Comparison of the reactivity of antimalarial 1,2,4,5-tetraoxanes with 1,2,4-trioxolanes in the presence of ferrous iron salts, heme, and ferrous iron salts/phosphatidylcholine

Article abstract of DOI:10.1021/jm200768h

Dispiro-1,2,4,5-tetraoxanes and 1,2,4-trioxolanes represent attractive classes of synthetic antimalarial peroxides due to their structural simplicity, good stability, and impressive antimalarial activity. We investigated the reactivity of a series of potent amide functionalized tetraoxanes with Fe(II)gluconate, FeSO₄, FeSO₄/TEMPO, FeSO ₄/phosphatidylcholine, and heme to gain knowledge of their potential mechanism of bioactivation and to compare the results with the corresponding 1,2,4-trioxolanes. Spin-trapping experiments demonstrate that Fe(II)-mediated peroxide activation of tetraoxanes produces primary and secondary C-radical intermediates. Reaction of tetraoxanes and trioxolanes with phosphatidylcholine, a predominant unsaturated lipid present in the parasite digestive vacuole membrane, under Fenton reaction conditions showed that both endoperoxides share a common reactivity in terms of phospholipid oxidation that differs with that of artemisinin. Significantly, when tetraoxanes undergo bioactivation in the presence of heme, only the secondary C-centered radical is observed, which smoothly produces regioisomeric drug derived-heme adducts. The ability of these tetraoxanes to alkylate the porphyrin ring was also confirmed with Fe ^IITPP and Mn^IITPP, and docking studies were performed to rationalize the regioselectivity observed in the alkylation process. The efficient process of heme alkylation and extensive lipid peroxidation observed here may play a role in the mechanism of action of these two important classes of synthetic endoperoxide antimalarial.

Full text of DOI:10.1021/jm200768h

ARTICLE
pubs.acs.org/jmc
Comparison of the Reactivity of Antimalarial 1,2,4,5-Tetraoxanes with
1,2,4-Trioxolanes in the Presence of Ferrous Iron Salts, Heme, and
Ferrous Iron Salts/Phosphatidylcholine
Fatima Bousejra-El Garah,*,† Michael He-Long Wong,‡ Richard K. Amewu,† Sant Muangnoicharoen,§
James L. Maggs,‡ Jean-Luc Stigliani,|| B. Kevin Park,‡ James Chadwick,† Stephen A. Ward,§ and
Paul M. O’Neill*,†
†Department of Chemistry, University of Liverpool, Liverpool L69 7ZD, U.K.
‡MRC Centre for Drug Safety Science, Department of Molecular and Clinical Pharmacology, University of Liverpool,
Sherrington Building, Liverpool L69 3GE, U.K.
§Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA, U.K.
Laboratoire de Chimie de Coordination, Universitꢀe de Toulouse, 31077 Toulouse Cedex 4, France
S Supporting Information
b
ABSTRACT:
Dispiro-1,2,4,5-tetraoxanes and 1,2,4-trioxolanes represent attractive classes of synthetic antimalarial peroxides due to their
structural simplicity, good stability, and impressive antimalarial activity. We investigated the reactivity of a series of potent amide
functionalized tetraoxanes with Fe(II)gluconate, FeSO4, FeSO4/TEMPO, FeSO4/phosphatidylcholine, and heme to gain knowl-
edge of their potential mechanism of bioactivation and to compare the results with the corresponding 1,2,4-trioxolanes. Spin-
trapping experiments demonstrate that Fe(II)-mediated peroxide activation of tetraoxanes produces primary and secondary
C-radical intermediates. Reaction of tetraoxanes and trioxolanes with phosphatidylcholine, a predominant unsaturated lipid present
in the parasite digestive vacuole membrane, under Fenton reaction conditions showed that both endoperoxides share a common
reactivity in terms of phospholipid oxidation that differs with that of artemisinin. Significantly, when tetraoxanes undergo
bioactivation in the presence of heme, only the secondary C-centered radical is observed, which smoothly produces regioisomeric
drug derived-heme adducts. The ability of these tetraoxanes to alkylate the porphyrin ring was also confirmed with FeIITPP
and MnIITPP, and docking studies were performed to rationalize the regioselectivity observed in the alkylation process. The efficient
process of heme alkylation and extensive lipid peroxidation observed here may play a role in the mechanism of action of these two
important classes of synthetic endoperoxide antimalarial.
’ INTRODUCTION
Artemisinin (Figure 1), a sesquiterpene lactone extracted from
the Chinese herb Artemisia annua, is highly active against both
chloroquine sensitive- and resistant-strains of Plasmodium
falciparum.3 It is known that the key pharmacophore of artemi-
sinin is the endoperoxide containing 1,2,4-trioxane unit.3 It is
also well established that iron(II) salts reductively activate the
According to the World Health Organization, more than one
million people die of malaria every year in Africa, countries where
children and pregnant women are especially at risk.1 The spread
of chloroquine and sulfadoxine-pyrimethamine resistance have
been implicated in the re-emergence of malaria in areas where the
disease had been eradicated. In order to control the burden of
malaria, artemisinin-based combination therapies are now re-
commended as a first-line treatment in many endemic regions.2
Received: September 7, 2010
Published: September 05, 2011
r
2011 American Chemical Society
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Figure 1. Fe(II)-mediated activation of artemisinin.
