Structural design, synthesis and pharmacological evaluation of 4-thiazolidinones against Trypanosoma cruzi

Article abstract of DOI:10.1016/j.bmc.2015.10.048

Chagas disease is an infection caused by protozoan Trypanosoma cruzi, which affects approximately 8-10 million people worldwide. Benznidazole is the only drug approved for treatment during the acute and asymptomatic chronic phases of Chagas disease; howev

Full text of DOI:10.1016/j.bmc.2015.10.048

Bioorganic & Medicinal Chemistry 23 (2015) 7478–7486
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Structural design, synthesis and pharmacological evaluation
of 4-thiazolidinones against Trypanosoma cruzi
a
Gevanio Bezerra de Oliveira Filho a, , Marcos Veríssimo de Oliveira Cardoso ,
⇑
José Wanderlan Pontes Espíndola a, Luiz Felipe Gomes Rebello Ferreira a, Carlos Alberto de Simone b,
Rafaela Salgado Ferreira c, Pollyanne Lacerda Coelho c, Cássio Santana Meira d,
Diogo Rodrigo Magalhaes Moreira d, Milena Botelho Pereira Soares d,e, Ana Cristina Lima Leite a
a Departamento de Ciências Farmacêuticas, Centro de Ciências da Saúde, Universidade Federal de Pernambuco, 50740-520 Recife, PE, Brazil
b Departamento de Física e Informática, Instituto de Física, Universidade de São Paulo, CEP 13560-970, São Carlos, SP, Brazil
c Departamento de Bioquímica e Imunologia, Universidade Federal de Minas Gerais, CEP 31270-901, Belo Horizonte, MG, Brazil
d Centro de Pesquisas Gonçalo Moniz, Fundação Oswaldo Cruz, CEP 40296-710, Salvador, BA, Brazil
e Centro de Biotecnologia e Terapia Celular, Hospital São Rafael, Salvador, BA, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
Chagas disease is an infection caused by protozoan Trypanosoma cruzi, which affects approximately 8–
10 million people worldwide. Benznidazole is the only drug approved for treatment during the acute
and asymptomatic chronic phases of Chagas disease; however, it has poor efficacy during the symp-
tomatic chronic phase. Therefore, the development of new pharmaceuticals is needed. Here, we
employed the bioisosterism to modify a potent antiparasitic and cruzain-inhibitor aryl thiosemicar-
bazone (4) into 4-thiazolidinones (7–21). Compounds (7–21) were prepared by using a straightforward
synthesis and enabled good to excellent yields. As a chemical elucidation tool, X-ray diffraction of com-
pound (10) revealed the geometry and conformation of this class compounds. The screening against cru-
zain showed that 4-thiazolidinones were less active than thiosemicarbazone (4). However, the
antiparasitic activity in Y strain trypomastigotes and host cell cytotoxicity in J774 macrophages revealed
that compounds (10 and 18–21) are stronger and more selective antiparasitic agents than thiosemicar-
bazone (4). Specifically, compounds (18–20), which carry a phenyl at position N3 of heterocyclic ring,
were the most active ones, suggesting that this is a structural determinant for activity. In infected macro-
phages, compounds (18–20) reduced intracellular amastigotes, whereas Benznidazole did not. In T. cruzi-
infected mice treated orally with 100 mg/kg of compound (20), a decreased of parasitemia was observed.
In conclusion, we demonstrated that the conversation of thiosemicarbazones into 4-thiazolidinones
retains pharmacological property while enhances selectivity.
Received 6 August 2015
Revised 20 October 2015
Accepted 31 October 2015
Available online 2 November 2015
Keywords:
Chagas disease
Trypanosoma cruzi
Thiosemicarbazones
Thiazolidinones
Bioisosterism
Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction
patients experience drug intolerance and adverse effects.6 Thus,
new drugs to treat Chagas disease are necessary.
It is estimated that 5–10% of the Latin American population has
Chagas disease, which is caused by the protozoan Trypanosoma
cruzi.1 This situation is alarming, because there are no vaccines
available and the current treatment using Benznidazole is of low
efficacy and followed by several side effects.2,3 Benznidazole has
antiparasitic effects against bloodstream parasites, but its activity
is limited against parasites in tissues.4,5 Additionally, many
To address this need, a number of molecular targets for anti-T.
cruzi drugs have been investigated, increasing the quality of drug
identification for Chagas disease treatment. Examples of such tar-
gets are the lanosterol 14
a
-demethylase,7 trypanothione reduc-
tase,8 cruzain,9 trans-sialidase,10 phosphatidylinositol 3-kinase11
and cytochrome b.12 By using structure-based drug design, two
small-molecules were developed and are considered strong drug
candidates: K11777, a vinyl sulfone peptide that inhibits cruzain,13
and VNI, an oxadiazole derivative inhibitor of 14-a-demethylase
activity.14,15
⇑
Corresponding author. Tel.: +55 81 21268511; fax: +55 81 21268510.
More recently, compound VFV reveals a broader antiprotozoal
spectrum of action. It has stronger antiparasitic activity in cellular
E-mail
addresses:
gev_filho@hotmail.com,
acllb2003@yahoo.com.br
(G.B. de Oliveira Filho).
http://dx.doi.org/10.1016/j.bmc.2015.10.048
0968-0896/Ó 2015 Elsevier Ltd. All rights reserved.

G. B. de Oliveira Filho et al. / Bioorg. Med. Chem. 23 (2015) 7478–7486
7479
(A)
Thiazolidinones described over the years
O
N
S
S
N
O
S
N
N
N
N
H
Thiazolidinone (1)
2
Thiazolidinone ( )
O
=
IC50 0.4 µM (cruzain)
=
IC50 6.6 µM (cruzain)
=
IC50 10 µM (T. cruzi)
IC50 = 6.1 µM (T. cruzi)
Proposed Thiazolidinones
(B)
H
bioisosterism
N
S
N
S
N
N
R1
NH2
N
Cl
Cl
R
O
Cl
Cl
4
Thiosemicarbazone ( )
Thiazolidinones (7-21)
=
IC50 50 nM (cruzain)
Compd.
R
H
H
H
H
H
R1
H
Compd.
12
R
R1
H
Compd.
R
C6H5
C6H5
R1
H
7
8
17
18
19
CH3
CH3
CH3
CH3
CH3
13
CH3
CH3
CH3
9
10
11
CH2CH3
14
CH2CH3
CH(CH3)2 20
C6H5 21
C6H5 CH2CH3
CH(CH3)2 15
C6H5 CH(CH3)2
C6H5
16
C6H5
C6H5
Figure 1. Bioisosterism-based design of novel thiazolidinones as potential anti-parasitic compounds. (A) Thiazolidinones described over the years (compounds 135 and 238
were previously investigated), (B) thiazolidinones proposed here.
experiments, and cures the experimental Chagas disease with 100%
of efficacy, and suppresses visceral leishmaniasis by 89% (vs 60%
for VNI).16 These compounds are likely to enter into clinical trials;
however, the chances of any drug being approved in the clinical
stage are very low. Given the outcomes observed for K11777,
VNI and VFV as anti-T. cruzi agents, the design of compounds to
the presence of catalytic H2SO4 or HCl (thiosemicarbazone 6). Thi-
azolidinones (7–21) were prepared by reacting the respective aryl
thiosemicarbazone with commercially available ethyl 2-bromoac-
etate or by preparing the desired 2-substituted-2-bromoacetates.
These reactions were carried out in the presence of an excess of
anhydrous NaOAc under reflux. This afforded compounds (7–21)
in variable yields (38–84%) and acceptable purity (>95%).
