Synthesis and in vitro evaluation of palladium(II) salicylaldiminato thiosemicarbazone complexes against Trichomonas vaginalis

Article abstract of DOI:10.1016/j.jinorgbio.2011.07.023

Eight mononuclear Pd(II) complexes containing salicylaldiminato thiosemicarbazones (saltsc-R; where R = H (1), 3-OMe (2), 3-^tBu (3) and 5-Cl (4)) as dinegative tridentate ligands were prepared by the reaction of the corresponding thiosemicarbazone with the precursor Pd(L)₂Cl ₂ (L = phosphatriazaadamantane or 4-picoline) in the presence of a weak base. These complexes (9-16) were characterised by a range of spectroscopic and analytical techniques including NMR spectroscopy and X-ray diffraction. These complexes along with four other Pd(II) analogues (5-8) were screened for activity in vitro against the Trichomonas vaginalis parasite. Preliminary results show that the type of ancillary ligand as well as the substituents on the aromatic ring of the salicylaldiminato thiosemicarbazone ligand influences the antiparasitic activity of these complexes.

Full text of DOI:10.1016/j.jinorgbio.2011.07.023

Journal of Inorganic Biochemistry 105 (2011) 1562–1568
Contents lists available at ScienceDirect
Journal of Inorganic Biochemistry
journal homepage: www.elsevier.com/locate/jinorgbio
Synthesis and in vitro evaluation of palladium(II) salicylaldiminato
thiosemicarbazone complexes against Trichomonas vaginalis
Prinessa Chellan a, Tameryn Stringer a, Ajit Shokar b, Padraick J. Dornbush b, Guillermo Vazquez-Anaya b,
Kirkwood M. Land b, Kelly Chibale a,c, Gregory S. Smith a,
⁎
a
Department of Chemistry, University of Cape Town, Private Bag, Rondebosch 7701, South Africa
Department of Biological Sciences, University of the Pacific, Stockton, CA 95211, United States
b
c
Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Rondebosch 7701, South Africa
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 6 May 2011
Received in revised form 29 July 2011
Accepted 29 July 2011
Available online 6 August 2011
Eight mononuclear Pd(II) complexes containing salicylaldiminato thiosemicarbazones (saltsc-R; where R=H
(1), 3-OMe (2), 3-tBu (3) and 5-Cl (4)) as dinegative tridentate ligands were prepared by the reaction of the
corresponding thiosemicarbazone with the precursor Pd(L)2Cl2 (L=phosphatriazaadamantane or 4-picoline)
in the presence of a weak base. These complexes (9–16) were characterised by a range of spectroscopic and
analytical techniques including NMR spectroscopy and X-ray diffraction. These complexes along with four
other Pd(II) analogues (5–8) were screened for activity in vitro against the Trichomonas vaginalis parasite.
Preliminary results show that the type of ancillary ligand as well as the substituents on the aromatic ring of
the salicylaldiminato thiosemicarbazone ligand influences the antiparasitic activity of these complexes.
© 2011 Elsevier Inc. All rights reserved.
Keywords:
Thiosemicarbazones
Pd(II) complexes
Antiparasital activity
Trichomonas vaginalis
1. Introduction
and the parasites, but also some compounds that were parasiticidal
despite a lack of activity against the proteases, suggesting that other
Trichomonas vaginalis is a flagellated facultative anaerobic protozoan,
which causes the sexually transmitted infection trichomoniasis in
humans. Metronidazole (Fig. 1), a 5-nitroimidazole drug derived
azomycin, is the current FDA-approved treatment [1]. Approximately 5%
of all cases are resistant to this compound, with that percentage increasing
[2]. There is therefore a need to find alternative forms of treatment due to
this increase in resistance. The use of thiosemicarbazones (TSCs) as
potential therapeutics is an active area of research. These Schiff-base type
compounds are noted for their pharmacological properties, particularly as
antiparasitic [3–8], antibacterial [9–11] and antitumoral agents [12–16].
As anticancer agents, it is believed that their mechanism of action is
through the inhibition of ribonucleotide reductase [17]. These com-
pounds have also been shown to inhibit a Trypanosoma cruzi derived
cysteine protease, cruzain [8] and are also effective against the parasites
Plasmodium falciparum and Trypanosoma brucei [18]. Thus thiosemicar-
bazones may be an alternative therapy against the parasite T. vaginalis,
since it also secretes cysteine proteases [19–21].
mechanisms besides cysteine protease inhibition are involved. This
data suggested that thiosemicarbazones represent validated drug
leads that kill several species of protozoan parasites through the
inhibition of cysteine proteases as well as other novel targets. The
thiourea moiety of thiosemicarbazones contains several donor atoms
and is thus capable of acting as a multidentate ligand toward a metal.
Their metal chelating abilities are believed to partially account for
their biological activity and numerous studies on the biological
activity of TSC metal complexes have been published [22–28]. The
study of the coordination chemistry of thiosemicarbazones has long
been of interest with the earliest review being published in 1974
[29]. Since then there have been extensive reports on the synthesis
of thiosemicarbazone complexes with metals including vanadium
[30,31], zinc [32], cobalt [33], gold [34], nickel [26,35], silver [36],
copper [37–39] and iron [40].
The synthesis and study of tridentate salicylaldiminato thiosemi-
carbazone Pd(II) complexes as potential biological agents have
previously been reported by us [41,42] as well as others [43]. The
complexes were found to be moderately cytotoxic as chemothera-
peutics when tested in vitro [41–43]. However, our studies into their
use against the malarial parasite P. falciparum in vitro revealed these
complexes to be promising inhibitors [41]. Coupled with our earlier
studies on parasiticidal thiosemicarbazone cysteine protease inhibitors
against P. falciparum, T. brucei and T. cruzi [18], these results have thus
prompted us to study the effects of these types of salicylaldiminato
In previous work, we synthesised a library of thiosemicarbazones
and screened them against three parasitic cysteine proteases, cruzain,
falcipain-2, and rhodesain and against the respective parasite sources
of these three proteases, T. cruzi, P. falciparum and T. brucei [18]. The
screens identified compounds that were effective against the enzymes
⁎
Corresponding author. Tel.: +27 21 650 5279; fax: +27 21 650 5195.
E-mail address: gregory.smith@uct.ac.za (G.S. Smith).
0162-0134/$ – see front matter © 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.jinorgbio.2011.07.023

P. Chellan et al. / Journal of Inorganic Biochemistry 105 (2011) 1562–1568
1563
filtration, washed with ethanol and diethyl ether, and dried in vacuo. All
of the products were recrystallised from DCM-Hexane.
OH
N
O2N
2.2.2. Spectroscopic data for 9
Salicylaldimine thiosemicarbazone 1 (0.055 g, 0.28 mmol) and Pd
(PTA)2Cl2 (0.14 g, 0.28 mmol) were refluxed in ethanol for 10 h. The
product obtained was a yellow powder (0.098 g, 77%). M.p.: 262 °C. 1
H
N
NMR (300.08 MHz, DMSO–d6, s=singlet, m=multiplet, d=doublet,
dd=doublet of doublet): δ (ppm)=4.31 (6H, s, H-PTA); 4.42 (3H,
d, 2JH–H=13.07, H-PTA); 4.57 (3H, d, 2JH–H=12.98, H-PTA); 6.56(3H, m,
Fig. 1. Structure of Metronidazole, the current FDA-approved treatment of T. vaginalis.
