Structure-activity relationships of mononuclear metal-thiosemicarbazone complexes endowed with potent antiplasmodial and antiamoebic activities

Article abstract of DOI:10.1016/j.bmc.2010.07.039

A useful concept for the rational design of antiparasitic drug candidates is the complexation of bioactive ligands with transition metals. In view of this, an investigation was conducted into a new set of metal complexes as potential antiplasmodium and an

Full text of DOI:10.1016/j.bmc.2010.07.039

Bioorganic & Medicinal Chemistry 18 (2010) 6857–6864
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Structure–activity relationships of mononuclear metal–thiosemicarbazone
complexes endowed with potent antiplasmodial and antiamoebic activities
Deepa Bahl a, Fareeda Athar b, Milena Botelho Pereira Soares c,d, Matheus Santos de Sá c,
a,
Diogo Rodrigo Magalhães Moreira e, Rajendra Mohan Srivastava e, Ana Cristina Lima Leite f, , Amir Azam
*
*
a Department of Chemistry, Jamia Millia Islamia, Jamia Nagar, New Delhi 110025, India
b Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, New Delhi 110025, India
c Center of Research Gonçalo Moniz, Oswaldo Cruz Foundation, Rua Waldemar Falcão, 121, Candeal, Salvador 40296-710, BA, Brazil
d São Rafael Hospital, Av. São Rafael, 2152, São Marcos, Salvador 41253-190, BA, Brazil
e Department of Fundamental Chemistry, Centre for Natural Sciences (CCEN), Federal University of Pernambuco, 50740-540 Recife, PE, Brazil
f Department of Pharmaceutical Sciences, Centre for Health Sciences, Federal University of Pernambuco, 50740-520 Recife, PE, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
A useful concept for the rational design of antiparasitic drug candidates is the complexation of bioactive
ligands with transition metals. In view of this, an investigation was conducted into a new set of metal
complexes as potential antiplasmodium and antiamoebic agents, in order to examine the importance
of metallic atoms, as well as the kind of sphere of co-ordination, in these biological properties. Four func-
tionalized furyl-thiosemicarbazones (NT1–4) treated with divalent metals (Cu, Co, Pt, and Pd) to form the
mononuclear metallic complexes of formula [M(L)2Cl2] or [M(L)Cl2] were examined. The pharmacological
characterization, including assays against Plasmodium falciparum and Entamoeba histolytica, cytotoxicity
to mammalian cells, and interaction with pBR 322 plasmid DNA was performed. Structure–activity rela-
tionship data revealed that the metallic complexation plays an essential role in antiprotozoal activity,
rather than the simple presence of the ligand or metal alone. Important steps towards identification of
Received 10 July 2010
Accepted 16 July 2010
Available online 21 July 2010
Keywords:
Antiparasitic drugs
DNA
Entamoeba histolytica
Metal complexes
Plasmodium falciparum
Thiosemicarbazones
novel antiplasmodium (NT1Cu, IC50 of 4.6 lM) and antiamoebic (NT2Pd, IC50 of 0.6 lM) drug prototypes
were achieved. Of particular relevance to this work, these prototypes were able to reduce the prolifera-
tion of these parasites at concentrations that are not cytotoxic to mammalian cells.
Ó 2010 Published by Elsevier Ltd.
1. Introduction
of valid molecular targets, such as the falcipain-2,8 CDK,9 purine
nucleoside phosphorylases,10 and protein serine/threonine phos-
According to the World Health Organization (WHO), there are
300–500 million clinical cases of malaria each year, resulting in
an alarming rate of about 1.5–2.0 million deaths annually.1 How-
ever, it is estimated that this death rate is likely to increase further
because of the high level of drug resistance to most of the clinically
used antimalarials.2 Given the evidence of the global spread of
drug resistance,3 there is a need for the identification of new anti-
plasmodial drugs. After malaria, amebiasis (caused by Entamoeba
histolytica) is the second leading cause of death from a protozoan
parasite.4 Metronidazole, the only WHO-recommended drug for
treating amebiasis, is toxic and of questionable effectiveness in
eliminating the parasite.5 New safe and affordable amoebicidal
drugs are therefore also urgently needed.
phatases11 of Plasmodium falciparum have contributed to more ra-
tional design of drug candidates. In combination with this
knowledge, the employment of modern concepts of medicinal
chemistry, such as bioisosterism,12 molecular hybridization,13 bio-
inspired design in potent hit-compounds,14 and the metallic com-
plexation of plasmodicidal compounds15 have accelerated the
discovery of antiplasmodium drug candidates.
A significant number of transition metal-containing compounds
have been either recently launched on the pharmaceutical market
or entered into clinical trials, as exemplified by Ferrocifen, NAMI-A,
Picoplatin, Ferroquine, and AMD3100.16 In the specific case of anti-
plasmodium agents, in addition to Ferroquine that has recently
entered in clinical trial, other metallic structures (complexes of
co-ordination and organometallics) have shown promising
in vitro and in vivo properties (Fig. 1).17 It can thus be concluded
that one successful drug development strategy is the complexation
of transition metals with plasmodicidal agents, as it is possible to
enhance the pharmacological and chemical properties (such as po-
tency, selectivity, chemical stability, and lipophilicity) of the anti-
plasmodium agent employed.
The elucidation of metabolic pathways of fundamental impor-
tance (e.g., fatty acid biosynthesis6 and heme detoxification7) and
* Corresponding authors. Tel.: +91 11 26981717x3250; fax: +91 11 26980229
(A.A.).
