5-(1H-Indol-3-ylmethylene)-4-oxo-2-thioxothiazolidin-3-yl)alkancarboxylic Acids as Antimicrobial Agents: Synthesis, biological evaluation, and molecular docking studies

Article abstract of DOI:10.3390/molecules25081964

Background: Infectious diseases symbolize a global consequential strain on public health security and impact on the socio-economic stability all over the world. The increasing resistance to the current antimicrobial treatment has resulted in crucial need for the discovery and development of novel entity for the infectious treatment with different modes of action that could target both sensitive and resistant strains. Methods: Compounds were synthesized using classical methods of organic synthesis. Results: All 20 synthesized compounds showed antibacterial activity against eight Gram-positive and Gram-negative bacterial species. It should be mentioned that all compounds exhibited better antibacterial potency than ampicillin against all bacteria tested. Furthermore, 18 compounds appeared to be more potent than streptomycin against Staphylococcus aureus, Enterobacter cloacae, Pseudomonas aeruginosa, Listeria monocytogenes, and Escherichia coli. Three the most active compounds 4h, 5b, and 5g appeared to be more potent against MRSA than ampicillin, while streptomycin did not show any bactericidal activity. All three compounds displayed better activity also against resistant strains P. aeruginosa and E. coli than ampicillin. Furthermore, all compounds were able to inhibit biofilm formation 2- to 4-times more than both reference drugs. Compounds were evaluated also for their antifungal activity against eight species. The evaluation revealed that all compounds exhibited antifungal activity better than the reference drugs bifonazole and ketoconazole. Molecular docking studies on antibacterial and antifungal targets were performed in order to elucidate the mechanism of antibacterial activity of synthesized compounds. Conclusion: All tested compounds showed good antibacterial and antifungal activity better than that of reference drugs and three the most active compounds could consider as lead compounds for the development of new more potent agents.

Full text of DOI:10.3390/molecules25081964

molecules
Article
5-(1H-Indol-3-ylmethylene)-4-oxo-2-thioxothiazolidin-
3-yl)alkancarboxylic Acids as Antimicrobial Agents:
Synthesis, Biological Evaluation, and Molecular
Docking Studies
Volodymyr Horishny 1, Victor Kartsev 2, Athina Geronikaki 3,
*
, Vasyl Matiychuk 4,
Anthi Petrou 3 , Jasmina Glamoclija 5, Ana Ciric 5 and Marina Sokovic 5
1
Department of Chemistry, Danylo Halytsky Lviv National Medical University, Pekarska 69, 79010 Lviv,
Ukraine; vgor58@ukr.net
InterBioScreen, 85355 Moscow, Russia; vkartsev@ibscreen.chg.ru
Department of Phram Chemistry, School of Pharmacy, Aristotle University of Thessaloniki,
2
3
54124 Thessaloniki, Greece; anthi.petrou.thessaloniki1@gmail.com
Department of Chemistry, Ivan Franko National University of Lviv, Kyryla i Mefodia 6, 79005 Lviv, Ukraine;
4
v_matiychuk@ukr.net
5
Mycological Laboratory, Institute of Biological Research Sinisa Stankovic, National Institute of Republic of
Serbia, Belgrade University, 11000 Belgrade, Serbia; jasna@ibiss.bg.ac.rs (J.G.); rancic@ibiss.bg.ac.rs (A.C.);
mris@ibiss.bg.ac.rs (M.S.)
*
Correspondence: geronik@pharm.auth.gr; Tel.: +30-2310997616
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editors: Marco Catto and Cosimo Damiano Altomare
Received: 2 April 2020; Accepted: 21 April 2020; Published: 23 April 2020
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: Background: Infectious diseases symbolize a global consequential strain on public health
security and impact on the socio-economic stability all over the world. The increasing resistance to the
current antimicrobial treatment has resulted in crucial need for the discovery and development of novel
entity for the infectious treatment with different modes of action that could target both sensitive and
resistant strains. Methods: Compounds were synthesized using classical methods of organic synthesis.
Results: All 20 synthesized compounds showed antibacterial activity against eight Gram-positive
and Gram-negative bacterial species. It should be mentioned that all compounds exhibited better
antibacterial potency than ampicillin against all bacteria tested. Furthermore, 18 compounds appeared
to be more potent than streptomycin against Staphylococcus aureus, Enterobacter cloacae, Pseudomonas
aeruginosa, Listeria monocytogenes, and Escherichia coli. Three the most active compounds 4h, 5b, and
5g appeared to be more potent against MRSA than ampicillin, while streptomycin did not show
any bactericidal activity. All three compounds displayed better activity also against resistant strains
P. aeruginosa and E. coli than ampicillin. Furthermore, all compounds were able to inhibit biofilm
formation 2- to 4-times more than both reference drugs. Compounds were evaluated also for their
antifungal activity against eight species. The evaluation revealed that all compounds exhibited
antifungal activity better than the reference drugs bifonazole and ketoconazole. Molecular docking
studies on antibacterial and antifungal targets were performed in order to elucidate the mechanism
of antibacterial activity of synthesized compounds. Conclusion: All tested compounds showed good
antibacterial and antifungal activity better than that of reference drugs and three the most active
compounds could consider as lead compounds for the development of new more potent agents.
Keywords: indole; antimicrobial; antifungal; E. coli MurB; Candida albicans 14α-demethylase; CYP51;
molecular docking
Molecules 2020, 25, 1964; doi:10.3390/molecules25081964
www.mdpi.com/journal/molecules

Molecules 2020, 25, 1964
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1. Introduction
Infectious diseases symbolize a global consequential strain on public health security and impact on
the socio-economic stability all over the world [ ]. For centuries they have monopolized the prevailing
1
factors of death and disability of millions of humans and are presently plaguing and even ravaging
populations worldwide each year, far surpassing the impact of wars. The growing challenges on health
and human economic progresses posed by infectious diseases is further aggravated by the continuous
emergence of new, obscure, and old endemic infections of global impact [1]. Indeed, during the
past two decades, the world’s scientific community was besieged by tremendous concerns caused by
infectious diseases whose incidence in humans has augmented for reasons such as the emergence of
new pathogens and the development of antimicrobial resistance [2]. At least 30 new infections have
risen insidiously and scattered to threaten the health of billions of humans across the planet especially
in low-income countries. Unfortunately, for many of them, there is no effective treatment or vaccine
alongside with limited scope of control or prevention strategies [3].
Despite the achievements in treatment of infective diseases during the last 50 years, unfortunately
the new infections and diseases affecting large populations, are instigating significant morbidity and
mortality, most recently as the syndrome of acquired immunodeficiency.
The increasing resistance to the current antimicrobial treatment has resulted in crucial need for
the discovery and development of novel entity for the infectious treatment with different modes
of action that could target both sensitive and resistant strains [4]. This need is even greater for
patients suffering from chronic inflammatory bowel diseases. During an inflammatory response in the
gut, some commensally microorganisms such as Escherichia coli and Candida albicans can thrive and
contribute to illness [5].
One of the promising methods for solving the resistance problem is screening of potential
antimicrobial agents among new classes of chemical compounds [6].
Rhodanine (2-thioxo-4-thiazolidone) derivatives during last years attracted the interest of scientists
due to their wide range of biological activities mainly to control human immunodeficiency virus (HIV),
hepatitis C virus (HCV), and dengue virus proteins [7].
5-Arylidene derivatives of rhodanines were found to possess various types of activity, in particular,
antitumor [
cholinesterase inhibitory activities [14
On the other hand, 5-arylidene-2-thioxo-4-thiazolidinones
8
], antiviral [
7
,
9
], anti-inflammatory, antidiabetic [10
–
12], antioxidant [13], LOX and
,
15] as well as aldose reductase inhibitory activities inhibitory [16].
1
are highly selective inhibitors
of UDP-MurNAc/L-Ala ligase, which are characterized by the influence on gram-positive
methicillin-resistant strains on the bacterial wall formation process of Staphylococcus aureus (MRSA) and
are promising for in-depth studies [17]. A number of 5-benzylidene-2,4-thiazolidinediones 2 exhibit high
effect against Gram-positive microorganisms (Staphylococcus aureus, Enterococcus faecalis, Streptococcus
pneumoniae). These microorganism are among the six pathogens with growing multidrug resistance
and virulence, named ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae,
Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) [18]. They are among recently
listed ESKAPE pathogens in the list of 12 bacteria by World Health Organization (WHO) against which
new antibiotics are urgently needed [19].
There are many references in the literature regarding antimicrobial activity of rhodanine
derivatives [20–25]. In particular, the high potential of 5-arylidenerodanine-3-carboxylic acids was
reported [4,5,7,8]. It could be noticed that, the rhodanine cycle is considered to be privileged [11–14].
Another interesting class of heterocyclic compounds is 5-(1H-Indol-3-ylmethylene)-2-
thioxothiazolidin-4-ones with wide spectrum of biological activities as well. Among them are
antitumor [26,27] and antimicrobial [28,29], inhibitors of proteases anthrax lethal factor, inhibitors
against neurotoxin type A [28], aldose reductase [30], PIM kinase [31], PI3K
and GSK-3 [34] enzymes.
α [32], IKKβ [33],