3
3
palladium(II) complexes on the causative agents of other parasitic
diseases exemplified by T. vaginalis.
NH2, H4); 6.89 (1H, d, JH–H=8.34, H2); 7.23 (1H, t, JH–H=8.58, H3);
4
3
4
7.37 (1H, dd, JH–H=1.77, JH–H=7.93, H5); 8.21 (1H, d, JP–H=13.18,
H7). 31P NMR (121.47 MHz, DMSO–d6): δ (ppm)=−40.70 (1P, s, PTA).
13C NMR (75.46 MHz, DMSO–d6): δ (ppm)=49.94 (PTA); 71.87 (PTA);
114.17 (C2); 117.83 (C6), 120.59 (C4); 132.25 (C5); 134.00 (C3); 147.57
(C7); 161.70 (C1); 171.17 (C8). IR (KBr, cm−1, m=medium, s=strong)
υ=3396 (m, N–H), 3285 (m, N–H), 1615 (m, C_N), 1595 (s, C_N),
1531 (s, C_C aromatics). Elemental analysis for C14H19N6SPOPd: found
C 36.77, H 4.24, N 17.97, S 6.87%; calculated C 36.81, H 4.19, N 18.40, S
7.02%. ESI–MS: m/z 457 (calcd. 456.82) ([M]+, 100%).
Herein, we report the synthesis, structural characterisation and
inhibitory investigation of a series of tridentate [O,N,S] salicylaldiminato
thiosemicarbazone Pd(II) complexes containing functionalised salicyl-
aldehyde moieties as well as various ancillary ligands. The objectives of
our study were to determine if coordination of these compounds to
palladium enhances their inhibitory effects and to ascertain if variation
of the ancillary ligand as well as the functional groups of the
thiosemicarbazone ligand would beneficially modify biological activity.
2.2.3. Spectroscopic data for 101
2. Experimental
3-Methoxy salicylaldimine thiosemicarbazone 2 (0.053 g, 0.23 mmol)
and Pd(PTA)2Cl2 (0.11 g, 0.233 mmol) were refluxed in ethanol for 8 h.
The product obtained was a yellow powder (0.092 g, 81%). M.p.: no
melting or decomposition up to 300 °C. 1H NMR (300.08 MHz, DMSO–
d6): δ (ppm)=3.84 (3H, s, OCH3); 4.41 (6H, s, H-PTA); 4.51 (3H,
2.1. Materials and methods
All complexation reactions were performed under a nitrogen or
argon atmosphere using a dual vacuum/nitrogen line and standard
Schlenk-line techniques unless otherwise stated. All reaction solvents
were dried by refluxing under an inert atmosphere over the
appropriate drying agent and all samples were dried under vacuum.
Reagents and solvents were purchased from commercial suppliers
and used without further purification. PdCl2 was kindly donated
by Johnson-Mathey Inc. NMR Spectra were recorded on a Varian
Unity XR400 MHz (1H at 399.95 MHz, 13C at 100.58 MHz, 31P at
161.90 MHz) or Varian Mercury XR300 (1H at 300.08 MHz, 13C at
75.46 MHz, 31P at 121.47 MHz) MHz spectrometer at ambient
temperature. Chemical shifts for 1H and 13C{1H} NMR signals are
2
2
d, JH–H=13.17, H-PTA); 4.64 (3H, d, JH–H=12.69, H-PTA); 6.56 (1H,
t, 3JH–H=7.79, H4); 6.64 (2H, s, NH2); 6.95 (1H, dd, 4JH–H=1.53, 3JH–H
=
4
3
7.59, H3); 7.07 (1H, dd, JH–H =1.50, JH–H =8.13, H5); 8.27 (1H,
4
d, JP–H =13.20, H7). 31P NMR (121.47 MHz, DMSO–d6): δ (ppm)=
−39.37 (1P, s, PTA). 13C NMR (100.58 MHz, DMSO–d6): δ (ppm)=
50.83 (OCH3); 57.00 (PTA); 72.68 (PTA); 114.17 (C4); 114.89 (C3);
118.50 (C6); 126.79 (C5); 148.16 (C7); 151.72 (C2); 153.74 (C1);
172.01 (C8). IR (KBr, cm−1, br m=broad medium) υ=3422 (br m,
N–H), 1623 (m, C_N), 1596 (s, C_N), 1540 (s, C_C aromatics).
Elemental analysis for C15H21N6SPO2Pd: found C 37.18, H 4.38, N
16.14, S 6.01%; calculated C 37.00, H 4.35, N 17.27, S 6.58%. ESI–MS:
m/z 487 (calcd. 486.76) ([M]+, 100%); 975 ([2 M+H]+, 20%).
reported using tetramethylsilane (TMS) as the internal standard and 31
P
{1H} spectra were measured relative to H3PO4 as the external standard.
IR spectra were measured on a Perkin-Elmer Spectrum One FT-IR
Spectrometer as KBr pellets. Microanalyses for C, H, N and S were carried
out using a Thermo Flash 1112 Series CHNS-O Analyser and melting
points were determined using a Kofler hot stage microscope (Reichert
Thermovar). Mass Spectrometry determinations were carried out on all
new compounds using electrospray ionisation on a Waters API Quattro
Micro instrument in the positive or negative mode.
2.2.4. Spectroscopic data for 111
3-tert Butyl salicylaldimine thiosemicarbazone 3 (0.050 g, 0.20 mmol)
and Pd(PTA)2Cl2 (0.096 g, 0.19 mmol) were refluxed in ethanol for 8 h.
The product obtained was a yellow powder (0.080 g, 80%). M.p.: no
melting or decomposition up to 300 °C. 1H NMR (300.08 MHz, DMSO–
d6): δ (ppm)=1.51 (9H, s, C(CH3)3); 4.43 (6H, s, H-PTA); 4.53 (3H,
d, 2JH–H=13.38, H-PTA); 4.62 (3H, d, 2JH–H=12.68, H-PTA); 6.60(3H, m,
4
NH2, H4); 7.32 (2H, d, 3JH–H=7.91, H5, H3); 8.28 (1H, d, JP–H=13.48,
2.2. Synthesis
H7). 31P NMR (121.47 MHz, DMSO–d6): δ (ppm)=−43.77 (1P, s, PTA).
13C NMR(100.58 MHz, DMSO–d6): δ (ppm)=30.04 (C(CH3)3); 35.88(C
(CH3)3); 48.92 (PTA); 72.66 (PTA); 114.46 (C4); 118.84 (C6); 131.67
(C5); 133.89 (C3); 139.36 (C2); 149.51 (C7); 161.85 (C1); 170.95 (C8).
IR (KBr, cm−1, br m=broad medium) υ=3437 (br m, N–H), 3293 (br
w, N–H), 1643 (m, C_N), 1593 (s, C_N), 1536 (s, C_C aromatics).
Elemental analysis for C18H27N6SPOPd: found C 42.24, H 5.35, N 17.45, S
5.65%; calculated C 42.15, H 5.30, N 16.39, S 6.25%. ESI–MS: m/z 513
(calcd. 512.84) ([M]+, 100%).
