In vitro and in vivo trypanosomicidal action of novel arylimidamides against trypanosoma cruzi

Article abstract of DOI:10.1128/AAC.01667-15

Arylimidamides (AIAs) have been shown to have considerable biological activity against intracellular pathogens, including Trypanosoma cruzi, which causes Chagas disease. In the present study, the activities of 12 novel bis-AIAs and 2 mono-AIAs against dif

Full text of DOI:10.1128/AAC.01667-15

AAC Accepted Manuscript Posted Online 8 February 2016
Antimicrob. Agents Chemother. doi:10.1128/AAC.01667-15
Copyright © 2016, American Society for Microbiology. All Rights Reserved.
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In vitro and In vivo Trypanosomicidal Action of Novel Arylimidamides
Against Trypanosoma cruzi
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Guedes-da-Silva, FH ; Batista DGJ ; Meuser, MB , Fulco, TO , Araújo, JS ; Da
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Silva, PB ; Da Silva CF ; Patrick, DA ; Bakunova, SM ; Bakunov, AS ; Tidwell,
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RR ; Oliveira, GM ; Britto, C ; Moreira, OC and Soeiro, MNC *
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Laboratório de Biologia Celular, Instituto Oswaldo Cruz, Fundação Oswaldo
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Cruz, Rio de Janeiro, RJ, Brazil, University of North Carolina, Chapel Hill,
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North Carolina, USA, Laboratório de Biologia Molecular e Doenças Endêmicas
Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Rio de Janeiro, RJ, Brazil
Running title: Trypanocidal effect of arylimidamides
*Correspondent footnote
E-mail: soeiro@ioc.fiocruz.br
Phone Number: 55 21 25621368
Fax Number: 55 21 25984469

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Summary
Arylimidamides (AIA) showed considerable biological activity against
intracellular pathogens, including Trypanosoma cruzi. Presently, the analysis of
twelve novel bis-AIAs and two mono-AIAs was performed against different
strains of T. cruzi in vitro and in vivo. The most active was m-terphenyl bis-AIA
35DAP073, with EC50 value of 0.5 µM on trypomastigotes (Y strain), being 26-
fold more effective than benznidazole (Bz, 13 μM). It was also active against
Colombiana strain, (EC50 = 3.8 μM). The analysis against intracellular forms
(Tulahuen strain) showed that this bis-AIA (EC50 = 0.04 µM) was about 100-fold
more active than Bz (2 µM). Trypanocidal effect was dissociated to their ability
to trigger intracellular lipid bodies within host cells detected by oil red labeling.
Both active (35DAP073) and inactive (26SMB060) compounds displayed similar
activation profiles. Due to high selectivity index, two AIAs (35DAP073 and
35DAP081) were moved to in vivo, but acute toxicity assays excluded the
following tests using 35DAP081. Findings with 35DAP073 using Y strain
revealed that two-days of therapy induced a dose-dependent action, leading to
96-46 % reduction of parasitemia. However, ten daily doses using Colombiana
strain resulted in animal toxicity, impairing longer periods of treatment. Then the
association of 0.5 mg/kg/day 35DAP073 with 100 mg/kg/day Bz was assayed
for 10 consecutive days. The association resulted in a suppression of the
parasitemia, elimination of neurological toxic effects and 100% survival of
animal. Quantitative polymerase chain reaction showed considerable reduction
of parasite load (60%) as compared to Bz or amidine alone. Our results support
further investigations of this class aiming development of novel alternatives for
Chagas disease therapy.

