Synthesis, Characterization, Molecular Docking, and Anticancer Evaluation of 4-Thiazolidinone Analogues

Article abstract of DOI:10.1002/jhet.3549

Scaffolds containing one or more heteroatoms are ubiquitous in nature and are responsible for almost every biological processes in flora and fauna. The fact that heterocyclic cores have high propensity for biological targets, several nitrogen, oxygen, and

Full text of DOI:10.1002/jhet.3549

Month 2019
Synthesis, Characterization, Molecular Docking, and Anticancer
Evaluation of 4-Thiazolidinone Analogues
a
Md Mushtaque,
Fernando Avecilla,b Zubair Bin Hafeez,c and Mohammad Moshahid A. Rizvic
*
aDepartment of Chemistry, School of Physical and Molecular Sciences, Faridabad, Haryana 121004, India
bGrupoXenomar, Centro de Investigacións Científicas Avanzadas (CICA), Departamento de Química, Facultade de
Ciencias, Universidade da Coruña, Campus de A Coruña, Coruña 15071 A, Spain
cDepartment of Biosciences, Jamia Millia Islamia, New Delhi 110025, India
*E-mail: mush.chem@gmail.com
Received November 17, 2018
DOI 10.1002/jhet.3549
Published online 00 Month 2019 in Wiley Online Library (wileyonlinelibrary.com).
Scaffolds containing one or more heteroatoms are ubiquitous in nature and are responsible for almost ev-
ery biological processes in flora and fauna. The fact that heterocyclic cores have high propensity for biolog-
ical targets, several nitrogen, oxygen, and sulfur-containing heterocyclic compounds have been reported in
the last few decades. One such intriguing class is 4-thiazolidinone that exhibit diverse range of pharmacolog-
ical activities. In the present study, we report synthesis, characterization, molecular docking, and anticancer
evaluation of 10 new 4-thiazolidinone analogues (5–14). One of the compounds (11) was structurally char-
acterized using single crystal X structure. Anticancer evaluation against hepatocellular carcinoma cell line
(HepG2) revealed that compound (7) was the most potent (IC50 = 75 μM) while other showed moderate
to low activity (85–530 μM). To underpin the druggability and possible modes of action, drug-likeness
was determined and docking studies were carried out against β-carbonic anhydrase receptor (PDB ID:
3IA). Attempts have also been made to establish a structure–activity relationship of the reported compounds.
J. Heterocyclic Chem., 00, 00 (2019).
INTRODUCTION
available to the clinicians [4]. However, limitations such
as high cost, varying activity with time (resistance issue),
lack of selectivity, and secondary toxicity pose a big
challenge to the clinicians as well as researchers.
Cancer, a group of diseases, is one of the leading causes
of deaths around the globe [1]. Not only it is devastating to
the individual (patient) and families but it also causes
terrible loss to the nation economy [2]. Although
several modalities and state-of-the art technologies are
available to treat different cancers [3], complete removal
is still a daunting task, especially in the late stage.
Interdisciplinary researchers are working continuously to
develop new drugs, formulations, methods, and
techniques to get rid of deadly disease. One such effort is
the development of new chemotherapeutic agent with
improved activity and easy accessibility. Indeed, several
synthetic and natural products-based drugs are already
In the pursuit of new drugs that could surpass these
issues (at least some if not all), several heterocyclic
scaffolds have been developed. Last few decades
witnessed an exponential rise in the development of
bioactive three-membered to six-membered heterocyclic
cores. Scaffolds embedding one or more heteroatoms
such as nitrogen (N), oxygen (O), or sulfur (S) are
integral part of drug discovery program [5,6]. It is these
electronegative heteroatoms that endow activity as well
as balanced lipophilicity and hydrophilicity to a drug [7].
Besides, the presence of heteroatoms assists drugs to
© 2019 Wiley Periodicals, Inc.

M. Mushtaque, F. Avecilla, Z. B. Hafeez, and M. M. A. Rizvi
Vol 000
Scheme 1. Toluene, room temperature (b) ClCH2COOH, anhydrous
CH3COONa, ethanol reflux (c) different aldehydes, piperidine, and
ethanol reflux.
interact with the target/receptor to furnish its activity [8].
Among plethora of classes and subclasses of
heterocyclic cores, 4-thiazolidinone has attracted
a
a
significant interest of the researchers [9]. The presence of
three heteroatoms in one core, easy and high yielding
synthesis, low to moderate molecular weight (MW),
broad range of activities through unique mechanism, and
so forth are some of the unique features offered by these
five-membered cores [10–18]. In the past, we have
demonstrated that 4-thiazolidinones can effectively check
the amoebic growth [15] and show moderate anticancer
activity in vitro [17,19]. Several other leading researchers
also demonstrated that 4-thiazolidinone is a privileged
scaffold in modern medicinal chemistry. For example, it
was found that compounds a and b (Chart 1) exhibit
potent anti-proliferative activity (IC50 in micromolar
range) [18]. It was argued that these compounds show
activity via targeting carbonic anhydrase (CAIX).
1
(5–14) were characterized by FTIR, H NMR, 13C NMR,
and mass spectral studies. The stereochemistry of
compounds was confirmed by 2D-NMR 1H-1H NMR
(COSY and NOESY) and X-ray single structure.
The appearance and disappearance of significant bands
confirmed the formation of compounds. In IR study, the
characteristic IR spectra showed significant bands for the
formation of 1-(2-chlorobenzyl)-3-phenylisothiourea (3).
The presence of the two significant peaks at 3244.71 and
1225.68 cmꢀ1 because of N─H and C═S groups,
respectively, confirmed the formation of compound (3).
In the view of versatile biological importance of 4-
thiazolidinone derivatives and to evaluate their anticancer
effect, we report the synthesis, characterization, molecular
docking, and anticancer evaluation of 4-thiazolidinone
analogues (5–14). The results of the findings are
discussed herein.
