Synthesis, antimalarial activity, biomimetic iron(II) chemistry, and in vivo metabolism of novel, potent C-10-phenoxy derivatives of dihydroartemisinin

Article abstract of DOI:10.1021/jm000987f

The combination of TMSOTf and AgClO₄ promotes the efficient C-10-phenoxylation of dihydroartemisinin (3) in good chemical yield and excellent stereoselectivity. All of the new phenoxy derivatives have potent in vitro antimalarial activity. On t

Full text of DOI:10.1021/jm000987f

58
J . Med. Chem. 2001, 44, 58-68
Syn th esis, An tim a la r ia l Activity, Biom im etic Ir on (II) Ch em istr y, a n d in Vivo
Meta bolism of Novel, P oten t C-10-P h en oxy Der iva tives of Dih yd r oa r tem isin in
Paul M. O’Neill,*,† Alison Miller,† Laurence P. D. Bishop, Stephen Hindley,† J ames L. Maggs, Stephen A. Ward,‡
Stanley M. Roberts,† Feodor Scheinmann,§ Andrew V. Stachulski,§ Gary H. Posner,# and B. Kevin Park*,‡
Robert Robinson Laboratories, University of Liverpool, Liverpool L69 7ZD, England, Department of Pharmacology and
Therapeutics, University of Liverpool, Liverpool L69 3GE, England, UFC Ltd., Synergy House, Guildhall Close, Manchester
Science Park, Manchester M15 6SY, England, and Department of Chemistry, The J ohns Hopkins University, Remsen Hall,
Baltimore, Maryland 21218
Received May 15, 2000
The combination of TMSOTf and AgClO4 promotes the efficient C-10-phenoxylation of
dihydroartemisinin (3) in good chemical yield and excellent stereoselectivity. All of the new
phenoxy derivatives have potent in vitro antimalarial activity. On the basis of the excellent
yield and stereoselectivity obtained for the p-trifluoromethyl derivative 7b, this compound and
the parent phenyl-substituted derivative 5b were selected for in vivo biological evaluation
against Plasmodium berghei in the mouse model and for metabolism studies in rats. Compound
7b demonstrated excellent in vivo antimalarial potency with an ED50 of 2.12 mg/kg (cf.
artemether ) 6 mg/kg) versus P. berghei. Furthermore, from preliminary metabolism studies,
this compound was not metabolized to dihydroartemisinin; suggesting it should have a longer
half-life and potentially lower toxicity than the first-generation derivatives artemether and
arteether. From biomimetic Fe(II)-catalyzed decomposition studies and ESR spectroscopy, the
mechanism of action of these new lead antimalarials is proposed to involve the formation of
both primary and secondary C-centered cytotoxic radicals which presumably react with vital
parasite thiol-containing cellular macromolecules.
In tr od u ction
The spread of the malaria parasite’s resistance to
chloroquine (1) has led the WHO to predict that without
new antimalarial drug intervention, the number of cases
of malaria will have doubled by the year 2010.1,2
Artemisinin (2, qinghaosu) is an unusual 1,2,4-trioxane
which has been used clinically in China for the treat-
ment of multidrug-resistant Plasmodium falciparum
malaria. However the therapeutic value of 2 is limited
to a great extent by its low solubility in both oil and
water. Consequently, in the search for more effective
and soluble drugs, a number of derivatives of the parent
drug have been prepared. Reduction of artemisinin
produces dihydroartemisinin (3, DHA), which has in
turn led to the preparation of a series of semisynthetic
first-generation analogues which include artemether
and short half-life (Scheme 1).4 Replacement of the
oxygen at the C-10 position with carbon would be
expected to produce compounds not only with greater
metabolic stability but also with a longer half-life and
(4a , R ) -Me) and arteether (4b, R ) -Et) and the
water-soluble derivative sodium artesunate (4c, R )
-C(O)CH2CH2CO2Na).
Although these derivatives are potent antimalarial
potentially lower toxicity. Consequently, several groups
agents in vitro, they have poor bioavailability principally
have developed synthetic and semisynthetic approaches
as a result of the metabolic instability of the acetal
function. One of the principal routes of metabolism of
to C-10-carba analogues.5-8 An alternative approach to
increasing the metabolic stability of artemisinin deriva-
tives involves incorporation of a phenyl group in place
of the alkyl group (in the ether linkage) of first-
arteether (4b), for example, involves oxidative dealkyl-
ation to DHA (3), a compound associated with toxicity3
generation analogues, e.g. 4a and 4b.
* To whom correspondence should be addressed. Tel: 0151-794-
This modification would be expected to block oxidative
8216. Fax: 0151-794-8218. E-mail: p.m.oneill01@liv.ac.uk.
† Robert Robinson Laboratories, University of Liverpool.
metabolic formation of DHA in vivo (e.g. Scheme 1). This
paper deals with the synthesis, in vitro and in vivo
biological activity, metabolism, and biomimetic Fe(II)
reactions of a new series of C-10-phenoxy derivatives
‡ Department of Pharmacology and Therapeutics, University of
Liverpool.
§ UFC Ltd.
#
The J ohns Hopkins University.
10.1021/jm000987f CCC: $20.00 © 2001 American Chemical Society
Published on Web 11/23/2000

C-10-Phenoxy Derivatives of Dihydroartemisinin
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 1 59
Sch em e 3
Sch em e 1
The reactions compete for the intermediate oxonium
ion. Similar results were obtained using either TMSOTf
or TMSCl as Lewis acid with phenol as nucleophile.
Further studies involving the use of the Mitsunobu
reaction10,11 (DEAD, Ph3P, phenol) on 3 gave high
stereoselectivity (12.5/1 â/R) but low overall yield (27.5%).
Ch a r t 1. Structures of C-10-Phenoxy Derivatives of
DHA
The dihydroartemisinin lactol can be recognized as a
relative of a pyranose sugar with a free anomeric
hydroxyl group.12 There are many papers detailing
reactions of glycosides, including C-C bond formation
at the anomeric site.13,14 Recently, studies by Suzuki
investigated the O to C glycoside rearrangement of
phenoxy glycosides.15 Different Lewis acid promoters
have been used, including BF3 etherate, SnCl4, and Cp2-
HfCl2-AgClO4 with varying success. In 1992, Toshima
discovered the efficient â-stereoselective C-aryl glycosi-
dation of 1-O-methyl sugars by the use of a TMSOTf-
AgClO4 catalyst system.16 This procedure gives excellent
yields and diastereoselectivity in favor of the â-isomer
by rearrangement of the preformed phenoxy glycoside
as shown (Scheme 3).
Sch em e 2
Such intriguing results led us to explore the use of
TMSOTf-AgClO4 catalysis in our DHA-phenol cou-
pling reactions. This approach involves dissolving 1
equiv of DHA, approximately 2 equiv of the desired
phenol and 0.20 equiv of AgClO4 in anhydrous dichloro-
methane, under nitrogen at -78 °C. TMSOTf (1 equiv)
is then added to the reaction mixture. In every case,
the reaction provided excellent yields with good dias-
tereoselectivity in favor of the â-isomers (Scheme 4).
Notably, only minor quantities of AHA were observed
(Scheme 3) and no O- to C-aryl glycoside rearrangment
was noted for any of the phenoxy derivatives obtained
(in contrast to the situation depicted in Scheme 3).
