Structure–Activity relationship of piplartine and synthetic analogues against schistosoma mansoni and cytotoxicity to mammalian cells

Article abstract of DOI:10.3390/ijms19061802

Schistosomiasis, caused by helminth flatworms of the genus Schistosoma, is an infectious disease mainly associated with poverty that affects millions of people worldwide. Since treatment for this disease relies only on the use of praziquantel, there is an urgent need to identify new antischistosomal drugs. Piplartine is an amide alkaloid found in several Piper species (Piperaceae) that exhibits antischistosomal properties. The aim of this study was to evaluate the structure–function relationship between piplartine and its five synthetic analogues (19A, 1G, 1M, 14B and 6B) against Schistosoma mansoni adult worms, as well as its cytotoxicity to mammalian cells using murine fibroblast (NIH-3T3) and BALB/cN macrophage (J774A.1) cell lines. In addition, density functional theory calculations and in silico analysis were used to predict physicochemical and toxicity parameters. Bioassays revealed that piplartine is active against S. mansoni at low concentrations (5–10 μM), but its analogues did not. In contrast, based on 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and flow cytometry assays, piplartine exhibited toxicity in mammalian cells at 785 μM, while its analogues 19A and 6B did not reduce cell viability at the same concentrations. This study demonstrated that piplartine analogues showed less activity against S. mansoni but presented lower toxicity than piplartine.

Full text of DOI:10.3390/ijms19061802

International Journal of
Molecular Sciences
Article
Structure–Activity Relationship of Piplartine and
Synthetic Analogues against Schistosoma mansoni
and Cytotoxicity to Mammalian Cells
Yuri Campelo 1,2,3, Alicia Ombredane 4, Andreanne G. Vasconcelos 5, Lucas Albuquerque 6,
Daniel C. Moreira 5 ID , Alexandra Plácido 7, Jefferson Rocha 2, Harold Hilarion Fokoue 8,
Lydia Yamaguchi 9, Ana Mafud 10 ID , Yvonne P. Mascarenhas 10, Cristina Delerue-Matos 7,
Tatiana Borges 6, Graziella A. Joanitti 4, Daniel D. R. Arcanjo 11 ID , Massuo J. Kato 9,
Selma A. S. Kuckelhaus 5, Marcos P. N. Silva 12, Josué de Moraes 12 and
José Roberto S. A. Leite 1,5,
*
ID
1
Núcleo de Pesquisa em Biodiversidade e Biotecnologia, Biotec, Universidade Federal do Piauí, UFPI,
Parnaíba-PI, 64202-020 Brazil; yd.dias@hotmail.com
Programa de Pós-Graduação em Biotecnologia, RENORBIO, Ponto focal Universidade Federal do Piauí,
UFPI, Teresina, PI, 64049-550, Brazil; jeffersonbiotec@gmail.com
Instituto de Educação Superior do Vale do Parnaíba, FAHESP/IESVAP, Parnaíba-PI, 64212-790, Brazil
Laboratório de Nanobiotecnologia, Instituto de Biologia, Campus Darcy Ribeiro, Universidade de Brasília,
UnB, Brasília-DF 70910-900, Brazil; aliciaombredane@gmail.com (A.O.); bygra1@gmail.com (G.A.J.)
Área de Morfologia, Faculdade de Medicina, Universidade de Brasília, UnB, Brasília-DF 70910-900, Brazil;
andreannegv@gmail.com (A.G.V.); danielcarmor@gmail.com (D.C.M.); selmask@gmail.com (S.A.S.K.)
Laboratorio de Imunologia, Faculdade de Medicina, Universidade de Brasília, UnB, Brasília-DF 70910-900,
Brazil; lucasfriaca@hotmail.com (L.A.); tatianakarlab@gmail.com (T.B.)
LAQV/REQUIMTE, GRAQ, Instituto Superior de Engenha do Porto, ISEP, Porto 4200-072, Portugal;
alexandra.placido@gmail.com (A.P.); cmm@isep.ipp.pt (C.D.-M.)
Laboratório de Avaliação e Síntese de Substâncias Bioativas, Universidade Federal do Rio de Janeiro, CCS,
Cidade Universitária, Rio de Janeiro-RJ 21941-902, Brasil; hfokoue@gmail.com
Instituto de Química, Universidade de São Paulo, São Paulo-SP 01005-010, Brazil;
lydyama@gmail.com (L.Y.); majokato@iq.usp.br (M.J.K.)
2
3
4
5
6
7
8
9
10
11
12
Instituto de Física de São Carlos, Universidade de São Paulo-SP 01005-010, Brazil;
carolmafud@gmail.com (A.M.); yvonne@ifsc.usp.br (Y.P.M.)
Núcleo de Pesquisas em Plantas Medicinais, NPPM, Universidade Federal do Piauí, UFPI,
Parnaíba-PI 64202-020, Brazil; daniel.arcanjo@ufpi.edu.br
Núcleo de Pesquisa em Doenças Negligenciadas, Universidade de Guarulhos, Guarulhos-SP 07023-070,
Brazil; marcos.p.bio@gmail.com (M.P.N.S.); josuem@usp.br (J.d.M.)
*
Correspondence: jrsaleite@gmail.com or jrsaleite@pq.cnpq.br; Tel.: +55-61-3107-1891
Received: 30 December 2017; Accepted: 27 February 2018; Published: 19 June 2018
Abstract: Schistosomiasis, caused by helminth flatworms of the genus Schistosoma, is an infectious
disease mainly associated with poverty that affects millions of people worldwide. Since treatment
for this disease relies only on the use of praziquantel, there is an urgent need to identify new
antischistosomal drugs. Piplartine is an amide alkaloid found in several Piper species (Piperaceae)
that exhibits antischistosomal properties. The aim of this study was to evaluate the structure–function
relationship between piplartine and its five synthetic analogues (19A, 1G, 1M, 14B and 6B) against
Schistosoma mansoni adult worms, as well as its cytotoxicity to mammalian cells using murine fibroblast
(NIH-3T3) and BALB/cN macrophage (J774A.1) cell lines. In addition, density functional theory
calculations and in silico analysis were used to predict physicochemical and toxicity parameters.
Bioassays revealed that piplartine is active against S. mansoni at low concentrations (5–10 µM),
but its analogues did not. In contrast, based on 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) and flow cytometry assays, piplartine exhibited toxicity in mammalian cells at
785
µM, while its analogues 19A and 6B did not reduce cell viability at the same concentrations.
Int. J. Mol. Sci. 2018, 19, 1802; doi:10.3390/ijms19061802
www.mdpi.com/journal/ijms

