Molecular modeling of drug-pathophysiological Mtb protein targets: Synthesis of some 2-thioxo-1, 3-thiazolidin-4-one derivatives as anti-tubercular agents

Article abstract of DOI:10.1016/j.molstruc.2017.07.009

Twenty novel 2-thioxo-1, 3-thiazolidin-4-one derivatives (5a-5t) were synthesized and evaluated for their antitubercular activity. The structure of the compounds was confirmed by IR, NMR and Mass Spectroscopy methods. In addition, single-crystal X-ray diffraction was performed for compound 5a. All the synthesized compounds were screened for their in-vitro antimycobacterial activity against MTB (H37RV, ATCC No: 27294) by Alamar Blue assay method. Compounds 5r, 5k, 5t displayed most potent in-vitro activity with MICs of 0.05, 0.1, 0.2 μg/ml concentrations respectively which are comparatively potent than the standards. Molecular docking and dynamics simulations were performed to find out the plausible mechanism of the titled compounds.

Full text of DOI:10.1016/j.molstruc.2017.07.009

Accepted Manuscript
Molecular modeling of drug-pathophysiological Mtb protein targets: Synthesis of some
2-thioxo-1, 3-thiazolidin-4-one derivatives as anti-tubercular agents
Kaveripakkam Mohammed Ali Noorulla, Ayyadurai Jerad Suresh, Vinod Devaraji, Bijo
Mathew, Devi Umesh
PII:
S0022-2860(17)30930-4
10.1016/j.molstruc.2017.07.009
MOLSTR 24036
DOI:
Reference:
To appear in: Journal of Molecular Structure
Received Date: 10 March 2017
Revised Date: 5 June 2017
Accepted Date: 7 July 2017
Please cite this article as: K.M. Ali Noorulla, A.J. Suresh, V. Devaraji, B. Mathew, D. Umesh, Molecular
modeling of drug-pathophysiological Mtb protein targets: Synthesis of some 2-thioxo-1, 3-thiazolidin-4-
one derivatives as anti-tubercular agents, Journal of Molecular Structure (2017), doi: 10.1016/
j.molstruc.2017.07.009.
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ACCEPTED MANUSCRIPT
Molecular Modeling of Drug-Pathophysiological Mtb Protein Targets:
Synthesis of some 2-thioxo-1, 3-thiazolidin-4-one derivatives as anti-
tubercular agents
Kaveripakkam Mohammed Ali Noorullaa, Ayyadurai Jerad Suresha*, Vinod Devarajia,
Bijo Mathewb, Devi Umesha
aDepartment of Pharmaceutical Chemistry, College of Pharmacy, Madras Medical College,
Chennai-600003, Tamil Nadu, India.
bDivision of Drug Design and Medicinal Chemistry Research Lab, Department of
Pharmaceutical Chemistry, Ahalia School of Pharmacy, Palakkad-678553, Kerala, India.
*Corresponding author: Ayyadurai Jerad Suresh
Tel: +919381037921
Email address: ajsuresh2001@yahoo.co.uk
Abstract
Twenty novel 2-thioxo-1, 3-thiazolidin-4-one derivatives (5a-5t) were synthesized and evaluated
for their antitubercular activity. The structure of the compounds was confirmed by IR, NMR and
Mass Spectroscopy methods. In addition, single-crystal X-ray diffraction was performed for
compound 5a. All the synthesized compounds were screened for their in-vitro antimycobacterial
activity against MTB (H37RV, ATCC No: 27294) by Alamar Blue assay method. Compounds
5r, 5k, 5t displayed most potent in-vitro activity with MICs of 0.05, 0.1, 0.2 µg/ml
concentrations respectively which are comparatively potent than the standards. Molecular
docking and dynamics simulations were performed to find out the plausible mechanism of the
titled compounds.
Keywords: Mycobacterium tuberculosis, 4-thiazolidinones, Malononitrile, Molecular dynamics.

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Graphical Abstract
Highlights
•
•
•
•
•
Twenty 2-thioxo-1, 3-thiazolidin-4-one derivatives were synthesized.
Exploration of the synthetic route by Computational Distance Analysis.
In-vitro Anti-tubercular activity by Alamar blue assay was performed.
Ligand Target Interaction mechanism using in-silico methods were explored.
Molecular docking and dynamics calculations were performed.
1. Introduction
Tuberculosis, MTB, or TB is an infectious disease caused by various strains of mycobacteria,
usually Mycobacterium tuberculosis [1]. It has become an important world-wide public health
problem with one-third of the world’s population infected by the TB bacillus. Tuberculosis
typically attacks the lungs, but can also affect other parts of the body. It is spread through the air
when people who have an active TB infection cough, sneeze or otherwise transmit respiratory
fluids through the air [2]. According to World Health Organization (WHO),one-third of the
world's population is thought to have been infected with M. tuberculosis [3]and new infections
occur in about 1% of the population each year [4]. In 2007, an estimated 13.7 million chronic
cases were active globally [5], while in 2013, an estimated 9 million new cases occurred [6]. In

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2013 about 1.3 and 1.5 million associated deaths were reported [6, 7], most of which occurred in
the developing countries [8]. People with weak immune systems (those with HIV/AIDS, those
receiving immunosuppressive drugs and chemotherapy) are at a greater risk for developing TB
disease. There is currently a growing concern about the development and discovery of multidrug
which extensively would be drug-resistant tuberculosis (MDR/XDR-TB) with potential to
paralyze TB care programs.
Furthermore, no new drugs have been introduced in the last four decades, except the recently
introduced fluoroquinolones [9, 10], which testifies to the lack of significant research in this area
in the pharmaceutical industry. Hence the development of new drugs, capable of overcoming
MDR- and XDR-TB, to efficiently treat this disease is imperative.
Small ring heterocycles containing nitrogen, sulfur and oxygen have been under investigation for
a long time for their medicinal properties. Of these, the 4-thiazolidinones were found to possess
various biological activities such as antibacterial, antifungal, diuretic, antihistaminic,
anticonvulsant, anticancer, antiviral, anti-HIV, anti-inflammatory and analgesic properties [11-
22]. Further, 4-thiazolidinones were found to possess potent antitubercular properties [23-26]. In
the view of above facts and in the continuation of our search for new class of anti-tubercular
heterocyclic compounds, we report herein synthesis, anti-tubercular activity, in-silico ADME
properties, molecular docking studies and molecular dynamic studies of some 2-thioxo-1, 3-
thiazolidin-4-one derivatives.
2. Results and discussion
2.1 Chemistry
In our initial endeavor, we have investigated a sequential three component reaction of
benzaldehyde 1a, malononitrile 2 and 2-thioxo-1, 3-thiazolidin-4-one 3 in ethanol and in
presence of triethylamine base under room temperature and stirring to afford functionalized
pyranothiazole derivative 5-amino-7-phenyl-2-thioxo-3, 7-dihydro-2H-pyrano [2, 3-d] [1, 3]
thiazole-6-carbonitrile 4a. However, the reaction did not give the expected pyranothiazole
derivative 4a and instead, only one compound by simple filtration was isolated in good yield and
identified as 5-benzylidene-2-thioxo-1, 3-thiazolidin-4-one5a (Scheme 1).
The structure of 5a was further verified by TLC, m.p. and mixed m.p after independent synthesis
via direct condensation of benzaldehyde 1a with 2-thioxo-1, 3-thiazolidin-4-one 3 in the
presence of bases like triethylamine and NaOH. However, the independent synthetic method
suffers from longer reaction times and requires aqueous workup procedures for the isolation of
compound (Scheme 2).

