Antiplasmodial activity of alkyl-substituted 1,2-dioxetanes against Plasmodium falciparum

Article abstract of DOI:10.1016/j.bmcl.2016.08.096

This article reports the in vitro antiplasmodial activity of two endoperoxides of the class 1,2-dioxetanes against Plasmodium falciparum: bis(adamantyl)-1,2-dioxetane and 3,3,4,4-tetramethyl-1,2-dioxetane. The results reveal that bis(adamantyl)-1,2-dioxetane displays substantial antiplasmodial activity, at least two orders of magnitude higher than that of artemisinin, while 3,3,4,4-tetramethyl-1,2-dioxetane is less active.

Full text of DOI:10.1016/j.bmcl.2016.08.096

Bioorganic & Medicinal Chemistry Letters xxx (2016) xxx–xxx
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Antiplasmodial activity of alkyl-substituted 1,2-dioxetanes against
Plasmodium falciparum
Adriana F. Silva a, Vani X. Oliveira Jr. a, Leandro S. Silva b, Ana A. S. Pinheiro b, Luiz F. M. L. Ciscato a,
⇑
a Centro de Ciências Naturais e Humanas, Universidade Federal do ABC, Avenida dos Estados 5001, Santo André, SP 09210-580, Brazil
b Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
This article reports the in vitro antiplasmodial activity of two endoperoxides of the class 1,2-dioxetanes
against Plasmodium falciparum: bis(adamantyl)-1,2-dioxetane and 3,3,4,4-tetramethyl-1,2-dioxetane.
The results reveal that bis(adamantyl)-1,2-dioxetane displays substantial antiplasmodial activity, at least
two orders of magnitude higher than that of artemisinin, while 3,3,4,4-tetramethyl-1,2-dioxetane is less
active.
Received 15 August 2016
Revised 29 August 2016
Accepted 31 August 2016
Available online xxxx
Ó 2016 Published by Elsevier Ltd.
Keywords:
Antimalarials
Peroxides
1,2-Dioxetane
Peroxidic antiplasmodial drugs like artemisinin (1) are essential
in frontline therapies capable of destroying or inactivating the
malaria-causing Plasmodium parasite;1 systematic studies
observed that several other peroxides with different structural pat-
terns also displayed significant antiplasmodial activity.2 The exact
mechanism of action of peroxidic drugs remains controversial,
however it is well established that redox-active iron(II) species
play an important role, supposedly through a mechanistic pathway
based on the Fenton-reaction, that produces reactive radical
species (Fig. 1).3
1,2-Dioxetanes are four-membered cyclic organic peroxides
that have the unique ability to produce light upon thermal or
chemical activation;4 therefore, they find extensive applications
as chemiluminescent reactants, mainly in bioanalytical assays.5
The antiplasmodial activity of 1,2-dioxetanes has been originally
briefly reported by Sharma and cooworkers;6 recently, our group
conducted a more extensive examination of the activity of a series
of 3-methoxy-1,2-dioxetanes, and found a moderate in vitro
antiplasmodial activity.7 This has encouraged us to test other
1,2-dioxetanes against Plasmodium falciparum cultures.
of the tested compounds in different concentrations.8 The percent-
age of parasite invasion9 (determined by dividing the number of
parasites presenting intracellular rings by the total number of par-
asites after 24 hours of incubation) yielded the parasitemia levels,
in the presence or absence of the tested substances. The presence
of rings is easily observed and indicates that the parasite was
active at the time of measurement.10
Also, the hemolytic activity of the substances was evaluated;
uninfected erythrocytes (kept in the same conditions used in the
invasion assay) were treated with 10À8 M of each tested compound
at 37 °C for 24 h. After this time, the supernatant was collected and
clarified at 900 g for 8 min and the free hemoglobin amount was
determined by absorption at 530 nm, using untreated cells as
control for non-specific hemolysis. The hemolytic activity was
determined calibrating the level of 100% hemolysis treating the
cells with distilled water (Table 1).
The therapeutic index (TI) – Minimum Hemolytic Concentra-
tion/Minimum Inhibitory Concentration (MHC/MIC) ratio – is a
parameter used to represent the specificity of the antiplasmodial
The antiplasmodial activity of the 1,2-dioxetanes was evaluated
observing their efficiency in deactivating Plasmodium falciparum
cultures in vitro, using artemisinin as reference; this data is
reported as IC50 values (Table 1). Schizont forms of the parasite
were incubated with a fresh culture of erythrocytes in the presence
⇑
Corresponding author. Tel.: +55 (11) 49968375.
E-mail address: luiz.ciscato@ufabc.edu.br (L.F.M.L. Ciscato).
Figure 1. Structure of artemisinin (1), bis(adamantyl)-1,2-dioxetane (2) and
3,3,4,4-tetramethyl-1,2-dioxetane (3).
http://dx.doi.org/10.1016/j.bmcl.2016.08.096
0960-894X/Ó 2016 Published by Elsevier Ltd.
Please cite this article in press as: Silva, A. F.; et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.08.096