RESULTS AND DISCUSSION
1
Besides, in H NMR, the presence of chemical shifts at
8.00 and 6.545 ppm [because of (─N─H) and
(S═C─NH) protons] further confirmed the formation of
compound (3). Bands at 1724.29 (O═C─N─), 1638
(C═N─), 1343.55 (tert. N), and 750–741 (C─S─C)
cmꢀ1 in IR spectra indicated successful formation of the
cyclized product (4). In 1H NMR, chemical shift at
3.90 ppm (because of S─CH2─ protons) as singlet
further indicated the formation of (4). Finally, 4-
thiazolidonone derivatives (5–14) showed alkenic (C═C)
bond stretching frequency in the region of 1550–
1600 cmꢀ1 while alkenic (H─C═C) proton appeared as
singlet between 7.6 and 8.3 ppm that confirmed the
formation of compounds. Additionally, formation of
compounds (5–14) was confirmed by 13C NMR because
of the presence of the characteristic carbonyl (─C═O)
peak in the range of 164–168 ppm. Moreover, mass
spectral studies (data in experimental section) also
support the formation of compounds.
Synthesis. Present study was undertaken to synthesize
the analogues of 4-thiazolidinone derivatives. Target
compounds (5–14) were obtained in a three step reaction
procedure (Scheme 1). First step involves the synthesis of
1-(2-chlorobenzyl)-3-phenylthiourea (3) by the reaction
of 2-chlorobenzylamine (1) and phenyl isothiocyanate (2)
in toluene at room temperature. In the second step,
anhydrous sodium acetate assisted cyclization of (3) in
absolute ethanol and chloroacetic acid yielded 3-(2-
chlorobenzyl)-2-(phenylimino)-1,3-thiazolidin-4-one (4).
The lead compounds (5–14) were obtained in the third
step by Knoevenagel condensation of 3-(2-chlorobenzyl)-
2-(phenylimino)-1,3-thiazolidin-4-one (4) with different
aromatic aldehydes in absolute ethanol. All compounds
Stereochemistry and conformational properties.
Final
compounds (5–14) possess exocyclic alkenic bond and
exocyclic nitrogen atom having aromatic ring that lead to
arise the question related to the stereochemistry of
the molecules, that is, whether the molecules occupy Z-
configuration or E-configuration. Therefore, in order to
confirm the stereochemistry of the molecules, 1H-1H
1
COSY and H-1H NOESY (2D-NMR) have been carried
Chart 1. Carbonic anhydrase inhibitors (CAIX).
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Synthesis, Characterization, Molecular Docking and Anticancer Evaluation of
4-Thiazolidinone Analogues
out. In H-1H COSY-NMR, compound (11) showed the
presence of strong coupling of off-diagonal peaks. The
singlet of CH2 at 5.32(ppm) coupled with singlet of
OCH3 at 3.977 (ppm) attached to aromatic ring. The
singlet of OCH3 at 3.977(ppm) attached to the aromatic
ring coupled with the aromatic protons substituted at
exocyclic nitrogen (C═N) at 7.39 (ppm). The singlet of
CH2 at 5.32 (ppm) coupled with 3-N substituted benzylic
aromatic protons at 6.68 (ppm). Besides, 5-arylidene
substituted protons at 7.75–7.41 (ppm) coupled with
exocyclic C═N-substituted aromatic protons at 7.39–7.32
(ppm) chemical shift, which confirmed that 2-exocyclic
nitrogen substituted and 5-arylidene-substituted aromatic
protons occupied Z-configuration. The sharp singlet at
downfield chemical shift at 8.20 (ppm) showed that 5-
arylidene aromatic protons occupied Z-configuration.
In 2D-NOESY 1H-1H NMR, strong interaction was
observed. The singlet of CH2 at 5.32(ppm) interacted
with the singlet of OCH3 at 3.977(ppm) attached to the
aromatic ring. The singlet of CH2 at 5.32 (ppm)
coupled with 3-N substituted benzylic aromatic protons
at 6.68 (ppm). The singlet of OCH3 at 3.977(ppm)
interacted with exocyclic C═N-substituted aromatic
protons at 7.32 (ppm), which confirmed that exocyclic
C═N-substituted aromatic ring occupied Z-configuration.
Moreover, 5-arylidene substituted protons at 7.75–7.41
(ppm) interacted with exocyclic C═N-substituted
aromatic protons at 7.39–7.32 (ppm), which confirmed
that the 2-exocyclic nitrogen-substituted aromatic
ring and 5-arylidene-substituted aromatic ring occupied
Z-configuration. Furthermore, the sharp singlet at 8.20
(ppm) because of methylene proton clearly showed that
5-substituted arylidene ring occupied Z-configuration.
Moreover, the stereochemistry was determined by X-ray
single crystal structure as shown in Figure 1. 1H-1H
COSY-NMR of compound (13) showed that the singlet
of CH2 at 5.38 (ppm) coupled with benzylic aromatic
protons at 7.29 (ppm). 5-Arylidene substituted protons
at 7.98–7.51 (ppm) coupled with exocyclic C═N-
substituted aromatic protons at 7.39–7.32 (ppm), which
displayed that 2-exocyclic nitrogen-substituted aromatic
ring and 5-arylidene-substituted aromatic ring occupied
Z-configuration. Furthermore, the appearance of
singlet at 8.40 (ppm) because of methylene proton
showed that 5-arylidene-substituted aromatic ring
occupied Z-configuration. In 2D-NOESY 1H-1H NMR,
it was observed that singlet of CH2 at 5.38 (ppm)
interacted with 6.78 (ppm) of the aromatic ring. 5-
Arylidene-substituted aromatic protons at 7.98–7.51
(ppm) interacted with exocyclic C═N-substituted
aromatic protons at 7.39–7.32 (ppm), which confirmed
that 2-exocyclic nitrogen-substituted aromatic ring and
5-arylidene-substituted aromatic ring occupied Z-
configuration.
