Applications of electron spin resonance and spin trapping in tropical parasitic diseases.

Abstract

Free radicals may be reaction intermediates in biological systems in more situations than are presently recognized. However, progress in detecting such species by Electron Spin Resonance (ESR) has been relatively slow. ESR is a very sensitive technique for free radical detection and characterization. It can be used to investigate very low concentrations of radicals provided that they are stable enough for their presence to be detected. For unstable radicals special techniques have to be employed. One of these methods is called Spin Trapping. Parasitic diseases in tropical and subtropical areas constitute a major health and economic problem. The range of antiparasitic drugs varies widely in structural complexity and action at the subcellular and molecular levels. However, a number of these drugs are thought to exert their action by generating free radicals. Most of the free radical producing drugs used against parasites are: quinones, naphtoquinones, quinone-imines, aminoquinolines, N-oxides and nitroheterocyclic compounds. This review summarizes some of the more relevant achievements of ESR and Spin Trapping applications in parasitic diseases studies. The use of ESR spectroscopy to obtain relevant information about free radical characterization and the analysis of the mechanisms of action of drugs involved in several parasitic diseases is also presented.

Full text

Page 1

211
Applications of Electron Spin Resonance and Spin Trapping in Tropical
Parasitic Diseases
C. Olea-Azar*1, C. Rigol1, F. Mendizábal2 and R. Briones1
1Departamento de Química Inorgánica y Analítica, Facultad de Ciencias Químicas y Farmacéuticas, Universidad
de Chile
2Departamento de Química, Facultad de Ciencias Universidad de Chile
Abstract: Free radicals may be reaction intermediates in biological systems in more situations than are
presently recognized. However, progress in detecting such species by Electron Spin Resonance (ESR) has been
relatively slow. ESR is a very sensitive technique for free radical detection and characterization. It can be used
to investigate very low concentrations of radicals provided that they are stable enough for their presence to be
detected. For unstable radicals special techniques have to be employed. One of these methods is called Spin
Trapping.
Parasitic diseases in tropical and subtropical areas constitute a major health and economic problem. The range
of antiparasitic drugs varies widely in structural complexity and action at the subcellular and molecular levels.
However, a number of these drugs are thought to exert their action by generating free radicals. Most of the free
radical producing drugs used against parasites are: quinones, naphtoquinones, quinone-imines,
aminoquinolines, N-oxides and nitroheterocyclic compounds.
This review summarizes some of the more relevant achievements of ESR and Spin Trapping applications in
parasitic diseases studies. The use of ESR spectroscopy to obtain relevant information about free radical
characterization and the analysis of the mechanisms of action of drugs involved in several parasitic diseases is
also presented.
Keywords: ESR, spin, trapping, free radical, parasitic diseases.
1. INTRODUCTION
Parasitic diseases in tropical and subtropical areas
constitute an economic problem. Considering the progress of
mankind at the end of the twentieth century, parasitic
diseases are synonymous with ignorance and low level of
education and income. For example Chagas’ disease is
endemic in Latin America, affecting 16-18 million people,
with more than 100 million exposed to the risk of infection
(WHO 1997) [1].
The last three decades research into free radicals has led
to a better understanding of physiological and drug induced
production of free radicals in biological systems, including
the parasites’. The drugs used for treatment in parasitic
diseases are known to act through several mechanisms.
However the most studied drugs, and the ones with
apparently better antiparasitic results are those which involve
a free radical generation mechanism. The families studied as
free radical producing drugs include quinones, quinone-
imines, aminoquinolines and nitroheterocyclic compounds.
The progress in detecting such species by Electron Spin
Resonance (ESR) has been relatively slow. ESR
spectroscopy is a technique that allows the detection and
quantification of paramagnetic species i.e. compounds with
unpaired electrons. It can be used to investigate very low
concentrations of radicals provided that they are stable
*Address correspondence to this author at the Departamento de Química
Inorgánica y Analítica, Facultad de Ciencias Químicas y Farmacéuticas,
Universidad de Chile; Tel: +56-26782834; Fax: +56-2-7370567; E-mail:
colea@uchile.cl
enough for their presence to be detected [2]. In biological
systems the direct detection of free radicals is often not
possible because of their high reactivity and transient nature.
At this time, Spin Trapping is used as a technique to make
this feasible [3,4].
This review summarizes some of the most relevant
achievements of interest about ESR and Spin Trapping
applications in the study of tropical parasitic diseases. We
also present the use of ESR spectroscopy to obtain relevant
information about the mechanism of action of drugs in some
parasitic diseases.
2. ESR AND SPIN TRAPPING GENERAL
PRINCIPLES
2.1 Electron Spin Resonance (ESR)
Paramagnetism arises as a consequence of the presence of
unpaired electrons within an atom or a molecule. It can be
said that Electron Spin Resonance (ESR), often called
Electron Paramagnetic Resonance (EPR), is the most direct
and sensitive technique to investigate paramagnetic
materials.
ESR is similar to Nuclear Magnetic Resonance (NMR),
the fundamental difference being that ESR is concerned with
the magnetically induced splitting of electronic spin states,
while NMR describes the splitting of nuclear spin states. In
both ESR and NMR, the sample material is immersed in a
strong static magnetic field and exposed to an orthogonal
low-amplitude high-frequency field. ESR usually requires
microwave-frequency radiation (GHz), while NMR is

