Antimalarial activity of 10-alkyl/aryl esters and -aminoethylethers of artemisinin

Article abstract of DOI:10.1016/j.bioorg.2012.10.002

A series of n-alkyl/aryl esters were synthesized and their in vitro antiplasmodial activity was measured alongside that of previously synthesized aminoethylethers of artemisinin ozonides against various strains of Plasmodium falciparum. The cytotoxicity against human cell lines was also assessed. The esters were synthesized in a one-step reaction by derivatization on carbon C-10 of dihydroartemisinin. Both classes were active against both the 3D7 and K1 strains of P. falciparum, with all compounds being significantly more potent than artemether against both strains. The majority of compounds possessed potency either comparable or more than artesunate with a high degree of selectivity towards the parasitic cells. The 10α-n-propyl 11 and 10α-benzyl 18 esters were the most potent of all synthesized ozonides, possessing a moderate (～3-fold) and significant (22- and 12-fold, respectively) potency increases against the 3D7 and K1 strains, respectively, in comparison with artesunate.

Full text of DOI:10.1016/j.bioorg.2012.10.002

Bioorganic Chemistry 46 (2013) 10–16
Contents lists available at SciVerse ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Antimalarial activity of 10-alkyl/aryl esters and -aminoethylethers of artemisinin
Theunis T. Cloete a, Henk J. Krebs a, Julie A. Clark b, Michele C. Connelly b, Amy Orcutt b, Martina S. Sigal b,
R. Kiplin Guy b, David D. N’Da a,
⇑
a Department of Pharmaceutical Chemistry, North-West University, Potchefstroom 2520, South Africa
b Department of Chemical Biology and Therapeutics, St. Jude Children’s Research Hospital, 262 Danny Thomas Place, Mailstop 1000, Memphis, TN 38105-3678, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 10 August 2012
Available online 3 November 2012
A series of n-alkyl/aryl esters were synthesized and their in vitro antiplasmodial activity was measured
alongside that of previously synthesized aminoethylethers of artemisinin ozonides against various strains
of Plasmodium falciparum. The cytotoxicity against human cell lines was also assessed. The esters were
synthesized in a one-step reaction by derivatization on carbon C-10 of dihydroartemisinin. Both classes
were active against both the 3D7 and K1 strains of P. falciparum, with all compounds being significantly
more potent than artemether against both strains. The majority of compounds possessed potency either
comparable or more than artesunate with a high degree of selectivity towards the parasitic cells. The
Keywords:
Ozonide
Artemether
Artesunate
Plasmodium falciparum
Cytotoxicity
10a-n-propyl 11 and 10a-benzyl 18 esters were the most potent of all synthesized ozonides, possessing
a moderate (ꢀ3-fold) and significant (22- and 12-fold, respectively) potency increases against the 3D7
and K1 strains, respectively, in comparison with artesunate.
Ó 2012 Elsevier Inc. All rights reserved.
1. Introduction
problem with these semi-synthetic compounds, however, is their
short pharmacological half-lives, a reflection of their acid lability,
Each year roughly 800,000 people die of malaria, with 95% being
African children [1]. The development and spread of multidrug
resistant (MDR) Plasmodium falciparum has led to the adoption of
artemisinin-based combination therapies (ACTs) as the first-line
treatment for falciparum malaria in most malaria-endemic coun-
tries of the world [2]. However, the recently confirmed emergence
of artemisinin resistance in western Cambodia is a major threat for
current initiatives to control and eliminate malaria [3–5]. While
artemisinin resistance has not yet spread to other areas [6], the
World Health Organization (WHO) is coordinating a large-scale
elimination campaign in this region aiming to contain the spread
of resistance [7,8].
The ACTs combine fast-acting artemisinin (ART) derived drugs
with other antimalarials possessing longer half-lives such as
mefloquine. Because the utility of artemisinin is limited by its sol-
ubility in both oil and water, this sesquiterpene has been structur-
ally modified by derivatization into short-chain oil soluble ether
derivatives of dihydroartemisinin (DHA, 2), such as artemether
(AM, 2a) and arteether (AE, 2b), and the water soluble sodium
artesunate (AS, 2c) (Fig. 1).
and facile metabolism to DHA [9–12]. In particular, artesunate is
hydrolytically unstable, even at neutral pH, and has a half-life of
only a few minutes [13].
A large amount of work has been carried out with the aim of
generating new derivatives [14–16]. Artemisone (Fig. 2) is thus
far the only second-generation semi-synthetic artemisinin deriva-
tive that has been found suitable for further clinical development,
progressing through Phase II but showing no major benefits over
existing derivatives [17]. Arterolane [18], a synthetic ozonide
(Fig. 2, OZ277) has a longer half-life, is fast acting, well tolerated,
and effective. It is now in Phase III clinical trials in combination
with piperaquine [19,20]. OZ439 (Fig. 2), a purely synthetic ozon-
ide, is a drug candidate designed to provide a single-dose oral cure
in humans. It has successfully completed Phase I clinical trials,
where it was shown to be safe at doses up to 1.6 g, and is currently
undergoing Phase IIa trials in malaria patients [21]. In in vitro and
vivo animal studies, OZ439 has been reported to have a better anti-
malarial drug profile than OZ277 and all other semi-synthetic arte-
misinin derivatives. On the basis of their structural differences, it is
believed that these synthetic ozonides may replace the standard
artemisinins if they become obsolete [17]. Nevertheless, the short-
comings of the current drugs, the emergence of P. falciparum resis-
tance to the artemisinin class of compounds and the fact that the
mechanism by which the parasite acquires resistance is still largely
unknown, warrant the search for new classes or derivatives.
In this communication, we report the synthesis of series of alkyl/
aryl esters and their antimalarial activity as well as cytotoxicity.
Thus, all derivatives currently in use are either alkyl acetals or
an ester acetal derivative of dihydroartemisinin (DHA, 2). The
⇑
Corresponding author. Address: Pharmaceutical Chemistry, School of Pharmacy,
North-West University, Private Bag X 6001, Internal Box 304, Potchefstroom 2520,
South Africa. Fax: +27 18 299 4243.
E-mail address: david.nda@nwu.ac.za (D.D. N’Da).
0045-2068/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.bioorg.2012.10.002

T.T. Cloete et al. / Bioorganic Chemistry 46 (2013) 10–16
11
Fig. 1. Structures of artemisinin 1 and its clinically used derivatives; dihydroartemisinin 2, artemether 2a, arteether 2b and sodium artesunate 2c.
