Schiff bases of 4-Phenyl-2-Aminothiazoles as hits to new antischistosomals: Synthesis, in vitro, in vivo and in silico studies

Article abstract of DOI:10.1016/j.ejps.2020.105371

The treatment of schistosomiasis is based on a single drug, the praziquantel (PZQ), an oral bioavailable and efficient agent which causes minimal side effects. The main concern about this approach, however, is that relying on only one drug to treat a helminthic disease is a dangerous strategy since history shows that pathogens easily evolve to resistant forms. Actually, reports about experimental strains exhibiting low sensibility to PZQ can be found in literature. The search for new antischistosomals, consequently, is urgent. Here we report the synthesis of seventeen Schiff bases of 4-(4-Substituted phenyl)-N-(4-substituted benzylidene)thiazole-2-amines which were tested in vitro and in vivo against Schistosoma mansoni adult worms. Moreover, in silico studies to propose potential macromolecular targets and to predict the oral bioavailability were also performed. The analog GPQF-108 exhibited the best in vitro performance (IC₅₀: 29.4 μM, SI:6.1) associated with promising in vivo activity, with a significant decrease in the adult life forms and oviposition. Oral bioavailability could be impaired by the predicted low water solubility of GPQF-108, although it also exhibited good membrane permeability. The water solubility, however, could be improved by decreasing the particles size. Serine/Threonine- and Tyrosine Kinases, Carbonic Anhydrase, Tyrosine Phosphatase and Arginase were predicted as potential macromolecular targets through which the GPQF-108 could be acting against the helminth. This class of compounds exhibited an interesting initial therapeutic profile with the advantage of being chemically diverse from the PZQ and be easily synthesized from commercial reagents which could lead to low-cost drugs. These aspects make this class of compounds interesting hits to be explored against schistosomiasis.

Full text of DOI:10.1016/j.ejps.2020.105371

EuropeanJournalofPharmaceuticalSciences150(2020)105371
Contents lists available at ScienceDirect
European Journal of Pharmaceutical Sciences
journal homepage: www.elsevier.com/locate/ejps
Schiff bases of 4-Phenyl-2-Aminothiazoles as hits to new antischistosomals:
Synthesis, in vitro, in vivo and in silico studies
Carina R. Amorima, Thais F.A. Pavania, Andrey F.S. Lopesa, Marcelo D. Duquea,
⁎
Ana C.A. Mengardab, Marcos P. Silvab, Josué de Moraesb, Daniela G.G. Randoa,
a Departamento de Ciências Farmacêuticas, Instituto de Ciências Ambientais, Químicas e Farmacêuticas da Universidade Federal de São Paulo, UNIFESP, Rua São Nicolau,
210, Centro, Diadema, SP, Brasil
b Núcleo de Pesquisas em Doenças Negligenciadas da Universidade Guarulhos, Praça Teresa Cristina, 88, Guarulhos, SP, Brasil
A R T I C L E I N F O
A B S T R A C T
Keywords:
The treatment of schistosomiasis is based on a single drug, the praziquantel (PZQ), an oral bioavailable and
efficient agent which causes minimal side effects. The main concern about this approach, however, is that relying
on only one drug to treat a helminthic disease is a dangerous strategy since history shows that pathogens easily
evolve to resistant forms. Actually, reports about experimental strains exhibiting low sensibility to PZQ can be
found in literature. The search for new antischistosomals, consequently, is urgent. Here we report the synthesis
of seventeen Schiff bases of 4-(4-Substituted phenyl)-N-(4-substituted benzylidene)thiazole-2-amines which
were tested in vitro and in vivo against Schistosoma mansoni adult worms. Moreover, in silico studies to propose
potential macromolecular targets and to predict the oral bioavailability were also performed. The analog GPQF-
108 exhibited the best in vitro performance (IC50: 29.4 µM, SI:6.1) associated with promising in vivo activity, with
a significant decrease in the adult life forms and oviposition. Oral bioavailability could be impaired by the
predicted low water solubility of GPQF-108, although it also exhibited good membrane permeability. The water
solubility, however, could be improved by decreasing the particles size. Serine/Threonine- and Tyrosine Kinases,
Carbonic Anhydrase, Tyrosine Phosphatase and Arginase were predicted as potential macromolecular targets
through which the GPQF-108 could be acting against the helminth. This class of compounds exhibited an in-
teresting initial therapeutic profile with the advantage of being chemically diverse from the PZQ and be easily
synthesized from commercial reagents which could lead to low-cost drugs. These aspects make this class of
compounds interesting hits to be explored against schistosomiasis.
4-Phenyl-2-aminothiazoles
Schistosoma mansoni
Schiff bases
Antischistosomal
Target fishing
1. Introduction
pyrazino-isoquinoline drug praziquantel. Commonly administered as
tablets of 600 mg (40-60 mg/kg), this drug is effective against the adult
Schistosomiasis is a flatworm-caused disease responsible for 250
million cases worldwide, being 700 to 800 million people at risk of
infection. Commonly associated with poverty, schistosomiasis occurs
mainly in tropical and sub-tropical areas, most of the cases reported
only in Africa. The etiological agents are helminths from Schistosoma
genus, of which six are known to be human-pathogenic species
(McManus et al., 2018).
worms, exhibiting minimal temporary side effects. For these reasons,
the praziquantel has been considered a safe and efficient drug for more
than 40 years (Lago et al., 2018).
Important, however, is the fact that relying on one single drug to
treat a parasitic disease could lead to selection for resistance (Cioli
et al., 2014). There are no evidence for widespread praziquantel re-
sistance but reports of experimental isolates exhibiting diminished
susceptibility to the drug as well as case reports about failures in
treatment can easily be found in literature (Fenwick and Webster, 2006;
Gryseels et al., 2001).