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This study demonstrated that piplartine analogues showed less activity against S. mansoni but
presented lower toxicity than piplartine.
Keywords: piplartine; analogues; Schistosoma mansoni; cytotoxicity
1. Introduction
Schistosomiasis, caused by helminth flatworms of the genus Schistosoma, is a chronic and often
painful disease mainly associated with poverty that affects more than 200 million people worldwide,
resulting in major economic and personal impacts from the years of healthy life lost to morbidity [
Recent global burden of disease estimates indicate that approximately 10,000 schistosomiasis-related
deaths occur each year [ ]. Moreover, the current estimate of yearly disability adjusted life-years
(DALYs) for schistosomiasis is 3.4 million [ ]. Three species (Schistosoma mansoni, Schistosoma
1].
2
3
haematobium and Schistosoma japonicum) account for the majority of human infections. The major
aetiological agent of human schistosomiasis is S. mansoni and intestinal schistosomiasis caused by this
species is present in Africa, the Middle East, the Caribbean, and South America [4].
Human treatment with praziquantel is playing a central role in the control and prevention of
schistosomiasis, being the only effective drug currently available [5]. However, praziquantel does not
prevent re-infection and its continued extensive use may result in the future emergence of drug-resistant
parasites. Having a single drug to treat a disease that affects millions of people is a real concern and,
consequently, it is imperative to develop new effective antischistosomal drugs [6].
The use of natural products has a long tradition in medicine, and they have proven to be an
important source of lead compounds in the development of new drugs. In order to provide new
compounds for use in drug development to control schistosomiasis, the search for anthelmintic
compounds, mainly from natural sources, has intensified in recent years [7–11]. Indeed, as reviewed
elsewhere, natural compounds have been recognized as promising candidates for antischistosomal
drugs [12].
In the north-eastern region of Brazil, several plants with active metabolites have been discovered,
among them is the Piperaceae family, which includes several compounds with antimicrobial
activities [13]. Piplartine (3,4,5-trimethoxycinnamoyl-N-dihydropyridin-2-one), also known as
piperlongumine were described in several species of Piper genus, and anti-inflammatory, antitumor,
antifungal, and antiparasitic effects were reported (for review see [14]). Our group previously
demonstrated that piplartine at 10
schistosomula and adult worms [15
µ
M possesses in vitro schistosomicidal activity against S. mansoni
,
16]. We also showed that piplartine caused morphological alterations
in the tegument of parasites in a concentration-dependent manner. The antischistosomal effect of piplartine
in association with other molecules (e.g., praziquantel) had also been described [17,18].
The activity of a molecule is characterised not only by its biological activity against
microorganisms, but also by its toxicity in various mammalian cells. Toxicity studies are, therefore,
performed prior to the administration of a molecule to humans. In order to establish a relationship
between the chemical structure of piplartine and its biological effects, in this study new analogues
of piplartine were synthesised and evaluated against adult S. mansoni ex vivo. The cytotoxicity of
compounds was also evaluated on murine fibroblast (NIH-3T3) and BALB/cN macrophage (J774A.1)
cell lines. Additionally, we also performed theoretical calculations and ultraviolet–visible absorption
spectra to characterize the obtained compounds. Furthermore, physicochemical and toxicological
parameters of piplartine and its derivatives were obtained by a computer-aided prediction.

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2. Results
2.1. Theoretical Calculations and Ultraviolet–Visible Absorption Spectra
The electron density data for the tested molecules are shown in Figure 1, and indicate that
piplartine had the highest dipole moment (5.47). Regions coloured red tended to have a more negative
charge, being polar, while those in blue were more positive. Figure 2 represents the ultraviolet–visible
(UV–Vis) light absorption spectrum of piplartine and its analogues, showing maximum absorbance
from 280 to 400 nm, with dislocation at the bands of the analogues in relation to the original molecule.
Figure 1. Electron density: (a) piplartine; (b) 19A; (c) 1G; (d) 14B; (e) 6B and (f) 1M. The colours
represent negative (red) and positive (blue). The dipole moments of all compounds in debye values are as
follows: piplartine: 5.4728337; 1G: 1.9658996; 1M: 4.4568136; 6B: 4.0062111; 14B: 3.9403826; 19A: 3.7385640.
Figure 2. Ultraviolet and visible light absorption spectra of piplartine and its analogues in arbitrary
units (A.U.).

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2.2. In Silico Studies Information
To investigate the differences between piplartine and its analogues, a structural and
physicochemical analysis was performed to calculate the van der Waals surface area (vdW), molecular
electrostatic potential (MEP) surface, molecular lipophilicity potential (MLP) and polar surface area
(PSA), LogP values, predicted solubility, and molecular weight of the compounds, using the ChemAxon
method. The results are shown in Table 1. Despite significant differences in surface area, the most
significant difference between the compounds was in the topological polarised surface area (TPSA).
TPSA of a molecule was defined as the surface sum over all polar atoms and is based simply on the
summation of polar fragments representing tabulated surface contributions, i.e., bonding patterns of a
molecule [19]. The fragments present in the molecules were compared with a library of substructure
fragments to determine the toxic characteristics of each of the studied molecules as well as to predict
toxicity activity. The results of this analysis are shown in Table 2, and indicate that piplartine shows
greater toxicity and increased ability to bind to nuclear receptors than its analogues, effects that can be
related to its cytotoxic effects observed during in vitro tests in mammalian cells. In addition, the Ames
mutagenicity prediction revealed only positive for piplartine.
Table 1. Basic, geometric, structural, and solubility parameters of the parent and analogue compounds.
Compounds
Piplartine
19A
14B
6B
1M
1G
Basic properties
Atom count
Mw
40
51
31
48
56
38
315.32
287.31
432.48
387.39
239.27
359.42
Structural properties
Asymmetric atom count
Rotatable bond count
Ring count
Aromatic ring count
Hetero ring count
0
5
2
2
1
0
5
0
0
6
2
1
1
0
5
0
0
2
3
2
2
0
2
0
0
5
3
3
0
3
5
0
0
7
3
3
0
1
5
0
0
5
2
2
1
0
4
0
Hydrogen bond donor count
Hydrogen bond acceptor count
Formal charge
Fsp3 (a)
0.18
65.07
87.42
32.54
0.40
65.07
101.15
38.29
0.13
31.23
70.63
26.66
0.00
93.11
112.82
41.46
0.12
77.10
123.64
46.66
0.19
49.69
79.89
30.59
Topological polar surface area (A2)
Molar refractivity cm3/mol
Polarisability (A3)
Solubility parameters
logP
Milogp(b)
logD pH range 1.7–8.0
Intrinsic solubility
Solubility category
logS pH range 1.7–8.0
1.89
2.19
1.89
−3.01
High
−3.01
2.71
2.78
2.71
−4.53
Moderate
−4.53
2.36
4.69
2.36
−2.87
High
−2.87
3.55
5.02
3.54
−4.73
Low
−4.73
4.63
2.86
4.63
−5.48
Low
−5.48
2.26
2.90
2.26
−2.81
High
−2.81
Geometry parameters
Van der Waals volume (Å3)
Volume (Å3) (b)
281.06
337.61
263.86
543.27
659.24
65.07
213.99
341.62
340.76
515.78
631.17
93.11
390.54
218.97
620.14
725.57
77.10
260.80
339.43
423.69
577.96
49.69
282.84
425.62
549.15
65.07
394.07
333.75
445.82
31.2
Van der Waals surface area (Å2)
Solvent accessible surface area (Å2)
Topological polar surface area (Å2)
Absorbance
322 nm
329 nm
321 nm
312 nm
306.5 nm 301.5 nm
(a) Number of sp3 carbons/number of carbons; (b) Molinspiration method. 19A, 14B, 6B, 1M, and 1G are analogue
compounds of piplartine. Fraction of sp3 carbon atoms (Fsp3).

