Novel potent metallocenes against liver stage malaria

Article abstract of DOI:10.1128/AAC.05345-11

Novel conjugates of the antimalarial drug primaquine (compound 1) with ferrocene, named primacenes, have been synthesized and screened for their activities against blood stage and liver stage malaria in vitro and host-vector transmission in vivo. Both transmission-blocking and blood-schizontocidal activities of the parent drug were conserved only in primacenes bearing a basic aliphatic amine group. Liver stage activity did not require this structural feature, and all metallocenes tested were comparable to or better than primaquine in this regard. Remarkably, the replacement of primaquine's aliphatic chain by hexylferrocene, as in compound 7, led to a ～45-fold-higher level activity against liver stage parasitemia than that of primaquine. Copyright

Full text of DOI:10.1128/AAC.05345-11

Novel Potent Metallocenes against Liver Stage Malaria
Joana Matos,a Filipa P. da Cruz,b Élia Cabrita,c Jiri Gut,d Fátima Nogueira,c Virgílio E. do Rosário,c Rui Moreira,e Philip J. Rosenthal,d
Miguel Prudêncio,b and Paula Gomesa
CIQUP-Centro de Investigação em Química da Universidade do Porto, Departamento de Químicae Bioquímica, Faculdade de Ciências, Universidade do Porto, Porto,
Portugala; Unidade de Malária, Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Lisbon, Portugalb; Centro de Malária e Outras Doenças
Tropicais, IHMT-Universidade Nova de Lisboa, Lisbon, Portugalc; Department of Medicine, San Francisco General Hospital, University of California, San Francisco, California,
USAd; and iMed.UL, CECF, Faculdade de Farmácia, Universidade de Lisboa, Lisbon, Portugale
Novel conjugates of the antimalarial drug primaquine (compound 1) with ferrocene, named primacenes, have been synthesized
and screened for their activities against blood stage and liver stage malaria in vitro and host-vector transmission in vivo. Both
transmission-blocking and blood-schizontocidal activities of the parent drug were conserved only in primacenes bearing a basic
aliphatic amine group. Liver stage activity did not require this structural feature, and all metallocenes tested were comparable to
or better than primaquine in this regard. Remarkably, the replacement of primaquine’s aliphatic chain by hexylferrocene, as in
compound 7, led to a ϳ45-fold-higher level activity against liver stage parasitemia than that of primaquine.
esistance to antimalarials, especially in Plasmodium falcipa- than PQ against liver stage parasites (31), which led us to seek
R
rum, is a serious problem that threatens to undermine the alternatives. Previous reports of enhanced antimalarial activity
efficacy of even the artemisinin components of new first-line upon the introduction of ferrocene-based moieties (4, 7, 9, 10) led
us to synthesize a new set of PQ metallocene derivatives, named
primacenes (compounds 3 to 8) (Fig. 1). These were screened for
their activities in vivo as transmission-blocking agents and in vitro
against liver stage P. berghei malaria parasites. Encouraging results
included the discovery of a metallocene with a level of liver stage
activity about 45-fold higher than that of the parent drug.
artemisinin-based combination therapies (ACTs) (5, 7, 24). In
addition to P. falciparum, Plasmodium vivax malaria is an
increasing source of concern (1, 3, 11, 12, 14, 21, 23, 28, 32, 34).
The benign nature once ascribed to P. vivax is being questioned, as
we have gained an appreciation of the potential for severe and
even fatal P. vivax malaria (1, 11). Furthermore, P. vivax is the
plasmodial species most adaptable to temperate climate condi-
tions (3, 14, 21). The biological robustness of P. vivax parasites is
also reflected in their ability to form hypnozoites, dormant liver
MATERIALS AND METHODS
Chemistry. N␣-protected amino acids were purchased from Bachem
forms that can cause relapses after the initial eradication of blood (Switzerland). Solvents of high quality and thin-layer chromatography
(TLC) aluminum foil plates covered with silica 60 F254 (0.25 mm) and
silica gel 60 (70 to 230 mesh; ASTM) for preparative column chromatog-
raphy were all obtained from Merck (VWR International, Portugal).
