Synthesis, docking, and biological evaluation of thiazolidinone derivatives against hepatitis C virus genotype 4a

Article abstract of DOI:10.1007/s00044-021-02721-w

Hepatitis C virus (HCV) genotype 4a (GT4a) is prevalent in Egypt. It did not gain the necessary scientific focus despite its high resistance. Since the crystal structure NS5B (RNA-dependent RNA polymerase) of HCV GT4a has not been resolved until now, homology modeling was conducted to build and validate the 3D model of the enzyme. Ligand binding sites including the allosteric thumb II pocket were detected and used in lead optimization. Sixty new 4-thiazolidinone derivatives have been virtually designed and docked into thumb II site of HCV NS5B GT4a using rigid docking approach. Eighteen compounds (7a–r) that show good docking scores were synthesized and tested in vitro against NS5B GT4a. Compounds 7b and 7n showed the best inhibitory activity (IC₅₀ = 0.338 and 0.342 μM, respectively). Compounds 7a, 7b, 7c, 7d, 7k, 7n, 7q, and 7r that have IC₅₀ values less than 2 μM were assessed for cellular anti-HCV GT4a activity using human hepatoma cell line (Huh 7.5). The percentages of viral growth inhibition are between 79.67 and 94.77%. Compound 7b is the most active in the in vitro and cellular assays and could be considered a potential new lead for future anti-HCV studies. [Figure not available: see fulltext.]

Full text of DOI:10.1007/s00044-021-02721-w

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research (2021) 30:1151–1165
https://doi.org/10.1007/s00044-021-02721-w
ORIGINAL RESEARCH
Synthesis, docking, and biological evaluation of thiazolidinone
derivatives against hepatitis C virus genotype 4a
1
Kamel M. Elberembally1 Mohammed A. Eldawy1
●
●
Ahmed S. Al-Behery
Received: 23 December 2020 / Accepted: 15 March 2021 / Published online: 26 March 2021
© The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature 2021
Abstract
Hepatitis C virus (HCV) genotype 4a (GT4a) is prevalent in Egypt. It did not gain the necessary scientific focus despite its
high resistance. Since the crystal structure NS5B (RNA-dependent RNA polymerase) of HCV GT4a has not been resolved
until now, homology modeling was conducted to build and validate the 3D model of the enzyme. Ligand binding sites
including the allosteric thumb II pocket were detected and used in lead optimization. Sixty new 4-thiazolidinone derivatives
have been virtually designed and docked into thumb II site of HCV NS5B GT4a using rigid docking approach. Eighteen
compounds (7a–r) that show good docking scores were synthesized and tested in vitro against NS5B GT4a. Compounds 7b
and 7n showed the best inhibitory activity (IC50 = 0.338 and 0.342 µM, respectively). Compounds 7a, 7b, 7c, 7d, 7k, 7n,
7q, and 7r that have IC50 values less than 2 µM were assessed for cellular anti-HCV GT4a activity using human hepatoma
cell line (Huh 7.5). The percentages of viral growth inhibition are between 79.67 and 94.77%. Compound 7b is the most
active in the in vitro and cellular assays and could be considered a potential new lead for future anti-HCV studies.
Graphical Abstract
●
●
●
Keywords HCV NS5B GT4a Thiazolidinones Homology modeling Molecular docking
Introduction
and hepatocellular carcinoma [1]. The high cost of treatment is
the major challenge for HCV-infected patients in developing
countries. In Egypt, where HCV genotype 4a (GT4a) infection
hits 30% of the population and represents 15% of the global
HCV infections, the treatment of HCV has become a national
health concern [2]. Boceprevir, Telaprevir, Simeprevir,
Ombitasvir, Sofosbuvir, and Dasabuvir are directly acting
antivirals (DAA), which have been used in the form of com-
bination therapies for HCV treatment [3–5]. HCV therapy has
been progressed rapidly with particular focus on GT1a and
GT1b, while the battle against GT4a requires more efforts
because of the high resistance, progressive mutations, and
pharmacoeconomic aspects [6, 7].
Hepatitis C virus (HCV) infection is considered as an immense
burden on global health because it is the leading cause of liver
cancer. Globally, an estimated 71 million people are infected
with chronic HCV and susceptible to develop liver cirrhosis
Supplementary information The online version contains
supplementary material available at https://doi.org/10.1007/s00044-
021-02721-w.
* Ahmed S. Al-Behery
ahmed.samir.chem@pharm.tanta.edu.eg
HCV NS5B is the responsible enzyme for genome
replication of the HCV [8]. DAA drugs can manipulate the
polymerase function of viruses either through the active
1
Faculty of Pharmacy, Tanta University, El Geish Street,
Tanta 31527, Egypt

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Medicinal Chemistry Research (2021) 30:1151–1165
(orthosteric) site by providing false nucleotide substrates
leading to RNA chain termination or through the allosteric
sites to inhibit RNA synthesis [8]. The NS5B structure
features a classical “right hand” architecture of the poly-
merase family, which is characterized by the GDD func-
tional motif (Gly317-Asp318-Asp319), which contains the
orthosteric site. The fingers, palm, and thumb domains hold
the allosteric sites (Fig. 1) [8, 9].
Non-nucleoside inhibitors are a class of small molecules
that target the NS5B allosteric sites that are thumb I, thumb
II, palm I, palm II, and palm III [8]. The binding of inhi-
bitors to these sites results in a conformational change of the
active site. Thumb II site is shallow and hydrophobic pocket
at the base of the thumb domain. It is 35 Å away from the
orthosteric site [10].
engage the thumb II site by hydrogen bonding with the
backbone NH of Ser476 and Tyr477 and the side chain of
Arg501. While right side substituents (blue color) provide
hydrogen bond donor and acceptor atoms, the left side
substituents (green color) offer hydrophobic contacts with
the lipophilic amino acids of the pocket [14, 15].
Filibuvir, with a dihydropyrone scaffold, is a potent and
selective inhibitor with IC50 of 0.019 μM against GT1b [16].
The triazolopyrimidine ring forms a π–π stacking interac-
tion with His475; this ring is linked to the dihydropyrone
core by a methylene group. Hydrophobic interaction
between the thumb II site and one of the ethyl groups of the
pyridine has provided good enzymatic potency [14]. Phase
II clinical candidate Radalbuvir (GT1b EC50 = 0.006 µM)
discovery led by the introduction of heterocyclic ethers on
the N-alkyl substituent hydroxyl groups. The heterocyclic
ether modulates protein binding and enhances the pharma-
cokinetic profile [15].
Thumb II inhibitors (Fig. 2) include dihydropyrone [11],
thiophene carboxylic acid [12], and 2-arylimino-4-
thiazolidinone [13] scaffolds (pink color). These scaffolds
Both compounds 1 (GT1b EC50 = 0.6 µM) [17] and 2
(GT1b IC50 = 11 µM) [17] have similar hydrophobic sub-
stituents (green color), while they differ in the right side
substituents. Compound 1 has extended pyridine-2-yl and
compound 2 contains 1,2,3-thiadiazole to provide hydrogen
bond acceptor atoms. Thiazolidinones were reported to have
versatile biological activities including antiproliferative,
antibacterial, antifungal [18–20]. Cyclooxygenase enzymes
can be targeted by these compounds, which make thiazo-
lidinones good scaffold to develop anti-tumor and anti-
inflammatory agents [21]. Furthermore, thiazolidinone
compounds have been shown to inhibit the HIV-1 reverse
transcriptase [22].
Recently, it has been reported that coupling of the 1,3,4-
thiadiazole ring with 4-thiazolidinone resulted in leads that
strongly inhibit HCV GT1b NS5B [23, 24]. These studies
have indicated that bioactivity was improved by increasing
the lipophilicity of the scaffold. Also studies indicated that
Fig. 1 HCV NS5B crystal structure (PDB: 3FRZ) showing palm (red),
thumb (blue), and finger (green) domain. The image generated by
PyMOL4.0.4 [43]
Fig. 2 HCV NS5B thumb II
inhibitors