Figure 2. Structures of antimalarial tetraoxanes 1ꢀ6 and their activities against 3D7 Plasmodium falciparum.
peroxide bond of artemisinin leading to the formation of a pair of
oxyl radical intermediates that rapidly rearrange via either a 1,5
H-shift or β-scission to produce the more stable carbon-centered
radicals.4ꢀ6 These alkyl radicals can be readily formed in vivo by
the reaction of artemisinin with iron(II)-heme,7,8 the most
abundant source of iron in Plasmodia.7,9 It is proposed that these
reactive C-radicals interact with cellular components such as
heme and parasite proteins10 resulting in the death of the parasite
(Figure 1). One such protein is PfATP6, the sarco/endoplasmic
reticulum calcium ATPase of P. falciparum, reported by Krishna
and us to be inhibited by artemisinin in Xenopus laevis oocytes.11
However, recent studies on purified PfATP612 and modeling
calculations13 have questioned the role of PfATP6 as a drug
target. As noted above, heme has also been proposed to play a
role in the mechanism of action of endoperoxides, and supportive
in vivo evidence was provided in a study where heme-artemisinin
adducts were detected in infected mice treated with artemisinin.
Inspite of this work, the mechanism of action of artemisinin and
synthetic endoperoxides remains controversial, and very re-
cently, a non iron-mediated mechanism of bioactivation has been
proposed by Haynes and Monti.14,15 While these studies provide
an attractive alternative mechanism of activation, additional
biological support for this proposal is urgently needed.
and highly potent dispiro-tetraoxanes that incorporate the adamantyl
group, known to bring stability to the endoperoxide motif.17,18
From a library of over 200 tetraoxanes, 3 (RKA182) (Figure 2)
has been selected as a candidate for full formal preclinical
development. Compound 3 has outstanding in vitro and
in vivo activity against P. falciparum and shows improved
pharmacokinetic characteristics compared to those of other
peroxide drugs.19
The first aim of the present study was to investigate the radical
pathway after reductive activation of highly potent tetraoxanes
(selected from 2ꢀ6, vide infra) with inorganic iron(II). Second,
we set out to investigate whether the oxidative degradation of
phospholipids, recently reported for water-soluble tetraoxanes,20
was observed with adamantane functionalized tetraoxanes and
their corresponding trioxolane analogues. In our model studies,
we employed phosphatidylcholine (PC) on the basis of research
by Tilley that clearly demonstrates that PC is a major constituent
of the food vacuole cell membranes of P. falciparum.21 Our third
aim was to explore the reactivity of 1,2,4,5-tetraoxanes with heme
for comparison with that of 1,2,4-trixolanes and to rationalize
differences in reactivity with inorganic iron sources. While these
studies may be fundamental to the mechanism of action, the
importance of understanding the ferrous ion reactivity of these
systems is also key to producing molecules that provide good
exposures in malaria infected patients. As seen recently with 15
(OZ277), degradation in the plasma of infected patients, by iron
Work by the Vennerstrom group in the early 1990s showed
thatdispiro-tetraoxanespossesshighin vitroantimalarialactivity.16
More recently, we have designed and prepared simple, achiral,
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Figure 3. (a) Fe-mediated activation of 2 and TEMPO spin-trapping; (b) structures of adducts 8b and 8c; and (c) fragmentations of adducts 7 and 8b.
released during infection, leads to lower than expected drug
exposure, and by studying the stability in the presence of infected
red blood cells and Fe(II) salts, more stable analogues can be
designed and synthesized as exemplified by 16 (OZ439).22
TEMPO in acetonitrile led to the formation of the two TEMPO
adducts 7 (m/z 324.3 MH+, Figure 3) and 8a (m/z 454.5 MH+).
The reactivity of the tetraoxanes occurs by coordination of Fe2+
with O2 resulting in the formation of the oxyl-radical 2a
(Figure 3). Ring-opening via β-scission produces the secondary
C-radical 2c, which was trapped with TEMPO to give the adduct
7, along with the keto-amide 9 (m/z 281.3 MH+). Alternatively,
coordination on O1 leads to the formation of the oxy radical 2b
that rearranges to the primary C-radical 2d, also trapped with
TEMPO to give the product 8a. Similar experiments were carried
out with tetraoxanes 1 and 5 in the presence of FeBr2 in THF.
From these experiments, the adduct 7 was identified in both
cases. Analogous to the adduct 8a, adducts 8b and 8c were also
’ RESULTS AND DISCUSSION
Reactivity with Fe(II) and TEMPO Spin-Trapping. In order
to confirm the proposition that carbon-centered free radical
intermediates are readily produced from our antimalarially active
lead 1,2,4,5-tetraoxanes,19 we set out to perform spin-trapping
experiments with 2,2,6,6-tetramethyl-1-piperidine 1-oxyl (TEMPO).
Reaction of tetraoxane 2 with ferrous sulfate in the presence of
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Figure 4. Tetraoxanes 2 and 3 and their trioxolane analogues 13 and 14, respectively, as well as 15 (OZ277) and 16 (OZ439).
C-radical 6d has the ability to alkylate heme, react with nitroso
spin-traps, and potentially abstract hydrogens from lipids, as
shown in Figure 8, an intramolecular attack on the peroxy ester
function by the C-radical center of 6d can readily yield the lactone
11a with loss of FeO3+ with the formation of the observed amide
12. Closer examination of the LCMS data revealed that lactone
11a was indeed generated under these reaction conditions
(Figure S1, Supporting Information). To be absolutely certain
that the H-abstraction product 11b was not produced under
these conditions, an authentic synthesis of 11b was performed to
provide a standard for LCMS analysis (Supporting Information).
With an authentic sample of 11b, we were able to rule out the
generation of this product under the reaction conditions. Addi-
tional studies with FeSO4 in acetonitile-water also revealed that
10, 12, and adamantanone are readily produced under aqueous
conditions (Figure S2, Supporting Information).
Figure 5. Time-dependence profile for the decomposition of peroxides
by 18 equiv of FeSO4 in aqueous acetonitrile.