When required, solvents were previously dried with preactivated molec-
ular sieves (4 Å), also from Merck. Racemic primaquine bisphosphate
(product no. 160393) and all remaining chemicals were obtained from
Sigma-Aldrich (Germany).
General synthetic procedures and chromatographic/spectroscopic
data were reported elsewhere previously for all compounds (19), except
for compounds 4f, 4g, and 8, for which relevant procedures and data
are available upon request (www.fc.up.pt/pessoas/pgomes/Matos-et-al
_AAC_SuppInfo.pdf).
The purities of all compounds were confirmed to be at least 98%, as
determined by high-performance liquid chromatography (HPLC) using a
Merck Hitachi Elite LaChrom instrument equipped with an L-2130
pump, an L-2200 autosampler, and an L-2455 diode array detector. Sam-
ples were injected onto a Merck Purospher Star RP-18e 125-cm by
4.6-mm (5-␮m) column equipped with a Merck Lichrocart precolumn
(Merck, Germany). Elution programs (eluent A consisted of H2O with
0.05% trifluoroacetic acid [TFA]; eluent B consisted of acetonitrile with
stage parasites (18, 33), a feature shared with Plasmodium ovale
(3). Importantly, hypnozoites elude the action of almost any avail-
able antimalarial drugs, which makes hypnozoite reservoirs a se-
rious obstacle to malaria eradication (33).
Recently, there has been new enthusiasm for malaria eradica-
tion. Eradication efforts will be greatly facilitated by the availabil-
ity of drugs that act against multiple stages of the parasite, includ-
ing the liver forms of all malaria parasites and the dormant liver
hypnozoites of P. vivax and P. ovale (3, 14, 33). Unfortunately,
primaquine (PQ) (compound 1) (Fig. 1) is currently the only
clinically available 8-aminoquinoline active against all liver stages
of plasmodia (3, 30). PQ also offers activity against gametocytes,
thereby blocking transmission to mosquitoes. However, PQ has
low oral bioavailability and is hemotoxic, causing hemolytic ane-
mia in glucose-6-phosphate dehydrogenase (G-6PD)-deficient
individuals after the primary induction of methemoglobinemia
(30). Many PQ derivatives have been developed over the past 2
decades, including tafenoquine and bulaquine, but none, except
for bulaquine in India, has yet been approved for clinical use (30).
With the aim of minimizing the drawbacks of PQ while conserv-
ing its valuable antimalarial properties, we recently developed
imidazoquines (Fig. 1, compound 2, where X is H or an amino
acid residue), which were shown to be promising leads for novel
transmission-blocking antimalarials, given their significant activ-
ity in blocking the transmission of Plasmodium berghei malaria
from mice to mosquitoes and their remarkable stability in physi-
ological media (2, 31). However, imidazoquines are less active
Received 21 July 2011 Returned for modification 30 September 2011
Accepted 4 December 2011
Published ahead of print 12 December 2011
Address correspondence to Paula Gomes, pgomes@fc.up.pt.
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
doi:10.1128/AAC.05345-11
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Potent Metallocenes against Liver Stage Malaria
FIG 1 Synthetic routes for primacenes 3 to 8. (i) Ferrocene-carboxylic acid (FcCOOH) (1 molar equivalent, eq.), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
hydrochloride (EDC.HCl) (1.1 eq.), triethylamine (TEA) (1.1 eq.), and dry dichloromethane (DCM) for 90 min in an ultrasound bath (USB) at room
temperature (RT), to give compounds 3, 5, and 6 (15) (a) or 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium hexafluorophosphate (HBTU) (1 eq.),
N-ethyl-N,N-diisopropylamine (DIEA) (2 eq.), and dry DCM at 0°C at RT for 24 h, to give compounds 4a to 4g. (ii) N␣-tert-Butoxycarbonyl-protected amino
acid succinimidyl ester (Boc-AA-OSu) (1.1 eq.), TEA (1.1 eq.), and dry DCM for 24 h at RT. (iii) Neat trifluoroacetic acid (TFA) for 30 min at RT and then 30%
aqueous (aq.) Na2CO3 dropwise to pH 11, followed by extraction with DCM. (iv) Dry acetone (2 eq. per day), 3-Å molecular sieves, and refluxing methanol for
3 days. (v) Same as described above for step ii but using Boc-Gly-OSu. (vi) SnCl2 (5 eq.) and dropwise concentrated HCl for 24 h at RT and then 8 M aq. NaOH
dropwise to pH 11, followed by extraction with DCM (15). (vii) 6-Bromohexylferrocene (0.6 eq.) and TEA at 120°C for 24 h.
