Synthesis and antibacterial activity of new thiazolidine-2,4-dione-based chlorophenylthiosemicarbazone hybrids

Article abstract of DOI:10.3390/molecules23051023

Series of new thiazolidine-2,4-dione-based chlorophenylthiosemicarbazone hybrids (17–40) were synthesized by the reaction of condensation chlorophenylthiosemicarbazides with formylphenyl 2-(2,4-dioxothiazolidin-5-yl/ylidene)acetates. New compounds were te

Full text of DOI:10.3390/molecules23051023

molecules
Article
Synthesis and Antibacterial Activity of New
Thiazolidine-2,4-dione-Based
Chlorophenylthiosemicarbazone Hybrids
1
,
2
1
3
2
Nazar Trotsko *, Urszula Kosikowska , Agata Paneth , Tomasz Plech , Anna Malm and
Monika Wujec 1
1
Department of Organic Chemistry, Faculty of Pharmacy with Medical Analytics Division,
Medical University of Lublin, 4A Chod z´ ki, Lublin 20-093, Poland; agatapaneth@umlub.pl (A.P.);
monika.wujec@umlub.pl (M.W.)
Department of Pharmaceutical Microbiology with Laboratory for Microbiological Diagnostics,
2
Faculty of Pharmacy with Medical Analytics Division, Medical University of Lublin, 1 Chod z´ ki,
Lublin 20-093, Poland; urszula.kosikowska@umlub.pl (U.K.); anna.malm@umlub.pl (A.M.)
Department of Pharmacology, Faculty of Health Sciences, Medical University of Lublin, 4A Chod z´ ki,
3
Lublin 20-093, Poland; tomasz.plech@umlub.pl
*
Correspondence: nazar.trotsko@umlub.pl; Tel.: +48-81-448-7243
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀ
ꢁꢂꢃꢄꢅꢆ
Received: 28 March 2018; Accepted: 24 April 2018; Published: 26 April 2018
Abstract: Series of new thiazolidine-2,4-dione-based chlorophenylthiosemicarbazone hybrids (17
were synthesized by the reaction of condensation chlorophenylthiosemicarbazides with formylphenyl
-(2,4-dioxothiazolidin-5-yl/ylidene)acetates. New compounds were tested on reference strains
–40)
2
of Gram-positive and Gram-negative bacteria. The antibacterial activity of target compounds
was determined by broth dilution method. Most active compounds possess minimum inhibitory
concentration (MIC) = 3.91 mg/L. These compounds were non-toxic at concentrations close to
their antibacterial effect. The antibacterial activity of some compounds was similar to or higher
than the activity of used reference drugs such as oxacillin and cefuroxime. The structure–activity
relationships (SARs) analysis collectively suggests that at least two different molecular mechanisms
of their antibacterial activity should be expected.
Keywords: thiazolidine-2,4-dione; chlorophenylthiosemicarbazones; antibacterial activity
1
. Introduction
The appearance of drug-resistant strains of pathogenic microorganisms is a serious medical
problem in worldwide. There is an urgent need to discover novel effective antimicrobial agents.
Discovery of new compounds with antimicrobial activity or finding new mechanisms of action for
known compounds is an important task of medicinal chemistry [1].
The 4-thiazolidinone derivatives and their narrow group—thiazolidine-2,4-diones (TZDs)—are
the object of special scientific studies. This is confirmed by numerous reviews [2–6]. This attention
is due not only to the wide possibilities for chemical modification of these derivatives, but also
to a diverse spectrum of pharmacological properties and affinity for various biological targets.
Consequently, TZD derivatives are the object of great interest as sources of novel drug candidates
with anti-inflammatory [7,8], antimicrobial [9–11], antidiabetic [12,13], and anticancer [14–16] effects.
Design of new compounds based on preferential scaffolds is one of the favourable directions in
drug discovery.
As we previously reported, substances with an N-N-C(=S)-N structural fragment exhibit
antimicrobial activity at low non-toxic concentrations. This activity is similar to or better than
Molecules 2018, 23, 1023; doi:10.3390/molecules23051023
www.mdpi.com/journal/molecules

Molecules 2018, 23, 1023
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reference substances: cefuroxime, ampicillin, and vancomycin [17
–
21]. Combination of these two
mentioned scaffolds in one molecule seems to be a promising “hybrid pharmacophore” approach to
new antibacterial agents.
In the present research, we used a fragment of 4-(chlorophenyl)thiosemicarbazone as a structural
motif containing N-N-C(=S)-N. This fragment was connected with TZD derivatives in five positions of
a heterocyclic ring, leading to new TZD-chlorophenylthiosemicarbazone hybrids. New compounds
were studied as antibacterial agents.
2
. Results and Discussion
2
.1. Rationale
Our research group was focused on developing new antibacterial agents composed of three-core
TZD-based scaffold (Figure 1). Previously reported compounds [22] were characterized by two
nitrogenous five-membered heterocyclic rings connected by alkyl–phenyl–ester linker. The most
potent of them had minimum inhibitory concentrations (MICs) against Gram-positive strains in the
range of 3.91 mg/L to 15.63 mg/L, thereby indicating in some cases more effective action than those
standard drug cefuroxime and ampicillin. Close inspection of the structure–activity relationships
(
SARs) revealed no direct effect of terminal TZD, rhodanine or 2-thiohydantoin core on the antibacterial
response. Hence, we decided to modify initial structures by replacing the azole rings with the
chlorophenylthiosemicarbazone fragment. Selection of such substituent was dictated by two factors;
as we reported previously, compounds with N-N-C(=S)-N structural motif have the potential to display
antibacterial activity at low, non-toxic concentration, similar to or even better than those for standard
antibiotics cefuroxime, ampicillin or vancomycin [17–21]. Among them, the best antibacterial response
was noted for those with the chlorophenyl substitution (Figure 1) [17]; the most active compounds had
MICs as low as 0.49 mg/L (against Micrococcus luteus and Bacillus subtilis), making them promising
starting structures for the development of new antibacterials. Thus, in this article, the active moieties
for previously reported TZDs and those for thiosemicarbazides were combined together in one scaffold
with the hope to obtain potent and non-toxic antibacterial agents (Figure 1).
Figure 1. Design of novel three-core thiazolidine-2,4-diones (TZD)-based compounds with
chlorophenyl substitution.

Molecules 2018, 23, 1023
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2
.2. Chemistry
TZD-chlorophenylthiosemicarbazone hybrid compounds were obtained in a three-step procedure
starting with appropriate (2,4-dioxothiazolidin-5-yl/ylidene)acetic acids ( ). Compounds
and were synthesized by the procedure described earlier [23]. The acids ( ) by
the reaction with thionyl chloride in anhydrous 1,4-dioxane medium were transformed into
acid chlorides ( ). Then (2,4-dioxothiazolidin-5-yl/ylidene)acetic acid chlorides (3, 4)
1, 2
1
2
1, 2
3,
4
in the presence of pyridine were reacted with different hydroxybenzaldehydes (namely:
salicylaldehyde, 3-hydroxybenzaldehyde, 4-hydroxybenzaldehyde and vanillin) to produce
corresponding formylphenyl (2,4-dioxothiazolidin-5-yl/ylidene) acetates (5–11). The pathway of
the reaction is illustrated in Scheme 1.
Scheme 1. Synthesis of formylphenyl (2,4-dioxothiazolidin-5-yl/ylidene)acetates. Reagents and
conditions: (a) pyridine, 1,4-dioxane, rt, after two hours acidified of solution of hydrochloric acid;
yields 75–85%.
The condensation of formylphenyl (2,4-dioxothiazolidin-5-yl/ylidene) acetates (
-(chlorophenyl)-3-thiosemicarbazides (12 16) in anhydrous ethanol medium in the presence of
catalytic amounts of acetic acid led to obtaining corresponding chlorophenylthiosemicarbazones
17–40). The route of reaction is presented in Scheme 2.
5–11) with
4
–
(

