Artemisinin and a series of novel endoperoxide antimalarials exert early effects on digestive vacuole morphology

Article abstract of DOI:10.1128/AAC.00609-07

Artermisinin and its derivatives are now the mainstays of antimalarial treatment; however, their mechanism of action is only poorly understood. We report on the synthesis of a novel series of epoxy-endoperoxides that can be prepared in high yields from simple starting materials. Endoperoxides that are disubstituted with alkyl or benzyl side chains show efficient inhibition of the growth of both chloroquine-sensitive and -resistant strains of Plasmodium falciparum. A trans-epoxide with respect to the peroxide linkage increases the activity compared to that of its cis-epoxy counterpart or the parent endoperoxide. The novel endoperoxides do not show a strong interaction with artemisinin. We have compared the mechanism of action of the novel endoperoxides with that of artemisinin. Electron microscopy reveals that the novel endoperoxides cause the early accumulation of endocytic vesicles, while artemisinin causes the disruption of the digestive vacuole membrane. At longer incubation times artemisinin causes extensive loss of organellar structures, while the novel endoperoxides cause myelin body formation as well as the accumulation of endocytic vesicles. An early event following endoperoxide treatment is the redistribution of the pH-sensitive probe LysoSensor Blue from the digestive vacuole to punctate structures. By contrast, neither artemisinin nor the novel endoperoxides caused alterations in the morphology of the endoplasmic reticulum nor showed antagonistic antimalarial activity when they were used with thapsigargin. Analysis of rhodamine 123 uptake by P. falciparum suggests that disruption of the mitochondrial membrane potential occurs as a downstream effect rather than as an initiator of parasite killing. The data suggest that the digestive vacuole is an important initial site of endoperoxide antimalarial activity. Copyright

Full text of DOI:10.1128/AAC.00609-07

Artemisinin and a Series of Novel
Endoperoxide Antimalarials Exert Early
Effects on Digestive Vacuole Morphology
Maria del Pilar Crespo, Thomas D. Avery, Eric Hanssen,
Emma Fox, Tony V. Robinson, Peter Valente, Dennis K.
Taylor and Leann Tilley
Antimicrob. Agents Chemother. 2008, 52(1):98. DOI:
1
0.1128/AAC.00609-07.
Published Ahead of Print 15 October 2007.
Updated information and services can be found at:
http://aac.asm.org/content/52/1/98
These include:
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ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Jan. 2008, p. 98–109
Vol. 52, No. 1
0
066-4804/08/$08.00ϩ0 doi:10.1128/AAC.00609-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Artemisinin and a Series of Novel Endoperoxide Antimalarials Exert
ᰔ
Early Effects on Digestive Vacuole Morphology ‡
1
2
1,3
1
2
Maria del Pilar Crespo, † Thomas D. Avery, † Eric Hanssen, Emma Fox, Tony V. Robinson,
2
2
1,3
Peter Valente, Dennis K. Taylor, and Leann Tilley *
1
3
Department of Biochemistry and ARC Centre of Excellence for Coherent X-ray Science, La Trobe University, Melbourne,
2
Victoria 3086, Australia, and Department of Chemistry, Adelaide University, Adelaide, South Australia 5005, Australia
Received 8 May 2007/Returned for modification 9 July 2007/Accepted 9 September 2007
Artermisinin and its derivatives are now the mainstays of antimalarial treatment; however, their mechanism
of action is only poorly understood. We report on the synthesis of a novel series of epoxy-endoperoxides that
can be prepared in high yields from simple starting materials. Endoperoxides that are disubstituted with alkyl
or benzyl side chains show efficient inhibition of the growth of both chloroquine-sensitive and -resistant strains
of Plasmodium falciparum. A trans-epoxide with respect to the peroxide linkage increases the activity compared
to that of its cis-epoxy counterpart or the parent endoperoxide. The novel endoperoxides do not show a strong
interaction with artemisinin. We have compared the mechanism of action of the novel endoperoxides with that
of artemisinin. Electron microscopy reveals that the novel endoperoxides cause the early accumulation of
endocytic vesicles, while artemisinin causes the disruption of the digestive vacuole membrane. At longer
incubation times artemisinin causes extensive loss of organellar structures, while the novel endoperoxides
cause myelin body formation as well as the accumulation of endocytic vesicles. An early event following
endoperoxide treatment is the redistribution of the pH-sensitive probe LysoSensor Blue from the digestive
vacuole to punctate structures. By contrast, neither artemisinin nor the novel endoperoxides caused alterations
in the morphology of the endoplasmic reticulum nor showed antagonistic antimalarial activity when they were
used with thapsigargin. Analysis of rhodamine 123 uptake by P. falciparum suggests that disruption of the
mitochondrial membrane potential occurs as a downstream effect rather than as an initiator of parasite killing.
The data suggest that the digestive vacuole is an important initial site of endoperoxide antimalarial activity.
Artemisinin is a sesquiterpene lactone antimalarial with a
,2,4-trioxane heterocyclic core that incorporates a peroxide
65). However only 10 structural classes of synthetic endoper-
oxides are currently under investigation (26), which is rather
few, given that artemisinin derivatives are effectively the only
drugs left in our antimalarial armory.
1
linkage that is essential for its activity (44). Artemisinin is of
major importance as a frontline treatment for malaria, partic-
ularly as it is active against chloroquine (CQ)-resistant strains
of Plasmodium falciparum (20, 23, 24, 45, 66, 74). Several
groups have reported on pathways for the synthesis of arte-
misinin, but all require numerous steps and have low yields (5,
Recently, Vennerstrom et al. reported on the synthesis of a
new synthetic peroxide antimalarial called OZ277 (70). OZ277
contains a five-membered 1,2,4-trioxolane, more commonly
known as a secondary ozonide, that is amenable to large-scale
synthesis. OZ277 has pharmacokinetic properties that are su-
perior to those of artemisinin derivatives; however, its half-life
is still only about 2 h (40, 51). OZ277 has recently been sub-
jected to phase II trials (70) but apparently showed a poor
half-life in vivo and is no longer supported by the Medicines
for Malaria Venture (43). This setback, coupled with the in-
evitable development of resistance to any antimalarial that is
employed in the field, makes it important to continue the
development of novel endoperoxides, especially compounds
with potentially longer half-lives or different molecular targets.
We have developed a simple synthetic pathway for the prep-
aration of a novel series of epoxy-endoperoxides (65). The
epoxide and endoperoxide moieties are designed to mimic the
oxygen atom arrangement in artemisinin, and we have previ-
ously generated compounds with considerable antimalarial ac-
tivity (65). In an effort to improve on the antimalarial activity
of this series, we have now investigated the epoxides of a series
of bicyclic endoperoxides in which the carbons adjacent to the
peroxide linkage are tetrasubstituted. This substitution is
known to increase the lifetime of the alkoxy radicals generated
upon homolytic cleavage of the peroxide unit (48) over cases in
which the substituent is H (65) or OH (53). Indeed, it is well
3
5). As a result, artemisinin-based antimalarials are produced
semisynthetically from extracts of Artemisia annua. A period of
over 1 year is needed for the horticultural, harvesting, extrac-
tion, and manufacturing processes (34, 41, 45), which limits the
ease of scale-up of production and makes artemisinin deriva-
tives much more expensive than traditional antimalarials, such
as CQ and sulfadoxine-pyrimethamine. Indeed, artemisinin
combinations are too costly for many patients, and the supply
of counterfeit or inferior drugs is a major problem (4, 45, 71).
Another issue is the fact that artemisinin and its derivatives are
not suitable for prophylaxis or for use as a monotherapy due to
their very short half-lives in vivo. These factors have been the
driving force behind a large number of analogue syntheses (52,
*
Corresponding author. Mailing address: Department of Biochem-
istry, La Trobe University, Plent Rd., Melbourne, 3086 Victoria, Aus-
tralia. Phone: 61-3-94791375. Fax: 61-3-94792467. E-mail: L.Tilley
@
latrobe.edu.au.
†
‡
These authors contributed equally to this work.
Supplemental material for this article may be found at http://aac
.
asm.org/.
ᰔ
Published ahead of print on 15 October 2007.
