3-amino-5-(Indol-3-yl)methylene-4-oxo-2-thioxothiazolidine derivatives as antimicrobial agents: Synthesis, computational and biological evaluation

Article abstract of DOI:10.3390/ph13090229

Herein we report the design, synthesis, computational, and experimental evaluation of the antimicrobial activity of fourteen new 3-amino-5-(indol-3-yl) methylene-4-oxo-2-thioxothiazolidine derivatives. The structures were designed, and their antimicrobial activity and toxicity were predicted in silico. All synthesized compounds exhibited antibacterial activity against eight Gram-positive and Gram-negative bacteria. Their activity exceeded those of ampicillin and (for the majority of compounds) streptomycin. The most sensitive bacterium was S. aureus (American Type Culture Collection ATCC 6538), while L. monocytogenes (NCTC 7973) was the most resistant. The best antibacterial activity was observed for compound 5d (Z)-N-(5-((1H-indol-3-yl)methylene)-4-oxo-2-thioxothiazolidin-3-yl)-4-hydroxybenzamide (Minimal inhibitory concentration, MIC at 37.9–113.8 μM, and Minimal bactericidal concentration MBC at 57.8–118.3 μM). Three most active compounds 5d, 5g, and 5k being evaluated against three resistant strains, Methicillin resistant Staphilococcus aureus (MRSA), P. aeruginosa, and E. coli, were more potent against MRSA than ampicillin (MIC at 248–372 μM, MBC at 372–1240 μM). At the same time, streptomycin (MIC at 43–172 μM, MBC at 86–344 μM) did not show bactericidal activity at all. The compound 5d was also more active than ampicillin towards resistant P. aeruginosa strain. Antifungal activity of all compounds exceeded those of the reference antifungal agents bifonazole (MIC at 480–640 μM, and MFC at 640–800 μM) and ketoconazole (MIC 285–475 μM and MFC 380–950 μM). The best activity was exhibited by compound 5g. The most sensitive fungal was T. viride (IAM 5061), while A. fumigatus (human isolate) was the most resistant. Low cytotoxicity against HEK-293 human embryonic kidney cell line and reasonable selectivity indices were shown for the most active compounds 5d, 5g, 5k, 7c using thiazolyl blue tetrazolium bromide MTT assay. The docking studies indicated a probable involvement of E. coli Mur B inhibition in the antibacterial action, while CYP51 inhibition is likely responsible for the antifungal activity of the tested compounds.

Full text of DOI:10.3390/ph13090229

pharmaceuticals
Article
3-Amino-5-(indol-3-yl)methylene-4-oxo-2-
thioxothiazolidine Derivatives as Antimicrobial
Agents: Synthesis, Computational and
Biological Evaluation
Volodymyr Horishny 1, Victor Kartsev 2, Vasyl Matiychuk 3, Athina Geronikaki 4,
* ,
Petrou Anthi 4 , Pavel Pogodin 5, Vladimir Poroikov 5 , Marija Ivanov 6 , Marina Kostic 6,
6
Marina D. Sokovic´
and Phaedra Eleftheriou 7
1
Department of Chemistry, Danylo Halytsky Lviv National Medical University, Pekarska 69, 79010 Lviv,
Ukraine; vgor58@ukr.net
InterBioScreen, 142432 Chernogolovka, Moscow Region, Russia; vkartsev@ibscreen.chg.ru
Department of Chemistry, Ivan Franko National University of Lviv, Kyryla i Mefodia 6, 79005 Lviv, Ukraine;
v_matiychuk@ukr.net
2
3
4
5
6
School of Pharmacy, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece;
anthi.petrou.thessaloniki1@gmail.com
Institute of Biomedical Chemistry, Pogodinskaya Street 10 Bldg.8, 119121 Moscow, Russia;
pogodinpv@gmail.com (P.P.); vladimir.poroikov@ibmc.msk.ru (V.P.)
Mycological Laboratory, Department of Plant Physiology, Institute for Biological Research, Siniša,
Stankovic´-National Institute of Republic of Serbia, University of Belgrade, Bulevar Despota Stefana 142,
11000 Belgrade, Serbia; marija.smiljkovic@ibiss.bg.ac.rs (M.I.); marina.kostic@ibiss.bg.ac.rs (M.K.);
mris@ibiss.bg.ac.rs (M.D.S.)
7
Department of Biomedical Sciences, School of Health Sciences, International Hellenic University, Sindos,
57400 Thessaloniki, Greece; eleftheriouphaedra@gmail.com
*
Correspondence: geronik@pharm.auth.gr; Tel.: +30-23-1099-7616
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 27 July 2020; Accepted: 28 August 2020; Published: 1 September 2020
Abstract: Herein we report the design, synthesis, computational, and experimental evaluation of the
antimicrobial activity of fourteen new 3-amino-5-(indol-3-yl) methylene-4-oxo-2-thioxothiazolidine
derivatives.
The structures were designed, and their antimicrobial activity and toxicity
were predicted in silico. All synthesized compounds exhibited antibacterial activity against
eight Gram-positive and Gram-negative bacteria. Their activity exceeded those of ampicillin
and (for the majority of compounds) streptomycin.
The most sensitive bacterium was
S. aureus (American Type Culture Collection ATCC 6538), while L. monocytogenes (NCTC
7973) was the most resistant. The best antibacterial activity was observed for compound 5d
(Z)-N-(5-((1H-indol-3-yl)methylene)-4-oxo-2-thioxothiazolidin-3-yl)-4-hydroxybenzamide (Minimal
inhibitory concentration, MIC at 37.9–113.8
57.8–118.3 M). Three most active compounds 5d, 5g, and 5k being evaluated against three resistant
strains, Methicillin resistant Staphilococcus aureus (MRSA), P. aeruginosa, and E. coli, were more
potent against MRSA than ampicillin (MIC at 248–372 M, MBC at 372–1240 M). At the same
time, streptomycin (MIC at 43–172 M, MBC at 86–344 M) did not show bactericidal activity at
µM, and Minimal bactericidal concentration MBC at
µ
µ
µ
µ
µ
all. The compound 5d was also more active than ampicillin towards resistant P. aeruginosa strain.
Antifungal activity of all compounds exceeded those of the reference antifungal agents bifonazole
(MIC at 480–640
µM, and MFC at 640–800 µM) and ketoconazole (MIC 285–475 µM and MFC
380–950 M). The best activity was exhibited by compound 5g. The most sensitive fungal was
µ
T. viride (IAM 5061), while A. fumigatus (human isolate) was the most resistant. Low cytotoxicity
against HEK-293 human embryonic kidney cell line and reasonable selectivity indices were shown
for the most active compounds 5d
,
5g
,
5k
,
7c using thiazolyl blue tetrazolium bromide MTT assay.
Pharmaceuticals 2020, 13, 229; doi:10.3390/ph13090229
www.mdpi.com/journal/pharmaceuticals

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The docking studies indicated a probable involvement of E. coli Mur B inhibition in the antibacterial
action, while CYP51 inhibition is likely responsible for the antifungal activity of the tested compounds.
Keywords: indole; thioxothiazolidine; antibacterial activity; antifungal activity; computer-aided
prediction; docking; Mur B; CYP 51
1. Introduction
Infectious diseases affect large populations and cause significant morbidity and mortality [1].
They represent a global indirect load on public health security and an impact on socio-economic
stability worldwide. Bacterial, fungal, and viral infections have monopolized the dominant factors
of death and disability of millions of humans for centuries. They are presently plaguing and even
ravaging populations worldwide each year with performances far surpassing wars [2].
It should be mentioned that several dozen new infections have grown and affected the health
of billions of people over the world, mainly in developing countries [3]. Unfortunately, there are no
successful pharmaceuticals or vaccines for many of these new infections [3].
The treatment of infectious disease is still an imperative and demanding problem due to the
growing number of multi-drug resistant pathogens, especially Gram-positive bacteria. Due to this, the
lack of effective antimicrobial drugs, morbidity, and mortality notably increased [4].
Drug resistance causes vast human suffering, and now it is one of the most significant challenges
of the twenty-first century. Species such as the methicillin-resistant S. aureus and vancomycin-resistant
enterococci have emerged due to the irrational or overuse of antimicrobial agents [5].
The pathogens, including Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae,
Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp. which also called ESCAPE
pathogens, are of particular importance since they play a significant role affecting several human
organs including the lung and urinary system. Besides, they exhibited increased resistance to clinically
used antibiotics [6].
Numerous of these pathogens are Gram-negative bacteria, which are of specific concern due
to their resistance of up to 50% against carbapenems that have been reported in some developing
countries [6]. Despite the availability of some new antibiotics against Gram-positive pathogens,
no treatment of these pathogens with a new class of compounds has been introduced in the last 40 years.
Therefore, to overcome the resistance, the discovery of safer and more effective antimicrobial agents
with a different mechanism of action is still an urgent need [7].
The interest in thiazolidine-based compounds attracted the attention of medicinal chemists,
and a plethora of them have been studied to evaluate pharmacological properties [8–10]. Despite the
appearance of some controversial opinions regarding the analysis of the molecular mechanism of
their action, prominent representatives among the developed drug-like molecules are thiazolidinone
derivatives [11
molecules [13–15].
N-(4-oxo-2-thioxothiazolidin-3-yl)carboxamides exhibit antimicrobial [16
actions, are dual COX-1/2 and 5-LOX inhibitors [24 25], non-nucleoside inhibitors of Hepatitis C NS5b
,12] since they are a valuable source of building blocks for the development of novel
–20] and antitumor [21–23]
,
RNA polymerase [26,27] and HIV-1 reverse transcriptase inhibitors [28].
The combination of the thiazolidinone ring with other pharmacologically promising heterocycles
has been a warranted approach for developing new “drug-like” molecules with the desired activity
profile [29–31]. Our previous studies showed that thiazolidinone core with indole fragment in one
molecule gave the compounds with high antimicrobial activity [19].
On the other hand, indole derivatives represent another scaffold widely spread in nature
with a broad spectrum of biological activities.
natural compounds but also in diverse semisynthetic and synthetic drug-like molecules [32
The indole ring was found not only in
33].
,

Pharmaceuticals 2020, 13, 229
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They exhibit antimicrobial [34–39], anti-inflammatory [40,41], COX inhibitory [42,43] anticancer [44–46],
antiviral [47,48], anti-HIV [49,50], and antidiabetic [51] activities. Among the natural compounds
containing the indolene fragment, several imidazoline and imidazolidine alkaloids are known,
which have a wide spectrum of biological activity, including antibacterial. Thus, indole-containing
azahydantoins 1-6 from sponges and streptomycetes have a potent antibacterial and antiseptic action
(Figure 1) [52–54]:
Aplysianopsine
from sponge Aplysinopsis
Aplysinopsine
from Streptomyces spp
6-Bromoaplysinopsin
from Smenospongia aurea
6-Bromo-4'-N-
demethylaplysinopsin
from Smenospongia aurea
Rhopaladin-C
from marine tunicate Rhopalaea sp.
Figure 1. Structure of indole-containing azahydantoins 1-5 from sponges and streptomycetes.
It is also known that synthetic thiohydantoin (rhodanine) analogs
antibacterial properties [55].
7, 8 (Figure 2), exhibit pronounced
Figure 2. Synthetic thiohydantoin analogues.
Therefore, the design and development of hybrid molecules combining thiazolidinone and indole
cores in the same structure is a promising approach. Taking into account all issues mentioned above
and encouraging results obtained in our earlier studies [19], in this paper, we present the synthesis and
biological evaluation of new (1H-indole-3-yl-methylene)-4-oxo-2-thioxothiazolidin derivatives with
potent antimicrobial activity.
2. Results and Discussion
2.1. In Silico Antimicrobial Activity Estimation
2.1.1. Antibacterial Activity
Using AntiBac-Pred [56] one of the predictive web services of Way2Drug platform [57],
activity against at least one strain of bacteria was predicted for each of the fourteen designed

Pharmaceuticals 2020, 13, 229
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compounds with Pa-Pi values in the range from 0.001 to 0.309. According to the prediction results,
the highest probability of antibacterial activity against the Bacillus subtilis subsp. subtilis str. 168 was
estimated for derivatives 7a and 5b (Pa-Pi values are 0.309 and 0.305, respectively).
Similarly, we estimated in silico the probability of antibacterial activity for the reference drugs
streptomycin and ampicillin. For both reference drugs, wide antibacterial action was predicted. For the
top-10 predictions of streptomycin Pa-Pi values vary from 0.905 to 0.947; for ampicillin—from 0.712 to
0.989. Contrary, for relatively new antibacterial agent trifolirhizin, which structure was disclosed only
on July 7, 2020 (Clarivate Analytics Integrity [58]), the top-10 predictions Pa-Pi values vary from 0.369
to 0.552.
2.1.2. Antifungal Activity
Using web service AntiFun-Pred [59], activity against at least one of the fungal species was
predicted for six of the fourteen studied compounds with Pa-Pi values ranging from 0.001 to 0.112.
The results show that among the studied compounds, derivatives 5a (Pa-Pi against Trichophyton
mentagrophytes equals 0.112) and 7a (Pa-Pi against Candida equals 0.101) have better chances to be found
active in biological evaluation of the antifungal activity.
The results of in silico antimicrobial activity assessment are given in the supplementary file
PASSweb_results_13mols.xlsx. Small Pa-Pi values reflect the novelty of the analyzed compounds
compared to those included in the PASS training set.
Similarly, for the reference drug ketoconazole wide antifungal action was predicted with Pa-Pi
values in the range 0.622–0.812 (top-10 predictions), while for the new antifungal agent drimenin
disclosed on 12 June 2020 (Clarivate Analytics Integrity [58]), only two antifungal activity were
predicted with Pa-Pi values 0.007 and 0.030.
2.1.3. Acute Rat Toxicity
Using web service based on GUSAR software [60,61], acute rat toxicity with regards to different
administration routes was estimated for the studied compounds. LD50 values and toxicity classes are
given in Table 1. Most of the predictions indicate that the studied compounds belong to the fifth or
fourth rodent toxicity classes.
Table 1. In silico assessments of acute rat toxicity.
LD50, mg/kg
Toxicity Class
Compound ID
IP
IV
Oral
SC
IP
IV
Oral
SC
5a
5b
5c
5d
5e
5f
5g
5h
5i
809.8
680.4
980.3
402.5
309
311.5
1218
1266
1325
780.4 *
1434
619.9 *
5
5
5
5
5
5
4
4
4
4 *
5
4 *
1263
1266
1010 *
1282
1299
1258
1031 *
1033
1061
466
843.9
469.2
192.4 *
1001
440 *
477.7 *
397.6 *
1588
545.4 *
422 *
NT
NT
5 *
NT
NT
NT
5 *
5
5
5
5
5
5
5
5
4
4
4
4
4
3 *
4
4 *
4 *
4 *
5
4 *
4 *
4 *
5 *
4 *
4 *
502.2
371.9
448.5
476.5
381.6
398.3
236.6
287.2
210.7
732.3
4
5j
196.2 *
202.8 *
593.7
720 *
765.1 *
3 *
3 *
4
4 *
4 *
5k
7a
7b
7c
442 *
1644 *
862.1 *
682 *
5
5 *
1180 *
Notes: *: Calculated for compounds that do not correspond to the model’s applicability domain; thus, they are less
reliable than unmarked ones. NT: Non-Toxic.

