Design, synthesis, antimicrobial evaluation and molecular modeling study of 1,2,4-triazole-based 4-thiazolidinones

Article abstract of DOI:10.3390/molecules21050568

A series of 3-(2H-1,2,4-triazol-5-yl)-1,3-thiazolidin-4-one derivatives (7c-l) was designed and synthesized. Their structures have been elucidated based on analytical and spectral data. They were evaluated for their antibacterial and antifungal activities. Compound 7h showed the highest activity against all tested strains, except P. vulgaris, with MIC 8 μg/mL and 4 μg/mL against S. aureus and C. albicans, respectively. Furthermore, Compounds 7c, 7h, and 7j demonstrated moderate anti-mycobacterium activity. The binding mode of the synthesized thiazolidinones to bacterial MurB enzyme was also studied. Good interactions between the docked compounds to the MurB active site were observed primarily with Asn83, Arg310, Arg188 and Ser82 amino acid residues.

Full text of DOI:10.3390/molecules21050568

molecules
Article
Design, Synthesis, Antimicrobial Evaluation and
Molecular Modeling Study of 1,2,4-Triazole-Based
4-Thiazolidinones
Sahar Ahmed 1,2,*, Mohamed F. Zayed 1,3, Shahenda M. El-Messery 1,4
,
Mohamed H. Al-Agamy 5,6 and Hamdy M. Abdel-Rahman 2
1
Department of Pharmacognosy and Pharmaceutical Chemistry, College of Pharmacy, Taibah University,
Al-Madinah, Al-Munawarah 30001, Saudi Arabia; mfzayed25@yahoo.com (M.F.Z.);
shahenda76@yahoo.com (S.M.E.-M.)
2
3
4
5
6
Department of Medicinal Chemistry, Faculty of Pharmacy, Assiut University, Assiut 71526, Egypt;
hamdym01@yahoo.com
Pharmaceutical Chemistry, Department of Pharmaceutical Chemistry, Faculty of Pharmacy,
Al-Azhar University, Cairo 11884, Egypt
Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Mansoura University,
Mansoura 35516, Egypt
Department of Pharmaceutics, College of Pharmacy, King Saud University, P. O. Box 2457,
Riyadh 11451, Saudi Arabia; elagamy71@yahoo.com
Department of Microbiology and Immunology, Faculty of Pharmacy, Al-Azhar University,
Nasr City, Cairo 11884, Egypt
*
Correspondence: smahmed@taibahu.edu.sa; Tel.: +96-656-864-5454
Academic Editor: Peter J. Rutledge
Received: 17 March 2016; Accepted: 22 April 2016; Published: 30 April 2016
Abstract: A series of 3-(2H-1,2,4-triazol-5-yl)-1,3-thiazolidin-4-one derivatives (7c–l) was designed
and synthesized. Their structures have been elucidated based on analytical and spectral data.
They were evaluated for their antibacterial and antifungal activities. Compound 7h showed the
highest activity against all tested strains, except P. vulgaris, with MIC 8
µg/mL and 4
µg/mL against
S. aureus and C. albicans, respectively. Furthermore, Compounds 7c
,
7h, and 7j demonstrated
moderate anti-mycobacterium activity. The binding mode of the synthesized thiazolidinones to
bacterial MurB enzyme was also studied. Good interactions between the docked compounds to the
MurB active site were observed primarily with Asn83, Arg310, Arg188 and Ser82 amino acid residues.
Keywords: 4-thiazolidinones; 1,2,4-triazoles; antibacterial agents; antifungal agents; molecular docking
1. Introduction
Despite the significant advances in the field of antimicrobial agents ever since the discovery of
penicillin, infections are still the second-leading cause of death worldwide and remain an important
public health problem [
bacterial pathogens, particularly Mycobacterium tuberculosis and Staphylococcus aureus, highlights the
need for new classes of antimicrobial agents [ ]. These new classes would either be of a different
chemical structure or a modified structure compared to those of existing agents [ ]. Peptidoglycan is an
essential component of the bacterial cell wall. It confers mechanical resistance to higher internal osmotic
pressure and maintains a defined cell shape [ ]. Therefore, drugs interfering with the biosynthesis and
1,2]. Consequently, the rapid emergence of drug-resistant strains of different
3
4
5
assembly of peptidoglycan are effective antimicrobial agents. The structure of peptidoglycan is unique
to prokaryotic cells and, thus, optimal for the selective targeting of vital microbial pathways [6].
Uridine diphosphate UDP-N-Acetylenolpyruvylglucosamine reductase (Mur) enzymes are
integral components in bacterial peptidoglycan biosynthesis that provide valuable targets for specific
Molecules 2016, 21, 568; doi:10.3390/molecules21050568
www.mdpi.com/journal/molecules

Molecules 2016, 21, 568
2 of 17
bacterial cell wall inhibitors. There is a growing interest in MurB inhibitors, as it is essential for
the viability of bacterial cells without any homologue in eukaryotic cells. A variety of MurB small
molecule inhibitors with antibacterial activity have already been reported (Figure 1) [7]. Substituted
4-thiazolidinones and their analogues working as MurB inhibitors have received great attention.
Trisubstituted 4-thiazolidinones inhibited the Escherichia coli MurB enzyme at a low micromolar level,
most probably acting as diphosphate surrogates for the natural substrate (Figure 1) [8]. The related
imidazolidinone also inhibited MurB enzyme, showing good antibacterial activity (Figure 1) [9].
Figure 1. Examples of small molecule MurB inhibitors with their reported antibacterial activity.
The SAR of the antibacterial activity of the 1,3-thiazolidin-4-one scaffold has become the subject
of intense research projects [10,11]. Exploration of the 1,3-thiazolidin-4-ones showed that their
antibacterial activity not only depended on the 1,3-thiazolidin-4-one pharmacophore, but also on the
nature and position of its substituents; for example, electron withdrawing group substitutions on
aromatic rings of biphenyl-4-thiazolidinone derivatives showed improved antibacterial activity [11].
Numerous new derivatives with the pharmacophoric 4-thiazolidinone ring were substituted with
five-membered heterocyclic rings, [12] such as thiadiazole, [13] thiazoles [14] or 1,2,4-triazoles [15], as
seen in Figure 2. Moreover, 1,2,4-triazoles are core structures in medicinal chemistry research with
diverse biological activities [16–18].
Based on the studies mentioned above, and in continuance of our previous work [15],
herein, we describe the rational design, synthesis and characterization of new substituted
4-thiazolidinone-1,2,4-triazole hybrids towards searching for new compounds of promising
antimicrobial activity. The synthesized compounds were evaluated against different Gram-positive and
Gram-negative bacteria strains. Their antimycobacterial activity was evaluated against M. fortuitum.
The prepared compounds were also evaluated for their antifungal activity against Candida albicans.
Additionally, molecular modeling studies were also carried out to shed light on the binding mode of
the synthesized compounds to the MurB enzyme by docking into the crystal structure of S. aureus
MurB (PDB 1HSK) [19].

