Investigation of 8-methoxy-3-(4-nitrobenzoyl)-6-propyl-2H-chromen-2-one as a promising coumarin compound for the development of a new and orally effective antileishmanial agent

Article abstract of DOI:10.1007/s11033-020-05887-5

Changes in host immunity and parasite resistance to drugs are among the factors that contribute to decreased efficacy of antiparasitic drugs such as the antimonial compounds pentamidine, amphotericin (AMP B) and miltefosine. Bioactive natural products cou

Full text of DOI:10.1007/s11033-020-05887-5

Molecular Biology Reports
https://doi.org/10.1007/s11033-020-05887-5
ORIGINAL ARTICLE
Investigation of 8‑methoxy‑3‑(4‑nitrobenzoyl)‑6‑propyl‑2H‑chrom
en‑2‑one as a promising coumarin compound for the development
of a new and orally eꢀective antileishmanial agent
1
1
2
3
Eduardo de Figueiredo Peloso · Rafaella Junqueira Merli · Patrícia Ferreira Espuri · Juliana Barbosa Nunes ·
4
5
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Fábio Antônio Colombo · Elkin José Torres Sierra · Daniela Carvalho de Paulo · Marcelo Henrique dos Santos ·
Diogo Teixeira Carvalho · Marcos José Marques
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Received: 23 June 2020 / Accepted: 30 September 2020
©
Springer Nature B.V. 2020
Abstract
Changes in host immunity and parasite resistance to drugs are among the factors that contribute to decreased eꢀcacy of
antiparasitic drugs such as the antimonial compounds pentamidine, amphotericin (AMP B) and miltefosine. Bioactive natural
products could be alternatives for the development of new drugs to treat neglected human diseases such as leishmaniasis.
Natural coumarins and synthetic analogues have shown leishmanicidal activity, mainly in vitro. This study investigated
the in vitro and in vivo leishmanicidal activity of synthetic coumarin compounds (C1–C5) in parasites Leishmania (L.)
amazonensis and L. (L.) infantum chagasi. The cytotoxicity of these compounds in mammalian cells and their inꢁuence on
production of reactive oxygen species was also investigated. In vitro assays showed that 8-methoxy-3-(4-nitrobenzoyl)-6-
propyl-2H-chromen-2-one (C4) was as active as AMP B mainly in the amastigote form (p<0.05); C4 presented a selectivity
index (65.43) four times higher than C2 (15.4) in L. amazonensis and six times higher (33.94) than C1 (5.46) in L. infantum
chagasi. Additionally, coumarin C4 reduced the H O concentration 32.5% more than the control group in L. amazonensis
2
2
promastigotes during the lag phase of proliferation. No interference of C4 was observed on the mitochondrial membrane
potential of the parasites. In vivo, coumarin C4 in corn oil (oral route) led to a reduction in the number of amastigotes from
6
4
L. infantum chagasi to 1.31×10 and 4.09×10 in the spleen and liver, respectively (p<0.05). Thus, C4 represents a can-
didate for further studies aiming at new treatments of leishmaniasis.
Keywords Coumarins · Nitro compounds · Leishmanicidal activity · Hydrogen peroxide production · Oral route
4
*
*
Eduardo de Figueiredo Peloso
eduardofpeloso@yahoo.com.br
Laboratory of Clinical Parasitology, Department of Clinical
and Toxicological Analysis, Faculty of Pharmaceutical
Sciences, Universidade Federal de Alfenas, Alfenas, MG,
Brazil
Marcos José Marques
marques.prppg@gmail.com
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Laboratory of Research in Pharmaceutical Chemistry,
Faculty of Pharmaceutical Sciences, Universidade Federal de
Alfenas, Alfenas, MG, Brazil
1
2
Department of Biochemistry, Institute of Biomedical
Sciences, Universidade Federal de Alfenas, 700 Gabriel
Monteiro da Silva St, Alfenas, MG CEP 37130-001, Brazil
Department of Chemistry, Universidade Federal de Viçosa,
Vicosa, MG CEP 36570-900, Brazil
Laboratory of Parasitology, Department of Pathology
and Parasitology, Institute of Biomedical Sciences,
Universidade Federal de Alfenas, 700 Gabriel Monteiro da
Silva St, Alfenas, MG CEP 37130-001, Brazil
3
Laboratory of Pathology of Infectious Diseases, Department
of Pathology, Medical School, São Paulo University,
Sao Paulo, SP CEP 01246-903, Brazil
Vol.:(0
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Molecular Biology Reports
Introduction
development of cutaneous lesions caused by L. amazon-
ensis. Prenylated coumarins auraptene, umbelliprenin and
galbanic acid isolated from Ferula szowitsiana (Apiaceae)
[17] have shown activity against L. major. In the New
World, the L. panamensis was inhibited by isopropenyl
coumarins from the extracts of Galipea panamensis leaves
[18]. In L. amazonensis, there are reports of leishmani-
cidal activity of the coumarin (-) mammea A/BB isolated
from C. brasiliense leaves [19]. Rosa et al. [10] showed
the in vitro leishmanicidal potential of two 4-phenylcou-
marins (HPC4 and HPC7, Fig. 1) against the promastig-
ote and amastigote forms of L. amazonensis; however, the
in vivo leishmanicidal activity of these coumarins was not
evaluated.
