Antiprotozoal, anticancer and antimicrobial activities of dihydroartemisinin acetal dimers and monomers

Article abstract of DOI:10.1016/j.bmc.2009.10.019

Nine dihydroartemisinin acetal dimers (6-14) with diversely functionalized linker units were synthesized and tested for in vitro antiprotozoal, anticancer and antimicrobial activity. Compounds 6, 7 and 11 [IC₅₀: 3.0-6.7 nM (D6) and 4.2-5.9 nM (W2)] were appreciably more active than artemisinin (1) [IC₅₀: 32.9 nM (D6) and 42.5 nM (W2)] against the chloroquine-sensitive (D6) and chloroquine-resistant (W2) strains of the malaria parasite, Plasmodium falciparum. Compounds 10, 13 and 14 displayed enhanced anticancer activity in a number of cell lines compared to the control drug, doxorubicin. The antifungal activity of 7 and 12 against Cryptococcus neoformans (IC₅₀: 0.16 and 0.55 μM, respectively) was also higher compared to the control drug, amphotericin B. The antileishmanial and antibacterial activities were marginal. A number of dihydroartemisinin acetal monomers (15-17) and a trimer (18) were isolated as byproducts from the dimer synthesis and were also tested for biological activity.

Full text of DOI:10.1016/j.bmc.2009.10.019

Bioorganic & Medicinal Chemistry 17 (2009) 7949–7957
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Antiprotozoal, anticancer and antimicrobial activities of dihydroartemisinin
acetal dimers and monomers
a
Desmond Slade a, , Ahmed M. Galal , Waseem Gul a,b, Mohamed M. Radwan a, Safwat A. Ahmed a,c
,
*
Shabana I. Khan a, Babu L. Tekwani a, Melissa R. Jacob a, Samir A. Ross a,d, Mahmoud A. ElSohly a,b,e,
*
a National Center for Natural Products Research, School of Pharmacy, University of Mississippi, University, MS 38677, USA
b ElSohly Laboratories, Inc., 5 Industrial Park Drive, Oxford, MS 38655, USA
c Department of Pharmacognosy, Faculty of Pharmacy, Suez Canal University, Ismailia 41522, Egypt
d Department of Pharmacognosy, School of Pharmacy, University of Mississippi, University, MS 38677, USA
e Department of Pharmaceutics, School of Pharmacy, University of Mississippi, University, MS 38677, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Nine dihydroartemisinin acetal dimers (6–14) with diversely functionalized linker units were synthe-
sized and tested for in vitro antiprotozoal, anticancer and antimicrobial activity. Compounds 6, 7 and
11 [IC50: 3.0–6.7 nM (D6) and 4.2–5.9 nM (W2)] were appreciably more active than artemisinin (1)
[IC50: 32.9 nM (D6) and 42.5 nM (W2)] against the chloroquine-sensitive (D6) and chloroquine-resistant
(W2) strains of the malaria parasite, Plasmodium falciparum. Compounds 10, 13 and 14 displayed
enhanced anticancer activity in a number of cell lines compared to the control drug, doxorubicin. The
Received 17 July 2009
Revised 7 October 2009
Accepted 9 October 2009
Available online 30 October 2009
Keywords:
Artemisia annua
Artemisinin
Dihydroartemisinin
Acetal monomers, dimers and trimers
Antimalarial activity
Antileishmanial activity
Anticancer activity
Antifungal activity
Antibacterial activity
antifungal activity of 7 and 12 against Cryptococcus neoformans (IC50: 0.16 and 0.55 lM, respectively)
was also higher compared to the control drug, amphotericin B. The antileishmanial and antibacterial
activities were marginal. A number of dihydroartemisinin acetal monomers (15–17) and a trimer (18)
were isolated as byproducts from the dimer synthesis and were also tested for biological activity.
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
vaquone and pyrimethamine. In areas where malaria is endemic,
control is limited by drug resistance, cytotoxicity, cost and avail-
Malaria, a devastating infectious disease caused by highly
adaptable protozoan parasites of the genus Plasmodium, has im-
pacted on humans for more than 4000 years, causing illness and
an estimated 1.5–2.5 million deaths each year.1 Malaria is endemic
throughout the tropics, especially in sub-Saharan Africa and the
developing world, threatening about 40% of the world’s population.
Although four Plasmodium parasite species can infect humans, Plas-
modium falciparum causes the majority of illnesses and deaths. Se-
vere malaria, defined as acute malaria with major signs of organ
dysfunction or high levels of parasitemia, predominantly affects
children and pregnant women.2
ability of new drugs. The persistence, and in some cases, resur-
gence, of malaria, especially in the developing world, is almost
exclusively due to multiple drug resistance caused by mono-
therapy drug treatment with traditional antimalarials.4 Chloro-
quine resistance has become unequivocal in P. falciparum regions.
The WHO recommends the use of combination therapies for the
treatment of uncomplicated P. falciparum malaria to delay or pre-
vent drug resistance.
The emergence of artemisinin (1), derived from the Chinese
herb Qing Hao (Artemisia annua L., family: Asteraceae), as a fast-
acting effective antimalarial agent resulted in the development of
numerous derivatives as well as artemisinin-based combination
therapies (ACTs) to prevent parasite resistance.5,6 The advantages
of artemisinins over conventional antimalarial drugs include kill-
ing parasites more rapidly, toxicity at nanomolar concentrations,
short fever clearance time (32 h vs 2–3 days for conventional anti-
malarials), activity against the sexual and asexual parasite stages,
rapid action against all erythrocytic stages of parasite resulting in
decreased transmission, limited resistance, safer and simpler oral
Chemotherapy is still at the forefront in the fight against malar-
ia due to the unavailability of effective vaccines.3 Numerous drugs
have been developed for the treatment of uncomplicated malaria,
for example, mefloquine, primaquine, quinidine, proguanil, ato-
* Corresponding authors. Tel.: +1 662 915 1708; fax: +1 662 915 5587 (D.S.); tel.:
+1 662 915 5928; fax: +1 662 915 5587 (M.A.E.).
E-mail addresses: dslade@olemiss.edu (D. Slade), melsohly@olemiss.edu (M.A.
ElSohly).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.10.019

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D. Slade et al. / Bioorg. Med. Chem. 17 (2009) 7949–7957
Scheme 1. Synthesis of 6.
and parenteral administration, no significant side effects and
efficacy for uncomplicated and severe malaria. Artemisinins
have, however, short half-lives (1–4 h), resulting in recrudescence
of infecting parasites and recurrent illness within days to
weeks when used as a monotherapy over a standard three day
course.5–7 Artemisinin monotherapy was therefore banned by the
WHO in 2006, while ACT was adopted as the suggested first-line
treatment for uncomplicated P. falciparum malaria in all endemic
areas. Intravenous artesunate (dihydroartemisinin hemisuccinate)
became the treatment of choice for severe malaria, except for
children in Africa.8 ACT in conjunction with a long acting agent is
a highly efficacious first-line therapy used in most countries
where malaria is endemic. Disadvantages of ACTs are higher
4), antifungal and antibacterial activities of the artemisinins are
also presented.