The salicylaldimine thiosemicarbazone ligands 1[44], 2[45], 3[41],
4[46] and the palladium complexes, dichlorobis(4-picoline)palladium
(II) [47], dichlorobis(phosphatriaza-adamantane)palladium(II) [48],
5 and 13[43] were prepared according to the published literature
procedures. The synthesis and characterisation of complexes 6–8 have
previously been reported by us [41].
2.2.1. General procedure for preparation of palladium(II) complexes
(9–16)
2.2.5. Spectroscopic data for 121
5-Chlorosalicylaldimine thiosemicarbazone 4 (0.050 g, 0.22 mmol)
and Pd(PTA)2Cl2 (0.11 g, 0.22 mmol) were refluxed in ethanol for 24 h.
The appropriate thiosemicarbazone ligand (1–4, 1 mol equiv.) was
added to dry ethanol (40 mL) under argon. The solution was heated to
60 °C with stirring. Triethylamine (2.1 mol equiv.) was added followed
by either Pd(PTA)2Cl2 (1 mol equiv.) for complexes 9–12 or Pd(4-
pic)2Cl2 (1 mol equiv.) for complexes 14–16. The mixture was refluxed
over periods of time designated in the subsequent text. The products
precipitated as an orange or yellow solid. The product was isolated via
1
Although the elemental analyses data for “N” (compounds 10, 11, 12 and 16) and
“C” (compound 16) are somewhat unsatisfactory, the ESI–MS result and other
spectroscopic analysis data (1H- and 13C-NMR, IR), and the X-ray crystal structure
determination for compound 11, reasonably support their formula.

1564
P. Chellan et al. / Journal of Inorganic Biochemistry 105 (2011) 1562–1568
The product obtained was a yellow powder (0.097 g, 92%). M.p.: no
2.3. X-ray crystallography
melting or decomposition up to 300 °C. 1H NMR (300.08 MHz, DMSO–
2
d6): δ (ppm)=4.31 (6H, s, H-PTA); 4.42 (3H, d, JH–H=13.12, H-PTA);
X-ray single crystal intensity data was collected on a Nonius
Kappa-CCD diffractometer using graphite monochromated MoKα
radiation. Temperature was controlled by an Oxford Cryostream
cooling system (Oxford Cryostat). The strategy for the data collections
was evaluated using the Bruker Nonius “Collect” programme Data
were scaled and reduced using DENZO-SMN software [49]. An
empirical absorption correction was applied using the programme
SADABS [50]. The structure was solved by direct methods and refined
employing full-matrix least-squares with the programme SHELXL-97
refining on F2 [51]. Packing diagrams were produced using the
programme PovRay and graphic interface X-seed [52]. All the non-
hydrogen atoms were refined anisotropically. All the hydrogen atoms,
except the amino hydrogens on N3A and N3B, were placed in
idealised positions in a riding model with Uiso set at 1.2 or 1.5 times
those of their parent atoms and fixed C–H bond lengths. The amino
hydrogen atoms were located in the difference electron density maps
and refined with simple bond length constraints. Crystallographic
data and data collection parameters are listed in Table 1.
4.58 (3H, d, 2JH–H=12.91, H-PTA); 6.75 (2H, s, NH2); 6.97 (1H, d, 3JH–H
9.02, H2); 7.27 (1H, dd, 4JH–H=2.84, 3JH–H=9.00, H3); 7.55 (2H, d, 4JH–H
=
=
2.83, H5); 8.30 (1H, d, 4JP–H=13.00, H7). 31P NMR (121.47 MHz, DMSO–
d6): δ (ppm)=−40.06 (1P, s, PTA). 13C NMR (75.46 MHz, DMSO–d6): δ
(ppm)=49.94 (PTA); 71.86 (PTA); 117.03 (C2); 119.08 (C6); 122.39
(C4); 131.65 (C5); 132.03 (C3); 146.51 (C7); 160.36 (C1); 171.98
(C8). IR (KBr, cm−1) υ=3390 (br w, N–H), 3286 (br w, N–H), 1630
(m, C_N), 1595 (s, C_N), 1516 (s, C_C aromatics). Elemental
analysis for C14H18N6SOClPdP: found: C 34.37, H 3.77, N 17.96, S
5.92%; calculated C 34.23, H 3.69, N 17.11, S 6.53%. ESI–MS: m/z 493
(calcd. 491.24) ([M+H]+, 100%).
2.2.6. Spectroscopic data for 14
Dichlorobis(4-picoline)palladium(II) (0.50 g, 1.4 mmol) was reacted
with 3-methoxy salicylaldimine thiosemicarbazone 2 (0.31 g, 1.4 mmol).
The reaction mixture was heated to 50 °C for 2 h. The product precipitated
as a crystalline orange solid (0.16 g, 27%). M.p.: 225–227 °C. 1H NMR
3
(300.08 MHz, CDCl3): δ (ppm)=8.70 (d, JH–H=6.60 Hz, 2H, Hα-4-
picoline), 7.97 (s, 1H, H7), 7.14–7.31 (m, 1H, H5), 6.95–7.02 (m, 3H, H4,
Hβ-4-picoline), 6.52–6.68 (m, 1H, H3), 4.74 (s, 2H, NH2), 3.85 (s, 3H,
OCH3), 2.43 (s, 3H, CH3-4-picoline). 13C NMR (75.46 MHz, CDCl3): δ
(ppm)=170.5 (C7), 153.9 (C8), 152.2 (C2), 151.8 (Cα-4-picoline), 150.7
(C(CH3)-4-picoline), 149.0 (C1), 127.4 (Cβ-4-picoline), 126.2 (C5), 125.3
(C4), 119.1 (C6), 114.5 (C3), 56.7 (OCH3), 21.2 (CH3-4-picoline). IR
(KBr, cm−1) υ=3428 (br m, N–H), 3351 (br m, N–H), 1621(s, C_N),
1603 (s, C_N), 1525 (s, C_C aromatics). Elemental analysis for
2.4. In vitro assay
Cultures of T. vaginalis T1 strain were grown in 5 mL completed
TYM Diamond's media in a 37 °C incubator for 24 h. One hundred
millimolar stocks of the compounds were made by dissolving in
DMSO, and were screened against T1 stain of T. vaginalis. Cells
untreated and inoculated with 5 μL DMSO are used as controls. Five
microlitres of 100 mM stocks of compound library were inoculated for
a final concentration of 100 μM. Results were calculated based on
counts utilising a hemocytometre after 24 h. For IC50 values,
increasing concentrations of the compound (0–100 μM) were tested
for inhibitory activity and concentrations that inhibited at approxi-
mately 50% were then obtained by linear regression analysis. These
predicted IC50 values were then confirmed by direct testing on T.
vaginalis strain T1 as described above.
C
15H16N4O2PdS·⅓CHCl3·½C6H14: found C 43.81, H 4.20, N 11.88, S
6.38%; calculated C 43.54, H 4.65, N 11.88, S 6.34%. ESI–MS: m/z 423
(calcd. 422.80) ([M]+, 100%).
