Second Generation of Mannich Base-Type Derivatives with in Vivo Activity against Trypanosoma cruzi

Article abstract of DOI:10.1021/acs.jmedchem.8b00468

Chagas disease is a potentially life-threatening and neglected tropical disease caused by Trypanosoma cruzi. One of the most important challenges related to Chagas disease is the search for new, safe, effective, and affordable drugs since the current ther

Full text of DOI:10.1021/acs.jmedchem.8b00468

Subscriber access provided by Kaohsiung Medical University
Article
Second generation of Mannich base type derivatives
with in vivo activity against Trypanosoma cruzi
Rubén Martín-Escolano, Elsa Moreno-Viguri, Mery Jhenny Santiváñez Veliz, Álvaro Martín-
Montes, Encarnación Medina-Carmona, Rocío Paucar, Clotilde Marín, Amaya Azqueta,
Nuria Cirauqui, Angel Luis Pey, Silvia Pérez-Silanes, and Manuel Sánchez-Moreno
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00468 • Publication Date (Web): 08 Jun 2018
Downloaded from http://pubs.acs.org on June 8, 2018
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 79
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Second generation of Mannich base type derivatives with in vivo activity against
Trypanosoma cruzi.
1
2
2
1
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Rubén MartínꢀEscolano , Elsa MorenoꢀViguri , Mery SantivañezꢀVeliz , Alvaro MartinꢀMontes ,
3
2
1
4
Encarnación MedinaꢀCarmona , Rocío Paucar , Clotilde Marín *, Amaya Azqueta , Nuria
5
3
2
1
Cirauqui , Angel L. Pey , Silvia PérezꢀSilanes * and Manuel SánchezꢀMoreno *.
1
Departament of Parasitology, Instituto de Investigación Biosanitaria (ibs.Granada), Hospitales
Universitarios De Granada/University of Granada, Severo Ochoa s/n, Eꢀ18071 Granada, Spain.
2
Universidad de Navarra, Department of Organic and Pharmaceutical Chemistry, Instituto de Salud
Tropical, Pamplona 31008, Spain.
3
Department of Physical Chemistry, Faculty of Sciences, University of Granada, Av. Fuentenueva
s/n, 18071, Granada, Spain.
4
Universidad de Navarra, Department of Pharmacology and Toxicology, Pamplona 31008, Spain.
5
Department of Pharmaceutical Sciences, Federal University of Rio de Janeiro, Rio de Janeiro
21949ꢀ900, Brazil.
ABSTRACT
Chagas Disease is a potentially lifeꢀthreatening and neglected tropical disease caused by
Trypanosoma cruzi. One of the most important challenges related to Chagas disease is the search
for new, safe, effective and affordable drugs since the current therapeutic arsenal is inadequate and
insufficient. Here, we report a simple and costꢀeffective synthesis and the biological evaluation of
the second generation of Mannich baseꢀtype derivatives. Compounds 7, 9 and 10 showed improved
in vitro efficiency and lower toxicity than benznidazole, in addition to no genotoxicity; thus, they
were applied in in vivo assays to assess their activity in both acute and chronic phases of the
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 2 of 79
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
disease. Compound 10 presented a similar profile to benznidazole from the parasitological
perspective but also yielded encouraging data, as no toxicity was observed. Moreover, compound 9
showed lower parasitaemia and higher curative rates than benznidazole, also with lower toxicity in
both acute and chronic phases. Therefore, further studies should be considered to optimize
compound 9 to promote its further preclinical evaluation.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
KEYWORDS: arylaminoketone, Mannich Bases, Superoxide dismutase, Trypanosoma cruzi,
Chagas disease.
ACS Paragon Plus Environment

Page 3 of 79
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
INTRODUCTION
Chagas disease (CD) is a chronic parasitosis caused by the protozoan parasite Trypanosoma
1
ꢀ3
cruzi (T. cruzi). CD is endemic in 21 American countries ; however, the disease has become a
public health concern in nonꢀendemic countries, such as the United States, Canada and some
European countries, due to the appearance of nonꢀvector transmission through blood transfusions
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4,
and organ transplants and congenital transmission associated with massive migratory movements
5
.
According to World Health Organization (WHO) data, CD affects approximately 5.7 million
people to cause approximately 7,000 deaths annually, and almost 70 million people are at risk of
6, 7
infection . The disease also causes approximately 0.6 million disabilityꢀadjusted life years with
further economic depression of the affected areas.
Progress in the control of infection transmission routes has not been accompanied by progress in
the development of new treatments for the disease due to different factors, such as a lack of
knowledge about the pathophysiology of the disease, the high level of biological and genetic
polymorphism presented by the parasite depending on the geographical region and the lack of
pharmaceutical company interest. In addition, the experimental tools available for in vitro and in
8
ꢀ12
vivo testing have had a low predictive value
.
For these reasons, there is no effective chemotherapy; indeed, only two drugs developed in the
0s, nifurtimox (NFX, Lampit®, Bayer) and benznidazole (BZN, Rochagan®, Radanil®, Roche),
6
are currently available. Both drugs present important limitations, such as low efficacy during the
chronic phase of the disease, different activities against various genetic lineages of T. cruzi,
numerous side effects in patients and a complicated treatment dosage. Based on its relatively better
toxicity profile and accumulated experience, BZN is considered the current standard of treatment;
1
3,
however, it remains inefficient in the chronic phase of the disease and has unacceptable toxicity
14
.
Moreover, no drugs have been authorized for the treatment of adults, and only a paediatric
15
formulation of BZN has recently been approved by the Food and Drug Administration (FDA)
.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 4 of 79
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Furthermore, the most promising nitroimidazoles, such as posaconazole and ravuconazole, have not
16ꢀ18
shown the expected good results in clinical trials, and their development has been discontinued
.
In recent years, important advances have been achieved in the field of molecular biology and
genomics of T. cruzi and in CD pathophysiology. As a result, more research groups and
pharmaceutical companies are investing the development of new drugs. However, no success has
been achieved in developing a drug for CD that fulfils the target product profile (TPP) proposed by
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
19, 20
the DNDi
.
In a previous study, our group identified new Mannich baseꢀtype derivatives that showed high in
2
1
vitro activity against different life cycle stages and parasite strains of T. cruzi . The lead
compounds showed high selectivity indexes (SIs), curative activity during the acute phase of the
disease and prevention of parasitaemia reactivation after immunosuppressive treatment,
demonstrating curative rates by polymerase chain reaction (PCR) higher than BZN in a murine in
21
vivo model. In addition, the compounds were not mutagenic or genotoxic .
Here we present the synthesis, in vitro antiꢀT. cruzi activity and cytotoxicity of a second
generation of arylaminoketone Mannich base derivatives designed following our previous study,
with the aim of identifying a costꢀeffective route that may allow the broad study of substituents.
IC values lower than 10 µM, SI higher than 50 and an absence of genotoxicity in the SOS/umu
5
0
screening test have been established as the cutꢀoff to further evaluate compounds for in vivo
activity, in both acute and chronic phases of the disease. Moreover, the possible mechanisms of
action of the new compounds have been studied in terms of the variations in excreted metabolite
patterns, alterations of mitochondrial membrane potential and FeꢀSuperoxide dismutase (FeꢀSOD)
inhibition. Taken together, these new series led us to an interesting new lead for the development of
antiꢀChagas treatment, which demonstrated both a good activity/selectivity profile and a costꢀ
effective synthetic route.
COMPOUND DESIGN
ACS Paragon Plus Environment

