Dithiocarbazate complexes with the [M(PPh3)]2+ (M=Pd or Pt) moiety. Synthesis, characterization and anti-Tripanosoma cruzi activity.

Article abstract of DOI:10.1016/j.jinorgbio.2010.08.009

New neutral Pd(II) and Pt(II) complexes of the type [M(L)(PPh₃)] (MPd or Pt) were prepared in crystalline form in high-yield synthesis with the S-benzyldithiocarbazates and S-4-nitrobenzyldithiocarbazates derivatives from 2-hydroxyacetophenone,

Full text of DOI:10.1016/j.jinorgbio.2010.08.009

Journal of Inorganic Biochemistry 104 (2010) 1276–1282
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Journal of Inorganic Biochemistry
journal homepage: www.elsevier.com/locate/jinorgbio
Dithiocarbazate complexes with the [M(PPh3)]2+ (M=Pd or Pt) moiety
Synthesis, characterization and anti-Tripanosoma cruzi activity
a
b
b
Pedro I. da S. Maia a, , André G. de A. Fernandes , Jean Jerley N. Silva , Adriano D. Andricopulo ,
⁎
Sebastião S. Lemos c, Ernesto S. Lang d, Ulrich Abram e, Victor M. Deflon a,
Instituto de Química de São Carlos, Universidade de São Paulo, 13566-590 São Carlos, SP, Brazil
⁎
a
b
Instituto de Física de São Carlos, Universidade de São Paulo, 13560-970 São Carlos, SP, Brazil
c
Instituto de Química, Universidade de Brasília, 70919-970 Brasília, DF, Brazil
d
Departamento de Química, Universidade Federal de Santa Maria, Santa Maria, RS, Brazil
Freie Universität Berlin, Institute of Chemistry and Biochemistry, Fabeckstr. 34-36, D-14195 Berlin, Germany
e
a r t i c l e i n f o
a b s t r a c t
Article history:
New neutral Pd(II) and Pt(II) complexes of the type [M(L)(PPh3)] (M Pd or Pt) were prepared in crystalline
form in high-yield synthesis with the S-benzyldithiocarbazates and S-4-nitrobenzyldithiocarbazates
derivatives from 2-hydroxyacetophenone, H2L1a and H2L1b, and benzoylacetone, H2L2a and H2L2b. The new
complexes [Pt(L1a)(PPh3)] (1), [Pd(L1a)(PPh3)] (2), [Pt(L1b)(PPh3)] (3), [Pd(L1b)(PPh3)] (4), [Pt(L2a)(PPh3)]
(5), [Pd(L2a)(PPh3)] (6), [Pt(L2b)(PPh3)] (7) and [Pd(L2b)(PPh3)] (8) were characterized on the basis of
elemental analysis, conductivity measurements, UV–visible, IR, electrospray ionization mass spectrometry
(ESI-MS), NMR (1H and 31P) and by X-ray diffraction studies. The studies showed that differently from what
was observed for the H2L1a and H2L1b ligands, H2L2a and H2L2b assume cyclic forms as 5-hydroxypyrazolinic.
Upon coordination, H2L2a and H2L2b suffer ring-opening reaction, coordinating in the same manner as H2L1a
and H2L1b, deprotonated and in O,N,S-tridentate mode to the (MPPh3)2+ moiety. All complexes show a quite
similar planar fourfold environment around the M(II) center. Furthermore, these complexes exhibited
biological activity on extra and intracellular forms of Trypanosoma cruzi in a time- and concentration-
dependent manner with IC50 values ranging from 7.8 to 18.7 μM, while the ligand H2L2a presented a
trypanocidal activity on trypomastigote form better than the standard drug benznidazole.
Received 20 February 2010
Received in revised form 13 August 2010
Accepted 13 August 2010
Available online 21 August 2010
Keywords:
Platinum(II) complexes
Palladium(II) complexes
Dithiocarbazates
Pyrazolines
Anti-trypanosomal activity
Crystal structures
© 2010 Elsevier Inc. All rights reserved.
1. Introduction
apeutical agents for chagasic patients, especially using inorganic
complexes [4–7].
American Trypanosomiasis or Chagas disease is endemic in Latin
America. Its etiological agent is the Trypanosoma cruzi, a hemoflagel-
late protozoan, whose life cycle involves vertebrate and invertebrate
hosts [1]. According to the World Health Organization (WHO),
18 million people are infected with the T. cruzi, resulting in an annual
death toll of 50,000 [2]. The current treatment of Chagas disease is
unsatisfactory, depending on two nitroheterocycles, nifurtimox and
benznidazole. Although effective for acute infections, they may cause
undesirable side effects, frequently leading to the abandonment of the
treatment [3]. Their efficacy during the chronic phase is still
controversial, with poor indices of apparent cure and a lack of
consensus regarding a parasitological cure in the future [3]. Thus,
considerable efforts are being directed to developing new chemother-
Metal chelates of dithiocarbazic acid, its S-alkyl/arylesters and
their Schiff bases have been studied, mainly due to their potential
anticancer, antibacterial, antifungal, antiamoebic and insecticidal
activities [8–14]. Although the synthesis and complexation of S-
benzyldithiocarbazate and its derivatives have been under study for
many years, considerable attention continues to be given to these and
related ligands and their metal complexes, since their properties can
be greatly modified by introducing different substituents [10]. As
reported for acylhydrazones and of 1,3-diketones [14], S-dithiocarba-
zate derivatives are also supposed to undergo cycle-chain equilibrium
(Fig. 1) and can exist in several distinct isomeric forms. In contrast,
thiosemicarbazone derivatives from 1,3-diketones were reported to
suffer cyclization only in the presence of a metal substrate to form
pyrazolate compounds, besides they were just found to act as
bidentate ligands upon complexation with Pd(II) and Pt(II) [15].
