Two-step synthesis of achiral dispiro-1,2,4,5-tetraoxanes with outstanding antimalarial activity, low toxicity, and high-stability profiles

Article abstract of DOI:10.1021/jm701435h

A rapid, two-step synthesis of a range of dispiro-1,2,4,5-tetraoxanes with potent antimalarial activity both in vitro and in vivo has been achieved. These 1,2,4,5-tetraoxanes have been proven to be superior to 1,2,4-trioxolanes in terms of stability and to be superior to trioxane analogues in terms of both stability and activity. Selected analogues have in vitro nanomolar antimalarial activity and good oral activity and are nontoxic in screens for both cytotoxicity and genotoxicity. The synthesis of a fluorescent 7-nitrobenza-2-oxa-1,3-diazole (NBD) tagged tetraoxane probe and use of laser scanning confocal microscopy techniques have shown that tagged molecules accumulate selectively only in parasite infected erythrocytes and that intraparasitic formation of adducts could be inhibited by co-incubation with the iron chelator desferrioxamine (DFO).

Full text of DOI:10.1021/jm701435h

2170
J. Med. Chem. 2008, 51, 2170–2177
Two-Step Synthesis of Achiral Dispiro-1,2,4,5-tetraoxanes with Outstanding Antimalarial
Activity, Low Toxicity, and High-Stability Profiles†
Gemma L. Ellis,‡ Richard Amewu,‡ Sunil Sabbani,§ Paul A. Stocks,‡ Alison Shone,# Deborah Stanford,‡ Peter Gibbons,‡
Jill Davies,# Livia Vivas,| Sarah Charnaud,| Emily Bongard,| Charlotte Hall,# Karen Rimmer,# Sonia Lozanom, María Jesús,
Domingo Gargallo, Stephen A. Ward,# and Paul M. O’Neill*,‡
Department of Chemistry, UniVersity of LiVerpool, LiVerpool, L69 7ZD, U.K., LiVerpool School of Tropical Medicine, Pembroke Place,
LiVerpool, L3 5QA, U.K., Chemistry, Department of Natural Sciences, Mid Sweden UniVersity, SE-85170 SundsVall, Sweden, Department of
Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel Street, London, WC1E 7HT, U.K., and
GlaxoSmithKline, S.A. Parque Tecnológico de Madrid, SeVero Ochoa, 2, 28760 Tres Cantos, Madrid, Spain
ReceiVed NoVember 14, 2007
A rapid, two-step synthesis of a range of dispiro-1,2,4,5-tetraoxanes with potent antimalarial activity both
in vitro and in vivo has been achieved. These 1,2,4,5-tetraoxanes have been proven to be superior to 1,2,4-
trioxolanes in terms of stability and to be superior to trioxane analogues in terms of both stability and
activity. Selected analogues have in vitro nanomolar antimalarial activity and good oral activity and are
nontoxic in screens for both cytotoxicity and genotoxicity. The synthesis of a fluorescent 7-nitrobenza-2-
oxa-1,3-diazole (NBD) tagged tetraoxane probe and use of laser scanning confocal microscopy techniques
have shown that tagged molecules accumulate selectively only in parasite infected erythrocytes and that
intraparasitic formation of adducts could be inhibited by co-incubation with the iron chelator desferrioxamine
(DFO).
Artemisinin 1 is an extract of the Chinese wormwood
Artemisia annua and has been used since ancient times to treat
malaria. Artemisinin is a highly potent antimalarial that exhibits
little or no cross-resistance with other antimalarials.1,2 It does,
however, have a limited availability, high cost, and poor
bioavailabilty. Semi-synthetic derivatives such as artesunate 2
and artemether 3 also display poor pharmacokinetic properties
(Figure 1).3–5 The key pharmacophore within these drugs is the
1,2,4-trioxane functionality. Recently several fully synthetic
trioxanes and 1,2,4-trioxolanes/ozonide (OZa) analogues have
Figure 1. Artemisinin and its semisynthetic analogues.
been synthesized incorporating this key peroxide bridge
pharmacophore.6–14
In this article we describe for the first time a series of stable,
orally active 1,2,4,5-tetraoxane based antimalarials that can be
prepared in only two synthetic steps. To our knowledge, this
represents the shortest sequence reported for the synthesis of
nonsymmetrical tetraoxanes with activity profiles similar to that
of the natural product artemisinin. While impressive activity
profiles have been recorded for molecules within the 1,2,4-
trioxolane class, we reasoned that the achiral tetraoxane template
5 would provide molecules with enhanced stability compared
with ozonides such as 4 and 6. This is based on the observation
that simple 1,2,4-trioxolanes such as 4 are antimalarially inactive
and unstable, whereas the close 1,2,4,5-tetraoxane 5 is relatively
stable and expresses antimalarial activity in the nanomolar range
(IC50 ) 25 nM).7 Given the observation that the adamantane
Figure 2. Stability and reactivity of endoperoxides 4-6.
