Identification of Phenylpyrazolone Dimers as a New Class of Anti-Trypanosoma cruzi Agents

Article abstract of DOI:10.1002/cmdc.201900370

Chagas disease is becoming a worldwide problem; it is currently estimated that over six million people are infected. The two drugs in current use, benznidazole and nifurtimox, require long treatment regimens, show limited efficacy in the chronic phase of infection, and are known to cause adverse effects. Phenotypic screening of an in-house library led to the identification of 2,2′-methylenebis(5-(4-bromophenyl)-4,4-dimethyl-2,4-dihydro-3H-pyrazol-3-one), a phenyldihydropyrazolone dimer, which shows an in vitro pIC₅₀ value of 5.4 against Trypanosoma cruzi. Initial optimization was done by varying substituents of the phenyl ring, after which attempts were made to replace the phenyl ring. Finally, the linker between the dimer units was varied, ultimately leading to 2,2′-methylenebis(5-(3-bromo-4-methoxyphenyl)-4,4-dimethyl-2,4-dihydro-3H-pyrazol-3-one (NPD-0228) as the most potent analogue. NPD-0228 has an in vitro pIC₅₀ value of 6.4 against intracellular amastigotes of T. cruzi and no apparent toxicity against the human MRC-5 cell line and murine cardiac cells.

Full text of DOI:10.1002/cmdc.201900370

Accepted Article
Title: Identification of Phenylpyrazolone Dimers as a New Class of Anti-
Trypanosoma cruzi Agents
Authors: Maarten Sijm, Julianna Siciliano de Araújo, Alba Ramos
Llorca, Kristina Orrling, Lydia Stiny, An Matheeussen, Louis
Maes, Iwan J.P. de Esch, Maria de Nazaré Correia Soeiro,
Geert Jan Sterk, and Rob Leurs
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: ChemMedChem 10.1002/cmdc.201900370
Link to VoR: http://dx.doi.org/10.1002/cmdc.201900370
A Journal of
www.chemmedchem.org

10.1002/cmdc.201900370
ChemMedChem
Identification of Phenylpyrazolone Dimers as a New
Class of Anti-Trypanosoma cruzi Agents
Maarten Sijm1, Julianna Siciliano de Araújo2, Alba Ramos Llorca1, Kristina Orrling1 , Lydia
Stiny1, An Matheeussen3, Louis Maes3, Iwan J.P. de Esch1, Maria de Nazaré Correia Soeiro2,
Geert Jan Sterk1 and Rob Leurs1*
1Division of Medicinal Chemistry, Faculty of Sciences, Amsterdam Institute for Molecules,
Medicines and Systems (AIMMS), Vrije Universiteit Amsterdam, De Boelelaan 1108
1081 HZ, Amsterdam, The Netherlands,
2Laboratório de Biologia Celular, Oswaldo Cruz Institute (Fiocruz), Av. Brasil, 4365 -
Manguinhos, RJ, 21040-900, Rio de Janeiro, Brazil,
3Laboratory for Microbiology, Parasitology and Hygiene (LMPH), University of Antwerp,
Universiteitsplein 1, 2610, Antwerpen, Belgium.
1
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201900370
ChemMedChem
Abstract
Chagas disease is becoming a worldwide problem with currently estimated that over 6 million
people are infected. The currently used drugs benznidazole (1) and nifurtimox (2) require long
treatment regimens, show limited efficacy in the chronic phase of infection and are known to cause
adverse drug effects. Phenotypic screening of an in-house library led to the identification of 2,2'-
methylenebis(5-(4-bromophenyl)-4,4-dimethyl-2,4-dihydro-3H-pyrazol-3-one) (3), a phenyl-
dihydropyrazolone dimer, showing an in vitro pIC50 of 5.4 against Trypanosoma cruzi. Initial
optimization was done by varying substituents of the phenyl ring, after which attempts were made
to replace the phenyl ring. Finally, the linker between the dimeric units was varied, ultimately
leading to NPD-0228 (7s) as the most potent analogue. 7s has an in vitro pIC50 of 6.4 against
intracellular amastigotes of T. cruzi and no apparent toxicity against the human MRC-5 cell line
and murine cardiac cells.
2
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201900370
ChemMedChem
Introduction
Chagas disease, a neglected parasitic disease mainly endemic in Latin America, is caused by the
kinetoplast Trypanosoma cruzi.1 This protozoan parasite can be transmitted by different routes,
e.g. during the bloodmeal of infected insect vectors belonging to the triatomine family, through
iatrogenic practices, mother-to-child transmission, laboratory accidents and by oral ingestion of
contaminated beverages.2 Over 6 million people are estimated as being infected, causing over
10.000 deaths each year.3 The impact of this disease is not only limited to Latin America, but has
lately been spreading towards North America as well as other parts of the globe.4 While the insect
vector is still only present in Latin and parts of North America, the disease can enter blood banks
via (labour)-migrants or infected travelers.5 Chagas disease occurs in two distinct phases. During
the acute phase, which lasts about 6-8 weeks, there is positive parasitemia but the symptoms are
generally mild and/or flu-like. As such this stage often passes without diagnosis of this disease.6
Due to the host immune response, the parasite load is controlled and the majority of the patients
move to a chronic stage after two months, which is generally asymptomatic (the patient is infected
and able to transmit, but without clear clinical pathological signs of the disease).1 However, after
years or even decades, approximately 30% of the infected patients develops a progressive chronic
cardiomyopathy, while another 10% develops digestive, neurological or mixed clinical
symptoms.7
Current first-line treatments are the nitro-aromatics benznidazole (1, Figure 1) and nifurtimox
(2). Both drugs have proven their efficacy during the acute phase of the disease, however, efficacy
during the chronic phase is much less evident.8, 9 In addition, both have adverse effects that lead
to discontinuation of treatment and natural resistance has also been noted.10-13 Despite the need
for novel anti-T. cruzi chemotherapy, the drug discovery pipeline remains fairly empty. In this
3
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201900370
ChemMedChem
paper, we describe part of the research of the EU-funded, public-private consortium PDE4NPD
(www.PDE4NPD.eu) that focused on identifying novel chemical candidates for neglected parasitic
diseases with (amongst other) anti-T. cruzi activity potential.
Figure 1 Current drugs against Chagas disease: nitroimidazole benznidazole (1) and nitrofuran
nifurtimox (2) and the identified phenyl-dihydropyrazolone dimer hit (3).