On the other hand, transition metal-phosphine complexes, espe-
cially with the platinum group elements, are considerably important
for both industrial and laboratory scale catalytic applications [16–18].
In the literature there is a number of complexes of Pd(II) and Pt(II)
⁎
Corresponding authors. Tel.: +55 16 3373 8038; fax: +55 16 3373 9985.
E-mail addresses: pedovivo@iqsc.usp.br (P.I.S. Maia), deflon@iqsc.usp.br
(V.M. Deflon).
0162-0134/$ – see front matter © 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.jinorgbio.2010.08.009

P.I.S. Maia et al. / Journal of Inorganic Biochemistry 104 (2010) 1276–1282
1277
on a Varian MERCURY plus spectrometer, 7.05 T, operating at 300.07
and 121.47 MHz for 1H and 31P, respectively. The 31P{1H} NMR spectra
were externally referenced to H3PO4 (85%, δ=0). The 1H NMR spectra
were internally referenced to TMS (tetramethylsilane). NMR spectra of
the free ligands and of the complexes 3, 4 and 7 were taken using a JEOL
multinuclear spectrometer, operating at 399.65 and 161.70 MHz for 1H
and 31P, respectively. Abbreviations used for the reported 1H NMR
spectra are as follows: s = singlet, d = doublet, dd = doublet of
doublets, ddd = doublet of doublets, m = multiplet.
H
S
OH
N
S
N
OH
N
N
S
SH
H2L1a (R = H)
1b
H2L (R = NO2)
(A)
(B)
R
R
2.3. X-ray structure determination
Single crystals suitable for X-ray data collections regarding 1, 3, 4,
5, 6 and 8 were grown by slow concentration of their solutions in a
1:2 mixture of MeCN/CH2Cl2. Suitable crystals of 2∙CH2Cl2 were
obtained by recrystallization from CH2Cl2. The data collections were
performed with Mo-Kα radiation (λ=71.073 pm) on a BRUKER
KAPPA APEX II CCD or on a STOE IPDS 2 T instrument. Standard
procedures were applied for data reduction and absorption
correction. The structures were solved by direct methods with
SHELXS-97 and refined with SHELXL-97 [26]. The positions of the
hydrogen atoms were calculated at idealized positions using the
riding model option of SHELXL-97 [26]. Additional crystal data and
more information about the X-ray structural analyses are shown in
Table S1 as Supplementary data.
H
OH
N
HO
S
N
N
N
S
S
S
2a
H2L (R = H)
2b
H2L (R = NO2)
(C)
(D)
R
R
Fig. 1. Possible equilibrium in solution of the ligands used: (A) Thione tautomeric form
of H2L1a and H2L1b; (B) thiol tautomeric form of H2L1a and H2L1b; (C) open-chain form of
H2L2a and H2L1b; (D) cycle-chain form of H2L2a and H2L2b, 5-hydroxypyrazolic form.
2.4. Preparation of the compounds
2.4.1. Preparation of 4-nitrobenzyldithiocarbazate
with similar ligands, but few studies have been carried out on
complexes having tridentate O,N,S-donor dithiocarbazate ligands and
having triphenylphosphine in the fourth coordination site [19–23].
In the present work we describe the synthesis, spectral properties,
crystal structures and the anti-trypanosomal activity of a series of
The previously reported method for preparing substituted dithio-
carbazate [6] was modified by reaction with 4-nitrobenzylchloride.
Potassium hydroxide (11.4 g, 0.2 mol) was dissolved completely in
90% ethanol (70 mL) and the mixture was cooled in ice. To the cold
solution, hydrazine hydrate (10 g, 0.2 mol) was added slowly, under
stirring. Carbon disulfide (15.2 g, 0.2 mol) in ethanol (25 mL) was
then added dropwise under vigorous stirring for about 1 h. The
temperature of the reaction mixture was kept around −5 °C during
the addition by adding nitrogen to the ice. Two layers formed at this
time. The resulting yellow oil (lower layer) was separated using a
separatory funnel, dissolved in 40% ethanol (40 mL) and cooled in ice.