Molecules 2018, 23, 1023
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Scheme 2. Synthesis of chlorophenylthiosemicarbazones (17–40). Reagent and conditions: (a) ethanol,
reflux for 15 min; yields 62–92%.
1
The structure of target compounds (17
–
40) was confirmed by elemental analysis, H NMR and
13
1
C NMR spectra. In H NMR spectra generally, the protons of CH=N group of all new synthesized
compounds show singlet signal at
two singlets in the range of 10.08–10.23 ppm and 11.89–12.11 ppm correspondingly. The protons of
NH group of thiazolidine ring appeared in the 12.12–12.19 ppm region for compounds (17 29) and the
δ ~8.15–8.31 ppm. The protons of NHCSNH group resonated as
–
1
2.91–13.04 ppm region for compounds as a singlet (30–40).
The presence of all carbon atoms for compounds (17
–
40) is confirmed by 13C NMR spectra.
The signal of C=S group of thiosemicarbazone fragment appeared in the 176.3–177.8 ppm region.
For the compounds 17 29, which are derivatives of 2-(2,4-dioxothiazolidin-5-yl)acetic acid, carbon
–
signal of two C=O group of thiazolidine ring appeared in the 166.4–172.7 ppm and 169.4–176.0 ppm
regions. Signals of two C=O group of thiazolidine ring for the 2-(2,4-dioxothiazolidin-5-ylilidene)acetic
acid derivatives 30
–
40 were visible at
δ
~166.3–166.5 ppm and 169.2–169.4 ppm ranges correspondingly.
1
13
The detailed results of H NMR and C NMR spectra are presented in Section 3.

Molecules 2018, 23, 1023
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2
.3. Antibacterial Screening
Initially, two series of TZD-chlorophenylthiosemicarbazones (17
–
29 and 30
–
40) were synthesized.
The antibacterial results for MICs are reported in Table 1. The ciprofloxacin, cefuroxime and
oxacillin were included as a control. The data from the assay showed that none of tested
compounds had an inhibitory effect on the growth of Gram-negative bacteria examined up to
a concentration of 1000 mg/L. In contrast, with the exception of 17
compounds possessed good to limited antibacterial activity. Among them, the best antibacterial
response was observed for 38; followed by 20 23; moderately active 21 26 33 34; and almost
inactive 35 39 and 40 From these results, together with those for three derivatives of the
most potent 38—compounds 41 (4-[(2,5-dioxoimidazolidin-4-ylidene)methyl]-2-methoxyphenyl
2,4-dioxo-1,3-thiazolidin-5-ylidene)acetate), 42 (4-[(2,5-dioxoimidazolidin-4-ylidene)methyl]
2-methoxyphenyl (2,4-dioxo-1,3-thiazolidin-5-yl)acetate) and 43 (2-ethoxy-4-[(5-oxo-
-thioxoimidazolidin-4-ylidene) methyl]phenyl (2,4-dioxo-1,3-thiazolidin-5-ylidene)acetate)—the
, 18, 25, 27, 28, 32, 37, all tested
,
,
,
,
,
.
(
-
2
following comments can be made: (i) since tested compounds varied widely in antibacterial activity,
the chlorophenylthiosemicarbazone segment is not an optimized moiety for their activity; (ii) SARs are
rather complex and cannot be easily explained. For example, compound 38 was a potent antibacterial
agent, with even more effective action than those standard drug cefuroxime against B. subtilis and
Bacillus cereus. The replacement of the chlorophenylthiosemicarbazone core with the hydantoin 41
abolished activity. In turn, the reduction of double bond in 41 furnished compound 42 with weak
inhibitory activity against all bacterial strains while increasing the carbon chain from methyl to
ethyl, providing compound 43 with mild activity. Further, slight change in geometry and electronic
properties of 38 (38 → 37) also abolished activity, while the reduction of 37 (37 → 25) does not result
in an active compound as was observed for 41 → 42.

Molecules 2018, 23, 1023
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Table 1. The activity of chlorophenylthiosemicarbazones (17
–40) against Gram-positive bacteria determined on the basis of MIC (minimal inhibitory concentration, in
mg/L and µM) and MBC (minimal bactericidal concentration, in mg/L and µM).
S. aureus
S. aureus
S. epidermidis
B. subtilis
B. cereus
M. luteus
Compound
ATCC 6538
ATCC 25923
ATCC 12228
ATCC 6633
ATCC 10876
ATCC 10240
MIC
25
51.3
.91
MBC
MIC
MBC
>1000
>2010.6
62.5
MIC
62.5
MBC
>1000
>2010.6
62.5
MIC
MBC
500
MIC
MBC
>1000
>2010.6
125
MIC
MBC
1000
1
>1000
>2010.6
3.91
62.5
125.7
3.91
8.4
62.5
125.7
31.25
67.5
62.5
125.7
3.91
8.4
62.5
125.7
7.81
16.9
15.63
33.8
3.91
8.4
1
9
0
2
125.7
31.25
67.5
1005.3
125
2010.6
125
3
2
8
.4
8.4
135
135
270
270
270
3
1
1.25
>1000
>2160.2
1000
7.81
16.9
31.25
67.5
3.91
7.9
1000
31.25
67.5
>1000
>2160.2
1000
62.5
250
31.25
67.5
7.81
16.9
7.81
15.7
62.5
125.7
62.5
126.8
15.63
29.6
3.91
8.5
250
15.63
33.8
2
1
6
7.5
2160.2
1000
135
540
540
5.63
15.63
33.8
31.25
67.5
>1000
>2160.2
62.5
>1000
>2160.2
>1000
>2010.6
>1000
>2010.6
>1000
>2028.6
1000
500
2
2
2
2
2
3
2
3
4
6
9
0
3
3.8
.91
2160.2
>1000
>2010.6
>1000
>2010.6
>1000
>2028.6
>1000
>1896.1
250
2160.2
>1000
>2010.6
>1000
>2010.6
>1000
>2028.6
>1000
>1896.1
250
2160.2
1000
1080.1
7.81
3
7.81
7.81
7.81
15.7
62.5
125.7
3.91
7.9
7
.9
15.7
2010.6
>1000
>2010.6
>1000
>2028.6
>1000
>1896.1
250
15.7
125.7
250
15.7
1
25
62.5
125.7
15.63
31.7
62.5
118.5
7.81
16.9
7.81
16.9
15.63
33.9
7.81
16.9
1000
2169.6
31.25
62.8
125
>1000
>2010.6
1000
2
1
51.3
5.63
251.3
15.63
31.7
502.6
500
31.25
63.4
3
1.7
1014.3
500
2028.6
1000
1
25
37
31.25
59.3
31.25
59.3
7.81
14.8
7.81
16.9
7.81
16.9
62.5
135.6
62.5
135.6
1000
2169.6
2
948
1896.1
500
1896.1
250
7
.81
6.9
.81
6.9
7.81
7.81
250
1
7
1
542.4
542.4
500
16.9
542.4
500
16.9
542.4
500
1084.8
500
542.4
1000
500
7.81
7.81
3.91
8.5
3
1
1084.8
1000
1084.8
250
16.9
1084.8
500
16.9
1084.8
500
1084.8
>1000
>2169.6
1000
2169.6
>1000
>2169.6
500
1
5.63
31.25
67.8
15.63
33.9
62.5
135.6
7.81
16.9
1000
2169.6
3
3
3
3
3
4
5
6
3
3.9
2169.6
500
542.4
500
1084.8
500
1084.8
500
3
1.25
15.63
33.9
15.63
33.9
6
7.8
00
084.8
25
52.3
1084.8
1000
1084.8
500
1084.8
500
1084.8
500
2169.6
1000
1084.8
1000
5
500
500
1
2169.6
1084.8
1084.8
1084.8
1084.8
1084.8
2169.6
2169.6
1
2
500
1009.4
7.81
15.8
125
252.3
7.81
15.8
62.5
126.2
250
504.7
1000
2018.7
7.81
15.8
1000
2018.7
7.81
15.8
250
504.7

