Evidence for a common non-heme chelatable-iron-dependent activation mechanism for semisynthetic and synthetic endoperoxide antimalarial drugs

Article abstract of DOI:10.1002/anie.200604697

(Chemical Equation Presented) The iron-y of fate: Isobole analyses of fluorescently labeled antimalarial endoperoxides with chelators selective for non-heme iron, and laser confocal microscopy studies within living malaria parasites have shown that the semisynthetic analogues of artemisinin and synthetic endoperoxides (see example) share a common mechanism of action involving chelatable-iron-mediated bioactivation (see images) and irreversible alkylation of parasite targets.

Full text of DOI:10.1002/anie.200604697

Communications
DOI: 10.1002/anie.200604697
Bioorganic Chemistry
Evidence for a Common Non-Heme Chelatable-Iron-Dependent
Activation Mechanism for Semisynthetic and Synthetic Endoperoxide
Antimalarial Drugs**
Paul A. Stocks, Patrick G. Bray,* Victoria E. Barton, Mohammed Al-Helal, Michael Jones,
Nuna C. Araujo, Peter Gibbons, Stephen A. Ward, Ruth H. Hughes, Giancarlo A. Biagini,
Jill Davies, Richard Amewu, Amy E. Mercer, Gemma Ellis, and Paul M. OꢀNeill*
Herbal extracts of Artemisia annua have been used to treat
malaria since ancient times. Artemisinin (1), the refined
natural product, is extremely potent and displays little or no
cross-resistance with other antimalarial compounds.[1,2] How-
ever, it is relatively expensive, has poor bioavailability, and
has poor pharmacokinetics, properties that are shared by
semisynthetic derivatives such as artesunate (2) and artem-
ether (3).[3,4] Recently, the development of several structural
intriguing questions about the mechanism of antimalarial
selectivity of such diverse chemical structures. Here, we
present evidence that all endoperoxide antimalarial com-
pounds share a common free-iron-dependent mechanism of
activation in malaria parasites, regardless of their other
structural features. None of these drugs are activated in
mammalian cell lines, which is consistent with the proposal
that selectivity stems from a parasite-specific iron-dependent
activation mechanism, rather than recognition of a specific
parasite protein target.
The critical common pharmacophore for the entire drug
class is an endoperoxide bridge. Analogues without a
peroxidic oxygen atom (desoxo derivatives) are essentially
devoid of antimalarial activity.[6,7] This simple observation
indicates that all of the endoperoxides must be activated in
some way to exert their antimalarial effect. Some investiga-
tors have attributed the potency of these compounds to a
random “dirty bomb” mechanism centered on the production
of oxyl radicals that rearrange to more stable carbon-centered
radical (CCR) intermediates.[8–10] These CCR intermediates
are proposed to kill the parasites by alkylating vital parasite
macromolecules and heme. In this case, the antimalarial
selectivity stems from parasite-specific drug activation.[11]
Other researchers have focused on the interactions of
artemisinins with specific target proteins.[12–14]
classes of totally synthetic derivatives has raised the real
prospect of cheaper alternatives which retain effectiveness
and tolerability.[1,5] This promising work has raised some
An early clue to the involvement of iron in the activation
mechanism came from studies which showed that the
antimalarial activity of artemisinin is strongly antagonized
by iron chelators.[15] Since the chelators used in this earlier
study are selective for non-heme sources of iron, it is apparent
that chelatable parasite iron sources may have an important
role to play in the mechanism of action of endoperoxide-
based antimalarial drugs. Meshnick and co-workers also
demonstrated that heme is alkylated by artemisinin,[16] and
elegant work by the Meunier research group has elucidated
and described the chemical structures of several drug–heme
adducts.[17–21] Furthermore, metabolites of hematin–artemisi-
nin adducts have been detected in malaria-infected mice
treated with artemisinin, thus indicating the potential impor-
tance of heme alkylation in the mechanism of action of 1,2,4-
trioxanes.[22] In this study, we set out to demonstrate the
importance of non-heme sources of iron for expression of
antimalarial activity and irreversible drug accumulation for
the three most important classes of endoperoxide antimalarial
drugs. In contrast to the recent research published by the
Haynes research group, where conclusions on the mechanism
[*] Dr. P. A. Stocks, Dr. P. G. Bray, M. Al-Helal, Prof. S. A. Ward,
R. H. Hughes, Dr. G. A. Biagini, J. Davies, Dr. G. Ellis
Liverpool School of Tropical Medicine
Pembroke Place, Liverpool L35QA (UK)
Fax: (+44)151-794-3588
E-mail: p.g.bray@liv.ac.uk
V. E. Barton, M. Jones, Dr. N. C. Araujo, Dr. P. Gibbons, R. Amewu,
Dr. A. E. Mercer, Prof. P. M. O’Neill
University of Liverpool
Department of Chemistry
Liverpool L697ZD (UK)
Fax: (+44)151-794-3553
E-mail: p.m.oneill01@liv.ac.uk
[**] This work was supported by grants from the BBSRC (UK) (P.A.S.,
P.O.N., P.G.B., V.B., M.J., S.A.W.; BB/C006321/1, BBS/B/05508,
BBS/Q/Q/2004/06032, and BBS/S/P/2003/10353), Romark, Flor-
ida (R.A.), and in part by the EU (Antimal. FP6 Malaria Drugs
Initiative).