E-mail addresses: acllb2003@yahoo.com.br (A.C.L. Leite), amir_sumbul@yahoo.
co.in (A. Azam).
0968-0896/$ - see front matter Ó 2010 Published by Elsevier Ltd.
doi:10.1016/j.bmc.2010.07.039

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D. Bahl et al. / Bioorg. Med. Chem. 18 (2010) 6857–6864
(Et)2
Cl
N
Ru
HN
Cl
HN
N
N
N
Cl
Fe
Cl
N
Cl
Cl
Ru
NH
N
Cl
(Et)2
Ru-chloroquine [17c]
Ferroquine [16d]
O
O
H
N
+
-
H
N
N
N
N
H
O
N
Cu
Cl
Cl
Cl
Cl
Ru
N
N
O
S
O
O
O
Cu-naphthoquinone[17d]
NAMI-A [17f]
Figure 1. Examples of metallic compounds endowed with potent anti-Plasmodium falciparum activity.
Studies of thiosemicarbazone-based libraries have proved that
In light of these findings, we decided to investigate the antipro-
tozoal properties of a new set of mononuclear metal–thiosemicar-
bazone complexes. In our design, the furfuryl ring was explored, in
view of previous observation of its antiparasitic properties.26 We
have excluded attachment of the nitro group, as it is well-known
that this induces toxicity, which is a drawback of the medicinal
chemistry of antiparasitic drugs.26c Later, we selected platinum,
palladium, cobalt, and copper, in search of complexes bearing
one (MLCl2) or two (ML2Cl2) ligands on each co-ordination sphere,
thereby aiming to investigate how the ligand sphere contributes to
antiprotozoal activity. Therefore, the main achievement of this
study was to have gathered, for the first time, valuable SAR data
on antiprotozoal metal–thiosemicarbazone complexes.
they are useful ligands for the building of metal complexes with
a wide variety of biological targets, including protozoan para-
sites.8,18 Such ligands constitute an ideal source of bioactive li-
gands because they are endowed with the unique capacity of
metallic co-ordination, semi-labile, chemically stables, and are
synthetically treatable-features which make them suitable for per-
forming structure–activity relationships (SAR) studies.19 It is thus
reasonable to believe that thiosemicarbazones are authentic privi-
leged structures.20
According to this line of reasoning, metal–thiosemicarbazone
complexes previously developed by us were found to improve
anti-E. histolytica activity against the HK-9 and HM1:1MSS strains,
when compared to the metal-free thiosemicarbazones.21 On the
one hand, we demonstrated that the cyclic bioisosters of thiosemi-
carbazones (thiocarboxamide-2-pyrazolines) are less effective
against E. histolytica than thiosemicarbazones, suggesting that the
replacement of a flexible backbone by a more rigid backbone re-
2. Results
2.1. Synthesis and general remarks of structural elucidation
sults in
a
distinct interaction with the protozoan targets.22
The synthesis of ligands NT1–4 was straightforward and pro-
ceeded moderate to good yields (41–90%). NT1–4 were further
used as chelating ligands to complex with [Pd(DMSO)2Cl2],
[Pt(DMSO)2Cl2], CoCl2ꢀ6H2O or CuCl2ꢀ2H2O to generate the respec-
tive mononuclear metal–thiosemicarbazone complexes. Pt (NT1Pt,
NT2Pt) and Pd (NT1Pd–NT4Pd) complexes were obtained as
monomers by heating the ligand and appropriate metallic
precursor under reflux, while the Co (NT1Co–NT4Co) and Cu
Moreover, insertion of bulk groups at the 4-position of thiosemi-
carbazones helps to improve the chemical stability of metal
complexes and their lipophilicity, resulting in more potent anti-
amoebic complexes (Fig. 2).23 Although there is a large number
of thiosemicarbazones endowed with antiplasmodium activity,24
investigations of the antiplasmodium activities for complexes of
co-ordination containing thiosemicarbazones are scarce.25
Insertion of
bulk groups
S
S
S
Classical
bioisosteres
Ar(Het)
N
Ar(Het)
N
H
Ar(Het)
N
N
NH2
N
H
N
H
N
H
N
H
H
N4-substituted thiosemicarbazones
thiosemicarbazones
thiocarboxamide- 2-pyrazolines
Figure 2. Our design concepts for thiosemicarbazones, their bioisosters classical (thiocarboxamide-2-pyrazolines) and their analogs containing bulk groups. Ar(Het) means
either aromatic or heterocyclic rings.

D. Bahl et al. / Bioorg. Med. Chem. 18 (2010) 6857–6864
6859
(NT1Cu–NT4Cu) were obtained as bis-chelated complexes under
similar conditions (Scheme 1). Elemental analysis data (C, H, N,
and Cl) confirmed this metal–ligand ratio and accorded well with
the calculated values.
shifts and the number of hydrogen atoms, in accordance with the
proposed structure, allowing the assignment of each proton for the
complexes. Regarding the ligands, it is worth reporting that even
in DMSO-d6 they were obtained in the thione form, which was
confirmed by the absence of signal at ca. 4.0 ppm (–SH proton).
