Antiparasitic activities of novel ruthenium/lapachol complexes

Article abstract of DOI:10.1016/j.jinorgbio.2014.03.009

The present study describes the synthesis, characterization, antileishmanial and antiplasmodial activities of novel diimine/(2,2′- bipyridine (bipy), 1,10-phenanthroline (phen), 4,4′-methylbipyridine (Me-bipy) and 4,4′-methoxybipyridine (MeO-bipy)/phosphi

Full text of DOI:10.1016/j.jinorgbio.2014.03.009

Journal of Inorganic Biochemistry 136 (2014) 33–39
Contents lists available at ScienceDirect
Journal of Inorganic Biochemistry
journal homepage: www.elsevier.com/locate/jinorgbio
Antiparasitic activities of novel ruthenium/lapachol complexes
Marília I.F. Barbosa a, Rodrigo S. Corrêa a, Katia Mara de Oliveira a, Claudia Rodrigues a, Javier Ellena b,
Otaciro R. Nascimento b, Vinícius P.C. Rocha c, Fabiana R. Nonato c, Taís S. Macedo c, José Maria Barbosa-Filho e,
Milena B.P. Soares c,d, Alzir A. Batista a,
⁎
a
Departamento de Química, Universidade Federal de São Carlos, CP 676, CEP 13565-905, São Carlos (SP), Brazil
Instituto de Física de São Carlos, Universidade de São Paulo, CP 369, CEP 13560-970, São Carlos (SP), Brazil
Laboratório de Engenharia Tecidual e Imunofarmacologia, Fiocruz, CEP: 40296-710, Salvador (BA), Brazil
Centro de Biotecnologia e Terapia Celular, Hospital São Rafael, CEP 41253-190, Salvador (BA), Brazil
b
c
d
e
Universidade Federal da Paraíba Laboratório de Tecnologia Farmacêutica, CEP 58051-900, João Pessoa (PB), Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
The present study describes the synthesis, characterization, antileishmanial and antiplasmodial activities of novel
diimine/(2,2′-bipyridine (bipy), 1,10-phenanthroline (phen), 4,4′-methylbipyridine (Me-bipy) and 4,4′-
methoxybipyridine (MeO-bipy)/phosphine/ruthenium(II) complexes containing lapachol (Lap, 2-hydroxy-3-
(3-33 methyl-2-buthenyl)-1,4-naphthoquinone) as bidentate ligand. The [Ru(Lap)(PPh3)2(bipy)]PF6 (1),
[Ru(Lap)(PPh3)2(Me-bipy)]PF6 (2), [Ru(Lap)(PPh3)2(MeO-bipy)]PF6 (3) and[Ru(Lap)(PPh3)2(phen)]PF6 (4)
complexes, PPh3 = triphenylphospine, were synthesized from the reactions of cis-[RuCl2(PPh3)2(X-bipy)] or
cis-[RuCl2(PPh3)2(phen)], with lapachol. The [RuCl2(Lap)(dppb)] (5) [dppb = 1,4-bis(diphenylphosphine)butane]
was synthesized from the mer-[RuCl3(dppb)(H2O)] complex. The complexes were characterized by elemental
analysis, molar conductivity, infrared and UV–vis spectroscopy, 31P{1H} and 1H NMR, and cyclic voltammetry.
The Ru(III) complex, [RuCl2(Lap)(dppb)], was also characterized by the EPR technique. The structure of the com-
plexes [Ru(Lap)(PPh3)2(bipy)]PF6 and [RuCl2(Lap)(dppb)] was elucidated by X-ray diffraction. The evaluation of
the antiparasitic activities of the complexes against Leishmania amazonensis and Plasmodium falciparum demon-
strated that lapachol–ruthenium complexes are more potent than the free lapachol. The [RuCl2(Lap)(dppb)] com-
plex is the most potent and selective antiparasitic compound among the five new ruthenium complexes studied in
this work, exhibiting an activity comparable to the reference drugs.
Received 29 November 2013
Received in revised form 19 March 2014
Accepted 19 March 2014
Available online 27 March 2014
Keywords:
Ruthenium (II) and (III) lapachol complex
Cytotoxicity
Antileishmanial and antiplasmodial activities
© 2014 Published by Elsevier Inc.
