Synthesis, structure-activity relationship and trypanocidal activity of pyrazole-imidazoline and new pyrazole-tetrahydropyrimidine hybrids as promising chemotherapeutic agents for Chagas disease

Article abstract of DOI:10.1016/j.ejmech.2019.111610

Drug therapy for Chagas disease remains a major challenge as potential candidate drugs have failed clinical trials. Currently available drugs have limited efficacy and induce serious side effects. Thus, the discovery of new drugs is urgently needed in the fight against Chagas' disease. Here, we synthesized and evaluated the biological effect of pyrazole-imidazoline (1a-i) and pyrazole-tetrahydropyrimidine (2a-i) derivatives against relevant clinical forms of Trypanosoma cruzi. The structure-activity relationship (SAR), drug-target search, physicochemical and ADMET properties of the major active compounds in vitro were also assessed in silico. Pyrazole derivatives showed no toxicity in Vero cells and also no cardiotoxicity. Phenotypic screening revealed two dichlorinated pyrazole-imidazoline derivatives (1c and 1d) with trypanocidal activity higher than that of benznidazole (Bz) against trypomastigotes; these were also the most potent compounds against intracellular amastigotes. Replacement of imidazoline with tetrahydropyrimidine in the pyrazole compounds completely abolished the trypanocidal activity of series 2(a-i) derivatives. The physicochemical and ADMET properties of the compounds predicted good permeability, good oral bioavailability, no toxicity and mutagenicity of 1c and 1d. Pyrazole nucleus had high frequency hits for cruzipain in drug-target search and structure activity relationship (SAR) analysis of pyrazole-imidazoline derivatives revealed enhanced activity when chlorine atom was inserted in meta-positions of the benzene ring. Additionally, we found evidence that both compounds (1c and 1d) have the potential to interact non-covalently with the active site of cruzipain and also inhibit the cysteine proteinase activity of T. cruzi. Collectively, the data presented here reveal pyrazole derivatives with promise for further optimization in the therapy of Chagas disease.

Full text of DOI:10.1016/j.ejmech.2019.111610

European Journal of Medicinal Chemistry 182 (2019) 111610
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Synthesis, structure-activity relationship and trypanocidal activity of
pyrazole-imidazoline and new pyrazole-tetrahydropyrimidine hybrids
as promising chemotherapeutic agents for Chagas disease
,
,
,
1
b
b
M.E. Monteiro a 1, G. Lechuga a 1, L.S. Lara a , B.A. Souto , M.G. Vigano ,
ꢀ
S.C. Bourguignon c, C.M. Calvet a, F.O.R. Oliveira Jr. a, C.R. Alves d, F. Souza-Silva d
,
,
e
M.S. Santos b **, M.C.S. Pereira a
,
, *
a
ꢀ
Laboratorio de Ultraestrutura Celular, Instituto Oswaldo Cruz, Fiocruz, Av. Brasil 4365 Manguinhos, 21040-900, Rio de Janeiro, RJ, Brazil
Laboratorio de Síntese Organica (LABSINTO), Instituto de Física e Química, Universidade Federal de Itajuba, Avenida BPS, 1303, Pinheirinho, Itajuba,
b
ꢀ
^
ꢀ
ꢀ
37500-903, Minas Gerais, Brazil
c
ꢀ
~
~
~
Laboratorio de Interaçao Celular e Molecular, Departamento de Biologia Celular e Molecular, Universidade Federal Fluminense, Rua Outeiro Sao Joao
ꢀ
Batista, 24020-141, Niteroi, Rio de Janeiro, Brazil
d
ꢀ
^
Laboratorio de Biologia Molecular e Doenças Endemicas, Instituto Oswaldo Cruz, Fiocruz, Av. Brasil 4365 Manguinhos, 21040-900, Rio de Janeiro, RJ, Brazil
e
ꢀ
Centro de Desenvolvimento Tecnologico em Saúde, Fiocruz, Avenida Brasil 4365, Manguinhos, 21040-900, Rio de Janeiro, RJ, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
Drug therapy for Chagas disease remains a major challenge as potential candidate drugs have failed
clinical trials. Currently available drugs have limited efficacy and induce serious side effects. Thus, the
discovery of new drugs is urgently needed in the fight against Chagas' disease. Here, we synthesized and
evaluated the biological effect of pyrazole-imidazoline (1a-i) and pyrazole-tetrahydropyrimidine (2a-i)
derivatives against relevant clinical forms of Trypanosoma cruzi. The structure-activity relationship (SAR),
drug-target search, physicochemical and ADMET properties of the major active compounds in vitro were
also assessed in silico. Pyrazole derivatives showed no toxicity in Vero cells and also no cardiotoxicity.
Phenotypic screening revealed two dichlorinated pyrazole-imidazoline derivatives (1c and 1d) with
trypanocidal activity higher than that of benznidazole (Bz) against trypomastigotes; these were also the
most potent compounds against intracellular amastigotes. Replacement of imidazoline with tetrahy-
dropyrimidine in the pyrazole compounds completely abolished the trypanocidal activity of series 2(a-i)
derivatives. The physicochemical and ADMET properties of the compounds predicted good permeability,
good oral bioavailability, no toxicity and mutagenicity of 1c and 1d. Pyrazole nucleus had high frequency
hits for cruzipain in drug-target search and structure activity relationship (SAR) analysis of pyrazole-
imidazoline derivatives revealed enhanced activity when chlorine atom was inserted in meta-positions
of the benzene ring. Additionally, we found evidence that both compounds (1c and 1d) have the po-
tential to interact non-covalently with the active site of cruzipain and also inhibit the cysteine proteinase
activity of T. cruzi. Collectively, the data presented here reveal pyrazole derivatives with promise for
further optimization in the therapy of Chagas disease.
Received 20 March 2019
Received in revised form
8 August 2019
Accepted 9 August 2019
Available online 10 August 2019
Keywords:
Chagas disease
Trypanosoma cruzi
Chemotherapy
Pyrazolederivatives
Cruzipain inhibitor
SAR analysis
© 2019 Elsevier Masson SAS. All rights reserved.
1. Introduction
Chagas disease (CD), a silent disease caused by Trypanosoma
cruzi, is a serious global public health problem [1]. CD is endemic in
21 countries of Latin America, with a prevalence of 62% of cases (3,5
million) in countries of the Southern Cone [2]. The expansion of its
geographical distribution to non-endemic regions is related to
* Corresponding author.
** Corresponding author.
E-mail addresses: mauriciosantos@unifei.edu.br (M.S. Santos), mirian@ioc.
fiocruz.br (M.C.S. Pereira).
emigration of infected individuals [3]. Mild symptoms or no med-
ical problems are mostly observed in the acute phase of the disease,
1
These authors contributed equally to the work.
https://doi.org/10.1016/j.ejmech.2019.111610
0223-5234/© 2019 Elsevier Masson SAS. All rights reserved.

