Synthesis and evaluation of anti-Toxoplasma gondii and antimicrobial activities of thiosemicarbazides, 4-thiazolidinones and 1,3,4-thiadiazoles

Article abstract of DOI:10.1016/j.ejmech.2010.05.017

In this work we reported the synthesis and evaluation of anti-Toxoplasma gondii and antimicrobial activities in vitro of three new compound series obtained from ethyl(5-methyl-1-H-imidazole-4-carboxylate): acylthiosemicarbazide analogues 3a-d, 4-thiazolidinone analogues 4a-d and 1,3,4-thiadiazole analogues 5a-d. All synthesized compounds were characterized by IR, ¹H, ¹³C NMR and HRMS. The majority of the tested compounds show excellent anti-T. gondii activity when compared to hydroxyurea and sulfadiazine. In addition it was also shown that most of the compounds in this study have a better performance against intracellular tachyzoites. The results for antimicrobial activity evaluation showed weak antibacterial and antifungal activities for all the tested molecules, when compared with the standard drugs (chloramphenicol and rifampicin for antibacterial activity; nistatin and ketoconazole for antifungal activity).

Full text of DOI:10.1016/j.ejmech.2010.05.017

European Journal of Medicinal Chemistry 45 (2010) 3685e3691
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and evaluation of anti-Toxoplasma gondii and antimicrobial activities
of thiosemicarbazides, 4-thiazolidinones and 1,3,4-thiadiazoles
André P. Liesen a, Thiago M. de Aquino a, Cristiane S. Carvalho c, Vânia T. Lima a, Janete M. de Araújo a,
José G. de Lima b, Antônio R. de Faria b, Edésio J.T. de Melo c, Antonio J. Alves b, Elias W. Alves c,
,
Anselmo Q. Alves b, Alexandre J.S. Góes a
*
a Departamento de Antibióticos, Universidade Federal de Pernambuco, Recife 50670-901, Brazil
b Departamento de Ciências Farmacêuticas, Universidade Federal de Pernambuco, Recife 50670-901, Brazil
c Laboratório de Biologia Celular e Tecidual, Universidade Estadual do Norte Fluminense, Rio de Janeiro 28013-620, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
In this work we reported the synthesis and evaluation of anti-Toxoplasma gondii and antimicrobial
activities in vitro of three new compound series obtained from ethyl(5-methyl-1-H-imidazole-4-
carboxylate): acylthiosemicarbazide analogues 3aed, 4-thiazolidinone analogues 4aed and 1,3,4-thia-
diazole analogues 5aed. All synthesized compounds were characterized by IR, 1H, 13C NMR and HRMS.
The majority of the tested compounds show excellent anti-T. gondii activity when compared to
hydroxyurea and sulfadiazine. In addition it was also shown that most of the compounds in this study
have a better performance against intracellular tachyzoites. The results for antimicrobial activity eval-
uation showed weak antibacterial and antifungal activities for all the tested molecules, when compared
with the standard drugs (chloramphenicol and rifampicin for antibacterial activity; nistatin and keto-
conazole for antifungal activity).
Received 29 November 2007
Received in revised form
3 May 2010
Accepted 10 May 2010
Available online 31 May 2010
Keywords:
Imidazole
1,3,4-Thiadiazole
4-Thiazolidinone
Antimicrobial activity
Toxoplasma gondii
Ó 2010 Elsevier Masson SAS. All rights reserved.
1. Introduction
gondii and treatment of toxoplasmosis [10e16]. In recent work, our
group reported the synthesis and evaluation of anti-T. gondii
Toxoplasma gondii infections are subclinical, unless located in
the uterus or developed in patients with serious immunosuppres-
sive condition. However, early studies also showed that they are
associated with lymphadenopathy, fever, weakness and debilita-
tion and ophthalmitis in immunocompetent individuals [1]. In
addition toxoplasmic encephalitis has been increasing worldwide
since the 1980s [2]. The treatments of toxoplasmic infections use
effective combination of pyrimethamine and sulphadiazine (or
clindamycin) with folinic acid [3]. Unfortunately, up to 40e50% of
patients treated develop severe adverse effects that force the
change or interruption of the therapy [4].
In immunocompetent patients, the infection with T. gondii can
cause symptoms as fever, headache or myalgia. However, serious
cases can result in toxoplasmic encephalitis (characterized by
intracerebralmasslesions)withmortalityratesexceeding30% [5e9].
Therefore becomes necessary the discovery of less-toxic and
more-efficacious parasite-specific drugs against the infection by T.
activity of 4-thiazolidinones substituted at arylhydrazone moiety
with electron-withdrawing or electron-donating groups, and at N-
3 position with H, methyl, ethyl and phenyl substituents (Fig. 1).
According to biological results, various compounds showed best
values of LD50 for both infected cells and intracellular parasites,
when compared with hydroxyurea and sulfadiazine (reference
substances) [17,18]. Thiosemicarbazones and 4-thiazolidinones are
known to inhibit the cell cycle in the G1/S phase, mainly inacti-
vating ribonucleotide reductase, an enzyme that mediates the
conversion of ribonucleotides to deoxyribonucleotides [19].
Compounds containing imidazole ring have been described as
antiprotozoal agents [20e22]. Most of these works report an
antitrypanosomal activity for these molecules. Megazol (2-amino-
5-(1-methyl-5-nitro-2-imidazolyl)-1,3,4-thiadiazole)
(Fig.
2),
synthesized in 1968 by Berkelhammer and Asato [23] as an anti-
microbial agent and characterized later as a powerful trypanocide
agent, was discarded because of its mutagenic risk [24]. Carvalho
et al. [25] reported new megazol analogues, containing a 2-aryl-
hydrazone moiety, where one of these compounds showed to be
a new potent trypanocide prototype. Imidazole derivatives are
* Corresponding author. Tel.: þ55 81 21268866; fax: þ55 81 21268346.
