Malaria-infected mice live until at least day 30 after a new monomeric trioxane combined with mefloquine are administered together in a single low oral dose

Article abstract of DOI:10.1021/jm9005934

In only five simple steps and 48% overall yield from the natural trioxane artemisinin, the thermally and hydrolytically stable trioxane fluoroanilide 4b has been prepared. Upon one oral dose of only 6.8 mg/kg of monomeric trioxane 4b combined with 20 mg/k

Full text of DOI:10.1021/jm9005934

7458 J. Med. Chem. 2009, 52, 7458–7462
DOI: 10.1021/jm9005934
Malaria-Infected Mice Live until at Least Day 30 after a New Monomeric Trioxane Combined with
Mefloquine Are Administered Together in a Single Low Oral Dose†
Lauren E. Woodard,‡ Wonsuk Chang,‡ Xiaochun Chen,§ Jun O. Liu,§ Theresa A. Shapiro, ,^ and Gary H. Posner*,‡,^
‡Department of Chemistry, School of Arts and Sciences, The Johns Hopkins University, 3400 North Charles Street, Baltimore, Maryland
21218-2685, §Department of Pharmacology and Oncology, School of Medicine, The Johns Hopkins University, Baltimore, Maryland 21205,
Division of Clinical Pharmacology, Department of Medicine, School of Medicine, The Johns Hopkins University, Baltimore, Maryland 21205,
and ^The Johns Hopkins Malaria Research Institute, Bloomberg School of Public Health, Baltimore, Maryland 21205
Received May 7, 2009
In only five simple steps and 48% overall yield from the natural trioxane artemisinin, the thermally and
hydrolytically stable trioxane fluoroanilide 4b has been prepared. Upon one oral dose of only 6.8 mg/kg
of monomeric trioxane 4b combined with 20 mg/kg of mefloquine hydrochloride, all of the malaria-
infected mice lived until at least day 30 post infection. Of the five mice in this surviving group, four (80%)
were completely cured (no parasites in their blood) and one mouse had 4% blood parasitemia.
Importantly, the efficacy of this ACT chemotherapy using monomeric trioxane 4b plus mefloquine
hydrochloride is considerably better than the efficacy under the same conditions using the popular
trioxane drug artemether plus mefloquine hydrochloride.
Introduction
fluoroanilide 4b is the most efficacious; using only one single-
digit mg/kg oral dose of this new trioxane monomer combined
with a 3-fold higher amount of mefloquine hydrochloride
prolonged survival of malaria-infected mice until at least day
30. The total amount of fluoroanilide 4b and mefloquine
hydrochloride needed to achieve this single oral dose high
efficacy compares favorably with the amounts of the anti-
malarial trioxane drugs 2b and 2c plus amine currently used
clinically in repeated oral dose human ACT chemotherapy
(Scheme 1).8
Resistanceof malaria parasites, especially tochloroquine, is
now widespread, seriously compromising the efficacy of this
antimalarial drug that has been so widely and successfully
used during the past 60 years.1 Today, as recommended by the
World Health Organization (WHO)2 and as adopted by most
countries where malaria is endemic, artemisinin (1) combina-
tion therapy (ACTa) is popular.3-7 Typically, naturaltrioxane
1 derivatives like artemether (2b) or sodium artesunate (2c) are
used. One leading ACT drug for chemotherapy of people sick
with malaria is a fixed 1:6 combination of the trioxane 2b with
the amino-alcohol lumefantrine.8 Typically, a six-dose adult
regimen requires a total of approximately 320 mg of trioxane
2b and 1920 mg of lumefantrine. A second ACT drug requires
a three-dose adult regimen totaling 600 mg of trioxane 2c and
750 mg of the quinoline antimalarial mefloquine. Patient
compliance with adhering to a repeated dose regimen, how-
ever, is often a serious problem. Therefore, a single dose oral
cure is highly desirable. Toward this goal, we have recently
reporteda“proof of principle”advance in malaria chemother-
apy:asingle144mg/kg oraldose cureofmalaria-infectedmice
by a new trioxane dimer sulfone carbamate.9 We have now
prepared and tested the in vivo antimalarial efficacies of the
fluorobenzyl amide trioxane dimer 3, of the corresponding
trioxane monomer fluorobenzyl amide 4a, and of the trioxane
monomer fluoroanilide4b (artefanilide). Monomeric trioxane
Results and Discussion
Chemistry. Monomeric trioxane dihydroartemisin C-10
acetate (2d) has been converted into dimeric trioxane 5 and
then into dimer carboxylic acid 6 (Scheme 2).10 Facile
amidation produced trioxane dimer fluorobenzyl amide 3.