2. Results and discussion
2.1. Chemistry
Artemisinin (1) was obtained commercially.13 Dihydroartemisi-
nin (DHA) (2) was prepared by sodium borohydride (NaBH4) reduc-
tion of 1 (Scheme 1).14 Anhydrodihydroartemisinin (3)15 was
isolated during the purification of 4.12b Dimers 4–5 were prepared
and purified as previously described.12b,16 Dimers 6–10 (Fig. 1)
were synthesized by reacting DHA (2) with 3,5-dihydroxybenzyl
alcohol, 3-aminophenol, cis-2-butene-1,4-diol, (S)-(ꢀ)-1-butene-
3,4-diol and (S)-(ꢀ)-1,2,4-butanetriol, respectively, under mild
acidic conditions using borontrifluoride etherate (BF3ꢁOEt2) as a cat-
alyst in dry ether (Scheme 1, Table 1). The reaction mixtures were
chromatographed to isolate 6–10, as well as monomers 15 (byprod-
uct from 412b), 16 (byproduct from 6), 17 (byproduct from 817) and
trimer 18 (byproduct from 10). The vicinal diols 11 and 12 were
prepared by osmium tetroxide syn-hydroxylation of 8 and 9,
respectively. Prilezhaev stereospecific cis-epoxidation of 8 with
m-chloroperoxybenzoic acid (m-CPBA) yielded 13, while methane-
sulfonyl chloride sulfonation of 512b yielded 14.
cost and
a
shorter shelf-life compared to traditional
antimalarial drugs.5,9 There is currently no ACT available in the
United States.2a
The mechanism(s) of action of artemisinins have not been fully
elucidated, with parasite specific and non-specific mechanisms
hypothesized. The former includes interference with sarcoplas-
mic/endoplasmic reticulum Ca2+-ATPases (SERCA) through homo-
lytic cleavage of the endoperoxide bond, forming free radicals,
and subsequent alkylation of heme and Plasmodium-specific pro-
teins. Non-specific mechanisms include iron mediated production
of reactive species via a reductive scission or an open peroxide
model.4a,10
Recent reports of artemisinin-resistant malaria warrants the
development of new artemisinin based drugs that can be used as
ACTs.11 Artemisinin dimers are known to possess not only signifi-
cant antimalarial activity, but also remarkable cytotoxicity against
tumor cells, with inhibitory activity in the nano- to micromolar
range.12 This manuscript reports the synthesis (Scheme 1, Table 1)
of nine dihydroartemisinin acetal dimers (6–14) with diversely
functionalized linker units (Fig. 1). A number of monomers (15–
17) and a trimer (18) were isolated as byproducts from the dimer
synthesis. The antimalarial, antileishmanial, anticancer (Tables 2–
2.2. Biological activity
2.2.1. Antimalarial activity
The in vitro antimalarial activity of 1–18 against chloroquine-
sensitive (D6) and chloroquine-resistant (W2) clones of P. falcipa-
rum was evaluated based on the determination of plasmodial
LDH activity.18 In order to determine the selectivity index (S.I.) of
antimalarial activity, cytotoxicity of 1–18 was also determined
against mammalian Vero cells (Table 2).19 S.I. is calculated as the
ratio of TC50 cytotoxicity and IC50 antimalarial activity values,
Table 1
Reaction details for synthesis of 6–10
Compound
DHA (2)
Linker
mmol
Ether (mL)
BF3ꢁOEt2
(
lL)
MSa (g)
Crudeb (mg)
Product
mg
mmol
equiv
mg
equiv
mg
28.0
%
6
7
8
9
10
360
510
402
480
540
1.27
1.79
1.41
1.69
1.90
2.11
18.82
2.22
3.10
2.08
84.0
10.4
56.2
48.0
97.0
0.60
0.10
0.64
0.54
0.91
1
1
1
1
1
120
75
75
50
400
700
220
410
450
1.7
—
—
—
—
330
410
335
416
575
6.9
44.0
88.0
135.0
55.0
71.9
22.2
39.9
9.4
60
a
MS: molecular sieve.
Crude: crude reaction mixture after workup.
b

D. Slade et al. / Bioorg. Med. Chem. 17 (2009) 7949–7957
7951
Figure 1. Artemisinin (1), dihydroartemisinin (DHA) (2), anhydrodihydroartemisinin (3), DHA acetal dimers 4–14, monomers 15–17 and trimer 18.
and measures the therapeutic index of the compound under inves-
tigation to malaria parasites in comparison to its toxicity to the
host cells (if there is any). An S.I. P10 is generally considered sig-
nificant, indicating that antimalarial activity is not due to cytotox-
icity.20 The synthesized analogs 6–17 exhibited strong in vitro
antimalarial activities, with IC50 values in the nanomolar range
(3.0–45.5 nM for D6 clone and 4.2–47.1 nM for W2 clone) (Table 2).
Dimers 6, 7, 8 and 11, and monomers 15 and 17 displayed excellent
activity against both clones (IC50 <10 nM), while trimer 18 dis-
played weak activity compared to the monomers and dimers.
The S.I. for all compounds was >10, indicating that there is at least
a 10-fold or higher difference in the effective dose for antimalarial
activity and cytotoxicity; however, no significant difference was
observed in the antimalarial efficacy towards D6 and W2 clones
(Table 2). The activities of monomers 15 and 17 were comparable
to their dimer analogs 4 and 8; however, monomer 16 was 5–6

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D. Slade et al. / Bioorg. Med. Chem. 17 (2009) 7949–7957
Table 2
latter being potentially lethal. Leishmaniasis is caused by various
Antimalarial and cytotoxic activity of 1–18
species of the protozoan parasite Leishmania, which is transmitted
by female sandflies.21 Artemisinin (1) has been shown to have
promising antileishmanial activity that is mediated by induction
of apoptosis and therefore warrants further study as a possible
therapeutic option for the treatment of leishmaniasis.12b,22 Com-
pounds 1–18 were tested against the protozoan parasite Leish-
mania donovani, using pentamidine and amphotericin B as drug
controls (Table 3).18 The in vitro antileishmanial activity of 6,
10–13, 16 and 18 was in the low micromolar range (IC50: 2.5–
Compound
Plasmodium falciparum
D6 clone W2 clone
Cytotoxicity
Vero
IC50 (nM)
S.I.
IC50 (nM)
S.I.
TC50 (nM)
1
2
3
4
5
6
7
8
32.9
14.8
15.8
5.3
11.2
3.0
>51
95
>113
49
42.5
14.1
25.5
5.8
>40
100
>70
44
>1686
1407
>1787
257
34
3.8
100
384
>2380
>1287
113
4.2
5.9
6.1
>1700
>1253
116
>7075
>7417
709
6.4
(2.5
l
l
M; IC90: 9.4–47.1
M) and IC90 (9.4
l
M), with 10 possessing excellent IC50
5.8
6.3
lM) values. Dimers 10 and 13, which
showed relatively weak antimalarial activity (Table 2), were the
most active compounds in the antileishmanial assay, with IC50 val-
ues comparable to the control drug, pentamidine.
