Tetraoxanes as inhibitors of apicomplexan parasites Plasmodium falciparum and Toxoplasma gondii growth and anti-cancer molecules

Article abstract of DOI:10.2298/JSC150430063O

New cyclohexylidene 1,2,4,5-tetraoxanes with polar guanidine and urea based groups were synthesized and evaluated for their antimalarial activity against chloroquine resistant and susceptible Plasmodium falciparum strains. The derivatives showed moderate, nM range antimalarial activities and low cytotoxicity. The N-phenylurea derivative 24 exhibited the best resistance indices (RIW2 = 0.44, RITM91C235 = 0.80) and was not toxic against human normal peripheral blood mononuclear cells (IC50 > 200 μM). Seven derivatives were tested in vitro against four human cancer cell lines and they demonstrated high selectivity toward leukaemia K562 cells. One compound, derivative 21 with a primary amino group, was the first tetraoxane tested in vivo against Toxoplasma gondii as another apicomplexan parasite. Subcutaneous administration at a dose of 10 mg kg^-1 day^-1 for 8 days allowed the survival of 20 % of infected mice, thus demonstrating the high potential of tetraoxanes for the treatment of apicomplexan parasites.

Full text of DOI:10.2298/JSC150430063O

J. Serb. Chem. Soc. 80 (11) 1339–1359 (2015)
JSCS–4801
UDC *Plasmodium falciparum*Toxoplasma
gondii+547.495.9+541.459:576+615.9:615.9:
615.285:615.277
Original scientific paper
Tetraoxanes as inhibitors of apicomplexan parasites Plasmodium
falciparum and Toxoplasma gondii growth and
anti-cancer molecules
DEJAN M. OPSENICA1*#, JELENA RADIVOJEVIĆ2, IVANA Z. MATIĆ3,
TIJANA ŠTAJNER4, SLAVICA KNEŽEVIĆ-UŠAJ5, OLGICA DJURKOVIĆ-DJAKOVIĆ4
and BOGDAN A. ŠOLAJA6#
1Institute of Chemistry, Technology, and Metallurgy, University of Belgrade, Njegoševa 12,
11000 Belgrade, Serbia, 2Institute of Molecular Genetics and Genetic Engineering, University
of Belgrade, Vojvode Stepe 444a, P. O. Box 23, 11010 Belgrade, Serbia, 3Institute for
Oncology and Radiology of Serbia, Pasterova 14, 11000 Belgrade, Serbia, 4National
Reference Laboratory for Toxoplasmosis, Institute for Medical Research, University of
Belgrade, Dr. Subotića 4, P. O. Box 102, 11129 Belgrade, Serbia, 5Institute for Pathology,
Medical Faculty, University of Novi Sad, Hajduk Veljkova 3, 21000 Novi Sad, Serbia and
6Faculty of Chemistry, University of Belgrade, Studentski trg 12–16, P. O. Box 51, 11158,
Belgrade, Serbia
(Received 30 April, revised and accepted 22 June 2015)
Abstract: New cyclohexylidene 1,2,4,5-tetraoxanes with polar guanidine and
urea based groups were synthesized and evaluated for their antimalarial activity
against chloroquine resistant and susceptible Plasmodium falciparum strains.
The derivatives showed moderate, nM range antimalarial activities and low
cytotoxicity. The N-phenylurea derivative 24 exhibited the best resistance
indices (RIW2 = 0.44, RITM91C235 = 0.80) and was not toxic against human nor-
mal peripheral blood mononuclear cells (IC50 > 200 μM). Seven derivatives
were tested in vitro against four human cancer cell lines and they demonstrated
high selectivity toward leukaemia K562 cells. One compound, derivative 21
with a primary amino group, was the first tetraoxane tested in vivo against
Toxoplasma gondii as another apicomplexan parasite. Subcutaneous adminis-
tration at a dose of 10 mg kg-1 day-1 for 8 days allowed the survival of 20 % of
infected mice, thus demonstrating the high potential of tetraoxanes for the
treatment of apicomplexan parasites.
Keywords: antimalarials; antiparasitic; peroxides; cancer; cytotoxicity.
*Corresponding author. E-mail: dopsen@chem.bg.ac.rs
# Serbian Chemical Society member.
doi: 10.2298/JSC150430063O
1339

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OPSENICA et al.
INTRODUCTION
Apicomplexan protozoa are single-celled parasites, with significant medical,
veterinary and economic effects worldwide. From the aspect of human medicine,
two apicomplexan infections are of major health concern – malaria, caused by
Plasmodium spp., and toxoplasmosis, caused by Toxoplasma gondii.
A potentially devastating disease, malaria represents an enormous public
health problem in the majority of developing countries, further emphasized by
1
the fact that nearly half of the world population is exposed to the infection.
Malaria may be caused by five Plasmodium species, P. falciparum, P. ovale, P.
vivax, P. malariae and P. knowlesi, of which P. falciparum that causes cerebral
malaria is the major threat. On a cellular level, all Plasmodium parasites contain
acidic food vacuoles (FV), where digestion of haemoglobin occurs and it is gen-
erally accepted that the FV are the site of action for a number of quinoline-like
drugs. The haem obtained from haemoglobin degradation is toxic to the parasite
and is transformed into insoluble haemozoin pigment, while the globin is hydro-
lysed into individual amino acids. Antimalarial drugs active within FV appear to
2
kill the parasite either by producing toxic free radicals or by blocking haemo-
3
zoin formation, as in the case of the 4-amino-7-chloroquinolines (ACQs). The
development of widespread drug-resistance to chloroquine (CQ), the most suc-
cessful antimalarial drug, has resulted in severe health issues in malaria endemic
regions. Although thorough investigation led to the realization that mutations in
P. falciparum chloroquine resistant transporter (PfCRT), multidrug resistance
protein 1 (PfMDR1) and multidrug resistance-associated protein (PfMRP) are
responsible for the development of resistance of the malaria parasite against chlo-
4
5
roquine and its analogues, additional analysis appears necessary. Therefore,
significant focus has been placed on the synthesis of peroxide antimalarials
6
active in FV or interfering in NADPH balance acting as leuco-methylene blue
(LMB) and FADH oxidisers, as well as on the development of new 4-amino-
quinolines, acridines and other molecules that prevent haem polymerisation.
7
2
8
9
10
T. gondii is an obligatory intracellular apicomplexan protozoan with world-
wide distribution. Globally, it has been estimated that one in three persons is
infected with T. gondii. In the United States, nearly 25 % of the adult population
11
has been infected with this organism, while most studies of European popul-
12
ations report 20−35 % seropositivity. However, in South America, these rates
13
can reach 50−75 %. Individuals with a compromised immune system, such as
HIV-infected persons and transplant recipients, are particularly prone to severe T.
gondii induced disease, mainly as a result of reactivation of a previously latent
14
infection. When primary T. gondii infection occurs during pregnancy, it can be
vertically transmitted, which may result in intrauterine death, birth defects, or to
late sequellae, such as ocular disease and mental retardation.

