Synthesis and antimalarial activity of cyclic peroxides, 1,2,4,5,7- pentoxocanes and 1,2,4,5-tetroxanes

Article abstract of DOI:10.1021/jm990014j

A variety of 1,2,4,5,7-pentoxocane and 1,2,4,5-tetroxane derivatives were prepared as potential peroxide antimalarial agents. In both series of cyclic peroxides, the steric and electronic effects of the substituents attached to the peroxide ring exert a r

Full text of DOI:10.1021/jm990014j

2604
J . Med. Chem. 1999, 42, 2604-2609
Syn th esis a n d An tim a la r ia l Activity of Cyclic P er oxid es, 1,2,4,5,7-P en toxoca n es
a n d 1,2,4,5-Tetr oxa n es
Hye-Sook Kim,† Yasuharu Shibata,† Yusuke Wataya,*,† Kaoru Tsuchiya,‡ Araki Masuyama,‡ and
Masatomo Nojima‡
Faculty of Pharmaceutical Sciences, Okayama University, Okayama 700-8530, J apan, and Department of Materials Chemistry,
Faculty of Engineering, Osaka University, Suita, Osaka 565-0871, J apan
Received J anuary 11, 1999
A variety of 1,2,4,5,7-pentoxocane and 1,2,4,5-tetroxane derivatives were prepared as potential
peroxide antimalarial agents. In both series of cyclic peroxides, the steric and electronic effects
of the substituents attached to the peroxide ring exert a remarkable influence on the
antimalarial activity. For some cyclic peroxides, which were found to be highly effective in
vitro, the study in vivo has been also conducted.
Sch em e 1
In tr od u ction
As a result of an apparent association between the
peroxide functional group and antimalarial activity, a
substantial effort has been devoted to developing new
peroxide antimalarials.1 Of these, we have been par-
ticularly interested in the fact that dispiro-1,2,4,5-
tetroxanes, e.g., 3,12-dimethyl-7,8,15,16-tetraoxadispiro-
[5.2.5.2]hexadecane, easily prepared by the acid-catalyzed
condensation of cyclohexanones and hydrogen peroxide,
exhibit remarkable antimalarial activities in vitro and
in vivo.2 To understand the detailed structure-activity
relationships, therefore, we have prepared a number of
3,6-disubstituted 1,2,4,5-tetroxanes and the related
1,2,4,5,7-pentoxocanes and have found that these cyclic
peroxides show significant antimalarial activities in
vitro, the activity being a marked function of the
substituent steric and electronic effects of these cyclic
peroxides. Some of the cyclic peroxides have been found
to be active in vivo, too.
Resu lts a n d Discu ssion
Syn th esis of 1,2,4,5,7-P en toxoca n es. About 2 de-
cades ago, we found that acidolysis of 1-phenylcyclo-
pentene ozonide (1a ) gives the novel 1,2,4,5,7-pentoxo-
cane derivative 3a , together with two stereoisomeric
tetroxanes, trans- and cis-4a .3 Also, the acidolysis of a
mixture of 1a , benzaldehyde, and hydrogen peroxide in
acetic acid led to the formation of the phenyl-substituted
1,2,4,5,7-pentoxocane 5a (Scheme 1).4 To see the pos-
sibility of 1,2,4,5,7-pentoxocanes as a potential anti-
malarial drug, we prepared in this study a series of new
pentoxocane derivatives 5b-f. Although the yields were
poor, pure compounds could be easily isolated by re-
crystallization and/or column chromatography on silica
gel (Scheme 1). Also, pentoxocanes 3b,c were prepared
by the acid-catalyzed dimerization of aryl-substituted
cyclopentene ozonides 1b,c. By the acid-catalyzed cyclo-
condensation of (4-fluorophenyl)cyclopentene ozonide
(1c) and aldehydes in the presence of H2O2, the corre-
sponding pentoxocanes 6a -c were obtained (Scheme 2).
Syn t h esis of 1,2,4,5-Tet r oxa n es. By the acid-
catalyzed dimerization of ozonides 1b,c, trans-3,6-bis-
(aryloxypropyl)-1,2,4,5-tetroxanes 4b,c were obtained
together with 3b,c (Schemes 1 and 2). As an alternative
and more general method for the synthesis of 1,2,4,5-
tetroxanes, J efford and co-workers5 reported that tri-
methylsilyl trifluoromethanesulfonate (TMSOTf)-cata-
lyzed condensation of an aldehyde and dioxybis[trimethyl-
silane] provides the symmetrically substituted 1,2,4,5-
tetroxane in high yield (Scheme 3). We repeated this
reaction to prepare a series of 3,6-dialkyl-substituted
1,2,4,5-tetroxanes 8a -d .
* Corresponding author: Yusuke Wataya. Phone: +81-86-251-7976.
Fax: +81-86-251-7974. E-mail: wataya@cc.okayama-u.ac.jp.
† Okayama University.
‡ Osaka University.