Molecules 2020, 25, 1964
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S
OH
O
O
O
O
Cl
S
NH
S
NH
S
Cl
Cl
O
NO2
1
2
Taking all mentioned above into account, herein we present the synthesis of new rodanin
derivatives and evaluation their antimicrobial activity. as well as their effect on biofilm formation since
it is one of the most considerable bacterial virulence factors. It was found that biofilm formation is
involved in a wide range of microbial infections in the body and is responsible for the serious chronic
diseases (80%) resistant to the most antibiotics used for therapy [35].
2. Results and Discussion
2.1. Chemistry
The title compounds were synthesized according to Scheme 1. As starting reagents for the
synthesis of the target (Z)-[5-(1-R1,5-R2,6-R3-1H-indol-3-ylmethylene)-4-oxo-2-thioxothiazolidin -3-yl]
alkanecarboxylic acids 4a
–i and 5a–k the amino acids glycine, β-alanine, GABA, ε-aminocaproic acid
and a number of -amino acids such as l-alanine, d, l-norvaline, methionine, d, l-amino(phenyl)acetic
α
acid, l-phenylalanine, d, l-aspartic and l-glutamic acid were used. Interaction of amino acids with
carbon disulfide in alkaline medium converted them to dithiocarbamic salts. After alkylation of the latest
with monochloracetic acid and subsequent cyclization, 4-oxo-2-thioxothiazolidin-3-ylalkanecarboxylic
acids 2a–d and 3a–g were obtained.
At the final stage of the synthesis of the target products, compounds 2a
–d and 3a–g
underwent the condensation with indole-3-carbaldehydes (1a ). The interaction was carried out
–d
in boiling alcohol in the presence of ammonium acetate. As a result, (Z)-[5-(1-R1,5-R2,6-R3-1H-
indol-3-ylmethylene)-4-oxo-2-thioxothiazolidin-3-yl]alkanecarboxylic acids 4a–i and 5a–k were obtained.
The structure of all synthesized compounds was confirmed by NMR spectroscopy. In the
1 H NMR spectra, signals of all protons are observed in regions corresponding to the structure
of the synthesized substances. In particular, the signals of the methylene group at position 5 of
4-oxo-2-thioxothiazolidin-3-ylalkanecarboxylic acids 2a–d and 3a–g are in the range 4.43–4.22 ppm.
Signals of NH protons of the indole cycle are observed at 11.96–12.43 ppm. The signals of the protons of
the methylidene group CH = appear as a singlet at 8.10–7.95 ppm, which indicates the Z-configuration
of these compounds. Protons NCH2 of the group of compounds 2a–d and 4a–i resonate at 2.63–2.19
ppm. and protons of the CH2COOH group of the same compounds at 4.72–3.76 ppm. At the same time,
protons of the NCH group in compounds 3a–g and 5a–k are observed in the range of 6.76–5.30 ppm.

Molecules 2020, 25, 1964
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Scheme 1. Synthesis of title compounds.
2.2. Biological Evaluation
2.2.1. Antibacterial Activity
Synthesized compounds were evaluated for their antibacterial activity using microdilution method
for the determination of their minimal inhibitory and minimal bactericidal/fungicidal concentrations.
All compounds showed antibacterial activity and results are shown in Table 1.The antibacterial
potential of these compounds can be presented as follows: 5b
>
4h
5j. The best antibacterial activity was observed for compound
10−2 and MBC at 2.08–16.67 10−2, while compound 9 5j was
> 5g > 5d > 4g > 5e > 4b > 5c > 5k > 5h
>
4e
>
4d
>
4i
>
4c
>
5f
>
4f
>
5a
>
4a
> 5i >
19 5b with MIC at 0.56–12.50
µ
M
×
µM
×
the less active with MIC and MBC at 7.68–30.74 µM × 10−2 and 15.37–61.48 µM × 10−2 respectively.
It was observed that bacteria in general showed different sensitivity towards compounds tested.
Thus, the most sensitive bacterium appeared to be S. aureus followed by P. aeruginosa, while L.
monocytogenes and E. coli were the most resistant representatives of Gram-positive and Gram-negative
bacteria. The antibacterial potency of compounds against S. aureus can be presented as: 5b
> 5g
>
4h 4b 4i 5h 5d 5k 4g 5i 4e 4f 5a 5f 4a 5e 5c 5j 4c 4d, while
>
>
>
>
>
>
>
>
>
>
>
>
>
>
>
>
>

Molecules 2020, 25, 1964
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against E. coli as: 4g
> 5d > 5c > 4h > 5b > 4i > 5k = 5e > 5f > 4f > 4b > 5g > 4d > 5h > 5i >
4e
> 4c > 5a > 4a > 5j. Compounds 5b and 5g exhibited very good activity against Bacillus cereus,
S. aureus, L. monocytogenes, En. cloacae, and S. typhimirium with MIC at 0.56–4.17 µM × 10−2 and MBC
at 2.08–3.68
µM
×
10−2.Good activity was observed for −co2 mpound 4h against M. flavus and S. aureus
with MIC and MBC at 1.99
with MIC value at 3.69
activity with compound 4h with MIC and MBC value at 3.69
µM
×
10−2and 3.98
µ
M
×
10 respectively and against all other bacteria
µM
×
10−2and MBC at 7.38
µM
×
10−2. Compound 5d showed the same good
10−2and 7.38 10−2 respectively
µM
×
µM
×
against all bacteria tested except of L. monocytogenes and S typhimirium.
Table 1. Antibacterial activity of compounds (µM × 10−2).
R.br
4a
B.c
M.f
S.a
L.m
En.cl
P.a
S.t
E. coli
MIC
4.72
28.30
37.72
13.54
18.05
12.91
6.92
9.43
2.26
4.51
8.61
9.43
18.86
9.03
18.05
8.61
4.72
9.43
2.26
4.51
4.31
8.61
4.33
8.66
8.28
4.72
9.43
2.26
4.51
4.31
8.61
4.33
8.66‘
4.14
8.28
4.16
8.32
3.98
7.97
3.98
7.97
5.87
8.01
4.52
9.04
2.22
8.33
7.64
4.72
9.43
18.05
36.10
4.31
28.30
37.72
7.47
MBC 9.43
MIC 4.51
MBC 9.03
MIC 8.61
4b
4c
18.05
12.91
17.22
8.66‘
17.32
12.42
16.57
8.32
16.64
1.59
1.99
3.98
7.97
5.87
8.01
18.07
36.14
4.17
8.33
3.82
7.64
3.69
MBC 17.22 17.22 17.22 17.22
MIC 8.66‘ 8.66 8.66‘
MBC 17.32 17.32 17.32
MIC 8.28
MBC 16.57
MIC 4.16
MBC 8.32
MIC 7.97
MBC 15.94
MIC 3.98
MBC 7.97
MIC 4.00
MBC 8.01
MIC 6.63
MBC 9.04
MIC 1.11
MBC 2.08
MIC 7.64
MBC 15.29
MIC 3.69
MBC 7.38
MIC 7.10
MBC 14.20
8.61
4.33
8.66‘
4.14
8.28
8.32
16.64
3.98
7.97
1.99
3.98
5.87
8.01
18.07
36.14
12.50
16.67
3.82
7.64
3.69
7.38
5.21
7.10
8.66‘
17.32
8.28
16.57
16.64
33.29
14.07
15.94
3.98
4d
4e
4.14
8.28
4.16
8.32
3.98
7.97
1.99
3.98
2.94
4.00
6.63
9.04
0.56
2.08
7.64
8.28
16.57 16.57
16.64
33.2
11.95
15.94
3.98
4.16
8.32
3.98
7.97
3.98
7.97
5.87
8.01
3.31
4.52
1.67
2.08
3.82
7.64
3.69
7.38
3.55
7.10
5.39
7.34
3.68
7.36
7.10
4f
4g
4h
4i
7.97
7.97
16.02
32.04
9.04
18.07
2.22
16.02
32.04
9.04
18.07
2.22
5a
5b
5c
5d
5e
4.17
7.64
4.17
7.64
15.29 15.29
15.29 15.29
3.69
7.38
7.10
7.38
14.76
7.10
3.69
7.38
3.55
7.10
5.39
7.34
1.96
3.68
7.10
7.38
14.76
3.55
7.38
7.10
14.20 14.20
7.10
14.20
11.02
14.69
11.03
14.72
7.10
14.20
11.96
15.94
30.74
61.48
11.25
14.88
37.20
49.20
17.20
34.40
MIC 11.02 11.02
MBC 14.69 14.69
5.39
7.34
1.96
3.68
3.55
7.10
3.99
7.97
7.68
7.34
14.69
1.96
3.68
7.10
14.69
29.38
1.96
3.68
7.10
5f
MIC
MBC 3.68
MIC 7.10
1.96
11.03
14.72
7.10
5g
5h
5i
MBC 14.20 14.20
MIC 7.97 11.96
MBC 15.94 15.94
MIC 15.37 23.05
MBC 30.74 30.74 15.37 61.48 15.37 15.37 30.74
3.71
MBC 7.42
MIC 24.80 24.80 24.80 37.20 24.80 74.40 24.80
MBC 37.20 37.20 37.20 74.40 37.20 124.0 49.20
4.30
MBC 8.60
14.20 14.20 14.20 14.20
15.94 7.97 7.97 15.94
31.88 15.94 15.94 31.88
30.74 7.68 11.53 15.37
5j
MIC
7.42
14.88
3.71
7.42
7.42
14.88 14.88
7.42
5.44
7.42
7.42
14.88
5k
Amp.
Strept.
MIC
8.60
17.20 25.80
4.30
8.60
17.20 17.20
34.40 34.40
17.20 34.40 51.60
B.c.—Bacillus cereus, M.f.—M. flavus, S.a.—Staphylococcus aureus, l.m.—Listeria monocytogenes, E.c.—Escherichia coli,
En.c.—Enterobacter cloacae, P.a.—Pseudomonas aeruginosa, S.t.—Salmonella typhimurium. Relative standard deviations
were all < 2.0. Amp.: Ampicillin, Strept.: Streptomycin.