†
This paper is dedicated to Kevin Barry, deceased October 3, 2007.
* To whom correspondence should be addressed. Phone: 0151-794-3553.
Fax: 0151-794-3588. E-mail: p.m.oneill01@liv.ac.uk.
group is capable of stabilizing an ozonide 6, it seemed logical
to explore 1,2,4,5-tetraoxanes that also incorporate this endop-
eroxide stabilizing motif (Figure 2).
‡
University of Liverpool.
Mid Sweden University.
Liverpool School of Tropical Medicine.
London School of Hygiene and Tropical Medicine.
§
#
|
1,2,4,5-Tetraoxanes were initially used industrially for the
production of macrocyclic hydrocarbons and lactones. They are
achiral and easily prepared from inexpensive materials. There
are many methods available for the synthesis of 1,2,4,5-tetraoxanes,
and these reactions are highly dependent on substrate, temperature,
GlaxoSmithKline.
a Abbreviations: OZ, ozonide; NBD, 7-nitrobenza-2-oxa-1,3-diazole;
HFIP, hexafluoroisopropanol; MTO, methyltrioxorhenium(VII); AM, amo-
diaquine; CQ, chloroquine; TI, therapeutic index; 4NNQO, 4-nitroquinoline
1-oxide; 2Aan, 2-aminoanthracene; DFO, desferrioxamine.
10.1021/jm701435h CCC: $40.75
2008 American Chemical Society
Published on Web 03/15/2008

Synthesis of Tetraoxanes
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7 2171
commercially available tropinone 13 was demethylated,31 sul-
fonated, and subjected to the one-pot procedure described above
to give tetraoxane 16.
This one-pot methodology was also applied to the synthesis
of a urea based dispiro 1,2,4,5-tetraoxane analogue of 8, a
derivative previously prepared in our group (Scheme 3).
Piperidinone 9 was reacted with acid chloride 17 to give urea
18. The one-pot methodology was then applied to give tetraox-
ane 19 in a 6% yield. Because of the poor yield of this reaction,
no further ureas were synthesized. Alternative routes into these
urea based compounds are currently under investigation.
Antimalarial Activity. Table 3 lists the in vitro antimalarial
activity of all the dispiro-1,2,4,5-tetraoxanes synthesized versus
the 3D7 strain of Plasmodium falciparum.32 Most of these
compounds have activity in the 3–30 nM region. There are clear
trends in the SAR required for maximum activity. The presence
of an adamantyl group greatly increases activity. Smaller alkyl
groups at R1 as opposed to larger aromatic groups also increase
activity.
From this in vitro data and taking into account other factors
such as cost and ease of synthesis, compound 11b was selected
as a lead for further biological studies with 11d selected as a
backup. 11b was then tested versus seven strains of Plasmodium
falciparum (Table 4).
In vivo antimalarial activity of 11b and 11d was measured
in P. berghei ANKA infected mice.33 The results can be seen
in Table 5. While the activities of 11b and 11d are slightly
lower than artesunate and 8, it is clear that these molecules are
potent antimalarials.
When this is taken into consideration along with their rapid
two-step synthesis (tetraoxane 8 is a six-step synthesis),27 they
show excellent potential as candidate antimalarial drugs. The
calculated log P and PSA of the two lead tetraoxanes 11b and
11d can be seen in Figure 4.
Cytotoxicity and Genotoxicity Screens. Tetraoxanes 11b
and 11d were subjected to cytotoxicity and therapeutic index
screens (Table 6). In the screens cytotoxicity was measured by
Resazurin reduction. Single full dose response curves were
generated using 10 independent drug concentrations. The 100
therapeutic index (TI) is the ratio of the TOX50 to the IC50 for
the specific compound against the 3D7 P. falciparum isolate.34
The tetraoxane derivatives 11b and 11d are remarkably nontoxic
in these screens with in vitro TI values between 5000 and
17 000.
Figure 3. Structures of artemisone and 8 (RKA216).
concentration, pH, addition mode, solvent, and type of
substrate.15–27
Here, we present a two-step synthesis of a range of achiral
dispiro 1,2,4,5-tetraoxanes with highly potent antimalarial
activity. These compounds are rapidly synthesized from inex-
pensive, readily available starting materials. The stability of the
1,2,4,5-tetraoxane pharmacophore compared to other synthetic
endoperoxides has been investigated. This chemistry has also
been employed to synthesize a fluorescent 4-chloro-7-ni-
trobenza-2-oxa-1,3-diazole (NBD) tagged compound which has
been used to probe the non-heme “chelatable iron” dependent
accumulation mechanism of 1,2,4,5-tetraoxanes.
