Antischistosomal properties of aurone derivatives against juvenile and adult worms of Schistosoma mansoni

Article abstract of DOI:10.1016/j.actatropica.2020.105741

Schistosomiasis is a neglected disease caused by helminth flatworms of the genus Schistosoma, affecting over 240 million people in more than 70 countries. The treatment relies on a single drug, praziquantel, making urgent the discovery of new compounds. A

Full text of DOI:10.1016/j.actatropica.2020.105741

Acta Tropica 213 (2021) 105741
Contents lists available at ScienceDirect
Acta Tropica
journal homepage: www.elsevier.com/locate/actatropica
Antischistosomal properties of aurone derivatives against juvenile and
adult worms of Schistosoma mansoni
,
,
Vinicius R.D. Pereiraa 1, Lígia S. da Silveirab 1, Ana C. Mengardad, Ismael J. Alves Júniora,
a
b
d
c
´
Ohana Oliveira Zuza da Silva , Fabio Balbino Miguel , Marcos P. Silva , Ayla das C. Almeida ,
c
c
c
d
´
Daniel da Silva Torres , Priscila de F. Pinto , Elaine S. Coimbra , Josue de Moraes , Mara R.
C. Courib *, Ademar A. da Silva Filhoa
,
,*
a
´
ˆ
ˆ
´
´
Faculdade de Farmacia, Departamento de Ciencias Farmaceuticas, Universidade Federal de Juiz de Fora, R. Jose Lourenço Kelmer s/n, Campus Universitario, Juiz de
Fora, MG, 36036-900, Brazil
b Departamento de Química, Universidade Federal de Juiz de Fora, Juiz de Fora, MG, 36036-900, Brazil
c
ˆ
´
Instituto de Ciencias Biologicas, Universidade Federal de Juiz de Fora, Juiz de Fora, MG, 36036-900, Brazil
d Núcleo de Pesquisa em Doenças Negligenciadas, Universidade Guarulhos, Guarulhos, SP, 07023-070, Brazil
A R T I C L E I N F O
A B S T R A C T
Keywords:
Schistosomiasis is a neglected disease caused by helminth flatworms of the genus Schistosoma, affecting over 240
million people in more than 70 countries. The treatment relies on a single drug, praziquantel, making urgent the
discovery of new compounds. Aurones are a natural type of flavonoids that display interesting pharmacological
activities, particularly as chemotherapeutic agents against parasites. In pursuit of treatment alternatives, the
present work conducted an in vitro and in vivo antischistosomal investigation with aurone derivatives against
Schistosoma mansoni. After preparation of aurone derivatives and their in vitro evaluation on adult schistosomes,
the three most active aurones were evaluated in cytotoxicity and haemolytic assays, as well as in confocal laser-
scanning microscope studies, showing tegumental damage in parasites in a concentration-dependent manner
with no haemolytic or cytotoxic potential toward mammalian cells. In a mouse model of schistosomiasis, at a
single oral dose of 400 mg/kg, the selected aurones showed worm burden reductions of 35% to 65.0% and egg
reductions of 25% to 70.0%. The most active thiophenyl aurone derivative 18, unlike PZQ, had efficacy in mice
harboring juvenile S. mansoni, also showing significant inhibition of oviposition by parasites, giving support for
the antiparasitic potential of aurones as lead compounds for novel antischistosomal drugs.
Anthelminthic
Schistosomicidal
Natural product
Schistosoma mansoni
Aurones
1. Introduction
et al., 2018). Although PZQ is safe, its major drawbacks include poor
activity against juvenile worms. Also, the variable bioavailability of PZQ
Schistosomiasis, also known as bilharzia, is a chronic, endemic and
after oral administration may require an increase of the dose, which may
¨
neglected disease caused by blood flukes of the genus Schistosoma
result in increased incidence of side effects (Mader et al., 2018; Mayoka
¨
(Mader et al., 2018), affecting over 240 million people in more than 70
et al., 2019; Zoghroban et al., 2019). In addition, the use of just a single
drug, over the last decades, may contribute to emerging PZQ-resistance
(Mayoka et al., 2019). Therefore, the lack of any other effective and safe
schistosomicidal compound, as well as the concern of PZQ resistance
(Cowan et al., 2015; Wang et al., 2012), have raised the urgent need for
new antischistosomal drugs that could either complement or replace
PZQ chemotherapy (De Farias Santiago et al., 2014; Dziwornu et al.,
2020; Liu et al., 2017.). Considerable efforts are ongoing in the devel-
opment of novel schistosomicidal agents in the last ten years (Guerra
countries, mainly in tropical and sub-tropical regions of South America,
¨
Africa and Asia (Lago et al., 2018; Mader et al., 2018; Siqueira et al.,
2017). Schistosomiasis continues to rank second – following malaria – in
the world’s parasitic diseases in terms of prevalence, morbidity and
mortality rates and, at least 200,000 people die each year of schistoso-
¨
miasis (Lago et al., 2018; Mader et al., 2018; Siqueira et al., 2017).
There is currently no vaccine for schistosomiasis and chemotherapy
relies on one drug only, praziquantel (PZQ) (Doenhoff et al., 2008; Lago
* Corresponding authors.
E-mail addresses: mara.rubia@ufjf.edu.br (M.R.C. Couri), ademar.alves@ufjf.edu.br (A.A. da Silva Filho).
1
These authors contributed equally to this work
https://doi.org/10.1016/j.actatropica.2020.105741
Received 11 February 2020; Received in revised form 1 September 2020; Accepted 14 October 2020
Available online 5 November 2020
0001-706X/© 2020 Elsevier B.V. All rights reserved.

V.R.D. Pereira et al.
Acta Tropica 213 (2021) 105741
et al., 2019; Lago et al., 2019, 2018; Roquini et al., 2019). As a result,
many substances with promising antischistosomal properties have been
identified, especially natural compounds (Lago et al., 2018).
Parana). Chemical shifts of 1H NMR and 13C NMR are given in ppm and
coupling constant (J) are given in Hz. TMS signal was used as internal
reference.
Aurones are a natural type of flavonoids found mainly in flowers and
fruits as bright yellow pigments (Alsayari et al., 2019; Hassan et al.,
2018). Along with their important role in pigmentation of plants,
aurones also display interesting biological activities, such as antimi-
crobial, antioxidant, and anticancer (Hassan et al., 2018; Mughal et al.,
2017; Uesawa et al., 2017). In addition, the antiparasitic potential of
aurones has been highlighted by studies that showed their anti-
leishmanial and antimalarial properties (Badavath et al., 2017; Kayser
et al., 2002; Mughal et al., 2017). To the best of our knowledge, the
antischistosomal activity of aurones has not been previously studied.
In this regard, we conducted an antischistosomal investigation with
aurone derivatives against S. mansoni and identified three schistosomi-
cidal compounds that effectively killed S. mansoni adult worms with no
haemolytic or cytotoxic activity to mammalian cells. Subsequently,
these compounds were selected for the in vivo assessment in mice
infected with adult worms and, the promising aurone compound 18 was
further investigated against the juvenile stages of S. mansoni.
2.1.1. Preparation of chalcone derivatives
2-hydroxychalcones were synthesized following the Clai-
sen–Schmidt condensation, according to a previously published pro-
cedure (Pereira et al., 2018). This method furnished 2-hydroxychalcones
(compounds 1-9) poor to good yields (18-91%).
(E)-1-(2-hydroxyphenyl)-3-(3,4,5-trimethoxyphenyl)prop-2-en-1-one
(1): Obtained from 2’-hydroxy-acetophenone and 3,4,5-trimethoxyben-
zaldehyde. The yellow solid was washed with ethanol/cold water. Yield
= 22%. The NMR spectroscopic data are according to the literature
(Tran et al., 2008). 1H NMR (300 MHz, CDCl3) δ (ppm): 3.92 and 3.94 (s,
9H, OCH3); 6.89 (s, 2H, H-2/H-6); 6.95 (t, 1H, J= 8.1 Hz, H-5’); 7.03 (d,
1H, J= 8.1 Hz, H-3’); 7.48-7.56 (m, 2H, H-4’ and H-α); 7.85 (d, 1H, Jβ,
α
= 15.6 Hz, H-β); 7.93 (d, 1H, J= 8.1 Hz, H-6’); 8.81 (s, 1H, OH). 13
C
NMR (75 MHz, CDCl3) δ (ppm): 56.5 (2 x OCH3); 61.2 (OCH3);
106.2-153.7 (C-α, C-β and Ar); 163.7 (C-2’); 193.7 (C=O).