Molecules 2018, 23, 1023
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Table 1. Cont.
S. aureus
S. aureus
S. epidermidis
B. subtilis
B. cereus
M. luteus
Compound
ATCC 6538
ATCC 25923
ATCC 12228
ATCC 6633
ATCC 10876
ATCC 10240
3
1.25
3.7
>1000
>2036.9
1000
7.81
>1000
>2036.9
1000
7.81
15.9
500
250
509.2
1000
3.91
62.5
127.3
500
3.91
62.5
3.91
125
3
3
4
8
9
0
6
15.9
250
8
8
127.3
1000
8
254.6
1000
2
50
500
1000
2036.9
1000
1903.4
31.25
73.6
1000
2036.9
1000
1903.4
0.98
2.3
5
09.2
2036.9
1000
509.2
250
2036.9
1000
1018.5
250
2036.9
1000
1018.5
500
1018.5
1000
2036.9
1000
2036.9
1000
5
00
51.7
.98
9
1903.4
475.8
0.49
1.2
1903.4
475.8
0.24
0.6
1903.4
951.7
15.63
36.8
0.015
0.05
0.06
0.15
1903.4
1903.4
1903.4
0
-
-
-
-
-
-
-
-
Cefuroxime
2
.3
0
.49
0.49
1.5
0.49
1.5
0.49
1.5
0.12
0.4
0.12
0.12
0.4
0.98
3.0
1.95
5.9
Ciprofloxacin
-
1
.5
0.4
0
0
.06
.15
0.06
0.15
0.12
0.3
0.12
0.3
0.12
0.3
62.5
62.5
155.7
0.98
2.4
0.98
2.4
-
Oxacillin
155.7
-
not determined, compounds 17, 18, 25, 27, 28, 32 and 37—not active.

Molecules 2018, 23, 1023
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With this information in hand, we started exploring the proper substitution pattern on both
terminal and central phenyl rings by initially adding the chloro substituent to potent antibacterial 20.
The chloro substitution at ortho position provided compound 24 with significantly lower activity; with
the exception of S. epidermidis, all Gram-positive bacterial strains were able to grow at much higher
concentrations as compared to 20 or were even almost insensitive. Low or even a lack of antibacterial
activity was also noted for its structural isomers 19 and 18, respectively. Important to note, compounds
18, 19 and 24 with negligible activity are all structural isomers of 23; one of the most potent among
all tested compounds. In turn, substitution of weakly active 24 with the methoxy group afforded
compound 29 with quite potent activity against M. luteus and B. cereus, whereas such modification in
3
3
(
33 → 37) abolished activity. Finally, the effect of isomerization at the central phenyl ring was tested.
For this purpose, compounds 17 30 31 and 32 were synthesized. Consistent with previous studies,
,
,
compounds with meta substitution 17 and 32 were inactive. Compounds with ortho substitution 30
and 31, however, showed an activity similar to or even better against S. aureus than the most potent
antibacterial 38, which in turn is in line with the trend in activity observed previously.
Summing up, the SARs analysis collectively suggest that at least two different molecular
mechanisms of antibacterial activity for TZD-based hybrid compounds are expected. Such observation,
however, is not surprising: a similar trend was observed previously for thiosemicarbazide derivatives;
one mechanism for their activity was associated with inhibition of bacterial topoisomerases, while the
nature of the other is still unrecognized [18,19,21,24,25].
2
.4. Bactericidal and Bacteriostatic Activity
Antibacterial studies were completed by the evaluation of MBCs (minimum bactericidal
concentrations) that allowed to determine the bactericidal or bacteriostatic mechanism of action.
On the basis of the MBC/MIC ratio, all compounds were mainly bacteriostatic in effect (MBC/MIC
≥
8), with the exception of potent 20 that exhibited bactericidal activity (MBC/MIC
the Gram-positive bacterial strains tested, further highlighting the concept of at least two molecular
targets for antibacterial activity of title compounds.
≤
4) to half of
2
.5. Computational Studies
Finally, efforts were undertaken to correlate antibacterial activity with physicochemical properties,
such as geometry of molecule, lipophilicity, refractivity, polarizability, surface area, volume, dipole
moment, hardness, Mulliken electronegativity, total energy, EHOMO, ELUMO, HOMO–LUMO gaps, ESP
charges, core–core interaction, heat of formation, electrostatic potential surfaces, electronic distribution
of the frontier orbitals HOMO and LUMO orbitals. As we assumed, analysis of the SARs did not
provide valuable information about the observed trend in bioactivity; the results showed that varying
the substitution pattern of the chloro subunit or desaturation process leads to relatively small changes in
geometry or physicochemical characterization but causes significant changes in antibacterial response
(data not shown).
2
.6. Cytotoxicity Studies
After confirming compounds 20, 23, 30, 31 and 38 as potent antibacterial agents, we moved
to examine their toxicity profile against human embryonic kidney cells (HEK-293). Compound 20
exhibited the best toxicity profile as it was non-toxic to HEK-293 cells to a concentration of 30.82 mg/L
(Table 2); this represents an eight-fold difference between the MICs and the concentration where its
significant toxicity is observed.

Molecules 2018, 23, 1023
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Table 2. Cytotoxic effect of compounds 20, 23, 30, 31 and 38 on human embryonic kidney cells
(HEK-293) after 24 h of incubation.
Compound
EC70 ± SD (mg/L) (Toxicity Threshold)
20
23
30
31
38
30.82 ± 3.73
21.28 ± 3.84
14.86 ± 1.12
10.94 ± 1.35
19.01 ± 3.80
Cytotoxic effect of the compounds was measured using MTT assay. Data are expressed as
mean SD from three independent experiments. Summing up, all compounds were non-toxic at
concentrations close to their antibacterial effect.
±
3
. Materials and Methods
3
.1. Chemistry
Melting points were determined by using Fisher–Johns apparatus (Fisher Scientific, Schwerte,
Germany) and are uncorrected. The purity of the compounds was checked by TLC on plates with
1
13
silica gel Si 60 F254, produced by Merck Co. (Darmstadt, Germany). The H NMR and C NMR
spectra were recorded by a Bruker Avance 300 MHz instrument using DMSO-d as solvent and TMS as
6
an internal standard. Chemical shifts were expressed as
δ (ppm). Elemental analyses were performed
by AMZ 851 CHX analyser and the results were within ±0.4% of the theoretical value.
3
0
.1.1. Synthesis of Chlorophenylthiosemicarbazones (17–40)
To the mixture of 0.001 mol formylphenyl (2,4-dioxothiazolidin-5-yl/ylidene)acetate (5–11) and
.001 mol of the appropriate 4-(chlorophenyl)-thiosemicarbazides (12–16) was added 5 mL of ethanol.
The reaction mixture was heated under reflux for 15 min. After cooling, the precipitate was filtered
off and washed with ethanol. After drying, precipitate was recrystallized from appropriate solvents.
The compounds (17
–
24
,
26,
27
,
30, 31, 33, 35–40) were crystallized from butanol, compounds 25, 29,
from propanol; compounds 28
,
34, from acetic acid; and compound 32, from mixture of DMF:H O
2
(2:1).
3-[{2-[(4-Chlorophenyl)carbamothioyl]hydrazinylidene}methyl]phenyl
(2,4-dioxo-1,3-thiazolidin-5-yl)acetate (17).
◦
1
Yield 80%, mp 210–212 C. H NMR (DMSO-d )
δ
(ppm): 3.42–3.45 m (2H, CH-CH2); 4.85 dd (1H,
6
C
H-CH , J = 6.8, 5.2 Hz); 7.05 s, 7.28–7.31 m, 7.41–7.61 m, 7.73–7.77 m, 7.92 t (8H, 3-O-C H and
2
6
4
4
-Cl-C H , J = 1.8 Hz); 8.16 s (1H, CH=N); 10.20 s, 11.97 s (2H, NHCSNH); 12.16 s (1H, NH, thiazolidine).
6 4
1
3
C NMR (DMSO-d6)
δ (ppm): 36.4; 46.8; 116.3; 120.1; 123.7; 128.3; 128.4; 130.0; 130.5; 136.3; 138.5;
1
42.5; 151.0; 169.6; 172.7; 175.9; 176.7. Anal. calc. for C H ClN O S (%): C 49.30; H 3.27; N 12.10.
19
15
4
4 2
Found: C 49.17; H 3.19; N 12.09.
3-[{2-[(2,4-Dichlorophenyl)carbamothioyl]hydrazinylidene}methyl]phenyl
(2,4-dioxo-1,3-thiazolidin-5-yl)acetate (18)
◦
1
Yield 83%, mp 224–225 C. H NMR (DMSO-d )
δ
(ppm): 3.42–3.44 m (2H, CH-CH2); 4.85 dd (1H,
6
C
H-CH , J = 6.8, 5.3 Hz); 7.06 s, 7.27–7.30 m, 7.45–7.62 m, 7.71–7.73 m, 7.90 t (7H, 3-O-C H and
2
6
4
2
,4-diCl-C H , J = 1.8 Hz); 8.17 s (1H, CH=N); 10.14 s, 12.00 s (2H, NHCSNH); 12.12 s (1H, NH,
6 3
13
thiazolidine). C NMR (DMSO-d6)
δ (ppm): 36.3; 46.6; 116.3; 120.0; 123.8; 127.9; 129.3; 130.5; 132.1;
1
32.4; 133.1; 136.3; 136.5; 142.4; 150.9; 164.4; 166.8; 169.5; 177.6. Anal. calc. for C H Cl N O S (%):
19
14
2
4
4 2
C 45.88; H 2.84; N 11.26. Found: C 45.83; H 2.85; N 11.18.

