Rational modification of Mannich base-type derivatives as novel antichagasic compounds: Synthesis, in vitro and in vivo evaluation

Article abstract of DOI:10.1016/j.bmc.2019.07.029

The current chemotherapy against Chagas disease is inadequate and insufficient. A series of ten Mannich base-type derivatives have been synthesized to evaluate their in vitro antichagasic activity. After a preliminary screening, compounds 7 and 9 were sub

Full text of DOI:10.1016/j.bmc.2019.07.029

Accepted Manuscript
Rational modification of Mannich base-type derivatives as novel antichagasic
compounds: synthesis, in vitro and in vivo evaluation
Rocío Paucar, Rubén Martín-Escolano, Elsa Moreno-Viguri, Amaya Azqueta,
Nuria Cirauqui, Clotilde Marín, Manuel Sánchez-Moreno, Silvia Pérez-Silanes
PII:
S0968-0896(19)30711-4
https://doi.org/10.1016/j.bmc.2019.07.029
BMC 15013
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
2 May 2019
10 July 2019
17 July 2019
Please cite this article as: Paucar, R., Martín-Escolano, R., Moreno-Viguri, E., Azqueta, A., Cirauqui, N., Marín,
C., Sánchez-Moreno, M., Pérez-Silanes, S., Rational modification of Mannich base-type derivatives as novel
antichagasic compounds: synthesis, in vitro and in vivo evaluation, Bioorganic & Medicinal Chemistry (2019), doi:
https://doi.org/10.1016/j.bmc.2019.07.029
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Rational modification of Mannich base-type derivatives as novel
antichagasic compounds: synthesis, in vitro and in vivo evaluation
Rocío Paucara, Rubén Martín-Escolanob, Elsa Moreno-Viguria, Amaya Azquetac, Nuria
Cirauquid, Clotilde Marínb, Manuel Sánchez-Morenob, Silvia Pérez-Silanesa*
a Universidad de Navarra, Department of Pharmaceutical Technology and Chemistry, Instituto
de Salud Tropical, Pamplona 31008, Spain.
b
Department of Parasitology. Instituto de Investigación Biosanitaria (ibs.GRANADA),
Hospitales Universitarios De Granada/University of Granada, Granada 18071, Spain.
c Universidad de Navarra, Department of Pharmacology and Toxicology, Pamplona 31008,
Spain.
d Department of Pharmaceutical Sciences, Federal University of Rio de Janeiro, Rio de Janeiro
21949-900, Brazil.
*Corresponding author
Dr. Silvia Pérez-Silanes, E-mail: sperez@unav.es. Universidad de Navarra, Department of
Pharmaceutical Technology and Chemistry, Faculty of Pharmacy, 31008, Pamplona, Spain.
Abstract
The current chemotherapy against Chagas disease is inadequate and insufficient. A series of ten
Mannich base-type derivatives have been synthesized to evaluate their in vitro antichagasic
activity. After a preliminary screening, compounds 7 and 9 were subjected to in vivo assays in
a murine model. Both compounds caused a substantial decrease in parasitemia in the chronic
phase, which was an even better result than that of the reference drug benznidazole. In addition,
compound 9 also showed better antichagasic activity during the acute phase. Moreover,
metabolite excretion, effect on mitochondrial membrane potential and the inhibition of
superoxide dismutase (SOD) studies were also performed to identify their possible mechanism
of action. Finally, docking studies proposed a binding mode of the Fe-SOD enzyme similar to
our previous series, which validated our design strategy. Therefore, the results suggest that these
compounds should be considered for further preclinical evaluation as antichagasic agents.
Keywords: Chagas disease, Mannich bases, Superoxide dismutase, Trypanosoma cruzi.

1. Introduction
Chagas disease is a neglected and potentially lethal tropical parasitic disease that continues to
be a serious public health problem worldwide.1 According to the WHO, approximately 6 to 7
million people are infected worldwide and causing more than 7500 deaths annually2-3; 70
million people are at risk of infection,1 and there is an estimated global cost of US$ 7-9 billion
per year.2, 4 Trypanosoma cruzi (T. cruzi) is the causative agent of this chronic parasitic illness,
and its infection could develop into irreversible cardiac or digestive system damage or both in
up to 30% of cases.5-7 Although different strategies have been performed to combat and
eradicate this disease in the last 25 years, there are still some gaps that need to be resolved.8-12
Benznidazole (BZN) and nifurtimox (NFX) were discovered more than 45 years ago and are
the only drugs prescribed to treat Chagas disease (CD).13-14 Additionally, BZN is considered as
the current drug of choice because it has a better safety profile and efficacy. In the acute phase
and during congenital transmission, both drugs produce a parasitological cure if they are
administered soon after infection. However, in the chronic phase and disease reactivation, their
efficiencies are variable. Furthermore, treatment with these drugs has important drawbacks,
including lengthy treatment, severely toxic side effects and different susceptibilities against T.
cruzi strains that can lead to treatment failure.7, 15-18 In the last decade, different research groups
have been identified potential agents to treat this parasitosis.19-25 Interestingly, posaconazole
and E1224 (ravuconazole prodrug) triggered a great expectation about their antiparasitic
profile, however, clinical trials results confirmed the superiority of BZN over these
candidates.26-30 Therefore, further research is needed for the discovery of more effective and
safer antichagasic agents.
Superoxide dismutase (SOD) metalloenzymes play crucial roles in antioxidant defense in
aerobic organisms.31 According to their metal selectivity, the following four isoforms have been
identified: iron-SOD (Fe-SOD), copper/zinc-SOD (Cu/Zn-SOD), manganese-SOD (Mn-SOD)
and nickel-SOD (Ni-SOD). An Fe-SOD isoform is found in trypanosomatids, some protozoan
parasites and plants.32-33 A Cu/Zn-SOD isoform is found in humans and mammalian hosts.34
Due to the biochemical and structural differences that Fe-SOD presents with respect to its
human ortholog Cu/Zn-SOD, it is considered a potential therapeutic target for the treatment of
CD.34
An attractive scaffold for new antiparasitic agents are Mannich base derivatives because of their
potential biological activity against different parasites responsible for tropical diseases such as

trypanosomiasis, leishmaniasis, and malaria.34-39 Hence, our research group has investigated a
variety of Mannich base molecules, finding promising trypanocidal candidates for further
preclinical studies.40-41 Moreover, we confirmed the Fe-SOD enzyme as the main target for
these derivatives which showed high selectivity compared to the human Cu/Zn-SOD ortholog.
By using molecular docking, we were able to propose a binding mode for the target, suggesting
that the carbonyl group, piperazine and its linked phenyl ring would be the most important
scaffolds for binding this enzyme (Figure 1).40 Nonetheless, our results suggest that the
presence of small para-substituents with either donor or acceptor hydrogen bond properties on
the aromatic ring next to the carbonyl group may improve binding via interactions with Gln72.
Finally, the literature reveals that Mannich bases containing amines in their structure, such as
phenethylamines42 or anilines,43 confer anti-parasitic and anti-bacterial activity, respectively.
(a)
(b)
Figure 1. Lead compound (I) from Martin-Escolano et al40, (a) Chemical synthesis and (b) docking
results showing the binding mode of the compound to Fe-SOD enzyme (PDB ID 4DVH). Oxygen (red),
nitrogen (blue), and chlorine (green).
Based on these premises, ten new derivatives were designed, starting from our previous lead
compound (compound I in Figure 1), and taking into account the following strategy.
Derivatives 1-6 were prepared by conserving the para-methoxy substituent in the aromatic ring
linked to the carbonyl group (Scheme 1) but substituting the piperazine by different amines
(phenethylamine or aniline). Furthermore, different substituents were attempted in this second
aromatic group to investigate their effects on pharmacological activity. The other group of
derivatives (7-10) were synthetized by replacing the methoxyl group in lead compound I to a
hydroxyl group and retaining the piperazine linked to the aromatic ring (Scheme 1), substituted
in the para position by F, Cl, CF3 or NO2. This design, as well as the previously designed