Also, C-10 acetate 2d has been converted into C-10-allyl
derivative 7 and then, via hydroboration followed by oxi-
dation, into monomeric trioxane carboxylic acid
8
(Scheme 3).11 Monomeric trioxane carboxylic acid 8 can be
transformed in one step directly into a diverse library of
monomeric trioxane amides.11 For example, one-step ami-
dation of carboxylic acid 8 produced trioxane fluorobenzyl
amide 4a, a monomeric version of dimer fluorobenzyl amide
3 (Scheme 3). In the same way, monomer trioxane fluoroa-
nilide 4b was prepared directly and in good yield from
carboxylic acid 8 (Scheme 3). The overall yield of 4b is
approximately 48% from natural trioxane 1, and scale up
to multigram or even kilogram amounts is expected to be
straightforward. Fluoroanilide 4b is stable as a solid in the
absence of solvent for at least 7 days at 60 ꢀC and for at least
1 day at 70 ꢀC. Because amides 3, 4a, and 4b are also C-10
nonacetal trioxanes, they are all more hydrolytically stable
than the C-10 acetal trioxane drugs 2b and 2c.
† In honor of the 100th anniversary of the Division of Medicinal
Chemistry
*To whom correspondence should be addressed. Phone: 410-516-
4670. Fax: 410-516-8420. E-mail: ghp@jhu.edu.
a Abbreviations: ACT, artemisinin combination therapy; DMSO,
dimethyl sulfoxide; DMS, dimethyl sulfide; EDC, N-(3-dimethyl-
aminopropyl)-N0-ethylcarbodiimide hydrochloride; HOBT, 1-hydroxy-
benzotriazole.
r
pubs.acs.org/jmc
Published on Web 07/08/2009
2009 American Chemical Society

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23 7459
Scheme 1
blood at that time. Average survival results are summarized in
Table 1, including single oral doses of 13 mg/kg of trioxane
combined with 13 mg/kg of mefloquine hydrochloride. The
clinically used monomeric water-soluble trioxane drug 2c and
the synthetic trioxolane peroxide drug development candidate
OZ277 (9) maleate are included as monotherapy reference
compounds.
Scheme 2
It is clear from the data in Table 1 that all three of the new
trioxane fluorinated amides 3, 4a, and 4b plus mefloquine
hydrochloride prolonged average survival time much more
effectively than monotherapy using trioxolane 9, which is in
phase II clinical trials.13 A nonfluorinated version of dimeric
trioxane fluorobenzyl amide 3 was much less antimalarially
efficacious than fluorobenzyl amide 3 (data not shown). It is
also apparent from the data in Table 1 that the mono-
meric trioxane fluoroanilide 4b, at a single oral dose of only
6.8 mg/kg plus 20 mg/kg of mefloquine hydrochloride, was
the most efficacious at prolonging survival. Of the five mice
in this 30-day surviving group, four (80%) were completely
cured (no parasites in their blood) on day 30 post infection
and one mouse had 4% blood parasitemia. This trioxane
fluoroanilide 4b caused a 99.0% suppression of parasitemia
on day 3 post infection. A single oral dose of 13 mg/kg of
trioxane fluoroanilide 4b combined with 13 mg/kg of me-
floquine hydrochloride also prolonged survival of all of the
mice until day 30, with four mice cured and one mouse
having 2% blood parasitemia. Reinfection of the four cured
mice on day 32 after the original infection caused an ex-
tended survival time, approximately four days longer than
the average survival time of a newly infected control group;
the mechanism(s) for this protective effect of trioxane fluor-
oanilide 4b against malaria reinfection is not clear at this
time. In monotherapy control experiments, a single high oral
dose (72 mg/kg) of the trioxane fluoroanilide 4b prolonged
average survival until day 24.4, and a single oral dose
(20 mg/kg) of mefloquine hydrochloride alone prolonged