9
24.2
43.8
6.7
11.7
45.5
11.8
7.9
20.9
6.2
298.3
32
25.8
45.4
5.2
14.0
47.1
11.8
7.0
20.7
7.6
243.1
30
773
10
11
12
13
14
15
16
17
18
>170
>1082
71
>164
542
>1700
>560
>2164
18
>164
>1400
59
>159
542
>1904
>567
>1763
22
>7452
>7270
825
>7475
6403
>13,355
>11,711
>13,430
5259
2.2.3. Anticancer activity
Artemisinins have shown promising anticancer properties in
various cell lines and animal models, displaying activity against a
variety of unrelated tumor cells lines, for example, colon, breast,
lung, leukemia and pancreatic cancer.23 Artemisinins cause de-
creased proliferation, apoptosis-induction, angiogenesis-inhibition
and increased levels of oxidative stress to cancer cells.23 The selec-
tivity of artemisinins towards cancer cells coupled with its non-
cytotoxicity towards normal cells makes it an ideal candidate for
targeted delivery via, for example, the transferrin receptor mecha-
nism, since artemisinins only become toxic after reacting with
iron.23 The in vitro cytotoxicity of 4–18 was determined against
four human solid tumor (SK-MEL, KB, BT-549 and SK-OV-3) and
two noncancerous mammalian cell lines (Vero and LLC-PK11) (Ta-
ble 4).18,19 The anti-cell proliferative activity of 8–10, 13–14 and
18 was significantly enhanced compared to the control drug, doxo-
rubicin, with 14 showing selectivity towards epidermal carcinoma
(KB). The observation that 10, 13 and 18 exhibited relatively weak
antimalarial activity, strong antileishmanial activity and higher
toxicity to all six cell lines indicates that the observed antileishma-
nial activity for these compounds could be related to their cyto-
toxic effect, while potential antimalarial activity of the other
analogs is more specific.
Chloroquinea
41.6
>357.9
378.3
>39.3
>14,881
IC50: concentration causing 50% growth inhibition. TC50: concentration toxic to 50%
of the cells. Vero: African monkey kidney fibroblast. S.I.: selectivity index [TC50
(Vero)/IC50 (P. falciparum)].
a
Control.
times less active than the corresponding dimer 6. Also, dimer 8 was
ca. 4 times more active than its structural isomer 9. Dihydroxyla-
tion of 8 and 9 yielded 11 and 12, respectively, with 8 and 11 hav-
ing similar activity, while 12 was twice as active as 9. Dimer 5 was
appreciably more active than its methylene homologue, dimer 10;
however, the hydroxy-analog of 10, namely 11, had activity com-
parable to 5. These results, taken together with previously pub-
lished data,12b indicate that increasing the chain length between
two DHA units decreases antimalarial activity, while hydroxylation
of the linker-chain increases the activity.
2.2.2. Antileishmanial activity
The three major forms of human leishmaniasis are normally dif-
ferentiated as cutaneous, mucocutaneous and visceral, with the
The antitumor activity of 6 was evaluated against the National
Cancer Institute’s 60 cultured human tumor cell lines (leukemia,
non-small cell lung cancer, colon cancer, central nervous system
cancer, melanoma, ovarian cancer, renal cancer, prostate cancer
and breast cancer).24 The cytostatic activity of 6 was evaluated at
Table 3
Antileishmanial activity of 1–18
five concentrations (100, 10, 1.0, 0.1 and 0.01 lM) via the sulforho-
Compound
IC50
(
lM)
IC90 (lM)
damine B (SRB) protein assay25 to estimate cell growth. Anticancer
activity, inferred from dose–response curves, is expressed through
GI50 (drug concentration resulting in a 50% reduction in the net
protein increase), TGI (drug concentration of total growth inhibi-
tion) and LC50 (concentration of drug resulting in a 50% reduction
in the measured protein at the end of the drug treatment as com-
pared to that at the beginning) values for each cell line. The mean
value for all tested cancer cell lines [MG_MID (mean graph mid-
point)] was also calculated. Dimer 6 displayed MG_MID GI50, TGI
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
40.7
87.9
30.0
7.4
12.8
5.9
24.9
NA
20.9
2.5
6.0
6.4
3.9
22.8
25.8
5.7
42.3
5.0
NA
NA
NA
38.5
47.9
32.7
62.3
NA
62.8
9.4
22.9
29.7
47.1
55.5
and LC50 values of 24.5 nM, 2.82 lM and 15.1 lM, respectively.
2.2.4. Antifungal activity
112.2
24.6
NA
The in vitro antifungal activity of 1–18 indicated selective activ-
ity against Cryptococcus neoformans, with 7, 12 and 17 displaying
enhanced activity compared to the control drug, amphotericin B,
confirming previous reports (see Supplementary data).26
44.2
Pentamidinea
2.8
0.2
6.4
0.4
Amphotericin Ba
2.2.5. Antibacterial activity
The in vitro antibacterial assay of 1–18 showed almost com-
plete inactivity to a panel of bacteria (see Supplementary data). A
IC50: concentration causing 50% growth inhibition. IC90: concentration causing 90%
growth inhibition. NA: not active.
a
Control.

D. Slade et al. / Bioorg. Med. Chem. 17 (2009) 7949–7957
7953
Table 4
Anticancer and cytotoxic activity of 4–18
Compound
Cancer cell lines
Noncancerous cell lines
SK-MEL
KB
BT-549
SK-OV-3
Vero
LLC-PK11
IC50
(l
M)
TC50 (lM)
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
0.56
8.00
NA
NA
NA
12.93
0.24
NA
NA
0.22
NA
0.24
0.26
NA
0.24
0.48
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
>8.03
5.60
NA
0.10
0.15
NA
NA
NC
>16.11
0.10
NA
NA
0.68
NC
NA
NA
NA
0.32
0.27
0.16
NA
1.53
2.58
0.08
NA
NA
0.30
NA
NA
NA
NA
0.06
0.18
0.43
>1.57
NA
NA
NA
0.47
0.28
NA
NA
NA
>1.57
2.13
NA
NA
NA
NA
NA
NA
0.24
20.76
2.46
NA
0.11
>1.10
0.07
Doxorubicina
Paclitaxela
1.73
4.10
2.04
0.02
1.96
0.02
2.04
0.53
NC
0.53
1.29
4.39
IC50: concentration causing 50% growth inhibition. TC50: concentration toxic to 50% of the cells. SK-MEL: human malignant melanoma. KB: human epidermal carcinoma. BT-
549: human breast carcinoma (ductal). SK-OV-3: human ovary carcinoma. Vero: African monkey kidney fibroblast. LLC-PK11: pig kidney epithelial. NA: not active. NC: not
cytotoxic.
a
Control.
cursory electronic literature search did not reveal any reports of
significant antibacterial activity for artemisinins.
