Synthesis, antimalarial activity and cytotoxicity of substituted 3,6-diphenyl-[1,2,4,5]tetraoxanes

Article abstract of DOI:10.1016/j.bmc.2009.06.020

Substituted tetraoxanes with different substitution pattern on the aromatic ring were synthesized in order to explore the influence of different substituents in the antimalarial activity. Antimalarial activity of these compounds improves by the introducti

Full text of DOI:10.1016/j.bmc.2009.06.020

Bioorganic & Medicinal Chemistry 17 (2009) 5632–5638
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, antimalarial activity and cytotoxicity of substituted
3,6-diphenyl-[1,2,4,5]tetraoxanes
Nitin Kumar a, Shabana I. Khan b, Beena a, G. Rajalakshmi c, P. Kumaradhas c, Diwan S. Rawat a,
*
a Department of Chemistry, University of Delhi, Delhi 110 007, India
b National Centre for Natural Products Research, University of Mississippi, MS-38677, USA
c Department of Physics, Periyar University, Salem 636 011, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Substituted tetraoxanes with different substitution pattern on the aromatic ring were synthesized in
order to explore the influence of different substituents in the antimalarial activity. Antimalarial activity
of these compounds improves by the introduction of ethyl, iso-propyl or n-propyl groups in the aromatic
ring but substitution with n-butyl or t-butyl leads decrease in antimalarial activity. Some of these com-
pounds exhibit promising antimalarial activity. None of the compounds shows any toxicity against vero
cells and three compounds (2a–2c) were tested against panel of six cell lines and none of these com-
pounds showed any toxicity. X-ray crystal structure of compound 2w showed that tetraoxane ring is
in the chair conformation with both the phenyl rings in the equatorial position. In addition, FeCl3 med-
iated O–O bond scission of tetraoxanes (2a–2c) was also examined.
Received 6 May 2009
Revised 4 June 2009
Accepted 11 June 2009
Available online 16 June 2009
Keywords:
Plasmodium falciparum
Artemisinin
Tetraoxane
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
compound is not available.11 Various synthetic methods have been
reported for the preparation of 1,2,4,5-tetraoxanes. The most com-
Malaria remains one of the most devasting tropical parasitic
disease after tuberculosis, causes infection to 100–300 million peo-
ple.1 Every year 1–3 million people die world wide and mortality
has increased in recent years because many Plasmodium falciparum
strains have developed resistance against most of the drugs,
including chloroquine (CQ),2 which has been drug of choice for
its treatment. The incidence of chloroquine and multidrug resistant
Plasmodium falciparum malaria has led the significant efforts to
search a novel molecule for its treatment.3
Artemisinin and other endoperoxide based antimalarials have
been most effective in the treatment of CQ-resistant P. falciparum.
However limited availability, high cost and poor bioavailability
have been the major drawbacks of artemisinin.4 Other semi-syn-
thetic derivatives of artemisinin such as artesunate and artemether
also shows poor pharmacokinetic properties.5 The endoperoxide
bridge present in artemisinin and its semi-synthetic or synthetic
derivatives is considered to be essential for the antimalarial activ-
ity.1b,6 Tetraoxanes (1,2,4,5-tetraoxacyclohexane) moiety has
become an interesting pharmacophore since ever antimalarial
activity of 1,10-dimethyl-7,8,15,16-tetraoxa-dispiro[5.2.5.2]hexa-
decane7 was reported to be identical to that of artimisinin,8 its
semi-synthetic9 or synthetic derivatives.10 In spite of huge medic-
inal potential structural diversity of this important class of
mon methods have been the reaction of ketones with acidic hydro-
gen peroxide12 or dimerization of carbonyl oxides obtained from
tetrasubstituted alkenes, enol ethers or oximes,13 cyclocondensa-
tion of bis(trimethylsilyl)peroxide with carbonyl compounds cata-
lyzed by trimethylsilyl trifluoromethanesulfonate (TMSOTf),14 and
cyclocondensation of carbonyl compounds with gem-hydroperox-
ides in presence of MeReO3–HBF4.15 Most of these methods are
highly dependent on several factors, such as the structure of
carbonyl compounds, temperature, concentration, pH, mode of
addition, solvent and the equilibrium between ketone and the pre-
cursors of cyclic peroxides,16 which lead to variable yields of the
tetraoxanes. Very recently, Dussault et al.17 have reported an effi-
cient synthesis of substituted tetraoxanes. Most of the tetraoxanes
reported in the literature have been prepared using cyclic ketones
as a starting material, hence there is a limited opportunity for fur-
ther functionalization. So there is always a need for the synthesis of
structurally diverse tetraoxanes, so that structure–activity rela-
tionship study can be carried out. Structural diversity in this class
of compounds can be generated if aromatic aldehydes are used as a
starting material rather than cyclic ketones. Careful literature sur-
vey revealed that limited number of tetraoxanes having aromatic
ring as a part of the active pharmacophore have been evaluated
for their antimalarial activity. As part of our ongoing efforts to-
wards the synthesis of tetraoxane based antimalarials,18 recently
we have reported iodine catalyzed synthesis of symmetrical and
asymmetrical tetraoxanes.18c This paper deals with the synthesis,
* Corresponding author. Tel.: +91 11 27667465; fax: +91 11 27667501.
E-mail address: dsrawat@chemistry.du.ac.in (D.S. Rawat).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.06.020

N. Kumar et al. / Bioorg. Med. Chem. 17 (2009) 5632–5638
5633
Table 1
antimalarial activity, cytotoxicity of symmetrically and asymmetri-
cally substituted tetraoxane. X-ray structure of one of the asym-
metrical tetraoxane was also determined.