Page 5 of 79
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Mannich bases are considered important pharmacophores with high potential in the field of
2
0, 22
medical chemistry and have shown a wide spectrum of biological activities
as antiꢀ
2
3
24ꢀ25
26
27
28
inflammatory , anticancer
, antibacterial , antifungal , antituberculosis and antimalarial
29
activities . In addition, Mannich bases endowed with biological activity against T. brucei
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
30
multiresistant strains as well as with inhibitory activity against T. cruzi trypanothione reductase
31
(
TRase) have also been described .
The simplicity and costꢀeffectiveness of the synthetic methodologies used in the drug discovery
process against neglected tropical diseases (NTD) play a key role. Taking advantage of the Mannich
reaction as a powerful tool for CꢀC bonding in organic reaction and considering the great potential
of Mannich base scaffolds as antiparasitic agents, we designed a second generation of βꢀ
21
aminoketones by modification of the previously identified lead compounds (Figure 1).
Our previous docking results on the FeꢀSOD enzyme (compounds A and B in Figure 1A)
showed that the carbonyl group (in red in Figure 1B), cyclic amine (in blue in Figure 1B) and the
following phenyl group (in green in Figure 1B) bind similarly in all compounds, with the phenyl
ring performing πꢀstacking interactions with Phe123 and the cyclic amine bound close to Glu166.
Therefore, we believe that these groups are the most important for binding to the SOD enzyme;
thus, they were retained in the new series we present herein. In contrast, the benzothiophene ring
was substituted with a mono or diꢀsubstituted benzene ring (in purple in Figure 1B), which allows
easier and lowerꢀcost coverage of modifications by including different electronic character
substituents. For the some reason we included only piperazine ring as cyclic amine. Fluorine atoms
have been substituted in different positions of the phenyl rings because fluorine is a wellꢀknown
hydrogen mimic. This strategy is one of the most classical bioisosteric replacements used in
medicinal chemistry. The trifluoromethyl moiety was also included with the same purpose of
enhancing metabolic stability. Nevertheless, some other halogens, methyl and methoxy moieties
were also included to explore their influence on the efficacy, toxicity and pharmacokinetic
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 6 of 79
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
(
PK)/pharmacodinamic (PD) properties so that a structureꢀactivity relationship could be explored.
We decided to retain the piperazine scaffold as constant in the general chemical skeleton
considering that the incorporation of cyclic amines is widely used in the search for biologically
active molecules, and previous studies by our group have confirmed interest in this amine. Several
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
21, 32ꢀ35
examples of antiꢀtrypanosomatid agents based on cyclic amines have been reported
, and
antifungal drugs with antiꢀkinetoplastid activity, such as ketoconazole, itraconazole or
36ꢀ39
posaconazole, present piperazine rings in their structures
. Moreover, we examined an
increasing length of the linker between the piperazine and the phenyl group, which may allow
improved binding of piperazine to the negative chain of Glu166 (in magenta in Figure 1B).
Considering that the predicted pKa of our compounds suggests equilibrium between a neutral and a
positively charged piperazine (at the nitrogen closer to the carbonyl group, in bold in Figure 1B),
we believe that its interaction with the negative side chain of Glu166 must be important for affinity.
This design also took into consideration the increment of chemical variability of βꢀaminoketones,
costꢀefficient chemistry and synthetic simplicity, which are essential factors for attracting the
attention and interest of pharmaceutical companies.
Figure 1. A) Docking results of our previous leads (in pale pink and black compounds 4 and 10
respectively from MorenoꢀViguri et al., 2016) on the FeꢀSOD enzyme, showing the conserved
binding mode for the carbonyl, cyclic amine and phenyl scaffolds, while the benzothiophene ring
ACS Paragon Plus Environment

Page 7 of 79
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
presents different positioning in each compound. B) Design of the new series of arylaminoketone
Mannich Base type derivatives. Simplified scheme of the new series design (down) based on our
previous leads (up). The different groups are colored as mentioned on the text.
RESULTS AND DISCUSSION
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Chemistry.
Figure 2. Synthesis of new arylaminoketone derivatives.
Fortyꢀfour out of fortyꢀnine compounds (2ꢀ17, 22ꢀ49) were obtained by Mannich reaction as
21
previously reported . Compounds 18ꢀ21 were obtained by an aliphatic nucleophilic substitution in
which the corresponding piperazine was condensed with a primary alkyl halide in the presence of
K
2
CO
3
as base and tetrahydrofuran (THF) as solvent (Figure 2). The synthetic methodology to
40
obtain compound 1 has been previously described . Therefore, 48 new derivatives have been
synthesized using a simple, fast and economically affordable methodology.
Table 1. Structure and molecular properties of the new compounds calculated with the DataWarrior
41
program .
Molecular properties
b
c
d
e
f
g
Comp
R
1
R
H
H
2
n
0
0
0
0
R
3
MW
clogP
clogS
ꢀ3.4
ꢀ3.9
ꢀ3.8
ꢀ4.2
PSA
HBA
HBD
a
1
H
H
H
H
4ꢀF
313.4
363.4
327.4
377.4
0.8
24.7
24.7
24.7
24.7
3
3
3
3
1
1
1
1
2
3
4
4ꢀCF
3
3
1.6
CH
CH
4ꢀF
1.2
3
3
4ꢀCF
1.9
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 8 of 79
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
5
6
7
8
9
H
CH
3
3
0
0
0
0
0
0
0
0
0
0
1
1
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
4ꢀCl
4ꢀNO
4ꢀF
343.9
354.4
343.4
393.4
359.9
370.4
373.4
423.4
389.9
400.4
373.4
355.5
369.5
327.4
331.4
343.4
358.4
392.3
442.3
408.7
419.3
410.3
460.3
426.7
437.3
349.3
399.4
1.7
1.5
0.7
1.5
1.3
1.1
0.7
1.4
1.2
1.0
0.7
0.6
0.9
1.2
0.9
0.8
1.2
1.6
2.3
2.1
1.9
1.7
2.4
2.2
2.0
1.0
1.8
ꢀ4.2
ꢀ3.9
ꢀ3.4
ꢀ3.9
ꢀ3.9
ꢀ3.6
ꢀ3.4
ꢀ3.9
ꢀ3.9
ꢀ3.6
ꢀ3.5
ꢀ3.1
ꢀ3.5
ꢀ3.8
ꢀ3.7
ꢀ3.4
ꢀ3.9
ꢀ4.2
ꢀ4.7
ꢀ4.7
ꢀ4.4
ꢀ4.6
ꢀ5.0
ꢀ5.0
ꢀ4.7
ꢀ4.0
ꢀ4.5
24.7
70.5
34.0
34.0
34.0
79.8
43.2
43.2
43.2
89.0
43.2
43.2
43.2
24.7
24.7
34.0
70.6
24.7
24.7
24.7
70.6
24.7
24.7
24.7
70.6
24.7
24.7
3
6
4
4
4
7
5
5
5
8
5
5
5
3
3
4
6
3
3
3
6
3
3
3
6
3
3
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
H
CH
2
2
2
H
OCH
OCH
OCH
OCH
OCH
OCH
OCH
OCH
OCH
OCH
OCH
F
3
3
3
3
3
3
3
3
3
3
3
H
4ꢀCF
4ꢀCl
4ꢀNO
4ꢀF
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
H
1
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
H
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
OCH
OCH
OCH
OCH
OCH
OCH
OCH
H
3
3
3
3
3
3
3
4ꢀCF
4ꢀCl
4ꢀNO
3ꢀF
3
H
3ꢀCH
3
2ꢀOCH
3
3
H
F
2ꢀF
H
F
4ꢀOCH
H
F
4ꢀNO
4ꢀF
2
2
2
H
Br
H
Br
4ꢀCF
4ꢀCl
4ꢀNO
4ꢀF
3
H
Br
H
Br
Br
F
Br
F
4ꢀCF
4ꢀCl
4ꢀNO
4ꢀF
3
Br
F
Br
F
F
F
F
F
4ꢀCF
3
ACS Paragon Plus Environment