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Table 2. Toxicity analysis and bioactivity prediction of the parent and analogue compounds against
various drug targets.
Compounds
Toxicity prediction (c)
Piplartine
19A
14B
6B
1M
1G
AMES toxicity
Yes
1.006
No
No
0.677
No
No
1.043
No
No
1.095
No
No
1.051
No
Max. tolerated dose (human) (0.505 log mg/kg/day)
hERG I inhibitor
1.114
hERG II inhibitor
No
No
No
Yes
No
Oral Rat Acute Toxicity (LD50) (2.661 mol/kg)
Oral Rat Chronic Toxicity (LOAEL) (3.402 log mg/kg bw/day)
Hepatotoxicity
2.268
1.64
No
2.295
1.787
Yes
2.413
1.812
No
2.195
2.455
Yes
2.37
2.388
Yes
2.389
1.592
No
Skin sensitisation
No
No
No
No
No
No
T. pyriformis toxicity(0.285 × log µg/L)
Minnow toxicity (5.577 × log mM)
Predictive bioactivity (b)
GPCR ligand
1.138
1.293
1.359
0.446
2.004
1.179
0.437
0.658
1.591
−0.172 −0.914 1.027
0.13
0.17
0.07
−0.11
−0.27
−0.20
−0.31
−0.38
−0.17
−0.04
−0.29
−0.09
−0.15
−0.54
−0.14
−0.31
−0.48
−0.00
Ion channel modulator
−0.51
−0.13
−0.32
−0.40
−0.02
−0.16 −0.53
−0.12 −0.13
−0.07 −0.12
Kinase inhibitor
Nuclear receptor ligand
Protease inhibitor
0.02
−0.39
0.24
−0.33
−0.12
Enzyme inhibitor
0.05
(a) Number of sp3 carbons/number of carbons; (b) Molinspiration method; (c) pkCSM. The Ames test is
a widely employed method that uses bacteria to test whether a given chemical can cause mutations in the
DNA of the test organism (Ames); The human ether-a-go-go-related gene (hERG); median lethal dose (LD50);
Lowest-Observed-Adverse-Effect Level (LOAEL); G-protein-coupled receptors (GPCR).
2.3. Cytotoxicity Assay
The cytotoxicity of piplartine and its analogues was evaluated by MTT assay in a murine fibroblast
(NIH-3T3) cell line at concentrations from 25 to 800 µg/mL. The results demonstrated that piplartine
decreased cell viability after 24 h of exposure, exhibiting considerable toxicity (Figure 3). Analogues 1G,
14B, and 1M promoted a considerable reduction in cell viability at concentrations of 400 and 800 µg/mL
when compared with piplartine. Furthermore, 1G and 14B presented the highest pD2 and Emax values
when compared with the other analogues (Table 3). However, analogues 19A and 6B did not exert
significant effects on mammalian cell viability at the tested concentrations when compared with the
effects of piplartine (Figure 3). The pD2 values of 19A and 6B were estimated based on their respective
Emax values, which were lower than 50% (Table 3), indicating lower cytotoxicity than piplartine and
the other analogues. Thus, among the tested compounds, piplartine presented the highest toxicity.
The dimethylsulphoxide (DMSO) vehicle control displayed no effect on cell viability.
Table 3. Determination of pD2 and Emax for the cytotoxic effect of piplartine and its analogues in
murine fibroblast (NIH-3T3) cells.
a
Emax (%) b
Compounds
pD2 (−logIC50
)
19A
1G
1M
14B
6B
−3.06 ± 0.15
−2.00 ± 0.03
−2.31 ± 0.04
−1.95 ± 0.04
−2.80 ± 0.08
5.68 ± 2.95
37.93 ± 2.85
95.69 ± 0.79
70.94 ± 1.20
83.64 ± 0.47
47.93 ± 2.52
96.94 ± 0.12
Piplartine
Negative logarithm of the mean inhibitory concentration (IC50); b Emax: decrease in cell viability at the maximum
a
concentration tested (800 µg/mL).

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Figure 3. Cytotoxicity evaluation using the MTT method in murine fibroblast (NIH3T3) cells after
exposure for 24 h to piplartine analogues ( ) 19A (72–2331 M), ( ) 6B (62–1998 M), ( ) 1G
(85–2738 M), ( ) 1M (56–1796 M), ( ) 14B (101–3250 M) and ( ) piplartine (78–2514 M). Molecules
were used at the same concentrations range at g/mL for all samples (25–800). Dimethyl sulfoxide
A
µ
B
µ
C
µ
D
µ
E
µ
F
µ
µ
(DMSO) was used as a negative control. The values are expressed as mean
DMSO control group. The dotted lines mark the 100% viability level.
±
SEM. * p < 0.05 vs.
The cytotoxicity of piplartine and its analogues was also evaluated by flow cytometry in a mouse
BALB/cN macrophage (J774A.1) cell line at concentrations of 25, 100 and 400 g/mL with exposure
µ
for 24 h (Figure 4). The DMSO vehicle control caused a significant (p < 0.05) reduction in cell viability,
similar to the hydrogen peroxide (H2O2) cell death control. Piplartine significantly (p < 0.05) affected
cell viability at concentrations of 25 and 100
µg/mL when compared with a DMEM untreated control.
Analogue 1M, at all tested concentrations, and analogue 1G at 100
µ
g/mL (342 M) significantly
µ
altered (p < 0.05) cell viability in comparison with the negative control, but not in a toxic manner.
Other analogues did not show significant effects on cell viability. Interestingly, treatment with
piplartine and its analogues appeared to reduce the cytotoxicity of the DMSO vehicle on J774A.1 cells.

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Figure 4. Cytotoxicity evaluation in the mouse BALB/cN macrophage (J774A.1) cell line following
exposure for 24 h to piplartine analogues ( ) 19A (72, 291 and 1165 M), ( ) 6B (62, 249 and 999 M),
) 1G (85, 342 and 1369 M), ( ) 1M (56, 224 and 898 M), ( ) 14B (101, 406 and 1625 M) and (
piplartine (78, 314 and 1257 M). Molecules were used at the same concentrations range at g/mL for
A
µ
B
µ
(
C
µ
D
µ
E
µ
F)
µ
µ
all samples (25, 100 and 400). Dulbecco’s Modified Eagle Medium (DMEM) was used as a negative
control. Cells were analysed by flow cytometry (20,000 events/sample). The values are expressed as
mean ± SEM. * p < 0.05 and **** p < 0.0001 vs. DMEM control group.
In the same cell viability assay, the percent of non-viable cells was analysed regarding the cytotoxic
mechanism in mammalian cells using annexin-V conjugated to green-fluorescent FITC and propidium
iodide (PI) staining to distinguish apoptotic cells (annexin-V FITC) from necrotic cells (PI) by flow
cytometry (Figure 5 and Figure S2). As expected, the control of cell death by apoptosis (H2O2)
showed a significant (p < 0.05) increase in annexin-V FITC fluorescence intensity compared with the
DMEM negative control. A significant (p < 0.05) increase in annexin-V FITC staining was observed in
J774A.1 cells following treatment with piplartine 25
(1625 M) when compared with the DMEM negative control. Furthermore, significant (p < 0.05)
differences between the annexin-V FITC and PI fluorescent signals were observed for each group of
µg/mL (785 µM) and analogue 14B at 400 µg/mL
µ

Int. J. Mol. Sci. 2018, 19, 1802
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J774A.1 cells treated with piplartine and its analogues, indicating that apoptosis was the principal
mechanism of cell death.
Figure 5. Evaluation of the cell death mechanism mediated by (
its analogues ( ) 1G (85, 342 and 1369 M), ( ) 1M (56, 224 and 898
) 14B (101, 406 and 1625 M) and ( ) 19A (72, 291 and 1165 M) in J774A.1 cells following treatment
for 24 h at the same concentrations range at g/mL for all samples (25, 100 and 400) using annexin-V
FITC (apoptosis marker) and propidium iodide (PI, necrosis marker) staining. Cells were analysed
by flow cytometry (20,000 events/sample). The values are expressed as mean SEM. * p < 0.05 vs.
A
) piplartine (78, 314 and 1257
µM) and
B
µ
C
µ
M), ( ) 6B (62, 249 and 999
D
µM),
(
E
µ
F
µ
µ
±
DMEM untreated control group. # p < 0.05 vs. apoptosis staining from each respective group.
2.4. In Vitro Experiments in Adult Worms of S. mansoni
The activity of the amide piplartine and its analogues against adult worms of S. mansoni aged
49 days was most prominent at the concentration of 10 µM and incubation time of 96 h (Table 4).
The amide piplartine showed activity against the parasite 24 h after incubation, while its analogues
showed no activity at the tested concentrations when compared with piplartine, as noted in Table S1.