Molecules 2020, 25, 1964
6 of 19
It was observed that for Gram-positive bacteria the range of MIC and MBC was
0.56–30.74
10−2 and 3.68–61.48 10−2 respectively, while for Gram-negative bacteria
this range was MIC at 1.67–28.5
10−2 and MBC at 3.68–37.72 10−2. It seems that
µM
×
µ
M
×
µM
×
µM
×
Gram-negative bacteria appeared to be more sensitive to compounds tested than Gram-positive.
Finally, it should be mentioned that all compounds exhibited better antibacterial potency than
ampicillin against all bacteria tested. Furthermore, all compounds appeared to be more potent than
streptomycin against S. aureus, En. cloacae, P. aeruginosa, L. monocytogenes (except of compound 5j),
and E. coli (except of 5j and 4a). Compounds 4f
than streptomycin against B. cereus while compound 4b
antibacterial potential than streptomycin against M. flavus (Table 1).
Ampicillin exhibited showed an inhibitory potential at 24.8–74.4
,
4h
,
4i, and 5b
,
,
5d
5d
,
,
5g, and 5k were more potent
5h 5i, and 5k exhibited better
,
4d 4i 5c
–
,
,
×
10−2 µM and bactericidal at
37.2–124.0 × 10−2 µM, while MIC/MBC of streptomycin is 4.3–25.8/8.6–34.4× 10−2 µM.
Three the most active compounds were tested against three resistant strains: Methicillin resistant
S. aureus, MRSA, P. aeruginosa and E. coli. (Table 2) All compounds appeared to be more potent against
MRSA, which is in the list of high priority group according to the urgency of need for new antibiotics,
than ampicillin, while streptomycin did not show any bactericidal activity. All three compounds
displayed better activity also against resistant strains P. aeruginosa and E. coli than ampicillin, which did
not show any bactericidal effect. These compounds were tested also for their effect on biofilm formation.
The evaluation revealed that all compounds were able to inhibit biofilm formation 2-to 4 times more
than both reference drugs (Table 2). The best ability was achieved for compound 4h followed by 5g
and the most active compound 5b.It should be mentioned that two compounds (5b, 5g) displayed
better effect on biofilm formation than reference drugs even in concentration of 0.5 MIC. The best effect
was observed for compound 5g.
Table 2. Antibacterial activity against resistant strains and effect on biofilm formation(mg/mL).
Resistant Strains
Biofilm Formation
Compounds
4h
MRSA
P.a.
E.c.
MIC
17.14
37.93
30.59
71.94
67.36
0.5MIC
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
0.5
1.0
0.25
0.5
0.5
1.0
0.1
/
0.12
0.25
0.12
0.25
0.12
0.25
0.05
0.1
0.5
1.0
0.25
0.5
0.5
1.0
0.1
0.2
0.2
/
NE
5b
5g
22.97
11.02
55.42
30.35
Streptomycin
Ampicilline
/
0.2
/
/
MRSA—methicillin resistant S. aureus.
A structure-activity relationship study revealed that the presence of pentanoic acid as substituent on
the nitrogen of 2-thioxothiazolidin-4-one ring of 5-(1H-indol-3-ylmethylene) (5b) was beneficial for the
antibacterial activity. Replacement of pentanoic acid by butyric and introduction of methoxy group in
position 6 of indole ring gave the second most active compound (4h). The presence of 3-phenylpropanoic
acid (5g) decreased a little activity. Introduction of 4-(methylthio) butanoic acid to the nitrogen of
2-thioxothiazolidin-4-one ring and at the same time methyl group to the nitrogen of 1H-indole resulted
less active compound (5d) than compound 5g, but still being among the most active compounds. On the
other hand, replacement of methyl indole in compound 5d by indole led to compound (5c) which is in the
middle of activity order. The presence of butyric acid in 2-thioxothiazolidin-4-one moiety in combination
with 5-OCH3 group in indole ring (12 4g) decrease more activity compared to 11 5d. The replacement
of butyric acid 2-methyl-4-(methylthio)butanoic acid of 11 5d by 4-(methylthio)butanoic acid (5e
)
did not improved activity, decreasing it more. Finally, the presence of dicarboxylic glutaric acid

Molecules 2020, 25, 1964
7 of 19
(5j) as a substituent of (Z)-5-(1H-indol-3-ylmethylene)-2-thioxothiazolidin-4-one was detrimental for
antibacterial activity.
From all mentioned above, it is obvious that the activity depends not only from the nature
of substituents in 2-thioxothiazolidin-4-one moiety and indole ring but also from their position
(compounds 4h and 4e). The presence of dicarboxylic acids is not beneficial for antibacterial activity,
no for N-methylindole derivatives, no for indole derivatives. In this case the activity decreased in the
following order: pentanoic (valeric) acid > 3-phenyl propanoic acid > propionic acid > acetic acid.
In case of N-methylindole derivatives the activity decreased from acetic acid as substituent to butyric
acid. In case of metoxy subastitution in positions 5 and 6 of the indole ring as longer is the chain of
acids as better is activity.
2.2.2. Antifungal Activity
Compounds were tested also against of panel of eight fungi, using ketoconazole and bifonazole
as reference drugs. The antifungal potential of synthesized compounds is presented in Table 3 and the
order of activity is: 5f
5d 4a 4i. Compound 5f appear to be the most potent, achieving inhibitory activity with MIC values
ranging at 2.31–4.33
antifungal activity with MIC and MFC at 4.00–32.04
> 4d > 4h > 4g > 5e > 5i > 4b > 5j > 4c > 5g > 5c > 5k > 5b > 5a > 5h > 4e > 4f >
>
>
µ
M
×
10−2 and MFC at 3.67–7.34
µ
M
M
×
×
10−2, while compound 4i showed the lowest
µ
10−2 and 8.01–64.09
µ
M
×
×
10−2 respectively.
10−2 and MFC
10−2 and MFC
Ketoconazole showed antifungal potential at MIC 38.00–475.00
57.00–570.00
at 48.30–80.00
×
10−2 respectively, while bifonazole showed MIC at 32.00–64.00
10−2 µ
×
×
M
×
10−2 respectively. The obtained results revealed that all tested compounds
exhibited higher antifungal activity than both drugs tested (Table 3).
The most sensitive fungal species is Trichoderma viride whereas Aaspergillus fumigatus appeared to
be the most resistant one (Table 3).
It should be mentioned that fungi, as in case of bacteria showed different sensitivity towards
compounds tested. Thus, the sensitivity of T. viride can be presented as follows: 5e
>
4g
4a, while of A. fumigates
4a 5d 4i 4f 4e.
> 5c > 5h > 5b
>
5f
>
4c
>
4d
>
5j
>
4h
>
4e
>
4b
>
5g
>
5d
>
5k
>
5c
>
5i
>
4i
>
4f
>
5a
>
was: 4h
>
4d
>
4b
>
5f
>
5i
=
4g
>
5k
>
5e
>
5g
>
5b
>
5j
>
4c
>
5c
>
5h
>
5a
>
>
>
>
>
Nevertheless, despite different sensitivity of fungi towards compounds tested, all of them, except A.
fumigatus appeared to be sensitive to compound 5e and in most cases to compound 5f. Compound
4h showed very good activity against A. fumigatus with MIC at 1.98–3.98
µ
M
×
10−2 and MFC at
10−2
M and
10−2 respectively.
10−2 and
3.98–7.97
3.31–4.51
µ
M
×
×
10−2, followed by compounds 4d and 4b with MIC at 2.31–4.33
10−2 respectively and MFC at 4.33–8.66 10−2 and 4.51–9.03
µ
M
×
µ
µM
µM
×
µM
×
Good activity was exhibited by compound 4g against T. viride with MIC at 2.12–3.98
µ
M
×
MFC at 3.96-7.97 µM × 10−2
.
From the study of structure-activity relationships it is obvious that the presence of phenylacetic
acid as substituent in (Z)-5-(1-methyl-1H-indol-3-ylmethylene)-2-thioxothiazolidin-4-one (5f) is the
most beneficial for antifungal activity. Replacement of phenylacetic acid by propionic acid gave a
little less active compound (4d), while introduction of butyric acid (4h) decreased more the antifungal
activity. In general, for (Z)-5-(1-methyl-1H-indol-3-ylmethylene)-2-thioxothiazolidin-4-one derivatives
the substituent such as 4-(methylthio) butanoic acid and butyric acid had negative effect on antifungal
activity followed by 3-phenyl propanoic acid. On the other hand, for not beneficial for activity for
(Z)-5-(1H-indol-3-ylmethylene)-2- thioxothiazolidin-4-one derivatives appeared to be the 5-methoxy
propanoic acid, acetic and hexanoic acid. The presence of the last one, as already mentioned, was very
negative for antifungal activity.
It should be mentioned, that as in case of antibacterial activity, antifungal activity
depends not only on the nature of substituent in 2-thioxothiazolidin-4-one moiety but also
on its nature and position in indole ring. Thus, the replacement of acetic acid substituent in
(Z)-5-(5-methoxy-1H-indol-3-ylmethylene)-2-thioxothiazolidin-4-one (4c) by propanoic acid remarkably
decreased the activity. On the other hand, shifting the methoxy group from position 5 of indole ring of