2
M.E. Monteiro et al. / European Journal of Medicinal Chemistry 182 (2019) 111610
whereas the chronic phase may have a silent clinical course which
progresses ultimately to serious cardiac or gastrointestinal com-
plications [4]. Negligence in the diagnosis and treatment of the
disease contributes to high morbidity and mortality, since it is
estimated that 80% of affected individuals do not have access to
treatment [5]. Additionally, the lack of public policies to eradicate
transmission in Latin America, where few countries have policies in
place to control vectorial and transfusional transmission and pro-
vide health-care access to the at-risk population, are obstacles to
attaining World Health Organization (WHO) 2020 goals to control
CD [6,7].
The etiological treatment is still based on two nitroaromatic
heterocycle compounds, benznidazole and nifurtimox, introduced
more than 50 years ago, which have both low efficacy in the chronic
phase and serious adverse effects. So far, little success has been
achieved in drug discovery and development due to failure of po-
tential azole-based candidates and combination therapy [8]. Pos-
aconazole monotherapy resulted in therapeutic failure [9]. The
STOP-CHAGAS trial demonstrated the trypanostatic action of pos-
aconazole but no advantage over combined treatment with benz-
nidazole (Bz), and showed lower efficacy than Bz monotherapy
[10]. Furthermore, the Benznidazole Evaluation for Interrupting
Trypanosomiasis (BENEFIT) trial also revealed that Bz was unable to
prevent the progression of cardiomyopathy [11]. New clinical trials
to improve drug safety and effectiveness are underway, including
(i) Benznidazole New Doses Improved Treatment and Associations
(BENDITA), which focuses on different therapeutic regimens of Bz
as well as combinations of Bz with E1224, and (ii) Fexinidazole
(FEXI12), also based on low dose and short duration treatment
schedules (DNDi 2018) [12]. The current scenario for drug discovery
for Chagas disease emphasizes the challenges of finding effective
new drugs to combat this neglected disease.
Leishmania (L.) amazonensis was demonstrated. The compound 5-
amino-1-(3,5-dichlorophenyl)-4-(4,5-dihydro-1H-imidazol-2-yl)-
1H-pyrazole showed a reduction of cutaneous lesion in Leishmania
(L.) amazonensis-infected mice [27].
The promising activity of pyrazole, imidazoline and tetrahy-
dropyrimidine led us to synthetize a series of 5-amino-1-aryl-4-
(4,5-dihydro-1H-imidazol-2-yl)-1H-pyrazoles and 2-(-5-amino-1-
aryl-1H-pyrazol-4-yl)-1,4,5,6-tetrahydropyrimidines to evaluate
their trypanocidal activity as well as to investigate the structure-
activity relationship. Together, the data we generated and report
here revealed the effect of pyrazole-imidazoline derivatives as in-
hibitors of cysteine protease with anti-T. cruzi activity, highlighting
their potential as hit compounds for treatment of Chagas disease.
2. Results and discussion
2.1. Chemical synthesis of compounds
The synthesis of the compounds 5-amino-1-aryl-4-(4,5-
dihydro-1H-imidazol-2-yl)-1H-pyrazoles 1(aei) and 2-(-5-amino-
1-aryl-1H-pyrazol-4-yl)-1,4,5,6-tetrahydropyrimidines 2(aei) is
summarized in Scheme 1. In the first step, arylhydrazine hydro-
chlorides 3(aei) were unprotonated with sodium acetate in
ethanol,
under
reflux,
for
20 min.
Then
ethox-
ymethylenemalononitrile was added and the reaction was kept
under reflux for 1 h to obtain 5-amino-1-aryl-1H-pyrazole-4-
carbonitriles 4(aei) [28]. These key intermediates 4(aei) were
the raw materials to access the targets 1(aei) and 2(aei) from 1,2-
diaminoethane (ethylenediamine) and 1,3-diaminopropane,
respectively, using carbon disulfide (CS2) as catalyst [27]. All 2(aei)
derivatives are published for the first time as well as compounds 1e,
1f and 1i.
Pyrazole is a five-membered aromatic heterocyclic ring con-
taining two adjacent nitrogen atoms. It is found in many com-
pounds with biological activity, including anti-inflammatory,
antidepressant, anticancer, analgesic, antioxidant, antifungal, anti-
viral and antileishmanial drugs [13e16]. The potent pharmacolog-
ical properties of nitrogen-containing heterocyclic compounds
have stimulated efforts to design, synthesize and screen com-
pounds containing a pyrazole nucleus against many infectious
agents [16e19]. Microbicidal activity against pathogenic bacteria by
coumarin-pyrazole carboxamide derivatives, a topoisomerase II
and IV inhibitor, was higher than strong antibiotics such as novo-
biocin and ciprofloxacin [20]. Potent antimalarial activity, including
against parasites resistant to currently used antimalarials, has been
shown by pyrazoleamide derivatives, whose mechanism of action
involves the disruption of Naþ regulation [21]. Anti-
trypanosomatid activity of some pyrazole derivatives has been re-
ported to inhibit iron superoxide dismutase (Fe-SOD), disturbing
antioxidant defense of the parasites [22].
We also drew attention to imidazoline and tetrahydropyr-
imidine. Both heterocyclic systems can be found in pharmacological
compounds used to treat hyperglycemia, psychiatric disorders,
cancer and bacterial infections [23,24]. Trypanocidal activity has
also been reported for imidazoline and tetrahydropyrimidine de-
rivatives. N-hydroxy imidazoline derivatives have demonstrated
potent activity against Trypanosoma brucei in central nervous sys-
tem stage (late-stage) sleeping sickness [25]. A pentamidine-
related imidazoline series has also been evaluated against
T. brucei, showing growth inhibition of the pentamidine-resistant
isolates [26]. However, pyrazole-imidazoline and pyrazole-
tetrahydropyrimidine hybrids have not yet been evaluated
against Trypanosoma species. The only work relating to the
pyrazole-imidazoline system was published by Santos and co-
workers [27], in which an in vitro leishmanicidal effect against
2.2. Biological activity of the pyrazole-imidazoline 1(aei) and
pyrazole-tetrahydropyrimidine 2(aei) derivatives
Both series of synthetic compounds, pyrazole-imidazoline
1(aei) and pyrazole-tetrahydropyrimidine 2(aei) derivatives,
were evaluated for cytotoxicity in Vero cells. Metabolic active cells
were determined by quantitating the amount of ATP as an indicator
of cell viability. All derivatives showed no toxicity in Vero cells
(Tables 1 and 2; concentration of compound that reduces 50% of
mammalian cell viability - CC50) but the compounds of the
pyrazole-imidazoline 1c (257.1 22.7
and 1h (352.2 43.4 M), showed lower CC50 values than pyrazole-
tetrahydropyrimidine 2c, 2d and 2h analogs (CC50 > 500 M). The
mM), 1d (243.9 51.6 mM)
m
m
in vitro phenotypic screening of 1(aei) and 2(aei) derivatives was
performed on parasite forms relevant to human infection (trypo-
mastigotes and intracellular amastigotes) (Tables 1 and 2). The
biological activity, evaluated on bloodstream and culture-derived
trypomastigote forms of T. cruzi Y strain and Dm28c-Luc clone,
respectively, revealed a greater activity of the most active com-
pounds 1c (IC50 ¼ 9.5 1.2
against T. cruzi Dm28c-Luc clone when compared to T. cruzi Y strain
(1c - IC50 ¼ 30.9 1.1 M and 1d e IC50 ¼ 28.3 2.7 M) and also to
other analogs of the pirazole-imidazoline series against both T. cruzi
stocks and Bz (Dm28c-Luc M;
IC50 ¼ 19.1 3.3
IC50 ¼ 23.3 4.5), the reference drug (Table 1). Additionally, 1c
(28.9 1.6 M) and 1d (29.0 2.3 M) were 3.2-fold more effective
than Bz (94.1 5.3 M) at IC90 level (Table 1).
mM) and 1d (IC50 ¼ 10.5 1.1
mM)
m
m
-
m
Y
-
m
m
m
The effect of the pyrazole-imidazoline derivatives was also
evaluated against intracellular amastigotes. Three compounds of
the series, 1b (IC50 ¼ 20.6 1.7
(IC50 ¼ 13.7 1.5 M), showed moderate activity against intracel-
lular forms of T. cruzi Dm28c-Luc clone (SI ꢀ 15) while Bz was more
mM), 1c (IC50 ¼ 16.5 1.6
mM) and 1d
m

M.E. Monteiro et al. / European Journal of Medicinal Chemistry 182 (2019) 111610
3
Scheme 1. Reagents and conditions: i) sodium acetate, ethanol, 20 min, reflux; ii) ethoxymethylenemalononitrile, 1h, reflux; iii) 1,2-diaminoethane, CS2, 12e14 h, 110e115 ꢁC; iv)
1,3-diaminopropane, CS2, 14e16 h, 85e95 ꢁC.
Table 1
Cytotoxicity and trypanocidal effect of pyrazole derivatives.
Compounds Trypanocidal effect (mean SD
Trypomastigotes
m
M)
Vero cells toxicity CC500 (mean SD Selectivity index (SI)
m
M)
Intracellular
amastigote Dm28c-
Luc
Trypomastigotes Intracellular amastigote Dm28c-
Luc
Dm28c-Luc
Y strain
Dm28c-
Luc
Y
strain
IC50
IC90
IC50
IC90 IC50
IC90
1a
>100
>100
>100
>100 84.2 11.1 >100
>500
nd
nd
>5.9
1b
1c
1d
1e
53.3 4.2 81.1 7.5 31.3 1.9 >100 20.6 1.7 55 3.7 >500
9.5 1.2* 28.9 1.6* 30.9 1.9 >100 16.5 1.6 29.9 0.5 257.1 22.7
10.5 1.1* 29.0 2.3* 28.3 2.7 >100 13.6 1.5 30.5 1.2 243.9 51.6
9.4
>15.9 >24.3
27.1
23.3
>6.3
nd
nd
nd
nd
nd
>26.3
8.3
8.6
>5.9
nd
15.6
17.9
nd
78.9 9.3 >100
84.2 7.7 >100 >100
>100 >100 >100
52.3 1.0 >100 84.2 5.4 >100
27.7 1.6 >100 67.4 6.7 >100
>100
>100
>500
>500
>500
352.2 43.4
>500
1f
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
nd
1g
1h
1i
2(a-i)
Bz
>9.5
12.7
nd
>5.9
5.2
>7.9
nd
>100
>100
>100 63.4 3.5 >100
>100 >100 >100
>500
nd
19.1 3.3 94.1 5.3 23.3 4.5 >100 1.9 0.2
6.9 0.4 >500
>21.4 >263.1
Average values of three independent experiments standard deviations; (*) Student t-test (*) p ꢂ 0.001.
IC50 and IC90: concentration that produces 50% and 90% inhibitory effect, respectively. CC50: concentration that reduce 50% of Vero cells viability.
SI ¼ CC50 Vero cells/IC50 Trypomastigotes and intracellular amastigote forms of T. cruzi.
nd ¼ Not determined.
SD ¼ Standard deviations.
Table 2
Cardiotoxic effect and trypanocidal activity of pyrazole-imidazoline analogs and Bz.
Compounds
Intracellular amastigote Y strain (IC50
m
M)
Toxicity in cardiomyocytes (CC50 mM)
SI
Bz
1c
1d
0.9 0.1
12.5 1.1
12.7 0.7
>500
378.9 12.1
411.5 23.7
>555
30.3
32.4
Average values of three independent experiments standard deviations.
CC50: concentration that reduce 50% of cardiomyocyte viability.
Selectivety index (SI) ¼ CC50 Cardiomyocytes/IC50 intracellular amastigote forms of T. cruzi.

