J. Med. Chem. 2008, 51, 4104–4114
4104
Complexation of the anti-Trypanosoma cruzi Drug Benznidazole Improves Solubility and
Efficacy
Jean Jerley Nogueira Silva,† Wander Rogério Pavanelli,‡ Fredy R. Salazar Gutierrez,‡ Francisco Chagas Alves Lima,†
Albérico Borges Ferreira da Silva,† João Santana Silva,‡ and Douglas Wagner Franco*,†
Departamento de Quı́mica e Fı́sica Molecular, Instituto de Quı́mica de São Carlos, UniVersidade de São Paulo (USP), São Carlos, SP, Brazil,
Departamento de Bioquı́mica e Imunologia, Faculdade de Medicina de Ribeirão Preto, UniVersidade de São Paulo (USP),
Ribeirão Preto, SP, Brazil
ReceiVed October 17, 2007
The ruthenium complex, trans-[Ru(Bz)(NH3)4SO2](CF3SO3)2 1, Bz ) benznidazole (N-benzyl-2-(2-nitro1H-imidazol-1-yl)acetamide), is more hydrosoluble and more active (IC50try/1 h ) 79 ( 3 µM) than free
benznidazole 2 (IC50try/1 h > 1 mM). 1 also exhibits low acute toxicity in vitro (IC50macrophages > 1 mM) and
in vivo (400 µmol/kg < LD50 < 600 µmol/kg) and the formation of hydroxylamine is more favorable in 1
than in 2 by 9.6 kcal/mol. In murine acute models of Chagas’ disease, 1 was more active than 2 even when
only one dose was administrated. Moreover, 1 at a thousand-fold smaller concentration than the considered
optimal dose for 2 (385 µmol/kg/day ) 100 mg/kg/day), proved to be sufficient to protect all infected mice,
eliminating the amastigotes in their hearts and skeletal muscles as observed in H&E micrographics.
Introduction
a
American trypanosomiasis or Chagas’ disease (ChD) remains
a significant public health problem and is still one of the major
causes of morbidity and mortality from cardiovascular diseases
in Latin America despite nearly one century of research.1 The
American Red Cross has recently published a study showing
that one in 4655 blood donors in the United States was
confirmed as positive for T. cruzi antibodies.2 Furthermore,
actions for preventing transmission of T. cruzi infection by organ
transplant or blood transfusion have also been taken in nonendemic areas such as the European countries,3 thus showing that
this disease is a cause for concern to developed countries as
well.3
Despite the progress achieved in the study of T. cruzi’s
biochemistry and physiology in which several enzymes have
been identified as potential new targets, a definitive chemotherapy for this parasitic infection remains undeveloped.4,5 The
compounds benznidazole 2 (Bz), (N-benzyl-2-(2-nitro-1Himidazol-1-yl)acetamide) and gentian violet 3 (Gv), (N-[4-(bis[4(dimethylamino)-phenyl]methylene]-2,5-cyclohaxadien-1-ylidene]* To whom correspondence should be addressed. Phone: +55 16 3373
9976. Fax: +55 16 3373 9976. E-mail: douglas@iqsc.usp.br. Address: Av.
Trabalhador São-Carlense, 400 CP 780, 13560-970 São Carlos, SP, Brazil.
This paper is based on part of the J. J. N. Silva, Ph.D. Thesis and is presented
as the Brazilian patent of invention PI 0704577-8.
†
Departamento de Quı́mica e Fı́sica Molecular, Instituto de Quı́mica de
São Carlos, Universidade de São Paulo.
‡
Departamento de Bioquı́mica e Imunologia, Faculdade de Medicina
de Ribeirão Preto, Universidade de São Paulo.
a
Abbreviations: BT, bloodstream trypomastigote forms of Trypanosoma
cruzi; Bz, benznidazole; ChD, Chagas’ disease; DFT, density functional
theory; Eaq, total energy in aqueous phase; Es, solvation energy; ∆E NO2,
difference in reduction potential of NO2 moiety between 1 and 2; GAP )
ELUMO - EHOMO; IC50try/1 h, inhibitory concentration on trypomastigote forms
after 1 h incubation; IC50try/4 h, inhibitory concentration on trypomastigote
forms after 4 h incubation; IC50try/24 h, inhibitory concentration on trypomastigote forms after 24 h incubation; MLCT, metal-to-ligand chargetransfer transition; MO, molecular orbital; NBO, natural bond orbital
analysis; PBS, phosphate-buffered saline; Protocol A, treatment for 15
consecutive days; Protocol B, treatment only on the fifth, sixth, and seventh
days preceding the parasitaemic peak; Saq, water solubility; SCE, aqueous
saturated calomel electrode; % TA, percentage of trypanocidal activity; T.
cruzi, Trypanosoma cruzi.
N-methyl-methanaminium chloride) are only clinically used for
the treatment of ChD and blood sterilization, respectively.
However, the Bz treatment is unsatisfactory and exhibit undesirable side effects such as anorexia, dermatotoxicity, digestive
disorders, peripheral polyneuropathy, genotoxicity, and allergic
dermopathy attributed to the redox damage to the host’s
tissue.6–10 If administrated in the acute phase, 2 could cure
50-70% of the patients, but a very low cure rate (8-20%) was
reported for chronically infected patients even when treated for
more than 10 years.6 Furthermore, several strains of T. cruzi
(e.g., Y, Colombiana, CS-28, and VL-10) are naturally Bzresistant.11 2 is claimed to act through the formation of free
radicals and/or electrophilic metabolites.9 The nitro group in 2
is reduced to an amino group through the nitroreductases, and
this process begins with a reaction catalyzed by NADPHcytochrome P-450 reductase, producing a nitro anion radical
(R-NO2•-),9 which is involved in its trypanocidal effect by
covalent bonding to macromolecules such as the DNA parasite.9
More recently, efforts have been devoted to the synthesis and
evaluation of platinum, palladium, rhenium, copper, gold, and
ruthenium derivatives as trypanocidal drugs.12–14 However, these
metal complexes have limited pharmacological application due
to their low hydrosolubility and toxic effects.12–15 An interesting
series of ruthenium complexes,14,15 type [Ru(H2O)2L2](PF6)2,
[RuL2Cl2], [RuL3Cl3] · 2CH3OH, [RuL2(H2O)Cl3] · 2H2O, and
[Ru(bpy)L2](PF6)2, L ) clotrimazole (Ctz) or ketoconazole (Ktz)
less toxic than the ones mentioned above, has been used in the
rational design of new antiparasitic agents. This species can
block specific lipid biosynthesis pathways by, e.g., inhibiting
the enzyme cytochrome P450 14R-demethylase involved in the
production of ergoesterol, which are essential for the parasite.12,14,15
According to the authors,14,15 these ruthenium complexes are
able to inhibit 70% of proliferation of epimastigote forms of T.