Structures of compounds were determined by nuclear magnetic
resonance (NMR, 1H and 13C), infrared (IR) and mass (HR-MS) spec-
tral, while purity was determined by elemental analysis (E.A.). To
define the relative configuration of the iminic bond, we executed
X-ray diffraction of single crystals of compound (10). As observed
in Figure 2, this compound adopts an E-geometry. In addition, it
was observed that iminic bond in C1 is located in an exocyclic posi-
tion in regard to the heterocyclic ring.
inhibit cruzain and the 14-
a-demethylase activity have received
special attention.17,18
Additionally, with the goal of avoiding the development of
resistant T. cruzi strains, an enhanced of the number of potential
drug candidates by combined drug therapy is necessary.19,20
Thiosemicarbazones have been largely investigated as anti-T.
cruzi agents.21–27 Among this class, the 3,4-dichlorophenyl
thiosemicarbazone (4) was identified as one of the most potent
cruzain inhibitor. The structure–activity relationships studies iden-
tified that the 3,4-dichlorophenyl is an important structural deter-
minant for trypanocidal activity.28
2.2. Pharmacological evaluation
Due to the poor drugability of thiosemicarbazones, many
attempts have been made to identify thiosemicarbazone-like com-
pounds with improved antiparasitic effects.29–34 Following this line
of research, our research group demonstrated an existing bioisos-
teric relationship between thiosemicarbazones and thiazolidi-
nones, which led to the identification of strong antiparasitic
thiazolidinones.35–38
Based on these findings, here new thiazolidinones (7–21) were
planned by employing the thiosemicarbazone (4) as a structural
prototype (Fig. 1). We synthesized compounds (7–21) and evalu-
ated their activity against cruzain and T. cruzi. The choice of sub-
stituents attached in compounds (7–21) was oriented by
observing previously described structure–activity relationships.38
We assessed the host cell cytotoxicity in J774 macrophages,
while the in vitro anti-T. cruzi activity was determined against
bloodstream trypomastigotes of Y strain (Table 1). Compounds that
showed IC50 values comparable to Benznidazole were considered
active.
The inhibitory activity for thiazolidinones (5–19) against cru-
zain was also investigated. We measured cruzain mediated enzy-
matic activity inhibition by using an assay based on competition
with the substrate Z-Phe-Arg-aminomethylcoumarin (Z-FR-AMC).
Compounds were screened at 100 lM, the maximum concentra-
tion at which they were soluble in the assay buffer. However, cru-
zain inhibition by these compounds was not observed (Table S1).39
The structure–activity relationship study began with two mod-
ifications on 4-thiazolidinone ring: (a) substituents at C5 position
and (b) attachment of a methyl or phenyl group at nitrogen N3.
These modifications yielded compounds (7–21). Among the tested
compounds, compound (20) was the most potent antiparasitic,
2. Results
2.1. Synthesis and chemical characterization
with an IC50 value of 1.7 0.17 lM for bloodstream trypomastig-
otes. This compound presented better antiparasitic properties than
Benznidazole. Importantly, this did not affect host cells viability in
concentrations up to 100 lM.
The general route to preparing thiazolidinones is shown in
Scheme 1. Firstly, thiosemicarbazones (4–6) were prepared by
reacting commercially available 30,40-dichloroacetophenone (3)
with the appropriate thiosemicarbazide in an ultrasound bath in

7480
G. B. de Oliveira Filho et al. / Bioorg. Med. Chem. 23 (2015) 7478–7486
O
H
N
H
N
N
S
N
R
N
R1
A
B
N
S
4-6
Cl
Cl
Cl
R
O
7-21
Cl
3',4'-dichloroacetophenone ( )
Cl
Cl
3
Tiosemicarbazones 4-6
Thiazolidinones 7-21
Compd.
R
Compd.
R
H
H
H
H
H
R1
H
Compd.
12
R
R1
H
Compd.
17
R
R1
4
5
6
H
7
8
CH3
CH3
CH3
CH3
CH3
C6H5
H
CH3
C6H5
CH3
13
CH3
18
C6H5
CH3
9
10
11
14
19
CH2CH3
CH2CH3
C6H5 CH2CH3
(
15
(
20
(
CH CH3)2
CH CH3)2
C6H5 CH CH3)2
16
21
C6H5
C6H5
C6H5 C6H5
Scheme 1. Synthetic procedures for thiazolidinones (7–21). Reagents and conditions: (A) thiosemicarbazides, EtOH, H2SO4, or HCl, ultrasound bath, rt, 1–2 h, yields of 80–
89%; (B) ethyl 2-bromoacetate or ethyl 2-substituted-2-bromoacetates; NaOAc, EtOH, reflux, overnight, yields of 38–84%.
H
Cl
Cl
N
N
N
O
S
Cpd. 10
Figure 2. (Left) chemical structure of compound (10). (Right) ORTEP-3 projection of compound (10) showing atom-numbering and displacement ellipsoids at the 50%
probability level.
Comparing thiosemicarbazones (4–6), it is observed that with
the increase of lipophilicity in substituents at N3, with methyl
(5) or phenyl (6), the trypanocidal activity decrease with an
improvement in toxicity for macrophages. This trend is not
observed for cyclic derivatives (7–21), otherwise an improvement
in toxicity profile is observed with the cyclization of the thioamide
into thiazolidinone moiety.
The good trypanocidal activity observed in unsubstituted
thiosemicarbazone (4) was not kept in their cyclic derivatives (7,
12 and 17), however, their present lower cytotoxicity for macro-
phages, in opposition to (4). Among cyclic derivatives, only com-
pounds (18), (19) and (20) present better trypanocidal activity
than thiosemicarbazone (4).
In general, substitutions at N3 improved the antiparasitic activ-
ity, with N-phenyl compounds (17–21) exhibiting the strongest
activity among all tested compounds. For instance, the methyl
(18), ethyl (19), and isopropyl (20) compounds were, respectively,
12-, 16- and 25-times more potent antiparasitic agents than
unsubstituted compound (17).
Given the selectivity of these compounds against bloodstream
trypomastigotes of T. cruzi, we next examined their activity against
intracellular parasites. To this end, we assessed an in vitro model of
parasite infection using mouse macrophages infected with Y strain
trypomastigotes. Three days after infection, 35–40% of the
untreated macrophages were infected, and a high number of
amastigotes per 100 macrophages were observed. Treatment with
against intracellular amastigotes. IC50 values of 5.9 0.52,
3.2 0.3, and 3.1 0.64 M for compounds (18), (19) and (20)
were found, respectively. These values were lower than Benznida-
zole (IC50 = 13.9 0.39 M).
l
l
Finally, we evaluated the in vivo efficacy of compound (20).
Starting on day 5 post-infection (dpi), compound (20) was admin-
istered at 100 mg/kg orally once per day for five consecutive days
in BALB/c mice infected with 104 Y strain trypomastigotes. Controls
included untreated and Benznidazole-treated mice. The course of
infection was monitored by counting blood parasites and animal
survival was followed for 1 month. As seen in Figure 4, compound
(20) reduced blood parasitemia (P <0.001) compared to untreated
control. At dose of 100 mg/kg, administration of (20) caused a
reduction in blood parasitaemia of 86.7 (10 days post-infection
(dpi)) and 97.1% (12 dpi) (Table 2). In the group treated with Ben-
znidazole, it was observed >99% of inhibition of blood para-
sitaemia, indicating that eradication of infection was achieved.
Treatment with (20), similar to the treatment with Benznidazole,
had a protective effect on mortality. No signs of toxicity or body
weight loss were observed in animals in the treatment group (data
not shown).
3. Discussion
Here, we examined the chemistry and pharmacology of thiazo-
lidinones derived from a thiosemicarbazone which is a highly
potent cruzain inhibitor but a poor selective antiparasitic agent.
