New oxidovanadium(IV) N-acylhydrazone complexes: Promising antileishmanial and antitrypanosomal agents

Article abstract of DOI:10.1016/j.ejmech.2012.12.036

Searching for new promising metal-based hits against Trypanosoma cruzi and Leishmania parasites, two related oxidovanadium(IV) N-acylhydrazone complexes, [V^IVO(LASSBio1064-2H)(H₂O)], 1, and [V ^IVO(LASSBio1064-2H)(phen)]·(H

Full text of DOI:10.1016/j.ejmech.2012.12.036

European Journal of Medicinal Chemistry 62 (2013) 20e27
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
New oxidovanadium(IV) N-acylhydrazone complexes: Promising
antileishmanial and antitrypanosomal agents
,
e
Julio Benítez a, Aline Cavalcanti de Queiroz b, Isabel Correia c, Marina Amaral Alves d
,
,
,e
Magna S. Alexandre-Moreira b, Eliezer J. Barreiro d e, Lidia Moreira Lima d
,
Javier Varela f, Mercedes González f, Hugo Cerecetto f, Virtudes Moreno g,
,
João Costa Pessoa c, Dinorah Gambino a
*
a Cátedra de Química Inorgánica, Facultad de Química, UDELAR, Gral. Flores 2124, 11800 Montevideo, Uruguay
b LaFI e Laboratório de Farmacologia e Imunidade, Instituto de Ciências Biológicas e da Saúde, Universidade Federal de Alagoas, Maceió, AL, Brazil
c Centro Química Estrutural, Instituto Superior Técnico, Universidade Técnica de Lisboa, Av Rovisco Pais, 1049-001 Lisboa, Portugal
d LASSBio e Laboratório de Avaliação e Síntese de Substâncias Bioativas, Faculdade de Farmácia, Universidade Federal do Rio de Janeiro,
P.O. Box 68024, 21944-971, Rio de Janeiro, RJ, Brazil1
e Pós-graduação em Química, Instituto de Química, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil
f Grupo de Química Medicinal, Laboratorio de Química Orgánica, Facultad de Ciencias-Facultad de Química, Universidad de la República, Iguá 4225,
Montevideo 11400, Uruguay
g Departamento de Química Inorgánica, Universitat Barcelona, Martí i Franquès 1-11, 08028 Barcelona, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
Searchingfornewpromising metal-based hitsagainstTrypanosoma cruziandLeishmaniaparasites,tworelated
oxidovanadium(IV) N-acylhydrazone complexes, [VIVO(LASSBio1064-2H)(H2O)], 1, and [VIVO(LASSBio1064-
2H)(phen)]$(H2O), 2, where LASSBio1064 ¼ (E)-N0-(2-hydroxybenzylidene-4-chlorobenzohydrazide and
phen ¼ 1,10-phenanthroline, were synthesized and characterized in the solid state and in solution by
elemental analysis, conductimetric measurements and ESI-MS, FTIR, EPR and 51V NMR spectroscopies and
were evaluated on T. cruzi and Leishmania major. In addition, their unspecific cytotoxicity was tested against
murine macrophages. Furthermore, to provide insight into the possible mechanism of its antiparasitic action,
[VO(LASSbio1064-2H)(phen)].(H2O) was tested for its DNA interaction ability on plasmid DNA by atomic force
microscopy (AFM) and on CT DNA by using DNAviscosity measurements and fluorescence spectroscopy. Both
complexes were active in vitro against the epimastigote form of T. cruzi (Tulahuen 2 strain) showing IC50 values
of the same orderorsignificantlylowerthanthatof thereference trypanosomicidal drug Nifurtimox. However,
only the mixed-ligand oxidovanadium(IV) complex 2, which includes phen in its coordination sphere, showed
Received 28 August 2012
Received in revised form
7 November 2012
Accepted 19 December 2012
Available online 29 December 2012
Keywords:
Oxidovanadium(IV) compounds
N-Acylhydrazones
Heteroleptic complexes
Chagas disease
Leishmaniasis
Oxidovanadium(IV) phenanthroline
complexes
activity on L. major promastigotes with a IC50 value of 22.1 ꢀ 0.6
mM. The compounds show low toxicity on
mammalian cells (IC50 > 100 M). DNA interaction studies showed that the mixed-ligand complex is able to
m
interact with this biomolecule probably through an intercalative mode, pointing out at DNA as a potential
target in the parasite. The results suggest that [VIVO(LASSBio1064-2H)(phen)]$(H2O) may be a promising
compound for further drug development stages.
Ó 2012 Elsevier Masson SAS. All rights reserved.
1. Introduction
trypanosomatid genus and kinetoplastid order, Trypanosoma cruzi
and Leishmania spp., respectively, and are mainly transmitted to the
Chagas disease (American Trypanosomiasis) and Leishmaniasis
are considered by the World Health Organization as neglected
diseases (NTDs) and constitute important health problems concen-
trated in the poorest regions of the planet [1e3]. Both are among the
NTDs with the highest statistics of death [1]. They are produced by
genetically related protozoan parasites that belong to the
mammalian host by the bite of certain insects [4e6].
The chemotherapy of both diseases is far from being adequate and
mostly relies on drugs that date back to over 40e50 years having poor
efficacy and/or high toxicity, and increasing resistance development
[2e4,7]. New drugs against both diseases are thus urgently needed
and efforts are being made by medicinal chemists to identify new
organic or metal-based compounds that may be leads or hits for
further antitrypanosomal or antileishmanial drug development [8,9].
The N-acylhydrazones (RCONHN]CHR0), NAH, are simple molec-
ular frameworks synthetically obtained from functional group
* Corresponding author. Tel.: þ598 29249739; fax: þ598 29241906.