4-nitrobenzylchloride (32.8 g, 0.2 mol) was partially dissolved in
100 mL of 80% ethanol and added slowly to the above solution under
vigorous mechanical stirring. This solution was kept under stirring for
1 h. The resulting pale yellow product was separated by filtration,
washed with water and dried. The product was used without further
recrystallization. Yield: 85%. Anal. Calcd for C8H9N3O2S2:
(243.30 g mol−1): C, 39.49; H, 3.73; N, 17.27; S, 26.35. Found: C,
40.25; H, 3.44; N, 16.95; S, 25.69%.
dithiocarbazates derivatives (chart 1) and their complexes: [Pt(L1a
)
(PPh3)] (1), [Pd(L1a)(PPh3)] (2), [Pt(L1b)(PPh3)] (3), [Pd(L1b)(PPh3)]
(4), [Pt(L2a)(PPh3)] (5) and [Pd(L2a)(PPh3)] (6), [Pt(L2b)(PPh3)] (7) and
[Pd(L2b)(PPh3)] (8), where L1a, L1b, L2a and L2b represent the dianions of
the ligands H2L1a=S-benzyl-β-N-(2-hydroxyphenylethylidine)dithio-
carbazate, H2L1b=S-4-nitrobenzyl-β-N-(2-hydroxyphenylethylidine)
dithiocarbazate, H2L2a=5-hydroxy-3-methyl-5-phenyl-pyrazoline-1-
(S-benzyldithiocarbazate) and H2L2b=5-hydroxy-3-methyl-5-phenyl-
pyrazoline-1-(S-4-nitrobenzyldithiocarbazate), respectively.
2. Experimental
2.1. Materials
[PtCl2(PPh3)2], benzyl dithiocarbazate, H2L1a and H2L2b were all
prepared according to literature procedures [13,17,24,25]. [PdCl2
(PPh3)2], 4-phenyl-2,4-butanedione, 2-hydroxyacetophenone and all
solvents used were of reagent grade, purchased from commercial
sources and used without further purification.
2.4.2. Preparation of H2L1b
4-nitrobenzyldithiocarbazate (4 mmol) dissolved in MeOH
(10 mL) was added to a solution of the desired 2-hydroxyacetophe-
none (4.1 mmol) in MeOH (5 mL). The mixture was refluxed for 2 h.
The solutions were cooled to room temperature, after which yellow
precipitates were obtained. The solids were filtered off, washed with
n-hexane and dried in air. Yield: 73%. Anal. Calcd for C16H15N3O3S2:
(361.43 g mol−1): C, 53.17; H, 4.18; N, 11.63; S, 17.74. Found: C,
54.50; H, 4.43; N, 11.57; S, 17.28%. 1H NMR (DMSO-d6, ppm): 2.53 (s,
3H, CH3), 4.74 (s, 2H, CH2), 6.90–7.00 (m, 2H, Ph), 7.33–7.65 (m, 2H,
Ph), 7.92 (d, J=8.5 Hz, 2H, Ph), 8.22 (d, J=8.5 Hz, 2H, Ph), 11.23 (s,
1H, NH), 12.89 (s, 1H, OH).
2.2. Physical measurements
IR spectra were measured as KBr pellets on a Shimadzu FTIR-
spectrometer in the4000–400 cm−1 region. Theelectronic spectra were
recorded with a Shimadzu UV-1800 spectrophotometer. The conduc-
tivities of the complexes were measured in DMSO with a Orion Star
Series conductimeter. Positive electrospray ionization mass spectrom-
etry (ESI-MS) data were measured using an Agilent 6210 ESI-TOF (time
of flight) spectrometer. All MS results aregivenin thefollowingform:m/
z, assignment. Elemental analyses (CHNS) were determined using
a FISONS EA-1108 micro analyzer or a Heraeus vario EL elemental
analyzer. NMR spectra for the complexes 1, 2, 5, 6 and 8 were acquired
2.4.3. Preparation of H2L2a
A solution of 4-phenyl-2,4-butanedione (0.811 g, 5.0 mmol) in
MeOH (10 mL) was added to a solution of the S-benzyldithiocarbazate
(0.991 g, 5.0 mmol) in MeOH (10 mL). The mixture was refluxed for 2 h

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P.I.S. Maia et al. / Journal of Inorganic Biochemistry 104 (2010) 1276–1282
and a yellow solution of H2L2a was obtained afterward. After filtration, a
clear solution was obtained and the slow evaporation of the solvent led
to the appearance of a colorless crystalline product. The crystals were
filtered off, washed with n-hexane and dried in air. Yield: 82%. Anal.
Calcd for C18H18N2OS2: (342.47 g mol−1): C, 63.13; H, 5.30; N, 8.18; S,
18.72. Found: C, 62.81; H, 5.42; N, 8.30; S, 19.08%. 1H NMR (DMSO-d6,
ppm): 2.05 (s, 3H, CH3), 3.18 (d, J=19.2 Hz, 1H, CH2), 3.34 (d,
J=19.2 Hz, 1H, CH2), 4.19 (d, J=13.4 Hz, 1H, SCH2), 4.31 (d,
J=13.4 Hz, 1H, SCH2), 7.09 (s, 1H, OH), 7.21–7.38 (m, 10H, Ph).
