Synthesis of thiosemicarbazone and 4-thiazolidinone derivatives and their in vitro anti-Toxoplasma gondii activity

Article abstract of DOI:10.1016/j.bmcl.2005.03.048

Thiosemicarbazone and 4-thiazolidinone derivatives were synthesized in one and two step, respectively, from thiosemicarbazide, in satisfactory yields. Then, the synthesized compounds were submitted to evaluation against host cells infected with Toxoplasma

Full text of DOI:10.1016/j.bmcl.2005.03.048

Bioorganic & Medicinal Chemistry Letters 15 (2005) 2575–2578
Synthesis of thiosemicarbazone and 4-thiazolidinone derivatives
and their in vitro anti-Toxoplasma gondii activity
a
Romulo P. Tenorio, Cristiane S. Carvalho, Carla S. Pessanha, Jose G. de Lima,
b
b
c
ˆ
´
´
Antonio R. de Faria, Antonio J. Alves, Edesio J. T. de Melo and Alexandre J. S. Goes
c
c
b
a,
*
ˆ
ˆ
´
´
a
´
Departamento de Antibioticos, Universidade Federal de Pernambuco, Recife 50670-901, Brazil
b
´
Laboratorio de Biologia Celular, Universidade Estadual do Norte Fluminense, Rio de Janeiro 28015-620, Brazil
c
ˆ ˆ
Departamento de Ciencias Farmaceuticas, Universidade Federal de Pernambuco, Recife 50670-901, Brazil
Received 2 December 2004; revised 8 March 2005; accepted 14 March 2005
Available online 9 April 2005
Abstract—Thiosemicarbazone and 4-thiazolidinone derivatives were synthesized in one and two step, respectively, from thiosemi-
carbazide, in satisfactory yields. Then, the synthesized compounds were submitted to evaluation against host cells infected with Toxo-
plasma gondii. The present studies showed that thiosemicarbazones 2 and 4-thiazolidinone derivatives 3 were effective against
intracellular T. gondii.
Ó 2005 Elsevier Ltd. All rights reserved.
1. Introduction
cell cycle in the G1/S phase and, then, inactivating the
DNA synthesis.2 However, HU has a low affinity for
the ribonucleotide reductase and a short half-life in hu-
mans.8 On the other hand, thiosemicarbazones are
known inhibitors of this enzyme, being approximately
1000 times more potent than hydroxyurea (Fig. 1b).9
Toxoplasma gondii is an obligate intracellular protozoa
able to infect a large number of vertebrate cells.1 After
host cell invasion, the infected form of T. gondii, called
tachyzoite, localized in the cytoplasm within a mem-
brane-bounded vacuole, known as parasitophorous vac-
uole (PV), multiplying at intervals of 6 h.2 In humans, it
is responsible for toxoplasmosis, a disease known for its
great importance to immunodepressed patients due to
its opportunistic character.3 With the advent of AIDS,
the worldwide incidence of infections caused by T. gon-
dii has been rising.4 In the USA, between 18% and 25%
of patients with AIDS will suffer from symptomatic
toxoplasmosis during the course of their illness.3 Besides
this, the current chemotherapy against human toxoplas-
mosis involve undesirable side effects and it is not effec-
tive in eliminating the parasite located in the central
nervous system.5,6
Biological activities of these thiosemicarbazones are re-
lated to their abilities to form complex with metal cations,
by bonding through the sulfur and azomethine nitrogen
atoms.10 Several works demonstrate their activity against
extracellular protozoan as Plasmodium falciparum, Tricho-
monas vaginalis, T. cruzi and other parasites.10–12 How-
ever, no study about their biological properties against
intracellular T. gondii was even related.
H
NH2
O
N
R1
N
H
O
O
a
Recent studies using hydroxyurea (HU) demonstrate its
efficacy, in vitro, in eliminating the intracellular para-
sites of T. gondii, Trypanosoma cruzi and Leishmania
amazonensis (Fig. 1a).7 HU acts by blocking the activity
of the enzyme ribonucleotide reductase, arresting the
N
N
1
NH
S
R
H
N
S
c
N
Ar
CO2H
b
Ar
Ar = aryl group; R1
= H, alkyl, aryl
Keywords: Thiosemicarbazones; 4-Thiazolidinones; Toxoplasma gondii.
*
Figure 1. Hydroxyurea (a), thiosemicarbazone (b) and 4-thiazolidi-
none (c).
Corresponding author. Tel.: +55 81 21268886; fax: +55 81
21268346; e-mail: ajsg@ufpe.br
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.03.048

