Phenotypic evaluation and in silico ADMET properties of novel arylimidamides in acute mouse models of Trypanosoma cruzi infection

Article abstract of DOI:10.2147/DDDT.S120618

Arylimidamides (AIAs), previously termed as reversed amidines, present a broad spectrum of activity against intracellular microorganisms. In the present study, three novel AIAs were evaluated in a mouse model of Trypanosoma cruzi infection, which is the causative agent of Chagas disease. The bis-AIAs DB1957, DB1959 and DB1890 were chosen based on a previous screening of their scaffolds that revealed a very promising trypanocidal effect at nanomolar range against both the bloodstream trypomastigotes (BTs) and the intracellular forms of the parasite. This study focused on both mesylate salts DB1957 and DB1959 besides the hydrochloride salt DB1890. Our current data validate the high activity of these bis-AIA scaffolds that exhibited EC₅₀ (drug concentration that reduces 50% of the number of the treated parasites) values ranging from 14 to 78 nM and 190 to 1,090 nM against bloodstream and intracellular forms, respectively, also presenting reasonable selectivity indexes and no mutagenicity profile predicted by in silico absorption, distribution, metabolism, excretion, and toxicity (ADMET). Acute toxicity studies using murine models revealed that these AIAs presented only mild toxic effects such as reversible abdominal contractions and ruffled fur. Efficacy assays performed with Swiss mice infected with the Y strain revealed that the administration of DB1957 for 5 consecutive days, with the first dose given at parasitemia onset, reduced the number of BTs at the peak, ranging between 21 and 31% of decrease. DB1957 was able to provide 100% of animal survival, while untreated animals showed 70% of mortality rates. DB1959 and DB1890B did not reduce circulating parasitism but yielded >80% of survival rates.

Full text of DOI:10.2147/DDDT.S120618

Drug Design, Development and erapy
Dovepress
open access to scientific and medical research
Open access FullText article
O r i g i n a l r e s e a r c h
Phenotypic evaluation and in silico aDMeT
properties of novel arylimidamides in acute
mouse models of Trypanosoma cruzi infection
This article was published in the following Dove Press journal:
Drug Design, Development andTherapy
3 April 2017
Number of times this article has been viewed
cristiane França da silva1
Denise da gama Jaén
Batista1
Abstract: Arylimidamides (AIAs), previously termed as reversed amidines, present a broad
spectrum of activity against intracellular microorganisms. In the present study, three novel
AIAs were evaluated in a mouse model of Trypanosoma cruzi infection, which is the causative
agent of Chagas disease. The bis-AIAs DB1957, DB1959 and DB1890 were chosen based on
a previous screening of their scaffolds that revealed a very promising trypanocidal effect at
nanomolar range against both the bloodstream trypomastigotes (BTs) and the intracellular forms
of the parasite. This study focused on both mesylate salts DB1957 and DB1959 besides the
hydrochloride salt DB1890. Our current data validate the high activity of these bis-AIA scaffolds
that exhibited EC50 (drug concentration that reduces 50% of the number of the treated parasites)
values ranging from 14 to 78 nM and 190 to 1,090 nM against bloodstream and intracellular
forms, respectively, also presenting reasonable selectivity indexes and no mutagenicity profile
predicted by in silico absorption, distribution, metabolism, excretion, and toxicity (ADMET).
Acute toxicity studies using murine models revealed that these AIAs presented only mild toxic
effects such as reversible abdominal contractions and ruffled fur. Efficacy assays performed
with Swiss mice infected with the Y strain revealed that the administration of DB1957 for
5 consecutive days, with the first dose given at parasitemia onset, reduced the number of BTs
at the peak, ranging between 21 and 31% of decrease. DB1957 was able to provide 100% of
animal survival, while untreated animals showed 70% of mortality rates. DB1959 and DB1890B
did not reduce circulating parasitism but yielded .80% of survival rates.
Julianna siciliano de araújo1
edézio Ferreira cunha-
Junior2
chad e stephens3
Moloy Banerjee4
abdelbasset a Farahat4,5
senol akay4
Mary K Fisher3
David W Boykin4
Maria de nazaré correia
soeiro1
1laboratory of cellular Biology,
2laboratory of Biochemistry
of Trypanosomatids, Fundação
Oswaldo cruz, rio de Janeiro,
Brazil; 3Department of chemistry
and Physics, augusta University,
augusta, 4Department of chemistry,
georgia state University, atlanta, ga,
Usa; 5Department of Pharmaceutical
Organic chemistry, Faculty of
Pharmacy, Mansoura University,
Mansoura, egypt
Keywords: Chagas disease, arylimidamides, experimental chemotherapy, in vivo assays
Introduction
American trypanosomiasis or Chagas disease (CD) caused by Trypanosoma cruzi
affects .6 million people worldwide, mostly in Latin America.1 Another public health
concern is that the prevalence of T. cruzi infection in non-endemic countries such as
Australia, Canada, Japan, Spain, and the US is increasing due to global migration of
Latin American immigrants unknowingly carrying the infection.2,3
After more than a hundred years since its discovery, this pathology still presents
several challenges. The silent chronic morbidities and high mortalities, the prolonged
and expensive treatment courses without being effective for the late-stage disease and
the current barriers to the access to essential medicines demand urgent attention for
this neglected disease.4,5
correspondence: Maria de nazaré
correia soeiro
laboratory of cellular Biology, Fundação
Oswaldo cruz, Maria de nazaré correia
soeiro, avenue Brasil 4365, Manguinhos,
rio de Janeiro, rio de Janeiro ceP, Brazil
Tel +55 21 2562 1368
Only two drugs are available for the treatment of CD: nifurtimox (Nf), a nitrofuran
with a fast plasmatic clearance profile (half-life of 3 h),2 and benznidazole (Bz), a nitroim-
idazole, both of which show variable activity across different T. cruzi strains.6
Fax +55 21 2562 1432
email soeiro@ioc.fiocruz.br
submit your manuscript | www.dovepress.com
Dovepress
Drug Design, Development andTherapy 2017:11 1095–1105
1095
© 2017 da Silva et al. This work is published and licensed by Dove Medical Press Limited. The full terms of this license are available at https://www.dovepress.com/terms.php
and incorporate the Creative Commons Attribution – Non Commercial (unported, v3.0) License (http://creativecommons.org/licenses/by-nc/3.0/). By accessing the work you
hereby accept the Terms. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed. For permission
for commercial use of this work, please see paragraphs 4.2 and 5 of our Terms (https://www.dovepress.com/terms.php).
http://dx.doi.org/10.2147/DDDT.S120618

da silva et al
Dovepress
The occurrence of T. cruzi strains exhibiting a naturally to increase solubility. In this study, the bis-AIAs DB1957,
resistant profile to Bz and Nf may be related, at least in part, to DB1959 and DB1890B were evaluated using an experimental
ABCG-like transporters, which are overexpressed in several mouse model for acute T. cruzi infection with the goal of
naturally resistant strains.7 Other significant drawbacks in identifying novel antiparasitic drug candidates for possible
the current medicines for CD are that both drugs require future alternative therapies for CD.