[Pd(L1b)(PPh3)] (4). Yield: 84% (0.122 g). Anal. Calc. for C34H28N3
O3PPdS2 (728.13): C, 56.09; H, 3.88; N, 5.77; S, 8.81%. Found: C, 56.08;
H, 3.96; N, 5.53; S, 8.99%. 1H NMR (CDCl3; ppm): 2.94 (s, 3H), 4.40 (s,
2H), 6.57–6.63 (m, 2H), 7.13 (ddd, J=7.6 Hz, 1H), 7.33–7.46 (m, 9H),
7.50 (d, J=8.5 Hz, 2H), 7.57–7.63 (m, 6H), 7.68 (d, J=7.5 Hz, 1H),
8.08 (d, J=8.5 Hz, 2H). 31P NMR (CDCl3; ppm): 22.65. Positive ESI-MS
(m/z): 728 ([M+H]+), 750 ([M+Na]+), 766 ([M+K]+). Molar
conductivity (10−3 M, DMSO): 0.9 S cm2 mol−1
.
[Pd(L2a)(PPh3)] (6). Yield: 75% (0.106 g). Anal. Calc. for C36H31N2
OPPdS2 (709.17): C, 60.97; H, 4.41; N, 3.95; S, 9.04. Found: C, 60.57; H,
4.49; N, 4.09; S, 9.13. 1H NMR (CDCl3; ppm): 2.49 (s, 3H), 4.38 (s, 2H),
5.86 (s, 1H), 7.12–7.48 (m, 19H), 7.62–7.72 (m, 6H). 31P NMR (CDCl3;
ppm): 27.20. Positive ESI-MS (m/z): 709 ([M+H]+), 731 ([M+Na]+),
2.4.4. Preparation of the platinum complexes
[PtCl2(PPh3)2] (0.080 g, 0.1 mmol) and the desired ligand
(0.1 mmol) were diluted separately in CH2Cl2 (10 mL) and MeCN
(10 mL), respectively, and then mixed. After 10 min of stirring, 4
drops of Et3N were added, forming yellow solutions. The mixtures
were then kept under stirring for 4 h more. After slow evaporation of
the solutions at room temperature over a period of 3–4 days, yellow
or brown crystals that deposited were filtered off, washed with n-
hexane and dried in air.
747 ([M+K]+). Molar conductivity (10−3 M, DMSO): 1.5 S cm2 mol−1
.
[Pd(L2b)(PPh3)] (8). Yield: 91% (0.121 g). Anal. Calc. for C36H30N3
O3PPdS2 (754.17): C, 57.33; H, 4.01; N, 5.57; S, 8.50%. Found: C, 56.79;
H, 4.15; N, 6.06; S, 8.31%. 1H NMR (CDCl3; ppm): 2.43 (s, 3H), 4.43 (s,
2H), 5.86 (s, 1H), 7.10–7.30 (m, 5H), 7.40–7.72 (m, 17H), 8.13 (d,
J=8.7 Hz, 2H). 31P NMR (CD2Cl2; ppm): 27.04. Positive ESI-MS (m/z):
754 ([M+H]+), 776 ([M+Na]+), 792 ([M+K]+). Molar conductivity
[Pt(L1a)(PPh3)] (1).Yield: 67% (0.052 g). Anal. Calc. for C34H29N2
OPPtS2 (771.79): C, 52.91; H, 3.79; N, 3.63; S, 8.31. Found: C, 53.46; H,
4.05; N, 3.74; S, 8.45. 1H NMR (CDCl3; ppm): 2.97 (s, 3H), 4.50 (s, 2H),
6.70–6.77 (m, 2H), 7.20–7.50 (m, 15H), 7.66–7.74 (m, 6H), 7.90 (dd,
J=8.0, J=2.0 Hz, 1H). 31P NMR (CDCl3; ppm): 10.33 [J(195Pt–31P):
3637 Hz]. Positive ESI-MS (m/z): 772 ([M+H]+), 794 [M+Na]+).
(10−3 M, DMSO): 1.7 S cm2 mol−1
.
2.5. Trypanocidal activity
2.5.1. Parasites and culture procedures
The experiments described herein were according to a well-
established approach using the T. cruzi Tulahuen-Lac Z strain [27,28].
Trypomastigotes were grown on monolayers of mouse fibroblasts
(L929) and epimastigotes were grown in liver infusion tryptone (LIT)
with 10% fetal calf serum, penicillin, and streptomycin as previously
described [29]. The cultures to be assayed for β-galactosidase activity
were grown in RPMI 1640 medium without phenol red plus 10% fetal
calf serum, glutamine, penicillin, and streptomycin.
Molar conductivity (10−3 M, DMSO): 1.4 S cm2 mol−1
.
[Pt(L1b)(PPh3)] (3). Yield: 80% (0.065 g). Anal. Calc. for C34H28N3O3
PPtS2 (816.79): C, 50.00; H, 3.46; N, 5.14; S, 7.85%. Found: C, 50.17; H,
3.52; N, 4.99; S, 7.87%. 1H NMR (CDCl3; ppm): 2.84 (s, 3H), 4.46 (s, 2H),
6.65–6.73 (m, 2H), 7.15–7.23 (m, CHCl3+1H) 7.34–7.41 (m, 9H), 7.51
(d, J=8.8 Hz, 2H), 7.55–7.65 (m, 6H), 7.82 (dd, J=7.0 Hz, 1H), 8.07 (d,
J=8.8 Hz, 2H). 31P NMR (CH2Cl2; ppm): 6.31 [J(195Pt–31P): 3609 Hz.
