Tetroxanes as New Agents against Leishmania amazonensis

Article abstract of DOI:10.1002/cbdv.202000142

Leishmaniasis is a neglected disease, caused by a parasite of Leishmania genus and widespread in the tropical and subtropical areas of the world. Currents drugs are limited due to their toxicity and parasite resistance. Therefore, the discovery of new treatment, more effective and less toxic, is urgent. In this study, we report the synthesis of six gem-dihydroperoxides (2a–2f), with yields ranging from 10 % to 90 %, utilizing a new methodology. The dihydroperoxides were converted into ten tetroxanes (3a–3j), among which six (3b, 3c, 3d, 3g, 3h and 3j) showed activity against intracellular amastigotes of Leishmania amazonensis. The cytotoxicity of all compounds was also evaluated against canine macrophages (DH82), human hepatoma (HepG2) and monkey renal cells (BGM). Most compounds were more active and less toxic than potassium antimonyl tartrate trihydrate, used as positive control. Amongst all tetroxanes, 3b (IC₅₀=0.64 μm) was the most active, being more selective than positive control in relation to DH82, HepG2 and BGM cells. In summary, the results revealed a hit compound for the development of new drugs to treat leishmaniasis.

Full text of DOI:10.1002/cbdv.202000142

Title: Tetraoxanes as new agents against Leishmania amazonensis
Authors: Luiz Claudio Almeida Barbosa, Isabel V. Antolínez, Tatiane F.
Borgati, Almodvar Baldaia, Raquel M. Almeida, Sebastião R.
Ferreira, and Ricardo T. Fujiwara
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To be cited as: Chem. Biodiversity 10.1002/cbdv.202000142
Link to VoR: https://doi.org/10.1002/cbdv.202000142

10.1002/cbdv.202000142
Chemistry & Biodiversity
Chem. Biodiversity
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Tetraoxanes as new agents against Leishmania amazonensis
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Isabel V. Antolínez,a Luiz C. A. Barbosa, a,* Tatiane F. Borgati,a,1 Almodvar Baldaia,a Sebastião R. Ferreira,b
Raquel M. Almeida,c Ricardo T. Fujiwarac,*
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a Department of Chemistry, Universidade Federal de Minas Gerais, Av. Pres. Antônio Carlos, 6627, Campus
Pampulha, CEP 31270-901, Belo Horizonte, MG, Brazil, e-mail: lcab@ufmg.br
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b Health Science Center, Universidade Federal do Sul da Bahia, Praça Joana Angélica, 250 São José, CEP 45988-
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058, Teixeira de Freitas, BA, Brazil.
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c Department of Parasitology, Universidade Federal de Minas Gerais, Av. Pres. Antônio Carlos, 6627, Campus
Pampulha, CEP 31270-901, Belo Horizonte, MG, Brazil, e-mail: fujiwara@icb.ufmg.br
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Leishmaniasis is a neglected disease, caused by a parasite of Leishmania genus and widespread in the tropical and
subtropical areas of the world. Currents drugs are limited due to their toxicity and parasite resistance. Therefore,
the discovery of new treatment, more effective and less toxic, is urgent. In this study we report the synthesis of
six gem-dihydroperoxides (2a-f), with yields ranging from 10% to 90%, utilizing a new methodology. The
dihydroperoxides were converted into ten tetraoxanes (3a-j), among which six (3b, 3c, 3d, 3g, 3h and 3j) showed
activity against intracellular amastigotes of Leishmania amazonensis. The cytotoxicity of all compounds was also
evaluated against canine macrophages (DH82), human hepatoma (HepG2) and monkey renal cells (BGM). Most
compounds were more active and less toxic than potassium antimonyl tartrate trihydrate, used as positive control.
Amongst all tetraoxanes, 3b (IC50 = 0.64 µM) was the most active, being more selective than positive control in
relation to DH82, HepG2 and BGM cells. In summary, the results revealed a hit compound for the development of
new drugs to treat leishmaniasis.