The employed route to preparing thiazolidinones was efficient to
produce compounds in acceptable yield and purity. X-ray diffrac-
tion revealed the conformation and geometry of iminic bound as
well as the tautomerism of heterocyclic ring. By varying sub-
stituents attached at heterocyclic ring, it was possible to screen
active compounds and to identify the main structural determi-
50 lM Benznidazole reduced the number of infected cells and the
number of intracellular amastigotes (P <0.001). We then tested the
three most potent compounds, thiazolidinones (18), (19), and (20)
at concentration of 50 lM. As shown in Figure 3, these compounds
inhibited T. cruzi infection with potency higher than Benznidazole
(P <0.001). When tested at different concentrations, it was possible
to calculate the IC50 values for compounds (18), (19) and (20)

G. B. de Oliveira Filho et al. / Bioorg. Med. Chem. 23 (2015) 7478–7486
7481
Table 1
compounds (1–2 and 8, 20) are the substituents linked at N1
(phenylthiopropyl for (1), phenoxypropan-2-yl for (2) and 3,4-
dichlorophenyl for (8) and (20). Comparing compound 1 with com-
pound (8), it was not observed an improvement in trypanocidal
activity. However, an improvement in trypanocidal activity was
observed for compound (20), being 3-times more potent than com-
pound (2), the lead compound identified by Moreira et al.38
Anti-T. cruzi activity of thiazolidinones (7–21)
Compd R1
Trypomastigotes, Y
strain T. cruzi IC50 SDa CC50 SDb
M) M)
Macrophages
Selectivity
indexc
(l
(l
(6.1 lM vs 1.7 lM). This result highlight the importance of the
3,4-dichloro moiety for the trypanocidal activity (Fig. 5).
Regarding the mechanism of action, the thiazolidinones did not
inhibit cruzain activity, while the thiosemicarbazone prototype
did. These findings show that the mechanism of action of these
antiparasitic thiazolidinones is different from thiosemicar-
bazones-inhibiting cruzain. In addition to displaying activity
against axenic parasite, the thiazolidinones exhibited strong activ-
ity against intracellular amastigotes. In comparison to untreated
infected macrophages, the compounds not only reduced the
in vitro infection but also the parasite burden. Importantly, this
activity was superior to Benznidazole-treated cells. Compound
(20), the most potent in vitro thiazolidinone, was efficient in reduc-
ing the blood parasitemia in acutely-infected mice.
4
5
6
H
7.1 0.76
11.2 1.62
>50
13.4 0.82
>100
>100
1.7
8.8
—
Methyl
Phenyl
7
8
9
10
11
H
>50
49.5 0.51
>50
76.5 3.18
>100
76.7 0.46
>100
—
2.0
—
5.9
—
Methyl
Ethyl
Isopropyl 16.9
In overall, the substitution at N3 with phenyl and C5 with alkyl
groups were benefic for the trypanocidal activity, generating com-
pounds (18), (19) and (20), the most potent compounds presented
in this study. It is worth to mention that compounds (18)
Phenyl
>50
96.9 1.37
(4.2 0.28
respectively, 2-, 3- and 6-times more potent than the reference
drug, Benznidazole (10.6 0.87 M) for trypomastigote form of
lM), (19) (2.9 0.91 lM) and (20) (1.7 0.17 lM) were,
l
the parasite. About intracellular amastigotes, compounds (18),
(19) and (20) also present good trypanocidal activity, being 2-, 4-
and 4-times more potent than Benznidazole, the unique drug avail-
able for treatment.
12
13
14
15
16
H
>50
>50
48.5
>100
>100
>100
>100
97.6
—
—
2.0
—
—
Methyl
Ethyl
Isopropyl >50
Phenyl
41.5
4. Conclusions
The search for antiparasitic compounds against T. cruzi based on
heterocyclic chemistry led us to develop a new series of thiazolidi-
nones. This study enabled molecular modifications at positions N3
and C5 on the thiazolidinic ring. The X-ray diffraction of compound
(10) revealed that an E-geometry for iminic bond is observed. The
in vitro screening showed that the presence of a phenyl group at
position N3 improves the antiparasitic activity. This led to the
identification of the potent N-phenyl substituted compounds
(18–20), which were more potent trypanocidal agents than Ben-
znidazole and previously described thiazolidinones (1–2). Com-
pound (20) reduced parasitemia in both in vitro and in an
acutely-infected mice model, thereby corroborating to the general
notion that this class of heterocyclic compounds are strong
antiparasitic agents.
17
18
19
20
21
Bdz
GV
H
>50
4.2 0.28
2.9 0.91
>100
—
6
8
>58
1.5
>9.4
—
Methyl
Ethyl
Isopropyl 1.7 0.17
Phenyl
—
—
26.1 0.90
23.3 0.96
>100
27.1 0.51
>100
17.7 0.09
10.6 0.87
—
0.45 0.04
ND = not determined. Bdz = Benznidazole, GV = violet gentian. SD = standard
deviation.
a
Determined after 24 h of incubation in the presence of compounds.
Determined in J774 cells for 72 h after incubation.
(CC50 macrophages)/(IC50 trypomastigotes).
b
c
5. Experimental section
5.1. Reagents and spectra analysis
nants of thiazolidinones for antiparasitic activity. We observed that
the nonsubstituted or those containing a methyl at N3 presented
poor antiparasitic activity. In contrast, thiazolidinones containing
a phenyl group at N3 exhibited trypanocidal properties and were
active against intracellular parasites, corroborating with a previ-
ously work of our group.38 In addition, it is worth mentioning that
these compounds presented low cytotoxicity in host cell viability,
therefore showing good selectivity indexes.
Compounds (1) (Fig. 1) and (2) were previously synthesized by
our group, with good trypanocidal activities (10 lM and 6.1 lM,
respectively). The compounds with similar structure are com-
pounds (8) and (20), with the same substituents at N3 and C5,
respectively. The main structural difference between these
All reagents were used as purchased from commercial sources
(Sigma–Aldrich, Vetec, or Fluka). Progress of the reactions was
followed by thin-layer chromatography (silica gel 60 F254 in alu-
minum foil). Purity of the target compounds was confirmed by
combustion analysis (for C, H, N and S) performed by a Carlo-
Erba instrument (model EA 1110). IR was determined in KBr pel-
lets. For NMR, we used a Varian UnityPlus 400 MHz (400 MHz
for 1H and 100 MHz for 13C) and Bruker AMX-300 MHz
(300 MHz for 1H and 75.5 MHz for 13C) instruments. DMSO-d6,
acetone-d6, or D2O were purchased from CIL. Chemical shifts
are reported in ppm, and multiplicities are given as s (singlet),

7482
G. B. de Oliveira Filho et al. / Bioorg. Med. Chem. 23 (2015) 7478–7486
Figure 3. Thiazolidinones (18), (19), and (20) affect intracellular parasite development. The percentage of infected macrophages (A) and the relative number of amastigotes
per 100 macrophages (B) are higher in untreated controls than in cultures treated with test compounds (18), (19), and (20), or Bdz. Macrophages were infected with Y strain
trypomastigotes for 2 h, and test inhibitors were then added at 50
were tested in triplicate, and two independent experiments were performed. Bars indicate standard deviation. Bdz was used as a positive control.
l
M. Cell cultures were incubated for 3 days. ***P <0.001 compared to untreated controls. All compounds
40
30
20
10
0
100
80
60
40
20
0
A
B
***
***
***
6
8
10
12
0
10
20
30
Days post infection
Days
20 - 100 mg/Kg
Vehicle
Bdz - 100 mg/Kg
Figure 4. (A) Compound (20) substantially reduced parasitemia in mice. Female BALB/c mice were infected with 104 Y strain trypomastigotes. Five days after infection, mice
were treated orally with compound (20) or Bdz with one daily dose (100 mg/kg) for five consecutive days. Parasitemia was monitored by counting the number of
trypomastigotes in fresh blood samples. Values represent the mean SEM of 6 mice per group. ***P <0.001 compared to untreated-infected group (vehicle). (B) Treatment with
compound (20) increases survival rates in infected mice. The animals were monitored for 30 days after infection. Results are from one experiment done with 6 mice per
group.
