Design and evaluation of primaquine-artemisinin hybrids as a multistage antimalarial strategy

Article abstract of DOI:10.1128/AAC.05133-11

It is widely accepted that the struggle against malaria depends on the development of new strategies to fight infection. The "magic bullet" thought to be necessary to reach eradication should not only provide treatment for all Plasmodium spp. that infect

Full text of DOI:10.1128/AAC.05133-11

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Oct. 2011, p. 4698–4706
0066-4804/11/$12.00 doi:10.1128/AAC.05133-11
Copyright © 2011, American Society for Microbiology. All Rights Reserved.
Vol. 55, No. 10
Design and Evaluation of Primaquine-Artemisinin Hybrids as a
Multistage Antimalarial Strategyᰔ†
Rita Capela,1‡ Ghislain G. Cabal,2‡ Philip J. Rosenthal,3 Jiri Gut,3 Maria M. Mota,2
Rui Moreira,1 Francisca Lopes,1* and Miguel Prudˆencio2*
iMed.UL—Faculdade de Farma´cia, Universidade de Lisboa, Av. Prof. Gama Pinto, 1649-003 Lisbon,1 and Unidade de
Mala´ria, Instituto de Medicina Molecular, Faculdade de Medicina da Universidade de Lisboa, Av. Prof. Egas Moniz,
1649-028 Lisbon,2 Portugal, and Department of Medicine, San Francisco General Hospital, University of
California, San Francisco, Box 0811, San Francisco, California 941433
Received 21 June 2011/Returned for modification 18 July 2011/Accepted 26 July 2011
It is widely accepted that the struggle against malaria depends on the development of new strategies to fight
infection. The “magic bullet” thought to be necessary to reach eradication should not only provide treatment
for all Plasmodium spp. that infect human red blood cells but should also eliminate the replicative and dormant
liver forms of the parasite. Moreover, these goals should ideally be achieved by using different mechanisms of
action so as to avoid the development of resistance. To that end, two hybrid molecules with covalently linked
primaquine and artemisinin moieties were synthesized, and their effectiveness against the liver and blood
stages of infection was compared in vitro and in vivo with those of the parent compounds. Both hybrids
displayed enhanced in vitro activities, relative to those of the parent compounds, against Plasmodium berghei
liver stages. Both compounds were about as potent as artemisinin against cultured Plasmodium falciparum
(50% inhibitory concentration [IC50], ϳ10 nM). When used to treat a murine P. berghei infection, one of the
molecules displayed better efficacy than an equimolar mixture of the parent pharmacophores, leading to
improved cure and survival rates. These results reveal a novel approach to the design and evaluation of
antimalarials based on the covalent combination of molecules acting on different stages of the parasite life
cycle.
The combination of pharmacological agents may enable syn-
ergistic interactions, where the efficacy of a single agent is
enhanced by the addition of a second compound. Hybrid (or
bifunctional ligand) compounds, where two pharmacophores
are combined in a single molecule, can be considered an ex-
tension of the concept of drug combinations in which the
challenges of ensuring compliance with complex regimens or
coformulating different compounds into a single tablet can be
avoided. Hybrid molecules have been designed for use in var-
ious therapeutic areas, such as inflammation (6, 16), allergy
(2), and depression (20). Typically, two pharmacophores are
conjugated through a linker unit, creating a single chemical
entity that is able to modulate multiple targets. This covalent
linkage may present advantages over combinations of the two
constituent drugs. These include enhanced uptake of a com-
ponent due to the physicochemical properties of the other
component, stronger synergism between the two components
due to their proximity, or improvement of individual pharma-
cokinetic, stability, or side effect profiles (35).
Malaria remains the most important vector-borne disease in
the world, threatening ϳ40% of the human population and
causing hundreds of millions of infections and about 1 million
deaths each year (39). Malaria is caused by protozoan Plasmo-
dium parasites that infect their mammalian hosts in two con-
secutive stages. The hepatic (liver) stage is initiated when
sporozoites injected through the bite of an Anopheles mosquito
travel to the liver and infect hepatocytes, where a clinically
silent asexual multiplication phase takes place, generating
thousands of merozoites. The release of merozoites into the
bloodstream marks the beginning of the erythrocytic (blood)
stage of infection, during which parasites infect red blood cells
(RBCs), undergo repeated asexual replication cycles, and give
rise to clinical illness (28).
Plasmodium falciparum is responsible for most malaria-as-
sociated mortality worldwide, and it is by far the predominant
species in Africa. The other Plasmodium species that infect
humans are P. vivax, P. ovale, P. malariae, and P. knowlesi. The
asexual blood stages of malaria parasites, which are solely
responsible for illness, are the targets of most available drugs.
Among these, artemisinin (ART) and its derivatives are highly
effective, fast-acting antimalarials and are particularly useful
against strains of P. falciparum resistant to other agents. They
have proven effective against all species capable of causing
human malaria and are generally advocated for the treatment
of uncomplicated P. falciparum malaria as components of
ART-based combination therapies (ACTs) in which the ART
* Corresponding authors. Mailing address for Francisca Lopes:
iMed.UL—Faculdade de Farm´acia, Universidade de Lisboa, Av. Prof.
Gama Pinto, 1649-003 Lisbon, Portugal. Phone: 351 217946476. Fax:
351 217946470. E-mail: fclopes@ff.ul.pt. Mailing address for Miguel
Prudˆencio: Unidade de Mal´aria, Instituto de Medicina Molecular,
Faculdade de Medicina da Universidade de Lisboa, Av. Prof. Egas
Moniz, 1649-028 Lisbon, Portugal. Phone: 351 217999509. Fax: 351
217999504. E-mail: mprudencio@fm.ul.pt.
† Supplemental material for this article may be found at http://aac
.asm.org/.
‡ R.C. and G.G.C. contributed equally to this work.
ᰔ Published ahead of print on 1 August 2011.
