Synthesis of Tetraoxane Antimalarials
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7 2265
EtOAc eluent (7/3). Yield 1.92 g (93%). Colorless foam, softness
136–137 °C. [R]20D +55.5 (c 0.2, CHCl3). Anal. (C36H56O10) C, H.
7r,12r-Diacetoxy-5ꢀ-cholan-24-ol-3-spiro-6′-(1′,2′,4′,5′-tet-
raoxacyclohexane)-3′-spirocyclohexane (15). Hydrolysis of Tri-
acetate 14. Triacetate 14 (1.67 g, 2.57 mmol) was dissolved in
dry methanol (50 mL), followed by addition of anhydrous K2CO3
(640 mg, 4.63 mmol). The suspension was stirred at room
temperature for 5 h. The mixture was evaporated to dryness,
dissolved in CH2Cl2 and H2O, and the layers were separated. The
organic layer was washed with brine, dried over anhydrous Na2SO4,
and evaporated to dryness. The crude monoalcohol 15 was purified
using dry flash chromatography using a heptane/EtOAc eluent (4/
In Vitro Antimalarial Activity. The in vitro antimalarial drug
susceptibility screen is a modification of the procedures first
published by Desjardins et al.,15 with modifications developed by
Milhous et al.,16 and the details are given in ref 5a.
In Vivo Antimalarial Activity. The P. berghei mouse efficacy
tests were conducted using a modified version of the Thompson
test. Groups of five mice were inoculated intraperitoneally with
erythrocytes infected with a drugsensitive strain of P. berghei on
day 0. Drugs were suspended in 0.5% hydroxyethylcellulose/0.1%
Tween-80 (for po administration) or in sesame oil (for sc
administration). Drugs were administered orally once a day
beginning on day 3 postinfection. Dosings are given in Table 2.
Cure was defined as survival until day 31 posttreatment. Untreated
control mice die on day 7–9 postinfection.
6). Yield 1.50 g (96%). Colorless foam, softness 207–210 °C. [R]20
D
+57.0 (c 0.2, CHCl3). Anal. (C34H54O9) C, H.
In Vitro Metabolism Studies. The metabolic stability assay
sample preparation was performed in a 96-well plate on a TECAN
Genesis robotic sample processor. All incubations were carried out
in 0.1 M sodium phosphate buffer (pH 7.4) in the presence of an
NADPH-regenerating system (NADP+ sodium salt, MgCl2 ·6H2O,
and glucose 6-phosphate). Test drug (10 µM), microsomes (1 mg/
mL total protein), buffer, and NADPH-regenerating system were
warmed to 37 °C, and the reaction was initiated by the addition of
glucose 6-phosphate dehydrogenase (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 gradient or isocratic methods. Percentages of
parent drug remaining at each time point were calculated using the
ratio of the peak area 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 generated, where the slope of that line was -k.
The half-life was calculated as 0.693/k.
Via Mixed Anhydride. Acid 19 (50 mg, 0.08 mmol) was
dissolved in dry THF (5 mL) and treated with Et3N (23 µL, 0.16
mmol) and ClCO2Et (15.34 µL, 0.16 mmol). After 3 h of stirring
at 0 °C, NaBH4 (30.5 mg, 0.8 mmol) was added. After an additional
24 h of stirring at room temperature, the mixture was diluted with
H2O and CH2Cl2, and the layers were separated. The water layer
was further extracted with CH2Cl2(2 × 50 mL) and the combined
organic layers were dried over anh. Na2SO4 and evaporated to
dryness. The crude alcohol 15 was purified using dry flash
chromatography using a heptane/EtOAc eluent (1/1). Yield 37 mg
(76%).
7r,12r-Diacetoxy-5ꢀ-cholan-24-al-3-spiro-6′-(1′,2′,4′,5′-tet-
raoxacyclohexane)-3′-spirocyclohexane (16). Alcohol 15 (100 mg,
0.16 mmol) was dissolved in dichloromethane (20 mL) followed
by the addition of pyridinium chlorochromate (53 mg, 0.25 mmol).
After 2 h the mixture was transferred to a silica gel column and
eluted with CH2Cl2 to afford 83 mg (83%) of 16 as a colorless
solid.
N-(n-Propyl)-7r,12r-diacetoxy-5ꢀ-cholan-24-amine-3-spiro-
6′-(1′,2′,4′,5′-tetraoxacyclohexane)-3′-spirocyclohexane (17a). To
a mixture of crude aldehyde 16 (83 mg, 0.14 mmol) and n-PrNH2
(23 µL, 0.28 mmol) in dichloromethane (20 mL), sodium triac-
etoxyborohydride (58 mg, 0.28 mmol) was added. The mixture was
stirred at room temperature for 18 h. The mixture was then poured
onto water and extracted with CH2Cl2 (2 × 50 mL). The combined
organic layers were dried over anhydrous Na2SO4 and evaporated
to dryness. The crude amine 17a was purified by dry flash
chromatography using an eluent of EtOAc/MeOH/NH3aq ) 8/1/1.