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Keywords: tetraoxane, antileishmanial peroxides, cytotoxicity, macrophage cell lines
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Introduction
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Leishmaniasis is a neglected disease caused by different species of parasite Leishmania that are transmitted
by bite of infected female sandflies.[1] The disease is widespread in the tropical and subtropical areas of the world,
and 90% of new cases are concentrated in 13 countries (Afghanistan, Algeria, Bangladesh, Bolivia, Brazil, Colombia,
Ethiopia, India, Iran, Peru, South Sudan, Sudan and Syria).[2] According to World Health Organization (WHO) about
1 Current address: Department of Exact Sciences, State University of Minas Gerais, Av. Paraná, 3001, Jardim Belvedere I,
Campus Divinópolis, CEP 35501-170, Divinópolis, MG, Brazil.
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700,000 to 1 million of new cases and some 26,000 to 65,000 deaths occur annually around the world.[3]
Leishmaniasis affect mainly people that are living in the most impoverished parts of developing countries because
of poor house conditions, poor sanitation and irregular garbage collection provide sandflies breeding grounds.[1]
The main clinical forms of the disease are cutaneous leishmaniasis (CL) and visceral leishmaniasis (VL), that
are caused by different etiologic agents.[4] CL happen when the parasites infect macrophages resident in the skin,
and if the disease spreads to the mucous membranes of the nose, mouth and throat it is characterized as
mucocutaneous leishmaniasis (MCL). VL occurs when parasites are spread by the blood circulation and infect cells
of the mononuclear phagocyte system of liver, spleen, bone marrow, lymph nodes and the intestine, and if not
treat can be fatal.[4]
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The main drugs in current use to treat leishmaniasis are Glucantime®, amphotericin B, pentamidine, and
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miltefosine (Figure 1).[5] Despite the effectiveness of these drugs to combat the parasite, they have severe side
effects on the cardiac, kidney and liver systems, and can also be teratogenic.[6] Besides that, other drawbacks
include their high cost, long therapies, and necessity to perform the treatment in a hospital environment.[2] In
addition, the emergence of parasite resistance to drugs has limited the use of these medicines in many places.[7,8]
Therefore, it is urgent the development of new drugs for the treatment of leishmaniasis. Such new compounds
must have a minimal side effects and, desirably, be orally administered and present low cost of production.
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Figure 1. Structures of pentavalent antimonial drug Glucantime®,
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amphotericin B, pentamidine, miltefosine, artemisinin and a
tetraoxane.
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Artemisinin (Figure 1), a sesquiterpene endoperoxide isolated from Artemisia annua, is an established drug
for treating malaria, including many drug-resistant strain parasites. The mechanism of action of artemisinin against
Plasmodium falciparum is believed to involve free radical intermediates generated in a reaction activated by
heme.[9] Avery et al.[10] related the activities of artemisinin derivatives against Leishmania donovani strain, and
highlighted the importance of peroxide group to this action, suggest as potential drug candidates against
leishmaniasis. Tetraoxanes or 1,2,4,5-tetraoxanes (Figure 1) are a class of compounds containing two peroxide
groups in a six-membered heterocycle, and have a large spectrum of biological action, such as antimalarial,[11–13]
antitumor[14] and herbicide.[15] It has been proposed that tetraoxanes have a similar mode of action of artemisinin
and derivatives,[16] that is believe to involve the formation of free radicals intermediates toxic to parasite and
cancer cells.
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Antileishmanial activity from tetraoxanes against intramacrophage forms of L. donovani was recently
reported by Cabral et al.[17], that showed the importance of endoperoxide bridge to biological activity. Our
research group have been searching for new molecules with antileishmanial activity, as illustrated by the
triterpene hederagenin, a natural compound isolated from Sapindus saponaria, and its synthetic derivatives that
exhibited IC50 against L. infantum ranging from 2-12 μM.[18,19]
Therefore, considering the urgency to discovery new treatment for leishmaniasis, in this work we report the
synthesis and the in vitro effect on the proliferation of intracellular amastigote forms of L. amazonensis of a
tetraoxanes series, highlighting the high selectivity index and low toxicity of this class of compounds.