10.1021/jm990014j CCC: $18.00 © 1999 American Chemical Society
Published on Web 06/29/1999

Antimalarial Pentoxocanes and Tetroxanes
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 14 2605
Ta ble 1. In Vitro Antimalarial Activities of Pentoxocanes
against P. Falciparum and Cytotoxicities against FM3A Cellsa
Sch em e 2
EC50 values (M)
pentoxocane
3a , Ar ) Ph
3b, Ar ) 4-MeOC6H4
3c, Ar ) 4-FC6H4
5a , R ) Ph
5b, R ) heptyl
5c, R ) cyclohexyl
5d , R ) tert-butyl
5e, R ) 3-pentyl
5f, R ) 4-FC6H4
6a , R ) Ph
6b, R ) cyclohexyl
6c, R ) 4-FC6H4
artemisinin
P. falciparumb
FM3Ac
selectivityd
1.3 × 10-6
9.3 × 10-7
7.1 × 10-7
1.4 × 10-6
1.5 × 10-6
2.1 × 10-7
1.7 × 10-5
5.1 × 10-7
2.8 × 10-6
2.9 × 10-6
1.5 × 10-7
1.9 × 10-6
7.8 × 10-9
NTe
>77
19
>141
36
8
1.8 × 10-5
NTe
5.0 × 10-5
1.2 × 10-5
1.0 × 10-5
2.6 × 10-5
3.3 × 10-5
1.7 × 10-5
8.1 × 10-6
2.5 × 10-6
9.0 × 10-6
1.0 × 10-5
48
2
65
6
4
17
5
1280
a
In vitro antimalarial activities and cytotoxicities are described
in the Experimental Section. b Chloroquine-sensitive (FCR-3 strain).
c Mouse mammary tumor FM3A cells in culture as a control for
d
mammalian cell cytotoxicity. Selectivity ) (mean of EC50 value
for FM3A cells)/(mean of EC50 value for P. falciparum). e NT, not
toxic at a 1 × 10-4 M concentration.
Sch em e 3
Ta ble 2. In Vitro Antimalarial Activities of Tetroxanes against
P. Falciparum and Cytotoxicities against FM3A Cellsa
EC50 values (M)
tetroxane
P. falciparumb
FM3Ac
selectivityd
trans-4a , Ar ) Ph
cis-4a , Ar ) Ph
4b, Ar ) 4-MeOC6H5
4c, Ar ) 4-FC6H4
8a , R ) heptyl
8b, R ) cyclohexyl
8c, R ) phenyl
8d , R ) benzyl
8e, R ) 4-MeOC6H4
8f, R ) 4-FC6H4
8g, R ) 2-CF3C6H4
8h , R ) 4-CF3C6H4
4.0 × 10-7
1.2 × 10-5
2.0 × 10-5
5.1 × 10-7
1.5 × 10-6
2.0 × 10-7
1.1 × 10-6
1.4 × 10-5
7.9 × 10-7
5.0 × 10-7
6.0 × 10-7
NTe
NTe
>250
>8
NTe
NTe
>5
NTe
>169
>67
55
>91
>7
>127
>200
5
NTe
1.1 × 10-5
NTe
NTe
Sch em e 4
NTe
NTe
3.0 × 10-6
NTe
a
In vitro antimalarial activities and cytotoxicities are described
in the Experimental Section. b Chloroquine-sensitive (FCR-3 strain).
c Mouse mammary tumor FM3A cells in culture as a control for
d
mammalian cell cytotoxicity. Selectivity ) (mean of EC50 value
for FM3A cells)/(mean of EC50 value for P. falciparum). e NT, not
toxic at a 1 × 10-4 M concentration.
In the case of the 3,6-diaryl-substituted tetroxanes,
the more conventional method was found to be effective,
i.e., H2SO4-catalyzed cyclocondensation of aldehydes and
hydrogen peroxide provided the expected tetroxanes
8e-h (Scheme 4).2 In the case of 3,6-bis(4-methoxyl-
phenyl)-1,2,4,5-tetroxane (8e), the yield was very poor.
Our trial to prepare the same compound 8e by the
method of J efford5 also failed. The reason is obscure.
An tim a la r ia l Activities of 1,2,4,5,7-P en toxoca n es
a n d 1,2,4,5-Tetr oxa n es in Vitr o. With a series of
1,2,4,5,7-pentoxocanes in hand, we tested the anti-
malarial activities against P. falciparum and cytotox-
icities against FM3A cells (Table 1).6,7 The results are
summarized as follows. (a) EC50 values of pentoxocanes
3a -c and 5a ,c,e against P. falciparum are in the ranges
of (1.4 × 10-6)-(2.1 × 10-7) M, and moreover, the
selectivities are notable. (b) The nature of the alkyl
substituent at C-4 shows a remarkable influence on the
activity, the cyclohexyl- and 3-pentyl-substituted ones,
5c,e, being the most attractive. (c) When 3a -c are
compared, it is noticed that substitution by the electron-
withdrawing fluorine increases the activity. Also, the
selectivity of 3c was remarkable (>141). In this respect,
Porter and co-workers8 have found a similar fluorine
atom effect on the antimalarial activity for 1,4-diaryl-
substituted 3,4-dioxabicyclo[2.2.2]octanes. In the case
of fluorophenyl-substituted pentoxocanes, 5f and 6c,
however, the activity was moderate and, moreover, the
selectivity was poor. In summary, 1,2,4,5,7-pentoxo-
canes are a new class of antimalarial peroxides; con-
sidering the ease of the preparation, the modification
of the structure would lead to the discovery of the
promising antimalarial drugs.
