In Vitro and in Vivo Antischistosomal Activities of Chalcones

Article abstract of DOI:10.1002/cbdv.201800398

In this study, we evaluated the in vitro and in vivo schistosomicidal activities of chalcones against Schistosoma mansoni worms. In vitro assays revealed that chalcones 1 and 3 were the most active compounds, without affecting significantly mammalian cells. Confocal laser scanning microscopy and scanning electron microscopy studies revealed reduction on the numbers of tubercles and morphological alterations in the tegument of S. mansoni worms after in vitro incubation with chalcones 1 and 3. In a mouse model of schistosomiasis, the oral treatment (400 mg/kg) with chalcone 1 or 3 significantly caused a total worm burden reduction in mice. Chalcone 1 showed significant inhibition of the S. mansoni ATP diphosphohydrolase activity, which was corroborated by molecular docking studies. The results suggested that chalcones could be explored as lead compounds with antischistosomal properties.

Full text of DOI:10.1002/cbdv.201800398

Title: In vitro and in vivo antischistosomal activities of chalcones
Authors: Vinícius R. D. Pereira, Ismael J. Alves Junior, Lígia S. da
Silveira, Reinaldo B. Geraldo, Priscila de F. Pinto, Fernanda
S. Teixeira, Maria C. Salvadori, Marcos P. Silva, Lara A.
Alves, Priscila V. S. Z. Capriles, Ayla das C. Almeida, Elaine
S. Coimbra, Pedro L.S. Pinto, Mara R. C. Couri, Josué de
Moraes, and Ademar Alves Da Silva Filho
This manuscript has been accepted after peer review and appears as an
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to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
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To be cited as: Chem. Biodiversity 10.1002/cbdv.201800398
Link to VoR: http://dx.doi.org/10.1002/cbdv.201800398

10.1002/cbdv.201800398
Chemistry & Biodiversity
Chem. Biodiversity
In vitro and in vivo antischistosomal activities of chalcones
Vinícius R. D. Pereiraa, Ismael J. Alves Juniora, Lígia S. da Silveirab, Reinaldo B. Geraldoa, Priscila de
F. Pintoc, Fernanda S. Teixeirad, Maria C. Salvadorid, Marcos P. Silvae, Lara A. Alvesf, Priscila V. S. Z.
Caprilesf, Ayla das C. Almeidag, Elaine S. Coimbrag, Pedro L.S. Pintoh, Mara R. C. Courib, Josué de
Moraese, Ademar A. Da Silva Filho a,*
aFaculdade de Farmácia, Departamento de Ciências Farmacêuticas, Universidade Federal de Juiz de Fora, R. José Lourenço Kelmer s/n, Campus
Universitário, 36036-900 Juiz de Fora, MG, Brazil, e-mail: ademar.alves@ufjf.edu.br.
bDepartamento de Química, Universidade Federal de Juiz de Fora, Juiz de Fora, MG, Brazil.
cDepartamento de Bioquímica, Instituto de Ciências Biológicas, Universidade Federal de Juiz de Fora, Juiz de Fora, MG, Brazil.
dInstituto de Física, Universidade de São Paulo, São Paulo, SP, Brazil.
eNúcleo de Pesquisa em Doenças Negligenciadas, Universidade Guarulhos, Guarulhos, SP, Brazil.
fPrograma de Pós-graduação em Modelagem Computacional, Departamento de Ciência da Computação, ICE, Universidade Federal de Juiz de
Fora, Juiz de Fora, MG, Brazil.
gDepartamento de Parasitologia, Microbiologia e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Juiz de Fora, Juiz de
Fora, MG, Brazil.
hNúcleo de Enteroparasitas, Instituto Adolfo Lutz, São Paulo, SP, Brazil.
In this study, we evaluated the in vitro and in vivo schistosomicidal activities of chalcones against Schistosoma mansoni worms. In vitro
assays revealed that chalcones 1 and 3 were the most active compounds, without affecting significantly mammalian cells. Confocal laser
scanning microscopy and scanning electron microscopy studies revealed reduction on the numbers of tubercles and morphological alterations
in the tegument of S. mansoni worms after in vitro incubation with chalcones 1 and 3. In a mouse model of schistosomiasis, the oral treatment
(400 mg/kg) with chalcone 1 or 3 significantly caused a total worm burden reduction in mice. Chalcone 1 showed significant inhibition of the
S. mansoni ATP diphosphohydrolase activity, which was corroborated by molecular docking studies. Results suggested that chalcones could
be explored as lead compounds with antischistosomal properties.