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Results and Discussion
Synthesis
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Although there are several methodologies to prepare 1,2,4,5-tetraoxanes, the overall yields are usually low,
especially in the case of asymmetric tetraoxanes.[20] In the current work the required compounds were obtained
from cyclic ketones and also aromatic aldehydes. The general methodology involves the conversion of a carbonyl
compound into the corresponding gem-dihydroperoxides, that is further reacted with another carbonyl (the same
type or a different one). In the current work four 1,1-dihydroperoxycycloalkanes (2a-d), previously prepared in
good yields,[15,21,22] were obtained, using an aqueous solution of H2O2 (56%), SnCl2 as catalyst and THF as solvent.
The compounds were isolated in pure forms in 77-90% yields (Scheme 1).
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Scheme 1. Synthesis of 1,1-dihydroperoxicycloalkanes (2a-d) derived from ketones and their conversion
into symmetric (3a-c) and asymmetric tetraoxanes (3d-h).
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In a previous work of our research group,[15] employing different experimental conditions (an aqueous
solution of H2O2 30%, and AlCl3.H2O as catalyst), compound 2d was obtained in only 36% and 2b and 2c in yields
around 10% lower than those herein reported. So, with the current optimized conditions (employing SnCl2 as a
catalyst and hydrogen peroxide at 56% v/v) the products are formed in a shorter reaction time (1.5 h against 6 h),
with higher yields (77% to 80%) and in a reproducible way (standard deviation of three repetitions of ± 2.5%).
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It is important to highlight that several works reported the synthesis of gem-dihydroperoxides under different
[24]
reaction conditions (AlCl3.6H2O;[21] H2WO4[22]; 30% H2O2, HCl;[23] NaHSO4.SiO2 and CAN[25] as catalyst, and
different aqueous solutions of H2O2), resulting in products with high yields (up to 98%). However, we found that
for some reported catalysts and experimental protocols, repeating the reaction several times under controlled
conditions, the yield can have a large variation.[26,27]
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With a reliable source of 2a-d, these compounds were reacted with cyclic ketones and benzaldehyde, under
catalysis of H2SO4 (50 mol%),[15] affording the tetraoxanes 3a-3h in yields in the range of 10 to 50%. Previous
studies reported the synthesis of some of these tetraoxanes in yields in the range of 30% to 86%, using
perfluoropropan-2-ol and 2,2,2-trifluoroethanol as solvent, and methyltrioxorhenium as catalyst.[28–31] Although
efficient, the catalyst and solvents are much more expensive than those (H2SO4 and CH2Cl2) used in current work.
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In general, our yields were higher than those previously reported for the same compounds and similar reaction
conditions.[15,31] Besides that, in the case of asymmetric tetraoxanes 3d-f, lowering the temperature to -78 ºC
resulted in better yields.
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The tetraoxanes 3i and 3j were obtained from benzaldehyde and 4-ethylbenzaldehyde, respectively (Scheme
2). The initial reaction of benzaldehyde with hydrogen peroxide and using SnCl2 resulted in 50% of the
dihydroperoxide 2e after isolation by silica-gel column chromatography. It was observed from the crude reaction
mixture that some benzoic acid was also formed (not isolated). The formation of this acid could involve a direct
oxidation of the aldehyde via a Baeyer-Villiger oxidation,[32] or by the Kornblum-DeLaMare rearrangement[33] of
the 1,1-dihydroperoxide resulting in the perbenzoic acid, that is further reduced by reaction with another
benzaldehyde molecule.
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Several attempts were made to optimize this reaction and the best conditions involved the use of SnCl2 (20%)
as catalyst and THF as solvent, at room temperature, resulting in 2e in 23%. The same procedure was then
employed in the preparation of 2f, but during purification there was a decomposition, and the required compound
was isolated in only 10%. Both 1,1-dihydroperoxides were fully characterized by spectroscopic analyses.
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Scheme 2. Synthesis of dihydroperoxides (2e-f) and their conversion into corresponding symmetric tetraoxanes.