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2.2. Chemistry
The starting N-(4-oxo-2-thioxothiazolidin-3-yl) -carbamides 3a-d was prepared by reacting the
acid hydrazides 1a-d with trithiocarbonyl diglycolic acid (Scheme 1). The reaction was carried out in a
medium of boiling aqueous alcohol. The yield of the products was 83–97%.
X
Y
O
O
R
H
S
N
H2N
N
N
HOOCCH2S
SCH2COOH
+
R
H
O
S
Y
S
1a-d
X
2
3a-d
1a, 3a R = 2-OH, X = Y = CH; 1b, 3b R = 4-OH, X = Y = CH; 1c, 3c X = N, R = H, Y = CH;
1d, 3d Y = N, R = H, X = CH
Scheme 1. Synthesis of initial compounds.
The titled compounds were synthesized according to the process shown in Scheme 2.
Scheme 2. Synthesis of final compounds.
The reaction of N-(4-oxo-2-thioxothiazolidin-3-yl)carbamides 3a–d with indole-3-carbaldehydes
4a-d in acetic acid in the presence of an ammonium acetate catalyst afforded with high yield
5-[(R-1H-indol-3-yl)methylene]-4-oxo-2-thioxothiazolidin-3-ylcarbamides 5a–k, while upon reaction of
indole-3-carbaldehydes 4a–d with 3-morpholino-2-thioxothiazolidin-4-one
6 in the same conditions
5-[(R-1H-indol-3-yl) methylene] -3-morpholin-4-yl-2-thioxothiazolidin-4-ones 7a–c were obtained.

Pharmaceuticals 2020, 13, 229
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1
All compounds were characterized by IR, H and 13C NMR spectroscopy. In the IR spectra
of compounds 3a–d, 5a–k, and 7a, 7c, the carbonyl group of the 4-thiazolidone ring absorbs at
1753.21–1690.53 cm−1, and the thiocarbonyl group—at 1608.56–1556.48 cm−1. The absorption band of
the carbonyl group of the amide fragment of 3a–d and 5a–k is located at 1689.56–1654.84 cm−1
.
In the starting 3-substituted 2-thione-4-thiazolidones, the amide proton NH-CO of the compounds
3a–d appears as a singlet in the range 11.95-10.91 ppm, and the cyclic methylene group resonates as a
singlet or quartet at 4.55–4.48 ppm. etc. In the target products 5a–k, the amide proton is in the range of
11.85–11.12 ppm. The 5-methylidene proton CH = of compounds 5a–k and 7a, 7c resonates in the form
of a singlet at 8.20–7.94 ppm, which, according to the literature [9,62], is characteristic of the Z isomer.
The singlet NH of the protons of the indole ring appeared in the range 12.31–12.06 ppm.
2.3. Biological Evaluation
2.3.1. Antibacterial Activity
Compounds 5a–k and 7a–c were evaluated for antibacterial activity, by microdilution method to
determine the minimal bacteriostatic and bactericidal concentrations. As reference compounds, we
used ampicillin and streptomycin, which are both broad-spectrum antibiotics commonly applied to
treat different conditions. Antibacterial activity of tested compounds is shown in Table 2 with MIC
values in the range of 36.5–211.5
which can be presented as: 5d 5g
activity is achieved for compound 5d with MIC at 37.9–113.8
antibacterial activity was observed for compound 5i with MIC values in the range of 76.1–152.1
and MBC at 152.1–304.2 M. The most sensitive bacterium appeared to be S.aureus (ATCC 6538), En.
µ
M and MBC at 73.3–282.0
µ
M. According to the order of activity
5f 5a 7c 7b 5b 7a 5i the best
M and MBC at 75.9–151.7 M. The lowest
>
>
5k 5j 5c 5h 5e
>
>
>
>
>
>
>
>
>
>
>
µ
µ
µM
µ
cloacae (ATCC 35,030) was the second most sensitive, while S.Typhimirium was the most resistant one.
Another resistant strain was Gram-negative bacterium S. Typhimurium (ATCC 13,311).
Compound 7b exhibited good activity against B. cereus with MIC and MBC at 41.7 and 83.4
respectively. Compound 5d appeared to be potent against S. aureus (ATCC 6538), P. aeruginosa (ATCC
27,853), and En. cloacaei (ATCC 35,030) with MIC at 37.9 M and MBC at 75.9 M. It also showed
good activity against B. cereus with MIC and MBC at 55.6 and 75.9 M respectively. Compound 5h
appeared to be potent against En. cloacae and P. aeruginosa (ATCC 27,853) with MIC and MBC at 39.4
and 78.9 M. Good activity against these two species and S. aureus (ATCC 6538) was also shown by
compound 5j (MIC/MBC 58.6/73.1 M). Good activity against S. aureus (ATCC 6538), also exhibited by
compound 7b with MIC at 41.7 M and MBC at 83.4 M. On the other hand, compound 5g exhibited
good activity against En. cloacae (ATCC 35030), S. aureus (ATCC 6538), and S. typhimurium (ATCC
13311) with MIC/MBC values 36.5/73.1 and 53.6/73.1 M, respectively. It is worth to notice that all
µM
µ
µ
µ
µ
µ
µ
µ
µ
compounds appeared to be more potent than ampicillin against all bacteria used and more active than
streptomycin against all bacteria except B. cereus and S. typhimurium (ATCC 13,311).
The structure-activity studies revealed that the most beneficial for antibacterial activity is the
presence of hydroxybenzamide (5d) on the N-atom of (Z)-5-((5-methoxy-1H-indol-3-yl)methylene)-3-
morpholino-2-thioxothiazolidin-4-one. Introduction of the 5-methoxy group to indole ring and
replacement of hydroxybenzamide by nicotinamide (5g) decreased a little activity while shifting
of methoxy group from position 5 to position 6 of indole ring and replacement of nicotinamide by
isonicotinamide led to less active compound 5k compared to compound 5g.
On the other hand, the isonicotinamide derivative of (Z)-5-((1-methyl-indol-3-yl)methylene)-2-
thioxothiazolidin-4-one (5i) appeared to be the less active compound. It was observed that for
(Z)-5- [(1H-indol-3-yl)methylene]-2-thioxothiazolidin-4-one (5h) as well as for (Z)-N-5-[(1-methyl-1H-
indol-3-yl)methylene]-4-oxo-2-thioxothiazolidin-4-one (5g) derivatives isonicotinamide substituent
is endowed with better activity. The opposite was observed for 6-methoxy indole derivatives where
more preferable is nicotinamide as a substituent (5k). Between methylindole derivatives (5a, 5f,
5i), more favorable for activity was nicotinamide substituent (5f), followed by benzamide, (5a
)

Pharmaceuticals 2020, 13, 229
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while isonicotinamide (5i) had a negative effect on antibacterial activity. For -2-hydroxybenzamides
derivatives more preferable for antibacterial activity appeared to be 6-methoxy substitution of indole
ring (5c) followed by methylidole (5a) while 5-methoxy substitution on indole ring was negative
leading to one of the less active compounds (5b). In the case of 3-morpholino-2-thioxothiazolidin-4- one
derivatives (7a–c), which were among the less active compound, it seems that 5-methoxy substitution
on indole ring is preferable than methylindole or indole ring.
Thus, it can be concluded that the most favorable effect on the antibacterial activity of the
target compounds is provided by the introduction into the molecule of an unsubstituted indolidene
and 6-methoxyindolidene fragment. In addition, the nature of the substituent at position 3 of the
thiazolidine ring has a direct influence on the enhancement of the antibacterial action. An increase in the
antibacterial effect is observed from the use of 4-hydroxybenzamide and isonicotinamide substitutes.
From all mentioned above, it is evident that the antibacterial activity of these compounds depends
not only on substituent and its position in the indole ring but also on substituent on the N-atom of
2-thioxothiazolidin-4-one ring.
Table 2. Antibacterial activity of compounds 5a–k and 7a–c (MIC/MBC in µM).
Com/d ID
B.c
M.f
S.a
L.m
En.cl
P.a
S.T
E.coli
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
73.3 ± 0.4
146.5 ± 1.0
70.5 ± 0.4
141.0 ± 1.0
68.2 ± 0.8
136.8 ± 1.0
55.6 ± 0.2
75.9 ± 0.4
78.9 ± 0.2
157.7 ± 0.8
114.1 ± 1.0
152.1 ± 2.0
73.1 ± 1.0
146.2 ± 1.0
78.9 ± 0.5
157.7 ± 1.0
114.1 ± 1.0
152.1 ± 1.0
73.1 ± 0.5
146.2 ± 1.0
73.1 ± 0.5
146.2 ± 1.0
130.3 ± 1.0
173.7 ± 2.0
41.7 ± 0.2
83.4 ± 0.4
79.9 ± 0.4
159.8 ± 1.0
248.0 ± 3.0
372.0 ± 4.0
43.0 ± 0.8
86.0 ± 1.0
109.9 ± 0.1
146.5 ± 2.0
105.8 ± 0.8
141.0 ± 2.0
102.6 ± 1.0
136.8 ± 1.5
113.8 ± 0.8
151.7 ± 2.0
118.3 ± 1.0
157.7 ± 2.0
114.1 ± 8.0
152.1 ± 1.0
73.1 ± 1.0
73.3 ± 0.3
146.5 ± 2.0
70.5 ± 0.8
141.0 ± 1.0
68.4 ± 0.5
136.8 ± .1.0
37.9 ± 0.2
75.9 ± 0.5
57.8 ± 0.4
78.9 ± 0.8
114.1 ± 1.0
152.1 ± 1.5
53.6 ± 0.4
73.1 ± 0.8
118.3 ± 1.0
157.7 ± 2.0
76.1 ± 0.8
152.1 ± 2.0
58.6 ± 0.4
73.1 ± 0.8
58.6 ± 0.4
73.1 ± 0.8
63.7 ± 0.4
86.9 ± 0.8
41.7 ± 0.2
83.4 ± 0.8
79.9 ± 1.0
159.8 ± 1.4
248.0 ± 2.0
372.0 ± 2.0
172.0 ± 2.0
344.0 ± 4.0
146.5 ± 1.0
293.0 ± 4.0
105.8 ± 1.5
141.0 ± 2.0
68.4 ± 0.4
73.3 ± 0.8
146.5 ± 1.0
70.5 ± 0.8
141.0 ± 1.0
68.4 ± 0.4
136.8 ± 1.0
37.9 ± 0.4
75.9 ± 0.8
78.9 ± 0.1
157.7 ± 2.0
76.1 ± 0.8
152.1 ± 1.0
36.5 ± 0.5
73.1 ± 1.0
39.4 ± 0.5
78.9 ± 0.8
76.1 ± 0.8
152.1 ± 1.2
58.6 ± 0.6
73.1 ± 0.6
58.6 ± 0.6
73.1 ± 0.8
86.9 ± 1.0
173.7 ± 1.5
83.4 ± 0.9
166.9 ± 1.0
58.6 ± 0.4
79.9 ± 1.0
248.0 ± 3.0
372.0 ± 3.0
43.0 ± 0.3
86.0 ± 0.6
73.3 ± 0.08
146.5 ± 1.0
70.5 ± 0.8
141.0 ± 0.1
68.4 ± 0.4
136.8 ± 1.0
37.9 ± 0.2
75.9 ± 0.6
78.9 ± 0.6
157.7 ± 1.5
76.1 ± 0.3
152.1 ± 1.0
109.6 ± 1.0
146.2 ± 1.2
39.4 ± 0.6
78.9 ± 0.8
76.1 ± 0.8
152.1 ± 1.2
58.6 ± 0.8
73.1 ± 0.06
58.6 ± 0.6
73.1 ± 0.8
86.9 ± 1.0
173.7 ± 1.5
61.2 ± 0.5
83.4 ± 1.0
58.6 ± 0.4
79.9 ± 1.0
744.0 ± 9.0
1240 ± 2
73.3 ± 0.08
146.5 ± 1.0
141.0 ± 1.2
282.0 ± 3.0
102.6 ± 1.0
136.8 ± 1.5
113.8 ± 1.0
151.7 ± 1.0
78.9 ± 0.8
109.9 ± 0.1
146.5 ± 2.0
211.5 ± 2.0
282.0 ± 2.0
102.6 ± 1.5
136.8 ± 2.0
75.6 ± 0.4
5a
5b
5c
136.8 ± 1.0
113.8 ± 0.8
151.7 ± 2.0
78.9 ± 0.5
5d
5e
151.7 ± 1.0
118.3 ± 1.0
157.7 ± 2.0
114.1 ± 1.0
152.1 ± 1.0
109.6 ± 1.0
146.2 ± 2.0
118.3 ± 1.5
157.7 ± 1.0
114.1 ± 1.0
152.1 ± 1.0
109.6 ± 1.0
146.2 ± 2.0
109.6 ± 2.0
146.2 ± 2.0
130.3 ± 2.0
173.7 ± 1.5
125.2 ± .1.0
166.9 ± 2.0
119.8 ± 1.5
159.8 ± .2.0
372.0 ± 4.0
492.0 ± 8.0
172.0 ± 2.0
344.0 ± 2.0
157.7 ± 1.5
76.1 ± 0.4
157.7 ± 1.0
114.1 ± 1.5
152.1 ± 2.0
53.6 ± 0.6
5f
152.1 ± 1.0
73.1 ± 0.8
5g
146.2 ± 1.0
118.3 ± 1.5
157.7 ± 2.0
114.1 ± 1.5
152.1 ± 2.0
109.6 ± 1.0
146.2 ± 2.0
109.6 ± 1.0
146.2 ± 2.0
130.3 ± 1.5
173.7 ± 2.0
125.2 ± 1.0
166.9 ± 2.0
119.8 ± 1.5
159.8 ± 2.0
248.0 ± 2.0
372.0 ± 4.0
86.0 ± 1.0
146.2 ± 1.6
78.9 ± 0.8
73.1 ± 1.0
78.9 ± 0.6
5h
5i
157.7 ± 1.0
152.1 ± 1.0
304.2 ± 4.0
146.2 ± 0.8
292.3 ± 0.2
109.6 ± 1.5
146.2 ± 1.0
86.9 ± 0.4
157.7 ± 1.2
152.1 ± 1.0
304.2 ± 2.0
109.6 ± 1.0
146.2 ± 1.0
109.6 ± 1.5
146.2 ± 2.0
173.7 ± 2.0
347.4 ± 4.0
166.9 ± 2.0
333.9 ± 4.0
119.8 ± 1.0
159.8 ± 2.0
248.0 ± 3.0
492.0 ± 6.0
172.0 ± 3.0
344.0 ± 3.0
5j
5k
7a
173.7 ± 1.5
166.9 ± 1.0
333.9 ± .2.0
159.8 ± 1.0
319.6 ± 2.0
372.0 ± 4.0
744.0 ± 8.0
258.0 ± 4.0
516.0 ± 4.0
7b
7c
Am.
Str.
172.0 ± 3.0
344.0 ± 3.0
172.0 ± 2.0
MIC–minimal inhibitory concentration, MBC–minimal bactericidal concentration, B.c.-B.cereus (clinical isolate),
M.f.-M.flavus (ATCC 10,240), S.a.-S.aurues (ATCC 6538), l.m.-L.monocytogenes (NCTC 7973), E.c.-E.coli (ATTC 35210,
En.c.-En.cloaca (ATCC 3503), P.a.-P.aeruginosa (ATCC 27,853), S.T.-S.Typhimurium (ATCC 13,311).
Three most active compounds were also evaluated against the resistant strains, including MRSA,
P. aeruginosa, and E. coli, (Table 3). From the obtained results, it is evident that all three compounds
were more active against MRSA than ampicillin, while streptomycin did not show any bactericidal
activity. The compound 5d was also more active than ampicillin towards resistant P. aeruginosa strain.