(â-series 5b-11b and R-series 5a -11a ; Chart 1). ESR
spectroscopy has also been utilized to provide evidence
for the existence of potentially toxic C-centered radicals.
Ch em istr y
As can be seen, compared with earlier methods
described above, the increase in yield upon using the
new catalyst combination was dramatic. Noticeably,
when R ) OMe, the yield increased by 55% (the yield
was only 18% using BF3‚Et2O catalysis). There is no
obvious explanation for the success of this reaction. It
may be that under these conditions the oxonium inter-
mediate is stabilized. Alternatively the conditions may
catalyze slow oxonium ion formation. Hence at any
given time there are larger quantities of nucleophile
available to react with the intermediate oxonium species
(Figure 1).
Several synthetic approaches have been investigated
for the production of target C-10 derivatives. The initial
approach was to couple DHA with various phenols using
boron trifluoride diethyl etherate catalysis. Many re-
searchers have used this method to form first-generation
endoperoxides. The method involved reacting 1 equiv
of DHA with 4 equiv of the phenol in anhydrous ether
at room temperature in the presence of BF3 etherate.
In every case, the major product obtained was the
anhydro derivative (AHA) in high yield. This suggests
the involvement of an oxonium ion (Scheme 2) inter-
mediate. The oxonium ion, being a chemically reactive
intermediate either reacts with a phenol to give the
phenyl ether or loses a proton to give the byproduct,
anhydroartemisinin (AHA).
The reaction was repeated for a range of phenols, and
yields are recorded in Scheme 4. The diastereoselectivity
ratios were calculated from NMR data and the stereo-

60 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 1
O’Neill et al.
Sch em e 4
Ta ble 1. In Vitro Antimalarial Activity of C-10-R-Phenoxy
(5a -12a ) and C-10-â-Phenoxy (5b-11b) Derivatives of DHA
versus P. falciparum (K1 strain)
compd
IC50 (nM)
SE
compd
IC50 (nM)
SE
5a
6a
7a
8a
9a
2.97
3.18
4.62
3.86
5.70
2.88
11.15
0.02 5b
0.05 6b
0.06 7b
0.24 8b
1.01 9b
3.66
3.92
5.29
4.58
4.58
8.89
4.55
1.88
0.11
0.08
0.04
0.04
0.05
0.12
11a
artemisinin
0.04 11b
0.32 artemether
Ta ble 2. In Vitro Antimalarial Activity of C-10-R-Phenoxy
(5a -12a ) and C-10-â-Phenoxy (5b-11b) Derivatives of DHA
versus P. falciparum (HB3 strain)
compd
IC50 (nM)
SE
compd
IC50 (nM)
SE
5a
6a
7a
8a
9a
2.61
2.87
3.42
3.32
4.04
2.88
9.67
0.31 5b
0.10 6b
0.05 7b
0.14
0.03 9b
0.04 11b
3.24
3.24
3.90
0.05
0.16
0.07
F igu r e 1. X-ray crystal structure of phenoxy compound 8b.
chemistry of the phenoxy derivatives was confirmed by
X-ray crystallography.17
2.88
7.06
3.42
0.21
0.04
1.14
11a
artemisinin
Recent studies by Wu et al. have described the boron
trifluoride-catalyzed Friedel Crafts alkylation of a C-10-
acetate derivative of 3 with naphth-2-ol.18 The reaction
is proposed to proceed through the intermediacy of the
â- and R-naphthoxy derivatives. The fact that no O to
C rearrangement was observed in our system may
reflect the much lower temperatures (-78 °C) employed
compared with those required to induce glycosidic
rearrangement. Currently, we are investigating the use
of the TMSOTf-AgClO4 activating system and higher
temperatures to induce O to C rearrangement of these
phenoxy derivatives.
0.24 artemether
Ta ble 3. In Vivo Antimalarial Activity of DHA, Artemether,
5b, and 7b versus P. berghei
compd
ED50 (mg/kg)
SD (()
5b
7b
3.09
2.12
6.02
1.95
0.30 (3)
0.20 (2)
0.15 (3)
0.32 (2)
artemether
DHA
slightly more potent than artemether. In the R-series,
the phenyl (5a ), 4-methylphenyl (6a ), 4-methoxyphenyl
(8a ), and naphthoxy derivatives of DHA were all more
potent than artemether.
Biology
In Vitr o a n d in Vivo An tim a la r ia l Activity. The
C-10-phenoxy derivatives were tested in vitro against
the K-1 chloroquine-resistant strain of P. falciparum.
The IC50 values are shown in Table 1.
On the basis of the excellent chemical yield and
stereoselectivity obtained for the p-trifluoromethyl de-
rivative 7b, this compound along with the parent
phenyl-substituted derivative 5b were selected for in
vivo biological evaluation against Plasmodium berghei
in the mouse model of malaria.
Table 3 lists the in vivo biological activity of lead
compounds 5b and 7b. Trioxane 7b has outstanding in
vivo antimalarial activity: equal to that of dihydroar-
temisinin and superior to that of artemether. Further
studies conducted by the WHO also demonstrate that
7b is active orally with an ED50 of 2.7 mg/kg and ED90
of 5.4 mg. The data shown in Table 4, which also has
data versus Plasmodium yoelli ssp. (NS), confirm that
It is immediately clear that compounds in this class
are potent antimalarials. The parent derivative (5a and
5b) and naphthyl derivative (11a ), for instance, have
similar antimalarial activity to artemether. In general
the R-series have similar biological activity to their
â-diastereomers against this strain. The phenoxy de-
rivatives were also tested against the chloroquine-
sensitive HB3 strain (Table 2). The most potent â-iso-
mers were the phenyl (5b), 4-methylphenyl (6b), and
4-fluorophenyl (9b) DHA derivatives. These were all

C-10-Phenoxy Derivatives of Dihydroartemisinin
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 1 61
Sch em e 5
Ta ble 4. In Vivo Comparison of 7b with Sodium Artesunate
versus P. berghei and P. yoelli ssp. NS
Sch em e 6
P. berghei (po)
P. yoelli (sc)
ED50
ED90
ED50
ED90
compd
(mg/kg)
(mg/kg)
(mg/kg)
(mg/kg)
7b
sodium artesunate
2.7
4.0
5.4
13.0
2.2
3.6
3.1
13.5
this new lead trioxane is superior to the clinically used
derivative, sodium artesunate.
Dr u g Meta bolism Stu d ies
The structure-metabolism relationships of the se-
lected phenoxy derivatives were investigated in the bile-
duct-cannulated rat. Cannulated and anesthetized male
Wistar rats were administered DHA and the following
O-ether derivatives of DHA (35 µmol/kg) by either iv
injection or gastric intubation: phenylDHA (iv) (5b ),
p-trifluoromethylphenylDHA (iv and po) (7b), and p-
methoxyphenylDHA (iv) (8b).
Com p ou n d 5b. Early bile fractions (hourly or half-
hourly) from iv-dosed animals contained DHA glucu-
ronide (13) and highly variable proportions of the furano
acetate glucuronide (14) (both identified by coelution
with the previously identified biliary metabolites of
DHA in rats), and also a metabolite (tR 21 min; it was
absent from the bile collected immediately before injec-
tion of the drug) corresponding to a glucuronide of the
hydroxylated drug (15) (m/z 570 for ammonium adduct)
(Scheme 6). The proportions of both metabolites in
sequential half-hourly bile collections decreased rapidly,
but they were still present in the 1.5-2.0 half-hourly
collection. Dearylation of phenylDHA to give DHA
should also yield phenol, which, in part, will be conju-
gated with glucuronic acid. However, no phenol glucu-
ronide was located in rat bile following administration
of 5b.
