Novel indol-3-yl-thiosemicarbazone derivatives: Obtaining, evaluation of in vitro leishmanicidal activity and ultrastructural studies

Article abstract of DOI:10.1016/j.cbi.2019.108899

Parasitic diseases still represent serious public health problems, since the high and steady emergence of resistant strains is evident. Because parasitic infections are distributed predominantly in developing countries, less toxic, more efficient, safer and more accessible drugs have become desirable in the treatment of the infected population. This is the case of leishmaniasis, an infectious disease caused by a protozoan of the genus Leishmania sp., responsible for triggering pathological processes from the simplest to the most severe forms leading to high rates of morbidity and mortality throughout the world. In the search for new leishmanicidal drugs, the thiosemicarbazones and the indole fragments have been identified as promising structures for leishmanicidal activity. The present study proposes the synthesis and structural characterization of new indole-thiosemicarbazone derivatives (2a-j), in addition to performing in vitro evaluations through cytotoxicity assays using macrophages (J774) activity against forms of Leishmania infantum and Leishmania amazonensis promastigote as well as ultrastructural analyzes in promastigotes of L. infantum. Results show that the indole-thiosemicarbazone derivatives were obtained with yield values varying from 32.09 to 94.64%. In the evaluation of cytotoxicity, the indole-thiosemicarbazone compounds presented CC₅₀ values between 53.23 and 357.97 μM. Concerning the evaluation against L. amazonensis promastigote forms, IC₅₀ values ranged between 12.31 and > 481.52 μM, while the activity against L. infantum promastigotes obtained IC₅₀ values between 4.36 and 23.35 μM. The compounds 2d and 2i tested against L. infantum were the most promising in the series, as they showed the lowest IC₅₀ values: 5.60 and 4.36 respectively. The parasites treated with the compounds 2d and 2i showed several structural alterations, such as shrinkage of the cell body, shortening and loss of the flagellum, intense mitochondrial swelling and vacuolization of the cytoplasm leading the parasite to cellular unviability. Therefore, the indole-thiosemicarbazone compounds are promising because they yield considerable synthesis, have low cytotoxicity to mammalian cells and act as leishmanicidal agents.

Full text of DOI:10.1016/j.cbi.2019.108899

Journal Pre-proof
Novel indol-3-yl-thiosemicarbazone derivatives: Obtaining, evaluation of in vitro
leishmanicidal activity and ultrastructural studies
Paula Roberta da Silva, Jamerson Ferreira de Oliveira, Anekécia Lauro da Silva,
Camila Marques Queiroz, Ana Paula Sampaio Feitosa, Denise Maria Figueiredo
Araújo Duarte, Aline Caroline da Silva, Maria Carolina Accioly Brelaz de Castro,
Valéria Rêgo Alves Pereira, Rosali Maria Ferreira da Silva, Luiz Carlos Alves, Fábio
André Brayner dos Santos, Maria do Carmo Alves de Lima
PII:
S0009-2797(19)31482-6
https://doi.org/10.1016/j.cbi.2019.108899
CBI 108899
DOI:
Reference:
To appear in: Chemico-Biological Interactions
Received Date: 1 September 2019
Accepted Date: 12 November 2019
Please cite this article as: P.R. da Silva, J.F. de Oliveira, Aneké.Lauro. da Silva, C.M. Queiroz, A.P.S.
Feitosa, Denise.Maria.Figueiredo.Araú. Duarte, A.C. da Silva, M.C.A.B. de Castro, Valé.Rê.Alves.
Pereira, R.M.F. da Silva, L.C. Alves, Fá.André.Brayner. dos Santos, M.d.C.A. de Lima, Novel indol-3-yl-
thiosemicarbazone derivatives: Obtaining, evaluation of in vitro leishmanicidal activity and ultrastructural
studies, Chemico-Biological Interactions (2019), doi: https://doi.org/10.1016/j.cbi.2019.108899.
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Novel indol-3-yl-thiosemicarbazone derivatives: obtaining, evaluation
of in vitro leishmanicidal activity and ultrastructural studies
Paula Roberta da Silvaa; Jamerson Ferreira de Oliveiraa; Anekécia Lauro da Silvab; Camila
Marques Queirozc; Ana Paula Sampaio Feitosac; Denise Maria Figueiredo Araújo Duartea;
Aline Caroline da Silvad; Maria Carolina Accioly Brelaz de Castrod,e; Valéria Rêgo Alves
Pereirad; Rosali Maria Ferreira da Silvaf; Luiz Carlos Alvesc; Fábio André Brayner dos
Santosc; Maria do Carmo Alves de Limaa,*
aUniversidade Federal de Pernambuco (UFPE), Departamento de Antibióticos, 50670-901,
Recife, PE (Brasil);
bUniversidade Federal do Vale do São Francisco (UNIVASF), Departamento de Medicina,
48607-190, Paulo Afonso, BA (Brasil);
cInstituto Aggeu Magalhães, Fundação Oswaldo Cruz (IAM-FIOCRUZ), Departamento de
Parasitologia, 50670-420, Recife, PE (Brasil);
dInstituto Aggeu Magalhães, Fundação Oswaldo Cruz (IAM-FIOCRUZ), Departamento de
Imunologia, 50670-420 Recife, PE (Brasil);
eUniversidade Federal de Pernambuco (UFPE), Núcleo de Enfermagem, 55608-680, Vitória
de Santo Antão, PE (Brasil);
fUniversidade Federal de Pernambuco (UFPE), Departamento de Ciências Farmacêuticas
(DCFAR), 50670-901, Recife-PE (Brazil).
To whom correspondence should be addressed: Maria do Carmo Alves de Lima
*Email: nenalima.mariadocarmo@gmail.com
Phone number: +55 8121268347
Fax: +55 8121268346
1

Abstract
Parasitic diseases still represent serious public health problems, since the high and steady
emergence of resistant strains is evident. Because parasitic infections are distributed
predominantly in developing countries, less toxic, more efficient, safer and more accessible
drugs have become desirable in the treatment of the infected population. This is the case of
leishmaniasis, an infectious disease caused by a protozoan of the genus Leishmania sp.,
responsible for triggering pathological processes from the simplest to the most severe forms
leading to high rates of morbidity and mortality throughout the world. In the search for new
leishmanicidal drugs, the thiosemicarbazones and the indole fragments have been identified as
promising structures for leishmanicidal activity. The present study proposes the synthesis and
structural characterization of new indole-thiosemicarbazone derivatives (2a-j), in addition to
performing in vitro evaluations through cytotoxicity assays using macrophages (J774) activity
against forms of Leishmania infantum and Leishmania amazonensis promastigote as well as
ultrastructural analyzes in promastigotes of L. infantum. Results show that the indole-
thiosemicarbazone derivatives were obtained with yield values varying from 32.09 to 94.64%.
In the evaluation of cytotoxicity, the indole-thiosemicarbazone compounds presented CC50
values between 53.23 and 357.97 µM. Concerning the evaluation against L. amazonensis
promastigote forms, IC50 values ranged between 12.31 and> 481.52 µM, while the activity
against L. infantum promastigotes obtained IC50 values between 4.36 to 23.35 µM. The
compounds 2d and 2i tested against L. infantum were the most promising in the series, as they
showed the lowest IC50 values: 5.60 and 4.36 respectively. The parasites treated with the
compounds 2d and 2i showed several structural alterations, such as shrinkage of the cell body,
shortening and loss of the flagellum, intense mitochondrial swelling and vacuolization of the
cytoplasm leading the parasite to cellular unviability. Therefore, the indole-thiosemicarbazone
2