From the in vitro phenotypic screening of an in-house library against our parasite panel, a phenyl-
dihydropyrazolone dimer (3, Figure 1) was identified as a moderately potent anti-trypanosomal
agent (pIC50 = 5.4) against T. cruzi amastigotes, without toxicity towards human MRC-5 cells
(pIC50 = <4.2).14 The in-house library consisted of previously synthesized compounds in previous
projects focused on anti-parasitic drugs. In this paper, we report on the optimization of this new
class of compounds as T.cruzi inhibitors.
4
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201900370
ChemMedChem
Chemistry
The initial two steps of the synthetic route were adapted and modified from the work of Orrling
et al. and were previously described in Sijm et al.15, 16 The first step is the acid chloride formation
of corresponding benzoic acids (4a-w, Scheme 1) which are subsequently condensed with the
lithium enolate of methylisobutyrate. The formed keto-esters (5a-w) were condensed with
hydrazine to yield the ring-closed dihydropyrazolones 6a-w. These monomeric pyrazolones were
then linked together by a methylene linker by refluxing in a two-phasic system of DCM and
aqueous NaOH in the presence of TBAB, yielding the desired pyrazolone dimers 7a-w.
Scheme 1 Reagents and conditions: i: (COCl)2, DMF, DCM, 2h, ii: methylisobutyrate, LDA, THF,
-78oC - rt, 18h, iii: N2H4·H2O, EtOH, 18h, rt, 17-82% (over 2 steps), iv: TBAB, aq. NaOH, DCM,
60oC, 18h, 40-97%.
Synthesis of the heterocyclic substituted pyrazolones (11a-g, Scheme 2) was done using the
same synthetic route as for the benzoic acids, although differences in solubility generally required
different isolation methods. The synthesis of 11g was initiated with the goal to obtain an oxazole
5
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201900370
ChemMedChem
substituted pyrazole. However, formation of the acid chloride proved to be extremely slow and
extra DMF had to be added. The subsequent addition of the lithium enolate of methylisoburate to
the remaining Vilsmeier reagent can be hypothesized to result into the formation of the
unsubstituted pyrazolone.
Scheme 2 Reagents and conditions: i: (COCl)2, DMF, DCM, 2h, ii: methylisobutyrate, LDA, THF,
-78oC - rt, o.n., iii: N2H4·H2O, EtOH, rt, 18h, 16-64% (over 2 steps), iv: TBAB, aq. NaOH, DCM,
60oC, 18h, 38-69%.
Installing the aliphatic linkers of 2 to 5 carbons on the 3-bromo-4-methoxypyrazolone (6s,
Scheme 3) was done by deprotonation of the monomeric dihydropyrazolone unit, followed by
addition of the corresponding dihalide, yielding analogues 12a-d. The ethylether analogue (12e)
was synthesized by a microwave reaction of the monomeric unit in the presence of 2-
bromoethylether and KOH.
6
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201900370
ChemMedChem
Scheme 3 Reagents and conditions: i: alkyldihalide, NaH, DMF, 18h, 39-84%, ii: 2-
bromoethylether, KOH, dioxane, 110oC, 18h, microwave 45%.
Introduction of a 2-propanol linker was done using a biphasic mixture of epichlorohydrin and
aqueous NaOH, yielding epoxide substituted dihydropyrazolone 13 (Scheme 4). Reaction of this
epoxide with a second monomeric unit in the presence of K2CO3 yielded the desired 2-propanol
linked dimer (14). Oxidation of this dimer with Dess-Martin periodane (DMP) yielded the closely
analogous 2-propanone linked dihydropyrazolone (15).
7
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201900370
ChemMedChem
Scheme 4 Reagents and conditions: i: epichlorohydrin, TBAB, aq. NaOH, 60oC, 18h, 52%, ii: 6s,
K2CO3, DMF, 100ºC, 18h, 53%, iii: DMP, DCM, rt, 2h, 40%.
Phenotypic screening
All compounds were initially tested for their in vitro anti-trypanosomal activity against
intracellular forms of T. cruzi (Tulahuen CL2, β-galactosidase, a drug sensitive strain, belonging
to the discrete typing units, DTU VI), T. brucei and Leishmania infantum as well as for cytotoxicity
on MRC-5SV2 cells (human lung fibroblasts) and on primary mouse cardiac cell cultures.17 The full
dataset is presented in Supporting Info Table 1. In-depth profiling with selected compounds was
performed using intracellular amastigotes from the Tulahuen strain transfected with β-
galactosidase and from the Y-strain (DTU II). Additionally, bloodstream trypomastigotes of the
Y-strain were obtained from infected Swiss Webster donor mice.16
8
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201900370
ChemMedChem
Results and discussion
With the discovery of the 4-bromophenyl pyrazolone dimer (3, Figure 1) as a phenotypic hit
against T. cruzi, SAR studies were initially focusing on the substituents of the phenyl ring, leading
to compounds 7a-7z (Table 1). Shifting the bromine to the 3- or 2-position (7a-b) resulted in two
inactive compounds (pIC50 <4.2), whereas the unsubstituted phenyl analog was also inactive (7c).
To decrease the molecular weight, the bromine was replaced by other halogen substituents. The
analog with a chlorine atom on the 4-position (7d) shows similar activity as the 4-bromophenyl
hit. Yet, 7d is equally toxic against MRC-5 cells. As observed with the bromine analogs 7a-b, the
2- and 3-chloro analogues 7e-f are inactive. The 2- and 4-fluoro analogues 7g, 7i are also inactive,
while the 3-fluorine analog 7h has a pIC50 of 5.2. Both the 3- and 4-methoxyphenyl analogues (7j-
k) showed pIC50’s around 5.0, while the 2-methoxy analog is inactive (7l). The 3- and 4-
dimethylamino and cyano derivatives 7m-p are all inactive.
9
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201900370
ChemMedChem
Table 1 In vitro activity of pyrazolone dimers against intracellular amastigotes of T. cruzi
(Tulahuen strain) and on MRC-5 cell growth by phenyldihydropyrazolone dimers with
modification on phenyl ring.
