Antimalarial effect of 3-methoxy-1,2-dioxetanes on the erythrocytic cycle of Plasmodium falciparum

Article abstract of DOI:10.1111/cbdd.12599

The antimalarial activity of peroxides most likely originates from their interaction with iron(II) species located inside the malaria parasite, which forms destructive radical species through a Fenton-like mechanism. This article reports the first evaluat

Full text of DOI:10.1111/cbdd.12599

Chem Biol Drug Des 2015; 86: 1373–1377
Research Article
Antimalarial Effect of 3-Methoxy-1,2-Dioxetanes on the
Erythrocytic Cycle of Plasmodium falciparum
1
1
Nicolas S. Lopes , Ariane M. Yoshitake ,
dence supports the occurrence of a Fenton-like reaction
(4) followed by the formation of highly reactive oxygen-
and carbon-centered radical species upon the irreversible
one-electron reduction of the peroxides by iron(II) species
1
1
Adriana F. Silva , Vani X. Oliveira Jr ,
2
2
Leandro S. Silva , Ana A. S. Pinheiro and
1
,
Luiz Francisco M. L. Ciscato *
(5). These radicals can cause non-specific destruction and
1
Centro de Ci e^ ncias Naturais e Humanas, Universidade
the alkylation of biomolecules, resulting in parasite inactiva-
tion or death. It is therefore clear that peroxidic drugs
interact primarily with the parasite phases containing sig-
nificant quantities of iron, that is, after the invasion of red
blood cells, in their erythrocytic stage (6).
Federal do ABC, Avenida dos Estados 5001, Santo Andr eꢀ ,
SP 09210-580, Brazil
2
Instituto de Bio fꢀı sica Carlos Chagas Filho, Universidade
Federal do Rio de Janeiro, Ilha do Fund a~ o, Rio de Janeiro,
RJ 21944-970, Brazil
*Corresponding author: Luiz Francisco M. L. Ciscato,
luiz.ciscato@ufabc.edu.br
1
,2-Dioxetanes are four-membered cyclic peroxides that
have been studied primarily for their chemiluminescent
properties, because they can undergo highly exergonic
cleavage to form carbonyl fragments in the triplet or in the
singlet excited states (7); they have also been studied for
their applications as valuable synthetic intermediates (8).
The most widely accepted mechanism for the formation of
excited states in high yields from 1,2-dioxetanes postu-
lates a one-electron reduction of the peroxide bond fol-
lowed by the formation of a paired radical cation and
anion, which can form carbonyl fragments in the excited
state through intra- or intermolecular recombination (9).
Because radical formation occurs upon the reduction of
The antimalarial activity of peroxides most likely origi-
nates from their interaction with iron(II) species located
inside the malaria parasite, which forms destructive
radical species through a Fenton-like mechanism. This
article reports the first evaluation of the in vitro antima-
larial activity of three peroxides of the class 1,2-dioxe-
tanes against Plasmodium falciparum; the results
reveal that the studied 3-methoxy-1,2-dioxetanes dis-
play significant antimalarial activity, at a similar level
as artemisinin and also that their reactivity toward iron
(II) correlate linearly with their antimalarial activity.
1
,2-dioxetanes, a central question is now under investiga-
Key words: 1,2-dioxetanes, antimalarial peroxides, Fenton
reaction, Plasmodium falciparum
tion: Do they also possess antimalarial activity analogous
to other peroxides?
Received 28 April 2015, revised 19 May 2015 and accepted
for publication 27 May 2015
It is expected that 1,2-dioxetanes form reactive species
upon reduction by iron(II) species, a result that has never
been directly observed by EPR but that is highly probable
(10). Because 1,2-dioxetanes have a high energy content,
they can spontaneously generate highly reactive species,
such as carbon-centered radicals; they certainly have suffi-
cient energy to generate singlet excited states. Following
this hypothesis, Ram P. Sharma and coworkers described,
in a communication in 1992, the use of two 1,2-dioxe-
tanes (prepared at the time as synthetic intermediates) as
antimalarials (11). They briefly reported a ‘moderate anti-
malarial activity when tested in vitro against the African D-
6 clone and Indo-China W-2 clone of P. falciparum’.