Keywords: Schistosomiasis • Chalcones • Natural products • Schistosoma mansoni ATP diphosphohydrolase
Introduction
Schistosomiasis is a neglected tropical disease (NTD) caused by trematode flukes of the genus Schistosoma, affecting over 200 million people
worldwide, mainly poor and marginal populations in Asia, Africa, and Latin America, causing about 300,000 deaths annually.[1-3] According
to the World Health Organization, there are more than seven hundred million people at risk of infection [1] and, only in Brazil, there are around
7 million people infected.[4-5] In addition, schistosomiasis usually leads to chronic disabilities and morbidities, which involve decreased quality
of life.[1-3] In contrast, there are no availability of vaccines to schistosomiasis and its chemotherapy is dependent on praziquantel (PZQ), which
does not act on immature worms and is the only drug in the market.[3, 4] In addition, the reliance on a single schistosomicidal drug, as well as
the emergence of worm strains less sensitive to PZQ have raised the urgent need for new therapeutic antischistosomal drugs.[3-6]
In this context, chalcones (1,3-diaryl-2-propen-1-ones) are natural plant metabolites that display antiparasitic activities against
Leishmania, Trypanosoma, and Plasmodium species.[7-9] In addition, some reports have highlighted the potential of chalcones as lead structures
against Schistosoma species. [10, 11] Recently, licochalcone A, a chalcone isolated from the roots of licorice (Glycyrrhiza inflata), was active in
vitro against S. mansoni adult worms. [10] Also, it was shown that cardamonin, a chalcone isolated from Piper aduncum (Piperaceae), displays
in vitro schistosomicidal activity against S. mansoni by inhibiting ATP-diphosphohydrolases.[11] However, in vivo studies to determine the
therapeutic potential of chalcones in the treatment of schistosomiasis have not been described.
Herein, we present the synthesis and the in vitro schistosomicidal activity of a series of chalcones and selected the most active to
be in vivo evaluated in mice infected with S. mansoni. In addition, the S. mansoni ATP diphosphohydrolase inhibitory activity of chalcone 1,
as well as, its molecular docking with S. mansoni ATPDase 1, were investigated.
Results and Discussion
1
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Several natural and synthetic compounds have been evaluated against schistosomes in the last few years.[3] Among natural
substances, chalcones constitute a class that displays a wide spectrum of biological activities [12, 13], including their actions against parasites that
cause neglected tropical diseases. [7, 8] First, chalcones 1-15 were synthesized (Scheme 1) following the Claisen–Schmidt condensation reaction.
Purity of all the isolated compounds was estimated to be higher than 95% by 13C and 1H NMR analysis.
(Insert Scheme 1 here)
Scheme 1. Preparation of chalcones derivatives
After purification and characterization of compounds, all chalcones were in vitro incubated with S. mansoni adult worms in the preliminary
test (Table 1). Incubated at 100 M, chalcones 1, 2, 3, 4, 5, 6 and 12 exhibited noticeable schistosomicidal in vitro activity, causing 100%
mortality in all schistosomes after 24 hours of incubation (Table 1), while no significant results were found for chalcones 7-11 and 13-15 at
100 M (data not shown). Among the active compounds, chalcones 1, 3, and 5 were able to kill all adult worms at 50 µM, while at 25 µM,
only compounds 1 and 3 were active after 24h of incubation. Both 1 and 3 exhibited considerably anthelmintic activity against schistosomes,
reducing motility, causing tegumental alterations and death at concentration of 25 휇M. PZQ (2 M) caused 100% mortality within 24h, whereas
no effect was observed in worms in the control groups (RPMI 1640 medium or RPMI medium plus 0.5 % DMSO).
(Insert Table 1 here)
Table 1. In vitro schistosomicidal and cytotoxic activities of chalcones against adult worms of S. mansoni
The active compounds were further tested at lower concentrations, as presented in Table 1. Among all tested compounds, chalcones 1
and 3 were the most active, causing mortality, tegumental alterations, as well as reduction in motor activity in adult parasites after 24 h of drug
exposure. Previous reports in literature showed that some chalcones, isolated from Astragalus englerianus, also possess in vitro
schistosomicidal activity, but when tested only at 700 µM or higher.[14] Comparing the structures of all tested chalcones, some structural features
seems to be responsible for improving the schistosomicidal activity. One of them is that all active chalcones possess hydroxyl groups at orto
or para position on the A ring. In this way, chalcones 9-11, which do not possess this substitution pattern, were inactive. According to literature,
considering the antileishmanial activity, substitutions with electron donor-groups on the A ring of chalcones are important to improve activity.[7-
9] However, even featuring hydroxyl groups at orto position on the A ring, chalcones 7-8 and 14 were not active. On this aspect, we may infer
that steric effects of substituents on the B ring, mainly at para position, may be related to the loss of schistosomicidal activity. In addition,
considering that chalcones 13 and 15 were inactive, substitutions with withdrawing electron groups at para position of B ring could reduce the
schistosomicidal activity. In this regard, our hypothesis may be corroborated with previous SAR studies with chalcones against Leshmania
amastigotes, which suggests that almost all substitutions at the para position of B ring, including volumous substituents, may reduce the
antiparasitic activity of chalcones. [7-9] It is interesting to highlight that there is no substituents on the B ring in the most active chalcones 1 and
3.
