Synthesis and antimalarial activity of 11 dispiro-1,2,4,5-tetraoxane analogues of WR 148999. 7,8,15,16-tetraoxadispiro[5.2.5.2]hexadecanes substituted at the 1 and 10 positions with unsaturated and polar functional groups

Article abstract of DOI:10.1021/jm980698f

Eleven novel dispiro-1,2,4,5-tetraoxanes 3 bearing unsaturated and polar functional groups were designed to enhance the oral antimalarial activity of the prototype tetraoxane 2 (WR 148999). With the exception of 3g and 3h, tetraoxanes 3 were available via

Full text of DOI:10.1021/jm980698f

J . Med. Chem. 1999, 42, 1477-1480
1477
Syn th esis a n d An tim a la r ia l Activity of 11 Disp ir o-1,2,4,5-tetr a oxa n e An a logu es
of WR 148999. 7,8,15,16-Tetr a oxa d isp ir o[5.2.5.2]h exa d eca n es Su bstitu ted a t th e 1
a n d 10 P osition s w ith Un sa tu r a ted a n d P ola r F u n ction a l Gr ou p s
Yuxiang Dong,† Hugues Matile,‡ J acques Chollet,‡ Ronald Kaminsky,§ J ames K. Wood,| and
J onathan L. Vennerstrom*,†
College of Pharmacy, University of Nebraska Medical Center, 986025 Nebraska Medical Center, Omaha, Nebraska 68198-6025,
Pharma Division, Preclinical Research, F. Hoffmann-LaRoche Ltd., CH-4070 Basel, Switzerland, Swiss Tropical Institute,
CH-4002 Basel, Switzerland, Department of Chemistry, University of Nebraska at Omaha, 60th and Dodge Street,
Omaha, Nebraska 68192-0109
Received December 11, 1998
Eleven novel dispiro-1,2,4,5-tetraoxanes 3 bearing unsaturated and polar functional groups
were designed to enhance the oral antimalarial activity of the prototype tetraoxane 2 (WR
148999). With the exception of 3g and 3h , tetraoxanes 3 were available via the peroxidation
of corresponding cyclohexanone derivatives in H2SO4/CH3CN. Tetraoxanes 3g and 3h were
prepared by hydrolysis of ester tetraoxanes 3e and 3i, respectively. Five of the 11 tetraoxanes
were inactive, but six tetraoxanes had IC50 values of 6-26 nM against the K1 and NF54 strains
of Plasmodium falciparum compared to corresponding IC50 values of 28 and 39 nM for 2, and
10 and 12 nM for artemisinin (1). Ester tetraoxane 3e was the most active in vitro, some 2-fold
more potent than 1. However, none of the six tetraoxanes active in vitro were as effective as
either 1 or 2 in vivo; at single doses of 100 mg/kg most possessed little to no vivo activity in
mice infected with Plasmodium berghei. Unsaturated tetraoxane 3a was uniquely more active
when administered per os (po) than subcutan (sc). For this series of tetraoxanes, the discrepancy
between vitro and vivo activities underscores the limitations of conclusions drawn solely from
in vitro antimalarial data and illustrates a practical benefit of complementary single-dose in
vivo antimalarial screens.
The discovery of artemisinin (qinghaosu, 1), a natu-
rally occurring endoperoxide sesquiterpene lactone,
stimulated a substantial effort to elucidate its structure-
activity relationship (SAR) and initiated an ongoing
quest to identify structurally simple and synthetically
accessible antimalarial peroxides.1 Among synthetic
antimalarial peroxides, dispiro-1,2,4,5-tetraoxanes such
as 2 (WR 148999) are notable in that they differ
considerably in structure from 1, are readily prepared
in one step from substituted cyclohexanones, and pos-
sess antimalarial activity comparable to 1.2,3
mechanism for drugs from the peritoneal cavity is by
way of the portal circulation.5 It follows that the low
oral activity of 2 may be due either to inactivating gut
metabolism or to an inadequate dissolution rate which
is a function of its intrinsic solubility, stability to
stomach acid, and formulation.
