Synthesis and in vitro anti-toxoplasma gondii activity of novel thiazolidin-4-one derivatives

Article abstract of DOI:10.3390/molecules24173029

Recent findings on the biological activity of thiazolidin-4-ones and taking into account the lack of effective drugs used in the treatment of toxoplasmosis, their numerous side effects, as well as the problem of drug resistance of parasites prompted us to

Full text of DOI:10.3390/molecules24173029

molecules
Article
Synthesis and In Vitro Anti-Toxoplasma gondii
Activity of Novel Thiazolidin-4-one Derivatives
Nazar Trotsko 1,*, Adrian Bekier 2 , Agata Paneth 1 , Monika Wujec 1 and
Katarzyna Dzitko 2,
*
1
Department of Organic Chemistry, Faculty of Pharmacy with Medical Analytics Division, Medical
University of Lublin, 4A Chodz´ki, 20-093 Lublin, Poland
Department of Immunoparasitology, Faculty of Biology and Environmental Protection, University of Lodz,
Banacha 12/16, 90-237 Lodz, Poland
2
*
Correspondence: nazar.trotsko@umlub.pl (N.T.); katarzyna.dzitko@biol.uni.lodz.pl (K.D.)
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 18 July 2019; Accepted: 19 August 2019; Published: 21 August 2019
Abstract: Recent findings on the biological activity of thiazolidin-4-ones and taking into account the
lack of effective drugs used in the treatment of toxoplasmosis, their numerous side effects, as well
as the problem of drug resistance of parasites prompted us to look for new agents. We designed
and synthesized a series of new thiazolidin-4-one derivatives through a two-step reaction between
4-substituted thiosemicarbazides with hydroxybenzaldehydes followed by the treatment with ethyl
bromoacetate; maleic anhydride and dimethyl acetylenedicarboxylate afforded target compounds.
The thiazolidin-4-one derivatives were used to assess the inhibition of Toxoplasma gondii growth
in vitro. All active thiazolidine-4-one derivatives (12 compounds) inhibited T. gondii proliferation
in vitro much better than used references drugs both sulfadiazine as well as the synergistic effect
of sulfadiazine + trimethoprim (weight ratio 5:1). Most active among them derivatives 94 and 95
showed inhibition of proliferation at about 392-fold better than sulfadiazine and 18-fold better than
sulfadiazine with trimethoprim. All active compounds (82–88 and 91–95) against T. gondii represent
values from 1.75 to 15.86 (CC30/IC50) lower than no cytotoxic value (CC30).
Keywords: thiazolidin-4-ones; anti-Toxoplasma gondii activity; cytotoxicity
1. Introduction
Toxoplasma gondii belongs to the cosmopolitan species of the parasite that causes toxoplasmosis in
all species of mammals and birds. It is estimated that up to 1/3 of the human population are infected by
this parasite [1]. The developed humoral and cellular response in immunocompetent persons quickly
limits its intense proliferation but does not completely eliminate it from the host, resulting in the
long-term presence of tissue cysts mainly located in the central nervous system (CNS), muscles, and
eyes. In turn, the long-term presence of a parasite carries the risk of permanent damage to the visual
organ and (or) the brain and is also correlated with the occurrence of serious nervous disorders such
as schizophrenia, Parkinson’s disease or epilepsy [2,3]. Infection with protozoan is also associated
with serious effects on the health of the fetus and people with immune dysfunction. Newborns with
congenital toxoplasmosis are characterized by pathological changes within the nervous system, with
the effects of congenital T. gondii invasion often noticeable after several years and include damage to
the eye or CNS, endocrine disorders, or abnormal sexual development [4]. In immunocompromised
persons, toxoplasmosis may manifest itself, among others headaches and around the chest, a sense of
confusion, expectoration of blood, problems with breathing because immune system’s lack of ability to
fight acute phase of infectious [
5]. In addition, there is a risk of permanent damage to the visual organ or
the brain in these patients. In extreme cases, this disease is fatal. Currently available drugs not only do
Molecules 2019, 24, 3029; doi:10.3390/molecules24173029
www.mdpi.com/journal/molecules

Molecules 2019, 24, 3029
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not completely eliminate parasitic cysts from the infected organism, and moreover, they show serious
side effects, which include hematological and neurological disorders, skin and mucous membrane
changes, gastrointestinal disorders, bone marrow damages, teratogenicity, and nephrolithiasis [6,7].
There are also cases of multidrug resistance. There is, therefore, a real need for the development of
new drugs that are capable of effective treatment of toxoplasmosis but without side effects.
In recent years, the interest in thiazolidine-4-one derivatives has been increased among scientists
due to their broad spectrum of biological activities, including antidiabetic, antibacterial, antifungal,
anticancer, and anti-inflammatory, confirmed by numerous reviews on the activity and mechanisms of
action of thiazolidine-4-ones [8–13].
In addition, the lack of 100% of the effectiveness of drugs used in the treatment of toxoplasmosis,
their numerous side effects, as well as the problem of increasing drug resistance of parasites prompted
us to look for new synthetic compounds that could be used in the future to fight this common
parasite. Literature report on the antiparasitic activity of thiazolidine-4-one derivatives [14–16] was an
inspiration to undertake research to obtain effective low-toxicity compounds with activity against T.
gondii, belonging to the thiazolidin-4-one group.
2. Results and Discussion
2.1. Rationale
Hitherto, our research group identified thiazolidine-chlorophenylhydrazone hybrids with
antiproliferative activity comparable to that of irinotecan [17] and thiazolidine-thiohydantoin and
thiazolidine-chlorophenylthiosemicarbazone hybrids with antibacterial activity comparable to that of
cefuroxime [18,19]. The results of the latest research work [20–22] in the field of inhibitory activity of
thiosemicarbazide derivatives against protozoan T. gondii, identified that in particular those compounds
which in position 4 contain halogen(alkyl)phenyl group are characterized by antiparasitic activity much
more advantageous than the commonly used drug sulfadiazine (IC50 25.67–68.18
µM vs. 10,873.71
µM). These results prompted us to start a pilot synthesis of a series of thiazolidin-4-one derivatives
containing in their structure the above-mentioned a thiosemicarbazide moiety (shown in red in Figure 1)
to confirm (or exclude) their inhibitory potential against T. gondii tachyzoites; from our research to
date, it appears that, although there are objective indications of anti-toxoplasmic activity for the
proposed group of compounds, strengthened by reports by G
óes and colleagues [14–16], in practice
these compounds may be quite significantly different in bioactivity or even inactive.
R
R
O
Z
N
OR
1
HN
HN
S
O
S
N
N
thiazolidine ring
NH
O
Z-single or double bond
R=H, Cl; R1=H, CH3;
azole
R2=OH, Cl, Br, OAlk
R
2
Figure 1. Design of novel thiazolidin-4-one derivatives.
2.2. Chemistry
We designed novel thiazolidin-4-one derivatives in which N4 and C3 atoms of thiosemicarbazide
incorporated in the heterocyclic system creating thiazolidine ring. Others N1 and N2 atoms of
thiosemicarbazide were as substitutions in position 2 of thiazolidin-4-ones. The target thiazolidine-4-one

Molecules 2019, 24, 3029
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derivatives were obtained in the two-step procedure starting with appropriate thiosemicarbazide
derivatives. In the first step were obtained thiosemicarbazones ( 40) by the condensation of
8–
thiosemicarbazide derivatives (
illustrated in Scheme 1.
1
–
7) with corresponding hydroxybenzaldehydes. The reactions are
O
NH NH
R
1
NH NH
S
NH
2
+
N
CH
R
R
S
8-40
R
1-7
1
8: R=H, R1=2-OH; 9: R=H, R1=3-OH; 10: R=H, R1=4-OH; 11: R=H,
R1=3-OCH3, 4-OH; 12: R=H, R1=3-OC2H5, 4-OH; 13: R=H, R1=3-Cl,
4-OH; 14: R=H, R1=3-Br, 4-OH; 15: R=2-Cl, R1=2-OH; 16: R=2-Cl,
R1=3-OH; 17: R=2-Cl, R1=4-OH; 18: R=2-Cl, R1=3-OC2H5, 4-OH; 19:
R=3-Cl, R1=2-OH; 20: R=3-Cl, R1=3-OH; 21: R=3-Cl, R1=4-OH; 22:
R=3-Cl, R1=3-OCH3, 4-OH; 23: R=3-Cl, R1=3-OC2H5, 4-OH; 24:
R=4-Cl, R1=2-OH; 25: R=4-Cl, R1=3-OH; 26: R=4-Cl, R1=4-OH; 27:
R=4-Cl, R1=3-OCH3, 4-OH; 28: R=4-Cl, R1=3-OC2H5, 4-OH; 29:
R=4-Cl, R1=3-Cl, 4-OH; 30: R=4-Cl, R1=3-Br, 4-OH; 31: R=2,4-diCl,
R1=3-OCH3, 4-OH; 32: R=4-Br, R1=3-OH; 33: R=4-Br, R1=4-OH; 34:
R=4-F, R1=2-OH; 35: R=4-F, R1=3-OH; 36: R=4-F, R1=4-OH; 37:
R=4-F, R1=3-OCH3, 4-OH; 38: R=4-F, R1=3-OC2H5, 4-OH; 39: R=4-F,
R1=3-Cl, 4-OH; 40: R=4-F, R1=3-Br, 4-OH.
Scheme 1. Synthesis of thiosemicarbazone derivatives (8–40); Reagents and conditions: Ethanol,
catalytic amounts of acetic acid, reflux for 0.5–2 h.
In the second step were obtained 3 series of compounds. The first series were thiazolidin-4-one
derivatives (41 73), which were obtained by the reaction of thiosemicarbazones ( 40) with ethyl
bromoacetate in anhydrous ethanol and presence of anhydrous sodium acetate. The second series were
(4-oxothiazolidin-5-ylo)acetic acid derivatives (74 88), which were synthesized from corresponding
–
8–
–
thiosemicarbazones by thia-Michael reaction, involving maleic anhydride as Michael acceptor. And the
third series was obtained from appropriate thiosemicarbazones and dimethyl acetylenedicarboxylate,
similarly as second group, by Michael addition of the sulfur atom to the triple bond and then cyclization
to give (4-oxothiazolidin-5-ylidene)acetic acid derivatives (89–104). The Scheme 2 was presented
synthetic route and reaction condition of these groups of compounds.
The structure of new compounds was confirmed by elemental analysis, 1H-NMR and
13C-NMR spectra.
All protons from the phenyl fragment of new compounds showed signals at δ ~6.82–7.96 ppm. For
para-substituted derivatives observed two doublets in above ranges with J = 8.7–8.8 Hz. Tri-substituted
derivatives showed signals as two doublets and doublet of doublets with coupling constant 1.8–2.1
and 7.8–8.7 Hz in above ranges.
In 1H-NMR spectra generally, the protons of CH=N group of all new synthesized compounds
show singlet signal at δ ~8.15–8.66 ppm.
For the derivatives 41–73 signal from protons of CH2 group of thiazolidine ring appearing as a
singlet at ranges 4.06–4.16 ppm (compounds 41–47, 52–63 and 65–73) and as a doublet of doublets at δ
~4.18–4.28 ppm for compounds 48–51 and 64.
1H-NMR spectra showed resonance assigned to the CH group of (4-oxothiazolidin-5-yl)acetic
acid derivatives (74–88) appearing as triplet or doublet of doublets at 4.53–4.68 ppm (ABX spin system)
due to the interaction with methylene group of these derivatives. These results agreed with the data of
De Aquino et al. for similar structures [16].