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Keywords: Arylimidamides, experimental chemotherapy, Chagas disease, lipid
bodies, combination therapy.
Introduction
Chagas disease (CD) is caused by the obligate intracellular protozoan
Trypanosoma cruzi. More than a hundred years after its discovery (1), CD is still
an important public health problem, affecting about 6 to 7 million people are
estimated to be infected worldwide, mostly in Latin America (2). Of these
infected individuals, 30-40% develops cardiomyopathy and / or digestive
syndromes (3). For over four decades, Nifurtimox (3-methyl-4-(5'-
nitrofurfurylideneamine
tetrahydro-4H-1,4-tiazine-1
,1-dioxide)
and
benznidazole (N-benzyl-2-nitroimidazole acetamide) introduced empirically in
therapy to CD still remain the sole current options (4). Both have several
limitations, including a variety of adverse effects and limited efficacy in later
chronic phase (5,6), which strengthen the need for new trypanocidal
compounds that overcome these limitations.
Classic aromatic diamidines (AD) are DNA minor groove binders with
recognized antimicrobial and antitumor broad spectrum activity (7). ADs such
as pentamidine, diminazene and propamidine have been used for decades in
medicine and veterinary therapy, but despite their excellent anti-parasitic effects
have relevant drawbacks that include low bioavailability and side effects. To
overcome these limitations, new analogues have been synthesized and tested
in vivo and in vitro (7). Several AD and related compounds have been screened
against T. cruzi showing promising results at least in part due to their capacity

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to alter the KDNA molecule (8, 9). Arylimidamides (AIAs, formerly called
“reversed” amidines because of the reversed position of nitrogen and carbon
atoms compared to classic aromatic amidines) are the most effective amidine
analogues tested against T. cruzi in vitro and in vivo (9,10,11,12).
Lipid bodies (LB) consist of a nucleus of cholesteryl esters and
triglycerides surrounded by a single monolayer of phospholipids. LB are
considered not only lipid storage compartments but also intracellular sites for
many biological events like cellular signaling and activation, regulation of lipid
metabolism, membrane trafficking and regulation of inflammatory mediators
(13,14). Recent studies proposed a dynamic role of LB in the control of
pathogens infection due to their localization inside the parasitophorous vacuoles
lodging intracellular parasites like T.cruzi (15).
Our present study focused on the analysis of twelve novel bis-AIAs and
two mono-AIAs against bloodstream trypomastigotes and intracellular forms of
T. cruzi (Tulahuen, Y and Colombiana strains) performed in vitro. The most
potent and most selective compound, 35DAP073 underwent further screening
in mouse experimental acute models of parasite infection (Y and Colombiana)
alone or combined with the reference drug (Bz) aiming to contribute for the
search of novel alternative therapeutic protocols for CD.
Materials and methods
Synthesis of the arylimidamides
The syntheses of the four m-terphenyl bis-arylimidamides 35DAP069,
35DAP073, 35DAP077, and 35DAP081, and the two mono-arylimidamides
35DAP092 and 38DAP096 (Figure 1) have been described previously (16),
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wherein the final step was the reaction of the appropriate terphenyl amine or
diamine with benzonitrile or 2-cyanopyridine using sodium
bis(trimethylsilyl)amide in tetrayhydrofuran. Similar reactions involving 1,4-
diphenylenediamine and 2,5- or 2,6-diaminopyridine were employed for the
syntheses of 19SAB003, 19SAB005, 19SAB007, 28SMB008 (17) (Figure 2).
Compounds 23SMB046, 23SMB050, 26SMB060, and 27SMB005 (Figure 3)
were prepared by previously described methodology from the methyl imidate
derivatives of 2- or 4-cyanopyridine and the appropriate α,ω-diaminoalkane
(18). All fourteen compounds were isolated as their hydrochloride salts.
Experimental details and physical data for the compounds shown in Figures 2
and 3 are given in the supplemental material (S1).
Stock Solutions of the tested compounds
The studied molecules were prepared in dimethyl sulfoxide (DMSO) -
(Sigma Aldrich) - with the final concentration (dilution using RPMI) of the solvent
never exceeding 0.6% and 10% for in vitro and in vivo analysis, respectively,
which do not exert cellular toxicity upon on parasites and mammalian host cells
(data not shown). Benznidazole (Bz) was purchased from Laboratório
Farmacêutico do Estado de Pernambuco, LAFEPE, Brazil. Bz was dissolved in
distilled and sterile water supplemented with 3% tween 80, which does not
cause any detectable effect on mice (19).
Mammalian cell cultures
Primary cultures of embryonic cardiac cells (CC) were obtained from
Swiss mice as previously reported (20). After purification, the CC were seeded
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at a density of 0.2 X 10 and 0.05 X 10 cell/well, respectively, into 24 and 96-