The hydroxyphenylDHA (hpDHA) glucuronide frag-
mented to the same lower mass ions as DHA glucu-
ronide, i.e. m/z 267, 221, and 163. From this, it is
deduced that the hydroxyl substituent is located on the
phenyl ring; the interpreted fragmentations of DHA
glucuronide and the hpDHA glucuronide are listed in
Table 5.
incubated with Helix pomatia (H-2) â-glucuronidase-
arylsulfohydrolase preparation (12.5 µL) at 37 °C for 5
h. This incubation was performed in parallel with an
incubation of bile from a rat given DHA. The incuba-
tions were analyzed by LC-MS without processing. The
prominent peaks of m/z 478 representing the two
metabolites of DHA (DHA glucuronide and its furano
acetate isomer) were absent after 5 h. The peaks
corresponding to DHA glucuronide and hpDHA glucu-
ronide in bile from the animal given phenylDHA were
likewise absent. The conversion of 13 to 14 may occur
in vivo, but isomerization followed by conjugation cannot
be excluded.
Com p ou n d 7b. Bile fractions (0-0.5 h to 1.5-2.0 h)
from iv-dosed rats contained three major (I, II, III on
Ultracarb column chromatograms) and two minor me-
tabolites (ion current peaks were absent from bile
collected immediately before administration of the drug)
corresponding to glucuronides of the hydroxylated drug
(m/z 638 for ammonium adduct). Superior resolution of
these compounds was obtained at shorter retention
times with the Nucleosil column. The proportions of the
major metabolites varied considerably with time but not
An aliquot (100 µL) of 0-1 h bile was diluted (1:1,
v/v) with sodium acetate buffer (0.1 M, pH 5.0) and

62 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 1
O’Neill et al.
Ta ble 5. Electrospray Mass Spectraa of Biliary Metabolites of 5b in the Rat
proposed metabolite
MS m/ z
DHA glucuronide (13)
478 ([M + NH4]+, 100), 267 ([M + NH4 - NH3 - DHG - H2O]+, 11), 221 ([267 - CO - H2O]+,
4), 163 ([221 - (CH3)2CO]+, 22)
OH-phenylDHA glucuronide (15)
570 ([M + NH4]+, 100), 267 ([M + NH4 - NH3 - DHG - dihydroxybenzene]+, 37),
221 ([267 -CO - H2O]+, 6), 163 ([221 - (CH3)2CO]+, 36)
a
Spectra were acquired at a cone voltage of 50 V; DHG ) dehydroglucuronic acid.
Ta ble 6. Electrospray Mass Spectraa of Biliary Metabolites of Compound 7b in the Rat
proposed metabolite
MS m/ z
II (16) (furano acetate gluc)
638 ([M + NH4]+, 100), 384 ([M + NH4 - NH3 - CH3CO2H - DHG - H2O]+, 60),
338 ([384 -CO - H2O]+, 55)
III (17) (OH-TFMphenylDHA gluc)
638 ([M + NH4]+, 57), 381 ([M + NH4 - NH3 - DHG - H2O - CO - H2O]+, 100),
219 ([381 - (p-TFMphenol)]+, 81)
a
Spectra were acquired at a cone voltage of 50 V; gluc ) glucuronic acid.
consistently from one animal to another. DHA glucu-
ronide was not seen.
doperoxide drugs such as mefloquine and lumefantrine
have been investigated.23
Cone-voltage fragmentation of metabolite II (16) (tR
23.5 min) at 50 V included loss of acetic acid and thereby
suggested a furano acetate structure (Table 6). The
fragmentation of III (17) (24 min) was indicative of a
conjugate with an intact endoperoxide function. The
parent ion was notable for the ease with which it
fragmented extensively even at very low cone voltages
(<30 V). Bile (80 µL) which contained a very high
proportion of metabolite II (16) was diluted (1:1, v/v)
with sodium acetate buffer and incubated with H-2
preparation (12 µL) at 37 °C for 24 h. The incubation
was analyzed by LC-MS after 3 and 24 h. The minor
m/z 638 peaks disappeared within 3 h, but metabolite
II was still present at a considerable intensity after 24
h. As in the case of 5b, isomerization of the hydroxylated
parent drug may precede glucuronylation.
Com p ou n d 8b. Bile fractions (hourly collections)
from an iv-dosed animal contained a peak in the mass
chromatogram for m/z 570 which was not present in
predosing bile and coeluted with the putative glucu-
ronide of hydroxyphenylDHA excreted by the rats given
phenylDHA. It was present, in diminishing amounts,
in the first and second hourly fractions but not in the
third. Another possible metabolite (tR 20.0 min), which
again was absent from the predose bile, yielded m/z 600,
corresponding to the glucuronide of the hydroxylated
drug. DHA glucuronide was not found.
Artemether is regarded as a prodrug of DHA in
humans though the two endoperoxides have similar
antiparasitic activities in vitro. Oral doses are absorbed
rapidly and converted extensively to DHA in humans
and rats. Systemic drug exposure is generally greater
when artemether is given intramuscularly, but the
bioavailability in severe malaria may be highly vari-
able.24 DHA in plasma is the only known human
metabolite of artemether, and plasma DHA has been
the only identified metabolite in rats.25
Since oxidative metabolism of artemether yields DHA,
a metabolite associated with neurotoxicity in high doses
and rapid O-glucuronidation to an inactive conjugate,
several groups have investigated the syntheses and
biological activity of C-10-carba analogues of the parent
drug. For example, J ung5,26 and Haynes and Vonwiller27
have shown that several C-10-alkyl and -aryl anlogues
of C-10-deoxoartemisinin have significant in vitro an-
timalarial activity. Pu and Ziffer, using allylsilane
chemistry, were able to prepare C-10-deoxoalkyl deriva-
tives which had potent antimalarial activity in vivo.28
Further studies have improved the synthesis of al-
lyldeoxo artemisinin, and we have recently prepared 10
fluorinated C-10-carba analogues, some of which were
more than 15 times as active as the parent drug.29
Further major advances in the preparation of C-10-
deoxo derivatives were recently reported by Posner.30
On the basis of the mechanism for the metabolic
conversion of artemether to DHA, we proposed that the
incorporation of an aryl group in place of the methyl in
4a would prevent oxidative formation of DHA in vivo.31
For this approach to be considered a viable alternative
to the incorporation of carbon at the C-10 position, in
terms of retarding metabolism to DHA and thereby
increasing the drug’s half-life, it was necessary to
demonstrate (i) that phenoxy derivatives are potent
antimalarials both in vitro and in vivo and (ii) that these
compounds are resistant to oxidative dearylation.
The data shown in Tables 1 and 2 confirm that all of
the phenoxy derivatives of 3 are potent antimalarials
with IC50 values comparable to that of artemether. The
parent derivatives (5a and 5b) and naphthyl derivative
(11a ), for instance, are slightly more potent than arte-
mether. In general compounds in the R-series have
similar biological activity to their â-diastereomers against
the K1 strain. The phenoxy derivatives were also tested
against the chloroquine-sensitive HB3 strain (Table 2).
