Synthesis and antimalarial activities of novel 3,3,6,6-tetraalkyl-1,2,4,5-tetraoxanes

Article abstract of DOI:10.1016/j.bmc.2006.07.069

The oxidative system H₂O₂/fluorinated alcohol (TFE, HFIP) was used for direct acid- and MeReO₃-catalyzed synthesis of 1,2,4,5-tetraoxanes from cyclic (C6, C7, and C12) and acyclic ketones. The influence of ring size and alkyl chain length were studied and antimalarial activities of synthetic 3,3,6,6-tetraalkyl-1,2,4,5-tetraoxanes were determined. Variations in their antimalarial activities were significant, although they share similar electrochemical properties of the peroxide bond.

Full text of DOI:10.1016/j.bmc.2006.07.069

Bioorganic & Medicinal Chemistry 14 (2006) 7790–7795
Synthesis and antimalarial activities of novel
3,3,6,6-tetraalkyl-1,2,4,5-tetraoxanes
a
a
a
b
ˇ
Katja Zmitek, Stojan Stavber, Marko Zupan, Daniele Bonnet-Delpon,
Sebastien Charneau,c Phillipe Grellierc and Jernej Iskraa,*
aLaboratory of Organic and Bioorganic Chemistry, ‘Jozˇef Stefan’ Institute and Faculty of Chemistry and
Chemical Technology of University of Ljubljana, Jamova 39, 1000 Ljubljana, Slovenia
bBIOCIS UPRES-A 8076, Faculte de Pharmacie, Universite Paris-Sud, Chatenay-Malabry, France
cUSM 504 Biologie fonctionnelle des protozoaires, Museum National d’Histoire Naturelle, CP52, 61 Rue Buffon, 75231 Paris, France
Received 23 June 2006; revised 27 July 2006; accepted 31 July 2006
Available online 17 August 2006
Abstract—The oxidative system H2O2/fluorinated alcohol (TFE, HFIP) was used for direct acid- and MeReO3-catalyzed synthesis
of 1,2,4,5-tetraoxanes from cyclic (C6, C7, and C12) and acyclic ketones. The influence of ring size and alkyl chain length were stud-
ied and antimalarial activities of synthetic 3,3,6,6-tetraalkyl-1,2,4,5-tetraoxanes were determined. Variations in their antimalarial
activities were significant, although they share similar electrochemical properties of the peroxide bond.
Ó 2006 Elsevier Ltd. All rights reserved.
1. Introduction
lyzed by boron trifluoride etherate.24 There are a few
reports of the synthesis of 1,2,4,5-tetraoxanes (TO) from
acyclic ketones by acid-catalyzed peroxidation. This
reaction requires the use of highly concentrated hydro-
gen peroxide or a tenfold excess of sulfuric and glacial
acetic acid.16,25–27
Malaria is still one of the major communicable diseases,
estimated to cause or contribute to as many as 3 million
deaths per year.1 Mortality caused by malaria has in-
creased in recent years due mainly to the pathogen hav-
ing developed resistance to available antimalarial
drugs.2–4 Artemisinin and its semi-synthetic and synthet-
ic endoperoxides that are active against drug-resistant
Plasmodium have emerged as potent alternative non-al-
kaloidal drugs. While the mechanism of their antimalar-
ial activity is still unclear, it is known that the peroxide
unit in these compounds is essential for their activity.5–11
Dispiro-1,2,4,5-tetraoxanes (TO), having two endoper-
oxide bonds, have been found to be potent and inexpen-
sive antimalarial agents.12–15 The easiest path for their
synthesis is acid-catalyzed cyclization of cyclic ketones
with hydrogen peroxide, but this is successful only with
selected substrates, and leads to mixture of compounds
only one of which is the desired tetraoxane.16–19 Alter-
native synthetic routes to tetraoxanes include ozonolysis
of cycloalkylidene-cycloalkanes,20 enol ethers,21,22 and
O-methyl oximes,19,23 and the recently reported reaction
of gem-bishydroperoxides with acetals or ketals cata-
Fluorinated alcohols such as hexafluoro-2-propanol,
(CF3)2CHOH or HFIP, and 2,2,2-trifluoroethanol,
TFE, are known activators of hydrogen peroxide in var-
ious oxidation reactions by virtue of their high hydrogen
bond donor strength, combined with their promotion of
template catalysis.28–30 We have used fluorinated alco-
hols in the methyltrioxorhenium (MTO)-catalyzed per-
oxidation of 4-methylcyclohexanone with 30% aqueous
H2O2 in neutral conditions and shown that the reaction
leads to the formation of the gem-dihydroperoxide,
which, with another carbonyl compound under acid
conditions, is further transformed to a non-symmetric
TO.31 We showed that by proper modification of the
catalyst, concentration, and temperature 4-substituted
cyclohexanones could be selectively transformed into
corresponding dispiro-TOs with good yields. The reac-
tion can be controlled in order to avoid the formation
of trimeric by-products.32
Keywords: Malaria; Tetraoxane; Peroxide; Artemisinin.
