Synthesis and biological evaluation of extraordinarily potent C-10 carba artemisinin dimers against P. falciparum malaria parasites and HL-60 cancer cells

Article abstract of DOI:10.1016/j.bmc.2008.12.017

A series of artemisinin dimers incorporating a metabolically stable C-10 carba-linkage have been prepared, several of which show remarkable in vitro antimalarial activity (as low as 30 pM) versus Plasmodium falciparum and in vitro anticancer activity in t

Full text of DOI:10.1016/j.bmc.2008.12.017

Bioorganic & Medicinal Chemistry 17 (2009) 1325–1338
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of extraordinarily potent C-10 carba
artemisinin dimers against P. falciparum malaria parasites
and HL-60 cancer cells
James Chadwick a, Amy E. Mercer b, B. Kevin Park b, Richard Cosstick a, Paul M. O’Neill a,b,
*
a Department of Chemistry, University of Liverpool, Liverpool, L69 7ZD, UK
b Department of Pharmacology and Therapeutics, University of Liverpool, Liverpool, L69 3GE, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of artemisinin dimers incorporating a metabolically stable C-10 carba-linkage have been pre-
pared, several of which show remarkable in vitro antimalarial activity (as low as 30 pM) versus Plasmo-
dium falciparum and in vitro anticancer activity in the micromolar to nanomolar range versus HL-60 cell
lines.
Received 1 August 2008
Revised 3 December 2008
Accepted 8 December 2008
Available online 24 December 2008
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Artemisinin
Cancer
Dimer
Malaria
1. Introduction
anticancer activity apart from two phosphate ester dimers 2 and
3 (Fig. 1), which expressed nanomolar growth inhibitory (GI50) val-
Artemisinin 1, also known as qinghaosu, is a tetracyclic 1,2,4-tri-
oxane occurring in Artemisia annua.1 Artemisinin and its deriva-
tives are currently recommended as front-line antimalarials for
regions experiencing Plasmodium falciparum resistance to tradi-
tional antimalarial drugs. In addition to their well-known antima-
larial activity, artemisinin derivatives possess potent activity
against cancer cells.2–6
A potential drawback with many of these derivatives is the
presence of a metabolically susceptible C-10 acetal linkage; which
is unstable in vivo due to the potential for metabolism to dihyd-
roartemisinin7 and subsequent rapid clearance from the body by
glucuronidation.8 It has been noted that the replacement of oxygen
at the C-10 position with carbon produces compounds not only
with greater hydrolytic stability but also with a longer half-life
and potentially lower toxicity.9,10
The observation that artemisinin dimers possess significant
antimalarial and anticancer activity2 prompted previous efforts,
both within our group11 and elsewhere,12–17 to synthesize C-10
non-acetal artemisinin dimers. We prepared a series of dimers,
which were all found to display low nanomolar antimalarial activ-
ity against K1 and HB3 strains of P. falciparum.11 In contrast to their
potent antimalarial activities, most of the dimers possessed poor
ues against a range of cancer cells in the NCI 60-cell line assay. De-
tailed investigations in human promyelocytic leukemia HL-60 and
Jurkat cell lines demonstrated that these compounds possessed
similar activity to doxorubicin, which is used clinically to treat
acute leukemias.
These promising results encouraged us to prepare a further ser-
ies of C-10 dimers to explore what effects varying the nature and
length of linker would have upon activity.
H
O
H
H
O
O
O
O
O
O
O
O
O
H
H
O
H
O
O
O
O
O
P
1
OR
2 R=Me
3 R=Ph
* Corresponding author. Tel.: +44 151 794 3553, fax: +44 151 794 3588.
E-mail address: P.M.Oneill01@liv.ac.uk (P.M. O’Neill).