Recent work on semisynthetic artemisinin derivatives by the
Haynes group has revealed that incorporation of metabolically
inert polar groups into the artemisinin framework provides
analogues (for example, artemisone 7 (Figure 3)) with signifi-
cantly reduced neurotoxicity and superior activity profiles when
compared with currently used artemisinin derivatives.2 Ven-
nerstrom et al. have also explored polar, synthetic 1,2,4-
trioxolanes containing sulfonamide, urea, and amide function-
alities. Previously within the O’Neill group the synthesis of a
range of 1,2,4,5-tetraoxanes with the amide functionality has
been achieved including 8 (RKA216),26 a highly potent anti-
malarial (IC50 ) 5.2 nM, ED50 ) 3.18 mg/kg). The synthesis
of 8 is a six-step process. Following from these initial studies,
we were keen to develop a shorter synthetic route. In order to
achieve the minimal number of synthetic steps, the sulfonamide
functional group and targeted derivatives 11a-9i and 12a-c,f
were selected.28
Results and Discussion
The potential genotoxicity of selected lead compounds 11b
and 11d was determined by the Salmonella typhimurium SOS/
umu assay in two strains: TA1535/pSK1002 and NM2009
(Table 7). This assay is based on the ability of DNA damaging
agents to induce the expression of the umu operon. The
Salmonella strains have a plasmid pSK1002 that carries an
umuC-lacZ fused gene that produces a hybrid protein with
ꢀ-galactosidase activity and whose expression is controlled by
the umu regulatory region. Since many compounds do not exert
their mutagenicity effect until they have been metabolized, the
assay was also performed in the presence of rat liver S9-mix.
Positive control agents (4-nitroquinoline 1-oxide (4NNQO) and
2-aminoanthracene (2Aan)) were used to test the response of
the tester strains. Negative results were obtained for tetraoxanes
11b and 11d at the highest concentration tested (50 µM), both
in the absence and in the presence of an in vitro metabolic
activation system (S-9 mix).35 (For detailed figures, see Sup-
porting Information.)
Dispiro 1,2,4,5-tetraoxanes 11a-i and 12a-c,f were prepared
using the two-step synthesis depicted in Scheme 1. Sulfonylpi-
peridones 10a-i were prepared from piperidinone 9 using the
respective sulfonyl chloride and K2CO3 in a biphasic reaction.29
Conversion to the tetraoxanes 11a-i was achieved in a one-
pot reaction. Ketones 10a-i were treated with hydrogen
peroxide and methyltrioxorhenium(VII) (MTO) using a method
developed by Iskra and co-workers to give the gem-dihydro-
peroxide. Adamantanone and HBF4 were then added to complete
conversion to the dispiro-1,2,4,5-tetraoxanes 11a-i in reason-
able to good yields (Tables 1 and 2), the key to the selectivity
of this reaction being the use of the fluorous solvent hexa-
fluoroisopropanol (HFIP).23,30 Tetraoxanes 12a-c,f were pre-
pared in a similar manner using cyclododecanone rather than
adamantanone.
From preliminary in vitro data it was rapidly apparent that
the presence of the adamantyl ring greatly improved activity.
One possible explanation for this is that the rigidity of the ring
imparts greater stability. With this in mind the tropinone
derivative below was synthesized (Scheme 2). In this case
Stability and Reactivity Studies. To investigate the activity,
stability, and reactivity of the 1,2,4,5-tetraoxane pharmacophore

2172 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7
Scheme 1. Two-Step Synthesis of Dispiro-1,2,4,5-tetraoxanes
Ellis et al.