Regarding the metallic complexes, the CH@N protons resonated at
6.6–7.3 ppm (singlet), owing to a deshielding effect observed for
those protons that are in close proximity to the co-ordinating atoms
(azomethine nitrogen), while, on free ligands, CH@N protons reso-
nated at 7.8–8.0 ppm. On the basis of these comparisons, we suggest
that an N,S-bidentate co-ordination is involved in these complexes,
as proposed in Scheme 1.
Attempts to obtain single crystals of these metallic complexes
suitable for X-ray diffraction were unsuccessful. To overcome this,
additional analyzes were recorded and carefully studied. The mass
spectra of the ligands and their metal complexes have showed the
expected fragment ions, confirming the respective molecular
weights. The overall splitting pathways of selected metal complexes
are summarized in Supplementary data (Scheme S1). According to
the electronic spectra, the NT1–4 ligands exhibited three bands in
The IR spectral signatures of NT1–NT4 thiosemicarbazones
show bands in the region at 1059–1109 cmꢁ1 due to (
mC@S), while
no absorption appeared due to (mC–SH) in the region 2500–
2600 cmꢁ1, suggesting that these NT1–NT4 remain in its thione
tautomeric form. For the metal complexes, although no shift was
observed in the vibrations attributed to (
m
C–O–C) of furan ring,
absorptions attributed to (
m
C@S) at 1059–1109 cmꢁ1 were shifted
to lower frequency (12–30 cmꢁ1) after the metallic complexation,
indicating that thionic sulfur does participate in the sphere of
co-ordination. More notably, strong bands at 1576–1603 cmꢁ1
were observed in all the complexes, which can be attributed to
chelation of (m mCH@N)
C@N) with the metal.19a,26b By contrast, the (
bands for the metal-free ligands are generally of higher frequency
(1613–1615 cmꢁ1) and are of weaker intensity than those ob-
served in the metal complexes. Bands observed in the region of
3100 cmꢁ1
(mN–H) were also slightly shifted in the complexes,
probably by any kind of adjustment of current around the thioam-
ide group. Taking into account previous assignments for similar
metal–thiosemicarbazone complexes, bands corresponding to me-
tal-to-ligand stretching modes were tentatively assigned.21 Thus,
bands in the region of low wave-number (472–522 and 347–
the region at 220–207, 276–263, and 348–330 nm. The most proba-
*
ble assignments for these bands are the n?
p (thiosemicarbazone),
*
*
p?p
(thiophene), and
U
?U
(thiophene) transitions, respectively.
A careful comparison of these bands with the electronic spectra of
metallic complexes showed that there was an increase in intensity
and a decrease in frequency due to the extended conjugation of
ligands after the complexation. The observation of strong bands
between 660 and 560 nm for the Pt (NT1Pt, NT2Pt) and Pd
435 cmꢁ1) were assigned to
mM–N and mM–S, respectively.
The 1H NMR spectra of the ligands and their complexes were con-
sistent with their corresponding protons both in terms of chemical
O
S
S
Cl
R1
ONa
CS2, KOH (1:3)
R1
R1
ONa
N
R
S
N
R
SK
NH
R
EtOH:H2O, reflux
O
O
S
S
O
NH2NH2.H2O
Aq. NaOH
R1
H
R1
NH2
HCl (conc.)
OH
N
R
N
H
N
R
S
EtOH:H2O (1:1)
O
O
Cl
Pd(DMSO)2Cl2 or
Pt(DMSO)2Cl2
M
Cl
N
HN
EtOH, reflux
S
M= PtII or PdII
R
N
S
R1
O
O
R1
N
R
N
R
N
H
N
R1
Cl
M
S
NT1-4
CoCl2 or CuCl2
EtOH, reflux
NH
N
N
HN
Cl
O
S
R
N
M= CuII or CoII
R
NH2
NH
R1
NH
=
R
N
H
N
H
NT-1
NT-2
NT-3
NT-4
Scheme 1. Preparation of ligands (NT1–4) and their metal complexes.

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D. Bahl et al. / Bioorg. Med. Chem. 18 (2010) 6857–6864
(NT1Pd–NT4Pd) complexes were assigned to 1A1g?1A2g that are
typically of a square planar geometry. For these complexes, the
ground state is 1A1g and the excited states are 1A2g, 1B1g, and 1Eg
in order of increasing energy. Intense bands were also observed at
433–470 nm and assignable to a combination of S?Pdk and
1A1g?1B1g transitions.27 The Pt (NT1Pt, NT2Pt) and Pd (NT1Pd–
NT4Pd) complexes were diamagnetic, as expected for a kind of com-
plex that assumes the planar geometry.
inum (NT1Pt, NT2Pt) complexes is not retained in DMSO solution,
taking place an fast transformation of [MLCl2] to [ML(DMSO)Cl]
complexes. All complexes were suspended in DMSO, thereby the
biological activity reflects the presence of (DMSO)chlorometal
complexes for the Pt and Pd ones.34
2.2. Pharmacological and physicochemical assays
For the Co (NT1Co–NT4Co) and Cu (NT1Cu–NT4Cu), two-spin
was observed to allowed transitions at 575–550 nm and at 475–
All the ligands and complexes were tested against the erythro-
cytic stage (bloodstream form of clinical relevance) of P. falciparum
(W2 clone, Chloroquine-resistant and Mefloquine-sensitive), HM-
1:IMSS strain of E. histolytica trophozoites and also for cytotoxicity
using BALB/c mouse splenocytes. The antiprotozoal properties
450 nm and attributed to 4T1g(F)?4T2g(F) ( 1) and 4T1g(F)?4T1g(P)
m
(m3). These transitions are suggestive of an octahedral geometry.