*
Herein we show that fluorinated alcohols permit synthe-
sis of 1,2,4,5-tetraoxanes directly from cyclic, as well as
Corresponding author. Tel.: +386 1 4773 631; fax: +386 1 4773
822; e-mail: jernej.iskra@ijs.si
0968-0896/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.07.069

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K. Zmitek et al. / Bioorg. Med. Chem. 14 (2006) 7790–7795
7791
the less reactive acyclic ketones. The simple structure of
the products, with a tetraoxane ring and alkyl groups
of different length and branching, enables modification
of the polarity of TOs and the steric hindrance around
the O–O bond without affecting the properties of phar-
macophoric peroxide unit. This offers an insight into
the correlation between the structure of TOs and their
antimalarial activity.
The differing behavior of the various cycloalkanones in
these peroxidations led us to examine the reactions of
acyclic dialkyl ketones and the influence on TO yield
of length and branching of the alkyl chain at positions
a- and b- to the carbonyl group.
This method (H2O2/HBF4/MTO in TFE) is successful
for the transformation of acyclic ketones to TO. Table 2
shows the effect of extension of the alkyl chains on the
yield of TO. It can be seen that extension of both the
alkyl chains of diethyl ketone 3a to dipropyl ketone 3b
resulted in a lower TO yield, which remained unchanged
by additional chain lengthening leading to dibutyl
ketone 3c. Comparison of results for given family of eth-
yl ketones (3a and 3d–3h) shows that extension of one of
the alkyl chains from C2H5 to C7H15 has no significant
influence on TO yield. The best yield of TO was ob-
served when the ethyl group in 3h was replaced with a
methyl group, in 3i. Tetraoxanes from unsymmetrical
ketones form cis and trans isomers, however, they could
not be separated. Tetraoxane ring inversion is very fast
at room temperature leading to broad signals in NMR
spectra. When the spectra were recorded at lower tem-
perature (ꢀ40 °C), conformation of tetraoxane ring
freezes and reveals that a mixture of isomers was
formed.
2. Results and discussion
First, the effect of ring size on the formation of TO by
acid-catalyzed peroxidation using 30% H2O2/ MTO/
fluorinated alcohols was tested. Previous results of such
peroxidation of 4-substituted cyclohexanones in fluori-
nated alcohols at room temperature showed that
reactions in HFIP produce lactones as products of Bae-
yer–Villiger rearrangement, while in TFE, tetraoxanes
are formed selectively.32 We mixed equimolar amounts
of the ketone 1, H2O2 (30% aqueous solution), and
HBF4 (54% ethereal solution), and treated the mixture
with 0.1 mol% MTO in TFE for one hour at room tem-
perature. Cyclopentanone 1a, with a ring that is strained
compared to that of cyclohexanone, gave under these
conditions tetrahydro-2H-pyran-2-one, while TO was
formed only in a trace amount (Table 1). The less
strained cyclohexanone 1b and cycloheptanone 1c were
successfully transformed into corresponding TOs 2b
and 2c in TFE without noticeable lactone formation.
