Synthesis, thermal stability, antimalarial activity of symmetrically and asymmetrically substituted tetraoxanes

Article abstract of DOI:10.1016/j.bmcl.2007.12.069

Symmetrically and asymmetrically substituted 1,2,4,5-tetraoxanes were synthesized by the oxidative system H₂O₂/TFE in presence of MeReO₃ as a catalyst. All of the synthesized compounds were characterized spectroscopically,

Full text of DOI:10.1016/j.bmcl.2007.12.069

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 1446–1449
Synthesis, thermal stability, antimalarial activity
of symmetrically and asymmetrically substituted tetraoxanes
Himanshu Atheaya,a Shabana I. Khan,b Ritu Mamgaina and Diwan S. Rawata,*
aDepartment of Chemistry, University of Delhi, Delhi 110007, India
bNational Center for Natural Products Research, School of Pharmacy, University of Mississippi, MS 38677, USA
Received 6 October 2007; revised 8 December 2007; accepted 28 December 2007
Available online 1 January 2008
Abstract—Symmetrically and asymmetrically substituted 1,2,4,5-tetraoxanes were synthesized by the oxidative system H2O2/TFE in
presence of MeReO3 as a catalyst. All of the synthesized compounds were characterized spectroscopically, and evaluated for cyto-
toxicity, and antimalarial activity. Several of these tetraoxanes exhibited in vitro antimalarial activity without showing any cytotox-
icity. Thermal stability of these compounds was studied by differential scanning calorimetry.
Ó 2007 Elsevier Ltd. All rights reserved.
Since last two decades endoperoxides have attracted
considerable amount of attention of chemist and biolo-
gist due to their potent activity against malarial parasite,
which affects more than 500 million people, causing
deaths of 1–3 million people every year.1–7 Artemisinin
and its semisynthetic derivatives represent the endoper-
oxide class of compounds, which shows antimalarial
activity against chloroquine-resistant strain of Plasmo-
dium falciparum.8 The impact of drug resistance is acute
for malaria chemotherapy because of the availability of
limited number of clinically useful antimalarial drugs.
Thus synthesis of new chemical entities for the antima-
larial therapy remains a challenging task for the scien-
tists involved in the malaria research.6 Structure–
activity relationship studies conducted on artemisinin
and its semisynthetic derivatives revealed that the perox-
ide linkage is the most crucial pharmacophore in these
molecules.9
The most common method of synthesis of symmetrical
tetraoxane is acid catalyzed cyclocondensation of hydro-
gen peroxide with ketones or aldehydes,13–17 ozonolysis
of olefins,18 enol ethers,19,20 O-ether oxime,21 and cyclo-
condensation of bis(trimethylsilyl) peroxide with car-
bonyl compounds catalyzed by trimethylsilyl
trifluoromethanesulfonate (TMSOTf).22,23 A number
of other methods have also been reported for the synthe-
sis of these compounds.24 The unsymmetrical tetraox-
anes have been synthesized by cyclocondensation of
ketones and aldehydes with steroidal gem-bis-hydroper-
oxides (H2SO4 catalyst),23 aliphatic and alicyclic
gem-hydroperoxides (MeReO3–HBF4, catalyst),25 and
gem-bis(trimethylsilyldioxy)alkanes (TMSOTf, catalyst).26
As part of our ongoing efforts toward the synthesis of
biologically active compounds,27,28 and due to the fact
that a very limited number of tetraoxanes are known,
this study was aimed to synthesize, characterize, and
evaluate the antimalarial activity of symmetrically and
asymmetrically substituted tetraoxanes.
This discovery was the beginning of a significant effort
to identify synthetically accessible antimalarial perox-
ides. Dispiro-tetraoxane is one of such class of com-
pounds which was found to be equally potent as
artemisinin.10 However, the structural diversity of this
important class of compounds is not available.11,12
The tetraoxanes reported in this paper have been synthe-
sized by a previously published method.25 Symmetrical
tetraoxanes were synthesized in moderate to good yield.
