Alkyl and aryl derivatives based on p-coumaric acid modification and inhibitory action against leishmania braziliensis and plasmodium falciparum

Article abstract of DOI:10.3390/molecules25143178

In low-income populations, neglected diseases are the principal cause of mortality. Of these, leishmaniasis and malaria, being parasitic, protozoan infections, affect millions of people worldwide and are creating a public health problem. The present work evaluates the leishmanicidal and antiplasmodial action of a series of twelve p-coumaric acid derivatives. Of the tested derivatives, eight presented antiparasitic activities 1-3, 8-12. The hexyl p-coumarate derivative (9) (4.14 ± 0.55 μg/mL; selectivity index (SI) = 2.72) showed the highest leishmanicidal potency against the Leishmania braziliensis amastigote form. The results of the molecular docking study suggest that this compound inhibits aldehyde dehydrogenase (ALDH), mitogen-activated kinase protein (MPK4), and DNA topoisomerase 2 (TOP2), all of which are key enzymes in the development of Leishmania braziliensis. The data indicate that these enzymes interact via Van derWaals bonds, hydrophobic interactions, and hydrogen bonds with phenolic and aliphatic parts of this same compound. Of the other compounds analyzed, methyl p-coumarate (64.59 ± 2.89 μg/mL; IS = 0.1) demonstrated bioactivity against Plasmodium falciparum. The study reveals that esters presenting a p-coumarate substructure are promising for use in synthesis of derivatives with good antiparasitic profiles.

Full text of DOI:10.3390/molecules25143178

molecules
Article
Alkyl and Aryl Derivatives Based on p-Coumaric Acid
Modification and Inhibitory Action against
Leishmania braziliensis and Plasmodium falciparum
Susiany P. Lopes 1, Lina M. Yepes 2, Yunierkis Pérez-Castillo 3 , Sara M. Robledo 2 and
Damião P. de Sousa 1,4,
*
1
PostGraduation Program in Technological Development and Innovation in Medicines, Federal University of
Paraíba, João Pessoa CEP 58051-970, Brazil; susiany.lopes@academico.ufpb.br
PECET-Facultad de Medicina, Universidad de Antioquia, Medellín Calle 70 # 52-21, Colombia;
lina.yepes@udea.edu.co (L.M.Y.); sara.robledo@udea.edu.co (S.M.R.)
Escuela de Ciencias Físicas y Matemáticas, Universidad de Las Américas, Quito 170504, Ecuador;
yunierkis.perez@udla.edu.ec
2
3
4
Department of Pharmaceutical Sciences, Federal University of Paraíba, João Pessoa CEP 58051-970, Brazil
Correspondence: damiao_desousa@yahoo.com.br
*
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 22 May 2020; Accepted: 17 June 2020; Published: 11 July 2020
Abstract: In low-income populations, neglected diseases are the principal cause of mortality. Of these,
leishmaniasis and malaria, being parasitic, protozoan infections, affect millions of people worldwide
and are creating a public health problem. The present work evaluates the leishmanicidal and
antiplasmodial action of a series of twelve p-coumaric acid derivatives. Of the tested derivatives, eight
presented antiparasitic activities
1
–
3
,
8
–
12. The hexyl p-coumarate derivative (
9
) (4.14 ± 0.55 µg/mL
;
selectivity index (SI) = 2.72) showed the highest leishmanicidal potency against the Leishmania
braziliensis amastigote form. The results of the molecular docking study suggest that this compound
inhibits aldehyde dehydrogenase (ALDH), mitogen-activated kinase protein (MPK4), and DNA
topoisomerase 2 (TOP2), all of which are key enzymes in the development of Leishmania braziliensis.
The data indicate that these enzymes interact via Van der Waals bonds, hydrophobic interactions, and
hydrogen bonds with phenolic and aliphatic parts of this same compound. Of the other compounds
analyzed, methyl p-coumarate (64.59
Plasmodium falciparum. The study reveals that esters presenting a p-coumarate substructure are
±
2.89 µg/mL; IS = 0.1) demonstrated bioactivity against
promising for use in synthesis of derivatives with good antiparasitic profiles.
Keywords: hydroxycinnamic acids; natural products; leishmanicidal activity; antiplasmodial activity;
cytotoxicity; neglected diseases
1. Introduction
Neglected diseases typically affect tropical and subtropical regions, principally developing
countries [
1
,2
], with poor and marginalized people [3] being the most affected. According to the
World Health Organization, neglected diseases affect more than 1 billion people around the world [
4].
These diseases cause great impacts on public health and include Chagas disease, leishmaniasis, malaria,
schistosomiasis, onchocerciasis, lymphatic filariasis, trypanosomiasis and dengue [5].
Leishmaniasis, caused by parasitic protozoans of the Leishmania genus, is a neglected disease that
affects both humans, and domestic and wild mammals [
(V.) guyanensis, L. (V.) lainsoni, L. (V.) shawi, L. (V.) naiffi, L. (V.) lindenbergi and L. (Leishmania) amazonensis
affect more than 98 countries worldwide [11 12]. Caused by unicellular eukaryotic protozoans of the
Plasmodium genus such as Plasmodium falciparum and Plasmodium vivax, leishmaniasis quickly develops
6–10]. These parasites: L. (Viannia) braziliensis, L.
,
Molecules 2020, 25, 3178; doi:10.3390/molecules25143178
www.mdpi.com/journal/molecules