Symmetrical and asymmetrical tetraoxanes
Entry
R
R0
Mp (°C)
Yield (%)
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
2m
2n
2o
2p
2q
2r
p-Et
p-Et
190–192
155
156–157
142–145
222–224
205
180–183
165
205
193
197
206
215–216
188–190
206
208
112
148
195
210
140
206
120
112
120
44
38
41
40
53
41
39
37
25
32
38
28
26
46
25
38
33
37
31
43
30
25
29
22
20
24
p-n-Pr
p-i-Pr
p-n-But
p-t-But
p-Et
p-Et
p-Et
p-Et
p-t-But
p-t-But
p-n-Pr
p-Me
p-Me
p-Me
p-Me
o-Me
p-Me
p-Me
p-Me
m- Me
p-Me
m-Me
p-t-But
p-n-Pr
p-i-Pr
p-n-Pr
p-i-Pr
p-n-But
p-t-But
p-Me
m-Me
p-i-Pr
p-t-But
p-n-Pr
p-i-Pr
p-i-Pr
p-i-Pr
p-t-But
o-Me
2. Results and discussion
Due to the possibility of selective incorporation of various sub-
stituents in the phenyl ring, we decided to use substituted benzal-
dehydes as a starting material for the preparation of tetraoxanes.
Our preliminary work in this direction was very encouraging, as to-
lyl based tetraoxanes exhibit good antimalarial activity.18a In order
to study the role of various alkyl substituents in the antimalarial
activity of resulting tetraoxanes, we incorporated ethyl, n-propyl,
iso-propyl, n-butyl, or tert-butyl groups in the tetraoxane scaffold
and studied their antimalarial activity. Use of substituted benzal-
dehydes gave a handle to generate wide range of tetraoxanes.
Bis-hydroperoxides have been used as an intermediate for the
preparation of tetraoxanes and it has been prepared via various
methods19 and iodine-catalyzed synthesis has been the most re-
cent method.20 Recently, we have developed a novel method for
the synthesis of substituted tetraoxanes using iodine as a cata-
lyst.18c This method offers a great opportunity to generate a wide
range of tetraoxanes with structural diversity. Bis-hydroperoxides
were prepared as described by Iskara et al.,20 and used as such for
the preparation of tetraoxanes. In a typical reaction conditions,
substituted benzaldehyde (1.0 equiv) was added to a stirred solu-
tion of H2O2 (6 equiv) and I2 (0.1 equiv) in 10 mL of acetonitrile.
Bis-hydroperoxide formation was confirmed by TLC and 1H NMR.
After this, same or different substituted benzaldehyde (1.0 equiv)
was added followed by the addition of 1.0 mL of HBF4ꢀEt2O
(Scheme 1).
After the completion of reaction as evident by TLC, excess of sol-
vent from the reaction mixture was removed under vacuum. The
crude product was quenched with NaHCO3, and compound was ex-
tracted with CHCl3 and washed with cold methanol. Solid thus ob-
tained was purified over SiO2 column. Next we studied the role of
varying amount of iodine in the outcome of the reaction. It was ob-
served that best yield of tetraoxanes was obtained when iodine
was used in 0.1 equiv. Increasing or decreasing the molar ratio of
iodine has negative effect on the yield of the product. This syn-
thetic protocol is of particular interest, as the functional groups
can be further manipulated chemically, if required. One such tetra-
oxane 2s has been synthesized and functionalization of this com-
pound is under progress. The reaction condition works well for
all kinds of substituted benzaldehydes having electron donating,
or neutral functional groups (Table 1). Notably, electron donating
OMe group favors the formation of tetraoxane (entries 2v–2z),
while electron withdrawing NO2 group inhibits the reaction, as
bis-hydroperoxide formation was not observed in this case.20 It is
important to mention here that during the synthesis of asymmet-
rical tetraoxanes (2f–2z), formation of symmetrical tetraoxane was
not noticed by TLC, however mass spectral analysis of crude reac-
tion mixture of 2a revealed that reaction mixture contains
symmetrical tetraoxane along with other non-characterizable
products. Efforts to isolate pure symmetrical tetraoxane from this
m-Me
m-Me
p-n-Pr
p-CHO
H
2s
2t
2u
2v
2w
2x
2y
2z
H
p-OMe
p-OMe
p- OMe
p-OMe
p-OMe
176
reaction mixture by column chromatography was unsuccessful. It
has been well documented in the literature that yield of the tetra-
oxanes depends on several factors.16
It is well documented that trioxanes and other endoperoxides
exerts their antimalarial activity due to C-radical generation under
physiological conditions.1b,21 Fe(II) induced O–O bond scission of
tetraoxanes have been reported by Solaja et al. and they have dem-
onstrated that only ROꢀ radical is responsible for their antimalarial
activity.22 In order to study the radical generation ability of these
tetraoxanes,23 we studied reaction of some tetraoxanes (2a–c)
with FeCl3 using reported reaction conditions.24 Tetraoxanes (2a–
2c) were subjected to FeCl3 mediated O–O bond scission in aceto-
nitrile at room temperature (Scheme 2). In a typical reaction con-
dition tetraoxanes (2a–2c; 0.37 mmol) was dissolved in 5 mL of
acetonitrile. To this, 10% solution of FeCl3 was added followed by
L-cysteine (0.74 mmol) and reaction mixture was stirred at room
temperature for 5 h. After completion of reaction as evident by
TLC, water was added to the reaction mixture and solid thus ob-
tained was purified over silica gel column. It is important to men-
tion here that under identical reaction conditions, bis-steroidal
tetraoxanes leads to the formation of ketones,22 but in this case
we isolated substituted benzoic acids (60–70% yield), which might
be formed by the oxidation of substituted benzaldehydes formed
as an intermediate during this reaction. The structures of these
compounds (3a–3c) were determined by comparing their melting
point and spectral data with authentic samples. This reaction fur-
ther confirms that tetraoxanes exhibit their antimalarial activity
due to their ability to form radicals.
O O
O O
2a-2z
(i), (ii)
O
O
O
O
CHO
(i)
COOH
R
1
R
R`
R
R
3a-3c
R`
2a-2c
Scheme 1. Reagents and conditions: (i) I2, H2O2, CH3CN; (ii) aromatic aldehyde,
HBF4ꢀEt2O, rt.
Scheme 2. (i) FeCl3, CH3CN, cysteine.

