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

Article abstract of DOI:10.1021/jm801484v

A new series of 6 dimeric trioxane sulfones has been prepared from the natural trioxane artemisinin in five or six chemical steps. One of these thermally and hydrolytically stable new chemical entities (4c) completely cured malaria-infected mice via a sin

Full text of DOI:10.1021/jm801484v

1198
J. Med. Chem. 2009, 52, 1198–1203
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
Andrew S. Rosenthal,† Xiaochun Chen,‡ Jun O. Liu,‡ Diana C. West,§ Paul J. Hergenrother,§ 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, DiVision of Pharmacology, Department of Medicine, School of Medicine, The Johns Hopkins UniVersity, Baltimore,
Maryland 21205, Department of Chemistry, UniVersity of Illinois, Urbana, Illinois 61801, DiVision of Clinical Pharmacology, Department of
Medicine, School of Medicine, The Johns Hopkins UniVersity, Baltimore, Maryland 21205, The Johns Hopkins Malaria Research Institute,
Bloomberg School of Public Health, Baltimore, Maryland 21205
ReceiVed NoVember 25, 2008
A new series of 6 dimeric trioxane sulfones has been prepared from the natural trioxane artemisinin in five
or six chemical steps. One of these thermally and hydrolytically stable new chemical entities (4c) completely
cured malaria-infected mice via a single oral dose of 144 mg/kg. At a much lower single oral dose of only
54 mg/kg combined with 13 mg/kg of mefloquine hydrochloride, this trioxane dimer 4c as well as its parent
trioxane dimer 4b also completely cured malaria-infected mice. Both dimers 4c and 4b were potently and
selectively cytotoxic toward five cancer cell lines.
Scheme 1
Introduction
Even in 2009, one child dies of malaria approximately every
30 seconds.1-3 No vaccine is yet available to prevent malaria
infection in humans.4 Chemotherapy of malaria-infected patients
has been used broadly with great success. Now, unfortunately,
there is widespread malaria parasite resistance to many of the
standard amine antimalarial drugs like chloroquine.5 During the
past few decades,6-13 a new, nonamine class of antimalarial
1,2,4-trioxanes has emerged from traditional Chinese herbal
remedies. Natural 1,2,4-trioxane artemisinin (1) and its semi-
synthetic daughter trioxanes dihydroartemisinin (1a), artemether
(1b), and water-soluble sodium artesunate (1c) are the fastest-
acting antimalarial drugs known.6-13 They are now often
combined, as recommended by the World Health Organization
(WHO), with standard antimalarial amines like lumefantrine or
amodiaquine; such artemisinin combination therapy (ACTa), the
current chemotherapeutic method of choice where malaria is
endemic, requires a repeated dosing regimen.14-17 For example,
a 1:6 fixed combination of the trioxane artemether and lume-
fantrine is curative in a three-day six-dose regimen.18 A single
dose cure would involve better patient compliance and lower
cost. Toward this challenging and urgent goal, we have reported
a series of trioxane dimers able to cure malaria-infected mice
after only a single subcutaneous dose19 as well as a related series
of trioxane dimers curative after three oral doses.20-22 Such
trioxane dimers are significantly more efficacious in curing
malaria-infected mice than twice the dose of the corresponding
trioxane monomers, as well as being significantly more cytotoxic
than the corresponding monomeric trioxanes toward cancer
cells.20-22 Here, as proof of principle, we describe a new series
of dimeric trioxane sulfones 4 (Scheme 1), two of which are
curative after only a single oral dose to malaria-infected mice.
Some of these new dimeric trioxane sulfones 4 also have
selective and potent anticancer activity.23 Increasingly wide-
spread evidence indicates that human cancer cells, richer than
normal cells in iron-transport transferrin receptors,24,25 selec-
tively activate trioxanes to produce various cytotoxic intermedi-
ates; this process is similar to that in the triggering of trioxanes
by heme iron in malaria-infected human erythrocytes.8 The
anticancer properties of trioxanes have been reviewed.26-29 Here
we disclose that some of the new dimeric trioxane sulfones 4
powerfully inhibit the growth (submicromolar IC50 values) of
various cancer cells in vitro without strongly affecting noncan-
cerous fibroblasts.
* To whom correspondence should be addressed. Phone: 410-516-4670.
Fax: 410-516-8420. E-mail: ghp@jhu.edu.
†
Department of Chemistry, School of Arts and Sciences, The Johns
Hopkins University.
‡
Division of Pharmacology, Department of Medicine, School of
Medicine, The Johns Hopkins University.
§
Department of Chemistry, University of Illinois.
Division of Clinical Pharmacology, Department of Medicine, School
|
of Medicine, The Johns Hopkins University.
The Johns Hopkins Malaria Research Institute, Bloomberg School of
Public Health.
a Abbreviations: ACT, artemisinin combination therapy; mCPBA, meta-
chloroperbenzoic acid; DMSO, dimethyl sulfoxide; MTS, 3-(4,5-dimeth-
ylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazo-
lium; SRB, sulforhodamine B; DMF, dimethyl formamide; NaH, sodium
hydride; EtOAc, ethyl acetate; LAH, lithium aluminum hydride; DMDO,
dimethyldioxirane.