2.2.7. Spectroscopic data for 15
Dichlorobis(4-picoline)palladium(II) (0.50 g, 1.4 mmol) was reacted
with 3-tert Butyl salicylaldimine thiosemicarbazone 3 (0.35 g,
1.4 mmol). The reaction mixture was refluxed for 5 h and the product
precipitated as a fluffy yellow solid (0.29 g, 46%). M.p.: no melting or
decomposition up to 300 °C. 1H NMR (399.95 MHz, CDCl3): δ (ppm)=
3
8.69 (d, JH–H=6.50 Hz, 2H, Hα-4-picoline), 7.97 (s, 1H, H7), 7.32–7.36
(m, 1H, H5), 7.18–7.25 (m, 3H, H4, Hβ-4-picoline), 6.79–6.42 (m, 1H,
H3), 4.73 (s, 2H, NH2), 2.47 (s, 3H, (CH3-4-picoline)), 1.33 (s, 9H, C(CH3)3).
13C NMR (75.46 MHz, CDCl3): δ (ppm)=169.8 (C7), 161.8 (C8), 152.0
(C1), 151.7 (Cα-4-picoline), 150.8 (C(CH3)-4-picoline), 138.9 (C2), 132.6
(C3), 130.3 (C5), 125.9 (Cβ-4-picoline), 118.7 (C4), 114.3 (C6), 35.2 (C C
(CH3)3), 29.4 (C(CH3)3), 21.2 (CH3-4-picoline). IR (KBr, cm−1) υ=3421
(m, N–H), 3310 (s, N–H), 1621 (br w, C_N), 1609 (s, C_N), 1591 (s,
C_N), 1527 (s, C_C aromatics). Elemental analysis for C18H22N4OPdS:
found C 47.81, H 5.03, N 13.01, S 6.96%; calculated C 48.16, H 4.94, N 12.48,
S 7.14%. ESI–MS: m/z 449 (calcd. 448.88) ([M]+, 100%).
Table 1
Selected crystallographic and refinement data for 11.
Empirical formula
Formula weight
T (K)
Wavelength (Å)
Crystal system
Space group
a (Å)
b (Å)
c (Å)
α (°)
C18H27N6OSPPd
512.89 g mol−1
173.2
0.71073
Triclinic
P 1
9.4573 (4)
12.318 (5)
18.679 (7)
92.746 (2)
β (°)
104.01 (2)
94.480 (2)
2099.8 (15)
4
1.622
1.081
0.20×0.14×0.12
2.07–25.37
2.2.8. Spectroscopic data for 161
γ (°)
Volume (Å3)
Dichlorobis(4-picoline)palladium(II) (0.39 g, 1.1 mmol) was reacted
5-chlorosalicyl-aldimine thiosemicarbazone 4 (0.25 g, 1.1 mmol). The
product precipitated out of solution as a yellow solid (0.14 g, 30%). M.p.:
decomposition without melting 238–240 °C. 1H NMR (300.08 MHz,
Z
Dcalc (Mg/m3)
Absorption coefficient (mm−1
Crystal size (mm3)
θ range (°)
)
3
CDCl3): δ (ppm)=8.59 (d, JH–H=6.60 Hz, 2H, Hα-4-picoline), 7.83 (s,
1H, H7), 7.16–7.44 (m, 3H, H5, Hβ-4-picoline), 6.94–6.96 (m, 2H, H2,
H3), 4.78 (s, 2H, NH2), 2.44 (s, 3H, CH3-4-picoline). 13C NMR (75.46 MHz,
CDCl3): δ (ppm) 172.71 (C7), 161.09 (C8), 152.3 (C1), 151.8 (Cα-4-
picoline), 147.9 (C(CH3)-4-picoline), 132.7 (C4), 132.3 (C5), 127.5 (Cβ-
4-picoline), 121.9 (C3), 120.5 (C6), 117.9 (C2), 21.29 (CH3-4-picoline).
IR (KBr, cm−1) υ=3403 (w, N–H), 3273 (w, N–H), 1622 (s, C_N), 1596
(s, C_N), 1518 (s, C_C aromatics). Elemental analysis for C14H13N4-
OPdS: found C 40.76, H 3.40, N 13.86, S 7.43%; calculated C 39.36, H 3.07,
N 13.11, S 7.51%. ESI–MS: m/z 429 (calcd. 427.22) ([M+2H]2+, 100%).
Limiting indices
Reflections collected
Unique reflections
Absorption correction
Data/restraints/parameters
Goodness-of-fit on F2
Final R indices [IN2σ(I)]
R indices (all data)
−11≤h≤11, −14≤k≤14, −22≤l≤22
14,900
7684 [R(int)=0.0541]
Semi-empirical from equivalents
7684/4/521
1.018
R1 =0.0368, wR2 =0.0593
R1 =0.0786, wR2 =0.0688
0.810
Max. residual density (eÅ−3
Min. residual density (eÅ−3
)
)
−0.662

P. Chellan et al. / Journal of Inorganic Biochemistry 105 (2011) 1562–1568
1565
3. Results and discussion
13.00 and 13.48 Hz and is consistent with coordination of the
phosphorus to the metal centre trans to the imine nitrogen.
3.1. Synthesis and characterisation of tridentate [O,N,S] Pd(II) complexes
The aliphatic protons of the PTA ancillary ligands for 9–12 are found
in the region of 4.5 ppm. There are two types of methylene protons
present in the PTA ligand. One type is assigned to the P–CH2–N protons,
which occur as a singlet in the region of 4.4 ppm. The protons of the
N–CH2–N moiety possess an AB spin system and appear as two doublets,
due to geminal coupling, at 4.5 ppm. One doublet corresponds to the
three N–CHaxial–N, while the other is observed for the N–CHequitorial–N
protons. This is consistent with other PTA transition metal complexes
[55–57]. The protons of the methyl substituent of the picolyl ring
resonate as a singlet at ca. 2.40 ppm for 14–16. The signal of one
aromatic proton of the thiosemicarbazone ligand overlaps with the
signal of the protons beta to nitrogen in the picolyl ring of the co-ligand
to occur as a multiplet in complexes 14–16.
For complexes 9–12, the carbon-13 NMR spectra show that the
signals accounting for the phenolic carbon atoms appear to shift
downfield upon coordination, from approximately 156 ppm in the
free ligands to approximately 160 ppm in the complexes. This same
phenomenon is observed for the azomethine carbon signals (140 ppm
in the ligands vs 146 ppm in the complexes). In contrast, complexes
14–16, where the ancillary ligand is 4-picoline, the azomethine
carbon of the coordinated thiosemicarbazone ligand resonates at
approximately 170.0 ppm in all of the complexes. This shift is
characteristic of coordination of the imine nitrogen to palladium
leading to a downfield shift of this carbon relative to the resonance
observed for the free ligand [58–60]. The thiolate carbon generally
occurs between 161.0 and 162.0 ppm for the complexes 15 and 16. In
the case of complex 14 the thiolate carbon resonates at 153.8 ppm. It
seems that in this complex there is greater shielding of the C–S carbon
due to the methoxy substituent of the thiosemicarbazone ligand.
Overall, the upfield shift of the C–S carbon in all complexes compared
to the thione carbon of the corresponding free ligand is evidence of
coordination of sulphur to palladium in the thiolato form since
shielding of the thiolate carbon is characteristic of this type of bonding
[58,61–63].