Bile (0-1 h collection) was incubated with H-2 â-glu-
curonidase-arylsulfohydrolase preparation in the same
manner as bile from the rats administered either DHA
or phenylDHA. The peaks of m/z 570 and 600 corre-
sponding, respectively, to the putative hpDHA and
hydroxylated p-methoxyphenylDHA glucuronides were
absent from the enzymic incubations after 5 h.
Discu ssion
Artemether (Paluther, 4a ; Rhone-Poulenc-Rorer) is
the O-methyl ether of DHA and a first-generation
derivative of artemisinin (quinghaosu), the principal
antimalarial constituent of the medicinal plant Artemi-
sia annua.21,22Artemether is active against the eryth-
rocytic stage of multidrug-resistant P. falciparum.22
Oral, rectal, and intramuscular regimens are generally
rapidly effective and well-tolerated for both severe and
uncomplicated falciparum malaria, though short-term
monotherapy has been associated with high rates of
recrudesence. Combinations of artemether with nonen-

C-10-Phenoxy Derivatives of Dihydroartemisinin
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 1 63
Sch em e 7
Ta ble 7. In Vitro Fe(II)-Mediated Degradation of Phenoxy
Derivative 8b
The most potent â-isomers were the phenyl (5b ), 4-
methylphenyl (6b), and 4-fluorophenyl (9b) DHA de-
rivatives which were all more potent than artemether.
In the R-series, the phenyl (5a ), 4-methylphenyl (6a ),
4-methoxyphenyl (8a ), and naphthoxy derivatives of
DHA were all more potent than artemether. On the
basis of the excellent chemical yields and excellent
stereoselectivities obtained for the â-phenoxy trioxane
7b, this compound and the parent â-substituted ana-
logue 5b were selected for evaluation versus P. berghei
in the mouse model. Compound 7b demonstrated potent
activity, with an ED50 value directly comparable to that
of DHA and superior to that of artemether (Table 3).
Good activity was also displayed by the parent deriva-
tive 5b. In in vivo screens performed by the WHO this
derivative was significantly more potent than the clini-
cally used sodium artesunate, regardless of the route
of administration (Table 4).
ratio reaction
yield of
Fe(II) salt
solvent
T (°C) 18:19
time
products (%)
FeSO4‚7H2O CH3CN
rt
3:1 5 days
1:1 48 h
9:1 30 min
80a
65a
78
FeSO4‚7H2O CH3CN/H2O 37
FeCl2‚4H2O CH3CN
rt
a
Starting material was recovered in these reactions.
demonstrated that the C-4 radical can be efficiently
spin-trapped by MNP.38
The p-methoxy analogue 8b was allowed to react with
FeCl2‚4H2O, and in line with the previous work of
J efford on artemether, the two main products obtained
from the reaction were 18 (R ) p-MeO-Ar) and 19 (R )
p-MeO-Ar). Notably, when FeCl2‚4H2O was utilized, 18
was always the major product with a typical ratio of
9:1 (18:19). In contrast, degradation of 8b with FeSO4
resulted in a much slower reaction: with 20% of the
starting material still present after 5 days at ambient
temperature. Heating the reaction to 37 °C produced
65% turnover after 48 h. Interestingly, in this latter
reaction, the ratio of products was 1:1 (Table 7), indicat-
ing that both primary and secondary radicals are formed
as intermediates.
To obtain evidence for the generation of the radical
intermediates shown in Scheme 7, we investigated the
electron-transfer reactions with ESR spin-trapping
techniques using conditions recently reported by Butler
and co-workers for artemether.34 Using ferrous sulfate
and the water-soluble spin-trap DBNBS (sodium 3,5-
dibromo-4-nitrosobenzenesulfonate), the ESR signal
observed after 4 h indicated two spin-trapped radicals.
From the intensities of the signals, the secondary
C-spin-trapped radical 19a was the major product
(Scheme 8).
To gain insight into the potential mechanism of
peroxide antimalarials, several workers have investi-
gated the biomimetic Fe(II)-mediated degradation of a
range of 1,2,4-trioxanes, artemisinin derivatives, and
synthetic endoperoxides.32-34 These studies provide
surrogate chemical markers of radical formation. The
two major products obtained from the Fe(II)-mediated
degradation of artemisinin and artemether 18 (R ) Me)
are a ring-contracted THF acetate 18 (R ) Me for
artemether) and the hydroxydeoxo derivative 19 (R )
Me for artemether).34 The formation of the former
product is indicative of the formation of a primary
C-centered radical intermediate, whereas the formation
of 19 (R ) Me) indicates a secondary C-centered radical
intermediate (Scheme 7).
Significantly in the presence of very low concentra-
tions of non-heme Fe(II), the primary radical derived
from 1 forms a covalent adduct with cysteine.35 It was
suggested that this mode of reactivity may be respon-
sible for alkylation of parasite proteins by artemisinin.36
Alternatively, based on mechanistic work of Posner and
Oh, the radical at the C-4 position of the carbon
framework of artemisinin has been implicated as a
potential mediator of antimalarial activity.37 Recently,
evidence for the formation of this radical during ferrous-
mediated reductive activation was provided by Wu, who
However, in contrast to the study by Wu et al. on
artemisinin, where MNP was used as the spin-trapping
agent, the spin-trapped secondary radical obtained here
was always accompanied by quantities of the corre-
sponding primary spin-trapped product 18a . This dif-
ference could be a result of subtle differences in the
reactivity of MNP (2-methyl-2-nitrosopropane) and DB-
NBS with carbon radical species. The measured hyper-
fine constants are listed in Table 8 and are compared

64 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 1
O’Neill et al.
Sch em e 8
system to the C-10 substituent but also restricts the
regioselectivity of hydroxylation to the extent that only
one hydroxyphenyl DHA glucuronide (15) was located
in bile. These results are in sharp contrast to the
metabolism of artemether in vivo, in which several
hydroxylated metabolites were observed.
A p-trifluoromethyl group on the phenyl ring not only
blocks dearylation entirely but also has a profound effect
on hydroxylation, which now occurs at several sites (at
least five) on the molecule. The most plausible inter-
pretation is that the hydroxylation reactions have been
redirected to the artemisinin ring system; thereby the
oxidative metabolism of 5b resembles that of artemether
and arteether. A notable feature of this derivative’s fate
in the rat is the complete absence of metabolites from
bile following oral administration. This appears to be a
biological phenomenon rather than an artifact of the
experimental system since DHA administered in the
same manner (as well as by iv injection) is eliminated
extensively in bile as its glucuronide.
A p-methoxy group on the phenyl ring also precludes
dearylation but does not have such a marked facilitating
effect on hydroxylation because a competing reaction,
namely, O-demethylation, appears to unmask an hy-
droxyl function which is readily glucuronylated in vivo.
The metabolite obtained by this process was also formed
from 5b, presumably in this case by seqeuential oxida-
tion at the para position and glucuronidation.
The fact that no dearylation of either 7b or 8b
occurred in these studies is significant since it indicates
that para-substituted phenoxy derivatives in general
may not form DHA metabolically in vivo. Consequently,
compounds such as 7b would be expected to have longer
half-lives and potentially lower neurotoxicity. Further
work is required to determine the in vivo pharmaco-
kinetics and hydrolytic stability41 of these lead phenoxy
derivatives of dihydroartemisinin which appear to be
promising new candidates with potential advantages
over existing derivatives.
