Structural design, synthesis and anti-Trypanosoma cruzi profile of the second generation of 4-thiazolidinones chlorine derivatives

Article abstract of DOI:10.1016/j.cbi.2021.109514

Chagas disease causes more deaths in the Americas than any other parasitic disease. Initially confined to the American continent, it is increasingly becoming a global health problem. In fact, it is considered to be an “exotic” disease in Europe, being virtually undiagnosed. Benznidazole, the only drug approved for treatment, effectively treats acute-stage Chagas disease, but its effectiveness for treating indeterminate and chronic stages remains uncertain. Previously, our research group demonstrated that 4-thiazolidinones presented anti-T. cruzi activity including in the in vivo assays in mice, making this fragment appealing for drug development. The present work reports the synthesis and anti-T. cruzi activities of a novel series of 4-thiazolidinones derivatives that resulted in an increased anti-T. cruzi activity in comparison to thiosemicarbazones intermediates. Compounds 2c, 2e, and 3a showed potent inhibition of the trypomastigote form of the parasite at low cytotoxicity concentrations in mouse splenocytes. Besides, all the 2c, 2e, and 3a tested concentrations showed no cytotoxic activity on macrophages cell viability. When macrophages were submitted to T. cruzi infection and treated with 2c and 3a, compounds reduced the release of trypomastigote forms. Results also showed that the increased trypanocidal activity induced by 2c and 3a is independent of nitric oxide release. Flow cytometry assay showed that compound 2e was able to induce necrosis and apoptosis in trypomastigotes. Parasites treated with the compounds 2e, 3a, and 3c presented flagellum shortening, retraction and curvature of the parasite body, and extravasation of the internal content. Together, these data revealed a novel series of 4-thiazolidinones fragment-based compounds with potential effects against T. cruzi and lead-like characteristics.

Full text of DOI:10.1016/j.cbi.2021.109514

Chemico-Biological Interactions 345 (2021) 109514
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Chemico-Biological Interactions
journal homepage: www.elsevier.com/locate/chembioint
Research paper
Structural design, synthesis and anti-Trypanosoma cruzi profile of the
second generation of 4-thiazolidinones chlorine derivatives
Gevanio Bezerra de Oliveira Filhoa, Marcos Veríssimo de Oliveira Cardosob,
c
c
´
Aline Caroline da Silva Santos , Thiago Andre Ramos dos Santos ,
c
a
a
˜
´
Ana Catarina Cristovao-Silva , Laura Gonzalez Rubio , Luiz da Silva Maia Neto ,
d
d
e
,
f
e,f
˜
´
Paulo Gaio Leite , Fabiana Simao Machado , Luiz Carlos Alves , Fabio Andre Brayner
,
c
a,*
´
ˆ
Valeria Rego Alves Pereira , Ana Cristina Lima Leite
a Department of Pharmaceutical Sciences, Health Sciences Center, Federal University of Pernambuco, 50740-520, Recife, PE, Brazil
b Laboratory of Prospecting Bioactive Molecules, University of Pernambuco, 56328-903, Petrolina, PE, Brazil
c Department of Immunology, Laboratory of Immunopathology and Molecular Biology, IAM / FIOCRUZ, 50740-465, Recife, PE, Brazil
d Department of Biochemistry and Immunology, Institute of Biological Sciences, Federal University of Minas Gerais, 31270-901, Belo Horizonte, MG, Brazil
e Laboratory of Immunopathology Keizo Asami-LIKA / UFPE, 50670-420, Recife, PE, Brazil
f Department of Parasitology, Cellular and Molecular Biology Laboratories, Leishmaniasis, and Mutagenesis, IAM / FIOCRUZ, 50740-465, Recife, PE, Brazil
A R T I C L E I N F O
A B S T R A C T
Keywords:
Chagas disease causes more deaths in the Americas than any other parasitic disease. Initially confined to the
American continent, it is increasingly becoming a global health problem. In fact, it is considered to be an “exotic”
disease in Europe, being virtually undiagnosed. Benznidazole, the only drug approved for treatment, effectively
treats acute-stage Chagas disease, but its effectiveness for treating indeterminate and chronic stages remains
uncertain. Previously, our research group demonstrated that 4-thiazolidinones presented anti-T. cruzi activity
including in the in vivo assays in mice, making this fragment appealing for drug development. The present work
reports the synthesis and anti-T. cruzi activities of a novel series of 4-thiazolidinones derivatives that resulted in
an increased anti-T. cruzi activity in comparison to thiosemicarbazones intermediates. Compounds 2c, 2e, and 3a
showed potent inhibition of the trypomastigote form of the parasite at low cytotoxicity concentrations in mouse
splenocytes. Besides, all the 2c, 2e, and 3a tested concentrations showed no cytotoxic activity on macrophages
cell viability. When macrophages were submitted to T. cruzi infection and treated with 2c and 3a, compounds
reduced the release of trypomastigote forms. Results also showed that the increased trypanocidal activity
induced by 2c and 3a is independent of nitric oxide release. Flow cytometry assay showed that compound 2e was
able to induce necrosis and apoptosis in trypomastigotes. Parasites treated with the compounds 2e, 3a, and 3c
presented flagellum shortening, retraction and curvature of the parasite body, and extravasation of the internal
content. Together, these data revealed a novel series of 4-thiazolidinones fragment-based compounds with po-
tential effects against T. cruzi and lead-like characteristics.
Chagas disease
Trypanosoma cruzi
Trypanocidal activity
Thiazolidinones
1. Introduction
humans is a triatomine bug, also known as a “kissing bug”. 8 million
people are estimated to be infected worldwide, mainly in Latin America
Chagas disease, also known as American trypanosomiasis, is one of
the main neglected diseases worldwide and a potentially life-threatening
illness caused by the protozoan parasite Trypanosoma cruzi (T. cruzi) [1].
It is found mainly in 21 Latin American countries, where it is primarily
vector-born. The primary vector involved in transmitting the parasite to
[1,2]. Other means of transmission involve transfusion of contaminated
blood and infected mothers to children during pregnancy or delivery
(congenital transmission). Less frequently, organ transplantation or
laboratory accidents can also result in transmission [3]. Population
migration and tourism have distributed the disease into the United
* Corresponding author. laboratory of planning in medicinal chemistry - lpqm, Federal University Of Pernambuco, Health Sciences Center, Department Of
´
Pharmaceutical Sciences, Arthur de Sa, S / N. University City. CEP, 50740-520, Recife, PE, Brazil.
E-mail address: ana.lleite@ufpe.br (A.C. Lima Leite).
https://doi.org/10.1016/j.cbi.2021.109514
Received 22 December 2020; Received in revised form 28 April 2021; Accepted 7 May 2021
Available online 21 May 2021
0009-2797/© 2021 Elsevier B.V. All rights reserved.

G. Bezerra de Oliveira Filho et al.
Chemico-Biological Interactions 345 (2021) 109514
Fig. 1. Anti-T. cruzi agents based in 4-thiazolidinones (IC50 for trypomastigote form and CC50 for macrophages).
Fig. 2. Bioisosterism-based design of novel thiazolidinones as potential antiparasitic compounds. Thiazolidinones are described over the years, and thiazolidinones
proposed here.
States, Europe, and Asia, which poses a growing risk to public health.
Globalization and increased international travel and trade between
endemic and non-endemic countries make Chagas disease a growing
concern globally [1,3–5].
treatment. From more than 1200 new drugs discovered between 1975
and 1997, only 4 resulted from a directed research and development
effort by the pharmaceutical industry on treating human tropical dis-
ease, and 1 of those 4 (nifurtimox) is no longer in widespread use [7,8].
The urgent need for new therapeutic approaches is being met by a
combined effort from the academic and commercial sectors, together
with significant input from not-for-profit drug development consortia.
With the disappointing outcomes of recent clinical trials against chronic
Chagas disease, it has become clear that an incomplete understanding of
parasite biology and disease pathogenesis impacts the development of
more effective drugs. Also, technical issues, including difficulties in
The only drugs accepted for clinical use are the two nitroheterocyclic
compounds, Benznidazole (BDZ) and Nifurtimox (NFX), which are
inadequate due to toxicity and low cure rates during the chronic stage of
the disease. The lack of pharmaceutical company interest in developing
anti-T. cruzi drugs make Chagas one of the major “neglected” diseases of
the world [6,7]. The toxicity of these drugs is linked to their mode of
action, and multiple undesired side effects inevitably accompany the
2