A similar substituent electronic effect was observed
for 1,2,4,5-tetroxanes 4a -c, too: i.e., the activity follows
the sequence: trans-4a (Ar ) Ph) ) 4c (Ar ) 4-FC6H4)
. 4b (Ar ) 4-MeOC6H4) (Table 2). It was surprising to
note that in contrast to trans-4a , the stereoisomeric cis-
4a was inactive, suggesting that the difference in
structure of the tetroxanes plays an important role. For
a series of 3,6-diaryl- or 3,6-dialkyl-substituted tetrox-
anes 8a -h , the diaryl-substituted tetroxanes 8c,e,f
were found to show remarkable activities against P.
falciparum. In marked contrast, 3,6-bis(trifluorometh-
ylphenyl)-1,2,4,5-tetroxanes 8g,h were ineffective. Also,
the dialkyl-substituted 1,2,4,5-tetroxanes 8a ,b,d showed

2606 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 14
Kim et al.
Ta ble 3. Antimalarial Activities of 1,2,4,5,7-Pentoxocanes and
to artemisinin (ED50: 5.4 mg/kg; data will be published
elsewhere). Although growth of the parasites was sup-
pressed during administration with 8b,f (each of 50 mg/
kg/day), malaria parasites were still observed in blood
stream after the 4-day suppressive test. Thus, all of the
mice treated with 8b,f died due to P. berghei infection.
The similar trend was observed for artemisinin. Our
experiment disclosed that artemisinin at a dose of 50
mg/kg/day also failed to resolve infection in mice.
Particularly interesting is the remarkably low toxicity
of these two agents (8b,f): no signs, including weight
loss and diarrhea, were observed at a total dose of 200
mg/kg (ip). 1,2,4,5,7-Pentoxocanes were either inactive
or less active than were 1,2,4,5-tetroxanes. It is also
interesting to note that the fluorine-substituted pent-
oxocane 3c and tetroxane 4c were ineffective in vivo,
although they showed notable characteristics in vitro.8
In summary, we have prepared a number of new
1,2,4,5-tetroxanes and 1,2,4,5,7-pentoxocanes to test
antimalarial activity. The study in vitro demonstrates
that these types of cyclic peroxides are active, the extent
being a marked function of the steric and electronic
effects of the substituents attached to the peroxide ring.
By the study in vivo, 3,6-dicyclohexyl- and 3,6-bis(4-
fluorophenyl)-1,2,4,5-tetroxanes have been found to be
promising as new antimalarial agents. These results
may help in the design of better chemotherapeutic
1,2,4,5-tetroxanes in the worldwide fight against ma-
laria.
1,2,4,5-Tetroxanes in Vivoa
growth inhibition (%)
of P. berghei in miceb
compd
20 mg/kg/day
50 mg/kg/day
Tetroxane
trans-4a , Ar ) Ph
4c, Ar ) 4-FC6H4
8b, R ) cyclohexyl
8c, R ) phenyl
30
0
50
6
40
30
96
50
90
8f, R ) 4-FC6H4
50
Pentoxocane
3c, Ar ) 4-FC6H4
5c, R ) cyclohexyl
5e, R ) 3-pentyl
0
0
15
0
15
18
artemisinin
93
100
a
In vivo antimalarial activity is described in the Experimental
b
Section. Growth inhibition (%) ) [(mean % parasitemia in
controls - mean % parasitemia in treated)/mean % parasitemia
in controls] × 100.
moderate to poor activities against P. falciparum, the
activity following the sequence 8b (R ) cyclohexyl) >
8a (R ) heptyl) > 8d (R ) benzyl) (Table 2).
By the intensive study on the origin of the notable
antimalarial activity of artemisinin and some related
synthetic organic endoperoxides, Posner and co-workers9
have proposed that the first step of the action involves
homolytic cleavage of the peroxide bridge by single
electron transfer from an Fe(II) species to produce an
oxy radical; subsequent intramolecular 1,5-hydride shift
leads to the formation of the carbon radical which, as
an active intermediate, would kill malaria parasites. For
an active antimalarial drug, arteflene, too, intervention
of a radical intermediate, derived from the O-O and
the subsequent C-C bond scission, has been sug-
gested.10 The structure-activity relationship observed
for 1,2,4,5,7-pentoxocanes and 1,2,4,5-tetroxanes, how-
ever, may suggest that a different factor would be also
important. If the activity were controlled by the stability
of the carbon radical, the tert-butyl-substituted pent-
oxocane 5d and dibenzyl-substituted tetroxane 8d might
have been most active. This is clearly inconsistent with
the experimental observations (Tables 1 and 2). To-
gether with the remarkable difference in activity be-
tween trans- and cis-4a , we prefer to consider that the
substituent-dependent ease in access of an Fe(II) species
such as heme to the peroxide bridge would play an
important role, particularly when complexation precedes
activation.11 Perhaps in accordance with this, Avery12
has demonstrated that large substituents at C-3 in
artemisinin homologues suppress activity, irrespective
of their electronic qualities.
