Study of in vitro biological activity of thiazoles on Leishmania (Leishmania) infantum

Article abstract of DOI:10.1016/j.jgar.2020.02.028

Objectives: In the prospection of possible agents against neglected diseases, thiazole compounds are presented as promising candidates and are known to have activity against trypanosomatid parasites. Thus, this work aimed to evaluate the effects of thiazo

Full text of DOI:10.1016/j.jgar.2020.02.028

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Study of in vitro biological activity of thiazoles on Leishmania
(Leishmania) infantum
Vin´ıcius Vasconcelos Gomes de Oliveira, Mary Angela Aranda de
´
Souza, Rafaela Ramos Mororo Cavalcanti, Marcos Ver´ıssimo de
Oliveira Cardoso, Ana Cristina Lima Leite, Valdemiro Amaro da
´
Silva Junior, Regina Celia Bressan Queiroz de Figueiredo
PII:
S2213-7165(20)30057-6
https://doi.org/10.1016/j.jgar.2020.02.028
JGAR 1182
DOI:
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Journal of Global Antimicrobial Resistance
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Accepted Date:
20 May 2019
18 December 2019
22 February 2020
´
Please cite this article as: de Oliveira VVG, de Souza MAA, Ramos Mororo Cavalcanti R, de
Oliveira Cardoso MV, Lima Leite AC, da Silva Junior VA, de Figueiredo RCBQ, Study of in
vitro biological activity of thiazoles on Leishmania (Leishmania) infantum, Journal of Global
Antimicrobial Resistance (2020), doi: https://doi.org/10.1016/j.jgar.2020.02.028
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Study of in vitro biological activity of thiazoles on Leishmania (Leishmania)
infantum
Vinícius Vasconcelos Gomes de Oliveiraa,g*, Mary Angela Aranda de Souzab,
Rafaela Ramos Mororó Cavalcantic, Marcos Veríssimo de Oliveira Cardosod; Ana
Cristina Lima Leitee, Valdemiro Amaro da Silva Juniorf, Regina Célia Bressan Queiroz
de Figueiredob
a Departamento de Morfologia e Fisiologia Animal, Universidade Federal Rural
de Pernambuco (UFRPE), Rua Dom Manoel de Medeiros s/n, Dois Irmãos,
Recife, PE, 52171-900, Brasil. E-mail: vinicius-vasconcelos@hotmail.com.
*Autor para correspondência.
b Departamento de Microbiologia, Centro de Pesquisas Aggeu Magalhães,
Recife, PE, Brasil.
c Departamento de Biofisica. Universidade Federal de Sao Paulo (Unifesp). Sao
Paulo, Brasil
d Colegiado de Nutrição, Universidade de Pernambuco, PE, Brasil.
e Departamento de Ciências Farmacêuticas, Centro de Ciências da Saúde,
Universidade Federal de Pernambuco, Recife, PE, Brasil.
f Departamento de Medicina Veterinária, Universidade Federal Rural de
Pernambuco (UFRPE), Recife, Brasil.
g Centro Acadêmico de Vitória, Universidade Federal de Pernambuco (UFPE),
Vitória de Santo Antão, PE, Brasil.
⁎ Corresponding author.
E-mail addresses: vinicius-vasconcelos@hotmail.com (V.V.G. Oliveira)
1