Page 2

212 Olea-Azar et al.
observed at lower radio frequencies (MHz). In ESR
spectroscopy, energy is absorbed by the sample when the
frequency of the radiation is coincident with the energy
difference between two electronic states in the sample, but
only if the transition satisfies the appropriate selection rules.
Splitting can occur only when the electron is in a state with
non-zero total angular momentum, i.e. electrons in atoms
with closed atomic shells cannot show this behavior [2].
For a free electron the spin angular momentum can have
two possible orientations and these give rise to two spin
states of opposite polarity. In the absence of an external
magnetic field the two spin states are degenerate. However,
if an external magnetic field is applied the degeneracy is
lifted, resulting in two states of different energy. This
splitting is called the Zeeman effect. A peak in the
absorption will occur when the magnetic field "tunes" the
two spin states so that their energy difference matches the
energy of the radiation.
Knowledge of the g values and the detailed hyperfine
interactions (a values) allow to identify radical species, and
these parameters contain information about the electron
distribution within the molecule. Radicals are often present
as intermediates during a reaction; consequently their
identification will give information concerning the reaction
mechanism and measurement of how their concentration
changes with time will give kinetic data.
2.2 Spin Trapping
ESR spin trapping techniques have successfully been
applied to determine and identify free radical intermediates
in biology. Spin trapping allows one to determine if short-
lived free radicals are involved as reaction intermediates by
scavenging the reactive radical to produce more stable
radicals, detectable by ESR.
The technique of spin trapping was developed in the late
1960s to facilitate the detection of reactive free radicals by
ESR spectroscopy [5-8]. This method involves the addition
of the spin trap, typically an organic nitrone or nitroso
compound (Fig. 1), to the radical generating system in a
concentration sufficient to ensure rapid reaction with any
radicals present to give stable, detectable, nitroxide radical
adducts. Nitroso spin traps, of which 2-methyl-2-
nitrosopropane (MNP) and 3,5-dibromo-4-nitrosobenzene
sulfonic acid (DBNBS) (Fig. 2) are the most commonly
employed, have the advantage that the reactive radical
attaches directly to the nitroso nitrogen atom, and is
therefore in close proximity to the unpaired electron which is
located primarily on the nitroxide function. This usually
results in the detection of additional distinctive hyperfine
couplings from magnetic nuclei present in the added radical.
The size and nature of these couplings make adduct
identification easier and more definitive. A number of
compilations of data for such adducts are available; see also
the spin trap database at the NIEHS website1. The relatively
small amount of kinetic data available on the rates of
trapping of radicals is also consistent with the rate of
addition of radicals to nitroso traps being more rapid than
1 (http://EPR.niehs.nih.gov)
with nitrones [4,9]. These traps do, however, have the
disadvantage that they form long-living readily detectable
adducts with a more limited range of radicals (usually
limited to carbon-centered species) than nitrone traps. The
nitroso compounds give an adduct in which the radical
added is bonded directly to the nitroxide nitrogen, so that
the hyperfine splittings in the ESR signal are more
diagnostic of the original radical. However the adducts are
less stable than nitrones’ and may decay within minutes.
CH N
R2
R3
O
R
K1
R1
NO
R
K1
R. = .OH, O2.-, LOO., LO., RS., .CCl3, etc
NO
R
R1
Spin Adduct
CHN
R2
R3
O
R
K2
K2
+
Degradation
Products
Spin TrapRadical
+
Degradation
Products
Fig. (1). Mechanism of the addition of free radical with of a
spin trap typically an organic nitrone or nitroso compounds.
N
C(CH3)3
O
N
H3C
H3C
O
N
CH NC(CH3)3
O
O
N
(HOEt)2P
H3C
O
O
(H3C)3C NO
N
Br Br
SO3Na
O
a)
b)
c)
d)
e)
f)
_
_
_
_
_
Fig. (2). Nitroso and nitrone spin traps molecular structure, a)
PBN; b) DMPO; c) POBN; d) DEMPO; e) MNP; f) DBNBS.
In contrast, nitrone spin traps, including 5,5-dimethyl-1-
pyrroline N-oxide (DMPO), 5-diethoxyphosphoryl-5-methyl-
1-pyrroline N-oxide (DEPMPO), N-tert-butyl-α
phenylnitrone (PBN), and α-(4-pyridyl-1-oxide)-N-tert-
butylnitrone (POBN) (Fig. 2), often form long lived adducts
with a wider range of radical species (e.g., carbon-, oxygen-,
sulfur-, and nitrogen-centered species). However, with
nitrone spin traps the reactive radical adds to the carbon
atom adjacent to the incipient nitroxide group, and is
therefore more distant from the molecular orbital containing
the unpaired electron. As a result it is often not possible to
resolve any hyperfine couplings from the added radical itself.
This makes the definitive assignment of the observed
spectral lines to a particular species much more complicated.
However, the magnitude of the hyperfine couplings arising
from the spin trap-derived nitroxide nitrogen and especially
the β-hydrogen, are dependent, particularly with the cyclic
nitrones, on the nature and structure of the added radical as a
result of the influence of this species on the conformation of

