2-(phenylthio)ethylidene derivatives as anti-Trypanosoma cruzi compounds: Structural design, synthesis and antiparasitic activity

Article abstract of DOI:10.1016/j.ejmech.2019.07.018

Chagas disease is an illness caused by the protozoan parasite Trypanosoma cruzi. The current chemotherapy is based on benznidazole, and, in some countries, Nifurtimox, which is effective in the acute phase of the disease, but its efficacy in the chronic p

Full text of DOI:10.1016/j.ejmech.2019.07.018

Accepted Manuscript
2-(phenylthio)ethylidene derivatives as anti-Trypanosoma cruzi compounds: Structural
design, synthesis and antiparasitic activity
Marcos Veríssimo de Oliveira Cardoso, Gevanio Bezerra de Oliveira Filho, Lucianna
Rabelo Pessoa de Siqueira, José Wanderlan Pontes Espíndola, Elany Barbosa
da Silva, Andresa Pereira de Oliveira Mendes, Valéria Pereira Hernandes, Maria
Carolina Accioly Brelaz de Castro, Rafaela Salgado Ferreira, Filipe Silva Villela,
Francielly Morais Rodrigues da Costa, Cassio Santana Meira, Diogo Rodrigo
Magalhães Moreira, Milena Botelho Pereira Soares, Ana Cristina Lima Leite
PII:
S0223-5234(19)30639-7
DOI:
https://doi.org/10.1016/j.ejmech.2019.07.018
EJMECH 11515
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 29 May 2019
Accepted Date: 6 July 2019
Please cite this article as: Marcos.Verí.de. Oliveira Cardoso, G.B.d. Oliveira Filho, L.R.P.d. Siqueira,
José.Wanderlan.Pontes. Espíndola, E.B.d. Silva, A.P.d. Oliveira Mendes, Valé.Pereira. Hernandes,
M.C.A.B.d. Castro, R.S. Ferreira, F.S. Villela, F.M.R.d. Costa, C.S. Meira, Diogo.Rodrigo.Magalhã.
Moreira, M.B.P. Soares, A.C.L. Leite, 2-(phenylthio)ethylidene derivatives as anti-Trypanosoma cruzi
compounds: Structural design, synthesis and antiparasitic activity, European Journal of Medicinal
Chemistry (2019), doi: https://doi.org/10.1016/j.ejmech.2019.07.018.
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ACCEPTED MANUSCRIPT
2-(phenylthio)ethylidene derivatives as anti-Trypanosoma cruzi
compounds: Structural design, synthesis and antiparasitic
activity
Marcos Veríssimo de Oliveira Cardoso1,*, Gevanio Bezerra de Oliveira Filho2, Lucianna
Rabelo Pessoa de Siqueira2, José Wanderlan Pontes Espíndola2, Elany Barbosa da Silva2,
Andresa Pereira de Oliveira Mendes3, Valéria Pereira Hernandes3, Maria Carolina Accioly
Brelaz de Castro3,4, Rafaela Salgado Ferreira5, Filipe Silva Villela5, Francielly Morais
Rodrigues da Costa5, Cassio Santana Meira6,7, Diogo Rodrigo Magalhães Moreira6 Milena
,
Botelho Pereira Soares6,7 and Ana Cristina Lima Leite2
1
Universidade de Pernambuco, Laboratório de Prospecção de Moléculas Bioativas,
Programa de Pós-Graduação em Ciência e Tecnologia Aplicadas no Semiárido, 56328-903,
Petrolina, PE, Brazil
2
Universidade Federal de Pernambuco, Laboratório de Planejamento e Síntese de
Fármacos, Departamento de Ciências Farmacêuticas, 50740-520, Recife, PE, Brazil
3
Fundação Oswaldo Cruz, Centro de Pesquisas Aggeu Magalhães, CEP, 50670-420,
Recife, PE, Brazil
4 Universidade Federal de Pernambuco, Laboratório de Parasitologia, Centro Acadêmico de
Vitória - CEP 55608-680, Vitória de Santo Antão, PE, Brazil
5
Universidade Federal de Minas Gerais, Departamento de Bioquímica e Imunologia, CEP
31270-901, Belo Horizonte, MG, Brazil
6 Fundação Oswaldo Cruz, Centro de Pesquisas Gonçalo Moniz, CEP 40296-750, Salvador,
BA, Brazil
7
Hospital São Rafael, Centro de Biotecnologia e Terapia Celular, CEP 41253-190,
Salvador, BA, Brazil
*To whom correspondence should be addressed: phone +55 (87)3866-6491; e-mail:
marcos.cardoso@upe.br

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Abstract
Chagas disease is an illness caused by the protozoan parasite Trypanosoma cruzi. The
current chemotherapy is based on benznidazole, and, in some countries, Nifurtimox, which
is effective in the acute phase of the disease, but its efficacy in the chronic phase remains
controversial. It can also cause serious side effects that lead sufferers to abandon treatment.
In the present work, is reported the synthesis and trypanocidal activity of new 2-
(phenylthio)ethylidene thiosemicarbazones (4-15) and 1,3-thiazoles (16-26). The
cyclization of thiosemicarbazones into 1,3-thiazoles presents an improvement in the
cytotoxic profile for T. cruzi parasite, denoting selective compounds. Compound 18 was
identified as the most promising of all compounds tested, showing an IC50 of 2.6 µM for
the trypomastigote form and a non-cytotoxic effect on mouse spleen cells, reaching a
selective index of 95.1. Among the 22 compounds tested, six compounds present a better
trypanocidal activity, and five compounds have an equipotent activity compared to
benznidazole. Flow cytometry and ultrastructural analysis were performed and indicate that
compound 18 causes parasite cell death through apoptosis and acts via an autophagic
pathway.
Keywords: Chagas disease; Trypanosoma cruzi; cruzain; thiazoles.

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1. Introduction
Chagas disease, a life-threatening illness caused by the protozoan parasite Trypanosoma
cruzi (T. cruzi) has afflicted humanity since its earliest presence in the New World[1] and
remains the most significant parasitic disease burden of the American continent[2–4]. T.
cruzi is a significant public health problem in tropical and subtropical countries, and new
chemotherapies are highly needed. It is estimated that approximately 6-7 million people
worldwide are infected with T. cruzi, mainly in Latin America[5].
Current specific chemotherapy is based only on two compounds, nifurtimox (Nfx) and
benznidazole (Bdz), which were developed empirically over 40 years ago[6] and are
associated with long-term treatments, severe side effects and low access worldwide [7,8].
Nfx and Bdz can eliminate patent parasitemia and reduce serological titers in acute and
early chronic infections[9,10], but they have low efficacy in prolonged chronic
infections[11,12].
Among the drug targets being investigated for Chagas disease, cruzain[13–16] is its most
abundant cysteine protease and is essential for parasite survival[17]. Cruzain is a cathepsin-
L-like protease of the papain family, relevant for intracellular replication and differentiation
of T. cruzi parasites[18]. Activity levels are highest in epimastigotes and, depending on the
life cycle stage, cruzain can be found on the cell surface of T. cruzi amastigotes or
lysosome-like organelles present on T. cruzi epimastigotes called reservosomes[19]. In
addition to its role in parasite nutrition, cruzain has been implicated in many cellular
processes, including cell invasion, proliferation, parasite differentiation, metacyclogenesis
and evasion of the host immune response[18].