Table 1. Yields of Adamantyl Dispiro-1,2,4,5-tetraoxanes 11a-i
Scheme 3. Two-Step Synthesis of Morpholine Urea
1,2,4,5-Tetraoxane
compd
R1
% yield
% yield 11
11a
11b
11c
11d
11e
11f
11g
11h
11i
Me
62
59
52
59
62
98
98
99
95
61
60
56
53
51
36
41
38
25
Et
iPr
Cp
CH3CF3
Ph
p-ClPh
p-FPh
p-CF3Ph
Table 2. Yields of Cyclododecyl Dispiro-1,2,4,5-tetraoxanes 12
compd
R1
% yield 12
12a
12b
12c
12f
Me
Et
36
32
38
20
Table 3. In Vitro Antimalarial Activities of the
Dispiro-1,2,4,5-tetraoxanes versus 3D7 Plasmodium falciparum
iPr
Ph
tetraoxane drug
R1
mean IC50 (nM) vs 3D7
artemether
artemisinin
11a
11b
11c
11d
11e
11f
11g
11h
11i
12a
12b
12c
12f
16
19
3.20 ( 1.97
9.20 ( 1.97
10.18 ( 6.80
5.55 ( 4.09
5.87 ( 4.34
3.52 ( 3.45
14.35 ( 5.42
8.10 ( 1.21
22.73 ( 4.04
16.73 ( 4.80
20.73 ( 7.79
27.75 ( 9.84
29.13 ( 15.50
86.37 ( 17.50
131.07 ( 33.18
60.57 ( 22.00
4.70 ( 3.04
Scheme 2. Synthesis of Tropinone Derived
Dispiro-1,2,4,5-tetraoxane
Me
Et
iPr
Cp
CH2CF3
Ph
p-ClPh
p-FPh
p-CF3
Me
Et
iPr
Ph
Et
N/A
Table 4. In Vitro Antimalarial Activities of Tetraoxane 11b versus
Seven Strains of Plasmodium falciparuma
IC50 ( SD (nM)
strain
tetraoxane 11b
artesunate
AM
CQ
DD2
K1
3.0 ( 1.0
3.0 ( 0.9
3.0 ( 0.6
2.7 ( 1.6
3.8 ( 2.7
1.9 ( 0.4
2.4 ( 0.3
1.5 ( 0.9
0.7 ( 0.5
1.1 ( 0.3
0.7 ( 0.3
1.7 ( 0.3
1.9 ( 0.4
0.5 ( 0.1
6.1 ( 2.9
10.2 ( 1.3
4.5 ( 1.0
7.7 ( 1.9
5.9 ( 0.4
4.7 ( 0.7
6.4 ( 0.7
80.5 ( 3.1
73.9 ( 2.5
8.1 ( 2.6
83.7 ( 7.8
6.6 ( 1.2
72.0 ( 10.8
91.3 ( 10.5
compared to other synthetic endoperoxides, a series of structur-
ally similar endoperoxides were synthesized varying only in the
nature of their endoperoxide cores (Figure 5). Compounds 20
and 21 were prepared by literature methods; for synthetic routes
see Supporting Information (phenyl derivatives selected because
of UV chromophore to aid TLC analysis).28
GC03
V1S
HB3
PH3
TM4
a AM ) amodiaquine. CQ ) chloroquine.
The synthesis of 1,2,4-trioxane 22 can be seen below (Scheme
4). BOC-protected piperidinone 23 was converted to epoxide
24 in 82% yield, and the synthesis of the peroxide 25 was
achieved using Mo(acac)2 and H2O2. The 1,2,4-trioxane core
26 was formed using p-toluenesulfonic acid and 2-adamantanone
in 83% yield. The BOC protecting group was then removed to
give the hydrochloride salt 27 in 62% yield. Subsequent reaction
with benzenesulfonyl chloride gave the desired 1,2,4-trioxane
22 in 92% yield.
In vitro antimalarial activity of the endoperoxides shows that
tetraoxane 11f and OZ analogue 20 are potent antimalarials with
comparable activity. Interestingly both 11f and 20 are nearly
100-fold more active than both the 1,2,4-trioxanes 21 (>1000
nM) and 22 (710 nM) (Table 8). This observation underscores
the advantage of the 1,2,4,5-tetraoxane core over the 1,2,4-
trioxane core structure.

Synthesis of Tetraoxanes
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7 2173
Table 5. In Vivo (Oral) Antimalarial Activities of Tetraoxanes 11b and
11d versus Plasmodium berghei
tetraoxane drug
ED50 (mg/kg)
11b
11d
8
artesunate
artemisinin
6.61
7.93
3.18
3.20
8.42
Figure 4. Calculated log P and PSA of lead compounds 11b and 11d.
The stability and reactivity of the endoperoxides in the
presence of iron were investigated. Tetraoxane 11f, OZ analogue
20, and trioxane 22 were subjected to 1.0 equiv of FeBr2 in
THF for the set time periods layed out in the table (this
combination leads to complete degradation of artemisinin after
24 h).36,37 The resulting residue was purified by flash column
chromatography and the percent recovery of starting endoper-
oxide calculated (Table 9).
Table 9 shows that the 1,2,4,5-tetraoxanes exhibit remarkable
stability with 69% recovery of starting material after 48 h. At
this time the OZ analogue 20 has been completely degraded.
The 1,2,4,5-tetraoxanes also have greater stability than the
trioxane 22 because only 43% of the starting trioxane was
recovered after 48 h. Tetraoxane 11f was also treated with
FeSO4 ·7H2O and FeCl2 ·4H2O, and no degradation was seen
after 24 h, further confirming the remarkable stability of these
1,2,4,5-tetraoxanes. Chemical stability tests in aqueous solution
and acid were also carried out and again resulted in no
significant degradation (see Supporting Information).