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well microplates containing gelatin-coated cover slips as reported (18). The
cardiac cultures were then sustained at 37° C in Dulbecco`s modified medium
(DMEM - without phenol red - from Sigma Aldrich) supplemented with 10%
2
horse serum, 5% fetal bovine serum, 2.5 mM CaCl , 1 mM L-glutamine and 2%
chicken embryo extract. Additionally, mouse L929 fibroblasts were cultivated (4
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X 10 cell/well into 96-well microplates) at 37° C in RPMI-1640 medium (pH 7.2-
7.4) without phenol red (Gibco BRL) supplemented with 10% fetal bovine serum
and 2 mM glutamine (RPMI), as reported (21).
Cytotoxicity in vitro tests
CC and L929 cell cultures were incubated at 37°C for different periods of
time (24-96 hours) with increasing concentrations of each compound (up to 96
µM) diluted in DMEM (without phenol red). Next, mammalian cells morphology
and spontaneous contractibility (CC) were evaluated by light microscopy, and
the cellular viability determined by a colorimetric assay using 10 μL AlamarBlue
(Invitrogen) added to each well. After incubation for 24 h, the absorbance was
determined (at 570 and 600 nm) and the results were determined following the
manufacturer's instructions. Then, the values of LC50, which corresponds to the
concentration that reduces in 50 % the cellular viability calculated as reported
(21).
Parasites
Bloodstream trypomastigote (BT) forms of Y and Colombiana strains of
T. cruzi were obtained from the blood of infected male Swiss mice at the peak
of parasitemia (12). Immediately after the purification step, the parasites were

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ressuspended in RPMI-1640 medium (pH 7.2-7.4) without phenol red (Gibco
BRL) supplemented with 10% fetal bovine serum (FBS) and 2 mM glutamine,
as reported previously (18). The effect against intracellular forms was
investigated through the use of L929 cell lineages infected with tissue culture-
derived trypomastigotes (Tulahuen strain expressing the E. coli β-galactosidase
gene), using a 10:1 parasite: host cell ratio. The incubation with the tested
compounds was performed for 96 h, following previously established protocols
(21). Alternatively, CC were infected (ratio 10:1) with bloodstream
trypomastigotes (Y strain). After 24 h of interaction, the infected-cultures were
rinsed and then exposed for 48 h with nontoxic concentrations of the amidines
previously screened on mammalian host cells.
Trypanocidal analysis
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BT forms of the Y and Colombiana strains (5 X 10 per mL) were
o
incubated for up to 24 h at 37 C in RPMI in the presence of serial dilutions of
the compounds (0 to 32 µM), the parasites incubated with culture medium alone
were used as control. After compound incubation, the parasite death rates were
determined by light microscopy through the direct quantification of the number
of live parasites using a Neubauer chamber, and the EC50 (compound
concentration that reduces 50 % of the number of parasites) calculated. Also,
the EC90 (compound concentration that reduces 90 % of the number of
parasites) was further calculated in the intracellular assays using the Y strain
(18). For the assay on intracellular forms, cardiac cell cultures and L929 cell
lines were used as hosts for the infection using the Y and Tulahuen strains,
respectively. Briefly, Tulahuen-infected-L929 cultures were exposed to 10 μM

