Novel Endoperoxide Antimalarials: Synthesis, Heme Binding, and Antimalarial Activity

Article abstract of DOI:10.1021/jm0305319

We report the synthesis of a series of novel epoxy endoperoxide compounds that can be prepared in high yields in one to three steps from simple starting materials. Some of these compounds inhibit the growth of Plasmodium falciparum in vitro. Structure-act

Full text of DOI:10.1021/jm0305319

J . Med. Chem. 2004, 47, 1833-1839
1833
Novel En d op er oxid e An tim a la r ia ls: Syn th esis, Hem e Bin d in g, a n d An tim a la r ia l
Activity
Dennis K. Taylor,*,† Thomas D. Avery,† Ben W. Greatrex,† Edward R. T. Tiekink,‡ Ian G. Macreadie,§
Peter I. Macreadie,§ Adam D. Humphries,# Martha Kalkanidis,# Emma N. Fox,# Nectarios Klonis,# and
Leann Tilley*,#
Department of Chemistry, Adelaide University, South Australia, 5005, Australia, Department of Chemistry,
National University of Singapore, 117543, Singapore, CSIRO Health Sciences and Nutrition, 343 Royal Parade, Parkville,
Victoria, 3052, Australia, and Department of Biochemistry, La Trobe University, Melbourne, Victoria, 3086, Australia
Received October 21, 2003
We report the synthesis of a series of novel epoxy endoperoxide compounds that can be prepared
in high yields in one to three steps from simple starting materials. Some of these compounds
inhibit the growth of Plasmodium falciparum in vitro. Structure-activity studies indicate that
an endoperoxide ring bisubstituted with saturated cyclic moieties is the pharmacophore. To
study the molecular basis of the action of these novel antimalarial compounds, we examined
their ability to interact with oxidized and reduced forms of heme. Some of the compounds
interact with oxidized heme in a fashion similar to chloroquine and other 4-aminoquinolines,
while some of the compounds interact with reduced heme. However, the level of antimalarial
potency is not well correlated with these activities, suggesting that some of the endoperoxides
may exert their antimalarial activities by a novel mechanism of action.
In tr od u ction
Malaria is a debilitating parasitic disease that is
responsible for the deaths of about two million children
each year. Artemisinin (Figure 1) has become increas-
ingly important as a malaria treatment because of the
fact that cheap alternatives such as chloroquine (CQ,
Figure 1) and Fansidar (S/P, sulfadoxine/pyrimethamine)
F igu r e 1. Structures of artemisinin and chloroquine.
have become ineffective with the emergence of drug-
resistant organisms. However, the use of artemisinin
is somewhat limited because of its relatively high cost,
parasite enzymes and lipids.2,9-11 Very recently, Eck-
limited production to GMP standards, and reports of
toxicity.1,2 The current routes for the total chemical
synthesis of artemisinin (qinghaosu) remain too complex
for commercial production.3-6 It is currently prepared
by large-scale extraction from Artemisia annua (sweet
wormwood), and derivatives such as artemether, arte-
sunate, arteether, and dihydroartemisinin are prepared
semisynthetically from the purified extract. Difficulties
have been encountered in the production of high-quality
material.1
Artemisinin is a sesquiterpene lactone that contains
a 1,2,4-trioxane ring system. The endoperoxide bond is
an essential feature of artemisinin and its active deriva-
tives. It has been proposed that active endoperoxides
accumulate in the parasite cytosol and membranes and
interact with the reduced form of heme (ferroprotopor-
phyrin IX, FP-Fe(II)7-9). In this reducing environment,
the peroxide moiety is thought to react with FP-Fe(II)
to form a cytotoxic carbon-centered radical intermediate
that further reacts with susceptible groups within
stein-Ludwig et al.12 showed that the P. falciparum
homologue of the sarco/endoplasmic reticulum Ca2+
ATPase (SERCA) is likely to be the major downstream
target of activated artemisinin in the parasite cytosol.