Figure 1. Artemisinin (1) and phosphate ester dimers (2 and 3).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.12.017

1326
J. Chadwick et al. / Bioorg. Med. Chem. 17 (2009) 1325–1338
H
H
H
H
O
O
O
O
i
ii
O
O
O
O
iii,iv
O
O
O
O
H
H
H
H
O
O
O
O
OH
OBz
OH
4
5 100%
6 92%
7 53%
H
H
O
O
O
O
O
O
v
H
O
H
O
O
O
O
P
R
2 R=OMe 58%
3 R=OPh 49%
8 R=Me 31%
Scheme 1. Reagents and conditions: (i) BzCl, py, CH2Cl2, 0 °C, 16 h; (ii) allyl trimethylsilane, ZnCl2, 4 Å molecular sieves, DCE, 0 °C, 3 h; (iii) O3, CH2Cl2, ꢀ78 °C, 1 h; (iv) NaBH4,
THF/MeOH (9:1), 0 °C, 4 h; (v) P(O)(R)Cl2, NaHMDS, THF, ꢀ78 °C?t, 16 h.
of 10b-vinyldeoxoartemisinin 16 and ozonolysis-reduction of the
double bond offered an attractive route. Initially, we endeavored
to employ our optimized procedure for the synthesis of 619 to
the synthesis of 10b-vinyldeoxoartemisinin 16, by replacing allyl
trimethylsilane with vinyl trimethylsilane (Scheme 4). Unfortu-
nately, vinyl trimethylsilane is not nucleophilic enough and as such
allows the competing dehydration reaction to predominate, mean-
ing that the only product isolated from the reaction was anhyd-
roartemisinin 17.
The C-10 position of artemisinin can be regarded as a sugar ano-
meric center. The synthesis of C-glycosyl allenes by the addition of
propargyl trimethylsilane to carbohydrate derivatives is well
known in carbohydrate chemistry; ozonolysis of the allene leading
directly to aldehydes without the need for an additional reducing
step.20,21 Based on this idea, it was decided to employ this route
2. Chemistry
The synthesis of dimers 2 and 3 from dihydroartemisinin is out-
lined in Scheme 1.11 Acylation of dihydroartemisinin 4 with ben-
zoyl chloride employing pyridine as a nucleophilic catalyst gave
dihydroartemisinin 10a-benzoate 5. Reaction of 5 with allyl tri-
methylsilane using zinc chloride as catalyst gave 10b-allyldeoxoar-
temisinin 6, ozonolysis of the terminal double bond in 6 followed
by sodium borohydride reduction of the ozonide giving alcohol 7.
Deprotonation of this alcohol with NaHMDS and reaction with
the appropriate dichlorophosphate gave dimers 2 and 3 in reason-
able yield.
The potential of methyl phosphate ester dimer 2 as an antican-
cer agent may be somewhat limited by the stability of the phos-
phate ester linkage. To address this potential problem we
decided to replace this linkage with a methyl phosphonate group,
which should result in a compound with increased hydrolytic sta-
bility.18 Deprotonation of 7 with NaHMDS and subsequent reaction
with methanephosphonic dichloride gave methyl phosphonate
linked dimer 8.
To determine whether removal of the endoperoxide bridge
would have an effect on the activity, the corresponding deoxygen-
ated methyl phosphonate linked dimer 10 was also synthesized
(Scheme 2). Reduction of the endoperoxide bridge of C-10 carba
alcohol 7 with zinc powder in acetic acid gave 9, which was subse-
quently used to prepare 10.
H
H
O
O
O
ii
i
O
O
H
H
O
O
OH
In order to examine whether the length of the spacer between
artemisinin moieties has an effect on activity we decided to syn-
thesize the analogs of 2 and 3 containing one less and one more
carbon atom, respectively, in the linker either side of the phos-
phate ester group.
OH
7
9 81%
H
H
O
O
The synthesis of the chain lengthened analogs 13 and 14 are
outlined in Scheme 3. Hydroboration of the terminal double bond
in 6 with borane dimethyl sulfide complex followed by oxidation
of the resulting organoborane with sodium perborate gave alcohol
11 as the major product along with a minor product 12. Alcohol 11
was allowed to react with the appropriate dichlorophosphate to
give 13 and 14, or with methanephosphonic dichloride to give 15.