G. Bezerra de Oliveira Filho et al.
Chemico-Biological Interactions 345 (2021) 109514
establishing parasitological cures in human and animal models, have
complicated drug efficacy assessments [9,10]. Therefore, it is of pri-
mordial importance the search for new compounds more active and
selective against T. cruzi.
for 2 h. After cooling back to room temperature, the precipitate was
permeated in a Büchner funnel with a sintered disc filter, washed with
cold ethanol, and dried over SiO2. The product was purified by recrys-
tallization in hot toluol. Colorless crystals, m.p.: 230–232 ◦C; yield: 1.83
g (69%); IR (KBr): 3386 and 3286 (NH), 3172 (C–H, Ar), 3030 (C–H),
Over the years, our research group has investigated small molecules
based on hydrazones (thiosemicarbazones, 4-thiazolidinones, and 1,3-
thiazoles) to obtain novel and potent anti-T. cruzi agents [11–19]. In
this way, 4-thiazolidinones derivatives have been largely investigated as
anti-T. cruzi agents [13,18,19]. In recent work, we employed bio-
isosterism to modify a potent antiparasitic and cruzain-inhibitor
dichlorinated phenyl-thiosemicarbazone (1) in a new series of 4-thiazo-
lidinones (Fig. 1).
1602 (C N) cmꢀ 1. 1H NMR (300 MHz, DMSO‑d6): δ 2.18 (s, 3H, CH3),
–
–
7.29 (d, 1H, Ar), 7.41 (d, 1H, Ar), 7.72 (s, 1H, NH2), 8.17 (s, 1H, NH),
10.44 (s, 1H, NH2). 13C NMR (75.5 MHz, DMSO‑d6): δ 18.26 (CH3),
127.95 (CH, Ar), 129.50 (CH, Ar), 130.54 (C, Ar), 131.10 (C, Ar), 132.87
–
–
–
(C, Ar), 136.40 (C, Ar), 147.40 (C N), 179.31 (C S). Anal. calcd for
–
C9H8Cl3N3S: C, 36.44; H, 2.72; N, 14.17; S, 10.81. Found: C, 36,16; H,
3,01; N, 14,09; S, 10,28.
The antiparasitic activity on Y strain trypomastigotes form and host
cell cytotoxicity in J774 macrophages revealed that compounds are
more potent (3–5) and more selective (2–5) antiparasitic agents than
thiosemicarbazone (1). In infected macrophages, compounds (3–5)
reduced intracellular amastigotes, whereas Benznidazole did not. In
T. cruzi-infected mice treated orally with 100 mg/kg of compound (5), a
decrease of parasitemia was observed. These compounds present a
dichlorinated phenyl ring as a structural feature, and the studies of the
structure-activity relationship identified that the 3,4-dichlorophenyl
moiety is a structural determinant for trypanocidal activity [13,20]. In
fact, over the years, our research group has described generations of
4-thiazolidinones with promising anti-T. cruzi activity, including the in
vivo assays in mice [13,19,21].
1-(2,5-dichlorophenyl)ethylidenethiosemicarbazone (2). Recrystal-
lization in hot toluol afforded colorless crystal, m.p.: 160 ◦C; yield: 1.77
g (64%); IR (KBr): 3441 and 3216 (NH), 3155 (C–H, Ar), 3020 (C–H),
1601 (C N) cmꢀ 1. 1H NMR (300 MHz, DMSO‑d6): δ 2.19 (s, 3H, CH3),
–
–
7.44 (d, 1H, Ar), 7.47 (d, 1H, Ar), 7.53 (s, 1H, Ar), 8.30 (s, 1H, NH), 9.50
(s, 1H, NH), 10.40 (s, 1H, NH2). 13C NMR (75.5 MHz, DMSO‑d6): δ 18.27
(CH3), 129.84 (CH, Ar), 129.98 (CH, Ar), 130.35 (CH, Ar), 131.35 (C,
–
–
–
–
Ar), 135.15 (C, Ar), 140.17 (C, Ar), 147.40 (C N), 179.31 (C S).
HRMS (ESI): 262.0520 [M+H]+. Anal. calcd for C9H9Cl2N3S: C, 41.23;
H, 3.46; N, 16.03; S, 12.23. Found: C, 41.52; H, 2.95; N, 16.17; S, 12.28.
1-(2,4-dichlorophenyl)ethylidenethiosemicarbazone (3). Recrystal-
lization in hot toluol afforded colorless crystal, m.p.: 210 ◦C; yield: 2.48
g (90%); IR (KBr): 3326 and 3286 (NH), 3172 (C–H, Ar), 3022 (C–H),
1601 (C N) cmꢀ 1. 1H NMR (300 MHz, DMSO‑d6): δ 2.28 (s, 3H, CH3),
–
–
Based on this, in the present work, we have developed a new gen-
eration of 4-thiazolidinones, having as main structural characteristics:
maintenance of thiazolidinone heterocycle, a di or tri-chlorophenyl-
ethylidenehydrazone, and H or alkyl substituents in C5 position of
thiazolidinone ring (Fig. 2). Compounds (1a-e, 2a-e, and 3a-e) were
assayed for their in vitro anti-T. cruzi activity against trypomastigote and
epimastigote forms. Their cytotoxicity in mammalian cell cultures was
also investigated. The most active compounds were also evaluated
against the intracellular form of the parasite, and the cell viability and
confocal microscopy studies were also undertaken.
7.63 (d, 1H, Ar), 7.89 (dd, 1H, Ar), 8.23 (d, 1H, H–Ar), 8.60 (s, 1H, NH),
8.62 (s, 1H, NH), 10.33 (s, 1H, NH2). 13C NMR (75.5 MHz, DMSO‑d6): δ
13.85 (CH3), 126.75 (CH, Ar), 128.14 (CH, Ar), 130.24 (CH, Ar), 131.35
–
–
–
–
(C, Ar), 131.56 (C, Ar), 138.32 (C, Ar), 145.03 (C N), 178.66 (C S).
Anal. calcd for C9H9Cl2N3S: C, 41.23; H, 3.46; N, 16.03; S, 12.23. Found:
C, 41.68; H, 2.58; N, 16.21; S, 11.69.
2.1.2. General procedure for the synthesis of thiazolidinones (1a-3e)
Example for compound (1a). Synthesis of 2-[1-(2,3,4-trichloro-
phenyl)ethylidenohydrazone]thiazolidin-4-one (1a). In
a 100 mL
round-bottom flask, 1.7 mmol (0.5 g) of thiosemicarbazone (1) was
dissolved in 30 mL of ethanol, followed by adding 6.8 mmol (0.557 g)
anhydrous sodium acetate under magnetic stirring and warming. After
15 min, 3.4 mmol (0.567 g) of ethyl 2-bromoacetate was added in
portions, and the mixture was maintained under reflux for 24 h. After
cooling back to rt, the precipitate was filtered in a Büchner funnel with a
sintered disc filter, washed with cold ethanol and water, and dried over
SiO2. The product was purified by recrystallization in hot cyclohexane.
2. Materials and methods
2.1. Chemistry
All reagents were used as purchased from commercial sources
(Sigma-Aldrich, Vetec, or Fluka). The progress of the reactions was
followed by thin-layer chromatography (silica gel 60 F254 in aluminum
foil). The 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 pellets. For NMR, we used
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) in-
struments. DMSO‑d6, acetone-d6, and D2O were purchased from CIL.
Chemical shifts are reported in ppm, and multiplicities are given as s
(singlet), d (doublet), t (triplet), q (quartet), m (multiplet), and dd
(double doublet), with 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, and 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 (QUIMIS) were
carried out under a frequency of 40 kHz (180 W) and without external
heating.
Colorless crystals were obtained. M.p: 210 ◦C; yield: 0.31 g (54%); IR
ꢀ 1
(KBr): 3117 (NH), 2968 (C–H), 1715 (C O), 1637 (C N) cm
.
1H
–
–
–
–
NMR (300 MHz, DMSO‑d6): δ 2.29 (s, 3H, CH3), 3.83 (s, 2H, S–CH2,
heterocycle), 7.41 (d, 1H, Ar), 7.62 (d, 1H, Ar), 11.99 (s, 1H, NH). 13
C
NMR and DEPT (75.5 MHz, DMSO‑d6): 18.67 (CH3), 32.7 (CH2, het-
erocycle), 127.95 e (CH, Ar), 128.94 e (CH, Ar), 129.50 (C, Ar), 131.11
–
–
–
–
(C, Ar), 133.00 (C, Ar), 135.40 (C, Ar), 160.64 (C N), 166.31 (S–C N),
–
–
174.18 (C O). Anal. calcd for C11H8Cl3N3OS: C, 39.25; H, 2.40; N,
12.48; S, 9.52. Found: C, 39.06; H, 2.47; N, 12.54; S, 9.11.
2-[1-(2,3,4-trichlorophenyl)ethylidenohydrazone]-5-methyl-
thiazolidin-4-one (1b). Recrystallization in toluol \ cyclohexane 2:1
afforded colorless crystals. M.p: 210 ◦C; yield: 0.29 g (49%); IR (KBr):
3153 (NH), 2963 (C–H), 1744 (C O), 1623 (C N) cmꢀ 1. 1H NMR (300
–
–
–
–
MHz, DMSO‑d6): δ 1.55 (d, 3H, CH3 heterocycle), 2.33 (s, 3H, CH3), 4.10
(q, 1H, CH heterocycle), 4.51 (s, NH), 7.40 (d, 1H,Ar), 7.61 (d, 1H,Ar).
13C NMR (75.5 MHz, DMSO‑d6): δ 19.36 (CH3 heterocycle), 23.01 (CH3),
43.21 (CH, heterocycle), 129.60 (CH, Ar), 130.98(CH, Ar), 132.65 (C,
2.1.1. General procedure for the synthesis of thiosemicarbazones (1–3)
Example for compound (1): Synthesis of 1-(2,3,4-trichlorophenyl)-
ethylidenethiosemicarbazone (1). Under ultrasound irradiation: in a
100 mL round-bottom flask, 8.95 mmol (0.815 g) of thiosemicarbazide,
8.95 mmol (2.0 g) of 2′,3′,4′-trichloroacetophenone, 7 drops of H2SO4,
and 30 mL of ethanol were added and maintained in an ultrasound bath
–
Ar), 134.37 (C, Ar), 135.15 (C, Ar), 140.17 (C, Ar), 161.71 (C N),
–
+
–
–
–
–
179.31 (S–C N), 175.01 (C O). HRMS (ESI): 350.0332 [M+H] . Anal.
calcd for C12H10Cl3N3OS: C, 41.11; H, 2.87; N, 11.78; S, 9.14. Found: C,
41.46; H, 3.13; N, 12.16; S, 9.58.
2-[1-(2,3,4-trichlorophenyl)ethylidenohydrazone]-5-
3