1. Introduction
The Tabebuia genus, belonging to the bignoniaceae plant family, is
widely used in the traditional medicine in South America [5,6]. Among
Leishmaniasis and malaria are diseases caused by protozoan
parasites and are characterized by high morbidity. It is estimated that
leishmania disease causes about seventy thousand deaths annually
and malaria kills around 1 million children only in Africa [1]. The first
line treatment for leishmaniasis still relies on the use of pentavalent
antimonials, although other drugs are also used for the treatment of
Leishmania infection, such as pentamidine isethionate, amphotericin B
and miltefosine [2,3]. Malaria treatment relies on the use of quinoline-
based drugs, such as chloroquine, primaquine and mefloquine, as well
as antifolates and artemisinin derivatives, depending on the parasite's
susceptibility [4]. Common problems with these antiparasitic drugs
are severe side effects and development of drug resistance. Based on
this scenery, the research of new active compounds against these para-
sites is pivotal.
the active secondary metabolites present in this genus, 2-hydroxy-3-
(3-methyl-2-buthenyl)-1,4-naphthoquinone (lapachol, Fig. 1) is one
of the most studied. Lapachol is endowed with anticancer and antimi-
crobial properties [7,8]. Because of its antiproliferative activity, lapachol
has been employed as a prototype for the design and synthesis of new
anticancer and antimicrobial agents. This has led to the identification
of fewer lapachol derivatives with an enhanced activity [9–12].
Like other naphthoquinones [13,14], lapachol is a feasible ligand for
the preparation of coordinating or organometallic compounds. In fact,
there are some findings showing that lapachol–metal complexes are bi-
ologically more active than the free molecule [15–18]. Ruthenium com-
plexes are considered to be one of the most promising types of metal
compounds for cancer treating, due its interesting chemical properties,
such as: versatility in ligand exchange, octahedral geometry and vari-
ability of oxidation states [19,20]. Recently it was observed that the
lapachol–Ru(II) complex is a more potent anticancer agent than
lapachol–Os(II) and Rh(III) complexes [18], suggesting that the use of
ruthenium is promising to improve the biological activity of lapachol.
⁎
Corresponding author. Tel.: +55 1633518285; fax: +55 1633518350.
E-mail address: daab@power.ufscar.br (A.A. Batista).
http://dx.doi.org/10.1016/j.jinorgbio.2014.03.009
0162-0134/© 2014 Published by Elsevier Inc.

34
M.I.F. Barbosa et al. / Journal of Inorganic Biochemistry 136 (2014) 33–39
using the software Collect [21] and Scalepack [22], and the final cell pa-
rameters were obtained on all reflections. Data reduction was carried
out using the software Denzo-SMN and Scalepack [22]. The structures
were solved by the Direct method using SHELXS-97 [15] and refined
using the software SHELXL-97 [23]. A Gaussian method implemented
was used for the absorption corrections [24]. Non-hydrogen atoms of
the complexes were unambiguously located, and a full-matrix, least-
square refinement of these atoms with anisotropic thermal parameters
was carried out. The aromatic C\H hydrogen atoms were positioned
stereochemically and were refined with fixed individual displacement
parameters [Uiso(H) = 1.2 Ueq(Csp2)] using a riding model with an aro-
matic, C\H bond length fixed at 0.93 Å. Methylene groups of the dppb
ligand in the complex (5), and methine group of the lapachol were set as
isotropic with a thermal parameter 20% greater than the equivalent
isotropic displacement parameter of the atom to which each one
was bonded, whereas methyl groups were set with Uiso(H) values of
1.5Ueq(Cmethyl). Tables were generated by WinGX [25] and the structure
representations by ORTEP-3 [18] and MERCURY [21]. The main crystal
data collections and structure refinement parameters for (1) and (5)
are summarized in Table 1.
Fig. 1. Lapachol structure.
Therefore, the present study describes the synthesis, characterization,
antileishmanial and antiplasmodial activities of novel diimines (2,2′-
bipyridine (bipy), 1,10-phenantroline (phen), 4,4′-methylbipyridine
(Me-bipy) and 4,4′-methoxybipyridine (MeO-bipy) and monophosphine
ruthenium(II) and (III) complexes containing lapachol as a bidentate
ligand.
2. Experimental section
2.4. Synthesis
2.1. Materials for synthesis
All the solvents used in this work were of reagent quality and used
without further purification. Lapachol was obtained according to the
procedure described in [24]. The precursors cis-[RuCl2(PPh3)2(X-bipy)]
(X = H, methyl (Me) and methoxy (MeO)) and cis-[RuCl2(PPh3)2
(phen)] were prepared according to literature [26,27]. Typically
[100.0 mg; 0.1 mmol] of the [RuCl2(PPh3)3] was dissolved in degassed
20 mL of dichloromethane (Merck) and N-heterocyclic (X-bipy or
Solvents were purified by standard methods. All chemicals used were
of reagent grade or comparable purity. The RuCl3∙3H2O was purchased
from Degussa or Aldrich. The ligands 1,4-bis(diphenylphosphino)butane
(dppb), triphenylphosphine (TPP), bipy, Me-bipy, MeO-bipy and phen
were used as received from Aldrich.