compounds are promising because they yield considerable synthesis, have low cytotoxicity to
mammalian cells and act as leishmanicidal agents.
Keywords: Leishmania infantum; Leishmania amazonensis; thiosemicarbazone; indole
derivatives; ultrastructural studies.
1. Introduction
World health remains affected by the consequences of infectious diseases, especially
parasitic diseases, since they are one of the major causes of high morbidity and mortality.
Therefore, it is necessary to search a form of controlling these diseases and, most of all, their
outbreaks [1]. According to the World Health Organization (WHO), leishmaniasis, a parasitic
disease caused by protozoan Leishmania spp. Is still classified as emerging and uncontrolled
disease with an annual incidence of 1.5 to 2 million cases, posing risk of infection for more of
350 million people [2,3].
It is estimated that there are more than 22,000 cases of individuals infected with
leishmaniasis in Brazil, predominantly in the Amazon region, where the insect vectors
belonging to the genus Phlebotomus and Lutzomyia are found. These insects are responsible
for the transmission of parasitosis [4]. Depending on the species at the time of infection, the
parasite and the immune response of the infected host, the clinical manifestations may vary in
cutaneous, mucocutaneous and visceral, the latter being the most aggressive, becoming fatal
in 85- 90% of untreated patients [5-7].
The ideal treatment for leishmaniasis is still chemotherapy, through the use of
pentavalent antimonial compounds (sodium stibogluconate and meglumine antimoniate),
including second-choice compounds such as amphotericin B, pentamidine and miltefosine
[8,9]. However, these therapies are quite toxic at the same time as they exhibit other
3

limitations such as the high cost and necessity of daily parenteral administration, which
characterizes an invasive intervention in the long term [10-12]. In addition to the
aforementioned drawbacks, there are also reports indicating the emergence of strains resistant
to the treatment of leishmaniasis [13-15].
Hence, the search for new, more effective, less toxic, safe and more suitable
leishmanicide chemotherapeutics is severely necessary. And it is above all in this context that
several in vitro and in vivo studies have been developed using synthetic and leishmanicidal
potential compounds already known.
A class of molecules that has been widely studied due to its chemical and
pharmacological properties are the thiosemicarbazones, which stand out due to their
leishmanicidal biological activity. Thiosemicarbazone derivatives have shown efficacy
against promastigotes and amastigotes of Leishmania with possible mechanisms of action
elucidated, acting in the depolarization of the mitochondria, interacting with the DNA,
causing the death of the parasite by apoptosis, besides inhibiting proteins of the parasite [16-
18].
Another fragment of interest in medicinal chemistry is the indole nucleus. As a
privileged structure, it stands out in the antiparasitic activity because it constitutes several
promising molecules of biological interest [19,20]. On the antimalarial activity (Plasmodium
falciparum), the indole is indicated as a key point for the active site binding and histone
deacetylase inhibition [21]. In addition, indole-containing compounds demonstrated activity
against trypanosomatida trypanothione reductase (Trypanosoma brucei, Trypanosoma cruzi
and Leishmania spp.) [22].
Recently, our research group has pointed to the leishmanicidal activity of new indole-
thiophene hybrids to verify the action of these compounds, against promastigotes and
amastigotes forms, in the immunomodulation and their role in the inhibition of trypanothione
4

reductase [23,24].This work investigated the chemical synthesis of new indole-
thiosemicarbazone derivatives and evaluated their cytotoxic and biological profile through in
vitro leishmanicidal investigation against the Leishmania infantum and Leishmania
amazonensis.
2. Results
2.1 Chemistry
Synthesis of new indole-thiosemicarbazone derivatives was conducted in two steps
(Scheme 1). First, intermediary compounds were obtained (thiosemicarbazide) through
nucleophilic addition between substituted hydrazine and isothiocyanate. The second step
consisted of a condensation reaction in which the synthesized thiosemicarbazide reacted to the
substituted indole-carboxaldehyde in the presence of a catalyst quantity of hydrochloric acid
forming interesting compounds with results that ranged from 32.09 to 94.64% of yield.
Analysis of the infrared spectrum showed that ligation (C-S) appears in the region
(1197.67-1450.51) cm-1, the band (C=N) was found in the region (1541-1628.36) cm-1, which
corroborated data from the literature in which the imino function appears in 1556-1630 cm-1
and the band (N-H) in the region (3105.8-3496). The possibility of tautomerism (thione-thiol)
due to absence of bands in the region 2500-2600 cm-1 [25] was excluded.
1
NMR spectrum H evidenced the presence of hydrogen in the ligation (HC=N) that is
presented as singlet between. Signals referring to hydrogen in the methyl (-CH3) group of
indole between 2.41-2.50 ppm were also observed. In addition, signals between 11.22–11.64
and 11.18-12.35 ppm were attributed to hydrogen in the groups NH hydrazine and NH indole,
respectively. The signal referring to NH phenyl appears between 8.01-9.69 ppm. This signal is
more protected than the other NH signals due to the resonance effect likely to happen between
Nitrogen and Caron in the thiocarbonyl (-NH-C=S; -N=C-SH) [26]. Therefore, after the
5

structural elucidation, the compounds that were planned and synthesized in this study were
subjected to biological evaluation.
PLEASE, INSERT SCHEME 1 HERE
2.2 Biological assays in vitro
2.2.1 Cytotoxicity assay
Cytotoxicity analysis employed macrofage (J774) and allowed observation of
cytotoxic variation between the compounds (53.23 to 357.97µM) (Table 1). Compounds 2a
and 2b substituted in the N-4 position of the thiosemicarbazone with hydrogen were the least
cytotoxic in the series, showing values of respectively. These compunds displayed greater
toxicity than drugs of reference that showed CC50 values of 37.6 and 219.05µM (AmB and
Mtf).
Substitution of N-4 position of the thiosemicarbazone with p-methyl-phenyl showed
decrease in CC50 with values of 118,31 and 135,25µM for compounds 2g and 2h respectively.
The other replacements (hydrogen, phenyl, p-ethyl-phenyl p-methoxy-phenyl) also showed
decrease in toxicity, however they also displayed values of CC50 higher than 50µM.
2.2.2 Biological activity in Leishmania infantum promastigotes
All ten compounds assessed in the present investigation are structurally different in
two aspects. The first differentiating aspect for compounds in the series are the substituents
found bound to N-4 position of the thiosemicarbazone (hydrogen, phenyl, p-methyl-phenyl, p-
ethyl-phenyl p-methoxy-phenyl). The additional structural difference concerns the pattern of
substitution of the indole ring (series 5-bromine-7-methyl/series 5-methyl-7-bromine). The
biological repercussion promoted by these molecular modifications were investigated.
6

Leishmanicidal evaluation verified the efficacy of indole thiosemicarbazone on the
promastigote forms of Leishmania infantum. All compounds in the series in the current
investigation were able to inhibit the promastigote forms of with IC50 that ranged from de 4.36
to 23.25µM. Non-substitution (hydrogen) in the N-4 position of the thiosemicarbazone
displayed better results for compounds 2a and 2b with significant IC50 values of 16.70 and
13.51µM. However, substitution with p-methyl-phenyl group did not favor activity,
significantly increasing as occurred for compound 2g that obtained value of 23.35µM. The
other substitutions with (phenyl, p-ethyl-phenyl, p-methoxy-phenyl) exhibited better activity
in the molecule, particularly compound 2d with phenyl substituent and 2i with p-ethyl-phenyl
substituent, both with bromine at C-5 and methyl at C-7 of the indole, displaying better
leishmanicidal activity with IC50 values of 5.60 and 4.36µM respectively (Table 1).
Leishmanicidal evaluation concerning the promastigote forms of de L. amazonensis
for all compounds in the series were also capable of inhibiting promastigote forms with IC50
values that ranged from 12.31 to > 481.52µM (Table 1). Substitution in the N-4 position of the
thiosemicarbazone with hydrogen, p-methyl-phenyl did not effectively increased
Leishmanicidal activity displaying values of 87.76, 75.24 and 134.38µM for compounds 2a,
2f and 2g. Similar to L. infantum testing, phenyl substitutions and p-ethyl-phenyl displayed
better results with IC50 values of 13.61 and 12.31µM for compounds 2c and 2i.
Evaluation of the selectivity index (SI) for compounds tested against L. infantum
displayed better results for compounds 2a; 2b; 2d; 2f and 2i, which displayed SI values of
14.24; 26.49; 17.97; 13.45; and 12,21 respectively. However, the compounds with the best
results were the compounds 2d and 2i because in addition to displaying better selectivity to
the parasite than a mammalian cell, they also displayed lower IC50. Evaluation of compounds
against L. amazonensis showed better results for derivatives 2b, 2c, 2h and 2i with values of
7