Results and Discussion
Chemistry. As outlined in Scheme 1, trioxane dimer primary
alcohol 2 was prepared in three steps from natural artemisinin,
10.1021/jm801484v CCC: $40.75
2009 American Chemical Society
Published on Web 02/02/2009

Malaria-Infected Mice Cured by Dimeric Trioxane Sulfones
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4 1199
Table 1. Antimalarial Efficacy Using a Single Oral Dose of Dimeric Trioxane Sulfones 4 in P. berghei-Infected Mice
average survival (days) after infection;
% suppression of parasitemia
(on day 3 post infection)
trioxane dose (mg/kg)
dimeric trioxane
sulfone
1 × 54
1 × 72
1 × 144
1 × 54
1 × 72
1 × 144
4a
4b
4c
4d
4e
4f
14.6
-
-
97.1
99.8
99.5
99.2
92.4
2.0
-
99.6
98.6
-
-
>99.9
99.3
-
20.4 (11, 12, 19, 30, 30)a
25.0 (12, 25, 27, 30, 30)a
30.0 (2 sick, 3 cured)
30.0 (all cured)
22.8 (11, 24, 26, 26, 27)a
23.0 (11, 14, 30, 30, 30)a
16.4
14.8
11.0
-
-
-
-
-
-
-
-
-
-
-
vehicle (no drug)
6.4 (6, 6, 6, 7, 7)a
9.8 (9, 9, 10, 10, 11)a
7.6b
-
-
-
-
1b
1c
7
11 (8, 10, 11, 13, 13)a
12.4 (12, 12, 12, 13, 13)a
99.4
-
98.7
-
99.8
-
-
-
-
-
10.7b
-
99.95b
-
a Actual mouse survival until day. b Data from Supporting Information in ref 39 using a single oral dose of 30 mg/kg. A dash in the table means not
tested.
as described previously.30 Primary bromide 3 was easily
dose of 72 mg/kg and a single oral dose of 144 mg/kg were
also used. Blood parasitemia levels as well as monitoring the
duration of animal survival compared to survival time of animals
receiving no drug are both widely accepted as a measure of a
drug’s efficacy in antimalarial drug development. Three days
after infection, an average of 16.2% blood parasitemia was
observed in the control (no drug) group. Animals receiving no
drug die typically 6-8 days postinfection. A widely accepted
yardstick of cure (i.e., 100% efficacy) is survival of animals to
day 30 postinfection, with no detectable malaria parasites in
the animal’s blood at that time. Average survival results are
summarized in Table 1. The clinically used monomeric trioxane
drugs 1b and 1c and the synthetic trioxolane peroxide drug
development candidate OZ277 maleate (7) are included as
standards.
It is clear from the data in Table 1 that all of the dimeric
trioxane sulfones 4a-4f prolonged average survival time at least
as effectively as trioxolane 7, which is in phase II clinical trials.39
It is also apparent from the data in Table 1 that the dimeric
trioxane sulfones 4b and 4c, at a single oral dose of 54 mg/kg,
were the most efficacious among sulfones 4a-4f at prolonging
survival. Therefore, sulfones 4b and 4c were tested further using
a higher single oral dose of 72 mg/kg and separately using an
even higher single oral dose of 144 mg/kg. As shown in Table
1, dimeric sulfones 4b and 4c at a single oral dose of 72 mg/kg
prolonged average survival to days 23-25. At a higher oral
dose, if 144 mg/kg, the benzyl alcohol sulfone 4b prolonged
average survival to day 30, but two of the five mice had 7-9%
parasitemia at day 30 and appeared sick. In contrast, carbamate
sulfone 4c at a single oral dose of 144 mg/kg completely cured
all of the malaria-infected mice with no adverse effects. Using
the same experimental protocol but combining benzylic alcohol
dimer 4b (40 mg/kg) with mefloquine hydrochloride (13 mg/
kg) in a single oral dose caused a 99.9% suppression of
parasitemia on day 3 post infection and raised average survival
to 28 days; a slightly higher single oral dose of dimer 4b (54
mg/kg) along with mefloquine hydrochloride (13 mg/kg) caused
>99.9% parasitemia suppression on day 3 post infection and
completely cured the mice. Similar curative results were
obtained by combining sulfone carbamate dimer 4c (54 mg/
kg) with mefloquine (13 mg/kg). In a control experiment, a
single oral dose of mefloquine hydrochloride (13 mg/kg) alone
prolonged average survival to only day 11. Neither overt toxicity
nor behavioral change attributable to trioxane drug administra-
tion was observed in any of the malaria-infected animals cured
by carbamate 4c or cured by the dimer plus mefloquine
hydrochloride combination. The standard monomeric trioxane
antimalarial drugs 1b and 1c and the trioxolane drug candidate
prepared in good yield by treating alcohol 2 with CBr4 and
Ph3P.31 Displacement of the bromide anion by a mercaptide
anion, followed by sulfidefsulfone oxidation with meta-
chloroperbenzoic acid (mCPBA) proceeded also in good yields
without disruption of the crucial peroxide pharmacophore. This
is an especially noteworthy result because most peroxides are
cleaved by mercaptide anions. Jung and co-workers reported
the first example of mercaptide displacement of a trioxane
primary bromide leading to a dimeric trioxane sulfone.22 We
prepared dimeric trioxane sulfones 4a-f from natural artemisi-
nin in 5-6 chemical steps and good overall yields. The
calculated octanol/water partition coefficient (log P) of these
sulfone dimers ranged from 7.0 to 9.5. Scale up to multigram
synthesis and even to kilogram manufacture of sulfones 4 is
not expected to be a problem. Some endoperoxide sulfones like
synthetic bicyclic sulfone 532 and semisynthetic sulfone 633
among others34-37 have excellent antimalarial activities. The
extraordinary antimalarial efficacy of dimeric trioxane sulfone
benzylic alcohol 4b as well as the chemical versatility of its
primary hydroxyl group prompted us to prepare the correspond-
ing carbamates 4c and 4d and phosphate 4e as potential
prodrugs. It was expected that, in vivo, esterase enzymes would
convert carbamates 4c and 4d and phosphate 4e back into their
parent alcohol 4b. Benzylic alcohol 4b and dimethyl carbamate
4c are stable in the solid state at 60 °C for g24 h; carbamate
4c is stable in the solid state at 60 °C even for one week. Both
dimers 4b and 4c are stable for at least 12 h at room temperature
in 80/20 DMSO/water at pH 7.4.
Biology. Each trioxane dimer 4a-4f (7.2 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 Plasmo-
dium berghei, ANKA strain (2 × 107 parasitized erythrocytes).38
Each of five mice in a group was treated orally with a single
dose of 0.20 mL (0.20 mL/1.21 mL × 7.2 mg ) 1.2 mg) of
diluted compound solution, corresponding to a dose of 54 mg/
kg, 24 h postinfection. In separate experiments, a single oral

1200 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4
Rosenthal et al.