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sufficiently pure to be used in the next step (yield, 78%). 1H NMR (CDCl3) ␦
10.02 (1H, s), 7.88 (2H, d, J ϭ 8.0), 7.49 (2H, d, J ϭ 8.0), 5.47 (1H, s), 5.00 (1H,
d, J ϭ 13.6), 4.94 (1H, d, J ϭ 3.6), 4.62 (1H, d, J ϭ 13.6), 2.72 (1H, m), 2.39 (1H,
m), 2.07 (1H, m), 1.94 to 1.80 (3H, m), 1.65 (1H, m), 1.56 to 1.48 (2H, m), 1.47
(3H, s), 1.39 to 1.25 (3H, m), 0.99 (3H, d, J ϭ 7.2), 0.96 (3H, d, J ϭ 6.0). To a
solution of the aldehyde compound 6 (64.0 mg; 0.16 mmol) in methanol (MeOH)
(2 ml) and glacial acetic acid (9.15 ␮l; 0.16 mmol) at 0°C were sequentially added
primaquine, compound 1 as a free base (46 mg; 0.176 mmol), and NaBH3CN (11
mg; 0.176 mmol). The reaction mixture was allowed to warm up to room tem-
perature. After the completion of TLC, the reaction mixture was diluted with
water (10 ml) and acidified to pH ϳ2 to 3 with a 10% (wt/vol) aqueous hydro-
chloric acid solution. This solution was washed with ethyl ether, neutralized with
a 30% (wt/vol) sodium hydroxide solution, and extracted three times with ethyl
ether (10 ml). The combined ether extracts were dried over Na2SO4; the solvent
was removed under reduced pressure; and the residue was purified by prepara-
tive chromatography using ethyl acetate (AcOEt) as an eluent to give the final
compound 7 as a yellow syrup (yield, 39%). 1H NMR (CDCl3) ␦ 8.54 (1H, dd, J ϭ
1.5, 4.2), 7.94 (1H, dd, J ϭ 1.5, 8.2), 7.32 (1H, dd, J ϭ 4.2, 8.2), 7.28 (4H, m), 6.35
(1H, d, J ϭ 2.4), 6.29 (1H, d, J ϭ 2.4), 6.03 (1H, brs), 5.48 (1H, s), 4.92 (1H, d,
J ϭ 4.0), 4.90 (1H, d, J ϭ 12.4), 4.52 (1H, d, J ϭ 12.4), 3.91 (3H, s), 3.81 (2H, brs),
3.62 (1H, m), 3.03 (1H, brs), 2.74 to 2.63 (3H, m), 2.40 (1H, m), 2.11 to 2.01 (2H,
m), 1.95 to 1.56 (7H, m), 1.55 to 1.20 (5H, m), 1.48 (3H, s), 1.31 (3H, d, J ϭ 6.4),
0.95 (6H, brd); m/zϩ 646.38.
(iii) Synthesis of hybrid compound 10. Sodium periodate (278 mg; 1.3 mmol)
and potassium permanganate (31 mg; 0.2 mmol) were added to a stirred solution
of 10␤-allyldeoxoartemisinin, compound 8 (99 mg; 0.32 mmol) in 20 ml acetone,
and 20 ml water at room temperature. On completion of the reaction, the
reaction mixture was filtered, and the filtrate was concentrated under reduced
pressure, treated with 10 M NaOH until basic, and washed with ethyl ether. The
aqueous phase was then acidified to pH 1 with concentrated HCl. The aqueous
phase was extracted three times with ethyl ether (20 ml). The combined organic
extracts were dried over Na2SO4 and were concentrated to give the carboxylic
acid derivative, compound 9, as a yellow oil (yield, 67%). 1H NMR (CDCl3) ␦
5.39 (1H, s), 4.88 (1H, m), 2.77 to 2.63 (2H, m), 2.52 (1H, m), 2.35 (1H, m), 2.1
to 1.92 (2H, m), 1.82 (1H, m), 1.77 to 1.66 (2H, m), 1.48 to 1.2 (5H, m), 1.43 (3H,
s), 0.99 (3H, d, J ϭ 5.6), 0.90 (3H, d, J ϭ 7.6). To a solution of compound 9 (133
mg; 0.407 mmol) in dimethylformamide (3 ml) stirred at 0°C were added TBTU
(142 mg; 0.425 mmol), triethylamine (57 ␮l; 0.409 mmol), and a solution of
primaquine diphosphate salt, compound 1 (183 mg; 0.403 mmol), and triethyl-
amine (115 ␮l; 0.826 mmol) in dimethylformamide (3 ml). The reaction mixture
was allowed to warm up to room temperature, and the reaction was monitored
by TLC. After completion, the reaction mixture was diluted with ethyl acetate
(25 ml) and was then poured into saturated NaHCO3 (25 ml). The layers were
separated, and the aqueous layer was extracted twice with ethyl acetate (25 ml).
The combined organic layers were treated with saturated NaHCO3 and brine and
were then dried over Na2SO4. The solvent was removed, and the crude product
was purified by column chromatography using ethyl acetate–hexane (1:1) to give
compound 10 as a yellow solid (yield, 43%), with a melting point (mp) of 127 to
129°C. 1H NMR (CDCl3) ␦ 8.54 (1H, dd, J ϭ 1.6, 4.0), 7.94 (1H, dd, J ϭ 1.6, 8.0),
7.32 (1H, dd, J ϭ 4.0, 8.0), 7.07 (1H, m), 6.34 (1H, d, J ϭ 2.4), 6.29 (1H, d, J ϭ
2.4), 6.03 (1H, d, J ϭ 8.0), 5.35 (1H, s), 4.75 (1H, m), 3.91 (3H, s), 3.65 (1H, m),
3.44 (1H, m), 3.18 (1H, m), 2.62 to 2.48 (2H, m), 2.37 to 2.23 (2H, m), 2.03 to 1.90
(2H, m), 1.82 to 1.65 (7H, m), 1.30 to 1.10 (5H, m), 1.32 (3H, d, J ϭ 6.4), 1.32
(3H, s), 0.98 (3H, d, J ϭ 5.6), 0.87 (3H, d, J ϭ 7.6). Analysis (C, H, N) calculated
for C32H45N3O6 ⅐ AcOEt: C, 65.93; H, 8.15; N, 6.41. Found: C, 65.21; H, 7.72; N,
6.73; m/zϩ 567.33.
component is partnered with a second, longer-acting agent
(37).
P. vivax, which is about as prevalent as P. falciparum, and P.
ovale uniquely produce chronic liver forms called hypnozoites,
which can remain dormant for extended periods before initi-
ating a blood-stage infection (relapse) (25). Primaquine (PQ)
is the only approved drug against hepatic stages of malaria
parasites, including parasites acutely infecting the liver and
hypnozoites. No other available drugs reliably clear hypnozo-
ites. PQ also acts against sexual stages, known as gametocytes;
this activity disrupts the transmission of infection to mosqui-
toes (21). Thus, PQ is provided in combination with an agent
that clears blood-stage parasites to achieve a radical cure of
infections with P. vivax or P. ovale and thereby prevent relapses
due to the development of subsequent blood-stage infections
from hypnozoites (3).