Molecules 2019, 24, 3029
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R
NH NH
S
1
N
CH
R
8-40
c
a
R
N
R
O
O
N
b
R
N
1
R
N
CH
1
S
H C
N
N
CH
3
S
O
O
89-104
41-73
R
N
41: R=H, R1=2-OH; 42: R=H, R1=3-OH; 43: R=H,
R1=4-OH; 44: R=H, R1=3-OCH3, 4-OH; 45: R=H,
R1=3-C2H5O, 4-OH; 46: R=H, R1=3-Cl, 4-OH; 47:
R=H, R1=3-Br, 4-OH; 48: R=2-Cl, R1=2-OH; 49:
R=2-Cl, R1=3-OH; 50: R=2-Cl, R1=4-OH; 51:
R=2-Cl, R1=3-OC2H5,4-OH; 52: R=3-Cl,
89: R=H, R1=2-OH; 90: R=H,
O
R1=3-OH; 91: R=H, R1=4-OH; 92:
R=H, R1=3-OCH3, 4-OH; 93: R=H,
R1=3-OC2H5, 4-OH; 94: R=H,
N
R
1
R1=3-Cl, 4-OH; 95: R=H, R1=3-Br,
4-OH; 96: R=2-Cl, R1=3-OC2H5,
4-OH; 97: R=4-Cl, R1=2-OH; 98:
R=4-Cl, R1=3-OH; 99: R=4-Cl,
R1=4-OH; 100: R=4-Cl, R1=3-OCH3,
4-OH; 101: R=4-Cl, R1=3-OC2H5,
4-OH; 102: R=4-Cl, R1=3-Cl, 4-OH;
103: R=4-Cl, R1=3-Br, 4-OH; 104:
R=2,4-diCl, R1=3-OCH3, 4-OH.
N
CH
S
R1=2-OH; 53: R=3-Cl, R1=3-OH; 54: R=3-Cl,
R1=4-OH; 55: R=3-Cl, R1=3-OCH3, 4-OH; 56:
R=3-Cl, R1=3-OC2H5, 4-OH; 57: R=4-Cl,
HO
74-88
O
74: R=H, R1=2-OH; 75: R=H,
R1=2-OH; 58: R=4-Cl, R1=3-OH; 59: R=4-Cl,
R1=4-OH; 60: R=4-Cl, R1=3-OCH3, 4-OH; 61:
R=4-Cl, R1=3-OC2H5, 4-OH; 62: R=4-Cl, R1=3-Cl,
4-OH; 63: R=4-Cl, R1=3-Br, 4-OH; 64:
R1=3-OH; 76: R=H, R1=4-OH; 77:
R=H, R1=3-OCH3, 4-OH; 78: R=H,
R1=3-OC2H5, 4-OH; 79: R=H,
R1=3-Cl, 4-OH; 80: R=H, R1=3-Br,
4-OH; 81: R=4-Cl, R1=2-OH; 82:
R=4-Cl, R1=3-OH; 83: R=4-Cl,
R1=4-OH; 84: R=4-Cl, R1=3-OCH3,
4-OH; 85: R=4-Cl, R1=3-OC2H5,
4-OH; 86: R=4-Cl, R1=3-Cl, 4-OH;
87: R=4-Cl, R1=3-Br, 4-OH; 88:
R=2,4-diCl, R1=3-OCH3, 4-OH.
R=2,4-diCl, R1=3-OCH3, 4-OH; 65: R=4-Br,
R1=3-OH; 66: R=4-Br, R1=4-OH4; 67: R=4-F,
R1=2-OH; 68: R=4-F, R1=3-OH; 69: R=4-F,
R1=4-OH; 70: R=4-F, R1=3-OCH3, 4-OH; 71:
R=4-F, R1=3-OC2H5, 4-OH; 72: R=4-F, R1=3-Cl,
4-OH; 73: R=4-F, R1=3-Br, 4-OH.
Scheme 2. Synthesis of thiazolidin-4-one derivatives (41–104); Reagents and conditions: (a) Ethyl
bromoacetate, anhydrous sodium acetate, anhydrous ethanol, reflux for 2 h; (b) maleic anhydride,
glacial acetic acid, reflux for 2 h; (c) dimethyl acetylenedicarboxylate, methanol, reflux for 1.5 h.
Protons signals of =CH-COO group of compounds 89
ppm range.
In the 13C-NMR
–104 were visible as a singlet at 6.80–6.89
δ
values of the carbons of the all thiazolidin-4-one derivatives (41
–104) showed
the carbon signals of C=O groups in the range 171.6–174.2 ppm (compounds 41
ppm for (4-oxothiazolidin-5-ylidene)acetic acid derivatives (89–104).
–88) and at 163.7–166.6
The detailed results of 1H-NMR and 13C-NMR spectra are presented in Section 3 and
supplementary materials.
2.3. Cytotoxicity of the Thiazolidin-4-one Derivatives against L929 Cells
According to the main assumption, compounds that have been tested for activity against T. gondii
proliferation should have low cytotoxicity or even they should not be toxic to the host cells. Therefore,
in the first stage of the study, a cytotoxicity screen against L929 cells was performed. To described
cytotoxicity we use CC50 value (cytotoxicity concentration 50%), in accordance with applicable rules,
defined as the concentration of test samples that causes 50% destruction of cells, but also CC30 value
was calculated to determine the initial dose of each compound to anti-Tg study (data not shown).
Among the 43 compounds tested derivatives 81, 96, 97, 100–104 were insoluble under the protocol
conditions and, consequently, they were eliminated from further experiments.
The first series of investigated compounds, that are thiazolidin-4-one derivatives (42
,
49
25.60
µM but the
, 50, 53,
54
,
58
,
59
,
65
,
66
,
68, and 69), showed high cytotoxicity CC50 = 83.54
±
8.27
−
385.61
20.35
±
µM
(Table 1), besides compound 43. The value of CC50 for compound 43 was 484.97
±
compound 43 with quite satisfactory cytotoxicity cannot alone represented a group of this derivatives.
All compounds of the first group were excluded from further anti-T. gondii activity study primarily
due to the low CC50 value which means as cytotoxicity.

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Table 1. Cytotoxicity of the thiazolidin-4-one derivatives (42
and 69), (4-oxothiazolidin-5-ylo)acetic acid derivatives (74 88), (4-oxothiazolidin-5-ylidene)acetic acid
derivatives (89–95, 98, and 99) and drugs against L929 cells; SD —standard deviation.
, 43, 49, 50, 53, 54, 58, 59, 65, 66, 68,
–
Compound or Drug
CC50 (µM)
SD (µM)
42
43
49
50
53
54
58
59
65
66
68
69
74
75
76
77
78
79
80
82
83
84
85
86
87
88
89
90
91
92
93
94
233.81
484.97
188.26
225.85
113.07
289.18
173.22
318.10
83.54
±12.51
±20.35
±7.72
±13.97
±4.79
±15.53
±8.39
±11.07
±8.27
±19.71
±8.23
±25.60
±22.24
–
247.02
180.36
385.61
500.82
≥1000
≥1000
≥1000
≥1000
≥1000
≥1000
656.20
906.30
827.45
759.22
≥1000
424.66
565.86
304.14
234.40
492.91
486.11
463.03
473.73
427.98
166.89
229.89
≥10,000
–
–
–
–
–
±14.26
±16.80
±13.16
±20.70
–
±8.96
±19.81
±11.40
±9.83
±19.89
±37.43
±30.61
±22.30
±21.90
±9.43
±9.46
–
95
98
99
Sulfadiazine
Sulfadiazine + Trimethoprim
(weight ratio 5:1)
9580.25
±44.21
The second series of compounds that are (4-oxothiazolidin-5-ylo)acetic acid derivatives (74
–88
)
were less cytotoxic than first series. Their CC50 were within 424.66 ± 8.96 – ≥ 1000 µM (Table 1).
The third series of compounds that are (4-oxothiazolidin-5-ylidene)acetic acid derivatives (89
–95
,
98, and 99) (Table 1). In this series, two derivatives with 3-phenyl substituent and two compounds with
3-(4-chlorophenyl) substituent in thiazolidine ring were considered as cytotoxic. The CC50 values were
166.89
showed not high cytotoxicity (CC50 = 427.98
the similar level (CC30 = 315.97.98 21.91, 404.08
14.94 µM, data not shown). Data of cytotoxicity of references drugs were also presented in Table 1.
±
9.43
−
304.14
±
11.40
µ
M. The remaining (4-oxothiazolidin-5-ylidene)acetic acid derivatives
21.90 492.91 19.89 M) and were no cytotoxic at
12.59, 440.06 15.78, 448.93 19.48, and 471.94
±
−
±
µ
±
±
±
±
±
To the next step of our studies forwarded 18 new substances from the second (75
–80, 82–88) and
third series (91–95).

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2.4. In Vitro Anti-T. gondii Activity of the Thiazolidin-4-ones (75–99)
In the next step of our studies, the obtained thiazolidin-4-ones (series 2 and 3) were used to assess
the inhibition of T. gondii growth in vitro. For this purpose, intracellular parasites (tachyzoites) of RH
strain were incubated with different concentrations of the thiazolidin-4-one derivatives. Therefore, that
T. gondii is an intracellular parasite and host cells should not be destroyed by the cytotoxic concentration
of tested compounds, the initial dose of each compound was optimized using CC30 value. Inhibition
of the parasite growth was monitored by measuring the specific incorporation of [3H]uracil in the
parasite’s nucleic acids.
Because of absence of detailed information about molecular targets and privileged scaffolds among
thiazolidin-4-one derivatives for anti-T. gondii activity, we chose group of non-cytotoxic derivatives
from series 2 and 3 to study the inhibition of T. gondii growth in vitro.
According to results presented in Table 2, the (4-oxothiazolidin-5-yl)acetic acid derivatives (75–80)
with the phenyl substituent at the position 3 of the thiazolidine system were inactive. Change the
phenyl substituent to 4-chlorophenyl at the position 3 caused the activity against T. gondii proliferation
for compounds 82
M. Added another chlorine atom at the phenyl ring in position 3 (84→88) led to increased activity
against T. gondii from IC50 = 219.93 ± 19.22 µM to IC50 = 129.42 ± 14.14 µM (Figure 2).
–88. Inhibitory concentration (IC50) was at the range 115.92
±
21.68 – 271.15
±
24.96
µ
Table 2. Anti-T. gondii tachyzoites activity of (4-oxothiazolidin-5-yl)acetic acid (75–80 and 82–88),
(4-oxothiazolidin-5-ylidene)acetic acid (91 95) and drugs; na—not active against T. gondii in the tested
–
concentration range; SD—standard deviation; SR—selectivity ratio.
Compound or Drug
IC50 (µM)
SD (µM)
SR
75
76
na
na
–
–
–
–
77
na
–
–
78
na
–
–
79
na
–
–
80
na
–
–
82
83
84
85
86
87
88
91
92
93
94
95
263.22
271.15
219.93
165.40
177.72
115.92
129.42
92.88
180.27
191.21
27.74
28.32
10,873.71
±19.31
±24.96
±19.22
±16.02
±18.59
±21.68
±14.14
±14.94
±21.91
±27.32
±4.27
±5.83
±122.76
2.49
3.34
3.76
4.59
≥5.63
3.66
4.37
5.31
2.69
2.42
17.07
15.11
≥0.92
Sulfadiazine
Sulfadiazine + Trimethoprim
(weight ratio 5:1)
510.22
±35.87
18.78
SR—selectivity ratio values were calculated as the ratio of the 50% cytotoxic concentration (CC50) to the 50%
antiparasitic concentration (IC50).

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Figure 2. Dose response curves and IC50 values (
tachyzoites proliferation.
µM) of 77, 84, 88, and 92 on T. gondii
On the contrary, it seems that the type of substituents (either electron withdrawing or electron
donating groups) in the hydroxybenzylidene fragment at the position 2 of the thiazolidine system
for compounds 82–88 did not have any significant effect on the values of activity against T. gondii
proliferation (Table 2).
Among the (4-oxothiazolidin-5-ylidene)acetic acid derivatives (91–95) all compounds showed an
activity at the range 27.74
activity of their saturated analogues 75–80 and 82–88 (Figures 2–4).
±
4.27 – 191.21
±
27.32 µM (Table 2), which was generally better than the
(a)
(b)
83, and 91 on T. gondii proliferation.
Figure 3.
(a
): Dose response curves and IC50 values (
µ
M) of 76
,
(b): Dose response curves and IC50 values (µM) of 78, 85, and 93 on T. gondii proliferation.

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(b)
µM) of 79, 86, and 94 on T. gondii proliferation.
(a)
): Dose response curves and IC50 values (
(b): Dose response curves and IC50 values (µM) of 80, 87, and 95 on T. gondii proliferation.
Figure 4.
(a
In case of derivatives 91
the substituent’s type at the position 3 (phenyl
cytotoxicity these derivatives (90
–
95, it was not possible to observe the dependence of activity on
4-chlorophenyl 2,4-dichlorophenyl) due high
98, and 99) or insolubility under the protocol conditions (compounds
→
→
,
96, 97, and 100–104).
On the other hand, it was the noticed the dependence between substituent’s type in the
hydroxybenzylidene fragment at the position 2 of the thiazolidine system and increasing activity
against T. gondii. Compounds 92 and 93 with electron donating group (methoxy and ethoxy) showed
activity with IC50 ~180–190
led to increased activity (IC50 = 92.88
(derivatives 94 and 95) strongly increased the activity (IC50 ~27–28 ± 5.05 µM).
All active (4-oxothiazolidin-5-yl)acetic acid derivatives
(4-oxothiazolidin-5-ylidene)acetic acid derivatives (91 95) inhibited T. gondii proliferation
in vitro in concentration lower than used references drugs both sulfadiazine (10,873.71 122.76 M) as
well as synergistic effect of sulfadiazine + trimethoprim (5:1) (510.22 35.87 M) (Table 2). In addition,
±
24.50
µ
M. The absence of the substituent in this fragment (compound 91
)
±
21.91 M) and added electron withdrawing group Cl or Br
µ
(
82
–
88
)
and
–
±
µ
±
µ
compounds 87 and 88 showed inhibition of proliferation at about 90-fold lower IC50 value relative to
sulfadiazine and more than four times lower relative to sulfadiazine with trimethoprim. Most active
among (4-oxothiazolidin-5-ylidene)acetic acid derivatives 94 and 95 showed inhibition of proliferation
at about 392-fold better than sulfadiazine and 18-fold better than synergistic effect of sulfadiazine +
trimethoprim (5:1).
3. Materials and Methods
3.1. Chemistry
All commercial reactants and solvents were purchased from either Alfa Aesar (Kandel, Germany)
or Sigma-Aldrich (St. Louis, MO, USA) with the highest purity and used without further purification.
The 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 silica
gel Si 60F254, produced by Merck Co. (Darmstadt, Germany). The 1H-NMR and 13C-NMR spectra
were recorded by a Bruker Avance 300 MHz instrument using DMSO-d6 as solvent and TMS as an
internal standard. Chemical shifts were expressed as
δ (ppm). Elemental analyses were performed by
AMZ 851 CHX analyzer and the results were within ±0.4% of the theoretical value.