TETRAOXANES AS INHIBITORS OF APICOMPLEXAN PARASITES GROWTH
1341
As intracellular pathogens, Plasmodium spp. and T. gondii both rely on the
invasion of host cells for survival and proliferation. Since both parasites belong
to the same apicomplexan phylum, they have common metabolic pathways and
15–17
thus may be susceptible to same drugs for prevention or treatment.
How-
ever, there are a number of differences between the two pathogens. During the
asexual reproduction stage, P. falciparum exclusively targets human erythro-
17
cytes, whereas T. gondii can infect any cell in most vertebrates. And, while
malaria is transmitted only via the mosquito vector or blood exchange, infection
with T. gondii occurs via ingestion of sporulated oocysts shed into the environ-
ment by infected cats, or by consuming bradyzoites in the form of tissue cysts
from infected animals. Current therapies for T. gondii include drugs which inhibit
18,19
folate metabolism, protein synthesis, or disrupt electron transport.
For the
inhibition of folate metabolism, a combination of diaminopyrimidine antimal-
arials, such as trimethoprim 1 or pyrimethamine 2 (Fig. 1), and sulfonamides,
such as sulfadiazine 3 (Fig. 1) or sulfamethoxazole, is the recommended therapy.
This combination acts synergistically not only against T. gondii, but also against
various bacterial and other parasitic microorganisms. In patients sensitive to sul-
fonamides, protein synthesis inhibitors such as macrolide and lincosamide anti-
biotics are the second line of drugs. Another antimalarial, atovaquone, an inhi-
bitor of mitochondrial electron-transport processes, is the drug of choice as third
20
line therapy. In all these treatments, drug resistance, high cost, limited efficacy,
21
and side effects often result in discontinuation of therapy. Therefore, new
agents with better activity and safety profiles and that are less expensive are
Fig. 1. Various inhibitors of the T. gondii parasite.

1342
OPSENICA et al.
19
urgently needed. A variety of T. gondii inhibitors designed for different targets
have been reported in the last few years, such as inhibitors of Ca-dependent
22,23
protein kinase-1 (TgCDPK1),
compounds which cause the disappearance of
16
17
apicoplasts and plastid-like organelles, phosphodiesterase, purine nucleoside
phosphorylase, and biosynthesis of pantothenic acid. Of these, outstanding
activities were exhibited by TgCDPK1 inhibitors 4 and 5,
of the vitamin B5 biosynthetic pathway, derivative 6 (Fig. 1).
18
24
22,23
and the inhibitor
24
Outstanding examples of drugs active against both Plasmodium spp. and T.
gondii parasites include artemisinin (7, ART, Fig. 2) and its semi-synthetic
derivatives. It was established that their main mechanism of action is interfering
25,26
in calcium homeostasis and triggering of micronema.
ART and artemether 8
exhibited 100 times better in vitro activity against T. gondii than trimethoprim 1,
19
the current front-line drug. Other semi-synthetic artemisinin derivatives, inc-
19
luding 9, artemisone 10 or artemiside 11 have shown even better activities in
27
the nM region (Fig. 2). These derivatives inhibit multiple steps in the T. gondii
19,28
cycle,
and the absence of activity of deoxyartemisinine confirmed the perox-
25
23
ide group as the pharmacophore. In vivo results are rare and incomplete, and
only few studies are reliable. Thus, artemisinin showed complete clearance of
–1
–1 29
parasitaemia after a 5-day treatment with 1.3 µg mL day . An 8-day admin-
–1
–1
istration of artemiside at 10 mg kg day 3 days after sulfadiazine treatment
prolonged survival in 80 % of the mice, without toxic side-effects.
24
Fig. 2. Structures of artemisinin derivatives.
30
31
Artemisinin and its derivatives, artemisone and other synthetic perox-
32,33
ides
also exert potent in vitro and in vivo anticancer activities affecting
diverse signalling pathways that regulate cell cycle, differentiation, apoptosis,
30
invasion and angiogenesis in cancer cells. Artemisinins can functionally be
clearly distinguished from the synthetic peroxides, in spite of the fact that they all
share a common peroxide pharmacophore, which suggests that these molecules
33
have multiple modes of action. Furthermore, it was found that synthetic and
semi-synthetic peroxides exhibited specific activities against cancer cells. Thus,
steroidal mixed tetraoxanes show highly specific activity toward UO31 renal

TETRAOXANES AS INHIBITORS OF APICOMPLEXAN PARASITES GROWTH
1343
2b
cancer cells, and artesunate against non-small cell lung cancer and colorectal
34
cancer.
The high antimalarial activities of dicyclohexylidene tetraoxanes that pos-
35
sess polar end groups attached to one of the cyclohexane units encouraged us to
explore the influence of modifications to the polar groups on their antimalarial
and cytotoxic activities. For this, compound 21 was chosen since it showed the
35c
most promising in vivo activity against the malaria parasite.
RESULTS AND DISCUSSIONS
Synthesis
Synthesis of targeted derivatives is presented in Schemes 1 and 2. The key
intermediary tetraoxane amine 21 was prepared according to a previously des-
35c
cribed procedure (Scheme 1). In brief, cyclohexane was transformed into gem-
36
dihydroperoxide 17 using 50 % H O in the presence of Re O as catalyst,
which was further coupled with benzyl 4-oxocyclohexanecarboxylate 15
2
2
2 7
35a,37
producing tetraoxane benzyl ester 18. The presence of the benzyl-group enabled
a more efficacious purification of the crude product using a Biotage SP
chromatography system. Reduction with LiAlH produced alcohol 19, which was
4
transformed via the corresponding azide 20 into amine 21. Final derivatives 22–
25 (Scheme 2) were obtained from 21 and corresponding coupling reactants, in
high to excellent yield (72–98 %). All derivatives were fully characterized and
their purity, as determined by HPLC, was ≥95 %, with the exception of
compound 23 that was 79–81 % pure. Full details are given in the Experimental
and the Supplementary material to this paper.
Scheme 1. Reaction pathway for the synthesis of derivative 21.