cruzi.12 Furthermore, the ruthenium complexes [Ru(Ctz)2(H2O)2](PF6)2 and [Ru(Ktz)2(H2O)Cl3] acting in synergism with antifungal drugs have proved to be more active than the corresponding free ligands.14,15
Following this approach, the [Ru(NH3)4L]n+ moiety has been
successfully tested as NO carrier in vitro and in vivo16,17 because
the NO production has been ascribed as a responsible for the
10.1021/jm701306r CCC: $40.75 2008 American Chemical Society
Published on Web 06/21/2008
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 14 4105
A Benznidazole Ruthenium DeriVatiVe
Scheme 1. General Procedures Used to Prepare the Desired Complex Ion 1a
a
The precursor complex ion was prepared according to the literature.19
trypanocidal effect in activated macrophages.18 In fact, in a
recent related work, trans-[Ru(NO)(NH3)4L]n+, L ) N-heterocyclic, SO32-, or P(OEt)3 exhibited not only low cytotoxicity
but also anti-T. cruzi activity due to NO action.17
In this context, ruthenium(II) tetraammine compounds are
good models for tailoring carriers for Bz because these species
are water soluble due to the presence of the NH3 ligands and
readily react with N-heretocyclic ligands yielding quite robust
compounds.19–21 Moreover, it is likely that these complexes,
upon coordination to the Bz, shift the reduction potential of the
nitro group in 2 to more positive values, benefiting the formation
of the corresponding nitro radical anion and subsequent hydroxylamine derivative. Because activity of 2 is attributed to
the generations of reduced metabolites, which are involved in
its trypanocidal effects,22 it is expected that the RuBz complex,
which the Bz ligand would be more accessible to reduction,
exhibits higher anti-T. cruzi activity than free Bz.
Using this general strategy and aiming to develop a more
water soluble and more active anti-T. cruzi compound with
reduced toxicity, we describe here the trans-[Ru(Bz)(NH3)4SO2](CF3SO3)2 complex 1, where Bz ) benznidazole, which
in biological media yields trans-[Ru(Bz)(NH3)4SO3]. Assays of
acute toxicity for 1 were then evaluated on female Swiss mice,
and comparison of the trypanocidal activity in vitro and in vivo
of 1 and 2 was performed using a partially Bz-resistant strain.
Results are explained based on both experimental and theoretical
parameters and the Ru(II) moiety influence on the chemical
properties of Bz ligand.19,20
Results
Chemistry. The New Ruthenium Complex, trans-[Ru(Bz)(NH3)4SO2](CF3HSO3)2. The first experiments were carried out
with [Ru(H2O)(NH3)5](PF6)2 complex and 2 in acetone. As
predicted from the redox potentials of Ru(III)/Ru(II) couple in
the aquopentaamine ruthenium species23 and of the nitro group
in 2,24 a redox reaction leading to metal center oxidation was
observed. The chemical reactivity of Ru(II) center is attributed
to its strong back-bonding ability in the binding to unsaturated
ligands.19–21 Because of the π acidity of the sulfur atom in SO2,
HSO3-, and SO32- ligands, the Ru(II) center in the trans[Ru(H2O)(NH3)4SO2]2+ complex ion becomes a weaker reductant agent than in [Ru(H2O)(NH3)5]2+. It is well-known that
the substitution of one of the NH3 ligands by SO2 in
[Ru(NH3)5Cl]2+ complex ion yields a well-defined and less
reductant species (Scheme 1) than in [Ru(H2O)(NH3)5]2+.19,20,25
Furthermore, the trans labilizing and trans influence effects of
SO2 increase the lability of the trans aquo ligand in trans[Ru(H2O)(NH3)4SO2]2+, facilitating their substitution by entering
Figure 1. Optimized structure for trans-[Ru(Bz)(NH3)4(SO3)] 1 and
structure of the currently used drug for the treatment of ChD,
benznidazole 2. Both compounds were optimized by ab initio DFT
calculations in aqueous phase.
ligands such as 2.26 Additionally, through cis delabilizing effect
of SO2 ligand, NH3 equatorial ligands still become more inert
regarding the substitution reactions,19,27 thus resulting in the
predictable quite robust trans-[Ru(Bz)(NH3)4SO2]2+ complex
ion 1 (Scheme 1).19,28 Compound 1 was isolated as trans[Ru(Bz)(NH3)4SO2](CF3HSO3)2 from the reaction between
trans-[Ru(H2O)(NH3)4SO2]2+ and 2 in acetone and characterized
by elemental analyses, spectroscopic techniques (1H and 13C
NMR, IR, and UV-vis), and density functional theory (DFT)
calculations.
Structural Analysis. So far it has not been possible to obtain
a good crystal for complex 1. Therefore, a structural analysis
was performed using an optimized geometry calculated through
DFT (Figure 1). The main objective was to evaluate the bond
lengths Ru-NH3, Ru-N(Bz), and Ru-S, and the angles
H3N-Ru-NH3 and O-S-O. Ru-N interatomic distance for
the equatorial ammines does not change significantly when L
is SO2 (2.182 Å), HSO3- (2.194 Å), or SO32- (2.201 Å).
However, this is not true for Ru-N(Bz) bond distance considering 2.151, 2.106, and 2.072 Å for L ) SO2, HSO3-, or SO32-,
respectively. Thus, Ru-N(Bz) bond strengthening is observed
when L ) SO2, HSO3-, or SO32-, respectively, which is
4106 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 14
consistent with the simultaneous Ru-S weakening, thus reflecting the increase of the biphilic character of L ligand. Additionally, H3N-Ru-NH3 (90.1 ( 1.2) and O-S-O (116.6 ( 2.5)
angles are approximately the same as in trans-[Ru(NH3)4SO2Cl]Cl.28
Molecular Orbital Analysis. The molecular orbital (MO)
composition of 1 was performed considering L ) SO2 representing the isolated species. LUMO and the two empty MOs
LUMO + 2 and LUMO + 3 have a combination of SO2 ligands
orbitals (20-40%), Bz antibonding orbitals (11–55%), and
ruthenium dσ orbitals (16-49%). However, the empty MO
LUMO + 1 has 100% Bz antibonding character centered in
the imN-NO2 moiety. In the same way, instead of a single
HOMO, there are three close-lying highest occupied orbitals,
which have a combination of ruthenium (3%), SO2 (10%), and
Bz (87%) orbitals for HOMO, 100% Bz phenyl group for
HOMO-1 and HOMO-2, and 100% ruthenium dπ orbital for
HOMO-3. Their energies would be ∼2.7 eV below the set of
π* orbitals of the Bz imidazole ring. Therefore, in addition to
intraligand band (n f π*) in the ultraviolet region,29 a metalto-ligand charge-transfer (MLCT) transition (Ru f Bz) in the
visible region (approximately 470-500 nm = 2.5 eV) is also
predicted. According to time-dependent DFT (TD-DFT) calculations, this transition would be spin forbidden and would
occur nearly 540 nm (∼2.3 eV) with low intensity (oscillator
strength (f) ) 5.5 × 10-3).