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3.2. General Method of Synthesis of Thiosemicarbazones (8–40)
A mixture of 0.01 mol thiosemicarbazide derivatives (
1–
7
) and 0.01 mol corresponding
hydroxybenzaldehydes in 15 mL anhydrous ethanol and presence catalytic amount of glacial acetic
acid was refluxed for 0.5 2h. The solid obtained after cooling of reaction mixture was filtered off, dried
–
and then recrystallized from acetic acid. Compounds 15
compounds 24–30 from mixture ethanol-water (1:1).
–18 were recrystallized from isopropanol and
1-[(2-Hydroxyphenyl)methylidene]-4-phenyl-3-thiosemicarbazide (
Number: 14938-70-6.
8
): Yield: 76%, CAS Registry
): Yield: 78%, CAS Registry
1-[(3-Hydroxyphenyl)methylidene]-4-phenyl-3-thiosemicarbazide (
9
Number: 76572-75-3.
1-[(4-Hydroxyphenyl)methylidene]-4-phenyl-3-thiosemicarbazide (10): Yield: 64%, CAS Registry
Number: 76572-74-2.
>1-[(4-Hydroxy-3-methoxyphenyl)methylidene]-4-phenyl-3-thiosemicarbazide (11): Yield: 80%, CAS Registry
Number: 20158-15-0.
1-[(3-Ethoxy-4-hydroxyphenyl)methylidene]-4-phenyl-3-thiosemicarbazide (12): Yield: 77%, CAS Registry
Number: 301350-49-2.
1-[(3-Chloro-4-hydroxyphenyl)methylidene]-4-phenyl-3-thiosemicarbazide (13): Yield: 70%, CAS Registry
Number: 340229-91-6.
1-[(3-Bromo-4-hydroxyphenyl)methylidene]-4-phenyl-3-thiosemicarbazide (14): Yield: 83%, CAS Registry
Number: 1799010-24-4.
4-(2-Chlorophenyl)-1-[(2-hydroxyphenyl)methylidene]-3-thiosemicarbazide (15): Yield: 80%, mp = 188–190 ◦C.
1H-NMR (DMSO-d6)
8.03 d (J = 7.8 Hz) (8H, 2-Cl-C6
δ
(ppm): 6.81–6.90 m, 7.22–7.40 m, 7.55 dd (J = 7.8, 1.5 Hz), 7.75 d (J = 7.8 Hz),
4 and 2-HO-C6 4); 8.49 s (1H, CH=N); 10.01 s (1H, OH); 10.05 s, 11.96
H
H
s (2H, 2xNH). Anal. calc. for C14H12ClN3OS (%): C 54.99; H 3.96; N 13.74. Found: C 55.03; H 4.00;
N 13.71.
4-(2-Chlorophenyl)-1-[(3-hydroxyphenyl)methylidene]-3-thiosemicarbazide (16): Yield: 75%, mp = 200–202
oC. 1H-NMR (DMSO-d6)
δ
(ppm): 6.82–6.86 m, 7.22–7.41 m, 7.56 dd, 7.77 dd, (8H, 2-Cl-C6H4 and
4, J = 7.8, 1.5 Hz); 8.08 s (1H, CH=N); 9.61 s (1H, OH); 10.08 s, 11.97 s (2H, 2xNH). 13C-NMR
(DMSO-d6) (ppm): 114.1; 117.9; 119.4; 127.6; 128.1; 129.8; 129.9; 130.2; 130.8; 135.7; 137.0; 143.8; 158.1;
3-HO-C6
H
δ
176.9. Anal. calc. for C14H12ClN3OS (%): C 54.99; H 3.96; N 13.74. Found: C 54.94; H 3.94; N 13.70.
4-(2-Chlorophenyl)-1-[(4-hydroxyphenyl)methylidene]-3-thiosemicarbazide (17): Yield: 84%, CAS Registry
Number: 1097214-18-0.
4-(2-Chlorophenyl)-1-[(3-ethoxy-4-hydroxyphenyl)methylidene]-3-thiosemicarbazide (18): Yield: 81%, mp =
194–196 oC. 1H-NMR (DMSO-d6)
δ
(ppm): 1.34 t (3H, C
6.9 Hz); 6.82 d (J = 8.1 Hz), 7.12 dd (J = 8.1, 1.8 Hz), 7.30 td (J = 7.8, 1.5 Hz), 7.38 td (J = 7.8, 1.5 Hz), 7.49 d
(J = 1.8 Hz), 7.56 dd (J = 7.8, 1.5 Hz), 7.85 dd (J = 8.1, 1.5 Hz) (7H, 2-Cl-C6 4 and 3-C2H5O-4-HO-C6 3);
8.04 s (1H, CH=N); 9.51 s (1H, OH); 9.98 s, 11.88 s (2H, 2xNH). 13C-NMR (DMSO-d6)
(ppm): 15.2;
H3CH2O, J = 6.9 Hz); 4.07 q (2H, CH3CH2O, J =
H
H
δ
64.4; 111.1; 115.9; 123.2; 125.7; 127.6; 128.0; 129.6; 129.8; 130.6; 137.0; 144.2; 147.2; 149.9; 176.3. Anal. calc.
for C16H16ClN3O2S (%): C 54.93; H 4.61; N 12.01. Found: C 54.94; H 4.56; N 12.10.
4-(3-Chlorophenyl)-1-[(2-hydroxyphenyl)methylidene]-3-thiosemicarbazide (19): Yield: 82%, CAS Registry
Number: 67804-97-1.
4-(3-Chlorophenyl)-1-[(3-hydroxyphenyl)methylidene]-3-thiosemicarbazide (20): Yield: 74%, CAS Registry
Number: 2006219-81-2.

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4-(3-Chlorophenyl)-1-[(4-hydroxyphenyl)methylidene]-3-thiosemicarbazide (21): Yield: 79%, CAS Registry
Number: 1097214-19-1.
4-(3-Chlorophenyl)-1-[(4-hydroxy-3-methoxyphenyl)methylidene]-3-thiosemicarbazide (22): Yield: 83%, CAS
Registry Number: 70161-62-5.
4-(3-Chlorophenyl)-1-[(3-ethoxy-4-hydroxyphenyl)methylidene]-3-thiosemicarbazide (23): Yield: 78%, CAS
Registry Number: 769153-34-6.
4-(4-Chlorophenyl)-1-[(2-hydroxyphenyl)methylidene]-3-thiosemicarbazide (24): Yield: 76%, CAS Registry
Number: 14121-95-0.
4-(4-Chlorophenyl)-1-[(3-hydroxyphenyl)methylidene]-3-thiosemicarbazide (25): Yield: 71%, CAS Registry
Number: 93535-31-0.
4-(4-Chlorophenyl)-1-[(4-hydroxyphenyl)methylidene]-3-thiosemicarbazide (26): Yield: 77%, CAS Registry
Number: 16434-23-4.
4-(4-Chlorophenyl)-1-[(4-hydroxy-3-methoxyphenyl)methylidene]-3-thiosemicarbazide (27): Yield: 82%, CAS
Registry Number: 16434-24-5.
4-(4-Chlorophenyl)-1-[(3-ethoxy-4-hydroxyphenyl)methylidene]-3-thiosemicarbazide (28): Yield: 73%, mp =
182–184 ◦C. 1H-NMR (DMSO-d6)
δ
(ppm): 1.35 t (3H, C
7.2 Hz); 6.83 d, 7.19 dd, 7.50 d (3H, 3-C2H5O-4-HO-C6
J = 8.7 Hz); 8.05 s (1H, CH=N); 9.48 s (1H, OH); 10.02 s, 11.76 s (2H, 2xNH). 13C-NMR (DMSO-d6)
H
3CH2O, J = 7.2 Hz); 4.09 q (2H, CH3C
H2O, J =
H
3, J = 8.1, 1.8 Hz); 7.42 d, 7.62 d (4H, 4-Cl-C6H4
,
δ
(ppm): 15.2; 64.5; 112.0; 115.9; 123.1; 125.7; 128.1; 128.4; 129.7; 138.7; 144.5; 147.6; 149.9; 175.9. Anal.
calc. for C16H16ClN3O2S (%): C 54.93; H 4.61; N 12.01. Found: C 54.84; H 4.55; N 12.03.
1-[(3-Chloro-4-hydroxyphenyl)-4-(4-chlorophenyl)methylidene]-3-thiosemicarbazide (29): Yield: 80%, mp =
179–181 ◦C. 1H-NMR (DMSO-d6)
δ
(ppm): 6.99 d, 7.55 dd, 8.09 d (3H, 3-Cl-4-HO-C6
4, J = 9.0 Hz); 8.04 s (1H, CH=N); 10.14 s (1H, OH); 10.72 s, 11.82 s (2H,
2xNH). 13C-NMR (DMSO-d6)
(ppm): 116.9; 121.1; 126.7; 128.3; 128.4; 128.7; 129.1; 129.8; 138.6; 142.7;
H3, J = 8.4, 2.1 Hz);
7.43 d, 7.59 d (4H, 4-Cl-C6
H
δ
155.3; 176.2. Anal. calc. for C14H11Cl2N3OS (%): C 49.42; H 3.26; N 12.35. Found: C 49.50; H 3.25;
N 12.16.
1-[(3-Bromo-4-hydroxyphenyl)-4-(4-chlorophenyl)methylidene]-3-thiosemicarbazide (30): Yield: 81%, mp =
188–190 ◦C. 1H-NMR (DMSO-d6)
δ
(ppm): 6.98 d (J = 8.4 Hz), 7.42 d (J = 8.7 Hz), 7.55–7.61 m, 8.21 d
(J = 1.8 Hz) (7H, 3-Br-4-HO-C6H3 and 4-Cl-C6 4); 8.04 s (1H, CH=N); 10.14 s (1H, OH); 10.78 s, 11.81 s
H
(2H, 2xNH). Anal. calc. for C14H11BrClN3OS (%): C 43.71; H 2.88; N 10.92. Found: C 43.69; H 2.81;
N 10.94.
4-(2,4-Dichlorophenyl)-1-[(4-hydroxy-3-methoxyphenyl)methylidene]-3-thiosemicarbazide (31): Yield: 84%,
CAS Registry Number: 70161-69-2.
4-(4-Bromophenyl)-1-[(3-hydroxyphenyl)methylidene]-3-thiosemicarbazide (32): Yield: 81%, mp = 201–203
◦C. 1H-NMR (DMSO-d6)
δ
(ppm): 6.82–6.86 m, 7.20–7.30 m (4H, 3-HO-C6
J = 9.9, 8.7 Hz); 8.08 s (1H, CH=N); 9.57 s (1H, OH); 10.11 s, 11.85 s (2H, 2xNH). 13C-NMR (DMSO-d6)
(ppm): 114.3; 117.8; 118.0; 119.4; 128.2; 130.1; 131.3; 135.7; 139.0; 144.0; 158.1; 176.3. Anal. calc. for
H4); 7.55 dd (4H, 4-Br-C6H4,
δ
C14H12BrN3OS (%): C 48.01; H 3.45; N 12.00. Found: C 48.09; H 3.41; N 11.94.
4-(4-Bromophenyl)-1-[(4-hydroxyphenyl)methylidene]-3-thiosemicarbazide (33): Yield: 85%, mp = 200–202
◦C. 1H-NMR (DMSO-d6)
δ
(ppm): 6.81 d, 7.73 d (4H, 4-HO-C6
J = 16.2, 9.0 Hz); 8.07 s (1H, CH=N); 9.94 s (1H, OH); 10.01 s, 11.74 s (2H, 2xNH). 13C-NMR (DMSO-d6)
(ppm): 116.0; 117.8; 125.3; 128.0; 130.0; 131.3; 139.1; 144.1; 160.0; 175.74. Anal. calc. for C14H12BrN3OS
(%): C 48.01; H 3.45; N 12.00. Found: C 48.07; H 3.39; N 11.92.
H4, J = 8.7 Hz); 7.56 dd (4H, 4-Br-C6H4,
δ

