In vitro and in vivo biological effects of novel arylimidamide derivatives against Trypanosoma cruzi

Article abstract of DOI:10.1128/AAC.02353-14

Chagas disease (CD), a neglected tropical disease caused by Trypanosoma cruzi, remains a serious public health problem in several Latin American countries. The available chemotherapies for CD have limited efficacy and exhibit undesirable side effects. Aro

Full text of DOI:10.1128/AAC.02353-14

AAC Accepts, published online ahead of print on 21 April 2014
Antimicrob. Agents Chemother. doi:10.1128/AAC.02353-14
Copyright © 2014, American Society for Microbiology. All Rights Reserved.
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In vitro and In vivo Biological Effect of Novel Arylimidamide Derivatives Against
Trypanosoma cruzi
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Bruno Lisboa Timm,a Patrícia Bernadino da Silva,a Marcos Meuser Batista,a Francisca Hildemagna
Guedes da Silva,a Cristiane França da Silva,a Richard R. Tidwell,b Donald A. Patrick,b Susan
Kilgore Jones,b Stanislav A. Bakunov,b Svetlana M. Bakunova,b Maria de Nazaré C. Soeiroa #
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aLaboratório de Biologia Celular, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Rio de Janeiro,
RJ, Brazil a; Department of Pathology and Laboratory Medicine, University of North Carolina,
Chapel Hill, North Carolina, USAb
Running Head: Trypanocidal activity of arylimidamides
#Address correspondence to Maria de Nazaré Correia Soeiro, soeiro@ioc.fiocruz.br

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ABSTRACT
Chagas disease (CD), a neglected tropical disease caused by Trypanosoma cruzi, remains a serious
public health problem in several Latin American countries. The available chemotherapies for CD
have limited efficacy and exhibit undesirable side effects. Aromatic diamidines and arylimidamides
(AIAs) have shown broad-spectrum activity against intracellular parasites including T. cruzi.
Therefore our aim was to evaluate the biological activity of eight novel AIAs (16DAP002;
16SAB079, 18SAB075, 23SMB022, 23SMB026, 23SMB054, 26SMB070 and 27SMB009) against
experimental models of T. cruzi infection in vitro and in vivo. Our data show that none of the
compounds induced loss of cellular viability up to 32 µM. Two AIAs, 18SAB075 and 16DAP002,
exhibited good in vitro activity against different parasite strains (Y and Tulahuen) and against the
two relevant forms of the parasite for mammalian hosts. Due to the excellent selective indexes of
18SAB075, this AIA was moved to in vivo tests for acute toxicity and parasite efficacy, the later,
employing non-toxic doses (NOAEL = 50mg/kg). In experimental acute models of T. cruzi
infection, 18SAB075 reduced only up to 50% the parasitemia levels and led to 40% of protection
against mortality (at 5 mg/kg), being less effective than the reference drug, benznidazole.

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INTRODUCTION
Chagas disease is a neglected tropical disease caused by the intracellular flagellate protozoan
Trypanosoma cruzi, which presents a complex life cycle with distinct morphological stages in its
obligatory passage through vertebrate and invertebrate hosts [1]. Currently, there are approximately
10 million infected individuals in endemic areas of Latin America and many reports also point to
the occurrence in non-endemic geographical areas such as the United States and Europe, mainly
attributed to migration of infected people [2-6]. CD can be transmitted by Triatominae insect feces,
blood transfusion, organ transplantation, laboratory accidents, and oral and congenital routes [7,8].
This pathology has the following two successive phases: a short acute phase characterized by a
patent parasitemia, followed by a chronic phase in which most of the infected individuals remain
asymptomatic (indeterminate form), but about one third may later on manifest cardiac and/or
digestive complications, developed progressively for years or decades after infection [9,10].
Benznidazole (Bz) and Nifurtimox, introduced into clinical therapy about 40 years ago, are the only
available drugs. Both have several shortcomings related to their required long periods of treatment,
the high toxicity, variable results and low efficacy during the chronic phase, justifying the
identification of novel therapies [11-13]. Aromatic diamidines and analogues exhibit broad
spectrum activity against pathogenic microorganisms including T. cruzi [14]. Among the different
tested derivatives of amidine compounds, the most effective against T. cruzi have been the called
bis-arylimidamides (AIAs) like DB766 [15] and DB1831 [16]. Thus, presently, the biological
activities of eight novel AIAs (16DAP002; 16SAB079, 18SAB075, 23SMB022, 23SMB026,
23SMB054, 26SMB070 and 27SMB009) were assayed against experimental models of T. cruzi
infection in vitro and in vivo, using different parasite strains and also exploring toxicity aspects
towards cardiac cell cultures and mouse models of acute toxicity.