14.97; 4.67; 6,91 and 4, 32 respectively; all of the mentioned compounds displayed SI values
higher than the standard drug (Miltefosine SI of 3,87).
The best compounds in the series were 2d and 2i as they presented good SI and the
lowest IC50. Thus, they were chosen for ultrastructural investigation of L. infantum and L.
amazonensis.
PLEASE, INSERT TABLE 1
2.2.3 Structural analysis of L. infantum promastigotes
Ultrastructural evaluation used the compound with best leishmanicidal activity,
compound 2d, with substituent phenyl and compound 2i with p-ethyl-phenyl both with
bromine at C-5 and methyl at C-7 of the indole nucleus.
Ultrastructural analysis through scanning electron microscope (SEM) showed that
untreated L. infantum promastigotes display fusiform morphology typical of the parasite in
addition to a body surface topologically normal with long and free scourge (Figure 1 A-B).
Promastigotes subjected to amphotericin B at 0.05 µM presented ultrastructural alterations
such as shrinkage and deformation of the cell body and flagellum, alterations in the membrane
with cell destruction (Figure 1 C-D).
PLEASE, INSERT FIGURE 1
L. infantum promastigotes treated with the compound 2d at 5.6 µM and 11.2 µM
concentrations displayed alterations on the parasite surface (Figure 2 A-D). Morphological
alterations could be noted such as body cell shrinkage, which changed the fusiform shape to
oval (Figure 2 A-B). In addition, the parasite membrane displayed undulation and
8

disintegration of the membrane (Figure 2 B-C) as well as shrinkage and flagellum loss (Figure
2 C-D). These alterations were similar to the ones observed in promastigotes subjected to
amphotericin B at 0.05 µM.
PLEASE, INSERT FIGURE 2
Compound 2i was also capable of causing alterations in L. infantum promastigotes for
both concentrations 4.36 µM and 8.72 µM. Alterations were similar to the ones described
after treatment with amphotericin B and compound 2d. When subjected to 4.36 µM
concentration, alterations in shape and size of promastigotes were noted with pronounced
shrinkage of the body cell and flagellum retraction (Figure 3A), and the presence of several
grooves in the outside surface of the protozoan was also noted (Figure 3B). Grooves were also
noted at concentration 8.72 µM (Figure 3C) in addition to alterations in cell morphology and
atypical cell division (Figure 3D).
PLEASE, INSERT FIGURE 3
Transmission electron microscopy (TEM) analysis showed that in the untreated group,
L. infantum promastigotes displayed normal shape and morphology with well-preserved
mitochondria, nucleus, flagellum pouch and flagellum (Figure 4 A-C). In cells treated with
amphotericin B at 0.05 µM, it was noted the presence of mitochondrial swelling, pyknotic
nucleus, intense vacuolization of the cytoplasm, electrodense particles and increase in
chromatin condensation (Figure 4 D-F).
PLEASE, INSERT FIGURE 4
9

L. infantum promastigotes when subjected to treatment with compound 2d at
concentration (Figure 5 A-C) displayed alterations similar to the ones caused by amphotericin
B at 0.05 µM. It was noted pyknotic nucleus, intense vacuolization of the cytoplasm,
electrodense particles, intense mitochondrial swelling and disorganization of the kinetoplast.
Subjected to treatment at 11.2 µM concentration, it was possible to observe the presence of
electrodense particles, loss of cytoplasmatic content leading to cell destruction (Fig.5 D-F)
PLEASE, INSERT FIGURE 5
Compound 2i for both 4.36 µM and 8.72 µM concentrations presented alterations in
the cell cytoplasm, mitochondrial swelling with severe decrease in electrodensity of the
matrix and appearance of concentric membrane structures within the organelles (Figure 6 A-
F). Intense vacuolization was noted in the cytoplasm (Figure 6 A-B, D-E). The presence of
autophagy vacuole (Figure 6 A-B) was noted at the smallest concentration, and at the highest
it was noted the pyknotic nucleus with increase in condensation of chromatin, noting its
marginalization.
PLEASE, INSERT FIGURE 6
3. Discussion
Cytotoxicity analysis of a compound is one of the criteria used to develop new drugs.
A promising compound is classified when presenting low toxicity in mammalian cells [27].
All compounds in the indole-thiosemicarbazone tested in this investigation displayed low
toxicity regarding mammal cells. Similar to Moreira et al. [28] who also tested cytotoxicity of
10

thiosemicarbazone derivatives and noted low toxicity values. Therefore, it can be inferred that
thiosemicarbazone derivatives present potential in the production of new drugs.
All indole-thiosemicarbazone derivatives tested in this investigation were capable of
inhibiting L. infantum and L. amazonensis promastigote forms. Such activity can be related to
the presence of the indole group and substitutions at position N-4 of thiosemicarbazones. The
indole group has gained attention of the scientific community due to its therapeutic use [29].
Indole is commonly found in nature, in animal hormones as serotonin and melatonin and also
present in plants in the constitution of some alkaloids [30]. Therefore, some natural and
synthetical compounds containing indole have displayed antitumor [31], antiviral [32],
antimicrobial [33], anti-inflammatory [34] and leishmanicidal [35].
Compounds 2c, 2d and 2i presented the best values of IC50 when tested against L.
infantum: 6.12; 5.60 and 4.36µM respectively. Compounds 2c and 2i also presented lower
values when tested against L. amazonensis, with IC50 value of 13.61 and 12.31µM
respectively. The compounds evaluated in this study have the disubstituted indole nucleus (5-
CH3/7-Br or 5-Br/7-CH3). However, based on the results mentioned above we can conclude
that the position of bromine or methyl group at the indole nucleus did not influence directly
the leishmanicidal activity. Nonetheless, the importance of halogens as substituents has been
described in the literature, such as the study by Hernandes et al. [36] with thiazolyl hydrazone
against a T. cruzi, when the presence of halogens was noted to increase the interaction
between the receptor and the drug, in addition to conserving permeability of the plasmatic
membrane thus allowing the entrance of the drug into the cell. This justifies the greater
activity of compounds with these groupings [37]. Furthermore, the presence of aromatic
sessions and halogens in the molecules showed overall improvement in biological activity of
the molecules against trypanosomatids [18, 38-41].
11

The compounds evaluated also presented structural difference with substituents in the
N-4 position of the thiosemicarbazone such as hydrogen and phenyl, p-methyl-phenyl, p-
methoxy-phenyl, p-ethyl-phenyl and p-methoxy-phenyl groups. In the present study,
substitutions with hydrogen, p-methoxy-phenyl and p-methyl-phenyl were not efficient to
promote compound activity. Carvalho et al. [42] studied derivatives of thiosemicarbazone
substituted in the N-4 position with hydrogen and tested against L. amazonensis also not
finding significant reduction in promastigotes. Substitution in the position N-4 with p-
methoxy-phenyl in works with thiosemicarbazone derivatives tested for L. major
promastigotes such as ours, also did not present reduction in IC50 values [17]. In Melos et al.
[43] thiosemicarbazone substituted in the position N-4 with p-methyl-phenyl presented the
best IC50 values but in the present study this substitution did not increase significantly the
action of compounds 2g and 2h when tested against L. infantum. However, assessment against
L. amazonensis showed compound 2h displayed IC50 values of 19.58 and SI de 6.91, a higher
selectivity index than the standard drug (Mtf 3.87).
Derivatives with substituents phenyl and p-ethyl-phenyl displayed the best activity for
both strains (L. infantum compounds 2d and 2i) and (L. amazonensis compounds 2c and 2i),
thus allowing the inference that the action is related to these substituents. The presence of
substituents on the phenyl group at the N-4 position of thiosemicarbazones was described in
the literature and showed significant results against forms of T. brucei trypomastigotes [44].
Regarding the p-ethyl-phenyl, Saad et al. [45] noted moderate activity of compounds with this
substituent. These results corroborate data found in the present study, in which compounds
were also active. This can be related to substitution in the N-4 position of the
thiosemicarbazone, as also described by Pervez et al. [17].
Investigations on the ultrastructural alterations was conducted with two compounds
chosen by their SI values as well as lower IC50. Based on these criteria, two compounds stand
12