2.2. Instrumentation
Elemental analyses were performed in a Fisons EA 1108 model
(Thermo Scientific). The IR spectra of the powder complexes were re-
corded using CsI pellets in the 4000–200 cm−1 region in a Bomen–
Michelson FT MB-102 instrument. The UV–Visible (UV–vis) spectra of
the complex were recorded in CH2Cl2 solution, in a Hewlett Packard
diode array—8452A. The electron paramagnetic resonance (EPR)
spectrum was measured in solid state at −160 °C using a Varian
E-109 instrument, recorded at the X band frequency, within a rectangu-
lar cavity (E-248) fitted with a temperature controller. Cyclic voltamm-
etry (CV) experiments of the complexes in solution were promoted in
an electrochemical analyzer BAS model 100B. These experiments
were carried out at room temperature, in CH2Cl2 containing 0.10 M
Bu4N+ClO−4 (TBAP) (FlukaPurum) as support electrolyte, and using
an one-compartment cell, with both working and auxiliary electrodes,
which were stationary Pt foils, while the reference electrode was
Ag/AgCl, 0.10 M TBAP in CH2Cl2. Under these conditions, the ferro-
cene is oxidized at 0.43 V (Fc+/Fc).
Table 1
Crystal data and structure refinement for complex [Ru(Lap)(PPh3)2(bipy)]PF6 (1) and
[RuCl2(Lap)(dppb)] (5).
[Ru(Lap)(PPh3)2(bipy)]PF6 [RuCl2(Lap)(dppb)]
Empirical formula
Formula weight
Crystal system
Space group
Unit cell dimensions
a (Å)
[RuC61H51N2O3P2]PF6
1168.02
[RuC43H41Cl2O3P2]
839.67
Monoclinic
P21/c
Monoclinic
P21/c
15.950(5)
16.744(5)
20.316(5)
93.151(5)
5418(3)
4
9.1790(1)
29.6950(5)
14.7120(3)
104.564(1)
3881.20(11)
4
b (Å)
c (Å)
β (o)
Volume (Å3)
Z
Density calculated
1.432
1.437
(Mg/m3)
μ (mm−1
)
0.447
0.663
F(000)
2392
1724
Crystal size (mm3)
θ range (°)
0.26 × 0.28 × 0.53
2.96 to 26.76°
−20 ≤ h ≤ 20
−19 ≤ k ≤ 21
−25 ≤ l ≤ 23
36,197
0.11 × 0.19 × 0.29
2.94 to 26.75°
−11 ≤ h ≤ 8
−37 ≤ k ≤ 37
−18 ≤ l ≤ 18
27,401
All NMR experiments were run on a BRUKER, DRX400 MHz equip-
ment, in a BBO 5 mm probe, at 298 K, and TMS (tetramethylsilane) for
internal reference. For 1H and 13C NMR, DMSO-d6 was used as solvent,
while CH2Cl2 was used as solvent for (31P{1H}) NMR. The splitting of
proton, carbon and phosphorus resonances was reported as s = singlet
and m = multiplet.
Index ranges
Reflections collected
Independent reflections
Completeness to θ
Max. and min.
11,479 [R(int) = 0.0423]
99.4%
8251 [R(int) = 0.0617]
99.7%
0.942 and 0.795
0.947 and 0.867
2.3. X-ray crystallography
transmission
Data/restraints/
parameters
11,479/0/687
8251/0/462
Blue single crystals of complexes (1) and (5) were grown by slow
evaporation of a dichloromethane/n-hexane solution. X-ray diffraction
experiments were carried out using a suitable crystal mounted on
glass fiber, and positioned on the goniometer head. Intensity data
were measured with the crystal at room temperature on an Enraf–
Nonius Kappa-CCD diffractometer with graphite monochromated
MoKα radiation (λ = 0.71073 Å). The cell refinements were performed
Goodness-of-fit on F2
Final R indices
1.209
1.129
R1 = 0.0566,
wR2 = 0.1321
R1 = 0.0669,
wR2 = 0.1386
0.553 and −0.641
R1 = 0.0376,
wR2 = 0.0724
R1 = 0.0692,
wR2 = 0.0776
0.557 and −0.541
[I N 2sigma(I)]
R indices (all data)
Δρmax.andΔρmin.
(e.Å−3
)

M.I.F. Barbosa et al. / Journal of Inorganic Biochemistry 136 (2014) 33–39
35
phen) [22.0 mg; 0.11 mmol] ligand was added. The reaction mixture
was stirred for 30 min at room temperature and the volume of the
resulting blue solution was reduced, under vacuum, to ca. 2 mL and
diethyl ether (Merck) was then added to precipitate a red solid, which
was filtered off, washed several times with diethyl ether, and dried
under vacuum. Yield: ~78 mg (80–90%).
hydrogen of PPh3 and 18H aromatic hydrogen for phen and Lap); 4.91
(m, 1H, CH of Lap); 3.26 (m, 2H, CH2 of Lap); 1.83 (s, 3H, CH3 of Lap);
1.55 (s, CH3 of Lap).13CNMR (400.21 MHz, DMSO-d6, 298 K): δ(ppm)
198.4 (C1_O of Lap), 180.6 (C4_O of Lap), 167.4 (C2\O of Lap). UV–
vis (CH2Cl2, 10−5 M): λ/nm (ε/M−1 L cm−1) 290 (shoulder), 300
(2.66 × 104), 408 (5.25 × 103).