The most prevalent species are the S. mansoni, endemic in sub-
Saharan Africa and in Central-South American countries such as
Suriname, Venezuela and Brazil, and the S. haematobium, prevalent in
54 countries of sub-Saharan Africa and Middle East (World Health
Organization 2019).
The loss of effectiveness of praziquantel could lead to a step back in
the control of this helminthiasis and, for this reason, search for new
therapeutic alternatives is urgent.
The pharmacological treatment for schistosomiasis is based on the
⁎ Corresponding author.
E-mail address: dgrando@unifesp.br (D.G.G. Rando).
https://doi.org/10.1016/j.ejps.2020.105371
Received 20 February 2020; Received in revised form 13 April 2020; Accepted 30 April 2020
Availableonline07May2020
0928-0987/©2020ElsevierB.V.Allrightsreserved.

C.R. Amorim, et al.
EuropeanJournalofPharmaceuticalSciences150(2020)105371
In the last years, our group have been screening classes of chemical
compounds, preferably those chemically diverse from Praziquantel
(PZQ), to their antischistosomal activity. Among the assayed chemical
classes, the Schiff bases from N-acylhydrazones and from 4-phenyl-2-
aminothiazoles exhibited the best biological activities. The purpose is to
explore different chemical scaffolds as starting points to be rationally
optimized in the search for alternatives to antischistosomal therapy
since this still relies in only one drug.
2.2. Methods
2.2.1. Synthesis
4-(4-Substituted phenyl)-N-(4-substituted benzylidene)-1,3-thia-
zole-2-amines were synthesized following
a two-step reactional
pathway. First, the intermediate 4-Substituted phenyl-1,3-thiazole-2-
amines were obtained through the classical methodology of Hantzsch
and Weber approach (Hantzsch and Weber, 1887). The Schiff bases
were obtained, then, from the reaction of the corresponding 4-Sub-
stituted phenyl-1,3-thiazole-2-amine with different aldehydes at equi-
molar ratio and employing piperidine as catalyst (Geronikaki et al.,
2004). Liquid chromatography in silica gel (hexane: ethylacetate, 7:3)
was employed to purify the final compounds.
Schiff bases from 4-phenyl-2-aminothiazoles have been explored for
several potential biological applications such as antibacterial, anti-
fungal, antitumoral, anti-inflammatory, antitubercular activities
(Brodowska and Łodyga-Chruścińska, 2014; Khan et al., 2012; Rana
et al., 2012). These compounds are structurally related to the N-acyl-
hydrazones, being a more rigid and restrict analog of the latter. Both
the nitrogen atoms of the structure, the one within the ring and the
other at the thiazole 2-position, can establish hydrogen bonds as ac-
ceptor with amino acid residues as well as the sulfur atom at the ring,
which is a isostere from oxygen atom present in carbonyl groups. The 4-
phenyl ring car be an important point for hydrophobic interactions,
such as observed by de Moraes Gomes et al. in their studies with 1,3-
thiazoles as potential trypanocidal agents (de Moraes Gomes et al.,
2016). The similarity of these groups with those present in peptide
substrate of several enzymes could explain the set of different biological
activity observed to 2-amiothiazole derivatives.
Structural characterization was performed by melting point range
determination, carbon and hydrogen nuclear magnetic resonance, in-
frared spectroscopy. Data can be found at Supplementary Material.
2.2.2. Animals and parasite maintenance
This study was approved by the Comissão de Ética no Uso de
Animais (CEUA), from Universidade Guarulhos (protocol no. 31/2017,
Brazil). The animal studies are reported in compliance with the “Animal
research: reporting of in vivo experiments” (ARRIVE) guidelines. Three-
week-old female Swiss mice were purchased from Laboratory Animals
Anilab (São Paulo, Brazil). They were housed in individually vented
caging systems in groups of five mice per cage. Mice were kept under a
12-h light/12-h dark environment cycle and maintained at uniform
temperature and humidity, with food and water available ad libitum
(Lago et al., 2019). Mice were infected with S. mansoni (BH strain) by
subcutaneous injection of ~80 cercariae. Cercariae were harvested
from infected intermediate host snails Biomphalaria glabrata by ex-
posure to light for 3 h, following standard procedures of our laboratory
(Guimarães et al., 2015; Silva et al., 2017).
How far we have searched, however, there are few reports in the
scientific literature about such compounds being exploited against
parasites or helminths. Rajmane and Ubale, for instance, demonstrated
that thiazole Schiff Bases derived from 4-(2’-halophenyl)-2-aminothia-
zoles exhibited nematocidal activity at very low concentrations against
the plant parasite Meloidogyne javanica as well as molluscicidal activity
against the freshwater helminth vector snail Lymnea auricularia
(Rajmane et al., 2013).
In this work, seventeen 4-(4-Substituted phenyl)-N-(4-substituted
benzylidene)thiazole-2-amines were synthesized, characterized and
tested in vitro on S. mansoni adult worms. Compounds were next tested
on toxicity and further studied in mice harboring a chronic S. mansoni
infection. Finally, in silico studies were also performed to predict po-
tential macromolecular targets, as well as to foresee the oral absorption
behavior of the best compound.
2.2.3. In vitro antischistosomal assay
Adult schistosomes were harvested from the hepatic portal veins
and mesenteric veins of infected mice (7 weeks post-infection) and in
vitro antischistosomal assay was performed as previously described (De
Castro et al., 2015; Mafud et al., 2016). Briefly, parasites were placed in
RPMI 1640 culture medium supplemented with 10% fetal bovine serum
containing 100 IU/ml, penicillin and 100 µg/ml streptomycin at 37°C
and 5% CO2. All compounds were dissolved in DMSO and were initially
tested at 100 μM in triplicate on adult S. mansoni. The compounds that
produced an effect superior to 80% after 24 h post-exposure were
considered as hits and underwent determination of their half-maximum
lethal concentrations, LC50, using 1:2 serial dilutions from 3.12 to 100
μM. Each concentration was tested in triplicate and the entire assay was
repeated at least three times. Parasites were also incubated in the
presence of the highest DMSO concentration (0.5%) as a negative
control. LC50 value of praziquantel, used a positive control, was de-
termined using 1:2 serial dilutions from 0.15 to 2.5 μM. The LC50 for
active compounds were calculated with GraphPad Prism software using
sigmoid dose-response curves and the 95% confidence intervals.