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4-(4-Fluorophenyl)-1-[(2-hydroxyphenyl)methylidene]-3-thiosemicarbazide (34): Yield: 83%, CAS Registry
Number: 16113-37-4.
4-(4-Fluorophenyl)-1-[(3-hydroxyphenyl)methylidene]-3-thiosemicarbazide (35): Yield: 77%, CAS Registry
Number: 908817-47-0.
4-(4-Fluorophenyl)-1-[(4-hydroxyphenyl)methylidene]-3-thiosemicarbazide (36): Yield: 81%, CAS Registry
Number: 16113-39-6.
4-(4-Fluorophenyl)-1-[(4-hydroxy-3-methoxyphenyl)methylidene]-3-thiosemicarbazide (37): Yield: 79%, CAS
Registry Number: 1494-02-6.
1-[(3-Ethoxy-4-hydroxyphenyl)-4-(4-fluorophenyl)methylidene]-3-thiosemicarbazide (38): Yield: 73%, CAS
Registry Number: 769148-51-8.
1-[(3-Chloro-4-hydroxyphenyl)-4-(4-fluorophenyl)methylidene]-3-thiosemicarbazide (39): Yield: 84%, mp =
193–195 ◦C. 1H-NMR (DMSO-d6)
δ
(ppm): 6.99 d (J = 8.4 Hz), 7.21 t (J = 8.7 Hz), 7.49–7.56 m, 8.10 d (J
4 and 3-Cl-4-HO-C6 3); 8.03 s (1H, CH=N); 10.11 s (1H, OH); 10.71 s, 11.77 s
(2H, 2xNH). 13C-NMR (DMSO-d6)
(ppm): 115.0; 115.3; 116.9; 121.1; 126.7; 128.6; 128.9; 129.0; 129.1;
= 2.1 Hz) (7H, 4-F-C6
H
H
δ
142.4; 155.3; 176.6. Anal. calc. for C14H11ClFN3OS (%): C 51.94; H 3.42; N 12.98. Found: C 51.88; H
3.40; N 12.94.
1-[(3-Bromo-4-hydroxyphenyl)-4-(4-fluorophenyl)methylidene]-3-thiosemicarbazide (40): Yield: 83%, mp
1
= 190-192oC. H-NMR (DMSO-d6)
δ
(ppm): 6.97 d, 7.20 t, 7.49-7.60 m, 8.21 s (7H, 4-F-C6H4 and
3, J = 8.5 Hz); 8.02 s (1H, CH=N); 10.10 s (1H, OH); 10.75 s, 11.74 s (2H, 2xNH). 13C-NMR
(ppm): 108.9; 113.1; 113.5; 114.7;125.3; 127.1; 127.8; 129.9; 134.2; 140.5; 154.4; 174.8. Anal.
3-Br-4-HO-C6
(DMSO-d6)
H
δ
calc. for C14H11BrFN3OS (%): C 45.67; H 3.01; N 11.41. Found: C 45.61; H 3.02; N 11.37.
3.3. General Method of Synthesis of Thiazolidin-4-ones (41–73)
A mixture of 0.003 mol thiosemicarbazones (8–40), 0.003 mol ethyl bromoacetate and 0.003 mol
anhydrous sodium acetate in 5 ml anhydrous ethanol was refluxed for 2h. After cooling of reaction
mixture, the solid obtained was filtered off, dried and then recrystallized from glacial acetic acid.
2-{[(2-Hydroxyphenyl)methylidene]hydrazinylidene}-3-phenyl-1,3-thiazolidin-4-one (41): Yield 75%, CAS
Registry Number: 97636-38-9.
2-{[(3-Hydroxyphenyl)methylidene]hydrazinylidene}-3-phenyl-1,3-thiazolidin-4-one (42): Yield 87%, mp =
235–236 ◦C. 1H-NMR
(9H, C6H5 and 3-HO-C6
δ
(ppm) (DMSO-d6): 4.10 s (2H, CH2); 6.83–6.87 m, 7.12–7.26 m, 7.37–7.55 m
H
4); 8.23 s (1H, CH=N); 9.67 s (1H, OH). 13C-NMR
δ
(ppm) (DMSO-d6): 32.8;
113.9; 118.6; 119.9; 128.8; 129.2; 129.6; 130.3; 135.5; 135.8; 158.0; 158.4; 165.7; 172.5. Anal. calc. for
C16H13N3O2S (%): C 61.72; H 4.21; N 13.50. Found: C 61.67; H 4.17; N 13.49.
2-{[(4-Hydroxyphenyl)methylidene]hydrazinylidene}-3-phenyl-1,3-thiazolidin-4-one (43): Yield 99%, CAS
Registry Number: 97738-29-9.
2-{[(4-Hydroxy-3-methoxyphenyl)methylidene]hydrazinylidene}-3-phenyl-1,3-thiazolidin-4-one (44): Yield
96%, CAS Registry Number: 98052-64-3.
2-{[(3-Ethoxy-4-hydroxyphenyl)methylidene]hydrazinylidene}-3-phenyl-1,3-thiazolidin-4-one (45): Yield 73%,
mp = 229–230 ◦C. 1H-NMR
δ
(ppm) (DMSO-d6): 1.35 t (3H, OCH2C
2CH3); 6.84 d, 7.15 dd, 7.30 d (3H, 3-C2H5O-4-HO-C6
m (5H, C6H5); 8.16 s (1H, CH=N); 9.59 bs (1H, OH). 13C-NMR
(ppm) (DMSO-d6): 15.2; 32.7; 64.3;
H
3, J = 6.9 Hz); 3.99–4.08 m (4H,
C
H2 and OC
H
H
3, J = 8.1, 1.8 Hz); 7.36–7.54
δ
112.2; 116.1; 122.8; 126.0; 128.7; 129.1; 129.5; 135.6; 147.4; 150.4; 158.2; 164.1; 172.4. Anal. calc. for
C18H17N3O3S (%): C 60.83; H 4.82; N 11.82. Found: C 60.80; H 4.77; N 11.81.
2-{[(3-Chloro-4-hydroxyphenyl)methylidene]hydrazinylidene}-3-phenyl-1,3-thiazolidin-4-one (46): Yield 83%,
mp = 215–216 ◦C. 1H-NMR
δ (ppm) (DMSO-d6): 4.09 s (2H, CH2); 7.03 d (J = 8.4 Hz), 7.36–7.56 m,

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7.70 d (J = 2.1 Hz) (8H, C6H5 and 3-Cl-4-HO-C6H δ
3); 8.20 s (1H, CH=N); 10.87 s (1H, OH). 13C-NMR
(ppm) (DMSO-d6): 32.8; 117.4; 120.7; 126.8; 128.2; 128.7; 129.1; 129.6; 129.7; 135.5; 156.0; 156.9; 165.0;
172.4. Anal. calc. for C16H12ClN3O2S (%): C 55.57; H 3.50; N 12.15. Found: C 55.61; H 3.47; N 12.13.
2-{[(3-Bromo-4-hydroxyphenyl)methylidene]hydrazinylidene}-3-phenyl-1,3-thiazolidin-4-one (47): Yield 88%,
mp = 216–218 ◦C. 1H-NMR
7.85 d (J = 1.8 Hz) (8H, C6H5 and 3-Br-4-HO-C6
δ
(ppm) (DMSO-d6): 4.09 s (2H, CH2); 7.01 d (J = 8.4 Hz), 7.36–7.60 m,
H
3); 8.20 s (1H, CH=N); 10.90 s (1H, OH). 13C-NMR
δ
(ppm) (DMSO-d6): 32.8; 110.2; 117.0; 127.3; 128.8; 128.9; 129.1; 129.6; 132.8; 135.5; 156.8; 156.9; 165.0;
172.5. Anal. calc. for C16H12BrN3O2S (%): C 49.24; H 3.10; N 10.77. Found: C 49.21; H 3.03; N 10.74.
3-(2-Chlorophenyl)-2-{[(2-hydroxyphenyl)methylidene]hydrazinylidene}-1,3-thiazolidin-4-one (48): Yield 78%,
mp = 168–170 ◦C. 1H-NMR
δ
(ppm) (DMSO-d6): 4.28 dd (2H, CH2, J = 33.6, 17.4 Hz); 6.89–6.95 m,
7.31–7.37 m, 7.53–7.62 m, 7.68–7.72 m (8H, 2-Cl-C6 4 and 2-HO-C6 4); 8.56 s (1H, CH=N); 10.79 s (1H,
OH). 13C-NMR
(ppm) (DMSO-d6): 33.0; 116.8; 118.7; 120.0; 128.9; 130.5; 131.1; 131.5; 131.7; 132.1;
H
H
δ
132.9; 133.0; 158.6; 159.9; 163.7; 171.6. Anal. calc. for C16H12ClN3O2S (%): C 55.57; H 3.50; N 12.15.
Found: C 55.55; H 3.49; N 12.07.
3-(2-Chlorophenyl)-2-{[(3-hydroxyphenyl)methylidene]hydrazinylidene}-1,3-thiazolidin-4-one (49): Yield 75%,
mp = 192–194 ◦C. 1H-NMR
δ
(ppm) (DMSO-d6): 4.21 dd (2H, CH2, J = 29.7, 17.4 Hz); 6.83–6.87 m,
7.12–7.26 m, 7.51–7.60 m, 7.67–7.70 m (8H, 2-Cl-C6H4 and 3-HO-C6 4); 8.23 s (1H, CH=N); 9.69 s (1H,
OH). 13C-NMR
(ppm) (DMSO-d6): 32.7; 113.9; 118.7; 120.0; 128.9; 130.3; 130.5; 131.5; 131.6; 132.1;
H
δ
133.1; 135.6; 158.0; 158.9; 164.2; 171.8. Anal. calc. for C16H12ClN3O2S (%): C 55.57; H 3.50; N 12.15.
Found: C 55.54; H 3.44; N 12.10.
3-(2-Chlorophenyl)-2-{[(4-hydroxyphenyl)methylidene]hydrazinylidene}-1,3-thiazolidin-4-one (50): Yield 85%,
mp = 178–180 ◦C. 1H-NMR
δ
(ppm) (DMSO-d6): 4.18 dd (2H, CH2, J = 27.6, 17.1 Hz); 6.82 d, 7.51–7.58
m, 7.67–7.70 m (8H, 2-Cl-C6H4 and 4-HO-C6 4, J = 8.7 Hz); 8.18 s (1H, CH=N); 10.06 s (1H, OH).
13C-NMR
(ppm) (DMSO-d6): 32.6; 116.2; 125.4; 128.9; 130.2; 130.5; 131.5; 131.6; 132.1; 133.2; 158.6;
H
δ
160.6; 162.5; 171.8. Anal. calc. for C16H12ClN3O2S (%): C 55.57; H 3.50; N 12.15. Found: C 55.58; H
3.46; N 12.16.
3-(2-Chlorophenyl)-2-{[(3-ethoxy-4-hydroxyphenyl)methylidene]hydrazinylidene}-1,3-thiazolidin-4-one (51):
Yield 81%, mp = 196–198 ◦C. 1H-NMR
δ
(ppm) (DMSO-d6): 1.34 t (3H, OCH2C
2CH3, J = 6.9 Hz); 4.18 dd (2H, CH2, J = 26.7, 17.4 Hz); 6.83 d, 7.15 dd, 7.30 d (3H,
3-C2H5O-4-HO-C6 3, J = 8.1, 1.8 Hz); 7.51–7.58 m, 7.66–7.70 m (4H, 2-Cl-C6H4); 8.16 s (1H, CH=N);
9.61 s (1H, OH). 13C-NMR
(ppm) (DMSO-d6): 15.1; 32.6; 64.2; 112.1; 116.1; 122.9; 125.8; 128.9; 130.5;
H3, J = 6.9 Hz); 4.02
q (2H, OC
H
H
δ
131.5; 131.6; 132.1; 133.1; 147.4; 150.4; 158.7; 162.6; 171.8. Anal. calc. for C18H16ClN3O3S (%): C 55.46;
H 4.14; N 10.78. Found: C 55.48; H 4.11; N 10.68.
3-(3-Chlorophenyl)-2-{[(2-hydroxyphenyl)methylidene]hydrazinylidene}-1,3-thiazolidin-4-one (52): Yield 81%,
mp = 194–196 ◦C. 1H-NMR
(8H, 3-Cl-C6H4 and 2-HO-C6
δ
(ppm) (DMSO-d6): 4.15 s (2H, CH2); 6.90–7.00 m, 7.31–7.44 m, 7.53–7.59 m
H
4); 8.59 s (1H, CH=N); 10.82 s (1H, OH). 13C-NMR
δ
(ppm) (DMSO-d6):
33.3; 116.8; 118.8; 120.0; 127.8; 128.9; 129.4; 131.1; 131.2; 132.9; 133.6; 136.6; 158.6; 159.7; 165.0; 172.1.
Anal. calc. for C16H12ClN3O2S (%): C 55.57; H 3.50; N 12.15. Found: C 55.49; H 3.43; N 12.11.
3-(3-Chlorophenyl)-2-{[(3-hydroxyphenyl)methylidene]hydrazinylidene}-1,3-thiazolidin-4-one (53): Yield 78%,
mp = 198–200 ◦C. 1H-NMR
m, 7.55–7.57 m (8H, 3-Cl-C6H4 and 3-HO-C6
δ
(ppm) (DMSO-d6): 4.08 s (2H, CH2); 6.83–6.87 m, 7.12–7.26 m, 7.39–7.42
H
4); 8.25 s (1H, CH=N); 9.66 s (1H, OH). 13C-NMR
δ
(ppm) (DMSO-d6): 32.9; 113.9; 118.7; 119.9; 127.8; 128.9; 129.3; 130.3; 131.2; 133.5; 135.7; 136.8; 158.0;
158.6; 165.4; 172.3. Anal. calc. for C16H12ClN3O2S (%): C 55.57; H 3.50; N 12.15. Found: C 55.51; H
3.51; N 12.13.
3-(3-Chlorophenyl)-2-{[(4-hydroxyphenyl)methylidene]hydrazinylidene}-1,3-thiazolidin-4-one (54): Yield 83%,
mp = 262–264 ◦C. 1H-NMR
δ
(ppm) (DMSO-d6): 4.06 s (2H, CH2); 6.82 d, 7.37–7.41 m, 7.54–7.59 m
(8H, 3-Cl-C6
H4 and 4-HO-C6
H
4, J = 8.7 Hz); 8.21 s (1H, CH=N); 10.05 s (1H, OH). 13C-NMR
δ
(ppm)