Molecules 2020, 25, 1964
8 of 19
(Z)-4-[5-(5-methoxy-1H-indol-3-ylmethylene)-4-oxo-2-thioxothiazolidin-3-yl)butanoic acid to position
6 led to small increase in activity.
Table 3. Antifungal activity of compounds (µM × 10−2).
R.br
4a
A.f
A.v
A.o
A.n
T.v
P.o
P.f
Pvc
MIC
18.87 18.87
MFC 44.03 37.74 18.87 37.74
9.43
18.87
6.92
9.43
3.31
4.51
2.30
4.31
2.31
4.33
8.28
16.57
8.32
6.92
18.87 18.87
9.43
18.87
37.74
4.51
9.03
4.31
8.61
2.89
4.33
9.14
17.08
10.12
19.76
3.98
7.97
5.84
MIC
MFC
MIC
3.31
4.51
8.61
4.51
9.03
4.31
8.61
4.33
8.66
4.14
8.28
4.16
8.32
3.98
7.97
3.98
7.97
3.31
4.51
3.16
4.31
2.89
4.33
6.07
8.28
6.10
8.32
2.12
3.98
2.92
3.98
4.00
8.01
9.04
4.51
9.03
4.31
8.61
4.33
8.66
3.04
4.14
4.16
8.32
3.98
7.97
3.98
7.97
32.04
64.09
9.04
4.51
9.03
3.16
4.31
4.33
8.66
6.07
8.28
8.32
4.51
9.03
6.31
8.61
4.33
8.66
8.28
16.57
8.32
4b
4c
MFC 17.22
MIC
MFC
MIC
MFC
MIC
2.31
4.33
4.14
8.28
8.32
4d
4e
4f
MFC 16.64
16.64 16.64 16.64
MIC
MFC
MIC
MFC
MIC
3.98
7.97
1.99
3.98
2.12
3.98
2.92
3.98
4.00
8.01
4.52
9.04
2.08
4.16
3.82
7.64
3.69
7.38
1.89
3.55
2.69
3.67
3.68
7.36
2.60
3.55
3.99
7.97
2.82
3.84
3.71
7.42
3.98
7.97
3.98
7.97
32.04 32.04
64.09 64.09
3.98
7.97
5.84
7.97
4g
4h
4i
7.97
32.04 16.02
MFC 64.09 32.04
32.04
64.09
9.04
18.07
8.32
16.67
7.64
15.29
14.76
29.52
3.55
MIC
MFC 36.14
MIC 8.32
MFC 16.67
MIC 11.46
MFC 15.29
MIC
MFC 59.04 59.04
18.07
4.52
9.04
4.16
8.32
3.82
7.64
4.52
9.04
6.11
8.32
5.61
7.64
10.07
14.76
3.55
7.10
3.67
3.67
3.68
7.36
14.20
28.40
3.99
7.97
3.84
7.68
14.88
29.67
9.04
18.07
8.32
16.67
5.61
7.64
5.41
7.38
3.55
7.10
5.39
3.67
7.36
14.72
3.55
7.10
3.99
7.97
3.84
7.68
5.44
7.42
5a
5b
5c
5d
5e
18.07 18.07
4.16
8.32
2.80
3.82
1.97
3.69
1.89
3.55
2.69
3.67
3.68
7.36
3.55
7.10
2.13
3.99
2.82
3.84
2.72
3.71
38.0
4.16
8.32
5.61
7.64
5.41
7.38
3.55
7.10
3.67
7.34
3.68
7.36
7.10
14.20
3.99
7.97
3.84
7.68
5.44
7.42
29.52 29.52
MIC
7.10
MFC 14.20
3.55
7.10
3.67
7.34
5.39
7.36
7.10
7.10
3.67
3.67
3.68
MIC
MFC
MIC
3.67
7.34
7.36
5f
5g
MFC 14.72
MIC 21.30
MFC 28.40 14.20
7.36
14.20
28.40
3.99
7.97
5.63
5h
5i
MIC
MFC
MIC
3.99
7.97
7.68
3.99
7.97
3.84
7.68
3.71
7.42
285.0
5j
MFC 15.37
7.68
7.42
MIC
MFC
MIC
5.44
7.42
38.0
5k
14.88
37.60
94.00
32.20
48.30
38.00 475.0 38.00 380.0
Ketoconazole
MFC 95.00 380.0 95.00 95.00 570.0 95.00 380.0
48.00
MFC 64.00
MIC
48.0
64.0
48.00 48.00 64.00 64.00 48.00
80.00 64.00 80.00 80.00 64.00
Bifonazole
A.fum.—A. fumigatus, A.v.—A. versicolor, A.o.—A. ochraceus, A.n.—A. niger, T.v.—T. viride, P.f.—P. funiculosum,
P.o.—P. ochrochloron, C.a.—C. albicans, P.v.c.—P. cyclpoium var verucosum. Relative standard deviations were
all < 2.20.
2.3. Docking Studies
In order to elucidate the probable mechanism of antibacterial and antifungal activity of tested
compounds docking studies were performed on three bacterial targets; DNA Gyrase, Thymidylate

Molecules 2020, 25, 1964
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kinase and E. coli MurB enzymes. Compounds were also docked to lanosterol 14a-demethylase of C.
albicans for antifungal activity mechanism.
2.3.1. Docking to Antibacterial Targets
The docking studies revealed that Free Energy of Binding to DNA Gyrase (
as well as to Thymidylate kinase ( 2.26– 4.66 kcal/mol), were higher than that to E. coli MurB
5.73– 12.33), therefore it may be resolved that E. coli MurB is the most suitable enzyme where
binding scores were consistent with biological activity (Table 4).
−
1.28– 7.15kcal/mol)
−
−
−
(−
−
Table 4. Molecular docking binding affinities on antibacterial targets.
Est. Binding Energy (kcal/mol)
Gyrase
1KZN
Thymidylate Kinase
4QGG
E. coli MurB
1-H
E. coli MurB
Residues
E. coli MurB
Comp.
E. coli MurB
2Q85
4a
4b
4c
4d
4e
4f
4g
4h
4i
5a
5b
5c
5d
5e
5f
5g
5h
5i
−1.28
−6.22
−4.36
−4.19
−5.10
−4.07
−6.25
−7.09
−5.28
−3.65
−7.15
−5.13
−6.92
−6.20
−4.32
−7.00
−5.87
-
-
−6.25
−9.84
−7.15
−8.10
−8.17
−7.11
−10.08
−11.25
−7.70
−6.88
−12.33
−8.25
−10.51
−9.82
−7.14
−11.28
−8.75
−5.73
−5.77
−8.67
−23.74
−30.42
−26.71
−28.22
−28.36
−26.55
−31.16
−33.49
−27.11
−24.79
−36.27
−28.41
−31.44
−30.71
−26.58
−33.42
−28.98
−20.75
−20.86
−28.33
1
2
2
2
2
2
2
3
2
2
3
2
2
2
2
3
2
-
Arg158
Ser228
−2.69
-
-
Arg158, Arg213
Arg158, Arg213
Ser228, Arg213
Arg158, Arg213
Gly122, Ser228
Arg158, Ser228, Asn232
Tyr189, Ser228
Arg158, Tyr189
Ser228, Ala226
Ser228, Arg213
Gly122, Ser228
Ser228, Ala226
Arg158, Arg213
Arg158, Ser228, Asn232
Gly122, Ser228
-
−2.41
-
−3.14
−4.66
-
−3.27
−4.19
−2.26
−3.27
-
-
−4.11
−3.15
-
5j
5k
−2.55
-
1
2
Arg213
Gly122, Ser228
−5.84
−3.11
The docking pose of the most active compound 5b in E. coli MurB enzyme showed three favorable
hydrogen bond interactions. The first one between the hydrogen atom of OH group of the compound
and the oxygen of the side chain of Ser228, the second between the oxygen atom of the C=O group
of the compound and the side chain of Ser228 (distance 2.70 Å and 2.42 Å respectively), and the last
one hydrogen bond between the hydrogen atom of OH group of the compound and side chain of
Ala226 (distance 2.87 Å). The benzothiazole imidazole moiety interacts hydrophobically with Arg158,
Tyr124, Tyr189, Gly122, Asn232, Ala123, and Leu289, while the thiazolidinone interact hydrophobically
with the residues Arg213, Gln287 and Leu217 (Figure 1). These interactions stabilize the complex
compound-enzyme and play a crucial role to the increased inhibitory action of the compound 5b.
2.3.2. Docking to Lanosterol 14α-Demethylase of C. albicans
As already mentioned in order to study the probable mechanism of antifungal activity all the
synthesized compounds and reference drug were docked to lanosterol 14
(Table 5).
α-demethylase of C. albicans
Docking results showed that the most active compound 5f take place inside the enzyme alongside
to heme group, interacting with the heme group of CYP51Ca throughout its benzene ring and –NO2
group its forms pi and negative ionizable interactions with heme group respectively. A hydrogen bond
interaction is formed between the oxygen atom of thiazolidinone moiety and the hydrogen atom of
the side chain of Tyr134. Moreover, hydrophobic interactions between Tyr118, Leu376, and Thr311
and the benzene rings of the compound 5f were detected. Interaction with the heme group was also
observed with the benzene ring of ketoconazole which forms positive ionizable interactions (Figures 2
and 3). However, compound 5f forming more interaction than ketoconazole and more stable complex

Molecules 2020, 25, 1964
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of ligand with enzyme. This is probably the reason why compound 8 5f have better antifungal activity
than ketoconazole.
Table 5. Molecular docking binding affinities for antifungal targets.
Est. Binding Energy
(kcal/mol) CYP51 of C.
albicans
Binding Affinity Score
CYP51 of C. albicans
PDB ID: 5V5Z
Residues
CYP51 of C. albicans
PDB ID: 5V5Z
No
I-H
PDB ID: 5V5Z
17 4a
4 4b
13 4c
−3.15
−8.14
−7.15
−10.89
−15.18
−27.22
−26.02
−31.08
-
1
1
-
Tyr132
Tyr132
HEM601
(ionizable)
5 4d
-
14 4e
2 4i
12 4g
−4.18
−3.15
−9.66
−10.14
−3.15
−5.12
−1.14
−5.16
−5.17
−15.21
−13.57
−29.47
−30.25
-
-
1
-
Tyr132
HEM601
(ionizable)
15 4h
-
2 4i
18 5a
19 5b
7 5c
−13.57
−20.96
−6.29
-
-
-
1
-
-
-
-
−20.85
−21.30
Tyr118
-
Tyr132
HEM601
(ionizable, pi)
-
11 5d
8 5f
−11.13
−32.56
1
1 5h
3 5i
9 5j
−6.68
−8.74
−8.14
−6.25
−24.79
−27.58
−26.97
−23.88
-
1
1
1
Tyr64
Tyr64
Tyr118
10 5k
Figure 1. Docked conformation of the most active compound 5b in E. coli MurB.