R1
T. cruzi MRC-5
Cmpd
a
a
pIC50
pIC50
4-
3-
2-
3
Br
H
H
5.4
< 4.2
< 4.2
< 4.2
5.3
< 4.2
< 4.2
< 4.2
< 4.2
5.1
7a
7b
7c
7d
7e
7f
H
Br
H
H
H
Cl
H
H
F
H
H
Br
H
H
Cl
H
H
H
< 4.2
< 4.2
< 4.2
5.2
< 4.2
< 4.2
< 4.2
4.4
H
Cl
H
7g
7h
7i
F
H
H
H
H
H
F
4.3
< 4.2
< 4.2
4.4
7j
OMe
H
H
5.0
7k
7l
OMe
H
H
5.1
H
OMe
H
< 4.2
< 4.2
< 4.2
< 4.2
< 4.2
5.2
< 4.2
< 4.2
< 4.2
< 4.2
< 4.2
< 4.2
< 4.2
7m
7n
7o
7p
7q
7r
NMe2
H
H
NMe2
H
H
CN
H
H
CN
F
H
OMe
OMe
H
Cl
H
5.9
10
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201900370
ChemMedChem
7s
OMe
Cl
Br
H
H
H
H
H
6.4
< 4.2
< 4.2
4.3
< 4.2
7t
OMe
OMe
OMe
F
< 4.2
< 4.2
< 4.2
< 4.2
7u
7v
7w
Br
OMe
F
5.1
a all reported values are within a standard deviation of ± 0.2
Combining halogen and methoxy substituents on the 3- and 4- position of the phenyl ring was
more successful. The 3-fluoro-4-methoxyphenyl analogue 7q (Table 1) shows a pIC50 of 5.2 while
the 3-chloro-4-methoxyphenyl dimer 7r shows an increased activity with a pIC50 of 5.9. The most
active molecule in this series is the 3-bromo-4-methoxyphenyl (7s, NPD-0228) which has a pIC50
of 6.4, with no apparent cytotoxicity towards MRC-5 cells. Switching the position of both
substituents, as with compound 7t and 7u, resulted in totally inactive compounds (pIC50’s <4.2)
which was surprising as the 4-chloro and 4-bromo substituted phenyl analogs (3, 7c) performed
better than their 3-substituted counterparts (7a, 7d). Finally, the 3,4-dimethoxyphenyl (7v) and
3,4-difluorophenyl (7w) analogues showed only weak anti-T. cruzi activity (pIC50 of 4.3 and 5.1,
respectively).
While the SAR study on the phenyl moiety yielded an improved hit 7s (Table 1, pIC50 = 6.4), its
drug-like properties are not ideal with a molecular weight of 606 and a cLogP of 6.1. To optimize
these parameters, several heterocycles were installed instead of the phenyl moiety. The 2- and 3-
thiophene (11a-b, Table 2), 2- and 3-furan (11c-d) and 4-thiazole (11e) were unfortunately all
inactive with pIC50’s <4.2. As the original hit (3) contained a bromine substituent, this moiety was
included on a 2-thiophene (11f), and some moderate activity (pIC50 = 5.1) was observed. In an
11
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201900370
ChemMedChem
attempt to introduce an oxazole substituent, the unsubstituted dihydropyrazolone (11g), obtained
via ring-closure of the intermediate Vilsmeier reagent, was tested, but also proved inactive.
Table 2 In vitro activity of pyrazolone dimers against intracellular amastigotes of T. cruzi
(Tulahuen strain) and on MRC-5 cells by dihydropyrazolone dimers with modification of the
aromatic ring
a
a
Cmpd
R2
T. cruzi pIC50
MRC-5 pIC50
11a
11b
11c
11d
11e
< 4.2
< 4.2
< 4.2
< 4.2
< 4.2
< 4.2
< 4.2
< 4.2
< 4.2
< 4.2
11f
5.1
< 4.2
< 4.2
11g
H
< 4.2
a all reported values are within a standard deviation of ± 0.2
As the activities of the heterocyclic-substituted pyrazolones were low, it was decided to continue
further investigations with the 4-methoxy-3-bromophenyl substituted dihydropyrazolone (7s).
Installing several aliphatic linkers with 2 to 5 carbons (12a-12d, Table 3) showed that the
12
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201900370
ChemMedChem
analogues with linkers of 2 to 4 carbons (12a-12c) are all inactive with pIC50’s below 4.3. Instead,
the 5-carbon linker analogue 12d shows quite good activity with a pIC50 of 5.7. The other 5-atom
linker, diethyl ether 12e shows a pIC50 just above 5. However, this compound is equally toxic
against MRC-5 cells. While the 2-propanol linker analogue 14 has a similar pIC50 as the 5-carbon
linked analogue (5.7), it also showed quite high cytotoxicity (pIC50 = 5.2). The closely related
ketone analogue 15 is totally inactive (Table 3).
Table 3 In vitro activity of pyrazolone dimers against intracellular amastigotes of T. cruzi
(Tulahuen strain) and on MRC-5 cell growth by dihydropyrazolone dimers with modification of
the linker.
a
a
Entry
1
Compound
7q
X
T. cruzi pIC50
MRC-5 pIC50
< 4.2
6.4
2
3
4
5
6
8
7
12a
12b
12c
12d
12e
14
< 4.2
4.3
< 4.2
< 4.2
< 4.2
4.3
< 4.2
5.7
5.1
5.2
5.7
5.2
15
< 4.2
< 4.2
a all reported values are within a standard deviation of ± 0.2
13
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201900370
ChemMedChem
Overall, optimization of the substituents on the phenyl ring gave valuable SAR data and most
importantly, the 3-bromo-4-methoxy analogue (7s, Table 1) was confirmed as the most promising
hit with sub-micromolar potency (pIC50 = 6.4) against T. cruzi and over 100-fold selectivity upon
MRC-5 cells. Introduction of aromatic heterocycles on the dihydropyrazolone dimer did not result
in potent compounds, only the 2-bromothiophene (11f, Table 2) showed some activity (pIC50 5.1).
Finally, installing linkers with different lengths showed that the dimer with a 5-carbon linker (12d,
Table 3) shows a pIC50 of 5.7. All compounds reported in Table 1-3 were screened against all
parasites in a parasite panel (T. brucei, T. cruzi and L. infantum), this data can be found in SI Table
1.
Next, the optimized hit (7s, Table 4) was screened in different assays against the intracellular
amastigote form of T. cruzi (Tulahuen and Y strains) as well as the bloodstream form of the Y-
strain. In both intracellular amastigote assays, NPD-0228 (7s) shows sub-micromolar potencies
with pIC50’s of 6.8 and 7.0 respectively. Additionally, measured pIC90’s are low micromolar (Table
4), while selectivity over mouse cardiac cells is >450-fold. The hit 7s did not show any activity
against the bloodstream trypomastigotes. In the same experiments, benznidazole (1, Figure 1)
shows a pIC50 of 5.5 against the intracellular form and 4.9 against the bloodstream form of the Y-
strain.