4
M.E. Monteiro et al. / European Journal of Medicinal Chemistry 182 (2019) 111610
effective (IC50 ¼ 1.9 0.2
m
M; SI > 263.1) (Table 1). At IC90 level, the
2.3. Physicochemical and ADMET properties
1c (29.9 0.5 mM) and 1d (30.5 1.2 mM) remained the most
effective compounds of the pyrazole-imidazoline series, but still
4.4-fold less active than Bz (6.9 0.4 M) (Table 1).
Although small structural changes in compounds may alter
physicochemical parameters, our in silico prediction revealed no
significant differences in physicochemical properties between
1(aei) and 2(aei) derivatives (Table 3). Both 1(aei) and 2(aei)
derivatives respected Lipinski's rule of five (Ro5) as they displayed a
relatively low molecular weight (MW) ꢂ 320.19, low lipophilicity
(cLogP ꢂ 2.12 and LogD ꢂ 1.26), Hydrogen-bond donors (HBDs) < 5,
Hydrogen-bond acceptors (HBAs) < 10 and also low polar surface
area (PSA < 79 Å2) (Table 3), filling up most of the drug-likeness
criteria [30]. Differences in the average of the fraction of carbons
that are sp3 hybridized (Fsp3) were evidenced between series 1
(Fsp3 ¼ 0.17) and 2 (Fsp3 ¼ 0.23e0.29), except the derivative 1i
(Fsp3 ¼ 0.29) (Table 3). It has been proposed that increased carbon
saturation may be related to improvement in the compound's
solubility, increasing the chances for success in drug discovery [31],
but this concept is still controversial [32]. Neither 1(aei) nor 2(aei)
series are an inducer of phospholipidosis (Table 3), suggesting no
adverse effect due to accumulation of drug-phospholipid com-
plexes in lysosomes. Interestingly, the physicochemical properties
comparison of the most effective compounds, the 1c and 1d de-
rivatives, and drugs that evolved to phase I of clinical trials for
Chagas disease, revealed that these compounds have physical pa-
rameters (MW and cLogP) closely related to those of Bz, nifurtimox
and fexinidazole, except for HBDs, HBAs and PSA, influencing
intermolecular interactions, hydrophobicity, drug absorption and
permeability (Fig. 1). In this context, the ADMET prediction
revealed that 1c and 1d, as well as Bz, are more likely to be
permeable to blood-brain barrier (BBB) and human intestinal ab-
sorption (Table S1). Regarding permeability in Caco-2 cells, a posi-
tive absorption was predicted only for compounds 1c and 1d. With
respect to drug metabolism, the 1c and 1d derivatives are inhibitors
of CYP450 1A2, which metabolizes xenobiotics and endogenous
compounds such as steroid hormones [33]. In contrast to Bz, 1(aei)
and 2(aei) derivatives have low risk of cardiotoxicity (weak in-
hibitor of hERG potassium channels), mutagenicity (no induction of
Ames mutation) and carcinogenicity (Table S1).
m
The most active compounds of pyrazole-imidazoline series were
selected to evaluate cardiotoxic effect and their activity against
T. cruzi amastigotes Y strain. To assess the cardiotoxic potential of the
compounds, primary cultures of cardiac muscle cells were treated for
72 h with 1c and 1d derivatives. The results revealed no cardiotox-
icity showing CC50 values of 378.9 12.1 mM and 411.5 23.7 mM, for
1c and 1d, respectively (Table 2). A selective effect against intracel-
lular amastigotes (SI ꢀ 30), T. cruzi Y strain, was observed on both
derivatives, reaching a 50% of effective dose at 12.5 1.1 mM and
12.7 0.7
observation is that the in vitro leishmanicidal activity of the 1c de-
rivative, which has similar potency (IC50 ¼ 15.5 6.8 M) against
mM for 1c and 1d, respectively (Table 2). An interesting
m
Leishmania (L.) amazonensis promastigotes forms, is still effective in
reducing the cutaneous lesion in vivo [27]. These data further
encourage the development of the pyrazole-imidazoline derivative
as a hit compound using rational drug design. Anti-trypanosomatid
activity was also evidenced with simple dialkyl pyrazole-3,5-
dicarboxylates and their sodium salts (pyrazolates) which showed
biological activity against T. cruzi, L. (L.) infantum and L. (V.) brazil-
iensis by inhibition of iron superoxide dismutase [22]. Anti-T. cruzi
activity was also recently reported for pyrazolo [3,4-e] [1,4] thiaze-
pine with potent inhibition of CYP51 [29].
As shown in Table 1, the substitution of the imidazoline nucleus
in the pyrazoles 1(aei) by tetrahydropyrimidine scaffold in the new
series of synthetic compounds 2(aei), completely abolished their
trypanocidal activity, suggesting the relevance of the imidazoline
ring within the molecular structure. The basicity of tetrahy-
dropyrimidine is quite similar to that of the imidazoline ring. The
nitrogen atoms at 1,3-positions are separated from each other by
just one carbon atom in each heterocyclic system. The main dif-
ference regards the ring size, where the 6-membered cyclic chain
shows more conformers than the 5-membered analogs; conse-
quently, the tetrahydropyrimidine presents more flexibility than
imidazoline. Since the 6-membered ring has one additional
methylene group (CH2), the hydrophobicity is greater than in the 5-
membered nucleus. These molecular structural characteristics are
likely to influence interactions in the drug-target binding site.
Table 3
In silico Physicochemical parameters of pyrazole-imidazoline 1(a-i) and pyrazole-tetrahydropyrimidine 2(a-i) derivatives.
Compounds
Physicochemical properties
MW
logP
logD
logSw
tPSA
HBD
HBA
Solubility
Phospholipidosis
Fsp3
1a
1b
1c
1d
1e
1f
1g
1h
1i
2a
2b
2c
2d
2e
2f
2g
2h
2i
227.27
261.71
296.16
296.16
261.71
245.26
306.16
306.16
257.29
241.29
275.74
310.18
310.18
275.74
259.28
320.19
320.19
271.32
0.5
0.05
0.66
1.26
1.26
0.66
0.2
0.82
0.82
ꢃ0.1
ꢃ0.47
0.13
0.74
0.74
0.13
ꢃ0.33
0.3
ꢃ1.7
69.84
69.84
69.84
69.84
69.84
69.84
69.84
69.84
69.84
69.84
69.84
69.84
69.84
69.84
69.84
69.84
79.07
69.84
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
5
5
5
5
5
5
5
5
6
5
5
5
5
5
5
5
5
6
Good
Good
Good
Good
Good
Good
Good
Good
Good
Good
Good
Good
Good
Good
Good
Good
Good
Good
NonInducer
NonInducer
NonInducer
NonInducer
NonInducer
NonInducer
NonInducer
NonInducer
NonInducer
NonInducer
NonInducer
NonInducer
NonInducer
NonInducer
NonInducer
NonInducer
NonInducer
NonInducer
0.17
0.17
0.17
0.17
0.17
0.17
0.17
0.17
0.29
0.23
0.29
0.23
0.23
0.29
0.29
0.29
0.29
0.29
1.13
1.76
1.76
1.13
0.6
ꢃ2.31
ꢃ2.92
ꢃ2.92
ꢃ2.31
ꢃ1.88
ꢃ2.63
ꢃ2.63
ꢃ1.78
ꢃ1.99
2.6
1.2
1.2
0.48
0.86
1.49
2.12
2.12
1.49
0.96
1.55
1.55
0.83
ꢃ3.21
ꢃ3.21
2.6
ꢃ2.17
ꢃ2.92
ꢃ2.92
2.07
0.3
ꢃ0.63
MW - Molecular weight: logP - The logarithm of the partition coefficient between n-octanol and water, characterizing lipophilicity; logD - Represents the logP of compounds at
physiological pH (7.4); logSw - represents the logarithm of compounds water solubility computed by the ESOL method; tPSA - Topological Polar Surface Area; HBD - Hydrogen
Bond Donnors; HBA - Hydrogen Bond Acceptors; Solubility - Water Solubility estimated by Hill method; Fsp3 - Fraction of sp3 carbon.