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
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of action were drawn based on the reactivity of various C-10
amino analogues with ferrous salts, we have restricted our
observations and interpretations to data obtained using living
parasites.[23]
We have employed a combination of isobole analysis and
analysis of IC50 values on a series of representative semi-
synthetic (1–4) and synthetic endoperoxides (5, 6) as well as
single-cell confocal imaging of malaria-infected erythrocytes
and endoperoxide-insensitive mammalian cell lines with two
subsets of purposely designed fluorescent-tagged probe
peroxide structures. The latter cell lines were included to
demonstrate the exquisite selectivity of malaria parasites for
this class of drug.
The isobole technique employed here is accepted by the
majority of investigators as the best way of depicting
synergistic or antagonistic interactions of a drug combina-
tion.[24,25] A marked antagonism was observed for all the
endoperoxide compounds in combination with the iron
chelator DFO (7, Figure 1). For example Figure 1A shows
raised that DFO exerts it effects by chelating extracellular
rather than intracellular iron despite data demonstrating that
a totally impermeable high-molecular-weight dextran DFO
derivative could not mimick the biological effects of DFO
iron, thus suggesting an intracellular site for DFO action.[27]
Nonetheless, we have addressed any doubts over the use of
DFO by employing the additional, more lipophilic iron
chelator deferipone (8, DFP).[28] The results with DFP were
similar to those with DFO (not shown). However, the
antagonism was more pronounced for some compounds; for
example, the combination of artemisone (4) and DFO was
found to be moderately (but reproducibly) antagonistic,
whilst the combination of artemisone and DFP produced a
significantly greater degree of antagonism (Figure 1D). In
view of the apparent resistance of artemisone to iron
activation in vitro[29] and because the iron chelators them-
selves display antimalarial activity, we needed to rule out any
possible nonspecific effects of the iron chelators (it is possible,
for example, that the endoperoxides could antagonize the
activity of the iron chelators). To address this issue we
examined the antagonism of artemisone (and artesunate) by
DFP over the much shorter time period of three hours,
conditions which had zero effect on parasite viability. By
using this protocol we found that the sensitivity of the
parasites to artemisone and artesunate was decreased signifi-
cantly (by tenfold and fivefold, respectively) in the presence
of 500 mm DFP relative to controls (two representative dose–
response graphs are shown in Figure S2 of the Supporting
Information). These data strongly suggest that non-heme
chelatable-free-iron activation is essential for the activity of
artemisone (and artesunate).