Reaction of the aldehydes or ketones, H2O2 (30% aque-
ous solution), and HBF4 (54% freshly prepared ethereal
solution), in presence of 0.1 mol% MTO in TFE led to
the formation of symmetrical tetraoxanes at room tem-
perature. p-Substituted aromatic aldehydes led to the
formation of symmetrical tetraoxanes in good yield
(Scheme 1), while o-substituted benzaldehyde led to a
Keywords: Tetraoxane; Antimalarial activity; Cytotoxicity; Differential
scanning calorimetry.
*
Corresponding author. Tel.: +91 11 27667465; fax: +91 11
27667501; e-mail: dsrawat@chemistry.du.ac.in
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.12.069

H. Atheaya et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1446–1449
1447
CHO
antimalarial activity due to their ability to form diradi-
cals.38 In order to study the thermal stability of these tet-
raoxanes, we recorded DSC of these compounds on a
neat material. DSC traces of the unsymmetrical tetraox-
anes clearly show an exothermic peak followed by neg-
ative endotherm corresponding to the melting points
of the respective tetraoxanes, while in case of symmetri-
cal tetraoxanes, the radical formation temperature was
found to be very high (Table 1). As for example, com-
pound 2b shows an endothermic peak at 157 °C attrib-
uted to the melting of the tetraoxane. For same
compound an exothermic peak was observed corre-
sponding to the ring-opening polymerization39 with on-
set at 190.57 °C and maximum at 210.35 °C (Fig. 1). The
amount of heat released during this process is about
À2135.4 J/g. The DSC traces clearly demonstrated that
these compounds are thermally stable.
O
O
O
O
H2O2, MTO, TFE
HBF4, RT
R
R
R
1
2a, R = p-CH3; 2b, R = m-CH3
2c, R = o-CH3; 2d, R = p-Br
2e, R = m-Br; 2f, R = o-Br
2g, R = p-Cl; 2h, R = m-Cl
2i, R = o-Cl; 2j, R = p-F
2k, R = p-CF3; 2l, R = m-CF3
Scheme 1.
poor yield of the product. However, reaction of benzal-
dehyde substituted by OH, OR, or NH2 functionality
did not give the desired tetraoxanes.
Assay for in vitro antimalarial activity: The antimalarial
activity was determined by measuring plasmodial LDH
activity as described earlier.40 A suspension of red blood
cells infected with D6 or W2 strains of P. falciparum
(200 lL, with 2% parasitemia and 2% hematocrit in
RPMI 1640 medium supplemented with 10% human ser-
um and 60 lg/mL Amikacin) was added to the wells of a
96-well plate containing 10 lL of serially diluted test
samples. The plate was flushed with a gas mixture of
90% N2, 5% O2, and 5% CO2 and incubated at 37 °C
for 72 h in a modular incubation chamber (Billups-
Rothenberg, CA). Parasitic LDH activity was deter-
mined by using MalstatTM reagent (Flow Inc., Portland,
OR) according to the procedure of Makler and Hin-
richs.41 Briefly, 20 lL of the incubation mixture was
mixed with 100 lL of the MalstatTM reagent and incu-
bated at room temperature for 30 min. Twenty microli-
ters of a 1:1 mixture of NBT/PES (Sigma, St. Louis,
MO) was then added and the plate was further incu-
bated in the dark for 1 h. The reaction was then stopped
by the addition of 100 lL of a 5% acetic acid solution.
The plate was read at 650 nm. Artemisinin and chloro-
quine were included in each assay as the drug controls.