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H
N
N
N
O
N
N
S
S
S
Ar
5 a-t
N H
O
2
S
2
Ar
N
N(Et)
RT
3
3
RT, N(Et)
2.5-3 hrs
O
H
N
3
S
Ar
2c
N H
O
S
1a-t
Ar
N
N H 2
4 a-t
Scheme 1: Synthesis of 2-thioxo-1, 3-thiazolidin-4-one derivatives.
RT, N(Et)3, 12hrs
S
S
O
H
S
NH
O
S
+
NH
3
1a
O
5a
RT, NaOH, 8hrs
Scheme 2: Synthesis of compound 5a via direct condensation.
A plausible mechanism was proposed for the formation of compound 5a and for the non-
formation of the compound 4a (Scheme 3). Initially, the reaction proceeded through the
generation of malonyl adduct 2c via the knoevenagel condensation of benzaldehyde 1a and
malononitrile 2 in the presence of triethylamine base. The intermediate 2c can be isolated and its
formation was identified by TLC. Compound 2c a key intermediate undergone Michael addition
with 3a to give 3c, which upon enolization gave 3d. The enol group and cyano group of the
intermediate 3d was expected to undergo nucleophilic addition in order to facilitate a
intramolecular 6-exo-dig cyclization process followed by a rearrangement via proton transfer to
yield compound 4a. Such an intramolecular cyclization process in 3d was not occurred to
generate compound 4a instead elimination of malononitrile took place leading to the formation
of compound 5a and the structure was confirmed by IR, NMR and Mass Spectroscopy and also
by X-Ray Crystallography.The non-formation of the compound 4a is likely the result of the non -
optimal distance between the reactive centres (enol group and cyano group) of 3d for
nucleophilic attack due to geometrical constraints (Figure 1).

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Figure 1:Distance between the reactive centres (enol and cyano group) of intermediate 3d.

ACCEPTED MANUSCRIPT
H
C
H2
C
(C2H5)3NH
(C2H5)3N
NC
CN
NC
CN
CN
CN
O
-H2O
H
C
(C2H5)3NH
(C2H5)3N
C
H
C
H
C
OH
H
C
O
H
NC
CN
CH
CN
CN
NC
NC
(2c)
(1a)
(2b)
(2)
(2a)
H
H
S
S
(C2H5)3NH
H
O
S
S
+
(C2H5)3N
+
N
H
N
H
O
(3a)
(3)
CN
NC
C
S
S
H
H
S
HC
NH
+
S
N
H
O
O
NC
CN
(3b)
(3a)
(2c)
(C2H5)3N
(C2H5)3NH
S
S
S
S
H
NH
NH
OH
O
NC
NC
C
CN
N
(3c)
(3d)
NC
CN
S
S
NC
S
NH
H
S
H
N
H
HN
O
O
(3c)
(3e)
H2
C
NC
CN
S
S
HN
NC
S
S
O
N
H
H2N
O
(4a)
(5a)
Scheme 3: Plausible mechanism for the formation of compound 5a and non-formation of
compound 4a.
In the view of intramolecular 6-exo-dig cyclization process to take place, the above reaction was
also attempted in different solvent systems like methanol, toluene and acetonitrile and in

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presence of different catalysts like InCl3, sodium hydroxide and pyridine under reflux conditions.
In all cases, no evolution of compound 4a was detected. Compound 5a was detected by TLC and
isolated by simple filtration.
An attempt of reaction under microwave radiation was used to induce intramolecular cyclization
on intermediate 3d. It was expected that microwave would assist the interconversion of
conformers through an efficient bond rotation to generate the pyran ring through a favoured 6-
exo-dig cyclization process. The success of this reaction was expected to depend on the
formation of the appropriate conformational isomer. The reaction was carried out using synthetic
microwave equipment between the isolated malonyl adduct 2c and 2-thioxo-1, 3-thiazolidin-4-
one 3, in the presence of catalytic amount of triethylamine in ethanol. The reaction mixture was
exposed to microwave at 300W intermittently at 1 min intervals for 15 mins and then at 600W
for 15 mins. In both cases, there was no evidence of the intramolecular cyclization for the
formation of compound 4a (Scheme 4).
S
NH
O
S
S
N(Et) , Microwave irradiation
3
Ar
N
Ar
N
NH
O
+
S
N
NH2
300W for 15 mins
600W for 15 mins
3
2c
4 a-t
Scheme 4: Reaction of malonyl adduct 2c and 2-thioxo-1, 3-thiazolidin-4-one 3.
The attempt to synthesize compound 4a through a favoured intramolecular cyclization process
was unsuccessful leading us to conclude that the in situ formed intermediate 3d would be a more
stable conformer which averts from cyclization process. Hence understanding the scope and
limitations of the reaction, further attention was made in the synthesis of 2-thioxo-1, 3-
thiazolidin-4-one derivatives 5 a-t with various substituted aldehyde derivatives. Under the
above reaction conditions, the reaction proceeded smoothly with various aldehydes, including
those containing electron withdrawing and electron releasing groups to provide 2-thioxo-1, 3-
thiazolidin-4-one derivatives 5 a-t in good yields (84–95%). The results are given in Table 1.

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Table: 1 Synthesis of 2-thioxo-1, 3-thiazolidin-4-one derivatives 5 a-t
Entry
Product
Ar-CHO
Structurеs
IUPAC namе
Yield
(%)
92
O
1
5a
“(Z)-5-bеnzylidеnе-2-
thioxothiazolidin-4-onе”
CHO
HN
S
S
S
CHO
2
3
4
5
5b
5c
5d
5e
“(Z)-5-(4-chlorobеnzylidеnе)-2-
thioxothiazolidin-4-onе”
86
95
89
90
Cl
S
S
HN
Cl
O
CHO
S
“(Z)-5-(4-
(dimеthylamino)bеnzylidеnе)-2-
thioxothiazolidin-4-onе”
HN
O
N
N
CHO
S
“(Z)-5-(4-(bеnzyloxy)bеnzylidеnе)-2-
thioxothiazolidin-4-onе”
S
S
O
HN
O
O
OH
O
“(Z)-5-(4-hydroxy-3-
mеthoxybеnzylidеnе)-2-
thioxothiazolidin-4-onе”
OH
O
S
HN
OHC
O