Fig. 2. Structures of the semi-synthetic artemisinin derivative artemisone and the synthetic ozonide OZ277 and OZ439.
Previously synthesized aminoethylethers of artemisinin were also
screened alongside the esters to highlight their inhibitory potential
against a variety of P. falciparum strains.
1100 series isocratic pump. A Zorbax Eclipse XDB C18, 5
l
m
(150 ꢁ 4.60 mm) column was used and the Agilent Chemstation
reverse A08.03 for LC systems software package for data analysis.
The compounds were quantified using
(A = 0.2% triethylamine in H2O, pH 7.0, B = acetonitrile) at a flow
rate of 1 ml/min with 20 l standard sample injections. The gradi-
a gradient method
2. Materials and methods
l
2.1. Materials
ent consisted of 25% of solvent B (ACN) until 1 min, then increased
linearly to 95% of B after 8 min, and held for 15 min, where after
the instrument was re-equilibrated to the starting conditions for
5 min. A calibration plot of peak area versus drug concentration
for each compound showed excellent linearity (0.993 < r2 6 1 over
All the acyl chlorides used in the synthesis were purchased from
Fluka (Johannesburg, South Africa). Anhydrous dichloromethane
was purchased from Sigma–Aldrich (Johannesburg, South Africa).
Dihydroartemisinin (
a and b racemates) was purchased from
the concentration range (0–2000 lg/ml) employed for the assays.
HuBei Enshi TianRanYuan Science and Technology Herbal Co.,
Ltd. (Hangzhou, China). HPLC grade methanol was obtained from
Labchem Ltd. (Johannesburg, South Africa). All the reagents and
chemicals were of analytical grade.
The absorption maximum for dihydroartemisinin and all its deriv-
atives was at 205 nm; this wavelength was consequently used for
the HPLC detection.
3. Experimental procedures
2.2. General procedures
3.1. Synthesis of esters 11–18
Thin-layer chromatography (TLC) was performed using silica
gel plates (60F254 Merck). Preparative flash column chromatogra-
phy was carried out on silica gel (230–240 mesh, G60 Merck)
and silica gel 60 (70–230 mesh ASTM, Fluka).
The synthesis of artemisinin esters is depicted in Scheme 1 and
was achieved as follows: dihydroartemisinin 2 (7 mmol) and acyl/
aryl chlorides 3–10 (14 mmol, 2.0 equiv.) were dissolved in anhy-
drous dichloromethane (65 ml) together with triethylamine
(14 mmol, 2.0 equiv.) and was stirred at room temperature for
24 h. The progress of the reaction was monitored by thin layer
chromatography (TLC). After completion, the reaction mixture
was evaporated to dryness in vacuo. The resulting residue was
purified by flash chromatography using dichloromethane. All the
synthesized compounds were solids, except 17 and 18, which were
oils. 1H and 13C NMR chemical shifts as well as low and high reso-
lution MS data of compounds 11–18 are reported.
The 1H and 13C NMR spectra were recorded on a Bruker Avance
III 600 spectrometer (at a frequency of 600.17 and 150.913 MHz,
respectively) in deuterated chloroform (CDCl3). Chemical shifts
are reported in parts per million (d ppm) using tetramethylsilane
(TMS) as internal standard. The splitting pattern abbreviations
are as follows: s (singlet), d (doublet), dd (doublet of doublets),
ddd (doublet of doublet of doublets), dt (doublet of triplets), t (trip-
let), td (triplet of doublets), tt (triplet of triplet) and m (multiplet).
Low resolution mass spectra (LRESI) were collected on the Ther-
mo Finnigan LXQ ion trap mass spectrometer in ESI mode. NaCl
was added as a modifier to stabilize the compounds in ESI mode.
The high resolution ESI (HRESI) mass spectra (MS) were recorded
on a Thermo Electron DFS LXQ ion mass spectrometer in ESI mode
and [M + Na]+ were recorded.
3.1.1. 10a-Dihydroartemisinyl-propanoate 11
Yield: 47.2%; off-white powder; mp: 70–72 °C; 1H NMR
(600 MHz, CDCl3): d 6.16 (s, 1H, H-10), 5.51 (s, 1H, H-12), 2.09–
1.95 (m, 2H, H-18), 2.59–1.74 (m, 5H, H-5a, -9, -8a and H-8),
2.16–1.96 (m, 2H, H-7), 1.88 (ddd, J = 25.2, 14.5, 10.9 Hz, 1H, H-
6), 1.74–1.10 (m, 10H, H-14, -4, -5 and H-19), 0.98 (d, J = 10.5 Hz,
3H, H-16), 0.88 (d, J = 10 Hz, 3H, H-15); 13C NMR (151 MHz, CDCl3)
d 165.5 (C-17), 134.94 (C-3), 108.07 (C-10), 104.48 (C-12), 68.10
2.3. High performance liquid chromatography (HPLC)
The HPLC system consisted of an Agilent 1100 series auto sam-
pler, Agilent 1100 series variable wave detector (VWD) and Agilent

12
T.T. Cloete et al. / Bioorganic Chemistry 46 (2013) 10–16
(C-21); LRESI m/z: 433 (M + 23, 100%); HRESI m/z: 433.2662
(M + 23) (M + 23, C23H38O6Na, 433.2566 calc.).
3.1.4. 10a-Dihydroartemisinyl decanoate 14
Yield: 78.45%, clear oil, 1H NMR (600 MHz, CDCl3): d 5.77 (d,
J = 9.8 Hz, 1H, H-10), 5.42 (s, 1H, H-12), 2.62-2.45 (m, 3H, H-5a),
2.42–2.27 (m, 3H, H-18 and H-4a), 2.01 (d, J = 14.5 Hz, 1H, H-4b),
1.93–1.83 (m, 1H, H-9), 1.79–1.66 (m, 2H, H-30), 1.66–1.51 (m,
5H, H-6, -7 and H-7), 1.50–1.34 (m, 5H, H-8a, H-19 and H-14),
1.34–1.15 (m, 14H, H-5 and H-20), 1.05–0.90 (m, 3H, H-16), 0.9–
0.76 (m, 6H, H-15 and H-21); 13C NMR (151 MHz, CDCl3): d
172.51 (C-17), 104.25 (C-3), 91.45 (C-10), 91.18 (C-12), 80.05 (C-
12a), 77.96 (C-5a), 77.05 (C-8a), 77.02 (C-4), 37.96 (C-9), 36.37
(C-7), 34.48 (C-6), 34.19 (C-18), 31.92 (C-6), 31.79 (C-20), 29.25
(C-14), 25.56 (C-8), 24.54 (C-5), 21.57 (C-15), 21.00 (C-19), 14.85
(C-16), 10.11 (C-21); LRESI m/z: 461 (M + 23, 20%); HRESI m/z:
461.2875 (M + 23) (M + 23, C25H42O6Na, 461.2879 calc.).
