Mixed tetraoxanes containing the acetone subunit as antimalarials

Article abstract of DOI:10.1016/j.bmc.2008.05.017

Eleven new tetraoxanes possessing cholic acid-derived carrier and isopropylidene moiety were synthesized and were tested in vitro and in vivo. In vitro screening revealed that nine of them were more potent against CQ-resistant W2 than CQ-susceptible D6 st

Full text of DOI:10.1016/j.bmc.2008.05.017

Bioorganic & Medicinal Chemistry 16 (2008) 7039–7045
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Mixed tetraoxanes containing the acetone subunit as antimalarials q
a
a
b
b
b
´
Dejan M. Opsenica , Nataša Terzic , Philip L. Smith , Youngsun Yang , Lalaine Anova ,
Kirsten S. Smith b, Bogdan A. Šolaja c,
*
a Institute of Chemistry, Technology and Metallurgy, Belgrade, Serbia
b Division of Experimental Therapeutics, Walter Reed Army Institute of Research, Washington, DC 20307-5100, USA
c Faculty of Chemistry, University of Belgrade, Studentski trg 16, PO Box 158, 11001 Belgrade, Serbia
a r t i c l e i n f o
a b s t r a c t
Article history:
Eleven new tetraoxanes possessing cholic acid-derived carrier and isopropylidene moiety were synthe-
sized and were tested in vitro and in vivo. In vitro screening revealed that nine of them were more potent
against CQ-resistant W2 than CQ-susceptible D6 strain and that two of them were equally or more potent
than artemisinin and mefloquine against multi-drug resistant TM91C235 strain. Amine 8 cured all mice
at the dose of 160 mg/kg/day, while the anilide 9 exhibited MCD 6 20 mg/kg/day. The diol 13 was most
potent antiproliferative with GI50, TGI, LC50 MG_MID 0.98 lM, 3.80 lM, 11.22 lM, respectively. All tested
compounds showed no toxic effects.
Received 3 April 2008
Revised 30 April 2008
Accepted 7 May 2008
Available online 10 May 2008
Keywords:
Mixed steroidal tetraoxanes
Cholic acid
Ó 2008 Elsevier Ltd. All rights reserved.
Antimalarials
Antiproliferatives
Metabolic stability
1. Introduction
this carrier is based on the expectation that the introduction of an
amphiphilic lipid carrier that is spiro bound to a 1,2,4,5-tetraoxa-
Malaria is one of the most deadly diseases of the developing
world, affecting 300–500 million people annually with an esti-
mated death toll of 1–3 million people. The most affected regions
are tropical Africa, Southeast Asia, and the Amazon region. Malaria
is caused by a one-celled parasite of the genus Plasmodium. Of the
four species that infect humans, Plasmodium falciparum is the most
dangerous, accounting for half of all clinical cases of malaria and
90% of deaths from the disease. Malaria kills one African child
every 30 s, and the emergence of multi-drug resistant strains of
plasmodia has rendered many affordable antimalarials such as
chloroquine (CQ) much less effective in addressing the severe
health issues in endemic regions.
Great efforts were recently expended to discover a new and effi-
cacious endoperoxide-based antimalarial,2 primarily because no
resistance to this type of drugs has yet been observed.3 Recently,
MMV-financed research on trioxolanes afforded the first drug can-
didate for Phase II human trials4 while researchers at WRAIR are
rapidly advancing a GMP version of Artesunate through Phase II
trials.
cyclohexane in the form of a mixed tetraoxane6 will afford similar
activity to the artemisinins,2c or to the 1,2,4-trioxolanes,7 but with
improved physicochemical properties. Although peroxide antimal-
arials have a rapid onset of action, their half-life is relatively short
and recrudescence often occurs.4 Therefore, the efforts to develop
these new antimalarials are needed to sustain the future introduc-
tion of new drug candidates.
In this paper, we report on a series of new steroidal mixed tet-
raoxanes containing the acetone subunit and analyze their antima-
larial activity. The idea was to introduce a simple subunit that had
little or no effect on any other part of the spirotetraoxacyclohexane
moiety, specifically, on the steroidal part, in order to examine the
influence of a cholic acid-derived carrier on the antimalarial activ-
ity. A review of the literature revealed syntheses of several tetraox-
ane structures by other groups; however, none of the compounds
had appreciable activity.8,9
2. Results and discussion
One part of our research in this field is focused on the develop-
ment of a new type of peroxide antimalarial called a 1,2,4,5-tetra-
oxacyclohexane (tetraoxane) that utilizes a steroid carrier.5 Use of
Mixed tetraoxanes 3–12 were prepared using the procedure
previously described.6a gem-Dihydroperoxide 2 was directly cou-
pled to acetone to give the parent methyl ester 3 (Scheme 1). Ester
3 was selectively hydrolyzed at C(24) into the corresponding acid
4, which was further transformed into amides 5–12 using a mixed
anhydride procedure. The overall yield starting from cholic acid
was ca. 31% (calculated, e.g., to amide 9).
q
See Ref. 1.
* Corresponding author. Tel.: +381 11 263 86 06; fax: +381 11 263 60 61.
E-mail address: bsolaja@chem.bg.ac.yu (B.A. Šolaja).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.05.017

7040
D. M. Opsenica et al. / Bioorg. Med. Chem. 16 (2008) 7039–7045
OH
AcO
AcO
AcO
CO2H
72%
CO2CH3
i)
CO2CH3
CO2H
ii)
O
O
O
HOO
HOO
O
O
HO
OH
OAc
OAc
OAc
H
O
H
H
O
H
O
1
2
3
4
iii)
O
AcO
O
HO
W
5: W = NH2, 91 %; 6: W = NHCH3, 92%; 7: W= NHPrn, 86%;
8: W = NHCH2CH2N(CH3)2, 86%; 9: W = NHPh, 82%;
10: W = NHCH2CO2CH3, 82%; 11: W = N( CH3)2, 86%;
12: W = NHNH2, 59%;
NH2
iv)
O
5
O
OAc
5-12
O
O
H
O
O
OH
O
H
O
13
Scheme 1. Reagents and conditions: (i) acetone, H2SO4/CH3CN, 53%; (ii) NaOH/i-PrOH/H2O, 80 °C, 99%; (iii) Et3N/ClCO2Et/CH2Cl2, amine; (iv) NaOH/MeOH/H2O, 70 °C, 96%.
Tetraoxanes were assayed in vitro against four P. falciparum
strains: D6, W2, RCS, and TM91C235 (Tables 1 and 2). In general,
tetraoxanes were more active against CQ-resistant strains (W2,
RCS, and TM91C235) than the CQ-susceptible D6 strain. As noticed
before,5,6 amides exhibit better in vitro activity than the corre-
sponding esters and acids.
