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

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. ŠOLAJA^6#

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 (RI_W2= 0.44, RI_TM91C235= 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^-1day^-1for 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

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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.

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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

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 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.

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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

D6^aW2^bTM91C235^cTM90C2B^d(–)PHA^e,f(+)PHA^g

PBMC, IC₅₀/ μM RI^h

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

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/–

ART^j

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.

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 IC₅₀values for the CQR versus CQS strain, W2/D6 and

TM91C235/D6, respectively; ⁱselectivity index (SI) is defined as the ratio of the IC₅₀values 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

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 vitro^aactivities (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

SI_Fem-x^aSI_HeLaSI_K562SI_MDA-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

166.10 168.70

>200 >200

32.74 32.01

2

6

2

4

2

4

2

8

3

7

5

3

5

3

8

6

11

9

2

15

5

2

3

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 IC₅₀for 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.

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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.

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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 IC₅₀and 2IC₅₀values).

C: control K562 cell sample.

Fig. 6. Photomicrographs of acridine orange/ethidium bromide-stained control K562 cells and

K562 cells treated with 2×IC₅₀concentrations 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

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) 10²and b) 10⁶mL^-1.

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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 F₂₅₄and RP-18 F₂₅₄plates. 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.⁴²with 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^-1L-glutamine, 100

µg mL^-1streptomycin, 100 IU mL^-1penicillin, 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^-1gelatine 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^-1of 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 Mosmann⁴⁴as modified by Ohno and

Abe.⁴⁵Briefly, 10 µL of MTT solution (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium

bromide, 5 mg mL^-1in phosphate buffered saline) was added to each well.⁴³The 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 IC₅₀concentration 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 IC₅₀and 2IC₅₀values 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^-1and 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.⁴³The 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^-1AO and 10 μg mL^-1EB 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 2×10⁶mL^-1with saline. The suspensions were also serially 10-fold

diluted, and 0.5-mL aliquots of 2×10²and 2×10⁶mL^-1dilutions 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^-1day^-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^-1day^-1) and ART (10 mg kg^-1day^-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 (RI_W2

=

= 0.44, RI_TM91C235= 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

SI_K562index. 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^–1day^–1allowed 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 pK_aand 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 (RI_W2= 0,44; RI_TM91C235= 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^-1омогућила 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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Tetraoxanes as inhibitors of apicomplexan parasites Plasmodium falciparum and Toxoplasma gondii growth and anti-cancer molecules

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