Further enlargement of the size of the ring resulted in
a decrease of conversion and selectivity for TO forma-
tion; reaction of the cyclooctanone 1d, cyclodecanone
1e, and cyclododecanone 1f yielded complex reaction
mixtures with minimal TO formation. Use of HFIP, a
more activating solvent,32 failed to lead to improvement
in the formation of TO from ketones 1d and 1e, but in
contrast, use of HFIP provided selective conversion of
cyclododecanone 1f to the corresponding tetraoxane 2f
in 55% yield.
Table 3 shows the influence on TO yield of alkyl chain
branching by introduction of a methyl substituent at
the a- and b-positions on one or both sides of the car-
bonyl group. It can be observed that introduction of
a-methyl group into diethyl ketone 3a to give ethyl
iso-propyl ketone (5a) reduces the yield by 50% and a
second methyl group at the a0 position (di-iso-propyl ke-
tone 5b) totally inhibits TO formation. Introduction of
methyl substituents to the b- and b0-positions (5c and
5d) has a similar effect on the formation of TO. Steric
influence therefore has an important role in the forma-
tion of TO, although it proved to be possible to trans-
Table 2. Influence on tetraoxane yield of the length of alkyl chains in
acyclic ketones
Table 1. The effect of ring size of cycloalkanone on tetraoxane yield
O O
O
R1
R2
R2
R1
H2O2/ HBF4/ MTO
TFE, rt, 1h
O O
H2O2/ HBF4/ MTO
(CH2)n
O
(CH2)n
(CH2)n
R1
R2
O O
4
fluorinated alcohol
O O
2
3
1
Ketone
R1
TO yielda (%)
Ketone
Fluorinated alcohol
TO yielda (%]
n
R2
1a
1b
1c
1d
1e
1f
4
5
6
7
9
TFE
TFE
TFE
TFE
TFE
HFIP
2a: <5b
2b: 88
2c: 45
3a
3b
3c
3d
3e
3f
Et
Pr
Bu
Et
Et
Et
Et
Et
Me
Et
Pr
4a: 57
4b: 45
4c: 44
4d: 56
4e: 55
4f: 59
4g: 55
4h: 57
4i: 72
Bu
Pr
c
—
c
—
2f: 55
Bu
11
Pent
Hex
Hept
Hept
3g
3h
3i
a React. cond.: solution of equimolar amounts of 1, H2O2, and HBF4,
0.1 mol% MTO was stirred for 1 h at room temp. Yields refer to
isolated products.
b Tetrahydro-2H-pyran-2-one was the major product as determined by
1H NMR of crude reaction mixture.
c Complex reaction mixture with low conversion.
a React. cond.: solution of equimolar amounts of 1, H2O2, and HBF4,
0.1 mol% MTO was stirred for 1 h at room temp. Yields refer to
isolated products.

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K. Zmitek et al. / Bioorg. Med. Chem. 14 (2006) 7790–7795
7792
Table 3. The effect of branching of the alkyl chain at the a- and
b-positions to carbonyl group on TO yield
differences in the antimalarial activities of the various
compounds were observed. The measured IC50 values
of tetraalkyl-TOs 4 ranged from 151.4 nM for 3,6-dibu-
tyl-3,6-diethyl-1,2,4,5-tetraoxane 4e to 387.1 nM for
3,6-diethyl-3,6-dihexyl-1,2,4,5-tetraoxane 4g, while tet-
raoxane with methyl substituent 4i was essentially inac-
tive (IC50 > 1 lM).