Int. J. Mol. Sci. 2018, 19, 1802
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Table 4. In vitro effects of compounds in 49-day-old S. mansoni worms.
Motor Activity Reduction (%) a
Slight Significant
Dead Worms (%) a
Period of Incubation
(h)
Group
M
F
M
F
M
F
24
48
72
96
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Control b
24
48
72
96
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0.5% DMSO
Praziquantel
24
100
100
0
0
100
100
48
72
96
100
100
100
100
100
100
0
0
0
0
0
0
100
100
100
100
100
100
2 µM
Amide piplartine
24
48
100
100
100
100
0
0
0
0
100
100
100
100
10 µM
72
96
24
48
72
96
100
100
0
0
0
100
100
0
0
0
0
0
100
100
100
0
0
0
100
100
100
0
100
100
0
0
0
100
100
0
0
0
5 µM
60
60
60
60
a
Percentages relative to the 20 worms investigated; b RPMI 1640. The effect of the compounds on motor activity of
adult S. mansoni was assessed qualitatively. Male (M) and Female (F).
3. Discussion
Schistosomiasis is a global public health problem, and the search is ongoing for drugs to treat this
neglected disease and meet the requirements of the standard treatment, praziquantel. Because it is a
safe medication when administered to children, adults, the elderly and pregnant women, with low
toxicity and few side effects, the use of praziquantel has been increasing, despite the number of
patients who are resistant to the drug. Thus, the development of new anthelmintic compounds to treat
schistosomiasis has become a major research objective [6,20].
Natural products represent an alternative to conventional therapeutic compounds because of
the diversity in molecules and biological activities that these products present. Biodiversity results
a large number of compounds with activities that are as yet unknown. Despite advancements in
biotechnology, genomics and medicinal chemistry, the discovery of new drugs for the treatment of
schistosomiasis remains challenging, since in addition to biological activity, drugs are required to
have low toxicity [6,21]. In this context, the objective of this work was to study the schistosomicidal
activity and cytotoxicity of piplartine and its synthetic analogues, and to compare the analogues to the
parent compound.
An alternative strategy to minimise drug resistance is the chemical synthesis of established drugs
or natural compounds with activities previously described in the literature, where chemical-structural
modifications are made in functional groups that may be responsible for biological activity [6].
These modifications can increase, decrease or abolish biological activity; in some instances,
these changes can promote decreased toxicity, but with an associated loss of biological activity.
The structural changes made to piplartine to produce the analogues in this study resulted in a loss of
activity against S. mansoni and a decrease in toxicity. Studies show that phenotypic analysis remains the
optimal approach to the screening of anthelmintics as it is a simple and low-cost, qualitative strategy,
widely used in in vitro testing [22,23].
Several studies have demonstrated the activity of a range of natural products against S. mansoni,
although the mechanism of action of these compounds remains unknown, such as, piplartine, which is
highly active against this parasite [15]. Previous results have shown that piplartine promotes changes
in motility, instigating the death of the parasite, and that no difference in response was observed

Int. J. Mol. Sci. 2018, 19, 1802
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between male and female parasites [15]. According to the theoretical data shown in Figure 1, piplartine
presents a higher electron density around the molecule, with a dipole moment of 5.47, which may
influence its anthelmintic activity.
It is necessary for a drug to go to the market that the drug has good ADMET (absorption,
distribution, metabolism, excretion, toxicity) properties. Drug toxicology is one of the crucial research
fields in the preclinical study. Toxicity is a leading cause of attrition at all stages of drug development.
In silico prediction of compound toxicity which can reduce the expenses of the company and save a lot
of time has attracted considerable attention [24].
Compound 1G did not exhibit antischistosome activity at the concentrations tested. As shown
in Figure 1, this compound had a low electron density, presenting a very low dipole moment
when compared with piplartine. Changes in the functional groups of a molecule can influence
its biological activity, and in this compound, modifications were observed in the dihydropiperidinone
ring, which was replaced by piperidinyl [25].
Compounds 14B and 19A have similar electron densities, based on their dipole moment values,
which are close to that of piplartine. The 19A molecule has a double bond between the rings
that underwent modifications, as well as a change in the aliphatic chain between these two rings.
Compound 14B underwent a dihydropiperidinone modification, being replaced with a piperidinyl
ring, and in the trimethoxybenzene moiety, substitution with a benzodioxol ring quashed the
schistosomicidal activity of this derivative [25]. These compounds have high electron densities and are
highly similar to piplartine but are large and bulky compounds when compared with their prototype.
These features may explain their lack of interaction with enzymes and ion channel receptors in the
parasite, promoting the loss of biological activity against S. mansoni when compared with piplartine,
whereas piplartine at low concentrations promotes alteration of the parasite tegument. Compound 14B
has a double bond between the two altered portions, thus increasing the distance between the two rings
forming the compound, which may have influenced the loss of this biological activity.
Compounds 1M and 6B present electronic densities similar to piplartine, according to Figure 1,
but did not present biological activity against the S. mansoni parasite. This loss in activity may be
related to the large size of these molecules, which prevents binding to enzymes that are in the tegument,
meaning that these molecules are unable to promote the necessary modifications in the membrane of the
parasite tegument leading to the death of the parasite. In the compound 1M, the dihydropiperidinone
moiety has been replaced by a trimethoxybenzene ring and the trimethoxybenzene moiety has been
replaced by a dicyclohexyl ring. This change in the position of the trimethoxybenzene ring may
influence the biological response by causing this compound to lose its activity against the parasite.
In compound 6B, the trimethoxybenzene is replaced by a dicyclohexyl ring, as in compound 1M,
which may promote the loss of activity against S. mansoni parasite when compared with piplartine,
because both of these compounds, with this same alteration, lost biological activity.
Complementary studies are still required to determine the relationship between the chemical
structure and biological activities of piplartine and its analogues to facilitate the discovery of
new prototypes from piplartine. The substitution of a hydrogen atom with a different functional
group such as methoxyl, nitro, halogen or others may alter the biological activity, and this may
subsequently promote changes in the duration and nature of the pharmacological effect as well
as in the physicochemical characteristics of the molecules, thus contributing to the production of
new molecules. From this study, it can be observed that functional groups trimethoxybenzene and
dihydropiperidinone are important for promoting schistosomicidal activity, because their removal or
alteration in modified analogues resulted in the loss of biological activity as well as the toxicity of the
compound [26].
In murine fibroblast cells, piplartine showed toxicity at all concentrations tested while compounds
1G and 14B, but not the other derivatives, showed toxicity at 200, 400 and 800 µg/mL. As shown by
the solubility parameters (Table 1), ultraviolet and visible light absorption spectra of piplartine and
its analogues it was possible to observe that the analogues present values close to that of piplartine,