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Table 3. Antibacterial activity against resistant strains (MIC/MBC in µM).
Resistant Strains
P.aeruginosa
Compound ID
MRSA
E.coli
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
1260 ± 0.8
2520 ± 0.1
1220 ± 18
2440 ± 0.2
1220 ± 0.6
2440 ± 22
172.0 ± 21
-
315 ± 9.0
630 ± 8.0
610 ± 5.0
1202 ± 21
610 ± 10
1220 ± 21
86 ± 12
172 ± 14
572 ± 64
/
1260 ± 21
2502 ± 22
1220 ± 19
2440 ± 16
1220 ± 0.6
2440 ± 22
172 ± 21
344 ± 42
572 ± 78
/
5d
5g
5k
Streptomycin
-
/
Ampicilline
2.3.2. Antifungal Activity
All compounds also showed antifungal activity with MIC values ranging from 9.7 to 347.4
MFC at 19.5–694.8
5g > 7c > 7b > 5d > 5b > 5e > 5k > 5f > 5j > 5c > 5i > 5a > 7a > 5h. Compound 5g displayed the best
activity with MIC values in the range of 9.7–73.1 M and MFC at 36.5–146.2 M, while compound 5h
exhibited the lowest potential with MIC and MFC at 28.9–315.5 M and 39.4–630.9 M respectively. It
µM and
µM.The antifungal activity of compounds is shown in Table 4 and follows the order:
µ
µ
µ
µ
was observed that similar to bacteria, fungi showed different sensitivity towards compounds tested.
Thus, the most sensitive fungal strain appeared to be T. viride (IAM 5061), while the most resistant
filamentous A. fumigatus. The behavior of compounds towards fungi species was different, too.
Several compounds showed very good activity against some species. For example, compound
5d exhibited good activity against the most resistant A. fumigatus (MIC/MFC at .20.2/37.9
compound 7b against T. viride (IAM 5061), P. cyclpoium var verucosum (food isolate) and all Aspergillus
species except A. fumigatus (human isolate) with MIC at 22.3 M and MFC at 41.4 M. Compound
5g exhibited excellent activity against T. viride (IAM 5061) (MIC/MFC at 0.97/1.95 mol/mL
10−2).
Additionally, good activity was achieved for compound 5g against A. versicolor (ATCC 11730),
A. ochraceus (ATCC 12066), P. funiculosum (ATCC 36839) with MIC and MFC at 19.5 M and 36.5
respectively. Compound 5c appeared to be potent against A. ochraceus (ATCC 12066) and T. viride (IAM
5061) (MIC/MFC at 18.8/35.3 M whereas compound 7c exhibited very good activity against T. viride
(IAM 5061)with MIC at 10.7 M and MFC at 21.3 M and also good activity against A. ochraceus (ATCC
12066) and P. funiculosum (ATCC 36839 (MC/MFC at 23.1/39.9 M. The potential of ketoconazole was
at MIC 285-475 M and MFC at 380–950 M. Bifonazole displayed MIC at 480-640 M and MFC at
640–800 M. It should be mentioned that all compounds appeared to be more potent than ketoconazole
µM, while
µ
µ
µ
×
µ
µM
µ
µ
µ
µ
µ
µ
µ
µ
and bifonazole. Only compound 7a against A. fumigatus (human isolate) was less active than bifonazole.
According to the analysis of the structure-activity relationships, the most beneficial for antifungal
activity is the presence of the 5-methoxy group in indole ring as well as nicotinamide as a substituent
of the side chain (5g). In contrast, the presence of isonicotinamide in methylindole (5i) derivative
appeared to be detrimental. Shifting of 5-OMe of compound 5g to position 6 of indole and replacement
of nicotinamide by 2-hydroxybenzamide resulted in compound 5c with decreased activity. Removal of
methoxy group and introduction of morpholino moiety to the N atom of thioxothiazolidinone (7a)
decreased more activity.
In indole derivatives (5d
activity, while isonicotinamide substituent had a negative effect. On the contrary, for methylindole
derivatives (5a 5f 5i), the negative impact was observed with the presence of 2-hydroxybenzamide,
while in the case of the 5-methoxy indole derivatives (5b 5j) it was the opposite. Finally, for the
, 5e, 5h), the presence of 4-hydroxybenzamide was favorable for antifungal
,
,
,
derivatives with morpholino moiety, the best activity was observed with the presence of the 5-methoxy
group in the indole ring. The indole derivative was one of the less potent.

Pharmaceuticals 2020, 13, 229
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Thus, as in the case of antibacterial activity, antifungal activity depends not only on substitution
in the indole ring but also on substituent on the N-atom of the 2-thioxothiazolidinone ring.
In the series of (Z)-5-((5-methoxy-1H-indol-3-yl)methylene)-3-morpholino-2-thioxothiazolidin-4-one
derivatives the most important structural features which enhanced the antifungal activity are again
4-hydroxybenzamide and 1H-indole moiety as well as nicotinamide and 5- and 6-methoxyindole
moieties. On the other hand, in the series of indole 3-methylene morpholino-2-thioxothiazolidin-4-one
derivatives, the presence of the 5-OCH3 group in the indole ring enhance the antifungal activity.
Table 4. Antifungal activity of compounds 5a–k and 7a–c (MIC/MFC in µM).
Com. ID
A.f
A.v
A.o
A.n
T.v
P.o
P.f
Pvc
MIC
MFC
MIC
MFC
MIC
MFC
MIC
MFC
MIC
MFC
MIC
MFC
MIC
MFC
MIC
MFC
MIC
MFC
MIC
MFC
MIC
MFC
MIC
MFC
MIC
MFC
MIC
MFC
MIC
MFC
MIC
MFC
293.0 ± 2.2
586.1 ± 7.0
35.2 ± 0.6
70.5 ± 0.6
282.0 ± .2.0
564.1 ± 4.0
202 ± 0.1
36.6 ± 0.4
73.3 ± 0.8
35.2 ± 0.2
70.5 ± 0.4
35.3 ± 0.2
68.2 ± 0.4
37.9 ± 0.2
75.6 ± 0.4
39.4 ± 0.2
78.9 ± 0.4
38.0 ± 0.2
76.1 ± 0.4
19.5 ± 0.2
36.5 ± 0.4
78.9 ± 1.0
157.7 ± 1.0
38.0 ± 0.4
76.1 ± 0.0
35.5 ± 0.2
73.1 ± 0.8
35.5 ± 0.2
73.1 ± 0.8
43.4 ± 0.2
86.9 ± 1.0
22.3 ± 0.1
41.7 ± 0.2
21.3 ± 0.1
39.9 ± 0.5
2850 ± 68
3800 ± 84
480 ± .2
26.9 ± 0.1
36.6 ± 0.2
25.9 ± 0.2
35.2 ± 0.4
18.8 ± 0.2
35.3 ± 0.2
27.8 ± 0.1
37.9 ± 0.5
21.0 ± 0.1
39.4 ± 0.2
27.9 ± 0.1
38.0 ± 0.5
19.5 ± 0.1
36.5 ± 0.5
39.4 ± 0.2
78.9 ± 0.8
38.0 ± 0.0
76.1 ± 1.0
53.6 ± 0.4
73.1 ± 0.8
35.5 ± 0.2
73.1 ± 1.0
43.4 ± 0.2
86.9 ± 0.5
22.3 ± 0.1
41.7 ± 0.5
16.0 ± 0.1
21.3 ± 0.1
380 ± 12
53.7 ± 0.6
73.3 ± 0.8
35.2 ± 0.2
70.5 ± 0.8
25.9 ± 0.1
35.3 ± 0.2
37.9 ± 0.0
75.6 ± 0.5
39.4 ± 0.5
78.9 ± 1.0
38.0 ± 0.5
76.1 ± 0.8
14.6 ± 0.1
19.5 ± 0.1
78.9 ± 0.5
157.7 ± 1.0
38.0 ± 0.0
76.1 ± 0.8
26.8 ± 0.2
35.5 ± 0.5
35.5 ± 0.5
73.1 ± 0.5
43.4 ± 0.2
86.9 ± 1.0
41.7 ± 0.5
83.4 ± .1.0
21.3 ± 0.2
39.9 ± 0.2
380 ± 8.0
950 ± 6.0
480 ± 12
26.9 ± 0.1
36.6 ± 0.3
25.9 ± 0.2
35.2 ± 0.2
18.8 ± 0.2
35.3 ± 0.5
27.8 ± 0.2
37.9 ± 0.5
21.0 ± 0.1
39.4 ± 0.5
27.9 ± 0.2
38.0 ± 0.5
9.7 ± 0.01
19.5 ± 0.08
28.9 ± 0.1
39.4 ± 0.2
27.9 ± 0.2
38.0 ± 0.5
35.5 ± 0.5
73.1 ± 1.0
35.5 ± 0.2
73.1 ± 0.8
31.8 ± 0.2
43.4 ± 0.5
22.3 ± 0.2
41.7 ± 0.8
10.7 ± 0.2
21.3 ± 0.5
475 ± 58
36.6 ± 0.2
73.3 ± 0.5
35.2 ± 0.5
70.5 ± 0.5
35.3 ± 0.2
68.2 ± 0.5
37.9 ± 0.5
75.6 ± 0.5
39.4 ± 0.5
78.9 ± 0.5
55.8 ± 0.5
76.1 ± 1.0
36.5 ± 0.5
73.1 ± 0.8
39.4 ± 0.5
78.9 ± 0.5
76.1 ± 0.5
152.1 ± 1.0
35.5 ± 0.4
73.1 ± 1.0
35.5 ± 0.5
73.1 ± 0.8
43.4 ± 0.2
86.9 ± 1.0
41.7 ± 0.5
83.4 ± 1.0
21.3 ± 0.1
39.9 ± 0.2
3800 ± 58
3800 ± 48
480 ± 20
36.6 ± 0.2
73.3 ± 0.8
51.7 ± 0.5
70.5 ± 0.5
35.3 ± 0.2
68.2 ± 0.5
37.9 ± 0.2
75.6 ± 0.5
39.4 ± 0.5
78.9 ± 0.5
55.8 ± 0.5
76.1 ± 0.8
19.5 ± 0.1
36.5 ± 0.5
78.9 ± 0.5
157.7 ± 1.0
55.8 ± 0.5
76.1 ± 0.8
35.5 ± 0.4
73.1 ± 1.0
35.5 ± 0.4
73.1 ± 0.5
43.4 ± 0.4
86.9 ± 1.0
41.7 ± 1.0
83.4 ± 1.0
21.3 ± 1.0
39.9 ± 1.0
380 ± 16
109.9 ± 0.1
146.5 ± 0.2
35.2 ± 0.2
70.5 ± 0.5
35.3 ± 0.2
68.2 ± 0.5
37.9 ± 0.2
75.6 ± 0.5
39.4 ± 0.5
78.9 ± 0.5
55.8 ± 0.5
76.1 ± 1.0
26.8 ± 0.1
36.5 ± 0.2
118.3 ± 1.0
157.7 ± 2.0
76.1 ± 0.5
152.1 ± 1.0
35.5 ± 0.6
73.1 ± 1.0
35.5 ± 0.2
73.1 ± 0.8
86.9 ± 1.0
173.7 ± 2.0
22.3 ± 0.1
41.7 ± 0.4
58.6 ± 0.4
79.9 ± 1.0
380 ± 12
5a
5b
5c
5d
5e
37.9 ± 0.2
39.4 ± 0.2
78.9 ± 0.4
76.1 ± 0.4
152.1 ± 0.1
73.1 ± 0.4
146.2 ± 0.1
315.5 ± 2.5
630.9 ± 8.0
152.1 ± 1.0
304.2 ± .2.0
146.2 ± 1.0
292.3 ± .2.0
35.5 ± 0.4
73.1 ± 0.8
347.4 ± 2.0
694.8 ± 4.0
22.3 ± 0.1
41.7 ± 0.2
79.9 ± 0.4
159.8 ± 1.0
380 ± 12
5f
5g
5h
5i
5j
5k
7a
7b
7c
Ket.
Bif.
950 ± 23
950 ± 12
570 ± 86
950 ± 26
950 ± 23
480 ± 22
480 ± 28
640 ± 28
640 ± 12
480 ± 22
640 ± 3.4
640 ± 0.8
800 ± 1.8
640 ± 2.3
800 ± 3.8
640 ± 1.6
800 ± 2.1
640 ± 3.4
MIC–minimal inhibitory concentration, MFC–minimal fungicidal concentration. A.fum.-A.fumigatus (human isolate),
A.v.-A.versicolor (ATCC 11730), A.o.-A.ochraceus (ATCC 12066), A.n.-A.niger (ATCC 6275), T.v.-T.viride (IAM 5061),
P.f.-P.funiculosum (ATCC 36839), P.o.-P.ochrochloron (ATCC 9112), P.v.c.-P.cyclpoium var. verucosum (food isolate).
2.3.3. Cytotoxicity Assessment
Low toxicity and selectivity of action of antimicrobial compounds is a crucial pre-requisite for
further development. Thus, we studied the cytotoxicity of the most active compounds. MTT analysis
was performed on the HEK-293 human embryonic kidney cell line. The cells were cultured in DMEM
medium supplemented with 10% fetal bovine serum. The cells were inoculated into a 96-well plate
at a concentration of 5.104/mL (5.103 per well, 100
preparations were added, and the results were obtained after a 72 h culture period. The compounds
were added at four concentrations (25, 50, 100, and 250 M). Since compound solutions contained
µL each). After one day of culture, compound
µ
DMSO, control cultures containing only DMSO at the final concentration obtained when the appropriate
volume of compound solution was added were performed.
Although the compounds do not exhibit statistically significant concentration-dependent toxicity
up to 100 µM (Figure 3), they show some toxicity at higher concentrations. The average CC50 values
obtained from three different experiments are given in Tables 5 and 6. The SI index is also shown in
Tables 5 and 6.
Compound 5g and 7c exhibited the best SI index for anti-fungal activity while compound 5d
exhibited the best SI index for anti-bacterial activity.
We compared the CC50 values of compounds 5d
, 5k, 5g, 7c with cytotoxicity of the reference
drugs obtained in the HEK-293 human embryonic kidney cell line. For antibacterials streptomycin,