9
8

VOL. 52, 2008
ENDOPEROXIDE ANTIMALARIALS DISRUPT DIGESTIVE VACUOLES
99
known that metal (Fe)-induced cleavage of the peroxide link-
age to initially form alkoxy radicals followed by ␤-scission to
form downstream carbon radicals is key to the activity of ar-
temisinin (59, 70). Thus, peroxide analogues with hydrogen
atoms on the carbons adjacent to the peroxide linkage will not
have the same propensity to form damaging carbon radicals
downstream, as intramolecular hydrogen atom abstraction will
be dominant in these cases, quenching the oxygen-centered
radicals formed by peroxide bond cleavage (6, 22).
We have determined the antimalarial parasite activities of
the novel epoxy-endoperoxides and investigated their mecha-
nisms of action. Active endoperoxide antimalarials are as-
sumed to interact with reduced heme (ferroprotoporphyrin
IX) or iron to form free-radical products of both the drug and
the heme (27, 43, 49, 52, 60). The radicals are thought to react
with susceptible groups within parasite enzymes and lipids (10,
cells were rinsed and pelleted at 1,650 ϫ g before being postfixed for 1 h with 1%
osmium tetroxide. After the cells were washed, they were stained en block with
% aqueous uranyl acetate and further rinsed before serial dehydration and
embedding in LR-White. In some cases the cells were embedded in 10% gelatin
and refixed with 2.5% glutaraldehyde in 0.1 M sodium cacodylate prior to
dehydration. Sections (70 nm) were prepared, recovered on 200-mesh Formvar-
coated copper grids, and then stained with aqueous uranyl acetate and lead
citrate for examination at 80 kV on a Jeol 2010HC transmission electron micro-
scope.
Digestive vacuole integrity. The pH-sensitive probe LysoSensor Blue-DND192
(Molecular Probes) was used to visualize acidic compartments. Trophozoite-
stage parasite-infected RBCs (strain 3D7) were incubated without drug or were
treated with artemisinin, compound 3a, or compound 3c at 2, 20, or 40 times their
respective IC50 values for 4 or 8 h. The parasitized RBCs were resuspended in
complete medium (3% hematocrit) and incubated with the pH-sensitive probe
LysoSensor Blue (1 ␮M) at 37°C for 20 min. The parasitized RBCs were
mounted wet on a glass slide, covered with a glass coverslip, and imaged within
20 min at ambient temperature (maintained at 20°C) by using an Olympus IX81
inverted microscope with DAPI filter settings. Three hundred infected RBCs
were classified according to whether their digestive vacuole morphology was
2
1
1, 37, 44, 47, 55, 56); however, the exact site of action is still
“
normal” or “redistributed.”
a matter of debate.
ER morphology. Trophozoite-stage parasite-infected RBCs (strain D10) were
A recent report implicates the P. falciparum homologue of
incubated without drug or were treated with thapsigargin, artemisinin, com-
pound 3a, or compound 3c at 10 times their respective IC50 values for 12 h.
Cultures were incubated with 500 nM ER Tracker Blue-White DPX probe for 30
min at 37°C. The cells were resuspended in fresh complete medium, and the ER
fluorescence pattern was examined with a DAPI filter set. Three hundred in-
fected RBCs were classified according to whether their ER morphology was
“normal” or “condensed.”
Mitochondrial integrity. Ring-stage parasite-infected RBCs (strain D10) were
incubated without drug or were treated with artemisinin, compound 3a, or
compound 3c at 2, 20, or 40 times their respective IC50 values for 24 h. Alter-
natively, trophozoite-stage parasites were treated for 4 h. As a control, samples
of untreated parasites were incubated with the ionophores nigericin and mo-
nensin (20 ␮M each) to collapse the membrane potential. An aliquot of rhoda-
mine 123 (final concentration, 0.2 ␮M) was added for 30 min at 37°C; and then
the infected RBCs were washed three times in HEPES-buffered saline, resus-
pended in complete medium, and imaged with a Leica SP2 confocal microscope
or an Olympus BX81 microscope with a ϫ100 oil immersion lenses (1.4 numer-
ical aperture) and fluorescein settings. One thousand infected RBCs were clas-
sified as normal or rhodamine 123 depleted.
2ϩ
the sarco/endoplasmic reticulum (ER) Ca ATPase (SERCA)
as a major downstream target of activated artemisinin in the
parasite cytoplasm (15); however, the synthetic endoperoxide
OZ277 inhibits SERCA much less potently (69). In Saccharo-
myces cerevisiae, components of the mitochondrial electron
transport chain have also been shown to be susceptible to
artemisinin (36); however, it has not been demonstrated if
these are important targets in plasmodia. In this work we have
examined the effects of artemisinin and the most active of the
novel endoperoxide antimalarials on the morphology of the
parasite’s mitochondrion, ER, and digestive vacuole. We con-
clude that disruption of the digestive vacuole function is an
early event in the action of endoperoxide antimalarials.
MATERIALS AND METHODS
General synthetic methods. Solvents were dried by appropriate methods wher-
ever needed. Thin-layer chromatography (TLC) used aluminum sheets (40 by 80
mm) were coated with silica gel 60 F254, and the gels were visualized under
254-nm light or were developed in vanillin or permanganate dip. Flash chroma-
tography was conducted with silica gel 60 with particle sizes of 0.040 to 0.063 mm.
Parasites and assessment of antimalarial activity. D10 and 3D7 are CQ-
sensitive strains of P. falciparum, and K1 is a CQ-resistant strain of P. falciparum
17, 19). P. falciparum-infected erythrocytes (RBCs) were cultured in medium
(
containing 4% human serum and 0.5% GIBCO AlbuMAX I (Invitrogen) in
RPMI 1640 supplemented with hypoxanthine and glutamate, as described pre-
viously (18), by using blood donated by the Red Cross Blood Service, Melbourne,
Australia. The parasites were plated at about a 1% parasitemia (2% hematocrit)
in 96-well trays, and different concentrations of the compounds were added as
serial dilutions in hypoxanthine-free medium from concentrated stocks in meth-
anol or dimethyl sulfoxide. The final solvent concentration was less than 0.1%.
The parasites were incubated for 48 h, with daily replacement of the drug-
1
Melting points (Mps) were uncorrected. H nuclear magnetic resonance (NMR)
1
3
and C NMR spectra were recorded in CDCl
3
solution at either 300 MHz or 600
(77.0 ppm) were used as inter-
MHz, and tetramethylsilane (0 ppm) and CDCl
3
nal standards. All yields reported refer to isolated material judged to be homo-
geneous by TLC and NMR spectroscopy.
1,4-Di-(3-methylphenyl)-1,3-cyclohexadiene (diene 1b). Diene 1b was synthe-
sized by the general procedure outlined by Posner et al. (54). Mp, 99 to 101°C;
3
1
supplemented medium. Growth curves based on the uptake of [ H]hypoxanthine
3
H NMR (300 MHz, CDCl ) ␦ 2.37 (s, 6H), 2.76 (s, 4H), 6.51 (s, 2H), 7.00 to 7.40
1
3
were obtained in triplicate, as described previously (58), and the concentration of
compound required to produce 50% inhibition of growth (IC50) was determined.
Drug interactions were assessed by isobologram analysis (1, 7, 12). Briefly, four
different combinations of artemisinin or the novel endoperoxides (compounds 3a
and 3c) with each other or with thapsigargin (a SERCA inhibitor) were serially
diluted at a fixed ratio, and their effects on parasite growth (strain D10 or 3D7;
(m, 8H); C NMR (75 MHz, CDCl
128.4, 136.0, 138.8, 140.8.
3
) ␦ 21.4, 26.1, 121.6, 122.1, 125.7, 127.9,
1-Bromo-4-fluorobenzene. To a two-necked round-bottom flask equipped with
a condenser and a dropping funnel were added fluorobenzene (5 g; 52 mmol)
and FeCl (100 mg). The mixture was cooled to Ϫ8°C with an ice-salt bath.
3
Bromine (8.5 g; 53 mmol) was then added dropwise over 2 h. Following addition
of bromine, the reaction mixture was heated to 60°C for 1 h. The crude product
was distilled at atmospheric pressure to yield 1-bromo-4-fluorobenzene (6.37 g,
70%) as a colorless oil. Boiling point, 148 to 152°C (150°C in the literature [J.
Oren, 12 March 1997, European patent application EP 761627]).