Molecules 2018, 23, 1023
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3-[{2-[(2,6-Dichlorophenyl)carbamothioyl]hydrazinylidene}methyl]phenyl
(2,4-dioxo-1,3-thiazolidin-5-yl)acetate (19)
◦
1
Yield 81%, mp 203–204 C. H NMR (DMSO-d )
δ
(ppm): 3.41–3.44 m (2H, CH-CH2); 4.85 dd (1H,
6
CH-CH , J = 6.7, 5.4 Hz); 7.06 s, 7.28–7.31 m, 7.35–7.41 m, 7.49–7.57 m, 7.72 d, 7.94 t (7H, 3-O-C H
2 6 4
and 2,6-diCl-C H , J = 7.5, 1.8 Hz); 8.16 s (1H, CH=N); 10.16 s, 12.08 s (2H, NHCSNH); 12.10 s (1H,
6
3
13
NH, thiazolidine). C NMR (DMSO-d6)
δ (ppm): 35.2; 46.8; 116.4; 119.8; 123.7; 128.8; 129.9; 130.5;
1
35.7; 136.4; 142.1; 145.7; 150.8; 164.4; 166.6; 169.4; 177.8. Anal. calc. for C H Cl N O S (%): C 45.88;
19
14
2
4
4 2
H 2.84; N 11.26. Found: C 45.77; H 2.80; N 11.29.
-[{2-[(2-Chlorophenyl)carbamothioyl]hydrazinylidene}methyl]phenyl
2,4-dioxo-1,3-thiazolidin-5-yl)acetate (20)
4
(
◦
1
Yield 82%, mp 190–192 C. H NMR (DMSO-d )
δ
(ppm): 3.42–3.45 m (2H, CH-CH2); 4.86 dd (1H,
6
C
H-CH , J = 7.0, 5.2 Hz); 7.21 d, 7.96 d (4H, 4-O-C H , J = 8.7 Hz); 7.32 td, 7.39 td, 7.56 dd 7.67 dd
2
6
4
(
4H, 2-Cl-C H , J = 7.8, 1.6 Hz); 8.16 s (1H, CH=N); 10.13 s, 12.02 s (2H, NHCSNH); 12.17 s (1H, NH,
6
4
1
3
thiazolidine). C NMR (DMSO-d6)
δ (ppm): 36.4; 46.8; 122.5; 127.7; 128.4; 129.3; 129.8; 130.6; 131.4;
1
32.5; 137.1; 142.4; 151.8; 169.5; 172.7; 176.0; 177.3. Anal. calc. for C H ClN O S (%): C 49.30; H 3.27;
19 15 4 4 2
N 12.10. Found: C 48.98; H 3.19; N 12.11.
-[{2-[(3-Chlorophenyl)carbamothioyl]hydrazinylidene}methyl]phenyl
2,4-dioxo-1,3-thiazolidin-5-yl)acetate (21)
4
(
◦
1
Yield 83%, mp 193–194 C. H NMR (DMSO-d )
δ
(ppm): 3.42–3.45 m (2H, CH-CH2); 4.86 dd (1H,
6
C
H-CH , J = 6.8, 5.3 Hz); 7.21 d, 8.01 d (4H, 4-O-C H , J = 8.7 Hz); 7.25–7.43 m, 7.57–7.63 m, 7.74–7.77
2
6
4
m (4H, 3-Cl-C H ); 8.17 s (1H, CH=N); 10.20 s, 11.98 s (2H, NHCSNH); 12.16 s (1H, NH, thiazolidine).
6
4
13
C NMR (DMSO-d6)
δ (ppm): 36.5; 46.8; 122.4; 124.8; 125.5; 125.8; 129.5; 130.1; 132.4; 132.6; 141.0;
1
42.8; 151.8; 169.4; 172.7; 176.0; 176.3. Anal. calc. for C H ClN O S (%): C 49.30; H 3.27; N 12.10.
19
15
4
4 2
Found: C 49.03; H 3.21; N 12.09.
4-[{2-[(4-Chlorophenyl)carbamothioyl]hydrazinylidene}methyl]phenyl
(2,4-dioxo-1,3-thiazolidin-5-yl)acetate (22)
◦
1
Yield 85%, mp 186–188 C. H NMR (DMSO-d )
δ (ppm): 3.42–3.45 m (2H, CH-CH2); 4.86 dd (1H,
6
C
H
-CH , J = 7.0, 5.2 Hz); 7.21 d, 7.98 d (4H, 4-O-C H , J = 8.7 Hz); 7.43 d, 7.61 d (4H, 4-Cl-C H ,
2
6
4
6
1
4
3
J = 8.8 Hz); 8.16 s (1H, CH=N); 10.17 s, 11.94 s (2H, NHCSNH); 12.15 s (1H, NH, thiazolidine).
NMR (DMSO-d6)
72.7; 176.0; 176.6. Anal. calc. for C H ClN O S (%): C 49.30; H 3.27; N 12.10. Found: C 49.35;
C
δ
(ppm): 36.5; 46.9; 122.5; 128.1; 128.4; 129.4; 129.9; 132.5; 138.6; 142.6; 151.8; 169.5;
1
19
15
4
4 2
H 3.25; N 12.00.
-[{2-[(2,4-Dichlorophenyl)carbamothioyl]hydrazinylidene}methyl]phenyl
2,4-dioxo-1,3-thiazolidin-5-yl)acetate (23)
4
(
◦
1
Yield 83%, mp 200–202 C. H NMR (DMSO-d )
δ
(ppm): 3.42–3.45 m (2H, CH-CH2); 4.86 dd (1H,
6
C
H-CH , J = 7.0, 5.2 Hz); 7.21 d, 7.95 d (4H, 4-O-C H , J=8.7 Hz); 7.47 dd, 7.67 d, 7.73 d (3H,
2
6
4
2
,4-diCl-C H , J = 8.6, 2.4 Hz); 8.16 s (1H, CH=N); 10.11 s, 12.08 s (2H, NHCSNH); 12.16 s (1H,
6 3
13
NH, thiazolidine). C NMR (DMSO-d6)
δ (ppm): 36.5; 46.9; 122.5; 127.8; 129.3; 129.4; 131.9; 132.0;
1
32.4; 132.7; 136.4; 142.7; 151.9; 169.4; 172.7; 175.9; 177.4. Anal. calc. for C H Cl N O S (%): C 45.88;
19
14
2
4
4 2
H 2.84; N 11.26. Found: C 45.67; H 2.80; N 11.23.
-[{2-[(2,6-Dichlorophenyl)carbamothioyl]hydrazinylidene}methyl]phenyl
2,4-dioxo-1,3-thiazolidin-5-yl)acetate (24)
4
(
◦
1
Yield 89%, mp 188–190 C. H NMR (DMSO-d )
δ
(ppm): 3.42–3.45 m (2H, CH-CH2); 4.86 dd (1H,
6
C
=
H-CH , J = 6.9, 5.3 Hz); 7.21 d, 7.98 d (4H, 4-O-C H , J = 8.7 Hz); 7.38 dd, 7.56 d (3H, 2,6-diCl-C H , J
2
6
4
6
3
1
3
8.6, 7.7 Hz); 8.15 s (1H, CH=N); 10.13 s, 12.03 s (2H, NHCSNH); 12.16 s (1H, NH, thiazolidine).
C