The thiosemicarbazone ligands, salicylaldimine thiosemicarbazone
(1), 3-methoxy salicylaldimine thiosemicarbazone (2), 3-tert-butyl
salicylaldimine thiosemicarbazone (3) and 5-chlorosalicylaldimine
thiosemicarbazone (4) were prepared by Schiff base condensation
reactions of the appropriate salicylaldehyde with thiosemicarbazide
(Scheme 1). The ligands 1 [44], 2 [45], 3 [41] and 4 [46] are known
compounds and their spectroscopic data and melting points correlate
with the literature. The palladium(II) complexes 5–16 were prepared by
reaction of the thiosemicarbazone ligand with different palladium(II)
precursors of the type, Pd(L)2Cl2 (where L=triphenylphosphine (PPh3)
for 5–8, phosphatriazaadamantane (PTA) for 9–12 and 4-picoline
(4-pic) for 13–16), in refluxing ethanol with triethylamine as base
(Scheme 1). This weak base promotes deprotonation of the phenolic
oxygen as well as the hydrazinic nitrogen thus allowing the thiosemi-
carbazone ligand to coordinate to palladium as a tridentate dinegative
donor via the phenolate oxygen, imine nitrogen and thiolato sulphur
atoms.
All of the palladium complexes were isolated as air, moisture and
thermally stable yellow or orange solids in moderate to high yields. The
synthesis and characterisation of complexes 5, 13 [43] and 6–8 [41] has
previously been reported.
For complexes 9–16, the absence of the resonances assigned to the
hydroxyl and hydrazinic protons of the free ligand in the proton NMR
spectra confirms that the ligand coordinates to palladium via the
thiolato sulphur and phenolic oxygen. A slight downfield shift in the
resonance for the imine proton to between 8.20 and 8.30 ppm for
complexes 9–12 and a significant upfield shift to between 7.80 and
8.00 ppm for complexes 14–16 compared to their corresponding free
ligands suggest coordination of the imine nitrogen to the metal centre.
This shielding of the imine proton in complexes 14–16 is expected as
the 4-picoline ancillary ligand is a better sigma-donor towards
palladium than PTA. For 9–12, the signal for the imine proton splits
to form a doublet. This splitting is attributed to coupling of the
phosphorus nucleus of the PTA ligand to the nucleus of the imine
proton situated four bonds away (4JPH =13.0–13.5 Hz). This phenom-
enon is documented in the literature for similar triphenylphosphine
complexes [53,54]. The coupling constants obtained range between
In the infrared spectra for complexes 9–12 two absorption bands
are observed in the imine region. This is consistent with the formation
of a new imine bond within the thiosemicarbazone ligand upon
coordination of sulphur in the thiolate form. The lower frequency
band, observed between 1592 and 1598 cm−1, is assigned to the
Scheme 1. Synthetic route to Pd(II) salicylaldiminato thiosemicarbazone complexes 5–16.

1566
P. Chellan et al. / Journal of Inorganic Biochemistry 105 (2011) 1562–1568
imine bond coordinated to palladium [44,64,65]. When the nitrogen
of the imine coordinates to the metal, a loss of double bond character
occurs due to stronger electron donation towards the metal, thus this
bond will vibrate at a lower frequency. The absorption band observed
at higher frequency, between 1615 and 1643 cm−1, are thus assigned
to the newly formed imine bond.
For complexes 14–16, high frequency bands observed between
1625 and 1620 cm−1 are assigned to the new imine bond formed. The
vibration of the coordinated imine for these complexes is harder to
assign in 14 and 16 as the signal for this vibration may overlap with
the imine vibration of the 4-picolyl ring. In complex 15 there are three
distinct bands in the imine region and the coordinated imine vibration
is assigned to the band observed at 1609 cm−1. The C_N stretch of
the 4-picolyl ring is allocated to the lowest frequency band observed.
In the spectra of complexes 14 and 16, the absorption bands observed
at 1603 cm−1 for 14 and 1596 cm−1 for 16 are slightly broad and this
may indicate the overlapping of two C_N vibrations resulting in only
one absorption band being observed.
3.2. X-ray diffraction studies
To further confirm the integrity of these complexes, crystals suitable
for X-ray structure determination were obtained for complex 11.
Crystals were grown from dimethylsulfoxide at room temperature.
Table 1 (see Experimental section) summarises the crystallographic
data and Table 2 lists some relevant bond parameters. The molecular
structure depicts coordination of the metal centre (Pd) to the 3-tert-
butyl salicylaldimine monothiosemicarbazone ligand via the phenolic
oxygen, imine nitrogen and sulphur atom in a slightly distorted square-
planar geometry. The structure confirms that the fourth coordination
site is occupied by a PTA ligand (Fig. 2).
The molecular structure of 11 shows that the thiosemicarbazone
ligand is coordinated to palladium in the expected tridentate (O–N–S)
fashion. The structure shows the formation of a six- and a five-
membered chelate ring upon coordination. The structure displays O
(1A)–Pd(1A)–N(1A) and N(1A)–Pd(1A)–S(1A) bond angles of 93.40
(9) and 84.45(8)°, respectively. Trans to N(1A), we find a PTA ligand
coordinated to the metal centre. O(1A)–Pd(1A)–P(1A) and S(1A)–Pd
(1A)–P(1A) bite angles of 91.85(7) and 90.19(3)°, respectively, can
also be seen. These angles indicate a square planar geometry with
slight distortion around the palladium centre, as these angles deviate
from ideal behaviour.
In comparison to similar monomeric and dimeric palladium
triphenylphosphine thiosemicarbazone complexes, Pd–O, Pd–N, Pd–S
and Pd–P bond distances appear quite similar [41–43,66,67]. In addition
to this, it can be seen that coordination of the thiosemicarbazone occurs
via the thiolate form, rather than the thione form due to the formation of
a second imine bond between C(1A) and N(2A). A value of 1.313(4)Å is
assigned to this bond, further supporting the notion that the C(1A)–N
(2A) bond tends towards double-bond character upon coordination.
Comparison of this length to the C(1A)–N(3A) bond length, shows that
the latter is slightly longer (1.349(4)Å) and therefore tends towards
single-bond character. The C–S bond length is also found to be
consistent with coordination of sulphur in the thiolate form, with a
value of 1.754(4)Å. A previous study by Halder et al. [43], based on
similar monomeric complexes, reported C–S values of approximately
1.74 Å. In simple systems it would be expected that a single C–S bond
Fig. 2. Molecular structure of complex 11 at 50% probability level with all H atoms
omitted for clarity.
length would be approximately 1.82 Å and a double bond would exhibit
a length of 1.56 Å. The length obtained, therefore appears to be an
intermediate length between the two [68].
Examination of the crystal packing arrangement revealed an
extensive arrangement of hydrogen bonding between each molecule
(Fig. 3). Closer inspection of this packing arrangement revealed
hydrogen bonding of one molecule to two adjacent molecules via the
N2 atoms, amino hydrogens and a nitrogen atom belonging to the PTA
group.