sustained periods of administration (30–60 days) and give
low curative rates for the late-chronic stage.8–10
Materials and methods
Pentamidine, an aromatic diamidine, has been exten- compounds
sively used as an antiparasitic drug for sleeping sickness and The syntheses of DB1957, DB1959 and DB1890B (Figure 1)
leishmaniasis.8 Several reports have demonstrated its effect were performed according to methods (Scheme S1) pre-
toward T. cruzi in vitro and in vivo leading to a reduction viously described.23–25 Bz, purchased from Laboratório
in parasitemia levels and providing enhanced mice survival Farmacêutico do Estado de Pernambuco (Laboratório
compared to untreated controls.11 Many of its derivatives, Farmacêutico do Estado de Pernambuco), Brazil, was used
especially the arylimidamides (AIAs), show potent efficacy as the reference drug.18 Stock solutions were prepared in
against intracellular parasites such as T. cruzi.12
dimethyl sulfoxide (DMSO), with the final concentration
AIAs have extraordinary biological action toward dif- for the in vitro experiments never exceeding 0.6%, which
ferent trypanosomatids, both in vitro13–16 and in vivo.17–19 An did not exert any toxicity toward the parasite or mammalian
important structural characteristic of AIAs is that the imino host cells. For in vivo studies, a stock solution of the AIA
group is linked to the “anilino” nitrogen, while in classical compounds was first prepared in DMSO and then diluted
amidines, it is directly attached to an aryl ring.15 Recent using distilled and sterile water. The final concentration of
findings demonstrated that bis-AIAs are more effective than DMSO never exceeded 10%, which did not provide detect-
mono-AIAs and that some of these bis-AIAs are able to reduce able mice toxicity.18,21 Bz was dissolved in sterilized distilled
the parasitemia in addition to protecting against mortality water supplemented with 3% Tween 80, which did not cause
induced by acute infection with T. cruzi.18–21 In a previous any detectable effect on the mice.
report, our group demonstrated the in vitro efficacy of several
bis-AIAs, including DB1867, DB1862 and DB1890B, which Parasites
exhibited high trypanocidal activity against bloodstream and The Y strain of T. cruzi was used throughout the study.
intracellular forms of T. cruzi superior to that of Bz.22
Bloodstream trypomastigote (BT) forms were obtained from
The excellent trypanosomicidal action of these molecules T. cruzi-infected Swiss mice at the peak of parasitemia.26
encouraged us to perform further studies to ascertain in vivo During the analyses using intracellular forms, the parasites
safety and potency of these three bis-AIAs by using mesylate lodged within cardiac cell (CC) cultures were employed, as
salts for two of them (DB1957 and DB1959) in an effort previously reported.20
2
2
2
2
6
2
1+
+1
+1
1+
+1
1+
1+
+1
1
1
1
1
'%ꢀꢁꢂꢃ%
'%ꢀꢂꢄꢅ
&)ꢀ
2
)ꢀ&
2
2
1+
+1
+1
1+
1
1
'%ꢀꢂꢄꢂ
Figure 1 structures of compounds.
submit your manuscript | www.dovepress.com
Drug Design, Development andTherapy 2017:11
1096
Dovepress

Dovepress
aias effect on T. cruzi
in silico study of the druglikeness and
absorption, distribution, metabolism,
excretion, and toxicity (aDMeT)
using Bouin and stained with Giemsa solution. The infective
index (II) was used to compare the compound activity calcu-
lated by multiplying the percentage of infected cells by the
mean number of parasites per infected cell.17 Next, the EC50
values were calculated based on the drug concentration that
reduced 50% of the II.
properties
In silico analysis of the pharmacokinetics properties
(absorption, distribution, metabolism, and excretion
[ADME]) and toxicity of the AIAs were evaluated using
the pkCSM approach,27 which uses graph-based signatures
to predict these parameters. The platform validation was
performed using cross-validation scheme and sets of external
validation data. pkCSM presents a statistically significant
improvement in predictive power in comparison with the
methods available.27
The selectivity index (SI) was calculated as follows:
SI = LC50/EC50. The SI was calculated using the data for 24
and 48 h of incubation of the host cells with the three AIAs
for BT and intracellular parasites, respectively.
acute toxicity for mice
Acute toxicity levels were evaluated using female and male
Swiss Webster mice (20–23 g) obtained from the animal
facilities of the Oswaldo Cruz Foundation (CECAL, Rio de
Janeiro, Brazil). Mice were housed per cage and were kept in
a conventional animal room at 20°C–24°C under a 12/12-h
light/dark cycle. The animals were provided with sterilized
water and chow ad libitum. On day 1, one female mouse was
treated with each compound by injection via intraperitoneal
(ip) using escalating doses every 2 h, starting at 20 up to
200 mg/kg, and thus, the final accumulative dose reached a
total of 400 mg/kg. The mice were inspected, 48 h post-final
injection, for toxic and sub-toxic symptoms according to
OECD guidelines as reported, and at least two assays were
conducted with male and female animals.22
cc cultures and cytotoxicity assays
For the evaluation of cytotoxicity and compound activity
against intracellular parasites, primary cultures of embryonic
CCs were obtained from Swiss mice and purified following
the method previously described.26 In order to rule out toxic
effects upon mammalian host cells, uninfected CC cultures
were exposed to each compound studied at 37°C for 24
and 48 h (1.18–32 µM). Untreated cultures were used as
control samples. The cell death rates were measured by
the MTT colorimetric assay allowing the determination of
LC50 values (compound concentration that reduces 50% of
cellular viability).22 All cell cultures were maintained in an
atmosphere of 5% CO2 and air, and the assays were run at
least three times in duplicates.
Mice infection and treatment schemes
Five male Swiss mice (18–21 g) were housed per cage and
were maintained as described earlier. Infection was performed
Trypanocidal assays
The effect of the compounds against BT was evaluated by ip injection of 104 (Y strain) BTs.18 The animals were
through assaying 5×106 parasites/mL for 24 h at 37°C in divided into the following groups: uninfected (noninfected
Roswell Park Memorial Institute (RPMI) 1640 medium and untreated), untreated (infected with T. cruzi but treated
supplemented with 10% of fetal bovine serum, in the presence only with vehicle), and treated (infected and treated – ip –
of serial dilutions of the AIAs (0.001–10.6 µM). Untreated with 25 mg/kg/day AIA or orally (po) 100 mg/kg/day Bz).
parasites were used as control samples. Parasite death rates Treated mice received daily dose (0.1 mL via ip) starting
were determined by light microscopy using a Neubauer at 5 days postinfection (dpi) that corresponded to the para-
chamber that allowed the direct visualization and quantifica- sitemia onset up to 9 dpi (1 day after the parasitemia peak)
tion of the number of motile and live parasites and allowed for all compounds (AIAs and Bz). For Bz treatment, infected
EC50 (drug concentration that reduce 50% of the number of mice received 0.2 mL oral dose (po) following the same
the treated parasites) values to be calculated.22
therapeutic scheme as described earlier.