G. Bezerra de Oliveira Filho et al.
Chemico-Biological Interactions 345 (2021) 109514
ethylthiazolidin-4-one (1c). Recrystallization in hot toluol afforded
2.26 (s, 3H, CH3), 3.53 (dd, 1H, CH heterocycle), 7.22 (d, 1H, Ar), 7.46
(d, 1H, Ar), 7.53 (s, 1H, Ar), 8.30 (s, 1H, NH). 13C NMR (75.5 MHz,
DMSO‑d6): δ 11.27 (CH3), 16.98 (CH3), 27.45 (CH2), 51.14 (CH, het-
erocycle), 128.16 (CH, Ar), 129.33 (CH, Ar), 129.89 (CH, Ar), 131.46 (C,
colourless crystals. M.p: 183 ◦C; yield: 0.35 g (57%); IR (KBr): 3149
(NH), 2974 (C–H), 1717 (C O), 1627 (C N) cmꢀ 1. 1H NMR (300 MHz,
–
–
–
–
DMSO‑d6): δ 2.20 (t, 3H, CH3), 2.20 (m, 1H, CH2), 2.52 (s, 3H, CH3),
3.83 (dd, 1H, CH heterocycle), 7.58 (d, 1H, Ar), 7.81 (d, 1H, Ar), 10.40
(s, 1H, NH). 13C NMR (75.5 MHz, DMSO‑d6): δ 10.88 (CH3), 18.87
(CH3), 26.88 (CH2), 50.20 (CH, heterocycle), 129.63 (CH, Ar), 130.02
(CH, Ar), 132.67 (C, Ar), 133.77 (C, Ar), 134.43 (C, Ar), 141.10 (C, Ar),
–
–
–
Ar), 131.45 (C, Ar), 141.17 (C, Ar), 155.87 (C N), 172.22 (S–C N),
–
–
182.4 (C O). Anal. calcd for C13H13Cl2N3OS: C, 47.28; H, 3.97; N,
–
12.72; S, 9.71. Found: C, 47.11; H, 3.77; N, 12.88; S, 9.51.
2-[1-(2,5-dichlorophenyl)ethylidenohydrazone]-5-iso-
–
–
–
–
162.31 (C N), 164.40 (S–C S), 176.12 (C O). HRMS (ESI): 364.0919
propylthiazolidin-4-one (2d). Recrystallization in hot toluol/cyclo-
–
–
[M+H]+. Anal. calcd for C13H12Cl3N3OS: C, 42.82; H, 3.32; N, 11.52; S,
8.79. Found: C, 42.56; H, 3.23; N, 11.36; S, 8.83.
hexane 2:1 afforded colorless crystals. M.p: 190 ◦C; yield: 0.30 g (45%);
ꢀ 1
–
–
–
–
IR (KBr): 3418 (NH), 1703 (C O), 1645 (C N) cm
.
1H NMR (300
2-[1-(2,3,4-trichlorophenyl)ethylidenohydrazone]-5-iso-
MHz, DMSO‑d6): δ 0.87 (d, 3H, CH3), 0.95 (d, 3H, CH3), 2.27 (s, 3H,
CH3), 3.41 (m, 1H, CH), 4.57 (d, 1H, CH heterocycle), 7.40 (d, 1H, Ar),
7.71 (d, 1H, Ar), 7.88 (s, 1H, Ar), 12.09 (s,1H, NH). 13C NMR (75.5 MHz,
DMSO‑d6): δ 15.33 (CH3), 17.69 (CH3), 21.27 (CH3), 27.78 (CH), 53.66
(CH, heterocycle), 128.31 (CH, Ar), 129.03 (CH, Ar), 130.99 (C, Ar),
propylthiazolidin-4-one (1d). Recrystallization in hot toluol afforded
colorless crystals. M.p: 232 ◦C; yield: 0.51 g (80%); IR (KBr): 3141 (NH),
ꢀ 1
.
1H NMR (300 MHz,
–
–
–
–
2964 (C–H), 1716 (C O), 1631 (C N) cm
DMSO‑d6): δ 0.85 (d, 3H, CH3), 0.95 (d, 3H, CH3), 2.35 (s, 3H, CH3),
3.31 (m, 1H, CH), 4.27 (d, 1H, CH heterocycle), 7.43 (d, 1H, Ar), 7.69 (d,
1H, Ar), 12.02 (s,1H, NH). 13C NMR (75.5 MHz, DMSO‑d6): δ 16.36
(CH3), 18.69 (CH3), 20.29 (CH3), 29.78 (CH), 54.65 (CH, heterocycle),
127.31 (CH, Ar), 129.03 (CH, Ar), 130.73 (C, Ar), 131.12 (C, Ar), 133.04
–
132.12 (C, Ar), 133.04 (C, Ar), 135.15 (C, Ar), 162.12 (C N), 165.06
–
–
–
–
–
(S–C N), 175.35 (C O). Anal. calcd for C14H15Cl2N3OS: C, 48.85; H,
4.39; N, 12.21; S, 9.31. Found: C, 48.44; H, 4.18; N, 12.57; S, 9.38.
2-[1-(2,5-dichlorophenyl)ethylidenohydrazone]-5-phenyl-
–
–
–
–
(C, Ar), 135.15 (C, Ar), 161.12 (C N), 164.06 (S–C N), 175.35 (C O).
thiazolidin-4-one (2e). Recrystallization in hot toluol afforded colorless
crystals. M.p: 160 ◦C; yield: 0.36 g (50%); IR (KBr): 3275 (NH), 2998
–
–
HRMS (ESI): 378.0726 [M+H]+. Anal. calcd for C14H14Cl3N3OS: C,
44.40; H, 3.73; N, 11.10; S, 8.47. Found: C, 44.25; H, 3.63; N, 10.77; S,
8.04.
(C–H), 1750 (C O), 1602 (C N) cmꢀ 1. 1H NMR (300 MHz, DMSO‑d6):
–
–
–
–
δ 2.27 (s, 3H, CH3), 4.07 (s, NH), 4.75 (s, 1H, CH heterocycle), 7.33 (m,
5H, Ar), 7.40 (d, 1H, Ar), 7.71 (d, 1H, Ar), 7.88 (s, 1H, Ar). 13C NMR
(75.5 MHz, DMSO‑d6): δ 18.33 (CH3), 54.85 (CH, heterocycle), 126.71
(CH, Ar), 128.62 (CH, Ar), 128.88 (CH, Ar), 128.99 (C, Ar), 129.92 (CH,
Ar), 129.97 (CH, Ar), 131.45 (CH, Ar), 141.41 (C, Ar), 141.98 (C, Ar),
2-[1-(2,3,4-trichlorophenyl)ethylidenohydrazone]-5-phenyl-
thiazolidin-4-one (1e). Recrystallization in hot toluol afforded colorless
crystals. M.p: 202 ◦C; yield: 0.45 g (64%); IR (KBr): 3428 (NH), 1711
(C O), 1625 (C N) cmꢀ 1. 1H NMR (300 MHz, DMSO‑d6): δ 2.32 (s, 3H,
–
–
–
–
–
–
–
–
CH3), 3.45 (s, NH), 5.31 (s, 1H, CH heterocycle), 7.33 (m, 5H, Ar), 7.41
(d, 1H,Ar), 7.70 (d, 1H,Ar). 2.41 (s, 3H, CH3), 5.49 (s, 1H, CH hetero-
cycle), 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): δ 21.07 (CH3), 52.16 (CH, heterocycle), 127.71
(CH, Ar), 128.62 (CH, Ar), 129.12 (CH, Ar), 130.34 (C, Ar), 131.17 (CH,
Ar), 132.19 (CH, Ar), 132.77 (CH, Ar), 137.41 (C, Ar), 137.98 (C, Ar),
142.97(C, Ar), 152.68 (C N), 172.35 (S–C N), 179.88 (C O). HRMS
– –
(ESI): 376.3217 [M+H]+. Anal. calcd for C17H13Cl2N3OS: C, 53.98; H,
3.46; N, 11.11; S, 8.48. Found: C, 53.88; H, 3.10; N, 11.34; S, 8.20.
2-[1-(2,4-dichlorophenyl)ethylidenohydrazone]thiazolidin-4-one
(3a). Recrystallization in hot cyclohexane afforded colorless. M.p: 203
◦
–
–
C; yield: 0.35 g (62%); IR (KBr): 3171 (NH), 2950 (C–H), 1721 (C O),
1633 (C N) cmꢀ 1. 1H NMR (300 MHz, DMSO‑d6): δ 2.43 (s, 3H, CH3),
–
–
–
–
–
–
139.97(C, Ar), 158.12 (C N), 171.99 (S–C N), 177.87 (C O). HRMS
3.95 (s, 2H, S–CH2, heterocycle), 7.72 (d, 1H, Ar), 7.83 (dd, 1H, Ar),
8.03 (d, 1H, Ar). 13C NMR and DEPT (75.5 MHz, DMSO‑d6): 14.24
(CH3), 32.01 (CH2, heterocycle) 126.35 (CH, Ar), 127.91 (CH, Ar),
130.54 (CH, Ar), 131.26 (C, Ar), 132.31 (C, Ar), 138.18 (C, Ar), 159.42
–
–
(ESI): 412.0927 [M+H]+. Anal. calcd for C17H12Cl3N3OS: C, 49.47; H,
2.93; N, 10.18; S, 7.77. Found: C, 49.68; H, 3.16; N, 10.01; S, 7.54.
2-[1-(2,5-dichlorophenyl)ethylidenohydrazone]thiazolidin-4-one
(2a). Recrystallization in hot toluol/cyclohexane 1:1 afforded colorless.
M.p: 180 ◦C; yield: 0.4 g (70%); IR (KBr): 3415 (NH), 2915 (C–H), 1715
–
–
–
–
–
–
(C N), 164.53 (S–C N), 172.04 (C O). Anal. calcd for C11H9Cl2N3OS:
C, 43.72; H, 3.00; N, 13.91; S, 10.61. Found: C, 43.33; H, 3.78; N, 13.44;
(C O), 1631 (C N) cmꢀ 1. 1H NMR (300 MHz, DMSO‑d6): δ 2.30 (s, 3H,
S, 10.78.
–
–
–
–
CH3), 3.85 (s, 2H, S–CH2, heterocycle), 7.49 (d, 1H, Ar), 7.52 (d, 1H, Ar),
7.57 (s, 1H, Ar) 11.98 (s,1H, NH). 13C NMR and DEPT (75.5 MHz,
DMSO‑d6): 18.74 (CH3), 33.35 (CH2, heterocycle), 128.61 (CH, Ar),
129.85 (CH, Ar), 129.98 (CH, Ar), 131.37 (C, Ar), 131.81 (C, Ar), 147.42
2-[1-(2,4-dichlorophenyl)ethylidenohydrazone]-5-methyl-
thiazolidin-4-one (3b). Recrystallization in hot toluol/cyclohexane 2:1
afforded colorless crystals. M.p: 170 ◦C; yield: 0.31 g (51%); IR (KBr):
ꢀ 1
–
–
–
–
3141 (NH), 1741 (C O), 1627 (C N) cm
.
1H NMR (300 MHz,
–
–
–
–
–
–
(C, Ar), 152.40 (C N), 172.31 (S–C N), 179.7 (C O). HRMS (ESI):
DMSO‑d6): δ 1.38 (d, 3H, CH3), 2.25 (s, 3H, CH3), 3.81 (q, 1H, CH
heterocycle), 4.20 (s, 1H, NH), 7.40 (d, 1H, Ar), 7.48 (d, 1H, Ar), 7.64 (s,
1H, Ar). 13C NMR (75.5 MHz, DMSO‑d6): δ 18.54 (CH3), 21.47 (CH3),
43.59 (CH heterocycle), 127.30 (CH, Ar), 129.16(CH, Ar), 131.67 (CH,
302.0331 [M+H]+. Anal. calcd for C11H9Cl2N3OS: C, 43.72; H, 3.00; N,
13.91; S, 10.61. Found: C, 43.53; H, 3.28; N, 13.64; S, 10.58.
2-[1-(2,5-dichlorophenyl)ethylidenohydrazone]-5-methyl-
–
thiazolidin-4-one (2b). Recrystallization in hot cyclohexane afforded
Ar), 132.12 (C, Ar),133.36 (C, Ar), 138.32(C, Ar), 156.26 (C N),
–
colorless crystals. M.p: 182 ◦C; yield: 0.36 g (60%); IR (KBr): 3141 (NH),
172.21 (S–C N), 183.08 (C O). Anal. calcd for C12H11Cl2N3OS: C,
–
–
–
–
ꢀ 1
–
–
–
–
2968 (C–H), 1716 (C O), 1630 (C N) cm
.
1H NMR (300 MHz,
45.58; H, 3.51; N, 13.29; S, 10.14. Found: C, 45.32; H, 3.17; N, 13.02; S,
10.47.
DMSO‑d6): δ 1.39 (d, 3H, CH3 heterocycle), 1.88 (s, 3H, CH3), 3.81 (q,
CH heterocycle), 7.44 (d, 1H, Ar), 7.51 (d, 1H, Ar), 7.60 (s, 1H, Ar), 9.50
(s, 1H, NH). 13C NMR (75.5 MHz, DMSO‑d6): δ 18.42 (CH3 heterocycle),
21.40 (CH3), 43.70 (CH heterocycle), 129.34 (CH, Ar), 129.80(CH, Ar),
129.88 (CH, Ar), 131.49 (C, Ar),131.56 (C, Ar), 141.04(C, Ar), 155.78
2-[1-(2,5-dichlorophenyl)ethylidenohydrazone]-5-ethylthiazolidin-
4-one (3c). Recrystallization in hot toluol afforded colorless crystals. M.
p: 120 ◦C; yield: 0.30 g (47%); IR (KBr): 3155 (NH), 2964 (C–H), 1726
(C O), 1625 (C N) cmꢀ 1. 1H NMR (300 MHz, DMSO‑d6): δ 0.92 (t, 3H,
–
–
–
–
–
–
–
–
(C N), 172.16 (S–C N), 183.54 (C O). Anal. calcd for
CH3), 1.80 (m, 1H, CH2), 1.94 (m, 1H, CH2), 2.23 (s, 3H, CH3), 4.16 (dd,
1H, CH heterocycle), 7.30 (d, 1H, Ar), 7.46 (d, 1H, Ar), 7.68 (s, 1H, Ar),
11.94 (s, 1H, NH). 13C NMR (75.5 MHz, DMSO‑d6): δ 10.35 (CH3), 18.73
(CH3), 25.45 (CH2), 48.75 (CH, heterocycle) 127.84 (CH, Ar), 128.98
(CH, Ar), 130.05 (CH, Ar), 131.75 (C, Ar), 135.15 (C, Ar), 140.17 (C, Ar),
–
–
C
12H11Cl2N3OS: C, 45.58; H, 3.51; N, 13.29; S, 10.14. Found: C, 45.12;
H, 3.57; N, 13.49; S, 10.35.
2-[1-(2,5-dichlorophenyl)ethylidenohydrazone]-5-ethylthiazolidin-
4-one (2c). Recrystallization in hot toluol/cyclohexane 2:1 afforded
colorless crystals. M.p: 175 ◦C; yield: 0.55 g (87%); IR (KBr): 3126 (NH),
159.06 (C N), 163.81 (S–C N), 175.92 (C O). Anal. calcd for
–
–
–
–
–
–
ꢀ 1
–
–
–
2961 (C–H), 1709 (C O), 1601 (C N) cm
.
1H NMR (300 MHz,
C
13H13Cl2N3OS: C, 47.28; H, 3.97; N, 12.72; S, 9.71. Found: C, 47.02; H,
–
DMSO‑d6): δ 0.89 (t, 3H, CH3), 1.63 (m, 1H, CH2), 1.91 (m, 1H, CH2),
3.98; N, 12.62; S, 9.32.
4

