Novel Peroxides as Promising Anticancer Agents with ...

DOI: 10.1002/cmdc.201700804

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Novel Peroxides as Promising Anticancer Agents with Unexpected Depressed Antimalarial Activity Paolo Coghi+,[a] Ivan A. Yaremenko+,[c, d, e] Parichat Prommana+,[b] Peter S. Radulov,[c, e] Mikhail A. Syroeshkin,[c] Yu Jun Wu,[a] Jia Ying Gao,[a] Floria M. Gordillo,[a] Simon Mok,[a] Vincent Kam Wai Wong,*[a] Chairat Uthaipibull,*[b] and Alexander O. Terent’ev*[c, d, e] Twenty six peroxides belonging to bridged 1,2,4,5-tetraoxanes, bridged 1,2,4-trioxolanes (ozonides), and tricyclic monoperoxides were evaluated for their in vitro antimalarial activity against Plasmodium falciparum (3D7) and for their cytotoxic activities against immortalized human normal fibroblast (CCD19Lu), liver (LO2), and lung (BEAS-2B) cell lines as well as human liver (HepG2) and lung (A549) cancer-cell lines. Synthetic ozonides were shown to have the highest cytotoxicity on HepG2 (IC50 = 0.19–0.59 mm), and some of these compounds selectively targeted liver cancer (selectivity index values for compounds 13 a and 14 a are 20 and 28, respectively) at levels that, in some cases, were higher than those of paclitaxel, arte-

misinin, and artesunic acid. In contrast some ozonides showed only moderate antimalarial activity against the chloroquinesensitive 3D7 strain of P. falciparum (IC50 from 2.76 to 24.2 mm; 12 b, IC50 = 2.76 mm; 13 a, IC50 = 20.14 mm; 14 a, IC50 = 6.32 mm). These results suggest that these derivatives have divergent mechanisms of action against cancer cells and malaria-infected cells. A cyclic voltammetry study of the peroxides was performed, but most of the compounds did not show direct correlation in oxidative capacity–activity. Our findings offer a new source of antimalarial and anticancer agents through structural modification of peroxide compounds.

Introduction Malaria is one of the world deadliest diseases with increasing clinical concern owing to the development of antimalarial drug resistance by the Plasmodium parasites that are transmitted by mosquitoes of the genus Anopheles. It is estimated that more than 200 million people are affected by the disease in subtropical and tropical countries with a global mortality rate of 0.2 %.[1] Chloroquine (Figure 1) is one of the most widely used [a] Dr. P. Coghi,+ Y. J. Wu, Dr. J. Y. Gao, Dr. F. M. Gordillo, Dr. S. Mok, Dr. V. K. W. Wong State Key Laboratory of Quality Research in Chinese Medicine, Macau University of Science and Technology, Avenida Wai Long, Taipa, Macau (China) E-mail: [email protected]

Figure 1. Structures of classical antimalarial drugs.

[b] P. Prommana,+ Dr. C. Uthaipibull National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), 113 Thailand Science Park, Pathum Thani, 12120 (Thailand) E-mail: [email protected]

and effective antimalarial drugs, which has been substituted by artemisinin (ART) and its synthetic derivative artesunic acid after the appearance of drug-resistant parasites.[2] The successful exploitation of semisynthetic ART derivatives[3] was a major breakthrough in malaria chemotherapy because of the profound rapid therapeutic response of these derivatives against malaria parasites. The World Health Organization recommends that the deadly species P. falciparum should be treated with artemisinin-based combination therapies (ACTs), in which the ART-based component is combined with a second, longeracting agent.[4] However, ART-resistance cases are currently emerging, with the first case documented in Cambodia,[5] which suggests the need for the search of novel pharmaceutical interventions for malaria. In fact, endoperoxide compounds represent an attractive alternative to ART and its derivatives. Similar to ART, endoperox-