(E)-3-(4-(hexyloxy)phenyl)-1-(2-hydroxyphenyl)prop-2-en-1-one (2):
Obtained from 2’-hydroxy-acetophenone and 4-hexyloxybenzaldehyde.
The orange solid was recrystallized from ethanol/water. Yield = 62%.
The NMR spectroscopic data are according to the literature (Cabrera
et al., 2007). 1H NMR (300 MHz, CDCl3) δ (ppm): 0.93 (t, 3H, J= 6.9 Hz,
-CH3); 1.34-1.53 (m, 6H, -CH2-); 1.81 (qui, 2H, J= 6.9 Hz, -OCH2CH2-);
4.01 (t, 2H, J= 6.9 Hz, -OCH2-); 6.92-6.97 (m, 3H, H-5’, H-3/H-5); 7.03
2. Materials and methods
2.1. Chemistry
Aurones were prepared according to a method previously described
(Detsi et al., 2009) and the chemical structures of all compounds (1-18)
are presented in Fig. 1. Reagents and solvents were purchased as reagent
grade and used without purification. Thin layer chromatography (TLC)
was performed on glass plates and silica gel sheets (silica gel F254;
Merck), visualized with iodine vapor, and/or ultraviolet light at 264 nm.
Column chromatography was carried out on silica-gel 60G
0.063-0.200mm (70-230 mesh). Melting points were determined on a
MQAPF-Microquimica apparatus. IR spectra were acquired using a
Bruker Alpha-E ATR (Attenuated total reflection) spectrometer. NMR
data were recorded on a Bruker 500 Advance spectrometer. The struc-
tures of the synthesized known aurones, which were previously
described, were confirmed by 1H and 13C NMR in comparison with
(d, 1H, J= 8.4 Hz, H-3’); 7.46-7.51 (m, 1H, H-4’); 7.54 (d, 1H, Jα,β=
15.3 Hz, H-α); 7.62 (d, 2H, J= 8.7 Hz, H-2/H-6); 7.88-7.94 (m, 2H, H-β
and H-6’); 12.99 (s, 1H, OH). 13C NMR (75 MHz, CDCl3) δ (ppm):
14.2-31.8 (Caliphatic); 68.5 (-OCH2-); 115.2-145.7 (C-α, C-β and Ar);
161.2 (C-4); 163.8 (C-2’); 193.9 (C=O).
(E)-1-(2-hydroxyphenyl)-3-(4-(octyloxy)phenyl)prop-2-en-1-one (3):
Obtained from 2’-hydroxy-acetophenone and 4-octyloxybenzaldehyde.
The yellow solid was recrystallized from ethanol (Cabrera et al.,
2007). Yield = 51%. IR (KBr)
ν
(cmꢀ 1): 3424 (OH); 2925 and 2854 (C-H
aliphatic); 1634 (C=O); 1603, 1557 and 1512 (C=C); 1275 (νas CAr-O-C).
1H NMR (300 MHz, CDCl3) δ (ppm): 0.91 (t, 3H, J= 6.6 Hz, -CH3);
1.31-1.50 (m, 8H,-CH2-); 1.82 (qui, 2H, J= 6.6 Hz, -OCH2CH2-); 4.01 (t,
2H, J= 6.6 Hz, -OCH2-); 6.93-6.96 (m, 3H, H-5’, H-3/H-5); 7.03 (d, 1H,
literature. The unpublished aurones were characterized by 1H NMR, 13
C
NMR, and High-resolution mass spectra (HRMS). HRMS of compounds
12, 13, and 14 were performed on LTQ Orbitrap XL ETD (mass spec-
trometry facility RPT02H PDTIS/Carlos Chagas Institute, Fiocruz
J= 8.4 Hz, H-3’); 7.47-7.52 (m, 1H, H-4’); 7.54 (d, 1H, Jα,β= 15.3 Hz,
H-α); 7.62 (d, 2H, J= 8.7 Hz, H-2/H-6); 7.89-7.94 (m, 2H, H-β and H-6’);
8.69 (s, 1H, OH). 13C NMR (75 MHz, CDCl3) δ (ppm): 14.3-32.0
Fig. 1. General information for the synthesis and chemical structures of all tested aurones
2

V.R.D. Pereira et al.
Acta Tropica 213 (2021) 105741
(Caliphatic); 68.5 (-OCH2-); 115.2-145.7 (C-
α
, C-β and Ar); 161.9 (C-4);
(C-1); 165.8 (C-2’); 188.5 (C=O).
163.8 (C-2’); 193.9 (C=O). MS (ESI) m/z: 353.2097 [M+H+].
(E)-1-(2-hydroxyphenyl)-3-(4-(nonyloxy)phenyl)prop-2-en-1-one (4):
Obtained from 2’-hydroxy-acetophenone and 4-nonyloxybenzaldehyde.
The yellow solid was recrystallized from ethanol. Yield = 91%. The NMR
spectroscopic data are according to the literature (Cabrera et al., 2007).
1H NMR (300 MHz, CDCl3) δ (ppm): 0.90 (t, 3H, J= 6.6 Hz, -CH3);
1.30-1.32 (m, 12H, -CH2-); 1.82 (qui, 2H, J= 6.6 Hz, -OCH2CH2-); 4.01
(t, 2H, J= 6.6 Hz, -OCH2-); 6.93-6.97 (m, 4H, H-4’, H-5’, H-3/H-5); 7.03
2.1.2. Preparation of aurone derivatives
Aurones (compounds 10-18) were synthesized by cyclization of
corresponding 2-hydroxychalcones, according to a method described
below (Detsi et al., 2009), affording the selected aurones in 14-98%
yield. To a solution of mercuric acetate (2.61 mmol) dissolved in pyri-
dine was added chalcone (2.61 mmol) and the mixture was stirred at
110◦C for 1-48 h. The reaction mixture was cooled and acidified with
HCl (10% aqueous solution) (Pereira et al., 2018). The solution was
taken up in CH2Cl2 and was washed successively with water. The
organic layer was then dried over anhydrous sodium sulfate, filtered,
and was concentrated under reduced pressure. The crude product was
purified by recrystallization in appropriate solvent and aurones were
obtained as yellow solids.
(d, 1H, J= 8.4 Hz, H-3’); 7.54 (d, 1H, Jα,β= 15.6 Hz, H-α); 7.62 (d, 2H,
J= 8.7 Hz, H-2/H-6); 7.88-7.94 (m, 2H, H-β and H-6’); 13.00 (s, 1H,
OH). 13C NMR (75 MHz, CDCl3) δ (ppm): 14.3-32.1 (Caliphatic); 68.5
(-OCH2-); 115.2-145.7 (C-α, C-β and Ar); 161.9 (C-4); 163.8 (C-2’);
193.9 (C=O).
