The design, synthesis, and: In vitro trypanocidal and leishmanicidal activities of 1,3-thiazole and 4-thiazolidinone ester derivatives

Article abstract of DOI:10.1039/d0ra06994a

Chagas and leishmaniasis are both neglected tropical diseases, whose inefficient therapies have made them remain the cause for millions of deaths worldwide. Given this, we synthesized 27 novel 1,3-thiazoles and 4-thiazolidinones using bioisosteric and est

Full text of DOI:10.1039/d0ra06994a

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The design, synthesis, and in vitro trypanocidal and
leishmanicidal activities of 1,3-thiazole and 4-
thiazolidinone ester derivatives
Cite this: RSC Adv., 2021, 11, 2487
Muhammad Haroon,a Mabilly Cox Holanda de Barros Dias,b
Aline Caroline da Silva Santos,c Valeria Rego Alves Pereira,c Luiz Alberto Barros
´
ˆ
d
Freitas,b Rodolfo Bento Balbinot,
Vanessa Kaplum,d Celso Vataru Nakamura,d
Luiz Carlos Alves,ef Fabio Andre Brayner,ef Ana Cristina Lima Leite
b
*
´
´
a
*
and Tashfeen Akhtar
Chagas and leishmaniasis are both neglected tropical diseases, whose inefficient therapies have made them
remain the cause for millions of deaths worldwide. Given this, we synthesized 27 novel 1,3-thiazoles and 4-
thiazolidinones using bioisosteric and esterification strategies to develop improved and safer drug
candidates. After an easy, rapid and low-cost synthesis with satisfactory yields, compounds were
structurally characterized. Then, in vitro assays were performed, against Leishmania infantum and
Leishmania amazonensis promastigotes, Trypanosoma cruzi trypomastigotes and amastigotes, for
selected compounds to determine IC50 and SI, with cytotoxicity on LLC-MK2 cell lines. Overall, 1,3-
thiazoles exhibited better trypanocidal activity than 4-thiazolidinones. The compound 1f, an ortho-
bromobenzylidene-substituted 1,3-thiazole (IC50 ¼ 0.83 mM), is the most potent of them all. In addition,
compounds had negligible cytotoxicity in mammalian cells (CC50 values > 50 mM). Also noteworthy is the
examination of the cell death mechanism of T. cruzi, which showed that compound 1f induced necrosis
and apoptosis in the parasite. Scanning electron microscopy analysis demonstrated that the treatment of
Trypanosoma cruzi trypomastigote cells with the compound 1f at different IC50 concentrations
promoted alterations in the shape, flagella and body surface, inducing parasite death. Together, our data
revealed a novel series of 1,3-thiazole structure-based compounds with promising activity against
Trypanosoma cruzi and Leishmania spp., broadening ways for scaffold optimization.
Received 14th August 2020
Accepted 25th October 2020
DOI: 10.1039/d0ra06994a
rsc.li/rsc-advances
(NTDs) due to various characteristics regarding their occur-
rence, to be noted poor housing conditions, lack of an adequate
Introduction
sanitary system, treatment, control scheme, malnutrition and
scarce resources for investment in research.2
Leishmaniasis consists of a group of diseases caused by the
Leishmania protozoan, a component of the Trypanosomatidae
family, which also includes Trypanosoma cruzi, the etiological
agent of chagas disease. Beyond other characteristics, they have
the presence of differentiated mitochondria called kinetoplas-
tid in common.1 They are so-called Neglected Tropical Diseases
The current therapy of chagas disease is mainly based on two
compounds, Nifurtimox (NFX) and Benznidazole (BZD), which
were developed about 40 years ago,3 and are associated with
long-term treatments, severe side effects and low access
worldwide.4,5 In fact, NFX and BZD can eliminate latent para-
sitemia and reduce serological titters in acute and early chronic
infections.6,7 However, they have low efficacy in prolonged
chronic infections.8
aDepartment of Chemistry, Mirpur University of Science and Technology (MUST),
Mirpur, Allama Iqbal Road, 10250-Mirpur, AJK, Pakistan. E-mail: tashfeenchem@
must.edu.pk
The treatment of choice for leishmaniasis has been based on
pentavalent antimonials.9 The second line treatment of chagas
disease includes drugs, such as Amphotericin B, Pentamidine
and Miltefosine.10 Most treatments are inadequate, owing to
factors like a low therapeutic index, which leads to high toxicity
and side effects, the emergence of resistant parasites, high costs
beyond the means of the affected countries and others. These
disadvantages, together with the lack of an effective vaccine,
indicate the need to investigate new drugs for NTDs.10
b
´
´
Laboratorio de Planejamento em quımica medicinal, Department of Pharmaceutical
Sciences, Health Sciences Centre, Federal University of Pernambuco, 50740-520,
Recife, PE, Brazil. E-mail: acllb2003@yahoo.com.br
c
˜
˜
Aggeu Magalhaes Research Center, Fundaçao Oswaldo Cruz, 50670-420, Recife, PE,
Brazil
d
´
˜
´
´
´
Laboratorio de Inovaçao Tecnologica no Desenvolvimento de Farmacos e Cosmeticos,
´
´
State University of Maringa, Parana, Brazil
e
´
Laboratorio de Imunopatologia Keizo Asami (LIKA), Campus UFPE, 50670-901, Recife,
PE, Brazil
f
˜
˜
Instituto Aggeu Magalhaes, Fundaçao Oswaldo Cruz, 50670-420, Recife, PE, Brazil
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Drug discovery approaches, for NTDs such as the afore- showed potent trypanocidal activity and low toxicity to mammalian
mentioned ones, have been a hot topic in medicinal chemistry cells.27
in the last years since their last annual occurrence was of more
than 1 billion people with signicant mortality of 500 000,11 isosteric strategy on thiosemicarbazones cyclization to 1,3-
calling for urgent attention to this topic. thiazoles/4-thiazolidinones rings and developing a novel deriv-
Herein, we present two novel series, exploring the bio-
Considering the lack of effective, affordable, or easy-to-use atives from the carboxylic acids produced by our group.27 The
drug treatments for NTDs, the adoption of the Privileged Struc- hypothesis was based on the concept of whether the classical
tures strategy has been advantageous to provide new chemical medicinal chemistry using esterication could be used to
entities with good ‘drug-like’ properties. Privileged motifs are simultaneously improve the solubility and permeability to
recurring in a wide range of biologically active compounds that obtain acetates. In two different series, we used a simple two-
reach different pharmaceutical targets and pathways. The drug- step synthesis approach to obtain novel 1,3-thiazoles (1a–k)
like properties of privileged structures and substructures might and 4-thiazolidinones (2a–p) from the thiosemicarbazones
produce many drug-like compound libraries and leads.12,13
nucleus, as summarized in Fig. 1. Following the synthesis, we
For example, 1,3-thiazole and 4-thiazolidinone nuclei are characterized these molecules and evaluated their trypanocidal,
privileged structures that are found in several natural products, leishmanicidal and cytotoxic activities. The utmost promising
and have also been used to develop synthetic drugs and drug- molecule was chosen to assess necrosis development on try-
like molecules with a variety of pharmacological effects.14–22
pomastigotes by ow cytometry assay and evaluation of activity
Taking account of their biological diversity and potential, against the intracellular forms of T. cruzi, as well as evaluation
together with the easy and rapid synthesis, 4-thiazolidinones and of its ultrastructural impacts on T. cruzi trypomastigotes, seen
1,3-thiazoles are outlined scaffolds in medicinal chemistry. A good by scanning electron microscopy (SEM). All data were collected
strategy for optimizing bioactive compounds is the bioisosterism. to establish the structure–activity relationships (SAR), and
This approach has been considered efficient through thio- provide a comparison with the previous results of their bio-
semicarbazones cyclization to 1,3-thiazoles/4-thiazolidinones, isosteric analogues27 and reference drugs. Hence, our work
taking to anti-T. cruzi and anti-Leishmania spp. activity demonstrates that 1,3-thiazoles and 4-thiazolidinone acetates
enhancement.23–26
are a versatile building block for lead generation, easily yielding
Indeed, we have used the bioisosteric strategy on previously access to diverse derivatives for drug optimization.
synthesized compounds by our group. The thiosemicarbazones
cyclization to 4-thiazolidinones obtained 23 different bioactive 4-
oxo-thiazolidinones-5-acetic-acids. As a common structural feature,
Results and discussion
this series of compounds presented an acetic acid in the 5th posi-
tion of the 4-oxo-thiazolidinones ring system, and a varied ring core
at the hydrazone moiety. Some of these reported compounds
Chemistry
1,3-Thiazoles synthesis and characterization. Firstly, ethyl 2-
(2-(arylidene)hydrazinyl)thiazole-4-carboxylates (1a–k) were
synthesized, as shown in Scheme 1. Different substituted
Fig. 1 Summary of the structural planning of the novel 1,3-thiazoles and 4-thiazolidinones reported in this work, based on previous results.27
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aldehydes were condensed with thiosemicarbazide via Schiff The formation of the synthesized 1,3-thiazole-4-carboxylates was
base condensation to obtain respective thiosemicarbazones, as in conrmed by high-resolution mass spectrometry technique.
our previous work.26 The thiosemicarbazones were then cyclized to
4-Thiazolidinones synthesis and characterization. To
their respective 1,3-thiazole-4-carboxylates (1a–k) with ethyl bromo- produce our second series of compounds, we used the esteri-
pyruvate using absolute ethanol under reux for about 4–6 hours. cation strategy of preceding acetic acids published by our
Aer reux, the solids were precipitated when put on ice. The group26 in novel alkyl 2-((2-(arylidene)hydrazinyl)-4-oxo-4,5-
products were obtained with satisfactory yields, ranging from 73% dihydrothiazol-5-yl)acetates (2a–p), which were synthesized as
to 90%. Only compounds 1d (51%) and 1g (61%) had lower yields, shown in Scheme 2. The 4-oxothiazolidine-5-acetic acids were then
with the possibility of optimization. The purity of all compounds esteried using thionyl chloride and the respective alcohol. Aer
was ascertained by observing a single spot on TLC. The synthesized almost one hour of reux, a solid precipitated when the reaction
ethyl 2-(2-(arylidene)hydrazinyl)thiazole-4-carboxylates were initially mixture was poured onto ice. The synthesized 2a–p were char-
characterized by observing changes in their physical parameters acterized, and conrmed by physical parameters analysis and
relating to the color, retardation factor (Rf), and melting point different spectroscopic techniques. In FT-IR, the characteristic
values. The compounds 1a–k were further veried and conrmed by alkyl chain –CH2– stretching was observed at 2981–3093 cmꢀ1. In
1
different spectroscopic techniques. FT-IR, H-, 13C-NMR and high- the 1H-NMR spectra, three protons triplet at 1.17–1.19 ppm and
resolution mass spectrometry results revealed that the proposed two protons quartet at 4.03–4.17 ppm due to –CH3 and –OCH2–,
structures of the synthesized 1,3-thiazoles were in good agreement respectively, were assigned to the ethyl group of the ester moiety,
with the actual structures. In FT-IR, a carbonyl group stretching was verifying the successful esterication of the 2-(2-(arylidene)
observed within the range of 1699–1724 cmꢀ1. In the 1H-NMR hydrazono)-4-oxothiazolidine-5-acetic acids. The aromatic and
spectra, three protons triplet of –CH3 and two protons quartet of other protons appeared in their respective regions, depending on
–CH2– were observed around 1.26–1.28 ppm and 4.22–4.28 ppm, their substitution patterns. In the 13C-NMR spectra, carbon
respectively, and were assigned to the ethyl group of the ester signals observed at the upeld region around 14.4–14.6 and 60.8–
moiety. One proton singlet appeared at 7.73–7.79 ppm, and was 61.2 ppm, and were assigned to the ethyl carbons of the ester
assigned to the proton of the thiazole ring. The multiplicity of the moiety. The structures of the synthesized 4-thiazolidinones were
aromatic protons appeared at their respective position in the further conrmed by mass spectrometry techniques.
aromatic region with regards to the substitution patterns. In the 13C-
NMR spectra, two carbon signals upeld at around 14.2–14.6 and
60.8–60.9 ppm were assigned to the ethyl group of the ester moiety.