Table 2. Anticancer Activity of Dimeric Trioxane Sulfones 4b and 4c
in Human Cancer Cell Lines
10 Hz, J2 ) 5.0 Hz, 1H), 3.82-3.78 (dd, J1 ) 10 Hz, J2 ) 5.0 Hz,
1H), 2.73-2.68 (q, 1H), 2.62-2.57 (q, 1H), 2.36-2.27 (m, 2H),
2.19-2.17 (m, 1H), 2.04 (m, 1H), 2.00 (m, 1H), 1.91-1.23 (m,
27H including singlets for 3H each at 1.41 and 1.39), 0.99-0.95
(m, 7H), 0.88-0.86 (m, 6H). 13C NMR (100 MHz, CDCl3) δ 103.3,
102.9, 89.6, 89.0, 81.2, 81.2, 74.7, 70.6, 52.5, 52.1, 44.6, 44.1, 40.7,
37.5, 37.4, 36.7, 36.6, 36.2, 34.5, 34.4, 32.1, 31.9, 30.6, 30.5, 26.1,
26.1, 24.9, 24.9, 24.8, 24.7, 20.3, 20.1, 13.3, 12.7. HRMS (ESI)
m/z calcd for C34H53BrO8Na (M + Na)+ 691.2816, found 691.2797.
Synthesis of Dimeric Fluorobenzylic Sulfone 4a. Bis-trioxane
bromide 3 (10 mg, 0.015 mmol) was dissolved in DMF (0.5 mL)
under argon. To the solution was added 4-fluorobenzyl mercaptan
(2 µL, 0.018 mmol) and NaH (0.5 mg, 0.018 mmol) consecutively
and the reaction was heated at 90 °C for 1 h. (Note: the reaction
went from colorless to pink almost instantly upon heating. The pink
color faded to a very light yellow over the course of the hour.)
The reaction was allowed to cool and was diluted with EtOAc (1
mL). The organic layer was then washed with ice-cold distilled
water (1 mL) and brine (1 mL). The organic layer was dried with
MgSO4, filtered, and concentrated in vacuo. The crude product was
dissolved in CH2Cl2 (0.5 mL) and mCPBA (4 mg, 0.020 mmol)
was added. The reaction was stirred at room temperature for 5 h
and then washed with saturated sodium bisulfite solution (1 mL)
and saturated sodium bicarbonate solution (1 mL) sequentially. The
organic layer was dried with MgSO4, filtered, and concentrated in
vacuo. The crude product was purified by flash silica gel column
chromatography (0 f 20% EtOAc/hexanes) to yield ASR-isobu-
SO2-CH2Ph-4-F 9 (6.5 mg, 0.010 mmol, 66%) as an amorphous
white solid. [R]D21.3 +67.7° (c ) 0.35, CHCl3). IR (thin film) 2938,
2876, 1509, 1456, 1377, 1309, 1226, 1118, 1099, 1056, 1007, 941,
878, 843 cm-1. 1H NMR (400 MHz, CDCl3) δ 7.48-7.44 (m, 2H),
7.08-7.04 (m, 2H), 5.48 (s, 1H), 5.31 (s, 1H), 4.40-4.38 (m, 1H),
4.35 (s, 2H), 4.15-4.13 (m, 1H), 3.54-3.50 (dd, J1 ) 14.3 Hz, J2
) 10.1 Hz, 1H), 3.04-2.98 (dd, J1 ) 14.4 Hz, J2 ) 6.4 Hz, 1H),
2.78-2.72 (m, 1H), 2.65-2.56 (m, 2H), 2.39-2.28 (m, 3H),
2.06-1.95 (m, 2H), 1.92-1.88 (m, 3H), 1.81-1.73 (m, 2H),
1.69-1.19 (m, 21H including singlets for 3H each at 1.42 and 1.37),
0.96-0.93 (dd, J1 ) 6.1 Hz, J2 ) 3.0 Hz, 7H), 0.86 (s, 3H), 0.84
(s, 3H). 13C NMR (100 MHz, CDCl3) δ 133.0, 132.9, 123.7, 123.7,
115.9, 115.7, 103.4, 103.0, 89.5, 88.7, 81.3, 81.2, 77.2, 74.1, 70.5,
58.6, 54.6, 52.6, 52.1, 44.6, 44.2, 37.5, 37.2, 36.6, 34.6, 34.4, 31.5,
31.4, 30.4, 30.3, 30.1, 26.3, 26.1, 24.8, 24.8, 24.7, 24.7, 20.3, 20.1,
13.4, 12.8. HRMS (FAB) m/z calcd for C41H60FO10S (M + H)+
763.3891, found 763.3850.
IC50 values (µM)a
cancer cell line
U-937-lymphoma
HL-60-leukemia
SK-MEL-5-melanoma
UACC-62-melanoma
HeLa-cervical
control: WT-MEF noncancerous
mouse embryonic fibroblasts
Hs888Lu noncancerous human
lung fibroblasts
dimer 4b
dimer 4c
0.28 ( 0.15
0.13 ( 0.09
0.14 ( 0.09
0.03 ( 0.01
1.1 ( 0.95
>50
0.28 ( 0.13
0.06 ( 0.02
0.06 ( 0.03
0.04 ( 0.02
0.11 ( 0.06
>50
>50
>50
a Average of at least three independent experiments with standard error
of the mean. Compound toxicity was determined by the MTS assay (for
U-937 and HL-60) or the SRB assay (for all other cell lines) after 72 h of
cell treatment with the trioxane dimer as described in the experimental
section. The IC50 value is the concentration, at which 50% of the cells are
no longer viable (MTS assay), or where 50% of the cell biomass has been
reduced (for the SRB assay).
7, although able to lower parasitemia levels considerably by
day 3 post infection, were not efficacious in prolonging the
average survival time beyond day 11.
Table 2 summarizes the in vitro cytotoxic activity of dimeric
trioxane sufones 4b and 4c in five different cancer cell lines.
