Systematic Identification of Thiosemicarbazides for Inhibition of Toxoplasma gondii Growth In Vitro

Article abstract of DOI:10.3390/molecules24030614

One of the key stages in the development of new therapies in the treatment of toxoplasmosis is the identification of new non-toxic small molecules with high specificity to Toxoplasma gondii. In the search for such structures, thiosemicarbazide-based compo

Full text of DOI:10.3390/molecules24030614

molecules
Article
Systematic Identification of Thiosemicarbazides for
Inhibition of Toxoplasma gondii Growth In Vitro
1
,
1
2
1
1
Agata Paneth * , Lidia W e˛ gli n´ ska , Adrian Bekier , Edyta Stefaniszyn , Monika Wujec ,
Nazar Trotsko 1 and Katarzyna Dzitko *
2,
1
Department of Organic Chemistry, Medical University, Chod z´ ki 4a, 20-093 Lublin, Poland;
lidia.weglinska@umlub.pl (L.W.); edyta.stefaniszyn@umlub.pl (E.S.); monika.wujec@umlub.pl (M.W.);
nazar.trotsko@umlub.pl (N.T.)
Department of Immunoparasitology, Faculty of Biology and Environmental Protection, University of Lodz,
2
Banacha 12/16, 90-237 Lodz, Poland; adrian.bekier@unilodz.eu
*
Correspondence: agata.paneth@umlub.pl (A.P.); katarzyna.dzitko@biol.uni.lodz.pl (K.D.)
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀ
ꢁꢂꢃꢄꢅꢆ
Received: 22 January 2019; Accepted: 7 February 2019; Published: 10 February 2019
Abstract: One of the key stages in the development of new therapies in the treatment of toxoplasmosis
is the identification of new non-toxic small molecules with high specificity to Toxoplasma gondii.
In the search for such structures, thiosemicarbazide-based compounds have emerged as a novel and
promising leads. Here, a series of imidazole-thiosemicarbazides with suitable properties for CNS
penetration was evaluated to determine the structural requirements needed for potent anti-Toxoplasma
gondii activity. The best 4-arylthiosemicarbazides
3 and 4 showed much higher potency when
compared to sulfadiazine at concentrations that are non-toxic to the host cells, indicating a high
selectivity of their anti-toxoplasma activity.
Keywords: thiosemicarbazides; anti-Toxoplasma gondii activity; toxicity; SAR analysis
1
. Introduction
According to the World Health Organization, approximately up to one third of the world’s
population is infected with Toxoplasma gondii (T. gondii). This protozoan is an obligate intracellular
parasite with a complex life cycle which requires intermediate and definitive hosts. Intermediate hosts
are especially all warm-blooded animals including most livestock, and humans. While T. gondii infect
intermediate hosts and asexually reproduce in them, the only known definitive hosts in which this
parasite may complete life cycle and sexually reproduce are members of the family Felidae. Cats become
infected mainly through predation of intermediate hosts with latent parasite invasion. The parasite’s
sexual replication takes place in the intestines and results in formation of oocysts. Cats shed large
numbers of unsporulated oocysts with feces, for 1–3 weeks. In the environment oocysts sporulate
within 1–5 days and become infective [1,2].
In T. gondii life cycle we can identify three main infective stages: tachyzoites, tissue cysts
with bradyzoites and above-mentioned mature oocyst containing sporozoites. The main rout of
parasite transmission to humans involves ingestion of either raw or underprepared meat containing
tissue cysts or water or vegetables contaminated with soil containing oocysts. Additionally, people
can become infected horizontally (iatrogenic) via blood transfusion or organ transplantation and
vertically from mother to fetus via placenta. The parasite is also responsible for livestock infections.
Farm animals, also these bred for human consumption, can acquire T. gondii infection through ingestion
of sporulated oocysts with water or plants. After the release from tissue cysts and oocysts, which
takes place in the intestines, sporozoites and bradyzoites, respectively, transform into the rapidly
dividing tachyzoite (from tachos—speed in Greek) that is responsible for acute toxoplasmosis. In the
Molecules 2019, 24, 614; doi:10.3390/molecules24030614
www.mdpi.com/journal/molecules

Molecules 2019, 24, 614
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immunocompetent individuals tachyzoites under the pressure of immune component convert into
slow-dividing bradyzoites (brady—slow in Greek) enclosed within tissue cysts localized in various
tissues e.g., neural or/and muscle. In people with a fully effective immune response those tissue cysts
do not possess a direct threat to health and even life [
the rupture of tissue cysts leads to the release of bradyzoites. Their transformation to tachyzoites, in
the absence of: nitric oxide, INF- , TNF- , T cells and IL-12, results in disease reactivation. The process
of tachyzoite–bradyzoite conversion is central to the pathogenesis and longevity of infection [ ].
2]. In contrast, in immunocompromised patients
γ
α
3,4
Therefore, the biggest challenge in the treatment of toxoplasmosis is related with acute phase, when
rapidly multiplying tachyzoites are responsible for the numerous of necrotic changes and destruction
of the host cells.
As mentioned earlier, although most human infections resolve without complications, they can
be fatal or lead to serious problems in fetuses and immunocompromised patients [5–8]. Active
T. gondii infection in HIV patients or those submitted for to cancer chemotherapy or organ transplants
primarily leads to encephalitis, pneumonia or chorioretinitis but tissue destruction in other organs
may occur as well [9,10]. Besides, when T. gondii primary infection is acquired during pregnancy, the
vertical transmission may occur, resulting in birth defects such as hydrocephalus, epilepsy and mental
retardation or even neonatal death [11,12].
Current first-line therapy for toxoplasmosis relies on inhibition of the folate pathway in the
parasite, although antibacterial drugs have also been used with some success [13–15]. The most
commonly used treatment is a combination of sulfonamides with 2,4-diaminopyrimidines, i.e.,
sulfadiazine with pyrimethamine—the principal drug combination or sulfamethoxazole with
trimethoprim—alternate first line therapy [15,16]. These combinations are highly synergistic as the
sulfonamide component inhibits dihydropteroate synthase—essential key enzyme for the utilization
by the microorganism of 4-aminobenzoic acid in vital biosynthesis of dihydropteroic acid while
2,4-diaminopyrimidine component blocks dihydrofolate reductase—the enzyme essential for the
conversion of dihydropteroic acid to tetrahydrofolate. Collectively, these components inhibit the
parasite growth by blocking the biosynthesis of tetrahydrofolate, an essential factor needed for the
production of nucleic acids which are required for DNA synthesis [17–23].
The combination of the sulfonamide with pyrimethamine, however, is highly effective in blocking
replication of tachyzoites but has not activity on the latent bradyzoite form and therefore does
not eliminate chronic infection [23
adverse reactions including anemia due to bone marrow suppression that requires coadministration
of leucovorin [26 28]. As well, many patients experience intolerance or allergic reaction to the
–25]. Furthermore, pyrimethamine is associated with significant
–
sulfa component [24]. Additionally, this therapeutic regiment requires long dosing periods and
is contraindicated during the first two trimesters of pregnancy due to the potential for inducing
development defects [16
incidence of relapses after discontinuation of therapy [25
,
29]. Other serious problems are the emergence of drug resistance and the
30]. Although alternative drugs such as
,
clindamycin, azithromycin have also been used to treat acute toxoplasmosis, they do not a clear chronic
infection as well [14]. Atovaquone is also now in use and unlike to sulfonamide and pyrimethamine,
this drug is effective against tachyzoite [31], besides other authors demonstrated that extended
incubation of isolated brain cysts of T. gondii with atovaquone resulted in the inactivation of intracystic
bradyzoite form [32,33]. Unfortunately, owing to the low-yield method for the synthesis and poor
bioavailability, the cost of treatment with atovaquone is not affordable by patients in need, particularly
in the third world countries [34].
Summarizing, currently, the only effective mean of avoiding T. gondii infection is a preventive
healthcare, especially raising the awareness of future mothers and early diagnosis of pregnant women,
and new-borns. An efficient method of complete elimination of the parasite from an infected organism
has not yet been developed, so new agents or combinations of agents with greater therapeutic efficacy
are necessary. Also, develop of safe and efficient tools for immunoprophylaxis of toxoplasmosis is still
needed. Nowadays, only one vaccine containing live attenuated tachyzoites of T. gondii S48 strain, are