Leishmaniasis is a neglected tropical disease that still
requires a great deal of eꢂort from control and elimination
programmes [1]. Leishmaniasis is endemic to 98 countries
and aꢂects mainly the vulnerable groups from a social,
environmental and economic point of view, living in
Africa, Asia and Latin America [2]. Meanwhile, the clini-
cal presentations of the disease can be determined by the
parasite features and the host’s immune response [3]. The
disease outcome can show both visceral and cutaneous
forms [4]. Leishmaniasis is a parasitic disease transmitted
to the vertebrate host by an insect (phlebotomine sandꢁy)
that can inoculate the protozoan Leishmania spp.[5].
The ꢃrst line of drugs for the leishmaniasis treatment
have been the antimonial compounds, used for more than
In this work, it was described the evaluation of the cou-
marins HPC4 and HPC7 on parasite burden in an in vivo
model of leishmaniasis by L. (L.) infantum chagasi. In
addition, we report herein for the ꢃrst time the in vitro and
in vivo leishmanicidal activities of a synthetic nitro cou-
marin (C4, Fig. 1), previously described as an anti-Trypa-
nosoma cruzi agent [20]. We also investigate its inꢁuence on
the parasite membrane potential and production of reactive
oxygen species by the parasite cells.
6
0 years [6]. Pentamidine and AMP B are included in the
second line of this treatment [7]. Miltefosine has been used
as an oral drug for visceral leishmaniasis in some coun-
tries, such as India, Nepal and Bangladesh [8]. However,
many factors have contributed to decrease the eꢀcacy of
these drugs, such as changes in the host immunity and par-
asite resistance by diꢂerent mechanisms [9]. There are also
other diꢀculties inherent to chemotherapy of leishmania-
sis in relation to diagnosis, drug toxicity, patient compli-
ance to treatment, healing criterion and patient follow-up.
Natural products have been a source of underexplored
bioactive molecules that could be used as models in the
discovery and development of new drugs for the treatment
of neglected tropical diseases. The uses of natural com-
pounds for the treatment of various parasitic diseases such
as trypanosomiasis, leishmaniasis, schistosomiasis and
ꢃlariasis has encouraged the survey for novel molecules
Methods
Chemistry
Generalities
Reagents and solvents were obtained from Sigma-Aldrich
(Saint Louis, MO, USA) and were used without further
puriꢃcation. For the evaluation of purity and progress of
the reaction, it was used thin-layer chromatography (TLC)
on silica gel TLC plates (ALUGRAM® Xtra Sil G/UV254)
and detection by exposure to UV light at 254 nm. For the
determination of melting point values, a PFM-II melting-
point apparatus was employed (MS Tecnopon, Piracicaba,
Brazil) and the obtained results were not corrected. Infra-
red (IR) spectra were obtained on an FT-IR-Affinity−
[
10]. The classes of natural compounds such as alkaloids,
chalcones, lactones, saponins and coumarins have been
studied as antiparasitic agents [11–13]. Natural and syn-
thetic coumarins present a wide range of biological activ-
ity [14, 15]. Tiuman et al. [16] reported that the coumarin
obtained from Calophyllum brasiliense showed in vivo
leishmanicidal activity and could be used to control the
1
Fig. 1 Coumarins previously described as leishmanicidal ((HPC4 and HPC7) and trypanocide (C4)
1
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Molecular Biology Reports
equipment operating with an ATR dispositive (Shimadzu®,
Kyoto, Japan). For the obtaining of Nuclear Magnetic Reso-
nance (NMR) spectra it was used an AC-300 spectrometer
117.11, 118.67, 119.80, 127.28, 128.66, 129.70, 133.86,
136.35, 137.25, 143.14, 145.76, 147.19, 158.12, 191.89.
1
13
operating at 300 MHz for H-NMR and 75 MHz for C-
NMR spectra (Bruker®, Billerica, USA). TMS was used
as the internal standard in NMR analysis and the coupling
constants (J) were registered in Hertz. NMR signals were
assigned as: s (singlet), d (doublet), dd (doublet of doublets),
t (triplet), tt (triplet of triplets), q (quartet), sex (sextet) and
m (multiplet). ClogP values of synthesized compounds were
calculated using ChemDraw® Ultra 11.0.
3‑benzoyl‑8‑methoxy‑6‑propyl‑2H‑chromen‑2‑one
(C2) Light yellow solid; Yield 65%; CLogP: 4.36; M.p.:140–
–
1
141 °C; F.M.: C H O ; IR (ν /cm ) 3051 (aromatic
2
0
18
4
max
3
C–H), 2953 (sp C–H), 1716 (ester C=O), 1676 (ketone
1
C=O), 1579, 1466 (aromatic C=C); H NMR (DMSO-
d ) 0.97 (t, J
H14.H15
=7.35, 3H), 1.68 (sex, J
=7.58,
6
H15.H14
=7.35, 2H), 2.65 (t, J
H14.H13
J
=7.58, 2H), 3.98 (s,
H13.H14
3H), 6.95 (s, 1H), 7.00 (s, 1H), 7.44–7.49 (m, 2H), 7.60 (tt,
J
=7.39, J =2.65, 1H), 7.85–7.89 (m, 2H), 8.02
H4′.H3′
H4′.H2′
1
3
(
s, 1H); C NMR (DMSO-d ) 13.68, 24.46, 37.70, 56.36,
6
General procedure for the synthesis of the coumarins C1‑C5
1
1
16.06, 118.50, 119.54, 127.16, 128.56, 129.62, 133.74,
36.31, 139.76, 142.85, 145.82, 146.98, 191.92.