average survival to day 17.6. In an ACT control experiment,
Biology. Each trioxane 2b, 3, 4a, and 4b (0.90 mg) was
dissolved in 0.11 mL of 7:3 Tween 80:ethanol and then diluted
with 1.10 mL of water for oral administration to 5-week old
C57BL/6J male mice (from the Jackson Laboratory) weighing
about 22 g that were infected intraperitoneally on day 0 with
the Plasmodium berghei, ANKA strain (2 ꢀ 107 parasitized
erythrocytes).12 Each of five mice in a group was treated orally
24 h postinfection with a single dose of 0.20 mL (0.20 mL/
1.21 mL ꢀ 0.9 mg = 0.15 mg) of diluted compound solu-
tion, corresponding to a dose of 6.8 mg/kg, combined with
20 mg/kg of mefloquine hydrochloride. Determining blood
parasitemia levelsas well asmonitoring the duration of animal
survival compared to survival time of animals receiving no
drug are both widely accepted as measures of a drug’s efficacy
in antimalarial drug development. Three days after infection,
an average of 16% blood parasitemia was observed in the
control (no drug) group. Animals receiving no drug died on
days 6-7 post infection. A widely accepted yardstick of cure
(i.e., 100% efficacy) is survival of animals to day 30 post
infection, with no detectable malaria parasites in the animal’s

7460 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23
Woodard et al.
Scheme 3
Table 1. Antimalarial Efficacy Using a Single Oral Dose of Trioxane Combined with Mefloquine Hydrochloride in Plasmodium berghei-Infected Mice
a Actual mouse survival until day. b Data from Supporting Information in ref 13 using a single oral dose of 30 mg/kg. c Dash indicates “not measured”.
6.8 mg/kg of the popular trioxane drug 2b combined with
20 mg/kg of mefloquine hydrochloride prolonged average
survival to only day 19.8 (Table 1). Neither overt toxicity nor
behavioral change attributable to trioxane drug administra-
tion was observed in any of the malaria-infected animals
cured by trioxane fluoroanilide 4b plus mefloquine hydro-
chloride combination. The water-soluble monomeric triox-
ane antimalarial drug 2c and the trioxolane antimalarial
drug candidate 9, although able to lower parasitemia levels
considerably by day 3 post infection, were not efficacious in
prolonging the mouse average survival time beyond day 11
when used as monotherapy at a dose of 30 mg/kg.
Conclusions
In conclusion, a single-digit oral dose of any one of the
three new trioxane fluorinated amides 3, 4a, and 4b
combined with mefloquine hydrochloride is considerably
more antimalarially efficacious than the popular ACT
trioxane drug 2b combined with mefloquine hydro-
chloride.14,15 Monomer trioxane fluoroanilide 4b stands
out as being the most powerful antimalarial in this series
of semisynthetic trioxane fluorophenyl amides. Preclini-
cal drug development is continuing on trioxane lead
optimization and on use of other amine combination
partners.

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23 7461
Experimental Section
then quenched with 1 N HCl, extracted with dichloromethane
(3ꢀ5 mL), washed with aqueous NaHCO3 and brine, dried over
magnesium sulfate, and evaporated. The crude product was
purified by flash column chromatography (silica gel, 30% ethyl
acetate/hexanes) to afford 4b (artefanilide) as an amorphous,
white solid (51 mg, 0.12 mmol, 75%). IR (thin film) 3313, 2939,
2874, 1663, 1614, 1543, 1509, 1451, 1406, 1377, 1212, 1124, 1091,
High-pressure liquid chromatography (HPLC) was performed
on a Rainin HPLX system equipped with two 25 mL pump heads
and a Rainin Dynamax UV-C dual-beam variable wavelength
detector set at 254 using a Phenomenex Luna 5 μ C18 250 mmꢀ
10 mm column. The purity of analogs 3, 4a, and 4b was g98%
based on HPLC analysis.