E-mail address: dgambino@fq.edu.uy (D. Gambino).
1
LASSBio, http://www.farmacia.ufrj.br/lassbio/.
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.12.036

J. Benítez et al. / European Journal of Medicinal Chemistry 62 (2013) 20e27
21
Table 1
interconversions, exploiting carboxylic acid derivatives as starting
materials, that have been reported as ‘privileged structures’ [10,11]. In
this context several pharmacological activities were reported for NAH
derivatives including antimicrobial, anticonvulsant, analgesic, anti-
inflammatory, antiplatelet, antiviral, antibacterial, antitumoral and
antiparasitic activities, among others [12]. Pursuing treatment of
parasiticdiseases changes inthe nature of the substituentsattached to
the acyl and imine groupsof N-acylhydrazone subunit hasallowed the
identification of lead-compounds for the treatment of leishmaniasis,
sleeping sickness and Chagas disease [13e15].
Tentative assignment of selected IR bands of LASSBio-1064 and complexes 1 and 2.
Band positions are given in cmꢁ1. See Fig. 1 for the structures.
Compound
n(VO) n(C]O) n n(OeH) n(NeH)
(C]N)a
LASSBio-1064
e
1652
e
1624
1608
1607
3436
e
3215
e
1, [VIVO(LASSBio1064-2H)(H2O)]
954
2, [VIVO(LASSBio1064-2H)(phen)]$ 955
e
e
e
(H2O)
a
The bands assigned to n(C]N) are coupled with the aromatic (C]C) stretching
bands [43,44].
The development of bioactive metal complexes is currently also
considered a promising approach in the search for new potential
drugs for the therapy of parasitic illnesses, particularly malaria,
leishmaniasis and trypanosomiasis [6,7,16e21]. In this path, some
bioinorganic chemistry groups are orienting their research toward
the development of trypanosomicidal metal-based compounds
[6,7,18e27]. In particular, some of us have been successfully
working on potential antitrypanosomal and antileishmanial
agents through different approaches using different metal centers
[Pd(II), Pt(II), Ru(II,III),V(IV,V), Au(I), and different 3d M(II) ions]
[19e21,28e33]. One of the strategies is based on the knowledge
that highly-proliferative cells such as kinetoplastid parasites
(Leishmania and Trypanosoma parasites) and tumor cells show
metabolic similarities that lead in many cases to a correlation
between antitrypanosomal and antitumor activities. Moreover, it
has been observed that some compounds that efficiently interact
with DNA in an intercalative mode also show antileishmanial
and/or antitrypanosomal activity [6,20,34]. The aim of this strategy
is to develop antitrypanosomatid metal compounds by including
DNA intercalators as ligands in the metal ion coordination sphere,
pointing out to DNA as potential target [19e21].
antitrypanosomatid drugs. Several homoleptic and heteroleptic
oxidovanadium(IV) complexes including DNA intercalators as
ligands (dppz ¼ dipyrido[3,2-a: 20,30-c]phenazine, bipy ¼ 2,20-
bipyridine, phen ¼ 1,10-phenanthroline) have been developed.
[VIVO(SO4)(H2O)2(dppz)]$2H2O and mixed-ligand oxidovanadiu-
m(IV) complexes, [VIVO(L2-2H)(L1)], including a bidentate poly-
pyridyl DNA intercalator (L1 ¼ dppz, bipy, phen) and a tridentate
salicylaldehyde semicarbazone derivative (L2) as ligands showed
IC50 values in the micromolar range against Dm28c strain (epi-
mastigotes) of T. cruzi, being slightly more active than the reference
trypanocidal drug Nifurtimox. By using atomic force microscopy,
gel electrophoresis, circular dichroism and fluorescence spectro-
scopic experiments and DNA viscosity measurements evidence was
obtained pointing out at DNA as a potential target of these
compounds [20,32,37,38].
Further work in this research area led to the development of two
related oxidovanadium(IV) N-acylhydrazone complexes including a
NAH designed by the LASSBio group, code LASSBio-1064, as ligand
(Fig. 1). A binuclear Zn(II) LASSBio-1064 complex, [Zn2(LASS-
Bio1064)2], was previously reported [37], and in the present
-
work two new complexes [VIVO(LASSBio1064-2H)(H2O)] and [VIV
Although the potentiality of vanadium compounds in medicinal
chemistry and medicinal applications has been extensively
explored, research has been mainly focused whether on improving
biodistribution and tolerability of the vanadium insulin-enhancing
core or on developing potential anti-tumor compounds [35,36].
Work on vanadium compounds for the treatment of some parasitic
diseases of high incidence in human health has only arisen in
a systematic way in recent years [20]. In particular, our research in
this area led to really encouraging results for the vanadium
complexes when compared to other metal compounds [20,28,29].
Having this background in mind, some of us have focused
their research on the development of potential vanadium-based
O(LASSBio1064-2H)(phen)]$(H2O) were prepared and character-
ized in the solid state and in solution and tested in vitro on T. cruzi
epimastigotes and Leishmania major promastigotes. In addition,
their unspecific cytotoxicity was tested against murine macro-
phages.Furthermore, to provide insight into the possible mecha-
nism of antiparasitic action, [VIVO(LASSbio1064-2H)(phen)]$(H2O)
was tested for its DNA interaction ability by using different
techniques.