Molecules 2019, 24, 614
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available, but the potential use of the vaccine is restricted to the veterinary purposes because of the
possible reversion of the attenuated mutant to the virulent strain [35].
One of the key steps in developing new therapies for the treatment of toxoplasmosis is to identify
new non-toxic small molecules with high specificity to T. gondii for therapeutic investigation. In the
search for such structures, thiosemicarbazides have emerged as a novel and promising leads. We
have recently [36] tested a small series of 4-aryl(alkyl)thiosemicarbazides and found that all of them
at non-toxic concentrations for the host cells were more effective than sulfadiazine. Compound
1
(Figure 1, left), the best of this group, was at least 15-fold more potent than sulfadiazine. Therefore,
this class of compounds could offer a new way to combat the problem of infectious diseases induced
by T. gondii. The conclusion from these studies was that the presence of five-membered heterocyclic
moiety at the N1 terminal position and aryl moiety at the N4 terminal position of thiosemicarbazide
core are the key functionalities required for potent anti-T. gondii activity. In this report, the systematic
explorations of these initial findings are presented.
2
. Results and Discussion
2
.1. Rationale and Chemistry
As mentioned in the Introduction, in our preliminary study on the anti-Toxoplasma gondii activity
of thiosemicarbazides we identified compound
1 (Figure 1, left) as the most potent in this respect [36].
The bioactivity of was substantially lost when the thiophene ring was replaced with a similar in size
1
furan or thiadiazole moiety. Although the thiosemicarbazides obtained were not enough to conduct
an exhaustive SAR analysis, data reported there allowed us to make some preliminary observations
regarding the effects of the type of substitution on bioactivity and other significant modifications.
In particular, the obtained data highlighted that the need for the presence of a five-membered
heterocyclic ring at the N1 terminal position and an aryl moiety at the N4 terminal position as
the key functionalities required for potent anti-Toxoplasma gondii activity of thiosemicarbazides. Based
on DFT studies, in turn, we have assumed that the steric hindrance between a substituent on the
heterocyclic ring and the carbonyl group of thiosemicarbazide core is the factor reducing bioactivity.
With this information in our hand, a systematic SAR study was started with the aim of
understanding better the substitution pattern at the N4 aryl moiety of azole-thiosemicarbazides on their
anti-toxoplasma activity. As model compounds, series of 4-arylthiosemicarbazides with imidazole
rings at the N1 position
2–7, 10–22 was selected. Most of these compounds are close structural
analogues of –they possess similar molecular geometry (Figure 1, right) while the steric interactions
1
between the methyl group of the imidazole ring and the carbonyl group of the thiosemicarbazide core
were computationally shown to not be significant (Figure 1, middle). For comparison reasons, two
imidazole-thiosemicarbazides with a benzyl group (compound
the N4 position were also included in our SAR study.
8) or an alkyl chain (compound 9) at
Figure 1. Left: Structure of previously reported 4-(4-fluorophenyl)-1-(thiophen-2-oyl)-Table 1. with
potent inhibitory activity against proliferation of Toxoplasma gondii (IC50 = 33.17 g/mL vs. > 500 µg/mL
for sulfadiazine) [36]. Middle: Geometry of representative model compound 18; remaining
-arylthiosemicarbazides 10 17 19 22 share similar conformations. All structures were obtained
µ
4
2
–7,
–
,
–
from the Amber force field (HyperChem 8.0.3.) that was found to provide correct structures for this
class of compounds [37]. Right: Superposition of 1 and 18.