G. Bezerra de Oliveira Filho et al.
Chemico-Biological Interactions 345 (2021) 109514
2-[1-(2,4-dichlorophenyl)ethylidenohydrazone]-5-iso-
2.2.4. Macrophage culture, T. cruzi infection, MTT and nitric oxide (NO)
evaluation
propylthiazolidin-4-one (3d). Recrystallization in hot toluol/cyclo-
hexane 2:1 afforded colorless crystals. M.p: 190 ◦C; yield: 0.25 g (38%);
Macrophages (MO) were harvested from the peritoneal cavity of
eight to ten weeks old female C57BL/6 mice three days after injection of
2 ml of 3% sodium thioglycolate. The cells were collected by washing
the peritoneal cavity using sterile phosphate-buffered saline (PBS),
centrifuged at 290 xg for 10 minutes, resuspended in a volume of 1mL of
medium (RPMIc). The cells were counted in Neubauer’s chamber and
cultured (2x106 cells/well) in RPMIc. The adherent cells were obtained
after 3 hours of incubation of single-cell suspensions in tissue culture
plates (24 wells) at 37 o C in 5%. Nonadherent cells were removed and
trypomastigotes forms were added at a 5:1 parasite-to-cells ratio to
macrophages for 2 hours after the extracellular parasites were removed,
and the cells were incubated at 37 o C in 5% CO2 in the presence or
ꢀ 1
–
–
–
–
IR (KBr): 3420 (NH), 1752 (C O), 1640 (C N) cm
.
1H NMR (300
MHz, DMSO‑d6): δ 0.88 (d, 3H, CH3), 0.97 (d, 3H, CH3), 2.33 (s, 3H,
CH3), 3.51 (m, 1H, CH), 5.07 (d, 1H, CH heterocycle), 7.50 (d, 1H, Ar),
7.81 (d, 1H, Ar), 7.98 (s, 1H, Ar), 8.2 (s,1H, NH). 13C NMR (75.5 MHz,
DMSO‑d6): δ 16.33 (CH3), 18.29 (CH3), 22.87 (CH3), 27.78 (CH), 86.08
(CH, heterocycle), 127.31 (CH, Ar), 129.03 (CH, Ar), 130.08 (C, Ar),
–
132.13 (C, Ar), 133.04 (C, Ar), 135.15 (C, Ar), 160.12 (C N), 164.06
–
+
–
–
–
(S–C N), 176.35 (C O). HRMS (ESI): 376.0030 [M ꢀ H] . Anal. calcd
–
for C14H15Cl2N3OS: C, 48.85; H, 4.39; N, 12.21; S, 9.31. Found: C, 48.22;
H, 4.08; N, 12.77; S, 9.38.
2-[1-(2,4-dichlorophenyl)ethylidenohydrazone]-5-phenyl-
thiazolidin-4-one (3e). Recrystallization in hot toluol afforded colour-
less crystals. MP: 170 ◦C; yield: 0.32 g (45%); IR (KBr): 3141 (NH), 1920
absence of 2c, 2e, and 3a (3.125, 6.25, 12.5, 25 and 50 μM) or with
recombinant murine IFN-γ (100 ng/mL - Sigma). They were maintained
throughout the remainder of the oven experiment at 37◦C, 5% CO2 . The
supernatants were harvested 72 hours or 7 days post-infection (p.i.)
and/or stimuli and assayed for nitrite concentration (72 h p.i.) by mixing
0.1 mL of culture supernatant with 0.1 mL of Griess reagent. Absorbance
at 540 nm was read 10 minutes later, and NO2 - concentration was
determined. The MTT was evaluated by adding methanesulfonyl and
methane dimethyl (oxide) sulfur at a 2mg/mL MTT solution to super-
natants (7 days p.i.) for 90 min at 37◦C. After this period, the MTT was
(C–H), 1721 (C O), 1628 (C N) cmꢀ 1. 1H NMR (300 MHz, DMSO‑d6):
–
–
–
–
2.32 (s, 3H, CH3), 5.24 (s, 1H, CH heterocycle) 7.55 (m, 5H, Ar), 7.67 (d,
1H, Ar), 7.89 (d, 1H, Ar), 8.2 (s, 1H, Ar), 9.5 (s,1H, NH). 13C NMR (75.5
MHz, DMSO‑d6): δ 19.74 (CH3), 54.50 (CH, heterocycle), 126.71 (CH,
Ar), 127.62 (CH, Ar), 129.12 (CH, Ar), 131.34 (C, Ar), 131.57 (CH, Ar),
132.19 (CH, Ar), 132.87 (CH, Ar), 137.41 (C, Ar), 137.98 (C, Ar), 139.97
–
–
–
–
(C, Ar), 152.40 (C N), 168.31 (S–C N), 172.07 (C O). Anal. calcd for
–
–
C
17H13Cl2N3OS: C, 53.98; H, 3.46; N, 11.11; S, 8.48. Found: C, 53.70; H,
3.00; N, 11.01; S, 8.78.
withdrawn from the wells, and 1.500
μ
L of dimethyl sulfoxide (DMSO)
(Synth) was added. Triplicates of all samples (200
μ
L/well) were placed
2.2. Biology
in a 96-well plate, and the absorbance at 490nm was read, with MTT
concentration determined. Macrophages were infected, the extracellular
parasites were removed, and the cells were incubated with or without
2c, 2e, and 3a. Parasite growth was evaluated by daily counting the
trypomastigotes in the supernatant of infected macrophages on days 3 to
7 post-infection.
2.2.1. Parasites
Epimastigotes of a Dm28c strain were maintained at 27 ◦C in liver
infusion tryptose (LIT) medium supplemented with 10% fetal bovine
serum (FBS, Cultilab). T. cruzi trypomastigotes (Y strain) were cultured
on Macaca mulatta fibroblast cells line (LLCMK2) in culture flasks (TPP)
with Roswell Park Memorial Institute 1640 medium (RPMI) supplemented
with 5% of FBS, 1% L-glutamine (200 mM), and 1% penicillin and 1%
streptomycin.
2.2.5. Cell death assessment
After confirmation of trypanocidal activity, Annexin-FITC/
Propidium Iodide labeling was used to characterize cell death modal-
ities induced by incubation with compounds. Metacyclic trypomasti-
gotes were collected and seeded at 4x105 cells/well in RPMI-1640
medium. All compounds were dissolved in DMSO and added to wells at
IC50 and 2x IC50 concentrations. Benznidazole (IC50 and 2x IC50) and
culture medium were used as positive and negative control, respectively.
Plates were incubated at the same conditions used for anti-
trypomastigote activity (37 ◦C, 24 hours). Briefly, after treatment, par-
asites were washed with PBS and resuspended in binding buffer
(Annexin V Binding Buffer- BD Pharmingen™, USA). For labeling, 10 mL
of propidium iodide (50 mg/mL) and 5 mL of Annexin-FITC (BD Phar-
mingen™, USA) were added for 15 min, at room temperature, in dark.
Flow cytometry was conducted in FACSCalibur (Becton & Dickinson,
USA). For each sample we acquire 20,000 events and the data were
analyzed using Cell Quest software (Becton & Dickinson, USA). Assays
were conducted in triplicate. For significance analysis was used ANOVA
and Dunnett’s test, considering p < 0.05.
2.2.2. Activity against epimastigotes and trypomastigotes
Epimastigotes were counted in a hemocytometer and dispensed into
96-well plates at 105 cells/well. Trypomastigotes were counted and
adjusted to 4 × 105 cells/well into a 96-well plate. Compounds were
added in ten dilutions (0.19–100 μg/mL) in triplicate. Epimastigotes
plates were incubated for 96 h at 27 ◦C, trypomastigotes plates, for 24 h,
at 37 ◦C. In both assays, wells without compounds and treated with
Benznidazole were used as negative and positive controls, respectively.
After incubation, parasites were quantified using a Neubauer chamber.
The percentage of inhibition was calculated using untreated cultures,
and IC50 was calculated using non-linear regression on Prism 5.0
(GraphPad).
2.2.3. Cytotoxicity in mice spleen cells
Splenocytes were collected from BALB/c mice and cultivated in
RPMI- 1640 medium supplemented with 10% FBS. The cells were placed
into 96-well plates at 5 × 106 cells/well. Compounds were added in six
2.2.6. Ultrastructural analysis
dilutions (1, 5, 10, 25, 50, and 100
μ
g/mL), and wells without com-
The parasites were cultured for 24 h in RPMI 1640 medium (Sigma-
Aldrich, St. Louis, MO, USA) buffered to pH 7.5 and supplemented with
HEPES (20 mM), 10% fetal bovine serum, penicillin (100 U/mL), and
streptomycin (100 mg/mL) containing the compound at the IC50 con-
centration and twice the value of IC50. The parasites were collected,
washed in PBS, and fixed with 2.5% glutaraldehyde, 4% formaldehyde,
and 0.1 M cacodylate buffer at pH 6.8. They were then postfixed in 2%
osmium tetroxide (OsO4) in a 0.1 M cacodylate buffer at pH 6.8 and
processed for routine scanning electron microscopy (SEM). The parasites
were dehydrated in graded ethanol and dried by the critical point
method with CO2. The samples were mounted on aluminum stubs,
coated with gold, and examined under a JEOL-5600LV microscope.
pounds and with saponin were used as negative and positive controls,
respectively. [3H]-thymidine (1.0
μCi/mL, PerkinElmer, Waltham, MA,
USA) was added to each well, and plates were incubated for 24 h at 37 ◦C
and 5% CO2. Cells were then transferred to a filter paper using a cell
harvester and quantified using a liquid scintillation counter (WALLAC
1209, Rackbeta Pharmacia, Stockholm, Sweden). [3H]-Thymidine
incorporation [%] was measured, and the cytotoxic concentration
(DL50) was determined using non-linear regression on Prism 5.0
(GraphPad).The assay was conducted in triplicate.
5