Both chelators employed in our studies are selective for
ferric iron, thus suggesting that either bioactivation of the
endoperoxide bridge is mediated by a ferric iron source in
combination with an endogeneous reducing agent or that
these chelators alter the equilibrium between ferrous and
ferric iron stores within the parasite. (It is well documented
that artemisinin derivatives are relatively stable to ferric iron
sources in the absence of reducing agents.)[30]
Figure 1. Isobolograms for: A) artemether (1; &), the ozonide ester (5;
~
*
), and the tetraoxane (6; ) in combination with DFO; B) artemisi-
What is clear is that since the chelators employed are
accepted to be selective for non-heme free iron it must be this
species of iron that is involved in the activation process. To
our knowledge, this is the first direct evidence in malaria
parasites that support the role of non-heme iron in the
intraparasitic activation process of all endoperoxides, includ-
ing the artemisone-like semisynthetic compounds which
appear to be resistant to degradation mediated by biomimetic
ferrous sulfate.[29] Having demonstrated clear antagonism for
both fluorescently tagged and untagged probe derivatives, we
then set about investigating the intraparasitic sites of accu-
mulation of representative examples as well as the role of iron
chelators in modulating cellular accumulation.
*
*
nin–acridine adduct 9 ( ) and ozonide–acridine adduct 10 ( ) with
DFO; C) artemisinin–NDBadduct ( 12) in combination with DFO; and
D) artemisone (4) in combination with DFO ( ) or DFP ( ). FIC=
fractional inhibitory concentration.
*
*
clear antagonism between DFO and artemether (3), DFO and
ozonide ester (5), as well as DFO and tetraoxane (6).
DFO (7) is a bulky trishydroxamic acid derivative that
chelates ferric iron in a 1:1 molar ratio.[26] Concerns have been
In a recent study in collaboration with the Krishna
research group a fluorescently labeled artemisinin was
employed in combination with confocal laser scanning micro-
scopy (CLSM), and the results demonstrated that the labeled
drug accumulates in the parasite cytoplasm with exclusion
from the digestive vacuole.[31] We also demonstrated that the
activation of labeled artemisinin can be prevented by DFO. In
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this fluorescent probe an acridine moiety was conjugated to
the artemisinin framework through a C-10 ether linkage. To
allay concerns that this linkage may be prone to intraparasitic
hydrolysis (as is the case for several C-10 ether-linked
derivatives in acidic media[32,33]) we have repeated these
studies using a more stable C-10 carba-linked acridine probe.
We have extended our initial observations to include appro-
priately labeled ozonides 10 and 13, and tetraoxane deriva-
tives 11.
Scheme 1. Synthesis of: A) C-10 carba-artemisinin conjugate 9 and
B) OZ conjugate 10. a) NaIO4 (0.004 equiv), acetone/water 1:1, 258C,
60%; b) oxalyl chloride (2 equiv), CH2Cl2, 08C, 100%; c) Et3N
(0.3 equiv), 6-aminoethylamino-9-chloro-2-methoxyacridine (2 equiv),
CH2Cl2, 08C, 258C, 75%; d) ClCH2CH2Cl, NaBH(OAc)3, 258C, 43%.
The synthesis of acridine artemisinin and OZ probe
molecules is depicted in Scheme 1. The C-10 allyldeoxoarte-
misinin 14 was converted into the carboxylic acid with NaIO4/
KMnO4. The carboxylic acid 15 was quantitatively converted
into the acid chloride 16 with two equivalents of oxalyl
chloride. Coupling of N-1-(6-chloro-2-methoxyacridin-9-
yl)ethane-1,2-diamine to 16, in the presence of triethylamine
provided the acridine conjugate 9 in 43% yield. The OZ
conjugate 10 was prepared by reductive amination of 17 with
the appropriate amino acridine side chain as shown in
Scheme 1B.
Tetraoxane conjugate 11 was prepared in a similar manner
by reductive amination of 6 with N-1-(6-chloro-2-methoxya-
cridin-9-yl)ethane-1,2-diamine in 68% yield (not shown, but
included in the Supporting Information). Figure 2 shows data
obtained with fluorescent acridine adducts of artemisinin,
tetraoxane, and ozonide ester, and it can be seen that
accumulation is selective only for infected erythrocytes. In
each case, there is a marked accumulation of fluorescent
probe in the parasite cytoplasm, both in the presence and
absence of DFO. These data suggest that there may be a
parasite-specific accumulation mechanism for all of these
lipophilic endoperoxides, perhaps the vacuolar membrane
Figure 2. Confocal microscope images of infected red blood cells with
acridine conjugates (1mm) in the presence or absence of DFO
(100mm). All fluorescent-labeled compounds were added 10 min before
imaging. For imaging experiments using DFO and DFP (at 100mm in
media), the parasite cultures were preincubated at 378C for
30 minutes with the appropriate iron chelator prior to use. After
uptake of the drug, the perfusion chamber was washed with 250” its
volume of media to examine the retention of fluorescence. Artemisi-
nin–acridine adduct 9 without DFO before (1a) and after wash (1b),
and with DFO (100mm) before (1c) and after wash (1d). Ozonide–
acridine adduct 10 without DFO before (2a) and after wash (2b), and
with DFO (100mm) before (2c) and after wash (2d). Tetraoxane–
acridine adduct 11 without DFO before (3a) and after wash (3b), and
with DFO (100mm) before (3c) and after wash (3d).