For the analysis of the effect against intracellular para-
sites, after 24 h of parasite–host cell interaction (ratio 10:1), Parasitemia and mortality rates
the infected CC cultures were washed to remove the free Parasitemia was individually checked by direct microscopic
parasites and then maintained at 37°C in an atmosphere of counting of parasites in 5 µL of blood, as described
5% CO2 and air in the presence of nontoxic concentrations of earlier.18 Mortality levels were checked until 56 dpi (cor-
each AIA (from 0.043 to 3.5 µM). Untreated infected cultures responding to 47 days after the last dose) and expressed as
were used as control. After 48 h, all CC cultures were fixed survival rates.20
submit your manuscript | www.dovepress.com
Drug Design, Development andTherapy 2017:11
1097
Dovepress

da silva et al
Dovepress
Table 1 The trypanocidal effect (µM) and si of aias and of Bz
against T. cruzi (Y strain)
Biochemical analysis
At the end point (56 dpi), the biochemical analysis of the
plasmatic levels of creatine kinase (CK), blood urea nitrogen
(BUN), andalanineaminotransferase(ALT)wasdeterminedin
theCECAL/Fiocruzplatform, usingVitros250(OrthoClinical,
Johnson & Johnson, Raritan, NJ, EUA) as reported.20
Compound
BTs (24 h/37°C)
Intracellular
amastigotes
(48 h/37°C)
EC50 (µM)
SI
EC50 (µM)
SI
DB1957
DB1959
DB1890B
Bz
0.068±0.0014*
0.078±0.01**
0.014±0.007***
12.94±1.93
17
0.19±0.12#
0.72±0.28#
1.09±0.03#
3.7±1.72
6
45
1.6
9.7
.360
statistical analysis
760
.77
Statistical analysis was carried out using an analysis of
variance (ANOVA) program with the level of significance
set at P#0.05. The data were representative of a minimum
of two experiments run in duplicate.
Notes: ec50 values, compound concentration that reduces the number of parasites
by 50%; si, that corresponds to the ratio of lc50/ec50 – for BT and intracellular
parasites calculated on lc50 values of 24 and 48 h of incubation at 37°c, respectively.
statistical analysis (aia vs Bz): *P=0.003, **P=0.003, ***P=0.0003, and #P.0.05. all
assays were run at least two times in duplicate.
All procedures were carried out in accordance with
the guidelines established and approved by the Fiocruz
Committee of Ethics for the Use of Animals (CEUA
LW16/14 and CEUA 0099/01).
Abbreviations: aia, arylimidamide; Bz, benznidazole; T. cruzi, Trypanosoma cruzi;
BT, bloodstream trypomastigote; si, selectivity index.
Next, in silico analysis of physicochemical parameters
and Lipinski’s rule of five (Table S1) besides the ADMET
properties of these AIAs were predicted using the pkCSM
Results
Previous in vitro data22 showed the excellent activity of three methodology. The findings (Tables S1 and S2) showed that
AIAs (DB1867, DB1862, and DB1890B), providing EC50 the three AIAs have quite similar physicochemical profile,
values of 0.02, 0.06, and 0.013 µM, respectively, against with low probability of permeability of Caco-2 cells for
BTs of T. cruzi after 24 h of incubation at 37°C.22 These DB1890B and DB1959 and a medium probability profile
AIAs did not exhibit toxicity against primary culture CCs achieved for DB1957. However, all three compounds showed
and reduced the infection of T. cruzi-infected CCs, yielding similar predicted permeability profiles (.95%) for human
EC50 values of 0.058, 0.016, and 0.9 µM, respectively, after intestinal absorption, which were greater than those for Bz
48 h of treatment.22 Thus, due to the low aqueous solubility (69%). The high volume of distribution of DB1957 compared
profile for HCl salts of AIAs previously noted, we decided to DB1959 and DB1890B may reflect the importance of the
to explore mesylate salts for two of the compounds studied thiophene group as the central ring instead of furan. These
(DB1957 and DB1959). The pkCSM-calculated solubility compounds are predicted to be metabolized by CYP3A4 and
for DB1957 (mesylate salt) was greater than that of the are predicted to be inhibitors of the same enzyme. Toxicity
corresponding HCL salt: 69.4 vs 8.89 µM, respectively (data predictions (Table S3) suggest that these compounds are
not shown). Similar results were observed with DB1959 expected neither to be mutagenic nor to inhibit hERG1, but
(data not shown) and its corresponding HCl salt: 188.36 vs all were predicted to inhibit hERG2. DB1890B and DB1959
24.43 µM, respectively (data not shown).
were not predicted to be hepatotoxic; however, both DB1957
Prior moving to in vivo models, the in vitro activity of and Bz were predicted to be hepatotoxic. These predictions
these three compounds was validated against bloodstream indicate the need for evaluation of hepatic markers in bio-
and intracellular forms of T. cruzi (Table 1). The findings con- chemical analysis of treated animals that was next conducted
firmed that these AIAs presented good antiparasitic effects in the efficacy mouse models.
on intracellular forms with EC50 values ranging from 0.19
Prior to this, to avoid toxic doses of the AIAs, preliminary
to 1.09 µM, all superior to that of Bz 3.7 µM (Table 1). The acute toxicity studies were conducted in female and male
AIAs were more effective and selective against the trypo- (data not shown) mice, which displayed similar results. After
mastigotes (BTs) compared to the amastigotes (intracellular 48 h of administration of the last dose, only mild toxic events
forms), exhibiting EC50 values of 0.068, 0.078, and 0.014 µM were detected, such as reversible abdominal contractions and
and SI of 17, 45, and 760 after 24 h of incubation at 37°C for ruffled fur (data not shown). Subsequently, the effectiveness
bloodstream forms using DB1957, DB1959, and DB1890B, of the AIAs was investigated in male Swiss mice inoculated
respectively (Table 1). Statistical analysis demonstrated that with 104 trypomastigotes (Y strain), using nontoxic doses
all AIAs were more potent than Bz on BT forms (P=0.003, of the three AIAs (25 mg/kg), via ip. Only animals that
P=0.003, and P=0.0003, respectively).
presented positive parasitemia were used in these studies.
submit your manuscript | www.dovepress.com
Drug Design, Development andTherapy 2017:11
1098
Dovepress

Dovepress
aias effect on T. cruzi
$
%
ꢆꢂꢅ
ꢀꢅꢅ
ꢄꢅ
ꢈꢅ
ꢉꢅ
ꢇꢅ
ꢅ
ꢆꢅꢅ
ꢇꢂꢅ
ꢇꢅꢅ
ꢀꢂꢅ
ꢀꢅꢅ
ꢂꢅ
ꢅ
ꢅ
ꢂ
ꢀꢅ
ꢀꢂ
ꢇꢅ
ꢇꢂ
ꢆꢅ
ꢅ
ꢉ
ꢄ
ꢀꢇ ꢀꢈ ꢇꢅ ꢇꢉ ꢇꢄ ꢆꢇ ꢆꢈ ꢉꢅ ꢉꢉ ꢉꢄ ꢂꢇ ꢂꢈ
GSL
GSL
8QWUHDWHG
%]
'%ꢀꢁꢂꢃ
'%ꢀꢁꢂꢁ
'%ꢀꢄꢁꢅ%
Figure 2 Treatment of T. cruzi-infected mice (inoculum of 104 per animal using BT from Y strain) with DB1957, DB1959, and DB1890B.
Notes: The activities of 25 mg/kg of compounds and 100 mg/kg of the reference drug Bz (by per oral) were also evaluated using similar therapeutic schemes at the 5–9 dpi.