Table 2
In vitro antimalarial activities of tetraoxanes 3–13 against P. falciparum RCSa and
TM91C235b strains
Compound
W
IC50 (nM)
TM91C235 RCS
IC90 (nM)
TM91C235
RCS
3
4
6
8
9
11
12
13
OCH3
OH
NHCH3
NDc
ND
11.55
25.99
ND
ND
57.27
231.16
74.82
19.82
20.89
50.93
38.07
Tetraoxanes 6, 9, and 11–13 were tested against a CQ-resistant
P. falciparum isolate from South America (RCS) and were found to
be more active against this strain than against the Southeast
Asian strains, W2 and TM91C235. In addition, the N-(2-dimethyl-
amino)ethyl and N-phenyl amides, 8 and 9, respectively, were
also potent against the latter two CQ-resistant strains. As it is
believed that peroxide antimalarials exert their activity in the
parasite food vacuole (FV),10 which has a pH ꢀ 5.5, the N-(2-
dimethylamino)ethyl derivative 8 was designed to potentially
concentrate at the site of action through suppressed efflux of
the protonated form through the FV membrane. Although tetra-
oxane 8 is less active than mefloquine against W2 strain, it exhib-
ited an IC90/IC50 ratio of 1.71 as compared to mefloquine’s ratio of
2.72. Moreover, 8 is 2–3 times more active against the multi-drug
resistant TM91C235 strain than mefloquine with a IC90/IC50 ratio
of about 1.5 times lower. N-Phenyl derivative 9 and mefloquine
have similar activities against all tested strains; however, 9 exhib-
ited a threefold better IC90/IC50 ratio against TM91C235. The
60.44
31.28
7.16
15.75
25.13
28.47
227.70
13.04
16.44
22.26
ND
4.45
14.00
19.49
167.17 498.31
17.40
5.00
NHCH2CH2N(CH3)2 ND
NHPh
N(CH3)2
NHNH2
NH2
1.89
7.19
7.03
38.70
Artemisinind
Mefloquinee
Chloroquinee
2.33
65.71
192.45 144.90
397.09 268.76
a
RCS is strain from Brazil, resistant to chloroquine, susceptible to mefloquine.
TM91C235 (Thailand) is highly resistant to mefloquine, chloroquine, and
b
quinine.
c
ND, not determined.
Average of >8 replicates.
Control drugs.
d
e
activity of both 8 and 9 is comparable to, or better than that of
artemisinin (Tables 1 and 2).
Table 1
In vitro antimalarial activities of tetraoxanes 3–13 against P. falciparum D6a and W2b strains, and metabolic half-lives
Compound
W
IC50 (nM)
IC90 (nM)
Metabolic t1/2
(min) (human)
Metabolic t1/2
(min) (mouse)
D6
14.98
46.50
12.21
46.87
7.40
W2
11.67
36.04
9.40
22.35
18.14
9.79
D6
26.68
96.28
ND
78.28
16.16
26.24
20.84
W2
20.70
94.67
ND
43.65
29.25
16.76
14.35
3c
4
5
6
7
8
9
OCH3
OH
NH2
NDd
>60
>60
>60
24
ND
>60
>60
>60
25
NHCH3
NHPrn
NHCH2CH2N(CH3)2
NHPh
11.60
6.97
>60
19
>60
16
5.02
10
11
12
13
NHCH2CO2CH3
N(CH3)2
NHNH2
NH2
14.28
35.54
28.10
215.25
9.0
5.98
ND
ND
70
49
ND
>60
—
32
17
ND
>60
—
17.29
16.36
153.31
6.7
3.09
349.35
71.47
46.31
346.51
12.8
21.65
17.63
31.62
22.95
233.95
11.5
8.41
491.53
Artemisinine
Mefloquinef
Chloroquinef
10.52
13.72
—
—
—
—
a
D6 is a clone from the Sierra I/UNC isolate and is susceptible to chloroquine and pyrimethamine, but has reduced susceptibilities to mefloquine and halofantrine.
b
c
d
e
f
W2 is a clone of the Indochina I isolate and is resistant to chloroquine and pyrimethamine, but susceptible to mefloquine.
Performed in duplicate, average reported.
ND, not determined.
Average of >8 replicates.
Control drugs.

D. M. Opsenica et al. / Bioorg. Med. Chem. 16 (2008) 7039–7045
7041
In attempt to increase the water solubility, we prepared tetra-
oxane 13 with free hydroxyl groups at C(7) and C(12). Unfortu-
nately, despite much better calculated solubility (Advanced
Chemistry Development, ACD/logD Solubility Suite, Version 8.0;
50 lg/mL, pH 2.0, logP 2.42) the in vitro activity of derivative 13
sharply decreased in comparison to parent tetraoxane 5 (calculated
solubility 0.00354 lg/mL, pH 2.0, logP 4.21), and it is the least ac-
tive compound in the series.
6–31. All mice were considered to be cured, and no significant
gross lesions were observed at necropsy. In the 40 mg/kg/day dose
group, all mice had negative blood smears on days 6 and 10. One
mouse was cured, and the other four mice recrudesced on day
13. Three of the 4 mice died by day 27: one on day 18, one on
day 20, and the other on day 27, with all showing typical malaria
gross lesions at necropsy. The other mouse maintained low posi-
tive blood smear results during days 13–20 and cleared from days
24–31. Mean parasitemia results on days 10, 13, 17, 20, 24, 27, and
31 were 0%, 0.004%, 15.48%, 3.9%, 12.53%, 0%, and 0%, respectively.
No gross lesions were seen in this mouse at necropsy, which was
consistent with the presence of negative blood smears from days
27 to 31. Finally, at the low dose of 20 mg/kg/day, a delay in pa-
tency was observed in all mice: four mice were negative on day
6 but parasite recrudesced on days 10 and 13. The other mouse
had a very low positive parasitemia (0.002%) on day 6. All mice
died between days 14 and 17 (1, 1, 1, and 2 mice on days 14, 15,
16, and 17, respectively). Mean parasitemia results on days 10,
13, and 17 were 0.032%, 8.72%, and 26.8%, respectively.
In vitro metabolism studies were performed on compounds 5–
8, 10, 11, and 13 to assess the bioavailability of possible drug can-
didates after oral administration. The metabolic stability assays
were done using human and mouse liver microsomes.6c Stable
compounds were defined as having half-lives >60 min and the rel-
evant data are given in Table 1. The in vitro metabolic half-life can
be used to approximate an in vivo half-life/hepatic clearance rate.