E-mail address: ajsg@ufpe.br (A.J.S. Góes).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.05.017

3686
A.P. Liesen et al. / European Journal of Medicinal Chemistry 45 (2010) 3685e3691
The synthetic route for the preparation of these molecules is out-
R
lined in Scheme 1. The acylthiosemicarbazide analogue 3aed and
1,3,4-thiadiazole analogue 5aed were prepared with satisfactory
yield, but for the synthesis of 4-thiazolidinones 4aed, the yields
ranged from 10 to 16%, due to formation of considerable quantities
of polar subproducts, which remain as residues in the column
chromatography on silica gel.
O
N
3
O
4
5
2
N
1
S
N
OH
O2N
The chemical structures were established using NMR, IR and
HRMS spectroscopy. The 1H NMR spectra for acylth-
iosemicarbazides 3aed showed signals at 12.41e9.48 ppm, attrib-
uted to NH, NH (imidazole) and NH-aromatic groups. On the other
hand, 13C NMR spectra exhibited resonated at 181.13e181.01 and
163.13e162.57 ppm assigned for C]S and C]O moieties, respec-
tively. For 4-thiazolidinones 4aed the IR spectra showed bands
around 1397e1378 cmꢀ1 characteristic for NCS bending vibration,
providing evidence for ring closure [28e31]. Further confirmation
was obtained from the 1H NMR spectra, which exhibited resonance
assigned to the SCH group appearing as triplet or double doublet
(ABX pattern) at 4.56e4.62 ppm, due to the interaction with
methylene protons of the acetyl group [32]. The formation of the
1,3,4-thiadiazoles 5aed was confirmed through the presence of
bands at 1104e1082 cmꢀ1 attributed to N]CeSeC]N vibrations.
Moreover, the absence of the signals at 181.13e181.01 and
163.13e162.57 ppm in 13C NMR spectra, attributed to C]S and C]
O moieties, was also a parameter considered for the confirmation of
the cyclodehydration reaction.
R = H, Alquil ou Aril
Fig. 1. 2-hydrazolyl-4-thiazolidinones.
known to possess a large range of biological activities, amongst
which are antiprotozoal (metronidazole, benznidazole, and meg-
azol) and antifungal (miconazole and ketoconazole) (Fig. 2).
Considering this panorama and perspectives, our research group
decided to synthesize new 4-thiazolidinones 4 and 1,3,4-thiadia-
zoles 5 from substituted acylthiosemicarbazides 3, containing the
imidazole ring, utilizing ethyl(5-methyl-1-H-imidazole-4-carbox-
ylate) 1 as a start compound. All compounds were screened for in
vitro anti-T. gondii activity, and evaluated for antimicrobial activity.
The aim of these biological assays is to discover new drugs with
antimicrobial and antiprotozoal potential, once the available drugs
are inefficient due to parasites elimination and show an increasing
number of resistant properties developed by the pathogens [26].
2. Results and discussion
2.2. Biological activities
2.1. Chemistry
Cultures of Vero cells were previously infected with T. gondii
and incubated for 24 h at 37 ꢁC. The synthesized and purified
acylthiosemicarbazides 3aed, 4-thiazolidinones 4aed and 1,3,4-
thiadiazoles 5aed were added to the cultures and incubated for
another 24 h at 37 ꢁC. Untreated cultures showed intracellular
proliferation of T. gondii leading to the lysis of some cells after
48 h. In the same time the cultures treated with the compounds
showed decreased percentage of infection with intracellular
tachyzoites elimination. During this drugs incubation the
The acylthiosemicarbazide analogues 3aed were synthesized by
an addition reaction between the hydrazide 2 (previously obtained
by condensation reaction between ethyl(5-methyl-1-H-imidazole-
4-carboxylate) 1, and hydrazidehydrate 80%) and the substituted
isothiocyanates. From these analogues were obtained two new
series of compounds: 4-thiazolidinones 4aed, by thia-Michael
reaction, involving maleic anhydride as Michael acceptor [18] and
1,3,4-thiadiazoles 5aed by cyclodehydration with sulfuric acid [27].
N
N
N
NO2
NO2
N
N
S
NO2
N
H
N
H2N
N
CH3
N
OH
O
Megazol
Cl
Benznidazole
Metronidazole
Cl
N
N
N
N
O
O
O
Cl
O
N
N
O
Cl
Cl
H
Ketoconazole
Cl
Miconazole
Fig. 2. Drugs containing Imidazole ring.

A.P. Liesen et al. / European Journal of Medicinal Chemistry 45 (2010) 3685e3691
3687
H
1
H
H
S
C
N
N
N
5
i
ii
O
NH NH2
NH NH
NH
R
2
4
N
N
N
3
O
O
O
1
2
3a-d
R
H
H
R
N
N
O
N
iii
iv
O
a
b
H
NH
S
N
N
N
S
OCH3
OH
O
3a-d
4a-d
c
d
Cl
F
NH
R
N
N
5a-d
Scheme 1. Reagents and conditions: (i) EtOH, NH2NH2$H2O 80%, reflux, 6 h; (ii) EtOH, RPhNCS, reflux, 4e8 h; (iii) dried toluene, maleic anhydride, reflux, 8e41 h; (iv) H2SO4,
50 ꢁC, 2 h.
tachyzoites showed complete morphological disorganization
before were completely eliminated. As a consequence the mean
number of tachyzoites decreased (Table 1). The acylth-
iosemicarbazides 3aed and 1,3,4-thiadiazoles 5aed were the most
active compounds on the infection at 0.1 mM concentration, while
the 4-thiazolidinones 4aed compounds had an efficient effect only
at 1 mM concentration. In spite of the compounds 3c and 5bed
had been less-toxic at 10 mM, all the other compounds showed
high toxicity at 10 mM concentration and cells were eliminated
from the culture (LD50).