Microanalyses suggested the formation of the complexes with
general formula [Ru(Lap)(PPh3)2(bipy)]PF6 (1), [Ru(Lap)(PPh3)2(Me-
bipy)]PF6 (2), [Ru(Lap)(PPh3)2(MeO-bipy)]PF6 (3), [Ru(Lap)(PPh3)2
(phen)]PF6 (4) and [RuCl2(Lap)(dppb)] (5). The molar conductivity
data reveal that the complex 5 (3.46 μS cm−1) is non-electrolyte and
complexes 1–4 (129.1, 146.8, 166.2 and 125.0 μS cm−1 respectively)
are 1:1 electrolytes (CH2Cl2), in accordance with the proposed
formulations.
2.4.1.5. [RuCl2(Lap)(dppb)], (5). The ruthenium (III) complex [RuCl2
(Lap)(dppb)] (5) was prepared dissolving (0.137 mmol; 33.0 mg) of
lapachol ligand in a mixture of CH2Cl2:MeOH (50:50) solvent and the
same equivalent of triethylamine (Et3N) and then added the mer-
[RuCl3(dppb)(H2O)] [28] precursor (0.137 mmol; 33.0 mg). The reac-
tion mixture was refluxed and stirred for 24 h, under Ar atmosphere.
The final purple solution was concentrated to ca. 2 mL, and 10 mL of
diethyl ether was added in order to obtain dark purple precipitate.
The solid was filtered off, well rinsed with diethyl ether and dried in
vacuo. Yield: 189 mg (98%). Anal. calc. for C43H41Cl2O3P2Ru: exp.
(calc) C, 61.40 (61.50); H, 4.80 (4.92). UV–vis (CH2Cl2, 10−5 M): λ/nm
(ε/M−1 L cm−1) 315 (shoulder), 330 (shoulder), 356 (2.77 × 103) and
558 (5.6 × 103).
2.4.1. [Ru(Lap)(PPh3)2(X-bipy)] and [Ru(Lap)(PPh3)2(phen)]
The ruthenium(II) complexes with N-N = bipy(1), Me-bipy(2),
MeO-bipy(3) and phen(4) were prepared by reacting an excess of
lapachol ligand (0.137 mmol; 33.0 mg), previously dissolved in
degassed mixture of CH2Cl2:MeOH (50:50) solvent, and the same equiv-
alent of triethylamine Et3N, and the cis-[RuCl2(PPh3)2(N-N)] precursors
(0.114 mmol; ≅100.0 mg). The reaction mixture was refluxed and
stirred for about 72 h, under Ar atmosphere. The final blue solutions
were concentrated to ca. 2 mL and 10 mL of water was added in order
to obtain dark blue precipitates. The solids were filtered off, well rinsed
with water and diethyl ether and dried in vacuum.
2.5. Biological experiments
2.5.1. Cells and cultures
Antiparasitic activity was performed with Leishmania amazonensis
(MHOM/BR88/BA-125) and W2 strain Plasmodium falciparum, while
hemolysis assays were done using O+ human erythrocytes and cytotox-
icity assays were done in J774 macrophages. The L. amazonensis
promastigotes were maintained in Schneider's insect medium (Sigma-
Aldrich, St. Louis, USA) supplemented with 10% fetal bovine serum
(Gibco Laboratories, Gaithersburg, USA) and 50 μg/mL of gentamicin
(Hipolabor, Belo Horizonte, Brazil). J774 macrophages were cultivated
in RPMI-1640 medium (Sigma-Aldrich, St. Louis, USA) supplemented
with 10% fetal bovine serum and 50 μg/mL of gentamicin. W2 strain
P. falciparum was maintained in continuous culture of human erythro-
cytes (blood group O+) using RPMI-1640 medium supplemented with
10% human plasma without hypoxanthine.
2.4.1.1. [Ru(Lap)(PPh3)2(bipy)]PF6 (1). Yield: 121 mg (88%). Anal. calcd
for C61H51F6N2O3P3Ru: exptl (calc) C, 62.30 (62.72); H, 4.20 (4.40); N,
2.18 (2.40). 31P{1H} NMR: δ(ppm) 29.3 (s); 1H NMR (400.21 MHz,
DMSO-d6, 298 K): δ(ppm) 9.80–7.00 (overlapped signals, 30H aromatic
hydrogen for PPh3 and 14H aromatic hydrogen for bipy and Lap) 4.88
(m, 1H, CH of Lap); 3.22 (m, 2H, CH2 of Lap); 1.83 (s, 3H, CH3 of Lap);
1.56 (s, 3H, CH3 of Lap). 13C NMR (400.21 MHz, DMSO-d6, 298 K):
δ(ppm) 198.1 (C1_O of Lap), 180.6 (C4_O of Lap), 167.2 (C2\O of
Lap). UV–vis (CH2Cl2, 10−5 M): λ/nm (ε/M−1 L cm−1) 370 (shoulder),
573 (6.30 × 103).