Synthesis of an analog of C-10 carba alcohol 7 with one fewer
carbon atom in the side chain proved more problematic. Synthesis
O
O
H
O
H
O
O
O
P
O
Me
10 47%
Scheme 2. Reagents and conditions: (i) Zn, AcOH, rt, 72 h; (ii) P(O)MeCl2, NaHMDS,
THF, ꢀ78 °C?rt, 24 h.

J. Chadwick et al. / Bioorg. Med. Chem. 17 (2009) 1325–1338
1327
H
H
H
H
H
O
O
O
O
O
O
O
O
O
O
i, ii
iii
O
O
O
O
O
+
H
H
H
H
O
H
O
O
O
O
O
O
OH
O
P
R
OH
6
11 62%
12 30%
13 R=OMe 25%
14 R=OPh 20%
15 R=Me 28%
Scheme 3. Reagents and conditions: (i) BH3ꢁSMe2, THF, 0 °C?rt, 24 h; (ii) NaBO3ꢁ4H2O, THF/H2O, rt, 24 h; (iii) P(O)(R)Cl2, NaHMDS, THF, ꢀ78 °C?rt, 16 h.
H
H
O
O
R'MgBr
ZnCl2
i
O
O
O
O
5
R
R
H
H
O
O
O
SO2Ph
O
R'
SO2Ph
20
i
16 0%
Figure 2. Substitution reaction of 2-benzenesulfonyl ethers with carbon nucleo-
philes and Posner’s artemeisinin C-10 sulfone (20).
H
O
O
O
thin layer chromatography of the reaction mixture showed almost
complete consumption of the allene and the 1H and 13C NMR spec-
tra of the concentrated reaction mixture showed signals at 10 ppm
and 202 ppm, respectively, indicating the presence of an aldehyde
functionality. However, following reaction workup and silica gel
flash column chromatography, none of the aldehyde 19 was
isolated.
H
O
17 80%
Scheme 4. Reagents and conditions: (i) vinyl trimethylsilane, ZnCl2, 4 Å molecular
sieves, DCE, 0 °C, 18 h.
The comparatively high cost of propargyl trimethylsilane and
the modest yield of 18 obtained, prompted us to look at an
alternative approach. Brown et al. have employed the benzenesul-
fonyl moiety as a leaving group for direct nucleophilic replacement
by organozinc reagents on 2-benzene sulfonyl cyclic ethers derived
from tetrahydropyrans or tetrahydrofurans (Fig. 2).22 Posner et al.
prepared a series of sulfide, sulfone and sulfonamide trioxanes and
assayed them for antimalarial activity.23 One of these derivatives
20, prepared by reacting anhydroartemisinin with benzenesulfinic
acid, incorporated the benzenesulfonyl group at the C-10 position
of artemisinin.
It was decided to prepare 20 and react it with the organozinc re-
agent derived from vinyl magnesium bromide and zinc chloride to
hopefully give 10b-vinyl artemisinin which could then be con-
verted into the required alcohol. Reacting DHA 4 with boron tri-
fluoride diethyl etherate gave anhydroartemisinin 17, which was
subsequently allowed to react with benzenesulfinic acid (Scheme
6). Unfortunately, this reaction led to epimerization at C-9 and
the product obtained was 10b-benzenesulfonyl-9-epi-dihydroarte-
misinin 21. Nucleophilic replacement of the sulfone with the
organozinc reagent, derived from vinylmagnesium bromide and
zinc chloride, introduced the required vinyl substituent at the C-
10 position. In addition to 22, significant amounts of anhydroarte-
misinin 17 were also formed and since it proved impossible to sep-
arate these products by chromatography the mixture was
subjected to ozonolysis and reduction. The required alcohol 23
was cleanly isolated in reasonable yield (32% over two steps) and
allowed to react with the appropriate dichlorophosphate to give
24a and 24b.