Molecules 2020, 25, 3178
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into a very serious human infection that can cause death [13
–
15]. Approximately 12 million people
are currently affected with the disease [16]. Malaria however, remains the most prevalent parasitic
infection in the whole world with more than 3.4 billion people infected, and roughly 1.2 billion cases per
year [17
development of drug resistance, it has become necessary to investigate new drug candidates [20–23].
Hydroxycinnamic acids are natural phenolic compounds found in fruits, cereals, and coffee [24 25].
Caffeic acid, p-coumaric acid and ferulic acid are principal representatives of this class [26 32]. Various
pharmacological activities for p-coumaric acid have been noted, stimulating many studies using both
in vivo (animal) and in vitro models to investigate this compound and its synthetic derivatives [33 36].
–19]. Chloroquine is the current drug of choice for treatment of malaria. However, due to the
,
–
–
In addition to its applicability in the food, cosmetics, chemical, and pharmaceutical products, reports
of its antioxidant, antimicrobial, anti-inflammatory, antineoplastic, antimalarial, anti-Alzheimer’s and
neuroprotective effects are abundant [37–40]. p-Coumaric acid presents antiparasitic activity against
L. amazonensis [41]. A derivative of p-coumaric acid, methyl p-coumarate, presents activity against
P. falciparum [42]. There are also reports regarding p-coumaric acid and inhibition of P. falciparum
dihydropteroate synthase [43]. The aim of the study was to prepare p-coumaric acid derivatives and
evaluate their antiprotozoal activity. To deduce possible sites of antiparasitic action, in silico modeling
was also performed.
2. Results and Discussion
2.1. Chemistry of the Compounds 1–12
In this study, twelve compounds (Scheme 1), containing the (E)-3-(4-hydroxyphenyl) acrylic
acid substructure were synthesized; exclusively changing the radical R to methyl (Me (
(Et ( )), propyl (n-Pr ( )), isopropyl (i-Pr ( )), methoxyethyl (MeOEt ( )), butyl (Bu ( )), pentyl (n-Pent
)), isopentyl (i-pent( )), hexyl (Hex ( )), dodecyl (Dodec (10)), 4-methylbenzyl (MBz (11)), and
1)), ethyl
2
3
4
5
6
(7
8
9
4-isopropylbenzyl (i-PrBz (12)) esters. Acrylic acid and alcohol were used in the reactions with
(E)-3-(4-hydroxyphenyl) (Scheme 1). Products were prepared by Fischer esterification and Mitsunobu
reaction (Scheme 1). The compounds were characterized by infrared spectra (Figure 1) and Nuclear
Magnetic Resonance (NMR) spectra, as evidently published by Lopes et al. [44].
Scheme 1. Preparation of the p-coumaric acid derivatives: (I) alcohol, H2SO4, reflux; (II) alcohol, THF,
TPP, DIAD, 0 ◦C to room temperature.

Molecules 2020, 25, 3178
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Figure 1. Infrared spectrum (cm−1) of methyl p-coumarate (1).
2.2. Antileishmanial and Antiplasmodial Activity of Compounds 1–12
The results were evaluated using the 50% effective concentration (EC50) for parasitic activity.
We analyzed the collection (twelve compounds) for activity against Leishmania braziliensis in its
amastigote form, against Plasmodium falciparum, and for cytotoxicity against U-937 macrophages using
50% lethal concentration (LC50). Of the derivatives analyzed in Table 1, eight compounds (
1–3, and
8
–
12) showed moderate to good activity against Leishmania braziliensis amastigotes. However, the
twelve compounds exhibited weak antiplasmodial activity.
Table 1. Antileishmanial and antiplasmodial activity, cytotoxicity and molar mass of compounds
p-Coumaric Acid Esters
1–12.
Leishmania braziliensis
U-937
Macrophages
Plasmodium falciparum
Molar Mass
(g/mol)
Amastigotes
EC50 (µg/mL) a
SI
SI
LC50 (µg/mL)
EC50 (µg/mL) b
1
2
3
4
5
6
7
8
9
8.28 ± 0.14
4.91 ± 0.64
6.3 ± 1.14
0.76
0.21
1.89
3.4
1.46
0.1
64.59 ± 2.89
110.31 ± 30.84
139.04 ± 4.07
106.08 ± 9.97
98.66 ± 17.7
198.84 ± 7.9
95.48 ± 1.84
156.37 ± 8.2
206.35 ± 46.4
81.19 ± 8.54
83.98 ± 15.34
93.66 ± 14.17
0.1
6.26 ± 0.03
1.06 ± 1.08
11.89 ± 0.8
96.23 ± 18.89
126.71 ± 16.5
30.9 ± 6.32
16.09 ± 3.81
19.58 ± 1.76
11.24 ± 0.86
15.43 ± 1.84
20.81 ± 3.47
35.2 ± 13.94
38.4 ± 6.3
178.18
192.21
206.23
206.23
222.23
220.26
234.29
234.29
248.31
332.47
268.30
296.36
0.01
0.09
0.91
1.28
0.16
0.17
0.13
0.05
0.19
0.25
0.38
28.33 ± 0.27
86.98 ± 18.13
323.29 ± 50.04
32.03 ± 5.05
6.02 ± 0.22
4.14 ± 0.55
16.34 ± 0.49
5.69 ± 0.3
0.5
3.25
2.72
0.94
3.66
3.14
128
10
11
12
11.21 ± 0.2
0.3 ± 0.07
AMB c
CQ d
3.35 ± 0.39
14.1
47.26 ± 4.9
a
Data represent mean value
±
standard deviation. LC50: median lethal concentration; b EC50: median effective
concentration; c AMB: amphotericin B, antileishmanial drug control d CQ: chloroquine diphosphate salt, antimalarial
drug control.
2.3. Antileishmanial Activity
The simplest structure, (compound
activity (EC50 of 8.28 0.14 g/mL). Substitution with an ethyl group (compound
the inhibitory effect and a decrease in EC50 (4.91 0.64 g/mL) was observed when compared
to compound . Increases in side chain length help to increase lipophilicity and consequently
decrease EC50 values. These results are in agreement with a study by N brega et al. (2019) [45],
1) methyl p-coumarate demonstrated excellent leishmanicidal
±
µ
2), potentiated
±
µ
1
ó
demonstrating that ethyl 3,4,5-trimethoxycinnamate presented better leishmanicidal activity than
methyl 3,4,5-trimethoxycinnamate. However, Lima et al. (2016) [46] demonstrated that ethyl caffeate
presents no activity against Leishmania amazonensis. In contrast, excellent cytotoxic activity was