In this work, we describe the synthesis of hybrid molecules
containing PQ and ART pharmacophoric units. We report on
their efficacies against Plasmodium liver and blood stages, both
in vitro and in animal models of malaria, and demonstrate their
potential as lead compounds for the development of novel
antimalarial drugs.
MATERIALS AND METHODS
Chemical synthesis. (i) General description. Melting points were determined
using a Kofler camera Bock monoscope M and are uncorrected. Nuclear mag-
netic resonance (NMR) spectra were recorded on a Bruker 400 Ultra-Shield
spectrometer. Chemical shifts are reported in ppm using either tetramethylsilane
or the solvent peak as an internal standard. Data are reported as follows:
chemical shift, integration, multiplicity (s, singlet; brs, broad singlet; d, doublet;
t, triplet; dd, double doublet; brd, broad doublet; m, multiplet), and coupling
constant (J), reported in hertz. High-resolution mass spectra were recorded by
electron impact using a Hewlett-Packard HP5988A system (University of Santi-
ago de Compostela, Santiago de Compostela, Spain). Low-resolution mass spec-
tra were recorded using a Micromass Quattro micro API mass spectrometer.
Elemental analyses were performed using a CE Instruments EA 1110 automatic
analyzer (University of Santiago de Compostela). Column chromatography was
performed with a silica gel (230/400 mesh ASTM; Merck), and preparative
thin-layer chromatography (TLC) was performed on silica gel GF254 (Merck).
Analytical TLC was performed on precoated silica gel F254 (Merck). Artemisinin
was purchased from Fraken Company, China, and was 99% pure. Dihydroarte-
misinin and artelinic acid were synthesized from artemisinin according to refer-
ence 5. 10␤-Allyldeoxoartemisinin was prepared according to reference 14. All
the other reagent-grade chemicals were bought from Sigma-Aldrich (Spain),
Merck (Spain), or Alfa Aesar (Spain). Tetrahydrofuran (THF) was dried by
distillation from sodium benzophenone.
(ii) Synthesis of hybrid compound 7. A suspension of O,N-dimethylhydroxy-
lamine hydrochloride (90.4 mg; 0.93 mmol) in dichloromethane (1 ml) and
triethylamine (129 ␮l; 0.93 mmol) was added to a solution of artelinic acid,
compound 4 (352 mg; 0.84 mmol) in dichloromethane (3 ml), O-(benzotriazol-
1-yl)-N,N,NЈ,NЈ-tetramethyluronium tetrafluoroborate (TBTU) (270 mg; 0.84
mmol), and triethylamine (118 ␮l; 0.84 mmol). After stirring for 7 h, the reaction
mixture was diluted with dichloromethane (20 ml), and the organic phase was
washed with saturated NaHCO3, treated with brine, and dried with anhydrous
Na2SO4. Removal of the solvent under reduced pressure gave the hydroxamate
compound 5 as a yellow oil (yield, 91%). 1H NMR (CDCl3) ␦ 7.68 (2H, d, J ϭ
8.0), 7.37 (2H, d, J ϭ 8.0), 5.48 (1H, s), 5.02 to 4.90 (2H, m), 4.58 (1H, d, J ϭ
12.8), 3.58 (3H, s), 3.39 (3H, s), 2.71 (1H, m), 2.40 (1H, m), 2.07 (1H, m), 1.93
to 1.82 (3H, m), 1.66 (1H, m), 1.57 to 1.43 (2H, m), 1.48 (3H, s), 1.37 to 1.23 (3H,
m), 0.98 (3H, d, J ϭ 6.4), 0.97 (3H, d, J ϭ 5.6). Lithium aluminum hydride (33.6
mg; 0.89 mmol) was added to a solution of the hydroxamate compound 5 (327
mg; 0.71 mmol) in dry tetrahydrofuran (8 ml) at 0°C. The reaction mixture was
stirred for 1 h at 0°C, after which it was quenched using a solution of KHSO4 (191
mg; 1.4 mmol) in water (2 ml), diluted with water (40 ml), and extracted three
times with ethyl ether (30 ml). The combined organic extracts were washed with
saturated NaHCO3 and brine, dried (with anhydrous NaSO4), and evaporated
under reduced pressure to give the aldehyde compound 6 as a colorless oil
Parasites, cells, and mice. In vitro blood schizonticidal activity assays were
performed as reported elsewhere (8). Briefly, synchronized ring-stage P. falcip-
arum strain W2 parasites were cultured with multiple concentrations of test
compounds (added from 1,000ϫ stocks in dimethyl sulfoxide [DMSO]) in RPMI
1640 medium with 10% human serum. After 48 h of incubation, when control
cultures contained new rings, parasites were fixed with 1% formaldehyde in
phosphate-buffered saline (PBS), pH 7.4, for 48 h at room temperature; then
they were labeled with YOYO-1 (1 nM; Molecular Probes) in 0.1% Triton X-100
in PBS. Parasitemia was determined from dot plots (forward scatter versus
fluorescence) acquired on a FACSort flow cytometer using CellQuest software
(Becton Dickinson). Fifty percent inhibitory concentrations (IC50s) for growth
inhibition were determined with GraphPad Prism software from plots of the
percentage of parasitemia of the control relative to the inhibitor concentration.
In each case, the goodness of the curve fit was documented by R2 values of Ͼ0.95.
Plasmodium berghei ANKA sporozoites expressing luciferase or green fluores-
cent protein (GFP) (parasite lines 676m1cl1 and 259cL2, respectively), were
obtained from 21- to 28-day infected female Anopheles stephensi mosquitoes (22,

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CAPELA ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
26). Mosquitoes bred at the Insectary of the Instituto de Medicina Molecular,
Lisbon, Portugal, were previously infected by feeding on infected mice using
standard methods. When required, the salivary glands of the mosquitoes were
collected by hand dissection in Dulbecco’s modified Eagle medium (DMEM;
GIBCO), homogenized with a plastic pestle, and filtered on a cell strainer
(70-␮m-pore-size nylon; BD Falcon). The free sporozoites were counted in a
Neubauer counting chamber using phase-contrast microscopy. For infections by
mosquito bite, infected mosquitoes were starved overnight and were then al-
lowed to feed on anesthetized mice for 30 min, at a proportion of 10 mosquitoes
per mouse.
phosphoribosyltransferase (HGPRT) housekeeping gene. Primer sequences spe-
cific to each gene were as follows: for the P. berghei ANKA 18S rRNA gene,
5Ј-AAG CAT TAA ATA AAG CGA ATA CAT CCT TAC-3Ј and 5Ј-GGA
GAT TGG TTT TGA CGT TTA TGT G-3Ј; for the mouse HGPRT gene,
5Ј-TGC TCG AGA TGT GAT GAA GG-3Ј and 5Ј-TCC CCT GTT GAC TGG
TCA TT-3Ј; and for the human HGPRT gene, 5Ј-TGC TCG AGA TGT GAT
GAA GG-3Ј and 5Ј-TCC CCT GTT GAC TGG TCA TT-3Ј.