derivatives, takes into consideration the priority of using simple and cost-effective synthetic
routes to obtain new compounds to combat this parasitic illness.
Herein, we report the synthesis and biological evaluation of ten Mannich base derivatives,
which represent an interesting therapeutic alternative against this parasitosis. Additionally, the
study of the possible mechanism of action was carried out for the most potent compounds.
2. Results and discussion
2.1. Chemistry
The general reaction pathways towards Mannich base derivatives are outlined in Scheme 1.
Vinyl ketone i1 was prepared by the -methylenation of 4'-methoxyacetophenone using an
ammonium catalyst in THF as described by Bugarin et al.44 Compounds 1-6 were synthesized
via the Michael addition reaction, in which vinyl ketone (i1) will undergo a Michael addition
with the corresponding aryl amine. Compounds 7-10 were synthesized by the Mannich reaction
based on our previous experience41 and published literature,45-46 where 4'-hydroxyacetophenone
was condensed with the corresponding arylamine in acidic medium using 1,3-dioxolane instead
of paraformaldehyde and as a solvent. All new derivatives were fully characterized by IR
spectroscopy and NMR (1H and 13C) as well as elemental microanalysis (C, H, N).
Scheme 1. General procedure for the synthesis of compounds 1-10. Reagents and conditions: (i)
paraformaldehyde, diisopropylammonium trifluoroacetate, trifluoroacetic acid, THF, reflux, 8 h, (ii)
CH2Cl2, rt, from 24 to 48 h; (iii) 1,3-dioxolane, HCl, reflux, 20 to 40 min.
2.2. In vitro assessment
Considering the genetic diversity of T. cruzi strains and their variable sensitivities to clinical
drugs,47-48 compounds 1-10 and BZN, as the reference drug, were evaluated against three
different strains of T. cruzi (Arequipa, SN3 and Tulahuen strain) belonging to different discrete

type units (DTUs) mainly associated with this human parasitosis. Additionally, the effect on
the growth and viability of host cells for T. cruzi was assessed using mammalian Vero cells.
The preliminary screening started with the in vitro evaluation of all compounds and BZN facing
up to the extracellular epimastigote forms. Additionally, mammalian cell cytotoxicity assays
were performed as described in the Experimental section. The results of these assays are
expressed as the concentration required to inhibit 50% of the parasites or mammalian cells
growth (IC50). Likewise, the selectivity index (SI) was calculated as follows: SI = IC50 Vero
cells/IC50 epimastigote forms. The results are summarized in Table 1. Compounds with an SI
higher than 5 in at least two T. cruzi strains progressed to the in vitro screening in amastigote
and trypomastigote forms of the parasite.
Table 1. In vitro activity of the studied compounds (1-10) in epimastigote forms.
Activity IC50 (µM)a
Toxicity IC50
Selectivity Indexc
Comp
Vero Cell
(µM)b
Arequipa
SN3
Tulahuen
Arequipa
SN3
Tulahuen
26.2 ± 7.3
17.8 ± 7.0
14.8 ± 2.6
15.3 ± 1.9
76.1 ± 20.3
25.4 ± 8.2
25.3 ± 0.9
24.6 ± 13.1
14.4 ± 7.7
16.4 ± 5.9
13.2 ± 2.6
20.3 ± 5.5
15.2 ± 2.7
14.8 ± 7.9
13.0 ± 1.6
10.6 ± 0.9
43.0 ± 8.1
7.6
1.8
9.0
7.8
1.5
1.9
1.0
4.7
2.1
5.6
1.4
13.1
0.4
5.2
3.8
6.9
3.9
3.7
8.5
5.0
8.6
2.7
7.9
1.3
9.3
8.9
6.9
3.3
3.5
7.2
3.9
9.8
0.8
BZN
200
32.6 ± 7.0
133.4 ± 6.0
145.7 ± 22.4
91.1 ± 2.5
66.2 ± 4.6
53.5 ± 4.4
106.9 ± 11.0
50.0 ± 5.3
103.7 ± 10.6
34.6 ± 11.2
1
2
3
18.7 ± 14.4 38.4 ± 12.3
59.7 ± 22.1
35.6 ± 6.1
51.6 ± 8.9
22.7 ± 1.4
24.4 ± 9.5
18.6 ± 5.9
24.8 ± 3.5
13.2 ± 0.9
16.9 ± 1.0
14.6 ± 4.2
12.6 ± 2.2
10.1 ± 0.6
12.1 ± 0.9
13.0 ± 1.1
4
5
6
7
8
9
10
IC50: Compound concentration that reduces the number of parasites or cells growth by 50%, calculated
through Graphpad prism 7 software. Results are presented as the mean and the standard deviation (SD),

correspond to triplicates from three independent experiments. Compounds screened against T. cruzi
epimastigotes had r2 values  0.7. (a) IC50 values against T. cruzi epimastigote forms. (b) IC50 Vero cells
cytotoxicity. (c) Selectivity index (SI) corresponds to the ratio of IC50 values of compounds against Vero
cells relative to those against T. cruzi epimastigote forms.
Therefore, compounds 2-4, 7 and 9 were selected to determine the IC50 values and their
respective SIs in the amastigote and trypomastigote forms (Table 2 and Table 3). The criteria
established for this screening as the cut-off of this step were the same as those according to the
literature49-51: IC50 values approximately 10 µM and SI  50 in at least two strains and one
parasitic form. Compounds 7 and 9 met these criteria, with IC50 values ranging from 0.7 - 6.2
µM in both amastigote and trypomastigote forms and SI  50 in the trypomastigote forms of
two T. cruzi strains. It is interesting to note that the lead compounds 7 and 9 contain an aryl
piperazine and a hydroxyl group in the para position of the aromatic ring linked to the ketone
group.
Therefore, more exhaustive assessments of their trypanocidal activity and genotoxic capacity
were performed.
Table 2. In vitro activity of the compounds 2-4, 7 and 9 in both amastigote and trypomastigote forms of
T. cruzi.
Activity IC50 (µM)
T. cruzi Arequipa strain
T. cruzi SN3 strain
T. cruzi Tulahuen strain
Comp.
Amastigote Trypomastigote Amastigote Trypomastigote Amastigote Trypomastigote
forms
forms
forms
forms
forms
forms
3.0 ± 0.4
6.2 ± 1.7
4.4 ± 0.2
33.3 ± 1.6
3.9 ± 0.2
6.7 ± 1.0
BZN
2
3
4
7
9
4.7 ± 1.0
7.3 ± 1.3
9.1 ± 0.8
6.2 ± 0.3
5.1 ± 0.5
2.1 ± 0.2
13.0 ± 3.5
3.6 ± 1.1
0.9 ± 0.1
0.7 ± 0.1
5.4 ± 1.6
6.0 ± 0.6
4.5 ± 0.4
6.6 ± 0.6
4.5 ± 0.3
3.4 ± 0.8
63.0 ± 12.3
5.5 ± 2.1
2.1 ± 0.4
2.0 ± 0.4
6.7 ± 0.1
26.3 ± 0.7
3.3 ± 0.1
3.9 ± 0.4
3.0 ± 0.3
2.7 ± 0.4
14.2 ± 1.7
3.5 ± 0.7
2.3 ± 0.3
2.1 ± 0.2

In vitro activity against amastigote and trypomastigote forms, expressed as IC50. Data presented as mean
 standard deviation (SD) of triplicates and at least two independent assays. Compounds screened
against T. cruzi trypomastigotes and amastigotes had r2 values  0.7.
Table 3. Selectivity index of the compounds 2-4, 7 and 9 in both amastigote and trypomastigote forms
of T. cruzi.
Selectivity Index
T. cruzi Arequipa strain
T. cruzi SN3 strain
T. cruzi Tulahuen strain
Comp
Amastigote Trypomastigote Amastigote Trypomastigote Amastigote Trypomastigote
forms
forms
forms
forms
forms
forms
32.5
45.6
6.0
51.7
29.9
BZN
67.2
27.6
19.9
10.0
17.4
20.4
64.8
11.2
24.7
24.1
20.2
16.2
23.2
39.4
2.3
20.1
5.5
48.8
10.2
26.1
45.7
49.6
2
3
4
7
9
25.0
16.7
51.0
50.9
27.7
27.4
34.5
123.1
140.9
Selectivity index (SI = IC50 Vero cells/IC50 of T. cruzi forms).*Lead compound (I) from Martin-
Escolano et al.40
Before the in vivo assessment, to obtain more information about the antichagasic activity of
compounds 2-4, 7, and 9 as well as BZN, the infection rates in Vero cells were evaluated by
counting the infected cells after 72 h of exposure to different concentrations (from 3.1 to 50 µM
for compound 2-4 and 7, and from 1.6 to 25 µM for compound 9). The results are presented in
Figure 2, together with the data used to determine the IC50 of the compounds against the
amastigote and trypomastigote forms.
In general, a gradual decrease in the ratio of infected cells was observed, while the concentration
of the tested compounds increased. Thus, the compounds reduced the ratio of infected cells to
18% (BZN), 10% (compound 2), 28% (compound 3), 17% (compound 4) and 7% (compound
7), with respect to the ratio of infected cells at 50 μM. Compound 9 decreased the rate of
infected cells to 19% and BZN to 18% at 25 μM. In addition, it is remarkable that compounds
7 and 9 showed a similar or higher infection rate at these concentrations compared to the

