Design, synthesis, antibacterial evaluation, and computational studies of hybrid oxothiazolidin–1,2,4-triazole scaffolds

Article abstract of DOI:10.1002/ardp.202000473

Bacterial infections are a serious threat to human health due to the development of resistance against the presently used antibiotics. The problem of growing and widespread antibiotic resistance is only getting worse with the shortage of new classes of antibiotics, creating a substantial unmet medical need in the treatment of serious bacterial infections. Therefore, in the present work, we report 18 novel hybrid thiazolidine–1,2,4-triazole derivatives as DNA gyrase inhibitors. The derivatives were synthesized by multistep organic synthesis and characterized by spectroscopic methods (¹H and ¹³C nuclear magnetic resonance and mass spectroscopy). The derivatives were tested for DNA gyrase inhibition, and the result emphasized that the synthesized derivatives have a tendency to inhibit the function of DNA gyrase. Furthermore, the compounds were also tested for antibacterial activity against three Gram-positive (Bacillus subtilis [NCIM 2063], Bacillus cereus [NCIM 2156], Staphylococcus aureus [NCIM 2079]) and two Gram-negative (Escherichia coli [NCIM 2065], Proteus vulgaris [NCIM 2027]) bacteria. The derivatives showed a significant-to-moderate antibacterial activity with noticeable antibiofilm efficacy. Quantitative structure–activity relationship (QSAR), ADME (absorption, distribution, metabolism, elimination) calculation, molecular docking, radial distribution function, and 2D fingerprinting were also performed to elucidate fundamental structural fragments essential for their bioactivity. These studies suggest that the derivatives 10b and 10n have lead antibacterial properties with significant DNA gyrase inhibitory efficacy, and they can serve as a starting scaffold for the further development of new broad-spectrum antibacterial agents.

Full text of DOI:10.1002/ardp.202000473

Received: 15 December 2020
DOI: 10.1002/ardp.202000473
Revised: 1 February 2021
Accepted: 4 February 2021
|
|
F U L L P A P E R
Design, synthesis, antibacterial evaluation, and
computationalstudiesofhybridoxothiazolidin–1,2,4‐triazole
scaffolds
1
1
2,3
Prateek Pathak
| Jurica Novak
| Vladimir Potemkin
| Parjanya K. Shukla
|
1
1
3
Maria Grishina
| Amita Verma
1
Laboratory of Computational Modeling of
Drugs, Higher Medical and Biological School,
South Ural State University, Chelyabinsk,
Russia
Abstract
Bacterial infections are a serious threat to human health due to the development of
resistance against the presently used antibiotics. The problem of growing and
widespread antibiotic resistance is only getting worse with the shortage of new
classes of antibiotics, creating a substantial unmet medical need in the treatment of
serious bacterial infections. Therefore, in the present work, we report 18 novel
hybrid thiazolidine–1,2,4‐triazole derivatives as DNA gyrase inhibitors. The deri-
vatives were synthesized by multistep organic synthesis and characterized by
2
Krishnarpit Institute of Pharmacy, Dr. A. P. J.
Abdul Kalam Technical University, Prayagraj,
Uttar Pradesh, India
3
Bioorganic and Medicinal Chemistry
Research Laboratory, Department of
Pharmaceutical Sciences, Sam Higginbottom
University of Agriculture, Technology &
Sciences, Prayagraj, Uttar Pradesh, India
1
13
spectroscopic methods ( H and C nuclear magnetic resonance and mass spec-
troscopy). The derivatives were tested for DNA gyrase inhibition, and the result
emphasized that the synthesized derivatives have a tendency to inhibit the function
of DNA gyrase. Furthermore, the compounds were also tested for antibacterial
activity against three Gram‐positive (Bacillus subtilis [NCIM 2063], Bacillus cereus
Correspondence
Prateek Pathak, Laboratory of Computational
Modeling of Drugs, Higher Medical and
Biological School, South Ural State University,
Chelyabinsk, Russia 454080.
Email: patkhakp@susu.ru
and prateekpharm05@gmail.com
[NCIM 2156], Staphylococcus aureus [NCIM 2079]) and two Gram‐negative (Escher-
Amita Verma, Bioorganic and Medicinal
Chemistry Research Laboratory,
ichia coli [NCIM 2065], Proteus vulgaris [NCIM 2027]) bacteria. The derivatives
showed a significant‐to‐moderate antibacterial activity with noticeable antibiofilm
efficacy. Quantitative structure–activity relationship (QSAR), ADME (absorption,
distribution, metabolism, elimination) calculation, molecular docking, radial
distribution function, and 2D fingerprinting were also performed to elucidate fun-
damental structural fragments essential for their bioactivity. These studies suggest
that the derivatives 10b and 10n have lead antibacterial properties with significant
DNA gyrase inhibitory efficacy, and they can serve as a starting scaffold for the
further development of new broad‐spectrum antibacterial agents.
Department of Pharmaceutical Sciences,
Sam Higginbottom University of Agriculture,
Technology & Sciences, Prayagraj,
Uttar Pradesh 211007, India.
Email: amitaverma.dr@gmail.com and
amita.verma@shiats.edu.in
Funding information
Government of the Russian Federation,
Grant/Award Numbers: Act 211, contract
02.A03.21.0011; Ministry of Science and
Higher Education of Russia,
K E Y W O R D S
Grant/Award Number: FENU‐2020‐0019
antibacterial activity, DNA gyrase inhibitors, docking study, hybrid 1,2,4‐triazole, QSAR study
Prateek Pathak and Parjanya K. Shukla contributed equally to this study.
Arch Pharm. 2021;e2000473.
wileyonlinelibrary.com/journal/ardp © 2021 Deutsche Pharmazeutische Gesellschaft
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https://doi.org/10.1002/ardp.202000473

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PATHAK ET AL.
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1
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INTRODUCTION
different positions within the ring can be changed according to the
desired derivatives designing, but the maximum antimicrobial prop-
erties are exerted by the groups attached to the nitrogen atom at the
Bacterial infections are a persistent and increasing threat to human
health due to the development of resistance against presently used
[
30]
first position.
Therefore, during the designing of novel 1,2,4‐
[
1–3]
antibiotics.
The problem of growing and widespread antibiotic
triazole‐based compounds, the position of the nitrogen atom should
be fixed according to its suggested structure–activity relationship
(SAR). 4‐Oxo‐thiazolidines are the derivatives of thiazolidinone with
a carbonyl group at position 4 formed by the attack of sulfur nu-
cleophile on imine carbon, followed by intramolecular cyclization
resistance is only getting worse with the shortage of new classes of
antibiotics, creating a substantial unmet medical need in the treat-
[
4,5]
ment of serious bacterial infections.
In a survey, worldwide, ap-
proximately half a million people have been infected by multidrug‐
resistant Mycobacterium tuberculosis, Neisseria gonorrhoeae, or
[
31]
with the elimination of water.
The nucleus of 4‐oxo‐thiazolidine
[
3,6,7]
Pseudomonas aeruginosa infections.
Therefore, the need for the
derivatives has a distinctive place in medicinal chemistry due to
its wide range of diverse pharmacological activities such as
development of effective antibacterials has become essential to
control the spread of superbug microorganisms. The designing of
novel antibacterial agents with a diverse mechanism of action could
[
32,33]
[34]
[35]
antimicrobial,
antidiabetic,
[37]
anti‐HIV,
enzyme murB
[
36]
[32]
inhibition,
antitubercular,
and anticancer.
[
8]
be a possible approach to overcome this resistance. DNA topoi-
In this study, we considered the abovementioned phenomena of
both heterocyclic skeletons and decided to synthesize 1,2,4‐triazole
derivatives via the hybridization with thiazolidin skeleton as poten-
tial antibacterial compounds. Moreover, the hybrid 1,2,4‐triazole‐
conjugated 4‐oxo‐thiazolidins were evaluated for their antibacterial
efficacy against five bacterial strains, followed by ADMET (absorp-
tion, distribution, metabolism, elimination) properties calculations,
molecular docking, and quantitative structure–activity relationship
(QSAR) studies.
somerase enzymes participate in interconversion, relaxation, and
[9,10]
supercoiling of DNA fragments.
They also act remarkably in
various physiological functions such as replication, transcription re-
[
11,12]
combination repair, and chromosome decondensation.
Struc-
turally, DNA gyrase is a type IIA topoisomerase, belonging to the
family of heat‐shock protein 90 (Hsp 90), histidine kinase, MutL
(
GHKL), protein kinases, and the DNA mismatch repair protein MutL
[
12,13]
(
mismatch from replication recognized by MutL).
It consists of
two subunits, that is, GyrA and GyrB. DNA gyrase functions involve
the coupling of GyrB subunit to supercoiling of DNA through ATP
hydrolysis and maintain the DNA topology during the replication
2
|
RESULTS AND DISCUSSION
Chemistry
[
14]
process.
Generally, DNA gyrase employs inhibition of bacterial
growth via two main mechanisms: stabilization of the covalent
2.1
|
enzyme–DNA complex (as ciprofloxacin) or blocking the ATP binding
[
15]
site of DNA gyrase B (as novobiocin).
It is considered as one of the
We have synthesized a series of derivatives by conjugating 1,2,4‐
triazole and the thiazolidine scaffolds to probe the SAR of hybrid
1,2,4‐triazole–thiazolidins. The complete synthetic procedure is
composed of the traditional seven steps of organic synthesis
(Schemes 1 and 2). The first step (reaction i) includes Fischer es-
terification, where substituted aromatic carboxylic acids 1a–c were
refluxed with methanol in the presence of sulfuric acid to afford the
intermediate aromatic acid esters (2a–c). Furthermore, in the next
step (reaction ii), the intermediates (2a–c) participated in the for-
mation of acid hydrazides (3a–c) through the attachment of the
hydrazide group via a classical addition reaction. The third synthetic
protocol (reaction iii) corresponds to the formation of substituted
dithiocarbazinic acid salts (4a–c) through the addition of carbon
crucial enzymes across the entire bacterial species and its inhibition
results in disruption of DNA synthesis, followed by cell death. Thus,
DNA gyrase inhibition is one of the most validating approaches in
[
16]
antibacterial chemotherapy.
Nowadays, the concept of hybrid
molecules is very popular, in which two or more structural domains
are conjugated and novel compounds possess different or dual
[
5,17]
pharmacological activity.
The drugs developed by this approach
have the ability to reduce toxicity, overcome drug resistance, and
[18,19]
improve optimal pharmacokinetic profiles.
Till date, several
compounds synthesized by the hybrid approach are under clinical
studies for their potential against various health‐related issues
such as abdominal infections, urinary tract infections, and
[
5,17,20]
pneumonia.
Heterocyclic skeletons are the main fragments of
2
disulfide (CS )/KOH with 3a–c and dithiocarbazinic acid salts. In the
many marketed drugs, and they also play an important role in anti-
next reaction step (reaction iv), 1,2,4‐triazole scaffolds (5a–c) were
[
5]
[26]
bacterial activity, as found in contemporary drug discovery. Com-
pounds containing 1,2,4‐triazole scaffold can impact lipophilicity,
polarity, hydrogen bonding capacity, pharmacological, pharmacoki-
synthesized under reflux using hydrazine hydrate.
Furthermore, in
the reaction v, 2‐chloro‐N‐(5‐mercapto‐3‐substituted phenyl‐1,5‐
dihydro‐4H‐1,2,4‐triazol‐4‐yl) (6a–c) were synthesized by the elec-
trophilic substitution of chlorine atom from the chloroacetyl chloride
in the presence of sodium acetate and glacial acetic acid. In reaction
vi, the addition of hydrazine hydrate with N‐(5‐aryl‐1,2,4‐triazole‐2‐
yl)‐2‐chloroacetamide (6a–c) in the presence of methanol offered the
synthesis of 2‐hydrazinyl‐N‐(5‐mercapto‐3‐substituted phenyl‐1,5‐
dihydro‐4H‐1,2,4‐triazol‐4‐yl)acetamides (7a–c). In the final step
[
5,21]
netic, toxicological, and physicochemical properties.
Triazole‐derived compounds customarily represent vital therapeutic
classes that parade broad spectrum of pharmacological
actions such as anticancer,
1,2,4‐
a
[22,23]
[24]
[25]
antiviral,
antitubercular,
[
26]
[27,28]
[29]
anti‐inflammatory,
antifungal,
and antibacterial
activities.
Studies show that substituents on the triazole nucleus at the