2
Likewise, the 2Eg and T2g microstates of the octahedral Cuk ion
(of configuration d9) split under the influence of the tetragonal dis-
tortion and such distortion causes the transitions 2B1g?2B2g and
2B1g?2A1g, although this remains unresolved in the electronic
spectra.28 These transitions lie within the single broad envelope
centered on the range mentioned above. But these assignments ac-
corded well with the general observation that transitions of the Cuk
d–d kind are often very similar in terms of energy.29 The N–Cuk and
S–Cuk ligand-to-metal charge transfer transitions were also ob-
served at 460 nm (Table S1, supplementary data). The Cu and Co
were expressed in terms of the IC50 (lM) values, the cytotoxicity
being expressed as the highest concentration tested that was
non-cytotoxic for the splenocytes (Table 2). Mefloquine (MQF)
and Metronidazole (MNZ) were used as reference drugs.
Recently, Sanchez-Delgado and co-workers have disclosed a
number of correlations between physicochemical parameters and
the antiplasmodium activity for a number of ruthenium–chloro-
quine complexes.17c,35 From these studies, important insights into
the antiplasmodium action mechanism were provided. Therefore,
the saline/n-octanol partition coefficient (log P), studies with the
pBR 322 plasmid DNA, and the inhibitory effects on the b-hematin
formation for our most potent antiplasmodium metal–thiosemi-
carbazone complexes were also recorded (Table 3, Figs. 3 and 4).
complexes exhibited room temperature magnetic moments (leff
)
values in the range of 1.92–1.97 and 5.05–5.12 B.M., respectively
(Table 1). These values are expected for magnetically diluted Cuk
and Cok complexes having unpaired electrons in the d-orbital.30
The EPR spectra of the Cuk and Cok complexes were measured
and the parameters are summed in Table 1. The general trend
2.3. Structure–activity relationships (SAR)
g > g\ > 2.0 for the Cu complexes suggests that the unpaired elec-
k
tron is located on the d(x2 ꢁ y2) ground state orbitals on the Cuk31
and can also be taken as evidence of an octahedral environment.32
Likewise, the Co complexes exhibiting the g value between 2.05
and 2.08, which also suggests an octahedral environment around
the metal.33 To sum up, a square planar geometry is suggested
for both the Ptk (NT1Pt, NT2Pt) and Pdk (NT1Pd–NT4Pd) com-
plexes, while Cuk complexes (NT1Cu–NT4Cu) and Cok complexes
(NT1Co–NT4Co) probably are of octahedral geometry.
The first analysis of the pharmacological results showed that
the NT1–4 ligands were only weak P. falciparum inhibitors. Like-
wise, they were only modestly active against E. histolytica, the most
potent being NT2, which is still four times less potent than MNZ.
Although it was not possible to draw SAR data from these results,
they displayed generally low cytotoxicity against mammalian cells,
which suggests an attractive application for the building of metal-
lic complexes.
These complexes are insoluble in water, sparingly soluble in
methanol and ethanol, but soluble in DMF and DMSO, producing
intense reddish, greenish or brownish solutions. The 1H NMR spec-
tra of metal complexes in DMSO-d6 remained unchanged at room
temperature for several days, showing no evidence of displace-
ment of the thiosemicarbazones by the solvent. The solid-state
geometry of dichloropalladium (NT1Pd–NT4Pd) and dichloroplat-
As Cu complexes (NT1Cu–NT4Cu) are isoelectronic and iso-
structural with the Co complexes (NT1Co–NT4Co), while a square
planar geometry is adopted by both Pt (NT1Pt, NT2Pt) and Pd
(NT1Pd–NT4Pd) complexes, we concluded that the most appropri-
ate discussion of the biological results is through the comparison
between the similar metals, along the comparative analysis of
the complex and the respective metal-free ligand.
Table 1
Analytical and physical data for the metal complexes
b
Compd
Color
Yielda (%)
Mp (°C)
(
leff)
EPR parametersc
g
g\
g
k
NT1Pt
NT2Pt
NT1Pd
NT2Pd
NT3Pd
NT4Pd
NT1Co
NT2Co
NT3Co
NT4Co
NT1Cu
NT2Cu
NT3Cu
NT4Cu
Yellowish solid
Pale yellow
Pale orange
Brick red
Pale yellow
Pale orange
Brownish solid
Wine red
34
43
56
30
30
55
42
35
33
45
54
51
32
40
260
200
268
257
260
0
0
0
0
0
0
5.10
5.05
5.12
5.07
1.92
1.97
1.92
1.95
—
—
—
—
—
—
—
—
—
—
2.30
2.28
2.10
2.20
—
—
—
—
—
—
—
—
—
—
2.07
2.05
2.05
2.06
—
—
—
—
—
—
2.08
2.05
2.05
2.06
—
—
—
—
220
262
165–170
265
Wine red
Chocolate brown
Greenish solid
Pale green
Greenish solid
Pale orange
240
158
154
160
164
a
b
c
Isolated products.
Effective magnetic moment expressed as Bohr Magneton (B.M.) and recorded at room temperature.
From frozen solutions (77 K) in DMSO.