Molecules 2019, 24, 614
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As listed in Table 1, all designed imidazole-thiosemicarbazides
2
–22 have suitable properties for
CNS penetration; positive attributes include: (i) satisfaction of Lipinski’s rule of five (molecular mass
less than 500 Da, logP not greater than 5, no more than 10 hydrogen bond acceptors, and no more than
5
hydrogen bond donors) [38], (ii) satisfaction of Veber’s descriptors (rotatable bond count not greater
2
than 10 and polar surface area not greater than 140 Å ) [39], (iii) solubility expressed as logS within
the range of
−
5 to
−
1 [40] and predicted absorption percentage (%ABS) within the range of 64.96 to
22 meet the requirements for good
8
0.77 [41]. Thus, since designed imidazole-thiosemicarbazides 2–
membrane permeability, all of them were subsequently prepared under routine protocol [42], in the
one-step reaction of 4-methylimidazole-5-carbohydrazide with isothiocyanate (Scheme 1).
Table 1. Physicochemical properties of 2–22 important for CNS penetration. HBA—number of
hydrogen bond acceptors; HBD—number of hydrogen bond donors; logS—solubility; PSA—polar
surface area in Å; %ABS—percentage of absorption.
Compound
Mass
logP
HBA
HBD
logS
PSA
%ABS
2
3
4
5
6
7
8
9
320.33
320.33
320.33
305.36
305.36
305.36
289.36
261.74
309.78
293.33
317.37
335.39
323.81
311.32
289.36
289.36
289.36
318.41
346.46
319.39
359.50
0.50
0.53
0.55
0.60
0.62
0.65
0.93
0.14
1.25
0.73
0.47
0.24
1.65
0.85
0.99
1.02
1.04
0.69
1.45
1.02
1.64
9
9
9
7
7
7
6
6
6
6
7
8
6
6
6
6
6
7
7
7
6
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
−4.15
−4.17
−4.18
−4.05
−4.06
−4.06
−4.04
−3.29
−4.28
−4.19
−4.24
−4.21
−4.50
−4.22
−4.09
−4.10
−4.11
−3.88
−3.99
−4.23
−5.07
127.66
127.66
127.66
91.07
91.07
91.07
81.83
81.83
81.83
81.83
98.91
100.30
81.83
81.83
81.83
81.83
81.83
85.07
85.07
91.07
81.83
64.96
64.96
64.96
77.58
77.58
77.58
80.77
80.77
80.77
80.77
74.88
74.40
80.77
80.77
80.77
80.77
80.77
79.65
79.65
77.58
80.77
1
1
1
1
1
1
1
1
1
1
2
2
2
0
1
2
3
4
5
6
7
8
9
0
1
2
RNCS
R
Scheme 1. Synthetic route for the imidazole-thiosemicarbazides
), 4-NO Ph ( ), 2-MeOPh ( ), 3-MeOPh ( ), 4-MeOPh ( ), Bz ( ), 2-ClEt (
-AcPh (12), 3,4-diMeOPh (13), 3-Cl-4-MePh (14), 2,5-diFPh (15), 2-MePh (16), 3-MePh (17), 4-MePh
18), 4-diMeaminoPh (19), 4-diEtaminoPh (20), 4-EtOPh (21), 2,6-di-iso-PrPh (22).
2–22. R = 2-NO Ph (2), 3-NO Ph
2 2
(
3
4
5
6
7
8
9), 3-ClPh (10), 3-FPh (11),
2
3
(
2
.2. In Vitro Anti-Toxoplasma gondii Activity of the Imidazole-Thiosemicarbazides 2–22
The first step of structure-activity relationship (SAR) study was to confirm the impact
of the N4 aryl moiety and its substitution pattern on anti-Toxoplasma gondii activity of
imidazole-thiosemicarbazides 22. For this purpose, compounds with an electron withdrawing
nitro, and electron donating methoxy ( ), benzyl ( ), and 2-chloroethyl ( ) substituents were prepared
and tested. As presented in Figure 2, compound with an aliphatic chain had only weak activity
IC50 > 125 g/mL) thereby confirming our initial assumption regarding the favorable role of the N4
phenyl ring on activity. When the IC50 values of the remaining compounds were analyzed it was clear
2–
2–4
5–
7
8
9
9
(
µ

Molecules 2019, 24, 614
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that the additional methylene bridge between the thiocarboxamide group and the phenyl scaffold
had a negative effect on anti-Toxoplasma activity as well. Indeed, all tested 4-arylthiosemicarbazides
2
–
7
were more potent than the benzyl analogue
8
(IC50 ~ 149.59
, IC50 = 14.78
4, IC50 = 21.62 µg/mL) showed higher activity than those with strong electron donating meta-methoxy
6, IC50 = 79.65 µg/mL) and para-methoxy substitution ( , IC50 = 113.99 g/mL). This might be either
µ
g/mL). Among them, compounds
having strong electron-withdrawing meta-nitro group (
(
(
3
µg/mL) or a para-nitro group
7
µ
due to the differential uptake and distribution of the compounds into the tachyzoites’ cells or due to
electronic effects of the substituents. In turn, the substitution pattern on the phenyl ring appears not to
play a crucial role. Indeed, whilst within nitro series
2–4 isomers meta-3 and para-4 were the most potent,
the best activity for methoxy series 5–7 was detected for the ortho-5 isomer (IC50 = 33.11 µg/mL).
Figure 2. Dose response curves and IC50 values [µg/mL] of compounds 2–9 on T. gondii proliferation.
With the aim of synthesizing new analogues that would significantly contribute to our envisaged
SAR study, compounds 10 15 were synthesized and tested. As illustrated in Figure 3, the introduction
of extra electron donating methoxy groups in the meta or para positions of weak inhibitors or leads to
a further reduction in activity (13, IC50 ~ 174.98 g/mL), thereby confirming our initial assumption that
meta and para positions are not tolerated for substituents with electron donating character. Contrary
to expectations, however, replacement of the electron withdrawing meta-chloro group of 10 (IC50
5.70 g/mL) by small in size electron withdrawing meta-fluoro group (11, IC50 ~ 117.92 g/mL)
or a bulky electron withdrawing meta-acetyl group (12, IC50 = 95.50 g/mL) reduced the activity
while adding an extra para electron donating methyl group to the structure of increased its potency
significantly (compound 14, IC50 = 18.62 g/mL). The results of the bioassay for 15 were also not
–
6
7
µ
=
2
µ
µ
µ
6
µ
unambiguous. Indeed, the introduction of extra fluorine into the ortho position of 11 leads to increased
activity, despite the fact that this position seemed to be unfavourable for substituents with electron
withdrawing character as noted for the nitro series 2–4.

Molecules 2019, 24, 614
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Figure 3. Dose response curves and IC50 values [µg/mL] of compounds 10–15 on T. gondii proliferation.
Since the influence of chemical substitution and electronic effects on anti-Toxoplasma gondii activity
of studied 4-arylthiosemicarbazides seems to be more complicated than expected, we also prepared a
series of methyl isomers 16–18, three compounds with para-substitution, i.e., N,N-dimethylamino 19,
N,N-diethylamino 20, ethoxy 21, and compound 22 with two isopropyl groups at the ortho position.
According to the results presented in Figure 4, in the 4-arylthiosemicarbazide structure there is
sufficient space at the para position for the chain extension, most likely for its potential interaction with
the molecular target(s). Indeed, in contrast to weak inhibitor 22 (IC50 = 97.72
isopropyl groups at the ortho position, compounds with para substitution 20 (IC50 = 25.70
and 21 (IC50 = 95.28 g/mL) displayed improved T. gondii inhibition compared to their shorter by
a methylene linker precursors 19 (IC50 ~ 164.70 g/mL) and (IC50 = 113.99 g/mL). Surprisingly,
similarly to the nitro series , within the electron donating methyl series 16 18 the lowest antiparasitic
effect was also noted for the ortho isomer 16 (IC50 ~ 150.31 g/mL). The antiparasitic activity of the
two remaining isomers, i.e., meta-17 (IC50 = 37.15 g/mL) and para-18 (IC50 ~ 57.58 g/mL), was
approximately two times lower compared to nitro analogs 3 and 4, respectively.
µg/mL) with two bulky
µg/mL)
µ
µ
7
µ
2
–4
-
µ
µ
µ
Summing up, SAR analysis indicates that steric theory alone, i.e., the interaction between
thiosemicarbazide and molecular target(s), cannot fully describe the observed trend in bioactivity
and that the non-correlation between the results of bioassay and substitution pattern at the N4 aryl
moiety of imidazole-thiosemicarbazide is likely due to the combination of both steric and specific, i.e.,
hydrophobic and electrostatic, intermolecular interactions. Evidently, further studies are needed to
understand the molecular basis of the anti-toxoplasma activity of this class of compounds.