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MATERIALS AND METHODS
Synthesis of the arylimidamides. The eight arylimidamides, all isolated as their
hydrochloride salts, were prepared by three general methods (see Supplementary data at AAC
Online). Briefly, phenylimidamides 16DAP022 and 16SAB079 (Figure 1) were prepared by the
reaction of with 2,7-diaminocarbazole (prepared by reduction of 2,7-dinitroocarbazole) [17] or m-
xylylenediamine with (2-naphthyl)methyl benzothioimidate hydrobromide [18]. The 2-
pyridylimidamide 18SAB075 (Fig. 2) was prepared by the reaction of 4,4ʹ-
diaminodiphenylacetylene [19,20] with 2-cyanopyridine by the method of Lange et al. [21] but
using lithium (rather than sodium) bis(trimethylsilyl)amide. Reaction of the methyl imidate
derivatives of 2- or 4-cyanopyridine with the appropriate α,ω-diamines or spermidine gave 2-
pyridylimidamides 23SMB022, 23SMB026, and 23SMB054, and 4-pyridylimidamides 26SBM070
and 27SMB009 (Fig. 3).
Mammalian cell cultures. For the in vitro analysis of compound toxicity and effect against
intracellular parasites (Y strain), primary cultures of embryonic cardiomyocytes (CM) were
obtained from Swiss mice as previously reported [22]. After purification, the CM were seeded at a
density of 0.2 X 106 and 0.05 X 106 cell/well, respectively, into 24 and 96-well microplates
containing gelatin-coated cover slips. The cultures were then maintained at 37° C in Dulbecco`s
modified medium supplemented with 10% horse serum, 5% fetal bovine serum, 2.5 mM CaCl2, 1
mM L-glutamine and 2% chicken embryo extract (DMEM). For the further analysis of the effect on
intracellular parasites for Tulahuen strain (parasites expressing the Escherichia coli -galactosidase
gene), monolayers of mouse L929 fibroblasts were cultivated (4 X 103 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 (FBS) and 2 mM glutamine (RPMIS), as reported [23].
Parasites. Bloodstream trypomastigote forms (BT) of the Y strain were obtained from the
blood samples of infected albino Swiss mice at the peak of parasitemia. The purified parasites were

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resuspended in Eagle medium modified by Dulbecco (DME) supplemented with 10% FBS (DMES)
as reported [22]. The effect on intracellular forms was investigated by both the infection of L929
cell lineages with tissue culture derived trypomastigotes (Tulahuen strain expressing the E. coli -
galactosidase gene) and the infection of CM with bloodstream trypomastigotes (Y strain) using a
10:1 ratio, following previously established protocols [15,23]. Stock solutions were prepared in
dimethyl sulfoxide (DMSO) with the final concentration of the solvent never exceeding 0.6% and
10% for in vitro and in vivo analysis, which did not exert any toxicity (data not shown).
Benznidazole (Bz) (2-nitroimidazole; Laboratório Farmacêutico do Estado de Pernambuco
[LAFEPE], Brazil) was used as reference drug.
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Cytotoxicity in vitro tests. CM were incubated for 24 h at 37° C with different concentrations
of each compound (up to 96 µM) diluted in DMEM (without phenol red), their morphology and
spontaneous contractibility evaluated by light microscopy and then their cellular viability
determined by the alamarBlue® assay. For this colorimetric bioassay, 10 L alamarBlue®
(Invitrogen) was added to each well and the plate further incubated for 24 h, after which the
absorbances at 570 and 600 nm were measured. As negative controls, AlamarBlue® assay was also
performed in the lack of cells, running only DMEM and DMEM containing each tested compound
(at 96 µM). The results were expressed as percent difference in reduction between compound
treated and vehicle treated cells by following the manufacturer’s instructions and the value of EC50
corresponds to the concentration that reduces in 50 % the cellular viability [23].
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Trypanocidal analysis. Bloodstream trypomastigotes of the Y strain (5 X 106 per mL) were
incubated for 24 h at 37oC in RPMI in the presence or absence of serial dilutions of the compounds
(0 to 32 µM). 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) was
then calculated.