reference drug. Additionally, it is important to note that compounds 2, 7 and 9 showed the best
IC50 values in trypomastigotes, amastigotes and infected Vero cells (Figure 2).
Finally, considering the results of activity and selectivity in different strains, as well as the
different forms and ratios of infected Vero cells, compounds 7 and 9 were selected as good
candidates for the in vivo study. Prior to the in vivo evaluation, the lead compounds were
evaluated to determine their genotoxic capacity.
Figure 2. Effect of the compounds in the propagation of the amastigotes and trypomastigotes and
infected Vero cells. The graphs are obtained from the mean and standard deviation of at least two
independent assays. IC50 values (in parenthesis) that were calculated using Graphpad prism 7 software.
2.3. In vitro genotoxicity screening. SOS/UMU-test

This assay was conducted to investigate the potential genotoxicity of compounds 7 and 9. The
results displayed that none of our compounds were genotoxic either in the presence or absence
of metabolic activation (see S2 section of the supporting information), whereas BZN induced
genotoxicity under the same conditions.41 Therefore, the results led us to examine these
compounds in in vivo studies in BALB/c mice infected with the parasite.
2.4. In vivo anti-T. cruzi activity in BALB/c female mice
Compounds 7 and 9 were selected for in vivo assays based on encouraging results of their
trypanocidal activity in vitro, SI and non-genotoxicity in the preliminary SOS/UMU tests. The
currently used chemotherapy has variable activity in the acute and chronic phase of this
disease52; therefore, test compounds were evaluated in both phases according to the experiments
established in Scheme 2. First, BALB/c mice were infected intraperitoneally according to the
literature.53 Compounds 7 or 9 or BZN were administered orally, which is the route of
administration ideally recommended to treat this parasitosis according to the target product
profile (TPP) established by the Drugs for Neglected Diseases Initiative (DNDi).54 The mice
were treated with the compounds from day 8 post-infection (pi), at 20 mg/kg/day for 5
consecutive days. This dosage represents a subcurative dose of BZN; therefore, this experiment
demonstrates whether the studied compounds are more effective or not than the reference drug
during the progress of the disease.
Parasitemia levels of different study groups during the acute phase were determined by counting
bloodstream trypomastigotes as described in the experimental section. Nonsignificant
differences were evidenced between the untreated mice and the mice treated with compound 7.
Compound 9 showed an antiparasitic activity similar to BZN (Figure 3). Compound 9 showed
lower parasitemia levels from the beginning of the treatment, and this behavior was maintained
until the end of the phase (48 days pi), noting that blood parasitemia levels were reduced to
undetectable levels four days before the reference drug (BZN) levels. Furthermore, it is
noteworthy that, during this phase, compound 9 decreased the parasitemia peak by
approximately 70% compared to the untreated control.

Figure 3. Parasitemia levels during acute phase of Chagas disease in BALB/c mice non-treated (control)
and treated with compound 7, 9 and BZN. All compounds were orally administered with 20 mg/kg/day
for 5 consecutive days. Days of treatment are represented in gray. Values are the means of results for
three mice  standard deviation.
On day 80 pi, the mice were immunosuppressed with cyclophosphamide to evaluate the
effectiveness of the treatment; this procedure expands the residual foci of infection to detectable
levels (if after being treated there was still presence of parasites in the vertebrate host).55-56 In
addition, this reactivation of parasitemia in the chronic phase has great relevance since it can
occur in immunocompromised patients, where the efficacy of current chemotherapy is
unknown, although it is presumed to be limited.16, 57
Figure 4 shows the reactivation percentages of infection compared to those of the untreated
(control) group after the mice were immunosuppressed with cyclophosphamide. As observed,
mice treated with compounds 7 or 9 or BZN showed a decrease in parasitemia in both phases.
The reactivation obtained in the acute phase could be proportional to the survival rate observed
in the parasitemia curve (mentioned above). It is also important to note that all mice treated
with the compounds in the chronic phase showed a lower percentage of reactivation than treated
mice in the acute phase. It is noteworthy to mention that mice treated with compound 9 showed
a reactivation of approximately 25% and 20% in the acute and chronic phases, respectively, and
this compound was more effective than the reference drug (approximately 65% and 40% in the

acute and chronic phase, respectively). Although there was a higher percentage of reactivation
(approximately 85%) in the groups treated with compound 7 in the acute phase, this value
decreased to 30% in the chronic phase. These results lead us to hypothesize that treatment with
compound 9 could be effective for both the acute and chronic phases of CD. This effectiveness
was confirmed later with the results obtained from tissue PCR.
100
Control
90
BZN
80
7
70
9
60
50
40
30
20
10
0
Acute phase
Chronic phase
Figure 4. Immunosuppression experiment in the in vivo assay for BALB/c mice non-treated and treated
with compound 7, 9 and BZN. The figure shows the reactivation parasitemia (%), after cycles
administration of the immunosuppressant cyclophosphamide. Values are the means of results for three
mice  standard deviation.
In accordance with previous studies, tissue PCR in animals has better sensitivity because it can
detect T. cruzi DNA in mice that are considered cured, as evaluated by other blood
methodologies.58 Therefore, in addition to evaluating the effectiveness of the compounds after
immunosuppression, the presence of parasites in target organs was evaluated through PCR after
necropsy (100 day). Figure 5 shows the PCR results for the target organs in all studied groups
in both the acute and chronic phases. Untreated mice (control group) in both phases showed the
presence of parasites in all target organs/tissues: adipose, bone marrow, brain, esophageal,
heart, muscle, spleen and stomach tissues. Mice treated with BZN demonstrated 25% and 38%
of organs and tissues free of parasites in the acute and chronic phases, respectively. A low
percentage was observed in the mice treated with compound 7 in the acute phase (13%),
although 50% was obtained in the chronic phase of this compound. Notably and in accordance
with the abovementioned results, compound 9 showed a better antiparasitic activity, even better

than that of the reference drug, with 50% (acute phase) and 63% (chronic phase) of organs and
tissues free of parasites, respectively.
Figure 5. PCR analysis of 8 tissues with the T. cruzi splice leader (SL) gene at the final day of
experiment in mice untreated and treated with 20 mg/kg/day for 5 consecutive days. Lanes: M, base pair
(bp) 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 muscle
tissue; 7, PCR spleen tissue; 8, PCR stomach tissue. *, It means that 1/3 of the corresponding organ PCR
■
products showed 300 bp band on electrophoresis; , It means that 2/3 of the corresponding organ PCR
products showed 300 bp band on electrophoresis; no * nor ■, it means that 3/3 or 0/3 of the corresponding
organ PCR products showed 300 bp band on electrophoresis.
However, the immune response to T. cruzi infection was evaluated by determining the levels of
immunoglobulin G (IgG) via ELISA using Fe-SOD isolated in the laboratory59 as the antigen.60
The titer of IgG is associated with the infection ratio (parasitic load), which would allow us to
evaluate the effectiveness of the studied compounds in combination with the innate protection
of mice.40, 61 Figure 6 shows the titer of anti-T. cruzi IgG in the different groups and the
respective controls. As observed, all treated mice, both acute and chronic, had lower levels of
IgG than untreated mice (control). Mice treated with compounds 7 or 9 had mostly similar IgG
values to those treated with BZN; however, during the acute phase, the efficacy of BZN was