D. Bahl et al. / Bioorg. Med. Chem. 18 (2010) 6857–6864
6861
Table 2
Biological results of ligands and their metal complexes
Compd complexes
P. falciparum W2 strain
IC50
M) after 24 ha
E. histolytica HM-1:IMSS strain
IC50
M) after 72 ha
Cytotoxicity to mammalian
b
(l
(l
cells (l )
g mLꢁ1
NT1
NT2
NT3
NT4
20.3 0.2
36.2 0.06
21.8 0.4
35.8 0.9
37.0 0.1
42.3 0.6
10.0 0.08
10.9 0.1
18.3 0.3
20.5 0.01
10.12 00.5
8.02 00.1
9.07 00.4
12.08 00.3
3.47 00.1
1.44 00.3
0.99 00.3
0.6 00.5
2.42 00.5
1.66 00.1
2.28 00.1
3.40 00.2
7.50 00.5
2.00 00.3
1.11 00.2
4.05 00.1
1.80 00.2
1.06 00.4
ꢁ
33
100
>100
>100
33
>100
3.3 (7.7)
33
33
33
33
33
33
100
33
11
33
3.3 (5.1)
N.d.
N.d.
NT1Pt
NT2Pt
NT1Pd
NT2Pd
NT3Pd
NT4Pd
NT1Co
NT2Co
NT3Co
NT4Co
NT1Cu
NT2Cu
NT3Cu
NT4Cu
MQNc
MNZc
Nd
41.0 0.4
21.4 0.2
58.7 1.7
4.6 0.1
5.2 0.1
7.8 2.0
4.6 0.1
0.039 0.01
ꢁ
1.80 00.3
N.d., not determined at concentrations tested.
a
Calculated from five concentrations using data obtained from at least three independent experiments. Values are mean standard deviation of
three determinations.
b
Expressed as the highest non-cytotoxic concentration for BALB/c mouse splenocytes. Values given in parentheses are expressed in
MQN is Mefloquine and MNZ is Metronidazole.
lM.
c
Table 3
Inhibitory effects on the b-hematin formation and partition
coefficient (log P) for the copper complexes
a
Compd complexes
IC50
(l
g mLꢁ1
)
log Pb
NT1Cu
NT2Cu
NT3Cu
NT4Cu
CPc
>50
48
43
>50
1.3
ꢁ1.73
ꢁ1.54
ꢁ1.49
ꢁ0.89
—
a
Calculated from six concentrations using data obtained
from two independent experiments. Values mean 1.0.
b
Performed as described in Section 4.
CP is chloroquine diphosphate.
c
Figure 4. Effect of complexes NAMI-A, NT1Cu, NT3Cu, and NT2Pd (50
mobility of pBR 322 plasmid DNA. DNA was incubated in 20
(pH 7.5) to a final volume of 20 L, and incubated in the absence or presence of
lM) on the
lM phosphate buffer
l
tested complexes at 37 °C in the dark for 24 h.
100
showed near IC50 values against P. falciparum. On the one hand,
the Pt complexes showed improved potency against P. falciparum
compared with than the metal-free ligands (NT1 and NT2). On
the other hand, the replacement of Pt by Pd led to only a slight in-
75
50
25
0
flciparum
.
P
crease in potency against P. falciparum (IC50 of 10.0–20.5 lM), far
lower than the potency of MQN, which is active in nanomolar con-
centrations. Apart from NT1Pd, which was cytotoxic at low doses,
the other Pt and Pd complexes retained their low cytotoxicity in
mammalian cells. Noticeably, most of Pt and Pd complexes were
highly potent in inhibiting the growth of E. histolytica, exhibiting
the same range of potency as the reference drug, MNZ. A compar-
ison of the inhibitory activity of ligand NT2 and the NT2Pd com-
plex, the latter proved to be 13 times more potent in inhibiting
the proliferation of E. histolytica. By contrast, the replacement of li-
gand NT2 by NT3 resulted in the NT3Pd complex, which was only
three times more potent than the free-ligand in inhibiting the
growth of E. histolytica.
NT1
PdCl2(DMSO)2
NT1 + Pd
NT1 + Pd
NT1 + Pd
1.0µM
5.0µM
10µM
administration
Figure 3. In vitro inhibition of P. falciparum growth at alone or concomitant
addition of ligand (NT1) plus metal [PdCl2(DMSO)2, represented as Pd]. NT1 was
added at fix concentration of 20 lM, while Pd was used at 10 lM (alone) or at
increasing concentrations (in concomitant with NT1). Data were measured in
triplicate after 24 h of incubation, and the percentages of inhibitions were
established in comparison to non-treated cells (control).
Regarding the antiplasmodial activity, the screening of Co and
Cu complexes demonstrated more promising results than the Pt
and Pd complexes. Analysis of these Co and Cu complexes bearing
two molecules of ligands gave rise to interesting SAR observations.
The complexation of NT1–4 ligands with Co led a new set of
We first analyzed the Pt and Pd series. Although the investiga-
tion of Pt complexes was limited by synthetic considerations and
only two complexes (NT1Pt and NT2Pt) were prepared, these

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D. Bahl et al. / Bioorg. Med. Chem. 18 (2010) 6857–6864
complexes that were (at least) one order of magnitude less active
against P. falciparum (or in some cases virtually inactive, e.g., for
NT1Co and NT4Co) when compared with the metal-free ligands.
Conversely, the complexation with Co resulted in antiamoebic
complexes more potent than the corresponding metal-free ligands.
