A single, low, oral dose of a 5-carbon-linked trioxane dimer orthoester plus mefloquine cures malaria-infected mice

Article abstract of DOI:10.1016/j.bmcl.2010.09.123

Four 5-carbon-linked trioxane dimer orthoesters (6a-6d) have been prepared in 4 or 5 chemical steps from the natural trioxane artemisinin (1). When administered orally to malaria-infected mice using a single dose of only 6 mg/kg body weight along with 18

Full text of DOI:10.1016/j.bmcl.2010.09.123

Bioorganic & Medicinal Chemistry Letters 21 (2011) 2773–2775
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
A single, low, oral dose of a 5-carbon-linked trioxane dimer orthoester
plus mefloquine cures malaria-infected mice
Deuk Kyu Moon a, Abhai Tripathi b,c, David Sullivan b,c, Maxime A. Siegler a, Sean Parkin d, Gary H. Posner a,c,
⇑
a Department of Chemistry, Johns Hopkins University, Baltimore, MD 21218, United States
b W Harry Feinstone Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD 21205, United States
c Malaria Research Institute, Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD 21205, United States
d Department of Chemistry, University of Kentucky, Lexington, KY 40506, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Four 5-carbon-linked trioxane dimer orthoesters (6a–6d) have been prepared in 4 or 5 chemical steps
from the natural trioxane artemisinin (1). When administered orally to malaria-infected mice using a sin-
gle dose of only 6 mg/kg body weight along with 18 mg/kg of mefloquine hydrochloride, trioxane dimer
orthoester sulfone 6d completely and safely cured the mice; after 30 days, the cured mice showed no
detectable parasitemia, gained at least as much weight as the control mice (no infection), and behaved
normally.
Received 13 August 2010
Revised 22 September 2010
Accepted 23 September 2010
Available online 29 September 2010
Keywords:
Ó 2010 Elsevier Ltd. All rights reserved.
Antimalarial chemotherapy
Trioxane dimer orthoesters
Single oral dose ACT cure
Many standard antimalarial drugs like chloroquine are no long-
er effective due to widespread resistance.1 A new non-alkaloid
class of antimalarial artemisinin trioxanes,2–9 based on ancient
Chinese folk medicine, is now recommended by the World Health
Organization (WHO) and is being adopted widely10–16; artemisinin
combination therapy (ACT) features the very rapid clearance of
most of the parasites by the trioxane drug followed by the pro-
longed antimalarial action of the partner drug. Typically, fixed dose
combinations are used,17 with the most popular example being a
curative six dose regimen of a 1:6 fixed combination of artemether
(total 320 mg) and lumefantrine (total 1920 mg). Often, however,
patient compliance with a repeated-dose regimen is problematic.
Therefore, a single dose cure of malaria-infected humans is highly
desirable. Toward this goal, we have developed some trioxane
monomers18 and dimers19 able to cure malaria-infected mice using
only a single low oral dose combined with mefloquine. We recently
reported a new series of 5-carbon-linked trioxane dimers 4 which,
combined with mefloquine, cure malaria-infected mice.20 We re-
port here a new series of 5-carbon-linked trioxane dimer orthoes-
ters 6a–6d.
of bis-allylic alcohol 4 followed by basic oxidation produced 1,3,5-
triol 5 as the major product in 50% yield. Acyclic 1,3,5-triol 5 reacted
with three equivalents of a series of commercial orthoesters to form
trioxanedimer orthoesters 6a–6c in 75–91%yields. The stereochem-
istry of triol 5 was clarified by X-ray crystallography of phenyl ortho-
ester 6b, establishing its structure as shown in Figure 2. X-ray
coordinates have been deposited with the Cambridge Crystallo-
graphic Data Center (CCDC# 789228). Trioxane dimer orthoester
sulfone 6d was prepared by thiophenoxide displacement of the
bromine atom in bromo orthoester 6c followed by sulfide?sulfone
oxidation. It is noteworthy that thiophenoxide accomplishes this
substitution reaction without cleaving the antimalarially crucial
peroxide bond in trioxane 6c; peroxides generally are easily cleaved
by thiolate anions.22 Displacement of bromo orthoester 6c with so-
dium benzene-sulfinate gave orthoester sulfone 6d directly in 84%
yield and in >99% purity by HPLC analysis. Trioxane dimer orthoester
sulfone 6d in the absence of solvent is stable at 60 °C for at least one
week, with less than 5% decomposition detected by 1H NMR spec-
troscopy. Orthoesters like 6, derived from 1,3,5-triols, are consider-
ably more stable toward acid hydrolysis than the corresponding
trialkyl orthoesters.23
C-10 Acetoxy artemisinin 2b, prepared in nearly quantitative
yield by reducing and then acetylating artemisinin ((1?2a?2b),21
reacted with silylated 5-carbon-linker 3 to form 10b,10b-dimer
allylic alcohol 4 as the major product in 65% yield (Fig. 1).20 Scale
up is not expected to be problematic. Dithexylborane hydroboration
Orthoester sulfone 6d is stable to simulated stomach acid24 (pH 2)
for at least 24 h at 25 °C as determined by analytical thin layer
chromatography.
Each trioxane dimer 6a–6d (0.48 mg) was dissolved in 0.08 mL
of 7:3 Tween 80/ethanol and then diluted with 0.73 mL of distilled
water for oral administration to 5-week old, approximately 20 g
C57BL/6 J male mice (from the Jackson Laboratory) weighing
⇑
Corresponding author.
E-mail address: ghp@jhu.edu (G.H. Posner).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.09.123