2.4.1.2. [Ru(Lap)(PPh3)2(Me-bipy)]PF6.CH3OH (2). Yield: 115 mg (84%).
Anal. calc. for C64H59F6N2O4P3Ru: exp (calc) C, 62.70 (62.59); H, 4.61
(4.84); N, 2.32 (2.28). 31P{1H} NMR: δ(ppm) 29.1 (s); 1H NMR
(400.21 MHz, DMSO-d6, 298 K): δ(ppm)2.30 (s, 3H, CH3); 2.42 (s, 3H,
CH3′) (aliphatic hydrogen for Me-bipy); 8.09–7.00 (overlapped signals,
30H aromatic hydrogen for PPh3 and 8H aromatic hydrogen of Me-
bipy); 4.87 (m, 1H, CH of Lap); 3.19 (m, 2H, CH2 of Lap); 1.81 (s, 3H,
CH3 of Lap); 1.55 (s, CH3 of Lap).13C NMR (400.21 MHz, DMSO-d6,
298 K): δ(ppm) 198.7 (C1_O of Lap), 182.3 (C4_O of Lap), 168.0
(C2\O of Lap). UV–vis (CH2Cl2, 10−5 M): λ/nm (ε/M−1 cm−1) 297
(shoulder), 572 (6.40 × 103).
2.5.2. Cytotoxicity assays
J774 macrophages (5 × 104 cells/mL) were distributed in 96-well
plate (100 μL/well) and incubated for 24 h at 37 °C in 5% CO2. Each
drug was solubilized in DMSO as a stock solution and diluted in culture
media in the tested concentrations ranging from 0.1 to 10 μg/mL
(100 μL/well). The final concentration of DMSO was 0.1%. Each concen-
tration was tested in triplicate. After incubation for 72 h, 20 μL of Alamar
blue (Invitrogen, CA, USA) was added to each well and incubated for 24
h in the dark. Gentian violet was used as control. The absorbance was
evaluated at 570 and 600 nm according to manufacturer's instructions.
The LC50 values were calculated using a non-linear regression curve fit
in the Prism version 5.03 (GraphPad Software).
2.4.1.3. [Ru(Lap)(PPh3)2(MeO-bipy)]PF6 (3). Yield: 110 mg (84%). Anal.
calcd for C63H55F6N2O5P3Ru: exp.(calc) C, 61.97 (61.61); H, 4.39
(4.51); N, 2.43 (2.28). 31P{1H} NMR: δ(ppm) 29.8 (s). 1H NMR
(400.21 MHz, DMSO-d6, 298 K):δ(ppm) 3.91 (s, 3H, CH3); 3.84 (s, 3H,
CH3′) (aliphatic hydrogen of MeO-bipy); 9.45–7.00 (overlapped signals,
30H aromatic hydrogen for PPh3 and 12H aromatic hydrogen for MeO-
bipy and Lap); 4.85 (m, 1H, CH of Lap); 3.16 (m, 2H, CH2 of Lap); 1.80
(s, 3H, CH3 of Lap); 1.54 (s, CH3 of Lap).13C NMR (400.21 MHz, DMSO-
d6, 298 K): δ(ppm) 198.2 (C1_O of Lap), 180.4 (C4_O of Lap), 167.6
(C2\O of Lap). UV–vis (CH2Cl2, 10−5 M): λ/nm (ε/M−1 cm−1) 297
(shoulder), 586 (6.11 × 103).
For the hemolysis assay, human erythrocytes type O+ were washed
three times in phosphate buffered saline and 100 μL of this suspension
(1% hematocrit) was distributed into a 96-well plate. Then, 100 μL of
each drug, previously dissolved in phosphate buffered saline, was
added in triplicate to the plate and incubated for 1 h. Saponin (Sigma-
Aldrich, St. Louis, USA) was used as reference drug at 1% v/v. After incu-
bation the cells were centrifuged (1500 rpm for 10 min) and 100 μL of
each supernatant was transferred to another microtiter plate. Released
haemoglobin was monitored by measuring the absorbance at 540 nm
in a spectrophotometer. The percentage of hemolysis was determined
in comparison to untreated cells.