5.1.1. General procedure for the synthesis of thiosemicar-
bazones (4–6). Example for compound (4): Synthesis of
1-(3,4-dichlorophenyl)-ethylidenethiosemicarbazone (4)
Table 2
Parasitemia and mortality evaluation in mice infected with Y strain T. cruzi and
treated daily with (20) or Benznidazole for 5 days
Compounds
Dose (mg/kg)
% Blood parasitemia
reduction in micea
Mortalityb
Under magnetic stirring: in
a 100 mL round-bottom flask,
1.06 mmol (0.097 g) of thiosemicarbazide, 1.06 mmol (0.2 g) of
30,40-dichloroacetophenone (3), 4 drops of H2SO4 and 20 mL of
ethanol were added and maintained under magnetic stirring and
reflux for 20 h. After cooling back to rt, the precipitate was filtered
in a Büchner funnel with a sintered disc filter, washed with cold
ethanol, and then dried over SiO2. Product was purified by recrys-
tallization in hot toluol. Colorless crystals, mp: 187–189 °C; yield:
0.10 g (38%).
10 dpi
12 dpi
20
Bdz
Vehicle
100
100
—
86.7
>99
—
97.1
>99
—
0/6
0/6
6/6
dpi = days post-infection. Bdz = Benznidazole. Vehicle = untreated and infected
group.
a
Calculated as ([average vehicle group ꢀ average treated group)/average vehicle
group] ꢁ 100%).
Under ultrasound irradiation: in a 10 mL round-bottom flask,
1.06 mmol (0.097 g) of thiosemicarbazide, 1.06 mmol (0.2 g) of
30,40-dichloroacetophenone (3), 4 drops of H2SO4 and 20 mL of
ethanol were added and maintained in a ultrasound bath for
30 min. After cooling back to rt, the precipitate was filtered in a
Büchner funnel with a sintered disc filter, washed with cold etha-
nol, and then dried over SiO2. Product was purified by recrystalliza-
tion in hot toluol. Colorless crystals, mp: 187–189 °C; yield: 0.22 g
(80%); Rf = 0.56 (toluol/ethyl acetate 6:4). IR (KBr): 3427 (NH2),
b
Mortality was monitored until 30 days after treatment.
d (doublet), t (triplet), q (quartet), m (multiplet), and dd (double
doublet), and coupling constants (J) in hertz. NH signals were
localized in each spectrum after the addition of a few drops of
D2O. Structural assignments were corroborated by DEPT analysis.
Mass spectrometry experiments were performed on a Q-TOF
spectrometer LC-IT-TOF (Shimadzu). When otherwise specified,
ESI was carried out in the positive ion mode. Reactions in an
ultrasound bath were carried out under frequency of 40 kHz
(180 W) and without external heating.
3399 (NH), 3138 (CAH), 1591 (C@N) cmꢀ1 1H NMR (300 MHz,
.
DMSO-d6): d 2.27 (s, 3H, CH3), 7.58 (d, 1H, J 9.37 Hz, Ar), 7.86
(dd, 1H, J 9.37 and 3.75 Hz, Ar), 8.18 (s, 1H, NH2), 8.26 (d, 1H, J
3.75 Hz, Ar), 8.36 (s, 1H, NH2), 10.29 (s, 1H, NH). Signals at d

G. B. de Oliveira Filho et al. / Bioorg. Med. Chem. 23 (2015) 7478–7486
7483
Lower Cytotoxicity
Improvement in
trypanocidal activity
H
N
S
N
N
S
S
N
N
N
NH2
HN
N
Cl
Cl
Cl
O
O
Cl
Cl
Thiazolidinone ( )
= >
Cl
O
20
Thiazolidinone (
IC50 1.7 µM (T. cruzi)
CC50 = >100 µM (J774)
)
4
Thiosemicarbazone ( )
7
=
=
IC50 7.1 µM (T. cruzi)
IC50
50 µM (T. cruzi)
CC50 = 13.4 µM (J774)
CC50 = 76.5 µM (J774)
N
S
N
N
O
Thiazolidinone (2)
IC50 = 6.1 µM (T. cruzi)
Figure 5. Structural improvements of proposed compounds.
8.18, 8.36 and 10.29 ppm disappear after adding D2O. 13C NMR
(75.5 MHz, DMSO-d6): d 13.8 (CH3), 126.7 (CH, Ar), 128.2 (CH,
Ar), 130.2 (CH, Ar), 131.3 (C, Ar), 131.6 (3ClC, Ar), 138.2 (4ClC,
Ar), 145.312 (C@N), 179.0 (C@S). HRMS (ESI): 259.9565 [MꢀH]+.
Anal. calcd for C9H9Cl2N3S: C, 41.23; H, 3.46; N, 16.03; S, 12.23.
Found: C, 41.21; H, 3.61; N, 16.36; S, 12.78.
filter, washed with cold ethanol, and then dried over SiO2. Product
was purified by recrystallization in hot cyclohexane. Colorless crys-
tals were obtained. Mp: 234–236 °C; yield: 0.75 g (65%); Rf: 0.53
(toluol/ethyl acetate 6:4). IR (KBr): 3143 (NH), 2985 (CAH), 1715
(C@O), 1628 (C@N) cmꢀ1 1H NMR (300 MHz, DMSO-d6): d 2.36
.
(s, 3H, CH3), 3.89 (s, 2H, SACH2), 7.69 (d, 1H, J 10 Hz, Ar), 7.80 (d,
1H, J 10 Hz, Ar), 8.00 (s, 1H, Ar), 12.06 (s, 1H, NH). 13C NMR and
DEPT (75.5 MHz, DMSO-d6): d 14.8 (CH3), 33.3 (CH2, heterocycle),
126.8 (CH, Ar), 128.4 (CH, Ar), 131.0 (CH, Ar), 131.8 (C, Ar), 132.7
(3ClC, Ar), 138.7 (4ClC, Ar), 158.6 (C@N), 166.0 (SAC@N), 174.3
(C@O). HRMS (ESI): 301.9364 [M+H]+. Anal. calcd for C11H9Cl2N3-
OS: C, 43.72; H, 3.00; N, 13.91; S, 10.61. Found: C, 44.00; H, 3.20;
N, 13.47; S, 10.11.
5.1.1.1.
carbazone (5).
1-(3,4-Dichlorophenyl)ethylideno-4-methylthiosemi-
Recrystallization in hot toluol afforded color-
less crystal, mp: 195–197 °C; yield: 0.24 g (84%); Rf = 0.7 (toluol/
ethyl acetate 6:4). IR (KBr): 3343 and 3219 (NH), 1547 (C@N)
cmꢀ1 1H NMR (300 MHz, DMSO-d6): d 2.28 (s, 3H, CH3), 3.04 (d,
.
3H, J 6 Hz, NACH3, coupling with NAH), 7.63 (d, 1H, J 8.57 Hz,
Ar), 7.88 (dd, 1H, J 8.57 and 2.14 Hz, Ar), 8.23 (d, 1H, J 2.14 Hz,
Ar), 8.61 (broad d, 1H, J 6 Hz, CH3ANH), 10.33 (s, 1H, NH). 13C
NMR (75.5 MHz, DMSO-d6): d 13.8 (CH3), 31.1 (NACH3), 126.7
(CH, Ar), 128.1 (CH, Ar), 130.2 (CH, Ar), 131.3 (C, Ar), 131.5 (3ClC,
Ar), 138.3 (4ClC, Ar), 145.0 (C@N), 178.6 (C@S). HRMS (ESI):
275.9475 [M+H]+. Anal. calcd for C10H11Cl2N3S: C, 43.49; H, 4.01;
N, 15.21; S, 11.61. Found: C, 43.34; H, 3.98; N, 15.55; S, 11.74.