4-Fluorophenyllithium. To a stirred solution of butyllithium (16 ml; 1.9 M in
hexane) in dry diethyl ether (20 ml) under anhydrous nitrogen at Ϫ30°C was
added dropwise 1-bromo-4-fluorobenzene (5.25 g, 30 mmol) in dry diethyl ether
(20 ml). After addition, the reaction mixture was allowed to warm to room
temperature. The product was used without further purification.
1
% parasitemia) were examined. The IC50 values were calculated for each drug
as though it had been added in isolation. These “apparent” IC50 values were
divided by the IC50 values for the drugs used alone to determine the fractional
inhibitory concentration (FIC). The FIC values for the two drugs used in the
combination were added to give the sums of the FIC values (SFICs). The
experiments were performed in duplicate on 2 or 3 days.
Electron microscopy. Ring-stage parasite-infected RBCs (strain D10 or 3D7)
were incubated without drug or were treated with artemisinin or the novel
endoperoxides (compounds 3a and 3c) at twice their respective IC50 values for
2
4 h. Alternatively, trophozoite-stage parasites were treated with 40 times the
1,4-Di-(4-fluorophenyl)-1,4-cyclohexadiol. To a solution of 4-fluorophenyl-
lithium in diethyl ether (40 ml; 30 mmol) was added a solution of 1,4-cyclohex-
anedione (0.84 g; 7.5 mmol) in diethyl ether (30 ml) over 30 min. The reaction
mixture was brought to reflux for 30 min and cooled in an ice bath, and 10%
IC50 values for 4 or 8 h. The parasites were harvested at the trophozoite stage by
floatation on a Percoll cushion (3), washed, and fixed in 0.1 M sodium cacodylate,
pH 7.4, containing 1% glutaraldehyde and 0.5% paraformaldehyde for 18 h. The

1
00
CRESPO ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
hydrochloric acid (100 ml) was added. The organic layer was separated, and the
aqueous layer was extracted with ethyl acetate. The combined organics were
General procedure for the synthesis of epoxy-endoperoxides (21). To a solu-
tion of endoperoxide 2b (1 equivalent in CH Cl [20 ml/g]) was added 70%
2 2
dried (MgSO
product was purified by flash chromatography to yield 1,4-di-(4-fluorophenyl)-
,4-cyclohexadiol (1.25 g; 55%) as a colorless solid; R , 0.6 (2:3 ethyl acetate-
) ␦ 1.55 (brs, 2OH), 1.69 to 1.82 (m, 4H),
.31 to 2.45 (m, 4H), 7.08 to 7.03 (m, 4H), 7.57 to 7.53.
,4-Di-(4-fluorophenyl)-1,3-cyclohexadiene and 1,4-di-(4-fluorophenyl)-1,4-
4
) and filtered, and the solvent was removed in vacuo. The crude
m-chloroperbenzoic acid (2 equivalents), and the reaction mixture was stirred at
ambient temperature until it was complete by TLC. Dichloromethane was then
1
f
added and the solution was extracted with saturated Na
extraction with saturated NaHCO . The organic layer was dried over MgSO
4
2
S
2
O
3
, followed by
1
hexane); H NMR (300 MHz, CDCl
3
3
and
2
filtered, and the volatile compounds were removed in vacuo. The crude epoxides
were purified by column chromatography.
1
cyclohexadiene (isomer mixture). To a solution of 1,4-di-(4-fluorophenyl)-1,4-
cyclohexadiol (1.1 g; 3.6 mmol) in benzene (50 ml) was added p-toluenesulfonic
acid (20 mg). The resulting reaction mixture was refluxed for 15 min, while the
water that formed was removed by azeotropic distillation. The crystalline product
that formed on cooling was recrystallized from ethanol to give a mixture of the
(؎)(1S,2R,4R,5S)1,5-Di-(3-methylphenyl)-3,6,7-trioxatricyclo[3.2.2.02,4]nonane
(compound 3b). The first isomer was the major product. Yield, 312 mg (59%);
colorless solid; Mp, 143 to 145°C (hexane-dichloromethane); R
, 0.38 (1:9 ethyl
f
1
acetate-hexane); H NMR (600 MHz, CDCl
(m, 2H), 2.39 (s, 6H), 7.17 to 7.18 (m, 2H), 7.30 to 7.33 (m, 6H); C NMR (75
MHz, CDCl ) ␦ 21.5, 30.3, 54.9, 82.3, 122.1, 125.8, 128.6, 129.2, 138.4, 139.6; IR
) ␦ 2.09 to 2.14 (m, 2H), 2.23 to 2.25
3
1
3
1
,3- and 1,4-di-(4-fluorophenyl)-1,3-cyclohexadiene (70:30) (0.84 mg; 87%) as a
yellow solid (G. H. Posner, 30 September 1997, U.S. patent application
,672,624).
,4-Di-(4-fluorophenyl)-1,3-cyclohexadiene (compound 1c). The isomeric mix-
3
Ϫ1
ϩ
(Nujol) 1607, 1588, 890, 879, 784 cm ; MS m/z (ϩEI) 308 (M , 12), 291 (ϩEI,
11), 276 (ϩEI, 36), 248 (ϩEI, 34), 119 (ϩEI, 66), 91 (ϩEI, 100), 65 (ϩEI, 53);
5
ϩ
ϩ
1
HRMS (ϩEI) (M
Na, 331.1310.
(؎)(1S,2S,4R,5R)1,5-Di-(3-methylphenyl)-3,6,7-trioxatricyclo[3.2.2.02,4]nonane
(compound 4b). The second isomer was the minor product. Yield, 141 mg (27%);
colorless solid; Mp, 143 to 145°C (hexane-dichloromethane); R , 0.13 (1:9 ethyl
) ␦ 2.22 to 2.34 (m, 2H), 2.39 (s,
ϩ Na) found, 331.1305; (M ϩ Na) calculated for
ture of 1,3- and 1,4-di-(4-fluorophenyl)-1,3-cyclohexadiene (0.2 g) was refluxed
for 4 h in t-butanol (50 ml) containing potassium t-butoxide (1.4 g). After the
mixture was cooled to room temperature, most of the t-butanol was removed and
water (30 ml) was added. The mixture was extracted with diethyl ether, dried
C H O
20 20 3
f
1
(MgSO
4
), and filtered; and the solvent was removed in vacuo. The crude product
acetate-hexane); H NMR (300 MHz, CDCl
6H), 2.68 to 2.86 (m, 2H), 3.68 (s, 2H), 7.22 to 7.36 (m, 6H), 7.44 (brs, 2H);
NMR (75 MHz, CDCl ) ␦ 21.4, 26.8, 54.3, 77.2, 123.6, 127.8, 128.6, 130.0, 137.0,
3
1
3
was recrystallized from benzene to yield 1,4-di-(4-fluorophenyl)-1,3-cyclohexa-
C
1
diene (compound 1c; 0.19 g; 95%) as a pale yellow solid; H NMR (300 MHz,
3
Ϫ1
ϩ
CDCl
Posner, U.S. patent application 5,672,624).
Methyl 3-(4-methylcyclohexa-1,3-dienyl)propanoate (compound 1d). A solu-
tion of 3-(4-methylcyclohexa-1,4-dienyl)propionic acid (1.833 g, 11.03 mmol)
13) in methanol (30 ml) and concentrated H SO (1 ml) was refluxed overnight.
The majority of the methanol was then removed in vacuo, and ether (50 ml) was
then added and extracted with concentrated NaHCO solution. The organic
compounds were then dried (MgSO ) and filtered, and the volatile compounds
3
) ␦ 2.75 (s, 4H), 6.44 (s, 2H), 7.08 to 7.01 (m, 4H), 7.45 to 7.42 (m, 4H)
138.5; IR (Nujol) 1607, 1588, 881, 786 cm ; MS m/z (ϩEI) 308 (M , 3), 276
(ϩEI, 70), 248 (ϩEI, 28), 119 (ϩEI, 100), 91 (ϩEI, 21), 65 (ϩEI, 50); HRMS
(ϩEI) (M ϩ Na) found, 331.1310; (M ϩ Na) calculated for C20H O Na,
(
ϩ
ϩ
20 3
331.1310.