The tetraoxanes demonstrated a wide range of stability with half-
lives from 24 min to greater than 60 min. Metabolite ID was per-
formed in order to identify differences in Phase I metabolism
across species, as well as to aid in interpretation of future animal
testing. The primary routes of metabolism for the tetraoxanes in-
clude hydroxylation, dihydroxylation, demethylation, and desatu-
ration; species differences were sometimes observed (Table 4).
In trying to correlate the metabolic profile with the in vitro and
in vivo efficacy data, no consistent or direct correlation could be
made between the metabolic profile and in vitro and in vivo test-
ing. Specifically, there did not appear to be a correlation between
the hepatic clearance rate, as estimated by metabolic stability,
and efficacy or toxicity. Even so, the ADME data collected are cur-
rently being used in conjunction with physicochemical and efficacy
data to identify structural liabilities within the compounds that
may be easily modified to improve drug-like properties without
Derivatives 3, 4, 8, and 9 were screened in vivo (po) against the
drug-sensitive Plasmodium berghei KBG 173 strain following the
modified Thompson test.6b,11 The data presented in Table 3 show
that compounds 3 and 4 extended mouse survival with a minimum
active dose (MAD) of 320 and 80 mg/kg/day, respectively. The N-
phenyl amide 9 cured 2 mice on day 31 at all three administered
doses; surviving mice showed complete clearance of parasitemia
and surviving mice were considered cured even at the low dose
of 20 mg/kg/day (0.035 mmol/kg/day). Compound 8, administered
orally at 320, 80, and 20 mg/kg, cured 4 of 5 mice at the high dose,
had a minimum active dose of 80 mg/kg/day, and was inactive at
the low dose. However, some observations merit further comment:
out of 5 mice in the group receiving 320 mg/kg/day, all were para-
site free from days 6–13. Parasite recrudesced in 2 mice on day 17.
One of the two mice died on day 20; however, the second one had
positive blood smears on days 17, 20, and 24 but negative blood
smears on days 27 and 31. The other three mice had negative blood
smears from days 6–30. Mean parasitemia results on days 17, 20,
24, 27, and 31 for that mouse were 2.36%, 1.9%, 1.4%, and 0%,
respectively. The other three mice had negative blood smears on
days 27 and 31. There was no indication of toxicity of the examined
tetraoxanes on tested animals.
It was calculated that tetraoxane 8 was appreciably more solu-
ble than the other acetone-derived tetraoxanes (calculated solubil-
ity 5380 lg/mL, pH 2.0), and since it was metabolically quite stable
with half-lives >60 min in human and mouse liver microsomes (Ta-
ble 1), the activity of this compound was examined in sc screen.
Tetraoxane 8 was administered sc at 10, 20, and 80 mg/kg twice
per day at 12 h apart for 3 days. Necropsies were performed in
all animals, and the eight mice that died prior to day 31 showed
typical gross lesions typical of a fatal malaria infection, such as
gray swollen liver, dark spleen, and pale emaciated carcass (similar
to the control group mice). In the 160 mg/kg/day group, 5 of 5 mice
survived through day 31 and had negative blood smears from days
Table 3
In vivo antimalarial activity of tetraoxanes 3, 4, 8, and 9 after administration against drug-sensitive P. berghei (KBG 173 strain)
Compound
mg/kg/day
Mice dead/day died
Mice alive day 31/total
Survival timea (days)
3, pob
320
80
20
320
80
20
320
80c
20
160
40
20
320
80
1/9, 1/16, 3/21
3/9, 1/21
1/7, 2/9, 2/21
2/14, 2/16, 1/25
1/7, 1/12, 1/16, 2/21
4/9, 1/12
0/5
1/5
0/5
0/5
0/5
0/5
4/5
0/5
0/5
5/5
2/5
0/5
2/5
2/5
2/5
0/5
18
16
13
17
15
10
29
14
8
31
25
16
23
25
23
7
4, po
8
po
1/20
1/10, 1/12, 1/14, 1/20
5/7–9
scd
1/18, 1/20, 1/27
1/14, 1/15, 1/16, 2/17
1/13, 1/17, 1/21
3/21
9, po
20
1/14, 1/17, 1/21
5/7
Infected control group
a
Including cured mice.
b
Groups of five P. berghei (KBG 173 strain) infected CD-1 mice were treated on days 3, 4, and 5 postinfection with tetraoxanes suspended in 0.5% hydroxyethylcellulose–
0.1% Tween 80. Mice alive on day 31 with no parasites in a blood film are considered cured.
c
One mouse excluded.
d
Drugs were suspended in sesame oil, and administered beginning on day 3 postinfection two times a day.

7042
D. M. Opsenica et al. / Bioorg. Med. Chem. 16 (2008) 7039–7045
Table 4
4. Experimental
Metabolites tentatively identified by LC–MS/MS in human and mouse microsomes
Compound
Human metabolite formationa
Mouse metabolite formation
4.1. General
[MH+16]+ [MHꢃ2]+ [MH+2]+ [MH+16]+ [MHꢃ2]+ [MH+2]+
Melting points were determined on a Boetius PMHK apparatus
and were not corrected. Optical rotation measurements were per-
formed on a Perkin-Elmer 341 polarimeter, at the given tempera-
tures. Concentrations are expressed in g/100 mL. IR spectra were
recorded on Perkin-Elmer spectrophotometer FT-IR 1725X. 1H
and 13C NMR spectra were recorded on a Varian Gemini-200 spec-
trometer (at 200 and 50 MHz, respectively) in the indicated solvent
using TMS as internal standard. Chemical shifts are expressed in
ppm (d) values and coupling constants (J) in Hz. ESI mass spectra
were acquired on a TSQ (Thermo Quest; Finnigan) quadrupole
instrument. Samples were dissolved in HPLC grade acetonitrile. Po-
sitive ions: capillary temperature 250 °C; ESI spray voltage 4.5 kV;
multiplier: 1500 V; loop injection into a solution of H2O/CH3CN/
HCOOH (1%) = 18:80:2 (v/v/v); flow rate: 0.3 mL/min. FAB mass
spectra were recorded with a JEOL JMS-SX 102 A spectrometer
and on a VG-ZAB-T instrument equipped with Cs ion gun. Acceler-
ating voltage was set to 8 kV using MNBA as matrix. Probe accurate
mass measurements were performed in the presence of PEG inter-
nal calibrant at 5000 resolution. Metabolic stability experiments:
ThermoFinnigan TSQ triple quadrupole mass spectrometer coupled
to a ThermoQuest Surveyor MS pump LC system using Xcalibur
software, Version 1.3. ThermoFinnigan LTQ ion trap mass spec-
trometer coupled to Agilent 1100 LC system using Xcalibur soft-
ware, Version 1.4. ESI positive ion. 0.1% formic acid, acetonitrile,
water. Waters XTerra MS C18 column, 2.1 ꢁ 50 mm, 3.5 lm parti-
cle size with 2.1 ꢁ 10 mm corresponding guard column. Gradient
95:5 formic:ACN to 25:75 over 6 min, returning to 95:5 at 6.01
at equilibrating for 3 min. Flow rate 300 lL/min. ESI negative ion.