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network, as has been suggested for artemisinin.[31,34] Also of
significance is the fact that the drugs can be completely
washed out in the presence of DFO (Figure 2, 1c versus 1d,
2c versus 2d and 3c versus 3d), but remain irreversibly bound
when the iron chelator is absent (Figure 2, 1a versus 1b, 2a
versus 2b, and 3a versus 3b). The simplest interpretation of
these data is that the labeled compounds are being accumu-
lated by the parasite and then activated by iron to form stable
covalent adducts with parasite macromolecules.
It is also apparent from the images in Figure 2 that
acridine-labeled compounds are accumulated in the cyto-
plasm and excluded from the digestive vacuole. At first glance
this seems to rule out drug activation in the digestive vacuole.
However, the fluorescence of acridine-based fluorophores is
quenched in lysosomes because of their acidic pH value[35]
(the digestive vacuole is a secondary lysosome that maintains
an acidic pH at around 4.7).[36] In addition, we have previously
demonstrated that hematin readily quenches the fluorescence
of the acridine conjugates 9 (see Figure S3 in the Supporting
Information, MJ51) while acridine-based probes demonstrate
a propensity to bind to DNA.[37] These problems raise a
number of concerns based on the quenching of the probe in
the food vacuole as a result of the low pH value and the
presence of hematin as well as the potential for an exagger-
ated nonvacuolar signal arising from acridine/DNA binding in
the relatively large and poorly defined parasite nucleus. To
completely eliminate these concerns from the data set we
synthesized an additional set of fluorescent probes based on
the nitrobenzyldiazole (NBD) fluorochrome. These com-
pounds provide an important control since NBD does not
bind to DNA, is insensitive to pH changes in the physiological
range,[38] and is very poorly quenched by hematin.
Scheme 2. Synthesis of: A) C-10 carba–NBD conjugate 12 and B) OZ–
NBD conjugate 13. a) 1. DCC (1.25 equiv), HOBt (1.2 equiv), 4-NMM
(2.5 equiv), CH2Cl2, RT, 24h; 2. H2N(CH2)4NHBoc (2 equiv), 258C,
16h, 58% for 18; b) HCl (4m), 1,4-dioxane, 08C, 24h; c) NBD-Cl
(1.1 equiv), NaHCO3 (0.1m), MeOH, 608C, 2h, 70% for 13;
d) 1. NaOH (15%), EtOH (95%), 258C, 24h; 2. HCl (3m) (70%).
Boc=tert-butoxycarbonyl.
Table 1: In vitro antimalarial activities of selected endoperoxides versus
the 3D7 strain of plasmodium falciparum and insensitivity of human
PBMC cells to 1 and 3.
The syntheses of NBD–artemisinin conjugate 12 and OZ
probe 13 are depicted in Scheme 2. The carboxylic acid 15 was
activated in situ with dicyclohexyldicarbodiimide (DCC),
1-hydroxybenzotriazole (HOBt), and N-methylmorpholine
(4-NMM). Subsequent reaction with tert-butyl 4-aminobutyl-
carbamate provided the Boc-protected derivative 18. Amine
deprotection with 4m HCl and coupling of the hydrochloride
with 4-chloro-7-nitrobenzo[c][1,2,5] oxadiazole (NBD-Cl)
provided the target molecule 12 in 70% yield. The corre-
sponding OZ conjugate 13 was prepared in a similar manner
from ozonide ester 5. Base-catalyzed hydrolysis provided
carboxylic acid 19; this was then allowed to couple with tert-
butyl 4-aminobutylcarbamate. Cleavage of the Boc group on
this amide-coupled derivative provided the free amine which
was then coupled with NBD-Cl to give the conjugate 13 (see
the Supporting Information).