G. Bezerra de Oliveira Filho et al.
Chemico-Biological Interactions 345 (2021) 109514
Scheme 1. Synthetic procedures for thiazolidinones (1a-3e). Reagents and conditions: (A) thiosemicarbazide, EtOH, H2SO4, ultrasound bath, rt, 1–2 h, yields of
64–90%; (B) ethyl 2-bromoacetate or ethyl 2-substituted-2-bromoacetates; NaOAc, EtOH, reflux, overnight, yields of 38–87%.
3. Results and discussion
(DL50= 3.48μM). In general, we observed that cyclization is beneficial
since cyclic compounds presented less cytotoxic than their non-cyclic
bioisosteres. This data corroborates with our previous studies, once
the cyclization generates less toxic and more selective compounds [13,
3.1. Synthesis and chemical characterization
The general route to preparing 4-thiazolidinones is shown in Scheme
1. First, thiosemicarbazones (1–3) were prepared by reacting commer-
cially available di- and trichloroacetophenones with the thio-
semicarbazide in an ultrasound bath in the presence of catalytic H2SO4.
The 4-thiazolidinones (1a-3e) were prepared by reacting to the
respective aryl thiosemicarbazone (1–3) with commercially available
ethyl 2-bromoacetate or by preparing [22] 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
(1a-3e) in variable yields (38–87%) and acceptable purity (>95%).
Structures of compounds were determined by nuclear magnetic
resonance (NMR-1H, and 13C), infrared (IR), and mass (HR-MS) spectra,
while purity was determined by elemental analysis (EA).
23]. The only exceptions are thiazolidinones 1e (DL50 = 24.33 μM) and
2e (DL50 = 13.26 μM), which presents a phenyl ring at C5 position
heterocyclic ring, suggesting that the lipophilic profile could influence
this cytotoxic property of these compounds.
3.3. Antiparasitic activity against extracellular form
After assessing the host cell cytotoxicity in mouse splenocytes, the in
vitro anti-T. cruzi activity was determined against trypomastigotes of Y
strain and epimastigotes of DM28c strain (Table 1). Compounds that
showed IC50 values comparable to Benznidazole were considered active.
The observed trypanocidal activity in unsubstituted thio-
semicarbazones (1–3) was kept in their cyclic derivatives. They also
presented lower cytotoxicity for splenocytes in opposition to thio-
semicarbazones (2 and 3). Among cyclic derivatives, compounds (1a,
2c, 2e, and 3a-c) present the same or better trypanocidal activity than
thiosemicarbazones (2–3). We can analyze the structure-activity re-
lations by dividing the sub-series of compounds (1a-e, 2a-e, and 3a-e).
For the sub-series (1a-e), we noted that substitutions at the C5 po-
sition of the heterocyclic ring were not beneficial for trypanocidal ac-
tivity. This data may be related to the presence of the tri-chlorinated
aromatic ring of this sub-series, which may be causing some steric hin-
drance in the target site, causing substitutions that increase the size of
the analog to render it inactive. We can highlight the 1a analog of this
sub-series, which presents the unsubstituted thiazolidinone ring at C5
An important structural feature of the compounds (1a-3e) is the
position of thedouble bond involving the C2 carbon of the heterocyclic
ring. Depending on the imino lactam tautomerism, the double bond of
the C2 carbon present in the compounds may be endocyclic or exocyclic
to the heterocycle (Fig. 3).
For an unambiguous assignment of this connection, we tried to
obtain crystals of the compounds (1a-3e) suitable for the diffraction of
X-rays, but we did not succeed. Taking account of our previous work
(2015) [13] identified by X-ray crystallography that the position of the
iminic bond in thiazolidinone heterocycle ring is located in an exocyclic
position and adopts an E-geometry; therefore, we suggest that for these
new compounds (1a-3e) this should be the accepted (Fig. 3) as observed
for the 4-thiazolidinones previous crystallized [13].
position and an IC50 = 9.20
For sub-series (2a-e), we observed that the analogs 2c (IC50 = 4.99
μM) and 2e (IC50 = 0.84 μM) were the most active of this sub-series; they
μM, equipotent to Benznidazole.
3.2. Cytotoxicity
present an ethyl and phenyl group respectively at the C5 position of the
thiazolidinone ring. We can suppose that these groups have improved
trypanocidal activity by increasing hydrophobic interactions in the
target site of the parasite. This data corroborates with our previous
studies, where the insertion of hydrophobic groups in the C5 position of
First, thiosemicarbazones 1-3 and 4-thiazolidinones 1a-3e toxicity to
splenic cells of BALB/c mice were evaluated. As observed in Table 1, the
cytotoxicity of the thiosemicarbazone 3 (2,4-dichloro) for splenic cells
was low (DL50= 3.83
μ
M) and could be compared to that of nifurtimox
–
Fig. 3. Example of the C N bond position on the C2 carbon of compound 1c.
–
6