(E)-1-(2-hydroxyphenyl)-3-(4-(tetradecyloxy)phenyl)prop-2-en-1-one
(5): Obtained from 2’-hydroxy-acetophenone and 4-tetradecyloxyben-
zaldehyde. The yellow solid was recrystallized from ethanol/dichloro-
methane. Yield = 34%. The NMR spectroscopic data are according to the
literature (Ngaini et al., 2009). 1H NMR (300 MHz, CDCl3) δ (ppm): 0.89
(t, 3H, J= 6.6 Hz, -CH3); 1.25-1.31 (m, 22H, -CH2-); 1.82 (qui, 2H, J=
6.6 Hz, -OCH2CH2-); 4.02 (t, 2H, J= 6.6 Hz, -OCH2-); 6.92-6.97 (m, 3H,
H-5’, H-3/H-5); 7.03 (d, 1H, J= 8.1 Hz, H-3’); 7.47-7.52 (m, 1H, H-4’);
(Z)-2-(3,4,5-trimethoxybenzylidene)benzofuran-3(2H)-one (10): Ob-
tained from compound 1. The yellow solid was recrystallized from ethyl
acetate/ methanol. Yield = 43%, m.p.: 132.5-134.8◦C. The NMR spec-
troscopic data are according to the literature (Morimoto et al., 2007). 1H
NMR (300 MHz, CDCl3) δ (ppm): 3.92 and 3.94 (s, 9H, OCH3); 6.82 (s,
1H, H-10); 7.18 (s, 2H, H-2’/H-6’); 7.22 (t, 1H, J= 7.5 Hz, H-5); 7.30 (d,
1H, J= 7.5 Hz, H-7); 7.65 (td, 1H, J6,5= J6,7= 7.5 Hz and J6,4= 1.2 Hz,
H-6); 7.81 (d, 1H, J= 7.5 Hz, H-4). 13C NMR (75 MHz, CDCl3) δ (ppm):
56.4 (2 x OCH3); 61.2 (OCH3); 109.2-153.5 (C-1, C-10, Ar); 166.1 (C-8);
184.7 (C=O).
7.54 (d, 1H, Jα,β= 15.6 Hz, H-α); 7.62 (d, 2H, J= 8.7 Hz, H-2/H-6);
7.89-7.94 (m, 2H, H-β and H-6’); 12.97 (s, 1H, OH). 13C NMR (75 MHz,
CDCl3) δ (ppm): 14.3-32.2 (Caliphatic); 68.5 (-OCH2-); 115.2-145.7 (C-
C-β and Ar); 161.9 (C-4); 163.8 (C-2’); 193.9 (C=O).
α,
(Z)-2-(4-(hexyloxy)benzylidene)benzofuran-3(2H)-one (11): Ob-
tained from compound 2. The orange solid was recrystallized from
ethanol. The NMR spectroscopic data are according to the literature
(E)-1-(2-hydroxy-4-methoxyphenyl)-3-(3,4,5-trimethoxyphenyl)prop-
2-en-1-one (6): Obtained from 2’-hydroxy-4’-methoxyacetophenone and
3,4,5-trimethoxyaldehyde. The yellow solid was recrystallized from
ethanol. Yield = 42%. The NMR spectroscopic data are according to
literature (Ducki et al., 2009). 1H NMR (300 MHz, CDCl3) δ (ppm): 3.86
(s, 3H, OCH3); 3.91 (s, 3H, OCH3); 3.93 (s, 6H, OCH3); 6.48 – 6.51 (m,
(Anitha et al., 2016). Yield = 63%, m.p.: 60.2-63.0◦C. IR (KBr)
ν
(cmꢀ 1):
2935 and 2856 (C-Haliphatic); 1697 (C=O); 1655 and 1597 (C=C); 1259
(
νas CAr-O-C). 1H NMR (300 MHz, CDCl3) δ (ppm): 0.93 (t, 3H, J= 6.6 Hz,
-CH3); 1.63 (s, 6H, -CH2-); 1.83 (qui, 2H, J= 6.6 Hz, -OCH2CH2); 4.04 (t,
2H, J= 6.6 Hz, -OCH2-); 6.92 (s, 1H, H-10); 6.99 (d, 2H, J= 8.7 Hz,
H-3’/H-5’); 7.23 (t, 1H, J= 7.5 Hz, H-5); 7.35 (d, 1H, J= 7.5 Hz, H-7);
7.66 (t, 1H, J= 7.5 Hz, H-6); 7.83 (d, 1H, J= 7.5 Hz, H-4); 7.91 (d, 2H,
J= 8.7 Hz, H-2’/H-6’). 13C NMR (75 MHz, CDCl3) δ (ppm): 14.2-31.7
(Caliphatic); 68.4 (-OCH2-); 113.0-136.6 (C-10 and Ar); 146.0 (C-1);
160.9 (C-4’); 166.0 (C-8); 184.6 (C=O).
2H, H-3’, H-5’); 6.87 (s, 2H, H-2, H-6); 7.45 (d, 1H, Jα,β = 15.4 Hz, H-α);
7.81 (d, 1H, Jβ,α = 15.4 Hz, H-β); 7.84 (d, 1H, J = 8.8 Hz, H-6’); 13.47
(s, 1H, OH). 13C NMR (75 MHz, CDCl3) δ (ppm): 55.76; 56.41; 61.18 (4 x
OCH3); 101.20 – 166.88 (C-α, C-β, Ar); 191.80 (C=O).
(E)-1-(2-hydroxy-4,6-dimethoxyphenyl)-3-(3,4,5-trimethoxyphenyl)
prop-2-en-1-one (7): Obtained from 2’-hydroxy-4’,6’-dimethox-
yacetophenone and 3,4,5-trimethoxyaldehyde. The yellow solid was
recrystallized from ethanol. Yield = 52%. The NMR spectroscopic data
are according to the literature (Mateeva et al., 2002). 1H NMR (300
MHz, CDCl3) δ (ppm): 3.84 (s, 3H, OCH3); 3.90 (s, 3H, OCH3); 3.91 (s,
9H, OCH3); 5.97 (d, 1H, J = 2.4 Hz, H-3’); 6.12 (d, 1H, J = 2.4 Hz, H-5’);
(Z)-2-(4-(octyloxy)benzylidene)benzofuran-3(2H)-one (12): Obtained
from compound 3. The yellow solid was recrystallized from ethanol.
Yield = 31%, m.p.: 83.5-85.2◦C. IR (KBr)
ν
(cmꢀ 1): 2942, 2921 and 2845
(C-Haliphatic); 1700 (C=O); 1649 and 1597 (C=C); 1255 (νas CAr-O-C). 1H
NMR (300 MHz, CDCl3) δ (ppm): 0.90 (t, 3H, J= 6.6 Hz, -CH3); 1.30-
1.34 (m, 10H, -CH2-); 1.81 (qui, 2H, J= 6.6 Hz, -OCH2CH2-); 4.01 (t,
2H, J= 6.6 Hz, -OCH2-); 6.89 (s, 1H, H-10); 6.97 (d, 2H, J= 8.7 Hz, H-3’/
H-5’); 7.21 (t, 1H, J= 7.5 Hz, H-5); 7.32 (d, 1H, J= 7.5 Hz, H-7); 7.64 (t,
1H, J= 7.5 Hz, H-6); 7.81 (d, 1H, J= 7.5 Hz, H-4); 7.88 (d, 2H, J= 8.7 Hz,
H-2’/H-6’). 13C NMR (75 MHz, CDCl3) δ (ppm): 14.3-32.0 (Caliphatic);
68.4 (-OCH2-); 113.1-136.7 (C-10 and Ar); 146.0 (C-1); 160.9 (C-4’);
166.0 (C-8); 184.7 (C=O). MS (ESI) m/z: 351.1928 [M+H+] found,
351.1882 [M+H+] calculated.
6.84 (s, 2H, H-2, H-6); 7.71 (d, 1H, Jα,β = 15.5 Hz, H-α); 7.80 (d, 1H, Jβ,
α
= 15.5 Hz, H-β); 14.31 (s, 1H, OH). 13C NMR (75 MHz, CDCl3) δ (ppm):
55.76; 55.96; 56.30; 61.16 (5 x OCH3); 91.49 – 168.58 (C-
192.51 (C=O).
α, C-β, Ar);
(E)-3-(4-bromophenyl)-1-(2-hydroxy-4,6-dimethoxyphenyl)prop-2-en-
1-one (8): Obtained from 2’-hydroxy-4’,6’-dimethoxyacetophenone and
4-bromoaldehyde. The yellow solid was recrystallized from ethanol.