5.1.2.1.
2-[1-(3,4-Dichlorophenyl)ethylidenohydrazone]-5-
Recrystallization in toluol/cy-
methylthiazolidin-4-one (8).
clohexane 2:1 afforded colorless. Mp: 208–210 °C; yield: 0.68 g
(56%); Rf: 0.52 (toluol/ethyl acetate 6:4). IR (KBr): 3422 (NH),
3139 (CAH), 1704 (C@O), 1627 (C@N) cmꢀ1 1H NMR (300 MHz,
.
DMSO-d6): d 1.48 (d, 3H, J 7.5 Hz, CH3 heterocycle), 2.27 and 2.35
(s, 3H, CH3), 4.15 (q, 1H, J 7.5 Hz, CH heterocycle), 7.60 and 7.68
(d, 1H, J 8.75 Hz, Ar), 7.78 and 7.87 (d, 1H, J 8.75 Hz, Ar), 7.99
and 8.26 (s, 1H, Ar), 8.36 and 10.30 (s, 1H, NH). 13C NMR
(75.5 MHz, DMSO-d6): d 13.8 and 14.3 (CH3 heterocycle), 18.8
(CH3), 41.0 (CH, heterocycle), 126.4 and 126.8 (CH, Ar), 127.8 and
128.2 (CH, Ar), 130.2 (CH, Ar), 131.3 and 131.6 (C, Ar), 132.2
(3ClC, Ar), 138.2 and 138.3 (4ClC, Ar), 145.3 (C@N), 157.9 (SAC@N),
5.1.1.2. 1-(3,4-Dichlorophenyl)ethylidene)-4-phenylthiosemi-
carbazone (6).
Recrystallization in hot toluol afforded color-
less crystal, mp: 200–202 °C; yield: 0.32 g (89%); Rf = 0.73 (toluol/
ethyl acetate 6:4). IR (KBr): 3309 and 3239 (NH), 3046 (CAH),
1523 (C@N) cmꢀ1 1H NMR (300 MHz, DMSO-d6): d 2.36 (s, 3H,
.
CH3), 7.23 (t, 1H, J 7.21 Hz, Ar), 7.38 (t, 2H, J 7.21 Hz, Ar), 7.51 (d,
2H, J 7.21 Hz, Ar), 7.64 (d, 1H, J 9.09 Hz, Ar), 7.97 (dd, 1H, J 9.09
and 2.72 Hz, Ar), 8.34 (d, 1H, J 2.72 Hz, Ar), 10.18 (s, 1H, NH),
10.66 (s, 1H, ArNH). 13C NMR (75.5 MHz, DMSO-d6): d 14.2 (CH3),
125.6 (CH, Ar), 126.5 (CH, Ar), 127.0 (CH, Ar), 128.1 (CH, Ar),
128.4 (CH, Ar), 130.2 (CH, Ar), 131.3 (C, Ar), 131.8 (3ClC, Ar),
138.1 (4ClC, Ar), 139.2 (CAN, Ar), 146.3 (C@N), 177.3 (C@S). HRMS
(ESI): 337.9678 [M+H]+. Anal. calcd for C15H13Cl2N3S: C, 53.26; H,
3.87; N, 12.42; S, 9.48. Found: C, 53.23; H, 3.84; N, 12.31; S, 9.32.
177.3 (C@O). HRMS (ESI): 315.9491 [M+H]+. Anal. calcd for C12H11
-
Cl2N3OS: C, 45.58; H, 3.51; N, 13.29; S, 10.14. Found: C, 45.86; H,
3.68; N, 12.90; S, 10.00.
5.1.2.2.
ethylthiazolidin-4-one (9).
2-[1-(3,4-Dichlorophenyl)ethylidenohydrazone]-5-
Recrystallization in hot toluol
afforded colorless. Mp: 175–177 °C; yield: 0.94 g (75%); Rf: 0.63
(toluol/ethyl acetate 6:4). IR (KBr): 2975 (CAH), 1704 (C@O),
5.1.2. General procedure for the synthesis of thiazolidinones
7–21. Example for compound (7)
1623 (C@N) cmꢀ1 1H NMR (300 MHz, DMSO-d6): d 0.95 (broad s,
.
3H, CH3), 1.88 (m, 2H, CH2), 2.36 (s, 3H, CH3), 4.20 (broad s, 1H,
CH heterocycle), 7.69 (d, 1H, J 8 Hz, Ar), 7.80 (d, 1H, J 8 Hz, Ar),
7.99 (s, 1H, Ar), 12.05 (s, 1H, NH). 13C NMR (75.5 MHz, DMSO-
d6): d 10.3 (CH3), 14.3 (CH3), 25.4 (CH2), 49.0 (CH, heterocycle),
126.4 (CH, Ar), 127.9 (CH, Ar), 130.6 (CH, Ar), 131.3 (C, Ar), 132.2
(3ClC, Ar), 138.3 (4ClC, Ar), 158.2 (C@N), 164.1 (SAC@N), 176.0
(C@O). HRMS (ESI): 329.9624 [M+H]+. Anal. calcd for C13H13Cl2N3-
OS: C, 47.28; H, 3.97; N, 12.72; S, 9.71. Found: C, 47.20; H, 3.60; N,
12.34; S, 9.55.
Synthesis of 2-[1-(3,4-dichlorophenyl)ethylidenohydrazone]thiazo-
lidin-4-one (7). in a 100 mL round-bottom flask, 3.81 mmol (1.0 g)
of thiosemicarbazone (4) was dissolved in 30 mL of ethanol, fol-
lowed by adding 15.24 mmol (1.25 g) anhydrous sodium acetate
under magnetic stirring and warming. After 15 min, 5.73 mmol
(0.8 g) of ethyl 2-bromoacetate was added in portions and the mix-
ture was maintained under reflux for 20 h. After cooling back to rt,
the precipitate was filtered in a Büchner funnel with a sintered disc

7484
G. B. de Oliveira Filho et al. / Bioorg. Med. Chem. 23 (2015) 7478–7486
5.1.2.3. 2-[1-(3,4-Dichlorophenyl)ethylidenohydrazone]-5-iso-
propylthiazolidin-4-one (10). Recrystallization in toluol/cy-
DMSO-d6): d 0.95 (t, 3H, CH3), 1.83 (m, 1H, CH2), 2.02 (m, 1H,
CH2), 2.42 (s, 3H, CH3), 3.19 (s, 3H, NACH3), 4.27 (m, 1H, CH hete-
rocycle), 7.70 (d, 1H, J 8 Hz, Ar), 7.83 (dd, 1H, J 8 and 2 Hz, Ar), 8.02
(d, 1H, J 2 Hz, Ar). 13C NMR (100 MHz, DMSO-d6): d 10.1 (CH3), 14.2
(CH3), 25.3 (CH2), 29.2 (NACH3), 48.1 (CH heterocycle), 126.3 (CH,
Ar), 127.9 (CH, Ar), 130.4 (CH, Ar), 131.2 (C, Ar), 132.3 (3ClC, Ar),
138.1 (4ClC, Ar), 159.5 (C@N), 162.9 (SAC@N), 174.0 (C@O). Anal.
calcd for C14H15Cl2N3OS: C, 48.84; H, 4.39; N, 12.21; S, 9.31. Found:
C, 48.83; H, 4.09; N, 12.15; S, 9.58.
clohexane 2:1 afforded colorless. Mp: 193–195 °C; yield: 0.73 g
(56%); Rf: 0.6 (toluol/ethyl acetate 6:4). IR (KBr): 2953 (CAH),
1707 (C@O), 1616 (C@N) cmꢀ1 1H NMR (300 MHz, DMSO-d6): d
.