2. Results and discussion
Two novel oxidovanadium(IV) complexes including the NAH
-
HO
ligand LASSBio-1064, [VIVO(LASSBio1064-2H)(H2O)], 1, and [VIV
Cl
O
O(LASSBio1064-2H)(phen)]$(H2O), 2, were synthesized with high
purity and reasonable yields (Fig. 1). Although both include the
same NAH ligand, one of them also includes 1,10-phenanthroline as
co-ligand. Both of them are neutral non conducting compounds in
DMF. Analytical, ESI mass spectrometry and FTIR and EPR
N
N
H
LASSBio-1064 (H2L)
Cl
N
O
O
V
N
O
V
O
H2O
O
O
N
N
N
N
[VO(LASSBio1064-2H)(phen)].(H O)
2
2
, compound
[VO(LASSBio1064-2H)(H2O)], compound 1
Cl
Fig. 1. Structural formulas of LASSBio-1064 and those proposed for the oxidovana-
dium(IV) complexes: [VIVO(LASSBio1064-2H)(H2O)], 1, and [VIVO(LASSBio1064-
2H)(phen)]$(H2O), 2.
Fig. 2. First derivative EPR spectra of frozen solutions (77 K) of complex 1 (ca. 1 mM) in
DMF and DMSO.

22
J. Benítez et al. / European Journal of Medicinal Chemistry 62 (2013) 20e27
Table 3
In vitro activity of the vanadyl complexes on T. cruzi (Tulahuen 2 strain). LASSBio-
1064 and Nifurtimox were included for comparison.
Compound
IC50 (mM)
[VIVO(LASSBio1064-2H)(H2O)]
[VIVO (LASSBio1064-2H)(phen)]$(H2O)
LASSBio1064
1.7 ꢀ 0.9
6.0 ꢀ 0.5
2.1 ꢀ 0.3
7.7a ꢀ 0.3
Nifurtimox
a
Ref. [59].
2.2. Characterization of the complexes in solution
Solutions of both compounds were studied by EPR and 51V NMR
spectroscopies. For this purpose ca. 1 mM solutions of the complexes
were prepared in DMF (and DMSO), kept at room temperature in
contact with air and the change over time of their spectroscopic (EPR
and 51V NMR) properties was studied.
Upon dissolving the complexes the solutions showed an
orange-brown color. In DMF solution compound 1 showed very
weak ded bands in the visible range at ca. 580 and 700 nm.
Compound 2 is more soluble in DMF than 1 and ded bands were
Fig. 3. Changes observed with time (hours) in the central region of the first derivative
EPR spectra for 1 mM solutions of complex 2 in DMF.
observed at ca. 800 nm (shoulder,
3
¼ 49 Mꢁ1 cmꢁ1), 707 nm
(
3
¼ 54 Mꢁ1 cmꢁ1), ca. 500 nm (shoulder,
3
¼ 92 Mꢁ1 cmꢁ1) and
a charge transfer band at 422 nm (
overlaps the band at 500 nm.
3
¼ 1000 Mꢁ1 cmꢁ1) which
spectroscopic results are in agreement with the proposed struc-
tures. The ESI-MS spectra confirm the formulas proposed for the
synthesized complexes. These experiments allowed the clear
detection of the protonated complex ion, M þ Hþ, for compound 2.
For the solution of complex 1 we assign the peak observed at
m/z ¼ 379.3 (100%) to [VVO2(LASSBio-1064-2H)$H]$Naþ formed by
oxidation of complex 1 during the experiment.
The EPR spectra of the complexes dissolved in DMF (and DMSO)
were measured at 77 K. The spectra exhibit a hyperfine pattern
typical of VIVO-complexes, consistent with the presence of mono-
meric VIVO-bound species with d1 ground-state configuration.
xy
Fig. 2 shows the spectra of complex 1 in both solvents and Fig. 3 the
variation of the EPR spectra of complex 2 (in DMF) with time.
Both complexes slowly oxidize. As expected, compound 2, con-
taining phen as co-ligand, is significantly more stable toward
oxidation and after 5 days it still showed the presence of a consid-
erable amount of VIVO. Table 2 contains the spin Hamiltonian
parameters obtained by simulation of the experimental spectra [45].
Once a particular binding mode is assumed, the values of Az
can be estimated (Aezst) using the additivity relationship proposed
by Wüthrich [46] and Chasteen [47], with estimated accuracy
of ꢀ3 ꢂ 10ꢁ4 cmꢁ1. Assuming that OCO binding contributes
with Az(OCO) ¼ 41.6 ꢂ 10ꢁ4 cmꢁ1, taking an average value for
2.1. IR spectroscopic studies
As previously described for related mixed-ligand VIVO-complexes
that simultaneously include a salicylaldehyde semicarbazone and
phenanthroline in the vanadium(IV) coordination sphere, N-acyl-
hydrazone complexes 1 and 2 show quite complex spectra [32]. In
particular, several bands corresponding to n(C]C) and n(C]N) in
heterocyclic compounds lie in the 1650e1550 cmꢁ1 region
[32,37,40]. Table 1 summarizes the tentative assignments made.
Az(NHZ) ¼ 41.6 ꢂ 10ꢁ4 cmꢁ1 [48], Az(Ophenolate ¼ Oph) ¼ 38.9 ꢂ 10ꢁ4 cmꢁ1
,
The non-observation of the n(C]O) band, indicates the enoli-
Az(Nphenanthroline ¼ Nphen ¼ Npyridine) ¼ 40.4 ꢂ 10ꢁ4 cmꢁ1 [52], Az
(ODMF) ¼ 43.7 ꢂ 10ꢁ4 cmꢁ1 and Az (ODMSO) z 43 ꢂ 10ꢁ4 cmꢁ1 [53] we
obtained for both complexes the values for Aezst presented in Table 2.