The potential cytotoxic prole of novel compounds against
Cytotoxic studies
mammalian cells was investigated by MTT assay28 using LLC-MK2
Scheme 1 Synthetic route for ethyl 2-(2-(arylidene)hydrazinyl)thiazole-4-carboxylates (1a–h; 1i–k27).
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Scheme 2 Synthetic route for alkyl 2-((2-(arylidene)hydrazinyl)-4-oxo-4,5-dihydrothiazol-5-yl)acetate (2a–p).
cells. Compounds 1a–k and 2a–p were added in increasing than to the mammalian cells (SI ¼ 153.6, 103.3 and 121.2, respec-
concentrations (10 to 1000 mM), and incubated for 96 hours for the tively), outlining 1f (o-Br-benzylidene) that performed a selectivity of
determination of the 50% cytotoxic concentration (CC50). As we can 303.2 in comparison with 17.8 of BZD. Concerning the 4-thiazoli-
see in Table 1, no compounds were considered cytotoxic to dinones derivatives, it was veried that among the 2a–p series,
mammalian cells since all of the CC50 values were higher than 50 compound 2d exhibited the best IC50 value (8.45 mM). Regarding the
mM. However, compounds 1d, 1j, 1k, 2b and 2n exhibited higher selectivity index, we can highlight that compound 2d (p-Br-
CC50 values than the reference BZD with highlights to the o-methyl benzylidene, SI ¼ 47), 2e (m-Br-benzylidene, SI ¼ 24.2), 2i (p-
substituted 2n (CC50 ¼ 770.37; SITrypo ¼ 61) and the p-methyl CH3O-benzylidene, SI ¼ 31.4), 2j (o-CH3O-benzylidene, SI ¼ 22.3),
substituted 2b (CC50 ¼ 659.08; SIL.inf ¼ 19.8) being less cytotoxic.
2m (p-CH3-benzylidene, SI ¼ 20.8), and 2n (o-CH3-benzylidene, SI ¼
61), with 2p (m-methoxy and p-hydroxy benzylidene, SI ¼ 15.3)
performed better than BZD. The methyl substitution on the
aromatic ring was evidenced as a good change, resulting in satis-
factory IC50 values in both synthesized positions (ortho and para), as
well as the methoxy substitution. In addition, the two leading
compounds were ortho-substituted in terms of the trypanocidal
activity of both series, evidencing the importance of this position
and corroborating our previous results with the non-cyclized 1g*
(IC50 ¼ 5.65 mM),27 which presents an o-chlorine substitution.
In general, the 1,3-thiazole derivatives (1a–k) were more
effective than the 4-thiazolidinones (2a–p) ones. The
compounds 1f and 1h can be highlighted as the hit compounds,
Trypanocidal activity evaluated against trypomastigote form
The compounds 1a–k and 2a–p were evaluated in vitro against T.
cruzi trypomastigote (strain Y). As shown in Table 1, most of the
tested compounds showed a promising IC50 value and selectivity in
comparison to the reference drug, BZD (IC50 ¼ 34.5 mM and SI ¼
17.8). The compounds that had outstanding performance with
minimal inhibitory concentration for 50% of the parasite cells (less
than 10 mM) were 1f, 1h, 1g, 1a and 2d (0.83 mM, 2.75 mM, 2.83 mM,
4.44 mM and 8.45 mM, respectively). The synthesized compounds 1h
(a furan moiety derivative), 1g (benzylidene) and 1a (m-Cl-
benzylidene) were over a hundred times more selective to T. cruzi
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Table 1 Determination of the in vitro cytotoxic, trypanocidal and leishmanicidal activity of 1a–k and 2a–p derivatives in comparison with the
d
standard drugs BZD28 and Miltefosine29
Trypomastigotes
Promastigotes
IC50b (mM)
IC50L.Ama
SIc
Compound code
LLC-MK2, CC50a (mM)
EC50b (mM)
SIc
IC50L.Inf
SIL.Ama
SIL.Inf
1a
1b
1c
1d
1e
1f
1g
1h
1i
1j
1k
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
2m
538.34
258.4
4.44
121.2
20.7
>48.8
15.5
12.8
303.2
103.3
153.6
24.1
NA
189.27
27.89
391.68
159.19
245.55
24.34
42.96
451.2
145.07
484.67
359.02
366.79
91.35
113.86
14.63
119.76
57.5
308.54
17.01
169.44
185.58
363.77
84.3
13.35
267.54
179.75
232.76
NA
164.32
14.39
332.08
103.85
170.28
23.46
2.8
9.3
2.6
4.0
2.4
10.3
6.8
0.9
4.1
1.9
1.8
1.3
7.2
2.0
27.2
3.7
12.6
1.4
33.9
2.4
2.9
1.7
0.6
45.7
2.9
1.6
2.3
NA
0.3
3.3
18.0
3.0
6.2
3.5
10.7
7.8
1.1
5.5
3.2
4.2
1.3
19.8
1.8
27.4
3.9
2.2
1.3
2.0
2.7
2.3
1.7
0.8
32.4
4.6
1.6
1.7
NA
0.2
12.50
20.51
12.91
46.73
0.83
2.83
2.75
24.87
ND
ND
33.83
ND
ND
8.45
18.24
ND
>1000
640.79
600.46
251.66
292.4
37.43
422.4
374.16
108.11
289.74
158.9
365.48
33.24
598.56
926.07
661.54
471.36
659.08
229.11
397.45
445.76
722.89
430.47
577.05
399.41
538.62
613.59
53.45
NA
13.9
NA
NA
128.72
14.49
47.0
24.4
NA
NA
NA
31.4
22.3
NA
115.55
324.99
329.38
295.13
146.52
236.03
370.31
67.27
ND
ND
12.72
24.15
ND
19.76
29.26
12.62
27.87
35.49
34.5 ꢁ 7.6
NA
2.7
609.45
770.37
279.57
544.65
614.7
20.8
61.0
10.0
15.3
17.8
NA
18.82
2n
2o
167.85
176.27
320.16
NA
2p
BZD10
Miltefosine29
12.27
36.31
53.71
a
CC50 ¼ cytotoxic concentration for 50% of the cells. b EC50 ¼ effective concentration for 50% of the parasites. c SI ¼ selectivity index (CC50/IC50).
NA ¼ non-applicable; ND ¼ no difference.
d
given their enhanced trypanocidal activity with high selectivity was the best among the tested compounds, with an IC50 ¼ 16.85
to parasite cells, and the low concentrations needed to kill half mM and SI ¼ 31.9. Nevertheless, it was weaker than the standard
of the parasite population, with 1f at the nanomolar scale (830 drug BZD (IC50 ¼ 5.65 mM, SI ¼ 108.8).
nM).
Cell death assessment
Trypanocidal activity evaluated against the amastigote forms
Aer the outstanding trypanocidal activity, the cell death mech-
The compounds 1a, 1g, 1h and 1f were tested in six different anism rates of the nanomolar-active compound 1f was investi-
concentrations (0.625, 1.25, 2.5, 5, 10 and 20 mg mLꢀ1) against T. gated using BZD as a standard. This was assessed through
cruzi amastigotes (Tulahuen strain). The analysis of the ob- conventional staining with Propidium Iodide (PI) and Annexin-V
tained results, displayed in Table 2, indicate that compound 1a in the ow cytometry method. First, PI is a stain that passes
Table 2 Results of the trypanocidal activity evaluation of compounds 1a, 1g, 1h, 1f and BZD against the amastigote forms of the Tulahuen strain
of Trypanosoma cruzi. Negative control—RPMI-1640 medium plus 1% DMSO displayed 0% lysis
Compound
code
LLC-MK2, CC50a (mM)
Amastigote, IC50b (mM)
Amastigote, SIc
1a
1g
1h
1f
BZD
538.34
292.4
422.4
251.66
614.7
16.85
40.71
75.39
56.46
5.65
31.9
7.2
5.6
4.5
108.8
a
CC50 ¼ cytotoxic concentration for 50% of cells. b IC50 ¼ inhibitory concentration for 50% of cells. c SI ¼ selectivity index (CC50/IC50).
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within the cell membrane whenever it is damaged, reaching and that T. cruzi trypomastigotes in the control group had preserved
binding directly to the DNA. On the other hand, Annexin V binds morphologies in the body and agella (Fig. 3A), while cells
to the phospholipid phosphatidylserine that is found in the inner treated with BZD had their bodies contorted with the extrava-
side of the cell membrane. There, it stains whenever this phos- sation of cytoplasmic content (Fig. 3B). In contrast, cells treated
pholipid is exposed, which happens when the cells go through with 1f at IC50, 2 ꢂ IC50, and 4 ꢂ IC50 (0.8 mM, 1.66 mM and 3.32
the apoptosis process. Moreover, when the apoptotic process is mM) concentrations, respectively, promoted dose-dependent
late or involves necrosis, there is PI staining on DNA.31
alterations in the shape, agella and surface of the parasite's
As shown in Fig. 2, compound 1f (at 1.66 mM and 3.32 mM) body (Fig. 3C–E) when compared to the untreated control
was able to induce signicant labeling, which is consistent with group. Furthermore, the compound 1f promoted body writhing
necrosis in trypomastigotes. Similar results were found in parasites with a swollen and rounded appearance, shortening of the
treated with BZD (34.5 mM, 70.8 mM, and 141.6 mM), the reference agellum, plus a disruption of the membrane with extravasa-
drug, and the positive control used in this assay (Fig. 2A). Further- tion of the cytoplasmic content. The activity of compound 1f can
more, at 3.32 mM concentration of 1f, an equivalent necrosis was be observed through ultrastructural alterations caused in T.
observed (17.58% double-stained) as that for the 70.8 mM of BZD cruzi, which certainly makes the cell unfeasible.
(19.56% double-stained), reinforcing the potency of the 1,3-thiazole
derivatives in comparison with the standard drug (Fig. 2B).
Leishmanicidal activity evaluated against promastigote forms
Ultrastructural studies for T. cruzi
The ability of the new derivatives (1a–k and 2a–p) to inhibit the
To evaluate the effects of 1,3-thiazoles on the parasite growth of the promastigote forms of L. amazonensis and L.
morphology, the most active compound of this work (1f) was infantum was investigated using Miltefosine as a standard.30 It
selected. Scanning electron microscopy analysis (Fig. 3) showed was veried that all of the synthesized compounds were less
Fig. 2 Flow cytometry results on T. cruzi trypomastigotes untreated and treated with 1f at 2 ꢂ IC50 (1.66 mM) and 4 ꢂ IC50 (3.32 mM); and treated
with BZD at 1 ꢂ IC50 (34.5 mM), 2 ꢂ IC50 (70.8 mM), and 4 ꢂ IC50 (141.6 mM), following staining with PI and Annexin-V. (A) Plotted flow cytometry
graphs with FITC on the X-axis and PI on the Y-axis. (B) Bar chart illustrating the proportions of each cell death mechanism among all
concentrations of 1f and BZD tested, and comparison with the untreated group. The percentage of populations can be separated on double-
negative (live) cells, Annexin-V positive (early apoptotic cells), PI-positive (late apoptotic), and double-positive (necroptotic) cells. All p values of
the shown concentrations were below 0.05. The graphs were plotted with the GraphPad Prism ® software.