Medicinal Chemistry Research (2021) 30:1151–1165
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condensation of arylidene moiety on 1,3-thiazolidine-4-one
ring could be essential for anti-HCV NS5B activity [23, 24].
However, this scaffold has not been tested yet for the
resistant GT4a. In this study, the HCV GT4a NS5B model
showed a polar region composed of Arg501, Arg505,
Arg498, Lys531, and Lys533 that can be targeted by
compounds with hydrogen bond acceptor functional groups.
Compounds 7b, c, f, g, j, l, m, n, q, and r were synthesized
and biologically evaluated to test this hypothesis. Further-
more, compound 7e with an extended alkyl side chain was
synthesized and explored for better fitting in the lipophilic
pocket. Compound 7a (Fig. 3) was reported to have good
activity on HCV NS5B GT1b (IC50 = 25.3 µM) [23] and it
is used in this study as a lead for the design of the new
compounds.
Accordingly, a set of compounds with diverse sub-
stitutions on ring A and B (Fig. 3) has been designed,
taking into consideration the features of the thumb II GT4a
binding site and previous reports. Ring A (green color)
should have halogens, while ring B (blue color) could be
derivatized with halogens, hydrogen bond donor/acceptor
functional groups, or extended alkyl side chains. A
homology model of HCV NS5B GT4a was constructed and
the compounds were docked into thumb II binding site in
order to validate the binding affinity of the designed set of
compounds. Based on the docking results, 18 compounds
were selected for synthesis and were further evaluated for
their activity.
A BLAST search of HCV NS5B GT4a against PDB
sequence entries shows a match with 77.54 sequence
identity. The SWISS-MODEL server was used to generate
homology model for HCV NS5B GT4a. Global Model
Quality Estimation (GMQE) and Qualitative Model Energy
Analysis (QMEAN) scoring functions were used as an
initial criterion to discriminate good from bad models. The
model shows –0.09 QMEAN score (Supplementary Fig.
S45a, Supplementary Material), which indicates good
agreement between the homology model and experimental
structures of the same size. The local quality estimate plot
(Supplementary Fig. S45b, Supplementary Material) shows
that there are no residues that gave a score below 0.6, which
indicates high model quality. In the comparison plot (Sup-
plementary Fig. S45c, Supplementary Material), the
homology model is shown as a red star, and it is aligned
with QMEAN score within one standard deviation of the
mean |Z-score| as the experimental structures represented in
black dots.
After building the homology model, it was subjected to
protein refinement using the ModRefiner server [26]. Vali-
dation was carried out to explore the stereochemical quality
of dihedral angles Ф against Ѱ of amino acid residues in the
homology model and identify sterically allowed regions for
these angles. Validation was carried out using SWISS-
MODEL structure assessment online server [27] (Fig. 4).
The Ramachandran plot of modeled HCV NS5B protein
(Fig. 4A) shows 0.75 MolProbity score, 98.04% Rama-
chandran favored, and 0.18% Ramachandran outliers. The
glycine and proline Ramachandran plot (Fig. 4B, C) show
no outliers in disallowed regions.
Results and discussion
Homology modeling
Molecular docking
The sequence of HCV NS5B GT4a was obtained from the
UniProt database [25]. SWISS-MODEL server has been
used to build the homology model (Supplementary Fig.
S46, Supplementary Material).
The docking procedure was validated by using crystal
structure of HCV NS5B GT1b in complex with Filibuvir
(PDB: 3FRZ) [14]. The redocked binding pose of Filibuvir
in GT1b observed overlays the original crystal structure
Fig. 3 The designed set of
compounds with diverse
substitutions on rings A and B

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Fig. 4 The Ramachandran plot
of modeled HCV NS5B protein
using SWISS-MODEL structure
assessment online server,
A General Ramachandran plot,
B Glycine Ramachandran plot,
C Proline Ramachandran plot.
Favored regions are shown in
green, additional allowed region
shown in light green, generously
allowed regions are shown in
pale green, and disallowed
regions shown in white
Fig. 5 A Docking pose of Filibuvir to HCV NS5B GT1b thumb II site overlaying on the original crystal structure binding pose. B Docking pose of
Filibuvir to HCV NS5B GT4a thumb II site and showing the new features of the allosteric pocket in GT4a
binding pose with the same binding interactions as reported
(Fig. 5A). The same established grid box coordinates in
HCV NS5B GT1b was taken as a model to expect the
binding site of HCV NS5B GT4a.
The docking score of Filibuvir was decreased from
–12.78 against HCV NS5B GT1b to –8.84 against HCV
NS5B GT4a. This decline indicates that the features of
the binding site in the HCV NS5B GT4a model changed
(Fig. 5B). Two lipophilic surfaces were detected in thumb II
GT4a, one of them was similar to GT1b. Hydrophilic
surface, also, was detected in thumb II GT4a that can be
targeted for hydrogen bonding and electrostatic interactions
by the studied compounds (Fig. 5B).
The alignment of the 3D structure of GT1b crystal
structure and the GT4a homology model was carried out to
analyze the differences in the amino acids and the spatial
arrangement of the critical residues of interaction (Fig. 6).
HCV NS5B GT1b thumb II site consists of Leu497,
Leu419, Ile482, Val376, His475, Trp528, Ser476, and
Arg501. These amino acids are the key amino acids for

