2-Amino-thiophene derivatives present antileishmanial activity mediated by apoptosis and immunomodulation in vitro

Article abstract of DOI:10.1016/j.ejmech.2015.10.011

This study evaluated the effects of 2-amino-thiophene derivatives on the promastigote and amastigote forms of Leishmania (Leishmania) amazonensis and their possible mechanisms of action. Initially, we evaluated the antileishmanial activity of ten 2-amino-thiophene derivatives on promastigote and axenic amastigote forms of Leishmania amazonensis and their cytotoxicity against murine macrophages and human red blood cells. Three promising compounds were selected for studies of the cell death process using flow cytometry analysis and a DNA fragmentation assay. The effects of the compounds were assessed on intramacrophagic amastigotes, and the modulation of cytokine and NO production was investigated. All thiophene derivatives showed antileishmanial activity against promastigotes and axenic amastigotes with less toxicity for murine macrophages and human red blood cells. The best values were obtained for compounds containing a lateral indole ring. Docking studies suggested that these compounds played an important role in inhibiting trypanothione reductase (TryR) activity. The selected compounds SB-200, SB-44, and SB-83 induced apoptosis in promastigotes involving phosphatidylserine externalization and DNA fragmentation in a pattern similar to that observed for the positive control. Additionally, SB-200, SB-44, and SB-83 significantly reduced the infection index of macrophages by the parasites; for compounds SB-200 and SB-83 this reduction was associated with increased TNF-α, IL-12, and NO levels. This study demonstrated the effective and selective action of 2-amino-thiophene derivatives against L. amazonensis, resulting in apoptosis-like cell death and immunomodulation in vitro. The results suggest that they are promising compounds for the development of new leishmanicidal drugs.

Full text of DOI:10.1016/j.ejmech.2015.10.011

Accepted Manuscript
2-Amino-thiophene derivatives present antileishmanial activity mediated by apoptosis
and immunomodulation in vitro
Klinger Antonio da Franca Rodrigues, Cínthia Nóbrega de Sousa Dias, Patrícia Lima
do Nascimento Néris, Juliana da Câmara Rocha, Marcus Tullius Scotti, Luciana
Scotti, Sandra Rodrigues Mascarenhas, Robson Cavalcante Veras, Isac Almeida de
Medeiros, Tatjana de Souza Lima Keesen, Tiago Bento de Oliveira, Maria do Carmo
Alves de Lima, Tatiane Luciano Balliano, Thiago Mendonça de Aquino, Ricardo
Olímpio de Moura, Francisco Jaime Bezerra Mendonça Junior, Márcia Rosa de
Oliveira
PII:
S0223-5234(15)30296-8
10.1016/j.ejmech.2015.10.011
EJMECH 8146
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 2 September 2015
Revised Date: 28 September 2015
Accepted Date: 6 October 2015
Please cite this article as: K.A. da Franca Rodrigues, C.N. de Sousa Dias, P.L. do Nascimento Néris,
J. da Câmara Rocha, M.T. Scotti, L. Scotti, S. Rodrigues Mascarenhas, R.C. Veras, I.A. de Medeiros,
T. de Souza Lima Keesen, T.B. de Oliveira, M.d.C.A. de Lima, T.L. Balliano, T.M. de Aquino, R.O. de
Moura, F.J.B. Mendonça Junior, M.R. de Oliveira, 2-Amino-thiophene derivatives present antileishmanial
activity mediated by apoptosis and immunomodulation in vitro, European Journal of Medicinal Chemistry
(2015), doi: 10.1016/j.ejmech.2015.10.011.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
2-Amino-thiophene derivatives present antileishmanial activity mediated by apoptosis
and immunomodulation in vitro
Klinger Antonio da Franca Rodriguesa, Cínthia Nóbrega de Sousa Diasa, Patrícia Lima do
Nascimento Nérisa, Juliana da Câmara Rochab, Marcus Tullius Scottia, Luciana Scottia,
Sandra Rodrigues Mascarenhasa, Robson Cavalcante Verasc, Isac Almeida de Medeirosa,c,
Tatjana de Souza Lima Keesena,d, Tiago Bento de Oliveirae, Maria do Carmo Alves de Limae,
Tatiane Luciano Ballianof, Thiago Mendonça de Aquinof, Ricardo Olímpio de Mourag,
Francisco Jaime Bezerra Mendonça Juniora,g, Márcia Rosa de Oliveirab,h*
aPrograma de Pós-Graduação em Produtos Naturais e Sintéticos Bioativos, Centro de
Ciências da Saúde, Universidade Federal da Paraíba, 58051-970, João Pessoa, PB, Brazil
bPrograma de Pós-Graduação em Biologia Molecular, Centro de Ciências Exatas e da
Natureza, Universidade Federal da Paraíba, 58051-970, João Pessoa, PB, Brazil
cDepartamento de Ciências Farmacêuticas, Centro de Ciências da Saúde, Universidade
Federal da Paraíba, 58051-970 João Pessoa, PB, Brazil
dDepartamento de Biologia Celular e Molecular, Centro de Biotecnologia, Universidade
Federal da Paraíba, 58051-970 João Pessoa, PB, Brazil
eDepartamento de Antibióticos, Laboratório de Planejamento e Síntese de Fármacos,
Universidade Federal de Pernambuco, 50670-901 Recife, PE, Brazil
fInstituto de Química e Biotecnologia, Universidade Federal de Alagoas, 57072-970, Maceió,
AL, Brazil
gLaboratório de Síntese e Vetorização de Moléculas, Departamento de Ciências Biológicas,
Universidade Estadual da Paraíba, 58058-420, João Pessoa, PB, Brazil
hDepartamento de Biologia Molecular, Centro de Ciências Exatas e da Natureza,
Universidade Federal da Paraíba, João Pessoa, Paraíba CEP 58059-900, Brazil
Running title: Antileishmanial activity of new 2-amino-thiophene derivatives
* Corresponding author
Márcia Rosa de Oliveira
Departamento de Biologia Molecular, Centro de Ciências Exatas e da Natureza, Universidade
Federal da Paraíba, Campus I, Castelo Branco, João Pessoa, Paraíba, CEP 58059-900, Brazil
Tel: +55 83 3216 7436
E-mail: mrosajp@gmail.com