Page 3

213
O
O2N
N HNCONH(CH2)3CH3
1
O
O2N
NHNCONH(CH2)6CH3
O
O2N
NHNCOO(CH2)6CH3
OO2N
CONH(CH2)5CH3
4
OO2N
NNSO2
N
N
O2N
NH
O
5b
N
N
CH3
O2N
N
N
S
NH2
N
N
O2N
OH
CH3
O
O2N
N HNCSNHR
OO2N
CHCHNNHCSNHR
N
O
N
CH NNHCONHR
N
R
NH
NH3
N Cl
NH
N
O
O
O
H
H
O
O
O
O
H
H
OCH3
O
O
O
O
SG
2
3
5a
6a
6b
7
8
7,8a R= Ethyl
7,8b R= Phenyl
7,8c R= Methyl
7,8e R= H
9a R= Buthyl
9b R= Hexyl
9c R= 3-(dimethylamino)propyl
9d R= 3-(diethylamino)propyl
10a R= OCH3
10b R= OH
10c
11a
11b
12a
12b
Fig. (3). Molecular structures of various antiparasitic compounds.
the nitrone. The size of these couplings can therefore provide
valuable information on the nature of the radical trapped
[10]. The use of spin traps to detect radicals in biological
systems has been reviewed extensively [4,7].
3. BIOLOGICAL APPLICATIONS OF ESR AND
SPIN TRAPPING TECHNIQUE
As introduced previously a number of antiparasitic agents
have been shown to exert their actions through a free radical
metabolism: nitro and N-oxide compounds used against
trypanosomatids, anaerobic protozoa and helminths; the
antimalarials primaquine, chloroquinine, and quinghaosu;
and quinones active in vitro and in vivo against different
parasites [11].
Nifurtimox (Nfx) and benznidazole ((5a,5b), (Fig. 3) are
currently used to treat Chagas’ disease. In general, the
biological effects of nitroheterocyclic compounds, especially
in T. cruzi, are believe to involve redox cycling of the
compounds and oxygen radical production, two processes in
which the nitroanion radicals play an essential role [12-15].
A characteristic ESR signal corresponding to the nitro
radical appears when Nfx is added to intact Trypanosoma
cruzi cells, the causative agent of Chagas' disease (American
trypanomiasis) [16]. This and other experiments [17] suggest
that intracellular reduction of Nfx followed by redox cycling
yielding O2.- and H2O2, may be the major mode of action
against T. cruzi. However, the use of these drugs has
disadvantageous side effects like fever, muscle weakness,
abdominal or stomach pain, vomiting, etc [18], frequently
forcing the treatment to be stopped. The mechanisms of
these side-effects were not fully understood, particularly in
the case of Nfx. Most studies available on the toxicology of
Nfx correlate the occurrence of Nfx-induced deleterious
effects with the nitroreductive biotransformation of this
nitroheterocyclic compound. However, the hypothesis was
advanced that peroxynitrite formation from Nfx resulting
from the interaction of nitric oxide and superoxide generated
during biotransformation of the Nfx might play a role in Nfx
toxicity [19].
Derivatives (1) and (2) for instance (Fig. 3) showed
interesting in vitro trypanocidal activity but failed to be
good trypanocidal agents in vivo because of their inherent
toxicity (mostly (2)) [13]. The undesirable toxicity of (1)
and (2) on the host is probably due to the nitro moiety
which acts as a nonselective redox-damaging function.
Olea-Azar et al. also reported a series of nitrocompounds
that generated nitro anion radicals, which was proved by
ESR spectroscopy [20,21]. Recently, Olea-Azar et al. [22]
studied new analogues of Nfx ((3), (4) (Fig. 3). The free
radical ESR spectra of these compounds is shown in (Fig.
4). Compound (4) showed better or at least similar
biological activity against T. cruzi than Nfx, and it produced
oxygen redox cycling in T. cruzi epimastigotes. The ESR

Page 4

214 Olea-Azar et al.
Fig. (4). (A) ESR experimental spectrum of the radical-anion of (3) (Fig. (4)) in DMSO and computer simulation of the same spectrum.
(B) ESR experimental spectrum of the radical-anion of (4) in DMSO and computer simulation of the same spectrum.
signal intensities were consistent with the trapping of both
hydroxyl radical ((*) (Fig. 5) and the nitrofurane ((#) (Fig. 5)
derivative radical by DMPO. These results were in
agreement with the observation of increasing the oxygen
uptake caused by the presence of the compound (4) in a T.
cruzi incubation solution, meaning that the anti-Chagas
activity of this compound was achieved by an oxidative
stress mechanism.
Tsuhako et al. [23] studied the bioreductive activation of
nitroimidazole derivative megazol (compound (6a), (Fig. 3)
promoted by ferrodoxin: NADP+ oxidoreductase, rat liver
microsome and cellular fractions of. T. cruzi. Direct ESR
detection and characterization by computer simulation of
megazol anion radical were possible in the presence of
NADPH and ferrodoxin: NADP+ oxidoreductase under
anaerobic conditions. However, the megazol anion radical
was not detected in the presence of either rat liver
microsomes or cellular fractions of T. cruzi. These results
indicate a restricted bioreductive metabolism of megazol and
suggested that the trypanocidal activity is unrelated to a
redox cycling process.
Viode et al. reported that megazol is a highly active
compound used against several strains of T. cruzi. With the
aim of determining the probable mode of action against the
parasite, the interaction of megazol with different redox
enzymes was studied and compared with that of Nfx and
metronidazole (compound (6b), (Fig. 3). The three
nitroaromatic compounds are reduced by L-lactate
cytochrome c-reductase, adrenodoxin reductase, and
NADPH:cytochrome P-450 reductase, the efficiencies of the
enzymatic reductions being roughly related to the reduction
potentials of these pseudo-substrates. As the enzyme
responsible for the reduction of megazol within the parasite
has not yet been identified, the nitroimidazole was assayed
with T. cruzi lipoamide dehydrogenase and trypanothione
reductase. Megazol did not inhibit the physiological