PATHAK ET AL.
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SCHEME 1 The reaction scheme for the synthesis of the 1,2,4‐triazol precursors. Reagents and conditions: (i) carboxylic acid, methanol,
conc. H SO , reflux for 8–10 h, (ii) hydrazine hydrate, methanol, reflux for 3 h, (iii) KOH, carbon disulfide, stirring for 16 h at room temperature,
iv) hydrazine hydrate, H O, stirring, and reflux for 3 h, (v) glacial acetic acid, saturated solution of sodium acetate, chloroacetyl chloride, stirring
2
4
(
2
for 3 h, (vi) hydrazine hydrate, ethanol, stirring for 20–30 min
SCHEME 2 Scheme for the synthesis of the final hybrid oxo‐thiazolidin–1,2,4‐triazole compounds. Reagents and conditions: (vii)
substituted benzaldehydes, substituted thioglycolic acid, ZnCl2, ethanol, reflux for 6–7 h
1
3
(
reaction vii), the heterocyclic skeletons 2‐hydrazinyl‐N‐(5‐mercapto‐
‐substituted phenyl‐1,5‐dihydro‐4H‐1,2,4‐triazol‐4‐yl)acetamides
7a–c) were condensed with benzaldehydes (8a–c) and thioglycolic
acid derivatives (9a–c) under reflux in the presence of ZnCl . Finally,
The C NMR spectrum of all the derivatives showed the presence
of six carbon atoms of the aromatic ring in the region of
115.1–167.2 ppm, five pyridine carbon peaks in the range of
3
(
2
2
121.3–149.6 ppm, and one carbon of CH at 66.6 ppm. Elemental
the aimed derivatives 2‐[2‐(substituted‐phenyl)‐5‐substituted‐4‐oxo‐
thiazolidin‐3‐ylamino]‐N‐[5‐mercapto‐3‐(substituted‐phenyl)‐1,5‐
dihydro‐1,2,4‐triazol‐4‐yl]acetamides (10a–r) were obtained and the
analysis confirmed that the presence of carbon, hydrogen, and nitrogen
in all derivatives is in accordance with expected values.
1
structural conformations were performed by H nuclear magnetic
1
3
1
resonance (NMR),
showed a singlet at δ 1.52 and δ 3.943 ppm, which are attributed to
the presence of SH and CH protons. CH peaks of the thiazolidine
C NMR, and elemental analysis. H NMR
2.2 Antibacterial activity
|
2
The entire series of derivatives were screened for their minimum
inhibitory concentration (MIC, μg/ml) against a panel of three Gram‐
positive and two Gram‐negative bacterial strains (details are pro-
vided in Section 4) for determination of antibacterial efficacy, and
results are depicted in Table 1. The present study included
and triazole appeared at 6.296 and 6.426 ppm. CH groups of aro-
matic rings appeared as doublets between δ 7.16–7.12 and δ
8
.12–8.09 ppm. Two different characteristic singlets in the range of δ
.87–9.13 ppm confirmed the presence of various NH groups.
8

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TABLE 1 Antibacterial and antibiofilm activity of the derivatives
Minimum inhibitory concentration (μg/ml)
Antibiofilm activity (μg/ml)
Staphylococcus aureus
Compound
Bacillus subtilis
Bacillus cereus
Staphylococcus aureus
Escherichia coli
12.5
25
Proteus vulgaris
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0a
0b
0c
0d
0e
0f
25
>50
25
25
50
6.25
12.5
12.5
6.25
6.25
12.5
6.25
12.5
25
12.5
25
3.125
12.5
12.5
6.25
6.25
6.25
6.25
12.5
25
6.25
25
25
ND
75
50
25
25
75
6.25
12.5
6.25
25
6.25
12.5
25
25
100
50
12.5
25
0g
0h
0i
100
75
50
12.5
50
25
25
100
100
50
0j
>50
6.25
25
50
0k
0l
12.5
>50
12.5
ND
12.50
>50
>50
12.5
25
12.5
12.5
25
100
75
0m
0n
0o
0p
0q
0r
12.5
6.25
6.25
25
6.25
3.125
6.25
25
12.5
3.125
6.25
12.5
25
12.5
25
12.5
25
12.5
12.5
ND
50
25
25
12.5
6.25
3.125
>50
25
100
50
12.5
6.25
12.50
3.125
25
Ciprofloxacin
6.25
3.125
25
Abbreviation: ND, not determined.
ciprofloxacin as a reference antibacterial agent for comparing the
effectiveness of synthesized derivatives. On the basis of SAR
compound 10a. However, the replacement of nitro group with chloro
(in compound 10c) slightly lowers the antibacterial potency against
all tested bacterial species. Compound 10d was derived through the
slight alteration (addition of methyl group at R″) in the thiazolidine
skeleton of 10a, and it shows a slight improvement in the activity
against Bacillus subtilis (MIC = 12.5 μg/ml) and Staphylococcus aureus
(
Figure 1), it is important to indicate that the presence of ni-
trobenzene substituent (at the position R) has a noticeable effect on
the activity profile, as seen in the case of compound 10b. Its activity
has been increased as compared with its pyridine counterpart,
FIGURE 1 General structure of the hybrid
derivatives

PATHAK ET AL.
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showed a radical drop in activity against an entire set of tested mi-
croorganisms. The overall results depicted that the entire set of
synthesized hybrid derivatives exhibited mild‐to‐moderate activity,
whereas derivatives 10b and 10n showed potent inhibition against
tested bacterial strains. On the basis of the antibacterial evaluation
outcomes, SAR studies suggested that the nature of substituents has
a strong impact on the antibacterial activity. It has been demon-
strated that the introduction of strong electron‐withdrawing groups
makes derivatives more active against tested bacterial strains. Thus,
it could be understood that these perturbations caused by electron‐
withdrawing groups (such as halogen) are very useful to modulate
the steric effect on the phenyl ring of the drug and facilitate the
[
38]
molecules to enter the bacterial cell wall.
FIGURE 2 Antibiofilm activity of the hybrid derivatives.
CPF, ciprofloxacin
2.3 Antibiofilm activity
|
The hybrid derivatives exhibiting antibacterial properties were also
screened for bacterial biofilm inhibition against S. aureus. The result
is displayed in Table 1 and Figure 2.
(
MIC = 12.5 μg/ml), but lowers the activity against Escherichia coli
MIC = 25 μg/ml). Hydrogen to methyl group mutation at the position
(
R″ does not influence the activity against Bacillus cereus (MIC = 50
μg/ml) and Proteus vulgaris (MIC = 25 μg/ml) as compared with 10a,
and it is found as slightly more active compared with the standard
and 10c. Compound 10e was constructed through the substitution of
hydrogen by a methyl group (at R″) on the thiazolidine ring of 10b,
and it illustrates a higher potency against B. cereus (MIC = 6.25 μg/ml)
and E. coli (MIC = 6.25 μg/ml). A significant upsurge in antibacterial
activity against tested microorganisms was reported by 10f having
electron‐withdrawing groups at both the positions (at R and Rʹ). The
addition of chlorobenzene to the 1,2,4‐triazole skeleton (at Rʹ) and
substitutions by different fragments introduced to the thiazolidine
ring at position R resulted in the derivatives 10g–i.
The main result is that the derivatives inhibit the biofilm for-
mation with IC50 values ranging between 25 and 100 μg/ml with
mild‐to‐moderate potential. Compounds 10n and 10b were found to
be the most effective (12.5 and 25 μg/ml, respectively) among the
entire set of tested derivatives, whereas derivative 10n has higher
potential as equated with ciprofloxacin.
2.4
|
Inhibition of DNA gyrase supercoiling
activity
In this section, we present the determination of the inhibitory
potential of the newly synthesized hybrid derivatives against DNA
gyrase enzyme. The results are given in terms of IC50, compared
with the ciprofloxacin as standard (Figure 3). The hybrid
The assay showed that the derivatives 10g–i have moderate
antibacterial potency (ranging from 6.25 to 25 μg/ml) against the
3
tested microorganism. The introduction of CH group on the thia-
zolidine ring of compounds 10g–i resulted in derivatives 10j–l and is
accompanied by a slight reduction in the antibacterial potency. The
derivatives 10m–o contain common fluorobenzene ring on thiazoli-
dine fragment (at position Rʹ). The different substitution occurs on
1
,2,4‐traizole scaffolds (pyridine, nitrobenzene, chlorobenzene at
position R, respectively). The antibacterial evaluation showed
that compound 10m has moderate inhibition potency. Compound
1
0n was found equipotent as standard, ciprofloxacin, against
B. subtilis, B. subtilis, and S. aureus, and less active against P. vulgaris
and E. coli. Hybrid compound 10o displayed equipotent efficacy as a
standard against B. subtilis and moderate activity against the rest of
the tested bacterial strains. Addition of methyl group on the thia-
zolidine fragment of the derivative 10m constructed 10p, a molecule
that displayed a notable drop in antibacterial potency. Repeatedly, it
was found that the addition of methyl side chain on thiazolidine ring
leads to lowering the antibacterial efficacy. Compound 7q was syn-
thesized through the introduction of nonhalogenated electron‐
withdrawing group on 1,2,4‐triazole (addition of nitrobenzene), and it
FIGURE 3 Staphylococcus aureus DNA gyrase inhibitory activity
of the hybrid derivatives. CPF, ciprofloxacin