better than that of both compounds. These results allow us to confirm the antichagasic activity
of both compounds (7 and 9), especially their effectiveness in the chronic phase.
1.0
Control
BZN
7
9
0.8
0.6
0.4
0.2
0.0
14th dpi (2 days 79th dpi (before Necropsy day 76st dpi (2 days Necropsy day
after treatment) IS) (after IS) after treatment) (after IS)
Treatment in acute phase
Treatment in chronic phase
Figure 6. Differences in the Ig G levels of anti-T. cruzi antibodies, expressed in absorbance units, optical
densities (OD) at 490 nm, between non-treated (control) and treated groups of mice. Values constitute
means of three mice ± standard deviation.
Furthermore, splenomegaly is manifested in both phases of the infection induced by T. cruzi62;
therefore, this phenomenon indicates the link between the parasitic load and the enlargement of
the spleen.40 Figure 7 shows the weight percentage of the spleens in the different groups
studied, including the nontreated and noninfected groups. The nontreated mice demonstrated
an increase in the weight of the spleen of more than twice the weights of noninfected mice,
ranging from 0.28% to 0.47% of the total weight of the mice. In addition, all treated mice, even
at subcurative doses of BZN, demonstrated a smaller increase in the weight of the spleen
compared to untreated mice, either in the acute or chronic phases, highlighting a decrease in the
splenomegaly by compound 9 of 44% and 81% in the acute and chronic phases, respectively,
and by compound 7 of 66% in the chronic phase compared to the reference drug. Therefore,
these results were consistent with the results described above, where the tested compounds
reduced the infection rates, emphasizing the antichagasic activity of compound 9.

1.0
0.8
0.6
0.4
0.2
0.0
Uninfected
Control
BZN
7
9
Acute phase
Chronic phase
Figure 7. Weight percentage of spleens (%) of different groups of mice at the end of experiment. Values
constitute means of three mice ± standard deviation.

Table 4. Biochemical clinical parameters of groups of Balb/c mice infected with T. cruzi.
Kidney marker profile
Heart marker profile
Liver marker profile
Uric acid
(mg·DL-1)
Alkaline
phosphatase
a]
b]
c]
d]
CK-MB[
(U·L-1)
LDH[
AST/GOT[
ALT/GPT[
Total bilirubin
(mg·DL-1)
Urea (mg·DL-1)
(U·L-1)
(U·L-1)
(U·L-1)
(U·L-1)
372 [150–
630]
3180 [2505–
3851]
153 [132–
177]
0.28 [0.22–
0.31]
Uninfected mice (n = 6)
14th dpi (Control) (n = 3)
14th dpi and BZN (2 days after treatment) (n =
35 [32–40]
4.5 [4.0–5.1]
55 [46–62]
169 [141–192]
37
4.0
365
3285
177
45
0.31
158
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-
--
33
---
----
---
-
----
----
----
528
=
-
---
---
3809
+
++
=
-
156
+
++++
++++
++++
64
-
=
----
--
176
=
3)
14th dpi and 7 (2 days after treatment) (n = 3)
++++
Treatment
in acute
phase
14th dpi and 9 (2 days after treatment) (n = 3)
Necropsy day of mice (Control) (n = 3)
Necropsy day of mice and BZN (n = 3)
+
0.30
=
4.4
--
=
Necropsy day of mice and 7 (n = 3)
=
----
----
=
=
=
++++
----
Necropsy day of mice and 9 (n = 3)
--
35
=
4.6
---
505
=
3166
=
188
++
58
=
0.25
-
76th dpi (Control) (n = 3)
76th dpi and BZN (2 days after treatment) (n =
164
---
=
----
33
----
---
=
----
----
----
528
-
--
---
-
+
--
--
156
=
++++
++++
+
64
=
-
=
--
=
176
-
3)
76th dpi and 7 (2 days after treatment) (n = 3)
++++
Treatment
in chronic
phase
76th dpi and 9 (2 days after treatment) (n = 3)
Necropsy day of mice (Control) (n = 3)
Necropsy day of mice and BZN (n = 3)
-
0.30
-
4.4
3809
----
----
--
Necropsy day of mice and 7 (n = 3)
Necropsy day of mice and 9 (n = 3)
=
-
--
=
----
---
----
=
=
--
=
=
++
--
----
=
[a] CK-MB, creatine kinase-muscle/brain. [b] LDH, lactate dehydrogenase. [c] AST/GOT, aspartate aminotransferase. [d] ALT/GPT, alanine aminotransferase.
dpi = day post-infection.
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.

Finally, biochemical parameters were tested to evaluate the possible metabolic abnormalities
related to treatment. According to the results summarized in Table 4, biochemical parameters
in groups treated with compounds 7 or 9 were less altered than those in the group treated with
the reference drug. However, the biochemical results obtained from necropsy showed that most
of these alterations returned to normal levels, with approximately 50% of the values obtained
in both phases (acute and chronic). With the encouraging results obtained in the in vivo
experiment, we could consider compound 9 as a potential candidate to treat both phases of the
illness. In addition, these results allow us to consider compound 7 for further studies of
combined treatment due to its antiparasitic activity in the chronic phase. In addition, considering
the activity and cytotoxicity values, although compound 9 is more cytotoxic than compound I,
the IC50 values against different T. cruzi strains and forms are similar.
2.5. Studies of the mechanism of action
2.5.1. Metabolite excretion
It is known that, for energy generation, T. cruzi requires a high rate of glucose consumption.
This parasite, which is produced and excreted into medium, reduces catabolites such as
pyruvate, acetate, and succinate, which are products of glucose catabolism, even under aerobic
conditions, instead of completely degrading glucose to CO2 and water.63-66 1H-NMR spectra
show the presence of these catabolites in fermentative medium.65 Therefore, epimastigote forms
of the T. cruzi Arequipa strain, untreated or treated with compounds 7 or 9 or BZN, were
analyzed by this technique to compare the effect of these compounds on glucose catabolism at
IC50 concentrations. Figure 8 shows the percentage variation among peaks of excreted
catabolites in treated parasites compared with that in nontreated parasites. As observed, the
excretion of all metabolites was altered in the treatment groups, with succinate being the most
altered metabolite, with values from more than 30% of variation with respect to the control
group. Compound 7 caused a remarkable decrease in succinate excretion ( 80%). To avoid an
energy deficit, other glycolytic routes could have been activated, producing a greater amount
of pyruvate to obtain acetate, D-lactate, ethanol and L-alanine as final products. Additionally,
the glycerol pathway could be activated.66 Thus, an increase in these metabolites was detected
in the medium by 1H-NMR. In contrast, in 9-treated parasites, a remarkable increase in excreted
succinate and pyruvate was found of 34% and 36%, respectively. A significant variation in
excreted succinate was also found in BZN. According to previous studies, an increase in
excreted succinate may be related to mitochondrial dysfunction.66-67 For this reason,
mitochondrial assays were performed.

BZN
Glycerol
Succinate
Pyruvate
Acetate
7
9
L-Alanine
D-Lactate
Ethanol
-100% -80% -60% -40% -20% 0% 20% 40% 60% 80%
Figure 8. Variation (%) among peaks of catabolites excreted by epimastigote forms of T. cruzi Arequipa
strain exposed to compound 7, 9 and BZN at IC50 concentrations in comparison to untreated parasites
incubated 72 h.
2.5.2. Mitochondrial membrane potential
Mitochondria play important roles in cell sustenance preservation. One of tasks of mitochondria
is the maintenance of an electrochemical gradient through the inner membrane. Disturbances
in membrane potential could generate a decrease in ATP levels as well as less DNA replication
and RNA transcription, leading to cell apoptosis and/or necrosis.68-69
Therefore, to continue with the metabolite excretion studies, where a possible mitochondrial
malfunction was found, probably due to an alteration in the glycolytic pathway, rhodamine 123
(rho 123) and acridine orange (AO) staining were performed to evaluate the function and
integrity of this organelle by flow cytometry.40 Figure 9 summarizes the percentage variation
obtained after the evaluation of the membrane potential. As shown, there was a reduction in the
mitochondrial membrane potential in parasites treated with BZN (15.2%), which can be
attributed to its mechanism of action.70 Compound 7-treated parasites reached a mitochondrial
membrane depolarization of 54.4%, while parasites treated with compound 9 reached the
highest depolarization (67.6%). Therefore, these results suggest that the trypanocidal activity
of compounds 7 and 9 may be related to an effect at the mitochondrial level.