Page 5

215
Fig. (5). ESR spectra of DMPO-OH· and DMPO-compound (4) (Fig. (4)) radical adducts obtained with T. cruzi extracts. The ESR spectra
were observed 10 min after incubation at 37°C with T. cruzi microsomal fraction (4 mg protein/mL), NADPH (1mM), EDTA (1mM), in
phophate buffer (20mM), pH 7,4 and (A) acetonitrile (10 v/v) and DMPO (100mM), (B) Nitro 2 (1mM in acetonitrile 10 v/v) and (C)
Nitro 2 (1mM in acetonitrile 10 v/v) and DMPO (100mM) (DMPO-OH adduct (*): aN=aH = 14.78 G; DMPO-Nitro 2 adduct (#): aN=
15.21 G, aH= 23.48 G. Spectrometer conditions: microwave frequency 9.68 GHz microwave power 20 mW, modulation amplitude
0.4G, scan rate 0.83 G/s , time constant 0.25 s number scans: 10.
reactions but proved to be a weak substrate of both
flavoenzymes. The single electron reduction of this
compound, as well as of Nfx and metronidazole, by
NADPH:cytochrome P-450 reductase, by rat liver as well as
by trypanosome microsomes was confirmed by ESR
experiments. The interest relies in the fact that megazol
interferes with the oxygen metabolism of the parasite as Nfx
and metronidazole, but its extra activity when compared to
Nfx may be related to other features not yet identified [24].
These results are in agreement with Tsuhako’s in terms of
the fact that it seems likely that because of its high
biological activity against T. cruzi, megazol acts on more
than one single target. This acquires particular interest taking
into account the severe side effects of nitrocompounds and
their carcinogenicity.
Aguirre et al., [25] studied the in vitro activity and the
mechanism of action against T. cruzi of 5-nitrofuryl
containing thiosemicarbazones ((7), (8) (Fig. 3). Free radical
production was detected when the compounds were
incubated in presence of mammalian liver microsomes. All
the 5-nitrofuryl thiosemicarbazone derivatives were capable
to produce free radicals in biological medium. So, the
microsomal incubations of all the compounds gave an ESR
spectrum after a brief induction period of 1–2min. The
authors analyzed theoretically the biological behaviour of the
studied compounds. All derivatives showed similar values
of atomic charge on NO2 nitrogen. This fact confirmed the
results obtained in the ESR experiments. The compounds
possess similar electrochemical behaviour, so they could act
biologically in an initial redox pathway.

Page 6

216 Olea-Azar et al.
Fig. (6). A Up: ESR spectrum of (9a) (Fig. (4)) N-oxide derivate generated by microsomal system. Bottom: Computer simulation of the
same spectrum
B Up: ESR spectrum of (9c) (Fig. 4) N-oxide derivate generated by microsomal system. Bottom: Computer simulation of the same
spectrum.
Like the nitro pharmacophore of antitrypanosomal drugs,
the N-oxide moiety has proved to be responsible for the
biological activity of different families of drugs (with
antitumor or antibacterial activities) through the production
of free radical species [26,27].
Cerecetto et al. reported studies on the 1,2,5-oxadiazole
N-oxide family (compound (9), (Fig. 3) in order to
determine their antitrypanosomal activities, tested in vitro
against the epimastigote form of T. cruzi [28]. Moreover,
they have shown ESR spectra that prove the facile
electronation of the N-oxide moiety. Besides, these new
structures were based on the conjunction of N-oxide systems
and the semicarbazide moieties similar to Trypanothione,
substrate of Trypanothione reductase involved in the defense
mechanism of trypanosomatids, against oxidative stress.
Olea-Azar et al. characterized the free radical species of N-
oxide families generated by microsomal reduction, using
ESR spectroscopy (Fig. 6) [29]. The hyperfine splitting
pattern of these biochemically generated free radicals was the
same as that obtained by electrochemical reduction. Also,
the ESR spectra proved that the reduction mechanism of
these compounds involves the protonation of the N-oxide
group, as suggested by the cyclic voltammetric results.
As we can see, oxidative stress might play a key role in
many fatal endpoints caused by other diseases and —at the
same time— it represents a most promising rationale for e.g.
antimalarial chemotherapy. The detoxification of reactive
oxygen species (ROS) is a challenge for erythrocytes infected
with Plasmodia. In this regard it is interesting to note that a
number of drugs currently in clinical use exert their
activities, at least in part, by increasing oxidative stress in
the parasitized erythrocyte [30]. That is the case of
primaquine ((10a) (Fig. 3), an 8-aminoquinoline, which is
the only tissue schizontocide currently available for free
radical treatment of malarial infections. Its utility is
compromised by its toxic effects on erythrocytes, and indeed
primaquine was one of the first agents recognized to produce
oxidative stress [31,32]. Despite its importance, years ago it
was not clear whether the pharmacological effects of
primaquine were due to the parent compound or to its
metabolites. In 1988, Ohara et al. [33] detected, by ESR
spectroscopy, during enzymatic oxidation of primaquine, a
drug-derived radical. The results showed the generation of a
radical species during to the oxidation of primaquine
catalyzed by horseradish peroxidase-H202 or
methemoglobin-H202. A complex product distribution is
expected during aromatic amine oxidations, as the initial
products are more easily oxidized than the parent compound,
undergoing rearrangements and addition reactions. However,
comparison of the obtained ESR parameters with those
reported in the literature indicated that a benzidine-like
rearrangement is the most plausible to happen. The further
generation of a benzidine-like radical in the presence of
nucleophilic groups can lead to further condensation
reactions accounting for the polymeric nature of the reaction
products. A similar spectrum was detected during enzymatic
oxidation of 6-hydroxyprimaquine ((10b) (Fig. 3) at pH 9.0.
Simulations of ESR spectra indicated that the free radicals
contain two primaquine moieties and the authors stated that
this in vitro oxidation of primaquine to a free radical
intermediate stable in the presence of oxygen might be
considered as a new mechanistic route for analyzing the
pharmacological effects of primaquine.
Various mechanisms of action have been found for the
different drugs used in antimalarial chemotherapy. For
example, chloroquine ((10c) (Fig. 3) acts by preventing toxic
haem (ferri/ferroprotoporphyrin IX, FP) detoxification and
its activity can be enhanced by depletion of GSH. In this
work the authors observed that the redox cycling of the
metabolites of primaquine [34] (Fig. 8) exerts a substantial
oxidative stress, also artemisinin ((11a), (Fig. 3) commonly
known as quinghaosu is thought to react with haem moieties
forming cytotoxic radicals [35].