Ten millimolar ammonium acetate (pH 9.2), acetonitrile, water.
Waters symmetry C8 column, 2.1 ꢁ 50 mm, 5 lm particle size.
Gradient 95:5 Am acetate:ACN to 5:95 over 3 min, hold for
5 min, returning to 95:5 at 8.10 min and equilibrating for 4 min.
Flow rate 300 lL/min, equilibration at 350 lL/min. Thin-layer chro-
matography (TLC) has been performed on precoated Merck silica
gel 60 F254 plates, using N,N-dimethyl-p-phenylene-diammonium
dichloride peroxide reagent for peroxide moiety detection,12 and
Lobar LichroPrep Si 60 (40–63 lm) columns coupled to Waters RI
401 detector were used for column chromatography. Where appro-
priate, the compounds are listed according to their elution order.
gem-Dihydroperoxide (2) and tetraoxanes 3–12 were synthe-
sized according to procedure described earlier.6a
4
5
6
7
ꢄ
ꢄ
ꢄ
ꢄ
ꢄ
ꢄ
ꢄ
ꢄ
ꢄ
ꢄ
ꢄ
ꢄ
ꢄ
ꢄ
ꢄ
ꢄ
ꢄ
ꢄ
8
9b
10
11
13
ꢄ
ꢄ
ꢄ
ꢄ
ꢄ
Tentative assignment: [MH+16]+ = hydroxylation; [MHꢃ2]+ = desaturation;
a
[MH+2]+ = demethylation + hydroxylation.
b
Undergoes dihydroxylation in both species.
Table 5
Inhibitory and lethal concentration in lM calculated from MG_MID point values
Compound
4
5
6
8
10
8.51
33.88
77.62
11
13
0.98
3.80
11.22
a
GI50
15.49
37.15
75.86
8.71
34.67
79.43
4.47
18.20
47.86
3.39
16.98
41.69
75.86
TGIb
10.23
22.39
c
LC50
a
GI50, the concentration of the drug that inhibits percentage growth by 50%.
TGI, total growth inhibition (concentration of the drug to achieve cytostasis).
LC50, concentration of the compound at which 50% of the cells are killed.
b
c
sacrificing compound potency. Since the in vitro activity of these
compounds is comparable to or better than artemisinin and meflo-
quine, continuing efforts will focus on strategies to improve in vivo
potency. Future work will continue to explore this promising class
of antimalarial tetraoxane derivatives.
Although the in vivo tested compounds showed no toxicity on
tested animals, based on previous results,5b,c it was of interest to
examine present tetraoxanes’ cytotoxicity against diverse cancer
cell lines. Seven compounds were tested in vitro by the National
Cancer Institute (NCI), Bethesda, in a panel of 60 human tumor cell
lines. Selected results are given as in Table 5. In general, the tested
tetraoxanes exhibit good total growth inhibitory activity, and low
overall cytotoxicity against cancer cell lines as indicated by
MG_MID (meangraph midpoint) values (Table 5).
The most active compound was the primary amide 13 that has
free hydroxyl groups at C(7) and C(12) and exhibits strong antipro-
liferative activity against the renal cancer cell line A498
(GI50 = 0.092 lM, TGI = 0.71 lM, and LC50 = 11.50 lM). It is more
potent in inhibiting the cell growth than in cell-killing, that is, its
GI50 value is 125 times lower than respective LC50. It is interesting
to note that the compound being the least active against malaria
parasites is the most active against cancer cell lines, suggesting
that, in this case, the structural features disfavoring antimalarial
activity stimulate the antiproliferative activity.
4.1.1. Methyl 7a,12a-diacetoxy-5b-cholan-24-oate-3-spiro-60-
(30,30-dimethyl-10,20,40,50-tetraoxacyclohexane) (3)
To the cold solution (ice-bath) of dihydroperoxide 2 (500.0 mg,
0.9 mmol) in dichloromethane (12.5 mL), acetone (132 lL,
1.80 mmol) was added and after 30 min, 650 lL of ice-bath cooled
(H2SO4/CH3CN) mixture (1:10, v/v) was added dropwise. The reac-
tion mixture was stirred at 0 °C for 15 min, and after usual work-up
the crude mixture was purified by column chromatography (Lobar,
LichroPrep RP-18, eluent MeOH/H2O = 8:2) to afford tetraoxane 3.
3. Conclusion
In this work, we reported the results of our study on mixed tetra-
oxanes possessing cholic acid-derived carrier and ‘open chain’ iso-
propylidene moiety originating from acetone. The obtained results
indicate that the compounds are efficient antimalarials in vitro and
in vivo. Acetone-derived tetraoxanes are more potent against CQ-
resistant W2 and multi-drug resistant TM91C235 strains, than
against CQ-susceptible D6 strain. These results, taken together with
the ease of the compounds’ synthesis (amide derivatives were ob-
tained in ca. 44% from cholic acid), their in vivo potential, and estab-
lished non-toxicity, reveals this class of tetraoxanes as promising
alternative to other peroxide antimalarials.
20
Yield 284 mg (53%). Colorless foam, softening at 77–80 °C. ½aꢂD
+48.50 (c 1.04, CHCl3). IR(KBr): 2953s, 2872s, 1738s, 1606w,
1440s, 1377s, 1240s, 1165s, 1126s, 1103s, 1076m, 1026s, 966w,
943w cmꢃ1 1H NMR (200 MHz, CDCl3): 5.09 (br s, H–C(12)),
.
4.95–4.90 (m, H–C(7)), 3.66 (s, CH3O2C(24)), 2.13 (br s, CH3COO–),
2.08 (br s, CH3COO–), 0.95 (H3C–C(10)), 0.81 (d, J = 6.0 Hz,
H3C–C(20)), 0.74 (H3C–C(13)). 13C NMR (50 MHz, CDCl3): 174.44,
170.51, 108.15, 107.82, 75.18, 70.57, 51.40, 47.21, 44.96, 43.23,
37.55, 34.58, 34.45, 32.03, 30.72, 30.61, 28.37, 27.02, 25.60, 22.65,
22.02, 21.49, 21.31, 20.58, 17.37, 12.09. FAB-MS (m/z): 1189.8

D. M. Opsenica et al. / Bioorg. Med. Chem. 16 (2008) 7039–7045
7043
([2M]+, 23), 733.5 (28), 663.5 (14), 613.2 ([M+NH4]+, 17), 595.4
([M+H]+, 100). Anal. Calcd for C32H50O10+H2O: C, 62.72; H, 8.55.