3.1.5. 10a-Dihydroartemisinyl benzoate 15
Yield: 70.5%; clear crystal; mp: 115–18 °C; 1H NMR (600 MHz,
CDCl3): d 8.09 (dd, J = 8.2 and 1.0 Hz, 2H, H-19), 7.60–7.48 (m,
1H, H-21), 7.42 (t, J = 7.8 Hz, 2H, H-20), 5.98 (d, J = 9.8 Hz, 1H, H-
10), 5.50 (s, 1H, H-12), 2.84-2.60 (m, 1H, H-9), 2.36 (ddd, J = 17.7,
13.9 and 4.0 Hz, 1H, H-8a), 2.02 (ddd, J = 14.6, 4.6 and 3.1 Hz, 1H,
H-5a), 1.94–1.57 (m, 2H, H-4), 1.57–1.16 (m, 8H, H-6, -7, -8 and
H-14), 1.14–0.7 (m, 8H, H-5, -15 and H-16); 13C NMR (151 MHz,
CDCl3): d 165.27 (C-17), 133.27 (C-21), 130.09 (C-18), 129.58 (C-
19), 128.26 (C-20), 104.40 (C-3), 92.49 (C-10), 91.56 (C-12), 80.17
(C-12a), 51.62 (C-5a), 45.31 (C-8a), 37.25 (C-4), 36.22 (C-9), 34.09
(C-7), 31.96 (C-6), 25.93 (C-14), 24.55 (C-8), 22.03 (C-5), 20.21 (C-
15), 12.21 (C-16); LRESI m/z: 411 (M + 23, 20%); HRESI m/z:
411.1773 (M + 23) (M + 23, C22H28O6Na, 411.1784 calc.).
Scheme 1. A general reaction scheme to illustrate the synthesis of 10-alkyl/aryl
esters 11–18 of artemisinin from dihydroartemisinin and various acyl/aryl
chlorides. Reagents and conditions: (a) dihydroartemisinin 2, acyl/aryl chlorides
3–10, dichloromethane, room temperature, 24 h.
(C-12a), 51.39 (C-5a), 44.39 (C-8a), 38.68 (C-4), 37.43 (C-9), 36.18
(C-7), 34.06 (C-6), 30.31 (C-18), 29.95 (C-14), 28.88 (C-8), 25.82 (C-
5), 20.25 (C-15), 16.15 (C-16), 10.92 (C-19); LRESI m/z: 363 (M + 23,
3.1.6. 10a-Dihydroartemisinyl 4-nitrobenzoate 16
Yield: 94%; yellow solid; mp: 94–98 °C; 1H NMR (600 MHz,
CDCl3): d 8.27 (s, 4H, H-19 and H-20), 6.00 (d, J = 9.8 Hz, 1H, H-
10), 5.51 (s, 1H, H-12), 2.84–2.64 (m, 1H, H-9), 2.37 (dt, J = 14.1
and 3.7 Hz, 1H, H-5a), 1.94–1.60 (m, 2H, H-4), 1.57–1.24 (m, 8H,
H-6, -7, -8 and H-14), 1.11–0.81 (m, 8H, H-5, -15 and H-16); 13C
NMR (151 MHz, CDCl3): d 163.49 (C-17), 150.76 (C-21), 135.03
(C-18), 131.21 (C-19), 123.5 (C-20), 104.55 (C-3), 93.37 (C-10),
91.69 (C-12), 80.10 (C-12a), 51.58 (C-5a), 45.27 (C-8a), 37.28 (C-
4), 36.20 (C-9), 34.06 (C-7), 31.89 (C-6), 25.90 (C-14), 24.56 (C-8),
22.03 (C-5), 20.20 (C-15), 12.22 (C-16); LRESI m/z: 456 (M + 23,
50%); HRESI m/z: 363.1700 (M + 23) (M + 23,
C18H28O6Na,
363.1784 calc.).
3.1.2. 10a-Dihydroartemisinyl butanoate 12
Yield: 18.7%; off-white powder; mp: 60–63 °C; 1H NMR
(600 MHz, CDCl3): d 5.78 (d, J = 9.8 Hz, 1H, H-10), 5.42 (s, 1H, H-
12), 2.64–2.24 (m, 3H, H-5a and H-18), 2.09–1.92 (m, 1H, H-4a),
1.90–1.81 (m, 1H, H-9), 1.8–1.15 (m, 14H, H-8a, -8, -5, 4b, -6, -7,
-14 and H-19), 1.06–0.88 (m, 6H, H-16 and H-20), 0.82 (d,
J = 7.1 Hz, H-15); 13C NMR (151 MHz, CDCl3): d 172.41 (C-17),
104.42 (C-3), 91.63 (C-10), 91.47 (C-12), 80.14 (C-12a), 77.22
(C-5a), 77.00 (C-8a), 77.00 (C-4), 37.25 (C-9), 36.19 (C-7), 34.08
(C-6), 31.81 (C-18), 25.96 (C-14), 24.56 (C-8), 21.99 (C-5), 20.21
(C-15), 18.15 (C-19), 13.63 (C-16), 12.1 (C-20); LRESI m/z: 377
(M + 23, 30%); HRESI m/z: 377.1935 (M + 23) (M + 23, C19H30O6Na,
377.1940 calc.).
40%); HRESI m/z: 456.1808 (M + 23) (M + 23,
456.1634 calc.).