Int. J. Mol. Sci. 2018, 19, 1802
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indicating that there are similarities of functional groups between these molecules (Table 1 and
Figure 2),; these compounds presented values close to that of piplartine, and the toxicity analysis
and bioactivity prediction (Table 2) showed that these synthetic compounds were again similar to
piplartine, thus explaining their toxicity. Similarly, flow cytometry cytotoxicity assays in mouse
BALB/cN macrophages showed that piplartine was more toxic to mammalian cells than the analogues.
However, a marked reduction in the cytotoxicity mediated the by DMSO vehicle was observed in cells
treated with piplartine and the analogues, indicating not only that the molecules modulated the toxic
effect in mammalian cells but also that they had a protective effect.
Annexin-V FITC/PI staining was used to determine the cell death mechanism observed in the
cytotoxicity assays by flow citometry. Recombinant annexin-V conjugated to FITC identifies exposed
phosphatidylserine on the inner membrane and is used to detect the initial phase of apoptosis [27],
while PI, a red-fluorescent nucleic acid probe to which living cells are impermeable, can be used to
detect necrotic cells [28]. In this study, piplartine- and analogue-treated J774A.1 cells showed increased
annexin-V FITC staining after 24 h compared to that after PI staining, suggesting that the mechanism
of cell death was related to apoptosis [29]. Apoptosis is characterized by cell shrinkage, chromatin
condensation and systematic DNA cleavage in a controlled way which prevents the inflammatory
process [30]. These data indicate the potential of a rational design of bioactive molecules with low
toxicity from piplartine, while further studies are required.
Studies show that the biological and toxicological activity of piplartine can be attributed to
the presence of the
unsaturated groups leads a molecule without cytotoxicity and to the loss of biological activity [14
Research using this compound has indicated that the unsaturated groups in the aliphatic chain
α
,
β
unsaturated carbonyl groups, where the substitution of any of these
,
31,32].
α, β
present between the two rings are important for the biological activity against S. mansoni as they
maintain the conformational arrangement of the two rings, whereas changes in this arrangement
decrease cytotoxicity. This effect was observed in the compounds synthesised without the double bond.
This study shows that the modification of piplartine to produce its derivatives resulted in
the loss of schistosomicidal activity, indicating that the functional groups trimethoxybenzene
and dihydropiperidinone are essential. The synthesis approach used promoted alterations in
conformational arrangements, the hydrophilic-lipophilic balance, and the distribution of electronic
density compared to those of piplartine. Modifications to trimethoxybenzene and the aliphatic chain
were the most significant, as changes to the methoxyl radical in the meta position of the aromatic ring
promoted the loss of schistosomicidal activity and subsequent reduction in toxicity.
4. Materials and Methods
4.1. Piplartine and Synthetic Analogues
Roots of Piper tuberculatum were harvested on the campus of the University of São Paulo (USP),
São Paulo, Brazil. Botanical classification was performed by Elsie Franklin Guimarães (Instituto de
Pesquisas Jardim Botânico do Rio de Janeiro). A voucher specimen (Kato-0169) has been deposited
at the herbarium of the same institute. Piplartine was isolated from the dry roots of P. tuberculatum
according to a published procedure [25]. Purity was determined by reversed-phase chromatography
(RP-HPLC) and the structure by proton nuclear magnetic resonance (1H-NMR) as described also in
the supplemental material section (Figure S1).
The cinnamides 1G and 14B were synthesised by adding triethylamine (3 equivalent) and amine
(pyrrolidine) to a solution of acid chloride (1 equiv) in CH2Cl2. To prepare the acid chloride, a solution
of 3,4,5-trimethoxycinnamic acid and 3,4-methylenedioxy cinnamic acid (1 equiv.) (Sigma Aldrich,
St. Louis, MO, USA) in dry tetrahydrofuran (THF) (10 mL), kept under nitrogen atmosphere,
and oxalyl chloride (5 equiv.) was added dropwise and stirred at room temperature for 5–6 h.
Excess oxalyl chloride was then removed under reduced pressure, yielding the corresponding acid
chloride. The reaction mixture was stirred overnight at room temperature, and quenched with

Int. J. Mol. Sci. 2018, 19, 1802
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saturated aqueous NH4Cl, and extracted with CH2Cl2 (three times). The combined organic phases
were washed with brine and dried over MgSO4. After filtration and concentration, the residue was
purified by flash chromatography to provide the desired amide [33].
Compounds 1M and 6B were synthesised by adding to a solution of 2E-3,4,5-trimethoxycinnamic
acid and 4-nitrocinnamic acid (1 equiv.) in THF 0.9 equiv. of N,N0-dicyclohexylcarbodiimide
(DCC). The reaction mixture was stirred overnight at room temperature and dried on a rotary
evaporator to remove any remaining solvent. The product was dissolved in (dichloromethane) DCM,
a saturated solution of NaHCO3 was added, and the resulting solution was washed three times
with DCM. The organic phase was combined, dried with MgSO4, filtered, and concentrated under
pressure. The obtained crystalline product was then recrystallised on hot methanol or purified by
chromatography [25,33].
Compound 19A was obtained by adding to a solution of acid chloride (1 equiv.) in THF 1 equiv.
butanol. The 2E,4E-5-3,4,5-trimethoxyphenyl penta-2,4-dienoic acid was synthetised as described
previously [34].
4.2. Theoretical Calculations
Density Functional Theory (DFT) [35] calculations were performed to determine the energy of the
frontier orbitals with b3lyp [36] and basis set 6-311g with two polarisation functions (d,p) and diffuse
functions for hydrogen (++), using Gaussian 09 software (Gaussian Inc., Wallingford, CT, USA) for
theoretical calculations [37]. Dipole moment structures were calculated to determine electron density
using GaussView 5.0 software (Gaussian Inc., Wallingford, CT, USA).
4.3. UV–Vis Absorption Spectra
Ultraviolet and visible light (UV–Vis) absorption spectra of piplartine and its analogues were
obtained with a UV-1280 spectrophotometer (Shimadzu, Kyoto, Japan). Purified piplartine and
its analogues (1G, 1M, 6B and 14B) were solubilised in HPLC-grade acetonitrile and diluted to a
concentration of 15
µg/mL. Because of its high absorbance and low solubility, the 19A analogue was
further diluted to 7.5
µg/mL before measurement. Scans were performed using a quartz cuvette
with a path length of 1 cm in the 200–450 nm range against a baseline recorded with acetonitrile at
room temperature.
4.4. In Silico Physicochemical and Toxicity Studies
Two-dimensional structures were designed using Marvin Sketch 17.4.3, 2017, ChemAxon
(Available online: http://www.chemaxon.com). 3D structures were geometrically optimised
using Another Molecular Modeling Program—Vis (AMMP-Vis) (AMMP 2.0, Monterey, CA,
USA), a full-featured molecular dynamics program for manipulating both small molecules and
macromolecules including proteins, nucleic acids, and other polymers [38]. Ammp-Mon charges were
added and an assisted model building with energy refinement (AMBER) field force was applied to
minimise the energy conformation using a genetic algorithm with 3000 steps, applying the water
dielectric constant. The conformational search was performed using the Boltzmann jump method,
with flexible and psi torsions, at 300 K, and with dielectric constant equal to 80,000 and root mean
squared deviation RMSD equal to 60.00.
Physicochemical properties were also calculated, including molecular weight (MW), lipophilicity
(logP), octanol-water coefficient solubility (logD), water solubility (logS), rotatable bonds, topological
polar surface area (tPSA), number of hydrogen bond acceptors and number of hydrogen bond donors
using Chemicalize, a web tool to predict basic, geometric, structural and solubility parameters,
Calculation, August/2017, (Available online: https://chemicalize.com/; developed by ChemAxon
(Available online: http://www.chemaxon.com)).

Int. J. Mol. Sci. 2018, 19, 1802
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The toxicity analyses were performed by pkCSM [39
,40]. The biological activity of the parent
drug and related compounds was predicted using the Molinspiration web server (http://www.
molinspiration.com/).
4.5. Cytotoxicity Assay
4.5.1. NIH3T3 and J774A.1 Cell Culture
Murine fibroblast (NIH3T3) cells were acquired from the cell bank of Rio de Janeiro (Rio de Janeiro
,
Brazil), and the murine BALB/cN macrophage (J774A.1, TIB-67
™
) (ATCC®, Washington, DC, USA).
Cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (Gibco BRL, Grand Island, NY,
USA) supplemented with 10% heat inactivated foetal bovine serum (Life, USA) and 1% antibiotic
solution (100 IU/mL penicillin/100 µg/mL streptomycin, Life, USA) at 37 ◦C and 5% CO2.
4.5.2. Cell Viability Assays
Viability assays were performed by the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium
bromide) dye reduction method. NIH3T3 cells were seeded into 96-well culture plates at a density
of 3
Next, the medium was removed and various concentrations (25–800
in 3% DMSO were applied. DMSO (3%) was used as a control. The plates were incubated for
24 h [28]. Next, 15 L of MTT solution (5 mg/mL in phosphate buffer) was added to each well and
the plates were incubated for 2 h. Medium was discarded and 100 L of DMSO was added to each
×
103 cells in DMEM culture medium overnight at 37 ◦C and 5% CO2 in a humid atmosphere.
µg/mL) of samples reconstituted
µ
µ
well. Absorbance was determined using a spectrophotometer with a microplate reader at 595 nm
(Molecular Devices Corporation, Sunnyvale, CA, USA).
For viability assays by flow cytometry method, J774A.1 cells were seeded into 96-well culture
◦
plates at a density of 1.0
×
105 cells in DMEM culture medium for 2 h at 37 C and 5% CO2 in a
humid atmosphere, and samples reconstituted in 1% DMSO were added to final concentrations of 25,
100 and 400 g/mL. The plates were incubated for 24 h [28], washed in phosphate buffered saline,
and resuspended in 100 L of binding buffer (10 mM (4-(2-hydroxyethyl)-1-piperazineethanesulfonic
acid) HEPES buffer pH 7.4, 140 mM NaCl, and 2.5 mM CaCl2). Cells were then treated with 2
of annexin-V FITC (Apoptosis Detection Kit; BD Biosciences, Franklin Lakes, NJ, USA) and 2 L of
L binding
µ
µ
µL
µ
propidium iodide (PI, 50 µg/mL) for 15 min in the dark at room temperature. Next, 100 µ
buffer was added and samples were analysed by flow cytometry (BD LSFortessaTM, Franklin Lakes,
NJ, USA). A total of 20,000 events were collected per sample.
4.6. Animals and Parasites
The Belo Horizonte (BH) strain of S. mansoni was maintained in Biomphalaria glabrata snails
and Mesocricetus auratus hamsters at the Adolfo Lutz Institute (São Paulo, Brazil) according to
standard procedures. Detailed methods for the infection of molluscs and hamsters, as well as for
the recovery of parasites, were as described previously [41]. Briefly, the intermediate host snails
were exposed to light for approximately 3 h and cercariae of S. mansoni were subsequently harvested.
Hamsters (aged 3 weeks, weight 25 g) were infected subcutaneously with 150 cercariae and, 49 days
post-infection, adult S. mansoni specimens were recovered from each hamster by perfusion with
Roswell Park Memorial Institute (RPMI) medium 1640 medium (Invitrogen, São Paulo, Brazil) and
heparin. This study was approved by the Institutional Review Board of the Guarulhos University
(São Paulo, Brazil; approval number 31/17, 20 March 2017). All animals were handled in strict
accordance with good animal practice as defined by the Animal Use Ethics Committee.
4.7. Adult Schistosomes
The preparations and culture of schistosomes was performed as previously described [42
,43].
Briefly, hamsters were killed 49 days after infection for the recovery of adult schistosomes. Next, fresh