5634
N. Kumar et al. / Bioorg. Med. Chem. 17 (2009) 5632–5638
Table 2
3. X-ray crystallographic study of 3-(4-methoxy-phenyl)-6-m-
tolyl-[1,2,4,5]tetraoxane (2w)
Selected bond lengths and angles
Bond length
[Å]
In Figure 1, the thermal ellipsoids were drawn at 50% probabil-
ity and hydrogen atoms were shown as spherical. The selected
bond lengths and bond angles are listed in Table 2. X-ray intensity
data collection, crystal data and the refinement details are pre-
sented in Table 3.
C(4)–C(8)
C(9)–C(10)
C(8)–O(5)
C(8)–O(2)
C(9)–O(3)
C(9)–O(4)
O(2)–O(3)
O(4)–O(5)
1.496(4)
1.487(4)
1.409(4)
1.420(4)
1.403(4)
1.412(4)
1.476(3)
1.477(3)
The Ortep view of the tetraoxane molecular structure with ther-
mal ellipsoid atoms is shown in Figure 1. The compound 3-(4-
methoxy-phenyl)-6-m-tolyl-[1,2,4,5]tetraoxane (2w) crystallizes
with four molecules in the unit cell. The asymmetric unit of the
molecule consists of two six-membered rings connected by a tetra-
oxane ring. The C–C bond distances of both aromatic rings are
found normal, except the bonds around the C(1) and C(12) atoms,
where methoxy and methyl groups are attached. The distances of
the bridge bonds which connects the aromatic and tetraoxane
rings are Csp3–Csp2 [C(4)–C(8): 1.496(4) Å, and C(9)–C(10):
1.487(4) Å. In the tetraoxane ring, the C–O bond lengths are vary-
ing, the value ranges 1.403(4)–1.420(4) Å. Notably, the distances of
the bonds C(8)–O(5):1.409(4) and C(9)–O(3):1.403(4) Å are nearly
equal and shorter than the other two similar bonds C(8)–
O(2):1.420(4) and C(9)–O(4): 1.412(4) Å in the ring. As expected,
the distances O(2)–O(3):1.476(3) Å and O(4)–O(5):1.477(3) Å are
found equal. The C–C–C bond angles of aromatic rings vary from
119.0(3)° to 121.2(3)° and average bond angle is 120°, while the
O–C–O angles are nearly equal (Table 2).
Bond angle
[°]
C(2)–C(1)–O(1)
C(6)–C(1)–O(1)
O(5)–C(8)–O(2)
O(5)–C(8)–C(4)
O(2)–C(8)–C(4)
O(3)–C(9)–O(4)
O(3)–C(9)–C(10)
O(4)–C(9)–C(10)
C(7)–O(1)–C(1)
C(8)–O(2)–O(3)
C(8)–O(5)–O(4)
C(9)–O(3)–O(2)
C(9)–O(4)–O(5)
C(11)–C(12)–C(13)
115.4(4)
123.4(4)
109.2(2)
108.0(3)
107.4(3)
109.0(3)
106.8(3)
108.8(3)
119.8(4)
106.6(2)
107.1(2)
107.4(2)
106.6(2)
117.5(3)
Table 3
Crystal data and structure refinement
The six-membered tetraoxane ring adopts chair conformation.
Evidently, four O-atoms in the ring lies in the same plane and
the two carbon atoms C(8) and C(9) are lie above/below the plane.
The torsion angle of C(8)–O(5)–O(4)–C(9) and C(8)–O(2)–O(3)–
C(9) bonds are ꢁ63.4(3) and 63.1(3)°, respectively, shows the ori-
entation of the C–O–O–C bonds in the tetraoxane ring. The dihedral
angle between the two aromatic rings is 170.1(1)°, confirm that
these planes are almost coplanar in the molecule. Further, the
dihedral angle between the aromatic ring (individually) and the
tetraoxane ring are 93.1(1)° and 101.1(1)°, respectively. There is
no significant hydrogen bonding interactions noticed in the crystal.
However, on the whole, the molecular packing in the crystal is sta-
bilized by large number of weak intermolecular interactions.25
Empirical formula
Formula weight
Temperature (K)
Wavelength (Å)
Crystal system
C16H16O5
288.29
293(2)
0.71073
Orthorhombic
P212121
a = 6.057(2) Å
b = 7.618(2) Å
c = 31.179(8) Å
1438.74(7)
4
1.331
0.099
608
Space group
Unit cell dimensions
Volume (Å3)
Z
Calculated density (Mg/m3)
Absorption coefficient (mmꢁ1
F(000)
)
Crystal size (mm)
h Range for data collection
Limiting indices
0.3 ꢂ 0.25 ꢂ 0.2
1.31–25.7°
ꢁ7 6 h P 7, ꢁ9 6 k P 9,
ꢁ37 6 l P 37
4. Assay for in vitro antimalarial activity and cytotoxicity
Reflections collected/unique
Completeness to h = 25.7° (%)
Absorption correction
Refinement method
13,146/2727 [R(int) = 0.0913]
99.7
In vitro antimarialal activity of the substituted tetraoxanes was
determined against chloroquine sensitive (D6) and chloroquine
resistant (W2) strains of Plasmodium falciparum. Although, synthe-
sis and antimalarial activity of 2a–2f, 2m, 2n and 2l have been
published in an earlier communication18c but the activity data is
included in Table 4 for comparison with newly synthesized mole-
cules. It is evident from Table 4 that symmetrical tetraoxanes (en-
tries 2a–2c) having ethyl, n-propyl or iso-propyl groups at the para
position of the phenyl ring showed good antimalarial activity with
IC50 value ranging from 0.61 to 0.99 against D6 clone and 0.76–
None
Full-matrix least-squares on F2
2727/0/193
Data/parameters
Goodness-of-fit on F2
0.985
Final R indices [I > 2
r
(I)]
R1 = 0.048, wR2 = 0.109
R1 = 0.123, wR2 = 0.146
0.25 and ꢁ0.14
R indices (all data)
Largest differences in peak and hole (e Åꢁ3
)
butyl or n-butyl at the para position of the phenyl ring (entries
2d, 2e) have negative effect on the antimalarial activity against
both strains of Plasmodium falciparum. Among the series of asym-
metrical tetraoxanes (2f–2z) compounds with p-ethyl group at
one end and p-methyl or m-methyl or p-iso-propyl group at the
other end of the tetraoxane (entries 2f–2h), were found to be most
active in terms of IC50 value and high selectivity index. These three
compounds (entries 2f–2h) were effective against both strains of
Plasmodium falciparum (D6 and W2) with IC50 values in the range
1.03lM against W2 clone. Addition of bulky groups such as tert-
of 0.35–0.57 lM and selectivity index of 26–48. Similarly methyl
group at one of the phenyl group and para, meta-methyl, p-iso-pro-
pyl, n-propyl, tert-butyl, at the other phenyl group (entries 2m, 2o,
2p, 2q) also showed promising antimalarial activity. Interestingly,
Figure 1. Molecular structure of the tetraoxane compound (2w) showing atom
labeling scheme with thermal ellipsoids drawn at 50% probability level.