14
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201900370
ChemMedChem
Table 4 Activity and selectivity index (SI) of NPD-0228 (7s) against various T. cruzi strains.
a
a
Strain (form) of T. cruzi
pIC50
pIC90
SIb
Tulahuen (intracellular amastigotes)
Y (intracellular amastigotes)
Y (bloodstream trypomastigotes)
6.8
7.0
<4.3
6.2
5.6
ND
>599
>455
ND
a all reported values are within a standard deviation of ± 0.2, b selectivity index is calculated against
the pIC50 of mouse cardiac cells. ND stands for “not determined”.
To investigate if the different activity against intracellular forms and bloodstream
trypomastigotes is due to the influence from the mammalian host cell triggering a mechanism to
control and clear the parasite intracellularly, L929 cells were pre-treated for 24 h with 7s at
0.17 µM (corresponding to IC50 on intracellular forms of Tulahuen). Next, the cultures were
washed to remove compound 7s and subsequently monitored for parasite infection. Our data show
that drug administration only prior to the in vitro infection does not control the parasite load,
leading to a maximum parasite reduction of 8.8 %, suggesting that the higher efficacy of NPD-
0228 (7s) against the intracellular forms was due to the activity against the intracellular parasite
itself and not related to an improved microbicidal capacity of the mammalian host cell, as already
reported for other antiparasitic drugs.18 As NPD-0228 (7s) is only active against intracellular forms
of the tested strains, it can be hypothesized that 7s interferes with intracellular processes essential
for the T. cruzi multiplication and viability of the highly metabolic proliferative form.
15
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201900370
ChemMedChem
Conclusion
Following an in vitro phenotypic screening and the identification of
a
4-
bromophenyldihydropyrazole-dimer (3, Figure 1) as hit with micromolar anti-T. cruzi activity, a
series of analogues was synthesized with variations on the phenyl ring (7a-7w, Table 1). The
resulting SAR led to the identification of the 3-bromo-4-methoxyphenyl dimer (7s) as optimized
hit with a pIC50 of 6.4 and no apparent toxicity towards the human MRC-5 cell line. Attempts to
introduce heterocycles (11a-11f, Table 2) instead of the phenyl moiety resulted in mostly inactive
compounds, whereas changing the linker length (12a-12e, 14, 15, Table 3) generally led to a
decrease in activity. Testing of NPD-0228 (7s) on different strains revealed that the
bromophenyldihydropyrazole-dimer is inactive against the bloodstream (trypomastigote) form of
the Y-strain, while having sub-micromolar potency on the intracellular (amastigote) forms of the
different parasite strains that are clinically relevant for human infection. As benznidazole (1) is
most active against the bloodstream form, NPD-0228 (7s) might be interesting to try in
combination treatments. However, for this series to continue further, drug-like properties have to
be improved substantially, as the current cLogP (6.1) and molecular weight (606) are far from
optimal.
Experimental section biology
T. cruzi cellular assay. Bloodstream trypomastigotes (BT) of the Y strain of T. cruzi were
obtained by cardiac puncture of infected Swiss Webster mice on the parasitemia peak.19, 20 For the
standard in vitro susceptibility assay on intracellular amastigotes, T. cruzi Tulahuen CL2, β-
galactosidase strain (nifurtimox-sensitive) was used. The strain is maintained on MRC-5SV2
(human lung fibroblast) cells7 in MEM, supplemented with 200 mM L-glutamine, 16.5 mM
16
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201900370
ChemMedChem
NaHCO3, and 5% inactivated fetal calf serum (FCS). All cultures and assays are conducted at 37°C
under an atmosphere of 5% CO2.14
MRC-5 cytotoxicity cellular assay. MRC-5SV2 cells were cultivated in MEM, supplemented with
L-glutamine (20 mM), 16.5 mM sodium hydrogen carbonate and 5% FCS. For the assay, 104
cells/well were seeded onto 96-well test plates containing the pre-diluted sample and incubated at
37°C and 5% CO2 for 72 h. Cell viability was assessed fluorometrically 4 h after addition of
resazurin (excitation 550 nm, emission 590 nm). The results are expressed as percentage reduction
in cell viability compared to untreated controls.14
Cytotoxicity assays on cardiac cells. Non-infected cultures were incubated at 37°C up to 48 h
with increasing concentrations of each compound diluted in RPMI. Morphology and spontaneous
contractibility were evaluated by light microscopy and cellular viability was determined by
PrestoBlue® tests.21 The results are expressed as the difference in reduction between treated and
non-treated cells according to the manufacturer instructions and the LC50 value (minimum
concentration that reduces in 50% the cellular viability) was determined.21
Trypanocidal activity. Intracellular amastigotes of the T. cruzi strain were screened according to
methodology previously reported by Romanha et al.22 Bloodstream forms from Y strain
(5x106/mL) obtained from Swiss male infected donor mice (5x104/animal, i.p.) at the parasitaemia
peak were incubated for 24 h at 37°C in RPMI in the presence or absence of 1:3 serial dilutions of
the compounds (0 - 50 μM) to determine parasite death rates through the direct quantification of
live parasites by light microscopy. The IC50 (compound concentration that reduces 50% of the
number of parasites) was calculated. Selected compounds (IC50 ≤ benznidazole (1)) were evaluated
on intracellular amastigotes of the Y strain in infected cardiac cell cultures.19 After 24h (10:1
parasite:cell infection ratio), the cultures were treated for 48h at 37°C with non-toxic
17
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201900370
ChemMedChem
concentrations of the compounds. Following the treatment, the cultures were washed with saline,
fixed with Bouin for 5 min, stained with Giemsa and evaluated by light microscopy. The
percentage of infected host cells and the number of parasites per cell were determined for the
calculation of the infection index and the pIC50 values.20 Selectivity index (SI) is expressed by the
ratio between pLC50 (toxicity for mammalian cells) and the pIC50 (activity on the parasite). At least
two independent assays were done in duplicate. In some assays, L929 cell cultures were incubated
for 24 h at the corresponding IC50 of NPD-0228 (7s), the cultures were rinsed using RPMI to
remove the compound, and then infected and processed as described above.22 All procedures
followed the guidelines as established by the Fiocruz Committee of Ethics for the Use of Animals
(CEUA LW16/14).