The majority of the tested compounds were more effective than
hydroxyurea and sulfadiazine (standard drugs). In general, among
the tested derivatives, the acylthiosemicarbazides 3aed and 1,3,4-
thiadiazoles 5aed were the most active against T. gondii. The values
of LD50 (Table 2), which corresponded to the concentration that
leads to 50% lethal death, showed a low cytotoxicity in the unin-
fected cells for all compounds (ꢂ10 mM), except for 3a, 3b (1 mM),
and 4aec (5 mM). All compounds had a range of IC50 values
between 0.05e1 mM for infected cells and 0.05e1.5 mM for para-
sites, indicating a more effective action than those standard drugs
(sulfadiazine and hydroxyurea). In accordance with the obtained
results, the derivative 5a was the most selective compound against
T. gondii intracellular parasite, among the molecules with low
cytotoxicity. According to SAR exploration, in both the compound
series, the presence of electron-withdrawing or electron-donating
substituents on the phenyl moiety did not dramatically alter the
anti-T. gondii activity.
The synthesized derivatives were also tested for antimicrobial
activity by the disc diffusion method. In general, these results
indicated weak antimicrobial activities for all compounds.
However, some compounds showed significant mean zone inhibi-
tion (MZI), for bacterial strains: 3a (18 mm and 27 mm), 3b (19 mm
and 22 mm), 3c (25 mm and 29 mm), 3d (20 mm and 28 mm) and
4a (19 mm and 27 mm) against Staphylococcus aureus and Bacillus
subtilis, respectively; 3d (21 mm) against Escherichia coli; 3c
(21 mm) and 3d (20 mm) against Mycobacterium smegmatis; for
Table 1
Effect of 3aed, 4aed, and 5aed on cultures of Vero cells infected and intracellular multiplication of T. gondii.
Compound
% Infected Vero cellsa
Untreated (control)
Mean number of intracellular parasitesa
Treated (mM)
0.1
Untreated (control)
Treated (mM)
0.1
1
10
1
10
3a
3b
3c
3d
4a
4b
4c
4d
5a
5b
5c
5d
59 ꢃ 7
73 ꢃ 4
80 ꢃ 1
78 ꢃ 4
74 ꢃ 6
76 ꢃ 5
65 ꢃ 5
58 ꢃ 14
72 ꢃ 7
56 ꢃ 8
70 ꢃ 7
56 ꢃ 4
59 ꢃ 9
79 ꢃ 11
18 ꢃ 8
22 ꢃ 6
11 ꢃ 3
14 ꢃ 3
69 ꢃ 5
73 ꢃ 4
63 ꢃ 8
59 ꢃ 26
13 ꢃ 4
28 ꢃ 9
12 ꢃ 2
22 ꢃ 7
53 ꢃ 7
64 ꢃ 11
6 ꢃ 2
0
0
788 ꢃ 107
730 ꢃ 98
739 ꢃ 148
706 ꢃ 26
748 ꢃ 87
806 ꢃ 53
513 ꢃ 56
861 ꢃ 142
780 ꢃ 76
444 ꢃ 75
595 ꢃ 45
507 ꢃ 56
487 ꢃ 69
716 ꢃ 191
77 ꢃ 31
76 ꢃ 28
14 ꢃ 4
25 ꢃ 4
12 ꢃ 5
0
0
12 ꢃ 2
8 ꢃ 3
1 ꢃ 1.8
26 ꢃ 4
3.06 ꢃ 3.09
8 ꢃ 2
0
0
0
51 ꢃ 15
7.02 ꢃ 2
413 ꢃ 37
123 ꢃ 33
308 ꢃ 49
193 ꢃ 36
6 ꢃ 1.6
0
0
0
42 ꢃ 5
17 ꢃ 6
51 ꢃ 6
22 ꢃ 6
10 ꢃ 1
14 ꢃ 4
8 ꢃ 1
718 ꢃ 64
750 ꢃ 104
440 ꢃ 57
439 ꢃ 43
103 ꢃ 32
194 ꢃ 40
147 ꢃ 46
139 ꢃ 52
452 ꢃ 58
570 ꢃ 101
0.19 ꢃ 0.2
0.06 ꢃ 0.07
0
0
0.36 ꢃ 0.2
0
2 ꢃ 1.9
0
11 ꢃ 2.5
13 ꢃ 1.2
3 ꢃ 2.8
115 ꢃ 68
79 ꢃ 16
42 ꢃ 12
34 ꢃ 15
6 ꢃ 1.1
1 ꢃ 0.3
3 ꢃ 0.5
1 ꢃ 0.3
36 ꢃ 18
6 ꢃ 2
Hydroxyurea
Sulfadiazine
4 ꢃ 0.3
48 ꢃ 15
259 ꢃ 114
a
Values are mean ꢃ SD (n ¼ 3).

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A.P. Liesen et al. / European Journal of Medicinal Chemistry 45 (2010) 3685e3691
Table 2
3. Conclusion
LD50 values of 3aed, 4aed, and 5aed for uninfected cells, infected cells, and
intracellular parasites, respectively, in millimolar concentration.
The acylthiosemicarbazides 3aed, 4-thiazolidinones 4aed and
1,3,4-thiadiazoles 5aed, obtained from ethyl(5-methyl-1-H-imid-
azole-4-carboxylate), were synthesized and characterized based on
their physical, analytical and spectral data. The compounds of these
three series were evaluated in vitro against T. gondii intracellular.