2. Material and methods
2.1. Material
All synthetic reagents are commercially available and were pur-
chased from Sigma-Aldrich/Merck company (São Paulo-SP/Brazil).
Solvents were obtained from LabSynth (Diadema-SP/Brazil) and used
without previous treatment. Three-week-old female Swiss mice were
purchased from Laboratory Animals Anilab (São Paulo, Brazil). Roswell
Park Memorial Institute (RPMI 1640) culture medium containing
phenol red and L-glutamine, M199 medium, inactivated fetal bovine
serum (FBS), penicillin G/streptomycin sulfate, and HEPES buffer were
obtained from Vitrocell (Campinas, SP, Brazil). DMSO employed to
prepare compound's solutions was obtained from Sigma-Aldrich (São
Paulo- SP/Brazil). The NMR spectra were recorded in a Bruker Advance
300 operating at 300 MHz for 1H and 75 MHz for 13C analysis. Chemical
shifts (δ) were measured from tetramethylsilane (TMS) as internal re-
ference and are reported in ppm. Coupling constants (J) are reported in
Hz, when applicable. Infrared data were acquired in an IR Affinity-1
Shimadzu spectrometer, as KBr pellets and considering the wave-
2.2.4. Cytotoxicity assay
The determination of cytotoxicity was performed with Vero cells
(ATCC CCL-81; Manassas, VA) according to a previously reported pro-
cedure (de Brito et al., 2017). Briefly, cells were seeded in 96-well
microtiter plates at a density of 5 × 104 cells/mL in DMEM medium
with 10% fetal bovine serum and L-glutamine (2 mM). Compounds
serially diluted 2-fold ranging from 15.62 to 500 μM in test medium
were added. The plates were incubated at 37°C at an atmosphere of 5%
CO2. After 72 h, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) was added and incubation was continued for another 3
h. The plate was then read using an Epoch™ 2 Microplate Spectro-
photometer (BioTek) at 595 nm. Values were expressed as percentage of
the control. Experiments were performed at least three times and IC50
numbers from 4000 to 400 cm−1
.
Freeware and web-based softwares were employed in the Target
Fishing studies, Autodock v.1.5.6 was used to perform the molecular
docking studies while the pharmacokinetics predictions were per-
formed with ADMET Predictor™ and GastroPlus™ softwares, both pro-
vided by Simulations Plus, Inc., Lancaster, CA, USA.
2

C.R. Amorim, et al.
EuropeanJournalofPharmaceuticalSciences150(2020)105371
Fig. 1. Synthetic route of the compounds.
values calculated as averages. The selectivity indices (SI) of tested
compounds were calculated by dividing CC50 values obtained on Vero
cells with LC50 values determined on adult schistosomes (Rodrigues
et al., 2018).
were prepared by adding the missing hydrogen atoms, removing all
water molecules, setting the atom types to AD4 force field and by re-
moving all non-essential ligands. Co-crystalized ligands were used as
reference to the redocking studies and to the comparative analysis
performed with GPQF-108. In the case of Carbonic Anhydrase, the
known inhibitor ethoxzolamide (ETZ) was employed to this compara-
tive analysis instead of glycerol, the co-crystalized ligand found in
6QQM model.
2.2.5. In vivo studies
In vivo antischistosomal assay was performed in a mouse model of
schistosomiasis according to the operating procedures for experimental
chemotherapy for antischistosomal drug (Lago et al., 2019). Groups of
five infected mice characterized by a chronic schistosome infection (49
days post-infection) were treated orally with the test drug using single
oral doses of 400 mg of compound per kg body weight. Five untreated
mice served as controls. Two weeks post treatment, animals were eu-
thanized with CO2, dissected, and worms were sexed and counted (De
Lima et al., 2018). Assessment of therapeutic efficacy was also based on
the technique of quantitative and qualitative oograms using a fragment
(10 mm) of the ascending colon, as well as the Kato-Katz method for
quantitative feces examination. The compound-treated group and
control group were compared using a parametric Dunnett's multiple-
comparison test, where statistical significance was set to P<0.05 in
GraphPad Prism software (de Moraes et al., 2014). The animals were
randomly assigned to the experimental groups, and pharmacological
treatments were counterbalanced randomly as well. The animals were
also euthanized in a random manner inside a group (Mengarda et al.,
2020).
All ligands were prepared by setting the atom types, adding missing
hydrogens, geometric optimization followed by energy minimization
and had their Geisteiger charges calculated (Gaussian 09W software).
Boron parameters were added to the AD4 parameter file (.dat). The
specific parameter data is provided at Supplementary Material. Grid
boxes of 50 × 50 × 50 Å, with centers at the zinc metal atom of
carbonic anhydrase (X:-43.963; Y: 19.849; Z:-35.532) and in the center
of the catalytic site of the Arginase 1, Chain A (X: -42.019; Y: -18.985; Z:
49.527) were employed as reference to the ligand docking simulation.
Calculated energies of binding were used in the comparative analysis
with GPQF-108.
Absorption predictions were performed with the ADMET Predictor™
software version 9.5 (Simulations Plus, Lancaster, CA, USA) using mol
file of GPQF-108 employed as input. GastroPlus™ software 9.6
(Simulations Plus, Lancaster, CA, USA) was also used to predict the
pharmacokinetic (PK) profile of the compound.