1344
OPSENICA et al.
Scheme 2. Reaction pathway for the synthesis of derivatives 22–25.
1
In the H-NMR spectra, all derivatives showed a characteristic signal at
around 3.0 ppm for the methylene groups bonded to the polar groups (imida-
zoline, guanidine, urea or thiourea), and characteristic signals for the introduced
polar groups: singlet at 3.38 ppm for the ethylene group in 22 and multiplets in
13
the region 7.50–6.80 ppm for the aromatic protons in 24 and 25. In the C-NMR
spectra, all derivatives showed signals for peroxy-acetal carbons in 107–108 ppm
region and signals in the 45–50 ppm region for the methylene groups bonded to
2
the polar groups. Additionally, a signal at 160 ppm for sp carbon in polar groups
in 22, 23 and 24, and a signal at 180 ppm for 25 were present. Furthermore,
aromatic carbons from the phenyl-groups in 24 and 25 appeared in the 117–140
ppm region.
Antimalarial activity
Compounds were screened in vitro against four P. falciparum strains: D6
(CQ susceptible (CQS) strain), W2 (CQ resistant (CQR) strain), TM91C235 (CQ
and MFQ resistant) and TM90C2B (atovaquone resistant), following well-
38
established protocols. In brief, the Malaria SYBR Green I based fluorescence
(MSF) assay is a microtiter plate drug sensitivity assay that uses the presence of
malarial DNA as a measure of parasitic proliferation in the presence of anti-
malarial drugs or experimental compounds. The intercalation of SYBR Green I
dye, and its resulting fluorescence, is relative to parasite growth, and a compound
that inhibits the growth of the parasite will result in lower fluorescence inten-
sities.
35c
The antimalarial activities are presented in Table I. Tetraoxane 21
was
also screened here in order to enable a more reliable comparison to be drawn

TETRAOXANES AS INHIBITORS OF APICOMPLEXAN PARASITES GROWTH
1345
with the activities of the new derivatives. All derivatives, with the exception of
23, showed low nanomolar activities against all four strains.
TABLE I. In vitro antimalarial activities against P. falciparum strains and cytotoxicity
(PBMC) of tetraoxanes 21–25; n.t. – not tested
Compd.
P. falciparum, IC50 / nM
D6a W2b TM91C235c TM90C2Bd (–)PHAe,f (+)PHAg
PBMC, IC50 / μM RIh
SIi
21
13.99 7.26
10.81
7.03
85.89
n.t.
185.61 182.22 0.52/ 13265/25560/
0.77 17165/26388
166.10 168.70 0.79/ 1791/2280/
22
92.75 72.86
208.54
2.25
>200 2.03/
6.11
>200 0.44/ 5480/12525/
0.80 6863/9343
32.01 0.61/ 2008/3295/
796/1934
820/400/
134/–
23
243.84 494.37 1489.80
>200
>200
32.74
n.t.
24
36.50 15.97
16.30 9.94
6.66 3.79
29.14
28.28
16.90
21.40
n.t.
25
1.73
0.57/
2.54
1158/–
ARTj
CQ
MQ
6.23
n.t.
7.63 462.99 198.70
14.69 6.52 80.28
181.08
11.60
n.t.
n.t. 60.70/
26.05
n.t.
n.t.
0.44/
5.47
aP. falciparum African D6 strain; bP. falciparum Indochina W2 strain; P. falciparum TM91C235 strain; dP.
falciparum TM90C2B strain; enon-stimulated with PHA; fPHA = phytohaemagglutinin, gstimulated with PHA;
hresistance index (RI) is defined as the ratio of the IC50 values for the CQR versus CQS strain, W2/D6 and
TM91C235/D6, respectively; iselectivity index (SI) is defined as the ratio of the IC50 values for PBMC
(–)PHA/D6, PBMC (–)PHA/W2, PBMC (–)PHA/TM91C235 and PBMC (–)PHA/TM90C2B, respectively;
javerage of greater than eight replicates, CQ = chloroquine, MQ = mefloquine
c
Simultaneously, all derivatives except 23 were more active against the chlo-
roquine-resistant (CQR) W2 strain than against the chloroquine-susceptible
(CQS) D6 strain, which could be seen from the favourable RI values. The deri-
vatives were somewhat less active against the multi-drug resistant TM91C235
(CQR and MQR strain) in comparison with the activities exhibited toward the
W2 strain. In addition, the corresponding RI values were less favourable, with the
exceptions 21 and 25. However, three of the five derivatives were 7.4–2.8 times
more active than MQ against the TM91C235 strain – 21, IC = 10.81 nM, 24,
50
IC = 29.14 nM and 25, IC = 28.28 vs. MQ, IC = 80.28 nM. The most active
50
50
50
derivative was 21 that showed exceptional activities against all the resistant
strains. Even against the atovaquone resistant strain TM90C2B, this amine
showed activities comparable with those of MQ. Removing the basic character of
the terminal amino-group attenuates the anti-malarial activity, as seen in the case
of compounds 21, 24 and 25. Transformation of the primary amino-group into
N-phenyl urea or thiourea reduced the antimalarial activity. However, 25 pre-