M.E. Monteiro et al. / European Journal of Medicinal Chemistry 182 (2019) 111610
5
Fig. 1. Physicochemical properties of compounds 1c and 1d and drugs used to treat Chagas disease that advance at least to phase I clinical trials. (A) Comparison of molecular weight
(MW) and lipophilicity (cLogP), color scale represents drug-likeness. (B) Distribution of drugs based on hydrogen bond acceptors (HBA) and topological surface area (PSA); color
scale represents hydrogen bond donors (HBD). Physicochemical properties were calculated using DataWarrior software and graphics were created using R. (For interpretation of the
references to color in this figure legend, the reader is referred to the Web version of this article).
2.4. Structure-activity relationship (SAR) analysis
1b and 1e, against intracellular amastigotes (Dm28c-Luc). Potency
was preserved when two chlorine atoms at two meta-positions (1c;
The impact of modifications in the benzene ring associated with
pyrazole-imidazoline derivatives was evaluated by the structure-
activity relationship analysis (SAR). Compound optimization was
performed by addition of halogens, in different positions of the
benzene ring, and methoxy group (para-position) in both pyrazole-
imidazoline and pyrazole-tetrahydropyrimidine series. The results
highlight the influence of the chlorine atom in the biological ac-
tivity of the pyrazole-imidazoline derivatives (Fig. 2). Dichlorinated
derivatives, 1c and 1d, were more effective than monochlorinateds,
IC50 ¼ 16.5 1.6
m
M) were switched between the meta- and para-
positions (1d; IC50 ¼ 13.6 1.5
m
M). In monochlorinated com-
pounds, the substitution of chlorine in the meta-to para-position
induced loss of activity. Replacement of chorine atom at para-po-
sition by other halogens, bromine and fluorine, or methoxy group
reduced the activity of the compounds (Fig. 2). Switching the
chlorine (1b) for bromine (1h) atom at meta-position and also
methoxy group at para-position reduced approximately 3-fold the
trypanocidal activity. Curiously, all compounds with halogens,

6
M.E. Monteiro et al. / European Journal of Medicinal Chemistry 182 (2019) 111610
Fig. 2. Structure-activity relationship (SAR) analysis of pyrazole-imidazoline and pyrazole-tetrahydropyrimidine derivatives. The amino-pyrazole center was used to separate
substituents in R1 (halogen and methoxy group insertions and position in benzene ring) and R2 (imidazoline or tetrahydropyrimidine rings). Bars represent mean values of IC50
achieved against intracellular amastigotes; color scale denotes the drug-likeness of each compound. Chlorine-containing compounds of pyrazole-imidazoline derivatives (1b-d) at
meta-position were the most active compounds of the series. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this
article).
including chlorine atom, and methoxy group at para-position of the
benzene ring linked to pyrazole-imidazoline derivatives showed
trypanothione reductase and tubulin alpha chain. Next, the com-
pounds’ scaffolds were queried in this small library of compounds
using the core-based SAR analysis tool available in the program,
low trypanocidal activity (IC50 ꢀ 63.3
m
M), except for meta- and
para-position of dichloride derivative (1d; IC50 ¼ 13.6 1.5
mM).
which identified 302 molecules, with activity ꢂ10
mM, containing
Overall, these data suggest that chlorination at the meta-position
may be crucial to the enhancement of trypanocidal activity by
pyrazole-imidazoline derivatives. In contrast, the electronic in-
fluences of the halogen and methoxy substituents did not result in
an increase of potency of pyrazole-tetrahydropyrimidine de-
rivatives (Fig. 2), suggesting a steric rather than an electronic effect.
The role of the halogen bond has been highlighted in drug dis-
covery, with organohalogens achieving 34% of pre- or clinical trials
stages [34]. But even though chlorine atom has been prominent as a
promoter of biological activity, chlorination may also abolish drug
activity or induce toxicological properties [35].
the selected fragments (Fig. 3). Only 2 targets of T. cruzi, 6-phospho-
1-fructokinase and cruzipain, were spotted by compounds con-
taining the selected fragments in their structures (Fig. 3). Notably,
cruzipain, the major cysteine proteinase of T. cruzi that belongs to
the papain-like clan CA, was the most recorded target in this set of
compounds. Pyrazole and imidazoline scaffolds were identified in
93.7% and 2.6%, respectively, of the total active compounds iden-
tified for cruzipain after fragment-based screening (Fig. 3). Only
few compounds containing pyrazole scaffold (0.09%) identified 6-
phospho-1-fructokinase in the library (Fig. 3). Although the
pyrazole-tetrahydropyrimidine derivatives have not been active, 11
tetrahydropyrimidine-containing compounds capable of recog-
nizing cruzipain are registered in the chembl database, suggesting
that the tetrahydropyrimidine scaffold may influence pharmaco-
logical activity when linked to a heterocyclic compounds such as
thiophene, imidazole or furan [36].
Cruzipain has been highlighted as an important target for drug
design due to its relevance in parasite metabolism and survival [37].
Cruzipain is expressed in all development stages of T. cruzi and its
inhibition leads to blockage of parasite invasion [38], metacyclo-
genesis and intracellular development [39]. K777 (N-[(2 S)-1-
[[(E,3 S)-1-(benzenesulfonyl)-5-phenylpent-1-en-3-yl]amino]-1-
oxo-3-phenylpropan-2-yl]-4-methylpiperazine-1-carboxamide, an
irreversible vinyl sulfone cruzipain inhibitor, showed potent effi-
2.5. In silico drug-target search
Subsequent analyses were performed to identify possible
binding targets for 1(aei) and 2(aei) compounds. Computational
analyses using DataWarrior software were based on the available
data of trypanocidal compounds integrated into Chembl database.
A total of 34.154 compounds were identified with activity against
16 targets of T. cruzi, including 6-phospho-1-fructokinase, bifunc-
tional dihydrofolate reductase-thymidylate synthase, cruzipain,
cyclic nucleotide specific phosphodiesterase, deoxyuridine tri-
phosphatase, farnesyl diphosphate synthase, farnesyl synthetase,
farnesyltransferase, glyceraldehyde-3-phosphate dehydrogenase,
glycosomal hexokinase, spermidine synthase, sterol 14-alpha
cacy in preclinical trials, validating cruzipain as
a potential
chemotherapeutic target [40,41]. Tetrafluorophenoxy ketone
demethylase,
trans-sialidase,
triosephosphate
isomerase,

M.E. Monteiro et al. / European Journal of Medicinal Chemistry 182 (2019) 111610
7
Fig. 3. In silico fragment-based search of molecular targets. Matched scaffolds of pyrazole-imidazoline and pyrazole-tetrahydropyrimidine to trypanocidal compounds with activity
M), against annotated targets in Chembl database. Analysis were made using DataWarrior software and retrieved two T. cruzi targets, cruzipain and putative 6-phospho-1-
(ꢂ10
m
fructokinase, N represents the number of compounds with each matched scaffold. (For interpretation of the references to color in this figure legend, the reader is referred to the
Web version of this article).
analogs have also been identified as promising cruzipain inhibitors,
showing trypanocidal activity in a mouse model of acute T. cruzi
infection [42]. Optimization of tetrafluorophenoxy ketone by
addition of the pyrazolopyrimidine ring potentially improved try-
panocidal activity and oral bioavailability [43]. Additionally, pyr-
azole derivatives, such as N-(1H-benzimidazol-2-yl)-1,3-dimethyl-
pyrazole-4-carboxamide (PDB code: 4W5B) [44] and N-(1H-ben-
zimidazol-2-yl)-3-(4-fluorophenyl)-1H-pyrazole-4-carboxamide
(PDB code: 4W5C) [45], also bind to cruzipain and have been
considered promising T. cruzi chemotherapeutic agents. Accord-
ingly, we docked compounds 1c and 1d in the 4W5B cruzipain
active site to predict binding poses and interactions between cru-
zipain and pyrazole-imidazoline derivatives.
this way, for the subsequent docking tests we adopted the ligand
flexible configuration with the residue and histidine protonated.
Docking results showed that the compound 1c ligand presented a
score ꢃ7.7 kcal/mol and RMSD 4.0 Å, and the 1d ligand had a
score ꢃ7.8 kcal/mol and RMSD 4.4 Å.
The binding modes of the crystallized complexes as 4W5B, 3iut
and 3kku with their respective 3H5, KB2 and B95 ligands, were
analyzed and the amino acid residues, that participate in the
binding site with the different ligands, identified. This analysis
allowed us to recognize that different ligands crystallized in the
active site of cruzipain have common binding residues in PDBs
4W5B, 3uit and 3kku including GLY23, CYS25, GLY65, GLY66,
ASP161, HIS162 and GLY163 (Fig. 4). We also noticed that the res-
idues of TRP26, LEU67 and LEU160 perform interactions only with
3uit and 3kku available in PDB while GLN19 with 3uit and 4W5B in
PDB. Except for LEU67 and LEU160, we have estimated that the
remaining amino acid residues also interact with the compounds
1c and 1d.
2.6. Prediction of compounds binding-mode
Compound N-(1H-benzimidazol-2-yl)-1,3-dimethyl-pyrazole-
4-carboxamide (3H5) from the 4W5B protein data base (PDB) file
was used as default and compared with 1c and 1d derivatives. In
the initial stage of these experiments, 4W5B redocking assays were
performed under both rigid and flexible binder conditions and with
protonation of the active center histidine residue. The rigid 3H5
linker: deprotonated histidine ꢃ7.85 kcal/mol and RMSD 1.15 Å;
histidine protonated ꢃ7.85 kcal/mol and RMSD 1.3 Å. The flexible
3H5 linker: deprotonated histidine ꢃ7.54 kcal/mol and RMSD 3.5 Å;
histidine protonated ꢃ7.85 kcal/mol and RMSD 1.3 Å (Fig. S1). In
Through the docking assays, we observed that the complexes of
the PDB files and the docked compounds showed hydrophobic
bonds with amino acid residues at the active site of cruzipain
(Fig. 4). In addition, the chlorine atom of both compounds possibly
binds to amino acids residues, 1c interacts with SER35 residue,
while 1d chlorine atoms closely pose to GLY66 and SER35 in active
site of the enzyme by hydrogen bond of 2.8 Å. This finding suggests
that chlorine atoms may contribute favorably to the stability of the