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1,3-Thiazole is one of the most remarkable scaffolds in heterocyclic chemistry, drug design
and discovery. It is found in a plethora of pharmacologically active substances and naturally
occurring compounds. Thiazole is a versatile building block for lead generation, easily
yielding access to diverse derivatives for subsequent lead optimization. In recent years,
thiazole derivatives have been synthesized and shown to possess varied biological
activities[20]. 1,3-thiazoles and their analogues have been examined as antiparasitic agents,
especially against Chagas disease[21–23]. As seen in compounds previously synthesized by
our group (Scheme 1), the first generation of thiazolin-4-ones was planned with a 2-
(phenylthio)ethylidene linked at N1. Further compounds explored a halogen exchange and
the modification of sulfur to oxygen. The next structural improvement was the scaffold
hopping from thiosemicarbazone (9) to the thiazolin-4-one moiety (6f), providing better
trypanocidal activity (IC50: 101.9 vs 0.3 µM)[24]. The importance of the bioisosteric
replacement of thiazolin-4-one (18) by a 1,3-thiazole (29) was also investigated, leading to
less cytotoxic and more active compounds against parasites (trypomastigote and
epimastigote forms)[25]. The use of the spacer group “-CH2-CH2-CH=” between the 2-
(phenylthio)ethylidene and N1 was also explored, however, no improvement in
trypanocidal activity was observed[26].

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Scheme 1. Structural planning of previously compounds synthesized by our group.
The good results achieved by thiazolin-4-one and 1,3-thiazole derivatives as anti-T. cruzi
agents motivate us to investigate the trypanocidal activity of new 1,3-thiazoles possessing
different 2-(phenylthio)ethylidene groups attached at N1 (Scheme 01)[23–30].
The search for less toxic and more potent compounds is a constant goal of the medicinal
chemistry. In this work, it was explored the trypanocidal activity of new 1,3-thiazoles,
applying substitutions with different 2-(phenylthio)ethylidenes at N1 and the scaffold
hopping of thiosemicarbazone to 1,3-thiazole, substituted with aromatic rings attached at
C4 (Fig 01).

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Figure 1. Main structural modifications proposed in this work.
In this work, is report the synthesis, trypanocidal activity, and primary structure-activity
relationships (SAR) of a series of 2-(phenylthio)ethylidene thiosemicarbazones (4-15) and
functionalized 1,3-thiazoles (16-26). Compounds (4-26) were assayed for their in vitro anti-
T. cruzi activity against the epimastigote and trypomastigote forms. The most active
compounds were also evaluated against the intracellular form of the parasite. Their
cytotoxicity in mammalian cell cultures was also investigated. The investigation of cruzain
as a possible target for (4-26) was also performed. To a better understanding of the
mechanism of action of the proposed compounds, cell viability, and confocal microscopy
studies were also undertaken.
2. Results and Discussion
2.1.Chemistry
Thiosemicarbazones (4-15) were prepared by reacting commercially available
thiosemicarbazide with the respective 2-(phenylthio)acetaldehyde (3) (1:1.2 mol ratio)
under ultrasound irradiation in the presence of a catalytic amount of acetic acid. This

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protocol led to yields in the range of 30-40%. 1,3-thiazoles (16-26) were prepared via
Hantsch cyclization between thiosemicarbazone (4) and substituted α-bromoacetophenones
(Scheme 2). These reactions proceed upon refluxing with ethanol (2-4 h), but we adapted
them under ultrasound conditions[31] using 2-propanol as solvent[32] and observed yield
ranges of 50-85% and shorter reaction times (60 min in most cases), compared with the
reflux protocol.
Scheme 2. Synthetic procedures for compounds 4-15 and 16-26.
Reagents and conditions: (a) K2CO3, bromoacetaldehyde diethyl acetal, ultrasound, 40°C, 120 min;
(b) H2SO4 10%, ultrasound, r.t., 120 min. (c) thiosemicarbazide, ethanol, acetic acid (3 drops),
ultrasound, 40°C, 120 min; (d) respective α-bromoacetophenone (2,4´-dibromopropiophenone for
compd. 18), calcium carbonate, 2-propanol, ultrasound, r.t., 60 min.
2.2. Cytotoxicity
After chemical characterization, the cytotoxic profile against BALB/c mouse splenocytes
3
(Table 1) was measured as the per cent of H-thymidine incorporation for treated cells in
comparison to untreated cells. The highest non-cytotoxic concentration was determined by
the average of triplicates (Table 1).

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Table 1. Anti T. cruzi activities of thiosemicarbazones 04-15 and 1,3-thiazoles 16-26.
T. cruzi, IC50 (µM)
trypomastigotea epimastigoteb splenocytesc
Toxicity
Compd.
Ar
SId
Ph
4-FPh
4-BrPh
89.0
5.7
6.6
39.7
6.1
1.8
ND
5.9
6.2
14.1
14.1
8.2
101.9
6.1
15.4
32.1
15.3
6.3
9.6
6.2
47.7
5.5
6.2
22.2
20.5
<3.3
<3.9
41.8
<4.2
4.2
<3.9
<3.8
96.2
<3.8
85.0
0.25
3.6
0.5
0.1
6.9
2.3
ND
0.7
0.6
6.8
0.3
10.4
04
05
06
07
08
09
10
11
12
13
14
15
4-CH3OPh
2-CH3Ph
3-CH3Ph
4-CH3Ph
2,5-diCH3Ph
2-ClPh
3-ClPh
4-ClPh
2,5-diClPh
6.1
Ph
4-CH3Ph
4-BrPh
4-BrPh
4-FPh
5.2
5.7
2.6
14.3
41.7
18.5
4.1
59.4
9.4
4.1
153.6
147.3
>247.3
>239.0
>291.2
>281.3
>277.8
126.8
126.8
135.0
67.5
29.5
25.8
95.1
16.7
7.0
15.2
67.8
30.9
8.3
16
17
18
19
20
21
22
23
24
25
26
Bdz
5.4
ND
62.7
6.8
7.9
56.5
8.4
4-CH3OPh
4-ClPh
2,4-diClPh
3,4-diClPh
3-NO2Ph
4-NO2Ph
---
4.1
15.2
19.2
8.4
7.0
8.0
15.5
16.0
6.6
6.2
96.1
a Determined 24 h after incubation of trypomastigotes with the compounds. b Determined five days after
incubation of epimastigotes with the compounds. IC50 was calculated from at least five concentrations, in
c
duplicate (SD ± 10%). Highest nontoxic concentration for mouse splenocytes after 24 h of incubation
in the presence of the compounds. d Selectivity index = toxicity splenocytes / IC50 trypomastigotes. Bdz:
benznidazole. ND: not determined.