The respective aromatic aldehyde (1 eq), a beta-ketoester
(
1 eq) and piperidine as a catalyst (3 drops) were added to
3
‑benzoyl‑8‑methoxy‑2H‑cromen‑2‑one (C3) Light Orange
a 50-mL round bottom ꢁask containing ethanol (15 mL).
This reaction mixture was stirred under heating at 85 °C
with a reꢁux apparatus for two hours (Fig. 2). The progress
of the reaction was checked by TLC (hexane:ethyl acetate,
solid; Yield 80%; CLogP: 2.84; M.p.: 130–131 °C; F.M.:
–
1
3
C H O ; IR (ν /cm ) 3052 (aromatic C–H), 3008 (sp
1
7
12
4
max
C–H), 1712 (ester C=O), 1656 (ketone C=O), 1572;
1
1
7
J
471 (aromatic C=C); H NMR (CDCl ) 3.94 (s, 3H),
3
7
:3, v/v). The mixture was cooled to 25 °C; the solid product
.10–7.15 (m, 2H), 7.20–7.23 (m, 1H), 7.42 (t, J
7.36;
H3′,H2′
was collected by vacuum ꢃltration, washed throughout with
cold ethanol and dried in a desiccator. All products were
suꢀciently pure for further evaluations.
=7.66, 2H), 7.56 (t, J
=7.66, 1H), 7.83 (d,
H3′,H4′
H4′,H3′
1
3
JH2′,H3′ =7.36, 2H), 8.00 (s, 1H); C NMR (CDCl ) 56.47,
3
1
1
15.38, 118.90, 120.49, 124.96, 127.37, 128.68, 129.71,
33.91, 136.29, 144.56, 145.70, 147.35, 158.01, 191.82.
Characterization data
8
‑methoxy‑3‑(4‑nitrobenzoyl)‑6‑propyl‑2H‑chromen‑2‑one
6
‑allyl‑3‑benzoyl‑8‑methox y‑2H‑ chromen‑2‑ one
(C4) Light yellowish solid; Yield 91%; CLogP: 4.32; M.p.:
–
1
(
C1) White solid; Yield 83%; CLogP: 3.86; M.p.: 159–160
143–144 °C; F.M.: C H NO ; IR (ν /cm ) 3069 (aro-
20 17 6 max
–
1
2
°
2
1
C; F.M.: C H O ; IR (ν /cm ) 3048 (aromatic C–H),
matic C–H), 3049 (sp C–H), 1582 (aromatic C=C), 1705
(ester C=O), 1682 (ketone C=O), 1513 (NO ), 1347 (NO );
2
0
16
4
max
3
962 (sp C–H), 1713 (ester C=O), 1659 (ketone C=O),
2
2
1
1
578; 1476 (aromatic C=C); H NMR (CDCl ) 3.43 (d, J
H NMR (DMSO-d ) 0.98 (t, J
=7.37, 3H), 1.69 (sex,
3
6
H15.H14
,
=6.63, 2H), 3.96 (s, 3H), 5.09–5.16 (m, 2H), 5.89–
J
=7.5; J
=7.37, 2H), 2.67 (t, JH13,H14 =7.5,
H13 H14
H14,H13
H14.H15
6
7
2
.02 (m, 1H), 6.96 (s, 1H), 7.00 (s, 1H), 7.42–7.47 (m, 2H),
2H), 3.99 (s, 3H), 7.01 (d, J
=1.71, 1H), 7.05 (d,
H7,H5
.59 (tt, J =7.41, J =2.61, 1H), 7.84–7.87 (m,
J =1.71, 1H), 7.99–7.95 (m, 2H), 8.23 (s, 1H), 8.28–
H4′,H3′
H4′,H2′
H5,H7
1
3
13
H), 8.00 (s, 1H); C NMR (CDCl ) 39.82, 56.46, 116.14,
8.33 (m, 2H); C NMR (DMSO-d ) 13.67, 24.43, 37.8,
3
6
Fig. 2 General scheme for the synthesis of coumarins C1-C5
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Molecular Biology Reports
5
1
1
6.40, 116.86, 118.40, 119.91, 123.68, 125.58, 130.18,
40.20, 141.52 (2C), 143.24, 147.05, 148.12, 150.39,
90.74.
using Prism software (version 5.0; GraphPad Software, Inc.,
La Jolla, California, USA), by nonlinear regression to obtain
the values of IC and CC50, followed by variance analyses
5
0
and Tukey’s test. Diꢂerences of IC among standard drug
5
0
8
‑methoxy‑3‑(4‑nitrobenzoyl)‑2H‑chromen‑2‑one
and coumarin derivatives were considered signiꢃcant when
(
C5): Light greenish solid; Yield: 89%; CLogP: 2.80; M.p.:
the p-value was less than 0.05.