Figure 9. Cytometry analysis of the mitochondrial membrane potential from epimastigote forms of T.
cruzi Arequipa strain exposed to BZN and compounds 7 and 9 at their IC50 in comparison to non-treated
parasites incubated 72 h. Percentage of variation in mitochondrial membrane potential in parenthesis.
2.5.3. DNA and RNA levels
The alteration of the mitochondrial membrane potential induces a decrease in ATP levels and
generates an imbalance in the NADH/NAD+ ratio,66 affecting DNA replication and RNA
transcription in parasitic genes. Therefore, DNA and RNA levels were quantified by flow
cytometry, employing AO as the fluorescent dye. The percentage change results are shown in

Figure 10. Nonsignificant differences were observed among the parasites treated with BZN, 7
or 9 and the nontreated parasites.
Figure 10. Flow cytometry analysis of the inhibition in DNA and RNA levels of epimastigote forms of
T. cruzi Arequipa strain exposed to compound 7, 9 and BZN at their IC50 in comparison to control
parasites incubated during 72 h. Percentage change at DNA and RNA levels in parenthesis.
2.5.4. T. cruzi Fe-SOD enzyme
Previous studies from our research group showed the selective inhibitory capacity of these
Mannich base derivatives on Fe-SOD and their possible binding mode for this enzyme by
computational studies.40-41 Therefore, inhibition studies of compounds 7 and 9 were carried out
on Fe-SOD and Cu/Zn-SOD enzymes to determine their selective potential inhibition against
the enzyme present in the parasite.
Figure 11 shows the inhibition curve of compounds 7 and 9, with the corresponding IC50 of
Fe-SOD from epimastigote forms of the Arequipa T. cruzi strain and its human homologue
Cu/Zn-SOD from erythrocytes (Sigma-Aldrich). As observed in Figure 11, compounds 7 and
9 showed a significant inhibitory capacity for the Fe-SOD enzyme, whereas an inhibitory effect
by the human enzyme was not detected up to a 50 µM concentration. Both tested compounds
(7 and 9) reached more than 50% inhibition of Fe-SOD at a 6.3 μM concentration with IC50

values of 4.3 and 3.9 μM, respectively. It is noteworthy to mention that compound 7 reached
total inhibition at a concentration of 25 μM. Hence, a remarkable selective inhibition of
compounds 7 and 9 against Fe-SOD allows us to propose this enzyme as a possible target of
action of these agents. Moreover, the significant variation in metabolites or these compounds
(Figure 8) may be due to the inhibition of the Fe-SOD enzyme, generating oxidative stress and
final damage of the parasite. However, we should not rule out other secondary routes of action
for our compounds.
1 0 0
8 0
6 0
F e-S O D -7 (4 .3 M )
4 0
F e-S O D -9 (3 .9 M )
2 0
0
C uZ n-S O D (> 50 M )
-1
0
1
2
L o g C ( M )
Figure 11. In vitro inhibition of Fe-SOD (activity 32.3 ± 6.7 U/mg) and CuZn-SOD (activity 781.5 ±
3.0 U/mg) for compounds 7 and 9. Values are the mean of three independent determinations ± standard
deviation. In brackets: IC50 value that were calculated using Graphpad prism 7 software.
2.6. Mutagenicity assay
2.6.1. Ames test
Considering the promising results obtained from the biological assays of the compound 9,
prompted us to evaluate its mutagenic capacity. Though the compound was negative in the
SOS/UMU test, it is known that some compounds can induce mutations without activating the
SOS pathway.71 In order be sure about the safety of compound 9, a miniaturized version of the
Ames test, using 6 well plates, was performed following the principles of the OECD guideline
for the Ames test.72 The highest concentration tested, 600 µg/well, was determined in a previous
solubility study. Results showed that compound 9 was not mutagenic in the presence or the
absence of external metabolic activation and in any of the strains used (Table 5). The compound
9 was toxic in some of the conditions tested. At this stage, when comparing the toxicological
results obtained from compound 9 with the published reports about BZN and NFX,73-75 in which
positive results in the Ames test were reported, compound 9 seems to have a better toxicological

profile. Therefore, the results led us to consider the compound 9 as an interesting candidate for
further preclinical studies.
Table 5. Results of the Ames test
Mean number of relevant colonies/well (Mean  SD of 3 wells)
TA97a
-S9
TA98
TA 100
-S9 S9
35.7  47.3  78.7 
TA 102
TA1535
-S9 S9
13.3  5.0 
S9
-S9
S9
-S9
S9
37 
55 
3.0 
72.0 
Negative control
4.0  2.0
6.5
7.1
2.6
0.6
5.7
7.8
1.0
2.5
3.6
Test dose
(µg/plate)
32.3
34.4  44.3  4.0 
5.0 6.8 1.7
44.7  72.0 
2.3 5.0
75.0 
2.3 
5.5 
7.4
6.0  1.0
4.0  1.7
6.3  0.6
1.5
5.0
0.6
0.7
35.7  38.3  4.7 
1.2 4.5 0.6
34.3  47.3  69.7 
5.8 5.1 2.5
84.7 
4.0 
3.0 
22.2
66.7
7.6
2.0
1.0
31.3  44.0  4.0 
38.0  54.7  73.3 
72.3 
3.7 
3.3 
5.7
7.0
0.0
toxa
toxa
3.6
5.9
4.0
toxa
toxa
0.6
2.3
0.6
32.3 +
8.3
41 
42.3 
78.0 
4.0 
7.0 
200.0
600.0
tox
2.7  1.2
2.6
6.7
12.3
1.7
2.0
toxa
toxa
toxa
toxa
toxa
toxa
toxa
toxa
196.0 242.0 238.7
261.3
235.3
289.3 
225  319 
15.0 12.2
133.7 
96.0 
Positive Control
 7.2  35.2  18.0
 40.9
 31.3
25.7
1.2
7.2
a Tox = Toxicity
Before to docking studies, it is interesting to note that, throughout the in vitro and in vivo
evaluation, compound 9 has shown an interesting antiparasitic profile and although the
cytotoxicity values are higher than BZN and lead compound I, it has an encouraging safety
profile.
2.7. Docking study of the new compounds on the T. cruzi Fe-SOD enzyme
To study the binding mode of the new compounds and to observe if it agrees with the hypotheses

used for their rational design, we performed docking studies for all ten derivatives on the Fe-
SOD enzyme. Our results suggest a binding mode similar to that of our previous series (Figure
1b).40-41 The full docking results can be found as supplementary information (Supporting
information Figure S3.1). In Figure 12, we show the proposed binding mode for compounds
7 and 9, where we can observe possible hydrogen bonds between: i) the compound carbonyl
group and both His32 and Tyr36, and ii) the new hydroxyl group proposed in our design
strategy and Gln72. Therefore, the inclusion of hydrogen bond donors substituted in para in the
aromatic ring next to the carbonyl group may actually increase the affinity of compounds by
allowing interactions with Gln72. Also, electrostatic interactions are observed between the
amino acid Lys39 (and for some compounds also Asn121), and the substituent in para in the
other aromatic ring (the one bound to the piperazine scaffold). Similar interactions are observed
for compounds 7 to 10. For compounds 1-6, our results suggest that the substitution of a
piperazine by phenethylamines or anilines may modify the binding mode of the compounds on
the dimer interface. Those compounds are expected to bind in an inversed conformation, where
the aromatic scaffolds will still bind between Phe123 and Tyr36, and the carbonyl group could
perform hydrogen bonds with Lys39 (Supporting information Figure S3.1). This agrees with
the good activity observed for them (Table 1).
Figure 12. Lowest scored binding pose obtained for compounds 7 (A) and 9 (B) by molecular docking
on the Fe-SOD enzyme (PDB ID 4DVH),76 showing the compounds bound at the dimer interface. The
figures were created with the software chimera.77 Non-polar hydrogen atoms are not displayed. The
following color scheme was used: protein chain A in yellow and chain B in green, compound 7 in cyan
and compound 9 in pale pink.
3. Conclusion
In conclusion, a series of ten Mannich base-type derivatives were rationally designed,
synthesized, characterized and evaluated for their antichagasic activity. The in vitro assays
allowed us to select compounds 7 and 9 based on activity, selectivity and non-genotoxicity for