Ferrous Salt-Mediated Reactivity: Tetraoxane vs Trioxo-
lanes. For the purposes of direct comparison, we prepared the
trioxolanes 13 and 14, analogues of tetraoxanes 2 and 3, res-
pectively, in order to compare the stability and the reactivity of
the 1,2,4,5-tetraoxane pharmacophore to the ozonide core in the
presence of iron(II) using standard conditions (Figure 4).
Iron-mediated decomposition was observed for both tetraox-
anes and trioxolanes. The time-dependent degradation studies,
conducted by monitoring the loss of parent endoperoxide by
LC-MS, showed a linear decrease in tetraoxane concentration
(Figure 5). Similar reaction profiles were observed for tetraox-
anes and trioxolanes, indicating that the stability of the two classes
of drugs with inorganic Fe(II) is comparable. The results observed
for these pairs of endoperoxides is in sharp contrast to studies
comparing the reactivity of sulfonamide based tetraoxanes where
tetraoxanes proved to be considerably more stable than their
trioxolane counterparts (note that this previous study used lower
equivalents of Fe(II) salts).18 This observation emphasizes the
important role of the side chain in influencing endoperoxide drug
stability, and such effects have been seen in the C-10 amino semi-
synthetic artemisinin derivatives studied by Haynes.25 Clearly by
design, it is possible to tune the reactivity of endoperoxides by the
incorporation of appropriate side-chains; a point exemplified by
the discovery of 16, a molecule with enhanced Fe(II) stability
preventing plasma-mediated decomposition in infected patients
and an outstanding antimalarial activity profile.22
characterized after trapping of the primary radical derived from 1
and 5, respectively (Figure 3b,c).
The identification of the two TEMPO adducts 7 and 8a
provided evidence for the formation of two carbon-centered
radicals after iron(II) activation of tetraoxanes, a similar feature
observed with artemisinin. Our experiments provide results that
are clearly distinct from previous work on steroidal 1,2,4,5-
tetraoxanes where Opsenica et al. reported the generation of oxyl
radical species that do not undergo any further rearrangement to
form carbon-centered radicals.23 Second, while experiments with
trioxolanes like 15 (Figure 4) resulted in the identification of only
secondary radicals,24 we observed the formation of both primary
and secondary carbon centered radicals in the presence of
inorganic ferrous salts. Thus, while the overall rates of degrada-
tion with excess FeSO4 are similar (Figure 5), tetraoxanes can
generate two C-radical species following activation.
While spin-trapping and LC-MS analysis of adducts provides a
qualitative view of reactivity, quantitative measurements of each
spin-trapped adduct proved difficult due to the high polarity of
adducts 8aꢀ8c. To achieve a quantitative assessment, we em-
ployed a more lipophilic fluorinated tetraoxane derivative 6 using
iron(II) gluconate as the reductant (Figure 6). In this study,
performed in the absence of a spin-trap, we obtained 10 and 12 in
yields of 44% and 51%, both of which were purified by column
chromatography. We propose that H-abstraction from DMF by
6c facilitates the formation of 10. While we demonstrate that the
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Figure 6. Fe(II)-mediated degradation of fluorophenyl amide tetraoxane 6.
H-abstract from biologically relevant lipid targets as an alter-
native potential mechanism of action. Both the primary and
secondary C-radicals, produced after tetraoxane activation, may
react in vivo with lipids of the membrane bilayer, a well-known
target for reactive oxygen species (ROS), leading to cell damage
and death. Kumura and colleagues have recently reported that
water-soluble structurally distinct tetraoxane salts (Figure 7) can
induce olefin oxidative degradation in the presence of an Fe(II)
salt. Several PC degradation products were identified, but no mecha-
nisms were proposed for their formation.20 Since the structure of
this salt is very different from our unsymmetrical more lipid soluble
1,2,4,5-tetraoxanes, it was considered vital to examine the reactivity
of our lead molecules in this system. The validity of using PC as a
target lipid is exemplified by the work of Tilley, who examined
the nature of the lipid environment within Plasmodium falciparum,
which included analysis of the food vacuole and associated food
vacuole lipid bodies.21 These studies reveal the presence of PC
in the parasite food vacuole, the site of heme production.
We studied the reactivity of tetraoxanes 2 and 3 with PC, under
Fenton reaction conditions, and we compared this reactivity with
that of trioxolane analogues, 13 and 14, respectively.
Figure 7. Structures of the symmetrical salt previously studied and
2-linoleoyl-1-palmitoyl-sn-phosphatidylcholine (PC).
Reactivity of Tetraoxanes with Fe(II) and Phosphatidyl-
choline (PC). Since the proposal that C-centered radicals can
alkylate proteins has been met with scepticism, we were keen
to investigate the capacity of tetraoxanes and trioxolanes to
2-Linoleoyl-1-palmitoyl-sn-phosphatidylcholine (PC) is com-
posed of a phosphocholine polar head linked to the glycerol
moiety with palmitic acid, a saturated fatty acid chain at the sn-1
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Figure 8. Proposed mechanisms for tetraoxane-mediated lipid peroxidation with structures suggested by Kumura et al.20
position, and linoleic acid, an unsaturated fatty acid chain at the
sn-2 position (Figure 7).
gem-dihyroperoxide, which ultimately collapses to provide the
observed product P3 with the expulsion of hydrogen peroxide.
The reaction with PC was carried out with trioxolanes 13 and
14 under the same conditions, and product analysis indicated
that the same PC degradation products were produced indicating
that in these conditions the two classes of antimalarials exhibit
similar reactivity toward phospholipids.