Molecules 2018, 23, 1023
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NMR (DMSO-d6)
δ
(ppm): 36.4; 46.8; 122.4; 128.8; 129.3; 129.9; 132.5; 135.6; 135.7; 142.3; 151.7; 169.4;
1
72.7; 175.9; 177.8. Anal. calc. for C H Cl N O S (%): C 45.88; H 2.84; N 11.26. Found: C 45.89; H
19 14 2 4 4 2
2
.77; N 11.18.
4-[{2-[(2-Chlorophenyl)carbamothioyl]hydrazinylidene}methyl]-2-methoxyphenyl
(2,4-dioxo-1,3-thiazolidin-5-yl)acetate (25)
◦
1
Yield 82%, mp 204–205 C. H NMR (DMSO-d )
δ
(ppm): 3.40–3.42 m (2H, CH-CH2); 3.83 s (3H,
6
OCH ); 4.84 dd (1H, C
H-CH , J = 6.7, 5.5 Hz); 7.16 d, 7.56 dd, 7.66 d (3H, 4-O-C H , J = 7.8, 1.7 Hz);
3
2
6
3
7
.29–7.43 m, 7.74 dd (4H, 2-Cl-C H , J = 7.8, 1.7 Hz); 8.14 s (1H, CH=N); 10.10 s, 12.06 s (2H, NHCSNH);
6 4
2.13 s (1H, NH, thiazolidine). 1 C NMR (DMSO-d6)
3
δ (ppm): 36.1; 46.9; 56.6; 111.0; 121.5; 123.4; 127.7;
1
1
28.4; 129.8; 130.4; 131.3; 133.6; 137.0; 140.9; 142.6; 151.5; 168.8; 172.7; 175.8; 177.1. Anal. calc. for
C H ClN O S (%): C 48.73; H 3.48; N 11.37. Found: C 48.67; H 3.50; N 11.33.
20
17
4
5 2
4-[{2-[(3-Chlorophenyl)carbamothioyl]hydrazinylidene}methyl]-2-methoxyphenyl
(2,4-dioxo-1,3-thiazolidin-5-yl)acetate (26)
◦
1
Yield 62%, mp 156–158 C. H NMR (DMSO-d )
δ
(ppm): 3.40–3.42 m (2H, CH-CH2); 3.85 s (3H,
6
OCH ); 4.84 dd (1H, C
H-CH , J = 6.6, 5.6 Hz); 7.17 d, 7.48 dd, 7.65 d (3H, 4-O-C H , J = 8.2, 1.8 Hz);
3
2
6
3
7
.26–7.30 m, 7.40 t, 7.56–7.59 m, 7.76 t (4H, 3-Cl-C H , J = 8.0, 2.0 Hz); 8.15 s (1H, CH=N); 10.18 s, 12.03
6 4
13
s (2H, NHCSNH); 12.13 s (1H, NH, thiazolidine). C NMR (DMSO-d6)
21.3; 123.4; 125.1; 125.7; 126.0; 130.1; 132.7; 133.6; 140.9; 141.0; 143.1; 151.4; 168.8; 172.8; 176.0; 176.4.
Anal. calc. for C H ClN O S (%): C 48.73; H 3.48; N 11.37. Found: C 48.70; H 3.43; N 11.38.
δ (ppm): 36.1; 47.0; 56.7; 111.7;
1
20
17
4
5 2
4-[{2-[(4-Chlorophenyl)carbamothioyl]hydrazinylidene}methyl]-2-methoxyphenyl
(2,4-dioxo-1,3-thiazolidin-5-yl)acetate (27)
◦
1
Yield 90%, mp 200-203 C. H NMR (DMSO-d ) δ (ppm): 3.40–3.42 m (2H, CH-CH2); 3.85 s (3H, OCH3);
6
4
.84 dd (1H, C
J = 8.2 Hz); 8.15 s (1H, CH=N); 10.16 s, 11.98 s (2H, NHCSNH); 12.14 s (1H, NH, thiazolidine).
NMR (DMSO-d6)
42.9; 151.4; 168.8; 172.7; 175.8; 176.6. Anal. calc. for C H ClN O S (%): C 48.73; H 3.48; N 11.37.
H
-CH , J = 6.6, 5.5 Hz); 7.17 d, 7.42–7.49 m, 7.60–7.66 m (7H, 4-O-C H & 4-Cl-C H ,
2
6
3
6
1
4
3
C
δ
(ppm): 36.1; 46.9; 56.7; 111.6; 121.3; 123.4; 128.3; 128.5; 130.0; 133.6; 138.5; 140.9;
1
20
17
4
5 2
Found: C 48.62; H 3.44; N 11.29.
-[{2-[(2,4-Dichlorophenyl)carbamothioyl]hydrazinylidene}methyl]-2-methoxyphenyl
2,4-dioxo-1,3-thiazolidin-5-yl)acetate (28)
4
(
◦
1
Yield 92%, mp 196–198 C. H NMR (DMSO-d )
δ
(ppm): 3.40–3.43 m (2H, CH-CH2); 3.84 s (3H,
6
OCH ); 4.84 dd (1H, C
H-CH , J = 6.6, 5.4 Hz); 7.17 d, 7.43 dd, 7.64 d (3H, 4-O-C H , J = 8.2, 1.8 Hz);
3
2
6
3
7
.48 dd, 7.72–7.75 m (3H, 2,4-diCl-C H , J = 8.6, 2.4 Hz); 8.14 s (1H, CH=N); 10.08 s (1H, NHCSNH);
6 3
13
1
1
2.13 s (2H, NHCSNH and NH thiazolidine). C NMR (DMSO-d6)
21.4; 123.4; 127.9; 129.3; 131.8; 131.9; 132.6; 133.6; 136.3; 140.9; 142.9; 151.5; 168.8; 172.7; 175.8; 177.2.
δ (ppm): 36.1; 46.9; 56.7; 111.2;
Anal. calc. for C H Cl N O S (%): C 45.55; H 3.06; N 10.62. Found: C 45.67; H 3.08; N 10.59.
20
16
2
4
5 2
4-[{2-[(2,6-Dichlorophenyl)carbamothioyl]hydrazinylidene}methyl]-2-methoxyphenyl
(2,4-dioxo-1,3-thiazolidin-5-yl)acetate (29)
◦
1
Yield 92%, mp 206–207 C. H NMR (DMSO-d )
δ
(ppm): 3.41 d (2H, CH-CH2, J = 6.3 Hz); 3.84 s (3H,
6
OCH ); 4.84 t (1H, C
H-CH , J = 6.3 Hz); 7.17 d, 7.45 dd, 7.65 d (3H, 4-O-C H , J = 8.2, 1.6 Hz), 7.39 dd,
3
2
6
3
7
.57 d (3H, 2,6-diCl-C H , J = 8.7, 7.6 Hz); 8.13 s (1H, CH=N); 10.06 s, 12.07 s (2H, NHCSNH); 12.13 s
6
3
3
1
(
1H, NH, thiazolidine). C NMR (DMSO-d6)
δ
(ppm): 36.1; 46.9; 56.8; 111.4; 121.4; 123.4; 128.9; 130.0;
1
33.7; 135.6; 135.8; 140.8; 142.8; 151.5; 168.8; 172.8; 175.9; 177.7. Anal. calc. for C H Cl N O S (%): C
20
16
2
4
5 2
4
5.55; H 3.06; N 10.62. Found: C 45.57; H 3.03; N 10.61.
2-[{2-[(2-Chlorophenyl)carbamothioyl]hydrazinylidene}methyl]phenyl
(2,4-dioxo-1,3-thiazolidin-5-ylidene)acetate (30)