Assessment of parasitemia. Giemsa staining followed by microscopic analysis
was performed daily to assess the presence or absence of parasites in the blood,
and parasitemia was quantified by flow cytometry as previously described (26).
Cells of the Huh7 human hepatoma cell line were cultured in RPMI medium
(Gibco/Invitrogen) supplemented with 10% fetal calf serum, 1% nonessential
amino acids, 1% penicillin-streptomycin, 1% glutamine, and 10 mM HEPES (pH
7) (all from Gibco/Invitrogen) and were maintained at 37°C under 5% CO2. For
infection, P. berghei sporozoites obtained from freshly dissected mosquito sali-
vary glands were added directly to the culture medium, and cell culture plates
were centrifuged for 5 min at 1,800 ϫ g to promote contact of the parasites with
the Huh7 cell monolayer.
RESULTS
Design and synthesis of primaquine-artemisinin hybrids.
Hybrid compound 7 was synthesized by starting from artelinic
acid (Fig. 1), a derivative of ART previously used in our lab-
oratory to prepare hybrid molecules, including vinyl sulfone
inhibitors of falcipain-2 (5). Artelinic acid has been shown to
be the most metabolically stable of the derivatives of ART
(17). It was converted to the corresponding hydroxamate, com-
pound 5, by reaction with N,O-dimethylhydroxylamine, which
was then reduced to the aldehyde compound 6. Reductive
amination of compound 6 using PQ in methanol gave the
desired hybrid compound 7, with an overall yield of 28%.
Hybrid compound 10 was designed to replace the oxygen at the
C-10 position with a carbon, a modification expected to pro-
duce a compound with greater hydrolytic stability, a longer
half-life, and potentially lower toxicity (23). Hybrid compound
10 was synthesized from 10␤-allyldeoxoartemisinin, compound
8, in two steps, involving oxidation of the allyl moiety and
coupling of the resulting acid with PQ, with an overall yield of
29% (Fig. 1). The structural identification of the hybrid
compounds was performed using mass spectrometry and nu-
clear magnetic resonance spectrometry (correlation spectros-
copy [COSY] and heteronuclear correlation spectroscopy
[HETCOR] experiments).
C57BL/6 mice were bred and housed in the pathogen-free facility of the
Instituto de Medicina Molecular, Lisbon, Portugal. All protocols were approved
by the Animal Care Committee of the Instituto de Medicina Molecular.
Luciferase assay for liver-stage infection in vitro. Huh7 cells at about 50 to
60% confluence were incubated with the test compounds for 1 h and were then
infected in 96-well plates at a multiplicity of infection of ϳ0.4 with luciferase-
expressing P. berghei sporozoites obtained from freshly dissected mosquito sali-
vary glands. The medium was replaced 24 h after infection by new medium
containing freshly diluted drugs. At 46 h after infection, cell confluence was
assessed by the alamarBlue (Biosource/Invitrogen) assay. Briefly, this assay is
based on an oxidation-reduction indicator that both fluoresces and changes color
in response to chemical reduction of the culture medium resulting from cell
growth. Cells were incubated for 1.5 h with fresh medium containing 5%
alamarBlue, and fluorescence was measured (excitation wavelength, 530 nm;
emission wavelength, 590 nm) with a Tecan Infinite M200 microplate reader.
Following alamarBlue measurement, cells were washed once with PBS and were
lysed with 75 ␮l of cell culture lysis reagent obtained from the Promega luciferase
assay system kit. Samples in lysis buffer were either stored at Ϫ20°C or processed
immediately to measure luminescence intensity. In the latter case, 35 ␮l of the
cell lysate was transferred to white 96-well plates (Nunc) and was mixed with 50
␮l of luciferase assay substrate, freshly reconstituted with luciferase assay buffer
(Promega luciferase assay system kit). Luminescence was measured over 100 ms
with a Tecan Infinite M200 microplate reader within the 3 min following the
addition of the luciferase assay substrate.
Inhibition of hepatic P. berghei infection by primaquine-
artemisinin hybrids. To evaluate the abilities of compounds 7
and compound 10 to inhibit Plasmodium infection of liver cells,
we tested them on an established in vitro infection system that
employs a human hepatoma cell line (Huh7) and the rodent
parasite P. berghei. Huh7 cells were incubated with various
amounts of each compound or compound mixture prior to the
addition of transgenic, luciferase-expressing P. berghei sporo-
zoites, and total infection loads were quantified 48 h later by
luminescence measurements of cell lysates (22). Effects on cell
proliferation and survival were assessed by fluorescence mea-
surements following 1.5 h of incubation with alamarBlue.
Compounds 7 and 10 had potent parasite-inhibitory effects,
displaying IC50 values ϳ66 and ϳ18 times lower, respectively,
than that of a 1:1 PQ-ART mixture (Fig. 2). Treatment with
ART had no detectable effect on P. berghei liver-stage infec-
tion. Importantly, at the concentrations used in this experi-
ment, none of the hybrid compounds showed any effect on
Huh7 cell proliferation, as measured by alamarBlue fluores-
cence (Fig. 2). Flow cytometry-based analysis of GFP-express-
ing P. berghei 48 h after the addition of P. berghei to Huh7 cells
showed that compounds 7 and 10 and a PQ-ART mixture
caused dose-dependent decreases in parasite development, as
assessed by the geometrical means of the GFP intensities of
infected cells (26) (see Fig. S1 in the supplemental material).
Thus, the hybrid molecules acted by preventing intracellular
FACS analysis of in vitro liver-stage infection. Huh7 cells at about 60%
confluence were treated in 24-well plates with the different compounds and were
infected 1 h later with GFP-expressing P. berghei sporozoites obtained from
freshly dissected mosquito salivary glands at a multiplicity of infection of ϳ0.3.
Fluorescence-activated cell sorting (FACS) analysis at 4 h and 48 h after infec-
tion was performed as previously described (26, 29).