Pharmaceuticals 2020, 13, 229
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ampicillin and antifungal bifonazole CC50 exceeded 100
µM [63
,64]; for antifungal ketoconazole
CC50 = 60
µM [65]. Thus, cytotoxicity of the most active compounds in our study is comparable or
lower than cytotoxicity of the reference antimicrobial drugs.
Table 5. Antibacterial activity (MIC), cytotoxicity (CC50), and selectivity indices (SI) of compounds 5d,
5g, 5k, 7c.
ID
5d
5g
CC50
B.c
M.f
S.a
L.m
En.cl
P.a
S.T
E.coli
MIC
SI
MIC
SI
MIC
SI
MIC
SI
55.6 ± 0.2
113.8 ± 0.8
37.9 ± 0.2
113.8 ± 0.8
37.9 ± 0.4
37.9 ± 0.2
113.8 ± 1.0
75.6 ± 0.4
252 ± 1.5
256 ± 6.21
252 ± 1.89
225± 1.87
4.5
2.2
6.7
2.2
6.7
6.7
2.2
3.3
73.1 ± 0.1
3.5
73.1 ± 1.0
3.5
53.6 ± 0.4
4.8
73.1 ± 0.8
3.5
36.5 ± 0.5
7.0
109.6 ± 1.0
2.3
53.6 ± 0.6
4.8
109.6 ± 1.0
2.3
73.1 ± 0.5
109.6 ± 1.0
58.6 ± 0.4
109.6 ± 1.5
58.6 ± 0.6
58.6 ± 0.6
109.6 ± 1.5
109.6 ± 2.0
5k
7c
3.5
2.3
4.3
2.3
4.3
4.3
2.3
2.3
79.9 ± 0.4
2.8
119.8 ± 1.5
1.9
79.9 ± 1.0
2.8
159.8 ± 1.0
1.4
58.6 ± 0.4
3.8
58.6 ± 0.4
3.8
119.8 ± 1.0
1.9
119.8 ± 1.5
1.9
Table 6. Antifungal activity (MIC), cytotoxicity (CC50), and selectivity indices (SI) of compounds 5d,
5g, 5k, 7c.
Com.
5d
CC50 (µM)
A.f
A.v
A.o
A.n
T.v
P.o
P.f
Pvc
MIC
SI
MIC
SI
MIC
SI
MIC
SI
202 ± 0.1
37.9 ± 0.2
27.8 ± 0.1
37.9 ± 0.2
27.8 ± 0.2
37.9 ± 0.5
37.9 ± 0.2
37.9 ± 0.2
252 ± 1.5
256 ± 6.21
252 ± 1.89
225 ± 1.87
60 *
1.3
6.7
9.1
6.7
9.1
6.7
6.7
6.7
73.1 ± 0.2
3.5
19.5 ± 0.2
13.1
19.5 ± 0.2
13.1
14.6 ± 0.1
17.5
9.7 ± 0.1
26.4
36.5 ± 0.5
7.0
19.5 ± 0.1
13.1
26.8 ± 0.1
9.6
5g
35.5 ± 0.4
35.5 ± 0.2
35.5 ± 0.2
35.5 ± 0.5
35.5 ± 0.2
35.5 ± 0.5
35.5 ± 0.4
35.5 ± 0.2
5k
7c
7.1
7.1
7.1
7.1
7.1
7.1
7.1
7.1
79.9 ± 0.4
2.8
21.3 ± 0.1
10.6
16.0 ± 0.1
14.1
21.3 ± 0.2
10.6
10.7 ± 0.2
21.0
21.3 ± 0.1
10.6
21.3 ± 1.0
10.6
58.6 ± 0.4
3.8
MIC
SI
380 ± 12
285 ± 68
380 ± 12
380 ± 8.0
475 ± 58
380 ± 58
380 ± 16
380 ± 12
Ket.
0.158
0.210
0.158
0.158
0.126
0.158
0.158
0.158
* 24 h.
Figure 3. MTT assay results for compounds 5d, 5k, 5g, 7c. According to the results, all compounds did
not show statistically significant, concentration-dependent cytotoxicity at concentrations up to 100 M.
µ
The stable decrease in viability observed can be attributed to dimethyl soulfoxide (DMSO,) present at
stable concentration at all compound samples.

Pharmaceuticals 2020, 13, 229
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2.4. Docking Studies
Since the mechanism of antimicrobial action of our compounds is not known, to choose the
proteins as potential targets, we based on the literature. It was found that benzothiazole derivatives are
mentioned as Gyrase inhibitors [66
–68]. On the other hand, according to the literature, thiazolidinones
act as MurB inhibitors [69 72]. Furthermore, prediction of the mechanism of action by computer
–
program PASS indicated Thymidylate kinase as the probable antibacterial target. On the other hand,
several publications mentioned thiazolidinone and indole derivatives as 14α-lanosterol demethylase
inhibitors [73–75]. Thus, taking all these into account, we proposed E. coli DNA Gyrase, Thymidylate
kinase, and E. coli MurB enzymes as antibacterial targets, with CYP51 as the antifungal target.
2.4.1. Docking to Antibacterial Targets
The docking studies revealed that estimated binding energy to E. coli DNA Gyrase (
6.54 kcal/mol) as well as to thymidylate kinase ( 1.55 to 4.12 kcal/mol), were higher than that to
E. coli MurB ( 7.07 to 10.93 kcal/mol). Therefore, it may be resolved that E. coli MurB is the most
−
2.59 to
−
−
−
−
−
suitable enzyme where binding scores were consistent with biological activity (Table 7).
Table 7. Molecular docking binding energies.
Est. Binding Energy (kcal/mol)
Comp.
I-H
E. coli MurB
Residues
E. coli MurB
E.coli
DNA Gyrase
1KZN
Thymidylate
Kinase
E. coli MurB
2Q85
4QGG
5a
5b
5c
5d
5e
5f
−4.63
−3.12
−5.39
−6.21
−6.28
−5.46
-
-
−8.22
−7.70
-9.16
−10.93
−8.97
−8.74
2
2
2
2
2
2
Gly122, Ser228
Arg213, Asn232
Gly122, Ser228
Ser228, Arg326
Arg213, Ser228
Gly122, Ser228
Gly122, Ser228,
Asn232
−2.13
−4.12
−2.39
−1.55
5g
−6.54
−3.26
−10.88
3
5h
5i
5j
5k
7a
7b
7c
−6.11
−3.69
−5.52
−5.63
−2.59
−3.67
−4.28
−1.24
−9.12
−7.07
−9.21
−9.83
−7.28
−7.75
−7.88
2
2
2
2
2
2
2
Gly122, Ser228
Arg213, Arg326
Gly122, Ser228
Arg213, Ser228
Gly122, Arg213
Ser228, Asn232
Gly122, Ser228
−1.15
−3.25
−2.96
-
-
-
The docking pose of the most active compound 5d in E. coli MurB enzyme showed two favorable
hydrogen bond interactions. The first one is between the oxygen atom of the C=O group of the
compound and the hydrogen of the side chain of Ser228. The second one between the oxygen atom of
-OH group of the compound and the side chain of Arg326 (distances 2.17 Å and 1.99 Å, respectively).
The fused rings interact hydrophobically with the residues Tyr189, Asn232, Leu289, Ala123, Leu217,
and Arg213, while the benzene ring interacts hydrophobically with the residues Asn50, Ser115, Ile118,
Ile121, Gln119 and Glu324 (Figure 4). These interactions stabilize the complex compound-enzyme and
play a crucial role in the increased inhibitory activity of compound 5d Moreover, the hydrogen bond
formation with the residue Ser228 is essential for the inhibitory action of the compounds; thus, this
residue takes part in the proton transfer at the second stage of peptidoglycan synthesis [76].
The second most active compound, 5g, also forms the hydrogen bond interaction with the residue
Ser228 that explains its high inhibitory action (Figure 2). Detailed analysis of the docking pose of the
two most active compounds showed that they similarly bind MurB, and they insert deeper to the
binding center of the enzyme than FAD, forming a hydrogen bond with the residue Ser228 (Figure 5).

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Figure 4. Docked conformation of the most active compound 5d in E.coli MurB (Left). 2D diagrams of
the most active compounds 5d (up) and 5g (down) in E.coli MurB (Right).
The same behavior was observed in the case of docking of the most active compound
among 5-(1H-indol-3-ylmethylene)-4-oxo-2-thioxothiazolidin-3-yl)alkane carboxylic acids [19] and
5-adamantane thiadiazole-based thiazolidinones [70]. Again, the formation of the hydrogen bond
between the C=O group and Ser228 was observed. Thus, the obtained results support previous
data [69
–72] that MurB maybe is the most appropriate target for the antibacterial activity for this
chemical series.
Figure 5. Docked conformation of compounds 5d (green), 5g (red) and FAD (blue) in E.Coli MurB.
2.4.2. Docking to Lanosterol 14α-demethylase of C. albicans
All the synthesized compounds and the reference drug ketoconazole were docked to lanosterol
14α-demethylase of C. albicans (Table 8).

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Table 8. Molecular docking binding energies.
Est. Binding Energy
(kcal/mol) CYP51 of
C. albicans
PDB ID: 5V5Z
Residues
CYP51 of
C. albicans
PDB ID: 5V5Z
Interactions with
HEM601
N/N
I-H
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
7a
7b
7c
−7.65
−9.74
−8.13
−10.22
−9.15
−8.79
−11.55
−7.11
−7.84
−8.72
−9.24
−7.08
−10.36
−10.84
−8.23
1
1
2
2
1
2
1
-
1
2
2
1
1
2
1
Tyr132
Tyr132
Tyr64, Tyr132
Tyr118, Tyr132
Tyr132
Hydrophobic
Ionizable, Hydrophobic
Hydrophobic
Ionizable, Hydrophobic
Ionizable, Hydrophobic
Hydrophobic
Tyr118, Tyr132
Tyr132
Fe binding, Ionizable, Hydrophobic
Ionizable, Hydrophobic
Ionizable, Hydrophobic
Hydrophobic
-
Tyr132
Tyr118, Met508
Tyr64, Tyr118
Tyr132
Tyr132
Tyr118, Tyr132
Tyr64
Hydrophobic
Hydrophobic
Ionizable, Hydrophobic
Ionizable, Hydrophobic
Ionizable, Hydrophobic
ketoconazole
Docking results showed that all the synthesized compounds might bind to CYP51Ca close to those
of the reference drug ketoconazole. Compound 5g is located inside the enzyme alongside to heme
group, interacting with the Fe of the heme group of CYP51Ca throughout its atom N of the pyridine
ring. Moreover, compound 5g forms a hydrogen bond between the oxygen of –OCH3 substituent and
the hydrogen of the side chain of Tyr132. Hydrophobic interactions were detected between residues
Thr122, Phe126, Tyr132, and Ile131 and the fused rings of the compound 5g, also between Leu376,
Thr311 and the benzene ring of the compound. Furthermore, compound 5g interacts hydrophobically
throughout its benzene ring with the heme group of the enzyme, and also it forms a positive ionizable
bond with it (Figure 6). Interaction with the heme group was also observed with the benzene ring of
ketoconazole, which forms positive ionizable interactions (Figures 6 and 7). However, compound 5g
forms a more stable complex of the ligand with enzyme indicating its interaction with the Fe, which is
probably why compound 5g showed high antifungal activity.
Figure 6. Docked conformation of ketoconazole in lanosterol 14alpha-demethylase of C. albicans
(CYP51ca).

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Figure 7. Docked conformation of compound 5g in lanosterol 14alpha-demethylase of C. albicans
(CYP51ca).
It should be mentioned that the tested compounds interact more strongly with the heme group of
the enzyme CYP51Ca because the heme’s Fe is involved in this interaction. In the case of our previous
work [19], the most active compound interacts with the heme but throughout its benzene ring and the
–NO2 group, forming pi and negative ionizable interactions with the heme group, respectively. In the
case of 5-adamantane thiadiazole-based thiazolidinones [72], again, the most active compound form
positive interactions between the heme group and heterocyclic rings of the compound. Thus, it can be
concluded that thiazolidinone derivatives, in general, can interact with the heme of CYP51Ca in the
same way as ketoconazole interacts.
3. Materials and Methods
All starting materials were purchased from Merck and used without purification. NMR spectra
were determined with Varian Mercury VX-400” (Varian Co., Palo Alto, CA, USA) and AM-300
Bruker 300 MHz. spectrometers in DMSO-d6. MS (ESI) spectra were recorded on an LC-MS
system-HPLC Agilent 1100 (Agilent Technologies Inc., Santa, Clara, CA USA) equipped with a diode
array detector Agilent LC
PN 821975-932), solvent water–acetonitrile mixture (95:5), 0.1% of aqueous trifluoroacetic acid; eluent
flow 3 mL min–1; injection volume 1
L; IR spectra were recorded on a Vertex 70 Bruker” (Bruker,
\MSD SL. Parameters of analysis: Zorbax SB-C18 column (1.8 µM, 4.6–15 mm,
µ
Karlsruhe, Germany) spectrometer in KBr pellets. Melting points were determined in open capillary
tubes and are uncorrected.
3.1. In Silico Biological Activity Evaluation
Antimicrobial activity and toxicity of the designed compounds have been estimated in silico
using web services available on the Way2Drug portal [56]. These services are based on the PASS
(Prediction of Activity Spectra for Substances) and GUSAR (General Unrestricted Structure-Activity
Relationships) software, which is described in detail elsewhere [60,61]. It is essential to mention that
PASS-based services provide the assessments of the compound’s activity as the difference between the
probabilities for the chemical compound with a particular structure to display activity (Pa) and do not
display this activity (Pi). By default, in PASS, all activities with Pa > Pi are considered as probable.
High Pa-Pi values reflect the high structural similarity of the analyzed compound to the structures
included in the training set with those activities. Since our goal was not finding close analogs of the