-
By contrast, CQ and other quinoline antimalarial
drugs accumulate in the acidic vacuole of the malaria
parasite where they are thought to interact with oxi-
dized heme (ferriprotoporphyrin IX, FP-Fe(III)) and to
inhibit the formation of â-hematin (the form of FP
present in the malaria pigment, hemozoin).13-15 Thus,
the quinoline antimalarial drugs are thought to exert
their activities by inhibiting FP detoxification, causing
a buildup of toxic FP molecules that eventually inhibit
parasite enzymes and destroy the integrity of the
membranes.
In this work, we extend our recently published
procedure for the synthesis of the relatively unknown
epoxy endoperoxides that can be readily generated from
inexpensive starting materials.16 We have examined the
abilities of these compounds to inhibit the growth of
malaria parasites in vitro and shown that a subset of
compounds possess substantial antimalarial activity.
We have shown that some of the compounds interact
with oxidized or reduced FP, which may provide some
information about their mechanism of action.
* To whom correspondence should be addressed. For D.K.T.: phone,
+61-8-83035494; fax, +61-8-83034358; e-mail, dennis.taylor@adelaide.
edu.au. For L.T.: phone, +61-3-94791375; fax, +61-3-94792467; e-mail,
l.tilley@latrobe.edu.au.
† Adelaide University.
‡ National University of Singapore.
§ CSIRO Health Sciences and Nutrition.
#
La Trobe University.
10.1021/jm0305319 CCC: $27.50 © 2004 American Chemical Society
Published on Web 02/27/2004

1834 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 7
Taylor et al.
Sch em e 1a
a
(i) O2, Rose Bengal bis(triethylammonium) salt, CH2Cl2, 7 h; (ii) m-CPBA, CH2Cl2, room temp; (iii) Co(II)salen or NEt3.
Resu lts
Syn th esis. We have developed a robust synthetic
procedure for the ready construction of endoperoxides
of types 4a -n , 9, and 11 and epoxy endoperoxides (5,
6, 10, and 12) according to generalized Scheme 1. Key
features include a [4 + 2] cycloaddition of singlet oxygen
with the appropriate 1,3-butadiene 3a -n to generate
the core of the endoperoxide ring and afford compounds
of type 4a -n . Further oxidation with m-CPBA at
ambient temperature furnishes the epoxy endoperoxides
5 and 6.16 This latter conversion affords from a single
precursor, both endoperoxides 5 and 6 which have the
epoxide oxygen atom either on the opposite face of
(compounds 5) or on the same face (compounds 6) as
the alkyl or aryl substituents. Therefore, the epoxide
moiety of these epoxy endoperoxides mimics the oxygen
atom (3) of artemisinin (Figure 1). Single X-ray analysis
of epoxy endoperoxide 5f not only confirmed our struc-
tural assignments but also highlights the proximity of
the epoxy oxygen to those of the endoperoxide moiety
(Figure 2). Consequently, two unique versions of these
new prodrugs are readily available for probing structure-
activity relationships. Additionally, it is well established
that exposure of artemisinin to Fe(II) results in the
cleavage of the peroxide linkage and the generation of
free radical intermediates, which ultimately lead to ring-
opened products in which the peroxide linkage has been
destroyed.17,18 Furthermore, we have also reported that
exposure of endoperoxides of type 4a -n and their epoxy
counterparts (5 and 6) to catalytic amounts of cobalt-
(II) salen complexes results in a clean free radical
rearrangement resulting in the formation of the down-
stream products (7 or 8).16,19 Consequently, given that
exposure of the epoxy endoperoxides (5 and 6) to Fe(II)
will result in significant formation of these downstream
products (7 or 8), we also independently prepared pure
samples of several of these ring-opened products (7a ,
7j, and 8a ) in order to evaluate their activities.