1
Result and discussion of X-ray crystal structure.
ORTEP diagram of compound (11) is shown in Figure 1,
and selected bond distances and angles have been given in
Table S1. C─H/π interactions are present in the structure
in the crystal packing (Figs 2 and 3). Compound (11)
presents weak C─H/π interactions between centroids
and carbon atoms, dc1-C(9)
dc2-C(18) = 3.860(2)Å, [c1, C(18L)-C(19L)-C(20L)-
C(21L)-C(22L)-C(23L), c2, C(9L)-C(10L)-C(11L)-
=
3.588(2)Ǻ and
C(12L)-C(13L)-C(14L)] (Fig. 3). Atropoisomers by
rotation around C(6)-C(7) and N(2)-C(14) bonds are
present in the structure [18]. Figure 4 shows two
enantiomers observed in the crystal structure. Hydrogen
bonds are not present in the structures. Thiazolidinone
Figure 1. ORTEP plot for the compound (11). All the non-hydrogen atoms are presented by their 50% probability ellipsoids. Hydrogen atoms are omitted
for clarity. [Color figure can be viewed at wileyonlinelibrary.com]
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M. Mushtaque, F. Avecilla, Z. B. Hafeez, and M. M. A. Rizvi
Vol 000
Figure 2. Crystal packing of the compound (11). View along edge that contains inversion center. Enantiomers are grouped in layers by van der Waals
forces and C─H–π interactions. All the non-hydrogen atoms are presented by their 50% probability ellipsoids. Hydrogen atoms are omitted for clarity.
[Color figure can be viewed at wileyonlinelibrary.com]
Figure 3. Partial view of the crystal packing in the compound (11). C─H–π interactions are showed by dashed black lines. [Color figure can be viewed at
wileyonlinelibrary.com]
ring is plane [mean deviation from planarity for C(8)-
C(15)-C(16)-N(1)-S(1), 0.0073 Ǻ] and the angle with
dimethoxybenzyl ring is 23.79(3)°.
and play leading role in the understanding of drug–drug
and drug–receptor interaction at supramolecular level
[20]. Low cost, freely available tools and software’s,
rapid and meaningful results, and so forth are some of the
important features that have attracted the researchers [21].
Both organic and organometallic pharmacophores are
being designed and studied using docking, simulation,
and other programs to delineate the mechanism of action
of new compounds [22–24].
COMPUTATIONAL STUDIES
Molecular docking study. In the modern drug discovery
program, computational tools emerged as an important tool
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Synthesis, Characterization, Molecular Docking and Anticancer Evaluation of
4-Thiazolidinone Analogues
Figure 4. Partial view of the crystal packing in the compound (11). Enantiomers are present in the crystal packing. They are generated by rotation around
C(6)-C(7) and N(2)-C(14) bonds. [Color figure can be viewed at wileyonlinelibrary.com]
Table 1
As stated earlier, among a number of targets, 4-
Binding energy of molecular docking of compounds (5–14) with protein
CAIX (PDBID = 3IAI) and residues of protein involved in binding.
thiazolidinones also have ability to inhibit the activity of
CAIX [18]. To check the affinity of developed
compounds against this protein, we carried out docking
studies against CAIX protein (PDB ID: 3IAI). The active
binding pocket of CAIX has been exploited to interpret
the interactions [25]. The results of the study (binding
energy and the residues involved in the interaction) are
depicted in Figure 5, Figure S1, and Table 1. Among all
the studied compound, two compounds (7 and 13)
showed highest binding energy (ꢀ8.8 and ꢀ9.0 kcal/mol,
respectively). Both compounds interacted within the
active site (binding pocket) of the target via polar and
nonpolar interactions with moderate strength, indicating
that the developed compounds can also be used as CA-
inhibitors. Nevertheless, the mode and strength of
interaction can further be optimized/modified through
structural variation.
Binding energy
kcal/mol
Residue involved
in binding with protein
S.N Compounds
1
2
5
6
ꢀ8.2
ꢀ8.3
THR0200
Hydrophobic
interactions
(see Supporting
Information)
(i) HIS094
3
7
ꢀ8.8
(ii) HIS0119
(iii) THR0199
THR 200
4
5
6
8
9
10
ꢀ8.0
ꢀ8.4
ꢀ7.9
GLY 8
(i) ARG0102 (ii)
GLY08
7
8
11
12
ꢀ8.4
ꢀ8.0
(i) TRP05 (ii) THR0200
Hydrophobic
interactions
Hydrophobic
interactions
GLY08
9
13
14
ꢀ9.0
ꢀ8.0
Correlation between in vitro activity and drug-likeliness.
In order to predict the druggability and to establish the
structure–activity relationship of the synthesized
10
Figure 5. 3D-molecular interactions of compound (7) with carbonic anhydrase CAIX (PDBID: 3IAI). [Color figure can be viewed at wileyonlinelibrary.