Experimental section chemistry
Chemicals and reagents were obtained from commercial suppliers and were used without further
purification. Anhydrous DMF, THF and DCM were obtained by passing them through an activated
alumina column prior to use. Microwave reactions were executed using a Biotage® Initiator
microwave system. 1H NMR spectra were recorded on a Bruker AVANCE 250 (250 MHz), Bruker
AVANCE 400 (400 MHz), Bruker AVANCE 500 (500 MHz) or Bruker 600 AVANCE (600 MHz)
spectrometer. Data are reported as follows: chemical shift, integration, multiplicity (s = singlet, d
= doublet, dd = double doublet, t = triplet, dt = double triplet, q = quartet, p = pentet, h = heptet,
bs = broad singlet, m = multiplet), and coupling constants (Hz). Chemical shifts are reported in
ppm with the natural abundance of deuterium in the solvent as the internal reference (CDCl3: δ
7.26, (CD3)2SO: δ 2.50). 13C NMR spectra were recorded on a Bruker AVANCE 500 (126 MHz)
or Bruker AVANCE 600 (150 MHz). Chemical shifts are reported in ppm with the solvent
18
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201900370
ChemMedChem
resonance resulting from incomplete deuteration as the internal reference (CDCl3: δ 77.16 or
(CD3)2SO: δ 39.52). Systematic names for molecules according to IUPAC rules were generated
using the Chemdraw AutoName program. LC-MS data was gathered using a Shimadzu HPLC/MS
workstation with a LC-20AD pump system, SPD-M20A diode array detection, and a LCMS-2010
EV mass spectrometer. The column used is an Xbridge C18 5 μm column (100 mm × 4.6 mm).
Solvents used were the following: solvent B = ACN, 0.1% formic Acid; solvent A = water, 0.1%
formic acid. The analysis was conducted using a flow rate +of 1.0 mL/min, start 5% B, linear
gradient to 90% B in 4.5 min, then 1.5 min at 90% B, linear gradient to 5% B in 0.5 min and then
1.5 min at 5% B, total run time of 8 min. All reported compounds have purities >95%, measured
at 254 nm, unless otherwise mentioned. All HRMS spectra were recorded on a Bruker microTOF
mass spectrometer using ESI in positive-ion mode. Column purifications were either carried out
automatically using Biotage equipment or manually, using 60-200 mesh silica. TLC analyses were
performed with Merck F254 alumina silica plates using UV visualization. All reactions were done
under N2 atmosphere, unless specifically mentioned.
19
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201900370
ChemMedChem
General methods
General method I: synthesis of β-keto-esters
Benzoic acid 4s (25.0 g, 108 mmol) was suspended in DCM (50 mL) while cooling to 0oC.
Subsequently oxalyl dichloride (13.7 mL, 162 mmol) and DMF (0.08 mL, 1.08 mmol) were added
and the mixture was allowed to warm up to room temperature. The mixture was stirred for 2 h
after which volatiles were evaporated. The remaining solids were dissolved in 50 ml of THF. In a
separate flask methyl isobutyrate (18.6 mL, 162 mmol) was stirred in THF (50 mL) at -78°C and
a 2M LDA (65 mL, 130 mmol) was added dropwise while maintaining -78°C. Upon full addition,
the mixture was stirred for 45m after which the acid chloride of 6 in THF was added dropwise,
again maintaining the temperate at -78°C. The reaction was allowed to warm up to room
temperature after which crude was quenched with water and extracted with diethyl ether. The
organic phase was washed twice with water and once with brine. The organic layer was then dried
with MgSO4, filtered and evaporated to dryness. The crude was used in the next step without
further purification.
General method II: ring closure of β-keto-esters
Crude keto-ester 5s (34 g, 79 mmol) was dissolved in ethanol (75 mL) and hydrazine hydrate
(64%) (38.6 mL, 793 mmol) was added. The reaction was stirred at room temperature for 48h after
which white precipitation was visible. To the stirred solution 20 mL of water was added to allow
further precipitation, after which solids were filtered off. Collected solids were dried in vacuo
yielding the desired product.
General method III: Installing the methylene linker
20
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201900370
ChemMedChem
Pyrazolone 6s (2.0 g, 6.73 mmol), was added to a microwave vial followed by TBAB (0.11 g,
0.34 mmol), 12M NaOH (3 mL) and DCM (8 mL) resulting in a white/yellow suspension. The
vial was sealed and refluxed in a sand bath at 60°C overnight after which the reaction mixture was
a clear solution. The solution was diluted with DCM (30 mL), and the organic layer was extracted
with water (3 x 30 mL). The organic layer was dried over MgSO4 and the solvent evaporated under
vacuo to give a white/yellow solid. The obtained solids were recrystallized from MeOH.
General method IV: Installing aliphatic linkers
Pyrazolone 6s (1.0 g, 3.4 mmol) was added to a flask and DMF (14 mL) was added after which
the mixture was cooled to 0°C. Subsequently, NaH (60% in mineral oil)(0.14 g, 3.4 mmol) was
added. The reaction mixture was stirred at room temperature for 30 min, after which 1,2-
dibromoethane (0.145 mL, 1.68 mmol) was added dropwise at 0°C. The reaction was stirred
overnight at room temperature. Upon completion, the reaction was quenched with sat. aqueous
NH4Cl and extracted with EtOAc (50 mL). The organic layer was washed with water (50 mL) and
brine (40 mL). After this, the organic layer was dried over MgSO4 and volatiles were evaporated
under vacuo. The crude product was purified over SiO2 using a gradient from 20% EtOAc in n-
heptane towards 65% EtOAc to give the title compound as an off-white solid.