C22H27NNaO8,
3.1.7. 10a-Dihydroartemisinyl 2-(acetyloxy) benzoate 17
Yield: 84.5%; off-white solid; mp: 80–83 °C; 1H NMR (600 MHz,
CDCl3): d 8.09 (dd, J = 17.7 and 1.6 Hz, 1H, H-19), 7.86 (ddd,
J = 17.7, 7.9 and 1.6 Hz, 1H, H-21), 7.55 (td, J = 7.9 and 1.7 Hz, 1H,
H-22), 7.30 (td, J = 7.7 and 0.9 Hz, 1H, H-20), 5.98 (d, J = 9.8 Hz,
1H, H-10), 5.48 (s, 1H, H-12), 2.73–2.56 (m, 1H, H-9), 2.41–2.22
(m, 4H, H-8a and H-25), 2.09–1.96 (m, 1H, H-5a), 1.92–1.84 (m,
1H, H-4a), 1.80–1.54 (m, 4H, H-7 and H-8), 1.54–1.20 (m, 3H, H-
6 and H-14), 0.99–0.92 (m, 3H, H-15), 0.86 (d, J = 7.1 Hz, H-3);
13C NMR (151 MHz, CDCl3): d 169.93 (C-24), 163.10 (C-17),
151.01 (C-23), 134.21 (C-21), 132.29 (C-19), 125.95 (C-20),
124.44 (C-22), 122.80 (C-18), 104.4 (C-3), 92.54 (C-10), 91.51
(C-12), 80.07 (C-12a), 53.42 (C-5a), 51.56 (C-8a), 45.33 (C-4),
37.29 (C-9), 36.23 (C-7), 34.09 (C-6), 32.01 (C-14), 25.56 (C-8),
21.07 (C-5), 20.22 (C-15), 12.20 (C-16); LRESI m/z: 469 (M + 23,
3.1.3. 10a-Dihydroartemisinyl octanoate 13
Yield: 65.7%), clear oil, 1H NMR (600 MHz, CDCl3): d 5.77 (d,
J = 9.9 Hz, 1H, H-10), 5.42 (s, 1H, H-12), 2.62–2.46 (m, 1H, H-5a),
2.42-2.27 (m, 3H, H-18 and H-4a), 2.01 (m, 1H, H-9), 1.92–1.81
(m, 1H, H-4b), 1.79–1.66 (m, 2H, H-19), 1.66–1.51 (m, 4H, H-8
and H-7), 1.49–1.36 (m, 4H, H-8a, H-6 and H-14), 1.35–1.19 (m,
10H, H-5 and H-20), 1.1–0.95 (m, 3H, H-16), 0.9–0.74 (m, 6H, H-
15 and H-21); 13C NMR (151 MHz, CDCl3): d 172.60 (C-17),
104.43 (C-3), 91.63 (C-10), 91.48 (C-12), 80.15 (C-12a), 77.22
(C-5a), 77.00 (C-8a), 77.00 (C-4), 37.26 (C-9), 36.22 (C-7), 34.33
(C-6), 34.09 (C-18), 31.82 (C-6), 31.63 (C-19), 29.03 (C-14), 25.96
(C-8), 24.62 (C-5), 22.57 (C-15), 22.00 (C-20), 14.05 (C-16), 12.11
100%); HRESI m/z: 469.1833 (M + 23) (M + 23,
469.1838 calc.).
C24H30O8Na,

T.T. Cloete et al. / Bioorganic Chemistry 46 (2013) 10–16
13
3.1.8. 10
a
-Dihydroartemisinyl 2-phenylacetate 18
compound. The average results are reported in Table 1. Further-
more, solubility in n-octanol was used to mimic the solubility in
a lipophilic environment. The lipid solubility (SL) values were de-
duced from the equation logSL = logD + logSW, where SW and logD
are experimental.
Yield: 40.1%; off-white solid; mp: 101–105 °C; 1H NMR
(600 MHz, CDCl3): d 7.36–7.19 (m, 5H, H-20, 21 and H-22), 5.76
(d, J = 9.8 Hz, 1H, H-10), 5.42 (s, 1H, H-12), 3.77–3.55 (m, 1H,
H-18), 2.64–2.43 (m, 1H, H-5a), 2.36 (td, J = 14.1 and 3.8 Hz, 2H,
H-4), 1.77–1.62 (m, 1H, H-9), 1.62–1.53 (m, 8H, H-6, -7, -8 and
H-14), 1.5–1.15 (m, 3H, H-16), 0.89 (d, J = 7.1 Hz, 1H, H-15); 13C
NMR (151 MHz, CDCl3): d 170.35 (C-17), 133.58 (C-19), 129.39
(C-20), 128.50 (C-21), 127.13 (C-22), 104.46 (C-3), 92.20 (C-10),
91.53 (C-12), 80.12 (C-12a), 51.54 (C-5a), 45.23 (C-8a), 41.28
(C-18), 37.23 (C-4), 36.20 (C-9), 34.05 (C-7), 31.85 (C-6), 25.96
(C-14), 24.56 (C-8), 21.94 (C-5), 20.19 (C-15), 11.88 (C-16); LRESI
m/z: 425 (M + 23, 40%); HRESI m/z: 425.1938 (M + 23) (M + 23,
3.3.2. Distribution coefficient (logD)
Equal volumes of n-octanol and PBS (pH 5.5 and 7.4) were
mixed with vigorous stirring for at least 24 h. 2 mg of each deriva-
tive was dissolved in 0.75 ml of this solution, the solution was then
stoppered and agitated for 10 min in 2 ml graduated tubes (0.5 ml
division). Subsequently 0.75 ml of pre-saturated buffer was trans-
ferred to the tubes containing the before mentioned solutions. The
tubes were stoppered and agitated for 45 min after which they
were centrifuged at 4 000 rpm (1503 g) for 30 min. The n-octanol
and aqueous phases were allowed to separate at room temperature
for 5 min, where after their volume ratio (v/v; n-octanol:buffer)
was determined. The volume ratio was found in all cases to be 1.
The n-octanol and aqueous phases were then analyzed by HPLC.
From this data, the concentrations of the derivative in both phases
were determined. The logD values were calculated as logarithmic
ratios of the concentrations in the n-octanol phase compared to
the concentrations in the buffer [23]. The experiment was per-
formed in triplicate and the results, expressed as means, are listed
in Table 1. As oxalate salts, the aminoethylethers 19–29 were not
subjected to experimental aqueous solubility and distribution
coefficient determinations.
C23H30O6Na, 425.1940 calc.).
3.2. Synthesis of 10-aminoethylethers of artemisinin 19-29
The 10-aminoethylethers of artemisinin 19–29 were previously
synthesized using a two-step process using a method reported by
Li et al. [15]. The synthesis and characterization of these com-
pounds have been reported elsewhere [22] and their structures
are delineated in Fig. 3.