The following abbreviations are used for NMR multiplicities: sin-
glet (s), broad singlet (br s), doublet (d), broad doublet (br d), dou-
blet of doublet (dd), triplet (t), broad triplet (br t) and multiplet
(m). Chemical shifts (d) are reported in ppm and coupling con-
stants (J) in hertz. HRESIFTMS were obtained on an Agilent Series
1100 SL mass spectrometer.
3. Conclusion
The remarkable chemistry and biological activity of artemisinin
(1) and its derivatives, especially towards malaria and cancerous
tumors, have been demonstrated in numerous reports, including
the current communication. However, the disconcerting accounts
of parasite resistance to artemisinins indicate that this valuable
natural product might follow the same fate as numerous other
drugs, that is, worldwide clinical resistance. Careful management
of the current artemisinins and newly developed derivatives, such
as the dimers reported herein, by health officials is critical in
ensuring the longevity of these drugs in the global fight against dis-
ease. The excellent in vitro antimalarial and anticancer activity of
the selected analogs presented in this study warrants further study
as potential drug leads through in vivo assays and pharmacokinetic
and pharmacodynamic analysis.
4.2. Synthesis
See Supplementary data for IUPAC nomenclature of 1–18.
4.2.1. Artemisinin (1), DHA (2), anhydrodihydroartemisinin (3)
and dimers 4–5
Artemisinin (1) was obtained commercially.13 DHA (2) was pre-
pared by NaBH4 reduction of 1 (Scheme 1).14 Anhydrodihydroarte-
misinin (3) was isolated during the purification of 4.15 Dimers 4–5
were prepared and purified as previously described.12b,16
4.2.2. Dimers 6–10
To a solution of DHA (2) (360 mg, 1.27 mmol, 2.11 equiv) in dry
ether (120 mL) and molecular sieves (1.7 g) was added 3,5-
dihydroxybenzyl alcohol (84 mg, 0.60 mmol, 1 equiv) and
BF3ꢁOEt2 (400
4. Experimental
l
L) (Scheme 1, Table 1).12b The reaction mixture
4.1. General experimental conditions
was stirred (2 h, rt, Ar) and quenched with 2% aqueous NaHCO3,
followed by addition and extraction with ether. The combined or-
ganic layer was washed with water, dried over anhydrous Na2SO4
and concentrated under reduced pressure to yield a crude product
(330 mg) which was purified by column chromatography (hex-
anes/EtOAc, 1:0–1:1, stepwise) to yield 6 (28 mg, 6.9% yield).
Dimers 7–10 were similarly prepared as described for 6 using
DHA (2) and 3-aminophenol, cis-2-butene-1,4-diol, (S)-(ꢀ)-1-bu-
tene-3,4-diol and (S)-(ꢀ)-1,2,4-butanetriol, respectively, as linkers
(Table 1). Molecular sieves were, however, not utilized in these
reactions.
All reactions were carried out in oven dried glassware. Diethyl
ether (anhydrous) was dried over molecular sieves prior to use.
All chemicals were purchased from Sigma–Aldrich or Acros Organ-
ics and used without further purification. Flash column chromato-
graphy was conducted with silica gel (particle size 230–400 mesh;
SiliCycle). Analytical thin-layer chromatography (TLC) was per-
formed with Silica Gel 60 F254 plates (250 lm thickness; SiliCycle)
using hexanes/EtOAc mixtures as solvent systems. Visualization
was accomplished by spraying with p-anisaldehyde spray reagent
followed by heating with a hot-air gun. IR spectra were obtained
using an AATI Mattson Genesis Series FTIR. Optical rotations were
recorded at ambient temperature using a Rudolph Research Ana-
lytical Autopol IV automatic polarimeter. 1D and 2D NMR spectra
were obtained on Varian AS400 and Bruker DRX 400 spectrometers
at 400 MHz (1H) and 100 MHz (13C) using the solvent peak as inter-
nal standard. The spectra were recorded in CDCl3 and pyridine-d5.
4.2.3. Dimers 11–12
Dimer 8 (30 mg, 48.3
lmol, 1 equiv) was dissolved in dry pyri-
dine (1 mL), osmium tetroxide (47 mg, 184.9
l
mol, 3.83 equiv)
added and the mixture stirred (105 min, rt). The reaction was sub-
sequently quenched by addition of 10% aqueous Na2S2O5 and

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D. Slade et al. / Bioorg. Med. Chem. 17 (2009) 7949–7957
stirring (30 min), followed by addition and extraction with CH2Cl2.
The organic layer was sequentially washed with 4% HCl and 2%
NaHCO3, dried over anhydrous Na2SO4 and concentrated under
high vacuum to yield 11 (30 mg, 94.8% yield). Dimer 9 (42 mg,
ture was quenched by the addition of water, followed by addition
and extraction with CH2Cl2. The organic layer was sequentially
washed with 4% HCl and distilled water, dried over anhydrous
Na2SO4 and concentrated under high vacuum to yield 14 (21 mg,
77.8% yield).
67.7
lmol, 1 equiv) was similarly oxidized (osmium tetroxide,
60 mg, 236.0
l
mol, 3.49 equiv), yielding 12 (44 mg, 99.3% yield).
4.2.6. Monomers 15–17 and trimer 18
4.2.4. Dimer 13
Dimer 8 (22 mg, 35.4
(2 mL), was epoxidized with m-CPBA (15 mg, 86.9
Monomers 15 (80 mg), 16 (99 mg) and 17 (46 mg), and trimer
18 (155 mg) were isolated during the purification of 4,12b,16 6, 8
and 10, respectively
l
mol, 1 equiv), dissolved in dry CH2Cl2
mol,
l
2.45 equiv) (500 min, rt). The reaction mixture was quenched with
2% aqueous NaHSO3, followed by addition and extraction with
CH2Cl2. The crude product was purified via preparative TLC (hex-
anes/EtOAc, 75:25) to afford 13 (15 mg, 66.5% yield).
4.3. Spectroscopic and physical data for 6–18
NMR spectroscopic data are given in Table 5 (1H) and Table 6
(
13C), and HRESIFTMS data are given in Table 7.
4.2.5. Dimer 14
Dimer 5 (24 mg, 38.4
lmol, 1 equiv), dissolved in dry pyridine
4.3.1. Dimer 6
(2 mL), was sulfonated with methanesulfonyl chloride (31 mg,
White solid; Rf 0.28 (hexanes/EtOAc, 1:1); [
a
]
D
+181.5 (c 0.13,
270.6
l
mol, 7.04 equiv) (180 min, 0–6 °C, N2). The reaction mix-
MeOH); IR (neat) m .