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R. P. Tenorio et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2575–2578
2576
In addition, thiosemicarbazones may also be seen as
structure analogues of 4-thiazolidinones carrying a
hydrazone moiety in the two position (Fig. 1c). Com-
pounds bearing a 4-thiazolidinone heterocycle are usu-
ally of biological interest, due to their large spectrum
of activity.13–17 In this communication, we described a
cost-effective synthesis, in one and two steps, of thiosemi-
carbazone and 4-thiazolidinone derivatives, their struc-
ture elucidation and in vitro biological activities
against T. gondii.
1
H
N
H2N
R1
NH
S
R
NH
S
H
N
I
N
1
2
Ar
II
R1
N
O
N
N
S
Scheme 1 shows the synthetic route employed for the
preparation of the compounds. As can be seen, a simple
synthesis was carried out, initiating from the commer-
cially available benzaldehyde derivatives and the
appropriate thiosemicarbazide substitutes 1, in
water/ethanolic media containing a few drops of acetic
acid as the catalyst. At the end of the reaction, the iso-
lated benzaldehyde thiosemicarbazone derivatives 2
were obtained as solids of yellow colour. This procedure
was adapted from an article previously reported by
Bharti et al.12 and gave us satisfactory yields.
Ar
3
CO2H
Scheme 1. Reagents and conditions: (I) ArCHO, H2O, EtOH, acid
acetic, reflux (56–97%); (II) dry toluene, anhydride maleic, DMF,
Dean–Stark, reflux (26–95%).
introduces an additional diversity point to further struc-
tural tuning, we utilized a thia-Michael addition reac-
tion with the employment of anhydride maleic as the
acceptor of Michael.18 The reaction was carried out in
dry toluene and DMF and, as expected, gave us the 4-
thiazolidinones substituted 3.
Furthermore, the obtained benzaldehyde thiosemicarb-
azone derivatives 2 were utilized for the preparation of 4-
thiazolidinones substituted in the five position with an
acetic acid group. Since the presence of an acid function
The synthesized compounds were obtained in satisfac-
tory yields (Table 1). The structures of all compounds
Table 1. Synthesized compounds, their yields, melting points and in vitro activities against T. gondii
R1
1
O
NH
H
N
R
N
N
N
N
S
S
R2
R2
2
3
CO2H
Compounds
R1
R2
Yield (%)
Mp (°C)
% Infected Vero cells
Mean number of
intracellular parasites
Untreated
76
65 12
Treated
Untreated
Treated
2a
2b
2c
2d
2e
2f
–H
–H
m-NO2
p-NO2
o-NO2
m-NO2
p-NO2
o-NO2
m-NO2
p-NO2
o-NO2
m-NO2
m-NO2
p-NO2
o-NO2
m-NO2
p-NO2
o-NO2
m-NO2
p-NO2
o-NO2
m-NO2
—
93
56
75
70
87
60
60
96
96
97
95
52
26
36
26
33
60
56
65
50
—
226–228
229–230
234–235
233–234
230–232
232–233
207–208
228–229
197–198
189–191
249–250
267–268
247–248
194–196
202–204
213–215
187–188
220–221
238–240
215–217
—
9
4
5
2
2
5
3
4
2
3
3
3
4
8
5
5
4
6
5
5
4
3
4
6
768 142
698 94
783 84
642 92
683 60
783 84
768 142
852 140
642 92
442 69
840 116
732 69
888 143
628 74
726 115
663 16
732 69
627 81
687 103
786 79
913 123
3
4
2
1
3
6
3
1
–CH3
–CH3
–CH3
81
84
89
81
76
86
84
4
3
6
4
9
4
3
9
9
12
8
72 28
–CH2CH3
–CH2CH3
–CH2CH3
–C6H5
–C6H5
–H
1
5
9
3
2
6
2
2
2g
2h
2i
7
17
7
2j
65 15
7
11 12
3a
3b
3c
3d
3e
3f
80
85
84
85
87
86
85
80
83
6
3
3
2
4
4
3
2
4
15
16
15
11
13
14
19
7
4
4
3
2
2
3
3
2
2
2
3
3
3
3
2
2
2
2
2
2
–H
–CH3
–CH3
–CH3
–CH2CH3
–CH2CH3
–CH2CH3
–C6H5
–C6H5
—
3g
3h
3i
8
3j
HUa
72 22
80
13
26
5
186 52
a Hydroxyurea; data is reported as mean SD of four independent experiments (p < 0.05) compared to untreated cells (Student t-test).