Design
To address the hypothesis that tetraoxane oral bio-
availability is a function of oral absorption, we envi-
sioned the synthesis of more water soluble tetraoxanes
bearing polar functional groups.6 These analogues might
be more easily absorbed than their alkyl-substituted
counterparts, an important consideration if lack of
absorption is a significant factor in the low oral activity
of tetraoxane 2. Using tetraoxane 2 as a prototype, we
designed target dispiro-1,2,4,5-tetraoxanes (7,8,15,16-
tetraoxadispiro[5.2.5.2]hexadecanes) 3 bearing unsatur-
The poor to modest oral bioavailability4 of artemisinin
and its semisynthetic derivatives has been attributed
to a high first-pass drug metabolism. Whether the poor
oral antimalarial activity of tetraoxane 2 is due to poor
absorption or rapid inactivating metabolism is un-
known. However, the excellent ip activity of 23 suggests
that this tetraoxane has some resistance to inactivating
first-pass hepatic drug metabolism as a major exit
ated and functional groups at the 1 and 10 positions.
As exemplified by 2, dispiro-1,2,4,5-tetraoxanes derived
from 2-alkyl cyclohexanones are always formed as single
centrosymmetric stereoisomers.2,7 We anticipated that
this predictable stereochemical outcome would hold true
for tetraoxanes 3 as well.
† University of Nebraska Medical Center.
‡ F. Hoffmann-LaRoche Ltd.
§ Swiss Tropical Institute.
| University of Nebraska at Omaha.
10.1021/jm980698f CCC: $18.00 © 1999 American Chemical Society
Published on Web 03/25/1999

1478 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 8
Brief Articles
Ta ble 1. Antimalarial Activity against P. falciparum in Vitro
Ta ble 2. Antimalarial Activity against P. berghei in Vivo
activity (%)b survival (days)c
sc po
IC50, nMa
cmpd
R
K1
NF54
cmpda
sc
po
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
2
CHdCH2
CtCH
C6H5
CH2COOEt
COOEt
CH2COOH
COOH
OH
OCOC6H5
OMe
OCH2C6H5
H
19
13
>200
23
13
>200
3a
3b
3d
3e
3j
3k
2
15
9
0
0
100
0
93
0
0
0
18
0
7
14
6.2
16
6.5
24.7
>200
>200
>200
>200
15
>200
>200
>200
>200
16
100
100
99.7
98
27.7
14
7.7
7.3
1
a
Compounds in a solution or a suspension containing 3%
ethanol and 7% Tween 80 were administered on day 1 post-
18
28
10
26
39
12
b
infection at a single dose of 100 mg/kg. Reduction of parasitemia
on day 3 post-infection. c On average, mice in a control group
survive 5.2 days post-infection.
1
a
Average of n ) 2.
3h , was inactive. We also note that the six active
tetraoxanes, like artemisinin, were more potent against
the chloroquine-resistant K1 strain than against the
drug-sensitive NF54 strain of Plasmodium falciparum,
although in each case, the potency difference was small.
The six active tetraoxanes, along with 1 and 2 as
controls, were selected for in vivo antimalarial evalua-
tion in mice infected with the ANKA strain of Plasmo-
dium berghei. Consistent with existing multiple-dose
Thompson test data,2,3 the vivo antimalarial efficacies
for 1 and 2 differed only slightly. From the vivo data
(Table 2), it is clear that none of these functionalized
tetraoxanes possessed useful oral antimalarial activity
and all were less effective than either 1 or 2. We
attribute the complete lack of vivo activity for ester
tetraoxanes 3d and 3e to in vivo chemical or enzymatic
hydrolysis to the inactive tetraoxane acids 3f and 3g,
respectively. Although methyl ether tetraoxane 3j had
good vivo activity sc, it was almost completely inactive
po due probably to first-pass O-dealkylative metabolism
to the inactive tetraoxane alcohol 3h . Unsaturated
tetraoxane 3a was uniquely more active po than sc, due
possibly to formation of some active metabolite.