Electronic Absorption Spectra. UV-vis spectra of 1 were
recorded in water, ethanol, and acetone. Spectrum of 2 in ethanol
shows an intraligand band at 314 nm, ε ) 8.2 × 103 M-1 cm-1
ascribed to n f π* transition.29 This same transition, upon
coordination to Ru(II) center, is shifted to 323 nm, ε ) 8.1 ×
103 M-1cm-1 and 322 nm, ε ) 4.5 × 103 M-1 cm-1 in ethanol
and water, respectively. The spectrum of 1 in acetone, ethanol,
and water exhibits a second band at 477 nm, ε ) 71 M-1 cm-1,
507 nm, ε ) 89 M-1 cm-1, and at 530 nm, ε ) 84 M-1 cm-1,
respectively, which, according to the DFT calculations, could
be assigned as MLCT. According to MO composition for 1,
HOMO-3 has 100% Rudyz character, whereas LUMO + 1 has
100% imN-NO2pxπ* character. Although this transition has small
intensity due to the asymmetry of the orbitals, it shows
solvatochromism (C3H6O, λabs ) 477 nm; C2H6O, λabs ) 507
nm; H2O, λabs ) 530 nm), which would suggest a charge-transfer
character. Thus, the experimental data for the electronic
spectrum are on agreement with DFT calculations predictions.
Vibrational Frequencies. The infrared absorption bands
νNO2, 1371 cm-1, νCN, 1523 cm-1, and νCO, 1633 cm-1 for
1 are coherent, with the Bz axial coordination to R(II) center
through N23 unsubstantiated nitrogen atom (Figure 1). These
frequencies were shifted to smaller values, considering the free
ligand, upon coordination. As previously mentioned, DFT
calculations suggest that the back-bonding Ru(II)-N(Bz)
involves the ruthenium dyz orbitals (100% Rudyz, HOMO-3) and
the imidazole ring π* orbitals in Bz (100% imN-NO2px π*,
LUMO + 1). Therefore, it is expected that, upon coordination,
bonds of the imidazole ring become weaker and, consequently,
these frequencies are shifted to smaller wavenumbers. Furthermore, the infrared absorption bands νSO2sym ) 1107 cm-1 and
νSO2asym ) 1281 and 1249 cm-1 are consistent with the SO2
ligand coordination through sulfur atom, as in trans-[Ru(NH3)4SO2Cl]Cl.28
NMR Spectra. Compound 1 was also well characterized by
1
H and 13C NMR spectroscopy. A small 1H shift considering
the free ligand was observed for H21 (∆δ 0.09) and H19 (∆δ
0.05) atoms located in the vicinity of the coordinated nitrogen.
SilVa et al.
Figure 2. Cyclic voltammograms for 1 (s) and 2 (---) in a protic
media (NaTFA, 0.1M, pH ) 2.2). V ) 100 mV/s. Cathodic wave
corresponding to the hydroxylamine formation from NO2 moiety
according to Bz-NO2 + 4e + 4H f Bz-NHOH + H2O.
Correspondingly, a small 13C shift considering the free ligand
was observed for C20 (∆δ 0.73) and C21 (∆δ 0.34) atoms, as
expected for Ru-N(Bz) bond.29 All other protons and carbons
shifted by <0.1 ppm. Additionally, the carbonyl group of 1 was
detected in 13C resonance (δ167.3), and the ammonia ligand
and ammine group detected in 1H resonance at δ3.1 and δ7.9,
respectively.
Cyclic Voltammetry. Comparison of cyclic voltammetry data
of both the ruthenium species and the ligand itself was used to
reveal the formation of the corresponding hydroxylamine
derivative, which is the stable product of the electrochemical
nitro reduction of 2 in protic medium.24 2 exhibits an irreversible
cathodic wave at -0.475 V vs SCE (pH ) 2.2) attributed to a
four-electron and four-proton transfer to form the hydroxylamine
derivate.24
Bz - NO2 + 4e + 4H+ f Bz - NHOH + H2O
(1)
After coordination, under the same conditions, this reduction
wave was shifted by +0.104 V, benefiting the formation of the
nitroanion radical and subsequent hydroxylamine by 9.6 kcal/
mol. Furthermore, a reversible redox couple with E1/2 ) 0.273
V vs SCE was also observed and ascribed to Ru(III)/Ru(II)
couple (Figure 2).
Titration of 1 by Alkali. From the titration of compound 1
with NaOH (Figure 3), two pKa values can be determined
corresponding to the equilibria:
trans-[Ru(Bz)(NH3)4(SO2)]2+ +
2H2O H trans-[Ru(Bz)(NH3)4(HSO3)]+ + H3O+pKa1 )
1.2 ( 0.1
trans-[Ru(Bz)(NH3)4(HSO3)]+ +
H2O H trans-[Ru(Bz)(NH3)4(SO3)]+ + H3O+pKa2 ) 4.3 ( 0.1
These values are smaller than the ones reported19 for the
trans-[Ru(H2O)(NH3)4(SO2)]2+ and trans-[Ru(H2O)(NH3)4(HSO3)]+, which are 2.1 and 5.0, respectively. This is coherent
with the higher basicity of Bz in relation to H2O and the
consequent increase of the electron density along the ruthenium-sulfur axis. Indeed, this has been anticipated by DFT
calculations.
As it can be observed in Figure 3, at physiological pH,
the trans-[Ru(Bz)(NH3)4SO3] species is the predominant form
of 1.
A Benznidazole Ruthenium DeriVatiVe
Figure 3. Distribution of trans-[Ru(Bz)(NH3)4L]2+/+/0, L ) SO2
(square), HSO3- (circle), or SO32- (triangle) species as a function of
hydrogen concentrations and titration curve (inside) with NaOH at 25
°C.