Yield = 61%. The NMR spectroscopic data are according to the literature
(Boeck et al., 2005). 1H NMR (300 MHz, CDCl3) δ (ppm): 3.84 (s, 3H,
OCH3); 3.92 (s, 3H, OCH3); 5.96 (d, 1H, J = 2.4 Hz, H-3’); 6.11 (d, 1H, J
= 2.4 Hz, H-5’); 7.46 (d, 2H, J = 8.5 Hz, H-3, H-5); 7.53 (d, 2H, J = 8.5
(Z)-2-(4-(nonyloxy)benzylidene)benzofuran-3(2H)-one (13): Obtained
from compound 4. The yellow solid was recrystallized from methanol.
Yield = 98%, m.p.: 56.5-58.7◦C. IR (KBr)
ν
(cmꢀ 1): 2921 and 2848 (C-
1
Hz, H-2, H-6); 7.70 (d, 1H, J
α
,β = 15.6 Hz, H-
α
); 7.88 (d, 1H, Jβ,
α
= 15.6
H
aliphatic); 1700 (C=O); 1651 and 1592 (C=C); 1254 (νas CAr-O-C). H
Hz, H-β); 14.21 (s, 1H, OH). 13C NMR (75 MHz, CDCl3) δ (ppm): 55.78;
NMR (300 MHz, CDCl3) δ (ppm): 0.90 (t, 3H, J= 6.6 Hz, -CH3); 1.29 (s,
12H, -CH2-); 1.81 (qui, 2H, J= 6.6 Hz, -OCH2CH2-); 4.02 (t, 2H, J= 6.6
Hz, -OCH2-); 6.90 (s, 1H, H-10); 6.98 (d, 2H, J= 8.7 Hz, H-3’/H-5’); 7.21
(t, 1H, J= 7.5 Hz, H-5); 7.33 (d, 1H, J= 7.5 Hz, H-7); 7.65 (t, 1H, J= 7.5
Hz, H-6); 7.81 (d, 1H, J= 7.5 Hz, H-4); 7.89 (d, 2H, J= 8.7 Hz, H-2’/H-
6’). 13C NMR (75 MHz, CDCl3) δ (ppm): 14.3-32.1 (Caliphatic); 68.4
(-OCH2-); 113.1-136.7 (C-10 and Ar); 146.0 (C-1); 161.0 (C-4’); 166.0
(C-8); 184.7 (C=O). MS (ESI) m/z: 365.2100 [M+H+] found, 365.2117
[M+H+] calculated.
56.05; (2 x OCH3); 91.49 – 168.60 (C-α, C-β, Ar); 192.49 (C=O).
(E)-1-(2-hydroxyphenyl)-3-(thiophen-2-yl)prop-2-en-1-one (9): Ob-
tained
from
2’-hydroxy-acetophenone
and
thiophene-2-
carboxyaldehyde. The yellow solid was recrystallized from ethanol.
Yield = 18%. The NMR spectroscopic data are according to the literature
(Zheng et al., 2011). 1H NMR (300 MHz, CDCl3) δ (ppm): 6.96 (t, 1H, J=
7.5 Hz, H-5’); 7.03 (d, 1H, J= 7.5 Hz, H-3’); 7.12 (t, 1H, J= 4.3 Hz, H-3);
7.41-7.53 (m, 4H, H-α, H-4’, H-2 and H-4); 7.89 (d, 1H, J= 7.5 Hz, H-6’);
8.06 (d, 1H, Jβ,
α
= 15.2 Hz, H-β); 8.78 (s, 1H, OH). 13C NMR (75 MHz,
(Z)-2-(4-(tetradecyloxy)benzylidene)benzofuran-3(2H)-one (14): Ob-
tained from compound 5. The yellow solid was recrystallized from
CDCl3) δ (ppm): 116.1-142.1 (C-α, C-β, Ar and Cheteroaromatic); 162.5
3

V.R.D. Pereira et al.
Acta Tropica 213 (2021) 105741
methanol. Yield = 14%, m.p.: 74.3-75.7◦C. IR (KBr)
ν
(cmꢀ 1): 2921 and
concentration of 100
μ
M, using DMSO stock solutions (10 mM) diluted
2850 (C-Haliphatic); 1702 (C=O); 1653 and 1598 (C=C); 1255 (νas CAr-O-
C). 1H NMR (300 MHz, CDCl3) δ (ppm): 0.89 (t, 3H, J= 6.6 Hz, -CH3);
1.25-1.32 (m, 22H, -CH2-); 1.81 (qui, 2H, J= 6.6 Hz, -OCH2CH2-); 4.01
(t, 2H, J= 6.6 Hz, -OCH2-); 6.89 (s, 1H, H-10); 6.97 (d, 2H, J= 8.7 Hz, H-
3’/H-5’); 7.21 (t, 1H, J= 7.5 Hz, H-5); 7.33 (d, 1H, J= 7.5 Hz, H-7); 7.64
(t, 1H, J= 7.5 Hz, H-6); 7.81 (d, 1H, J= 7.5 Hz, H-4); 7.88 (d, 2H, J= 8.7
Hz, H-2’/H-6’). 13C NMR (75 MHz, CDCl3) δ (ppm): 14.3-32.1 (Caliphatic);
68.4 (-OCH2-); 113.1-136.7 (C-10 and Ar); 146.0 (C-1); 161.0 (C-4’);
166.0 (C-8); 184.7 (C=O). MS (ESI) m/z: 435.2883 [M+H+] found,
435.2821 [M+H+] calculated.
in supplemented RPMI 1640 medium within 24 flat bottom well plates
(Tissue Culture plastics, TPP, St. Louis, MO) with a final volume of 2 mL
per well (Mafud et al., 2016). Compounds were tested in triplicate with
two worms of both sexes placed into each well. Wells with the highest
concentration of DMSO in medium (0.5%) served as controls. Prazi-
quantel (2 μM) served as a positive control. Parasites were kept for 72 h
(37◦C, 5 % CO2) and their viability was assessed via microscopic readout
(Leica Microsystems, Wetzlar, Germany) (de Castro et al., 2015). Next,
compounds presenting antischistosomal activity were tested at 100, 50,
25, 10, and 5 μM as described above and each experiment was per-
(Z)-6-methoxy-2-(3,4,5-trimethoxybenzylidene)benzofuran-3(2H)-one
(15): Obtained from compound 6. The yellow solid was recrystallized
from methanol. The NMR spectroscopic data are according to the liter-
ature (Boussafi et al., 2016). Yield = 56%, m.p.: 178.0-179.0◦C. 1H NMR
(300 MHz, CDCl3) δ (ppm): 3.92 (s, 3H, OCH3); 3.94 (s, 3H, OCH3); 3.95
(s, 6H, OCH3); 6.73 (d, 1H, J = 2.1 Hz, H-7); 6.75 (s, 1H, H-10); 6.77 (dd,
1H, J5,4 = 8.6 Hz, J5,7 = 2.1 Hz, H-5); 7.16 (s, 2H, H-2’, H-6’); 7.72 (d,
1H, J = 8.6 Hz, H-4). 13C NMR (75 MHz, CDCl3) δ (ppm): 56.23; 56.39;
61.17 (4 x OCH3); 96.81 – 168.49 (C-9, C-10, Ar); 183.00 (C=O).
(Z)-4,6-dimethoxy-2-(3,4,5-trimethoxybenzylidene)benzofuran-3(2H)-
one (16): Obtained from compound 7. The yellow solid was recrystal-
lized from methanol. The NMR spectroscopic data are according to the
formed at least three times (Silva et al., 2015).
2.4. Microscopy studies
Adult schistosomes were monitored by light microscopy using a
Leica Microsystems EZ4E (Wetzlar, Germany) (Mafud et al., 2018). Also,
tegumental alteration and quantification of the number of tubercles
were performed in adult male schistosomes for the most active aurone
derivatives 10, 16 and 18 (10, 25, 50, and 100 μM) using a confocal laser
scanning microscope (Laser Scanning Microscope, LSM 510 META, Carl
¨
Zeiss, Gottingen, Vertrieb, Germany) (Mafud et al., 2018). For confocal
1
literature (Zhang et al., 2015). Yield = 91%, m.p.: 182.0-183.0◦C. H
studies, after the occurrence of death, helminths were fixed in a
formalin-acetic acid-alcohol solution and analyzed under a confocal
microscope with autofluorescence excited at 488 nm and emitted light at
505 nm (Mafud et al., 2018).