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14.39 mM, SI ¼ 18). Furthermore, the ortho-substitution with
a halogen group on the benzylidene moiety was demonstrated
to be benecial for the bioactivity of the substituted 1,3-
thiazole-4-carboxylates, whereas the 4-thiazolidinone-5-acetates
displayed enhanced leishmanicidal activity when para-
substituted (2m, 2d, 2b). In addition, the compound 2d (p-Br-
benzylidene) is outlined as a promising structure with satis-
factory activity against both T. cruzi (SI ¼ 47) and Leishmania sp.
(SIL.ama ¼ 27.2; SIL.inf ¼ 27.4), having the same atom allocated on
the benzylidene ring as 1f (o-bromine). Moreover, our strategy of
esterication was proven to be efficient since the outstanding
carboxylic acids synthesized by our group had their activity greatly
improved, to enumerate: 1b* (IC50L.ama ¼ 34.48 mM, IC50L.inf
¼
59.52 mM) p-bromine substituted that was esteried to 2d, and 1c*
(IC50L.inf ¼ 85.30 mM), p-chlorine that originated from 2b.27
The summarized SAR evaluation for both 1,3-thiazoles and 4-
thiazolidinones can be seen in Fig. 4, while the trypanocidal and
leishmanicidal activities regarding the SI values are compiled in Fig. 5.
Experimental
Synthesis and characterization of novel 1,3-thiazole and
thiazolidin-4-one analogues
High-purity grade reagents and solvents were used for the
synthesis
of
ethyl
2-(2-(arylidene)hydrazinyl)thiazole-4-
carboxylates (1a–k) and alkyl 2-((2-(arylidene)hydrazinyl)-4-oxo-
4,5-dihydrothiazol-5-yl)acetates (2a–p). All synthesized esters
of 1,3-thiazoles and thiazolidin-4-ones were initially character-
ized by their physical parameters, like the change in color,
Fig. 3 Ultrastructural alterations in trypomastigote forms of T. cruzi
treated with BZD and compound 1f by SEM. (A) Control untreated
trypomastigotes showing the typical elongated body with the slick cell
surface and preserved flagellum. (B) Treated trypomastigotes with BZD
(IC50 ¼ 34.5 mM) showing a contorted and swollen body, with
disruption displaying internal cell content. (C) Treated trypomastigotes
with 1f (IC50 ¼ 0.8 mM) showing body writhed with appearance swollen
and rounded, beyond flagellum shortening. (D) Treated trypomasti-
gotes with 1f (2 ꢂ IC50 ¼ 1.66 mM), showing a rounded body, rupture of
the cytoplasmic membrane, and exposure of internal cellular content.
(E) Treated trypomastigotes with 1f (4 ꢂ IC50 ¼ 3.32 mM) showing
alterations in the flagella form and cell body, with total membrane
destruction and extravasation of the cytoplasmic content.
toxic and more selective than the standard drug Miltefosine, as
shown in Table 1.
The compounds 1b, 1f, 2d and 2m exhibited better IC50
values for both L. amazonensis (27.89 mM, 23.34 mM, 14.63 mM
and 13.35 mM) and L. infantum (14.39 mM, 23.46 mM, 14.49 mM
and 18.81 mM), in comparison to the standard Miltefosine
(36.31 mM and 53.71 mM). They also displayed good selectivity,
with a focus on 2d (around 27 times, both) and 2m (SI ¼ 45.7
and SI ¼ 32.4) that had high SI for both Leishmania spp. tested.
In addition, 2m performed the highest selectivity value for L.
amazonensis of all tested compounds, being 46 times more
selective to the parasite than to the LLC-MK2 cells (and 32.4 to
L. infantum). Among the 1,3-thiazoles, the compound 1f was
once again evidenced for its bioactivity (L. amazonensis – IC50
¼
24.34 mM, SI ¼ 10.3; L. infantum – IC50 ¼ 23.46 mM, SI ¼ 10.7),
Fig. 4 Summary of the SAR evaluation between the top compounds
of each 1,3-thiazole series (1f) and 4-thiazolidinone series (2m),
regarding the trypanocidal and leishmanicidal activity.
together with 1g (L. infantum – IC50 ¼ 37.43 mM, SI ¼ 7.8) and 1b
(L. amazonensis – IC50 ¼ 27.89 mM, SI ¼ 9.3; L. infantum – IC50
¼
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Fig. 5 Improvement in the SI (selectivity index) values was observed by the change in the radicals of 1,3-thiazoles and 4-thiazolidinones.
melting points, and the Rf (retardation factor) values. Spectro- ethanol for 4 to 6 hours. Thin-layer chromatography (TLC,
scopic techniques, like FT-IR, NMR and high-resolution mass acetone : n-hexane; 1 : 2) was used to check the completion and
spectrometry, were used for the conrmation of the synthesized purity of the reaction. The reaction mixture solidied when put
compounds. To evaluate the purity of all synthesized on ice. The solid was ltered and washed with plenty of water to
compounds, the Rf values were calculated by thin-layer chro- obtain a pure product, and dried at room temperature.
matography using silica gel 60 HF254 pre-coated aluminum
Ethyl
2-(2-(3-chlorobenzylidene)hydrazinyl)thiazole-4-
sheets (Merck). Melting points were recorded on DMP-300 A&E carboxylate (1a). Light yellow solid; yield: 89%; melting point:
Lab UK, apparatus. Characteristic functional groups were 249–251 C; Rf: 0.54 (acetone/n-hexane, 1 : 2); IR (ATR, cmꢀ1):
ꢃ
determined by the FT-IR spectrophotometer using ATR. Bruker 1093 (C–O stretching, ester), 1446 (C–H bending, aliphatic),
300 MHz, Varian VNMRS 400 MHz, and 500 MHz spectrometers 1573 (C]C ring stretching), 1716 (C]O stretching), 2987 (C–H
1
were used to check protons and carbon signals. HRMS was stretching, aliphatic); H-NMR (300 MHz): d 1.28 (3H, t, –CH3
recorded using a MALDI-TOF mass spectrometer, Bruker. The ester, J ¼ 7.20 Hz), 4.25 (2H, q, –CH2–CH3 ester, J ¼ 7.20 Hz),
purity of the selected compound was calculated by qualitative 7.44 (2H, m, Ar-H), 7.61 (1H, m, Ar-H), 7.70 (1H, m, Ar-H), 7.78
HNMR method using qHNMR normalization (100%) method, (1H, s, 1,3-thiazole ring C-5), 7.90 (1H, s, –CH]N- azomethine),
established by Pauli et al. The spectra were recorded using this 12.49 (1H, s, –N–NH–C–); 13C-NMR (75 MHz): d 14.6 (–CH3
optimized protocol.32
ester), 60.9 (–OCH2– ester), 119.7 (1,3-thiazole ring C-5), 125.5,
126.1, 129.5, 131.2, 134.2, 136.9 (Ar-C), 140.6 (–CH]N– azo-
methine), 143.6 (1,3-thiazole ring C-4), 161.4 (C]O), 168.4 (1,3-
thiazole ring C-2); HRMS (MALDI-TOF): calcd C13H12ClN3NaO2S
(M + Na) ¼ 332.02364, found: 332.01055.
Synthesis and characterization of ethyl 2-(2-(arylidene)
hydrazinyl)thiazole-4-carboxylates (1a–k)
Ethyl
2-(2-(2-chlorobenzylidene)hydrazinyl)thiazole-4-
Different aryl-substituted thiosemicarbazones (1.0 mmol) and
ethyl bromopyruvate (1 : 1 equivalent) were reuxed in absolute
carboxylate (1b). Off white solid; yield: 90%; melting point:
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213–215 C; Rf: 0.60 (acetone/n-hexane, 1 : 2); IR (ATR, cmꢀ1): 168.4 (1,3-thiazole ring C-2); HRMS (MALDI-TOF): calcd C13-
1091 (C–O stretching, ester), 1423 (C–H bending, aliphatic), H12BrN3NaO2S (M + Na) ¼ 375.97313, found: 376.16224.
ꢃ
1585 (C]C ring stretching), 1687 (C]N stretching), 1724 (C]O
Ethyl
2-(2-(2-bromobenzylidene)hydrazinyl)thiazole-4-
stretching), 2985 (C–H stretching, aliphatic); 1H-NMR (300 MHz): carboxylate (1f). Light yellow solid; yield: 81%; melting point:
d 1.27 (3H, t, –CH3 ester, J ¼ 7.20 Hz), 4.28 (2H, q, –CH2–CH3 226–228 C; Rf: 0.59 (acetone/n-hexane, 1 : 2); IR (ATR, cmꢀ1):
ꢃ
ester, J ¼ 7.20 Hz), 7.39 (2H, m, Ar-H), 7.47 (1H, m, Ar-H), 7.79 1103 (C–O stretching, ester), 1431 (C–H bending, aliphatic),
(1H, s, 1,3-thiazole ring C-5), 7.90 (1H, m, Ar-H), 8.34 (1H, s, 1575 (C]C ring stretching), 1687 (C]N stretching), 1699 (C]O
–CH]N– azomethine), 12.48 (1H, s, –N–NH–C–); 13C-NMR (75 stretching), 3045 (C–H stretching, aliphatic); 1H-NMR (300
MHz): d 14.6 (–CH3 ester), 60.9 (–OCH2– ester), 119.8 (1,3-thiazole MHz): d 1.27 (3H, t, –CH3 ester, J ¼ 7.09 Hz), 4.23 (2H, q, –CH2–
ring C-5), 126.7, 128.1, 130.4, 131.3, 131.8, 132.7 (Ar-C), 138.0 CH3 ester, J ¼ 7.10 Hz), 7.30 (1H, ddd, Ar-H, J ¼ 1.72 Hz, J ¼
(–CH]N– azomethine), 143.3 (1,3-thiazole ring C-4), 161.4 (C] 7.23 Hz, J ¼ 8.16 Hz), 7.44 (1H, m, Ar-H), 7.65 (1H, dd, Ar-H, J ¼
O), 168.3 (1,3-thiazole ring C-2); HRMS (MALDI-TOF): calcd C13- 1.17 Hz, J ¼ 8.05 Hz), 7.78 (1H, s, 1,3-thiazole ring C-5), 7.87 (1H,
H12ClN3NaO2S (M + Na) ¼ 332.02364, found: 331.99641.
dd, Ar-H, J ¼ 1.69 Hz, J ¼ 7.91 Hz), 8.30 (1H, s, –CH]N– azo-
Ethyl 2-(2-(4-methoxybenzylidene)hydrazinyl)thiazole-4-carboxylꢃate methine), 12.50 (1H, s, –N–NH–C–); 13C-NMR (75 MHz): d 14.6
(1c). Yellowish brown solid; yield: 80%; melting point: 213–215 C; (–CH3 ester), 60.9 (–OCH2– ester), 119.8 (1,3-thiazole ring C-5),
Rf: 0.58 (acetone/n-hexane, 1 : 2); IR (ATR, cmꢀ1): 1091 (C–O stretching, 123.1, 127.1, 128.6, 131.5, 133.3, 133.6 (Ar-C), 140.4 (–CH]N–
ester), 1423 (C–H bending, aliphatic), 1585 (C]C ring stretching), 1687 azomethine), 143.3 (1,3-thiazole ring C-4), 161.4 (C]O), 168.3
(C]N stretching), 1724 (C]O stretching), 2985 (C–H stretching, (1,3-thiazole ring C-2); HRMS (MALDI-TOF): calcd C13H12BrN3-
aliphatic); 1H-NMR (300 MHz): d 1.28 (3H, t, –CH3 ester, J ¼ 7.20 Hz), NaO2S (M + Na) ¼ 375.97313, found: 376.12838.