in vivo testing in both the acute and chronic phases of Chagas disease. After performing
different assays, such as PCR and immunosuppression, both compounds were of interest
because they both caused a substantial decrease in parasitemia in the chronic phase when
compared with the reference drug. Furthermore, compound 9 also showed remarkable
antichagasic activity during the acute phase. The possible mechanism of action via different
assays, including metabolite excretion, mitochondrial membrane potential and the inhibition of
SOD, was investigated. These in vitro studies demonstrated that both compounds may act by
the selective inhibition of Fe-SOD. Furthermore, docking studies were done to identify the
binding mode of the Fe-SOD enzyme. We found that these compounds act similarly to the
previously studied series. Therefore, these results allow us to consider both compounds for
further studies in the development of promising candidates in Chagas therapy.
4. Experimental
4.1. Chemistry
4.1.1. Material and methods
All the chemicals were purchased by commercial vendors and were used without further
purification. The synthesis of final compounds was followed by thin layer chromatography
(TLC) using SIL G/UV254, 0.2 mm thickness (ALUGRAM® Xtra SIL G, Macherey-Nagel
GmbH & Co. KG) as the stationary phase, solvent mixtures (CH2Cl2/methanol or hexane/ethyl
acetate) as mobile phase, and UV lamp (254 nm) were employed to spots detection. Purification
of compounds were carried out on a CombiFlash®RF instrument (Teledyne Isco) employing
Silica RediSep Rf gold® columns in a normal phase gradient. For the chemical characterization
an Infrared spectra (IR), proton nuclear magnetic resonance (1H NMR), carbon nuclear
magnetic resonance (13C NMR) and elemental microanalyses (CHN) were employed. IR
spectrums were analysed on a Nicolet Nexus FTIR (Thermo Madison) employing KBr pellets.
Absorption values are given in cm-1 and reported only significant absorption bands as medium
1
13
(m) and strong (s). 400 MHz H and 100 MHz C NMR spectroscopy were carried out on a
Bruker 400 Ultrashield using TMS as an internal reference. Chemical shifts (δ), expressed in
ppm, were referenced according to DMSO-d6 or CDCl3 solvents.78 Coupling constants (J) were
given in hertz (Hz) and multiplicities detected in 1H NMR were reported as singlet (s), bs (broad
singlet), doublet (d), doublet of doublets (dd), triplet (t) and multiplet (m). In some cases, 2D
NMR were performed for assigning peaks (HMBC and HMQC). Purity of final compounds

was 95% and it was carried out on a CHN-900 elemental analyzer (Leco, TresCantos).
Elemental analysis of C, H, and N values were within the range of ± 0.4 with respect to
theoretical values. Melting points were conducted using a Mettler FP82 + FP80 (Greifensee)
and were uncorrected.
4.1.2. Synthesis of the intermediate compound
4.1.2.1. Synthesis of 1-(4-methoxyphenyl)prop-2-en-1-one (i1). i1 was synthesized according to
44
Bugarin et al with some modifications . Briefly, a mixture of 4′-methoxyacetophenone (1.0
mmol), paraformaldehyde (8.0 equiv.), and diisopropylammonium 2,2,2-trifluoroacetate (1.0
equiv.) were dissolved in THF (10 mL), then, trifluoroacetic acid (0.1 equiv.) was added. The
mixture reaction was stirred under reflux for 8 h. After that, THF was removed and the residue
was diluted in diethyl ether. The organic phase was washed with HCl 1M, NaOH 1M and brine
(saturated NaCl solution), dried over anhydrous Na2SO4, filtered, and evaporated.
4.1.3. Synthesis and Chemical characterization of Mannich bases-type derivatives (1-10)
The general procedure for the synthesis of compounds 1-6 was following Michael reaction
between vinyl ketones and arylamines. A mixture of i1 (3.0 mmol) and the corresponding amine
(3.0 mmol) was diluted in CH2Cl2 (20 mL). The reaction was stirred at room temperature and
the reaction completion was determined by TLC (24 to 48 h). The crude was purified by
automated flash chromatography with CH2Cl2/methanol solvent system as the eluent (in
gradient). The final compound was precipitated with cold diethyl ether.
On the other hand, the synthesis of compounds 7-10 was performed by Mannich reaction
following the procedures previously reported with some modifications.41, 45 A mixture of 4′-
hydroxyacetophenone (2.0 mmol), the corresponding aryl-piperazine (2.0 mmol), 1,3-
dioxolane (5 mL) and concentrated HCl (0.5 mL) was refluxed until TLC proved that the
reaction did not go (20 to 40 min). Products were obtained by vacuum filtration and they were
purified either by automated flash chromatography, eluting in gradient with CH2Cl2/methanol,
and finally washed with acetone or only washing with acetone.
4.1.3.1. 1-(4-Methoxyphenyl)-3-((4-(trifluoromethyl)phenyl)amino)propan-1-one (1). Yield:
20%. Mp: 154-155 ºC. IR (KBr) ν cm-1: 3399 (s, N-H); 1665 (s, C=O); 1170 (s, C-F); 1110 (s,
1
C-F); 1069 (s, C-F). H NMR (DMSO-d6, 400 MHz) δ ppm: 7.96 (d, 2H, H2 + H6, J2-3 = 8.8
Hz); 7.37 (d, 2H, H3' + H5', J3'-2' = 8.6 Hz); 7.04 (d, 2H, H3 + H5, J3-2 = 8.8 Hz); 6.69 (d, 2H,
H2' + H6', J2'-3' = 8.6 Hz); 3.84 (s, 3H, OCH3); 3.43 (t, 2H, CH2, JCH2-CH2 = 6.5 Hz); 3.26 (t, 2H,

CH2, JCH2-CH2 = 6.5 Hz). 13C NMR (APT, DMSO-d6, 100MHz) δ ppm: 197.29; 163.64; 152.05;
130.69 (2C); 130.10; 126.70 (2C, q, 3J= 3.7 Hz); 125.87 (q, 1J= 269.7 Hz); 115.61 (q, 2J = 31.9
Hz); 114.35 (2C); 111.71 (2C); 55.98; 38.14; 37.40. Anal. Calc. for C17H16F3NO2: C 63.15%,
H 4.99%, N 4.33%. Found: C 63.45%, H 5.01%, N 4.55%.
4.1.3.2. 3-((4-Fluorophenyl)amino)-1-(4-methoxyphenyl)propan-1-one (2). Yield: 36%. Mp:
144-145 ºC. IR (KBr) ν cm-1: 3371 (s, N-H); 1665 (s, C=O). 1H NMR (DMSO-d6, 400 MHz) δ
ppm: 7.94 (d, 2H, H2 + H6, J2-3 = 8.9 Hz); 7.04 (d, 2H, H3 + H5, J3-2 = 8.9 Hz); 6.91 (d, 2H, H3'
+ H5', J3'-2' = 8.9 Hz); 6.57 (dd, 2H, H2' + H6', J2'-3' = 9.0 Hz, J2'-F = 4.6 Hz); 5.49 (t, 1H, NH,
JNH-CH2 = 5.7 Hz); 3.84 (s, 3H, OCH3); 3.37-3.30 (m, 2H, CH2 + H2O); 3.21 (t, 2H, CH2, JCH2-
13
1
CH2 = 6.5 Hz). C NMR (APT, DMSO-d6, 100MHz) δ ppm: 197.63; 163.60; 154.75 (d, J =
230.9 Hz); 145.88, 130.68 (2C); 130.17; 115.72 (2C, d, 2J = 21.9 Hz); 114.35 (2C); 113.24 (2C,
d, 3J = 7.3 Hz); 55.98; 39.30; 37.70. Anal. Calc. for C16H16FNO2: C 70.31%, H 5.90%, N 5.12%.
Found: C 70.34%, H 5.50%, N 5.02%.
4.1.3.3. 3-((4-Chlorophenyl)amino)-1-(4-methoxyphenyl)propan-1-one (3). Yield: 26%. Mp:
154-155 ºC. IR (KBr) ν cm-1: 3369 (s, N-H); 1665 (s, C=O). 1H NMR (DMSO-d6, 400 MHz) δ
ppm: 7.94 (d, 2H, H3 + H5, J3-2 = 8.8 Hz); 7.13-6.99 (m, 4H, H2 + H6 + H3' + H5'); 6.59 (d, 2H,
H2' + H6', J2'-3' = 8.8 Hz); 5.80 (t, 1H, NH, JNH-CH2 = 5.5 Hz); 3.84 (s, 3H, OCH3); 3.41-3.30 (m,
2H, CH2 + H2O); 3.23 (t, 2H, CH2, JCH2-CH2 = 6.5 Hz). 13C NMR (APT, DMSO-d6, 100MHz)
δ ppm: 197.50; 163.61; 148.06; 130.69 (2C); 130.14; 129.06 (2C); 119.32; 114.36 (2C); 113.79
(2C); 56.00; 38.71; 37.56. Anal. Calc. for C16H16ClNO2: C 66.32%, H 5.57%, N 4.83%. Found:
C 66.47%, H 5.39%, N 4.90%.
4.1.3.4. 1-(4-Methoxyphenyl)-3-((4-(trifluoromethyl)phenethyl)amino)propan-1-one
Yield: 19%. mp: 88-89 ºC. IR (KBr) ν cm-1: 3297 (m, N-H); 1667 (s, C=O); 1170 (m, C-F);
1120 (m, C-F); 1069 (s, C-F). 1H NMR (CDCl3, 400 MHz) δ ppm: 7.86 (d, 2H, H3 + H5, J3-2
(4).
=
8,9 Hz); 7.47 (d, 2H, H3' + H5', J3'-2' = 8.1 Hz); 7.25 (d, 2H, H2 + H6, J2-3 = 8.0 Hz); 6.86 (d,
2H, H2' + H6', J2'-3' = 6.9 Hz); 3.80 (s, 3H, OCH3); 3.06 (t, 2H, CH2, JCH2-CH2 = 6.3 Hz); 2.96 (t,
2H, CH2, JCH2-CH2 = 6.1 Hz); 2.89-2.83 (m, 2H, CH2); 6.79 (t, 2H, CH2, JCH2-CH2 = 6.8 Hz). 13C
NMR (APT, CDCl3, 100 MHz) δ ppm: 198.02; 163.60; 144.26; 130.29 (2C); 129.99; 129.00
(2C); 128.47 (q, 2J = 32.6 Hz); 125.34 (2C, q, 3J = 3.7 Hz); 124.33 (q, 1J = 271.4 Hz); 113.76
(2C); 55.45; 51.00; 44.69; 38.27; 36.29. Anal. Calc. for C19H20F3NO2: C 64.95%, H 5.74%, N
3.99%. Found: C 64.94%, H 5.65%, N 3.91%.

