Cyclic peroxides as promising anticancer agents: in vitro cytotoxicity study of synthetic ozonides and tetraoxanes on human prostate cancer cell lines

Article abstract of DOI:10.1007/s00044-016-1736-2

Abstract: Synthetic ozonides and tetraoxanes were shown to have high cytotoxicity in vitro when tested on androgen-independent prostate cancer cell lines DU145 and PC3, which is in some cases was higher than that of doxorubicin, cisplatin, etoposide, arte

Full text of DOI:10.1007/s00044-016-1736-2

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-016-1736-2
ORIGINAL RESEARCH
Cyclic peroxides as promising anticancer agents: in vitro
cytotoxicity study of synthetic ozonides and tetraoxanes on human
prostate cancer cell lines
Ivan A. Yaremenko1,2 Mikhail A. Syroeshkin1 Dmitri O. Levitsky2,3
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Fabrice Fleury3 Alexander O. Terent’ev1,2
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Received: 22 June 2016 / Accepted: 25 October 2016
© Springer Science+Business Media New York 2016
Abstract Synthetic ozonides and tetraoxanes were shown
to have high cytotoxicity in vitro when tested on androgen-
independent prostate cancer cell lines DU145 and PC3,
which is in some cases was higher than that of doxorubicin,
cisplatin, etoposide, artemisinin, and artesunate. Activity of
ozonide stereoisomers differs from each other. This differ-
ence in activity and absence of correlation between activity
of stereoisomers and their oxidative properties allow us to
suggest existence of a quite specific mechanism of cyto-
toxicity of these endoperoxides different from a traditional
mechanism based mainly on oxidative properties of per-
oxides.
Graphical Abstract
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Keywords Ozonide Tetraoxane Peroxide Cytotoxicity
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Prostate Cancer
Introduction
Organic peroxides based on ketones are produced by dozens
of companies on a large scale and are used as initiators for
the radical polymerization of unsaturated compounds
(Denisov et al. 2003; Moad and Solomon 2006) and for
cross-linking of polymer chains (Ray 2002). The interest in
inexpensive and efficient radical polymerization initiators
and technologically available biologically active com-
pounds gave impetus to the development of new methods
for the synthesis of peroxides starting from ketones, their
derivatives, H2O2, and hydroperoxides (Azarifar et al. 2009;
Bunge et al. 2009; Das et al. 2007; Differding 2014; Gahlot
2012; Griesbeck et al. 2005, 2013; Li et al. 2009; Ravi et al.
2013; Solaja et al. 2002; Terent’ev et al. 2005, 2008a, b,
2006, 2007).
Electronic supplementary material The online version of this article
(doi:10.1007/s00044-016-1736-2) contains supplementary material,
which is available to authorized users.
* Fabrice Fleury
Fabrice.Fleury@univ-nantes.fr
Alexander O. Terent’ev
terentev@ioc.ac.ru
1
Zelinsky Institute of Organic Chemistry, Russian Academy of
Sciences, Leninsky Prospect 47, Moscow 119991, Russian
Federation
2
All-Russian Research Institute of Phytopathology, Bol’shie
Vyazemy, Moscow region 143050, Russian Federation
In the past three decades, the chemistry of organic per-
oxides was extensively developed because these com-
pounds were found to have high antimalarial (Ghorai et al.
3
Mechanism and regulation of DNA repair team Université de
Nantes, UFIP CNRS UMR 6286, 2 rue de la Houssinière,
Nantes 44322, France

Med Chem Res
H
CH3
H
H
CH3
O
H
O
H3C
H
O
O
O
H
H3C
H
Fig. 2 Structure of arterolane maleate
O
H
O
O
H
CH3
H
O
O
H
O
CH3
2011; Mankil et al. 2003; Nakase et al. 2008; Posner et al.
2006, 2004, 2003; Reiter et al. 2015; Rosenthal et al. 2009;
Vennerstrom et al. 2004; Xie et al. 2011; Zhou et al. 2008;
Zizak et al. 2009).
In our previous study we have developed a convenient
two-step approach for the synthesis of ozonides (1,2,4-
trioxolanes) 2a–e, 3a–e by the reaction of β,δ-triketones
1a–e (the products of Michael addition of vinyl ketones to
β-diketones) with H2O2 catalyzed by phosphomolybdic acid
(PMA) (Scheme 1) (Yaremenko et al. 2014).
OH
O
Artemisinin
Artesunate
H
H
CH3
CH3
H
H
O
O
CH3
H3C
H
H
O
O
O
H
O
H
O
H
H
O
CH3
CH3
HO
H
MeO
H
Dihydroartemisinin
Artemether
In this work, the cytotoxicity of the ozonides 2a–e, 3a–e
and two bridged tetraoxanes with adamantane unit 5a, b
(see Table 1) was evaluated on androgen-independent
prostate cancer cell lines DU145 and PC3 and on a normal
prostate cell line. Previously, we found the existence of high
cytotoxicity and antishistosomal activity of 5a (Ingram et al.
2012). Cyclic peroxides 5a, b were prepared by the PMA-
catalyzed reaction of β-diketones 4a, b with hydrogen
peroxide (Scheme 2) (Terent’ev et al. 2013).