Int. J. Mol. Sci. 2018, 19, 1802
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adult worm pairs (male and female) were washed and placed in a 24-well culture plate (TPP, St. Louis,
MO, USA) containing RPMI 1640 medium supplemented with 10% foetal bovine serum (Gibco-BRL,
Life Technologies do Brazil Ltda, Sao Paulo, SP, Brazil), 200 µg/mL streptomycin, and 200 IU/mL
penicillin (Invitrogen, São Paulo, SP, Brazil) at 37 ◦C in a 5% CO2 atmosphere. Adult schistosomes
were maintained continuously in medium (with or without drugs) for 96 h as described below.
4.8. In Vitro Experiments with Adult Worms of S. mansoni
In vitro assays in S. mansoni adults were performed with mated males and females. The worm
pairs obtained from the infusion hamsters were washed twice with RPMI 1640 medium containing
200 U/mL penicillin, 200 µg/mL streptomycin and 2 µg/mL amphotericin B (Vitrocell, Campinas,
Brazil). The mated parasites were then transferred into 24-well cell culture plates (TPP, St. Louis,
MO, USA) containing, per well, 1 pair of worms in 2 mL of RPMI 1640 medium supplemented with
10% foetal bovine serum and buffered with 25 mM HEPES. Worms were incubated with the amide
pyramethrin analyzes at different concentrations (50, 25, 10 and 5
to 96 h; 2 M praziquantel was used as positive control and culture medium was used as a negative
control [44].
µM); 5 and 10 µM piplartine for up
µ
Cultures of adult worms were monitored by microscopy or magnifying glass. The effect of the
drug was assessed with emphasis on changes in worm motor activity and death of worms as previously
described [45–47]. The mortality of worms was judged by the absence of movement for 2 min or when
touched with forceps.
4.9. Statistical Analysis
Statistical analysis was performed by analysis of variance (ANOVA) and Bonferroni post hoc
test using Graph Pad Prism 5.03 (GraphPad Software, La Jolla, CA, USA) to evaluate the statistical
significance of differences between control and treated cells in cytotoxicity assays. All values were
expressed as means
experiments, and a value of p < 0.05 was considered statistically significant.
±
standard error of the mean (SEM) from triplicates in three independent
5. Conclusions
This study demonstrated that analogues of piplartine, when compared with the parent molecule,
showed a loss of activity against the S. mansoni parasite, but showed decreased toxicity compared to
that of piplartine. Alterations in the trimethoxybenzene ring of piplartine, following unsaturation of the
aliphatic chain and the dihydropiperidinone ring, were shown to be highly sensitive to modification,
promoting the loss of biological activity and decreased toxicity. These findings indicate that analogous
molecules within the same series do not present the same physicochemical and biological activity.
Supplementary Materials: Supplementary materials can be found at http://www.mdpi.com/1422-0067/19/6/
1802/s1.
Acknowledgments: This work was partially supported by grants from the Fundação de Amparo à Pesquisa do
Estado de São Paulo (FAPESP grant number 2016/22488-3), the authors are grateful to CAPES/FAPEPI for the
scholarship granted. ACM is grateful to FAPESP (2014/02282-6). YPM is grateful to CNPq (Grant 302674/2010-1).
Financial Support: FCT (Fundação para Ciência e Tecnologia) by grant no. UID/MULTI/04378/2013
POCI/01/0145/FEDER/007728, Fapesp (2014/50316-7). The work at UCIBIO/Requimte was supported by
the Fundação para a Ciência e a Tecnologia (FCT) with financial support from FCT/MEC through national funds
and co-financed by FEDER, under the Partnership Agreement PT2020. Alexandra Plácido is grateful to FCT by
her grant SFRH/BD/97995/2013, financed by POPH–QREN–Tipologia 4.1–Formação Avançada, subsidized by
Fundo Social Europeu and Ministério da Ciência, Tecnologia e Ensino Superior.
Author Contributions: Conceived and designed the experiments: Yuri Dias Macedo Campelo,
Andreanne G. Vasconcelos
,
Alexandra Plácido, Lydia Fumiko Yamaguchi Yvonne P. Mascarenhas,
José Roberto S. A. Leite
;
Performed the experiments: Yuri Dias Macedo Campelo, Alicia Ombredane,
Andreanne G. Vasconcelos, Lucas F. F. Albuquerque, Daniel C. Moreira, Alexandra Plácido, Lydia Fumiko
Yamaguchi, Ana Carolina Mafud, Tatiana K. S. Borges, Massuo J. Kato, Josué de Moraes, Jefferson A. Rocha
,
Harold Hilarion Fokoue, and, Marcos P. Silva; Analyzed the data: Yuri Dias Macedo Campelo, Alicia Ombredane,