Mechanism of Accumulation. In order to investigate the
intraparasitic sites of accumulation, it was necessary to synthe-
size an NBD tagged dispiro-1,2,4,5-tetraoxane for confocal
microscopy studies (Scheme 5).38 Tetraoxane 29 was synthe-
sized using sulfonation followed by one-pot synthesis of the
tetraoxane as described earlier. The methyl ester 29 was then
converted to acid 30 using NaOH in ethanol. Coupling of the
acid 30 with the mono-BOC protected diamine and removal of
the BOC group provided the free amine that was coupled with
NBD-Cl to give the desired NBD tagged tetraoxane 33. This
compound was found to have an IC50 of 16 nM versus the 3D7
strain of Plasmodium falciparum.39
The NBD tagged tetraoxane 33 was then used in single-cell
confocal imaging of malaria infected erythrocytes, the results
of which can be seen in Figure 6.38 It can be seen from these
images that the NBD tagged 1,2,4,5-tetraoxane accumulates only
in infected erythrocytes (see panel a) within the cytoplasm and
the digestive vacuole of the parasite; washing the cells with
buffer failed to remove the tagged molecule as depicted in panel
b. The formation of stable adducts produced within the parasite
was inhibited by co-incubation with the iron chelator desferri-
oxamine (DFO). In the presence of DFO, there is significant
intraparasitic accumulation (see panel c), the majority of which
can be removed by cell washing (see panel d). This implies
that DFO, by chelation of non-heme iron, reduces the reductive
bioactivation and covalent adduct formation between the drug
and parasite protein targets. It is also noted that accumulation
of the probe can be seen within the acidic hematin-containing
food vacuole and that there is evidence of some association of
33 with malaria pigment as seen in Figure 6d.
Table 6. Cellular Cytotoxicity Screens (µM) and Therapeutic Index (TI)
(µM) for Tetraoxanes 11b and 11da
drug
Hep2G
L6
MRC-5
VERO
H9c(2-1)
11b TOX50
TI
11d TOX50
TI
>50
>9090
>50
31
5636
>50
>50
>9090
>50
>50
>9090
>50
>50
>9090
>50
>14285
3
>14285
>14285 >14285 >14285
doxorubicin
0.3
>5
2
>5
a HepG2: human Caucasian hepatocyte carcinoma. H9c2(2-1): myocar-
dium, heart, rat. L6: skeletal muscle myoblast, rat. Vero: kidney, African
green monkey, Cercopithecus aethiops. MRC-5: embryonal lung, diploid,
male, human.
Table 7. Mutagenic Potential on Salmonella typhimurium Strains TA
1535/pSK1002 and NM2009 in the Absence and Presence of S9-Mix
TA1535/pSK1002
NM2009
+rat S9
compd
-S9
+rat S9
-S9
11b
11d
4NNQO
2Aan
neg
neg
pos
neg
neg
neg
pos
pos
neg
neg
pos
neg
neg
neg
pos
pos
exhibit no genotoxicity. The 1,2,4,5-tetraoxanes have been
proven to be superior to the ozonide analogues in terms of
stability and to be superior to 1,2,4-trioxane analogues in terms
of both stability and activity. The synthesis of a fluorescent
NBD-tagged tetraoxane probe and subsequent single-cell con-
focal imaging have shown that the NBD probe accumulates in
the parasite and that formation of stable adducts was inhibited
by co-incubation with the iron chelator DFO. These results
encourage further preclinical development of these candidates
as cost-effective alternatives to the artemisinin derivatives.
Experimental Section
All reactions that employed moisture sensitive reagents were
performed in dry solvent under an atmosphere of nitrogen in oven-
dried glassware. All reagents were purchased from Sigma Aldrich
or Alfa Aesar chemical companies and were used without purifica-
Conclusions
To conclude, rapid two-step syntheses of a range of dispiro-
1,2,4,5-tetraoxanes with potent antimalarial activity both in vitro
and in vivo have been reported. The lead compounds within
this series have been proven to be remarkably nontoxic and to
Figure 5. Tetraoxane 11f, OZ analogue 20, and trioxanes 21 and 22
used in stability studies.

2174 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7
Scheme 4. Synthesis of 1,2,4-Trioxane 22
Ellis et al.
11b: R ) Et, 60%, white solid. 1H NMR (CDCl3), δ: 3.22–3.45
(4H, m, 4H1), 2.95 (2H, q, J ) 7.4 Hz, CH2CH3), 2.50 (2H, s,
2H2a), 1.51–2.23 (16H, m, 2H2b and adamantane), 1.35 (3H, t, J
) 7.4 Hz, CH2CH3). 13C NMR (CDCl3), δ: 111.5, 106.0, 44.9,
37.2, 36.2, 33.5, 31.35, 30.6, 27.4, 8.3. MS m/z (ES, +ve, CH3OH),
396 ([M + Na]+, 100%). Found [M + Na]+, 396.1447.