Reactions of PC (m/z 758 M+) with tetraoxanes 2 and 3 in the
presence of ferrous sulfate led to the formation of several
products, different from the native PC. LC-MS analysis showed
the predominant degradation products had m/z 622.4, 666.4,
788.6 (810.5), and 808.5. All of these ions, except m/z 808.5,
were detected by Kumura.20 The structures proposed for the PC
oxidation products are depicted in Figure 8. The ketone (9 when
tetraoxane 2 was used) was also identified as a byproduct of the
reaction. The bis-allylic hydrogen at C11 of the linoleic acid is
more likely to be abstracted by the tetraoxane-derived radicals,
and we have rationalized the formation of each of these products
according to the mechanisms depicted in Figure 8 on the basis of
considerations of the extensive mechanistic studies of Spiteller26
and Domingues.27,28 Product P1 is derived from the C-9 capture
of the triplet oxygen followed by the peroxyl radical H-abstrac-
tion from another molecule of PC. Homolytic cleavage of the
hydroperoxide and fragmentation provides a primary PC derived
lipid C-radical on C-8, which abstracts a hydrogen to produce the
observed product. Product P2 is derived via Hock cleavage and
generation of an aldehyde, which we proposed is oxidized under
the reaction conditions to provide P2. Product P3 with m/z
788.6 is produced from PC by C-11 O2 capture, reductive
activation by Fe2+ to produce a peroxy epoxide that can readily
undergo further H-abstraction, and oxidation to produce a
Having performed these initial studies with the tetraoxane
amide 2, we extended our mechanistic work to the fluoro analogue
6 in order to pinpoint which of the carbon radical species might be
responsible for lipid peroxidation via hydrogen atom abstraction
from the C-11 of PC. This molecule is currently of interest since
it has a potent in vitro antimalarial activity of 3.0 nM versus the
3D7 strain and oralactivity versus Plasmodium berghei (Supporting
Information). An experiment was performed using 6 with PC
and monitored using ESI/MS to determine the tetraoxane
degradation products. ESI/MS revealed the same lipid peroxida-
tion products depicted in Figure 8 along with acid 10, adaman-
tanone, and lactone 11a; notably, acid 11b was not detected. The
data obtained from this experiment suggests strongly that the
primary radical 6c is the likely mediator of hydrogen abstraction
in these reactions, although we cannot rule out the role of 6d as
the initiator since only catalytic quantities may be required to set
in motion the pathway of PC breakdown depicted in Figure 8.
Under the same reaction conditions with artemisinin, native
PC was found intact, confirming that artemisinin does not
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Figure 9. (a) UVꢀvis trace; (b) extracted ion chromatogram at m/z 783.2 of the reaction of heme with tetraoxane; and (c) mass spectra of the heme
adducts.
produce phospholipid oxidation in a manner similar to the other
endoperoxides examined. With heme as the activator, artemisinin
has been shown to enhance the heme-mediated lipid membrane
damage29 but did not affect PC in our nonheme conditions. This
difference of reactivity between artemisinin and tetraoxanes/
trioxolanes here may be due to the difference in the stability of
the radical intermediates. The artemisinin-derived radicals are
more reactive, and the intramolecular rearrangement of these
radicals may be too rapid for intermolecular H-bond abstraction
from lipids.
Reactivity of Tetraoxanes with Heme. Several studies have
shown that iron(II)-heme quickly reacts with the peroxide bond
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Figure 10. Proposed mechanism for the alkylation of heme (Fe(II)-PPIX) by tetraoxane 2. Adapted with permission from ref 31. Copyright 2008
American Society for Microbiology.
of various synthetic endoperoxide antimalarials. The pathways
are mechanistically similar to those described with ferrous ions
with the exception that the generated C-centered radicals tend to
react in an intramolecular fashion with the porphyrin macrocycle
to form heme-drug adducts. Such adducts have been isolated
and/or characterized with artemisinin8 and other antimalarial
peroxides.30ꢀ32
We investigated the reaction of tetraoxanes 2 and 3 with
ferrous-heme and found that the peroxides rapidly react with the
porphyrin. In both cases, HPLC analysis of the crude mixture
showed that most of the starting heme had reacted within 30 min,
giving three products with higher retention times than the heme
itself (Figure 9a) and a maximum absorption of the Soret band at
Figure 11. Time-dependence profile for the conversion of heme with
peroxides.
430 nm, instead of 398 nm for heme. LC-MS analysis showed
that these three compounds exhibit a m/z 782.3 (M+), consistent
with covalent coupling products (17, Figure 10) formed between
heme (mass 616) and the tetraoxane-derived secondary C-cen-
tered radical 2c. The same alkylated heme adduct has been
reported with trioxolanes in similar conditions by Creek et al.31
The extracted ion chromatogram at m/z 782.3 showed four
peaks (Figure 9b) expected to be the four possible regio-isomers
of the alkylated heme adduct 17, as reported by Robert et al. for
heme-artemisinin adducts.33
Similar results were obtained with all the active antimalarial
tetraoxanes tested, including 3 and 4 (results not shown). The
ketoamide compound, a coproduct of the reaction, was also
detected in all cases. We monitored the conversion of heme
during the reaction (Figure 11). As a control, the reaction was
carried out without the drug, to estimate the loss of heme due to
the degradation by residual oxygen. Reaction with artemisinin
showed that c.a. 70% of the starting heme was converted within
15 min. Reaction with tetraoxanes 2 and 3 was also rapid and
resulted in the conversion of the starting heme of 53% and 38%,
respectively, after only 5 min. Concentrationꢀtime profiles of
heme showed that the reaction is complete within 15 min, as the
absorbance of the Soret band remains constant beyond this
time point.
Reaction of the direct trioxolane analogues with heme in the
same conditions showed a very similar profile, and the same
coupling products were identified (Figure 12). However, the
adduct formation appeared to be more rapid with trioxolane 13,
and the peak ratio was different. The reaction with heme occurs
much faster than the reaction with ferrous sulfate; indeed,
tetraoxanes were degraded in a few minutes, while reaction with
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Figure 12. LC trace of the reaction mixture of tetraoxane 2 (top) and trioxolane 13 (bottom) with heme.