H
H
O
O
O
O
i
ii
O
O
5
H
H
O
O
O
•
18 42%
19
Scheme 5. Reagents and conditions: (i) propargyl trimethylsilane, ZnCl2, 4 Å
molecular sieves, DCE, 0 °C, 24 h; (ii) O3, CH2Cl2, ꢀ78 °C, 1 h.
to synthesise the required aldehyde 19 which could then be re-
duced to the corresponding alcohol.
Initially, the conditions employed in carbohydrate chemistry
were used; dihydroartemisinin was converted into its acetate
and allowed to react with propargyl trimethylsilane in the pres-
ence of TMSOTf. Unfortunately, these conditions led exclusively
to the formation of anhydroartemisinin with none of the required
product being observed.
The failure of the conditions employed in carbohydrate chemis-
try prompted us to combine this approach with our procedure for
controlled oxonium ion formation (Scheme 5). Dihydroartemisinin
10a-benzoate 5 was allowed to react with propargyl trimethylsi-
lane in the presence of ZnCl2 to give the required allene 18 in mod-
erate yield along with trace amounts of anhydroartemisinin.
Ozonolysis of the allene appeared to give the required aldehyde;

1328
J. Chadwick et al. / Bioorg. Med. Chem. 17 (2009) 1325–1338
H
H
H
H
O
O
O
O
i
ii
iii
O
O
O
O
O
O
O
O
H
H
H
H
O
O
O
O
OH
SO2Ph
4
17 70%
21 66%
22
H
H
H
O
O
O
O
O
O
O
O
iv, v
vi
H
O
H
H
O
O
O
O
O
P
O
OH
23 32% from 21
OR
24a R=Me 15%
24b R=Ph 32%
Scheme 6. Reagents and conditions: (i) BF3ꢁOEt2, THF, 66 °C, 2.5 h; (ii) PhSO2H, CH2Cl2, rt, 30 min; (iii) vinylmagnesium bromide, ZnCl2, THF, rt, 48 h; (iv) O3, CH2Cl2, ꢀ78 °C,
1 h; (v) NaBH4, THF/MeOH (9:1), 0 °C, 4 h; (vi) P(O)(OR)Cl2, NaHMDS, THF, ꢀ78 °C?rt, 16 h.
to lead to product degradation and led to very complicated reac-
H
H
tion mixtures which proved impractical to purify. Oxidation with
Dess–Martin periodinane gave aldehyde 27 without the problems
of degradation (Scheme 8).
O
O
O
O
i
ii
O
O
Aldehydes can be prepared directly from alkenes by a number
of procedures; ozonolysis of the alkenes and in situ reduction of
the ozonide with dimethyl sulfide or triphenyl phosphine is a pop-
ular route. However, previous attempts to prepare aldehyde 27 by
ozonolysis-reduction proved unsuccessful. Simply carrying out the
ozonolysis in methanol, rather than the customary dichlorometh-
ane, and employing triphenyl phosphine as the reducing agent
led to 27 being isolated in 76% yield (Scheme 9). Aldehyde 27
was also prepared directly from 6 by the oxidative cleavage of
the double bond with OsO4 and NaIO4 in the presence of 2,6-luti-
dine. Oxidation of 27 with sodium chlorite gave 28 in quantitative
yield. Conversion of 28 to its acid chloride with oxalyl chloride and
reaction with 26 gave 29 (Scheme 9).
7
H
H
O
O
OMs
NH2
25 100%
26 98%
Scheme 7. Reagents and conditions: (i) MsCl, NEt3, CH2Cl2, 0 °C, 2 h; (ii) NH4OH,
EtOH, rt, five days.
As well as looking at varying the length of the linker between
the artemisinin moieties we also wished to look at varying the nat-
ure of the linker to see the effects that this might have on cytotox-
icity. It is noteworthy that the most active dimers previously
synthesized contain polar groups in the linker. For example, phos-
phate ester dimers 2 and 311 and several of the dimers prepared by
Posner14,15 and Jung16 either contain water solubilizing groups or
polar functionality capable of acting as a H-bond donor or acceptor
in the linker. Such functionality may enhance binding to the bio-
logical target molecule(s).