Molecules 2016, 21, 568
3 of 17
Figure 2. Rationale and design of title Compounds 7c–l based on the thiazolidin-4-one and the
triazole scaffold.
2. Results and Discussion
2.1. Chemistry
Simple and straightforward synthetic procedures were adopted for the synthesis of our target
4-thiazolidinone derivatives. The key intermediates 3-amino-5-substituted-1,2,4-triazoles 5a were
synthesized in accordance with the previously-developed chemistry (Scheme 1) [20 21]. Synthesis
of 1,3-thiazolidin-4-ones involves three main components: an amine, a carbonyl compound and a
mercapto-acid, the synthetic process either prepared in a one-pot three-component condensation [15
or in a two-step process, which we followed (Scheme 2). The reaction pathway involves the in situ
formation of intermediate 5-triazolylimines (6c ) by condensation of the amine and the appropriate
aldehydes [22]. The produced imines 6c undergo attack by generated sulfur nucleophile when
refluxed with mercaptoacetic acid followed by the intramolecular cyclization to yield the substituted
1,3-thiazolidin-4-ones 7c . All of the synthesized compounds were characterized by spectroscopic
techniques (1H-NMR, 13C-NMR and mass spectra), as well as elemental analyses. The spectral data
–b
,
]
–l
–
l
–
l
of the synthesized compounds (7c–l) were in agreement with their proposed structures. The IR
data clearly showed a strong C=O stretching band around 1730 cm´1 and 1259 cm´1, which we´re1
characteristic for 4-thiazolidinones, in addition to the aromatic C-H absorption band around 3030 cm
along with the absence of the characteristic fork-shaped band of the free amino group for 5a–b. The
1H-NMR data indicated the presence of characteristic peaks for 4-thiazolidinone protons of the doublet
peak CHb of CH2 around 4.15 ppm and a doublet of the doublet peak for CHa at around 4.08 ppm of
CH2 for 4-thiazolidinone. A more downfield doublet peak of the CH proton of the 4-thiazolidinone
ring was observed at about 5.6 ppm. Characteristic peaks for aromatic protons appear in their expected
range of 6–7 ppm. 13C-NMR of synthesized compounds showed characteristic peaks for carbon 2 and
carbon 5 of the thiazolidinone ring at their expected chemical shift δ 34 and 73 ppm, respectively, while
its C4 appeared as a downfield signal at around δ 170 ppm.

Molecules 2016, 21, 568
4 of 17
O
O
O
NH2
OH
i
N
ii
OCH3
R
R
R
H
1
3
2
O
N
NH
H
N
NH2
NH
N
H
NH2
iii
iv
R
R
N
4
5 a-b
Scheme 1. Synthesis of starting compounds (5a
,
b). Reagents and conditions: (i) HCl gas, methanol;
(
ii) H2NNH2, absolute ethanol, reflux 5 h; (iii) S-methylisothiourea sulfate; (iv) 5% aqueous NaOH
reflux 3 h; neutralize with HCl.
O
Ha
NH
N
N
Hb
N
S
NH
NH
N
N
ii
i
N
NH2
R
N
N
R1
R1
(7c-l)
R
R
(6c-l)
(5a-b)
(6 & 7)c R = 3-Cl, R1 = 4-Cl
(6 & 7)d R = 3-Cl, R1 = 4-F
(6 & 7)e R = 3-Cl, R1 = 4-NO2
(6 & 7)f R = 3-Cl, R1 = 4-OCH3
(6 & 7)g R = 3-Cl, R1 = 4-CH3
(6 & 7)h R = 4-OCH3, R1 = 4-Cl
(6 & 7)i R = 4-OCH3, R1 = 4-F
(6 & 7)j R = 4-OCH3, R1 = 4-NO2
(6 & 7)k R = 4-OCH3, R1 = 4-OCH3
(6 & 7)l R = 4-OCH3, R1 = 4-CH3
a: R = 3-Cl
b: R = 4-OCH3
Scheme 2. Synthesis of target compounds (7c–l); Reagents and conditions: (i) Ar-CHO, HOAc, reflux
10–12 h; (ii) mercaptoacetic acid, THF, pinch ZnCl2; reflux, 12 h.
2.2. Biological Studies
2.2.1. Antimicrobial Activity
Primary screening for antimycobacterial activity of all newly-synthesized compounds was
performed against Mycobacterium fortuitum at a concentrations ranging from 0.0625–128 g/mL using
moxifloxacin as the standard drug (Table 1). Compound 7h was found to be the most active against
Mycobacterium fortuitum with an MIC value of 32 g/mL with a diameter of growth of inhibition zone
(IZ) of 25 mm compared to 26 mm (IZ) of moxifloxacin. On the other hand, Compounds 7c showed
variable activities. Weak activity was observed for Compounds 7d with an IZ range from 9–12 mm.
Good activities were observed for Compounds 7c 7j and 7h with the IZ varying from 20–25 mm
with an MIC between 32 and 64 g/mL. Different variations on R and R1 gave compounds with a
µ
µ
–
j
–
g
,
µ
range of antimycobacterial activity. The antimycobacterial activity of the synthesized compounds
against Mycobacterium fortuitum was observed when R was 4-methoxy, and R1 groups were in the
order: 4-chloro > 4-nitro > 4-fluoro. Furthermore, when R was 3-chloro, R1 groups show in the order:
4-chloro > 4-methoy > 4-nitro and 4-fluoro (Scheme 2).

Molecules 2016, 21, 568
5 of 17
Table 1. In vitro antimicrobial activity of the test compounds using the inhibition zone (IZ) and MIC.
Gram-negative Bacteria Gram-Positive Bacteria
Fungi
Pseudomonas
aeruginosa
ATCC 27857
Staphylococcus
aureus
ATCC 29213
Enterococcus
faecalis
ATCC 29212
Mycobacterium
fortuitum
ATCC 6841
Compound
cLogP a
Escherichia coli
ATCC 25922
Proteus vulgaris
Bacillus subtilis
ATCC 10400
Candida albicans
No.
atcc 6380
ATCC 2091
IZ
MIC
IZ
MIC
IZ
MIC
IZ
MIC
IZ
MIC
IZ
MIC
IZ
MIC
IZ
MIC
7c
7d
7e
7f
7g
7h
7i
7j
7k
5.12
4.55
4.15
4.32
4.41
3.84
3.44
3.62
4.19
4.91
17
Nil
Nil
Nil
Nil
30
19
22
17
23
64
ND
ND
ND
ND
16
64
32
64
32
Nil
Nil
Nil
Nil
Nil
Nil
Nil
Nil
Nil
Nil
25
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ď0.25
ND
ND
Nil
Nil
Nil
Nil
Nil
20
18
16
17
18
ND
ND
ND
ND
ND
32
32
32
32
32
16
Nil
Nil
Nil
Nil
21
14
16
15
17
128
ND
ND
ND
ND
8
128
16
128
64
Nil
Nil
Nil
Nil
Nil
19
Nil
Nil
Nil
Nil
ND
25
ND
ND
ND
ND
ND
16
ND
ND
ND
ND
ND
0.5
Nil
Nil
Nil
Nil
Nil
15
Nil
Nil
Nil
Nil
ND
27
ND
ND
ND
ND
ND
16
ND
ND
ND
ND
ND
0.094
ND
20
9
9
10
9
25
12
22
Nil
Nil
ND
26
ND
64
>128
>128
>128
>128
32
>128
64
ND
ND
ND
0.125
ND
Nil
Nil
Nil
Nil
Nil
20
19
18
16
20
ND
ND
ND
ND
ND
4
16
16
16
16
7l
CIPRO
MOX
FL
27
ND
ND
ď0.25
ND
ND
29
ND
ND
ď0.25
ND
ND
ND
28
ND
ND
<0.002
ND
ND
ND
28
ND
ND
0.5
ND
ND
ND
ND
ND
Antimicrobial activities were expressed as the inhibition zone (IZ) in millimeters (mm); Nil means (no activity) <3 mm; (weak) = 6–12 mm; (moderate) = 13–15 mm; (strong) = 16–20 mm
;
and (very strong) >21 mm; the experiment was carried out in triplicate, and the average zone of inhibition was calculated at a concentration of 512 g/100 L; MIC = minimum
µ
µ
inhibitory concentration; MIC expressed in µg/mL; MIC was done by the cup plate method according to European Committee on Antimicrobial Susceptibility testing (EUCAST);
CIPRO = ciprofloxacin; MOX = moxifloxacin, FL = fluconazole; ND = not determined, Nil = no zone of inhibition = no antimicrobial activity, a Calculated values using ChemDraw
Ultra 8.0 software.