F igu r e 2. Molecular structure and crystallographic number-
ing scheme for C14H20O3. Key geometric parameters are the
following: O1-C1a 1.443(2) Å, O1-C5a 1.437(2) Å, C1a-C5a
1.461(2) Å, C2-O3 1.434(2) Å, O3-O4 1.462(2) Å, O4-C5
1.411(2) Å, C5-C5a 1.486(3) Å; C1a-O1-C5a 60.9(1)°, C2-
O3-O4 106.9(1)°, O3-O4-C5 106.4(1)°.
(Table 1) and compared with data for CQ and artemisi-
nin. A number of the compounds prepared in this study
showed some antimalarial activity, with compounds 5a ,
5b, 6a , and 6b showing the highest activity. These
compounds comprise epoxy endoperoxide ring systems
with saturated five- or six-member ring systems at-
tached in the 3 and 6 positions. The dicyclobutyl
derivatives (5c, 6c) were less active, indicating that the
larger ring system is favored. The presence of the epoxy
group appears to be important for good antimalarial
activity but is not essential because compounds 4a and
4b retained significant activity. Ring-opened derivatives
of the cyclohexyl derivatives (7a , 8a ) showed no activity
in the range tested, indicating that the endoperoxide
ring system is critical for activity. The equivalent
compounds with phenyl substituents (4j, 5j) or alkyl
chain substituents (4h ) did not show activity, indicating
that the nature of the substituent groups is critical.
Similarly, compounds with a single cyclohexyl or cyclo-
pentyl moiety (4d , 4e, 5e, 5d , 5g, 6g) also had much
lower activities, indicating that the double substitution
is preferred. However, the monosubstituted cis-epoxy
An tim a la r ia l Activities. The abilities of the endo-
peroxides to inhibit the growth of the D10 strain of P.
falciparum during in vitro culture were determined

Novel Endoperoxide Antimalarials
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 7 1835
Ta ble 1. Biological Activities of Some of the Novel
Endoperoxides
antimalarial activity,b
compd
hemolysisa (%)
IC50 (µM)
ART
CQ
4a
4b
4c
4d
4e
4f
4
1
0.01
0.03
1.2
100
52
32
100
11
100
100
2
62
4
9
32
27
0
0.6
>10
3.2
>10
1.3
4g
4h
4i
9.5
>10
>10
6.0
8.1
>10
4jc
4k
4lc
4m
4n
5a
1.0
2.5
0.5
6
5b
5c
8
1
0.5
2.9
5d
5ed
5f
5g
5i
5j
5k
5l
2
1
41
3
47
5
2
2
6
5
9
1
1
6
51
4
8
2
14
2
>10
1.4
4.0
>10
>10
>10
>10
>10
1.3
F igu r e 3. Spectral analysis of the interactions of CQ, arte-
misinin, and some novel endoperoxides with FP-Fe(III): ab-
sorption spectra of 50 µM FP-Fe(III) in the presence of (A) CQ,
(B) artemisinin, (C) 4b, and (D) 6a under nonreducing condi-
tions. The spectra in the presence of the highest concentration
of compound (390, 710, 900, and 750 µM compound, respec-
tively) are represented by the thick curves, whereas the spectra
for FP-Fe(III) alone are the dashed curves. The dilution factor
for intermediate concentrations is 2.5. CQ and 4b possess some
absorption below 400 nm and contribute to the absorption in
this region. Spectra were normalized to that of FP in the
absence of compound. The absorbance ratio at 430 and 400
nm (A430/A400) is a useful indicator of changes to the structure
of the Soret band in the presence of compounds, and the dose
dependence of this parameter for a number of compounds is
shown in (E) and (F).
5m
6a
0.32
0.5
1.6
1.4
2.0
6b
6c
6ee
6g
6i
>10
6m
7a
5.0
>10
>10
>10
>10
5.0
7jc
8a
10
12c
1
a
Hemolysis of erythrocytes induced by
1 mM compound.
b
Antimalarial activity of compounds against the D10 parasite
strain. c Compounds were not completely soluble in ethanol.