Page 7

217
N
H3CO
NH
NH3
N
HO
NH
NH3
N
HO
NH
NH3
OH
N
H3CO
NH
OH
5-HPQ
N
H3CO
NH2
MAQ
PMQ
5-H-DPQ
5-H-6-DPQ
Fig. (8). Molecular structure of Primaquine metabolites with oxidative activity. PMQ = Primaquine; 5-HPQ = 5-hydroxyprimaquine;
5-H-DPQ = 5-hydroxydemethylprimaquine; 5-H-6-DPQ = 5,6-dihydroxydemethylprimaquine; MAQ = aminoquinoline.
N
O
13a R= H
13b R= OH
13c R= O
R
ON
CH2CH2COOCH3
O
H3C(H2C)11H2C
N
•
HO
OH
N
O
O
NNH
N
13d
14
+
Fig. (7). Molecular structure of spin probes and iron chelator
L2-9.
Deslauriers et al. [36] showed another mechanism of
action concerning primaquine and studied it using ESR.
Erythrocytes from normal mice and mice infected with the
malarial parasite Plasmodium berghei reduce the water-
soluble spin probes, stable paramagnetic compounds,
usually nitroxides, 2,2,6,6-tetramethylpiperidine-N-oxyl
(TEMPO (13a), (Fig. 7), 2,2,6,6-tetramethylpiperidine-4-
hydroxy-N-oxyl (TEMPOL (13b), (Fig. 7), and 2,2,6,6-
tetramethylpiperidine-4-keto-N-oxyl (TEMPONE (13c),
(Fig. 7) at similar rates under both air and N2 atmospheres.
The ESR signal of the lipid-soluble spin probe 5-
doxylstearate ((13d), (Fig. 7) is stable on incorporation into
erythrocytes from normal mice. In contrast, parasitized red
cells reduce this nitroxide probe, at a rate which increases
with the level of parasitemia. Inhibitors of electron transport
such as KCN and NaN3, increase the rate of reduction. It is
proposed that nitroxide reduction occurs via the electron
transport chain in the parasite. The antimalarial drug
primaquine causes reduction of both water-soluble and lipid-
soluble spin probes. This action of primaquine is
independent of its ability to release H2O2 from
oxyhemoglobin. The increased production of NADPH
results in increased rates of reduction of the nitroxide
radicals. Chloroquine, however has no such effect.
Parasitized mice treated with chloroquine six hours prior to
ESR measurements show less nitroxide reducing capacity
than do untreated mice. The metabolic influences of the two
antimalarial drugs are, thus, quite different.
Malaria parasites have been shown to be more susceptible
to oxidative stress than their host erythrocytes. A
chloroquine resistant malaria parasite, Plasmodium
falciparum (FCR-3) was found to be susceptible in vitro to
a pyridoxal based iron chelator-(1-[N-ethoxycarbonylmethyl-
pyridoxylidenium]-2-[2'-pyridyl]hydrazine bromide (code
named L2-9, (14) (Fig. 7). 2 h exposure to 20 microM L2-9
was sufficient to irreversibly inhibit parasite growth.
Desferrioxamine blocked the drug effect, indicating the
requirement for iron. Oxygen however, was not essential.
Spectrophotometric analysis showed that under anoxic
conditions, L2-9-Fe(II) chelate undergoes an intramolecular
redox reaction which presumably involves a one electron
transfer and is expected to result in the formation of free
radicals. Spin trapping coupled to ESR studies of L2-9-iron
chelate showed that L2-9-Fe(II) produced free radicals both
in the presence and absence of cells, while L2-9-Fe(III)
produced free radicals only in the presence of actively
metabolising cells [30].
Another group of molecules, quinones, are naturally
occurring pigments in a variety of plants and fungi, and
some are clinically important antitumor drugs [37]. They are
substrates for flavoenzymes and can undergo either one- or
two- electron reduction, a property of importance in
determining the cytotoxic and antitumor effects of quinones
[38-40].
The bioactivation of exogenous compounds with quinone
structures, like many chemotherapeutic agents, has been
demonstrated to proceed via one-electron reduction to
semiquinone radicals, which, in a redox-cycle with the
quinones under aerobic conditions, may reduce molecular
oxygen to superoxide anions. Redox-cycling of quinones
and their semiquinones is thought to be responsible for the
concomitant oxygen toxicity often observed. Van de Straat
et al. investigated the possible role of cytochrome P-450 in
the one-electron reduction of quinoid compounds as well as