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Matos et al.
0.05% TFA) were as follows: 45 to 60% solvent B in solvent A (0 to 10 RESULTS
min), 60% solvent B in solvent A (10 to 18 min), 60 to 100% solvent B in
Chemistry. Primacenes were prepared, as depicted in Fig. 1, ac-
solvent A (18 to 19 min), and 100% solvent B in solvent A (19 to 23 min)
for compound 4 and 70 to 100% solvent B in solvent A (0 to 5 min) and
100% solvent B in solvent A (5 to 15 min) for compound 8. Nuclear
magnetic resonance (NMR) spectra of compounds dissolved in either
deuterated chloroform (CDCl3) or hexadeuterated dimethyl sulfoxide
(DMSO-d6), containing tetramethylsilane (TMS) as an internal refer-
ence, were acquired on a Bruker AMX-300 spectrometer. Mass spectrom-
etry (MS) was performed by use of an electrospray ionization-ion trap
(ESI-IT) technique with a Finnigan Surveyor LCQ Deca XP Max quadru-
pole mass spectrometer.
Biology. (i) In vivo transmission-blocking activity assays. BALB/c
mice were infected by intraperitoneal inoculations of 107 erythrocytes
parasitized with a green fluorescent protein (GFP)-expressing strain of P.
berghei ANKA. After 4 days, the presence of gametocytes was observed by
the microscopic observation of Giemsa-stained blood films and microg-
amete exflagellation. For each compound and PQ, mice were randomly
separated into groups of three animals, and each group was treated by the
intraperitoneal administration of one single dose of each compound and
PQ (10 and 50 ␮mol/kg of body weight in inoculation volumes of 0.1 to
0.2 ml; controls consisted of infected mice given a phosphate-buffered
saline [PBS] solution). Two hours after administration, mice were anes-
thetized and placed on top of individual cages containing ca. 50 glucose-
starved Anopheles stephensi female mosquitoes, which were allowed to
feed for 1 h. After the blood meal, unfed female mosquitoes were removed
from each cage. Ten days after the blood meal, mosquitoes were dissected
for the microscopic detection of oocysts in midguts.
(ii) In vitro blood-schizontocidal activity assays. In vitro blood-
schizontocidal activity assays were conducted as previously reported (31).
Briefly, synchronized ring stage W2 strain P. falciparum parasites were
cultured with multiple concentrations of test compounds (added from
1,000ϫ stocks in DMSO) in RPMI 1640 medium with 10% human serum
or 0.5% Albumax serum substitute. After 48 h of incubation, when con-
trol cultures contained new rings, parasites were fixed with 1% formalde-
hyde in PBS (pH 7.4) for 48 h at room temperature (RT) and then labeled
with YOYO-1 (1 nM; Molecular Probes) in 0.1% Triton X-100 in PBS.
Parasitemias were determined from dot plots (forward scatter versus flu-
orescence) acquired on a FACSort flow cytometer using CELLQUEST
software (Becton Dickinson). Fifty-percent inhibitory concentrations
(IC50s) for growth inhibition were determined from plots of percentages
of the control parasitemia relative to the inhibitor concentration by use of
GraphPad Prism software. In each case, the goodness of the curve fit was
documented by R2 values of Ͼ0.95.
(iii) In vitro Plasmodium liver stage infection assays. The inhibition
of liver stage infection was determined by measuring the luminescence
intensity in Huh-7 cells infected with a firefly luciferase-expressing P.
berghei line, PbGFP-Luccon, as previously described (27). Huh-7 cells, a
human hepatoma cell line, were cultured in RPMI 1640 medium supple-
mented with 10% (vol/vol) fetal calf serum, 1% (vol/vol) nonessential
amino acids, 1% (vol/vol) penicillin-streptomycin, 1% (vol/vol) glu-
tamine, and 10 mM HEPES (pH 7) and maintained at 37°C with 5% CO2.