Endoperoxide drug
3D7 Plasmodium falciparum
mean IC50 [nm][a]
artemisinin 1
artemether 3
OZ ester 5
tetraoxane 6
artemisinin–acridine conjugate 9
OZ–acridine conjugate 10
tetraoxane–acridine conjugate 11
artemisinin–NBD conjugate 12
OZ–NBD conjugate 13
7.5Æ0.2
0.9Æ0.2
1.7Æ1.1
6.7Æ3.2
10.7Æ5.2
9.1Æ5.6
12.3Æ0.9
13.2Æ1.9
5.2Æ2.3
human PBMC (mm)[b]
>250
artemisinin 1
artemether 3
>250
[a] Drug susceptibilities were assessed by the measurement of fluores-
cence after the addition of SYBR Green I as previously described by
Smilkstein et al.[39a] Drug IC50 values were calculated from the log of the
dose/response relationship, as fitted with Grafit software (Erithacus
Software, Kent, United Kingdom). Results are given as the means of at
least three separate experiments. [b] IC50 values were calculated using
the MTT assay following 72 h incubation with the endoperoxides
according to the method of Holl et al.[39b]
Table 1 lists the in vitro antimalarial activity[39] of all the
analogues used in this study. All of the compounds express
antimalarial activity at less than 13 nm, and it is clear that the
presence of the fluorescent group (for example, NBD) has
little effect on the inherent drug potency, thereby validating
the use of this probe in the experiments that follow. Data were
also recorded for artemisinin and artemether versus human
peripheral blood mononuclear type B cells, where the
IC50 values are greater than 250 mm.
Figure 3. In most respects, the images obtained using the
NBD probes are similar to those obtained using acridine
probes; both sets of probes were accumulated in the parasite
Results of confocal experiments using artemisinin–NBD
conjugate 12 and ozonide ester–NBD 13 are presented in
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these data;[31] indeed it is difficult to explain the potency of
artemisone without proposing an interaction with this target.
Nevertheless, we believe that parasite-specific mechanisms of
activation are indespensible and they may be all that is
required to explain the antimalarial effects of distant endo-
peroxide structures such as the ozonide and tetraoxane
derivatives. The fact that these compounds are not excluded
from the digestive vacuole leaves the door open for a role for
chelatable non-heme iron mediated activation in this parasite
compartment. As noted, several studies have demonstrated
that artemisinin and simplified 1,2,4-trioxanes readily alkylate
heme but the impact of this event in the mechanism of action
remains unclear, particularly given the fact that other
endoperoxides such as arteflene, which fail to produce
covalent adducts with heme, express antimalarial activity in
the nanomolar range.[1,40] Chelatable sources of parasite iron
play an important role in the mechanism of action of all the
important classes of endoperoxide antimalarial drugs. This
includes totally synthetic compounds that may lack the
structural motifs for binding to PfATPase6, namely the
tetraoxanes,[41] the tetraoxane dimers,[42] and ozonide deriva-
tives.[43,44]
Figure 3. Confocal microscope images of infected red blood cells with
fluorescent NBD conjugates (1mm) in the presence or absence of iron
chelators DFO and DFP (100mm). The same protocols as described in
Figure 2 were used. Artemisinin–NBD adduct 12 without DFO before
(1a) and after wash (1b), and with DFO (100mm) before (1c) and after
wash (1d). Artemisinin–NBD adduct 12 without DFP before (2a) and
after wash (2b), and with DFP (100mm) before (2c) and after wash
(2d). Ozonide–NBD adduct 13 without DFP before (3a) and after
wash (3b), and with DFP (100mm) before (3c) and after wash (3d).
Peripheral mononuclear type Bcells (PBMC) cells with adduct 12
before (4a) and after wash (4b), and with adduct 13 before (4c) and
after wash (4d).
Received: November 17, 2006
Revised: May 31, 2007
Published online: July 19, 2007
Keywords: antimalarial agents · chelates · confocal imaging ·
.
fluorescence · iron
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