Medicinal Chemistry Research (2021) 30:1151–1165
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Fig. 6 Alignment of the 3D
structure of HCV NS5B GT1b
crystal structure (green color)
(PDB: 3FRZ) and HCV NS5B
GT4a homology model (cyan
color). Ser476, Leu419, Ile482,
and Arg501 (red color) are of
HCV NS5B GT1b, Gly476,
Ile419, Leu482, and
Arg501amino acids (blue color)
are of HCV NS5B GT4a. The
image was generated by
PyMOL4.0.4 [43]
interaction with the reported active compounds of dihy-
dropyrone, thiophene carboxylic acid, and 2-arylimino-4-
thiazolidinone scaffolds [28]. However, in HCV NS5B
GT4a thumb II there are three amino acid mutations
reported, S476G, I482L, and L419I that render the binding
site more lipophilic and confer a high level of resistance
[6, 7]. These findings indicate that the binding site of HCV
NS5B GT4a lost the features of interactions that afforded in
HCV NS5B GT1b. Thus, the designed compounds should
have functional groups that afford more hydrogen bond
interactions with optimal lipophilicity. Also, compounds
with extended side chains will be investigated for a suitable
fitting of the molecule to the binding site.
Docking results showed that the thiadiazole ring with
substituted halogenated phenyl derivatives interacts with the
hydrophobic region of thumb II site, which indicates that
these compounds can be active against HCV NS5B GT4a.
The top 18 compounds were selected for synthesis and
biological evaluation (Table 1).
thiocyanate [23]. Compounds 5a–e react with ammonium
thiocyanate to form intermediates that undergo intramole-
cular cyclization to generate compounds 6a–e as indicated
in Scheme 3 [30].
The target compounds 7a–r were obtained by the reac-
tion of compounds 6a–e with different aldehydes in the
presence of sodium methoxide [23].
Compounds 3a–e, 4a–e, 5a–e, and 6a–e were previously
reported [23, 24], while compounds 7a–r are new and con-
firmed through elemental and spectroscopic analysis. Elemental
analysis (within the accepted limits ( 0.4 %)) confirms the
purity of the final compounds and the spectroscopic data of the
synthesized final compounds 7a–r agreed with the expected
chemical structures as given in the experimental section.
IR of compounds 7a–r shows one broad peak in the
range (3450–3300 cm−1) of the lactam NH and peak at
1720 cm−1 of the carbonyl functional group in the thiazo-
1
lidinone ring. H NMR shows the characteristic singlet at
(12.5–13.5 ppm) that is assigned to NH of 1,3-thiazolidin-4-
one ring and the peaks of the aromatic protons and exo-
cyclic =CH- proton were found in the range of (7–8 ppm).
Chemistry
1
The H NMR spectra of the compounds 6a–e were char-
Compounds 7a–r were synthesized according to Scheme 1.
Thiosemicarbazide 2 and different aldehydes 1a–e reacted
with each other to give the thiosemicarbazone derivatives
3a–e.
Compounds 3a–e undergo oxidative cyclization into 2-
Amino-1,3,4-thiadiazole derivatives 4a–e [23]. A suggested
mechanism for the oxidative cyclization is shown in
Scheme 2 [23, 29]. According to this mechanism, reaction
is initiated by deprotonation, forming a radical at thiose-
micarbazone N2. The resonance hybrid thiol radical attacks
azomethine carbon to form the ring. The expulsion of
another hydrogen radical finally yields 5-aryl-1,3,4-thia-
diazole-2-amines [23, 29].
acterized by the (singlet, 2H) peak of the active methylene
in 1,3-thiazolidin-4-one ring at 4.1 ppm [23]. The absence
of this peak in H NMR of compounds 7a–r confirms the
condensation of the different aldehydes and formation of the
target final compounds (7a–r). The exocyclic =CH- proton
in compounds 7a–r showed singlet peak with resonances at
7.70–8.10 ppm within the aromatic peaks range of 7–8 ppm.
5-Arylidene-1,3-thiazolidinones exist as Z configurational
isomer as evidenced by previous literature reports of the 1H
NMR [31] and X-ray crystallography [32].
1
The 13C NMR spectra of the reported compounds 6a–e
characterized by the thiazolidinone C5 signal at the range of
(30–38 ppm) [23], while the corresponding carbon signals
in the final compounds 7b–f, 7n, and 7q were shifted to the
range of (135–155 ppm).
Compounds 4a–e were converted to compounds 5a–e by
reaction with chloroacetyl chloride in the presence of tri-
methylamine. The synthesis of compounds 6a–e was carried
out by the reaction of compounds 5a–e with ammonium
1H-1H correlation spectroscopy (COSY) and heteronuclear
single quantum correlation (HSQC) experiments were carried

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Table 1 The docking scores and
IC50 results of the synthesized
compounds (7a–r)
a
Compound
X
R
Chemgauss4 scores (HCV IC50 SDb (µM)
NS5B GT4a)
(HCV NS5B GT4a)
7a
7b
7c
7d
2-Chloro- 3-Fluorophenyl
6-fluoro
–9.70
1.029 0.02
2-Chloro- 1,3-benzodioxol-5-yl
6-fluoro
–9.82
–9.9
0.338 0.01
0.634 0.01
1.882 0.04
2-Chloro- 4-Dimethylaminophenyl
6-fluoro
2-Chloro- 3-Chlorophenyl
–9.74
6-fluoro
7e
7f
4-Fluoro
4-Fluoro
4-Chloro
Phenylethyl
–10.28
–9.14
–9.92
5.006 0.11
4.128 0.09
11.760 0.26
3,4-Dimethoxyphenyl
7g
4-(benzyloxy)-3-
methoxyphenyl
7h
7i
3-Fluoro
3-Fluoro
3-Fluoro
3-Fluoro
3-Fluoro
2-Chloro
3-Bromophenyl
–9.97
–10.19
–10.11
–9.86
2.401 0.05
6.660 0.15
5.770 0.13
0.564 0.01
2.670 0.06
8.565 0.19
2-Bromophenyl
7j
4-Dimethylaminophenyl
3-Chlorophenyl
7k
7l
4-Methoxyphenyl
–9.8
7m
4-(benzyloxy)-3-
methoxyphenyl
–10.06
7n
7o
7p
7q
7r
2-Chloro
2-Chloro
2-Chloro
2-Chloro
2-Chloro
4-Dimethylaminophenyl
3-Bromophenyl
–10.23
–10.22
–10.2
0.342 0.01
2.193 0.05
2.954 0.06
0.642 0.01
0.936 0.02
3-Chlorophenyl
3-Nitrophenyl
–10.02
–10.09
4-Methoxyphenyl
aThe half-maximal inhibitory concentration
bThe standard deviation of triplicate experiments
out for compound 7b as a representative example for further
structural confirmation. The atom numbering of compound 7b
was generated by ChemBioDraw software [33] specifically for
confirms the assignment of the signal at 7.3 ppm to H22. Signal
at 115.07 ppm (C17) is correlated with 7.5 ppm (H17), signal at
126.21 ppm (C19) is correlated with 7.6 ppm (H19), and signal
at 135.86 ppm (C18) is correlated with 7.7 ppm (H18).
1
NMR analysis. The H-1H COSY (Supplementary Fig. S5,
Supplementary Material) confirms the 1H NMR assignment as
follow; signal at 7.2 ppm (H21) is correlated with signal at
7.3 ppm (H22), signal at 7.7 ppm (H18) is correlated with
signals at 7.5 ppm (H17) and 7.6 ppm (H19). HSQC (Sup-
plementary Fig. S6, Supplementary Material) spectrum indi-
Biological evaluation and molecular modeling
studies
In vitro HCV NS5B GT4a inhibition assay
1
cates that the exocyclic H13 singlet signal at 7.8 ppm in H
NMR is correlated with 134.83 ppm (C13) in 13C NMR that is
more de-shielded than C21, C22, and C25, while the signal at
126.18 ppm (C25) is correlated with 7.3 ppm (H25) that
The HCV NS5B GT4a inhibition assay is carried out on
18 synthesized compounds 7a–r (Table 1). The results
show that compound 7a (IC50 = 1.029 µM) is 25 folds more