[c] Dr. I. A. Yaremenko,+ P. S. Radulov, Dr. M. A. Syroeshkin, Prof. A. O. Terent’ev N.D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Leninsky Prospect 47, Moscow 119991 (Russian Federation) E-mail: [email protected] [d] Dr. I. A. Yaremenko,+ Prof. A. O. Terent’ev Faculty of Chemical and Pharmaceutical Technology and Biomedical Products, D.I. Mendeleev University of Chemical Technology of Russia, 9 Miusskaya Square, Moscow 125047 (Russia) [e] Dr. I. A. Yaremenko,+ P. S. Radulov, Prof. A. O. Terent’ev All Russian Research Institute for Phytopathology, 143050 B. Vyazyomy, Moscow Region (Russia) [+] These authors contributed equally to this work. Supporting information for this article can be found under: https://doi.org/10.1002/cmdc.201700804.

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T 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers ides such as trioxolane[6] and tetraoxane[7] (Figure 1) are highly active against the asexual erythrocytic stage of malaria infection. ART contains a 1,2,4-trioxane core that is reductively activated (electron transfer) by iron(II) heme, a by-product of host hemoglobin degradation, to form carbon-centered radicals capable of reacting with heme and proteins.[8] An alternative model for the antimalarial mechanism of endoperoxides was put forward in our previous papers,[9] whereby the endoperoxides mediate their antimalarial activity through interaction with cofactors. Intriguingly, various semisynthetic and synthetic peroxide classes such as 1,2,4,5-tetraoxanes,[10] ozonides[11] (or 1,2,4-trioxolanes), and 1,2-dioxanes[12] have also been studied recently for their effect on different in vitro models, including cancer cell lines. A convenient approach was recently developed for the synthesis of bridged ozonides,[13] bridged 1,2,4,5tetraoxanes,[14] tricyclic monoperoxides,[15] and some of them were evaluated for their anticancer[16] and antischistosomal activity.[17] In this study, we evaluated the in vitro anticancer and antimalarial activity of a set of peroxides including bridged 1,2,4,5tetraoxanes, bridged 1,2,4-trioxolanes, and tricyclic monoperoxides (Figure 2). The effects of these compounds were also examined on human liver and lung cancer cell lines, HepG2 and A549, by comparing with control hepatic LO2 and bronchial epithelial BEAS-2B cells, respectively. The cytotoxicity of such compounds toward normal human fibroblast-like

CCD19Lu was also examined. We found that some ozonides showed promising selectivity toward liver cancer cells. All compounds were tested against P. falciparum, and selected compounds with high anticancer activity were further subjected to cyclic voltammetry analysis. To our surprise, only some of the ozonides showed moderate antiplasmodial activity. To our knowledge, tricyclic monoperoxides were tested herein for the first time for their antimalarial activity.

Results and Discussion In vitro activity against Plasmodium falciparum Peroxides were screened against the chloroquine-sensitive P. falciparum strain 3D7 to assess their potential as blood-stage antimalarials (Table 1). Of these compounds, trioxolanes, in particular 12 b, 14 a, and 14 b, revealed moderate antimalarial activities (1–10 mm), whereas peroxides of the other two classes (tricyclic monoperoxides and bridged 1,2,4,5–tetraoxanes) were not active, except for compounds 7, 9, 10, and 15, which showed low activity (IC50 values of 50 to 70 mm). To our knowlTable 1. Antimalarial activity of peroxides against P. falciparum strain 3D7. Peroxide class

Compd

tetraoxanes

1 2 3 4 5 6 7 8 9 10 11

trioxolanes

12 a 12 b 13 a 13 b 14 a 14 b

monoperoxides

15 16 17 18 19 20 21 22 23

chloroquine artemether[18] artemisinin[19] artesunic acid[20]

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147.78 : 28.88 192.21 : 20.12 124.95 : 12.81 119.90 : 26.06 112.85 : 50.84 195.20 : 23.79 67.34 : 35.64 276.72 : 8.96 69.21 :10.35 52.80 : 1.97 339.40 : 41.58 8.52 : 1.60 2.76 : 0.99 20.14 : 6.52 13.25 : 3.14 6.32 : 2.50 4.77 : 0.61 51.27 : 2.83 > 1000 232.68 : 33.04 128.06 : 16.86 171.34 : 16.18 104.05 : 12.47 146.49 : 33.17 187.85 : 7.17 205.26 : 56.58 0.0124 : 0.0009 0.0037 0.0039 : 0.08 0.0005