Molecules 2016, 21, 568
6 of 17
2.2.2. Antibacterial Activity
Antibacterial activities of Compounds 7c
–
l
were evaluated against six different groups of
bacterial strains. The utilized microorganisms were Enterococcus faecalis, Bacillus subtilis and
Staphylococcus aureus as Gram-positive strains and Pseudomonas aeruginosa, Proteus vulgaris and
Escherichia coli as Gram-negative strains. Ciprofloxacin and Moxifloxacin were used as positive
control drugs. The antibacterial activity was assessed using both the inhibition zone (IZ) and the
minimal inhibitory zone (MIC). As shown in Table 1, Compounds 7h–l showed moderate to very good
broad-spectrum antibacterial activity, while Compounds 7d showed no activity against all tested
–
g
strains. Furthermore, it was noted that none of the tested compounds showed activity against the
Gram-negative microorganism Proteus vulgaris. Compound 7h showed the highest activity, and it was
effective against all tested strains, except Proteus vulgaris. This compound was the most active against
Escherichia coli with an inhibition zone (IZ = 30 mm) greater than the standard drug ciprofloxacin
(IZ = 27 mm) at the same tested concentration. Compound 7c showed moderate activity with ~61%
inhibition zone compared to the standard drugs against Staphylococcus aureus and Escherichia coli. The
MIC, as well as the zone of inhibition values for each compound are explained in Table 1 and Figure 3.
All MIC values of the active compounds were found to be higher than those of ciprofloxacin and
moxifloxacin. Most of the 1,2,4-triazole derivatives described here revealed activity that was found to
have good MIC values (8–128
µg/mL), as shown in Table 1. The best MIC value observed for 7h was
8
µg/mL against Staphylococcus aureus. Lipophilicity is an important physicochemical parameter in
the development of an antibacterial agent, because it is known to be closely related to the permeation
through the lipid coat of bacteria. The title compounds showed very good lipophilic characteristics
with p-values of 3.4–5.2 (Table 1). From the structural point of view, it seems that the presence of a
methoxy substituent at position R and fluorine, nitro, methoxy and methyl at the R1 position produce
derivatives with promising antibacterial activity (Scheme 2). The 3-chloro substituent at position R
produced promising antibacterial activity when R1 = Cl, while other substitutions did not give any
antibacterial activity.
Figure 3. Graphical representation of the zone of inhibition (mm) of the compounds (7c–l) and reference
antimicrobial drugs against different bacterial and fungal strains.

Molecules 2016, 21, 568
7 of 17
2.2.3. Antifungal Activity
Preliminary screening of Compounds 7c
–l
revealed good antifungal activity of the five derivatives
7h–l against Candida albicans. The best activity was observed for Compounds 7h and 7l with IZ of
20 mm followed by 7i (IZ = 19 mm) then 7g (IZ = 18 mm), while 7k showed the least activity with
IZ of 16 mm compared to the 28-mm IZ of the standard drug fluconazole (Figure 3). The MIC of
Compound 7h against Candida albicans is 4
µg/mL compared to 0.5
µg/mL of fluconazole. On the
other hand, Compounds 7i showed MIC values of 16 µ
–
l
g/mL, whereas the rest of the compounds
did not show any antifungal activity at the tested concentration (Table 1).
2.2.4. Molecular Modeling Study
The X-ray crystallographic data of MurB, with UDP-sugar substrate, were used as guide surrogates
of the diphosphate moiety. MurB confirmed that both substrates use the same binding pocket of the
enzyme; this result is consistent with the role of the bound FAD cofactor in mediating hydride transfer
from the NADPH to the UDP-sugar substrate [23]. The maximum number of poses per ligand was set
to 30, and no constraints were used to perform molecular docking. The docking complex assumed to
represent ligand-receptor interactions was selected based on three criteria: (i) binding affinity score
of the pose; (ii) its orientation into the active site in a similar manner as the co-crystallized ligands
orientation; and (iii) the preservation of binding interactions, especially hydrogen bond formation and
lipophilic contacts. The estimated binding affinity of the co-crystallized ligand was
´
13.5795 Kcal/mol
with of complex hydrogen network with a plethora of amino acids including, Arg310, Val199, Ala117,
Gly153, Gly70, Asn83, Ser142, Ser82, Arg188, Gly133 and Arg228, giving a clue about the importance
of hydrogen bond formation for effective enzyme binding.
The synthesized compounds were docked into the binding site of the MurB enzyme pocket
and will be presented according to their better MIC values; however, there was a lack of accordance
with matching MIC, which is logical, since the MIC is complex and unrelated to specific inhibition
to any target enzyme. Modeling studies suggest that Compound 7h binds to the MurB active site
with a binding affinity of
´
12.18234 Kcal/mol via the hydrogen bonding interaction of the NH of
triazole ring group with Asn83 with (36%, 1.75 Å) and Arg225 with the oxygen of methoxy group
with (34% 2.38 Å), while it also shows cationic-arene interaction with the Arg188 residue (Figure 4).
Compound 7j showed a hydrogen bond interaction network with a triad of amino acids Asn83 (13%,
2.06 Å), Ser82 (23%, 1.91 Å) and Gly81 (40%, 1.61 Å) via the NH group of the triazole ring and the
carbonyl group of the thiazolidine ring, respectively, with a binding affinity of
addition to a cationic arene interaction with the aromatic ring with the Arg310 amino acid (Figure 4).
Compound 7l has a binding affinity of 12.42222 Kcal/mol through the hydrogen bond interaction of
´12.81985 Kcal/mol; in
´
(61%, 1.4 Å) and (17%, 2.22 Å) with Arg310 and Ser82 via methoxy oxygen and the carbonyl group of
the thiazolidine ring, respectively (Figure 5). Compound 7c gives two kinds of interactions: one is the
hydrogen bonding interaction of (35%, 2.32 Å) with the Ser143 amino acid; the second is a cationic
arene interaction with Arg188 (Figure 5) with a binding affinity of
showed a triad hydrogen bonding interaction with Gly81, Ser82 and Gln193 with 44%, 14% and 12%,
respectively (Figure 6).
´
11.64368, while Compound 7i
Compound 7k provided a hydrogen bond interaction via Gln193 (28%) and Arg310 a
cationic-arene interaction with a binding energy of
Compounds 7d and 7e are examples of inactive antibacterial agents that did not show any binding to
´
12.71658 Kcal/mol (Figure 6). On the other hand,
the MurB active site (Figure 7).

Molecules 2016, 21, 568
8 of 17
Figure 4. 2D binding mode interaction and residues involved in the recognition of the most promising
Compounds 7h and 7j into MurB binding site interaction showing the percentage of hydrogen bonds.
(7l)
(7c)
Figure 5. 2D binding mode interaction and residues involved in the recognition of moderate
active Compounds 7l and 7c into the MurB binding site interaction showing the percentage of
hydrogen bonds.

Molecules 2016, 21, 568
9 of 17
Figure 6. 2D binding mode interaction and residues involved in the recognition of moderate
active Compounds 7i and 7k into the MurB binding site interaction showing the percentage of
hydrogen bonds.
(7d)
(7e)
Figure 7. 2D binding mode interaction and residues involved in the recognition of the non-active
Compounds 7d and 7e, into the MurB binding site.