Found: C, 62.29; H, 8.28. Note: in one attempt we obtained a sample
with correct microanalytical data; however, it is difficult to obtain a
H2O free sample without significant heating in vacuo. Then, decom-
position is probable.
(KBr): 3430s, 2954m, 2875w, 1736s, 1651m, 1554w, 1445w,
1377w, 1245s, 1204m, 1164w, 1126w, 1076w, 1027m, 944w,
895w cmꢃ1 1H NMR (200 MHz, CDCl3): 5.60–5.45 (m, HN–C(24)),
.
5.09 (br s, H–C(12)), 4.95–4.85 (br s, H–C(7)), 2.80 (d, J = 4.8 Hz,
H3C–NH), 2.13 (br s, CH3COO–), 2.08 (br s, CH3COO–), 0.94 (H3C–
C(10)), 0.81 (d, J = 6.0 Hz, H3C–C(20)), 0.73 (H3C–C(13)). 13C NMR
(50 MHz, CDCl3): 173.91, 170.64, 108.22, 107.90, 75.27, 70.63,
47.43, 44.99, 43.24, 37.56, 34.69, 34.62, 33.34, 31.46, 28.41,
27.11, 26.24, 25.68, 22.71, 22.05, 21.58, 21.42, 17.50, 12.17.
(+)ESI-MS (m/z): 658.29 (30), 657.29 (75), 616.29 ([M+Na]+, 85),
594.29 ([M+H]+, 100). Anal. Calcd for C32H51NO9+0.5H2O: C,
63.76; H, 8.70. Found: C, 63.49; H, 8.89.
4.1.2. 7a,12a-Diacetoxy-5b-cholan-24-oic acid-3-spiro-60-(30,30-
dimethyl-10,20,40,50-tetraoxacyclohexane) (4)
Methyl ester 3 (300.0 mg, 0.5 mmol) was hydrolyzed at 80 °C
with NaOH (30.26 mg, 0.7 mmol) in i-PrOH/H2O mixture (120 mL,
3:1 v/v). After 15 min the reaction mixture was cooled and diluted
with 50 mL H2O and 100 mL CH2Cl2. Water layer was acidified to
pH 2 with diluted HCl, and layers were separated. Water layer
was further extracted with CH2Cl2 (3ꢁ 30 mL), combined organic
layers were washed with water and brine, dried over anhyd Na2SO4
and evaporated to dryness. Yield 291.2 mg (99%). Mp = 195–197 °C
4.2.3. N-(n-Propyl)-7a,12a-diacetoxy-5b-cholan-24-amide-3-
spiro-60-(30,30-dimethyl-10,20,40,50-tetraoxacyclohexane) (7)
Acid 4 (300 mg, 0.52 mmol) was transformed into amide 7
(284.6 mg, 86%) according to general procedure using a 6 equiv
n-PrNH2 (251 lL, 3.10 mmol) in dry CH2Cl2 (20 mL). Column
chromatography: Lobar B, LichroPrep Si 60, eluent EtOAc/hep-
20
(colorless prisms, diisopropylether). ½aꢂD +47.95 (c 1.20, CHCl3).
IR(KBr): 3453m, 2965s, 2869w, 1735s, 1702m, 1452w, 1378s,
1256s, 1245s, 1203m, 1182m, 1163w, 1128w, 1103w, 1075w,
20
tane = 95:5. Colorless foam, softening at 101–103 °C. ½aꢂD
1026m, 968w cmꢃ1
.
1H NMR (200 MHz, CDCl3): 5.09 (br s, H–
+36.05 (c 0.92, CHCl3). IR (KBr): 3404m, 2958s, 2875m, 1737s,
C(12)), 4.95–4.90 (m, H–C(7)), 2.13 (br s, CH3COO–), 2.09 (br s,
CH3COO–), 0.95 (H3C–C(10)), 0.82 (d, J = 5.8 Hz, H3C–C(20)), 0.74
(H3C–C(13)). 13C NMR (50 MHz, CDCl3): 179.72, 170.67, 108.22,
107.92, 75.27, 70.70, 47.27, 45.03, 43.30, 37.62, 34.63, 34.47,
30.74, 30.45, 28.42, 27.08, 25.68, 22.73, 22.07, 21.58, 21.40,
20.69, 17.41, 12.17. FAB-MS (m/z): 1161.68 (2M+, 3), 603.28
([M+Na]+, 5), 580.70 (M+, 4). Anal. Calcd for C31H48O10+0.5H2O: C,
63.14; H, 8.38. Found: C, 63.58; H, 8.29.
1649s, 1547m, 1444m, 1377s, 1244s, 1204m, 1163w, 1126w,
1076w, 1027m, 964w, 944w cmꢃ1 1H NMR (200 MHz, CDCl3):
.
5.60–5.45 (m, HN–C(24)), 5.09 (br s, H–C(12)), 4.91 (br s, H–
C(7)), 3.20–3.15 (m, CH3CH2CH2–NH), 2.13 (br s, CH3COO–),
2.08 (br s, CH3COO–), 1.60–1.40 (m, CH3CH2CH2–NH–), 1.00–
0.85 (m, H3C–C(10), and CH3CH2CH2–NH–), 0.81 (d, J = 6.0 Hz,
H3C–C(20)), 0.73 (H3C–C(13)). 13C NMR (50 MHz, CDCl3):
173.17, 170.62, 108.19, 107.86, 75.25, 70.61, 47.41, 44.98,
43.23, 41.08, 37.55, 34.65, 34.60, 33.52, 31.52, 28.37, 27.10,
25.64, 22.80, 22.69, 22.03, 21.56, 21.40, 17.48, 12.15, 11.27.
FAB-MS (m/z): 1243.8 ([2M]+, 7), 757.5 (3), 622.4 ([M+H]+,
100), 562.4 (6). Anal. Calcd for C34H55NO9+0.5H2O: C 64.74; H
8.95. Found: C, 64.68; H, 8.92.
4.2. General procedure for preparation of amides 5–12
A solution of 4 (600.0 mg, 1.03 mmol), in dry CH2Cl2 (40 mL),
with added Et3N (143 lL, 1.03 mmol) and ClCO2Et (99 lL,
1.03 mmol) was stirred for 60 min at 0 °C. Given amount of amine
was added, and after 30 min of stirring the reaction mixture was
warmed to rt. After 90 min it was diluted with H2O, the layers were
separated and the reaction mixture was worked-up in a usual man-
ner.6a Crude product was purified by column chromatography.