1346
OPSENICA et al.
served high activity against the W2 strain, with an IC of 9.94 nM, which is
50
comparable to those of MQ and 21 with values of 6.52 nM and 7.26 nM,
respectively. On the other hand, increasing basic character of the terminal group
did not increase the activity unconditionally. Introduction of more basic groups,
such as imidazolidine or guanidine, led to a sharp decrease in activity. This was
even more striking with the guanidine derivative 23, which was 17, 68 and 138
times less active than 21 against D6, W2 and TM91C235 strains, respectively.
Such sharp decreases in activity caused by more basic groups is in accordance to
39
the behaviour observed with trioxolanes. Derivative 23 was also less active
than 22, which makes it the least active compound within this series. Derivatives
22 and 23 have same pK values (Table S-I of the Supplementary material to this
a
paper), which are two orders of magnitude higher than the pK for 21, and
a
correlations between pK or log P values (Table S-I) with antimalarial activity
a
40
could not be established. The derivatives were in general less active than ART
against all four strains. Tetraoxane 21 was the only one with activities
comparable with those of ART. However, two tetraoxanes 21 and 25 had better
RI values compared to that of ART against multi-drug resistant strain TM91C235
– 21, RI = 0.77 and 24, RI = 0.80 vs. ART, RI = 2.54.
The toxicity of the compounds was estimated using human normal periphe-
ral blood mononuclear cells (PBMC), non-stimulated (PBMC (–)PHA) and sti-
mulated (PBMC (+) PHA) with PHA (Table I). In general, the PBMC assay for
cytotoxicity estimation revealed that all compounds are well tolerated by normal
immunocompetent cells, possessing IC ≥ 32 µM. In addition, high selectivity
50
indices (SI) were obtained for all compounds toward all P. falciparum strains.
Derivative 21 had the best SI indices, which were in the range 13000–26000,
depending on the strain. The second best SI was exhibited by the urea derivative
24, which had the lowest cytotoxicity >200 µM and moderate antimalarial
activities in the range 16–36 nM.
In vitro cytotoxic activity
Cytotoxic activities of the new tetraoxanes and the previous compounds 21
35c
and 20 were evaluated against four human cancer cell lines: cervix adenocar-
cinoma HeLa, melanoma Fem-x, breast adenocarcinoma MDA-MB-361 and
chronic myelogenous leukaemia K562 cells.
The intensities of the cytotoxic activities are given in Table II and Fig. 3.
First, it should be emphasized that the tested tetraoxanes showed the strongest
cytotoxic activities against myelogenous leukaemia K562 cells. The highest cyto-
toxicity against K562 cells was exerted by derivatives 25 with an IC of 6.15
50
µM, 24 with 13.23 µM, and 18, 20 and 22 with an IC of ca. 18.5 µM.
50

TETRAOXANES AS INHIBITORS OF APICOMPLEXAN PARASITES GROWTH
1347
TABLE II. In vitroa activities (IC50 / μM) of tetraoxanes against cancer cell lines (Fem-x,
human melanoma cell line; HeLa, human cervical adenocarcinoma cell line; K562, myelo-
genous leukaemia cell line; MDA-MB-361, human breast adenocarcinoma cell line) and peri-
pheral blood mononuclear cells (PBMC), non-stimulated or stimulated with PHA (phyto-
haemagglutinin)
PBMC PBMC
(–)PHA (+)PHA
MDA-MB-
-361
Compd. Fem-x HeLa K562
SIFem-xa SIHeLa SIK562 SIMDA-MB-361
21
18
20
22
23
24
25
cis-Pt
79.75 63.56 33.68
31.86 27.01 18.84 73.92
98.82 37.67 18.43 94.7
43.84 34.56 18.38 80.29
>100 75.66 81.49 90.91
52.68 39.85 13.23 96.85
>100
185.61 182.22
>200 >200
>200 >200
166.10 168.70
>200 >200
>200 >200
32.74 32.01
2
6
2
4
2
4
2
8
3
7
5
5
3
5
3
8
6
11
11
9
2
15
5
2
3
2
2
2
2
1
2
15.67 10.51 6.15
8.10 7.89 5.64 >33.34 >66.67 >66.67
41.13
12
aThe selectivity index (SI) is defined as the ratio of the IC50 for PBMC (–)PHA / cancer cell line
Fig. 3. Survival (S%) of: A) cervical adenocarcinoma HeLa, B) melanoma Fem-x, C) myelo-
genous leukaemia K562 and D) breast adenocarcinoma MDA-MB-361 cells grown for 72h in
the presence of increasing concentrations of the investigated tetraoxanes,
determined by MTT test.

1348
OPSENICA et al.
Against cervix adenocarcinoma HeLa cells, compounds 18, 20, 22, 24 and
25 exerted pronounced cytotoxic actions with IC values in the range of 10.51 and
50
39.85 µM. The highest cytotoxic effect was exhibited by derivative 25 with
IC = 10.51 µM. Against melanoma Fem-x cells, compound 25 was the most
50
active with an IC of 15.67 µM; derivative 18 exerted pronounced cytotoxic
50
effects with IC value of 31.86 µM. Compounds 22 and 24 exerted moderate
50
cytotoxic action, while the cytotoxic effects of the other derivatives were lower,
with derivative 23 being the least active, showing an IC value higher than 100
50
µM. All tested tetraoxanes exerted low cytotoxic actions against breast adeno-
carcinoma MDA-MB-361 cells with IC values >73.92 µM, except derivative
50
25 which had moderate activity against this cell line, with an IC value of 41.13
50
µM. Tetraoxane 23 with a guanidine group was the least active compound in this
series, showing lowest activities towards all cell lines in comparison with other
members of the series.
All tetraoxanes exerted selective concentration-dependent cytotoxic activity
against specific malignant cell lines, which was especially high against myelo-
genous leukaemia K562 cells. It is noteworthy that the cytotoxic actions of com-
pounds 21, 18, 22, 24 and 25 were notably higher against K562, HeLa and Fem-x
cells than against MDA-MB-361 breast cancer cells, indicating selectivity in
their anticancer action against specific malignant cell types.
To further evaluate the anticancer potential of the tested tetraoxanes, their
cytotoxic activities were examined against normal healthy peripheral blood
mononuclear cells (PBMC), both resting and stimulated to proliferate by mitogen
phytohaemagglutinin (PHA), Table II and Fig. 4. Each of the examined tetraox-
anes exerted remarkably higher cytotoxic activities against K562, HeLa and
Fem-x malignant cell lines than against both non-stimulated and stimulated
PBMC, indicating a significant specificity in their anticancer action. The highest
Fig. 4. Survival of (A) resting PBMC and (B) PBMC stimulated to proliferate by PHA grown
for 72h in the presence of increasing concentrations of the investigated tetraoxanes,
determined by the MTT test.