Regarding the metal-free thiosemicarbazones (IC50 of 20.3–
sured after 24 h of treatment. From the data on Figure 3, it is pos-
sible to suggest that the increasing addition of Pd enhances the
antiplasmodium activity of NT1 ligand at certain point. However,
at high concentrations of Pd (more than 10 lM), no differences of
inhibitions are observed, may because of metal toxicity (data not
shown). Although preliminary, the role of metal facilitating the
intracellular delivery of thiosemicarbazones into cellular compart-
ments may occur in the complexes described here, providing a
means for drug activation or formation of reactive species that
could eventually interact with the protozoan targets.
36.2
lM), their Cu complexes were proven to be good antiplasmo-
dial agents (IC50 of 4.6–7.8
lM), exhibiting the following order of
potency: NT1Cu = NT4Cu > NT2Cu > NT3Cu. Altering the structure
of the cyclohexyl ring on the ligands of C-methyl (NT2Cu) to N-
methyl (NT3Cu) led to a slight reduction in potency (IC50 = 5.2 vs
As previously cited, there are various examples of drug targets of
P. falciparum. Following the findings provided by Sanchez-Delgado
and co-workers, the DNA and hematin were considered as potential
drug targets.35 Therefore, studies of DNA interaction in cell-free
media was conducted, employing pBR 322 plasmid DNA (composed
of the supercoiled form of higher mobility and an open circular re-
laxed form) and CT-DNA (denatured form). We chose three of the
most potent and non-cytotoxic complexes to be tested, NT1Cu,
NT3Cu, and NT2Pd. These complexes are insoluble in pure water,
and DMSO was thus used as a co-solvent in concentrations that do
not affect the CT-DNA. The addition of the metal complexes to the
CT-DNA solution resulted in small changes in the UV–vis absorption
spectra. The observed changes were almost identical in the presence
of each of these three complexes, suggesting that the complexes
interact with CT-DNA in the same way (data not shown).
7.8 lM). Moreover, it was observed that, although the ring expan-
sion of cyclohexyl (NT1Cu) by cycloheptane (NT4Cu) retains the
antiplasmodial activity, NT4Cu was cytotoxic against mammalian
cells.
2.4. Advanced biological experiments
On the basis of the SAR data presented in Table 2, it seems that
the antiplasmodial activity of these complexes is dependent on the
substituents (R) on the structures of thiosemicarbazones (NT1–4).
In general, the lipophilicity of metallic structures is a relevant
descriptor for explaining the cell uptake, and for explaining the dif-
ference in potency between the metal complex and its metal-free
ligand. In the case of anti-malarial complexes, such as Ferroqu-
ine16d and ruthenium–chloroquine,17c,35 the antiplasmodium
activity is governed by the influence of lipophilicity. The lipophil-
icity (log P) of our most potent antiplasmodium complexes
(NT1Cu–NT4Cu) was thus measured, in order to check any kind
of correlation with the biological activity. Comparing the experi-
mental values of log P (Table 3) with the IC50 values against the
W2 strain P. falciparum, a correlation between the potency and
lipophilicity is not observed. However, comparing the lipophilicity
of NT1 (log P = 2.16) ligand and its copper complex (NT1Cu), it is
possible to conclude that the copper complexes are in fact more
hydrophilic than the free thiosemicarbazones.
More valuable informations were gathered by way of agarose
gel electrophoresis assays using pBR 322 plasmid DNA. In this as-
say, the NAMI-A (an imidazole–rutheniumk complex, Fig. 1) of rec-
ognized in vitro antiplasmodium property17e was used, so as to
establish a point of reference for the metal entity. At concentra-
tions below 50 lM, the complexes (NT1Cu, NT3Cu, and NT2Pd)
did not alter either the migration of plasmid DNA bands or other
interaction processes (cleavage, for instance) to a significant de-
gree, while the NAMI-A showed interactions as soon as a concen-
tration of 10 lM was reached. However, at 50 lM, a new pattern
was observed after the incubation of these complexes with plasmid
DNA. Figure 4 suggests that the DNA interaction of these com-
plexes occurs through the conversion of the supercoiled form,
probably to the relaxed form, while the NAMI-A-treated DNA be-
haved quite differently, exploiting the cleavage process.
To ascertain whether complexation with a ligand is an essential
factor for antiprotozoal activity, the same culture of W2 strain
P. falciparum was simultaneously treated with stoichiometric quan-
tities (20 lM) of metal-free ligand NT1 and its metallic precursor
[PdCl2(DMSO)2], with the percentage of cell growth inhibition being
measured after 24 h of treatment. In this condition, only 56% of the P.
falciparum proliferation was inhibited, approximately twice lower
than that of the corresponding PdNT1 complex (98% of inhibition
Inhibition of b-hematin formation was also measured for our
more potent copper complexes (Table 3). All the tested complexes
did not inhibit the b-hematin formation to a significant extent,
while chloroquine did. This suggests these complexes use a differ-
ent action mechanism than quinoline-based antimalarial drugs. In
view of the current problem of resistance to anti-malarial drugs,3 it
is especially important to identify novel anti-malarial drug candi-
dates not based on quinolines.