The cytotoxic activities of the most active chalcones were evaluated by MTT assay. As shown in Table 1, chalcone 1 had a low
cytotoxic effect (CC50> 250 µM), while 3 showed a CC50 of 163.5 ± 5.2 µM. Considering only the in vitro anthelmintic assays, 3 was more
effective than 1 in causing dead in S. mansoni adult worms. However, although the in vitro schistosomicidal activity of 1 may be lower than
3, chalcone 1 may also be considered a promising lead schistosomicidal compound, since it has no cytotoxicity to the mammalian cells (Table
1).
Next, considering the active chalcones 1 and 3 as promising compounds, morphological effects of chalcones 1 and 3 on the tegument
of schistosomes were analyzed by SEM (Figure 1A-E). In the negative control group, adult worms of S. mansoni showed intact surface
structure (Figure 1A). In contrast, chalcones 1 (Fig 1D-E) and 3 (Fig 1B-C) caused damages and prominent alterations at the tegumental
surfaces of schistosomes with tubercles appearing collapsed and disrupted.
(Insert Figure 1 here)
Figure 1. Scanning electron microscopy investigation of adult S. mansoni male worms.
Additional morphological studies with confocal microscopy revealed that 1 and 3 reduced the number of tubercles in a dose-
dependent manner (Figure 2). At treatment with 100 µM of chalcone 1, no intact tubercles were observed, while in the groups exposed to 100
µM of 3, the number of intact tubercles was 16 ± 5, which was significantly different from the negative control group (P< 0.001). In the PZQ
group (2 µM), the number of intact tubercles was 17 ± 4 (Figure 2). The tegument of Schistosoma, with its numerous invaginations, has been
involved in nutrient absorption and secretion of products. [15] Also, the tegument has key role in the protection of schistosome against the host
2
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immune system, being the major interface between its external environment. [3] Because of its importance to schistosomes survival, the tegument
is the major target for the development of antischistosomal drugs. [3]
(Insert Figure 2 here)
Figure 2. Effects of the active chalcones 1 and 3 on tubercles of S. mansoni male worms.
Afterwards, we analyzed the in vivo schistosomicidal effects of chalcones 1 and 3 in a mouse model of schistosomiasis. As presented
in Figures 3 and 4, there were significant reductions (P<0.05) in worm burden upon oral treatment with 1 and 3 to mice 49 days post-infection.
Total worm burden reductions of 32.8% and 31.8% were observed with a single oral dose (400 mg/kg) of 1 and 3, respectively, in comparison
with negative control group. In addition, egg production was also analyzed after oral treatment with 1 and 3. However, no statistically
significant effects were observed on oogram, as shown in Figure 4. In comparison with other in vivo schistosomicidal studies with adult S.
mansoni infection, our present results indicated a moderate schistosomicidal activity for chalcones 1 or 3. Similar in vivo schistosomicidal
efficacy was found to other synthetic and natural compounds that showed a total worm burden reductions about of 20% to 30% when evaluated
at a single oral dose of 400 mg/kg. [16, 17] To our knowledge, the schistosomicidal activity of some synthetic chalcones, as well as their in vivo
activities are being reported for the first time in this study. Also, none of the active chalcones have been previously reported in literature as
schistosomicidal compounds. Furthermore, considering that PZQ is the only drug available and is poorly active on larval stages and immature
worms [3, 4], it could be interesting to evaluate the activities of chalcones on schistosomula and juvenile worms.
(Insert Figure 3 and 4 here)
Figure 3. Effect on worm burden of a single oral dose (400 mg/kg) of chalcones 1 and 3 administered to mice harboring a 49-day-old adult
S. mansoni infection, stratified by sex.
Figure 4. Effect on egg development stages (oogram) of a single oral dose of chalcones 1 and 3 administered to mice harboring a 49-day-old
adult S. mansoni infection.
The mechanism by which chalcones 1 and 3 exerts their effects on S. mansoni is still not clear, since chalcones may act on several
parasitic targets. [12] However, considering that 1 and 3 could act notably on tegument, some important enzymes are expressed at the external
surface of the S. mansoni, such as the ATP diphosphohydrolases. [18, 19] Then, we speculated if 1 and 3 could also act, like the chalcone
cardamonin [11], inhibiting S. mansoni ATP diphosphohydrolases. In this regard, the in vitro inhibitory activities of the S. mansoni ATP
diphosphohydrolases were determined for 1 and 3. Chalcone 1 inhibited the S. mansoni ATPase activity showing an IC50 of 30.62 µM, whereas
the ATPase activity remained unaffected (ca. <10%) after incubation with 3 (data not shown).