Despite their plans to prepare and study other 1,2-dioxe-
tanes more extensively, there has been no such report in
the literature. In this work, we prepared and studied the
antimalarial activity of the 1,2-dioxetanes 2, 3 and 4 in an
initial examination of the biological effects of this class of
compounds on Plasmodium falciparum.
The antimalarial activity of artemisinin (Figure 1, 1), a natural
peroxide extracted from the Chinese herb Artemisia annua, has
led to the development of modern therapies (artemisinin-based
combination therapies, ACTs) capable of destroying new qui-
nine-resistant plasmodium strains in vivo (1). However, the
majority of artemisinin is obtained from a natural source with
erratic availability, and its synthesis from commercial com-
pounds is considered too complex and costly, despite signifi-
cant recent progress in this field (2). To analyze the antimalarial
activity of artemisinin and its semi-synthetic derivatives, system-
atic studies have tested a variety of organic peroxides, many of
which also display significant antimalarial activity, particularly
the peroxides 1,2,4-trioxolane and 1,2,4,5-tetraoxanes (3).
The exact mechanism of action of peroxidic drugs against
the plasmodium is still under debate, but accumulated evi-
ª 2015 John Wiley & Sons A/S. doi: 10.1111/cbdd.12599
1373

Lopes et al.
enriched in the schizont form were obtained after synchro-
nization with 5% sorbitol, as described elsewhere (13).
H
O O
O
O
O
O
O
H
Erythrocyte hemolysis assay
2
O
The bioassay experiment was carried out in duplicate and
generated equivalent results. To perform the hemolysis
assay, uninfected erythrocytes were kept in the same con-
ditions used in the invasion assay. Briefly, cells were trea-
1
O
O O
O O
ꢀ8
ted with each compound tested (10 M), at 37 °C for
2
4 h. After incubation, the supernatant was collected, clar-
O
ified at 900 g for 8 min and the hemoglobin content was
detected in a spectrophotometer at 530 nm. Non-specific
hemolysis was discounted using untreated cells as control.
The hemolytic activity was determined as percentage of
the control, which was prepared treating the cells with dis-
tilled water (100% hemolysis).
3
4
Figure 1: Structure of artemisinin (1) and the 1,2-dioxetanes
studied in this work (2, 3 and 4).
Methods and Materials
Statistical analysis
1
,2-dioxetanes and artemisinin
The experiment was performed in independent cell sus-
pensions. The data were analyzed by one-way analysis of
variance (ANOVA), considering the treatments as factors.
The significance of the differences was verified by the
Bonferroni t-test. Statistical analysis was performed using
absolute values; the data are expressed as the
mean ꢁ standard deviation of the parasitemia percentage/
blade (n = 10) and were considered statistically significant
compared with the control value if p < 0.05. The software
GraphPad Prism version 5.00 for Windows was used
(GraphPad Software, San Diego, CA, USA, www.graph-
pad.com).
The synthesis and characterization of all 3-methoxy-1,2-di-
oxetanes studied in this work are described in detail at the
supporting information. Artemisinin (Sigma, Saint Louis,
MO, USA, 98%) was used as received.
Parasite culture in vitro
Erythrocytic asexual stages of the P. falciparum W2 strain,
characterized as chloroquine resistant and mefloquine sen-
sitive, were maintained in continuous culture in RPMI 1640
medium (Invitrogen) in the presence of A-type human
blood and 10% A positive human serum, 50 mg/mL gen-
tamicin (Invitrogen) at 4–5% hematocrit, as described by
Trager and Jensen (12). A thick blood smear was pre-
pared for parasitemia determination by Diff-Quick staining,
and the percentage parasitemia was described as the
number of parasitized red blood cells (PRBC) in 100 ery-
throcytes after the analysis of at least five random micro-
scopic fields.
Kinetic assays
A solution containing 0.20 M of ammonium iron(II) sulfate
hexahydrate was prepared freshly each day in hydrochlo-
ric acid solution at pH = 2.0. A stock solution of artemisi-
nin or the 1,2-dioxetanes in DME with concentration of
ca. 20 mM was stored in the refrigerator; this solution had
its exact concentration determined by iodometric assay.