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(corresponding the EC90 value of Bz, as reported by 18) of each compound
diluted in RPMI and the compounds that presented ≥ 50% of reduction on the
parasite infection index were further screened under increasing concentrations
aiming to determine the EC50 values (18). After 96 h of compound incubation at
37°C, chlorophenol red glycoside (500 µM ) - (Sigma Aldrich) in 0.5% Nonidet
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P40 was added to each well and the plate incubated for 18 h at 37°C. Next the
absorbance was measured at 570 nm. Uninfected and T. cruzi-infected cultures
submitted to vehicle and Bz exposure were run in parallel. The results are
expressed as the percentage of T. cruzi growth inhibition in compound-tested
cells as compared to the infected cells and untreated cells (21). Triplicate
samples were run in the same plate and at least two assays performed in each
analysis. For the analysis of the effect against intracellular amastigotes from Y
strain, after 24 h of parasite-host cell interaction, the infected CC cultures were
washed to remove free parasites and then incubated for another 48 h with
increasing concentrations of the test compounds. CC were maintained at 37°C
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in an atmosphere of 5% CO and air and the medium replaced every 24 h.
Then, the samples were fixed and stained with Giemsa solution - (Sigma
Aldrich) and the number of infected host cell and the number of parasite per
infected host cell determined through light microscopy analysis (18). Only
characteristic T. cruzi nuclei and kinetoplasts were counted as surviving
parasites since irregular structures could mean parasites undergoing death. The
compound activity was estimated by calculating the infection index (percentage
of infected cells multiplied by the average number of intracellular amastigotes
per infected host cell) (12). At least two assays were performed in duplicates in
each analysis.

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Lipid Bodies labeling
To determine the biogenesis of lipid bodies (LB) in cardiac cell cultures
exposed to amidines, untreated CC were incubated for 2-48 h with the
corresponding EC50 values of the studied compounds (previously determined
against intracellular forms from Y strain) and then fixed for 10 min with 3.7%
2
+
2+
formaldehyde in Ca -Mg -free HBSS (pH 7.4). Next, the samples were rinsed
twice using distilled water, incubated for 5 min in absolute propylene glycol and
stained for 10 min in 0.5% oil red O (Sigma-Aldrich). The cultures were
incubated for 3 min with 85% propylene glycol, and finally rinsed twice using
distilled water. The samples were incubated or not with 1 µg/mL 4,6-diamidino-
2-phenylindole (DAPI) - (Sigma Aldrich) for DNA staining (host cells nuclei),
rinsed with saline buffer, dried and mounted using aqueous mounting medium
2.5% 1.4-diazabicyclo-(2.2.2)octane (DABCO) and the fluorescence analyzed
with a x63 oil objective in a Zeiss photomicroscope (Axiocam) equipped with
epifluorescence (Zeiss Inc, Thornwood, New York), using a filter set for UV
excited probes. Images were captured using the software AnalySIS OPTI. As
positive control, 2 µM of Oleic acid (Sigma-Aldrich) was used to trigger lipid
bodies’ biogenesis (22).
In vivo acute toxicity
In order to determine the no-observed-adverse-effect level (NOAEL),
increasing doses of the tested compounds (up to 200 mg/kg of body weight)
were injected by intraperitoneal (ip) route individually in Swiss Webster female
mice (20 to 23 g, n = 2 per assay, two assays). Treated animals were inspected

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for toxic and sub-toxic symptoms according to the Organization for Economic
Cooperation and Development (OECD) guidelines. Forty-eight hours after
compound injection, the NOAEL values were determined as reported previously
(23).
Biochemical analysis
Forty-eight hours after compound administration, mouse blood was
collected and immediately submitted to biochemical analysis for determination
of plasma tissue markers including Urea (BUN), Alanine aminotransferase
(ALT), Aspartate aminotransferase (AST) and Creatine Kinase (CK) that was
performed at animal facilities of the Oswaldo Cruz Foundation (Rio de Janeiro,
Brazil, CECAL/Fiocruz platform) using Vitros 250 (Ortho Clinical-Johnson &
Johnson), as reported previously (23).
In vivo infection
Swiss Webster male mice (18-20 g) obtained from the animal facilities of
CECAL were housed at a maximum of 6 per cage and kept in a specific
pathogen free (SPF) room at 20–24°C under a 12/12 h light/dark cycle and
provided with sterilized water and chow ad libitum. The animals were allowed to
acclimate for 7 days before starting the experiments. Infection was performed
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by intraperitoneal (ip) injection of 10 and 5 x 10 bloodstream trypomastigotes
(Y and Colombiana strain, respectively). Age-matched non-infected mice were
maintained under identical conditions (12)
Treatment schemes