(3i-j)
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Considering that 2e-f were decomposing during the purification by flash column chromatography, we then
reacted the crude mixture of 2e-f, after the removal of THF, with another equivalent of the corresponding
aldehyde, in the presence of H2SO4 (50% mol) in acetonitrile (1:9). This procedure afforded the required
tetraoxanes 3i and 3j in 20% and 24%, respectively, what correspond approximately the same result previously
reported.[15] All compounds had their structures proved by spectroscopic analyses and the data are in agreement
with the literature.[15]
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Biological Assay
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The leishmanicidal activities of compounds 3a-j were evaluated by in vitro ELISA assay employing intracellular
amastigotes of L. amazonensis parasite of canine macrophages DH82. L. amazonensis is a dermotropic species,
very common in Brazil, and often associated with drug resistance.[34,35] In a preliminary experiment, the percentage
of parasite growth inhibition was determined for each compound at 1000 µM in order to select those with some
potential activity for further evaluation. As observed from the data displayed at Table 1, from the ten compounds
evaluated six (3b, 3c, 3d, 3g, 3h and 3j) caused parasite burden reduction greater than 50%.
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Table 1. Percentages of L. amazonensis parasite burden reduction at 1000 M.
Compound
% Inhibition
3a
3b
3c
3d
3e
3f
3g
3h
3i
49
50
52
55
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39
58
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58
3j
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At this stage we did not attempt to correlate the structure with activity, but it can be seen that for the most
active compounds the percentage of L. amazonensis reduction varied from 50% to 58%, a very narrower interval.
From these initial results, the six most active compounds were subjected to the same assay at concentrations
varying from 0.1 µM to 1000 µM, in order to prepare a dose-response curves log (substance concentration) versus
percentage inhibition of intracellular amastigote forms of L. amazonensis, as illustrated for compound 3g (Figure
2), and calculate the IC50 values, as displayed in Table 2.
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Figure 2. Inhibition of intracellular amastigote forms of L. amazonensis caused by 3g. The dose-response
curve log (concentration of 3g) versus inhibition of L. amazonensis was performed in four replicates and
the results are represented by the mean ± SD.
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Although potassium antimonyl tartrate pentahydrate (Glucantime®) is a first-line drug in the treatment of
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the leishmaniasis, potassium antimonyl tartrate trihydrate was used as positive control of the test. It is suggested
that pentavalent antimony may be a pro-drug, that undergoes a bio-reduction of Sb5+ to its trivalent form Sb3+,
this one responsible for the toxicity and therapeutic activity of the drug.[36]
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Table 2. Antileishmanial activity of some tetraoxanes against the intracellular amastigotes of L.
amazonensis parasite of canine macrophages DH82.
Compound
IC50 ± SD (µM) [a]
Intracellular
CC50 ± SD(µM) [b]
Toxicity DH82
SI [c]
amastigotes forms
Canine macrophages
3b
0.64 ± 0.14
221.00 ± 0.17
345.3
>50.8
2.7
3c
19.70 ± 0.19
99.50 ± 0.10
16.00 ± 0.17
22.60 ± 0.13
18.00 ± 0.13
94.00 ± 0.10
>1000
3d
268.00 ± 0.17
150.00 ± 0.09
141.00 ± 0.11
175.00 ± 0.15
3.20 ± 0.11
3g
3h
9.4
6.2
3j
10.0
0.03
Control [d]
[a] IC50 – concentration of active ingredient able to inhibit by 50% the in vitro growth of L. amazonensis.
SD – standard deviation.
[b] CC50 – concentration of active ingredient that inhibits 50% of canine macrophages DH82.
[c] SI – selectivity index corresponding to the ratio CC50/IC50.
[d] Potassium antimonyl tartrate trihydrate, used as positive control.