3. Results and discussions
2.2.6. Molecular modeling and in silico absorption studies
Target fishing studies were performed employing a combination of
GaussView 5.0, Gaussian 09W (Frisch et al., 2016), Pharmmapper
Webserver (Liu et al., 2010) softwares and the Basic Local Alignment
Search Tool, BLAST (Altschul et al., 1990).
3.1. Synthesis
Seventeen compounds were obtained through a fast and simple
synthetic pathway (Fig. 1) but with low to moderate yields (Table 1).
The low obtained yields were probably related to the low nucleo-
philic power of the amino group of the 4-substituted-phenyl-1,3-thia-
zole-2-amine since, for all compounds, a certain amount of this starting
material was observed by the end of the reactions. Liquid chromato-
graphy was, then, performed to purify the final Schiff bases.
Derivatives of the 5-nitrothiophene carboxaldehyde exhibited the
lower yields values since the chromatography of nitro compounds is
recognizable difficult due to their high affinity for silica gel. The same
was true for the 4-nitrobenzaldehyde derivatives as well as better yields
were obtained for the non-nitrated ones.
Compounds were sketched in GaussView 5.0 and the tridimensional
geometries optimized using PM6 semi-empirical force field. Following
the structural optimization, energy optimization was performed by ab
initio approach, employing method HF/6.31G* and ChelpG charges
calculation. The files containing the geometric coordinates of the atoms
and their ChelpG charges were, then, used as mol2 input in the
Pharmmapper Web-based. This software was adjusted to search for all
potential macromolecular targets (humans and not humans), and to
perform 300 conformers generation.
A set of 100 results for each compound was retrieved from
Pharmmapper and analyzed by their Z'Score, Fit Score and Normalized
Fit Score values. The results of each compound were also compared to
select only targets common to all seven analogues and, with the se-
lected targets, a BLAST search was done to find similar proteins ex-
pressed by Schistosomatidae family, with special attention to those ex-
pressed by S. mansoni. Seven homolog proteins from S. mansoni
proteome were selected as the best regarding the percentage of identity
with the orthologue protein (more than 40%) and the statistics of the
alignment, such as the E-value (lower than 10−50) and Max-Score.
Docking studies were performed employing Autodock v. 1.5.6 free
software and the structures of the known crystallized S. mansoni pro-
teins, the Carbonic Anhydrase (PDB ID: 6QQM, resolution: 1.75 Å) and
S. mansoni Arginase (PDB ID:4Q3Q, resolution: 2.00 Å). The proteins
Compounds were structurally characterized by infrared spectro-
scopy, hydrogen and carbon nuclear magnetic resonance (1H- and 13C-
NMR). Purities were accessed by a combination of melting point ranges
(up to 4 °C) and 1H-NMR spectra analysis, by considering the absence of
signals other than the expected ones.
Results also prove the preferential synthesis of the E isomers, when
regarding the azomethine double bond, since the NH signal appeared at
10 to 12 ppm, as well as no duplicated signal, suggesting the presence
of the Z isomer, was observed. GPQF-102 and GPQF-118, conversely,
presented duplicated signals, but in intensities coherent with the pre-
valence of the E isomers.
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C.R. Amorim, et al.
EuropeanJournalofPharmaceuticalSciences150(2020)105371
Table 1
Schiff bases of 4-substituted-phenyl-1,3-thiazole-2-amines.
General Structure
Ar/Het
GPQF
R1
Yield* (%)
Melting Point oC
108
109
110
113
115
101
102
107
116
117
118
122
123
124
132
133
134
H
9
150–154
152–156
137–141
164–168
115–119
152–155
127–130
149–153
155–157
116–118
88–91
4-Me
4-Et
4-TertBu
4-Cl
4-Me
4-Et
H
12
10
10
4
19
9
22
17
55
19
54
22
52
30
23
51
4-Cl
4-Me
3,4-Cl
H
94–98
4-Et
4-Cl
4-Et
4-Me
4-Cl
103–106
115–118
92–94
133–136
129–132
⁎
Yields obtained after liquid chromatography purification.
3.2. In vitro assays
One could conclude that the most common mechanism of action of
such compounds, the nitro reduction by unspecific nitroreductases,
would be the most probable explanation for the observed anti-
schistosomal activity (Race et al., 2005). This hypothesis, however, is
uncertain since all the 5-nitrobenzaldehyde analogs tested were also
inactive against the adult worms. Moreover, nitro reduction is a me-
chanism associated with some toxicity toward mammalian cells, which
was not observed in the VERO cytotoxicity assays, even with the active
compounds, which presented CL50 values above 150 μM.
Once characterized, the seventeen compounds were evaluated to
their potential to inhibit the S. mansoni adult worms through in vitro
screening as well as had their cytotoxicity toward VERO cells assessed
(Table 2). Selectivity indexes were calculated as the proportion be-
tween the CL50 and IC50 values.
GPQF-108 was notably the most interesting analog with an IC50
value of 29.44 μM associated to a CL50 value of 179.44, which corre-
sponds to a selectivity index of approximately 6.1. GPQF-110 and 113
presented a very similar biological behavior, being the second most
active compounds of the series. GPQF-115 was the third in potency
(IC50: 51.31 μM) against adult worms but showed a selectivity index
(3.72) lower than GPQF-109, which was less active, but seems to be
more selective (IC50 value of 60.17 μM but SI of 5.11). All compounds
were considerably less active than PZQ when tested against schisto-
somes in vitro.
The nitro group alone, thus, is not responsible for the total activity
of these compounds. It seems it must be bonded to a heterocyclic aro-
matic ring. The need for a heteroatom in the ring can point to some
electronic contribution of this atom. Studies with a larger series of ac-
tive compound could elucidate this point.