Results showed a significant decrease in the percentage of infected
cells and in the mean number of tachyzoites per cell from the
concentrations of 0.1, 1 and 10 mM, when compared with
hydroxyurea and sulfadiazine (standard drugs). Acylth-
iosemicarbazides 3aed and 1,3,4-thiadiazoles 5aed showed the
best results. The majority presented high toxicity for both infected
cells and intracellular parasites, where the determined DL50 values
ranged from 0.05 to 1 mM. The derivative 5a was the most selective
against intracellular parasites and showed low toxicity. On the
other hand, the synthesized compounds were evaluated in vitro
against bacteria and fungal species, showing weak antimicrobial
activities. Finally, it can be concluded that thiosemicarbazides, 4-
thiazolidinones and 1,3,4-thiadiazoles, obtained from ethyl(5-
methyl-1-H-imidazole-4-carboxylate), provide interesting lead for
anti-T. gondii drugs discovery. Modifications to improve the potency
for these derivatives by structural diversification are currently
under progress in our laboratory.
Compound
LD50 (mM)a
Uninfected cells
Infected cells
Intracellular parasites
3a
3b
3c
3d
1
1
>10
10
0.05
0.07
0.06
0.06
5
0.05
0.06
0.06
0.05
1.5
4a
5
4b
5
1
0.5
4c
4d
>10
5
1
1
1.5
0.1
5a
5b
5c
5d
>10
>10
>10
>10
>10
>10
0.6
0.1
0.08
0.07
>10
>10
0.05
0.05
0.05
0.05
0.5
Hydroxyurea
Sulfadiazine
6
a
Values are mean ꢃ SD (n ¼ 3).
Table 3
Minimum inhibitory concentration (MIC) in
m
g mLꢀ1 of selected compounds.
Organism
Compound
MICa g mLꢀ1
(m )
4. Experimental
S. aureus
3a
3b
3c
3d
190
210
190
190
250
35
4.1. Chemistry protocols
4a
The melting points were determined on BÜCHI-535 apparatus
and are uncorrected. IR spectra were measured on BRUKER IFS-66
IR spectrophotometer. NMR spectra were recorded on UNITY PLUS-
300 MHz-VARIAN spectrometer by using tetramethylsilane as an
internal standard. HRMS were recorded on Varian MAT 711 spec-
trometer 70 eV electron impact, Kratos profile spectrometer 70 eV
Chloramp.
B. subtilis
3a
3b
3c
3d
140
160
130
130
270
30
4a
Chloramp.
electron impact. The chemical shifts are reported in
d units, and
E. coli
3d
Chloramp.
250
14
coupling constants (J) are reported in hertz (Hz). TLC development
was conducted on 0.25-mm silica gel plates (60F254, Merck). The
purification of 4-thiazolidinones 4aed was achieved by passage
through column chromatography on silica gel 60 (mesh 230e400,
E. Merck) with the indicated solvent system.
M. smegmatis
3c
3d
150
160
55
Rifampicin
C. albicans
4c
280
50
25
Nistatin
Ketoconazole
4.1.1. Representative procedure for (3aed)
4.1.1.1. 1-[(5-Methyl-1H-imidazole-4-yl)carbonyl]-4-phenyl-thio-
semicarbazide (3a). To a solution of hydrazide 2 (1 g, 0.0071 mol) in
30 mL of EtOH, was added, dropwise a 0.71 mL (0.007 mol) of
phenylisothiocyanate. The mixture was stirred under reflux for 6 h
and cooled to room temperature. The solvent was removed under
reduced pressure to get a crude product that was purified by
recrystallization from EtOH. White fine powder; yield 80%; mp
160e162 ꢁC; IR (KBr): nmax 3178 cmꢀ1 (NeH), 1657 cmꢀ1 (C]O),
1583 and 1536 cmꢀ1 (NC]O), 1200 and 656 cmꢀ1 (C]S); 1H NMR
Candida sp. (UFPEDA 2224)
4c
300
50
40
Nistatin
Ketoconazole
A. niger
4d
Nistatin
350
50
a
MIC, minimum inhibitory concentration (the lowest concentration that inhibi-
ted the bacterial growth).
(300 MHz, DMSO-d6):
d 12.41 (br s, 1H, NH imidazole), 9.84 (s, 1H,
fungal strains: 4c (20 and 20 mm) against Candida albicans and
Candida sp. (UFPEDA 2224); 4d (18 mm) against Aspergillus niger.
The values of minimum inhibitory concentration (MIC) of the
compounds with MZI above or equal to 18 mm are reported in Table
3. The results showed that the tested molecules possess weak
antibacterial and antifungal activities, when compared with the
standard drugs (chloramphenicol and rifampicin for antibacterial
activity; and nistatin and ketoconazole for antifungal activity). The
NNHCS), 9.59 (br s, 2H, CONHN and CSNHPh), 7.62 (s, 1H, CH
imidazole), 7.49 (d, 2H, J ¼ 9 Hz, Ar-H), 7.30 (t, 2H, J ¼ 9 Hz, Ar-H),
7.12 (t, 2H, J ¼ 9 Hz, Ar-H), 2.45 (s, 3H, CH3); 13C NMR (75.4 MHz,
DMSO-d6): dC 180.93 (C]S), 162.84 (C]O), 139.32 (Cq Ar), 133.70
(C2 imidazole), 132.10 (C4 imidazole), 128.56 (C5 imidazole), 128.01
(CH Ar), 124.00 (CH Ar), 124.62 (CH Ar), 10.54 (CH3); HRMS (EIþ):
calcd. (C12H13N5OS) 275.0840; found: 275.0924.