ABSTRACT
Objectives: In the prospection of possible agents against neglected diseases, thiazole
compounds are presented as promising candidates and are known to have activity
against trypanosomatid parasites. Thus, this work aimed to evaluate the effects of
thiazole compounds on Leishmania infantum, etiological agent of visceral
leishmaniasis.
Methods: Thiazole compounds, being 5 thiazoacetylpyridines (TAPs-01; 04; 05; 06; 09)
and 5 thiazopyridines (TPs-01; 04; 05; 06; 09) were tested regarding its leishmanicidal
activity on both promastigote and amastigote forms of L. infantum. Its cytotoxicity was
tested using peritoneal macrophages of BALB/c mice. Ultrastructural analyzes were
performed to identify possible intracellular targets of the most effective compound on
promastigote forms. In order to observe routes that can clarify the possible mechanism
of action of the compounds on the intracellular amastigote forms, the Nitrite dosage was
performed.
Results: All compounds inhibited the growth of promastigote and presented low
cytotoxicity, being more selective to the parasite than to mammalian cells. All
compounds tested were able to decrease macrophage infection. There was a significant
decrease in the survival rate of the amastigote when compared to the untreated cells,
with TAP-04 presenting the best index. TAP-04 compound induced ultrastructural
changes that are relted to cell death by apoptosis. None of the macrophage groups
infected with L. infantum and subsequently treated showed increased nitrite release.
Conclusions: The low toxicity to mammalian cells and the leishmanicidal activity
observed demonstrate that the synthesis of drugs based in thiosemicarbazone nucleus,
thiazole and pyridine derivatives are promising to the treatment of VL.
Keywords: Ultrastructure; cytotoxicity; leishmaniasis
1. Introduction
Visceral leishmaniasis (VL) is an important parasitic zoonosis caused by the
protozoan Leishmania infantum [1, 2]. It is known that dogs are the main reservoirs of
the parasite in the urban environment in Brazil [3]. The transmission of VL among
mammals is essentially through vectors [4], although there is a possibility of direct dog-
dog transmission, such as transplacental vertical transmission in female dogs [5], as well
as sexual transmission [6].
Despite of the existence of several canine leishmaniasis (CanL) treatment
protocols [7], the combination of allopurinol with either meglumine antimonate or
miltefosine is currently considered the treatment of choice. These drugs trigger a
number of side effects, are difficult to administer and can lead to parasite resistance to
the treatment [8]. These substances may also further potentiate kidney damage [7].
Thus, alternative drugs, such as aminosidine (paromomycin) has been shown to have
2

leishmanicidal activity. However, these drugs are expensive and are also reported to be
toxic to mammals [9]. It is clear that new alternatives for the treatment against CanL
and by extension also against VL in humans, are necessary through the use of safer and
effective drugs.
Among other chemical groups that have been studied for anti-protozoal
activities, thiosemicarbazones [10] are promising. In addition, the cyclization of the
thiosemicarbazone in thiazole and the condensation of the pyridine ring with this new
molecule have shown anti-Trypanosoma activity [11]. The thiazole is an important class
of heterocyclic compounds that exhibit a broad biological activity spectrum, such as
antitumor [12], antibacterial [13], anti-inflammatory [14] and leishmanicidal activities
[15]. It has been demonstrated that cysteine-protease enzymes of L. infantum are
identified as targets of thiazolic compounds [16, 11]. These enzymes play an important
role in Leishmania sp. virulence, in its viability maintenance, in its morphology, and in
the host mononuclear phagocytic system (SFM) immune response [17].
Thus, the goal of this work was to evaluate the in vitro biological activity of
different thiazoles compounds in L. infantum.
2. Material and methods
2.1. Thiazolic derivatives
Five thiazol derivatives of the thiazopyridines series (TP) and five of the
thiazolacepyridines series (TAP) were used. They are N- (2-methyl-pyridine) -N '-
(phenylthiazol-2-yl) -hydrazine (TP-01); N- (2-methyl-pyridine) -N '- (p-
bromophenylthiazol-2-yl) -hydrazine (TP-04); N- (2-methyl-pyridine) -N '- (P-
fluorophenylthiazol-2-yl) -hydrazine (TP-05); N- (2-methyl-pyridine) -N '- (p-anisal
thiazol-2-yl) -hydrazine (TP-06); N- (2-methyl-pyridine) -N '- (3,4-
dichlorophenylthiazol-2-yl) -hydrazine (TP-09); N- (1-methyl-2-methyl-pyridine) -N '-
(phenylthiazol-2-yl) -hydrazine (TAP-01); N- (1-methyl-2-methyl-pyridine) -N '- (p-
bromophenylthiazol-2-yl) -hydrazine (TAP-04); N- (1-methyl-2-methyl-pyridine) -N '-
(P-fluorophenylthiazol-2-yl) -hydrazine (TAP-05); N- (1-methyl-2-methylpyridine) -N
'- (p -anisaltiazol-2-yl) -hydrazine (TAP-06) and N- (1-methyl-2-methylpyridine) -N' -
(3,4-dichlorophenylthiazol-2-yl) -hydrazine (TAP-09)]. The synthesis of these
compounds was developed by using high purity chemical reagents described by
Cardoso et al [11] (Fig. 1).
2.2. Parasites
Promastigote forms of L. infantum (Strain BH 46) were maintained at 26°C in
Schneider's medium (Sigma) supplemented with 10% fetal bovine serum (FBS) and
used in the exponential growth phase. Amastigote forms were obtained from the
inoculation of infective promastigote forms in cultures of peritoneal macrophages of
BALB/c mice.
3

