C-16 artemisinin derivatives and their antimalarial and cytotoxic activities: Syntheses of artemisinin monomers, dimers, trimers, and tetramers by nucleophilic additions to artemisitene

Article abstract of DOI:10.1021/jm0103007

Nucleophilic additions of lithium keto and ester enolates and mono- and bifunctional Grgnard reagents to artemisitene provided C-16-derived artemisinin monomers, dmers, trimers, and tetramers whose antimalarial and cytotoxic have been evaluated.

Full text of DOI:10.1021/jm0103007

4
688
J . Med. Chem. 2001, 44, 4688-4695
C-16 Ar tem isin in Der iva tives a n d Th eir An tim a la r ia l a n d Cytotoxic Activities:
Syn th eses of Ar tem isin in Mon om er s, Dim er s, Tr im er s, a n d Tetr a m er s by
Nu cleop h ilic Ad d ition s to Ar tem isiten e
†
‡
†
‡
Sanchai Ekthawatchai, Sumalee Kamchonwongpaisan, Palangpon Kongsaeree, Bongkoch Tarnchompoo,
,
†,‡
‡
Yodhathai Thebtaranonth,* and Yongyuth Yuthavong
Department of Chemistry, Faculty of Science, Mahidol University, Rama 6 Road, Bangkok 10400, Thailand, and
National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency,
Rama 6 Road, Bangkok 10400, Thailand
Received J une 29, 2001
Nucleophilic additions of lithium keto and ester enolates and mono- and bifunctional Grignard
reagents to artemisitene provided C-16-derived artemisinin monomers, dimers, trimers, and
tetramers whose antimalarial and cytotoxic activities have been evaluated.
In tr od u ction
compounds that are highly active against malaria
parasites with low toxicity to the hosts.
1
4,15
Malaria, the disease caused by the protozoan parasite
Plasmodium falciparum, is still one of the major en-
demic infectious diseases in many developing countries,
especially in Asia, Africa, and South America, and kills
over a million people each year. The prevalence of a
multidrug resistant P. falciparum strain against anti-
folates and standard quinoline antimalarial drugs,
together with the decline of support for antimalarial
development from pharmaceutical companies, makes it
1
imperative to search for new antimalarial drugs.
The new clinically valuable antimalarial agent,
artemisinin (qinghaosu), 1, isolated from Artemisia
annua L. (Asteraceae),2 is a sesquiterpene lactone
containing a unique endoperoxide bridge responsible for
the antimalarial activity. It is generally believed that
mechanism of action of artemisinin involves the forma-
tion of free radical intermediates resulting from interac-
tion of the endoperoxide moiety with heme.4 However,
it is not entirely clear on how the free radicals cause
the parasite death.8 The effectiveness of artemisinin,
however, is impaired by its low solubility and poor oral
bioavailability. Chemical modifications of artemisinin
have resulted in new derivatives that combine higher
antimalarial efficacy and compounds’ solubility. For
example, derivatization of artemisinin at C-10 (see
numbering in 1) has yielded compounds 2-5, of which
,3
An oxidized form of artemisinin, artemisitene (6), was
found to be the minor product isolated together with
the more abundant 1 from the American variant A.
-7
,9
1
6
annua L. Chemically, 6, possessing an R-methylene
lactone moiety, is most susceptible to nucleophilic attack
in a Michael addition fashion to give the corresponding
adduct (e.g., 8). The overall process can be regarded as
an easy access to C-16 derivatives of 1. However,
because of the fact that 6 coexists with 1 in the plant
in low quantities and also that its isolation and purifi-
cation from the plant had proved not to be a straight-
forward process, 6 was, therefore, not utilized as start-
ing material in the preparation of artemisinin derivatives.
Semisynthesis of 6 from the naturally abundant 1 would
provide the solution to this drawback, and after unsuc-
2
-4 are now in use clinically. Compounds of this type
have been shown to have ∼10-folds increase in their
antimalarial activities as compared to the parent com-
pound 1.10
Lately, there were reports describing the synthesis
of C-10-derived artemisinin and other trioxane dimers
having linkers between the two core molecules. These
compounds were much more potent against malaria
parasites than artemisinin but also process antitumor
17
18
cessful attempts, 6 was synthesized in 1990. There-
after, we reported the convenient one-pot synthesis of
19a
6
via a selenoxide elimination route together with its
reactions with nucleophiles in our study on correlation
of antimalarial activity of artemisinin derivatives with
1
1-13
activities.
Therefore, optimizing types and length,
the linkage of the trioxane dimers may bring about
19b
their binding affinities with ferroprotoporphyrin IX.
Similar methods have also been subsequently pub-
lished.
We now report on our continued effort to prepare C-16
derivatives of 1 from the reactions of 6 with various
*
To whom correspondence should be addressed. Tel: 662-02201-
20,21
5
135. Fax: 662-02247-7050. E-mail: scytr@mahidol.ac.th.
†
Mahidol University.
National Science and Technology Development Agency.
‡
1
0.1021/jm0103007 CCC: $20.00 © 2001 American Chemical Society
Published on Web 11/22/2001

Synthese of Artemisinin by Additions to Artemisitene
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 26 4689
Sch em e 1
Sch em e 2a
types of nucleophiles and the in vitro antimalarial and
cytotoxicity evaluations of products obtained against P.
falciparum, human epidermoid carcinoma (KB), human
breast cancer (BC), and African green monkey kidney
fibroblast (vero cells), respectively.