Molecules 2020, 25, 1964
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Figure 2. Docked conformation of ketoconazole in lanosterol 14α-demethylase of C. albicans (CYP51ca).
Figure 3. Docked conformation of compound 5f in lanosterol 14α-demethylase of C. albicans (CYP51ca).
3. Experimental Part
3.1. General Procedure for the Synthesis of 4-Oxo-2-thioxothiazolidin-3-ylalkanecarboxylic Acids 2a–d and
3a–g
A mixture of a corresponding amino acid (50 mmol), cooled solution of KOH in water (20 mL)
(150 mmol in case of dicarboxylic acids) and CS2 (55 mmol) were stirred in a flat-bottomed flask
until a solution was formed. A solution of monochloracetic acid (55 mmol) was added with stirring,
pre-neutralized with sodium bicarbonate (55 mmol) in water (25 mL) and left at room temperature for
2 days.
Then, to the formed solution a 6N HCl solution (20 mL) was added and heated to boiling and kept
at a slow boil for 1 h. After cooling, the precipitate formed was filtered off, dried and recrystallized,
alternately, from diluted acetic acid, ethanol and toluene.
(4-Oxo-2-thioxothiazolidin-3-yl)acetic acid (2a). Yield 79%; m.p. 146–149 ◦C. 1H-NMR (400 MHz, DMSO-d6,
ppm)
δ
13.33 (s, 1H, COOH), 4.56 (s, 2H, CH2COOH), 4.41 (s, 2H, CH2). MS (ESI): m/z = 190.0 [M
−
H]−. Anal. Calcd. for C5H5NO3S2 (%): C, 31.41; H, 2.64; N, 7.32; S, 33.53 Found (%): C, 31.53; H, 2.59;
N, 7.38; S, 33.46.
3-(4-Oxo-2-thioxothiazolidin-3-yl)-propionic acid (2b). Yield 80%; m.p. 158–160 ◦C. 1H-NMR (400 MHz,
DMSO-d6, ppm) δ 12.50 (s, 1H, COOH), 4.22 (s, 2H, CH2), 4.05 (t, 2H, J = 8.0 Hz, CH2COOH), 2.51 (t,

Molecules 2020, 25, 1964
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J = 8.0 Hz, 2H, NCH2). MS (ESI): m/z = 204.0 [M
−
H]−. Anal. Calcd. for C6H7NO3S2 (%): C, 35.11; H,
3.44; N, 6.82; S, 31.24 Found (%): C, 35.23; H, 3.51; N, 6.71; S, 31.18.
4-(4-Oxo-2-thioxothiazolidin-3-yl)butyric acid (2c). Yield 91%; m.p. 121–122 C. H-NMR (400 MHz,
DMSO-d6, ppm) 12.07 (s, 1H, COOH), 4.18 (s, 2H, SCH2), 3.86 (t, J = 7.0 Hz, 2H, CH2COOH), 2.21 (t,
J = 7.4 Hz, 2H, NCH2), 1.79–1.71 (m, 2H, NCH2CH2CH2COOH).). MS (ESI): m/z = 218.0 [M
H]−.
◦
1
δ
−
Anal. Calcd. for C7H9NO3S2 (%): C, 38.34; H, 4.14; N, 6.39; S, 29.24 Found (%):C, 38.25; H, 4.01; N,
6.45; S, 29.32
◦
1
6-(4-Oxo-2-thioxothiazolidin-3-yl)-hexanoic acid (2d). Yield 88%; m.p. 86–89 C. H-NMR (400 MHz,
DMSO-d6, ppm) 12.01 (s, 1H, COOH), 4.25 (s, 2H, SCH2), 3.88–3.76 (m, 2H, CH2COOH), 2.19 (t,
δ
J = 7.3 Hz, 2H, NCH2), 1.61–1.40 (m, 4H, 2CH2), 1.34–1.18 (m, 2H, CH2). MS (ESI): m/z = 246.0 [M
−
H]−. Anal. Calcd. for C9H13NO3S2 (%): C, 43.71; H, 5.30; N, 5.66; S, 25.93 Found (%): C, 43.84; H, 5.24;
N, 5.72; S, 26.04.
2-(4-Oxo-2-thioxothiazolidin-3-yl)propionic acid (3a). Yield 64%; m.p. 148–151 ◦C. 1H-NMR (400 MHz,
DMSO-d6, ppm)
Hz, 3H, CH3). MS (ESI): m/z = 204.0 [M
6.82; S, 31.24 Found (%): C, 35.04; H, 340; N, 6.88; S, 31.31.
δ
13.12 (s, 1H, COOH), 5.42 (q, J = 7.1 Hz, 1H, NCH), 4.31 (s, 2H, CH2), 1.43 (d, J = 7.1
−
H]−. Anal. Calcd. for C6H7NO3S2 (%): C, 35.11; H, 3.44; N,
2-(4-Oxo-2-thioxothiazolidin-3-yl) pentanoic acid (3b). Yield 67%; m.p. 87–90 ◦C. 1H-NMR (400 MHz,
DMSO-d6, ppm)
2.03 (dd, J = 15.5, 7.5 Hz, 2H, CH2), 1.31–1.13 (m, 2H, CH2), 0.85 (t, J = 7.3 Hz, 3H, CH3). MS (ESI):
m/z = 232.0 [M
H]−. Anal. Calcd. for C8H11NO3S2 (%): C, 41.19; H, 4.75; N, 6.00; S, 27.49 Found (%):
δ 13.16 (s, 1H, COOH), 5.44–5.30 (m, 1H, NCH), 4.37 (q, J = 18.7 Hz, 2H, SCH2),
−
C, 41.03; H, 4.82; N, 6.11; S, 27.41.
4-Methylsulfanyl-2-(4-oxo-2-thioxothiazolidin-3-yl)butyric acid (3c). Yield 65%; m.p. 116–119 ◦C. 1H-NMR
(400 MHz, DMSO-d6, ppm)
4H, 2CH2), 2.01 (s, 3H, CH3). MS (ESI): m/z = 264.0 [M
36.21; H, 4.18; N, 5.28; S, 36.25 Found (%): C, 36.14; H, 4.27; N, 5.28; S, 36.19.
δ
13.27 (s, 1H, COOH), 5.55 (s, 1H, CH), 4.32 (s, 2H, SCH2), 2.47–2.22 (m,
−
H]−. Anal. Calcd. for C8H11NO3S3 (%): C,
(4-Oxo-2-thioxothiazolidin-3-yl)-phenylacetic acid (3d). Yield 57%; m.p. 169–171 ◦C. 1H-NMR (400 MHz,
DMSO-d6, ppm)
4.43 (s, 2H, CH2). MS (ESI): m/z = 266.0 [M
N, 5.24; S, 23.99 Found (%): C, 49.31; H, 3.45; N, 5.35; S, 24.06.
δ
13.49 (s, 1H, COOH), 7.47–7.42 (m, 2H, Ph), 7.37–7.29 (m, 3H, Ph), 6.63 (s, 1H, NCH),
−
H]−. Anal. Calcd. for C11H9NO3S2 (%): C, 49.42; H, 3.39;
2-(4-Oxo-2-thioxothiazolidin-3-yl)-3-phenylpropionic acid (3e). Yield; 58%; m.p. 102–105 ◦C. 1H-NMR (400
MHz, DMSO-d6, ppm)
2H, SCH2), 3.49–3.15 (m, 2H, CH2). 13C-NMR (101 MHz, DMSO-d6, ppm)
137.15, 129.48, 128.69, 127.08, 58.50, 35.12, 33.39. MS (ESI): m/z = 280.0 [M
δ 13.25 (s, 1H, COOH), 7.29–7.04 (m, 6H, C6H5), 5.64 (s, 1H, CH), 4.40–4.05 (m,
δ
203.07, 174.40, 169.31,
−
H]−. Anal. Calcd. for
C12H11NO3S2 (%): C, 51.23; H, 3.94; N, 4.98; S, 22.79 Found (%):C, 51.32; H, 3.89; N, 4.91; S, 22.86.
2-(4-Oxo-2-thioxothiazolidin-3-yl) succinic acid (3f). Yield 53%; m.p. 197–199 ◦C. 1H-NMR (400 MHz,
DMSO-d6, ppm)
δ
13.33 (s, 1H, COOH), 12.59 (s, 1H, COOH), 5.79 (s, 1H, NCH), 4.32 (s, 2H, CH2), 3.17
(dd, J = 16.2, 10.0 Hz, 1H, CH2), 2.69 (dd, J = 16.6, 3.3 Hz, 1H, CH2). MS (ESI): m/z = 248.0 [M
Anal. Calcd. for C7H7NO5S2 (%): C, 33.73; H, 2.83; N, 5.62; S, 25.73 Found (%): C, 33.83; H, 2.92; N,
5.55; S, 25.64.
−
H]−.
2-(4-Oxo-2-thioxothiazolidin-3-yl)pentanedioic acid (3g). Yield 79%; m.p. 146–149 ◦C. 1H-NMR (400 MHz,
DMSO-d6, ppm)
[M
H]−. Anal. Calcd. for C8H9NO5S2 (%): C, 36.50; H, 3.45; N, 5.32; S, 24.36 Found (%): C, 36.62; H,
3.38; N, 5.35; S, 24.27.
δ 5.44 (s, 1H, NCH), 4.30 (s, 2H, SCH2), 2.42–2.18 (m, 4H, 2CH2). MS (ESI): m/z = 262.0
−