Molecules 2020, 25, 3178
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observed against U-937 macrophage cells for both compounds
1
and
2
(respectively 6.26
±
0.03
µ
g/mL
and 1.06 ± 1.08 µg/mL).
Compounds presenting three side chain carbons (
3,
4
, and
g/mL, and 86.98 18.13
) to isopropyl ( ), contributing to the decrease in leishmanicidal
activity seen in further branching in compound 47
than compound . The presence of the oxygen atom in the compound
a reduction in antiparasitic activity. Meanwhile, N
side chain oxygen in a methoxy-ethyl derivative (3,4,5-trimethoxycinnamate) increases leishmanicidal
activity. The leishmanicidal activity values of both compounds and were lower (more potent)
than their cytotoxic activity values against U-937 macrophage cells (respectively 96.23 18.89 g/mL,
126.71 16.5 g/mL. These same cytotoxicity values were also much higher (less potent) than that of
compound 3 (11.89 ± 0.8 µg/mL) which had high cytotoxicity.
Butyl p-coumarate ( ) being compared to compound
potency, with an EC50 of 323.29 50.04 g/mL. Interestingly, Bernal et al. (2019) [49] found that groups
with a medium length linear alkyl chain exhibited higher activity against L. donovani when compared
to groups presenting short chains [50]. Meanwhile, pentyl p-coumarate ( ) was five times less potent
than isopentyl p-coumarate ( ) (32.03 5.054.3 g/mL, 6.02 0.22 g/mL, respectively), possibly due to
compound branching. When compared as to cytotoxicity, neither expressed a significant difference
(respectively 16.09 3.81 g/mL and 19.58 1.76 g/mL). Steverding et al. (2016) [51] also observed
antiparasitic activity for isopentyl caffeate against Trypanosoma brucei.
Hexyl p-coumarate ( ), dodecyl p-coumarate (10), 4-methylbenzyl p-coumarate (11) and
4-isopropylbenzyl p-coumarate (12), (respectively 4.14 0.55 g/mL, 16.34 0.49 g/mL,
5.69 ± 0.3 µg/mL and 11.21 0.2 g/mL) displayed high leishmanicidal activity, likely due to their
relatively high lipophilicity. Yet Otero et al. (2014) [52] found in their study that hexyl cinnamate
presents less leishmanicidal activity than dodecyl cinnamate. However, in the present study was
5) exhibited differing EC50 values
(respectively 6.3
alteration in the n-propyl radical (
±
1.14
µ
g/mL, 28.33
±
0.27
µ
±
µg/mL). This may be due to an
3
4
4
[
,
48]. Compound
3
was fourteen times more active
5
5 side chain likely contributes to
ó
brega et al. (2019) [45] observed that the presence of
4
5
±
µ
±
µ
6
1, presented the lowest leishmanicidal
±
µ
7
8
±
µ
±
µ
8
±
µ
±
µ
9
±
µ
±
µ
±
µ
9
considered the most potent compound compared to both compound 10 and the other derivatives
analyzed. The results obtained are in accordance with the preparation plan for the compounds, in which
alkyl and benzyl substituents were added to increase the lipophilicity of the compounds. In this way,
compounds with good absorption were obtained to cross the parasite membrane resulting in greater
bioavailability [53–55]. Several mechanisms of action can occur, such as greater access to organelles
that are biological targets for leishmanicidal activity [56]. As suggested in the molecular docking
study, hexyl p-coumarate should inhibit the enzymes ALDH, MPK4, and TOP2 (see Table 2) which are
essential for Leishmania braziliensis growth. Regarding the substitution of the aliphatic chain in the para
position of the aromatic ring in compounds 11 and 12, a difference in leishmanicidal activity was noted
since compound 11 was twice as potent as compound 12 which possessed the more bulky substituent.
These same results were also observed by Lopes et al. (2019) [44] in relation to trypanocidal activity.
2.4. Antiplasmodial Activity
Methyl p-coumarate (compound
P. falciparum. An increase in the side chain resulted in a decrease in antiplasmodial activity, since
ethyl p-coumarate showed a higher EC50 (110.31 30.84 g/mL). Alson et al. (2018) [57] observed in
their study that methyl caffeate and ethyl caffeate expressed the best antiplasmodial activities against
P. falciparum. On the other hand, for Arsianti et al. (2018) [58], propyl p-coumarate (compound ) had low
antiplasmodial activity when compared to isopropyl p-coumarate ( ) and methoxy-ethyl p-coumarate ( ),
(respectively 139.04 4.07 g/mL, 106.08 9.97 g/mL, 98.66 17.7 g/mL). Furthermore, when
with butyl p-coumarate ( ), an increase in EC50 of 198.84 7.9 g/mL
1
) exhibited the lowest EC50 (64.59
±
2.89 µg/mL) against
±
µ
3
4
5
±
µ
±
µ
±
µ
comparing compound
was observed.
1
6
±
µ
If the side chain of the p-coumarate derivative is increased, an increase in the lipophilicity of the
compounds is observed for pentyl p-coumarate of ( ), isopentyl p-coumarate of ( ), hexyl p-coumarate
7
8

Molecules 2020, 25, 3178
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of (
between these compounds; compound 8 (95.48
(156.37 8.2 g/mL). Yet Arsianti et al. (2017) [59] observed that alkyl ester groups presenting linear
chains such as pentyl gallate, present better inhibitory action in relation to antiplasmodial activity
than compounds with branched structures. When comparing compounds and 10, there was a
significant difference (respectively 206.35 46.4 g/mL, 81.19 8.54 g/mL); and hexyl p-coumarate
9) and dodecyl p-coumarate (10). As for antiplasmodial activity, there was a difference in EC50
±
1.84 g/mL) was more potent than compound
µ
7
±
µ
9
±
µ
±
µ
showed the least antiplasmodial activity against P. falciparum [60]. In compounds 11 and 12 the
substituent is a substituted aromatic ring; the antiplasmodial potency of these compounds was greater
than the majority of the derivatives with carbon chains; both 4-methylbenzyl p-coumarate (11) and
4-isopropylbenzyl p-coumarate (12) presented weak antiplasmodial activity with respective EC50 of
83.98 ± 15.34 µg/mL and 93.66 ± 14.17 µg/mL.
Table 2. Potential targets of compound 9 in Leishmania braziliensis identified by the homology-based
target fishing approach.
L. braziliensis Target (a)
Description
ID
A4HED7_LEIBR
A4HE56_LEIBR
A4HP40_LEIBR
A4HG21_LEIBR
A4HGT2_LEIBR
A4H9D1_LEIBR
A4HBR4_LEIBR
A4HDK5_LEIBR
A4HA94_LEIBR
A4H9X2_LEIBR
A4HJJ7_LEIBR
A4HK26_LEIBR
A4H9L8_LEIBR
Serine/threonine-protein kinase
Aldehyde dehydrogenase, mitochondrial
Aldehyde dehydrogenase
PKC
ALDH2
ALDH
AKR
TOP2
GSK3
HDA1
HDA2
MPK4
CBPKC
PGFS
PGFS2
PKAC3
PKAC2a
CRK7
Aldo-keto reductase protein
DNA topoisomerase 2
Putative glycogen synthase kinase
Histone deacetylase
Histone deacetylase
Mitogen-activated protein kinase
Mitogen-activated protein kinase
Prostaglandin-F synthase protein
Prostaglandin f synthase
Protein kinase A catalytic subunit
A4HN71_LEIBR
A4HEL2_LEIBR
A4H8N9_LEIBR
Protein kinase A catalytic subunit isoform 1
Protein kinase domain-containing protein
Tyrosyl or methionyl-tRNA synthetase-like protein
YARS1
(a)
UniProt entry name.
2.5. Computational Methods
Computational target fishing approaches rely on the similarity principle: if a query compound is
similar to a reference which binds a certain receptor, then the query might also bind the same receptor.
Reference ligand–receptor interactions are generally extracted from large publicly available databases.
However, the information in these databases is biased against human receptor–ligand associations.
For this reason, the homology-based target fishing approach described in the Methods section was
implemented to identify potential Leishmania braziliensis targets for compound
9. The 16 identified
potential Leishmania braziliensis targets for compound are provided in Table 2. Of them, seven are
9
annotated in the UniProt database as confirmed or predicted protein kinases, suggesting a subset of
possible targets enriched with this function.
Molecular docking of compound
as described in the Methods section. It has been shown that GSK3 inhibitors are effective in binding to
ATP and substrate binding sites in both Leishmania spp. and in the human homolog [61 62]. Based
9 (to the 16 potential targets listed in Table 2) was thus performed
,
on these findings, we explored GSK3 for ligand binding to these two sites in Leishmania braziliensis.
TOP2 is an ATP-dependent DNA topoisomerase enzyme that contains ATP and nucleotide binding
sites [63
of the consensus docking protocol are summarized in Table 3. Ten (10) of the 16 modeled targets,
(for compound ), predicted more than one possible binding mode, leading to 33 ligand–receptor
,64]. For the TOP2 docking calculations, only the ATP binding cavity was explored. The results
9