As a result of these assays, the plasmodicidal action mechanism
must, in part, be related to action on the DNA structure. As for the
antiamoebic properties of these metal complexes, it is not clear
whether the antiamoebic action mechanism involves their interac-
tion with DNA. Although additional experimental data is not avail-
able at present, a very recent survey of the literature indicates that
the sequestration of Fe via either chelator compounds or exchange
of endogenous Fe by exogenous metal (transmetalation) affects
E. histolytica viability.36 In light of these studies, it is reasonable to
propose that, given the recognized ability of thiosemicarbazones
to act as Fe chelators, as well as the feasibility of the complexes de-
scribed here to participate in the processes of dissociation, they may
use this action mechanism to inhibit the growth of E. histolytica.
at 20 lM). Since the bioactivity of simultaneously treatment (ligand
plus metallic precursor) and its corresponding metallic complex are
distinct, the antiplasmodium activity of these complexes is not gov-
erned by synergistic effects alone. Cumulatively, it is supposed that
the formation of a metal complex, rather than the simple presence of
the ligand or transition metal alone, plays a crucial role in determin-
ing its antiprotozoal properties.
Regarding the lability of thiosemicarbazones, Bernhardt and
coworkers have proposed that thiosemicarbazones from metallic
complexes are displaced (dissociated) under physiological condi-
tions or, in other words, the metals act as lipophilic vehicles,
facilitating the intracellular delivery of the metal-free thiosemicar-
bazones into cellular compartments.19b,c In light of these findings,
it is supposed that the processes of precomplexation and dissocia-
tion play a pivotal role in determining the pharmacological
potency for the metallic complexes described here. To gather addi-
tional evidences, additional biological assays were performed with
palladium, which is kinetically more labile than Cu, Co, or Pt.
The same culture of W2 strain P. falciparum was simultaneously
3. Conclusions
treated with metal-free ligand NT1 (20
cursor [PdCl2(DMSO)2] at increasing concentrations (1.0, 5.0, and
10 M), with the percentage of cell growth inhibition being mea-
lM) and its metallic pre-
We were able to identify NT2Pd as the most potent antiamoebic
agent tested in this study, being twice as effective as MNZ. NT2Pd
l

D. Bahl et al. / Bioorg. Med. Chem. 18 (2010) 6857–6864
6863
did not show cytotoxicity against splenocytes at a concentration 10
times higher than that capable of inhibiting E. histolytica growth. In
other words, it was effective at concentrations that do not overtly
affect mammalian cells. This represents the kind of profile, that is,
generally required for antiparasitic agents, with selective toxicity
against parasites, although future investigations in animal models
are necessary.
Our most potent antiplasmodial complex, NT1Cu, is far less po-
tent than MNQ. However, the copper complexes, particularly
NT1Cu and NT2Cu, represent good starting points for further
medicinal chemistry programs aiming to discover antimalarial
drug candidates based on metallic structures. Our future goals
are to study Pd and Cu complexes bearing mixed-ligands to further
validate the hypothesis that the process of dissociation may occur
and could be exploited to produce complexes more active com-
plexes against the aforementioned parasites.
lowed by a small quantity of methanol and dried in a vacuum des-
iccator over anhydrous silica gel to give amorphous solids.
4.1.3. Synthesis of Cuk and Cok complexes of thiosemicarbazones
A stirred solution of hydrated metal chloride (2 mmol) dis-
solved in minimal quantity of methanol was added to a stirring
hot solution of ligand (4 mmol) in methanol (20 mL) and the reac-
tion mixture was heated under reflux for 1–3 h. This solution was
kept to room temperature overnight, when the precipitate was fil-
tered out, washed with hot water followed by small quantity of
methanol, and dried. Recrystallization was carried out from
methanol.
4.2. Pharmacological procedures
4.2.1. Cytotoxicity to mammalian cells
The cytotoxicity of the compounds and metal complexes was
determined using BALB/c mouse splenocytes (5 ꢂ 106 cells well
ꢁ1) cultured in 96-well plates in Dulbecco’s Modified Eagle’s Med-
ium (DMEM, Sigma Chemical Co., St. Louis, MO) supplemented
with 10% of fetal calf serum (FCS; Cultilab, Campinas, SP, Brazil)
4. Experimental section
4.1. Chemistry
and 50
Each compound was evaluated in five concentrations (1.1, 3.3,
11, 33, and 100
g mLꢁ1), in triplicate. Cultures were incubated
in the presence of 3H-thymidine (1 Ci wellꢁ1) for 24 h at 37 °C
l
g mLꢁ1 of gentamycin (Novafarma, Anápolis, GO, Brazil).
General remarks: reactions were monitored by TLC analysis
using Merck pre-coated aluminum plate silica gel 60F254 thin layer
plates. All the chemicals were purchased from Aldrich Chemical
Company (USA). Elemental analysis (C, H, N) was carried out by
Central Drug Research Institute, Lucknow (India). Chlorine was
estimated by decomposing the complexes with Na2O2/NaOH and
precipitating as AgCl with AgNO3 after dissolving in diluted
HNO3. Melting points were recorded on KSW melting point appa-
ratus and are uncorrected. Electronic spectra were recorded in
methanol on a Shimadzu UV-1601 PC UV-visible spectrophotome-
ter. IR spectra on KBr disks were recorded on a Perkin Elmer model
1620 FT-IR spectrophotometer. 1H NMR spectra were obtained at
l
l
and 5% CO2. After this period, the content of the plate was har-
vested to determine the 3H-thymidine incorporation using a
b-radiation counter (Multilabel Reader, Hidex, Turku, Finland).
The cytotoxicity of the compounds was determined comparing
the percentage of 3H-thymidine incorporation (as indicator of via-
bility cell) of drug-treated wells in relation to untreated wells.