G. Bezerra de Oliveira Filho et al.
Chemico-Biological Interactions 345 (2021) 109514
Table 1
Anti-T. cruzi activity of thiazolidinones (1a-3e).
Compd.
R1
Cytotoxicity DL50
(
μ
M)a
Trypomastigotes
Epimastigotes [DM28c] IC50
(
μ
M)c
Y strain T. cruzi IC50
(μ
M)b
1
2
3
–
–
–
84.76
38.32
3.83
41.80
2.97
3.66
29.02
6.6
24.79
1a
1b
1c
1d
1e
H
74.64
9.20
66.66
ND
CH3
71.64
65.47
34.14
24.21
30.14
CH2CH3
CH(CH3)2
C6H5
68.88
7.16
47.12
1.96
>265.26
24.33
2a
2b
2c
2d
2e
H
83.06
79.37
151.97
145.76
13.26
78.49
41.50
4.99
ND
CH3
60.33
58.74
ND
CH2CH3
CH(CH3)2
C6H5
57.83
0.84
36.21
3a
H
83.06
79.37
30.39
29.15
132.62
96.06
3.48
1.12
7.14
2.57
ND
ND
3b
CH3
60.33
3.42
3.45
6.63
6.26
ND
3c
CH2CH3
3d
CH(CH3)2
3e
C6H5
67.35
6.26
2.64
BDZ
NFX
–
–
ND = Not determined.
a
Cytotoxic concentration for 50% of mouse splenocytes culture after 24h incubation in the presence of the compounds.
b
Determined 24 h after incubation of the trypomastigote forms with the compounds. IC50 was calculated from five concentrations; IC50 values were calculated using
at least seven data point points using non-linear regression.
c
Determined 24 h after incubation of the epimastigote forms with the compounds. IC50 was calculated from five concentrations; IC50 values were calculated using at
least seven data-points using non-linear regression. BDZ: Benznidazole. NFX: Nifurtimox.
Fig. 4. Summary of the biological structure-activity relationships of the new generation of thiazolidinones.
heterocycle benefited the trypanocidal activity [13,23].
thiazolidinone ring. Although, compound 3a (IC50 = 1.12 μM), which is
For sub-series (3a-e), it was observed that the analogs 3b (IC50
=
the most active analog of this sub-series, does not show substitution at
the C5 position of the heterocyclic ring, which leads us to suggest that if
the 4-position of the aromatic ring is substituted with chlorine, the
7.14 μM) and 3c (IC50 = 2.57 μM) showed promising activity. They
present a methyl and ethyl group, respectively, at the C5 position of the
7