SI[b] 0.081 0.024 0.0085 0.0084 0.0061 0.0013 0.0040 0.36 0.029 0.0066 0.012 0.1 0.15 0.58 0.14 1.61 0.33 0.58 – 0.42 0.43 0.58 0.144 0.42 0.17 0.39 > 1000 n/a n/a n/a

[a] Median inhibitory concentration (IC50) values are the mean : SD calculated from at least three experiments. [b] Selectivity index: (IC50 for liver cytotoxicity)/(IC50 for antimalarial activity); n/a: not available.

Figure 2. Structures of investigated compounds 1–23.

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Full Papers edge, this is the first report of antimalarial activity for tricyclic monoperoxides that show low antimalarial activity but a good selectivity index (66 % of compounds showed IC50 > 50 mm on normal liver cells).

acid, those of tetraoxanes 3, 4, 5,and 9 and ozonide 14 a were slightly increased (Table 2). These compounds were also evaluated for cytotoxicity against normal human fibroblast-like CCD19Lu (Table S1 and Figure S5), and 24 compounds (92 %) had a higher selectivity index than artemisinin.

In vitro activity against cancer and normal cell lines Electroanalytical study of peroxides

The cytotoxicity of the peroxides was examined on human liver and lung cancer cell lines, HepG2 and A549, respectively, and then on the immortalized normal liver LO2 and lung BEAS2B cell lines, and the results were compared with those obtained for the natural peroxides artemisinin, artesunic acid, and chloroquine and the widely used anticancer agent paclitaxel (Table 2). Of the 26 compounds tested, 14 compounds (53 %) showed higher in vitro activity against liver HepG2 cancer cell lines than artesunic acid in addition to a good selectivity index (Table 2; also see Figures S1 and S2 in the Supporting Information). All cyclic peroxides showed better selectivity index (SI) values for lung A549 cancer cell lines than artemisinin, chloroquine, and paclitaxel (Table 2, Figures S3 and S4). The selectivity index of tetraoxane 4 was 540 times higher than that of the reference compound paclitaxel. Relative to the SI of artesunic

The reduction potential was measured for the 15 most-active peroxides from three different classes in CH3CN with 0.1 m Bu4NClO4 as the supporting electrolyte. We found that tetraoxane 3 had the highest oxidative capacity (Ep = @0.797 V), and ozonide 12 a had the lowest oxidative capacity (Ep = @1.440 V) of the three classes examined. The oxidative capacities of ozonide 12 a and monoperoxides 16 and 21 were found to be close. Artemisinin showed the lowest oxidative capacity (Ep = @1.580 V) of all the compounds, whereas the synthetic derivative artesunic acid showed a capacity that was similar to those of 13 a and 14 a (Ep = @1.270 V) (Table 3 and Figure S6 a, b). 1,2,4-Trioxolanes (ozonides) and 1,2,4,5-tetraoxanes are currently considered to be promising classes of organic peroxides for the development of antiparasitic[22] and anticancer drugs.[23]

Table 2. Cytotoxicity values of tetraoxanes 1–11, ozonides 12–14 (a, b), and tricyclic monoperoxides 15–23 against human liver HepG2 and lung A549 cancer cell lines and the immortalized liver LO2 and lung BEAS-2B cell lines.

