Synthesis and evaluation of new antimalarial phenylurenyl chalcone derivatives

Article abstract of DOI:10.1021/jm058208o

Phenylurenyl chalcone derivatives have been synthesized and tested as inhibitors of in vitro development of a chloroquine-resistant strain of Plasmodium falciparum, activity of the cysteine protease falcipain-2, in vitro globin hydrolysis, β-hematin forma

Full text of DOI:10.1021/jm058208o

3654
J. Med. Chem. 2005, 48, 3654-3658
Synthesis and Evaluation of New Antimalarial Phenylurenyl Chalcone
Derivatives
Jose´ N. Dom´ınguez,*,§ Caritza Leo´n,§ Juan Rodrigues,† Neira Gamboa de Dom´ınguez,† Jiri Gut,‡ and
Philip J. Rosenthal‡
Laboratorio de Sı´ntesis Orga´nica and Laboratorio de Bioquı´mica, Facultad de Farmacia, Universidad Central de Venezuela,
Caracas, Venezuela, and Department of Medicine, San Francisco General Hospital, University of California, San Francisco,
Box 0811, San Francisco, California 94143
Received March 11, 2005
Phenylurenyl chalcone derivatives have been synthesized and tested as inhibitors of in vitro
development of a chloroquine-resistant strain of Plasmodium falciparum, activity of the cysteine
protease falcipain-2, in vitro globin hydrolysis, â-hematin formation, and murine Plasmodium
berghei malaria. The most active antimalarial compound was 1-[3′-N-(N′-phenylurenyl)phenyl]-
3(3,4,5-trimethoxyphenyl)-2-propen-1-one 49, with an IC50 of 1.76 µM for inhibition of P.
falciparum development. Results suggest that chalcones exert their antimalarial activity via
multiple mechanisms.
Introduction
Results and Discussion
Malaria, the most severe human parasitic disease,
causes 200-500 million cases and 0.7-2.7 million
deaths each year.1-4 Although the greatest burden of
malaria and the focus of its control are in Africa, it is
also an important problem in other tropical areas such
as Asia, South America, and Oceania.
Synthesis. A procedure based on a Claisen-Schmidt
condensation was developed for syntheses of all phenyl-
urenyl chalcones and their derivatives. Part of the work
presented here was undertaken to elaborate structure-
activity relationships (SARs) with respect to variations
of R, R5, and R6 (Scheme 1). Syntheses of methyl ketone
derivatives 6-9 were obtained by reaction of p- or
m-amino acetophenones 4 and 5 with the corresponding
isocyanate derivatives, where R ) H, Cl, OMe. The
subsequent treatment of these derivatives with solid
sodium hydroxide as a catalyst in methanol at room
temperature and the corresponding substituted aro-
matic aldehydes by the use of Claisen-Schmidt con-
densation13 yielded 10-56. The above reaction condi-
tions were found to be optimal, whereas organic bases
such as triethylamine or piperidine generally gave lower
yields under the same reaction conditions. Sodium
methoxide did not work as well as the sodium hydroxide
pellet. If the starting material was insoluble in metha-
nol, then tetrahydrofuran (THF) or 1, 4-dioxane was
used as a cosolvent. The vinylic protons of cis alkenes
have coupling constants smaller than those of their
trans isomers. Some studies12,14 on chalcone derivatives
have shown exclusively E configuration; however, NMR
techniques allowed us to conclude that the compounds
obtained almost always yielded the trans alkenes (E
form). To assess the specificity and selectivity of these
derivatives, they were tested in different malaria assays.
Plasmodium falciparum and Plasmodium vivax are
the two major human malaria parasites. P. falciparum
is responsible for most deaths, and it has developed
resistance to nearly all available drugs.5 The search for
novel antimalarial drugs against specific parasitic tar-
7
gets is thus an urgent priority.6, The antimalarial
activity of chalcones has generated interest.8-12 Their
antimalarial activity was first reported when licochal-
cone A, a natural product isolated from Chinese liquo-
rice root, exhibited potent antimalarial activity.8 Sub-
sequently, a synthetic analogue was reported to have
antimalarial activity.9 Some chalcones inhibit falcipain
cysteine proteases, but it is unclear if the antimalarial
activity of this class is primarily due to protease
inhibition.10 Several oxygenated chalcones and bischal-
cones were reported to have antimalarial activity.11 In
a recent study the synthesis and antimalarial activity
of some quinolinyl chalcone analogues have been re-
ported.12
Our work now focuses on the synthesis and charac-
terization of a series of novel phenylurenyl chalcone
compounds, including evaluation of effects on develop-
ment of cultured P. falciparum parasites, cysteine
protease activity, globin hydrolysis, and heme polym-
erization and in a murine malaria model.