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Abstract
This study evaluated the effects of 2-amino-thiophene derivatives on the promastigote and
amastigote forms of Leishmania (Leishmania) amazonensis and their possible mechanisms of
action. Initially, we evaluated the antileishmanial activity of ten 2-amino-thiophene
derivatives on promastigote and axenic amastigote forms of L. amazonensis and their
cytotoxicity against murine macrophages and human red blood cells. Three promising
compounds were selected for studies of the cell death process using flow cytometry analysis
and a DNA fragmentation assay. The effects of the compounds were assessed on
intramacrophagic amastigotes, and the modulation of cytokine and NO production was
investigated. All thiophene derivatives showed antileishmanial activity against promastigotes
and axenic amastigotes with less toxicity for murine macrophages and human red blood cells.
The best values were obtained for compounds containing a lateral indole ring. Docking
studies suggested that these compounds played an important role in inhibiting trypanothione
reductase (TryR) activity. The selected compounds SB-200, SB-44, and SB-83 induced
apoptosis in promastigotes involving phosphatidylserine externalization and DNA
fragmentation in a pattern similar to that observed for the positive control. Additionally, SB-
200, SB-44, and SB-83 significantly reduced the infection index of macrophages by the
parasites; for compounds SB-200 and SB-83 this reduction was associated with increased
TNF-α, IL-12, and NO levels. This study demonstrated the effective and selective action of 2-
amino-thiophene derivatives against L. amazonensis, resulting in apoptosis-like cell death and
immunomodulation in vitro. The results suggest that they are promising compounds for the
development of new leishmanicidal drugs.
Keywords: Leishmaniasis, drug development, mechanisms of action, synthetic chemistry

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1. Introduction
Leishmaniasis is a complex of infectious diseases caused by more than 20
kinetoplastid protozoan parasites that belong to the Trypanosomatidae family and genus
Leishmania. It affects approximately 12 million people in more than 98 countries distributed
in five continents and is included in the neglected tropical diseases (NTD) group [1,2]. The
clinical manifestations can vary from nodular and ulcerative skin lesions to the progressive
mucocutaneous and visceral form, which is the most severe form and can potentially result in
fatal disease [3]. Leishmania (Leishmania) amazonensis is found in the New World primarily
in Latin America and is associated with different clinical forms of cutaneous leishmaniasis. It
is the main agent of diffuse cutaneous leishmaniasis, which is commonly refractory to
available treatments [4].
Currently, leishmaniasis chemotherapy has a reduced arsenal of drugs and is far from
satisfactory. Most of the drugs exhibit severe toxic side effects, and increased
chemoresistance of the parasite greatly reduces its sensitivity to these drugs [3]. The first-line
and most widely used drugs for the treatment of leishmaniasis are pentavalent antimonials
such as meglumine antimoniate (Glucantime®) and sodium stibogluconate (Pentostan®).
Pentavalent antimonials have been used since 1945; they require parenteral administration and
are toxic and poorly tolerated drugs [5]. Second-line drug such amphotericin B, pentamidine,
paromomycin and miltefosine are used in refractory and resistant cases, but these drugs have
high costs and may be even more toxic than the antimonials [6]. Therefore, there is a dire
need globally for the development of drugs that are more effective against the parasite and
less toxic than conventional drugs.
Planning and synthesizing new drugs based on known structures is founded on the
main objective of obtaining derivatives with greater therapeutic interest and higher activity,

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bioavailability and metabolism that are more suited to therapeutic use. These drugs should
exhibit less toxicity and may even acquire more appropriate pharmacotechnical features [7,8].
Among the compounds that have been tested as potential antileishmanial agents,
thiophene derivatives are particularly interesting [9]. Thiophene derivatives have been
identified as secondary metabolites in various organisms including fungi and plants, such as
Tagetes patula and Tagetes erecta (Asteraceae) [10]. In medicinal chemistry, thiophenes are a
versatile class of sulphur-containing heterocyclic compounds that meet the characteristics of a
“privileged structure” in which a simple molecular framework is able to provide ligands for
diverse targets [11]. Several reports confirm that small substitutions in the thiophene structure
cause large differences in the biological and pharmacological profiles [12]. Properties
described in the literature include: anti-inflammatory, antitumor, antinociceptive,
anticonvulsant, and antiarrhythmic [13]. These structures also exhibit promising activity
against microorganisms due to their antibacterial, antifungal, and antiprotozoal properties
[12,14]. Some plant-extracted thiophene derivatives have been reported to be effective against
the promastigote forms of L. donovani, L. infantum, L. braziliensis, and L. amazonensis,
[9,15] thereby highlighting the potential of these molecules as agents for the treatment of
leishmaniasis. However, studies of naturally occurring substances commonly present
limitations in terms of the availability of the substance to be tested.
In this context, the present study evaluated the antileishmanial activity of 10 synthetic
2-amino-cycloalkyl[b]thiophene-3-carbonitrile derivatives against L. amazonensis, one of the
etiological agents of American cutaneous leishmaniasis (ACL). These molecules present
simple chemical structures and are easily synthesized using a low cost method via a two-step
reaction. The reaction starts with the one pot reaction between malononitrile with a cyclic
ketone and elemental sulphur in the presence of morpholine, followed by the Gewald reaction
[16]. Among the molecules tested, three showed better selectivity indices to the parasite and

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were evaluated on amastigotes internalized into macrophages. Finally, possible mechanisms
of action responsible for the antileishmanial activity were evaluated.
2. Results
2.1 Synthetic Chemistry
Scheme 1 shows the reaction sequence used to obtain the thiophene derivatives and
the chemical structures of the studied compounds. For the evaluation of the antileishmanial
activity, all compounds were re-synthesized and their chemical structures were confirmed by
1
comparison with their physico-chemical characteristics and signals in the NMR H spectra.
All compounds were obtained with a good yield (greater than 70%), demonstrating the good
synthetic feasibility and choice of standardization of reaction conditions employed in the
synthetic strategy.
2.2 X-Ray crystal structures of SB-83 and SB-200
The crystal structures of two of the thiophene derivatives (compounds SB-200 and SB-
83) were solved by X-ray crystallography. The crystallographic parameters, crystal data, data
collection details and structure refinement results for SB-200 and SB-83 were presented in
Table 1.