Fig. 1 Antimalarial natural peroxide artemisinin and its semisynthetic
derivatives
2008; Hao et al. 2013; Jefford 2012; Opsenica and Solaja
2009; Slack et al. 2012; Vennerstrom et al. 1992; Yadav
et al. 2014) and anthelminthic (Boissier et al. 2012; Ingram
et al. 2012; Keiser et al. 2012, 2010; Xiao et al. 2002)
activities. Natural 1,2,4-trioxane artemisinin and its semi-
synthetic derivatives dihydroartemisinin, artemether, and
artesunate have been used for malaria treatment for several
decades (Fig. 1) (WHO 2010; 2013).
Material and methods
The interest to natural and synthetic peroxides con-
siderably increased when the first antimalarial drug Synriam
containing completely synthetic peroxide received market
approval (Vennerstrom et al. 2004). Synriam is the arter-
olane maleate in combination with the piperaquine phos-
phate (Fig. 2). Ozonide cycle is the key pharmacophore part
of the drug (Balganesh et al. 2014; Differding 2014; Gahlot
2012; Wang et al. 2013; Wongsrichanalai and Sibley 2013).
Recently, it has been shown that synthetic peroxides,
1,2,4-trioxanes, have cytotoxic effect on distinct cell lines,
such as human lung cancer cell line A549, human prostate
cancer cell line PC3, canine osteosarcoma D17 (Rubush
et al. 2012; van Huijsduijnen et al. 2013). 1,2,4,5-tetra-
oxanes have antiproliferative activity against broad spec-
trum of cancer cell lines (Opsenica et al. 2003a, b, 2006;
Terzić et al. 2007), and synthetic endoperoxides, 1,2-diox-
anes, have exhibited cytotoxic activity against DU145
human prostate cancer cell line (Parrish et al. 2012). Quite a
few data obtained on different cancer cell lines reveal high
cytoxicity of artemisinin analogs and other types of endo-
peroxides (Alagbala et al. 2006; Chaturvedi et al. 2010;
Chen et al. 2003, 2004; Dembitsky 2008, 2007; Dwivedi
et al. 2015; Efferth 2005; Kim and Park 2002; Kumar et al.
NMR spectra of peroxides 2a–e, 3a–e, 5a, and 5b were
recorded on a commercial instrument (300.13 MHz for Н,
1
75.48 MHz for 13С in СDCl3). The TLC analysis was car-
ried out on standard silica gel chromatography plates. The
melting points were determined on a Kofler hot-stage
apparatus. Chromatography was performed on silica gel
(0.060–0.200 mm, 60 A, CAS 7631-86-9). Elemental ana-
lysis on C, H, and N atoms was carried out using a 2400
Perkin-Elmer CHN analyzer. Purity of all peroxides was
assessed by elemental (combustion) analysis, deviation
from the theoretical values for C, H, and N content was less
than 0.4 %. These data confirm >95 % purity of compounds
2a–e, 3a–e, 5a and b. Doxorubicin, cisplatin, etoposide,
artemisinin, and artesunate were purchased from Sigma-
Aldrich. Tetrabutylammonium perchlorate and MeCN
(“extra dry” grade) were supplied by Acros Organics.
General procedure for the synthesis of ozonides 2a–e
and 3a–e
The synthesis of ozonides 2a–e and 3a–e was performed
according to published procedure (Yaremenko et al. 2014).
An ethereal solution of H2O2 (1.5 mol of H2O2 per mole of

Med Chem Res
Scheme 1 Two-step synthesis
of the ozonides 2a–e, 3a–e
Table 1 Structures of tested peroxides
Ozonide
2a
2b
2c
2d
2e
3a
3b
3c
3d
3e
Tetraoxane
5a
5b
triketone 1a–e, M = 1.10 mol L−1) and PMA (5 mol%) were
successively added with stirring to a solution of β,
δ–triketone 1a–e (0.30 g) in CH3CN (4 mL) at room tem-
perature. The reaction mixture was stirred at 20–25 °C for 8
h. Then CH2Cl2 (20 mL) was added. The organic layer was
washed with water (5 mL), a 5 % aqueous solution of
NaHCO3, and again with water (5 mL). The mixture was
dried over Na2SO4 and filtered. The solvent was removed
with a water-jet vacuum pump. Products 2a–e, 3a–e were
isolated by silica gel column chromatography by gradient
elution with 10–80 vol. % ethyl acetate in petroleum ether.
1-((1R,2R,5S)-2-Benzyl-1,5-dimethyl-6,7,8-trioxabicyclo
[3.2.1]oct-2-yl)ethanone (2a)
This compound was prepared by the reaction of β,
22
δ–triketone 1a with H2O2. Colorless oil, yield 15 %; nD
=
1
1.5162; Н NMR (CDCl3, 300.13 MHz): δ = 1.47 (s, 3H,

Med Chem Res
CH3C=O), 2.64–2.79 (m, 1H, CCCH2), 3.00 (d, 1H,
CH2Ar, J = 13.2 Hz), 3.66 (d, 1H, CH2Ar, J = 13.2 Hz),
7.27 (d, 2H, 2 × o-CHAr, J = 8.8 Hz), 8.08 (d, 2H, 2 × m-
CHAr, J = 8.8 Hz); 13С NMR (CDCl3, 75.48 MHz,): δ =
18.2 (CH3, CH3CC), 20.7 (CH3, CH3CCH2), 22.2 (CH2,
CCCH2), 29.9 (CH3, CH3C=O), 30.8 (CH2, CH2CCH3),
35.4 (CH2, CH2Ar), 59.6 (C, CH2CCH2), 109.2 (C,
OOCCH2), 110.8 (C, OOCC), 123.4 (CH, m-CHAr), 131.2
(CH, o-CHAr), 145.8 (C, CHArCCHAr), 146.8 (C, CNO2),
210.3(C, CH3C=O); Anal. Calcd. for C16H19NO6: C, 59.81;
H, 5.96; N, 4.36. Found: C, 59.77; H, 5.90; N, 4.41.