Under identical reaction conditions, unsymmetrical tet-
raoxanes were prepared (Scheme 2). After completion
of the reaction chloroform was added to the reaction
mixture and the organic layer was washed with NaH-
SO3, followed by saturated solution of NaHCO3. Or-
ganic layer was dried over Na2SO4 and excess of
solvent was removed under reduced pressure. The puri-
fication of tetraoxanes has been very difficult by column
chromatography due to the formation of uncharacteriz-
able side products. The crude product was washed with
cold hexanes followed by mixture of cold methanol and
hexanes (1:1). Pure tetraoxanes were obtained by wash-
ing the reaction mixture two to three times.
This synthetic methodology is being extended to the syn-
thesis of functionalized tetraoxanes, which in turn can
be transformed to tetraoxane-aminoquinoloine conju-
gates. This work is in progress and will be published
in due course of time. Compounds 2a, 2g, 2j, and 2k
have been prepared earlier.29–31 All of the compounds
reported in above schemes have been characterized
spectroscopically.32
Thermal stability: Thermal stability of these tetraoxanes
was studied by differential scanning calorimetry (DSC).
DSC has been used to determine a number of character-
istic properties of a sample such as fusion, crystalliza-
tion, and glass transition temperatures (Tg). It can also
be used to study oxidation, radical formation processes,
and many other chemical reactions.33–37 Recently, Sola-
ja et al. have demonstrated that tetraoxanes exert their
Table 1. DSC temperature of tetraoxanes
Compound
DSC exothermic (°C)
On set temp. (°C)
Peak max. (°C)
2a
2b
2c
2d
2e
2g
2h
2i
237.87
190.57
163.76
221.18
160.60
174.34
118.93
233.69
57.02
240.97
210.35
170.23
224.46
165.52
186.67
124.95
236.98
64.90
CHO
O
O
O
O
O
H2O2, MTO, TFE
HBF4, RT
n
+
4a
4b
4d
4e
4f
n
65.02
83.21
67.94
88.30
R
R
3
1
4a, R = H, n = 1; 4b, R = p-Br, n = 1
4c, R = p-Cl, n = 1; 4d, R = H, n = 2
4e, R = p-Cl, n = 2; 4f, R = m-Cl, n = 2
4g, R = o-Cl, n = 2; 4h, R = p-Br, n = 2
4i, R = H, n = 3; 4j, R = p-Br, n = 3
4k, R = p-Cl, n = 3
97.20
90.75
100.19
97.67
4g
4h
4i
108.40
117.12
57.02
117.30
120.87
64.90
4j
52.75
58.09
Scheme 2.

1448
H. Atheaya et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1446–1449
Table 2. Antimalarial activity and cytotoxicity of substituted
tetraoxanes
Compound
P. falciparum
P. falciparum
Cytotoxicity
(Vero cells)
(D6 Clone)
(W2 Clone)
IC50
(lM)
SI
IC50
(lM)
SI
2a
2b
2c
2d
2e
2f
1.47
0.96
2.46
1.39
NA
>11.9
>18.2
>7.1
1.18
0.77
1.29
0.67
NA
>14.9
>22.7
>13.6
>13.6
NC
NC
NC
NC
>8.5
NA
NA
2g
2h
2i
4.47
1.37
NA
>3.4
>11.1
3.19
0.70
NA
>4.8
>21.6
NC
NC
2j
1.64
NA
>10.4
>2.6
1.14
NA
>14.9
>2.8
NC
NC
2k
2l
NA
NA
Figure 1. DSC curve of compound 2b.
4a
4b
4c
4d
4e
4f
8.09
NA
7.65
NA
IC50 values were computed from the dose–response
curves. To determine the selectivity index of antimalarial
activity of compounds, their in vitro cytotoxicity to
mammalian cells was also determined. The assay was
performed in 96-well tissue culture-treated plates as de-
scribed earlier.42 Vero cells (monkey kidney fibroblasts)
were seeded to the wells of 96-well plate at a density of
25,000 cells/well and incubated for 24 h. Samples at dif-
ferent concentrations were added and plates were again
incubated for 48 h. The number of viable cells was deter-
mined by Neutral Red assay. IC50 values were obtained
from dose–response curves. Doxorubicin was used as a
positive control.