Tetraoxanes 3 were designed as derivatives of 2 by
replacing one methyl hydrogen atom of each of the two
methyl groups in 2 with unsaturated (3a -3c), carboxy-
lic (3d -3g, 3i), hydroxylic (3h ) and ether (3j, 3k )
substituents (Table 1). Five of the target tetraoxanes
could also conceivably function as prodrugs in vivo. For
example, 3j and 3k could both be converted to 3h via
metabolic O-dealkylation reactions. In addition, 3d , 3e,
and 3i could be converted to 3f, 3g, and 3h , respectively,
via plasma esterase enzymes.
Ch em istr y
Nine tetraoxanes were prepared according to the
peroxidation method developed by McCullough et al.8
in which cyclohexanones are treated with hydrogen
peroxide in H2SO4/CH3CN. The unoptimized yields
ranged from 4 to 12%. Tetraoxanes 3g and 3h were not
formed using this method and were instead indirectly
prepared by the hydrolysis of tetraoxane esters 3e and
3i in yields of 72 and 60%, respectively. The failure to
form 3g and 3h via acid-catalyzed peroxidation is
possibly associated with formation of persistent perhy-
drates, in agreement with reported data9 in which
2-chloro- and 2-bromocyclohexanone afforded isolable
perhydrates instead of tetraoxanes due to the formation
of an intramolecular hydrogen bond between the hy-
droperoxide hydrogen and the halogen substituent.
Tetraoxanes 3a -3k are white crystalline solids stable
Discu ssion
In this work, we identified six functionalized tetraox-
anes with in vitro potency equal or superior to prototype
tetraoxane 2 and one diester tetraoxane (3e) with in
vitro potency superior to 1. It is conceivable that some
of the tetraoxanes were well absorbed orally but suffered
from a lack of metabolic stability. Nevertheless, the poor
vivo antimalarial activity of these tetraoxanes provides
no justification for additional experiments (i.e., blood
level determinations) to assess their oral bioavailability.
For this series of tetraoxanes, the discrepancy between
vitro and vivo activities underscores the limitations of
conclusions drawn solely from in vitro antimalarial data
and illustrates how the single-dose in vivo antimalarial
screen used in this study provided a necessary and more
rigorous test of antimalarial potential.
As these functionalized tetraoxanes were based on
tetraoxane 2 as a structural template, they manifest the
advantage of both defined and predictable stereochem-
istry but also the liability of their centrosymmetric
symmetry in which each tetraoxane (ketone diperoxide)
necessarily bears two functional groups present singly
in the starting material cyclohexanones. Existing tet-
raoxane synthetic methods10 do not allow for the syn-
thesis of nonsymmetrical tetraoxanes. Even with this
1
at room temperature for months if not years. In H NMR
spectra of these tetraoxanes, as is also the case for
tetraoxane 2,2 a characteristic peak for the two methine
protons at the 1 and 10 positions falls between 2.7 and
3.4 ppm. In the corresponding 13C NMR spectra, a
diagnostic signal is present at 108-110 ppm, corre-
sponding to the spiro quaternary carbon atoms.2,7
An tim a la r ia l Activity
The in vitro antimalarial data (Table 1) reveal that
six of the tetraoxanes were at least as potent as the
parent tetraoxane 2. Of these, 3b, 3d , and 3j were
nearly as potent as artemisinin, and 3e was notably
more potent than artemisinin. From these data, we can
conclude that the presence of alkene (3a ), alkyne (3b),
and ether (3j, 3k ) moieties maintain or enhance anti-
malarial potency whereas carboxylic acid (3f, 3g) and
alcohol (3h ) moieties decrease antimalarial potency. The
situation for esters is less clear; ethyl esters 3d and 3e
had good activity, whereas 3i, the dibenzoate ester of

Brief Articles
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 8 1479
1H NMR (DMSO-d6) 1.05-1.80 (m, 18H), 1.81-2.05 (bs, 2H),
2.06-2.40 (m, 4H), 2.70-2.95 (bs, 2H); 13C NMR (DMSO-d6)
22.19, 23.00, 24.25, 27.16, 29.64, 32.06, 42.58, 109.51, 174.89.