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As observed from Table 2, the positive control presented IC50 of 94 µM and the tetraoxanes had IC50 values in
the range of 0.64 to 99.50 µM. Only 3d was slightly less active (IC50 = 99.5 µM) than the positive control. It is
interesting to note that all tetraoxanes derived from cyclopentanone (3a, 3d and 3e) were amongst the less potent
ones. On the other hand, 3b was the most active (IC50 = 0.64 µM), being 147 more potent compared to the positive
control. Tetraoxanes 3c, 3g, 3h, and 3j have IC50 in the range of 16 to 22.6 µM, being 4-6 times more potent in
comparison with the positive control. We should note that 3c, a symmetric tetraoxane derived from a seven-
membered ring ketone, was 31 times less potent than 3b, derived from cyclohexanone. Although the number of
compounds is too small to allow us to draw a conclusive structure-activity relationship, such results are in line
with a report that tetraoxanes derived from cyclohexanone are more potent as antimalarial than the
corresponding ones with smaller or larger rings.[29]
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Having demonstrated that the tetraoxanes are active against L. amazonensis parasite, their toxicity (CC50) and
selectivity index (SI) towards canine macrophages DH82 were also determined (Table 2). According to Weniger et
al [37], biological activity of the tested compounds is not attributable to cytotoxicity when the SI ≥ 10. From Table
2, it can be observed that the commercial standard drug has the lowest selectivity index (SI = 0.03), compared
with all six tetraoxanes. The SI values of tetraoxanes range from 2.7 to 345.5. Even 3d, the less active compound,
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has a SI of 2.7, a value 90 times higher than the positive control. Tetraoxane 3b, the most potent compound, has
the highest SI (345.5), being 11,510 times more selective in comparison with the positive control. Hence, 3b can
be considered a hit candidate to develop new antileishmanial drugs.
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Once we have shown that the tetraoxanes are promising candidates for the development of a new
therapeutic agent for L. amazonensis, and considering that the principle side effect of drugs to treat leishmaniasis
are nephrotoxicity and hepatotoxicity,[38] the compounds were tested on two strains of human hepatoma (HepG2)
and monkey renal (BGM) cells to evaluate their effects on liver and kidney (all results, including the corresponding
SI values, are depicted in Table 3).
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Table 3. Cytotoxic concentration (CC50) of some tetraoxanes and a standard antimonial drug against HepG2 and
BGM cells and their corresponding selectivity index (SI).
Compound
CC50 ± SD(µM) [a]
HepG2
172.00 ± 0.13
SI [b]
HepG2
268.8
CC50 ± SD (µM) [c]
BGM
101.00 ± 0.43
SI [c]
BGM
157.8
3b
3c
>1000
>50.8
1.0
>1000
>50.8
0.4
3d
98.00 ± 0.45
89.00 ± 0.12
57.00 ± 0.07
>1000
39.00 ± 0.20
82.00 ± 0.31
10.00 ± 0.08
>1000
3g
3h
5.6
5.1
2.5
0.4
3j
>55.6
0.3
>55.6
0.02
Control [d]
32.00 ± 0.05
2.00 ± 0.12
[a] CC50 – concentration of active ingredient that inhibits 50% of human hepatoma cells HepG2.
SD – standard deviation.
[b] SI – selectivity index corresponding to the ratio between CC50 and the IC50 values presented
in Table 2.
[c CC50 – concentration that kills 50% of monkey renal cells BGM.
[d Potassium antimonyl tartrate trihydrate, used as positive control.
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As expected,[18] the data from Table 3 confirms that the antimony salt, used as positive control, is very toxic
towards the human hepatoma (HepG2) and the monkey renal (BGM) cells, with selectivity index of 0.3 and 0.02,
respectively. In line with the data presented in Table 2, all tetraoxanes are less toxic in comparison with positive
control for both cell lines. In relation to the HepG2 cells, the CC50 values are in the range of 57 µM (3h) to >1000
µM (3c and 3j). All tetraoxanes were more selective than the positive control, with the lowest SI = 1.0 for 3d and
the best result was observed for 3b (SI = 268.8). This last compound is 896 times more selective in comparison
with the antimony reference.