Molecules 2018, 23, 1023
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◦
1
Yield 81%, mp 222–223 C. H NMR (DMSO-d )
δ
(ppm): 7.14 s (1H, CH=); 7.29–7.42 m, 7.50–7.63 m,
6
8
.34 dd (8H, 2-O-C H and 2-Cl-C H , J = 7.8, 1.5 Hz); 8.30 s (CH=N); 10.06 s, 11.89 s (2H, NHCSNH);
6 4 6 4
13
1
1
3.02 s (1H, NH, thiazolidine). C NMR (DMSO-d6)
28.6; 129.8; 130.7; 131.5; 131.7; 136.9; 137.9; 145.8; 149.2; 164.3; 166.3; 169.2; 177.3. Anal. calc. for
δ (ppm): 116.3; 123.2; 126.5; 127.3; 127.4; 127.7;
C H ClN O S (%): C 49.51; H 2.84; N 12.16. Found: C 49.56; H 2.87; N 12.11.
19
13
4
4 2
2-[{2-[(3-Chlorophenyl)carbamothioyl]hydrazinylidene}methyl]phenyl
(2,4-dioxo-1,3-thiazolidin-5-ylidene)acetate (31)
◦
1
Yield 85%, mp 220–222 C. H NMR (DMSO-d )
δ
(ppm): 7.15 s (1H, CH=); 7.25–7.44 m, 7.50–7.61 m,
6
7
.71–7.75 m, 8.38 dd (8H, 2-O-C H and 3-Cl-C H , J = 7.8, 1.4 Hz); 8.31 s (CH=N); 10.18 s, 11.85 s (2H,
6
4
6
4
13
NHCSNH); 13.04 s (1H, NH, thiazolidine). C NMR (DMSO-d6)
25.7; 126.4; 127.3; 127.6; 130.1; 131.8; 132.7; 138.2; 140.9; 145.8; 149.3; 164.3; 166.4; 169.2; 176.4. Anal.
calc. for C H ClN O S (%): C 49.51; H 2.84; N 12.16. Found: C 49.36; H 2.67; N 12.13.
δ (ppm): 116.3; 123.2; 124.7; 125.6;
1
19
13
4
4 2
3-[{2-[(2-Chlorophenyl)carbamothioyl]hydrazinylidene}methyl]phenyl
(2,4-dioxo-1,3-thiazolidin-5-ylidene)acetate (32)
◦
1
Yield 93%, mp 222–224 C. H NMR (DMSO-d )
δ
(ppm): 7.05 s (1H, CH=); 7.27–7.41 m, 7.49–7.61 m,
6
7
.71 d, 7.91–7.95 m (8H, 3-O-C H and 2-Cl-C H , J = 7.8 Hz); 8.17 s (1H, CH=N); 10.16 s, 12.06 s (2H,
6
4
6
3
4
1
NHCSNH); 12.91 s (1H, NH, thiazolidine). C NMR (DMSO-d6)
28.6; 129.8; 130.5; 131.0; 131.8; 136.4; 137.1; 142.0; 145.5; 150.8; 162.8; 164.4; 166.4; 169.4; 177.6. Anal.
calc. for C H ClN O S (%): C 49.51; H 2.84; N 12.16. Found: C 49.53; H 2.81; N 12.07.
δ (ppm): 116.5; 119.9; 123.6; 127.7;
1
19
13
4
4 2
4-[{2-[(2-Chlorophenyl)carbamothioyl]hydrazinylidene}methyl]phenyl
(2,4-dioxo-1,3-thiazolidin-5-ylidene)acetate (33)
◦
1
Yield 83%, mp 224–226 C. H NMR (DMSO-d )
δ
(ppm): 7.07 s (1H, CH=); 7.31–7.42 m, 7.56 dd, 7.67
6
dd (6H, 4-O-C H and 2-Cl-C H , J = 7.8, 1.5 Hz); 7.99 d (2H, 4-O-C H , J = 8.8 Hz) 8.18 s (CH=N);
6
4
6
4
6
4
13
1
0.15 s, 12.04 s (2H, NHCSNH); 12.93 s (1H, NH, thiazolidine). C NMR (DMSO-d6)
δ (ppm): 116.7;
122.4; 127.7; 128.4; 129.3; 129.8; 130.6; 131.4; 132.8; 137.1; 142.3; 145.3; 151.6; 164.4; 166.5; 169.4; 177.3.
Anal. calc. for C H ClN O S (%): C 49.51; H 2.84; N 12.16. Found: C 49.45; H 2.77; N 12.14.
19
13
4
4 2
4-[{2-[(3-Chlorophenyl)carbamothioyl]hydrazinylidene}methyl]phenyl
(2,4-dioxo-1,3-thiazolidin-5-ylidene)acetate (34)
◦
1
Yield 84%, mp 214–216 C. H NMR (DMSO-d )
δ
(ppm): 7.07 s (1H, CH=); 7.25–7.43 m, 7.57–7.60
6
m, 7.73–7.77 m, 8.01 d (8H, 3-Cl-C H and 4-O-C H , J = 8.7 Hz); 8.18 s (1H, CH=N); 10.22 s, 12.00
6
4
6
1
4
3
s (2H, NHCSNH); 12.93 s (1H, NH, thiazolidine). C NMR (DMSO-d6)
25.5; 125.8; 129.5; 130.1; 132.6; 132.7; 141.0; 142.7; 145.3; 151.7; 164.2; 166.4; 169.4; 176.4. Anal. calc. for
C H ClN O S (%): C 49.51; H 2.84; N 12.16. Found: C 49.47; H 2.74; N 12.04.
δ (ppm): 116.8; 122.4; 124.8;
1
19
13
4
4 2
4-[{2-[(4-Chlorophenyl)carbamothioyl]hydrazinylidene}methyl]phenyl
(2,4-dioxo-1,3-thiazolidin-5-ylidene)acetate (35)
◦
1
Yield 86%, mp 214–216 C. H NMR (DMSO-d ) δ (ppm): 7.06 s (1H, CH=); 7.32 d, 8.01 d (4H, 4-O-C H ,
6 4
6
J = 8.7 Hz); 7.43 d, 7.61 d (4H, 4-Cl-C H , J = 8.7 Hz); 8.18 s (CH=N); 10.20 s, 11.97 s (2H, NHCSNH);
6
4
1
1
2.97 s (1H, NH, thiazolidine). 13C NMR (DMSO-d6)
32.7; 138.5; 142.5; 145.3; 151.6; 164.2; 166.4; 169.4; 176.5. Anal. calc. for C H ClN O S (%): C 49.51;
δ (ppm): 116.8; 122.3; 128.1; 128.4; 129.5; 129.9;
19
13
4
4 2
H 2.84; N 12.16. Found: C 49.41; H 2.64; N 12.02.
4-[{2-[(2,4-Dichlorophenyl)carbamothioyl]hydrazinylidene}methyl]phenyl
(2,4-dioxo-1,3-thiazolidin-5-ylidene)acetate (36)
◦
1
Yield 79%, mp 230–232 C. H NMR (DMSO-d ) δ (ppm): 7.07 s (1H, CH=); 7.32 d, 7.98 d (4H, 4-O-C H ,
6 4
6
J = 8.7 Hz); 7.47 dd, 7.64-7.70 m, 7.73 d (3H, 2,4-diCl-C H , J = 8.6, 2.4 Hz); 8.17 s (CH=N); 10.13 s, 12.09
6
3
s (2H, NHCSNH); 12.98 s (1H, NH, thiazolidine). 13C NMR (DMSO-d6)
δ (ppm): 116.8; 122.4; 127.8;