Medicinal Chemistry Research (2021) 30:1151–1165
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Scheme 1 Synthetic route for
compounds 7a–r. (i) Acetic
acid, reflux, 12 h; (ii) Ferric
chloride, ethanol, reflux,20 h;
(iii) Chloroacetyl chloride,
triethylamine, dichloromethane,
4 h; (iv) Ammonium
thiocyanate, reflux, 20 h; (v)
Different aldehydes, CH3ONa,
methanol, reflux, 12 h
Scheme 2 The mechanism of
1,3,4-thiadiazole ring formation
Scheme 3 The mechanism of
thiazolidin-4-one ring formation
potent on HCV NS5B GT4a than reported on HCV NS5B
GT1b (IC50 = 25.3 µM) [23], from which we conclude
that 2-[(5-substituted phenyl-1,3,4-thiadiazol-2-yl)imino]-5
(substituted phenylmethylidene)-1,3-thiazolidin-4-one scaf-
fold is more potent on HCV NS5B GT4a.
Compound 7b is considered to be three folds more potent
than the lead compound 7a (IC50 = 1.029 µM on HCV
NS5B GT4a). Also, compound 7b is considered to be 74
folds more potent than the lead compound 7a (IC50
=
25.3 µM on HCV NS5B GT1b).
The NS5B inhibitory assay results illustrate that com-
pounds 7a–d and 7n–r having 2-Chloro-6-fluorophenyl and
2-Chlorophenyl ring A substitution on ring A respectively
exhibit better HCV NS5B inhibition activity than 3-Fluoro,
4-Fluoro, and 4-chloro substitutions. Among the series
7a–d and 7n–r, compounds 7b and 7n (IC50 = 0.338 and
0.342 µM, respectively) are the most potent compounds.
Analysis of the results focusing on ring B and con-
sidering compounds 7a–d, 7h, 7k, 7l, 7n–r reveals that the
substitution on meta or para positions is more favorable
than substitution on ortho position, as exhibited by the IC50
values of compounds 7h and 7i showing IC50 2.401 and
6.66 μM, respectively. The results show that the most active
four compounds are 7b, 7c, 7k, 7n, which can be explained

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Fig. 7 The 3D (left) and 2D (right) of the docking interactions of compound 7b with HCV NS5B GT4a thumb II site
Fig. 8 The 3D (left) and 2D (right) of the docking interactions of compound 7c with HCV NS5B GT4a thumb II site
by the docking studies. Generally, the most prominent
interaction type of compounds 7a–r (Figs. 7–10, and S47-
S60, Supplementary Material) with the surrounding amino
acids is in the form of hydrogen bonding through the car-
bonyl oxygen and the NH group of the thiazolidinone ring.
Analysis of the binding interactions of 7b, 7c, 7k, and 7n
with the thumb II site is summarized in Table 2.
The most active compound 7b forms two hydrogen
bonds with Lys533 and Thr473, and it shows hydrophobic
contacts with many hydrophobic amino acids in the binding
site (Fig. 7 and Table 2).
The decrease in the biological activity of compound
7c could be explained by the inability to form two
hydrogen bonds compared to compound 7b. Compound
7c forms one hydrogen bond with His475 amino
acid. Also, it has fewer nearby hydrophobic interactions
(Fig. 8 and Table 2).
Compound 7k forms three hydrogen bonds with
His475, Arg422, Thr473 amino acids, and shows less
hydrophobic interactions through aromatic rings. Also,
hydrogen bond distance with Arg422 is less than optimum
(Fig. 9 and Table 2).

Medicinal Chemistry Research (2021) 30:1151–1165
1159
Fig. 9 The 3D (left) and 2D (right) of the docking interactions of compound 7k with HCV NS5B GT4a thumb II site
Fig. 10 The 3D (left) and 2D (right) of the docking interactions of compound 7n with HCV NS5B GT4a thumb II site
Table 2 Analysis of binding interactions of 7b, 7c, 7k, and 7n with HCV NS5B GT4a thumb II site
Compound IC50
(µM)
Hydrogen bonding interactions
(distance Å)
Hydrophobic interaction
Pi-cation
interaction
7b
0.338
Lys533 (2.32), Thr473 (2.12)
Ile419, Leu482, Leu497, Leu474, Val376, Leu536, Leu469,
Gly476, Thr477
Lys533
7c
0.634
0.564
His475 (2.21)
Ile419, Leu497, Leu474, Val376, Lys533, Trp528
Leu474, Ile419, Val376, Trp528
Lys533
Lys533
7k
His475 (2.08), Thr473 (2.58),
Arg422 (1.70)
7n
0.342
His475 (2.24), Thr473 (2.61),
Arg422 (2.41)
Leu474, Leu497, Ile419, Val376, Lys533, Trp528, Ala529
Lys533
Also, compound 7n forms three hydrogen bonds with
Antiviral activity on HCV GT4a
His475, Arg422, Thr473 amino acids, and this can explain
its high activity. 7n shows hydrophobic interactions through
aromatic rings with hydrophobic amino acids (Fig. 10 and
Table 2).
Compounds 7a–d, 7k, 7n, 7q, and 7r showed the most
potent activities with IC50 < 2 µM. Thus, they have been
selected for antiviral activity testing. The results illustrated