Biological Evaluation. Inhibition of Parasite
Development and Enzyme Inhibition. Compounds
were tested for their ability to inhibit parasite develop-
ment by incubating different concentrations with para-
sites for 48 h, beginning at the ring stage, counting new
ring forms by fluorescence-activated cell sorting (FACS)
analysis, and comparing parasitemias with those of
untreated controls (Table 1). Twelve compounds inhib-
ited parasite development at or below 10 µM (Table 1).
* To whom correspondence should be addressed. Phone: +58 212
6052722. Fax: +58 212 6052707. E-mail: jdomingu@cantv.net.
§ Laboratorio de S´ıntesis Orga´nica, Universidad Central de Ven-
ezuela.
† Laboratorio de Bioqu´ımica, Universidad Central de Venezuela.
‡ San Francisco General Hospital, University of California, San
Francisco.
10.1021/jm058208o CCC: $30.25 © 2005 American Chemical Society
Published on Web 04/21/2005

Brief Articles
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 10 3655
Scheme 1a
Figure 2. The % parasitemia at 4th day postinfection and
survival days on mice treated with 47, 49, 51, 55, 25, 26, 28,
31 (20 mg/kg) and CQ (25 mg/kg): (/) p < 0.05, (//) p < 0.01,
(///) p < 0.001, comparing with saline-treated mice (n ) 6).
a R5: (a) 2,4-di-OMeC6H3; (b) 4-FC6H4; (c) 3,4-OCH2OC6H3; (d)
4-ClC6H4; (e) 2,4-di-ClC6H3; (f) 2,4-di-FC6H3; (g) 3,4,5-tri-OMeC6H2;
(h) C6H5; (i) 4-MeC6H4; (j) 4-OMeC6H4; (k) C5H4N. R6: (a-j) same
as for R5; (l) 2,3-di-OMeC6H3; (m) 3,4-di-OMeC6H3; (n) 4-BrC6H4;
(o) N(Me)2C6H4, (i) Me2CO, room temp, (ii) MeOH, NaOH, R5CHO
or R6CHO.
Table 2. Inhibition of Hemozoin Formation, Hemoglobin
Hydrolysis, and Development in a P. berghei Murine Model
(Peter’s Test) by the Most Active Compoundsa
Table 1. Effect of Compounds on P. falciparum Development
inhibition inhibition of
of hemozoin hemoglobin
formationb hydrolysisc,e
and Inhibition of Falcipain-2 Activity
compda
FACSb IC50, µM
falcipain-2c IC50, µM
compd
(%)
(%)
Peter’s testd,e (%P/SD)
23
10
2.5
2.6
2.5
2.6
1.8
3.8
3.4
12.5
d
saline
25
(23.62 ( 1.36)/(10.50 ( 1.12)
24
10
<5
60.31 ( 1.41 (11.60 ( 1.57)/(12.00 ( 1.26)
33.04 ( 3.23 (18.60 ( 3.81)/(9.50 ( 1.25)
98.64 ( 0.73 (16.25 ( 0.84)/(12.66 ( 1.56)
60.72 ( 1.73 (7.40 ( 1.64)/(15.50 ( 2.37)
51.45 ( 2.26 (6.80 ( 2.17)/(13.66 ( 1.64)
74.93 ( 2.53 (8.40 ( 3.40)/(13.50 ( 1.61)
66.58 ( 1.80 (15.50 ( 2.57)/(6.16 ( 0.77)
5.15 ( 1.85
25
5
26
<5
89.2
<5
<5
<5
f
26
8
31
27
3
47
28
9
49
31
9
51
42
10
55
47
2.14
1.76
2.74
2.10
6.26
2.50
0.059
CQ
LEU
PEP
86.6
f
f
49
d
87.02 ( 0.69
51
d
d
91.91 ( 0.67
55
LEU
E-64
CQ
0.0058
0.058
d
a CQ ) chloroquine; LEU ) leupeptin; PEP ) pepstatin
compared to LEU; P ) parasitemia; SD) survival days. b % of
inhibition of hemozoin formation. c Hemoglobin hydrolysis. d Peter’s
test results of the most active drugs. e The results are expressed
by the mean ( standard error of the mean. p < 0.0001 f Unknown.