Molecules 2019, 24, 3029
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(DMSO-d6): 32.8; 116.2; 125.5; 127.8; 128.9; 129.2; 130.1; 131.2; 133.5; 136.9; 158.4; 160.6; 163.7; 172.3.
Anal. calc. for C16H12ClN3O2S (%): C 55.57; H 3.50; N 12.15. Found: C 55.53; H 3.47; N 12.12.
3-(3-Chlorophenyl)-2-{[(4-hydroxy-3-methoxyphenyl)methylidene]hydrazinylidene}-1,3-thiazolidin-4-one (55):
Yield 81%, mp = 210–212 ◦C. 1H-NMR
7.17 dd, 7.32 d (3H, 3-CH3O-4-HO-C6
δ
(ppm) (DMSO-d6): 3.79 s (3H, OCH3); 4.07 s (2H, CH2); 6.83 d,
3, J = 8.4, 1.8 Hz), 7.38–7.41 m, 7.54–7.56 m (4H, 3-Cl-C6H4);
8.20 s (1H, CH=N); 9.65 s (1H, OH). 13C-NMR
(ppm) (DMSO-d6): 32.8; 56.0; 111.0; 116.1; 122.9; 125.9;
H
δ
127.8; 128.9; 129.2; 131.1; 133.5; 136.9; 148.3; 150.2; 158.5; 163.8; 172.3. Anal. calc. for C17H14ClN3O3S
(%): C 54.33; H 3.75; N 11.18. Found: C 54.34; H 3.71; N 11.13.
3-(3-Chlorophenyl)-2-{[(3-ethoxy-4-hydroxyphenyl)methylidene]hydrazinylidene}-1,3-thiazolidin-4-one (56):
Yield 78%, mp = 214–216 ◦C. 1H-NMR
δ
(ppm) (DMSO-d6): 1.35 t (3H, OCH2C
2CH3); 6.84 d, 7.16 dd, 7.31 d (3H, 3-C2H5O-4-HO-C6 3, J = 8.4, 1.8 Hz), 7.37–7.41
m, 7.54–7.56 m (4H, 3-Cl-C6H4); 8.18 s (1H, CH=N); 9.58 s (1H, OH). 13C-NMR
(ppm) (DMSO-d6):
H3, J = 6.9 Hz); 3.99–4.06
m (4H, C
H
2 and OC
H
H
δ
15.2; 32.6; 63.9; 116.1; 122.8; 125.9; 127.8; 128.4; 129.2; 131.1; 133.5; 136.9; 147.4; 150.4; 155.2; 158.5; 162.1;
172.3. Anal. calc. for C18H16ClN3O3S (%): C 55.46; H 4.14; N 10.78. Found: C 55.41; H 4.10; N 10.75.
3-(4-Chlorophenyl)-2-{[(2-hydroxyphenyl)methylidene]hydrazinylidene}-1,3-thiazolidin-4-one (57): Yield 72%,
mp = 238–240 ◦C. 1H-NMR
δ
(ppm) (DMSO-d6): 4.16 s (2H, CH2); 6.89–6.95 m, 7.31–7.37 m, 7.54 dd
4, J = 7.8, 1.8 Hz); 7.46 d, 7.61 d (4H, 4-Cl-C6H4, J = 8.8 Hz); 8.57 s (1H, CH=N); 10.82 s
(1H, OH). 13C-NMR
(ppm) (DMSO-d6): 33.2; 116.8; 118.8; 120.0; 129.7; 130.7; 131.1; 132.9; 133.9; 134.2;
(4H, 2-HO-C6
H
δ
158.6; 159.6; 165.1; 172.2. Anal. calc. for C16H12ClN3O2S (%): C 55.57; H 3.50; N 12.15. Found: C 55.53;
H 3.44; N 12.09.
3-(4-Chlorophenyl)-2-{[(3-hydroxyphenyl)methylidene]hydrazinylidene}-1,3-thiazolidin-4-one (58): Yield 77%,
mp = 230–232 ◦C. 1H-NMR
δ
(ppm) (DMSO-d6): 4.09 s (2H, CH2); 6.85 dd, 7.12–7.26 m (4H, 3-HO-C6H4
J = 7.8, 1.8 Hz); 7.44 d, 7.60 d (4H, 4-Cl-C6H4, J = 8.7 Hz); 8.24 s (1H, CH=N); 9.80 s (1H, OH). 13C-NMR
(ppm) (DMSO-d6): 32.9; 113.9; 118.7; 119.9; 129.6; 130.3; 130.7; 133.8; 134.4; 135.7; 158.1; 158.6; 165.5;
,
δ
172.4. Anal. calc. for C16H12ClN3O2S (%): C 55.57; H 3.50; N 12.15. Found: C 55.50; H 3.46; N 12.08.
3-(4-Chlorophenyl)-2-{[(4-hydroxyphenyl)methylidene]hydrazinylidene}-1,3-thiazolidin-4-one (59): Yield 75%,
mp = 280–282 ◦C. 1H-NMR
Hz), 7.56–7.60 m (8H, 4-Cl-C6
δ
(ppm) (DMSO-d6): 4.07 s (2H, CH2); 6.83 d (J = 8.7 Hz), 7.43 d (J = 8.8
H
4 and 4-HO-C6 δ
H
4); 8.20 s (1H, CH=N); 10.05 s (1H, OH). 13C-NMR
(ppm) (DMSO-d6): 32.8; 116.2; 125.5; 129.6; 130.1; 130.7; 133.7; 134.4; 158.3; 160.6; 163.8; 172.3. Anal.
calc. for C16H12ClN3O2S (%): C 55.57; H 3.50; N 12.15. Found: C 55.56; H 3.51; N 12.11.
3-(4-Chlorophenyl)-2-{[(4-hydroxy-3-methoxyphenyl)methylidene]hydrazinylidene}-1,3-thiazolidin-4-one (60):
Yield 80%, mp = 226–228 ◦C, 1H-NMR
δ
(ppm) (DMSO-d6): 3.79 s (3H, OCH3); 4.07 s (2H, CH2); 6.83 d,
3, J = 8.2, 1.8 Hz); 7.43 d, 7.59 d (4H, 4-Cl-C6H4, J = 8.9 Hz); 8.18
s (1H, CH=N); 9.67 s (1H, OH). 13C-NMR
(ppm) (DMSO-d6): 32.8; 55.9; 110.8; 116.0; 122.9; 125.9;
7.16 dd, 7.31 d (3H, 3-CH3O-4-HO-C6
H
δ
129.6; 130.7; 133.7; 134.4; 148.3; 150.1; 158.4; 163.9; 172.3. Anal. calc. for C17H14ClN3O3S (%): C 54.33;
H 3.75; N 11.18. Found: C 54.29; H 3.74; N 11.16.
3-(4-Chlorophenyl)-2-{[(3-ethoxy-4-hydroxyphenyl)methylidene]hydrazinylidene}-1,3-thiazolidin-4-one (61):
Yield 83%, mp = 218–220 ◦C, 1H-NMR
δ
(ppm) (DMSO-d6): 1.35 t (3H, OCH2C
2CH3 and CH2); 6.84 d, 7.15 dd, 7.30 d (3H, 3-C2H5-4-HO-C6 3, J = 8.1, 1.8 Hz); 7.43 d,
7.59 d (4H, 4-Cl-C6H4, J = 8.7 Hz); 8.17 s (1H, CH=N); 9.59 s (1H, OH). 13C-NMR
(ppm) (DMSO-d6):
H3, J = 6.9 Hz); 3.99–4.07
m (4H, OC
H
H
δ
15.2; 32.8; 64.3; 112.2; 116.1; 122.8; 125.9; 129.6; 130.7; 133.7; 134.4; 147.4; 150.4; 158.4; 163.9; 172.3. Anal.
calc. for C18H16ClN3O3S (%): C 55.46; H 4.14; N 10.78. Found: C 55.43; H 4.15; N 10.69.
2-{[(3-Chloro-4-hydroxyphenyl)methylidene]hydrazinylidene}-3-(4-chlorophenyl)-1,3-thiazolidin-4-one (62):
Yield 71%, mp = 231–233 ◦C, 1H-NMR
δ
(ppm) (DMSO-d6): 4.08 s (2H, CH2); 7.03 d, 7.55 dd, 7.70 d
3, J = 8.5, 2.0 Hz); 7.43 d, 7.59 d (4H, 4-Cl-C6H4, J = 8.8 Hz); 8.21 s (1H, CH=N);
10.86 s (1H, OH). 13C-NMR
(ppm) (DMSO-d6): 32.8; 117.4; 120.7; 126.8; 128.3; 129.6; 129.7; 129.9;
(3H, 3-Cl-4-HO-C6
H
δ

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130.4; 130.7; 134.4; 157.1; 164.8; 172.3. Anal. calc. for C16H11Cl2N3O2S (%): C 50.54; H 2.92; N 11.05.
Found: C 50.44; H 2.88; N 10.99.
2-{[(3-Bromo-4-hydroxyphenyl)methylidene]hydrazinylidene}-3-(4-chlorophenyl)-1,3-thiazolidin-4-one (63):
Yield 74%, mp = 235–236 ◦C, 1H-NMR
δ
(ppm) (DMSO-d6): 4.08 s (2H, CH2); 7.01 d (J = 8.4 Hz), 7.43 d
(J = 8.7 Hz), 7.58-7.66 m, 7.85 d (J = 1.8 Hz) (7H, 3-Br-4-HO-C6H3 and 4-Cl-C6 4); 8.20 s (1H, CH=N);
10.91 s (1H, OH). 13C-NMR
(ppm) (DMSO-d6): 32.8; 110.2; 117.0; 126.8; 127.2; 128.9; 129.6; 130.7;
H
δ
132.8; 133.7; 134.4; 157.0; 164.8; 172.3. Anal. calc. for C16H11BrClN3O2S (%): C 45.25; H 2.61; N 9.89.
Found: C 45.23; H 2.59; N 9.90.
3-(2,4-Dichlorophenyl)-2-{[(4-hydroxy-3-methoxyphenyl)methylidene]hydrazinylidene}-1,3-thiazolidin-4-one
(
64): Yield 83%, mp = 140–142 ◦C, 1H-NMR
δ
(ppm) (DMSO-d6): 3.79 s (3H, OCH3); 4.18 dd (2H, CH2,
J = 32.1, 17.7 Hz); 6.83 d, 7.16 dd, 7.31 d (3H, 3-CH3O-4-HO-C6 3, J = 8.4, 1.8 Hz), 7.63 d, 7.90 t (3H,
2,4-diCl-C6H3, J = 1.2 Hz); 8.18 s (1H, CH=N); 9.67 s (1H, OH). 13C-NMR
(ppm) (DMSO-d6): 32.7;
H
δ
56.0; 110.9; 116.0; 123.0; 125.8; 129.1; 130.2; 132.3; 132.8; 133.4; 135.4; 148.3; 150.2; 158.9; 162.2; 171.6.
Anal. calc. for C17H13Cl2N3O3S (%): C 49.77; H 3.19; N 10.24. Found: C 49.74; H 3.12; N 10.23.
3-(4-Bromophenyl)-2-{[(3-hydroxyphenyl)methylidene]hydrazinylidene}-1,3-thiazolidin-4-one (65): Yield 83%,
◦
1
mp = 242–244 C. H-NMR
3-HO-C6
4); 7.38 d, 7.73 d (4H, 4-Br-C6H4, J = 8.7 Hz); 8.24 s (1H, CH=N); 9.67 s (1H, OH). 13C-NMR
(ppm) (DMSO-d6): 32.9; 113.9; 118.7; 119.9; 122.3; 130.3; 131.0; 132.6; 134.8; 135.7; 158.0; 158.6; 165.4;
δ (ppm) (DMSO-d6): 4.09 s (2H, CH2); 6.83–6.87 m, 7.12–7.26 m (4H,
H
δ
172.3. Anal. calc. for C16H12BrN3O2S (%): C 49.24; H 3.10; N 10.77. Found: C 49.22; H 3.08; N 10.76.
3-(4-Bromophenyl)-2-{[(4-hydroxyphenyl)methylidene]hydrazinylidene}-1,3-thiazolidin-4-one (66): Yield 74%,
mp = 288–290 ◦C. 1H-NMR
δ
(ppm) (DMSO-d6): 4.07 s (2H, CH2); 6.82 d, 7.57 d (4H, 4-HO-C6
H4, J =
8.7 Hz); 7.37 d, 7.72 d (4H, 4-Br-C6H4, J = 8.7 Hz); 8.19 s (1H, CH=N); 10.04 s (1H, OH). 13C-NMR
δ
(ppm) (DMSO-d6): 32.8; 116.2; 122.2; 125.5; 130.1; 131.0; 132.6; 134.9; 158.3; 160.6; 163.7; 172.3. Anal.
calc. for C16H12BrN3O2S (%): C 49.24; H 3.10; N 10.77. Found: C 49.19; H 3.06; N 10.78.
3-(4-Fluorophenyl)-2-{[(2-hydroxyphenyl)methylidene]hydrazinylidene}-1,3-thiazolidin-4-one (67): Yield 97%,
CAS Registry Number: 16113-43-2.
3-(4-Fluorophenyl)-2-{[(3-hydroxyphenyl)methylidene]hydrazinylidene}-1,3-thiazolidin-4-one (68): Yield 85%,
◦
1
mp = 204–206 C. H-NMR
δ
(ppm) (DMSO-d6): 4.09 s (2H, CH2); 6.83–6.86 m, 7.12–7.49 m (8H,
(ppm) (DMSO-d6): 32.8;
4-F-C6 4 and 3-HO-C6 δ
H
H
4); 8.23 s (1H, CH=N); 9.66 s (1H, OH). 13C-NMR
113.9; 116.3; 116.7; 118.6; 119.9; 130.3; 131.0; 131.7; 135.8; 158.1; 158.5; 165.7; 172.5. Anal. calc. for
C16H12FN3O2S (%): C 58.35; H 3.67; N 12.76. Found: C 58.29; H 3.66; N 12.78.
3-(4-Fluorophenyl)-2-{[(4-hydroxyphenyl)methylidene]hydrazinylidene}-1,3-thiazolidin-4-one (69): Yield 86%,
CAS Registry Number: 16113-47-6.
3-(4-Fluorophenyl)-2-{[(4-hydroxy-3-methoxyphenyl)methylidene]hydrazinylidene}-1,3-thiazolidin-4-one (70):
Yield 79%, mp = 236–238 ◦C. 1H-NMR
δ
(ppm) (DMSO-d6): 3.79 s (3H, OCH3); 4.07 s (2H, CH2); 6.83 d,
7.17 d, 7.32–7.48 m (7H, 4-F-C6H4 and 3-CH3O-4-HO-C6 3, J = 8.2 Hz); 8.18 s (1H, CH=N); 9.65 s (1H,
OH). 13C-NMR
(ppm) (DMSO-d6): 32.7; 56.0; 111.0; 116.1; 116.3; 116.6; 122.9; 126.0; 130.9; 131.0; 148.3;
H
δ
150.2; 158.3; 164.1; 172.4. Anal. calc. for C17H14FN3O3S (%): C 56.82; H 3.93; N 11.69. Found: C 56.79;
H 3.95; N 11.68.
2-{[(3-Ethoxy-4-hydroxyphenyl)methylidene]hydrazinylidene}-3-(4-fluorophenyl)-1,3-thiazolidin-4-one (71):
Yield 69%, mp = 240–242 ◦C. 1H-NMR
δ
(ppm) (DMSO-d6): 1.35 t (3H, OCH2C
2CH3); 6.84 d, 7.16 d, 7.30–7.47 m (7H, 4-F-C6 4 and 3-C2H5O-4-HO-C6
8.2 Hz); 8.17 s (1H, CH=N); 9.57 s (1H, OH). 13C-NMR
(ppm) (DMSO-d6): 15.2; 32.7; 64.3; 112.3; 116.1;
H
3, J = 6.9 Hz); 3.99–4.06
m (4H, C
H
2 and OC
H
H
H
3, J =
δ
116.3; 116.6; 122.8; 126.0; 130.9; 131.1; 147.4; 150.4; 158.3; 164.1; 172.5. Anal. calc. for C18H16FN3O3S
(%): C 57.90; H 4.32; N 11.25. Found: C 57.87; H 4.33; N 11.18.