16.72
2.20
2.66
2.14
14.04
3.81
9.99
NA
>1.0
>9.2
>6.6
>8.2
>1.3
>4.0
>1.9
16.72
1.82
2.59
2.47
9.24
5.39
7.59
NA
>1.0
>11.1
> 6.8
>7.1
>1.9
>2.8
>2.5
NC
NC
NC
NC
NC
NC
NC
4g
4h
4i
4j
4k
CQ
ART
16.72
0.05
0.035
>1.0
>298
>476
16.72
0.41
0.015
>1.0
>42
NC
NC
NC
>1400
NC, no cytotoxicity up to 16.72 lM.
NA, no activity up to 16.72 lM.
SI, selectivity index (IC50 for cytotoxicity/IC50 for antimalarial
activity).
In vitro antimalarial activity of these tetraoxanes was
determined against chloroquine sensitive (D6) and chlo-
roquine-resistant (W2) strains of P. falciparum. Among
the series, compounds 2a, 2b, 2c, 2d, 2h, 2j, 4d, 4e, and
4f exhibited the most potent antimalarial activity with
IC50 values ranging from 0.95 to 2.66 lM for D6 and
from 0.67 to 2.59 lM for W2 strain as shown in Table
2. Other compounds also showed moderate to mild anti-
malarial activities (IC50 3.81–16.72 lM). None of these
tetraoxanes showed any cytotoxicity (Table 2) to mam-
malian kidney fibroblast (Vero cells). A higher selectiv-
ity index (SI > 10) for antimalarial activity was
observed for compounds 2a, 2b, 2c, 2d, 2h, 2j, and 4d
in either D6 or W2 strain. It is interesting to note that
most of the active compounds like 2a, 2b, 2c, 2d, 2h,
2j, and 4d have shown equal or better activity in chloro-
quine-resistant (W2) strain in comparison to chloro-
quine sensitive (D6) strain. The activity profile of these
compounds clearly indicates that the tetraoxanes with
substituent present at meta or para position of the ben-
zene ring show better antimalarial activity (entry 2b, 2h,
4d, 4e, and 4f) than the tetraoxanes having substituent at
ortho position (entry 2i and 4g), which is in good agree-
ment with the observation made by Iskra et al.43 Fur-
thermore, smaller or larger ring size than cyclohexane
in the tetraoxane has negative effect on the antimalarial
activity of these tetraoxanes (entry 4a, 4b, 4i, 4j, and 4k).
Interestingly, symmetrical tetraoxanes having methyl
substituent at ortho, para, and meta position exhibited
very good antimalarial activity. Among these, com-
pound with methyl group at meta position of the tetra-
oxane (entry 2b) was the most potent against both
strains of P. falciparum (D6 and W2). However, when
methyl group of compounds 2a and 2b was substituted
by CF3 group, the resulting tetraoxane (entry 2k and
2l) lost antimalarial activity.
Caution: We have not encountered any difficulties in
working with these compounds, routine precautions
such as shields, fume hoods, and avoidance of transition
metal salts should be observed whenever possible, as or-
ganic peroxides are explosive in nature.
Acknowledgments
D.S.R. thanks CSIR, New Delhi, for financial support
and H.A. is thankful to CSIR for the award of junior
and senior research fellowships. USDA Agricultural Re-
search Service Specific Cooperative Agreement No. 58-
6408-2-0009 is also acknowledged for partial support of
this work.
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solution of 2 mmol of cycloalkanone, 4 mmol of H2O2,
and 0.002 mmol of MTO in 4 mL of TFE was stirred for
2 h at room temperature. Into the solution, 4 mmol of
desired aldehyde was added, followed by the addition of
2 mmol of a 54% ethereal solution of HBF4. The reaction
mixture was left under stirring for an additional hour.
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(CH), 108.92 (Cquart), 127.02 (CH) 128.79 (CH), 129.88
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