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thiazolidine–triazole derivatives have moderate‐to‐significant in-
hibitory potential, with derivatives 10b, 10n, 10o, and 10r being the
maximum effective (with IC50 values of 26.36 ± 0.33, 25.06 ± 0.4,
Radial distribution function (RDF) weighted by the number of va-
lence shell electrons for all synthesized molecules have two well‐
resolved maxima at 1.4 and 2.5 Å (Figure 5, left). This corresponds to
the approximate carbon–carbon bond length (1.4 Å) and the distance
between two carbon atoms sharing the same neighbor (2.5 Å). The
highest standard deviation of the RDF is at the distance of 8.6 Å,
reaching 63% of the mean RDF value at that distance (Figure 5, right).
This fact reflects a different orientation of the same (or similar)
groups in space. For example, compounds 10b and 10n differ only by
the type of halogen atom on para position of benzene ring on thia-
zolidine skeleton. By overlaying the nitrobenzene moieties of the two
molecules (Figure S1), one can see that plains defined by ni-
trobenzene and fluorobenzene rings in 10n are almost parallel, but
are directed in opposite sides as compared with thiazolidin–1,2,4‐
triazole backbone. In the case of 10b, both phenyl groups are from
the same side of thiazolidin–1,2,4‐triazole backbone. The changes in
the geometry are captured by the RDF, as can be seen from Figure 5,
where RDF for 10b and 10n is highlighted. The intrinsic property of
the RDF is that it unambiguously describes the arrangement of the
atoms in 3D space, plus it is invariant against the translation and
rotation of a molecule. Therefore, it is a perfect candidate as a
structure‐related descriptor in the investigation of the relationship
between structure and relevant properties in drug design. A three‐
parameter (plus intercept) regression model correlating the RDF
descriptors and IC50 values is developed:
2
5.09 ± 0.5, and 24.84 ± 0.71 μg/ml, respectively).
Furthermore, a slight decline in inhibition was reported for
derivatives 10a (IC50 = 27.03 ± 0.24 μg/ml), 10f (IC50 = 29.25 ± 0.15
μg/ml), 10k (IC50 = 29.42 ± 0.22 μg/ml), 10m (IC50 = 28.15 ± 0.25
μg/ml), 10p (IC50 = 28.28 ± 0.5 μg/ml), and 10q (IC50 = 27.03 ±
0.24 μg/ml). For derivatives 10c, 10d, 10e, 10g, 10h, 10i, 10j and 10l, IC50
decreases and displayed above 30 μg/ml. The standard drug ciprofloxacin
that was used for the comparison of efficacy exhibited IC50 at
2
6.15 ± 0.25 30 μg/ml. Other researchers also illustrated the DNA gyrase
[39]
inhibition potency of ciproflaxacin at similar concentration. This study
suggests that the introduction of electron‐withdrawing group increases
the DNA gyrase inhibitory potential, which is found to be in accordance
with the in vitro antibacterial activity assay. On the basis of the similarity
of antibacterial activity and DNA gyrase inhibition results, it can be
concluded that the synthesized derivatives may act as DNA gyrase in-
hibitors and produce their antibacterial activity typically through its DNA
gyrase inhibition mechanism.
2.5
|
Three‐dimensional (3D) QSAR studies
3
D QSAR studies are important methods in modern novel drug de-
sign and their results can guide the synthesis of future compounds
with desired properties. In the current study, one such method was
used to elucidate key derivatives features, important for their anti-
bacterial activity. The distribution of pharmacophore (red), anti-
pharmacophore (blue), and inactive parts (gray) of the most
promising compounds, 10b and 10n, are displayed in Figure 4. The
gray part of the ring does not participate in pharmacological activity,
but is important for configuring the chemical structure. The assay
illustrates that hydrogen atoms within the entire section of com-
pounds act as pharmacophoric parts. The sulfur and oxygen atoms in
the thiazolidine ring and oxygen atom of the acetamide bridge act as
antipharmacophoric parts. As suggested by directional structural
changes aimed at increasing biological activities, these anti-
pharmacophoric atoms can be replaced by any suitable pharmaco-
phoric group.
IC50 = 42.29226372 − 0.01683116g5.5 + 0.05702761g6.0
−
0.06652460g6.2
.
(1)
2
The model has the coefficient of determination (R ) of .661 and
standard error of the estimate of 2.8. g represents the value of the
r
RDF descriptor at distance r. The plot of the experimentally de-
termined versus IC50 values predicted by Equation (1) is graphically
presented in Figure 6.
2.6
|
Absorption, distribution, metabolic liability
prediction, and excretion studies
From the last few decades, in silico ADME studies are found to be
more beneficial than wet experiments, due to reduction of cost and
time. In the current section, the derivatives were evaluated for their
ADME properties to elucidate key features, which have importance
in antibacterial activity (Table 2).
Briefly, the results showed that all synthesized derivatives have
log P values <5, low tendency to be absorbed through the gastro-
2
intestinal tract, optimal TPSA values (in the range of 180–212 Å ),
[
40]
low number of violations of Lipinki's rule of five,
and they do not
cross the blood–brain barrier. It is important to note that Lipinski's
rules represent statistical guidelines for orally administered drugs,
and there are numerous cases of orally available drugs that do not
comply with all the rules, for example, cefozopran (Firstcin®) as a
[
41]
[42]
representative of cephalosporin
and azithromycin
and roxi-
FIGURE 4 Pharmacophore (red), antipharmacophore (blue), and
ballast (gray) parts of the derivatives 10b (left) and 10n (right)
[
43]
thromycin
as representatives of other well‐known antibacterial

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FIGURE 5 Radial distribution functiong (r) (left) and its SD (right) for a series of hybrid oxothiazolidin–1,2,4‐triazole compounds
cytochrome P450 isozyme (3A4, 1A2, 2C19, 2C9, and 2D6), and the
results are displayed in Table 2. Metabolism prognosis for different CYP
enzymes showed that the entire set of compounds is not metabolized at
the site of CYP2D6 and CYP1A2. For other CYP enzymes (namely 3A4,
2C19, and 2C9), the results are mixed, but what is important is that the
least active compounds (10a, 10g, 10j, and 10m) are not predicted to be
metabolized by CYP3A4, which was found in accordance with other
standard chemotherapeutic agents clarithromycin, erythromycin, itra-
[46]
conazole, ketoconazole, ritonavir, and verapamil.
Therefore, there is
a large possibility that these active molecules can serve as a good
starting point for the further development of active antibacterial
[45]
agents. The RS‐Predictor
probabilities to metabolize at each CYP isozyme (1A2, 2A6, 2B6, 2C8,
C9, 2C19, 2D6, 2E1, and 3A4). The most probable SOM of the most
was utilized to identify the SOM and its
2
FIGURE 6 Experimentally observed versus predicted IC50 values
using Equation (1), with 95% confidence interval
active compound 10b and the rest of the compounds are displayed in
Figures 7 and S2, respectively. In the entire series, the sulfur atom of
thiazolidine is highly labile as the SOM, except in the case of CYP2AB6
isozyme. Other fragments of the molecule were identified as moder-
ately and fairly labile. Organic cation transporter 2 is a renal uptake
transporter that plays an important role in the disposition and renal
clearance of drugs and endogenous compounds. Thus, the assessment
of OCT2 of any drug candidate provides useful information about
drugs. Moreover, clinically used drugs are subjected to metabolism in
vivo, which increases the degradation of the molecule into the var-
ious fragments and increases polarity for excretion. The metabolic
processes are also related to the production of toxic byproducts. In
the early stage of drug discovery, the site of metabolism (SOM) of
drug candidate is very vital, and its prediction could prevent re-
capitulation in the subsequent stages and reduce the possible liabilities
[44]
clearance.
The prognosis showed that most effect derivatives 10b
and 10n have a lower tendency to transport from OCT2 pathway,
which need to be investigated further from in vivo studies. Due to
lipophilic character and minimal tendency to transport from OCT2
pathway, it can be predicted that derivatives 10a, 10b, 10d, 10g, 10h,
10j, 10m, 10n, 10p, and 10q can effectively bind with serum/plasma and
could participate in long‐lasting therapeutic effect.
[38]
and risks coupled with biotransformation.
As we found that the
synthesized scaffolds have a promising antibacterial activity, it was
worthy to define its metabolic liability. Although the metabolites of our
synthesized compounds and their reactivity are still unknown (a step
performed in later stages in the drug development), it is important to
assess the metabolism of the parent compounds by CYP enzymes, to
avoid pharmacokinetic interactions. In the present study, we have uti-
2.7
|
Molecular docking experiments
[44]
[45]
lized two diverse programs, pkCSM
and RS‐Predictor,
for the
prediction of metabolism. The pkCSM was used to recognize the pos-
sibility of metabolism of synthesized compounds at different sites of
Being aware of the advantages and issues of molecular docking
[
47]
experiments,
we performed docking of synthesized hybrid