Molecules 2020, 25, 3178
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complexes needing further analysis Visual inspection of these 33 predicted complexes revealed
meaningful binding modes for all of them. In all cases, the ligand was inside the receptor binding site,
and either interaction or blocking of functionally relevant residues was noted.
Table 3. Results of the molecular docking of compound
Leishmania braziliensis.
9 to its predicted potential molecular targets in
CHEMPLP
GoldScore
ChemScore
ASP
Z-Score
Consensus
Z-Score
Target
Conformer
Score Z-Score Score
Z-Score Score
Z-Score Score
1
2
1
1
2
3
4
1
2
3
1
2
1
2
1
2
1
1
1
2
3
1
1
1
1
2
3
1
1
2
3
1
2
64.76
60.60
63.77
64.64
59.38
62.51
62.25
60.32
61.32
51.69
57.19
57.11
70.94
67.43
50.52
48.54
65.54
67.10
58.40
55.89
57.14
54.89
50.11
32.76
58.29
57.86
56.43
61.50
66.15
65.15
63.92
61.62
57.26
2.69
1.31
2.22
1.97
0.56
1.40
1.33
2.04
2.17
0.90
1.26
1.24
2.37
1.62
1.64
1.17
1.28
0.94
2.11
1.23
1.67
2.04
1.89
1.06
1.47
1.33
0.84
2.15
2.13
1.86
1.53
2.08
0.97
29.03
29.05
34.27
66.89
69.68
66.04
65.00
10.04
−8.41
−2.36
33.54
32.47
34.80
29.45
25.70
27.61
29.53
40.20
32.17
26.35
33.77
24.42
24.68
13.87
36.02
31.27
36.64
32.33
36.34
34.07
33.69
21.92
16.22
1.06
1.07
1.46
1.54
2.24
1.32
1.06
1.10
0.13
0.45
1.29
1.01
1.27
0.22
0.50
0.72
0.57
1.55
1.73
0.12
2.17
1.07
1.70
1.33
1.16
0.57
1.24
1.09
2.04
1.38
1.27
1.15
0.80
24.23
23.04
21.22
20.48
20.25
17.22
17.98
25.09
21.15
18.34
19.27
16.63
24.32
23.34
13.30
14.49
24.30
27.36
20.59
24.14
20.50
17.09
13.91
7.74
1.32
0.71
0.76
1.65
1.54
0.09
0.45
2.50
1.84
1.37
1.92
0.85
1.97
1.40
0.87
1.32
0.79
1.03
0.66
1.92
0.63
0.06
1.14
1.27
1.83
1.01
0.73
1.72
1.60
1.36
0.62
1.56
1.24
24.27
23.60
25.99
27.38
27.18
29.32
29.19
37.69
26.77
29.66
23.73
22.31
32.04
29.85
18.51
17.12
31.58
35.93
20.43
21.72
18.31
19.03
30.81
20.71
24.29
26.05
24.93
20.13
25.98
24.01
25.55
19.98
23.33
1.68
1.35
0.47
0.79
0.71
1.63
1.57
2.35
1.04
1.39
1.36
0.92
2.70
1.66
1.87
1.34
1.40
1.33
0.70
1.27
−0.25
0.89
1.82
1.51
1.08
1.82
1.35
−0.17
1.43
0.65
1.26
0.23
1.52
1.69
1.11
1.23
1.49
1.26
1.11
1.10
2.00
1.30
1.03
1.46
1.00
2.08
1.22
1.22
1.14
1.01
1.21
1.30
1.14
1.05
1.01
1.64
1.29
1.39
1.18
1.04
1.20
1.80
1.31
1.17
1.26
1.13
PKC
ALDH2
ALDH
AKR
TOP2
GSK3 (a)
GSK3 SBS (a)
HDA1
HDA2
MPK4
CBPKC
PGFS
PGFS2
23.65
21.71
21.06
24.27
24.01
23.39
21.55
21.95
21.32
PKAC3
PKAC2a
CRK7
YARS1
(a)
Two different binding sites were explored for GSK3: the ATP binding site (GSK3) and the substrate binding
pocket (GSK3 SBS).
Considering the same consensus protocol employed for ligand pose selection, the best docking
results in GSK3 targets were obtained for the HDA2 and the ATP binding sites. However, the
worst docking performance for GSK3 targets was observed for the PGFS and substrate binding sites.
Interestingly, the GSK3 docking results presented large differences in scores for the ATP and substrate
binding sites, which could be an indicator of selectivity towards ATP binding. Overall, the docking
results did not allow for confident selection of the most probable compound
of predicted docking complexes using Molecular Dynamics (MD) simulations along with estimations of
free energies of binding can help refine target prioritization [44 65]. Calculations were thus performed
(described in the Methods section) to estimate the free energies of binding for compound to its
predicted targets using the MD simulations data. These simulation results are demonstrated in Table 4.
9 targets. Post-processing
,
9