max: 3393 (OH) cmꢀ1
Table 5
1H NMR data for 5 and 6–18 (dH in ppm, J values in Hz)
Position
5/50
5a
6a
7a
8a
9a
10a
11a
12a
5.35 (s)
5.45 (s)
5.40 (s)
5.49 (s)
5.40 (s)
5.36 (s)
5.41 (s)
2.58 (m)
5.37 (s)
5.43 (s)
2.60 (m)
5.8 (s)
5.9 (s)
2.65 (m)
5.44 (s)
5.43 (s)
2.66 (m)
11/110
12/120
13/130
2.61 (m)
2.64 (m)
2.76 (m)
4.87 (d, 3.6)
5.42 (d, 3.2)
0.97 (d, 7.6)
0.99 (d, 7.2)
0.93 (d, 6.0)
1.41 (s)
2.78 (m)
2.54 (br s)
4.773 (d, 3.8)
4.766 (d, 3.8)
0.88 (d,7.4)
5.45 (d, 4.0)
6.48 (d, 1.2)
0.99 (d, 7.2)
4.80 (d, 3.2)
0.90 (d, 7.6)
4.74 (d, 2.8)
4.84 (d, 3.6)
0.86 (d, 7.2)
0.83 (d, 7.2)
0.92 (d, 6.4)
1.39 (s)
4.78 (d, 3.2)
4.76 (d, 3.2)
0.88 (d, 7.2)
4.80 (d, 3.6)
4.82 (d, 3.6)
0.89 (d, 7.2)
4.85 (d, 3.2)
4.94 (d, 3.2)
0.93 (d, 7.2)
14/140
15/150
0.91 (d, 6.3)
1.38 (s)
0.95 (d, 6.0)
1.43 (s)
0.94 (d, 6.4)
1.43 (s)
0.91 (d, 6.0)
1.38 (s)
0.93 (d, 6.8)
1.40 (s)
0.95 (d, 6.4)
1.40 (s)
1.44 (s)
1.43 (s)
16
3.47 (dd, 10.3, 4.3)
3.84 (m)
3.84 (m)
3.40 (dd, 9.8, 5.4)
3.84 (m)
4.40 (d, 12.4)
4.74 (d, 12.4)
—
6.45 (s)
6.56 (s)
—
4.01 (d, 10.8)
4.26 (d, 10.8)
5.59 (br s)
3.43 (m)
3.89 (m)
4.33 (m)
5.61 (m)
3.48 (m)
3.68 (m)
3.88
4.01 (dd, 10.8, 3.2)
3.91 (m)
3.73 (m)
3.80 (m)
17
18
6.48 (d, 1.2)
—
3.80 (m)
3.80 (m)
5.59 (br s)
1.72 (m)
3.73 (m)
19
—
—
6.32 (d, 7.6)
4.01 (d, 10.8)
5.23 (m)
3.52 (m)
3.66 (dd, 11.2, 3.6)
4.14 (br d, 8.8)
4.26 (d, 10.8)
3.93 (m)
3.56 (m)
3.66 (m)
20
21
22
—
—
—
6.57 (s)
—
6.45 (s)
6.56 (s)
7.03 (d, 8.0)
6.50 (br d)
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
Position
5/50
5a
13a
14b
15a
16b
17a
18a
5.35 (s)
5.39 (s)
5.65 (s)
5.72 (s)
2.78 (br s)
4.92 (d, 2.8)
4.98 (d, 3.2)
5.42 (s)
5.69 (s)
5.35 (s)
5.33 (s)
5.40 (s)
11/110
12/120
2.61 (m)
4.773 (d, 3.8)
4.766 (d, 3.8)
2.62 (br s)
4.77 (d, 3.2)
4.84 (d, 3.2)
2.70 (m)
4.83 (d, 3.6)
2.81 (br m)
5.06 (br s)
2.54 (br s)
4.80 (d, 3.6)
2.62 (m)
4.72 (d, 3.2)
4.79 (d, 2.8)
4.81 (d, 3.2)
0.86 (d, 7.2)
0.90 (d, 7.6)
0.925 (d, 6.4)
0.932 (d, 6.0)
1.36 (s)
13/130
14/140
15/150
0.88 (d, 7.4)
0.91 (d, 6.3)
1.38 (s)
0.91 (d, 7.2)
0.92 (d, 6.4)
1.40 (s)
0.90 (d, 7.2)
0.85 (d, 6.8)
1.48 (s)
0.94 (d, 6.0)
0.99 (d, 6.4)
1.43 (s)
0.87 (d, 7.2)
0.80 (d, 6.0)
1.51 (s)
0.85 (d, 7.6)
0.89 (d, 6.4)
1.37 (s)
1.39 (s)
1.40 (s)
16
3.47 (dd, 10.3, 4.3)
3.84 (m)
3.84 (m)
3.40 (dd, 9.8, 5.4)
3.84 (m)
—
3.96 (dd, 3.2, 2.8)
3.57 (m)
3.19 (m)
3.76 (dd, 5.6, 10.8)
4.37 (dd, 3.6, 10.8)
5.30 (m)
3.80 (dd, 4.4, 11.2)
4.37 (dd, 3.6, 10.8)
—
4.23 (d, 17.6)
4.42 (s)
—
4.53 (d, 17.2)
4.42 (s)
—
4.62 (d, 12.0)
5.04 (d, 13.6)
—
5.59 (m)
5.75 (m)
5.59 (m)
5.75 (m)
3.71 (dd, 3.6, 6.8)
3.86 (m)
3.86 (m)
1.73 (m)
1.86 (m)
3.44 (m)
3.86 (m)
—
17
18
3.19 (m)
6.90 (s)
19
3.83 (m)
3.57 (m)
—
—
—
—
5.59 (m)
5.75 (m)
—
—
—
20
21
22
—
—
—
—
—
—
—
—
—
6.96 (s)
—
6.90 (s)
—
—
a
CDCl3.
Pyridine-d5.