–
1
2
2
60–261 °C; F.M.: C H NO ; IR (ν /cm ) 3105 (sp
17 11 6 max
C–H), 3075 (aromatic C–H), 1574 (aromatic C=C), 1698
Determination of hydrogen peroxide release
and mitochondrial membrane potential (ΔΨ)
(
ester C=O), 1660 (ketone C=O), 1514 (NO ), 1345 (NO );
2
2
1
H NMR (DMSO-d ) 3.95 (s, 3H), 7.37 (t, J
7.65;
6
H6,H5=
8
J
7.92, 1H), 7.43–7.48 (mm, 2H), 8.15 (d, J
9.00,
Promastigotes (10 cells/mL) of L. (L.) amazonensis at lag
H6,H7=
H3′,H2′=
1
3
2
H), 8.33 (d, J
9.00, 2H), 8.56 (s, 1H); C NMR
(2 days) and log (4 days) phases of the proliferation curve
were treated or not (control). The coumarin derivative was
selected among C1–C5 (according to in vitro activity); they
were incubated in reaction buꢂer 1X from Amplex Red®
kit in the presence of 40 μM digitonin, 5 mM succinate,
0.1 U/mL horseradish peroxidase and 25 μM Amplex Red
H2′,H3′=
(
DMSO-d ) 11.31, 56.28, 118.81, 121.20, 123.70, 124.97,
6
1
1
25.45, 130.64, 141.41, 143.86, 146.48, 147.65, 150.09,
57.81, 190.95.
Biological assays
(
Molecular Probes®). The ꢁuorescence was monitored at the
Leishmanicidal activity against promastigotes
and amastigotes; cytotoxicity assay
emission and excitation wavelengths of 585 nm and 571 nm,
respectively, in a 96-well plate using a Varian Cary Eclipse
Fluorescence Spectrophotometer. The quantitative correla-
tion between the ꢁuorescence and the H O released by the
Promastigotes of L. (L.) amazonensis (strain MHOM/
BR/71973/M2269) and L. (L.) infantum chagasi (strain
MHOM/BR/1972/BH46) assays were performed using LIT
medium with foetal bovine serum (10%; Gibco®, USA)
and antibiotics. The compounds C1–C5 were dissolved in
dimethyl sulfoxide (DMSO 0.6%, v/v in all wells), added
2
2
cells was determined as previously described [22]. The ΔΨ
was performed as previously described [23]. Brieꢁy, L. ama-
7
zonensis promastigotes (1×10 cells/mL) in the lag phase
of the proliferation curve were treated or not (control) with
30 μM of a coumarin derivative (selected among C1–C5,
according to their in vitro activity) for 48 h. After incuba-
tion, the cells were washed and resuspended in phosphate
buꢂer (pH 7.4); cells were incubated for thirty minutes with
a JC-10 probe at a concentration of 10 mg/mL at 25 ºC.
After this period, the promastigotes were washed twice and
resuspended in phosphate buꢂer (pH 7.4); ΔΨ was deter-
mined at emission wavelengths of 530 nm and 590 nm and
excitation wavelength of 480 nm using a spectroꢁuorimeter
(Spectrophotometer of Varian Cary Eclipse Fluorescence®).
As a positive control of uncoupling, the parasites were
incubated with 20 μM CCCP (Carbonyl cyanide m-chlo-
rophenyl hydrazone). The data obtained correspond to the
mean ± standard deviation of at least three independent
experiments carried out in triplicate. The comparison was
determined using Student’s t-test with Origin 6.0 software,
and p<0.05 was considered statistically signiꢃcant.
6
to promastigote cultures (1×10 cells/mL) in the range of
0
.10–40.00 µg/mL, and incubated at 25 °C. The tests were
performed in triplicate and amphotericin B (AMP B; Sigma)
was used as the reference drug. The maintenance of murine
peritoneal macrophages was in RPMI 1640 medium (Sigma,
USA) with 10.0% heat-inactivated foetal bovine serum at
3
7 °C in a 5.0% CO incubator. The cells were infected with
2
late log-phase promastigotes at a 10:1 parasite:macrophage
ratio and incubated at the same conditions. The compounds
C1–C5 (from 0.10 to 40.00 µg/mL in DMSO) were added
after the removal of non-phagocytosed promastigotes. The
calculation of inhibition ratio (IC value) was did in com-
5
0
parison to DMSO alone. All assays were performed in trip-
licate on independent occasions using AMP B (Sigma) as
5
the reference drugs. For the cytotoxicity evaluation, 8×10
murine peritoneal macrophages in RPMI 1640 suspen-
sion with 10.0% heat-inactivated foetal bovine serum and
antibiotics were added to each well in 96-well plates. The
compounds C1–C5 and reference drug ranging from 3.91 to
In vivo leishmanicidal assay
5
00.00 µg/mL in DMSO were added to the wells contain-
Parasites and animals
ing the cells. The leishmanicidal activity was evaluated by
the MTT method [21]. The best coumarin derivative for the
in vitro leishmanicidal tests was selected to carry out the
inhibition of reactive oxygen species production and in vivo
leishmanicidal assays. The complete protocols were previ-
ously described [21]. All statistical analysis was performed
All experimental procedures involving animals were
approved by the Research Ethics Commission of the Fed-
eral University of Alfenas (CEUA/UNIFAL-MG 566/2014)
and were performed according to the Guide for the Care
and Use of Laboratory Animals. Leishmania (L.) infantum
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Molecular Biology Reports
chagasi (strain MHOM/BR/1972/BH46) promastigotes
were maintained in M-199 medium and amastigotes were
maintained by passaging in golden hamsters (Mesocricetus
auratus). The organs (spleens and liver) of infected animals
were removed and macerated using a tissue grinder; and the
number of amastigotes was determined as described previ-
ously [24].