Hydrophilic and Electronic Properties. Differences in the
hydrophilic and electronic properties between 1 and 2 were
analyzed through DFT calculations considering L ) SO32-, the
predominant form for 1 in physiological medium. The molecular
properties determined and examined in this study were LUMO
energy, GAP (ELUMO - EHOMO), total energy in aqueous phase
(Eaq), solvation energy in water (Es), and atomic charges
obtained by using the natural bond orbital analysis (NBO)
method.7,8 Table 1 summarizes the main electronic and hydrophilic differences between 1 and 2. As predicted,22,24 electrochemical reduction can occur in the nitro group because this
moiety is the most susceptible part of 1 and 2 to nucleophilic
attack. These data are also in agreement with the NBO charges
calculated, showing that the NO2 moiety is more electrophilic
in 1 (N45, 0.43; O46/47, -0.32) than in 2 (N25, 0.42; O26/27,
-0.37). Furthermore, according to GAP and LUMO energies,
the electronic affinity of the nitro group in 1 would be higher
than in 2 because the calculated energies in 1 (GAP ) 1.85
eV) are smaller than in 2 (GAP ) 2.54 eV)7,8 (Table 1).
In agreement with the experimental data for hydrosolubity,
the calculated solvation energy and total energy in aqueous phase
have pointed out that 1 would be more water soluble than 2.
Whereas 2 is not highly soluble in water (0.4 mg/mL ) 0.2
µmol/mL, at 37 °C),30 the experimentally measured water
solubility of 1 is 3.7 mg/mL ) 4.4 µmol/mL in the same
medium (Table 1).
Biology. Toxicity Assays. Cell viability was the experimental
model used to evaluate cytotoxicity in vitro at concentrations
of 1 ranging from 10 nM to 1 mM and using macrophages or
spleen cells from Balb/c mice. Lymphocytes, which are the most
abundant types of cells present in spleen cells, are a highly
susceptible group of cells, and their proliferation depends on
the interaction with viable macrophages. Because the concentrations assayed for 1 did not reduce the viability of macrophages
or splenocytes, it is reasonable to assume that IC50 against
macrophages is higher than those concentrations. Thus, considering the IC50try ) 79 µM and IC50macrophages > 1 mM for 1,
its in vitro therapeutic window (tw ) IC50macrophages/IC50try) is
higher than 12. According to the reported data,33 the IC50 value
for 2 on Chinese hamster lung fibroblasts (V-79 cells) is 2 mM.
Therefore, its IC50 against trypomastigotes (IC50try > 1 mM) is
very proximal of the value against V-79 cells (2 mM),
suggesting a quite narrow therapeutic window for 2.
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 14 4107
At concentrations up to 400 µmol/kg, no acute toxic effect
was observed for 1 when using an up-and-down method in
vivo.34 However, at 600 µmol/kg concentrations, the mice died,
thus suggesting that LD50 for 1 ranges between 400 and 600
µmol/kg.
In Vitro Trypanocidal Activity on Trypomastigotes. As
expected,35,36 2 after 1 h of incubation at 37 °C at 1 mM
concentrations presented very low percentage of lysis (<15%)
of bloodstream trypomastigotes (BT) of the Y strain of T. cruzi.
However, under the same conditions, 1 showed higher activity
than 2 over this partially Bz-resistant strain, with %TA equal
to 63 ( 7, 75 ( 4, 95 ( 6, and 100 (mean ( SEM, n ) 4-7)
at concentrations of 0.1, 0.25, 0.5, and 1.0 mM, respectively.
Accordingly, 1 exhibits smaller inhibitory concentration (IC50try/1
h ) 79 ( 3 µM) than 2 (IC50try/1 h > 1 mM) after 1 h of
incubation (Table 1). Furthermore, when 1 is compared with
the current blood sterilization drug 3, IC50try/24 h on BT for 1
(IC50try/24 h ) 58 ( 4 µM) is about 1 order of magnitude smaller
than the value reported for 3 (IC50try/24 h ) 536 µM).37
In Vitro Antiproliferative Activity on Epimastigotes. Experiments carried out to determine the in vitro antiproliferative
activity of compounds 1 and 2 were set up using cultures of
epimastigote forms. Table 2 summarizes the antiproliferative
activity data of these compounds in the exponential phase of T.
cruzi growth expressed as the percentage of growth inhibition
(% GI). According to these data, 1 is shown to exhibit greater
antiproliferative activity (IC50epi ) 127 ( 8 µM) than 2 (IC50epi
> 1 mM) under the conditions tested.
In Vivo Trypanocidal Activity (Acute Model). Preliminary
assays were conducted with trans-[Ru(H2O)(NH3)4SO2](CF3HSO3)2 complex, a precursor in the synthesis of 1 (Scheme
1). No in vivo activity was observed for this compound when
administrated in doses up to 350 µmol/kg for 15 consecutive
days in T. cruzi-infected Swiss mice. After that, different in
vivo murine models for acute ChD were used to compare the
activity of compounds 1 and 2. Groups of six female Swiss
mice were inoculated and intraperitoneally treated for 15
consecutive days (protocol A) or for only three days, the fifth,
sixth, and seventh days that precede the parasitaemic peak
(protocol B) with doses of 1. As Figure 4b shows, 1 at 100
nmol/kg dose provided good level of protection against death
(60%) when mice were treated according to protocol A, whereas
no protection was observed for the nontreated group, not even
when treated with 2 at 100 nmol/kg dose for 15 consecutive
days. Furthermore, the parasitemia levels were significantly
smaller (P > 0.05) than the control group levels when mice
were treated with 1 according to protocols A or B (Figure 4a).
With the purpose of comparison, other groups of mice were
inoculated with 1.0 × 103 BT per mouse and orally treated with
1 at 100 nmol/kg and 385 nmol/kg and with 2 at 385 µmol/kg
for 15 consecutive days. Note that 1 at 385 nmol/kg dose is a
thousand-fold smaller than the considered optimal dose (385
µmol/kg ) 100 mg/kg) for 2.38,39 Figure 5a shows that, similarly
to the group intraperitoneally treated, 1 at 100 nmol/kg dose
orally administrated also decreases the parasitaemic peak by
65-70%. As a consequence, this dose assured 60% survival of
the treated animals (Figure 5b). However, when the infected
mice were treated with either 385 nmol/kg dose of 1 or 385
µmol/kg dose of 2, 100% survival was observed with a
significant decrease (P > 0.001) of the parasitaemic peak for 1
and 2. In another experiment, mice were inoculated with 1.0 ×
103 BT per mouse and orally treated with 1 or 2 at 385 µmol/
kg only on the first day after infection. While 1 was able to
4108 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 14
SilVa et al.