3.3. Biological studies
The four thiosemicarbazone ligands (1–4) along with their
corresponding palladium complexes (5–16) were tested for inhibitory
activity in vitro against T. vaginalis. Table 3 lists the data ascertained
for the initial percent inhibition screening carried at 100 μM. Two
thiosemicarbazone ligands (1 and 3) as well as five of the Pd(II)
complexes (8 and 9, 12–16) exhibited a percent inhibition greater
than 80% at 100 μM. The free ligand 1 displayed the best inhibitory
activity (88.9%) out of all four thiosemicarbazone ligands. However,
it's corresponding Pd(II) complex 5, where the ancillary ligand is
Table 2
Selected bond lengths (Å) and angles (°) for complex 11.
Pd(1A)–O(1A)
Pd(1A)–N(1A)
Pd(1A)–S(1A)
Pd(1A)–P(1A)
N(1A)–C(2A)
N(2A)–C(1A)
2.028(2)
2.012(3)
2.2389(9)
2.2461(10)
1.293(4)
1.313(4)
O(1A)–Pd(1A)–N(1A)
N(1A)–Pd(1A)–S(1A)
O(1A)–Pd(1A)–P(1A)
S(1A)–Pd(1A)–P(1A)
O(1A)–Pd(1A)–S(1A)
N(1A)–Pd(1A)–P(1A)
93.40(9)
84.45(8)
91.85(7)
90.19(3)
177.08(7)
173.71(8)
Fig. 3. Two dimensional H-bond network of complex 11 projected along [100]. All
hydrogen atoms except the amino hydrogen atoms were omitted. The hydrogen bonds
are shown as dotted lines.

P. Chellan et al. / Journal of Inorganic Biochemistry 105 (2011) 1562–1568
1567
Table 3
Average percentage inhibition determined against Trichomonas vaginalis.
Table 4
IC50 determination for Pd(II) complexes 9 and 12–16.
Compound no.
Average % inhibition
Compound
IC50 [μM]
Ligand 1
Ligand 2
Ligand 3
Ligand 4
89 ( 6)
60 ( 15)
82 ( 11)
25 ( 3)
14 ( 8)
25 ( 12)
59 ( 13)
81 ( 7)
97 ( 3)
35 ( 15)
69 ( 6)
95 ( 4)
97 ( 3)
96 ( 4)
97 ( 3)
92 ( 8)
100 ( 6)
9
12
13
14
15
16
21 ( 1)
17 ( 1)
23 ( 1)
37 ( 1)
23 ( 1)
21 ( 3)
0.72 ( 1)
Complex 5
Complex 6
Complex 7
Complex 8
Complex 9
Complex 10
Complex 11
Complex 12
Complex 13
Complex 14
Complex 15
Complex 16
Metronidazolea
Metronidazolea
a
Current FDA approved treatment for T. vaginalis infections.
proteases) in T. vaginalis [20,71], other proteases must be validated as
potential targets for compounds described in this paper by using a
proteomics approach to confirm the identity of the targets of some
potent compounds.
4. Conclusions
a
Current FDA approved treatment for T. vaginalis infections.
Four thiosemicarbazone ligands and their corresponding tridentate
Pd(II) complexes were studied for antiparasital activity against
T. vaginalis. It was found that when the co-ligand was either PTA or
4-picoline, good inhibition was observed. The presence of a substituent
in the 5-position of the aromatic ring of the thiosemicarbazone ligand
clearly enhances the biological activity of these complexes and further
investigation of this observation is ongoing. This preliminary study
has established that a selection of these Pd(II) thiosemicarbazone
complexes display inhibitory activity against T. vaginalis. Currently, we
are investigating their antiparasitic effect against strains resistant to
Metronidazole.
triphenylphosphine, exhibited a value approximately six times lower.
Where the ancillary ligand was PTA (9) or 4-picoline (13), a percent
inhibition of 97% was observed suggesting that these co-ligands
enhance the activity of these complexes.
The thiosemicarbazone Pd(II) complexes with triphenylphosphine
as ancillary ligand do not show appreciable inhibition with the
exception of 8 (81% inhibition) and this may be attributed to the
presence of a chlorido group at the 5-position of the aromatic ring in
the thiosemicarbazone ligand. Our previous studies of this ligand (4)
and its complex (8) for antimalarial activity showed a similar trend in
activity [41]. It is unclear if this is a steric or electronic effect and
mechanistic studies into the significance of this observation are
ongoing.
For the complexes where PTA is the ancillary ligand, only
complexes 9 and 12 exhibited good inhibition. Complexes 13–16,
where 4-picoline are the co-ligand demonstrated consistently high
percent inhibitions, irrespective of the substituents present on the
aromatic ring of the thiosemicarbazone ligand. Overall, six complexes
(9, 12–16) were very effective at inhibiting the growth of the parasite,
displaying inhibition greater than 90%. Based on this, these compounds
were selected for IC50 determination. Table 4 lists the IC50 determina-
tions along with the reference drug Metronidazole, which is the current
FDA approved treatment for T. vaginalis.
Abbreviations
tBu
ESI–MS Electrospray Ionisation Mass Spectrometry
tertiary butyl
4-pic
PTA
saltsc
TMS
TSC
4-picoline
phosphatriazaadamantane
salicylaldiminato thiosemicarbazone
trimethylsilane
thiosemicarbazone
Acknowledgements
The complexes tested displayed IC50 values in the low micromolar
range (17–37 μM). The best activity was displayed by complex 12
(R=5-Cl and L=PTA), where an IC50 value of 17 μM was observed.
Complex 9 (R=H and L=PTA) also displayed good activity giving an
IC50 value of 21 μM. It is apparent that the presence of the 5-Cl moiety
enhances the activity of the complexes as complex 16 (R=5-Cl and
L=4-picoline) also displays the best activity in the series of the
4-picoline complexes, while complex 12 shows the best activity with
regards to the PTA complexes. PTA is a water soluble compound that is
widely used in the preparation of metallotherapeutics as they
enhance the water solubility or overall hydrophilicity of the metal
complex [69,70]. The enhanced activity of these PTA complexes in
comparison to some of the 4-picoline derivatives may be due to the
enhanced hydrophilicity of the phosphane in comparison to the
4-picoline ancillary ligand. The PTA moiety is also able to be protonated
at low pH which may increase the overall hydrophilicity of the complex
which may have a favourable effect on the activity. As previously noted
[18], the mechanism of action of thiosemicarbazones is complex and
may be mediated through inhibition of multiple targets. Some
compounds described in this paper may target T. vaginalis cysteine
proteases while others may act against other cysteine protease targets.
Since there are a number of putative cysteine proteases (as well as other
Financial support from the University of Cape Town, the National
Research Foundation (NRF) of South Africa, and a donation of metal
salts from the Anglo Platinum Corporation is gratefully acknowledged.
KML would like to thank the Department of Biological Sciences and
the Office of Sponsored Programs, the University of the Pacific for
providing funding for this project. P.J.D. was supported by a Hunter-
Nahas Fellowship.
Supplementary material
CCDC 794243 contains the supplementary crystallographic data
for this paper. These data can be obtained free of charge at www.ccdc.
cam.ac.uk/conts/retrieving.html [or from the Cambridge Crystallo-
graphic Data Centre, 12, Union Road, Cambridge CB2 1EZ, UK; fax:
(internat.) +44-1223/336-033; E-mail: deposit@ccdc.cam.ac.uk].