Ketone
TO yielda (%)
O
5a
6a: 26
O
O
5b
5c
5d
5e
6b: 0
The small amount of experimental data on the effect of
lipophilicity on the in vivo antimalarial activity suggests
that within a given peroxide chemical family, the more
lipophilic compounds possess superior antimalarial
activity.15,33,34 In this respect, TOs are interesting proto-
types with which to seek correlations between the struc-
ture of TO (lipophilicity and steric effects around
peroxide bond) and in vitro antimalarial activities. The
alkyl chains in tetraalkyl TO are a major part of the
structure and their variation has no significant effect
on the electronic properties of pharmacophoric peroxide
bond because of their similar electron-donating abilities
and absence of other functional groups. Accordingly, we
correlated the antimalarial activity of selected tetraox-
anes with calculated values for their lipophilicity (logP).
It was found that as the alkyl chains in a given TO fam-
ily become longer, the calculated lipophilicity, expressed
as logP, increases and the antimalarial activity decreas-
es, as shown in Figure 1A. A good correlation between
logP and IC50 was observed for related tetraoxanes with
linear alkyl chains over a polarity range of more than
three orders of magnitude (Fig. 1B), but the TO 4d with
a shorter alkyl substituent was not so correlated and the
TOs with a methyl substituent on TO ring (4i, 6c) have
IC50 values above 1 lM. A further comparison with TOs
with branched alkyl chains that influence the sterical
hindrance around O–O bond indicates that polarity, or
lipophilicity might not be the only significant parameter.
Comparison of the antimalarial activity of diethyl dipro-
pyl TO 4d with that of diethyl di-iso-propyl TO (6a) re-
veals that the methyl substituent at the position a to the
ring carbon drastically decreases the activity; the tetra-
oxane 4d is more than twice as potent than its branched
analog 6a, although they share similar lipophilicity
(logP 4.88 and 4.62, respectively).
6c: 20
6d: 0
O
O
6e: 15
a React. cond.: solution of equimolar amounts of 1, H2O2, and HBF4,
0.1 mol% MTO was stirred for 1 h at room temp. Yields refer to
isolated products.
form methyl tert-butyl ketone 5e into the corresponding
TO 6e in 15% yield.
Dispiro-1,2,4,5-tetraoxanes are known antimalarials,
but there are no published data on the antimalarial
activity of 3,3,6,6-tetraalkyl-1,2,4,5-tetraoxanes. There-
fore, we have determined in vitro antimalarial activities
of synthesized TOs using FCB1 strain of Plasmodium
falciparum, which is resistant to chloroquine (IC50
=
115 25 nM). The dispiro TO 2c showed the highest
antimalarial activity (Table 4). Extension of the alkyl
chain in TO does not influence the electronic properties
of the peroxide bond as alkyl chains have similar
electron-donating abilities. In spite of this, notable
Table 4. In vitro antimalarial activities against Plasmodium falciparum
strain FCB1 for synthesized tetraoxanes and their logP values
IC50 (nMa)
logPb
Tetraoxane
O O
R1
R2
O
R2
R1
A
O O
O
8
4g
9
R1
–(CH2)6–
R2
O
B
B
O
2c
2f
82.9
Totally insoluble
183.4
4.73
10.32
5.94
4.88
5.94
7.00
8.06
8.06
4.62
4.36
6.74
8
4g
O
4f
7
6
–(CH2)11
n-Pr
Et
–
O
R= 0.98
7
4f
4b
6c
4b
4d
4e
4f
n-Pr
n-Pr
O
O
6
5
4
4e
2c
179.4
Et
Et
n-Bu
n-Pent
n-Hex
151.4
243.7
O
O
4e
4b
0
200
400
4g
4i
Et
387.1
>1000
478.1
5
4
O
O
O
O
Me
Et
n-Hept
i-Pr
t-Bu
O
O
6a
6e
6c
2c
4d
6a
Me
Et
>1000
135.8
3.5
CH2CH(CH3)2
0
100
200
300
400
500
600
IC50[nM]
Artemether (ref)
a IC50 values were means of three independent experiments.
b logP values were calculated using program http://www.daylight.com.