Parasitemia curve (A) and survival rates (B) are shown. all assays were run at least two times. statistical analysis: all groups with P#0.05.
Abbreviations: T. cruzi, Trypanosoma cruzi; BT, bloodstream trypomastigote; Bz, benznidazole.
In this experimental acute mouse model that peaks at 8 dpi (Table 2). Regarding CK measurements, DB1890B showed
in the untreated and infected groups, the administration of the higher levels although still under the limits of the refer-
DB1959 and DB1890B gave no reduction in the parasitemia ence values and only demonstrated statistical significance
levels (Figure 2A). However, treatment of the infected mice compared with DB1959 (P=0.03; Table 2).
with DB1957 led to 21%–31% reduction in parasitemia peak
(Figure 2A). DB1890B and DB1959 gave 80% survival
Discussion
rates, whereas DB1957 yielded 100% animal survival. The
CD is one of the leading causes of infection-induced heart
infected and untreated mice groups experienced only 30%
failure in Latin America28 and remains a serious health
survival (Figure 2B). The reference drug used in the same
problem.29 The drugs currently used lead to adverse events
therapeutic scheme resulted in parasitemia suppression and
that frequently prevent the completion of the therapy regi-
gave mice survival rates of 80% (Figure 2A and B). Statistical
men of the infected patients.10 Nf is currently produced and
analysis demonstrated that all groups demonstrated no dif-
used predominantly in Central America,30 and Bz is now
ferent significance (P#0.05).
being produced only by Fundación Mundo Sano and ELEA
Biochemical analysis performed at the end point (56 dpi)
Laboratory in Argentina. A new pediatric formulation of Bz
showed only mild alterations in BUN levels when the mice
is available improving the dose accuracy given to infected
were exposed to 25 mg/kg of the three AIAs (Table 2). Also,
children.3 Unfortunately, a recent report from the BENEFIT
at 56 dpi (46 days after the last drug dose), only DB1890B
clinical trial (a prospective, multicenter, randomized study
resulted in a slight increase in the ALT plasma biochemical
involving 2,854 patients with Chagas’ cardiomyopathy who
values, while the other two AIAs did not lead to altered levels
received Bz or placebo [up to 80 days] and followed for a
mean of 5.4 years) demonstrated that although Bz reduced
Table 2 Biochemical analysis (mean ± sD values) at the 56 dpi the parasitism, it did not alter the cardiopathology clinical
progression.31 Another important challenge is that, despite
of mice blood samples from T. cruzi-infected groups treated with
25 mg/kg/day of each aia
current recommendations, ,1% of infected individuals are
treated with the available drugs.32 Thus, there is an urgent
need for new safer and more selective drugs and new strate-
gies for treatment of this illness, especially for the chronic
late phase.5,31
Many AIAs such as DB76618 and DB1831,20 among
others,12 have superior in vitro trypanocidal activity to that of
the related classical aromatic amidines as well as the reference
Compound
BUN
ALT
CK
DB1957
DB1959
DB1890B
61.2±6.51
44.8±11.17
47.05±19.87
36.88±6.08
37.5±3.54
141±147.08
414.67±295.16
151.5±67.18*
1,051.5±212.84*
Notes: reference values for healthy mice (cecal/Fiocruz): BUn: 18–29, alT:
28–132, and cK: 68–1,070. statistical analysis: *P=0.03 (DB1959/DB1980B).
Abbreviations: sD, standard deviation; T. cruzi, Trypanosoma cruzi; aia, arylimi-
damide; BUn, blood urea nitrogen; alT, alanine aminotransferase; cK, creatine
kinase.
submit your manuscript | www.dovepress.com
Drug Design, Development andTherapy 2017:11
1099
Dovepress

da silva et al
Dovepress
drug for CD. Our previous study of the in vitro biological need for new candidates for alternative therapy is urgent.
efficacy of seven novel AIAs on BTs and intracellular amas- In this sense, aromatic amidines represent a promising class
tigotes of T. cruzi gave very promising results.22 In view of of antiparasitic compounds that deserve further optimization
these positive data, three AIAs were advanced to in vivo and deeper pre-clinical analysis.
studies. In addition, theoretical analysis of the druglikeness
was predicted by in silico analysis. The development of
Acknowledgments
This work was supported by Fundação Carlos Chagas Filho
computational approaches to estimate pharmacokinetic and
toxicity properties facilitates the progression of novel drug
de Amparo a Pesquisa do Estado do Rio de Janeiro (FAPERJ;
candidates.33 The prediction of metabolism or inhibition by
E-26/102.839/2011; 203636 and 200381), Conselho
the CYP3A4 indicates the need to perform further studies of
Nacional de Desenvolvimento Científico e Tecnológico
interaction with drugs that inhibit the enzymes such as ritona-
vir and lopinavir.34 These compounds are predicted neither to
Oswaldo Cruz, Program for Technological Development
(CNPq; 555010/2008-2, 301372/2015-2), Fundação
be mutagenic nor to inhibit hERG I; however, interaction with
in Tools for Health, PAEF/CNPq/Fiocruz, and CAPES
hERG2 is predicted. It is interesting to point out that these
(23038.009424/2012-50, 23038008979201/10-1). MDNCS
predictions are in line with the experimental data on cardio-
myocytes, since hERG2 is not expressed in the heart.35
thank the Program for Technological Development in Tools
is a research fellow of CNPq and CNE research. The authors
In vivo studies demonstrated a low acute toxicity pro-
for Health-PDTIS-Fiocruz for use of its facilities. The
file for these three compounds using both healthy female
present study was supported by grants from, in part by, the
and male mice, which only displayed mild and reversible
Bill & Melinda Gates Foundation through a subcontract
effects up to 400 mg/kg. Despite showing in vitro potency,
with the Consortium for Parasitic Drug Development (to
DB1890B, DB1957, and DB1959 did not suppress the para-
DWB – R01AI64200). The authors also thank Marcos
sitemia peak when nontoxic doses were used by the ip route
Meuser Batista for his assistance in their experiments and
(25 mg/kg), while the reference drug (dose of 100 mg/kg)
Eduardo Caio Torres-Santos for his assistance in ADMET
suppressed it completely. It is worthy to note that DB1890B
in silico analysis.
and DB1959 provided $80% mice survival rates, similar
to that of Bz, while untreated animals presented with 30%
Disclosure
survival. More importantly, DB1957, which had the high-
The authors report no conflicts of interest in this work.
est predicted VDss, showed a 21%–31% reduction in peak
parasitemia and provided a 100% survival rate, which is
References
consistent with the control of the replication of the intracel-
1. World Health Organization [webpage on the Internet]. WHO Fact
lular parasitism into the tissues.
Sheet No 340; 2015. Available from: http://www.who.int/mediacentre/
factsheets/fs340/en/. Accessed November 3, 2016.
As previously discussed, a novel anti-T. cruzi entity
must be highly active against intracellular parasites (the
proliferative forms in the vertebrate hosts) and bloodstream
forms.12,36,37 The three AIAs studied here range from 2.7 to
77 times less active against intracellular amastigotes than the
bloodstream forms. It is intriguing that DB1957 is the most
active compound in vivo (regarding survival rates of 100%)
and more potent (0.19 µM) against the intracellular forms.