In vivo analysis of liver-stage development by luminescence. Luciferase activity
in animals was visualized at 44 h after infection with luciferase-expressing P.
berghei sporozoites, as previously described (22). Briefly, D-luciferin (Caliper Life
Sciences) dissolved in PBS (100 mg/kg of body weight) was injected subcutane-
ously in the necks of the animals. Five minutes later, mice were anesthetized by
intraperitoneal (i.p.) injection of xylazine (20 mg/kg) and ketamine (120 mg/kg)
dissolved in PBS. Bioluminescence measurements were performed in an IVIS
Lumina imaging system (Caliper Life Sciences) 10 min after D-luciferin injection.
Assessment of liver parasite loads by qRT-PCR. Determination of liver par-
asite loads in vivo was carried out as previously described (27). Briefly, total RNA
was isolated from livers by using Qiagen’s RNeasy Mini kit according to the
manufacturer’s instructions. Livers were collected and homogenized in a dena-
turing solution (4 M guanidine thiocyanate; 25 mM sodium citrate [pH 7], 0.5%
sarcosyl, and 0.7% ␤-mercaptoethanol in diethyl pyrocarbonate-treated water)
40 h after sporozoite injection. Total RNA was extracted using the Qiagen
RNeasy Mini kit according to the manufacturer’s instructions. RNA for infection
measurements was converted into cDNA by using the Roche Transcriptor first-
strand cDNA synthesis kit according to the manufacturer’s protocol. The quan-
titative real-time PCRs (qRT-PCRs) used the Applied Biosystems Power SYBR
green PCR master mix and were performed according to the manufacturer’s
instructions on an ABI Prism 7500 Fast system (Applied Biosystems). Amplifi-
cation reactions were carried out in a total reaction volume of 25 ␮l, containing
0.8 pmol/ml or 0.16 pmol/ml of the P. berghei ANKA 18S rRNA gene- or
housekeeping gene-specific primers, respectively. Relative amounts of P. berghei
ANKA mRNA were calculated against the amount of the hypoxanthine guanine

VOL. 55, 2011
MULTISTAGE ANTIMALARIAL HYBRIDS
4701
FIG. 1. Syntheses. Shown are the chemical structures of primaquine (compound 1), artemisinin (compound 2), dihydroartemisinin (compound
3), and artelinic acid (compound 4) and synthetic schemes for hybrid compounds 7 and 10.
parasite replication, in accordance with our previous data for
the parent compound PQ (34). Compound 10 also showed
modest inhibition of cell invasion by the parasites, as shown by
the infection rate measured 4 h after the addition of parasites
to the cells (see Fig. S1A in the supplemental material). Al-
though the IC50s determined by this method were ϳ1.5- to
2.5-fold higher than those determined by luminescence, they
indicated similar relative efficacies for the three sets of com-
pounds and confirmed that compound 7 displayed a much
stronger inhibitory effect than the PQ-ART mixture.
We next tested the abilities of the hybrid compounds to
inhibit P. berghei liver infection in mice. Mice were infected by
intravenous (i.v.) injection of 10,000 luciferase-expressing P.
berghei sporozoites, and liver parasite loads were assessed 42 h
later following the injection of luciferin (22). We observed
significant decreases in parasite loads in the livers of mice
treated with a single intraperitoneal (i.p.) injection of 26.5
␮mol/kg (equivalent to 12 mg/kg of PQ) of compound 7 or 10,
delivered 3 h after infection, although this effect was less
marked than that observed in mice similarly treated with PQ
(see Fig. S2A in the supplemental material). We then investi-
gated whether administration of the compounds during the
liver stage would affect the subsequent appearance of para-
sitemia. Mice infected with GFP-expressing P. berghei sporo-
zoites and treated 3 h later by i.p. injection of 26.5 ␮mol/kg of
compound 7, compound 10, or PQ were monitored daily for
the appearance of parasitemia (see Fig. S2B in the supplemen-
tal material) and disease symptoms. Remarkably, while com-
pound 10 had no effect on infection, administration of com-
pound 7 during the liver stage delayed or prevented the
appearance of parasites to an extent similar to that of PQ (see
Fig. S2B). Importantly, these results are consistent with the
relative in vitro efficacies of compounds 7 and 10 against he-
patic infection (Fig. 2).
Having established the efficacy of i.p. administration of com-
pound 7 during liver infection, we then assessed efficacy when
the compounds were administered orally. We compared the
activities of the hybrid molecules with that of an equimolar
mixture of PQ and ART. Four 65.9-␮mol/kg (equivalent to 30
mg/kg of PQ) doses of the compounds were administered by
oral gavage 30 min, 15 h, 23 h, and 39 h after infection of mice
with GFP-expressing P. berghei sporozoites. Parasite loads in

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FIG. 2. In vitro inhibition of hepatic Plasmodium berghei infection by compounds 7 and 10. (A to C) Compounds were added to Huh7 hepatoma
cells 1 h before infection with luciferase-expressing sporozoites. An amount of DMSO equivalent to that in the highest compound concentration
tested was used as a control. Forty-eight hours after the addition of P. berghei sporozoites, cell confluence (red lines on bar plots) was assessed by
alamarBlue fluorescence, and the infection rate (bars) was measured by quantifying the luciferase activity by luminescence. The effects of different
concentrations of compound 7 (A), compound 10 (B), and a 1:1 mixture of primaquine and artemisinin (C) are shown. Results are expressed as
means Ϯ standard deviations. (D) For each compound, the IC50 was calculated by sigmoidal fitting. Red, compound 7; orange, compound 10;
green, primaquine plus artemisinin.
livers collected 44 h postinfection were then determined by
quantitative real-time PCR (Fig. 3A). The results were in
agreement with those obtained following i.p. treatment (see
Fig. S2A in the supplemental material), with oral administra-
tion of compound 7 resulting in near-abrogation of liver infec-
tion. Finally, in a parallel experiment, mice were infected with
GFP-expressing P. berghei sporozoites and were treated 4 h
later with a single 65.9-␮mol/kg oral dose of the compounds,
followed by daily monitoring of blood for parasitemia. As ex-
pected, no parasitemia was detected in mice treated with the
PQ-ART mixture (Fig. 3B), presumably due to parasite clear-
ance in the liver by PQ. Four out of 6 mice treated with
compound 7 were also blood-stage negative at the end of the
experiment. Importantly, parasites were detected in the blood
of three mice in this group up to day 5 postinfection but were
then cleared, suggesting that the hybrid may remain in the
circulation for a considerable time or that an immune response
contributed to parasite clearance.