Figure 13. Expected structures of Mn-TPC- and H2-TPC-tetraoxane adducts 18 and 19.
inorganic iron requires several hours. This increased reaction rate
is also observed with trioxanes and trioxolanes.31,32
Because of their low stability, quantification of the adducts was
not possible. However, the estimated yield of adducts after 15
min for 2 is 34%, which is about half of the yield observed with 13
in the same conditions (71%).34 No significant differences were
observed between 3 and 14 (data not shown).
As reported with artemisinin, we also studied the alkylating
activity of tetraoxanes with iron(II) tetraphenylporphyrin and
manganese(II) tetraphenylporphyrin, two symmetrical synthetic
porphyrins, to confirm the results observed with heme.35 Reac-
tion of tetraoxane 2 with MnIITPP gave a porphyrin-drug adduct
with m/z 835.10 (M+), consistent with a chlorin-type adduct
(18). This adduct results from alkylation on one of the eight β-
pyrrolic positions of the macrocycle by the tetraoxane-derived
secondary radical. The same chlorin-type adduct was identi-
fied after reaction with FeIITPP. The demetalated adduct 19
(Figure 13) was also identified (m/z 783.10, MH+) after workup
and Mn removal. These results with the tetraphenylporphyrins
confirm the alkylating capacity of antimalarial tetraoxanes ob-
served with heme.
One can note that, although the primary and the secondary
radical can possibly be generated after reduction by Fe2+ salts,
only the tetraoxane-derived secondary radical (2c) was found
to react with heme and tetraphenylporphyrins. In the case of
artemisinin and other trioxanes, only adducts resulting from the
primary radical were reported.8,32 This could be explained by the
relative positions of the peroxide bond and the porphyrin ligand.
Contrary to free iron, which can coordinate both oxygen atoms,
iron in the heme porphyrin would less easily coordinate O1,
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in the association of the endoperoxide bridge with a preference
for the association of the least hindered atom O2 with heme Fe2+.
The high reactivity with heme and the formation of covalent
heme-drug adducts are features shared by all tested active semi-
synthetic artemisinins and trioxolane and tetraoxane derivatives;
the role of this event in the antimalarial mechanism remains to be
clarified. The establishment that the front line synthetic artemi-
sinin replacements efficiently promote lipid peroxidation has
potential implications for both mechanism of action and potential
drug safety, and studies in Plasmodium falciparum and susceptible
human cell lines (e.g., neuronal cells, embryonic cell lines) are
warranted in the near future.
’ EXPERIMENTAL SECTION
LC-MS Analysis. Mass spectrometry analysis was performed on a
Thermo TSQ Quantum Access triple quadrupole mass spectrometer
connected to a LC device Thermo Accela high pressure pump, auto
sampler, and PDA detector. Analytical separations were performed on a
150 ꢁ 3 mm Thermo Hypersyl HyPURITY C18 column (5 μm). Data
were captured in full MS scan mode and processed using Xcalibur
software (version 2.0.7) and a Thermo Quan browser (version 2.0.7).
Chemistry. All reagents were purchased from Sigma Aldrich UK
and were used without purification. NMR spectra were recorded on a
Bruker AMX 400 (1H, 400 MHz; 13C, 100 MHz) spectrometer, using
CDCl3 or d4-MeOD as the solvent. Chemical shifts are described in
ppm (δ) downfield from an internal standard of trimethylsilane. Tetra-
oxanes 1, 4, 5, and 6 were prepared according to the procedure reported
by Amewu et al.17 The purity of all target compounds was >95% as
confirmed by combustion analysis.
Synthesis of Tetraoxanes 2 and 3. Tetraoxanes 2 and 3 were
prepared according to the method reported by O’Neill et al.19
Synthesis of Tetraoxane 6. Tetraoxane 6 was prepared according
to the procedure reported by Amewu et al.17 using 4-fluorobenzylamine
in the coupling step. White solid (0.190 g, 426 μmol, 71%); 1H NMR
(400 MHz, CDCl3) δ 7.29ꢀ7.20 (m, 2H), 7.01ꢀ6.98 (m, 2H), 5.70
(s, 1H), 4.41 (d, J = 5.7 Hz, 2H), 2.11 (d, J = 7.1 Hz, 2H), 2.07 ꢀ 1.91
(m, 5H), 1.90ꢀ1.83 (m, 3H), 1.82ꢀ1.66 (m, 7H), 1.66ꢀ1.50 (m, 4H),
and 1.45ꢀ1.15 (m, 2H). 13C NMR (101 MHz, CDCl3) δ 171.8, 162.3
(d, J = 245.0 Hz), 129.6 (d, J = 8.1 Hz), 115.7 (d, J = 21.5 Hz), 110.6,
107.8, 43.5, 43.0, 37.1, 34.2, 33.3, and 27.2. Mp = 146 °C; IR 3242, 3072,
2918, 2856, 2370, 2341, 1631, 1545, 1510, 1448, 1375, 1302, 1225, 1176,
1101, 1061, 999, 928, 829, 741, 685, and 613 cmꢀ1. HRMS calculated for
C25H32FNO5 [M + Na]+, 468.2162; found, 468.2149. Microanalysis
calculated for C25H32FNO5: C, 67.40%; H, 7.24%; N, 3.14%. Found: C,
67.66%; H, 7.28%; N, 3.15%.
Figure 14. Docking configuration between heme and RKA182 (3).
which is close to the bulky adamantane group. However, we
cannot rule out the possibility that the primary radical is also
formed but does not react with the porphyrin.