Page 8

218 Olea-Azar et al.
in the formation of reduced oxygen species and used ESR
combined with spin trapping to detect the free radicals
formed [41]. Although menadione (2-methyl-1,4-
naphthoquinone; vitamin K3 (12a) (Fig. 3) is used
therapeutically, it is also cytotoxic and causes a marked
decrease of intracellular thiols such as GSH and protein
sulfhydryl groups and the formation of O2.- in large
amounts. Takahashi et al. [42] reported the identification of
the transient semiquinone-type radicals by ESR spectroscopy
during the non-enzymatic reaction of both menadione ((12a)
(Fig. 3) and 1,4-naphthoquinone with the biological
reducing agents GSH and NADPH. The menadione-induced
loss of cellular thiols occurs by their reaction with these
active oxygen species and by the direct arylation of protein
sulfhydryl groups and GSH. Reaction with GSH forms a
menadione-GSH conjugate (2-methyl-3-S-glutathionyl-1,4-
naphthoquinone; thiodione ((12b) (Fig. 3) at the 3-position
[43]. The authors observed ESR spectra obtained by reaction
of both naphthoquinones and their GSH conjugates in boric
acid-borax buffer, pH 8.5, under a nitrogen atmosphere,
generated by the reaction of Menadione semiquinone with
different concentrations of GSH and of thiodione with
NADPH. The spectra proved to be identical. This experiment
led the authors to discuss that the incubation of menadione
or 1,4-naphthoquinone with the reducing agent, NADPH,
led to the formation of the corresponding semiquinone-free
radical in buffer at pH 7.4,8.5, or 9.0 under a nitrogen
atmosphere. In the presence of GSH as a reducing agent,
menadione and 1,4-naphthoquinone underwent conjugation
with GSH at either or both of the 2- and 3-positions,
depending upon the ratio of the quinone to GSH and net
one-electron reduction to form the corresponding
semiquinones. At lower GSH concentrations only reduction
to the respective semiquinones was detected, as was reported
for 1,4- naphthoquinone [44].
Artemisinin ((11a) (Fig. 3) and its derivatives represent a
very important new class of antimalarials; they are becoming
more and more commonly used throughout the world.
Artemisinin structure is unlike those of any other known
antimalarial and is thus likely to have a different mechanism
of action. The first clue to its mechanism came from
synthetic chemists who demonstrated that the endoperoxide
bridge was necessary for antimalarial activity [45,46]. Since
peroxides are a known source of reactive oxygen species such
as hydroxyl radicals and superoxide [47], this observation
suggested that free radicals might be involved in the
mechanism of action. The role of free radicals in the
biological mechanism of action of artemisinin derivatives
was demonstrated in the late 1980s [48].
The mechanism of action of these compounds appears to
involve the heme-mediated decomposition of the
endoperoxide bridge to produce carbon-centred free radicals.
Reaction of the antimalarial and anti-schistosomal drug
artemether (11b), an artesimnin derivative, (Fig. 3) and
catalytic amount of ferrous ion in the presence of excess
cysteine gave two adducts of cysteine and previously
postulated carbon-centred free radicals that were trapped with
2-methyl-2-nitrosopropane (MNP) and provided the very
first direct evidence for the involvement of radicals in the in
vitro cleavage of artemisinin-type compounds [49]. This
piece of further evidence for the presence of carbon-centered
radicals, especially the secondary carbon-centered free radical
detected for the first time by the isolation of its coupling
adduct, is helpful to understand the mechanism of action of
artemether and other qinghaosu derivatives against parasites
[50].
Two important pathogens of developing countries,
Mycobacterium leprae, the etiologic agent of leprosy, and
Leishmania donovani, the protozoal parasite that causes
kalaazar, persist in the human host primarily in mononuclear
phagocytes. The mechanisms by which they survive in these
otherwise highly cytocidal cells are presently unknown.
Since the best understood cytocidal mechanism of these cells
is the oxygen-dependent system that provides lethal oxidants
including the superoxide anion (O2-.), hydrogen peroxide
(H2O2), hydroxyl radical (OH.), and singlet oxygen (1O2),
Chan et al. [51] sought specific microbial products of these
organisms that might enable them to elude oxidative
cytocidal mechanisms. Phenolic glycolipid I of M. leprae
and lipophosphoglycan of L. donovani are unique cell-wall-
associated glycolipids produced in large amounts by the
organisms. In this study, phenolic glycolipid I derivatives
and lipophosphoglycan were examined for their ability to
scavenge potentially cytocidal oxygen metabolites in vitro.
ESR and spin-trapping indicated that phenolic glycolipid I
derivatives and lipophosphoglycan are highly effective in
scavenging hydroxyl radicals and superoxide anions. The
results suggested that complex glycolipids and carbohydrates
of intracellular pathogens that can scavenge oxygen radicals
may contribute to their pathogenicity and virulence [51].
The cytotoxins produced by phagocytic cells lacking
peroxidases such as macrophages remain elusive. To
elucidate macrophage microbicidal mechanisms in vivo,
Linares et al. compared the lesion tissue responses of
resistant (C57Bl/6) and susceptible (BALB/c) mice to
Leishmania amazonensis infection. This comparison
demonstrated that parasite control relied on lesion
macrophage activation with inducible nitric oxide synthase
expression (iNOS), nitric oxide synthesis, and extensive
nitration of parasites inside macrophage phagolysosomes at
an early infection stage. Nitration and iNOS expression were
monitored by confocal microscopy; nitric oxide synthesis
was monitored by ESR. The main macrophage nitrating
agent was shown to be peroxynitrite-derived because parasite
nitration occurred in the virtual absence of
polymorphonuclear cells (monitored as peroxidase activity)
and was accompanied by protein hydroxylation (monitored
as 3-hydroxytyrosine levels). In vitro studies confirmed that
peroxynitrite is cytotoxic to parasites whereas nitric oxide is
cytostatic. The results indicated that peroxynitrite is likely
to be produced close to the parasites and most of it reacts
with carbon dioxide to produce carbonate radical anion and
nitrogen dioxide whose concerted action leads to parasite
nitration. In parallel, some peroxynitrite decomposition to
the hydroxyl radical should occur due to the detection of
hydroxylated proteins in the healing tissues. Consequently,
peroxynitrite and derived radicals are likely to be important
macrophage-derived cytotoxins [52].
While some groups studying parasitic diseases focus
their work on free radical generation as the mechanisms of
action, some have concentrated on the synthesis of new
pharmaceutical agents by the coordination of antiprotozoal
organic drugs [53]. Others have tested antitumor metal-