Molecules 2018, 23, 1023
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1
29.3; 129.4; 131.9; 132.0; 132.7; 132.8; 136.4; 142.6; 145.3; 151.6; 164.2; 166.4; 169.4; 177.4. Anal. calc. for
C H Cl N O S (%): C 46.07; H 2.44; N 11.31. Found: C 46.09; H 2.44; N 11.22.
19
12
2
4
4 2
4-[{2-[(2-Chlorophenyl)carbamothioyl]hydrazinylidene}methyl]-2-methoxyphenyl
(2,4-dioxo-1,3-thiazolidin-5-ylidene)acetate (37)
◦
1
Yield 84%, mp 212–214 C. H NMR (DMSO-d )
δ (ppm): 3.84 s (3H, OCH ); 7.08 s (1H, CH=); 7.27 d,
3
6
7
.56 dd, 7.69 d (3H, 4-O-C H , J = 8.1, 1.7 Hz); 7.32–7.47 m, 7.71–7.78 m (4H, 2-Cl-C H ); 8.16 s (CH=N);
6 3 6 4
13
10.11 s, 12.07 s (2H, NHCSNH); 12.93 s (1H, NH, thiazolidine). C NMR (DMSO-d6)
111.1; 116.0; 121.5; 123.5; 127.7; 128.4; 129.8; 130.4; 131.4; 134.0; 137.0; 140.5; 142.6; 146.0; 151.4; 163.7;
166.4; 169.3; 177.2. Anal. calc. for C H ClN O S (%): C 48.93; H 3.08; N 11.41. Found: C 46.09; H
2
δ (ppm): 56.7;
20
15
4
5 2
.44; N 11.22.
4-[{2-[(3-Chlorophenyl)carbamothioyl]hydrazinylidene}methyl]-2-methoxyphenyl
(2,4-dioxo-1,3-thiazolidin-5-ylidene)acetate (38)
◦
1
Yield 80%, mp 190–192 C. H NMR (DMSO-d )
δ (ppm): 3.86 s (3H, OCH ); 7.08 s (1H, CH=); 7.28 d,
3
6
7
.40 t, 7.52 dd, 7.55–7.61 m, 7.69 d, 7.75–7.79 m (7H, 4-O-C H and 3-Cl-C H , J = 8.1, 1.4 Hz); 8.17 s
6 3 6 4
13
(1H, CH=N); 10.20 s, 12.04 s (2H, NHCSNH); 12.96 s (1H, NH, thiazolidine). C NMR (DMSO-d6)
δ
(ppm): 56.7; 111.8; 116.0; 121.3; 123.4; 125.1; 125.7; 126.0; 130.1; 132.7; 133.9; 140.5; 141.0; 143.0; 145.9;
1
51.3; 163.7; 166.3; 169.3; 176.4. Anal. calc. for C H ClN O S (%): C 48.93; H 3.08; N 11.41. Found: C
20
15
4
5 2
4
8.89; H 3.10; N 11.37.
4-[{2-[(4-Chlorophenyl)carbamothioyl]hydrazinylidene}methyl]-2-methoxyphenyl
(2,4-dioxo-1,3-thiazolidin-5-ylidene)acetate (39)
◦
1
Yield 82%, mp 196–198 C. H NMR (DMSO-d )
δ (ppm): 3.87 s (3H, OCH ); 7.08 s (1H, CH=); 7.27 d,
3
6
7
.53 dd, 7.69 d, (3H, 4-O-C H , J = 8.1, 1.8 Hz); 7.42 d, 7.60 d (4H, 4-Cl-C H , J = 8.7 Hz); 8.16 s (CH=N);
6 3 6 4
13
10.20 s, 12.05 s (2H, NHCSNH); 12.94 s (1H, NH, thiazolidine). C NMR (DMSO-d6)
111.4; 116.0; 121.4; 123.5; 128.4; 128.6; 130.0; 133.6; 138.4; 140.8; 142.9; 145.8; 151.4; 163.6; 166.4; 169.4;
176.5. Anal. calc. for C H ClN O S (%): C 48.93; H 3.08; N 11.41. Found: C 48.90; H 3.05; N 11.38.
δ (ppm): 56.7;
20
15
4
5 2
4-[{2-[(2,4-Dichlorophenyl)carbamothioyl]hydrazinylidene}methyl]-2-methoxyphenyl
(2,4-dioxo-1,3-thiazolidin-5-ylidene)acetate (40)
◦
1
Yield 79%, mp 202–204 C. H NMR (DMSO-d ) δ (ppm): 3.85 s (3H, OCH ); 7.09 s (1H, CH=); 7.46
3
6
dd, 7.62–7.67 m, 7.73 d (3H, 2,4-diCl-C H , J = 8.6, 2.4 Hz); 7.28 d, 7.53 dd, 7.69 d, (3H, 4-O-C H ,
6
3
6
1
3
3
J = 8.1
,
1.8 Hz); 8.18 s (CH=N); 10.21 s, 12.03 s (2H, NHCSNH); 12.95 s (1H, NH, thiazolidine).
C
NMR (DMSO-d6)
δ
(ppm): 56.6; 111.2; 115.9; 121.5; 123.5; 127.9; 129.3; 131.7; 131.8; 132.6; 133.6; 136.4;
1
2
40.9; 142.9; 146.0; 151.3; 163.7; 166.5; 169.5; 176.6. Anal. calc. for C H Cl N O S (%): C 45.72; H
.69; N 10.66. Found: C 45.57; H 2.59; N 10.63.
20
14
2
4
5 2
3
.1.2. Synthesis of 4-[(2,5-dioxoimidazolidin-4-ylidene)methyl]-2-methoxyphenyl
(
2,4-dioxo-1,3-thiazolidin-5-yl/ylidene)acetates (41, 42)
The solution of 0.01 mol acid chloride ( or ) in 3 mL of anhydrous dioxane was added
3
4
to a solution of 0.01 mol of 5-(4-hydroxy-3-methoxybenzylidene)-imidazolidine-2,4-dione in 5 mL
anhydrous pyridine. After 2 h, water was added and the mixture was acidified of diluted hydrochloric
acid solution to pH = 3–4 and left at room temperature for 24 h. The precipitate was filtered off and
then crystallized from n-butanol.
4
-[(2,5-Dioxoimidazolidin-4-ylidene)methyl]-2-methoxyphenyl (2,4-dioxo-1,3-thiazolidin-5-ylidene)
acetate (41)
◦
1
Yield 71%, mp 187–189 C. H NMR (DMSO-d ) δ (ppm): 3.85 s (3H, OCH ); 6.44 s (1H, CH=); 7.07 s
3
6
(
1H, =CH-COO); 7.20–7.31 m (3H, 4-O-C H ); 10.67 s, 11.29 s (2H, 2NH, hydantoin); 12.95 bs (1H, NH,
6 3