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O
OH
6
7
5f
5g
5h
5i
“(Z)-5-(2-hydroxybеnzylidеnе)-2-
thioxothiazolidin-4-onе”
88
85
92
93
88
87
HN
CHO
S
HO
S
O
O
N+
“(Z)-5-(4-nitrobеnzylidеnе)-2-
thioxothiazolidin-4-onе”
S
HN
N+
O-
O-
S
OHC
O
CHO
Br
S
8
“(Z)-5-(2-bromobеnzylidеnе)-2-
thioxothiazolidin-4-onе”
Br
S
HN
O
F
9
“(Z)-5-(2,3-difluoro-6-
mеthoxybеnzylidеnе)-2-
thioxothiazolidin-4-onе”
O
O
F
HN
S
F
CHO
O
S
F
S
O
10
11
5j
“(Z)-5-((bеnzo[b]thiophеn-3-
yl)mеthylеnе)-2-thioxothiazolidin-4-
onе”
HN
S
CHO
S
S
S
CHO
5k
“(Z)-5-(4-mеthoxybеnzylidеnе)-2-
thioxothiazolidin-4-onе”
O
S
HN
O
O

ACCEPTED MANUSCRIPT
O
12
13
14
5l
5m
5n
“(Z)-5-((furan-2-yl)mеthylеnе)-2-
thioxothiazolidin-4-onе”
85
92
84
O
HN
CHO
S
S
O
S
S
“(Z)-5-((thiophеn-2-yl)mеthylеnе)-2-
thioxothiazolidin-4-onе”
S
HN
CHO
S
O
S
“(Z)-5-(3,4,5-
trimеthoxybеnzylidеnе)-2-
thioxothiazolidin-4-onе”
O
O
S
O
O
O
O
HN
OHC
O
CHO
S
15
16
5o
5p
“(Z)-5-((naphthalеn-2-yl)mеthylеnе)-
2-thioxothiazolidin-4-onе”
89
90
S
HN
O
O
CHO
“(Z)-5-((anthracеn-9-yl)mеthylеnе)-
2-thioxothiazolidin-4-onе”
NH
S
S

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S
O
N+
17
5q
“(Z)-5-(2-nitrobеnzylidеnе)-2-
thioxothiazolidin-4-onе”
85
S
O-
HN
CHO
N+
O
-O
O
O
N+
O
18
19
20
5r
5s
5t
“(Z)-5-(3-nitrobеnzylidеnе)-2-
thioxothiazolidin-4-onе”
94
86
88
O
N+
OHC
OHC
O-
O-
HN
S
S
O
N+
“(Z)-5-((1H-indol-3-yl)mеthylеnе)-2-
thioxothiazolidin-4-onе”
NH
O
O-
S
S
HN
S
O
H
“(Z)-5-((1H-pyrrol-2-yl)mеthylеnе)-
2-thioxothiazolidin-4-onе”
N
CHO
N
H
HN
S
1
The structures of the 2-thioxo-1, 3-thiazolidin-4-one derivatives 5 a-t were established from IR, H NMR, 13C NMR and Mass
spectroscopic data. IR spectra in general showed =CH peak in the regions of 3109-2839 cm-1. In the 1H NMR spectra the signals of
the respective protons of the synthesized compounds were confirmed based on their chemical shifts, multiplicities and coupling
constants. These spectra showed a singlet at around δ 7.60 ppm, which corresponds to the -CH=. The 13C NMR and еlеmеntal analysis
were in accordance to the synthesized structure. Elemental analysis results were within ±0.4% of the theoretical values. The
appearance of their rеspеctivе molecular ion peaks (M+) in the mass spectroscopy also confirms the formation of the compounds. The
relative stereochemistry of the product 5a was established through single-crystal X-ray analysis (Figure 2).

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Figure 2: ORTEP diagram of compound 5a
2.2 Pharmacology
All the synthesized compounds were screened for their in-vitro antimycobacterial activity against
MTB (H37RV, ATCC No: 27294) by Alamar Blue assay (MABA) method [27]. Pyrazinamide,
Ciprofloxacin and streptomycin were used as reference drugs. The results were represented as
minimum inhibitory concentration (MIC) (Figure 3) and are presented in Table 2.
When screened against MTB the synthesized thiazolidinone derivatives 5 a-t showed moderate
to potent in vitro activity against MTB with MIC range 0.05-50 µg/ml. Compounds 5r, 5k, 5t
displayed most potent in-vitro activity with MICs 0.05, 0.1, 0.2 µg/ml concentrations
respectively. Further, compounds 5n, 5q, 5s, 5m, 5o, 5p, 5l showed potent in-vitro activity with
MIC range 0.4 to 1.6 µg/ml concentrations, while other compounds shown moderate to good
activity.
The influence of the different chemical groups on the observed antimycobacterial activity against
MTB deserves comment. Incorporation of nitro group at 3rd position of the phenyl ring
(Compound 5r) led to show potent inhibition of M tuberculosis at 0.05 µg/ml concentration,
while nitro position at 2nd and 4th position of the phenyl ring (Compound 5q and 5g) led to
reduction of the activity 8 and 100 times respectively. Introduction of mono-methoxy group at 4th
position of the phenyl ring (Compound 5k) also exhibited potent inhibition at 0.1 µg/ml
concentration, while tri-methoxy group at 3rd, 4th and 5th positions (Compound 5n) reduced the
activity 8 times. The phenyl substitution (Compound 5a) showed moderate activity at 50 µg/ml
concentration whereas there is a tenfold increase in activity in case of naphthalene and
anthracene aromatic hydrocarbon substitution (Compound 5o and 5p). Conversely five
membered heterocyclic rings in the place of six membered rings (Compound 5j, 5l, 5m, 5s and
5t) increased the activity remarkably. Furthermore halogen substitution of the phenyl ring
(Compound 5b, 5h and 5i) showed moderate activity.

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Figure 3: A) Antimycobacterial activity of standard drugs Pyrazinamide, Ciprofloxacin and
Streptomycin. B) Antimycobacterial activity of compounds 5 a-j tested at 100-0.8 µg/ml
concentrations. C) Antimycobacterial activity of compounds 5 k-t tested at 100-0.8 µg/ml
concentrations. D) Antimycobacterial activity of compounds 5 k-t tested at 0.4-0.003 µg/ml
concentrations.
Table 2: Antimycobacterial activity of 2-thioxo-1, 3-thiazolidin-4-one derivatives
Entry
1.
Product
5a
MIC (µg/ml)
Rankinga
50
50
20
19
13
18
15
14
17
2.
5b
3.
5c
12.5
50
4.
5d
5.
5e
50
6.
5f
12.5
50
7.
5g