2.4.1.4. [Ru(Lap)(PPh3)2(phen)]PF6(4). Yield: 128 mg (94%). Anal. calcd
for C63H51F6N2O3P3Ru: exp.(calc) C, 63.97 (63.48); H, 3.99 (4.31); N,
2.39 (2.35). 31P{1H} NMR: δ(ppm) 32.6 (s). 1H NMR (400.21 MHz,
DMSO-d6, 298 K):δ(ppm)10.00–7.00 (overlapped signals, 30H aromatic
2.5.3. Antileishmanial activity against promastigotes
L. amazonensis promastigotes (2 × 106 cells/mL) in stationary
growth phase were distributed in a 96-well plate (100 μL/well) at
24 °C. Each drug was solubilized in DMSO as described above,

36
M.I.F. Barbosa et al. / Journal of Inorganic Biochemistry 136 (2014) 33–39
Fig. 2. X-ray structures for (a) [Ru(Lap)(PPh3)2(bipy)]PF6 (1) and (b) [RuCl2(Lap)(dppb)] (5), showing atoms labeling and 50% of probability ellipsoids.
diluted in the culture medium and added in serial dilution from 0.1
to 10 μg/mL (100 μL/well). The final DMSO concentration was 0.1%.
Amphotericin B (Gibco Laboratories, Gaithersburg, USA) was used as
reference drug. After 72 h incubation at 24 °C, the number of viable par-
asites was counted in a Neubauer chamber. The IC50 values were calcu-
lated in Prism version 5.03 (GraphPad Software) using non-linear
regression.
promastigotes was determined by adding Alamar Blue (20 μL/well) and
incubated for 24 h. The absorbance was evaluated at 570 and 600 nm
according to the manufacturer's instructions. The IC50 values were cal-
culated in Prism version 5.03 (GraphPad Software) using non-linear
regression.
2.5.5. Antimalarial activity
The antimalarial effects of the compounds were measured with the
[3H]-hypoxanthine (PerkinElmer, Boston, USA) incorporation assay.
W2 P. falciparum grown at 1–2% parasitemia and 2.5% hematocrit were
aliquoted in a 96-well plate. Drugs were solubilized as described above
in a concentration range of 0.1 to 10 μg/mL; each concentration was per-
formed in triplicates. Mefloquine (Farmanguinhos, Rio de Janeiro, RJ,
Brazil) was used as reference drug. After 24 h of incubation with the test-
ed compounds, 25 μL of medium containing [3H]hypoxanthine (0.5 μCi/
well) was added per well, followed by another 24 h of incubation. The
parasites were harvested using a cell harvester to evaluate the [3H]-hy-
poxanthine incorporation in a β-radiation counter (Multilabel Reader;
Hidex, Turku, Finland). Inhibition of parasite growth was evaluated by
comparing the [3H]-hypoxanthine uptake in untreated versus treated
cells. IC50 values were calculated in a Graph Pad Prism version 5.03
(Graph Pad Software, San Diego, CA) using non-linear regression.
2.5.4. In vitro leishmania infection
J774 macrophages (2 × 105cells/mL) were plated in 96-well plate
(100 μL/well) and incubated overnight at 37 °C in 5% CO2.
L. amazonensis promastigotes in the stationary growth phase were
added to the cell culture (100 μL/well) at a parasite/macrophage ratio
of 10:1 and incubated for 24 h. Plates were washed to remove non-
phagocytosed parasites. Each drug, solubilized as described above, was
added and incubated for 72 h. Amphotericin B was used as reference
drug. Infected macrophages were lysed by addition of 0.01% sodium do-
decyl sulfate (Sigma-Aldrich, St. Louis, USA) in PBS (phosphate-buffered
saline) at 37 °C for 30 min.
Amastigotes from lysed macrophages were incubated at 24 °C for
48 h, which then differentiated in promastigotes. The number of viable
Table 2
Selected bond length (Ả) and angles (°) for complexes (1) and (5).
3. Results and discussion
Fragment
Complex (1)
Complex (5)
In this work the lapachol acted as bidentate ligand and monoanionic
species, coordinating with the ruthenium atoms through its ortho oxy-
gens (O1, O2—Fig. 1). The structures of the complexes [Ru(Lap)(PPh3)2
(bipy)]PF6 (1) and [RuCl2(Lap)(dppb)] (5) were confirmed based on
X-ray diffraction data (see Fig. 2). These compounds crystallize in the
Ru(1)–O(1)
Ru(1)–O(2)
Ru(1)–P(2)
Ru(1)–P(1)
Ru(1)–N(2)
Ru(1)–N1(1)
Ru(1)–Cl(1)
Ru(1)–Cl(2)
O(1)–C(1)
2.0710(19)
2.133(2)
2.3952(11)
2.4104(10)
2.038(2)
2.050(2)
–
2.1707(15)
2.0580(15)
2.3728(6)
2.2910(6)
–
–
2.3308(7)
2.3343(7)
1.235(3)
1.309(3)
1.230(3)
77.85(6)
91.69(6)
171.11(5)
93.39(5)
92.01(2)
168.99(3)
–
Table 3
1.250(3)
1.308(3)
1.236(4)
76.22(7)
174.56(5)
90.02(6)
89.95(6)
178.25(3)
–
Cyclic voltammetry data for complexes (1)–(4) (TBAP 0.1 M; CH2Cl2; Ag/AgCl; work
O(2)–C(2)
O(3)–C(4)
electrode Pt; 100 mVs−1).