Scheme 2 Synthesis of the tetraoxanes 5a, b from β-diketones 4a, b
CH3CC), 1.52–1.62 (m, 4H, CH3CCH2, CCCH2),
1.75–1.85 (m, 1H, CH2CCH3), 1.90–2.03 (m, 1H,
CH2CCH3), 2.14 (s, 3H, CH3C=O), 2.56–2.70 (m, 1H,
CCCH2), 2.88 (d, 1H, CH2Ar, J = 13.2 Hz), 3.53 (d, 1Н,
CH2Ar, J = 13.2 Hz), 7.03–7.09 (m, 2Н, 2 × o-CHAr),
7.15–7.26 (m, 3Н, 2 × m-CHAr, p-CHAr); 13C NMR
(CDCl3, 75.48 MHz,): δ = 18.4 (CH3, CH3CC), 20.8 (CH3,
CH3CCH2), 22.3 (CH2, CCCH2), 30.3 (CH3, CH3C=O),
31.0 (CH2, CH2CCH3), 36.2 (CH2, CH2Ar), 59.2 (C,
CH2CCH2), 109.2 (C, OOCCH2), 111.4 (C, OOCC), 126.6
(CH, p-CHAr), 128.3 (CH, m-CHAr), 130.2 (CH, o-CHAr),
137.7 (C, CHArCCHAr), 210.9 (C, CH3C=O); Anal. Calcd.
for C16H20O4: C, 69.54; H, 7.30. Found: C, 69.49; H, 7.35.
1-((1R,2S,5S)-1,5-Dimethyl-2-(4-nitrobenzyl)-6,7,8-
trioxabicyclo[3.2.1]oct-2-yl)ethanone (3b)
This compound was prepared by the reaction of β,
δ–triketone 1b with H2O2. White crystals, yield 12 %; m.p.:
133–134 °С; 1Н NMR (CDCl3, 300.13 MHz): δ =
1.48–1.61 (m, 4H, CH3CCH2, CCCH2), 1.66 (s, 3H,
CH3CC), 1.89–2.09 (m, 2H, CH2CCH3), 2.13–2.31 (m, 4H,
CH3C=O, CCCH2), 2.67 (d, 1H, CH2Ar, J = 13.2 Hz), 3.54
(d, 1H, CH2Ar, J = 13.2 Hz), 7.25 (d, 2H, 2 × o-CHAr, J =
8.8 Hz), 8.09 (d, 2H, 2 × m-CHAr, J = 8.8 Hz); 13С NMR
(CDCl3, 75.48 MHz,): δ = 18.5 (CH3, CH3CC), 20.6 (CH3,
CH3CCH2), 25.5 (CH2, CCCH2), 30.1 (CH3, CH3C=O),
32.8 (CH2, CH2CCH3), 40.6 (CH2, CH2Ar), 58.3 (C,
CH2CCH2), 109.0 (C, OOCCH2), 110.6 (C, OOCC), 123.3
(CH, m-CHAr), 131.1 (CH, o-CHAr), 144.3 (C, CHArC-
CHAr), 147.0 (C, CNO2), 209.5 (C, CH3C=O).; Anal.
Calcd. for C16H19NO6: C, 59.81; H, 5.96; N, 4.36. Found:
C, 59.85; H, 5.91; N, 4.40.
1-((1R,2S,5S)-2-Benzyl-1,5-dimethyl-6,7,8-trioxabicyclo
[3.2.1]oct-2-yl)ethanone (3a)
This compound was prepared by the reaction of β,
δ–triketone 1a with H2O2. White crystals, yield 10 %; m.p.:
89–90 °С; 1Н NMR (CDCl3, 300.13 MHz): δ = 1.50 (s, 3H,
CH3CCH2), 1.61–1.78 (m, 4H, CH3CC, CCCH2),
1.86–1.96 (m, 2H, CH2CCH3), 2.07 (s, 3H, CH3C=O),
2.18–2.34 (m, 1H, CCCH2), 2.59 (d, 1H, CH2Ar, J = 13.2
Hz), 3.35 (d, 1H, CH2Ar, J = 13.2 Hz), 7.00–7.10 (m, 2H,
2 × o-CHAr), 7.15–7.29 (m, 3H, 2 × m-CHAr, p-CHAr); 13С
NMR (CDCl3, 75.48 MHz,): δ = 18.9 (CH3, CH3CC), 20.6
(CH3, CH3CCH2), 25.8 (CH2, CCCH2), 30.0 (CH3,
CH3C=O), 33.0 (CH2, CH2CCH3), 41.4 (CH2, CH2Ar),
58.3 (C, CH2СCH2), 109.0 (C, OOCCH2), 111.3 (C,
OOCC), 126.7 (CH, p-CHAr), 128.3 (CH, m-CHAr), 130.1
(CH, o-CHAr), 136.0 (C, CHArCCHAr), 210.3 (C,
CH3C=O); Anal. Calcd. for C16H20O4: C, 69.54; H, 7.30.
Found: C, 69.49; H, 7.37.