Resu lts a n d Discu ssion
Ch em ica l Syn th eses. Three types of nucleophiles
have been employed in the 1,4-nucleophilic additions to
a
Key: 10a , n ) 2 (86%); 10b, n ) 3 (91%); 10c, n ) 4 (97%);
1
0d , n ) 5 (93%).
6
.
(
a ) Or ga n olith iu m s. Artemisitene (6), prepared from
1
9b
shift of the carbon attached to C-9 on the R-side appears
1
by the reported selenoxide elimination approach,
1
0
at a much lower field than the opposite. For example,
the proton at C-9 and carbon at C-16 absorptions of 1
are observed at δ 3.39 and 12.50 ppm while those of
epi-artemisinin appear at δ 2.28 and 20.48 ppm, respec-
tively. These differences in chemical shifts have been
used in the stereochemical identification of all com-
pounds reported here. For compound 8c, the R-H (â-
isomer) at C-9 resonated at δ 3.31 ppm and that of the
opposite isomer, the â-H, appeared at δ 2.30 ppm.
Reaction of the anion derived from 1 to 6 was
exceptionally clean and only one product, the dimer 8e,
was obtained in near quantitative yield (it was the only
product identified by TLC of the crude reaction mixture,
readily underwent 1,4-addition reactions with alkyl-
lithium and lithium keto and ester enolates at -78 °C
in tetrahydrofuran (THF) to give the corresponding
enolate 7, which, upon protonation during workup,
provided the corresponding C-16 derivatives, 8a -e, of
artemisinin (Scheme 1).
The above reactions, except for 8c, gave only thermo-
dynamically less favorable C-9R stereochemical isomers
of 8 due to the fact that protonation of the intermediate,
lithium enolate (7), at low temperature took place on
the more favorable â-face of the molecule. In the case
of 8c, the dianion generated from 2-acetylpyrrole with
2
equiv of lithium diisopropylamide (LDA) was insoluble
8
8% after crystallization). The structure of 8e (m/e 563.7
in THF solution at -78 °C, and after the addition of 6,
the reaction turned into a clear yellow solution which,
upon protonation with saturated aqueous NH4Cl solu-
tion at low temperature followed by preparative liquid
chromatography (PLC) separation (silica gel, EtOAc/
hexane ) 25/75 as the developing solvent), provided two
products, the R- and â-stereoisomers of 8c in 45 and
+
(M + 1) ) has been deduced from its spectral data, and
the complete stereostructure was confirmed by X-ray
crystallographic analysis. Here, the sole 1,4-addition
reaction to 6 surprisingly took place on the apparently
more hindered R-side of 7 followed by protonation on
the less hindered â-face of the molecule.
Related nucleophilic addition reactions between 6 and
dithiolates, 9a -d , derived from the corresponding di-
thiols, were also investigated. As shown in Scheme 2,
dimeric artemisinin analogues 10a -d with a linker,
S-(CH2)n-S, of various length and flexibility were
obtained in very good yields when the above reactants
were allowed to react in THF at -78 °C. However,
compounds 10a -d were not very stable and spontane-
2
8%, respectively.
Because of the fact that nuclear magnetic resonance
(NMR) data of many artemisinin derivatives have been
well-documented, stereochemical identification of prod-
ucts described was straightforward, where upon the
chemical shift of protons at C-9 situated on the R-face
of the molecule resonates at about 1 ppm downfield from
that of the â-counterpart. Also, a 13C NMR chemical

4
690 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 26
Ekthawatchai et al.
Sch em e 3
ously decomposed in solutions or upon storage at room
temperature giving complex mixtures. Chemical yields
of 10a -d shown were calculated from the pure samples
obtained directly from PLC purification without crystal-
lization. NMR spectra of the products, dimer 10, indi-
synthesis of novel artemisinin analogues. The reaction
involved Michael addition of 1 equiv of the bis-Grignard
reagent, 14, to the exo double bond of 6 (2 equiv) at
-78 °C to yield a mixture of artemisinin derivatives,
which after PLC separation (silica gel, EtOAc/hexane
) 30/70 as the developing solvent) afforded two isomeric
artemisinin adducts (15 (C-9, â-) and 16 (C-9, R-)), two
unsymmetrical and one symmetrical bis adducts (17, 18
(C-9, R-; C-9′, â-), and 19 (C-9, R-; C-9′, R-)), respectively,
and tris- (20) and tetrakis- (21) adducts as shown in
Scheme 4.
cated that they are symmetrical molecules. For example,
+
1
0a (m/e 677.5 (M + Na) for C32H46O10S2) showed only
one absorption at δ 2.31 ppm (dd, J ) 11.5, 3.5 Hz) for
the two identical protons at C-9 and C-9′ and also one
singlet at δ 5.96 ppm assignable for equivalent protons
at C-12 and C-12′. Chemical shifts of the protons
described led to the conclusion for the R-stereochemistry
at both C-9 and C-9′ of the dimers 10a -d .