Mann–Whitney (unpaired, two-tailed) for the signiꢃcance
test (p<0.05), as described elsewhere [27].
Results
Chemistry
In vivo testing of experimental compounds
against Leishmania
The reactions took an average of 2 h to complete, and their
equilibrium was favoured for the product formation since
they are insoluble in ethanol. This also helped in the reaction
workup by ꢃltration and there was no need for additional
treatments. Coumarin formation was certiꢃed by the analy-
For the in vivo assay were used female golden hamsters that
had been recently weaned (120 g) and were infected intra-
7
1
13
peritoneally with 1×10 amastigotes of L. (L.) infantum cha-
sis of their IR, H and C NMR spectra and comparison to
previously reported data, with which they had total agree-
ment. The main ꢃndings in IR spectra were the two carbonyl
stretching bands of ketone and lactone groups typical for
all these coumarins. In coumarins C4–C5, the asymmetric
and symmetric stretching bands were observed for the nitro
group.
gasi (MHOM/BR/1972/BH46). After 50 days of infection
the animals were divided into 5 groups (5 per group) and
subjected for 10 consecutive days to one of the following
treatments: 0.5% of carboxymethyl cellulose (CMC) suspen-
sion, administered orally (untreated, or UTG group); 50 mg/
kg/day of Glucantime (GLU), by intraperitoneal injec-
tion (GLU group); 20 mg/kg/day of compounds HPC4,
HPC7 and the best coumarin derivative selected among
C1–C5 (according to their in vitro activity), administered
orally as suspensions in 0.5% of CMC. In addition, a choice
coumarin derivative (according logP and in vitro activity)
was assayed via the oral route with corn oil in a ꢃnal solu-
tion undiluted of 137.0 μL each animal/day, using 20 mg/
kg/day. After 10 days of treatment, animals were sacriꢃced
1
By the analysis of H-NMR spectra, a singlet was
observed in the range 8.56–8.0 ppm attributed to H-4, which
is shared by all coumarins. Ketone and lactone carbonyl
signals were observed near 190 and 158 ppm, respectively.
Coumarins C4 and C5 exhibited the expected pattern for
para-substituted phenyl rings, and a deshielding eꢂect was
evident on the hydrogens ortho to the nitro group. TLC
and NMR were used to infer about the purities of products,
which were suꢀciently pure for the biological evaluations.
in a CO chamber, and a sample of the spleen and the liver
2
(
approximately 50 mg) was removed, weighed and used for
total RNA extraction, as previously described [25]. Were
used standard curves assays by quantitative real-time PCR
In vitro leishmanicidal activity
(
qPCR) of parasite DNA as described previously [26]. A
The data obtained in the in vitro leishmanicidal assays
are shown in Table 1. The coumarin C4 showed a similar
leishmanicidal activity to AMP B against promastigote and
amastigote forms of L. amazonensis with IC values of 12.0
parasite load estimation by qPCR was performed using the
TaqMan® probe double-labelled with FAM and the primers
LinJ31. From the linear regression data from the standard
curve performed with promastigote DNA were calculated
the number of parasites per gram of organs tissues and the
statistical analysis was performed by Student’s t-test with
50
and 3.53 μM, respectively (p<0.05). In L. infantum chagasi
promastigotes, the IC for AMP B was the lowest, but most
5
0
compounds had IC values less than 27.0 μM.
5
0
Table 1 Biological activity of
Compound Promastigote
Amastigote
Cytotoxicity
SI
coumarin derivatives against
IC μM (SD)
IC μM (SD)
IC μM (SD)
5
0
50
50
both forms of Leishmania
amazonensis (L. am.) and L.
infantum chagasi (L. inf.),
compared to amphotericin
L. am
L. inf
L. am
L. inf
L. am L. inf
C1
438.59 (132.6) 24.3 (1.55) >125.00
55.5 (0.62)
>164.11
>124.1
303.2 (1.09)*
272.5 (3.25)*
334.4 (5.8)
231.0 (2.3)
300.5 (1.74)*
–
5.46
–
–
(
AMP B)
C2
82.0 (20.3)
122.7 (12.4)
12.0 (3.2)*
322.2 (91.8)
5.1 (0.36)*
26.7 (0.31) 17.7 (3.2)
18.73 (0.9) 43.8 (3.1)
15.4
C3
7.63
C4
18.0 (3.8)
63.5 (44.1) >123.00
3.5 (0.02)
3.53 (0.7)* 6.8 (1.08)*
65.43 33.97
C5
>123.00
–
–
AMP B
1.3 (0.11)* 4.02 (0.60)* 270.5 (11.25)* 208.1 67.3
SI selectivity index
IC50 values statistically similar when compared to Amphotericin (AMP B)
*
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Molecular Biology Reports
The coumarin C4 had an activity against L. infantum
chagasi amastigote similar to AMP B. The coumarin nitro
compound C4 was as active as AMP B, mainly in amastigote
forms (p<0.05), and presented a selectivity index (65.43)
four times higher than C2 (15.4) in L. amazonensis and six
times higher (33.94) than C1 (5.46) in L. infantum chagasi.