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8.
5h
5i
50
25
16
12
11
2
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
5j
12.5
0.1
1.6
0.8
0.4
0.8
0.8
0.4
0.05
0.4
0.2
5k
5l
10
7
5m
5n
5o
5p
5q
5r
5s
5t
6
8
9
4
1
5
3
Pyrazinamide
Ciprofloxacin
Streptomycin
3.12
3.12
6.25
-
-
-
a- ranking of synthetic compounds based on the in-vitro antitubercular activity data
2.3 Computational Chemistry
Computational chemistry approach is the most dynamic technology, which had gained lot of
prominence in modern drug discovery [28]. So we were inquisitive in understanding and
knowing the features in our synthesized compounds that are accountable for inhibitory activity.
2.3.1 In-silico ADME Properties
The QikProp (Schrodinger®) is the prediction tool used to obtain the pharmacokinetics and
pharmacodynamics of the analogues by assessing the drug like properties. The structures of the
synthesized compounds were built using Maestro (Schrodinger®) build panel. All the ligands
were prepared by using Ligprep (Schrodinger®) which uses OPLS_2005 (Optimized Potential for
Liquid Simulations) force field and gave the corresponding low energy 3D conformers of the

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ligands. The low energy 3D conformers of the ligands were checked for their ADME properties
using QikProp (Schrodinger®).
QikProp (Schrodinger®) prediction gave information about descriptors like molecular weight
(mol_MW), octanol/water partition coefficient (QPlogPo/w), brain/blood partition coefficient
(QPlogBB), % human oral absorption (PercentHumanOralAbsorption), human serum albumin
binding (QPlogKhsa), IC50 for HERG K+ Channel blockage (QPlogHERG), Lipinski’s rule of
five violations (Rule ofFive). All the compounds were found to be within the range and
recommended values for 95% of known drugs. The results are given in Table 3.
Table 3: In-silico ADME properties of the synthesized compounds
MW
QPlog QPlog QPlog QPlog
Percent
Ligands
Po/w HERG
BB
Khsa HumanOral
Absorption
Rule ofFive
221.29
2.28
2.91
2.72
4.38
1.98
1.77
1.82
2.91
2.78
3.29
2.54
1.86
2.41
2.75
3.38
4.38
1.90
1.82
2.60
1.72
-2 to
6.5
-4.51
-4.47
-4.61
-6.34
-4.43
-4.39
-4.48
-4.40
-4.35
-4.74
-4.44
-4.20
-4.17
-4.25
-5.23
-5.31
-4.43
-4.48
-4.79
-4.09
<-5
-0.06
0.10
-0.19
-0.08
-0.03
0.41
-0.31
-0.34
-0.22
-0.07
-0.09
0.08
-0.17
-0.42
-0.27
-0.15
0.17
100
100
100
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
5p
5q
5r
255.74
264.36
327.42
267.32
237.29
266.29
300.19
287.30
277.37
251.32
211.25
227.31
311.37
271.35
321.41
266.29
266.29
260.33
210.27
130-725
-0.19
-0.37
-0.53
-0.52
-1.01
0.08
100
89.84
86.94
78.54
100
100
100
100
95.25
100
100
100
0.01
0.04
-0.14
-0.04
0.04
-0.29
-0.10
0.09
-0.89
-1.01
-0.33
-0.25
0.58
100
-0.23
-0.22
-0.05
-0.38
80.97
78.55
94.77
90.29
0
0
0
0
5s
5t
Range*
-3.0 to -1.5 to <25 is poor,
1.2 1.5 >80 is high
Maximum
upto 4
* - For 95% known drugs

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2.3.2 Molecular docking
2.3.2.1 Pose Docking RMSD (Root mean Square deviation):
In order to understand the accuracy of the programs we had carried out docking pose RMSD
calculation (Table 4) of co-crystal proteins, where the crystal compound is re-docked using Glide
(citation) software and checked for atom to atom with the co-crystal pose. The predictive
capability of software is confirmed if the deviation is less. We had done considered all proteins
which had co-crystal organic molecules using XP docking mode with all default settings
provided by the Glide software. We found that most of the proteins gave good pose
reproducibility with crystal interaction retained in all, except 1YK3 as BOD. High flexibility in
case of 1YK3/BOD could be the reason for the deviation. Pose reproducibility of protein pdb:
2PZI with ligand id AXX is shown in Figure 4.
Figure 4: Pose reproducibility of protein pdb: 2PZI with ligand id AXX in thin tube form where
light brown is crystal pose and light blue is docked pose.

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Table 4: RMSD values of co-crystal proteins
SI.No
PDB_ID
Ligand ID
PLP
Pose RMSD
1.43
1HKV
1L1E
1YK3
1ZVW
2AF6
2NSD
1G3U
2PZI
1
2
SAH
1.23
BOD
3.45
3
PRP
1.98
4
BRU
1.8
5
4PI
2.2
6
TMP
0.76
7
AXX
0.79
8
3UC1
3VAE
APO
No ligand
No ligand
9
APO (PEG)
10
APO (No crystal Ligand)
In order to understand the nature of interactions and to explain the plausible mechanism of the
synthesized compounds for in-vitro antitubercular activity, we carried out molecular docking
between all the ligands and the active sites of ten pathophysiological tubercular target enzymes.
The target enzymes were selected from the target identification pipeline for Mycobacterium
tuberculosis, which comprises a total of 451 high-confidence targets [29]. The ten high-
confidence targets with their PDB ids were selected based on their structural similarity with the
synthesized compounds, good X-ray resolution and without structural issues or breaks and are as
follows, Diaminopimelate Decarboxylase [30] (PDB id – 1HKV), Cyclopropane Synthase [31]
(PDB id – 1L1E), Antibiotic Resistance Protein [32] (PDB id – 1YK3), TrpD essential for lung
colonization [33] (PDB id – 1ZVW), Thymidylate Synthase X [34] (PDB id – 2AF6), InhA, the
enoyl acyl carrier protein reductase [35] (PDB id – 2NSD), Thymidylate Kinase [36] (PDB id –
1G3U), Protein Kinase G [37] (PDB id – 2PZI), GyraseTypeIIA Topoisomerase [38] (PDB id –
3UC1), L, D Transpeptidase 2 [39] (PDB id – 3VAE). All the targets were retrieved from Protein
Data Bank (PDB) and the molecular docking studies were performed using Glide (Schrodinger®)
model (Extra Precision XP) of the default parameter settings and the docking scores were
summarized in Table 5. The overall glide docking scores ranges from -10.62 to -2.00 for all the
ligands on all the target enzymes.