Molecules 2020, 25, 3178
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Table 4. Predicted free energies of binding of compound
9 to its predicted targets. The components of
the free energies of binding are also included. All energies are expressed in kcal/mol.
MM-PBSA Components
∆G TOTAL (h)
Target
Conformer
VDWAALS(a) EEL (b)
EPB (c)
ENPOLAR (d) EDISPER(e) ∆G Gas (f)
∆G Solv (g)
1
2
1
1
2
3
4
1
2
3
1
2
1
2
1
2
1
1
1
2
3
1
1
1
1
2
3
1
1
2
3
1
2
−31.35
−33.93
−29.85
−38.10
−37.59
−34.69
−38.78
−30.88
−28.48
−29.47
−42.66
−46.13
−28.68
−34.81
−18.60
−27.34
−28.68
−25.51
−30.88
−36.54
−39.58
−32.78
−26.09
−22.24
−36.72
−38.00
−37.74
−33.06
−40.13
−34.61
−38.51
−25.99
−24.81
−27.22
−15.64
−26.61
−15.73
−22.81
−23.13
−30.30
−13.99
−7.63
37.23
29.63
36.34
36.01
36.17
37.63
40.58
28.85
22.63
32.42
42.55
35.84
31.25
30.70
23.44
26.18
31.32
35.37
35.63
31.88
32.41
35.50
19.97
20.66
57.32
33.67
57.82
27.04
38.07
30.38
35.40
23.28
28.89
−27.27
−28.73
−25.56
−29.50
−30.23
−28.16
−30.43
−25.79
−22.28
−23.82
−31.82
−31.37
−21.69
−28.87
−16.13
−20.94
−22.81
−22.39
−26.12
−29.36
−30.01
−27.42
−20.90
−18.94
−29.95
−28.84
−30.26
−25.35
−31.07
−27.47
−30.09
−21.71
−22.35
43.17
46.01
43.08
48.51
49.25
46.95
50.68
40.67
36.82
39.14
51.87
52.34
37.74
46.12
26.89
35.99
37.85
35.83
42.49
46.60
47.48
44.34
34.35
30.60
47.33
47.80
47.61
43.07
48.14
45.17
48.31
35.63
34.35
−58.57
−49.57
−56.46
−53.83
−60.40
−57.82
−69.08
−44.87
−36.11
−43.24
−67.37
−62.96
−40.02
−52.11
−32.81
−36.56
−44.88
−49.88
−55.60
−50.23
−57.02
−55.67
−35.42
−33.87
−71.70
−50.25
−71.74
−40.92
−58.86
−47.56
−53.78
−37.83
−42.43
53.12
46.91
53.86
55.02
55.19
56.43
60.83
43.74
37.17
47.74
62.60
56.80
47.30
47.95
34.20
41.23
46.36
48.80
51.99
49.11
49.88
52.42
33.41
32.32
74.69
52.63
75.18
44.76
55.14
48.08
53.62
37.19
40.89
−5.45
−2.66
−2.60
1.19
−5.21
−1.39
−8.25
−1.13
1.06
PKC
ALDH2
ALDH
AKR
−13.77
−24.71
−16.83
−11.34
−17.30
−14.21
−9.22
4.50
−4.76
−6.15
7.27
TOP2
GSK3
−4.16
GSK3
(SBS)
HDA1
HDA2
1.39
4.67
1.47
−16.20
−24.37
−24.72
−13.69
−17.44
−22.89
−9.33
−1.08
−3.61
−1.11
−7.14
−3.25
−2.01
−1.55
2.99
MPK4
CBPKC
PGFS
PGFS2
−11.63
−34.98
−12.24
−34.00
−7.86
2.38
3.44
3.84
PKAC3
PKAC2a
CRK7
−18.73
−12.95
−15.27
−11.84
−17.62
−3.72
0.52
−0.16
−0.64
−1.54
YARS1
(a) van der Waals energy, (b) electrostatic energy, (c) polar solvation energy, (d) non-polar solvation energy, (e) dispersion
solvation energy, (f) total gas phase free energy, (g) total solvation free energy, (h) total free energy.
The MM-PBSA calculations reveal that due to the predicted positive free energies of binding,
the complexes formed by compound
Though negative, the Gs of binding predicted for AKR, HDA2, PGFS, PGFS2, and YARS1, range from
1.08 kcal/mol to 2.01 kcal/mol. The values predict low stability for these complexes. Slightly better
free energies of binding are predicted for the ALDH2, GSK3, CBPKC, CRK7, and PKC targets. Despite
stable binding, ( G TOTAL < 0) is predicted for most of the predicted compound targets, as seen
9 with HDA1, PKAC3, PKAC2, and GSK3 (SBS) are unfeasible.
∆
−
−
∆
9
in the results in Table 4, where the most probable compound
One interesting result is that of the predicted likely compound
9
9
targets are ALDH, MPK4, and TOP2.
targets, the binding sites of TOP2 and
the kinases (MPK4, PKC, GSK3, CBPKC, and CRK7) are all ATP binding cavities. This suggests that
compound might interfere with ATP binding in various proteins related to diverse cellular functions.
9
Based on the results thus obtained, we selected ALDH, MPK4, and TOP2 as targets for additional
evaluation concerning compound 9 potential anti-leishmanial mechanism of action.
The interactions between compound 9 and its most probable targets were analyzed in detail along
with the 200 snapshots employed for Molecular Mechanics with Poisson–Boltzmann and Surface Area
solvation (MM-PBSA) calculations. The binding modes of the ligand to these receptors as predicted are
shown in Figure 2. To choose a representative binding mode for each receptor, the 200 conformations
of compound 9 were clustered and the centroid mode from the most populated cluster was selected.
Figure 2 includes the network of interactions observed during the MD simulations between the ligand
and the investigated proteins. Only the interactions taking place in more than 30% of the MD snapshots
used for free energy of binding estimation are included in the figure. Given that our structural analyses
are preformed over an ensemble of 200 conformations for each complex, not all hydrogen bond