3.3. Physicochemical properties
3.3.1. Aqueous solubility (SW
)
The aqueous solubility (SW) of crystalline compounds 11, 12,
and 15–18 as well as DHA 2 were obtained by preparing solutions
in PBS (pH 5.5 and pH 7.4). The slurries were stirred in a water bath
at 37 °C for 24 h. An excess of solute was present at all times to
provide saturated solutions. After 24 h, the solutions were filtered
and analyzed directly by HPLC to determine the concentration of
solute dissolved in the solvent. For the oils 13 and 14, after 24 h
stirring, the mixtures were allowed to stand at room temperature
then the aqueous layers were carefully separated and analyzed by
HPLC [23]. The experiment was performed in triplicate for each
3.4. Methodology for in vitro biological evaluation
3.4.1. Determination of antimalarial effective concentration (EC)
The esters and aminoethylethers of artemisinin were screened
against the chloroquine-sensitive (CQS, 3D7) and chloroquine-
resistant (CQR, K1) strains of P. falciparum. Continuous in vitro
cultures of asexual erythrocyte stages of P. falciparum were main-
tained using a modified method of Trager and Jensen [24]. Asyn-
chronous parasites were grown in the presence of fresh group
O-positive erythrocytes (Lifeblood, Memphis, TN) in Petri dishes
at a hematocrit of 4–6% in RPMI-based medium consisting of RPMI
1640 supplemented with 0.5% AlbuMAX II, 25 mM HEPES, 25 mM
NaHCO3 (pH 7.3), 100
cin. Cultures were incubated at 37 °C in a gas mixture of 90% N2,
5% O2, and 5% CO2. For EC50 determinations, 20 l of RPMI 1640
with 5 g/ml gentamicin were dispensed per well in an assay plate
lg/ml hypoxanthine, and 5 lg/ml gentami-
l
l
(384-well microtiter plate, clear-bottom, tissue-treated). Next,
40 nl of each compound, previously serial diluted in a separate
384-well white polypropylene plate, were dispensed in the assay
plate, and then 20 ll of a synchronized culture suspension (1%
rings, 10% hematocrit) were added per well to make a final hemat-
ocrit and parasitemia of 5% and 1%, respectively. Assay plates were
incubated for 72 h, and the parasitemia was determined by a meth-
od previously described [25]. Briefly, 10 ll of 10X Sybr Green I,
0.5% v/v Triton, and 0.5 mg/ml saponin solution in RPMI were
added per well. Assay plates were shaken for 30 s, incubated in
the dark for 4 h, and then read with the Envision spectrofluorome-
ter at Ex/Em 485 nm/535 nm. EC50s were calculated using proprie-
tary software developed in house (RISE) in the Pipeline Pilot
environment that pools data from all replicates of the experiment
and fits a consensus model.
3.4.2. In vitro cytotoxicity against HEK293 and HEP G2 cells
HEK293 and HepG2 cell lines were purchased from the Ameri-
can Type Culture Collection (ATCC, Manassas, VA) and were cul-
tured according to recommendations. Cell culture media were
purchased from ATCC. Cells were routinely tested for mycoplasma
contamination using the MycoAlert Mycoplasma Detection Kit
Fig. 3. Structures of 10-aminoethylethers of artemisinin 19–29.

14
T.T. Cloete et al. / Bioorganic Chemistry 46 (2013) 10–16
Table 1
Aqueous solubility, distribution coefficients and lipid solubility of esters 11–18. The aqueous solubility (SW) was determined in PBS (pH 5.5 and 7.4) at 37 °C after incubation in a
water bath with stirring for 24 h; distribution coefficient (logD) was obtained after n-octanol/PBS (pH 5.5 and 7.4) partition, and lipid solubility (SL) was deduced from
experimental SW and SL.
J (Hz)a
Isomer
SW7.4
(
l
M)b
SD
logD7.4
SL7.4
(l
M)d
SW5.5
(
l
M)a
SD
logD5.5
SL5.5 (l
M)b
c
a
Comp.
DHA 2
11
12
13
14
15
16
17
18
ND
ND
9.8
9.9
9.8
9.8
9.8
9.8
9.8
1.2
0.3
0.3
0.2
0.5
0.3
1.0
0.7
0.7
0.09
0.01
0.01
0.04
0.01
0.07
0.08
0.02
0.01
0.1
0.8
0.7
0.9
0.9
0.5
0.4
0.3
0.7
1.5
1.9
1.5
1.6
4.0
1.0
2.5
1.4
3.5
3.2
0.4
0.9
0.5
0.5
0.6
1.4
0.9
0.9
0.08
0.04
0.01
0.05
0.03
0.07
0.05
0.03
0.01
0.3
0.3
0.7
0.2
0.9
0.2
0.6
0.2
0.3
6.4
0.8
2.3
0.8
4.0
1.0
5.6
1.4
1.8
a
a
a
a
a
a
a
a
b
c
Coupling constant (J) between H-10 and H-9.
Experimental aqueous solubility (SW), each value represents the mean of three independent measurements.
Experimental distribution coefficient, logD (n-octanol: PBS, pH 5.5 and 7.4), each value represents the mean of three independent measurements.
Lipid solubility (SL) deduced using logSL = logD + logSW where logD and SW are experimental data; standard deviation (SD).
d
(Lonza). Exponentially growing cells were plated in Corning 384
well white custom assay plates, and incubated overnight at 37 °C
in a humidified, 5% CO2 incubator. DMSO inhibitor stock solutions
4. Results and discussion
4.1. Chemistry
were added the following day to a top final concentration of 25 lM,
0.25% DMSO and then diluted 1/3 for a total of ten testing concen-
trations. Cytotoxicity was determined following a 72-h incubation
using Promega Cell Titer Glo Reagent according to the manufac-
turer’s recommendation. Luminescence was measured on an Envi-
sion plate reader (Perkin Elmer). EC50s were calculated using
proprietary software developed in house (RISE) in the Pipeline Pilot
environment that pools data from all replicates of the experiment
and fits a consensus model.
The esters were obtained by acylation of C-10 of dihydroarte-
misinin. The configuration at that stereocenter was assigned based
on the vicinal coupling constant JH-9:H-10 [28]. A large coupling con-
stant (9–10 Hz) is generally found for the 10a-isomer [29], indicat-
ing the relative trans configuration. The 10b-isomer, on the other
hand has a smaller coupling constant (3.6–5 Hz) [30]. Thus, the
esters 12–18, all with JH-9:H-10 values of 9.8 Hz were the 10a-iso-
mers. Compound 11 showed no detectable coupling constant thus,
the stereochemistry at the C-10 could not be deduced on the basis
of that parameter.
3.4.3. Dose–response curve fitting
In the 1H spectra of these 10
a-esters, the doublet due to reso-
Dose–response curves were calculated from percent activity
values and log10-transformed concentrations, the proprietary
robust interpretation of screening experiments (RISE) application
written in Pipeline Pilot (Accelrys, v. 8.5) and the R program [26].