(
2
4
(؎)(1S,2R,4R,5S)1,5-Di-(4-fluorophenyl)-3,6,7-trioxatricyclo[3.2.2.02,4]nonane
(compound 3c). The first isomer was the major product. Yield, 77 mg (36%); pale
1
yellow solid; Mp, 142 to 144°C (benzene); R
MHz, CDCl
7.50 (m, 2H); C NMR (75 MHz, CDCl
127.4, 135.3, 135.4, 161.3, 165.4; IR (solid) 1610, 1508, 1232, 1225, 1159, 823
, 0.65 (benzene); H NMR (300
3
f
3
) ␦ 2.09 to 2.25 (m, 4H), 3.82 (s, 2H), 7.09 to 7.14 (m, 2H), 7.46 to
4
1
3
were removed in vacuo to give a crude mixture of 1,3- and 1,4-dienes. The diene
mixture was then purified by column chromatography to give a mixture of methyl
3
) 30.2, 55.0, 82.2, 115.8, 116.1, 127.3,
Ϫ1
ϩ
3
-(4-methylcyclohexa-1,3-dienyl)propanoate (compound 1d) and methyl 3-(4-
methylcyclohexa-1,4-dienyl)propanoate (60:40) (1.039 g; 52%) as a colorless oil;
, 0.5 (1:9 ethyl acetate-hexane).
Methyl 3-(4-methylcyclohexa-1,3-dienyl)propanoate (compound 1d). H NMR
300 MHz, CDCl ) ␦ 1.76 (s, 3H), 2.11 (bs, 4H), 2.35 to 2.49 (m, 4H), 3.67 (s,
H), 5.59 (bs, 2H).
Methyl 3-(4-methylcyclohexa-1,4-dienyl)propanoate. 1H NMR (300 MHz,
CDCl ) ␦ 1.67 (s, 3H), 2.27 to 2.32 (m, 2H), 2.42 to 2.48 (m, 2H), 2.58 (bs, 4H),
.67 (s, 3H), 5.40 (bs, 1H), 5.44 (bs, 1H).
General procedure for the synthesis of endoperoxides (42). A solution of the
appropriate 1,3-butadiene (ϳ4 g) in CH Cl (30 ml/g) was photolyzed with three
00-W halogen lamps in the presence of Rose Bengal bis(triethylammonium) salt
100 mg) and oxygen for 6 h. The reaction was performed in a flask fitted with
cm ; MS m/z (ϩEI) 316 (M , Ͻ1), 282 (ϩEI, 16), 271 (ϩEI, 15), 167 (ϩEI, 25),
123 (ϩEI, 100), 109 (ϩEI, 17), 95 (ϩEI, 25); HRMS (ϩEI) (M ϩ H) found,
ϩ
ϩ
R
f
317.0996; (M ϩ H) calculated for C18H F O , 317.0989.
15 2 3
1
(؎)(1S,2S,4R,5R)1,5-Di-(4-fluorophenyl)-3,6,7-trioxatricyclo[3.2.2.02,4]nonane
(
3
(compound 4c). The second isomer was the minor product. Yield, 41 mg (19%);
1
3
pale yellow solid; R
f
, 0.22 (benzene); H NMR (300 MHz, CDCl
3
) ␦ 2.24 to 2.41
(m, 2H), 2.69 to 2.86 (m, 2H), 3.68 (s, 2H), 7.10 to 7.17 (m, 2H), 7.56 to 7.60 (m,
1
3
2H); C NMR (75 MHz, CDCl
3
) 27.1, 54.3, 77.1, 115.9, 116.2, 129.1, 129.2,
3
Ϫ1
3
132.8, 160.8, 165.1; IR (solid) 1759, 1686, 1598, 1504, 1224, 1159, 841, 833 cm
MS m/z (ϩEI) 316 (M , Ͻ1), 282 (ϩEI, 12), 266 (ϩEI, 13), 167 (ϩEI, 25), 123
(ϩEI, 100), 109 (ϩEI, 17), 95 (ϩEI, 25); HRMS (ϩEI) (M ϩ Na) found,
339.0808; (MϩNa) calculated for C18H F O Na, 339.0809.
;
ϩ
ϩ
2
2
ϩ
5
(
14 2 3
(؎)(1R,2S,4R,5R)Methyl 3-(5-methyl-3,6,7-trioxatricyclo[3.2.2.02,4]nonane)pro-
an external cooling jacket, and the temperature was maintained at below 5°C for
the duration of the reaction. The solution was concentrated in vacuo, and the
resulting residue was purified by flash chromatography.
panoate (compound 3d). The first isomer was the minor product. Yield, 103 mg
1
(39%); colorless oil; R
CDCl
2
, 0.45 (3:7 ethyl acetate-hexane); H NMR (300 MHz,
f
3
) ␦ 1.34 (s, 3H), 1.64 to 1.85 (m, 4H), 1.95 to 2.09 (m, 2H), 2.42 to 2.47 (m,
H), 3.34 to 3.39 (m, 2H), 3.70 (s, 3H); C NMR (75 MHz, CDCl ) ␦ 21.9, 25.8,
3
1
3
1
,4-Di-(3-methylphenyl)-2,3-dioxabicyclo[2.2.2]oct-5-ene (compound 2b).
Yield, 0.47 g (21%); colorless solid; Mp, 99 to 101°C (hexane-dichloromethane);
, 0.38 (1:9 ethyl acetate-hexane); 1H NMR (300 MHz, CDCl
) ␦ 1.92 to 2.09
m, 2H), 2.39 (s, 6H), 2.57 to 2.71 (m, 2H), 6.86 (s, 2H), 7.17 to 7.19 (m, 2H), 7.26
2
7.8, 27.9, 30.6, 51.8, 52.8, 53.8, 78.5, 79.9, 172.9; IR (neat) 1737, 1438, 1294,
Ϫ1
ϩ
1197, 1118, 877 cm
1
Na) calculated for C11H16O Na, 251.0895.
; MS m/z (ϩEI) 228 (M , 4), 195 (ϩEI, 30), 137 (ϩEI, 27),
R
f
3
ϩ
09 (ϩEI, 74), 43 (ϩEI, 100); HRMS (ϩEI) (MϩNa) found, 251.0892; (M ϩ
(
ϩ
1
3
to 7.36 (m, 6H); C NMR (75 MHz, CDCl
3
) ␦ 21.5, 29.4, 78.3, 123.1, 127.0,
5
Ϫ1
(؎)(1R,2R,4S,5R)Methyl 3-(5-methyl-3,6,7-trioxatricyclo[3.2.2.02,4]nonane)pro-
1
28.4, 129.2, 136.6, 138.3, 139.4; infrared (IR; Nujol) 1608, 1587, 1077, 931 cm
;
ϩ
panoate (compound 4d). Yield, 113 mg (43%); colorless oil; R , 0.13 (3:7 ethyl
mass spectrum (MS) m/z (with electron ionization [ϩEI]) 292 (M , 11), 260
f
1
acetate-hexane); H NMR (300 MHz, CDCl
3
) ␦ 1.33 (s, 3H), 1.67 to 2.09 (m,
(
(
ϩEI, 100), 160 (ϩEI, 13), 119 (ϩEI, 33); high-resolution mass spectrometry
HRMS) (ϩEI) (M ϩ Na) found, 315.1359; (M ϩ Na) calculated for
1
3
ϩ
ϩ
6H), 2.54 (t, J ϭ 8.0 Hz, 2H), 3.27 (dd, J ϭ 12.0, 5.0 Hz, 2H), 3.69 (s, 3H);
NMR (75 MHz, CDCl ) ␦ 21.5, 27.8, 27.9, 29.0, 31.1, 51.7, 52.1, 52.6, 74.0, 75.6,
73.4; IR (neat) 1737, 1438, 1303, 1197, 885, 725 cm ; MS m/z (ϩEI) 228 (M
12), 196, (ϩEI, 56), 168 (ϩEI, 22), 115 (ϩEI, 44), 43 (ϩEI, 100); HRMS (ϩEI)
C
20 20 2
C H O
Na, 315.1361.
3
Ϫ1
ϩ
1
,
1
,4-Di-(4-fluorophenyl)-2,3-dioxa-bicyclo[2.2.2]oct-5-ene (compound 2c) (54).