TETRAOXANES AS INHIBITORS OF APICOMPLEXAN PARASITES GROWTH
1349
selectivity in antitumor action was observed against myelogenous leukaemia
K562 cells in comparison with normal healthy PBMC with tetraoxanes 18, 20
and 24, which showed the highest SI values, 11, 11 and 15 respectively. In addi-
tion, the tested tetraoxanes showed good selectivity as follows: 18 and 24 against
cervix adenocarcinoma HeLa and melanoma Fem-x cells, 20 against HeLa cells,
and 22 against K562, HeLa and Fem-x cells. Low selectivity in the anticancer
action of the tested tetraoxanes was observed against breast adenocarcinoma
MDA-MB-361 cells, which showed the lowest sensitivity to the cytotoxic action
of these compounds.
In contrast to antimalarial activity, it could not be concluded that activities
depend on the presence of a specific functional group. Rather, the activity was
specific towards particular cell lines. However, in general, the obtained results
showed that derivatives 21–23 with basic groups have lower activities. Deri-
vatives were less active than cisplatin with the exception of derivative 25. The
thiourea derivative 25 was the most active tetraoxane in this series, with IC
50
values in the range 41–6.0 µM, which are the same as for cisplatin. However, the
low SI of 25 for all tested cell lines makes this derivative less interesting, in spite
of having good cytotoxic activities. Conversely, derivatives 18, 20 and 24 had SI
values for the K562 cell line in the same range as that for cisplatin, which makes
them very interesting candidates for further examinations.
The mechanisms of cytotoxic action of 18, 20 and 24 were examined by cell
cycle analysis and morphological assessment of cell death modalities induced by
these tetraoxanes using fluorescence microscopy. The tested compounds at IC
50
concentrations induced time-dependent percentage increases of the target K562
cells in the subG1 phase, as could be seen in Fig. 5. It is noteworthy that com-
pound 20 at the IC concentration caused the most remarkable increase in the
50
percentage of K562 cells in the subG1 cell cycle phase after 48 h treatment. In
addition, treatment with 2IC concentrations of three tetraoxanes resulted in an
50
increase in the percentages of apoptotic subG1 K562 cells after 24 h exposure.
After 48h, the percentages of subG1 K562 cells exposed to 2IC concentrations
50
of 18, 20 and 24 were approximately 12 times higher compared to the control cell
sample, pointing to a strong proapoptotic activity of these compounds. Exam-
ination of changes in the morphological features confirmed that the tested tetra-
oxanes triggered apoptosis in K562 leukaemia cells after 24 h treatment. Typical
hallmarks of apoptotic K562 cells induced by tetraoxanes, such as shrinkage of
the nucleus, chromatin condensation and fragmentation of the nucleus in addition
to orange–red stained cells in the final stages of apoptosis are presented in Fig. 6.
Prominent proapoptotic effects of tetraoxanes 18, 20 and 24 in addition to
high selectivity in their anticancer activity against myelogenous leukaemia K562
cells in comparison to normal immunocompetent PBMC, suggest significant
anticancer potential of these compounds.

1350
OPSENICA et al.
Fig. 5. Changes in the cell cycle phase distribution of K562 cells induced by the investigated
tetraoxanes 18, 20 and 24 (tested concentrations corresponded to IC50 and 2IC50 values).
C: control K562 cell sample.
Fig. 6. Photomicrographs of acridine orange/ethidium bromide-stained control K562 cells and
K562 cells treated with 2×IC50 concentrations of 18, 20 and 24, for 24 h.

TETRAOXANES AS INHIBITORS OF APICOMPLEXAN PARASITES GROWTH
1351
In vivo activity against T. gondii
Derivative 21 was chosen for examination of in vivo activity against T.
gondii. The activity was examined in murine models of infection with tachyzoites
of the highly virulent RH strain of T. gondii following a well-established pro-
41
tocol. The compound exhibited no visible toxicity as judged by the quality of
fur, gait and general activity of the uninfected treated mice. The activity of 21
was compared to that of ART.
The results obtained in the treated animals are presented in Fig. 7. In the
2
infection model where infection was established with 10 per mL, all untreated
mice died between day 6 and day 9 p.i. (mean 8 days). Most importantly, treat-
–1
–1
ment with both 21 and ART in 10 mg kg day doses allowed survival of 20 %
of infected treated mice after 15 days (Fig. 7a). Compared with the control mice,
treatment with both drugs significantly prolonged survival, but the effect of 21
was more pronounced (P = 0.014) than that of ART (P = 0.0413), whereas the
effects of the two compounds were mutually comparable (P = 0.9086).
Fig. 7. Survival of infected mice after treatment with 21 or ART; parasite concentration:
a) 102 and b) 106 mL-1.