For infection assays, Huh-7 cells (1.2 ϫ 104 cells per well) were seeded
cording to previously described methods (19). Detailed synthetic
and spectroscopic data on these compounds were previously re-
ported (19), except for primacenes 4f, 4g, and 8, for which relevant
data are available upon request. In all cases, target compounds
were successfully isolated with a high level of purity, and their
structures were conveniently confirmed.
All compounds whose synthesis involved the coupling of race-
mic primaquine to enantiomerically pure L-amino acids (com-
pounds 4b to 4g) were isolated as mixtures of coeluting diaste-
reomers. All attempts to separate the two diastereomers of
compounds 4b to 4g using column chromatography, flash chro-
matography, and preparative TLC were unsuccessful.
Also, reverse-phase HPLC using Merck RP-8 and RP-18 col-
umns (both 125 and 250 mm) consistently gave single peaks for
derivatives 4b to 4g (not shown). Similar observations were re-
ported previously for the N␣-aminoacyl-primaquine precursors
(2, 31).
Biology. (i) In vivo transmission-blocking activity. The abil-
ities of primacenes 3, 4a, 4f, 4g, 6, and 8 to inhibit the sporogonic
cycle of plasmodia within the mosquito gut were studied by using
a model consisting of P. berghei ANKA-GFP-infected BALB/c
mice and Anopheles stephensi mosquitoes and were compared to
that of PQ (2, 31). Activities were measured at two different com-
pound concentrations by determining the percentage of mosqui-
toes with oocysts (infection rate) and the mean number of oocysts
per infected mosquito (oocyst burden) (Table 1). Although this
model cannot distinguish gametocytocidal (inhibiting the stage
infective for mosquitoes) from sporontocidal (inhibiting mos-
quito stages) activity, it can clearly elucidate whether a compound
is effective at interrupting the transmission of infection from
mammalian hosts to mosquitoes. Of all the compounds tested,
only compounds 4a, 4f, and 8 showed an ability to decrease the
level of parasitemia in mosquitoes. Compound 8 was the only
compound, at a concentration of 50 ␮mol/kg, able to completely
inhibit the sporogonic cycle of Plasmodium.
(ii) In vitro activity against blood stage P. falciparum. Com-
pounds 4f and 8 were evaluated for in vitro antiplasmodial activity
against chloroquine-resistant P. falciparum strain W2 (Table 1), as
previously described for compounds 3, 4a to 4e, and 7 (19). Unlike
what was found previously for compounds 3, 4a to 4e, and 7,
which were completely devoid of blood-schizontocidal activity
(19), compounds 4f (IC50 ϭ 3.48 ␮M) and 8 (IC50 ϭ 1.25 ␮M)
were moderately active, matching or even slightly outdoing the
activity of the parent drug (IC50 ϭ 3.3 ␮M).
(iii) Anti-Plasmodium liver stage activity. The ability of pri-
macenes to inhibit the development of P. berghei schizonts in
into 96-well plates the day before drug treatment and infection. The me- Huh-7 human hepatoma cells was assessed according to previ-
dium in the cells was replaced with medium containing the appropriate
concentration of each compound approximately 1 h prior to infection
with sporozoites freshly obtained through the disruption of the salivary
glands of infected female Anopheles stephensi mosquitoes. The addition of
sporozoites was followed by centrifugation at 1,700 ϫ g for 5 min. At 24 h
postinfection, the medium was replaced with fresh medium containing
the appropriate concentration of each compound. The inhibition of par-
asite development was measured 48 h after infection. The effect of the
compounds on the viability of Huh-7 cells was assessed by the AlamarBlue
assay (Invitrogen, United Kingdom) according to the manufacturer’s
protocol.
ously reported methods (27), as described in Materials and
Methods.