Assays were performed using suspensions and activities expressed
d
drug and the FP µ-oxo dimer.20-22 In contrast, artemisi-
nin produces a detectable change in the absorption
spectrum only at a 100-fold greater concentration than
FP-Fe(III) (Figure 3B,E), although a time-dependent
decrease in the absorption of the Soret band has been
reported in dimethyl sulfoxide-water mixtures over a
24 h period.23 The addition of some of the novel endo-
peroxides (especially those lacking the epoxy moiety
such as 4a , 4b, 4c, 4m ) resulted in dose-dependent
changes in the absorption spectrum of FP that re-
sembled the changes observed with CQ (Figure 3C-F).
This suggests that the compounds are binding to FP in
a manner that produces a similar spectral change.
Interestingly, some of the endoperoxides that do not
contain π bonds such as 5d and 6b also exhibited this
intermediate strength binding (Table SI, Supporting
Information). It is important to note, however, that the
binding of the endoperoxides to FP-Fe(III) did not seem
to correlate with antimalarial activity because some of
the most active antimalarials, such as 5a , 5b, 6a , and
6b, do not appear to interact with FP-Fe(III) or interact
only very weakly under the conditions of the assay
(Figure 3B and Table SI, Supporting Information).
using the total concentration. Used as a 75:25 ratio of trans/cis
isomers. e Used as a 75:25 ratio of cis/trans isomers.
cycloalkyl derivatives (6e, 6g) did show moderate activ-
ity. Indeed the cis-epoxy derivatives (6a , 6b, 6c, 6e, 6g)
in general showed higher activity than their trans-epoxy
counterparts. Similarly, compounds with mixed sub-
stituents (5l, 10, 12, 4l) had variable activity. Some of
the adamantane-linked compounds did show activity
(4f), which was improved when the adamantane group
was attached to the ring by an ester linkage (4m , 5m ).
In ter a ction w ith F P . In an effort to determine the
likely mode of action of these novel endoperoxides, we
have examined their interaction with oxidized and
reduced FP. The binding of different quinoline antima-
larial drugs to FP-Fe(III) has previously been shown to
be important for the activity of these drugs. The binding
of drugs such as CQ to FP modifies the spectral
characteristics of FP, producing a decrease in the
absorption and a broadening of the main Soret band at
400 nm even when it is present in stoichiometric
quantities (Figure 3A). These effects are also observed
in the presence of phenothiazines and may reflect the
formation a π-π donor-acceptor complex between the

1836 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 7
Taylor et al.
strating the usefulness of this assay for screening for
artemisinin-like activity.
We found that a number of the novel endoperoxides
(e.g., 4f, Figure 4C) appeared to react with FP-Fe(II) in
a manner similar to artemisinin, albeit at much higher
concentrations. In general, compounds containing the
epoxy group and two cyclic substituents showed the
highest activity (Table SI, Supporting Information).
Interestingly, some of the endoperoxides such as 6a
(Figure 4D) induced a blue shift in the Soret band rather
than its ablation, indicating a specific interaction with
FP-Fe(II). The endoperoxides exhibiting this binding
mode tended to be those lacking the epoxy group (Table
SI, Supporting Information). Other endoperoxides ap-
peared to produce both effects, i.e., a reduction in the
Soret band occurring in concert with a blue shift (Table
SI, Supporting Information). Of the endoperoxides
exhibiting the greatest antimalarial activity, 5a , 5b, and
6b exhibited artemisinin-like activity whereas 6a ex-
hibited a shift in the Soret band (Figure 4 and Table
SI, Supporting Information). As with the binding to
oxidized FP, no clear correlation was observed with the
antimalarial activity. For example, 6e and 6g exhibited
moderate antimalarial activity but no detectable inter-
action with FP-Fe(II). In contrast, 4c exhibited some
interaction with FP-Fe(II) but no detectable antima-
larial activity (Table SI, Supporting Information).