8
M.E. Monteiro et al. / European Journal of Medicinal Chemistry 182 (2019) 111610
Fig. 4. Molecular docking analysis of 1c, 1d and inhibitors of cruzipain into enzyme binding pocket. (A) Binding mode of 3H5 [1 (N-(1H-benzimidazol-2-yl)-1,3-dimethyl-pyrazole-
4-carboxamide)](PDB: 4W5B), compounds 1c (B) and 1d (C), showing hydrophobic (gray) interactions with amino acid residues inside cruzipain active site. (D) Docking analysis of
1c and 1d binding poses, showing that compounds interact mostly with the same residues and are tightly superimposed. The residues GLY66 and SER65 interact closely (2.8 Å) to
chlorine atoms of 1d, whereas, only Ser65 interact with chlorine atom at para position in 1c. (E) Mapping of important amino acid residues in binding pocket of cruzipain
crystallographic structures available in PDB 4W5B, 3IUT and 3KKU. Mapping performed considering interactions with respective ligands 3H5, KB2 and B95, compound 1c, 1d and E-
64, a cysteine protease inhibitor. Most common residues that interact with ligands in cruzipain active site are GLY23, GLY65, GLY66, ASP161, HIS162 and GLY163. Docking visu-
€
alization of binding poses and amino acid residue interactions were performed using PyMOL and Maestro (Schrodinger). (For interpretation of the references to color in this figure
legend, the reader is referred to the Web version of this article).
residues of the enzyme in the interaction with this compound.
Among the 33 compounds identified in the virtual screening, 4
compounds (Flucloxacillin sodium, Cefoperazone sodium, Piper-
acillin sodium, Etofyllin clofibrate), which interact with the cata-
lytic amino acids SER25, HIS162 and TRP184, were identified as
potential inhibitors of cruzipain [47].
Thus, considering the binding prediction for 1c and 1d in the
active site of cruzipain, an open question was whether this mainly
hydrophobic interaction could lead to inhibition of the cysteine
proteinase activity of T. cruzi. In this way, we performed a kinetics of
the enzymatic activity using a fluorogenic substrate. The results
indicated that 1c and 1d derivatives inhibit cysteine proteinase
activity from T. cruzi protein extract, assessed with Z-FR-AMC that is
an excellent substrate for the fluorometric assay of cysteine pro-
teinase [48]. The inhibition rate reached levels of 50% enzymatic
activity (44 ꢄ 105
m mM and
mol minꢃ1 mg of proteinꢃ1) with 24
180 mM for 1c and 1d derivatives, respectively. Compared with the
IC50 value of E-64 (3.0 mM), an irreversible and selective inhibitor of
Fig. 5. Proteinase activities of Trypanosoma cruzi trypomastigotes proteins extract. For
the enzyme quantification, about 5 g of the protein was incubated with 30 M of Z-
FR-AMC, in enzyme activation buffer pH 5.0, at 37 ꢁC. The activities of the samples
were followed by co-incubation with different concentrations ( M) of E-64 (black
cysteine proteinase [49], the 1c and 1d compounds showed a lower
performance of 8-fold and 60-fold, respectively. (Fig. 5). Interest-
ingly, although derivatives 1c and 1d have similar anti-T. cruzi ac-
tivity, they have quite distinct inhibitory activity for cysteine
protease. The difference between enzymatic activity and trypano-
cidal effect suggests that these derivatives may act as a multi-target
drug. Further studies will be conducted to evaluate the multi-target
profile of both derivatives and also their inhibitory activity of pu-
rified T. cruzi cruzipain for molecular docking validation.
m
m
m
circle), 1c (white circle) and 1d (gray circle) compounds. Results presented as
percentile of proteinase activity (%) and represent the median of three analyses. In
these assays, 100% of activity is relative to 88 ꢄ 105
m .
mol minꢃ1 mg of proteinꢃ1
ligand-target complex at the binding site of cruzipain, a property
that is also exploited for some experimental and approved drugs in
clinical development [46].
For comparison purposes, the binding mode of E-64, a classical
inhibitor of cysteine proteinase, was accessed (ꢃ7.0 kcal/mol). The
results also indicated the partitioning of GLY23 and ASP161
Pyrazole derivatives have been described as inhibitors of iron
superoxide dismutase (65e98% inhibition) [50] and CYP51
(IC50 ¼ 0.1
mM) [29] of T. cruzi and reported as potential anti-
cathepsin B, H and L agents [51]. Hybrid compounds based on