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Table 1 – Crystal data, data collection details and structure refinement results for SB-200 and
SB-83.
Parameter
Molecular Weight (g/mol)
SB-200 - C19H16BrN3S
398.32
SB-83 - C18H14BrN3S
384.29
Space Group
P21/n
Pmnb
a (Å)
b (Å)
c (Å)
11.370(1)
11.195(1)
14.275(1)
106.026(1)
1746.5(3)
4
6.769(1)
10.160(2)
24.968(4)
90.000 (0)
1717.3(1)
4
β (°)
V (Å3)
Z
Dc(g.cm-3)
1.515
2.478
4,143
3,265
1.486
2.518
2,206
1,731
Absorbance (µ.mm-1)
Independent reflections
Reflections I>2σ(I)
R-factor
0.0396
0.0429
The fractional atomic coordinates and isotropic temperature parameters (Å2) for SB-
200 and SB-83 are presented in the supplementary data. Figures 1 and 2 represent the
stereodiagrams of the crystal structures of SB-200 and SB-83. The nonhydrogen atoms are
drawn as ellipsoids that represent the probability (50%) that the atoms are located within the
designated volumes. The interatomic bond distances (Å) and angles (°) for SB-200 and SB-83
are presented in the supplementary data. The view of the packing of SB-200 and SB-83
showing the intermolecular interactions that stabilize the crystal structures are presented in the
supplementary data.
Figure 1. Projection of SB-200, showing atom labeling and 50% probability displacement
ellipsoids.

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Figure 2. Projection of SB-83, showing atom labeling and 50% probability displacement
ellipsoids.
2.3 In vitro antileishmanial efficacy of 2-amino-thiophenes and their cytotoxicity
In a preliminary experiment, ten 2-amino-thiophene derivatives were investigated.
Their antileishmanial efficacy on the promastigote and axenic amastigote forms of L.
amazonensis and their cytotoxicity against murine peritoneal macrophages and human red
blood cells were evaluated to identify the most active and selective molecules against the
parasite. Their IC50, EC50, CC50 and HC50 calculated values are shown in Table 2; meglumine
antimoniate and amphotericin B were included as reference drugs.
All thiophene derivatives showed antileishmanial activity on promastigotes, with IC50
values ranging from 3.37 µM (SB-83) to 189.3 µM (SB-63) and EC50 values ranging from
15.82 µM (SB-44) to 212.73 µM (6CN10) when assayed against axenic amastigotes. Among
the evaluated compounds, SB-200, SB-44, and SB-83 (which contain a lateral indole ring)
showed higher antileishmanial activity. The antileishmanial activity was higher than
meglumine antimoniate and lower than amphotericin B. With regards to their cytotoxic
effects, all of the compounds showed lower toxicity for macrophages than for the different
parasite forms (CC50 values ranging from 32.02 µM for SB-200 to 721.76 µM for SB-63).
The haemolytic assays showed that the 2-amino-thiophene derivatives did not exhibit toxicity
towards human red blood cells (HC50 values ranging from 624.8 µM for SB-81 to > 800 µM
for the compounds 6CN10, 6CN09, SB-80, SB-63, SB-200, and SB-83). In contrast,

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meglumine antimoniate was less toxic to the parasite than the macrophages (CC50 of 1035
mg/L) and human erythrocytes (HC50 of 429.5 mg/L), and amphotericin B presented high
levels of cytotoxicity (CC50 of 0.18 µM and HC50 of 11.6 µM).
The best selectivity index values were again obtained for the compounds containing a
lateral indole ring (SB-200, SB-44, and SB-83). The selective indices of these compounds
compared to the reference drugs were particularly interesting because the selectivity of SB-
200, SB-44, and SB-83 for the parasites was much more pronounced. For example, SB-83
was 840- and >14,836-fold more benign than meglumine antimoniate towards macrophages
and erythrocytes, respectively. These values are highly representative of the in vitro potential
efficacy of thiophenes compared to the reference drugs. In view of the promising results
obtained with the thiophene derivatives containing an indole ring (the SB-200, SB-44, and
SB-83 compounds) that showed the best selectivity indices for the parasite (Table 3), these
molecules were selected for further study.