21
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201900370
ChemMedChem
Experimental data
5-(3-bromo-4-methoxyphenyl)-4,4-dimethyl-2,4-dihydro-3H-pyrazol-3-one (6s)
Prepared according to the general method I and II using 25 g (43.3 mmol) of 3-bromo-4-
methoxybenzoic acid. The formed precipitate was filtered off dried at 40°C in vacuo to give 25.4
1
g (85.0 mmol, 79%) of the title compound as a white solid. H NMR (500 MHz, DMSO-d6) δ
11.54 (s, 1H), 7.96 (d, J = 2.2 Hz, 1H), 7.77 (dd, J = 8.6, 2.2 Hz, 1H), 7.16 (d, J = 8.7 Hz, 1H),
3.89 (s, 3H), 1.34 (s, 6H). 13C NMR (126 MHz, DMSO) δ 181.0, 160.6, 156.8, 130.3, 127.3, 125.3,
113.1, 111.8, 56.9, 46.8, 22.3. LC-MS (ESI) m/z found: 297 [M+H]+; retention time: 4.51 minutes
2,2'-methylenebis(5-(3-bromo-4-methoxyphenyl)-4,4-dimethyl-2,4-dihydro-3H-pyrazol-3-
one) (7s) (NPD-0228)
Prepared according to general method III using 2.0 g (6.7 mmol) of pyrazolone 6s. The white solid
obtained was recrystallized from MeOH to give 1.8 g (3.0 mmol, 88%) of the title compound as a
white solid. 1H NMR (600 MHz, CDCl3) δ 7.98 (d, J = 2.1 Hz, 2H), 7.68 (dd, J = 2.1, 8.7 Hz, 2H),
6.89 (d, J = 8.7 Hz, 2H), 5.67 (s, 2H), 3.92 (s, 6H), 1.50 (s, 12H). 13C NMR (151 MHz, CDCl3) δ
179.2, 161.3, 157.4, 131.5, 126.9, 124.7, 112.4, 111.7, 56.5, 51.9, 48.4, 22.8. LC-MS (ESI) m/z
found: 605 [M+H]+; retention time: 5.23 minutes. HRMS-ESI [M+H]+ calculated for
+
C25H27Br2N4O4 : 605.0321, found:605.0394.
1,1'-(propane-1,3-diyl)bis(3-(3-bromo-4-methoxyphenyl)-4,4-dimethyl-1H-pyrazol-5(4H)-
one) (12b)
Prepared according to general method IV using 1.0 g (3.37 mmol) of pyrazolone 6a and 0.171 mL
(1.68 mmol) of 1,2-dibromopropane. The crude was purified over SiO2 using a gradient from 20%
EtOAc in n-heptane towards 60% EtOAc to give 0.90 g (0.66 mmol, 84%) of the title compound
as a white solid. 1H NMR (600 MHz, CDCl3) δ 8.03 (d, J = 2.1 Hz, 2H), 7.69 (dd, J = 2.1, 8.6 Hz,
2H), 6.91 (d, J = 8.7 Hz, 2H), 3.94 (s, 6H), 3.83 (t, J = 7.0 Hz, 4H), 2.26 (p, 7.0 Hz, 2H), 1.48 (s,
12H). 13C NMR (151 MHz, CDCl3) δ 178.6, 160.9, 157.4, 131.6, 126.9, 125.3, 112.7, 111.9, 56.7,
48.7, 41.8, 27.3, 23.0. LC-MS (ESI) m/z found: 633 [M+H]+; retention time: 5.45 minutes. HRMS-
+
ESI [M+H]+ calculated for C27H31Br2N4O4 : 633.0634, found: 633.0707.
2,2'-(oxybis(ethane-2,1-diyl))bis(5-(3-bromo-4-methoxyphenyl)-4,4-dimethyl-2,4-dihydro-
3H-pyrazol-3-one) (12e)
To mixture of pyrazolone 6a (200 mg, 0.67 mmol) and KOH (28.3 mg, 0.51 mmol) in dioxane (3
mL) was added 1-bromo-2-(2-bromoethoxy)ethane (78 mg, 0.334 mmol). The reaction mixture
was heated at 110oC for 18h in the microwave. Upon completion the reaction was quenched with
sat. aqueous NH4Cl and extracted with EtOAc. The organic layer was washed with water and brine.
After this the organic later was dried over MgSO4 and volatiles were evaporated under vacuo. The
crude product was purified over SiO2 using a gradient from 30% EtOAc in n-heptane towards 80%
22
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201900370
ChemMedChem
1
EtOAc to give 99 mg (0.15 mmol, 45%) of the title compound as a white solid. H NMR (600
MHz, CDCl3) δ 8.00 (d, J = 2.1 Hz, 2H), 7.65 (dd, J = 2.1, 8.6 Hz, 2H), 6.89 (d, J = 8.7 Hz, 2H),
3.93 (s, 6H), 3.91 (t, J = 5.9 Hz, 4H), 3.80 (t, J = 5.9 Hz, 4H), 1.44 (s, 12H). 13C NMR (151 MHz,
CDCl3) δ 178.7, 160.4, 157.0, 131.2, 126.6, 124.9, 112.3, 111.6, 67.6, 56.4, 48.3, 43.8, 22.6. LC-
MS (ESI) m/z found: 663 [M+H]+; retention time: 5.24 minutes. HRMS-ESI [M+H]+ calculated
+
for C28H33Br2N4O5 : 663.0739, found: 663.0813.
5-(3-bromo-4-methoxyphenyl)-4,4-dimethyl-2-(oxiran-2-ylmethyl)-2,4-dihydro-3H-pyrazol-
3-one (13)
Pyrazolone 6s (2.0 g, 6.7 mmol), TBAB (0.18 g, 0.34 mmol), 50% aq. NaOH (4 mL) and 2-
(chloromethyl)oxirane (8 mL, 102 mmol) were added to a microwave tube resulting in a white
suspension. The mixture is refluxed at 60°C overnight after which the reaction mixture was a clear
suspension. The two layers were separated, and the organic layer was extracted with water (3 x 10
mL). The organic layer was dried over MgSO4 and the solvent evaporated under vacuo to give a
colourless oil. The obtained oil was purified over SiO2 using a gradient from 20% EtOAc in n-
heptane towards 60% EtOAc to give 1.22 g (3.47 mmol, 52%) of the title compound as a colourless
oil. 1H NMR (300 MHz, CDCl3) δ 8.04 (d, J = 2.1 Hz, 1H), 7.69 (dd, J = 2.1, 8.6 Hz, 1H), 6.92 (d,
J = 8.7 Hz, 1H), 3.96 – 3.95 (m, 2H), 3.94 (s, 3H), 3.32 – 3.24 (m, 1H), 2.86 (t, J = 4.4 Hz, 1H),
2.70 (dd, J = 2.5, 4.8 Hz, 1H), 1.49 (s, 6H). LC-MS (ESI) m/z found: 353 [M+H]+; retention time:
4.23 minutes.
1,1'-(2-hydroxypropane-1,3-diyl)bis(3-(3-bromo-4-methoxyphenyl)-4,4-dimethyl-1H-
pyrazol-5(4H)-one) (14)
Pyrazolone 6a (1.0 g, 3.47 mmol), oxirane 20 (1.22 g, 3.47 mmol) and K2CO3 (1.44 g, 10.4 mmol)
in DMF (0.2 M) were added to a microwave tube. The mixture was stirred at 100°C overnight.