References
[1] S.L. Cudmore, K.L. Delgaty, S.F. Hayward-McClelland, D.P. Petrin, G.E. Garber, Clin.
Microbiol. Rev. 17 (2004) 783–793.
[2] J.R. Schwebke, D. Burgess, Clin. Microbiol. Rev. 17 (2004) 794–803.
[3] R.B. de Oliveira, E.M. de Souza-Fagundes, R.P.P. Soares, A.A. Andrade, A.U. Krettli,
C.L. Zani, Eur. J. Med. Chem. 43 (2008) 1983–1988.

1568
P. Chellan et al. / Journal of Inorganic Biochemistry 105 (2011) 1562–1568
[4] K.J. Duffy, A.N. Shaw, E. Delorme, S.B. Dillon, C. Erickson-Miller, L. Giampa, Y.
Huang, R.M. Keenan, P. Lamb, N. Liu, S.G. Miller, A.T. Price, J. Rosen, H. Smith, K.J.
Wiggall, L. Zhang, J.I. Luengo, J. Med. Chem. 45 (2002) 3573–3575.
[5] M. Abid, S.M. Agarwal, Amir Azam, Eur. J. Med. Chem. 43 (2008) 2035–2039.
[6] A. Walcourt, M. Loyevsky, D.B. Lovejoy, V.R. Gordeuk, D.R. Richardson, Int. J.
Biochem. Cell Biol. 36 (2004) 401–407.
[7] N. Fujii, J.P. Mallari, E.J. Hansell, Z. Mackey, P. Doyle, Y.M. Zhou, J. Gut, P.J.
Rosenthal, J.H. McKerrow, R.K. Guy, Bioorg. Med. Chem. Lett. 15 (2005) 121–123.
[8] X. Du, C. Guo, E. Hansel, P.S. Doyle, C.R. Caffery, T.P. Holler, J.H. McKerrow, F.E.
Cohen, J. Med. Chem. 45 (2002) 2695–2707.
[35] V.M. Leovac, L.S. Jovanović, V.S. Jevtović, G. Pelosi, F. Bisceglie, Polyhedron 26
(2007) 2971–2978.
[36] G. Parameswaran, S. Laly, Asian J. Chem. 5 (1993) 712–718.
[37] B. Garcia, J. Garcia-Tojal, R. Ruiz, R. Gil-Garcia, S. Ibeas, B. Donnadieu, J.M. Leal, J.
Inorg. Biochem. 102 (2008) 1892–1900.
[38] M.B. Ferrari, S. Capacchi, G. Pelosi, G. Reffo, P. Tarasconi, R. Albertini, S. Pinelli, P.
Lunghi, Inorg. Chim. Acta 286 (1999) 134–141.
[39] J.P. Scovill, D.L. Klayman, C.F. Franchino, J. Med. Chem. 25 (1982) 1261–1264.
[40] J. Patole, S. Padhye, M.S. Moodbidri, N. Shirsat, Eur. J. Med. Chem. 40 (2005)
1052–1055.
[9] S.A. Khan, M. Yusuf, Eur. J. Med. Chem. 44 (2009) 2597–2600.
[10] S.A. Khan, P. Kumar, R. Joshi, P.F. Iqbal, K. Saleem, Eur. J. Med. Chem. 43 (2008)
2029–2034.
[11] C. Costello, T. Karpanen, P.A. Lambert, P. Mistry, K.J. Parker, D.L. Rathbone, J. Ren, L.
Wheeldon, T. Worthington, Bioorg. Med. Chem. Lett. 18 (2008) 1708–1711.
[12] D. Kovala-Demertzi, M.A. Demertzis, E. Filou, A.A. Pantazaki, P.N. Yadav, J.R. Miller,
Y. Zheng, D.A. Kyriakidis, Biometals 16 (2003) 411–418.
[13] J.M. Kolesar, W.R. Schelman, P.G. Geiger, K.D. Holen, A.M. Traynor, D.B. Alberti, J.P.
Thomas, C.R. Chitambar, G. Wilding, W.E. Antholine, J. Inorg. Biochem. 102 (2008)
693–698.
[41] P. Chellan, N. Shunmoogam-Gounden, D.T. Hendricks, J. Gut, P.J. Rosenthal, C.
Lategan, P.J. Smith, K. Chibale, G.S. Smith, Eur. J. Med. Chem. (2010) 3520–3528.
[42] T. Stringer, P. Chellan, B. Therrien, N. Shunmoogam-Gounden, D.T. Hendricks, G.S.
Smith, Polyhedron 28 (2009) 2839–2846.
[43] S. Halder, S.M. Peng, G.H. Lee, T. Chatterjee, A. Mukherjee, S. Dutta, U. Sanyal, S.
Bhattacharya, N. J. Chem. 32 (2008) 105–114.
[44] I.D. Kostas, F.J. Andreadaki, D. Kovala-Demertzi, C. Prentjas, M.A. Demertzis,
Tetrahedron Lett. 46 (2005) 1967–1970.
[45] A.P. Kumar, Anal. Lett. 41 (2008) 1022–1037.
[46] I. Yilmaz, Heteroatom Chem. 14 (2003) 617–621.
[14] H.G. Petering, H.H. Buskirk, G.E. Underwood, Cancer Res. 24 (1964) 367–372.
[15] M.-C. Liu, T.-S. Lin, A.C. Sartorelli, J. Med. Chem. 35 (1992) 3672–3677.
[16] I. Dilovic, M. Rubcic, V. Vrdoljak, S.K. Pavelic, M. Kralj, I. Piantanida, M. Cindric,
Bioorg. Med. Chem. 16 (2008) 5189–5198.
[17] K.C. Agrawal, B.A. Booth, E.C. Moore, A.C. Sartorelli, J. Med. Chem. 15 (1972)
1154–1158.
[18] D.C. Greenbaum, Z. Mackey, E. Hansell, P. Doyle, J. Gut, C.R. Caffrey, J. Lehman, P.J.
Rosenthal, J.H. McKerrow, K. Chibale, J. Med. Chem. 47 (2004) 3212–3219.
[19] D.A. Scott, M.J. North, G.H. Coombs, Int. J. Parasitol. 25 (1995) 657–666.
[20] U. Sommer, C.E. Costello, G.R. Beach, R.O. Gilbert, J.J. Lucas, B.N. Singh, J. Biol. Chem.
280 (2005) 23853–23860.
[47] W.A. Hermann, A. Slazer, Synthetic Methods of Organometallic and Inorganic
Chemistry, vol. 1, Georg Thieme Verlag, 1996.
[48] A.M.M. Meij, S. Otto, A. Roodt, Inorg. Chim. Acta 358 (2005) 1005–1011.
[49] Z. Otwinowski, W. Minor, Methods in Enzymology, Macromolecular Crystallog-
raphy, vol. 276, Academic Press, 1997, pp. 307–326, (Part A).
[50] G.M. Sheldrick, SADABS, University of Göttingen, Germany, 1996.
[51] G.M. Sheldrick, SHELXL-97 and SHELXS-97, Program for Crystal Structure
Refinement, University of Göttingen, Germany, 1997.