An tim a la r ia l Activities of 1,2,4,5,7-P en toxoca n es
a n d 1,2,4,5-Tetr oxa n es in Vivo. In vivo antimalarial
activity for 1,2,4,5,7-pentoxocanes and 1,2,4,5-tetrox-
anes against P. berghei NK 65 strain was determined.13
Results shown in Table 3 indicate that 1,2,4,5-tetrox-
anes had potent antimalarial activities compared with
those of 1,2,4,5,7-pentoxocanes in vivo. The activity of
1,2,4,5-tetroxanes follows the sequence: 8b (R ) cyclo-
hexyl) ) 8f (R ) 4-FC6H4) > trans-4a (Ar ) Ph) > 8c
(R ) phenyl) > 4c (Ar ) 4-FC6H4). Especially, tetrox-
anes 8b,f seem to be potent antimalarial agents against
P. berghei (ED50: approximately 20 mg/kg). Regretfully,
8b,f have, however, weak antimalarial activity relative
Exp er im en ta l Section
Gen er a l P r oced u r e. 1H (270 MHz) and 13C (67.5 MHz)
NMR spectra were obtained in CDCl3 with SiMe4 as standard.
Ozonides 1a -c,3 pentoxocanes 3a 3 and 5a ,4 and tetroxanes
trans- and cis-4a 3 and 8c5 were prepared by the reported
method.
(4-Meth oxyp h en yl)cyclop en ten e ozon id e (1b): mp 64-
1
65 °C (from hexane/ether); H NMR δ 1.8-2.3 (m, 6 H), 3.82
(s, 3 H), 5.98 (s, 1 H), 6.92 (d, J ) 8.9 Hz, 2 H), 7.48 (d, J )
8.9 Hz, 2 H); 13C NMR δ 16.08, 29.09, 32.61, 55.27, 103.66,
107.92, 113.71, 127.55, 129.85, 130.35, 160.48, 163.48. Anal.
(C12H14O4) C, H.
(4-F lu or op h en yl)cyclop en ten e ozon id e (1c): an oil; 1H
NMR δ 1.8-2.4 (m, 6 H), 5.99 (s, 1 H), 7.07 (t, J ) 8.8 Hz, 2
H), 7.53 (dd, J ) 8.8 and 5.3 Hz, 2 H); 13C NMR δ 16.06, 29.00,
32.97, 103.79, 107.60, 115.37 (d, J ) 20.8 Hz), 128.12, 131.82,
163.38 (d, J ) 249 Hz). Anal. (C11H11FO3) C, H.
Ca u tion : Since organic peroxides are potentially hazardous
compounds, they must be handled with due care; avoid
exposure to strong heat or light, mechanical shock, oxidizable
organic materials, and transition-metal ions. No particular
difficulties were experienced in handling any of the new
peroxides synthesized in this work using the reaction scales
and procedures described below together with the safeguard
mentioned above.
Syn t h esis of 1,2,4,5,7-P en t oxoca n es b y t h e Acid -
Ca ta lyzed Cyclocon d en sa tion of Ar ylcyclop en ten e Ozo-
n id es 1a -c, Ald eh yd es, a n d Hyd r ogen P er oxid e. The
reaction of 1-phenylcyclopentene ozonide (1a ) with octanal is
representative. An equimolar mixture of 1a (600 mg, 3.13
mmol) and hydrogen peroxide (30%; 355 mg, 3.13 mmol) in
the presence of chlorosulfonic acid (36 mg, 0.31 mmol) in acetic
acid-CH2Cl2 (35 mL, 5:2, v/v) was stirred at room temperature
for 30 min. Then, octanal (400 mg, 3.13 mmol) was added, and
the mixture was stirred for a further 90 min. Workup of the
reaction mixture was carried out by pouring it into water,
extracting the latter with ether, and washing the organic
extracts with portions of aqueous NaHCO3 and saturated
brine. After evaporation of the solvent, the neutral products

Antimalarial Pentoxocanes and Tetroxanes
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 14 2607
1
were isolated by column chromatography on silica gel, using
benzene-hexane (3:2) as an eluent. The first fraction contained
pentoxocane 5b (184 mg, 18%).
132-133 °C (from methanol); H NMR δ 1.5-2.1 (m, 10 H),
2.89 (t, J ) 6.9 Hz, 2 H), 3.80 (s, 3 H), 3.86 (s, 3 H), 5.62 (s, 1
H), 5.74 (t, J ) 5.6 Hz, 1 H), 6.8-7.0 (m, 4 H), 7.46 (d, J ) 8.6
Hz, 2 H), 7.87 (d, J ) 8.90 Hz, 2 H); 13C NMR δ 14.79, 19.57,
24.76, 28.01, 34.07, 37.52, 55.24, 55.44, 98.44, 103.02, 108.88,
113.39, 113.64, 122.28, 126.68, 130.28, 134.57, 159.28, 163.36,
198.16. Anal. (C24H24O8) C, H.
1,4-Dip h en yl-2,3,5,6,11-p en t a oxa b icyclo[5.3.1]u n d ec-
1
a n e (5a ):3 mp 134-135 °C (from methanol); H NMR δ 1.4-
2.4 (m, 6 H), 5.73 (s, 1 H), 6.62 (s, 1 H), 7.1-7.7 (m, 10 H).