4.2.4. N-(2-Dimethylamino)ethyl-7a,12a-diacetoxy-5b-cholan-
24-amide-3-spiro-60-(30,30-dimethyl-10,20,40,50-tetraoxacyclohex-
ane) (8)
Acid 4 (312.1 mg, 0.54 mmol) was transformed into amide 8
(300.4 mg, 86%) according to general procedure using a 6 equiv
N,N-dimethylethane-1,2-diamine (343 lL, 3.23 mmol) in dry
CH2Cl2 (20 mL). Column chromatography: Lobar B, LichroPrep Si
60, eluent CHCl3/MeOH/NH3 = 9:1:1. Colorless foam, softening at
4.2.1. 7a,12a-Diacetoxy-5b-cholan-24-amide-3-spiro-60-(30,30-
dimethyl-10,20,40,50-tetraoxacyclohexane) (5)
Using a suspension of 10 equiv NH4Cl and 10 equiv Et3N in dry
CH2Cl2 (20 mL), 541.7 mg (91%) of 5 was obtained. Column chro-
matography: Lobar B, LichroPrep Si 60, eluent EtOAc. Colorless
20
86–88 °C. ½aꢂD 48.5 (c 0.194, CHCl3). IR(KBr): 3443m, 2951s,
2873m, 1734s, 1653w, 1544m, 1449m, 1376m, 1247s, 1203w,
1125w, 1076w, 1028m, 957w cmꢃ1 1H NMR (200 MHz, CDCl3):
.
20
foam, softening at 122–124 °C. ½aꢂD +42.12 (c 1.02, CHCl3). IR(KBr):
6.20–6.10 (m, HN–C(24)), 5.09 (br s, H–C(12)), 4.91 (br s, H–
C(7)), 3.40–3.25 (m, –NH–CH2CH2N(CH3)2), 2.50–2.42 (m, –NH–
CH2CH2N(CH3)2), 2.27 (s, –N(CH3)2), 2.13 (br s, CH3COO–), 2.09
(br s, CH3COO–), 0.94 (s, H3C–C(10)), 0.82 (d, J = 6.0 Hz, H3C–
C(20)), 0.73 (H3C–C(13)). 13C NMR (50 MHz, CDCl3): 173.44,
170.62, 108.21, 107.88, 75.27, 70.63, 57.83, 47.41, 44.99, 44.96,
43.24, 37.60, 36.42, 34.62, 33.27, 31.37, 30.54, 28.41, 27.10,
25.66, 22.71, 22.05, 21.58, 21.40, 17.50, 12.17. (+)ESI-MS (m/z):
725.42 (10), 699.49 (5), 652.56 (35), 651.45 ([M+H]+, 100). FAB-
MS (m/z): 651.3 ([M+H]+, 100), 649.3 (11), 561.3 (7), 307.0 (12),
289.0 (7), 154.0 (64), 59.2 (60), 44.4 (9). HRMS-ESI: m/z 651.4236
corresponds to C35H59O9N2 (error in ppm: 2.3).
3456m, 2957s, 2877m, 1736s, 1674s, 1618w, 1445m, 1378s,
1244s, 1204m, 1164w, 1127w, 1076w, 1027m, 965w, 944w
cmꢃ1 1H NMR (200 MHz, CDCl3): 5.73 (br s, H2N–C(24)), 5.52 (br
.
s, H2N–C(24)), 5.09 (br s, H–C(12)), 4.91 (br s, H–C(7)), 2.13 (br s,
CH3COO–), 2.09 (br s, CH3COO–), 0.94 (H3C–C(10)), 0.82 (d,
J = 6.0 Hz, H3C–C(20)), 0.73 (H3C–C(13)). 13C NMR (50 MHz, CDCl3):
175.90, 170.62, 108.19, 107.88, 75.23, 70.61, 47.36, 44.99, 43.24,
37.55, 34.60, 32.63, 31.26, 28.37, 27.11, 25.66, 22.67, 22.03,
21.56, 21.40, 17.48, 12.17. (+)ESI LC–MS/MS (m/z): 579.9 (MH+),
519.68 (MHꢃ60)+, 459.99 (MHꢃ120)+. Anal. Calcd for
C31H49NO9+0.5H2O: C, 63.24; H, 8.56. Found: C, 63.32; H, 8.89.
4.2.2. N-Methyl-7a,12a-diacetoxy-5b-cholan-24-amide-3-spiro-
60-(30,30-dimethyl-10,20,40,50-tetraoxacyclohexane) (6)
4.2.5. N-Phenyl-7a,12a-diacetoxy-5b-cholan-24-amide-3-spiro-
60-(30,30-dimethyl-10,20,40,50-tetraoxacyclohexane) (9)
Acid 4 (300.0 mg, 0.52 mmol) was transformed into amide 9
(277.3 mg, 82%) according to general procedure using a 6 equiv
PhNH2 (288 lL, 3.12 mmol) in dry CH2Cl2 (20 mL). Column chro-
matography: Lobar B, LichroPrep Si 60, eluent EtOAc/heptane = 3:7.
Acid 4 (300 mg, 0.52 mmol) was transformed into amide 6
(281 mg, 92%) according to general procedure using a suspension
of 6 equiv MeNH3Cl and 6 equiv Et3N in dry CH2Cl2 (20 mL). Col-
umn chromatography: Lobar B, LichroPrep Si 60, eluent EtOAc. Col-
20
orless foam, softening at 117–118 °C. ½aꢂD +59.8 (c 0.132, CHCl3). IR

7044
D. M. Opsenica et al. / Bioorg. Med. Chem. 16 (2008) 7039–7045
20
Colorless foam, softening at 126–128 °C. ½aꢂD +46.3 (c 0.082,
CHCl3). IR (KBr): 3453m, 2949m, 2876w, 1736s, 1669w, 1602m,
1541m, 1498m, 1442m, 1377m, 1245s, 1204w, 1126w, 1077w,
4.92 (br s, H–C(7)), 2.13 (br s, CH3COO–), 2.08 (br s, CH3COO–),
0.94 (H3C–C(10)), 0.81 (d, J = 5.6 Hz, H3C–C(20)), 0.73 (H3C–
C(13)). 13C NMR (50 Hz, CDCl3): 170.62, 170.20, 108.21, 107.90,
75.20, 70.61, 47.32, 45.01, 43.26, 37.56, 34.62, 31.05, 30.88,
28.39, 27.13, 25.68, 22.69, 22.38, 22.07, 21.58, 21.43, 20.63,
17.48, 12.18. Anal. Calcd for C31H50N2O9: C, 62.61; H, 8.47. Found:
C, 62.70; H, 8.35.