In a quartz cuvette of capacity 3.0 mL and 1.0 cm of
optical path (Hellma, QS Suprasil), 2.0 mL of 0.1 M ace-
tate buffer solution at pH = 3.8 was added, and the cuv-
ette was inserted in a thermostatized cell holder inside a
spectrophotometer and allowed to stabilize at the desired
temperature. Following this, peroxide solution was added
to obtain a final concentration of [peroxide] = 0.05 mM.
The assay was initiated by injection of 2.5–25 lL of a
0.2 M solution of the ammonium iron(II) sulfate hexahy-
drate, and the increase in absorption intensity at 280 nm
was followed over at least three half-life times. The
observed rate constants (kobs) were obtained by fitting the
absorption versus time curves using a monoexponential
associated (ExpAssoc) growth function. In all cases, a
clear first-order dependency was observed. The mean of
at least three assays is reported followed by its standard
deviation.
Parasite invasion assay
Mature forms of the parasites were collected using a Per-
coll/sorbitol gradient. Briefly, a suspension of PRBC at
2
0% hematocrit was centrifuged at 2200 9 g for 30 min
at room temperature in a bench top centrifuge over a
0%, 70%, and 90% Percoll/sorbitol gradient. A brown
4
band containing more than 90% PRBC formed at the 40%
and 70% interface and was harvested and used in interac-
tion assays with fresh erythrocyte cultures. Parasite schiz-
ont forms were incubated with a fresh erythrocyte culture
at 2% parasitemia in the absence or in the presence of ar-
temisinin and the 1,2-dioxetanes 2, 3 and 4 at the desired
concentration. Parasite invasion was determined by the
number of intracellular rings observed after 24 h incuba-
tion, at indicated parasitemia levels in the presence or in
the absence of specific compounds. Alternatively, cultures
1
374
Chem Biol Drug Des 2015; 86: 1373–1377

Antimalarial Effect of 1,2-Dioxetanes
O
MB, DCM
O2, hν
2
3
8%
45%
O
7
O
MB, DCM
O2, hν
NaHMDS
PPh3
PPh3
E/Z 2:1
THF
3
4
o
15%
O
–
20 C
O
33%
O
O
8
9
5
6
O
MB, DCM
O2, hν
Scheme 1: Synthesis of the 3-methoxy-
,2-dioxetanes 2, 3 and 4 from the
corresponding enol ethers.
42%
37%
1
Table 1: Antimalarial and hemolytic activity of artemisinin and the
,2-dioxetanes 2, 3 and 4 and bimolecular rate constant for their
reaction with iron(II)
1
8
a
Entry
IC50 9 10 (M)
Hemolytic activity (%)
k
2
(M/s)
b
1
2
3
4
2.0 ꢁ 0.2
1.0 ꢁ 0.2
2.8 ꢁ 0.6
0.60 ꢁ 0.05
1.1 ꢁ 0.1
1.5 ꢁ 0.2
4.4 ꢁ 0.4
1.1 ꢁ 0.1
–
57 ꢁ 2
40.2 ꢁ 0.4
60 ꢁ 1
a
Conditions: 0.1 M acetate buffer pH 3.8 [2, 3, and
] = 2.5 9 10 M.
ꢀ5
4
b
The autooxidation of iron(II) is faster than the rate constant for
the reaction of it with the artemisinin; therefore, it could not be
confidently determined.
Figure 2: Antimalarial and hemolytic activity of artemisinin and
the tested 1,2-dioxetanes.
Results
Chemistry
1,2-Dioxetanes are considered thermally unstable sub-
stances because of the existence of a highly exothermic
reaction pathway, combined with a weak O–O bond,
causing a low activation energy. However, this instability
can be circumvented by the placement of bulky, sterically
hindered hydrocarbon substituents adjacent to the perox-
ide (14). The adamantyl group has been shown to be suit-
able for the thermal and chemical stabilization of
1,2-dioxetanes; the fenchyl group is also very bulky and
hindered, conferring a stability similar to that of the ada-
mantyl group (15).