best results for antibacterial activity were 3c (MIC ¼ 130
and 3d (MIC ¼ 130
g mLꢀ1) against B. subtilis. For antifungal
properties, the low activities of the tested molecules ranged from
280 g/mL to 350 g/mL.
m )
g mLꢀ1
4.1.1.2. 1-[(5-Methyl-1H-imidazole-4-yl)carbonyl]-4-(4-methox-
yphenyl])-thiosemicarbazide (3b). White fine powder; yield 97%;
mp 161e162 ꢁC; IR (KBr): nmax 3339 cmꢀ1 (NeH), 1668 cmꢀ1 (C]
O), 1594 and 1555 cmꢀ1 (NC]O), 1244 and 643 cmꢀ1 (C]S); 1H
m
m
m

A.P. Liesen et al. / European Journal of Medicinal Chemistry 45 (2010) 3685e3691
3689
NMR (300 MHz, DMSO-d6):
d
12.39 (br s, 1H, NH imidazole), 9.77 (s,
(KBr): nmax 1731 (C]O), 1643 (NC]O), 1505 (C]N), 1397 (NCS),
1295 (NeN]C); 1H NMR (300 MHz, DMSO-d6):
12.54 (br s, 1H,
NH imidazole), 10.49 (br s, 1H, CO2H), 10.28 (br s, 1H, CONHN), 7.68
(s, 1H, H-imidazole), 6.92 (d, 2H, J ¼ 6 Hz, Ar-H), 6.82 (d, 2H,
J ¼ 6 Hz, Ar-H), 4.60 (dd, 1H, J ¼ 3 and 12 Hz, CH), 3.73 (s, 3H, OCH3),
3.18 (m, 1H, CH2a), 2.73 (m, 1H, CH2b), 2.44 (s, 3H, CH3); 13C NMR
1H, NNHCS), 9.48 (br s, 2H, CONHN and CSNHPh), 7.61 (s, 1H, CH
imidazole), 7.31 (d, 2H, J ¼ 9 Hz, Ar-H), 6.86 (d, 2H, J ¼ 9 Hz, Ar-H),
3.73 (s, 3H, OCH3), 2.45 (s, 3H, CH3); 13C NMR (75.4 MHz, DMSO-
d
d6): dC 181.09 (C]S), 162.57 (C]O), 156.51 (Cq Ar), 136.90 (Cq Ar),
133.66 (C2 imidazole), 132.14 (C4 imidazole), 128.41 (C5 imidazole),
126.79 (CH Ar), 123.20 (CH Ar), 55.20 (OCH3), 10.61 (CH3); HRMS
(EIþ): calcd. (C13H15N5O2S) 305.0946; found: 305.0937.
(75.4 MHz, DMSO-d6): dC 171.47 (CO2H), 170.55 (CO 4-thiazolidi-
none), 159.38 (CH Ar), 151.69 (CONH), 149.24 (C]N), 138.71 (Cq Ar),
134.14 (C4 imidazole), 133.74 (C2 imidazole), 128.66 (CH Ar), 128.32
(C5 imidazole), 119.97 (CH Ar), 55.25 (OCH3), 42.24 (CH2), 34.03
(CH), 10.42 (CH3); HRMS (EIþ): calcd. (C17H17N5O5S) 403.0950;
found: 403.0875.
4.1.1.3. 1-[(5-Methyl-1H-imidazole-4-yl)carbonyl]-4-(4-chlorophenyl)-
thiosemicarbazide (3c). White fine powder; yield 73%; mp
217e218 ꢁC; IR (KBr): nmax 3355 cmꢀ1 (NeH), 1670 cmꢀ1 (C]O),
1593 and 1547 cmꢀ1 (NC]O), 1263 and 640 cmꢀ1 (C]S); 1H NMR
(300 MHz, DMSO-d6):
d
12.42 (br s, 1H, NH imidazole), 9.84 (s, 1H,
4.1.2.3. 3-(4-Chlorophenyl)-2-{[(5-methyl-1H-imidazole-4-yl)
NNHCS), 9.69 (br s, 2H, CONHN and CSNHPh), 7.63 (s, 1H, CH
carbonyl]hydrazono}-4-oxo-1,3-thiazolidin-5-yl-acetic acid (4c).
imidazole), 7.54 (d, 2H, J ¼ 9 Hz, Ar-H), 7.36 (d, 2H, J ¼ 9 Hz, Ar-H),
Yellow oil; yield 10%; 1H NMR (300 MHz, DMSO-d6):
d 12.51 (br s,
2.46 (s, 3H, CH3); 13C NMR (75.4 MHz, DMSO-d6):
dC 181.01 (C]S),
1H, NH imidazole), 10.52 (br s, 1H, CO2H), 10.32 (br s, 1H, CONHN),
7.66 (s, 1H, H-imidazole), 7.39 (d, 2H, J ¼ 9 Hz, Ar-H), 6.88 (d, 2H,
J ¼ 9 Hz, Ar-H), 4.64 (dd, 1H, J ¼ 3 and 9 Hz, CH), 3.17 (m, 1H, CH2a),
2.73 (m, 1H, CH2b), 2.44 (s, 3H, CH3); 13C NMR (75.4 MHz, DMSO-
163.13 (C]O), 138.38 (Cq Ar), 133.71 (C2 imidazole), 132.21 (C4
imidazole), 128.56 (C5 imidazole), 127.82 (CH Ar), 127.01 (CH Ar),
124.44 (CH Ar), 10.57 (CH3); HRMS (EIþ): calcd. (C13H15N5O2S)
309.0451; found: 309.0398.
d6): dC 171.53 (CO2H), 170.69 (CO 4-thiazolidinone), 152.94 (CONH),
146.62 (C]N), 161.00 (Cq Ar), 142.27 (Cq Ar), 134.09 (C4 imidazole),
133.25 (C2 imidazole), 129.33 (C5 imidazole), 128.41 (CH Ar), 127.63
(CH Ar), 40.96 (CH2), 35.60 (CH), 10.48 (CH3); HRMS (EIþ): calcd.