To confirm the hypothesis that the two oxygens (O2 and O4)
preferentially coordinate to the heme iron center, we performed a
modeling study of the interaction between heme and tetraoxane
3. The objective here was to see whether the regioselectivity of
the alkylation reaction could be predicted by docking calcula-
tions. For this purpose, we used the AutoDock 4.0 software36
with two different protocols: in the first procedure, the heme
receptor was kept fully rigid, while in the second protocol, the
two propionate chains of the protoporphyrin were left flexible. In
bothcases, dockedconformations showed thathemepreferentially
interacts with the tetraoxane via the O2 atom, rather than the
alternative O1 atom. For 3, conformation depicted in Figure 14,
the O1ꢀO2 bond and the amide side chain are at equatorial
positions of the cyclohexyl ring, e.g., trans to each other, making
this peroxide accessible to the Fe2+ center of the porphyrin
(Figure 14). In all docking conformations, the shortest distance
between iron heme and 3 was found to be with O2, ranging from
2.4 Å to 2.8 Å for the lowest energy conformations (27/50)
suggesting a preferred coordination of Fe on the sterically less-
hindered oxygen atom O2 of the peroxide bond of 3, confirming
the regioselectivity observed in alkylation experiments.
’ CONCLUSIONS
Iron(II)-mediated activation of tetraoxanes results in the
formation of a primary and a secondary C-centered radical, in
contrast to 1,2,4-trioxolanes and the stability of 1,2,4,5-tetraox-
anes studied here, in the presence of nonheme ferrous iron, is
dependent on the nature of the amide side chain. Overall the
stability of tetraoxanes is comparable with that of their trioxolane
counterparts (when substituted with identical side chains), and
both classes of drugs react with PC to give peroxidation products
after reductive activation by Fe2+. On the basis of products
identified from the activation chemistry in these reactions, it
appears that for tetraoxanes the pathway of lipid peroxidation
involves lipid H-abstraction by C-radical intermediates. All of the
tetraoxanes used in this study were shown to rapidly react with
Fe(II)-heme; the major reaction products are alkylated heme
adducts, resulting from the addition of the adamantane-derived
secondary C-radical intermediate. Docking studies with heme
provide a plausible explanation for the regioselectivity observed
Synthesis of Trioxolanes 13 and 14. cis-Adamantane-2-spiro-
30-80-(carboxymethyl)-10,20,40-trioxaspiro[4.5]decane was prepared ac-
cording to the method reported by Vennerstrom.37
Preparation of cis-adamantane-2-spiro-30-80-[1-(2-methylpropyl)pip-
erazino]-10,20,40-trioxaspiro[4.5]decane (13): To a solution of cis-ada-
mantane-2-spiro-30-80-(carboxymethyl)-10,20,40-trioxaspiro[4.5]decane
(0.5 g, 1.55 mmol) in dry CH2Cl2 (50 mL) at 0 °C were added Et3N
(0.44 mL, 3.10 mmol, 2 equiv) and methyl chloroformate (0.12 mL,
1.55 mmol, 1 equiv) under N2. The mixture was stirred at 0 °C for 1 h,
and 1-(2-methylpropyl)piperazine was added (0.22 mL, 1.55 mmol,
1.0 equiv). The reaction mixture was stirred at 0 °C for 45 min and at rt
for another 2 h. The reaction mixture was diluted with 50 mL of water
and extracted with CH2Cl2 (2 ꢁ 60 mL). The organic layers were com-
bined and washed with water (40 mL) and brine (40 mL), dried on
Na2SO4, and evaporated in vaccuo. The crude product was purified by
flash chromatography (silica gel, eluent, ethyl acetate/CH2Cl2 50:50) to
afford trioxolane 13 (0.48 g, 70%) as a white solid.
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1H NMR (500 MHz) δ 3.64 (bs, 2H), 3.48 (bs, 2H), 2.39 (bs, 4H),
2.23 (d, J = 6.9 Hz, 2H), 2.10ꢀ1.60 (m, 24H), 1.26 (dq, J = 13.2 and 5.4
Hz, 2H), 0.93 (d, J = 6.6 Hz, 6H); 13C NMR (125 MHz) δ 170.4, 111.1,
108.6, 66.7, 53.9, 53.3, 45.8, 41.6, 39.1, 36.9, 36.5, 34.8, 34.1, 33.4, 30.3,
26.9, 26.6, 25.4, 20.8. HRMS calculated for C26H43N2O4 [M + H]+:
447.3223. Found: 447.3221. Elemental analysis, C: 70.16, H: 9.61, N:
6.40 (required values, C: 69.92, H: 9.84, N: 6.40).
37.8, 32.8, 29.7, 27.0, 11.0; MS (ES+) HRMS calculated for C15H20F-
NO3 [M + Na]+, 304.1325. Found, 304.1319.