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Overall, tested thiosemicarbazones showed toxicity higher than Bdz, except for 13 and 15,
that present similar toxicity. Otherwise, 1,3-thiazoles present a significant improvement in
the toxicity profile by comparison to thiosemicarbazones 4-15, probably due to the
cyclization of thiocarbonyl to a 1,3 thiazole group (Table 1), these findings are in
agreement with previous results[23,24,26,33–35]. The most of 1,3-thiazole derivatives
tested present lower toxicity than Bdz, except for compound 26 (4-nitro derivative) (Table
1).
2.3. Antiparasitic activity against extracellular forms
Derivatives 4-15 and 16–26 were evaluated in vitro against the epimastigote and
trypomastigote forms of T. cruzi in a screening scheme that included five different
concentrations (1.2, 3.7, 11.1, 33.3, and 100 µg/mL). Their ability to inhibit parasite growth
was tested in comparison to the standard drug (Bdz). The selective index was measured
only for compounds that present cytotoxicity higher than 100 µM.
In view to state the influence of the arylthio group in the trypanocidal activity, a
comparison between previously synthesized compounds bearing arylthio and alkoxy group
was made, as expressed in Fig. 2 that present potent arylthio and alkoxy derivatives.
HERNANDES et al. demonstrated the synthesis and anti. T. cruzi profile of 23 compounds,
being twelve thiosemicarbazones and eleven 1,3-thiazoles, and identified potent
compounds for anti-T. cruzi activity, however, none of them was more potent than lead
compound (18). Another important work in this field is described by MOREIRA et al.,
which presented 38 compounds, exploring thiazolin-4-one and 1,3-thiazole ring. Those
molecules possess an alkoxy group instead arylthio group, performing high activity against
epimastigotes and trypomastigotes of T. cruzi; however, the most active compounds did not

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overlap compound 18, denoting the importance of the arylthio group. The compound 34
presented by Moreira et al. have significant structural similarity to compound 18, differing
only by a methyl at C3 and an alkoxy instead of an arylthio group; This demonstrates the
benefits of the arylthio group over alkoxy group, due to a 10-fold more potency against
epimastigote form.
Figure 2. Comparison of T. cruzi activity of arylthio and alkoxy groups.
To discuss the importance of the cyclization of thiocarbonyl into 1,3-thiazole is first
compared to the results of anti-T. cruzi activity against the trypomastigote form of the non-
substituted thiosemicarbazone 4 (89.0 µM) with the non-substituted 1,3-thiazole 16 (5.2
µM). It can be observed that compound 16 is 17-fold more potent than 4, demonstrating the
importance of the cyclization. Indeed, compound 4 is 14-fold less potent than Bdz,
followed by thiosemicarbazones 7, 13, and 14. However, compounds 5 (5.7 µM), 6 (6.6
µM), 8 (6.1 µM), 11 (5.9 µM), 12 (6.1 µM) and 15 (8.2 µM) present equipotent
trypanocidal profile to Bdz (6.2 µM) (Table 1). Analyzing para-substituted compounds 5-
7, 10 and 14, is noted that the insertion of substituents at C4 provides a significant

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improvement in trypanocidal activity, in comparison to the non-substituted compound 4.
The insertion of fluorine at C4 (5) leads to the most active compound, giving a significant
improvement in anti-T. cruzi activity (trypomastigote form), being 15-fold more potent than
compound 4 (IC50 5.7 vs 89.0 µM). About methyl-substituted compounds 8-11, it was
noticed that 8 is equipotent to dimethyl-substituted 11, but it has 10-fold less toxicity.
Compound 9 is the most active methyl substituted compound and, compared with
compound 4, it exhibits an incredible 49-fold improvement of the IC50, being the most
active thiosemicarbazone evaluated in this work. However, it also presents toxicity to
BALB/c splenocytes, leading to a low selective index. For chlorine-substituted 12-15, it
was observed a good improvement in the trypanocidal activity, with 13 being the most
active for the epimastigote form. Comparing compounds 11 and 15, both disubstituted at
2,5 positions with methyl (11) and chlorine (15), respectively, is observed an equipotency in
the trypanocidal activity, but derivative 15 shows lower cytotoxicity.
Among the twelve thiosemicarbazones derivatives assayed, seven are equipotent to
benznidazole, with compound 9 being the most active, exhibiting an IC50 of 1.8 µM against
the trypomastigote form (Table 1). However, compound 15 presents similar toxicity and
trypanocidal activity to benznidazole, making it the most promising thiosemicarbazone
evaluated in this study. About the 1,3-thiazole derivatives, trypanocidal activities
(trypomastigote) of para-substituted compounds were first compared with that of
compound 16 (non-substituted).
Compound 16 shows a trypanocidal activity equivalent to Bdz, with lower cytotoxicity.
Comparing para-substituted compounds (17-22 and 26) with 16 was observed an
improvement in the cytotoxicity profile, except for compound 17, which shows similar

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trypanocidal and toxicity activities, and 26, which shows increased toxicity. With respect to
the trypanocidal activity, a substantial increment was observed for compounds 18 (2.6 µM)
and 22 (4.1 µM), while 17 (5.7 µM), 19 (14.3 µM), 20 (41.7 µM), 21 (18.5 µM) and 26 (8.4
µM) were less effective than 16 (5.2 µM).
Predominantly, the insertion of substituents at the para position did not increase the
trypanocidal activity, but maintained or decreased the toxicity, especially the chlorine (22)
and bromine-substituted (18) compounds, which showed both low toxicity levels (>100
µM) and potent trypanocidal activity, leading to a high selective index (SI). Nitro
compounds, meta (25) and para (26) substituted, show lower trypanocidal activities than
16, with compound 25 presenting a better toxicity level (135 µM against 67.5 µM) and
lower trypanocidal activity (19.22 µM against 8.45 µM) than compound 26. Attachment of
a methyl group at C5 in the 1,3-thiazole ring improved the trypanocidal activity, as shown
by the comparison between compounds 18 and 19, both bromine-substituted.
In overall, about the trypanocidal activity of 1,3-thiazoles 16-26 (Table 1), the most viable
compounds were 18 and 22, presenting higher SI than Bdz. Aim In Fig. 2, the relationships
between compounds 18, 22, and 23 and their drug likenesses[36] are measured; however,
no clear correlation can be established.
2.4.Antiparasitic activity against the intracellular form
Aiming at a better understanding of the trypanocidal activity of 1,3-thiazoles, was evaluated
the effect of the compounds on trypomastigote invasion and development, as these events
are crucial for the establishment of the disease in mammalian hosts[37,38]. Chemically
inhibiting parasite invasion and growth are desirable functional properties for any Chagas

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disease drug candidate. For that reason, it was performed in vitro experiments on Y strain
trypomastigotes in mouse macrophages[39]. Non-cytotoxic compounds with trypanocidal
activities higher than that of Bdz were selected for the evaluation of in vitro assays of
infection by T. cruzi (Fig. 3).
Figure 3. T. cruzi-infected macrophages were treated and incubated for three days. A) Mean the
number of intracellular amastigotes per 100 infected macrophages. B) Infected cells in per cent. C)
Macrophages were simultaneously exposed to trypomastigotes and treatment. The cell culture was
incubated for 2 h and washed, and the per cent of infected cells was determined after 2 h. Bdz=
benznidazole, AmpB = amphotericin B. Data are the mean ± S.E.M. (error bars) of two independent
experiments performed. Panels A and B: ***, P <0.001; panel C: ***,
P <0.001; **, P <0.01.
Macrophages previously infected with Y strain trypomastigotes were treated with 20 µM of
compounds 17, 18, 22 and 26. Cells were stained with Giemsa and observed by optical
microscopy to evaluate the percentage of infected macrophages and the relative number of
amastigotes per 100 cells. As shown in Fig. 3, the treatment with 1,3-thiazoles significantly
decreased the percentage of infected macrophages (P < 0.05) and the relative number of