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Table 5: Glide docking scores of 2-thioxo-1, 3-thiazolidin-4-one analogs (5a-t)
Glide docking scores in kcal/mol
Ligands
1HKV 1L1E 1YK3 1ZVW 2AF6 2NSD 1G3U 2PZI 3UCI 3VAE
-2.0
-2.0
-1.8
-2.3
-4.1
-2.9
-1.5
-2.0
-2.0
-1.8
-1.4
-3.2
-2.8
-2.6
-3.0
-2.0
-2.3
-1.8
-0.6
-2.7
-7.0
-6.0
-6.3
-8.8
-6.7
-7.5
-5.6
-5.3
-7.2
-6.2
-5.7
-4.4
-6.4
-6.4
-6.4
-5.9
-4.8
-7.1
-8.3
-6.0
-5.3
-6.1
-4.6
-7.9
-5.5
-5.2
-5.7
-5.4
-5.2
-5.4
-5.2
-4.7
-4.9
-5.2
-5.2
-6.6
-4.8
-4.7
-4.3
-4.8
-3.8
-5.2
-5.2
-5.9
-5.3
-4.9
-5.0
-4.3
-4.5
-4.7
-4.8
-3.5
-3.8
-4.6
-4.8
-5.3
-4.3
-3.8
-5.6
-3.8
-4.2
-4.0
-4.4
-8.3
-9.1
-8.7
-7.1
-6.3
-3.8
-5.2
-5.8
-3.4
-6.6
-2.8
-2.5
-3.5
-7.6
-7.3
-6.8
-4.5
-5.3
-3.4
-4.3
-6.9
-5.3
-7.4
-7.1
-6.8
-6.8
-6.4
-6.9
-8.4
-5.7
-5.9
-5.5
-7.5
-6.8
-7.3
-6.9
-5.2
-7.7
-8.9
-5.9
-5.2
-6.9
-6.9
-2.9
-4.7
-2.6
-4.5
-4.6
-5.3
-3.7
-3.3
-3.5
-4.2
-3.2
-3.9
-4.0
-3.6
-3.6
-3.9
-4.3
-4.5
-5.4
-4.2
-4.1
-4.3
-4.4
-5.2
-5.5
-5.0
-4.2
-4.3
-5.8
-4.4
-3.2
-5.4
-3.2
-4.7
-4.1
-6.3
-6.3
-4.5
-5.0
-5.1
5a
5b
5c
5d
5e
5f
-5.4 -10.6
-4.6
-3.4
-4.0
-4.0
-3.7
-3.3
-3.8
-3.7
-3.6
-4.8
-8.8
-8.1
-8.1
-8.9
-8.7
-9.5
-8.4
-7.2
-7.2
-8.7
5g
5h
5i
5j
5k
5l
5m
5n
5o
5p
5q
5r
5s
-3.4 -10.3
-3.8
-3.7
-3.7
-4.4
-4.0
-9.9
-7.9
-8.3
-8.7
-5.8
5t
As per the docking results, all the ligands showed better interactions with the active sites of
target enzymes. On comparing the docking results with in-vitro antitubercular activity results,
top-ranked compounds of in-vitro antitubercular activity shows deviation from docking

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pose/score rankings in all the studied target enzymes. In order to rationalize the correlation
between the in-vitro antitubercular activity and docking results a cross observational analysis
was performed. The top-ranked three compounds (5r, 5k and 5t) of in-vitro antitubercular
activity were cross observed with their docking ranks on all the studied target enzymes for their
deviations in ranks. The results are given in Table 6. Based upon the results, the docking pose
ranking of all the three compounds (5r, 5k and 5t) showed marginal deviation in thymidylate
kinase (PDB id – 1G3U) target enzyme at 5th, 1st and 2nd ranks respectively when compared with
docking pose rankings of other target enzymes that were studied. Additionally top three ranked
in-vitro antitubercular compounds lie within top five positions in thymidylate kinase (PDB id –
1G3U) target enzyme. Thus the study warrants the elimination of false negatives and rationalizes
that the enzyme thymidylate kinase would be the plausible target for the tested compounds for
their in-vitro antitubercular activity.
Table 6: Cross observational analysis of top three ranked compounds with docking ranks
Ranking positions based on
docking scores
Target Enzymes (PDB id)
5ra
5ka
5ta
17
5
19
16
10
10
12
12
1
6
13
18
18
8
Diaminopimelate Decarboxylase (1HKV)
Cyclopropane Synthase (1L1E)
17
16
16
13
5
Antibiotic Resistance Protein (1YK3)
TrpD essential for lung colonization (1ZVW)
Thymidylate Synthase X (2AF6)
20
2
InhA, the enoyl acyl carrier protein reductase (2NSD)
Thymidylate Kinase (1G3U)
20
7
12
18
20
9
Protein Kinase G (2PZI)
10
7
GyraseTypeIIA Topoisomerase (3UC1)
L, D Transpeptidase 2 (3VAE)
18
a - top three ranked compounds (5r-1st, 5k-2nd and 5t-3rd) in in-vitro antitubercular activity.
The docked pose of the synthesized compound 5d at the active site of thymidylate kinase is
shown in Figure 5. The major interactions by the ligands with thymidylate kinase can be
categorized as hydrogen bonding, hydrophobic, electrostatic interactions, π-π-stacking and π-

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cation-stacking, which are critical for stabilizing the inhibitors inside the binding pocket of the
receptor. The important interactions are shown in Table 7. The amino acid Asn100 displays
strong hydrogen bond interaction with the inhibitors. Arg74 and Tyr39 participate in hydrogen
bond interaction in only one inhibitor each, along with Asn100. Other amino acids like Asp163,
Arg95, Lys13 and Gly59 also contribute to the hydrogen bonding in some inhibitors. The
inhibitors had successfully influenced hydrophobic effect with Tyr39, Tyr103, Tyr165, Pro37,
Phe70 and Leu52. Other amino acids like Ala67, Ala35, Ala49, Met66, Val63 and Phe36 also
contribute to the hydrophobic interactions. Electrostatic interactions are predominant in
inhibitors having interactions with amino acids like Arg74, Arg95, Arg160, Glu166, Asp9 and
Asp163. Other weak interactions like π-cation-stacking and π-π-stacking are witnessed in most
of the inhibitors with their aromatic group positioned near Arg95, Tyr103 and Phe70. The three
top ranked compounds with their ligand interactions are shown in Figure 6.
Figure 5: The docked pose of the synthesized compound 5d at the active site of thymidylate
kinase
Table 7: Residue interaction pattern for the synthesized compounds against thymidylate kinase
Important Interactions of Ligands with amino acids of binding site of
thymidylate kinase (1G3U)
Ligands
Hydrogen
Bonding
Hydrophobic
Positive
Ionizable
Negative
Ionizable
Polar
Asn100
Tyr103, Phe70, Pro37,
Tyr39, Leu52, Ala67,
Tyr165
Arg74,
Arg95
Glu166,
Asp9,
Asp163
Ser104,
Asn100,
Ser99,
5a