Int. J. Mol. Sci. 2018, 19, 1802
15 of 17
Andreanne G. Vasconcelos, Daniel C. Moreira, Alexandra Plácido, Ana Carolina Mafud, Tatiana K. S.
Borges, Graziella A. Joanitti Daniel E.R. Arcanjo, Massuo J. Kato, Josué de Moraes, José Roberto S. A. Leite.
,
Contributed reagents/materials/analysis tools: Yvonne P. Mascarenhas, Cristina Delerue-Matos, Tatiana K. S.
Borges, Graziella A. Joanitti, Massuo J. Kato, Selma S. Kucklhaus, Josué de Moraes, José Roberto S. A. Leite.
Wrote the paper: Yuri Dias Macedo Campelo, Alicia Ombredane, Andreanne G. Vasconcelos, Daniel C. Moreira,
Alexandra Plácido, Graziella A. Joanitti, Daniel D. R. Arcanjo, Massuo J. Kato, Josué de Moraes, José Roberto S.
A. Leite.
Abbreviations
DMSO
THF
Dimethyl sulfoxide
Tetrahydrofuran
DCC
Dicyclohexylcarbodiimide
References
1.
World Health Organization. Schistosomiasis; WHO: Geneva, Switzerland, 2016; Available online: http:
//who.int/mediacentre/factsheets/fs115/en/ (accessed on 20 November 2017).
2.
GBD 2016 Causes of Death Collaborators. Global, regional, and national age-sex specific mortality for 264
causes of death, 1980–2016: A systematic analysis for the Global Burden of Disease study 2016. Lancet 2017
,
390, 1151–1210. [CrossRef]
3.
GBD 2015 DALYs and HALE Collaborators. Global, regional, and national disability-adjusted life-years
(DALYs) for 315 diseases and injuries and healthy life expectancy (HALE), 1990–2015: A systematic analysis
for the Global Burden of Disease Study 2015. Lancet 2016, 388, 1603–1658. [CrossRef]
Colley, D.G.; Bustinduy, A.L.; Secor, W.E.; King, C.H. Human schistosomiasis. Lancet 2014, 383, 2253–2264.
[CrossRef]
4.
5.
6.
7.
Cioli, D.; Pica-Mattoccia, L.; Basso, A.; Guidi, A. Schistosomiasis Control: Praziquantel forever?
Mol. Biochem. Parasitol. 2014, 195, 23–29. [CrossRef] [PubMed]
Lago, E.M.; Xavier, R.P.; Teixeira, T.R.; Silva, L.M.; da Silva Filho, A.A.; de Moraes, J. Antischistosomal agents:
State of art and perspectives. Future Med. Chem. 2018, 10, 89–120. [CrossRef] [PubMed]
De Almeida, L.M.; de Carvalho, L.S.; Gazolla, M.C.; Silva Pinto, P.L.; da Silva, M.P.; de Moraes, J.; Da Silva
Filho, A.A. Flavonoids and sesquiterpene lactones from Artemisia absinthium and Tanacetum parthenium
against Schistosoma mansoni worms. Evid Based Complement Alternat Med. 2016, 2016, 9521349. [CrossRef]
[PubMed]
8.
9.
De Moraes, J.; Almeida, A.A.; Brito, M.R.; Marques, T.H.; Lima, T.C.; Sousa, D.P.; Nakano, E.; Mendonça, R.Z.;
Freitas, R.M. Anthelmintic activity of the natural compound (+)-limonene epoxide against Schistosoma
mansoni. Planta Med. 2013, 79, 253–258. [CrossRef] [PubMed]
Silva, M.P.; Oliveira, G.L.; de Carvalho, R.B.; de Sousa, D.P.; Freitas, R.M.; Pinto, P.L.; de Moraes, J.
Antischistosomal activity of the terpene nerolidol. Molecules 2014, 19, 3793–3803. [CrossRef] [PubMed]
10. Silva, A.P.; Silva, M.P.; Oliveira, C.G.; Monteiro, D.C.; Pinto, P.L.; Mendonça, R.Z.; Costa Júnior, J.S.;
Freitas, R.M.; de Moraes, J. Garcinielliptone FC: Antiparasitic activity without cytotoxicity to mammalian
cells. Toxicol. In Vitro 2015, 29, 681–687. [CrossRef] [PubMed]
11. De Castro, C.C.; Costa, P.S.; Laktin, G.T.; de Carvalho, P.H.; Geraldo, R.B.; de Moraes, J.; Pinto, P.L.;
Couri, M.R.; Pinto Pde, F.; Da Silva Filho, A.A. Cardamonin, a schistosomicidal chalcone from Piper aduncum
L. (Piperaceae) that inhibits Schistosoma mansoni ATP diphosphohydrolase. Phytomedicine 2015, 22, 921–928.
[CrossRef] [PubMed]
12. De Moraes, J. Natural products with antischistosomal activity. Future Med. Chem. 2015, 7, 1–20. [CrossRef]
[PubMed]
13. Parmar, V.S.; Jain, S.C.; Bisht, K.S.; Jain, R.; Taneja, P.; Jha, A.; Tyagi, O.M.; Prasad, A.K.; Wengel, J.; Olsen, C.E.;
et al. Phytochemistry of the genus Piper. Phytochemistry 1997, 46, 597–673. [CrossRef]
14. Bezerra, D.P.; Pessoa, C.; de Moraes, M.O.; Saker-Neto, N.; Silveira, E.R.; Costa Lotufo, L.V. Overview of
the therapeutic potential of piplartine (Piperlongumine). Eur. J. Pharm. Sci. 2013, 48, 453–463. [CrossRef]
[PubMed]

Int. J. Mol. Sci. 2018, 19, 1802
16 of 17
15. De Moraes, J.; Nascimento, C.; Lopes, P.O.; Nakano, E.; Yamaguchi, L.F.; Kato, M.J.; Kawano, T. Schistosoma
mansoni: In vitro schistosomicidal activity of piplartine. Exp. Parasitol. 2011, 127, 357–364. [CrossRef]
[PubMed]
16. Moraes, J.; Nascimento, C.; Yamaguchi, L.F.; Kato, M.J.; Nakano, E. Schistosoma mansoni: In vitro
schistosomicidal activity and tegumental alterations induced by piplartine on schistosomula. Exp. Parasitol.
2012, 132, 222–227. [CrossRef] [PubMed]
17. Campelo, Y.D.M.; Mafud, A.C.; Véras, L.M.C.; Guimarães, M.A.; Yamaguchi, L.F.; Lima, D.F.; Arcanjo, D.D.R.;
Kato, M.J.; Mendonça, R.Z.; Pinto, P.L.S.; et al. Synergistic effects of in vitro combinations of piplartine,
epiisopiloturine and praziquantel against Schistosoma mansoni. Biomed. Pharmacother. 2017, 88, 488–499.
[CrossRef] [PubMed]
18. De Moraes, J.; Keiser, J.; Ingram, K.; Nascimento, C.; Yamaguchi, L.F.; Bittencourt, C.R.; Bemquerer, M.P.;
Leite, J.R.; Kato, M.J.; Nakano, E. In vitro synergistic interaction between amide piplartine and antimicrobial
peptide dermaseptin against Schistosoma mansoni schistosomula and adult worms. Curr. Med. Chem. 2013, 20,
301–309. [CrossRef] [PubMed]
19. Ertl, P.; Rohde, B.; Selzer, P. Fast calculation of molecular polar surface area as a sum of fragment-based
contributions and its application to the prediction of drug transport properties. J. Med. Chem. 2000, 43,
3714–3717. [CrossRef] [PubMed]
20. Moraes, J. Antischistosomal natural compounds: Present challenges for new drug screens. In Current Topics
in Tropical Medicine; Rodriguez-Morales, A.J., Ed.; InTech: Rijeka, Croatia, 2012; pp. 333–358.
21. Silva, M.P.; de Oliveira, R.N.; Mengarda, A.C.; Roquini, D.B.; Allegretti, S.M.; Salvadori, M.C.; Teixeira, F.S.;
de Sousa, D.P.; Pinto, P.L.S.; da Silva Filho, A.A.; et al. Antiparasitic activity of nerolidol in a mouse model of
schistosomiasis. Int. J. Antimicrob. Agents 2017, 50, 467–472. [CrossRef] [PubMed]
22. Mafud, A.C.; Ferreira, L.G.; Mascarenhas, Y.P.; Andricopulo, A.D.; de Moraes, J. Discovery of novel
antischistosomal agents by molecular modeling approaches. Trends Parasitol. 2016, 32, 874–886. [CrossRef]
[PubMed]
23. De Moraes, J.; Dario, B.S.; Couto, R.A.; Pinto, P.L.; da Costa Ferreira, A.M. Antischistosomal activity of
oxindolimine-metal complexes. Antimicrob. Agents Chemother. 2015, 59, 6648–6652. [CrossRef] [PubMed]
24. Shen, J.; Cheng, F.; Xu, Y.; Li, W.; Tang, Y. Estimation of ADME Properties with Substructure Pattern
Recognition. J. Chem. Inf. Model. 2010, 50, 1034–1041. [CrossRef] [PubMed]
25. Uretsky, N.; Robertson, L. Academic Preparation for Modern Drug Discovery. 2004. Available online:
http://www.sciencemag.org/careers/2004/03/academic-preparation-modern-drug-discovery (accessed
on 20 November 2017).
26. Araújo-Vilges, K.M.; Oliveira, S.V.; Couto, S.C.P.; Fokoue, H.H.; Romero, G.A.S.; Kato, M.J.; Romeiro, L.A.S.;
Leite, J.R.S.A.; Kuckelhaus, S.A.S. Effect of piplartine and cinnamides on Leishmania amazonensis,
Plasmodium falciparum and on peritoneal cells of Swiss mice. Pharm. Biol. 2017, 55, 1601–1607. [CrossRef]
[PubMed]
27. Jain, M.V.; Paczulla, A.M.; Klonisch, T.; Dimgba, F.N.; Rao, S.B.; Roberg, K.; Schweizer, F.; Lengerke, C.;
Davoodpour, P.; Palicharla, V.R.; et al. Interconnections between apoptotic, autophagic and necrotic
pathways: Implications for cancer therapy development. J. Cell. Mol. Med. 2013, 17, 12–29. [CrossRef]
[PubMed]
28. Atale, N.; Gupta, S.; Yadav, U.C.; Rani, V. Cell-death assessment by fluorescent and nonfluorescent
cytosolicand nuclear staining techniques. J. Microsc. 2014, 255, 7–19. [CrossRef] [PubMed]
29. Mukhopadhyay, S.; Panda, P.K.; Sinha, N.; Das, D.N.; Bhutia, S.K. Autophagy and apoptosis: Where do they
meet? Apoptosis 2014, 19, 555–566. [CrossRef] [PubMed]
30. Dive, C.; Gregory, C.D.; Philips, D.J.; Evans, D.L.; Milner, A.E.; Wylle, A.H. Analysis and discrimination of
necrosis and apoptosis (programmed cell death) by multiparameter flow cytometry. Biochem. Biophys. Acta
1992, 1133, 275–285. [CrossRef]
31. Bezerra, D.P.; de Castro, F.O.; Alves, A.P.; Pessoa, C.; de Moraes, M.O.; Silveira, E.R.; Lima, M.A.; Elmiro, F.J.;
de Alencar, N.M.; Mesquita, R.O.; et al. In vitro and in vivo antitumor effect of 5-FU combined with piplartine
and piperine. J. Appl. Toxicol. 2008, 28, 156–163. [CrossRef] [PubMed]
32. Ponte-Sucre, A.; Bruhn, H.; Schirmeister, T.; Cecil, A.; Albert, C.R.; Buechold, C.; Tischer, M.; Schlesinger, S.;
Goebel, T.; Fuß, A.; et al. Piperamides and their derivatives as potential anti-trypanosomal agents.
Med. Chem. Res. 2015, 18, 703–711. [CrossRef]