Perhaps, in the future, more attention should be focused on
optimization of in vitro activity of target compounds against
intracellular amastigotes, which are the multiplying forms
present in the host cell cytoplasm that may be distributed to
different organs and tissues forming residual parasite nests.
2. Barrett MP, Croft SL. Management of trypanosomiasis and
leishmaniasis. Br Med Bull. 2012;104:175–196.
3. Drugs for Neglected Disease initiative (DNDi, 2016) [webpage on the
Internet]. Paediatric dosage form of benznidazole (Chagas). Available
from: http://www.dndi.org/diseases-projects/portfolio/benznidazole-
paedriatric-dosage-form.html. Accessed November 3, 2016.
4. Hotez PJ, Dumonteil E, Woc-Colburn L, et al. Chagas disease: “the new
HIV/AIDS of the Americas”. PLoS Negl Trop Dis. 2012;6:e1498.
5. Chatelain E. Chagas disease drug discovery: toward a new era. J Biomol
Screen. 2015;20(1):22–35.
6. Teston AP, Monteiro WM, Reis D, et al. In vivo susceptibility to
benznidazole of Trypanosoma cruzi strains from the western Brazilian
Amazon. Trop Med Int Health. 2013;18(1):85–95.
7. Zingales B, Araujo RG, Moreno M, et al. A novel ABCG-like transporter
of Trypanosoma cruzi is involved in natural resistance to benznidazole.
Mem Inst Oswaldo Cruz. 2015;110(3):433–444.
8. Soeiro MN, Dantas AP, Daliry A, et al. Experimental chemotherapy
for Chagas disease: 15 years of research contributions from in vivo and
in vitro studies. Mem Inst Oswaldo Cruz. 2009;104(suppl 1):301–310.
9. Apt W. Current and developing therapeutic agents in the treatment of
Chagas disease. Drug Des Devel Ther. 2010;24:243–253.
10. Maya JD, Orellana M, Ferreira J, Kemmerling U, López-Muñoz R,
Morello A. Chagas disease: present status of pathogenic mechanisms
and chemotherapy. Biol Res. 2010;43(3):323–331.
Conclusion
Since the current treatment for CD is far from being ideal,
presenting serious drawbacks related to efficacy, especially
at the later chronic stage, and low safety profile, a compelling
submit your manuscript | www.dovepress.com
Drug Design, Development andTherapy 2017:11
1100
Dovepress

Dovepress
aias effect on T. cruzi
11. Díaz MV, Miranda MR, Campos-Estrada C, et al. Pentamidine exerts
in vitro and in vivo anti Trypanosoma cruzi activity and inhibits the
polyamine transport in Trypanosoma cruzi. Acta Trop. 2014;134:1–9.
12. Soeiro MN, Werbovetz K, Boykin DW, Wilson WD, Wang MZ,
Hemphill A. Novel amidines and analogues as promising agents against
intracellular parasites: a systematic review. Parasitology. 2013;140(8):
929–951.
13. Silva CF, Batista MM, Mota RA, et al. Activity of “reversed” diamidines
against Trypanosoma cruzi in vitro. Biochem Pharmacol. 2007;73:
1939–1946.
14. Silva CF, Meuser MB, De Souza EM, et al. Cellular effects of reversed
amidines on Trypanosoma cruzi. Antimicrob Agents Chemother. 2007;
51(11):3803–3809.
15. Rosypal AC, Werbovetz KA, Salem M, et al. Inhibition by Dications of
in vitro growth of Leishmania major and Leishmania tropica: causative
agents of old world cutaneous leishmaniasis. J Parasitol. 2008;94(3):
743–749.
24. Wang MZ, Zhu X, Srivastava A, et al. Novel arylimidamides for treat-
ment of visceral Leishmaniasis. Antimicrob Agents Chemother. 2010;
54:2507–2516.
25. Chai Y, Munde M, Kumar A, et al. Structure-dependent binding of
arylimidamides to the DNA minor groove. Chembiochem. 2014;15(1):
68–79.
26. Meirelles MN, de Araujo-Jorge TC, Miranda CF, de Souza W,
Barbosa HS. Interaction of Trypanosoma cruzi with heart muscle
cells: ultrastructural and cytochemical analysis of endocytic vacuole
formation and effect upon myogenesis in vitro. Eur J Cell Biol. 1986;
41(2):198–206.
27. Pires DE, Blundell TL, Ascher DB. pkCSM: predicting small-molecule
pharmacokinetic and toxicity properties using graph-based signatures.
J Med Chem. 2015;58(9):4066–4072.
28. Roman-Campos D, Sales-Júnior P, Duarte HL, et al. Cardiomyocyte
dysfunction during the chronic phase of Chagas disease. Mem Inst
Oswaldo Cruz. 2013;108(2):243–245.
16. Timm BL, da Silva PB, Batista MM, et al. In vitro and in vivo biological
effects of novel arylimidamide derivatives against Trypanosoma cruzi.
Antimicrob Agents Chemother. 2014;58(7):3720–3726.
17. Da Silva CF, Batista MM, Batista DG, et al. In vitro and in vivo
studies of the trypanocidal activity of a diarylthiophene diamidine
against Trypanosoma cruzi. Antimicrob Agents Chemother. 2008;
52(9):3307–3314.
29. Soeiro MN, de Castro SL. Screening of potential anti-Trypanosoma
cruzi candidates: in vitro and in vivo studies. Open Med Chem J.
2011;5:21–30.
30. Marin-Neto JA, Rassi A Jr, Avezum A Jr, et al; BENEFIT Investigators.
The BENEFIT trial: testing the hypothesis that trypanocidal therapy
is beneficial for patients with chronic Chagas heart disease. Mem Inst
Oswaldo Cruz. 2009;104(suppl 1):319–324.
18. Batista DG, Batista MM, de Oliveira GM, et al. Arylimidamide DB766,
a potential chemotherapeutic candidate for Chagas’ disease treatment.
Antimicrob Agents Chemother. 2010;54(7):2940–2952.
19. De Araújo JS, Da Silva CF, Batista DG, et al. In vitro and in vivo studies
of the biological activity of novel arylimidamides against Trypanosoma
cruzi. Antimicrob Agents Chemother. 2014;58(7):4191–4195.
20. Da Silva CF, Batista DG, Oliveira GM, et al. In vitro and in vivo inves-
tigation of the efficacy of arylimidamide DB1831 and its mesylated salt
form – DB1965 – against Trypanosoma cruzi infection. PLoS One. 2012;
7(1):e30356.
21. Guedes-da-Silva FH, Batista DG, Meuser MB, et al. In vitro and in vivo
trypanosomicidal action of novel arylimidamides against Trypanosoma
cruzi. Antimicrob Agents Chemother. 2016;60(suppl 4):2425–2434.
22. Da Silva CF, Daliry A, Silva PB, et al. The efficacy of arylimid-
amides against Trypanosoma cruzi in vitro. Parasitology. 2011;138:
1863–1869.
31. Morillo CA, Marin-Neto JA, Avezum A, et al. Randomized trial of
benznidazole for chronic Chagas’ cardiomyopathy. N Engl J Med. 2015;
373:1295–1306.
32. Maguire JH. Treatment of Chagas’ disease – time is running out. N Engl
J Med. 2015;373:1369–1370.