Peroxide class

Compd

tetraoxanes

1 2 3 4 5 6 7 8 9 10 11

trioxolanes

12 a 12 b 13 a 13 b 14 a 14 b

monoperoxides

15 16 17 18 19 20 21 22 23

artemisinin artesunic acid chloroquine paclitaxel

BEAS-2B

IC50 [mm][a]

19.3 : 0.4 14.3 3.36 : 0.5 4.97 : 0.9 6.51 : 0.1 0.76 1.93 : 0.3 > 100 16.1 : 0.5 0.82 18.9 : 1.4

100 7.53 3.07 < 0.1

LO2

IC50 [mm][a]

HepG2

SI[c]

36.7 : 0.6 38.5 : 1.2 2.24 : 0.1 3.05 : 0.1 5.62 : 0.5 3.47 : 0.5 5.13 : 0.7 > 100 14.5 : 0.5 1.32 : 0.04 47.9 : 3.5

0.52 0.37 1.5 1.62 1.15 0.21 0.37 – 1.11 0.62 0.39

12.11 : 0.7 4.68 : 1.1 1.07 : 0.1 1.01 : 0.1 0.69 0.27 : 0.1 0.33 > 100 2.03 : 0.1 0.35 4.33

7 : 0.3 7.08 : 0.1 0.39 : 0.06 0.55 : 0.05 0.62 : 0.07 0.63 : 0.06 1.01 : 0.2 > 100 1.74 : 0.1 0.37 : 0.1 5.89 : 1.2

1.73 0.66 2.74 1.83 1.11 0.42 0.32 – 1.16 0.94 0.73

4.57 : 0.4 3.94 : 0.05 20.2 : 1.9 5.01 : 0.2 7.24 : 0.6 3.85 : 0.4

0.33 0.42 0.67 0.67 1.13 0.39

0.88 : 0.1 0.42 11.7 : 2.2 1.92 : 0.1 10.2 : 3.4 1.6 : 0.1

0.49 : 0.6 0.19 : 0.01 0.59 : 0.01 0.39 : 0.08 0.36 : 0.08 0.22 : 0.03

1.79 2.21 19.83 4.92 28.33 7.27

1.55 : 0.2 1.69 13.7 : 3 3.39 : 0.5 8.25 : 4.21 1.51 : 0.1 5.33 : 0.5 > 100 > 100 42.3 : 0.6 > 100 9.58 : 0.2 22.2 : 3.8 90.78 : 3.7 > 100

SI[b]

A549

78.5 : 14.5 > 100 > 100 > 100 > 100 59.6 : 1.3 > 100 > 100 > 100 100 9.85 100 33.2

0.067 – – 0.42 – 0.16 0.22 0.90 –

30 : 15.5 > 100 > 100 56.1 :10.5 > 100 15 : 0.6 62.6 : 5.1 32.14 : 3.4 81.37 : 16.6

10.2 : 2.1 > 100 68.4 : 10.9 15 : 3.5 > 100 3.39 : 0.4 11.4 : 1.8 50 : 5.8 > 100

2.94 – 1.46 3.74 – 4.42 5.49 0.64 –

– 0.76 0.030 0.003

> 100 8.25 15 < 0.1

> 100 4.09 : 0.4 49.02 : 0.4 0.19 : 0.4

– 2.01 0.30 0.52

[a] Values are the mean : SD calculated from at least three experiments. [b] Selectivity index lung: (IC50 for cytotoxicity of normal cells)/(IC50 for cytotoxicity of cancer cells). [c] Selectivity index liver: (IC50 for cytotoxicity of normal cells)/(IC50 for cytotoxicity of cancer cells).