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For the assay on intracellular forms, different parasite strain and protocols were performed
using Y and Tulahuen strains. For Tulahuen-infected-L929 cells, the cultures were exposed to 1.0
g/mL diluted in RPMIS and those compounds those that presented ≥ 50% of reduction on the
parasite infection index were further screened under increasing concentrations aiming to determine
the EC50 values [23]. After 96 h of compound incubation at 37C, chlorophenol red glycoside (500
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µM) in 0.5% Nonidet 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 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 [23].
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For the analysis of the effect against intracellular amastigotes from Y strain, after 24 h of
parasite-host cell interaction, the infected cultures were washed to remove free parasites and then
incubated for another 48 h with increasing concentrations of the test compounds. CM were
maintained at 37°C in an atmosphere of 5% CO2 and air and the medium replaced every 24 h. Then,
untreated and treated infected CM were fixed and stained with Giemsa solution and the mean
number of infected host cells and of parasites per infected cells scored as reported [16]. 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 (II - percentage of infected cells times the average number of
intracellular amastigotes per infected host cell) [24]. Triplicate assays were run in the same plate
and at least two assays performed in each analysis.
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Mouse acute toxicity. In order to determine the NOAEL (No Observed Adverse Effect
Level), each dose of the tested compounds (12.5 up to 200 mg/kg) was injected by intraperitoneal
route (ip) individually in g Swiss Webster female mice (20-23 g). On days 2 and 3, mice were
inspected for toxic and sub-toxic symptoms according to OECD guidelines (Organization for

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Economic Co-operation and Development). Forty-eight hours after compound injection, the
NOAEL values were determined and plasma biochemical analysis performed for alanine
aminotransferase (ALT), urea and creatine kinase (CK) as reported [16].
Mouse infection and treatment schemes. Male Swiss mice were obtained from the
Fundação Oswaldo Cruz (FIOCRUZ) animal facilities (Rio de Janeiro, Brazil). Mice were housed
at maximum 6 per cage and kept in a conventional room at 20-24ºC under a 12/12-h light/dark
cycle. The animals were provided with sterilized water and chow ad libitum. Infection was
performed by ip injection of 104 bloodstream trypomastigotes (Y strain). The animals (18-21 g)
were divided into the following groups: uninfected (non-infected and untreated); untreated (infected
with T. cruzi and treated with vehicle); and treated – infected and treated (ip) with 0.5 up to 20
mg/kg/day test compound (up to 0.2 mL) or with 100 mg/kg/day Bz (po). The mouse treatment
started at 5 days post infection (dpi) followed by (i) one second dose at 8 dpi or (ii) five consecutive
daily doses. For Bz treatment, infected mice received 0.2 mL oral dose (gavage) following the same
therapeutic schemes as above described [15].
Parasitemia, mortality rates and ponderal curve analysis. Parasitemia was individually
checked by direct microscopic counting of parasites in 5 µL of blood, as described before [25].
Body weight was weekly evaluated, and mortality checked daily until 30 days post treatment and
expressed as percentage of cumulative mortality (% CM) [15].
Ethics. All procedures were carried out in accordance with the guidelines established by the
FIOCRUZ Committee of Ethics for the Use of Animals (CEUA LW-16/2013).
RESULTS