In addition to this experimental ATPDase inhibition, we also performed molecular docking studies to analyze the interaction of 1
with the active site of SmATPDase1 (Figure 5). Molecular docking studies showed that the best conformation of 1 in the active site of the
enzyme SmATPDase1 had an energy of -7.3 Kcal/mol, presenting hydrogen-bonding interactions with residues S81, T82, S83, H87 and G235
and a calculated Ki (cKi) of 4.45 μM, as shown in Figure 5. The negative energy values (-7.3 Kcal/mol to -6.0Kcal/mol) suggest that the
interaction between the active site and 1 may occur spontaneously. Also, based on the catalytic mechanism proposed for ATP
diphosphohydrolase of Solanum tuberosum (apyrase)[20], it is suggested that the two polar residues (S54 and T55) could participate in the
conversion of ATP to AMP by water-mediated interaction. These polar residues are represented by S81 and T82 in SmATPDase1.
(Insert Figure 5 here)
Figure 5. The best conformation of chalcone 1 (blue carbon sticks) docked in the active site of SmATPDase1 (blue cartoon).
[7-9]
Chalcones possess a wide range of biological activities, mainly against parasites
and, several antiparasitic mechanisms have
been reported and proposed. [12, 21] Considering that chalcones 1 and 3 causing extensive morphological alteration on the tegument of adult
schistosomes, ATPDase inhibition may not be the only suggested target of schistosomicidal action and other mechanisms may be involved. In
this regard, it is reported that the therapeutic potential of chalcones is related to their structure as α,β-unsaturated ketones and, therefore, to
their action as Michel acceptors, which could capture nucleophiles, such as cysteine, and bind covalently to proteins and enzymes. [12, 21] It is
also reported that some chalcones may act on parasite mitochondria.[22] Licochalcone A, for instance, was found to inhibit parasite mitochondrial
dehydrogenases, causing mitochondrial alteration, as well as morphological alterations on the S. mansoni tegument. [10]
3
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Conclusions
In summary, our results demonstrated that some chalcones, especially those that possess hydroxyl groups at orto or para position on the A
ring, were able to kill adult schistosomes and damage the worm’s tegument without affecting mammalian cells. Chalcones 1 and 3 were also
active in a mouse model of schistosomiasis, showing significant reductions on worm burden in comparison with infected untreated controls.
In addition, chalcone 1 inhibited the in vitro activity of S. mansoni ATP diphosphohydrolase, which was corroborated with molecular docking
studies. Overall, these findings of this study open the route to further schistosomicidal studies of natural and synthetic chalcones.
Experimental Section
Instrumentation
1
IR spectra were recorded using a BOMEM-FTIR MB102 spectrometer (AABB Bomem Inc., Quebec, Canada). H and 13C NMR
spectra were recorded on Bruker Advance DRX300 spectrometer (Bruker Corporation, Billerica, MA, USA). Thin-layer chromatography
(TLC) was performed on glass plates and silica gel sheets (Silica Gel F254; Merck, Whitehouse Station, NK, USA), visualized with iodine
vapor, and/or revealed with ethanolic H2SO4 solution. Solvents were purchased from Vetec Química (Vetec, Xerem, RJ, Brazil) and were
distilled prior to use. Reagents were purchased from Aldrich (Sigma Aldrich, Saint Louis, MI, USA) and used without further purification.
Synthesis of chalcones
Chalcones (1-15) were synthesized following according to Claisen–Schmidt condensation reaction [23], in which to 27 mL of solution
of the NaOH (4M aqueous solution) in 25 mL of EtOH was added to the appropriate acetophenone (11 mmol) and the mixture was stirred for
30 min, in ice-water bath. Then, substituted benzaldehyde (11 mmol) was added and the mixture was stirred at room temperature for 3.5-120
h. The reaction mixture was kept overnight at 0–5°C and after diluted with chilled water and then acidified with HCl (10% aqueous solution).
The solution was taken up in CH2Cl2 and, after, it was washed successively with water. Then, the organic layer was dried with Na2SO4, filtrated
and concentrated under reduced pressure. Crude products were purified by recrystallization in appropriate solvent in order to obtain yellow
isolated solids.