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earlier discovered antimicrobial agents, we considered compounds with small Pa-Pi values as the
promising hits for experimental testing. If the experiment will confirm their activity, there is a chance
to find a New Chemical Entity. GUSAR-based service [60,61] provides the quantitative assessment
of acute rat toxicity expressed as LD50 values for four routes of administration: intraperitoneal (IP),
intravenous (IV), oral, and subcutaneous (SC).
3.2. Chemistry
3.2.1. General Procedure for the Preparation of N-(4-oxo-2-thioxothiazolidin-3-yl) carbamides 3a–d
In a round-bottom flask equipped with a reflux condenser, 0.05 mol of trithiocarbonyl diglycolic
acid, 0.05 mol of the corresponding hydrazide and alcohol-water mixture (1:1) were placed and boiled
for 3 h. The reaction mixture is cooled, the precipitate is filtered off and recrystallized.
2-Hydroxy-N-(4-oxo-2-thioxothiazolidin-3-yl)benzamide 3a. Yield 97%; m.p. 104–106 ◦C
(CH3COOH-H2O 2:1). IR (cm–1): 3342.48 (OH), 1751.28 (C=O), 1657.74 (C=O), 1608.56 (C=S).).
1H NMR (400 MHz, DMSO-d6, ppm)
δ
11.62–10.91 (br.s, 2H, NH, OH), 7.88 (dd, J = 8.0, 1.6 Hz, 1H, H6
benzene), 7.52–7.46 (m, 1H, H3 benzene), 7.05–6.95 (m, 1H, 2H, H4 +H5, aromatic), 4.48 (q, J = 18.7 Hz,
2H, CH2). 13C NMR (101 MHz, DMSO, ppm)
199.90, 170.25, 164.91, 157.93, 134.62, 129.83, 119.44,
δ
117.19, 115.07, 33.38. Anal. Calcd. for C10H8N2O3S2 (%): C, 44.77; H, 3.01; N, 10.44; S, 23.90 Found
(%):C, 44.88; H, 3.09; N, 10.37; S, 23.95.
4-Hydroxy-N-(4-oxo-2-thioxothiazolidin-3-yl)benzamide 3b. Yield 87%; m.p. 207–209 ◦C
(CH3COOH).). IR (cm–1): 3259.54 (OH), 3166(NH), 1739.71 (C=O), 1667.38(C=O), 1583.48 (C=S).
1H NMR (400 MHz, DMSO-d6, ppm)
δ
11.25 (s, 1H, NH), 10.29 (s, 1H, OH), 7.81 (dd, J = 9.1, 2.3 Hz,
2H, H2 +H6, benzene), 6.88 (dd, J = 9.1, 2.3 Hz, 2H, H3 +H5, benzene), 4.51 (s, 2H, CH2). 13C NMR
(101 MHz, DMSO, ppm) 200.33, 170.54, 163.93, 161.44, 129.96, 121.44, 115.24, 33.32. Anal. Calcd. for
δ
C10H8N2O3S2 (%): C, 44.77; H, 3.01; N, 10.44; S, 23.90 Found (%):C, 44.69; H, 2.95; N, 10.36; S, 23.81.
N-(4-Oxo-2-thioxothiazolidin-3-yl)nicotinamide 3c. Yield 83%; m.p. 190 ◦C decomp.(C2H5OH).
IR (cm–1): 1753.21 (C=O), 1687.63 (C=O), 1556.48 (C=S). 1H NMR (400 MHz, DMSO-d6, ppm)
δ
11.95
(s, 1H, NH), 8.97–8.73 (m, 2H, H2 +H4, pyridine), 7.99–7.70 (m, 2H, H5 +H6, pyridine), 4.55 (s, 2H,
CH2).). 13C NMR (101 MHz, DMSO, ppm)
199.81, 170.20, 163.25, 150.76, 137.93, 121.31, 119.56, 33.55.
δ
Anal. Calcd. for C9H7N3O2S2 (%): C, 42.68; H, 2.79; N, 16.59; S, 25.32 Found (%):C, 42.79; H, 2.70; N,
16.48; S, 25.45.
N-(4-Oxo-2-thioxothiazolidin-3-yl)isonicotinamide 3d
(C2H5OH). IR (cm–1): 1753.21(C=O), 1678.95 (C=O), 1556.48 (C=S).1H NMR (400 MHz, DMSO-d6,
ppm) 11.95 (s, 1H, NH), 8.87–8.79 (m, 2H, H3 +H5, pyridine), 7.85–7.80 (m, 2H, H2 +H6, pyridine),
4.55 (s, 2H, CH2). 13C NMR (101 MHz, DMSO, ppm)
199.81, 170.19, 163.25, 150.76, 137.93, 121.39,
.
Yield 85%; m.p. 193 ◦C decomp.
δ
δ
119.56, 33.58. Anal. Calcd. for C9H7N3O2S2 (%): C, 42.68; H, 2.79; N, 16.59; S, 25.32 Found (%):C, 42.77;
H, 2.85; N, 16.76; S, 25.26.
3.2.2. General Procedure 5-[(R-1H-indol-3-ylmethylene)-4-oxo-2-thioxothiazolidin-3-yl] carbamides
5a–k and 5-(R-1H-indol-3-ylmethylene)-3-morpholin-4-yl-2-thioxothiazolidin-4-ones 7a–c
In a round-bottom flask equipped with a reflux condenser, 2.5 mmol of 3-substituted
2-thioxo-4-oxothiazolidine 3a-d or 6, 3.3 mmol of the corresponding aldehyde 1a-d, 2.5 mmol of
ammonium acetate and 5 mL of acetic acid are placed. The reaction mixture is boiled for 2 h, cooled,
the precipitate is filtered off, washed with acetic acid and water, dried and recrystallized.
2-Hydroxy-N-{(5Z)-5-[(1-methyl-1H-indol-3-yl)methylene]-4-oxo-2-thioxothiazolidin-3-
◦
yl}benzamide 5a. Yield 98%; m.p. 265–267 C (DMFA-CH3COOH). IR (cm–1): 3272.08 (OH),
1
1700.17 (C=O), 1656.77 (C=O), 1588.3 (C=C), 1573.84 (C=S). H-NMR (300 MHz, DMSO-d6, ppm)
δ
11.40 (s, 1H, NH-CO), 11.31 (s, 1H, OH), 8.12 (s, 1H, CH=), 8.01 (d, J = 7.9 Hz, 1H, H6 benzene),
7.91 (d, J = 4.2 Hz, 2H, H4 +H7, indole), 7.54–7.41 (m, 2H, H3 benzene + H2 indole), 7.37–7.22 (m,
2H, H5 +H6, indole), 6.97 (dd, J = 17.3, 8.1 Hz, 2H, H4 +H5, benzene), 4.00 (s, 3H, CH3N). 13C NMR

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(101 MHz, DMSO, ppm)
δ
189.71, 164.99, 163.08, 158.00, 136.96, 134.65, 134.45, 129.90, 127.29, 127.01,
123.54, 122.01, 119.46, 118.78, 117.22, 115.13, 111.01, 109.93, 33.50. ESI-MS [m/z]: [M + H]+ = 411.0; [M
−
H]− = 408.2. Anal. Calcd. for C20H15N3O3S2 (%): C, 58.66; H, 3.69; N, 10.26; S, 15.66 Found (%):
58.75 H, 3.62; N, 10.21; S, 15.52.
2-Hydroxy-N-{(5Z)-5-[(5-methoxy-1H-indol-3-yl)methylene]-4-oxo-2-thioxothiazolidin-3-
yl}benzamide 5b. Yield 77%; m.p. 239–241 ◦C (DMFA-CH3COOH).). IR (cm–1): 3234.47 (OH), 1699.21
(C=O), 1654.84 (C=O), 1585.41 (C=C, C=S). 1H-NMR (300 MHz, DMSO-d6, ppm)
δ 12.15 (s, 1H, NH),
11.45 (s, 1H, NH-CO), 11.35 (s, 1H, OH), 8.18 (s, 1H, CH=), 8.00 (d, J = 7.8 Hz, 1H, H6 benzene), 7.74 (d,
J = 2.2 Hz, 1H, H4 indole), 7.50–7.33 (m, 3H, H3 benzene + H2 +H7, indole), 7.04–6.90 (m, 2H, H4 +H5,
benzene), 6.83 (d, J = 8.4 Hz, 1H, H6 indole), 3.87 (s, 3H, CH3O 13C NMR (101 MHz, DMSO, ppm)
δ
189.72, 165.10, 163.07, 158.10, 155.45, 134.64, 131.12, 131.09, 129.80, 128.22, 127.84, 119.43, 117.25, 115.07,
113.67, 113.38, 111.09, 110.44, 100.58, 55.85. ESI-MS [m/z]: [M + H]+ = 426.0; [M H]− = 424.0. Anal.
−
Calcd. for C20H15N3O4S2 (%): C, 56.46; H, 3.55; N, 9.88; S, 15.07 Found (%):C, 56.57; H, 3.49; N, 9.96; S,
15.15.
2-Hydroxy-N-{(5Z)-5-[(6-methoxy-1H-indol-3-yl)methylene]-4-oxo-2-thioxothiazolidin-3-
yl}benzamide 5c. Yield 80%; m.p. 268–270 ◦C (DMFA-CH3COOH). IR (cm–1): 3227.72 (OH), 1705.96
1
(C=O), 1670.27 (C=O), 1591.2 (C=C), 1576.73 (C=S). H-NMR (300 MHz, DMSO-d6, ppm)
δ 12.06
(s, 1H, NH), 11.45 (s, 1H, NH-CO),), 11.34 (s, 1H, OH), 8.10 (s, 1H, CH=), 7.99 (d, J = 7.9 Hz, 1H, H6
benzene), 7.75 (d, J = 8.7 Hz, 1H, H4 indole), 7.69 (d, J = 1.9 Hz, 1H, H3 benzene), 7.48-7,42 (m, 1H, H2
indole), 7.05–6.91 (m, 3H, H7 indole +H4 + H5, benzene), 6. 83 (d, J = 8.5 Hz, 1H, H5 indole), 3.84 (s,
3H, CH3O13C NMR (101 MHz, DMSO, ppm)
δ
189.71, 165.00, 163.10, 158.01, 156.93, 137.32, 134.64,
130.31, 129.87, 127.93, 120.68, 119.49, 119.44, 117.22, 115.11, 111.63, 111.13, 111.06, 95.41, 55.28. ESI-MS
[m/z]: [M + H]+ = 426.0; [M H]− = 424.0.. Anal. Calcd. for C20H15N3O4S2 (%): C, 56.46; H, 3.55; N,
−
9.88; S, 15.07 Found (%):C, 56.39; H, 3.51; N, 9.80; S, 15.01.
4-Hydroxy-N-[(5Z)-5-(1H-indol-3-ylmethylene)-4-oxo-2-thioxothiazolidin-3-yl]benzamide 5d.
Yield 90%; m.p. > 275 ◦C (DMFA-CH3COOH). IR (cm–1): 3369.48 (OH), 3225.79 (NH), 1694.38 (C=O),
1668.35 (C=O), 1591.2 (C=C), 1573.84 (C=S). 1H-NMR (300 MHz, DMSO-d6, ppm)
δ 12.23 (s, 1H, NH),
11.12 (s, 1H, NH-CO), 9.89 (s, 1H, OH), 8.13 (s, 1H, CH=), 7.92–7.81 (m, 3H, H2 + H6, benzene + H4
indole), 7.79 (d, J = 3.0 Hz, 1H, H7 indole), 7.53–7.45 (m, 1H, H2 indole), 7.28–7.15 (m, 2H, H5 + H6,
indole), 6.85 (d, J = 8.7 Hz, 2H, H3 +H5, benzene). 13C NMR (101 MHz, DMSO, ppm)
δ
190.12, 164.06,
163.36, 161.49, 136.48, 131.20, 129.97, 127.80, 126.77, 123.49, 121.69, 121.50, 118.64, 115.30, 112.61, 111.16,
110.97. ESI-MS [m/z]: [M + H]+ = 396.0; [M H]− = 394.0. Anal. Calcd. for C19H13N3O3S2 (%): C,
−
57.71; H, 3.31; N, 10.63; S, 16.22 Found (%):C, 57.62; H, 3.37; N, 10.55; S, 16.16.
N-[(5Z)-5-(1H-Indol-3-ylmethylene)-4-oxo-2-thioxothiazolidin-3-yl]nicotinamide 5e. Yield
90%; m.p. > 275 ◦C (DMFA-CH3COOH). IR (cm–1): 3485.2 (NH), 1696.31 (C=O), 1674.13 (C=O), 1596.98
(C=C), 1577.7 (C=S). 1H-NMR (500 MHz, DMSO-d6, ppm) δ 12.31 (s, 1H, NH), 11.75 (s, 1H, NH-CO),
9.15 (d, J = 1.7 Hz, 1H, H4 pyridine), 8.77 (dd, J = 4.8, 1.4 Hz, 1H, H2 pyridine), 8.33 (d, J = 8.0 Hz, 1H,
H6 pyridine), 8.17 (s, 1H, CH=), 7.90 (d, J = 7.6 Hz, 1H, H5 pyridine), 7.84 (d, J = 3.0 Hz, 1H, H4 indole),
7.57–7.48 (m, 2H, H2 +H7, indole), 7.27–7.18 (m, 2H, H5 +H6, indole). 13C NMR (101 MHz, DMSO,
ppm)
δ 189.54, 163.32, 162.94, 150.78, 137.89, 136.41, 131.49, 128.38, 126.78, 123.56, 121.78, 121.38, 118.68,
112.65, 110.98, 110.72. ESI-MS [m/z]: [M + H]+ = 381.0; Anal. Calcd. for C18H12N4O2S2 (%): C, 56.83;
H, 3.18; N, 14.73; S, 16.86 Found (%):C, 56.71; H, 3.24; N, 14.80; S, 16.79.
N-{(5Z)-5-[(1-Methyl-1H-indol-3-yl)methylene]-4-oxo-2-thioxothiazolidin-3-yl}nicotinamide
5f. Yield 94%; m.p. 270–272 ◦C (DMFA-CH3COOH). IR (cm–1): 3241.22 (NH), 1706.92 (C=O), 1681.85
(C=O), 1589.27 (C=C), 1572.87 (C=S). 1H-NMR (300 MHz, DMSO-d6, ppm)
δ 11.75 (s, 1H, NH-CO),
9.16 (d, J = 1.9 Hz, 1H, H pyridine), 8.77 (dd, J = 4.8, 1.3 Hz, 1H, H2 pyridine), 8.36–8.31 (m, 1H, H6
4
pyridine), 8.12 (s, 1H, CH=), 7.96 (s, 1H, H5 pyridine), 7.91 (d, 1H, J = 7.9 Hz, H4 indole), 7.58–7.47 (m,
2H, H2 + H7, indole), 7.32 (t, J = 7.5 Hz, 1H, H6 indole), 7.26 (t, J = 7.4 Hz, 1H, H5 indole), 4.00 (s,
3H, CH3N). 13C NMR (101 MHz, DMSO, ppm)
δ
189.64, 163.34, 163.01, 153.40, 148.60, 136.99, 135.59,
134.68, 127.57, 127.30, 126.77, 123.97, 123.60, 122.10, 118.80, 111.08, 110.55, 109.95, 33.54. ESI-MS [m/z]:

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[M + H]+ = 395.0; [M
16.26 Found (%):C, 57.94; H, 3.51; N, 14.15; S, 16.35.
−
H]− = 394.0. Anal. Calcd. for C19H14N4O2S2 (%): C, 57.85; H, 3.58; N, 14.20; S,
N-{(5Z)-5-[(5-Methoxy-1H-indol-3-yl)methylene]-4-oxo-2-thioxothiazolidin-3-yl}nicotinamide
5g. Yield 00%; m.p. 199–201 ◦C. IR (cm–1): 3254.72 (NH), 1718.49 (C=O), 1681.85 (C=O), 1585.41 (C=C),
1576.73 (C=S). 1H-NMR (300 MHz, DMSO-d6, ppm) 1H-NMR (300 MHz, DMSO-d6, ppm)
δ 12.17 (s,
1H, NH), 11.72 (s, 1H, NH-CO), 9.17 (s, 1H, H4 pyridine), 8.77 (d, J = 3.0 Hz, 1H, H2 pyridine), 8.35 (d,
J = 7.5 Hz, 1H, H6 pyridine), 8.19 (s, 1H, CH=), 7.74 (s, 1H, H5 pyridine), 7.59–7.49 (m, 1H, H2 indole),
7.46–7.30 (m, 2H, H4 +H7 indole), 6.83 (d, J = 8.5 Hz, 1H, H6 indole), 3.86 (s, 3H, CH3O). 13C NMR
(101 MHz, DMSO, ppm)
127.86, 126.82, 123.94, 113.69, 113.41, 111.12, 110.01, 100.64, 55.54. ESI-MS [m/z]: [M + H]+ = 411.0; [M
H]− = 409.0. Anal. Calcd. for C19H14N4O3S2 (%): C, 55.60; H, 3.44; N, 13.65; S, 15.62 Found (%): C,
δ 189.67, 163.30, 163.01, 155.51, 153.36, 148.61, 135.57, 131.33, 131.15, 128.76,
−
55.49; H, 3.39; N, 13.58; S, 15.67.
N-[(5Z)-5-(1H-Indol-3-ylmethylene)-4-oxo-2-thioxothiazolidin-3-yl]isonicotinamide 5h. Yield
86%; m.p. > 275
(C=O), 1672.2 (C=O), 1594.09 (C=C), 1576.73 (C=S). 1H-NMR (300 MHz, DMSO-d6, ppm)
º
C Yield 86%; m.p. > 275 ◦C (DMFA-CH3COOH). IR (cm–1): 3196.86 (NH), 1718.49
δ
12.27 (s,
1H, NH), 11.79 (s, 1H, NH-CO), 8.78 (d, J = 5.8 Hz, 2H, H2 +H6, pyridine), 8.17 (s, 1H, CH=), 7.94–7.86
(m, 3H, H3 +H5, pyridine +H4 indole), 7.82 (s, 1H, H7 indole), 7.51 (d, J = 7.1 Hz, 1H, H2 indole),
7.30–7.15 (m, 2H, H5 +H6, indole). 13C NMR (101 MHz, DMSO, ppm) δ 189.53, 163.32, 162.94, 150.76,
137.94, 136.42, 131.46, 128.34, 126.77, 123.54, 121.76, 121.38, 118.66, 112.65, 110.99, 110.78. ESI-MS [m/z]:
[M + H]+ = 381.0; [M
16.86 Found (%):C, 56.89; H, 3.26; N, 14.65; S, 16.88.
−
H]− = 379.0. Anal. Calcd. for C18H12N4O2S2 (%): C, 56.83; H, 3.18; N, 14.73; S,
N-{(5Z)-5-[(1-Methyl-1H-indol-3-yl)methylene]-4-oxo-2-thioxothiazolidin-3-
yl}isonicotinamide 5i. Yield 95%; m.p. 269–271 ◦C (DMFA-CH3COOH). IR (cm–1): 3217.11
(NH), 1710.78 (C=O), 1674.13 (C=O), 1587.34 (C=C), 1570.95 (C=S).1H-NMR (300 MHz, DMSO-d6,
ppm)
(m, 4H, H3 +H5, pyridine + H4 +H7, indole), 7.51 (d, J = 7.9 Hz, 1H, H2 indole), 7.37–7.20 (m, 2H, H5
+H6, indole), 4.00 (s, 3H, CH3N). 13C NMR (101 MHz, DMSO, ppm)
189.49, 163.32, 162.90, 150.78,
137.88, 137.00, 134.73, 127.68, 127.30, 123.61, 122.12, 121.38, 118.81, 111.09, 110.46, 109.95, 33.55. ESI-MS
[m/z]: [M + H]+ = 395.0; [M H]− = 393.0. Anal. Calcd. for C19H14N4O2S2 (%): C, 57.85; H, 3.58; N,
δ 11.81 (s, 1H, NH-CO), 8.79 (d, J = 5.9 Hz, 2H, H2 +H6, pyridine), 8.13 (s, 1H, CH=), 7.99–7.86
δ
−
14.20; S, 16.26 Found (%):C, 57.78; H, 3.53; N, 14.28; S, 16.17.
N-{(5Z)-5-[(5-Methoxy-1H-indol-3-yl)methylene]-4-oxo-2-thioxothiazolidin-3-
yl}isonicotinamide 5j. Yield 89%; m.p. 261–263
º
C (CH3COOH).). IR (cm–1): 3199.75 (NH),
1706.92 (C=O), 1676.06 (C=O), 1588.3 (C=C, C=S). 1H-NMR (300 MHz, DMSO-d6, ppm)
δ
12.20 (s, 1H,
NH), 11.83 (s, 1H, NH-CO), 8.78 (d, J = 5.4 Hz, 2H, H2 +H6, pyridine), 8.20 (s, 1H, CH=), 7.90 (d, J = 5.4
Hz, 2H, H3 +H5, pyridine), 7.76 (d, J = 3.0 Hz, 1H, H4 indole), 7.44–7.33 (m, 2H, H2 +H7, indole), 6.83
(dd, J = 8.9, 1.7 Hz, 1H, H6 indole), 3.86 (s, 3H, CH3O). 13C NMR (101 MHz, DMSO, ppm)
δ
189.50,
163.29, 162.91, 155.49, 150.78, 137.91, 131.39, 131.12, 128.89, 127.88, 121.39, 113.72, 113.42, 111.12, 109.87,
100.58, 55.51. ESI-MS [m/z]: [M + H]+ = 411.0; [M H]− = 409.0. Anal. Calcd. for C19H14N4O3S2 (%):
−
C, 55.60; H, 3.44; N, 13.65; S, 15.62 Found (%): C, 55.52; H, 3.47; N, 13.73; S, 15.55.
N-{(5Z)-5-[(6-Methoxy-1H-indol-3-yl)methylene]-4-oxo-2-thioxothiazolidin-3-
yl}isonicotinamide 5k. Yield 89%; m.p. 275–277 ◦C (CH3COOH). IR (cm–1): 3550.78 (NH),
1
3346.34 (NH), 1725.24 (C=O), 1689.56 (C=O), 1596.98 (C=C), 1576.73 (C=S). H-NMR (300 MHz,
DMSO-d6, ppm)
δ 12.11 (s, 1H, NH), 11.85 (s, 1H, NH-CO), 8.78 (d, J = 5.3 Hz, 2H,H2 +H6, pyridine),
8.11 (s, 1H, CH=), 7.89 (d, J = 5.3 Hz, 2H, H3 +H5, pyridine), 7.74 (dd, J = 13.7, 5.6 Hz, 2H, H2 +H4,
indole), 6.96 (s, 1H, H7 indole), 6.83 (d, J = 8.6 Hz, 1H, H5 indole), 3.84 (s, 3H, CH3O). 13C NMR
(101 MHz, DMSO, ppm)
δ
189.49, 163.30, 162.91, 156.97, 150.78, 137.88, 137.36, 130.66, 128.61, 121.38,
H]− = 409.0.
120.66, 119.52, 111.71, 111.17, 110.52, 95.46, 55.29. ESI-MS [m/z]: [M + H]+ = 411.0; [M
−
Anal. Calcd. for C19H14N4O3S2 (%): C, 55.60; H, 3.44; N, 13.65; S, 15.62 Found (%): C, 55.73; H, 3.49; N,
13.57; S, 15.54.

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(5Z)-5-(1H-Indol-3-ylmethylene)-3-morpholin-4-yl-2-thioxothiazolidin-4-one7a. Yield 86%;
◦
m.p. 273–275 C (DMFA:CH3COOH). IR (cm–1): 3247.97 (NH), 1690.53 (C=O), 1594.09 (C=C),
1575.77 (C=S).1H-NMR (300 MHz, DMSO-d6, ppm)
δ
12.10 (s, 1H, NH), 7.98 (s, 1H, CH=), 7.85 (d,
J = 6.8 Hz, 1H, H4 indole), 7.66 (d, J = 2.8 Hz, 1H, H7 indole), 7.51–7.45 (m, 1H, H2 indole), 7.27–7.13 (m,
2H, H5 +H6, indole), 3.81 (s, 6H, morpholine), 3.06 (s, 2H, morpholine). 13C NMR (101 MHz, DMSO,
ppm)
δ
189.92, 165.21, 136.32, 130.51, 126.72, 126.03, 123.34, 121.50, 118.48, 112.53, 111.68, 110.95, 66.56,
H]− = 344.2. Anal. Calcd. for C16H15N3O2S2 (%): C,
50.14. ESI-MS [m/z]: [M + H]+ = 346.2; [M
−
55.63; H, 4.38; N, 12.16; S, 18.56 Found (%):C, 55.74; H, 4.32; N, 12.24; S, 18.49.
(5Z)-5-[(1-Methyl-1H-indol-3-yl)methylene]-3-morpholin-4-yl-2-thioxo-thiazolidin-4-one 7b was
prepared according to [42].
(5Z)-5-[(5-Methoxy-1H-indol-3-yl)methylene]-3-morpholin-4-yl-2-thioxo-thiazolidin-4-one7c.
Yield 82%; m.p. 250–252 ◦C (CH3COOH). IR (cm–1): 3163.11 (NH), 1690.53 (C=O), 1580.59 (C=C, C=S).
1H-NMR (300 MHz, DMSO-d6, ppm)
δ
12.08 (s, 1H, NH), 7.99 (s, 1H, CH=), 7.61 (s, 1H, H4 indole),
7.34 (d, J = 3.4 Hz, 2H, H2 +H7, indole), 6.81 (d, J = 8.7 Hz, 1H, H6 indole), 4.05–3.57 (m, 9H, CH3O,
morpholine), 3.03 (s, 2H, morpholine). 13C NMR (101 MHz, DMSO, ppm)
189.87, 165.20, 155.29,
131.04, 130.51, 127.75, 126.61, 113.52, 113.30, 111.04, 110.74, 100.34, 66.56, 55.46, 50.11. ESI-MS [m/z]: [M
+ H]+ = 376.0; [M H]− = 374.0. Anal. Calcd. for C17H17N3O3S2 (%): C, 54.38; H, 4.56; N, 11.19; S,
δ
−
17.08 Found (%):C, 54.31; H, 4.62; N, 11.04; S, 17.15.
3.3. Antibacterial Activity Evaluation
Bacterial strains utilized include Gram-negative: Salmonella typhimurium (ATCC 13311)
Pseudomonas aeruginosa (ATCC 27853), Escherichia coli (ATCC 35210), Enterobacter cloacae (ATCC
35030) and Gram-positive bacteria: Micrococcus flavus (ATCC 10240), Bacillus cereus (isolated clinically),
Staphylococcus aureus (ATCC 6538), and Listeria monocytogenes (NCTC 7973) bacteria. Pathogens were
provided from the Mycological Laboratory, Institute for Biological Research “Siniša Stankovic”
National institute of Republic of Serbia Belgrade. Resistant strains used were MRSA, E. coli,
and P. aeruginosa [77,78].
For the determination of minimum inhibitory (MIC) and minimum bactericidal concentrations, the
microdilution method, as previously described [77
bactericidal (MBC) concentrations were determined by the modified microdilution method as previously
reported [77 79]. Briefly, the fresh overnight culture of bacteria was adjusted to a concentration of
105 CFU/mL. The tested compounds were dissolved in 5% DMSO and serially diluted in tryptic
soy broth (TSB) medium with bacterial inoculum (1.0
104 CFU per well). The microplates were
–79]. The minimum inhibitory (MIC) and minimum
–
1
×
×
incubated for 24 h at 37 ◦C. The MIC of the samples was detected following the addition of 40
µL
of iodonitrotetrazolium chloride (INT) (0.2 mg/mL) and incubation at 37 ◦C for 30 min. The lowest
concentration that produced a significant inhibition of the growth of the bacteria in comparison with
the positive control was identified as the MIC. MBC was determined by serial sub-cultivation of
10 µL into microplates containing 100 µL of TSB. The lowest concentration that shows no growth
after this sub-culturing was identified as the MBC, indicating 99.5% death of the original inoculum.
Streptomycin and ampicillin were used as positive controls.
3.4. Antifungal Evaluation
The following fungi were used: Aspergillus niger (ATCC 6275), Aspergillus ochraceus (ATCC
12066), Aspergillus fumigatus (human isolate), Aspergillus versicolor (ATCC 11730), Penicillium
funiculosum (ATCC 36839), Penicillium ochrochloron (ATCC 9112), Trichoderma viride (IAM 5061),
Penicillium verrucosum var. cyclopium (food isolate). The organisms were obtained from the
Mycological Laboratory, Department of Plant Physiology, Institute for Biological Research “Siniša
Stankovic”, National institute of Republic of Serbia, Belgrade, Serbia. All experiments were performed
in duplicate and repeated three times, as previously described [80,81].