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Full Papers as the supporting electrolyte. Such a technique was used in the past for the electrochemical analysis of antimalarial drugs[28] and for mechanistic study of the hemin-mediated opening of the peroxide bond induced by ART.[21] Upon comparing with the class of ozonides containing a structural core similar to that of the 1,2,4-trioxolanes of artemisinin, we found that 12 b and 13 b possessed higher oxidative capacity than ozonides 12 a and 13 a and showed better activity against P. falciparum than their isomers. Instead, the oxidative property–activity relationship among the isomers of 14 did not show a direct correlation. In addition, tetraoxane 3, which had the highest oxidative capacity of the tested compounds, showed very low antimalarial activity. Parental artemisinin and its derivative artesunic acid did not show direct correlation in oxidative capacity–activity (Tables 1 and 3 and Figure S6 b). On the basis of the results obtained in the antiplasmodial assay and CV, we are currently continuing our research on evaluating the oxidation of dihydroflavin by tetraoxanes 1– 11, ozonides 12–14 (a, b), and monoperoxides 15–23 in acetonitrile/aqueous buffer (1:1, pH 7.4), as previously reported by us,[29] and we are repeating the CV experiments under the same conditions. A study by O’Neill et al. in 2005[30] revealed that the enantiomers of trioxanes showed the same antimalarial activity, and this confirmed the absence of a specific interaction at an enzyme active site. The same conclusions were reached by Jefford et al.[31a] and Najjar et al.[31b] for the activities of the enantiomeric pairs of synthetic cyclopentotrioxanes and the enantiomeric benzyl ether analogues of the natural endoperoxide G3 factor, respectively. The biological results of our enantiomeric pairs of ozonides 12–14 appear to confirm these findings. The cytotoxicity of peroxides was examined on different liver cancer and normal cell lines. An increase in the length of the alkyl chain of the tetraoxane (from four to seven carbon atoms) slightly decreased the activity on liver cancer cells (IC50 from 0.39 to 1.01 mm). For the class of ozonides 12–14, all tested compounds showed good activities (IC50 < 0.6 mm), the IC50 values of which were higher than those of artemisinin and its semisynthetic derivative artesunic acid (7–21-fold higher), whereas compounds 12 b and 14 b displayed activity similar to that of paclitaxel (IC50 < 0.22 mm). A tendency of the structural features among highly active ozonide could be noted. We previously reported that peroxides were cytotoxic toward androgen-independent prostate cancer cell lines PC3, whereas cyclic voltammetry did not show a direct correlation (oxidative property–activity).[15] Here, we observed a similar situation for human liver cancer cell lines. Ozonides 12 b and 13 b possess higher oxidative capacity than ozonides 12 a and 13 a, but in the case of the pair of stereoisomers 14 a/14 b the situation is reverse—ozonide 14 a has higher oxidative capacity than ozonide 14 b. In the case of the stereoisomers, the cytotoxicity induced by 12 b, 13 b, and 14 b on human liver cancer cell lines HepG2 is higher than that induced by their isomers 12 a, 13 a, and 14 a. Compounds 13 a and 14 a present better selectivity indexes (20 and 28 times) than all of the reference compounds, and in particular, the selectivity index of 14 a is 54

Table 3. Reduction potentials of the peroxides. EpV[a]

Peroxide class

Compd

tetraoxanes

1 3 4 9

@1.067 @0.797 @1.101 @0.947

trioxolanes

12 a 12 b 13 a 13 b 14 a 14 b

@1.440 @1.375 @1.270 @1.191 @1.271 @1.352

monoperoxides

15 16 18 20 21

@1.185 @1.415 @1.171 @1.200 @1.402

artemisinin[21] artesunic acid

@1.580 @1.270

[a] Ep = cathodic peak potential.

The molecular targets of artemisinin and peroxides in P. falciparum and in tumor cells are still under debate.[24] In our previous studies,[9, 25, 26] we described that artemisinin and semisynthetic peroxide analogues acted in malaria parasites by oxidizing dihydroflavin cofactors of redox-active flavoenzyme. Perturbation of redox homeostasis coupled with the generation of reactive oxygen species (ROS) contributed to the death of the parasites. On the basis of this discovery, our current study measured the oxidative capacities of three classes of compounds, and for the first time, the correlation between the oxidative properties and antimalarial activity among these compounds was examined. First, the in vitro activity of the candidate compounds (depicted in Figure 2) against the chloroquine-sensitive P. falciparum 3D7 strain (Table 1) was studied. Most of our compounds, including ozonides 12–14, showed low antimalarial activity. This observation could be explained by hypothesizing that the methylene bridge in 1–11 and the hindered structures of 15– 23 could influence the bioavailability and reactivity of the peroxide bond for oxidation of the cofactors. Previously, Ellis et al. showed bridged achiral 1,2,4,5-tetraoxanes with good in vitro antimalarial activity,[14b] while in another study,[27] bicyclic trioxanes structurally similar to 12–14 demonstrated antimalarial activity against the D8 strain at the micromolar level, comparable to our results in Table 1. Taking into account that electron transfer can play a significant role in the activities of cyclic peroxides, accurate reduction potentials were examined by cyclic voltammetry (CV), which is an electrochemical technique used to quantify the antioxidant capacity of compounds in blood or tissue homogenates; it is the most commonly used characterization method for redox systems. CV measures the current in an electrochemical cell under conditions for which a solvent, typically water or acetonitrile, contains NaCl or tetrabutylammonium perchlorate ChemMedChem 2018, 13, 902 – 908