Overall, these results indicate that the PQ-ART hybrid mol-
ecules display strong antiplasmodial effects during the liver
stage of infection. In particular, compound 7 displayed strong
in vivo efficacy as a liver-stage antimalarial. Since our in vitro
results showed that ART alone does not possess significant
activity against Plasmodium liver stages, these data suggest that
the hybrid molecule constitutes a modification of PQ that
potentiates its effect, rendering it more effective at lower molar
amounts.
became the standard means of treating malaria in nearly all
countries where the disease was endemic (18). Thus, we as-
sessed the in vitro activities of compounds 7 and 10 against the
erythrocytic stage of P. falciparum and compared these activi-
ties with those of PQ and ART. While PQ showed only modest
activity in these assays, as expected, compounds 7 and 10 and
ART all yielded potent inhibitory effects, with IC50s in the
nanomolar range (Table 1).
We then compared the in vivo therapeutic efficiencies of the
hybrid compounds and ART in the treatment of an established
murine blood infection. C57BL/6 mice were infected by i.p.
injection of 106 P. berghei-infected RBCs (iRBCs), and their
parasitemia was monitored daily until their percentages of
iRBCs reached 2 to 3%. At this point, mice were treated daily,
for 4 days, by subcutaneous injection of 31.8 ␮mol/kg (equiv-
alent to 9 mg/kg of ART) of compound 7, compound 10, or
ART, an amount corresponding to the reported 90% effective
dose (ED90) for ART administered by this route (24), while
control mice were treated with equivalent amounts of a sol-
vent. Mice were then monitored daily for parasitemia and
disease symptoms (see Fig. S3 in the supplemental material).
As expected, the parasitemia of solvent-treated control mice
increased steadily until they perished with symptoms of exper-
imental cerebral malaria (19) between days 7 and 8 postinfec-
tion. Conversely, the parasitemia of compound-treated mice
decreased soon after the initiation of treatment, approaching
zero for all mice around day 9 postinfection. However, para-
sitemia recurred at days 12 to 13 postinfection in mice treated
with ART or compound 7 (see Fig. S3A and B in the supple-
mental material), and all mice in these two groups eventually
Treatment of Plasmodium parasitemia with primaquine-ar-
temisinin hybrids. As P. falciparum resistance to chloroquine
became increasingly widespread, artemisinin-based therapies

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MULTISTAGE ANTIMALARIAL HYBRIDS
4703
FIG. 3. In vivo inhibition of Plasmodium berghei liver-stage infection and blood patency by oral administration of compounds 7 and 10. C57BL/6
mice (n ϭ 6) were infected by intravenous injection of 10,000 GFP-expressing P. berghei sporozoites. (A) Mice were treated by oral administration
of the different compounds in the amounts and at the times noted in the text. Parasite liver loads (P. berghei ANKA 18S rRNA; relative quantity
normalized against that of the host HGPRT gene) were assessed by quantitative real-time PCR 44 h after infection. (B) Mice were treated by a
single administration of the compounds in the amounts and at the time noted in the text, and the appearance of parasites in the blood was
monitored daily. The percentage of mice with parasitemia under the detection limit (parasite-free mice) is shown.
succumbed on days 15 to 16 and 16 to 17 postinfection, respec-
tively. These outcomes were presumably due to hyperpara-
sitemia, since the mice did not display symptoms of experimen-
tal cerebral malaria (see Fig. S3C in the supplemental
material). Importantly, mice treated with compound 10 did not
experience recurrent parasitemia after initial clearance of in-
fections. Instead, they remained blood-stage negative through-
out the duration of the experiment (see Fig. S3A and B) and
had a 100% survival rate (see Fig. S3C).
In view of these results, we investigated the efficacy of oral
administration of the hybrid molecules in the treatment of a
patent blood infection. In this experiment, mice were infected
by i.v. injection of 10,000 GFP-expressing P. berghei sporozo-
ites, and treatment was initiated when the level of parasitemia
reached 2 to 3%. Both hybrid molecules and an equimolar
mixture of PQ and ART were administered daily by oral ga-
vage for 10 days, at 31.8 ␮mol/kg per dose. We selected a
dosage expected to be suboptimal for orally administered ar-
temisinin (24) in order to facilitate comparison of the efficacies
of the different treatments. Mice were monitored daily for
parasitemia, disease symptoms, and survival (Fig. 4). As ex-
pected, all control mice died with symptoms of experimental
cerebral malaria between days 7 and 8 after infection. Most
noticeably, parasitemia and survival rates were remarkably in-
creased in mice treated with compound 10 over those for the
PQ-ART mixture (Fig. 4). These results are in agreement with
those obtained after i.p. dosing (see Fig. S3 in the supplemen-
tal material) and demonstrate that the hybrid molecules can be
administered orally to treat malaria.
TABLE 1. In vitro blood schizonticidal activity, molecular weight,
and ClogP for primaquine, artemisinin, and
hybrid compounds 7 and 10
Compound
IC50 (nM)a
Mol wt
ClogPb
Primaquine
Artemisinin
Compound 7
Compound 10
3,300 Ϯ 55
8.2 Ϯ 0.9
12.5 Ϯ 1.1
9.1 Ϯ 0.6
259
282
646
568
2.76
2.90
5.88
5.25
a Determined against the chloroquine-resistant P. falciparum strain W2.
b ClogP is an estimate of the value of the logarithm of the partition coefficient
between 1-octanol and water, calculated using ALOGPS software (http://www
.vcclab.org/lab/alogps/). The logP values measure a compound’s lipophilicity
(higher values mean higher lipophilicity).
Overall, our results show that both hybrid molecules, and
particularly compound 10, can be used to control an ongoing
blood-stage infection with greater efficacy than that of the

4704
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FIG. 4. In vivo treatment of Plasmodium berghei blood infection by oral administration of compounds 7 and 10. C57BL/6 mice (n ϭ 5) were
infected by intravenous injection of 105 GFP-expressing sporozoites. Oral administration of the compounds was initiated when parasitemia reached
2 to 3% and was carried out daily for 10 days. (A) Blood parasite patency, monitored daily by flow cytometry. The percentages of mice with
parasitemia under the detection limit are shown. (B) Survival plot showing the percentages of live mice throughout the duration of the experiment.
parent compound ART or ART-PQ. Interestingly, although
compound 10 had a lower IC50 than compound 7 in vitro, it was
more active in vivo, suggesting enhanced pharmacokinetic
properties or stability. Indeed, compound 10 was designed to
replace the oxygen at the C-10 position with a carbon, a mod-
ification expected to produce a compound that was both less
toxic and more stable (23).
the PQ and ART pharmacophores. We found that these com-
pounds displayed antimalarial efficacies that in some instances
were superior to those of the parent compounds.