3.79 (3H, s, –OCH3), 4.23 (2H, q, –CH2–CH3 ester, J ¼ 7.20 Hz), 6.99
Ethyl
2-(2-(1-phenylethylidene)hydrazinyl)thiazole-4-
(2H, d, Ar-H, J ¼ 8.70 Hz), 7.59 (2H, d, Ar-H, J ¼ 8.70 Hz), 7.73 (1H, s, carboxylate (1g). Dark yellow solid; yield: 61%; melting point:
1,3-thiazole ring C-5), 7.95 (1H, s, –CH]N– azomethine), 12.14 (1H, s, 108–110 C; Rf: 0.60 (acetone/n-hexane, 1 : 2); IR (ATR, cmꢀ1):
ꢃ
–N–NH–C–); 13C-NMR (75 MHz): d 14.6 (–CH3 ester), 55.7 (–OCH3), 60.8 1087 (C–O stretching, ester), 1442 (C–H bending, aliphatic),
(–OCH2– ester), 119.1 (1,3-thiazole ring C-5), 114.8, 127.2, 128.4, 160.8 1573 (C]C ring stretching), 1699 (C]N stretching), 1712 (C]O
(Ar-C), 142.4 (-CH]N- azomethine), 143.3 (1,3-thiazole ring C-4), 161.5 stretching), 2978 (C–H stretching, aliphatic); 1H-NMR (500
(C]O), 168.7 (1,3-thiazole ring C-2); HRMS (MALDI-TOF): calcd C14- MHz): d 1.27 (3H, t, –CH3 ester, J ¼ 7.14 Hz), 2.29 (3H, s, –CH3),
H15N3NaO3S (M + Na) ¼ 328.0732, found: 327.97609.
Ethyl
4.23 (2H, q, –CH2–CH3 ester, J ¼ 7.08 Hz), 7.39 (3H, m, Ar-H),
2-(2-(3-methoxybenzylidene)hydrazinyl)thiazole-4- 7.75 (2H, m, Ar-H + 1,3-thiazole ring C-5), 7.91 (1H, s, –CH]
carboxylate (1d). Yellowish brown solid; yield: 51%; melting N– azomethine), 12.31 (1H, s, –N–NH–C–); 13C-NMR (126 MHz):
point: 188–190 ꢃC; Rf: 0.60 (acetone/n-hexane, 1 : 2); IR d 14.2 (–CH3 ester), 14.9 (–CH3), 60.8 (–OCH2– ester), 119.7 (1,3-
(ATR, cmꢀ1): 1071 (C–O stretching, ester), 1456 (C–H bending, thiazole ring C-5), 126.2, 128.7, 128.9, 129.3 (Ar-C), 138.2 (–CH]
aliphatic), 1573 (C]C ring stretching), 1617 (C]N stretching), N– azomethine), 147.4 (1,3-thiazole ring C-4), 161.6 (C]O),
1708 (C]O stretching), 2980 (C–H stretching, aliphatic); 1H- 170.1 (1,3-thiazole ring C-2); HRMS (MALDI-TOF): calcd C14-
NMR (300 MHz): d 1.28 (3H, t, –CH3 ester, J ¼ 7.20 Hz), 3.79 H15N3NaO2S (M + Na) ¼ 312.0783, found: 311.86282.
(3H, s, –OCH3), 4.24 (2H, q, –CH2–CH3 ester, J ¼ 6.90 Hz), 6.95
Ethyl 2-(2-(furan-2-ylmethylene)hydrazinyl)thiazole-4-carboxylate
ꢃ
(1H, m, Ar-H), 7.22 (2H, m, Ar-H), 7.33 (1H, t, Ar-H, J ¼ 7.80 Hz), (1h). Dark green solid; yield: 73%; melting point: 215–217 C; Rf:
7.76 (1,3-thiazole ring C-5), 7.97 (1H, s, –CH]N– azomethine), 0.52 (acetone/n-hexane, 1 : 2); IR (ATR, cmꢀ1): 1091 (C–O stretch-
12.34 (1H, s, –N–NH–C–); 13C-NMR (75 MHz): d 14.6 (–CH3 ing, ester), 1431 (C–H bending, aliphatic), 1573 (C]C ring
ester), 55.5 (–OCH3), 60.9 (–OCH2– ester), 119.4 (1,3-thiazole stretching), 1622 (C]N stretching), 1720 (C]O stretching), 2976
ring C-5), 111.6, 115.8, 119.4, 130.4, 136.0, 159.9 (Ar-C), 142.1 (C–H stretching, aliphatic); 1H-NMR (500 MHz): d 1.26 (3H, t,
(–CH]N– azomethine), 143.3 (1,3-thiazole ring C-4), 161.5 (C] –CH3 ester, J ¼ 7.12 Hz), 4.22 (2H, q, –CH2–CH3 ester, J ¼ 7.05
O), 168.6 (1,3-thiazole ring C-2); HRMS (MALDI-TOF): calcd Hz), 6.59 (1H, m, Ar-H), 6.81 (1H, d, Ar-H, J ¼ 3.41 Hz), 7.73 (1H, s,
C
14H15N3NaO3S (M + Na) ¼ 328.0732, found: 327.96025.
1,3-thiazole ring C-5), 7.78 (1H, m, Ar-H), 7.86 (1H, s, –CH]N–
Ethyl
2-(2-(3-bromobenzylidene)hydrazinyl)thiazole-4- azomethine), 12.54 (1H, s, –N–NH–C–); 13C-NMR (126 MHz):
carboxylate (1e). Light yellow solid; yield: 88%; melting point: d 14.6 (–CH3 ester), 60.8 (–OCH2– ester), 119.7 (1,3-thiazole ring
266–268 C; Rf: 0.54 (acetone/n-hexane, 1 : 2); IR (ATR, cmꢀ1): C-5), 112.5, 113.1, 145.1, 149.5 (Ar-C), 132.6 (–CH]N– azome-
ꢃ
1095 (C–O stretching, ester), 1446 (C–H bending, aliphatic), thine), 143.2 (1,3-thiazole ring C-4), 161.4 (C]O), 168.3 (1,3-
1579 (C]C ring stretching), 1687 (C]N stretching), 1724 (C]O thiazole ring C-2); HRMS (MALDI-TOF): calcd C11H11N3NaO3S (M
stretching), 2999 (C–H stretching, aliphatic); 1H-NMR (300 + Na) ¼ 288.0419, found: 287.63438.
MHz): d 1.28 (3H, t, –CH3 ester, J ¼ 7.20 Hz), 4.24 (2H, q, –CH2–
CH3 ester, J ¼ 7.20 Hz), 7.38 (1H, t, Ar-H, J ¼ 7.80 Hz), 7.56 (1H,
Synthesis of alkyl 2-(2-(arylidene)hydrazono)-4-
oxothiazolidine-5-acetates (2a–p)
d, Ar-H, J ¼ 8.70 Hz), 7.65 (1H, d, Ar-H, J ¼ 7.80 Hz), 7.78 (1H, s,
1,3-thiazole ring C-5), 7.83 (1H, s, Ar-H), 7.96 (1H, s, –CH]N–
azomethine), 12.44 (1H, s, –N–NH–C–); 13C-NMR (75 MHz): Thionyl chloride (2.0 equivalent) and the respective alcohol (20
d 14.6 (–CH3 ester), 60.9 (–OCH2– ester), 119.7 (1,3-thiazole ring mL) were initially stirred together at 0 ꢃC, followed by the
C-5), 122.7, 125.8, 128.9, 131.5, 132.4, 137.1 (Ar-C), 140.5 (–CH] addition of 2-(2-(arylidene)hydrazono)-4-oxothiazolidine-5-
N– azomethine), 143.3 (1,3-thiazole ring C-4), 161.41 (C]O), acetic acids (1.0 mmol). The reaction mixture was further
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stirred at room temperature for 30 to 50 minutes. The reaction
mixture was then reuxed for 30 to 50 minutes. Conversion of
acids into respective esters was observed by taking TLC
(acetone : n-hexane; 1 : 3). The solid appeared when the reac-
tion mixture was put on ice, which was ltered and washed with
an excess of water. The esters were dried at room temperature.
Ethyl 2-(2-(4-bromobenzylidene)hydrazono)-4-oxothiazolidine-5-
acetate (2d). White solid; yield: 90%; melting point: 208–210 ꢃC;
Rf: 0.41 (acetone/n-hexane, 1 : 3); IR (ATR, cmꢀ1): 1010 (C–O
stretching, ester), 1334 (C–H bending, aliphatic), 1512 (C]C ring
stretching), 1643 (C]N stretching), 1712 (C]O stretching), 2981
(C–H stretching, aliphatic); 1H-NMR (300 MHz): d 1.18 (3H, t, –CH3, J
¼ 6.90 Hz), 2.98 (1H, dd, –CH2–, J ¼ 7.80 Hz, J ¼ 17.40 Hz), 3.08 (1H,
dd, –CH2–, J ¼ 4.50 Hz, J ¼ 17.85 Hz), 4.09 (2H, q, –CH2–CH3 ester, J
¼ 6.60 Hz), 4.41 (1H, dd, 1,3-thiazolidin-4-one ring C-5, J ¼ 4.50 Hz, J
¼ 7.80 Hz), 7.69 (4H, m, Ar-H), 8.40 (1H, s, –CH]N– azomethine),
12.09 (1H, s, –NH); 13C-NMR (75 MHz): d 14.4 (–CH3), 36.8 (–CH2–),
43.8 (1,3-thiazolidin-4-one ring C-5), 61.1 (–OCH2– ester), 124.5,
129.7, 129.9, 130.7, 132.1, 132.3 (Ar-C), 132.4 (–CH]N– azome-
thine), 133.9 (1,3-thiazolidin-4-one ring C-2), 155.7 (C]O), 170.6
(1,3-thiazolidin-4-one ring C-4); HRMS (MALDI-TOF): calcd C14H14-
BrN3NaO3S (M + Na) ¼ 405.98369, found: 406.43054.