The IC50 values are shown as an average of at least three
experiments with standard error of the mean using a standard
MTS assay or sulforhodamine B (SRB) assay. Clearly, both
dimers 4b and 4c are strongly cytotoxic, often comparing well
with the anticancer potency of doxorubicin, a clinically used
anticancer drug.40 Doxorubicin, however, has serious side effects
in humans.41 In this study, we have found that doxorubicin is
toxic toward the WT-MEF and Hs888Lu noncancerous im-
mortalized fibroblast cell lines (with IC50 values of 3.4 ( 1.3
µM and 1.4 ( 0.7 µM, respectively). Neither dimer 4b or 4c
significantly affected these noncancerous fibroblast cell lines
(see Table 2). This selective cytotoxicity of both dimers 4b and
4c is of special importance for potential anticancer drug
development of this class of dimeric trioxane sulfones.
In conclusion, at only a single oral dose of 144 mg/kg, dimeric
trioxane sulfone carbamate 4c completely cured malaria-infected
mice. Combining a much lower amount (54 mg/kg) of dimer
alchol 4b or of dimer carbamate 4c with a small amount of
mefloquine hydrochloride (13 mg/kg) in a single oral dose also
completely cured the malaria-infected mice. Both dimers 4b
and 4c have powerful and selective anticancer activities. This
desirable combination42 of high antimalarial activity and high
anticancer activity encourages further lead optimization and
preclinical drug development of trioxane dimer sulfones like
4b and 4c.
Synthesis of Dimeric Sulfone Benzylic Alcohol 4b. Bis-trioxane
bromide 3 (39 mg, 0.058 mmol) was dissolved in acetonitrile (3
mL) under argon at room temperature. To the solution was added
4-hydroxymethyl thiophenol (16 mg, 0.116 mmol), easily prepared
by LAH reduction of the commercially available parent carboxylic
acid, and NaH (3.0 mg, 0.116 mmol) consecutively, and the reaction
was stirred at room temperature overnight. The reaction was
quenched with sat. aq sodium bicarbonate (2 mL) and extracted
with EtOAc (2 mL). The organic layer was then washed with ice
cold distilled water (2 × 2 mL) and brine (2 mL). The organic
layer was dried with MgSO4, filtered, and concentrated in vacuo.
The crude product was dissolved in dichloromethane (3 mL).
mCPBA (10 mg, 0.057 mmol) was added at room temperature and
stirred for 2 h. The reaction was quenched with saturated aqueous
sodium bisulfite (2 mL), and the organic layer was extracted with
sat. aq sodium bicarbonate (2 mL). The crude product was purified
by flash silica gel column chromatography (20 f 30% EtOAc/
hexanes) to yield dimeric sulfone benzylic alcohol 4b (25 mg, 0.033
mmol, 57%) as an amorphous white solid; mp ) 109-112 °C;
[R]D21.3 +55° (c ) 0.55, CHCl3). IR (thin film) 3511, 2948, 2848,
1454, 1408, 1306, 1197, 1142, 1094, 1051, 1008, 936, 880, 837,
Experimental Section
Synthesis of Dimer Primary Bromide 3. Dimeric trioxane
primary alcohol30 2 (76 mg, 0.13 mmol) was dissolved in CH2Cl2
(4 mL) under argon. Triphenylphosphine (49 mg, 0.19 mmol) and
carbon tetrabromide (62 mg, 0.19 mmol) were added to the solution
at room temperature and allowed to stir overnight. After 18 h, the
reaction was quenched with distilled water (2 mL). The aqueous
layer was extracted with CH2Cl2 (2 × 3 mL) and washed with brine
(3 mL). The organic layer was dried with MgSO4, filtered, and
concentrated in vacuo. The crude product was purified by flash silica
gel column chromatography (0 f 20% EtOAc/hexanes) to yield
dimeric trioxane bromide 3 as an amorphous white solid (72 mg,
0.11 mmol, 86%). [R]D21.9 +70.8° (c ) 0.56, CHCl3). IR (thin film)
2951, 2875, 1451, 1377, 1252, 1206, 1119, 1055, 1007, 942, 879,
1
763 cm-1. H NMR (400 MHz, CDCl3) δ 7.97-7.95 (d, 2 7.49-
7.47 (d, 2H), 5.44 (s, 1H), 5.32 (s, 1H), 4.76 (s, 2H), 4.11-4.06
(m, 1H), 3.63-3.58 (dd, J1 ) 14 Hz, J2 ) 8.0 Hz, 1H), 3.34-3.29
(dd, J1 ) 14 Hz, J2 ) 8.0 Hz, 1H), 2.71-2.64 (m, 1H), 2.57-2.49
(m, 1H), 2.51-2.44 (m, 1H), 2.37-2.25 (m, 2H), 2.20-1.16 (m,
29H, including singlets at 1.42 and 1.34 for 3H each), 0.95-0.91
1
735 cm-1. H NMR (400 MHz, CDCl3) δ 5.37 (s, 1H), 5.30 (s,
1H), 4.41-4.37 (m, 1H), 4.24-4.20 (m, 1H), 3.91-3.88 (dd, J1 )

Malaria-Infected Mice Cured by Dimeric Trioxane Sulfones
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4 1201
(m, 8H), 0.83-0.80 (m, 7H). 13C NMR (100 MHz, CDCl3) δ 146.8,
139.2, 128.3, 127.0, 103.4, 102.9, 89.5, 88.8, 81.3, 81.2, 73.9, 71.0,
62.2, 60.4, 58.9, 52.5, 52.1, 44.6, 44.1, 37.4, 36.6, 36.6, 34.5, 34.4,
31.2, 31.1, 30.7, 30.4, 26.2, 26.1, 24.8, 24.8, 21.1, 20.3, 20.1, 14.2,
13.3, 12.7. HRMS (FAB) m/z calcd for C41H60O11SNa (M + Na)+
783.3749, found 783.3707.
layer was dried with MgSO4, filtered, and concentrated in vacuo.