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The screening of the eight novel arylimidamides (Figs. 1-3) against bloodstream
trypomastigotes (Y strain) revealed that after 2 h of incubation at 37oC, 18SAB075 and 16DAP002
showed considerable activity, displaying EC50 values of 11 and 14 µM (Table 1). After 24 h of
incubation, both were more effective than Bz, displaying EC50 values of 0.3 and 3 µM, respectively,
with the former molecule exhibiting about 43 fold higher trypanocidal effect than the reference drug
Bz (Table 1). In vitro analysis of cytotoxicity on cardiac cell cultures performed by the
alamarBlue® assay showed that up to 32 µM, none of the studied compounds caused loss of
cellular viability (Table 1). AlamarBlue® performed in the lack of cells, with DMEM containing or
not each tested compound (at 96 µM) showed none alteration in the absorbance measurements (data
not shown). When tested on T. cruzi-infected host cells (Tulahuen strain transfected with β-
galactosidase gene) under a standardized screening protocol using a fixed concentration of 1 µg/mL
[23], again both 16DAP002 and 18SAB075 were effective (Table 2). Next, both AIAs were again
screened against Tulahuen-infected host cells in order to determine the EC50 values. The findings
showed that 18SAB075 was the most active with values of 1.5 µM after 96 h of incubation,
comparable to Bz and 16DAP002 (Table 3). The further analysis of the effect of 18SAB075 against
T. cruzi-infected cardiac cells using another strain (Y strain) confirmed the promising activity (EC50
= 0.9 µM) of this AIA, especially noticed at the mean number of infected cardiac cells data, without
causing loss of host cell viability when the infected cultures were incubated with concentrations up
to 32 µM (Table 4, A and B). Although no loss of viability was noticed up to ≤ 32 µM screened by
alamarBlue® method, the light microscopy analysis demonstrated that 16SAB002 at concentrations
> 3.5 µM induced cellular vacuolization and impaired the contractibility of the cardiac cells in vitro.
Besides, no effect on the levels of T. cruzi infection was observed when non-toxic doses (≤ 3.5 µM)
of this AIA were tested (data not shown).

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Next, due to the higher selectivity indexes of 18SAB075 on bloodstream trypomastigotes
(>106 – Table 1) and on intracellular forms (40 – Table 4), this compound was moved to in vivo
analysis.
Our first in vivo step was to evaluate aspects of acute toxicity of 18SAB075 aiming to
determine its NOAEL value. The administration of this AIA via the ip route to Swiss female mice
(12.5 up to 200 mg/kg) followed for 48 h showed that none of the doses caused death. Up to 50
mg/kg no alteration on animal behavior (as compared to vehicle treated mice) was found. However,
increased doses induced visible neurological disorders like tremors, ataxia and hyperactivity (data
not shown). The gross pathology performed at 48 h after drug administration showed that 200
mg/kg induced hemorrhagic hepatic signs (data not shown). This hepatic injury was confirmed by
the biochemical plasma analysis of the higher dose as increased levels of AST and ALT were found
as compared to the animals from the other groups (data not shown). Then, the efficacy of
18SAB075 was tested in Swiss male mice inoculated with 104 bloodstream parasites using a
therapeutic scheme employing doses administrated once a day, at 5 and 8 dpi (Fig. 4) that
corresponds to the onset and parasitemia peak, respectively in this animal model (Batista e cols.,
2010). Only parasitemia positive mice were used and Bz treatment was run in parallel using
standard protocol (100mg/kg via p.o.), following the same therapeutic scheme as above described
(Fig. 4). Although 20 and 10 mg/kg did not reduce or only slightly impaired (20%), respectively,
the parasitemia levels, the lower dose (5 mg/kg) resulted in 50% of decrease in the parasitism peak
decrease, also leading to 40% of animal survival while the other mice (except for Bz group), all
died (Fig. 4).
Then, aiming to allow a longer plasma exposure of the compound but taking into
consideration the use of maximum non-toxic doses (up to 50 mg/kg), additional studies were
performed providing the AIA for 5 daily consecutive days, starting at 5 dpi (Fig. 5). As expected for
this experimental mouse model of T. cruzi acute infection, infected and vehicle-treated animals

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presented high parasitemia and no animal survival (Figs. 4 and 5). When 18SAB075 was
administrated via ip route, none of the doses reduced parasitemia or protected against mortality. As
expected, Bz completely suppressed the parasitemia and gave 100% of survival (Fig. 5). Also, none
of the doses were able to protect the animals against the body weight loss (data not shown) induced
by T. cruzi infection in this experimental acute model [26].
DISCUSSION
Presently, structural variations include seven different linkers between the two AIA moieties
and three different outer aromatic rings. Those molecules containing multiple or fused aromatic
rings in their linkers were active against the bloodstream and intracellular parasites. The most active
compound, 18SAB075, contains a diphenylacetylene linker, which offers extended conjugation.
The second most active, 16DAP002, contains a carbazole system as its linker, which also offers
extended conjugation but a more rigid conformation due to the fused rings. Both compounds
displayed AIA nitrogen atoms attached directly to aromatic rings. Xylene derivative 26SMB070,
which was less active, bears an aromatic ring in its linker, but the nitrogen atoms are attached to
aliphatic carbons. Analogues of the two most active compounds containing the same linkers but
different outer rings were not included in this study.
Chemotherapeutic options for treating chagasic patients is currently dependent on only two
nitro derivative drugs, namely benznidazole (2-nitroimidazole; Laboratório Farmacêutico do
Estado de Pernambuco [LAFEPE], Brazil) and nifurtimox (5-nitrofuran, Bayer 2502; Bayer,
Germany) [27]. Both exhibit severe side effects resulting in discontinuation of the treatment, low
effectiveness, especially in the later chronic phase, demanding the identification of novel drugs to
treat this devastating chronic pathology. However, there is a lack of interest from most
pharmaceutical companies for drug discovery of new anti-T. cruzi agents mainly due to the long
time and high costs associated to a drug pipeline for this as well as other neglected parasitic