Molecules 2019, 24, 614
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Figure 4. Dose response curves and IC50 values [µg/mL] of compounds 16–22 on T. gondii proliferation.
2
.3. Cytotoxicity of the Imidazole-Thiosemicarbazides 2–22 against L929 Cells and Selectivity Index
To ensure the imidazole-thiosemicarbazides 22 were targeting the parasite and not the host
cells, a cytotoxicity screen against mammalian L929 cells was performed. Cytotoxicity, expressed as
CC , was defined as the highest dilution of test samples that causes 30% or greater destruction of cells.
2–
30
In Table 2 the results expressed as the percent of viable cells
±
standard deviation in the concentration
11 14 18 19)
range between 3.91 to 500 g/mL and the CC30 values are presented. As seen, only 5 (
µ
8
,
,
,
,
of 21 imidazole-thiosemicarbazides have unfavorable cytotoxic concentration close to or lower than
IC50 values.
Table 2. The viability of L929 cells [in % of viable L929 cells
between 3.91 to 500 µg/mL.
±
SD] in the concentration range of 2–22
Concentration [µg/mL]
CC30
µg/mL]
Compound
[
5
00.00
9.12
.17
250.00
125.00
62.50
31.25
15.63
7.81
3.91
2
2
53.38
5.01
75.33
3.36
89.73
3.34
94.61
4.82
96.73
5.88
100.57
1.44
102.91
2.54
2
3
4
5
6
7
181.55
141.25
238.78
138.04
165.96
389.05
1
0
5.27
.77
55.81
3.57
72.38
0.53
79.00
1.12
89.38
1.07
94.61
2.18
94.64
0.54
95.37
2.85
2
5
5
6
3.19
.70
66.91
1.52
79.58
3.46
88.86
3.30
93.11
2.35
95.08
1.81
95.71
2.11
98.52
5.26
0
3.64
.97
63.12
2.25
69.69
6.41
78.51
7.89
86.52
2.83
89.00
1.17
91.87
3.39
95.80
4.83
6
6.79
.65
67.32
1.50
68.79
1.42
79.66
1.61
83.28
0.40
86.17
1.58
88.60
1.16
92.82
1.56
0
8.56
.33
70.70
2.04
83.97
3.30
85.77
2.66
87.65
3.44
89.47
2.38
90.31
2.17
95.08
2.23
2

Molecules 2019, 24, 614
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Table 2. Cont.
Concentration [µg/mL]
CC30
µg/mL]
Compound
[
5
00.00
6.58
.33
250.00
125.00
62.50
31.25
15.63
7.81
3.91
4
5
53.26
2.57
64.92
11.22
88.77
2.60
92.04
3.92
96.52
2.13
99.36
1.44
100.77
1.48
8
9
123.03
8
2
4
5
7
1
5
6
4
5
5
1
7
5
7.44
.13
92.82
2.87
94.44
2.43
95.57
1.91
95.97
2.17
97.07
2.82
98.34
2.85
100.37
4.66
>500.00
112.20
95.50
4
2.09
.67
61.82
1.35
69.89
2.32
72.12
0.89
74.63
1.33
93.54
0.87
94.09
1.01
95.74
0.51
1
0
1
2
3
4
5
6
7
8
9
0
0
6.32
.94
57.77
5.36
66.05
3.52
75.56
3.25
92.73
2.41
99.82
3.16
101.38
4.18
103.55
4.22
1
1
1
1
1
1
1
1
1
2
5
6.21
.57
76.77
2.63
96.70
2.35
99.30
2.00
101.38
2.15
102.71
1.00
104.30
1.38
106.32
3.04
206.54
>500.00
28.18
0
7.00
.51
94.18
2.70
95.74
2.82
97.51
3.18
99.85
2.78
102.62
2.64
105.08
3.60
107.45
3.17
4
0.41
.40
21.05
3.40
49.27
11.56
61.53
1.68
67.49
0.48
76.63
3.12
91.49
2.92
101.21
4.46
0
1.18
.69
65.18
3.28
74.84
2.03
84.32
3.56
96.20
2.33
100.77
0.35
101.96
1.05
104.65
2.38
158.49
288.40
50.12
0
3.38
.14
71.48
1.77
79.69
1.29
91.23
2.91
93.83
2.62
99.96
3.23
102.28
1.75
103.23
1.35
4
0.66
.30
50.05
2.40
56.53
6.98
64.89
4.98
74.78
2.22
85.74
4.54
88.72
5.67
94.09
2.69
8
2.95
.69
60.44
2.04
63.73
1.01
64.60
0.45
69.05
2.20
82.04
2.35
91.69
0.61
99.10
1.48
34.67
2
3.32
.55
60.78
2.37
68.47
0.63
78.57
1.03
80.76
2.41
85.71
3.61
91.75
4.92
93.08
5.38
112.20
63.10
1
2.87
.20
53.35
15.09
65.96
1.34
67.92
1.38
71.31
3.07
85.65
4.03
93.60
6.55
101.55
5.20
0
2.03
.17
82.27
1.30
84.35
0.63
100.83
3.08
106.18
5.33
111.59
2.84
113.96
3.30
114.94
3.43
2
1
2
500.00
190.55
4
8.84
.96
68.18
2.65
76.60
1.19
79.81
2.27
80.71
2.48
101.27
3.40
110.20
3.49
115.81
3.10
2
2
To calculate the reduction of viability compared to the untreated blank the equation was used: viability (%) =
00% sample OD570 (the mean value of the measured optical density of the treated cells)/blank OD570 (the
1
×
mean value of the measured optical density of the untreated cells). CC30—represents the concentration of tested
compounds that was required for 30% cells proliferation inhibition in vitro. The effect of tested compounds on the
cell lines was measured using MTT assay according to the international standard: ISO 10993-5:2009(E). CC30 values
were determined based on the plotted curves using GraphPad Prism version 8.0.1 for macOS (GraphPad Software,
San Diego, CA, USA)
Based on the toxicity against L929 cells, the selectivity index (SI) was also calculated as the
logarithm of the ratio of the CC30 and the IC50 values. The selectivity index is a widely accepted
parameter used to express a compound’s in vitro efficacy in the inhibition of T. gondii proliferation
and only compounds with SIs higher than 0 could be considered as selective. The higher SI ratio, the
theoretically more effective and safe a drug would be during in vivo treatment for toxoplasmosis.
As shown in Table 3, except for
parasite, thus confirming their safety profile. Important to note, the best inhibitors of Toxoplasma gondii
proliferation , and presented also the highest selective index, making them a good candidate for
future in vivo studies.
8, 11, 18 and 19, all compounds showed selective inhibition of the
3
4

