
among other compounds (4a-m, 5a-f, 6a-f). The native li-
gand, 7PY, had the highest docking score, and it fitted the
target by making hydrogen bonds with Cysteine 502 of FAK
(Figure 2). As a comparison, 2D binding interaction diagrams
were also depicted for one active and one inactive compound,
6d and 4h, respectively. As can be seen from the Figure 2, 6d
makes the same hydrogen bond with the amino acid residue,
CYS 502.
In order to get more insight into the difference between the
binding modes of the active and inactive compounds, the elec-
trostatic potential map surface of the receptor and the binding
surface of the ligands were drawn (Figure 3). Blue colour indi-
cates low electron density regions, while red colour illustrates
high electron density zones. The binding conformation of ac-
tive compound 6d and inactive compound 4h look thoroughly
different. The active compound 6d has favourable electrostatic
interactions with the receptor. For instance, high electron den-
sity oxygen atom of carbonyl group interacts with low electron
density region of the target (Figure 3).
However, compounds 4c, 4h, 4l and 5d (20.58
3.31%,
21.74 2.87%, 5.93 0.04% and 37.29 2.85%, respec-
tively) did not show considerable FAK inhibition in C6 can-
cer cells. These results pointed out the importance of the
phenyl, propyl, tert-butyl, methyl moieties at the eighth and
second position of the spirothiazolidinone ring for FAK in-
hibitory activity.
When all results are evaluated, spirothiazolidinone deriv-
atives seem to have higher anticancer activity than hydrazone
derivatives, and interestingly, the hydrazone derivatives carry-
ing tetramethyl and phenyl substitutions show great anticancer
activity. Propyl substitution at 8th position on spirothiazolid-
inone ring increases activity. Additionally, small alkyl groups
such as methyl boost the activity. 4-Chlorophenyl and phe-
nyl derivatives show slightly different anticancer activity.
Electronegativity might affect anticancer activity, especially
for hydrazone derivatives.
In our recent studies, we have used a number of computa-
tional approaches against some important therapeutic targets
(Chan et al., 2020; Ece, 2020; Ece & Pejin, 2015; Mustafa
Er et al., 2018; Maryam et al., 2020; Maryam et al., 2020;
Yamali et al., 2021). Hence, molecular docking simula-
tions were implemented to see probable binding modes of
the synthesized compounds. A high resolution (2.3 Å) X-
ray crystal structure of focal adhesion kinase domain (PDB
ID: 2ETM) in complex with 7H-Pyrrolo[2,3-d]pyrimidine
derivative (7PY, native ligand) was used in calculations.
As an internal validation, co-crystallized native ligand was
docked in the binding site and then both conformations were
The binding free energies of macromolecules were cal-
culated by MM/GBSA (Molecular Mechanics/Generalized
Born Surface Area) methods using combining continuum
solvation models and molecular mechanics calculations to

10
BAŞOĞLU et AL.
|
perform more rigorous binding affinity calculations (Hou
et al., 2011). Hou et al. (2011) assessed the performance of
MM/GBSA on the binding free energies. The authors re-
ported MM/GBSA to serve as a powerful tool in ranking of
inhibitors (Hou et al., 2011).
The combination of computational study and experi-
mental findings on SAR works disclosed that the presence
of spiro[4.5]decane scaffold with -CH3 at 2nd position and
lipophilic groups at 8th position on spiro[4.5]decane ring has
significant effects on the anticancer activity.
The calculated molecular descriptors related to drug like-
ness and absorption, distribution, metabolism and excretion
(ADME) were all in acceptable ranges (Table 4).
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CONCLUSION
|
The fact that focal adhesion kinase signalling pathways
can accelerate tumour progression and metastasis forma-
tion that makes it a promising therapeutic target prompted
us to study novel small molecule inhibitors of this target.
In the present study, novel imidazo[2,1-b]thiazole deriva-
tives were obtained using simple and practical methods.
Their structure was characterized using various spec-
troscopic methods such as FT-IR, H-NMR, 13C-NMR,
1
HSQC, elemental analysis and mass spectroscopy. The in
vitro anticancer activities were also investigated against
focal adhesion kinase. Most of the compounds, especially
the derivatives which carry 3-oxo-1-tiya-4-azaspiro[4.5]
decane moiety, showed considerable anticancer activity.
Furthermore, compounds 6b, 6d, 6f carrying methyl, pro-
pyl, phenyl moieties at the eighth and second position of
the spirothiazolidinone ring, showed high FAK inhibitory
activities. Additionally, according to the results, com-
pound 4k can also be considered as a promising antican-
cer lead compound due to its high SI value and apoptotic
effect. However, it does not show anticancer activity by
inhibiting FAK. Molecular docking studies were imple-
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probable binding modes.
The biological assay data, along with the computational
results that shed light on the binding motifs and critical inter-
actions, might provide insight into designing new inhibitors
against focal adhesion kinase.
ACKNOWLEDGEMENT
The present research was financially supported by the
Research Fund of Istanbul University. Project No. 24304 and
TDK-2016-20280.
ORCID
Faika Başoğlu
Abdulilah Ece
https://orcid.org/0000-0002-4890-3124
https://orcid.org/0000-0002-3087-5145

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How to cite this article: Başoğlu, F., Ulusoy-
Güzeldemirci, N., Akalın-Çiftçi, G., Çetinkaya, S., &
Ece, A. (2021). Novel imidazo[2,1-b]thiazole-based
anticancer agents as potential focal adhesion kinase
inhibitors: Synthesis, in silico and in vitro evaluation.
Chemical Biology & Drug Design, 00, 1–13. https://
doi.org/10.1111/cbdd.13896
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