Molecules 2020, 25, 3178
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interactions are present in all snapshots. For this reason, only the hydrogen bonds observed in the
selected representative structure of each complex are depicted as black dashed lines in Figure 2.
Figure 2. Predicted binding modes of compound
9
to its more probable receptors: (A) ALDH, (B) MPK4
and ( ) TOP2. Receptors are depicted gray and compound
C
9
cyan. Non-carbon ligand and receptors
atoms are colored following the scheme: red for oxygen, blue for nitrogen and yellow sulfur. Only
receptors residues interacting with the ligand in more than 50% of the selected MD snapshots are
labelled in the complexes. Hydrogen bonds are represented as black dashed lines. The observed
networks of interactions along the MD snapshots used for MM-PBSA calculations are presented at the
left. Darker lines in the interaction networks indicate more frequent interactions.
In the case of ALDH, the ligand binds to the substrate binding channel making contact with E128,
E132, Y135, Y139, F182, A185, M186, R189, R307, S471 and F477 in more than 50% of the analyzed MD
snapshots. As shown in Figure 2A, the hexyl tail occupies a sub-cavity close to the NAD cofactor,
and hence completely blocks access, leading to enzyme blockage. In this predicted complex, the
hydroxyphenyl ring of the compound interacts with residues at the entrance of the binding pocket.
Most of the interactions observed were Van der Waals and hydrophobic. One factor adding stability to
this complex is the presence of hydrogen bonds between the ligand’s hydroxyl moiety and E128 of

Molecules 2020, 25, 3178
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ALDH. Compound
9
is also predicted to accept hydrogen bonds from the side chain of Y139 and the
backbone of R189 through its carbonyl group. It is noteworthy that the hydrophobic hexyl fragment of
the ligand localizes in a region containing hydrogen bond accepting capable residues such as T256,
E279, T312, and the NAD cofactor. We postulate that inclusion of hydrogen bond donor substitutions
in this part of compound 9 would improve the stability of this ALDH complex.
In the complex predicted for compound
9 with MPK4 (Figure 2B), interactions with I36, V44, A57,
K59, E77, M81, I90, F109, M111, Y114, T116, A120, R120, L163 and D174 occur in the majority of the
MD snapshots analyzed. The interactions are mainly Van der Waals and electrostatic in nature, and
as in the complex predicted with ALDH, the ligand’s hexyl moiety orients toward the bottom of the
cavity and the hydroxyphenyl substituent occupies the entrance of the binding site. In most of the MD
snapshots studied, the hydroxyl group of the latter is predicted to achieve hydrogen bonding with
either the backbone of Y114 or with side chains of R120 and D117. Another similarity between its
predicted binding pose and that of ALDH is that the hydrophobic tail of compound 9 fits closely to
two hydrogen bond acceptor amino acids at the receptor: E77 and D174.
The compound
9 complex predicted for the TOP2 ATP binding site (Figure 2C) reveals a large
network contacts that includes N67, A68, N71, N95, E98, I100, I116, F117, T122, S123, S124, G136, G139,
F140, G141, A142 and T190. Similar to its ALDH and MPK4 targets, the compound poses in TOP2
with its hexyl part oriented towards the bottom of the binding cavity and its aromatic substituent
oriented towards the cavity entrance. The phenyl substituent is also capable of forming hydrogen
bonds at the drug receptor site in the TOP2 complex, either through backbones E98 and T190 or via
side chains of Q79 and N95. The cavity region where the aliphatic tail of compound 9 binds contains
hydrogen bonding capable residues that include N67, S123, S124 and N138. This suggests that addition
of hydrogen bond donor groups to the hydrophobic tail of the compound might increase its multitarget
affinity against Leishmania braziliensis through ALDH, MPK4, and the TOP2–ATP binding site.
Of the three Leishmania braziliensis targets selected as the most likely for compound 9, the aldehyde
dehydrogenase function (ALDH) has been proposed as a process which (through inhibition) could
be useful for development of therapeutic agents against Leishmania spp. [66]. Furthermore, there are
several reports in the literature demonstrating the importance of MPK4 to Leishmania spp. in both
development and infectious capability, making it an attractive for drug discovery efforts [67–69].
Finally, the importance of DNA topoisomerases in the life cycle of this type of parasite, and their use as
drug targets has already been established in the scientific literature [70].
3. Conclusions
Twelve compounds were evaluated for antiparasitic potential against L. braziliensis amastigotes,
against Plasmodium falciparum, and for cytotoxicity in U-937 macrophages. Eight of the derivatives were
bioactive against Leishmania braziliensis, such as hexyl p-coumarate (compound
IS = 2.72) which indicated the most potent inhibitory effect. The studies performed using molecular
docking suggested that compound interacts with the enzymes ALDH, MPK4, and TOP2, which are
9) (4.14
±
0.55 µg/mL;
9
essential to Leishmania braziliensis development. Some of the compounds tested were bioactive against
P. falciparum yet demonstrated little antiparasitic activity. These results may be useful to other studies
involving p-coumaric acid, to help obtain ever more potent and selective derivatives against these
protozoan species, which continue to compromise the quality of life of various world populations.
4. Materials and Methods
4.1. Reagents and Chemical Characterization
The derivatives were purified by column adsorption chromatography (CC) using silica gel
60, ART 7734—MERCK, St. Louis, Missouri, EUA. Infrared spectra were performed using FTIR
spectrophotometry and 1H and 13C NMR spectra were obtained using a Bruker-AscendTM machine,