(C16H14ClN5O4S) 407.0455; found: 407.0590.
4.1.1.4. 1-[(5-Methyl-1H-imidazole-4-yl)carbonyl]-4-(4-fluorophenyl)-
thiosemicarbazide (3d). White fine powder; yield 54%; mp
187e188 ꢁC; IR (KBr): nmax 3184 cmꢀ1 (NeH), 1663 cmꢀ1 (C]O),
1590 and 1541 cmꢀ1 (NC]O), 1200 and 656 cmꢀ1 (C]S); 1H NMR
(300 MHz, DMSO-d6):
NNHCS), 9.62 (br s, 2H, CONHN and CSNHPh), 7.62 (s, 1H, CH
d
12.44 (br s, 1H, NH imidazole), 9.78 (s, 1H,
4.1.2.4. 3-(4-Fluorophenyl)-2-{[(5-methyl-1H-imidazole-4-yl)carbonyl]
hydrazono}-4-oxo-1,3-thiazolidin-5-yl-acetic acid (4d). White fine
powder; yield 16%; mp 200e205 ꢁC (decomposed); IR (KBr): nmax
1742 (C]O), 1646 (NC]O), 1502 (C]N), 1388 (NCS), 1283 (NeN]
imidazole), 7.46 (t, 2H, J ¼ 6 Hz, Ar-H), 7.14 (t, 2H, J ¼ 6 Hz, Ar-H),
2.45 (s, 3H, CH3); 13C NMR (75.4 MHz, DMSO-d6):
dC 181.13 (C]S),
162.75 (C]O), 159.21 (d, J ¼ 240.5 Hz, Cq Ar), 135.67 (Cq Ar), 133.69
(C2 imidazole), 132.32 (C4 imidazole), 128.37 (C5 imidazole), 127.41
(CH Ar), 114.41 (d, J ¼ 23.3 Hz, CH Ar), 10.60 (CH3); HRMS (EIþ):
calcd. (C13H15N5O2S) 293.0746; found: 293.0686.
C); 1H NMR (300 MHz, DMSO-d6):
d 12.54 (br s, 1H, NH imidazole),
10.55 (br s, 1H, CO2H), 10.34 (br s, 1H, CONHN), 7.68 (s, 1H, H-
imidazole), 7.18 (t, 2H, J ¼ 9 Hz, Ar-H), 6.89 (dd, 2H, J ¼ 3 and 9 Hz,
Ar-H), 4.65 (dd, 1H, J ¼ 3 and 12 Hz, CH), 3.18 (m, 1H, CH2a), 2.75 (m,
1H, CH2b), 2.45 (s, 3H, CH3); 13C NMR (75.4 MHz, DMSO-d6):
dC
4.1.2. Representative procedure for (4aed)
171.80 (CO2H), 170.71 (CO 4-thiazolidinone), 161.38 (CONH), 159.10
(d, J ¼ 240 Hz, Cq Ar), 152.96 (C]N), 144.16 (Cq Ar), 133.90 (C2
imidazole), 133.31 (C4 imidazole), 127.66 (C5 imidazole), 122.46 (d,
J ¼ 8.2 Hz, CH Ar), 116.06 (d, J ¼ 22.3 Hz, CH Ar), 45.36 (CH2), 34.20
(CH), 10.53 (CH3); HRMS (EIþ): calcd. (C16H14FN5O4S) 391.0750;
found: 391.0464.
4.1.2.1. 3-Phenyl-2-[(5-methyl-1H-imidazole-4-yl)carbonyl]hydra-
zono-4-oxo-1,3-thiazolidin-5-yl-acetic acid (4a). A mixture of 1 g
(0.0036 mol) of 1-[(5-methyl-1H-imidazole-4-yl)carbonyl]-4-
phenyl-thiosemicarbazide 3a and 1.058 g (0.0108 mol) of maleic
anhydride in 50 mL of dried toluene was stirred under reflux. The
mixture was stirred in the same conditions till the completion of
the reaction (8 h). The solvent was evaporated at reduced pressure,
and the crude product was extracted with ethyl acetate. The
organic layer was treated with anhydrous sodium sulfate and
evaporated again. Finally the pure product was obtained by column
chromatography on silica gel using hexaneeethyl acetate and
methanoleethyl acetate as an eluent. White fine powder; yield
12%; mp 205e210 ꢁC (decomposed); IR (KBr): nmax 3504 cmꢀ1
(OeH), 3347 cmꢀ1 (NeH), 1709 (C]O), 1643 (NC]O), 1585 (C]N),
4.1.3. Representative procedure for (5aed)
4.1.3.1. N-Phenyl-5-(5-methyl-1H-imidazole-4-yl)-1,3,4-thiadiazole-
2-amine (5a). White fine powder; yield 93%; mp 249e250 ꢁC;
a mixture of 0.28 g (0.001 mol) of 2-[(5-methyl-1H-imidazole-4-yl)
carbonyl]-4-phenyl-thiosemicarbazide 3a and 0.5 mL of conc.