NMR (300 MHz, CDCl3) δ (ppm): 3.91 (s, 3H, OCH3); 3.93 (s, 3H, OCH3);
3.94 (s, 6H, OCH3); 3.96 (s, 3H, OCH3); 6.15 (d, 1H, J = 1.8 Hz, H-5);
6.34 (d, 1H, J = 1.8 Hz, H-7); 6.70 (s, 1H, H-10); 7.13 (s, 2H, H-2’, H-6’).
13C NMR (75 MHz, CDCl3) δ (ppm): 56.31; 56.36; 56.39; 61.15 (5 x
OCH3); 89.38 – 169.08 (C-9, C-10, Ar); 180.70 (C=O).
2.5. In vivo antischistosomal studies
(Z)-2-(4-bromobenzylidene)-4,6-dimethoxybenzofuran-3(2H)-one
(17): Obtained from compound 8. The yellow solid was recrystallized
from methanol. Yield = 74%, m.p.: 170.0-171.0◦C. The NMR spectro-
scopic data are according to literature (Beney et al., 2001). 1H NMR (300
MHz, CDCl3) δ (ppm): 3.92 (s, 3H, OCH3); 3.96 (s, 3H, OCH3); 6.15 (d,
1H, J = 1.8 Hz, H-5); 6.39 (d, 1H, J = 1.8 Hz, H-7); 6.69 (s, 1H, H-10);
7.55 (d, 2H, J = 8.6 Hz, H-3’, H-5’); 7.72 (d, 2H, J = 8.6 Hz, H-2’, H-6’).
13C NMR (75 MHz, CDCl3) δ (ppm): 56.19 and 56.28 (2 x OCH3);
89.33-169.17 (C-9, C-10, Ar); 180.52 (C=O).
Mice were infected percutaneously with 80 cercariae each. Twenty-
one days (juvenile stage, pre-patent infection) or 49 days (adult worm
stage, patent infection) post-infection, groups of five mice were treated
orally with single doses of 400 mg/kg of candidate compounds dissolved
in 2% ethanol in water (v/v) while untreated mice served as controls
(Guerra et al., 2019; Silva et al., 2017). PZQ, at a single oral dose of 400
mg/kg, was used as a positive control (de Brito et al., 2020). At 63 days
postinfection, animals were euthanized by the CO2 method and
dissected. Surviving schistosomes residing in the mesenteric veins and
the liver were counted and sexed as previously described (Lago et al.,
2019). Activity of test compounds was determined by comparing the
worm reduction in the treated animals relative to the worm burden in
the infected but untreated control groups. The difference was considered
(Z)-2-(thiophen-2-yl-methylene)benzofuran-3(2H)-one (18): Obtained
from compound 9. The yellow solid was purified by column chroma-
tography on alumina (hexane). The NMR spectroscopic data are ac-
cording to the literature (Xu et al., 2018). Yield = 23%. IR (KBr)
ν
(cmꢀ 1): 1696 (C=O); 1643 and 1592 (C=C); 710 (=C-S). 1H NMR (300
MHz, CDCl3) δ (ppm): 7.09-7.19 (m, 3H, H-4’, H-5 and H-10); 7.28 (d,
1H, J= 8.4 Hz, H-7); 7.50 (d, 1H, J= 3.6 Hz, H-2’); 7.56-7.62 (m, 2H, H-6
and H-3’); 7.74 (dd, 1H, J4,5= 8.4 Hz and J4,6= 1.2 Hz, H-4). 13C NMR
(75 MHz, CDCl3) δ (ppm): 107.0-136.7 (C-10, Ar); 145.4 (C-1); 165.6
(C-8); 183.8 (C=O).
statistically
significant
if
p<0.05
using
the
Dunnett’s
multiple-comparison test (de Moraes et al., 2014).
2.6. Randomization and blinding
2.2. Maintenance of the S. mansoni life cycle
For in vivo studies, the animals were randomly assigned to the
experimental groups, and pharmacological treatments were counter-
balanced randomly as well. The animals were euthanized in a random
manner inside a group and all parameters were conducted by different
people, done by two different investigators. Therefore, operators of ex-
periments were not the same as the data analysts, to eliminate bias in
interpretation (Lago et al., 2019).
Schistosoma mansoni (BH strain) was maintained by passage through
Biomphalaria glabrata, as the intermediate host, and Swiss female mice
˜
(Anilab, Sao Paulo, Brazil) as definitive host as previously described (de
Moraes et al., 2014). Both mice and snails were kept under environ-
mentally controlled conditions (temperature, 25◦C; humidity, 50%;
standard food and water ad libitum).
2.3. In vitro antischistosomal assay
2.7. In vitro cytotoxicity studies on macrophages
Seven weeks post infection S. mansoni were removed from the he-
patic portal system and cultured in RPMI 1640 culture medium (sup-
plemented with 5% inactivated fetal calf serum and 100 U/mL penicillin
Cytotoxicity of the active aurones was determined in murine peri-
toneal macrophages using the MTT assay (Mosmann, 1983), according
to a previous report (Antinarelli et al., 2016), using three independent
experiments in duplicate. The values of cytotoxic concentration
reducing 50% of viable cells (CC50) were obtained using GraFit Version
5 software.
and 100 μ
g/mL streptomycin (Vitrocell, Campinas, SP, Brazil) at 37◦C in
an atmosphere of 5% CO2 until usage. For the determination of activity
against adult schistosomes all compounds were initially tested at the
4

V.R.D. Pereira et al.
Acta Tropica 213 (2021) 105741
2.8. In vitro hemolytic activity
Among the tested compounds, aurones 10, 16 and 18 (Fig. 1) showed
the best in vitro antischistosomal activity, with no significant cytotox-
icity to macrophages or to red blood cells, as shown in Table 1. Also,
aurones 10, 16 and 18 were able to decrease the motor activity and to
increase the mortality rates of adult schistosomes in a dose dependent
manner (Table 1). Aurones 10, 16 and 18 caused 100% mortality at 100
Different concentrations (12.5 to 200.0 µM) of the most active
aurones 10, 16 and 18 were incubated in a 96-well microplate with a 1%
erythrocyte suspension for 3h at 37◦C. The microplate was centrifuged
at 1,000 × g for 10 min, and the cell lysis was determined spectropho-
tometrically (540 nm). SDS (sodium dodecyl sulfate) 0.01% and PBS
were used as positive and negative controls, respectively (Dagnino et al.,
2018). The results were presented as the percentage of hemolysis
compared to the positive control and expressed as the 50% hemolytic
concentration of red blood cells (RBC50) (Ahmad et al., 2019).
and 50
analysis and monitoring of cultures, remarkable changes in motor ac-
tivity on adult worms were found (aurones 10 and 16: 100 to 25 M; and
μM of all adult schistosomes (Table 1). After light microscopy
μ
aurone 18: 100 to 10 μM). Following incubation with aurones 10, 16
and 18, significant contractions and paralysis were observed. Indepen-
dent experiments showed a significant and concentration-dependent in
vitro schistosomicidal activity for aurones 10, 16 and 18.
2.9. Ethics statement
All experiments were conducted in conformity with the Brazilian law
for Guidelines for Care and Use of Laboratory Animals (Law 11790/
2008). The protocol for experimental design was approved by the
3.2. In vitro confocal laser-scanning microscope analysis with selected
aurones against S. mansoni
´
˜
Comissao de Etica no Uso de Animais (CEUA), Brazil (Protocols ∕= CEUA
031/2017 and ∕=CEUA 007/2018). Animal studies are reported in
compliance with the ARRIVE guidelines.