33. Cumming JG, Davis AM, Muresan S, Haeberlein M, Chen H. Chemical
predictive modelling to improve compound quality. Nat Rev Drug
Discov. 2013;12(12):948–962.
34. Li F, Lu J, Ma X. CPY3A4-mediated lopinavir bioactivation and its
inhibition by ritonavir. Drug Metab Dispos. 2012;40:18–24.
35. Gutman GA, Chandy KG, Grissmer S, et al. International Union of
Pharmacology. LIII. Nomenclature and molecular relationships of
voltage-gated potassium channels. Pharmacol Rev. 2005;57(suppl 4):
473–508.
36. Molina I, Gómez I, Prat J, et al. Randomized trial of posaconazole and
benznidazole for chronic Chagas’ disease. N Engl J Med. 2014;370:
1899–1908.
37. Patterson S, Wyllie S. Nitro drugs for the treatment of trypanosomatid
diseases: past, present, and future prospects. Trends Parasitol. 2014;
30(6):289–298.
23. Stephens CE, Brun R, Salem MM, et al. The activity of diguanidino
and ‘reversed’ diamidino 2,5-diarylfurans versus Trypanosoma cruzi
and Leishmania donovani. Bioorg Med Chem Lett. 2003;13(12):
2065–2069.
submit your manuscript | www.dovepress.com
Drug Design, Development andTherapy 2017:11
1101
Dovepress

da silva et al
Dovepress
Supplementary materials
a starting pressure of 50 psi. The consumption of hydrogen
gave a clear solution. The solution was filtrated over well-
packed Celite, and the filtrate was removed under reduced
pressure to give a light brown fluffy solid. This compound
was used in the next step without further characterization.
S-(2-Naphthylmethyl)-2-pyridylthioimidate hydrobromide
(2.2 mmol) was added to a cooled solution of 2,5-bis-(2-
2,5-Bis(2-isobutoxy-4-nitrophenyl)furan
2,5-Bis(trimethylstannyl)furan (606 mg, 1.5 mmol)
was injected into a solution of 1-bromo-2-isobutoxy-
4-nitrobenzene (880 mg, 3.1 mmol) and tetrakis-
(triphenylphosphine)palladium(0) (54 mg, 0.04 mmol) in
anhydrous 1,4-dioxane (10 mL) at room temperature under
nitrogen. The mixture was allowed to reflux under nitrogen isobutoxy-4-aminophenyl)furan (1 mmol) in a mixture of
overnight. The orange-colored suspension was diluted
with hexanes (8 mL) and cooled to room temperature. The
residue was filtered and washed with hexanes. The orange
fluffy residue was recrystallized from toluene (25 mL) to
give yellow fluffy solid (72%), mp: 230°C–232°C. 1H NMR
(400 MHz, CDCl3): δ 1.16 (d, J =6.8 Hz, 12H), 2.35–2.28
(m, 2H), 4.01 (d, J =6 Hz, 4H), 7.36 (s, 2H), 7.82 (s, 2H),
7.94 (d, J =8.4 Hz, 2H), and 8.10 (d, J =8.8 Hz, 2H); 13C
NMR (100 MHz, CDCl3): δ 19.5, 28.3, 75.8, 106.9, 116.1,
116.2, 124.9, 125.9, 147.1, 148.9, and 155.1; and ESI-MS:
m/z calculated for C24H26N2O7: 454.1 and found: 455.2
(M +1). Analysis was calculated for C24H26N2O7–0.7H2O:
C, 61.71; H, 5.91; and N, 5.99 and found: C, 61.61; H, 5.65;
and N, 5.59.
dry ethanol (30 mL) and dry acetonitrile (10 mL) in an ice
bath. The reaction mixture was stirred at room temperature
overnight. After the disappearance of the starting material,
the organic solvent was evaporated under reduced pressure to
yield a crude oil product. Dry ether (50 mL) was added to
the crude material, and the mixture was stirred at room
temperature for 4 h. The red precipitate was filtered and
washed with dry ether. The solid was dissolved in ethanol
(5 mL); the solution was cooled to 0°C in an ice bath, and
10% NaOH was added until pH reached ~10. The free base
was extracted with ethyl acetate (3×200 mL). The organic
layer was washed with distilled water, dried over dry K2CO3,
filtered, and concentrated under reduced pressure. The result-
ing suspension was crystallized by adding dry ether and then
filtered. The free base was suspended in dry ethanol (10 mL)
and cooled to 0°C in an ice bath. Freshly prepared ethanolic
HCl (2 mL) was added to the suspension, and the mixture
was stirred at room temperature overnight. The resulting red
solution was concentrated under reduced pressure. The red
crude solid was crystallized from dry ethanol and dry ether
and filtered (orange solid, yield [68%], mp 205°C–206°C).
1H NMR (400 MHz, DMSO-d6): 1.10 (d, J =5.6 Hz, 12H),
2,5-Bis[2-isobutoxy-4-(2-pyridylimino)
aminophenyl]furan hydrochloride
(DB 1890B)
To a suspension of 2,5-bis(2-isobutoxy-4-nitrophenyl)furan
(2.5 g, 5.21 mmol) in a mixture of ethyl acetate (50 mL) and
anhydrous ethanol (20 mL), Pd/C (500 mg, 10%) was added.
The suspension was bubbled with dry nitrogen for 15 min
and hydrogenated overnight using a Parr apparatus with 2.25 (m, 2H), 3.99 (d, J =5.6 Hz, 4H), 7.35 (brs, 2H), 7.87
5
2
5
2
5
5
2
2
D
;
;
2ꢀ1
12ꢀ
+ꢀ1
+1
1+ꢀ
E
5
2
5
2
1+
;
1
+
1
+
1
1
'%ꢁꢉꢂꢊ%ꢄ; 2ꢅꢄ5 LꢈEXW\O
'%ꢁꢂꢃꢇꢄ; 6ꢅꢄ5 LꢈSURS\O
'%ꢁꢂꢃꢂꢄ; 2ꢅꢄ5 &+ꢀ&)ꢆ
Scheme S1 reagents and conditions: (a) h2/Pd (c), etOh, and etaOc or sncl2-2h2O, etOh, and DMsO, 80°c and (b) (i) S-(2-naphthylmethyl)-2-pyridylthioimidate-hBr,
etOh, and ch3cn and (ii) etOh/hcl or chcl2/ch3sO3h.
Abbreviation: DMsO, dimethyl sulfoxide.
submit your manuscript | www.dovepress.com
Drug Design, Development andTherapy 2017:11
1102
Dovepress

Dovepress
aias effect on T. cruzi
1
Table S1 Physicochemical parameters and Lipinski’s rule of five
fluffy solid (3.21 g, 74%), mp 162°C–164°C. H NMR
Descriptor
DB1890B
DB1957
DB1959
(500 MHz, DMSO-d6): δ (ppm): 1.45 (d, J =5.7 Hz, 12H),
5.03 (m, 2H), 7.85–7.90 (m, 6H), and 8.08 (d, J =8.5 Hz, 2H)
and 13C NMR (125 MHz, DMSO-d6): δ (ppm): 21.7, 72.0,
108.7, 115.9, 127.8, 128.3, 129.7, 139.2, 146.9, and 153.0.
Analysis calculated for C22H22N2O6S: C, 59.72; H, 5.01; and
N, 6.33 and found: C, 59.57; H, 5.02; N, 6.23.