Anal. (C18H18O5) C, H.
tr a n s-4,4′-(1,2,4,5-Tetr oxa n e-3,6-d iyl)bis[1-(4-m eth oxy-
p h en yl)-1-bu ta n on e] (4b): mp 149-150 °C (from methanol);
1H NMR δ 1.5-2.0 (m, 8 H), 2.96 (t, J ) 7.3 Hz, 4 H), 3.87 (s,
6 H), 5.94 (t, J ) 5.3 Hz, 2 H), 6.92 (d, J ) 8.9 Hz, 4 H), 7.91
(d, J ) 8.9 Hz, 4 H); 13C NMR δ 18.26, 29.11, 37.32, 55.49,
108.59, 113.75, 129.85, 130.28, 163.48, 212.59. Anal. (C24H28O8)
C, H.
4-H ep t yl-1-p h en yl-2,3,5,6,11-p en t a oxa b icyclo[5.3.1]-
u n d eca n e (5b): mp 64-65 °C (from methanol); H NMR δ
0.85 (t, J ) 6.9 Hz, 3 H), 1.2-2.1 (m, 18 H), 5.64 (s, 1 H), 5.68
(t, J ) 5.9 Hz, 1 H), 7.3-7.6 (m, 5 H); 13C NMR δ 13.95, 22.48,
24.89, 28.29, 28.81, 28.92, 29.00, 31.52, 33.89, 43.78, 98.24,
102.86, 109.17, 125.28, 125.84, 127.92, 127.98, 142.08. Anal.
(C19H28O5) C, H.
1
1-(4-F lu or op h e n yl)-4-[1-(4-flu or op h e n yl)-2,3,5,6,11-
p en ta oxa bicyclo[5.3.1]u n d ec-4-yl]-1-bu ta n on e (3c): mp
126-127 °C (from methanol); H NMR δ 1.5-2.1 (m, 10 H),
4-Cycloh exyl-1-ph en yl-2,3,5,6,11-pen taoxabicyclo[5.3.1]-
u n d eca n e (5c): mp 121-122 °C (from methanol); 1H NMR δ
1.2-2.1 (m, 17 H), 5.44 (d, J ) 6.8 Hz, 1 H), 5.62 (s, 1 H),
7.3-7.6 (m, 5 H); 13C NMR δ 14.72, 24.76, 25.41, 25.52, 25.86,
27.80, 28.56, 33.87, 38.08, 98.24, 102.84, 111.50, 125.39,
125.89, 127.98, 128.32, 142.16. Anal. (C18H24O5) C, H.
4-ter t-Bu tyl-1-p h en yl-2,3,5,6,11-p en ta oxa bicyclo[5.3.1]-
1
2.92 (t, J ) 7.6 Hz, 2 H), 5.62 (s, 1 H), 5.74 (t, J ) 5.9 Hz, 1
H), 7.02 (t, J ) 8.7 Hz, 2 H), 7.11 (t, J ) 8.6 Hz, 2 H), 7.51
(dd, J ) 8.9 and 5.6 Hz, 2 H), 7.91 (dd, J ) 8.9 and 5.3 Hz, 2
H); 13C NMR δ 14.74, 19.21, 24.71, 27.91, 34.00, 37.75, 98.45,
102.82, 108.84, 114.93 (d, J ) 22 Hz), 115.63 (d, J ) 22 Hz),
127.28, 130.65, 133.26, 138.04, 161.68 (d, J ) 246.1 Hz), 162.52
(d, J ) 246.1 Hz), 197.75. Anal. (C22H22F2O6) C, H.
tr a n s-4,4′-(1,2,4,5-Tetr oxan e-3,6-diyl)bis[1-(4-flu or oph en -
yl)-1-bu ta n on e] (4c): mp 155-156 °C (from ether); 1H NMR
δ 1.5-2.0 (m, 8 H), 2.99 (t, J ) 7.3 Hz, 4 H), 5.95 (t, J ) 5.6
Hz, 2 H), 7.13 (t, J ) 8.9 Hz, 4 H), 7.95 (dd, J ) 8.9 and 5.3
Hz, 4 H); 13C NMR δ 17.99, 29.00, 37.57, 108.53, 115.72 (d, J
) 21.9 Hz), 130.53, 130.67, 133.13, 165.76 (d, J ) 253 Hz),
197.37. Anal. (C22H22F2O6) C, H.
Syn th esis of 1,2,4,5-Tetr oxa n es by TMSOTf-Ca ta lyzed
Cyclocon d en sa tion of Ald eh yd es a n d Dioxybis[tr im eth -
ylsila n e]. The synthesis of tetroxane 8b is representative. To
an ice-cold solution of TMSOTf (666 mg, 3.00 mmol) in CH3CN
(15 mL) was added dioxybis[trimethylsilane]14 (540 mg, 3.00
mmol) by a syringe over 4 min under argon. Then, a solution
of cyclohexanecaboxaldehyde (336 mg, 3.00 mmol) in CH3CN
(20 mL) was added by a syringe during 30 min at 0 °C. After
stirring for more than 90 min at the same temperature, the
mixture was poured into ether (70 mL). Then, the organic layer
was washed with ice-cold NaHCO3 and saturated brine and
dried over anhydrous MgSO4. After evaporation of the solvent
under vacuum, the residue was separated by column chroma-
tography on silica gel. Elution with ether-hexane (1:50) gave
the tetroxane 8b (223 mg, 58%).