In addition to the light microscopy, confocal laser-scanning micro-
scope analysis showed intense and prominent alterations in morphology
of the schistosomes tegument after treatment with aurones 10, 16 and
18 (Fig. 2). Male adult schistosomes showed tegumental damages, with
rupture and/or disintegration of tubercles along the surface of worms,
while non-treated adult worms (Fig. 2B) showed intact surface. In this
regard, a quantitative analysis of the number of intact tubercles on the
dorsal surface of male parasites was performed (Fig. 3). Results showed
a dose-response effect by aurones 10, 16 and 18 on the tubercles of the
male worm teguments. Also, no intact tubercles were seen after exposure
to 100 µM of aurones 10 and 16. Treatment with 10, 16, and 18 led to
extensive damage of the tegument, decreasing the number of intact tu-
bercles, which appeared eroded and deformed (Fig. 3).
2.10. Statistical analysis
All statistical analyses were performed using Graph Pad Prism soft-
ware 5.0 (Graphpad software Inc., La Jolla, CA, USA). For in vivo
experimental analysis, a parametric Dunnett’s multiple-comparison test
was used to analyze the statistical significance of differences between
mean experimental and control values (Roquini et al., 2019). P values of
< 0.05 were considered significant. The data and statistical analysis
comply with the recommendations on experimental design and analysis
in pharmacology (Roquini et al., 2019).
3.3. In vivo studies of selected aurones against S. mansoni in patent
infection
3. Results
3.1. In vitro preliminary studies against S. mansoni
Guided by the best in vitro findings against adult worms, aurones 10,
16, and 18 were in vivo evaluated in mice harboring adult S. mansoni. For
comparison, data obtained with the drug of reference PZQ are also
presented (Fig. 4). Using a single oral dose (400 mg/kg), aurones 10 and
16 achieved a moderate, but significant worm burden reductions of
19.9% (P<0.05) and 34.3% (P<0.001), respectively (Fig. 4). In addi-
tion, the treatment with a single oral dose of 18 (400 mg/kg) markedly
All produced compounds (Fig. 1) were incubated for 24h and tested,
at 100 µM, in a preliminary in vitro screening against S. mansoni. Com-
pounds 1-9, and 12-15 (Fig. 1) did not show any activity for schisto-
somes at the highest concentration tested (100 µM), while compounds
11 and 17 showed weak activities (See Supplementary Table S1).
Table 1
In vitro schistosomicidal and cytotoxic activities of aurone derivatives
Groups
Concentration (µM)
Dead worms (%)a,c
Motor activity reduction
Worms with tegumental
alterations (%)a
Cytotoxicity CC50 (µM)d
RBC50 (µM)e
(%)a
Slight
Significant
Partial
Extensive
Controlb
PZQ
-
-
-
-
-
-
-
-
2
-
100
-
100
-
25
-
75
-
DMSO 0.5%
Auronesc
10
100
50
100
100
-
-
-
-
100
100
100
-
100
100
50
> 150
>150
> 150
> 200
> 200
> 200
-
25
50
16
18
100
50
100
100
-
-
100
100
50
-
-
-
100
100
-
-
25
50
100
50
100
100
50
-
-
100
100
50
-
100
100
50
-
-
-
25
50
50
50
-
10
50
a
Percentages relative to the 20 untreated worms.
RPMI 1640.
b
c
Incubation period: 24h.
d
e
CC50 values (50% cytotoxic concentration) on macrophages.
RCB50 values (50% hemolytic concentration) on red blood cells, - = not determined.
5

V.R.D. Pereira et al.
Acta Tropica 213 (2021) 105741
Fig. 2. Confocal laser scanning microscopy observations of S. mansoni male worms after in vitro incubation with aurones 10, 16, and 18. (A) General view of the
anterior worm region showing, in red, the location where tegument was analyzed. (B) Control containing RPMI 1640 with DMSO 0.5%. (C) 2 μM PZQ. (D) 50 μM 10.
(E) 100
μ
M 10. (F) 50
μM 18. (G) 100
μM 18. (H) 50
μM 16. (I) 100
μ
M 16. Scale bars, 200 μm (A) and 50 μm (B-I).
6

V.R.D. Pereira et al.
Acta Tropica 213 (2021) 105741
Fig. 3. Effect of aurones 10, 16, and 18 on tubercles of S. mansoni male worms. Quantification of the number of tubercles was performed using confocal microscopy.
Intact tubercles numbers were measured in a 20,000
μ
m2 of area calculated with the Zeiss LSM Image Browser software. Praziquantel (PZQ, 2
μM) was used as
reference compound. A minimum of three tegument areas of each parasite were assessed. Values are means ± SD (bars) of ten male adult worms. *P < 0.05 and ***P
< 0.001 compared with untreated groups.
decreased worm load of the total and couple worms by 65.1% (P<0.001)
and 68.8% (P<0.001), respectively, in comparison with untreated con-
trol. PZQ resulted in total worm burden reduction of 93.3% (Fig. 4).
In addition, in feces collected from infected treated mice, the number
of eggs per gram was reduced by 22.4 % (P<0.05), 42.0 % (P<0.01), and
70.0 % (P<0.001) after the oral administration of 10, 16, and 18,
respectively, in comparison with the infected untreated control group
(Fig. 5). Also, considering egg development stages (oogram), only the
administration of 18 was able to reduce significantly the number of
immature and mature eggs by 87.7% (P<0.001) and 54.5% (P<0.01),
respectively (Fig. 5). Besides, the analysis of oogram showed that a
single dose administration of 18 was able to increase by seven-fold
(P<0.001) the amount of dead eggs in comparison with control group.
PZQ produced a highly significant egg reduction in the intestine and
feces (Fig. 5).
3.4. In vivo studies of the aurone 18 against juvenile S. mansoni in pre-
patent infection
Based on the results in patent infection, the efficacy of aurone 18 was
also assessed in juvenile (pre-patent infection) stages of S. mansoni, with
PZQ as a reference drug. The oral treatment with a single dose (400 mg/
kg) of aurone 18 to mice infected with juvenile worms revealed a high
efficacy, showing a significant reduction of 52.5% in the total worm
burden and a reduction of 57.9% in egg burden in comparison with the
infected untreated control group (Fig. 6). Moreover, analysis of the
oogram pattern (Fig. 6) showed that aurone 18 reduced the number of
immature and mature eggs by 70.5% and 39.7%, respectively, also
showing an increase in dead eggs compared with control group. In
contrast, PZQ had a modest antischistosomal property against juvenile
S. mansoni (Fig. 6).
7

V.R.D. Pereira et al.
Acta Tropica 213 (2021) 105741
Fig. 4. Effect on worm burden of a single dose (400 mg/kg, p.o.) of aurones 10 (A), 16 (B), 18 (C), and PZQ (D) administered to mice harboring a 49-day-old adult
S. mansoni infection, stratified by sex. Bars represent data from individual mice that were infected and treated with aurones or infected and untreated (control). *P <
0.05, **P < 0.01, *** P < 0.001 compared with untreated groups.
4. Discussion
antimalarial activities (Hassan et al., 2018, Roussaki et al., 2012). In this
regard, we have synthesized aurones which have methoxy and O-alkyl
groups in their structures to verify if methoxy groups and lipophilicity
could influence the activity. Then, aurone derivatives with methoxy
(aurones 10, 15, 16, and 17) or O-alkyl (aurones 11, 12, 13, and 14)
groups incorporated in the benzofuran and benzylidene rings were
produced. In addition, an aurone bearing a thiophene group (aurone
18), instead of a general benzylidene ring, was also produced.
Schistosomiasis is a neglected disease with a considerable and
serious impact on public health. Also, there is only one available drug to
treat schistosomiasis and no antischistosomal drug in the development
pipeline, with an urgent need to identify new schistosomicidal drugs (de
Brito et al., 2017; Lago et al., 2018). In this regard, aurones are prom-
ising antiparasitic compounds, displaying activities against Leishmania,
Plasmodium, and Trypanosoma species (Hassan et al., 2018; Kayser et al.,
2002). In this context, we have highlighted the antischistosomal activity
of selected aurones against S. mansoni, identifying the thiophenyl aurone
18 as a lead schistosomicidal drug that caused in vitro morphological
alterations in the tegument of adult schistosomes and showed significant
in vivo efficacy against both adult and juvenile worms of S. mansoni in
murine models.
ˆ
According to recent literature (Correa et al., 2019), in vitro assays are
essential tools to the initial selection of a potential anthelmintic drug.