a Only compounds expressing activity against P. falciparum
parasites (IC50 < 10 µM) were screened for activity against
recombinant falcipain-2. b IC50 for tested compounds as determined
by flow cytometry. c IC50 of selected compounds active against
d
recombinant falcipain-2. There was no significant effect on
hemoglobin degradation at of 5 µM. Six compounds (47,
49, 51, 55, 25, and 37) inhibited hemoglobin degradation
less completely (51-75%) (Figure 2, Table 2).
inhibition of falcipain-2 probably because of limited solubility.
Inhibition of Hemozoin Formation. We tested the
ability of the compounds to inhibit hemozoin formation.
Compound 31 inhibited this process (IC50 ) 0.73 mM)
with activity somewhat greater than that of chloroquine
(IC50 ) 1.33 mM) (Table 2).
Activity in a Mouse Malaria Model. Selected
compounds that were active in vitro (47, 49, 51, 25, 26,
28, and 31) were tested for activity in mice infected with
P. berghei ANKA, a chloroquine-susceptible strain of
murine malaria parasites. The mice were treated with
compounds (20 mg/kg) or chloroquine (25 mg/kg) intra-
peritoneally once daily for 4 consecutive days (days 0-3
postinfection), and their survival times and parasitemia
on day 4 were compared with those of control mice
receiving only saline. A number of compounds signifi-
cantly inhibited day 4 parasitemia and increased sur-
vival times (Figure 2).
Figure 1. Effects on globin hydrolysis of chalcone derivatives.
The samples were solubilized in SDS-sample buffer contain-
ing â-mercaptoethanol and boiled before electrophoresis in 15%
SDS-PAGE gels. The gels were stained with Coomassie blue.
The positions of molecular weight (MW) standards are shown
in kDa. Undegraded globin appears as a dimer at 14 kDa: (A)
control hemoglobin without enzyme; (B) control enzyme with
hemoglobin. Also shown are 47, 49, 51, 55, 25, 26, 28, and 31
(5 µM), PEP ) pepstatin (10 µM), LEU ) leupeptin (10 µM),
and CQ ) chloroquine (100 µM).
For the remaining compounds, the IC50 for the inhibition
of parasite development was >10 µM.
Inhibition of Globin Hydrolysis. Selected com-
pounds that inhibited parasite development were evalu-
ated for the inhibition of globin hydrolysis (Figure 1).
Compound 31 was most active, with 98% inhibition of
In P. falciparum, a number of chalcones have been
shown to exert antimalarial activity.15 We have syn-
thesized and evaluated the antimalarial activity of 47

3656 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 10
Brief Articles
new chalcone derivatives. We demonstrated in vitro and
in vivo antimalarial activity of a number of compounds.
To explore the mechanisms of action of these com-
pounds, we considered the effects of compounds that
inhibited parasite development on activity of the cys-
teine protease falcipain-2, catabolism of hemoglobin, and
hemozoin formation and in a murine malaria model.
Nine compounds inhibited the development of cul-
tured P. falciparum parasites with IC50 < 10 µM. The
most active compounds were 47, 49, 51, and 55. Com-
pounds 47 and 49 also showed good inhibition of globin
hydrolysis. These compounds also were active against
P. berghei infections in mice. However, the antimalarial
effect in vivo was modest, and all treated mice eventu-
ally died because of lethal infections.
activity. Selected compounds were also tested in vivo
in P. berghei infected mice. We found that compounds
47, 49, 51, and 55 were excellent inhibitors against
cultured P. falciparum parasites, with a good correlation
with activity in a murine malaria model except for 55.
The most active derivative was 1-[3’-N-(N′-phenylure-
nyl)phenyl]-3(3,4,5-trimethoxyphenyl)-2-propen-1-one 49
with an IC50 of 1.76 µM against cultured P. falciparum.