Figure 2 shows the results obtained upon the initial screening
of both cell viability and the inhibition of the P. berghei infection of
Huh-7 cells at two different concentrations. The parent drug, PQ,
was included at 15 ␮M as a positive control. None of the com-
pounds affected cell confluence, as assessed by AlamarBlue fluo-
rescence, except for compound 8 at 2.5 ␮M or above. At lower
concentrations, this compound was not active against liver stage
parasites. All other primacenes (compounds 3 to 7) were active
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Potent Metallocenes against Liver Stage Malaria
TABLE 1 Antiplasmodial activities of primacenes 3 to 8e
Transmission-blocking activity
(infection rate [%]) (mean oocyst
burden [no. of oocysts/infected
mosquito] ϮSEM)b
Charton’s ␯
constants for R1
(ref. 6)
Activity against blood Activity against liver
clogP for model
amides 9a–9fa
stage P. falciparum
W2 (IC50 [␮M])
stage P. berghei
(IC50 [␮M])
Compound R1
10 ␮mol/kg
50 ␮mol/kg
1
3
45.7 (8.8 Ϯ 3.40)
98.3 (46.5 Ϯ 6.10) 88.1 (57.9 Ϯ 8.10) Ͼ10c
33.8 (40.8 Ϯ 10.3) 41.8 (80.2 Ϯ 7.60) Ͼ10c
ND
ND
ND
ND
93.3 (106 Ϯ 4.40) 42.4 (26.0 Ϯ 4.40) 3.48
68.2 (62.5 Ϯ 6.70) 73.2 (57.6 Ϯ 4.00) ND
ND
75.6 (30.2 Ϯ 4.2)
80.0 (70.7 Ϯ 10.3) 95.8 (85.1 Ϯ 7.70) Ͼ10c
26.9 (10.0)d
3.3c
7.50
1.74
9.33
6.46
3.09
1.90
2.40
6.46
ND
7.41
2.82
0.17
ND
4af
4b
4c
4d
4e
4f
4g
5
H
0
Ϫ0.93
Ϫ0.52
0.28
0.74
0.75
Me
0.52
0.76
0.98
0.70
ND
ND
ND
ND
Ͼ10c
Ͼ10c
8.33c
Ͼ10c
iPr
iBu
Bzl
(CH2)4NH2
CH2NH2
Ϫ0.62
ND
86.7 (47.4 Ϯ 4.40) Ͼ10c
Ͼ10c
6
7
8
65.2 (61.7 Ϯ 6.30) 0.00 (0.00)
1.25
a Calculated logarithm of the partition coefficient; calculated by using the OSIRIS Property Explorer (http://www.organic-chemistry.org/prog/peo/).
b See the text.
c Data taken from reference 19.
d Only two data points were available.
e Primaquine (compound 1) is also included for comparison. ND, not determined.
f In 4a to 4g, R1 stands for amino acid side chain; H, hydrogen; Me, methyl; iPr, isopropyl; iBu, isobutyl; Bzl, benzyl.
against liver stage P. berghei, in contrast to their behavior as ence of a basic aliphatic amine group connected to a poly-
transmission-blocking or blood-schizontocidal agents. Pri- methylene chain: in compound 4f, this corresponds to the R1
macenes 4a, 4b, 4f, and 5 presented activities similar to that of PQ,
while primacene 4c had a 2.5-fold-higher level of activity, and
primacene 7 had a 45-fold-higher level of activity (Table 1).
lysine side chain [—(CH2)4NH2], whereas in compound 8, it cor-
responds to the PQ aliphatic amine bound to ferrocene through a
—(CH2)6— spacer. Transmission-blocking activity was lost if a
basic aliphatic amine was absent, as in compounds 3, 6, and 7, or
when this amine was not connected to a polymethylene chain, as
in compound 4g. Thus, it seems that the presence of a —(CH2)n—
NHR (where n is Ͼ1 and R is H or an alkyl group) motif is re-
quired for the preservation of transmission-blocking activity in
PQ-ferrocene conjugates.
Interestingly, a previously reported assessment of the activity
of primacenes 3, 4a to 4e, and 7 against blood stage P. falciparum
(chloroquine-resistant strain W2) also revealed that the moderate
activity of PQ against blood stage malaria was completely lost in
compounds lacking the basic aliphatic amine (19); in turn, the
present study (Table 1) showed that such blood-schizontocidal
activity was recovered in compounds 4f and 8, again with com-
pound 8 performing slightly better than the parent drug.
DISCUSSION
Concerning the ability of primacenes to block host-to-vector ma-
laria transmission, Table 1 shows that only compounds 4a, 4f, and
8 were able to decrease the level of parasitemia in mosquitoes.