Characterization of synthesized compounds:
(E)-2’-hydroxy-4-methoxychalcone (1): Obtained from 2-hydroxyacetophenone and 4-methoxybenzaldehyde. The yellow solid was
recrystallized from ethanol. Yield: 39%. The NMR spectroscopic data are according to the literature.[24]
(E)-2’-hydroxychalcone (2): Obtained from 2-hydroxyacetophenone and benzaldehyde. The yellow solid was recrystallized from
ethanol. Yield: 50%. The NMR spectroscopic data are according to the literature.[24, 25]
(E)-4’-hydroxychalcone (3): Obtained from 4-hydroxyacetophenone and benzaldehyde. The yellow solid was recrystallized from
ethanol. Yield: 36%. The NMR spectroscopic data are according to the literature. [25]
(E)-2’-hydroxy-4’,6’-dimethoxychalcone (4): Obtained from 2-hydroxy-4,6-dimethoxyacetophenone and benzaldehyde. The yellow
solid was recrystallized from ethanol. Yield: 59%. The NMR spectroscopic data are according to the literature.[26]
(E)-2’,6’-dihydroxychalcone (5): Obtained from 2,6-dihydroxyacetophenone and benzaldehyde. The yellow solid was recrystallized
from ethanol. Yield: 15%. The NMR spectroscopic data are according to the literature. [27]
(E)-2’-hydroxy-4’-methoxychalcone (6): Obtained from 2-hydroxy-4-methoxyacetophenone and benzaldehyde. The yellow solid
was recrystallized from ethanol. Yield: 38%. The NMR spectroscopic data are according to the literature.[27]
(E)-2’-hydroxy-4’,3,4,5-tetramethoxychalcone (7): Obtained from 2-hydroxy-4-methoxyacetophenone and 3,4,5-
trimethoxybenzaldehyde. The yellow solid was recrystallized from ethanol. Yield: 42%. The NMR spectroscopic data are according to the
literature. [28]
(E)-2’-hydroxy-4’,6’,3,4,5-pentamethoxychalcone (8): Obtained from 2-hydroxy-4,6-dimethoxyacetophenone and 3,4,5-
trimethoxybenzaldehyde. The yellow solid was recrystallized from ethanol. Yield: 52%. The NMR spectroscopic data are according to the
literature.[29]
(E)-4’-hydroxy-3,4,5-trimethoxychalcone (9): Obtained from 4-hydroxyacetophenone and 3,4,5-trimethoxybenzaldehyde. The
yellow solid was recrystallized from ethanol. Yield: 51%. The NMR spectroscopic data are according to the literature.[30]
(E)-4’-hydroxy-3’,3,4,5-tetramethoxychalcone (10): Obtained from 4-hydroxy-3-methoxyacetophenone and 3,4,5-
trimethoxybenzaldehyde. The yellow solid was recrystallized from ethanol. Yield: 48%. The NMR spectroscopic data are according to the
literature. [28]
(E)-3’,4’,5’-trimethoxychalcone (11): Obtained from 3,4,5-trimeyhoxyacetophenone and benzaldehyde. The yellow solid was
recrystallized from ethanol. Yield: 20%. The NMR spectroscopic data are according to the literature. [29-31]
4
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(E)-2’-hydroxy-4’,6’-dimethoxy-4-bromochalcone (12): Obtained from 2-hydroxy-4,6-dimethoxyacetophenone and 4-
bromobenzaldehyde. The yellow solid was recrystallized from ethanol. Yield: 61%. The NMR spectroscopic data are according to the literature.
[32, 33]
(E)-2’-hydroxy-4-trifluoromethylchalcone (13): Obtained from 2-hydroxyacetophenone and 4-trifluoromethylbenzaldehyde. The
yellow solid was recrystallized from hexane/methanol. Yield: 10%. The NMR spectroscopic data are according to the literature. [9]
(E)-2’-hydroxy-4-propyloxychalcone (14): Obtained from 2-hydroxyacetophenone and 4-propyloxybenzaldehyde. The yellow solid
1
was recrystallized from ethanol. Yield: 30%. H NMR (300 MHz, CDCl3). δ (ppm): 2.58 (t, 1H, J= 2.4 Hz, ≡CH); 4.76 (d, 2H, J= 2.4 Hz, -
OCH2-); 6.95 (t, 1H, J= 7.5 Hz, H-5’); 7.02-7.05 (m, 3H, H-3/H-5, H-3’); 7.47-7.57 (m, 1H, H-4’ and H-α); 7.65 (d, 2H, J= 8.4 Hz, H-2/H-6);
7.88-7.93 (m, 2H, H-β and H-6’); 12.93 (s, 1H, OH). 13C NMR (75 MHz, CDCl3) δ (ppm): 56.1 (-OCH2-); 76.3 (≡CH); 78.1(-C≡); 115.6-
145.2 (C-α, C-β and Ar); 160.0 (C-4); 163.8 (C-2’); 193.8 (C=O).