1027m, 965w cmꢃ1 1H NMR (200 MHz, CDCl3): 7.60–7.00 (m,
.
Ph–NHC(24)), 5.10 (br s, H–C(12)), 4.92 (br s, H–C(7)), 2.13 (br s,
CH3COO–), 2.08 (br s, CH3COO–), 0.94 (H3C–C(10)), 0.85 (d,
J = 5.6 Hz, H3C–C(20)), 0.73 (H3C–C(13)). 13C NMR (50 Hz, CDCl3):
171.51, 170.67, 137.94, 128.96, 124.16, 119.71, 108.21, 107.90,
75.31, 70.65, 47.52, 45.05, 43.26, 37.58, 34.69, 34.62, 31.28,
28.41, 27.15, 25.68, 22.73, 22.05, 21.58, 21.43, 17.59, 12.20.
(+)ESI-MS (m/z): 720.55 (30), 719.41 (85), 694.38 ([M+K]+, 30),
678.45 ([M+Na]+, 90), 657.35 (30), 656.31 ([M+H]+, 100). Anal.
Calcd for C37H53NO9: C, 67.76; H, 8.15. Found: C, 67.34; H, 8.37.
4.2.9. 7a,12a-Dihydroxy-5b-cholan-24-amide-3-spiro-60-(30,30-
dimethyl-10,20,40,50-tetraoxacyclohexane) (13)
Amide 5 (50 mg, 0.086 mmol) was hydrolyzed at 70 °C using
NaOH (20.7 mg, 0.52 mmol) in MeOH/H2O mixture (2 mL, 3:1, v/
v). After 6 h, reaction was cooled and diluted with 20 mL H2O
and 50 mL CH2Cl2. Water layer was acidified with diluted HCl to
pH 2, and layers were separated. Water layer was further extracted
with CH2Cl2 (3ꢁ 30 mL), combined organic layers were washed
with water and brine, dried over anhyd Na2SO4 and evaporated
to dryness. Column chromatography. Lobar A, LichroPrep, eluent
EtOAc/heptane = 9:1. Yield 42.7 mg (96%). Colorless foam, soften-
4.2.6. N-(Methoxycarbonylethyl)-7a,12a-diacetoxy-5b-cholan-
24-amide-3-spiro-60-(30,30-dimethyl-10,20,40,50-tetraoxacyclohex-
ane) (10)
Acid 4 (300.0 mg, 0.52 mmol) was transformed into amide 10
(276.1 mg, 82%) according to general procedure using a suspension
of 6 equiv methyl glycinate hydrochloride and 6 equiv Et3N in dry
CH2Cl2 (20 mL). Column chromatography: Lobar B, LichroPrep Si
60, eluent EtOAc/heptane = 95:5. Colorless foam, softening at 98–
20
ing at 117–119 °C. ½aꢂD +25.0 (c 0.112, CHCl3). IR(KBr): 3418m,
2940s, 2873m, 1666s, 1615w, 1460w, 1407w, 1377m, 1256w,
1204m, 1164w, 1123w, 1080m, 1042m, 980w, 946w, 922w
20
cmꢃ1 1H NMR (200 MHz, CDCl3): 6.24, 6.03 (both br s, H2N–
.
100 °C. ½aꢂD +36.09 (c 0.46, CHCl3). IR(KBr): 3350m, 2951s, 2876w,
1736s, 1661m, 1539m, 1442m, 1377s, 1244s, 1205s, 1127w,
C(24)), 3.98 (br s, H–C(12)), 3.84 (br s, H–C(7)), 0.99 (d, J = 5.6,
H3C–C(10)), 0.92 (s, H3C–C(20)), 0.68 (H3C–C(13)). 13C NMR
(50 MHz, CDCl3): 177.46, 108.73, 107.64, 73.05, 68.21, 46.40,
41.77, 39.17, 35.34, 35.05, 32.08, 31.39, 28.17, 27.57, 25.86,
23.16, 21.91, 17.30, 12.33. (+)ESI-MS (m/z): 1014.21 (40), 1013.39
([2M+Na]+, 100), 991.32 ([2M]+, 30), 559.39 (70), 543.31 ([M+K]+,
50), 496.36 ([M+H]+, 5). Anal. Calcd for C27H45NO7: C, 65.43; H,
9.15. Found: C, 65.13; H, 9.39.
1076m, 1027m, 965w cmꢃ1 1H NMR (200 MHz, CDCl3): 6.05–5.95
.
(m, HN–C(24)), 5.09 (br s, H–C(12)), 4.95–4.85 (m, H–C(7)), 4.04 (d,
J = 5.2 Hz, H2C–N), 3.77 (s, CH3O2C(26)), 2.14 (br s, CH3COO–), 2.09
(br s, CH3COO–), 0.95 (s, H3C–C(10)), 0.82 (d, J = 6.0 Hz, H3C–
C(20)), 0.73 (H3C–C(13)). 13C NMR (50 Hz, CDCl3): 173.41, 170.62,
108.22, 107.90, 75.27, 70.65, 52.35, 47.40, 45.01, 43.26, 41.11,
37.60, 34.63, 33.01, 31.24, 30.55, 28.40, 27.11, 25.67, 22.71, 22.07,
21.58, 21.0, 17.50, 12.16. (+)ESI LC–MS/MS (m/z): 652.2 (MH+),
270.20 (MHꢃ38)+, 261.10 (MHꢃ391)+, 235.30 (MHꢃ417)+. Anal.
Calcd for C34H53NO11: C, 62.65; H, 8.20. Found: C, 62.31; H, 8.37.
4.3. In vitro antimalarial activity
The in vitro antimalarial drug susceptibility screen is a mod-
ification of the procedures first published by Desjardins et al.,13
with modifications developed by Milhous et al.,14 and the details
are given in Ref. 4b. All 11 synthesized mixed tetraoxanes were
screened in vitro against P. falciparum strains: CQ- and MFQ-sus-
ceptible strain D6 (clone of Sierra I/UNC isolate), CQ-resistant
but MFQ-susceptible strain W2 (clone of Indochina I isolate),
and CQ- and MFQ-resistant strain TM91C235 (clone of South-
East Asian isolate). Compounds 9, 12, and 13 were additionally
screened against CQ-resistant strain RCS (clone of South America
isolate).