C17H27NO6NaS requires 396.1457. Anal. (C17H27NO6S) C, H, N.
11c: R ) iPr, 56%, white solid. 1H NMR (CDCl3), δ: 3.22–3.35
(4H, m, 4H1), 3.15 (1H, m, CH3CHCH3), 2.50 (2H, s, 2H2a),
1.51–2.10 (16H, m, 2H2b and adamantane), 1.31 (6H, d, J ) 6.9
Hz, CH3CHCH3). 13C NMR (CDCl3), δ: 111.5, 106.0, 54.1, 44.9,
37.3, 33.5, 32.0, 27.4, 23.0, 17.1, 14.5. MS m/z (ES, +ve, CH3OH),
410 ([M + Na]+, 100%). Found [M + Na]+, 410.1600.
C18H29NO6NaS requires 410.1613. Anal. (C18H29NO6S) C, H, N.
11d: R ) Cp, 53%, white solid. 1H NMR (CDCl3), δ: 3.30–3.50
(4H, m, 4H1), 2.50 (2H, s, 2H2a), 2.20 (1H, m, CH2CHCH2),
1.51–2.10 (16H, m, 2H2b and adamantane), 1.15 (2H, m,
CH2CHCH2), 0.95 (2H, m, CH2CHCH2). 13C NMR (CDCl3), δ:
111.5, 106.0, 54.1, 44.9, 37.3, 33.5, 32.0, 27.4, 26.5, 4.8. MS m/z
(ES, +ve, CH3OH), 408 ([M + Na]+, 100%). Found [M + Na]+,
408.1438. C18H27NO6NaS requires 408.1457. Anal. (C18H27NO6S)
C, H, N.
Table 8. In Vitro antimalarial activities of the phenyl sulfonamide
endoperoxides
Endoperoxide Drug
Mean IC50(nM)3D7
Artesunate
0.60 ( 0.035
5.46 ( 1.19
4.98 ( 0.60
>1000
11f
20
21
22
710 ( 115
Table 9. Iron degradation studies on endoperoxides 11f, 20 and 22
% Recovery of Endoperoxide
Drug
4 h
8 h
24 h
48 h
Tetraoxane 11f
OZ analogue 20
1,2,4-Trioxane 22
88.7
11.0
96.8
80.5
9.0
84.9
72.0
2.6
56.7
69.0
0.0
43.2
tion. Thin layer chromatography (TLC) was carried out on Merck
silica gel 60 F-254 plates, and UV inactive compounds were
visualized using iodine or anisaldehyde solution. Flash column
chromatography was performed on ICN Ecochrom 60 (32–63 mesh)
silica gel, eluting with various solvent mixtures and using an air
line to apply pressure. NMR spectra were recorded on a Brucker
AMX 400 (1H, 400 MHz; 13C, 100 MHz) spectrometer. Chemical
shifts are described in parts per million (δ) downfield from an
internal standard of trimethylsilane. Microanalyses were performed
in the University of Liverpool Microanalysis Laboratory. Mass
spectra were recorded on a VG analytical 7070E machine and
Fisons TRIO spectrometers using electron ionization (EI) and
chemical ionization (CI).
11e: R ) CH2CF3, 51%, white solid. 1H NMR (CDCl3), δ: 3.70
(2H, q, J ) 9.3 Hz, CH2CF3), 3.30–3.60 (4H, m, 4H1), 2.50 (2H,
s, 2H2a), 1.51–2.23 (16H, m, 2H2b and adamantane). 13C NMR
(CDCl3), δ: 110.2, 104.3, 52.2, 51.9, 35.9, 32.1, 31.35, 30.6, 27.4,
-1.0. MS m/z (ES, +ve, CH3OH), 450 ([M + Na]+, 100%). Found
[M + Na]+, 450.1156. C17H24NO6F3NaS requires 450.1174. Anal.
(C17H24NO6F3S) C, H, N.
11f: R ) Ph, 35%, white solid. 1H NMR (CDCl3), δ: 7.50–7.85,
(5H, aromatics), 3.01–3.25, (4H, m, 4H1), 2.51, (2H, s, 2H2a),
1.51–2.20 (16H, m, 2H2b and adamantane). 13C NMR (CDCl3),
δ: 136.6, 133.4, 129.6, 128.0, 111.5, 105.8, 47.4, 39.7, 37.2, 36.7,
33.4, 27.8, 27.3. MS m/z (ES, +ve, CH3OH), 444 ([M + Na]+,
100%). Found [M + Na]+, 444.1445. C21H27NO6NaS requires
444.1457. Anal. (C21H27NO6S) C, H, N.
General Procedure for Preparation of Adamantyl-1,2,4,5-
tetraoxanes 11a-i. A solution of 1-R1-sulfonylpiperidin-4-one 10
(1.13 mmol), 30% H2O2 (0.26 mL, 2.26 mmol, 2.0 equiv), and MTO
(trace) in HFIP (2.27 mL) was stirred at room temperature for 2 h.