Molecules 2019, 24, 614
9 of 17
Table 3. Presentation of CC , IC , SI values and compound structures of derivatives 2–22
30
50
and sulfadiazine.
Compound
Compound Structure
CC30 [µg/mL]
IC50 [µg/mL]
SI
2
3
4
181.55
43.05
0.63
0.98
1.05
141.25
238.78
14.78
21.62
5
138.04
33.11
0.62
6
7
165.96
389.05
79.65
0.54
0.53
113.99
8
9
123.03
~149.59
−0.08
>500
112.20
95.50
206.54
>500
>125
25.70
-
1
1
1
1
1
1
0
1
2
3
4
5
0.64
−0.09
0.34
<0.46
0.18
0.47
~117.92
95.50
~174.98
18.62
28.18
158.49
53.70

Molecules 2019, 24, 614
10 of 17
Table 3. Cont.
Compound Structure
Compound
CC30 [µg/mL]
IC50 [µg/mL]
SI
1
1
1
6
7
8
288.40
50.12
34.67
~150.31
0.28
37.15
0.13
~57.58
−0.22
1
2
9
0
112.20
63.10
~164.70
25.70
−0.17
0.39
2
2
1
2
500
95.28
97.72
0.72
0.29
190.55
Sulfadiazine
1251.12
~2721.45
−0.34
CC30—represents the concentration of tested compounds that was required for 30% of cells proliferation inhibition
in vitro. CC30 values were determined based on the plotted curves using GraphPad Prism version 8.0.1 for macOS
(
GraphPad Software, San Diego, CA, USA). IC50—represents the concentration of tested compounds that was
required for 50% inhibition of T. gondii proliferation in vitro. IC50 values were determined based on the plotted
curves using GraphPad Prism program (version 8.0.1). SI—selectivity index values were calculated as the logarithm
of the ratio of the CC30 and the IC50 values, a positive value represents more selectivity against T. gondii than L929
cells, and negative values (or near 0) indicate higher toxicity to L929 cells and low selectivity to the parasite.
3
. Materials and Methods
3
.1. Chemistry
All commercial reactants and solvents with the highest purity were purchased from either
Sigma-Aldrich (St. Louis, MS, USA) or Alfa Aesar (Karlsruhe, Germany) and used without further
purification. The melting points were determined on a Fischer-Johns block (Fisher Scientific, Schwerte,
Germany) and are uncorrected. Elemental analyses were determined by an AMZ-CHX elemental
analyzer (PG, Gda n´ sk, Poland) and are within
±
0.4% of the theoretical values. 1H-NMR spectra
were recorded on an Avance 300 MHz) spectrometer (Bruker BioSpin GmbH, Rheinstetten, Germany).
Analytical thin layer chromatography was performed with 60F254 silica gel plates (Merck, Darmstadt,
Germany) and visualized by UV irradiation (254 nm). The physicochemical characterization of

Molecules 2019, 24, 614
11 of 17
compounds
7
and 18 was presented in [43
,44]. The structure of
4
was presented in our previous
publication [45], however no physicochemical characterization data was reported.
3
.2. General Procedure for Synthesis of the Imidazole-Thiosemicarbazides 2–22
A solution of 4-methylimidazole-5-carbohydrazide (0.01 mol) and an equimolar amount of an
isothiocyanate (0.01 mole) in anhydrous ethanol (25 mL) was heated under reflux for 10–30 min.
After cooling, the solid formed was filtered off, dried and crystallized from ethanol.
◦
1
1
(
(
-(4-Methylimidazol-5-oyl)-4-(2-nitrophenyl)thiosemicarbazide (
DMSO-d6) (ppm): 2.45 (s, 3H, CH ); 7.35–7.40 (m, 1H, 1 CHar); 7.62–7.75 (m, 2H, 2
m, 1H, 1 CHar); 8.31–8.34 (m, 1H, 1 CHar); 9.61, 10.03, 10.25; 12.41 (4s, 4H, 4 NH). Anal. Calcd. for
2
). Yield: 84%. M.p. 215–217 C. H-NMR
δ
×
×
CHar); 8.02–8.05
3
×
×
×
C H N O S (320.33): C 44.99; H 3.78; N 26.24. Found: C 44.84; H 3.77; N 26.26.
12
12
6
3
◦
1
1
-(4-Methylimidazol-5-oyl)-4-(3-nitrophenyl)thiosemicarbazide (
3
). Yield: 80%. M.p. 210–212 C. H-NMR
CHar); 7.95–8.04 (m, 2H, 2 CHar); 8.53 (s,
NH). Anal. Calcd. for C H N O S (320.33): C 44.99; H 3.78;
(DMSO-d6)
δ
(ppm): 2.46 (s, 3H, CH ); 7.56–7.64 (m, 2H, 2
×
×
3
1
H, 1
×
CHar); 9.85, 9.98, 12.41 (3s, 4H, 4
×
12
12
6
3
N 26.24. Found: C 44.83; H 3.79; N 26.32.
-(4-Methylimidazol-5-oyl)-4-(4-nitrophenyl)thiosemicarbazide (
DMSO-d6) (ppm): 2.46 (s, 3H, CH ); 7.63 (s, 1H, 1 CHar); 7.91–7.96 (m, 2H, 2
NH); 12.36 (s, 1H, 1 NH). Anal. Calcd. for C H N O S (320.33):
◦
1
1
(
2
4
). Yield: 80%. M.p. 235–237 C. H-NMR
CHar); 8.16–8.21 (m,
δ
×
×
3
H, 2
×
CHar); 9.98–10.24 (m, 3H, 3
×
×
12
12
6
3
C 44.99; H 3.78; N 26.24. Found: C 44.82; H 3.77; N 26.33.
◦
4
-(2-Methoxyphenyl)-1-(4-methylimidazol-5-oyl)thiosemicarbazide (
5
)
.
Yield: 87%. M.p. 200–202 C.
CHar);
NH). Anal. Calcd. for C H N O S
1
H-NMR (DMSO-d6)
7
δ
(ppm): 2.46 (s, 3H, CH ); 3.73 (s, 3H, OCH ), 6.89–7.13 (m, 4H, 4
×
3
3
.64 (s, 1H, 1 CHar); 8.92–10.03 (m, 3H, 3
×
×
NH); 12.44 (s, 1H, 1
×
13
15
5
2
(305.36): C 51.13; H 4.95; N 22.94. Found C 51.47; H 4.96; N 22.98.
◦
4
-(3-Methoxyphenyl)-1-(4-methylimidazol-5-oyl)thiosemicarbazide (
6
)
.
Yield: 86%. M.p. 196–198 C.
CHar);
NH);
1
H-NMR (DMSO-d6)
.06–7.09 (m, 1H, 1 CHar); 7.18–7.24 (m, 2H, 2
2.40 (s, 1H, 1
H 4.96; N 22.89.
δ
(ppm): 2.45 (s, 3H, CH ); 3.72 (s, 3H, OCH ), 6.68–7.13 (m, 1H, 1
×
3
3
7
1
×
×
CHar); 7.62 (s, 1H, 1
×
CHar); 9.59–9.76 (m, 3H, 3
×
×
NH). Anal. Calcd. for C H N O S (305.36): C 51.13; H 4.95; N 22.94. Found C 51.35;
13 15 5 2
◦
1
4
-Benzyl-1-(4-methylimidazol-5-oyl)thiosemicarbazide (
8
)
.
Yield: 90%. M.p. 225–227 C. H-NMR
CH ); 7.26–7.32 (m, 5H, 5 CHar); 7.59 (s,
NH). Anal. Calcd. for C H N OS (289.36): C 53.96; H
(
DMSO-d6) (ppm): 2.44 (s, 3H, CH ); 4.70–4.71 (d, 2H, 1
δ
×
×
3
2
1
H, 1
×
CHar); 8.31, 9.26, 9.67, 12.36 (4s, 4H, 4
.23; N 24.20. Found C 54.01; H 5.22; N 24.11.
×
13
15
5
5
◦
4
-(2-chloroethylphenyl)-1-(4-methylimidazol-5-oyl)thiosemicarbazide (
9
×
)
.
Yield: 96%. M.p. 175–177 C.
CH ); 7.72 (s, 1H, 1 CHar); 10.66,
NH). Anal. Calcd. for C H ClN OS (261.73): C 36.71; H 4.62; N 26.76. Found C 36.59;
1
H-NMR (DMSO-d6)
1.59 (2s, 4H, 4
H 4.61; N 26.81.
δ
(ppm): 2.44 (s, 3H, CH ); 3.88–3.93 (m, 4H, 2
×
3
2
1
×
8
12
5
◦
4
-(3-Chlorophenyl)-1-(4-methylimidazol-5-oyl)thiosemicarbazide (10
)
.
Yield: 78%. M.p. 203–205 C.
CHar); 7.30–7.35 (t, 1H, 1
CHar); 9.71–9.81 (m, 3H, 3 NH); 12.40
NH). Anal. Calcd. for C H ClN OS (309.77): C 46.53; H 3.90; N 22.61. Found C 46.67; H
1
H-NMR (DMSO-d6)
7
δ
(ppm): 2.43 (s, 3H, CH ); 7.15–7.16 (d, 1H, 1
×
×
CHar);
3
.47–7.53 (m, 1H, 1 CHar); 7.63 (s, 1H, 1xCHar); 7.70 (s, 1H, 1
×
×
×
(
s, 1H, 1
×
12
12
5
3
.89; N 22.65.
◦
4
-(3-Fluorophenyl)-1-(4-methylimidazol-5-oyl)thiosemicarbazide (11
)
.
×
Yield: 81%. M.p. 191–192 C.
CHar); 7.31–7.34 (m, 2H, 2
CHar); 9.70, 9.82, 9.86, 12.40 (4s, 4H, 4 NH). Anal. Calcd. for
C H FN OS (293.32): C 49.14; H 4.12; N 23.88. Found C 49.29; H 4.13; N 23.79.
1
H-NMR (DMSO-d6)
7
δ
(ppm): 2.43 (s, 3H, CH ); 6.91–6.97 (m, 1H, 1
×
CHar);
3
.53 (s, 1H, 1 CHar); 7.63 (s, 1H, 1
×
×
×
12
12
5