Page 9 of 79
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
F
F
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
4ꢀCl
4ꢀNO
4ꢀF
365.8
376.4
365.8
415.8
382.3
392.8
365.8
415.8
382.3
392.8
381.4
413.4
397.8
408.4
397.4
447.4
413.9
424.4
1.5
1.3
1.5
2.3
2.1
1.8
1.5
2.3
2.1
1.8
1.7
2.3
2.2
2.0
1.9
2.7
2.4
2.2
ꢀ4.5
ꢀ4.2
ꢀ4.5
ꢀ4.9
ꢀ4.9
ꢀ4.6
ꢀ4.5
ꢀ4.9
ꢀ4.9
ꢀ4.6
ꢀ4.2
ꢀ4.6
ꢀ4.6
ꢀ4.3
ꢀ4.4
ꢀ4.9
ꢀ4.9
ꢀ4.6
24.7
70.6
24.7
24.7
24.7
70.6
24.7
24.7
24.7
70.6
24.7
24.7
24.7
70.6
34.0
34.0
34.0
79.8
3
6
3
3
3
6
3
3
3
6
3
3
3
6
4
4
4
7
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
F
F
2
2
2
2
2
Cl
Cl
Cl
Cl
F
F
F
4ꢀCF
4ꢀCl
4ꢀNO
4ꢀF
3
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
F
F
Cl
Cl
Cl
Cl
CF
CF
CF
CF
F
4ꢀCF
4ꢀCl
4ꢀNO
4ꢀF
3
F
F
H
H
H
H
H
H
H
H
3
3
3
3
4ꢀCF
4ꢀCl
4ꢀNO
4ꢀF
3
OCF
OCF
OCF
3
3
3
3
4ꢀCF
3
4ꢀCl
OCF
4ꢀNO
b
ꢀ1
c
a: Previously published. Total Molecular Weight (g·mol ). Logarithm of the partition coefficient between nꢀoctanol
d
ꢀ1
e
2
f
and water. Logarithm of the solubility measured in mol·L . Polar Surface Area (Å ). Number of hydrogenꢀbond
g
acceptors. Number of hydrogenꢀbond donors.
The results of the in silico study of some relevant molecular properties related to the drugꢀ
likeness of the compounds are shown in Table 1. It can be observed that all compounds fulfil the
ꢀ
1
Lipinski “rule of five”, with a molecular weight lower than 500 g·mol , an estimated partition
coefficient (logP) lower than 5, a number of hydrogenꢀbond acceptors lower than 10, and less than 5
hydrogen bond donors. Moreover, the polar surface area (PSA) and aqueous solubility (logS) values
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 10 of 79
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2
anticipate good oral bioavailability, with the first being lower than 140 Å and the second similar or
higher than ꢀ4.
In vitro trypanocidal evaluation.
The genetic diversity of T. cruzi is extensively acknowledged. Currently, T. cruzi is divided into
seven discrete typing units (DTUs), presenting different genotypes and phenotypes, ecological and
epidemiological associations, evolutionary relationships, pathogenesis, tropism and drug resistance
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4
2
.
Consequently, three different T. cruzi strains (TcI, TcV and TcVI), with different tropisms, hosts
and locations, were used to determine and select those compounds with good performance.
The extracellular epimastigoteꢀlike form is habitually used because of its simple culture and
maintenance in the laboratory. However, tests against the developed forms in vertebrate hosts,
bloodstream trypomastigotes and intracellular amastigote forms (responsible for the chronic phase
43
of CD), are more appropriate . For this reason, the in vitro trypanocidal activity of compounds 1ꢀ
9 and the reference drug BZN were evaluated in the epimastigote form with the objective of
4
establishing the inhibition concentrations 50 (IC50 values) as a primary screen.
Table 2 shows the IC50 values of the compounds on the epimastigote forms of the three T. cruzi
strains cited above, the cytotoxicity using mammalian Vero cells and the selectivity index (SI = IC50
Vero cells/IC epimastigote forms). Interestingly, almost all these Mannich baseꢀtype derivatives
50
were substantially less toxic than the reference drug BZN after 72 h of exposure at different
concentrations.
Table 2. In vitro activity, cytotoxicity and selectivity index for compounds on the epimastigote
form of Trypanosoma cruzi strains.
a
c
Activity IC50 (µM)
Selectivity Index
T. cruzi
Arequipa
strain
T. cruzi
T. cruzi
Tulahuen
strain
Toxicity IC50
T. cruzi
Arequipa
strain
T. cruzi T. cruzi
Comp
BZN
b
SN3
Vero Cell (µM)
SN3
Tulahuen
strain
strain
strain
16.9 ± 1.8
36.2 ± 2.4
19.7 ± 1.7
23.2 ± 2.1
1.4
0.6
1.2
ACS Paragon Plus Environment