1-((1R,2R,5S)-1,5-Dimethyl-2-(4-methylbenzyl)-6,7,8-
trioxabicyclo[3.2.1]oct-2-yl)ethanone (2c)
This compound was prepared by the reaction of β,
22
δ–triketone 1c with H2O2. Yellow oil, yield 18 %; nD
=
1
1.5214; Н NMR (CDCl3, 300.13 MHz): δ = 1.48 (s, 3H,
CH3CC), 1.51–1.65 (m, 4H, CH3CCH2, CCCH2),
1.74–1.86 (m, 1H, CH2CCH3), 1.90–2.04 (m, 1H,
CH2CCH3), 2.14 (s, 3H, CH3C=O), 2.28 (s, 3H, CH3Ar),
2.55–2.71 (m, 1H, CCCH2), 2.85 (d, 1H, CH2Ar, J = 13.2
Hz), 3.49 (d, 1H, CH2Ar, J = 13.2 Hz), 6.95 (d, 2H, 2 × o-
CHAr, J = 8.1 Hz), 7.03 (d, 2H, 2 × m-CHAr, J = 8.1 Hz);
13С NMR (CDCl3, 75.48 MHz,): δ = 18.4 (CH3, CH3CC),
20.8 (CH3, CH3CCH2), 20.9 (CH3, CH3Ar), 22.3 (CH2,
CCCH2), 30.3 (CH3, CH3C=O), 30.9 (CH2, CH2CCH3),
35.8 (CH2, CH2Ar), 59.1 (C, CH2CCH2), 109.2 (C,
OOCCH2), 111.4 (C, OOCC), 129.0 (CH, m-CHAr), 130.0
(CH, o-CHAr), 134.5 (C, CHCCH3), 136.1 (C, CHArC-
CHAr), 211.0 (C, CH3C=O); Anal. Calcd. for C17H22O4: C,
70.32; H, 7.64. Found: C, 70.35; H, 7.66.
1-((1R,2R,5S)-1,5-Dimethyl-2-(4-nitrobenzyl)-6,7,8-
trioxabicyclo[3.2.1]oct-2-yl)ethanone (2b)
This compound was prepared by the reaction of β,
δ–triketone 1b with H2O2. White crystals, yield 15 %; m.p.:
115–116 °С; 1Н NMR (CDCl3, 300.13 MHz): δ =
1.37–1.49 (m, 4H, CH3CC, CCCH2), 1.58 (s, 3H,
CH3CCH2), 1.77–2.03 (m, 2H, CH2CCH3), 2.21 (s, 3H,

Med Chem Res
1-((1R,2S,5S)-1,5-Dimethyl-2-(4-methylbenzyl)-6,7,8-
trioxabicyclo[3.2.1]oct-2-yl)ethanone (3c)
40.5 (CH2, CH2Ar), 58.2 (C, CH2CCH2), 109.0 (C,
OOCCH2), 111.0 (C, OOCC), 128.4 (CH, m-CHAr), 131.5
(CH, o-CHAr), 132.7 (C, CCl), 134.6 (C, CHArCCHAr),
210.0 (C, CH3C=O); Anal. Calcd. for C16H19ClO4: C,
61.84; H, 6.16; Cl, 11.41. Found: C, 61.90; H, 6.20; Cl,
11.45.
This compound was prepared by the reaction of β,
δ–triketone 1c with H2O2. White crystals, yield 10 %; m.p.:
90–91 °С; 1Н NMR (CDCl3, 300.13 MHz): δ = 1.50 (s, 3H,
CH3CCH2), 1.62–1.78 (m, 4H, CH3CC, CCCH2),
1.85–1.96 (m, 2H, CH2CCH3), 2.07 (s, 3H, CH3C=O),
2.13–2.35 (m, 4H, CH3Ar, CCCH2), 2.56 (d, 1H, CH2Ar, J
= 13.2 Hz), 3.30 (d, 1H, CH2Ar, J = 13.2 Hz), 6.93 (d, 2H,
2 × o-CHAr, J = 8.1 Hz), 7.04 (d, 2H, 2 × m-CHAr, J = 8.1
Hz); 13С NMR (CDCl3, 75.48 MHz,): δ = 18.9 (CH3,
CH3CC), 20.6 (CH3, CH3CCH2), 21.0 (CH3, CH3Ar), 25.8
(CH2, CCCH2), 30.0 (CH3, CH3C=O), 33.1 (CH2,
CH2CCH3), 41.0 (CH2, CH2Ar), 58.3 (C, CH2CCH2), 109.0
(C, OOCCH2), 111.3 (C, OOCC), 128.9 (CH, m-CHAr),
130.0 (CH, o-CHAr), 132.8 (C, CHCCH3), 136.3 (C,
CHArCCHAr), 210.4 (C, CH3C=O); Anal. Calcd. for
C17H22O4: C, 70.32; H, 7.64. Found: C, 70.35; H, 7.68.
1-((1R,2R,5S)-2-(4-Bromobenzyl)-1,5-dimethyl-6,7,8-
trioxabicyclo[3.2.1]oct-2-yl)ethanone (2e)
This compound was prepared by the reaction of β,
δ–triketone 1e with H2O2. White crystals, yield 15 %; m.p.:
109–110 °С; 1Н NMR (CDCl3, 300.13 MHz): δ =
1.35–1.63 (m, 7H, CH3CC, CH3CCH2, CH2), 1.74–2.00
(m, 2H, CH2CCH3), 2.18 (s, 3H, CH3C=O), 2.57–2.75 (m,
1H, CCCH2), 2.84 (d, 1H, CH2Ar, J = 13.2 Hz), 3.50 (d,
1H, CH2Ar, J = 13.2 Hz), 6.96 (d, 2H, 2 × o-CHAr, J = 8.1
Hz), 7.35 (d, 2H, 2 × m-CHAr, J = 8.1 Hz); 13С NMR
(CDCl3, 75.48 MHz,): δ = 18.3 (CH3, CH3CC), 20.8 (CH3,
CH3CCH2), 22.2 (CH2, CCCH2), 30.2 (CH3, CH3C=O),
30.9 (CH2, CH2CCH3), 35.3 (CH2, CH2Ar), 59.2 (C,
CH2CCH2), 109.2 (C, OOCCH2), 111.1 (C, OOCC), 120.6
(C, CBr), 131.4 (CH, m-CHAr), 132.0 (CH, o-CHAr), 136.8
(C, CHArCCHAr), 210.7 (C, CH3C=O); Anal. Calcd. for
C16H19BrO4: C, 54.10; H, 5.39; Br, 22.49. Found: C, 54.00;
H, 5.43; Br, 22.55.