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Table 2 – Antileishmanial activity, cytotoxic effects against mammalian cells, and selectivity
index values calculated for thiophene derivatives, meglumine antimoniate and amphotericin
B.
Cytotoxicity
Antileishmanial activity
Promastigotes Axenic amastigotes
Compounds
Macrophages
Red
blood cell
CC50
µM
HC50
µM
IC50
µM
SIm
SIrb
EC50
µM
SIm
SIrb
228.86
>800
187.1 1.22
>4.27
212.73 1.07 >3.76
167.2 1.26 >4.78
6CN10
6CN09
211.43
>800
88
2.4
>9.1
141.1
540.44
438.46
721.76
196.01
32.02
49.08
113.4
1035a
0.18
682.1
>800
716.3
>800
624.8
>800
721.3
>800
429.5a
11.6
61.5
2.29
11.09
>5.73
4.35
124.4 1.13
210.7 2.56 >3.79
132.37 3.31 5.41
192.15 3.75 >4.16
102.8 1.9
20.09 1.59 >39.8
5.4
6CN01
SB-80
SB-68
SB-63
SB-81
SB-200
SB-44
SB-83
139.44 3.87
164.66 2.66
189.3 3.81
49.12 3.99
>4.22
12.71
6
3.65
7.37
3.37
8.77 >291.17
6.65 97.86
33.6 >237.38
15.82
18.5
3.1
6.12 >43.24
0.42
0.78 14.87
45.5
25740a 0.04
0.17 1.05
0.016
68.23
1017.4a 1.01
0.23
Meglumine
antimoniate
Amphotericin B
SIm (selectivity index) = CC50/IC50
SIrb (selectivity index) = HC50/IC50
a Values in mg/L
2.4 Docking
To determine the molecular mechanism responsible for the antileishmania activity of
the 2-amino-thiophene derivatives, docking studies on trypanothione reductase (TryR) from
L. infantum (PDB id: 2jk6) were performed. Table 3 shows the results of docking the
thiophene derivatives into the catalytic site of TryR by presenting the binding energies (Edock).
The best ligands were compounds SB-200, SB-44 and SB-83, with Edock values of –11.17, –

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10.29 and –9.81 Kcal/mol, respectively. In contrast, the worse ligands were compounds SB-
80 and SB-81, with Edock values of –7.86 and –7.69 Kcal/mol, respectively.
Table 3 – Docking results of 2-amino-thiophene derivatives docked to Trypanothione
reductase from L. infantum (PDB id: 2jk6).
Compound
Afinity energy
(Kcal/mol)
-8,79
6CN01
6CN09
6CN10
SB63
-8,60
-9,36
-8,20
SB200
SB44
SB80
-11,17
-10,29
-7,86
SB81
-7,69
SB83
-9,81
SB68
-9,64
Docking analysis of the ligands into the TryR-binding site revealed that the 2-amino-
thiophene derivatives fit well into the active site pocket made up of key residues (Figure 3).
The main residues were Thr51 and Ser162, which interacted with all of the compounds
through hydrogen bonds and acted as H-bond donors. Residue Thr51 made an H-bond with
the sp nitrogen of the nitrile (–C≡N), and residue Ser162 stabilised the ligands though H-
bonding with the sp2 nitrogen of the imine group (–N=CH–). Other hydrophobic interactions
with residues Gly11, Gly13, Val36, Ala159, Thr160 and Asp327 helped to stabilise the guest-
host complexes and played an important role in the ligand binding affinity.

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Figure 3 – a) Interaction of SB-200 with the TryR-binding site. Blue lines represent hydrogen
bonds. b) Interaction of SB-81 with the TryR-binding site. Blue lines represent hydrogen
bonds. c) TryR binding sites cluster with compounds SB-44 (green), SB-200 (yellow), SB-81
(blue) and SB-80 (purple).

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2.5 Apoptotic-necrotic profiling in Leishmania promastigotes by selected thiophenes
To determine the cell death process in parasites exposed to compounds SB-200, SB-
44, and SB-83, conventional flow cytometry was performed using Annexin V-FITC and PI.
Viable cells remained unstained (Annexin V-FITC−/PI−). Early apoptotic cells showed
Annexin V-FITC+/PI− staining patterns, whereas late apoptotic cells exhibited Annexin V-
FITC+/PI+ staining patterns due to the loss of plasma membrane integrity [17]. Moreover,
DNA fragmentation was analysed using the agarose gel electrophoresis assay. Representative
dot-plots of Annexin V-FITC/PI staining are provided in Figure 4.
No death from initial apoptosis was observed (cells positive for annexin V and
negative for PI) after treatment with the compounds SB-200, SB-44, SB-83 and amphotericin
B (positive control). However, an increase in the number of promastigotes with late apoptosis
(parasites staining positive for annexin V and PI) was observed following treatment with SB-
200 (2x and 4x IC50), SB-44 (4x IC50), SB-83 (2x and 4x IC50), and amphotericin B (1x, 2x,
and 4x IC50). Furthermore, necrotic cell death (cells negative for annexin V and positive for
PI) was detected for compounds SB-200 (1x, 2x, and 4x IC50), SB-44 (1x, 2x, and 4x IC50),
SB-83 (2x, and 4x IC50), and amphotericin B (4x IC50) (Table 4).
In a qualitative analysis using the DNA fragmentation assay by agarose gel
electrophoresis, DNA fragmentation was detected in promastigote cells treated with SB-200,
SB-44, and SB-83 at concentrations of 1x, 2x, and 4x the IC50 (Figure 5). The result was
similar to that observed following treatment with amphotericin B at a concentration of 1x the
IC50 using cellular death by apoptosis as a positive control. These results suggest that the cell
death caused by the compounds SB-200, SB-44 and SB-83 in the L. amazonensis
promastigote forms is associated with endonucleases activity, which is responsible for the
DNA fragmentation that occurs as a result of apoptosis.

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Figure 4 - Representative dot plots showing staining of Leishmania amazonensis
promastigotes. L. amazonensis promastigotes were incubated at 26ºC for 4 h in the absence or
presence of 2-amino-thiophene derivatives or amphotericin B at IC50, 2x IC50 and 4x IC50
concentrations. Annexin/propidium iodide staining was performed and the samples were
analysed by flow cytometry.