After this time 2-adamantanone (339 mg, 2.26 mol, 2.0 equiv) was
added followed by dropwise addition of a 54% ethereal solution
of HBF4 (368 mg, 2.26 mmol, 2.0 eq). The reaction was then
allowed to stir at room temperature for 1 h. Dichloromethane (10
mL) was added. The organic layer was washed with a saturated
solution of NaHCO3 and dried over MgSO4 and the solvent removed
in vacuo. The resulting residue was purified by flash column
chromatography (SiO2, hexane/EtOAc ) 9:1) to give the desired
dispiro-1,2,4,5-tetraoxane 11a-i.
11g: R ) p-ClPh, 38%, white solid. 1H NMR (CDCl3), δ:
7.50–7.85, (4H, aromatics), 3.05–3.25, (4H, m, 4H1), 2.55, (2H, s,
2H2a), 1.55–2.10 (16H, m, 2H2b and adamantane). 13C NMR
(CDCl3), δ: 140.0, 129.9, 129.4, 124.0, 111.5, 105.6, 47.4, 39.7,
37.2, 36.7, 33.4, 32.0, 27.3. MS m/z (ES, +ve, CH3OH), 478 ([M
+ Na]+, 100%). Found [M + Na]+, 478.1081. C21H26NO6NaSCl
requires 478.1067. Anal. (C21H26NO6S) C, H, N.
11h: R ) p-FPh, 41%, white solid. 1H NMR (CDCl3), δ:
7.10–7.80, (4H, aromatics), 3.05–3.25, (4H, m, 4H1), 2.55, (2H, s,
2H2a), 1.55–2.10 (16H, m, 2H2b and adamantane). 13C NMR
(CDCl3), δ: 130.7, 130.6, 117.0, 116.7, 111.5, 105.6, 47.4, 39.7,
37.2, 36.7, 33.4, 32.0, 27.3. MS m/z (ES, +ve, CH3OH), 462 ([M
+ Na]+, 100%). Found [M + Na]+, 462.1341. C21H26NO6FNaS
requires 462.1363. Anal. (C21H26NO6FS) C, H, N.
11a: R ) Me, 61%, white solid. 1H NMR (CDCl3), δ: 3.22–3.45
(4H, m, 4H1), 2.80 (3H, s, CH3), 2.52 (2H, s, 2H2a), 1.51–2.23
(16H, m, 2H2b and adamantane). 13C NMR (CDCl3), δ: 111.6,
105.8, 41.6, 37.2, 36.2, 34.2, 33.5, 31.4, 27.4, 26.2. MS m/z (ES,
+ve, CH3OH), 382 ([M + Na]+, 100%). Found [M + Na]+,
382.1313. C16H25NO6NaS requires 382.1300. Anal. (C16H25NO6S)
C, H, N.
11i: R ) p-CF3Ph, 25%, white solid. 1H NMR (CDCl3), δ:
7.75–7.95, (4H, aromatics), 3.05–3.25, (4H, m, 4H1), 2.55, (2H, s,

Synthesis of Tetraoxanes
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7 2175
Scheme 5. Synthesis of NBD-Tetraoxane Conjugate 33
2H2a), 1.55–2.10 (16H, m, 2H2b and adamantane). 13C NMR
(CDCl3), δ: 128.4, 128.0, 127.3, 127.3, 126.9, 116.8, 111.4, 47.4,
39.7, 37.2, 36.7, 33.4, 32.0, 27.3. MS m/z (ES, +ve, CH3OH), 512
([M + Na]+, 100%). Found [M + Na]+, 512.1346. C22H26-
NO6F3NaS requires 512.1331. Anal. (C22H26NO6F3S) C, H, N.
General Procedure for Preparation of Cyclododecyl-1,2,4,5-
tetraoxanes 12a-c,f. A solution of 1-R1-sulfonylpiperidin-4-one
10 (1.13 mmol), 30% H2O2 (0.26 mL, 2.26 mmol, 2.0 equiv), and
MTO (trace) in HFIP (2.27 mL) was stirred at room temperature
for 2 h. After this time cyclododecanone (412 mg, 2.26 mol, 2.0
equiv) was added followed by dropwise addition of a 54% ethereal
solution of HBF4 (368 mg, 2.26 mmol, 2.0 equiv). The mixture
was then stirred at room temperature for 1 h. Dichloromethane (10
mL) was added, and the organic layer was washed with a saturated
solution of NaHCO3 and dried over MgSO4. The solvent was
removed in vacuo. The resulting residue was purified by flash
column chromatography (SiO2, hexane/EtOAc ) 9:1) to give the
desired dispiro-1,2,4,5-tetraoxane 12a-c,f.