(E)-2’-hydroxy-4-nitrochalcone (15): Obtained from 2-hydroxyacetophenone and 4-nitrobenzaldehyde. The brown solid was
recrystallized from ethanol. Yield: 24%. The NMR spectroscopic data are according literature. [24]
In vitro schistosomicidal assays
S. mansoni (BH strain) worms were obtained from Mesocricetus auratus hamsters, according to standard procedures previously
described by our group. [6] Briefly, hamsters (3 weeks old) were infected by subcutaneous injection of 150 S. mansoni cercariae. After 9 weeks,
adults S. mansoni worms were recovered from the animals by perfusion with RPMI 1640 medium supplemented with heparin. All experiments
were authorized by the Committee for Ethics in Animal Care of Institute Adolf Lutz (CEUA protocol no 11.794/08; CEUA protocol no.
05/2015) and conducted in strict adherence with good animal practice as defined by guidelines for animal husbandry, in accordance with
Brazilian legislation.
To study the in vitro schistosomicidal effects of chalcones, adult parasites were washed in RPMI 1640 medium kept at pH 7.5 with
HEPES 20 mM, supplemented with 10% fetal bovine serum and 200 μg/mL streptomycin and 200 IU/mL penicillin (Vitrocell). After washing,
adult worms (female and male) were incubated at 37 °C in a 5% CO2 atmosphere in a 24-well culture plate containing the same medium, as
previously described.[34] Chalcones were dissolved in dimethyl sulfoxide (DMSO; 0.5 % of final concentration) and evaluated initially at 100
M (preliminary screening). After, the most active chalcones were also tested at 50, 25 and 10 μM. Samples were added to the culture from a
4000 μg/mL stock solution in RPMI 1640 containing DMSO. The final volume in each well was 2 mL. Negative control group used worms
assayed in RPMI 1640 medium and RPMI 1640 with 0.5% DMSO, while PZQ (2 μM) was used as positive control group. All experiments
were repeated at least three times independently for each treatment, and plating was carried out in triplicate for each concentration. Parasites
were maintained for 48 h and monitored every 24 h using a light microscope in order to evaluate their general condition, such as changes in
motor activity, mortality rate, and tegumental alterations. [5, 6, 13]
Quantification of the number of tubercles by confocal laser scanning microscopy
The effects of the most active chalcones 1 and 3 (10, 25, 50 and 100 µM) on the tegument of adult S. mansoni was observed using a
confocal laser scanning microscope (Laser Scanning Microscope, LSM 510 META, Carl Zeiss, Standorf Gottingen, Vertrieb, Germany), which
was used to quantify the number of tubercles according to method previously published.[34] After the occurrence of death, helminths were fixed
in formalin–acetic acid-alcohol solution (FAA) and analyzed under a confocal microscope with autofluorescence excited at 488 nm and emitted
light at 505 nm. Three dimensional images of the tegument, obtained from confocal laser microscopy, were assessed. The number of intact
tubercles on the dorsal surface of male parasites, during the microscopic analysis, was counted in a 20,000 µm2 area.
Scanning electron microscopy (SEM) studies
Tegumental alterations in adult worms of S. mansoni were performed for the most active chalcones (1 and 3) using SEM, according
to method previously described.[5] After death, parasites were fixed in 1 mL 2.5% glutaraldehyde for 3–24 h at room temperature. Specimens
were air-dried, mounted on stubs and metalized with gold using a Sputter Coater. Samples were then visualized using a high-resolution SEM
accelerating voltage of 20 kV (Jeol-JSM-6460LV).
In vivo schistosomicidal assays
For in vivo studies, three-week-old Balb/c mice (Anilab, Paulínia, SP, Brazil) were infected with 80 cercariae of S. mansoni (BH
strain) and kept under environmentally controlled conditions (humidity: 70 %; temperature: 25 °C) and had free access to water and food. After
49 days post-infection, mice were treated with a single oral dose (400 mg/kg) of chalcones 1 or 3. Mice were divided into 3 experimental
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groups, in which each group consisted of 10 animals treated with a single oral dose, as follows: Group I, vehicle-treated (100 μL of 3% ethanol
in saline) control group; Group II, chalcone 1-treated group (400 mg/kg); Group III, chalcone 3-treated group (400 mg/kg). Two weeks after
oral treatment, mice were euthanized, and the surviving schistosomes located at the mesenteric veins and the liver were counted and sexed, as
previously described. 35, 36] The assessment of therapeutic efficacy was also based on the technique of quantitative and qualitative oograms,
using a fragment of the intestine (10 mm) [16, 17], as well as the Kato-Katz method for quantitative feces examination. 37] In the oogram pattern,
eggs were scored as immature, mature or dead. 36-38]
Schistosoma mansoni ATP diphosphohydrolase activity
The S. mansoni ATP diphosphohydrolase activity was determined according to previous report. [11] Briefly, the homogenized of the
tegument of S. mansoni adults worms (0.05 mg protein/mL), containing ATP diphosphohydrolases, was pre-incubated for 30 min at 37 °C
with chalcones 1 or 3 (5 μM to 40µM). Then, inorganic phosphate (Pi) liberated was determined.[11, 39] During the assay, in the absence of 10%
DMSO, the homogenized presented an ADPase activity of 76.49  11.47 nmol Pi mg-1 min-1 and an ATPase activity of 55.80 ± 8.36 nmol Pi
mg-1 min-1. The half inhibitory concentration (IC50) was calculated from dose-response curves by non-linear regression analysis.