Briefly, non-linear regression was performed using the R drc pack-
age with the four-parameter log–logistic function (LL2.4) [27]. The
data for all replicates for each compound was fit three separate
times by varying the parameters that were fixed during regression:
(1) all parameters free, (2) high response fixed to 100, (3) low
response fixed to 0. The best fit from these three nested models
was selected using the anova.drc function. 95% confidence intervals
were produced based upon this fit.
Dose–response curves were assigned a quality score according
to the following heuristic. Compounds which failed to fit to any
curve, or with curves having efficacy <25% or >150% or hill slope
<0.5 or >25 were designated class ‘D1’. Compounds passing this
first criteria with curves having efficacy <50%, calculated
EC50 > the highest concentration tested, lower and upper EC50
confidence limits > 10-fold EC50, or slope at the highest concentra-
tion tested >75% (non-saturating) were designated class ‘C1’. Com-
pounds passing previous criteria with curves having lower and
upper EC50 confidence limits > 5-fold EC50 or slope at the highest
concentration tested >25% (not completely saturating) were desig-
nated class ‘B1’. All remaining curves were designated ‘A1’, which
is indicative of ideal, well-behaved sigmoidal response. Curves that
were inverted (activity decreased as concentration increased) were
prefixed with the letter ‘N’, such as ‘NA1’ In tabulating data, EC50
values are only called for A1 and B1 class curves with a single value
and error being provided for A1 curves and a range provided for B1
curves. C1 and D1 curves were assigned an arbitrary value of either
greater than the highest concentration tested or lower than the
lowest concentration tested, when appropriate. All the biological
results are summarized in Table 2.
nance of H-10 appeared downfield compared to the singlet of
H-12. This as the result of the electron-withdrawing eddect of
the ester carbonyl. In the 13C spectra, the presence of carbonyl car-
bon C-17 is noticeable at 170–160 ppm. The C-10, which usually
resonates at ꢀ99 ppm for DHA, was further shielded for these com-
pounds resulting in the signal shifting up field to 92–90 ppm. All
signals due to DHA’s carbons were also found in the spectra of
the esters confirming the presence of that moiety in their
structures.
The chemical structures of the title esters 11–18 were further
confirmed by MS analysis. LRESI data showed the molecular ions
M + 23 m/z of compounds 11, 12, 13, 14, 15, 16, 17 and 18 at 363,
377, 433, 461, 411, 455, 425 and 469, respectively. The HRESI–MS
confirmed the exact mass of 11 [363.1700], which corresponds to
the formula C18H28O6Na, of 12 [377.1935] for C19H30O6Na, of 13
[433.2662] for C23H38O6Na, of 14 [461.2875] for C25H42O6Na, of
15 [411.1773] for C22H28O6Na, of 16 [456.1808] for C22H27O8Na,
of 17 [469.1838] for C24H30O8Na, and of 18 [425.1938] for the
formula C23H30O6Na.
The 10-aminoethylethers 19–29 were all 10b-isomers [22].
4.2. Aqueous solubility and logD
Aqueous solubility and lipophilicity influence the way a mole-
cule passes through biological membranes and barriers to ulti-
mately enter the systemic circulation. Drugs must thus possess
balanced lipophilic/hydrophilic properties to both permeate bio-
logical membranes and be taken up for systemic circulation. The
logarithm of ratio of octanol solubility to water solubility (logP)
is absorption good measure of this balance with values between
0 and 5 being targeted, and 0–3 being ideal [31,32]. The distribu-
tion coefficient (logD), a pH dependent version of the partition

T.T. Cloete et al. / Bioorganic Chemistry 46 (2013) 10–16
15
Table 2
In vitro antimalarial activity of esters 11–18 and aminoethylethers 19–29 of artemisinin against 3D7 and K1 strains of Plasmodium falciparum, and their cytotoxicity against
HEK293 and HepG2 cell lines. Cells were incubated with compounds at various concentrations for 72 h; antimalarial activity and cytotoxicity were determined using SYBR Green
I-based fluorescence and CellTiter-Glo luminescent cell viability assays, respectively.
Activity EC50 (nM)a
3D7
RIb
Cytotoxicity EC50
HEK293
(l
M)c
103 ꢁ SI1
103 ꢁ SI2
d
e
Comp.
K1
HepG2
11
12
13
14
15
16
17
18
3.1
13.3
9.4
21.3
3
3.1
5.1
2.4
0.3
6.2
5.0
17
0.9
1.1
3.3
0.5
0.1
0.5
0.5
0.8
0.3
0.4
0.7
0.2
>26.0
>26.0
>26.0
>26.0
>26.0
>23.8
>26.0
>26.0
>26.0
>26.0
>26.0
>26.0
>26.0
>23.8
>26.0
>26.0
>8.4
>2.0
>2.8
>1.2
>8.7
>7.7
>5.1
>10.9
>8.4
>2.0
>2.8
>1.2
>8.7
>7.7
>5.1
>10.9
19
20
21
22
23
24
25
26
27
28
29
AM 2a
AS 2c
8.7
3.1
6.3
5.5
6.6
4.1
3.6
5.8
26.1
10.8
4.0
2.4
1723.3
6.6
0.4
0.4
0.6
0.6
0.4
0.5
0.6
1.2
0.5
0.5
0.4
10.4
1
>27.5
>21.4
>31.7
>13.3
>18.6
>29.6
>26.6
>22.7
>30.1
>28.9
>16.0
>26.0
>26.0
>27.5
>21.4
>31.7
>13.3
>18.6
>29.6
>26.6
>22.7
>30.1
>28.9
>16.0
>26.0
>26.0
>3.2
>1.5
>3.1
>1.2
>1.7
>3.8
>3.0
>1.0
>1.3
>3.7
>2.4
>0.2
>3.9
>3.2
>1.5
>3.1
>1.2
>1.7
>3.8
>3.0
>1.0
>1.3
>3.7
>2.4
>0.2
>3.9
14.8
10.1
11.3
11.3
7.9
9
22.1
23.7
7.9
6.8
166
6.6
a
b
c
Effective concentration of compound inducing 50% reduction in parasitic cells count.
Effective concentration of compound selectively inducing 50% reduction in parasitic cells count in the presence of mammalian cells.
Resistance index (RI) = EC50 K1/EC50 3D7.
Selectivity index (SI1) = EC50 HEK293/EC50 3D7.
Selectivity index (SI2) = EC50 Hep G2/EC50 3D7.
d
e
coefficient (logP) was determined experimentally in a phosphate
buffer at two distinctive pH values (7.4 and 5.5), chosen to mimic
the cytosolic and parasite digestive vacuole environments,
respectively.