Page 11 of 79
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
2
3
4
5
6
7
8
9
65.7 ± 5.7
12.8 ± 1.3
43.5 ± 4.8
14.2 ± 1.7
31.7 ± 2.9
28.4 ± 3.1
1.4 ± 0.1
32.9 ± 2.9
16.2 ± 1.5
78.8 ± 6.7
17.6 ± 1.5
26.0 ± 2.4
18.2 ± 1.4
2.0 ± 0.1
94.1 ± 7.9
24.9 ± 3.1
25.0 ± 2.1
22.5 ± 2.3
29.5 ± 3.1
46.2 ± 4.5
1.8 ± 0.2
13.4 ± 1.4
15.7 ± 1.7
0.2 (0)
1.2 (1)
1.3 (1)
9.8 (7)
5.8 (4)
8.3 (6)
40.2 (29)
1.9 (1)
31.5 (23)
18.6 (14)
1.1 (1)
4.1 (3)
2.1 (1)
1.1 (1)
0.5 (0)
1.6 (1)
2.7 (2)
0.4 (0)
1.3 (1)
1.3 (1)
1.7 (1)
4.4 (3)
2.6 (2)
1.2 (1)
2.9 (2)
9.1 (7)
1.4 (1)
7.7 (6)
7.0 (5)
18.0 (13)
0.4 (0)
5.6 (4)
1.4 (1)
0.4 (1)
1.0 (2)
0.7 (1)
7.9 (12)
7.1 (11)
12.9 (20)
27.9 (44)
4.2 (7)
47.8 (75)
26.7 (4)
2.8 (4)
8.7 (14)
4.2 (7)
0.6 (1)
0.5 (1)
0.9 (1)
2.2 (3)
0.6 (1)
0.9 (1)
1.4 (2)
2.0 (3)
1.8 (3)
2.8 (4)
2.0 (3)
2.0 (3)
8.3 (13)
3.2 (5)
5.9 (9)
6.8 (11)
10.3 (16)
0.5 (1)
6.3 (10)
0.8 (1)
0.1 (0)
0.6 (1)
2.2 (2)
6.2 (5)
6.2 (5)
5.1 (4)
30.2 (26)
3.1 (3)
40.9 (35)
15.7 (13)
1.7 (1)
7.2 (6)
5.1 (4)
1.0 (1)
0.6 (0)
1.0 (1)
1.8 (2)
0.5 (0)
0.9 (1)
0.8 (1)
1.4 (1)
2.0 (2)
2.8 (2)
2.5 (2)
1.7 (1)
8.5 (7)
2.6 (2)
6.7 (6)
13.4 (11)
5.9 (5)
0.3 (0)
1.9 (2)
2.3 (2)
55.4 ± 4.2
139.7 ± 12.4
183.8 ± 157
234.6 ± 17.8
55.0 ± 4.9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
72.9 ± 6.7
9.5 ± 0.8
32.3 ± 3.5
6.3 ± 0.5
44.0 ± 4.9
7.3 ± 0.6
135.2 ± 11.0
300.0 ± 24.1
587.2 ± 37.6
174.8 ± 16.8
242.5 ± 21.0
185.4 ± 14.8
105.2 ± 8.9
22.4 ± 3.8
1
0
1
2
31.6 ± 0.3
153.8 ± 14.2
59.0 ± 6.2
90.3 ± 8.9
94.5 ± 9.7
49.0 ± 5.1
29.5 ± 3.0
19.4 ± 2.1
58.3 ± 4.9
37.9 ± 3.7
24.5 ± 2.1
65.8 ± 5.9
22.5 ± 2.7
30.0 ± 2.8
42.5 ± 3.9
27.2 ± 3.1
38.8 ± 3.7
46.6 ± 4.1
26.6 ± 2.7
36.5 ± 3.4
21.4 ± 2.0
33.4 ± 3.0
15.4 ± 1.2
85.2 ± 7.8
22.0 ± 0.2
61.5 ± 6.7
27.8 ± 3.2
43.9 ± 4.5
173.6 ± 19.8
48.0 ± 5.0
55.2 ± 6.1
24.1 ± 2.4
40.5 ± 3.4
52.6 ± 6.0
22.2 ± 2.0
54.5 ± 4.2
56.8 ± 6.1
28.3 ± 3.2
26.4 ± 2.7
39.7 ± 4.0
42.4 ± 4.3
20.8 ± 2.3
35.1 ± 3.1
37.5 ± 4.0
37.6 ± 4.2
28.8 ± 2.4
13.7 ± 1.1
160.1 ± 14.8
37.4 ± 0.4
103.3 ± 9.4
33.9 ± 3.1
36.6 ± 3.8
104.9 ± 11.2
40.8 ± 4.5
46.2 ± 4.1
28.4 ± 2.4
45.6 ± 3.8
53.5 ± 5.1
38.9 ± 3.4
78.1 ± 6.8
49.5 ± 4.5
28.5 ± 3.1
20.9 ± 1.7
46.2 ± 3.8
41.4 ± 3.4
25.6 ± 2.1
30.6 ± 2.4
19.1 ± 1.5
65.5 ± 6.1
54.1 ± 5.1
46.2 ± 4.2
53.1 ± 4.9
1
1
13
1
1
4
5
16
47.5 ± 5.7
1
1
1
2
7
8
9
0
52.0 ± 4.9
24.0 ± 2.7
49.5 ± 3.9
31.9 ± 3.4
21
110.5 ± 13.4
99.5 ± 7.4
2
2
2
3
78.8 ± 6.8
24
52.1 ± 6.2
2
2
2
2
5
6
7
8
79.8 ± 8.1
352.1 ± 40.0
66.8 ± 8.1
205.9 ± 17.0
256.2 ± 22.8
385.2 ± 31.9
14.0 ± 1.1
29
3
3
0
1
32
86.2 ± 7.8
3
3
122.3 ± 13.8
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 12 of 79
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
3
3
4
5
25.3 ± 2.4
21.6 ± 1.9
45.5 ± 4.1
34.2 ± 3.1
39.5 ± 3.8
27.6 ± 2.4
42.0 ± 3.9
23.8 ± 2.7
36.9 ± 3.8
24.3 ± 2.3
52.7 ± 5.4
13.5 ± 1.1
85.4 ± 8.1
51.2 ± 4.9
42.2 ± 4.0
41.1 ± 3.4
41.6 ± 3.8
45.0 ± 4.2
29.5 ± 3.4
51.0 ± 4.5
75.3 ± 6.4
49.1 ± 4.2
51.6 ± 5.4
32.4 ± 3.4
66.4 ± 5.9
34.7 ± 3.8
38.2 ± 4.1
20.0 ± 1.4
27.6 ± 2.8
38.4 ± 3.7
34.1 ± 3.9
95.0 ± 8.7
37.1 ± 3.1
29.0 ± 2.5
29.1 ± 2.9
41.5 ± 3.8
46.2 ± 5.1
66.7 ± 6.1
55.5 ± 5.1
15.5 ± 1.2
60.3 ± 5.4
43.0 ± 4.8
39.1 ± 4.2
15.1 ± 1.8
52.6 ± 5.8
86.3 ± 7.5
61.8 ± 5.1
52.1 ± 6.0
185.1 ± 21.6
74.5 ± 8.7
114.2 ± 12.9
115.7 ± 11.0
105.2 ± 9.1
74.3 ± 6.9
174.8 ± 15.6
89.2 ± 7.4
28.5 ± 3.1
49.2 ± 3.8
54.2 ± 5.0
75.7 ± 6.9
70.3 ± 7.2
64.9 ± 5.7
95.6 ± 8.6
93.2 ± 7.4
7.3 (5)
3.4 (3)
2.5 (2)
3.4 (2)
2.7 (2)
2.7 (2)
4.2 (3)
3.8 (3)
0.8 (1)
2.0 (1)
1.0 (1)
5.6 (4)
0.8 (1)
1.3 (1)
2.3 (2)
2.3 (2)
4.5 (7)
1.7 (3)
3.9 (6)
2.3 (4)
1.4 (2)
1.5 (2)
3.4 (5)
2.8 (4)
0.4 (1)
1.4 (2)
1.4 (2)
3.8 (6)
2.5 (4)
1.7 (3)
2.8 (4)
1.0 (2)
5.0 (4)
2.6 (2)
3.9 (3)
2.8 (2)
2.3 (2)
1.1 (1)
3.2 (3)
5.7 (5)
0.5 (0)
1.1 (1)
1.4 (1)
5.0 (4)
1.3 (1)
0.8 (1)
1.5 (1)
1.8 (2)
36
3
3
3
4
7
8
9
0
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
41
4
4
4
4
2
3
4
5
46
4
4
7
8
49
IC50 = the concentration required to give 50 % inhibition, calculated using GraphPad Prism. Each drug concentration
a
b
c
was tested in triplicate in four separate determinations. Towards Vero Cells after 72 h of culture. Selectivity Index =
IC50 Vero Cells/IC50 epimastigote form of parasite. In brackets: number of times that compound SI exceeds the
reference drug SI (on epimastigote form of T. cruzi).
SI values higher than 10 in the epimastigote form, in any of the T. cruzi strains, were established
as the cutꢀoff in this first preliminary stage. Thus, compounds 6, 7, 9, 10, 29 and 30 were selected as
potential trypanocidal agents. Subsequently, their activities were evaluated against the developed
forms in vertebrate hosts (amastigote and trypomastigote forms), which are the relevant forms from
the clinical perspective. The IC50 values and the SI for these selected compounds on extraꢀ and
intracellular forms of T. cruzi are shown in Table 3 and Table 4, respectively. In accordance with
some authors, potential antichagasic agents must meet certain criteria: IC50 value must not exceed
4
4
1
0 ꢁM, and the SI must be higher than 50 . Three out of the six tested compounds (7, 9 and 10)
fulfil those requirements at least for one of the parasite forms. Moreover, these three Mannich baseꢀ
type derivatives showed IC50 values in Vero cells and SI that were superior to those of the reference
ACS Paragon Plus Environment