1
u n d eca n e (5d ): mp 141-142 °C (from methanol); H NMR
δ 0.95 (s, 9 H), 1.5-2.1 (m, 6 H), 5.35 (s, 1 H), 5.61 (s, 1 H),
7.3-7.6 (m, 5 H); 13C NMR δ 14.74, 24.80, 25.84, 33.55, 34.92,
98.17, 102.70, 113.26, 125.43, 125.91, 127.89, 128.36, 128.85,
142.16. Anal. (C16H22O5) C, H.
4-(3-P en tyl)-1-p h en yl-2,3,5,6,11-p en ta oxa bicyclo[5.3.1]-
1
u n d eca n e (5e): mp 68-69 °C (from methanol); H NMR δ
0.86 (t, J ) 6.9 Hz, 6 H), 1.2-2.2 (m, 11 H), 5.60 (br s, 2 H),
7.2-7.4 (m, 5 H); 13C NMR δ 11.23, 11.28, 14.81, 21.62, 22.07,
24.83, 33.76, 42.01, 98.33, 102.84, 110.31, 125.42, 127.98,
129.88, 142.28. Anal. (C17H24O5) C, H.
4-(4-Flu or oph en yl)-1-ph en yl-2,3,5,6,11-pen taoxabicyclo-
1
[5.3.1]u n d eca n e (5f): mp 113-114 °C (from methanol); H
NMR δ 1.5-2.3 (m, 6 H), 5.73 (s, 1 H), 6.61 (s, 1 H), 6.96 (t, J
) 8.9 Hz, 2 H), 7.3-7.4 (m, 5 H), 7.58 (d, J ) 7.9 Hz, 2 H); 13
C
NMR δ 14.81, 24.80, 33.89, 98.69, 103.36, 107.83, 115.31 (d, J
) 21.9 Hz), 125.35, 127.15, 128.12, 129.23, 129.36, 142.03,
163.53 (d, J ) 249.1 Hz). Anal. (C18H17FO5) C, H.
1-(4-Flu or oph en yl)-4-ph en yl-2,3,5,6,11-pen taoxabicyclo-
1
[5.3.1]u n d eca n e (6a ): mp 122-123 °C (from methanol); H
NMR δ 1.5-2.2 (m, 6 H), 5.70 (s, 1 H), 6.62 (s, 1 H), 7.02 (t, J
) 8.8 Hz, 2 H), 7.3-7.8 (m, 7 H); 13C NMR δ 14.81, 24.78,
33.93, 98.65, 102.98, 108.53, 114.91 (d, J ) 22.0 Hz), 127.22,
127.27, 128.38, 128.46, 130.04, 131.07, 138.02, 162.52 (d, J )
245 Hz). Anal. (C18H17FO5) C, H.
3,6-Dih ep tyl-1,2,4,5-tetr oxa n e (8a ): mp 57-58 °C (from
methanol); 1H NMR δ 0.88 (t, J ) 6.3 Hz, 6 H), 1.2-1.6 (m, 24
H), 5.87 (t, J ) 5.6 Hz, 2 H); 13C NMR δ 14.05, 22.61, 23.49,
24.84, 29.15, 29.69, 31.69, 109.40. Anal. (C16H32O4) C, H.
3,6-Dicycloh exyl-1,2,4,5-tetr oxa n e (8b): mp 68-69 °C
4-Cycloh exyl-1-(4-flu or op h en yl)-2,3,5,6,11-p en t a oxa -
bicyclo[5.3.1]u n d eca n e (6b): mp 131-132 °C (from metha-
1
nol); H NMR δ 1.2-2.1 (m, 17 H), 5.42 (d, J ) 6.6 Hz, 1 H),
5.60 (s, 1 H), 7.03 (t, J ) 8.6 Hz, 2 H), 7.52 (dd, J ) 8.3 and
5.3 Hz, 2 H); 13C NMR δ 14.75, 24.78, 25.57, 25.93, 28.05,
28.59, 33.96, 38.17, 98.31, 102.60, 111.57, 114.70, 115.02,
1
(from methanol); H NMR δ 1.5-2.0 (m, 22 H), 5.66 (t, J )
6.3 Hz, 2 H); 13C NMR δ 25.34, 25.88, 26.81, 39.03, 110.96.
Anal. (C14H24O4) C, H.
127.33, 127.44, 138.17, 162.51 (d, J ) 247 Hz). Anal. (C18H23
FO5) C, H.
-
3,6-Diben zyl-1,2,4,5-tetr oxa n e (8d ): mp 112-113 °C
(from methanol); 1H NMR δ 2.85 (d, J ) 5.3 Hz, 4 H), 6.08 (t,
J ) 5.6 Hz, 2 H), 7.0-7.4 (m, 10 H); 13C NMR δ 36.24, 108.57,
127.20, 128.57, 129.45, 133.10. Anal. (C16H16O4) C, H.