O(2)–Ru(1)–O(1)
O(1)–Ru(1)–P(1)
O(2)–Ru(1)–P(2)
O(1)–Ru(1)–P(2)
P(1)–Ru(1)–P(2)
Cl(1)–Ru(1)–Cl(2)
Complex
Epa (V)
E1/2 (V)
pKa (N–N)
[Ru(Lap)(PPh3)2(bipy)]PF6 (1)
[Ru(Lap)(PPh3)2(Me-bipy)]PF6 (2)
[Ru(Lap)(PPh3)2(MeO-bipy)]PF6 (3)
[Ru(Lap)(PPh3)2(phen)]PF6 (4)
1.03
0.96
0.77
1.08
0.99
0.87
0.71
1.00
4.86
4.92
5.74
4.44

M.I.F. Barbosa et al. / Journal of Inorganic Biochemistry 136 (2014) 33–39
37
Fig. 3. Cyclic voltammogram of [Ru(Lap)(PPh3)2(phen)]PF6 (4) (TBAP 0.1 M; CH2Cl2; Ag/AgCl; work electrode Pt; 100 mV.s−1).
monoclinic system, with the space group P21/c. It is observed that the O1
and O2 atoms are involved in the coordination, where O2, is negatively
charged and O1, is neutral. A distorted octahedral geometry is observed
for both crystal structures, as observed by the bond angles (Table 2).
Some distance and selected angles in the X-ray structure of complex
(1) and (5) are shown in Table 2, which are, in general, in accordance
with values expected for similar phosphine complexes of Ru(II) and
Ru(III) for Ru–N, Ru–P and Ru–Cl [28–30]. But, it is interesting to point
out that the distances of Ru(II)–O for complex (1) are also in accordance
with the expected values, where the distance Ru–O2 [2.133(2) Ả] is lon-
ger than the distance Ru–O1 [2.0710(19) Ả], since the O2 has charge
minus one and its radius is bigger than the one for the neutral species.
Therefore, the same was not observed for complex (5), where the dis-
tance Ru–O1 [2.1707(15) Ả] is longer than the distance Ru–O2
[2.0580(15) Ả]. Probably in this case the strong trans effect of phospho-
rus atoms is more effective when it is trans to neutral atoms, and not
when it is trans to negatively charged atoms. As it can be seen in
Table 2 the distance of Ru(III)–O1 is 0.1 Ả longer than Ru(II)–O1, show-
ing the strong trans effect phosphorus atoms. On other hand the dis-
tance Ru(III)–O1, is shorter than Ru(II)–O1, as expected, considering
the size of the radius of Ru(III) and Ru(II).
redox pair Ru(III)/Ru(II), as can be seen from the Table 3, and Fig. 3 for
the case of complex (4). In the negative region a quasi-reversible one-
electron reduction process was observed in all cases, which most prob-
ably correspond to the ligand reduction to the semiquinone form [31].
As can be seen in Table 3 the redox potential of 1–4 decreases when
the diimine basicity is increased. Analyzing the complex [RuCl2
(Lap)(dppb)] (5), in the same experimental conditions, it is observed
a Ru(III)/Ru(II) reversible process with E1/2 of 0.18 V.
The IR spectra of complexes (1–5) confirm the presence of
the lapachol ligand coordinated to the metal. The band located at
3351 cm−1 in the free lapachol [31,32] assigned to OH, disappears upon
coordination, as expected. The characteristic ν(C_O) stretching bands,
found at ν(C1_O) 1664 and ν(C4_O) 1641 cm−1 in free lapachol
[11] shifted to lower frequencies in complexes, 1561–1591 cm−1 and
1581–1533 cm−1, respectively. This behavior was also observed for
other complexes like Ru(II), Co(II), Ni(II) and Cu(II) containing the
lapachol as ligand [5,11,12]. The characteristic ν(C2\O) stretching band
found in 1028 cm−1 in the free lapachol shifted to higher frequencies in
complexes (1065–1079 cm−1). Finally, new bands of medium intensities,
located below 500 cm−1 are present in the spectra of complexes, which
may be related to metal–ligand vibrations.
In the 31P{1H} NMR spectra of the complexes (1–4) just one singlet
at about 30 ppm is observed in all cases, indicating the magnetic equiv-
alence of the two trans phosphorus atoms, as expected. Also, each 31P
{1H} NMR spectra exhibit a heptet signal at −144 ppm, corresponding
to the phosphorus atoms of the PF−6 counter ion. The EPR spectra in
solid state, for complex (5), confirms the presence of Ru (III) paramag-
netic species, showing g1 = 2.578, g2 2.128 and g3 = 1.822 typical of
ruthenium (III) complexes [29].