There is consensus that efforts to eradicate malaria should
be multifaceted and that new and effective antimalarial drugs
will constitute essential tools in this struggle (1, 37). PQ is the
compound of reference against the liver stage of mammalian
infection by Plasmodium, while ART derivatives, such as arte-
sunate and artemether, are the compounds of reference
against blood-stage parasites. PQ is usually employed in com-
bination with blood schizonticides, in particular chloroquine
(12) or ART derivatives (9, 32, 33), to prevent relapses in P.
vivax infections and to reduce malaria transmission following
treatment (31, 33). Two studies on chimeric PQ-containing
molecules, in which PQ was linked to statin-based inhibitors of
plasmepsins, have been reported (10, 30). Although these dou-
DISCUSSION
Treatment of malaria relies on agents that eliminate blood-
stage infection, notably ART derivatives, and, in the case of P.
vivax and P. ovale infections, PQ to eradicate dormant liver-
stage parasites (7, 11). Optimal therapy might incorporate
agents active against both blood- and liver-stage parasites in
the same molecule. To this end, we have synthesized two hy-
brid molecules, compounds 7 and 10, that covalently combine

VOL. 55, 2011
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4705
ble drugs displayed improved in vitro blood schizonticidal ac-
tivities relative to those of the single compounds, no data were
provided on their in vitro effects against Plasmodium liver
stages or on their in vivo activities against liver and blood
stages. Both hybrids described in the present study displayed
marked antiplasmodial activity in vitro against liver stages, with
IC50s lower than those reported for PQ (22) (Fig. 2). Our in
vitro results suggest that the efficacy of PQ is increased by the
introduction of the ART pharmacophoric unit into the hybrid
molecules. Additionally, both hybrid molecules significantly
reduced parasite loads in the livers of mice following intrave-
nous injection of sporozoites, although this effect was lower
than that of PQ (Fig. 3A; see also Fig. S2A in the supplemental
material). Taken together, our data suggest that the greater in
vitro efficacy of the compounds was, to some extent, counter-
balanced by a decrease in their bioavailability, metabolic sta-
bility, or other factors necessary for in vivo activity. Activity
may be limited by the fact that the drug needs to cross several
barriers before reaching the intrahepatic parasite, which re-
sides inside a parasitophorous vacuole. Nevertheless, when
sporozoites were delivered by mosquito bite and infection was
allowed to proceed to the blood stage, compound 7 displayed
significant in vivo efficacy in controlling parasitemia (see Fig.
S2B in the supplemental material). Further, compound 7 was
also efficacious when administered orally following i.v. injec-
tion of sporozoites, although in this case its effect was not as
potent as that of the PQ-ART mixture (Fig. 3B). We therefore
speculate that the reduced bioavailability of compound 7 is
compensated for by its strong efficacy against the liver stage,
which, combined with its blood schizonticidal activity (see be-
low), renders it a promising compound for the treatment of P.
vivax malaria.
Antimalarial therapy increasingly involves the use of drug
combinations as a means of improving treatment efficacy and
reducing the development of resistance (4, 38). The emphasis
on fixed-dose antimalarial drug combinations has led logically
to the development of hybrid molecules that covalently join
two distinct antimalarial moieties. Most antimalarial hybrid
molecules have been designed to act solely on the asexual
blood stages of the parasite (reviewed in reference 35). Only
two of these designs included ART as one of the units in the
chimeric molecule, coupled with either mefloquine (13) or
quinine (36). The ART-mefloquine hybrids were more effec-
tive at controlling parasitemia in P. berghei-infected mice than
artemether (13). The ART-quinine chimera was tested only in
vitro, where it showed anti-P. falciparum activity superior to
that of ART alone, quinine alone, or a 1:1 mixture of ART and
quinine (36). The data obtained with our hybrid compounds
indicate that the covalent linkage of the PQ and ART units
leads to enhanced efficacy in the treatment of a patent blood-
stage infection over that of the parent compounds, particularly
in the case of compound 10 (Fig. 4). It is not clear why a similar
effect was not observed for compound 7, despite the similarity
of the in vitro activities of compounds 7 and 10 (Table 1).
Differences in activity might be explained by differences in
compound stability. Another possibility is that the differential
activities shown by compounds 7 and 10 against blood and liver
stages may result from different preferences of the two com-
pounds for RBCs and hepatocytes.
file presents several important challenges to medicinal chem-
ists, not the least of which is achieving a design that confers
“drug-like” properties on the resulting molecule (15). In this
work we describe, for the first time, the ability of hybrid PQ-
ART molecules to inhibit the liver and blood stages of mam-
malian Plasmodium infection. Our data constitute a proof of
principle that paves the way for the exploitation of a multistage
hybrid approach in drug-based strategies for malaria control
and eradication.
ACKNOWLEDGMENTS
Inˆes Albuquerque is gratefully acknowledged for help with the as-
sessment of parasite patency in Giemsa-stained blood smears.
M.P. holds a Cieˆncia 2007 position from the Portuguese Ministry of
Science and Technology. R.C. acknowledges FCT for Ph.D. grant
SFRH/BD/30418/2006, and G.G.C. acknowledges the FP7 of the Eu-
ropean Commission for Marie Curie fellowship FP7-PEOPLE-IEF-
2008-235864. This work was supported by Portuguese Foundation for
Science and Technology grant PTDC/SAUMII/099118/2008 to
M.P. P.J.R. is a Distinguished Clinical Scientist of the Doris Duke
Charitable Foundation.
REFERENCES
1. Alonso, P. L., et al. 2011. A research agenda for malaria eradication: drugs.
PLoS Med. 8:e1000402.
2. Aslanian, R., et al. 2003. Identification of a dual histamine H1/H3 receptor
ligand based on the H1 antagonist chlorpheniramine. Bioorg. Med. Chem.
Lett. 13:1959–1961.
3. Baird, J. K., and S. L. Hoffman. 2004. Primaquine therapy for malaria. Clin.
Infect. Dis. 39:1336–1345.
4. Bell, A. 2005. Antimalarial drug synergism and antagonism: mechanistic and
clinical significance. FEMS Microbiol. Lett. 253:171–184.
5. Capela, R., et al. 2009. Artemisinin-dipeptidyl vinyl sulfone hybrid mole-
cules: design, synthesis and preliminary SAR for antiplasmodial activity and
falcipain-2 inhibition. Bioorg. Med. Chem. Lett. 19:3229–3232.
6. Cena, C., et al. 2003. Antiinflammatory, gastrosparing, and antiplatelet prop-
erties of new NO-donor esters of aspirin. J. Med. Chem. 46:747–754.
7. Chattopadhyay, R., et al. 2010. Establishment of an in vitro assay for assess-
ing the effects of drugs on the liver stages of Plasmodium vivax malaria. PLoS
One 5:e14275.