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amastigotes per 100 macrophages (P < 0.05) when compared with untreated cultures. When
tested at different concentrations, it was possible to calculate the IC50 value of 1,3-thiazoles
against intracellular amastigotes. Compounds 18 and 22 presented IC50 values of 10.15
(±0.98) µM and 7.88 (±0.03) µM, respectively, demonstrating an activity superior to that of
Bdz in inhibiting infection (Fig 3A and 3B). Compounds 17 and 26 presented a moderate
activity, with IC50 values of 18.78 (±0.03) µM and 19.26 (±0.53) µM, respectively.
It was also evaluated the effect of 1,3-thiazoles on the invasion process. In this assay,
mouse macrophages were exposed to trypomastigotes and at the same time were treated
with the 1,3-thiazoles (20 µM) for 2 h. After this time, the cells were washed with saline
solution to remove extracellular parasites and incubated for 2 additional hours. Cells were
stained with hematoxylin and eosin and analyzed by optical microscopy. Amphotericin B
was used as a positive control for this experiment. As shown in Fig. 3C, 1,3-thiazoles
significantly inhibited the parasite invasion in comparison to untreated cells (P < 0.001) but
not as efficiently as the positive control amphotericin B. However, they were more effective
than benznidazole, which shows a weak activity on this assay.
2.5. Cruzain inhibition activity
The thiosemicarbazones and their cyclic analogues 1,3-thiazoles were tested against the
enzyme cruzain the T. cruzi in view to investigate the probable mechanism of action. The
inhibition of cruzain enzymatic activity by all compounds was measured using a
competition-based assay with the fluorescent substrate Z-Phe-Arg-aminomethylcoumarin
(Z-FR-AMC)[40]. All compounds were screened at 100 µM, and only compounds having
an inhibition value of >70% were chosen to determine their IC50 values. Therefore, in some

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cases, the discussion was performed in terms of the percentage of cruzain inhibition (Table
3).
Table 3. In vitro cruzain inhibition activity of compounds 04-26.
% Cruzain Inhibition
(100
M)a
% Cruzain Inhibition
(100
M)a
Compound
Compound
µ
µ
42.1 ± 5.6
27.5 ± 3.3
16.6 ± 3.5
34.6 ± 3.4
13.9 ± 3.2
ND
04
05
06
07
08
09
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
11.9 ± 2.8
27.0 ± 2.4
7.0 ± 2.2
22.9 ± 2.3
ND
17.6 ± 4.8
67.2 ± 5.4
62.9 ± 4.5
16.2 ± 2.8
18.9 ± 3.8
43.4 ± 5.2
31.4 ± 4.4
87.6 ± 2.3 (3.3 ± 2.6b)
46.0 ± 5.4
75.9 ± 2.3 (19.0 ± 15.2b)
25.2 ± 6.0
37.4 ± 5.8
a
Average values and standard errors were calculated based on five independent
b
experiments, performed in triplicate. Average and standard errors were calculated based
on two independent curves, each including at least seven compound concentrations in
triplicates. IC50 (µM) are given in parenthesis. Standard errors were calculated based on the
formula σ/√n, where σ is the standard deviation and n is the number of measurements.
Cruzain inhibition by thiosemicarbazones was first reported by Du et al.[41]. The suggested
mechanism of inhibition involves a nucleophilic attack at the thiocarbonyl group by the
catalytic Cys25 residue and electron delocalization to the sulfur atom, which is stabilized
by the neighbouring His159[41]. Based on the importance of the sulfur highlighted by this
work, it was evaluated several thiosemicarbazones with a spacer group containing a sulfur
atom to investigate a possible increment in the cruzain inhibition. Substitution of the

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unsaturated sulfur in the thiocarbonyl group with saturated cyclic sulfur on the 1,3-thiazole
ring, along with the cyclization of thiosemicarbazone to 1,3-thiazole, was also investigated.
Of thiosemicarbazones 4-15, only two compounds (11 and 13) show cruzain inhibition
higher than 70% at 100 µM (87.6 and 75.9%, respectively), presenting IC50 values of 3.3 ±
2.6 and 19.0 ± 15.2 µM, respectively. In comparison to compound 4, only compounds 11
and 13 show increments in the cruzain inhibition. The difference between compounds 11
and 13 is that 11 is substituted in positions 2,5 by a methyl group, while 13 is substituted by
a chloro group at the meta position. In addition to the orientation of the substituents, the
main difference is that 13 has an electron-withdrawing substituent, whereas 11 has donor
substituents. However, this trend is not observed in other compounds with
withdrawing/donor substituents.
Of cyclic compounds 16-26, none of them present inhibition rates greater than 70% at 100
µM, so the IC50 values were not measured. In the comparison of 1,3-thiazoles with the lead
thiosemicarbazone 4, it is noted that only compounds 23 and 24 present better inhibition
rates (67.2 ± 5.4 and 62.9 ± 4.5 vs 42.1 ± 5.6, respectively). These results indicate that the
trypanocidal activity of these compounds is due to another mechanism of action. This result
is not in agreement to previous findings that suggest that thiosemicarbazones and 1,3-
thiazoles are inhibitors of cruzain[28,30], which as not observed in this work. Furthermore,
studies of cell death and ultra-structural analysis were performed, aiming at a better
understanding of the mechanism of action.
2.6. Flow cytometry analysis

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After confirming that the compounds can kill parasites, the next step was to understand
how they affect parasite cells through assays with the trypomastigote form of T. cruzi
treated with MBHA3. Understanding the mechanism involved may provide insights into
the pathogenesis of Chagas disease and help to develop better therapies against this illness.
Evaluation of apoptosis and necrosis by flow cytometry is usually accomplished by the
combined use of annexin V (AV) - FITC and PI. Annexin V - FITC accesses
phosphatidylserine molecules exposed on the external membrane in the early stage of
apoptosis, and PI allows the identification of nuclear alterations in the late stages of
apoptosis or necrosis, as a consequence of the increase in membrane permeability[42–44].
Most cells positive for AV were also PI positive, suggesting that apoptotic cells evolved
into secondary necrosis. However, we cannot rule out the possibility that annexin might
also bind to the inner phosphatidylserine residues after the membrane integrity has been
lost. As shown in Fig. 4, when compared with untreated cells, most of the parasite cells
treated with thiosemicarbazones were positively stained for AV and PI. Therefore, we
suggest that 1,3-thiazole-based treatment causes parasite cell death through apoptosis.