2
A. F. Silva et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
Table 1
Antiplasmodial and hemolytic data for artemisinin (1), bis(adamantyl)-1,2-dioxetane (2) and 3,3,4,4-tetramethyl-1,2-dioxetane (3)
Entry
IC50 (Â108 M)
Relative antiplasmodial activity
Hemolytic activity (%)
Therapeutic index
1
2
3
1.0 0.2
0.019 0.001
4.5 0.1
1
105
0.45
1.5 0.2
2.0 0.4
1.4 0.4
108
108
107
Conditions: IC50 assay – compounds were diluted in seven concentrations giving 16–84% inhibition. Hemolytic assay – uninfected human red blood cells were treated with
distilled water and kept in the same conditions used in the invasion assay. After incubation, the supernatant was collected, centrifuged and the hemoglobin content was
detected in a spectrophotometer.
compound in parasite infection and it is associated with different
parasites growth stages in erythrocytes from blood.11 It was
calculated by the ratio of MHC and MIC; higher values in therapeu-
tic index represent greater antiplasmodial specificity. The MHC
values for all compounds were calculated and compared to com-
pound 1. Even if the experiments exhibited different results for
MIC and MHC values, the TIs for compounds 1 and 2 are equivalent,
and on the other hand, compound 3 presented different specificity
that is observed by TI value for compound 3 (Table 1 and Table S2
of Supplementary Data).
Despite all compounds presenting low hemolytic activity, their
antiplasmodial activity was remarkably diverse. Surprisingly, in
the conditions tested, bis(adamantyl)-1,2-dioxetane (2) displayed
a remarkable antiplasmodial activity, two orders of magnitude
higher then artemisinin on a molar basis. However, 3,3,4,4-
tetramethyl-1,2-dioxetane (3) had a much lower antiplasmodial
activity, about half of the artemisinin. Since 3 is much more
sensitive then 2 to thermal cleavage and chemical reactivity, it is
possible that it is affected and destroyed before entering the
parasite, while 2 is much more robust, it can enter and affect the
parasite more effectively.
In summary, this work supports the hypothesis that 1,2-
dioxetanes have significant antiplasmodial activity in vitro. The
effects of these compounds were studied in schizont forms of
Plasmodium falciparum of the W2 strain, and the statistical analysis
of the obtained data corroborates the existence of a significant
effect compared to the control experiment. The discovery of a
new class of peroxides with significant antiplasmodial activity
can open new horizons for the study of these compounds in several
other conditions, such as in different phases of the plasmodium or
using different concentrations or structures of 1,2-dioxetane to
tune the formation of a specific set of reactive radicals. It may also
be important in the development of new therapeutic strategies to
address the new artemisinin-resistant strains that are slowly
emerging worldwide.12
Supplementary data
Supplementary data (synthesis details, complete data sets
regarding the parasite counting and statistical analysis) associated
with this article can be found, in the online version, at http://
dx.doi.org/10.1016/j.bmcl.2016.08.096.
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This work was supported by FAPESP grants (LFMLC
2012/02428-5 and VXOJ 2011/10823-9).
Please cite this article in press as: Silva, A. F.; et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.08.096