Acknowledgment. This work was supported by the Ministry
of Science of Serbia (Grant No. 142022) and the Serbian
Academy of Sciences and Arts. Research was conducted in
compliance with the Animal Welfare Act and other federal
statutes and regulations relating to animals 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 contained
herein are the private views of the author and are not to be
construed as official or as reflecting the true views of the
Department of the Army or the Department of Defense.
Yield 64 mg (72%). Colorless foam, softness 76–78 °C. [R]20
D
+43.0 (c 0.2, CHCl3). Anal. (C37H61NO8) C, H, N.
N-(2-Dimethylamino)ethyl)-7r,12r-diacetoxy-5ꢀ-cholan-24-
amine-3-spiro-6′-(1′,2′,4′,5′-tetraoxacyclohexane)-3′-spirocyclo-
hexane (17b). Aldehyde 16 (200 mg, 0.33 mmol) was transformed
into amine 17b (168 mg, 75%) using Me2NCH2CH2NH2 (72.5 µL,
0.66 mmol) and NaBH(OAc)3 (140 mg, 0.66 mmol). The crude
product was purified using dry flash chromatography with an
Supporting Information Available: Analytical data of synthe-
sized/isolated compounds. This material is available free of charge
EtOAc/MeOH/NH3aq (8/1/1) eluent. Solid. [R]20 +45.0 (c 0.2,
D
CHCl3). Anal. (C38H64N2O8 ·5H2O) C, H, N.
References
7r,12r-Diacetoxy-5ꢀ-cholan-24-azido-3-spiro-6′-(1′,2′,4′,5′-
tetraoxacyclohexane)-3′-spirocyclohexane (18). To a solution of
alcohol 15 (200 mg, 0.33 mmol) in pyridine (4 mL) at room
temperature was added methanesulfonyl chloride (31 µL, 0.4 mmol).
The mixture was stirred at room temperature for 2 h, then diluted
with H2O and EtOAc. The water layer was acidified with diluted
HCl, and layers were separated. The water layer was further
extracted with EtOAc (3 × 50 mL), and the combined organic layers
were dried over anhydrous Na2SO4 and evaporated to dryness. The
obtained crude product was used in the following step. To a solution
of mesylate (226 mg, 0.33 mmol) in DMF (5 mL) was added NaN3
(214 mg, 3.3 mmol). The mixture was stirred at 50 °C for 16 h
before being quenched with water and EtOAc, and layers were
separated. The water layer was further extracted with EtOAc (3 ×
75 mL), and the combined organic layers were washed with brine,
dried over anhydrous Na2SO4, and evaporated to dryness. The crude
product was purified using dry flash chromatography using a
(1) Preliminary results were presented at the 43rd Meeting of the Serbian
Chemical Society, Belgrade, Serbia, January 24–25, 2005, and at the
1st European Chemistry Congress, Budapest, Hungary, August 27–
31, 2006.
(2) (a) For recent developments in artemisinin field, see the followin: Posner,
G. H.; Paik, I.-K.; Chang, W.; Borstnik, K.; Sinishtaj, S.; Rosenthal,
A. S.; Shapiro, T. A. Malaria-Infected Mice Are Cured by a Single
Dose of Novel Artemisinin Derivatives. J. Med. Chem. 2007, 50, 2516–
2519. (b) Haynes, R. K.; Chan, W. C.; Lung, C-M.; Uhlemann, A-C.;
Eckstein, U.; Taramelli, D.; Parapini, S.; Monti, D.; Krishna, S. The
Fe2+-Mediated Decomposition, PfATP6 Binding, and Antimalarial
Activities of Artemisone and Other Artemisinins: The Unlikelihood
of C-Centered Radicals as Bioactive Intermediates. ChemMedChem
2007, 2, 1480–1497.
(3) Vennerstrom, J. L.; Fu, H.-N.; Ellis, W. Y.; Ager, A. L.; Wood, J. K.,
Jr.; Andersen, S. L.; Gerena, L.; Milhous, W. K. Dispiro-1,2,4,5-
tetraoxanes: A New Class of Antimalarial Peroxides. J. Med. Chem.
1992, 35, 3023–3027. (b) Todorovic´, N. M.; Tinant, B.; Declercq,
J.-P.; Makler, M. T.; Šolaja, B. A. Steroidal Geminal Dihydroperoxides
and 1,2,4,5-Tetraoxanes: Structure Determination and Their Antima-
larial Activity. Steroids 1996, 61, 688–696.
heptane/EtOAc (7/3) eluent. Yield 198,5 mg (95%). Solid oil. [R]20
D
+44.5 (c 0.2, CHCl3). Anal. (C34H53N3O8 ·2H2O) C, H, N.