The Aezst agree well with the experimental values.
zation of the amide functionality upon coordination to the VIV-
center. Instead, strong bands at ca. 1620e1635 cmꢁ1 are observed,
which can be attributed to the asymmetric stretching vibration of
the conjugated CH]NeN]C group, characteristic of the coordi-
nated enolate form of the ligands [39,41]. These and other spectral
changes such as those summarized in Table 1 are in agreement with
LASSBio-1064 double deprotonation and tridentate coordination
For complex 2 in DMF the parent N-acylhydrazone (LASSBio-
1064) acts as a tridentate ligand binding with (Oph, NHZ, OCO equa-
)
torialand the phenanthroline (phen) binds as a bidentate ligand
through the two N donors, one N in the equatorial position and the
other trans to the oxo oxygen donor (Fig. 1). This axial-equatorial
through the carbonyl oxygen (OCO), the hydrazone N atom (NHZ
and the phenolate oxygen (Oph) (Fig. 1) [42].
)
Table 2
Spin Hamiltonian parameters obtained by simulation of the EPR spectra with the computer program of Rockenbauer and Korecz [43], and proposed equatorial binding sets.
a
Complex
Solvent
gx, gy
gz
Ax, Ay (ꢂ104 cmꢁ1
56.9
65.5
55.3
)
Az (ꢂ104 cmꢁ1
165.4
177.4
159.2
w164
)
Aezst (ꢂ104 cmꢁ1
165.8
174.7
162.5
165.1
)
Proposed equatorial binding seta
b
1
DMF
DMSO
DMF
1.980
1.976
1.982
1.950
1.937
1.953
Oph OCO NHZ ODMF
Owater 3 ꢂ ODMSO
Oph OCO NHZ Nphen
Oph OCO NHZ ODMSO
2
DMSO
a
The following Az,i values were used in the calculation of Aezst (see text): Ophenolate ¼ 38.9 ꢂ 10ꢁ4 cmꢁ1, Nphen ¼ 40.4 ꢂ 10ꢁ4 cmꢁ1, NHZ ¼ 41.6 ꢂ 10ꢁ4 cmꢁ1
,
OCO ¼ 41.6 ꢂ 10ꢁ4 cmꢁ1, ODMF ¼ 43.7 ꢂ 10ꢁ4 cmꢁ1, ODMSO z 43 ꢂ 10ꢁ4 cmꢁ1. For some of these their predicted contributions to the Az hyperfine coupling constant are not
straightforward, namely the contributions of NHZ and of OCO. In fact, the NHZ is probably close to the values of imine N atoms and these may vary between 38.1 and
43.7 ꢂ 10ꢁ4 cmꢁ1 [44,48,49], while OCO may vary between the contribution of a typical C]O carbonyl (43.7 ꢂ 10ꢁ4 cmꢁ1) [50], and of an O-enolate(ꢁ1) (37.6 ꢂ 10ꢁ4 cmꢁ1 [48]),
an average value in [VIVO(acac)2] being 41.6 ꢂ 10ꢁ4 cmꢁ1 [51].
b
The Aezst does not change much if the bound solvent is DMF, H2O or ethanol.

J. Benítez et al. / European Journal of Medicinal Chemistry 62 (2013) 20e27
23
Table 4
for both complexes showing some extent of ligand substitution/
oxidation in neat organic solvents which are capable to act as
ligands (DMSO or DMF). After oxidation in the neat solvents the
same type of complex is formed in both cases, but even after five
days of contact with the solvent and air at room temperature the
main resonance observed in the 51V NMR spectra corresponds
to species VVO2(N-acylhydrazone)(solvent) with binding set
VVO2(Oph, NHZ, OCO)(solvent). Conductivity measurements carried
out with 10ꢁ4 M solutions in 6% DMSOeH2O suggest that the
substitution/oxidation process occurs more slowly in this melieu
than in neat DMSO solution. After 24 h conductivity begins to
slowly increase probably due to generation of V(V) charged species.
In vitro activity of pentamidine (reference drug), LASSBio-1064 and its vanadium
compounds 1 and 2 against promastigotes of Leishmania major.
a
Compound
IC50
Highest
(
m
M ꢀ S.E.M.) efficacy (% ꢀ S.E.M.)
Pentamidine
0.8 ꢀ 0.2
72.6 ꢀ 2.0**
31.5 ꢀ 5.9
26.6 ꢀ 9.1
83.6 ꢀ 6.0**
LASSBio-1064
>100
1, [VIVO(LASSBio1064-2H)(H2O)]
>100
2, [VIVO(LASSBio1064-2H)(phen)]$(H2O) 22.1 ꢀ 0.6
Data are reported as means ꢀ S.E.M. Differences with a ##p value <0.01 were
considered significant in relation of medium group. Differences with a **p value
<0.01 were considered significant in relation of DMSO 0.1% group.
a
IC50 is the concentration required to give 50% inhibition, calculated by linear
regression analysis from the Kc values at concentrations employed (100, 30, 10, 3, 1,
0.3, 0.1 and 0.03 mM).
2.3. Biological evaluation of the complexes
binding geometry is found frequently in VIVO(
L-tridentate)(L1)
2.3.1. In vitro anti-T. cruzi activity
when L1 is bipy, phenanthroline, dppz or other similar hetero-
aromatic ligands [32,37,44,48,54,55].
Both complexes and the NAH-ligand LASSBio-1064 were eval-
uated in vitro for their anti-T. cruzi activities against epimastigotes
of Tulahuen 2 strain (Table 3). Results were compared to the
reference drug Nifurtimox. All complexes were active in vitro
against the epimastigote form of T. cruzi (Tulahuen 2 strain)
showing IC50 values lower or of the same order of those of the
reference drug Nifurtimox and the free ligand LASSBio1064.
In DMSO immediately after dissolution some aggregation of
molecules is maintained and the EPR spectra at 77 K show some
broadening of the lines and lower intensity of the spectra. This
behavior has been previously observed [56,57]. For this complex the
parameters obtained indicate significant solvolysis and substitution
of the phenanthroline ligand by DMSO. Due to the low intensity of the
spectra of complex 2 measured in DMSO it was not possible to carry
out adequate simulation of the spectra, but an estimate of the Az
values is given inTable 2. For complex 1 in DMSO, a solvent with good
coordinating ability, the spectrum indicates almost total replacement
of the N-acylhydrazone ligand by DMSO molecules, while in DMF the
EPR spectra are consistent with the formula given in Fig. 1.