Molecular weight (#500)
logP (#5)
602.74
590.75
8.32
7
654.57
8.16
7
8.34
no of acceptors (#10)
no of donors (#5)
no of rotatable bonds (#10)
surface area
7
4
4
4
14
12
14
263.244
255.36
262.77
2,5-Bis[2-isopropoxy-4-(2-pyridylimino)
(m, 2H), 8.16 (d, J =7.6 Hz, 2H), 8.24 (t, J =7.6 Hz, 2H), 8.55 aminophenyl]thiophene and dimesylate
(d, J =7.6 Hz, 2H), 8.90 (d, J =5 Hz, 2H), 9.41 (s, 2H), 10.19
(s, 2H), and δ 11.99 (s, 2H); 13C NMR (100 MHz, CD3OD):
(DB1957)
A mixture of 2,5-bis(2-isopropoxy-4-nitrophenyl)thiophene
δ 18.4, 28.1, 75.2, 109.3, 113.1, 117.2, 120.1, 122.9, 126.7,
128.4, 133.4, 138.2, 144.1, 148.4, 150.0, 156.1, and 160.3;
and ESI-MS: m/z calculated for C36H38N6O3 (base): 602.3 and
found: 603.4 (M + H)+. Analysis calculated for C36H38N6O3-
2HCl–2.85H2O: C, 59.47; H, 6.33; and N, 11.56 and found:
C, 59.45; H, 6.36; and N, 11.19.
(1.42 g, 3.2 mmol) and SnCl2 dihydrate (5.87 g, 26 mmol) in
EtOH (80 mL) and DMSO (20 mL) was heated overnight at
80°C (oil bath). The cooled reaction was then basified with
aqueous NaOH and extracted with ethyl acetate (with addi-
tion of brine). The extract was dried (Na2SO4), evaporated in
vacuo, and the residue purified by column chromatography
(SiO2) using hexanes:EtOAc (1:1) as eluent. The pure frac-
tions were combined and concentrated in vacuo to give, after
standing, the diamine [2,5-bis(4-amino-2-isopropoxyphenyl)
2,5-Bis(2-isopropoxy-4-nitrophenyl)
thiophene
1-Bromo-2-isopropoxy-4-nitrobenzene (5.23 g, 20 mmol), thiophene] as a green sold (0.52 g, 43%). 1H NMR (300 MHz,
2,5-bis(trimethylstannyl)thiophene (4.10 g, 10 mmol), and DMSO-d6): δ (ppm): 1.35 (d, 12H), 4.57 (m, 2H), 5.21 (s, 4H),
Pd(PPh3)4 (0.3 g, 0.26 mmol) were heated in 1,4 dioxane 6.17 (dd, J =8.2 and 1.6 Hz, 2H), 6.30 (d, J =1.5 Hz, 2H), 7.12
(25 mL) overnight at 100°C (oil bath) under nitrogen. (s, 2H), and 7.29 (d, J =8.2 Hz, 2H). Without further char-
The dark red solution was then diluted with excess hexanes acterization, the diamine (0.75 g, 1.95 mmol) was dissolved
to give an orange precipitate, and the filtered product was in acetonitrile (15 mL) and EtOH (8 mL) and treated with
recrystallized from DMF/MeOH to yield a bright orange S-(2-naphthylmethyl)-2-pyridylthioimidate hydrobromide
Table S2 in silico aDMe
ADME predictors
DB1890B
DB1957
DB1959
Bz
absorption
caco-2 permeability (log Papp in 106 cm/s)
intestinal absorption (human; %)
skin permeability (log Kp)
Distribution
0.385
94.957
−2.749
0.742
98.939
−2.859
0.133
98.031
−2.744
0.479
68.885
−2.893
VDss (human; log l/kg)
Fraction unbound (human; Fu)
BBB permeability (log BB)
cns permeability (log Ps)
Metabolism
−0.586
0
−1.424
−1.906
0.791
0.253
−0.104
0.503
−0.619
−2.995
0.127
−0.979
−2.044
0.145
−1.929
−2.439
cYP2D6 substrate
cYP3a4 substrate
cYP1a2 inhibitor
cYP2c19 inhibitor
cYP2c9 inhibitor
cYP2D6 inhibitor
cYP3a4 inhibitor
no
Yes
no
no
Yes
no
Yes
no
Yes
no
no
no
no
Yes
no
Yes
no
no
no
no
Yes
no
no
no
no
no
no
no
excretion
Total clearance (log ml/min/kg)
0.586
0.731
0.188
0.625
Abbreviations: ADME, absorption, distribution, metabolism, and excretion; Bz, benznidazole; Papp, apparent permeability coefficient; AMES, assay of the ability of a
chemical compound to induce mutations in Dna; Kp, skin permeability constant; Fu, Fraction Unbound; BBB, blood–brain barrier; BB, blood-brain; cns, central nervous
system; Ps, permeability-surface area.
submit your manuscript | www.dovepress.com
Drug Design, Development andTherapy 2017:11
1103
Dovepress

da silva et al
Dovepress
Table S3 in silico toxicity
Toxicity predictors
DB1890B
DB1957
DB1959
Bz
aMes toxicity
no
no
no
Yes
Maximum tolerated dose (human; log mg/kg/day)
herg i inhibitor
0.437
no
−0.64
no
−0.481
no
0.984
no
herg ii inhibitor
Yes
Yes
Yes
no
Oral rat acute toxicity (lD50; mol/kg)
Oral rat chronic toxicity (lOael; log mg/kg_bw/day)
hepatotoxicity
2.777
1.307
no
3.046
0.004
Yes
2.931
−0.152
no
2.454
1.649
Yes
skin sensitization
no
no
no
no
T. pyriformis toxicity (log µg/l)
Minnow toxicity (log mM)
0.29
−0.455
0.348
−0.714
0.302
−0.247
1.227
1.649
Abbreviations: Bz, benznidazole; T. pyriformis, Tetrahymena pyriformis; aMes, assay of the ability of a chemical compound to induce mutations in Dna; lD, lethal dose;
lOael, lowest-observed-adverse-effect level.
(1.41 g, 3.9 mmol). After stirring overnight, the red solution refluxed for 24 h under the exclusion of light after which the
was concentrated in vacuo and the resulting oil was triturated yellow solution was cooled and diluted with hexane to give a
with ether to give a solid, which was collected by suction. red precipitate. The precipitate was filtered and washed with
The solid was then dissolved in a minimal amount of EtOH hexane and dried to give 1.02 g (83%) of the title compound,
and basified with NaOH (1 N) to give a yellow orange solid mp 282°C–286°C. 1H NMR (400 MHz, DMSO-d6): δ (ppm):
after stirring. Recrystallization of this solid from EtOH gave 5.21 (q, J =8.8 Hz, 4H), 7.29 (s, 2H), 8.06–8.10 (m, 4H), and
the title compound as the free base as yellowish brown crys- 8.33 (d, J =8.4 Hz, 2H); 13C NMR (100 MHz, DMSO-d6):
tals (0.61 g, 79% from the diamine), mp 112°C–115°C. 1H δ (ppm): 65.4 (q, JC-F = 35 Hz), 108.4, 115.6, 117.2, 123.4 (q,
NMR (300 MHz, DMSO-d6): δ (ppm): 1.37 (d, 12H), 4.76 JC-F =276 Hz), 124.1, 126.4, 146.8, 147.9, and 152.4. Analysis
(m, 2H), 6.56 (dd, J =8.2 and 1.7 Hz, 2H), 6.64 (d, J =1.5 Hz, calculated for C20H12F6N2O7: C, 47.44; H, 2.39; and N, 5.53
2H), 7.45 (s, 2H), 7.55 (m, 2H), 7.66 (d, J =8.3 Hz, 2H), and found: C, 47.61; H, 2.34; and N, 5.44.