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Table 4 - Flow cytometry analysis of Leishmania amazonensis promastigotes without
treatment and treated with SB- 200, SB-44, SB-83 and the reference drug amphotericin B
showing the percentage of annexin-V and propidium iodide (PI)-positive cells. The results
represent the means ± S.E.M. of three experiments performed in triplicate. (a) P < 0.05 vs.
control; (b) P < 0.01 vs. control; (c) P < 0.001 vs. control.
Leishmania intracellular entities (% of cells)
Annexin
8.3
Annexin/PI
12.05
9.06
PI
Control
2.6
SB-200 1x IC50
SB-200 2x IC50
SB-200 4x IC50
SB-44 1x IC50
SB-44 2x IC50
SB-44 4x IC50
SB-83 1x IC50
SB-83 2x IC50
SB-83 4x IC50
Amph. B 1x IC50
Amph. B 2x IC50
Amph. B 4x IC50
4.8
5.6
4.3
4.07
3.5
4.06
7.1
5.2
5.03
6
10.4
10.3
6.1a
27.3b
27.1b
11.6
22.7c
53.2c
12.1a
31.6c
54.9c
3.5
15.6
25.2b
12.5
28.5b
27.2b
23.8a
38.8c
42.2c
21.7c
51.9c
2.8
5.2
8.6a
Figure 5 – The genomic DNA of Leishmania amazonensis promastigotes treated with
different concentrations of SB-83, SB-44, and SB-200 or amphotericin B. The agarose gel
(1%) was stained with gel red (1:500), and the analysis was performed under UV light. (M) –
molecular marker 1 kb DNA (300 – 10,000 bp – Axygen); (1) – control; (2) – amphotericin B
at a concentration of 1x IC50; SB-200 at concentrations of 1x IC50 (3), 2x IC50 (4), and 4x IC50
(5); SB-44 at concentrations of 1x IC50 (6), 2x IC50 (7), and 4x IC50 (8); SB-83 at
concentrations of 1x IC50 (9), 2x IC50 (10), and 4x IC50 (11).

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2.6 In vitro efficacy of thiophenes SB-200, SB-44, and SB-200 against intramacrophagic
amastigotes
To evaluate the action of selected thiophenes against intramacrophagic amastigotes, an
in vitro model of infection of murine peritoneal macrophages was used. The results of the
treatment with thiophenes SB-200, SB-44, and SB-83 on macrophages infected with L.
amazonensis are presented in Figures 6 and 7. The obtained values revealed a significant and
concentration-dependent reduction in the survival rate of amastigotes after 24h of treatment,
resulting in EC50 values of 7.84, 10.5, and 8.17 for compounds SB-200, SB-44, and SB-83,
respectively, while the reference drug meglumine antimoniate showed no effect. After 72 h of
treatment, SB-200, SB-44, and SB-83 induced an even greater concentration-dependent
reduction in the survival index, with EC50 values of 4.81, 9.52, and 6.4 ꢀM, respectively.
These thiophene derivatives had a much more significant effect than meglumine antimoniate
after 72 h of exposure under the same experimental conditions; this reference drug was only
able to induce a significant reduction in the survival index at a concentration of 300 mg/L and
had an EC50 of 169.43 mg/L. Additionally, Figure 7 shows a qualitative representation of the
effect of the thiophenes, which can be observed through the preserved integrity of
macrophages after 72 h of treatment with different concentrations.

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Figure 6 – Effects of SB-44, SB-200, SB-83, and the reference drug meglumine antimoniate
on the survival index of amastigotes internalized in macrophages after 24 and 72 h of
exposure. Murine macrophages were infected with promastigote forms of Leishmania
amazonensis and treated at different concentrations of SB-44 (A), SB-200 (B), SB-83 (C) and
meglumine antimoniate (D). The results represent the means ± S.E.M. of three experiments
performed in triplicate. (*) P < 0.05 vs. control; (**) P < 0.01 vs. control; (***) P < 0.001 vs.
control. C = control.

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Figure 7 – Optical microscopy images of macrophages infected with promastigotes of L.
amazonensis and treated with the 2-amino-thiophenes SB-200, SB-44, and SB-83 and the
reference drug meglumine antimoniate for 72 h. The slides were observed at 1000x
magnification.
2.7 Compounds SB-200, SB-44, and SB-200 induce a host-protective cytokine response
and provide stimulus to increase NO levels
Due to the reduction in the infection rate of macrophages treated with the thiophene
derivatives SB-44, SB-200 and SB-83, we investigated whether the antileishmanial activity
was associated with an in vitro immunomodulatory action. SB-200 and SB-83 increased the
production of TNF-α (Figure 8A), IL-12 (Figure 8B) and NO levels (Figure 8D) in murine
macrophages infected with L. amazonensis without altering the level of IL-10 (Figure 8C)
after 72 h of treatment. SB-44 did not affect the cytokine or NO levels under the evaluated
conditions.

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Figure 8 – Effects of SB-44, SB-200 and SB-83 on cytokine and nitric oxide (NO)
production. The cytokines TNF-α (A), IL-12 (B), IL-10 (C) and NO (D) concentrations were
measured in the supernatants from cultured macrophages infected with Leishmania
amazonensis and treated with SB-44, SB-200, and SB-83 for 72 h at 37 °C and 5% CO2. LPS
- Bacterial lipopolysaccharide (10 mg/L). Data are presented as the mean ± S.E.M. of three
experiments performed in triplicate. (*) P < 0.05 vs. control; (**) P < 0.01 vs. control; (***)
P < 0.001 vs. control.
3. Discussion
Synthetic compounds or those derived from medicinal plants have been extensively
evaluated for antileishmanial activity due to the urgent need for the discovery of new agents
that are more effective and less toxic than the conventional drugs used for the treatment of