Evidently, the bifunctional Grignard reagent reacted
either on one end with the first molecule of 6 (giving 15
and 16 after protonation) followed by trapping the
emerging enolate with another molecule of 6 (to give
17) or at both ends with two molecules of 6 (yielding 18
and 19) followed by further addition of the bis enolate
to one or two molecules of 6 to provide the trimer, 20,
(
b) Gr ign a r d Rea gen ts. Grignard reagents have
been employed as alternative Michael donors in the
addition reactions to 6 (Scheme 3). The reactions were
conducted in THF solution at -78 °C in the presence of
a catalytic amount of Cu(I) salt to afford, upon workup
at the same temperature, adduct 11 as a major product.
However, in contrast to those of organolithiums, reac-
tions of 6 with Grignard reagents also provided reason-
able amounts of the C-9â-stereoisomers (12), and the
dimer 13 resulted from further reaction of the emerging
enolate 7 with another molecule of 6 as shown in
Scheme 3. It should be noted that probably due to the
difference in nature of the enolates, the stereochemistry
at C-9 of dimer 13, obtained from the attack of 7 to 6,
is opposite to that observed in 8e.
1
13
or tetramer, 21, respectively. Mass, H, and C NMR
spectral data analyses were employed for structural and
stereochemical identification of all products obtained
above. For example, two types of protons at C-9, the
â-form at C-9 (δ 3.20 ppm) and R-form at C-9′ (δ 2.10
ppm) and two singlets at δ 5.86 and 5.92 ppm, assign-
able for two protons at C-12 and C-12′, were observed
+
for the dimer 18a (m/e 619.3569 (M + 1) ). On the other
+
hand, compound 19a (m/e 619.3563 (M + 1) ) showed
identical protons at C-9 and C-9′ (δ 2.05 ppm) and at
C-12 and C-12′ (δ 5.92 ppm). The trimer (m/e 900.6 (M
(
c) Bifu n ction a l Gr ign a r d Rea gen ts. Bifunctional
+
Grignard reagents are of our interest in the further
+ 1) ) showed two sets of C-9 and C-9′′ â-protons and

Synthese of Artemisinin by Additions to Artemisitene
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 26 4691
Sch em e 4
three singlets for three different protons at C-12, C-12′,
Because artemisinin is believed to require iron for its
antimalarial action, covalent linkage of artemisinin to
iron chelator yielded more potent artemisinin deriva-
1
and C-12′′ in the H NMR spectrum, while identification
+
of the tetramer (m/e 1178.6 (M )) was surprisingly
2
4
straightforward due to its symmetrical structural fea-
ture.
tives. The 1,4-addition of the anion derived from
methylene-bis(diethyldithiocarbamate), an iron chelator,
to 6 provided the artemisinin-iron chelator adduct, 8a ,
showing much better antimalarial activity against P.
falciparum than 1 as predicted with a good therapeutic
Biologica l Activities. All compounds prepared as
described above were subjected to in vitro malaria
screening system against P. falciparum (K1, multidrug
resistant strain), and the assay was performed follow-
ing the microculture radioisotope technique described
2
5
index of 3500 times of its cytotoxic effect to vero cells.
This supports the belief that iron is essential in the
2
2
4-7
by Desjardins using both artemisinin and dihydro-
artemisinin as standards. Cytotoxicity against KB, BC,
and vero cells was tested using the protocol described
mode of action of artemisinin.
It has been reported that certain artemisinin dimers
linked together at C-10 were much more potent against
2
3
14,15
by Skehan. IC50 values of the standard compound,
malaria parasites than artemisinin,
but as can be
ellipticine, were 2.44 ( 0.41, 2.03 ( 0.82, and 2.84 (
seen in Table 2, dimers 10a -d , 18, and 19 are not. It is
important, however, to note that stereochemical orien-
tations at C-9 and C-9′ of the two artemisinin molecules
in these dimers play almost insignificant roles with
regard to their biological activities, against both P.
falciparum and KB, BC, and vero cells (Table 2).
Antimalarial activities of artemisinin dimers (Table
3), trimers, and tetramers (Table 4) are quite impressive
because they are higher than artemisinin (with the
exception of 21b). Also, their differences in antimalarial
activities and toxicities are quite large, except for
compounds 13d -f, which show comparatively high
potency against vero cells.
0
.41 µM, respectively, for KB, BC, and vero cells. Re-
sults are grouped into four categories, i.e., artemisinin
derivatives substituted at C-16 by simple alkyls, aryls,
and iron chelator (Table 1); artemisinin dimers linked
by alkyl and alkyl disulfide chains (Table 2); other
artemisinin dimers (Table 3); and artemisinin trimers
and tetramers (Table 4).
It can be seen from Table 1 that activities against
malaria parasites of most C-16 substituted artemisinin
derivatives are comparable to artemisinin. In the cases
where both R-isomers and â-isomers were obtained from
1
,4-addition reactions of nucleophiles to 6, the â-isomers
are, in general, more potent against P. falciparum than
their counterparts, suggesting stereochemistry depend-
ent activity of these derivatives.10
Most interesting is compound 8e, obtained from the
addition of artemisinin anion to 6 (Scheme 1), whose
activity against P. falciparum is 1 order of magnitude

4
692 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 26
Ekthawatchai et al.