M.E. Monteiro et al. / European Journal of Medicinal Chemistry 182 (2019) 111610
9
1,4-dihydropyridine and pyrazole moieties showed potential anti-
malarial activity against chloroquine-sensitive Plasmodium falcip-
arum, whose falcipain-2 inhibition, a plasmodial cysteine protein-
ase, was proposed as mechanism of action [52]. These data
highlight that the structural optimization of pyrazole derivatives
may contribute to the identification of novel antiparasitic drugs.
4.1.1.1. 5-amino-4-(4,5-dihydro-1H-imidazol-2-yl)-1-phenyl-1H-
pyrazole (1a). Yield: 55%; mp: 138e140 ꢁC; FT-IR (cmꢃ1): 3351,
3264, 3149, 3074, 934, 2885, 2864, 1595, 1567; 1H NMR (500 MHz,
MeOH-d4):
¼ 7.69 (s, 1H), 7.53e7.54 (m, 4H), 7.42e7.45 (m, 1H),
n
d
3.65 (s, 4H); 13C NMR (125 MHz, MeOH-d4):
d
¼ 162.8, 148.9, 140.1,
139.4, 130.8, 129.4, 125.6, 95.6, 49.8; HRMS (ESI): m/z [MþH]þ:
calcd. for C12H13N5: 228.1249; found: 228.1261.
3. Conclusion
4.1.1.2. 5-amino-1-(3-chlorophenyl)-4-(4,5-dihydro-1H-imidazol-2-
yl)-1H-pyrazole (1b). Yield: 74%; mp: 160e162 ꢁC; FT-IR
n
(cmꢃ1):
3376, 3280, 3222, 3171, 2948, 2922, 2876,1632,1609,1594,1569; 1H
NMR (500 MHz, MeOH-d4):
¼ 7.70 (s, 1H), 7.61e7.62 (m, 1H),
7.52e7.53 (m, 2H), 7.42e7.44 (m, 1H), 3.65 (s, 4H); 13C NMR
Chagas disease chemotherapy remains an important challenge.
Only two drugs, each with significant limitations of efficacy and
presenting serious adverse effects, are currently used in the clinic
for treatment of Chagas disease. The identification of effective and
safe new drugs is essential to bring hope to millions of chronically
infected individuals, most with severe cardiomyopathies. Here, we
integrated in silico analyses and preclinical in vitro assays to eval-
uate the trypanocidal activity and predict drug-likeness properties
and drug-target interactions. In vitro phenotypical screening and
SAR analysis revealed the effectiveness of two dichlorinated
pyrazole-imidazoline derivatives, compounds 1c and 1d, against
T. cruzi, highlighting the influence of the chlorine atom at the meta-
position of the benzene ring in the compound's potency. These
compounds showed important physicochemical properties similar
to chemotherapeutic clinical agents (benznidazole and nifurtimox)
and also to drugs that advanced to phase I clinical trials. Structure-
based virtual screening predicted cruzipain as a potential biological
target of pyrazole-imidazoline derivatives. Theoretical evaluation
of the docking complexes revealed that both compounds (1c and
1d) have potential to bind to the active site of cruzipain as a
cysteine proteinase inhibitor of T. cruzi, highlighting them as
promising novel drug candidates for the control of Chagas disease.
d
(125 MHz, MeOH-d4):
d
¼ 162.7, 149.1, 140.7, 140.6, 136.2, 132.1,
129.1, 125.4, 123.4, 96.0; HRMS (ESI): m/z [MþH]þ: calcd. for
C12H12ClN5: 262.0859; found: 262.0877.
4.1.1.3. 5-amino-1-(3,5-dichlorophenyl)-4-(4,5-dihydro-1H-imida-
zol-2-yl)-1H-pyrazole (1c). Yield: 83%; mp: 96e98 ꢁC; FT-IR
(cmꢃ1): 3420, 3282, 3083, 2946, 2873, 1619, 1583, 1568; 1H NMR
n
(500 MHz, DMSO‑d6):
d
¼ 7.76 (s, 1H), 7.67 (d, J ¼ 1.8 Hz, 2H), 7.62 (t,
J ¼ 1.8 Hz, 1H), 6.85 (br, 2H), 3.53 (s, 4H); 13C NMR (125 MHz,
DMSO‑d6):
d
¼ 160.1, 148.6, 140.8, 140.2, 135.0, 126.7, 121.6, 94.6,
48.3; HRMS (ESI): m/z [MþH]þ: calcd. for C12H11Cl2N5: 296.0470;
found: 296.0474.
4.1.1.4. 5-amino-1-(3,4-dichlorophenyl)-4-(4,5-dihydro-1H-imida-
zol-2-yl)-1H-pyrazole (1d). Yield: 71%; mp: 197e199 ꢁC; FT-IR
n
(cmꢃ1): 3418, 3298, 3074, 2935, 2873, 1611, 1560, 1522; 1H NMR
(500 MHz, DMSO‑d6):
¼ 7.85 (d, J ¼ 2.5 Hz, 1H), 7.78 (d, J ¼ 8.7 Hz,
1H), 7.73 (s, 1H), 7.63 (dd, J ¼ 8.7, 2.5 Hz, 1H), 6.77 (br, 2H), 3.51 (s,
d
4H); 13C NMR (125 MHz, DMSO‑d6):
d
¼ 159.8, 147.9, 139.2, 138.3,
131.6, 131.1, 128.8, 124.0, 122.5, 94.8; HRMS (ESI): m/z [MþH]þ:
calcd. for C12H11Cl2N5: 296.0470; found: 296.0472.
4. Material and methods
4.1.1.5. 5-amino-1-(4-chlorophenyl)-4-(4,5-dihydro-1H-imidazol-2-
4.1. Compounds
yl)-1H-pyrazole (1e). Yield: 42%; mp: 179e181 ꢁC; FT-IR
n
(cmꢃ1):
3397, 3265, 3122, 2932, 2852, 1606, 1568, 1523; 1H NMR (500 MHz,
All commercial reagents were used as received unless otherwise
noted. The reaction progress was monitored by thin layer chro-
matography (TLC) with precoated 60 F254 silica gel plates. The
melting points were determined on a Fisatom 430 apparatus.
Infrared spectra (FT-IR) were recorded on a PerkinElmer Spectrum
100, ATR diamond-ZnSe apparatus, wave numbers expressed in
cmꢃ1. NMR spectra were recorded on a Bruker Avance (500 or
400 MHz), at 298 K, in CDCl3, methanol-d4 or DMSO‑d6. Chemical
MeOH-d4):
d
¼ 7.69 (s, 1H), 7.54 (s, 4H), 3.65 (s, 4H); 13C NMR
(125 MHz, MeOH-d4):
d
¼ 162.6, 148.9, 140.3, 138.0, 134.7, 130.8,
126.8, 95.8, 49.7; HRMS (ESI): m/z [MþH]þ: calcd. for C12H12ClN5:
262.0859; found: 262.0861.
4.1.1.6. 5-amino-4-(4,5-dihydro-1H-imidazol-2-yl)-1-(4-
fluorophenyl)-1H-pyrazole (1f). Yield: 66%; mp: 164e166 ꢁC; FT-IR
n
(cmꢃ1): 3280, 3075, 2943, 2872,1611, 1598, 1567; 1H NMR
shifts (d) are expressed in parts per million (ppm) and coupling
(500 MHz, MeOH-d4):
d
¼ 7.68 (s, 1H), 7.55 (dd, J ¼ 9.0, 4.8 Hz, 2H),
constants (J) in Hertz (Hz). The High-Resolution Mass Spectrometry
(HRMS) was performed using Micromass/Waters ZQ-4000 Spec-
trometer, capillary 3.0 kV, cone 30.0 V, extrator 1 V, RF lens 1.0 V,
source temperature 150 ꢁC, desolvation temperature 300 ꢁC (Elec-
trospray Ionization-ESI). The key intermediates 5-amino-1-aryl-
1H-pyrazole-4-carbonitriles 4(aei) were synthesized by our
research group according to described previously.
7.28 (t, J ¼ 9.0 Hz, 2H), 3.65 (s, 4H); 13C NMR (125 MHz, MeOH-d4):
d
¼ 163.6 (d, J ¼ 244.2 Hz), 162.5, 149.0, 140.1, 135.5 (d, J ¼ 2.9 Hz),
127.9 (d, J ¼ 8.9 Hz), 117.4 (d, J ¼ 23.3 Hz), 95.6, 49.7; HRMS (ESI): m/
z [MþH]þ: calcd. for C12H12FN5: 246.1155; found: 246.1162.
4.1.1.7. 5-amino-1-(4-bromophenyl)-4-(4,5-dihydro-1H-imidazol-2-
yl)-1H-pyrazole (1g). Yield: 84%; mp: 184e188 ꢁC; FT-IR
n
(cmꢃ1):
3387, 3260, 3119, 2926, 2852, 1605, 1566, 1523; 1H NMR (400 MHz,
MeOH-d4):
¼ 7.68e7.70 (m, 3H), 7.49 (d, J ¼ 8.9 Hz, 2H), 3.65 (s,
4.1.1. General procedure for synthesis of 5-amino-1-aryl-4-(4,5-
dihydro-1H-imidazol-2-yl)-1H-pyrazoles 1(aei)
d
4H); 13C NMR (100 MHz, MeOH-d4):
d
¼ 161.1, 147.5, 138.9, 137.1,
5-amino-1-aryl-1H-pyrazole-4-carbonitriles 4(aei) (0.001 mol),
2.0 mL of 1,2-diaminoethane (ethylenediamine) and carbon disul-
fide (0.004 mol) were added into a 50 mL round-bottom flask
adapted with a glass condenser. The mixture was kept at 110-115oC
for 12-14 hours. After that, the productwas poured into cold water,
the precipitate was filtered out and washed with cold water. The
reactions were accompanied by means of TLC and dichloromethane
as eluent.
132.4, 125.6, 121.1, 94.2; HRMS (ESI): m/z [MþH]þ: calcd. for
C12H12BrN5: 306.0354; found: 306.0365.
4.1.1.8. 5-amino-1-(3-bromophenyl)-4-(4,5-dihydro-1H-imidazol-2-
yl)-1H-pyrazole (1h). Yield: 47%; mp: 88e92 ꢁC; FT-IR (cmꢃ1):
3442, 3321, 3249, 3088, 3063, 2941, 2869, 1614,1592,1527; 1H NMR
n
(400 MHz, MeOH-d4):
d
¼ 7.76 (t, J ¼ 1.9 Hz, 1H), 7.70 (s, 1H),
7.56e7.60 (m, 2H), 7.45 (t, J ¼ 8.0 Hz, 1H), 3.65 (s, 4H); 13C NMR