Molecules 2016, 21, 568
10 of 17
The docking study indicated that the important interactions of the compounds under study have
resemblance to the binding mode with the FAD flavin ring system co-crystallized with enzyme with
Asn83 and Arg310 or Arg188 and Ser82 amino acids, so they appear to provide a structural platform
resulting in the tested compound’s proximity to FAD and its hydrogen bond to the main chain of these
amino acids.
Analysis of the docking results suggests that the promising antimicrobial activity of the tested
compounds could be attributed to: (i) higher binding affinity, but still less than the reference ligand;
(ii) the incorporation with amino acids is similar to the co-crystallized ligand, especially the hydrogen
bonding interactions. These findings are considered encouraging, providing a possible mechanism of
action for the tested compounds and indicating the possibility of developing novel antibacterial agents
targeting an early step in peptidoglycan biosynthesis.
It was reported that the lipophilic characteristic of the previously-tested compounds could play
an important role in their binding to the MurB active site [24]. Therefore, it was interesting to perform
a deeper investigation of our compounds for the MurB binding site by studying the hydrophobic
surface mapping of some tested candidates to get a clue about their lipophilic characteristic, which
may imply their binding affinity to the binding pocket. In Figure 8, the surface map of the active site
for the selected MurB enzyme along with the docked conformation of Compound 7h are illustrated,
showing a higher space of greener areas in the enzyme binding pocket, indicating a highly lipophilic
active site. Figure 9 shows the surface mapping of some promising antimicrobials, Compounds 7h
,
7j and 7l, where the phenyl group was chosen for its steric and/or hydrophobic interaction with the
protein residues. Moreover, the substitution on the phenyl ring of the parent molecule was selected
as the hydrogen bond acceptor or hydrogen bond donor to form a bond or to impart hydrophobicity.
Figures 8 and 9 are complementary to each other, showing the higher lipophilic characteristics of
both active sites and the most promising synthesized candidates that may be responsible for their
interaction with the amino acid residues of the enzyme active pocket, especially with the presence of
lipophilic moieties, such as the methoxy and chloro groups.
Figure 8. Surface map for the MurB active site with the docked conformation of Compound 7h
(red stick) (pink: hydrogen bond; blue: mild polar; green: hydrophobic region).

Molecules 2016, 21, 568
11 of 17
Figure 9. Surface map for Compounds 7h, 7j and 7l (pink: hydrogen bond; blue: mild polar; green:
hydrophobic region).
3. Materials and Methods
3.1. General
Melting points were measured on a Griffin melting point apparatus (Griffin, Valdosta, GA, USA)
and are uncorrected. The infrared spectra were recorded as KBr discs on a Nicolet IR 200 (Thermo
1
Fisher Scientific, Barrington, RI, USA). The H-NMR and 13C-NMR spectra were run using TMS
as an internal standard (Aldrich chemical Co., Milwaukee, WI, USA) on a Varian Mercury VXr-300
NMR (Varian, Palo Alto, CA, USA). Mass spectra were measured on a JEOL-SX-102 instrument using
electron impact ionization. Elemental analysis were performed on a Perkin-Elmer 240C analyzer
(Perkin-Elmer, Norwalk, CT, USA) at the Analytical Center, College of Science, Cairo University, Egypt.
All values were within
˘
0.4% of the theoretical values. The reactions were monitored by thin-layer
chromatography (TLC) using TLC sheets precoated with UV fluorescent silica gel Merck 60 F254 plates
and were visualized using a UV lamp and different solvents as mobile phases. All chemicals were
purchased from (Sigma-Aldrich, Milwaukee, WI, USA).
3.1.1. General Method
Title compounds 7c–l, as well as intermediates, were prepared in the following steps: synthesis of
3-amino-5-(substituted phenyl)-2H-1,2,4-triazol (5a,b); Compound 5a,b was prepared according to the
literature procedure [20,21].
3.1.2. General Method for Synthesis of Schiff Bases (6c–l)
A mixture of an equimolar amount of amine (5a,b) (0.01 mole) and the aromatic aldehyde
(0.01 moles) in 50 mL acetic acid was refluxed for about 10–12 h on an oil bath. The reaction mixture
was cooled, poured into ice water, extracted with ethyl acetate and, finally, dried over anhydrous
sodium sulfate. The solvent was evaporated to give the solid product that was crystallized from ethyl
acetate-hexane using decolorizing charcoal. The obtained compounds (6c–l) were pure enough to
proceed to the next steps without further purification.

Molecules 2016, 21, 568
12 of 17
3.1.3. General Method for the Synthesis of the Compounds (7c–l)
A mixture of Schiff bases (6c–l) (0.01 mol) in THF (30 mL) and mercapto acetic acid (thioglycolic
acid) (0.01 mol) with a pinch of anhydrous ZnCl2 was refluxed for 12 h. The solvent was then removed
to get a residue, which was dissolved in benzene and passed through a column of silica gel using
benzene:chloroform (8:2 v/v) mixture as the eluent. The eluate was concentrated and the product
crystallized using alcohol to give 4-thiazolidinones (7c–l).
2-(4-Chlorophenyl)-3-(5-(3-chlorophenyl)-2H-1,2,4-triazol-3-yl)thiazolidin-4-one (7c): Yield, 68%; m.p.:
˝
164–166 C. IR (KBr, cm´1): 3065 (CH aromatic), 1720 (C=O), 1259 (C-S). 1H-NMR (DMSO-d6, 300 MHz):
δ
= 3.22 (d,1H,-CHb of CH2 of thiazolidinone), 4.12 (dd, J = 1.4 Hz, 1H, -Cha of CH2 of thiazolidinone),
5.81 (d. 1H, C of thiazolidinone), 7.01–7.52 (m, 4H, Ar-H), 7.83 (dd, 4H, J = 7.9 Hz, 1.5, Ar-H), 8.6
(s, 1H, N = 34.22 (CH2-C3H3NOS), 68.23 (CH-C3H3NOS),
), 13C-NMR (DMSO-d6, 300 MHz):
H
H
δ
121.77 (CH-C6H5), 124.26 (CH-C6H5), 126.83 (CH-C6H5), 127.41 (CH-C6H5), 128.19 (CH-C6H5),
128.96 (CH-C6H5), 129.18 (CH-C6H5), 129.02 (CH-C6H5), 130.17 (C-C6H5), 131.81 (C-C6H5), 132.43
(C,4Cl-C6H5), 138.76 (C,3Cl-C6H5), 163.04 (C-C2HN3), 164.71 (C-C2HN3), 175.78 (CO-C3H3NOS), MS
(m/z): 391.01 [M]+. Analysis for C17H12Cl2N4OS (Molecular weight (m. w.) 390.01); Calcd.: C, 52.18;
H, 3.09; N, 14.32. Found: C, 52.44; H, 3.13; N, 14.51.
3-(5-(3-Chlorophenyl)-2H-1,2,4-triazol-3-yl)-2-(4-fluorophenyl)thiazolidin-4-one (7d): Yield, 65%; m.p.:
˝
1
170–172 C. IR (KBr, cm´1): 3068 (CH aromatic), 1730 (C=O), 1256 (C-S). H-NMR (DMSO-d6,
300 MHz):
thiazolidinone), 5.68 (d. 1H, C
1.4 Hz, Ar-H), 88.3 (s, 1H, N
), 13C-NMR (DMSO-d6, 300 MHz):
δ
= 4.21 (d,1H,-CHb of CH2 of thiazolidinone), 4.34 (dd, J = 1.2 Hz, 1H, -CHa of CH2 of
of thiazolidinone), 7.12–7.71 (m, 4H, Ar-H), 7.91 (dd, 4H, J = 7.7
=33.24 (CH2-C3H3NOS), 68.15
H
,
H
δ
(CH-C3H3NOS), 121.73 (CH-C6H5), 125.11 (CH-C6H5), 126.24 (CH-C6H5), 127.64 (CH-C6H5), 128.16
(CH-C6H5), 128.79 (CH-C6H5), 129.12 (CH-C6H5), 129.75 (CH-C6H5), 130.11 (C-C6H5), 131.21 (C-C6H5),
132.03 (C,3-Cl-C6H5), 155.76 (C-C2HN3), 163.12 (C-C2HN3), 164.04 (C,4F-C6H5), 176.71 (CO-C3H3NOS),
MS (m/z): 375.04 [M]+. Analysis for C17H12ClFN4OS (m. w. 374.04); Calcd.: C, 54.47; H, 3.23; N, 14.95.
Found: C, 54.51; H, 3.42; N, 14.82.
3-(5-(3-Chlorophenyl)-2H-1,2,4-triazol-3-yl)-2-(4-nitrophenyl)thiazolidin-4-one (7e): Yield, 66%; m.p.:
˝
178–180 C. IR (KBr, cm´1): 3085 (CH aromatic), 1735 (C=O), 1257 (C-S). 1H-NMR (DMSO-d6, 300 MHz):
δ
= 4.09 (d,1H,-CHb of CH2 of thiazolidinone), 4.17 (dd, J = 1.3 Hz, 1H, -CHa of CH2 of thiazolidinone),
5.74 (d. 1H, C of thiazolidinone), 7.11–7.62 (m, 4H, Ar-H), 7.87 (dd, J = 8.1, 1.7 Hz, 4H, Ar-H),
8.1 (s, 1H, N =34.51(CH2-C3H3NOS), 68.32 (CH-C3H3NOS),
), 13C-NMR (DMSO-d6, 300 MHz):
H
H
δ
121.41 (CH-C6H5), 124.52 (CH-C6H5), 125.94 (CH-C6H5), 127.41 (CH-C6H5), 128.34 (CH-C6H5), 128.81
(CH-C6H5), 129.21 (CH-C6H5), 129.42 (CH-C6H5), 130.37 (C-C6H5), 130.13 (C,3-Cl-C6H5), 141.31
(C-C6H5), 147.28 (C,4-NO2-C6H5), 164.47 (C-C2HN3), 165.68 (C-C2HN3), 173.82 (CO-C3H3NOS), MS
(m/z): 402.03 [M]+. Analysis for C17H12ClN5O3S (m. w. 401.03); Calcd.: C, 50.81; H, 3.01; N, 17.43.
Found: C, 50.62; H, 3.42; N, 17.24.
3-(5-(3-Chlorophenyl)-2H-1,2,4-triazol-3-yl)-2-(4-methoxyphenyl)thiazolidin-4-one (7f): Yield, 62%; m.p.:
˝
207–209 C. IR (KBr, cm´1): 3068 (CH aromatic), 1732 (C=O), 1255 (C-S). 1H-NMR (DMSO-d6, 300 MHz):
δ
= 3.74 (s, 3H, OC
of thiazolidinone), 5.84 (d. 1H, C
4H, Ar-H), 8.54 (s, 1H, N
), 13C-NMR (DMSO-d6, 300 MHz):
H
3), 4.24 (d,1H,-CHb of CH2 of thiazolidinone), 4.38 (dd, J = 1.3 Hz, 1H, -CHa of CH2
of thiazolidinone), 7.13–7.62 (m, 4H, Ar-H), 7.92 (dd, J = 8.2, 1.4 Hz,
= 34.2 (CH2-C3H3NOS), 54.23 (C-OCH3),
H
H
δ
68.24 (CH-C3H3NOS), 116.78 (CH-C6H5), 116.82(CH-C6H5), 126.41 (CH-C6H5), 127.41 (CH-C6H5),
128.61 (CH-C6H5), 128.96 (CH-C6H5), 129.21 (CH-C6H5), 129.47 (CH-C6H5), 131.31 (C-C6H5), 133.74
(C-C6H5), 136.25 (C,3-Cl-C6H5), 157.02 (C,4-OCH3-C6H5), 164.22 (C-C2HN3), 164.51 (C-C2HN3), 172.89
(CO-C3H3NOS), MS (m/z): 387.06 [M]+. Analysis for C18H15ClN4O2S (m. w. 386.06); Calcd.: C, 55.88;
H, 3.91; N, 14.48. Found: C, 55.93; H, 3.78; N, 14.27.