0.87 (d, 3H, J 6 Hz, CH3), 0.98 (d, 3H, J 6 Hz, CH3), 2.36 (s, 3H,
CH3), 2.50 (m, 1H, CH), 4.30 (d, 1H, J 3 Hz, CH heterocycle), 7.69
(d, 1H, J 10 Hz, Ar), 7.81 (d, 1H, J 10 Hz, Ar), 7.99 (s, 1H, Ar),
12.06 (s, 1H, NH). 13C NMR (75.5 MHz, DMSO-d6): d 14.4 (CH3),
16.4 (CH3), 20.3 (CH3), 29.9 (CH), 54.7 (CH, heterocycle), 126.4
(CH, Ar), 128.0 (CH, Ar), 130.6 (CH, Ar), 131.3 (C, Ar), 132.3 (3ClC,
Ar), 138.3 (4ClC, Ar), 158.6 (C@N), 163.7 (SAC@N), 175.4 (C@O).
HRMS (ESI): 343.9749 [MꢀH]+. Anal. calcd for C14H15Cl2N3OS: C,
48.84; H, 4.39; N, 12.21; S, 9.31. Found: C, 49.19; H, 4.56; N,
12.16; S, 9.49.
5.1.2.8. 2-[1-(3,4-Dichlorophenyl)ethylidenohydrazone]-5-iso-
propyl-3-methylthiazolidin-4-one (15).
Recrystallization in
cyclohexane/toluol 3:1 afforded colorless. Mp: 137–140 °C; yield:
0.70 g (53%); Rf: 0.84 (toluol/ethyl acetate 6:4). IR (KBr): 2947
(CAH), 1713 (C@O), 1610 and 1563 (C@N) cmꢀ1 1H NMR
.
(300 MHz, DMSO-d6): d 0.84 (d, 3H, J 6.42 Hz, CH3), 1.00 (d, 3H, J
6.42 Hz, CH3), 2.43 (s, 3H, CH3), 2.45 (m, 1H, CH), 3.21 (s, 3H,
NACH3), 4.38 (d, 1H, J 2.5 Hz, CH heterocycle), 7.72 (d, 1H, J
7.5 Hz, Ar), 7.85 (dd, 1H, J 7.5 and 1.25 Hz, Ar), 8.03 (d, 1H, J
1.25 Hz, Ar). 13C NMR (75.5 MHz, DMSO-d6): d 14.4 (CH3), 16.4
(CH3), 20.2 (CH3), 29.3 (CH), 30.1 (NACH3), 53.9 (CH heterocycle),
126.5 (CH, Ar), 128.1 (CH, Ar), 130.7 (CH, Ar), 131.3 (C, Ar), 132.4
(3ClC, Ar), 138.1 (4ClC, Ar), 159.9 (C@N), 163.2 (SAC@N), 173.8
(C@O). HRMS (ESI): 357.9853 [M+H]+. Anal. calcd for C15H17Cl2N3-
OS: C, 50.28; H, 4.78; N, 11.73; S, 8.95. Found: C, 50.29; H, 4.64; N,
11.71; S, 9.19.
5.1.2.4.
2-[1-(3,4-Dichlorophenyl)ethylidenohydrazone]-5-
Recrystallization in hot toluol
phenylthiazolidin-4-one (11).
afforded colorless. Mp: 245–247 °C; yield: 0.95 g (65%); Rf: 0.57
(toluol/ethyl acetate 6:4). IR (KBr): 1711 (C@O), 1618 (C@N)
cmꢀ1 1H NMR (300 MHz, DMSO-d6): d 2.41 (s, 3H, CH3), 5.49 (s,
.
1H, CH heterocycle), 7.39 (m, 5H, Ar), 7.68 (d, 1H, J 9 Hz, Ar),
7.81 (dd, 1H, J 9 and 2.25 Hz, Ar), 8.01 (d, 1H, J 2.25 Hz, Ar),
12.35 (s, 1H, NH). 13C NMR (75.5 MHz, DMSO-d6): d 14.9 (CH3),
51.4 (CH, heterocycle), 126.9 (CH, Ar), 128.4 (CH, Ar), 128.7 (CH,
Ar), 128.9 (CH, Ar), 129.3 (CH, Ar), 131.1 (CH, Ar), 131.8 (C, Ar),
132.8 (C, Ar), 137.2 (3ClC, Ar), 138.6 (4ClC, Ar), 159.2 (C@N),
162.0 (SAC@N), 175.0 (C@O). HRMS (ESI): 378.0310 [M+H]+. Anal.
calcd for C17H13Cl2N3OS: C, 53.98; H, 3.46; N, 11.11; S, 8.48. Found:
C, 53.75; H, 3.53; N, 11.12; S, 8.78.
5.1.2.9.
2-[1-(3,4-Dichlorophenyl)ethylidenohydrazone]-3-
Recrystallization in
methyl-5-phenylthiazolidin-4-one (16).
hot toluol afforded colorless. Mp: 194–196 °C; yield: 0.91 g
(87%); Rf: 0.82 (toluol/ethyl acetate 6:4). IR (KBr): 1709 (C@O),
5.1.2.5.
2-[1-(3,4-Dichlorophenyl)ethylidenohydrazone]-3-
Recrystallization in hot toluol
1603 and 1565 (C@N) cmꢀ1 1H NMR (300 MHz, DMSO-d6): d
.
methylthiazolidin-4-one (12).
2.47 (s, 3H, CH3), 2.50 (s, 3H, NACH3), 5.55 (s, 1H, CH heterocycle),
7.39 (m, 5H, Ar), 7.71 (d, 1H, J 8.57 Hz, Ar), 7.84 (dd, 1H, J 8.57 and
2.14 Hz, Ar), 8.03 (d, 1H, J 2.14 Hz, Ar). 13C NMR (75.5 MHz, DMSO-
d6): d 14.4 (CH3), 29.9 (NACH3), 50.1 (CH heterocycle), 126.5 (CH,
Ar), 128.0 (CH, Ar), 128.3 (CH, Ar), 128.5 (CH, Ar), 128.8 (CH, Ar),
130.6 (CH, Ar), 131.3 (C, Ar), 136.5 (3ClC, Ar), 138.0 (4ClC, Ar),
160.0 (C@N), 163.0 (SAC@N), 173.0 (C@O). HRMS (ESI): 392.0450
[M+H]+. Anal. calcd for C18H15Cl2N3OS: C, 55.11; H, 3.85; N,
10.71; S, 8.17. Found: C, 55.37; H, 3.92; N, 10.58; S, 8.44.
afforded colorless. Mp: 195–197 °C; yield: 0.70 g (61%); Rf: 0.73
(toluol/ethyl acetate 6:4). IR (KBr): 2942 (CAH), 1717 (C@O),
1613 and 1570 (C@N) cmꢀ1 1H NMR (400 MHz, DMSO-d6): d
.
2.43 (s, 3H, CH3), 3.20 (s, 3H, NACH3), 3.95 (s, CH2 heterocycle),
7.72 (d, 1H, J 11.42 Hz, Ar), 7.83 (dd, 1H, J 11.42 and 1.43 Hz, Ar),
8.03 (d, 1H, J 1.43 Hz, Ar). 13C NMR (100 MHz, DMSO-d6): d 14.2
(CH3), 29.2 (NACH3), 32.0 (CH2 heterocycle), 126.3 (CH, Ar),
127.9 (CH, Ar), 130.5 (CH, Ar), 131.2 (C, Ar), 132.3 (3ClC, Ar),
138.1 (4ClC, Ar), 159.4 (C@N), 164.5 (SAC@N), 172.0 (C@O). HRMS
(ESI): 315.9498 [M+H]+. Anal. calcd for C12H11Cl2N3OS: C, 45.58; H,
3.51; N, 13.29; S, 10.14. Found: C, 45.48; H, 3.33; N, 13.18; S, 9.90.