Molecules 2020, 25, 3178
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Bruker, Bremen, Germany, operating at 400 and 100 MHz. All compounds were structurally
characterized as published by Lopes et al., 2019 [44].
4.2. General Procedure for Preparation of Compounds 1–8
p-Coumaric acid (0.1 g; 0.61 mmol) in alcohol (20 mL) was dissolved in the presence of H2SO4
(0.2 mL) and refluxed for complete reaction (5–27 h), being observed using single spot thin-layer
chromatography (TLC) [71], as published by Lopes et al. [44].
4.3. Preparation of Compounds 9–12 by Mitsunobu Reaction
p-Coumaric acid (0.1 g; 0.61 mmol) and alcohol (0.61 mmol) were dissolved◦in 2.25 mL of
tetrahydrofuran. The reaction mixture was stirred under magnetic stirring at 0 C for 30 min.
Subsequently, diisopropyl azodicarboxylate (0.12 mL; 0.61 mmol) and triphenylphosphine (0.16 g;
0.61 mmol) were added. The mixture was stirred at room temperature for 48–52 h and monitored with
TLC [72], as published by Lopes et al. [44]. Spectroscopic data for the compounds in this study are
available in Supplementary Materials.
4.4. Cell Culture
As cells utilized were L. braziliensis amastigotes, Plasmodium falciparum, and U-937 (ATCC CRL-
1593.2TM) macrophages.
4.5. In Vitro Cytotoxicity
The compounds were analyzed for viability of the human pro-monocytic cell line U-937 (ATCC CRL-
1593.2TM) using an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide) assay based
on a previously described methodology, assessing cytotoxic activity [73]. The resulting compounds
were determined by measuring mitochondrial dehydrogenase activity, adding 10 mL/well of MTT
solution (0.5 mg/mL) and incubation at 37 ◦C for 3 h. Cell viability was defined from the quantity of
formazan produced and the intensity of color (absorbance) registered through optical density (O.D.)
obtained at 570 nm in a (VarioskanTM Flash Multimode Reader—Thermo Scientific, Waltham, MA,
USA) spectrophotometer. As a negative control, isolated cells were cultured (without the compounds)
to observe viability. Doxorubicin was used as a cytotoxicity control in a similar manner. We performed
two independent tests with each concentration, in triplicate [73].
4.6. In Vitro Antileishmanial Activity
The activities of the derivatives in L. braziliensis intracellular amastigotes, transfected with the
green fluorescent protein gene (MHOM/CO/88/UA301-EGFP) were evaluated. Initially, human U-937
cells at a density of 3
×
105 cells/mL in RPMI 1640 and 0.1
µg/mL PMA (phorbol-12-myristate-13-acetate)
were dispensed in a 24-well microplate and incubated at 37 ◦C, and 5% CO2. At 72 h of incubation, the
cells were infected with growing (stationary phase) promastigotes; 15 parasites per cell. The plates
were incubated at 34 ◦C and 5% CO2 for 3 h and the cells then washed twice with phosphate buffer
solution (PBS) to eliminate non-internalized parasites. Fresh RPMI-1640 was added to each well (1 mL)
and the plates were incubated again to complete the infection. At 24 h of infection, the RPMI-1640
medium was replaced by a fresh culture medium containing each compound in four serial dilutions,
the highest concentration being equivalent to twice the LC50, and the plates were incubated at 37 ◦C
and 5% CO2 for 72 h. The cells were then removed from the bottom plate with a trypsin/EDTA solution
(250 mg), and centrifuged at 1100 rpm for 10 min at 4 ◦C; the supernatant was discarded and the cells
were washed with 1 mL of cold PBS and centrifuged twice at 1100 rpm for 10 min at 4 ◦C. After this
final wash, the supernatant was discarded and the cells were suspended in 500 µL of PBS.
The cells were run in a flow cytometer (Cytomics FC 500MPL), reading at 488 nm (exciting) and
525 nm (emitting) over an argon laser, and counting 10,000 events. Infected cells were determined

Molecules 2020, 25, 3178
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according to green fluorescence events (parasites). Infected cells exposed to the control drug
(amphotericin B) were used as antileishmanial activity controls (positive control). Infected cells
incubated in the absence of any compound or drug were used as infection controls (negative
control). Non-specific fluorescence was corrected by subtracting fluorescence from unstained cells.
All determinations were performed in triplicate, in at least two independent experiments [74].
4.7. In Vitro Antiplasmodial Activity
Cultures of asynchronous P. falciparum 3D7 strains were adjusted for 0.5% parasitemia and
1% hematocrit in RPMI medium enriched with 3% lipid-rich bovine serum albumin—Albumax II.
Afterwards, 100
and subsequently exposed to 100
incubated for 48 h at 37 ◦C in an atmosphere of N2 (90%), CO2 (5%) and O2 (5%). After incubation, the
parasites were harvested and subjected to three 20-min freeze-thaw cycles. At the same time, 100 L of
Malstat reagent (400 L of Triton X-100 in 80 mL of deionized water, 4 g of L-lactate, 1.32 g of Tris buffer,
µL of the parasite suspension was dispensed to each well of a 96-well cell culture plate
µ
L of each compound at 100, 25, 6.25, or 1.56 g/mL. The plates were
µ
µ
µ
and 0.022 g of adenine acetylpyridine dinucleotide in 200 mL of deionized water; pH 9.0), and 25 mL
of NBT/PES solution (0.16 g of nitroblue-tetrazolium salt, and 0.08 g of phenazine sulfate in 100 mL of
deionized water) were added to each well of an additional 96-well plate. After freezing and thawing
cycles, the culture in each of the wells on the first plate was re-suspended by pipetting, and 15 µL was
removed from each well and added to the corresponding well on the additional plate (containing the
Malstat and NBT/PES reagents). After one hour of incubation in the dark, the color development of the
lactate dehidrogenase (LDH) reaction was read on a spectrofluorometer (Varioskan, Thermo, Waltham,
MA, USA) at 650 nm. The color intensity in each experimental condition was recorded in fluorescent
units (F.U). Non-specific fluorescence was corrected by subtracting F.U. from the blanks. The entire
experiment was performed in triplicate in at least two independent experiments. Chloroquine (QC)
was used as a positive control and the culture medium was used as a negative control [75].
4.8. Statistical Analysis
Cytotoxicity was determined according to the viability and mortality percentages obtained in
each experimental condition (synthesized compounds, amphotericin B, benznidazole, chloroquine,
and culture medium). Viability percentages were first calculated using Equation (1).
% Viability = (O.D exposed cells ÷ O.D Control cells) × 100
(1)
Then, cell growth inhibition percentage was calculated as 100 - % viability.
The cell growth inhibition percentages were used to calculate median lethal concentrations (LC50)
using Probit analysis [76]. Toxicity was defined according to LC50 values, according to the following
scale: toxic; LC50 < 100
nontoxic; LC50 > 200 µM.
µM; moderately toxic; LC50 > 100 µg/mL and <200 µM, and potentially
The antiplasmodial activity of each compound was evaluated through reduction in parasite
growth, calculated according to Equation (2):
% parasite inhibition = 100 − [(F.U exposed parasites ÷ F.U non exposed parasites) × 100]
(2)
Data for percentages of parasite reduction or inhibition were used to calculate the EC50 using
Probit linear regression modeling [76]. Activity was graded as high, moderate or low according to EC50
values as follows: high activity when the EC50 was <25
µg/mL; moderate activity when the EC50 was
in the range from 25 to 50 g/mL, and low activity when the EC50 was >50
µ
µg/mL. The selectivity index
(SI) was calculated dividing cytotoxic activity by anti-parasitic activity using the following formula:
SI = CL50 ÷ CE50.