Upon completion, the reaction mixture is allowed to cool towards rt. The reaction is diluted in
EtOAc and washed with water and brine. After this the organic layer was dried over MgSO4 and
volatiles were evaporated under vacuo. The obtained solids were recrystallized from MeOH to
give 1.2 g (1.9 mmol, 53%) of the title compound as a white solid. 1H NMR (600 MHz, CDCl3) δ
8.00 (d, J = 1.9 Hz, 2H), 7.67 (dd, J = 1.9, 8.6 Hz, 2H), 6.91 (d, J = 8.7 Hz, 2H), 4.44 (p J = 6.4
Hz, 1H), 4.03 – 3.83 (m, 10H), 1.51 (s, 6H), 1.49 (s, 6H). 13C NMR (151 MHz, CDCl3) δ 179.2,
161.3, 157.4, 131.4, 126.8, 124.6, 112.5, 111.7, 69.1, 56.5, 48.5, 48.1, 22.8. LC-MS (ESI) m/z
found: 649 [M+H]+; retention time: 4.97 minutes. HRMS-ESI [M+H]+ calculated for
+
C27H31Br2N4O5 : 649.0583, found: 649.0656.
1,1'-(2-oxopropane-1,3-diyl)bis(3-(3-bromo-4-methoxyphenyl)-4,4-dimethyl-1H-pyrazol-
5(4H)-one) (15)
To a solution of alcohol 18 (200 mg, 0.31 mmol) in DCM (1.5 mL), Dess Martin periodinane (157
mg, 0.37 mmol) was added. The mixture was stirred for 2h at rt. Next, diethyl ether (5 mL), sat.
23
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201900370
ChemMedChem
aqueous Na2S2O3 (5 mL) and sat. aqueous NaHCO3 (5 mL) were added and the resulting mixture
was stirred for 5 min. The organic phase was separated, dried over MgSO4 and volatiles were
evaporated under vacuo. The reaction crude was purified over SiO2 using a gradient from 40%
EtOAc in n-heptane towards 80% EtOAc and recrystallized from MeOH to give 80 mg (0.12
mmol, 40%) of the title compound as a white solid. 1H NMR (600 MHz, CDCl3) δ 8.01 (d, J = 2.1
Hz, 2H), 7.69 (dd, J = 2.1, 8.6 Hz, 2H), 6.92 (d, J = 8.7 Hz, 2H), 4.67 (s, 4H), 3.94 (s, 6H), 1.52
13
(s, 12H). C NMR (151 MHz, CDCl3) δ 197.6, 179.1, 161.5, 157.4, 131.6, 126.9, 124.6, 112.4,
111.8, 56.5, 51.7, 48.2, 22.8. LC-MS (ESI) m/z found: 647 [M+H]+; retention time: 5.13 minutes.
+
HRMS-ESI [M+H]+ calculated for C27H29Br2N4O5 : 647.0426, found: 647.0499.
Corresponding author
Rob Leurs, r.leurs@vu.nl
Author contributions
M.S., A.R.L., L.S and K.O., performed chemistry experiments; M.S., A.R.L., M. de N.C.S,
writing of the initial manuscript, delivering figures and schemes; J.S. de A., A.M., in vitro
screening and phenotypic experiments; L.M., M. de N.C.S. G.J.S, I.J.P. de E, R.L, supervision of
the project, design experiments, funding. All authors have contributed and read the final
manuscript.
Funding Sources
The PDE4NPD project was funded by by the European Union under the FP-7-Health program,
project ID: 602666. MNCS is CNE from FAPERJ and CNPq researcher. Fiocruz, CNPq and
FAPERJ are acknowledged
Acknowledgements
Authors would like to thank A. van der Stolpe and H. Custers for their technical assistance.
List of abbreviations
24
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201900370
ChemMedChem
bs: broad singlet, BT: bloodstream trypomastigotes, d: doublet, DCM: dichloromethane, dd:
double doublet, DMF: dimethylformamide, DMP: Dess-Martin periodane, dt: double triplet, DTU:
discrete typing unit, ESI: electron spray ionization, EtOAc: ethylacetate, EtOH: ethanol, FCS: fetal
calf serum, h: hour, h: heptet, HPLC: high pressure liquid chromatography, HRMS: high resolution
mass spectroscopy, Hz: Hertz, LC-MS: liquid chromatography mass spectroscopy, LDA: lithium
diisopropylamide, m: minute, m: multiplet, MeOH: methanol, MRC-5: medical research council
strain 5, human fibroblast from lung tissue, o.n.: overnight, p: pentet, PDE4NPD:
phosphodiesterase inhibitors for neglected parasitic disease, pIC50: -log of the value at which 50%
of growth is inhibited, the q: quartet, rt: room temperature, s: singlet, SAR: structure activity
relationship, t: triplet, TBAB: tetrabutyl ammonium bromide, THF: tetrahydrofuran, TLC: thin
layer chromatography, UV: ultraviolet.
Supporting information
All experimental data of the reported compounds can be found in the supporting information.
25
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201900370
ChemMedChem
References
1.
1402.
Rassi Jr, A.; Rassi, A.; Marin-Neto, J. A. Chagas disease. The Lancet 2010, 375, 1388-
Coura, J. R.; Vinas, P. A. Chagas disease: a new worldwide challenge. Nature 2010, 465,
2.
S6-S7.
3.
WHO.
Chagas
disease
(American
trypanosomiasis).
http://www.who.int/mediacentre/factsheets/fs340/en/ (05 November 2018).
4.
Lee, B. Y.; Bacon, K. M.; Bottazzi, M. E.; Hotez, P. J. Global economic burden of Chagas
disease: a computational simulation model. The Lancet Infectious Diseases 2013, 13, 342-348.
5.
Pinto Dias, J. C. Human chagas disease and migration in the context of globalization: some
particular aspects. J Trop Med 2013, 2013, 789758-789758.
6.
91.
Dias, J. C. P. Acute Chagas' disease. Memórias do Instituto Oswaldo Cruz 1984, 79, 85-
Dias, J. THe treatment of chagas disease (south american trypanosomiasis). Annals of
7.
Internal Medicine 2006, 144, 772-774.
8. Morillo, C. A.; Marin-Neto, J. A.; Avezum, A.; Sosa-Estani, S.; Rassi, A. J.; Rosas, F.;
Villena, E.; Quiroz, R.; Bonilla, R.; Britto, C.; Guhl, F.; Velazquez, E.; Bonilla, L.; Meeks, B.;
Rao-Melacini, P.; Pogue, J.; Mattos, A.; Lazdins, J.; Rassi, A.; Connolly, S. J.; Yusuf, S.