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illnesses that afflict millions of the poorest people worldwide [28]. In this context, the present aim
was to explore the possibility of finding new amidine derivatives with considerable activity against
T. cruzi in vitro and in vivo, which possess the potential to act as clinical candidates against this
parasitic neglected disease. In this vein, eight novel AIAs were screened against bloodstream and
intracellular forms of the parasite and two of them, 18SAB075 and 16 DAP002, demonstrated
higher activity, with EC50 values below 4 µM. Both compounds were effective against different
strains (Y and Tulahuen) and due to the high selectivity, 18SAB075 was further examined within in
vivo to ascertain preliminary acute toxicity and for efficacy determination using acute mouse model
of T. cruzi infection (male Swiss mice infected with Y strain). The follow up of acute toxicity signs
for 48 h after 18SAB075 administration via ip showed that although up to 50mg/kg none alterations
could be noticed, considerable toxic side effects like ataxia, hyperactivity and tremors were found
with doses 100 and 200 mg/Kg. The gross pathology confirmed that the higher dose also induced
hemorrhagic hepatic signs that were corroborated by increased plasma levels of hepatic markers
like AST. Thus, efficacy analysis was further explored only using doses ≤ 20mg/kg/day, never
exceeding cumulative doses of 50 mg/kg. Bz was included as a reference drug given at its effective
dose of 100 mg/kg/day [23]. The ip administration of 18SAB075, in all assayed therapeutic
schemes (two and five days of treatment) using non-toxic doses failed to demonstrate the activity of
this diphenylacetylene AIA in vivo while Bz not only suppressed the parasitemia but also induced
100% protection against mortality due to the experimental infection as described [26]. Previous data
using bis-AIAs like the 2,5-bis[2-(2-propoxy)-4-(2-pyridylimino)aminophenyl]furan - DB 766 [15]
and a related analog, DB 1831 [16] showed their high activity in vivo in the acute experimental
mouse models of T. cruzi, exhibiting similar trypanocidal effect as Bz. In fact, under the use of a
same highly stringent experimental in vivo model as presently performed for 18SAB075, both
previous studies reported that the presence of pyrimidine and pyridine units of bis-AIAs seemed to

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be advantageous for T. cruzi activity [14], supporting the continuity of pre-clinical studies of novel
related molecules aiming to find new alternatives for treating Chagas disease.
ACKNOWLEDGMENTS
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
e Tecnológico (CNPq) and Fundação Oswaldo Cruz, PDTIS, PROEP/CNPq/Fiocruz and CAPES.
Support was received from The Bill and Melinda Gates Foundation through a subcontract with the
Consortium for Parasitic Drug Development (CPDD).
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LEGENDS
Figure 1: Synthesis of 16DAP002 and 16SAB079. Reagents and conditions: (a) appropriate
diamine, ethanol (plus acetonitrile for 16DAP022), 0-25 °C, overnight (66-71%).
Figure 2: Synthesis of 18SAB075. Reagents and conditions: (a) 2-cyanopyridine, lithium
bis(trimethylsily)amide, tetrahydrofuran, 25 °C, 2 days, (16%).
Figure 3: Synthesis of 23SMB022, 23SMB026, 23SMB054, 26SMB070, and 27SMB009.
Reagents and conditions: (a) sodium methoxide, methanol, 25 °C, 1 week; (b) appropriate
α,ω-diamine (or spermidine for 23SMB054), 4 M HCl in dioxane, methanol, 25 °C (5-92%).
Figure 4: In vivo effect of 18SAB075 on T. cruzi infection using male Swiss mice inoculated with
104 bloodstream trypomastigotes (Y strain). The activity of 5, 10 and 20 mg/kg/day
18SAB075 (ip) and of benznidazole (100 mg/kg/day by gavage) given at 5 and 8 dpi was
evaluated through the (A) parasitemia levels and (B) cumulative mortality.
Figure 5: In vivo effect of 18SAB075 on T. cruzi infection using male Swiss mice inoculated with
104 bloodstream trypomastigotes (Y strain). The activity of 0.5, 1 and 5 mg/kg/day
18SAB075 (ip) and of benznidazole (100 mg/kg/day by gavage) given for 5 consecutive days
(5-9 dpi) was evaluated through the (A) parasitemia levels and (B) cumulative mortality.