1352
OPSENICA et al.
6
–1
In mice infected with 10 mL , all untreated mice died within 5 days. Com-
pared to these, treatment with either 21 or ART significantly prolonged survival
(P = 0.0092 and P = 0.0013, respectively) but did not afford complete protection.
The antitoxoplasmatic activity of the two compounds was similar (P = 0.8358).
Pathohistological analysis was performed on all surviving infected treated
mice (four treated with 21 and two treated with ART), as well as on represent-
ative controls (non-infected treated mice), which were all sacrificed day 24 p.i.
No pathological changes other than extramedullary haematopoiesis (character-
istic of these mice) and mild lymphocyte infiltration of the liver were revealed in
both 21 and ART-treated mice, except for the finding of focal myocarditis in one
ART-treated infected mouse. These results indicate no significant macroscopic or
microscopic toxicity of the tested compounds, and moreover, show the potential
of derivative 21 to clear T. gondii infection.
EXPERIMENTAL
Chemistry
Melting points were determined on a Boetius PMHK or a Mel-Temp apparatus and were
not corrected. Optical rotations were measured on a Rudolph Research analytical automatic
polarimeter, Autopol IV in dichloromethane (DCM) or methanol (MeOH) as solvent. The IR
spectra were recorded on a Perkin-Elmer FT-IR 1725X spectrophotometer. The 1H- and
13C-NMR spectra were recorded on a Varian Gemini-200 spectrometer (at 200 and 50 MHz,
respectively), and on a Bruker Ultrashield Advance III spectrometer (at 500 and 125 MHz,
respectively) employing the indicated solvents (Supplementary material) using TMS as the
internal standard. Chemical shifts are expressed in ppm (δ) values and coupling constants (J)
in Hz. The ESI-MS spectra were recorded on an Agilent Technologies 6210 Time-Of-Flight
LC-MS instrument in the positive ion mode with CH3CN/H2O 1/1 with 0.2 % HCOOH as the
carrying solvent solution. The samples were dissolved in CH3CN or MeOH (HPLC grade
purity). The selected values were as follows: capillary voltage, 4 kV; gas temperature, 350 °C;
drying gas, 12.l min-1; nebulizer pressure, 310 kPa and fragmentator voltage, 70 V. Elemental
analyses were realized on the Vario EL III- C,H,N,S/O elemental analyzer (Elementar
Analysensysteme GmbH, Hanau, Germany). Thin-layer chromatography (TLC) was per-
formed on precoated Merck silica gel 60 F254 and RP-18 F254 plates. Column chromatography
was performed on Lobar LichroPrep Si 60 (40–63 µm), or RP-18 (40–63 µm) columns
coupled to a Waters RI 401 detector, and on Biotage SP1 system with a UV detector and
FLASH 12+, FLASH 25+ or FLASH 40+ columns charged with KP-SIL (40–63 µm, pore
diameter 60 Å), or KP-C18-HS (40–63 µm, pore diameter 90 Å) as adsorbent. Compounds 18
and 20–25 were analyzed for purity (HPLC) using an Agilent 1200 HPLC system equipped
with Quat pump (G1311B), injector (G1329B) 1260 ALS, TCC 1260 (G1316A) and a 1260
Infinity refractive index detector (RID) was. HPLC analyses were performed in two diverse
systems. Method A: Zorbax Eclipse Plus C18, 4.6 mm×150 mm, 1.8 µm, S.N. USWKY01594
was used as the stationary phase. The compounds were eluted using an isocratic protocol and
eluent was made from water/MeOH, 30/70 (V/V). The compounds were dissolved in metha-
nol; the final concentrations were 0.5 mg mL-1. Method B: Poroshell 120 EC-C18, 4.6 mm×50
mm, 2.7 µm, S.N. USCFU07797 was used as the stationary phase. The compounds were

TETRAOXANES AS INHIBITORS OF APICOMPLEXAN PARASITES GROWTH
1353
eluted using an isocratic protocol and the eluent was made from water/MeOH, 30/70 (V/V).
The compounds were dissolved in methanol; the final concentrations were 0.5 mg mL-1.
Synthetic procedures
Benzyl 7,8,15,16-tetraoxadispiro[5.2.5.2]hexadecane-3-carboxylate (18). Into ice-cold
(ice–water mixture) solution of gem-dihydroperoxide 17 (412.7 mg, 2.53 mmol) and ketone
15 (374.8 mg, 2.53 mmol) in CH2Cl2 (5 mL), under intensive stirring, precooled freshly
prepared solution of conc. H2SO4/CH3CN (1/10, V/V, 1.82 mL) was added dropwise. After
one hour stirring at the same temperature, the reaction was quenched with water and the layers
were separated. Water layer was extracted with CH2Cl2 (3×50 mL). The combined organic
layers were washed with sat. NaHCO3 (3×20 mL), brine (2×10 mL) and dried over anh.
Na2SO4. The solvent was removed under reduce pressure and the product was isolated after
column chromatography purification (dry-flash, SiO2-column, eluent hexane/EtOAc, gradient
97/3 → 9/1). Yield: 241.9 mg (26 %), as an amorphous powder.
N-(7,8,15,16-Tetraoxadispiro[5.2.5.2]hexadec-3-ylmethyl)-4,5-dihydro-1H-imidazol-2-
-amine (22). A mixture of 21 (100.0 mg, 0.39 mmol), I (142.2 mg, 0.58 mmol) and Et3N (0.5
mL) in methanol (3 mL) was stirred under an inert atmosphere (Ar) for 3 days at 70 °C. The
solvent was evaporated, the residue dissolved in CH2Cl2, 10 % NaOH solution added and the
mixture intensively shaken. The layers were separated, the organic layer was washed once
with water, once with brine and dried over anh. Na2SO4. The solvent was removed under
reduce pressure and the product isolated after column chromatography purification (Lobar,
SiO2-column A, eluent CH2Cl2/CH3OH sat. NH3 = 1/1). Yield: 91 mg (72 %).
1-(7,8,15,16-Tetraoxadispiro[5.2.5.2]hexadec-3-ylmethyl)guanidine (23). A mixture of
21 (100.0 mg, 0.39 mmol), S-methylthiourea sulphate (162.0 mg, 0.58 mmol) and Et3N (0.5
mL) in methanol (3 mL) was stirred at 70 °C under an inert atmosphere (Ar) for 3 days. Into
the warm solution, methanol was added (10 mL) and the warm mixture was filtered, the
solvent removed under reduce pressure and product isolated after column chromatography
purification (dry-flash, SiO2-column, gradient CH2Cl2/MeOH, = 9/1 → 1/1) as a pale yellow
oil that became solid with time. Yield: 108.6 mg (93 %).
1-Phenyl-3-(7,8,15,16-tetraoxadispiro[5.2.5.2]hexadec-3-ylmethyl)urea (24). A mixture
of 21 (150.0 mg, 0.58 mmol) and phenyl-isocyanate (63.4 μL, 0.58 mL) in CH2Cl2 (10 mL),
was stirred at r.t. under an inert atmosphere (Ar) for 45 min. The solvent was evaporated and
product isolated after column chromatography purification (dry-flash, SiO2-column, gradient
hexane/EtOAc = 1/1 → EtOAc → EtOAc/MeOH = 7/3). Yield: 215 mg (98 %).
1-Phenyl-3-(7,8,15,16-tetraoxadispiro[5.2.5.2]hexadec-3-ylmethyl)thiourea (25). A mix-
ture of 21 (78.2 mg, 0.30 mmol) and phenyl-isothiocyanate (35.57 μL, 0.30 mL) in CH2Cl2
(15 mL) was stirred under an inert atmosphere at r.t. for 2 h. The solvent was removed under
reduce pressure and the product isolated after column chromatography purification (dry-flash,
SiO2-column, gradient hexane/EtOAc = 9/1 → EtOAc). Yield: 87.3 mg (73 %).
In vitro antimalarial activity
The in vitro antimalarial drug susceptibility screen is a modification of the procedures
first published by Desjardins et al.42 with modifications developed by Milhous et al., the
details of which are given in ref. 8b. All the synthesized aminoquinolines were screened in
vitro against the P. falciparum strains CQ and MFQ, as well as the susceptible strain D6
(clone of the Sierra I/UNC isolate), the CQ resistant but MFQ susceptible strain W2 (clone of
the Indochina I isolate), and the CQ and MFQ resistant strain TM91C235 (clone of the South–
East Asian isolate), and TM90C2B (clone of the Thailand isolate).