Mem br a n e Activity. To determine whether any of
the compounds showed membrane activity, we have
examined their ability to lyse human erythrocytes. None
of the compounds induced hemolysis at the highest
concentration (10 µM) used in the antimalarial drug
assays. However at a concentration of 1 mM and in the
absence of protein in the bathing medium, some of the
compounds did induce hemolysis (Table 1). This ap-
peared to be directly related to the nature of the
substituents. For example, alkyl-chain-substituted en-
doperoxides were significantly more active than ring-
substituted compounds. This probably reflects the ten-
dency of the alkyl chain to insert into the lipid bilayer.
Similarly, the adamantyl group in 4f appears to confer
detergent-like properties. The epoxy endoperoxides were
less membrane-active than their endoperoxide counter-
parts, presumably because of the additional polarity
offered by the epoxy substituent. Again, there was no
correlation between membrane activity and antima-
larial activity, indicating that the compounds do not act
by a nonspecific membrane perturbation mechanism.
F igu r e 4. Spectral analysis of the interactions of CQ, arte-
misinin, and some novel endoperoxides with FP-Fe(II): ab-
sorption spectra of 30 µM FP-Fe(II) in the presence of (A)
artemisinin, (B) CQ, (C) 4f, and (D) 6a under reducing
conditions. The spectra in the presence of the highest concen-
tration of compound (710, 390, 900, and 750 µM compound,
respectively) are represented by the thick curves, whereas the
spectra for FP-Fe(II) alone are the dashed curves. The dilution
factor for intermediate concentrations is 2.5. The spectra are
normalized to that of FP in the absence of compound and are
shown at wavelengths above 375 nm because of the large
absorbance of the dithionite ion at shorter wavelengths. The
dose dependence of the loss of the Soret absorption at 400 nm
is shown in (E). In some cases, compounds induce a change in
the structure of the Soret band rather than a loss of absor-
bance. The absorbance ratio at 430 and 400 nm (A430/A400
)
indicates changes to the structure of the Soret band. The dose
dependence of this parameter for 6a is shown in (F).
The antimalarial activity of artemisinin is thought to
arise from its interaction with FP-Fe(II), resulting in
the formation a cytotoxic carbon-centered radical inter-
mediate that further reacts with susceptible groups
within parasite enzymes and lipids and can result in
the alkylation of FP itself.2,9-11 The addition of arte-
misinin to FP in the presence of the reducing agent
dithionite produced dose-dependent decreases in the
Soret absorption band (Figure 4A,E) and the appearance
of a new absorption band evident as a shoulder at 475
nm (Figure 4A). A similar system has been previously
employed using artemisinin and its derivatives, and the
spectral changes were shown to correlate reasonably
well with the antimalarial activity.4 In this previous
study, the reduction in absorbance was attributed to a
binding interaction between the drug and FP. However,
the complete loss of the Soret absorbance suggests that
it in fact reflects the destruction of the FP chromophore
with the concomitant formation of a new chromophore
with different spectral characteristics. In contrast to
artemisinin, CQ, which acts via a different mechanism,
does not perturb the FP-Fe(II) spectrum under the
conditions of this assay (Figure 4B,E), further demon-
Discu ssion
During intraerythrocytic growth, the malaria parasite
feeds by degrading hemoglobin in an acidic food vacuole.
It digests about 75% of the 20 mM hemoglobin in its
host cell cytosol.21,23,24 The free FP, which is produced
as a byproduct of hemoglobin degradation, creates a
waste disposal problem for the parasite (see ref 25 for
review). At the pH of the food vacuole (estimated to be
∼pH 5.2),26-28 the FP in oxyhemoglobin is oxidized from
the Fe(II) state to the Fe(III). Thus, the parasite needs
to deal with FP-Fe(III) waste, which could reach a
cellular concentration of about 15 mM. A major route
for disposal of toxic FP is via a novel process known as
biomineralization. The FP-Fe(III) is sequestered into a
crystalline form, referred to as hemozoin.13 This leads

Novel Endoperoxide Antimalarials
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 7 1837
to the formation of the characteristic malaria pigment
that is visible in smears of infected blood.
whether addition of a basic side chain to compounds
such as 4a , 4m , 4b, and 4c would improve their
antimalarial activities. The different FP-Fe(II) binding
modes of these compounds and their different structures
compared to that of CQ may allow activity against CQ-
resistant strains of parasites.