Positive ESI-MS (m/z): 817 ([M+H]+), 839 ([M+Na]+), 855 ([M+K]+).
Molar conductivity (10−3 M, DMSO): 1.0 S cm2 mol−1
.
2.5.2. Evaluation of the activity on epimastigote forms
[Pt(L2a)(PPh3)] (5). Yield: 70% (0.056 g). Anal. Calc. for C36H31N2
OPPtS2 (797.81): C, 54.19; H, 3.92; N, 3.51; S, 8.02. Found: C, 54.60; H,
4.05; N, 3.44; S, 7.88. 1H NMR (CDCl3; ppm): 2.53 (s, 3H), 4.54 (s, 2H),
6.03 (s, 1H), 7.11–7.49 (m, 19H), 7.65–7.72 (m, 6H). 31P NMR (CDCl3;
ppm): 12.94 [J(195Pt–31P): 3500 Hz]. Positive ESI-MS (m/z): 798 ([M+
H]+), 820 ([M+Na]+), 837 ([M+K]+), 1618 ([2 M+Na]+). Molar
Epimastigote forms were grown in LIT medium, supplemented
with 10% fetal calf serum, harvested during the exponential phase of
growth, washed in phosphate-buffered saline (PBS) and resuspended
to 1.0×106 parasite mL−1. A volume of 200 μL of parasites was plated
onto 96-well plates (in triplicate) and treated with the complexes
diluted in acetonitrile/PBS (final concentration of acetonitrile smaller
than 1%) and incubated at 28 °C. Benznidazole from Aldrich Chemical
Company (ACC) was used as the reference trypanocidal drug (positive
control). Parasite viability was subsequently tested by determining
the number of motile forms in a Newbauer chamber [30] and the
percentage of antiproliferative activity (growth inhibition,% GI) was
calculated as follows:
conductivity (10−3 M, DMSO): 1.7 S cm2 mol−1
.
[Pt(L2b)(PPh3)] (7). Yield: 65% (0.055 g). Anal. Calc. for C36H30N3
O3PPtS2 (842.83): C, 51.30; H, 3.59; N, 4.99; S, 7.61. Found: C, 51.29; H,
3.84; N, 5.48; S, 7.34. 1H NMR (CDCl3; ppm): 2.39 (s, 3H), 4.42 (s, 2H),
5.96 (s, 1H), 7.02–7.15 (m, 4H), 7.25–7.45 (m, 10H), 7.50 (d,
J=8.8 Hz, 2H), 7.55–7.65 (m, 6H), 8.06 (d, 8.8 Hz, 2H). 31P NMR
(CDCl3; ppm): 9.42 [J(195Pt–31P): 3492 Hz]. Positive ESI-MS (m/z):
843 ([M+H]+), 865 ([M+Na]+), 882 ([M+K]+). Molar conductivity
(10−3 M, DMSO): 1.8 S cm2 mol−1
.
% GI = f1–½ðLDt−LDtoÞ= ðLCt−LCtoÞꢀg × 100
2.4.5. Preparation of the palladium(II) complexes
where LDt is the average of the number of motile forms in wells
containing the complexes at time t, LDto is the average of the number
of motile forms in wells containing the complexes at time t=zero, LCt
is the average of the number of motile forms in wells in the absence of
any compound at time t (negative control) and LCt is the average of the
number of motile forms in wells in the absence of any compound at
4 drops of triethylamine were added to a suspension of [PdCl2
(PPh3)2] (0.140 g, 0.2 mmol) in MeCN (10 mL) premixed with the
solution of the desired ligand (0.2 mmol) in CH2Cl2 (10 mL). The
resulting orange solutions were kept under reflux for 2 h. The
solutions were evaporated at room temperature over a period of 3–
4 days forming suitable crystals for X-ray diffraction, except for
complex 2. The crystals were filtered off, washed with a small portion
of n-hexane and dried in air.
time t=zero [6]. The concentration of compound corresponding to
epi
50% antiproliferative 72 h of incubation was expressed as IC
.
50
[Pd(L1a)(PPh3)] (2). Yield: 59% (0.080 g). Anal. Calc. for C34H29N2
OPPdS2 (683.13): C, 59.78; H, 4.28; N, 4.10; S, 9.39. Found: C, 58.91; H,
4.54; N, 3.98; S, 9.52. 1H NMR (CD2Cl2, ppm): 2.92 (s, 3H), 4.43 (s, 2H),
6.60–6.65 (m, 2H), 7.18–7.8 (m, 22H). 31P NMR (CD2Cl2, ppm): 25.88.
Positive ESI-MS (m/z): 683 ([M+H]+), 705 ([M+Na]+), 722 ([M+
2.5.3. Evaluation of the activity on amastigote forms
Ninety-six-well tissue culture plates were seeded with L929
fibroblasts at 2.0×103 per well in 80-μL volumes (RPMI 1640 without
phenol red) and incubated overnight. In the next morning, β-
galactosidase-expressing trypomastigotes (Tulahuen strain-Lac Z)
were then added at 1.0×104 per well in 20-μL volumes (RPMI 1640
K]+). Molar conductivity (10−3 M, DMSO): 1.3 S cm2 mol−1
.