b

D. Slade et al. / Bioorg. Med. Chem. 17 (2009) 7949–7957
7955
Table 6
13C NMR data for 5 and 6–18 (dC in ppm)
Position
5a
6a
7a
8a
9a
10a
11a
12a
1/10
2/20
3/30
4/40
5/50
6/60
7/70
8/80
9/90
10/100
11/110
12/120
13/130
14/140
15/150
16
52.9
25.0
36.9
104.5
88.3
81.4
44.8
25.0
35.0
37.8
31.3
103.2/103.1
13.5
20.8
26.5
70.2
70.1
70.3
—
52.6
52.8
24.7/24.9
36.6
104.4
88.5
81.3
44.7
24.7/24.9
34.9
37.6
31.3
52.7
24.6/24.8
36.6
104.2
88.1
81.3
44.6
24.6/24.8
34.8
37.5
31.0
101.7
13.2
20.6
26.3
64.1
129.2
129.2
64.1
—
52.8
52.7/52.8
24.69/24.85/24.87
36.59/36.63
104.27/104.32
88.07/88.12
81.1/81.2
44.5/44.6
24.69/24.85/24.87
33.9/34.2
37.66/37.67
31.1
102.4/102.6
13.19/13.21
20.50/20.53
26.29/26.34
73.3
52.7
24.8
36.6
104.4
88.1/88.2
81.1/81.2
44.4/44.5
24.8
34.8
37.7
31.1
102.8/103.0
13.3
20.6
26.3
70.6
71.4
52.65/52.71
24.8/24.9
36.6
104.3/104.5
88.3/88.2
81.2
44.5
24.8/24.9
34.8
37.6/37.7
31.0/31.4
103.2/103.3
13.2/13.5
20.5/20.6
26.4
63.7
78.5
71.6
69.5
—
—
—
24.4/24.5/24.67/24.71
36.4/36.5
104.2/104.3
88.1/88.3
24.5/24.7/24.9
36.62/36.64
104.3
88.27/88.30
81.3/81.4
44.59/44.64
24.5/24.7/24.9
34.9
37.56/37.64
30.9/31.2
98.9/103.0
13.2/13.3
20.58/20.62
26.4
71.8
76.4
135.9
119.3
—
—
—
81.0/81.2
44.42/44.44
24.4/24.5/24.67/24.71
34.66/34.69
37.41/37.46
31.0
100.6/101.4
13.0/13.2
20.36/20.39
26.0/26.2
100.6/103.8
13.2
20.6
26.3
69.4
141.2
158.9
103.8
148.0
109.2
130.0
107.2
—
17
18
19
20
21
22
68.7
33.9
65.7
—
—
—
108.0/108.2
156.9/158.5
103.3
156.9/158.5
108.0/108.2
71.4
70.2
—
—
—
—
—
—
—
—
Position
5a
13a
14b
15a
16b
17a
18a
1/10
2/20
3/30
4/40
5/50
6/60
7/70
8/80
9/90
10/100
11/110
12/120
13/130
14/140
15/150
16
52.9
25.0
36.9
104.5
88.3
81.4
44.8
25.0
35.0
37.8
31.3
52.7/52.8
24.7/24.9
36.6
104.3/104.4
88.1/88.2
81.3
44.55/44.56
24.7/24.9
34.8
37.6/37.7
31.0/31.1
102.4/103.0
13.2
20.5
26.3
67.1
54.4
55.1
66.5
—
—
53.00/53.03
25.1/25.3
37.1
52.4
24.4/24.6
53.0
25.11/25.38
37.1
104.5
88.5
81.5
45.1
25.11/25.38
35.0
37.6
31.7
101.7
13.6
20.7
26.5
52.6
24.6/24.8
36.5
104.5
88.1
81.3
44.5
24.6/24.8
34.7
37.6
31.0
52.8
24.6/24.7/24.8/24.90/24.91
36.7
104.17/104.27/104.30
88.11/88.15/88.17
81.2/81.3
44.64/44.69/44.74
24.6/24.7/24.8/24.87/24.91
34.8/34.9
37.65/37.69
31.1/31.2/31.6
102.3/102.6/103.1
13.2/13.4/13.7
36.3
104.3
88.1
80.9
44.1
24.4/24.6
34.5
37.5
30.7
102.7
13.0
20.3
26.0
71.5
207.8
66.7
—
104.0
88.5/88.6
80.2/81.4
44.9/45.0
25.1/25.3
35.1
37.5/37.7
31.56/31.64
102.5/103.2
13.39/13.45
20.77/20.81
26.3/26.5
67.3/67.8
80.2
103.2/103.1
101.6
13.14
20.54
26.2
13.5
20.8
26.5
70.2
70.1
70.3
—
—
—
—
20.49/20.53
26.4
71.7
75.6
33.0
64.6
—
70.4
58.3
17
18
19
20
21
22
142.0
106.6
160.8
103.4
160.8
106.6
132.7
128.0
63.6
—
—
67.3/67.8
—
—
—
—
—
—
—
—
—
—
—
a
CDCl3.
Pyridine-d5.
b
Table 7
HRESIFTMS (direct injection, positive mode) data for 6–18
Compound
Molecular formula
[M+Na]+
[M+K]+
Exact
Exp
Error
Exact
Exp
Error
6
7
8
C37H52O11
C36H51NO9
C34H52O10
C34H52O10
C34H54O11
695.3408
664.3462
643.3458
643.3458
661.3564
677.3513
677.3513
659.3408
725.3183
355.1757
429.1890
377.1940
927.5082
695.3284
664.3330
643.3391
643.3369
661.3624
677.3413
677.3416
659.3630
725.3140
355.1762
429.1888
377.1913
927.5106
17.8
19.9
10.4
13.8
9.1
14.8
14.3
33.7
5.9
1.5
0.5
7.2
2.6
—
—
—
680.3201
659.3197
659.3197
—
693.3252
693.3252
—
741.2922
—
445.1629
393.1679
943.4821
680.3117
659.3090
659.3101
—
693.3115
693.3172
—
741.2840
—
445.1613
393.1680
943.5021
12.3
16.2
14.6
—
19.8
11.5
—
11.1
—
3.6
0.3
9
10
11
12
13
14
15a
16
17
18
C
34H54O12
C34H54O12
C34H52O11
C34H54O13
C18H28O7
C22H30O7
C19H30O6
S
C
49H76O15
21.2
Exp: experimental. Error: error in ppm.
a
[MꢀH]ꢀ data.

7956
D. Slade et al. / Bioorg. Med. Chem. 17 (2009) 7949–7957
4.3.2. Dimer 7
Instruments, Winooski, VT). IC50 values were computed from the
Yellowish solid; Rf 0.43 (hexanes/EtOAc, 7:3); [
a
]
D
+176.0 (c
dose–response curves by plotting percent growth versus test con-
centration. Artemisinin (1) and chloroquine were included in each
assay as drug controls.
0.10, MeOH); IR (neat) m .
max: 3447 (NH), 3376 (NH) cmꢀ1
4.3.3. Dimer 8
White solid; Rf 0.59 (hexanes/EtOAc, 7:3); [
MeOH).
a
]
]
+160.0 (c 0.10,
+131.8 (c 0.10,
4.4.2. Antileishmanial assay
D
The antileishmanial activity was evaluated against a culture of
L. donovani promastigotes grown in RPMI 1640 medium supple-
mented with 10% GIBCO fetal calf serum at 26 °C.18 A three-day-
old culture was diluted to 5 ꢂ 105 promastigotes/mL. Drug
4.3.4. Dimer 9
White solid; Rf 0.67 (hexanes/EtOAc, 7:3); [
a
D
MeOH).
dilutions (50–3.1 lg/mL) were prepared directly in cell suspension
in a 96-well plate, followed by incubation (26 °C, 48 h). Growth of
leishmanial promastigotes was determined by the Alamar Blue as-
say (BioSource International, Camarillo, CA).28 Standard fluores-
cence was measured by a Fluostar Galaxy plate reader (excitation
wavelength, 544 nm; emission wavelength, 590 nm). Pentamidine
and amphotericin B were used as the drug controls. Percent growth
was calculated and plotted against the tested concentrations in or-
der to determine the IC50 and IC90 values.
4.3.5. Dimer 10
White solid; Rf 0.29 (hexanes/EtOAc, 7:3); [
a
]
a
a
D +134.8 (c 0.095,
MeOH); IR (neat) m .
max: 3503 (OH) cmꢀ1
4.3.6. Dimer 11
White solid; Rf 0.53 (hexanes/EtOAc, 3:7); [
MeOH).
]
]
+147.8 (c 0.10,
+135.8 (c 0.10,
D
D
4.3.7. Dimer 12
White solid; Rf 0.12 (hexanes/EtOAc, 3:7); [
MeOH).
4.4.3. Anticancer assay
The cytotoxicity was determined against a panel of cancer (SK-
MEL, KB, BT-549 and SK-OV-3) and noncancerous cell lines (Vero
and LLC-PK11) (American Type Culture Collection, Rockville, MD).