4.1.3.5. 3-((4-Fluorophenethyl)amino)-1-(4-methoxyphenyl)propan-1-one (5). Yield: 25%.
Mp: 75-76 ºC. IR (KBr) ν cm-1: 3297 (m, N-H); 1671 (s, C=O); 1216 (s, C-F). 1H NMR (CDCl3,
400 MHz) δ ppm: 8.17-7.86 (m, 2H, H3 + H5); 7.42-6.85 (m, 6H, H2 + H6 + H2' + H3' + H5' +
H6'); 3.87 (s, 3H, OCH3); 3.25-2.68 (m, 8H, CH2). 13C NMR (APT, CDCl3, 100 MHz) δ ppm:
197.97; 163.50; 161.40 (d, 1J = 239.9 Hz); 135.61; 130.27; 130.22 (2C); 129.96 (2C, d, 3J = 7.8
Hz); 115.12 (2C, d, 2J = 21.1 Hz); 113.68 (2C); 55.38; 51.40; 44.66; 38.28; 35.56. Anal. Calc.
for C18H20FNO2: C 71.74%, H 6.69%, N 4.65%. Found: C 71.34%, H 6.41%, N 4.63%.
4.1.3.6. 3-((4-Chlorophenethyl)amino)-1-(4-methoxyphenyl)propan-1-one (6). Yield: 20%.
Mp: 98-99 ºC. IR (KBr) ν cm-1: 3297 (m, NH); 1671 (s, C=O). 1H NMR (CDCl3, 400 MHz) δ
ppm: 7.85 (d, 2H, H3 + H5, J3-2 = 8,9 Hz); 7.18 (d, 2H, H3' + H5', J3'-2' = 8.4 Hz); 7.06 (d, 2H,
H2' + H6', J2'-3' = 8.4 Hz); 6.86 (d, 2H, H2 + H6, J2-3 = 8.9 Hz); 3.80 (s, 3H, OCH3); 3.05 (t, 2H,
CH2, JCH2-CH2 = 6.1 Hz); 2.95 (d, 2H, CH2, JCH2-CH2 = 6.2 Hz); 2.82 (t, 2H, CH2, JCH2-CH2 = 7.1
Hz); 2.70 (t, 2H, CH2, JCH2-CH2 = 7.2 Hz). 13C NMR (APT, CDCl3, 100 MHz) δ ppm: 197.93;
163.48; 138.44; 131.77; 130.20 (2C); 129.95 (2C); 128.45 (2C); 128.21; 113.67 (2C); 55.37;
51.14; 44.63; 38.24; 35.71. Anal. Calc. for C18H20ClNO2: C 68.03%, H 6.34%, N 4.41%.
Found: C 68.41%, H 6.30%, N 4.36%.
4.1.3.7.
1-(4-Hydroxyphenyl)-3-(4-(4-(trifluoromethyl)phenyl)piperazin-1-yl)propan-1-one
hydrochloride (7). Yield: 56%. Mp: 189-190 ºC. IR (KBr) ν cm-1: 1678 (s, C=O); 1170 (s, C-
F); 1104 (s, C-F); 1072 (s, C-F). 1H NMR (DMSO-d6, 400 MHz) δ ppm: 11.44-11.27 (bs, 1H,
HCl); 10.62 (s, 1H, OH); 7.90 (d, 2H, H2 + H6, J2-3 = 8.8 Hz); 7.56 (d, 2H, H3' + H5', J3'-2' = 8.8
Hz); 7.16 (d, 2H, H2' + H6', J2'-3' = 8.8 Hz); 6.92 (d, 2H, H3 + H5, J3-2 = 8.8 Hz); 4.10-3.97 (m,
2H, CH2); 3.73-3.57 (m, 4H, Hp); 3.52-3.42 (m, 2H, CH2); 3.35-3.11 (m, 4H, Hp). 13C NMR
(APT, DMSO-d6, 100 MHz) δ ppm: 194.56, 162.56, 151.93, 130.51 (2C), 127.39, 126.22 (2C,
q, 3J = 3.2 Hz), 124.72 (q, 1J = 270.7 Hz), 118.84 (q, 2J = 31.9 Hz), 115.26 (2C), 114.84 (2C),
50.84, 50.40 (2C), 44.20 (2C), 32.07. Anal. Calc. for C20H21F3N2O2.HCl: C 57.90%, H 5.35%,
N 6.75%. Found: C 58.26%, H 5.49%, N 7.00%.
4.1.3.8.
3-(4-(4-Fluorophenyl)piperazin-1-yl)-1-(4-hydroxyphenyl)propan-1-one
hydrochloride (8). Yield: 20%. Mp: 191-192 ºC. IR (KBr) ν cm-1: 1675 (s, C=O); 1170 (s, C-
F). 1H NMR (DMSO-d6, 400 MHz) δ ppm: 11.23-11.00 (bs, 1H, HCl); 10.59 (s, 1H, OH); 7.90
(d, 2H, H2 + H6, J2-3 = 8.4 Hz); 7.14-6.98 (m, 4H, H2' + H3' + H5' + H6'); 6.91 (d, 2H, H3 + H5,
J3-5 = 8.4 Hz); 3.79-3.69 (m, 2H, CH2); 3.69-3.56 (m, 4H, Hp); 3.52-3.41 (m, 2H, CH2); 3.28-
3.05 (m, 4H, Hp). 13C NMR (APT, DMSO-d6, 100 MHz) δ ppm: 194.59, 162.53, 156.51 (d, 1J