However, the activity of compound 4a must be seen skeptically, as
there was no dose-effect dependency. In turn, both compounds 4f
and 8 showed dose-dependent activity, with compound 8 being
able to fully impair the parasite’s sporogonic cycle at 50 ␮mol/kg,
performing better than the parent drug PQ at the same concen-
tration. The common feature of compounds 4f and 8 is the pres-
Overall, there is strong evidence that the absence of
—(CH2)n—NHR (where n is Ͼ1 and R is H or an alkyl group) is
detrimental to the activity of primacenes against both host-to-
vector transmission and blood stage malaria parasites (Table 1).
The relevance for the PQ antimalarial activity of a basic amino
group linked to the 8-aminoquinoline core through a carbon
chain of 2 to 6 carbons was established long ago (20, 25, 30). In
fact, most PQ derivatives that have lately been evaluated in clinical
trials as antiparasitics have the parent drug’s aliphatic chain either
conserved (e.g., tafenoquine) or modified at the amine terminus
in a way that reasonably preserves its basicity (e.g., bulaquine or
sitamaquine) (30). Many reports from different research groups
have demonstrated that the antimalarial activity of PQ is not abol-
ished by the acylation of its primary amine, but in all cases, an-
other basic amine was present elsewhere in the new molecule (2,
FIG 2 Evaluation of the activities of primacenes 3 to 8 against P. berghei liver
stages. Shown are data for the screening of anti-infective activity (bars) and
toxicity to hepatoma cells (line with square markers).
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Matos et al.
FIG 4 General structures of imidazoquines.
infected Huh-7 cells with compound 7 led to a remarkably potent
inhibition of liver stage parasites, as shown in Fig. 2. This exciting
finding was confirmed by the subsequent determination of IC50s
(Table 1), which substantiated that all primacenes have activity
against liver stage parasites equal to or better than that of PQ.
Specifically, compared to PQ, primacenes 4a, 4b, 4f, and 5 were
comparable in activity; primacene 4c had a 2.5-fold-higher level of
activity; and primacene 7 had a 45-fold-higher level of activity
(Table 1).
Another interesting observation was that for the subfamily of
compounds 4a to 4f, where natural amino acids are used as spacers
between PQ and the ferrocenoyl group, the IC50s correlated with
the nature of the amino acid side chains (Fig. 3A). These data
suggest a near-linear dependency between IC50s and Charton’s
steric constants, ␯ (6), for the hydrocarbon side chains present in
compounds 4a to 4e (H, Me, iPr, iBu, and Bzl); however, the phe-
nylalanine derivative compound 4e (Fig. 3A, dashed circle) falls
out of this linear correlation, which may indicate that side-chain
bulkiness is not the only structural factor affecting activity. In fact,
since bulkier hydrocarbon side chains are increasingly lipophilic,
compound activity may depend not (or not only) on their size but
(also) on their lipophilicity. In support of this hypothesis, clogP
values estimated for the model amides 9a to 9f (Fig. 3B), having
the same side-chain variation as that of primacenes 4a to 4f, were
found to correlate linearly (r2 ϭ 0.96) with IC50s for these com-
pounds (Fig. 3B). It is interesting that we have previously found a
similar trend for compounds 4a to 4e in terms of their activities
against another PQ-sensitive pathogen, Pneumocystis jirovecii
(19).
FIG 3 (A) Correlation between IC50s (liver stage activity) (Table 1) and Char-
ton’s steric parameters for amino acid side chains (R1) in compounds 4a to 4e.
(B) clogP values estimated for model amides 9a to 9f, whose side chains (R1)
match those of primacenes 4a to 4f.
The effect of ferrocene on the liver stage activity of primacenes,
compared to the parent drug, is not uniquely demonstrated by the
remarkable activity of compound 7: primacene 6 (IC50 ϭ 2.82
␮M) is not only 2.6-fold more active than PQ (7.5 ␮M) but also
3.5- to 11-fold more active than similar imidazoquines 10 possess-
ing natural amino acids instead of the ferrocenoyl group (31) (Fig.
4). Thus, it is sensible to assume that iron must be an important
player in the tissue-schizontocidal activity displayed by pri-
macenes.