2.3.2. In vitro anti-Leishmania activity and toxicity to mammalian
cells
To establish the leishmanicidal profile of LASSBio-1064 and its
vanadium complexes (1 and 2), these were evaluated in vitro
against promastigotes forms of L. major [60]. IC50 values obtained
from these studies are shown in Table 4. Complex 2 exhibited
antileishmanial activity (with the highest efficacy of 83.6 ꢀ 6.0%
The 51V NMR spectra measured at room temperature for the
1 mM solutions of the complexes in DMF confirm their progres-
sive oxidation over time. One hour after dissolution of the
complexes bands assignable to VV complexes could be already
observed at ca. ꢁ540 ppm. The intensity of the bands increased
with time and in the spectrum of complex 2 the resonances cor-
responding to vanadate oligomers (<10%) were found after 5 days
[ꢁ553 ppm (V1) and ꢁ576 ppm (V4)]. We can, therefore, conclude
that the same type of VV-species is being formed upon oxidation
of the two VIVO-complexes under air. Chemical shifts of VVO2-
semicarbazone complexes have been reported and theoretically
predicted in the ꢁ530 to ꢁ550 ppm range [58]. Thus the VV-
complexes formed probably correspond to species formulated as
VVO2(LASSBio1064)(solvent).
and IC50 values of 22.1 ꢀ 0.6
mM). However in molar units this
VIVO-complex was less potent than pentamidine (with the highest
efficacy of 72.6 ꢀ 2.0% and IC50 values of 0.8 ꢀ 0.2 M). In contrast,
LASSBio-1064 and complex 1 did not present activity against pro-
mastigotes forms of L. major (IC50 values > 100 M).
m
m
Fig. 4 shows the cytotoxicity results for the LASSBio-1064
compounds and pentamidine (positive control) tested against
murine macrophages using the MTT method [61]. The vanadium
complex 1 at concentration of 100
mM and the vanadium complex
2 up to 30 M showed deleterious activity on host cells evidenced
m
by the trials of cell viability in murine macrophages. However, the
LC50 values against peritoneal macrophages of complexes 1 and
2 are higher than 100 mM (Table 5). On the other hand, after 48 h of
Globally, we can conclude that both complexes show good
incubation, LASSBio-1064 and pentamidine did not affect the
viability of inflammatory macrophages.
stability in DMF, but lower in DMSO, the EPR and 51V NMR data
Fig. 4. Effect of pentamidine (reference drug), LASSBio-1064 and its vanadium compounds 1 and 2 against peritoneal macrophage cells after 48 h of treatment as assayed by the
MTT assay. The experiments were carried out in triplicate. The positive control consisted of dead cells lysed with 0.1% of Triton 100ꢂ. Data are reported as means ꢀ S.E.M.
Differences with a ##p value <0.01 were considered significant in relation of medium group and *p value <0.05 were considered significant in relation of vehicle group (DMSO).

24
J. Benítez et al. / European Journal of Medicinal Chemistry 62 (2013) 20e27
Table 5
Effect of pentamidine (reference drug), LASSBio-1064 and its vanadium compounds
1 and 2 against peritoneal macrophage cells after 48 h of treatment as evaluated by
the MTT assay.
a
Compound
LC50
Highest
(
mM ꢀ S.E.M.)
cytotoxicity
(% ꢀ S.E.M.)
Pentamidine
LASSBio-1064
>100
>100
>100
>100
20.2 ꢀ 3.4
28.0 ꢀ 5.1
42.8 ꢀ 5.1*
43.9 ꢀ 3.8*
1, [VIVO(LASSbio1064-2H)(H2O)]
2, [VIVO(LASSbio1064-2H)(phen)]$(H2O)
Data are reported as means ꢀ S.E.M. Differences with a *p value <0.05 were
considered significant in relation of DMSO 0.1% group.
a
LC50 is the concentration required to give 50% of mortality, calculated by linear
regression analysis from the Kc values at concentrations employed (100, 30, 10, 3, 1
and 0.3 mM).
Fig. 6. Relative viscosity e ri curve for [VIVO(LASSBio1064-2H)(phen)]$(H2O) (ri ¼ mol
of complex/mol of DNA base pairs) measured at 25 ꢃC.
2.4. DNA interaction studies
measurements also suggest an intercalating interaction between
compound 2 and DNA.
To provide insight into the possible mechanism of antiparasitic
action, the mixed-ligand compound [VIVO(LASSBio1064-
2H)(phen)]$(H2O) was tested for its DNA interaction ability on
plasmid DNA by atomic force microscopy (AFM) and on CT DNA by
using DNA viscosity measurements and fluorescence spectroscopy.
As previously described for structurally related oxidovanadium(IV)
complexes the presence of phen in the coordination sphere would
allow the intercalation between base pairs of the complex through
this planar moiety [32,37,38,62e64].
The fluorescence technique was not able to detect significant
effects due to interaction of the complex with CT DNA. These
studies showed only a very slight decrease of intensity, indicative of
non efficient displacement of intercalated ethidium bromide by the
mixed-ligand oxidovanadium(IV) compound (data not shown).
Probably complex 2 binds DNA less tightly than ethidium bromide,
therefore being unable to displace it.
The image obtained by AFM after incubation of plasmid DNA
with the complex for 24 h at 37 ꢃC is depicted in Fig. 5, together
with the image of the pBR322 plasmid alone incubated in the same
conditions. Effects due to the interaction are clearly observed, such
as kinks, crosslinking and supercoiling. Similar effects have been
previously observed for metal complexes capable to intercalate
DNA and, particularly, for other oxidovanadium(IV) complexes with
DNA intercalating ligands [32,37,38,62e64].