Figure 1. Correlation between antimalarial activity and lipophilicity
(logP) of selected tetraoxanes.

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K. Zmitek et al. / Bioorg. Med. Chem. 14 (2006) 7790–7795
7793
3. Conclusion
CH2Cl2 (20 mL) was added and the solution washed
twice with 20 mL of H2O. The organic phase was dried
with Na2SO4 and the solvent evaporated. Tetraoxanes
2a–2c, 4a–4i, and 6a–6e were purified by column chro-
matography (SiO2, CH2Cl2/petrol ether 4:1). They were
the first products eluted.
The oxidative system—H2O2/fluorinated alcohol (TFE,
HFIP)—has proved useful for direct acid- and MTO-cat-
alyzed synthesis of tetraoxanes from cyclic (C6, C7, and
C12) and acyclic ketones. Cyclopentanone was converted
mainly to a lactone, while cyclooctanone and cyclodeca-
none were less reactive. The reactions of acyclic ketones
revealed that the length of the alkyl chain and its branch-
ing around carbonyl group has a significant influence on
TO yield. As selected substrates have similar substituents
in terms of electron-donating properties, differences in
their reactivity were attributed to their geometry and lipo-
philicity. It was observed that chain length does not have
a great influence on TO yield as long as one of the two al-
kyl chains is short. Introduction of a- and b-substituents
leads to reduction of TO yield.
For synthesis of 2f HFIP (2 mL) was used as solvent and
2f was isolated by filtration of the reaction mixture and
washed with CH2Cl2.
4.2.1. 7,8,15,16-Tetraoxa-dispiro[5.2.5.2]hexadecane (2b).
Compound 2b was isolated as white solid in 88% yield
(201 mg): mp 128.8–129 °C (lit.,35 130–131 °C); 1H
NMR: d 1.37–1.51 (m, 4H), 1.51–1.70 (m, 12H), 2.27
(br s, 4H); 13C NMR: d 22.05 (br), 25.36, 29.54 (br),
31.80 (br), 108.12.
The 3,3,6,6-tetraalkyl-1,2,4,5-tetraoxanes have antima-
larial activities against a chloroquine-resistant strain of
P. falciparum similar to that of their dispiro analogs.
The variations in antimalarial activities of the TOs stud-
ied were significant, although they share similar electron-
ic properties of peroxide bonds. These can probably be
attributed to the combination of the influence of polarity
(higher lipophilicity, lower activity) and steric hindrance
(branching and substitution) of the peroxide bond.
4.2.2. 8,9,17,18-Tetraoxa-dispiro[6.2.6.2]octadecane (2c).
Compound 2c was isolated as white solid in 45% yield
1
(115 mg): mp 99–100 °C (lit.,26 98–100 °C); H NMR:
d 1.45–1.84 (m, 20H), 2.42 (br s, 4H); 13C NMR: d
22.43, 29.55, 30.21, 31.02, 36.00, 112.43.
4.2.3. 13,14,27,28-Tetraoxa-dispiro[11.2.11.2]octacosane
(2f). Compound 2f was isolated as white solid in 55%
yield (220 mg): mp 176–179 °C (dec); 1H NMR: d
1.05–1.76 (m, 40H), 2.23 (br s, 4H); 13C NMR: d 18.00
(br), 19.41 (br), 21.64 (br), 22.23, 25.89 (br), 25.99,
29.24 (br), 112.00; IR (cmꢀ1): 2940, 2840, 1470, 1450,
1440, 1160, 1175, 1080, 1060, 1000, 950, 910. Anal.
Calcd for C24H44O4 (414.62): C, 69.52; H, 11.18. Found
C,69.67; H, 10.89.
4. Experimental
4.1. General remarks
Ketones and methyltrioxorhenium were obtained from
commercial sources and were used as received. 30%
H2O2 (Perhydrol) was obtained from Merck KGaA.