3.3. In vivo assays
Noteworthy is the fact that only the derivatives of 5-nitro-2-thio-
phenecarboxaldehyde were active. The nitro group seems to be ex-
plicitly involved in the mechanism of action, since the corresponding
derivatives of thiophenecarboxaldehyde did not show any significant
activity.
Due to its significant performance in this first in vitro assay, GPQF-
108 was elected to be studied to its potential antischistosomal activity
in a mouse model. Worm burden, egg production and hepato- and
splenomegaly were examined after a single oral dose of 400 mg/kg,
administered 42 days after infection to mice harboring adult S. mansoni
(chronic infection). Fig. 2 shows that treatment with GPQF-108 resulted
in a significant reduction of ~54% (P < 0.01) for the total worm
Table 2
In vitro activities and selectivity indexes
GPQF
IC50 (μM) [*]
CL50 (μM) [*]
SI
108
109
110
113
115
101
102
107
116
117
118
122
123
124
132
133
134
PZQ
29.44 [20.04–37.16]
179.44 [148.93–202.47]
6.10
5.11
5.51
5.41
3.72
ND
60.17 [52.12–74.06]
307.54 [273.91–334.41]
33.96 [26.77–45.05]
187.15 [150.14–223.27]
38.02 [32.49–46.01]
205.68 [178.76–249.89]
51.31 [44.61–62.88]
191.03 [158.09–227.98]
>100
˃100
ND
ND
ND
˃100
ND
ND
˃100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
ND
˃100
ND
˃100
ND
˃100
ND
˃100
ND
˃100
ND
˃100
ND
˃100
ND
˃100
ND
1.08 [0.71–1.46]
>50
Fig. 2. Effect of GPQF-108 on the parasite burden of mice infected with S.
mansoni.
ND: Not determined; [*]: Confidence Interval. PZQ: praziquantel (positive
control).
4

C.R. Amorim, et al.
EuropeanJournalofPharmaceuticalSciences150(2020)105371
Fig. 4. Effect of GPQF- 108 on the liver and spleen pathology of mice infected
with S. mansoni.
manifested human pathology. The protective effect of GPQF-108 was
also found to lead to a reduction of hepato- and splenomegaly, as
measured by weight, compared to the control infected mice. In more
details, liver and spleen weights were significantly decreased by
24.05% (P ˂ 0.05) and 29.79% (P ˂ 0.05), respectively. This result
could be attributed to a reduction in the worm burden and oviposition
(Fig. 4).
Horizontal bars represent median values. *P ˂ 0.05, compared with
untreated groups. Squares: liver weigh, Circles: spleen weigh, Filled
symbols: control, Empty symbols: treated. OWR: Organ weight reduc-
tion.
In tandem, oral treatment with GPQF-108 in mice harboring chronic
infections significantly reduced worm burden, egg production, and
hepato- and splenomegaly. Comparatively, the antischistosomal effect
of GPQF-108 is weaker than praziquantel, which is known to reduce
~90% of the worm burden. (Roquini et al., 2019; Guerra et al., 2019)
Despite of not achieving the same potency presented by Praziquantel
and other known drugs, it is important to highlight that GPQF-108 is
herein considered as a starting point to be explored to find new anti-
schistosomal drugs with a scaffold different from the standard drug and,
for this reason, a good alternative in case of resistance emergence.
Fig. 3. Effect of GPQF-108 on the egg burden of mice infected with S. mansoni.
burden.
The sexually mature male and female worms pair up and the female
produces hundreds to thousands of eggs a day, half of which become
trapped in tissues while the other eggs are eliminated by the feces. With
respect to egg burden, samples of intestinal tissue were used to study
the percent egg developmental stages (oogram), including the number
of egg in fecal samples by the Kato-Katz method. As shown in Fig. 2, a
single oral dose of GPQF-108 led to a reduction of 38.72% (P ˂ 0.05) in
the number of immature eggs (Fig. 3A), whereas analysis in fecal
samples revealed a reduction of 56.38% (P ˂ 0.01) of eggs (Fig. 3B).
This finding could be attributed to a reduction in the worm burden as a
result of treatment with GPQF-108 and/or inhibition of oviposition by
adult helminths.
3.4. Target fishing studies
In order to propose possible macromolecular targets through which
the active 5-nitrothiophene derivatives could be acting (besides the
nitro reduction known mechanism), a target fishing study was per-
formed employing the Pharmmapper software. One-hundred targets
were retrieved to each analogue (active and inactive ones) and 12
macromolecules were selected to further studies since they were
common to all active 5-nitrothiophene derivatives (Table 1, Supple-
mentary Material).
Points represent data from individual mice. Horizontal bars re-
present median values. ** P ˂ 0.01 compared with untreated groups.
Squares: females, Circles: males, Triangles: Total, Filled symbols: con-
trol, Empty symbols: treated. WBR: Worm burden reduction.
A: Egg development stages (oogram). B: stool egg load. Points re-
present data from individual mice. Horizontal bars represent median
values. ** P ˂ 0.05, ** P ˂ 0.01 compared with untreated groups. In
A = Squares: Immature eggs, Circles: Mature eggs, Triangles: Dead
eggs, Filled symbols: control, Empty symbols: treated. In B = Squares:
eggs per gram. Filled and Empty symbols correspond to control and
treated group, respectively. EBR: Egg burden reduction.
Targets are listed following the best Z'scores obtained to the best
compound, GPQF-108. None of the found targets was exhibited ex-
clusively for GPQF-108, since this is a congeneric series and, as such,
the compounds present several structural similarities which could
guarantee the presence of the pharmacophores in more than one ana-
logue. Differences in activity should result, thus, from other char-
acteristics such as, the ability to be transported through the worm te-
gument or to achieve the target.