[52] L.J. Barbour, J. Supramol. Chem. 1 (2001) 189–191.
[53] J. Albert, J. Granell, J. Sales, M. Fon-Bardia, X. Solans, Organometallics 14 (1995)
1393–1404.
[21] J.M. Carlton, R.P. Hirt, J.C. Silva, A.L. Delcher, M. Schatz, Q. Zhao, J.R. Wortman, S.L.
Bidwell, U.C.M. Alsmark, S. Besteiro, T. Sicheritz-Ponten, C.J. Noel, J.B. Dacks, P.G.
Foster, C. Simillion, Y. Van de Peer, D. Miranda-Saavedra, G.J. Barton, G.D. Westrop,
S. Müller, D. Dessi, P.L. Fiori, Q. Ren, I. Paulsen, H. Zhang, F.D. Bastida-Corcuera, A.
Simoes-Barbosa, M.T. Brown, R.D. Hayes, M. Mukherjee, C.Y. Okumura, R.
Schneider, A.J. Smith, S. Vanacova, M. Villalvazo, B.J. Haas, M. Pertea, T.V.
Feldblyum, T.R. Utterback, C.-L. Shu, K. Osoegawa, P.J. de Jong, I. Hrdy, L.
Horvathova, Z. Zubacova, P. Dolezal, S.-B. Malik, J.M. Logsdon Jr., K. Henze, A.
Gupta, C.C. Wang, R.L. Dunne, J.A. Upcroft, P. Upcroft, O. White, S.L. Salzberg, P.
Tang, C.-H. Chiu, Y.-S. Lee, T.M. Embley, G.H. Coombs, J.C. Mottram, J. Tachezy, C.M.
Fraser-Liggett, P.J. Johnson, Science 315 (2007) 207–212.
[54] M. Lopez-Torres, P. Juanatey, J.J. Fernandez, A. Fernandez, A. Suarez, D. Vazquez-
Garcıa, J.M. Vila, Polyhedron 21 (2002) 2063–2069.
[55] R. Schibli, K.V. Katti, W.A. Volkert, C.L. Barnes, Inorg. Chem. 37 (1998) 5306–5312.
[56] P. Smolenski, C. Dinoi, M. Fatima, C. Guedes da Silva, A.J.L. Pombeiro, J. Organomet.
Chem. 693 (2008) 2338–2344.
[57] P. Smolenski, A.J.L. Pombeiro, Dalton Trans. (2008) 87–91.
[58] T.S. Lobana, R. Sharma, G. Bawa, S. Khanna, Coord. Chem. Rev. 253 (2009) 977–1055.
[59] L. Papathanasis, M.A. Demertzis, P.N. Yadav, D. Kovala-Demertzi, C. Prentjas, A.
Castineiras, S. Skoulika, D.X. West, Inorg. Chim. Acta 357 (2004) 4113–4120.
[60] S. Castro-Juiz, M. Lopez-Torres, A. Fernandez, R. Mosteiro, A. Suarez, J.M. Vila, J.J.
Fernandez, Polyhedron 20 (2001) 2925–2933.
[22] W.E. Antholine, J.M. Knight, D.H. Petering, J. Med. Chem. 19 (1976) 339–341.
[23] D. Singh, R.V. Singh, J. Inorg. Biochem. 15 (1993) 227–235.
[24] A. Maiti, A.K. Guha, S. Ghosh, J. Inorg. Biochem. 33 (1988) 57–65.
[25] C.R. Kowol, R. Eichinger, M.A. Jakupec, M. Galanski, V.B. Arion, B.K. Keppler, J.
Inorg. Biochem. 101 (2007) 1946–1957.
[26] Z. Afrasiabi, E. Sinn, W. Lin, Y. Ma, C. Campana, S. Padhye, J. Inorg. Biochem. 99
(2005) 1526–1531.
[27] N.C. Kasuga, K. Sekino, C. Koumo, N. Shimada, M. Ishikawa, K. Nomiya, J. Inorg.
Biochem. 84 (2000) 55–65.
[28] M.B. Ferrari, F. Bisceglie, G. Pelosi, M. Sassi, P. Tarasconi, M. Cornia, S. Capacchi, R.
Albertini, S. Pinelli, J. Inorg. Biochem. 90 (2002) 113–126.
[29] M.A. Ali, S.E. Livingstone, Coord. Chem. Rev. 13 (1974) 101–115.
[30] N.A. Mangalam, M.R.P. Kurup, Spectrochim. Acta A 71 (2009) 2040–2044.
[31] M. Cindrić, M. Rubčić, I. Đilović, G. Giester, B. Kamenar, Croat. Chem. Acta 80
(2007) 583–590.
[61] M.A. Demertzis, S.K. Hadjikakou, D. Kovala-Demertzi, A. Koutsodimou, M. Kubicki,
Helv. Chim. Acta 83 (2000) 2787–2801.
[62] T.S. Lobana, G. Bawa, G. Hundal, A.P.S. Pannu, R.J. Butcher, B.-J. Liaw, C.W. Liu,
Polyhedron 26 (2007) 4993–5000.
[63] V. Dokorou, D. Kovala-Demertzi, J.P. Jasinski, A. Galani, M.A. Demertzis, Helv.
Chim. Acta 87 (2004) 1940–1950.
[64] M.A. Demertzis, P.N. Yadav, D. Kovala-Demertzi, Helv. Chim. Acta 89 (2006)
1959–1970.
[65] D. Kovala-Demertzi, N. Kourkoumelis, M.A. Demertzis, J.R. Miller, C.S. Frampton,
J.K. Swearingen, D.X. West, Eur. J. Inorg. Chem. (2000) 727–734.
[66] S. Halder, R.J. Butcher, S. Bhattacharya, Polyhedron 26 (2007) 2741–2748.
[67] D. Kovala-Demertzi, A. Domopoulou, M.A. Demertzis, G. Valle, A. Papageorgiou, J.
Inorg. Biochem. 68 (1997) 147–155.
[68] Z. Afrasiabi, E. Sinn, S. Padhye, S. Dutta, S. Padhye, C. Newton, C.E. Anson, A.K.
Powell, J. Inorg. Biochem. 95 (2003) 306–314.
[32] P. Ren, T. Liu, J. Qin, C. Chen, Spectrochim. Acta A 59 (2003) 1095–1101.
[33] R.M. El-Shazly, G.A.A. Al-Hazmi, S.E. Ghazy, M.S. El-Shahawi, A.A. El-Asmy, J.
Coord. Chem. 59 (2006) 845–859.
[34] J.S. Casas, M.V. Castano, M.C. Cifuentes, J.C. Garcia-Monteagudo, A. Sanchez, J.
Sordo, U. Abram, J. Inorg. Biochem. 98 (2004) 1009–1016.
[69] A.D. Phillips, L. Gonsalvi, A. Romerosa, F. Vizza, M. Peruzzini, Coord. Chem. Rev.
248 (2004) 955–993.
[70] J. Bravo, S. Bolano, L. Gonsalvi, M. Peruzzini, Coord. Chem. Rev. 254 (2010)
555–607.
[71] D.A. Scott, M.J. North, G.H. Coombs, Int. J. Parasitol. 25 (2005) 657–666.