Cytotoxicity assay
Cytotoxicity in macrophages was determined with the MTT assay [40], using different concentrations (25, 50, 100, and 200 μM) of
the active chalcones using three independent experiments in duplicate. Peritoneal macrophages used in this study were obtained according to
previous report.[41] The values of cytotoxicity concentration to reduce 50% of viable macrophages (CC50) were obtained using GraFit Version
5 software.
In silico analysis and molecular docking of SmATPDase 1
The three-dimensional (3D) model of SmATPDase 1 used in this work was previously published. [42] The protein was prepared using
the MGLTools program version 1.5.6., setting as flexible the side chain of the catalytic residue E201, and of residues considered important for
the mechanism of reaction (D78, S81, T82, K85, T154, K279, and Y397). For docking analysis, the MGLTools program version 1.5.6. was
used to grid construction with dimensions x=y=z=30 Å, centered in the catalytic site at position of x = 22.268 Å, y = 34.516 Å and z = 21.621
Å. The structure of chalcone 1 was constructed using the program ChemSketch version 11.0 and prepared using the the MGLTools program
version 1.5.6.
Statistical analysis
Statistical tests were performed with Graphpad Prism version 5.0 (Graphpad software Inc., La Jolla, CA, USA). Dunnet's test was
used to analyze the statistical significance of differences between mean experimental and control values. [5] P values of < 0.05 were considered
significant.
Acknowledgements
The authors are grateful to FAPEMIG (Grant numbers #PPM-00296-16; CEX APQ 01287/14; CEX - RED-00010-14; APQ
02015/14; APQ 01622/13; RED-00010-14; BPD 00284-14), CNPq (Grant numbers # 487221/2012-5; 455551/2014-6; PDJ# 403233/2015-1),
and FAPESP (Grant number #2016/22488-3) for financial support, as well as to CAPES, PIBIC/CNPq/UFJF and CNPq for fellowships. We
are also grateful to Lorena Rodrigues Riani for technical assistance in UFJF, as well as to Silvia G. Chiodelli, Anderson B. Gonçalves, and
Yone C. Xavier for excellent technical assistance with S. mansoni life cycle maintenance at the Adolfo Lutz Institute (São Paulo, SP, Brazil).
Author Contribution Statement
V.R.D.P, I.J.A.J., L.S.S., and R.B.G. contributed by synthesizing and identifying the chalcones, which synthesis was planned and designed by
M.R.C.C; F.S.T and M.C.S performed SEM analysis; P.F.P, M.P.S., P.L.S.P., A.C.A., E.S.C., and J.M. contributed with the schistosomicidal,
enzymatic and cytotoxic assays. J.M. supervised the schistosomicidal assays. L.A.A. and P.V.S.Z.C. contributed with molecular and docking
studies. A.A.S.F. designed and drafted all experiments and the manuscript. All authors have read the final version.
6
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8
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Chem. Biodiversity
Scheme 1. Preparation of chalcones derivatives
R1
O
O
R1
O

NaOH,
EtOH
R2
R3
R6
R7
R2
R3
R6
3'
1
CH3
H

+
3
6'
6
0 °C-r.t.