com]
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M. Mushtaque, F. Avecilla, Z. B. Hafeez, and M. M. A. Rizvi
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compounds (5–14), pharmacokinetic properties have been
determined. Lipinski’s rule of five (Ro5) is a general set
of rules followed by most of the orally administered
drugs [26]. According to this rule, a molecule can act as
drug if it has (i) MW < 500 D, (ii) <5 hydrogen bond
donors (HBD), (iii) <10 hydrogen bond acceptor (HBA),
and (iv) octanol–water partition coefficient (logP) value
<5. Polar surface area (PSA) and number of rotatable
bonds (RBs) are also useful descriptors to optimize the
bioavailability [27]. According to Veber’s rule, for good
oral bioavailability, the number of RB must be ≤10 and
total PSA values should be ≤140 Å2 [28]. It has been
reported that a molecule violating more than one of the
rules is less likely to be a drug (because of poor
absorption, bioavailability, or solubility) [29]. In the
present study, we have determined the above-said
properties for only those compounds that showed
moderate activity, that is, 5, 6, 7, and 10 (Table S2). It
was noted that compounds 5 and 7 that exhibited highest
activity among the studied compounds (85 and 75 μM,
respectively) also followed the rule very well
(logP = 4.33; Mol. wt. = 464; HBA = 4; and HBD = 0
Figure 6. Representation of drug property with the help of simple colors, bars, and pie diagram. Light colors show more drug-likeliness. [Color figure can
be viewed at wileyonlinelibrary.com]
Figure 7. Percentage cells viability against HepG2 cells at the concentration range 0–640 μM of the compounds (5–14). [Color figure can be viewed at
wileyonlinelibrary.com]
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Synthesis, Characterization, Molecular Docking and Anticancer Evaluation of
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Table 3
for compound 5 and logP = 3.8; Mol. wt. = 449; HBA = 4;
and HBD = 0 for compound 7). Figure 6 depicts the PSA,
MW, logP, and number of RBs present in compounds (5–
14). It can be noted that compounds 5, 6, 7, and 10 possess
PSA (76.4, 58, 103.8, and 78.2 Å, respectively) and RBs
(6, 4, 5, and 4) within the limits. Hence, on the basis of
computational and experimental studies, it can be
concluded that compound (7) has the best activity as well
as drug-likeness.
Crystal data and structure refinement for the compound (11).
Compound
Formula
Formula weight
T, K
Wavelength, Å
Crystal system
Space group
a/Å
b/Å
c/Å
β/°
V/Å3
11
C25 H21 Cl N2 O3 S
464.95
100(2)
0.71073
Monoclinic
P21/n
12.9850(6)
7.6084(4)
22.7244(11)
100.759(2)
2205.59(19)
4
Table 2
The cytotoxicity of the compounds (5–14) performed against HepG2 cell
Z
lines and their IC50 values.
F000
968
1.400
0.299
Dcalc/g cmꢀ3
Cytotoxicity
(IC50 values in μM)
μ/mmꢀ1
S.N
R
θ/ (°)
Rint
1.98 to 27.88
0.0293
0.50 × 0.41 × 0.21
1.042
0.0281
0.0782
5
85
Crystal size/mm3
Goodness-of-fit on F2
R1[I > 2σ(I)] a
wR2 (all data) b
6
125
75
Largest differences peak and hole (eÅ-3) 0.356 and ꢀ0.214
aR1 = Σ∣|Fo|-|Fc|∣/Σ∣Fo∣. bwR2 = {Σ[w(∣|Fo|2-|Fc|2∣)2]∣/Σ[w
(Fo2)2]}1/2.
7
8
CYTOTOXICITY STUDY
(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl
bromide (MTT) is tetrazolium salt that is commonly used
to study the viability (toxicity) of
Mechanistically, MTT is reduced by succinate
dehydrogenase enzyme of living cells to produce water
insoluble purple formazan crystals [30,31]. The amount
of formazan produced is directly proportional to the
number of living cells in the culture [32,33].
tetrazolium
426
160
a compound.
9
10
11
130
460
In order to underpin the cytotoxicity study of
compounds (5–14), they have been screened against
HepG2 cell lines for 48 h at the concentration range 0–
640 μM (Fig. 7a and b). The cytotoxicity study revealed
that compound (7) displayed the best activity
(IC50
=
75 μM) followed by compound
5
(IC50 = 85 μM). Compounds 6, 9, 10, and 13 displayed
moderate activity and their IC50 values are in the range of
125–160 μM (Table 2). However, compounds 8, 11, 12,
and 14 were found to be nontoxic (Table 2). Hence, it
can be concluded that compound (7) is potent among the
series.
12
13
530
155
CONCLUSION
14
We herein conclude from our experimental results that
the synthesized compounds adopted (2Z, 5Z)-
configuration. The in vitro MTT-assay study revealed
that out of screened compounds (5–14) against HeG2 cell
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lines, compound (7) is the most cytotoxic (IC50 = 75 μM)
followed by compound (5) (IC50 = 85 μM). The binding
energy of the compounds (5 and 7) was observed ꢀ8.2
and ꢀ8.8 kcal/mol. The structure–activity relationship
study also revealed that compound (7) showed more
drug-likeliness. Hence, it can be concluded that
compound (7) is most potent among the series.
was taken and dissolved in absolute ethanol. After this,
anhydrous sodium acetate (7.14 g, 86.94 mmol) and
chloroacetic acid (5.07 g, 53.94 mmol) were added in
sequence. The suspension was refluxed for 12 h. The
progress of the reaction was monitored by TLC in 5%
methanol: chloroform system. After the completion of the
reaction, solvent was evaporated in rotatory vacuum
evaporator. Subsequent to this, water was added and the
aqueous layer was back-extracted with ethyl acetate. The
organic layer was washed with saturated sodium
bicarbonate and brine. The combined organic extracts
were dried over Na2SO4 filtered through cotton and
concentrated in rotatory vacuum evaporator; after the
concentration, the yellowish-white solid product was
obtained.
EXPERIMENTAL PROTOCOL
All the required chemicals were purchased from Merck
and Aldrich Chemical Company (USA). Precoated
aluminum sheets (Silica gel 60 F254, Merck Germany)
were used for thin-layer chromatography (TLC), and spots
were visualized under UV light. FTIR spectra were re-
corded on Perkin Elmer model 1600 FTIR RX1 spectro-
photometer as KBr discs. Elemental analysis was carried
out on CHNS Elemental analyzer (Vario EL-III), and the
3-(2-Chlorobenzyl)-2-(phenylimino)thiazolidin-4-one (4).