5.1.2.10.
phenylthiazolidin-4-one (17).
2-[1-(3,4-Dichlorophenyl)ethylidenohydrazone]-3-
Recrystallization in hot toluol
afforded colorless. Mp: 232–233 °C; yield: 0.8 g (71%); Rf: 0.79
(toluol/ethyl acetate 6:4). IR (KBr): 2970 (CAH), 1728 (C@O),
5.1.2.6.
2-[1-(3,4-Dichlorophenyl)ethylidenohydrazone]-3,5-
Recrystallization in cyclo-
dimethylthiazolidin-4-one (13).
1606 and 1562 (C@N) cmꢀ1 1H NMR (400 MHz, DMSO-d6): d
.
hexane/toluol 3:1 afforded colorless. Mp: 178–180 °C; yield:
0.90 g (75%); Rf: 0.8 (toluol/ethyl acetate 6:4). IR (KBr): 1704
2.16 (s, 3H, CH3), 4.10 (s, 2H, CH2 heterocycle), 7.41 (d, 2H, J
7.54 Hz, Ar), 7.45 (t, 1H, J 7.54 Hz, Ar), 7.53 (t, 2H, J 7.54 Hz, Ar),
7.70 (d, 1H, J 9.05 Hz, Ar), 7.79 (d, 1H, J 9.05 Hz, Ar), 7.99 (s, 1H,
Ar). 13C NMR (100 MHz, DMSO-d6): d 14.3 (CH3), 32.2 (CH2),
126.3 (CH, Ar), 127.8 (CH, Ar), 128.4 (CH, Ar), 128.7 (CH, Ar),
130.5 (CH, Ar), 134.9 (C, Ar), 136.8 (C, Ar), 163.0 (C@N), 171.6
(SAC@N), 198.6 (C@O). HRMS (ESI): 377.9950 [M+H]+. Anal. calcd
for C17H13Cl2N3OS: C, 53.98; H, 3.46; N, 11.11; S, 8.48. Found: C,
53.68; H, 3.42; N, 10.89; S, 7.94.
(C@O), 1606 and 1570 (C@N) cmꢀ1 1H NMR (400 MHz, DMSO-
.
d6): d 1.53 (d, 3H, J 7.14 Hz, CH3), 2.42 (s, 3H, CH3), 3.22 (s, 3H,
NACH3), 4.24 (q, J 7.14 Hz, CH heterocycle), 7.70 (d, 1H, J 8.51 Hz,
Ar), 7.82 (dd, 1H, J 8.51 and 2.12 Hz, Ar), 8.02 (d, 1H, J 2.12 Hz,
Ar). 13C NMR (100 MHz, DMSO-d6): d 14.1 (CH3, position 5 of the
heterocycle), 18.5 (CH3), 29.3 (NACH3), 41.1 (CH heterocycle),
126.3 (CH, Ar), 127.8 (CH, Ar), 130.4 (CH, Ar), 131.2 (C, Ar), 132.2
(3ClC, Ar), 138.1 (4ClC, Ar), 159.3 (C@N), 163.0 (SAC@N), 175.0
(C@O). HRMS (ESI): 329.9630 [M+H]+. Anal. calcd for C13H13Cl2N3-
OS: C, 47.28; H, 3.97; N, 12.72; S, 9.71. Found: C, 47.33; H, 3.81; N,
12.63; S, 9.69.
5.1.2.11.
2-[1-(3,4-Dichlorophenyl)ethylidenohydrazone]-3-
Recrystallization in
phenyl-5-methylthiazolidin-4-one (18).
cyclohexane/toluol 3:1 afforded colorless. Mp: 208–209 °C; yield:
0.78 g (67%); Rf: 0.83 (toluol/ethyl acetate 6:4). IR (KBr): 1712
5.1.2.7.
2-[1-(3,4-Dichlorophenyl)ethylidenohydrazone]-5-
Recrystallization in
(C@O), 1609 and 1571 (C@N) cmꢀ1 1H NMR (300 MHz, DMSO-
.
ethyl-3-methylthiazolidin-4-one (14).
d6): d 1.63 (d, 3H, J 6.9 Hz, CH3), 2.16 (s, 3H, CH3), 4.41 (q, 1H, J
6.9 Hz, CH heterocycle), 7.41–7.55 (m, 5H, Ar), 7.70 (d, 1H, J
8.4 Hz, Ar), 7.79 (dd, 1H, J 8.4 and 1.8 Hz, Ar), 7.99 (d, 1H, J
1.8 Hz, Ar). 13C NMR (75.5 MHz, DMSO-d6): d 14.3 (CH3), 18.7
hot cyclohexane afforded colorless. Mp: 167–168 °C; yield: 0.80 g
(64%); Rf: 0.81 (toluol/ethyl acetate 6:4). IR (KBr): 2970 (CAH),
1716 (C@O), 1610 and 1567 (C@N) cmꢀ1 1H NMR (400 MHz,
.

G. B. de Oliveira Filho et al. / Bioorg. Med. Chem. 23 (2015) 7478–7486
7485
(CH3), 41.0 (CH heterocycle), 126.4 (CH, Ar), 127.9 (CH, Ar), 128.5
(CH, Ar), 128.8 (CH, Ar), 130.6 (CH, Ar), 131.3 (C, Ar), 132.4 (3ClC,
Ar), 135.0 (C, Ar), 138.0 (4ClC, Ar), 159.7 (C@N), 163.2 (SAC@N),
174.8 (C@O). Anal. calcd for C18H15Cl2N3OS: C, 55.11; H, 3.85; N,
10.71; S, 8.17. Found: C, 55.36; H, 4.01; N, 10.44; S, 7.64.
mined in
a
Synergy
2
(Biotek), from the Center of Flow
Cytometry and Fluorimetry at the Biochemistry and Immunology
Department (UFMG). The filters employed were 340 nM for excita-
tion and 440 nM for emission. Activity was calculated based on ini-
tial velocity rates, compared to
compound stocks were prepared in DMSO. All compounds were
evaluated at 10 M and inhibition was measured in at least two
a DMSO control, since all
5.1.2.12.
2-[1-(3,4-Dichlorophenyl)ethylidenohydrazone]-3-
Recrystallization in
l
phenyl-5-ethylthiazolidin-4-one (19).
independent experiments, each case in triplicate.
hot cyclohexane afforded colorless. Mp: 154 °C; yield: 0.42 g
(35%); Rf: 0.86 (toluol/ethyl acetate 6:4). IR (KBr): 1708 (C@O),
4.3. Animals
1607 and 1568 (C@N) cmꢀ1 1H NMR (300 MHz, DMSO-d6): d
.
1.03 (t, 3H, CH3), 1.98 (m, 1H, CH2), 2.08 (m, 1H, CH2), 2.16 (s,
3H, CH3), 4.45 (m, 1H, CH heterocycle), 7.39–7.55 (m, 5H, Ar),
7.71 (d, 1H, J 8.7 Hz, Ar), 7.80 (dd, 1H, J 8.7 and 1.8 Hz, Ar), 7.99
(d, 1H, J 1.8 Hz, Ar). 13C NMR (75.5 MHz, DMSO-d6): d 10.1 (CH3),
14.4 (CH3), 25.5 (CH2), 48.2 (CH heterocycle), 126.4 (CH, Ar),
127.9 (CH, Ar), 128.5 (CH, Ar), 128.9 (CH, Ar), 130.6 (CH, Ar),
131.3 (C, Ar), 132.4 (3ClC, Ar), 134.9 (C, Ar), 138.0 (4ClC, Ar),
160.0 (C@N), 163.0 (SAC@N), 173.9 (C@O). HRMS (ESI): 405.9517
[M+H]+. Anal. calcd for C19H17Cl2N3OS: C, 56.16; H, 4.22; N,
10.34; S, 7.89. Found: C, 55.80; H, 4.23; N, 9.96; S, 7.97.