Four other chalcone derivatives, 25-28, had IC50 below
9 µM, and the most active of these compounds against
cultured P. falciparum parasites and the cysteine pro-
tease falcipain-2 was 1-[4′-N-(N′-p-chlorophenylurenyl)-
phenyl]-3(3,4,5-trimethoxyphenyl)-2-propen-1-one 27 (IC50
of 3 and 1.8 µM, respectively). However, its biological
study was incomplete because of poor solubility in polar
solvents. A very interesting result was obtained for
compound 1-[4′-N-(N′-p-chlorophenylurenyl)phenyl]-3-
(3-pyridinyl)-2-propen-1-one 31, which showed a good
antimalarial effect in vitro and in vivo. Nevertheless,
this compound was able to interfere with heme detoxi-
fication (inhibition 89.2%) and globin hydrolysis (inhibi-
tion 98.64%) (Table 2). This property could be attributed
to derivative 31 having an aromatic ring that has been
substituted by an aromatic pyridinyl group and refers
to a nitrogen-containing heterocyclic (Scheme 1) that
resembled the nitrogen of chloroquine and may be
somewhat concentrated in the food vacuoles of malaria
parasites for a given result.18 The comprehensive rela-
tionship between antiparasitic activity, inhibition of
hemozoin formation, and hemoglobin hydrolysis for this
type of compound has been observed. Therefore, a potent
inhibition of heme formation and hemoglobin hydrolysis
does not guarantee antimalarial activity. These results
provide an understanding of the structural features that
influence functional activity for this class of compounds
and also offer new possibilities for improvements in the
antimalarial performance of urenyl chalcones. We con-
cluded from our data that derivative 49 has the best
antimalarial properties, but its mechanism of action
does not appear to be related to hemoglobin hydrolysis
or heme detoxification, and if we compare it against
derivative 31, we found that at the same concentration
49 was the best to arrest the development of P. falci-
parum in culture (IC50 ) 1.76 µM) and to reduce the
parasitemia of P. berghei infected mice (from control
23-6.8%). Further studies are necessary to improve its
antimalarial activity.
The data suggest that activity in most cases was
governed to a large extent by groups attached to the
substituted aromatic ring assigned as R5 (difluoride,
dichloride, and trimethoxy) and R6 (dichloride and
trimethoxy). These groups played an important role
apparently because of electron-donating properties,
yielding the most active antimalarial compounds. In
general we speculate that electron-withdrawing groups
on the substituted aromatic ring for these urenyl
chalcones should favor the Michael addition to an
available nucleophilic side chain on the enzyme. Com-
pounds 23-28 and 31 were the most active as inhibitors
of falcipain-2. It appears that their effects were due to
inhibition of this important hemoglobinolytic enzyme
(Table 1). We observed some correlation for compounds
25-28 and 31 between inhibition of falcipain-2 and of
development of cultured parasites, suggesting that
plasmodial cysteine proteases are involved in their
antimalarial activity. None of the above active com-
pounds were found to inhibit hemozoin formation in
vitro in the same range compound 31, and five among
them, 25, 31, 47, 51, and 55, displayed strong inhibition
of globin hydrolysis (60-98%), suggesting a different
mechanism of action. Considerable experimental evi-
dence suggests that inhibition of hemozoin formation
is central to the mechanism of action of the quinoline-
containing antimalarial agents.16 Some results indicate
that the extent of drug accumulation at the site of the
heme is also a regulator of antimalarial activity.17
Within the range of solubility, hemozoin formation
inhibitory activity could be measured for most of the
studied chalcones. Only 31 showed an excellent inhibi-
tion of hemozoin formation (89.2%) and hemoglobin
hydrolysis (98.64%). However, 31 failed to demonstrate
a clear relationship between antiparasitic activity and
inhibition of hemozoin formation. Therefore, the inhibi-
tion of heme formation does not guarantee antimalarial
activity. Finally, it seems that para and meta positions
in the urenyl ring play an important role in their
antimalarial activity, as could be observed for aromatic
substituted trimethoxyl 27 (IC50 for P. falciparum is 3
µM) vs 49 (1.76 µM) and for dichloride 25 (5 µM)) vs 47
(2.14 µM). These results offer new possibilities for
further improvements in the antimalarial performance
of urenyl chalcones.
Experimental Section
General Procedure for Preparation of 6-9. A mixture
of the corresponding aminoacetophenone 4 or 5 (1 mmol) and
phenylisocyanates 1-3 recently distilled (1 mmol) was dis-
solved in dry acetone (5 mL). The mixture was stirred under
nitrogen atmosphere for 3-7 h at room temperature. The
resulting solid was filtered, and crystallization with the
appropriate solvent afforded the desired urenylacetophenone
in pure form.