Molecules 2019, 24, 614
12 of 17
◦
4
-(3-Acetylphenyl)-1-(4-methylimidazol-5-oyl)thiosemicarbazide (12
)
.
Yield: 78%. M.p. 206–208 C.
1
H-NMR (DMSO-d6)
δ
(ppm): 2.46 (s, 3H, CH ); 2.57 (s, 3H, COCH ); 7.43–7.48 (t, 1H, 1
×
CHar);
CHar);
3
3
7
9
4
.63 (s, 1H, 1
.70–9.80 (m, 3H, 3
.76; N 22.07. Found C 53.08; H 4.75; N 22.00.
×
CHar); 7.71–7.74 (m, 1H, 1
×
CHar); 7.81–7.86 (m, 1H, 1
×
CHar); 8.07 (s, 1H, 1
×
×
NH); 12.40 (s, 1H, 1
×
NH). Anal. Calcd. for C H N O S (317.37): C 52.98; H
14
15
5
2
◦
-(3,4-Dimethoxyphenyl)-1-(4-methylimidazol-5-oyl)thiosemicarbazide (13). Yield: 81%. M.p. 223–225 C.
4
1
H-NMR (DMSO-d6)
δ
(ppm): 2.45 (s, 3H, CH ); 3.72 (s, 3H, OCH ); 3.73 (s, 3H, OCH ), 6.87–6.90 (m,
3
3
3
1
2
H, 1
×
CHar); 6.96–6.99 (m, 1H, 1
×
CHar); 7.15 (s, 1H, 1
×
CHar); 7.62 (s, 1H, 1
×
CHar); 9.45–9.50 (m, 2H,
×
NH); 9.70, 12.40 (2s, 2H, 2
×
NH). Anal. Calcd. for C H N O S (335.38): C 50.14; H 5.11; N 20.88.
1
4
17
5
3
Found C 50.19; H 5.12; N 20.80.
◦
4
-(3-Chloro-4-methylphenyl)-1-(4-methylimidazol-5-oyl)thiosemicarbazide (14
)
.
Yield: 89%. M.p. 213–215 C.
CHar); 7.35–7.38
NH); 12.40 (s, 1H,
1
H-NMR (DMSO-d6)
δ
(ppm): 2.29 (s, 3H, CH ); 2.45 (s, 3H, CH ); 7.25–7.28 (m, 1H, 1
×
3
3
(
m, 1H, 1
×
CHar); 7.62 (s, 1H, 1
×
CHar); 7.64 (s, 1H, 1
×
CHar); 9.64–9.76 (m, 3H, 3
×
1
×
NH). Anal. Calcd. for C H ClN OS (323.80): C 48.22; H 4.36; N 21.63. Found C 48.06; H 4.37;
13 14 5
N 21.65.
◦
4
-(2,5-Difluorophenyl)-1-(4-methylimidazol-5-oyl)thiosemicarbazide (15
)
.
Yield: 79%. M.p. 218–220 C.
CHar); 9.35 (s, 1H, 1 CHar);
NH). Anal. Calcd. for C H F N OS (311.31): C 46.30; H 3.56; N 22.50.
1
H-NMR (DMSO-d6)
0.70, 12.41, 13.74 (3s, 4H, 4
Found C 46.45; H 3.57; N 22.54.
δ
(ppm): 2.43 (s, 3H, CH ); 7.57–7.69 (m, 3H, 3
×
×
3
1
×
12
11
2
5
◦
1
-(4-Methylimidazol-5-oyl)-4-(2-methylphenyl)thiosemicarbazide (16
)
.
Yield: 96%. M.p. 210–212 C.
(ppm): 2.40 (s, 3H, CH ); 2.43 (s, 3H, CH ); 7.56–7.64 (m, 4H, 4 CHar);
NH). Anal. Calcd for C H N OS (289.36): C 53.96;
1
H-NMR (DMSO-d6)
.34 (s, 1H, 1 CHar); 10.70, 12.35, 13.73 (3s, 4H, 4
H 5.23; N 24.20. Found C 53.76; H 5.22; N 24.17.
δ
×
3
3
9
×
×
13
15
5
◦
1
-(4-Methylimidazol-5-oyl)-4-(3-methylphenyl)thiosemicarbazide (17
)
.
Yield: 86%. M.p. 193–195 C.
1
H-NMR (DMSO-d6)
.16–7.21 (m, 1H, 1 CHar); 7.30–7.34 (m, 2H, 2
Anal. Calcd. for C H N OS (289.36): C 53.96; H 5.23; N 24.20. Found C 54.01; H 5.24; N 24.27.
δ
(ppm): 2.23 (s, 3H, CH ); 2.45 (s, 3H, CH ); 6.92–6.95 (m, 1H, 1 CHar);
×
3
3
7
×
×
CHar); 9.50–9.75 (m, 3H, 3
×
NH); 12.40 (s, 1H, 1
×
NH).
1
3
15
5
1
-(4-Methylimidazol-5-oyl)-4-(4-dimethyloaminophenyl)thiosemicarbazide (19
)
.
Yield: 94%. M.p. 215–217
◦
1
C. H-NMR (DMSO-d )
δ
(ppm): 2.45 (s, 3H, CH ); 2.87 (s, 6H, 2
×
CH ); 6.65–6.70 (m, 2H, 2 CHar);
×
6
3
3
7
.16–7.22 (m, 2H, 2
×
CHar); 7.61 (s, 1H, 1
×
CHar); 9.15, 9.32, 9.69, 12.39 (4s, 4H, 4
×
NH). Anal. Calcd.
for C H N OS (318.40): C 52.81; H 5.70; N 26.39. Found C 52.94; H 5.69; N 26.47.
14
18
6
◦
1
-(4-Methylimidazol-5-oyl)-4-(4-diethyloaminophenyl)thiosemicarbazide (20). Yield: 81%. M.p. 225–227 C.
1
H-NMR (DMSO-d6)
6
4
δ
(ppm): 1.05–1.10 (t, 6H, 2
CHar); 7.14–7.17 (dd, 2H, 2
NH). Anal. Calcd. for C H N OS (346.45): C 55.47; H 6.40; N 24.26. Found C 55.57; H 6.38;
×
CH ); 2.45 (s, 3H, CH ); 3.28–3.32 (m, 4H, 2
×
CH2);
3
3
.58-6.61 (dd, 2H, 2
H, 4
×
×
CHar); 7.61 (s, 1H, 1
×
CHar); 9.27, 9.36, 9.68, 12.38 (4s,
×
16
22
6
N 24.18.
◦
4
-(4-Ethoxyphenyl)-1-(4-methylimidazol-5-oyl)thiosemicarbazide (21
)
.
Yield: 84%. M.p. 210–212 C.
(ppm): 1.29–1.34 (t, 3H, CH ); 2.45 (s, 3H, CH ); 3.96–4.02 (q, 2H, 1 CH2);
CHar); 9.44, 9.72, 12.38 (3s,
1
H-NMR (DMSO-d6)
6
4
δ
×
3
3
.84–6.87 (dd, 2H, 2
H, 4
×
CHar); 7.30–7.33 (dd, 2H, 2
×
CHar); 7.61 (s, 1H, 1
×
×
NH). Anal. Calcd. for C H N O S (319.38): C 52.65; H 5.37; N 21.93. Found C 52.52; H 5.38;
14 17 5 2
N 22.00.
◦
4
-(2,6-Diisopropylphenyl)-1-(4-methylimidazol-5-oyl)thiosemicarbazide (22
)
.
Yield: 73%. M.p. 202–204 C.
1
H-NMR (DMSO-d6)
CHisopropyl); 7.05–7.34 (m, 3H, 3
Anal. Calcd. for C H N OS (359.49): C 60.14; H 7.01; N 19.48. Found C 60.01; H 7.02; N 19.54.
δ
(ppm): 1.29–1.31 (d, 12H, 4
×
CH ); 2.43 (s, 3H, CH ); 3.21–3.27 (sept., 2H,
3
3
2
×
×
CHar); 7.60 (s, 1H, 1
×
CHar); 9.00, 9.33, 9.42, 12.34 (4s, 4H, 4 NH).
×
18
25
5