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30
20
10
0
1xIC50 - PI
2xIC50 - PI
80
60
40
20
0
1xIC50 - AV+PI
2xIC50 - AV+PI
40
30
20
10
0
1xIC50 - AV
2xIC50 - AV
Figure 4. Effects of compound 06, 09, 11, 17, 18, 22 and 23 treatments on Annexin V/PI labelling.
2.7. Ultra-structural analysis
To investigate the mechanism of action of compound 18, ultrastructural alterations in
bloodstream trypomastigotes were also analyzed. As shown in Fig. 5A, thin sections of
untreated trypomastigotes observed by TEM revealed a typical appearance of organelles,
intact plasma membrane and parasite cytoplasm without alterations. However, the treatment
with compound 18 at 3.0 µM for 24 h caused disorganization of the Golgi complex (Fig.
5B), the appearance of numerous atypical vacuoles in the cytoplasm (Fig. 5C) and the

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appearance of myelin figures within the mitochondria and in the cytoplasm. Vacuole
formation and the presence of myelin-like figures are commonly associated with the
process of autophagy[45,46]. This might be indicative that the trypanocidal activity of
compound 18 can be, in part, assigned to the activation of autophagic pathways.
Figure 5. Transmission electron micrographs of trypomastigotes treated or not with compound 18
for 24 hours. (A) Shows an image of untreated trypomastigotes, presenting a typical morphology of
the nucleus (N), kinetoplast (K), mitochondria (M) and Golgi complex (GC). Treatment with
compound 18 at 3 µM (B-D) induces Golgi complex disorganization (B), appearance of atypical
vacuoles in the cytoplasm (C) and the appearance of myelin Figs within the mitochondria and in the

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cytoplasm (D). Black arrows indicate alterations from parasites. Scale bars: A = 1 µm; B-D = 0.5
µM.
2.8.Physicochemical properties
For a better analysis of the synthesized compounds, the online software
SwissADME (a free web tool to evaluate pharmacokinetics, drug-likeness and medicinal
chemistry friendliness of small molecules) was used to determine the physicochemical
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 [36,47]. This rule has essential determinants in providing better
pharmacokinetics and analyzes promising future drug development. Compound following
at least three of the four criteria are considered to adhere to the Lipinski Rule [36]. All
synthesized compounds are compatible with Lipinski Rule. Another attractive property is
the number of rotatable bonds and the polar surface area (PSA). A large number of rotatable
bonds (≥10) has been associated with poor oral bioavailability [48]. Compounds with a low
PSA (≤ 140 Å2) tend to have higher oral bioavailability [48,49]. All synthesized compounds
have appropriate PSA and number of rotatable bonds (Table 4).
Table 4: Physicochemical property profile of derivatives (4-26), calculated by SwissADME web
tool (http://www.swissadme.ch/index.php)
MW
CPD. (g/mol)
<500
MLog P
<=4.15
H-bond
H-bond
Lipinski #Rotatable
TPSA
<=140
donors <5 acceptors <10 violations bonds <10
225.33
243.32
304.23
255.36
239.36
239.36
239.36
253.39
259.78
259.78
259.78
294.22
325.45
1.69
2.11
2.41
1.41
1.99
1.99
1.99
2.28
2.26
2.26
2.26
2.82
3.37
2
2
2
2
2
2
2
2
2
2
2
2
1
1
2
1
2
1
1
1
1
1
1
1
1
2
0
0
0
0
0
0
0
0
0
0
0
0
0
5
5
5
6
5
5
5
5
5
5
5
5
6
107.8
107.8
107.8
117.0
107.8
107.8
107.8
107.8
107.8
107.8
107.8
107.8
90.8
4
5
6
7
8
9
10
11
12
13
14
15
16

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339.48
418.37
404.35
343.44
355.48
359.90
394.34
394.34
370.45
370.45
260.25
3.61
4.23
4
1
1
1
1
1
1
1
1
1
1
1
2
2
2
3
3
2
2
2
4
4
4
0
1
0
0
0
0
1
1
0
0
0
6
6
6
6
7
6
6
6
7
7
6
90.8
90.8
90.8
90.8
100.0
90.8
90.8
90.8
136.6
136.6
92.7
17
18
19
20
21
22
23
24
25
26
Bdz
3.76
3.03
3.88
4.38
4.38
3.17
3.17
0.37
As demonstrated in Table 5, the most active compounds shown variable permeability based
on gastrointestinal absorption (GI), according to the BOILED-Egg predictive model (Brain
Or IntestinaL EstimateD 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 permanent. These are
important as it would allow the drug to have high uptake. The most selective derivatives
showed high gastrointestinal absorption (18, 22, and 23). Concerning oral bioavailability, it
has expected 0.55 of the probability of oral bioavailability score > 10% in the rat for all
compounds, similar to Bdz. All these data suggest a good in silico drug-likeness profile and
high chemical stabilities for all compounds synthesized.
Table 5: ADME properties of most active compounds.
*BBB
**GI
Bioavailabilit
y Score
Compound
permeant absorption
18
22
23
No
No
No
No
High
High
High
High
0.55
0.55
0.55
0.55
Bdz
*BBB - blood-brain barrier.
**GI - Gastrointestinal absorption.
3. Conclusion
In conclusion, the functionalization of thiosemicarbazones (4-15) to thiazoles (16-26)
resulted in a better cytotoxicity profile, as the cyclic analogues showed an improved

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selectivity index. Comparing compounds 4 to 16, unsubstituted representatives of
thiosemicarbazone and 1,3-thiazole groups, there was observed an 18-fold improvement in
the trypanocidal activity (trypomastigote form), suggesting the importance of the
functionalization of thiocarbonyl to a thiazole moiety. Compound 18 is the most promising
candidate an anti-T. cruzi drug, being non-toxic for splenocytes at the highest dose tested
(247.8 µM) and keeping high trypanocidal activity. Besides, ultrastructural studies and flow
cytometry analysis revealed ultrastructural alterations induced by the 1,3-thiazoles, which
at first result in apoptotic parasite cell death.
4. Experimental section
4.1. General
All reagents were purchased from commercial sources (Sigma-Aldrich, Vetec, or Fluka).
Progression of the reactions was followed by thin-layer chromatography (TLC) analysis
(Merck, silica gel 60 F254 in aluminium foil). The purity of the target compounds was
confirmed by combustion analysis (for C, H, N, S) performed by a Carlo-Erba instrument
(model EA 1110). Melting points were determined on a Fisatom 430D electrothermal
capillary melting point apparatus and were uncorrected. NMR spectra were measured on
1
13
either a Varian UnityPlus 400 MHz (400 MHz for H and 101 MHz for C) or a Bruker
1
13
AMX-300 MHz (300 MHz for H and 75.5 MHz for C) instrument. DMSO-d6 and D2O
were purchased from CIL or Sigma-Aldrich. Chemical shifts (δ) are reported in ppm.
Multiplicities are given as s (singlet), d (doublet), t (triplet), m (multiplet), dd (double
doublet), and coupling constants (J) in Hertz. Mass spectrometry experiments were
performed on an LC-IT-TOF (Shimadzu). Unless otherwise specified, ESI was carried out
in positive ion mode. Typical conditions were a capillary voltage of 3 kV, a cone voltage of