8. Coteron, J. M., et al. 2010. Falcipain inhibitors: optimization studies of the
2-pyrimidinecarbonitrile lead series. J. Med. Chem. 53:6129–6152.
9. Dao, N. V., et al. 2007. Vivax malaria: preliminary observations following a
shorter course of treatment with artesunate plus primaquine. Trans. R. Soc.
Trop. Med. Hyg. 101:534–539.
10. Dell’Agli, M., et al. 2006. High antiplasmodial activity of novel plasmepsins
I and II inhibitors. J. Med. Chem. 49:7440–7449.
11. Dembele, L., et al. 2011. Towards an in vitro model of Plasmodium hypno-
zoites suitable for drug discovery. PLoS One 6:e18162.
12. Galappaththy, G. N., A. A. Omari, and P. Tharyan. 24 January 2007. Prim-
aquine for preventing relapses in people with Plasmodium vivax malaria.
Cochrane Database Syst. Rev. 1:CD004389. doi:10.1002/14651858.
CD004389.pub2.
13. Grellepois, F., P. Grellier, D. Bonnet-Delpon, and J. P. Begue. 2005. Design,
synthesis and antimalarial activity of trifluoromethylartemisinin-mefloquine
dual molecules. Chembiochem 6:648–652.
14. Hindley, S., et al. 2002. Mechanism-based design of parasite-targeted arte-
misinin derivatives: synthesis and antimalarial activity of new diamine con-
taining analogues. J. Med. Chem. 45:1052–1063.
15. Lipinski, C. A., F. Lombardo, B. W. Dominy, and P. J. Feeney. 2001. Exper-
imental and computational approaches to estimate solubility and permea-
bility in drug discovery and development settings. Adv. Drug Deliv. Rev.
46:3–26.
16. Lolli, M. L., et al. 2001. A new class of ibuprofen derivatives with reduced
gastrotoxicity. J. Med. Chem. 44:3463–3468.
17. Navaratnam, V., et al. 2000. Pharmacokinetics of artemisinin-type com-
pounds. Clin. Pharmacokinet. 39:255–270.
18. Nosten, F., and N. J. White. 2007. Artemisinin-based combination treatment
of falciparum malaria. Am. J. Trop. Med. Hyg. 77:181–192.
19. Pamplona, A., et al. 2007. Heme oxygenase-1 and carbon monoxide suppress
the pathogenesis of experimental cerebral malaria. Nat. Med. 13:703–710.
20. Perez, M., et al. 1998. Design and synthesis of new potent, silent 5-HT1A
antagonists by covalent coupling of aminopropanol derivatives with selective
serotonin reuptake inhibitors. Bioorg. Med. Chem. Lett. 8:3423–3428.
21. Peters, W. 1999. The evolution of tafenoquine—antimalarial for a new mil-
lennium? J. R. Soc. Med. 92:345–352.
The design of compounds with a predefined multitarget pro-

4706
CAPELA ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
22. Ploemen, I. H., et al. 2009. Visualisation and quantitative analysis of the
rodent malaria liver stage by real time imaging. PLoS One 4:e7881.
23. Posner, G. H., et al. 2003. Orally active, antimalarial, anticancer, artemisinin-
derived trioxane dimers with high stability and efficacy. J. Med. Chem.
46:1060–1065.
24. Posner, G. H., et al. 1999. Orally active, hydrolytically stable, semisynthetic,
antimalarial trioxanes in the artemisinin family. J. Med. Chem. 42:300–304.
25. Price, R. N., et al. 2007. Vivax malaria: neglected and not benign. Am. J.
Trop. Med. Hyg. 77:79–87.
26. Prudencio, M., C. D. Rodrigues, R. Ataide, and M. M. Mota. 2008. Dissect-
ing in vitro host cell infection by Plasmodium sporozoites using flow cytom-
etry. Cell. Microbiol. 10:218–224.
27. Prudencio, M., et al. 2008. Kinome-wide RNAi screen implicates at least 5
host hepatocyte kinases in Plasmodium sporozoite infection. PLoS Pathog.
4:e1000201.
28. Prudencio, M., A. Rodriguez, and M. M. Mota. 2006. The silent path to
thousands of merozoites: the Plasmodium liver stage. Nat. Rev. Microbiol.
4:849–856.
29. Rodrigues, C. D., et al. 2008. Host scavenger receptor SR-BI plays a dual role
in the establishment of malaria parasite liver infection. Cell Host Microbe
4:271–282.
30. Romeo, S., et al. 2004. Plasmepsin II inhibition and antiplasmodial activity of
Primaquine-Statine ‘double-drugs.’ Bioorg. Med. Chem. Lett. 14:2931–2934.
31. Shekalaghe, S., et al. 2007. Primaquine clears submicroscopic Plasmodium
falciparum gametocytes that persist after treatment with sulphadoxine-pyri-
methamine and artesunate. PLoS One 2:e1023.
32. Shekalaghe, S. A., et al. 2010. In Tanzania, hemolysis after a single dose of
primaquine coadministered with an artemisinin is not restricted to glucose-
6-phosphate dehydrogenase-deficient (G6PD AϪ) individuals. Antimicrob.
Agents Chemother. 54:1762–1768.
33. Smithuis, F., et al. 2010. Effectiveness of five artemisinin combination reg-
imens with or without primaquine in uncomplicated falciparum malaria: an
open-label randomised trial. Lancet Infect. Dis. 10:673–681.
34. Vale, N., R. Moreira, and P. Gomes. 2009. Primaquine revisited six decades
after its discovery. Eur. J. Med. Chem. 44:937–953.
35. Walsh, J. J., and A. Bell. 2009. Hybrid drugs for malaria. Curr. Pharm. Des.
15:2970–2985.
36. Walsh, J. J., D. Coughlan, N. Heneghan, C. Gaynor, and A. Bell. 2007. A
novel artemisinin-quinine hybrid with potent antimalarial activity. Bioorg.
Med. Chem. Lett. 17:3599–3602.
37. Wells, T. N., P. L. Alonso, and W. E. Gutteridge. 2009. New medicines to
improve control and contribute to the eradication of malaria. Nat. Rev. Drug
Discov. 8:879–891.
38. White, N. 1999. Antimalarial drug resistance and combination chemother-
apy. Philos. Trans. R. Soc. Lond. B Biol. Sci. 354:739–749.
39. World Health Organization. 2010. World malaria report 2010. World Health
Organization, Geneva, Switzerland. http://www.who.int/malaria/world
_malaria_report_2010/en/index.html.