N. Kumar et al. / Bioorg. Med. Chem. 17 (2009) 5632–5638
5635
Table 4
ative effect on the antimalarial activity. Same trend was observed
in the asymmetrical tetraoxanes. None of the reported compounds
shows toxicity against vero cells. X-Ray structure of one of the tet-
raoxane showed that the tetraoxane ring is in the chair conforma-
tion with both of the phenyl rings are in the equatorial position.
Antimalarial activity of symmetrical and asymmetrical tetraoxanes
Ent
R
R0
P. falciparum
(D6 clone)
P. falciparum
(W2 clone)
Cytotoxicity
(vero cells)
IC50
(
lM) S.I.
IC50
(
lM) S.I.
2a
2b
2c
2d
2e
2f
2g
2h
2i
p-Et
n-Pr
p-i-Pr
p-Et
p-n-Pr
p-i-Pr
0.99
0.61
0.67
>15.9 0.99
>23.8 0.76
>21.6 1.03
>15.9
>19
>14
>2.8
NC
NC
NC
NC
NC
NC
NC
NC
NC
NC
NC
NC
NC
NC
NC
NC
NC
NC
6. Experimental
p-n-But p-n-But 3.65
p-t-But
p-Et
p-Et
p-Et
>3.7
>6.1
4.77
3.93
6.1. X-ray crystallographic study
p-t-But
p-Me
2.19
0.38
0.35
0.45
5.48
4.09
13.9
0.96
0.59
0.60
3.05
0.67
1.03
1.99
ND
0.93
0.50
0.76
0.42
0.54
0.94
7.90
0.05
>3.4
>43.6 0.45
>47.6 0.56
>36.7
>29.8
>26.4
>4.0
>5.3
>1.5
>18.3
>20.9
>19.0
>7.9
>17.0
>12.9
>14.9
m-Me
p-i-Pr
p-t-But
p-n-Pr
p-i-Pr
p-i-Pr
p-i-Pr
p-t-But
o-Me
m-Me
m-Me
p-n-Pr
p-CHO
H
X-ray intensity diffraction data were collected at room temper-
ature on a Bruker-Nonius Kappa four-circle diffractometer26 fitted
with CCD Area detector using Mo K radiation (k = 0.71069 Å) in
>34
0.57
3.65
2.63
9.34
p-Et
>2.6
>3.4
>1.0
a
2j
2k
2l
p-t-But
p-t-But
p-n-Pr
the
x-scan mode. All the measured reflections were reduced and
corrected for Lorentz and polarization effects.27 The structure
was solved by direct methods using SHELXS9728 and refined by SHE-
LEXL9729 programs by full-matrix least-squares methods. All non-
hydrogen atoms were refined anisotropically. The H-atoms were
geometrically fixed and constrained to ride on the parent atom in
the model. The residual index after the full convergence of the
refinement was R(F) = 0.048 and wR(F2) = 0.109 for 193
parameters.
>15.4 0.8
>26.4 0.77
>25.1 0.79
2m p-Me
2n
2o
2p
2q
2r
p-Me
p-Me
p-Me
o-Me
p-Me
p-Me
p-Me
m- Me
p- Me
m-Me
p-t-But
p-n-Pr
p-i-Pr
>5.7
2.20
>25.1 1.03
>17
>7.9
1.36
1.06
ND
2s
2t
>19.8 1.08
>36.8 0.46
>21.6 1.08
>39.7 0.62
>26.4 0.64
>15.8 0.66
>17.0
>39.7
>15.4
>26.4
>22.7
>22.7
>2.6
NC
NC
NC
NC
NC
NC
NC
NC
2u
2v
2w
2x
2y
2z
H
p-OMe
p-OMe
p-OMe
p-OMe
p-OMe
6.2. In vitro antimalarial activity assay
The antimalarial activity was determined by measuring plasmo-
dial LDH activity as described earlier.30 A suspension of red blood
cells infected with D6 or W2 strains of P. falciparum (200
2% parasitemia and 2% hematocrit in RPMI 1640 medium supple-
mented with 10% human serum and 60 g/mL Amikacin) was
added to the wells of a 96-well plate containing 10 L 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 placed
in a modular incubation chamber (Billups-Rothenberg, CA). Para-
>1.9
>298
>476
5.69
0.41
0.015
CQ
Art
>42
>1400 NC
0.035
lL, with
NC: No cytotoxicity upto 16.72
NA: No activity upto 16.72
ND: Not determined.
SI: selectivity index (IC50 for cytotoxicity/IC50 for antimalarial activity).
CQ: Chloroquine.