2.3. Analysis of cytotoxicity in peritoneal macrophages of BALB/c mice
Peritoneal macrophages of BALB/c mice were treated with/without different
concentrations (2.78, 5.56, 11.12, 22.25, 44.5 μM) of the compounds to evaluate its
cytotoxicity in mammalian cells using the CellTiter-Glo® Luminescent Cell Viability
Assay method. Cells were incubated in 96-well plates (106 cells/mL). After a 48h-
treatment with the compounds, it was possible to determine the cytotoxic concentration
for 50% of the peritoneal macrophages (CC50). Its cytotoxicity and its activity against
promastigote parasites were compared to each other to determine the Selectivity Index
(SeI/pro= CC50 of macrophages divided by IC50 of promastigote forms).
2.4. Effect of the compounds on promastigate parasites
Promastigote forms of L. infantum were incubated in Schneider's medium
(Sigma) supplemented with 10% FBS at a concentration of 1x106 cells/mL. After
diluting the cells, they were incubated in the presence of different concentrations (2.78-
44.5 μM) of the compounds for 48h to determine the concentration that inhibited 50%
growth of the parasites (IC50/pro). Cell growth was monitored by CellTiter-Glo®
Luminescent Cell Viability Assay. Cells incubated with only Schneider's medium were
used as cell control. Each test was done in triplicate and in 3 independent experiments.
2.5. Effect of the compounds on amastigote forms
Peritoneal macrophages of BALB/c mice were incubated with RPMI-1640
medium (Sigma-Aldrich, Co., St. Louis, MO, USA) supplemented with 10% FBS in 24-
well plates containing coverslip (106 cells/mL). Macrophages were incubated at 37ºC
and 5% CO2 for 2 hours for adhesion and then infected with promastigote parasites
using a 1:15 parasite/cell ratio at 37ºC for 14h. Parasites that didn’t adhere on the
coverslips were washed out. Then, TAP-01, TAP-04 and TAP-06 (compounds selected
by previous tests) were added in the cell culture in different concentrations (0, 0.25, 0.5
and 1 μg/mL) for 24h. The coverslips were stained with Giemsa (Sigma-Aldrich, Co.,
St. Louis, MO, USA). The concentration capable of inhibiting 50% of macrophage
infection by amastigote forms (IC50/ama) was estimated from direct counting of
intracellular amastigote/100 infected cells by optical microscopy. Survival index was
determined by multiplying the number of amastigote by the number of infected
macrophages.
2.6. Ultrastructural analysis
4

In order to identify the drug’s possible intracellular target and also deleterious
effects of the compounds on the parasite membrane, ultrastructural analyzes using
transmission electron microscopy (MET) and scanning electron microscopy (MEV)
were performed. To perform MET, promastigotes forms of L. infantum with/without
TAP-04 (1/2 IC50 and IC50 incubation for 48h) were fixed for 60 min in a solution
containing 2.5% glutaraldehyde in sodium cacodylate buffer 0,1M (pH 7.2) and post-
fixed for one hour in a solution containing 1% osmium tetroxide (OsO 4), 0.8%
potassium ferricyanide, 5 mM CaCl 2 in 0.1M cacodylate buffer. After this step, the
samples were dehydrated in increasing concentrations of acetone, infiltrated and
included in epoxy resin (Fluka Analytical). Ultrafine cuts of approximately 70 nm
thickness were obtained in ultramicrotome (Leica EMUC6). Then, 5% uranyl acetate
and lead citrate were added for analysis on the Zeiss EM109B transmission microscope.
For MEV, promastigote forms of L. infantum treated with TAP-04 were fixed as
described above and placed to adhere to coverslips previously mentioned with poly-
lysine. After 20 minutes, the coverslips were washed with PBS to remove unbound and
post-fixed cells as described for MET. The cells were then washed in 0.1 M sodium
cacodylate buffer, dehydrated in increasing ethanol series and subjected to critical-point
drying at Critical Point dryer HCP-2 (Hitachi), metallized with 20 nm gold on the JFC
metallizer -1100 (Jeol) and viewed through the scanning electron microscope JEOL T-
200.
2.7. Nitric oxide production
To analyze the effect of the compounds using nitrite dosage, 100μL of the
culture’s supernatant from infected macrophages with and without treatment with the
compounds for 24 hours were incubated with 100μL of Griess reagent (1%
sulfanilamide and 0.1 % N - (1-naphthyl) -ethylenediamine dihydrochloride / 2.5% H 3
PO 4) at room temperature for 10 minutes. The absorbance was measured with a 540nm
filter in the Benchmark Plus ELISA reader (Bio-Rad Laboratories, Philadelphia, PA,
USA). The nitrite concentration was determined using a standard curve of sodium
nitrite.
2.8. Data analysis
Linear regression analyzes were performed in SPSS 8. (IBM Co., New York,
USA) for Windows and significance analyzes, considering p <0.05 as significant values,
were performed using the ANOVA test using the program GraphPad Prism 5.
(GraphPad, Calif., USA) for Windows.
3. Results
5