The antimalarial activities of other quinoline-derived metal-
locenes, as ferroquine, are thought to be multifactorial, including
the generation of reactive oxygen species (ROS), like hydroxyl
radicals, under the oxidizing conditions of the food vacuole of
blood stage parasites; these radicals may be produced by a Fenton-
like redox reaction, represented by equation 1 (10):
15–17, 26, 31). Therefore, structural requirements for the activity
of primacenes against both host-to-vector transmission and blood
stage malaria parasites fully agree with previously established
structure-activity relationships (SAR) for PQ. However, any SAR
on PQ-based structures must be taken cautiously, as (i) the mode
of action of PQ is not yet fully understood and (ii) most SAR
reported for PQ-related structures have been taken from in vitro
blood-schizontocidal activity assays (20, 25, 30) and cannot be
used to predict or explain the tissue-schizontocidal activities of
novel PQ derivatives. This is dramatically demonstrated by the
present work, where all primacenes, except for compound 8, were
found to be active against liver stage parasites (Table 1 and Fig. 2).
Therefore, the lack of structural requisites found to be crucial for
the action of primacenes as blood schizontocidals or transmission
blockers was not impeditive to the display of liver stage activity, in
some cases at a notably high level. In particular, the treatment of
Fe2ϩ ϩ H2O2 → Fe3ϩ ϩ HOϪ ϩ HO·
(1)
Although the mechanism of action of PQ has not yet been deter-
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Potent Metallocenes against Liver Stage Malaria
mined, the drug is known to interfere with electron transport in the affect P. vivax hypnozoites, since a suitable in vitro model of these
respiratory chain, possibly leading to oxidative stress and an impair- parasite forms for drug screening is still unavailable (8). Nonetheless,
ment of mitochondrial function (30). Therefore, it is reasonable to the remarkably potent activity of compound 7 makes it a highly
hypothesize that a ferrocene-based moiety, as in primacenes, might promising lead for novel agents against liver stage plasmodia. To the
increase the generation of hydroxyl radicals in the liver, reinforcing bestofourknowledge, theseareunprecedentedfindingsthatpavethe
the ROS-related tissue-schizontocidal activity of PQ. In fact, using a way toward the development of novel primaquine-derived metal-
ferrocene-based compound as an iron donor, Moon et al. recently locenes as highly potent agents against liver stage malaria.
established that iron potentiates acetaminophen-induced oxidative
ACKNOWLEDGMENTS
stress and mitochondrial dysfunction in cultured primary mouse
This work was supported mainly by the Fundação para a Ciência e a
hepatocytes, following earlier reports on the increased production of
Tecnologia (FCT) (Portugal) and FEDER (European Union), reference
hydroxyl radicals and lipid peroxidation in rats with an iron overload
no. PTDC-QUI-65142-2006 and FCOMP-01-0124-FEDER-007418 to
in the liver (22). While this indicates that the presence of iron in
P.G. and reference no. PTDC-BIA-BCM-71920-2006 to M.P. P.G. and
primacenes will eventually bring about a higher level of hepatotoxic-
R.M. thank the FCT for financial support to the CIQUP and iMED.UL-
ity than that of the parent drug PQ, it may also explain why pri-
CECF research units, respectively.
macenes like primacene 7 are so good at killing liver stage malaria
M.P. is a holder of a Ciência 2007 position of the Portuguese Ministry
parasites. Upcoming toxicological studies will provide the grounds to
establish a useful therapeutic window for primacenes; also, bioavail-
abilitystudiesmustbeundertakentoconfirmourbeliefthatthesePQ
derivatives do not undergo the metabolic oxidative deamination that
underlies the low bioavailability of PQ (19, 31). It is possible that a
highly active primacene, such as compound 7, will exhibit a signifi-
cantly improved therapeutic index over that of PQ.
of Science. P.J.R. is a distinguished clinical scientist of the Doris Duke
Charitable Foundation.
Compound mass and nuclear magnetic resonance spectra were ac-
quired on facilities funded by FCT, through projects CONC/REEQ/275/
Qui and REDE/1517/RMN/2005.
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A future definition of therapeutic windows/indices for pri-
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