8
of 18
PATHAK ET AL.
|
TABLE 2 Physiochemical properties (ADME) calculation of synthesized derivatives
Metabolism at CYP450
Renal OCT2
substrate
Compound
log P
1.4
1.9
2.7
1.8
2.3
3.1
1.3
1.8
2.6
1.7
2.2
2.9
0.81
1.3
2.1
1.1
1.7
2.4
GI abs
Lip vio
H acc
H don
TPSA
190
206
201
197
212
208
187
202
198
193
208
204
180
196
192
187
202
198
BBB per
−1.6
−1.6
−1.5
−1.6
−1.6
−1.5
−1.5
−1.5
−1.5
−1.6
−1.5
−1.4
−1.5
−1.5
−1.5
−1.6
−1.5
−1.5
3A4
N
Y
1A2
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
2C19
N
N
Y
2C9
N
Y
2D6
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0a
0b
0c
0d
0e
0f
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
1
2
1
1
2
1
0
2
0
0
2
1
0
1
0
0
2
0
9
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
N
N
Y
10
8
Y
Y
9
Y
N
N
Y
N
N
Y
N
Y
10
8
Y
Y
Y
0g
0h
0i
9
N
Y
N
N
N
N
N
N
N
N
N
N
N
N
N
Y
N
N
Y
10
8
Y
N
N
N
Y
0j
9
N
Y
N
Y
0k
0l
10
8
Y
Y
0m
0n
0o
0p
0q
0r
9
N
Y
N
Y
N
N
Y
10
8
Y
N
N
N
Y
9
Y
N
N
Y
10
8
Y
Y
Note: Log P is the logarithm of the octanol–water partition coefficient; GI abs, gastrointestinal absorption; Lip vio, total number of violation of Lipinski's
rule of five; H acc, H acceptor; H don, H donor; TPSA, total polar surface area; columns 1A2, 2C19 2C9, 2D6, and 3A4 showed the metabolism on
different isoforms of cytochrome‐P450; renal OCT2 substrate, organic cation transporter 2; L, low; Y, likely to be metabolized; N, not likely to be
metabolized.
molecules to the DNA gyrase's active site, with the intention to
identify binding poses and to compare the binding energy with the
known inhibitor ciprofloxacin. All docking results are compiled in
Table 3. The top three compounds according to experimentally de-
termined IC50 values against S. aureus DNA gyrase are 10r, 10n, and
ciprofloxacin in the active pocket (PDB ID: 2XCT), a similar pattern
can be observed. An additional hydrogen bond between guanine
(chain G) and carbonyl oxygen (10b) is formed. In compound 10n, the
bromine atom on the benzene ring of 10b is replaced by fluorine.
Due to this minor modification, in the most stable pose of 10n inside
the catalytic pocket, thiazolidine moiety with fluorobenzene is in-
tercalated between nucleobases thymine (chain E) and guanine
(chain G). 1,2,4‐Triazole with nitrobenzene is anchored to protein
pocket at the interface of two subunits. There are several FDA‐
approved drugs that exhibit a bactericidal action through the in-
hibition of topoisomerase II (DNA gyrase) and/or topoisomerase
1
0o, respectively, and together with 10b, they display a higher ac-
tivity as compared with standard (ciprofloxacin). These results are in
good agreement with our docking studies, where 10b, 10n, 10o, and
1
0r have the lowest predicted binding energy.
As compounds 10b and 10n are identified as hit molecules (they
also have the highest anti‐biofilm activity), their interaction pattern
[
9]
inside the DNA gyrase active pocket was analyzed in more detail
IV. Therefore, to validate our results additionally, we selected and
docked five known drugs and their precursor (nalidixic acid) to DNA
gyrase (PDB ID: 2XCT), following the same protocol as for novel
hybrid compounds (Table 3). All drugs demonstrate a similar affinity
toward the enzyme and the predicted binding constants are com-
parable to binding constants of newly synthesized compounds. The
binding score for nalidixic acid is −7.9 kcal/mol, showing how the
optimization of the lead compound could lead toward inhibitors that
are more efficient.
(
Figures 8 and S3). Derivative 10b is positioned in the active site in a
way that nitrobenzyl moiety is placed above nucleobases thymine
chain E) and adenine (chain H), and it establishes favorable aromatic
(
stacking
interactions with thymine and hydrogen bridge with adenine.
On top of nitrobenzyl is guanine (chain G), so substituted
1
,2,4‐triazole substructure is actually intercalated between two
subsequent base pairs. For crystal structure of DNA gyrase with

PATHAK ET AL.
9 of 18
|
FIGURE 7 Prediction of the site of metabolism by RS‐Predictor of compound 10b at (a) CYP1A2, (b) CYP2A6, (c) CYP2B6, (d) CYP2C8, (e)
CYP2C9, (f) CYP2C19, (g) CYP2D6, (h) CYP2E1, (i) CYP3A4. Color code: orange: highly labile; gray: moderately labile; and light green: fairly
labile
2
.8
|
Fingerprinting and molecular similarity
scaffolds are under investigation as potential DNA gyrase inhibitors,
a similarity search for newly synthesized hybrid compounds was
performed against molecules from the ChEMBL database tested for
DNA gyrase inhibition (from now on, referred to as ChEMBL
The ChEMBL database holds manually curated bioactive molecules
with drug‐like properties. To check whether any similar molecules/

10 of 18
PATHAK ET AL.
|
a
TABLE 3 Two‐dimensional (2D) structures of synthesized compounds and selected FDA‐approved drugs with the lowest binding score for
DNA gyrase (PDB ID: 2XCT)
Compound
2D structure
Score
Compound
10b
2D
Score
1
1
1
1
1
1
1
1
0a
0c
0e
0g
0i
−10.1
−10.2
−9.8
−9.9
−8.3
−9.2
−9.7
−9.6
−10.1
10d
10f
10h
10j
−9.4
−9.7
−9.7
−8.8
−9.4
−10.2
−9.6
0k
0m
0o
10l
10n
10p

PATHAK ET AL.
11 of 18
|
TABLE 3 (Continued)
Compound
0q
2D structure
Score
Compound
10r
2D
Score
1
−10.2
−10.0
CPF
LEV
NAL
−9.6
−10.0
−7.9
GEM
MOX
NOR
−9.9
−10.2
−9.1
a
CPF, ciprofloxacin; GEM, gemifloxacin; LEV, levofloxacin; MOX, moxifloxacin; NAL, nalidixic acid; NOR, norfloxacin.
molecules). The molecular similarity was estimated on the basis of
the similarity of 2D molecular fingerprints calculated using the ex-
tended connectivity fingerprint (FP) approach. The adopted method
was demonstrated to have the highest precision on average, ac-
3
|
CONCLUSION
Here, we report a synthesis of a series of hybrid 1,2,4‐triazole and
oxothiazolidin derivatives and their antibacterial activity. The
SAR indicated that the introduction of electron‐withdrawing
groups such as halobenzene moiety into the 1,2,4‐triazole ring
increases ligand potency in DNA gyrase inhibition. In vitro anti-
bacterial evaluation showed that compounds 10b and 10n have
the potential to inhibit a variety of Gram‐positive and ‐negative
bacteria through additional mechanisms of action, such as mod-
ulating the DNA gyrase activity. In this regard, 10b and 10n are
excellent lead compounds with efficiency similar to that of a
series of FDA‐approved drugs that act as DNA gyrase inhibitors.
Experimental findings are corroborated by docking studies.
Interaction pattern analysis revealed intercalation of aromatic
groups between two subsequent DNA base pairs, in a similar
fashion as the known DNA gyrase inhibitors ciprofloxacin, gemi-
floxacin, levofloxacin, moxifloxacin, and norfloxacin. The low
molecular similarity compared with 430 compounds from
ChEMBL, which were tested as DNA gyrase inhibitors, demon-
strates that molecules with oxothiazolidin–1,2,4‐triazole
scaffolds are a novel class of potential DNA gyrase inhibitors.
Their further investigation and targeted substitution might help
us fight against resistant bacteria.
[
48]
cording to database search by compound similarity based on FP.
The molecular similarity between newly synthesized and ChEMBL
molecules is presented as a heat map in Figure S4, with only those
compounds that have Tanimoto's coefficient for at least one hybrid
compound higher than 0.20. Three compounds (CHEMBL457095,
CHEMBL458404, and CHEMBL458367) were found to have the
highest Tanimoto molecular similarity index for the biggest subset of
C
our molecules. CHEMBL457095 has T equal to 0.27, when com-
pared with compound 10l. It also enters the molecules most similar
to hit compounds 10b and 10n to the top three compounds. The
value by itself is relatively low, meaning that they have only some
fragments in common, like halogen‐substituted benzene ring, car-
bonyl group, or five‐membered nitrogen‐containing ring. Figure S5
displays the 2D structures of most similar molecoules of ChEMBL
database and there Tanimoto's coefficient. For CHEMBL457095, the
experimentally measured IC50 value for DNA gyrase inhibition is
[
49]
0
.5 μg/ml.
This is 50 times lower as compared with 10n, corro-
borating that our compounds are members of the new class of po-
tential DNA gyrase inhibitors, whose activity could be increased by
targeted substitutions.

12 of 18
PATHAK ET AL.
|
FIGURE 8 Insight into the catalytic site neighborhood of DNA gyrase (PDB ID: 2XCT) for the best poses and two‐dimensional interaction of
0b (left) and 10n (right) obtained by docking
1
4
4
4
|
EXPERIMENTAL
|
Chemistry
The InChI codes of the investigated compounds, together with
some biological activity data, are provided as Supporting Information.
.1
.1.1
|
General
4.1.2
|
Synthesis of 1,2,4‐triazoles (5a–c)
All experiments were carried out using commercial reagents and
analytical grade solvents without further purification. Thin‐layer
chromatography (TLC) was performed on precoated silica gel plates
The derivatives 5a–c were synthesized from the reagents 1a–c as
[
26]
described in our previous study.
(
F254 grade, 0.2 mm thickness), using ethyl acetate/n‐hexane (3:7)
as an eluent, and visualization was carried out in an iodine chamber.
The completion of the reaction and purity of the synthesized deri-
vatives were evaluated on the basis of a sharp single spot. Melting
points of the derivatives were determined using Veego (MPI melting
4.1.3
|
Synthesis of 2‐chloro‐N‐(5‐mercapto‐3‐
substituted‐phenyl‐1,5‐dihydro‐4H‐1,2,4‐triazol‐4‐yl)
(6a–c)
1
13
point instrument). H NMR and C NMR analysis were performed
using JEOL 300 and Bruker Avance II 100 NMR spectrometers,
Here, 0.5 mol of substituted 1,2,4‐triazoles 5a–c was dissolved in the
glacial acetic acid (25 ml), which contained 25 ml of a saturated so-
lution of sodium acetate. If the substance did not dissolve com-
pletely, the mixture was heated to make the solution clear. The
reaction mixture was cooled on an ice bath, and chloroacetyl chloride
(0.06 mol, 5 ml) was added in a dropwise manner with continuous
stirring to avoid vigorous proceeding of reaction. After 30–45 min, an
3
respectively, with CDCl as the solvent and tetramethylsilane (TMS)
as the internal standard. Mass spectra were obtained using the
VG‐AUTOSPEC spectrometer equipped with an ESI source (Fisons
Instruments). Elemental analysis was carried out on a Vario EL‐III
CHNOS elemental analyzer (Elementar Analysensysteme).