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3.2. General Procedure for the Synthesis of 5-(1-R1,5-R2,6-R3-1H-Indol-3-ylmethylene)-4-oxo-2-
thioxothiazolidin-3-yl] Alkane Carboxylic Acids 4a–i and 5a–k
4-Oxo-2-thioxothiazolidin-3-ylalkanecarboxylic acid 2a–d or 3a–g (2 mmol), the corresponding
indole-3-carbaldehyde (2.5 mmol), ammonium acetate (2 mmol) and ethanol (7 mL) were placed in a
round-bottom flask under reflux. The reaction mixture was boiled for 2 to 3 h, cooled, the reaction
product filtered off, washed with ethanol, water, dried and recrystallized from acetic acid or acetic acid
- DMF.
(Z)-[5-(1H-Indol-3-ylmethylene)-4-oxo-2-thioxothiazolidin-3-yl] acetic acid (4a). Yield 99%; m.p. 277–278 ◦C.
1H-NMR (300 MHz, DMSO-d6, ppm)
δ
12.11 (s, 1H, NH), 8.10 (s, 1H, CH=), 7.86 (d, J = 6.8 Hz, 1H, Ar),
7.73 (d, J = 2.9 Hz, 1H, Ar), 7.53–7.43 (m, 1H, Ar), 7.29–7.13 (m, 2H, Ar), 4.72 (s, 2H, NCH2). 13C-NMR
(101 MHz, DMSO-d6, ppm)
δ 192.21, 167.42, 166.02, 136.43, 130.90, 126.97, 126.73, 123.42, 121.60, 118.62,
113.88, 112.50, 111.06, 44.94. MS (ESI): m/z = 319.0 [M + H]+. Anal. Calcd. for C14H10N2O3S2 (%): C,
52.82; H, 3.17; N, 8.80; S, 20.14 Found (%): C, 52.71; H, 3.25; N, 8.87; S, 20.08.
(Z)-[5-(1-Methyl-1H-indol-3-ylmethylene)-4-oxo-2-thioxothiazolidin-3-yl] acetic acid (4b). Yield 99%; m.p.
273–275 ◦C. 1H-NMR (300 MHz, DMSO-d6, ppm)
δ
8.05 (s, 1H, CH=), 7.89 (d, J = 7.7 Hz, 1H, Ar), 7.84
(s, 1H, Ar), 7.49 (d, J = 8.3 Hz, 1H, Ar), 7.37–7.20 (m, 2H, Ar), 4.71 (s, 2H, NCH2), 3.98 (s, 3H, CH3).
13C-NMR (101 MHz, DMSO-d6, ppm)
δ 192.11, 167.43, 165.94, 136.99, 134.21, 127.24, 126.23, 123.47,
121.91, 118.66, 113.61, 110.98, 110.03, 44.27, 33.45. MS (ESI): m/z = 333.2 [M + H]+. Anal. Calcd. for
C15H12N2O3S2 (%): C, 54.20; H, 3.64; N, 8.43; S, 19.29 Found (%): C, 54.33; H, 3.60; N, 8.34 S, 19.21.
(Z)-[5-(6-Methoxy-1H-indol-3-ylmethylene)-4-oxo-2-thioxothiazolidin-3-yl] acetic acid (4c). Yield 89%; m.p.
> 270 ◦C. 1H-NMR (300 MHz, DMSO-d6, ppm)
δ
12.00 (s, 1H, NH), 8.03 (s, 1H, CH=), 7.72 (d, J = 8.7
Hz, 1H, Ar), 7.62 (d, J = 2.8 Hz, 1H, Ar), 6.94 (d, J = 1.7 Hz, 1H, Ar), 6.85–6.78 (m, 1H, Ar), 4.70 (s,
2H, NCH2), 3.83 (s, 3H, CH3O). 13C-NMR (101 MHz, DMSO-d6, ppm)
192.19, 167.49, 166.04, 156.96,
δ
137.42, 129.99, 127.24, 120.70, 119.40, 113.72, 111.60, 111.29, 95.51 55.34 (s), 44.97. MS (ESI): m/z = 349.2
[M + H]+. Anal. Calcd. for C15H12N2O4S2 (%): C, 51.71%; H, 3.47; N, 8.04; S, 18.41 Found (%): C,
51.69%; H, 3.40; N, 8.11; S, 18.39.
(Z)-3-[5-(1-Methyl-1H-indol-3-ylmethylene)-4-oxo-2-thioxothiazolidin-3-yl] propionic acid (4d). Yield 83%;
m.p. 246–248 ◦C. 1H-NMR (300 MHz, DMSO-d6, ppm)
δ
8.01 (s, CH=), 7.88 (d, J = 7.6 Hz, 1H, Ar), 7.80
(s, 1H, Ar), 7.48 (d, J = 8.0 Hz, 1H, Ar), 7.33–7.21 (m, 2H, Ar), 4.30 (t, J = 9.0 Hz, 2H, CH2COOH), 3.97
(s, 3H, CH3), 2.63 (t, J = 9.0 Hz, 2H, NCH2). 13C-NMR (101 MHz, DMSO-d6, ppm)
192.02, 171.83,
δ
166.29, 136.94, 133.94, 127.25, 125.48, 123.41, 121.85, 118.60, 114.13, 110.95, 110.06, 33.42, 30.94. MS (ESI):
m/z = 347.0 [M + H]+. Anal. Calcd. for C16H14N2O3S2 (%): C, 55.47; H, 4.07; N, 8.09; S, 18.51 Found
(%):C, 55.38; H, 4.01; N, 7.98; S, 18.46.
(Z-)3-[5-(5-Methoxy-1H-indol-3-ylmethylene)-4-oxo-2-thioxothiazolidin-3-yl] propionic acid (4e). Yield 96%;
m.p. 226–228 ◦C. 1H-NMR (300 MHz, DMSO-d6, ppm)
δ 12.07 (s, 1H, NH), 8.05 (s, 1H, CH=), 7.62
(d, J = 3.1 Hz, 1H, Ar), 7.37–7.31 (m, 2H, Ar), 6.81 (dd, J = 8.8, 2.1 Hz, 1H, Ar), 4.27 (t, J = 9.0 Hz 2H,
CH2COOH), 3.86 (s, 3H, CH3O), 2.61 (t, J = 9.0 Hz, 2H, NCH2). 13C-NMR (101 MHz, DMSO-d6, ppm)
δ
192.01, 171.74, 155.31, 131.15, 130.55, 127.73, 126.72, 114.45, 113.53, 113.50, 113.31, 111.15, 100.37, 55.48,
30.93. MS (ESI): m/z = 363.0 [M + H]+. Anal. Calcd. for C16H14N2O4S2 (%): C, 53.03; H, 3.89; N, 7.73; S,
17.69 Found (%):C, 53.14; H, 3.91; N, 7.65; S, 17.58.
(Z)-4-[5-(1-Methyl-1H-indol-3-ylmethylene)-4-oxo-2-thioxothiazolidin-3-yl] butyric acid (4f). Yield 96%; m.p.
229–230 ◦C. 1H-NMR (300 MHz, DMSO-d6, ppm)
δ 8.00 (s, 1H, CH=), 7.88 (d, J = 7.3 Hz, 1H, Ar),
7.79 (s, 1H, Ar), 7.48 (d, J = 7.6 Hz, 1H, Ar), 7.33–7.22 (m, 2H, Ar), 4.14 (t, J = 7.1 Hz, 2H, CH2COOH),
3.97 (s, 3H CH3), 2.30 (t, J = 7.4 Hz, 2H, NCH2), 2.04–1.89 (m, 2H, NCH2CH2CH2COOH). 13C-NMR
(101 MHz, DMSO-d6, ppm)
δ 192.41, 173.71, 166.74, 136.95, 133.89, 127.26, 125.32, 123.40, 121.83 (s),
118.62, 114.31, 110.95, 110.10, 43.53, 33.42, 31.01, 22.13. MS (ESI): m/z = 361.2 [M + H]+. Anal. Calcd.
for C17H16N2O3S2 (%): C, 56.65; H, 4.47; N, 7.77; S, 17.79 Found (%): C, 56.53; H, 4.41; N, 7.85; S, 17.71.