1
f
Yield, 51%; white solid; Mp, 152 to 153°C; R , 0.57 (1:4 ethyl acetate-hexane); H
ϩ
ϩ
(
M ϩ Na) found, 251.0895; (M ϩ Na) calculated for C11H16O Na, 251.0895.
NMR (300 MHz) ␦ 1.92 to 2.28 (m, 2H), 2.55 to 2.71 (m, 2H), 6.84 (s, 2H), 7.18
to 7.08 (m, 4H), 7.54 to 7.50 (m, 4H).
5
Methyl 3-(4-methyl-2,3-dioxa-bicyclo[2.2.2]oct-5-en-1-yl)propanoate (com-
pound 2d). Yield, 411 mg (93%); colorless oil; R
f
, 0.28 (1:4 ethyl acetate-hexane);
RESULTS
1
H NMR (300 MHz, CDCl
3
) ␦ 1.38 (s, 3H), 1.44 to 1.60 (m, 2H), 1.98 to 2.17 (m,
1
3
4
H), 2.39 to 2.57 (m, 2H), 3.69 (s, 3H), 6.42 (s, 2H); C NMR (75 MHz, CDCl
3
)
Chemistry. Building on our previous research into epoxy-
␦
1
3
21.3, 28.15, 28.20, 29.5, 30.2, 51.7, 74.8, 76.2, 133.8, 136.6, 173.5; IR (neat) 1737,
endoperoxides as antimalarial drug candidates (64), we have
examined the epoxides of bicyclic endoperoxides 2a to 2d,
which contain tetrasubstituted carbons adjacent to the perox-
ide linkage. Endoperoxides 2a and 2c have previously been
Ϫ1
ϩ
637, 1438, 1378, 1197, 1172, 883 cm ; MS m/z (ϩEI) 212 (M , Ͻ1), 211 (ϩEI,
), 196 (ϩEI, 9), 181 (ϩEI, 100), 148 (ϩEI, 38), 123 (ϩEI, 67), 106 (ϩEI, 54);
ϩ
ϩ
HRMS (ϩEI) (M
ϩ Na) found, 235.0948; (M ϩ Na) calculated for
11 16 4
C H O Na, 235.0946.

VOL. 52, 2008
ENDOPEROXIDE ANTIMALARIALS DISRUPT DIGESTIVE VACUOLES
101
during a 48-h in vitro culture period were determined (Table 1)
and compared with data for CQ and artemisinin and previously
generated monocyclic endoperoxides 6a, 6b, 7a, and 7b (64)
and some novel derivatives, compounds 9c and 10c (62) (Fig. 1,
scheme 2). The most active compounds were also tested
against the CQ-resistant strain (strain K1) (Table 1). The com-
pounds showed activity against the CQ-resistant strain similar
to or higher than that against the CQ-sensitive strain. The
activities of the bicyclic epoxy-endoperoxides and their parent
endoperoxides were significantly better than those of similarly
substituted monocyclic versions; compare the data for com-
pounds 6a and 2b, 6b and 2a, 7a and 3b, or 7b and 3a.
Interaction of the novel endoperoxides with artemisinin. It
was previously reported that the synthetic trioxolane endoper-
oxide (OZ277) interacts antagonistically with artemisinin
against P. falciparum in vitro (69). We have examined the
interactions between artemisinin and compounds 3a and 3c.
The drugs were diluted over a 300-fold range starting with
different fixed ratios of the test compounds (Table 2). The IC50
values for the inhibition of parasite growth (strain D10) were
determined for each combination of drugs (Table 2), and the
data were used to estimate the SFIC values (Table 2) and to
prepare isobolograms (Fig. 2). A concave curve indicates a
synergistic interaction, a convex curve indicates an antagonistic
interaction, and a straight line indicates no interaction (7).
Similarly, the SFIC value gives a numerical indication of an
antagonistic interaction. An SFIC value of greater than 4 is
indicative of strong antagonism. The slightly convex data set
observed for the artemisinin combinations with compound 3a
or 3c and SFIC values slightly greater than 1 indicate no in-
teraction or an indeterminate interaction between these drugs.
Effect on parasite ultrastructure. In an attempt to obtain
more information about the mechanisms of action of the novel
endoperoxides, we examined the effects of artemisinin and the
novel endoperoxides (compounds 3a and 3c) on the parasite
ultrastructure. Trophozoite-stage parasite cultures (strain
FIG. 1. Schemes for the synthesis of endoperoxides. Me, methyl;
iPr, iso-propyl; MePh, methylphenyl; FPh, fluorophenyl; MCPBA,
meta-chloroperbenzoic acid; p-TSA, para-toluenesulfonic acid; NMo,
N-methylmorpholine-N-oxide.
TABLE 1. Biological activities of the novel endoperoxides
Antimalarial activity (IC50 [␮M])
assessed as antimalarials against the NF54 strain of P. falcip-
arum (54). Epoxidation of these endoperoxides gave two iso-
mers with the epoxide trans (compound 3) and cis (compound
Compound
Strain D10
Strain K1
Artemisinin
CQ
0.013 Ϯ 0.003
0.03 Ϯ 0.01
0.011
0.66 Ϯ 0.02
4
) to the peroxide linkage (Fig. 1, scheme 1). Ascaridole (com-
pound 2a) was synthesized by photo-oxidation of ␣-terpinene
28) and was epoxidized as outlined by Turner and Herz (67).
a
6
7
6
7
a
a
b
b
6
a
Ͼ10a
(
Ͼ10
Ͼ10
The stereochemistry of compounds 3a and 4a was then con-
clusively assigned through two-dimensional 2D ROESY (rota-
tional nuclear Overhauser effect spectroscopy) NMR of the
ring-opened derivative of compound 3a, confirming that it had
the stereochemistry tentatively assigned by Turner and Herz
a
2a
0.36 Ϯ 0.04
0.29 Ϯ 0.06
0.80 Ϯ 0.02
0.79 Ϯ 0.01
0.25 Ϯ 0.04
1.96 Ϯ 0.29
0.29 Ϯ 0.06
0.1 Ϯ 0.01
0.36 Ϯ 0.06
5 Ϯ 2.2
0.15
0.12 Ϯ 0.02
0.37
3
4
2
3
a
a
b
b
0.39 Ϯ 0.12
(67). Endoperoxide 2b was synthesized as outlined by Posner
4b
2c
c
c
d
d
4d
c
et al. (54), with epoxidation performed as was done with the
ascaridole derivatives (Fig. 1, scheme 1) and stereochemical
determination through two-dimensional ROESY NMR of the
epoxides. Synthesis of the 4-fluoro diene, compound 1c, was
achieved through a route alternative to that used by Posner et
al. (54).
0.23 Ϯ 0.01
0.08 Ϯ 0.02
0.16 Ϯ 0.04
3
4
2
3
1.4 Ϯ 0.46
7.2
6 ϩ 0.9
7.2 Ϯ 0.3
9
1
0c
Antimalarial activity. The abilities of the endoperoxides to
inhibit the growth of CQ-sensitive strain D10 of P. falciparum
a
Data are from Taylor et al. (65).

1
02
CRESPO ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
TABLE 2. Interaction of two of the novel endoperoxides with artemisinin
Assays with compound 3a
Apparent IC50 (␮M)
Assays with compound 3c
Starting conc (␮M)
of artemisinina
IC50 (␮M)
Starting conc (␮M)
Starting conc (␮M)
of compound 3c
of compound 3aa
SFIC
SFIC
Artemisinin Compound 3a
Artemisinin
Compound 3c
0.04
0.02
0.01
0.005
0.09
0.18
0.35
0.70
0.0016
0.0012
0.0010
0.0002
0.032
0.10
0.32
0.29
1.30 Ϯ 0.48
1.22 Ϯ 0.18
1.22 Ϯ 0.14
1.11 Ϯ 0.03
0.035
0.075
0.15
0.038 Art
0.023
0.051
0.092
0.095
1.84 Ϯ 0.43
1.36 Ϯ 0.11
1.25 Ϯ 0.13
0.98 Ϯ 0.03
0.048 ␮M Art
0.08 ␮M Art
0.15 ␮M Art
0.30
a
3
Twofold dilutions of the drugs combined in a fixed ratio were added to P. falciparum parasites (strain D10), and the incorporation of [ H]hypoxanthine was
determined over a 48-h incubation period. The apparent IC50 values for each of the drugs were estimated as though each drug had been added in isolation and were
divided by the IC50 for the drug used alone to give the FICs. The individual FICs were used to calculate the SFICs. The results represent the means Ϯ standard
deviations of experiments performed in triplicate on three separate days. The IC50 values for the individual drugs determined for the artemisinin and compound 3a
set were 0.011 Ϯ 0.003 ␮M for artemisinin and 0.24 Ϯ 0.05 ␮M for compound 3a, and for the IC50 values for the individual drugs determined for the artemisinin and
compound 3c set were 0.015 Ϯ 0.004 ␮M for artemisinin and 0.11 Ϯ 0.02 ␮M for compound 3c.