Upon interaction the complex increased the viscosity of CT
DNA solution in a concentration dependent manner as depicted in
Fig. 6. This type of behavior is usually attributed to intercalators as
base pairs are separated to accommodate the binding ligand,
leading to an increase of DNA length with a concomitant rise in
viscosity [65e67]. Therefore, the results of the DNA viscosity
2.5. Comments on the activity of the compounds
It is known that the biological activity of several metal based
drugs is due to species with structures distinct from those char-
acterized in the solid state. For example, the VIVO-compounds
selected for clinical trials as potential insulin-enhancing drugs have
demonstrated changes in the vanadium coordination sphere after
dissolution, and particularly after being absorbed in the gastro-
intestinal tract [36,68e70].
The present VIVO-compounds could act in biological media as
prodrugs leading to VIV-and/or VV-LASSBio-1064 species that would
be responsible of the observed activities. As the vanadium
compounds show higher activity than the ligand and/or the metal
salts, this is a clear indication that the activity is associated to
Fig. 5. a) AFM image of pBR322 DNA incubated 24 h at 37 ꢃC and b) AFM image of pBR322 DNA after interaction with [VIVO(LASSBio1064-2H)(phen)]$(H2O) at a molar ratio
compound: DNA base pairs 1:5 and 24 h incubation at 37 ꢃC.

J. Benítez et al. / European Journal of Medicinal Chemistry 62 (2013) 20e27
25
vanadium species containing the ligand at least till the compounds
4.3. Synthesis of the mixed-ligand vanadyl complex
are up-taken bycells.Thehigheractivityof themetal compoundsmay
be related to either an easier uptake and/or to their higher efficacy
toward their targets. On the other hand, inside cells the vanadium
speciation (not addressed in this work) may yield species containing
or not the original ligands. The phenanthroline moiety may indeed be
relevant in this respect; there are several other examples where phen
and other related compounds, which may act as DNA intercalators,
improve the biological activity of the metal-based systems [40,71].
[VIVO(LASSBio1064-2H)(phen)]$(H2O), 2
LASSBio-1064 (50 mg, 0.188 mmol),1,10-phenanthroline (34 mg,
0.188 mmol) and [VIVO(acac)2] (50 mg, 0.188 mmol) were sus-
pended in 20 mL of absolute ethanol previously purged with
nitrogen for 10 min. Nitrogen was kept passing through the solu-
tion for ca. 10 min. The suspension was heated at reflux under
nitrogen for 24 h and the reddish-brown solid formed was filtered
off from the hot mixture, and was washed three times with 2 mL
portions of ethanol:diethyl ether (1:1). Yield: 69% (70 mg). Anal (%)
calc. for C26H17ClN4O4V$H2O: C, 58.06; H, 3.56; N, 10.42. Found: C,
57.85; H, 3.53; N, 10.78. ESI-MS (MeOH) m/z [Found (calc.)]: 520.2
(520.0) (40%) (M þ Hþ).
3. Conclusions
Two new oxidovanadium(IV) complexes, [VIVO(LASSBio1064-
2H)(H2O)] and [VIVO(LASSBio1064-2H)(phen)]$(H2O), including
the N-acylhydrazone LASSBio-1064 ligand, were synthesized and
characterized in the solid state and in solution. Both complexes and
the N-acylhydrazone compound showed trypanosomicidal activity
on T. cruzi, the etiological agent of Chagas disease, slightly higher (in
a molar basis) than that of the trypanocidal drug Nifurtimox.
However, only the mixed-ligand vanadyl complex 2 containing
phen as co-ligand showed activity on L. major promastigotes.
DNA interaction studies showed that this mixed-ligand complex is
able to interact with the biomolecule probably through an inter-
calative mode, pointing out at DNA as a probable target. Other
potential targets could not be discarded. A higher uptake by cells and/
or the interaction with DNA could be responsible for the increased
antileshmanial activity of [VIVO(LASSBio1064-2H)(phen)]$(H2O) as
compared with [VIVO(LASSBio1064-2H)(H2O)] and LASSBio1064.
Both VIVO-compounds showed low toxicity on mammalian cells and,
therefore, a good selectivity toward the parasite.
4.4. Physicochemical characterization
C, H and N analyses were carried out with a Carlo Erba Model
EA1108 elemental analyzer. Conductimetric measurements were
carried out at 25 ꢃC in 10ꢁ3 M dimethylformamide (DMF) solutions
using a Conductivity Meter 4310 Jenway [75]. A 500-MS Varian Ion
Trap Mass Spectrometer was used to measure electrospray ioniza-
tion mass spectra (ESI-MS) of methanol solutions of the complexes in
the positive mode; the complexes were first dissolved in a minimum
amount of DMF and then diluted with MeOH. Each spectrum was
obtained as a combination of several scans for each sample. FTIR
spectra (4000e400 cmꢁ1) of the complexes and the free ligand were
measured as KBr pellets with a Bomen FTIR model MB102 instru-
ment. The UVeVis absorption spectra were measured with a Perkin
Elmer Lambda 35 spectrophotometer and 51V NMR spectra of ca.