Ta ble 8. ESR Parameters of the Carbon Radicals Formed on
Reaction of Phenoxy Derivative 8b with FeSO4 in the Presence
of DBNBS in Aqueous Acetonitrile (1:1)a
endoperoxide
carbon radical adduct
a N (G)
a H (G)
compound 8b
primary 18a
secondary 19a
primary
secondary
primary
13.45
13.45
13.6
13.30
13.60
13.50
11.50
6.00
12.0
6.80
12.20
7.50
artemisinin (2)
artemether (4a )
secondary
aValues are compared with those of artemether and artemisinin.
with those obtained for artemether and artemisinin.
Thus, it is likely that these phenoxy derivatives behave
in a manner similar to artemether and artemisinin, and
we propose that their potent antimalarial activity is
mediated by the formation of carbon radical species in
the parasite’s food vacuole.
Su m m a r y
The structure-metabolism relationships of the se-
lected phenoxy derivatives were investigated in the bile-
duct-cannulated rat with particular reference to the
following considerations: (i) the blocking of O-glucuro-
nylation, (ii) the redirection of hydroxylation from the
artemisinin ring system to the substituent, and (iii) the
stability of the endoperoxide bridge. With respect to
proof of concept particular importance was attached to
the ability of these derivatives to form DHA. The
glucuronylation of DHA, reduction of the endoperoxide
bridge, and with few exceptions hydroxylation of the
artemisinin ring system (in arteether) abolish parasiti-
cidal activity in vitro. Since our previous studies25 have
shown that the glucuronide of DHA is excreted exclu-
sively in bile, the model used in this study was consid-
ered a suitable means of investigating the capacity of
the phenoxy derivatives to resist metabolism to DHA.
Studies on 5b established that this compound under-
went dephenylation. The formation of DHA glucuronide
from 5b in vivo implies that the C-10 position is readily
hydroxylated which is, presumably, the necessary pre-
liminary step to dearylation. Substitution of a phenyl
ring for the C(O)-10-methyl of artemether not only
redirects hydroxylation from the endoperoxide ring
We have investigated the synthesis, in vitro and in
vivo antimalrial activity, and metabolism of a new series
of C-10-phenoxy derivatives of artemisinin. Biomimetic
degradation with Fe(II) salts and ESR spectroscopy
suggest that these derivatives readily form both second-
ary and primary C-centered radicals. Intraparasitic
formation of these potentially cytotoxic radicals might
be responsible for the derivatives high antimalarial
activity. Drug metabolism studies have shown that
chemical substitution at the para position of the phenyl
ring can prevent DHA formation in the rat. This finding,
coupled with the ease of synthesis and potent oral
antimalarial potency, makes this class of antimalarials
worthy of further investigation as next-generation lead
compounds.
Exp er im en ta l Section
Ch em istr y. Merck Kieselgel 60 F 254 precoated silica
plates for TLC were obtained from BDH, Poole, Dorset, U.K.
Column chromatography was carried out on Merck 938S silica
gel. Infrared (IR) spectra were recorded in the range 4000-
600 cm-1 using a Perkin-Elmer 298 infrared spectrometer.
Spectra of liquids were taken as films. Sodium chloride plates

C-10-Phenoxy Derivatives of Dihydroartemisinin
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 1 65
1
(Nujol mull) and solution cells (dichloromethane) were used
as indicated.
141-143 °C; [R]D +167° (c 1.0, CHCl3); H NMR (CDCl3, 300
MHz) δ 7.55 (2H, d, J ) 8.0 Hz); 7.20 (2H, d, J ) 8.0 Hz), 5.57
(1H, d, J ) 3.0 Hz), 5.45 (1H, s), 2.86 (1H, m), 2.36 (1H, dt),
1.21-2.05 (10H, m), 1.43 (3H, s), 1.02 (3H, d, J ) 8.0 Hz),
0.95 (3H, d, J ) 6.0 Hz); 13C NMR (75 MHz, CDCl3) δ 160,
127, 117, 104, 100, 88, 80, 52.5, 44, 37, 36, 35, 31, 26, 25, 24,
20, 13; IR (Nujol mull)/cm-1 2925 (CH), 1461, 1377, 1328, 1240,
1151, 1108, 981, 876 (O-O); MS (CI, NH3) 429 ([M + H]+, 64),
411 ([M + H]+ - F, 34), 289 (74), 221 (18); HRMS [M + H]+
m/z calcd for C22H28O5F3 429.18888, found 429.18818.
Data for R-isomer 7a : mp 161-163 °C; [R]D -39.5° (c 1.0,
CHCl3); 1H NMR (CDCl3, 300 MHz) δ 7.54 (2H, d, J ) 9.0 Hz),
7.17 (2H, d, J ) 8.0 Hz), 5.51 (1H, s), 5.10 (1H, d, J ) 9.0 Hz),
2.78 (1H, m), 2.41 (1H, dt, J ) 4.0 Hz), 1.26-2.09 (10H) 1.50
(3H, s), 0.98 (6H, d, J ) 5.0 Hz); 13C NMR (75 MHz, CDCl3) δ
127, 117, 105, 98.5, 91, 80, 52, 45, 37, 36, 34, 32, 26, 22, 24,
20, 12; IR (Nujol mull)/cm-1 2926 (CH), 1462, 1120, 1037, 879
(O-O), 838 (O-O); MS (CI, NH3) 429 ([M + H]+, 26), 383 (85),
289 (100), 221 (25), 43 (100).
1H NMR spectra were recorded using Perkin-Elmer R34
(220 MHz) and Bruker (400 and 200 MHz) spectrometers.
Solvents are indicated in the text, and tetramethylsilane was
used as the internal reference. Mass spectra were recorded at
70 eV using a VG7070E mass spectrometer. The samples were
introduced using a direct-insertion probe. In the text the
parent ion (M+) is given, followed by peaks corresponding to
major fragment losses with intensities in parentheses.
Gen er a l P r oced u r e 1. To a stirred solution of dihydroar-
temisinin (1.0 g, 3.54 mmol) in anhydrous dichloromethane
(25 mL) were added phenol (10.5 mmol) and silver perchlorate
(0.15 g, 0.72 mmol). The resulting solution was cooled to -78
°C (CO2/acetone) and trimethylsilyl triflate (1.0 mL, 5.00
mmol) added. The reaction was allowed to stir for 1 h. After
this time the reaction was quenched with triethylamine (1.5
mL) and allowed to warm to room temperature. The suspen-
sion was filtered, and the solvent removed in vacuo. Purifica-
tion by column chromatography gave the required products
as â- and R-isomers.