Table 1. Comparison of in Vitro Activity and Molecular Electronic (LUMO Energy, NBO Charges) in Nitro Group, and Difference of Energy between
the Frontier Orbital (GAP ) ELUMO - EHOMO) and Hydrophilic (Solvation Energy, Total Energy in Aqueous Phase, and Water Solubility) Descriptors
and Epc for 1 and 2
survival
(%)a
LUMO
(eV)
GAPb
(eV)
79 ( 3
60
-3.19
1.85
<1000
0
-3.94
2.54
compounds
IC50try/1 h
(µM)
1
2
NBOc
Epc
(V)d
EAq
(kcal/mol)e
Es
(kcal/mol)f
N45(0.43),
O46/47 (-0.32)
N25(0.42),
O26/27 (-0.37)
-0.371
-1.61 × 106
-306.69
-0.475
-5.70 × 105
-8.21
Saq
(mg/mL)g
3.7
0.430
a
% survival at 100 nmol/kg dose administrated (ip) for 15 consecutive days. b GAP ) ELUMO - EHOMO optimized by DFT calculations in aqueous phase
considering L as SO32-. c NBO: charges in nitro group. The charges in oxygen atom are the average between that of the nitro moiety. d Epc: reduction
potential according to reaction Bz-NO2 + 4e + 4H+ f Bz-NHOH + H2O, pH ) 2.2, µ ) 0.1 M of NaCF3COO, potential versus SCE. e Eaq: total energy
in aqueous phase considering L as SO32-. f Es: solvation energy in water considering L as SO32-. g Saq: experimental water solubility (pH ) 6.5 and t )
37 °C).52,53
Table 2. Antiproliferative Activity of 1 and 2 on Epimastigote Forms at Different Concentrations and Incubation Times
antiproliferative activity (% GI)
T)1h
compounds
1
2
0.1
a
33 ( 5
0
T)4h
T ) 24 h
0.5
1
0.1
0.5
1
0.1
0.5
1
56 ( 4
0
67 ( 3
7(3
63 ( 4
11 ( 3
75 ( 4
15 ( 4
80 ( 6
24 ( 2
69 ( 6
15 ( 4
79 ( 5
26 ( 4
92 ( 3
31 ( 5
IC50epi (µm)
127 ( 8
>1000
a
Values are express in mM. Results are mean ( SEM, n ) 3, P < 0.05. Antiproliferative activity expressed as the percentage of growth inhibition at
defined concentration. IC50epi corresponding to 50% antiproliferative activity after 24 h incubation.
protect 60% of the infected animals, 2 was able to protect only
20% (data not shown).
Histological Analysis. Experiments have been performed
according to a previously described protocol.18,36 Swiss mice
were intraperitoneally infected with 1.0 × 103 BT per mouse
and treated with different doses of 1 and 2 for 15 consecutive
days. On the 15th day after infection, all surviving mice from
the nontreated group, the group intraperitoneally treated with 1
at 100 nmol/kg (Figure 4b), and the group orally treated with 1
at 100 nmol/kg, 385 nmol/kg and 2 at 385 µmol/kg (Figure 5b)
were euthanized and their hearts (Figure 4c and 5c), skeletal
muscles (Figure 4c), and livers (Figure 6) processed for H&E
stain. Groups of uninfected mice were also euthanized and their
organs processed for comparison.
Microscopic analysis revealed that infected and nontreated
mice (control) showed several nests of amastigotes in their hearts
as well as in their skeletal muscles, whereas mice treated with
1 at 100 nmol/kg dose showed no nests either in their hearts or
in their skeletal muscles (Figure 4c). Furthermore, results
obtained from these experiments have also showed that chemotherapy with 1 decreases the occurrence of myocarditis
(Figure 4c and 5c). When considering the group treated with 2
at the same single dose as 1, that is 385 µmol/kg, the nests of
amastigotes in the mice’s hearts were not completely eradicated
for both of the compounds (data not shown). Figure 6 reveals
that the group treated with 1 showed a smaller number of
inflammatory cells in their livers than the group treated with 2.
Additionally, from liver histological analysis, no morphological
evidence for toxicity was found when Swiss mice were treated
with 1 at 385 nmol/kg for 15 consecutive days.
Discussion
Previous work40,41 has shown that some ruthenium complexes
transporting antifungal or semicarbazones are more active
against T. cruzi than the corresponding free ligands. However,
the low solubility of these complexes in water limits their
pharmacology potential.12 It has been reported that etanidazole
(N-(2-hydroxyethyl)-2-nitro-1-imidazoleacetamide), a new 2-nitroimidazole used as a clinical hypoxic cell radiosensitizer,
displays trypanocidal activity against isolated trypomastigotes
and amastigotes of T. cruzi not affecting host cell viability.
Although not completely equivalent to Bz, etanidazole takes
advantage in exerting its anti-T. cruzi activity for longer periods
with no serious toxic effects as those recorded for Bz-treated
patients. According to this report,42 the small toxic effects found
for etanidazole were ascribed to its higher hydrosolubility than
Bz. Similarly, active 2H-benzimidazole 1,3 dioxide derivatives
are more hydrosoluble, electrophilic, and easily reduced than
the inactive ones.7 The presence of the NH3 and SO2 ligands,
which make the complex 1 more soluble in water than 2, has
circumvented this inconvenience. Furthermore, as mentioned
here before, at physiological pH, the trans-[Ru(Bz)(NH3)4SO3]
is the predominant form of 1, a neutral species that also could
be facilitate the complex movement across the cellular
membranes.
It is well accepted that the biochemical mechanism of action
of 2 involves reduction of the nitro group to a nitro radical anion
as a key step.9,22,43 The reduced intermediates do not stimulate
redox cycling but act covalently, modifying biomacromolecules
such as DNA or cytochrome P450 of the parasite.9,44 Upon
coordination, the nitro radical anion formation in 1 would
become more easily accessible to nitroreductase enzymes
because the cathodic wave attributed to reduction of the nitro
group to the corresponding hydroxylamine derivative was shifted
to -0.371 V vs SCE in acid medium. Therefore, the coordination of 2 to the [Ru(NH3)4SO2]2+ moiety will activate the NO2
group toward reduction by +0.104 V favoring the nitro radical
anion formation and subsequent hydroxylamine derivative by
9.6 kcal/mol in terms of free energy. Accordingly, the molecular
electrostatic potential energy isosurfaces (MEP) and NBO
charges show that the nitro group in 1 (N45, 0.43; O46/47, -0.32)
is more electrophilic than in 2 (N25, 0.42; O26/27, -0.37), hence
more susceptible to nucleophilic attack upon coordination to
the Ru(II) center (Table 1). The difference of energy between
the frontier orbitals (GAP ) ELUMO - EHOMO) was claimed to
be associated with the trypanocidal activity in pyrazolo pyridine
and 5-ethenylbenzofuroxan derivatives.45,46 The smaller the
GAP value, the higher the electronic affinity in the electrophilic
groups (e.g., NO2 moiety) the more active are the compounds.45,46
The GAP for 1, optimized by DFT calculations in aqueous
phase, is 1.85 eV, whereas for 2 the GAP is 2.54 eV. This GAP
energy decrease is in agreement with the higher electronic
A Benznidazole Ruthenium DeriVatiVe
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 14 4109
Figure 4. Parasitemia (a), survival rate (b), and representative microphotographs of heart (c) and skeletal muscle (d) sections of Swiss mice
infected with T. cruzi and treated with 1 or 2. Mice were infected with T. cruzi (Y strain, 1.0 × 103 BT per mouse) and treated with 1 at 100
nmol/kg for 15 consecutive days (protocol A, open diamond), or only on the 5th, 6th, and 7th days (protocol B, triangle). Other groups of infected
mice received only PBS (control, square) or doses of 2 at 100 nmol/kg (closed diamond). 100 µL PBS were intraperitoneally administrated for all
treatments. In a similar experiment, mice were euthanized on the 15th day after infection and their heart and skeletal muscles processed for H&E
stain. Note the presence of amastigotes nests (arrows) and the intensity of the inflammatory process with mononuclear cell infiltrates (circles) in the
control panels and no occurrence in the micrographics treated with 1 according to protocol A. Data are representative of three independent experiments
with similar results, n ) 6. Final magnification: 200×.