1354
OPSENICA et al.
Cytotoxic activity
Reagents. Stock solutions of investigated tetraoxanes were prepared in dimethyl sul-
phoxide (DMSO) at concentrations of 10 mM and subsequently diluted with complete nutrient
medium (RPMI-1640 without phenol red) supplemented with 3 mmol L-1 L-glutamine, 100
µg mL-1 streptomycin, 100 IU mL-1 penicillin, 10 % heat inactivated foetal bovine serum
(FBS), and 25 mM HEPES, and adjusted to pH 7.2 with bicarbonate solution. RPMI-1640,
FBS, HEPES, and L-glutamine were purchased from Sigma–Aldrich, St. Louis, MO, USA.
Cell cultures. Human cervical adenocarcinoma HeLa, human melanoma Fem-x and
human breast adenocarcinoma MDA-MB-361 cells were cultured as a monolayer, while
human chronic myelogenous leukaemia K562 cells were grown in a suspension in the com-
plete nutrient medium, at 37 °C in a humidified air atmosphere with 5 % CO2, as previously
described.43
Preparation of peripheral blood mononuclear cells (PBMC). PBMC were separated
from whole heparinised blood (obtained from two healthy volunteers) by Histopaque®-1077
(Sigma–Aldrich) density gradient centrifugation. Interface cells, washed three times with
Haemaccel (aqueous solution supplemented with 145 mM Na+, 5.1 mM K+, 6.2 mM Ca2+,
145 mM Cl- and 35 g L-1 gelatine polymers, pH 7.4), were counted and suspended in nutrient
medium with 10 % FBS.43
Treatment of human malignant cell lines. HeLa (2,000 cells per well), Fem-x (5,000 cells
per well) and MDA-MB-361 cells (10,000 cells per well) were seeded into 96-well microtiter
plates. Twenty hours later, after cell adherence, five different concentrations of the inves-
tigated compounds in complete nutrient medium were added to the cells (range 6.25–100 µM
or 3.125–50 µM), except for the control cells to which nutrient medium only was added. K562
cells (5,000 cells per well) were seeded two hours before addition of the investigated com-
pounds to obtain final concentrations within the above-mentioned range. All experiments were
realised in triplicate. Cisplatin was used as a positive control.
Treatment of PBMC from healthy donors. PBMC cells were seeded at a density of
150,000 cells per well in nutrient medium only, or in the nutrient medium enriched with 5 µg
mL-1 of phytohaemagglutinin (PHA – INEP, Belgrade, Serbia) in 96-well microtiter plates.
Two hours later, five different concentrations of the investigated tetraoxanes were added to
the wells with non-stimulated and PHA-stimulated PBMC (in order to obtain five final con-
centrations within the range of 12.5–200 µM). All experiments were realised in triplicate. Cis-
platin was used as a positive control.
Determination of cell survival. Cell survival 72 h after addition of the drug was deter-
mined by the MTT test according to the method of Mosmann44 as modified by Ohno and
Abe.45 Briefly, 10 µL of MTT solution (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide, 5 mg mL-1 in phosphate buffered saline) was added to each well.43 The samples
were incubated for a further four hours at 37 °C in a humidified atmosphere with 5 % CO2.
Then, 100 µL of 10 % SDS solution was added to the wells. The absorbance was measured at
570 nm the next day. To obtain cell survival (S / %), the absorbance at 570 nm of a sample
with cells grown in the presence of various concentrations of agent was divided by the absorb-
ance of the control sample (the absorbance of cells grown only in nutrient medium), implying
that the absorbance of the blank was always subtracted from the absorbance of a corres-
ponding sample with target cells. The IC50 concentration was defined as the concentration of
an agent inhibiting cell survival by 50 %, compared to the corresponding control.
Cell cycle analysis. K562 cells were exposed to two different concentrations of the
examined compounds 18, 20 and 24 (corresponding to the IC50 and 2IC50 values determined

TETRAOXANES AS INHIBITORS OF APICOMPLEXAN PARASITES GROWTH
1355
after 72 h treatment) for 24 and 48 h. After incubation, the target cells were collected, washed
and fixed in 70 % ethanol on ice. The cell samples were stored at −20 °C for at least one week
before staining. The cells were collected by centrifugation, washed, resuspended in PBS
containing RNase A at a final concentration of 200 μg mL-1 and incubated for 30 min at 37
°C. Subsequently, the propidium iodide solution was added to the cells at a final concentration
of 40 μg mL-1.43
The phase distribution of the cell cycle was determined using a FACSCalibur flow
cytometer (BD Biosciences, Franklin Lakes, NJ, USA). The data (10,000 events collected for
each sample) were analysed using CELLQuest software (BD Biosciences).
Morphological evaluation of K562 cell death. To examine the mode of death of human
chronic myelogenous leukaemia K562 cells induced by the investigated tetraoxanes, morpho-
logical analysis by microscopic examination of acridine orange/ethidium bromide-stained
target cells was performed, as already described.43 The K562 cells were seeded in 6-well
plates (200,000 cells per well) in complete nutrient medium. After 2 h, the cells were treated
with the investigated compounds for 24 h at concentrations corresponding to double the IC50
values obtained after 72 h treatments. After this period, the target cells were collected by
centrifugation and stained with 20 µL of a mixture of acridine orange and ethidium bromide
(3 μg mL-1 AO and 10 μg mL-1 EB in PBS) dyes, and visualized under a fluorescence micro-
scope – Carl Zeiss PALM MicroBeam with Axio Observer.Z1 using AxioCam MRm (filters
Alexa 488 and Alexa 568).
Anti-toxoplasmatic activity
Mice. Female Swiss Webster mice (Medical Military Academy Animal Research Facil-
ity, Belgrade, Serbia) weighing 18 to 20 g at the beginning of each experiment were used. The
mice were housed six to a cage and offered drinking water ad libitum.
T. gondii. Tachyzoites of the virulent RH strain maintained through serial intraperitoneal
(i.p.) passages were used. For the experimental infections, tachyzoites were harvested from
mouse peritoneal fluids 72 h post infection and purified by centrifugation, cotton wool
filtration, and needle extraction. The parasites were counted in a haemocytometer, and their
numbers were adjusted to 2×106 mL-1 with saline. The suspensions were also serially 10-fold
diluted, and 0.5-mL aliquots of 2×102 and 2×106 mL-1 dilutions were inoculated i.p. into fresh
mice.
Infected non-treated mice served as infection controls. Mice were randomly assigned
into experimental groups and treated with 21 or ART. Both drugs were administered at a dose
of 0.2 mg per mouse per day (10 mg kg-1 day-1), subcutaneously (s.c.) for 8 consecutive days
starting from the day of infection (day 0). Survival of the mice was monitored for another
week after the end of treatment, meaning a total follow-up period of 15 days post infection
(p.i.). To control for drug side effects (toxicity), separate groups of non-infected animals were
given 21 (10 mg kg-1 day-1) and ART (10 mg kg-1 day-1) in the same manner and duration as
in the experimental groups.
Statistical analysis
The rates of survival in particular treatment groups were estimated by the Kaplan–Meier
product limit method and compared by the log-rank test. The level of statistical significance
was 0.05.