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Full Papers times higher than that of the most-active compound, paclitaxel. However, they have the same reducing potential (@1.270 and @1.271 V) if compared with the corresponding reference compounds (Table 3 and Figure S6 a). Of note, the results of these ozonides suggest that compounds of this class may serve clinically as a potential treatment strategy. Similar to our results, moreover, Abrams[32] et al. showed two enantiomerically pure forms of FINO2 (ferroptosis inducing), isomers containing a 1,2-dioxolane moiety, that showed a substantial difference in anticancer activity against fibroblast cancer cells. Additional experiments suggested that cell death was due to ferroptosis.[33] Ferroptosis is a nonapoptotic form of cell death that is induced by the accumulation of lipid peroxides, and it has also been demonstrated for ART derivatives.[34] These compounds that induce ferroptosis are proposed to be selective, because many cancer cells have an increased concentration of iron relative to healthy cells.[35] However, as previously described in our recent review[9a] and by others,[9c, 36] many ARTs and peroxide analogues do not react with iron in vitro despite their high biological activity. Iron may enhance the cytotoxicity of ARTs by redox cycling through the FeII state and reaction with oxygen.[9a] Upon comparing the cytotoxicity on lung cancer cell lines, tetraoxane 3 has a higher oxidative capacity (Ep = @0.797 V, Table 3 and Figure S6) and higher selectivity index than paclitaxel. However, we found that the data obtained from the cytotoxicity of the peroxides and cyclic voltammetry did not show a direct correlation (oxidative property–activity), as demonstrated in studies performed on human liver cancer cell lines HepG2. Recently, some papers[37] showed the synthesis of artemisinin–triphenylphosphonium (TPP) derivatives with potent antitumorigenic activity against liver cancer cell lines HepG2 (IC50 = 2.69 mm) and depressed antimalarial activity against the 3D7 strain of P. falciparum (IC50 = 320 nm) but modest selectivity against liver normal cell lines LO2 (IC50 = 2.78 mm). The authors synthesized a mitochondria-targeted artemisinin derivative by conjugating TPP to peroxide that selectively accumulated adduct in the mitochondria. The results here showed that a peroxide was highly active and selective against HepG2 (14 a, IC50 = 0.39 mm, 28-fold lower than that against LO2) with very low antimalarial activity (14 a, IC50 = 6.32 mm) without the need for conjugation. Further studies of reactivity are in progress with the aim to explain these different activities on cancer cells and P. falciparum.

ides revealed low antimalarial activity, but some of them showed good selectivity against cancer cells at levels that were higher than that of the anticancer agent paclitaxel and higher than those of artesunic acid and artemisinin. Taken together, the present findings show for the first time promising new peroxide derivatives with anticancer activities that are much higher than their antimalarial activities and with a considerable increase in selectivity. Our results provide a unique synthetic approach to yield a new class of structurally modified peroxides for both antimalarial and anticancer treatment.