Molecules 2016, 21, 568
13 of 17
3-(5-(3-Chlorophenyl)-2H-1,2,4-triazol-3-yl)-2-p-tolylthiazolidin-4-one (7g): Yield, 64%; m.p.: 225–227 ˝C.
IR (KBr, cm´1): 3070 (CH aromatic), 1745 (C=O), 1258 (C-S). 1H-NMR (DMSO-d6, 300 MHz):
= 2.36
(s, 3H, C 3), 4.16 (d,1H,-CHb of CH2 of thiazolidinone), 4.28 (dd, J = 1.2 Hz, 1H, -Cha of CH2 of
thiazolidinone), 5.57 (d. 1H, C of thiazolidinone) 7.08–7.79 (m, 4H, Ar-H), 7.95 (dd, 4H, J = 8.5, 1.8 Hz,
Ar-H), 8.14 (s, 1H, N = 22.78 (C-CH3), 35.32 (CH2-C3H3NOS),
), 13C-NMR (DMSO-d6, 300 MHz):
δ
H
H
H
δ
60.92 (CH-C3H3NOS), 123.77 (CH-C6H5), 124.41 (CH-C6H5), 126.42 (CH-C6H5), 126.78 (CH-C6H5),
127.1 (CH-C6H5), 127.44 (CH-C6H5), 130.29 (CH-C6H5), 130.71 (CH-C6H5), 131.05 (C-C6H5), 132.47
(C,3-Cl-C6H5), 135.72 (C-C6H5), 136.47 (C-C6H5), 157.71 (C-C2HN3), 158.94 (C-C2HN3), 175.48
(CO-C3H3NOS), MS (m/z): 371.07 [M]+. Analysis for C18H15ClN4OS (m. w. 370.07); Calcd.: C,
58.30; H, 4.08; N, 15.11. Found: C, 58.45; H, 4.19; N, 14.96.
2-(4-Chlorophenyl)-3-(5-(4-methoxyphenyl)-2H-1,2,4-triazol-3-yl)thiazolidin-4-one (7h): Yield, 65%; m.p.:
˝
211–213 C. IR (KBr, cm´1): 3079 (CH aromatic), 1740 (C=O) 1256 (C-S). 1H-NMR (DMSO-d6, 300 MHz):
δ
= 3.81 (s, 3H, OC
of CH2 of thiazolidinone), 5.71 (d. 1H, C
J = 8.7, 2.1 Hz, Ar-H), 8.4 (s, 1H, N
), 13C-NMR (DMSO-d6, 300 MHz):
H
3), 4.21 (d,1H,-CHb of CH2 of thiazolidinone), 4.36 (dd, J = 1.2 Hz, 1H, -CHa
of thiazolidinone), 7.11–7.64 (m, 4H, Ar-H), 7.87 (dd, 4H,
= 36.7 (CH2-C3H3NOS),
H
H
δ
52.43 (C-OCH3), 69.42 (CH-C3H3NOS), 121.57 (CH-C6H5), 123,89 (CH-C6H5), 127.98 (CH-C6H5),
128.71 (CH-C6H5), 128.89 (CH-C6H5), 129.24 (CH-C6H5), 129.41 (CH-C6H5), 130.87 (CH-C6H5), 131.61
(C-C6H5), 132.54 (C,4-Cl-C6H5), 137.97 (C-C6H5), 155.4 (C,4-OCH3-C6H5), 156.62 (C-C2HN3), 156.81
(C-C2HN3), 174.5 (CO-C3H3NOS), MS (m/z): 387.06 [M]+. Analysis for C18H15ClN4O2S (m. w. 386.06);
Calcd.: C, 55.88; H, 3.91; N, 14.48. Found: C, 56.04; H, 3.73; N, 14.51.
2-(4-Fluorophenyl)-3-(5-(4-methoxyphenyl)-2H-1,2,4-triazol-3-yl)thiazolidin-4-one (7i): Yield, 68%; m.p.:
˝
215–217 C. IR (KBr, cm´1): 3078 (CH aromatic), 1725 (C=O), 1259 (C-S). 1H-NMR (DMSO-d6, 300 MHz):
δ
= 3.81 (s, 3H, OCH3), 4.09 (d,1H,-CHb of CH2 of thiazolidinone), 4.18 (dd, J = 1.5 Hz, 1H, -CHa of CH2
of thiazolidinone), 5.61 (d. 1H, CH of thiazolidinone), 7.21–7.73 (m, 4H, Ar-H), 7.98 (dd, 4H, J = 8.5, 1.9,
Ar-H), 8.2 (s, 1H, N = 32.92 (CH2-C3H3NOS), 54.13 (C-OCH3),
), 13C-NMR (DMSO-d6, 300 MHz):
H
δ
67.83 (CH-C3H3NOS), 121.27 (CH-C6H5), 124.52 (CH-C6H5), 126.48 (CH-C6H5), 126.61 (CH-C6H5),
128.51 (CH-C6H5), 128.89 (CH-C6H5), 129.61 (CH-C6H5), 129.42 (CH-C6H5), 130.27 (C-C6H5), 132.84
(C-C6H5), 154.62 (C-C2HN3), 155.62 (C-C2HN3), 159.84 (C,4-F-C6H5), 163.81 (C,4-OCH3-C6H5), 174.18
(CO-C3H3NOS), MS (m/z): 371.09 [M]+. Analysis for C18H15FN4OS (m. w. 370.09); Calcd.: C, 58.37; H,
4.08; N, 15.13. Found: C, 58.46; H, 3.87; N, 15.47.
3-(5-(4-Methoxyphenyl)-2H-1,2,4-triazol-3-yl)-2-(4-nitrophenyl)thiazolidin-4-one (7j): Yield, 68%; m.p.:
˝
218–220 C. IR (KBr, cm´1): 3078 (CH aromatic), 1740 (C=O), 1257 (C-S). 1H-NMR (DMSO-d6, 300 MHz):
δ
= 3.87 (s, 3H, OC
of CH2 of thiazolidinone), 5.76 (d. 1H, C
J = 8.5, 1.6 Hz, Ar-H), 8.2 (s, 1H, N
), 13C-NMR (DMSO-d6, 300 MHz):
H
3), 4.15 (d,1H,-CHb of CH2 of thiazolidinone), 4.08 (dd, J = 1.5 Hz, 1H, -CHa
of thiazolidinone), 7.08–7.62 (m, 4H, Ar-H), 8.01 (dd, 4H,
= 30.92 (CH2- C3H3NOS),
H
H
δ
55.32 (C-OCH3), 65.93 (CH-C3H3NOS), 122.47 (CH-C6H5), 124.72 (CH-C6H5), 126.18 (CH-C6H5),
126.55 (CH-C6H5), 128.81 (CH-C6H5), 128.92 (CH-C6H5), 129.12 (CH-C6H5), 129.52 (CH-C6H5), 129.94
(C-C6H5), 129.98 (C-C6H5), 140.47 (C,4-NO2-C6H5), 153.52 (C-C2HN3), 154.87 (C-C2HN3), 164.61
(C,4-OCH3-C6H5), 176.28 (CO-C3H3NOS), MS (m/z): 398.08 [M]+. Analysis for C18H15N5O4S (m. w.
397.08); Calcd.: C, 54.40; H, 3.80; N, 17.62. Found: C, 54.64; H, 3.93; N, 17.41.
2-(4-Methoxyphenyl)-3-(5-(4-methoxyphenyl)-2H-1,2,4-triazol-3-yl)thiazolid-din-4-one (7k): Yield, 68%;
m.p.: 219–221 ˝C. IR (KBr, cm´1): 3085 (CH aromatic), 1730 (C=O), 1253 (C-S). 1H-NMR (DMSO-d6,
300 MHz):
4.23 (dd,1H, -CHa of CH2 of thiazolidinone), 5.79 (d. 1H, C
Ar-H), 8.09 (dd, 4H, J = 8.5, 1.6 Hz, Ar-H), 8.7 (s, 1H, N
), 13C-NMR (DMSO-d6, 300 MHz): δ = 32.26
δ
= 3.75 (s, 3H, OC
H
3), 3.97 (s, 3H, OC
H
3), 4.38 (d,1H,-CHb of CH2 of thiazolidinone),
H
of thiazolidinone), 7.21–7.72 (m, 4H,
H
(CH2-C3H3NOS), 52.44 (C-OCH3), 52.86 (C-OCH3), 67.28 (CH-C3H3NOS), 121.64 (CH-C6H5), 124.32
(CH-C6H5), 126.38 (CH-C6H5), 127.51 (CH-C6H5), 127.81 (CH-C6H5), 128.21 (CH-C6H5), 129.41