D10) were treated for 4 or 8 h with 40 times the IC50 value
determined from the 48-h growth assay) and examined by
transmission electron microscopy. Alternatively, ring-stage
parasite cultures were treated for 24 h with approximately
twice the IC50 value.
In control trophozoite-stage parasites (Fig. 3a and i; see Fig.
S1 in the supplemental material), a dominant feature is the
presence of hemozoin crystals contained within a clearly de-
lineated digestive vacuole. Large vesicles containing hemoglo-
bin taken up from the host cell cytoplasm (endocytic vesicles)
are seen as more darkly staining structures. The parasite cyto-
plasm is heavily dotted with ribosomes and contains a clearly
defined nucleus. Trophozoite-infected RBCs treated for 4 h
with high concentrations of either artemisinin or compound 3a
or 3c did not exhibit a major restructuring compared with the
structure of the control cells; however, there was a loss of
digestive vacuole integrity in some artemisinin-treated cells
After 8 h of treatment with the endoperoxide antimalarials,
more dramatic effects on parasite morphology were observed.
In many of the artemisinin-treated parasites, the digestive vac-
uole membrane was disrupted and the hemozoin crystals were
in contact with the parasite cytoplasm (Fig. 3b; see Fig. S1f in
the supplemental material). By contrast, trophozoites treated
with the novel endoperoxides showed an accumulation of un-
digested hemoglobin-containing endocytic vesicles, many of
which appeared to be located within the digestive vacuole
membrane (Fig. 3c and d; see Fig. S1g and h in the supple-
mental material). After the 8-h incubation period, some cells
had reached the schizont stage (Fig. 3e to h). Control schizonts
showed the developing merozoites with clearly defined nuclei,
darkly staining rhoptries, and dense granules (Fig. 3e). Arte-
misinin-treated schizonts again showed a loss of the digestive
vacuole membrane and aberrant merozoite morphology (Fig.
3f). Schizonts incubated with compounds 3a and 3c showed
highly misshapen merozoites with uneven staining of the nu-
cleus (Fig. 3g and h) and an accumulation of darkly stained
vesicular structures (Fig. 3g, asterisk).
(
(see Fig. S1b in the supplemental material) and a buildup of
endocytic vesicles in some compound 3a- and compound 3c-
treated cells (see Fig. S1c and d in the supplemental material).
FIG. 2. Interaction of artemisinin and two novel endoperoxides in inhibiting parasite growth. Isobolograms were constructed from the IC50
values in Table 2. For each drug combination, FICs were calculated by dividing the measured apparent IC50 values for the individual drugs in the
different combinations of artemisinin with compound (Cmpd) 3a (A) or compound 3c (B) by the IC50 values obtained when the drugs were used
alone.

VOL. 52, 2008
ENDOPEROXIDE ANTIMALARIALS DISRUPT DIGESTIVE VACUOLES
103
FIG. 3. Transmission electron microscopy analysis of endoperoxide-treated parasitized RBCs. Trophozoite-stage parasite-infected RBCs
strain D10) were incubated with no drug (a and e) or 40 times the IC50 values of artemisinin (b and f), compound 3a (c and g), or compound 3c
d and h) for 8 h. Alternatively, ring-stage parasite-infected RBCs were incubated with no drug (i) or two times the IC50 values of artemisinin (j),
(
(
compound 3a (k), or compound 3c (l) for 24 h. Sections through the control trophozoite-stage parasites (a and i) show endocytic vesicles (EV) and
typical hemozoin crystals (Hz) within a digestive vacuole (DV). The parasite cytoplasm is dotted with ribosomes and has a defined nucleus (N).
The mitochondrion (M) is visible in some sections (i). Schizonts (e) show well-defined nuclei and developing apical organelles, rhoptries (R), and
dense granules (DG). After an 8-h treatment with a high concentration of artemisinin, both trophozoite-stage parasites (b) and schizont-stage
parasites (j) show the loss of digestive vacuole integrity. Many of the infected RBCs treated for 24 h with artemisinin (j) show a substantial loss
of membranous features, with individual hemozoin crystals in contact with the parasite cytoplasm and the formation of vacuoles (V). Trophozoites
treated for 8 h with 40 times the IC50 value of compound 3a (c) or compound 3c (d) show an accumulation of undigested endocytic vesicles in the
digestive vacuole. Schizonts in the samples treated for 8 h with compound 3a (g) or compound 3c (h) show merozoites with an aberrant morphology
and uneven staining of the nucleus. There is an accumulation of dense vesicles (g, asterisk) in many cells. Infected RBCs treated for 24 h with two
times the IC50 value of compound 3a show an accumulation of endocytic vesicles (EV) and myelin bodies (MB) (see Fig. S1k in the supplemental
material). Compound 3c causes less obvious damage (l), but development seems to be inhibited and very few schizonts are observed. Bar, 1 ␮m.
Additional images are presented in Fig. S1 in the supplemental material.
Ring-stage parasite-infected RBCs treated for 24 h with
artemisinin at two times its IC50 value (Fig. 3j; see Fig. S1j in
the supplemental material) advanced to the trophozoite stage
but showed a marked loss of organellar structures accompa-
nied by the disintegration of many of the major membrane-
bound features. In particular, the loss of the digestive vacuole
membrane often left individual hemozoin crystals in direct
contact with the parasite cytoplasm. Vacuoles were observed in
some cells (Fig. 3j). Infected RBCs treated for 24 h with twice
the IC50 value of compound 3a or 3c (Fig. 3k and l) showed a
less obvious loss of membrane structures; however, a major
feature was the appearance of large vacuoles and myelin bod-
ies as well as hemoglobin-containing endocytic vesicles (Fig. 3k
and l; see Fig. S1k and l in the supplemental material). Similar
effects of the different drug treatments on parasite morphology
were observed with strain 3D7 parasites.
damage to different organelles within infected RBCs. In an
effort to visualize damage to the digestive vacuole we have
made use of the pH-sensitive probe LysoSensor Blue. This
probe is a weak base that accumulates in acidic compartments
(38, 72). In mid-trophozoite-stage parasites, the probe is con-
centrated in the digestive vacuole (as judged by the overlap
with the hemozoin location); however, there is also some weak
labeling of the parasite cytoplasm (Fig. 4A, panels a and e).
The fluorescence profile was independent of the time of incu-
bation with LysoSensor Blue, indicating that the weak base
probe does not induce a change in the pH of the labeled
compartment. The LysoSensor Blue fluorescence also colocal-
izes with the fluorescence of plasmepsin-green fluorescent
protein (31) in transfected parasites expressing this digestive
vacuole-located chimeric protein (data not shown).
The infected RBCs were treated with artemisinin, com-
pound 3a, or compound 3c for 4 or 8 h at different concentra-
tions. Even after only 4 h of treatment with artemisinin, a
substantial proportion of the parasite population showed al-
terations in LysoSensor Blue labeling. The probe was redis-
Effect of endoperoxides on digestive vacuole integrity. Be-
cause of the difficulty of quantitating the effects of the com-
pounds on the ultrastructural morphology, we have developed
a series of fluorescence microscopy-based techniques to assess

1
04
CRESPO ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
FIG. 4. Analysis of digestive vacuole integrity in endoperoxide-treated parasitized RBCs. Strain D10 parasites at the trophozoite stage were
incubated with no additions or 2, 20, or 40 times the IC50 values of artemisinin, compound 3a, or compound 3a for 4 or 8 h. LysoSensor Blue was
used to probe acidic compartments, with imaging performed by fluorescence microscopy. (A) Bright-field, fluorescence signals and overlays of
control and drug-treated parasites. (B and C) The LysoSensor Blue labeling pattern in infected RBCs (300 cells) in cultures that were drug treated
for 4 (B) or 8 h (C) was classified as normal or redistributed. Data are normalized relative to those for the control, to which no drug was added,
and represent the means Ϯ standard deviations of data from three separate experiments. cont, control; DV, digestive vacuole; Art, artemisinin.
tributed to punctate structures either associated with the di-
gestive vacuole or in the parasite cytoplasm (Fig. 4A, panel b).