Syn th esis of 1,2,4,5-Tetr oxa n es by th e H2SO4-Ca ta -
lyzed Cyclocon d en sa tion of Ar yla ld eh yd es w ith Hyd r o-
gen P er oxid e. Reaction of 4-fluorobenzaldehyde is repre-
sentative. Aqueous H2SO4 (36 wt %; 50 mL) was added by slow
dropwise addition to a solution of aqueous ethanol (1:1 v/v, 50
mL) at 0 °C. Then, a solution of 4-fluorobenzaldehyde (5.00 g,
40 mmol) and 30% H2O2 (2.5 mL, 22.06 mmol) in ethanol (10
mL) was added in one portion, and the mixture was stirred
for 1 day. After workup as described above, tetroxane 8f was
isolated by recrystallization (1.22 g, 22%).
1,4-Bis(4-flu or oph en yl)-2,3,5,6,11-pen taoxabicyclo[5.3.1]-
u n d eca n e (6c): mp 112-113 °C (from methanol); 1H NMR δ
1.5-2.2 (m, 6 H), 5.72 (s, 1 H), 6.60 (s, 1 H), 7.0-7.1 (m, 4 H),
7.3-7.4 (m, 2 H), 7.5-7.6 (m, 2 H); 13C NMR δ 14.81, 24.76,
33.94, 98.72, 103.07, 107.83, 114.96 (d, J ) 20.7 Hz), 115.54
(d, J ) 22.0 Hz), 127.24, 127.37, 129.22, 129.34, 137.93, 137.98,
162.57 (d, J ) 246 Hz), 163.61 (d, J ) 249 Hz). Anal.
(C18H16F2O5) C, H.
Acid-Catalyzed Dim er ization of Ar ylcyclopen ten e Ozo-
n id es. Reaction of (4-fluorophenyl)cyclopentene ozonide (1c)
is representative. A mixture of ozonide 1c (210 mg, 1.00 mmol)
and ClSO3H (0.3 equiv) in CH2Cl2 (10 mL) was stirred at room
temperature for 30 min. After workup as described above, the
mixture of the crude products was triturated with ether to give
the tetroxane 4c (41 mg, 10%). After evaporation of the ether,
the residue was column chromatographed on silica gel. Elution
with ether-hexane (1:9) gave the pentoxocane 3c (125 mg,
30%).
3,6-Bis(4-m eth oxyp h en yl)-1,2,4,5-tetr oxa n e (8e): mp
214-215 °C (from methanol); 1H NMR δ 3.83 (s, 6 H), 6.84 (s,
2 H), 6.94 (d, J ) 8.9 Hz, 4 H), 7.45 (d, J ) 8.9 Hz, 4 H). Anal.
(C16H16O6) C, H.
1-(4-Met h oxyp h en yl)-4-[1-(4-m et h oxyp h en yl)-2,3,5,6,
11-pen taoxabicyclo[5.3.1]u n dec-4-yl]-1-bu tan on e (3b): mp
3,6-Bis(4-flu or op h en yl)-1,2,4,5-tetr oxa n e (8f): mp 222-
1
223 °C (from methanol); H NMR δ 6.89 (s, 2 H), 7.14 (t, J )

2608 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 14
Kim et al.
8.7 Hz, 4 H), 7.52 (dd, J ) 8.9 and 5.3 Hz, 4 H). Anal.
(C14H10F2O4) C, H.
assigned to treated groups and housed in cages each containing
five individuals. Parasites were collected by cardiac puncture
in a heparinized syringe from a donor mouse harboring about
15% parasitemia. The blood was diluted with 0.9% NaCl
solution to a final concentrations of 1 × 106 infected erythro-
cytes/0.2 mL of infecting suspension. Test compounds were
prepared at doses of 20 and 50 mg/kg in dimethyl sulfoxide.
Five animals were treated with each dose. Initially, each
mouse was inoculated intravenously in the tail vein with 1 ×
106 parasitized erythrocytes (infecting suspension in 0.2 mL
of 0.9% NaCl solution). The compounds were administrated
once a day starting on day 0 and continued on day 1, day 2,
and day 3. The first administration of test compound intra-
peritoneally started 2 h after parasite inoculation. Parasitemia
levels were determined on the day following the last treatment
(on day 4). To evaluate the antimalarial activity of the
compounds, we prepared tail blood smears and stained them
with Giemsa (E. Merck, Germany). Total 1 × 104 erythro-
cytes/1 thin blood film were examined under microscopy. On
day 4, parasitemia of control mice were between 18% and 22%.
The suppression of parasitemia for the dose of 1,2,4,5,7-
pentoxocanes and 1,2,4,5-tetroxanes were caluated by the
formula: [(average % parasitemia in controls (sham-treated)
- average % parasitemia, in treated mice)/average % para-
sitemia in controls (sham-treated)] × 100. Five infected,
dimethyl sulfoxide-dosed mice were used as a control. Treat-
ment was considered curative when no parasites were detected
after 60 days. The data shown are the mean values from five
mice in one test. The care and treatment of mice were in
accordance with the guidelines (No. 141, 1987) issued by the
Science and International Affairs Bureau of the J apanese
Ministry of Education, Science, Culture and Sports.