3.5-120h
R5
R5
R7
5'
R4
R8
R4
R8
1) R1=OH; R7=OCH3; R2=R3=R4=R5=R6=R8=H
2) R1=OH; R2=R3=R4=R5=R6=R7=R8=H
3) R3=OH; R1=R2=R4=R5=R6=R7=R8=H
4) R1=OH; R3=R5=OCH3; R2=R4=R6=R7=R8=H
5) R1=R5=OH; R2=R3=R4=R6=R7=R8=H
6) R1=OH; R3=OCH3; R2=R4=R5=R6=R7=R8=H
7) R1=OH; R3=R6=R7=R8=OCH3; R2=R4=R5=H
8) R1=OH; R3=R5=R6=R7=R8=OCH3; R2=R4=H
9) R3=OH; R6=R7=R8=OCH3; R1=R2=R4=R5=H
10) R3=OH; R2=R6=R7=R8=OCH3; R1=R4=R5=H
11) R2=R3=R4=OCH3; R1=R5=R6=R7=R8=H
12) R1=OH; R3=R5=OCH3; R7=Br; R2=R4=R6=R8=H
13) R1=OH; R7=CF3; R2=R3=R4=R5=R6=R8=H
14) R1=OH; R7=O(CH2)3CH3; R2=R3=R4=R5=R6=R8=H
15) R1=OH; R7=NO2; R2=R3=R4=R5=R6=R8=H
9
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Table 1. In vitro schistosomicidal and cytotoxic activities of chalcones against adult worms of
S. mansoni
Worms with tegumental
Motor activity
reduction (%)a
alterations
(%)a
Dead worms
(%)a
Cytotoxicity
CC50 (µM)e
Groups
Concentration (µM)
Slight Significant
Partial
Extensive
Control b
PZQc
-
5
-
-
100
-
-
-
-
-
100
-
-
25
-
-
75
-
DMSO 0.5%
Chalconesd
1
2
3
100
50
100
100
50
-
-
-
100
100
50
-
-
-
100
100
50
-
>250.0
25
50
100
50
-
10
100
50
100
0
-
-
100
0
-
-
100
0
177.2 ± 27.8
163.5 ± 5.2
100
50
100
100
100
50
-
-
100
100
100
50
-
-
100
100
100
50
25
-
-
10
50
50
4
5
100
50
100
-
-
100
50
-
100
50
85.1 ± 5.8
50
50
100
50
100
100
-
-
-
-
100
100
-
-
-
-
100
100
-
142.1 ± 11.9
25
6
100
50
100
-
-
100
50
-
-
100
-
>250.0
50
12
100
50
100
-
-
100
50
-
-
100
-
58.8 ± 1.5
50
b
a Percentages relative to the 20 worms investigated. RPMI 1640. c Tested at 2 µM.d Incubation period: 24h. e CC50 values (50% cytotoxicity
concentration) on macrophages ± standard errors of the mean.
10
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A
B
C
D
E
Figure 1. Scanning electron microscopy investigation of adult S. mansoni male worms. A: worms non-treated (control). B and C: worms
treated with 3 (100 µM): tegumental surface showing blebs, swelling and shortening. D and E: worms treated with 1 (100 µM): dorsal
tegumental surface showing swelling, shortening, and collapse of the tubercles. Scale bar 10 μm.
11
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50
40
30
20
10
0
***
***
50
***
0
10
25
100
PZQ
_____________________
Chalcone 1 (M)
50
40
30
20
10
0
*
***
***
0
10
25
50
100
PZQ
____________________
Chalcone 3 (M)
Figure 2. Effects of the active chalcones 1 and 3 on tubercles of S. mansoni male worms. Quantification of the number of tubercles was
performed using confocal microscopy. The numbers of intact tubercles were measured in a 20,000 μm2 of area, calculated with the Zeiss LSM
Image Browser software. PZQ (2 μM) was used as positive control. A minimum of three tegument areas of each parasite was assessed. Data
are presented as means ± SD (bars) of ten male adult worms and were considered statistically significant results with *P< 0.01 and ***P<
0.001 in comparison with untreated groups (Dunnet's test).
12
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50
40
30
20
10
0
**
*
l
l
l
e
e
e
e
e
e
l
l
l
a
l
l
a
t
l
a
t
t
p
p
p
o
o
o
T
u
Co
u
Co
u
Co
Ma
Ma
Ma
male
male
male
T
T
e
e
e
F
F
F
Control
1
3
Figure 3. Effect on worm burden of a single oral dose (400 mg/kg) of chalcones 1 and 3 administered to mice harboring a 49-day-old adult S.
mansoni infection, stratified by sex. Points represent data from individual mice that were infected and treated with chalcones, or infected and
untreated (control). Data are presented as means ± SD (bars) (n=10) and were considered statistically significant results with *P < 0.05, ** P
< 0.01 in comparison with untreated groups (Dunnet's test).
13
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250
200
150
100
50
0
s
s
s
s
s
s
s
s
s
g
g
g
g
g
g
g
g
g
g
g
g
g
g
g
g
g
g
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
d
d
d
r
r
r
a
a
a
u
u
u
t
t
t
De
De
De
Ma
Ma
Ma
mmatur
mmatur
mmatur
I
I
I
1
3
Control
Figure 4. Effect on egg development stages (oogram) of a single oral dose of chalcones 1 and 3 administered to mice harboring a 49-day-old
adult S. mansoni infection. Points represent data from individual mice that were infected and treated with chalcones, or infected and untreated
(control). The horizontal bars represent median values. Data are presented as means ± SD (bars) (n=10) and were considered statistically
significant results with *P < 0.05 in comparison with untreated groups (Dunnet's test).
14
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Figure 5. The best conformation of chalcone 1 (blue carbon sticks) docked in the active site of SmATPDase1 (blue cartoon). Hydrogen bonds
and their lengths are represented using dashed lines (yellow). The image was generated using PyMOL, v.1.8.2, Schrödinger – LLC.
15
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