After 8 h at 20 to 40 times the IC50 value of artemisinin, most
of the cells in the population showed an altered distribution of
the fluorescent probe (Fig. 4A, panel f), and many parasites
showed a complete loss of the fluorescence signal (data not
shown). The novel endoperoxides, at 20 to 40 times their re-
spective IC50 values, also caused substantial changes to the
distribution of the LysoSensor Blue probe (Fig. 4A, panels c, d,
g, and h), with the LysoSensor Blue-stained compartments
often appearing fragmented or dispersed. This suggests that
compounds 3a and 3c also disrupt the function of the digestive
vacuole. The redistribution and loss of LysoSensor Blue may
indicate a restructuring of this compartment and the loss of the
digestive vacuole pH gradient, respectively. The punctate
structures that accumulate the pH probe in compound 3a- and
the different compounds on LysoSensor Blue staining (Fig. 4B
and C). After 8 h of treatment with 20 to 40 times the IC50
value of artemisinin, more than 80% of the parasites showed
altered digestive vacuole staining. Similar effects were ob-
served with the novel endoperoxides. Treatment with com-
pound 3a for 8 h at only two times its IC50 value resulted in an
altered distribution of the pH sensor in more than 80% of the
parasites (Fig. 4C), with a complete loss of the fluorescence
signal in many parasites (data not shown). The data further
indicate that disruption of the integrity of the digestive vacuole
may be an early event in the mechanism of action of endoper-
oxide antimalarials.
Effect of endoperoxides on ER morphology and interactions
2
؉
with ER Ca ATPase inhibitors. Recently, it was shown that
artemisinin inhibits the activity of the ER protein SERCA
(also known as PfATPase6) in an oocyte expression model
(68), and it was proposed that SERCA is a major downstream
target of artemisinin (15, 68). We have examined the abilities
of thapsigargin (a sesquiterpene lactone that is a specific
3
c-treated parasites may represent the accumulated endocytic
vesicles observed by electron microscopy.
We have made a semiquantitative analysis of the effects of

VOL. 52, 2008
ENDOPEROXIDE ANTIMALARIALS DISRUPT DIGESTIVE VACUOLES
105
FIG. 5. Analysis of morphology of the ER in endoperoxide-treated parasitized RBCs. Strain D10 parasites at the trophozoite stage were
incubated with (A) no additions or 10 times the IC50 values of (B) thapsigargin, (C) artemisinin, (D, upper panel) compound 3a, or (D, lower panel)
compound 3c for 12 h. ER Tracker was used to label the ER before imaging by fluorescence microscopy. (B) Fluorescence images of infected RBCs
(
300 cells) were classified as normal or condensed. Data are normalized relative to those for the control, to which no drug was added, and represent
the means Ϯ standard deviations of data from three separate experiments.
SERCA inhibitor) and the endoperoxides to alter the mor-
phology of the ER as defined by the ER-selective probe ER
Tracker Blue-White. The ER appears as a reticular network in
the cytoplasm of the parasite (Fig. 5A). After a 12-h treatment
of trophozoite-stage parasites with 10 times the IC50 value of
thapsigargin, a marked contraction and reorganization of the
ER was observed (Fig. 5B). By contrast, artemisinin, com-
pound 3a, and compound 3c at 10 times their respective IC50
values had no major effect on the morphology of the ER (Fig.
prepare isobolograms (Fig. 6). The slightly concave data set
observed for the thapsigargin-artemisinin combination and
SFIC values slightly less than 1 do not support the previously
reported antagonism between these drugs. Similar results were
obtained for a combination of artemisinin and thapsigargin
against strain 3D7 (data not shown). Similarly, we found no
evidence for an interaction between compounds 3a and 3c and
thapsigargin in strain D10 (Table 3; Fig. 6B and C). The same
result was obtained for compounds 3a and 3c with thapsigargin
in strain 3D7 (data not shown).
5
C and D). We made a semiquantitative analysis of the effects
of the different compounds (Fig. 5E). The effect of thapsigar-
gin on the morphology of the ER was much more marked than
that of the endoperoxides. This suggests that the endoperox-
ides may exert their activities by a mode of action different
from that of thapsigargin.
It was previously reported that thapsigargin antagonizes the
plasmocidal activity of artemisinin (68). We examined the in-
teractions between thapsigargin and artemisinin and between
thapsigargin and compounds 3a and 3c. We found that thap-
sigargin inhibited the parasite growth of strains D10 and 3D7
of P. falciparum, with IC50 values of 7.2 Ϯ 1.6 ␮M and 4.2 Ϯ 0.5
Effects of endoperoxides on mitochondrial morphology and
membrane potential. Components of the mitochondrial elec-
tron transport chain in yeast have been shown to be susceptible
to artemisinin (36). To further examine the mode of action of
artemisinin and the novel endoperoxides, we have used rho-
damine 123, a membrane potential-dependent probe, to detect
mitochondrial damage. In control infected RBCs, rhodamine
123 accumulated within the slender branched mitochondrion
of P. falciparum (Fig. 7A), as reported previously (14). Treat-
ment of infected RBCs with monesin and nigericin dissipates
the membrane potential and completely abrogates the fluores-
cence signal (Fig. 7B). By contrast, the treatment of infected
RBCs for 4 h with 40 times the IC50 values of artemisinin and
compound 3a had no obvious effect on the morphology of the
mitochondrion (Fig. 7C and D). To obtain a more quantitative
␮M, respectively. These values are slightly higher than the
value previously reported for strain 3D7 (68). We undertook
an analysis of the interactions of these compounds (against
strain D10) to estimate the SFIC values (Table 3) and to
TABLE 3. Interaction of artemisinin and two novel endoperoxides with thapsigargin
Assays with artemisinin
Assays with compound 3a
Assays with compound 3c
Starting
concn
Starting
concn
Starting
concn
(␮M) of
compound
3c
Apparent IC50 (␮M)
Apparent IC50 (␮M)
Apparent IC50 (␮M)
(
␮M) of a Artemisinin
SFIC
(␮M) of
compound
SFIC
SFIC
thapsigargin concn (␮M)
Compound
Compound
Thapsigargin
3c
Artemisinin Thapsigargin
Thapsigargin
3
a
3
a
2
5
.5
.0
0.04
0.02
0.01
0.010
0.0072
0.0054
0.31
0.83
2.33
1.04
0.83
0.85
1.6
0.8
0.4
0.14
0.14
0.079
0.22
0.93
2.0
0.8
0.6
0.10
0.068
0.048
0.42
1.12
3.02
0.97
0.77
0.84
0.8
0.3
1
0.0
0.71
0.15
a
Twofold dilutions of the drugs combined in a fixed ratio were added to P. falciparum parasites (strain D10). The apparent IC50 values for each of the drugs were
estimated and used to calculate the SFIC values. The results represent the mean values for an experiment performed in duplicate. The IC50 values for the individual
drugs determined for this data set were 7.4 ␮M for thapsigargin, 0.010 ␮M for artemisinin, 0.18 ␮M for compound 3a, and 0.11 ␮M for compound 3c.