The assay was performed in 96-well tissue culture-treated micro-
plates.18,19 Cells (25,000 cells/well) were seeded in the wells of
the plate and incubated (24 h), followed by addition of the samples
and further incubation (48 h). The number of viable cells was
determined using Neutral Red.29 IC50 values were determined from
logarithmic graphs of growth inhibition versus concentration.
Doxorubicin was used as a positive control drug, while DMSO
was used as the negative (vehicle) control.
4.3.8. Dimer 13
White solid; Rf 0.51 (hexanes/EtOAc, 75:25); [
0.095, MeOH).
a
]
+141.1 (c
+104.0 (c
D
4.3.9. Dimer 14
Yellowish solid; Rf 0.40 (hexanes/EtOAc, 7:3); [a]
D
0.10, MeOH); IR (neat) m .
max: 1361 (SO2Me) cmꢀ1
4.3.10. Monomer 15
White solid; Rf 0.47 (hexanes/EtOAc, 1:1); [
a
]
]
D +105.5 (c 0.165,
4.4.4. Antimicrobial assay
MeOH); IR (neat)
m
max: 3462 (OH), 1733 (C@O) cmꢀ1
.
All organisms were obtained from the American Type Culture
Collection (Manassas, VA) [fungi: Candida albicans (ATCC 90028),
Candida glabrata (ATCC 90030), Candida krusei (ATCC 6258), C. neo-
formans (ATCC 90113) and Aspergillus fumigatus (ATCC 90906); bac-
teria: Staphylococcus aureus (ATCC 29213), methicillin-resistant S.
aureus (MRSa) (ATCC 43300), Escherichia coli (ATCC 35218), Pseudo-
monas aeruginosa (ATCC 27853) and Mycobacterium intracellulare
(ATCC 23068)]. Susceptibility testing was performed for all organ-
isms, except M. intracellulare, using modified versions of the CLSI/
NCCLS methods.30 M. intracellulare susceptibility was tested using
a modified Franzblau-method.31 Samples dissolved in DMSO were
serially diluted in DMSO/saline (20%/0.9%) and transferred in dupli-
cate to 96-well flat bottom microplates. Microbial inocula were pre-
pared by correcting the OD630 of microbe suspensions in incubation
broth to afford final target inocula. Controls [fungi: amphotericin B;
bacteria: ciprofloxacin (ICN Biomedicals, OH)] were included in each
assay. All organisms were read at 630 or 544(ex)/590(em) nm (M.
intracellulare and A. fumigatus) prior to and after incubation. Percent
4.3.11. Monomer 16
White solid; Rf 0.06 (hexanes/EtOAc, 1:1); [
a
+134.2 (c 0.155,
D
MeOH); IR (neat) m .
max: 3385 (OH) cmꢀ1
4.3.12. Monomer 17
Oil; Rf 0.18 (hexanes/EtOAc, 7:3); [a]D +125.8 (c 0.10, MeOH); IR
(neat) m .
max: 3482 (OH) cmꢀ1
4.3.13. Trimer 18
White solid; Rf 0.41 (hexanes/EtOAc, 7:3); [
MeOH).
a
] +157.3 (c 0.15,
D
4.4. Biological assays
4.4.1. Antimalarial assay
The assay is based on the determination of plasmodial LDH
activity.18 A suspension of red blood cells infected with D6 or
growth was plotted versus test concentration to afford the IC50
.
W2 strains of P. falciparum [200
ocrit in RPMI 1640 medium supplemented with 10% human serum
and Amikacin (60 g/mL)] was added to a 96-well plate containing
10 L of serially diluted test samples. The plate was placed in a
lL, 2% parasitemia and 2% hemat-
Acknowledgments
l
l
The authors sincerely thank Mr. John M. Trott, Ms. Marsha A.
Wright, Rajnish Sahu, Dr. Bharathi Avula and Dr. Paulo Carvalho
for their assistance in biological and analytical analysis. The NCI’s
Developmental and Therapeutics Program is acknowledged for
the anticancer screening of some of these compounds. This work
was supported in part by the United States Department of Agricul-
ture, Agricultural Research Service Specific Cooperative Agreement
No. 58-6408-2-0009, and by the National Institutes of Health, Na-
tional Institute of Allergy and Infectious Diseases, Division of AIDS,
Grant No. AI 27094.
modular incubation chamber flushed with N2/O2/CO2 (90:5:5)
and incubated (37 °C, 72 h). Parasitic LDH activity was determined
by using MalstatTM reagent (Flow Inc., Portland, OR).27 The incuba-
tion mixture (20
(100 L) and incubated (30 min). Nitro blue tetrazolium (NBT)/
phenazine ethosulfate (PES) (20 L, 1:1) (Sigma, St. Louis, MO)
was added and the plate incubated in the dark (60 min). The reac-
tion was stopped by the addition of 5% acetic acid (100 L) and the
plate was read at 650 nm on an EL340 BioKinetics Reader (Bio-Tek
l
L) was mixed with the MalstatTM reagent
l
l
l

D. Slade et al. / Bioorg. Med. Chem. 17 (2009) 7949–7957
7957
15. Lin, A. J.; Lee, M.; Klayman, D. L. J. Med. Chem. 1989, 32, 1249.
16. ElSohly, M. A.; Gul, W. WO2006002105A1, US20080275106A1, 2006.
Supplementary data
17. Grellepois, F.; Crousse, B.; Bonnet-Delpon, D.; Begue, J.-P. Org. Lett. 2005, 7,
5219.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2009.10.019.
18. (a) Jain, M.; Khan, S. I.; Tekwani, B. L.; Jacob, M. R.; Singh, S.; Singh, P. P.; Jain, R.
Bioorg. Med. Chem. 2005, 13, 4458; (b) Bharate, S. B.; Khan, S. I.; Tekwani, B. L.;
Jacob, M.; Khan, I. A.; Singh, I. P. Bioorg. Med. Chem. 2008, 16, 1328.
19. Mustafa, J.; Khan, S. I.; Ma, G.; Walker, L. A.; Khan, I. A. Lipids 2004, 39, 167.
20. Vonthron-Senecheau, C.; Weniger, B.; Ouattara, M.; Bi Fezan, T.; Kamenan, A.;
Lobstein, A.; Brun, R.; Anton, R. J. Ethnopharmacol. 2003, 87, 221.
21. (a) Singh, I. P.; Lal, U. R.; Bodiwala, H. S.; Mahajan, R. C.; Bhutani, K. K. Recent
Prog. Med. Plants 2006, 13, 115; (b) Maltezou, H. C. Recent Patents Anti Infect.
Drug Disc. 2008, 3, 192.
22. (a) Sen, R.; Bandyopadhyay, S.; Dutta, A.; Mandal, G.; Ganguly, S.; Saha, P.;
Chatterjee, M. J. Med. Microbiol. 2007, 56, 1213; (b) Menon, R. B.; Kannoth, M.
M.; Tekwani, B. L.; Gut, J.; Rosenthal, P. J.; Avery, M. A. Comb. Chem. High
Throughput Screening 2006, 9, 729.