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Gln172
Asn100, π-
cation-
stacking-
Arg95
Phe36, Phe70, Pro37,
Tyr39, Leu52, Tyr103,
Tyr165, Ala67
Asp9,
Asp163,
Glu166
Arg74,
Arg95
Ser99,
Ser104,
Asn100
5b
5c
-
Phe70, Tyr39, Pro37,
Leu52, Tyr103, Tyr165,
π -π-stacking-Tyr103
Asp9,
Asp163,
Glu166
Arg74,
Lys13,
Arg160,
Arg153,
Arg95
Ser99,
Asn100
5d
5e
5f
Gly159
Tyr103, Pro37, Ala49,
Leu52, Tyr39, Ala161,
Phe70, Tyr165, π -π-
stacking-Phe70
Asp9,
Asp163,
Glu166
Arg95,
Arg160
Ser99,
Asn100
Asn100,
Arg74
Phe36, Tyr96, Pro37,
Tyr39, Leu52, Tyr165,
Phe70, Tyr103, π -π-
stacking-Phe70
Asp9,
Asp163,
Glu166
Arg74,
Arg95,
Arg160
Ser99,
Asn100
Asp163, π-
cation-
stacking-
Arg95
Phe70, Tyr96, Pro37,
Tyr39, Tyr165, Leu52,
Tyr103
Asp9,
Asp163,
Glu166
Arg74,
Arg95,
Arg160
Ser99,
Asn100
Asn100, π-
cation-
stacking-
Arg95
Tyr103, Phe36, Phe70,
Pro37, Tyr39, Tyr165,
Ala67
Asp9,
Asp163,
Glu166
Arg74,
Arg95,
Arg160
Ser99,
Asn100,
Ser104
5g
π-cation-
stacking-
Arg95
Phe70, Phe36, Pro37,
Tyr39, Leu52, Tyr103,
Tyr165
Asp9,
Asp163,
Glu166
Arg74,
Arg95,
Arg160
Ser99,
Asn100
5h
5i
Asp163, π-
cation-
stacking-
Arg95
Pro37, Ala35, Tyr39,
Phe36,Tyr165, Leu52,
Phe70, Ala11
Asp9,
Asp94,
Asp163,
Glu166
Arg14,
Lys13,
Arg153,
Arg95,
Arg160
-

ACCEPTED MANUSCRIPT
-
Phe70, Phe36, Pro37,
Tyr39, Leu52,
Asp9,
Asp163,
Glu166
Arg74,
Arg95,
Arg160
Ser99,
Asn100
5j
Tyr103,Tyr165
Asn100, π-
cation-
stacking-
Arg95
Tyr103, Phe70, Pro37,
Tyr39, Tyr165, Ala67
Asp9,
Asp163,
Glu166
Arg74,
Arg95,
Arg160
Ser99,
Asn100,
Ser104,
Gln172
5k
Asn100, π-
cation-
stacking-
Arg95
Tyr103, Phe70, Pro37,
Tyr39, Tyr165, Ala67
Asp9,
Asp163,
Glu166
Arg74,
Arg95
Ser99,
Asn100,
Ser104
5l
Asn100
Arg95
Tyr103, Phe70, Pro37,
Tyr39, Tyr165, Ala67
Asp9,
Asp163,
Glu166
Arg74,
Arg95
Ser99,
Asn100,
Ser104
5m
5n
Pro37, Phe70, Tyr96,
Ala49, Tyr39, Leu52,
Tyr103, Tyr165
Asp9,
Asp163,
Glu166
Arg74,
Arg95,
Lys13,
Arg160
Ser99,
Asn100,
Hie53
Asn100, π-
cation-
stacking-
Arg95
Phe70, Phe36, Pro37,
Tyr39, Leu52, Tyr103,
Tyr16, Ala67
Asp9,
Asp163,
Glu166
Arg74,
Arg95,
Arg160
Ser99,
Asn100,
Ser104
5o
5p
Lys13
Phe70, Tyr103, Tyr39,
Pro37, Ala35, Ala49,
Leu52, Tyr165
Asp9,
Asp163,
Asp94
Lys13,
Arg95,
Arg14,
Arg160
Ser99,
Hie53
-
Phe70, Tyr96, Pro37,
Phe36, Tyr39, Tyr103,
Tyr165
Asp9,
Asp163,
Glu166
Arg74,
Arg95,
Arg160
Ser99,
Asn100,
Ser104
5q
5r
Asn100,
Tyr39, π-
cation-
stacking-
Arg95
Tyr103, Phe36, Phe70,
Pro37, Tyr39, Ala67,
Tyr165
Asp9,
Asp163,
Glu166
Arg74,
Arg95,
Arg160
Ser99,
Asn100,
Ser104,
Gln172
Asn100, π-
Phe70, Phe36, Pro37,
Asp9,
Arg74,
Ser99,
5s

ACCEPTED MANUSCRIPT
cation-
stacking-
Arg95
Tyr39, Tyr103, Ala67,
Tyr165
Asp163,
Glu166
Arg95,
Arg160
Asn100,
Ser104
Asn100
Phe70, Pro37, Tyr165,
Tyr103, Met66, Val63,
Ala67
-
Arg74,
Arg95,
Arg107
Ser99,
Asn100,
Ser104
5t
Figure 6: A) Ligand interaction diagram of compound 5r. B) Ligand interaction diagram of
compound 5k. C) Ligand interaction diagram of compound 5t. D) Ligand interaction diagram
legend.
2.3.3 Molecular dynamic simulation
The obtained docking results allowed us to know a general and reasonable binding mode of the
ligands and to ascertain the residues involved in the ligand recognition. Nevertheless, we decided
to perform a molecular dynamic simulation of a ligand-receptor complex for further investigation
of binding modes of ligand and to explicate the effects of ligand binding on the conformation of
protein. Hence, the ligand 5k-1G3U (5k-thymidylate kinase) complex (top-ranked docking

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ligand-receptor complex) was selected as a representation for MD (Molecular Dynamic)
simulation. The aim of MD simulation was to get more appropriate ligand-receptor model in a
close state to the natural conditions and to further investigate the binding modes of the ligand, in
the view of the fact that MD simulation can account for even the smaller variances.
The conformational stability of the 5k-1G3U complex in the simulation procedure was assessed
by carrying out a 50ns (nanoseconds) molecular dynamic simulation using Desmond
(Schrodinger®) and analysed with Maestro’s trajectory Visualizer. The trajectories were stable
throughout the MD simulation run. The trajectory stability was checked and was substantiated by
the analysis of protein-ligand RMSD (Root Mean Square Deviation) Figure 7.
Figure 7: Plot showing Protein-Ligand RMSD evolution
Figure 7 reveals that the protein RMSD values (left Y-axis) have some acceptable fluctuations
which did not exceed ~2.5Å throughout the MD simulation run. This indicates that the protein is
stable without any notable conformational changes during the simulation run. In case of ligand
RMSD (right Y-axis) the fluctuations did not exceed ~2.1Å throughout the MD simulation run.
Moreover the observed ligand RMSD values are smaller than RMSD values of the protein,
which indicates the stability of the ligand with respect to the protein and its binding pocket.
At the end of the MD simulation, position and orientation of ligand in the introduced binding site
were changed (Figure 8) and this important observation indicates useful application of MD
simulation after docking of ligands in the binding site. Explorative run of the MD simulation on
the 5k-1G3U complex revealed that except for Asn100, Arg95, Tyr165 and Phe70 the rest of