1-((1R,2R,5S)-2-(4-Chlorobenzyl)-1,5-dimethyl-6,7,8-
trioxabicyclo[3.2.1]oct-2-yl)ethanone (2d)
This compound was prepared by the reaction of β,
δ–triketone 1d with H2O2. White crystals, yield 20 %; m.p.:
1
121–122 °С; Н NMR (CDCl3, 300.13 MHz): δ = 1.44 (s,
3H, CH3CC), 1.48–1.62 (m, 4H, CH3CCH2, CCCH2),
1.76–1.99 (m, 2H, CH2CCH3), 2.17 (s, 3H, CH3C=O),
2.59–2.73 (m, 1H, CCCH2), 2.85 (d, 1H, CH2Ar, J = 13.6
Hz), 3.51 (d, 1H, CH2Ar, J = 13.6 Hz), 7.01 (d, 2H, 2 × o-
CHAr, J = 8.4 Hz), 7.19 (d, 2H, 2 × m-CHAr, J = 8.4 Hz);
13С NMR (CDCl3, 75.48 MHz,): δ = 18.3 (CH3, CH3CC),
20.8 (CH3, CH3CCH2), 22.2 (CH2, CCCH2), 30.2 (CH3,
CH3C=O), 30.9 (CH2, CH2CCH3), 35.2 (CH2, CH2Ar),
59.3 (C, CH2CCH2), 109.2 (C, OOCCH2), 111.1 (C,
OOCC), 128.4 (CH, m-CHAr), 131.6 (CH, o-CHAr), 132.5
(C, CCl), 136.2 (C, CHArCCHAr), 210.7 (C, CH3C=O);
Anal. Calcd. for C16H19ClO4: C, 61.84; H, 6.16; Cl, 11.41.
Found: C, 61.90; H, 6.20; Cl, 11.48.
1-((1R,2S,5S)-2-(4-Bromobenzyl)-1,5-dimethyl-6,7,8-
trioxabicyclo[3.2.1]oct-2-yl)ethanone (3e)
This compound was prepared by the reaction of β,
δ–triketone 1e with H2O2. White crystals, yield 14 %; m.p.:
107–108 °С; 1Н NMR (CDCl3, 300.13 MHz): δ =
1.46–1.72 (m, 7H, CH3CCH2, CH3CC, CCCH2), 1.87–1.98
(m, 2H, CH2CCH3), 2.11 (s, 3H, CH3C=O), 2.13–2.28 (m,
1H, CCCH2), 2.52 (d, 1H, CH2Ar, J = 12.8 Hz), 3.33 (d,
1H, CH2Ar, J = 12.8 Hz), 6.93 (d, 2H, 2 × o-CHAr, J = 8.3
Hz), 7.35 (d, 2H, 2 × m-CHAr, J = 8.3 Hz); 13С NMR
(CDCl3, 75.48 MHz,): δ = 18.7 (CH3, CH3CC), 20.6 (CH3,
CH3CCH2), 25.6 (CH2, CCCH2), 30.1 (CH3, CH3C=O),
32.9 (CH2, CH2CCH3), 40.6 (CH2, CH2Ar), 58.1 (C,
CH2CCH2), 109.0 (C, OOCCH2), 111.0 (C, OOCC), 120.8
(C, CBr), 131.4 (CH, m-CHAr), 131.9 (CH, o-CHAr), 135.2
(C, CHArCCHAr), 210.0 (C, CH3C=O); Anal. Calcd. for
C16H19BrO4: C, 54.10; H, 5.39; Br, 22.49. Found: C, 54.19;
H, 5.47; Br, 22.55.
1-((1R,2S,5S)-2-(4-Chlorobenzyl)-1,5-dimethyl-6,7,8-
trioxabicyclo[3.2.1]oct-2-yl)ethanone (3d)
This compound was prepared by the reaction of β,
δ–triketone 1d with H2O2. White crystals, yield 10 %; m.p.:
105–106 °С; 1Н NMR (CDCl3, 300.13 MHz): δ =
1.48–1.73 (m, 7H, CH3CCH2, CH3CC, CH2), 1.87–2.01
(m, 2H, CH2CCH3), 2.08–2.29 (m, 4H, CH3C=O, CCCH2),
2.54 (d, 1H, CH2Ar, J = 12.9 Hz), 3.35 (d, 1H, CH2Ar, J =
12.9 Hz), 6.98 (d, 2H, 2 × o-CHAr, J = 8.3 Hz), 7.20 (d, 2H,
2 × m-CHAr, J = 8.3 Hz); 13С NMR (CDCl3, 75.48 MHz,):
δ = 18.7 (CH3, CH3CC), 20.6 (CH3, CH3CCH2), 25.6 (CH2,
CCCH2), 30.1 (CH3, CH3C=O), 32.9 (CH2, CH2CCH3),
General procedure for the synthesis of tetraoxanes
The synthesis of tetraoxanes 5a and 5b was performed
according to published procedure (Terent’ev et al. 2013).