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The fungal spores were washed from the surface of agar plates with sterile 0.85% saline containing
0.1% Tween 80 (v/v). The spore suspension was adjusted with sterile saline to a concentration of
approximately 1.0
×
105 in a final volume of 100
µL per well. MIC determinations were performed by
a serial dilution technique using 96-well microtiter plates. The examined compounds were serially
diluted in broth Malt medium (MA), after which inoculum was added. The microplates were incubated
for 72 h at 28 ◦C. The lowest concentrations without visible growth (at the binocular microscope) were
defined as MICs. The fungicidal concentrations (MFCs) were determined by serial subcultivation of
2 µL of tested fractions dissolved in medium and inoculation into microtiter plates containing 100 µL
of broth per well and further incubation 72 h at 28 ◦C. The lowest concentration with no visible growth
was defined as MFC, indicating 99.5% killing of the original inoculum. The fungicides bifonazole and
ketoconazole were used as positive controls.
3.5. Docking Studies
The program AutoDock 4.2® software was used for the docking simulation. The free energy of
binding (∆G) of E. coli DNA GyrB, Thymidylate kinase, E. coli MurA, E. coli primase, E. coli MurB,
DNA topo IV, and CYP51 of C. albicans, in complex with the inhibitors were generated using this
molecular docking program. The X-ray crystal structures data of all the enzymes used were obtained
from the Protein Data Bank (PDB ID: 1KZN, AQGG, 1DDE, JV4T, 2Q85, 1S16, and 5V5Z respectively).
All procedures were performed according to our previous paper [78].
3.6. Cytotoxicity
HEK 293 cells were cultured in DMEM medium, supplemented with 10% fetal calf serum (Sigma
Chemical Co., St. Louis, MO, USA), 50 µg/mL streptomycin (Sigma Chemical Co.), and 50 units/mL
penicillin (Sigma Chemical Co.) in 5% CO2-containing humidified atmosphere at 37◦C. Since compound
solutions contained DMSO, control cultures containing only DMSO at the final concentration obtained
when the appropriate volume of compound solution was added were performed.
MTT Assay for Determination of Cell Viability
MTT assay based on the colorimetric measurement of formazan formed after reducing MTT
by cellular NAD(P)H-dependent oxidoreductases was used to examine the cytotoxic activity of the
compounds. Briefly, the cells were seeded into 96-well plates in 100
a concentration of 5,000 substrate-dependent cells per well and left incubated overnight as described
above. The formulations to be tested (100 L aliquots) were added to the culture medium at different
concentrations and left incubated for 72 h. The MTT assay was performed following the manufacturer’s
µL of complete culture medium at
µ
recommendations and assessed using an EL
VT, USA).
×
800 absorbance reader (BioTek Instruments; Winooski,
4. Conclusions
Eleven 5-[(R-1H-indol-3-yl)methylene]-4-oxo-2-thioxo-thiazolidin-3-ylcarbamides 5a-k and three
5-[(R-1H-indol-3-yl) methylene] -3-morpholin-4-yl-2-thioxothiazolidin-4-ones 7a-c were designed,
synthesized and evaluated in silico and experimentally for their antimicrobial action against panel of
Gram positive, Gram negative bacteria and fungi.
It should be mentioned that all compounds appeared to be more potent than ampicillin against all
bacteria tested and then streptomycin against all bacteria except B. cereus (isolated clinically M. flavus
(ATCC 10240), and En. cloacae (ATCC 35030). The most sensitive bacteria was found to be S. aureus
(ATCC 6538), while L. monocytogenes (NCTC 7973) was the most resistant one. Compounds also
appeared to be active against three resistant strains MRSA, E. coli, and P. aeruginosa showing better
activity against MRSA than both reference drugs while against the other two resistant strains better
than ampicillin.

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Concerning antifungal action, the tested compounds exhibited very good activity against all the
fungal species tested, being more active than ketoconazole and bifonazole. The most sensitive fungal
strain appeared to be T. viride (IAM 5061), while the most resistant filamentous A. fumigatus (human
isolate).
It can be observed that the growth of both Gram-negative and Gram-positive bacteria and fungi
responded differently to the tested compounds, which indicates that different substituents may lead to
different modes of action or that the metabolism of some bacteria/fungi was better able to overcome
the effect of the compounds or adapt to it.
Docking analysis to DNA Gyrase, Thymidylate kinase and E.coli MurB indicated a probable
involvement of MurB inhibition in the antibacterial mechanism of compounds tested while docking
analysis to 14α-lanosterol demethylase (CYP51) and tetrahydrofolate reductase of Candida albicans
indicated a likely implication of CYP51 reductase at the antifungal activity of the compounds and
secondary involvement of dihydrofolate reductase inhibition at the mechanism of action of the most
active compounds.
Since the most active compounds 5d, 5g, 5k, 7c demonstrated the low cytotoxicity against HEK-293
human embryonic kidney cell line and reasonable selectivity index, this chemical series looks promising
for investigations as the antimicrobial agents.
Finally, compounds 5d (Z)-N-(5-((1H-indol-3-yl)methylene)-4-oxo-2-thioxothiazolidin-3
-yl)-4-hydroxybenzamide
and
5g
(Z)-N-(5-((5-methoxy-1H-indol-3-yl)methylene)-4-oxo-2-
thioxothiazolidin-3-yl)nicotinamide as well as 7c (Z)-5-((5-methoxy-1H-indol-3-yl)methylene)-3-
morpholino-2-thioxothiazolidin-4-one can be considered as lead compounds for further development
of more potent and safe antibacterial and antifungal agents.
Supplementary Materials: The following are available online at http://www.mdpi.com/1424-8247/13/9/229/s1,
Supplementary file PASSweb_results_13mols.xlsx: Predictions of antimicrobial activity and acute rat toxicity.
Author Contributions: Conceptualization, A.G. and V.K.; methodology, V.H.; software, P.A. and P.P.; formal
analysis, V.M.; investigation, M.I., M.K. and M.D.S; data curation, A.G., V.P., M.D.S. and P.E., original draft
preparation, A.G. and P.P.; review & editing, A.G. and V.P.; supervision, A.G. and V.P. All authors have read and
agreed to the published version of the manuscript.
Funding: This research was supported by the Serbian Ministry of Education, Science and Technological
Development for financial support (project No. 451-03-68/2020-14/200007).
Acknowledgments: Computational predictions of biological activity by AntiBac-Pred, AntiFun-Pred and AcuTox
web-services (P.P. and V.P.) were performed in the framework of the Russian State Academies of Sciences
Fundamental Research Program for 2013–2020.
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
2.
Michaud, C.M. Global Burden of Infectious Diseases. Encycl. Microbiol. 2009, 444–454.
Nii-Trebi, N.I. Emerging and neglected infectious diseases: Insights, advances, and challenges. Biomed. Res.
Int. 2017, 2017, 5245021. [CrossRef] [PubMed]
3.
Mukherjee, S. Emerging Infectious Diseases: Epidemiological Perspective. Indian J. Dermatol. 2017, 62,
459–467.
4.
5.
Ventola, C.L. The antibiotic resistance crisis: Part 1: Causes and threats. Pharm. Ther. 2015, 40, 277–283.
Michael, C.A.; Dominey-Howes, D.; Labbate, M. The antimicrobial resistance crisis: Causes, consequences,
and management. Front. Public Health 2014, 16, 145. [CrossRef]
6.
7.
8.
Rice, L.B. Federal funding for the study of antimicrobial resistance in nosocomial pathogens: No ESKAPE.
J. Infect. Dis. 2008, 197, 1079–1081. [CrossRef]
Holmes, A.H.; Moore, L.S.; Sundsfjord, A.; Steinbakk, M.; Regmi, S.; Karkey, A.; Guerin, P.J.; Piddock, L.J.
Understanding the mechanisms and drivers of antimicrobial resistance. Lancet 2016, 387, 176–187. [CrossRef]
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, 7, 52–57. [CrossRef]

Pharmaceuticals 2020, 13, 229
21 of 24
9.
Kaminskyy, D.; Kryshchyshyn, A.; Lesyk, R. 5-Ene-4-thiazolidinones–An efficient tool in medicinal chemistry.
Eur. J. Med. Chem. 2017, 140, 542–594. [CrossRef]
10. Kaminskyy, D.; Kryshchyshyn, A.; Lesyk, R. Recent developments with rhodanine as a scaffold for drug
discovery. Expert Opin. Drug Discov. 2017, 12, 1233–1252.
11. Baell, B. Observations on screening-based research and some concerning trends in the literature. Future Med.
Chem. 2010, 2, 1529–1546.
12. Mendgen, T.; Steuer, C.; Klein, C.D. Privileged scaffolds or promiscuous binders: A comparative study on
rhodanines and related heterocycles in medicinal chemistry. J. Med. Chem. 2012, 55, 743–753. [CrossRef]
[PubMed]
13. Morphy, R.; Rankovic, Z. Designed multiple ligands. An emerging drug discovery paradigm. J. Med. Chem.
2005, 48, 6523–6543. [CrossRef] [PubMed]
14. Fortin, S.; Bérubé, G. Advances in the development of hybrid anticancer drugs. Expert Opin. Drug Discov.
2013, 8, 1029–1047.
15. Kryshchyshyn, A.; Roman, O.; Lozynskyi, A.; Lesyk, R. Thiopyrano[2,3-d]thiazoles as new efficient scaffolds
in medicinal chemistry. Sci. Pharm. 2018, 86, 26. [CrossRef] [PubMed]
16. Cong, N.T.; Nhan, H.T.; Van Hung, L.; Thang, T.D.; Kuo, P.C. Synthesis and antibacterial activity of analogs
of 5-arylidene-3-(4-methylcoumarin-7-yloxyacetylamino)-2-thioxo-1,3-thiazoli-din-4-one. Molecules 2014, 19,
13577–13586. [CrossRef]
17. Song, M.-X.; Deng, X.-Q.; Li, Y.-R.; Zheng, C.-J.; Hong, L.; Piao, H.-R. Synthesis and biological evaluation of
(E)-1-(substituted)-3-phenylprop-2-en-1-ones bearing rhodanines as potent anti-microbial agents. J. Enzyme
Inhib. Med. Chem. 2014, 29, 647–653. [CrossRef]
18. Krátký, M.; Vinšová, J.; Stolarˇíková, J. Antimicrobial activity of rhodanine-3-acetic acid derivatives. Bioorg.
Med. Chem. 2017, 25, 1839–1845. [CrossRef]
19. Horishny, V.; Kartsev, V.; Geronikaki, A.; Matiychuk, V.; Petrou, A.; Glamoclija, J.; Ciric, A.; Sokovic, M.
5-(1H-Indol-3-ylmethylene)-4-oxo-2-thioxothiazolidin-3-yl)alkancarboxylic Acids as Antimicrobial Agents:
Synthesis, Biological Evaluation, and Molecular Docking Studies. Molecules 2020, 25, 1964. [CrossRef]
´
20. Incerti, M.; Vicini, P.; Geronikaki, A.; Eleftheriou, P.; Tsagkadouras, A.; Zoumpoulakis, P.; Fotakis, C.; Ciric´, A.;
Glamocˇlija, J.; Sokovic´, M. New N-(2-phenyl-4-oxo-1,3-thiazolidin-3-yl)-1,2-benzothiazole -3-carboxamides
and Acetamides as Antimicrobial Agents. Med. Chem. Commun. 2017, 8, 2142–2154. [CrossRef]
21. Ozen, C.; Ceylan-Unlusoy, M.; Aliary, N.; Ozturk, M.; Bozdag-Dundar, O. Thiazolidinedione or Rhodanine:
A Study on Synthesis and Anticancer Activity Comparison of Novel Thiazole Derivatives. Pharm. Pharm.
Sci. 2017, 20, 415–427. [CrossRef]
22. Fu, H.; Hou, X.; Wang, L.; Dun, Y.; Yang, X.; Fang, H. Design, synthesis and biological evaluation of
3-aryl-rhodanine benzoic acids as anti-apoptotic protein Bcl-2 inhibitors. Bioorg. Med. Chem. Lett. 2015, 25,
5265–5269. [CrossRef] [PubMed]
23. Havrylyuk, D.; Zimenkovsky, B.; Lesyk, R. Synthesis and Anticancer Activity of Novel Nonfused Bicyclic
Thiazolidinone Derivatives. Phosphorus Sulfur 2009, 184, 638–650. [CrossRef]
24. El-Miligy, M.; Hazzaa, A.; El-Messmary, H.; Nassra, R.A. El-Hawash, Soad, New hybrid molecules combining
benzothiophene or benzofuran with rhodanine as dual COX-1/2 and 5-LOX inhibitors: Synthesis, biological
evaluation and docking study. Bioorg. Chem. 2017, 72, 102–115. [CrossRef] [PubMed]
25. R. Atta-Allah, S.; Nassar, I.F.; El-Sayed, W.A. Design, synthesis and anti-inflammatoryvel
5-(Indol-3-yl)-thiazolidinone derivatives as COX-2 inhibitors. J. Pharmacol. Ther. Res. 2020, 5, 1–16.
26. Powers, J.P.; Piper, D.E.; Li, Y.; Mayorga, V.; Anzola, J.; Chen, J.M.; Jaen, J.C.; Lee, G.; Liu, J.; Peterson, M.G.;
et al. SAR and mode of action of novel non-nucleoside inhibitors of hepatitis C NS5b RNA polymerase.
J. Med. Chem. 2006, 49, 1034–1046. [CrossRef] [PubMed]
27. Ramkumar, K.; Yarovenko, V.N.; Nikitina, A.S.; Zavarzin, I.V.; Krayushkin, M.M.; Kovalenko, L.V.;
Esqueda, A.; Odde, S.; Neamati, N. Design, synthesis and structure-activity studies of rhodanine derivatives
as HIV-1 integrase inhibitors. Molecules 2010, 15, 3958–3992. [CrossRef]
28. Petrou, A.; Eleftheriou, P.; Geronikaki, A.; Akrivou, M.G.; Vizirianakis, I. Novel thiazolidin-4-ones as potential
non-nucleoside inhibitors of HIV-1 reverse transcriptase. Molecules 2019, 24, 3821. [CrossRef]
29. Kryshchyshyn, A.; Kaminskyy, D.; Roman, O.; Kralovics, R.; Karpenko, O.; Lesyk, R. Synthesis and
anti-leukemic activity of pyrrolidinedione-thiazolidinone hybrids. Ukr Biochem. J. 2020, 92, 108–119.