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1279
w/o phenol red). After 24 h, the complexes were added in serial
dilutions (100, 33.3, 11.1, 3.7, 1.2, 0.4 and 0.1 μM) in 50-μL volumes.
Each dilution was tested in triplicate. Previously the stock solutions
were prepared in acetonitrile and diluted in RPMI 1640 w/o phenol
red (final concentration of acetonitrile smaller than 1%). After 72 h,
when expanded numbers of trypomastigotes were easily visible in the
control wells, the assays were developed by adding the substrate
chloro-phenol red β-D-galactosidase (CPRG) (250 μM final concen-
tration) and Nonidet P-40 (0.1% final concentration). The plates were
incubated for 3–6 h at 37 °C. Wells with β-galactosidase activity
turned the media from yellow to red, and were measured at 570 nm.
The percentage of trypanocidal activity (%TA) was calculated as
follow:
reasonably assignable to a combination of sulfur→metal(II) and
nitrogen→metal(II) charge-transfer and M(II) d–d bands [21,22].
1H NMR spectra of the free ligands H2L1a, H2L1b, H2L2a and H2L2b
were measured in DMSO-d6 solutions. The spectra of the ligands H2L1a
and H2L1b show that they exist almost exclusively in the thione
tautomeric form A (see Fig. 1) in DMSO-d6 solution [24]. The ligands
H2L2a and H2L2b [25] are observed in the cyclic tautomeric form D,
with an insignificant percentage of them being present in the open
ring form C. The ligands H2L1a and H2L1b show a broad signal at δ 11.26
and 12.89 ppm, respectively, due to the phenolic hydroxilic group,
while the ligands H2L2a and H2L2b show signals related to the
pirazolinic OH at 7.09 and 7.16 ppm, respectively. The appearance of
a resonance for NH at δ 11.25 and 11.23 ppm in the spectra of H2L1a
and H2L1b, respectively, suggests the predominance of the thione
tautomer in solution. For the ligands H2L2a and H2L2b the NH
resonance is not observed, as expected for the cyclic pirazolinic
form [25]. The absence of both of these signals in the complexes is in
agreement with the coordination of phenolate oxygen and thiolate
sulfur, as observed in the IR spectra. The 1H NMR data are thus
consistent with the ONS dibasic tridentate binding mode of the
ligands.
% TA = ½1–ðLDt = LCtÞꢀ × 100
where LDt is the average of the absorbance at 570 nm in wells
containing the compounds and LCt is the average of the absorbance at
570 nm in wells in absence of any compounds.
3. Results and discussion
The 31P{1H} NMR signal of the precursor [PdCl2(PPh3)2] is
observed at δ 23.9 ppm [11], while the [PtCl2(PPh3)2] signal is found
at δ 11.39 ppm with a J1(195Pt–31P) of 3675 Hz. The 31P{1H} NMR
spectra of the Pd(II) complexes consist of sharp singlet signals at δ
25.88, 22.65, 27.20 and 27.04 ppm for 2, 4, 6 and 8, respectively.
However, in the 31P{1H} NMR spectra of the Pt(II) complexes, these
resonance signals are observed at δ 10.33, 6.31, 12.94 and 9.42 ppm,
for 1, 3, 5 and 7 respectively, with a J1(195Pt–31P) coupling of around
3500 Hz. According to the literature this coupling value is character-
istic of Pt–P bond trans to nitrogen bond system [37,38].
3.1. Synthesis
Triethylamine was used as supporting base in all reactions to
reduce the reaction time and improve the yields, by favoring the
deprotonation of the ligand and activation of the chlorine atoms of the
precursors [MCl2(PPh3)2], under formation of NEt3·HCl. The Pt(II)
complexes were prepared at room temperature, while the Pd(II)
complexes were refluxed to complete the reactions in a short time and
due to the poor solubility of the [PdCl2(PPh3)2] in the solvents used.
The residues of PPh3 were washed with n-hexane. The complexes
were obtained as pure crystalline solids and are air stable. They are
quite soluble in CHCl3, CH2Cl2 and DMSO, less soluble in MeCN and
insoluble in MeOH or EtOH.
3.3. Description of crystal structures
The complexes 1, 2, 3, 4, 5, 6 and 8 were studied by single crystal X-
ray diffraction analyses, confirming the spectroscopic results. The
molecular structures for complexes 1 and 8 with atomic numbering
scheme are shown in Figs. 2 and 3, respectively, and the ortep
drawings for complexes 2–6 can be obtained as Supplementary data.
Selected bond lengths and angles are given in Table S4 (Supplemen-
tary data).