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AIAs were first dissolved in DMSO and then freshly diluted with sterile
distilled water. The stock solution of benznidazole (N-benzyl-2-nitroimidazol
acetamide) was prepared in sterile distilled water with 3% Tween 80 (Sigma
Aldrich). The animals were divided into the following groups (5 animals per
group): uninfected (non-infected and non-treated); untreated (infected but
treated only with vehicle); and treated (infected and treated with the
compounds). The therapy (once a day) was performed through different
schemes (see Supplemental Material - S2): In the first set of assays, T. cruzi-
infected mice (Y strain) were treated for only two days (at 5 dpi and at 8 dpi that
corresponds to the parasitemia onset and peak in this experimental model),
using 5-20 mg/kg/day AIA (ip) and with 100 mg/kg/day Bz (po). In the second
set of experiments, mice were infected with Colombiana strain and at the 10 dpi
(parasitemia onset in this animal model) were treated for ten consecutive days
with 35DAP073 alone (5 mg/kg/day) or in association with Bz (0.5 mg/kg/day
AIA plus 100 mg/kg/day of Bz). In all assays, only mice with positive
parasitemia were used in the infected groups.
Parasitemia and mortality rates
Parasitemia was individually checked by direct microscopic counting of
parasites in 5 µL of blood, and mortality rates checked daily until 30 days post
treatment and expressed as percentage of cumulative mortality (% CM) as
described before (18).
Blood parasite load by qPCR

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For quantitative real time polymerase chain reaction (qPCR), 500 µL
blood were diluted in 1:2 volume of guanidine solution (Sigma Aldrich)
(Guanidine-HCl 6M/EDTA 0.2M), and heated for 90 sec. in boiling water.
Guanidine-EDTA Blood Samples (GEB) were processed using the QIAamp
DNA mini kit (QIAGEN) (24). Quantitative Real Time PCR Multiplex assays
using TaqMan probes were performed targeting the T. cruzi satellite nuclear
DNA and the internal amplification control - IAC (pZErO-2 plasmid containing an
insert from the A. thaliana aquaporin gene), as described by (25). The standard
curves for absolute quantification were constructed with serial dilutions of total
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DNA, obtained from a negative GEB sample spiked with 10 parasite
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equivalents per milliliter of blood (par. eq./mL), ranging from 10 to 0.5 par.
eq./mL.
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Ethics
All procedures were carried out in accordance with the guidelines
established by the FIOCRUZ Committee of Ethics for the Use of Animals
(CEUA LW16/14).
Statistical Analysis
The data represent means ± standard deviations from 2 experiments run
in duplicate and statistical analysis performed by the ANOVA test with the level
of significance set at p≤0.05 (23).
Results

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The phenotypic analysis against BT (Y strain, DTU II) showed that all
studied compounds were active against the parasite. 35DAP073, 35DAP081,
35DAP077 and 35DAP092 presented a time-dependent response, exhibiting
EC50 values ranging from 0.7-28 µM and 0.5-10 µM after 2 and 24 h of
incubation, respectively, while Bz resulted in EC50 value of 13 μM after 24 h
(Table 1). Among these four compounds, the most active were 35DAP073 and
35DAP081 and both also displayed the highest selectivity indexes (64 and 53,
respectively). The statistical analysis revealed that 35DAP073, 35DAP081 and
35DAP092 presented superior efficacy than Bz (p<0.05). When BT forms of
Colombiana strain (DTU I) were assayed, both amidines showed trypanocidal
effect showing EC50 values lower than 4µM after 24 h of exposure (Table 2).
35DAP073 and 35DAP081 were very active against intracellular forms from
both Tulahuen (Table 3) and Y (Table 4) strains of T. cruzi, representatives of
DTU VI and II, respectively, being more potent than Bz (p ≤ 0.05). The m-
terphenyl bis-AIA 35DAP073 showed the higher activity expressed by the lower
EC90 values (87nM) after 48 h of incubation, being about 126-fold more effective
than Bz, besides exhibiting the highest SI (2000) (Table 4).
As 35DAP073 was more effective against intracellular forms as
compared to BT (Y strain, Tables 1 and 4), and biogenesis of lipid bodies (LB)
may be induced by different stimuli (26), LB induction was evaluated during the
incubation of uninfected CC with this amidine with a head-to-head comparison
with another non active molecule against T. cruzi in vitro (26SMB060). After
incubation for different time periods (2-48 h) our data revealed that both
amidines were able to induce similar levels of LB biogenesis as a very fast
phenomenon since only 2 h post-treatment resulted in strong LB formation as