Yield 80%; mp 131°C; Anal. Calcd: C16H13ClN2OS: C
60.66, H 4.14, N 8.84, S 10.12%; found: C 60.62, H
4.12, N 8.81, S 10.10%: IR (νmax) (cmꢀ1): 1705.21
(C═O), 1625.74 (C═N), 761.63 (C─S─C), 1371.79 (Ter.
1
results were within 0.3% of the theoretical values. H
1
NMR and 13C NMR spectra were recorded on Bruker
Spectrospin DPX 300 MHz spectrometer respectively
using CDCl3 as a solvent and trimethylsilane as an internal
standard. Splitting patterns are designated as follows: s,
singlet; d, doublet; and m, multiplet. Chemical shift values
are given in ppm. X-ray data were collected on Bruker
SMART Apex CCD diffractometer (SAI, Universidade
da Coruña).
amine): H NMR (CDCl3) δ (ppm); 7.389–7.316 (m, 3H,
Ar─H), 7.246–7.201 (m, 2H, Ar─H), 7.181–7.092 (m,
1H, Ar─H), 6.914 (d, 2H, J = 7.2 Hz, Ar─H), 5.162 (s,
2H, CH2), 3.900 (s, 2H, CH2).
General procedure of synthesis of thiazolidinone
derivatives. To a round bottom flask of 50 mL, (1 mmol)
of 3-(2-chlorobenzyl)-2-(phenylimino) thiazolidin-4-one
(4) was taken in absolute ethanol (35 mL) and dissolved.
After this, (1 mmol) aldehyde was added followed by
addition of (1.15 mmol) hexahydropiperidine to the
reaction mixture. The reaction mixture was refluxed for
11–12 h. The progress of the reaction was monitored by
TLC in 10% (ethyl acetate: hexane). After the completion
of the reaction, the reaction mixture was cooled at room
temperature and the solid precipitate was collected by
filtration and washed with ethanol. The solid yellow
General procedure for the synthesis of 1-(2-chlorobenzyl)-
3-phenylyhiourea.
To a round bottom flask, 2-
chlorobenzylamine (14 g, 98.87 mmol) was dissolved in
toluene at room temperature. Subsequent to this,
phenylisothiocynate (13.36 g, 98.87 mmol) was added
slowly and stirred at room temperature. After 5 min, solid
white precipitate appeared. Stirring was continued for
1 h. The TLC of the reaction mixture was monitored in
5% methanol: chloroform system. After the formation of
compound, the precipitate was collected by filtration and
washed with toluene. The product obtained was dried to
afford final product as white powder (95.0%) (1).
product obtained was recrystallized in chloroform.
(2Z,5Z)-3-(2-chlorobenzyl)-5-(3,4-dimethoxybenzylidene)-2-
(phenylimino)thiazolidin-4-one (5). Yield 80%; mp 210°C;
Anal. Calcd: C25H21ClN2O3S: C 64.58, H 4.55, N 6.02, S
1-(2-Chlorobenzyl)-3-phenylyhiourea (3).
Yield 95%;
7.62%; found: C 64.50, H 4.50 N 6.01, S 7.60%; IR (νmax
)
(cmꢀ1): 2924.85 (─O─CH3), 2924.25 (Ar─H), 1146.52
(C─O─C), 1705.05 (C═O), 1638.63 (C═N), 1538.60
(C═C), 1375.56 (3°N), 738.82 (C─S─C); 1H NMR
(300 MHz, CDCl3) δ (ppm): 7.745 (s, 1H, H─C═C─),
7.407–7.327 (m, 3H, Ar─H), 7.257–7.160 (m, 4H,
Ar─H), 7.13–7.07 (m, 1H, Ar─H), 6.96–6.88 (m, 4H,
Ar─H), 5.307 (s, 2H, CH2), 3.903 (s, 6H, ─OCH3); 13C
NMR (DMSO-d6) δ (ppm): 165.73 (─C═O), 150.55
(─C═N), 148.88 (─CH═C─), 120.94 (C─S─C),
149.57, 147.42, 132.80, 131.60, 131.32, 129.42, 128.97,
127.47, 127.27, 125.93, 124.89, 122.49, 117.84, 114.31,
112.03 (aromatic), 55.56 (OCH3), 43.66 (CH2N). Calcd
mass = 464; observed mass: [M + 2H]+ = 466.7.
mp125°C; Anal. Calcd: C14H13ClN2S: C 60.75, H 4.37,
N 10.12, S 11.58%; found: C 60.72, H 4.38, N 10.10, S
11.54%; IR (νmax) (cmꢀ1): 3265.71 (NH), 3009.93
(C─H, Ar─H), 1245.23 (C═S); 1H NMR (CDCl3) δ
(ppm): 7.499–7.372 (m, 3H, Ar─H), 7.362–7.290 (m,
2H, Ar─H), 7.265–7.161 (m, 4H, Ar─H), 8.172 (bs, 1H,
NH), 6.550 (bs,1H, S═C─NH), 4.944 (d, 2H, J = 5.7 Hz,
CH2); 13C NMR (CDCl3) δ (ppm): 180.53 (C═S)
(135.77,124.67, 133.45, 130.59, 130.17, 129.45, 129.13,
127.31, 126.99, 125.12, aromatic), 46.95 (CN).