3.1. Cytotoxicity of compounds on peritoneal macrophages of balb/c
When tested against peritoneal macrophages, some compounds showed
cytotoxic effect at concentrations of CC50 close to the IC50/pro values determined for L.
infantum. However, calculation of SeI /pro demonstrated that all compounds were more
selective for the promastigote parasite than for the mammalian cells. Among the
compounds that presented the best selectivity index, we highlight TAP-01 (SeI = 12.73),
TAP-04 (SeI = 18.94) and TAP-06 (SeI = 7.79) (Table 1).
3.2. Effect of compounds on promastigotes forms of L. infantum
All thiazolic compounds were able to inhibit L. infantum promastigote growth.
The IC50 values ranged from 0.42 to 22.55 μM (Table 1). The most effective
compounds, ie those with the lowest IC50 concentrations, were TAP-05 (0.42 ± 0.03
μM), TAP-09 (2.73 ± 0.61 μM) and TAP-01 (3.57 ± 0.95 μM).
3.3. Effect of compounds on amastigotes forms of L. infantum
The amastigote parasites are responsible for the clinical manifestation of the
disease. In this way, the compounds TAP-01, TAP-04 and TAP-06 with highest SeI
values for L. infantum promastigote were chosen for the following tests. All compounds
were more effective against amastigotes than for promastigotes. TAP-06 and TAP-04
were about seven times more effective for intracellular forms of the parasite than for
promastigote forms, whereas for TAP-01 this difference was approximately 3 times.
The IC50/ama values regarding TAP-01, TAP-04 and TAP-06 were 0.99, 0.43 and 0.59
μM, respectively. The differences in the activity of the compounds on amastigotes were
directly related to the selectivity index of these parasites corresponding to the values of
46, 10, 137.37 and 59.05 for TAP-01, TAP-04 and TAP-06, respectively. Thus,
according to results TAP-04 had the best selectivity index for L. infantum amastigote
(Table 1). For the three concentrations tested (0.25, 0.5 and 1 μg / mL) there was a
significant (p <0.05) decrease in the survival index of the parasites inside the
macrophage when compared to the untreated cells. TAP-04 was the compound that
presented the highest rate of the amastigote viability inhibition inside the macrophage.
At the concentration of 1μg/mL the macrophage infection was almost 100% abolished
(Fig. 2).
3.4. Ultrastructural analysis
Considering that TAP-04 was the one that presented the best activity against
both forms of the parasite and was also the least toxic, we performed new experiments
to investigate possible intracellular targets of the compound as well as its effects on the
parasites’ cellular membrane using MET and MEV. As observed by MET,
promastigotes without addition of TAP-04 showed regular morphology, with the
nucleus well preserved, occupying the most central part of the cytoplasm and chromatin
6