Experimental Section General Plasmodium falciparum strain 3D7 was obtained from the Malaria Research and Reference Reagent Resource Center (MR4). Human O + erythrocytes were obtained from healthy, nonpregnant volunteers following the Red Cross National Blood Center protocol. Chloroquine diphosphate and saponin were purchased from Sigma–Aldrich (USA); dihydroartemisinin (DHA) was obtained from Dafra Pharma International (Belgium); SYBR Green I nucleic acid staining dye was purchased from Molecular Probes (USA). Human liver and lung cancer cell lines, HepG2 and A549, respectively, and immortalized normal liver LO2, lung BEAS-2B, and fibroblast-like CCD19Lu cells were all purchased from ATCC. Cells were cultured in RPMI-1640 medium supplemented with 10 % fetal bovine serum and the antibiotics penicillin (50 U mL@1) and streptomycin (50 mg mL@1; Invitrogen, UK). All cells were incubated at 37 8C in a 5 % humidified CO2 incubator. NMR spectra were recorded with a commercial instrument (300.13 MHz for 1H, 75.48 MHz for 13C) in CDCl3. High-resolution mass spectrometry (HRMS) was performed by using electrospray ionization (ESI).[38] The measurements was done in the positive-ion mode (interface capillary voltage: 4500 V); the mass ratio was from m/z 50 to 3000 Da; external/ internal calibration was done with electrospray calibration solution. A syringe injection was used for solutions in MeCN (flow rate: 3 mL min@1). Nitrogen was applied as a dry gas; the interface temperature was set at 180 8C. TLC analysis was performed on silica gel chromatography plates. Macherey– Nagel Alugram UV254; sorbent: Silica 60, specific surface (BET): & 500 m2 g@1, mean pore size: 60 a, specific pore volume: 0.75 mL g@1, particle size: 5–17 mm; binder: highly polymeric product, which is stable in almost all organic solvents and resistant toward aggressive visualization reagents. The melting points were determined with a Kofler hot-stage apparatus. Chromatography of tetraoxanes and monoperoxides was performed on silica gel (0.060–0.200 mm, 60 A, CAS 7631-86-9). Chromatography of ozonides was performed on silica gel (0.040–0.060 mm, 60 A, CAS 7631-86-9). A solution of H2O2 in Et2O (3.7 m) was prepared by extraction with Et2O (5 V 100 mL) from a 35 % aqueous solution (100 mL) followed by drying (MgSO4). For the synthesis of the ozonides, acetonitrile was distilled with P2O5.

Conclusions In the present work, synthetic ozonides were shown to have high cytotoxicity in vitro and selectivity, in particular ethyl (1R,2R,5S)-2-allyl-1,5-dimethyl-6,7,8-trioxabicyclo[3.2.1]octane-2carboxylate (13 a) and ethyl (1R,2S,5S)-2-hexyl-1,5-dimethyl6,7,8-trioxabicyclo[3.2.1]octane-2-carboxylate (14 a), against HepG2 and A549 cancer cell lines. Unexpectedly, the same class of compounds showed only moderate antimalarial activity in vitro. The anticancer activities of the ozonide stereoisomers differed from each other. Tetraoxanes and monoperoxChemMedChem 2018, 13, 902 – 908

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Chemistry Synthesis: Eleven bridged 1,2,4,5-tetraoxanes, six bridged 1,2,4-trioxolanes, and nine tricyclic monoperoxides (Figure 2) were prepared on the basis of the methods described in the literature.[13a, 39]

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Full Papers Electroanalytical instrumentation, experimental techniques, and reagents: Cyclic voltammetry (CV) was performed with an IPC-Pro computer-assisted potentiostat manufactured by Econix (scan rate error 1.0 %, potential setting 0.25 mV). The experiments were performed in a 10 mL five-necked glass conic electrochemical cell with a water jacket for thermostating. CV curves for peroxides (see Figure S6) were recorded by using a three-electrode scheme. The working electrode was a disc glassy-carbon electrode (d = 1.7 mm). A platinum wire served as an auxiliary electrode. A saturated calomel electrode (SCE, EoxFc1/2 = 409 mV; Fc = ferrocene) was used as the reference electrode and was linked to the solution by a bridge with a porous ceramic diaphragm filled with background electrolyte (0.1 m Bu4NClO4 in MeCN). The tested solutions were thermostated at (25 : 0.5) 8C. Deaeration of solutions was performed by passing argon. To prevent contact between the solution surface and ambient air during the experiment, argon was constantly fed to the cell’s free space above the solution surface. In a typical case, 5 mL solution was used, and the depolarizer concentration was 5 mm. The working electrode was polished, and the solution was agitated vigorously with argon before recording each CV curve.