Trifluoroethanol (TFE), 1,1,1,3,3,3-hexafluoro-2-propa-
nol (HFIP), and other solvents were distilled before use.
4.2.4. 3,3,6,6-Tetraethyl-[1,2,4,5]tetraoxane (4a). The tet-
raoxane 4a was isolated as colorless oil in 57% yield
1
(116 mg): H NMR: d 0.95 (t, 7.6 Hz, 12H), 1.64 (br s,
4H), 2.23 (br s, 4H); 13C NMR: d 6.60 (br), 7.93 (br),
23.48 (br), 26.39 (br), 110.89.25
1H and 13C NMR spectra were obtained with TMS and
CDCl3, respectively, as standards on a Varian Unity-300
spectrometer. Chemical shifts are reported in ppm from
the internal standard. Melting points were determined
4.2.5. 3,3,6,6-Tetrapropyl-[1,2,4,5]tetraoxane (4b). The
tetraoxane 4b was isolated as white solid in 45% yield
1
(116 mg): mp 52–53 °C (lit.,26 52–54 °C); H NMR: d
on a Buchi 535 apparatus and are uncorrected. Elemental
¨
0.8–1.04 (m, 12H), 1.28–1.5 (m, 8H), 1.55 (m, 4H),
2.13 (br s, 4H); 13C NMR: d 14.31, 15.71 (br), 17.00
(br), 33.26 (br), 35.99 (br), 110.47.
analyses were performed at the Microanalytisches Labor
Pascher. IR spectra were recorded on Perkin-Elmer 1310
spectrometer. Standard KBr pellet procedures were used
to obtain IR spectra of solids, while a film of neat material
was used to obtain IR spectra of liquid products.
4.2.6. 3,3,6,6-Tetrabutyl-[1,2,4,5]tetraoxane (4c). The tet-
raoxane 4c was isolated as white solid in 44% yield
1
(138 mg): mp 36.3–36.9 °C (lit.,26 37–39 °C); H NMR:
Caution. Although we have encountered no difficulties
in working with these relatively stable tetraoxanes, rou-
tine precautions (shields, fume hoods, and avoidance of
transition metal salts) should be observed whenever pos-
sible, as organic peroxides are potentially hazardous
compounds.
d 0.92 (m, 12H), 1.35 (m, 16H), 1.48–1.78 (m, 4H),
2.17 (br s, 4H); 13C d NMR: 13.87, 22.88, 24.40 (br),
25.66 (br), 30.67 (br), 33.55 (br), 110.63.
4.2.7. 3,6-Diethyl-3,6-dipropyl-[1,2,4,5]tetraoxane (4d).
The tetraoxane 4d was isolated as colorless oil in 56%
1
yield (130 mg): H NMR: d 0.95 (m, 12H), 1.30–1.53
4.2. Reaction procedure for synthesis of tetraoxanes
(m, 4H), 1.61 (br s, 4H), 2.21 (br s, 4H); 13C NMR: d
6.42 (br), 7.529 (br), 14.32, 15.50 (br), 16.60 (br), 23.97
(br), 26.68 (br), 32.80, 35.18 (br), 110.76; IR (cmꢀ1):
2960, 2860, 1460, 1160, 1140, 1000, 970, 950, 910. Anal.
Calcd for C12H24O4 (232.32): C, 62.04; H, 10.41. Found
C, 61.85; H, 10.33.
MTO (0.5 mg, 2 lmol), 30% H2O2 (0.227 mL, 2 mmol),
and 54% HBF4 solution in Et2O (0.275 mL, 2 mmol)
were dissolved in TFE (2 mL). Substrate (2 mmol) was
added and stirred at room temperature for one hour.

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7794
4.2.8. 3,6-Dibutyl-3,6-diethyl-[1,2,4,5]tetraoxane (4e).