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compared to untreated group, and with similar profile as the positive control
(oleic acid) (Figure 4). A great number of LB was also observed after 24 h of
amidine exposure; which levels were maintained up to 48 h despite the oleic
acid displayed a progressive and continuous formation of LB (increase in the
number and volume) (Figure 4).
Next, due to the high activity and selectivity of 35DAP073 and
35DAP081, Swiss mice were used to determine their NOAEL values.
35DAP073 proved to be less toxic at all doses tested, presenting NOAEL 25
mg/kg while 35DAP081 showed a value < 12.5 mg/kg (Table 5). The plasma
biochemical analysis (p ≤ 0.1) after 48 h of compound administration did not
show any statistical difference among all tested groups when ALT, AST, CK and
BUN measurements were evaluated (Table 6). Due to these preliminary acute
toxicity studies, only 35DAP073 was move to in vivo acute models of T. cruzi
infection using no-toxic doses (up to 25 mg/kg/day). In parallel, a control group
was orally treated with Bz (100 mg/kg/day) (Figure 5).
Our data demonstrated a dose-dependent effect, leading to 96-46 %
reduction of parasitemia with concentrations of up to 20 mg/kg/day (Figure 5).
The animal survival rates achieved 100 % when mice were treated with 5 and
10 mg/kg/day of 35DAP073, similarly as 100 mg/kg/day Bz (Figure 5). However,
as the tested compound did not result in complete suppression of the blood
parasite load, next, a longer period of therapy (10 days using 5 mg/kg/day) was
3
investigated on mice infected with 5x10 bloodstream trypomastigotes of the
Colombiana strain. Also, a combination with Bz (10 days using 0.5 mg/kg/day
35DAP073 and 100 mg/kg/day Bz) was assayed in parallel using the same
experimental model, starting the therapy at the parasitemia onset. Our findings

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showed that 5 mg/kg/day of 35DAP073 completely suppressed the parasitemia
and gave 100% animal survival, as did Bz (Figure 6). However, at the end of the
therapy period (last day of 35DAP073 administration), mice presented some
undesirable neurological toxic disorders (tremors, shaking, ataxia) that were
reverted after 48 h post-treatment (data not shown). The combination of 0.5
mg/kg/day of this amidine with Bz resulted in parasitemia suppression, 100%
animal survival and no toxic events (Figure 6). The analysis by qPCR showed
that although no statistical significance was found among the tested groups
(p>0.05), the monotherapy with Bz and 35DAP073 resulted in mean values of
204 and 265 par.Eq/mL respectively, while the association of Bz with the AIA
induced a higher reduction (60 %) of the blood parasite load (82 par.Eq/mL)
(Figure 7).
Discussion
The current therapy for Chagas disease still remains unsatisfactory due
to various side effects and limited efficacy (27). Amidines have been used for
decades in human and veterinary medicine as anti-parasitic agents but show
important limitations as parental administration and undesirable side effects (7).
In order to overcome these limitations, the synthesis and phenotypic analysis of
novel amidines have been conducted in vivo and in vitro (28, 12). Among them,
arylimidamides (AIA) showed considerable biological activity against several
intracellular pathogens, including T.cruzi (29), being the bis-AIAS the most
effective among the group (19).
A previous study reported the synthesis of dicationic m-terphenyl
derivatives and their biological effect against T. brucei rhodesiense,