Anal. (C18H28O8) C, H.
1,10-Bis(ben zoyloxym eth yl)-7,8,15,16-tetr a oxa d isp ir o-
[5.2.5.2]h exa d eca n e (3i): yield 10%; mp 195 °C dec (ether:
pentane, 1:1); 1H NMR 1.15-2.01 (m, 14H), 2.04-2.32 (bs, 2H),
2.90-3.25 (bs, 2H), 4.10-4.49 (bs, 2H), 4.50-4.80 (bs, 2H),
7.30-7.75 (m, 6H), 7.90-8.30 (m, 4H); 13C NMR 22.03, 24.55,
25.72, 30.04, 43.30, 62.90, 109.13, 128.32, 129.56, 130.10,
132.92, 166.42. Anal. (C28H32O8) C, H.
1,10-Bis(m et h oxym et h yl)-7,8,15,16-t et r a oxa d isp ir o-
[5.2.5.2]h exa d eca n e (3j): yield 8%; mp 117-118 °C; 1H NMR
1.10-2.05 (m, 16H), 2.75-3.00 (bs, 2H), 3.32 (s, 6H), 3.15-
3.40 (m, 2H), 3.55-3.80 (m, 2H); 13C NMR 22.17, 24.38, 25.84,
29.90, 43.91, 58.84, 70.36, 109.14. Anal. (C16H28O6) C, H.
1,10-Bis(b en zyloxym et h yl)-7,8,15,16-t et r a oxa d isp ir o-
[5.2.5.2]h exa d eca n e (3k ): yield 10%; mp 131 °C (pentane);
1H NMR 1.20-1.82 (m, 12H), 1.85-2.10 (bs, 4H), 2.70-3.00
(bs, 2H), 3.20-3.55 (bs, 2H), 3.60-3.95 (bs, 2H), 4.30-4.70 (m,
4H), 7.15-7.50 (m, 10H); 13C NMR 22.17, 24.39, 26.02, 29.93,
44.06, 68.22, 73.14, 109.18, 127.52, 127.54, 128.32, 138.35.
Anal. (C28H36O6) C, H.
P r oced u r e for P r ep a r a tion of 3g a n d 3h . A mixture of
diester tetraoxane 3e or 3i (1 mmol), dichloromethane (1 mL),
MeOH (10 mL), and KOH (6 mmol, 0.35 g) dissolved in water
(1 mL) was heated to 70 °C for 1 h. Diacid tetraoxane 3g was
obtained by adding concentrated HCl (1 mL) to the cooled
reaction mixture followed by filtration and recrystallization
from DMSO. Tetraoxane diol 3h was obtained by adding water
(20 mL) to the cooled reaction mixture followed by filtration
and recrystallization from MeOH. Tetraoxanes 3g and 3h were
isolated as white crystalline solids.
1,10-Bis(ca r b oxym e t h yl)-7,8,15,16-t e t r a oxa d isp ir o-
[5.2.5.2]h exa d eca n e (3g): yield 72%; mp 198-199 °C dec;
1H NMR (DMSO-d6) 1.10-1.80 (m, 14H), 1.90-2.25 (m, 4H),
2.00-2.20 (m, 2H), 2.75-3.00 (bs, 2H); 13C NMR (DMSO-d6,
30 °C) 21.72, 23.89, 27.96, 29.07, 32.79, 39.68, 108.44, 172.88.
Anal. (C16H24O8) C, H.