The 1,2-dioxetanes 2 (3-methoxy-1,2-dioxaspiro[3.5]non-
0
0
0
ane), 3 (4 -methoxyspiro[adamantane-2,3 -[1,2]dioxetane),
Figure 3: Linear correlation between the antimalarial activity of
the tested 1,2-dioxetanes and their reactivity toward iron(II).
and
4
(4 -methoxy-1,3,3-trimethylspiro[bicyclo[2.2.1]hep-
0
tane-2,3 -[1,2]dioxetane]), containing, respectively, cyclo-
hexyl, adamantyl, and fenchyl groups, were prepared
according to a known methodology used by our group,
which employs the Wittig reaction (Scheme 1) (16). First,
the Wittig betaine (5) was formed from the correspond-
ing phosphonium salt (6) using NaHMDS as the base;
then, the appropriate ketone (adamantanone or (ꢀ)-
fenchone) dissolved in 10 mL THF was added, forming
the enol ethers 7, 8, and 9 with yields of 38%, 15%
and 42%, respectively. The photooxygenation of these
compounds at ꢀ20 °C formed the 1,2-dioxetanes 2, 3
and 4 with 45%, 34% and 37% isolated yields, respec-
tively.
The reactivity of the 1,2-dioxetanes toward iron(II) was
determined using an optimized a protocol to follow the
rate of the production of iron(III) species in function of
time, using UV–vis spectrophotometry (17); as the oxida-
tion of the iron is concerted with the peroxide reduction,
Chem Biol Drug Des 2015; 86: 1373–1377
1375

Lopes et al.
it is clear that we followed the rate of the electron trans-
fer (ET), which is the rate-determining step. High-quality
kinetic data could be obtained for the reaction of the
Conflict of Interest
The authors declare no competing financial interest.
1
,2-dioxetanes with excess iron(II); the reactivity of each
compound with iron(II) is displayed as the bimolecular
rate constant (k ), which can be determined from the
References
2
slope of the plot of the observed rate constant (kobs
)
versus the concentration of iron(II). All these data are
listed in Table 1.
1. Miller L.H., Ackerman H.C., Su X.Z., Wellems T.E.
(2013) Malaria biology and disease pathogenesis:
insights for new treatments. Nat Med;19:156–167.
2
. Levesque F., Seeberger P.H. (2012) Continuous-flow
synthesis of the anti-malaria drug artemisinin. Angew
Chem Intl Edit;51:1706–1709.
Antimalarial and hemolytic activity
The percentage of parasite invasion was determined by
counting the number of parasites presenting intracellular
rings after 24 h of incubation relative to the total number
of parasites (18); the presence of rings is easily observed
and indicates that the plasmodium was active at the time
of measurement. Therefore, the effective deactivation of
the plasmodium leads to a lower percentage of observed
rings. The values of ring formation can be mathematically
converted to a relative activity, where the control has no
activity (0%). The values are listed in Table 1.
3. Jefford C.W. (2012) Synthetic peroxides as potent anti-
malarials. Curr Top Med Chem;12:373–399.
4. Salas P., Herrmann C., Orvig C. (2013) Metalloantimal-
arials. Chem Rev;113:3450–3492.
5. Bousejra-El Garah F., Wong M.H.L., Amewu R.K.,
Muangnoicharoen S., Maggs J.L., Stigliani J.L., Park
B.K., Chadwick J., Ward S.A., O’Neill P.M. (2011)
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/phos-
phatidylcholine. J Med Chem;54:6443–6455.
Discussion
6. Weissbuch I., Leiserowitz L. (2008) Interplay between
malaria, crystalline hemozoin formation, and antimalarial
drug action and design. Chem Rev;108:4899–4914.
7. Baader W.J., Bastos E.L., Stevani C.V. (2006) Chemilu-
minescence of organic peroxides. In: Rappoport Z.,
editor. The Chemistry of Peroxides. Chichester, UK:
Wiley; p. 1211–1278.
Observing the data gathered in this work (Figure 2), it
seems clear that the tested 1,2-dioxetanes have an anti-
malarial activity at least at the same level as artemisinin;
additionally, they have a low hemolytic activity. The linear
regression of the antimalarial activity (i.e. IC50) with the
1
,2-dioxetanes’ reactivity toward iron(II) displayed a very
8. Jefford C.W., Boukouvalas J., Kohmoto S., Bernardi-
nelli G. (1985) New chemistry of zwitterionic peroxides
arising by photooxygenation of enol ethers. Tetrahe-
dron;41:2081–2088.