Molecules 2016, 21, 568
14 of 17
(CH-C6H5), 129.92 (CH-C6H5), 130.17 (C-C6H5), 131.56 (C-C6H5), 157.62 (C-C2HN3), 158.14 (C-C2HN3),
163.41 (C,4-OCH3-C6H5), 164.77 (C,4-OCH3-C6H5), 174.78 (CO-C3H3NOS), MS (m/z): 391.01 [M]+.
Analysis for C19H18N4O3S (m. w. 382.11); Calcd.: C, 59.67; H, 4.74; N, 14.65. Found: C, 59.47; H, 4.93;
N, 14.52.
˝
3-(5-(4-Methoxyphenyl)-2H-1,2,4-triazol-3-yl)-2-p-tolylthiazolidin-4-one (7l): Yield, 68%; m.p.: 216–218 C.
IR (KBr, cm´1): 3070 (CH aromatic), 1728 (C=O), 1254 (C-S). 1H-NMR (DMSO-d6, 300 MHz): δ = 2.24
(s, 3H, C
CH2 of thiazolidinone), 5.91 (d. 1H, C
J = 8.3, 1.5 Hz, Ar-H), 8.4 (s, 1H, N
), 13C-NMR (DMSO-d6, 300 MHz):
H
3), 3.76 (s, 3H, OC
H
3), 4.28 (d,1H,-CHb of CH2 of thiazolidinone), 4.21 (dd,1H, -Cha of
of thiazolidinone), 7.21–7.72 (m, 4H, Ar-H), 8.03 (dd, 4H,
= 26.14 (C-CH3), 31.92
H
H
δ
(CH2-C3H3NOS), 57.21 C-OCH3), 66.42 (CH-C3H3NOS), 121.57 (CH-C6H5), 124,82 (CH-C6H5), 125.88
(CH-C6H5), 126.91 (CH-C6H5), 128.11 (CH-C6H5), 128.79 (CH-C6H5), 129.61 (CH-C6H5), 129.82
(CH-C6H5), 130.07 (C-C6H5), 130.81 (C-C6H5), 134.54 (C-C6H5), 154.47 (C-C2HN3), 155.44 (C-C2HN3),
165.31 (C,4-OCH3-C6H5), 170.8 (CO-C3H3NOS), MS (m/z): 367.12 [M]+. Analysis for C19H18N4O2S
(m. w. 366.12); Calcd.: C, 62.28; H, 4.95; N, 15.29. Found: C, 62.47; H, 5.14; N, 14.91.
3.2. Biological Evaluation
The initial antimicrobial screening (growth inhibition zone) and determination of minimum
inhibitory concentration (MIC) of the tested compounds was performed by cup plate and broth
dilution methods, respectively, with different strains [25,26]. Ten synthesized compounds, 7c–l,
were screened for their antibacterial activity against Escherichia coli ATCC 25922, Proteus vulgaris atcc
6380, Pseudomonas aeruginosa ATCC 27857, Staphylococcus aureus ATCC 29213, Enterococcus faecalis
ATCC 29212, Bacillus subtilis ATCC 10400, antimycobacterial activity against Mycobacterium fortuitum
ATCC 6841 and antifungal activity against Candida albicans ATCC 2091. The tested compounds were
dissolved in dimethyl sulfoxide (DMSO) to obtain a stock solution (5120 µg/mL).
3.2.1. Inhibition Zone Measurement Using the Cup Plate Method
Three to five pure colonies of each tested organism were taken from the overnight culture of
tryptone soy agar and suspended in 5 mL of sterile normal saline. The bacterial culture was measured
using the spectrophotometer (LKB® Ultrospec) at 625 nm to give absorbance 0.1–0.14 (1
ˆ
108 CFU/mL).
The suspension was diluted 1:100 in Cation Adjustment Mueller–Hinton (CAMH) broth (Merck®,
Darmstadt, Germany) to obtain 1
106 CFU/mL. The suspension was swabbed on a CAMH agar
plate (Merck®, Darmstadt, Germany) and allowed to dry thoroughly. Then, four cups per agar plate
were made using a cork borer. A 100 L (512 g) stock solution (5120 g/mL) was transferred into
ˆ
µ
µ
µ
˝
the cup using a sterile pipette. The plates were kept in the refrigerator at 4 C for 30 min for diffusion.
˝
Then, the plates were incubated at 37 C for 24 h, except for Mycobacterium fortuitum ATCC 6841, which
was incubated for 48 h. After the incubation period, the diameter of the inhibition zone (including the
cup diameter) was measured and recorded in mm. Ciprofloxacin, moxifloxacin and fluconazole disks
BD BBL™ Sensi-Disc™ (Beckton Dickinson BBL, Franklin Lakes, NJ, USA) (BBL) were used as positive
controls for antibacterial and antifungal activities, respectively. The assay was carried out in duplicate.
3.2.2. Determination of MIC
MIC values were determined for the active in the cup plate method. Briefly, 1 mL of sterilized
CAMH broth was dispensed into a sterile 7-mL bijou tube (Sterilin Limited, Birmingham, UK).
For each compound, 14 tubes were used. Tube Numbers 13 and 14 were used as the positive growth
control (no tested compound) and the negative control for the medium sterility (no microorganism),
respectively. A 1 mL stock solution (5120
512 g/mL. One milliliter of the tested compounds was transferred into Tube #1 and mixed well to
give a concentration of 256 g/mL. Then, 1 mL was pipetted from Tube #1 and transferred to Tube #2
to make a two-fold dilution (128 g/mL). This procedure was repeated down to Tube #12 to reach the
µg/mL) was 10-fold diluted in 9 mL CAMH to obtain
µ
µ
µ