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Plasmodium falciparum, Leishmania amazonensis and T. cruzi (16). Among
them, bis-pyridylimidamides were the most potent against the intracellular
amastigote form of the T. cruzi (Tulahuen strain, DTU VI). Presently, we further
explored the phenotypic activity in vitro and in vivo of fourteen bis-and mono-
arylimidamides against bloodstream trypomastigotes (BT) and intracellular
forms of T. cruzi, extending the analysis to other parasite strain (Y and
Colombiana strains, belonging to DTUs II and I, respectively).
Structural variations of the test compounds in this study included those in
AIA moieties as well as the central cores of the molecules. Maximum potencies
were observed in 35DAP073 and 35DAP081, which each have two 2-
pyridylimidamide groups attached to a m-terphenyl nucleus, with at least one of
the two AIA groups meta to the central ring. These two compounds were more
potent than the corresponding bis-phenylimidamides 35DAP069 and
35DAP077. The difference in potencies between 35DAP073 and 35DAP069
was greater than 60-fold, but less pronounced between 35DAP081 and
35DAP077. The bis-pyridylimidamides 35DAP073 and 35DAP081 were also
more potent than the mono-pyridylimidamides 35DAP092 and 38DAP096, thus
demonstrating the importance of two AIA moieties. Compound 38DAP092, in
which the AIA group is meta to the central ring, was more potent than its
regioisomer 38DAP096 which bears a para-AIA moiety. None of the bis-AIAs
with lone aromatic rings or aliphatic chains between the two AIA functionalities
(Figures 2 and 3) showed detectable activity.
The most active molecule, the m-terphenyl bis-AIA 35DAP073, showed
after 24 h an EC50 value of 0.5 µM on trypomastigotes (Y strain), being more
effective than the reference drug, Bz (13 μM). This amidine was also active on

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highly naturally resistant strain (Colombiana), exhibiting EC50 = 3.8 μM. The
analysis against intracellular forms (Tulahuen strain) showed that this bis-AIA
(EC50 = 0.04 µM), was about 50-fold more active than Bz (2 µM). The higher
effect against intracellular forms as compared to BT was dissociated to the
ability of amidines to induce LB accumulation within mammalian host cells.
Biogenesis of lipid bodies is a central event in several cellular homeostasis, as
well as during intracellular pathogen infection (30, 31). LB are recognized as
dynamic organelles composed of a triglyceride and cholesteryl ester core with a
surrounding monolayer of phospholipid, cholesterol, and a variety of associated
proteins with diverse functions in cell metabolism, signaling, and inflammation
(14,32). Under inflammatory and infectious conditions, prostaglandins and
others lipid mediators are mainly produced by LB (33). Also, recent findings
showed that antimicrobial compounds may induce an accumulation of lipid
droplets in the cytoplasm of yeasts (34) and protozoan (35). In M. leprae
infected-cells, LBs show high ability to fuse forming giant LDs (36). Since
amidines can modulate functional activities of mammalian host cells linked to
the control of parasite proliferation and/or survival (37, 38) and LB act on
cellular metabolism, inflammation and infectious conditions (14,32), presently
we investigated whether the studied AIAs could interfere with LB biogenesis
and parasitism control in vitro. Our data also demonstrate that 35DAP073 and
28SMB060 are able to induce LB biogenesis in cardiac cells in at all times
tested (2-48 h), exhibiting a similar morphological profile. No correlation was
found between trypanocidal effect of amidines and LB accumulation in CC but
further studies are desirable to better understand the consequences of LB