Molecules 2018, 23, 1023
14 of 17
thiazolidine). 13C NMR (DMSO-d6)
δ
(ppm): 56.6; 108.1; 114.1; 116.0; 122.6; 123.4; 128.7; 133.1; 139.0;
1
45.8; 151.1; 156.3; 163.7; 166.0; 166.3; 169.3.
4-[(2,5-Dioxoimidazolidin-4-ylidene)methyl]-2-methoxyphenyl (2,4-dioxo-1,3-thiazolidin-5-yl)acetate
(42)
◦
1
Yield 72%, mp 192–194 C. H NMR (DMSO-d )
δ
(ppm): 3.40–3.42 m (2H, CH-CH2); 3.84 s (3H,
6
OCH ); 4.85 dd (1H, C
H-CH , J = 6.7, 5.5 Hz); 6.42 s (1H, CH=); 7.08 d, 7.21–7.28 m (3H, 4-O-C H , J =
3
2
6
3
13
8.4 Hz); 10.65 s, 11.28 s (2H, 2NH, hydantoin); 12.13 s (1H, NH, thiazolidine). C NMR (DMSO-d6)
δ
(ppm): 36.1; 46.9; 56.3; 110.2; 113.7; 116.2; 123.9; 124.8; 125.9; 148.2; 156.2; 164.4; 166.1; 168.9; 172.6;
1
75.8.
2
-Ethoxy-4-[(5-oxo-2-thioxoimidazolidin-4-ylidene)methyl]phenyl (2,4-dioxo-1,3-thiazolidin-5-ylidene)
acetate (43)
was obtained by the method reported previously [21]. Spectral characteristics were presented in above
mentioned publication.
3
.2. Microbiology Tests
Twelve reference strains of bacteria from ATCC (American Type Culture Collection) were used.
Among Gram-positive bacteria there were: Staphylococcus aureus ATCC 25923, Staphylococcus aureus
ATCC 6538, Staphylococcus epidermidis ATCC 12228, Bacillus subtilis ATCC 6633, Bacillus cereus ATCC
1
0876, and Micrococcus luteus ATCC 10240. However, within Gram-negative bacteria there were species
with low growth and atmosphere requirements from Enterobacteriaceae family (Escherichia coli ATCC
5922, Klebsiella pneumoniae ATCC 13883, Proteus mirabilis ATCC 12453) and from Pseudomonadaceae
family (Pseudomonas aeruginosa ATCC 9027), as well as fastidious bacteria from Pasteurellaceae family
2
(
Haemophilus influenzae ATCC 10211 and Haemophilus parainfluenzae ATCC 51505) with high growth
6
and atmosphere requirements. Microbial suspensions with an optical density of 150
×
10 CFU/mL
(CFU—colony forming units, 0.5 McFarland standard) were prepared in sterile 0.85% NaCl.
The compounds’ stock solutions were dissolved in dimethyl sulfoxide (DMSO) with no inhibitory
concentration on the growth of bacteria (negative control). The medium with DMSO without the
tested compounds was used as negative control. A ciprofloxacin, cefuroxime and oxacillin were used
as reference antimicrobials.
The antibacterial activity of tested compounds was firstly screened by the agar dilution method
on the basis of the microbial growth inhibition zone (giz, in mm, with 1000 mg/L concentration of the
tested compounds). Then it was determined by broth microdilution technique with series of twofold
dilution (final concentrations from 0.007 to 1000 mg/L), of the tested compounds or ciprofloxacin,
cefuroxime and oxacillin as was described earlier [26]. The antimicrobial effect was detected with the
Mueller–Hinton medium.
The activity was expressed as the minimal inhibitory concentration (MIC) assayed
spectrophotometrically by optical density determination (OD600). The MBC (minimal bactericidal
concentration)—defined as the lowest concentration of each compound that resulted in >99.9%
reduction in CFU of the initial inoculum—was also determined by plating out the 5 µL contents
of wells with no visible growth of bacteria, onto Mueller–Hinton agar plates (incubation conditions
◦
3
5 C for 18 h). The compounds were classified as bacteriostatic when the MBC/MIC ratio was
≥
8
and bactericidal when the MBC/MIC ratio was ≤4 [27].
.3. Computational Details
Conformational search, physicochemical parameters, and HOMO/LUMO maps were calculated
3
using HyperChem 8.0.1 [28]. Extensive conformational searches were carried out using the molecular
mechanics level with OPLS force field [29]. The most stable structures obtained were subsequently
optimized to the closest local minimum at the semiempirical level using RM1 parametrization.

Molecules 2018, 23, 1023
15 of 17
Convergence criteria were set to 0.1 and 0.01 kcal mol 1
−
Å
−1
for OPLS and RM1 calculations,
respectively. Electrostatic potentials were calculated using Gaussian 03 and GaussView 5 at the
HF/6–31 G level.
3
.4. Cytotoxicity Studies
Cytotoxic properties of the investigated compounds were evaluated using human embryonic
kidney (HEK-293) cell line. HEK-293 cells were purchased from the American Type Culture
Collection (ATCC CRL-1573) and were cultured using DMEM (Sigma–Aldrich) supplemented with
1
1
0% fetal bovine serum (FBS, Sigma–Aldrich, St. Louis, MO, USA), 100 U/mL of penicillin and
00 mg/L of streptomycin (PenStrep, Sigma–Aldrich). Cells were incubated at 37 C in a humidified
◦
atmosphere of 5% CO . Stock solutions of the tested compounds were prepared dissolving in
2
a sterile dimethyl sulfoxide (DMSO) to the concentration of 100 mg/L. On the day of experiment,
5
HEK-293 cells were seeded into 96-well sterile plates (Nunc) at a cell density of 3
×
10 cells/well.
After 24 h of incubation, the medium was removed from each well and then cells were incubated
for the next 24 h with different concentrations of the tested compounds (1–250 mg/L) in DMEM
containing 2% FBS. Control cells were cultured only with medium containing 2% addition of FBS.
Toxicity of the compounds was evaluated using MTT assay, which is based on the conversion of
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) into dark-blue formazan crystals.
Briefly, after 24 h incubation of cells with varying concentrations of tested compounds, all culture media
were removed from the plates. Cells were washed with PBS, and then 100
µ
L of medium containing
◦
10% MTT solution (5 mg/L) was added to each well. Cells were incubated for the next 4 h at 37 C in
an atmosphere of 5% CO . Afterwards, 100
µ
L (per well) of 10% SDS buffer solution was added to
dissolve formazan crystals and after an overnight incubation the absorbance was measured at 540 and
20 nm using a microplate reader (Epoch, BioTek Instruments, Luzern, Switzerland). The obtained
results were expressed as EC70 SD from three independent experiments done in triplicate. EC70
represents the concentration of the compound that inhibited viability of cells by 30%.
2
6
±
4
. Conclusions
The studied new thiazolidine-2,4-dione-chlorophenylthiosemicarbazone hybrids showed
promising activity against Gram-positive bacterial strains. The most potent derivatives (20 23 30 31
,
,
,
and 38) exhibited good activity against all six Gram-positive strains used in microbiological studies.
The compound 38 showed more effective action than standard drug cefuroxime against B. subtilis
and B. cereus. These compounds were non-toxic in concentration close to their antibacterial effect.
Moreover, the SARs analysis collectively suggests that at least two different molecular mechanisms of
antibacterial activity for TZD-based hybrid compounds should be expected.
Author Contributions: N.T.: synthesis, writing of manuscript; U.K., A.M.: microbiological tests; A.P.:
computational studies, writing of manuscript; T.P.: cytotoxicity studies; M.W.: analyzed the data.
Acknowledgments: The project was funded by the National Science Centre, Poland (decision number:
2
012/05/D/NZ7/02278).
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Sample Availability: Samples of the compounds 17–40 are available from the authors.
©
2018 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/).
(