G. Bezerra de Oliveira Filho et al.
Chemico-Biological Interactions 345 (2021) 109514
Fig. 5. Potent trypanocidal activity of 2c and 3a on macrophages. Peritoneal macrophages of C57BL/6 mice were cultured and infected with T. cruzi trypomastigote
forms at the ratio of 5: 1 (parasite/cell) and stimulated or not, with IFN-ɣ (100 ng/ml) or 2c, 2e, 3a (3.125, 6.25, 12.5, 25 and 50 μM). Parasite growth was measured
by counting trypomastigote forms released in the supernatant at different times after infection (A-D = dose-response and D = 50 mM). Cell damage was evaluated by
measuring MTT cytotoxicity assay after 7 days of treatments with different 2c, 2e, and 3a (E–G) concentrations. Nitric Oxide was measured by Griess method in the
culture supernatant after 72 h of stimulation with 2c, 2e, 3a at the concentration of 50 mM. Data expressed as a mean ± SEM – Two-way ANOVA, post-test
Bonferroni; One-way ANOVA, post-test Tukey: ***p < 0.001, **p < 0.01, *p < 0.05.
8

G. Bezerra de Oliveira Filho et al.
Chemico-Biological Interactions 345 (2021) 109514
Fig. 6. Flow cytometry analysis for trypomastigote form of T. cruzi. Parasites were examined by flow cytometry with annexin V and PI staining. The percentage of
cells in each quadrant represents: lower left, double negative; upper left, PI single positive; lower right, annexin V single positive; upper right, PI and annexin V
double positive.
substitution at the C5 position of the heterocyclic becomes disadvanta-
geous. Fig. 4 shows a summary of the biological structure-activity
relationships.
3a (50, 25, and 12.5
μM), presented higher viability when compared
with unstimulated infected macrophages (Fig. 5D, E, and 5F). It is worth
mentioning that, among the tested compounds, the 3a at 50 μM pre-
Compounds 1a (IC50 = 9.20
7.14
compounds 2e (IC50 = 0.84
1.12
μM), 2c (IC50 = 4.99
μ
M) and 3b (IC50
=
sented higher trypanocidal activity associated (Fig. 5D) with lower
cellular toxicity (Fig. 5G).
μ
M) were equipotent to Benznidazole (IC50 = 6.26
μ
M), while
μ
M), 3c (IC50 = 2.57 M) and 3a (IC50
μ
=
Altogether, these results suggested that 2c and 3a can control the
release of T. cruzi by macrophages in vitro. The increased trypanocidal
activity induced by 2c and 3a was not related to increased nitric oxide
production since NO levels were increased only in the presence of IFN-γ
(Fig. 5H). This indicates that the trypanocidal activity of 2c and 3a is
independent of NO release.
μ
M) demonstrated greater activity than Benznidazole (IC50 = 6.26
μ
M). A highlight must be made for compound 3a (IC50 = 1.12 μM),
which has a selectivity index of 74.16, five times that of Benznidazole
(IS = 15.34). The compounds were also tested against the epimastigote
form of T. cruzi, but the results obtained did not present a significant
correlation with the data obtained on trypomastigotes.
3.5. Flow cytometry analysis for trypomastigote form of T. cruzi
3.4. Macrophage culture, T. cruzi infection, MTT and nitric oxide
evaluation
The flow cytometry analysis was released to compounds 2e, 3a, and
3c by using IC50 and twice IC50 values. It was observed that compound
2e (IC50 and 2x IC50) induced significantly labeling compatible with
necrosis and apoptosis in trypomastigotes. Similar results were found in
parasites treated with BDZ, the reference drug, and positive control used
in this assay (Fig. 6).
Macrophages are innate immune cells that play an essential role in
controlling the replication of parasites and stimulating adaptive im-
munity cells. In peritoneal macrophages submitted to T. cruzi infection
and treated with 2c and 3a, but not with 2e, in a dose-response curve
(Fig. 5A–C), it was possible to reduce the release of trypomastigote forms
relative to their control 5 days after infection at the concentration of 50
3.6. Ultrastructural studies of T. cruzi
μ
M of the 2c and 50 and 25 μM of the 3a (Fig. 5A, C, and 5D). In the cell
viability test measured by the MTT cytotoxicity assay, all the concen-
trations of 2c, 2e, and 3a tested showed no cytotoxic activity on mac-
rophages (Fig. 5E–G). Moreover, T. cruzi-infected macrophages
In order to investigate the effects of 4-thiazolidinones chlorine de-
rivatives on parasite morphology, the most active compounds of this
work were selected. Analysis by scanning electron microscopy showed
that Trypanosoma cruzi trypomastigote forms of the control group
stimulated with the concentrations of 2c (50 and 25 μM), 2e (50 μM), or
9