Page 13 of 79
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
drug BZN. Therefore, these three compounds were prioritized to determine their in vivo
trypanocidal activities. These in vivo assays were performed only in the T. cruzi Arequipa strain, as
there were no significant differences between the performances of the selected compounds for the
other evaluated strains.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 3. In vitro activity and cytotoxicity for selected compounds (6, 7, 9, 10, 29 and 30) on extraꢀ
and intracellular forms of Trypanosoma cruzi.
a
a
a
Activity IC50 (µM)
T. cruzi Arequipa strain
Trypo
Activity IC50 (µM)
Activity IC50 (µM)
T. cruzi Tulahuen strain
Trypo
T. cruzi SN3 strain
Toxicity IC50
Vero Cell
Comp
Trypo
m.
Epim.
Amast.
Epim.
forms
Amast.
forms
Epim.
Amast.
b
m.
m.
(µM)
forms
forms
Forms
Forms
forms
forms
forms
16.9 ±
12.4 ±
1.1
36.1 ±
3.1
19.7 ±
1.7
15.1 ±
1.3
BZN
8.3 ± 0.7
nd
36.2 ± 2.4 16.6 ± 1.4
18.2 ± 1.4 24.0 ± 2.7
10.0 ± 0.8
Nd
23.2 ± 2.1
234.6 ± 17.8
55.0 ± 4.9
1
.8
8.4 ±
.1
.4 ±
0.1
.5 ±
.8
31.6 ±
.3
6.5 ±
.4
2
25.3 ±
2.1
46.2 ±
4.5
6
7
9
nd
nd
3
1
1.1 ±
0.1
1.4 ±
0.1
1.8 ±
0.2
1.2 ±
0.1
0.6 ± 0.0
3.6 ± 0.3
29.4 ± 0.3
nd
2.0 ± 0.1
6.3 ± 0.5
3.1 ± 0.3
5.8 ± 0.5
1.6 ± 0.2
3.8 ± 0.3
33.6 ± 0.3
38.7 ± 3.4
Nd
9
3.4 ±
0.3
3.9 ±
0.4
7.3 ±
0.6
2.9 ±
0.3
300.0 ± 24.1
587.2 ± 37.6
256.2 ± 22.8
385.2 ± 31.9
0
11.7 ±
0.1
3.1 ±
0.3
37.4 ±
0.4
14.5 ±
0.1
1
0
22.0 ± 0.2 30.5 ± 0.3
0
3
19.1
±1.5
65.5 ±
6.1
32.1 ±
2.8
29
nd
37.5 ± 4.0
nd
nd
3
2
1.4 ±
2.0
31.6 ±
3.0
42.2 ±
4.1
3
0
28.3 ± 2.7
37.6 ± 4.2 51.1 ± 5.5
nd
a
IC50 = the concentration required to give 50 % inhibition, calculated using GraphPad Prism. Each drug concentration
b
was tested in triplicate in four separate determinations. Towards Vero Cells after 72 h of culture. nd, not determined.
Table 4. Selectivity Index for selected compounds (6, 7, 9, 10, 29 and 30) on extraꢀ and intracelular
forms of Trypanosoma cruzi.
Comp.
Selectivity index
Selectivity index
Selectivity index
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 14 of 79
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
a
a
a
T. cruzi Arequipa strain
T. cruzi SN3 strain
T. cruzi Tulahuen strain
Epim.
forms
Amast.
forms
Trypom.
forms
Epim.
forms
Amast.
forms
Epim.
Forms
Amast.
Forms
Trypom.
forms
Trypom. forms
BZN
1.4
2.8
nd
1.9
nd
0.6
1.4
0.6
9.3 (15)
39.9 (62)
76.1 (118)
187.0 (291)
nd
1.2
2.3
nd
1.5
nd
6
7
9
8.3 (6)
12.9 (20)
9.8 (7)
5.1 (4)
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
40.2 (29) 88.7 (32) 49.1 (26) 27.9 (44) 17.6 (13)
31.5 (23) 82.4 (29) 89.6 (48) 47.8 (75) 51.5 (37)
30.2 (26) 34.6 (15)
40.9 (35) 78.7 (34)
47.4 (31)
102.7 (67)
40.6 (26)
8.0 (5)
nd
10
29
30
18.6 (14)
7.0 (5)
20.0 (7)
nd
50.4 (27)
nd
26.7 (4)
6.8 (11)
10.3 (16)
19.2 (14)
nd
15.7 (13)
13.4 (11)
5.9 (5)
17.5 (8)
6.6 (3)
nd
18.0 (13)
13.6 (5)
12.2 (6)
7.5 (5)
9.1 (14)
a
Selectivity index = IC50 Vero Cells/(IC50 extracellular and intracelular form of parasite. In brackets: number of times
that compound SI exceeds the reference drug SI (on extracellular and intracellular forms of T. cruzi). nd, not
determined.
From the structural perspective, it must be mentioned that the three lead compounds (7, 9 and
10) presented the methoxy donor group substituted on the para position of the phenyl ring. The
substitution of an extra methoxy group did not influence the cytotoxicity in Vero cells; however,
this modification led to a reduction in in vitro activity and, as a consequence, a great decrement of
the SI (compounds 7 vs. 11, 9 vs. 13 and 10 vs. 14). Furthermore, the substitution of the methoxy
by the trifluoromethoxy moiety (compounds 7 vs. 46, 9 vs. 48 and 10 vs. 49) led to a clear increase
in cytotoxicity and decrease in in vitro activity.
Ultimately, the rates of infection in host Vero cells were measured by counting the infected cells
after 72 h of exposure at different concentrations to acquire more accurate information about the
most active compounds, including BZN. The data are shown in Figure 3, together with the data of
the amastigote and trypomastigote forms. The rates of infected cells gradually decreased in all
cases, declining to 7 % for compound 7, 15 % for compound 9 and 52 % for compound 10 at
concentrations of 50 µM with respect to the infected control Vero cells. We must highlight the
action of compounds 7 and 9, with IC50 values (relative to the % of infected cells) of 1.9 µM and
6.4 µM, respectively. These two compounds showed higher efficiency than the reference drug BZN
ACS Paragon Plus Environment