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Table 3 HCV NS5B GT4a antiviral assay of the selected compounds
Conclusion
Compound
Titer (IU/mL) Fold
% Inhibition
Our efforts are directed to develop potent inhibitors of HCV
NS5B GT4a for treating HCV GT4a infection. A set of
compounds with 4-thiazolidinone derivatives has been
designed. To validate the binding affinity of this set of
compounds, homology model of HCV NS5B GT4a was
constructed and the compounds were docked. Based on the
docking results, 18 compounds (7a–r) were selected for
synthesis and were further evaluated for their activity.
Compounds (7a–r) were subjected to in vitro HCV NS5B
GT4a inhibition assay. Compounds 7b and 7n showed the
highest inhibitory activity (IC50 = 0.338 and 0.342 µM,
respectively). Compounds 7a, 7b, 7c, 7d, 7k, 7n, 7q, and 7r
with IC50 < 2 µM were selected for the assessment of the
anti-HCV GT4a activity using human hepatoma cell line
(Huh 7.5), the percentage of inhibition of the tested com-
pounds was between 79.67 and 94.77%. Compounds 7a,
7b, and 7c were tested for their cytotoxicity on the Huh7.5
cell line and exhibited low cytotoxicity compared to
Beclabuvir reference compound. Compound 7b was iden-
tified through this study as a potential new lead for future
HCV inhibition studies.
7a
7b
7c
7d
7k
7n
7q
7r
337,790
202,786
148,856
426,019
270,760
578,547
295,742
556,282
0.118706 88.13
0.071263 92.88
0.052311 94.77
0.149712 85.03
0.095151 90.49
0.203313 79.67
0.103930 89.61
0.195489 80.46
0.072876 92.72
Beclabuvir (BMS-791325) 207,376
Positive control 2,845,597
1
0
Table 4 Cytotoxic activity (CC50) of 7a, 7b, and 7c against human
hepatoma (Huh7.5) cell line
CC50 SDb (μM)
a
Compound
7a
58.22 1.72
176.87 4.99
133.48 3.69
28.05 0.78
7b
7c
Beclabuvir (BMS-791325)
aThe half-maximal cytotoxic concentration
bThe standard deviation of triplicate experiments
Materials and methods
Chemistry
in Table 3 show that all eight tested compounds exhibited
more than 79% inhibition of the HCV GT4a viral activity,
which indicates the good activity of these compounds.
Compounds 7b (0.338 µM, 92.88%) and 7c (0.634 µM,
94.77%) have high % inhibition compared to Beclabuvir
reference compound (92.72%). Compound 7c exhibited the
highest % inhibition among the tested compounds, while 7b
showed higher IC50 with good percentage of inhibition too.
General
Chemical reagents and materials were supplied by Sigma-
Aldrich and Alfa Aesar companies and were used without
further purification. Electrothermal melting point device
(Stuart SMP30) was used for melting points detection, and the
results were reported uncorrected. TLC aluminum sheets
Silica 60 F254 (Merck Co.) were used for reactions monitoring
and were visualized with UV light at 254 nm, using TLC
system:chloroform/methanol/acetic acid (9.3:0.5:0.2 v/v/v).
The PerkinElmer 2400 series II CHNS elemental analyzer was
used for elemental analysis. IR spectra of all compounds were
recorded on a PerkinElmer FT-IR spectrophotometer using
Cytotoxicity study on human hepatoma (Huh7.5) cell line
Compounds 7b and 7c, the most active inhibitors, and the
lead compound 7a were subjected to cytotoxicity study on
Huh7.5 cell line along with Beclabuvir as a reference
compound (Table 4). The assay was carried out using MTT
assay kit, in which the detection of cell metabolic activity is
based on the purple color that results from biochemical
reaction. The developed color is directly proportional to the
cell number, which indicates the cell viability.
The results show that all tested compounds exhibited low
cytotoxicity on Huh7.5 cell lines compared to Beclabuvir
reference compound. Moreover, compound 7b exhibited the
least cytotoxicity by more than six folds compared to
Beclabuvir.
KBr discs with a frequency range of (4000–400 cm−1). H
1
NMR spectra were processed on Bruker FT-NMR (400 MHz)
spectrometer using dimethyl sulfoxide (DMSO) as a solvent,
and the chemical shifts were reported as ppm. 13C NMR
spectra of the representative examples (7b–f, 7n, 7q) were
recorded on Bruker FT-NMR (100 MHz) using DMSO-d6 as
a solvent with tetramethylsilane as an internal standard. Pyr-
idine was used to dissolve compounds 7b, 7n, and 7f duo to
their poor solubility in DMSO. All chemical shift values and
multiplicity were quoted in ppm.