The crude product was purified by flash silica gel column
chromatography (50% EtOAc/hexanes) to yield dimeric sulfone
benzylic diethyl phosphate 4e (8.2 mg, 0.009 mmol, 37%) as an
amorphous white solid. [R]D24.1 +27.1° (c ) 0.06, CHCl3). IR (thin
film) 2925, 2854, 1699, 1653, 1558, 1541, 1521, 1507, 1457, 1375,
1270, 1036 cm-1. 1H NMR (400 MHz, CDCl3) δ 8.06-8.03 (d, J
) 8.0 Hz, 2H), 7.54-7.52 (d, J ) 8.0 Hz, 2H), 5.49 (s, 1H), 5.35
(s, 1H), 5.14-5.12 (d, J ) 8.0 Hz, 2H), 4.48-4.43 (m, 1H),
4.16-4.08 (m, 5H), 3.66-3.61 (dd, J1 ) 14 Hz, J2 ) 6.8 Hz,
1H), 3.39-3.34 (dd, J1 ) 14 Hz, J2 ) 6.8 Hz, 1H), 2.70-2.66 (m,
1H), 2,58-2.53 (m, 2H), 2.38-2.28 (m, 2H), 2.23-2.16 (m, 1H),
2.06-2.00 (m, 2H), 1.93-1.90 (m, 2H), 1.79-1.21 (m, 29H
including singlets for 3H each at 1.44 and 1.35), 0.96-0.95 (m,
8H), 0.85-0.82 (m, 6H). 13C NMR (100 MHz, CDCl3) δ 140.5,
138.0, 128.6, 127.7, 103.3, 102.9, 89.6, 88.9, 81.3, 81.2, 74.0, 70.9,
67.8, 67.7, 64.1, 64.1, 58.8, 57.2, 57.1, 52.6, 52.2, 44.6, 44.1, 37.5,
37.4, 36.7, 36.7, 34.4, 31.3, 30.7, 30.4, 29.7, 26.2, 26.1, 24.8, 24.8,
20.2, 20.1, 16.2, 16.1, 14.2, 13.2, 12.6. HRMS (ESI) m/z calcd for
C45H69O14PSNa (M + Na)+ 919.4038, found 919.3999.
Synthesis of Dimeric Sulfone Benzylic Dimethyl Carbamate
4c. Dimeric sulfone benzylic alcohol 4b (20 mg, 0.026 mmol) was
dissolved in DMF (1 mL) under argon at room temperature. To
the solution was added dimethylcarbamyl chloride (5 µL, 0.052
mmol) and NaH (1.2 mg, 0.052 mmol) consecutively, and the
reaction was stirred at room temperature for 2 h. The reaction was
quenched with sat. aq sodium bicarbonate (1 mL) and extracted
with dichloromethane (2 × 2 mL). The organic layer was dried
with MgSO4, filtered, and concentrated in vacuo. The crude product
was purified by flash silica gel column chromatography (40%
EtOAc/hexanes) to yield dimeric sulfone benzylic dimethyl car-
bamate 4c (16 mg, 0.019 mmol, 73%) as an amorphous white solid.
[R]D23.4 +73.7° (c ) 0.12, CHCl3). IR (thin film) 2939, 2876, 1711,
1495, 1454, 1394, 1377, 1306, 1181, 1145, 1089, 1054, 1007, 878,
830, 767, 669 cm-1. 1H NMR (400 MHz, CDCl3) δ 8.02-8.00 (d,
J ) 8.0 Hz, 2H), 7.50-7.48 (d, J ) 8.0 Hz, 2H), 5.48 (s, 1H),
5.34 (s, 1H), 5.18 (s, 2H), 4.45-4.43 (m, 1H), 4.14-4.10 (m, 1H),
3.63-3.58 (dd, J1 ) 14.4 Hz, J2 ) 7.2 Hz, 1H), 3.38-3.32 (dd, J1
) 14.4 Hz, J2 ) 7.2 Hz, 1H), 2.95 (s, 6H), 2.71-2.65 (m, 1H),
2.58-2.50 (m, 2H), 2.39-2.26 (m, 2H), 2.24-2.15 (m, 1H),
2.09-1.97 (m, 2H), 1.95-1.84 (m, 3H), 1.82-1.16 (m, 22H
including singlets for 3H each at 1.43 and 1.35), 0.95-0.03 (m,
8H), 0.84-0.82 (m, 6H). 13C NMR (100 MHz, CDCl3) δ 174.7,
142.7, 139.9, 128.4, 127.9, 103.3, 102.9, 89.5, 88.8, 81.3, 81.2,
77.2, 74.0, 71.0, 66.0, 58.9, 56.6, 52.5, 52.1, 44.6, 44.1, 37.4, 37.3,
36.7, 36.6, 34.6, 34.4, 31.2, 31.1, 30.7, 30.4, 26.2, 26.1, 24.8, 24.8,
24.7, 24.7, 20.3, 20.1, 13.3, 12.7. HRMS (ESI) m/z calcd for
C44H65NO12SNa (M + Na)+ 854.4120, found 854.4100.