While most of the FP-Fe(III) appears to be efficiently
detoxified within the food vacuole, it is likely that a part
of the FP population escapes the crystallization process
and diffuses down the concentration gradient into the
parasite cytosol where it will be reduced to FP-Fe(II).
The redistribution of even a small fraction of the 15 mM
cellular load of FP could potentially damage host
proteins and membranes. Indeed, studies have shown
that nonsequestered FP can be present at concentra-
tions up to 100 µM in parasitized erythrocytes.21,29
Although this represents a small proportion (0.5%) of
the total FP in the parasitized erythrocyte, free FP at
levels much lower than this have been shown to inhibit
parasite enzymes,30,31 to lyse erythrocytes,32 and to
cause substantial redox damage.21,33 Thus, the parasite-
specific processes of hemoglobin degradation and FP
detoxification leave the parasite susceptible to quinoline
and endoperoxide drugs that interfere with aspects of
the detoxification process.
We also examined the abilities of the compounds to
react with FP-Fe(II). Activation of artemisinin by reac-
tion with reduced FP has long been thought to be critical
for its activity. Recent studies suggest that activated
artemisinin targets the SERCA calcium transporter in
the endoplasmic reticulum of P. falciparum.12 These
authors showed that desferoxamine antagonized the
action of artemisinin against SERCA and suggested that
Fe2+ is the likely activator of artemisinin. However, it
has previously been shown that desferoxamine also
interacts with FP,37 and given that the concentration
of free Fe2+ in the parasite is likely to be limited, it
seems more likely that reduced FP functions as the
activator of artemisinin in vivo. In this work we found
that the antimalarial activities of the novel endoperox-
ides is not correlated with their FP reactivity, a feature
which may reflect the inability of some of the compounds
to be located to the appropriate intracellular site. The
spectroscopic analysis indicated that artemisinin and
a subset of the endoperoxide compounds resulted in the
destruction of the FP chromophore under reducing
conditions. Intriguingly, some of the compounds did not
destroy the FP but appeared to form a complex with
the reduced FP that exhibits a spectral blue shift.
Although the novel endoperoxides undergo rapid scis-
sion upon interaction with metals as indicated by
analysis of the products formed during interaction with
the copper catalysts, it is possible that the interaction
of the bicyclic endoperoxides with FP-Fe(II) stabilizes
the porphyrin ring and thereby prevents transfer of the
radical intermediates to the porphyrin following scission
of the peroxide moiety. Another subset of the compounds
exhibit both FP destruction and FP binding, suggesting
in these cases that the reaction between the compound
and the FP is slowed sufficiently so that the complex
can be observed. In this respect, the novel compounds
may prove to be useful in elucidating the complex
interaction of artemisinin with FP and in the design of
new artemisinin-like compounds. In this work, we have
not examined the effect of the novel endoperoxides on
SERCA activity and it is possible that some of the
compounds target this enzyme. However, given the
range of interactions with FP, we suggest that the
compounds may act via a number of different molecular
mechanisms.
In this work, we have prepared a novel series of
endoperoxide compounds. The route for the construction
of the new targets is robust, can be performed on a large
scale in a safe manner, and is of low cost. These new
endoperoxides not only have the endoperoxide linkage
but also have a third oxygen atom that is positioned in
an environment similar to that of oxygen atom 3 within
artemisinin. We have also prepared a series of endo-
peroxides that lack the epoxide group but have ad-
ditional functional groups attached to the endoperoxide
ring. In addition, we have utilized cobalt catalysts to
induce ring opening of these endoperoxides, providing
a synthetic route to their isomeric ring-opened deriva-
tives. This free radical mechanism of ring opening is
likely to parallel the reaction of the endoperoxides with
FP-Fe(II). We were therefore able to test the possible
downstream products for activity.