PATHAK ET AL.
13 of 18
|
off‐white product was formed, which was separated through simple
filtration. Furthermore, the product was washed with 50% aqueous
acetic acid, followed by distilled water and recrystallization from
absolute alcohol.
thiazolidine), 6.463 (s, 1H, CH, triazole), 7.14–7.115 (d, 2H, J = 7.5 Hz,
Br–Ph), 7.682–7.652 (d, 2H, J = 9.0 Hz, NO –Ph), 7.630–7.604 (d, 2H,
J = 7.8 Hz, Br–Ph), 8.121–8.092 (d, 2H, J = 8.7 Hz, NO –Ph), 8.876
(s, 1H, NH), 9.134 (s, 1H, NH); C NMR (100 MHz, CDCl ) δ ppm:
3.89, 49.27, 66.24, 72.2, 124.02, 126.2, 127.85, 128.13, 129.65,
2
2
13
3
3
1
30.95, 136.95, 145.2, 147.98, 165.8, 167.26; MS‐ESI (m/z): 551.10
+
4.1.4
|
Synthesis of 2‐hydrazinyl‐N‐(5‐mercapto‐3‐
[M+H] ; elemental analysis: calculated C: 41.315%, H: 3.28%, N:
17.75%; found C: 41.40%, H: 3.42%, N: 17.65%.
substituted‐phenyl‐1,5‐dihydro‐4H‐1,2,4‐triazol‐4‐yl)‐
acetamides (7a–c)
2‐{[2‐(4‐Bromophenyl)‐4‐oxothiazolidin‐3‐yl]amino}‐N‐[3‐(4‐
Here, 0.01 mol of N‐(5‐aryl‐1,2,4‐triazole‐2‐yl)‐2‐chloroacetamides
chlorophenyl)‐5‐mercapto‐1,5‐dihydro‐4H‐1,2,4‐triazol‐4‐yl]‐
acetamide (10c)
6a–c was dissolved in 25 ml of ethanol. Furthermore, 0.01 mol (5 ml) of
hydrazine hydrate was added into the reaction mixture and the resultant
mixture was poured on the crushed ice to separate the product.
Light
yellow
solid;
; melting point: 253–255°C; molecular weight:
41.87; H NMR (300 MHz, CDCl , TMS) δ ppm: 1.524 (s, 1H, SH),
.602–3.562 (d, 1H, J = 12.0 Hz, CH , thiazolidine), 3.802–3.762 (d,
, thiazolidine), 3.493 (s, 2H, CH ), 4.243 (s, 1H,
yield:
80%;
molecular
formula:
C
19
H
18BrClN O S
6 2 2
1
5
3
3
2
4.1.5
|
Synthesis of 2‐[2‐(substituted‐phenyl)‐5‐
1H, J = 12.0 Hz, CH
2
2
substituted‐4‐oxo‐thiazolidin‐3‐ylamino]‐N‐[5‐
mercapto‐3‐(substituted‐phenyl)‐1,5‐dihydro‐1,2,4‐
triazol‐4‐yl]acetamides (10a–r)
NH), 6.296 (s, 1H, CH, thiazolidine), 6.463 (s, 1H, CH, triazole),
7.16–7.135 (d, 2H, J = 7.5 Hz, Br–Ph), 7.635–7.611 (d, 2H, J = 7.2 Hz,
Br–Ph), 7.674–7.649 (d, 2H, J = 7.5 Hz, Cl–Ph), 7.921–7.892 (d, 2H,
13
J = 8.7 Hz, Cl–Ph), 8.876 (s, 1H, NH), 9.134 (s, 1H, NH); C NMR
(100 MHz, CDCl ) δ ppm: 33.89, 49.27, 66.24, 72.7, 124.02, 126.2,
A
mixture of 2‐hydrazinyl‐N‐(5‐mercapto‐3‐substituted‐phenyl‐1,5‐
3
dihydro‐1,2,4‐triazol‐4‐yl)acetamides (7a–c) (0.01 mol), substituted ben-
zaldehydes (8a–c) (0.01 mol), thioglycolic acid derivatives (9a–c)
127.82, 128.09, 129.08, 130.95, 135.68, 136.95, 145.2, 165.8,
+
167.26; MS‐ESI (m/z): 540.12 [M+H] ; elemental analysis: calculated
(
0.01 mol), and a pinch of ZnCl
2
were refluxed in 15 ml of ethanol for
C: 42.12%, H: 3.35%, N: 15.51%; found C: 42.20%, H: 3.42%,
6–7 h or reflux until a distinct single point was obtained on TLC. As a
N: 15.62%.
result of the reaction, a solid was formed and separated on the bottom of
the vessel. Furthermore, the products were filtered, washed with water,
and recrystallized with ethanol to obtain the pure compound.
2‐{[2‐(4‐Bromophenyl)‐5‐methyl‐4‐oxothiazolidin‐3‐yl]amino}‐N‐[5‐
mercapto‐3‐(pyridin‐4‐yl)‐1,5‐dihydro‐4H‐1,2,4‐triazol‐4‐yl]‐
acetamide (10d)
2
3
‐{[2‐(4‐Bromophenyl)‐4‐oxothiazolidin‐3‐yl]amino}‐N‐[5‐mercapto‐
‐(pyridin‐4‐yl)‐1,5‐dihydro‐4H‐1,2,4‐triazol‐4‐yl]acetamide (10a)
Yellow solid; yield: 74%; molecular formula:
C
19
H
20BrN
7 2 2
O S ;
1
melting point: 193–195°C; molecular weight: 522.44; H NMR
Yellow solid; yield: 78%; molecular formula:
C
18
H
18BrN
1
7
O
2
S
2
;
(300 MHz, CDCl
3
, TMS) δ ppm: 1.264–1.241 (d, 3H, J = 6.9 Hz, CH
3
),
melting point: 251–253°C; molecular weight: 508.41; H NMR
1.524 (s, 1H, SH), 3.493 (s, 2H, CH
2
), 4.092 (m, 1H, CH, thiazolidine),
(
300 MHz, CDCl
J = 15.0 Hz, CH , thiazolidine), 3.802–3.762 (d, 1H, J = 12.0 Hz, CH
thiazolidine), 3.943 (s, 2H, CH ), 4.243 (s, 1H, NH), 6.296 (s, 1H, CH,
3
, TMS) δ ppm: 1.524 (s, 1H, SH), 3.612–3.562 (d, 1H,
4.243 (s, 1H, NH), 6.216 (s, 1H, CH, thiazolidine), 6.423 (s, 1H, CH,
triazole), 7.16–7.135 (d, 2H, J = 7.5 Hz, Br–Ph), 7.630–7.604 (d, 2H,
J = 7.8 Hz, Br–Ph), 7.664–7.649 (d, J = 4.5 Hz, CH, pyridine),
8.664–8.679 (d, 2H, J = 4.5 Hz, CH, pyridine), 8.876 (s, 1H, NH), 9.134
2
2
,
2
thiazolidine), 6.426 (s, 1H, CH, triazole), 7.16–7.135 (d, 2H, J = 7.5 Hz,
Br–Ph), 7.572–7.558 (d, 2H, J = 4.2 Hz, CH, pyridine), 7.630–7.604 (d,
13
3
(s, 1H, NH); C NMR (100 MHz, CDCl ) δ ppm: 33.89, 49.27, 66.24,
2
8
H, J = 7.8 Hz, Br–Ph), 8.679–8.664 (d, 2H, J = 4.5 Hz, CH, pyridine),
72.75, 121.33, 124.02, 126.80, 127.82, 127.82, 130.95, 136.95,
1
3
+
.876 (s, 1H, NH), 9.134 (s, 1H, NH); C NMR (100 MHz, CDCl
3
) δ
145.2, 149.65, 165.8, 167.26; MS‐ESI (m/z): 521.32 [M+H] ; ele-
ppm: 33.89, 49.27, 66.24, 72.5, 121.33, 124.02, 126.8, 127.82,
mental analysis: calculated C: 43.68%, H: 3.86%, N: 18.77%; found C:
1
30.95, 136.95, 145.2, 149.65, 165.8, 167.26; MS‐ESI (m/z): 508.25
43.45%, H: 3.82%, N: 18.72%.
+
[
M+H] ; elemental analysis: calculated C: 42.52%, H: 3.57%, N:
1
9.29%; found C: 42.40%, H: 3.42%, N: 19.65%.
2‐{[2‐(4‐Bromophenyl)‐5‐methyl‐4‐oxothiazolidin‐3‐yl]amino}‐N‐[5‐
mercapto‐3‐(4‐nitrophenyl)‐1,5‐dihydro‐4H‐1,2,4‐triazol‐4‐yl]‐
acetamide (10e)
2
3
‐{[2‐(4‐Bromophenyl)‐4‐oxothiazolidin‐3‐yl]amino}‐N‐[5‐mercapto‐
‐(4‐nitrophenyl)‐1,5‐dihydro‐4H‐1,2,4‐triazol‐4‐yl]acetamide (10b)
Yellow‐brown solid; yield: 72%; molecular formula: C20
H
20BrN
7 4 2
O S ;
1
Yellow‐brown solid; yield: 76%; molecular formula: C19
H
18BrN
1
7
O
4
S
2
;
melting point: 213–215°C; molecular weight: 566.45; H NMR
melting point: 184–186°C; molecular weight: 552.42; H NMR
(300 MHz, CDCl
3
, TMS) δ ppm: 1.264–1.241 (d, 3H, J = 6.9 Hz, CH
3
),
(
300 MHz, CDCl
J = 12.0 Hz, CH , thiazolidine), 3.802–3.762 (d, 1H, J = 12.0 Hz, CH
thiazolidine), 3.943 (s, 2H, CH ), 4.243 (s, 1H, NH), 6.302 (s, 1H, CH,
3
, TMS) δ ppm: 1.524 (s, 1H, SH), 3.602–3.562 (d, 1H,
1.524 (s, 1H, SH), 3.953 (s, 2H, CH
2
), 4.092 (m, 1H, J = 7.0 Hz, CH,
2
2
,
thiazolidine), 4.243 (s, 1H, NH), 6.423 (1H, s, CH, triazole),
2
7.16–7.135 (d, 2H, J = 7.5 Hz, Br–Ph), 7.682–7.652 (d, 2H, J = 9.0 Hz,