Ta ble 1. Antimalarial Activity and Cytotoxicity of Compounds
Ta ble 2. Antimalarial Activity and Cytotoxicity of Compounds
10a -d , 18a ,b, and 19a ,ba
a
8
a -e, 11a -g, and 12a -g
cytoxicity
cytoxicity
antimalarial activity
vero cells
IC50
antimalarial activity
EC50 (nM)
vero cells
IC50
(µM)
EC50 (nM)
KB IC50 BC IC50
KB IC50
(µM)
BC IC50
(µM)
b
(µM)c
(therapeutic index)b
c
cmpds
(therapeutic index)
(µM)
(µM)
cmpds
8
a
1.9d
2.5
2.4
6.6
10a
10b
10c
10d
18a
7.4
13.0
5.7
12.0
10.5
d
d
d
d
d
d
d
d
d
d
d
d
3
(
3.5 × 10 )
e
8
8
8
8
8
1
1
1
1
1
1
1
1
1
1
1
1
1
1
b
12.8
inactive inactive
inactive inactive
inactive inactive
inactive inactive
>130
>130
>130
>130
89
4
(
(
(
>1.0 × 10 )
d
c (C-9R)
24.8
1.8
1.6
10
3
2
>5.2 × 10 )
(9.5 × 10 )
e
c (C-9â)
d
8.8
19a
18b
19b
1.4
2.3
1.6
10
4
3
>1.5 × 10 )
(7.1 × 10 )
d
0.91e
>26.1
0.76
1.1
0.36
1.1
2.8
4.9
3
3
(
>5.0 × 10 )
(3.1 × 10 )
d
1.1e
e
1.0
8.9 × 10 )
11
23
13
4
3
(
(4.5 × 10 )
1a
inactive inactive
inactive inactive
>130
>130
>130
>130
19
a
4
Artemisinin, EC50 ) 11.9 nM; dihydroartemisinin, EC50 ) 2.96
(
(
(
(
>1.2 × 10 )
nM. b Therapeutic index ) IC50 of vero cells/EC50 of artemisinin
2a
6.3
c
4
derivatives. Maximum concentration ) 130 µM with ellipticine
>2.1 × 10 )
d
as positive control. Not performed due to compound’s instability.
1b
2b
1c
13.8
54
inactive inactive
11 11
inactive inactive
inactive
e
3
Dihydroartemisinin, EC50 ) 0.55 nM.
>9.4 × 10 )
11.3
Ta ble 3. Antimalarial Activity and Cytotoxicity of Compounds
4
>1.2 × 10 )
1
3a -ga
11.7
3
(
1.6 × 10 )
cytoxicity
2c
8.9
>130
14
4
antimalarial activity
EC50 (nM)
vero cells
IC50
(µM)
(
>1.5 × 10 )
KB IC50
(µM)
BC IC50
(µM)
1d
2d
1e
13.8
6.2
6.2
5.2
11
6.2
5.7
5.0
14
(therapeutic index)b
c
3
cmpds
(
1.0 × 10 )
12.9
1.2 × 10 )
10.3
16
13a
4.4
inactive inactive
>130
>130
63
3
4
(
(>3.0 × 10 )
13
13b
13c
13d
13e
13f
13g
4.3
inactive inactive
3
4
(
1.3 × 10 )
(>3.0 × 10 )
2e
8.4
16
3.9
23
36
3
4
(
1.9 × 10 )
(1.6 × 10 )
1f
12.9
35
41
120
5.0
4.0
5.7
7.7
4.0
1.3
1.2
7.4
3.5
2.5
3
2
(
9.3 × 10 )
(5.0 × 10 )
d
2f
6.7
inactive inactive
44 41
inactive inactive
>130
>130
>130
3.4
2.4
4
2
(
1.9 × 10 )
(7.1 × 10 )
2.3e
1.1
1g
2g
18.2
3
2
(
7.1 × 10 )
20.7
6.3 × 10 )
(4.8 × 10 )
2.1e
5.5
3
3
(
(2.6 × 10 )
a
a
Artemisinin, EC50 ) 12.1 nM; dihydroartemisinin, EC50 ) 3.0
Artemisinin, EC50 ) 12.1 nM; dihydroartemisinin, EC ) 3.0
50
nM. b Therapeutic index ) IC50 of vero cells/EC50 of artemisinin
nM. b Therapeutic index ) IC50 of vero cells/EC of artemisinin
50
c
c
derivatives. Maximum concentration ) 130 µM with ellipticine
derivatives. Maximum concentration ) 130 µM with ellipticine
d
as positive control. d Artemisinin, EC50 ) 7.81 nM; dihydroartemi-
sinin, EC50 ) 1.40 nM. e Dihydroartemisinin, EC50 ) 0.81 nM.
as positive control. Artemisinin, EC50 ) 11.7 nM; dihydroartemi-
e
sinin, EC50 ) 2.92 nM. Artemisinin, EC50 ) 7.81 nM; dihydro-
artemisinin, EC50 ) 1.40 nM.