We examined the antimalarial activities of the mem-
bers of this novel class of endoperoxides. We identified
a number of compounds with IC50 values in the range
300-500 nM. The minimum pharmacophore appears to
be an endoperoxide ring system with saturated cyclic
substituents at the 1 and 4 positions. In an effort to
determine the mechanism of action of the novel endop-
eroxides, we examined their interactions with FP. CQ
and other quinoline antimalarials have previously been
shown to form complexes with FP.34-36 The observed
spectral changes are thought to be derived from π-π
complexation of the porphyrin and quinoline ring sys-
tems.20 A number of the novel endoperoxides induce
similar changes to the spectral properties of FP-Fe(III)
as CQ. Interestingly, some of these compounds lacked
a π system, indicating they bind to FP in a manner
different from that of CQ. It is possible that these
compounds may cause the bridging of two FP mono-
mers, resulting in π-π stacking. We found no correla-
tion between the FP-Fe(III) binding and antimalarial
activity of the compounds, which may suggest that the
compounds are not efficiently targeted to the food
vacuole of the parasite. Quinoline antimalarials are
weak bases and are accumulated in the food vacuole,
at least in part, by a proton-trapping mechanism (see
ref 25 for review). It would be interesting to determine
In conclusion, we have identified a facile route for the
synthesis of a series of novel endoperoxides with anti-
malarial activities in the submicromolar range. The IC50
values are relatively high compared with values for
artemisinin (∼10 nM) and CQ (∼30 nM for a CQ-
sensitive strain); however, they approach the IC50 values
for CQ against CQ-resistant strains (300-600 nM)38 and
for compounds such as sulfadoxine (∼100 nM for sensi-
tive strains and up to 10 µM for resistant strains). The
efficacy of these compounds may be further improved
by adding substituents onto the cyclic ring systems. For
example, addition of an amino side chain has been
shown to improve the efficacy of artemisinin,39 presum-

1838 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 7
Taylor et al.
of [3H]-hypoxanthine were obtained in triplicate as described
previously,38 and the concentration of compound required to
produce 50% inhibition of growth (IC50) was determined.
F P Bin d in g Assa ys. The interaction of the compounds with
FP was examined spectrophotometrically under reducing and
nonreducing conditions by monitoring the Soret absorption
band of FP. Compound stocks were prepared in ethanol and
serially diluted in a 96-well microtiter plate into nonreducing
(50 µM FP, 100 mM sodium phosphate, pH 7.4, 1% SDS) or
reducing (30 µM FP, 100 mM sodium phosphate, pH 7.4, 1%
SDS, 35 mM sodium dithionite) buffer. The incorporation of
the SDS detergent in the buffers was necessary to produce a
stable absorption because FP has a tendency to aggregate in
aqueous solution and its absorption can exhibit a temporal
dependence. The concentration of ethanol in the assay was less
than 2% (v/v). Absorption spectra were collected using a
Spectromax 250 plate reader.
Er yth r ocyte Lysis Assa y. Washed human erythrocytes
(400 µL, 0.4 × 108 cells) in phosphate-buffered saline were
incubated with the compounds to a final concentration of 10
µM or 1 mM and incubated 30 min at 37 °C. Cells were
pelleted, and the absorbance of the supernatant was compared
with that for a sample of freeze-thawed cells to determine
the percentage of hemolysis.
ably by enhancing drug uptake. The fact that some of
the compounds appear to inhibit parasite growth by
what appears to be a novel mechanism of action
indicates that they are worth pursuing in an effort to
obtain novel compounds to replenish our rapidly dwin-
dling armory of useful antimalarials.
Exp er im en ta l Section
Gen er a l Syn th etic Meth od s. Solvents were dried by
appropriate methods wherever needed. Thin-layer chroma-
tography (TLC) used aluminum sheets coated with silica gel
60 F254 (40 mm × 80 mm) from Merck, visualized under 254
nm light or developed in vanillin or permanganate dip. Flash
chromatography was conducted using Merck silica gel 60 of
particle size 0.040-0.063 mm. Melting points were taken on
a Reichert Thermovar Kofler apparatus and are uncorrected.