1
Data for 12: H NMR (400 MHz, CDCl3) δ 7.28ꢀ7.22 (m, 2H),
7.08ꢀ6.97 (m, 2H), 5.77 (s, NH), 4.43 (d, J = 5.7 Hz, 2H), 2.48ꢀ2.32
(m, 6H), 2.24ꢀ2.17 (m, 3H), and 2.17ꢀ2.05 (m, 2H). 13C NMR
(100 MHz, CDCl3) δ 211.6, 171.5, 162.3 (d, J = 246.1 Hz), 134.1 (d, J =
3.2 Hz), 129.6 (d, J = 8.0 Hz), 115.7 (d, J = 21.5 Hz), 43.1, 42.8, 40.8,
33.6, 32.7; MS (ES+), HRMS calculated for C15H18FNO2 [M + Na]+,
286.1214. Found 286.1216.
Preparation of cis-adamantane-2-spiro-30-80-[1-methyl-4-(4-piperidino)-
piperazino]-10,20,40-trioxaspiro[4.5]decane (14): To a solution of cis-
adamantane-2-spiro-30-80-(carboxymethyl)-10,20,40-trioxaspiro[4.5]decane
(1.5 g, 4.65 mmol) in dry CH2Cl2 (150 mL) at 0 °C were added Et3N
(1.32 mL, 9.30 mmol, 2 equiv) and methyl chloroformate (0.36 mL,
4.65 mmol, 1 equiv) under N2. The mixture was stirred at 0 °C for 1 h, and
1-methyl-4-(4-piperidino)piperazine was added (860 mg, 4.69 mmol,
1 equiv). The reaction mixture was stirred at 0 °C for 30 min and at rt
for other 2 h. The reaction mixture was diluted with 150 mL of water and
extracted with CH2Cl2 (3 ꢁ 100 mL). The organic layers were combined
and washed with water (100 mL) and brine (100 mL), dried on Na2SO4,
and evaporated in vaccuo. The crude product was purified by flash
chromatography (silica gel, eluent, CHCl3/methanol 100:0 to 90:10) to
afford trioxolane 14 (418 mg, 20%) as a white solid.
The degradation reaction of tetraoxane 6 (0.100 g, 224 μmol) was
also performed using ferrous sulfate. We obtained 6, 10, 12, and
unknown products in yields of 8.9%, 47%, 27%, and 22 mg, which were
purified by column chromatography
Reaction of Antimalarial Peroxides with FeSO4 and PC.
Stock solutions of PC, FeSO4, and peroxides were degassed prior to use.
To a solution of PC in water (500 μL, 0.83 μmol) were added an
aqueous solution of ferrous sulfate (500 μL, 7.2 μmol, 8.7 equiv) and a
solution of peroxide in absolute ethanol (500 μL, 6.81 μmol, 8.2 equiv).
For the control reaction, the peroxide solution was replaced by absolute
ethanol (500 μL). The cloudy mixtures were stirred at 38 °C for 24 h
under nitrogen atmosphere. An aliquot of each crude mixture was taken
for ESI/MS analysis before reactions were quenched with 50% phytic
acid solution (10 μL) and a trace of BHT, according to the procedure
reported by Kumura et al.20 The reaction mixtures were extracted with
CHCl3/CH3OH (1:2, 1 mL), and the chloroform layers were analyzed
by ESI/MS.
General Procedure for the Reaction of Antimalarial Per-
oxides with Heme. Hemin stock solution (10 mM in 0.1 M NaOH)
was freshly prepared and degassed with acetonitrile prior to use. Hemin
solution (2.0 mL, 0.02 mmol) was added to a solution of peroxide
(0.02 mmol, 1 equiv) and dithionite (41 mg, 0.20 mmol, 10 equiv) in
acetonitrile (2.0 mL) under argon. The mixture was stirred at room
temperature and monitored by LC-MS.
LC-MS analysis: Compounds were eluted using a binary gradient
solvent system consisting of MeOH/1% TFA (solvent A) and H2O/1%
TFA (solvent B). The gradient used was 70% to 80% of solvent A over
10 min (flow rate: 0.5 mL/min). Retention times were 3.24 min for
heme and 5.39 to 8.15 min for the heme-tetraoxane adducts.
Molecular Docking. Docking calculations: The docking calcula-
tions were carried out using the automated docking program AutoDock
4.0.36 The grid maps for each atom type found in the ligand structure
were calculated using the auxiliary program AutoGrid 4.0. The grid size
was set to 100 ꢁ 100 ꢁ 100 points with 0.375 Å spacing. The Lamarckian
genetic algorithm (LGA) was selected for ligand conformational search-
ing. Default parameters were used, except for the number of docking
runs, which was set to 50 and the number of evaluations to 10,000,000.
Flexible torsion angles in the ligands were assigned with Autotors, an
auxiliary module of AutoDock Tools. The resulting docked conforma-
tions were clustered into groups of similar binding modes, with a root-
mean-square deviation (rmsd) clustering tolerance of 2.0 Å.
Heme and tetraoxane structures: The heme molecule was prepared
from hemoglobin crystallographic structure obtained from the Protein
Data Bank (pdb code: 2DN1). The Fe oxidation state was 2. RKA182
(3) was built using its X-ray structure. The conformation of the 6-
membered rings was kept to that of the X-ray structure, e.g., chair con-
formation. The atomic charges were calculated with the Gaussian suite.38
The UHF/6-31 g** and HF/6-31 g** basis sets were used for heme and
tetraoxane, respectively.
1H NMR (500 MHz) δ 4.65 (bd, J = 12.8 Hz, 2H, CH2), 3.92 (bd, J =
13.3 Hz, 2H, CH2), 3.01 (t, J = 12.8 Hz, 2H), 2.70ꢀ2.39 (m, 7H, CH/
CH2), 2.31 (s, 3H, NCH3), 2.23 (d, J = 6.9 Hz, 2H), 2.03ꢀ1.25 (m, 27H,
CH/CH2); 13C NMR (125 MHz) δ 170.2, 111.3, 108.7, 61.7, 55.4, 49.0,
45.9, 45.1, 41.1, 39.2, 36.9, 36.5, 34.8, 34.1, 33.4, 30.3, 29.2, 28.3, 26.9,
26.6. HRMS calculated for C28H46N3O4 [M + H]+, 488.3488. Found:
488.3487. Elemental analysis, C: 68.76, H: 9.00, N: 8.60 (required
values, C: 68.96, H: 9.30, N: 8.62).
General Procedure for the Reaction of Tetraoxanes with
Fe2+ and TEMPO. A solution of tetraoxane (0.5 mmol), FeSO4 or
FeBr2 (2 equiv), and TEMPO (2 equiv) in THF or CH2Cl2/CH3CN
50:50 (10 mL) was stirred at ambient temperature under nitrogen atmo-
sphere for 24 h and concentrated. The crude product was dissolved in
ethyl acetate, washed with water and brine, dried over MgSO4, filtered,
and concentrated. The crude product was analyzed by LC-MS, and
TEMPO adducts were identified in all cases.