Ethyl
2-((2-benzylidene)hydrazono)-4-oxothiazolidine-5-
acetate (2a). White solid; yield: 96%; melting point: 224–
226 C; Rf: 0.33 (acetone/n-hexane, 1 : 3); IR (ATR, cmꢀ1): 1018
ꢃ
(C–O stretching, ester), 1338 (C–H bending, aliphatic), 1516
(C]C ring stretching), 1643 (C]N stretching), 1708 (C]O
stretching), 2993 (C–H stretching, aliphatic); 1H-NMR (300
MHz): d 1.19 (3H, t, –CH3, ester, J ¼ 7.05 Hz), 2.68 (1H, dd,
–CH2–, J ¼ 9.70 Hz, J ¼ 16.95 Hz), 3.08 (1H, dd, –CH2-, J ¼
3.75 Hz, J ¼ 16.95 Hz), 4.10 (2H, q, –CH2–CH3 ester, J ¼ 6.45 Hz),
4.50 (1H, dd, 1,3-thiazolidin-4-one ring C-5, J ¼ 4.50 Hz, J ¼ 7.70
Hz), 7.42 (3H, m, Ar-H), 7.70 (2H, m, Ar-H), 8.29 (1H, s, –CH]N–
azomethine); 13C-NMR (75 MHz): d 14.5 (–CH3 ester), 38.6
(–CH2–), 45.8 (1,3-thiazolidin-4-one ring C-5), 60.9 (–OCH2–
ester), 127.5, 127.8, 129.2, 130.1 (Ar-C), 135.7 (–CH]N– azo-
methine), 152.6 (1,3-thiazolidin-4-one ring C-2), 171.5 (C]O),
181.9 (1,3-thiazolidin-4-one ring C-4); HRMS (MALDI-TOF):
calcd C14H15N3NaO3S (M + Na) ¼ 328.07318, found: 329.35526.
Methyl 2-(2-(4-chlorobenzylidene)hydrazono)-4-oxothiazolidinꢃ e-
5-acetate (2b). White solid; yield: 87%; melting point: 218–220 C;
Rf: 0.45 (acetone/n-hexane, 1 : 3); IR (ATR, cmꢀ1): 1087 (C–O
stretching, ester), 1442 (C–H bending, aliphatic), 1600 (C]C ring
stretching), 1643 (C]N stretching), 1712 (C]O stretching), 1735
Ethyl 2-(2-(3-bromobenzylidene)hydrazono)-4-oxothiazolidine-5-
acetate (2e). White solid; yield: 91%; melting point: 218–220 ꢃC;
Rf: 0.43 (acetone/n-hexane, 1 : 3); IR (ATR, cmꢀ1): 1021 (C–O
stretching, ester), 1341 (C–H bending, aliphatic), 1600 (C]C ring
stretching), 1647 (C]N stretching), 1710 (C]O stretching), 2981
(C–H stretching, aliphatic); 1H-NMR (300 MHz): d 1.19 (3H, t, –CH3, J
¼ 6.89 Hz), 2.99 (1H, dd, –CH2–, J ¼ 7.80 Hz, J ¼ 17.40 Hz), 3.08 (1H,
dd, –CH2–, J ¼ 4.50 Hz, J ¼ 17.85 Hz), 4.10 (2H, q, –CH2–CH3 ester, J
¼ 6.65 Hz), 4.42 (1H, dd, 1,3-thiazolidin-4-one ring C-5, J ¼ 4.52 Hz, J
¼ 7.80 Hz), 7.78 (4H, m, Ar-H), 8.42 (1H, s, –CH]N– azomethine),
11.51 (1H, s, –NH); 13C-NMR (75 MHz): d 14.4 (–CH3), 38.7 (–CH2–),
43.9 (1,3-thiazolidin-4-one ring C-5), 61.2 (–OCH2– ester), 124.5,
129.7, 129.9, 130.7, 132.1, 132.4 (Ar-C), 133.5 (–CH]N– azome-
thine), 135.9 (1,3-thiazolidin-4-one ring C-2), 159.7 (C]O), 172.6
(1,3-thiazolidin-4-one ring C-4); HRMS (MALDI-TOF): calcd C14H14-
BrN3NaO3S (M + Na) ¼ 405.98369, found: 406.89014.
1
(C]O stretching), 2985 (C–H stretching, aliphatic); H-NMR (300
MHz): d 3.01 (1H, dd, –CH2–, J ¼ 8.40 Hz, J ¼ 17.70 Hz), 3.11 (1H, dd,
–CH2-, J ¼ 4.30 Hz, J ¼ 17.30 Hz), 3.64 (3H, s, –OCH3), 4.42 (1H, dd,
1,3-thiazolidin-4-one ring C-5, J ¼ 4.20 Hz, J ¼ 8.30 Hz), 7.56 (2H, m,
Ar-H), 7.77 (2H, d, Ar-H, J ¼ 8.40 Hz), 8.41 (1H, s, –CH]N– azo-
methine), 12.08 (1H, s, –NH); 13C-NMR (75 MHz): d 36.6 (-CH2-), 43.8
(1,3-thiazolidin-4-one ring C-5), 52.4 (–OCH3), 129.4, 130.5, 133.1,
136.5 (Ar-C), 155.5 (–CH]N– azomethine), 165.3 (1,3-thiazolidin-4-
one ring C-2), 171.3 (C]O), 175.8 (1,3-thiazolidin-4-one ring C-4);
Ethyl 2-(2-(4-nitrobenzylidene)hydrazono)-4-oxothiazolidine-
5-acetate (2f). Yellow solid; yield: 80%; melting point: 226–
228 C; Rf: 0.29 (acetone/n-hexane, 1 : 3); IR (ATR, cmꢀ1): 1033
ꢃ
(C–O stretching, ester), 1342 (C–H bending, aliphatic), 1518
(C]C ring stretching), 1643 (C]N stretching), 1728 (C]O
stretching), 2985 (C–H stretching, aliphatic); 1H-NMR (300
MHz): d 1.19 (3H, t, –CH3, ester, J ¼ 6.90 Hz), 2.97 (1H, dd,
–CH2–, J ¼ 6.90 Hz, J ¼ 16.95 Hz) 3.09 (1H, dd, –CH2–, J ¼
3.30 Hz, J ¼ 17.70 Hz), 4.10 (2H, q, –CH2–CH3 ester, J ¼ 6.60 Hz),
4.40 (1H, dd, 1,3-thiazolidin-4-one ring C-5, J ¼ 4.10 Hz, J ¼ 7.70
Hz), 8.00 (2H, d, Ar-H, J ¼ 7.80 Hz), 8.30 (2H, d, Ar-H, J ¼ 8.10
Hz), 8.53 (1H, s, –CH]N– azomethine), 12.00 (1H, s, –NH); 13C-
NMR (75 MHz): d 14.4 (–CH3, ester), 37.0 (–CH2–), 44.2 (1,3-
thiazolidin-4-one ring C-5), 61.1 (–OCH2– ester), 124.6, 128.9,
141.0, 148.5 (Ar-C), 154.0 (–CH]N– azomethine), 168.7 (1,3-
thiazolidin-4-one ring C-2), 170.8 (C]O), 176.9 (1,3-thiazolidin-
4-one ring C-4); HRMS (MALDI-TOF): calcd C14H14N4NaO5S (M
+ Na) ¼ 373.05826, found: 373.37363.
HRMS (MALDI-TOF): calcd C12H13ClN3NaO3S (M
348.0186, found: 348.04657.
+
Na)
¼
Ethyl 2-(2-(2-chlorobenzylidene)hydrazono)-4-oxothiazolidine-
5-acetate (2c). White solid; yield: 89%; melting point: 210–212 ꢃC;
Rf: 0.47 (acetone/n-hexane, 1 : 3); IR (ATR, cmꢀ1): 1086 (C–O
stretching, ester), 1445 (C–H bending, aliphatic), 1601 (C]C ring
stretching), 1647 (C]N stretching), 1710 (C]O stretching), 1731
(C]O stretching), 2985 (C–H stretching, aliphatic); 1H-NMR (300
MHz): d 1.17 (3H, t, –CH3, ester, J ¼ 7.03 Hz), 2.67 (1H, dd, –CH2-,
J ¼ 9.69 Hz, J ¼ 16.95 Hz), 3.08 (1H, dd, –CH2–, J ¼ 3.70 Hz, J ¼
16.95 Hz), 4.08 (2H, q, –CH2–CH3 ester, J ¼ 6.45 Hz), 4.42 (1H, dd,
1,3-thiazolidin-4-one ring C-5, J ¼ 4.20 Hz, J ¼ 8.30 Hz), 7.56 (2H,
m, Ar-H), 7.77 (2H, d, Ar-H, J ¼ 8.40 Hz), 8.41 (1H, s, –CH]N–
azomethine), 12.02 (1H, s, –NH); 13C-NMR (75 MHz): d 14.5 (–CH3
ester), 38.7 (–CH2–), 45.7 (1,3-thiazolidin-4-one ring C-5), 60.8
(–OCH2– ester), 127.5, 127.8, 131.2, 132.1 (Ar-C), 136.7 (-CH]N-
azomethine), 153.6 (1,3-thiazolidin-4-one ring C-2), 173.5 (C]O),
181.9 (1,3-thiazolidin-4-one ring C-4); HRMS (MALDI-TOF): calcd
C14H14ClN3NaO3S (M + Na) ¼ 362.03421, found: 362.12087.
Ethyl 2-(2-(3-nitrobenzylidene)hydrazono)-4-oxothiazolidine-
5-acetate (2g). White solid; yield: 88%; melting point: 191–
193 C; Rf: 0.27 (acetone/n-hexane, 1 : 3); IR (ATR, cmꢀ1): 1080
ꢃ
(C–O stretching, ester), 1354 (C–H bending, aliphatic), 1523
(C]C ring stretching), 1627 (C]N stretching), 1732 (C]O
stretching), 3093 (C–H stretching, aliphatic); 1H-NMR (300
2496 | RSC Adv., 2021, 11, 2487–2500
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MHz): d 1.19 (3H, t, –CH3 ester, J ¼ 7.00 Hz), 3.03 (1H, dd, –CH2-, C-5, J ¼ 4.50 Hz, J ¼ 7.80 Hz), 7.01 (1H, t, Ar, J ¼ 7.50 Hz), 7.10
J ¼ 7.50 Hz, J ¼ 17.40 Hz), 3.12 (1H, dd, –CH2–, J ¼ 4.80 Hz, J ¼ (1H, d, Ar, J ¼ 8.40 Hz), 7.45 (1H, t, Ar-H, J ¼ 8.40 Hz), 7.82 (1H,
9.00 Hz), 4.10 (2H, q, –CH2–CH3 ester, J ¼ 6.60 Hz), 4.45 (1H, dd, d, Ar-H, J ¼ 7.80 Hz), 8.60 (1H, s, –CH]N– azomethine), 12.07
1,3-thiazolidin-4-one ring C-5, J ¼ 4.80 Hz, J ¼ 7.50 Hz), 7.76 (1H, s, –NH); 13C-NMR (75 MHz): d 14.5 (-CH3, ester), 36.9
(1H, t, Ar-H, J ¼ 8.00 Hz), 8.1 (1H, d, Ar-H, J ¼ 7.80 Hz), 8.29 (1H, (–CH2–), 43.9 (1,3-thiazolidin-4-one ring C-5), 56.2 (–OCH3), 61.1
dd, Ar-H, J ¼ 1.65 Hz, J ¼ 8.25 Hz), 8.50 (1H, s, Ar-H), 8.58 (1H, s, (–OCH2– ester), 112.4, 121.2, 122.5, 126.5, 132.7, 151.5 (Ar-C),
–CH]N– azomethine), 12.17 (1H, s, –NH); 13C-NMR (75 MHz): 158.5 (–CH]N– azomethine), 170.7 (1,3-thiazolidin-4-one ring
d 14.4 (–CH3 ester), 36.6 (–CH2–), 43.9 (1,3-thiazolidin-4-one ring C-2), 176.2 (C]O), 189.2 (1,3-thiazolidin-4-one ring C-4); HRMS
C-5), 61.1 (–OCH2– ester), 122.3, 125.3, 130.9, 133.9, 136.4, 148.6 (MALDI-TOF): calcd C15H17N3NaO4S (M + Na) ¼ 358.08375,
(Ar-C), 154.9 (–CH]N– azomethine), 166.3 (1,3-thiazolidin-4- found: 358.19232.