Ta ble 4. Antimalarial Activity and Cytotoxicity of Compounds
20a ,b and 21a ,ba
better than that of the parent compound, 1, and displays
low cytotoxicity. X-ray crystal data of this compound
indicated that in crystal form, 8e arranged itself such
that all oxygen atoms were engulfed by the hydrophobic
part of its molecule as shown in Figure 1. A plausible
explanation can be put forward that the antimalarial
activity of dimer 8e correlates with its lipophilicity due
to the compound’s better ability to penetrate the mem-
brane. However, this explanation should be treated with
caution because the conformation of 8e in solution is
still not known and also there is a report that the
increase in lipophilicity may lead to higher cytotoxicity
of artemisinin derivatives.26
X-r a y Cr ysta llogr a p h ic Stu d y of 8e. Crystal data
of 8e: C30H40O10, MW ) 560.62, monoclinic, P21, a )
9
1
density was 1.307 g cm . Data were collected from a
0
cytoxicity
antimalarial activity
vero cells
IC50
(µM)
EC50 (nM)
KB IC50
(µM)
BC IC50
(µM)
(therapeutic index)b
c
cmpds
0a
2
2.4
1.7
2.8
2.0
5.4
1.2
2.0
2.8
9.6
6.6
3
(
2.8 × 10 )
20b
3.1
10
3
(3.2 × 10 )
2
2
1a
1b
5.8
9.2
>42
3
(
1.6 × 10 )
>20
(>2.1 × 103)
a
Artemisinin, EC50 ) 12.1 nM; dihydroartemisinin, EC50 ) 3.0
nM. b Therapeutic index ) IC50 of vero cells/EC50 of artemisinin
c
derivatives. Maximum concentration ) 130 µM with ellipticine
as positive control.
.214(1) Å, b ) 11.282(1) Å, c ) 14.181(1) Å, V )
424.7(2) Å , F(000) ) 600. With Z ) 2, the calculated
3
-
3
radiation (λ ) 0.710 73 Å) in θ-2θ scan mode. A total
of 4761 independent reflections was measured (4292
observed, |Fo| > 4σ|Fo|) between 2 and 25°. There was
3
.30 × 0.30 × 0.50 mm crystal at room temperature
on an Enraf-Nonius CAD4 diffractometer with Mo KR

Synthese of Artemisinin by Additions to Artemisitene
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 26 4693
F igu r e 1. Computer-generated perspective drawing of 8e.
no crystal decay during data collection, and no correction
was made for absorption. The crystal structure was
solved by direct methods using SHELXS-97, and then,
all atoms except hydrogen atoms were refined aniso-
sodium/benzophenone) was used in all experiments. Reactions
were carried out under nitrogen atmosphere.
Typ ica l P r oced u r e. Com p ou n d 8a . To a solution of LDA
(
0.70 mmol) in THF (1 mL) at -78 °C was slowly added a
solution of methylene-bis(diethyldithiocarbamate) (183 mg,
.59 mmol) in THF (2 mL), and the reaction mixture was left
2
tropically on F using SHELXL-97 to give a final
0
R-factor of 0.0449 (wR ) 0.1253) with a goodness of fit
stirring at -78 °C for 45 min. A solution of 6 (150 mg, 0.54
mmol) in THF (2 mL) was added, and the reaction mixture
)
0.909 and a data-to-parameter ratio of 13.3:1. Atomic
coordinates, bond lengths, bond angles, and thermal
parameters have been deposited with the Cambridge
Crystallographic Data Center, 12 Union Road, Cam-
bridge CB2 1EZ, England (CCDC161500).
was left stirring at -78 °C for 5 h. Saturated aqueous NH
solution workup at -78 °C followed by extraction with CH Cl
drying (MgSO ), and evaporation gave the crude product,
4
Cl
2
2
,
4
which was purified by PLC (silica gel, 25% EtOAc/hexane as
eluent) to afford a single isomer (C-9â), 8a , as yellow solid,
2
5
28.5 mg (80%), mp 133-135 °C. MS (LC/(+)ESI-MS) m/e:
Con clu sion
+
91.9 (M + 1) . Anal. (C26
Com p ou n d 8b. Yield, 44%; white crystals (CH
mp 119-120.5 °C. ESITOF exact mass m/e: 401.1945 (M +
42 2 5 4
H N O S ) C, H, N.
Nucleophilic additions of alkyllithium, lithium keto
and ester enolates, Grignard, and bifunctional Grignard
reagents to 6 have led to various C-16-derived artemisi-
nin derivatives, which include artemisinin monomers,
dimers, trimers, and tetramers. Most of these com-
pounds showed antimalarial activity better than or
comparable to that of artemisinin with no or low
cytotoxicity against two tumor cell lines, KB, BC, and
vero cells. Although some of these compounds are toxic
to vero and also cancer cells at micromolar ranges, the
cytotoxicities are generally over 500 times higher than
their corresponding antimalarial activities (EC50 val-
ues). This indicates that these artemisinin derivatives
are highly selective to malaria parasites with a large
safety window over their cytotoxic effects.
2 2
Cl /hexane);
+
1
) . Anal. (C23
Com p ou n d 8c. The crude product was purified by PLC
silica gel, 25% EtOAc/hexane as eluent) to obtain two sepa-
28 6
H O ) C, H.
(
rable (â, R) C-9 stereoisomers in ratio of 1.0:1.6: â-isomer, 55.2
mg (28%), and R-isomer, 88.4 mg (45%), respectively.
Com p ou n d 8c (C-9â). White crystals (EtOAc/hexane); mp
+
65-67 °C (dec). ESITOF exact mass m/e: 309.1913 (M + 1) .
6
Anal. (C21H27NO ) C, H, N.
Com p ou n d 8c (C-9R). White crystals (EtOAc/hexane); mp
+
1
09.8 °C (dec). ESITOF exact mass m/e: 390.1614 (M + 1) .
6
Anal. (C21H27NO ) C, H, N.
Com p ou n d 8d . Yield, 82%; white crystals (CH
mp 103-105 °C. MS (LC/(+)ESI-MS) m/e: 383.5 (M + 1) .