10â-(p-Meth oxyp h en oxy)d ih yd r oa r tem isin in (8b) a n d
10r-(p-Meth oxyp h en oxy)d ih yd r oa r tem isin in (8a ). These
compounds were prepared by general procedure 1 to give the
products as white cystalline solids in 73% yield with a â/R ratio
of 4:1. Data for â-isomer 8b: mp 118-120 °C; [R]D +141.1° (c
10â-P h en oxyd ih yd r oa r tem isin in (5b) a n d 10r- P h en -
oxyd ih yd r oa r tem isin in (5a ). These compounds were pre-
pared by general procedure 1 to give the products as white
cystalline products in 68% yield with a â/R ratio of 4:1. Data
for â-isomer 5b: mp 104-106 °C; [R]D +204° (c 1.0, CHCl3);
1H NMR (CDCl3, 300 MHz) δ 7.29 (2H, t, J ) 8.81 Hz), 7.22
(2H, d, J ) 8.81 Hz), 6.99 (1H, t, J ) 7.9 Hz), 5.50 (1H, d, J )
3.30 Hz), 2.81 (1H, m), 2.39 (1H, dt), 1.23-2.07 (10H, m), 1.44
1
1.0, CHCl3); H NMR (CDCl3, 300 MHz) δ 7.03 (2H, m); 6.83
(2H, m), 5.53 (1H, s), 5.37 (1H, d, J ) 3.30 Hz), 3.76 (3H, s,
OMe), 2.78 (1H, m), 2.38 (1H, dt), 1.21-2.07 (10H, m), 1.43
(3H, s), 1.02 (3H, d, J ) 7.2 Hz,), 0.95 (3H, d, J ) 3.60 Hz);
13C NMR (75 MHz, CDCl3) δ 155, 152, 118, 115, 104, 102, 88,
81, 56, 53, 44, 37, 36, 35, 31, 26, 25, 24, 20, 13; IR (Nujol mull)/
cm-1 2990 (CH), 1421, 1262, 895 (O-O); MS (CI, NH3) 408
([M + NH4]+, 7%), 373 (24%), 348 (51%), 345 (100%), 221
(100%); HRMS [M + NH4]+ m/z calcd for C22H34NO6 408.23861,
found 408.23798.
(3H, s), 1.13 (3H, d, J ) 7.40 Hz), 0.96 (3H, d, J ) 6.0 Hz); 13
C
NMR (75 MHz, CDCl3) δ 157, 129, 122, 116, 104, 100, 88, 81,
52, 44, 37, 36, 34, 31, 26, 25, 24, 20, 12; IR (Nujol mull)/cm-1
2926(CH), 1463, 1193, 875(O-O), 832 (O-O); MS (EI) 361 (M+,
85), 267 (M+ - OPh, 33), 221 (25).
Data for R-isomer 8a : mp 147-149 °C; [R]D -43.5° (c 1.0,
CHCl3); 1H NMR (CDCl3, 300 MHz) δ 7.08 (2H, m), 6.80 (2H,
m), 5.44 (1H, s), 4.92 (1H, d, J ) 9.3 Hz), 3.76 (3H, s, OMe),
2.70 (1H, m), 2.40 (1H, dt), 1.22-2.07 (10H, m), 1.42 (3H, s),
1.02 (3H, d, J ) 3.5 Hz), 0.96 (3H, d, J ) 4.86 Hz); 13C NMR
(75 MHz, CDCl3) δ 155, 152, 118.5, 114.5, 104, 100, 91, 80,
56, 52, 45, 37, 36, 34, 33, 26, 25, 22, 20, 12.5; IR (Nujol mull)/
cm-1 2934 (CH), 1468, 1423, 1379, 1224, 1131, 1029, 879 (O-
O), 825 (O-O); MS (CI, NH3) 390 ([M + H]+, 16%), 221 (28%),
124 (80%).
10â-(p-F lu or op h en oxy)d ih yd r oa r tem isin in (9b) a n d
10r-(p-F lu or op h en oxy)d ih yd r oa r tem isin in (9a ). These
compounds were prepared by general procedure 1 to give the
products as white cystalline solids in 60% yield with a â/R ratio
of 4:1. Data for â-isomer 9b: mp 75-77 °C; [R]D +186° (c 1.0,
CHCl3); 1H NMR (CDCl3, 300 MHz) δ 6.91-7.10 (4H, m,
aromatic), 5.51 (1H, s), 5.39 (1H, d, J ) 3.3 Hz), 2.79 (1H, m),
2.38 (1H, dt), 1.21-2.07 (10H, m), 1.42 (3H, s, Me at C-3), 1.02
(3H, d, J ) 7.2 Hz, Me at C-6), 0.97 (3H, d, J ) 6.0 Hz, Me at
C-9);13C NMR (75 MHz, CDCl3) δ 160, 156.5, 154, 118, 116,
104, 101, 86, 81, 52.5, 44, 37, 36, 35, 31, 26, 25, 20, 13; IR
(Nujol mull)/cm-1 2932 (CH), 1503, 1378, 1139, 1121, 1095,
877 (O-O), 832 (O-O); MS (EI), 379 (M+, 39%), 361 (M+ - F,
61%), 333 (69%), 289 (74%), 221 (18%).
Data for R-isomer 9a : mp 154-156 °C; 1H NMR (CDCl3,
300 MHz) δ 7.05-7.09 (2H, m, aromatic), 6.93-6.98 (2H, m,
aromatic), 5.46 (1H, s), 4.95 (1H, d, J ) 9.3 Hz), 2.70 (1H, m),
2.40 (1H, dt, J ) 13.8, 3.8 Hz), 1.25-2.07 (10H, m), 1.44 (3H,
s, Me at C-3), 1.02 (3H, d, J ) 3.0 Hz, Me at C-6), 0.98 (3H, d,
J ) 1.8 Hz, Me at C-9); 13C NMR (75 MHz, CDCl3) δ 160, 157,
154, 118.5, 118, 116, 115.5, 104.5, 100, 91, 80, 51.5, 45, 37,
36, 34, 32, 26, 25, 22, 20, 12; IR (Nujol mull)/cm-1 2923 (CH),
1505, 1377, 1134, 1094, 881 (O-O), 833 (O-O). Anal. Calcd
for C21H27FO5: C 66.67, H 7.19. Found: C 66.49, H 7.19.
10â-(p-Ch lor op h en oxy)d ih yd r oa r tem isin in (10b) a n d
10r-(p-Ch lor op h en oxy)d ih yd r oa r tem isin in (10a ). This
mixture of compounds was prepared by general procedure 1.
NMR analysis revealed a diastereomeric mixture of 4:1 (â/R).
However, attempted separation of the mixture by silica gel
chromatography failed.