1,10-Bis(h yd r oxym e t h yl)-7,8,15,16-t e t r a oxa d isp ir o-
[5.2.5.2]h exa d eca n e (3h ): yield 60%; mp 182 °C dec; 1H
NMR (DMSO-d6) 1.05-2.10 (m, 16H), 2.56-2.89 (bs, 2H),
3.05-3.32 (m, 2H), 3.60-3.89 (m, 2H), 4.54 (t, J ) 5.3 Hz, 2H);
13C NMR (DMSO-d6) 21.93, 23.62, 25.13, 29.19, 45.85, 58.34,
108.99. Anal. C14H24O6: C, H.
An tim alar ial Scr een s. In vitro antimalarial activity against
the drug-sensitive NF54 and chloroquine-resistant K1 strains
of P. falciparum was determined by a modified tritiated
hypoxanthine uptake method of Desjardins et al.13 as previ-
ously described in detail.14 Data were expressed as IC50 values,
estimated as described by Huber and Koella.15 In vivo anti-
malarial activity was assessed using Moro SPF mice infected
with the ANKA strain of P. berghei as described by Ridley et
al.14 Groups of three mice were treated on day 1 post-infection
with tetraoxanes dissolved or suspended in 3% ethanol and
7% Tween 80. Tetraoxanes were administered as single 100
mg/kg doses sc and po. Antimalarial activity was measured
by percent reduction in parasitemia on day 3 post-infection
and by survival times compared to an untreated control group.
limitation, incorporation of unsaturated and polar func-
tional groups at other positions in dispiro-1,2,4,5-
tetraoxanes is possible, and this work provides a guide
to functional group selection in the search for more
orally active tetraoxane antimalarials.
Exp er im en ta l Section
Melting points are uncorrected. Unless noted otherwise, 1H
(300 MHz) and 13C (75 MHz) NMR spectra were recorded on
a Varian XL-300 spectrometer using CDCl3 as a solvent. All
chemical shifts are reported in parts per million (ppm) and
are relative to internal (CH3)4Si for 1H and CDCl3 (77.0 ppm)
for 13C NMR. Microanalyses were performed by M-H-W-
laboratories, Phoenix, AZ. 2-Allyl- and 2-carboethoxymethyl-
cyclohexanone were purcased from Aldrich Chemical Co.
2-Benzyl-, 2-propargyl-, 2-carboethoxyethyl-, and 2-benzyl-
oxymethylcyclohexanone were prepared by enamine alkylation
reactions described by Stork et al.11 2-Hydroxymethylcyclo-
hexanone was prepared from cyclohexanone and paraform-
aldehyde.12 2-Benzoyloxymethylcyclohexanone was synthe-
sized by acylation of 2-hydroxymethylcyclohexanone with
benzoyl chloride in pyridine/dichloromethane. 2-Carboxyethyl-
and 2-carboxymethylcyclohexanone were prepared by hydroly-
sis of the corresponding commercially available ethyl esters
in KOH/MeOH followed by acidification using concentrated
HCl.
Gen er a l P r oced u r e8 for t h e P r ep a r a t ion of Tet r a -
oxa n es 3a -3f a n d 3i-3k . A cold (-20 °C) solution of a parent
cyclohexanone (10 mmol) in acetonitrile (2 mL) was added
dropwise to a stirred, cold (-30 °C) solution of 50% hydrogen
peroxide (0.60 mL, 11 mmol) and concentrated sulfuric acid
(1 mL) in acetonitrile (4 mL). After being stirred for an
additional 1 h at -30 to -20 °C, the solution was kept at -20
°C for 2 days. If the required product precipitated or crystal-
lized out of the reaction solvent (3a , 3c, 3e, 3f, 3i, 3k ), a simple
filtration and recrystallization sufficed for purification. If the
product tetraoxane did not precipitate (3b, 3d , 3j), extraction
with dichloromethane and subsequent silica gel flash column
chromatography eluting with petroleum ether/ether (9:1) were
applied to isolate the product. In each case, product tetra-
oxanes were isolated as white crystalline solids.