4′-N-(N′-Phenylurenyl)acetophenone (6). Following the
general procedure, a white solid was obtained that was
recrystallized in EtOH: yield 52%; mp 175-177 °C; IR 3360
(NH), 1685 (CO) cm-1 1H NMR (DMSO-d6) δ 2.51 (s, 3H,
;
COMe), 6.98 (q, 2H, H4′′, J ) 8.91 Hz), 7.28 (q, 4H, H3′′5′′, J )
8.91 Hz), 7.43-7.48 (m, 4H, H2′′-6′′), 7.58 (d, 2H, H3′-5′, J )
8.88 Hz), 7.91 (d, 2H, H2′-6′, J ) 8.88 Hz), 8.64 (s, OH), 8.79
(br s, NH), 9.09 (br s, NH); 13C NMR (DMSO-d6) δ 26.87
(COMe), 117.69 (C3′-5′), 118.76 (C2′′-6′′), 119.02 (C2′′6′′), 122.38
(C4′′), 122.80 (C4′′), 129.33 (C3′′-5′′), 129.39, (C3′′-5′′), 130.20
Conclusion
A series of phenylurenyl chalcone derivatives have
been synthesized and evaluated for in vitro antimalarial

Brief Articles
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 10 3657
(C2′-6′), 130.98 (C1′), 139.81 (C4′), 140.23 (C1′′), 144.91 (C1′′),
Supporting Information Available: Synthesis and
analytical data for 10-56 and biological techniques. This
material is available free of charge via the Internet at http://
pubs.acs.org.
152.72 (CO(NH)2), 153.09 (NdCOH-NH2), 196.86 (CO).
4′-N-(N′-p-chlorophenylurenyl)acetophenone (7). The
solid was recrystallized in EtOH: yield 65%; mp 224-225 °C;
IR 3376 (NH), 1715 (COMe) cm-1, 1648 (CO); 1H NMR (DMSO-
d6) δ 2.51 (s, 3H, COMe), 7.33 (d, H3′′-5′′, J ) 8.67 Hz), 7.49 (d,
H2′′-6′′, J ) 8.67 Hz), 7.57 (d, H3′-5′, J ) 8.64 Hz), 7.90 (d, H2′-6′,J
) 8.64 Hz), 8.94 (br s, NH), 9.13 (br s, NH); 13C NMR (DMSO-
d6) δ 26.89 (COMe), 117.82 (C3′-5′), 120.56 (C2′′-6′′), 126.35 (C4′′),
129.22 (C3′′-5′′), 130.19 (C2′-6′),131.13 (C1′),138.84 (C1′′), 144.70
(C4′), 152.65 (CO(NH)2), 196.89 (COR,â).
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4′-N-(N′-p-Methoxyphenylurenyl)acetophenone (8). The
solid was recrystallized in a mixture of EtOH/H2O: yield 82%;
mp 210-212 °C; IR 3376 (NH), 1706 (CO), 1642 (CO), 1581
(CdC Ar) cm-1 1H NMR (DMSO-d6) δ 2.51 (s, 3H, COMe),
;
3.72 (s, 3H, 4′′-OMe), 6.88 (d, H3′′-5′′, J ) 8.91 Hz), 7.37 (d,
H2′′-6′′, J ) 8.91 Hz), 7.57 (d, H3′-5′, J ) 8.91 Hz), 7.89 (d, H2′-6′
,
J ) 8.91 Hz), 8.61 (br s, NH), 9.02 (br s, NH); 13C NMR
(DMSO-d6) δ 26.86 (COMe), 56.60 (4′′-OMe), 114.55 (C3′-5′),
117.55 (C2′′-6′′), 120.86 (C3′′-5′′), 130.20 (C2′-6′), 130.76 (C1′),
132.78 (C1′′), 145.12 (C4′), 152.88 (CO(NH)2), 155.28 (C4′′),
196.83 (CO).