It was thought that an amide linked dimer would be readily
accessible from our C-10 carba alcohol 7 via a few simple transfor-
mations to give a C-10 carba carboxylic acid and C-10 carba amine
26 which could then be joined by amide bond formation. Mesyla-
tion of 7 and reaction with ammonia solution gave the required C-
10 carba amine 26 (Scheme 7). Upon storage the peroxide bridge in
26 appeared to degrade and therefore for prolonged storage this
compound was formulated as its hydrochloride salt.
The required carboxylic acid 28 has been prepared previously
by oxidation of the terminal double bond in 6 with NaIO4 and
KMnO4, however the product obtained from this procedure ap-
peared contaminated with a number of other products and also de-
graded quite rapidly. Therefore, it was decided to oxidize alcohol 7
first to the aldehyde 27 and then to the required carboxylic acid 28.
Previously aldehyde 27 has been prepared within this group by
Swern oxidation of 7 but this procedure gave unsatisfactory re-
sults; the dimethyl sulfide generated during the reaction seemed
A carbonate-linked dimer 30 was prepared by reaction of 10b-
(2-hydroxyethyl)-deoxoartemisinin 7 with trichloromethyl chloro-
formate (Scheme 10). The corresponding reaction with 10b-(2-
aminoethyl)deoxoartemisinin 26 gave the urea-linked dimer 31,
albeit in low yield (18%) (Scheme 10).
A 4,40-bipiperidine-linked dimer 32 was prepared by reacting
4,40-bipiperidine with 10b-(2-methanesulfonylethyl)deoxoarte-
misinin 25 in refluxing benzene for 48 h (Scheme 11). One valuable
property of the 4,40-bipiperidine-linked dimer is the presence of
H
H
O
O
O
O
i
O
O
H
H
O
O
OH
O
27 68%
7
Scheme 8. Reagents and conditions: (i) Dess–Martin periodinane, CH2Cl2, rt,
30 min.

J. Chadwick et al. / Bioorg. Med. Chem. 17 (2009) 1325–1338
1329
H
H
O
H
H
O
i, ii
or iii
O
O
O
O
O
O
iv
v,vi
O
O
O
O
H
H
6
H
H
O
O
O
O
H
OH
N
O
O
O
28 100%
27 76%
29 74%
Scheme 9. Reagents and conditions: (i) O3, MeOH, ꢀ78 °C, 60 min; (ii) PPh3, MeOH, ꢀ78 °C?rt, 18 h or (iii) OsO4 (cat.), NaIO4, 2,6-lutidine, dioxane/H2O, rt, 24 h; (iv) NaClO2,
2-methyl-2-butene, NaH2PO4, t-BuOH/H2O, rt, 2 h; (v) (CO)2Cl2, CH2Cl2, 0 °C?rt, 90 min; (vi) 26, NEt3, CH2Cl2, 0 °C?rt, 16 h.
tion that the endoperoxide is essential was provided by the signif-
icant drop in activity for the corresponding deoxygenated
compound 8.
H
H
H
O
O
O
O
O
The influence of the linker length on activity was examined by
preparing dimers 13, 14 and 15 which contain one additional car-
bon atom on either side of the linker and also 24a and 24b which
has one fewer carbon atom on each side. The dimers with three
carbon atoms in each of the artemisinin C-10 side chains (13, 14
and 15) are of the same order of magnitude but slightly more ac-
tive than those with two (2, 3 and 8). On reducing the number of
carbon atoms in each C-10 side chain from two to one the activity
is decreased, although the stereochemistry at the C-9 position has
been inverted and the influence that this might have on the activity
is unknown.
The amide-linked dimer 29 also shows significant activity
against HL-60 cells. It is interesting to note the similarity of 29 to
one of the most potent compounds prepared by Jung et al.,16 which
shows activity against a range of murine and human cancer cell
lines.