out when tested against, 2d and 2i with IC50 values of 5.60 and 4.36µM. The activity of these
compounds can be seen through ultrastructural alterations that can lead to cell unviability thus
reinforcing claims by Menna-Barreto et al. [46] who suggest that a great number of
morphological alterations is always related to loss of cell viability and parasite death.
Rounding and shrinkage of the cell body are described in the literature as characteristic events
of death by apoptosis when despite alterations in the plasmatic membrane, it remains intact
[47].
The alterations seen in the morphology of the parasites such as undulation in the
membrane, changes in the form of promastigotes and shortening of the flagellum were also
described in different studies. Dos Santos-Aliança et al. [48] assessed chemical compounds
derivatives of phthalimido-thiazole against L. infantum promastigotes and observed the
presence of undulations in the membrane. Brita et al. [16] tested thiosemicarbazone derivative
of S-Limonene to find severe cell alterations in L. amazonenses at 13.9 µM concentrations.
This was also found in the present study when L. infantum promastigotes were also subjected
to compound 2i at 8.72 µM concentration. Bilbão-Ramos et al. [49] investigated sulfonamide
derivatives against L. infantum and observed changes in the morphology of the cell body and
shortening of the flagellum.
Compounds 2d and 2i displayed leishmanicidal activity affecting the parasite
mitochondria, an alteration that can be observed through TEM. Trypanosomatides are
characterized for having a single branched mitochondrion along the body in which a region
known as kinetoplast holds 30% of the DNA material of the protozoan known as k-DNA
[50,51]. Therefore, the proper functioning of this organelle is important for the production of
ATP and its alteration can lead to death which makes the mitochondria an important target for
synthetic drugs [52,53]. Savoia et al. [54] report that the activity of a synthetic peptide caused
severe mitochondrial swelling in strains of L. infantum and L. amazonensis as demonstrated
13

by the molecules in our study. Ferreira et al. [53] reported that piperine derivatives and
phenyl-amide induced death of L. amazonensis promastigotes thus affecting directly the
mitochondria physiology of the parasites. Based on both the excessive mitochondrial swelling
that the indole-thiosemicarbazone induced and the literature, it is possible to claim that the
parasite mitochondria is potential target for synthetic compounds.
Compounds 2d and 2i induced intense vacuolization and the presence of electrodense
material. Such alterations were also present in the study by Ramírez-Macías et al. [55] who
tested triazolopyrimidine against L. braziliensis promastigotes. De Mello et al. [56] used the
same leishmania species to analyze chalcones and noted intense vacuolization as well as
complete disorganization of the cytoplasm as occurred to compound 2d at 11.2 µM
concentration. Phenethylamine derivatives tested against L. infantum triggered the presence of
electrodense particles and vacuolization which suggests that the presence of such alterations is
related to cell death [57]. This finding corroborates investigations by Kaczanowski et al. [58]
who studied eukaryotic cells and described alterations in the morphology of cells with
vacuolization of cytoplasm as characteristic of cell death.
Therefore, it is possible to claim that compounds 2d and 2i are related to
leishmanicidal activity and their mechanism of action implicates alterations in cell
morphology and organization that leads the parasite to death. The death of parasites in this
study presented characteristics of apoptosis such as mitochondrial swelling, condensation of
chromatin and shrinkage of the cell body [59]. This is a significant type of cell death in
parasites because it promots the removal of the parasite without inflammatory processes in the
host organism [60, 61].
Finally, results allow the conclusion that indole-thiosemicarbazone compounds are
likely prototypes for the development of leishmanicidal compounds. Further investigation is
necessary to support the applicability of such compounds in the production of new drugs.
14

4. Experiment Section
4.1 Chemicals
The reagents used in this study are easily found and commercially available (Sigma-
Aldrich, Acros Organics, VETEC). The fusion points were determined by Quimis 340
(Quimis, Brazil). The development of the reactions was analyzed through thin layer
chromatography (TLC) (Merck, silica gel 60 F254 in aluminum foil) using ultraviolet light
reading (254 or 366 nm). RMN spectrum were registered with Varian Unityplus (400 MHz
1
for H and 100 MHz for 13C), the DMSO-d6 used as solvent was acquired from Sigma-
Aldrich. IV spectrum were registered with spectrophotometer model Bruker IFS66 FT-IR
(Bruker, Germany) using KBr disks. Chemical deviation is given in ppm and the multiplicities
are given as s (singlet), d (duplet), t (triplet) and coupling (C) in Hertz. Mass spectrometry
experiments were conducted in MALDI-TOF Autoflex III (Bruker Daltonics, Billerica, MA,
USA).
4.1.1 General Synthesis to obtain thiosemicarbazide
Equimolar amounts of hydrazine hydrate 0.8361 mL (0.00061 mol) were added slowly
in solution with equimolar amounts of substituted isothiocyanate 0.377 mL (0.00061 mol) and
20 of mL of dichloromethane. The reaction was kept in reflux for 3 hours at room
temperature. The product was filtered, rinsed with dichloromethane and dried in vacuum
desiccator [26].
4.1.2 General synthesis to obtain thiosemicarbazone
Equimolar amounts of substituted aromatic aldehydes 0,05 g (0.00021 mol), with
15mL of ethylic alcohol and hydrochloric acid as catalyst. After 20 minutes, the
thiosemicarbazides were added in equimolar amounts. The reaction was kept in reflux for 24
15

hours, the product was filtered and purified through washings with ethylic alchol and dried in
vacuum desiccator [26].
4.1.2.1 2-((5-bromo-7-methyl-1H-indol-3-yl)methylene)hydrazinecarbothioamide (2a)
1
Purple powder; MP: 239 °C; Yield: 37.54%; Rf: 0.46 (n-hexane/ethyl acetate 1:1). NMR H
(DMSO, d6, 400 MHz, ppm): δ 2.47 (s, 3H, CH3), δ 7.16 (s, 1H, indole), δ 7.49 (s, 1H, NH2),
δ 7.885 (s, 1H, indole), δ 8.01 (s, 1H, NH2), δ 8.12 (s, 1H, indole), δ 8.26 (s, 1H, indole), δ
13
11.18 (s, 1H, NH, indole), δ 11.82 (s, 1H, NH). NMR C (DMSO, d6, 100 MHz, ppm): δ
16.35, 111.09, 113.39, 121.27, 123.58, 125.13, 125.53, 131.78, 135.34, 140.39, 176.63. MS
m/z (ES+): 309.8850 [M]+. IR (KBr, cm−1): C=S (1367), C=N (1541), N-H (3428), N-H
(3275).
4.1.2.2 2-((7-bromo-5-methyl-1H-indol-3-yl)methylene)hydrazinecarbothioamide (2b)
1
Orange powder; MP: 234 °C; Yield: 39.08%; Rf: 0.47 (n-hexane/ethyl acetate 6:4). NMR H
(DMSO, d6, 400 MHz, ppm): δ 2.43 (s, 3H, CH3), δ 7.26 (s, 1H, indole), δ (s, 1H, NH2), δ
7.825 (s, 1H, indole), δ 7.98 (s, 1H, indole), δ 8.03 (s, 1H, NH2), δ 8.26 (s, 1H, CH), δ 11.19
13
(s, 1H, NH, indole), δ 11.71 (s, 1H, NH). NMR C (DMSO, d6,100 MHz, ppm): δ 20.71,
103.87, 111.79, 121.05, 125.75, 126.47, 131.79, 133.71, 140.39, 176.61. MS m/z (ES+):
309.8950 [M]+. IR (KBr, cm−1): C=S (1380), C=N (1546), N-H (3496), N-H (3366).
4.1.2.3 2-((7-bromo-5-methyl-1H-indol-3-yl)methylene)-N-phenylhydrazinecarbothioamide
(2c)
Dark yellow powder; MP: 174° C; Yield: 94.64%; Rf: 0.50 (n-hexane/ethyl acetate 4:6).
1
NMR H (DMSO, d6, 400MHz) δ: 2.41 (s, 3H, CH3), 7.18 (t, 1H, J= 7.2Hz, phenyl), 7.27 (s,
1H, indole), 7.37 (t, 2H, J=8Hz, phenyl), 7.69 (d, 2H, J=7.2Hz, phenyl), 7.96 (d, 1H, J=2.8Hz,
16