For the renal monkey cells, the CC50 values are in the range of 10 µM (3h) to >1000 µM (3c and 3j), being
compounds 3d and 3h most toxic (showing CC50 = 39 µM and 10 µM, respectively). Despite that, these compounds
are still much less toxic than the reference towards renal monkey cells. For this same cell line, compounds 3b, 3c
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and 3j presented SI > 50, with 3b being the one with the highest selective index (SI = 157.8, corresponding to 7,890
times more selective in comparison with the antimony salt). A possible explanation to the high selectivity of
tetraoxanes is the presence of the endoperoxide bridge that can be cleaved and activated in the presence of heme
iron or free Fe2+.[39] Leishmania gets iron from host macrophages for survival and growth. [40],[41] The accumulation
of iron within the parasite can generate oxyradicals. By Fenton reaction the oxyradical intermediates can be
degraded and produce hydroxyl radicals that are very reactive against a wide variety of molecules such as enzyme,
protein and nucleic acids, and takes the parasite to death.[39]
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During the course of our research, in an innovative work Cabral et al.[17] reported the antileishmanial activity
of some 1,2,4,5-tetraoxanes against intramacrophage amastigotes forms of L. donovani strain. They found IC50
values in the range of 2.0 to 45.0 µM, and the cytotoxicity macrophage with CC50 = 34.12 to 100 µM. The authors
also reported results of in vivo activity tests, with the most active compound causing a reduction of the liver
parasite burden by 37%. Since in the current work we report activity of different tetraoxanes against L.
amazonensis, a direct comparison of our data with those by Cabral et al.[17] is not straightforward. Despite that,
tetraoxanes 3a-j presented a comparable or better profile in terms of IC50 and SI, for L. amazonensis. Although we
have not yet carried out in vivo assay, we envisage that our compounds will possibly present high activity. In
addition, based on previous studies of compounds of the same class,[42] it is expected that the tetraoxanes
obtained in this work will also present low cytotoxicity in vivo.
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Based on all data presented on Tables 1 to 3, the most active and less toxic compound was 3b, but compounds
3c, 3g and 3j were also potent and selective towards the canine macrophage (DH82), human hepatoma (HepG2)
and monkey renal (BGM) cell lines. Considering that all tetraoxanes showed SI values greater than that of positive
control potassium antimonyl tartrate trihydrate, these compounds can be considered a good model for the
development of a new drug to combat L. amazonensis.
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Conclusions
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In this work six gem-dihydroperoxides (2a-f) were efficiently synthesized; these intermediates were further
converted into tetraoxanes 3a-j. The antileishmanial activity of tetraoxanes were evaluated against the
intracellular amastigotes of L. amazonensis parasitized macrophages (DH82). Five compounds (3b, 3c, 3g, 3h and
3j with IC50 ranged from 0.64 µM to 22.6 µM) were more active than positive control Sb3+ salt (IC50 = 94 µM). The
cytotoxicity and selectivity index were also screened using DH82, BGM and HepG2 line cells and, in comparison
with positive drug control, all tetraoxanes were less cytotoxic and more selective towards the parasite. We
highlight compound 3b, most active for leishmania (IC50 = 0.64 µM), and less toxic for all cell lines (DH82, HepG2,
BGM). In summary the results revealed a class of promising molecules and a hit compound (3b) for the
development of new drugs to treat leishmaniasis.
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Experimental Section
Experimental generation information
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All reagents and solvents were purchased by Sigma Aldrich (St. Louis, MO, United States) and LabSynth (São Paulo,
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Brazil), respectively, and were purified, when necessary. The H, C and DEPT 135 nuclear magnetic resonance
(NMR) spectra were recorded on a Bruker Avance DPX 200 and DPX 400 spectrometer at 200 and 400 MHz using
CDCl3 as the solvent and tetramethylsylane (TMS) as the internal standard ( = 0). The NMR data are presented as
follows: chemical shift (); J in Hz. Multiplicities are indicated by the following abbreviations: s (singlet), d (doublet),
dd (double doublet), t (triplet), q (quartet), m (multiplet), qn (quintet) and brs (broad singlet). Infrared spectra
(FTIR) were recorded on a Spectra One PerkinElmer spectrophotometer, fitted with a Paragon ATR accessory.