Preliminary in vitro assays against S. mansoni showed that amongst all
tested compounds, aurones 10, 16, and 18 were the most active anti-
schistosomal compounds, being able to cause death on all adult worms
without any significant apparent cytotoxicity against mammalian cells
when tested at the same range of schistosomicidal concentrations.
Although the number of aurone derivatives evaluated in our work is
limited, the analysis of structure-activity relationship indicated that
lipophilic compounds with O-alkyl chains at positions C4’ in the ben-
zylidene moiety (compounds 11 to 14) showed little or no
Then, keeping in view the above-mentioned antiparasitic importance
of aurones, we performed a synthesis of some aurone derivatives. Ac-
cording to previous literature, alkyl and methoxy groups in both A and B
rings of aurone derivatives may enhance their antileishmanial and
8

V.R.D. Pereira et al.
Acta Tropica 213 (2021) 105741
Given the importance of the worm’s tegument in the action of drugs,
confocal laser microscopy studies were performed to evaluate tegument
structures and their alterations at the male surface of worms exposed to
the selected aurones. Along with death, aurones 10, 16, and 18 also
caused noticeable damage at the tegument of male schistosomes with
tubercle structures at the surface of worms exposed being destroyed in a
dose dependent manner. Schistosoma tegument is well-recognized as an
˜
important drug target and study model in schistosomiasis (Guimaraes
et al., 2018). According to well recognized literature, the tegument is
pivotal for the survival of Schistosoma not only because it is one of the
major routes for nutrient absorption, but also because it is important for
the protection of schistosomes, since tegumental changes might result in
ˆ
exposure of parasite antigens to the host immune system (Correa et al.,
˜
2019; Doenhoff et al., 1987; Guimaraes et al., 2018; Xiao et al., 2000).
Due to these in vitro findings, aurones 10, 16, and 18 progressed into
additional evaluation in the S. mansoni mouse model.
In the murine model of schistosomiasis, the treatment with the
respective aurone (10, 16, and 18) had significant differences in efficacy
against adult worms in S. mansoni-infected mice after a patent infection.
Aurones 10 and 16 showed modest, but significant reductions in worm
burden after oral administration of a single dose. In contrast, aurone 18
achieved a high therapeutic efficacy in mice infected with adult
S. mansoni. Interestingly, but not statistically significant, it appeared
that adult female worms were more susceptible than male worms after
treatment with 18, since no female worms were found in treated mice
with 18 in comparison with infected untreated-mice. Considering that,
eggs are closely related to the immunopathogenesis of schistosomiasis
(Costain et al., 2018), a drug able to reduce females is of great
importance.
Also, in this regard, the efficacy on patent infection was also assessed
by oogram and egg load after oral administration of aurones to mice
harboring adult S. mansoni. Quantitative feces examination by the Kato-
Katz method was performed, showing a significant, but less intense,
reduction in the number of eggs in feces after oral treatment with
aurones 10 and 16, which is in agreement with their ability to reduce
worm burden. On the contrary, a significant reduction (~ 70%) in the
number of eggs eliminated in feces (per gram) after oral administration
of 18 in comparison with the infected untreated control group was
found.
Fig. 5. Effects on egg development stages (oogram) (A) and on stool egg load
(B) of a single dose (400 mg/kg, p.o.) of aurones 10, 16, 18 and PZQ admin-
istered to mice harboring a 49-day-old adult S. mansoni infection. Bars and
points represent data from individual mice that were infected and treated with
aurones or infected and untreated (control). *P < 0.05, ** P < 0.01, *** P <
0.001 compared with untreated groups.
In addition, considering the oogram pattern, only treatment with 18
caused significant reductions in immature and mature eggs, along with
significant increase in the number of dead eggs. Considering the oogram
pattern and the findings on egg production, it is possible that the
decrease in egg load was not only a consequence of reduction in worm
burden, but also due to a possible effect of 18 on the female parasite
reproductive system. However, this hypothesis should be further eval-
uated, considering mainly possible damages in the female parasite
ovaries and vitelline glands. We also found a significant reduction in the
number of eggs eliminated in feces from infected treated mice with 10,
16, and 18. Taken together, results on egg production and changes in the
oogram pattern showed a noticeable effect of 18 in egg load, which are
of great significance, since the number of eggs is directly associated to
transmission of schistosomiasis and, mainly, to the inflammatory
antischistosomal activity. On the other hand, most of the aurones having
methoxy groups as electron donating substituents in the benzylidene
moiety showed excellent in vitro activity, as exemplified by aurones 10
and 16. Also, together with three methoxy groups in the benzylidene
moiety (ring B), the incorporation of only one methoxy group at the C6
of the benzofuran ring (ring A) seemed to decrease the activity, as
observed with aurone 15 in comparison with aurone 16. In addition,
even keeping methoxy groups at benzofuran ring, the replacement of
methoxy groups with halogen substituent at the benzylidene moiety
(such as brome in aurone 17) also caused a decrease in the activity in
comparison with aurone 16. Interestingly, the best activity was identi-
fied in aurone 18, in which the benzylidene moiety was modified to the
electron-donating thiophenyl group.
ˆ
response in the host tissues and organs (Correa et al., 2019; de Oliveira
et al., 2017; Gryseels et al., 2001). Given the highest antischistosomal in
vivo efficacy against adult worms of S. mansoni on patent infection, 18
was selected for evaluation against juvenile worms on pre-patent
infection.
Taken together, our results suggest that the electronic nature of the
substituents and their positions played important roles in the anti-
schistosomal activity of aurones. Also, our findings are in agreement
with literature, which reported that aurones bearing methoxy groups at
positions 2 and 4 on ring A and electron donating groups on ring B may
exhibit a good antiparasitic activity (Hassan et al., 2018, Roussaki et al.,
2012).
Our results obtained with the reference drug PZQ (400 mg/kg) were
similar to those described in the literature (de Brito et al., 2020; Xavier
et al., 2020). Thus, when compared to PZQ, which is known to reduce ~
90% of the parasitic burden (Lago et al., 2018), compound 18 exhibited
a moderate antischistosomal efficacy in patent S. mansoni infection.
However, unlike PZQ, which has low efficacy against immature para-
sites (Lago et al., 2018; Xavier et al., 2020), our results showed that 18,
after a single oral dose, was significantly active against juvenile worms
Considering the best in vitro antischistosomal results, aurones 10, 16,
and 18 were selected for additional in vitro morphological studies on
Schistosoma’ tegument.
9

V.R.D. Pereira et al.
Acta Tropica 213 (2021) 105741
Fig. 6. Effect of a single oral dose of aurone 18 (400 mg/kg) and PZQ (400 mg/kg) administered to mice harboring a 21-day-old juvenile S. mansoni infection. A:
effect on worm burden, stratified by sex. B: Effect on egg development stages (oogram). C: Effect on stool egg load. Bars represent data from individual mice that were
infected and treated with 18, or infected and untreated (control). *P < 0.05, ** P < 0.01, *** P < 0.001 compared with untreated groups.
of S. mansoni. Interestingly, data showed that the treatment of 18
significantly reduced more coupled worms (P<0.001) than total, male or
female schistosomes. Also, in pre-patent infection, a remarkable reduc-
tion in egg load, as well as an impairment in the development and
maturity of eggs were observed after oral administration of 18 in mice
infected with juvenile S. mansoni. We also found a significant reduction
in the number of eggs eliminated in feces in mice infected with juvenile
S. mansoni after administration of 18. Moreover, treatment with 18
resulted in the appearance of high number of dead eggs, with significant
decrease in the number of both immature and mature ova in the oogram
assessment, while the untreated control showed a greater number of
immature and mature eggs. All results in egg load and oogram are of
main importance, since when an active drug interrupts or reduces
oviposition, there will be a reduction and a progressive change in the
percentage of viable eggs at different stages of development (Pellegrino
and Katz, 1968).