379

700
600
500
400
300
200
100
0
Vehicle
Bz
18SAB075 20 mg/kg
18SAB075 10 mg/kg
18SAB075 5 mg/kg
5
7
9
11
13
15
17
19
Days post infection
100
50
0
Vehicle
Bz
18SAB075 20 mg/kg
18SAB075 10 mg/kg
18SAB075 5 mg/kg
0
5
10
15
20
25
30
35
40
Days post Infection

600
500
400
300
200
100
0
Vehicle
Bz
18SAB075 5 mg/kg
18SAB075 1 mg/kg
18SAB075 0.5 mg/kg
5
7
9
11
13
15
17
19
Days post infection
100
50
Vehicle
Bz
18SAB075 5 mg/kg
18SAB075 1 mg/kg
18SAB075 0.5 mg/kg
0
0
5
10
15
20
25
30
35
40
Days post infection

TABLE I Trypanocidal activity (EC50) of the studied arylimidamides against bloodstream
trypomastigotes of T. cruzi (Y strain) and their selectivity index (SI) related to
cardiomyocytes (EC50)
Compounds
EC50 (µM)
SIa
Trypomastigotes
2 h
Cardiomyocytes
24 h
24 h
24 h
18SAB075
16DAP002
27SMB009
23SMB054
23SMB022
23SMB026
26SMB070
16SAB079
Benzinidazole
11 ± 1
14 ± 3
>100
>100
>100
>100
>100
>100
>100
0.3 ± 0
3 ± 1
>32
>32
>32
>32
>32
>32
>32
>32
1000
>106
>11
85 ± 21
>100
>0.4
-
-
>100
>100
-
>100
-
>100
-
13 ± 2b
77
aTreatment for 24 h at 37°C; Selectivity index (SI) = EC50 cardiomyocytes/EC50 parasite
b Silva et al 2011

TABLE II Activity of arylimidamides (at 1 µg/mL) against intracellular forms of
T. cruzi (Tulahuen strain transfected with β-galactosidase) after treatment for 96 h at 37°C
Compounds
% Inhibition of the host cell infection
58 ± 31
50± 7
15 ± 8
4 ± 5
3 ± 4
1 ± 1
0 ± 0
0 ± 0
82 ± 3
16DAP002
18SAB075
26SMB070
16SAB079
23SMB054
23SMB022
27SMB009
23SMB026
Benznidazole

TABLE III In vitro effect (EC50 values) of arylimidamides against intracellular forms of
T. cruzi (Tulahuen strain transfected with β-galactosidase) after treatment for 96 h at 37°C
Compounds
EC50 (µM)
4.1 ± 1.8
1.5 ± 0.2
2.6 ± 0.8
16DAP002
18SAB075
Benznidazole

TABLE IV Activity (EC50 values) and selective index (SI) of 18SAB075 against intracellular
forms of T. cruzi (Y strain) after treatment for 48 h at 37°C
18SAB075
EC50 (µM)
SI
0.8 ± 0.1
6.0 ± 2.0
0.9 ± 0.1
40
5
% Host cell Infection
Mean number of parasite/per infected host cell
Infection Index*
36
A
B
0.02 mm
0.02 mm
•
*Host cell infection x number of parasite/infected host cell as reported in Batista et al.. 2010
•
•
A: T.cruzi-infected cardiac cells incubated with vehycle
B: T.cruzi-infected cardiac cells incubated with 32 µM.
•
Arrow: Intracellular parasites