Pharmaceuticals 2020, 13, 229
22 of 24
30. Havrylyuk, D.; Zimenkovsky, B.; Vasylenko, O.; Gzella, A.; Lesyk, R. Synthesis of new 4- thiazolidinone-,
pyrazoline-, and isatin-based conjugates with promising antitumor activity. J. Med. Chem. 2012, 55, 8630–8641.
[CrossRef]
31. Havrylyuk, D.; Roman, O.; Lesyk, R. Synthetic approaches, structure activity relationship and biological
applications for pharmacologically attractive pyrazole/pyrazoline–thiazolidine– based hybrids. Eur. J. Med.
Chem. 2016, 113, 145–166. [CrossRef]
32. Saini, T.; Kumar, S.; Narasimhan, B. Central nervous system activities of indole derivatives: An overview.
Cent. Nerv. Syst. Agents Med. Chem. 2016, 16, 19–28. [CrossRef] [PubMed]
33. Singh, P.; Singh, O.M. Recent progress in biological activities of indole and indole alkaloids. Mini Rev. Med.
Chem. 2018, 18, 9–25.
34. Kaur, J.; Utreja, D.; Jain, N.; Sharma, S. Recent Developments in the Synthesis and Antimicrobial Activity of
Indole and Its Derivatives. Curr. Org. Synth. 2019, 16, 17–37. [CrossRef]
35. Bathula, C.; Tripathi, S.; Srinivasan, R.; Jha, K.K.; Ganguli, A.; Chakrabarti, G.; Singh, S.; Munshi, P.; Sen, S.
Synthesis of novel 5-arylidenethiazolidinones with apoptotic properties via a three component reaction
using piperidine as a bifunctional reagent. Org. Biomol. Chem. 2016, 14, 8053–8063. [CrossRef]
36. Sayed, M.; El-Dean, A.; Ahmed, M.; Hassanien, R. Synthesis of some heterocyclic compounds derived from
indole as antimicrobial agents. Synth. Commun. 2018, 48, 413–421. [CrossRef]
37. Jain, P.; Utreja, D.; Sharma, P. An efficacious synthesis of N-1–, C-3–substituted indole derivatives and their
antimicrobial studies. J. Hetrocyclic Chem. 2020, 57, 428–435. [CrossRef]
38. Shaikh, T.M.A.; Debebe, H. Synthesis and Evaluation of Antimicrobial Activities of Novel N-Substituted
Indole Derivatives. J. Chem. 2020, 1–9, Article ID 4358453, 9 pages.
39. Kumar, P.; Singh, S.; Rizki, M.; Pratama, F. Synthesis of some novel 1H-indole derivatives with antibacterial
activity and antifungal activity. Lett. Appl. NanoBioScience 2020, 9, 961–967.
40. Liu, Z.; Tang, L.; Zhu, H.; Xu, T.; Qiu, C.; Zheng, S.; Gu, Y.; Feng, J.; Zhang, Y.; Liang, G. Design,
Synthesis, and Structure Activity Relationship Study of Novel Indole-2-carboxamide Derivatives as
−
Anti-inflammatoryAgents for the Treatment of Sepsis. J. Med. Chem. 2016, 59, 4637–4650. [CrossRef]
41. Li, S.; Wang, Z.; Xiao, H.; Bian, Z.; Wang, J. Enantioselective synthesis of indole derivatives byRh/Pd relay
catalysis and their anti-inflammatory evaluation. Chem. Commun. 2020, 56, 7573–7576. [CrossRef]
42. Abdellatif, K.R.A.; Elsaady, M.Y.; Amin, N.H.; Hefny, A.A. Design, Synthesis and biological evaluation of
some novel indole derivatives as selective COX-2 inhibitors. J. Appl. Pharm. Sci. 2017, 7, 69–77.
43. Bhat, M.A.; Al-Omar, M.A.; Raish, M. Indole Derivatives as Cyclooxygenase Inhibitors: Synthesis, Biological
Evaluation and Docking Studies. Molecules 2018, 23, 1250. [CrossRef] [PubMed]
44. Sidhu, J.S.; Singla, R.; Jaitak, V. Indole Derivatives as Anticancer Agents for Breast Cancer Therapy: A Review.
Anticancer Agents Med. Chem. 2015, 16, 160–173. [CrossRef] [PubMed]
45. El-Sharief, A.M.S.; Ammar, Y.A.; Belal, A.; El-Sharief, M.A.S.; Mohamed, Y.A.; Ahmed, B.M.; Mehany, A.B.M.;
Elhag Ali, G.A.M.; Ragab, A. Design, synthesis, molecular docking and biological activity evaluation of some
novel indole derivatives as potent anticancer active agents and apoptosis inducers. Bioorg. Chem. 2019, 85,
399–412. [CrossRef]
46. Cascioferro, S.; Li Petri, G.; Parrino, B.; El Hassouni, B.; Carbone, D.; Arizza, V.; Perricone, U.; Padova, A.;
Funel, N.; Peters, G.J.; et al. 3-(6-Phenylimidazo [2,1-b][1,3,4]thiadiazol-2-yl)-1H-Indole Derivatives as New
Anticancer Agents in the Treatment of Pancreatic Ductal Adenocarcinoma. Molecules 2020, 25, 329. [CrossRef]
[PubMed]
47. Zhang, M.-Z.; Chen, Q.; Yang, G.F. A review on recent developments of indole-containing antiviral agents.
J. Med. Chem. 2015, 89, 421–441. [CrossRef] [PubMed]
48. Bardiot, D. Discovery of Indole Derivatives as Novel and Potent Dengue Virus Inhibitors. J. Med. Chem.
2018, 61, 8390–8401. [CrossRef]
49. Che, Z.; Tian,
Υ.; Liu, S.; Hu, M.; Che, G. Discovery of N-arylsulfonyl-3-acylindole benzoyl hydrazone
derivatives as anti-HIV-1 agents. Braz. J. Pharm. Sci 2019, 54, e17543. [CrossRef]
50. Sanna, G.; Madeddu, S.; Giliberti, G.; Piras, S.; Struga, M.; Wrzosek, M.; Kubiak-Tomaszewska, G.; Koziol, A.E.;
Savchenko, O.; Lis, T.; et al. Synthesis and Biological Evaluation of Novel Indole-Derived Thioureas. Molecules
2018, 23, 2554. [CrossRef]

Pharmaceuticals 2020, 13, 229
23 of 24
51. Ramya, V.; Vembu, S.; Ariharasivakumar, G.; Gopalakrishnan, M. Synthesis, Characterisation,
Molecular Docking, Anti-microbial and Anti-diabetic Screening of Substituted
4-indolylphenyl-6-arylpyrimidine-2-imine Derivatives. Drug Res. (Stuttg) 2017, 67, 515–526. [CrossRef]
52. Tymiak, A.A.; Rinehart, K.L.; Bakus, G.J. Constituents of morphologically similar sponges: Aplysina and
Smenospongia species. Tetrahedron 1985, 41, 1039–1047. [CrossRef]
53. Djura, P.; Stierle, D.B.; Sullivan, B. Some metabolites of the marine sponges Smenospongia aurea and
Smenospongia (ident.Polyfibrospongia) echina. J. Org.Chem. 1980, 45, 1435–1441. [CrossRef]
54. Fattorusso, E.; Lanzotti, V.; Magno, S.; Novellino, E. Tryptophan derivatives from a Mediterranean anthozoan,
Astroides calycularis. J. Nat. Prod. 1985, 48, 924–927. [CrossRef]
55. Buyukbingol, E.; Suzen, S.; Klopman, G. Studies on the synthesis and structure–activity relationships of
5-(30-indolyl)-2-thiohydantoin derivatives as aldose reductase enzyme inhibitors. Farmaco 1994, 49, 443–447.
[PubMed]
56. Pogodin, P.V.; Lagunin, A.A.; Rudik, A.V.; Druzhilovskiy, D.S.; Filimonov, D.A.; Poroikov, V.V. AntiBac-Pred:
A Web Application for Predicting Antibacterial Activity of Chemical Compounds. J. Chem. Inf. Model. 2019
59, 4513–4518. [CrossRef] [PubMed]
,
57. Poroikov, V.; Filimonov, D.; Gloriozova, T.; Lagunin, A.; Druzhilovskiy, D.; Rudik, A.; Stolbov, L.; Dmitriev, A.;
Tarasova, O.; Ivanov, S.; et al. Computer-aided prediction of biological activity spectra for organic compounds:
The possibilities and limitations. Russ. Chem. Bull. 2019, 68, 2143–2154. [CrossRef]
58. Clarivate Analytics Integrity. Available online: https://integrity.clarivate.com/integrity/ (accessed on 21 July
2020).
59. Antifungal Activity Predictor. Available online: http://www.way2drug.com/micf (accessed on 21 July 2020).
60. Filimonov, D.A.; Zakharov, A.V.; Lagunin, A.A.; Poroikov, V.V. QNA-based ‘Star Track’ QSAR approach.
SAR QSAR Env. Res. 2009, 20, 679–709. [CrossRef]
61. Lagunin, A.; Zakharov, A.; Filimonov, D.; Poroikov, V. QSAR Modelling of Rat Acute Toxicity on the Basis of
PASS Prediction. Mol. Inform. 2011, 30, 241–250. [CrossRef]
62. Bruno, G.; Costantino, L.; Curinga, C.; Maccari, R.; Monforte, F.; Nicolo, F.; Ottana, R.; Vigorita, M.G.
Synthesis and Aldose Reductase Inhibitory Activity of5-Arylidene-2,4-thiazolidinediones. Bioorg. Med.
Chem. 2002, 10, 1077–1084. [CrossRef]
63. Abdeen, S.; Kunkle, T.; Salim, N.; Ray, A.-M.; Mammadova, N.; Summers, C.; Stevens, M.; Ambrose, A.J.;
Park, Y.; Schultz, P.G.; et al. Sulfonamido-2-arylbenzoxazole GroEL/ES Inhibitors as Potent Antibacterials
against Methicillin-Resistant Staphylococcus aureus (MRSA). J. Med. Chem. 2018, 61, 7345–7357. [CrossRef]
64. Menozzi, G.; Merello, L.; Fossa, P.; Ranise, A.; Mosti, L.; Bondavalli, F.; Loddo, R.; Murgioni, C.; Mascia, V.;
La Colla, P.; et al. Synthesis, antimicrobial activity and molecular modeling studies of halogenated
4-[1H-imidazol-1-yl(phenyl)methyl]-1,5-diphenyl-1H-pyrazoles. Bioorg. Med. Chem. 2004, 12, 5465–5483.
[CrossRef] [PubMed]
65. Vieira, F.T.; de Lima, G.M.; Maia, J.R.; Speziali, N.L.; Ardisson, J.D.; Rodrigues, L.; Correa, A., Jr.;
Romero, O.B. Synthesis, characterization and biocidal activity of new organotin complexes of
2-(3-oxocyclohex-1-enyl)benzoic acid. Eur. J. Med. Chem. 2010, 45, 883–889. [CrossRef] [PubMed]
66. Gjorgjieva, M.; Tomasiccˇ, T.; Barancokova, M.; Katsamakas, S.; Ilas, J.; Tammela, P.; Masiccˇ, L.P.; Kikelj, D.
Discovery of Benzothiazole Scaffold-Based DNA Gyrase B Inhibitors. J. Med. Chem. 2016, 59, 8941–8954.
[CrossRef] [PubMed]
67. Parvathy, N.G.; Manju, P.; Mukesh, M.; Thomas, L. Design, synthesis and molecular docking studies of
benzothiazole derivatives as anti microbial agents. Int. J. Pharm. Pharm. Sci. 2013, 5, 101–106.
68. Ren, Y.; Zhang, L.; Zhou, C.H.; Geng, R.X. Recent Development of Benzotriazole-based Medicinal Drugs.
Med. Chem. 2014, 4, 640–662. [CrossRef]
69. Andres, C.J.; Bronson, J.J.; D’Andrea, S.V.; Walsh, A.W. 4-thiazolidinones: Novel inhibitors of the bacterial
enzyme MurB. Bioorg. Med. Chem. Lett. 2000, 10, 715–717. [CrossRef]
70. Ahmed, S.; Zayed, M.F.; El-Messery, S.M.; Al-Agamy, M.H.; Abdel-Rahman, H.M. Design, Synthesis,
Antimicrobial Evaluation and Molecular Modeling Study of 1,2,4-Triazole-Based 4-Thiazolidinones. Molecules
2016, 21, 568. [CrossRef]
71. Pitta, E.; Tsolaki, E.; Geronikaki, A.; Petrovic, J.; Glamocˇlija, J.; Sokovic, M.; Crespan, E.; Maga, G.; Bhunia, S.S.;
Saxena, A.K. 4-Thiazolidinone derivatives as potent antimicrobial agents: Microwave-assisted synthesis,
biological evaluation and docking studies. MedChemComm 2015, 6, 319–326. [CrossRef]

Pharmaceuticals 2020, 13, 229
24 of 24
B.K.
72. Karanth,
S.;
Narayana,
B.;
Kodandoor,
S.C.;
Sarojini,
2-{[(4-Hydroxy-3,5-dimethoxyphenyl)methylidene]hydrazinylidene}-4-oxo-1,3-thiazolidin-5-yl Acetic Acid.
Molbank 2018, 2018, 2-9 M1009. [CrossRef]
73. Stana, A.; Vodnar, D.C.; Tamaian, R.; Pîrnău, A.; Vlase, L.; Ionut, , I.; Oniga, O.; Tiperciuc, B. Design, Synthesis
and Antifungal Activity Evaluation of New Thiazolin-4-ones as Potential Lanosterol 14α-Demethylase
Inhibitors. Int. J. Mol. Sci. 2017, 18, 177. [CrossRef]
´
74. Incerti, M.; Vicini, P.; Geronikaki, A.; Eleftheriou, P.; Tsagkadouras, A.; Zoumpoulakis, P.; Fotakis, C.; Ciric´, A.;
Glamocˇlija, J.; Sokovic´, M. New N-(2-phenyl-4-oxo-1,3-thiazolidin-3-yl)-1,2-benzothiazole-3-carboxamides
and acetamides asantimicrobial agents. Med. Chem. Commun. 2017, 8, 2142. [CrossRef] [PubMed]
75. Can, N.O.; Çevik, U.A.; Sag˘lık, B.N.; Levent, S.; Korkut, B.; Özkay, Y.; Kaplancıklı, Z.A.; Koparal, A.S.
Synthesis, Molecular Docking Studies, and Antifungal Activity Evaluation of New Benzimidazole-Triazoles
as Potential Lanosterol 14
[CrossRef]
α-Demethylase Inhibitors. J. Chem. 2017, 1–15, Article ID 9387102, 15 pages.
76. Benson, T.E.; Walsh, C.T.; Massey, V. Kinetic characterization of wild-type and S229A mutant MurB: Evidence
for the role of Ser 229 as a general acid. Biochemistry 1997, 36, 796–805. [CrossRef] [PubMed]
77. Kartsev, V.; Lichitsky, B.; Geronikaki, A.; Petrou, A.; Smiljkovic, M.; Kostic, M.; Radanovic, O.; Sokovic´, M.
Design, synthesis and antimicrobial activity of usnic acid derivatives. MedChemComm 2018, 9, 870–882.
[CrossRef]
78. Fesatidou, M.; Zagaliotis, P.; Camoutsis, C.; Petrou, A.; Eleftheriou, P.; Tratrtat, C.; Haroun, M.; Geronikaki, A.;
Ciric, A.; Sokovic, M. 5-Adamantan thiadiazole-based thiazolidinones as antimicrobial agents. Design,
synthesis, molecular docking and evaluation. Bioorg. Med. Chem. 2018, 26, 4664–4676. [CrossRef]
´
79. Kostic´, M.; Smiljkovic´, M.; Petrovic´, J.; Glamocˇilija, J.; Barros, L.; Ferreira, I.C.F.R.; Ciric´, A.; Sokovic´, M.
Chemical, nutritive composition and a wide range of bioactive properties of honey mushroom Armillaria
mellea (Vahl: Fr.) Kummer. Food Function 2017, 8, 3239–3249. [CrossRef]
80. Kritsi, E.; Matsoukas, M.T.; Potamitis, C.; Detsi, A.; Ivanov, M.; Sokovic, M.; Zoumpoulakis, P. Novel Hit
Compounds as Putative Antifungals: The Case of Aspergillus fumigatus. Molecules 2019, 24, 3853. [CrossRef]
81. Aleksic´, M.; Stanisavljevic´, D.; Smiljkovic´, M.; Vasiljevic´, P.; Stevanovic´, M.; Sokovic´, M.; Stojkovic´, D.
Pyrimethanil: Between efficient fungicide against Aspergillus rot on cherry tomato and cytotoxic agent on
human cell lines. Ann. App. Bio. 2019, 175, 228–235. [CrossRef]
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