1 mM solutions of the complexes in DMSO and DMF (p.a. grade)
were recorded on a Bruker Avance III 400 MHz instrument: (i) after
dissolution, and (ii) during a 5 day period standing in aerobic
conditions at room temperature. 51V chemical shifts were referenced
relative to neat VOCl3 as external standard. EPR spectra were recor-
ded at 77 K with a Bruker ESP 300E X-band spectrometer coupled to
a Bruker ER041 X-band frequency meter (9.45 GHz). The complexes
were dissolved at room temperature in DMSO or DMF (p.a. grade),
previously degassed with N2 for 10 min, using ultrasound to assist
the dissolution of the solid, and the obtained solutions were then
frozen in liquid nitrogen. The spin Hamiltonian parameters were
obtained by simulation of the spectra with the computer program of
Rockenbauer and Korecz [76]. Stability of the complexes in 6%
DMSOeH2O$10ꢁ4 M solution at room temperature was followed by
conductivity measurements for 5 days.
The results suggest that [VIVO(LASSBio1064-2H)(phen)]$(H2O)
may be a promising compound for further drug development stages.
Moreover, further speciation studies would help in establishing the
actual species responsible of the detected biological activity.
4. Materials and methods
4.1. Materials
All common laboratory chemicals, including [VIVO(acac)2], where
acac ¼ acetylacetonate, 1,10-phenanthroline (phen), VOSO4$5H2O,
pentamidine, Schneider’s milieu, Fetal Bovine Serum and Dubbeco’s
Modified Eagle’s milieu (DMEM), were from commercial sources and
were used without further purification. LASSBio-1064, (E)-N0-
(2-hydroxybenzylidene-4-chlorobenzohydrazide), was synthesized
and characterized as previously described by reaction of salicy-
laldehyde (2-hydroxy-benzaldehyde) and 4-chlorobenzohydrazide
in 1:1 M ratio in ethanoleHCl milieu [72e74].
4.5. In vitro anti-T. cruzi activity
T. cruzi epimastigotes (Tulahuen 2 strain) were grown at 28 ꢃC in
an axenic milieu (BHI-Tryptose), as previously described, com-
plemented with 5%fetalcalfserum[28,29,77]. Cells were harvestedin
the late log phase, re-suspended in fresh milieu, counted in Neu-
bauer’s chamber and placed in 24-well plates (2 ꢂ 106/mL). Cell
growth was measured as the absorbance of the culture at 590 nm,
which was proved to be proportional to the number of cells. Before
inoculation the media were supplemented with the indicated
amount of the studied compound from a stock solution in DMSO
prepared immediately before use. The final concentration of DMSO in
the culture media never exceeded 1% and the control was run in the
presence of 1% DMSO as well as in the absence of any compound. No
effect on epimastigotes growth was observed by the presence of up to
1% DMSO in the culture media. Nifurtimox was used as the reference
trypanosomicidal drug. The percentage of growth inhibition was
calculated as follows {1 ꢁ [(Ap ꢁ A0p)/(Ac ꢁ A0c)]} ꢂ 100,
where Ap ¼ A590 of the culture containing the studied compound at
4.2. Synthesis of [VIVO(LASSBio1064-2H)(H2O)], 1
LASSBio-1064 (50 mg, 0.188 mmol) and VIVOSO4$5H2O (47 mg,
0.188 mmol) were suspended in 20 mL of absolute ethanol previ-
ously purged with nitrogen for 10 min. The suspension was heated at
reflux under nitrogen for 24 h and the reddish-brown solution ob-
tained was partially evaporated (final volume 3 mL). The brown solid
formed was filtered off from the mixture and washed with 2 mL
portions of diethyl ether, acetone and methanol. Yield: 40% (27 mg).
Anal (%) calc. for C14H11ClN2O4V: C, 47.02; H, 3.10; N, 7.83. Found: C,
46.79; H, 3.34; N, 7.78. ESI-MS (MeOH) m/z Found ¼ 379.3 (100%)
{[VIVO(LASSBio1064-2H)(H2O)]$Naþ, with m/z ¼ 380.6 does not fit}.
Probably the compound oxidized in this dilute solution and what is
detected is the corresponding VVO2-complex ([VVO2(LASSBio1064-
H)]$Naþ) for which the molecular weight is 378.62 and the m/z for
the molecular ion is 379.6, which fits well with the experimental.

26
J. Benítez et al. / European Journal of Medicinal Chemistry 62 (2013) 20e27
day 5; A0p ¼ A590 of the culture containing the studied compound
right after addition of the inocula (day 0); Ac ¼ A590 of the culture in
the absence of any compound (control) at day 5; A0c ¼ A590 in the
absence of the compound at day 0. To determine IC50 values parasite
growth was followed in the absence (control) and presence of
increasing concentrations of the corresponding compound. The IC50
values were determined as the drug concentrations required to
reduce by half the absorbance of that of the control (without
compound) [28,29,77].
the treated and the control groups were evaluated by ANOVA and
Dunnett hoc tests. Differences with a p value <0.05 or lower were
considered significant.
4.8. DNA interaction studies
4.8.1. Atomic force microscopy (AFM) studies
To optimize the observation of the conformational changes in the
tertiary structure of pBR322 plasmid DNA, the plasmid was heated at
60 ꢃC for 30 min to obtain mostly the open circular form. Then 15 ng
of pBR322 DNA were incubated in an appropriate volume with the
required compound concentration corresponding to the molar ratio
base pairs (bp):compound 5:1. The oxidovanadium(IV) complex was
dissolved in a minimal amount of DMSO and 4-(2-hydroxyethyl)-1-
piperazineethanesulfonic acid buffer (HEPES) pH 7.4 was then added
up to the required concentration. All the solutions as well as Milli-QÒ
4.6. In vitro activity against L. major
Promastigotes of L. major IOC/L0581 (MHOM/SU/1973/5-ASKH)
were obtained from Leishmania collection of the Oswaldo Cruz
Institute. The parasites were maintained in vitro in Schneider’s
milieu, supplemented with 10% FBS and 2% human urine. Stock
-
solutions of LASSBio-1064, [VIVO(LASSBio1064-2H)(H2O)], 1, [VIV
water were filtered with 0.2 mm FP030/3 filters (Schleicher & Schuell
O(LASSBio1064-2H)(phen)]$(H2O), 2, and pentamidine (reference
leishmanicidal drug) were prepared in DMSO immediately before
use. The cytotoxicity of LASSBio1064, complexes 1 and 2 and
pentamidine against promastigotes was then determined.