14 of 18
PATHAK ET AL.
|
NO
2
–Ph), 7.630–7.604 (d, 2H, J = 7.8 Hz, Br–Ph), 8.121–8.092 (d, 2H,
NMR (100 MHz, CDCl
3
) δ ppm: 33.89, 49.27, 66.24, 72.7, 123.98,
13
J = 8.7 Hz, NO
2
–Ph), 8.876 (s, 1H, NH), 9.134 (s, 1H, NH); C NMR
126.2, 127.82, 128.71, 130.66, 133.16, 134.10, 136.92, 145.2,
+
(
100 MHz, CDCl ) δ ppm: 17.8, 44.31, 49.27, 66.24, 72.75, 117.29,
3
147.98, 149.65, 165.8, 167.26; MS‐ESI (m/z): 507.45 [M+H] ; ele-
1
1
24.02, 126.2, 127.82, 128.1, 130.95, 136.95, 140.47, 145.2, 165.8,
mental analysis: calculated C: 44.93%, H: 3.57%, N: 19.30%; found C:
44.5%, H: 3.42%, N: 19.75%.
+
67.26; MS‐ESI (m/z): 565.52 [M+H] ; elemental analysis: calculated
C: 42.41%, H: 3.56%, N: 17.31%; found C: 42.5%, H: 3.75%,
N: 17.52%.
2‐{[2‐(2‐Chlorophenyl)‐4‐oxothiazolidin‐3‐yl]amino}‐N‐[3‐(4‐
chlorophenyl)‐5‐mercapto‐1,5‐dihydro‐4H‐1,2,4‐triazol‐4‐yl]‐
acetamide (10i)
2
‐{[2‐(4‐Bromophenyl)‐5‐methyl‐4‐oxothiazolidin‐3‐yl]amino}‐N‐[3‐
(
4‐chlorophenyl)‐5‐mercapto‐1,5‐dihydro‐4H‐1,2,4‐triazol‐4‐yl]‐
White solid; yield: 76%; molecular formula:
melting point: 241–243°C; molecular weight: 497.41; H NMR
(300 MHz, CDCl , TMS) δ ppm: 1.524 (s, 1H, SH), 3.602–3.562 (d, 1H,
J = 12.0 Hz, CH , thiazolidine), 3.802–3.762 (d, 1H, J = 12.0 Hz, CH
thiazolidine), 3.953 (s, 2H, CH ), 6.296 (s, 1H, CH, thiazolidine), 4.243
19 2 6 2 2
C H18Cl N O S ;
1
acetamide (10f)
Yellow solid; yield: 76%; molecular formula: C20
H
20BrClN
1
6
O
2
S
2
;
3
melting point: 241–243°C; molecular weight: 555.89; H NMR
2
2
,
(
300 MHz, CDCl
.524 (s, 1H, SH), 3.953 (s, 2H, CH
thiazolidine), 4.243 (s, 1H, NH), 6.296 (1H, s, CH, thiazolidine), 6.463
s, 1H, CH, triazole), 7.16–7.135 (d, 2H, J = 7.5 Hz, Br–Ph),
.630–7.605 (d, 2H, J = 7.5 Hz, Br–Ph), 7.674–7.648 (d, 2H,
J = 7.8 Hz, Cl–Ph), 7.921–7.892 (d, 2H, J = 8.7 Hz, Cl–Ph), 8.876 (s,
3
, TMS) δ ppm: 1.264–1.241 (d, 3H, J = 6.9 Hz, CH
3
),
2
1
2
), 4.092 (m, 1H, J = 7.0 Hz, CH,
(s, 1H, NH), 6.463 (s, 1H, CH, triazole), 7.215 (m, 1H, Ph–Cl), 7.334
(m, 1H, Ph–Cl), 7.398–7.373 (d, 2H, J = 7.5 Hz, Cl–Ph), 7.425–7.41 (d,
2H, J = 7.5 Hz, Cl–Ph), 7.524 (m, 1H, Cl–Ph), 7.921–7.892 (d, 2H,
(
13
7
J = 8.7 Hz, Cl–Ph), 8.872 (s, 1H, NH), 9.132 (s, 1H, NH); C NMR
(100 MHz, CDCl ) δ ppm: 33.89, 49.27, 66.24, 72.7, 126.2, 127.82,
3
1
3
1
4
1
H, NH), 9.134 (s, 1H, NH); C NMR (100 MHz, CDCl
3
) δ ppm: 17.8,
128.09, 128.71, 129.08, 130.66, 133.16, 135.68, 136.92, 145.2,
+
4.31, 49.27, 66.24, 72.5, 117.29, 124.02, 126.2, 127.82, 128.52,
149.62, 165.8, 167.26; MS‐ESI (m/z): 496.52 [M+H] ; elemental
30.95, 136.95, 140.47, 145.2, 165.8, 167.26; MS‐ESI (m/z): 554.10
analysis: calculated C: 45.88%, H: 3.65%, N: 16.90%; found C:
+
[
M+H] ; elemental analysis: calculated C: 43.21%, H: 3.63%, N:
45.58%, H: 3.78%, N: 16.70%.
1
5.12%; found C: 43.4%, H: 3.52%, N: 15.24%.
2‐{[2‐(2‐Chlorophenyl)‐5‐methyl‐4‐oxothiazolidin‐3‐yl]amino}‐N‐[5‐
2
3
‐{[2‐(2‐Chlorophenyl)‐4‐oxothiazolidin‐3‐yl]amino}‐N‐[5‐mercapto‐
‐(pyridin‐4‐yl)‐1,5‐dihydro‐4H‐1,2,4‐triazol‐4‐yl]acetamide (10 g)
mercapto‐3‐(pyridin‐4‐yl)‐1,5‐dihydro‐4H‐1,2,4‐triazol‐4‐yl]‐
acetamide (10j)
Brown solid; yield: 71%; molecular formula: C18
H
1
18ClN
7
O
2
S
2
; melt-
Reddish‐yellow
solid;
melting point: 243–245°C; molecular weight:
, TMS) δ ppm: 1.241–1.264 (d, 3H,
), 1.524 (s, 1H, SH), 3.953 (s, 2H, CH ), 4.092 (m, 1H,
yield:
72%;
molecular
formula:
ing point: 176–178°C; molecular weight: 463.96; H NMR (300 MHz,
C
19
H
20ClN
1
7 2 2
O S ;
CDCl
J = 12.0 Hz, CH
thiazolidine), 3.953 (s, 2H, CH
thiazolidine), 6.463 (s, 1H, CH, triazole), 7.215 (m, 1H, Ph–Cl), 7.334
m, 1H, Ph–Cl), 7.524 (m, 1H, Cl–Ph), 7.664–7.651 (d, 2H, J = 3.9 Hz,
CH, pyridine), 7.921–7.892 (d, 2H, J = 8.7 Hz, Cl–Ph), 8.679–8.664 (d,
3
, TMS) δ ppm: 1.524 (s, 1H, –SH), 3.602–3.562 (d, 1H,
, thiazolidine), 3.802–3.762 (d, 1H, J = 12.0 Hz, CH
), 4.243 (s, 1H, NH), 6.296 (s, 1H, CH,
477.99; H NMR (300 MHz, CDCl
3
2
2
,
J = 7.0 Hz, CH
3
2
2
CH, thiazolidine), 4.243 (s, 1H, NH), 6.213 (s, 1H, CH, triazole), 6.296
(s, 1H, CH, thiazolidine), 6.463 (s, 1H, CH, triazole), 7.215–7.19 (m,
1H, Ph–Cl), 7.334–7.303 (m, 1H, Ph–Cl), 7.524 (m, 1H, Cl–Ph),
7.664–7.649 (d, 2H, J = 4.5 Hz, CH, pyridine), 7.921–7.892 (d, 2H,
J = 8.7 Hz, Cl–Ph), 8.679–8.664 (d, 2H, J = 4.5 Hz, CH, pyridine),
(
13
2
H, J = 4.5 Hz, CH, pyridine), 8.876 (s, 1H, NH), 9.134 (s, 1H, NH);
C
13
NMR (100 MHz, CDCl
3
) δ ppm: 33.89, 49.27, 66.24, 72.15, 121.33,
3
8.876 (s, 1H, NH), 9.134 (s, 1H, NH); C NMR (100 MHz, CDCl ) δ
1
1
26.8, 127.82, 128.71, 130.66, 133.16, 134.10, 136.92, 145.2,
ppm: 17.8, 44.31, 49.27, 66.24, 72.7, 121.33, 122.2, 126.80, 127.82,
+
49.65, 165.8, 167.26; MS‐ESI (m/z): 463.42 [M+H] ; elemental
128.71, 130.66, 133.16, 136.92, 145.2, 149.65, 165.8, 167.26; MS‐
+
analysis: calculated C: 46.60%, H: 3.91%, N: 21.13%; found C:
6.25%, H: 3.92%, N: 21.2%.
ESI (m/z): 477.82 [M+H] ; elemental analysis: calculated C: 47.74%,
4
H: 4.22%, N: 20.51%; found C: 47.8%, H: 4.28%, N: 20.42%.
2
3
‐{[2‐(2‐Chlorophenyl)‐4‐oxothiazolidin‐3‐yl]amino}‐N‐[5‐mercapto‐
‐(4‐nitrophenyl)‐1,5‐dihydro‐4H‐1,2,4‐triazol‐4‐yl]acetamide (10h)
2‐{[2‐(2‐Chlorophenyl)‐5‐methyl‐4‐oxothiazolidin‐3‐yl]amino}‐N‐[5‐
mercapto‐3‐(4‐nitrophenyl)‐1,5‐dihydro‐4H‐1,2,4‐triazol‐4‐yl]‐
acetamide (10k)
Yellow solid; yield: 74%; molecular formula: C19
H
7
18ClN O
4
S
2
; melt-
ing point: 255°C; molecular weight: 507.97; H NMR (300 MHz,
CDCl , TMS) δ ppm: 1.524 (s, 1H, SH), 3.602–3.562 (d, 1H,
J = 12.0 Hz, CH , thiazolidine), 3.802–3.762 (d, 1H, J = 12.0 Hz, CH
thiazolidine), 3.953 (s, 2H, CH ), 6.296 (s, 1H, CH, thiazolidine), 4.243
1
Brown solid; yield: 79%; molecular formula: C20
H
20ClN
7
O
4
S
2
; melt-
ing point: 237–235°C; molecular weight: 522; H NMR (300 MHz,
CDCl , TMS) δ ppm: 1.241–1.264 (d, 3H, J = 7.0 Hz, CH ), 1.524 (s,
1H, SH), 3.953 (s, 2H, CH ), 4.092 (m, 1H, CH, thiazolidine), 4.243 (s,
1
3
2
2
,
3
3
2
2
(
s, 1H, NH), 6.463 (s, 1H, CH, triazole), 7.215 (m, 1H, Ph–Cl), 7.334
m, 1H, Ph–Cl), 7.524 (m, 1H, Cl–Ph), 7.921–7.892 (d, 2H, J = 8.7 Hz,
1H, NH), 6.296 (s, 1H, CH, thiazolidine), 6.463 (s, 1H, CH, triazole),
7.215 (m, 1H, Ph–Cl), 7.334 (m, 1H, Ph–Cl), 7.524 (m, 1H, Cl–Ph),
7.921–7.892 (d, 2H, J = 8.7 Hz, Cl–Ph), 8.12–8.092 (d, 2H, J = 8.4 Hz,
(
Cl–Ph), 8.12–8.092 (d, 2H, J = 8.4 Hz, NO
2
–Ph), 8.489–8.462 (d, 2H,
13
J = 8.1 Hz, CH, NO –Ph), 8.872 (s, 1H, NH), 9.132 (s, 1H, NH);
2
C
2 2
NO –Ph), 8.489–8.462 (d, 2H, J = 8.1 Hz, CH, NO –Ph), 8.872 (s, 1H,