Anal. (C20 ) C, H.
Com p ou n d 8e. Yield, 88%; white crystals (CH Cl /hexane).
2 2
Cl /hexane).
+
30 7
H O
2
2
+
mp 188-190 °C. MS (LC/(+)ESI-MS m/e: 563.7 (M + 1) . Anal.
Exp er im en ta l Section
(C H O ) C, H.
3
0
42 10
Com p ou n d 10a . Yield, 86%; white semisolid. MS (LC/
Ch em istr y. Melting points were determined by an Elec-
+
(
+)ESI-MS for C32
46 10 2
H O S m/e: 677.5 (M + Na) .
trothermal melting point apparatus and were uncorrected. The
1
13
Com p ou n d 10b. Yield, 91%; white semisolid. MS (LC/
H and C NMR spectra were recorded on a Bruker DPX 300
+
(
+)ESI-MS) for C33
48 10 2
H O S m/e: 691.8 (M + Na) .
and Bruker DRX 400. ESITOF mass spectra were obtained
from a Micromass LCT mass spectrometer, and lock mass
calibration was applied for the determination of the accurate
mass. IR spectra were recorded on an FT-IR system 2000
Com p ou n d 10c. Yield, 97%; white semisolid. MS (LC/
+
(+)ESI-MS) for C34
50 10 2
H O S m/e: 706.0 (M + Na) .
Com p ou n d 10d . Yield, 93%; white semisolid. MS (LC/
+
(
Perkin-Elmer) spectrometer. Elemental analyses were carried
52 10 2
(+)ESI-MS) for C35H O S m/e: 720.4 (M + Na) .
out by using a Perkin-Elmer elemental analyzer PE2400 series
II and a Perkin-Elmer elemental analyzer 2400 CHN. Merck
silica gel 60 PF254 was used for PLC. Solvents were distilled
before used. Dried, oxygen free THF (freshly distilled from
Gen er a l P r oced u r e for Syn th esis of Ar tem isin in Ad -
d u cts fr om th e Rea ction of Gr ign a r d Regen t w ith Ar -
t em isit en e. Grignard reagent27 was prepared in the usual
manner from magnesium turnings (3 equiv) and bromo

4
694 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 26
Ekthawatchai et al.
compound (1.5 equiv) in THF (0.7 M) at such a rate as to
maintain gentle reflux and stirred at room temperature for 2
h. The solution of Grignard reagent was added to a suspension
of Cu(I)Br in THF (1 mL), and the reaction mixture was left
stirring at -40 °C for 20 min. Then, a solution of 6 (1 equiv)
in THF (1 mL/50 mg) was added and the reaction mixture was
(5 equiv) are placed in a reaction flask. A mixture of 1,2-
dibromoethane (0.1 mL) in THF (5 mL) was added, and the
flask was warmed until gas evolution was apparent and then
further for approximately 2 min. The THF was removed by a
syringe and replaced by fresh THF (14 mL). 1,4-Dibromo-
alkane (1.1 equiv) was added, while the solution was stirred
magnetically. Stirring was continued for 6 h. A solution of 6
(2 equiv) at -78 °C was added, and the reaction mixture was
again left stirring at -78 °C for 2 h. Saturated aqueous NH
solution workup followed by extraction with CH Cl , drying
MgSO ), and evaporation gave crude product. The crude
4
Cl
2
2
(
4
left stirring for 24 h. Saturated aqueous NH
4
Cl solution
),
product was purified by PLC (silica gel, 25% EtOAc/hexane
as the eluent) to obtain two separable (R and â) C-9 stereo-
isomers (11 and 12) and a dimeric isomer (13), respectively.
workup followed by extraction with CH Cl , drying (MgSO
2
2
4
and evaporation gave the crude product. The crude product
was purified by PLC (silica gel, 30% EtOAc/hexane as the
eluent) to obtain monomeric, dimeric, trimeric, and tetrameric
adducts.
Com p ou n d 11a . Yield, 61%; white crystals (CH
2 2
Cl /hex-
ane); mp 126-127 °C. ESITOF exact mass m/e: 311.1869 (M
+
+
1) . Anal. (C17
H
26
O
5
) C, H.
2 2
Com p ou n d 15a . Yield, 3%; white crystals (CH Cl /hexane);
mp 122-124 °C (lit10 mp 122.5-123.5 °C). ESITOF exact mass
Com p ou n d 12a . Yield, 18%; white crystals (CH
2
Cl
2
/hex-
1
0
+
ane); mp 146-147 °C (lit mp 149-150 °C). MS (LC/(+)ESI-
m/e: 339.2170 (M + 1) . Anal. (C19
30 5
H O ) C, H.
+
MS) m/e: 311.4 (M + 1) . Anal. (C17
Com p ou n d 13a . Yield, 9%; white crystals (CH
mp 178-180 °C. MS (LC/(+)ESI-MS) m/e: 591.7 (M + 1) .
Anal. (C32 10) C, H.
Com p ou n d 11b. Yield, 41%; white crystals (CH
ane); mp 114.7-116.5 °C. MS (LC/(+)ESI-MS) m/e: 325.4 (M
H
26
O
5
) C, H.