Data for R-isomer 5a : mp 143-145 °C; [R]D -54.5° (c 1.0,
CHCl3); 1H NMR (CDCl3, 300 MHz) δ 7.25 (2H, t, J ) 4.4 Hz),
7.11 (2H, d, J ) 5.8 Hz), 7.00 (1H, t, J ) 7.1 Hz) 5.49 (1H, s),
5.05 (1H, d, J ) 9.5 Hz), 2.73 (1H, m), 2.42 (1H, dt), 1.26-
2.10 (10H, m), 1.43 (3H, s), 0.99 (3H, d, J ) 3.2 Hz), 0.97 (3H,
d, J ) 6.0 Hz); 13C NMR (75 MHz, CDCl3) δ 145, 129, 122,
117, 104.5, 100, 99, 91, 52, 45, 37, 36, 33, 26, 25, 22, 20, 12.5;
IR (Nujol mull)/cm-1 2925 (CH), 1040, 881 (O-O), 825 (O-
O); MS (CI, NH3) 378 ([M + NH4]+, 44), 332 (90), 301 ([M +
NH4]+ - Ph, 5), 221 (100); HRMS (CI, NH4+) m/z calcd for
C
21H32NO5 [M + NH4]+ 378.22805, found 378.22777
10â-(p-Meth ylp h en oxy)d ih yd r oa r tem isin in (6b) a n d
10r-(p-Meth ylp h en oxy)d ih yd r oa r tem isin in (6a ). These
compounds were prepared by general procedure 1 to give the
products as white cystalline solids in 84% yield with a â/R ratio
of 5:1. Data for â-isomer 6b: mp 78-80 °C; [R]D +175° (c 1.0,
CHCl3); 1H NMR (CDCl3, 300 MHz) δ 7.01 (4H, m, aromatic),
5.5 (1H, s), 5.43 (1H, d, J ) 3.4 Hz), 2.78 (1H, m), 2.37 (1H,
dt), 2.27 (3H, s, Me), 1.20-2.07 (10H, m), 1.40 (3H, s), 0.99
(3H, d, J ) 5.7 Hz), 0.95 (3H, d, J ) 6.0 Hz); 13C NMR (75
MHz, CDCl3) δ 156, 131, 129, 117, 104, 100, 88, 81, 77, 60, 53,
44.5, 37, 36, 35, 31, 26, 24, 21, 20, 14, 13; IR (Nujol mull)/
cm-1 2925 (CH), 1509, 1226, 1036, 979, 959, 876 (O-O); MS
(FAB) 375 ([M + H]+, 45), 329 (100), 267 ([M + H]+ - OC6H4-
Me, 22).
Data for R-isomer 6a : mp 160-162 °C; [R]D -59° (c 1.0,
CHCl3); 1H NMR (300 MHz, CDCl3) δ 7.05 (4H, m, aromatic),
5.46 (1H, s), 4.98 (1H, d, J ) 9.0 Hz), 2.71 (1H, m), 2.40 (1H,
dt), 2.28 (3H, s, Me), 1.23-2.07 (10H, m), 1.42 (3H, s), 0.97
(6H, d, J ) 4.7 Hz); 13C NMR (75 MHz, CDCl3) δ 156, 131.5,
130, 117, 104, 99, 91, 80, 52, 45, 37, 36, 34, 33, 26, 25, 22,
20.5, 20, 12.5; IR (Nujol mull)/cm-1 2925 (CH), 1509, 1225,
1038, 878 (O-O), 818 (O-O); MS (FAB) 375 ([M + H]+, 18),
329 (100), 267 ([M + H]+ - OC6H4Me, 30). Anal. C 70.62, H
8.11, requires C 70.55, H 8.09.
10â-(p -Tr iflu or om et h ylp h en oxy)d ih yd r oa r t em isin in
(7b) a n d 10r-(p-Tr iflu or om eth ylp h en oxy)d ih yd r oa r te-
m isin in (7a ). These compounds were prepared by general
procedure 1 to give the products as white cystalline solids in
68% yield with a â/R ratio of 8:1. Data for â-isomer 7b: mp

66 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 1
O’Neill et al.
10â-(Na p h th yloxy)d ih yd r oa r tem isin in (11b) a n d 11r-
(Naph th yloxy)dih ydr oar tem isin in (11a). These compounds
were prepared by general procedure 1 to give the products as
white cystalline solids in 84% yield with a â/R ratio of 4:1:
Data for â-isomer 11b: mp 138-140 °C; [R]D +240° (c 1.0,
CHCl3); 1H NMR (CDCl3, 300 MHz) δ 7.75 (1H, d, J ) 9.0 Hz,
1 × aromatic), 7.56 (1H, s, 1 × aromatic), 7.40 (3H, m, 3 ×
aromatic), 7.24 (2H, d, J ) 9.0 Hz, 2 × aromatic), 5.66 (1H, d,
J ) 3.0 Hz), 5.55 (1H, s), 2.86 (1H, m), 2.40 (1H, dt), 1.46 (3H,
s, Me at C-3), 1.23-2.12 (10H, m), 1.06 (3H, d, J ) 8.0 Hz, Me
at C-6), 0.97 (3H, d, J ) 6.0 Hz, Me at C-9); 13C NMR (75 MHz,
CDCl3) δ 155, 135, 129, 127, 126, 124, 119, 111, 100, 88, 81,
53, 44.5, 37, 36, 35, 31, 26, 25, 24, 20, 13; IR (Nujol mull)/
cm-1 2926 (CH), 1464, 1211, 977, 839 (O-O); MS (EI) 410 (M+,
22%), 364 (100%), 221 (25%), 163 (61%), 144 (100%), 43 (72%).
Found, M+ 410.20913, C25H30O5 requires 410.20932. Anal.
Calcd for C25H30O5: C 73.15, H 7.37. Found: C 72.92, H 7.41,
EP R Sp ectr oscop y. All spectra were recorded at ambient
temperatures. Typical spectrometer conditions were: center
field 3368 G; field sweep 70 G; field modulation amplitude 1
G; time constant 320 ms; microwave power 10 mW; microwave
frequency 9.45 GHz. Experiments involved the mixing of
solutions of 8b (typically 2 mM, concentrations throughout
being after mixing), the spin-trap (typically, 40 mM for
DBNBS) and finally the metal ion (typically 2 mM) in
unbuffered deoxygenated aqueous acetonitrile (1:1) at pH 7.
An tim a la r ia l Activity. Two strains of P. falciparum from
Thailand were used in this study: (a) the uncloned K1 strain
which is known to be CQ-resistant and (b) the HB3 strain
which is sensitive to all antimalarials. Parasites were main-
tained in continuous culture using the method of Trager and
J enson.39 Cultures were grown in flasks containing human
erythrocytes (2-5%) with parasitemia in the range of 1-10%
suspended in RPMI 1640 medium supplemented with 25 mM
HEPES and 32 mM NaHCO3 and 10% human serum (complete
medium). Cultures were gassed with a mixture of 3% O2, 4%
CO2 and 93% N2.
(a ) In Vitr o Testin g. Antimalarial activity was assessed
with an adaption of the 48-h sensitivity assay of Desjardins
et al.40 using [3H]hypoxanthine incorporation as an assessment
of parasite growth. Stock drug solutions were prepared in
100% dimethyl sulfoxide (DMSO) and diluted to the appropri-
ate concentration using complete medium. Assays were per-
formed in sterile 96-well microtiter plates, each plate contained
200 µL of parasite culture (2% parasitemia, 0.5% hematocrit)
with or without 10 µL drug dilutions. Each drug was tested
in triplicate and parasite growth compared to control wells
(which consituted 100% parasite growth). After 24-h incuba-
tion at 37 °C, 0.5 µCi hypoxanthine was added to each well.
Cultures were incubated for a further 24 h before they were
harvested onto filter mats, dried for 1 h at 55 °C and counted
using a Wallac 1450 Microbeta Trilux Liquid scintillation and
luminescence counter. IC50 values were calculated by inter-
polation of the probit transformation of the log dose-response
curve.
(b) In Vivo Testin g. Male, random Swiss albino mice
weighing 18-22 g were inoculated intraperitoneally with 107
parasitized erythrocytes with P. berghi NS strain. Animals
were then dosed daily via two routes (intraperitoneal or oral)
for 4 consecutative days beginning on the day of infection.