Int. J. Mol. Sci. 2018, 19, 1802
17 of 17
33. Adams, D.J.; Dai, M.; Pellegrino, G.; Wagner, B.K.; Stern, A.M.; Shamji, A.F.; Schreiber, S.L. Synthesis, cellular
evaluation, and mechanism of action of piperlongumine analogs. Proc. Natl. Acad. Sci. USA 2012, 109,
15115–15120. [CrossRef] [PubMed]
34. Marques, J.V.; de Oliveira, A.; Raggi, L.; Young, M.C.; Kato, M.J. Antifungal activity of natural and synthetic
amides from Piper species. J. Braz. Chem. Soc. 2010, 21, 1807–1813. [CrossRef]
35. Frisch, M.J.; Trucks, G.W.; Schlegel, H.B.; Scuseria, G.E.; Robb, M.A.; Cheeseman, J.R.; Scalmani, G.; Barone, V.;
Mennucci, B.; Petersson, G.A.; et al. Gaussian 09, Revision A.1; Gaussian, Inc.: Wallingford, CT, USA, 2009;
Available online: www.gaussian.com (accessed on 22 June 2017).
36. Lee, C.; Yang, W.; Parr, R.G. Development of the colle-salvetti correlation-energy formula into a functional of
the electron-density. Phys. Rev. B 1988, 37. [CrossRef]
37. Hohenberg, P.; Kohn, W. Inhomogeneous electron gas. Phys. Rev. 1964, 136. [CrossRef]
38. Pedretti, A.; Villa, L.; Vistoli, G. VEGA—An open platform to develop chemo-bio-informatics applications,
using plug-in architecture and script programming. J. Comput. Aided Mol. Des. 2004, 18, 167–173. [CrossRef]
[PubMed]
39. Pires, D.E.V.; Blundell, T.L.; Ascher, D.B. pkCSM: Predicting small-molecule pharmacokinetic and toxicity
properties using graph-based signatures. J. Med. Chem. 2015, 58, 4066–4072. [CrossRef] [PubMed]
40. Pires, D.E.V.; Blundell, T.L.; Ascher, D.B. Theory—How Tointerpret pkCSM Results; University of Cambridge:
Cambridge, UK, 2015; Available online: http://bleoberis.bioc.cam.ac.uk/pkcsm/theory (accessed on
20 November 2017).
´
41. Sliwka, L.; Wiktorska, K.; Suchocki, P.; Milczarek, M.; Mielczarek, S.; Lubelska, K. The Comparison of
MTT and CVS Assays for the Assessment of Anticancer Agent Interactions. PLoS ONE 2016, 11, e0155772.
[CrossRef] [PubMed]
42. Keiser, J.; Ingram, K.; Vargas, M.; Chollet, J.; Wang, X.; Dong, Y.; Vennerstrom, J.L. In vivo activity of aryl
ozonides against Schistosoma species. Antimicrob. Agents Chemother. 2012, 56, 1090–1092. [CrossRef]
[PubMed]
43. De Moraes, J.; de Oliveira, R.N.; Costa, J.P.; Junior, A.L.; de Sousa, D.P.; Freitas, R.M.; Allegretti, S.M.;
Pinto, P.L.S. Phytol, a diterpene alcohol from chlorophyll, as a drug against neglected tropical disease
Schistosomiasis mansoni. PLoS Negl. Trop. Dis. 2014, 8, e2617. [CrossRef] [PubMed]
44. Mafud, A.C.; Silva, M.P.; Monteiro, D.C.; Oliveira, M.F.; Resende, J.G.; Coelho, M.L.; de Sousa, D.P.;
Mendonça, R.Z.; Pinto, P.L.; Freitas, R.M.; et al. Structural parameters, molecular properties, and biological
evaluation of some terpenes targeting Schistosoma mansoni parasite. Chem. Biol. Interact. 2016, 244, 129–139.
[CrossRef] [PubMed]
45. De Brito, M.R.M.; Peláez, W.J.; Faillace, M.S.; Militão, G.C.G.; Almeida, J.R.G.S.; Argüello, G.A.; Szakonyi, Z.;
Fülöp, F.; Salvadori, M.C.; Teixeira, F.S.; et al. Cyclohexene-fused 1,3-oxazines with selective antibacterial
and antiparasitic action and low cytotoxic effects. Toxicol. In Vitro 2017, 44, 273–279. [CrossRef] [PubMed]
46. Rocha, J.A.; Andrade, I.M.; Véras, L.M.; Quelemes, P.V.; Lima, D.F.; Soares, M.J.; Pinto, P.L.; Mayo, S.J.;
Ivanova, G.; Rangel, M.; et al. Anthelmintic, antibacterial and cytotoxicity activity of imidazole alkaloids
from Pilocarpus microphyllus leaves. Phytother. Res. 2017, 31, 624–630. [CrossRef] [PubMed]
47. Dias, M.M.; Zuza, O.; Riani, L.R.; de Faria Pinto, P.; Pinto, P.L.S.; Silva, M.P.; de Moraes, J.; Ataíde, A.C.Z.;
de Oliveira Silva, F.; Cecílio, A.B.; et al. In vitro schistosomicidal and antiviral activities of Arctium lappa
L. (Asteraceae) against Schistosoma mansoni and Herpes simplex virus-1. Biomed. Pharmacother. 2017, 94,
489–498. [CrossRef] [PubMed]
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