Art: Artemisinin.
l
M.
lM.
l
l
TM
sitic LDH activity was determined by using Malstat reagent (Flow
Inc., Portland, OR) according to the procedure of Makler and Hin-
asymmetrical tetraoxanes having phenyl group at one end and
para or meta tolyl functionality at the other end of the tetraoxanes,
have shown promising antimalarial activity (entries 2t, 2u) with
high selectivity index. Tetraoxanes with para-tert-butyl at one
end and para-ethyl, para-n-propyl and para-iso-propyl on the other
end were not as effective (entries 2i–2k). Similarly, tetraoxanes
with iso-propyl group at para position of the phenyl ring and p-
methoxy at the other end showed poor antimalarial activity (entry
2z), while other compounds with methoxy group at para position
of the phenyl ring and para-methyl, meta-methyl, para-tert-butyl
and para-n-propyl exhibit potent antimalarial activity (entries
2v–2y).
richs.31 Briefly, 20
l
L of the incubation mixture was mixed with
TM
100 lL of the Malstat reagent and incubated at room temperature
for 30 min. Twenty microliters of a 1:1 mixture of NBT/PES (Sigma,
St. Louis, MO) was then added and the plate is further incubated 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 chloroquine were included in each assay as the
drug controls. 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 cul-
ture-treated plates as described earlier.32 Vero cells (monkey kid-
ney 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 determined by Neutral Red
assay. IC50 values were obtained from dose response curves. Doxo-
rubicin was used as a positive control. We assume that all com-
pounds were soluble at tested concentrations in the biological
assays since no visible precipitation appeared after sample dilu-
tions in aqueous medium.
Cytotoxicity of symmetrical tetraoxanes 2a–2c was also tested
against a panel of six cell lines viz SK-MEL, KB, BT-549, SK-OV-3,
Vero and LLC-PK11 and none of these compounds showed any cyto-
toxicity up to 30 lM.
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 organic peroxides are explosive in nature.
5. Conclusions
6.3. Chemical synthesis
In summary, wide array of substituted tetraoxanes have been
prepared using iodine as a catalyst and antimalarial activity and
cytotoxicity of these compounds were evaluated. From the struc-
tural subset of 26 compounds, it was observed that antimalarial
activity of these compounds generally improves when phenyl ring
contains ethyl, n-propyl, iso-propyl, OMe at the para position of the
phenyl ring, while bulky group like tert-butyl or n-butyl have neg-
All of the chemicals used in the syntheses were purchased
from Sigma–Aldrich and were used as such. Thin layer chroma-
tography was used to monitor the progress of the reactions. The
compounds were purified by silica gel column. Melting points
were determined on a melting point apparatus and are uncor-

5636
N. Kumar et al. / Bioorg. Med. Chem. 17 (2009) 5632–5638
rected. IR (KBr) spectra were recorded using Perkin–Elmer FT-IR
spectrophotometer and the values are expressed as mmax cmꢁ1
6.3.2. Synthesis of asymmetrical tetraoxanes (2f–2z): general
method
.
Mass spectral data were recorded in waters micromass LCT Mass
Spectrometer/Data system. The 1H NMR and 13C NMR spectra
were recorded on Bruker Spectrospin spectrometer at 300 MHz
and 75.5 MHz, respectively, using TMS as an internal standard.
The chemical shift values are recorded on d scale and the coupling
constants (J) are in hertz. Elemental analysis were performed on a
Carlo Erba Model EA-1108 elemental analyzer and data of C, H
and N is within 0.4% of calculated values.
Substituted benzaldehyde (5.82 mmol) was added to a stirred
solution of I2 (0.582 mmol), H2O2 (24.0 mmol) in 10 mL acetonitrile
and the reaction mixture was stirred at room temperature. After
the completion of the reaction, different substituted benzaldehyde
(5.82 mmol) was added followed by the addition of 1.0 mL of
HBF4ꢀEt2O. The reaction mixture was stirred at room temperature
for 2.0 h and progress of reaction was monitored by TLC. The crude
product was quenched with NaHCO3 after the removal of solvent
from the reaction mixture and compound was extracted with
CHCl3 and washed with cold methanol. Solid thus obtained was
purified by flash chromatography. Identities of all of the synthe-
sized compounds were confirmed by IR, NMR, and MS data.
6.3.1. Synthesis of symmetrical tetraoxanes (2a–2e): general
method
To a solution of I2 (0.582 mmol), H2O2 (24.0 mmol) in 10 mL
acetonitrile, substituted benzaldehyde (5.82 mmol) was added
and the reaction mixture was stirred at room temperature.
Progress of reaction was monitored by TLC and 1H NMR. After
the completion of the reaction, same aldehyde (5.82 mmol) was
added followed by the addition of 1.0 mL of HBF4ꢀEt2O. The
reaction mixture was stirred at room temperature for 2.0 h, and
progress of reaction was monitored by TLC. The excess of solvent
from the reaction mixture was removed under vacuum. The crude
product was quenched with NaHCO3 and compound was
extracted with CHCl3 and washed with cold methanol. Solid thus
obtained was purified by flash chromatography. Identities of all of
the synthesized compounds were confirmed by IR, NMR, and MS
data.
6.3.2.1. 3-(4-Ethyl-phenyl)-6-p-tolyl-[1,2,4,5]tetraoxane (2f). Yield:
41%; mp: 205 °C; IR (KBr, cmꢁ1): 2949, 1610, 1420, 1361, 1180,
1021, 909, 840; 1H NMR (300 MHz, CDCl3): 1.26 (t, J = 6 Hz, 3H),
2.40 (s, 3H), 2.66–2.74 (m, 2H), 6.91 (s, 2H), 7.28 (m, 4H), 7.43 (m,
4H); HR-MS (m/z): 286.1209 (M+); Anal. Calcd for C17H18O4: C,
71.31; H, 6.34. Found: C, 71.61; H, 6.60.
6.3.2.2. 3-(4-Ethyl-phenyl)-6-m-tolyl-[1,2,4,5]tetraoxane (2g). Yield:
39%; mp: 180–183 °C; IR (KBr, cmꢁ1): 2952, 1653, 1543, 1510, 1437,
1360, 1281, 1181, 1009, 887; 1H NMR (300 MHz, CDCl3): 1.23 (t,
J = 6H, 3H), 2.38 (s, 3H), 2.66 (q, J = 6 Hz, 2H), 6.88 (s, 2H), 7.26–7.28
(m, 4H), 7.41–7.43 (m, 4H); MS-FAB (m/z): 315 (M++1); Anal. Calcd
for C17H18O4: C, 71.31; H, 6.34. Found: C, 71.57; H, 6.69.