Medicinal Chemistry Research (2021) 30:1151–1165
1161
General synthetic procedure of 2-(arylmethylidene)
hydrazinecarbothioamides (3a–e) [23]
(1.8 mmol) was dissolved in methanol in excess amount and
added to the mixture. The reaction mixtures were heated
under reflux for 12 h. The reactions were left to cool at
25 °C. Then 40 mL ice-cold water was added. Then the
mixtures were neutralized with glacial acetic acid to give
compounds 7a–r. The precipitated products were filtered,
washed with water, and the recrystallization was performed
using Dimethylformamide.
Solutions of thiosemicarbazide 2 (30 mmol) were added to
solutions of aldehydes (1a–e) (30 mmol), and five drops of
acetic acid were added then the reaction mixtures were
heated under reflux for 12 h. The products (3a–e) were fil-
tered, and the recrystallization was performed using ethanol.
Compounds 3a, 3d, and 3e [23] and compounds 3b and
3c [34] were reported.
5-(3-Fluorobenzylidene)-2-{[5-(2-chloro-6-fluorophenyl)-1,3,4-
thiadiazol-2-yl]imino}-1,3-thiazolidin-4-one (7a) The results
of the characterization of the compound 7a (shown in Page
2, Supplementary Material) were as reported [23].
General synthetic procedure of 5-(substituted phenyl)-1,3,4-
thiadiazol-2-amines (4a–e) [23, 24]
Solutions of compounds 3a–e (2 mmol) in ethanol were
added to solutions of ferric chloride (8 mmol). Then the
reactions were heated under reflux for 20 h. The reactions
were neutralized by ammonia solution, filtered, dried, and
the recrystallization was performed using ethanol.
Compounds 4a, 4d, and 4e [23] and compounds 4b and
4c [24] were reported.
5-(1,3-Benzodioxol-5-ylmethylidene)-2-{[5-(2-chloro-6-fluoro-
phenyl)-1,3,4-thiadiazol-2-yl]imino}-1,3-thiazolidin-4-one
(7b) Pale yellow solid; yield (0.065 g, 20.2%) with mp
285–289 °C. IR (KBr, cm−1): 3449 (N-H str.), 3145 (Ar-H),
2902 (C-H str. of CH2), 1705 (C=O, lactam), 1593 (C=N
1
str.). H NMR (400 MHz, DMSO-d6) δ (ppm): 6.17 (s, 2H,
CH2), 7.17–7.19 (d, 1H, Ar-H21), 7.2 (s, 1H, Ar-H25),
7.27–7.29 (d, 1H, Ar-H22), 7.49–7.53 (t, 1H, Ar-H17),
7.59–7.61 (d, 1H, Ar-H19), 7.67–7.71 (q, 1H, Ar-H18),
7.78 (s, 1H, =CH13), 13.04 (broad s, 1H, NH of lactam).
13C NMR (100 MHz, pyridine) δ (ppm): 102.28 (C27),
109.16 (C21), 109.71 (C22), 114.85 (C6), 115.07 (C17),
122.78 (C15), 123.81 (C20), 126.18 (C25), 126.21(C19),
132.67 (C23), 132.79 (C24), 134.83 (C13), 135.86 (C18),
148.74 (C16), 149.08 (C10), 150.15 (C2), 159.70 (C8),
162.21 (C4), 198.20 (C11). Anal. Calcd. for
C19H10ClFN4O3S2: C, 49.51; H, 02.19; N, 12.16; S,
13.91%; Found: C, 49.80; H, 02.42; N, 12.35; S, 14.15%.
General synthetic procedure of 2-chloro-N-[5-(substituted
phenyl)-1,3,4-thiadiazol-2-yl]acetamides (5a–e) [23, 24]
Compounds 4a–e (10 mmol) were dissolved in dichlor-
omethane, and triethylamine (12 mmol) was added to the
reaction pots. Chloroacetyl chloride (20 mmol) was added
to the reaction pots slowly under anhydrous condition,
followed by stirring for 4 h under reflux. The reactions were
filtered then dried, and the recrystallization was performed
using ethanol.
Compounds 5a, 5d, and 5e [23] and compounds 5b and
5c [24] were reported.
5-(4-Dimethylaminobenzylidene)-2-{[5-(2-chloro-6-fluorophe-
nyl)-1,3,4-thiadiazol-2-yl]imino}-1,3-thiazolidin-4-one (7c)
Orang solid; yield (0.132 g, 38.27 %) with mp 270–273 °C.
IR (KBr, cm−1): 3439 (N-H str.), 3142 (Ar-H), 2926 (C-H
General synthetic procedure of 2-{[5-(substituted phenyl)-1,3,4-
thiadiazol-2-yl]imino-1,3-thiazolidin-4-ones (6a–e) [23, 24]
1
str. of CH3), 1699 (C=O, lactam), 1583 (C=N str.). H
Solutions of compounds 5a–e (2.5 mmol) in 1,4-dioxane were
added to ethanolic solutions of ammonium thiocyanate
(5 mmol). The reactions were stirred under reflux for 20 h. The
solvent was evaporated. The reactions were filtered then dried,
and the recrystallization was performed using ethanol.
Compounds 6a, 6d, and 6e [23] and compounds 6b and
6c [24] were reported.
NMR (400 MHz, DMSO-d6) δ (ppm): 3.04 (s, 6H, 2-CH3),
6.89–6.91 (d, 2H, Ar-H), 7.48–7.72 (m, 6H, Ar-H, =CH),
12.84 (s, 1H, NH of lactam). 13C NMR (100 MHz, DMSO)
δ (ppm): 40.04 (2 CH3), 112.66, 115.68, 115.89, 116.27,
120.29, 126.77, 133.05, 134.37 (Ar-C), 135.05 (thiazolidi-
none C5), 152.14 (=CH-Ar), 160.51, 161.90 (thiadiazole
C5, thiazolidinone C2), 154.40 (thiadiazole C2), 167.75
(thiazolidinone C4). Anal. Calcd. for C20H15ClFN5OS2: C,
52.23; H, 03.29; N, 15.23; S, 13.94%; Found: C, 52.51; H,
03.49; N, 15.45; S, 13.76%.
General synthetic procedure of 5-(substituted arylidene)-2-
{[5-(substituted phenyl)-1,3,4-thiadiazol-2-yl]imino}-1,3-
thiazolidin-4-ones (7a–r) [23, 24]
5-(3-Chlorobenzylidene)-2-{[5-(2-chloro-6-fluorophenyl)-
1,3,4-thiadiazol-2-yl]imino}-1,3-thiazolidin-4-one (7d) Yel-
low solid; yield (0.443 g, 65.44%) with mp 284–287 °C. IR
(KBr, cm−1): 3437 (N-H str.), 3095 (Ar-H), 1714 (C=O, str.),
Compounds 6a–e (1.5 mmol) were dissolved in methanol,
then sodium methoxide (3 mmol) in methanol was added,
and the mixture was heated for 10 min. The aldehyde