1,10-Bis(2-p r op en yl)-7,8,15,16-tetr a oxa d isp ir o[5.2.5.2]-
h exa d eca n e (3a ): yield 5%; mp 101-102 °C (CH3CN); 1H
NMR 1.05-1.45 (m, 4H), 1.46-1.82 (m, 12H), 1.85-2.15 (m,
2H), 2.40-2.70 (m, 2H), 2.80-3.10 (m, 2H), 4.90-5.17 (m, 4H),
5.55-5.90 (m, 2H); 13C NMR 22.31, 24.54, 27.01, 29.91, 32.06,
43.27, 109.28, 116.46, 136.84. Anal. (C18H28O4) C, H.
1,10-Bis(2-p r op yn yl)-7,8,15,16-tetr a oxa d isp ir o[5.2.5.2]-
h exa d eca n e (3b): yield 10%; mp 167-168 °C dec (CH3CN);
1H NMR 1.15-2.40 (m, 20H), 2.50-2.79 (m, 2H), 2.80-3.04
(bs, 2H); 13C NMR 17.54, 22.18, 24.59, 27.19, 29.83, 42.97,
69.44, 82.51, 108.67. Anal. (C18H24O4) C, H.
1,10-Dib en zyl-7,8,15,16-t et r a oxa d isp ir o[5.2.5.2]h exa -
d eca n e (3c): yield 13%; mp 182-184 °C dec (CH3CN); 1H
NMR 1.10-1.42 (m, 4H), 1.48-1.78 (m, 10H), 1.82-1.98 (m,
2H), 2.34-2.53 (m, 2H), 3.03-3.19 (m, 2H), 3.20-3.37 (m, 2H),
7.09-7.37 (m, 10H); 13C NMR 22.36, 24.61, 26.66, 30.08, 33.71,
45.65, 109.31, 125.99, 128.29, 129.21, 140.24. Anal. (C26H32O4)
C, H.
Ack n ow led gm en t. We gratefully acknowledge the
expert technical assistance of Ms. A. Luginbu¨hl with the
vitro and vivo antimalarial assays. This investigation
received financial support from the UNDP/WORLD
BANK/WHO Special Program for Research and Train-
ing in Tropical Diseases (TDR ID No. 960275) and
DHHS/NIH/NIAID (R15 AI39670-01).
1,10-Bis(ca r boeth oxyeth yl)-7,8,15,16-tetr a oxa d isp ir o-
[5.2.5.2]h exa d eca n e (3d ): yield 9%; mp 87 °C; 1H NMR 1.26
(t, J ) 7.2 Hz, 6H), 1.20-1.85 (m, 18H), 1.90-2.50 (m, 6H),
2.80-3.15 (bs, 2H), 4.13 (q, J ) 7.1 Hz, 4H); 13C NMR 14.17,
23.08, 24.39, 27.31, 29.64, 29.72, 32.59, 42.91, 60.24, 109.55,
173.40. Anal. (C22H36O8) C, H.
1,10-Bis(car boeth oxym eth yl)-7,8,15,16-tetr aoxadispir o-
[5.2.5.2]h exa d eca n e (3e): yield 4%; mp 110 °C (pentane);
1H NMR 1.26 (t, J ) 7.1 Hz, 6H), 1.20-1.85 (m, 14H), 2.05-
2.40 (m, 4H), 2.70-2.89 (m, 2H), 2.90-3.12 (bs, 2H), 4.13 (q,
J ) 7.1 Hz, 4H); 13C NMR 14.18, 22.17, 24.70, 28.36, 29.89,
33.34, 40.33, 60.50, 108.80, 172.38. Anal. (C20H32O8) C, H.
1,10-Bis(car boxyeth yl)-7,8,15,16-tetr aoxadispir o[5.2.5.2]-
h exa d eca n e (3f): yield 12%; mp 178 °C (CH3OH:H2O, 1:1);
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