Cells without drug treatment were used as control. Subsequently, 5 mg mL@1 MTT solution (10 mL) was added to each well and incubated at 37 8C for 4 h, which was followed by the addition of solubilization buffer [100 mL, 10 mm HCl in solution of 10 % of sodium dodecyl sulfate (SDS)] and overnight incubation. The A570 nm value was then determined in each well on the next day. The percentage of cell viability was calculated by using Equation (1): Cell viability ½%A ¼

Acknowledgements This work was supported by a FDCT grant from the Macao Science and Technology Development Fund to V.K.W.W. (Project code: 084/2013/A3) and by Thailand National Science and Technology Development Agency (Project code: P1450883), National Center for Genetic Engineering and Biotechnology and Thailand Research Fund (Project code: RSA5880064) to C.U. The synthesis of the peroxides was supported by the Russian Science Foundation (Grant No. 14-50-00126 to I.Y. and A.T).

In vitro P. falciparum growth inhibition assay: Asexual P. falciparum parasites were maintained in vitro in human O + erythrocytes at 4 % hematocrit in complete RPMI-1640 medium (Invitrogen, U.K.) supplemented with 2 mm l-glutamine, 25 mm HEPES, 2 g L@1 sodium bicarbonate, 5 g L@1 AlbuMAX I (Life Technologies, USA), 0.37 mm hypoxanthine, and 40 mg L@1 gentamicin under a gas mixture of 5 % O2, 5 % CO2, and 90 % N2 at 37 8C. Every 3–4 d, the parasite culture was synchronized with 5 % sorbitol and was transferred into fresh complete medium with uninfected erythrocytes. For growth inhibition assays, synchronized ring stage parasites at 1 % parasitemia and 2 % hematocrit were put into individual wells of a 96-well black plate, whereas non-parasitized erythrocytes at 2 % hematocrit served as reference controls. The test compounds were prepared at a stock concentration of 1 m in DMSO, serially diluted in complete medium, and dispensed into duplicate test wells to yield final concentrations ranging from 0 to 1000 mm. The plates were incubated at 37 8C with a gas environment of 5 % O2, 5 % CO2, and 90 % N2. After 48 h, the SYBR Green I solution [0.2 mL SYBR Green I per mL buffer solution consisting of 20 mm Tris 20 (pH 7.5), 5 mm ethylenediaminetetraacetic acid (EDTA), 0.008 % w/v saponin, and 0.08 % v/v Triton X-100] was added to each well and mixed at 1000 rpm for 30 s by a microplate mixer. After 1 h of incubation in the dark at room temperature, fluorescence signal was measured with a Spectramax M5 Multi-Mode Microplate Reader (Molecular Devices, USA) with excitation and emission wavelength bands centered at 485 and 535 nm, respectively. The background reading from wells of non-parasitized erythrocytes was subtracted to yield fluorescence counts for analysis. The SYBR Green I signal in drug-treated samples were normalized to untreated control parasite samples in the same experiment, which were taken as 100 %. Median inhibitory concentration (IC50) values with standard deviation (SD) were calculated from at least three experiments.

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Cytotoxicity drug assay: All test compounds were dissolved in DMSO at a final concentration of 50 mm and were stored at @20 8C before use. Cytotoxicity was assessed by using the 3-(4,5-dimethylthiazole-2yl)-2,5-diphenyltetrazolium bromide (MTT) (5 mg mL@1) assay, as previously described.[40] Briefly, 4 V 103 cells per well were seeded in 96-well plates before drug treatment. After overnight cell culture, the cells were then exposed to different concentrations of selected compounds (0.19–100 mm) for 72 h. www.chemmedchem.org

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Representative graphs of at least three independent experiments are shown in Figures S1–S5.

Biological methods

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Manuscript received: December 19, 2017 Revised manuscript received: February 15, 2018 Accepted manuscript online: February 22, 2018 Version of record online: March 15, 2018

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