= 236.8 Hz), 146.39, 130.53 (2C), 127.41, 117.79 (2C, d, 3J = 7.6 Hz), 115.41 (2C, d, 2J = 22.2
Hz), 115.26 (2C), 50.82 (3C), 46.10 (2C), 32.10. Anal. Calc. for C19H21FN2O2.HCl: C 62.55%,
H 6.08%, N 7.68%. Found: C 62.85%, H 6.12%, N 7.86%.
4.1.3.9.
3-(4-(4-Chlorophenyl)piperazin-1-yl)-1-(4-hydroxyphenyl)propan-1-one
1
hydrochloride (9). Yield: 26%. Mp: 194-195 ºC. IR (KBr) ν cm-1: 1674 (s, C=O). H NMR
(DMSO-d6, 400 MHz) δ ppm: 11.37-11.02 (bs, 1H, HCl); 10.62 (s, 1H, OH); 7.90 (d, 2H, H2
+ H6, J2-3 = 8.7 Hz); 7.28 (d, 2H, H3' + H5', J3'-2' = 8.9 Hz); 7.03 (d, 2H, H2' + H6', J2'-3' = 8.9
Hz); 6.91 (d, 2H, H3 + H5, J3-2 = 8.7 Hz); 3.92-3.74 (m, 2H, CH2); 3.71-3.53 (m, 4H, Hp); 3.52-
13
3.40 (m, 2H, CH2); 3.35-3.11 (m, 4H, Hp). C NMR (APT, DMSO-d6, 100 MHz) δ ppm:
194.64, 162.55, 148.41, 130.54 (2C), 128.73 (2C), 127.42, 123.45, 117.43 (2C), 115.28 (2C),
50.84, 50.63 (2C), 45.24 (2C), 32.14. Anal. Calc. for C19H21ClN2O2.HCl: C 59.85%, H 5.82%,
N 7.35%. Found: C 59.47%, H 6.00%, N 6.97%.
4.1.3.10.
hydrochloride (10). Yield: 22%. Mp: 196-197 ºC. IR (KBr) ν cm-1: 1677 (s, C=O); 1597 (s,
NO2); 1318 (s, NO2). 1H NMR (DMSO-d6, 400 MHz) δ ppm: 11.65-11.44 (bs, 1H, HCl); 10.64
1-(4-Hydroxyphenyl)-3-(4-(4-nitrophenyl)piperazin-1-yl)propan-1-one

(s, 1H, OH); 8.10 (d, 2H, H3' + H5', J3'-2' = 9.4 Hz); 7.89 (d, 2H, H2 + H6, J2-3 = 8.7 Hz); 7.14
(d, 2H, H2' + H6', J2'-3' = 9.4 Hz); 6.91 (d, 2H, H3 + H5, J3-2 = 8.7 Hz); 4.32-4.07 (m, 2H, CH2);
3.76-3.39 (m, 8H, Hp + CH2); 3.25-3.11 (m, 2H, Hp). 13C NMR (APT, DMSO-d6, 100 MHz)
δ ppm: 194.66, 162.57, 153.76, 137.89, 130.53 (2C), 127.41, 125.56 (2C), 115.28 (2C), 113.43
(2C), 50.89, 50.34 (2C), 43.63 (2C), 32.17. Anal. Calc. for C19H21FN3O4.HCl: C 58.24%, H
5.66%, N 10.72%. Found: C 57.85%, H 6.02%, N 10.51%.
4.2. In vitro biological assays
4.2.1. Mammalian Cell culture and cytotoxicity assay on Vero cells
Vero Cells from african green monkey kidney (EACC number 84113001), were grown in
RPMI-1640 medium (Gibco®) supplemented with 10% heat inactivated foetal bovine serum
(FBS, Gibco®), antibiotics, at 37 ºC and 5% CO2. Stock solutions of studied compounds
(synthesized Mannich base derivatives and BZN) were dissolved in 100% DMSO. So,
compounds were tested for cytotoxicity evaluation against Vero Cells to determine the IC50 as
previously described by Garnica et al.79 Briefly, cells during the logarithmic growth phase were
seeded in 96-well plates (0.8  104 cell/well) to be exposed to serial dilutions of compounds
(from 10.0 to 200.0 µM). After 72
h
MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-

diphenyltetrazolium bromide) was added and after a further 4 h, absorbances measurements
were determined using a Tecan SunriseTM Microplate Reader. Each studied concentration was
assayed in quadruplicate in three independent experiments. IC50 values were calculated through
nonlinear regression (log(inhibitor) vs. normalized response-variable slope) equation employing
GraphPad Prism 7 software.
4.2.2. Parasites and compounds
The studied compounds were tested against three different strains of T. cruzi, including SN3
strain (IRHOD/CO/2008/SN3; DTU I), obtained from domestic Rhodnius prolixus (Guajira,
Colombia)80; Arequipa strain (MHOM/Pe/2011/Arequipa; DTU V), isolated from a human
(Arequipa, Peru) and Tulahuen strain (TINF/CH/1956/Tulahuen; DTU VI) isolated from
Tulahuen, Chile. Furthermore, they were evaluated against three different morphological forms
of the parasite (epimastigote, trypomastigote and amastigote forms).
Compounds and BZN were dissolved in 100% DMSO to prepare stock solutions. Further work
dilutions were prepared on culture medium before the experiment.
4.2.3. Extracellular epimastigote forms assessment
Epimastigotes forms of T. cruzi were grown in medium trypanosomes liquid (MTL)
supplemented with 10% heat inactivated FBS at 28 ºC.60 Compounds were tested for in vitro
antiparasitic activity on epimastigote forms as procedure described.81 Compounds, at five levels
of concentration (from 6.3 μM to 50 μM), were added to suspensions of 5  105 parasites/ml in
96-well plates at 28ºC. After 48 h, resazurin sodium salt (Sigma-Aldrich) was added and
incubated for further 24 h. After this period the absorbances were measured employing a
microplate reader (SunriseTM, TECAN) at 570 and 600 nm.82 The IC50 were then generated for
each dose-response curve (nonlinear regression; log(inhibitor) vs. normalized response-variable
slope) using GraphPad Prism 7 software. Each level of concentration was carried out in
triplicate, and the experiments were performed three times.
4.2.4. Transformation of epimastigotes to metacyclic forms
Metacyclogenesis from epimastigotes into metacyclic forms, to infect BALC/c albino mice,
was tested according to Cardoso and Soares.83
4.2.5. Bloodstream trypomastigotes assessment
Blood containing the trypomastigote forms were extracted via cardiac puncture from BALB/c
albino mice and diluted in RPMI (Gibco®) supplemented with 10% heat-inactivated FBS

(Gibco®) according to previous publications.81 Compounds 2-4, 7, 9 and BZN were evaluated
against this parasite forms. The protocol was done as procedure previously described by
Moreno-Viguri et al, with certain modifications.84-85 Studied compounds and reference drug
were diluted to obtain different concentrations ranging from 3.1 to 50.0 μM, although, in some
compounds it was necessary to test lower doses to determine the IC50. By triplicate in at least
three separate experiments, parasites at 2  106 trypomastigotes/mL were seeded in culture
plates of 96-well and the compounds were added to be incubated for 24 h at 37 ºC in 5% CO2.
After this period, the trypomastigotes number were counted microscopically employing a
Neubauer
chamber.
Results
were
determined
by
nonlinear
regression
(log(inhibitor) vs. normalized response-variable slope) equation using Graphpad Prism 7 software.
4.2.6. Intracellular amastigote forms and infected Vero cells assessment
This in vitro activity was conducted as described previously in the literature.85-86 Vero cells
were maintained in supplemented RPMI (Gibco ®) as described above (see Mammalian Cell
culture and cytotoxicity assay on Vero cells section). During the exponential grown phase, 1 
104 Vero cells/well were distributed in 24-well cultured plates (each well contains a circular
coverslip) and incubated at 37 °C and 5% CO2 during 24 h. Afterwards, trypomastigotes were
added to infect the adhered Vero cells (multiplicity of infection ratio, 10:1). After 24 h,
extracellular parasites were removed by three successive washes with PBS and compounds 2-
4, 7 and 9 and BZN at different concentrations (3.1, 6.25, 12.5, 25 and 50 μM) were added.
After 72 h of incubation at the same conditions, the number of amastigote forms and infected
Vero cells were determined via Giemsa staining. Each studied concentration was made in
triplicate and the experiments were made in two separate determinations. The IC50 were
calculated using Graphpad Prism 7 software by nonlinear regression (log(inhibitor) vs. normalized
response-variable slope) equation.
4.2.7. In vitro Genotoxicity assessment
4.2.7.1. SOS/UMU-test
This screening was carried out to determine the genotoxic potential of selected compounds and
some of their metabolites using a Salmonella typhimurium TA1535/pSK1002 test strain
(DSMZ, Germany) in 96 well-plates with and without S9 metabolic activation as described
Perez-Silanes et al.87 Firstly, the bacterial survival percentage was measured by exposing this