Determination of hydrogen peroxide (H O ) release
2
2
The inꢁuence of the coumarin C4 (choice compound) on the
production of H O in L. amazonensis promastigotes in the
2
2
lag and log phases of the proliferation curve was evaluated.
Our results demonstrated under our experimental conditions
that treatment with the coumarin C4 reduced the concen-
tration of H O by approximately 32.5% compared to the
Fig. 4 Evaluation of the mitochondrial membrane potential (ΔΨ).
2
2
7
Leishmania amazonensis promastigotes (1×10 cells/mL) in the
control group (untreated) in the lag phase (Fig. 3).
lag phase of the proliferation curve were treated or not (Ctrl) with
the coumarin C4. The values are expressed as the ratio of the ꢁuo-
rescence measurements at 530 nm (for J-monomer) versus 590 nm
When analysing the eꢂect of this treatment on log-phase
promastigotes, there was no signiꢃcant diꢂerence between
the H O concentration in parasites treated and those not
(
J-aggregate). The experiments were carried out in triplicate on inde-
2
2
pendent occasions. *No statistical diꢂerence
treated (Fig. 3). We observed that treatment with the cou-
marin C4 did not inꢁuence ΔΨ, because there was no sig-
niꢃcant diꢂerence between treated parasites and the control
infection (clinical symptoms such as ascites and alopecia)
with L. (L.) infantum chagasi.
(
Fig. 4).
Treatment with compound HPC4 did not decrease the
number of amastigotes in the spleen and liver of infected
In vivo leishmanicidal activity evaluation
7
7
hamsters (1.67 × 10 and 1.25 × 10 ) when compared to
The coumarin C4, HPC4 and HPC7 were chosen to
UTG. However, the treatment with compounds C4 and
6
6
the in vivo assay. In the untreated group (UTG; vehicle-
HPC7 showed a parasite burden of 5.3×10 and 7.16×10
8
6
7
treated), the average number of parasites was 1.53×10 and
to spleen and 2.23×10 and 1.23×10 to the liver, respec-
tively, both in carboxymethyl cellulose (C4 CMC) by the
oral route; these values are comparable with UTG results.
Glucantime (50 mg/kg/day, intraperitoneal route) and C4
Corn Oil (20 mg/kg/day, oral route) were the most eꢂec-
tive compounds at reducing the parasite burden in infected
animals, since treatment with these drugs led to a reduction
6
3
.47×10 per gram of tissue in the spleen and liver, respec-
tively, conꢃrming that these animals had an established
4
6
in the number of amastigotes to 1.85× 10 and 1.31 × 10
in the spleen, respectively; the drugs reduced amastigotes
3
4
to 6.02×10 and 4.09×10 in the liver, respectively, when
compared to UTG (Fig. 5; p<0.05).
Discussion
The coumarins C1–C5 have already been synthesized and
described elsewhere for other biological purposes [20, 28].
In this work, all coumarin derivatives were obtained in yields
mostly above 80% by a Knoevenagel condensation meth-
odology as described by Vazquez-Rodriguez et al. [29] for
other coumarins. In the biological tests the compounds C2,
C3 and C4 were more potent against the amastigote form of
L. amazonensis (IC was 17.7, 43.8 and 3.53, respectively)
Fig. 3 Determination of H O release in Leishmania (L.) amazon-
2
2
5
0
ensis promastigotes at lag and log phases of the proliferation curve
treated or not (control) with the coumarin C4. *Indicates statistical
diꢂerence
compared to their action against the promastigote form (IC
50
was 82.0, 122.7 and 12.0, respectively); this diꢂerence in
1
3

Molecular Biology Reports
The metabolism of diꢂerent unicellular or multicellular
organisms is dependent on the synthesis of energetic mol-
ecules such as ATP. Speciꢃcally, protozoan Leishmania
has ATP synthesis, mainly in their single mitochondrion
[
32], which, in addition to this function, are involved in
other processes such as calcium homeostasis and apoptosis
33]. At the same time, the mitochondrion is the main site
[
for the generation of reactive oxygen species (ROS) [33],
molecules that must be kept in adequate concentrations
to prevent the oxidation of cellular components such as
DNA, lipids, proteins and carbohydrates due to oxidative
stress. This peculiarity of trypanosomatids in having only
one mitochondrion makes this organelle an interesting
therapeutic target since mitochondrial integrity must be
maintained so that trypanosomatids, such as Leishmania,
can survive.
It has also been reported that coumarins have a wide
range of biological activities, including antioxidant [34].
Feriani et al. [35] reported that the coumarin-derived
compound (E)-N′-(1-(7-hydroxy-2-oxo-2H-chromen-3-yl)
ethylidene)-benzohydrazide is a scavenger of free radicals,
which led to a reduction in oxidative stress, inꢁammation
and apoptosis. Thus, it was possible to notice the inꢁuence
of the coumarin C4 on the production of H O in L. ama-
Fig. 5 Quantiꢃcation of parasite burden in the spleen and liver from
hamsters infected with Leishmania (L.) infantum chagasi, as quanti-
ꢃed by quantitative reverse transcription PCR (qRT-PCR) for the
detection of the Linj31 marker. The numbers of parasites (amastig-
otes) per gram of tissue were estimated based on a qPCR standard
curve using promastigotes. UTG untreated group (vehicle-treated),
GLU animals treated with Glucantime (50 mg/kg/day), coumarin
derivatives HPC4, C4 CMC, C4 Corn Oil and HPC7 (20 mg/kg/
day). Data are represented as group mean and individual values for
each animal. Statistical diꢂerence *p<0.05; **p<0.01; ***p<0.001
2
2
zonensis promastigotes. This result corroborates with the
role of coumarin derivatives as a scavenger of free radicals
[
35], thereby reducing the concentration of oxidising agents
intracellular activity may be derived from the formation of
a more active metabolite in the infected cell [21].
such as H O .