indole), 7.97 (s, 1H, indole), 8.38 (s, 1H, =CH-), 9.69 (s, 1H, NH), 11.64 (s, 1H, NH), 11.79
(s, 1H, NH). NMR 13C (DMSO, d6, 100MHz) δ: 20.85, 104.02, 111.60, 120.72, 124.55,
124.78, 125.98, 126.42, 128.13, 128.13, 131.40, 131.78, 133.69, 139.25, 140.43, 174.46. IR
(KBr, cm−1): C=S (1197.67), C=N (1533.79), N-H (3326.4). MS m/z (ES+): 387,0210 [M]+.
4.1.2.4 2-((5-bromo-7-methyl-1H-indol-3-yl)methylene)-N-phenylhydrazinecarbothioamide
(2d)
1
Silver powder; MP: 199° C; Yield: 35.72%; Rf: 0.51 (ethyl acetate/n-hexane 7:3). NMR H
(DMSO, d6, 400MHz) δ: 2.48 (s, 3H, CH3), 7.17 (s, 1H, phenyl), 7.19 (s, 1H, indole), 7.37 (t,
2H, J=8Hz, phenyl), 7.71 (d, 2H, J=8Hz, phenyl), 7.98 (s, 1H, indole), 8.17 (s, 1H, indole),
13
8.38 (s, 1H, =CH-), 9.73 (s, 1H, NH), 11.62 (s, 1H, NH), 11.80 (s, 1H, NH). NMR C
(DMSO, d6, 100MHz) δ: 16.35, 110.99, 113.38, 121.25, 123.76, 124.22, 124.72, 125.34,
125.48, 128.18, 131.88, 135.33, 139.28, 140.50, 174.37. IR (KBr, cm−1): C=S (1267.33), C=N
(1557.99), N-H (3410.7). MS m/z (ES+): 387.0142 [M]+.
4.1.2.5 2-((7-bromo-5-methyl-1H-indol-3-yl)methylene)-N-(4-methoxyphenyl)hydrazinecarbo-
thioamide (2e)
1
White powder; MP: 206° C; Yield: 53.97%; Rf: 0.53 (n-hexane/ethyl acetate 5:5). NMR H
(DMSO, d6, 400MHz) δ: 2.41 (s, 3H, CH3), 3.76 (s, 3H, OCH3), 6.93 (d, 2H, J= 8.8 Hz,
phenyl), 7.26 (s, 1H, indole), 7.51 (d, 2H, J= 8.8 Hz, phenyl), 7.94 (d, 1H, J= 2.8 Hz, indole),
7.97 (s, 1H, indole), 8.37 (s, 1H, =CH-), 9.56 (s, 1H, NH), 11.53 (s, 1H, NH), 11.77 (s, 1H,
NH). NMR 13C (DMSO, d6, 100MHz) δ: 20.87, 55.23, 104.01, 111.68, 113.32, 120.74,
126.00, 126.41, 126.77, 126.77, 131.38, 131.62, 132.22, 133.69, 140.23, 156.70, 174.99. IR
(KBr, cm−1): C=S (1241.91), C=N (1556.29), N-H (3149.1), N-H (3300.6), N-H (3439.2). MS
m/z (ES+): 418.9989 [M]+.
17

4.1.2.6 2-((5-bromo-7-methyl-1H-indole-3-yl)methylene)-N-(4-methoxyphenyl)hydrazinecar-
bothioamide (2f)
1
White powder; MP: 192° C; Yield: 71.31%; Rf: 0.48 (ethyl acetate/n-hexane 7:3). NMR H
(DMSO, d6, 400MHz) δ: 2.47 (s, 3H, CH3), 3.76 (s, 3H, OCH3), 6.94 (d, 2H, J= 8.4 Hz,
phenyl), 7.16 (s, 1H, indole), 7.52 (d, 2H, J= 8.8 Hz, phenyl), 7.96 (s, 1H, indole), 8.16 (s, 1H,
indole), 8.37 (s, 1H, =CH-), 9.60 (s, 1H, NH), 11.51 (s, 1H, NH), 11.86 (s, 1H, NH). NMR
13C (DMSO, d6, 100MHz) δ: 16.40, 55.26, 111.08, 113.38, 121.28, 123.74, 125.37, 125.51,
126.55, 131.76, 132.26, 135.35, 140.37, 156.68, 174.91. IR (KBr, cm−1): C=-S (1239.92),
C=N (1555), N-H (3311.5). MS m/z (ES+): 419.0001 [M]+.
4.1.2.7 2-((5-bromo-7-methyl-1H-indole-3-yl)methylene)-N-(p-tolyl)hydrazinecarbothioamide
(2g)
Light green powder; MP: 195° C; Yield: 40.34%; Rf: 0.45 (n-hexane/ethyl acetate 6:4). NMR
1H (DMSO, d6, 400MHz) δ: 2.30 (s, 3H, CH3), 2.48 (s, 3H, CH3), 7.17 (d, 2H, J= 8.4 Hz,
phenyl), 7.23 (s, 1H, indole), 7.57 (d, 2H, J= 8.4 Hz, phenyl), 8.05 (s, 1H, indole), 8.06 (s, 1H,
indole), 8.34 (s, 1H, =CH-), 9.93 (s, 1H, NH), 9.93 (s, 1H, NH), 12.35 (s, 1H, NH). NMR 13C
(DMSO, d6, 100MHz) δ: 16.33, 20.51, 113.35, 117.79, 120.40, 121.23, 124.31, 124.47,
125.35, 126.30, 128.64, 135.33, 136.71, 138.77, 140.35, 174.45, 185.11. IR (KBr, cm−1): C=S
(1450.51), C=N (1628.36), N-H (3107.3), N-H (3171.7). MS m/z (ES+): 401.0279 [M]+.
4.1.2.8 2-((7-bromo-5-methyl-1H-indole-3-yl)methylene)-N-(p-tolyl)hydrazinecarbothioamide
(2h)
Light yellow powder; MP: 195° C; Yield: 32.09%; Rf: 0.60 (n-hexane/ethyl acetate 5:5).
NMR 1H (DMSO, d6, 400MHz) δ: 2.40 (s, 3H, CH3), 3.36 (s, 3H, CH3), 7.16 (d, 2H, J= 8 Hz,
18

phenyl), 7.32 (s, 1H, indole), 7.54 (d, 2H, J= 8 Hz, phenyl), 7.90 (s, 1H, indole), 8.28 (s, 1H,
indole), 8.29 (s, 1H, =CH-), 9.93 (s, 1H, NH), 11.59 (s, 1H, NH), 12.26 (s, 1H, NH). NMR
13C (DMSO, d6, 100MHz) δ: 20.85, 40.12, 104.32, 111.65, 118.51, 120.05, 124.62, 125.89,
125.99, 126.40, 127.24, 128.60, 131.38, 133.20, 133.79, 136.69, 139.11, 185.28. IR (KBr,
cm−1): C=S (1199.82), C=N (1618.41), N-H (3105.8). MS m/z (ES+): 401.0220 [M]+.
4.1.2.9 2-((5-bromo-7-methyl-1H-indole-3-yl)methylene)-N-(4-ethylphenyl)hydrazinecarbo-
thioamide (2i)
Light yellow powder; MP: 195° C; Yield: 87.79%; Rf: 0.60 (n-hexane/ethyl acetate 7:3).
1
NMR H (DMSO, d6, 400MHz) δ: 1.20 (t, 3H, J= 7.2 Hz, CH3), 2.50 (s, 3H, CH3), 2.61 (q,
2H, J= 7.2 Hz, CH2), 7.17 (s, 1H, indole), 7.21 (d, 2H, J= 8 Hz, phenyl), 7.59 (d, 2H, J= 8 Hz,
phenyl), 7.98 (s, 1H, indole), 8.17 (s, 1H, indole), 8.38 (s, 1H, =CH-), 9.65 (s, 1H, NH), 11.60
13
(s, 1H, NH), 11.86 (s, 1H, NH). NMR C (DMSO, d6, 100MHz) δ: 15.69, 16.37, 27.68,
111.02, 113.37, 121.25, 123.76, 124.37, 125.34, 125.47, 125.47, 127.46, 127.46, 131.83,
135.34, 136.90, 140.33, 140.38, 174.42. IR (KBr, cm−1): C=S (1266.85), C=N (1551.13), N-H
(3258.7), N-H (3623). MS m/z (ES+): 415,0590 [M]+.
4.1.2.10 2-((7-bromo-5-methyl-1H-indole-3-yl)methylene)-N-(4-ethylphenyl)hydrazinecarbo-
thioamide (2j)
1
Yellow powder; MP: 210° C; Yield: 59.17%; Rf: 0.52 (n-hexane/ethyl acetate 5:5). NMR H
(DMSO, d6, 400MHz) δ: 1.19 (t, 3H, J= 7.6 Hz, CH3), 2.41 (s, 3H, CH3), 2.61 (q, 2H, J= 7.6
Hz, CH2), 7.20 (d, 2H, J= 8.4 Hz, phenyl), 7.27 (s, 1H, indole), 7.57 (d, 2H, J= 8 Hz, phenyl),
7.95 (s, 1H, indole), 7.97 (s, 1H, indole), 8.38 (s, 1H, =CH-), 9.62 (s, 1H, NH), 11.60 (s, 1H,
13
NH), 11.78 (s, 1H, NH). NMR C (DMSO, d6, 100MHz) δ: 15.69, 20.85, 27.68, 104.03,
111.64, 120.72, 124.67, 125.97, 126.40, 127.41, 131.38, 131.73, 133.70, 136.88, 140.29,
19