The antiparasitic and toxicity of host cell were evaluated. For com-
parison, the metal-free lapachol was included in the pharmacological
evaluation. Firstly, compounds were evaluated on their ability to inhibit
the L. amazonensis promastigote proliferation, as well as against intra-
cellular amastigotes, according to standard methodology [33]. Secondly,
the antimalarial activity of the complexes was determined against the
erythrocytic stage of W2 strain P. falciparum. Host cell cytotoxicity in
J774 macrophages as well as the hemolysis in uninfected erythrocytes
was determined [34,35]. The results were expressed in terms of IC50
and LC50 values. Amphotericin B and mefloquine were respectively
Cyclic voltammograms of Ru(II) complexes (1–4) show a quasi-
reversible process between 0.71 and 1.0 V, which correspond to the
Table 4
Antiparasitic activity and cytotoxicity for the ruthenium complexes.
Compounds
L. amazonensis, IC50
SEM(μM)
P. falciparum(c)
Cytotoxicity(d)
SI(e)
IC50
SEM(μM)
LC50
SEM(μM)
Promastigotes(a)
In vitro infection(b)
N10
Lapachol
(1)
12.4
N10
0.18
0.42
1.6
0.69
11.3
43.5
0.35
0.53
0.19
0.21
0.04
–
4.1
N10
0.33
1.0
6.7
1.9
N.D.
4.7
0.07
0.17
N10
N.D.
0.57
–
0.002
0.01
0.71
0.26
0.28
0.17
0.10
0.01
0.08
0.46
1.3
1.3
(2)
0.04
0.03
0.44
5.9
(3)
(4)
(5)
N.D.
N.D.
17.5
N.D.
N.D.
–
0.14
–
0.04
0.01
N10
–
Mefloquine
Amphotericin B
Gentian Violet
0.13
–
0.23
N.D.
0.09
N10
0.60
N.D.
0.07
(a)Values determined 72 h after incubation with drugs. (b)Values determined for infected macrophages 72 h after incubation with drugs (c)Determined against W2 strain P. falciparum
(erythrocytic stage) 24 h after incubation with drugs. (d)Cytotoxicity was determined in J774 macrophages after 72 h incubation with drugs. (e)SI value is given from the ratio LC50 in
J774/IC50 (L. amazonensis, in vitro infection). IC50 and LC50 values were determined from two independent experiments, concentration in triplicates. SEM = standard error of the
mean; N.D. = not determined. SI = selectivity index.

38
M.I.F. Barbosa et al. / Journal of Inorganic Biochemistry 136 (2014) 33–39
new ruthenium complexes studied in this work, exhibiting an
activity comparable to the one of reference drugs. Specifically,
lapachol–ruthenium complexes displayed potent and selective
antileishmanial activity.
Acknowledgments
This study received support from CNPq, FAPESP, FAPESB and CAPES.
T.S.M. holds a FAPESB scholarship. R.S.C. thanks FAPESP for a PhD fel-
lowship (Grant number 2009/08131-1). The authors acknowledge the
assistance of Diogo Rodrigo de Magalhães Moreira for helpful discus-
sions during the preparation of the manuscript.
Appendix A. Supplementary data
Fig. 4. Hemolytic activity of lapachol and complexes. The hemolytic activity of the com-
pounds was assayed in fresh human erythrocytes type O+. Saponin was used as hemolytic
drug at 1% v/v. Released hemoglobin was monitored by measuring the absorbance at
540 nm in a spectrophotometer. Results shown are mean SD of one experiment per-
formed in triplicate.
Coordinates and other crystallographic data have been deposited
with the CCDC, deposition codes CCDC 973562 and 973365, for the com-
plexes (1) and (5), respectively. Copies of this information may be ob-
tained from The Director, CCDC, 12 Union Road, Cambridge, CB2 1EZ,
UK, Fax: +44 1233 336033, E-mail: deposit@ccdc.cam.ac.uk or www.
ccdc.cam.ac.uk. Supplementary data to this article can be found online
at http://dx.doi.org/10.1016/j.jinorgbio.2014.03.009.
used as reference drugs for Leishmania and Plasmodium tests respective-
ly, while gentian violet was used as control in host cell cytotoxicity.
Amphotericin B, which was used as a reference drug, exhibited an
IC50 = 0.13 0.01 μM, while lapachol was in practice, inactive against
L. amazonensis promastigotes. Complex (1) was inactive to inhibit
promastigotes, while complexes (2–5) were able to inhibit their prolif-
eration. Specifically, complexes (2) and (5) exhibited activity against
promastigotes similar to the observed for amphotericin B. Regarding
the inhibitory activity in L. amazonensis-infected macrophages,
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