G. Bezerra de Oliveira Filho et al.
Chemico-Biological Interactions 345 (2021) 109514
Fig. 7. (A–H). Electron micrographs of Trypanosoma cruzi trypomastigote forms.
(Fig. 7A) showed typical morphology with long flagella and preserved
body topography. The cells treated with BDZ (Fig. 7B) presented a
shortening of the flagellum, curvature of the body with extravasation of
the internal content. The cells treated with the compounds 2e (Fig. 7C
and D), 3a (Fig. 7E and F), and 3c (Fig. 7G and H) presented morpho-
logical alterations such as flagellum shortening, retraction, and curva-
ture of the parasite body and extravasation of the internal content.
In the present study, the activity of this compound (2e, 3a and 3c)
10

G. Bezerra de Oliveira Filho et al.
Chemico-Biological Interactions 345 (2021) 109514
Table 2
Physicochemical property profile of thiazolidinone derivatives, calculated by online software SwissADME (http://www.swissadme.ch/index.php).
CPD.
MW < 500
MLog P <=4.15
H-bond donors <5
H-bond acceptors <10
Lipinski violations
#Rotable bonds <10
TPSA <=140
1
296.6
3.1
2
2
2
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
3
3
3
2
2
3
3
3
2
2
3
3
3
2
2
3
3
3
82.5
2
262.16
262.16
336.62
350.65
364.68
378.7
2.55
2.55
3.12
3.38
3.63
3.87
4.34
2.59
2.86
3.11
3.36
3.84
2.59
2.86
3.11
3.36
3.84
82.5
3
82.5
1a
1b
1c
1d
1e
2a
2b
2c
2d
2e
3a
3b
3c
3d
3e
79.12
79.12
79.12
79.12
79.12
79.12
79.12
79.12
79.12
79.12
79.12
79.12
79.12
79.12
79.12
412.72
302.18
316.21
330.23
344.26
378.28
302.18
316.21
330.23
344.26
378.28
stabilities for all compounds synthesized.
Table 3
ADME properties of most active compounds.
4. Conclusion
aBBB permeant
bGI absorption
Bioavailability Score
Compound
Three thiosemicarbazones (1–3) and fifteen thiazolidinones de-
rivatives (1a-e, 2a-e, 3a-e), based on the bioisosteric strategy, were
obtained reasonable yields using a simple methodology. The new gen-
eration of 4-thiazolidinones presents a thiazolidinone heterocycle, a di
or tri-chlorophenyl-ethylidenehydrazone, and H or alkyl substituents in
the C5 position of thiazolidinone ring. Concerning cytotoxicity in mouse
splenocytes, in general, cyclization is beneficial since cyclic compounds
presented better toxicity indices when compared to their non-cyclic
bioisosteres. The in vitro anti-T. cruzi activity against trypomastigotes
of Y strain revealed that between all series, compounds 1a, 2c and 3b
were equipotent to Benznidazole, while compounds 2e, 3a, and 3c
demonstrated higher activity than Benznidazole, with compound 3a
2c
No
No
No
No
High
High
High
High
55
55
55
55
2e
3a
BDZ
a
BBB - blood-brain barrier.
b
GI - Gastrointestinal absorption.
can be observed through ultrastructural changes caused in Trypanosoma
cruzi, which certainly makes the cell unfeasible.
(IC50 = 1.12 μM) having a selectivity index of 74.16, five times that of
Benznidazole (IS = 15.34). In peritoneal macrophages submitted to
T. cruzi infection 2c and 3a, it was possible to reduce the release of
trypomastigote forms relative to their control 5 days after infection.
When T. cruzi-infected macrophages stimulated with 2c, 2e, 3a exhibi-
ted higher viability when compared with unstimulated infected mac-
rophages. Parasites treated with the compounds 2e, 3a and 3c presented
flagellum shortening, retraction and curvature of the parasite body, and
extravasation of the internal content. All synthesized compounds are
compatible with the Lipinski and Veber rules.
3.7. Physicochemical properties
To better understand the physicochemical contributions of the syn-
thesized compounds, the online software SwissADME [24] (http
://www.swissadme.ch/index.php) was used to determine the physico-
chemical descriptors and define the pharmacokinetic properties and
drug-like nature of all compounds. Physicochemical properties were
important to determine if they agree with Lipinski’s rule [25,26].
Compounds following at least three of the four criteria are considered to
respect the Lipinski Rule [26]. As we can see in Table 2, all synthesized
compounds are compatible with the Lipinski Rule. Another attractive
property is the number of rotatable bonds and the topological polar
surface area (TPSA). A large number of rotatable bonds (≥10) have been
associated with poor oral bioavailability [26]. Compounds with a low
TPSA (≤140 Å2) tend to have higher oral bioavailability [27,28]. All
synthesized compounds present appropriate TPSA and number of
rotatable bonds, agreeing with the Veber rule.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgment
As demonstrated in Table 3, the most active compounds shown
variable permeability based on gastrointestinal absorption (GI), ac-
cording to the BOILED-Egg predictive model (Brain Or IntestinaL Esti-
mateD permeation method). The toxicity result of the drug from
SwissADME shows it to be very soluble in the body, it also has a high
Gastrointestinal Tract (GI) absorption, and the drug was not blood-brain
barrier permeant. These are important as they would allow the drug to
have high uptake. The most selective derivatives showed high gastro-
intestinal absorption (2c, 2e, and 3a). Concerning oral bioavailability, it
has expected 0.55 of the probability of oral bioavailability score >10%
in the rate for all compounds, similar to Benznidazole. All these data
suggest a good in silico drug-likeness profile and exceptional chemical
This work was funded by Conselho Nacional de Desenvolvimento
´
˜
`
ˆ
Científico e Tecnologico (CNPq) and Fundaçao de Amparo a Ciencia e
Tecnologia de Pernambuco (FACEPE). A.C.L.L. is receiving a CNPq se-
nior fellowship. G.B.O.F. received a FACEPE PhD scholarship. M.V.O.C.
is thankful to CNPq (427078/2016–4) and FACEPE (APQ-0350-4.03/
18) for financial support. The authors are thankful to the Departamento
de Química Fundamental (DQF-UFPE) for recording the 1H NMR, 13C
NMR, and IR spectra of compounds. We are also thankful to Centro de
´
Tecnologias Estrategicas do Nordeste (CETENE) for recording the MS
spectra of compounds. All authors declare no competing financial
interest.
11

G. Bezerra de Oliveira Filho et al.
Chemico-Biological Interactions 345 (2021) 109514
[15] M.V.O. Cardoso, L.R.P. Siqueira, E.B. Silva, et al., 2-Pyridyl thiazoles as novel anti-
Trypanosoma cruzi agents : structural design , synthesis and pharmacological
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Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.cbi.2021.109514.
[16] P.A.T.M. Moraes, P.A.T. Gomes, M.O. Barbosa, E.F. Santiago, et al., New 1,3-
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[17] D.R.M. Moreira, A.D.T. Oliveira, P.A.T.M. Gomes, et al., Conformational restriction
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