Molecules 2019, 24, 614
13 of 17
3
.3. Cytotoxicity Assays
3
.3.1. Cell Culture
Cell line L929 mouse fibroblast (ATTC® CCL-1
™) was routinely cultured in IMDM
(
(
Iscove’s Modified Dulbecco Medium—Biowest, Nuaille, France) supplemented with 10% HIFBS
◦
Heat-Inactivated (1 h in 56 C) Fetal Bovine Serum—Biowest), 100 U/mL penicillin and 100
µ
g/mL
6
streptomycin. Cells were trypsinized twice a week, and seeded at density 1
flask, and incubated for 24–48 h at 37 C and 10% CO to achieve a confluent monolayer.
×
10 per T25 cell culture
◦
2
3
.3.2. Preparation of Compounds and Sulfadiazine
Compounds 22 were dissolved in dimethyl sulfoxide (DMSO—Sigma) to 50,000
The final concentration of DMSO in compounds dilutions was not higher than 1.00%. Sulfadiazine
4-amino-N-(2-pyrimidinyl)benzenesulfonamide] (S8626, Sigma) was dissolved in 1 M sodium
hydroxide (NaOH—Sigma) to 100 mg/mL. Final concertation of NaOH in sulfadiazine dilutions
2–
µg/mL.
[
was not higher than 2.5%. Dilution of the all compounds
the cells were exposed.
2–22 and drug were freshly prepared before
3
.3.3. Cell Viability Assay
The effects of tested compounds and drugs on the viability of mouse fibroblasts L929 cells
were evaluated using the MTT salt [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]
Sigma). The MTT assay was used according to international standards: ISO 10993-5:2009(E), Biological
(
evaluation of medical devices, Part 5: Tests for in vitro cytotoxicity. L929 cells were placed into
4
9
6-well plates at a density of 1
×
10 /100
µ
L/well in culture medium RPMI 1640 without phenol
red (Biowest) supplemented with 10% HIFBS, 2 mM L-glutamine (Sigma), 100 U/mL penicillin,
◦
100
µ
g/mL streptomycin, grown at 37 C in a 10% CO humidified environment and allowed to
2
attach and form a confluent monolayer for 24 h before treatment. Afterward, the culture medium
in the plates was replaced by 100 L of compounds and sulfadiazine suspension and the cells were
µ
exposed for 24 h. Then, 1 mg/mL of MTT solution in RPMI 1640 without phenol red was prepared and
◦
50
µ
L of the solution was added to each well and incubated at 37 C, 10% CO for 2 h. Mitochondrial
2
dehydrogenases of viable cells reduced the yellowish water-soluble MTT to water-insoluble formazan
crystals, which were solubilized with dimethyl sulfoxide (DMSO). The cell culture medium was
aspirated cautiously, after which 150
µL DMSO was added to each well, the plates were gently
mixed, then 25 L 0.1 M glycine buffer (pH = 10.5) (Sigma) was added. Optical density was read
µ
on the ELISA reader (Multiskan EX, Labsystems, Vienna, VA, USA) at 570 nm. The results were
expressed as the percentage of viability compared to untreated cells. All experiments were performed
in triplicate. Moreover, cells were treated with 4.0–0.03% concentration of DMSO as a compounds
solvent (data not shown).
3
.4. In Vitro Assay for Anti-T. gondii Activity
3
.4.1. Cell Line and Parasite Culture
®
Vero cell line (African green monkey kidney) (ATCC CCL-81
™
) were maintained in EMEM
®
(
EMEM/10%HIFBS/P/S) (Eagle’s Minimum Essential Medium—ATCC 30-2003
supplemented with 10% HIFBS (Heat-Inactivated (1 h in 56 C) Fetal Bovine Serum—ATCC), 100 U/mL
™) culture medium
◦
penicillin, 100
ATCC 30-2101
µ
g/mL streptomycin. The cell line was trypsinized (using Trypsin-EDTA Solution, 1X
®
6
™
) twice a week, and seeded at density 1
×
10 per T25 cell culture flask (Falcon),
◦
and incubated for 24–48 h at 37 C and 5 CO to achieve a confluent monolayer. The highly virulent
RH strain of Toxoplasma gondii, haplogroup I (ATCC PRA-310
according to the ATCC product sheet, in DMEM (Dulbecco’s Modified Eagle’s Medium—ATCC
2
®
™) were maintained as tachyzoites
®