3.2. IR, UV–visible and (1H, 31P) NMR spectroscopy
Selected infrared absorption frequencies of both the ligands and
their complexes are given in Table S2 (Supplementary data). The
comparisons of the spectra of the complexing agents with them of
their derivative complexes support the dianionic tridentate O,N,S-
chelation of the ligands. The disappearance of the ν(O–H) and ν(N–
H) ligands' bands upon complex formation, is in agreement with the
coordination of the thioenolate sulfur to Pt(II) or Pd(II) after
deprotonation. Strong ν(C N) bands at 1615 cm− 1 (in H2L1a),
1643 cm− 1 (in H2L1b), 1629 cm−1 (in H2L2a) and 1628 cm− 1 (in
H2L1b), are shifted to lower wavenumbers in the corresponding
complexes, hence indicating the coordination of the azomethine
nitrogen to the metal ion [31–35]. The coordination of the PPh3
group to the M(II) centers in 1–8 is observed by the presence of
strong bands assigned to ν(P–Ph) at 1098–1099 cm−1 and to phenyl
In all complexes, the M(II) ion (M Pd or Pt) is in a quite similar,
nearly planar, fourfold environment. The metal is coordinated to the
ring expansion and contraction β(ring, Ph) at 691–694 cm− 1
,
without significant changes when compared to the starting com-
plexes [PdCl2(PPh3)2] and [PtCl2(PPh3)2] (692 and 696 cm− 1
respectively).
,
Table S3 (Supplementary data) lists the absorptions in the
electronic spectra of the free dithiocarbazates and their Pd(II) and
Pt(II) complexes, recorded in acetonitrile. The electronic spectra for d8
predicts three d–d transitions. However, in complexes containing
sulfur donors strong charge-transfer transition invariably interferes
and prevents observing all the expected bands [22,36]. The absorption
spectra of 5–8 are almost identical with only one maximum in the
range 300–405 nm, while in the spectra of 1–4 the transitions were
observed with different maxima, in the range 277–433 nm, being
Fig. 2. Ellipsoid representation of [Pt(L1a)(PPh3)] (1) (50% probability level) showing
the atomic numbering scheme.

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P.I.S. Maia et al. / Journal of Inorganic Biochemistry 104 (2010) 1276–1282
Table 1
Antiproliferative activity of the free ligands and of the complexes 1–8 on epimastigotes.
epi
Compounds
IC (μM)
50
H2L1a
117.8
121.5
15.0
H2L1b
H2L2a
H2L2b
35.6
[Pt(L1a)(PPh3)] (1)
[Pd(L1a)(PPh3)] (2)
[Pt(L1b)(PPh3)] (3)
[Pd(L1b)(PPh3)] (4)
[Pt(L2a)(PPh3)] (5)
[Pd(L2a)(PPh3)] (6)
[Pt(L2b)(PPh3)] (7)
[Pd(L2b)(PPh3)] (8)
Bz
145.9
189.7
101.4
214.5
156.5
257.2
135.6
N500
1800
Antiproliferative activity expressed as the percentage of growth inhibition at given
epi
concentration. IC
corresponding to 50% antiproliferative activity after 72 h of
50
incubation. Bz, benznidazole, n=3–5, Pb0.05.
Fig. 3. Ellipsoid representation of [Pd(L2b)(PPh3)] (8) (50% probability level) showing
the atomic numbering scheme. The triphenylphosphine hydrogen atoms are omitted
for clarity.
compared to the Pd(II) complexes. Supposing the ligands to be the
active species, the higher activity of the Pt(II) complexes might be
correlated to their superior kinetic stability in comparison with Pd(II)
complexes, resulting in a better ability to carry the ligands to their
target or a different mechanism of action may even be present. These
results are different from those observed for other Pd(II) and Pt(II)
complexes of the type [MCl2(HL)] and [M(L)2] (L=bidentate
thiosemicarbazones), where the palladium complexes were more
active than the corresponding isostructural platinum complexes
against T. cruzi (Tulahuen 2 and Dm28c strains) in almost all the
cases [39,40]. It is well known that Pt(II) compounds, including
cisplatin, have shown anti-T. cruzi activity, acting through different
mechanisms, like DNA interaction and irreversible inhibition of T.
cruzi's trypanothione reductase [40–42]. Furthermore, when com-
pared to the standard drug, most of the compounds have a
comparable or higher activity in vitro on epimastigotes than other
previously reported complexes tested against other strains
[6,7,39,40,43–45].
binegatively charged organic molecule, which acts as a tridentate
ligand through the azomethine nitrogen atom [M–N(1) bond
distances in the range 2.01–2.04 Å], the oxygen atom [M–O distances
in the range 1.99–2.05 Å] and the thiolate sulfur atom [M–S(1)
distances around 2.24 Å]. The remaining binding site is occupied by a
phosphorous atom, with M–P distances in the range 2.25–2.28 Å,
which is within the expected range for Pt(II) or Pd(II) single bond
[38]. Apart from the S-Benzyl fragment for the H2L1a and H2L1b and
from both S-Benzyl and phenyl ring of the H2L2a and H2L2b, the
established six-membered and five-membered chelate rings are
nearly planar [maximum rms deviation of atoms from the mean
plane of 0.041 Å (1), 0.079 Å (2), 0.099 Å (3), 0.158 Å (4), 0.081 Å and
0.039 Å (for 5A and 5B, respectively), 0.062 Å (6) and 0.092 (8)]. A
considerable delocalization of π-electron density is indicated by the
observed bond lengths involving the chelate rings. The O(1)–M–S(1)
and N(1)–M–P bond angles present a deviation around 5° below the
expected value of 180°. No hydrogen bonds are observed for these
complexes.