Randomized Trial of Benznidazole for Chronic Chagas’ Cardiomyopathy. New England Journal
of Medicine 2015, 373, 1295-1306.
9.
Jannin, J.; Villa, L. An overview of Chagas disease treatment. Memórias do Instituto
Oswaldo Cruz 2007, 102, 95-98.
10.
Pinazo, M.-J.; Guerrero, L.; Posada, E.; Rodríguez, E.; Soy, D.; Gascon, J. Benznidazole-
Related Adverse Drug Reactions and Their Relationship to Serum Drug Concentrations in Patients
with Chronic Chagas Disease. Antimicrobial Agents and Chemotherapy 2013, 57, 390-395.
11.
Castro, J. A.; deMecca, M. M.; Bartel, L. C. Toxic Side Effects of Drugs Used to Treat
Chagas’ Disease (American Trypanosomiasis). Human & Experimental Toxicology 2006, 25, 471-
479.
12.
Clayton, J. Chagas disease: pushing through the pipeline. Nature 2010, 465, S12-S15.
13.
Mejia, A. M.; Hall, B. S.; Taylor, M. C.; Gómez-Palacio, A.; Wilkinson, S. R.; Triana-
Chávez, O.; Kelly, J. M. Benznidazole-Resistance in Trypanosoma cruzi Is a Readily Acquired
Trait That Can Arise Independently in a Single Population. The Journal of Infectious Diseases
2012, 206, 220-228.
26
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201900370
ChemMedChem
14.
Blaazer, A. R.; Orrling, K. M.; Shanmugham, A.; Jansen, C.; Maes, L.; Edink, E.; Sterk,
G. J.; Siderius, M.; England, P.; Bailey, D.; de Esch, I. J. P.; Leurs, R. Fragment-Based Screening
in Tandem with Phenotypic Screening Provides Novel Antiparasitic Hits. Journal of Biomolecular
Screening 2014.
15.
Orrling, K. M.; Jansen, C.; Vu, X. L.; Balmer, V.; Bregy, P.; Shanmugham, A.; England,
P.; Bailey, D.; Cos, P.; Maes, L.; Adams, E.; van den Bogaart, E.; Chatelain, E.; Ioset, J.-R.; van
de Stolpe, A.; Zorg, S.; Veerman, J.; Seebeck, T.; Sterk, G. J.; de Esch, I. J. P.; Leurs, R. Catechol
Pyrazolinones as Trypanocidals: Fragment-Based Design, Synthesis, and Pharmacological
Evaluation of Nanomolar Inhibitors of Trypanosomal Phosphodiesterase B1. Journal of Medicinal
Chemistry 2012, 55, 8745-8756.
16.
Sijm, M.; Siciliano de Araújo, J.; Kunz, S.; Schroeder, S.; Edink, E.; Orrling, K. M.;
Matheeussen, A.; van de Meer, T.; Sadek, P.; Custers, H.; Cotillo, I.; Martin, J. J.; Siderius, M.;
Maes, L.; Brown, D. G.; de Nazaré Correia Soeiro, M.; Sterk, G.; de Esch, I. J. P.; Leurs, R.
Phenyldihydropyrazolones as Novel Lead Compounds Against Trypanosoma cruzi. ACS Omega
2019, 4, 6585-6596.
17.
Higuera, S. L.; Guhl, F.; Ramírez, J. D. Identification of Trypanosoma cruzi Discrete
Typing Units (DTUs) through the implementation of a High-Resolution Melting (HRM)
genotyping assay. Parasites & Vectors 2013, 6, 112.
18.
Leepin, A.; Stüdli, A.; Brun, R.; Stephens, C. E.; Boykin, D. W.; Hemphill, A. Host Cells
Participate in the In Vitro Effects of Novel Diamidine Analogues against Tachyzoites of the
Intracellular Apicomplexan Parasites <em>Neospora caninum</em> and <em>Toxoplasma
gondii</em>. Antimicrobial Agents and Chemotherapy 2008, 52, 1999-2008.
19.
Meirelles, M. N.; de Araujo-Jorge, T. C.; Miranda, C. F.; de Souza, W.; Barbosa, H. S.
Interaction of Trypanosoma cruzi with heart muscle cells: ultrastructural and cytochemical
analysis of endocytic vacuole formation and effect upon myogenesis in vitro. European Journal
of Cell Biology 1986, 2, 9.
20.
Batista, D. d. G. J.; Batista, M. M.; Oliveira, G. M. d.; Amaral, P. B. d.; Lannes-Vieira, J.;
Britto, C. C.; Junqueira, A.; Lima, M. M.; Romanha, A. J.; Sales Junior, P. A.; Stephens, C. E.;
Boykin, D. W.; Soeiro, M. d. N. C. Arylimidamide DB766, a Potential Chemotherapeutic
Candidate for Chagas' Disease Treatment. Antimicrobial Agents and Chemotherapy 2010, 54,
2940-2952.
21.
Timm, B. L.; da Silva, P. B.; Batista, M. M.; da Silva, F. H. G.; da Silva, C. F.; Tidwell, R.
R.; Patrick, D. A.; Jones, S. K.; Bakunov, S. A.; Bakunova, S. M.; Soeiro, M. d. N. C. In Vitro and
In Vivo Biological Effects of Novel Arylimidamide Derivatives against Trypanosoma cruzi.
Antimicrobial Agents and Chemotherapy 2014, 58, 3720-3726.
22.
Romanha, A. J.; Castro, S. L. d.; Soeiro, M. d. N. C.; Lannes-Vieira, J.; Ribeiro, I.; Talvani,
A.; Bourdin, B.; Blum, B.; Olivieri, B.; Zani, C.; Spadafora, C.; Chiari, E.; Chatelain, E.; Chaves,
G.; Calzada, J. E.; Bustamante, J. M.; Freitas-Junior, L. H.; Romero, L. I.; Bahia, M. T.;
Lotrowska, M.; Soares, M.; Andrade, S. G.; Armstrong, T.; Degrave, W.; Andrade, Z. d. A. In
27
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201900370
ChemMedChem
vitro and in vivo experimental models for drug screening and development for Chagas disease.
Memórias do Instituto Oswaldo Cruz 2010, 105, 233-238.
28
This article is protected by copyright. All rights reserved.