Melting points were determined using a MQAPF-301 melting point apparatus (Microquímica, Brazil) and are
uncorrected. The progress of the reactions was monitored by thin layer chromatography (TLC) on aluminum
packed pre-coated silica gel plates. Column chromatography was performed over silica gel (60-230 mesh).
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Synthetic procedures
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1,1-dihydroperoxycyclopentane (2a)
To a two-neck round-bottom flask (25 mL) containing cyclopentanone (5 mmol; 420 mg) and tetrahydrofuran
(8 mL) was slowly added an aqueous solution of H2O2 56% v/v (3 ml) and SnCl2 (20 mol%; 190 mg). This mixture
was kept under magnetic stirring at 25 ºC for 90 min. Then the reaction mixture was extracted with
dichloromethane (3 x 10 mL). The organic layers were washed with saturated solution of NaHCO3 (5 mL), and dried
over Na2SO4 anhydride. The solvent was removed in a rotatory evaporator to afford the resulting crude product
which was purified by recrystallization employing the solvent mixture of hexane and diethyl ether. This procedure
afforded compound 2a as white solid (402 mg, 3.2 mmol, 60%).
The dihydroperoxides 2b-f were prepared employing a procedure similar to that described for compound 2a.
The physical and spectroscopic data of compounds 2a-f were in agreement with the previously reported [15] and
are reported in the supplementary information.
6,7,13,14-tetraoxadispiro[4.2.48.25]tetradecane (3a)
To a two-neck round-bottom flask were added cyclopentanone (0,22 mmol; 18,5 mg) and DCM (5 mL). This
mixture was cold until 0 ºC and under magnetic stirring. Then, after 10 min, 1,1-dihydroperoxycyclopentane (2a)
(0,2 mmol; 27 mg) was added to the reaction mixture. After 30 min, an aqueous solution of H2SO4 (0,11 mmol; 50
mol%) was dropwise added. The mixture was stirred for 6 h at room temperature. The reaction was quenched
with saturated solution of NaHCO3 (5 mL) and extracted with dichloromethane (3 x 5 mL). The organic layers were
collected and dried with Na2SO4 anhydride. The solvent was removed under reduced pressure to afford a dark oil.
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This oil was purified by column chromatography of silica gel eluting with a mixture of hexane/ chloroform (9:1).
This procedure afforded compound 3a as a white solid (20 mg, 0.11 mmol, 50%).
The tetraoxanes 3b-j were prepared employing a procedure similar to that described for compound 3a. The
[15]
physical and spectroscopic data of compounds 3a-j were in agreement with the previously reported
and are
reported in the supplementary information.
Caution: Since peroxides are potentially explosives, the work should be carried out taking all standard
precautions. The reactions should be carried out in a fume hood, behind a protective shield and other precautions
required by the safety committee of your institution should be considered. We had no problem or incident during
our work.
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Biological assay
Antileishmanial assay
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The leishmanicidal activities of compounds 3a-j were assessed in vitro against amastigote forms of L.
amazonensis by ELISA assay using canine macrophages (DH82), and based on previously work at the literature.[18]
About 5x104. macrophages/wellwere distributed in 96-well culture plates, and incubates at 37 ºC in 5% CO2. After
24 h, the adherent macrophages were infected with L. amazonensis amastigotes at 50:1 (parasite: cell host ratio).