Infrared spectra were recorded on an ATI Mattson Genesis
series FTIR spectrophotometer. 1H NMR and 13C NMR spectra
were recorded in CDCl3 solution on a Varian Gemini 2000 (200
MHz), Varian Gemini 2000 (300 MHz), or Varian INOVA (600
MHz) instrument, using TMS (0 ppm) and CDCl3 (77.0 ppm)
as internal standards. Electron impact mass spectra (EI-MS)
were recorded at 70 eV. Accurate mass measurements were
performed at the Central Science Laboratory, University of
Tasmania, Tasmania, Australia, or at the Department of
Chemistry, Monash University, Victoria, Australia. Microanal-
yses were performed in the Department of Chemistry, Uni-
versity of Otago, Dunedin, New Zealand. All yields reported
refer to isolated material judged to be homogeneous by TLC
and NMR spectrometry.
Ack n ow led gm en t. The National Health and Medi-
cal Research Council of Australia, the Brailsford Rob-
ertson Collaborative Grants Program, and the National
University of Singapore (Grant R-143-00-139-112) are
gratefully acknowledged.
Gen er a l Syn th esis of En d op er oxid es. All endoperoxides
(4, 9, and 11) were prepared via photolysis (3 × 500 W
tungsten/halogen lamps) of an oxygen-saturated dichlo-
romethane solution of the 1,3-butadiene in the presence of the
photosensitizer Rose Bengal, bis(triethylammonium) salt, for
7 h.40 In all cases, starting diene was reclaimed from the
photolysis reactions as the other major product.
Ap p en d ix
Abbr evia tion s. FP-Fe(III) ) oxidized heme ) fer-
riprotoporphyrin IX, FP-Fe(II) ) reduced heme ) fer-
roprotoporphyrin IX; CQ ) chloroquine; BSA ) bovine
serum albumin; SDS ) sodium dodecyl sulfate; THF )
tetrahydrofuran; m-CPBA ) m-chloroperoxybenzoic
acid.
Gen er a l Syn th esis of Ep oxy En d op er oxid es (5, 6, a n d
12).16 To a stirred solution of 1,2-dioxine (1 mmol) in CH2Cl2
(5 mL) was added 70% m-chloroperbenzoic acid (2 mmol). The
reaction was stirred at ambient temperature until complete
by TLC. Dichloromethane (10 mL) was then added, and the
solution was extracted with saturated Na2S2O3 (10 mL)
followed by NaHCO3 (10 mL). The organic layer was dried over
MgSO4 and filtered, and volatiles were removed in vacuo. The
crude epoxides were purified by column chromatography.
Epoxides 5a , 6a , 5j, 5l, and 10 have previously been reported.16
Cr ysta l Str u ctu r e Deter m in a tion of C16H14O3 (5f).
Crystal data for C14H20O3: M ) 236.30, T ) 223(2) K,
monoclinic, P21/c, a ) 15.2364(13) Å, b ) 7.2642(7) Å, c )
11.1369(10) Å, â ) 98.777(4)°, V ) 1218.20(19) Å3, Z ) 4, Dx
) 1.288, F(000) ) 512, µ ) 0.089 mm-1, no. of unique data
(Bruker AXS SMART CCD using Mo KR radiation so that θmax
) 30.1°) ) 3553, no. of parameters ) 154, R (2185 data with
I g 2σ(I)) ) 0.062, wR (all data) ) 0.193, F ) 0.39 e Å-3. The
structure was solved by direct methods (SIR92) and refined
Su p p or tin g In for m a tion Ava ila ble: Table SI containing
data for the interaction of the compounds in Table 1 with
oxidized and reduced FP; synthetic protocols and NMR data
for each of the compounds, and relevant references. This
material is available free of charge via the Internet at http://
pubs.acs.org.
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