9. Ciscato L.F.M.L., Bartoloni F.H., Weiss D., Beckert R.,
Baader W.J. (2010) Experimental evidence of the
occurrence of intramolecular electron transfer in cata-
ꢀ9
good fit (Figure 3), with a slope of (ꢀ1.09 ꢁ 0.03) 9 10
and a correlation coefficient R = 0.9995; however, there
is still no unequivocal evidence supporting the idea that
the two phenomena are directly connected. Former stud-
ies tried to observe a relationship between the iron(II)
reactivity and the antimalarial effect with dispiro-1,2,4-tri-
oxolanes (17), without obtaining any linear correlation or
obvious SAR. This article is perhaps the first time when
this kind of linear relationship has been confidently
observed.
lyzed 1, 2-dioxetane decomposition.
J
Org
Chem;75:1211–1278.
10. Bos R., Tonkin S.A., Hanson G.R., Hindson C.M., Lim K.F.,
Barnett N.W. (2009) In search of a chemiluminescence 1,4-
dioxy biradical. J Am Chem Soc;131:2770–2771.
11. Zaman S.S., Debnath J., Sarmah P., Barua N.C.,
Sharma R.P. (1992) 1,2-Dioxetanes as new antima-
larial agents. J Chem Soc Chem Commun;1992:
329–330.
12. Trager W., Jensen J.B. (1976) Human malaria para-
sites in continuous culture. Science;193:673–675.
13. Lambros C., Vanderberg J.P. (1979) Synchronization
of Plasmodium falciparum erythrocytic stages in cul-
ture. J Parasitol;65:418–420.
The plasmodium and other apicomplexan forms of life
have a somehow weak ability to cope with oxidative stress
(19); therefore, a high rate of formation of oxidative species
is indeed a way to kill this parasite. In summary, this work
supports the hypothesis that 1,2-dioxetanes have indeed
significant antimalarial activity in vitro, despite their struc-
tural simplicity and ease of preparation when compared
with artemisinin.
1
4. Bastos E.L., Baader W.J. (2007) Theoretical studies on
thermal stability of alkyl-substituted 1,2-dioxetanes.
Arkivoc;8:257–272.
Acknowledgments
This work was supported by FAPESP grants (LFMLC
15. Ciscato L.F.M.L., Weiss D., Beckert R., Baader W.J.
(2011) Fenchyl substituted 1,2-dioxetanes as an
2
012/02428-5 and VXOJ 2011/10823-9).
1
376
Chem Biol Drug Des 2015; 86: 1373–1377

Antimalarial Effect of 1,2-Dioxetanes
alternative to adamantyl derivatives for bioanalytical
applications. Photochem Photobiol A;218:41–
7.
19. van Dooren G.G., Kennedy A.T., McFadden G.I. (2012)
The use and abuse of heme in apicomplexan para-
sites. Antioxid Redox Sign;17:634–656.
J
4
1
6. Meijer E.W., Wynberg H. (1979) Singlet oxygen oxida-
tion of enol ethers – synthesis of optically-active 1,2-di-
oxetanes-II. Tetrahedron Lett;20:3997–4000.
Supporting Information
1
7. Creek D.J., Charman W.N., Chiu F.C.K., Prankerd
R.J., McCullough K.J., Dong Y., Vennerstrom J.L.,
Charman S.A. (2007) Iron-mediated degradation kinet-
ics of substituted dispiro-1,2,4-trioxolane antimalarials.
J Pharm Sci;96:2945–2956.
Additional Supporting Information may be found in the
online version of this article:
Figure S1. Structure of artemisinin (1) and the tested 3-
1
8. Saraiva V.B., Silva L.S., Da Silva C.T.F., Filho J.L.S.,
Ferreira A.T., Perales J., Souza M.C., Henriques
M.G., Neves C.C., Pinheiro A.A.S. (2011) Impairment
of the Plasmodium falciparum erythrocytic cycle
induced by angiotensin peptides. PLoS ONE;6:
e17174.
methoxy-1,2-dioxetanes.
Table S1. Raw data for the counting of reinfected erythro-
cytes upon incubation with schizont forms of Plasmodium
falciparum and the tested compounds; the method is
described in detail by Lambros and Vanderberg (13).
Chem Biol Drug Des 2015; 86: 1373–1377
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