Molecules 2016, 21, 568
15 of 17
concentration of 0.125
µ
g/mL. One milliliter was discarded from Tube #12. A volume of 1 mL of inocula
(1
ˆ
106 CFU/mL) was added to all tubes, except Tube #14, to give final inocula of 5
ˆ
105 CFU/mL,
and the concentration of the tested compounds was two-fold diluted. Ciprofloxacin, moxifloxacin
and fluconazole were used as positive c˝ontrols as the antibacterial and antifungal, respectively. The
inoculated tubes were incubated at 37 C for 20 h, except for Mycobacterium fortuitum ATCC 6841,
which was incubated for 48 h. After the incubation period, the results of the MIC were recorded
manually according to the guidelines of European Committee on Antimicrobial Susceptibility testing
(EUCAST), 2003.
3.3. Molecular Modeling Studies
The investigated compounds were subjected to surface mapping experiments using “Molecular
Operating Environment” software (MOE of Chemical Computing Group Inc., on a Core i7 workstation).
The molecules were built using the Builder module of MOE. Their geometry was optimized by using
the Merck Molecular Force Field (MMFF94x) force field. The molecular mechancis (MM) calculations
in vacuo, dipole bond option for electrostatics, Polak–Ribiere algorithm and root mean square deviation
(RMSD) gradient of 0.01 kcal/mol conformational searching in torsional space were performed using
the multi-conformer method. Energy minima for the above compounds were determined by a
semi-empirical method Austin Model 1 (AM1) (as implemented in MOE, 2009.10). The lowest energy
conformer of each new analogue “global-minima” that was docked into the MurB enzyme-binding
domain was used. The crystal structure of Staphylococcus Aureus MurB (PDB Entry Code 1HSK) [19
]
was extracted from the Brookhaven Protein Database (PDB http://www.rcsb.org/pdb). During the
docking process, water molecules and all of the ligands in the crystal structures were removed, except
the FAD co-factor for the protein PDB Code 1HSK. All of the hydrogens were added, and the enzyme
structure was subjected to a refinement protocol in which the constraints on the enzyme were gradually
removed and minimized until the RMSD gradient was 0.01 kcal/mol Å. The energy minimization
was carried out using the molecular mechanics force field “AMBER.” For each of the 4-thiazolidinone
analogues, energy minimizations (EM) were performed using 1000 steps of steepest descent, followed
by conjugate gradient minimization to an RMSD energy gradient of 0.01 Kcal/mol Å. The active
site of the enzyme was defined using a radius of 10.0 Å around FAD. The energy of binding was
calculated as the difference between the energy of the complex and individual energies of the enzyme
and ligands [27–30]. Lowest energy aligned conformation(s) were identified. The map is color coded
and indicates the following: pink, hydrogen bond; blue, mild polar; green, hydrophobic region.
4. Conclusions
We have reported here the design and synthesis of new 1,2,4-triazole-based thiazolidin-4-one
hybrids along with their spectral and antimicrobial evaluation data. Compound 7h followed by 7j
gave the most potent activity against Staphylococcus aureus. Compounds 7h–l showed good antifungal
activity against Candida albicans. The in vitro studies demonstrated that 7h 7j and 7c showed promising
,
inhibitory activities against Mycobacterium fortuitum. Despite that the MIC values of the tested
compounds were not as strong as the reported antibiotics, however, they possessed a broad spectrum
of activity against Gram-positive, Gram-negative, mycobacterium and fungal strains. Our compounds
are not selective, so they may be useful as moderate to high-level disinfectants. This suggests that
these compounds could be formulated as disinfectants or antiseptics if they are proven later to be
nontoxic to skin/mucous membranes. The search for new antibacterial agents directed towards
novel targets became highly imperative. Therefore, we performed a deeper modeling study on the
molecular level to clarify the proposed binding mode of these compounds into the MurB active site
and consequently provide straight forward information for further structural optimization. Our results
suggested that docked ligands 7h, 7j, 7l, 7c, 7i and 7k arranged in such a conformation with a proper
and stable complex with high lipophilic interaction where the p-chloro or p-methoxy phenyl ring
was embedded within the hydrophobic active site residues. Our new 1,2,4-triazole-thiazolidin-4-ones