1162
Medicinal Chemistry Research (2021) 30:1151–1165
1
1598 (C=N str.). H NMR (400 MHz, DMSO-d6) δ (ppm):
7.48–7.84 (m, 7H, Ar-H), 7.79 (s, 1H, =CH), 13.17 (s, 1H,
NH of lactam). 13C NMR (100 MHz, DMSO) δ (ppm):
115.68, 115.89, 126.23, 126.73, 128.42, 130.66, 131.77,
132.06, 134.02, 134.31, 134.34, 134.46, 135.76, 155.18 (Ar-
C, =CH-Ar, thiazolidinone C5), 159.39, 159.69 (thiadiazole
C5, thiazolidinone C2), 161.89 (thiadiazole C2), 167.33
(thiazolidinone C4). Anal. Calcd. for C18H9Cl2FN4OS2:
C, 47.90; H, 02.01; N, 12.42; S, 14.21%; Found: C, 47.73;
H, 01.83; N, 12.71; S, 13.97%.
5-(3-Bromobenzylidene)-2-{[5-(3-fluorophenyl)-1,3,4-thiadia-
zol-2-yl]imino}-1,3-thiazolidin-4-one (7h) Yellow solid;
yield (0.075 g, 21.74%) with mp 274–276 °C. IR (KBr, cm−1):
1721 (C=O st., lactam), 1610 (C=N str.). 1H NMR
(400 MHz, DMSO-d6) δ (ppm): 7.40–7.80 (m,8H, Ar-H), 7.90
(s,1H, =CH), 13.12 (s, 1H, NH of lactam). Anal. Calcd. for
C18H10BrFN4OS2: C, 46.86; H, 02.19; N, 12.15; S, 13.89%;
Found: C, 46.99; H, 01.92; N, 11.98; S, 14.21%.
5-(2-Bromobenzylidene)-2-{[5-(3-fluorophenyl)-1,3,4-thiadia-
zol-2-yl]imino}-1,3-thiazolidin-4-one (7i) Yellow solid;
yield (0.058 g, 16.76%) with mp 267–268 °C. IR (KBr,
cm−1): 3434 (N-H str.), 1724 (C=O st., lactam), 1603
(C=N str.). 1H NMR (400 MHz, DMSO-d6) δ (ppm):
7.41–7.85 (m, 8H, Ar-H), 7.90 (s, 1H, =CH), 13.18 (s, 1H,
NH of lactam). Anal. Calcd. for C18H20BrNO4: C, 46.86; H,
02.19; N, 12.15; S, 13.89%; Found: C, 46.57; H, 02.48; N,
12.42; S, 13.61%.
5-(3-Phenylpropylidene)-2-{[5-(4-fluorophenyl)-1,3,4-thiadia-
zol-2-yl]imino}-1,3-thiazolidin-4-one (7e) Yellow solid;
yield (0.063 g, 20.46%) with mp 224–226 °C. IR (KBr,
cm−1): 3447 (N-H str.), 3084 (Ar-H), 2923 (C-H str. of
CH2), 1728 (C=O, str.), 1585 (C=N str.). 1H NMR
(400 MHz, DMSO-d6) δ (ppm): 2.61–2.62 (m, 2H,
CH2CH2-Ph), 2.84–2.89 (m, 2H, CH2CH2-Ph), 6.92–6.94
(t, 1H, =CH), 7.20–7.42 (m, 7H, ArH), 7.97–7.99 (t, 2H,
ArH), 12.78 (s, 1H, NH of lactam). 13C NMR (100 MHz,
DMSO) δ (ppm): 33.09, 33.59 (2 CH2) 116.89, 117.11,
126.68, 127.06, 127.83, 128.83, 129.93, 138.09, 140.85
(Ar-C), 159.32 (thiazolidinone C5), 162.82 (=CH-Ar),
164.14, 165.30 (thiadiazole C5, thiazolidinone C2),
166.09 (thiadiazole C2), 170.32 (thiazolidinone C4).
Anal. Calcd. for C20H15FN4OS2: C, 58.52; H, 03.68; N,
13.65; S, 15.62%; Found: C, 58.78; H, 03.38; N, 13.89;
S, 15.37%.
5-(4-Dimethylaminobenzylidene)-2-{[5-(3-fluorophenyl)-1,3,4-
thiadiazol-2-yl]imino}-1,3-thiazolidin-4-one
(7j) Orange
solid; yield (0.093 g, 29.14%) with mp 255–258 °C. IR
(KBr, cm−1): 3448 (N-H str.), 3145 (Ar-H), 1699 (C=O st.,
1
lactam), 1583 (C=N str.). H NMR (400 MHz, DMSO-d6)
δ (ppm): 3.06 (s, 6H, 2 CH3), 7.71 (s, 1H, =CH), 6.90–7.82
(8H, Ar-H), 12.81 (s, 1H, NH of lactam). Anal. Calcd. for
C20H16FN5OS2: C, 56.45; H, 03.79; N, 16.46; S, 15.07%;
Found: C, 56.68; H, 04.10; N, 16.71; S, 15.32%.
5-(3,4-Dimethoxybenzylidene)-2-{[5-(4-fluorophenyl)-1,3,4-
5-(3-Chlorobenzylidene)-2-{[5-(3-fluorophenyl)-1,3,4-thiadia-
zol-2-yl]imino}-1,3-thiazolidin-4-one (7k) Yellow solid;
yield (0.158 g, 50.54%) with mp 275–278 °C. IR (KBr,
cm−1): 3448 (N-H str.), 3151 (Ar-H), 1709 (C=O str.,
lactam), 1593 (C=N str.). 1H NMR (400 MHz, DMSO-d6)
δ (ppm): 7.41–7.95 (m,9H, ArH, =CH), 13.13 (s, 1H, NH
of lactam). Anal. Calcd. for C18H10ClFN4OS2: C, 51.86;
H, 02.42; N, 13.44; S, 15.38%; Found: C, 51.56;
H, 02.14; N, 13.14; S, 15.11%.
thiadiazol-2-yl]imino}-1,3-thiazolidin-4-one
(7f) Yellow
solid; yield (0.170 g, 38.49%) with mp 266–268 °C. IR
(KBr, cm−1): 3452 (N-H str.), 1640 (C=O st., lactam) 1587
1
(C=N str.). H NMR (400 MHz, DMSO-d6) δ (ppm): 3.84
(s, 6H, CH3), 7.22–8.02 (m, 7H, Ar-H), 8.02 (s, 1H, =CH),
12.92 (s, 1H, NH of lactam); 13C NMR (100 MHz, Pyr-
idine) δ (ppm): 55.66 (2 OCH3), 112.17, 114.23, 116.26,
116.48, 123.83, 127.60, 129.53, 129.61, 134.84 (Ar-C,
=CH-Ar), 135.61 (thiazolidinone C5), 151.52 (thiazolidi-
none C2), 162.30 (thiazolidinone C4). Anal. Calcd. for
C20H15FN4O3S2: C, 54.29; H, 03.42; N, 12.66; S, 14.49%;
Found: C, 54.51; H, 03.62; N, 12.99; S, 14.98%.
5-(4-Methoxybenzylidene)-2-{[5-(3-fluorophenyl)-1,3,4-thia-
diazol-2-yl]imino}-1,3-thiazolidin-4-one (7l) Yellow solid;
yield (.050 g, 24.25%) with mp 267–270 °C. IR (KBr, cm−1):
3448 (N-H str.), 3100 (Ar-H), 1702 (C=O st., lactam), 1587
1
5-{[4-(Benzyloxy)-3-methoxyphenyl]methylidene}-2-{[5-(4-
chlorophenyl)-1,3,4-thiadiazol-2-yl]imino}-1,3-thiazolidin-4-
one (7g) Yellow solid; yield (0.085 g, 23.72%) with mp
259–261 °C. IR (KBr, cm−1): 3451 (N-H str.), 1641 (C=O
(C=N str.). H NMR (400 MHz, DMSO-d6) δ (ppm): 3.84
(s, 3H, CH3), 7.16–7.80 (m, 9H, Ar-H, =CH), 12.96 (s, 1H,
NH of lactam). Anal. Calcd. for C18H13FN4O2S2: C, 55.33;
H, 03.18; N, 13.59; S, 15.55%; Found: C, 55.02; H, 02.95;
N, 13.83; S, 15.23%.
1
st., lactam), 1587 (C=N str.). H NMR (400 MHz, DMSO-
d6) δ (ppm): 3.85 (s, 3H, CH3), 5.19 (s, 2H, CH2),
7.23–7.78 (m, 12H, Ar-H), 7.97 (d, 1H, =CH), 12.96 (s,
1H, NH of lactam). Anal. Calcd. for C26H19ClN4O3S2: C,
58.37; H, 03.58; N, 10.47; S, 11.99%; Found: C, 58.56; H,
03.79; N, 10.62; S, 12.26%.
5-{[4-(Benzyloxy)-3-methoxyphenyl]methylidene}-2-{[5-(2-
chlorophenyl)-1,3,4-thiadiazol-2-yl]imino}-1,3-thiazolidin-4-
one (7m) Yellow solid; yield (0.420 g, 52.33%) with mp
250–252 °C. IR (KBr, cm−1): 3450 (N-H str.), 1688 (C=O