An ethereal solution of H2O2 (3.0 mol of H2O2 per mole of
β-diketone 4a,b, M = 1.10 mol L−1) and PMA (14 mol%)

Med Chem Res
were successively added with stirring to a solution of
β-diketone 4a,b (0.30 g) in CH2Cl2 (5 mL). The hetero-
geneous reaction mixture was vigorously stirred at 20–25 °
C for 24 h in case of β-diketone 4a and 3 h in case of β-
diketone 4b. Then CH2Cl2 (30 mL) was added. The organic
layer was washed with water (5 mL), a 5 % aqueous solu-
tion of NaHCO3, and again with water (5 mL). The mixture
was dried over Na2SO4 and filtered. The solvent was
removed with a water-jet vacuum pump. Products 5a and
5b were isolated using silica gel column chromatography by
gradient elution with 10–40 vol.% ethyl acetate in petro-
leum ether.
Fig. 3 CV curve for 5 mmol L−1 of 2a in 0.1 М Bu4NClO4 in MeCN
at a potential scan rate of 0.1 V s−1
7-(1-Adamantyl)-1,4-dimethyl-2,3,5,6-tetraoxabicyclo
[2.2.1]heptane (5a)
This compound was prepared by the reaction of β–diketone
4a with H2O2. White crystals, yield 60 %; m.p.: 134–135 °C;
1Н NMR (CDCl3, 300.13 MHz): δ = 1.63–2.04 (m, 21H,
2 × CH3C, 6 × CH2CH, 3 × CHCH2), 2.38 (s, 1H, CHCO);
13С NMR (CDCl3, 75.48 MHz,): δ = 12.8 (CH3, CCH3),
28.5 (CH, CHCH2), 33.1 (C, CH2CCH2), 36.9 (CH2,
CHCH2), 40.8 (CH2, CHCH2), 67.0 (CH, CHCO), 110.8 (C,
OCO); Anal. Calcd. for C15H22O4: C, 67.64; H, 8.33.
Found: C, 67.60; H 8.40.
1-(1-Adamantyl)-4-methyl-2,3,5,6-tetraoxabicyclo[2.2.1]
Fig. 4 CV curve for 5 mmol L−1 of 3a in 0.1 М Bu4NClO4 in MeCN
heptane (5b)
at a potential scan rate of 0.1 V s−1
This compound was prepared by the reaction of β–diketone
4b with H2O2. White crystals, yield 57 %; m.p.: 113–115 °С;
1H NMR (CDCl3, 300.13 MHz): δ = 1.55–1.89 (m, 15 H,
3 × CHCH2, CCH3, 3 × CHCH2), 1.95–2.05 (m, 3 H, 3 ×
CHCH2), 2.65 (s, 2 H, CH2CO); 13С NMR (CDCl3, 75.48
MHz,): δ = 10.8 (CH3, CCH3), 28.0 (CH, CHCH2), 35.5 (C,
CH2CCH2), 36.5 (CH2, CHCH2), 37.9 (CH2, CHCH2), 46.4
(CH2CO), 109.3 (C, OC(CH3)O), 115.9 (C, OC(C)O); Anal.
Calcd. for C14H20O4: C, 66.65; H, 7.99. Found: C, 66.60;
H, 8.02.
Electroanalytical instrumentation, experiment
techniques and reagents
Fig. 5 CV curve for 5 mmol L−1 of 2e in 0.1 М Bu4NClO4 in MeCN
Cyclic voltammetry (CV) was performed on 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-neck glass
conic electrochemical cell with a water jacket for thermo-
statting. CV curves for 2a, 3a, 2e, 3e (Figs. 3–6) were
recorded 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 satu-
at a potential scan rate of 0.1 V s−1
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 solution in
MeCN). The tested solutions were thermostatted at 25
0.5 °С. Deaeration of solutions was performed by passing
argon. To prevent contact between the solution surface and
rated calomel electrode (SCE, Eox 1/2 = 409 mV) was used
Fc

Med Chem Res
regression analysis of the concentration–log response
curves using the software GraphPad InStat 3.0
Results and discussion
1,2,4-trioxanes (ozonides) and 1,2,4,5-tetraoxanes are cur-
rently considered as promising classes of organic peroxides
for the development of antiparasitic and anticancer drugs.
For example, very efficient antimalarial drug with trade
name Synriam manufactured since 2012 is characterized by
the presence of ozonide cycle in the structure of the
molecule.
Fig. 6 CV curve for 5 mmol L−1 of 3e in 0.1 М Bu4NClO4 in MeCN
at a potential scan rate of 0.1 V s−1
In this work, we synthesized peroxides 2a–e, 3a–e, 5a
and b (Table 1) which were isolated in individual form by
column chromatography. The structures of these peroxides
were confirmed by NMR data and elemental analysis (see
Material and methods). Stereoisomeric ozonides 2a–e, 3a–e
were chosen with two main purposes, firstly, to test their
cytotoxicity against cancer cell lines and, secondly, to
understand some aspects of their cytostatic mechanism of
action. Namely, we tried to find out the correlation between
cytotoxic properties and oxidative potential of the synthe-
sized molecules.