12a: R ) Me, 36%, white solid. 1H NMR (CDCl3), δ: 3.25–3.42,
(4H, m, 4H1), 2.80, (3H, s, CH3), 2.50 (2H, bs, 2H2a), 2.25 (2H,
m, 2H3a), 1.85 (2H, bs, 2H2b), 1.60 (2H, m, 2H3b), 1.21–1.49
(18H, m, dodecane ring). 13C NMR (CDCl3), δ: 113.5, 105.7, 43.3,
42.1, 35.5, 29.9, 29.5, 26.3, 22.6, 19.8, 18.4. MS m/z (ES, +ve,
CH3OH), 414 ([M + Na]+, 100%). Found [M + Na]+, 414.1926.
C18H33NO6NaS requires 414.1926. Anal. (C18H33NO6S) C, H, N.
12b: R ) Et, 32%, white solid. 1H NMR (CDCl3), δ: 3.31–3.50,
(4H, m, 4H1), 2.95, (2H, q, J ) 7.4 Hz, CH2CH3), 2.50 (2H, bs,
2H2a), 2.25 (2H, bs, 2H3a), 1.85 (2H, bs, 2H2b), 1.60 (2H, m,
2H3b), 1.28 (3H, t, J 7.4, CH2CH3), 1.31–1.49 (18H, m, dodecane
ring). 13C NMR (CDCl3), δ: 113.5, 106.0, 44.9, 42.1, 35.5, 29.9,
Figure 6. Confocal microscope images of infected red blood cells with NBD conjugates (1 µM) in the presence or absence of DFO (100 µM). The
NBD-labeled compound was added 10 min before imaging. For imaging experiments using DFO, the parasite cultures were preincubated at 37 °C
for 30 min with DFO prior to use. After uptake of drug, the perfusion chamber was washed with 5000× its volume of media to examine the
retention of fluorescence: Tetraoxane-NBD adduct 33 without DFO before (a) and after wash (b) and with DFO (100 µM) before (c) and after
wash (d).

2176 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7
Ellis et al.
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26.4, 26.2, 22.6, 19.8, 8.26. MS m/z (ES, +ve, CH3OH), 428 ([M
+ Na]+, 100%). Found [M + Na]+, 428.2100. C19H35NO6NaS
requires 428.2083. Anal. (C19H35NO6S) C, H, N.
12c: R ) iPr, 38%, white solid. 1H NMR (CDCl3), δ: 3.31–3.50,
(4H, m, 4H1), 3.15, (2H, m, CH3CHCH3), 2.50 (2H, bs, 2H2a),
2.25 (2H, bs, 2H3a), 1.85 (2H, bs, 2H2b), 1.60–1.20 (26H, m, 2H3b,
CH3CHCH3, dodecane ring). 13C NMR (CDCl3), δ: 113.5, 105.9,
54.2, 44.8, 42.0, 35.3, 29.9, 26.3, 26.1, 22.6, 19.7, 17.7. MS m/z
(ES, +ve, CH3OH), 442 ([M + Na]+, 100%). Found [M + Na]+,
442.2256. C20H37NO6NaS requires 442.2239. Anal. (C20H37NO6S)
C, H, N.
12f: R ) Ph, 20%, white solid. 1H NMR (CDCl3), δ: 7.95–7.45
(5H, m, aromatics), 3.05–3.35 (4H, m, 4H1), 2.50 (2H, bs, 2H2a),
2.25 (2H, bs, 2H3a), 1.85 (2H, bs, 2H2b), 1.62 (2H, m, 2H3b),
1.21–1.49 (18H, m, dodecane ring). 13C NMR (CDCl3), δ: 136.5,
133.4, 129.6, 128.0, 113.4, 105.7, 43.7, 42.4, 31.6, 29.7, 26.5, 26.2,
23.1, 19.7. MS m/z (ES, +ve, CH3OH), 476 ([M + Na]+, 100%).
Found [M + Na]+, 476.2097. C23H35NO6NaS requires 476.2083.
Anal. (C23H35NO6S) C, H, N.
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Acknowledgment. This work was supported by grants from
the EU (Antimal, FP6 Malaria Drugs Initiative) and the BBSRC
(S.A.W., P.O.N., P.G., Grant BB/C006321/1).
Supporting Information Available: Experimental details for
the synthesis of 10a-i and tetraoxane 33, combustion analysis
results, additional detailed information on cytotoxicity and ge-
neotoxicity studies, synthesis procedures for compounds 20-22
used in the section Stability and Reactivity Studies and additional
details on stability tests, and details of the confocal microscopy
procedure. This material is available free of charge via the Internet
at http://pubs.acs.org.
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JM701435H