6.3.1.1. 3,6-Bis-(4-ethyl-phenyl)-[1,2,4,5]tetraoxane (2a). Yield:
44%; mp: 190–192 °C; IR (KBr, cmꢁ1): 2969, 1610, 1512, 1458,
1422, 1362, 1181, 1117, 1022, 1003, 909, 840; 1H NMR
(300 MHz, CDCl3): 1.26 (t, J = 6 Hz, 6H), 2.66–2.94 (m, 4H), 6.91
(s, 2H), 7.28 (d, J = 8 Hz, 4H), 7.53 (d, J = 8 Hz, 4H); MS-FAB (m/z):
301 (M++1); Anal. Calcd for C18H20O4: C, 71.98; H, 6.71. Found: C,
71.77; H, 6.53.
6.3.2.3. 3-(4-Ethyl-phenyl)-6-(4-isopropyl-phenyl)-[1,2,4,5]tet-
raoxane (2h). Yield: 37%; mp: 165 °C; IR (KBr, cmꢁ1): 2966, 1610,
1511, 1460, 1361, 1181, 1020, 909, 838; 1H NMR (300 MHz, CDCl3):
1.23–1.28 (m, 9H), 2.66–2.74 (m, 2H), 2.93–2.98 (m, 1H), 6.91 (s,
2H), 7.32 (m, 4H), 7.47 ppm (m, 4H); HRMS calcd for C19H22O4:
314.1518 (M+), found: 314.1508; Anal. Calcd for C19H22O4: C,
72.59; H, 7.05. Found: C, 72.69; H, 6.90.
6.3.1.2. 3,6-Bis-(4-n-propyl-phenyl)-[1,2,4,5]tetraoxane (2b). Yield:
38%; mp: 155 °C; IR (KBr, cmꢁ1): 2956, 2927, 1613, 1421, 1359,
1179, 1018, 1001, 909, 835, 790; 1H NMR (300 MHz, CDCl3): 0.94 (t,
6H), 1.60–1.70 (m, 4H), 2.62 (t, 4H), 6.88 (s, 2H), 7.26 (d, J = 8 Hz,
4H), 7.43 (d, J = 8 Hz, 4H); MS-FAB (m/z): 321 (M++1); Anal. Calcd for
C20H24O4: C, 73.15; H, 7.37. Found: C, 73.37; H, 7.50.
6.3.2.4. 3-(4-tert-Butyl-phenyl)-6-(4-ethyl-phenyl)-[1,2,4,5]tet-
raoxane (2i). Yield: 25%; mp: 205 °C; IR (KBr, cmꢁ1): 3430, 2960,
2867, 1614, 1514, 1462, 1360, 1312, 1267, 1108, 1022, 1004,
911, 838; 1H NMR (300 MHz, CDCl3): 1.24 (t, 3H), 1.32 (s, 9H),
2.67 (q, 2H), 6.89 (s, 2H), 7.25–7.27 (m, 2H), 7.41–7.45 (m, 6H);
MS-FAB (m/z): 329 (M++1); Anal. Calcd for C20H24O4: C, 73.15; H,
7.37. Found: C, 73.39; H, 7.60.
6.3.1.3. 3,6-Bis-(4-iso-propyl-phenyl)-[1,2,4,5]tetraoxane (2c). Yield:
41%; mp: 156–157 °C; IR (KBr, cmꢁ1): 2959, 2932, 1611, 1510, 1462,
1411, 1362, 1182, 1021, 909, 841;1H NMR (300 MHz, CDCl3): 1.28 (d,
J = 6 Hz, 12H), 2.95 (sept, 2H), 6.91 (s, 2H), 7.30 (d, J = 8 Hz, 4H), 7.44
(d, J = 8 Hz, 4H); MS-FAB (m/z): 329 (M++1); Anal. Calcd for C20H24O4:
C, 73.15; H, 7.37. Found: C, 73.39; H, 7.70.
6.3.2.5. 3-(4-tert-Butyl-phenyl)-6-(4-propyl-phenyl)-[1,2,4,5]
tetraoxane (2j). Yield: 32%; mp: 193 °C; IR (KBr, cmꢁ1): 3442,
2959, 2861, 2354, 1614, 1514, 1461, 1361, 1266, 1180, 1140,
1022, 1005, 912, 839; 1H NMR (300 MHz, CDCl3): 0.92 (t, 3H),
1.32 (s, 9H), 1.53–1.65 (m, 2H), 2.60 (t, 2H), 6.89 (s, 2H), 7.23–
7.25 (m. 2H), 7.41–7.45 (m, 6H); MS-FAB (m/z): 343 (M++1); Anal.
Calcd for C21H26O4: C, 73.66; H, 7.65. Found: C, 73.77; H, 7.53.
6.3.1.4. 3,6-Bis-(4-butyl-phenyl)-[1,2,4,5]tetraoxane (2d). Yield:
40%; mp: 142–145 °C; IR (KBr, cmꢁ1): 2957, 2856, 1611, 1511,
1462, 1363, 1121, 1023, 1001, 909, 841, 794, 697 cmꢁ1 1H NMR
;
(300 MHz, CDCl3): 0.94 (t, 6H), 1.30–1.43 (m, 4H), 1.57–1.67 (m,
4H), 2.66 (t, 4H), 6.90 (s, 2H), 7.24 (d, J = 8 Hz, 4H), 7.42 (d,
J = 8 Hz, 4H); MS-FAB (m/z): 357 (M++1); Anal. Calcd for
C22H28O4: C, 74.13; H, 7.92. Found: C, 74.37; H, 7.73.