2 2
On the other hand, it was not possible to note the same
eꢂect on log-phase promastigotes. This could be related to
the diꢂerential expression of antioxidant enzymes from tryp-
anosomatids along the proliferation curve, as in Trypano-
soma cruzi [22]. In other words, less activity of the cytosolic
and mitochondrial triparedoxins peroxidases of Leishmania
in the log phase would allow a higher concentration of H O ,
Only the coumarin C4 showed signiꢃcant activity against
the promastigote and amastigote forms of L. amazonensis
and L. infantum chagasi. This coumarin derivative is the
only one tested that bears nitro and propyl groups in its
structure. Brancaglion et al. [20] evaluated the coumarin
C4 and congeners against Trypanosoma cruzi and suggested
that the presence of a propylic side chain on the coumarin
nucleus next to a nitro group positively aꢂects the trypano-
cidal activity. Hydrophobic interactions between this ꢁexible
propyl side chain and a molecular target from trypasonoso-
matid could be essential for the trypanocidal activity. In their
review, Patterson and Wyllie [30] highlighted a repurpos-
ing of nitroaromatic drugs, noting the requirements for new
drugs for trypanosomatid diseases.
2
2
so that when incubating this protozoan with an ROS scaven-
ger compound, the reduction in the concentration of H O
2
2
would not be detectable. However, since the H O concentra-
2
2
tion is the same in the lag and log phase control groups, this
hypothesis could not support this result.
The biological role of H O in trypanosomatids has been
2
2
widely explored. In this family of protozoa, through the
activities of antioxidant enzymes, the intracellular envi-
ronment is maintained in a reduced form, balancing, for
example, the concentration of reactive species such as H O
In this sense, the approach would be through reducing
the toxicity of nitro compounds. However, the cytotoxicity
of the nitro coumarin C4 was very low in our evaluation.
According to Lehnhardt Pires et al. [31], a selectivity index
higher than 10 is required to ensure safety for a drug. In the
present work, the coumarin nitro compound C4 showed the
best SI, which value indicated the high selective toxicity for
the parasite. Rosa et al. [10] also reported that 7-hydroxy-
2
2
[36]. At the same time, Peloso et al. [22] demonstrated that
the increase in H O production signals an increase in the
2
2
expression of cytosolic and mitochondrial peroxidases with
diꢂerential expression of these antioxidant enzymes along
the proliferation curve. Additionally, it has been reported
that a low concentration of H O stimulates cell prolifera-
2
2
4
-phenylcoumarin derivatives did not display toxicity in
tion [37]. Finzi et al. [37] demonstrated that 20 μM of H O
2 2
mammalian cells.
stimulated cell proliferation in T. cruzi while increasing
1
3

Molecular Biology Reports
the resistance of this parasite to sublethal doses of H O
platform. Besides a higher dosage, Glucantime was admin-
istered intraperitoneally, while the assayed compounds
HPC4, HPC7, C4 CMC and C4 Corn Oil were administered
orally. Theoretically, soluble and neutral compounds are
well absorbed after oral treatment. The absorbed dose may
therefore be diꢂerent from the administered dose because
of the nature of the compounds, which can lead to diꢂerent
absorption rates being faster for compounds in solution than
for those in suspension.
2
2
(
100 μM).
Another hypothesis for the reduction in H O concen-
2
2
tration observed in the lag phase of the proliferation curve
when the parasites were treated with the coumarin C4 would
be the diꢂerence in biochemical aspects between the lag,
log and stationary phases of the parasite. Along the prolif-
eration curve, there is a diꢂerence in the synthesis of vari-
ous compounds, such as polyamines, which are essential
for trypanosomatid metabolism [38]. Proteome studies,
such as the one reported by Alcolea et al. [39], have also
showed a high number of proteins with varied functions, dif-
ferentially expressed between the phases of the proliferation
curve. Thus, these biochemical aspects could exert a strong
inꢁuence on the activity of the coumarin C4 due to several
possibilities of chemical interaction, thus facilitating its role
as an antioxidant in the lag phase, and, consequently, reduce
the concentration of H O .
The level of hydrophobicity can indicate the choice of
the suitable vehicle for administering the test compounds
[47]. The coumarin C4 has a logP around 4.32 which makes
it suitable to be administered as a suspension using CM/
CMC or as a solution in corn oil. However, vehicles such as
CMC can retard the passive diꢂusion of the drug and, con-
sequently, the absorption from the gastrointestinal tract, but
the administration with corn oil can improve this absorption
according to the chemical nature of the compounds [48]. The
diꢂerence observed in the in vivo results with C4 CMC and
C4 Corn Oil can be explained by the distinct formulations
used our study and can represent an alternative to overcome
the diꢀculties in the oral administration of compounds from
natural origin [49].