4.2.7. N,N-Dimethyl-7a,12a-diacetoxy-5b-cholan-21-amide-3-
spiro-60-(30,30-dimethyl-10,20,40,50-tetraoxacyclohexane) (11)
Acid 4 (300.0 mg, 0.52 mmol) was transformed into amide 11
(276.1 mg, 86%) according to general procedure using a solution
of 6 equiv NH(CH3)2 (156 lL, 3.10 mmol) in dry CH2Cl2 (20 mL).
Column chromatography: Lobar B, LichroPrep Si 60, eluent
20
EtOAc/heptane = 95:5. Colorless foam, softening at 93–94 °C. ½aꢂD
+60.5 (c 0.076, CHCl3). IR(KBr): 3475m, 2954s, 2875m, 1735s,
1649s, 1446w, 1377m, 1244s, 1204m, 1163w, 1127w, 1076w,
1027m, 943w, 891w cmꢃ1 1H NMR (200 MHz, CDCl3): 5.10 (br s,
.
H–C(12)), 4.91 (br s, H–C(7)), 3.00 (s, H3C–N), 2.94 (s, H3C–N),
2.13 (br s, CH3COO–), 2.08 (br s, CH3COO–), 0.95 (H3C–C(10)),
0.83 (d, J = 6.2 Hz, H3C–C(20)), 0.74 (H3C–C(13)). 13C NMR (50 Hz,
CDCl3): 173.33, 170.66, 108.21, 107.86, 75.25, 70.61, 47.56, 44.99,
43.23, 37.55, 37.22, 35.33, 34.82, 34.60, 30.87, 30.28, 28.37,
27.10, 25.66, 24.06, 22.73, 22.40, 22.05, 21.58, 21.40, 20.61,
17.63, 12.17. (+)ESI-MS (m/z): 672.48 (10), 671.38 (30), 631.43
(10), 630.35 ([M+Na]+, 30), 608.38 ([M+H]+, 100). Anal. Calcd for
4.4. In vivo antimalarial activity
The P. berghei mouse efficacy tests were conducted using a
modified version of the Thompson test. Basically, groups of five
mice were inoculated intraperitoneally with erythrocytes infected
with a drug-sensitive strain of P. berghei on day 0. For po adminis-
tration, drugs were suspended in 0.5% hydroxyethylcellulose–0.1%
Tween 80 and administered beginning on day 3 postinfection once
a day. For sc administration, drugs were suspended in sesame oil,
and administered beginning on day 3 postinfection two times a
day. Dosings are given in Table 3. Cure was defined as survival until
day 31 posttreatment. Untreated control mice usually die on day
6–8 postinfection.
C33H53NO9: C, 65.21; H, 8.79. Found: C, 64.80; H, 9.04.
4.2.8. 7a,12a-Diacetoxy-5b-cholan-24-hydrazide-3-spiro-60-
(30,30-dimethyl-10,20,40,50-tetraoxacyclohexane) (12)
Acid 4 (300.0 mg, 0.52 mmol) was transformed into amide 11
(182,6 mg, 59%) according to general procedure using a solution
of 6 equiv NH2NH2 ꢁ 2HCl and 12 equiv Et3N in dry CH2Cl2
(20 mL). Colorless solid, m.p. 174–177 °C. ½aꢂ2D0 +58.6 (c 0.07, CHCl3).
IR (KBr):3528m, 3496m, 3386m, 2950s, 2876w, 1737s, 1638m,
1446m, 1377s, 1244s, 1204m, 1165w, 1126w, 1076w, 1027m,
4.5. In vitro metabolism studies
The metabolic stability assay sample preparation was per-
formed in a 96-well plate on a TECAN Genesis robotic sample pro-
cessor. All incubations were carried out in 0.1 M sodium phosphate
965w, 944w, 892w cmꢃ1
.
1H NMR (200 MHz, CDCl3): 8.62 (br s,
H2N–NH–C(24)), 7.28 (s, H2N–NH– C(24)), 5.08 (br s, H–C(12)),

D. M. Opsenica et al. / Bioorg. Med. Chem. 16 (2008) 7039–7045
7045
isolates in vitro to artemether: (b) Jambou, R.; Legrand, E.; Niang, M.; Khim,
N.; Lim, P.; Volney, B.; Ekala, M. T.; Bouchier, C.; Esterre, P.; Fandeur, T.;
Mercereau-Puijalon, O. Lancet 2005, 366, 1960; See also: Legrand, E.; Volney,
B.; Meynard, J.-P.; Mercereau-Puijalon, O.; Esterre, P. Antimicrob. Agents
Chemother. 2008, 52, 288. However, the resistance of P. falciparum strains to
artemisinins is not observed in vivo presumably because of applied
artemisinin-based combination therapies (ACTs).
buffer (pH 7.4) in the presence of an NADPH-regenerating system
(NADP+ sodium salt, MgCl2ꢄ6H2O, and glucose-6-phosphate). Test
drug (10 lM), microsomes (1 mg/mL total protein), buffer, and
NADPH-regenerating system were warmed to 37 °C, and the reac-
tion was initiated by the addition of glucose-6-phosphate dehydro-
genase (G6PD). Samples were quenched using an equal volume of
cold methanol. Samples were centrifuged to pellet the proteins,
and the supernatant was analyzed by LC–MS/MS using fast LC gra-
dient or isocratic methods. Percentages of parent drug remaining
at each time point were calculated using a ratio of the peak areas
at each time point to the area of the time zero point. To calculate
the half-life, a first-order rate of decay was assumed. A plot of
the natural log (LN) of the drug concentration versus time was gen-
erated, where the slope of that line was ꢃk. Half-life was calculated
as 0.693/k.
4. (a) Vennerstrom, J. L.; Arbe-Barnes, S.; Brun, R.; Charman, S. A.; Chiu, F. C. K.;
Chollet, J.; Dong, Y.; Dorn, A.; Hunziker, D.; Matile, H.; McIntosh, K.;
Padmanilayam, M.; Tomas, J. S.; Scheurer, C.; Scorneaux, B.; Tang, Y.;
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This work has been supported by the Ministry of Science of Ser-
bia (Grant No. 142022) and the Serbian Academy of Sciences and
Arts. Research was conducted in compliance with the Animal Wel-
fare Act and other federal statutes and regulations relating to ani-
mals and experiments involving animals and adheres to principles
stated in the Guide for the Care and Use of Laboratory Animals,
NRC Publication, 1996 edition. Material has been reviewed by the
Walter Reed Army Institute of Research. There is no objection to
its presentation or publication. The opinions or assertions con-
tained herein are the private views of the author and are not to
be construed as official or as reflecting true views of the Depart-
ment of the Army or the Department of Defense.
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