associated with the inner nuclear membrane. A single mitochondria containing a
kinetoplast, region where mitochondrial DNA (k-DNA) is concentrated, was found
close to the flagellar pocket where the flagellum emerges (Fig. 3a).
After adding 1/2x IC50 of TAP-04, promastigote started to present swollen
mitochondria, besides intense disorganization of the mitochondrial ridges and in the
kinetoplast region, cellular disorganization with displacement of the nucleus to the
periphery of the cell and an increased profile of the endoplasmic reticulum. In addition
to these alterations, disorganization of Golgi complex cisternae (Fig. 3b, c, d) was
observed. These changes were more dramatic in cells treated with IC50/pro. It was also
possible to observe a change in the acidocalcisome volume and in the electron-dense
deposits present in this organelle, loss of mitochondrial matrix material and cytoplasm
compatible with possible damage to the plasma membrane. (Fig. 3e, f).
MEV experiments without addition of TAP-04 showed normal promastigote
shape, presenting an elongated cell body and an intact membrane (Figure 4a, b). Cells
treated with 1/2x IC5/pro (Fig. 4c, d) showed changes in the shape of the cell body
which started to assume a rounded morphology and loss of the flagellum. In the most
drastically affected cells it was possible to observe loss of cell volume with intense cell
membrane wrinkling. However, it was not possible to observe the presence of cellular
debris or apparent damages in the cell membrane, suggesting a cell death by apoptosis.
3.5. Effect of compounds on the production of NO
Considering the effect of the selected compounds (TAP-01, TAP-04 and TAP-
06) on amastigotes, we ran the NO experiment to see if these effects could be related to
an activation of the microbicidal capacity of the macrophages through the production of
nitric oxide (NO). Nitrite dosage was developed using the supernatant of infected
cultures with and without addition of the drugs. The data indicated that none of the
groups of macrophages infected with L. infantum and subsequently treated with TAP-
01, TAP-04 and TAP-06 showed increased NO production when compared to the
untreated infected cells. In cells treated with TAP-04 there was a significant decrease in
the highest concentrations tested (Fig. 5).
4. Discussion
Several studies have demonstrated that thiazole-derived compounds may exhibit
an important activity against trypanosomatid parasites [18, 19, 20, 21]. To evaluate the
leishmanicidal potential of thiazole derivatives, we performed a screening of the
biological activity of ten compounds on L. infantum promastigote parasites. All
compounds inhibited promastigote growth of forms. However, treatment with TAP-01,
TAP-04 and TAP-06 were considered more effective because they presented lower
values of IC50/pro and higher survival index when compared to the other compounds,
with TAP-04 being the compound that presented highest SeI /ama
For the TP series, comparing the compound without modification (TP-01, <2.78
μM) to the substituted ones, it is possible to observe only TP-06 (OMe) showed
7

leishmanicidal activity. Compounds TP-04 (Br), TP-05 (F) and TP-09 (Cl) were less
potent than TP-01. For the TAP series, all compounds presented relevant leishmanicidal
activity, with TAP-05 (F, 0.42 μM) and TAP-09 (Cl, 0.99 μM) being the ones with the
best results. In fact, TAP-05 was up to 8 times more potent than the base compound
without substituent (TAP-01, 2.73μM). These results are very similar to those found in
trypomastigote and epimastigote of Trypanosoma cruzi [11]. These authors showed that
the TAP series were an excellent inhibitor of cruzaine activity, being greater than the TP
series. Both results demonstrate the importance of methyl for antichagasic and
leishmanicidal activity. In fact, the presence of methyl in the chemical structure usually
improves the liposolubility, thus improving the drug bioavailability with increasing of
the compounds biological activity [11, 20].
Similarly to the results observed by thiazolic compounds’ experiments, it was
possible to observe potent activity against naphthotiazoles L. braziliensis promastigotes;
also, the compounds tested by them reduced the survival rate of amastigote forms in
mammalian macrophages [22]. In addition, studies by Nava-Zuazo et al. [15] also
showed that compounds containing the thiazolic ring in their structure inhibited L.
amazonensis promastigote. These results demonstrated that thiazole derivatives have
promising leishmanicidal activity. It is important to emphasize that it’s the first time
that the compounds used in all experiments developed by us were tested for in vitro
biological activity on L. infantum.
The important requirement to develop new medicine routes with leishmanicidal
action is that they must not be toxic to mammalian cells [23]. Therefore, not only the
effect of the compounds on parasite growth was considered but also the cytotoxic
potential of these on mammalian cells was evaluated. The low toxicity of some thiazole
derivatives to mammalian cells when compared to reference drugs, such as Glucantime
(727.63 μM) [24], had a direct reflection on SeI /pro values, which were always more
selective for parasites than for mammalian cells. Even the drug with the highest CC50
value among the 10 ones tested so far, TP-05 (109.67 μM), was almost 7x less cytotoxic
than Glucantime. Cytotoxicity in peritoneal macrophages treated with thiazopyridine
derivatives can be attributed to the presence of the pyridine ring, since it has been
demonstrated that the thiazole group is non-toxic and it improves the cellular viability
of thiosemicarbazones through the functionalization of thiocarbonyl to thiazole [11].
TAP-04 was selected for ultrastructural analysis through MET after
demonstrating the highest index selectivity to the parasite when compared to the host
cell. After 48h-treatment, it was possible to observe different morphological features in
the parasites in a dose-dependent relation, such as complete disorganization of
mitochondrial ridges. In fact, mitochondria is a main target of several drugs [25, 26]
because this organelle is crucial for the survival of several cell types, especially for
trypanosomatids, which present only a single mitochondria. Modifications on the
trypanosomatid mitochondria structure caused by drugs are commonly observed,
showing that this organelle is chemotherapeutic sensitive [27]. In addition, there are
modifications in the endoplasmic reticulum profile, vesiculation and fragmentation of
the Golgi complex and appearance of structures typical of the autophagic process.
8