one ring C-2), 170.7 (C]O), 175.8 (1,3-thiazolidin-4-one ring
Ethyl
2-(2-(3-hydroxybenzylidene)hydrazono)-4-
C-4); HRMS (MALDI-TOF): calcd C14H14N4NaO5S (M + Na) ¼ oxothiazolidine-5-acetate (2k). Off white solid; yield: 80%;
373.05826, found: 373.27241.
melting point: 236–238 ꢃC; Rf: 0.26 (acetone/n-hexane, 1 : 3); IR
Ethyl 2-(2-(2-nitrobenzylidene)hydrazono)-4-oxothiazolidine- (ATR, cmꢀ1): 1022 (C–O stretching, ester), 1330 (C–H bending,
5-acetate (2h). Off white solid; yield: 86%; melting point: 189– aliphatic), 1600 (C]C ring stretching), 1647 (C]N stretching),
190 C; Rf: 0.37 (acetone/n-hexane, 1 : 3); IR (ATR, cmꢀ1): 1086 1732 (C]O stretching), 2993 (C–H stretching, aliphatic); 1H-
ꢃ
(C–O stretching, ester), 1387 (C–H bending, aliphatic), 1589 NMR (300 MHz): d 1.18 (3H, t, –CH3 ester, J ¼ 7.05 Hz), 2.99
(C]C ring stretching), 1643 (C]N stretching), 1730 (C]O (1H, dd, –CH2-, J ¼ 8.10 Hz, J ¼ 17.70 Hz), 3.08 (1H, dd, –CH2–, J
stretching), 3089 (C–H stretching, aliphatic); 1H-NMR (300 ¼ 4.50 Hz, J ¼ 17.70 Hz), 4.17 (2H, q, –CH2–CH3 ester, J ¼ 6.90
MHz): d 1.19 (3H, t, –CH3 ester, J ¼ 7.01 Hz), 3.09 (1H, dd, –CH2-, Hz), 4.40 (1H, dd, 1,3-thiazolidin-4-one ring C-5, J ¼ 4.50 Hz, J ¼
J ¼ 7.55 Hz, J ¼ 17.45 Hz), 3.13 (1H, dd, –CH2–, J ¼ 4.90 Hz, J ¼ 7.80 Hz), 6.87 (1H, m, Ar-H), 7.19 (3H, m, Ar-H), 8.30 (1H, s,
9.00 Hz), 4.12 (2H, q, –CH2–CH3 ester, J ¼ 6.65 Hz), 4.42 (1H, dd, –CH]N– azomethine), 9.74 (1H, s, –OH), 12.04 (1H, s, –NH);
1,3-thiazolidin-4-one ring C-5, J ¼ 4.85 Hz, J ¼ 7.55 Hz), 7.78 13C-NMR (75 MHz): d 14.5 (–CH3 ester), 36.9 (–CH2–), 43.8 (1,3-
(4H, m, Ar-H), 8.60 (1H, s, –CH]N– azomethine); 13C-NMR (75 thiazolidin-4-one ring C-5), 61.1 (–OCH2– ester), 113.7, 118.5,
MHz): d 14.4 (–CH3 ester), 36.8 (–CH2–), 42.9 (1,3-thiazolidin-4- 119.8, 130.3, 135.8, 158.1 (Ar-C), 156.9 (–CH]N– azomethine),
one ring C-5), 61.2 (–OCH2– ester), 123.4, 126.3, 131.9, 133.9, 170.7 (1,3-thiazolidin-4-one ring C-2), 175.8 (C]O), 189.2 (1,3-
136.5, 149.6 (Ar-C), 155.9 (–CH]N– azomethine), 165.3 (1,3- thiazolidin-4-one ring C-4); HRMS (MALDI-TOF): calcd C14H15
thiazolidin-4-one ring C-2), 171.7 (C]O), 176.9 (1,3-thiazolidin- N3NaO4S (M + Na) ¼ 344.0681, found: 344.16745.
-
4-one ring C-4); HRMS (MALDI-TOF): calcd C14H14N4NaO5S (M
Ethyl
2-(2-(2-hydroxybenzylidene)hydrazono)-4-
+ Na) ¼ 373.05826, found: 373.27236.
oxothiazolidine-5-acetate (2l). Light green solid; yield: 91%;
Ethyl 2-(2-(4-methoxybenzylidene)hydrazono)-4-oxothiazolidiꢃne- melting point: 248–250 ꢃC; Rf: 0.33 (acetone/n-hexane, 1 : 3); IR
5-acetate (2i). White solid; yield: 94%; melting point: 201–203 C; (ATR, cmꢀ1): 1014 (C–O stretching, ester), 1338 (C–H bending,
Rf: 0.36 (acetone/n-hexane, 1 : 3); IR (ATR, cmꢀ1): 1033 (C–O aliphatic), 1525 (C]C ring stretching), 1639 (C]N stretching),
stretching, ester), 1311 (C–H bending, aliphatic), 1512 (C]C ring 1716 (C]O stretching), 2989 (C–H stretching, aliphatic); 1H-
stretching), 1651 (C]N stretching), 1720 (C]O stretching), 2989 NMR (300 MHz): d 1.18 (3H, t, –CH3 ester, J ¼ 6.75 Hz), 3.03
(C–H stretching, aliphatic); 1H-NMR (300 MHz): d 1.18 (3H, t, –CH3 (1H, dd, –CH2–, J ¼ 8.10 Hz, J ¼ 17.40 Hz), 3.09 (1H, dd, –CH2-, J
ester, J ¼ 7.20 Hz), 2.97 (1H, dd, –CH2–, J ¼ 8.10 Hz, J ¼ 17.40 Hz), ¼ 4.20 Hz, J ¼ 7.10 Hz), 4.10 (2H, q, –CH2–CH3 ester, J ¼ 6.45
3.07 (1H, dd, –CH2-, J ¼ 4.35 Hz, J ¼ 17.55 Hz), 3.81 (3H, s, –OCH3), Hz), 4.50 (1H, dd, 1,3-thiazolidin-4-one ring C-5, J ¼ 4.20 Hz, J ¼
4.09 (2H, q, –CH2–CH3 ester, J ¼ 6.30 Hz), 4.38 (1H, dd, 1,3- 6.30 Hz), 6.95 (2H, m, Ar-H), 7.35 (1H, m, Ar-H), 7.65 (1H, m, Ar-
thiazolidin-4-one ring C-5, J ¼ 4.50 Hz, J ¼ 7.80 Hz), 7.03 (2H, d, Ar- H), 8.64 (1H, s, –CH]N– azomethine), 10.87 (1H, s, –OH), 12.16
H, J ¼ 9.00 Hz), 7.69 (2H, d, Ar-H, J ¼ 8.70 Hz), 8.33 (1H, s, –CH]N– (1H, s, –NH); 13C-NMR (75 MHz): d 14.4 (-CH3, ester), 36.6
azomethine), 12.03 (1H, s, –NH); 13C-NMR (75 MHz): d 14.4 (–CH3 (–CH2–), 44.2 (1,3-thiazolidin-4-one ring C-5), 61.2 (–OCH2–
ester), 36.9 (–CH2–), 43.8 (1,3-thiazolidin-4-one ring C-5), 55.8 ester), 116.8, 118.9, 119.9, 130.8, 132.7, 159.1 (Ar-C), 158.5
(–OCH3), 61.1 (–OCH2– ester), 114.8, 127.3, 129.9, 161.7 (Ar-C), 156.2 (–CH]N– azomethine), 170.6 (1,3-thiazolidin-4-one ring C-2),
(-CH]N- azomethine), 170.7 (1,3-thiazolidin-4-one ring C-2), 175.9 175.5 (C]O), 189.2 (1,3-thiazolidin-4-one ring C-4); HRMS
(C]O), 189.2 (1,3-thiazolidin-4-one ring C-4); HRMS (MALDI-TOF): (MALDI-TOF): calcd C14H15N3NaO4S (M + Na) ¼ 344.0681,
calcd C15H17N3NaO4S (M + Na) ¼ 358.08375, found: 359.64858.
Ethyl 2-(2-(2-methoxybenzylidene)hydrazono)-4-
found: 344.11239.
Ethyl
2-(2-(4-methylbenzylidene)hydrazono)-4-
oxothiazolidine-5-acetate (2j). White solid; yield: 96%; melting oxothiazolidine-5-acetate (2m). Light yellow solid; yield: 77%;
point: 189–191 ꢃC; Rf: 0.39 (acetone/n-hexane, 1 : 3); IR melting point: 253–255 ꢃC; Rf: 0.33 (acetone/n-hexane, 1 : 3); IR
(ATR, cmꢀ1): 1018 (C–O stretching, ester), 1334 (C–H bending, (ATR, cmꢀ1): 1014 (C–O stretching, ester), 1336 (C–H bending,
aliphatic), 1521 (C]C ring stretching), 1639 (C]N stretching), aliphatic), 1618 (C]C ring stretching), 1647 (C]N stretching),
1720 (C]O stretching), 2985 (C–H stretching, aliphatic); 1H- 1712, 1726 (C]O stretching), 2987 (C–H stretching, aliphatic);
NMR (300 MHz): d 1.18 (3H, t, –CH3, ester, J ¼ 7.20 Hz), 2.97 1H-NMR (300 MHz): d 1.19 (3H, t, –CH3 ester, J ¼ 7.00 Hz), 2.34
(1H, dd, –CH2–, J ¼ 8.10 Hz, J ¼ 17.40 Hz), 3.07 (1H, dd, –CH2–, J (3H, s, –CH3), 2.99 (1H, dd, –CH2–, J ¼ 8.10 Hz, J ¼ 17.70 Hz),
¼ 4.50 Hz, J ¼ 17.40 Hz), 3.88 (3H, s, –OCH3), 4.03 (2H, q, –CH2– 3.08 (1H, dd, –CH2-, J ¼ 4.50 Hz, J ¼ 17.40 Hz), 4.10 (2H, q,
CH3 ester, J ¼ 6.00 Hz), 4.38 (1H, dd, 1,3-thiazolidin-4-one ring –CH2–CH3 ester, J ¼ 6.30 Hz), 4.41 (1H, dd, 1,3-thiazolidin-4-one
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ring C-5, J ¼ 4.50 Hz, J ¼ 7.50 Hz), 7.27 (2H, d, Ar-H, J ¼ 8.10 Hz), Ar-H, J ¼ 8.10 Hz), 7.18 (1H, dd, Ar-H, J ¼ 1.50 Hz, J ¼ 8.40 Hz),
7.65 (2H, d, Ar-H, J ¼ 8.10 Hz), 8.36 (1H, s, –CH]N– azome- 7.31 (1H, d, Ar-H, J ¼ 1.50 Hz), 8.26 (1H, s, –CH]N– azome-
thine), 12.07 (1H, s, –NH); 13C-NMR (75 MHz): d 14.5 (–CH3 thine), 9.65 (1H, s, –OH), 11.94 (1H, s, –NH); 13C-NMR (75 MHz):
ester), 21.6 (–CH3), 36.8 (–CH2–), 43.7 (1,3-thiazolidin-4-one ring d solubility issue. HRMS (MALDI-TOF): calcd C15H17N3NaO5S
C-5), 61.1 (–OCH2–, ester), 128.1, 129.9, 131.9, 141.1 (Ar-C), (M + Na) ¼ 374.07866, found: 374.31122.