The cytotoxicity of ozonide stereoisomeric peroxides
2a–e, 3a–e and tetraoxanes 5a and b (Table 2) was exam-
ined on human prostate cancer cell lines DU145 and PC3
and on immortalized normal prostate epithelial cell line PZ-
HPV-7.
the 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 utilized and the depolarizer
concentration was 5 mmol ∙ L–1. The working electrode was
polished and the solution was agitated vigorously with
argon before recording each CV curve.
Determination of viability of PC3, DU145 and PZ-HPV-7
cells using MTT assay
Prostate cancer cell lines PC3 (adenocarcinoma), DU145
(carcinoma) and immortalized normal prostate epithelial
cell line PZ-HPV-7 were cultured in minimum essential
medium (RPMI 1640 medium) supplemented with 10 %
fetal bovine serum (FBS) and 1 % penicillin/streptomycin,
and were grown at 37 °C with 5 % CO2. Peroxides 2a–e,
3a–e, 5a and b were dissolved and stored in DMSO at 20
mM concentration. Briefly, DU145, PC3 and PZ-HPV-7
cells were seeded at 5 × 103 cells/well in 100 µl of medium
in standard 96-well plates. The plates were incubated 24 h at
37 °C at an atmosphere of 5 % CO2. After 24 h solutions of
tested compounds in DMSO (20 mM) were serially diluted
by RPMI 1640 medium from 60 µM to 0.02 µM were added
in 100 µl RPMI in quadruplicate. After 72 h of incubation,
20 μL of 5 mg mL−1 MTT was added to each well according
to the manufacturer’s instructions. After 2 h of incubation at
37 °C, the medium was removed, and 200 µl DMSO was
added to each well to dissolve formazan crystals. The
absorbance at 540 nm of each well was then monitored
using an EnSpire multimode plate reader. Absorbance of
each well was expressed as percentage of the control (wells
with untreated cells), and the 50 % inhibitory concentration
(IC50) was determined. IC50 values are defined as the con-
centration of drug causing 50 % growth inhibition. Results
represent the average values of at least three experiments.
All data were expressed as the mean values standard
deviation. The IC50 values were calculated by linear
Table 2 Cytotoxicity of ozonides 2a–e, 3a–e and tetraoxanes 5a, b
against cancer DU145, PC3 and normal PZ-HPV-7 prostate cell linesa
Peroxide DU145 cell
line
PC3 cell line
PZ-HPV-
7 cell line
IC50
[μM]
(SD)
SI
IC50 [μM] SI IC50 [μM]
(SD) (SD)
Ozonide
2a
3a
2b
3b
2c
3c
2d
3d
2e
3e
ND
ND
7.8 (0.4) 0.8 6.5 (0.7)
3.7 (0.3)
1.7 (0.4)
ND
2.5 12.4 (0.9) 0.7 9.1 (1,2)
6.5 6.8 (0.9) 1.6 11.1 (2.9)
ND
8.8 (1.8) 0.6 5.5 (0.9)
4.1 5.7 (0.4) 0.9 4.9 (0.2)
5.3 12.5 (0.8) 0.6 7.4 (0.5)
1.2 (0.4)
1.4 (0.3)
0.6 (0.1) 23.7 15.2 (0.8) 0.9 14.2 (1.1)
0.7 (0.3) 5.7 4.0 (0.7) 1.0 4.0 (0.4)
0.4 (0.1) 42.0 13.5 (0.9) 1.2 16.8 (0.5)
0.3 (0.1)
ND
8.7 2.0 (0.4) 1.3 2.6 (0.8)
Tetraoxane 5a
ND
ND
2.0 (0.1) 0.8 1.7 (0.1)
1.2 (0.1) 1.1 1.3 (0.1)
5b
ND
ND not done, SD standard deviation, SI selectivity index (cytotoxicity
IC50 for normal prostate cells/ IC50 for cancer cells)
a
Incubation time—72 h

Med Chem Res
Table 3 Cytotoxicity of ozonides 2c, 3e, tetraoxanes 5a, b and
reference compounds against cancer PC3 and normal PZ-HPV-7
prostate cell linesa
compared with those of natural peroxide artemisinin, arte-
sunate and widely used anticancer agents doxorubicin,
camptothecin, cisplatin, and etoposide (Table 3). As indi-
cated above, lower sensitivity of PC3 cells to peroxides (in
comparison to the cells DU145) after a long time treatment
could be due to their higher antioxidant capacity of the
former cells line. In the experiment presented in Table 3, the
time of cell incubation with the endoperoxides was shor-
tened to 24 h.
Peroxide
PC3 cell line
PZ-HPV-7 cell line
IC50 [μM] (SD)
SI
IC50 [μM] (SD)
2c
5.2 (0.8)
3.0 (0.7)
2.6 (0.6)
1.2 (0.2)
2.4 (0.6)
0.6 (0.2)
21.0 (4.6)
17.(7.6)
>30.0
3.0
1.6
2.1
3.3
0.5
1.5
0.8
0.3
ND
ND
15.5 (2.0)
4.9 (0.3)
5.6 (0.8)
3.9 (0.5)
1.2 (0.3)
0.9 (0.6)
17.3 (1.0)
6.1 (1.2)
ND
3e
5a
5b
Comparing the data in Tables 2 (72 h incubation time)
and 3 (24 h incubation time) we can see that SI increased for
2c from 0.9 to 3.0, for 3e from 1.3 to 1.6, for 5a from 0.8 to
2.1, and for 5b from 1.1 to 3.3. As was shown in Table 2, all
tested ozonides demonstrated high activity and selectivity
on DU145 cells. As to another cancer cell line PC3
(Table 3), ozonides 2b, 2c, 3d, 3e and tetraoxanes 5a,b
were found to be more efficient. The most active ozonide 3e
and tetraoxane 5a possessed similar activity in comparison
with doxorubicin and exceeded cisplatin, etoposide, arte-
misinin, and artesunate in their efficiency. Tetraoxane
5b was more active than doxorubicin, cisplatin,
etoposide, artemisinin, and artesunate. Activity of ozonide
stereoisomers 2 and 3 in each pair differs from each other.