6.3.2.6. 3-(4-tert-Butyl-phenyl)-6-(4-iso-propyl-phenyl)-[1,2,4,5]
tetraoxane (2k). Yield: 38%; mp: 197 °C; IR (KBr, cmꢁ1): 3427,
2959, 1613, 1511, 1481, 1421, 1309, 1184, 1108, 1022, 911, 838;
1H NMR (300 MHz, CDCl3): 1.24 (d, 6H), 1.32 (s, 9H), 2.91–2.95
(sept, 1H), 6.89 (s, 2H), 7.25–7.30 (m, 2H), 7.42–7.45 (m, 6H);
MS-FAB (m/z): 343 (M++1); Anal. Calcd for C21H26O4: C, 73.66; H,
7.65. Found: C, 73.69; H, 7.90.
6.3.1.5. 3,6-Bis-(4-tert-butyl-phenyl)-[1,2,4,5]tetraoxane (2e). Yield:
53%; mp: 222–224 °C; IR (KBr, cmꢁ1): 2924, 1614, 1461, 1370,
1312, 1267, 1187, 1021, 1003, 911, 838, 803; 1H NMR (300 MHz,
CDCl3): 1.31 (s, 18H), 6.92 (s, 2H), 7.50–7.62 (m, 8H); 13C NMR
(75.5 MHz, CDCl3): 31.16 (CH3), 34.91 (CH3), 108.10 (CH), 125.76
(CH), 127.57(CH), 128.06 (C), 154.67 (CH); MS-FAB (m/z): 357
(M++1); Anal. Calcd for C22H28O4: C, 74.13; H, 7.92. Found: C, 74.27;
H, 7.69.
6.3.2.7. 3-(4-iso-Propyl-phenyl)-6-(4-propyl-phenyl)-[1,2,4,5]
tetraoxane (2l). Yield: 28%; mp: 130 °C; IR (KBr, cmꢁ1): 3441,
2927, 2854, 1633, 1469, 1450, 1378, 1352, 1262, 1233, 1164,
1126, 1093, 1071, 1011, 964, 889; 1H NMR (300 MHz, CDCl3):
0.86–0.88 (m, 3H), 1.18 (d, 6H), 1.56–1.58 (m, 2H), 2.86 (m, 3H),

N. Kumar et al. / Bioorg. Med. Chem. 17 (2009) 5632–5638
5637
6.81 (s, 2H), 7.10–7.23 (m, 4H), 7.35 (m, 4H); MS-FAB (m/z): 329
(M++1); Anal. Calcd for C20H24O4: C, 73.15; H, 7.37. Found: C,
73.49; H, 7.50.
MS-FAB (m/z): 259 (M++1); Anal. Calcd for C18H20O4: C, 69.76; H,
5.46. Found: C, 69.97; H, 5.53.
6.3.2.17. 3-(4-Methoxy-phenyl)-6-p-tolyl-[1,2,4,5]tetraoxane
(2v). Yield: 25%; mp: 206 °C; IR (KBr, cmꢁ1): 2950, 1701, 1609,
1582, 1513, 1460, 1362, 1309, 1258, 1174, 1017, 907, 843, 824,
791; 1H NMR (300 MHz, CDCl3): 2.38 (s, 3H), 3.82 (s, 3H), 6.84 (s,
1H), 6.92 (s, 1H), 7.27 (m, 4H), 7.38 (m, 4H); MS-FAB (m/z): 289
(M++1); Anal. Calcd for C16H16O5: C, 66.66; H, 5.59. Found: C,
66.89; H, 5.80.
6.3.2.8. 3-(4-iso-Propyl-phenyl)-6-p-tolyl-[1,2,4,5]tetraoxane
(2m). Yield: 26%; mp: 215–216 °C; IR (KBr, cmꢁ1): 2947, 1609,
1359, 1021, 909, 828, 791; 1H NMR (300 MHz, CDCl3): 1.28 (d,
J = 6 Hz, 6H), 2.40 (s, 3H), 2.92 (septet, 1H), 6.89 (s, 2H), 7.27–
7.32 (m, 4H), 7.40–7.46 (m, 4H); MS-FAB (m/z): 315 (M++1); Anal.
Calcd for C18H20O4: C, 71.98; H, 6.71. Found: C, 71.77; H, 6.59.
6.3.2.9. 3-(4-tert-Butyl-phenyl)-6-p-tolyl-[1,2,4,5]tetraoxane
(2n). Yield: 46%; mp: 188–190 °C; 1H NMR (300 MHz, CDCl3): 1.35
(s, 9H), 2.41 (s, 3H), 6.90 (s, 1H), 6.93 (s, 1H), 7.28 (d, 4H), 7.42 (d,
4H); 13C NMR (75.5 MHz, CDCl3): 21.49, 31.17, 34.93, 108.14,
125.77, 127.57, 127.77, 128.11, 129.46, 141.61; MS-FAB (m/z):
315.4 (M++1); Anal. Calcd for C19H22O4: C, 72.59; H, 7.05. Found:
C, 72.27; H, 7.33.
6.3.2.18. 3-(4-Methoxy-phenyl)-6-m-tolyl-[1,2,4,5]tetraoxane
(2w). Yield: 29%; mp: 120 °C; IR (KBr, cmꢁ1): 2954, 1611, 1583,
1516, 1462, 1363, 1309, 1261, 1175, 1033, 1014, 839, 786; 1H
NMR (300 MHz, CDCl3): 2.38 (s, 3H), 3.82 (s, 3H), 6.83 (s, 1H),
6.92 (s, 1H), 7.31–7.35 (m, 6H), 7.42 (d, 2H); MS-FAB (m/z): 289
(M++1); Anal. Calcd for C16H16O5: C, 66.66; H, 5.59. Found: C,
66.49; H, 5.86.
6.3.2.10. 3-o-Tolyl-6-p-tolyl-[1,2,4,5]tetraoxane
(2o). Yield:
6.3.2.19. 3-(4-tert-Butyl-phenyl)-6-(4-methoxy-phenyl)-[1,2,4,5]
tetraoxane (2x). Yield: 22%; mp: 168 °C; IR (KBr, cmꢁ1): 3433,
2965, 1610, 1584, 1516, 1463, 1365, 1179, 1109, 1021; 1H NMR
(300 MHz, CDCl3): 1.32 (s, 9H), 3.83 (s, 3H), 6.85 (s, 1H), 6.92 (s,
1H), 7.43–7.46 (m, 8H); MS-FAB (m/z): 331 (M++1); Anal. Calcd
for C19H22O5: C, 69.07; H, 6.71. Found: C, 69.27; H, 6.93.
25%; mp: 206 °C; IR (KBr, cmꢁ1): 2953, 1608, 1512, 1459, 1361,
1311, 1198, 1181, 1018, 910, 828, 792; 1H NMR (300 MHz, CDCl3):
2.38 (s, 6H), 6.87 (s, 2H), 7.22–7.41 (m, 8H); MS-FAB (m/z): 273.2
(M++1); Anal. Calcd for C16H16O4: C, 70.57; H, 5.92. Found: C,
70.79; H, 6.23.