Synthesis of Dimeric Sulfone Hexanol 4f. Dimeric trioxane
bromide 3 (20 mg, 0.030 mmol) was dissolved in anhydrous
acetonitrile (2 mL) under argon at room temperature. To the solution
was added 6-mercapto-1-hexanol (9 µL, 0.066 mmol) and sodium
hydride (NaH, 2.0 mg, 0.066 mmol) consecutively, and the reaction
was stirred at room temperature for 2 h. The reaction was quenched
with sat. aq sodium bicarbonate (2 mL) and extracted with
dichloromethane (2 × 2 mL). The organic layer was dried with
MgSO4, filtered, and concentrated in vacuo. The crude product was
dissolved in freshly made dimethyldioxirane solution in acetone
(DMDO, 2 mL) at 0 °C and stirred for 1 h. The reaction was
concentrated the crude product was purified by flash silica gel
column chromatography (50% EtOAc/hexanes) to yield dimeric
sulfone hexanol 4f (12 mg, 0.015 mmol, 51%) as an amorphous
white solid. [R]D22.5 +58.1° (c ) 0.15, CHCl3). IR (thin film) 3520
(br), 2925, 2854, 1456, 1377, 1296, 1121, 1055, 1007, 878, 845
Synthesis of Dimeric Sulfone Benzylic Diethyl Carbamate
4d. Dimeric sulfone benzylic alcohol 4b (18 mg, 0.024 mmol) was
dissolved in DMF (1 mL) under argon at room temperature. To
the solution was added diethylcarbamyl chloride (4 µL, 0.026 mmol)
and NaH (0.62 mg, 0.026 mmol) consecutively, and the reaction
was stirred at room temperature for 2 h. The reaction was quenched
with sat. aq sodium bicarbonate (2 mL) and extracted with
dichloromethane (2 × 2 mL). The organic layer was dried with
MgSO4, filtered, and concentrated in vacuo. The crude product was
purified by flash silica gel column chromatography (30% EtOAc/
hexanes) to yield dimeric sulfone benzylic diethyl carbamate 4d
1
cm-1. H NMR (400 MHz, CDCl3) δ 5.41 (s, 1H), 5.34 (s, 1H),
4.46-4.42 (m, 1H), 4.20-4.16 (m, 1H), 3.65-3.62 (t, J ) 6.4 Hz,
2H), 3.49-3.42 (dd, J1 ) 14.2 Hz, J2 ) 7.2 Hz, 1H), 3.10-3.05
(m, 3H), 2.74-2.69 (m, 1H), 2.60-2.54 (m, 2H), 2.35-2.27 (m,
3H), 2.03-1.21 (m, 34H including singlets for 3H each at 1.40
and 1.25), 0.96-0.94 (m, 8H), 0.87-0.85 (m, 8H)). 13C NMR (100
MHz, CDCl3) δ 103.3, 102.9, 89.7, 88.8, 81.3, 81.2, 73.7, 70.3,
62.7, 55.7, 53.0, 52.6, 52.1, 44.5, 44.1, 37.5, 37.3, 36.7, 36.6, 34.6,
34.4, 32.3, 31.9, 31.6, 30.6, 30.3, 29.8, 29.7, 28.2, 26.2, 26.1, 25.1,
24.8, 24.8, 24.7, 24.7, 21.6, 20.3, 20.1, 13.2, 12.7. HRMS (ESI)
m/z calcd for C40H66O11SNa (M + Na)+ 777.4218, found 777.4211.
23.6
(15.5 mg, 0.018 mmol, 76%) as an amorphous white solid. [R]D
+41.6° (c ) 0.12, CHCl3). IR (thin film) 2924, 2853, 1703, 1457,
1377, 1313, 1274, 1168, 1146, 1089, 1053, 1007, 878, 825, 766
Cell Culture Conditions and Determination of IC50 Values.
HL-60, U-937, SK-MEL-5, UACC-62, and HeLa cell lines were
cultured in RPMI 1640 media supplemented with 10% fetal bovine
serum (FBS) and 1% penicillin/streptomycin. WT-MEF and
Hs888Lu cell lines were cultured in DMEM media supplemented
with 10% fetal bovine serum and 1% penicillin/streptomycin. All
cell lines were obtained from the American Type Culture Collection
(Manassas, VA), with the exception of the HL-60 line, which was
a generous gift from Professor Huimin Zhao (University of Illinois),
and were grown at 37 °C with CO2/air (5:95). 99 µL of cells in
media were plated in a 96-well plate. Adherent cell lines were
incubated at least 5 h to allow cells to attach to the plate. A volume
of 1 µL of a range of compound concentrations was added in to
the cells, and the plates were incubated for 72 h. Cell viability in
HL-60 and U-937 suspension cell lines were evaluated using the
CellTiter 96 AQueous nonradioactive cell proliferation assay (Prome-
ga, Inc.). The 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethox-
yphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS)/ phenazine meth-
osulfate (PMS) solution was added to the cells, and absorbance at
490 nm was measured in a Spectra Max Plus 384 plate reader
(Molecular Devices, Sunnyvale, CA) following dye development.
Biomass was quantitated in SK-MEL-5, UACC-62, HeLa, WT-
MEF, and Hs888Lu cells using the sulforhodamine B assay
(SRB).43 Briefly, the cells were fixed with 10% trichloroacetic acid
cm-1. H NMR (400 MHz, CDCl3) δ 8.03-8.01 (d, J ) 8 Hz,
1
2H), 7.49-7.47 (d, J ) 8 Hz, 2H), 5.48 (s, 1H), 5.34 (s, 1H), 5.19
(s, 2H), 4.47-4.44 (m, 1H), 4.16-4.12 (dd, J ) 4.4 Hz, 1H),
3.65-3.60 (dd, J1 ) 9.6 Hz, J2 ) 7.4 Hz, 1H), 3.38-3.33 (dd, J1
) 9.6 Hz, J2 ) 7.4 Hz, 1H), 3.35-3.31 (m, 4H), 2.71-2.67 (m,
1H), 2.58-2.53 (m, 2H), 2.37-2.27 (m, 2H), 2.22-2.17 (m, 1H),
2.05-1.99 (m, 2H), 1.92-1.82 (m, 2H), 1.79-1.73 (m, 4H),
1.65-1.18 (m, 19H including singlets for 3H each at 1.43 and 1.34),
1.15-1.12 (t, J ) 6.8 Hz, 6H), 0.96-0.94 (d, J ) 6.4 Hz, 8H),
0.84-0.82 (d, J ) 7.2 Hz, 6H). 13C NMR (100 MHz, CDCl3) δ
156.9, 142.9, 140.0, 128.4, 127.8, 103.3, 102.9, 89.5, 88.8, 81.3,
81.2, 73.9, 71.0, 65.7, 58.8, 52.6, 52.2, 44.6, 44.1, 37.4, 37.3, 36.7,
36.6, 34.6, 34.4, 31.3, 31.2, 31.2, 30.7, 30.4, 29.7, 26.2, 26.1, 24.8,
24.8, 24.7, 24.7, 20.2, 20.1, 13.2, 12.7. HRMS (ESI) m/z calcd for
C46H69NO12SNa (M + Na)+ 882.4433, found 882.4419.