affinity of NO2 moiety in 1 than in 2 and with the higher
accessibility to the reduction potential of the nitro group (NO2/
NO2•-) by nitroreductase enzymes (Figure 2, Table 1).7,9,22
Thus, the best performance of 1 than 2 could be a consequence
of a higher accessibility of the nitro group to reduction
(Table 2).
Compound 1 administrated at 385 nmol/kg exhibits 100%
protection against death when infected mice are treated for
15 consecutive days. It is important to remind that this dose
is a thousand-fold smaller than the optimal dose for 2 (385
µmol/kg ) 100 mg/kg).36 The data presented here for 2 are
also in agreement with those previously published, showing
that it also exhibited 100% protection against death when
infected mice were treated with a 385 µmol/kg dose for 20
consecutive days.38 However, in this work, when infected
mice were treated with 2 at 100 nmol/kg, no protection
against death was observed, whereas mice treated with 1
showed 60% protection up to 120 days (Figure 4b). This
indicates that this new benznidazole derivative 1 is more
active than 2 even in concentrations 4000 times smaller than
its data estimated in vivo acute toxicity (LD50 > 400 µmol/
kg). Furthermore, 1 was also more active than 2 even when
only one dose was administrated.
In Central and South America, the chronic phase of the ChD
is one of the main causes of death due to heart failure.47 In
addition to being highly toxic, 2 exhibits a very low efficacy in
4110 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 14
SilVa et al.
Figure 5. Parasitemia (a), survival rate (b), and representative microphotographs of heart (c) sections of Swiss mice infected with T. cruzi and
treated with 1, 2, or nontreated (control). Mice were infected with T. cruzi (Y strain, 1.0 × 103 BT per mouse) and treated with 1 at 100 nmol/kg
dose (open diamond), 385 nmol/kg (triangle), or with 2 385 µmol/kg (closed diamond). Another group of infected mice was not treated (control,
square). 100 µL of PBS were orally administrated for all treatments. In a similar experiment, mice were euthanized on the 15th day after infection
and their hearts processed for H&E stain. Note the presence of amastigotes nests (arrows) and the intensity of the inflammatory process with
mononuclear cell infiltrates (circles) in the nontreated group (control) and no occurrence in the micrographics treated with 1 at 100 nmol/kg, 1 at
385 nmol/kg, or 2 at 385 µmol/kg. 200 µL for 1 and 100 µL for 2 were orally administrated for all treatments. Data are representative of three
independent experiments with similar results, n ) 6. Final magnification: 200×.
the treatment of patients chronically infected with T. cruzi.47,48
It has recently been reported that treatment of mice with 2 with
a 385 µmol/kg dose prevents from severe chronic cardiomyophaty, but it does not lead to a complete parasite eradication.47
On the other hand, 1 with a 100 nmol/kg dose proved to
eliminate the nests of amastigotes in myocardium tissue and
skeletal muscles and decrease myocarditis in the mouse acute
model. In fact, preliminary experiments carried out to evaluate
the trypanocidal activity of 1 on amastigotes-infected Vero cells
showed that this compound is able to eliminate viable amastigotes inside the Vero cells (data not shown). This reinforces the
potential therapeutic use of this compound as chemoprophylaxis
as well as chemotherapeutic agents.
Conclusion
The new hydrosoluble ruthenium benznidazole derivative 1
reported here behaves as a promising anti-T. cruzi compound
displaying interesting activities either in vitro or in vivo studies.
The higher trypanocidal activity for compound 1 than 2 could
be ascribed to the coordination of 2 to the trans-[Ru(NH3)4L]2+/+/0 moiety. The potentialization of the benznidazole
through coordination to the metal center can be explained based
A Benznidazole Ruthenium DeriVatiVe
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 14 4111
Figure 6. Representative microphotographs of liver sections of mice infected with T. cruzi 1.0 × 103 BT per mouse and nontreated group (a) or
treated with 1 at 385 nmol/kg (b) or 2 at 385 µmol/kg (c). Panel (d) represents a liver from a noninfected group. 100 µL for 1 and 200 µL for 2
were orally administrated for all treatments. On the 15th day after infection, mice were euthanized and their livers processed for H&E stain. Circles
indicate the inflammatory cells. Microphotographs are representative of three independent experiments with similar results, n ) 6. Final magnification:
200×.
on the electron withdrawing ability of the Ru(II) center due to
of the L ligand and the increase on the Bz water solubility
promoted by the coordination to the quite hydrophilic trans[Ru(NH3)4L]2+/+/0 moiety. Furthermore, 1 does not exhibit any
acute toxicity in Swiss mice at doses up to 400 µmol/kg. Assays
to verify the activity of 1 on other pathogenic trypanosomatids
such as Leishimania major are on course and results will be
reported soon.
Experimental Section
Chemicals and Reagents. Ruthenium trichloride (Aldrich) was
the starting material for the synthesis of all ruthenium complexes
described herein. All solvents were purified following known
procedures49 and doubly distilled water was used throughout. All
syntheses and manipulations were carried out under argon atmosphere.50 2 was obtained from Sigma-Aldrich and used as reference
drug.