The tetraoxane 4e was isolated as colorless oil in 55%
(br), 11.25, 20.66 (br), 24.48 (br), 26.78 (br), 29.39
(br), 30.62, 36.68 (br), 40.21 (br), 111.65; IR: 2950,
2870, 1460, 1380, 1000, 980, 950, 930 cmꢀ1. Anal. Calcd
for C16H32O4 Æ 1/4H2O (292.92): C, 65.27; H, 11.18.
Found C, 64.98; H, 10.74.
1
yield (142 mg): H NMR: d 0.82–1.06 (m, 12H), 1.22–
1.48 (m, 8H), 1.60 (br s, 4H), 2.22 (br s, 4H); 13C
NMR: d 6.62 (br), 7.52 (br), 13.84, 22.87, 23.88 (br),
25.48 (br), 26.95 (br), 30.31 (br), 32.99 (br), 110.76; IR
(cmꢀ1): 2960, 2870, 1460, 1160, 1140, 990, 960, 950.
Anal. Calcd for C14H28O4 (260.37): C, 64.58; H, 10.84.
Found C, 64.57; H, 10.75.
4.2.15. 3,6-Di-tert-butyl-3,6-dimethyl-[1,2,4,5]tetraoxane
(6e). The tetraoxane 6e was isolated as white solid in
15% yield (34 mg): mp 123.8–124 °C (lit.,36 122.5–
1
123.5 °C); H NMR: d 1.04 (s, 18H), 1.74 (s, 6H); 13C
4.2.9. 3,6-Diethyl-3,6-dipentyl-[1,2,4,5]tetraoxane (4f).
The tetraoxane 4f was isolated as colorless oil in 59%
NMR: d 15.35, 24.70, 38.89, 111.79.
1
yield (171 mg): H NMR: d 0.81–1.03 (m, 12H), 1.18–
4.3. In vitro assays
1.50 (m, 12H), 1.61 (br s, 4H), 2.21 (br s, 4H); 13C
NMR: d 6.55 (br), 7.97 (br), 13.92, 21.57 (br), 22.42,
24.08 (br), 26.78 (br), 30.52 (br), 31.94, 33.13 (br),
110.93; IR (cmꢀ1): 2960, 2860, 1460, 1160, 1140, 1000,
940, 930. Anal. Calcd for C16H32O4 Æ 1/10H2O
(290.22): C, 66.17; H, 11.18. Found C, 66.08; H, 11.08.
Chloroquine-resistant P. falciparum strain FCB1
(Colombia) was maintained in a continuous culture
on human erythrocytes as described by Trager and
Jensen.37 In vitro antiplasmodial activity of com-
pounds was determined using a modification of the
semi-automated microdilution technique of Desjardins
et al.38 Stock solutions of tested compounds were pre-
pared in DMSO. Drug solutions were serially diluted
with the culture medium and added to parasite cul-
tures synchronized at the ring stages (1% parasitemia
and 1% final hematocrit) in 96-well plates. Parasite
growth was assessed by adding 0.5 lCi of [3H]hypo-
xanthine (10–30 Ci/mmol, Amersham Biosciences Eur-
ope GmbH) to each well. Plates were incubated for
48 h at 37 °C in the appropriate atmosphere. Immedi-
ately after incubation, the plates were frozen and
thawed to lyse erythrocytes. The contents of each well
were collected on filter microplates, washed using a
cell harvester, and dried. Scintillation cocktail was
added to each filter, and radioactivity incorporated
by the parasites was measured in a scintillation count-
er. The growth inhibition for each drug concentration
was determined by comparison of the radioactivity
incorporated in the treated culture with that in the
control culture (without drug). The drug concentration
causing 50% inhibition (IC50) was determined by non-
linear regression analysis of log(dose)–response curves.
Values are the average of three experiments. DMSO
introduced into the cultures never exceeded 0.1% and
did not affect parasite growth.
4.2.10. 3,6-Diethyl-3,6-dihexyl-[1,2,4,5]tetraoxane (4g).