H2SO4 was heated at 50 ꢁC for 2 h, according to procedure stated in
literature [26]. The resulting solution was cooled, poured into
crushed ice, and treated with sodium carbonate to pH 6. The
precipitate was collected by filtration under vacuum and washed
with water. IR (KBr): nmax 1657 cmꢀ1 (C]N), 1566 (C]C), 1100 (N]
1378 (NCS), 1278 (NeN]C); 1H NMR (300 MHz, DMSO-d6):
d 12.48
(br s, 1H, NH imidazole), 10.43 (br s, 1H, CO2H), 10.27 (br s, 1H,
CONHN), 7.65 (s,1H, H-imidazole), 7.35 (t, 2H, J ¼ 9 Hz, Ar-H), 7.11 (t,
1H, J ¼ 9 Hz, Ar-H), 6.83 (t, 2H, J ¼ 9 Hz, Ar-H), 4.59 (dd,1H, J ¼ 3 and
12 Hz, CH), 3.16 (m, 1H, CH2a), 2.73 (m, 1H, CH2b), 2.45 (s, 3H, CH3);
CeSeC]N); 1H NMR (300 MHz, DMSO-d6):
d 10.65 (br s, 2H, NHPh
and NH-imidazole), 7.66 (d, 2H, J ¼ 6 Hz, H-Ar), 7.65 (s, 1H, H-
imidazole), 7.33 (t, 2H, J ¼ 6 Hz, H-Ar), 6.97 (t, 1H, J ¼ 6 Hz, H-Ar),
13C NMR (75.4 MHz, DMSO-d6):
d
C 171.38 (CO2H), 170.38 (CO 4-
2.53 (s, 3H, CH3); 13C NMR (75.4 MHz, DMSO-d6):
dC 162.27 (Cq
thiazolidinone), 151.72 (CONH), 147.65 (C]N), 135.1 (Cq Ar), 133.90
(C4 imidazole), 133.40 (C2 imidazole), 129.22 (C5 imidazole), 124.21
(CH Ar), 120.63 (CH Ar), 120.36 (CH Ar), 42.50 (CH2), 36.70 (CH),
10.36 (CH3); HRMS (EIþ): calcd. (C16H15N5O4S) 373.0844; found:
373.0712.
thiadiazole), 155.80 (Cq thiadiazole), 140.95 (Cq Ar), 134.98 (C2
imidazole), 129.05 (CH Ar), 127.68 (C4 imidazole), 126.24 (C5 imid-
azole), 121.49 (CH Ar), 117.19 (CH Ar), 10.70 (CH3); HRMS (EIþ):
calcd. (C12H11N5S) 257.0735; found: 257.0924.
4.1.3.2. N-(4-Methoxyphenyl)-5-(5-methyl-1H-imidazole-4-yl)-1,3,
4-thiadiazole-2-amine (5b). White fine powder; yield 84%; mp
279e280 ꢁC; IR (KBr): nmax 1623 cmꢀ1 (C]N), 1546 cmꢀ1 (C]C),
4.1.2.2. 3-(4-Methoxyphenyl)-2-{[(5-methyl-1H-imidazole-4-yl)
carbonyl]hydrazono}-4-oxo-1,3-thiazolidin-5-yl-acetic acid (4b).
White fine powder; yield 15%; mp 205e210 ꢁC (decomposed); IR
1082 cmꢀ1 (N]CeSeC]N); 1H NMR (300 MHz, DMSO-d6):
d 10.47

3690
A.P. Liesen et al. / European Journal of Medicinal Chemistry 45 (2010) 3685e3691
(br s, 2H, NHPh and NH-imidazole), 7.92 (s, 1H, H-imidazole), 7.55
(d, 2H, J ¼ 9 Hz, H-Ar), 6.95 (d, 2H, J ¼ 9 Hz, H-Ar), 3.73 (s, 3H,
de Pernambuco, Brazil. Namely, S. aureus (UFPEDA 02), B. subtilis
(UFPEDA 16), Micrococcus luteus (UFPEDA 100), E. coli (UFPEDA
224), Klebisiella pneumoniae (UFPEDA 396), Pseudomonas aerugi-
nosa (UFPEDA 416), M. smegmatis (UFPEDA 71), Mycobacterium
tuberculosis (UFPEDA 82), Streptococcus faecalis (UFPEDA 138),
Saccharomyces cerevisiae (UFPEDA 1012), Candida sp. (URM 720),
Candida sp. (UFPEDA 2224), Candida krusei (UFPEDA 1002), C.
albicans (UFPEDA 1007), Malassezia furfur (URM 4849), A. niger
(UFPEDA 2003), Fusarium moniliforme (UFPEDA 2409), and Fusa-
rium oxysporum (UFPEDA 2414). The antibacterial and antifungal
activities were reported preliminary utilizing disc diffusion method
[35]. In this method, discs containing known amounts of an anti-
microbial agent were placed on the surface of an agar plate that has
been inoculated with a standardized suspension of microorganisms
to be tested. Paper discs with only DMSO or CH2Cl2 (tests involving
ketoconazole) were used as negative controls. The MZI for chlor-
amphenicol and rifampicin (antibacterial) and nistatin and keto-
conazole (antifungal) was used as reference values (millimeter). All
experiments were conducted in triplicate and repeated if the
results differed. All compounds used having MZI larger or equal to
18 mm were selected to MIC.
OCH3), 2.50 (s, 3H, CH3); 13C NMR (75.4 MHz, DMSO-d6):
dC 162.11
(Cq thiadiazole), 158.22 (Cq Ar), 151.95 (Cq thiadiazole), 137.40 (Cq
Ar), 134.92 (C2 imidazole), 127.66 (C4 imidazole), 124.47 (CH Ar),
126.35 (C5 imidazole), 123.45 (CH Ar), 49.03 (OCH3), 10.66 (CH3);
HRMS (EIþ): calcd. (C13H13N5OS) 287.0840; found: 287.0925.