O
O
H
X
H
X
O
O
i or ii
H
O
O
O
X
O
7 X=OH
26 X=NH2
30 X=O 73%
31 X=NH 18%
Scheme 10. Reagents and conditions: (i) 7, CCl3OC(O)Cl, py, Et2O, 0 °C?rt 48 h or
(ii) 26, CCl3OC(O)Cl, NEt3, Et2O, 0 °C?rt 16 h.
H
H
O
O
Of the remaining dimers synthesized in this study, the 4,40-bipi-
peridine-linked dimer 32 possesses good activity against HL-60
cells whereas the carbonate and urea-linked dimers, 30 and 31,
respectively, are essentially devoid of activity.
O
O
O
O
i
H
N
H
N
25
O
O
3.2. Antimalarial activity
32 63%
The antimalarial activity of artemisinin-dimers was evaluated
in vitro against either chloroquine-resistant K1 or chloroquine-
sensitive 3D7 strains of P. falciparum by [3H]-hypoxanthine incor-
poration using artemisinin and artemether as positive controls (Ta-
ble 2). Several of the dimers have remarkable activity against P.
falciparum malaria.
Scheme 11. Reagents and conditions: (i) 4,40-bipiperidine, benzene, 75 °C, 48 h.
two protonatable nitrogen atoms that could allow this compound
to be formulated as a water soluble salt.
As reported previously, dimers 2 and 3 are among the most po-
tent compounds to have been tested against the K1 strain of P.
falciparum.
3. Results and discussion
3.1. Anticancer activity
The other dimers prepared were assayed against the chloro-
quine-sensitive 3D7 strain of P. falciparum. Methyl phosphonate di-
mer 8 and amide dimer 29 are the two most active compounds to
have been tested and are among the most potent antimalarials that
have been prepared at The University of Liverpool. The necessity of
the endoperoxide bridge is demonstrated by the complete absence
of activity for the deoxygenated methyl phosphonate dimer 10.
The lack of activity observed for urea-linked dimer 31 may be
attributable to its poor solubility, which was observed during the
testing procedure.
The cytotoxic properties of artemisinin dimers were evaluated
against human promyelocytic leukemia HL-60 cells in vitro using
the MTT assay (Table 1). This cell line was chosen because leuke-
mic cell lines are known to be particularly sensitive to artemisinin
derivatives. The MTT assay measures the activity of dehydrogenase
enzymes within the mitochondria of metabolically active cells.
As we have previously reported, 2 and 3 were studied in detail
in HL-60 cells using dihydroartemisinin and doxorubicin as posi-
tive controls.11 Both demonstrated excellent activity with IC50 val-
ues comparable to doxorubicin and in terms of cytotoxicity to non-
cancerous mammalian cells these compounds were non-toxic to
4. Conclusion
peripheral blood mononuclear cells at doses above 100 lM.
To address the potential problem with the stability of the phos-
phate ester linkage in 2 the methyl phosphonate-linked dimer 8
was prepared and also proved to possess potent activity. Confirma-
In conclusion, we have prepared a series of C-10 carba-linked
artemisinin dimers, several of which show remarkable antimalarial
and anticancer activity and deserve closer examination as potential

1330
J. Chadwick et al. / Bioorg. Med. Chem. 17 (2009) 1325–1338
Table 1
Table 1 (continued)
In vitro anticancer activity of artemisinin-dimers against HL-60 cells
Compound
IC50
M)
SD
M)
Compound
IC50
M)
SD
M)
(
l
(
l
(
l
(
l
H
H
H
H
O
O
O
O
O
O
O
O
O
O
O
O
H
O
H
O
H
O
H
O
O
O
2
0.07
0.02
15
0.10
0.04
O
O
O
P
O
P
OMe
Me
H
H
H
H
O
O
O
O
O
O
O
O
O
O
O
O
O
O
H
H
H
H
O
3
0.27
0.11
24a
0.77
0.18
O
O
O
O
O
P
O
P
O
O
O
OPh
OMe
H
O
H
O
H
H
O
O
O
O
O
O
O
O
H
H
O
O
O
24b
1.04
0.28
O
H
O
H
8
0.16
0.06
O
O
O
O
P
O
OPh
P
O
Me
H
H
H
H
O
O
O
O
O
O
O
O
O
O
H
H
29
0.10
0.07
O
O
O
H
O
H
O
10
28.14
3.56
H
N
O
P
O
Me
H
H
H
H
O
O
O
O
O
O
O
O
O
O
H
O
H
H
O
H
O
O
O
O
13
0.05
0.02
30
>100
O
O
O
P
O
O
O
OMe
H
H
H
H
O
O
O
O
O
O
O
O
H
H
O
O
O
O
31
>100
O
O
H
O
H
O
O
14
0.25
0.05
HN
NH
O
O
O
P
OPh

J. Chadwick et al. / Bioorg. Med. Chem. 17 (2009) 1325–1338
1331
Table 1 (continued)
Trio 1000 quadrupole GC mass spectrometer (CI) or a Micromass
LCT mass spectrometer (ESI). Reported mass values are within er-
ror limits of 5 ppm. Elemental analyses (%C, %H, %N) were deter-
mined by the University of Liverpool Microanalysis Laboratory.