General procedure of synthesis of 3-(2-chlorobenzyl)-2-
(phenylimino)thiazolidin-4-one. In a round bottom flask,
1-(2-chlorobenzyl)-3-phenylthiourea (12 g, 43.47 mmol)
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

Month 2019
Synthesis, Characterization, Molecular Docking and Anticancer Evaluation of
4-Thiazolidinone Analogues
(2Z,5Z)-3-(2-chlorobenzyl)-5-(4-methylbenzylidene)-2-
(phenylimino)thiazolidin-4-one (6). Yield 79%; mp 231°C;
Anal. Calcd: C24H19ClN2OS: C 68.81, H 4.57, N 6.69, S
7.706 (s, 1H, H─C═C─), 7.392–7.341 (m, 3H, Ar─H),
7.257–7.177 (m, 4H, Ar─H), 7.019–6.944 (m, 4H,
Ar─H), 6.865 (d, 1H, J = 8.1 Hz, Ar─H), 6.012 (s, 2H,
piperonal, CH2), 5.302 (s, 2H, CH2); 13C NMR (DMSO-
d6) δ (ppm): 165.72 (─C═O), 149.41 (─C═N), 148.01
(─CH═C─), 120.91 (C─S─C), 147.39, 132.77, 131.61,
131.01, 129.47, 129.00, 127.47, 127.37, 125.25, 124.93,
118.32, 109.43, 109.12, (aromatic), 101.92 (CH2O2),
43.73 (CH2N).
7.65%; found: C 68.75, H 4.60, N 6.60, S 7.65%; IR
(νmax
)
(cmꢀ1): 2964.25 (Ar─H), 1705.92 (C═O),
1632.60 (C═N), 1367.52 (3°N), 747.48 (C─S─C); 1H
NMR (300 MHz, CDCl3) δ (ppm): 7.780 (s, 1H, Ar─H),
7.376–7.352 (m, 5H, Ar─H), 7.254–7.177 (m, 6H,
Ar─H), 6.970 (d, 2H, J = 8.1 Hz, Ar─H), 5.306 (s, 2H,
CH2), 2.367 (s, 3H, CH3); 13C NMR (DMSO-d6) δ
(ppm): 165.73 (─C═O), 149.42 (─C═N), 147.40
(─CH═C-), 120.90 (C─S─C), 140.40, 132.76, 131.64,
131.02, 130.43, 129.91, 129.49, 129.42, 129.02, 127.48,
127.43, 124.95, 119.59 (aromatic), 43.72 (CH2N), 21.07
(CH3).
(2Z,5Z)-3-(2-chlorobenzyl)-5-(4-hydroxybenzylidene)-2-
(phenylimino)thiazolidin-4-one (10).
Yield 80%; mp
273°C; Anal. Calcd: C23H17CN2O2S: C 65.63, H 4.07, N
6.67, S 7.62%; found: C 65.60 H 3.85 N 6.30 S 7.61; IR
(νmax
)
(cmꢀ1): 3392.95 (─OH), 2981.14 (Ar─H),
1701.41 (C═O), 1630.82 (C═N), 1577.21 (C═C),
1
1342.18 (3°N), 761.10 (C─S─C); H NMR (300 MHz,
3-(2-Chlorophenyl)-5-(4-nitrobenzylidene)-2-(phenylimino)
thiazolidin-4-one (7). Yield 81%; mp 229°C; Anal. Calcd:
CDCl3) δ (ppm): 7.748 (s, 1H, OH), 7.607(s, 1H,
H─C═C─), 7.467–7.346 (m, 5H, Ar─H), 7.228–7.188
(m, 4H, Ar─H), 6.971 (d, 2H, J = 7.2 Hz, Ar─H), 6.884
(m, 2H J = 8.7 Hz, Ar─H), 5.308 (s, 2H, CH2); 13C NMR
(DMSO-d6) δ (ppm): 165.92 (─C═O), 149.62 (─C═N),
147.62 (─CH═C─), 120.91 (C─S─C), 159.69, 132.85,
132.22, 131.60, 131.43, 129.47, 129.40, 128.95, 127.47,
127.28, 124.81, 124.09, 116.31, 116.20 (aromatic), 55.56,
43.65 (CH2N). Calcd mass: 420; observed mass:
C23H16ClN3O3S: C 61.87, H 4.33, N 9.02, S 7.13%; found:
C 61.78, H 4.30, N 9.01, S 7.02%; IR (νmax) (cmꢀ1):
2926.07 (Ar─H), 1711.15 (C═O), 1636.43 (C═N),
1586.45 (C═C), 1334.19 (Ar─NO2), 1374.94 (3°N),
1
750.34 (C─S─C); H NMR (300 MHz, CDCl3) δ (ppm):
8.277–8.248 (m, 2H, Ar─H), 7.809 (s, 1H, H─C═C─),
7.626–7.597 (d, 2H, Ar─H), 7.428–7.363 (m, 3H,
Ar─H), 7.265–7.188 (m, 5H, Ar─H), 6.967–6.939 (m,
2H, Ar─H), 5.334 (s, 2H, J = 10.2 Hz, CH2); 13C NMR
(DMSO-d6) δ (ppm): 165.53 (─C═O), 148.70 (─C═N),
147.21 (─CH═C─), 120.83 (C─S─C), 147.12, 139.50,
132.54, 131.78, 129.56, 129.43, 129.11, 128.18, 127.62,
127.49, 125.37, 125.17, 124.29 (aromatic), 44.06 (CH2N).
3-(2-Chlorobenzyl)-5-(4-(3-methyl-2-thiophen)benzylidene-
[M + H]+ = 421.3.
(2Z,5Z)-5-(2,5-dimethoxybenzylidene)-3-(2-chlorobenzyl)-2-
(phenylimino)thiazolidi-4-one (11). Yield 64%; mp 185°C;
Anal. Calcd: C25H21ClN2O3S: C 64.58, H 4.55, N 6.02, S
6.90%; found: C 64.50, H 4.86 N 6.10, S 6.81; IR (νmax
)
(cmꢀ1): 2984.14 (─O─CH3), 2920.34 (Ar─H), 1148.87
(C─O─C), 1709.03 (C═O), 1620.73 (C═N), 1581.11
(C═C), 1343.02 (3°N), 744.72 (C─S─C); 1H NMR
(300 MHz, CDCl3) δ (ppm): 8.149 (s, 1H, H─C═C─),
7.394–7.307 (m, 3H, Ar─H), 7.247–7.116 (m, 4H,
Ar─H), 6.947–6.891 (m, 3H, Ar─H), 6.871–6.810 (m,
2H, Ar─H), 5.301 (s, 2H, CH2), 3.807 (s, 3H, ─OCH3),
3.726 (s, 3H, ─OCH3); 13C NMR (DMSO-d6) δ (ppm):
165.58 (─C═O), 149.43 (─C═N), 147.30 (─CH═C─),
120.91 (C─S─C), 152.86, 152.16, 132.72, 131.63,
129.40, 128.99, 127.45, 125.35, 124.96, 122.31, 121.22,
116.20, 114.10, 112.72 (aromatic), 56.01 (OCH3), 55.38
(OCH3), 43.70 (CH2N). Calcd mass = 464; observed
mass: [M + 2H]+ = 466.7.