3.4.2. Activity of amastigote forms
Since the life cycle of T. cruzi involves vertebrate (mammals,
including man) and invertebrate (triatomine bugs) hosts and
following invasion of vertebrate host cells, the trypomastigotes
undergo differentiation into amastigotes, we decided to investigate
the action of these complexes on amastigotes in human fibroblasts.
Similarly to that observed with epimastigote forms, the complexes
exhibited smaller activity than the respective ligands (Table 2).
3.4. In vitro anti-trypanosomal activity
As part of our ongoing research program aimed at discovering new
metal complexes with substantial in vivo activity on T. cruzi so that we
have tested the compounds presented here against replicative and
infective forms of T. cruzi.
Table 2
Trypanocidal activity of the free ligands and of the complexes 1–8 on amastigote forms.
3.4.1. Activity on epimastigote forms
try
In order to better evaluate the biological activity of metal on
replicative forms of T. cruzi, these compounds were set up using in
vitro cultures of epimastigotes (Tulahuen WT strain). Table 1
summarizes the results of the antiproliferative activity of these
compounds in the exponential phase of T. cruzi growth expressed as
the percentage of growth inhibition (% GI). As can be seen, all the
Compounds
IC (μM)
50
H2L1a
69.3
41.2
0.6
H2L1b
H2L2a
H2L2b
9.6
[Pt(L1a)(PPh3)] (1)
[Pd(L1a)(PPh3)] (2)
[Pt(L1b)(PPh3)] (3)
[Pd(L1b)(PPh3)] (4)
[Pt(L2a)(PPh3)] (5)
[Pd(L2a)(PPh3)] (6)
[Pt(L2b)(PPh3)] (7)
[Pd(L2b)(PPh3)] (8)
Bz
7.8
11.5
16.1
18.7
18.4
13.2
16.9
15.1
2.5
complexes, except the palladium complex [Pd(L2b)PPh3], showed
significantly higher antiproliferative activity (IC range from 101.4 to
257.2 μM) than the standard drug Bz (IC =1,800 μM). On the other
epi
50
epi
50
hand, the ligands H2L2a and H2L2b, which have the cyclic 5-
hydroxypyrazolic form in their structures, showed higher antiproli-
ferative activity than the respective complexes when incubated for up
to 72 h. Additionally, it was also observed that the anti-T. cruzi activity
is dependent on the nature of the metal ion once all the Pt complexes
are more effective against the epimastigote forms of T. cruzi when
Trypanocidal activity expressed as the percentage number of lysed trypomastigotes
try
compared to the negative control. IC values correspond to 50% trypanocidal activity
50
after 24 h of incubation. Bz, benznidazole, n=2–4, Pb0.05.

P.I.S. Maia et al. / Journal of Inorganic Biochemistry 104 (2010) 1276–1282
1281
However, as can be seen in this table, the ligands and complexes were
less active than the reference drug Bz, except for the ligand H2L2a
Acknowledgements
.
Since the mechanism of the action of Bz is through reduced
intermediates (R–NO2•), it is reasonable to conclude that the medium
in which the epimastigotes were assayed exhibits a weak reduction.
However, our results suggest that the activity of the dithiocarbazates
cannot be attributable to the nitro group. Moreover, the values of IC50
on amastigotes obtained by the compounds studied in this work are
comparable or higher than other active compounds reported before
[6,7,45], while the compound H2L2a alone presents an impressive
good value.
The decrease of activity upon complexation, in the case of the
ligands H2L2a and H2L2b, could be related to a greater stability of the
dithiocarbazates in the complexes, avoiding its proper interaction
with the biological target, or to the breakdown of the pyrazolinic
ring, since H2L1a and H2L1b, which does not contain this ring in their
structure, presented lower activities, even in relation to their
derivative complexes. Studies on the coordination of the ligands
H2L2a and H2L2b to a metal center that is able to keep the pyrazolinic
ring will help to answer this question, being currently underway in
our laboratories. By the moment, it is clear that Pd or Pt per se is not
responsible for the studied anti-T. cruzi activity.
The authors thank CNPq, CAPES (PROBRAL) and FAPESP for
supporting this work.
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Abbreviations
BT
Bz
EF
bloodstream trypomastigote forms of Trypanosoma cruzi;
benznidazole;
epimastigote forms of Trypanosoma cruzi;
ESI-MS electrospray ionization mass spectrometry;
GV
gentian violet;
epi
IC
IC
concentration corresponding to 50% antiproliferative
activity on epimastigote forms after 72 h of incubation at
28 °C;
50
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ama
50
concentration corresponding to 50% trypanocidal activity on
amastigote forms into mouse fibroblasts after 72 h of
incubation;
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Biochem. 103 (2009) 411–418.
PBS
phosphate-buffered saline;
T. cruzi Trypanosoma cruzi

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