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R. P. Tenorio et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2575–2578
2577
Table 2. IC50 values for representative compounds of the two series
were established on the basis of their NMR spectro-
scopic data, IR and, only for the 4-thiazolidinones, mass
spectroscopy.19
Compounds
Infected cells
IC50 (mM)
Intracellular parasites
IC50 (mM)
2a
2b
2
0.05
1.5 0.05
0.06
1.5 0.04
0.1 0.06
0.5 0.06
0.5 0.05
0.5 0.05
2. Biological studies
2c
2d
2
2e
10
2
1
3
0.4
2.1. In vitro activity against T. gondii
2f
2g
0.05
0.05
0.5 0.05
0.08 0.002
0.5 0.05
0.5 0.05
0.08 0.004
0.5 0.06
0.5 0.04
0.5 0.06
0.5 0.06
0.1 0.003
0.1 0.07
1
The cultures infected with tachyzoites were treated with
the drugs as described by Melo et al.2 and Melo and
Beiral.7 Concisely Vero cells (kidney fibroblast from
African green monkeys) were infected with tachyzoites
of T. gondii (1:10) and incubated at 37 °C for 24 h. After
that the cultures were allowed in presence of thiosemi-
carbazone compounds 2 (1 mM), 4-thiazolidinone deri-
vates 3 (1 mM) and hydroxyurea (4 mM) for 24 h.
These concentrations were screened in early studies to
shown anti-parasite effects. After that the cultures were
processed in light microscopy and counted using AXIO-
PLAN microscope equipped with x63 objective. Statisti-
cal analysis was carried out using the Student t-test, with
the level of significance set at p < 0.05. The data shown
are representative of four experiments in triplicate.
2h
2i
2.5 0.05
1
1
2
0.05
0.05
0.05
2j
3a
3b
3c
2.5 0.04
2
2
0.07
0.06
3h
HUa
SULFb
0.1 0.004
0.09 0.04
a Hydroxyurea.
b Sulfadiazine; each single compound was tested in a series dilution
(0.1, 1, 10, 20, 30 mM) in triplicate per assay. The IC50 values
(mean SD) were determinate by a non-linear regression using
exclusion test with trypan blue.
In presence of thiosemicarbazones (2a–j) and 4-thiazo-
lidinone derivates (3a–j), a drastic decrease in infected
host cells with T. gondii was observed (Table 1). This
reduction of infected host cells was due to elimination
of intracellular parasites (Table 1). The treated host cells
not present morphologic alterations after 24 h of drugs
treatment and they continued their development after
drugs were withdrawn from the medium (data not
shown). This potentiality of the compounds as inhibitors
of reductase activity suggests further that arrest parasite
cycle induced to decrease of the infection and elimina-
tion of intracellular parasites. These new compounds
also were most effective than hydroxyurea (HU) to elim-
inate intracellular T. gondii (Table 1, Fig. 2). The two
series of compounds showed little differences in the
anti-T. gondii effect at 1 mM (Fig. 2). The IC50 of some
representative compounds was determined (Table 2).
After 24 h exposure in the concentrations range of 0.01–
30 mM, the IC50 values of most compounds were ob-
served (Table 2). In the infected cells, the IC50 values
of 2a–f,h were P1.5 mM whereas 2g,i,j were 1 mM.
However, all compounds showed lower cytotoxicity
than hydroxyurea and sulfadiazine. The toxicity of all
compounds were more effective against intracellular para-
sites (Table 2) where IC50 were 60.5 mM with excep-
tion to compounds 2g,j (0.08 mM) and 2a (0.1 mM).
Some studies showed that thiosemicarbazones have been
effective against extracellular parasites.20,21 However,
very little is known about the effect of thiosemicarbaz-
ones on intracellular parasites. The present studies
showed that the presence of thiosemicarbazones 2 and
4-thiazolidinone derivates 3 were also effective against
intracellular T. gondii. Studies using these compounds
Intracellular Parasites
Intracellular Parasites
200
200
180
160
140
120
100
80
180
160
140
120
100
80
60
60
40
40
20
20
0
0
3a 3b 3c 3d 3e 3f 3g 3h 3i
Compounds
3j HU
2a 2b 2c 2d 2e 2f 2g 2h 2i 2j HU
Compounds
Figure 2. Relative number of intracellular parasites in infected host cells after treatment with the compounds.