Overall the esters had aqueous solubility slightly lower than
that of DHA, regardless of the medium. While this may not be sig-
nificant enough to influence their relative absorption through bio-
logical membranes, it can be attributed to the fact that the
hydrogen bonding polar site of DHA, is masked by a lipophilic
group.
Comparison of SW, logD, and SL values in both media also re-
vealed no significant differences among the esters. They all dis-
played comparable aqueous and lipid solubility as well as
distribution coefficients in both environments. No significant
structure-physiochemical property relationship could thus be de-
duced from this study.
artemsinin derivatives synthesized during this study. They were
significantly more potent than AS against the K1 strain with 11
and 18 possessing 22- and 12-fold improvements, respectively.
No SAR could be deduced with the esters due to limited structural
diversity.
In the 10-aminoethylether series, ether 29 was the most active
of all. It was as potent as AS against 3D7 and possessed a moderate
potency improvement (ꢀ3-fold) against K1. Ether 26, on the other
hand was the least potent. It found 3-fold less potent than AS
against both the 3D7 and K1 strains. The series may be divided into
two sub-series viz. the monoamine series comprising ethers 19, 21,
24, 25, 28 and 29, and the polyamine one including 20, 22, 23, 26
and 27. Cloete et al. [22] reported the monoamine sub-series to
be more active than the polyamine in screening against CQS D10
and CQR Dd2 strains. This was also the case against the 3D7 and
K1 strains in this study. However, these ethers appear more active
against D10 than 3D7, and K1 than Dd2. Average 3- to 6-fold and 5-
to 10-fold variances were found between activity against D10 and
3D7, and K1 and Dd2, respectively, which shows that although both
are considered to be CQ sensitive, these strains are still different cell
lines and have different behaviors in the presence of the com-
pounds. It is, thus, noteworthy that these 10-aminoethylethers of
artemisinin are active against four different strains of P. falciparum.
Furthermore, the aminoethylethers are hydrophilic and weak
bases able to undergo protonation at lower pH value such as that
occurring in the parasitic digestive vacuole (pHꢀ5.5), and thus
accumulate in higher concentration by pH-trapping mechanism.
One would then expect a synergism between the action inherent
to the DHA endoperoxide and the pH-trapping mechanism to ren-
der these aminoethers more active than the esters, which lack the
amine(s) for pH-trapping mechanism. However, the EC50 data
against both strains showed the opposite, confirming that the
drug’s absorption is dependent upon multiple physicochemical
parameters [33].
4.3. In vitro antimalarial activity and cytotoxicity
The esters 11–18 and aminoethers 19–29 were tested in vitro
against both the chloroquine sensitive (CQS) 3D7, and chloroquine
resistant (CQR) K1 strains of P. falciparum (Table 2). Both com-
pound-types were active against both the 3D7 and K1 strains.
The esters were all significantly more potent than AM, up to 60-
and 5000-fold against 3D7 and K1, respectively. The aminoethyle-
thers also displayed significantly better potency than AM but to a
lesser extent, 20- and 700-fold.
In comparison with artesunate, however, the behavior was
quite different. Ester 14 and ethers 26 and 27 were 3-fold less po-
tent than AS, while all other derivatives had comparable to AS
against the 3D7 strain. This trend was also observed against the
K1 strain. Ester 14 and ethers 26 and 27 were still less potent than
AS but the remaining derivatives were either as active or more po-
tent than the reference drug. Thus, against this strain, the esters
were found still more active than the ethers. Compounds 11, 15,
16 and 18, all esters were distinctively the most active of all
To determine whether the synthesized compounds were selec-
tive in their action against the plasmodia cells, relative to host

16
T.T. Cloete et al. / Bioorganic Chemistry 46 (2013) 10–16
[6] H. Noedl, D. Socheat, W. Satimai, N. Engl. J. Med. 361 (2009) 540–541.
[7] WHO World Health Organization, Development of strategy towards
cells, a selectivity index (SI) was determined for multiple mamma-
lian cell types. All compounds 11–29 proved to be non-toxic to
both HEK293 and HepG2 cells as can be seen from the high SI1
and SI2 values, all above 1000.
a
elimination of Plasmodium falciparum parasites with altered responses to
artemisinins, 2000.
[8] WHO World Health Organization, Strategic Framework for Artemisinin
Resistance Containment in Myanmar (MARC) 2011–2015, 2011.
[9] A.J. Lin, D.L. Klayman, W.K. Milhous, J. Med. Chem. 30 (1987) 2147–2150.
[10] V. Leskovac, A.D. Theoharides, Comp. Biochem. Physiol. 99 (1991) 383–390.
[11] V. Leskovac, A.D. Theoharides, Comp. Biochem. Physiol. 99 (1991) 391–396.
[12] J.K. Baker, J.D. McChesney, H.-T. Chi, Pharm. Res. 10 (1993) 662–666.
[13] K.T. Batty, K.F. IIett, T.M.E. Davis, J. Pharm. Pharmacol. 48 (1996) 22–26.
[14] China Cooperative Research Group on Qinghaosu and Its Derivatives as
Antimalarials, J. Tradit. Chin. Med. 2 (1982) 9–16.
5. Conclusions
A series of 10a-n-alkyl/aryl esters were synthesized through
derivatization at C-10 of dihydroartemisinin. The structures of
the compounds were verified and confirmed by NMR and MS anal-
yses. The esters were subjected to distribution coefficient and
aqueous solubility experimental determinations in both weakly
acid (pH 5.5) and physiological (pH 7.4) media. The esters were
found moderately less hydrophilic than DHA with comparable
lipophilicity, and displayed similar distribution coefficients and
aqueous solubility in both media. The esters were screened for
antimalarial activity alongside 10-aminoethylethers of artemisinin
against the CQS 3D7 and CQR K1 strains of P. falciparum. Both com-
pound types were active with the esters having a moderate upper
edge over the ethers. They possessed significant potency improve-
ment over artemether against both strains, up to 500-fold on aver-
age. In comparison to artesunate, the majority of compounds
possessed either similar or a higher degree of potency against both
[15] Y. Li, Y.-M. Zhu, H.-J. Jiang, J.-P. Pan, G.-S. Wu, J.-M. Wu, J. Med. Chem. 43
(2000) 1635–1640;
R.K. Haynes, H.-W. Chan, M.-K. Cheung, W.-L. Lam, M.-K. Soo, H.-W. Tsang, A.
Voerste, I.D. Williams, Eur. J. Org. Chem. (2002) 113–132;
S. Hindley, S.A. Ward, R.C. Storr, N.L. Searle, P.G. Bray, B.K. Park, J. Davies, P.M.
O’Neill, J. Med. Chem. 45 (2002) 1052–1063;
R.K. Haynes, H.-W. Chan, M.-K. Cheung, W.-L. Lam, M.-K. Soo, H.-W. Tsang, A.