6.3.2.11. 3-m-Tolyl-6-p-tolyl-[1,2,4,5]tetraoxane
(2p). Yield:
6.3.2.20. 3-(4-Methoxy-phenyl)-6-(4-propyl-phenyl)-[1,2,4,5]
tetraoxane (2y). Yield: 20%; mp: 120 °C; IR (KBr, cmꢁ1): 2923,
2854, 1613, 1584, 1517, 1462, 1376, 1307, 1257, 1175, 1114,
1029, 998; 1H NMR (300 MHz, CDCl3): 0.91–0.95 (t, 3H), 1.55–
1.67 (m, 2H), 2.58–2.63 (t, 2H), 3.83 (s, 3H), 6.7–6.88 (m, 1H),
6.92–6.95 (s, 1H), 7.22–7.25 (m, 4H), 7.40–7.46 (m, 4H); MS-FAB
(m/z): 317 (M++1); Anal. Calcd for C18H20O5: C, 68.34; H, 6.37.
Found: C, 68.59; H, 6.80.
38%; mp: 208 °C; IR (KBr, cmꢁ1): 2925, 1610, 1459, 1359, 1181,
1017, 909, 827, 789; 1H NMR (300 MHz, CDCl3): 2.38 (s, 6H),
6.87 (s, 2H), 7.22–7.41 (m, 8H); MS-FAB (m/z): 273.2 (M++1); Anal.
Calcd for C16H16O4: C, 70.57; H, 5.92. Found: C, 70.69; H, 5.99
6.3.2.12. 3-m-Tolyl-6-o-tolyl-[1,2,4,5]tetraoxane
(2q). Yield:
33%; mp: 112 °C; IR (KBr, cmꢁ1): 2952, 1604, 1490, 1366, 1192,
1096, 942, 817, 757; 1H NMR (300 MHz, CDCl3): 2.38 (s, 3H),
2.53 (s, 3H), 6.83 (s, 2H), 7.22–7.50 (m, 8H); MS-FAB (m/z): 273.2
(M++1); Anal. Calcd for C16H16O4: C, 70.57; H, 5.92. Found: C,
70.77; H, 5.73.
6.3.2.21. 3-(4-iso-Propyl-phenyl)-6-(4-methoxy-phenyl)-[1,2,4,5]
tetraoxane (2z). Yield: 24% mp: 176 °C; 1H NMR (300 MHz,
CDCl3): 1.25 (d, 6H), 2.89–2.95 (m, 1H), 3.83 (s, 3H), 6.84 (s, 1H),
6.92 (d, 1H), 7.27–7.30 (m, 2H), 7.42–7.46 (m, 6H); MS-FAB (m/
z): 317 (M++1); Anal. Calcd for C18H20O5: C, 68.34; H, 6.37. Found:
C, 68.57; H, 6.53.
6.3.2.13. 3-(4-Propyl-phenyl)-6-p-tolyl-[1,2,4,5]tetraoxane
(2r). Yield: 37%; mp: 148 °C; 1H NMR (300 MHz, CDCl3): 0.86 (t,
3H), 1.47–1.61 (m, 2H), 2.38 (s, 3H), 2.54 (t, 2H), 6.81 (s, 2H),
7.15–7.19 (m, 6H), 7.44 (m, 2H); MS-FAB (m/z): 301 (M++1); Anal.
Calcd for C18H20O4: C, 71.98; H, 6.71. Found: C, 71.79; H, 6.90.
Acknowledgements
D.S.R. thanks Department of Science and Technology (SR/S1/OC-
08/2008), New Delhi and Council of Scientific and Industrial Re-
search, New Delhi, India for financial support. N.K. and Beena are
thankful to CSIR for the award of junior research fellowship. United
States Department of Agriculture (USDA), Agricultural Research
Service Specific Cooperative Agreement No. 58-6408-2-0009 is also
acknowledged for partial support of this work.
6.3.2.14. 4-(6-p-Tolyl-[1,2,4,5]tetroxan-3-yl)-benzaldehyde
(2s). Yield: 31%; mp: 195 °C; IR (KBr): 2924, 2845, 1707, 1423,
1358, 1202, 1018, 1007, 911, 835 cmꢁ1; 1H NMR (300 MHz, CDCl3):
2.38 (s, 3H), 6.88 (s, 1H), 6.94 (s, 1H), 7.26 (m, 2H), 7.39 (d, J = 8 Hz,
2H), 7.71 (d, J = 8 Hz, 2H), 7.97 (d, J = 8 Hz, 2H), 9.21 (s, 1H); 13C
NMR (75.5 MHz, CDCl3): 20.65, 106.15, 107.45, 126.32, 126.96,
127.61, 128.67, 129.01, 135.45, 137.32, 141.06, 190.75; MS-FAB
(m/z): 289 (M++1); Anal. Calcd for C16H14O5: C, 67.13; H, 4.99.
Found: C, 67.27; H, 4.79.
References and notes
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6.3.2.15. 3-Phenyl-6-p-tolyl-[1,2,4,5]tetraoxane (2t). Yield: 43%;
mp: 210 °C; IR (KBr, cmꢁ1): 2962, 1696, 1454, 1348, 1095, 940,
815, 783; 1H NMR (300 MHz, CDCl3): 2.38 (s, 3H), 6.87 (s, 2H),
7.22–7.50 (m, 9H); MS-FAB (m/z): 259(M++1); Anal. Calcd for
C15H14O4: C, 69.76; H, 5.46. Found: C, 69.89; H, 5.23.
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30%; mp: 140 °C; IR (KBr, cmꢁ1): 2958, 1608, 1456, 1363, 1160,
1018, 910, 817, 788, 755; 1H NMR (300 MHz, CDCl3): 2.38 (s, 3H),
6.89 (s, 1H), 6.93 (s, 1H), 7.28–7.31 (m, 4H), 7.41–7.53 (m, 5H);
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