Reported atomic percentages are within error limits of 0.5%. In in-
stances where purity was not determined by elemental analysis,
compounds displayed only one observable spot by T.L.C. at the re-
ported Rf.
Compound
IC50
M)
SD
M)
(
l
(
l
H
H
O
O
O
O
O
O
32
0.40
0.17
H
N
H
N
O
O
5.1.1. General procedures
5.1.1.1. Synthesis of bisphosphate ester dimers: general proce-
dure 1 (GP1). NaHMDS [1.0 M in THF] (1.2 equiv) was added to a
stirring solution of the appropriate alcohol (1.0 equiv) in anhy-
drous THF (0.05 M) at ꢀ78 °C. After stirring at ꢀ78 °C for 10 min,
the appropriate dichlorophosphate (0.5 equiv) was added. The
reaction mixture was allowed to warm up to room temperature
and stirred overnight, before being quenched at 0 °C with water
(5 mL). The mixture was extracted with ether (2 ꢂ 10 mL) and
the organic extracts washed with brine (10 mL), dried over MgSO4,
filtered and concentrated under reduced pressure to give the crude
product, which was purified by column chromatography on
Florisil.
4
Dihydroartemisinin
Doxorubicin
2.41
0.018
0.71
0.002
antimalarials or anticancer agents. Amide-linked dimer 29 was the
most potent compound assayed against 3D7 P. falciparum
(IC50 = 0.03 nM), as well as having significant activity against HL-
60 cancer cells (IC50 = 0.10
the most potent of the compounds assayed against HL-60 cancer
cells (IC50 = 0.05 M). In addition to their excellent antimalarial
and anticancer activity, these compounds also appear to be non-
toxic to normal cell lines; compounds 2 and 3 are not toxic to
peripheral blood mononuclear cells (PBMCs) at concentrations
lM). Methyl phosphate dimer 13 was
l
5.1.1.2. Synthesis of methylphosphonate dimers: general pro-
cedure 2 (GP2). NaHMDS [1.0 M in THF] (1.2 equiv) was added to
a stirring solution of the appropriate alcohol (1.0 equiv) in anhy-
drous THF (0.05 M) at ꢀ78 °C. After stirring at ꢀ78 °C for 10 min,
a solution of methanephosphonic dichloride (0.5 equiv) in anhy-
drous THF (0.05 M) was added and the reaction mixture allowed
to warm up to room temperature and stirred for 24 h. After 24 h,
the reaction mixture was concentrated under reduced pressure
and purified by flash column chromatography on silica gel.
>250 lM giving a therapeutic index of >500.
5. Experimental
5.1. Chemistry
Air- and moisture-sensitive reactions were carried out in oven-
dried glassware sealed with rubber septa under a positive pressure
of dry nitrogen or argon from a manifold or balloon, unless other-
wise indicated. Similarly sensitive liquids and solutions were
transferred via syringe or stainless steel cannula. Reactions were
stirred using Teflon-coated magnetic stir bars. Organic solutions
were concentrated using a Buchi rotary evaporator with a dia-
phragm vacuum pump.