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levels induced by amidines in the parasite as well mammalian cells physiology
since this modulation may influence the growth of intracellular parasites (36).
Next, due to the excellent selective indexes of 35DAP073 and
35DAP081, both were tested for acute toxicity analysis. The results showed
values of 25 and 12.5 mg/kg NOAEL for 35DAP073 and 35DAP081. Then, in
vivo efficacy studies were conducted with 35DAP073, using concentrations < 25
mg/kg. Our data demonstrated that the infection of mouse models using Y and
Colombiana strains resulted in a dose-dependent action of 35DAP073, leading
to 96-46 % reduction of parasitemia and 100 % of animal survival, with similar
effect of Bz. Unfortunately, as the ten-daily dose (using 5mg/kg via ip) resulted
in animal toxic aspects, the combined therapy of 35DAP073 with Bz was
performed aiming to reduce the toxic aspects of the studied amidine. The
monotherapy using only 0.5mg/kg/day was not approached as previous data
with related amidines demonstrated a mild effect of parasitemia control using
doses ≤ 5mg/kg/day (23). As also found by our group (28) using the
combination of Bz with the amidine DB289, presently the association of 0.5
mg/kg 35DAP073 with Bz improved the control of the parasite proliferation
providing a 9-fold enhancement of activity as compared to that of Bz alone.
qPCR analysis showed that the combination therapy was the best protocol
regimen exhibiting parasitemia suppression with no detectable animal toxicity.
In this sense, our results support further experimental investigations of this
compound class in association with licensed anti-parasitic drugs aiming to
contribute for the identification of novel alternatives to treat parasitic neglected
pathologies as Chagas disease.

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Financial support
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The present study was supported by grants from Fundação Carlos
Chagas Filho de Amparo a Pesquisa do Estado do Rio de Janeiro (FAPERJ),
Conselho Nacional Desenvolvimento científico Tecnológico (CNPq),
e
Fundação Oswaldo Cruz, PDTIS, PROEP/CNPq/Fiocruz, CAPES. MNCS and
CB are research fellows of CNPq. MNCS and CB are CNE researchers.
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Acknowledgments
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The authors thank the Program for Technological Development in Tools
for Health (PDTIS-Fiocruz) for the facilities on the Real Time PCR RPT09A
platform and RPT11G.
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Legend of the Figures
Figure 1: Structures of mono- and bis-arylimidamide derivatives of m-terphenyl.
Figure 2: Synthesis of bis-arylimidamide derivatives of benzene and pyridine.
Reagents and conditions: (a) sodium bis(trimethylsilyl)amide, terahdyrofuran,
25°C.
Figure 3: Synthesis of bis-arylamidamide deratives of n-alkanes. Reagents and
conditions: (a) 4 M HCl in dioxane, methanol, 25 °C.
Figure 4: Differential interference contrast (DIC) microscopy analysis of oil red
labeling of cardiac cells untreated (A-C) and submitted to oleic acid (D-F),
35DAP073 (G-I) and 26SMB060 (J-L) for 2 (A,D,G,J), 24 (B, E,H, K) and 48

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(C,F, I, L) hours of exposure at 37°C. Detailed oil red labeling of cardiac cells by
DIC (M, O, Q and S) and fluorescence microscopy using DAPI staining of
mammalian cell nuclei (N, P, R and T) after 24 h of incubation with 35DAP073
(Q and R) and 35DAP060 (S and T) showing the accumulation of lipid bodies
randomly distributed throughout the cytoplasm.
Figure 5: Effect of 35DAP073 (20-5 mg/kg/day) and of Benznidazole (100
mg/kg/day) administration at 5 and 8 day after infection (dpi) on mouse
experimental model of T. cruzi infection (Y strain). (A) Parasitemia levels and
(B) Percentage of cumulative mortality.
Figure 6: Effect of 10 daily administration of 35DAP073 (5 mg/kg/day),
Benznidazole (100 mg/kg/day) and 35DAP073 + Bz (0.5 mg/kg/day and 100
mg/kg/day, respectively) on mouse experimental models of T. cruzi infection
(Colombiana strain), starting the compound administration at the 10 dpi. (A)
Parasitemia levels and (B) Percentage of cumulative mortality.
Figure 7: Blood qPCR analysis of the parasite equivalent per milliliter (Par. Eq. /
mL) from uninfected and infected mice (using Colombiana strain) submitted to
each different treatment regimen: Monotherapies (benznidazole 100 mg/kg/day
and 35DAP073 5 mg/kg/day) and combined therapy (benznidazole (100
mg/kg/day) + 35DAP073 (0.5 mg/kg/day)). Each symbol represents an
individual value and the bars represent respective mean values.