2-(phenylimino)thiazolidin-4-one
(8).
Yield
83%;
mp184°C; Anal. Calcd: C22H17ClN2OS2: C 62.18, H
4.03, N 6.59, S 15.09%; found: C 62.15, H 4.08, N 6.75,
S15.30;. IR (νmax) (cmꢀ1); 2927.20 (Ar─H), 1711.51
(C═O), 1627.64 (C═N), 1583.23 (C═C), 1347.03 (3°N),
1
731.49 (C─S─C); H NMR (300,MHz,CDCl3) δ (ppm):
8.031 (s, 1H, H─C═C─), 7.465 (d, 1H, J = 4.8 Hz,
Ar─H), 7.384–7.333 (m, 3H, Ar─H), 7.245–7.110 (m,
4H, Ar─H), 6.985–6.941 (m, 3H, Ar─H), 5.300 (s, 2H,
CH2), 2.395 (s, 3H, CH3); 13C NMR (DMSO-d6) δ
(ppm): 165.52 (─C═O), 149.10 (─C═N), 147.41
(─CH═C─), 120.89 (C─S─C), 143.21, 132.77, 131.68,
131.43, 131.36, 129.47, 129.40, 128.99, 127.43, 124.97,
122.15, 117.31 (aromatic), 43.81 (CH2N), 15.02 (CH3).
Calcd mass: 418; observed mass: [M + H]+ = 419.4;
(2Z,5Z)-3-(2-chlorobenzyl)-5-(4-chlorobenzylidene)-2-
(phenylimino)thiazolidin-4-one (12).
Yield 75%; mp
222°C; Anal. Calcd: C23H16Cl2N2OS: C 62.88, H 3.67,
N 6.38 S 6.90%; found: C 62.80, H 3.60, N 6.30, S
6.82; IR (νmax) (cmꢀ1): 2972.84 (Ar─H), 1705.21
(C═O), 1630.58 (C═N), 1583.93 (C═C) 1334.89 (3°N),
744.06 (C─S─C); 1H NMR (300 MHz, CDCl3) δ
(ppm): 7.739 (s, 1H, H─C═C─), 7.386 (d, 6H,
J = 12.3 Hz, Ar─H), 7.294–7.122 (m, 5H, Ar─H),
6.960–6.892 (m, 2H, Ar─H), 5.311 (s, 2H, CH2); 13C
NMR (DMSO-d6) δ (ppm):165.52 (─C═O), 149.10
[M + Na]+ = 441.4.
(2Z,5Z)-3-(2-chlorobenzyl)-5-(benzo[d][1,3]dioxol-5-
ylmethylene)-2(phenylimino)thiazolidin-4-one (9).
Yield
80%; mp 218°C; Anal. Calcd: C24H17ClN2O3S: C 64.21,
H 3.82, N 6.24, S 7.14%; found: C 64.18, H 3.97, N
6.37, S 7.30; IR (νmax) (cmꢀ1): 2947.10 (Ar─H) 1704.16
(C═O), 1631.42 (C═N), 1583.59 (C═C), 1346.14 (3°N),
1
747.90 (C─S─C); H NMR (300 MHz, CDCl3) δ (ppm):
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

M. Mushtaque, F. Avecilla, Z. B. Hafeez, and M. M. A. Rizvi
Vol 000
(─C═N), 147.41 (─CH═C─), 120.89 (C─S─C),
143.21, 132.77, 131.68, 131.43, 131.36, 129.47,
129.40, 128.99, 127.43, 124.97, 122.15, 117.31
(aromatic), 43.81 (CH2N), 15.02 (CH3). Calcd mass:
438; observed mass: [M + H]+ = 439.3.
C(24) and C(25), were included in calculation position and
refined in the riding mode. Refinements were done with
allowance for thermal anisotropy of all non-hydrogen
atoms. A final difference Fourier map showed no residual
density outside: 0.356 and ꢀ0.214 e.Åꢀ3 for 8. A
(2Z,5Z)-3-(2-chlorobenzyl)-5-(anthracen-10ylmethylene)-2-
weighting scheme
w
=
1/[σ2(F2o)
+
(0.041400
(phenylimno)thiazolidin-4-one (13).
Yield 21%, yellow
P)2 + 0.878500 P] for (11), where P = (|Fo|2 + 2|Fc|2)/3,
was used in the latter stages of refinement. Further
details of the crystal structure determination are given in
Table 3. CCDC1844607 contains the supplementary
crystallographic data for the structure reported in this
paper. These data can be obtained free of charge via http://
www.ccdc.cam.ac.uk/conts/retrieving.html, or from the
Cambridge Crystallographic Data Centre, 12 Union Road,
Cambridge CB2 1EZ, UK; fax: (+44) 1223 336 033; or e-
mail: deposit@ccdc.cam.ac.uk. Supplementary data
associated with this article can be found, in the online
version, at doi: $$$$$.