Synthesis of Dimeric Sulfone Benzylic Diethyl Phosphate
4e. Dimeric sulfone benzylic alcohol 4b (20 mg, 0.026 mmol) was
dissolved in anhydrous dichloromethane (2 mL) under argon at
room temperature. To the solution was added diethyl chlorophos-
phate (19 µL, 0.13 mmol) and pyridine (11 µL, 0.13 mmol)
consecutively, and the reaction was stirred at room temperature
for 18 h. The reaction was quenched with sat. aq sodium bicarbonate
(2 mL) and extracted with dichloromethane (2 × 2 mL). The organic

1202 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4
Rosenthal et al.
(20) Posner, G. H.; Chang, W.; Hess, L.; Woodard, L.; Sinishtaj, S.; Usera,
A. R.; Maio, W.; Rosenthal, A. S.; Kalinda, A. S.; D’Angelo, J. G.;
Petersen, K. S.; Stohler, R.; Chollet, J.; Santo-Tomas, J.; Snyder, C.;
Rottmann, M.; Wittlin, S.; Brun, R.; Shapiro, T. A. Malaria-infected
mice are cured by oral administration of new artemisinin derivatives.
J. Med. Chem. 2008, 51, 1035–1042.
(21) For other trioxane dimers, see: Ekthawatchai, S.; Kamchonwongpaisan,
S.; Kongsaeree, P.; Tarnchompoo, B.; Thebtaranonth, Y.; Yuthavong,
Y. C-16 artemisinin derivatives and their antimalarial and cytotoxic
activities: Syntheses of artemisinin monomers, dimers, trimers, and
tetramers by nucleophilic additions to artemisitene. J. Med. Chem.
2001, 44, 4688–4695.
(22) For additional trioxane dimers, see: Jung, M.; Lee, S.; Ham, J.; Lee,
K.; Kim, H.; Kim, S. K. Antitumor activity of novel deoxoartemisinin
monomers, dimers, and trimer. J. Med. Chem. 2003, 46, 987–994.
(23) Alagbala, A. A.; McRiner, A. J.; Borstnik, K.; Labonte, T,; Chang,
W.; D-Angelo, J. G.; Posner, G. H.; Foster, B. A. Biological
mechanism of action of Novel C-10 non-acetal trioxane dimers in
prostate cancer cell lines. J. Med. Chem. 2006, 49, 7836–7842.
(24) Gatter, K. C.; Brown, G.; Townbridge, I. S.; Woolston, R. E.; Mason,
D. Y. Transferrin receptors in human tissues; their distribution and
possible clinical relevance. J. Clin. Pathol. 1983, 36, 539–545.
(25) Zhou, H.-J.; Wang, Z.; Li, A. Dihydroartemisinin induces apoptosis
in human leukemia cells HL60 via downregulation of transferrin
receptor expression. Anti-Cancer Drugs 2008, 19, 247–255.
(26) Lai, H.; Sasaki, T.; Singh, N. P. Targeted treatment of cancer with
artemisinin and artemisinin-tagged iron-carrying compounds. Expert
Opin. Ther. Targets 2005, 9, 995–1007.
(27) Posner, G. H.; D’Angelo, J.; O’Neill, P. M.; Mercer, A. Anticancer
activity of artemisinin-derived trioxanes. Expert Opin. Ther. Patents
2006, 16, 1665–1672.
(28) Jung, M.; Lee, K.; Kim, H.; Park, M. Recent advances in artemisinin
and its derivatives as antimalarial and antitumor agents. Curr. Med.
Chem. 2004, 11, 1265–1284.
(29) Efferth, T. Mechanistic perspectives for 1,2,4-trioxanes in anti-cancer
therapy. Drug Res. Update 2005, 8, 85–97.
(30) 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 efficacy. J. Med. Chem.
2003, 46, 1060–1065.
overnight at 4 °C, washed with water, and 100 µL of 0.057% w/v
sulforhodamine in 1% acetic acid was added to each well for 30
min. After rinsing the plates in 1% acetic acid, 200 µL of 10 mM
tris base (pH > 10) was added to each well for 30 min and
absorbance was read at 510 nm. Logistical dose response curves
and IC50 values were generated from the MTS and SRB data using
TableCurve 2D 5.01 (SYSTAT Software, Inc., Richmond, CA).
Acknowledgment. We thank Nirbhay Kumar (J.H.U.) 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 to J.O.L.).
1
Supporting Information Available: H, 13C NMR spectra for
all of the new trioxane dimers. This material is available free of
charge via the Internet at http://pubs.acs.org.
References
(1) Ridley, R. G. Medical need, scientific opportunity, and the drive for
antimalarial drugs. Nature 2002, 415, 686–693.
(2) Breman, J. G.; Alilio, M. S.; Mills, A. Conquering the intolerable
burden of malaria: what’s new, what’s needed: a summary. Am. J.
Trop. Med. Hyg. 2004, 71, 1–15.
(3) Snow, R. W.; Guerra, C. A.; Noor, A. M.; Myint, H. Y.; Hay, S. I.
The global distribution of clinical episodes of Plasmodium falciparum
malaria. Nature 2005, 434, 214–217.
(4) Troye-Blomberg, M.; Berzins, K. Rational vaccine development against
malaria. Microbes Infect. 2007, 9, 749–750.
(5) Olliaro, P. L.; Boland, P. B. Clinical public health implications of
antimalarial drug resistance. In Antimalarial Chemotherapy: Mecha-
nisms of Action, Resistance, and New Directions in Drug DiscoVery;
Rosenthal, P. J., Ed.; Humana Press: Totowa, NJ, 2001; pp 65-83.
(6) Shizhen, L. Compendium of Materia Medica (Bencao Gangmu);
Foreign Languages Press: Beijing, 2003; first published in Chinese in
1593, translation published in 2003.
(7) Klayman, D. L. Qinghaosu (artemisinin): an antimalarial drug from
China. Science 1985, 228, 1049–1055.
(8) O’Neill, P. M.; Posner, G. H. A medicinal chemistry perspective on
artemisinin and related endoperoxides. J. Med. Chem. 2004, 47, 2945–
2964.