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leishmaniasis [6,18]. Thus, we evaluated the antileishmanial efficacy of ten synthetic 2-
amino-thiophene derivatives and their potential mechanisms of action. The thiophenes
evaluated in this study were previously synthesized and shown to be promising antifungal
[12] and antitumor agents [14]. These thiophenes have an additional advantage over natural
compounds because they are synthesized with high purity and may be obtained on a large
scale, including an industrial scale. The crystallographic data of compounds SB-200 and SB-
83 confirms the chemical structures of the compounds in question and unequivocally provides
the stereochemistry of the imine bond (CH = N), in both cases confirming the achievement of
the E isomer. This information allowed us to obtain docking data with higher reliability and
showed that the compounds could be obtained with very high purity (an indispensable
condition to obtain single crystals of drugs).
As shown in Table 1, all ten evaluated thiophene derivatives inhibited the growth of L.
amazonensis promastigotes, with SB-200, SB-44, and SB-83 presenting the highest activity.
Studies with other thiophene derivatives also reported the antileishmanial potential of this
class of chemical compounds against promastigote forms [19]. The necessity to minimize the
use of laboratory animals in pharmacological and toxicological potential assessments of new
products has led to the development of alternative in vitro methods. Thus, models involving
axenic amastigotes are important for the screening of new drugs because the life stages of the
parasite responsible for the different clinical manifestations of leishmaniasis must be
considered [18]. Similar to the promastigote forms, all of the derivatives showed
antileishmanial activity against the axenic amastigote forms. The compounds that showed
enhanced antileishmanial activity against the promastigotes and axenic amastigotes (SB-200,
SB-44, and SB-83) had in common the presence of an indole moiety ring that might be
involved in the increase in antileishmanial activity. These compounds also showed increased
antileishmanial activity compared to meglumine antimoniate, which was used as a reference

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drug. Meglumine antimoniate showed higher antileishmanial activity against axenic
amastigotes (IC50 of 1017.4 µM) than against promastigotes (IC50 of 25740 µM). These
differences were also observed in previous studies and might be related to differences in the
metabolic machinery and biochemistry of both stages [20,21].
To investigate the safety of 2-amino-thiophene derivatives on mammalian cells, we
evaluated possible cytotoxic effects on murine peritoneal macrophages and human red blood
cells. Experimental cytotoxic models involving macrophages are ideal for studies on
antileishmanial activity because they are the major host cell type parasitized by Leishmania
spp [22,23]. All compounds tested in this study were safer for macrophages than the reference
drug, as evidenced by the CC50 values and selectivity indices. The presence of the indole ring
appeared to greatly increase the selectivity indices of SB-200, SB-44, and SB-83, thereby
contributing to the safety of these molecules. Other thiophenes that were evaluated for their
murine macrophage cytotoxicity also presented higher selectivity for the parasite [9]. The
selective action against L. amazonensis described for the thiophenes analysed in this work
was remarkably higher than meglumine antimoniate, which was the first choice drug for
leishmaniasis treatment; indeed, meglumine antimoniate presented higher cytotoxicity for the
macrophages than for the parasite. The reference drug amphotericin B also exhibited high
toxicity to macrophages despite having an antileishmanial activity superior to the evaluated
compounds. This result can be compared to the in vivo effects of amphotericin B, which has
high toxicity and induces many side effects (i.e., nephrotoxicity) despite being a drug of
second choice. Therefore, amphotericin B is not an ideal drug [2].
Other cytotoxicity models have been investigated to determine the safety of new
compounds with antileishmanial activity [21]. Cytotoxicity evaluations using red blood cells
represent an in vitro model that is correlated with cell damage in vivo based on the injury or
formation of pores in the plasma membrane [18]. Thus, the cytotoxicity of 2-amino-thiophene

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derivatives against human red blood cells was also evaluated. A safety level was
demonstrated in these cells because all thiophenes showed a lower percentage of haemolysis
(624.8 – >800 µM) compared to meglumine antimoniate (429.5 mg/L) and amphotericin B
(11.6 µM), which exhibited high toxicity. Other thiophene derivatives were found to be
weakly toxic to erythrocytes, [24,25] demonstrating the safety of this compound class. Similar
to the macrophage cytotoxicity results, the presence of the indolic ring increased the
selectivity indices of SB-44, SB-200, and SB-83 against the promastigote and axenic
amastigote forms with superior values compared to the reference drugs. The comparison with
other studies confirmed the selectivity of thiophenes for parasites of the genus Leishmania
compared to host cells [9,19]. Moreover, the safety of these molecules may be a strong
indication of low toxicity in vivo because many studies have reported a correlation between in
vitro cytotoxicity assays and acute toxicity in animals and humans [26].
Trypanothione reductase (TyrR) is an NADPH-dependent flavoprotein that is present
only in protozoan parasites from the genera Trypanosoma and Leishmania. The absence of
TyrR in humans makes it an attractive target for rational drug design targeting leishmaniasis
and trypanossomiasis [27]. The trypanothione system is essential for parasite survival because
the dithiol trypanothione system is required for the synthesis of DNA precursors, the
detoxification of hydroperoxides, and the sequestration/export of thiol conjugates [27–29].
Computational tools are typically used to aid in the development and discovery of new drugs
based on the chemical structures and properties of biological targets (enzymes) with the aim
of decreasing illness [30].
The enzyme TryR from L. infantum (PDB id: 2jk6) is broadly used as a leishmaniasis
target in docking studies. In our study, we found that several of our thiophene derivatives had
lower Edock values compared to other compounds described in the literature as promising
inhibitors of TryR with high affinity. The Edock values found for SB-200, SB-44 and SB-83 (–