PATHAK ET AL.
15 of 18
|
1
3
NH), 9.132 (s, 1H, NH); C NMR (100 MHz, CDCl
3
) δ ppm: 17.8,
calculated C: 46.43%, H: 3.69%, N: 19.95%; found C: 46.52%, H:
3.25%, N: 19.75%.
4
1
4.31, 49.27, 66.24, 72.2, 121.33, 122.2, 123.98, 126.2, 127.58,
28.13, 128.92, 130.55, 133.16, 145.2, 147.98, 165.8, 167.26; MS‐
+
ESI (m/z): 521.2 [M+H] ; elemental analysis: calculated C: 46.02%, H:
N‐[3‐(4‐Chlorophenyl)‐5‐mercapto‐1,5‐dihydro‐4H‐1,2,4‐triazol‐4‐
yl]‐2‐{[2‐(4‐fluorophenyl)‐4‐oxothiazolidin‐3‐yl]amino}‐
acetamide (10o)
3
.86%, N: 18.78%; found C: 46.25%, H: 3.92%, N: 18.85%.
N‐[3‐(4‐Chlorophenyl)‐5‐mercapto‐1,5‐dihydro‐4H‐1,2,4‐triazol‐4‐
yl]‐2‐{[2‐(2‐chlorophenyl)‐5‐methyl‐4‐oxothiazolidin‐3‐yl]amino}‐
acetamide (10l)
Brown solid; yield: 77%; molecular formula: C19
H
18ClFN
6 2 2
O S ;
1
melting point: 231–233°C; molecular weight: 480.96; H NMR
(300 MHz, CDCl
J = 15.0 Hz, CH , thiazolidine), 3.802–3.762 (d, 1H, J = 12.0 Hz, CH
thiazolidine), 3.943 (s, 2H, CH ), 4.243 (s, 1H, NH), 6.296 (s, 1H, CH,
3
, TMS) δ ppm: 1.524 (s, 1H, SH), 3.612–3.562 (d, 1H,
Yellow solid; yield: 68%; molecular formula: C20
H
20Cl
2
N
6
O
2
S
2
;
2
2
,
1
melting point: 167°C; molecular weight: 511.44; H NMR (300 MHz,
2
CDCl
3
, TMS) δ ppm: 1.241–1.264 (d, 3H, J = 7.0 Hz, CH
3
), 1.524 (s,
thiazolidine), 6.426 (s, 1H, CH, triazole), 7.162–7.134 (d, 2H,
J = 8.4 Hz, F–Ph), 7.398–7.373 (d, 2H, J = 7.5 Hz, Cl–Ph), 7.425–7.4
(d, 2H, J = 7.5 Hz, Cl–Ph), 7.620–7.591 (d, 2H, J = 8.6 Hz, F–Ph), 8.876
1
1
7
H, SH), 3.953 (s, 2H, CH ), 4.092 (m, 1H, CH, thiazolidine), 4.243 (s,
2
H, NH), 6.296 (s, 1H, CH, thiazolidine), 6.463 (s, 1H, CH, triazole),
13
.215 (m, 1H, Ph–Cl), 7.334 (m, 1H, Ph–Cl), 7.398–7.373 (d, 2H,
3
(s, 1H, NH), 9.134 (s, 1H, NH); C NMR (100 MHz, CDCl ) δ ppm:
J = 7.5 Hz, Cl–Ph), 7.425–7.392 (d, 2H, J = 8.4 Hz, Cl–Ph), 7.524 (m,
33.89, 49.27, 66.24, 72.2, 115.16, 126.2, 127.82, 128.09, 129.08,
1
9
4
1
5
1
H, Cl–Ph), 7.921–7.892 (d, 2H, J = 8.7 Hz, Cl–Ph), 8.872 (s, 1H, NH),
135.68, 136.95, 145.2, 163.35, 165.8, 167.22; MS‐ESI (m/z): 480.25
13
+
.132 (s, 1H, NH); C NMR (100 MHz, CDCl
3
) δ ppm: 17.8, 44.31,
[M+H] ; elemental analysis: calculated C: 47.45%, H: 3.77%, N:
9.27, 66.24, 72.2, 121.33, 122.2 126.2, 127.82, 128.15, 128.71,
17.47%; found C: 47.5%, H: 3.95%, N: 17.56%.
29.08, 130.66, 133.16, 135.68, 145.2, 165.8, 167.26; MS‐ESI (m/z):
+
10.2 [M+H] ; elemental analysis: calculated C: 46.97%, H: 3.94%, N:
2‐{[2‐(4‐Fluorophenyl)‐5‐methyl‐4‐oxothiazolidin‐3‐yl]amino}‐N‐[5‐
mercapto‐3‐(pyridin‐4‐yl)‐1,5‐dihydro‐4H‐1,2,4‐triazol‐4‐yl]‐
acetamide (10p)
6.43%; found C: 46.90%, H: 3.8%, N: 16.75%.
2
3
‐{[2‐(4‐Fluorophenyl)‐4‐oxothiazolidin‐3‐yl}amino]‐N‐[5‐mercapto‐
‐(pyridin‐4‐yl)‐1,5‐dihydro‐4H‐1,2,4‐triazol‐4‐yl]acetamide (10m)
Yellow solid; yield: 73%; molecular formula: C18
H
1
18FN
7
O
2
S
2
; melting
point: 234–236°C; molecular weight: 447.51; H NMR (300 MHz,
Yellow solid; yield: 75%; molecular formula: C18
H
18FN
7
O
2
S
2
; melting
CDCl
3
, TMS) δ ppm: 1.241–1.264 (d, 3H, J = 7.0 Hz, CH
3
), 1.524 (s,
1
point: 171–173°C; molecular weight: 447.51; H NMR (300 MHz,
2
1H, SH), 3.943 (s, 2H, CH ), 4.092 (m, 1H, CH, thiazolidine), 4.243 (s,
CDCl
J = 15.0 Hz, CH
thiazolidine), 3.943 (s, 2H, CH
3
, TMS) δ ppm: 1.524 (s, 1H, SH), 3.612–3.562 (d, 1H,
, thiazolidine), 3.802–3.762 (d, 1H, J = 12.0 Hz, CH
), 4.243 (s, 1H, NH), 6.296 (s, 1H, CH,
1H, NH), 6.296 (s, 1H, CH, thiazolidine), 6.426 (s, 1H, CH, triazole),
7.572–7.557 (d, 2H, J = 4.5 Hz, CH, pyridine), 7.620–7.591 (d, 2H,
J = 8.6 Hz, F–Ph), 8.664–8.679 (d, 2H, J = 4.5 Hz, CH, pyridine), 8.876
2
2
,
2
1
3
thiazolidine), 6.426 (s, 1H, CH, triazole), 7.162–7.134 (d, 2H,
J = 8.4 Hz, F–Ph), 7.572–7.561 (d, 2H, J = 3.3 Hz, CH, pyridine),
3
(s, 1H, NH), 9.134 (s, 1H, NH); C NMR (100 MHz, CDCl ) δ ppm:
17.8, 44.31, 49.27, 66.24, 72.2, 115.16, 121.33, 126.80, 127.82,
7
.620–7.592 (d, 2H, J = 8.6 Hz, F–Ph), 8.664–8.679 (d, 2H, J = 4.5 Hz,
136.95, 145.2, 149.65, 163.35, 165.8, 167.26; MS‐ESI (m/z): 447.52
1
3
+
CH, pyridine), 8.876 (s, 1H, NH), 9.134 (s, 1H, NH); C NMR
100 MHz, CDCl ) δ ppm: 33.89, 49.27, 66.24, 72.2, 115.16, 121.33,
26.8, 127.82, 136.95, 145.2, 149.65, 163.35, 165.8, 167.26; MS‐ESI
[M+H] ; elemental analysis: calculated C: 48.31%, H: 4.05%, N:
(
3
21.91%; found C: 48.15%, H: 3.98%, N: 21.75%.
1
+
(m/z): 447.12 [M+H] ; elemental analysis: calculated C: 48.31%, H:
2‐{[2‐(4‐Fluorophenyl)‐5‐methyl‐4‐oxothiazolidin‐3‐yl]amino}‐N‐[5‐
mercapto‐3‐(4‐nitrophenyl)‐1,5‐dihydro‐4H‐1,2,4‐triazol‐4‐yl]‐
acetamide (10q)
4
.05%, N: 21.91%; found C: 48.25%, H: 4.22%, N: 21.85%.
2
3
‐{[2‐(4‐Fluorophenyl)‐4‐oxothiazolidin‐3‐yl]amino}‐N‐[5‐mercapto‐
‐(4‐nitrophenyl)‐1,5‐dihydro‐4H‐1,2,4‐triazol‐4‐yl]acetamide (10n)
Yellow solid; yield: 69%; molecular formula: C20
H
20FN
1
7 4 2
O S ;
melting point: 232–234°C; molecular weight: 505.54; H NMR
Yellow‐brown solid; yield: 71%; molecular formula: C19
H
18FN
1
7
O
4
S
2
;
(300 MHz, CDCl
CH ), 1.524 (s, 1H, SH), 3.943 (s, 2H, CH
CH, thiazolidine), 3.943 (s, 2H, CH ), 4.243 (s, 1H, NH), 6.296
3
, TMS) δ ppm: 1.241–1.264 (d, 3H, J = 7.0 Hz,
melting point: 260–262°C; molecular weight: 491.52; H NMR
3
2
), 4.092 (m, 1H, J = 7.0 Hz,
(
300 MHz, CDCl
J = 15.0 Hz, CH , thiazolidine), 3.802–3.762 (d, 1H, J = 12.0 Hz, CH
thiazolidine), 3.943 (s, 2H, CH ), 4.243 (s, 1H, NH), 6.296 (s, 1H, CH,
3
, TMS) δ ppm: 1.524 (s, 1H, SH), 3.612–3.562 (d, 1H,
2
2
2
,
(s, 1H, CH, thiazolidine), 6.426 (s, 1H, CH, triazole), 7.162–7.134
(d, 2H, J = 8.4 Hz, F–Ph), 7.620–7.591 (d, 2H, J = 8.6 Hz, F–Ph),
2
thiazolidine), 6.426 (s, 1H, CH, triazole), 7.162–7.134 (d, 2H,
J = 8.4 Hz, F–Ph), 7.620–7.591 (d, 2H, J = 8.6 Hz, F–Ph),
7.662–7.633 (d, 2H, J = 8.5 Hz, NO
J = 8.7 Hz, NO –Ph), 8.876 (s, 1H, NH), 9.134 (s, 1H, NH); C NMR
(100 MHz, CDCl ) δ ppm: 17.8, 44.31, 49.27, 66.24, 72.2, 115.16,
2
–Ph), 8.121–8.092 (d, 2H,
1
3
2
7
.662–7.6336 (d, 2H, J = 8.5 Hz, NO
J = 8.7 Hz, NO –Ph), 8.876 (s, 1H, NH), 9.134 (s, 1H, NH); C NMR
100 MHz, CDCl ) δ ppm: 33.89, 49.27, 66.24, 72.2, 115.16,
2
–Ph), 8.121–8.092 (d, 2H,
3
1
3
2
123.98, 126.2, 127.82, 128.13, 136.95, 145.2, 147.98, 163.35,
+
(
3
165.8, 167.26; MS‐ESI (m/z): 505.62 [M+H] ; elemental analysis:
1
1
23.98, 126.2, 127.82, 128.13, 136.95, 145.2, 147.98, 163.35,
calculated C: 47.52%, H: 3.99%, N: 19.39%; found C: 47.85%,
+
65.8, 167.26; MS‐ESI (m/z): 491.25 [M+H] ; elemental analysis:
H: 3.92%, N: 19.45%.