Conclusion
This study has revealed, for the first time, the in vitro and in vivo
antischistosomal properties of some aurone-type compounds against
S. mansoni. Overall, our best results showed that a thiophenyl aurone
derivative 18 had potent in vitro activity with no cytotoxicity against
mammalian cells and, unlike PZQ, 18 may act in vivo against both adult
and juvenile S. mansoni worms. Finally, our findings identified aurone
derivatives as promising lead antischistosomal compounds, supporting
the potential of aurones as antiparasitic drugs.
Supplementary information
Additional details of the in vitro schistosomicidal activity data of all
1
compounds evaluated (Table S1), as well as HRMS, H and 13C NMR
spectra of aurones.
In the pre-patent infection, a positive relationship between the worm
burden and the egg output was observed, since the reduction in egg load
may correlate with the reduction of worm burden, mainly coupled
schistosomes, causing a noticeable impairment in the development and
maturity of Schistosoma eggs. We also observed that 18 may affect
pairing of coupled worms, which could affect fecundity and survival of
female worms and decreasing their ability to lay eggs. Paired schisto-
somes that remain in the blood system of vertebrate hosts produce
hundreds of eggs per day, resulting in immunopathological and in-
flammatory lesions in the target organ (Costain et al., 2018). However, it
is still unknown whether these anti-fecundity effects are related to a
specific inhibition of reproductive processes. Anti-fecundity effects were
observed with artesunate, which impaired the fecundity of adult female
schistosomes, resulting in significant egg count reduction, mainly of
immature eggs, as well as an increasing of the dead eggs proportion
(Shaohong et al., 2006).
CRediT authorship contribution statement
Vinicius R.D. Pereira: Methodology, Software, Formal analysis,
Investigation, Data curation, Writing - original draft. Lígia S. da Sil-
veira: Methodology, Investigation, Software, Formal analysis, Investi-
gation, Data curation, Writing - original draft. Ana C. Mengarda:
Methodology, Investigation. Ismael J. Alves Júnior: Methodology,
Investigation. Ohana Oliveira Zuza da Silva: Methodology, Investi-
´
gation. Fabio Balbino Miguel: Methodology, Investigation. Marcos P.
Silva: Methodology, Investigation. Ayla das C. Almeida: Methodology,
Investigation, Data curation. Daniel da Silva Torres: Methodology,
Investigation. Priscila de F. Pinto: Methodology, Investigation, Data
curation. Elaine S. Coimbra: Methodology, Resources, Investigation,
´
Data curation. Josue de Moraes: Methodology, Resources, Funding
acquisition, Writing - original draft. Mara R.C. Couri: Methodology,
10

V.R.D. Pereira et al.
Acta Tropica 213 (2021) 105741
Schistosoma mansoni. Antimicrob. Agents Chemother. 59, 1935–1941. https://doi.
org/10.1128/AAC.04463-14.
Resources, Funding acquisition, Writing - original draft. Ademar A. da
Silva Filho: Conceptualization, Methodology, Resources, Funding
acquisition, Writing - original draft, Writing - review & editing, Super-
vision, Project administration, Funding acquisition.
Dagnino, A.P.A., Mesquita, C.S., Dorneles, G.P., Teixeira, V.D.O.N., De Barros, F.M.C.,
Vidal Ccana-Ccapatinta, G., Fonseca, S.G., Monteiro, M.C., Júnior, L.C.R., Peres, A.,
˜
Von Poser, G.L., Romao, P.R.T., 2018. Phloroglucinol derivatives from hypericum
species trigger mitochondrial dysfunction in leishmania amazonensis. Camb. Law J.
145, 1199–1209. https://doi.org/10.1017/S0031182018000203.
´
˜
de Brito, M.R.M., Pelaez, W.J., Faillace, M.S., Militao, G.C.G., Almeida, J.R.G.S.,
Declarations of Competing Interest
The authors declare no conflicts of interest.
Acknowledgements
¨
Argüello, G.A., Szakonyi, Z., Fülop, F., Salvadori, M.C., Teixeira, F.S., Freitas, R.M.,
Pinto, P.L.S., Mengarda, A.C., Silva, M.P.N., Da Silva Filho, A.A., de Moraes, J., 2017.
Cyclohexene-fused 1,3-oxazines with selective antibacterial and antiparasitic action
and low cytotoxic effects. Toxicol. Vitr. 44, 273–279. https://doi.org/10.1016/j.tiv.
2017.07.021.
de Brito, M.G., Mengarda, A.C., Oliveira, G.L., Cirino, M.E., Silva, T.C., de Oliveira, R.N.,
Allegretti, S.M., de Moraes, J., 2020. Therapeutic effect of diminazene aceturate on
parasitic blood fluke Schistosoma mansoni infection. Antimicrob. Agents Chemother.
17. AAC.01372-20. https://doi.org/10.1128/AAC.01372-20.
The authors are grateful to FAPEMIG (Grant numbers PPM 00296/
16; BPD 00284-14; APQ 02015/14; CEX APQ 01287/14), CNPq (Grant
number 487221/2012-5; EDITAL MCTI/CNPq No: 14/2014), 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 Dr. Pedro L. Pinto for assistance with S. mansoni life cycle
de Castro, C.C.B., Costa, P.S., Laktin, G.T., de Carvalho, P.H.D., Geraldo, R.B., de
Moraes, J., Pinto, P.L.S., Couri, M.R.C., Pinto, P., de, F., Da Silva Filho, A.A., 2015.
Cardamonin, a schistosomicidal chalcone from Piper aduncum L. (Piperaceae) that
inhibits Schistosoma mansoni ATP diphosphohydrolase. Phytomedicine 22,
921–928. https://doi.org/10.1016/j.phymed.2015.06.009.
De Farias Santiago, E., De Oliveira, S.A., De Oliveira Filho, G.B., Magalhaes Moreira, D.
R., Teixeira Gomes, P.A., Da Silva, A.L., De Barros, A.F., Da Silva, A.C., Ramos Dos
Santos, T.A., Alves Pereira, V.R., Araújo Gonçalves, G.G., Brayner, F.A., Alves, L.C.,
Wanderley, A.G., Lima Leite, A.C., 2014. Evaluation of the anti-Schistosoma mansoni
activity of thiosemicarbazones and thiazoles. Antimicrob. Agents Chemother. 58,
352–363. https://doi.org/10.1128/AAC.01900-13.
˜
maintenance at the Adolfo Lutz Institute (Sao Paulo, SP, Brazil). We also
thank Dr. Henrique K. Roffato and Dr. Ronaldo Z. Mendonça (Butantan
˜
Institute, Sao Paulo, SP, Brazil) for expert help with confocal microscope
studies (FAPESP Grant number 00/11624-5). This study was financed in
de Moraes, J., de Oliveira, R.N., Costa, J.P., Junior, A.L.G., de Sousa, D.P., Freitas, R.M.,
Allegretti, S.M., Pinto, P.L.S., 2014. Phytol, a Diterpene Alcohol from Chlorophyll, as
a Drug against Neglected Tropical Disease Schistosomiasis Mansoni. PLoS Negl.
Trop. Dis. 8, 51. https://doi.org/10.1371/journal.pntd.0002617.
˜
part by the Coordenaçao de Aperfeiçoamento de Pessoal de Nível Su-
perior - Brazil (CAPES) - Finance Code 001. This work is a collaboration
research project of members of the Rede Mineira de Química (RQ-MG).
de Oliveira, C.N.F., Frezza, T.F., Garcia, V.L., Figueira, G.M., Mendes, T.M.F.,
Allegretti, S.M., 2017. Schistosoma mansoni: in vivo evaluation of Phyllanthus
amarus hexanic and ethanolic extracts. Exp. Parasitol. 183, 56–63. https://doi.org/
10.1016/j.exppara.2017.10.008.
Supplementary materials
Detsi, A., Majdalani, M., Kontogiorgis, C.A., Hadjipavlou-Litina, D., Kefalas, P., 2009.
Natural and synthetic 2’-hydroxy-chalcones and aurones: synthesis, characterization
and evaluation of the antioxidant and soybean lipoxygenase inhibitory activity.
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Supplementary material associated with this article can be found, in
the online version, at doi:10.1016/j.actatropica.2020.105741.
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