Com p ou n d 16a . Yield, 14%; white crystals (CH
2 2
Cl /hex-
2
2
Cl /hexane);
ane); mp 96.4-97 °C. MS (LC/(+)ESI-MS) m/e: 339.4 (M +
+
+
1) . Anal. (C19
Com p ou n d 17a . Yield, 7%; white crystals (CH
mp 190-192 °C. MS (LC/(+)ESI-MS) m/e: 619.7 (M + 1) .
Anal. (C34 10) C, H.
Com p ou n d 18a . Yield, 5%; white crystals (CH
mp 176-178 °C. ESITOF exact mass m/e: 619.3569 (M + 1) .
Anal. (C34 10) C, H.
Com p ou n d 19a . Yield, 4%; white crystals (CH
mp 187-189 °C. ESITOF exact mass m/e: 619.3563 (M + 1) .
Anal. (C34 10) C, H.
Com p ou n d 20a . Yield, 4%; white crystals (CH
mp 149-151 °C. MS (LC/(+)ESI-MS) m/e: 900.6 (M + 1) .
Anal. (C49 15) C, H.
Com p ou n d 21a . Yield, 7%; white crystals (CH
mp 179-180 °C. MS (LC/(+)ESI-MS) m/e: 1178.6 (M ). Anal.
20) C, H.
2
Com p ou n d 15b. Yield, 3%; white crystals (CH /hexane);
30 5
H O ) C, H.
H
46
O
2 2
Cl /hexane);
+
2
2
Cl /hex-
50
H O
+
+
1) . Anal. (C18
H
28
O
5
) C, H.
2 2
Cl /hexane);
+
Com p ou n d 12b. Yield, 9%; white crystals (CH
2
Cl
2
/hexane);
1
0
mp 130-132 °C (lit mp 129-130 °C). MS (LC/(+)ESI-MS)
50
H O
+
m/e: 325.4 (M + 1) . Anal. (C18
Com p ou n d 13b. Yield, 13%; white crystals (CH
ane); mp 199-201 °C. MS (LC/(+)ESI-MS) m/e: 605.7 (M +
H
28
O
5
) C, H.
2 2
Cl /hexane);
+
2
2
Cl /hex-
50
H O
+
1
) . Anal. (C33
Com p ou n d 11c. Yield, 55%; white crystals (CH
ane); mp 96.4-97 °C. MS (LC/(+)ESI-MS) m/e: 339.4 (M +
H
48
O
10) C, H.
2 2
Cl /hexane);
+
2
2
Cl /hex-
70
H O
+
1
) . Anal. (C19
H
30
O
5
) C, H.
2 2
Cl /hexane);
+
Com p ou n d 12c. Yield, 15%; white crystals (CH
2
Cl
2
/hex-
1
0
ane); mp 122-124 °C (lit mp 122.5-123.5 °C). ESITOF exact
(C64
90
H O
+
mass m/e: 339.2170 (M + 1) . Anal. (C19
Com p ou n d 13c. Yield, 21%; white crystals (CH
ane); mp 190-192 °C. MS (LC/(+)ESI-MS) m/e: 619.7 (M +
H
30
O
5
) C, H.
2
Cl
mp 52-54 °C (lit mp 80.5-82 °C). MS (LC/(+)ESI-MS) m/e:
1
0
2
Cl
2
/hex-
+
353.5 (M + 1) . Anal. (C20
32 5
H O ) C, H.
+
1
) . Anal. (C34
50
H O10) C, H.
Com p ou n d 16b. Yield, 12%; colorless oil. ESITOF exact
+
Com p ou n d 11d . Yield, 47%; colorless oil. ESITOF exact
mass m/e: 353.2311 (M + 1) . Anal. (C20
Com p ou n d 17b. Yield, 7%; white crystals (CH
mp 160-162 °C. MS (LC/(+)ESI-MS) m/e: 633.7 (M + 1) .
Anal. (C35 10) C, H.
Com p ou n d 18b. Yield, 6%; white crystals (CH
mp 70-72 °C. MS (LC/(+)ESI-MS) m/e: 633.7 (M + 1) . Anal.
10) C, H.
Com p ou n d 19b. Yield, 16%; white crystals (CH
ane); mp 62-64 °C. MS (LC/(+)ESI-MS) m/e: 633.7 (M + 1) .
Anal. (C35 10) C, H.
Com p ou n d 20b. Yield, 32%; white crystals (CH
ane); mp 126.5-128 °C. MS (LC/(+)ESI-MS) m/e: 915.4 (M +
H
32
O
5
) C, H.
+
mass m/e: 353.2311 (M + 1) . Anal. (C20
H
32
O
5
) C, H.
Cl
ane); mp 52-54 °C (lit mp 80.5-82 °C). MS (LC/(+)ESI-MS
2
Cl /hexane);
2
+
Com p ou n d 12d . Yield, 15%; white crystals (CH
2
2
/hex-
1
0
52
H O
+
m/e: 353.5 (M + 1) . Anal. (C20
Com p ou n d 13d . Yield, 9%; white crystals (CH
mp 160-162 °C. MS (LC/(+)ESI-MS m/e: 633.8 (M + 1) . Anal.
10) C, H.
Com p ou n d 11e. Yield, 35%; colorless oil. ESITOF exact
H
32
O
5
) C, H.
2 2
Cl /hexane);
+
2
2
Cl /hexane);
+
(C35
52
H O
(C
35
H
52
O
2 2
Cl /hex-
+
+
mass m/e: 401.2305 (M + 1) . Anal. (C24
H
32
O
5
) C, H.