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30 V, and a peak scan between 50-1000 m/z. IR spectra were recorded with a Bruker model
IFS66 FT-IR spectrophotometer using KBr pellets.
4.2.General procedure for the synthesis of thiosemicarbazones (4-15)
Example for compound (4): to a solution aldehyde (3) (0.76 g, 5 mmol) in ethanol (10 mL)
was added thiosemicarbazide (0.46 g, 5 mmol) and a few drops of acetic acid. The reaction
vessel was placed in an ultrasonic bath (40 MHz, 180 V) and irradiated for 120 min at r.t.
The precipitate was filtered, washed with hexane, and then dried in a desiccator under
vacuum. An additional amount of the desired compound could be recovered from the
filtrate after cooling.
4.2.1. 2-[(2-phenylthio)ethylidene] thiosemicarbazone (4)
Crystallization from toluene/hexane 7:3, afforded white crystals, yield = 86%. Mp (ºC):
1
125-126. IR (KBr): 1595 (C=N), 3247 (NH), 3390 (NH2) cm-1. H NMR (300 MHz,
DMSO-d6) δ 3.78 (d,
J
= 6.0 Hz, 2H, S-CH2), 7.19 (t,
J
= 7.2 Hz, 1H, N=CH), 7.31 (t,
J =
6.6 Hz, 2H, Ar), 7.34 (t,
J
= 6.1 Hz, 1H, Ar), 7.38 (d,
J
= 7.2 Hz, 2H, Ar), 7.54 (s, 1H,
NH2), 8.09 (s, 1H, NH2), 11.19 (s, 1H, NH). 13C NMR (75 MHz, DMSO-d6) δ 33.5 (S-
CH2), 126.1 (C-Ar), 128.4 (C-Ar), 129.0 (C-Ar), 134.6 (C-Ar), 141.5 (C=N), 177.8 (C=S).
HRMS (ESI): 226.0491 [M + H]+. Anal. Calcd for C9H11N3S2: C, 47.97; H, 4.92; N, 18.65;
S, 28.46; found: C, 47.80; H, 5.09; N, 18.61; S, 28.50.
4.2.2. 2-{2-[(4-fluorophenyl)thio]ethylidene} thiosemicarbazone (5)
Crystallization from toluene/hexane 7:3, afforded yellow crystals, yield = 39%. Mp (ºC):
1
106-108. IR (KBr): 1591 (C=N), 3258 (NH), 3404 (NH2) cm-1. H NMR (300 MHz,

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DMSO-d6) δ 3.74 (d,
J
= 6.0 Hz, 2H, S-CH2), 7.32 (t,
J
= 6.0 Hz, 1H, N=CH), 7.42 (d,
J =
5.3 Hz, 2H, Ar), 7.45 (d,
J = 5.3 Hz, 2H, Ar), 7.48 (s, 1H, NH2), 8.08 (s, 1H, NH2), 11.18
13
(s, 1H, NH). C NMR (75.5 MHz, DMSO-d6): 34.5 (S-CH2), 115.9 (C-Ar), 116.2 (C-Ar),
129.7 (C-Ar), 131.5 (C-Ar), 131.6 (C-S), 141.4 (C=N), 162.6 (C-Ar), 177.8 (C=S). HRMS
(ESI): 244.0405 [M + H]+. Anal. Calcd for C9H10FN3S2: C, 44.43; H, 4.14; N, 17.27; S,
26.35; found: C, 44.31; H, 4.11; N, 17.33; S, 26.31.
4.2.3. 2-{2-[(4-bromophenyl)thio]ethylidene} thiosemicarbazone (6)
Crystallization from toluene/hexane 7:3, afforded yellow crystals, yield = 37%. Mp (ºC):
1
104-106. IR (KBr): 1594 (C=N), 3239 (NH), 3387 (NH2) cm-1. H NMR (300 MHz,
DMSO-d6): 3.74 (d, 2H, S-CH2), 7.17 (t, 2H, Ar), 7.31 (t, 1H, N=CH), 7.42 (dd, 2H, Ar),
7.49 (s, 1H, NH2), 8.09 (s, 1H, NH2), 11.18 (s, 1H, NH). 13C NMR (75.5 MHz, DMSO-d6):
33.3 (S-CH2), 118.9 (C-Ar), 130.1 (C-Ar), 131.7 (C-Ar), 133.7 (C-Ar), 134.2 (C-Ar), 135.4
(C-Ar), 140.9 (C=N), 177.8 (C=S). HRMS (ESI): 303.9504 [M + H]+. Anal. Calcd for
C9H10BrN3S2: C, 35.53; H, 3.31; N, 13.81; S, 21.08; found: C, 35.62; H, 3.38; N, 13.85; S,
20.92.
4.2.4. 2-{2-[(4-methoxyphenyl)thio]ethylidene} thiosemicarbazone (7)
Crystallization from toluene/hexane 7:3, afforded orange crystals, yield = 30%. Mp (ºC):
124. IR (KBr): 1598 (C=N), 3255 (NH), 3404 (NH2) cm-1. 1H NMR (300 MHz, DMSO-d6):
δ 3.64 (d,
J = 6.0 Hz, 2H, S-CH2), 3.73 (s, 3H, CH3), 6.90 (d, J = 8.8 Hz, 2H, Ar), 7.31 –
7.37 (m, 3H, Ar, N=CH), 7.41 (s, 1H, NH2), 8.05 (s, 1H, NH2), 11.16 (s, 1H, NH). 13C
NMR (75.5 MHz, DMSO-d6): 35.7 (S-CH2), 55.2 (O-CH3), 114.7 (C-Ar), 124.0 (C-Ar),
132.5 (C-S), 158.6 (C-Ar), 142.1 (C=N), 177.7 (C=S). HRMS (ESI): 256.0612 [M + H]+.