The tetraoxane 4g was isolated as colorless oil in 55%
1
yield (173 mg): H NMR: d 0.81–1.05 (m, 12H), 1.15–
1.48 (m, 16H), 1.60 (br s, 4H), 2.20 (br s, 4H); 13C
NMR: d 6.36 (br), 7.96 (br), 14.03, 21.96 (br), 22.54,
23.63 (br), 26.84 (br), 29.45, 31.59 (br), 33.12 (br),
110.78; IR (cmꢀ1): 2950, 2920, 2850, 1460, 1160, 1140,
1000, 960, 940. Anal. Calcd for C18H36O4 (316.48): C,
68.31; H, 11.47. Found C, 68.33; H, 11.44.
4.2.11. 3,6-Diethyl-3,6-diheptyl-[1,2,4,5]tetraoxane (4h).
The tetraoxane 4h was isolated as colorless oil in 57%
1
yield (196 mg): H NMR: d 0.77–1.01 (m, 12H), 1.12–
1.47 (m, 20H), 1.60 (br s, 4H), 2.20 (br s, 4H); 13C
NMR: d 6.53 (br), 7.92 (br), 14.06, 21.86 (br), 22.63,
23.81 (br), 26.94 (br), 29.07, 29.75, 30.46 (br), 31.74,
33.05 (br), 110.76; IR (cmꢀ1): 2950, 2920, 2850, 1460,
1150, 1140, 1000, 970, 950. Anal. Calcd for C20H40O4
(344.53): C, 69.72; H, 11.70. Found C, 69.59; H, 11.64.
4.2.12. 3,6-Diheptyl-3,6-dimethyl-[1,2,4,5]tetraoxane (4i).
The tetraoxane 4i was isolated as colorless oil in 72%
1
yield (228 mg): H NMR: d 0.82–0.95 (m, 6H), 1.1–1.5
(m, 23H), 1.60 (br s, 2H), 1.74 (br s, 3H), 2.18 (br s,
2H); 13C NMR: d 14.04, 19.15 (br), 20.02 (br), 22.22
(br), 22.63, 23.92 (br), 29.07, 29.67, 31.73, 32.46 (br),
36.48 (br), 109.23; IR (cmꢀ1): 2950, 2920, 2850, 1460,
1380, 1200, 1150, 1100, 940. Anal. Calcd for C18H36O4
(316.48): C, 68.31; H, 11.47. Found C, 68.23; H, 11.34.
Acknowledgments
This research has been supported by the Ministry of
Higher Education, Science and Technology of the
Republic Slovenia, the Young Reasearcher program
4.2.13.
3,6-Diethyl-3,6-diisopropyl-[1,2,4,5]tetraoxane
ˇ
(6a). The tetraoxane 6a was isolated as colorless oil in
26% yield (60 mg): 1H NMR: d 0.73–1.13 (m, 18H),
1.25–2.65 (m, 4H), 3.23 (br s, 2H); 13C NMR: d 7.37
(br), 16.29, 22.58 (br), 31.70 (br), 111.76; IR (cmꢀ1):
2980, 2940, 2850, 1460, 1160, 1120, 1010, 970, 960,
940, 930. Anal. Calcd for C12H24O4 (232.32): C, 62.04;
H, 10.41. Found C, 62.15; H, 10.27.
(K.Z.) of the Republic Slovenia and conducted within
the SI-FR bilateral program PROTEUS (BI-FR/03-
003). We are thankful to the staff of the NMR Center
at the National Institute of Chemistry in Ljubljana
and Dr. G.W.A. Milne.
References and notes
4.2.14. 3,6-Diethyl-3,6-bis-(2-methyl-butyl)-[1,2,4,5]tetra-
oxane (6c). The tetraoxane 6c was isolated as colorless
oil in 20% yield (59 mg): 1H NMR: d 0.75–1.05 (m,
18H), 1.05–2.58 (m, 14H); 13C NMR: d 6.57 (br), 8.31
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