Ruthenium Complexes. The trans-[Ru(H2O)(NH3)4(SO2)](TFMS)2 complex was synthesized and characterized following
published procedures.19,28 1 was prepared by the reaction between
the trans-[Ru(H2O)(NH3)4(SO2)](TFMS)2 complex (35 mg, 64
µmol) and 2 (40 mg, 0.15 mmol) in degassed acetone. Upon 2 h of
reaction, under argon atmosphere, the solution was rotevaporated
and the pale-yellow solid collected, filtered inside of a glovebag,
washed with 50 mL of degassed hexane three times, and kept in
dessicator under vacuum. As suggested from electronic and infrared
spectra, this complex is stable for at least 2 months when stored
under these conditions. Yield better than 55%. Calcd for trans[Ru(Bz)(NH3)4(SO2)](TFMS)2 · C3H6O: H, 3.53; N, 13.19; C, 24.03;
S, 11.32; Ru, 11.90. Found: H, 3.36; N, 13.32; C, 24.78; S, 11.06;
Ru, 11.83. Thermogravimetric (TG) experiments were performed
using a TGA-951 thermogravimetric unit coupled to a TGA-2100
thermal analyzer, both from TA Instruments, using a sample mass
of ca. 7 mg and a platinum sample holder under 90 mL/min dynamic
air flow. Only under temperatures higher than 218 °C was mass
loss observed. Therefore, it is reasonable to assume that 1 has
thermal stability under temperatures up to 200 °C.
Instrumentation. Hydrogen, carbon, nitrogen, and sulfur elemental microanalyses were done using an EA 1110 CHNS-O CE
Instrument. Ruthenium analyses were performed according to the
method proposed in published procedures,51 using a Polarized
Zeeman atomic absorption spectrophotometer, Z-8100 Hitachi, with
a Hitachi hollow cathode lamp, 12 mA, and 349.9 nm wavelength.
UV-visible measurements were performed in a 1.0 cm quartz
cell on a 8452A Hewlett-Packard diode array spectrophotometer.
IR spectra were recorded on a MB-102 Bomem FTIR spectrophotometer at 400-4000 cm-1 range in KBr pellets (1 wt %). The 1H
and 13C NMR spectra were performed in acetone-d6 and recorded
on a Bruker DPX-200 instruments at 200 MHz.
A 264A Princeton Applied Research polarographic analyzer/
stripping voltammeter attached to a microcomputer and employing
Microquı́mica Eletrochemical Software was used for the electrochemical measurements. The electrochemical cell used was a
conventional three-electrode type with an aqueous saturated calomel
electrode (SCE) as a reference electrode and a glassy-carbon and
platinum wire as working and auxiliary electrodes, respectively.
The solutions were degassed with argon for 10 min before each
measurement at 25 °C.
Solubility Measurements. The solubility values for 1 were
determined simulating a “bio-relevant” medium (pH ) 6.5, t ) 37
°C), which simulates the proximal small intestine in the fasted state.
This medium was prepared as previously reported52,53 and contained
3 mM sodium taurocholate, 0.75 mM lecithin, 3.9 mg KH2PO4,
7.7 mg KCl, and had regulated pH of 6.5-7.4 with NaOH. A known
amount of 1 was added to 5 mL of the medium, and the resulting
suspension was stirred for 24 h and filtered through a 0.45 µm filter.
The filtered suspension was then diluted and assayed by UV-vis
at 323nm (ε ) 4.5 × 103 M-1 cm-1) to determine in triplicate52,53
the concentration of 1.
Titration of 1 by Alkali. The pKa experiments were performed
according to a previous report.19 Then 25 mL of 1 was transferred
to a cell holding a combination electrode. The titrating solution,
NaOH 0.1 N, measured by means of a glass micrometer calibrated
to read 0.05 mL, was conduced through a burette. The temperature
was 25 ( 0.1 °C, measured during the experiment. Standard buffers
4112 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 14
at pH 4.004, 7.006, and 10.008 (25.0 °C) were used to standardize
the pH meter.
Molecular Descriptors. DFT calculations were performed using
the Gaussian 03 program.54 Optimized molecular geometries were
obtained in vacuum by using the hybrid functional B3LYP,55,56
the standard 6-31G(d) basis set for H, C, N, S, and O, and the
LANL2DZ basis set for Ru. B3LYP includes the functional
developed by Becke et al.,55 which combines the Becke’s gradient
part of the exchange functional,57 the Lee-Yang-Parr,58 and the
Vosko-Wilk-Nusair correlation functional with part of the exact
Hartree-Fock exchange energy.56 All the minimal energies were
characterized by frequency calculation, which show all real
frequencies. Zero-point vibrational energies were added on the
B3LYP frequency calculations (unscaled) using the same basis set
as in the geometry optimizations. The electronic structure of 1 and
2 were examined by NBO analysis59 as implemented in the
Gaussian 03 package. A detailed conformational search for the
molecules 1 and 2 was performed using DFT calculations to find
the minimum energy of the highest stable conformer, and the
geometry of this conformer was fully optimized applying the DFT
method in aqueous phase (Figure 1). Because in physiological pH
the predominant form of 1 is the trans-[Ru(Bz)(NH3)4SO3] species,
all the calculations were performed considering L in 1 as a SO32ligand, except when mentioned in text. Solvation energies have been
calculated using the polarizable continuum model (PCM).60–62 The
PCM is based on a description of the solvent as macroscopic
continuum medium having suitable properties (dielectric constant,
thermal expansion coefficient, etc.). In this procedure, the solute is
embedded in a cavity in the dielectric medium defined above. The
solute-solvent interactions are described in terms of the reaction
field due to the presence of the dielectric medium, which acts as a
perturbation on the Hamiltonian of the solute through its reaction
potential. Single-point calculations with the B3LYP and the standard
6-311+G(d,p) basis sets for H, C, N, S, and O, and the LANL2DZ
basis set for Ru was calculated. A dielectric constant of ε ) 78.39
was used for the reference reaction of 1 and 2 in water. Vibrational
frequencies were calculated from analytic second derivatives to
check the minimum on the potential energy surface.
Parasites. The Y strain of T. cruzi, a partially Bz-resistant and
highly virulent strain,11 was used for all experiments. Swiss mice
were infected intraperitoneally with 1.0 × 103 bloodstream trypomastigote forms of T. cruzi (BT) obtained from an intermediary
strain-matched infected mouse. Before infection of intermediary
mice, parasites were grown in Schneider’s medium and purified
from a monkey kidney fibroblast cell line LLC-MK2.