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D. K. Moon et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2773–2775
Table 1
Antimalarial efficacy using a single oral dose (6 mg/kg) of trioxane combined with
mefloquine hydrochloride (18 mg/kg) in Plasmodium berghei-infected mice
Trioxane
Average survival
(days) after infection
% Suppression of
parasitemia on day
3 post infection
Artemether (2c)
Artemether (6 mg/kg) +
14.3 (11, 15, 17)
24.0 (23, 23, 26)
>99.9
>99.99
lumefantrine (18 mg/kg)
5
18.6 (12, 16, 30)
19.0 (11, 16, 30)
23.7 (19, 19, 30)
20.7 (12, 22, 30)
>99.9
>99.9
>99.9
>99.99
6a
6b
6c
6d
>99.9
Controls
Vehicle (no drug)
Mefloquine (18 mg/kg)
Lumefantrine (18 mg/kg)
7.3 (7, 7, 8)
20.0 (16, 17, 28)
11.7 (11, 12, 12)
0
>99.9
99.1
dose of 6 mg/kg, combined with 18 mg/kg of mefloquine hydrochlo-
ride. Both determining blood parasitemia levels as well as monitor-
ing the duration of animal survival compared to survival time of
infected animals receiving no drug were the malariometrics used.
Three days after infection, an average of 9% blood parasitemia
(Giemsa microscopy) was observed in the no-drug control group
of mice. The average survival time of the malaria-infected animals
receiving no drug was 7.3 days post infection.
All of the infected mice in this study receiving trioxane drug
artemether (2c) plus mefloquine died on the average on day 14 post
infection. Importantly, a single oral dose of artemether (6 mg/kg)
plus lumefantrine (18 mg/kg) was not curative (Table 1). With mef-
loquine hydrochloride alone at a single oral dose of 18 mg/kg, the
average survival time of the infected mice was 20 days. A widely
accepted indication of complete cure (i.e., 100% efficacy) is survival
of animals to day 30 post infection with no detectable malaria para-
sites in the animals’ blood at that time.
Fig. 1. Trioxane dimer orthoesters.
The average survival times of orthoester-treated infected mice
are shown in Table 1. Noteworthy is that orthoesters 6a–6c plus
mefloquine were not curative. In sharp contrast, orthoester sulfone
6d plus mefloquine was fully curative (Table 1). It is apparent from
the data presented in Table 1 that the trioxane dimer orthoester
sulfone 6d, at a single oral dose of only 6 mg/kg plus 18 mg/kg of
mefloquine hydrochloride, is much higher in efficacy than the anti-
malarial drug 2c plus mefloquine and is fully efficacious at curing
the malaria-infected mice; all three mice in this 30-day surviving
group were completely cured (no parasites in their blood on day
30 post infection), and they had gained at least as much weight as
the uninfected control mice (data not shown). All of the orthoesters
6a–6d, as well as trioxane drug 2c, caused at least 99.9% suppres-
sion of parasitemia on day 3 post infection. Neither overt toxicity
nor behavioral change attributable to trioxane drug administration
was observed in any of the malaria-infected animals cured by
trioxane orthoester sulfone 6d plus mefloquine hydrochloride
combination.
In conclusion, syntheses of trioxane dimer orthoesters 6 were
achieved in moderate overall yields from the natural trioxane arte-
misinin (1); scale-up synthesis to kilogram quantities of these
thermally and hydrolytically stable new chemical entities is ex-
pected to be straightforward. The single oral dose antimalarial effi-
cacy of trioxane dimer orthoester sulfone 6d combined with
mefloquine hydrochloride is superior to that of the popular clini-
cally used monomeric trioxane drug 2c.25,26 Investigation of the
preclinical pharmacology of trioxane dimer orthoester sulfone 6d
will allow a fuller comparison of the chemotherapeutic value of
this semi-synthetic endoperoxide versus that of the popular anti-
malarial trioxane drug 2c.
Fig. 2. X-ray crystallography of phenyl orthoester 6b.
about 20 g that were infected intraperitoneally on day 0 with the
Plasmodium berghei, ANKA malaria strain (5 Â 107 parasitized
erythrocytes).
Each of three mice in a group was treated orally 24 h post infec-
tion with
a
single dose of 0.20 mL (0.20 mL/0.81 mL Â 0.48
mg = 0.12 mg) of diluted compound solution, corresponding to a

D. K. Moon et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2773–2775
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Acknowledgments
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We thank the NIH (AI 34885 to G.H.P.), the Johns Hopkins
Malaria Research Institute, and the Bloomberg Family Foundation
for financial support (to G.H.P. and to D.S.).
Supplementary data
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