1
1055, 1012, 876, 835, 754 cm-1. H NMR (400 MHz, CDCl3)
Synthesis of Trioxane Dimer Fluorobenzyl Amide (3). To a
solution of acid 610 (600 mg, 0.967 mmol) in CH2Cl2 (10 mL)
were added N-(3-dimethylaminopropyl)-N0-ethylcarbodiimide
hydrochloride (222 mg, 1.16 mmol) and 1-hydroxybenzotriazole
(157 mg, 1.16 mmol) and it was stirred for 1 h at rt. To the
reaction were added 4-fluorobenzylamine (0.33 mL, 2.9 mmol),
and the solution was stirred for 5 h. It was quenched with water
(3 mL). Layers were separated and the aqueous layer was
extracted with EtOAc (2 ꢀ 4 mL). The combined organic
solution was dried (MgSO4) and concentrated. The residue
was purified by flash column chromatography (elution with
EtOAc:hexanes = 1:3) to provide 3 (651 mg, 93%) as a white
solid: [R]D24 = þ82.1 (c = 1.55, CHCl3); mp = 110 ꢀC. IR (thin
1H NMR (400 MHz, CDCl3) δ 7.32 (m, 2H), 6.97 (m, 2H), 6.23
(t, J = 5.6 Hz, 1H), 5.27 (s, 1H), 5.20 (s, 1H), 4.41 (s, 1H), 4.39
(s, 1H), 4.09 (m, 2H), 2.76 (dq, J = 13.2, 7.2 Hz, 1H), 2.66 (dq,
J = 13.6, 6.4 Hz, 1H), 2.54 (octet, J = 4.0 Hz, 1H), 2.31 (m, 2H),
2.18 (m, 1H), 2.01-1.95 (m, 3H), 1.92-1.18 (m, 24H including s
at 1.35 and 1.26), 0.98-0.79 (m, 14H including d at 0.95 with
J = 5.6 Hz, 0.93 with J = 6.0 Hz, 0.85 with J = 7.6 Hz, and 0.82
with J = 7.2 Hz). 13C NMR (100 MHz, CDCl3) δ 175.8, 160.8,
134.3, 129.8, 129.7, 115.3, 115.1, 103.4, 102.9, 100.8, 88.6, 88.4,
81.2, 81.1, 76.4, 73.7, 52.5, 52.4, 44.7, 44.5, 44.3, 43.3, 37.4, 37.2,
36.5, 34.5, 33.3, 32.9, 30.2, 29.9, 26.2, 26.0, 24.9, 24.8, 24.6, 24.5,
20.2, 13.5, 13.0. 19F NMR (282 MHz, CDCl3) δ -115.7. HRMS
(FAB) calculated for C41H59FNO9 [(M þ H)þ] 728.4174, found
728.4177.
δ 8.07 (br. s, 1H), 7.50 (m, 2H), 6.96 (m, 2H), 5.34 (s, 1H),
4.14 (m, 1H), 2.73 (m, 1H), 2.60 (m, 1H), 2.46 (m, 1H), 2.31 (m,
1H), 2.02-1.78 (m, 5H), 1.65-1.55 (m, 2H), 1.47-1.20 (m, 7H,
including singlet at 1.35), 0.97-0.93 (m, 4H), 0.87 (d, 3H,
J = 7.6 Hz). 13C NMR (100 MHz, CDCl3) δ 171.33, 157.89,
134.17, 121.63, 115.33, 103.42, 88.79, 81.08, 76.12, 52.30,
44.31, 37.34, 36.42, 35.62, 34.32, 30.85, 30.13, 25.98, 24.82,
22
24.55, 20.09, 13.04; [R]D = þ60 (c = 0.47, CHCl3). HRMS
(FAB) m/z calcd for C24H33FNO5 (M þ H)þ 434.2343, found
434.2335.
Acknowledgment. We thank Nirbhay Kumar (JHU) for a
gift of the P. berghei malaria parasites, 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 J.O.L.).
film) 3312, 2939, 1669, 1510, 1377, 1221, 1052, 1012, 735 cm-1
.
Supporting Information Available: 1H, 13C NMR spectra for
all of the new trioxanes 3, 4a, and 4b. This material is available
free of charge via the Internet at http://pubs.acs.org.