7.95 (m, 2H), 8.32 (d, J =7.9 Hz, 2H) and 8.63 (m, 2H) and
2,5-Bis[2-(2,2,2-trifluoroethoxy)-4-(2-
pyridylimino)aminophenyl]furan and
13C NMR (75 MHz, DMSO-d6): δ (ppm): 22.1, 70.0, 107.3,
114.2, 118.1, 121.4, 123.4, 125.4, 128.3, 137.1, 138.5,
148.1, 150.6, 151.4, 151.9, and 153.8. Finally, to prepare dimesylate (DB1959)
the title dimesylate salt (DB1957), a solution of the free 2,5-Bis(2-(2,2,2-trifluoroethoxy)-4-nitrophenyl)furan
base (0.296 g, 0.5 mmol) in EtOH (35 mL) was treated with (0.94 g, 1.86 mmol) and 150 mg of 10% Pd/C as a cata-
methanesulfonic acid (0.10 g, 1.05 mmol) and then diluted lyst was suspended in 150 mL of a degassed mixture of
with excess diethyl ether to give a yellow brown solid, which EtOAc–EtOH (9:1) and kept under a hydrogen pressure
was collected and dried under high vacuum, 90–95 mp dec. of 50 psi with shaking for 5 h. The solution was then
1H NMR (500 MHz, DMSO-d6): δ (ppm): 1.44 (d, 12H), 2.32 filtered to remove the catalyst by passing through a bed
(s, 6H), 4.86 (m, 2H), 7.11 (d, J =9.4 Hz, 2H), 7.32 (s, 2H), of Celite, and the bed was washed with dichloromethane.
7.69 (s, 2H), 7.86 (m, 2H), 8.24 (m, 2H), 8.36 (d, J =7.5 Hz, The filtrate was concentrated under reduced pressure to
2H), 8.91 (s, 2H), 9.34 (s, 2H), 10.03 (s, 2H), and 11.68 viscous oil, and hexane was added to give a yellowish-
(s, 2H); 13C NMR (125 MHz, DMSO-d6): δ (ppm): 22.0, 71.0, white precipitate of the diamine (yield: 0.8 g [96%], mp
111.6, 117.9, 123.5, 123.9, 125.4, 128.7, 128.8, 134.1, 138.5, 196°C–198°C). The diamine was used directly in the next
138.6, 144.5, 149.9, 153.8, and 159.5. Analysis calculated step. 2,5-Bis(2-(2,2,2-trifluoroethoxy)-4-aminophenyl)
for C34H34N6O2S-2CH3SO3H–1.5H2O: C, 53.38; H, 5.60; and furan (0.7 g, 1.58 mmol) was dissolved in a mixture of
N, 10.38 and found C, 53.47; H, 5.48; and N, 10.20.
40 mL of MeCN–EtOH (1:3) and cooled to 0°C, and
S-(2-naphthylmethyl)-2-pyridylthioimidate hydrobromide
(1.44 g, 4 mmol) was added to it and after 30 min allowed
to warm to room temperature. The yellow solution was
2,5-Bis(2-(2,2,2-trifluoroethoxy)-4-
nitrophenyl)furan
A mixture of 1-bromo-4-nitro-2-(2,2,2-trifluoroethoxy) stirred for 2 days after which it was concentrated in vacuo
benzene (1.54 g, 5.13 mmol) and 2,5-bis(trimethylstannyl)- and diethyl ether was added to provide a yellow precipitate
furan (0.96 g, 2.43 mmol) was added to 50 mL of degassed of the hydrobromide salt, which was filtered and washed
dioxane. Pd(PPh3)4 (0.3 g, 0.26 mmol) was added to it and with a mixture of 1:1 ethanol–diethyl ether. The solid was
submit your manuscript | www.dovepress.com
Drug Design, Development andTherapy 2017:11
1104
Dovepress

Dovepress
aias effect on T. cruzi
then dissolved in a minimum volume of ethanol, and a orange-red solid (yield: 0.62 g [96%], mp 273°C–275°C
1
dilute aqueous solution of sodium hydroxide was added dec). H NMR (400 MHz, DMSO-d6): δ (ppm): 2.32
to generate the free base. The organic layer was extracted (s, 6H), 5.01 (q, J =8.4 Hz, 4H), 7.11 (s, 2H), 7.30 (d,
with dichloromethane, dried with Na2SO4, filtered, and J =8.4 Hz, 2H), 7.51 (s, 2H), 7.87–7.90 (m, 2H), 8.24–8.28
concentrated in vacuo to yield the free base 0.82 g (79%) (m, 4H), 8.39 (d, J =8 Hz, 2H), 8.93 (d, J =4.4 Hz, 2H),
as a yellow solid, mp 207°C–209°C. The imidamide free 9.42 (brs, 2H), 10.15 (brs, 2H), and 11.85 (brs, 2H); 13C
base was dissolved in 20 mL dichloromethane–ethanol NMR (100 MHz, DMSO-d6): δ (ppm): 65.2 (q, JC-F =34 Hz),
(1:1) mixture, and (154 mg, 1.60 mmol) of methanesul- 111.1, 113.0, 118.7, 119.8, 123.9, 124.0 (q, JC-F =276 Hz),
fonic acid diluted with 5 mL diethyl ether was added to it 127.0, 128.7, 134.3, 138.5, 144.5, 147.8, 150.0, 153.1, and
with immediate darkening of the yellow solution to red. 159.6. LRMS ESI (+): calculated for C32H24F6N6O3 (base):
The mixture was stirred for 1 h, the solvents were evacu- 654.2 and found (M + H+): 655.3. Analysis calculated for
ated to a small volume, and diethyl ether was added to the C32H24F6N6O3-2CH3SO3H-2H2O: C, 46.26; H, 4.11, and
mixture to precipitate the bis(methanesulfonate) salt as an N, 9.52 and found C, 45.88; H, 3.98; and N, 9.26.
Drug Design, Development and erapy
Dovepress
Publish your work in this journal
Drug Design, Development and Therapy is an international, peer-
reviewed open-access journal that spans the spectrum of drug design
and development through to clinical applications. Clinical outcomes,
patient safety, and programs for the development and effective, safe,
and sustained use of medicines are the features of the journal, which
has also been accepted for indexing on PubMed Central. The manu-
script management system is completely online and includes a very
quick and fair peer-review system, which is all easy to use. Visit
http://www.dovepress.com/testimonials.php to read real quotes from
published authors.
Submit your manuscript here: http://www.dovepress.com/drug-design-development-and-therapy-journal
submit your manuscript | www.dovepress.com
Drug Design, Development andTherapy 2017:11
1105
Dovepress