crystals; mp 121°C; Anal. Calcd: C31H21ClN2OS: C
73.73, H 4.19, N 5.55, S 6.35%; found: C 73.70, H
4.19, N 5.80, S 6.40; IR (νmax) (cmꢀ1): 2925.35
(Ar─H), 171.40 (C═O), 1628.76 (C═N), 1586.08
(C═C), 1372.79 (3°N), 722.38 (C─S─C); 1H NMR
(300 MHz) (CDCl3)
δ
(ppm): 7.802 (s, 1H,
H─C═C─), 7.474 (d, 2H, J = 6.67 Hz, Ar─H), 7.403–
7.337 (m, 5H, Ar─H), 7.239–7.147 (m, 5H, Ar─H),
6.965–6.888 (m, 2H, Ar─H), 5.311 (s, 2H, CH2); 13C
NMR (DMSO-d6) δ (ppm): 164.48 (─C═O), 149.34
(─C═N), 147.12 (─CH═C─), 120.67 (C─S─C),
132.82, 131.71, 130.69, 129.82, 129.40, 129.22,
128.82, 128.93, 128.84, 127.50, 126.96, 125.75,125.06,
124.76 (aromatic), 43.95 (CH2N). Calculated mass: 505;
Molecular docking study.
Docking studies were
performed at Intel(R) Core (TM) i3 CPU (2.3 GHz) with
XP-based operating system (Windows 2007). 3D
Structure of the lead compounds (5–14) was drawn by
Marvin Sketch and saved in PDB file format. The
preparation of the compounds were carried out by
assigning Gastegier charges, merging nonpolar hydrogens,
and saving it in PDBQT file format using AutoDock
Tools (ADT) 4.2 [36,37]. The X-ray crystal structure of
CAIX (PDB ID: 3IAI) was obtained from the Protein
Data Bank [Using ADT 4.2], CAIX was saved in PDB
file format leaving heteroatoms (water). The molecular
docking study was performed with Auto-Dock-vina 4.2
(Scripps Research Institute, USA), considering all the RBs
of ligand as rotatable and receptor as rigid [38]. Grid box
size of 40 × 40 × 40 Å with 0.375 Å spacing was used
that included whole active site of CAIX. All the
interactions of the compounds (5–14) with protein have
been visualized by PYMOL (PYMOL molecular graphics
system, version1.7.4.4, Schrödinger, LLC, and Portland,
OR, USA) and Maestro (Maestro, Schrödinger, LLC,
New York, NY, 2017).
observed mass: [M]+ = 505.4.
(2Z,5Z)-3-(2-chlorobenzyl)-5-benzylidene-2-(phenylimino)
thiazolidin-4-one (14).
Yield 79%; mp 141°C; Anal.
Calcd: C23H17ClN2OS: C 68.22, H 4.23, N 6.92, S
7.92%; found: C 68.20, H 4.45, N 7.12,S 8.02%; IR
(νmax
)
(cmꢀ1): 2925.35 (Ar─H), 1707.40 (C═O),
1630.06 (C═N), 1585.98 (C═C), 1340.58 (3°N), 745.05
1
(C─S─C); H NMR (300 MHz) (CDCl3) δ (ppm): 7.474
(d, 3H, J = 6.6 Hz, Ar─H), 7.403–7.337 (m, 4H, Ar─H),
7.239–7.147 (m, 4H, Ar─H), 6.965 (t, 2H, J = 7.2 Hz,
Ar─H), 7.802 (s, 1H, CH═C─), 5.311 (s, 2H, CH2); 13C
NMR (DMSO-d6) δ (ppm): 165.71 (─C═O), 148.97
(─C═N), 147.06 (─CH═C─), 120.51 (C─S─C),
132.87, 132.35, 132.05, 130.83, 129.50, 129.40, 129.03,
128.84, 128.57, 128.16, 126.64, 126.48, 124.47, 120.44
(aromatic), 43.51 (CH2N). Calcd mass: 404; observed
mass: [M + H]+ = 405.2.
X-ray crystal structure determination.
Three-
dimensional X-ray data were collected on a Bruker Kappa
Apex CCD diffractometer at low temperature by the ϕ-ω
scan method. Reflections were measured from
a
Cytotoxicity studies (MTT assay). Cell culture. Human
hepatocellular carcinoma cell line (HepG2) cell lines were
procured from National Curator of Cell Sciences (NCCS)
Pune, India. Cells were cultured in Dulbecco’s modified
Eagle’s medium with 10% fetal bovine serum with
100 units/mL penicillin, 100-μg/mL streptomycin, and
2.5-μg/mL amphotericin B, at 37°C in a relative humidity
80%, 5% CO2 [39]. The cell lines were harvested
when they reached 80% confluence to maintain exponen-
tial growth.
hemisphere of data collected from frames, each of them
covering 0.3° in ω. A total of 62,318 for (11), reflections
measured were corrected for Lorentz and polarization
effects and for absorption by multi-scan methods based on
symmetry-equivalent and repeated reflections. Of the total,
4772 independent reflections exceeded the significance
level (∣F∣/σ∣F∣) > 4.0. After data collection, a multi-
scan absorption correction (SADABS) [34] was applied,
and the structure was solved by direct methods and refined
by full matrix least squares on F2 data using SHELX suite
of programs [35]. Hydrogen atoms were located in
difference Fourier map and left to refine freely, except for
MTT assay.
Cytotoxicity of compounds (5–14) was
evaluated through MTT-(3-(4,5-dimethylthiazole-2-yl)-
2,5-diphenyl tetrazolium bromide, M2128 Sigma Aldrich)
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

Month 2019
Synthesis, Characterization, Molecular Docking and Anticancer Evaluation of
4-Thiazolidinone Analogues
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Acknowledgments. One of the authors, Dr Md Mushtaque, is
highly thankful to the University Grants Commission-Basic
Scientific Research for financial support during my PhD and
also thankful to Professor Amir Azam (Head of the Dept. of
Chemistry) for guidance. This part of the work is of my thesis,
submitted to (2013) the Department of Chemistry, Jamia Millia
Islamia University, New Delhi 110025, India.
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