Page 15 of 79
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
(
approximately 20 % of infected cells at 50 µM and with an IC50 value of 15.1 µM). Likewise, the
average number of amastigote forms per cell decreased for all tested compounds (Supplementary
Figure S97). A decrease in the number of amastigote and trypomastigote forms was also observed.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 3. Reduction of the infection of T. cruzi Arequipa strain regarding the decrease of
amastigote and trypomastigote forms and infected cells treated with (a) BZN, (b) 7, (c) 9 and (d) 10.
Values are the means of the three separate determinations. In brackets: IC50 value, calculated using
GraphPad Prism.
In vitro genotoxicity screening ꢀ SOS/umu assay.
The SOS/umu genotoxicity assay has been used as a screening tool to determine the compounds
to prioritize for further studies. The three most active compounds in the in vitro evaluation
(compounds 7, 9 and 10) were included in the SOS/umu test to explore their genotoxicity. All
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 16 of 79
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
assessed compounds were nonꢀgenotoxic with or without metabolic activation in the tested
conditions (supp. Material). However, BZN showed in vitro genotoxicity with and without
metabolic activation. The obtained results confirm the promising profile of these series.
In vivo antiꢀT. cruzi activity in BALB/c female mice.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
To assess the effectiveness of the treatment in both the acute and chronic phases, experimental
tests during the in vivo evaluation were performed to examine the following: a) parasitaemia levels
by counting bloodstream trypomastigotes in the acute phase treatment; b) parasitaemia reactivation
in the chronic phase by counting bloodstream trypomastigotes after immunosuppression (IS); c)
parasites in target organs in the chronic phase by PCR after IS; d) levels of immunoglobulinꢀG (Igꢀ
G) by the enzymeꢀlinked immunosorbent assay (ELISA) and the splenomegaly as indicators of the
immune response; e) serum biochemistry parameters as an indicator of metabolic disturbances or
abnormalities related to the treatment.
Since compounds 7, 9 and 10 exhibited remarkable in vitro results towards trypomastigote and
4
4
amastigote forms (IC50 and SI values close to the criteria established by Nwaka et al. ) and the
screening test showed no genotoxicity, those Mannich baseꢀtype derivatives were evaluated in
BALB/c mice (in vivo studies).
As mentioned above, and because of the different effectiveness levels of current drugs in the
treatment against CD during the acute and chronic phases (where the performance of drugs is not as
45
effective as it should) , the effect of these compounds was evaluated by treating mice in each
phase: the treatment was carried out from day 10 postꢀinfection (pi) for the acute phase and from
day 75 pi (it was established that the animals entered the chronic phase, in which there are no
parasites remaining in the bloodstream) for the chronic phase, both during 5 consecutive days.
Drugs were administered by oral dose because it is the preferred route for the treatment of parasitic
diseases, in addition to the oral therapeutic route, which leads to better patient compliance and has a
4
6
low cost (critical aspects of human treatment in developing countries) .
ACS Paragon Plus Environment

Page 17 of 79
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Treatment for both compounds under study and the reference drug BZN was performed with
ꢀ1
subcurative doses for BZN (20 mg·kg per day for 5 days) to evaluate whether the compounds
demonstrated higher in vivo effectiveness than the reference drug. Noticeably, none of the treated
mice died during and/or after treatment, nor did they lose more than 10 % body mass. Those
compounds with higher effectiveness could be studied at higher doses since they do not present
toxicity, establishing a new treatment guideline based on pharmacokinetic studies, to achieve total
cure. It is proposed that the different effectiveness levels in the acute and chronic phases are related
to inadequate pharmacokinetics between the compounds and the location of the parasites in the
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4
7
tissues during the chronic phase of infection .
First, Figure 4 shows the parasitaemia levels in the different groups of mice that were untreated
and treated in the acute phase. A reduction of parasitaemia in mice treated with the tested Mannich
baseꢀtype derivatives with respect to the untreated mice was observed. Moreover, compound 9
exhibited higher in vivo trypanocidal activity in the acute phase than the reference drug BZN. This
activity was evident since the beginning of the treatment (day 10 pi) and was maintained until the
end of the acute phase (day 50 pi). The peak of parasitaemia (day 23 pi) caused a reduction of 68 %
for compound 10, 76 % for compound 9 and 55 % for compound 7, compared with the control
group; even compound 9 showed a higher reduction than BZN (76 % versus 70 %, respectively).
Furthermore, parasitaemia was not detected on day 40 pi for compound 9, while it was observed in
the group of mice that were untreated and treated with BZN up to day 49 and 47 pi, respectively.
This finding appears to indicate that parasites were eliminated or were depleted to undetectable
levels for counting.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 18 of 79
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 4. Parasitaemia in murine model of acute CD: control (untreated), BZN, compound 7,
compound 9 and compound 10. In all cases, mice were intraperitoneally infected with
trypomastigotes of T. cruzi Arequipa strain, and compounds were orally administered with a total
ꢀ1
dose of 100 mg·kg of body mass. Treatment days are represented in grey. Values constitute means
of six mice ± standard deviation.
Second, with the aim of determining the effectiveness of the treatment and the disease extent in
the chronic phase, parasitaemia reactivation was determined after IS up to day 120 pi (late chronic
phase, when the parasite is nested inside target organs in the amastigote form), as mentioned in the
experimental section. The objective was to reactivate the parasitaemia under the control of the
immunological system of mice, which was blocked using cyclophosphamide monohydrate (CP).
The reactivation is an important matter because seemingly cured immunocompromised individuals
and cured patients submitted to kidney or liver transplantation (treated with anticancer
chemotherapy or diagnosed with AIDS) exhibit clinically aggressive reactivation of the
parasitaemia. Hence, IS is performed as a first cure confirmation technique, with the second being
ACS Paragon Plus Environment

Page 19 of 79
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
PCR of the organs (which is mentioned below): those animals infected with T. cruzi and treated
with parasitaemia that does not reappear and negative PCR results for organs after IS are considered
48
cured . Figure 5 shows the percentage of reactivation of parasitaemia after IS in comparison with
the control groups, which is proportional to the survival rate of the parasites. Parasitaemia
reactivation was reduced for all mice treated with the tested compounds and with BZN in both the
acute and chronic phases. It is noteworthy that all tested compoundꢀtreated mice in the acute phase
showed reduced parasitaemia reactivation than those treated in the chronic phase; the opposite
occurred for mice treated with BZN. Regardless, compound 9ꢀtreated mice showed the lowest
reactivation of parasitaemia both in the acute and chronic phases (12 % and 20 %, respectively),
even with much lower values than those mice treated with BZN (75 % and 51 %, respectively). This
significant decrease in parasitaemia reactivation was expected in compound 9ꢀtreated mice in the
acute phase because of the obtained parasitaemia curve (mentioned above). Additionally, as
mentioned below, the presence of parasites in the target organs was negative for most of the
analysed organs in these mice treated in the acute phase. Moreover, the low parasitaemia
reactivation observed in compound 9ꢀtreated mice in the chronic phase indicated that the treatment
was also effective in this phase and confirmed the results obtained using PCR.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 20 of 79
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Figure 5. Immunosuppression in vivo assay for mice untreated (control) and treated with BZN, 7, 9
and 10. Figure shows the reactivation of parasitaemia after the treatment in both phases and the
immunosuppression cycles by fresh blood in comparison to the mice untreated (control). Values
constitute means of six mice ± standard deviation.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Finally, the presence of parasites in the target organs was determined by PCR after necropsy
(
day 127 postꢀinfection) to evaluate the curative effect of these compounds as a second technique of
confirmation of cure. PCR results for the target organs (positive for control groups) in the different
groups of mice, both treated in the acute phase and in the chronic phase, are shown in Figure 6.
PCR of the control groups, both in the acute and chronic phases, was positive for these 9 organs and
tissues: adipose, bone marrow, brain, oesophagus, heart, lung, muscle, spleen and stomach. As
observed and according to previous studies, compound 9 showed the best in vivo trypanocidal
activity, which was even better than that shown by the reference drug BZN: 89 % and 78 % of
parasiteꢀfree organs/tissues for compound 9ꢀtreated mice in the acute and chronic phases,
respectively, in comparison with BZNꢀtreated mice (33 % and 55 % of parasiteꢀfree organs/tissues),
thus confirming the partial curative effect of this compound at this dosage in both phases.
Assessment of clinical cure in T. cruzi infections is debatable due to the lack of a trustworthy test
49
to ensure parasite elimination . PCR techniques to identify cure raise critical questions, and their
main utility is to confirm the failure of clinical cure because even consistently negative results using
blood, for which this technique is capable of detecting a single parasite in 5 mL, are insufficient to
50, 51
confirm the complete elimination of tissue parasites
. Detection methodology has been
enhanced by specific PCR with the TaqMan technology, using fluorogenic probes, realꢀtime
amplification reaction and sequences of constant regions and variables of minicircles of kinetoplast
DNA. This technology is quite useful to assess the parasitic load in earlier and laterꢀtreated chronic
patients and for the future establishment of a suitable criterion for the healing of patients submitted
5
2
53
to therapy . Regarding animal testing models, IS is the formula applied to demonstrate cure . We
ACS Paragon Plus Environment