52
H O
1
0
Com p ou n d 12e. Yield, 8%; colorless oil. ESITOF exact
2
2
Cl /hex-
+
mass m/e: 401.2304 (M + 1) . Anal. (C24
Com p ou n d 13e. Yield, 35%; white crystals (CH
ane); mp 185.5 °C (dec). MS (LC/(+)ESI-MS m/e: 681.8 (M +
H
32
O
5
) C, H.
+
Cl
2
/hex-
1) . Anal. (C50
Com p ou n d 21b. Yield, 11%; white crystals (CH
ane); mp 172-174 °C. MS (LC/(+)ESI-MS) m/e: 1193.6 (M +
72
H O15) C, H.
2
2
2
Cl /hex-
+
1
) . Anal. (C39
Com p ou n d 11f. Yield, 75%; white crystals (CH
mp 122-123 °C. ESITOF exact mass m/e: 377.1765 (M + 1) .
Anal. (C21 ) C, H.
Com p ou n d 12f. Yield, 11%; white crystals (CH
mp 121-123 °C. ESITOF exact mass m/e: 377.1716 (M + 1) .
Anal. (C21 ) C, H.
Com p ou n d 13f. Yield, 4%; white crystals (CH
mp 127-129 °C. ESITOF exact mass m/e: 657.2866 (M + 1) .
Anal. (C36
Com p ou n d 11g. Yield, 65%; white crystals (CH
ane); mp 131-133 °C. ESITOF exact mass m/e: 377.1756 (M
52
H O10) C, H.
+
Cl
2
/hexane);
92
1) . Anal. (C65H O20) C, H.
2
+
An tim a la r ia l Activity. Continuous in vitro cultures of the
asexual erythrocytic stage of P. falciparum (K1, multidrug
resistant stain) were maintained. Quantitative assessment of
antimalarial activity in vitro was determined using the mi-
croculture radioisotope technique based upon the method
described by Desjardins.22 Effective concentration (EC50) rep-
resents the concentration that cause 50% reduction in parasite
H
25FO
5
2
2
Cl /hexane);
+
H
25FO
5
2
2
Cl /hexane);
+
3
growth as indicated by the in vitro uptake of [ H]hypoxanthine
H
45FO10) C, H.
by P. falciparum.
2
2
Cl /hex-
Cyt ot oxicit y Assa y. The cytotoxicity of the artemisinin
analogues against KB, BC, and vero cell lines was evaluated
employing the colorimetric method as by Skehan and co-
workers.23 Ellipticine was used as the reference substance,
exhibiting the activity toward KB and BC cell lines, with an
EC50 of 2.44 ( 0.41 and 2.03 ( 0.41 µM, respectively.
+
+
1) . Anal. (C21
Com p ou n d 12g. Yield, 8%; white crystals (CH
mp 104-106 °C. ESITOF exact mass m/e: 377.1758 (M + 1) .
Anal. (C21 ) C, H.
Com p ou n d 13 g. Yield, 15%; white crystals (CH
hexane); mp 188-190 °C. ESITOF exact mass m/e: 657.2883
5
H25FO ) C, H.
2
2
Cl /hexane);
+
H
25FO
5
2
2
Cl /
+
Ack n ow led gm en t. Student grants from National
Science and Technology Development Agency (NSTDA)
and Thailand Graduate Institute of Science and Tech-
(
M + 1) . Anal. (C36
Gen er a l P r oced u r e for th e Rea ction of Ar tem isiten e
a n d 1,n -Dim a gn esiu m br om oa lk a n e.28 Magnesium turnings
H45FO10) C, H.

Synthese of Artemisinin by Additions to Artemisitene
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 26 4695
nology (TGIST) to S.E. and NSTDA’s Senior Research
Scholar to Y.T. are gratefully acknowledged. This work
also received support from U.S. NIH ICIDR program.
We are indebted to the Biodiversity Research and
Training Program (BRT), Thailand-Tropical Diseases
Research Programme (T-2), and BIOTEC for the sup-
ports of biological screening facilities.
(14) Posner, G. H.; Ploypradith, P.; Hapangama, W.; Wang, D.;
Cumming, J . N.; Dolan, P.; Kensler, T. W.; Klinedinst, D.;
Shapiro, T. A.; Zheng, Q. Y.; Murray, C. K.; Pilkington, L. G.;
J ayasinghe, L. R.; Bray, J . F.; Daughenbaugh, R. Trioxane
Dimers have Potent Antimalarial, Antiproliferative and Anti-
tumor Activities in vitro. Bioorg. Med. Chem. 1997, 5, 1257-
1
265.
(15) Posner, G. H.; Ploypradith, P.; Parker, M. H.; O’Dowd, H.; Woo,
S.-H.; Northrop, J .; Krasavin, M.; Dolan, P.; Kensler, T. W.; Xie,
S.; Shapiro, T. A. Antimalarial, Antiproliferative, and Antitumor
Activities of Artemisinin-Derived, Chemically Robust, Trioxane
Dimers. J . Med. Chem. 1999, 42, 4275-4280.
Su p p or tin g In for m a tion Ava ila ble: NMR (1H and 13C)
and IR spectroscopic data of all artemisinin derivatives. This
material is available free of charge via the Internet at http://
www/pubs.acs.org.
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J M0103007