156.7 (–CH]N– azomethine), 163.9 (1,3-thiazolidin-4-one ring
C-2), 170.7 (C]O), 175.8 (1,3-thiazolidin-4-one ring C-4); HRMS
In vitro evaluation and mechanism of action assays
(MALDI-TOF): calcd C15H18N3NaO3S (M + Na) ¼ 342.08883,
found: 342.11165.
Cell assays. The trypomastigote forms of T. cruzi (Y strain)
Ethyl
2-(2-(2-methylbenzylidene)hydrazono)-4- were maintained in DMEM (Dulbecco's Modied Eagle
oxothiazolidine-5-acetate (2n). White solid; yield: 93%; Medium) medium, pH 7.2 supplemented with 10% SFB and
melting point: 226–228 ꢃC; Rf: 0.28 (acetone/n-hexane, 1 : 3); IR incubated at 37 ꢃC with 5% CO2. LLC-MK2 cells (Monkey kidney
(ATR, cmꢀ1): 1022 (C–O stretching, ester), 1338 (C–H bending, epithelial cells [Macaca mulatta]; CCL-7; American Type Culture
aliphatic), 1523 (C]C ring stretching), 1643 (C]N stretching), Collection, Rockville, MD, USA) were cultured in DMEM
1724 (C]O stretching), 2993 (C–H stretching, aliphatic); 1H- medium, pH 7.2, supplemented with 10% L-glutamine SFB, and
NMR (300 MHz): d 1.18 (3H, t, –CH3, ester, J ¼ 7.20 Hz), 2.49 incubated at 37 ꢃC in a 5% CO2 atmosphere. L. amazonensis and
(3H, s, –CH3), 2.98 (1H, dd, –CH2–, J ¼ 7.95 Hz, J ¼ 17.55 Hz), L. infantum (WHOM/00LTB0016 and MHOM/MA/67/ITMAP-263,
4.09 (2H, q, –CH2–CH3 ester, J ¼ 7.20 Hz), 4.39 (1H, dd, 1,3- respectively) promastigote forms were maintained in Schneid-
thiazolidin-4-one ring C-5, J ¼ 4.50 Hz, J ¼ 8.10 Hz), 7.31 (3H, m, er's insect medium, pH 7.2 supplemented with 10% SFB and
ꢃ
Ar-H), 7.76 (1H, m, Ar-H), 8.92 (1H, s, –CH]N– azomethine), incubated at 26 C.
12.09 (1H, s, –NH); 13C-NMR (75 MHz): d 14.5 (–CH3, ester), 20.4
Cytotoxicity on mammalian cells. Cytotoxicity was evaluated
(–CH3), 36.9 (–CH2-), 43.9 (1,3-thiazolidin-4-one ring C-5), 61.1 in LLC-MK2 cells using the MTT reduction cell viability assay
(–OCH2–, ester), 126.6, 128.4, 130.6, 131.6, 132.6, 137.9 (Ar-C), (MOSSMANN, 1983). This method is based on the ability of
155.8 (–CH]N– azomethine), 170.7 (1,3-thiazolidin-4-one ring mitochondrial dehydrogenase enzymes to convert the water-
C-2), 176.3 (C]O), 189.2 (1,3-thiazolidin-4-one ring C-4); HRMS soluble
tretazolium
(3-[4,5-dimethylthiazol-2-yl]-2,5-
(MALDI-TOF): calcd C15H18N3NaO3S (M + Na) ¼ 342.08883, diphenyltetra-zolium bromide) salt to an insoluble purple
found: 342.07265.
substance called Formazan.
Ethyl
2-((2-(3,4-dihydronaphthalen-1(2H)-ylidene))
For this purpose, LLC-MK2 cells (2.5 ꢂ 105 cells per mL) in
hydrazono)-4-oxo-thiazolidin-5-acetate (2o). Brown solid; yield: DMEM medium supplemented with 10% SFB were cultured in
69%; melting point: 246–248 ꢃC; Rf: 0.30 (acetone/n-hexane, 96-well plates, and maintained at 37 C and 5% CO2 for 24 h.
ꢃ
1 : 3); IR (ATR, cmꢀ1): 1031 (C–O stretching, ester), 1346 (C–H The substances were then added at increasing concentrations
bending, aliphatic), 1616 (C]C ring stretching), 1716 (C]O (10, 50, 100, 500, and 1000 mM), and the plate incubated for
stretching), 2909 (C–H stretching, aliphatic); 1H-NMR (300 96 hours. Aer treatment, cells were washed with PBS (pH 7.2)
MHz): d 1.18 (3H, t, –CH3 ester, J ¼ 7.20 Hz), 1.81 (2H, m, –CH2–, and incubated in the presence of MTT (2 mg mLꢀ1) for 4 hours.
tetralone), 2.79 (4H, m, tetralone), 2.98 (1H, dd, –CH2–, J ¼ The supernatant was removed, the Formazan crystals were
7.80 Hz, J ¼ 17.40 Hz), 3.07 (1H, dd, –CH2–, J ¼ 4.50 Hz, J ¼ solubilized in DMSO, and the absorbance reading was taken at
17.40 Hz), 4.09 (2H, q, –CH2–CH3 ester, J ¼ 6.90 Hz), 4.37 (1H, 570 nm on a plate spectrophotometer (Power Wave XS-BioTek).
dd, 1,3-thiazolidin-4-one ring C-5, J ¼ 4.50 Hz, J ¼ 7.80 Hz), 7.26 The percentage of viable cells was calculated concerning the
(3H, m, tetralone), 8.06 (1H, m, tetralone), 12.01 (1H, s, –NH); control. The CC50 values (50% cytotoxic concentration) were
13C-NMR (75 MHz): d 14.6 (–CH3 ester), 22.3, 27.4, 29.6 (3-CH2–, determined by nonlinear regression analysis.
tetralone), 36.9 (–CH2–), 43.4 (1,3-thiazolidin-4-one ring C-5),
Amastigote assay. For the amastigote assay, T. cruzi amasti-
61.1 (–OCH2–, ester), 125.0, 126.6, 129.3, 130.2, 132.6, 140.9 gote forms of Tulahuen strain expressing the Escherichia coli
(Ar-C), 160.3 (–C]N–), 162.9 (1,3-thiazolidin-4-one ring C-2), beta-galactosidase gene were grown on a monolayer of mouse L-
170.7 (C]O), 175.6 (1,3-thiazolidin-4-one ring C-4); HRMS 929 broblasts. Cultures assayed for beta-galactosidase activity
(MALDI-TOF): calcd C17H19N3NaO5S (M + Na) ¼ 368.01448, were grown in RPMI 1640 medium without phenol red plus 10%
found: 368.27513.
fetal bovine serum and glutamine. Ninety-six-well tissue culture
Ethyl 2-(2-(4-hydroxy-3-methoxybenzylidene)hydrazono)-4- microplates were seeded with L-929 broblasts at 4.0 ꢂ 103 per
oxothiazolidine-5-acetate (2p). Off white solid; yield: 91%; well in 80 mL, and incubated overnight at 37 ꢃC, 5% CO2. Beta-
melting point: 226–228 ꢃC; Rf: 0.30 (acetone/n-hexane, 1 : 3); IR galactosidase-expressing trypomastigotes were then added at
(ATR, cmꢀ1): 1026 (C–O stretching, ester), 1338 (C–H bending, 4.0 ꢂ 104 per well in 20 mL. Aer 2 hours, the medium with
aliphatic), 1595 (C]C ring stretching), 1637 (C]N stretching), trypomastigotes that have not penetrated the cells was dis-
1724 (C]O stretching), 2990 (C–H stretching, aliphatic), 3375 carded, and replaced by 200 mL of fresh medium. Aer 48 hours,
(–OH, stretching); 1H-NMR (300 MHz): d 1.18 (3H, t, –CH3, ester, the medium was discarded again, and replaced by 180 mL of
J ¼ 7.20 Hz), 3.04 (1H, dd, –CH2–, J ¼ 4.80 Hz, J ¼ 13.20 Hz), 3.01 fresh medium and 20 mL of test compounds dissolved in DMSO.
(1H, dd, –CH2–, J ¼ 4.80 Hz, J ¼ 13.20 Hz), 3.80 (3H, s, –OCH3), Each compound was tested in quadruplicate. Aer 7 days of
4.09 (2H, q, –CH2–CH3 ester, J ¼ 7.20 Hz), 4.38 (1H, dd, 1,3- culture development, chlorophenol red beta-D-galactopyrano-
thiazolidin-4-one ring C-5, J ¼ 4.50 Hz, J ¼ 7.50 Hz), 6.84 (1H, d, side at 100 mM and Nonidet P-40 at 0.1% were added to the
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ꢃ
plates, and incubated overnight at 37 C. The absorbance was binding buffer (Annexin V Binding Buffer- BD Pharmingen™,
measured at 570 nm in an automated microplate reader. BZD at USA). For labeling, 10 mL of propidium iodide (50 mg mLꢀ1) and
3.81 mM (IC50) was used as a positive control. The results are 5 mL of Annexin-FITC (BD Pharmingen™, USA) were added for
expressed as a percentage of parasite growth inhibition. Two 15 min at room temperature in the dark. Flow cytometry was
independent experiments were performed.
conducted in a FACS Caliber (Becton & Dickinson, USA). For
Trypomastigote assay. Trypomastigote forms (1 ꢂ 107 para- each sample, we acquired 20 000 events and the data were
sites per mL) in DMEM medium supplemented with 10% SFB analyzed using Cell Quest soware (Becton & Dickinson, USA).
were added to 96-well microplates in the presence and absence Assays were conducted in triplicate. For signicance analysis,
of increasing concentrations (1, 5, 10, 50, 100 mM) of the ANOVA and Dunnett's test was used, taking into account p <
ꢃ
substances, and incubated for 24 hours at 37 C in a 5% CO2 0.05.
atmosphere. The result was obtained by observing the
mortality, allowing the determination of parasite viability using
the Brener method (BRENER, 1962). It consists of the parasite
Conclusions
count of 50 elds of 5 mL of the slide-cover slip sample, and the
EC50 (effective concentration for 50% of the parasites) was
nally calculated by nonlinear regression.
Ultrastructural analysis. The samples of T. cruzi trypomas-
tigote were cultured for 24 hours in RPMI 1640 medium (Sigma-
Eleven 1,3-thiazole-4-carboxylates and sixteen 4-thiazolidinone-
acetates were obtained with reasonable yields using a simple
methodology by bioisosteric and esterication strategies. These
molecules were evaluated for their trypanocidal, leishmanicidal
and cytotoxicity. Some of them exhibited very good trypanocidal
Aldrich, St. Louis, MO, USA); buffered to pH 7.5 and supple-
activity, especially compounds 1f, 1h, 1g, 1a, and 2d. From the
mented with HEPES (20 mM), 10% fetal bovine serum, peni-
tested scaffolds, the 1,3-thiazoles exhibited better trypanocidal
cillin (100
U )
mLꢀ1 and streptomycin (100 mg mLꢀ1),
activity than 4-thiazolidinones. The ow cytometry study
showed that compound 1f (IC50 ¼ 0.83 mM) kills the parasite via
necrosis and apoptosis. However, the activity was not observed
in the amastigote form. Scanning electron microscopy analysis
showed that compound 1f at IC50 concentrations promoted
alterations in the shape, agella and surface of the body,
inducing parasite death. Besides, compounds 1b, 1f, 2d and 2m
exhibited better IC50 values for both L. amazonensis and L.
infantum in comparison to standard Miltefosine. Our work
corroborated the bioisosteric and esterication approaches for
drug optimization and discovery with regards to chagas disease
and leishmaniasis through 1,3-thiazole and 4-thiazolidinone
scaffolds.
containing the compound at the IC50 concentration (0.8 mM),
twice (1.66 mM) and four times (3.32 mM) the value of IC50. The
parasites were collected, washed in PBS and xed with 2.5%
glutaraldehyde, 4% formaldehyde and 0.1 M cacodylate buffer
at pH 6.8. They were then post xed in 2% osmium tetroxide
(OsO4) in a 0.1 M cacodylate buffer at pH 6.8, and processed for
routine scanning electron microscopy. 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 (NPT/FIOCRUZ-PE). The controls were composed
of untreated cells (negative control) and those treated with BZD
(positive control).