16 of 18
PATHAK ET AL.
|
N‐[3‐(4‐Chlorophenyl)‐5‐mercapto‐1,5‐dihydro‐4H‐1,2,4‐triazol‐4‐
yl]‐2‐{[2‐(4‐fluorophenyl)‐5‐methyl‐4‐oxothiazolidin‐3‐yl]amino}‐
acetamide (10r)
4.4
|
DNA gyrase inhibitory activity
The synthesized compounds were screened for their potency against
Brown solid; yield: 72%; molecular formula: C20
H
20ClFN
1
6
O
2
S
2
; melting
DNA gyrase‐A of S. aureus according to the previously reported
[57]
point: 189–191°C; molecular weight: 494.99; H NMR (300 MHz,
method.
Briefly, the colony of S. aureus was cultured in a medium
SO , 8 g
, 4 g glucose in 1 L distilled water).
The purification, supercoiling, and decatenation of S. aureus were exe-
CDCl
J = 7.0 Hz, CH, thiazolidine), 3.943 (s, 2H, CH
s, 1H, CH, thiazolidine), 6.426 (s, 1H, CH, triazole), 7.162–7.134 (d, 2H,
J = 8.4 Hz, F–Ph), 7.398–7.373 (d, 2H, J = 7.5 Hz, Cl–Ph), 7.425–7.40 (d,
3
, TMS) δ ppm: 1.524 (s, 1H, SH), 3.943 (s, 2H, CH
2
), 4.092 (m, 1H,
(consisting of 2 g yeast extract, 10 g polypeptone, 1.2 g (NH
4
)
2
4
2
), 4.243 (s, 1H, NH), 6.296
Na HPO, 2 g KH PO , 0.2 g MgSO
2
2
4
4
(
[58]
cuted according to the reported method of Blanche et al.,
and the
2
1
4
1
H, J = 7.5 Hz, Cl–Ph), 7.620–7.591 (d, 2H, J = 8.6 Hz, F–Ph), 8.876 (s,
IC50 (in μg/ml) was determined through a dose–response curve.
13
3
H, NH), 9.134 (s, 1H, NH); C NMR (100 MHz, CDCl ) δ ppm: 17.8,
4.31, 49.27, 66.24, 72.2, 115.16, 126.2, 127.82, 128.09, 129.08, 135.68,
+
36.95, 145.2, 163.35, 165.8, 167.26; MS‐ESI (m/z): 494.24 [M+H] ;
4.5
|
3D QSAR analysis
elemental analysis: calculated C: 48.53%, H: 4.07%, N: 16.98%; found C:
8.35%, H: 3.98%, N: 16.75%.
4
3D QSAR studies were performed using the “Cinderella Shoe” (CiS)
[
59]
[60,61]
method
and continual molecular interior (CoMIn) algorithms.
[59]
Cinderella Shoe
is a complementary pseudoatomic receptor model
4
.2
|
Antibacterial activity
used for calculating contributions of ligands' topographies and de-
fining potentials at the points of molecular space obtained in the
generalized superimposed lattice of the entire data set. In this way, a
reliable pattern for pharmacophore, antipharmacophore, and ballast
fragments of ligands can be obtained very fast. The CiS method uses
10 splits for validation, where each contains 80% training and 20%
for testing data set. Each split is randomly selected and follows the
The antimicrobial potency of newly synthesized hybrid triazole de-
rivatives was screened and expressed as MIC (μg/ml) against three
Gram‐positive (B. subtilis [NCIM 2063], B. cereus [NCIM 2156], S.
aureus [NCIM 2079]), and two Gram‐negative (E. coli [NCIM 2065]
and P. vulgaris [NCIM 2027]) bacterial strains using the broth dilution
method. Ciprofloxacin, a covalent DNA gyrase inhibitor, was used as
the standard to perform the comparison of efficacy between the
synthesized derivatives and standard antibacterial agent. Clinical and
Laboratory Standards Institute methodology with a minor modifica-
[
60,62–64]
decoy set in both training and testing data sets in this variant.
Recently introduced RDF descriptor weighted by the number of
valence shell electrons (g(r)) was used to design QSAR model correlating
[65,66]
it with the ligands' potential to inhibit DNA gyrase (IC50 values).
[
50]
tion was used for the antibacterial activity evaluation.
Different
For each optimized structure, hydrogen atoms were removed, and g(r)
concentrations of the test samples and standard compounds were
was calculated. It is represented as a vector of a predetermined size
[
51,52]
prepared according to our previous research.
N−1
N
−
2/3
(rij−r)2
−
a
g (r) = ∑∑
p
i
p
j
e
ij
(2)
i=1 j>i
4
.3
|
Antibiofilm activity
with elements calculated at a discrete distance r and a distance step
of 0.1 Å. aij is the sum of atomic polarizabilities of atoms i and j, rij the
distance between atoms i and j, N is the number of atoms in a mo-
The potential of the hybrid thiazolidine–1,2,4‐triazole derivatives to
prevent initial cell attachment was investigated through the biofilm in-
lecule, and the pre‐exponential factors p
i j
and p account for the
[53]
hibition assay.
The bacterial strain of S. aureus (NCIM 2079) was
number of outer electrons of the ith and jth atoms, correspondingly.
[
67–69]
cultured overnight in tryptone soya broth (supplemented with 0.5%
glucose). Furthermore, different concentrations of test derivatives and
standard ciprofloxacin (100 μl, ranging from 0 to 100 µg/ml) were mixed
with the predefined bacterial suspension (having 100‐μl aliquot of stan-
Atomic polarizabilities are calculated within the MERA model.
Multiple linear regression algorithm with the forward stepwise
method was exploited to build a QSAR model correlating the RDF‐
based descriptors and IC50 values.
6
dardized concentration of cultures with OD560 = 0.02, 1.0 × 10 CFU/ml
for each well and incubated at 37°C for 4 h without shaking) and then
[54,55]
incubated for 24 h at 37°C under sterile condition.
After the com-
4.6
|
Absorption, distribution, metabolic liability
pletion of the incubation period, the culture medium was discarded and
washed with phosphate‐buffered saline to remove nonadherent bacteria.
Now, each well of the microtiter plate was stained with 0.1% of crystal
violet solution (100 µl) and re‐incubated at room temperature for 30 min.
Furthermore, the additional crystal violet solution from the plate was
cast off and the disc was washed with distilled water, followed by air
drying. Then, 100 µl of 95% ethanol was added into the well to solubi-
lize stained crystal violet, and the absorbance was measured using a
prediction, and excretion studies
The ADME computations for log P, TPSA, Lipinski violation, H donor,
H acceptor, water solubility score, gastrointestinal absorption score,
and the possibility of metabolism at different sites of CYP450 were
[
70]
carried out using online platform www.chemosophia.com,
pkCSM
[
44]
[71]
predictor,
and Swissadme.ch.
Furthermore, participation of
different atoms of the derivatives in metabolic liability at cytochrome
[45]
[56]
TRIAD Multimode Reader at 540 nm against a blank.
P450 was determined using the web server of RS‐Predictor.

PATHAK ET AL.
17 of 18
|
4.7
|
Molecular docking
Vladimir Potemkin
http://orcid.org/0000-0002-5244-8718
Amita Verma
http://orcid.org/0000-0001-6653-7335
[
72]
AutoDockTools 4 script prepare_ligand4.py
was used to prepare
the optimized molecules for docking, saving them in the pdbqt file
format. The crystal structure of the DNA gyrase complexed with
drug ciprofloxacin and DNA (PDB ID: 2XCT) was downloaded from
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Jurica Novak
https://orcid.org/0000-0002-6160-9755
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Parjanya K. Shukla
Maria Grishina
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