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11.17, –10.29 and –9.81 Kcal/mol, respectively) were smaller than: Taxifolin (–8.82
Kcal/Mol) [28]; a series of various tricyclic and quinone derivatives (between –6.70 and –9.50
Kcal/Mol) [31]; and chromene-2-thione and benzo[f]chromene-2-thione analogues (between –
6.82 and –9.20 Kcal/Mol) [29]. These results indicate that these thiophene derivatives may be
potent inhibitors of TryR. The study of Gundampati and Jagannadham [28], confirmed our
docking results and also identified several amino acid residues including the Ser-162 residue
as important for ligand binding to the TryR binding pocket. The association between the
experimental data (in vitro antileishmanial activity of thiophenes; Table 2) with the docking
study results (Table 3) shows a high correlation between the results, providing additional
evidence that the possible mechanism of action of these molecules may occur through the
inhibition of TryR. However, additional biological studies using the enzyme need to be
performed to validate this hypothesis. In both studies (experimental and in silico), the
compounds that showed more pronounced activity (lower IC50 and Edock values) were the
same: compounds SB-200, SB-44 and SB-83. Likewise, compounds 6CN09, SB63 and SB80
were the seventh, eighth and ninth best ligands in the docking study and in the inhibition of
the axenic amastigote forms, respectively.
In the present study, compounds SB-200, SB-44, and SB-83 displayed a better
antileishmanial capability and an increased safety level for mammalian cells. Based on these
characteristics, we provide the first insights into the mechanism of cell death induced by 2-
amino-thiophene derivatives in L. amazonensis using the compounds SB-200, SB-44, and SB-
83. For this purpose, we initially investigated the apoptotic-necrotic profile using Annexin V-
FITC/PI staining. Annexin V is a Ca2+-dependent phospholipid-binding protein that is
typically used to investigate the externalization of phosphatidyl serine due to a high affinity
for this phospholipid. Double-staining with PI, which is a DNA intercalator that only travels
through damaged membranes, allows apoptotic cells to be distinguished from necrotic cells

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[17]. Our results demonstrated that our thiophene derivatives acted through a direct
mechanism on promastigote forms that was associated with apoptosis and accompanied by
secondary necrotic cell death. The reference drug amphotericin B, which was known to cause
cell death by apoptosis in Leishmania spp. [32] and was used in this study as a positive
control, showed the same profile. In a qualitative analysis, the DNA fragmentation assay
using agarose gel electrophoresis showed that the effect of the compounds SB-200, SB-44,
and SB-83 on promastigotes led to fragmentation of the oligonucleosomal DNA, which was
similar to the results observed with amphotericin B and confirmed the involvement of
apoptosis in the mechanism of action of these substances. Different studies have provided a
large number of promising substances that promote antileishmanial activity by inducing
apoptosis of the promastigote and amastigote forms of Leishmania spp. These mechanisms
are mainly related to the externalization of phosphatidyl serine, DNA fragmentation, and
depolarization of the mitochondrial membrane potential [17].
In addition to direct effects on the parasite, in this study we also investigated the
effects of the compounds SB-200, SB-44, and SB-83 in a model of in vitro infection using
murine peritoneal macrophages. We ascertained the potential of these molecules to modulate
the immune response of host cells. Experimental models with intracellular amastigotes
represent the most effective way to relate antileishmanial activity of a drug in vitro to its
potential in vivo because macrophages are the primary cells parasitized by Leishmania spp. in
mammalian hosts [33]. The 2-amino-thiophene derivatives SB-200, SB-44, and SB-83 were
more effective in treating macrophages infected with L. amazonensis than meglumine
antimoniate, and they were more sensitive against intramacrophagic amastigotes compared to
axenic amastigotes. The difference in antileishmanial activity may be indicative of
macrophage activation, which combines their microbicidal power with direct effects
previously demonstrated for the three compounds [34].

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Macrophages are the main phagocytic cells of the host. They perform their
microbicidal functions through structural (phagocytosis, vacuolization, spreading, and
increased lysosomal volume) and cellular changes (altered cytokine levels, changes in
reactive oxygen species (ROS) and NO profiles, and matrix metalloproteinase secretion) [18].
To investigate the mechanisms by which SB-200, SB 44, and SB-83 diminished the
macrophage infection rate, we determined the immunomodulator effect by analysing the
production of TNF-α, IL-10, IL-12 and NO by infected macrophages treated with the
thiophenes. Our results suggest that the anti-amastigote effect of compounds SB-200 and SB-
83 is associated with increased TNF-α, IL-12, and NO; in contrast, SB-44 appears to exert its
antileishmanial activity only through a direct parasite killing action. Successful elimination of
parasites of the genus Leishmania have been reported to be dependent on the stimulation of
the immune system by the test drug via activation of host-protective Th1-dominated immune
responses with the production of protective cytokines (IFN-γ, TNF-α, and IL-12) [35]. TNF-α
is a proinflammatory cytokine produced by many cell types, although it is primarily produced
by macrophages. It induces the activation of different signalling pathways, including MAPK
and NF-κB (a major transcription factor that modulates iNOS gene expression and results in
the subsequent production of NO) [36]. IL-12 is a proinflammatory cytokine produced by
various cells types such as NK cells, polymorphonuclear cells and macrophages that is involved
in macrophage activation and the induction of the production of INF-γ, TNF-α and NO [37].
Experimental models of L. amazonensis infection of murine macrophages showed that INF-γ and
IL-12 were essential for parasite control [38], which was in agreement with our findings. Gomes
et al. [39] demonstrated that the involvement of ecto-nucleoside triphosphate
diphosphohydrolase (E-NTPDase) activity of L. amazonensis in the increase in the parasite's
survival was linked to the down-modulation of macrophage production of IL-12, TNF-a and
NO but not IL-10. We observed in the present work that the decrease in the infection rate was

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dependent on NO production. In response to the signal initiated by the activation of protective
cytokines, infected cells produced microbicidal molecules such as ROS and NO, which
played a crucial role in the elimination of the parasite in vitro and in vivo [40]. In terms of
antileishmanial activity, NO is the principal effector molecule involved in the killing of
established intracellular amastigotes [41]. Previous works demonstrated that the control of L.
amazonensis in vitro and in vivo was associated with the combination of NO and the
superoxide anion, resulting in production of peroxynitrite (ONOO-), a powerful, highly
reactive oxidant that acts as a leishmanicide. This finding is in contrast to other species such
as L. major, in which NO acts alone with a high leishmanicidal effect [42].
4. Conclusions
In conclusion, our results reveal that 2-amino-thiophene derivatives exhibit selective
antileishmanial activity against both stages of L. amazonensis with lower levels of cytotoxicity
to host cells. The effects of the selected compounds SB-200, SB-44, and SB-83 against the
promastigote forms is associated with apoptosis involving phosphatidylserine externalization
and DNA fragmentation accompanied by secondary necrotic cell death. The three compounds
were effective against macrophage infection, and the anti-amastigote activity of SB-200 and
SB-83 was associated with the modulation of the host immune response. These results
encourage the progression of studies on these compounds for the development of new
leishmanicidal agents.
5. Experimental section
5.1 Chemicals
Schneider's medium, the antibiotics penicillin and streptomycin, tetrazolium salt (3-
(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide) (MTT), dimethylsulfoxide