Medicinal Chemistry Research (2021) 30:1151–1165
1163
1
st., lactam), 1577 (C=N str.). H NMR (400 MHz, DMSO-
167.18 (thiadiazole C2), 171.36 (thiazolidinone C4).
Anal. Calcd. for C18H10ClN5O3S2: C, 48.70; H, 02.28; N,
15.78; S, 14.45%; Found: C, 49.01; H, 02.56; N, 15.49;
S, 14.72%.
d6) δ (ppm): 3.85 (s, 3H, CH3), 5.19 (s, 2H, CH2),
7.26–8.20 (m, 12H, Ar-H), 8.2 (d, 1H, =CH), 12.98 (s, 1H,
NH of lactam). Anal. Calcd. for C26H19ClN4O3S2: C, 58.37;
H, 03.58; N, 10.47; S, 11.99%; Found: C, 58.68; H, 03.77;
N, 10.19; S, 12.28%.
5-(4-Methoxybenzylidene)-2-{[5-(2-chlorophenyl)-1,3,4-thia-
diazol-2-yl]imino}-1,3-thiazolidin-4-one (7r) Yellow solid;
yield (0.133 g, 41.36%) with mp 279–281 °C. IR (KBr,
cm−1): 3422 (N-H str.), 3097 (Ar-H), 1701 (C=O st.,
5-(4-Dimethylaminobenzylidene)-2-{[5-(2-chlorophenyl)-
1,3,4-thiadiazol-2-yl]imino}-1,3-thiazolidin-4-one
(7n)
1
Orange solid; yield (0.076 g, 22.98%) with mp 291–294 °C.
lactam), 1587 (C=N str.). H NMR (400 MHz, DMSO-
IR (KBr, cm−1): 3450 (N-H str.), 1697 (C=O st., lactam),
d6) δ (ppm): 3.84 (s, 3H, CH3), 7.17–7.78 (m, 8H, Ar-H),
8.10 (d, 1H, =CH), 12.98 (s, 1H, NH of lactam). Anal.
Calcd. for C19H13ClN4O2S2: C, 53.21; H, 03.06; N,
13.07; S, 14.95%; Found: C, 53.52; H, 02.85; N, 13.32;
S, 15.23%.
1
1577 (C=N str.). H NMR (400 MHz, DMSO-d6) δ (ppm):
3.04 (s, 6H, 2 CH3), 8.10 (d, 1H, =CH), 6.89–7.73 (8H, Ar-
H), 12.82 (s, 1H, NH of lactam). 13C NMR (100 MHz,
Pyridine) δ (ppm): 33.17 (2 CH3), 106.02, 114.73, 116.54,
117.58, 121.37, 123.7, 124.55, 125.32, 125.92(Ar-C), 145.4
(thiazolidinone C5), 126.52 (=CH-Ar), 129.6, 129.35
(thiadiazole C5, thiazolidinone C2), 128.28 (thiadiazole
C2), 153.91 (thiazolidinone C4). Anal. Calcd. for
C20H16ClN5OS2: C, 54.35; H, 03.65; N, 15.85; S, 14.51%;
Found: C, 54.59; H, 03.87; N, 16.10; S, 14.81%.
Biology
In vitro HCV NS5B inhibition assay
Scintillation proximity assay (SPA) was used to evaluate the
HCV NS5B inhibition using SPA beads. The beads emit an
amount of light that is detected using a top count plate
reader (Packard Top Count NXT scintillation counter,
PerkinElmer). The detailed assay protocol was carried out
as described (Page 31 and Supplementary Fig. S61–S78,
Supplementary Material), and as reported [35, 36].
5-(3-Bromobenzylidene)-2-{[5-(2-chlorophenyl)-1,3,4-thiadia-
zol-2-yl]imino}-1,3-thiazolidin-4-one (7o) Yellow solid;
yield (0.108 g, 30.14%) with mp 280–283 °C. IR (KBr,
cm−1): 3449 (N-H str.), 1704 (C=O st., lactam), 1594
(C=N str.). 1H NMR (400 MHz, DMSO-d6) δ (ppm):
7.57–7.91 (m, 8H, Ar-H), 8.20 (d, 1H, =CH), 13.15 (s,
1H, NH of lactam); Anal. Calcd. for C18H10BrClN4OS2:
C, 45.25; H, 2.11; N, 11.73; S, 13.42%; Found: C, 45.51;
H, 1.84; N, 11.44; S, 13.17%.
Antiviral activity
Huh-7.5 cells (ATCC code PTA-8561) were cultured as
reported [36], the detailed procedure was carried out as
described (Page 40, Supplementary Material). The infected
Huh-7.5 cells were cultured and then serial concentrations
of the tested compounds were added after incubation. The
level of HCV RNA in each sample was assessed by
quantitative real-time PCR on Rotor-Gene Cycler (Qia-
gen). The test was carried out using a commercially
available kit supplied by “DNA-Technology, Research &
Production” LLC (Moscow, Russia) according to the
manufacturer’s instructions. The primers used for HCV
and GAPDH internal control were previously reported
[37]. The percent inhibition was calculated for each sam-
ple (Supplementary Fig. S79, Supplementary Material) as
previously described [36].
5-(3-Chlorobenzylidene)-2-{[5-(2-chlorophenyl)-1,3,4-thiadia-
zol-2-yl]-imino}-1,3-thiazolidin-4-one (7p) Yellow solid;
yield (0.107 g, 32.92%) with mp 270–271 °C. IR (KBr,
cm−1): 3449 (N-H str.), 1709 (C=O st., lactam), 1592
(C=N str.). 1H NMR (400 MHz, DMSO-d6) δ (ppm):
7.57–7.81 (m, 8H, Ar-H), 8.20 (d, 1H, =CH), 13.14 (s,
1H, NH of lactam). Anal. Calcd. for C18H10Cl2N4OS2: C,
49.89; H, 02.33; N, 12.93; S, 14.79%; Found: C, 50.21;
H, 2.11; N, 12.75%; S, 15.10%.
5-(3-Nitrobenzylidene)-2-{[5-(2-chlorophenyl)-1,3,4-thiadia-
zol-2-yl]imino}-1,3-thiazolidin-4-one (7q) Yellow solid;
yield (0.165 g, 49.56%) with mp 272–274 °C. IR (KBr,
cm−1): 3419 (N-H str.), 3121 (Ar-H), 1726 (C=O st.,
1
lactam), 1596 (C=N str.). H NMR (400 MHz, DMSO-
Cytotoxicity study
d6) δ (ppm): 7.54–8.32 (m, 8H, Ar-H), 8.52 (s,1H, =CH),
13.19 (s, 1H, NH of lactam). 13C NMR (100 MHz,
DMSO) δ (ppm): 125.04, 125.15, 127.44, 128.47, 128.98,
131.05, 131.19, 131.46, 131.71, 132.79, 135.26, 136.00,
(Ar-C), 148.73 (thiazolidinone C5), 160.72 (=CH-Ar),
158.87 (thiadiazole C5), 158.80 (thiazolidinone C2),
Huh-7.5 cells were cultured, and cytotoxicity of the tested
compounds was measured using MTT kit (Sigma-Aldrich)
as described by the manufacturer. The detailed procedure
was carried out as described (Page 42, Supplementary Fig.
S80–S83, Supplementary Material) [35].

1164
Medicinal Chemistry Research (2021) 30:1151–1165
Computational studies
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Acknowledgements We are grateful for OpenEye Scientific Software
for supporting the academic license granted to MAE laboratory. We
also thank Dr. Khaled M. Elokely, Tanta University, for his help in
analyzing the computational data and Dr. Ahmed E. Goda, Tanta
University, for his help in the biological studies.
Author contributions MAE contributed to conceptualization and
design. ASA-B collected the data and carried out the experimental
section. All authors analyzed the data, wrote, and revised the
manuscript.
Compliance with ethical standards
Conflict of interest The authors declare no competing interests.
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Publisher’s note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.

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