Generally, biological activity of peroxides depends on
their oxidative properties. It is considered that the oxidation
potential of a peroxide directly correlates with its activity
(Donkers and Workentin 1998; Najjar et al. 2003,
2004; Workentin et al. 1995; Wu and Wu 2000). In
this work potential of reduction was measured for two
pairs of diastereoisomers 2a, 3a and 2e, 3e in CH3CN
with supporting electrolyte 0.1 M Bu4NClO4: 2a
(−1.732 V), 3a (−1.891 V), 2e (−1.716 V), and 3e
(−1.830 V). Cyclic voltammetry curves are presented in
Figs. 3–6.
We found that ozonides 2a, e possessed higher oxidative
capacity than ozonides 3a,e. In the case of stereoisomers,
cytotoxicity of 2a was higher than that of 3a, but cyto-
toxicity of 2e was lower than that of 3e. Thus, the data
obtained from cytotoxicity of peroxides against cancer cell
lines and cyclic voltammetry do not show a direct correla-
tion (oxidative property / antiproliferative activity). We
assume that the mechanism of action of peroxides is very
complicated and its elucidation requires further more
detailed study. We would like to mention that this study is
the first one describing antiproliferative activity of synthetic
bridged ozonides and bridged tetraoxanes. At present, about
sixty synthetic peroxides belonging to four different classes
are being tested on three cancer cell lines. The future
investigation will allow us to make more general conclu-
sions concerning structure/function relationships of the
synthetic peroxides not only as cytotoxic agents but also at
the level of ROS generation in the cells, DNA fragmenta-
tion and caspase cascade activation.
Doxorubicin
Camptothecin
Cisplatin
Etoposide
Artemisinin
Artesunate
>30.0
ND
ND not done, SD standard deviation, SI selectivity index
a
Incubation time–24 h
Ozonides 2b, 3a, 2c–e and 3c–e showed high cytotoxi-
city when tested on DU145 cells with IC50 varying from 0.3
to 3.7 µМ and demonstrated high selectivity comparing to
normal prostate cells. All tested peroxides also inhibited
PC3 cells, but only peroxides 2b, 2e, 3e, and 5b possessed a
moderate selectivity (Selectivity index > 1, Table 2). It is
important to note that changing time of PC3 cells incubation
with peroxides 2c, 3e, 5a and b from 72 to 24 h increased
selectivity index (SI > 2) (Table 3). Different sensitivity of
two cancer cell lines to tested molecules deserves further
comment. One has to take into account different origin that
may modify biochemical and physiological properties of
DU145 and PC3 cells. Indeed DU145 cells derived from a
brain metastasis while PC3 cells have their origin metastatic
brain. These two cells lines may also differ in their pro/
antioxidant status. Our data (not shown) indicated that
though PC3 and DU145 cell lines didn’t differ in super-
oxidase dismutase level, expression of another antioxidant
enzyme, catalase, was much higher in PC3 cells. We cannot
exclude that long treatment (72 h) of the cells PC3 with the
peroxides would diminish efficient concentrations of the
molecules.
It is important to mention that large variations of IC50
values, depending on the type of cancer cells treated with
endoperoxides have been demonstrated in a study per-
formed on 68 cell lines derived from solid tumors (van
Huijsduijnen et al. 2013). Potential synthetic anticancer
peroxides, artemisinin and its analogs differed considerably
in their antiproliferative efficiency when tested not only on
cells of distinct cancer types but as well on cancer cell lines
of the same tissue.
Cytotoxic effects of ozonides 2c, 3e and tetraoxanes 5a,
5b on PC3 and PZ-HPV-7 after 24 h treatment were

Med Chem Res
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Conclusion
Synthetic ozonides and tetraoxanes were shown to have
high cytotoxicity against androgen-independent prostate
cancer cell lines DU145 and PC3. The antiproliferative
action of the tested molecules was higher than that of
anticancer agents doxorubicin, cisplatin and etoposide, as
well as of artemisinin and artesunate. Activity of ozonide
stereoisomers differed from each other.
Gahlot M (2012) Indian drug approvals come under fire. Chem Ind-
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Acknowledgments The work on the synthesis and analysis of the
organic peroxides was supported by the Russian Science Foundation
(Grant № 14-23-00150) and biological experiments were funded by
the Ligue Contre le Cancer (Comités 44 and 85).
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Compliance with ethical standards
Conflict of interest The authors declare that they have no com-
Hao HD, Wittlin S, Wu YK (2013) Potent antimalarial 1,2,4-trioxanes
through perhydrolysis of epoxides. Chem-Eur J 19:7605–7619
Ingram K, Yaremenko IA, Krylov IB, Hofer L, Terent’ev AO, Keiser J
(2012) Identification of antischistosomal leads by evaluating
bridged 1,2,4,5-tetraoxanes, alphaperoxides, and tricyclic mono-
peroxides. J Med Chem 55:8700–8711
peting interests.
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