3,6-Bis(2-t r iflu or om e t h ylp h e n yl)-1,2,4,5-t e t r oxa n e
1
(8g): mp 172-173 °C (from methanol); H NMR δ 7.32 (s, 2
H), 7.6-7.9 (m, 8 H); 13C NMR δ 104.53, 123.54 (q, J ) 274
Hz), 126.18, 126.25, 129.38, 129.70, 131.57, 132.40. Anal.
(C16H10F6O4) C, H.
3,6-Bis(4-t r iflu or om e t h ylp h e n yl)-1,2,4,5-t e t r oxa n e
1
(8h ): mp 206-207 °C (from methanol); H NMR δ 7.00 (s, 2
H), 7.65 (d, J ) 8.6 Hz, 4 H), 7.73 (d, J ) 8.6 Hz, 4 H). Anal.
(C16H10F6O4) C, H.
An tim a la r ia l Activities of 1,2,4,5,7-P en toxoca n es a n d
1,2,4,5-Tetr oxa n es in Vitr o. Ma la r ia p a r a sites: Plasmo-
dium falciparum (ATCC 30932, FCR-3 strain) was used in our
study. P. falciparum was cultivated by a modification of the
method of Trager and J ensen15 using a 5% hematocrit of type
A human red blood cells suspended in RPMI 1640 medium
(Gibco, NY) supplemented with heat-inactivated 10% type A
human serum. The plates were placed in a CO2-O2-N2
incubator (5% CO2, 5% O2, and 90% N2 atmosphere) at 37 °C,
and the medium was changed daily until 5% parasitemia
(which means that 5 parasite-infected erythrocytes in every
100 erythrocytes were existing).
Ma m m a lia n Cells: Mouse mammary tumor FM3A cells
(wild-type, subclone F28-7)16 were supplied by the J apanese
Cancer Research Resources Bank (J CRB). FM3A cells were
maintained in suspension culture at 37 °C in a 5% CO2
atmosphere in plastic bottles containing ES medium (Nissui
Pharmaceuticals, Tokyo, J apan) supplemented with 2% heat-
inactivated fetal bovine serum (Gibco, NY).
In vitr o a n tim a la r ia l a ctivity of 1,2,4,5-tetr oxa n es a n d
1,2,4,5,7-p en toxoca n es: The following procedures were used
for assay of antimalarial activity.6, 7 Asynchronously cultivated
P. falciparum were used. Various concentrations of compounds
in dimethyl sulfoxide were prepared. Five microliters of each
solution was added to individual wells of a multidish, 24 wells.
Erythrocytes with 0.3% parasitemia were added to each well
containing 995 µL of culture medium to give a final hematocrit
level of 3%. The plates were incubated at 37 °C for 72 h in a
CO2-O2-N2 incubator (5% CO2, 5% O2, and 90% N2 atmo-
sphere). To evaluate the antimalarial activity of test com-
pound, we prepared thin blood films from each culture and
stained them with Giemsa (E. Merck, Germany). Total 1 ×
104 erythrocytes/1 thin blood film were examined under
microscopy. All of the test compounds were assayed in dupli-
cate at each concentration. Drug-free control cultures were run
simultaneously. All data points represent the mean of three
experiments. Parasitemia in control reached between 4% and
5% at 72 h. The EC50 value refers to the concentration of the
compound necessary to inhibit the increase in parasite density
at 72 h by 50% of control.
Toxicity a ga in st m a m m a lia n cell lin e: FM3A cells grew
with a doubling time of about 12 h. Prior to exposure to drugs,
cell density was adjusted to 5 × 104 cells/mL. A cell suspension
of 995 µL was dispensed to the test plate, and compound at
various concentrations suspended in dimethyl sulfoxide (5 µL)
was added to individual wells of a multidish, 24 wells. The
plates were incubated at 37 °C in a 5% CO2 atmosphere for
48 h. All of the test compounds were assayed in duplicate at
each concentration. Cell numbers were measured using a
microcell counter CC-130 (Toa Medical Electric Co., J apan).
All data points represent the mean of three experiments. The
EC50 value refers to the concentration of the compound
necessary to inhibit the increase in cell density at 48 h by 50%
of control. Selectivity refers to the mean of EC50 value for
FM3A cells per the mean of EC50 value for P. falciparum.
An tim a la r ia l Activities of 1,2,4,5,7-P en toxoca n es a n d
1,2,4,5-Tetr oxa n es in Vivo. In vivo antimalarial activities
of 1,2,4,5,7-pentoxocanes and 1,2,4,5-tetroxanes were deter-
mined in mice infected with P. berghei (NK 65 strain).13 Five-
week-old ICR male mice obtained in sterile containers from
Charles River Breeding Laboratories, Inc. (Yokohama, J apan)
weighing 22-25 g were used. They were housed under a
natural day-night cycle at 25 °C. The mice were randomly
Ack n ow led gm en t. This work was supported in part
by a Grant-in Aid for Scientific Research on Priority
Areas (10153238, 10166211, and 08281105) from the
Ministry of Education, Science, Culture and Sports of
J apan. We thank the British Council (Tokyo) for the
award of a travel grant to M.N.
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