References and notes
1. Pierce, S. K.; Miller, L. H. J. Immunol. 2009, 182, 5171.
2. (a) Rosenthal, P. J. N. Eng. J. Med. 2008, 358, 1829; (b) White, N. J. Science 2008,
320, 330.
3. (a) Genton, B. Expert Rev. Vaccines 2008, 7, 597; (b) Vekemans, J.; Ballou, W. R.
Expert Rev. Vaccines 2008, 7, 223.
4. (a) Turschner, S.; Efferth, T. Mini-Rev. Med. Chem. 2009, 9, 206; (b) Edwards, G.;
Biagini, G. A. Br. J. Clin. Pharmacol. 2006, 61, 690.
5. (a) Aweeka, F. T.; German, P. I. Clin. Pharmacokinet. 2008, 47, 91; (b) Plowe, C. V.
Clin. Infect. Dis. 2007, 44, 1075; (c) Kindermans, J.-M.; Pilloy, J.; Olliaro, P.;
Gomes, M. Malar. J. 2007, 6.
23. Krishna, S.; Bustamante, L.; Haynes, R. K.; Staines, H. M. Trends Pharmacol. Sci.
2008, 29, 520.
24. (a) Wang, H.; Klinginsmith, J.; Dong, X.; Lee, A. C.; Guha, R.; Wu, Y.; Crippen, G.
M.; Wild, D. J. J. Chem. Inf. Model. 2007, 47, 2063; (b) Huang, R.; Wallqvist, A.;
Covell, D. G. J. Med. Chem. 2006, 49, 1964; (c) Boyd, M. R.; Paull, K. D. Drug Dev.
Res. 1995, 34, 91.
25. Vichai, V.; Kirtikara, K. Nat. Protocols 2006, 1, 1112.
26. (a) Galal, A. M.; Ross, S. A.; Jacob, M.; ElSohly, M. A. J. Nat. Prod. 2005, 68, 1274;
(b) Lee, S. Mini-Rev. Med. Chem. 2007, 7, 411.
27. (a) Makler, M. T.; Hinrichs, D. J. Am. J. Trop. Med. Hyg. 1993, 48, 205; (b)
Nkhoma, S.; Molyneux, M.; Ward, S. Am. J. Trop. Med. Hyg. 2007, 76, 1107.
28. (a) Ma, G.; Khan, S. I.; Jacob, M. R.; Tekwani, B. L.; Li, Z.; Pasco, D. S.; Walker, L.
A.; Khan, I. A. Antimicrob. Agents Chemother. 2004, 48, 4450; (b) Mikus, J.;
Steverding, D. Parasitol. Int. 2000, 48, 265; (c) Hamid, R.; Rotshteyn, Y.; Rabadi,
L.; Parikh, R.; Bullock, P. Toxicol. In Vitro 2004, 18, 703.
6. (a) Efferth, T. Planta Med. 2007, 73, 299; (b) Namdeo, A. G.; Mahadik, K. R.;
Kadam, S. S. Phcog Mag. 2006, 2, 106.
7. (a) Gomes, M.; Ribeiro, I.; Warsame, M.; Karunajeewa, H.; Petzold, M. BMC
Infect. Dis. 2008, 8, 1; (b) Rydén, A.-M.; Kayser, O. Chemistry, Biosynthesis and
Biological Activity of Artemisinin and Related Natural Peroxides. In Gupta, R. R.,
Khan, M. T. H., Eds.; Bioactive Heterocycles III (Topics in Heterocyclic
Chemistry); Springer: Berlin, Heidelberg, 2007; Vol. 9, pp 1–31; (c) Sharma,
V. P. Curr. Sci. 2006, 90, 1323; (d) Pasvol, G. Br. Med. Bull. 2005, 75–76, 29.
8. Guidelines for the treatment of malaria. Global Malaria Programme. World
Health
Organization
2006
(www.who.int/malaria/docs/
TreatmentGuidelines2006.pdf).
9. Li, Q.; Weina, P. J.; Milhous, W. K. Curr. Drug Ther. 2007, 2, 210.
10. (a) Lelievre, J.; Berry, A.; Benoit-Vical, F. Curr. Opin. Invest. Drugs 2007, 8, 117;
(b) Golenser, J.; Waknine, J. H.; Krugliak, M.; Hunt, N. H.; Grau, G. E. Int. J.
Parasitol. 2006, 36, 1427; (c) Stockwin, L. H.; Han, B.; Yu, S. X.; Hollingshead, M.
G.; ElSohly, M. A.; Gul, W.; Slade, D.; Galal, A. M.; Newton, D. L. Int. J. Cancer
2009, 125, 1266.
11. (a) Maude, R. J.; Pontavornpinyo, W.; Saralamba, S.; Aguas, R.; Yeung, S.;
Dondorp, A. M.; Day, N. P. J.; White, N. J.; White, L. J. Malar. J. 2009, 8; (b) Noedl,
H.; Se, Y.; Schaecher, K.; Smith, B. L.; Socheat, D.; Fukuda, M. M. N. Eng. J. Med.
2008, 359, 2619.
12. (a) Chadwick, J.; Mercer, A. E.; Park, B. K.; Cosstick, R.; O’Neill, P. M. Bioorg. Med.
Chem. 2009, 17, 1325; (b) Galal, A. M.; Gul, W.; Slade, D.; Ross, S. A.; Feng, S.;
Hollingshead, M. G.; Alley, M. C.; Kaur, G.; ElSohly, M. A. Bioorg. Med. Chem.
2009, 17, 741; (c) Posner, G. H.; D’Angelo, J.; O’Neill, P. M.; Mercer, A. Expert
Opin. Ther. Patents 2006, 16, 1665.
29. Borenfreund, E.; Babich, H.; Martin-Alguacil, N. In Vitro Cell. Dev. Biol.: J. Tissue
Culture Assoc. 1990, 26, 1030.
30. (a) National Committee for Clinical Laboratory Standards (NCCLS) (Wayne,
Pa.): Reference method for broth dilution antifungal susceptibility testing of
yeasts; Approved Standard—Second Edition. Document M27-A2, 2002, 22; (b)
NCCLS: Methods for dilution antimicrobial susceptibility tests for bacteria that
grow aerobically; Approved Standard—Seventh Edition. Document M7-A7,
2006, 26; (c) NCCLS: Susceptibility testing of mycobacteria, nocardia, and other
aerobic actinomycetes; Tentative Standard—Approved Standard. Document
M24-A, 2003, 23; (d) NCCLS: Reference method for broth dilution antifungal
susceptibility testing of filamentous fungi; Approved Standard. Document
M38-A, 2002, 22.
31. Franzblau, S. G.; Witzig, R. S.; McLaughlin, J. C.; Torres, P.; Madico, G.;
Hernandez, A.; Degnan, M. T.; Cook, M. B.; Quenzer, V. K.; Ferguson, R. M.;
Gilman, R. H. J. Clin. Microbiol. 1998, 36, 362.
13. Holley Pharmaceutical Company, 1440 N. Harbor Blvd., # 900, Fullerton, CA
92835, USA (www.holleypharma.com).
14. Lin, A. J.; Klayman, D. L.; Milhous, W. K. J. Med. Chem. 1987, 30, 2147.