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residues of the active site determined by docking were changed. The residues such as Tyr103 and
Arg74 are newly positioned in proximity of ligand and participated in the interaction. At the end
of MD simulation a new hydrogen bonding was found to exist between docked molecule and
Arg74. Compound 5k is also stabilized by an extra hydrophobic π-π-stacking interaction with
Tyr103 which does not exist before MD simulation. On the other end, the hydrophobic
interactions (Ala67, Pro37 and Tyr39) and electrostatic interactions (Arg160, Glu66, Asp9 and
Asp163) with the ligand were vanished. Finally the difference in the orientation of ligand 5k in
binding modes after MD simulation was also noticed.
These results revealed that MD simulation obligate ligand to optimize its orientation and distance
to binding site for maximum interaction with receptor. On docking of compound 5k with 1G3U,
the lowest energy confirmation did not show any hydrogen bonding and hydrophobicπ-π-
stacking interactions with amino acid residues Arg74 and Tyr103 respectively due to the absence
of appropriate orientation and distance as was observed at the MD simulation. Ligand interaction
pattern with percentage of contacts (interactions that occur more than 20.0% of the simulation
time) of the ligand 5k against 1G3U & BAR representation of the conserved binding site
residues which influenced compound 5k against 1G3U were shown in Figure 8 & 9.
Figure 8: A) Ligand interaction pattern of the ligand 5k against 1G3U before MD simulation. B)
Ligand interaction pattern with percentage of contacts of the ligand 5k against 1G3U after MD
simulation.

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Figure 9: BAR representation of the conserved binding site residues which influenced
compound 5k against 1G3U.
3. Conclusion
In this work, the synthesis and in-vitro antimycobacterial activity evaluation and chemical
features responsible for inhibitory activity of 2-thixo-1, 3-thiazolidine-4-one derivatives have
been described. Compounds 5k, 5r and 5t exhibited good in-vitro antimycobacterial activity at
0.05, 0.1, 0.2 µg/ml concentrations respectively.
The results of Qikprop analysis gave information about the drug likeness properties of the
compounds and all the compounds were found to be within the recommended values for 95% of
known drugs. The molecular docking results and cross observational analysis suggested that the
protein thymidylate kinase (1G3U) could be the plausible target for the tested compounds. The
study also threw light about the various interaction patterns like hydrogen bonding, hydrophobic
and electrostatic interactions between the ligands and the amino acid residues in the binding site.
The docking results of ligands 5a-t with1G3U protein gave information about the general
binding mode and orientation of the ligands with respect to the receptor. In order to further
investigate the binding modes of ligands on the confirmation of protein, 5k-1G3U ligand-protein
complex was selected as representative for the MD simulation study. The MD study suggested

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the stability of both protein and ligand throughout the simulation time (50ns) as observed by the
analysis of RMSD values of the both ligand and protein.
At the end of the MD simulation, the position and orientation of ligands introduced into the
binding site were changed and this important observation indicates the useful application of MD
simulation. Explorative run of MD simulation on the 5k-1G3U complex revealed changes in
some of the active site residues which were determined by docking. A new hydrogen bonding
and an extra hydrophobic π-π stacking interaction was also noticed which did not exist before
MD simulation. These results strongly suggests that MD simulation obligate the ligand to
optimize its orientation and distance to the binding site for maximum interaction with receptor
which was not observed in the docked complex before the MD simulation.
The tested compounds constitute an interesting lead for the evaluation of antimycobacterial
activity. The optimized binding sites after MD simulation run can serve as a standard binding
model.
4. Experimental
Melting points were taken using open capillary tubes and are uncorrected. 1H, 13C NMR spectra
were recorded on a Bruker 500 MHZ instrument in DMSO-d6. Chemical shifts are given in parts
per million. IR spectra were recorded on a Perkin-Elmer Spectrometer using KBr pellets. MASS
Spectra were recorded on a Perkin Elmer Clarus 600 (EI) instrument. Elemental analyses were
performed on a Perkin Elmer 2400 Series II Elemental CHNS analyzer. X-ray crystallographic
study for one of the synthetic derivative was performed on a Bruker Kappa APEXII instrument.
4.1. Synthesis of 2-thioxo-1, 3-thiazolidin-4-ones 5
4.1.1. General procedure
A mixture of malononitrile (1.1 mmol) and substituted aldehydes (1.0 mmol) in ethanol in the
presence of triethylamine was stirred at room temperature. After the complete disappearance of
both starting materials (monitored by TLC), 2-thioxo-1, 3-thiazolidin-4-one(1.0 mmol) was
added and the stirring was continued for 2 -2½hrs. The completion of the reaction was monitored
by TLC. The solid formed in the reaction mixture was filtered, dried and recrystallized in ethanol
to obtain the pure product in good yields.

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4.1.1.1. (Z)-5-bеnzylidеnе-2-thioxothiazolidin-4-onе (5a). Solid; (Yield 92%); mp 200-202°C; IR
(KBr, νmax) cm-1: 1488.94 (Ar C-C), 1674.09 (C=O), 1704.95 (C=S), 2923.87 (=C-H), 3062.73
(Ar C-H), 3463.90 (N-H); 1H NMR (DMSO-D6) δ: 7.49-7.56 (m, 3H, Ar-H), 7.60 (d, 2H, Ar-H),
7.65 (s, 1H, =CH-), 13.83 (s, 1H, N-H); 13C NMR (DMSO-D6) δ: 116.17, 126.00, 129.91,
130.93, 131.19, 132.10, 133.44, 143.11, 169.81, 196.16; MS (EI): m/z221.05 [M+]; Anal. Calcd.
for C10H7NOS2: C, 54.27; H, 3.19; N, 6.33; Found: C, 54.18; H, 3.18; N, 6.34.
4.1.1.2. (Z)-5-(4-chlorobenzylidene)-2-thioxothiazolidin-4-one (5b). Solid; (Yield 86%); mp210-
212 °C; IR (KBr, νmax) cm-1: 825.47 (C-Cl), 1442.65 (Ar C-C), 1596.94 (C=O), 1704.95 (C=S),
1
2923.87 (=C-H), 3085.88 (Ar C-H), 3479.33 (N-H); H NMR (DMSO-D6) δ: 7.59-7.63 (m, 4H,
Ar-H), 7.64 (s, 1H, =CH-), 13.88 (s, 1H, N-H); 13C NMR (DMSO-D6) δ: 115.14, 126.78, 129.98,
130.65, 132.36, 132.53, 135.82, 140.43, 169.78, 195.88; MS (EI): m/z255.05 [M+]; Anal. Calcd.
for C10H6ClNOS2: C, 46.96; H, 2.36; N, 5.48; Found: C, 46.85; H, 2.35; N, 5.49.
4.1.1.3. (Z)-5-(4-(dimеthylamino)bеnzylidеnе)-2-thioxothiazolidin-4-onе (5c). Solid; (Yield
95%); mp272-274°C; IR (KBr, νmax) cm-1: 1249.78 (amine C-N), 1442.65 (Ar C-C), 1612.37
(C=O), 1681.80 (C=S), 2854.44 (alkane C-H), 2916.16 (=C-H), 3039.59 (Ar C-H), 3457.06 (N-
H); 1H NMR (DMSO-D6) δ: 3.03 (s, 3H, N-CH3), 3.10 (s, 3H, N-CH3), 6.81-6.85 (q, 2H, Ar-H),
7.41 (d, 1H, Ar-H), 7.51 (s, 1H, =CH-), 7.83 (d, 1H, Ar-H), 13.54 (s, 1H, N-H); 13C NMR
(DMSO-D6) δ: 68.19, 69.15, 115.99, 117.83, 119.23, 120.26, 133.36, 133.73, 134.06, 152.25,
169.88, 195.47; MS (EI): m/z264.16 [M+]; Anal. Calcd. for C12H12N2OS2: C, 54.52; H, 4.58; N,
10.60; Found: C, 54.44; H, 4.59; N, 10.58.
4.1.1.4. (Z)-5-(4-(bеnzyloxy)bеnzylidеnе)-2-thioxothiazolidin-4-onе (5d). Solid; (Yield 89%);
mp230-232 °C; IR (KBr, νmax) cm-1: 1249.78 (ether C-O), 1442.65 (Ar C-C), 1589.23 (C=O),
1
1689.52 (C=S), 2854.44 (alkane C-H), 2923.87 (=C-H), 3055.02 (Ar C-H), 3456.18 (N-H); H
NMR (DMSO-D6) δ: 5.22 (d, 2H, CH2), 7.19 (d, 2H, Ar-H), 7.33-7.37 (q, 1H, Ar-H), 7.41 (t, 2H,
Ar-H), 7.47 (d, 2H, Ar-H), 7.57 (d, 2H, Ar-H), 7.61 (s, 1H, =CH-), 13.74 (s, 1H, NH); 13C NMR
(DMSO-D6) δ: 70.04, 115.26, 126.16, 128.28, 128.43, 128.51, 128.66, 128.97, 129.01, 132.27,
133.14, 133.82, 136.51, 136.91, 141.24, 160.90, 163.89, 169.92, 195.98; MS (EI): m/z327.17
[M+]; Anal. Calcd. for C17H13NO2S2: C, 62.36; H, 4.00; N, 4.28; Found: C, 62.20; H, 4.01; N,
4.27.