Four hours following infection, cultures were washed three times with ice-cold PBS (phosphate-buffered saline)
and compound solutions in RPMI were added at the wells. After 48 h, the cultures were fixed at ice cold methanol,
dried at hot air, washed three times with 100 µL PBS containing 5% BSA (bovine serum albumin) and incubated
for 1 h at 37 ºC. After this period, the plates were washed for four times with PBS-Tween-20 (pH 7.4) and incubated
with serum of dog infected with Leishmania (1: 300) for 1 h at 37 ºC. Plates were washed four times and 100 μL
of HPR-conjugated anti-dog IgG (1: 2000) were added. The plates were incubated for 1 h at 37 ºC, and then washed
four times with PBS containing 0.05% Tween-20 (pH 7.4). The reaction proceeded by adding 100 μL of substrate
solution (10 mL citrate buffer, 10 mg of ortho-phenylenediamine, and 2 μL H2O2) to each well. After 15 min, the
reaction was stopped by the addition of 30 μL of H2SO4 (2 mol L-1). Optical density reading was measured at 492
nm (OD492nm) in an ELISA reader. The results of in vitro activity were expressed as an index of inhibition growth
compared to controls of macrophages infected with amastigotes and untreated, as follows: INDEX = (OD492nm
experimental wells / OD492nm untreated controls) x 100. To determine the activity of the compound, the index
response data was plotted and modeled by a dose-response curve based on four logistic adjustment parameters.
In the first time all the compounds were tested at concentration of 1000 μM, those compounds that inhibited
50% of in vitro parasites growths were tested at 0.1, 1, 10, 100 and 1000 μM. The experiment was performed in
quadruplicate. Potassium antimonyl tartrate trihydrate was used as positive drug control at same concentrations
of the compounds (0.1 – 1000 μM).
The inhibitory concentration of 50% of the parasite growth (IC50) was determined as the concentration of the
compound capable of reducing INDEX by 50%, and was obtained from non-linear regression analysis of the dose-
response curves obtained from of statistical software GraphPad Prism 7.0.
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Cytotoxicity assay
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Cytotoxicity determination was carrying out in monkey renal cell (BGM), human hepatoma (HepG2) and dog
macrophage cell lines (DH82), by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay.18 The
cells were grown in RPMI containing penicillin (100 U mL-1); streptomycin (100 μg mL-1) and inactivated fetal bovine
serum (10%). As BGM and HepG2 cells were plated at 1x104 cells per well, and DH82 were plated at 5x104 cells
per well. Then, 100 μL of solution of compounds 3a-j in RPMI were added in each well and incubated for 48 h at
37 ºC in a 5% CO2. Compounds were tested in the following concentrations: 0.1, 1, 10, 100 and 1000 μM. After the
incubation period, 10 μL of MTT solution (5 mg mL-1) was added and incubated again for 4 h, then the supernatant
was removed, and 50 µL of DMSO were added in each well. The experiments were performed in quadruplicate.
The culture plates were read by a spectrophotometer (Versamax, Molecular Devices, USA) at 560 nm.
Concentration values inhibiting 50% of cell metabolism, cytotoxic concentration (CC50) was determined from non-
linear regression analysis of the dose-response curves obtained from of the statistical software GraphPad Prism
7.0, and selective indexes (SI = CC50/IC50) were calculated for each compound.
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Supplementary Material
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Supporting information for this article is available on the http://dx.doi.org/10.1002/MS-number.
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Acknowledgment
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This study was financed in part by the Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG),
Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and the Coordenação de Aperfeiçoamento
de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001. IVA was granted FAPEMIG and CNPq scholarships,
AB was supported by a CAPES scholarship, TFB was granted a PNPD-CNPq fellowship, LCAB and RTF are supported
by a CNPq Research Fellowship.
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Author Contribution Statement
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Isabel V. Antolínez and Almodvar Baldaia, carried out the synthesis and purification of all compounds, and worked
on the manuscript preparation. Tatiane F. Borgati carried out literature reviews, data analyses, structural analyses
of the compounds, interpretation of the results, preparation of graphics as well as compilation and preparing the
manuscript. Luiz C. A. Barbosa conceived and coordinated the project “bioactive peroxides”, supervised the
synthesis work, analyzed the data and worked on the manuscript preparation. Raquel M. Almeida carried out the
biological assays. Ricardo T. Fujiwara and Sebastião R. Ferreira carried out biological assays, discussed the results
and contributed in preparing the manuscript.
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References
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Graphical Illustration
A highly active and selective tetraoxane against Leishmania amazonensis (IC50 = 0.64 ± 0.14 M; SI =
345.3) is reported. The compound is more potent and less toxic than the control Sb3+ salt.
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