Molecules 2016, 21, 568
16 of 17
hybrids presented in this work could provide a potential for further optimization towards more active
derivatives against Staphylococcus aureus, as well as Candida albicans. This study has also paved the way
for generating more useful thiazolidinone analogues in future studies as bacterial MurB inhibitors.
Acknowledgments: The authors extend their appreciation to the Deanship of Scientific Research at
Taibah University, Al-Madinah Al-Munawarah, Kingdom of Saudi Arabia, for the financial support of our
group research Project Number 6225/1435. We would also like to thank Mr. Mohamed Yasser Mahmoud for
English proof reading of the manuscript.
Author Contributions: S.A. and H.M.A.-R. designed the idea, and the protocol of the whole study, synthesized
the starting and intermediate compounds and wrote the manuscript. M.F.Z. synthesized the final compounds,
wrote their experimental parts and interpreted their spectral data. S.M.E.-M. performed and interpreted the
modeling study helped in interpretation of some NMR data, and helped in the introduction part about MurB
inhibitors and M.H.A.-A. performed the antimicrobial studies and wrote their experimental data. All the authors
revised the whole manuscript and responded to reviewers comments.
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
Carlet, J.; Collignon, P.; Goldmann, D.; Goossens, H.; Gyssens, I.C.; Harbarth, S.; Jarlier, V.; Levy, S.B.;
N’Doye, B.; Pittet, D.; et al. Society’s failure to protect a precious resource: Antibiotics. Lancet 2011, 378,
369–371. [CrossRef]
2.
3.
4.
5.
6.
7.
8.
World Health Organization. The World Health Report 2000—Health Systems: Improving Performance;
World Health Organization: Geneva, Switzerland, 2000.
Mark, W.; Jeremy, F. Policy: An intergovernmental panel on antimicrobial resistance. Nature 2014, 509,
555–557.
Anil, K.; Eric, A.; Nacer, L.; Jerome, G.; Koen, A. The challenge of new drug discovery for tuberculosis.
Nature 2011, 469, 483–490.
Rogers, H.J.; Perkins, H.R.; Ward, J.B. Biosynthesis of peptidoglycan. In Microbial Cell Walls and Membranes;
Chapman & Hall: London, UK, 1980.
Van Heijenoort, J. Recent advances in the formation of the bacterial peptidoglycan monomer unit.
Nat. Prod. Rep. 2001, 18, 503–519. [CrossRef] [PubMed]
Mengin-Lecreulx, D.; Flouret, B.; van Heijenoort, J. Cytoplasmic steps of peptidoglycan synthesis in
escherichia coli. J. Bacteriol. 1982, 151, 1109–1117. [PubMed]
Andres, C.J.; Bronson, J.J.; D’Andrea, S.V.; Despande, M.S.; Falk, P.J.; Grant-young, K.A.; Harte, W.E.;
Ho, H.T.; Misco, P.F.; Robertson, J.G.; et al. 4-Thiazolidinones: Novel inhibitors of the bacterial enzyme murB.
Bioorg. Med. Chem. Lett. 2000, 10, 715–717. [CrossRef]
9.
Bronson, J.J.; DenBleyker, K.L.; Falk, P.J.; Mate, R.A.; Ho, H.T.; Pucci, M.J.; Snyder, L.B. Discovery of the first
antibacterial small molecule inhibitors of murb. Bioorg. Med. Chem. Lett. 2003, 13, 873–875. [CrossRef]
10. Jain, A.K.; Vaidya, A.; Ravichandran, V.; Kashaw, S.K.; Agrawal, R.K. Recent developments and biological
activities of thiazolidinone derivatives: A review. Bioorg. Med. Chem. 2012, 20, 3378–3395. [CrossRef]
[PubMed]
11. Deep, A.; Jain, S.; Sharma, P.C.; Mittal, S.K.; Phogat, P.; Malhotra, M. Synthesis, characterization and
antimicrobial evaluation of 2,5-disubstituted-4-thiazolidinone derivatives. Arab. J. Chem. 2014, 7, 287–291.
[CrossRef]
12. Tripathi, A.C.; Gupta, S.J.; Fatima, G.N.; Sonar, P.K.; Verma, A.; Saraf, S.K. 4-Thiazolidinones: The advances
continue. Eur. J. Med. Chem. 2014, 72, 52–77. [CrossRef] [PubMed]
13. Patel, H.; Mishra, L.; Noolvi, M.; Karpoormath, R.; Cameotra, S.S. Synthesis, in vitro evaluation, and
molecular docking studies of azetidinones and thiazolidinones of 2-amino-5-cyclopropyl-1,3,4-thiadiazole as
antibacterial agents. Arch. Pharm. 2014, 347, 668–684. [CrossRef] [PubMed]
´
14. Omar, K.; Geronikaki, A.; Zoumpoulakis, P.; Camoutsis, C.; Sokovic´, M.; Ciric´, A.; Glamocˇlija, J. Novel
4-thiazolidinone derivatives as potential antifungal and antibacterial drugs. Bioorg. Med. Chem. 2010, 18,
426–432. [CrossRef] [PubMed]
15. El Bialy, S.A.; Nagy, M.M.; Abdel-Rahman, H.M. Efficient regioselective three-component domino synthesis
of 3-(1,2,4-triazol-5-yl)-1,3-thiazolidin-4-ones as potent antifungal and antituberculosis agents. Arch. Pharm.
2011, 344, 821–829. [CrossRef] [PubMed]

Molecules 2016, 21, 568
17 of 17
16. Abdel-Rahman, H.M.; Hussein, M.A. Synthesis of beta-hydroxypropanoic acid derivatives as potential
anti-inflammatory, analgesic and antimicrobial agents. Arch. Pharm. 2006, 339, 378–387. [CrossRef] [PubMed]
17. Zhang, F.; Wen, Q.; Wang, S.F.; Shahla Karim, B.; Yang, Y.S.; Liu, J.J.; Zhang, W.M.; Zhu, H.L. Design,
synthesis and antibacterial activities of 5-(pyrazin-2-yl)-4H-1,2,4-triazole-3-thiol derivatives containing schiff
base formation as fabh inhibitory. Bioorg. Med. Chem. Lett. 2014, 24, 90–95. [CrossRef] [PubMed]
18. Romagnoli, R.; Baraldi, P.G.; Salvador, M.K.; Prencipe, F.; Bertolasi, V.; Cancellieri, M.; Brancale, A.;
Hamel, E.; Castagliuolo, I.; Consolaro, F.; et al. Synthesis, antimitotic and antivascular activity of
1-(31,41,51-trimethoxybenzoyl)-3-arylamino-5-amino-1,2,4-triazoles. J. Med. Chem. 2014, 57, 6795–6808.
[CrossRef] [PubMed]
19. Benson, T.E.; Harris, M.S.; Choi, G.H.; Cialdella, J.I.; Herberg, J.T.; Martin, J.P., Jr.; Baldwin, E.T. A structural
variation for murb: X-ray crystal structure of staphylococcus aureus udp-n-acetylenolpyruvylglucosamine
reductase (murb). Biochemistry 2001, 40, 2340–2350. [CrossRef] [PubMed]
20. Dolzhenko, A.V.; Pastorin, G.; Dolzhenko, A.V.; Chui, W.K. An aqueous medium synthesis and tautomerism
study of 3(5)-amino-1,2,4-triazoles. Tetrahedron Lett. 2009, 50, 2124–2128. [CrossRef]
21. Bera, H.; Tan, B.J.; Sun, L.; Dolzhenko, A.V.; Chui, W.-K.; Chiu, G.N.C. A structure–activity relationship study
of 1,2,4-triazolo [1,5-a] [1,3,5]triazin-5,7-dione and its 5-thioxo analogues on anti-thymidine phosphorylase
and associated anti-angiogenic activities. Eur. J. Med. Chem. 2013, 67, 325–334. [CrossRef] [PubMed]
22. Sun, X.; Liu, Y.; Chen, S.; Chen, B.; Jia, Y.; Zeng, Z. Synthesis and biological activities of schiff bases of
3-amino-1h-1, 2, 4-triazole. Chin. J. Chem. 2009, 27, 949–952. [CrossRef]
23. Constantine, K.L.; Mueller, L.; Goldfarb, V.; Wittekind, M.; Metzler, W.J.; Yanchunas, J., Jr. Characterization
of nadp+ binding to perdeuterated murb: Backbone atom nmr assignments and chemical-shift changes.
J. Mol. Biol. 1997, 267, 1223–1246. [CrossRef] [PubMed]
24. Patel, S.V.; Patel, M.N. Insilico design, synthesis and evaluation of murb inhibitors as antibacterial agents.
Int. J. Pharm. Technol. 2011, 3, 3064–3082.
25. Arendrup, M.C.; Cuenca-Estrella, M.; Lass-Flörl, C.; Hope, W. Eucast technical note on the eucast definitive
document edef 7.2: Method for the determination of broth dilution minimum inhibitory concentrations of
antifungal agents for yeasts edef 7.2 (eucast-afst). Clin. Microbiol. Infect. 2012, 18, E246–E247. [CrossRef]
[PubMed]
26. European Committee for Antimicrobial Susceptibility Testing (EUCAST) of the European Society of Clinical
Microbiology and Infectious Diseases (ESCMID). Determination of minimum inhibitory concentrations
(mics) of antibacterial agents by broth dilution. Clin. Microbiol. Infect. 2003, 9, ix–xv.
27. Profeta, S., Jr.; Allinger, N. Molecular mechanics calculations on aliphatic amines. J. Am. Chem. Soc. 1985
107, 1907–1918. [CrossRef]
,
28. Allinger, N.L. Conformational analysis. 130. Mm2. A hydrocarbon force field utilizing v1 and v2 torsional
terms. J. Am. Chem. Soc. 1977, 99, 8127–8134. [CrossRef]
29. Labute, P.; Williams, C.; Feher, M.; Sourial, E.; Schmidt, J.M. Flexible alignment of small molecules.
J. Med. Chem. 2001, 44, 1483–1490. [CrossRef] [PubMed]
30. Kearsley, S.K.; Smith, G.M. An alternative method for the alignment of molecular structures: Maximizing
electrostatic and steric overlap. Tetrahedron Comput. Methodol. 1990, 3, 615–633. [CrossRef]
Sample Availability: Not available.
©
2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC-BY) license (http://creativecommons.org/licenses/by/4.0/).