References
(1) Olliaro, P. L.; Boland, P. B. Clinical public health implications
of antimalarial drug resistance. In Antimalarial Chemotherapy:
Mechanisms of Action, Resistance, and New Directions in Drug
Discovery; Rosenthal, P. J., Ed.; Humana Press: Totowa, NJ, 2001; pp
65-83.
(2) Guidelines for the Treatment of Malaria; World Health Organiza-
tion: Geneva, 2006.
(3) Ashley, E. A.; White, N. J. Artemisinin-based combinations. Curr.
Synthesis of Trioxane Monomer Fluorobenzyl Amide (4a).
Into a flame-dried 5 mL RBF was charged acid 811 (75 mg,
0.22 mmol), N-(3-dimethylaminopropyl)-N0-ethylcarbodiimide
hydrochloride (67 mg, 0.35 mmol), and 1-hydroxybenzotriazole
(35 mg, 0.26 mmol). Dichloromethane (2.5 mL) was then added,
and the mixture was stirred for an hour, at which time
4-fluorobenzylamine (95 μL, 0.84 mmol) was added by syringe.
The reaction was allowed to stir at room temperature for 3 h.
It was then quenched with 1 N HCl, extracted with dichlo-
romethane (3 ꢀ 5 mL), washed with aqueous NaHCO3 and
brine, dried over magnesium sulfate, and evaporated. The crude
product was purified by preparative thin layer chromatography
(silica gel, 100% diethyl ether) to afford 4a as an amorphous,
white solid (69 mg, 0.15 mmol, 70%). IR (thin film) 3321, 2947,
2875, 1648, 1546, 1510, 1453, 1378, 1223, 1127, 1095, 1052, 1011,
940, 879, 822, 756 cm-1. 1H NMR (400 MHz, CDCl3) δ 7.25 (m,
2H), 7.00 (m, 2H), 6.08 (br. s, 1H), 5.27 (s, 1H), 4.40 (m, 2H),
4.09 (m, 1H), 2.69 (m, 1H), 2.46 (m, 1H), 2.33 (m, 2H), 2.04-
1.77 (m, 5H), 1.64-1.55 (m, 2H), 1.48-1.20 (m, 7H, including
singlet at 1.36), 0.99-0.92 (m, 4H), 0.85 (d, 3H, J = 6.0 Hz).
13C NMR (100 MHz, CDCl3) δ 172.97, 163.40, 160.96, 134.27,
129.54, 115.41, 103.28, 88.91, 81.14, 75.75, 52.44, 44.45, 43.01,
37.43, 36.58, 34.64, 34.48, 30.22, 26.11, 25.15, 24.89, 24.68,
Opin. Infect. Dis. 2005, 18, 531–536.
(4) (a) Adjuik, M.; Babiker, A.; Garner, P.; Olliaro, P.; Taylor, W.;
White, N. Artesunate combinations for treatment of malaria:
meta-analysis. Lancet 2004, 363, 9–17. (b) Guthmann, J.-P.; Cohuet,
S.; Rigutto, C.; Fortes, F.; Saraiva, N.; Kiguli, J.; Kyomuhendo, J.;
Francis, M.; Noel, F.; Mulemba, M. Balkan, S. High efficacy of two
artemisinin-based combinations (artesunate þ amodiaquine and arte-
mether þ lumefantrine) in Caala, Central Angola. Am. J. Trop. Med.
Hyg. 2006, 75, 143–145.
(5) Myint, H. Y.; Ashley, E. A.; Day, N. P. J.; Nosten, F.; White, N. J.
Efficacy and safety of dihydroartemisinin-piperaquine. Trans. R.
Soc. Trop. Med. Hyg. 2007, 101, 858–866.
(6) Sirima, S. B.; Tiono, A. B.; Gansane, A.; Diarra, A.; Ouedraogo,
A.; Konate, A. T.; Kiechel, J. R.; Morgan, C. C.; Olliaro, P. L.;
Taylor, W. R. J. Malar. J. 2009, 8, 48.
(7) de Pilla Varotti, F.; Botelho, A. C. C.; Andrade, A. A.; de Paula, R.