3′-N-(N′-Phenylurenyl)acetophenone (9). The solid was
recrystallized in EtOH and afforded the title compound: yield
65%: mp 172-173 °C: IR 3.296 (NH), 1.680 (CO) cm-1 1H
;
NMR (DMSO-d6) δ 2.50 (s, 3H, COCH3), 6.99 (t, 1H, H4′, J )
6.43 Hz), 7.28 (t, 2H, H3′-5′, J ) 6.94 Hz), 7.46 (d, 2H, H2′-6′, J
) 6.94 Hz), 7.58 (d, 1H, H4, J ) 6.94 Hz), 7.66 (d, 1H, H6, J )
6.94 Hz), 8.06 (s, 1H, H2), 8.69 (br s, 1H, NH), 8.88 (br s, 1H,
NH); 13C NMR (DMSO-d6) δ 117.89, 1218.92, 122.48, 122.58,
123.34, 129.35, 129.74, 137.94, 140.05, 140.69, 153.10, 198.35.
General Procedure for Preparation of 10-56. Title
compounds were prepared by reacting equimolecular quanti-
ties of urenylacetophenone 6-9 and the corresponding sub-
stituted aldehydes in the presence of an excess sodium
hydroxide (2.5 mmol) in dry methanol (5 mL). The mixture
was stirred at room temperature, and the resulting solids were
collected on a filter and washed three times with cold metha-
nol. In most cases, off-white to bright-yellow solids were formed
within 3-5 h. The product was recrystallized from appropriate
solvents whenever necessary as indicated in Table 3. The yield
of pure product ranged from 54% to 99%.
1-[4′-N-(N′-p-Chlorophenylurenyl)phenyl]-3-(3-pyridi-
nyl)-2-propen-1-one (31). 1H NMR (DMSO-d6) δ 7.32 (d,
H3′′-5′′, J ) 8.91 Hz), 7.46-7.52 (m, H2′′-6′′), 7.67 (d, H3′-5′, J )
8.40 Hz), 7.73 (d, HR, J ) 15.82 Hz), 8.14 (d, H2′-6′, J ) 8.40
Hz), 8.34 (d, H4, J ) 7.91 Hz), 8.60 (d, H6, J ) 8.70 Hz), 9.01
(s, H2), 9.63 (br s, NH); 13C NMR (DMSO-d6) δ 118.05 (C3′-5′),
120.65 (C2′′-6′′), 124.45 (CR), 124.57 (C5), 126.30 (C4′′), 129.14
(C3′′-5′′), 130.71 (C2′-6′), 131.20 (C4), 135.63 (C1), 138.97 (C1′),
140.14 (C1′′), 145.35 (Câ), 150.63 (C2), 151.30 (C6), 152.88 (CO-
(NH)2), 187.77 (CO R,â); CIMS (m/z) 379 [M + H]; IR 3344 (NH),
1654 (CO) cm-1. Anal. (C21H16ClN3O2) C, H, N.
1-[3′-N-(N′-Phenylurenyl)phenyl]-3(3,4,5-trimethoxy-
1
phenyl)-2-propen-1-one (49). H NMR: (DMSO-d6) δ 3.71
(s, 3H, 4-OCH3), 3.86 (s, 6H, 3,5-OCH3), 6.97 (t, 1H, H4′′, J )
7.43 Hz), 7.22 (s, 2H, H2, H6), 7.28 (t, 2H, H3′′-5′′, J ) 7.43 Hz),
7.48 (d, 2H, H2′′-6′′, J ) 7.43 Hz), 7.66-7.85 (m, 3H, H6′, H4′,
Hâ), 8.12 (s, 1H, H2′), 9.01 (br s, 1H, NH), 9.19 (br s, 1H, NH);
13C NMR (DMSO-d6) δ 56.58, 60.81, 106.75, 119.31, 121.97,
123.02, 123.14, 129.39, 129.91, 130.67, 138.65, 139.58, 140.16,
140.43, 145.37, 153.49, 153.54, 190.59; CIMS (m/z) 433 [M +
H]; IR 3.296 (NH), 1.638 (CO) cm-1. Anal. (C25H24N2O5) C, H,
N.
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Acknowledgment. This work was supported by
Consejo de Desarrollo Cient´ıfico y Human´ıstico (CDCH)
Universidad Central de Venezuela (Grants PG 0630-
5126-2.003 and PG 0630-5125-2.003), the National
Institutes of Health (Grants AI 35800 and AI 35707),
and the Medicines for Malaria Venture. P.J.R. is a Doris
Duke Distinguished Clinical Scientist.
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