Despite good statistical data, none of the target fishing resulting
macromolecules corresponds to crystals of proteins expressed by worms
from Schistosomatidae family. For this reason, a BLAST (Basic Local
Alignment Search Tool, National Center for Biotechnology Information)
search was performed to find homologous proteins in the S. mansoni
The eggs are crucial for the maintenance of the schistosome life
cycle and disease transmission and are further responsible for
5

C.R. Amorim, et al.
EuropeanJournalofPharmaceuticalSciences150(2020)105371
Table 3
interesting target to new therapeutics (Da'dara et al., 2019). Re-
sponsible for catalyzing the reversible conversion of carbon dioxide and
water into bicarbonate ion and ionic hydrogen, this enzyme is found on
the tegument surface of the S. mansoni and seems to present a role in the
regulation of the acid-basic homeostasis at the parasite surface as well
as in the osmotic balance. Its inhibition could promote a lack of osmotic
balance (Castro-Borges et al., 2011). Interesting, this same target was
recently identified, by our group, as a potential target to a vanillin-
related N-acylhydrazone which presents structural similarities with the
Schiff bases herein reported (Rando et al., 2019).
Results from target fishing after BLAST analysis.
Target PDB Blast alignment results
S. mansoni Ortholog
Proteins
Identity (%) E-Value Max
State of Art
known
Score
374
1KE5
Serine/Threonine
Kinase
60.54
40.13
9e−129
3CP9
1BNV
1D3V
1VZC
Tyrosine kinase
6e−63
1e−61
3e−82
4e−81
211
196
255
250
known
known
known
known
Carbonic Anhydrase 40.23
Chain A Arginase-1
Uncharacterized
Protein Sm_200410
Tyrosine
41.72
42.72
Two of the pointed S. mansoni targets, the Carbonic Anhydrase and
the Arginase-1, had their crystal structure elucidated by X-Ray
Crystallography and, for this reason, molecular docking studies with
these two targets were performed employing Autodock 4 software.
Fig. 5A shows the simulated docking of the ethoxzolamide (ETZ)
within the S. mansoni Carbonic Anhydrase (SmCA). This compound was
demonstrated to be a good, but not selective, inhibitor of the SmCA by
Da'dara et al. (Da'dara et al., 2019) and, in fact, its simulation at the
enzyme's active site resulted in the best pose exhibiting an energy of
binding of -5.36 kcal/mol. It also showed a binding mode very similar
to those observed to this same compound when co-crystalized with the
human enzyme (hCAII, PDB ID: 3CAJ). Hydrogen bonds with the
Thr231 and Thr232 are observed as well as a pi-staking interaction with
thehis177 imidazole ring. The ETZ was employed here as the known
inhibitor of the SmCA since, the co-crystalized compound of the used
geometric model was the glycerol, a promiscuous ligand and not a good
alternative to serve as reference to SmCA binding mode.
2CNG
45.05
1e−83
265
putative
Phosphatase, non-
receptor type nt1
available proteome (Altschul et al., 1990).
Six S. mansoni proteins exhibited suitable structural correspondence
to the targets obtained with Pharmmapper. They are listed in Table 3
together with the statistical analyses which support their significance.
Five out of the six best BLAST results are known proteins of the S.
mansoni, while one of them is a putative protein. Kinase families were
pointed out as potential targets.
Serine/Threonine Kinases and Tyrosine Kinases are families of ki-
nases widely distributed among eukaryotes. It has already been re-
ported the expression of different serine/threonine and tyrosine kinases
by S. mansoni, such as Ctk, Src, Fyn and Syk as well as members of the
Fes family. These proteins are found at worm subtegument, par-
enchyma, gastrodermis, ovary and testicles (Bahia et al., 2007;
Grevelding et al., 2018; Kapp et al., 2004, 2001). Known inhibitors of
these kinases such as Herbimycin and Imatinib lead to oogenesis and
spermatogenesis alterations when assayed against S. mansoni adult
worms (Knobloch et al., 2006; Manley et al., 2002). Both studies, yet,
demonstrated significant morphological alterations and decrease in egg
production, the same effect obtained with the exposition to GPQF-108.
Since these are broad classes of proteins, further studies must be con-
ducted to elucidate if they could be genuine targets of this Schiff base.
Other interesting macromolecular target would be the
“Uncharacterized Protein Sm_200410”, which corresponds to the
Thymidylate synthase S. mansoni orthologue enzyme (Kouni, 2017).
Despite presenting a fundamental role in eukaryote nucleotides synth-
esis, this protein has never been explored as a macromolecular target
for schistosomiasis. The effect of its inhibition probably would be the
worm death, but selectivity toward helminth enzyme would be a
challenge. Nevertheless, several thymidylate synthase drugs are avail-
able in the market and selectivity against pathogens are a recognizable
possibility.
Under the same simulation conditions, GPQF-108 adjusted to the
SmCA catalytic site quite similarly to the observed with ETZ (Fig. 5B).
An energy of binding of -4.8 kcal/mol was calculated, a little bit higher
than the ETZ, but the compound established interactions with Gln115
residue through the nitrogen from the thiazole ring. Hydrogen bond is
also present between the nitro group and the His119. This result could
explain, in part, the nitro group importance to the observed activity.
This group approaches the Zn2+ metal cofactor, fundamental to the
SmCA activity, and established interactions within the cavity through
its oxygen atoms. This may be the reason why non-nitrated analogs did
not exhibited activity.
It is noteworthy the similar fitting of both ligands within in the
actives site of SmCA (Fig. 5C).
Fig. 5D, in turn, shows the results obtained to the docking simula-
tions with the Arginase-1, chain A, enzyme and the known and co-
crystallized ligand 2(S)-Amino-6-Boronohexanoic Acid (ABH). This
compound achieved an energy of binding of –8.98 kcal/mol and ex-
hibited the same interactions observed in the crystal structure (4Q3Q).