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(Z)-4-[5-(5-Methoxy-1H-indol-3-ylmethylene)-4-oxo-2-thioxothiazolidin-3-yl] butyric acid (4g). Yield 87%;
m.p. 214–216 ◦C. 1H-NMR (300 MHz, DMSO-d6, ppm) δ 11.96 (s, 1H, NH), 7.97 (s, 1H, CH=), 7.72 (d,
J = 8.7 Hz, 1H, Ar), 7.57 (d, J = 2.8 Hz, 1H, Ar), 6.93 (d, J = 1.6 Hz, 1H, Ar), 6.81 (dd, J = 8.7, 1.9 Hz, 1H,
Ar), 4.12 (t, J = 7.0 Hz, 2H, CH2COOH), 3.83 (s, 3H, CH3O), 2.29 (t, J = 7.3 Hz, 2H, NCH2), 1.95 (p,
J = 7.0 Hz, 2H, NCH2CH2CH2COOH). 13C-NMR (101 MHz, DMSO-d6, ppm)
δ 192.37, 173.66, 166.74,
155.27, 131.11, 130.50, 127.73, 126.58, 113.64, 113.52, 113.31, 111.17, 100.30, 55.45, 43.49, 30.95, 22.08. MS
(ESI): m/z = 377.2 [M + H]+. Anal. Calcd. for C17H16N2O4S2 (%): C, 54.24; H, 4.28; N, 7.44; S, 17.03
Found (%):C, 54.18; H, 4.22; N, 7.48; S, 17.15.
(Z)-4-[5-(6-Methoxy-1H-indol-3-ylmethylene)-4-oxo-2-thioxothiazolidin-3-yl] butyric acid (4h). Yield 84%;
m.p. 213–215 ◦C. 1H-NMR (300 MHz, DMSO-d6, ppm)
δ 11.96 (s, 1H, NH), 7.97 (s, 1H, CH=), 7.71
(d, J = 8.7 Hz, 1H, Ar), 7.56 (d, J = 2.5 Hz, 1H, Ar), 6.92 (s, 1H, Ar), 6.81 (d, J = 8.7 Hz, 1H, Ar), 4.12
(t, J = 6.9 Hz, 2H, CH2COOH), 3.83 (s, 3H, CH3), 2.28 (t, J = 7.3 Hz, 2H, NCH2), 2.01–1.87 (m, 2H,
NCH2CH2CH2COOH). 13C-NMR (101 MHz, DMSO-d6, ppm)
δ 192.39, 173.61, 166.73, 156.85, 137.31,
129.61, 126.24, 120.65, 119.26, 114.34, 111.44, 111.26, 95.41, 55.28, 43.53, 30.99, 22.09. MS (ESI): m/z = 377.2
[M + H]+. Anal. Calcd. for C17H16N2O4S2 (%): C, 54.24; H, 4.28; N, 7.44; S, 17.03 Found (%):C, 54.31;
H, 4.20; N, 7.38; S, 17.09.
(Z)-6-[5-(1H-Indol-3-ylmethylene)-4-oxo-2-thioxothiazolidin-3-yl] hexanoic acid (4i). Yield 98%; m.p. 205–207
◦C. 1H-NMR (300 MHz, DMSO-d6, ppm)
δ 12.17 (s, 1H, NH), 8.03 (s, 1H, CH=), 7.85 (d, J = 6.8 Hz, 1H,
Ar), 7.68 (d, J = 2.9 Hz, 1,H, Ar), 7.48–7.46 (m, 1H, 1H, Ar), 7.25–7.13 (m, 2H, Ar), 4.12–3.97 (m, 2H,
CH2COOH), 2.20 (t, J = 7.3 Hz, NCH2), 1.78–1.55 (m, 4H, 2CH2), 1.48–1.34 (m, 2H, CH2). 13C-NMR
(101 MHz, DMSO-d6, ppm)
δ 192.23, 174.33, 166.66, 136.43, 130.58, 126.77, 126.17, 123.38, 121.54 (s),
118.52, 114.47, 112.58, 111.15, 43.97, 33.44, 26.21, 25.73, 24.07. MS (ESI): m/z = 375.2 [M + H]+. Anal.
Calcd. for C18H18N2O3S2 (%): C, 57.73; H, 4.84; N, 7.48; S, 17.12 Found (%): C, 57.67; H, 4.79; N, 7.53; S,
17.01.
(Z)-2-[5-(1H-Indol-3-ylmethylene)-4-oxo-2-thioxothiazolidin-3-yl] propionic acid (5a) Yield 90%; m.p. > 270
◦C. 1H-NMR (400 MHz, DMSO-d6, ppm)
δ
12.43 (s, 1H, NH), 8.10 (s, 1H, CH=), 8.00–7.93 (m, 2H, Ar),
7.52 (d, J = 7.9 Hz, 1H, Ar), 7.25 (dt, J = 14.7, 7.1 Hz, 2H, Ar), 5.62 (q, J = 6.9 Hz, 1H, NCH), 1.55 (d,
J = 7.1 Hz, 3H, CH3). 13C-NMR (101 MHz, DMSO-d6, ppm)
191.83, 169.83, 165.96, 136.46, 130.94 (s),
δ
126.86, 126.79, 123.47, 121.66, 118.59, 113.38, 112.64, 111.12, 52.73, 13.58. Anal. MS (ESI): m/z = 333.2 [M
+ H]+. Calcd. for C15H12N2O3S2 (%): C, 54.20; H, 3.64; N, 8.43; S, 19.29 Found (%): C, 54.14; H, 3.72; N,
8.51; S, 19.20.
(Z)-2-[5-(1H-Indol-3-ylmethylene)-4-oxo-2-thioxothiazolidin-3-yl] pentanoic acid (5b). Yield 90%; m.p.
257–259 ◦C. 1H-NMR (300 MHz, DMSO-d6, ppm)
δ 12.10 (s, 1H, Ar), 8.03 (s, 1H, CH=), 7.85 (d, J = 7.0
Hz, 1H, Ar), 7.71 (d, J = 2.9 Hz, 1H, Ar), 7.52–7.46 (m, 1H, Ar), 7.27–7.16 (m, 2H, Ar), 5.55 (dd, J = 9.2, 5.6
Hz, 1H, NCH), 2.38–2.12 (m, 2H, CH2), 1.49–1.21 (m, 2H, CH2), 0.97 (t, J = 7.3 Hz, 3H, CH3). 13C-NMR
(101 MHz, DMSO-d6, ppm)
δ 192.59, 169.44, 166.27, 136.49, 131.02, 127.05, 126.79, 123.48, 121.67, 118.59,
113.06, 112.65, 111.16, 56.92, 29.71, 19.04, 13.65. Anal. MS (ESI): m/z = 361.0 [M + H]+. Anal. Calcd. for
C17H16N2O3S2 (%): C, 56.65; H, 4.47; N, 7.77; S, 17.79 Found (%): C, 56.55; H, 4.41; N, 7.84; S, 17.70.
(Z)-2-[5-(1H-Indol-3-ylmethylene)-4-oxo-2-thioxothiazolidin-3-yl]-4-methylsulfanylbutyric acid (5c). Yield
98%; m.p. 204–205 ◦C. 1H-NMR (400 MHz, DMSO-d6, ppm)
δ
12.43 (s, 1H, NH), 8.10 (s, 1H, CH=),
7.99–7.94 (m, 2H, Ar), 7.52 (d, J = 7.7 Hz, 1H, Ar), 7.26 (ddd, J = 14.9, 13.8, 6.6 Hz, 2H, Ar), 5.74 (s, 1H,
NCH), 3.34 (b. s, 4H, 2CH2), 2.02 (s, 3H, CH3). 13C-NMR (101 MHz, DMSO-d6, ppm)
192.54, 169.20,
δ
166.35, 136.41, 130.90, 126.83, 126.74, 123.42, 121.61, 118.54, 113.30, 112.59, 111.09, 56.08, 30.13, 27.16,
14.57. Anal. MS (ESI): m/z = 393.0 [M + H]+. Anal. Calcd. for C17H16N2O3S3 (%): C, 52.02; H, 4.11; N,
7.14; S, 24.51 Found (%): C 51.94; H, 4.08; N, 7.10; S, 24.42.
(Z)-2-[5-(1-Methyl-1H-indol-3-ylmethylene)-4-oxo-2-thioxothiazolidin-3-yl]-4-methylsulfanyl-butyric acid (5d).
Yield 94%; m.p. 238–240 ◦C. 1H-NMR (300 MHz, DMSO-d6, ppm)
δ 8.00 (s, 1H, CH=), δ 7.89 (s, 1H, Ar),

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7.86 (s, 1H, Ar), 7.49 (d, J = 8.0 Hz, 1H, Ar), 7.34–7.20 (m, 2H, Ar), 5.70 (d, J = 5.7 Hz, 1H, NCH), 3.98
(s, CH3N), 2.65–2.34 (m, 4H, CH2), 2.07 (s, 3H, CH3). 13C-NMR (101 MHz, DMSO-d6, ppm)
169.27,
δ
166.37, 137.04, 134.25, 127.30, 126.19, 123.53, 121.99, 118.73, 113.13, 111.07, 110.12, 56.13, 33.52, 30.20,
27.22, 14.62. MS (ESI): m/z = 407.0 [M + H]+. Anal. Calcd. for C18H18N2O3S3 (%): C, 53.18; H, 4.46; N,
6.89; S, 23.66 Found (%): C 53.23; H, 4.53; N, 6.78; S, 23.54.
(Z)-2-[5-(5-Methoxy-1H-indol-3-ylmethylene)-4-oxo-2-thioxothiazolidin-3-yl]-4-methylsulfanyl-butyric acid
(
5e). Yield 93%; m.p. 222–223 ◦C. 1H-NMR (300 MHz, DMSO-d6, ppm)
δ
12.11 (s, 1H, NH), 8.07 (s, 1H,
CH=), 7.66 (s, 1H, Ar), 7.35 (d, J = 8.8 Hz, 2H, Ar), 6.81 (d, J = 8.7 Hz, 1H Ar), 5.69 (s, 1H, NCH), 3.86 (s,
3H, CH3O), 2.61–2.33 (m, 4H, 2CH2), 2.06 (s, 1H, CH3). 13C-NMR (101 MHz, DMSO-d6, ppm)
192.50,
δ
169.18, 166.31, 155.38, 131.18, 130.85, 127.78, 127.38, 113.56, 113.36, 112.50, 111.20, 100.50, 56.08, 55.49,
30.17, 27.21, 14.59. MS (ESI): m/z = 423.0 [M + H]+. Anal. Calcd. for C18H18N2O4S3 (%): C, 51.17; H,
4.29; N, 6.63; S, 22.76 Found (%): C 51.25; H, 4.21; N, 6.69; S, 22.69.
(Z-)[5-(1-Methyl-1H-indol-3-ylmethylene)-4-oxo-2-thioxothiazolidin-3-yl]-phenylacetic acid (5f). Yield 90%;
m.p. > 270 ◦C. 1H-NMR (300 MHz, DMSO-d6, ppm)
δ
8.02 (s, 1H, CH=), 7.89–7.82 (m, 2H, Ar), 7.62–7.56
(m, 2H, Ar), 7.48 (d, J = 7.9 Hz, 1H, Ar), 7.38–7.21 (m, 5H, Ar), 6.76 (s, 1H, NCH), 3.97 (s, 3H, CH3).
13C-NMR (101 MHz, DMSO-d6, ppm)
191.89, 167.96, 165.89, 137.05, 134.46, 133.77, 129.70, 128.32,
δ
128.11, 127.31, 126.91, 123.57, 122.07, 118.74, 112.35, 111.07, 110.14, 60.06, 33.53. MS (ESI): m/z = 409.2
[M + H]+. Anal. Calcd. for C21H16N2O3S2 (%): C, 61.75; H, 3.95; N, 6.86; S, 15.70 Found (%): C 61.67;
H, 3.89; N, 6.94; S, 15.81.
(Z)-2-[5-(1H-Indol-3-ylmethylene)-4-oxo-2-thioxothiazolidin-3-yl]-3-phenylpropionic acid. (5g). Yield 97%;
1
m.p. 271 ◦C decomp. H-NMR (400 MHz, DMSO-d6, ppm)
δ
12.42 (s, 1H, NH), 8.06 (s, 1H, CH=), 7.96
(d, J = 7.6 Hz, 1H, Ar), 7.88 (d, J = 2.7 Hz, 1H, Ar), 7.51 (d, J = 7.7 Hz, 1H, Ar), 7.30–7.10 (m, 7H, Ar),
5.88 (s, 1H, NCH), 3.52 (d, J = 5.3 Hz, 2H, CH2). 13C-NMR (101 MHz, DMSO-d6, ppm)
δ
192.03, 168.99,
166.29, 136.72, 136.45, 131.00, 129.04, 128.24, 126.80, 126.75, 126.66, 123.49, 121.67, 118.60, 112.91, 112.63,
111.07, 58.04, 33.22. MS (ESI): m/z = 409.2 [M + H]+. Anal. Calcd. for C21H16N2O3S2 (%): C, 61.75; H,
3.95; N, 6.86; S, 15.70 Found (%): C 61.74; H, 3.99; N, 6.91; S, 15.64
(Z)-2-[5-(1-Methyl-1H-indol-3-ylmethylene)-4-oxo-2-thioxothiazolidin-3-yl]-3-phenylpropionic acid (5h). Yield
99%; m.p. 227–228 ◦C. 1H-NMR (300 MHz, DMSO-d6, ppm)
δ 7.95 (s, 1H, CH=), 7.87 (d, J = 7.4 Hz, 1H,
Ar), 7.78 (s, 1H, Ar), 7.48 (d, J = 7.7 Hz, 1H, Ar), 7.34–7.21 (m, 2H, Ar), 7.20–7.12 (m, 5H, Ar), 5.83 (dd,
J = 9.2, 6.7 Hz, 1H, NCH), 3.96 (s, 3H, CH3N), 3.65–3.46 (m, 2H, CH2). 13C-NMR (101 MHz, DMSO-d6,
ppm)
δ 191.94, 169.06, 166.24, 137.02, 136.70, 134.32, 129.05, 128.24, 127.25, 126.67, 126.16, 123.55, 122.00,
118.74, 112.64, 111.05, 110.03, 58.00, 33.49, 33.19. MS (ESI): m/z = 423.0 [M + H]+. Anal. Calcd. for
C22H18N2O3S2 (%): C, 62.54; H, 4.29; N, 6.63; S, 15.18 Found (%): C 62.47 H, 4.35; N, 6.56; S, 15.09.
(Z)-2-[5-(1H-Indol-3-ylmethylene)-4-oxo-2-thioxothiazolidin-3-yl] succinic acid (5i). Yield 99%; m.p. 241 ◦C
1
decomp. H-NMR (300 MHz, DMSO-d6, ppm)
δ 12.12 (s, 1H, NH), 8.05 (s, 1H, CH=), 7.86 (d, J = 6.8 Hz,
1H, Ar), 7.72 (d, J = 2.9 Hz, 1H, Ar), 7.53–7.46 (m, 1H, Ar), 7.28–7.15 (m, 2H, Ar), 5.98 (t, J = 6.9 Hz, 1H,
NCH), 3.30 (dd, J = 16.5, 7.7 Hz, 1H, CH2), 2.82 (dd, J = 16.5, 6.0 Hz, 1H, CH2). 13C-NMR (101 MHz,
DMSO-d6, ppm) δ 191.93, 171.24, 168.83, 166.19, 136.43, 130.92, 126.92, 126.73, 123.43, 121.62, 118.54,
113.32, 112.60, 111.07, 53.20, 33.14. MS (ESI): m/z = 377.0 [M + H]+. Anal. Calcd. for C16H12N2O5S2
(%): C, 51.06; H, 3.21; N, 7.44; S, 17.04 Found (%): C 51.12; H, 3.16; N, 7.38; S, 17.13.
(Z)-2-[5-(1H-Indol-3-ylmethylene)-4-oxo-2-thioxothiazolidin-3-yl]-pentanedioic acid (5j). Yield 99%; m.p.
253–255 ◦C. 1H-NMR (300 MHz, DMSO-d6, ppm)
δ 12.21 (s, 1H, NH), 8.04 (s, 1H, CH=), 7.86 (d, J = 6.8
Hz, 1H, Ar), 7.74 (d, J = 3.0 Hz, 1H, Ar), 7.53–7.47 (m, 1H, Ar), 7.27–7.15 (m, 2H, Ar), 5.59 (dd, J = 9.3,
5.1 Hz, 1H, NCH), 2.56–2.45 (m, 2H, CH2), 2.30–2.23 (m, 2H, CH2). 13C-NMR (101 MHz, DMSO-d6,
ppm)
δ 192.67, 173.56, 169.18, 166.27, 137.07, 134.22, 127.30, 126.14, 123.52, 121.98, 118.70, 113.18, 111.05,
110.14, 56.57, 33.50, 30.25, 23.09. MS (ESI): m/z = 391.0 [M + H]+. Anal. Calcd. for C17H14N2O5S2 (%):
C, 52.30; H, 3.61; N, 7.17; S, 16.42 Found (%): 52.21; H, 3.75; N, 7.12; S, 16.51.