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Anal. Calcd for C10H13N3OS2: C, 47.04; H, 5.13; N, 16.46; S, 25.11; found: C, 47.34; H,
5.55; N, 16.23; S, 25.21.
4.2.5. 2-{2-[(2-methylphenyl)thio]ethylidene} thiosemicarbazone (8)
Crystallization from toluene/hexane 7:3, afforded white crystals, yield = 33%. Mp (ºC):
1
104-105. IR (KBr): 1599 (C=N), 3260 (NH), 3375 (NH2) cm-1. H NMR (400 MHz,
DMSO-d6) δ 2.26 (s, 3H, CH3), 3.74 (d,
J
= 6.0 Hz, 2H, S-CH2), 7.10 (td,
J
J
= 1.3, 7.3 Hz,
= 6.0 Hz, 1H,
1H, Ar), 7.15 (d,
HC=N), 7.38 (d,
J
J
= 5.8 Hz, 1H, Ar), 7.17 (t, = 7.0 Hz, 1H, Ar), 7.31 (t,
J
= 6.5 Hz, 1H, Ar), 7.52 (s, 1H, NH2), 7.93 (s, 1H, NH2), 11.12 (s, 1H,
NH). 13C NMR (101 MHz, DMSO-d6) δ 20.3 (CH3), 33.7 (S-CH2), 126.4 (C-Ar), 127.0 (C-
Ar), 128.4 (C-Ar), 130.5 (C-Ar), 134.2 (C-Ar), 137.0 (C-Ar), 142.0 (C=N), 178.3 (C=S).
HRMS (ESI): 240.0663 [M + H]+. Anal. Calcd for C10H13N3S2: C, 50.18; H, 5.47; N,
17.56; S, 26.79; found: C, 50.23; H, 5.51; N, 17.46; S, 26.64.
4.2.6. 2-{2-[(3-methylphenyl)thio]ethylidene} thiosemicarbazone (9)
Crystallization from toluene/hexane 7:3, afforded yellow crystals, yield = 32%. Mp (ºC):
1
118-119. IR (KBr): 1604 (C=N), 3266 (NH), 3422 (NH2) cm-1. H NMR (400 MHz,
DMSO-d6): 2.26 (3H, CH3), 3.76 (d,
J = 5.7 Hz, 2H, S-CH2), 7.00 (d, J = 6.6 Hz, 1H, Ar),
7.24 – 7.10 (m, 3H, Ar), 7.33 (t,
J = 5.7 Hz, 1H, N=CH), 7.51 (s, 1H, NH2), 8.07 (s, 1H,
NH2, 11.17 (s, 1H, NH). 13C NMR (100 MHz, Chloroform-
d
): 21.5 (CH3), 35.8 (S-CH2),
128.3 (C-Ar), 128.4 (C-Ar), 129.1 (C-Ar), 131.9 (C-Ar), 133.5 (C-Ar), 139.1 (C-Ar), 142.5
(C=N), 178.7 (C=S). HRMS (ESI): 240.0638 [M + H]+. Anal. Calcd for C10H13N3S2: C,
50.18; H, 5.47; N, 17.56; S, 26.79; found: C, 50.12; H, 5.49; N, 17.46; S, 26.88.

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4.2.7. 2-{2-[(4-methylphenyl)thio]ethylidene} thiosemicarbazone (10)
Crystallization from toluene/hexane 7:3, afforded yellow crystals, yield = 36%. Mp (ºC):
99-101. IR (KBr): 1602 (C=N), 3261 (NH), 3412 (NH2).cm-1. 1H NMR (300 MHz, DMSO-
d6): 2.25 (3H, CH3), 3.72 (d, 2H, S-CH2), 7.19 (dd, 4H, Ar), 7.31 (t, 1H, N=CH), 7.51 (s,
1H, NH2), 8.08 (s, 1H, NH2), 11.18 (s, 1H, NH). 13C NMR (75.5 MHz, DMSO-d6): 20.6
(CH3), 34.1 (S-CH2), 129.2 (C-Ar), 129.7 (C-Ar), 130.7 (C-Ar), 135.8 (C-S), 141.8 (C=N),
177.8 (C=S). HRMS (ESI): 240.0643 [M + H]+. Anal. Calcd for C10H13N3S2: C, 50.18; H,
5.47; N, 17.56; S, 26.79; found: C, 50.21; H, 5.43; N, 17.42; S, 26.86.
4.2.8. 2-{2-[(2,5-dimethylphenyl)thio]ethylidene} thiosemicarbazone (11)
Crystallization from toluene/hexane 7:3, afforded yellow crystals, yield = 35%. Mp (ºC):
119-120. IR (KBr): 1243 (C=S), 1597 (C=N), 3252 (NH), 3377 (NH2) cm-1. 1H NMR (300
MHz, DMSO-d6) δ 2.24 (s, 6H, CH3), 3.76 (d,
J
= 5.7 Hz, 2H, S-CH2), 6.91 (d,
J = 7.4 Hz,
1H, Ar), 7.08 (d, = 7.5 Hz, 1H, Ar), 7.23 (s, 1H, Ar), 7.32 (t,
J
J
= 5.5 Hz, 1H, N=CH), 7.55
(s, 1H, NH2), 8.11 (s, 1H, NH2), 11.20 (s, 1H, NH). 13C NMR (75.5 MHz, DMSO-d6): 19.4
(CH3), 20.5 (CH3), 33.2 (S-CH2), 126.6 (C-Ar), 128.3 (C-Ar), 128.4 (C-Ar), 129.9 (C-Ar),
133.4 (C-Ar), 135.6 (C-Ar), 141.3 (C=N), 177.9 (C=S). HRMS (ESI): 254.0823 [M + H]+.
Anal. Calcd for C11H15N3S2: C, 52.14; H, 5.97; N, 16.58; S, 25.31; found: C, 52.28; H,
6.08; N, 16.65; S, 25.41.
4.2.9. 2-{2-[(2-chlorophenyl)thio]ethylidene} thiosemicarbazone (12)
Crystallization from toluene/hexane 7:3, afforded yellow crystals, yield = 31%. Mp (ºC):
1
125-126. IR (KBr): 1601 (C=N), 3241 (NH), 3376 (NH2) cm-1. H NMR (300 MHz,
DMSO-d6) δ 3.86 (d,
J = 5.7 Hz, 2H, S-CH2), 7.20 (t, J = 7.6 Hz, 1H, N=CH), 7.32 (t, J =

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6.0 Hz, 2H, Ar), 7.46 (d,
J = 7.8 Hz, 1H, Ar), 7.55 (d, J = 8.0 Hz, 1H, Ar), 7.67 (s, 1H,
NH2), 8.14 (s, 1H, NH2), 11.24 (s, 1H, NH). 13C NMR (75.5 MHz, DMSO-d6): 32.5 (S-
CH2), 126.9 (C-Ar), 127.8 (C-Ar), 128.0 (C-Ar), 129.5 (C-Ar), 131.2 (C-Ar), 134.2 (C-Ar),
140.6 (C=N), 177.9 (C=S). HRMS (ESI): 260.0119 [M + H]+. Anal. Calcd for
C9H10ClN3S2: C, 41.61; H, 3.88; N, 16.18; S, 24.68; found: C, 41.41; H, 3.93; N, 16.25; S,
24.73.
4.2.10. 2-{2-[(3-chlorophenyl)thio]ethylidene} thiosemicarbazone (13)
Crystallization from toluene/hexane 7:3, afforded yellow crystals, yield = 32%. Mp (ºC):
111. IR (KBr): 1598 (C=N), 3245 (NH), 3439 (NH2) cm-1.1H NMR (300 MHz, DMSO-d6
δ 3.94 (d, = 5.9 Hz, 2H, S-CH2), 7.25 (d, = 2.3 Hz, 1H, N=CH), 7.28 (d, = 2.3 Hz, 1H,
Ar), 7.32 (t, = 5.9 Hz, 1H, Ar), 7.46 – 7.54 (m, 1H, Ar), 7.57 (d, = 2.4 Hz, 1H, Ar), 8.22
)
J
J
J
J
J
(s, 1H, NH2), 8.65 (s, 1H, NH2), 11.28 (s, 1H, NH). 13C NMR (75,5 MHz, DMSO-d6): 32.4
(S-CH2), 126.5 (C-Ar), 126.7 (C-Ar), 129.5 (C-Ar), 130.9 (C-Ar), 132.4 (C-Ar), 136.8 (C-
Ar), 140.1 (C=N), 178.0 (C=S). HRMS (ESI): 260.0017 [M + H]+. Anal. Calcd for
C9H10ClN3S2: C, 41.61; H, 3.88; N, 16.18; S, 24.68; found: C, 41.55; H, 3.94; N, 16.27; S,
24.63.
4.2.11. 2-{2-[(4-chlorophenyl)thio]ethylidene} thiosemicarbazone (14)
Crystallization from toluene/hexane 7:3, afforded yellow crystals, yield = 38%. Mp (ºC):
1
133-135. IR (KBr): 1606 (C=N), 3249 (NH), 3411 (NH2) cm-1. H NMR (400 MHz,
DMSO-d6) δ 3.75 (d,
J
= 6.0 Hz, 2H, S-CH2), 7.27 (t,
J
= 6.0 Hz, 1H, N=CH), 7.32 (d,
J =
8.7 Hz, 2H, Ar), 7.36 (d,
J = 8.8 Hz, 2H, Ar), 7.56 (s, 1H, NH2), 7.88 (s, 1H, NH2), 11.09
13
(s, 1H, NH). C NMR (75,5 MHz, DMSO-d6): 34.0 (S-CH2), 129.3 (C-Ar), 130.5 (C-Ar),
131.1 (C-Ar), 134.1 (C-Ar), 141.0 (C=N), 177.9 (C=S). HRMS (ESI): 260.0119 [M + H]+.