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(DMSO, 99% purity) thioglycollate medium, sodium dodecyl sulphate (SDS), and Griess
reagent (1% sulphanilamide in H3PO4 10% (v/v) in Milli-Q water) were purchased from
Sigma Chemical (St. Louis, MO, USA). RPMI-1640 medium and foetal bovine serum (FBS)
were obtained from Cultilab (São Paulo, SP, Brazil). The chemotherapy drug amphotericin B
(Anforicin B®) was obtained from Cristália (São Paulo, SP, Brazil), and meglumine
antimoniate (Glucantime®) was purchased from Aventis Pharma (São Paulo, SP, Brazil). The
haematological stain kit Panótico Rápido was obtained from Laborclin (Curitiba, PR, Brazil).
The sandwich ELISA assay kit and Annexin V-FITC/Propidium Iodide (PI) staining kit were
obtained from eBioscience™ (San Diego, CA, USA).
5.2 Synthetic Chemistry
2-Amino-cycloalkyl[b]thiophene-3-carbonitrile derivatives (called simply thiophene
derivatives in this work) were synthesized via a two-step reaction. The reaction started with
the one pot reaction between malononitrile with a cyclic ketone and elemental sulphur in the
presence of morpholine, followed by the Gewald procedure [16]. Treatment of the Gewald
adducts with aromatic aldehydes in ethanolic medium and reflux afforded the target
compounds 6CN01, 6CN09, 6CN10, SB-63, SB-80, SB-68, SB-81, SB-44, SB-83 and SB-
200 according to previous procedures (Scheme 1). All compounds were previously
synthesized and described by our group [12,14].

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Scheme 1 - Synthesis and chemical structures of studied compounds.
Compound
n
R
R´
O
CN
6CN01
2
H
S
R´
+
(CH2)n
+
6CN09
2
2-Br-5-OMe
CN
EtOH
rt
6CN10
SB-63
SB-80
SB-68
SB-81
SB-44
SB-83
2
3
1
3
1
3
2
4-NO2
Morfoline
S
-
(CH2)n
CN
-
-
NH2
S
EtOH
-
H
N
RCHO
reflux
R´
(CH2)n
CN
5-Bromo
N
R
SB-200
3
5-Bromo
H
N
S
5.3 X-Ray crystal structures of SB-83 and SB-200
The crystals of the compounds SB-83 and SB-200 were obtained from ethanol solution
using the slow evaporation method at 30°C under saturating conditions. The obtaining
procedures of experimental data, cell refinements, data reduction and resolution of the crystal
structure were performed according as described in Franscisco et al. [43] and are briefly
described. The experimental data set was obtained using a Bruker Enraf - Nonius Kappa CCD
diffractometer. The cell refinements were performed using the software Collect and
Scalepack, and the final cell parameters were obtained on all reflections. Data were collected
up to 28.2° in θ for SB-83 and up to 27.9° for SB-200, resulting in 15,624 and 15,822 Bragg
reflections, respectively. Data reduction was performed using the software Denzo-SMN and
Scalepack. Absorption was found to have a significant effect on both compounds (2.517 mm-1
for SB-83 and 2.478 mm-1 for SB-200); therefore, absorption correction was applied. The
structure was solved using the software SHELXS-97 and refined using the software
SHELXL-97. The C, N and O atoms were clearly solved and the full matrix least-squares
refinement of these atoms with anisotropic thermal parameters was performed. The hydrogen

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atoms were positioned stereochemically and refined with the riding model, except when they
were found to be involved in hydrogen bonds on the electronic density map. The details
concerning data collection and structure refinement were prepared using WinGX (version
1.70.01). The ORTEP-3 program was used to prepare the figures. The complete
crystallographic data have been deposited at the Cambridge Crystallographic Data Center as
supplementary publications no. CCDC 1029425 (SB-83) and no. CCDC 1027323 (SB-200).
Copies of the data are available upon application to The Director, CCDC, 12 Union Road,
Cambridge CB2 1EZ, UK (e-mail: deposit@ccdc.cam.ac.uk or http://www.ccdc.cam.ac.uk).
5.4 Docking Studies
The molecular modelling was performed as described in Scotti et al. [44]. Using the
program Hyperchem v. 8.0.3, the chemical structures of the compounds of interest were
drawn and their geometry was optimised using the MM+ force field. Afterwards, we
performed a new geometry optimisation based on the semi-empirical method AM1 (Austin
Model 1). The optimised structures were subjected to conformational analyses using the
Spartan for Windows 10.0 software. We selected the Monte Carlo search method with 1,000
interactions, 100 cycles of optimisation, and 10 conformers with the lowest minimum energy.
The dihedrals were evaluated by rotation in accordance with the standard (default) conditions
of the program, in which the number of simultaneous variations was 1 to 8, acyclic chains
were subjected to rotations from 60 to 180° and torsion rings were in the range of 30 to 120°.
The conformers with the lowest minimum energy were selected and saved in .sdf format. The
docking simulations were performed on the AutoDock 4.2 software. Receptor and ligand
preparation was performed using VEGA ZZ 3.0.1 and MOLEGRO MOLECULAR VIEWER
2.5. Initially, the structures of the ligand and receptor were saved in pqbqt format to be used
for docking calculations. PyRx 0.9 software was used to aid the steps of job submission and