Page 21 of 79
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
evaluate the establishment of cure in the chronic phase, as mentioned above, using a double
confirmation based on IS and PCR of the target organs after necropsy. Therefore, we evaluate the
presence (or not) of parasites in both blood and tissue. At present, highly sensitive bioluminescence
imaging model techniques are used to prove cure, generating data with superior accuracy to other
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
53
methods, including PCR . However, we can provide evidence of cure (or, at least, a considerable
reduction in the parasitic load) based on the results of six independent samples by PCR and
reactivation after IS (double checking of cure).
Figure 6. PCR analysis of 9 tissues with the T. cruzi SOD gene at the final day of experiment in
ꢀ1
mice untreated and treated with 100 mg·kg body mass. (a) Shows untreated mice group, (b) Shows
the group of mice treated with BZN, (c) Shows the group of mice treated with 7, (d) Shows the
group of mice treated with 9, (e) Shows the group of mice treated with 10. Lanes: M, base pair (bp)
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 22 of 79
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
marker; ꢀ, PCR negative control; +, PCR positive control; 1, PCR adipose tissue; 2, PCR bone
marrow tissue; 3, PCR brain tissue; 4, PCR oesophagus tissue; 5, PCR heart tissue; 6, PCR lung
tissue; 7, PCR muscle tissue; 8, PCR spleen tissue; 9, PCR stomach tissue. *, It means that 2/6 of
■
the corresponding organ PCR products showed 300 bp band on electrophoresis; , It means that 4/6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
■
of the corresponding organ PCR products showed 300 bp band on electrophoresis; no * , it means
that 6/6 or 0/6 of the corresponding organ PCR products showed 300 bp band on electrophoresis.
To assess the immune status of the mice (untreated and treated in different phases) during
infection, IgꢀG levels were determined using the ELISA and FeꢀSOD enzyme (isolated in our
54
laboratory) like antigen . Detection of total IgꢀG reveals the infection rates and verifies the level of
protection (effectiveness) attributed to the tested compounds, in combination with the innate
55
protection of mice, because the titre of immunoglobulins is linked to the parasite load . Figure 7
shows the titre of antiꢀT. cruzi IgꢀG. All treated mice and samples analysed in the acute and chronic
phases showed decreased IgꢀG levels with respect to samples from untreated (control) mice,
excluding those of day 81 pi in the chronic phase, which is rational since those samples were
retrieved from mice only two days after treatment. Mice treated with compounds 7 and 10 showed
higher IgꢀG levels than those that received reference drug BZN in both acute and chronic phases,
suggesting a higher parasitic load and a lower trypanocidal effect, consistent with previous studies.
In contrast, compound 9ꢀtreated mice showed a significant reduction of IgꢀG levels and the lowest
increase in these levels after IS as a consequence of the low parasitaemia and the low reactivation
after IS, mainly due to the good in vivo trypanocidal activity of this compound, as demonstrated by
PCR.
ACS Paragon Plus Environment

Page 23 of 79
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 7. Differences in the IgꢀG levels of antiꢀT. cruzi antibodies, expressed in absorbance units
(optical densities (OD) at 490 nm), between control (untreated) and treated groups of mice at
different days postꢀinfection (pi). IS, immunosuppression.
Another feature that is linked to the parasite load is splenomegaly, since the spleen is an organ
that is implicated in defence against infection. Splenomegaly occurs in experimentally infected
mice in both acute and chronic phases, during which the spleen of chronic mice is frequently
5
3
approximately twice the mass of those from uninfected mice . Importantly, treatment with BZN
reduces infectionꢀinduced splenomegaly, even at subcurative doses and in the absence of
53
parasitological cure, since it is linked to a reduction of the parasite load . Accordingly, Figure 8
shows the weight percentage of spleens from different groups of mice in both acute and chronic
phases. We found that untreated (control) mice showed a weight percentage value that was twice
the value corresponding to uninfected mice: 0.28 % for uninfected mice and 0.56 % and 0.59 % for
untreated (control) mice in the acute and chronic phases, respectively. Moreover, untreated mice
showed a higher weight percentage value in both phases compared with the mice treated with the
tested compounds and BZN. This finding indicated that the tested Mannich baseꢀtype derivatives
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 24 of 79
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
and BZN reduced splenomegaly and, therefore, infection rates. Compound 9ꢀtreated mice showed
lower splenomegaly: these mice showed a reduction of splenomegaly of 52 % for mice treated in
the acute phase and 68 % for mice treated in the chronic phase compared with untreated mice. It
should be mentioned that the higher splenomegaly usually observed in the mice treated in the
chronic phase should not be related to a lower activity in this phase; these mice simply suffered an
acute phase without treatment with high levels of parasitaemia.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 8. Weight percent of spleens of different groups of mice at the final day of experiment.
Values constitute means of six mice ± standard deviation.
To confirm the metabolic abnormalities associated with the treatment, clinical chemistry
measurements were carried out as mentioned in the experimental section. Table 5 shows the
biochemical clinical parameters obtained in both untreated and treated mice in the acute and chronic
phases, where values for uninfected and untreated mice were also included. Most of the biochemical
parameters tested for the three studied compounds were not altered after compound administration,
and almost all those alterations returned to normal levels in the samples obtained on the day of mice
necropsy. Moreover, these tested compounds showed less toxicity than the reference drug BZN.
The lack of toxicity along with the high activity of compound 9 led us to consider this compound as
a promising candidate for the treatment of CD.
ACS Paragon Plus Environment