140.37, 174.53. IR (KBr, cm−1): C=S (1264.92), C=N (1549.13), N-H (3170), N-H (3294.2).
MS m/z (ES+): 415.0548 [M]+.
4.2 Biologic Assay
4.2.1 Macrophage Culture
Macrophage (J774) were cultivated in RPMI (Sigma Aldrich, St. Louis, USA)
supplemented with 10% fetal bovine serum (FBS) and kept in incubator stove at 5% CO2, at
37 ºC.
4.2.2 Parasite Culture
Promastigote forms of Leishmania infantum (strain MHOM/BR/70/BH46) and
Leishmania amazonensis (strain WHOM/00LTB0016) and were kept in culture medium
supplemented with 20% FBS and 1% penicillin-streptomycin (Sigma Aldrich, St. Louis,
USA) cultivated at 26ºC. Promastigote forms were used in the exponential growth phase in all
steps of the experiment [48]
4.2.3 Cytotoxicity Analysis of compounds
Cytotoxicity in mammal cells was evaluated through tests with 3-(4,5-
dimethylthiazole-2-yl)-2,5-diphenyltetrazolium (MTT) bromide. Macrophage were cultivated
in 96-well cell culture plate at 6 x 105 cell/plate concentrations and incubated in atmosphere of
5% CO2 at 37 °C. After 24h, the supernatant was removed and the cells were incubated in the
presence of several concentrations of the compounds (200 to 6µg/mL) for 72h. Absorbance
reading of the solubilized formazan crystals was conducted with ELISA Benchmark Plus
(Bio-Rad, California, USA) with wavelength of 490nm. The concentration was capable of
causing 50% of loss for cellular viability determined by linear regression analysis. Each
20

experiment was conducted in biological duplicate. Macrophage incubated in culture medium
and in standard drugs (Miltefosine and Amphotericin B) were used as negative and positive
control, respectively [62].
4.2.4 Biological activity in Leishmania infantum and Leishmania amazonensis
Promastigote forms L. infantum and L. amazonensis were collected, counted and
diluted in Schneider medium supplemented with 20% of FBS at 2 x 106 cell/mL
concentration. Next, the cells were incubated in different concentrations of the compounds
(25 to 1.56 µg/mL) for L. infantum promastigote forms and 200 a 6µg/mL for L. amazonensis
forms of both for 72h. Parasites incubated in medium culture and standard drug (Miltefosine
and Amphotericin B) were used as negative and positive control, respectively. Culture growth
was monitored through counting using Neubauer chamber (iNCYTO C-Chip DHC-N01,
Cheonan-Si, South Korea). The concentration that inhibited 50% of parasite growth (IC50)
was determined after 72h through linear regression. Biological duplicate was conducted for
each experiment. Selectivity Index (SI) was determined as the ratio between CC50 and IC50
values and obtained for each compound [48].
4.2.5 Ultrastructural Analysis
The compounds that displayed the best SI results associated to the best IC50 value were
selected for ultrastructural analysis. Therefore, given the established pattern, compounds 2d
and 2i tested against L. infantum were selected. Promastigote forms of untreated L. infantum
(negative control) treated with amphotericin B at 0.05 µM (positive control) and treated with
concentrations 5.6 and 11.2 µM to compound 2d and 4.36 and 8.72 µM to compound 2i, were
washed and fixed in solution 2.5% glutaraldehyde and 4% of formaldehyde in sodium
21

cacodylate buffer 0.1 M at pH 7.2. These were processed for transmission and scanning
electronic microscopy.
For SEM, promastigotes were placed to adhere in dishes previously covered with
poly-L-lysine (Sigma Aldrich, St. Louis, USA). After 20 minutes, the dishes were washed
with cacodylate buffer 0.1M to remove non-adherent cells and post-fixed for 1 hour in
solution 1% osmium tetroxide (OsO4) in cacodylate buffer. Next, cells were dehydrated in
crescent series of ethanol and critical-point-dried with HCP-2 (Hitachi, Tokyo, Japan)
assembled in metallic support, sputter-coated with 20nm of gold in JFC-1100 and examined
in scanning electron microscope JEOL T-200.
In TEM, parasites were washed and post-fixed for 1h 1% osmium tetroxide (OsO4) in
cacodylate buffer. Samples were then dehydrated in crescent series of acetone, infiltrated and
incorporated in EPON (Sigma Aldrich, St. Louis, USA). The material was sectioned in layers
of 70nm with ultramicrotome Leica EMUC6 (Leica, Wetzlar, Germany), and contrasted
against and analyzed in transmission electron microscope TecNai G2 Spirit TEM (FEI,
Hillsboro, USA) uranyl acetate and lead.
4.2.6 Statistical Analysis
Linear regression was conducted using software SPSS 8.0 (IBM Co., New York,
USA) for Windows.
Acknowledgements
This study was supported by Brazilian agencies Fundação de Amparo Pesquisa do
Estado de Pernambuco (FACEPE, Brazil) and Conselho Nacional de Desenvolvimento
Científico e Tecnológico (CNPq).
22

Conflict of Interest
The authors declare that there were no competing interests.
References
[1] K. Jain, N.K. Jain, Vaccines for visceral leishmaniasis: A review. J Immunol Methods.
422 (2015) 1-12. https://doi.org/10.1016/j.jim.2015.03.017
[2] World Health Organization (WHO), Leishmaniasis. http://www.who.int/leishmaniasis/en/
(accessed 27 April 2018).
[3] S. Pomel, S. Cojean, P. Loiseau, Targeting sterol metabolism for the development of
antileishmanials. Trends Parasitol. 31 (2015) 5-7. https://doi.org/10.1016/j.pt.2014.11.007
[4] J. Van Griensven, E. Diro, Visceral leishmaniasis. Infect. Dis. Clin. North Am. 26 (2012)
309-322. https://doi.org/10.1016/j.idc.2012.03.005.
[5] F.T. Silveira, R. Lainson, C.E.P. Corbett, Clinical and Immunopathological Spectrum of
American Cutaneous Leishmaniasis with special reference to the disease in Amazonian Brazil
- A Review. Mem. Inst. Oswaldo Cruz. 99 (2010) 239-251. http://dx.doi.org/10.1590/S0074-
02762004000300001.
[6] L. Stockdale, R. Newton, A review of preventative methods against human leishmaniasis
infection. PLoS Negl Trop Dis. 7 (2013) e2278. https://doi.org/10.1371/journal.pntd.0002278
[7] L.M. Antinarelli, N.C. Pinto, E. Scio, E.S. Coimbra, Antileishmanial activity of some
Brazilian plants, with particular reference to Casearia sylvestris. An. Acad. Bras. Ciênc. 87
(2015) 733-742. http://dx.doi.org/10.1590/0001-3765201520140288.
[8] F. Chappuis, S. Sundar, A. Hailu, H. Ghalib, S. Rijal, R.W. Peeling, J. Alvar, M. Boelaert,
Visceral leishmaniasis: what are the needs for diagnosis, treatment and control? Nat. Rev.
Microbiol., 5 (2007) 873-882. https://doi.org/10.1038/nrmicro1748
23