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4.9. Computational Methods
4.9.1. Target Selection
Compound
9 (the most active) was selected for modeling studies. The potential target set for this
compound was selected using a homology-based approach. Initially, potential targets of compound
9
were predicted using the similarity ensemble approach (SEA) web server [77], and the identified subset
of potential targets was used as input for a BLAST search [78], for Leishmania braziliensis (taxid:5660)
proteins found in the Reference Proteins Database (refseq_protein). The BLAST search was performed
using its NCBI server implementation (https://blast.ncbi.nlm.nih.gov/). The Leishmania braziliensis
proteins were covered (at a sequence minimum of 70%) by the BLAST sequence alignment. Those with
at least 35% identity with any query protein were regarded as potential compound 9 parasite targets.
4.9.2. Molecular Docking
Molecular docking followed the same protocol described in our previous publications [44
,65].
The initial three-dimensional conformation of compound was obtained with OpenEye Omega [79];
9
and AM1-BCC partial atomic charges were added to it using Molcharge [80]. Since none of the
predicted targets had their 3D structures solved, homology models were generated for all the studied
proteins with the SWISS-MODEL server [81]. Catalytically important cofactors, including metal ions,
were added to the homology models taking their templates as reference.
Gold software was used for molecular docking [82]. Receptors’ binding sites were defined from
the ligands present on the templates used for homology models. Any residue located at a distance
lower than 6 Å from the reference ligand was selected for the active site. In the absence of a reference
ligand, the binding site was defined as any amino acid within 10 Å of a point located at the center
of the cavity. For primary docking, 30 different solutions were explored for compound
9 using the
CHEMPLP scoring function with the Search efficiency of Gold set to 200%. The obtained 30 different
ligand conformations were rescored with the GoldScore, ChemScore, and ASP scoring functions.
Furthermore, during molecular docking the side chains of the residues pointing toward the binding
cavity were set to flexible.
The most probable binding modes of compound
9 (for each receptor) were selected from a
consensus scoring strategy. For this, the score of each ligand conformation according to scoring
function j was converted to Z-scores following the transformation:
´
Si,j − Sj
Zi,j
=
ꢀ
ꢁ
(3)
std Sj
´
where Zi,j is the Z-score of conformation i according to scoring function j, Sj indicates the average
ꢀ
ꢁ
value of scoring function j across all predicted binding modes and std Sj represents the standard
deviation of these values. The final aggregated score Zi of a predicted conformation was computed as
the average of its Z-scores across all the CHEMPLP, GoldScore, ChemScore and ASP scoring functions.
Any predicted binding mode with Zi > 1 was selected for further calculations.
4.9.3. Molecular Dynamics Simulations and MM-PBSA Calculations
Amber 18 [83] was employed for molecular dynamics (MD) simulations and MM-PBSA calculations.
These calculations were performed using the same protocol as in our previous publication [44]. For the
MD simulations, all predicted ligand–receptor complexes underwent the same modeling steps:
energy minimization, heating, equilibration, production runs, and free energy of binding prediction.
The Amber force field ff14SB and gaff force fields were respectively used to parameterize amino acids
and non-amino acid residues. MD simulations took place in an explicit solvent for which the complexes
were enclosed in truncated octahedron boxes and solvated with TIP3P water molecules. To neutralize
any excess net charge, either Na+ of Cl− ions were added to the solvated systems.

Molecules 2020, 25, 3178
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The prepared systems were energy minimized in two steps. First, 500 steps of the steepest descent
method followed by 500 cycles of conjugate gradient were executed while constraining all atoms
except solvent with a force constant of 500 kcal/mol
Å2. Then, all atoms were unconstrained for the
·
500 steps of the steepest descent algorithm followed by 1000 cycles of conjugate gradient. Long range
electrostatic interactions were treated using the particle mesh Ewald (PME) method implemented in
Amber, and a cutoff distance of 10 Å during all minimization steps.
The systems were then heated from 0 K to 300 K, keeping their volumes constant at 20 ps.
The solute was constrained with a force constant of 10 kcal/mol
Å2 and temperature was controlled
·
using a Langevin thermostat with a collision frequency of 1.0 ps−1 during heating. The PME cutoff
was set to 12 Å during this modeling stage. During all MD steps from this point onwards, the
bonds involving hydrogen atoms were constrained and their interactions omitted by means of the
SHAKE algorithm.
The heated systems were then equilibrated for 100 ps, at constant pressure (1 bar) and temperature
(300 K). For equilibration, temperature was controlled during heating, and pressure was controlled
with isotropic position scaling set at a relaxation time of 2 ps. The PME cutoff for equilibration was set
to 12 Å. The equilibrated systems were used as inputs for 20 production runs of 2 ns length each, using
the same parameters as those used during the equilibration of the systems. Atomic velocities were
randomly initialized before each production run.
Compound
9 free energy of binding to its potential receptors was estimated using MM-PBSA
calculations. These were performed with the MM-PBSA.py script provided with Amber 18 [84].
The MM-PBSA calculations were performed with 200 MD snapshots (one every 200 ps) selected from
20 production runs with the ionic strength set to 100 mM.
Supplementary Materials: Supplementary materials can be found at http://www.mdpi.com/1420-3049/25/14/3178/s1.
Author Contributions: Investigation and writing—original draft preparation, S.P.L.; methodology, Y.P.-C., L.M.Y.
and S.M.R.; data curation, S.M.R.; writing—review and editing and supervision, D.P.d.S. All authors have read
and agreed to the published version of the manuscript.
Funding: This research was funded by the Brazilian agencies Conselho Nacional de Desenvolvimento Cient
í
fico e
Tecnol
ó
gico (CNPq)—number of grant: 306661/2016-0, and Coordenaç
ã
o de Aperfeiçoamento de Pessoal de N vel
í
Superior (CAPES).
Conflicts of Interest: The authors declare no conflict of interest.
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