Molecules 2019, 24, 3029
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2-{[(3-Chloro-4-hydroxyphenyl)methylidene]hydrazinylidene}-3-(4-fluorophenyl)-1,3-thiazolidin-4-one (72):
Yield 76%, mp = 222–224 ◦C. 1H-NMR
(ppm) (DMSO-d6): 4.07 s (2H, CH2); 7.03 d, 7.55 dd, 7.70 d
(3H, 3-Cl-4-HO-C6 3, J = 8.4, 1.8 Hz); 7.32–7.47 m (4H, 4-F-C6H4); 8.21 s (1H, CH=N); 10.86 s (1H,
OH). 13C-NMR
(ppm) (DMSO-d6): 32.8; 116.3; 116.6; 117.4; 120.7; 126.8; 128.3; 129.7; 130.9; 131.1;
δ
H
δ
156.0; 157.0; 165.0; 172.5. Anal. calc. for C16H11ClFN3O2S (%): C 52.83; H 3.05; N 11.55. Found: C
52.86; H 3.03; N 11.56.
2-{[(3-Bromo-4-hydroxyphenyl)methylidene]hydrazinylidene}-3-(4-fluorophenyl)-1,3-thiazolidin-4-one (73):
Yield 80%, mp = 224–226 ◦C. 1H-NMR
δ
(ppm) (DMSO-d6): 4.07 s (2H, CH2); 7.01 d, 7.59 dd, 7.85 d
3, J = 8.2, 1.8 Hz); 7.32–7.48 m (4H, 4-F-C6H4); 8.20 s (1H, CH=N); 10.89 s (1H,
(ppm) (DMSO-d6): 32.8; 110.2; 116.3; 116.7; 117.1; 127.3; 128.9; 130.9; 131.1; 131.7;
(3H, 3-Br-4-HO-C6
OH). 13C-NMR
H
δ
132.8; 156.9; 165.0; 172.5. Anal. calc. for C16H11BrFN3O2S (%): C 47.07; H 2.72; N 10.29. Found: C
46.99; H 2.69; N 10.26.
3.4. General Method of Synthesis of (4-oxothiazolidin-5-ylo)Acetic Acid Derivatives (74–88)
A mixture of 0.0015 mol thiosemicarbazones (8–14 or 24–31), 0.0015 mol maleic anhydride in 4
mL glacial acetic acid was refluxed for 2h. After cooling of reaction mixture, the solid obtained was
filtered off, dried and then recrystallized from glacial acetic acid or isopropanol.
[2-{[(2-Hydroxyphenyl)methy◦lidene]hydrazinylidene}-4-oxo-3-phenyl-1,3-thiazolidin-5-yl]acetic acid (74):
1
Yield 64%, mp = 228–230 C. H-NMR
δ
(ppm) (DMSO-d6): 3.15 d (2H, C
H
2CH, J = 6.0 Hz);
4.64 t (1H, CH2C
H
, J = 6.0 Hz); 6.89–6.95 m, 7.31–7.57 m (9H, C6H5 and 2-HO-C6
H4); 8.56 s (1H,
CH=N); 10.83 s (1H, OH); 12.88 bs (1H, COOH). 13C-NMR
δ
(ppm) (DMSO-d6): 37.1; 43.5; 116.8; 118.8;
120.0; 128.7; 129.3; 129.6; 131.2; 132.9; 135.5; 158.6; 159.6; 164.6; 172.3; 174.0.
[2-{[(3-Hydroxyphenyl)methy◦lidene]hydrazinylidene}-4-oxo-3-phenyl-1,3-thiazolidin-5-yl]acetic acid (75):
1
Yield 59%, mp = 214–216 C. H-NMR
δ
(ppm) (DMSO-d6): 3.11 d (2H, C
H
2CH, J = 6.0 Hz);
4.55 t (1H, CH2C
1.8 Hz); 8.22 s (1H, CH=N); 9.67 s (1H, OH); 12.85 bs (1H, COOH). 13C-NMR
43.0; 113.7; 118.6; 120.0; 128.7; 129.2; 129.6; 130.3; 135.6; 135.7; 158.0; 158.3; 165.2; 172.3; 174.2.
H
, J = 6.0 Hz); 6.84 dd, 7.10–7.26 m, 7.37–7.55 m (9H, C6
H5 and 3-HO-C6
H4, J = 8.1,
δ
(ppm) (DMSO-d6): 37.3;
[2-{[(4-Hydroxyphenyl)methy◦lidene]hydrazinylidene}-4-oxo-3-phenyl-1,3-thiazolidin-5-yl]acetic acid (76):
1
Yield 77%, mp = 236–238 C. H-NMR
δ
(ppm) (DMSO-d6): 3.09 d (2H, C
H
2CH, J = 6.0 Hz);
4.53 t (1H, CH2C
CH=N); 10.05 s (1H, OH); 12.54 bs (1H, COOH). 13C-NMR
128.7; 129.5; 130.1; 131.3; 135.7; 158.1; 160.6; 166.6; 172.3; 174.2.
H
, J = 6.0 Hz); 6.82 d, 7.36–7.59 m (9H, C6H5 and 4-HO-C6
H4, J = 8.4 Hz); 8.18 s (1H,
δ
(ppm) (DMSO-d6): 37.4; 42.9; 116.2; 125.6;
[2-{[(4-Hydroxy-3-methoxyphenyl)methylidene]hydrazinylidene}-4-oxo-3-phenyl-1,3-thiazolidin-5-yl]acetic
acid (77): Yield 82%, CAS Registry Number: 1008533-45-6.
[2-{[(3-Ethoxy-4-hydroxyphenyl)methylidene]hydrazinylidene}-4-oxo-3-phenyl-1,3-thiazolidin-5-yl]acetic acid
(
78): Yield 73%, mp = 224–226 ◦C. 1H-NMR
δ
(ppm) (DMSO-d6): 1.35 t (3H, OCH2C
2CH, J = 6.0 Hz); 4.03 q (2H, OC 2CH3, J = 6.9 Hz); 4.53 t (1H, CH2C
6.83 d, 7.14 dd, 7.29 d (3H, 3-C2H5O-4-HO-C6 3, J = 8.4, 1.8 Hz); 7.35–7.55 m (5H, C6H5); 8.15 s (1H,
CH=N); 9.57 s (1H, OH); 12.81 bs (1H, COOH). 13C-NMR
(ppm) (DMSO-d6): 15.2; 37.3; 42.9; 64.3;
H
3, J = 6.9 Hz);
3.10 d (2H, C
H
H
H, J = 6.0 Hz);
H
δ
112.1; 116.1; 122.9; 125.9; 128.7; 129.1; 129.5; 135.8; 147.5; 150.4; 158.3; 163.3; 172.2; 174.2.
[2-{[(3-Chloro-4-hydroxyphenyl)methylidene]hydrazinylidene}-4-oxo-3-phenyl-1,3-thiazolidin-5-yl]acetic acid
(
79): Yield 76%, mp = 244–246 ◦C. 1H-NMR
δ
(ppm) (DMSO-d6): 3.11 d (2H, C
, J = 6.0 Hz); 7.03 d, 7.36–7.56 m, 7.69 d (8H, 3-Cl-4-HO-C6 3 and C6
8.20 s (1H, CH=N); 10.83 s (1H, OH); 12.66 bs (1H, COOH). 13C-NMR
(ppm) (DMSO-d6): 37.3; 42.9;
H
2CH, J = 6.0 Hz); 4.55 t
(1H, CH2C
H
H
H
5, J = 8.4, 1.8 Hz);
δ
117.4; 120.7; 126.9; 128.2; 128.7; 129.2; 129.5; 129.7; 135.7; 155.9; 156.9; 164.4; 172.3; 174.2.
[2-{[(3-Bromo-4-hydroxyphenyl)methylidene]hydrazinylidene}-4-oxo-3-phenyl-1,3-thiazolidin-5-yl]acetic acid
(
80): Yield 79%, mp = 252–254 ◦C. 1H-NMR
δ (ppm) (DMSO-d6): 3.11 d (2H, CH2CH, J = 6.0 Hz); 4.54 t

Molecules 2019, 24, 3029
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(1H, CH2C
H
, J = 6.0 Hz); 7.00 d, 7.58 dd, 7.84 d (3H, 3-Br-4-HO-C6
H
3, J = 8.4, 2.1 Hz); 7.35–7.55 m (5H,
C6H5); 8.19 s (1H, CH=N); 10.93 s (1H, OH); 12.63 bs (1H, COOH). 13C-NMR
δ (ppm) (DMSO-d6): 37.2;
42.9; 110.2; 117.0; 127.3; 128.7; 128.9; 129.2; 129.6; 132.8; 135.7; 156.7; 156.9; 164.4; 172.3; 174.2.
[3-(4-Chlorophenyl)-2-{[(2-hydroxyphenyl)methylidene]hydrazinylidene}-4-oxo-1,3-thiazolidin-5-yl]acetic acid
(
81): Yield 73%, mp = 234–236 ◦C. 1H-NMR
δ
(ppm) (DMSO-d6): 3.16 d (2H, C
, J = 6.0 Hz); 6.89–7.00 m, 7.31–7.37 m, 7.53–7.55 m (4H, 2-HO-C6
4-Cl-C6H4, J = 8.7 Hz); 8.57 s (1H, CH=N); 10.80 s (1H, OH); 12.76 bs (1H, COOH). 13C-NMR
H
H
2CH, J = 6.0 Hz); 4.63 t
4); 7.45 d, 7.63 d (4H,
(ppm)
(1H, CH2C
H
δ
(DMSO-d6): 37.0; 43.6; 116.8; 118.8; 120.0; 129.7; 130.6; 131.1; 133.0; 133.9; 134.3; 158.6; 159.6; 164.4;
172.3; 173.9.
[3-(4-Chlorophenyl)-2-{[(3-hydroxyphenyl)methylidene]hydrazinylidene}-4-oxo-1,3-thiazolidin-5-yl]acetic acid
(
82): Yield 68%, mp = 226–228 ◦C. 1H-NMR
δ
(ppm) (DMSO-d6): 3.12 d (2H, C
, J = 6.0 Hz); 6.83–6.87 m, 7.11–7.13 m, 7.20–7.26 m, (4H, 3-HO-C6
4-Cl-C6H4, J = 8.7 Hz); 8.23 s (1H, CH=N); 9.66 s (1H, OH); 12.79 s (1H, COOH). 13C-NMR
H
H
2CH, J = 6.0 Hz); 4.55 t
4); 7.43 d, 7.61 d (4H,
(ppm)
(1H, CH2C
H
δ
(DMSO-d6): 37.3; 43.1; 113.7; 118.7; 120.0; 129.7; 130.4; 130.6; 133.8; 134.4; 135.7; 158.0; 158.5; 164.9;
172.3; 174.1.
[3-(4-Chlorophenyl)-2-{[(4-hydroxyphenyl)methylidene]hydrazinylidene}-4-oxo-1,3-thiazolidin-5-yl]acetic acid
(
83): Yield 75%, mp = 234–235 ◦C. 1H-NMR
δ
(ppm) (DMSO-d6): 3.10 d (2H, C
, J = 6.0 Hz); 6.82 d, 7.57 d (4H, 4-HO-C6 4, J = 8.7 Hz); 7.42 d, 7.60 d (4H, 4-Cl-C6H4, J =
8.7 Hz); 8.19 s (1H, CH=N); 10.04 s (1H, OH); 12.84 s (1H, COOH). 13C-NMR
(ppm) (DMSO-d6): 37.3;
H2CH, J = 6.0 Hz); 4.52 t
(1H, CH2C
H
H
δ
43.0; 116.2; 125.5; 129.6; 130.1; 130.6; 133.7; 134.5; 158.3; 160.6; 163.2; 172.3; 174.0.
[3-(4-Chlorophenyl)-2-{[(4-hydroxy-3-methoxyphenyl)methylidene]hydrazinylidene}-4-oxo-1,3-thiazolidin-5-
yl]acetic acid (84): Yield 71%, mp = 254–256 ◦C. 1H-NMR
δ
(ppm) (DMSO-d6): 3.11 d (2H, C
, J = 6.0 Hz); 6.82 d, 7.15 dd, 7.31 d (3H,
3, J = 8.1, 1.8 Hz); 7.42 d, 7.61 d (4H, 4-Cl-C6H4, J = 8.7 Hz); 8.18 s (1H, CH=N); 9.66
s (1H, OH); 12.71 s (1H, COOH). 13C-NMR
(ppm) (DMSO-d6): 37.2; 43.0; 56.0; 110.7; 116.0; 123.1;
H2CH,
J = 6.0 Hz); 3.79 s (3H, OCH3); 4.53 t (1H, CH2C
4-HO-3-CH3O-C6
H
H
δ
125.9; 129.6; 130.6; 133.7; 134.5; 148.3; 150.2; 158.5; 163.1; 172.3; 174.0.
[(3-(4-Chlorophenyl)-2-{[(3-ethoxy-4-hydroxyphenyl)methylidene]hydrazinylidene}-4-oxo-1,3-thiazolidin-5-
yl]acetic acid (85): Yield 69%, mp = 224–226 ◦C. 1H-NMR
δ
(ppm) (DMSO-d6): 1.35 t (3H, OCH2C
H3, J
= 6.9 Hz); 3.10 d (2H, C
Hz); 6.83 d, 7.14 dd, 7.29 d (3H, 3-C2H5O-4-HO-C6
8.7 Hz); 8.16 s (1H, CH=N); 9.58 s (1H, OH); 12.80 s (1H, COOH). 13C-NMR
H
2CH, J = 6.0 Hz); 4.03 q (2H, OC
H
2CH3, J = 6.9 Hz); 4.52 t (1H, C 2CH, J = 6.0
H
H
3, J = 8.1, 1.8 Hz); 7.41 d, 7.60 d (4H, 4-Cl-C6H4, J =
(ppm) (DMSO-d6): 15.2;
δ
37.3; 43.0; 64.3; 112.1; 116.1; 123.0; 125.9; 129.6; 130.6; 133.7; 134.5; 147.5; 150.4; 158.5; 163.0; 172.2; 174.0.
[2-{[(3-Chloro-4-hydroxyphenyl)methylidene]hydrazinylidene}-3-(4-chlorophenyl)-4-oxo-1,3-thiazolidin-5-
yl]acetic acid (86): Yield 77%, mp = 256–257 ◦C. 1H-NMR
δ
(ppm) (DMSO-d6): 3.11 d (2H, C
, J = 6.0 Hz); 7.03 d, 7.55 dd, 7.69 d (3H, 3-Cl-4-HO-C6 3, J = 8.7, 1.8 Hz);
7.42 d, 7.61 d (4H, 4-Cl-C6H4, J = 8.7 Hz); 8.20 s (1H, CH=N); 10.86 s (1H, OH); 12.82 s (1H, COOH).
13C-NMR
(ppm) (DMSO-d6): 37.2; 43.0; 117.4; 120.7; 126.8; 128.3; 129.7; 129.8; 130.6; 133.8; 134.5;
H2CH, J =
6.0 Hz); 4.53 t (1H, CH2C
H
H
δ
156.0; 157.1; 164.2; 172.3; 174.0.
[2-{[(3-Bromo-4-hydroxyphenyl)methylidene]hydrazinylidene}-3-(4-chlorophenyl)-4-oxo-1,3-thiazolidin-5-
yl]acetic acid (87): Yield 62%, mp = 254–255 ◦C. 1H-NMR
δ
(ppm) (DMSO-d6): 3.11 d (2H, C
, J = 6.0 Hz); 7.01 d, 7.42 d, 7.57-7.62 m, 7.84 d (7H, 3-Br-4-HO-C6
4, J = 8.7 Hz); 8.20 s (1H, CH=N); 10.92 s (1H, OH); 12.82 s (1H, COOH). 13C-NMR
H
2CH, J =
6.0 Hz); 4.53 t (1H, CH2C
1.8 Hz and Cl-C6
H
H3, J = 8.4,
H
δ
(ppm) (DMSO-d6): 37.2; 43.0; 110.2; 117.0; 127.2; 128.9; 129.7; 130.6; 132.8; 133.8; 134.5; 156.9; 157.0;
164.2; 172.3; 174.1.
[3-(2,4-Dichlorophenyl)-2-{[(4-hydroxy-3-methoxyphenyl)methylidene]hydrazinylidene}-4-oxo-1,3-thiazolidin-5-
◦
1
yl]acetic acid (88): Yield 66%, mp = 226–228 C. H-NMR
δ (ppm) (DMSO-d6): 3.12–3.16 m (2H,
C
H
2CH); 3.79 s (3H, OCH3); 4.68 dd (1H, CH2C , J = 6.9, 4.2 Hz); 6.82 d, 7.13–7.17 m, 7.30 s, 7.54 d,
H