10
M.E. Monteiro et al. / European Journal of Medicinal Chemistry 182 (2019) 111610
(100 MHz, MeOH-d4):
d
¼ 161.1, 147.6, 139.2, 139.1, 130.7, 130.6,
1H NMR (400 MHz, MeOH-d4)
d
¼ 7.64 (s, 1H), 7.53 (s, 4H), 3.40 (t,
126.7, 122.3, 122.3, 94.4; HRMS (ESI): m/z [MþH]þ: calcd. for
J ¼ 5.8 Hz, 4H), 1.84 (quint, J ¼ 5.8 Hz, 2H); 13C NMR (100 MHz,
C
12H12BrN5: 306.0354; found: 306.0357.
MeOH-d4)
d
¼ 152.8, 147.0, 137.7, 136.8, 133.1, 129.3, 125.3, 99.0,
40.8, 20.6; HRMS (ESI): m/z [MþH]þ: calcd. for C13H14ClN5:
4.1.1.9. 5-amino-4-(4,5-dihydro-1H-imidazol-2-yl)-1-(4-
276.1016; found: 276.1025.
methoxyphenyl)-1H-pyrazole (1i). Yield: 50%; mp: 171e173 ꢁC; FT-
IR
n
(cmꢃ1): 3263, 2940, 2857, 2837, 1602, 1561, 1510; 1H NMR
4.1.2.6. 2-(5-amino-1-(4-fluorophenyl)-1H-pyrazol-4-yl)-1,4,5,6-
(500 MHz, MeOH-d4):
d
¼ 7.65 (s, 1H), 7.41 (d, J ¼ 8.9 Hz, 2H), 7.07
tetrahydropyrimidine (2f). Yield: 5%; mp: 146e147 ꢁC; FT-IR
n
(d, J ¼ 8.9 Hz, 2H), 3.85 (s, 3H), 3.64 (s, 4H); 13C NMR (125 MHz,
(cmꢃ1): 3392, 3281, 3098, 2951, 2936, 2855, 1610, 1593, 1567, 1532;
MeOH-d4):
d
¼ 162.8, 161.1, 148.8, 139.6, 131.8, 127.4, 115.8, 95.3,
1H NMR (400 MHz, CDCl3)
d
¼ 7.50e7.54 (m, 3H), 7.17 (t, J ¼ 8.3 Hz,
56.1, 49.7; HRMS (ESI): m/z [MþH]þ: calcd. for C13H15N5O:
2H), 3.46 (t, J ¼ 5.8 Hz, 4H), 1.87 (quint, J ¼ 5.8 Hz, 2H); 13C NMR
258.1355; found: 258.1366.
(100 MHz, CDCl3)
d
¼ 161.9 (d, J ¼ 248.0 Hz), 151.4, 146.6, 136.5,
134.5 (d, J ¼ 2.9 Hz), 125.9 (d, J ¼ 8.6 Hz), 116.7 (d, J ¼ 22.9 Hz), 99.0,
41.6, 21.3; HRMS (ESI): m/z [MþH]þ: calcd. for C13H14FN5: 260.1311;
found: 260.1319.
4.1.2. General procedure for synthesis of 2-(-5-amino-1-aryl-1H-
pyrazol-4-yl)-1,4,5,6-tetrahydropyrimidines 2(aei)
The experimental procedure was similar to synthesis of analogs
1(aei).
5-amino-1-aryl-1H-pyrazole-4-carbonitriles
4(aei)
4.1.2.7. 2-(5-amino-1-(4-bromophenyl)-1H-pyrazol-4-yl)-1,4,5,6-
(0.001 mol) were reacted with 2.0 mL of 1,3-diaminopropane and
carbon disulfide (0.004 mol). The temperature was 85-95oC and the
reaction time was 14-16 hours. The reaction mixture was poured
into cold water, the precipitate was filtered out and washed with
cold water. The reactions were accompanied by means of TLC and
dichloromethane as eluent.
tetrahydropyrimidine (2g). Yield: 42%; mp: 192e198 ꢁC; FT-IR
n
(cmꢃ1): 3411, 3225, 3098, 2951, 2934, 2850, 1610, 1575, 1560, 1511;
1H NMR (400 MHz, MeOH-d4)
¼ 7.69 (s, 1H), 7.66 (d, J ¼ 8.8 Hz,
d
2H), 7.47 (d, J ¼ 8.8 Hz, 2H), 3.42 (t, J ¼ 5.8 Hz, 4H), 1.88 (quint,
J ¼ 5.8 Hz, 2H). 13C NMR (100 MHz, MeOH-d4)
d
¼ 154.5, 148.4,
139.4, 138.5, 133.8, 127.1, 122.5, 99.6, 41.9, 21.7; HRMS (ESI): m/z
[MþH]þ: calcd. for C13H14BrN5: 320.0511; found: 320.0520.
4.1.2.1. 2-(5-amino-1-phenyl)-1H-pyrazol-4-yl)-1,4,5,6-
tetrahydropyrimidine (2a). Yield: 48%; mp:146e147 ꢁC; FT-IR
n
4.1.2.8. 2-(5-amino-1-(3-bromophenyl)-1H-pyrazol-4-yl)-1,4,5,6-
(cmꢃ1): 3313, 3184, 3039, 2972, 2876, 1612, 1596, 1556, 1529. 1H
tetrahydropyrimidine (2h). Yield: 52%; mp: 100e104 ꢁC; FT-IR
n
NMR (400 MHz, MeOD)
d
¼ 7.71 (s, 1H), 7.55e7.57 (m, 4H), 7.39 (s,
(cmꢃ1): 3250, 3103, 3063, 2951, 2865, 2825, 1605, 1590, 1532; 1H
NMR (400 MHz, MeOH-d4)
¼ 7.74 (t, J ¼ 1.9 Hz, 1H), 7.69 (s, 1H),
1H), 3.49 (t, J ¼ 5.7 Hz, 4H), 1.95 (quint, J ¼ 5.7 Hz, 2H). 13C NMR
d
(100 MHz, MeOD)
d
¼ 154.8, 148.8, 140.2, 139.0, 131.3, 129.5, 124.4,
7.54e7.60 (m, 2H), 7.42e7.45 (m, 1H), 3.48 (t, J ¼ 5.8 Hz, 4H), 1.95
99.8, 41.7, 21.4. HRMS (ESI): m/z [MþH]þ: calcd. for C13H16N5:
(quint, J ¼ 5.8 Hz, 2H); 13C NMR (100 MHz, MeOD):
¼ 155.0, 140.5,
d
242.1406; found: 242.1413.
136.5, 132.4, 131.8, 130.9, 128.7, 125.6, 121.5, 99.7, 41.4, 21.1; HRMS
(ESI): m/z [MþH]þ: calcd. for C13H14BrN5: 320.0511; found:
320.0517.
4.1.2.2. 2-(5-amino-1-(3-chlorophenyl)-1H-pyrazol-4-yl)-1,4,5,6-
tetrahydropyrimidine (2b). Yield: 56%; mp: 130e132 ꢁC; FT-IR
n
(cmꢃ1): 3306, 3260, 3194, 2962, 2869, 1611, 1594, 1552, 1533; 1H
NMR (400 MHz, MeOH-d4)
¼ 7.71 (s, 1H), 7.59e7.60 (m, 1H),
4.1.2.9. 2-(5-amino-1-(4-methoxyphenyl)-1H-pyrazol-4-yl)-1,4,5,6-
d
tetrahydropyrimidine (2i). Yield: 45%; mp: 177e179 ꢁC; FT-IR
n
7.51e7.52 (m, 2H), 7.45e7.46 (m, 1H), 3.49 (t, J ¼ 5.8 Hz, 4H), 1.96
(cmꢃ1): 3310, 3245, 3092, 2942, 2863, 2848, 1603, 1578, 1550, 1532;
1H NMR (400 MHz, MeOD)
¼ 7.68 (s, 1H), 7.42 (d, J ¼ 8.8 Hz, 2H),
(quint, J ¼ 5.8 Hz, 2H); 13C NMR (100 MHz, MeOH-d4)
d
¼ 155.0,
d
148.7, 140.4, 140.3, 136.3, 132.1, 129.5, 125.8, 123.9, 41.3, 21.0; HRMS
(ESI): m/z [MþH]þ: calcd. for C13H14ClN5: 276.1016; found:
276.1022.
7.10 (d, J ¼ 8.8 Hz, 2H), 3.87 (s, 3H), 3.48 (t, J ¼ 5.8 Hz, 4H), 1.95
(quint, J ¼ 5.8 Hz, 2H). 13C NMR (100 MHz, MeOD):
¼ 162.0, 155.1,
d
148.4, 137.8, 132.3, 127.4, 115.8, 99.5, 55.7, 41.7; 21.2; HRMS (ESI): m/
z [MþH]þ: calcd. for C14H17N5O: 272.1511; found: 272.1520.
4.1.2.3. 2-(5-amino-1-(3,5-dichlorophenyl)-1H-pyrazol-4-yl)-
1,4,5,6-tetrahydropyrimidine (2c). Yield: 64%; mp: 144 ꢁC; FT-IR
n
4.2. Cell cultures
(cmꢃ1): 3306, 3078, 3048, 2941, 2860, 1610, 1585, 1569, 1522; 1H
NMR (400 MHz, CDCl3)
¼ 7.56 (d, J ¼ 1.8 Hz, 2H), 7.50 (s, 1H), 7.32
d
Vero cells (ATCC® CCL81™), derived from the kidney of an Af-
rican green monkey, were subcultured with dissociation solution
(0.25% trypsin-EDTA) when cultures achieved 80e100% confluence.
Subsequently, the cells were washed and cultivated in RPMI-1640
medium with 10% fetal bovine serum (FBS).
(t, J ¼ 1.8 Hz, 1H), 4.13 (br, 2H), 3.45 (t, J ¼ 5.8 Hz, 4H), 1.86 (quint,
J ¼ 5.8 Hz, 2H); 13C NMR (100 MHz, CDCl3)
d
¼ 151.1, 146.9, 140.4,
137.2, 136.0, 127.3, 121.5, 99.8, 41.8, 21.4; HRMS (ESI): m/z [MþH]þ:
calcd. for C13H13Cl2N5: 310,0626; found: 310.0634.
Primary cultures of heart muscle cells were obtained from 18
days mouse fetuses as previously described [53]. Ventricular tissue
was dissociated with trypsin and collagenase type II solution. Iso-
lated cells were cultivated in Dulbecco's modified Eagle medium,
supplemented with 7% fetal FBS, 2% chicken embryo extract, 1 mM
L-glutamine and antibiotics, and maintained at 37 ꢁC in 5% CO2
atmosphere. All procedures with experimental animals were
approved by the Institutional Animal Care and Utilization Com-
mittee (license L15-17).
4.1.2.4. 2-(5-amino-1-(3,4-dichlorophenyl)-1H-pyrazol-4-yl)-
1,4,5,6-tetrahydropyrimidine (2d). Yield: 77%; mp: 188e190 ꢁC; FT-
IR
n
(cmꢃ1): 3448, 3296, 3230, 3068, 2936, 2860, 1621, 1595, 1555,
1501; 1H NMR (400 MHz, MeOH-d4)
d
¼ 7.78 (d, J ¼ 2.4 Hz, 1H),
7.67e7.68 (m, 2H), 7.54 (dd, J ¼ 8.7, 2.4 Hz, 1H), 3.42 (t, J ¼ 5.8 Hz,
4H), 1.87 (quint, J ¼ 5.8 Hz, 2H); 13C NMR (100 MHz, MeOH-d4)
d
¼ 154.4, 148.8, 139.8, 139.3, 134.3, 132.6, 132.5, 127.0, 124.7, 100.5,
42.3, 22.0; HRMS (ESI): m/z [MþH]þ: calcd. for C13H13Cl2N5:
310.0626; found: 310.0636.
4.3. Parasites
4.1.2.5. 2-(5-amino-1-(4-chlorophenyl)-1H-pyrazol-4-yl)-1,4,5,6-
tetrahydropyrimidine (2e). Yield: 50%; mp: 164e168 ꢁC; FT-IR
n
Bloodstream trypomastigote forms of T. cruzi, Y strain (TcII),
were obtained by cardiac puncture of Swiss webster infected mice at
(cmꢃ1): 3314, 3215, 3087, 2941, 2862, 2840, 1603, 1579, 1560, 1521;