General Procedure for the Reaction of Antimalarial Per-
oxides with Ferrous Sulfate. Stock solution of peroxide (10 mM in
absolute ethanol) and ferrous sulfate (100 mM in water) were freshly
prepared and degassed prior to use. A solution of ferrous sulfate (1.0 mL,
18.2 mM) was added to a solution of peroxide (1.0 mL, 1 mM) in ACN/
H2O 1:1 (final volume 5.5 mL). The reactions were allowed to stir at
room temperature under nitrogen with LC-MS monitoring.
LC-MS analysis: Compounds were eluted using a ternary solvent
system consisting of 50% MeOH, 35% acetonitrile, and 15% 0.1 M am-
monium acetate in isocratic mode (flow rate: 0.5 mL/min).
General Procedure for the Reaction of Tetraoxane 6 with
Iron(II) Gluconate. To a stirred solution of 6 (0.200 g, 449 μmol) in
water (5 mL) and dimethylformamide (20 mL) under nitrogen atmo-
sphere, Fe(gluconate)2 (0.400 g, 898 μmol, 2 equiv) was added. The
mixture was left to stir for 12 h at room temperature. Dimethylformamide
was removed in vacuo and the crude product extracted with dichloro-
methane (3 ꢁ 20 mL). The combined organic extracts were washed with
brine, dried over magnesium sulfate, and concentrated in vacuo to give
the crude product. Purification by flash column chromatography (silica
gel, 1:1 ethyl acetate/dichloromethane) obtained 10 (56.4 mg, 44.6%)
and 12 (61.2 mg, 51.7%) as white solids.
Data for 10: 1H NMR (400 MHz, d4-MeOH) δ 7.45ꢀ7.08 (m, 2H),
7.08ꢀ6.91 (m, 2H), 6.34 (s, NH), 4.35 (pseudo s, 2H), 2.28 (s, 2H,),
2.20 (s, 2H), 1.85 (s, 1H), 1.58 (s, 2H), 1.29 (s, 2H), and 0.84 (s, 3H);
13C NMR (100 MHz, d4-MeOH) δ 178.3, 175.4, 163.4 (d, J = 244 Hz),
136.3 (d, J = 3 Hz), 130.5 (d, J = 8 Hz), 116.1 (d, J = 22 Hz), 43.4, 41.3,
’ ASSOCIATED CONTENT
S
Supporting Information. Additional information includ-
b
ing the synthesis of 11b, LCMS methods and traces, determina-
tion of antimalarial activity, and the in vivo activity data for 6.
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Journal of Medicinal Chemistry
ARTICLE
This material is available free of charge via the Internet at http://
pubs.acs.org.
(12) Cardi, D.; Pozza, A.; Arnou, B.; Marchal, E.; Clausen, J. D.;
Andersen, J. P.; Krishna, S.; Moller, J. V.; le Maire, M.; Jaxel, C. Purified
E255L Mutant SERCA1a and Purified PfATP6 Are Sensitive to SERCA-
type Inhibitors but Insensitive to Artemisinins. J. Biol. Chem. 2010,
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’ AUTHOR INFORMATION
(13) Bousejra-El Garah, F.; Stigliani, J. L.; Cosledan, F.; Meunier, B.;
Robert, A. Docking Studies of Structurally Diverse Antimalarial Drugs
Targeting PfATP6: No Correlation between in Silico Binding Affinity
and in Vitro Antimalarial Activity. ChemMedChem 2009, 4, 1469–1479.
(14) Haynes, R. K.; Chan, W. C.; Wong, H. N.; Li, K. Y.; Wu, W. K.;
Fan, K. M.; Sung, H. H. Y.; Williams, I. D.; Prosperi, D.; Melato, S.;
Coghi, P.; Monti, D. Facile Oxidation of Leucomethylene Blue and
Dihydroflavins by Artemisinins: Relationship with Flavoenzyme Func-
tion and Antimalarial Mechanism of Action. ChemMedChem 2010,
5, 1282–1299.
(15) Haynes, R. K.; Cheu, K. W.; Tang, M. M. K.; Chen, M. J.; Guo,
Z. F.; Guo, Z. H.; Coghi, P.; Monti, D. Reactions of Antimalarial
Peroxides with Each of Leucomethylene Blue and Dihydroflavins: Flavin
Reductase and the Cofactor Model Exemplified. ChemMedChem 2011,
6, 279–291.
Corresponding Author
*(P.M.O.) Phone: 00-44-151-794-3553. Fax: 00-44-151-794-3588.
E-mail: p.m.oneill01@liv.ac.uk. (F.B.-E.G.) Phone: 00-33-561-333-
248. Fax: 00-33-561-553-003. E-mail: bousejra@lcc-toulouse.fr.
’ ACKNOWLEDGMENT
We thank Jill Davies (LSTM) for IC50 values and Allan Mills
(University of Liverpool) for MS analysis. F.B.-E.G. thanks
Dr. Anne Robert for helpful discussions and comments and
CALMIP (Calcul Intensif en Midi-Pyrꢀenꢀees, Toulouse) for
computing facilities. This work was supported by a grant from
the EU (Antimal PhD fellowship (to F.B.-E.G.) and FP6Malaria
Drugs Initiative (LSHPCT20050188)) and by the Medical
Research Council [grant number G0700654] (to M.H.-L.W.).
(16) Vennerstrom, J. L.; Fu, H. N.; Ellis, W. Y.; Ager, A. L.; Wood,
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Tetraoxanes: A New Class of Antimalarial Peroxides. J. Med. Chem.
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PC, phosphatidylcholine; TPP, tetraphenylporphyrin; ROS,
reactive oxygen species; WHO, World Health Organization
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