Molecules 2019, 24, 614
14 of 17
◦
™) (DMEM/3%HIFBS/P/S—parasite culture medium) under condition 37 C and 5 CO and
2
3
0-2002
passage twice per week.
3
.4.2. Influence of 2–22 and Sulfadiazine on T. gondii Proliferation
The Vero cells were seeded on 96-well plates (1
EMEM/10%HIFBS/P/S. After 24 h of incubation medium was removed and then T. gondii
RH tachyzoites were added to the cell monolayers at density 1
DMEM/3%HIFBS/P/S. One hour later different compounds and sulfadiazine dilutions (100
×
105 cells/100
µL/well) in
5
×
10 cells/100
µL/well in
µL/well)
in parasite culture medium were added to the cell monolayers with T. gondii. After subsequent 24 h of
3
incubation 1
µ
Ci/well [5,6- H] uracil (Moravek Biochemicals Inc., Brea, CA, USA) was applied to each
microculture for 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.
All experiments were performed in triplicate.
3
.5. 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). Additionally, to the established relationship between
cytotoxicity and antiparasitic activity typical selectivity indexing is calculated as the CC30 or
even CC50 over IC50 values. In this paper we also established this dependence, to prioritize
imidazole-thiosemicarbazides as therapeutic agents, through a modified selectivity index, which
was calculated as the logarithm of the ratio of the CC30 and the IC50 values, according to the formula:
SI = log(CC /IC ). A positive value represents more selectivity against T. gondii than L929 cells,
30
50
while negative ones (or close to zero) indicate higher toxicity to L929 cells and low selectivity to
the parasite [46 47]. With a CC30 range between 3.91 to 500 g/mL and IC50 values between 1 and
25 g/mL, the equation gives a SI value between 1.5 to 2.7. It worth underlining that in our
,
µ
1
µ
−
study we developed a rigorous approach to determine cytotoxicity by using the CC30 instead of CC50,
which makes our SI value stricter and more precise. The higher the SI value the more selective the
imidazole-thiosemicarbazides were towards inhibiting T. gondii proliferation vs. cytotoxicity. Therefore,
compounds with a positive SI value closer to 2.7 may have potential as agents against toxoplasmosis,
while derivatives with a negative SI possesses more toxic properties than specific T. gondii in vitro
inhibition and received low priority for follow-up. For compounds with IC50 values greater than
1
25
µg/mL, the highest concentration tested, IC50 values were calculated based on extrapolation of the
curves using GraphPad Prism program (version 8.0.1).
3
.6. Computational Details
Conformational search was performed using the Amber force field as implemented in
HyperChem8.0.3. [48] and default convergence criteria. The rule of five, the number of rotatable bonds,
and polar surface area (PSA) were determined using the Molinspiration program [49]. Solubility (logS)
was calculated using ALOGPS 2.1 program [50]. Absorption (%ABS) was calculated by equation
%
ABS = 109 − 0.345 × PSA [38].
. Conclusions
The results reported in this paper discusses our prior findings on the inhibitory activity of
4
thiosemicarbazides against Toxoplasma gondii proliferation. In particular, we have confirmed that
-arylthiosemicarbazides with a five-membered heterocyclic ring at the N1 position might be
4
considered as potential anti-Toxoplasma gondii agents. The best imidazole-thiosemicarbazides reported
here, 3 and 4, were 125 to 184 fold more potent than sulfadiazine, respectively.

Molecules 2019, 24, 614
15 of 17
An ideal anti-Toxoplasma drug should have efficient tissue penetration, especially through the
CNS and the placental barrier. Moreover, should possess activity against tachyzoites related with
acute phase, and ability to cyst penetration and bradyzoites inactivation which are responsible for
the latent stage of toxoplasmosis. Any drug approved for use also should be free from fetal toxicity
and teratogenicity. However, none of the currently used drugs fulfill these criteria. Promising
results obtained in this research of thiosemicarbazide derivatives which inhibit the tachyzoites
proliferation in vitro, at a concentration that is not cytotoxic for the host cells, are the basis for the
currently started experiments including in vivo tests on a mouse model with another T. gondii strain
(Me49 and DX) responsible for cyst formation. If found to be effective in our in vivo studies, such
compounds could represent leads for the development of novel drugs against toxoplasmosis. From
the other hand, due to slight differences between tachyzoites and bradyzoites, such as differences in
metabolism, morphological changes and stage-specific antigen expression [3,4], we can speculate that
our compounds will also have activity against bradyzoites.
Author Contributions: Conceptualization, A.P. and K.D.; methodology, L.W., A.B.; software, A.P.; formal analysis,
A.P. and K.D.; investigation, L.W., A.B.; resources, E.S.; data curation, L.W., E.S., M.W., N.T.; writing—original
draft preparation, L.W., A.B.; writing—review and editing, A.P., K.D.; visualization, L.W., A.B.; supervision, A.P.,
K.D.
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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article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).