Voerste, I.D. Williams, Eur. J. Org. Chem. (2003) 2098–2114.
[16] Few reviews on clinical use, chemistry, pharmacology, and mode of action S.R.
Meshnick, Int. J. Parasitol. 32 (2002) 1655–1660;
T. Gordi, E.-I. Lepist, Toxicol. Lett. 147 (2004) 99–107;
G. PrayGod, A. de Frey, M. Eisenhut, Malaria J. 7 (2008) 210;
D. Chaturvedi, A. Goswani, P.P. Sakia, N.C. Barua, P.G. Rao, Chem. Soc. Rev. 39
(2010) 435–454.
[17] M. Enserink, Science 328 (2010) 846.
[18] J.L. Vennerstrom, S. Arbe-Barnes, R. Brun, S.A. Charman, F.C. Chiu, J. Chollet, Y.
Dong, A. Dorn, D. Hunziker, H. Matile, K. McIntosh, M. Padmanilayam, T.J.
Santo, C. Scheurer, B. Scorneaux, Y. Tang, H. Urwyler, S. Wittlin, W.N. Charman,
Nature 430 (2004) 900–904.
strains. The 10a-n-propyl 11 and 10a-benzyl 18 esters were the
most potent of all synthesized derivatives with activity comparable
to that of AS against 3D7 but more importantly by their significant
potency (22- and 12-fold, respectively) over AS against the resis-
tant strain K1. Short biological half-life is a feature common to ozo-
nides. Thus, these two esters stand as good candidates to undergo
pharmacokinetic study to ascertain whether they possess half-lives
longer than those of the current clinically available artemisinin
derivatives.
[19] P. Olliaro, T.N.C. Wells, Clin. Pharmacol. Ther. 85 (2009) 584–595.
[20] N. Valecha, S. Krudsood, N. Tangpukdee, S. Mohanty, S.K. Sharma, P.K. Tyagi, A.
Anvikar, R. Mohanty, B.S. Rao, A.C. Jha, B. Shahi, J.P. Singh, A. Roy, P. Kaur, M.
Kothari, S. Mehta, A. Gautam, J.K. Paliwal, S. Arora, N. Saha, Clin. Infect. Dis.
(Epub 2012 June 18). doi:10.1093/cid/cis475.
[21] S.A. Charman, S. Arbe-Barnes, I.C. Bathurst, R. Brund, M. Campbell, W.N.
Charman, F.C.K. Chiu, J. Chollet, J.C. Craft, D.J. Creek, Y. Dong, H. Matile, M.
Maurer, J. Morizzi, T. Nguyen, P. Papastogiannidis, C. Scheurer, D.M.
Shackleford, K. Sriraghavan, L. Stingelin, Y. Tang, H. Urwyler, X. Wang, K.L.
White, S. Wittlin, L. Zhou, J.L. Vennerstrom, PNAS Early Ed. (2011) 1–6.
[22] T.T. Cloete, J.W. Breytenbach, C. de Kock, P.J. Smith, J.C. Breytenbach, D.D. N’Da,
Bioorg. Med. Chem. 20 (2012) 4701–4709.
Acknowledgments
[23] M. Steyn, D.D. N’Da, J.C. Breytenbach, P.J. Smith, S. Meredith, W.J. Breytenbach,
J. Pharm. Pharmacol. 63 (2011) 278–286.
[24] W. Trager, J.B. Jensen, Science 193 (1976) 673–675.
[25] M. Smilkstein, N. Sriwilaijaroen, J.X. Kelly, P. Wilairat, M. Riscoe, Antimicrob.
Agents Chemother. 48 (2004) 1803–1806.
The authors thank the National Research Foundation (NRF) and
the North-West University (NWU) for financial support.
[26] R Development Core Team, R. A language and environment for statistical
computing, R Foundation for Statistical Computing, Vienna, Austria, 2011,
ISBN 3-900051-07-0. <http://www.R-project.org>.
References
[27] C. Ritz, J.C. Streibig, Software 12 (2005).
[28] B. Venugopalan, P.J. Karnik, C.P. Bapat, D.K. Chatterjee, N. Iyer, D. Lepcha, Eur. J.
Med. Chem. 30 (1995) 697–706.
[29] A.J. Lin, A.B. Zikry, D.E. Kyle, J. Med. Chem. 40 (1997) 1396–1400.
[30] A.J. Lin, M. Lee, D.L. Klayman, J. Med. Chem. 32 (1989) 1249–1252.
[31] G. Caron, F. Raymond, P.-A. Carrupt, H.H. Girault, B. Testa, Pharm. Sci. Technol.
Today 2 (1999) 327–335.
[32] C.A. Lipinski, F. Lombardo, B.W. Dominy, P.J. Feeney, Adv. Drug Deliv. Rev. 46
(2001) 3–26.
[1] L. Liu, H.L. Johnson, S. Cousens, J. Perin, S. Scott, J.E. Lawn, I. Rudan, H. Campbell,
R. Cibulskis, M. Li, C. Mathers, R.E. Black, Lancet 379 (2012) 2151–2161. Epub
2012 May 11.
[2] N.J. White, Science 320 (2008) 330–334.
[3] A.M. Dondorp, F. Nosten, P. Yi, D. Das, A.P. Phyo, J. Tarning, K.M. Lwin, F. Ariey,
W. Hanpithakpong, S.J. Lee, P. Ringwald, K. Silamut, M. Imwong, K. Chotivanich,
P. Lim, T. Herdman, S.S. An, S. Yeung, P. Singhasivanon, N.P.J. Day, N.
Lindegardh, D. Socheat, N.J. White, N. Engl. J. Med. 361 (2009) 455–467.
[4] H. Noedl, Y. Se, K. Schaecher, B.L. Smith, D. Socheat, M.M. Fukuda, N. Engl. J.
Med. 359 (2008) 2619–2620.
[5] H. Noedl, Y. Se, S. Sriwichai, K. Schaecher, P. Teja-Isavadharm, S. Bryan, R.
Wiriya, B. Delia, S. Sittidech, M.M. Fukuda, D. Socheat, L.C. Thap, Clin. Infect.
Dis. 51 (2010) e82–89.
[33] F. Bigucci, T. Kamsu-Kom, C. Cholet, M. Besnard, D. Bonnet-Delpon, G. Ponchel,
J. Pharm. Pharmacol. 60 (2008) 163–169.