Page 25 of 79
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Table 5. Biochemical clinical parameters measured at different experimental situations and days postꢀinfection (pi) in groups of BALB/c mice
infected with T. cruzi.
Kidney marker profile
Heart marker profile
Liver marker profile
Group
Alkaline
phosphatase
(U/L)
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
c
d
Uric acid
mg/DL)
AST/GOT
ALT/GPT
Total bilirubin
(mg/DL)
a
b
Urea (mg/DL)
CKꢀMB (U/L)
LDH (U/L)
(
(U/L)
(U/L)
Uninfected mice
4.5 [4.0ꢀ
5.1]
3
5 [32ꢀ40]
372 [150ꢀ630]
3180 [2505ꢀ3851]
153 [132ꢀ177]
55 [46ꢀ62]
0.28 [0.22ꢀ0.31]
169 [141ꢀ192]
(
n = 15)
control
BZN
31
4.3
535
3275
167
60
0.23
180
ꢀꢀꢀ
ꢀꢀꢀꢀ
=
=
++++
++++
++
++
Compound 7
Compound 9
+++
ꢀꢀꢀ
++
ꢀꢀꢀ
=
=
ꢀꢀꢀ
ꢀꢀ
ꢀꢀꢀ
ꢀꢀ
ꢀ
ꢀ
ꢀꢀ
ꢀ
1
6 days pi*
ꢀꢀ
ꢀꢀ
Compound 10
control
ꢀꢀ
34
ꢀꢀꢀ
ꢀꢀ
4.0
=
=
496
=
=
2761
=
++++
179
++++
49
=
0.21
+
=
161
=
Treatment in
acute phase
BZN
++++
=
Compound 7
Compound 9
Compound 10
control
+
=
+
=
=
=
=
=
ꢀ
ꢀ
=
ꢀ
=
=
=
=
Necropsy day
=
=
=
=
+++
260
+++
57
=
=
Treatment in
81 days pi*
45
5.7
751
5951
0.25
167
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 26 of 79
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
chronic phase
BZN
ꢀꢀꢀ
+++
ꢀꢀꢀ
ꢀꢀꢀꢀ
ꢀꢀ
ꢀ
ꢀꢀꢀ
++
ꢀꢀꢀ
ꢀꢀ
++
ꢀꢀꢀ
ꢀꢀꢀ
+++
ꢀ
++++
Compound 7
Compound 9
+
ꢀ
ꢀ
ꢀꢀꢀ
ꢀꢀꢀ
ꢀꢀ
ꢀꢀ
=
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
Compound 10
control
ꢀ
ꢀꢀꢀ
4.8
ꢀꢀ
ꢀꢀ
538
=
=
6679
ꢀꢀ
+++
286
+
+++
64
=
+
37
ꢀꢀꢀ
0.20
++
149
++
BZN
=
Compound 7
Compound 9
Compound 10
+
=
ꢀ
=
=
=
ꢀ
=
=
=
ꢀꢀ
ꢀꢀ
ꢀ
ꢀ
=
=
=
=
=
=
Necropsy day
ꢀꢀꢀ
=
++
+
a
b
c
d
*2 days after treatment. CKꢀMB, creatine kinaseꢀmuscle/brain. LDH, lactate dehydrogenase. AST/GOT, aspartate aminotransferase. ALT/GPT, alanine aminotransferase.
pi, postꢀinfection.
Key: =, variation no larger than 10 %; +, up to 10 % increase over the range; ++, up to 20 % increase over the range; +++, up to 30 % increase over the range; ++++, more
than 40 % increase over the range; −, up to 10 % decrease over the range; − −, up to 20 % decrease over the range; − − −, up to 30 % decrease over the range; − − − −, more
than 40% decrease over the range.
ACS Paragon Plus Environment

Page 27 of 79
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
Metabolite excretion.
T. cruzi catabolizes glucose at a high rate, excreting into the medium a substantial portion of the
56
hexose skeleton as partly oxidized end products , and acidifying the medium in the presence of
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
7, 58
oxygen
. The final products in this catabolism are mainly pyruvate, acetate, succinate, Lꢀalanine,
59
Dꢀlactate and ethanol .
1
Proton nuclear magnetic resonance ( H NMR) spectra of treated and untreated T. cruzi Arequipa
epimastigotes forms were registered to obtain some information about the effects of compounds 7, 9
and 10, at IC25 concentrations, on glucose metabolism in T. cruzi (spectra not shown). The final
excretions were qualitatively and quantitatively analysed and compared with those found for the
control (untreated T. cruzi epimastigotes). Figure 9 shows the results obtained compared with this
control. The most remarkable differences were the altered amount of succinate, with value changes
higher than 20 % for the three tested Mannich baseꢀtype derivatives. It is interesting to note the effect
of compound 9, which showed an increase in succinate excretion of 200 %. Nonꢀsignificant alterations
were observed in the other metabolites. It is wellꢀknown that the increase in the amount of succinate
indicates catabolic changes that could be associated with a mitochondrial malfunction due to the redox
60
stress produced by inhibition of the mitochondrionꢀresident FeꢀSOD enzyme , and not by a direct
effect on the glycolytic pathway. Owing to the feasible mitochondrial dysfunction and that the main
role of succinate is to maintain the glycosomal redox balance via NADH reoxidation produced in the
glycosome catabolic pathway, it is possible that this route can be increased to maintain the balance,
5
7, 61
with the consequent increase in succinate as the final product
. In contrast, compound 7 produces a
reduction in succinate excretion along with a reduction of pyruvate and increase in glycerol. These
results suggest, in addition to alterations at the mitochondrial level, that some of the glyceraldehydeꢀ3ꢀ
phosphate pathwayꢀinvolved enzymes of glucose catabolism (like glyceraldehydeꢀ3ꢀphosphate
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 28 of 79
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
dehydrogenase or phosphoglycerate kinase enzymes) can be inhibited and the carbon skeleton directed
57
towards the glycerol pathway as the final product .
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 9. Percentages of variation among peaks of catabolites excreted by epimastigotes of T. cruzi
Arequipa strain exposed to compounds 7, 9 and 10 at their IC in comparison to a control (untreated)
25
incubated 72 h. Each drug was tested in three separate determinations.
Effects on the mitochondrial membrane potential and DNA replication and RNA synthesis in
T. cruzi.
It is wellꢀknown that mitochondria play an imperative role in cell death decisions. In normal cells,
+
active pumping of H is produced to maintain the electrochemical gradient and the integrity and
function of mitochondria. Disturbances in this potential lead to a decrease in ATP production and a
6
2, 63
reduction in DNA replication and RNA transcription
.
ACS Paragon Plus Environment

Page 29 of 79
Journal of Medicinal Chemistry
1
2
3
4
5
As mentioned above, T. cruzi catabolizes glucose at a high rate through the pentose phosphate
pathway (PPP). Subsequently, these metabolites are directed to the tricarboxylic acid (TCA) cycle in
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
6
4
the mitochondria for ATP synthesis, along with the electronꢀtransport chain . Mitochondrial
+
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
dysfunction can then produce an imbalance in the ATP/ADP and NADH/NAD ratios, in addition to a
cellular accumulation of pyruvate, malate and succinate, causing a blockade of the glycolytic pathway
5
7
.
BZN, for instance, kills T. cruzi through its reduction by type I nitroreductase activity that leads to
65
the formation of highly reactive metabolites : it causes respiratory chain inhibition, and thus we
observed a decrease in mitochondrial membrane potential when treating T. cruzi with BZN.
Therefore, to evaluate whether the alteration observed in glucose metabolism pathway in the
metabolite excretion study was a consequence of a mitochondrial dysfunction (and not a direct action
of the tested compounds at the glycosome or cytoplasmic level), studies were conducted on this
organelle using rhodamine 123 (Rho 123) and acridine orange (AO).
Figure 10 shows the results of the flow cytometry analysis of the mitochondrial membrane
potential. We observed a membrane potential reduction by 35.4 % when T. cruzi was treated with BZN,
which causes respiratory chain inhibition. Cells treated with compounds 7 and 9 and labelled with Rho
1
23 showed a decrease in membrane potential with an approximate value of 26 %, whereas the highest
depolarization of the mitochondrial membrane occurred in compound 10ꢀtreated cells (76.3 %). These
alterations could precede T. cruzi cell death via necrosis in a mitochondrionꢀdependent manner, which
could be the cause of their trypanocidal activities.
ACS Paragon Plus Environment