4.1.3.3. N-(4-Chlorophenyl)-5-(5-methyl-1H-imidazole-4-yl)-1,3,4-
thiadiazole-2-amine (5c). White fine powder; yield 71%; mp
256e257 ꢁC; IR (KBr): nmax 1618 cmꢀ1 (C]N), 1560 cmꢀ1 (C]C),
1095 cmꢀ1 (N]CeSeC]N); 1H NMR (300 MHz, DMSO-d6):
d 10.42
(br s, 2H, NHPh and NH-imidazole), 7.69 (s, 1H, H-imidazole), 7.64
(d, 2H, J ¼ 9 Hz, H-Ar), 7.35 (d, 2H, J ¼ 9 Hz, H-Ar), 2.50 (s, 3H, CH3);
13C NMR (75.4 MHz, DMSO-d6):
dC 161.90 (Cq thiadiazole), 156.32
(Cq thiadiazole), 139.79 (Cq Ar), 134.97 (C2 imidazole), 128.87 (CH
Ar), 127.78 (C4 imidazole), 126.09 (C5 imidazole), 124.87 (Cq Ar),
118.71 (CH Ar), 10.61 (CH3); HRMS (EIþ): calcd. (C12H10ClN5S)
291.0345; found: 291.0668.
4.1.3.4. N-(4-Fluorophenyl)-5-(5-methyl-1H-imidazole-4-yl)-
1,3,4-thiadiazole-2-amine (5d). White fine powder; yield 94%; mp
259e261 ꢁC; IR (KBr): nmax 1632 cmꢀ1 (C]N), 1572 cmꢀ1 (C]C),
For MIC assays [36,37], a stock solution (1 mg mLꢀ1) of each test
compound was prepared in dimethylsulfoxide solvent. Further, the
serial dilution of test compounds was carried out and the concen-
1104 cmꢀ1 (N]CeSeC]N); 1H NMR (300 MHz, DMSO-d6):
d 10.54
(br s, 2H, NHPh and NH-imidazole), 8.49 (s, 1H, H-imidazole), 7.68
(dd, 2H, J ¼ 6 and 9 Hz, H-Ar), 7.20 (t, 2H, J ¼ 9 Hz, H-Ar), 2.51 (s, 3H,
trations used ranged from 50 to 400 m
g mLꢀ1. Test compounds at
various concentrations were added to culture medium in a test tube
and different strains were inoculated at 108 bacteria mLꢀ1
concentration. Tryptic soy agar and nutrient agar (for antibacterial)
and Sabouraud liquid medium (for antifungal) were utilized as
culture medium. The tubes were incubated at 37 ꢁC (antibacterial)
or 30 ꢁC (antifungal) for 24e48 h and then examined for the
presence or absence of growth organisms tested. Chloramphenicol,
rifampicin, nistatin, and ketoconazole were used as antibacterial
and antifungal substances. The MIC values were obtained from the
lowest concentration of the test compounds where the tubes
remained clear, indicating that the bacterial or fungal growth was
completely inhibited at this concentration. MIC values were
CH3); 13C NMR (75.4 MHz, DMSO-d6):
dC 163.68 (Cq thiadiazole),
157.32 (d, J ¼ 238.1 Hz, Cq Ar), 150.03 (Cq thiadiazole), 137.06 (Cq
Ar), 134.90 (C2 imidazole), 127.62 (C4 imidazole), 123.48 (C5 imid-
azole), 119.17 (d, J ¼ 7.5 Hz, CH Ar), 115.69 (d, J ¼ 22.3 Hz, CH Ar),
10.52 (CH3); HRMS (EIþ): calcd. (C12H10FN5S) 275.0640; found:
275.0643.
4.2. Biological assays
4.2.1. Assay for anti-T. gondii activity
Tachyzoites from the virulent RH strain of T. gondii were
obtained by intraperitoneal passages in Swiss mice and collected in
Ringer’s solution at pH 7.2, 48 h after infection. Vero cells (kidney
fibroblasts from African green monkeys) were incubated (37ꢁ) with
T. gondii tachyzoites (parasite/host cell 10:1 relationship) for 1 h,
washed twice with phosphate-buffered saline solution (PBS) to
remove extracellular parasites, and incubated for 24 h at 37 ꢁC in
the presence of medium 199 supplemented with 5% fetal calf serum
(FCS). Cells infected with T. gondii were incubated with test
compounds in the concentrations of 0.1,1, and 10 mM for 24 h (37ꢁ).
Hydroxyurea and sulfadiazine were utilized as reference
substances. All compounds were added to the infected cells during
intense parasite proliferation. The infected cultures were washed
thrice with PBS, fixed with Bouin’s fixative, stained with Giemsa,
and observed under a light microscope (63ꢄ objective Axioplan,
Zeiss, Jena, Germany). The percentage of infected cells and the
mean number of intracellular parasites were determined by the
examination of at least 400 cells [33,34]. Statistical analysis was
carried out using the Student’s t-test. P values <0.05 were consid-
ered as significant. Data shown are representative of 13 assays in
triplicate. Finally, the LD50 values for infected cells and intracellular
parasites for all compounds were obtained after 24-h exposure in
the concentrations ranged of 0.01e10 mM, in triplicate per assay, by
a non-linear regression using exclusion test with trypan blue [20].
expressed in
m .
g mLꢀ1
Acknowledgements
The authors acknowledge Coordenação de Aperfeiçoamento de
Pessoal de Nível Superior (CAPES), Conselho Nacional de Desen-
volvimento Científico e Tecnológico (CNPq) and Fundação de
Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ).
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