(31) Kocienski, P. J.; Cernigliaro, G.; Feldstein, G. A synthesis of methyl
n-tetradeca-trans-2,4,5-trienoate, an allenic ester produced by the male
dried bean beetle acanthoscelides obtectus (say). J. Org. Chem. 1977,
42, 353–355.
(9) Tang, Y.; Dong, Y.; Vennerstrom, J. L. Synthetic peroxides as
antimalarials. Med. Res. ReV. 2004, 24, 425–448.
(32) Bachi, M. D.; Korshin, E. E.; Hoos, R.; Szpilman, A. M.; Ploypradith,
P.; Xie, S.; Shapiro, T. A.; Posner, G. H. A short synthesis and
biological evaluation of potent and nontoxic antimalarial bridged
bicyclic sulfonyl-endoperoxides. J. Med. Chem. 2003, 46, 2516–2533.
(33) Haynes, R. K.; Fugmann, B.; Stetter, J.; Rieckmann, K.; Heilmann,
H.-D.; Chan, H.-W.; Cheung, M.-K.; Lam, W-L.; Wong, H.-N.; Croft,
S. L.; Vivas, L.; Rattray, L.; Stewart, L.; Peters, W.; Robinson, B. L.;
Edstein, M. D.; Kotecka, B.; Kyle, D. E.; Beckermann, B.; Gerisch,
M.; Radtke, M.; Schmuck, G.; Steinke, W.; Wollborn, U.; Schmeer,
K.; Ro¨mer, A. Artemisone-A highly active antimalarial drug of the
artemisinin class. Angew. Chem., Int. Ed. 2006, 45, 2082–2088.
(34) Posner, G. H.; O’Dowd, H.; Ploypradith, P.; Cumming, J. N.; Xie,
S.; Shapiro, T. A. Antimalarial cyclic peroxy ketals. J. Med. Chem.
1998, 41, 2164–2167.
(35) Posner, G. H.; O’Dowd, H.; Caferro, T.; Cumming, J. N.; Ploypradith,
P.; Xie, S.; Shapiro, T. A. Antimalarial sulfone trioxanes. Tetrahedron
Lett. 1998, 39, 2273–2276.
(36) Posner, G. H.; Maxwell, J. P.; O’Dowd, H.; Krasavin, M.; Xie, S.;
Shapiro, T. A. Antimalarial sulfide, sulfone, and sulfonamide trioxanes.
Bioorg. Med. Chem. 2000, 8, 1361–1370.
(37) 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 efficacy. J. Med. Chem.
2003, 46, 1060–1065.
(38) Chen, X.; Chong, C. R.; Shi, l.; Yoshimoto, T.; Sullivan, D. J., Jr.;
Lin, J. O. Inhibitors of Plasmodium falciparum methionine aminopep-
tidase 1b possess antimalarial activity. Proc. Natl. Acad. Sci. U.S.A.
2006, 103, 14548–14553.
(39) 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.; Scor-
neaux, B.; Tang, Y.; Urwyler, H.; Wittlin, S.; Charman, W. N.
Identification of an antimalarial synthetic trioxolane drug development
candidate. Nature 2004, 430, 900–904.
(40) Harris, K. A.; Harney, E.; Small, E. J. Liposomal doxorubicin for the
treatment of hormone-refractory prostate cancer. Clin. Prostate Cancer
2002, 1, 37–41.
(10) Jefford, C. W. Synthetic peroxides as antimalarials. Curr. Opin. InVest.
Drugs 2004, 5, 866–872.
(11) Haynes, R. K. From artemisinin to new artemisinin antimalarials:
biosynthesis, extraction, old and new derivatives, stereochemistry and
medicinal chemistry requirements. Curr. Top. Med. Chem. 2006, 6,
509–537.
(12) Be´gue´, J.-P.; Bonnet-Delpon, D. Fluoroartemisinins: metabolically
more stable antimalarial artemisinin derivatives. ChemMedChem 2007,
2, 608–624.
(13) Gelb, M. H. Drug discovery for malaria: A very challenging endeavor.
Curr. Opin. Chem. Biol. 2007, 11, 440–445.
(14) Ashley, E. A.; White, N. J. Artemisinin-based combinations. Curr.
Opin. Infect. Dis. 2005, 18, 531–536.
(15) (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 + amodiaquine and artemether + lumefan-
trine) in Caala, Central Angola. Am. J. Trop. Med. Hyg. 2006, 75,
143–145.
(16) Guidelines for the Treatment of Malaria; World Health Organization:
Geneva, 2006.
(17) 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.
(18) 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 uncom-
plicated Plasmodium falciparum malaria in Mali. Am. J. Trop. Med.
Hyg. 2008, 79, 655–661.
(19) Posner, G. H.; Paik, I.-H.; Chang, W.; Borstnik, K.; Sinishtaj, S.;
Rosenthal, A. S.; Shapiro, T. A. Malaria-infected mice are cured by
a single dose of novel artemisinin derivatives. J. Med. Chem. 2007,
50, 2516–2519.

Malaria-Infected Mice Cured by Dimeric Trioxane Sulfones
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4 1203
(41) Takara, H.; Sakaeda, T.; Yagami, T.; Kobayashi, H.; Ohmoto, N.;
Horinouchi, M.; Nishiguchi, K.; Okumura, K. Cytotoxic Effects of
27 Anticancer Drugs in HeLa and MDR1-Overexpressing Derivative
Cell Lines. Biol. Pharm. Bull. 2002, 25, 771–778.
(42) Paik, I.-H.; Xie, S.; Shapiro, T. A.; Labonte, T.; Narducci Sargeant,
A. A.; Baege, A. C.; Posner, G. H. Second generation, orally active,
antimalarial, artemisinin-derived trioxane dimers with high stability,
efficacy and anticancer activity. J. Med. Chem. 2006, 49, 2731–
2734.
(43) Vichai, V.; Kirtikara, K. Sulforhodamine B colorimetric assay for
cytotoxic screening. Nat. Protoc. 2006, 3, 1112–1116.
JM801484V