Evaluation of the Antiproliferative and Trypanocidal Activities in Vitro. All the experiments against trypomastigotes and
epimastigotes were performed according to previous report.18,36 BT
were obtained from mice on the parasitemia peak and resuspended
to 1.0 × 106/mL. Epimastigote forms (EF) were grown in a
Schneider’s medium, supplemented with 20% fetal calf serum,
harvested during the exponential phase of growth, washed in
phosphate-buffered saline (PBS), and resuspended to 2.0 × 106
parasites/mL. Then 200 µL of parasites were plated in triplicate
onto 96-well microplates and treated with both 1 and 2 diluted in
PBS (0.1, 0.25, 0.5, and 1.0 mM) and incubated at 37 °C, 5% CO2.
The parasite viability was subsequently assayed by determining the
number motile forms in a hemocytometer. The percentage of
trypanocidal activity (% TA) and the percentage of antiproliferative
activity (growth inhibition, % GI) for both compounds 1 and 2
were calculated as follows: % TA ) [1 - (LDt/LCt)] × 100 and %
GI ) {1 - [(LDt - LDt0)/LCt - LCt0)]} × 100, where LDt is the
average of the number motile forms in a well containing the drug
at time t, LDt0 is the average of the number motile forms in well
containing the drug at time t ) zero, LCt is the average of the number
of motile forms in well in the absence of any compound at time t
(negative control), and LCt0 is the average of the number of motile
forms in well in the absence of any compound at time t ) zero.36
The compound concentration corresponding to 50% of the antiproliferative or trypanocidal activities was later expressed as IC50epi
and IC50try, respectively.36
SilVa et al.
Evaluation of the Trypanocidal Activity in Vivo (Acute
Models). Female Swiss mice, 5-6 weeks old, were bred and
maintained under standard conditions in the animal house of the
Department of Biochemistry and Immunology, School of Medicine
of Ribeirão Preto-USP (Ribeirão Preto, Brazil). These animals were
infected by intraperitoneal administration of 1.0 × 103 BT per
mouse and treated with the doses of 1 at 100 nmol/kg/day ) 85
µg/kg/day or at 385 nmol/kg/day ) 330 µg/kg/day for 15 consecutive days (protocol A) or for only three days, the fifth, sixth, and
seventh days that precede the parasitaemic peak (protocol B). Two
other groups were treated with 2 at 385 µmol/kg/day ) 100 mg/
kg/day or at 100 nmol/kg/day ) 26 µg/kg/day. Nontreated infected
mice or infected mice treated only with PBS were used as control.
In a model, compounds 1 and 2 were injected by intraperitoneal
administration in 100 µL of PBS. In a second model, the doses of
1 and 2 (2 dissolved in water containing gum arabic) were given
by oral administration in 100 or 200 µL of PBS. All procedures
performed in the studies described herein were approved by the
Ethics Committee on Animal Research of the University of São
Paulo. Moreover, it is committed to the 3R principle (replacement,
reduction, refinement), in which alternative methods to the use of
animals and the refinement of technicians aiming to diminish,
whenever possible, the suffering and the number of animals in the
project are proposed.63,64 The course of infection was monitored
by counting the number of motile trypomastigotes in 5 µL blood
samples drawn from the tail veins, as previously described.65
Histological Analysis. All the untreated infected mice died of
acute ChD 15-30 days postinfection and were immediately
necropsied.30 The majority of the treated mice survived infection
and were euthanized 15 days after infection with T. cruzi. Hearts,
livers, and skeletal muscles were fixed in 10% formaldehyde
solution in PBS, embedded in paraffin, sectioned, stained with
hematoxylin-eosin, and examined through light microscopy analysis.
Unspecific Cytotoxicity. In Vitro. Cell viability assay was
performed on CD11c- CD11b+ bone marrow-derived macrophages
by propidium iodide (PI) exclusion as described elsewhere.66,67
Briefly, after 24 h of incubation of 1 × 106 cells per cm2 in 48well culture plates (Costar) containing RuBz (0.1 to 1 mM
concentrations) or RPMI medium alone, cells were harvested in
separate assay tubes, adding PI (Sigma P 4170) to each tube (50
µM final concentration), and the cells were kept in the dark at room
temperature for 15 min. After that, the cells were analyzed by
fluorescence with a FACS Canto II cytometry system (BD biosciences). The total of cells acquired for each sample was 10000.
Analysis was performed in FL2-A units. Background readings were
obtained from cells incubated with RPMI medium alone and stained
with PI. Positive controls were obtained by incubating cells with
seriate dilutions of Triton (X-100) for 15 min before PI addition.
The frequency of PI positive cells was determined according to
these controls. Additionally, cell viability and proliferation was also
evaluated using spleen cells from two Balb/c mice. This cells were
cultured with different concentrations of 1 (10 nM up to 1 mM) or
medium, and 6, 12, 24, and 48 h later, the percentage of viable
cells were determined in an hemocytometer using trypan blue
staining for exclusion (dead cells).31 In another assay, spleen cells
from two Balb/c mice were stained with CFSE (Sigma 5 µM) and
cultured for 72 h on a 96-well plate (Nunc, 5.0 × 105 cells/well),
under the same concentrations of 1 or medium. A set of wells was
also added with ConA (2 µg/mL), for induction of nonspecific
(policlonal) proliferation. After this, the cells suspensions were
acquired in a FACscan flow cytometer and analyzed with Cellquest
software (both Becton-Dickinson Immunocytometry Systems Inc.,
San Jose, CA).32
In Vivo. Swiss female mice (5- to 6-week-old) were treated with
1 diluted in PBS following the up-and-down test protocol for acute
toxicity testing.30 The compound was intraperitoneally administrated
in a single dose to only one animal and a 48 h observation period followed. If death occurred, another animal would be chosen
and would receive 1/3 of the previous dose; if not, the next animal
would receive three times the previous dose.30 In this study the
starting concentration was 100 µmol/kg.
A Benznidazole Ruthenium DeriVatiVe
Statistical Analysis. All the results herein presented were
considered statistically significant (P > 0.05) and were expressed
as mean ( sem The Mann-Whitney and Kruskal-Wallis were
used to determine the statistical significance of the intergroup
comparison.
Acknowledgment. We acknowledge the financial support
from Coordenação de Aperfeiçoamento de Pessoal de Nı́vel
Superior (CAPES), the Millennium Institute for Vaccine
Development and Technology (MIVDT/CNPq), Conselho Nacional de Desenvolvimento Cientı́fico e Tecnológico (CNPq),
and Fundação de Amparo à Pesquisa do Estado de São Paulo
(FAPESP).
Supporting Information Available: Combustion analyses for
target compounds, parasitemia of Swiss mice infected with T. cruzi
and treated with a single dose of 1 or 2, characters and energies of
LUMOs and HOMOs for complex 1, Vero cells infected with T.
cruzi and treated with 1 and 2 compounds. This material is available
free of charge via the Internet at http://pubs.acs.org.
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