Female BALB/c mice (6–8 weeks old) were supplied by the ani-
mal breeding facility at Centro de Pesquisas Gonçalo Moniz (Fun-
dação Oswaldo Cruz, Bahia, Brazil) and maintained in sterilized
cages under a controlled environment, receiving a balanced diet
for rodents and water ad libitum. All experiments were carried
out in accordance with the recommendations of Ethical Issues
Guidelines, and were approved by the local Animal Ethics
Committee.
4.4. Parasites
5.1.2.13.
2-[1-(3,4-Dichlorophenyl)ethylidenohydrazone]-3-
Recrystallization
Bloodstream trypomastigotes forms of T. cruzi were obtained
from supernatants of LLC-MK2 cells previously infected and main-
tained in RPMI-1640 medium (Sigma–Aldrich, St. Louis, MO) sup-
plemented with 10% fetal bovine serum (FBS; Cultilab, Campinas,
phenyl-5-isopropylthiazolidin-4-one (20).
in hot cyclohexane afforded colorless. Mp: 158–160 °C; yield:
0.76 g (61%); Rf: 0.9 (toluol/ethyl acetate 6:4). IR (KBr): 2964
(CAH), 1720 (C@O), 1603 and 1564 (C@N) cmꢀ1
.
1H NMR
Brazil), and 50 lg/mL gentamycin (Novafarma, Anápolis, Brazil)
(300 MHz, DMSO-d6): d 0.98 (d, 3H, CH3), 1.06 (d, 3H, CH3), 2.16
(s, 3H, CH3), 2.36 (m, 1H, CH), 4.53 (d, 1H, CH heterocycle), 7.36–
7.56 (m, 5H, Ar), 7.71 (d, 1H, J 8.4 Hz, Ar), 7.81 (dd, 1H, J 8.4 and
1.8 Hz, Ar), 7.99 (d, 1H, J 1.8 Hz, Ar). 13C NMR (75.5 MHz, DMSO-
d6): d 14.5 (CH3), 16.5 (CH3), 20.1 (CH3), 30.5 (CH), 53.8 (CH hete-
rocycle), 126.5 (CH, Ar), 127.9 (CH, Ar), 128.4 (CH, Ar), 128.7 (CH,
Ar), 130.2 (CH, Ar), 131.3 (C, Ar), 132.5 (3ClC, Ar), 134.8 (C, Ar),
138.0 (4ClC, Ar), 160.3 (C@N), 163.1 (SAC@N), 173.5 (C@O). HRMS
(ESI): 419.9888 [M+H]+. Anal. calcd for C20H19Cl2N3OS: C, 57.15; H,
4.56; N, 10.00; S, 7.63. Found: C, 57.16; H, 4.49; N, 9.90; S, 7.68.
at 37 °C and 5% CO2.
4.5. Host cell toxicity
J774 macrophages were dispensed on 96-well plates at a cell
number of 5 ꢁ 104 cells/mL in 200
l
L of RPMI medium supple-
mented with 10% of FBS and 50
lg/mL of gentamycin and incu-
bated for 24 h at 37 °C and 5% CO2. After that time each
compound, dissolved in DMSO was added at six concentrations
(0.41–100 lM) in triplicate and incubated for 72 h. Cell viability
was determined by AlamarBlue assay (Invitrogen, Carlsbad, CA,
USA) according to the manufacturer instructions. Colorimetric
readings were performed after 6 h at 570 and 600 nm. Cytotoxic
concentration to 50% (CC50) was calculated using data-points gath-
ered from three independent experiments. Gentian violet (Synth,
São Paulo, Brazil) was used as positive control. The final concentra-
tion of DMSO was less than 1% in all in vitro experiments.
5.1.2.14. 2-[1-(3,4-Dichlorophenyl)ethylidenohydrazone]-3,5-
diphenylthiazolidin-4-one (21).
Recrystallization in hot
toluol afforded colorless. Mp: 210–212 °C; yield: 0.77 g (58%); Rf:
0.88 (toluol/ethyl acetate 6:4). IR (KBr): 3053 (CAH), 1715 (C@O),
1605 and 1566 (C@N) cmꢀ1 1H NMR (300 MHz, DMSO-d6): d
.
2.20 (s, 3H, CH3), 5.70 (s, 1H, CH heterocycle), 7.36–7.58 (m, 10H,
Ar), 7.69 (d, 1H, J 8.4 Hz, Ar), 7.80 (dd, 1H, J 8.4 and 2.1 Hz, Ar),
7.99 (d, 1H, J 2.1 Hz, Ar). 13C NMR (75.5 MHz, DMSO-d6): d 14.0
(CH3), 50.1 (CH heterocycle), 126.3 (CH, Ar), 127.8 (CH, Ar), 128.0
(CH, Ar), 128.1 (CH, Ar), 128.4 (CH, Ar), 128.7 (CH, Ar), 129.2 (CH,
Ar), 129.6 (CH, Ar), 130.5 (CH, Ar), 133.5 (C, Ar), 133.7 (C, Ar),
134.8 (C, Ar), 134.9 (C, Ar), 136.8 (C, Ar), 163.0 (C@N), 171.2
(SAC@N), 198.0 (C@O). HRMS (ESI): 452.0380 [MꢀH]+. Anal. calcd
for C23H17Cl2N3OS: C, 60.80; H, 3.77; N, 9.25; S, 7.06. Found: C,
60.68; H, 3.74; N, 9.26; S, 7.08.
4.6. Toxicity for Y strain trypomastigotes
Trypomastigotes collected from the supernatant of LLC-MK2
cells were dispensed into 96-well plates at a cell density of
4 ꢁ 105 cells/well. Test inhibitors, were diluted into five different
concentrations and added into their respective wells, and the plate
was incubated for 24 h at 37 °C and 5% of CO2. Aliquots of each well
were collected and the number of viable parasites, based on para-
site motility, was assessed in a Neubauer chamber. The percentage
of inhibition was calculated in relation to untreated cultures. IC50
calculation was also carried out using non-linear regression with
Prism 4.0 GraphPad software. Benznidazole was used as the refer-
ence drug.
5.3. Cruzain inhibition
Cruzain activity was measured by monitoring the cleavage of
the fluorogenic substrate Z-FR-AMC. Assays were performed in
0.1 M sodium acetate buffer pH 5.5, in the presence of 1 mM
beta-mercaptoethanol and 0.01% Triton X-100. The final concen-
trations of cruzain was 0.5 nM, and the Z-FR-AMC substrate con-
4.7. Intracellular parasite development
centration was 2.5
l
M (Km = 1
l
M) to a final volume of 200
l
L
Peritoneal exudate macrophages were obtained by washing,
with cold RPMI medium, the peritoneal cavity of BALB/c mice 4–
5 days after injection of 3% thioglycolate (Sigma) in saline
(1.5 mL per mice). Then, cells were seeded at a cell density of
2 ꢁ 105 cells/well in a 24 well-plate with rounded coverslips on
and at 25 °C. In all assays, the enzyme was pre-incubated with
the compounds for 10 min. before adding a solution containing
the Z-FR-AMC substrate. Enzyme kinetic was followed by continu-
ous reading for 5 min at 12 s intervals and readings were deter-

7486
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Acknowledgements
This work received support from CNPq (grant reference
471461/2011-3 to A.C.L.L.) and FAPESB (PRONEX program, grant
reference 6596 to M.B.P.S.). G.B.O.F. received a FACEPE Masters
scholarship. A.C.L.L., C.A.S. and M.B.P.S. hold a CNPq senior fellow-
ship. C.A.S also thanks the Instituto de Física de São Carlos (Univer-
sity of Sao Paulo, Brazil) for the use of KappaCCD diffractometer.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2015.10.048.
These data include MOL files and InChiKeys of the most important
compounds described in this article.
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