[9] S.A. Ejazi, N. Ali, Developments in diagnosis and treatment of visceral leishmaniasis
during the last decade and future prospects. Expert Rev Anti Infect Ther. 11 (2013) 79-98.
https://doi.org/10.1586/eri.12.148
[10] S.L. Croft, P. Olliaro, Leishmaniasis chemotherapy challenges and opportunities. Clin
Microbiol Infec. 17 (2011) 1478-1483. https://doi.org/10.1111/j.1469-0691.2011.03630.x
[11] A.G. Tempone, C.M. De Oliveira, R.S. Berlinck, Current Aproaches to Discover Marine
Antileishmanial Natural Products. Planta Med. 77 (2011) 572-585. https://doi.org/10.1055/s-
0030-1250663
[12] B. Singh, S. Sundar, Leishmaniasis: vaccine candidates and perspectives. Vaccine. 30
(2012) 3834-3842. https://doi.org/10.1016/j.vaccine.2012.03.068
[13] S. Sundar, P.K. Sinha, M. Rai, D.K. Verma, K. Nawin, S. Alam, J. Chakravarty, M.
Vaillant, N. Verma, K. Pandey, P. Kumari, C.S. Lal, R. Arora, B. Sharma, S. Ellis, N. Strub-
Wourgaft, M. Balasegaram, P. Olliaro, P. Das, F. Modabber, Comparison of short-course
multidrug treatment with standard therapy for visceral leishmaniasis in India: an open-label,
non-inferiority, randomised controlled trial. The Lancet. 377 (2011) 477-486.
https://doi.org/10.1016/S0140-6736(10)62050-8
[14] K. Rai, B. Cuypers, N.R. Bhattarai, S. Uranw, M. Berg, B. Ostyn, J.C. Dujardin, S. Rijal,
M. Vanaerschot, Relapse after treatment with miltefosine for visceral leishmaniasis is
associated with increased infectivity of the infecting Leishmania donovani strain. MBio. 4
(2013) 00611-13. https://dx.doi.org/10.1128%2FmBio.00611-13
[15] A. Cavalli, M.L. Bolognesi, Neglected tropical diseases: multi-target-directed ligands in
the search for novel lead candidates against Trypanosoma and Leishmania. J Med Chem. 52
(2009) 7339-7359. https://doi.org/10.1021/jm9004835
[16] E.A. Britta; A.P. Silva, T. Ueda-Nakamura, B.P. Dias-Filho, C.C. Silva, R.L. Sernaglia,
C.V. Nakamura, Benzaldehyde thiosemicarbazone derived from limonene complexed with
24

copper induced mitochondrial dysfunction in Leishmania amazonensis. PLoS One. 7 (2012)
e41440. https://doi.org/10.1371/journal.pone.0041440.
[17] H. Pervez, N. Manzoor, M. Yaqub, K.M. Khan, 5-Nitroisatin-derived
thiosemicarbazones: potential antileishmanial agents. J Enzyme Inhib Med Chem. 29 (2014)
628-632. https://doi.org/10.3109/14756366.2013.836641
[18] J.I. Manzano, F. Cochet, B. Boucherle, V. Gómez-Pérez, A. Boumendjel, F. Gamarro,
M. Peuchmaur, Arylthiosemicarbazones as antileishmanial agents. Eur J Med Chem. 123
(2016) 161-170. https://doi.org/10.1016/j.ejmech.2016.07.014.
[19] P.N. Kalaria, S.C. Karad, D.K. Raval, A review on diverse heterocyclic compounds as
the privileged scaffolds in antimalarial drug Discovery. Eur J Med Chem. 158 (2018) 917-
936. https://doi.org/10.1016/j.ejmech.2018.08.040.
[20] P. Ashok, S. Chander, A. Tejería, L. García-Calvo, R. Balaña-Fouce, S. Murugesan,
Synthesis and anti-leishmanial evaluation of 1-phenyl-2,3,4,9-tetrahydro-1H-β-carboline
derivatives against Leishmania infantum. Eur J Med Chem. 123 (2016) 814-821.
https://doi.org/10.1016/j.ejmech.2016.08.014
[21] S.L. Colletti, R.W. Myers, S.J. Darkin-Rattray, A.M. Gurnett, P.M. Dulski, S. Galuska,
J.J. Allocco, M.B. Ayer, C. Li, J. Lim, T.M. Crumley, C. Cannova, D.M. Schmatz, M.J.
Wyvratt, M.H. Fisher, P.T. Meinke, Broad spectrum antiprotozoal agents that inhibit histone
deacetylase: structure-activity relationships of apicidin. Bioorg Med Chem Lett. 11 (2001)
113-117. https://doi.org/10.1016/S0960-894X(00)00605-3
[22] G.A. Holloway, W.N. Charman, A.H. Fairlamb, R. Brun, M. Kaiser, E. Kostewicz, P.M.
Novello, J.P. Parisot, J. Richardson, I.P Street, K.G. Watson, J.B. Baell, Trypanothione
reductase high-throughput screening campaign identifies novel classes of inhibitors with
antiparasitic activity. Antimicrob Agents Chemother. 53 (2009) 2824-2833.
https://doi.org/10.1128/AAC.01568-08
25

[23] M.B. Félix, E.R. De Souza, M.D.C.A. De Lima, D.K.G. Frade, V.L. Serafim, K.A.D.F.
Rodrigues, P.L.D. N. Néris, F.F. Ribeiro, L. Scotti, M.T. Scotti, T.M. De Aquino, F.J.B.
Mendonça Junior, M.R. De Oliveira, Antileishmanial activity of new thiophene–indole
hybrids: Design, synthesis, biological and cytotoxic evaluation, and chemometric studies.
Bioorg Med Chem. 24 (2016) 3972-3977. https://doi.org/10.1016/j.bmc.2016.04.057
[24] K.A. Rodrigues, C.N. Dias, P.L. Néris, J.C. Rocha, M.T. Scotti, L. Scotti, S.R.
Mascarenhas, R.C. Veras, I.A. De Medeiros, T.S. Keesen, T.B. De Oliveira, M.C. De Lima,
T.L. Balliano, T.M. De Aquino, R.O. De Moura, F.J. Mendonça Junior, M.R. De Oliveira, 2-
amino-thiophene derivatives present antileishmanial activity mediated by apoptosis and
immunomodulation
in
vitro.
Eur
J
Med
Chem.
106
(2015)
1-14.
https://doi.org/10.1016/j.ejmech.2015.10.011
[25] R.P. Tenório, A.J. Góes, J.G. De Lima, A.R. De Faria, A.J. Alves, T.M. De Aquino,
Tiossemicarbazonas: métodos de obtenção, aplicações sintéticas e importância biológica.
Quim Nova. 28 (2005) 1030-1037. http://dx.doi.org/10.1590/S0100-40422005000600018.
[26] J.F. De Oliveira, A.L. Da Silva, D.B. Vendramini-Costa, C.A. Da Cruz Amorim, J.F.
Campos, A.G. Ribeiro, R.O. De Moura, J.L. Neves, A.L. Ruiz, J.C. Ernesto, M.C.A. Lima,
Synthesis of thiophene-thiosemicarbazone derivatives and evaluation of their in vitro and in
vivo
antitumor
activities.
Eur
J
Med
Chem.
104
(2015)
148-156.
https://doi.org/10.1016/j.ejmech.2015.09.036
[27] P. Bhargava, R. Singh, Developments in Diagnosis and Antileishmanial Drugs.
Interdiscip Perspect Infect Dis. 2012 (2012) 1-13. http://dx.doi.org/10.1155/2012/626838
[28] D.R.M. Moreira, A.D. De Oliveira, P.A.T.M. Gomes, C.A. De Simone, F.S. Villela, R.S.
Ferreira, A.C. Da Silva, T.A. Dos Santos, M.C.A.C. Brelaz, V.R. Pereira, A.C. Leite,
Conformational restriction of aryl thiosemicarbazones produces potent and selective anti-
26