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7.63–7.68 m, 7.89–7.92 m (6H, 4-HO-3-CH3O-C6H3 and 2,4-diCl-C6
H
3, J = 8.4 Hz); 8.17 s (1H, CH=N);
9.69 s (1H, OH); 12.85 bs (1H, COOH). 13C-NMR
δ (ppm) (DMSO-d6): 37.3; 42.8; 56.0; 110.8; 116.0;
123.2; 125.7; 129.2; 130.2; 132.4; 133.5; 133.6; 135.4; 148.3; 150.3; 159.0; 161.4; 172.2; 173.4.
3.5. General Method of Synthesis of (4-oxothiazolidin-5-ylidene)Acetic Acid Derivatives (89–104)
A mixture of 0.0015 mol thiosemicarbazones (8–14, 18 or 24–31), 0.0015 mol dimethyl
acetylenedicarboxylate in 10 mL methanol was reflux for 1.5h. After cooling of reaction mixture, the
solid obtained was filtered off, dried and then recrystallized from glacial acetic acid.
Methyl [2-{[(2-hydroxyphenyl)methylidene]hydrazinylidene}-4-oxo-3-phenyl-1,3-thiazolidin-5-ylidene]acetate
(
89): Yield 90%, mp = 242–244 ◦C. 1H-NMR
δ
(ppm) (DMSO-d6): 3.83 s (3H, COOCH3); 6.85 s (1H,
CH=); 6.91–6.96 m, 7.34–7.40 m, 7.50–7.57 m, 7.68 dd (9H, 2-HO-C6H4 and C6 5, J = 8.1, 1.8 Hz); 8.65 s
(1H, CH=N); 10.53 s (1H, OH). 13C-NMR
(ppm) (DMSO-d6): 53.2; 115.7; 116.9; 119.1; 120.2; 128.7;
H
δ
129.6; 129.7; 129.8; 133.6; 134.6; 141.7; 158.5; 159.6; 160.4; 164.4; 166.5.
Methyl [2-{[(3-hydroxyphenyl)methylidene]hydrazinylidene}-4-oxo-3-phenyl-1,3-thiazolidin-5-ylidene]acetate
(
90): Yield 84%, mp = 236–238 ◦C. 1H-NMR
δ
(ppm) (DMSO-d6): 3.83 s (3H, COOCH3); 6.82 s (1H,
CH=); 6.88–6.91 m, 7.17–7.19 m, 7.25–7.30 m, 7.49–7.58 m (9H, 3-HO-C6H4 and C6 5); 8.38 s (1H,
CH=N); 9.82 s (1H, OH). 13C-NMR
(ppm) (DMSO-d6): 53.1; 113.7; 115.4; 119.4; 120.6; 128.7; 129.5;
H
δ
129.6; 130.5; 134.7; 135.2; 142.3; 158.1; 160.6; 161.3; 164.7; 166.6.
Methyl [2-{[(4-hydroxyphenyl)methylidene]hydrazinylidene}-4-oxo-3-phenyl-1,3-thiazolidin-5-ylidene]acetate
(
91): Yield 81%, mp = 222–224 ◦C. 1H-NMR
δ
(ppm) (DMSO-d6): 3.83 s (3H, COOCH3); 6.80 s (1H,
4, J = 8.7 Hz); 7.47–7.57 m (5H, C6H5); 8.33 s (1H, CH=N); 10.22 s
(ppm) (DMSO-d6): 53.0; 115.1; 116.3; 125.0; 128.7; 129.5; 129.6; 130.6; 134.8; 142.5;
CH=); 6.86 d, 7.64 d (4H, 4-HO-C6
(1H, OH). 13C-NMR
H
δ
159.6; 160.3; 161.2; 164.6; 166.5.
Methyl [2-{[(4-hydroxy-3-methoxyphenyl)methylidene]hydrazinylidene}-4-oxo-3-phenyl-1,3-thiazolidin-5-
ylidene]acetate (92): Yield 79%, mp = 210–212 ◦C. 1H-NMR
δ
(ppm) (DMSO-d6): 3.81 s (3H, OCH3); 3.82
s (3H, COOCH3); 6.80 s (1H, CH=); 6.87 d, 7.25 dd, 7.35 d (3H, 4-HO-3-CH3O-C6 3, J = 8.1, 1.8 Hz);
7.49-7.58 m (5H, C6H5); 8.31 s (1H, CH=N); 9.84 s (1H, OH). 13C-NMR
(ppm) (DMSO-d6): 53.1; 56.0;
111.3; 115.1; 116.2; 123.4; 125.4; 128.7; 129.5; 129.6; 134.8; 142.5; 148.3; 150.8; 159.4; 160.6; 164.6; 166.5.
H
δ
Methyl [2-{[(3-ethoxy-4-hydroxyphenyl)methylidene]hydrazinylidene}-4-oxo-3-phenyl-1,3-thiazolidin-5-
ylidene]acetate (93): Yield 81%, mp = 190–192 ◦C. 1H-NMR
(ppm) (DMSO-d6): 1.36 t (3H, OCH2CH3
J = 6.9 Hz); 3.82 s (3H, COOCH3); 4.06 q (2H, OC 2CH3, J = 6.9 Hz); 6.80 s (1H, CH=); 6.88 d, 7.25 dd,
7.33 d (3H, 3-C2H5O-4-HO-C6 3, J = 8.1, 1.8 Hz); 7.47–7.57 m (5H, C6H5); 8.30 s (1H, CH=N); 9.74 s
(1H, OH). 13C-NMR
(ppm) (DMSO-d6): 15.2; 53.1; 64.4; 112.8; 115.1; 116.3; 123.3; 125.4; 128.7; 129.5;
129.6; 137.8; 142.4; 147.5; 151.0; 159.3; 160.6; 164.6; 166.5.
δ
,
H
H
δ
Methyl
[2-{[(3-chloro-4-hydroxyphenyl)methylidene]hydrazinylidene}-4-oxo-3-phenyl-1,3-thiazolidin-5-
ylidene]acetate (94): Yield 83%, mp = 238–240 ◦C. 1H-NMR
6.81 s (1H, CH=); 7.07 d, 7.63 dd, 7.74 d (3H, 3-Cl-4-HO-C6
δ
(ppm) (DMSO-d6): 3.83 s (3H, COOCH3);
H3, J = 8.4, 2.0 Hz); 7.49–7.57 m (5H, C6H5);
8.34 s (1H, CH=N); 11.00 s (1H, OH). 13C-NMR
δ
(ppm) (DMSO-d6): 53.1; 115.3; 117.5; 120.7; 126.3;
128.5; 128.7; 129.5; 129.6; 130.4; 134.7; 142.3; 156.5; 159.2; 160.5; 164.6; 166.5.
Methyl
[2-{[(3-bromo-4-hydroxyphenyl)methylidene]hydrazinylidene}-4-oxo-3-phenyl-1,3-thiazolidin-5-
ylidene]acetate (95): Yield 75%, mp = 236–238 ◦C. 1H-NMR
6.81 s (1H, CH=); 7.06 d, 7.67 dd, 7.90 d (3H, 3-Br-4-HO-C6
δ
(ppm) (DMSO-d6): 3.83 s (3H, COOCH3);
H
3, J = 8.4, 2.0 Hz); 7.47–7.57 m (5H, C6H5);
8.34 s (1H, CH=N); 11.06 s (1H, OH). 13C-NMR
δ (ppm) (DMSO-d6): 53.1; 110.3; 115.3; 117.2; 126.7;
128.7; 129.2; 129.5; 129.6; 133.4; 134.7; 142.3; 157.5; 159.0; 160.4; 164.6; 166.5.
Methyl [3-(2-chlorophenyl)-2-{[(3-ethoxy-4-hydroxyphenyl)methylidene]hydrazinylidene}-4-oxo-1,3-thiazolidin-
5-ylidene]acetate (96): Yield 63%, mp = 216–218 ◦C. 1H-NMR
δ
(ppm) (DMSO-d6): 1.36 t (3H, OCH2CH3
,
J = 6.9 Hz); 3.83 s (3H, COOCH3); 4.05 q (2H, OC
H2CH3, J = 6.9 Hz); 6.87 s (1H, CH=); 6.88 d, 7.25 dd,