C.; Fagundes, E. M. S.; Valverde, A.; Mayer, L. M. U.; Mendonca,
J. S.; de Souza, M. V. N.; Boechat, N.; Krettli, A. U. Synthesis,
antimalarial activity, and intracellular targets of MEFAS, a new
hybrid compound derived from mefloquine and artesunate. Anti-
microb. Agents Chemother. 2008, 52, 3868–3874.
(8) Sagara, I.; Diallo, A. D.; Kone, M.; Coulibaly, M.; Diawara, S. I.;
Guindo, O.; Maiga, H.; Niambele, M. B.; Sissoko, M.; Dicko, A.;
Djimde, A.; Doumbo, O. K. A randomized trial of artesunate-
mefloquine versus artemether-lumefantrine for treatment of
uncomplicated Plasmodium falciparum malaria in Mali. Am. J.
Trop. Med. Hyg. 2008, 79, 655–661.
22
20.12, 13.00; [R]D = þ72 (c = 0.97, CHCl3). HRMS (FAB)
m/z calcd for C25H34FNO5Na (M þ Na)þ 470.2313, found
470.2300.
Synthesis of Trioxane Monomer Fluoroanilide (4b). Into a
flame-dried 5 mL RBF was charged carboxylic acid 811 (55 mg,
0.16 mmol), N-(3-dimethylaminopropyl)-N0-ethylcarbodiimide
hydrochloride (50 mg, 0.26 mmol), and 1-hydroxybenzotriazole
(30 mg, 0.19 mmol). Dichloromethane (2.5 mL) was then added
and the mixture was stirred for an hour, at which time
4-fluoroaniline (60 μL, 0.61 mmol) was added by syringe. The
reaction was allowed to stir at room temperature for 3 h. It was
(9) Rosenthal, A. S.; Chen, X.; Liu, J. O.; West, D. C.; Hergenrother,
P. J.; Shapiro, T. A.; Posner, G. H. Malaria-infected mice are cured
by a single oral dose of new dimeric trioxane sulfones which are also
selectively and powerfully cytotoxic to cancer cells. J. Med. Chem.
2009, 52, 1198–1203.
(10) Posner, G. H.; Paik, I.-H.; Sur, S.; McRiner, A. J.; Borstnik, K.;
Xie, S.; Shapiro, T. A. Orally active, antimalarial, anticancer,
artemisinin-derived trioxane dimers with high stability and effi-
cacy. J. Med. Chem. 2003, 46, 1060–1065.

7462 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23
Woodard et al.
(11) Jung, M.; Lee, S.; Ham, J.; Lee, K.; Kim, H.; Kim, S. K. Antitumor
activity of novel deoxoartesmisinin monomers, dimers, and trimer.
J. Med. Chem. 2003, 46, 987–994.
(12) Chen, X.; Chong, C. R.; Shi, l.; Yoshimoto, T.; Sullivan, D. J., Jr.;
Lin, J. O. Inhibitors of Plasmodium falciparum methionine amino-
peptidase 1b possess antimalarial activity. Proc. Natl. Acad. Sci. U.
S.A. 2006, 103, 14548–14553.
(13) 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.; Santo, T. J.;
Scheurer, C.; Scorneaux, B.; Tang, Y.; Urwyler, H.; Wittlin, S.;
Charman, W. N. Identification of an antimalarial synthetic
trioxolane drug development candidate. Nature 2004, 430, 900–
904.
(14) Sagara, I.; Rulisa, S.; Mbacham, W.; Adam, I.; Sissoko, K.; Maiga,
H.; Traore, O. B.; Dara, N.; Dicko, Y. T.; Dicko, A.; Djimde, A.;
Jansen, F. H.; Doumbo, O. K. Efficacy and safety of a fixed dose
artesunate-sulphamethoxypyrazine-pyrimethamine compared
to artemether-lumefantrine for the treatment of uncomplicated
falciparum malaria across Africa: a randomized multicentre trial.
Malar. J. 2009, 8, 63.
(15) Gautam, A.; Ahmed, T.; Batra, V.; Paliwal, J. Pharmacokinetics
and Pharmacodynamics of Endoperoxide Antimalarials. Curr.
Drug. Metab. 2009, 10, 289–306.