2
2
Additionally, the production of ROS such as H O may
2
2
directly inꢁuence mild dysfunctions in the mitochondrial
membrane potential (ΔΨ) [40]. The maintenance of ΔΨ
is essential for the functionality of mitochondria since it is
directly involved with oxidative phosphorylation and ATP
synthesis, control of ROS production levels and intracel-
Solubility issues of coumarins were observed in other
studies considering an in vivo model of cutaneous leishma-
niasis [45, 50]. Kermani et al. [50] assayed osthole, a pre-
nylated coumarin extracted from Prangos asperula Boiss,
against L. major. These authors reported that osthole could
decline the progression of lesions compared to untreated
mice, and its eꢂect was restricted because it is a poorly solu-
ble coumarin. In another study, Mandlik et al. [45] devel-
oped a nanoliposomal formulation to circumvent the prob-
lem with an insoluble coumarin derivative. They observed a
healing eꢂect in the footpads of BALB/c mice infected with
L. major when treated with this nano formulated compound.
This approach is under investigation in our group to enhance
the in vivo activity of the presented coumarins.
+
2
lular Ca homeostasis [41]. Mitochondrial changes such
as swelling, alteration of ΔΨ and release of cytochrome c,
represent the main signs of the apoptosis-like processes in
Leishmania [42]. Additionally, the ΔΨ reduction is char-
acteristic of a cell undergoing programmed cell death [43].
In promastigotes from Leishmania, both hyperpolarisation
and depolarisation can result in cell death from apoptosis
[
44]. Mandlik et al. [45] reported that the coumarin derivate
C2 induces ΔΨ reduction in L. major promastigotes. In this
sense, we evaluated ΔΨ in parasites in the lag phase, treated
with the coumarin C4, to clarify whether the reduction in
H O production was the result of a mitochondrial alteration
2
2
caused by this compound. However, the coumarin C4 does
not alter the energetic functionality of the mitochondrion
since ΔΨ remained unchanged.
Colombo et al. [46] described a quantitative reverse tran-
scription PCR (qRT-PCR) assay that can be used for sensi-
tive and accurate quantiꢃcation of parasites in infected tis-
sues. For this purpose, we used the detection of the LinJ31
marker (Linj31-qPCR) for the quantiꢃcation of live amas-
tigotes of L. (L.) infantum chagasi in the spleen and liver of
infected hamsters after experimental treatment using benzo-
phenone derivatives.
Conclusion
In our study, coumarin derivatives showed leishmanicidal
activity in vitro and in vivo, especially C4 when compared to
reference drugs. The coumarin C4 could reduce the concen-
tration of H O produced in L. amazonensis promastigotes
2
2
and the parasite burden in the liver and spleen of infected
animals with L. infantum chagasi when given orally in a
corn oil solution. These results suggest that the presence
of a propylic side chain on the coumarin nucleus, as seen
in coumarin C4, can improve the leishmanicidal activity
probably by inꢁuencing solubility issues and hydrophobic
interactions with molecular targets from trypasonosomatid.
Probably, this feature enables the access to the macrophage
In this way, the compound C4 was chosen to assay using
corn oil as a vehicle because it showed the best in vitro activ-
ity, particularly against the L. amazonensis amastigote form.
In addition, coumarin C4 has a theoretical logP value that
is suitable for oral route evaluations; it has a good gastroin-
testinal absorption proꢃle, as predicted by the SwissADME
1
3

Molecular Biology Reports
and to the intracellular parasite. Thus, this series of cou-
marin derivatives, and especially the coumarin C4, could
be explored as candidates for the development of new drug
prototypes against leishmaniasis.
13. Ulloa JL, Spina R, Casasco A et al (2017) Germacranolide-
type sesquiterpene lactones from Smallanthus sonchifolius with
promising activity against Leishmania mexicana and Trypano-
soma cruzi. Parasites Vectors 10:567. https://doi.org/10.1186/
s13071-017-2509-6
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vanillin ethers from 4-(bromomethyl) coumarins as anti-
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org/10.1016/S0223-5234(03)00016-3
Funding This work was supported by PPM-00482-16 from FAPEMIG
(
Fundação de Amparo à Pesquisa do Estado de Minas Gerais), CAPES
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior),
5. Kontogiorgis C, Nicolotti O, Mangiatordi GF et al (2015)
Studies on the antiplatelet and antithrombotic proꢃle of anti-
inꢁammatory coumarin derivatives. J Enzyme Inhib Med Chem
(
INCT-INOFAR from CNPq (Conselho Nacional de Desenvolvimento
Cientíꢀco e Tecnológico) and FINEP (Financiadora de Estudos e Pro-
jetos), Núcleo de Estudos, Pesquisa e Assessoria à Saúde.
3
0:925–933. https://doi.org/10.3109/14756366.2014.995180
6. Tiuman TS, Brenzan MA, Ueda-Nakamura T et al (2012) Intra-
muscular and topical treatment of cutaneous leishmaniasis
lesions in mice infected with Leishmania amazonensis using
coumarin (-) mammea A/BB. Phytomedicine 19:1196–1199.
https://doi.org/10.1016/j.phymed.2012.08.001
Compliance with ethical standards
Conflict of interest The authors declare that they have no conꢁict of
interest.
17. Iranshahi M, Arfa P, Ramezani M et al (2007) Sesquiterpene
coumarins from Ferula szowitsiana and in vitro antileishma-
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