Page 9

219
containing complexes such as cisplatin against kinetoplastid
parasites [54]. These studies showed that the tested
compounds display biological activity against protozoa,
results that, along with the fact that many antiprotozoal
drugs bind to DNA leads to expect that in general every
DNA interacting compound could be active against parasites
and that one of the mechanisms possible is through free
radical generation [55].
Similar is the case of paramagnetic complexes
[Cu(dppz)(NO3)]NO3 (15), [Cu(dppz)2(NO3)]NO3 (16),
[Cu(dpq)(NO3)]NO3 (17), and [Cu(dpq)2(NO3)]NO3 (18)
(dppz: dipyrido[3,2-a:2’,3’-c]phenazine; dpq: dipyrido[3,2-
a:2’,3’-h]quinoxaline) whose molecular structure was
characterized by ESR, among other spectroscopies [56].
DNA interaction studies showed that intercalation is an
important way of interacting with DNA for these complexes.
The biological activity of these copper complexes was
evaluated on Leishmania braziliensis promastigotes, and the
results showed leishmanicidal activity. Preliminary
ultrastructural studies with the most active complex (16) at 1
h revealed parasite swelling and binucleated cells. This
finding suggests that the leishmanicidal activity of the
copper complexes could be associated with their interaction
with the parasitic DNA, results in agreement with the ESR
results indicating that the spectra for complexes (15) and
(17) showed a high symmetry in the plane of the copper,
which is indicative of a high electron density in the copper
plane. These results lead to propose a square planar structure
for those complexes. The spectrum of complex (16) showed
an asymmetry in the plane of the copper and the hyperfine
splitting resolution is almost totally lost. The spectrum of
compound (18) showed a slight distortion of the planar
symmetry, with loss of resolution in the lines corresponding
to the hyperfine splitting.
ACKNOWLEDGMENTS
This research was supported by FONDECYT 1030949,
7040037 grant, CONICYT AT-4040020 grant, U. de Chile
DID graduate grant PG-65, and CEPEDEQ.
REFERENCES
[1] (a) Schofeld, C. J. Br. Med. Bull. 1985, 41,187.
(b) http://www.who.int/ctd/chagas/disease.htm
Weil, J. A.; Bolton, J. R.; Wertz, J. E. Electron Paramagnetic
Resonance. Elementary Theory and Practical Applications. John
Wiley & Sons, Inc. 1994.
Rowlands, C. C.; Murphy, D. M. Encyclopedia of Spectroscopy
and Spectrometry, Magnetic Resonance, Chemical Applicatons of
EPR. Elsevier Ltd. 2004, pp. 190-198
Rosen, G.; Britigan, B.; Halpern, H.; Pou, S. Free Radicals,
Biology and Detection by Spin Trapping, Oxford University Press,
1999.
Lagercrantz, C. J. Phys. Chem. 1971, 75, 3466.
Perkins, M. J. Adv. Phys. Org. Chem. 1980, 17, 1.
Janzen, E.G.; Haire, D.L. In Advances in Free Radical Chemistry,
Tanner, D.D.; ed. Greenwich, C.T: JAI Press, 1990, pp. 253-295.
Gilbert, B. C.; Davies, M. J.; Murphy, D. M. Recent developments
in EPR spin trapping. eds. Electron Spin Resonance. Cambridge:
Royal Society of Chemistry, 2002, Vol. 18, pp. 47- 73.
Davies, M. J.; Timmins, G. S.; Clark, R. J. H.; Hester, R. E., EPR
spectroscopy of biologically relevant freer radicals in cellular, ex
vivo, and in vivo systems. Biomedical applications of
Spectroscopy. New York/London: Wiley; 1996, pp. 217- 266.
Buettner, G. R. Free Radic. Biol. Med. 1987, 3, 259.
[2]
[3]
[4]
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
Docampo, R. Chem. Biol. Interact. 1990 , 73, 1.
Cerecetto, H.; Mester, B.; Onetto, S.; Seoane, G.; González, M.;
Zinola, Z. Il Fármaco 1992, 47, 1207.
Cerecetto, H.; Di Maio, R.; Ibarruri, G.; Seoane, G.; Denicola, A.;
Peluffo, G.; Quijano, C. And Paulino, M. Il Farmaco 1998, 53, 89.
Di Maio, R.; Cerecetto, H.; Seoane, G.; Ochoa, C.; Arn, V. J.;
Pérez, E.; Gómez, A.; Muelas, S. and Martínez, A.R. Arzneimittel-
Forsch. Drug. Res. 1999, 49, 759.
Cerecetto, H.; Di Maio, R.; González, M.; Risso, M.; Sagrera, G.;
Seoane, G.; Denicola, A.; Peluffo, G.; Quijano, C.; Basombryo, M.
A.; Stoppani, A. O. M.; Paulino, M. and Olea-Azar, C. Eur. J.
Med. Chem. 2000, 35, 343.
Docampo, R.; Moreno S. N. J. Rev. Infect. Dis. 1984, 6, 223.
Hazra, B.; Sur, P.; Banerjee, A.; Roy D.K. Planta Med. 1984, 51,
295.
Estani, S.S.; Segura, E.L. Mem Inst Oswaldo Cruz, Rio de Janeiro
1999, 94, Suppl. I, 363.
Díaz, E. G.; Montalto de Mecca, M. and Castro, J. A. J. Appl.
Toxicol. 2004, 24, 189.
Olea-Azar, C.; Atria, A.; Di Maio, R.; Seoane, G.; Cerecetto H.
Spectroscopy Lett. 1998, 31, 849.
Olea-Azar, C.; Atria, A.; Mendizabal, F.; Di Maio, R.; Seoane, G.;
Cerecetto, H. Spectroscopy Lett. 1998, 31, 99.
Olea-Azar, C.; Rigol, C.; Mendizàbal, F.; Morello, A.; Maya, J.;
Moncada, C.; Cabrera, E.; Di Maio, R; González, M.; Cerecetto
H. Free Radic. Res. 2003, 37, 993.
Tsuhako, M.; Alves, M.; Colli, W.; Brener, Z.; Augusto, O.
Biochem. Pharm. 1989, 38, 4491.
Viode, C.; Bettache, N.; Cenas, N.; Krauth-Siegel, R.L.;
Chauviere, G.; Bakalara, N.; Perie, J. Biochem. Pharmacol. 1999,
57, 549.
Aguirre, G.; Boiani, L.; Cerecetto, H.; Fernandez, M.; Gonzalez,
M.; Denicola, A.; Otero, L.; Gambino, D.; Rigol, C.; Olea-Azar,
C.; Faundez, M. Bioorg. Med. Chem. 2004, 12, 4885.
Cahill, A.; Whit, I. N. Biochem. Soc. Trans. 1991, 19, 1275.
Brown, J. M. Selective Activation of Drugs by Redox Processes,
Plenum Press, Fermo, Italy 2000; pp. 137-148.
Cerecetto, H.; Di Maio, R.; González, M.; Risso, M.; Saenz, P.;
Seoane, G.; Denicola, A.; Peluffo, G.; Quijano, C.; Olea-Azar, C.
J. Med. Chem. 1999, 42, 1941.
Olea-Azar, C.; Rigol, C.; Mendizábal, F.; Briones, R.; Cerecetto,
H.; Di Maio, R.; González, M.; Porcal, W.; Risso, M.
Spectrochimica Acta Part A 2003, 59, 69.
Iheanacho, E. N.; Sarel, S.; Samuni, A.; Avramovici-Grisaru, S.;
Spira, D. T. Free Radic. Res. Commun. 1991, 15, 1.
Cohen, G.; Hochstein, P. Biochemistry 1964, 3, 895.
Holtzman, J. L. Life Sci. 1981, 30, 1.
Ohara, A.; Schreiber, J.; Mason, R. P. Biochem. Pharmacol. 1988,
37, 2791.
Vasquez-Vivar, J.; Ohara, A. Biochem. Pharmacol. 1994, 47,
309-316.
Becker, K.; Tilley, L.; Vennerstrom, J.; Roberts, D.; Rogerson, S.;
Ginsburg, H. Inter. J. Parasitol. 2004, 34 163.
Deslauriers, R.; Butler, K.; Smith, I. C. Biochim. Biophys. Acta
1987, 931, 267.
Driscoll, J. S.; Hazard, G. F.; Wood, H. B.; Goldin, A. Cancer
Chemoth. Rep. (Part 2) 1974, 4, 1.
Lind, C.; Hochstein, P.; Ernster, L. Arch. Biochem. Biophys. 1982,
216, 178.
Thor, H.; Smith, M. T.; Hartzell, P.; Bellomo, G.; Jewelj, S. A.;
Orrenius, S. J. Biol. Chem. 1982, 257, 12419.
Thor, H.; Smith, M. T.; Hartzell, P.; Orrenius, S. Cytochrome P-
450, Biochemistry,Biophysics, and Environmental Implications.
Elsevier Biomedical Press, New York. 1982; pp. 729-732.
Van de Straat, R.; de Vries, J.; Vermeulen, N. P. E. Biochem.
Pharmacol. 1987, 36, 613.
Takahashi, N.; Schreiber, J.; Fischer, V.; Mason, R. P. Arch.
Biochem. Biophys. 1987, 252, 41.
Wefers, H.; Sies, H. Arch. Biochem. Biophys. 1983, 224, 568.
Gant, T. W.; D’Arcy Doherty, M.; Odowole, D.; Sales, K. D.;
Cohen, G. M. FEBS Lett. 1986, 201, 296-300.
China Cooperative Reserach Group. J. Trad. Chinese Med. 1972,
2, 3.
Brossi, A.; Venugopalan, B.; Dominguez Gerpe L.; Yeh, H. J.;
Flippen-Anderson, J. L.; Buchs, P.; Luo, X. D.; Milhous, W.;
Peters, W. J. Med. Chem. 1988, 31, 64550.
[13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
[23]
[24]
[25]
[26]
[27]
[28]
[29]
[30]
[31]
[32]
[33]
[34]
[35]
[36]
[37]
[38]
[39]
[40]
[41]
[42]
[43]
[44]
[45]
[46]