These involves a series of hydrogen bond interacts mainly established
with the Gly157, Asp158, Ile159, Asp154, Asp264 and even with the
two manganese ions (Mn2+) present as cofactors of this enzyme.
The simulations with GPQF-108 showed that it docked in a similar
position (Fig. 5E) and exhibited an even better calculated energy of
binding, of -9.24 kcal/mol. It established interactions with Asp158,
Asp264, His131and Thr276 through its nitro group and a pi-stacking
interaction with the His156. Again, the nitro group approaches de ions
Mn2+ from the catalytic site in distances compatible with ion-dipole
interactions (1.63 and 1.65 Å from the Mn2+ 1 and 2 in Fig. 5E).
Legend: Carbon, nitrogen, oxygen, hydrogen and sulfur atoms are re-
presented in gray, blue, red, white and yellow tubes. Carbon atoms of the
ligands are represented in magenta for “known inhibitors” and in green for
GPQF-108. Thin tubes represent protein atoms, while bold tubes represent
the ligands. Three-letter amino acid code was employed.
Arginase-1, SmARG, is expressed at all life forms of the helminth
and seems to present an important role in host immune response eva-
sion. This enzyme is also present in other pathogens, such as Leishmania
sp, Helicobacter pylori as well as in cancer cells and had its role in
lowering host immune response well defined and could provide a
macromolecular target to be explored (Gaur et al., 2007; Hai et al.,
2014; Zabaleta et al., 2004).
Regarding the Tyrosine Phosphatase non-receptor type nt1, al-
though it had been extensively studied in several eukaryote, little is
known about their role to the S. mansoni helminth (Protasio et al.,
2012).
Recently elucidated by crystallography, the zinc-dependent me-
talloenzyme S. mansoni Carbonic Anhydrase could constitute an
6

C.R. Amorim, et al.
EuropeanJournalofPharmaceuticalSciences150(2020)105371
Fig. 5. Docking simulation studies with GPQF-108 and the Schistosome mansoni enzymes SmCA and SmArg.
Once more, both compounds exhibited similar fitting within the
active cavity of the SmArg enzyme (Fig. 5F).
Table 4
These results reinforce the potential of the GPQF-108, and maybe its
analogues, of act through inhibition of these enzymes. Only experi-
mental assays; however, could confirm the real inhibitory potential of
the GPQF-108 and its analogs toward these S. mansoni targets.
Absorption prediction parameters
Property
Predicted values
pKa (S+Basic_pKa)a,
LogP
*
1.71–2.27
3.191
3.5. In silico absorption and bioavailability studies
LogD7.4
3.191
Molecular Weigh (g/mol)
DiffCoefb
315.37
Finally, in silico studies were performed to predict key physical-
chemical and biopharmaceutical properties of GPQF-108 as well as its
oral bioavailability. Some properties, such as pKa, LogP, LogD7.4, Water
Diffusion Coefficient, Jejune Effective Permeability and Water solubi-
lity were calculated using ADMET Predictor™ software. Results pointed
out the dominance of the basic amino groups in the structure and the
expected inability of ionization of it, through identical LogP and
LogD7.4 values (Table 4).
0.804
S+Peffc,*
4.582
S+SW (mg/mL)d,
*
8.130 × 10−5
a
b
c
d
⁎
pKa predicted ionization coefficient
Water Diffusion Coefficient (cm/s x 10−5
)
Jejune Effective Permeability (cm2/s x 10−4
Water Solubility (mg/mL)
)
S+ = Simulations Plus algorithm
7

C.R. Amorim, et al.
EuropeanJournalofPharmaceuticalSciences150(2020)105371
Fig. 6. – BCS/DCS classification of GPQF-108.
Based on these values (Table 4), the BCS/DCS Explorer tool in
ADMET Predictor™ was used to predict the classification according to
the Biopharmaceutical Classification System (BCS) (Amidon et al.,
1995) and the Developability Classification System (DCS) (Butler and
Dressman, 2010) of the compound.
man with 70 Kg, was used in GastroPlus™ to predict PK values. Other
calculated parameters in GastroPlus™ include the expected half-life
[T1/2, h)] and plasma clearance (L/h) of GPQF-108. It could be ex-
pected a 12.13 h as T1/2 and a clearance of 3.04 L/h for GPQF-108,
which are values compatible with a once a day dose. Despite of the
intestinal solubility problems, the herein predicted pharmacokinetic
aspects indicate the druggability of GPQF-108.
The BCS classifies the drugs into four classes according to the aqu-
eous solubility in physiology pH range and intestinal permeability: class
I (high solubility and high permeability), class II (low solubility and
high permeability), class III (high solubility and low permeability) and
class IV (low solubility and low permeability). The volume of 250 mL of
buffer solutions is used for BCS solubility classification. The DCS pro-
poses changing the volume to 500 mL and the addition of two sub-
classes for BCS class II compounds: class IIa and class IIb. The BCS/DCS
classification of GPQF-108 obtained using ADMET Predictor™ software
is shown in Fig. 6.
In conclusion, GPQF-108 exhibited a promising activity/selectivity
profile, but it was still deficient when compared to the standard drug,
the praziquantel. This compound, however, posed as a good hit to be
optimized in the search for new antischistosomal drugs. By means of
rational chemical modifications, it would be possible to improve both
activity and the absorption aspects which, as presented, does not need
much effort to accomplish. If these compounds keep showing suitable
biological profile, extensive in vivo and off-target activities should be
performed to guarantee the efficacy and safety.
According to the results pointed out in Table 4 the estimated (Fig. 4)
BCS classification of GPQF-108 is class II (low solubility and high
permeability) and the DCS classification is class IIb. This indicates that
GPQF-108 could present absorption limited by its solubility. Moreover,
class IIb compounds tend to have incomplete intestinal absorption
(Butler and Dressman, 2010).