1356
OPSENICA et al.
CONCLUSIONS
Herein, the synthesis and biological activity of new cyclohexylidene
mixed 1,2,4,5-tetraoxanes containing polar guanidine and urea based
groups were reported. Four new tetraoxanes were tested in vitro against P.
falciparum CQR and CQS strains. The derivatives showed moderate nano-
molar antimalarial activities, and differences in activities were clearly inf-
luenced by changes in the structures of the introduced polar groups. The
N-phenylurea derivative 24 showed the best resistance indices (RIW2
=
= 0.44, RITM91C235 = 0.80), the highest SI score against all four tested P.
falciparum strains and the lowest toxicity against PBMC (IC50 > 200 μM).
Seven tetraoxanes were tested in vitro against four human cancer cell lines
and five of them showed pronounced cytotoxic effects against myelo-
genous leukaemia K562 cells in 6.15–18.84 μM concentrations with high
SIK562 index. The amino-tetraoxane 21 was the first one evaluated for its
anti T. gondii effect in vivo, in a murine model of acute toxoplasmosis. An
8-day treatment at a dose of 10 mg kg–1 day–1 allowed survival of 20 % of
the infected mice. The obtained results clearly showed the activity of the
investigated tetraoxanes against acute murine toxoplasmosis, suggesting
the potential of synthetic organic peroxides in T. gondii treatment.
SUPPLEMENTARY MATERIAL
Calculated pKa and log P values for derivatives 21–23, synthetic procedures for
derivatives 13–17 and 19–21 and analytical data for derivatives 13–25 and HPLC chroma-
tograms for determination of the purity of tested compounds are available electronically from
http://www.shd.org.rs/JSCS/, or from the corresponding author on request.
Acknowledgements. This work was supported by the Ministry of Education, Science and
Technological Development of the Republic of Serbia (Grants 172008 and 175011).
И З В О Д
ТЕТРАОКСАНИ КАО ИНХИБИТОРИ АПИКОМПЛЕКСНИХ ПАРАЗИТА Plasmodium
falciparum И Toxoplasma gondii И АНТИ-КАНЦЕРСКИ МОЛЕКУЛИ
1
2
3
4
ДЕЈАН М. ОПСЕНИЦА , ЈЕЛЕНА РАДИВОЈЕВИЋ , ИВАНА З. МАТИЋ , ТИЈАНА ШТАЈНЕР ,
5
4
6
СЛАВИЦА КНЕЖЕВИЋ-УШАЈ , ОЛГИЦА ЂУРКОВИЋ-ЂАКОВИЋ и БОГДАН А. ШОЛАЈА
1Институт за хемију, технолохгију и металургију, Универзитет у Београду, Његошева 12, 11000
Београд, 2Институт за молекуларну генетику и генетски инжењеринг, Универзитет у Београду,
Војводе Степе 444a, п. пр. 23, 11010 Београд, 3Институт за онкологију и радиологију Србије,
Пастерова 14, 11000 Београд, 4Национална референтна лабораторија за токсоплазмозу, Институт
за медицинска истраживања, Универзитет у Београду, др Суботића 4, п. пр. 102, 11129 Београд,
5Институт за патологију, Медицински факултет, Универзитет у Новом Саду, Хajдук Вељкова 3,
21000 Нови Сад и 6Хемијски факултет, Универзитет у Београду, Студентски трг 12–16,
п. пр. 51, 11158 Београд
Синтетисана је група нових циклохексилиденских 1,2,4,5-тетраоксана који имају
гванидино и уреидо поларне групе и испитана је њихова антималаријска активност
према хлорокивин-резистентним и осетљивим сојевима Plasmodium falciparum. Деривати

TETRAOXANES AS INHIBITORS OF APICOMPLEXAN PARASITES GROWTH
1357
показују умерене активности у nM опсегу и ниску цитотоксичност. Дериват N-фенил-
-урее 24 има најбољи индекс резистенције (RIW2 = 0,44; RITM91C235 = 0,80) и није
токсичан према хуманим нормалним мононуклеарним ћелијама периферне крви (IC50
> 200 μM). Седам деривата је тестирано in vitro према четири хумане малигне ћелијске
линије и показало је високу селективност према K562 ћелијама леукемије. Дериват 21
који има примарну амино групу је први тетраоксан тестиран in vivo према још једном
паразиту из филума Apicomplexa, Toxoplasma gondii. Приликом супкутаног администри-
рања, дневна доза од 10 mg kg-1 омогућила је преживљавање 20 % инфицираних
мишева, што показује висок потенцијал тетраоксана за терапију инфекција изазваних
апикомплексним паразитима.
(Примљено 30. априла, ревидирано и прихваћено 22. јуна 2015)
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