Molecules 2019, 24, 3029
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7.33 d (3H, 3-C2H5O-4-HO-C6
H
3, J = 8.1, 1.8 Hz); 7.55-7.60 m, 7.70–7.74 m (4H, 2-Cl-C6H4); 8.30 s (1H,
CH=N); 9.75 s (1H, OH). 13C-NMR
δ
(ppm) (DMSO-d6): 15.2; 53.2; 64.4; 112.7; 116.1; 116.3; 123.5; 125.2;
129.0; 130.6; 131.5; 131.9; 132.1; 132.2; 141.3; 147.5; 151.1; 157.7; 161.2; 163.8; 166.4.
Methyl [3-(4-chlorophenyl)-2-{[(2-hydroxyphenyl)methylidene]hydrazinylidene}-4-oxo-1,3-thiazolidin-5-
ylidene]acetate (97): Yield 93%, mp = 214–216 ◦C. 1H-NMR
(ppm) (DMSO-d6): 3.84 s (3H, COOCH3);
6.87 s (1H, CH=); 6.92–6.96 m, 7.34–7.40 m, 7.56–7.70 m (8H, 2-HO-C6 4 and 4-Cl-C6 4); 8.66 s (1H,
CH=N); 10.51 s (1H, OH). 13C-NMR
(ppm) (DMSO-d6): 53.2; 115.8; 117.0; 119.1; 120.2; 129.7; 130.6;
130.7; 133.4; 133.6; 134.3; 141.7; 158.5; 159.6; 160.2; 164.4; 166.5.
Methyl [3-(4-chlorophenyl)-2-{[(3-hydroxyphenyl)methylidene]hydrazinylidene}-4-oxo-1,3-thiazolidin-5-
ylidene]acetate (98): Yield 90%, mp = 222–224 ◦C. 1H-NMR
(ppm) (DMSO-d6): 3.82 s (3H, COOCH3);
6.81 s (1H, CH=); 6.88–6.92 m, 7.17–7.30 m (4H, 2-HO-C6 4); 7.56 d, 7.62 d (4H, 4-Cl-C6H4, J = 8.7 Hz);
8.38 s (1H, CH=N); 9.78 s (1H, OH). 13C-NMR
(ppm) (DMSO-d6): 53.1; 113.8; 115.5; 119.4; 120.6;
129.7; 130.5; 130.6; 133.5; 134.2; 135.2; 142.2; 158.1; 160.7; 161.1; 164.5; 166.5.
Methyl [3-(4-chlorophenyl)-2-{[(4-hydroxyphenyl)methylidene]hydrazinylidene}-4-oxo-1,3-thiazolidin-5-
ylidene]acetate (99): Yield 88%, mp = 250–251 ◦C. 1H-NMR
(ppm) (DMSO-d6): 3.82 s (3H, COOCH3);
6.80 s (1H, CH=); 6.86 d (2H, 4-HO-C6 4, J = 8.7 Hz); 7.55 d (2H, 4-Cl-C6 4, J = 8.7 Hz); 7.61–7.66 m
(4H, 4-HO-C6H4 and 4-Cl-C6 (ppm) (DMSO-d6):
4); 8.33 s (1H, CH=N); 10.19 s (1H, OH). 13C-NMR
53.1; 115.1; 116.4; 125.0; 129.7; 130.7; 133.6; 134.2; 142.4; 159.4; 160.4; 161.2; 164.5; 166.5.
Methyl [3-(4-chlorophenyl)-2-{[(4-hydroxy-3-methoxyphenyl)methylidene]hydrazinylidene}-4-oxo-1,3-
thiazolidin-5-ylidene]acetate (100): Yield 84%, mp = 228–230 ◦C. 1H-NMR
(ppm) (DMSO-d6): 3.82 s
(3H, OCH3); 3.83 s (3H, COOCH3); 6.81 s (1H, CH=); 6.87 d, 7.25 dd, 7.35 d (3H, 4-HO-3-CH3O-C6 3, J
= 8.1, 1.8 Hz); 7.56 d, 7.63 d (4H, 4-Cl-C6H4, J = 8.7 Hz); 8.32 s (1H, CH=N); 9.82 s (1H, OH). 13C-NMR
(ppm) (DMSO-d6): 53.1; 56.0; 111.3; 115.2; 116.2; 123.4; 125.3; 129.7; 130.7; 133.6; 134.2; 142.4; 148.4;
150.8; 159.2; 160.7; 164.5; 166.5.
δ
H
H
δ
δ
H
δ
δ
H
H
H
δ
δ
H
δ
Methyl
[3-(4-chlorophenyl)-2-{[(3-ethoxy-4-hydroxyphenyl)methylidene]hydrazinylidene}-4-oxo-1,3-
thiazolidin-5-ylidene]acetate (101): Yield %, mp = 221–223 ◦C. 1H-NMR
δ (ppm) (DMSO-d6): 1.37 t (3H,
OCH2C
d, 7.25 dd, 7.33 d (3H, 3-C2H5O-4-HO-C6
8.30 s (1H, CH=N); 9.73 s (1H, OH). 13C-NMR
H
3, J = 6.9 Hz); 3.83 s (3H, COOCH3); 4.06 q (2H, OC
3, J = 8.1, 1.8 Hz); 7.56 d, 7.63 d (4H, 4-Cl-C6H4, J = 9.0 Hz);
(ppm) (DMSO-d6): 15.2; 53.1; 64.4; 112.8; 115.2; 116.3;
H2CH3, J = 6.9 Hz); 6.81 s (1H, CH=); 6.88
H
δ
123.3; 125.3; 129.7; 130.7; 133.6; 134.2; 142.4; 147.5; 151.1; 159.1; 160.8; 164.5; 166.5.
Methyl [2-{[(3-chloro-4-hydroxyphenyl)methylidene]hydrazinylidene}-3-(4-chlorophenyl)-4-oxo-1,3-thiazolidin-
5-ylidene]acetate (102): Yield 85%, mp = 248–250 ◦C. 1H-NMR
δ
(ppm) (DMSO-d6): 3.83 s (3H, COOCH3);
3, J = 8.4, 2.1 Hz); 7.54-7.64 m (5H, 3-Cl-4-HO-C6H3
4); 8.34 s (1H, CH=N); 11.00 s (1H, OH). 13C-NMR
(ppm) (DMSO-d6): 53.1; 115.4; 117.5;
120.8; 126.2; 128.6; 129.7; 130.4; 130.7; 133.5; 134.2; 142.3; 156.6; 159.3; 160.3; 164.5; 166.5.
6.81 s (1H, CH=); 7.08 d, 7.75 d (2H, 3-Cl-4-HO-C6
and 4-Cl-C6
H
,
H
δ
Methyl [2-{[(3-bromo-4-hydroxyphenyl)methylidene]hydrazinylidene}-3-(4-chlorophenyl)-4-oxo-1,3-thiazolidin-
5-ylidene]acetate (103): Yield 88%, mp = 263–264 ◦C. 1H-NMR
(ppm) (DMSO-d6): 3.83 s (3H, COOCH3);
6.81 s (1H, CH=); 7.06 d, 7.67 dd, 7.90 d (3H, 3-Br-4-HO-C6 3, J = 8.4, 1.8 Hz); 7.55 d, 7.63 d (4H,
4-Cl-C6H4, J = 9.0 Hz); 8.34 s (1H, CH=N); 11.07 s (1H, OH). 13C-NMR
(ppm) (DMSO-d6): 53.1; 110.3;
δ
H
δ
115.3; 117.2; 126.7; 129.2; 129.7; 130.7; 133.4; 133.5; 134.2; 142.3; 157.6; 159.2; 160.3; 164.5; 166.5.
Methyl [3-(2,4-dichlorophenyl)-2-{[(4-hydroxy-3-methoxyphenyl)methylidene]hydrazinylidene}-4-oxo-1,3-
thiazolidin-5-ylidene]acetate (104): Yield 68%, mp = 214–216 ◦C. 1H-NMR
δ
(ppm) (DMSO-d6): 3.82 s
(3H, OCH3); 3.83 s (3H, COOCH3); 6.89 s (1H, CH=); 6.87 d, 7.25 dd, 7.35 d (3H, 4-HO-3-CH3O-C6 3, J
= 8.4, 1.8 Hz); 7.69 dd, 7.78 d, 7.96 d (3H, 2,4diCl-C6H3, J = 8.4, 2.1 Hz); 8.32 s (1H, CH=N); 9.83 s (1H,
OH). 13C-NMR
(ppm) (DMSO-d6): 53.2; 56.0; 111.3; 116.2; 116.3; 123.6; 125.2; 129.3; 130.3; 131.4; 132.8;
133.3; 135.9; 141.2; 148.4; 150.9; 157.5; 161.3; 163.7; 166.4.
H
δ

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3.6. Preparation of Compounds and Drugs
Compounds were dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich, St. Louis, MO, USA) to
125 mM. The final concentration of DMSO in the compounds dilutions was not higher than 1.00%. Drug,
sulfadiazine [4-amino-N-(2-pyrimidinyl)benzenesulfonamide] (S8626, Sigma-Aldrich) was dissolved in
1M sodium hydroxide (NaOH, Sigma-Aldrich) to 400 mM. Final concertation of NaOH in sulfadiazine
dilutions was not higher than 2.5%. Ready solution sulfadiazine and trimethoprim in weight ratio 5:1
(Sul-Tridin 24%, 200 mg/mL [800 mM] + 40 mg/mL [137.78 mM]– ScanVet Poland). Dilution of the all
compounds and drugs were freshly prepared before the cells were exposed in appropriate medium.
3.7. Cytotoxic Assay
The L929 mouse fibroblast (ATTC® CCL-1
the ATCC product sheet. Cells were trypsinized (Trypsin-EDTA Solution, 1X ATCC® 30-2101
a week and seeded at a density of 1
106 per T25 cell culture flask and incubated in a 37 ◦C and 5%
CO2 to achieve a confluent monolayer.
™
, Manassas, VA, USA) were culturing according to
) twice
™
×
The cell viability assay were performed precisely according to international standards (ISO
10993-5:2009(E)), using the tetrazolium salt [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide] (MTT, Sigma-Aldrich) and mouse fibroblasts L929 cells. Cells (1
×
104 per well) were
treated 24 h with compounds or drug in different concentration range. The optical density at 570 nm
on the ELISA reader (Multiskan EX, Labsystems, Vienna, VA, USA), was read. The results were
expressed as a percentage of viability compared to untreated cells. To calculate the reduction of viability
compared to the untreated blank the equation was used: viability (%) = 100%
×
sample OD570 (the
mean value of the measured optical density of the treated cells)/blank OD570 (the mean value of the
measured optical density of the untreated cells). The CC30/CC50 represents the concentration of tested
compounds or drugs that was required for 30% or 50% cells proliferation inhibition in vitro. According
to the previously mentioned ISO 10993-5:2009(E) norm, the CC30 value is recognized as non-cytotoxic
concentration for the cell line. All experiments were performed in triplicate.
3.8. Assay In Vitro for Anti-T. gondii Activity
The Hs27 (human foreskin fibroblast) (ATCC® CRL-1634
product sheet. The cell line, when it achieved a confluent monolayer, was trypsinized and seeded
at a density of 1
106 per T25 cell culture flask and incubated for 48–72 h in a 37 ◦C and with 5%
CO2. The RH strain of Toxoplasma gondii (ATCC® PRA-310
) is highly virulent were maintained as
™) were culturing according to the ATCC
×
™
tachyzoites according to the ATCC product sheet. Infected tissue culture cells were incubated in a
37 ◦C and 5% CO2.
The influence of compounds and drugs on T. gondii proliferation was described in our previous
work [20–22]. Due to the fact that T. gondii is an intracellular parasite and host cells should not be
destroyed by the cytotoxic concentration of compounds, to determine the initial dose of each compound
to antiparasitic study CC30 value was calculated (data not shown). In our study we use a [3H]-uracil
incorporation assay, which is specific for the labelling of the nucleic acids of the parasite. Shortly, 1
104
×
per well of Hs27 cells were seeded on 96-well plates. After 72 h of incubation, tachyzoites of the RH
strain were added to the cell monolayers, ratio 1:10, and incubate for 1h. Then compounds or drug in
different concentration range, were added to the Hs27 cells with T. gondii. After a subsequent 24 h of
incubation, 1 µ
Ci/well [5,6-3H] uracil (Moravek Biochemicals Inc., Brea, CA, USA) was added to each
microculture for a further 72 h. The amount of the isotope incorporated into the parasite nucleic acid
pool, corresponding to the parasite growth, was measured by liquid scintillation counting using 1450
Microbeta Plus Liquid Scintillation Counter (Wallac Oy, Turku, Finland). The results were expressed
as counts per minute (CPM) and transformed to the percentage of viability compared to untreated
cells. The IC50 represents the concentration of tested compounds or drugs that was required for 50%
inhibition of T. gondii proliferation in vitro. All experiments were performed in triplicate.

Molecules 2019, 24, 3029
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3.9. Graphs and Statistical Analyses
Statistical analyses and graphs were performed using GraphPad Prism version 8.0.1 for macOS
(GraphPad Software, San Diego, CA, USA). For compounds with CC30/CC50 or IC50 values greater
than the highest concentration tested, values were calculated based on extrapolation of the curves
using the GraphPad Prism program (version 8.0.1). The standard deviation (SD) were calculated from
a sample of values, also using the GraphPad Prism. Selectivity ratio (SR) values were calculated as the
ratio of the 50% cytotoxic concentration (CC50) to the 50% antiparasitic concentration (IC50).
4. Conclusions
We evaluated a series of new thiazolidin-4-one derivatives for their ability to inhibit in vitro
growth of the parasite T. gondii. All active compounds inhibit proliferation of T. gondii tachyzoites
better than used references drugs both sulfadiazine as well as synergistic effect of sulfadiazine +
trimethoprim (5:1). The most active of them (compounds 94 and 95) showed anti-T. gondii activity over
392-fold higher than sulfadiazine and 18-fold higher than sulfadiazine + trimethoprim (5:1). All active
compounds (82–88 and 91–95) against T. gondii represent selectivity ratio values from 1.75 to 15.86
(CC30/IC50) which shows that were more towards inhibiting parasite proliferation vs. cytotoxicity.
The results of these studies also collectively suggest that thiazolidin-4-ones containing in their
structure halogenphenylthiosemicarbazide moiety was useful for their antitoxoplasmic activity that
confirmed their inhibitory potential against T. gondii tachyzoites. Received funding from the National
Science Centre allow (project: Antiparasitic properties and molecular target identification of new
thiosemicarbazide and thiazolidinone derivates in Toxoplasma gondii invasions) to determine a molecular
target(s) for selected new compounds from thiosemicarbazide and thiazolidinone derivatives possessing
the most potent anti-T. gondii activity.
Supplementary Materials: The following are available online, Figures S1–S116: 1H and 13C-NMR spectra for
compounds 42, 45–66, 68, 70–76 and 78–104.
Author Contributions: Conceptualization, N.T.; methodology, K.D.; formal analysis, N.T. and K.D.; investigation,
N.T. and A.B.; resources, N.T. and K.D.; data curation, N.T., A.P., A.B., and K.D.; writing—original draft preparation,
N.T. and K.D.; writing—review and editing, K.D., A.P., and M.W.; visualization, N.T., A.B., and K.D.; supervision,
N.T., K.D., and M.W.
Funding: This research was funded by Medical University of Lublin (DS 15) and Faculty of Biology and
Environmental Protection, University of Lodz (B1711000000263.01).
Conflicts of Interest: The authors declare no conflict of interest.
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2019 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/).