Bilbao-Ramos et al [28] developed ultrastructural experiments on L. infantum
promastigote with another class of thiazole derivatives. However, they observed similar
leishmanicidal effect, such as damage to the nucleus, mitochondria and kinetoplast.
Finally, electron microscopy studies demonstrated that the parasites treated with TAP-
04 clearly showed ultrastructural modifications compatible with programmed cell death
[29], such as, cell rounding, chromatin condensation and mitochondrial swelling.
Taking into account that the thiazole derivatives showed good leishmanicidal
activity against L. infantum promastigotes and low toxicity in host cells, we expected
that intracellular amastigote might be also sensitive to treatment with the chosen drugs.
In this case, the compounds might have two mechanism of action: a direct effect on the
host cells making them more efficient in combating infection [30] or an effect on
therapeutic targets important to the survival of amastigotes in the macrophage. In
addition, the compounds selected for evaluation in amastigotes present cysteine-
protease inhibition activity [11]. This enzyme plays an important role on the parasite
viability, such as in its morphology maintenance [31, 17]. In fact, cysteine-protease is
found in large quantities in lysosomes, which are particularly abundant in Leishmania
amastigotes [16].
The current progress within drug development is possible because it was
established a few criteria that classify the compound efficiency [32]. These criteria
include biological, physicochemical and pharmacokinetic aspects, as well as safety and
toxicological aspects of drug discovery for several neglected diseases, including
leishmaniasis [32, 33]. Among these criteria, there are also the definition of chemical
structure; the synthetic route establishment; and the amastigote selectivity index value
that has to be higher than 20 [32]. In fact, TAP-01, TAP-04 and TAP-06 fulfill those last
three criteria [11], highlighting TAP-04 for presenting the highest selectivity index (SeI
/ama=137.37), which was approximately 7 times higher than the minimum established
for a promising drug [32].
In the vertebrate host, the early stages of the trypanosomatid parasites’ infection
are capable of triggering inflammation mediators from infected cells, such as Th1 or
Th2 cytokines, as well as reactive oxygen species (ROS) and nitrogen species (eg NO)
[34]. Data from the present study showed a significant reduction of peritoneal exudate
(PEC) cells infected by L. infantum after treatment with thiazole derivatives. However,
analysis of nitric oxide production by these cells demonstrated that there was no
significant increase in NO in infected macrophages and in macrophages treated with the
compounds. Several authors describe that the release of cytokines from the Th2 profile
triggered by the activation of inflammatory regulation factors are the defense
mechanism against Leishmania sp. [35, 36]. Bcl-2 family members are known as
apoptosis regulators in mammalian cells and therefore mediators of parasite infection
response. They may be related to the elimination of parasites by the activation of
inflammatory cytokines [37]. Thus, the activation of such regulatory mediators could
modulate the inflammatory process that normally occurs during Leishmania infection
and thus inhibit the exacerbated production of nitric oxide in these infected
9