L. amazonensis and L. infantum assay. Promastigotes were
incubated (1 ꢂ 106 cells per mL, Schneider's medium with 10%
Fetal Bovine Serum) in a 96-well microplate with compounds in
eight different concentrations (1.5 to 200 mg mLꢀ1) for 72 hours
at 26 ꢃC. Cells without treatment were used as negative controls,
and Miltefosine was used as the standard. Cell growth was
assessed and IC50/72 hours was determined by regression
analyses. Assays were conducted in triplicate.
Conflicts of interest
The authors declare no conict of interest.
Acknowledgements
Ana Cristina Lima Leite is receiving a CNPq senior fellowship.
The authors are thankful for the Centro de Tecnologias
´
Estrategicas do Nordeste (CETENE) for recording LCMS of all
Cell death assessment through ow cytometry
compounds. All authors declare no competing nancial
interest. The author Muhammad Haroon is grateful to Prof. Dr
Alan S. Goldman. Department of chemistry and chemical
biology, Rutgurs University New Brunswick 610 Taylor Road,
Piscataway, NJ 08854-8087 USA for providing NMR facility. The
corresponding author is thankful to Prof. Dr Shahid Hameed,
Department of Chemistry, QAU, Islamabad-45320, Pakistan for
providing NMR facility.
Aer conrmation of trypanocidal activity, Annexin-FITC/
propidium iodide labeling was used to characterize cell death
modalities induced by incubation with compounds. Metacyclic
trypomastigotes were collected from the supernatant of infected
L929 cells, and then seeded at 4 ꢂ 105 cells per well in RPMI-
1640 medium. Compound 1f was dissolved in DMSO, and
added to wells at IC50, 2 ꢂ IC50, and 4 ꢂ IC50 concentrations.
Benznidazole (IC50, 2 ꢂ IC50, and 4 ꢂ IC50) and the culture
medium were used as a positive and negative control, respec-
tively. Plates were incubated at the same conditions used for
anti-trypomastigote activity (37 ꢃC, 24 hours). Briey, aer
treatment, parasites were washed with PBS and resuspended in
References
1 Laboratory of immunomodulation, Department of
Protozoology/IOC – FIOCRUZ, The Leishmaniasis, 1997,
© 2021 The Author(s). Published by the Royal Society of Chemistry
RSC Adv., 2021, 11, 2487–2500 | 2499

View Article Online
RSC Advances
Paper
accessed on 28th August 2019, http://www.dbbm.ocruz.br/ 19 G. Cihan-Ustundag, E. Gursoy, L. Naesens, N. Ulusoy
¨
˘
¨
¨
¨
tropical/leishman/leishext/html/morfologia.html.
Guzeldemirci and G. Çapan, Bioorg. Med. Chem., 2016,
2 Pan American Health Organization, Leishmaniasis:
24(2), 240–246.
Epidemiological Report in the Americas, Pan American 20 D. Secci, S. Carradori, B. Bizzarri, P. Chimenti, C. De Monte,
Health
www.paho.org/leishmaniasis.
3 J. A. Urbina, J. Eukaryotic Microbiol., 2015, 62, 149–156.
Organization,
Washington,
2019,
http://
A. Mollica, D. Rivanera, A. Zicari, E. Mari, G. Zengin and
A. Aktumsek, Eur. J. Med. Chem., 2016, 117, 144–156.
21 D. R. Moreira, A. C. Leite, M. V. Cardoso, R. M. Srivastava,
M. Z. Hernandes, M. M. Rabello, L. F. da Cruz,
R. S. Ferreira, d. C. A. Simone, C. S. Meira,
E. T. Guimaraes, A. C. da Silva, T. A. dos Santos,
V. R. Pereira and M. B. Soares, ChemMedChem, 2014, 9(1),
177–188.
´
4 M. J. Pinazo, L. Guerrero, E. Posada, E. Rodrıguez, D. Soy and
J. Gascon, Antimicrob. Agents Chemother., 2013, 57, 390e395.
5 D. H. Molyneux, L. Savioli and D. Engels, Lancet, 2017, 389,
312–325.
6 J. A. Urbina and R. Docampo, Trends Parasitol., 2003, 19,
495–501.
22 G. B. de Oliveira Filho, M. V. de Oliveira Cardoso,
´
7 J. Urbina, Chemotherapy of chagas disease, Curr. Pharm.
Des., 2002, 8, 287–295.
8 A. L. Ribeiro, M. P. Nunes, M. M. Teixeira and
M. O. C. Rocha, Nat. Rev. Cardiol., 2012, 9, 576–589.
J. W. Espındola, L. F. Ferreira, C. A. de Simone,
R. S. Ferreira, P. L. Coelho, C. S. Meira, M. D. R. Moreira,
M. B. Soares and A. C. Lima Leite, Bioorg. Med. Chem.,
2015, 23(23), 7478–7486.
´
´
´
9 C. Marın, I. Ramırez-Macıas, M. J. Rosales, B. Muro, 23 G. B. de Oliveira Filho, M. V. d. O. Cardoso,
´
´
´
F. Reviriego, P. Navarro, V. J. Aran and M. Sanchez-
J. W. P. Espındola, D. A. Oliveira e Silva, R. S. Ferreira,
Moreno, Acta Trop., 2015, 148, 170–178.
10 S. Espuelas, D. Plano, P. Nguewa, M. Font, J. A. Palop,
P. L. Coelho, P. S. dos Anjos, E. d. S. Santos, C. S. Meira,
D. R. M. Moreira, M. B. P. Soares and A. C. L. Leite, Eur. J.
Med. Chem., 2017, 141, 346–361.
´
J. M. Irache and C. Sanmartın, Curr. Med. Chem., 2012, 19,
4259–4288.
24 M. V. O. Cardoso, G. B. Oliveira Filho, L. R. P. Siqueira,
´
11 World Health Organization, Defeating Neglected Tropical
Diseases: Progress, Challenges and Opportunities, WHO/CDS/
NTD/2019.01, 2019, access in 8th September 2019, https://
apps.who.int/iris/handle/10665/325755.
J. W. P. Espındola, E. B. D. Silva, A. P. O. Mendes,
V. R. A. Pereira, M. C. A. B. Castro, R. S. Ferreira,
F. S. Villela, F. M. R. D. Costa, C. S. Meira,
D. R. M. Moreira, M. B. P. Soares and A. C. L. Leite, Eur. J.
Med. Chem., 2019, 180, 191–203.
12 R. W. DeSimone, K. S. Currie, S. A. Mitchell, J. W. Darrow
and D. A. Pippin, Comb. Chem. High Throughput Screening, 25 A. S. dos Santos Aliança, A. R. Oliveira, A. P. S. Feitosa,
2004, 7(5), 473–494.
K. R. C. Ribeiro, M. C. A. B. de Castro, A. C. L. Leite and
13 J. Polanski, A. Kurczyk, A. Bak and R. Musiol, Curr. Med.
Chem., 2012, 19(13), 1921–1945.
F. A. Brayner, Eur. J. Pharm. Sci., 2017, 105, 1–10.
26 C. M. Queiroz, G. B. de Oliveira Filho, J. W. P. Espındola,
´
´
14 D. Gonzalez Cabrera, F. Douelle, T.-S. Feng, A. T. Nchinda,
A. V. do Nascimento, A. S. dos Santos Aliança, V. M. B. de
Lorena, A. P. S. Feitosa, P. R. da Silva, L. C. Alves,
A. C. L. Leite and F. A. Brayner, Med. Chem. Res., 2020, 29,
2050–2206.
Y. Younis, K. L. White, Q. Wu, E. Ryan, J. N. Burrows,
D. Waterson, M. J. Witty, S. Wittlin, S. A. Charman and
K. J. Chibale, Med. Chem., 2011, 54(21), 7713–7719.
15 P. A. de Moraes Gomes, M. de Oliveira Barbosa, E. Farias 27 M. Haroon, T. Akhtar, A. C. d. S. Santos, V. R. A. Pereira,
Santiago, M. V. de Oliveira Cardoso, N. T. Capistrano
Costa, M. Z. Hernandes, D. R. Moreira, A. C. da Silva,
T. A. Dos Santos, V. R. Pereira, F. A. Brayner Dos Santos,
G. A. do Nascimento Pereira, R. S. Ferreira and A. C. Leite,
Eur. J. Med. Chem., 2016, 121, 387–398.
L. F. G. R. Ferreira, M. Z. Hernandes, R. E. O. Rocha,
R. S. Ferreira, P. A. T. d. M. Gomes, F. A. de Sousa,
M. C. H. d. B. Dias, M. N. Tahir, S. Hameed and
A. C. L. Leite, ChemistrySelect, 2019, 4, 13163–13172.
28 C. da Silva, T. A. R. Dos Santos, I. V. B. da Silva, M. V. G. de
Oliveira, D. R. M. Moreira, A. C. L. Leite and V. R. A. Pereira,
Exp. Parasitol., 2017, 177, 57–65.
16 G. C. Moraski, N. Seeger, P. A. Miller, A. G. Oliver,
H. I. Boshoff, S. Cho, S. Mulugeta, J. R. Anderson,
S. G. Franzblau and M. J. Mille, ACS Infect. Dis., 2016, 2, 29 E. Izumi, T. V. J. V. F. Ueda-Nakamura, A. C. Pinto and
393–398. C. V. Nakamura, J. Med. Chem., 2012, 55, 2994–3001.
17 M. V. Cardoso, L. R. de Siqueira, E. B. da Silva, L. B. Costa, 30 L. P. Borba-Santos, T. Gagini, K. Ishida, W. de Souza and
M. Z. Hernandes, M. M. Rabello, R. S. Ferreira, L. F. da S. Rozental, J. Med. Microbiol., 2015, 64(4), 415–422.
Cruz, D. R. Moreira, V. R. Pereira, M. C. de Castro, 31 S. Pietkiewicz, J. H. Schmidt and I. N. Lavrik, J. Immunol.
P. V. Bernhardt and A. C. Leite, Eur. J. Med. Chem., 2014,
86, 48–59.
18 H. M. Ashour, I. M. El-Ashmawy and A. E. Bayad, Monatsh.
Chem., 2016, 147, 605–618.
Methods, 2015, 423, 99–103.
¨
32 G. F. Pauli, S.-N. Chen, C. Simmler, D. C. Lankin, T. Godecke,
B. U. Jaki, J. B. Friesen, J. B. McAlpine and J. G. Napolitano, J.
Med. Chem., 2014, 57, 9220–9231.
2500 | RSC Adv., 2021, 11, 2487–2500
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