Page 10

220 Olea-Azar et al.
[47]Halliwell, B.; Gutteridge, J. M. C.; Free Radicals in Biology and
Medicine, Clarendon Press, Oxford. 1999.
Meshnick, S. R. Int. J. Parasitol. 2002, 32 , 1655.
Wen-Min, W.; Yikang, W.; Yu-Lin, W.; Zhu-Jun, Y.; Cheng-
Ming, Z.; Ying L.; Feng, S. J. Am. Chem. Soc. 1998, 120, 3316.
Wen-Min, W.; Yan-Li, C.; Zhai, Z.; Shu-Hua, X.; Yu-Lin, W.
Bioorg. Med. Chem. Lett. 2003, 13, 1645.
Chan, J.; Fujiwara, T.; Brennan, P.; McNeil, M.; Turco, S.; Sibille,
J.; Snapper, M.; Aisen, P.; Bloom, B. Proc. Natl. Acad. Sci. USA
1989 , 86 , 2453.
Linares, E.; Giorgio, S.; Mortara, R.A.; Santos, C.X.; Yamada,
A.T.; Augusto, O. Free Radic Biol. Med. 2001, 30, 1234.
[48]
[49]
[50]
[51]
[52]
[53]Navarro, M.; Lehmann, T.; Cisneros-Fajardo, E. J.; Fuentes, A.;
Sánchez-Delgado, R. A.; Silva, P.; Urbina, J. A. Polyhedron 2000,
19, 2319.
Farrel, N. P. Biochem. Pharmacol. 1984, 33, 961.
Hugo Cerecetto, Mercedes González, Mariela Risso, Patricio
Saenz, Claudio Olea-Azar, Ana M. Bruna, Amaia Azqueta.
Adela López de Ceráin and Antonio Monge. Archiv der
Pharmazie 2004, 337, 271.
Navarro, M.; Cisneros-Fajardo, E.J.; Sierralta, A.; Fernandez-
Mestre, M.; Silva, P.; Arrieche, D.; Marchan, E. J. Biol. Inorg.
Chem. 2003, 8, 401.
[54]
[55]
[56]