Trypanosoma cruzi compounds which induce apoptotic parasite death. Eur J Med Chem. 75
(2014) 467-478. https://doi.org/10.1016/j.ejmech.2014.02.001
[29] N.K. Kaushik, N. Kaushik, P. Attri, N. Kumar, C.H. Kim, A.K. Verma, E.H. Choi,
Biomedical
importance
of
indoles.
Molecules.
18
(2013)
6620-6662.
https://doi.org/10.3390/molecules18066620
[30] N. Chadha, O. Silakari, Indoles as therapeutics of interest in medicinal chemistry: Bird's
eye view. Eur J Med Chem.134 (2017) 159-184. https://doi.org/10.1016/j.ejmech.2017.04.003
[31] J.F., De Oliveira, T.S., Lima, D.B., Vendramini-Costa, S.C.B.P., Lacerda, E.A.,
Lafayette, R.M.F., Silva, S.M.V., Almeida, R.O., Moura, A.L.T.G., Ruiz, J.E., Carvalho,
M.C.A., Lima, Thiosemicarbazones and 4-thiazolidinones indole-based derivatives:
Synthesis, evaluation of antiproliferative activity, cell death mechanisms and topoisomerase
inhibition
https://doi.org/10.1016/j.ejmech.2017.05.023
[32] M.Z. Zhang, Q. Chen, G.F. Yang, A review on recent developments of indole-containing
antiviral agents. Eur Med Chem. 89 (2015) 421-441.
https://doi.org/10.1016/j.ejmech.2014.10.065.
assay.
Eur
J
Med
Chem.
136
(2017)
305-314.
J
[33] R.A. Al-Qawasmeh, M. Huesca, V. Nedunuri, R. Peralta, J. Wright, Y. Lee, A. Young,
Potent antimicrobial activity of 3-(4, 5-diaryl-1H-imidazol-2-yl)-1H-indole derivatives
against methicillin-resistant Staphylococcus aureus. Bioorg Med Chem Lett. 20 (2010) 3518-
3520. https://doi.org/10.1016/j.bmcl.2010.04.137
[34] A.D.T.M. De Oliveira, M.D.S. Miranda, I.T.T. Jacob, C.A.D.C. Amorim, R.O. Moura,
S.A.S.D. Silva, M.B.P. Soares, S.M.V. Almeida, T.R.C.L. Souza, J.F. Oliveira, T.G.D. Silva,
C.M.L. Melo, D.R.M. Moreira, M.D.C.A. Lima, Synthesis, in vitro and in vivo biological
evaluation, COX-1/2 inhibition and molecular docking study of indole-N-acylhydrazone
27

derivatives.
Bioorg
Med
Chem.
26
(2018)
5388-5396.
https://doi.org/10.1016/j.bmc.2018.07.024
[35] S. Pandey, S.S. Chauhan, R. Shivahare, A. Sharma, S. Jaiswal, S. Gupta, J. Lal, P.M.
Chauhan, Identification of a diverse indole-2-carboxamides as a potent antileishmanial
chemotypes.
Eur
J
Med
Chem.
110
(2016)
237-245.
https://doi.org/10.1016/j.ejmech.2016.01.028
[36] M.Z. Hernandes, M.M. Rabello, A.C. Leite, M.V. Cardoso, D.R. Moreira, D.J.
Brondani, C.A. Simone, L.C. Reis, M.A. Souza, V.R. Pereira, R.S. Ferreira, J.H. McKerrow,
Studies toward the structural optimization of novel thiazolylhydrazone-based potent
antitrypanosomal agents. Bioorganic & medicinal chemistry. Bioorg Med Chem 18 (2010a)
7826-7835. https://doi.org/10.1016/j.bmc.2010.09.056
[37] M.Z. Hernandes, S.M. Cavalcanti, D.R. Moreira, W.F. Azevedo Junior, A.C. Leite,
Halogen atoms in the modern medicinal chemistry: hints for the drug design. Curr Drug
Targets. 11 (2010b) 303-314. https://doi.org/10.2174/138945010790711996
[38] A.C. Da Silva, T.A.R. Dos Santos, I.V.B. Silva, M.V.G. Oliveira, D.R.M. Moreira,
A.C.L. Leite, V.R.A. Pereira, Aryl thiosemicarbazones: In vitro and immunomodulatory
activities
against
L.
amazonensis.
Exp
Parasitol.
177
(2017)
57-65.
https://doi.org/10.1016/j.exppara.2017.04.003
[39] W. Yi, C. Dubois, S. Yahiaoui, R. Haudecoeur, C. Belle, H. Song, R. Hardré, M.
Réglier, A. Boumendjel, Refinement of arylthiosemicarbazone pharmacophore in inhibition
of
mushroom
tyrosinase.
Eur
J
Med
Chem.
46
(2011)
4330-4335.
https://doi.org/10.1016/j.ejmech.2011.07.003
[40] M.G. Temraz, P.A. Elzahhar, A. El-Din, A. Bekhit, A.A. Bekhit, H.F. Labib, A.S.F.
Belal, Anti-leishmanial click modifiable thiosemicarbazones: design, synthesis, biological
28

evaluation and in silico studies. Eur J Med Chem. 151 (2018) 585-600.
https://doi.org/10.1016/j.ejmech.2018.04.003
[41] P. Linciano, C.B. Moraes, L.M. Alcantara, C.H. Franco, B. Pascoalino, L.H. Freitas-
Junior, S. Macedo, N. Santarem, A. Cordeiro-da-Silva, S. Gul, G. Witt, M. Kuzikov, B.
Ellinger, S. Ferrari, R. Luciani, A. Quotadamo, L. Costantino, M.P., Costi, Aryl
thiosemicarbazones for the treatment of trypanosomatidic infections. Eur J Med Chem. 146
(2018) 423-434. https://doi.org/10.1016/j.ejmech.2018.01.043
[42] L.P. Carvalho, R.C.C. Pereira, H.C. Castro, M.A.G.B. Gomes, R.R. Oliveira, J.
Edmilson, E.J. Maria, E.J.T. Melo, Thiosemicarbazones and thiazolidinones, SAR-studies and
anti-extra and intracellular parasitic elimination of leishmania amazonensis. Eur J Pharm Med
Res. 4 (2017) 799-812. https://www.ejpmr.com/admin/assets/article_issue/1499161850.pdf
[43] J.L. Melos, E.C. Torres-Santos, V.S. Faiões, C.N. Del Cistia, C.M. Sant'Anna C.E.
Rodrigues-Santos, A. Echevarria, Novel 3,4-methylenedioxyde-6-X-benzaldehyde-
thiosemicarbazones: Synthesis and antileishmanial effects against Leishmania amazonensis.
Eur. J. Med. Chem. 103 (2015) 409-417. https://doi.org/10.1016/j.ejmech.2015.09.009
[44] B. Glinma, S.D. Kpoviessi, F.A. Gbaguidi, C.N. Kapanda, J. Bero, J. Quetin-Leclercq,
M. Moudachirou, J. Poupaert, G.C. Accrombessi, E.W. Gachomo, L. Baba-Moussa, S.O.
Kotchoni, Trypanocidal and cytotoxic evaluation of synthesized thiosemicarbazones as
potential drug leads against sleeping sickness. Mol Biol Rep. 41 (2014) 1617-1622.
https://doi.org/10.1007/s11033-013-3008-2
[45] S.M. Saad, N. Ghouri, S. Perveen, K.M. Khan, M.I. Choudhary, 4-Arylamino-6-
nitroquinazolines: Synthesis and their activities against neglected disease leishmaniasis. Eur J
Med Chem. 108 (2016) 13-20. https://doi.org/10.1016/j.ejmech.2015.11.016
[46] R.F. Menna-Barreto, K. Salomão, A.P. Dantas, R.M. Santa-Rita, M.J. Soares, H.S.
Barbosa, S.L. Castro, Different cell death pathways induced by drugs in Trypanosoma cruzi:
29