Chemical diversity is exactly what is desired when looking for al-
ternative drugs to a previous one which is under the risk of resistance
emergence. Moreover, the Schiff bases of 4-Phenyl-2-aminothiazoles
presents a completely unexplored chemical structure, mainly regarding
activity against the Schistosomatidae family, which can be obtained
through a fast and economic synthetic pathway, an aspect to be con-
sidered in the search for drugs to treat neglected diseases.
The parameters DiffCoef and S+SW corroborate the difficult water
media diffusion of GPQF-108. Globular compounds are more suitable to
diffuse in water media, but GPQF-108, as all other 4-(4-Substituted
phenyl)-N-(4-substituted benzylidene)thiazole-2-amines, is planar due
to its extended electronic conjugation.
The results herein reported open a new chemical field to be explored
in the search for alternatives drugs to treat Schistosomiasis.
ADMET Predictor™ has the Lipinski's Rule of 5 (Ruleof5) descriptor.
This score indicates the number of potential oral absorption problems a
compound would have. The predicted value for GPQF-108 was 0.000,
which means that this compound appeared to be Rule of 5 compliant.
Predictions of oral absorption with GastroPlus™ software, however,
revealed that the lower the particle size, the higher its oral absorption
and bioavailability (Table 2, Supplementary material). This compound
presented low predicted bioavailability values, which can be improved
by formulation strategies to enhance its solubility.
Funding sources
This work was supported by São Paulo Research Foundation
(FAPESP, Brazil, grant 2013/01875-0) and Brazilian National Council
for Scientific and Technological Development (CNPq, grant 408581/
2018-2). TFAP, ACM and MPS were supported by a fellowship from the
Brazilian Federal Agency for Support and Evaluation of Graduate
Education (CAPES). J.M. also received established investigator fellow-
ship from CNPq.
MaxRTD (mg/Kg/day), the maximum recommended therapeutic
dose administered as oral dose, estimated by ADMET Predictor™ was <
3.16. Based on this result, a daily oral dose of 2.5 mg/Kg, for an adult
8

C.R. Amorim, et al.
EuropeanJournalofPharmaceuticalSciences150(2020)105371
CRediT authorship contribution statement
Mascarenhas, Y.P., Moraes, J.De, De Souza De Almeida Leite, J.R., Jiang, C.S.,
Azevedo, R.B., Figueiró Longo, J.P., 2018. Self-nanoemulsifying drug-delivery sys-
tems improve oral absorption and antischistosomal activity of epiisopiloturine.
Nanomedicine 13, 689–702. https://doi.org/10.2217/nnm-2017-0308.
de Moraes Gomes, P.A.T., de Oliveira Barbosa, M., Farias Santiago, E., de Oliveira
Cardoso, M.V., Capistrano Costa, N.T., Hernandes, M.Z., Moreira, D.R.M., da Silva,
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their biological and ultrastructural effects on Trypanosoma cruzi. Eur. J. Med. Chem.
121, 387–398. https://doi.org/10.1016/j.ejmech.2016.05.050.
Carina R. Amorim: Investigation, Methodology, Data curation.
Thais F.A. Pavani: Methodology, Data curation, Writing - original
draft, Writing - review & editing. Andrey F.S. Lopes: Investigation,
Methodology, Data curation, Software. Marcelo D. Duque:
Conceptualization, Methodology, Data curation, Software, Writing -
original draft. Ana C.A. Mengarda: Investigation, Methodology, Data
curation. Marcos P. Silva: Investigation, Methodology, Data curation.
Josué de Moraes: Conceptualization, Writing - original draft, Funding
acquisition. Daniela G.G. Rando: Supervision, Conceptualization,
Validation, Project administration, Writing - review & editing, Funding
acquisition.
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
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Krajneva, V., Lagunin, A., Macaev, F.Z., Molodavkin, G., Poroikov, V.V., Pogrebnoi,
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Declaration of Competing Interest
The authors of this study declare no conflicts of interest.
Acknowledgment
Grevelding, C.G., Langner, S., Dissous, C., 2018. Kinases: molecular stage directors for
schistosome development and differentiation. Trends Parasitol. 34, 246–260. https://
doi.org/10.1016/j.pt.2017.12.001.
We thank all of J. de Moraes's laboratory for help in maintaining the
S. mansoni life cycle. We also thank Daniel B. Roquini and Tais C. Silva
for support during in vivo studies.
Gryseels, B., Mbaye, A., De Vlas, S.J., Stelma, F.F., Guissé, F., Van Lieshout, L., Faye, D.,
Diop, M., Ly, A., Tchuem-Tchuenté, L.A., Engels, D., Polman, K., 2001. Are poor
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Senegal due to resistance? An overview of the evidence. Trop. Med. Int. Heal. 6,
864–873. https://doi.org/10.1046/j.1365-3156.2001.00811.x.
Portions of these results were generated by ADMET Predictor™ and
GastroPlus™ software provided by Simulations Plus, Inc., Lancaster, CA,
USA.
Guerra, R.A., Silva, M.P., Silva, T.C., Salvadori, M.C., Teixeira, F.S., De Oliveira, R.N.,
Rocha, J.A., Pinto, P.L.S., De Moraes, J., 2019. In vitro and in vivo studies of spir-
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S.A., Kuckelhaus, S.A.S., Pinto, P.L.S., Eaton, P., Mafud, A.C., Mascarenhas, Y.P.,
Allegretti, S.M., de Moraes, J., Lolić, A., Verbić, T., Leite, J.R.S.A., 2015. Anthelmintic
activity in vivo of epiisopiloturine against juvenile and adult worms of Schistosoma
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Supplementary materials
Supplementary material associated with this article can be found, in
the online version, at doi:10.1016/j.ejps.2020.105371.
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