ACCEPTED MANUSCRIPT
4.1.1.5. (Z)-5-(4-hydroxy-3-mеthoxybеnzylidеnе)-2-thioxothiazolidin-4-onе (5e). Solid; (Yield
90%); mp220-222°C; IR (KBr, νmax) cm-1: 1280.64 (alcoholic C-O), 1442.65 (Ar C-C), 1589.23
(C=O), 1712.66 (C=S), 2854.44 (alkane C-H), 2923.87 (=C-H), 3008.73 (Ar C-H), 3340.46 (N-
1
H), 3610.48 (O-H); H NMR (DMSO-D6) δ: 3.82 (s, 3H, O-CH3), 6.93 (d, 1H, Ar-H), 7.06 (d,
1H, Ar-H), 7.11 (s, 1H, Ar-H), 7.54 (s, 1H, =CH-), 10.13 (s, 1H, Ar-OH ), 13.62 (s, 1H, N-H);
13C NMR (DMSO-D6) δ: 56.11, 114.73, 121.06, 124.86, 125.55, 133.25, 134.90, 137.44, 148.59,
169.88, 195.90; MS (EI): m/z267.15 [M+]; Anal. Calcd. for C11H9NO3S2: C, 49.42; H, 3.39; N,
5.24; Found: C, 49.29; H, 3.38; N, 5.23.
4.1.1.6. (Z)-5-(2-hydroxybеnzylidеnе)-2-thioxothiazolidin-4-onе (5f). Solid; (Yield 88%);
mp158-160°C; IR (KBr, νmax) cm-1: 1280.64 (alcoholic C-O), 1442.65 (Ar C-C), 1653.52 (C=O),
1
1743.52 (C=S), 2862.15 (=C-H), 3116.74 (Ar C-H), 3340.46 (N-H), 3633.62 (O-H); H NMR
(DMSO-D6) δ: 5.03 (s, 1H, O-H), 7.02-7.04 (q, 1H, Ar-H), 7.17-7.22 (m, 2H, Ar-H), 7.30-7.34
(m, 1H, Ar-H), 7.44 (d, 1H, =CH-), 13.32 (s, 1H, N-H); 13C NMR (DMSO-D6) δ: 116.65,
120.22, 120.81, 125.54, 128.92, 129.77, 130.89, 149.68, 176.34, 203.70; MS (EI): m/z237.26
[M+]; Anal. Calcd. for C10H7NO2S2: C, 50.61; H, 2.97; N, 5.90; Found: C, 50.77; H, 2.96; N,
5.91.
4.1.1.7. (Z)-5-(4-nitrobеnzylidеnе)-2-thioxothiazolidin-4-onе (5g). Solid; (Yield 85%); mp240-
242 °C; IR (KBr, νmax) cm-1: 1342.36 (nitro N-O), 1411.79 (Ar C-C), 1604.66 (C=O), 1720.38
(C=S), 2923.87 (=C-H), 3016.45 (Ar C-H), 3440.76 (N-H); 1H NMR (DMSO-D6) δ: 7.73 (s, 1H,
13
=CH-), 7.86 (t, 2H, Ar-H), 8.32-8.34 (m, 2H, Ar-H), 14.00 (s, 1H, NH); C NMR (DMSO-D6)
δ: 116.96, 124.79, 129.03, 130.44, 131.76, 135.33, 139.67, 147.99, 169.70, 195.73; MS (EI):
m/z266.11 [M+]; Anal. Calcd. for C10H6N2O3S2: C, 45.10; H, 2.27; N, 10.52; Found: C, 45.31;
H, 2.26; N, 10.55.
4.1.1.8. (Z)-5-(2-bromobеnzylidеnе)-2-thioxothiazolidin-4-onе(5h). Solid; (Yield 92%); mp236-
238 °C; IR (KBr, νmax) cm-1: 604.87 (C-Br), 1450.36 (Ar C-C), 1604.66 (C=O), 1728.09 (C=S),
1
2923.87 (=C-H), 3109.02 (Ar C-H), 3440.76 (N-H); H NMR (DMSO-D6) δ: 7.40-7.45 (m, 1H,
Ar-H), 7.51-7.59 (m, 2H, Ar-H), 7.72 (s, 1H, =C-H), 7.81 (t, 1H, Ar-H), 13.96 (s, 1H, N-H); 13C
NMR (DMSO-D6) δ: 117.18, 128.86, 129.11, 129.40, 132.21, 132.54, 133.71, 142.54, 169.05,
195.58; MS (EI): m/z300.01 [M+]; Anal. Calcd. for C10H6BrNOS2: C, 40.01; H, 2.01; N, 4.67;
Found: C, 40.19; H, 2.00; N, 4.68.