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R. P. Tenorio et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2575–2578
2578
14. Rao, A.; Balzarini, J.; Carbone, A.; Chimirri, A.; Clerq,
E.; Monforte, A. M.; Monforte, P.; Pannecouque, C.;
against other intracellular parasites are also currently
being conducted in our laboratory.
´
Zappala, M. Il Farmaco 2004, 59, 33.
´
15. Vigorita, M. G.; Ottana, R.; Monforte, F.; Maccari, R.;
Monforte, M. T.; Trovato, A.; Taviano, M. F.; Miceli, N.;
Luca, G.; Alcaro, S.; Ortuso, F. Bioorg. Med. Chem. 2003,
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Acknowledgements
This work was supported by Conselho Nacional de
¨
16. Ergenc¸, E.; C¸ apan, G.; Gunay, N. S.; Ozkirimli, S.;
¨
¨
Gungor, M.; Ozbey, S.; Kendi, E. Arch. Pharm. Med.
´
´
Desenvolvimento Cientıfico e Tecnologico (CNPq). We
also thank CAPES and the Fundac¸a˜o de Amparo a Pes-
quisa do Estado do Rio de Janeiro (FAPERJ) for the
student fellowships.
¨
Chem. 1999, 332, 343.
17. Alves, A. J.; Leite, A. C. L.; Santana, D. P.; Beltra˜o, T.
M.; Coelho, M. R. D. Il Farmaco 1993, 48, 1167.
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References and notes
19. All the compounds were fully characterized. Structural
data for 2c and 3c is present here. Compound 2c: FTIR (m
cmÀ1 KBr): 3370 (NHCH3), 1513 (C@N), 1098 (C@S);
1H NMR (DMSO-d6, 300 MHz/ppm): 11.8 (1H, s, NH),
8.66 (1H, br q, J = 4.5 Hz, NHCH3), 8.47 (1H, s,
CH@N), 8.42 (1H, dd, J = 8.1 Hz and J = 1.2 Hz, CH
arom), 8.03 (1H, dd, J = 8.1 Hz and J = 1.2 Hz, CH
arom), 7.74–7.79 (1H, m, CH arom), 7.60–7.65 (1H, m,
CH arom), 3.02 (3H, br d, J = 4.5 Hz, NHCH3). Com-
pound 3c: FTIR (m cmÀ1 KBr): 1714 (C@O; CO2H), 1617
(C@O; lactam), 1356 (NCS); 1H NMR (DMSO-d6,
300 MHz/ppm): 12.73 (1H, br s, CO2H), 8.74 (1H, s,
CH@N), 8.07 (1H, dd, J = 8 Hz and J = 1.2 Hz, CH
arom), 8.02 (1H, dd, J = 8 Hz and J = 1.2 Hz, CH arom),
7.79–7.85 (1H, m, CH arom), 7.68–7.74 (1H, m, CH
arom), 4.43 (1H, dd, J = 9 Hz and J = 4 Hz, CH), 3.19
(3H, s, CH3), 3.08 (1H, dd, J = 18 Hz and J = 4 Hz,
CH2), 2.91 (1H, dd, J = 18 Hz and J = 9 Hz, CH2); 13C
NMR (DMSO-d6, 75.4 MHz/ppm): 29.50 (CH3), 36.64
(CH2), 42.79 (CH), 124.62 (CH arom), 128.22 (Cq, arom),
129.05 (CH arom), 131.31 (CH arom), 133.65 (CH arom),
148.36 (Cq, arom), 153.58 (CH, CH@N), 166.44 (Cq,
C@N), 171.69 (Cq, C@O lactam), 173.81 (Cq, CO2H);
Mass m/e, M+ = 335.
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