Compounds were dissolved or suspended in the vehicle solution
consisting of methanol, phosphate-buffered saline and DMSO
(2:5:3 v/v). The parasitemia was determined on the day
following the last treatment and the ED50 (50% suppression
of parasites when compared to vehicle only treated controls)
calculated from a plot of log dose against parasitemia. The data
shown in Table 4 were obtained from the WHO.
Dr u g Meta bolism Stu d ies. The structure-metabolism
relationships of the selected phenoxy derivatives were inves-
tigated in the bile-duct-cannulated rat. Cannulated and anes-
thetized male Wistar rats were administered DHA and the
following O-ether derivatives of DHA (35 µmol/kg) by either
iv injection or gastric intubation. Aliquots of the bile (hourly
or half-hourly collections) were analyzed by LC-electrospray
MS using (i) a Columbus or Ultracarb 5-µm C-8 column (0.46
× 25 cm; Phenomenex, Macclesfield, Cheshire) with gradients
of acetonitrile in 0.1 M ammonium acetate, pH 6.9: 20-35%
over 15 min and 35-70% over 10 min (the gradient employed
unless otherwise indicated); or 20-35% over 15 min, at 35%
for 10 min and 35-70% over 10 min; (ii) a Nucleosil 5-µm C-8
column (0.32 × 25 cm; Phenomenex) with a gradient of
acetonitrile, 20-35% over 15 min, 35-70% over 10 min (for
7b metabolites only). The flow rate in all cases was 0.9 mL/
min.
Data for R-isomer 11a : mp 148-150 °C; [R]D +16° (c 1.0,
CHCl3); 1H NMR (CDCl3, 300 MHz) δ 7.75 (2H, m, 2 ×
aromatic), 7.40 (5H, m, 5 × aromatic), 5.58 (1H, s), 5.33 (1H,
d, J ) 9.30 Hz), 2.78 (1H, m), 2.43 (1H, dt), 1.56 (3H, s, Me at
C-3), 1.26-2.08 (10H, m), 1.06 (3H, d, J ) 12.9 Hz, Me at C-6),
1.00 (3H, d, J ) 7.2 Hz, Me at C-9); 13C NMR (75 MHz, CDCl3)
δ 129, 128, 127, 126, 124, 119.5, 111, 99, 80, 52, 45, 37, 36, 34,
32.5, 26, 22, 20, 12.5; IR (Nujol mull)/cm-1 2926 (CH), 1465,
1377, 1038, 879 (O-O), 839 (O-O); MS (EI) 410 (M+, 100%),
364 (97%), 346 (82%), 321 (38%), 304 (54%), 221 (24%), 163
(93%), 144 (100%), 115 (58%), 43 (87%). Calcd for C25H30O5
410.20932, found 410.20913.
F er r ou s-Med ia ted Degr a d a tion Rea ction s. To a room
temperature stirred solution of 8b (0.200 g, 0.51 mmol) in
acetonitrile and water (1:1, 10 mL) under nitrogen atmosphere
was added ferrous sulfate (0.140 g, 0.51 mmol, 1 equiv). The
reaction mixture was heated to 37 °C and left for 2 days. The
reaction mixture was then diluted with water (10 mL) and
extracted with ethyl acetate (3 × 20 mL). The combined
organic extracts were dried over anhydrous magnesium sulfate
and concentrated in vacuo. Flash column chromatography
using 25% ethyl acetate/75% n-hexane as the eluent afforded
the THF acetate 18 (0.045 g, 22%), 19 (0.046 g, 23%) and
recovered starting material 8b (0.080 g, 40%).
Data for THF acetate 18: 1H NMR (CDCl3, 300 MHz) δ
7.07-7.02 (2H, m, Ar), 6.82-6.79 (2H, m, Ar), 6.47 (1H, s, 12-
H), 5.20 (1H, d, J ) 4.5 Hz, 10-H), 4.28 (1H, ddd, J ) 9.3 Hz,
8.1 Hz, 2.1 Hz), 3.94 (1H, ddd, J ) 16.5 Hz, 7.8 Hz, 7.8 Hz),
3.76 (3H, s, OMe), 2.55 (1H, m), 2.12 (3H, s, Me), 2.11 (1H,
m), 1.96-1.73 (5H, m), 1.57 (1H, m), 1.43-1.26 (2H, m), 1.04
(3H, d, J ) 7.5 Hz, Me), 0.94 (3H, d, J ) 6.3 Hz, Me); 13C NMR
(75 MHz, CDCl3) δ 168.8, 155.3, 152.2, 119.4, 114.5, 103.1,
88.9, 80.6, 68.7, 55.8, 55.6, 47.1, 35.9, 33.5, 30.6, 27.8, 24.7,
21.5, 20.5, 12.6; IR (Nujol mull)/cm-1 2925, 1759, 1506, 1457,
1368, 1222, 1065, 1038, 969, 940. Anal. C 67.40, H 7.50;
requires C 67.67, H 7.74.
Data for 19: 1H NMR (CDCl3, 300 MHz) δ 7.05-7.00 (2H,
m, Ar), 6.84-6.80 (2H, m, Ar), 5.37 (1H, s, 12-H), 5.34 (1H, d,
J ) 4.2 Hz, 10-H), 3.76 (3H, s, OMe), 3.60 (1H, m, CHOH),
2.62 (1H, m), 2.02-1.75 (6H, m), 1.55 (3H, s, Me), 1.45 (1H,
m), 1.28-1.23 (2H, m), 1.07 (3H, d, J ) 7.2 Hz, Me), 0.89 (3H,
d, J ) 6.3 Hz, Me); 13C NMR (75 MHz, CDCl3) δ 154.9, 151.8,
118.0, 114.3, 108.2, 99.6, 94.2, 84.2, 69.8, 55.7, 42.4, 40.8, 34.8,
34.7, 30.5, 30.4, 25.0, 20.9, 18.4, 12.2; IR (Nujol mull)/cm-1
3504, 2929, 1462, 1378, 1225, 1032, 979 and 871. Anal. C
67.45, H 7.38; requires C 67.67, H 7.74.
The reaction was repeated in acetonitrile using 8b (0.200
g, 0.51 mmol) and ferrous sulfate (0.140 g, 0.51 mmol, 1 equiv).
After 5 days, the reaction mixture was filtered through Celite
and the filtrate concentrated in vacuo. Flash column chroma-
tography using 25% ethyl acetate/75% n-hexane as the eluent
afforded 18 (0.133 g, 66%), 19 (0.028 g, 14%) and recovered
starting material (0.020 g, 10%). All analytical data agreed
with those obtained for the products of the above reaction. The
reaction was also carried out using the conditions of J efford32c
to afford a 9:1 mixture of 18:19.
Ack n ow led gm en t. We thank the Wellcome Trust
(B.K.P. (Wellcome Principal Fellow), L.P.D. (Wellcome
Trust Studentship), A.M. (EPSRC/UFC), and S.H. (UN-
DP/World Bank/WHO Special Programme for Research
and Training in Tropical Diseases (TDR) ID 980176).
The authors also acknowledge the Wellcome Trust for

C-10-Phenoxy Derivatives of Dihydroartemisinin
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 1 67
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a grant for the LC-MS mass spectrometer used in this
work. We also thank the WHO for head-to-head in vivo
comparisons of lead trioxane 7b with sodium artesuante
and Dr. F. Mabbs, National EPSRC EPR Centre,
University of Manchester, Manchester, U.K., for the
EPR studies described in this work.
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