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(Z-)2-[5-(1-Methyl-1H-indol-3-ylmethylene)-4-oxo-2-thioxothiazolidin-3-yl]-pentanedioic acid (5k). Yield 76%;
m.p. 256–258 ◦C. 1H-NMR (300 MHz, DMSO-d6, ppm)
8.00 (s, 1H, CH=), 7.90–7.83 (m, 2H, Ar), 7.49
(d, J = 7.9 Hz, 1H Ar), 7.35–7.20 (m, 2H Ar), 5.59 (dd, J = 9.2, 5.0 Hz, 1H, NCH), 2.56–2.45 (m, 2H, CH2),
2.31–2.22(m, 2H, CH2). 13C-NMR (101 MHz, DMSO-d6, ppm)
192.60, 173.49, 169.11, 166.21, 137.02,
δ
δ
134.16, 127.24, 126.09, 123.47, 121.92, 118.65, 113.13, 111.00, 110.09, 56.51, 33.45, 30.20, 23.04. MS (ESI):
m/z = 405.0 [M + H]+. Anal. Calcd. for C18H16N2O5S2 (%): C, 53.45; H, 3.99; N, 6.93; S, 15.85 Found
(%): 53.52; H, 4.05; N, 6.99; S, 15.74.(Spectra see in supplementary)
3.3. Biological Evaluation
3.3.1. Antibacterial Activity
The following Gram-negative bacteria: Escherichia coli (ATCC 35210), Enterobacter cloacae (clinical
isolate), Salmonella typhimurium (ATCC 13311), as well as Gram-positive bacteria: Listeria monocytogenes
(NCTC 7973), Bacillus cereus (clinical isolate), and Staphylococcus aureus (ATCC 6538) were used.
The organisms were obtained from the Mycological Laboratory, Department of Plant Physiology,
Institute for Biological Research “Siniša Stankovic”, Belgrade, Serbia.
The minimum inhibitory (MIC) and minimum bactericidal (MBC) concentrations were determined
by the modified microdilution method as previously reported [36–38].
Resistant strains used in microdilution assay were isolates of S. aureus (strain isolated from cow),
E. coli (strain isolated form pig) and P. aeruginosa (strain isolated from cat) obtained as described in
Kartsev et al. [39].
3.3.2. Inhibition of Biofilm Formation
Method was performed as described [40] with some modifications. Briefly, P. aeruginosa resistant
strain was incubated with MIC and subMIC of tested compounds in Triptic soy broth enriched with
2% glucose at 37 ◦C for 24 h. After 24 h, each well was washed twice with sterile PBS (Phosphate
buffered saline, pH 7.4) and fixed with methanol for 10 min. Methanol was then removed and the
plate was air dried. Biofilm was stained with 0.1% crystal violet (Bio-Merieux, Craponne, France) for
30 min. Wells were washed with water, air dried and 100
µL of 96% ethanol (Zorka, Serbia) was added.
The absorbance was read at 620 nm on a Multiskan FC Microplate Photometer, Thermo Scientific
™
™
(Waltham, MA, USA). The percentage of inhibition of biofilm formation was calculated by the formula:
[(A620 control − A620 sample)/A620 control] × 100.
3.3.3. Antifungal Activity
For the antifungal bioassays, six fungi were used: Aspergillus niger (ATCC 6275), Aspergillus
fumigatus (ATCC 1022), Aspergillus versicolor (ATCC 11730), Penicillium funiculosum (ATCC 36839),
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,” Belgrade, Serbia. All experiments were performed in duplicate and
repeated three times [35,39].
3.4. Docking Studies
The program AutoDock 4.2® software (version 4.2.6, San Diego, California, CA, USA) was used
for the docking stimulation. The free energy of binding (∆G) of E. coli DNA GyrB, Thymidylate kinase,
E. coli MurA, E. coli primase, E. coli MurB, DNA topoIV 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 [38].

Molecules 2020, 25, 1964
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4. Conclusions
The range of twenty new 5-(1H-Indol-3-ylmethylene)-4-oxo-2-thioxothiazolidin-3-yl)
alkancarboxylic acid derivatives were synthesized and evaluated for their antimicrobial activity
exhibiting a remarkable inhibition of the growth of a wide spectrum of Gram-positive and
Gram-negative bacteria and fungi. All compounds displayed better antibacterial activity than ampicillin
against all bacteria tested, while eighteen out of twenty showed better activity than streptomycin
against S. aureus, En. cloacae, P. aeruginosa, L. monocytogenes, and E. coli. The best antibacterial activity
was achieved for compound 5b (Z)-2-[5-(1H-Indol-3-ylmethylene)-4-oxo-2-thioxothiazolidin-3-yl]
pentanoic acid. Three the most active compounds 4h, 5b and 5g tested against three resistant strains:
Methicillin resistant S. aureus, MRSA, P. aeruginosa and E. coli appeared to be more potent against MRSA
than ampicillin, while streptomycin did not show any bactericidal activity. All three compounds
displayed better activity also against resistant strains P. aeruginosa and E. coli than ampicillin. These
compounds were tested also for their effect on biofilm formation. All compounds were able to inhibit
biofilm formation 2 to 4 times more than both reference drugs. The most sensitive bacterium was
found to be P. aeruginosa, while M. flavus was the most resistant.
The evaluation of antifungal activity revealed that all compounds appeared to be more potent
than ketoconazole and bifonazole used as reference drugs. The most potent compound appeared to be
5f (Z-)[5-(1-Methyl-1H-indol-3-ylmethylene)-4-oxo-2-thioxothiazolidin-3-yl]-phenylacetic acid.
The most sensitive fungal to compounds tested was T. viride, while A fumigatus was found to be the
most resistant one. It should be mentioned that the growth of both Gram-positive and Gram-negative
bacteria as well as fungi showed different sensitivity towards compounds tested.
Docking analysis to different antibacterial targets (MurB, Gyrase, Thymidylate kinase)
demonstrated that E. coli Mur B inhibition, probably, is involved in antibacterial mechanism of
action of compounds tested. On the other hand, docking analysis to 14α-lanosterol demethylase
(CYP51) and tetrahydrofolate reductase of Candida albicans specified a probable implication of CYP51
reductase at the antifungal activity of the compounds.
Supplementary Materials: The following are available online.
Author Contributions: V.H.—synthesis of compounds; V.K.—NMR spectra interpretation; V.M.—NMR and
HRMS spectra; A.G.—general design of experiment and preparation of the paper; A.P.—docking; J.G.—antibacterial
activity; A.C.—antifungal activity; M.S.—antimicrobial activity. All authors have read and agreed to the published
version of the manuscript.
Funding: This research received no external funding.
Acknowledgments: This work has been supported by Ministry of Education, Science and Technological
Development of Republic of Serbia (451-03-68/2020-14/200007).
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Some samples of the compounds are available from the authors.
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2020 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/).