Stationary phase L. major promastigotes were plated in 96-well
vessels (Nunc) at 1 ꢂ 105 cells per well, in Schneider’s milieu,
supplemented with 10% FBS and 2% human urine, and increasing
GmbH, Germany). Immediately after preparing the solutions incu-
bations were carried out at 37 ꢃC for 24 h. Samples were prepared by
placing a drop of DNA solution or DNA-compound solution onto
mica (TED PELLA, INC. California, USA). After adsorption for 5 min at
room temperature, the samples were rinsed for 10 s in a jet of
deionized water (18 M
system) directed onto the surface, blow dried with compressed
argon and then imaged by AFM.
U
cmꢁ1 from a Milli-QÒ water purification
concentrations (0.3 mMe100 mM) of each compound solution were
added. Cells were also cultured in a milieu free of compounds or
vehicle (basal growth control) or with DMSO 0.1% (vehicle control).
After 48 h extracellular load of L. major promastigotes was esti-
mated by counting the promastigotes in Schneider’s milieu in
a CELM automatic cell counter (model CC530) [60].
The samples were imaged by a Nanoscope III Multimode AFM
(Digital Instrumentals Inc., Santa Barbara, CA) operating in tapping
mode in air at a scan rate of 1e3 Hz. The AFM probe was 125 mm-
long monocrystalline silicon cantilever with integrated conical
shaped Si tips (Nanosensors GmbH Germany) with an average
resonance frequency fo ¼ 330 kHz and spring constant K ¼ 50 N/m.
The cantilever was rectangular and the tip radius given by the
supplier was 10 nm, a cone angle of 35ꢃ and high aspect ratio. The
images were obtained at room temperature (t ¼ 23 ꢀ 2 ꢃC) and the
relative humidity was usually lower than 40% [32,38].
4.7. Cytotoxicity against host cells
Inflammatory macrophages were obtained using adult Swiss
mice weighing 20e30 g each, males or females, of about 6e8
weeks of age. All animals came from the breeding unit of the
BIOCEN e UFAL. The animals were maintained with free access to
food and water and kept at 25e28 ꢃC under a controlled 12 h
light/dark cycle. All animals used in this work were treated
according to the regulations established by the Ethics Committee
of Federal University of Alagoas e UFAL (number: 23065.024392/
2009-90) for animal handling. To evaluate the cytotoxic activity
against the murine macrophages the animals were injected with
1.0 mL of thioglycolate 4%. After 4 days the animals were killed
by cervical dislocation. The peritoneal cavity was washed with
5.0 mL of Hank’s buffer salt solution (HBSS, Sigma), and after
gentle manual massage the exudate was retrieved to obtain
inflammatory macrophages. The peritoneal macrophages were
plated in 96-well vessels at 2 ꢂ105 cells per well in complete
culture milieu 10% FBS at 37 ꢃC. After 1 h wells were washed
with warm HBSS to remove non-adherent cells, leaving
approximately 1 ꢂ105 adherent macrophages. All cultures were
done in DMEM complete supplemented with 10% FBS. The
compounds and pentamidine were added at serial concentra-
4.8.2. Fluorescence studies
To a 50
L of a 5 mM ethidium bromide solution was added to get
m
M calf thymus DNA (CT DNA) solution in Milli-QÒ water,
30
m
a 1:1 M ratio of ethidium bromide to DNA. The mixture was incu-
bated for 30 min at 37 ꢃC. Increasing amounts of a 1.5 mM
DMSO/Milli-QÒ water stock solution of the complex under study
were added to reach the following final concentrations of the
complex: 0, 10, 20, 30, 40 and 50
mM. Fluorescence spectra
(
lex ¼ 520 nm) were recorded at room temperature with a HORI-
BA Nanolog iHR 320 spectrophotometer in the wavelength range
530e670 nm after a short incubation time [48].
4.8.3. Viscosity measurements
Viscosity experiments were conducted at 25 ꢃC on an automated
and viscometer model SV-10. Stock solutions of each complex were
prepared in DMSO/water. A 1 mM CT DNA solution was diluted 1:4
with TE buffer. For each complex increasing amounts of complex
stock solution were added to the DNA solution to reach com-
plex/DNA molar ratios in the range 0e2.0. The DMSO amount in the
samples never exceeded 2%. After thermal equilibrium was ach-
ieved (15 min), the viscosity of each sample was repeatedly
measured. Mean values of five measurements carried out at inter-
vals of 1 min were used to obtain the viscosity of each sample [48].
tions (0.3e100 mM). The cells were also cultured with milieu free
from compounds or vehicle (basal growth control) or in media
with DMSO 0.1% (vehicle control). Positive control (dead cells)
was obtained by cellular lysis with 1% of Triton 100ꢂ in DMEM
complete. After 48 h, the cytotoxicity was evaluated by the MTT
assay [61].
Acknowledgments
4.7.1. Statistical analysis
Data obtained from in vitro experiments were expressed as the
mean ꢀ standard error of the mean (Mean ꢀ S.E.M.) of triplicate
cultures of representative assays. Statistical differences between
Authors wish to thank CYTED networks RIIDFCM and RID-
IMEDCHAG, Prosul-CNPq Proc. 490.600/2007-8, Brazil, PEDECIBA
Química, Uruguay and Fundação para a Ciência e Tecnologia (FCT,

J. Benítez et al. / European Journal of Medicinal Chemistry 62 (2013) 20e27
27
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