M.E. Monteiro et al. / European Journal of Medicinal Chemistry 182 (2019) 111610
11
the parasitemia peak (7ꢁ days post infection; dpi). Tissue culture-
derived trypomastigotes, Dm28c-Luc clone (genetically modified
to express the firefly luciferase) (TcI), were harvested from infected
Vero cells cultures supernatants at 4ꢁ dpi. The selection of the
T. cruzi lineages, TcI and TcII, is related to their frequent association
with human infection. All animal procedures were approved by the
institutional ethics committee (license L15-17).
human infection. Trypomastigotes (1 ꢄ 106 parasites/well), Y strain
and Dm28c-Luc clone, were treated for 24 h at 37 ꢁC with the pyr-
azole derivatives and Bz at different concentrations (0.41e100 mM).
Viable parasites of T. cruzi Y strain were measured by ATP-based
quantification usig CellTiter Glo whereas for clone Dm28c-Luc the
viability was determined by the activity of the enzyme luciferase
[56]. The luminescent signal was detected in the FlexStation 3
reader. The IC50 and IC90 values (concentration capable of reducing
the number of viable parasites by 50% and 90%, respectively) was
calculated by linear regression. Bz and DMSO (concentration ꢂ1%)
were used as positive and negative controls, respectively.
The susceptibility of intracellular amastigotes to compounds
was first tested on Vero cells infected with T. cruzi Dm28c-Luc clone
(10:1 parasites/host cell). T. cruzi-infected cultures (24 h) were
treated for 72 h at 37 ꢁC with serial dilution of pyrazole derivatives
4.4. Parasite protein extraction and quantification
Trypomastigotes of T. cruzi (1 ꢄ 108) were washed three times by
centrifugation (3500 g, 10 min, 4 ꢁC) in PBS (pH 7.2) and then sub-
jected to 4 cycles of vortex-mixing for 30 min in lysis buffer
(100 mM Tris-HCl pH 8.0, 150 mM NaCl, 10% glycerol, 0.6% Triton X-
100). The soluble protein fraction was obtained by centrifugation
(25,000 g, 30 min, 4 ꢁC) and stored (ꢃ20 ꢁC) until further use. Pro-
tein concentration was determined by colorimeter assay as previ-
ously described [54], using BSA as standard protein.
and Bz (0.41e100
mM). Intracellular amastigotes viability, evaluated
by addition of luciferin (300
mg/mL), was analyzed using FlexStation
3 reader. Effective compounds were also screened against T. cruzi-
infected heart muscle cells (Y strain; 72 h). After staining with Gi-
emsa, the IC50 and IC90 values of compounds were determined by
counting intracellular amastigotes and infected cells under optical
microscope. A minimum of three independent experiments were
performed in duplicate.
4.5. Proteinase activity in solution
The proteinase activities of parasite proteins (5 mg), which was
used as positive activity control, were accessed in activation buffer
[CH3COONa 10 mM, pH 5.0 containing 1 mM DTT, final volume of
100
mL], using fluorogenic peptide substrates [30
m
M N-benzylox-
4.8. Physicochemical and ADMET prediction
ycarbonyl-L-phenylalanyl-
L
-arginine 7-amino-4-methylcoumarin
(Z-FR-AMC)]. Samples were incubated for 45 min at 37 ꢁC, and the
variance in the relative fluorescence units was monitored with a
spectrophotometer SpectraMaxM2e (Molecular Devices). Inhibition
assays were performed by coincubation with different concentra-
Physicochemical properties associated with compounds were
calculated using Datawarrior software version 4.7.3 [36] and FAF-
Drugs4 (http://fafdrugs4.mti.univ-paris-diderot.fr/). For target
search Datawarrior was used to retrieve molecules within ChEMBL
database annotated to target T. cruzi proteins. Compounds were
tion of the compounds (1c and 1d) and transepoxysuccinyl-
cylamido-(4-guanidino)butane (E-64) separately. The substrate
cleavage rate was defined as follow: v ¼ s/ t, where v ¼ velocity,
t ¼ total reaction time
L-leu-
filtered by IC50 and potency (ꢂ10
mM), any duplicity were removed,
D
D
D
then, fragments of pyrazole-imidazoline and pyrazole-
tetrahydropyrimidine were queried against the small library of
compounds. ADMET parameters (adsorption, distribution, meta-
bolism, elimination and toxicity) were acquired inserting com-
pounds molecular structures in ADMETsar plataform (http://lmmd.
ecust.edu.cn/admetsar1).
D
s ¼ substrate concentration variation and
[55]. The assays were controlled verifying the self-degradation of
the fluorescent peptide substrate at the same time interval. The
releasing amount of Amino Methyl Coumarin during the reaction
was calculated by using
a
Molar Extinction Coefficient
(ε ¼ 1.78 ꢄ 104 Mꢃ1 cmꢃ1). Additional control was performed at low
concentration (ꢂ1%) of dimethyl sulfoxide (DMSO). The enzymatic
4.9. Molecular docking
activity is expressed as m .
mol minꢃ1 mg of proteinꢃ1
The binding mode of 1c and 1d compounds into T. cruzi cysteine
proteinase were assessed by docking using the DockThor program
[57,58]. The crystal structure of cruzipain complexed with 3H5
compound [1 (N-(1H-benzimidazol-2-yl)-1,3-dimethyl-pyrazole-
4-carboxamide)] was obtained from the Protein Data Bank (PDB
accession number 4W5B). The docking was established in a cubic
grid box of 10 by 10 by 10 Å3, and the parameters are referred to as
defaults in DockThor. Structures with positional root mean square
deviation (RMSD) of up to 2 Å were clustered together, and the
results with the most favorable free energy of binding were
selected as the resultant complex structures. We also performed
redocking of 3H5 to the crystal structure of cruzipain, with a suc-
cess rate (RMSD of 2.0 Å for the interface backbone atoms) of 53%.
The docking assays were repeated three times.
4.6. Cytotoxicity assay
Vero cells, seeded in 96-well white culture plates at a density of
1.5 ꢄ 104 cells/well, were used to analyze the toxic effect of pyrazole
derivatives to mammalian cells. Twenty-four hours later, cells were
exposed to a range of Bz and pyrazole analogs concentration
(1.95e500 m
M) for 72 h at 37 ꢁC. CellTiter-Glo kit (Promega Corpo-
ration, Madison, WI, USA) was used to evaluate cell viability based
on ATP measurement and luminescent signal was read on Flex-
Station 3 reader (Molecular Devices, Sunnyvale, CA, USA). The most
active compounds against T. cruzi were further evaluated for car-
diotoxicity. Thus, primary cultures of heart muscle cells
(5 ꢄ 104 cells/well) were also applied in the viability assays after
treatment with the most effective compounds. The concentration
of compound that reduces 50% of mammalian cell viability (CC50
)
Acknowledgements
was determined by linear regression. Control was performed at low
concentration (ꢂ1%) of dimethyl sulfoxide (DMSO). At least three
independent assays were performed in duplicate.
The authors thank the Multi-user Research Facility of Bioassay
Platform of Instituto Oswaldo Cruz, Fiocruz, Rio de Janeiro, Brazil.
This work was supported by research funds from the Oswaldo Cruz
Institute of the Oswaldo Cruz Foundation (Fiocruz), Programa
4.7. Antiparasitic assay
ꢀ
ꢁ
Estrategico de Apoio a Pesquisa em Saúde (Papes VI)/Conselho
ꢀ
Screening of active compounds against T. cruzi was performed
Nacional de Desenvolvimento Científico e Tecnologico (CNPq),
on trypomastigotes and intracellular amastigotes, relevant forms in
Brazil (grant 421856/2017-3 and 424015/2018-8 to M.C.S.P.),

12
M.E. Monteiro et al. / European Journal of Medicinal Chemistry 182 (2019) 111610
pyrazole sulfonamide series of Trypanosoma brucei N-myristoyltransferase
inhibitors: identification and evaluation of CNS penetrant compounds as po-
tential treatments for stage 2 human African trypanosomiasis, J. Med. Chem.
57 (2014) 9855e9869.
~
ꢁ
Fundaçao de Amparo a Pesquisa do Estado do Rio de Janeiro
(FAPERJ) (grant E-26/010.001548/2014 and E-26/110.553/2014 to
~
M.C.S.P.) and Coordenaçao de Aperfeiçoamento de Pessoal de Nível
[19] N. Kumar, S. Sreenivasa, V. Kumar, N.R. Mohan, 3-(3,5-Difluorophenyl)-1-
phenyl-1H-pyrazole-4-carbaldehyde, Molbank 3 (2018) M1011.
[20] H. Liu, Z.L. Ren, W. Wang, J.X. Gong, M.J. Chu, Q.W. Ma, J.C. Wang, X.H. Lv,
Novel coumarin-pyrazole carboxamide derivatives as potential topoisomerase
II inhibitors: design, synthesis and antibacterial activity, Eur. J. Med. Chem.
157 (2018) 81e87.
Superior - Brasil (CAPES) - Finance Code 001. This work is a
collaboration research project of members of the Rede Mineira de
Química (RQ-MG) supported by FAPEMIG-Brazil (Project: CEX-RED-
00010-14 to M.S.) and Programa Primeiros Projetos (Project: CEX-
APQ-01014-14 to M.S.). We thank The Program for Technological
Development in Tools for Health-RPT-Fiocruz for use of its facilities.
[21] A.B. Vaidya, J.M. Morrisey, Z. Zhang, S. Das, T.M. Daly, T.D. Otto, N.J. Spillman,
M. Wyvratt, P. Siegl, J. Marfurt, G. Wirjanata, B.F. Sebayang, R.N. Price,
A. Chatterjee, A. Nagle, M. Stasiak, S. Charman, I. Angulo-Barturen, S. Ferrer,
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M. Belen Jimenez-Díaz, M.S. Martínez, F.J. Gamo, V.M. Avery, A. Ruecker,
M. Delves, K. Kirk, M. Berriman, S. Kortagere, J. Burrows, E. Fan, L.W. Bergman,
Pyrazoleamide compounds are potent antimalarials that target Naþ homeo-
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Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2019.111610.
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