VOL. 55, 2011
MULTISTAGE ANTIMALARIAL HYBRIDS
4699
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,