1
06
CRESPO ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
FIG. 6. Interaction of endoperoxide and thapsigarin in parasite killing. Isobolograms were constructed from the IC50 values in Table 3. For each
drug combination, the FICs were calculated by dividing the measured apparent IC50 values for individual drugs in the different combinations of
thapsigargin with artemisinin (A), compound 3a (B), or compound 3c (C) by the IC50 values obtained when the drugs were used alone.
analysis of the effects of the endoperoxides, artemisinin and
compound 3a were added at concentrations corresponding to
sites were then returned to culture for 24 and 4 h, respectively,
before they were stained with rhodamine 123. Trophozoite-
stage parasites treated for 4 h with the drugs showed relatively
little effect on mitochondrial potential even at the highest drug
2
, 20, and 40 times their IC50 values to parasites in the ring or
trophozoite stage, and the ring- and trophozoite-stage para-
FIG. 7. Analysis of mitochondrial function in endoperoxide-treated parasitized RBCs. Strain D10 trophozoite-stage parasite-infected RBCs were
incubated with (A) no additions, (B) the ionophores nigericin and monensin (20 ␮M each), or 40 times the IC50 values of (C) artemisinin or
(D) compound 3a for 4 h. Rhodamine 123 (0.2 ␮M) was used to stain the mitochondrion for confocal microscopy. (E) Trophozoite-stage parasites were
incubated for 4 h or (F) ring-stage infected RBCs were incubated for 24 h with no drug or with 2, 20, or 40 times the IC50 values of artemisinin or
compound 3a. Infected RBCs (1,000 cells) were counted and classified as normal or rhodamine 123 depleted by fluorescence microscopy. Data are
normalized relative to those for the control, to which no drug was added.

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ENDOPEROXIDE ANTIMALARIALS DISRUPT DIGESTIVE VACUOLES
107
concentrations examined (Fig. 7E), indicating that the loss of
mitochondrial function is not an early event in the action of
either artemisinin or compound 3a. After treatment of the
ring-stage parasites for 24 h with artemisinin or compound 3a
at twice the IC50 value, the mitochondrial membrane potential
appeared to be at least partially intact (Fig. 7F), despite the
significant disruption to the membrane structures observed by
electron microscopy. This suggests that disruption of the mi-
tochondrion is probably not the mode of action of either drug.
Higher concentrations of the drug did impair rhodamine 123
accumulation (Fig. 7F), but this is probably consequent upon
rather than responsible for parasite killing.
of an ester group on the alkyl side chain significantly decreased
the level of activity (compounds 2 to 4d).
The main advantage of our novel class of compounds is their
ease of synthesis (and, thus, low cost). We have shown that the
trans-epoxide derivatives exhibit maximum activity, indicating
that the position of the epoxide oxygen plays an important role
in determining the activity of the endoperoxide. This is in
keeping with the suggestion that the activated endoperoxides
exert their lethal effects by binding to and damaging particular
proteins rather than by causing more general damage as rap-
idly diffusing free radical species. These epoxy-endoperoxides
are readily synthesized and represent good lead compounds for
the development of highly effective and inexpensive endoper-
oxide antimalarials; however, further development of these
compounds will require the demonstration of their activity in
vivo.
DISCUSSION
We have generated a novel series of bicyclic endoperoxides
that inhibit parasite growth at IC50 values down to 80 nM. This
is a significant improvement compared with the findings for
our previously synthesized monocyclic compounds, compounds
The compounds provide us with a useful tool with which the
mechanism(s) of action of endoperoxide antimalarials can be
studied. The precise mode of action of artemisinin and its
derivatives is still a matter of some debate; however, it has
1
4
6
a, 6b 7a, and 7b (Table 1), and supports our suggestion that
been shown that [ C]artemisinin accumulates in the digestive
vacuole and mitochondria and that artemisinin treatment in-
duces morphological changes to the mitochondria, rough ER,
nuclear envelope, and digestive vacuole (39, 44).
tetrasubstitution of the carbons adjacent to the peroxide link-
age is important for antimalarial activity. The most active com-
pounds (compounds 3a to 3c; 0.08 to 0.29 ␮M) were less potent
than artemisinin (ϳ10 nM) and were also about threefold less
active than CQ against the CQ-sensitive strain (0.03 ␮M);
however, they were much more active than CQ against CQ-
resistant strains (IC50 value, 0.66 ␮M; Table 1) and their ac-
tivity was similar to or better than that of sulfadoxine (ϳ0.1
To investigate the likely mode of action of the novel en-
doperoxides, we have examined their effects on the cellular
ultrastructure. Given that activated endoperoxide antimalari-
als are thought to exert their activity by alkylating specific
protein or lipid targets, we reasoned that this is likely to cause
early morphological alterations to the organelle in which the
target is located. Indeed, when trophozoite-infected RBCs
were treated for 4 to 8 h with high concentrations of artemisi-
nin, we observed a loss of digestive vacuole integrity in many
cells. This suggests that artemisinin exerts its activity by alkyl-
ating the protein and lipid components of the digestive vacuole
membrane. This suggestion is supported by the findings of a
number of previous studies (8, 9, 39, 50, 61). A longer incuba-
tion of infected RBCs with two times the IC50 value of arte-
misinin caused a marked disintegration of many of the major
membrane-bound features. This general loss of membrane
structure in artemisinin-treated cells has also been noted pre-
viously (29, 30, 39).
Interestingly, trophozoites treated for 4 to 8 h with high
concentrations of compound 3a or 3c did not show a loss of
digestive vacuole integrity but did exhibit a buildup of undi-
gested endocytic vesicles within the digestive vacuole. Schiz-
ont-stage parasites also showed abnormal nuclear staining and
morphology. A buildup of hemoglobin-containing vesicles in
the parasite cytoplasm or in the digestive vacuole has been
observed previously upon the treatment of infected RBCs with
quinolines (16, 25, 73), and hemoglobin accumulation in the
digestive vacuole is observed upon treatment with protease
inhibitors (63). The buildup of intact endocytic vesicles within
the confines of the digestive vacuole suggests that the endoper-
oxides may inhibit the phospholipases that are needed to de-
grade the membranes of endocytic vesicles (32, 33). In contrast
to artemisinin, longer periods of incubation with lower con-
centrations of the novel endoperoxides did not cause an ex-
tensive loss of membranous features, indicating that they kill
parasites without complete disruption of the cells. A major
␮
(
2
M for sensitive strains and up to 10 ␮M for resistant strains)
57). Two of the parent endoperoxide compounds (compounds
a and 2c) have previously been tested for their antimalarial
activities against a CQ-sensitive strain (54). The IC50 value
reported here for compound 2c (0.23 to 0.29 ␮M) is somewhat
higher than the value (0.07 ␮M) reported previously. This may
reflect the shorter growth inhibition time and different strain of
P. falciparum used in our assay, as our IC50 value for CQ
against D10 was also higher (0.03 ␮M) than the value obtained
by Posner et al. (0.005 ␮M) (54). The IC50 value reported here
for compound 2a (0.36 ␮M) is somewhat lower than the value
(0.65 ␮M) reported previously, but the authors noted that this
compound was volatile and that its activity may have been
underestimated (54).
Interestingly, the trans isomers of the bicyclic endoperoxides
were more active than the cis isomers. By contrast, we previ-
ously found that cis-epoxides of the monocyclic endoperoxides
were more active than their trans counterparts (64). This sug-
gests that the spatial placement of the epoxide oxygen is im-
portant for binding to a putative endoperoxide target but that
the optimal position depends on other substituents on the
endoperoxide ring system. The endoperoxide parents of the
bicyclic epoxy-endoperoxides showed significantly lower levels
of activity than the trans-epoxides but levels of activity similar
to or even better than those of the cis-epoxides. This suggests
that it is the position of the endoperoxide oxygen rather than
its electron-withdrawing effects that determines its increased
activity. Dihydroxy compound 9c and its acetonide derivative,
compound 10c (64), showed much lower levels of activity,
indicating that an epoxy substituent is more effective at pro-
moting activation of the endoperoxide. Similarly, the inclusion

1
08
CRESPO ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
ultrastructural feature was the accumulation of endocytic ves-
icles, vacuoles. and myelin bodies. Multilamellar structures
have also been reported previously following the treatment of
parasites with quinoline antimalarials (46). Thus, the data in-
dicate that both artemisinin and the novel endoperoxides have
an early effect on digestive vacuole function; however, this
effect may have a different molecular basis.
We also used a fluorescent pH probe to monitor the integrity
of the digestive vacuole. We observed substantial alterations in
the distribution of the probe after only a few hours of treat-
ment with physiologically relevant concentrations of artemisi-
nin or the novel endoperoxides. While it remains possible that
the reorganization of the digestive vacuole is a downstream
consequence of damage to a protein in another compartment,
our work supports the findings of previous studies implicating
the digestive vacuole as a possible site of action of artemisinin
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