Non-cytotoxic concentrations were defined as those causing a
reduction of 3H-thymidine incorporation below 10% in relation to
untreated controls. Note: BALB/c mice were handled according to
the NIH guidelines for animal experimentation. All procedures de-
scribed here had prior approval from the animal ethics committee
of FIOCRUZ (Brazil).
ambient temperature using
a Brucker spectroscopin DPX-
300 MHz spectrophotometer in DMSO-d6 using TMS as an internal
standard. Splitting patterns are designated as follows: s, singlet, d,
doublet, m, multiplet. The FAB mass spectra of all the complexes
were recorded on a JEOL SX 102/DA-6000 Mass Spectroscopy/Data
System, using argon/xenon (6 kV, 10 mA) as the FAB gas and
m-nitrobenzyl alcohol (NBA) as the matrix. Room temperature
magnetic susceptibility was measured at 298 K by a Vibrating sam-
ple Magnetometer 155, E-112 ESR Spectrometer, Varian, USA using
nickel as standard and such values were expressed as effective
4.2.2. Antimalarial activity
It was performed using the [3H]-hypoxanthine incorporation
assay, as previously described.39 Briefly, parasites were maintained
in continuous culture of human erythrocytes (blood group O+)
using RPMI 1640 medium supplemented with 10% human plasma.
Parasites grown at 1–2% parasitemia and 2.5% hematocrit were
incubated with the pure substances tested at five different concen-
trations, diluted with 4% DMSO in culture medium (RPMI 1640)
without hypoxanthine. Cultures containing parasites were har-
vested using a cell harvester to evaluate the [3H]-hypoxanthine
incorporation in a b-radiation counter. Inhibition of parasite
growth was evaluated by comparison with [3H]-hypoxanthine up-
take in drug treated versus untreated wells after 24 h of incubation
with the tested compounds. IC50 values were calculated triplicates,
comparing with the Mefloquine (MQN) as standard drug.
magnetic moment (leff) in Bohr Magneton (B.M.). The electron
paramagnetic resonance (EPR) spectra were recorded with a
Bruker ESP 300E X-band spectrometer.
4.1.1. Synthesis of ligands
The thioglycolic acids were prepared as outlined in reference.37
The thiosemicarbazones were synthesized by heating at reflux an
aqueous solution of respective aminothiocarbonylhydrazines
(0.003 mol in 10 mL) with ethanolic solution of furan-2-carboxal-
dehyde (0.228 g, 0.003 mol in 10 mL) for 3 h with continuous stir-
ring. After cooling, the precipitated compound was filtered and
recrystallized from appropriate solvent.
4.2.3. Antiamoebic activity
Thiosemicarbazones and their metal complexes were screened
against the HM-1:IMSS strain of E. histolytica by using the micro-
plate method.40 All the experiments were carried out in triplicates
at each concentration level and repeated thrice. E. histolytica tro-
phozoites were cultured in TYI-S-33 growth medium in wells of
4.1.2. Synthesis of Pdk and Ptk complexes of thiosemicarbazones
The synthesis of Pdk and Ptk complexes [M(DMSO)2Cl2, where M
is Pd or Pt] were prepared in accordance the procedure outlined in
the literature.38
A solution of [Pd(DMSO)2Cl2]/[Pt(DMSO)2Cl2]
96 well microtiter plates.41 DMSO (40
lL) was added to all the
(2 mmol) dissolved in methanol (5 mL) was added to a solution
of respective ligand (2 mmol) previously dissolved in a minimum
quantity of methanol and the reaction mixture was heated under
reflux for 1–3 h. After keeping the solution at 0 °C overnight, the
colored solid was filtered out. This was washed with hot water fol-
samples (1 mg) followed by enough culture medium to obtain con-
centration of 1 mg/mL. The maximum concentration of DMSO in
the test did not exceeded 0.1%, and at this level no inhibition of
amoebal growth has occurred. Compounds were further diluted
with medium to a concentration of 0.1 mg mLꢁ1. Twofold serial

6864
D. Bahl et al. / Bioorg. Med. Chem. 18 (2010) 6857–6864
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test included Metronidazole (MNZ) as the standard amoebicidal
drug, control (culture medium plus parasite) and a blank (culture
medium only). The cell suspension was then diluted to 105 organ-
ism/mL by adding fresh medium and 170 lL of this suspension was
added to the test and control well in the plate. Plate was sealed and
gassed for 10 min with nitrogen before incubation at 37 °C for 72 h.
After incubation, the growth of amoebae in the plate was checked
with a low power microscope and the optical density of the solu-
tion in each well was determined at 490 nm with a microplate
reader. The% inhibition of amoebal growth was calculated from
the optical densities of the control and test wells and plotted
against the logarithm of the dose of the drug tested. Linear regres-
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This study received support from Council of Scientific and
Industrial Research (grant #01(2278)/08/EMR-II New Delhi, India),
Pernambuco State Foundation of Science (FACEPE, grant #APQ-
0123-4.03/08, Brazil), and Bahia State Foundation of Science
(FAPESB, PRONEX project, Brazil). Diogo Moreira holds a CNPq
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Supplementary data
Supplementary data (compound characterization of the com-
pounds outlined in Scheme 1. Pharmacological protocols con-
ducted can also be found in the Supporting Information)
associated with this article can be found, in the online version, at
doi:10.1016/j.bmc.2010.07.039.
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