macrophages [38, 39]. However, further studies are necessary to understand the
mechanism of action of these compounds in the intracellular L. infantum amastigote.
5. Conclusions
These results demonstrated that thiazole compounds of the series thiazopyridines
(TP) and thiazoacetylpyridines (TAP) have promising leishmanicidal activity. In
addition, it is important to emphasize its high selective activity against L. infantum with
low toxicity against host cells.
Funding
This work was supported by the FACEPE (APQ/FACEPE-0354-5.05/15).
Ethical approval
The present study was carried out in accordance with the ethical principles adopted by
Brazilian law 11.794/2008 and approved by the Ethical Committee for Animal Research
of the Instituto Aggeu Magalhães/Fundação Oswaldo Cruz (N° 039/12).
Competing interests
None declared.
Acknowledgments
The authors thank the Fundação de Amparo à Ciência e Tecnologia do Estado de
Pernambuco (FACEPE) for its fi nancial support.
10

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Figure legends
Fig. 1. Synthetic procedures for thiosemicarbazones (2a-b) and 2- (pyridin-2-yl) 1,3-thiazole
(TPs and TAPs). Reagents and conditions: (a) thiosemicarbazide, 2-propanol, acetic acid (3
drops), ultrasonic irradiation, r.t., 120 min; (B) Substituted 2-bromoacetophenones, 2-propanol,
CaCO 3, ultrasonic irradiation, r.t., 60 min. Adapted from Cardoso et al [11].
Fig. 2. Survival Index of Leishmania infantum within macrophages after 24 hours of treatment
with TAP-01, TAP-04 and TAP-06. Note: Asterisks correspond to significant values (p <0.05)
18

Fig. 3. Transmission Electron Microscopy (MET) of Leishmania infantum promastigote
parasites after treatment with TAP-04. A: promastigote without addition of TAP-04
demonstrating preserved morphology of mitochondria (M), nucleus (N), kinetoplast (C),
flagellar purse (BF), flagellum (F) and glycosome (*). B, C, D: Promastigote treated with 0.5x
IC50. Note disorganization of the mitochondrial ridges and kinetoplast, cellular disorganization
with displacement of the nucleus to the periphery of the cell and increase of the endoplasmic
reticulum (ER) profiles and formation of atypical vesicles in cisterns of the Golgi complex. E,
F: Promastigote treated with IC50 showing condensation of the chromatin and greater
disorganization of the mitochondrial ridges.
19

Fig. 4. Scanning Electron Microscopy (MEV) of Leishmania infantum promastigote parasites
after treatment with TAP-04. A, B: Promastigote without addition of TAP-04 presenting an
elongated cell body and an integral membrane. C, D: Promastigote treated with 0.5x IC50
showing changes in morphology, rounded forms and loss of flagellum. E, F: Parasite treated
with IC50 showing loss of membrane integrity with exposure of the cytoplasmic material
demonstrated with the appearance of electron-sparing regions in the cytoplasm region.
20

Fig. 5. Nitric oxide dosage in the supernatant of the infected cultures cells with and without
addition of TAP-01, TAP-04 and TAP-06.
21

22

Table 1
Effect of the thiazole derivatives on the promastigotes growth and amastigotes growth of
Leishmania infantum and on peritoneal macrophages of BALB/c mice.
Promastigotes Macrophages
amastigotes
Promastigotes
Amastigotes
IC50
CC50
IC50
SeI
SeI
μM
μM
3.57 ± 0.95
3.12 ± 0.86
0.42 ± 0.03
4.44 ± 1.27
2.73 ± 0.61
<2.78
45.46 ± 11.66
59.09 ± 15.19
2.95 ± 0.29
34.62 ± 13.21
4.31 ± 0.86
11.71 ± 1.21
41.51 ± 1.70
109.67 ±
12.73
18.94
7.07
7.79
1.57
*
0.99 ± 0.03
46.10
TAP-01
TAP-04
TAP-05
TAP-06
TAP-09
TP-01
0.43 ± 0.11
137.37
-
-
0.59 ± 0.09
59.05
-
-
-
-
-
-
7.01 ± 3.72
5.92
TP-04
22.55 ± 0.97
4.86
-
-
TP-05
39.15
<2.78
1.35 ± 0.19
4.68 ± 1.87
*
-
-
-
-
TP-06
TP-09
5.52 ± 0.29
0.84
* it was not possible to determine.
23