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Anal. Calcd for C9H10ClN3S2: C, 41.61; H, 3.88; N, 16.18; S, 24.68; found: C, 41.81; H,
3.94;N, 16.17; S, 24.69.
4.2.12. 2-{2-[(2,5-dichlorophenyl)thio]ethylidene} thiosemicarbazone (15)
Crystallization from toluene/hexane 7:3, afforded yellow crystals, yield = 36%. Mp (ºC):
1
149-150. IR (KBr): 1601 (C=N), 3241 (NH), 3402 (NH2) cm-1. H NMR (300 MHz,
DMSO-d6) δ 3.94 (d,
J
= 5.9 Hz, 2H, S-CH2), 7.25 (d,
J = 2.3 Hz, 1H, Ar), 7.28 (d, J = 2.3
Hz, 1H, Ar), 7.32 (t,
J = 5.9 Hz, 1H, N=CH), 7.47 (s, 1H, Ar), 8.22 (s, 1H, NH2), 8.65 (s,
1H, NH2), 11.28 (s, 1H, NH). 13C NMR (75.5 MHz, DMSO-d6): 32.4 (S-CH2), 126.5 (C-
Ar), 126.7 (C-Ar), 129.5 (C-Ar), 130.9 (C-Ar), 132.4 (C-Ar), 136.8 (C-Ar), 140.1 (C=N),
178.0 (C=S).
HRMS (ESI): 293.8745 [M + H]+. Anal. Calcd for C9H9Cl2N3S2: C, 36.74; H, 3.08; N,
14.28; S, 21.79; found: C, 36.78; H, 3.17; N, 14.31; S, 21.84.
4.3. General procedure for the synthesis of 1,3-thiazoles (16-26)
Example for compound (16): Thiosemicarbazone (4) (0.45 g, 2 mmol) was dissolved in 2-
propanol (10 mL), and then 2-bromoacetophenone (0.48 g, 2.4 mmol) and calcium
carbonate (0.24 g, 2.4 mmol) were added to a glass tube. The tube was placed in an
ultrasonic bath (40 MHz, 180 V) and irradiated for 60 min at r.t. Hexane was added, and the
mixture was cooled in a freezer overnight. The precipitate was filtered off and washed with
hexane and then dried in a desiccator under vacuum. An additional amount of the desired
compound was obtained from the filtrate after cooling. Pure products were obtained after
recrystallization using the solvent system detailed below for each compound.

ACCEPTED MANUSCRIPT
4.3.1. 2-{2-[2-(phenylthio)ethylidene]hydrazinyl}-4-phenyl-1,3-thiazole (16)
Crystallization from toluene/hexane 7:3, afforded brown crystals, yield = 72%. Mp (ºC):
1
148-149. IR (KBr): 1581 (C=N), 1611 (C=N), 3089 (N-H) cm-1. H NMR (300 MHz,
DMSO-d6) δ 3.85 (d,
J
= 5.9 Hz, 2H, S-CH2), 5.89 (bs, 1H, NH), 7.19 (t,
J
= 7.1 Hz, 2H,
Ar), 7.27 (s, 1H, Ar), 7.32 (t,
J
= 6.4 Hz, 2H, Ar), 7.35 – 7.49 (m, 5H, Ar, CH), 7.79 (d,
J =
13
7.4 Hz, 2H, Ar). C NMR (75.5 MHz, DMSO-d6): 33.8 (S-CH3), 103.7 (S-CH), 125.7 (C-
Ar), 126.2 (C-Ar), 127.8 (C-Ar), 128.3 (C-Ar), 128.6 (C-Ar), 129.0 (C-Ar), 133.8 (C-Ar),
134.7 (S-C), 141.6 (C-Ar), 148.9 (N=CH), 168.2 (N=C). HRMS (ESI): 326.0610 [M + H]+.
Anal. Calcd for C17H15N3S2: C, 62.74; H, 4.65; N, 12.91; S, 19.70; found: C, 62.72; H,
4.68; N, 12.96; S, 19.63.
4.3.2. 2-{2-[2-(phenylthio)ethylidene]hydrazineyl}-4-(p-tolyl)-1,3-thiazole (17)
Crystallization from toluene/hexane 7:3, afforded yellow crystals, yield = 68%. Mp (ºC):
1
148-149. IR (KBr): 1568 (C=N), 1625 (C=N), 3083 (N-H) cm-1. H NMR (400 MHz,
DMSO-d6) δ 2.27 (s, 3H, CH3), 3.79 (d,
1H, Ar), 7.18 (d, = 8.4 Hz, 2H, Ar), 7.31 (t,
Ar), 7.63 (d, = 7.9 Hz, 3H, Ar), 7.76 (d,
J = 6.0 Hz, 2H, S-CH2), 4.19 (s, 1H, NH), 7.11 (s,
J
J
= 7.4 Hz, 1H, Ar), 7.36 (t,
J = 6.4 Hz, 2H,
J
J
= 8.0 Hz, 2H, Ar). 13C NMR (75.5 MHz,
DMSO-d6): 20.8 (CH3), 33.8 (S-CH2), 102.6 (S-CH), 125.5 (C-Ar), 126.1 (C-Ar), 126.9 (C-
Ar), 128.6 (C-Ar), 128.9 (C-Ar), 129.1 (C-Ar), 129.6 (C-Ar), 131.3 (C-Ar), 134.7 (S-C),
136.9 (C-Ar), 141.6 (C-Ar), 151.2 (N=CH), 168.0 (N=C). HRMS (ESI): 340.0950 [M +
H]+. Anal. Calcd for C18H17N3S2: C, 63.69; H, 5.05; N, 12.38; S, 18.89; found: C, 63.74; H,
5.15; N, 12.32; S, 18.91.