Anhydrous solvents were either obtained from commercial
sources or dried and distilled immediately prior to use under a
constant flow of dry nitrogen. THF and diethyl ether were distilled
from Na, CH2Cl2 and NEt3 from CaH2. All other reagents were used
as received from commercial sources unless otherwise indicated.
Analytical thin layer chromatography was performed with
0.25 mm silica gel 60F plates with 254 nM fluorescent indicator
from Merck. Plates were visualised by ultraviolet light or by treat-
ment with iodine, p-anisaldehyde, ninhydrin or potassium per-
manganate followed by gentle heating. Chromatographic
purification of products was accomplished by flash chromatogra-
phy, as described by Still et al.24
Melting points were determined on a Gallenkamp apparatus
and are uncorrected. NMR spectra were measured on Bruker
(400 MHz and 250 MHz) nuclear magnetic resonance spectrome-
ters. Solvents are indicated in the text. Data for 1H NMR spectra
are reported as follows: chemical shift (d, ppm) relative to tetra-
methylsilane as the internal reference, integration, multiplicity
(s = singlet, br s = broad singlet, d = doublet, t = triplet, q = quartet,
sex = sextet, td = triplet of doublets, m = multiplet), coupling con-
stant (J, Hz), assignment. Data for 13C NMR are reported in terms
of chemical shift (d, ppm) relative to residual solvent peak. Infrared
spectra were recorded on a PerkinElmer RX1 FT-IR spectrometer
and are reported in wavenumbers (cmꢀ1). Mass spectra (MS) and
high resolution mass spectra (HRMS) were recorded on either a
5.1.2. Procedures
5.1.2.1. Dihydroartemisinin 10a-benzoate 5. Benzoyl chloride
(3.2 mL, 27.6 mmol) was added to a stirring solution of dihydroar-
temisin 4 (5.00 g, 17.6 mmol) in anhydrous CH2Cl2 (54 mL) and
anhydrous pyridine (9 mL) at 0 °C. After stirring at room tempera-
ture for 16 h, 7% aq citric acid solution (50 mL) was added. The or-
ganic layer was separated and the aqueous layer extracted with
EtOAc (2 ꢂ 50 mL). The combined organic layers were washed with
7% aq citric acid solution (2 ꢂ 50 mL), saturated NaHCO3 (50 mL)
and water (50 mL). The organic layer was dried over MgSO4, fil-
tered and concentrated under reduced pressure to give a yellow
oil. Purification by flash column chromatography (silica gel,
10:90 EtOAc/n-Hex) gave 5 (6.83 g, 100%) as a white crystalline so-
lid. 5: Mp 111–112 °C; 1H NMR (400 MHz, CDCl3) d 8.13 (2H, m,
Ar–H,), 7.57–7.45 (3H, m, Ar–H), 6.02 (1H, d, J = 9.8 Hz, H-10b),
5.53 (1H, s, H-12), 2.76 (1H, sex, H-9), 2.40 (1H, td, J = 14.0,
4.1 Hz, H-4a), 2.08–0.93 (19H, m) including 1.43 (3H, s, 3Me),
0.99 (3H, d, J = 6.0 Hz, 6Me) and 0.93 (3H, d, J = 7.2 Hz, 9Me); 13C
NMR (100 MHz, CDCl3) d 165.4, 133.3, 130.2, 129.8, 128.4, 104.5,
92.6, 91.7, 80.2, 51.7, 45.4, 37.3, 36.3, 34.2, 32.0, 25.9, 24.6, 22.1,
20.2 and 12.2; IR (Nujol)/cmꢀ1 2924, 1738 (C@O), 1491, 1452,
1377, 1272, 1114, 1100, 1037, 877 (O–O) and 831 (O–O); HRMS
(CI) C23H32NO6 [M+NH4]+ requires 406.2230, found 406.2232;
Anal. calcd C22H28O6 requires: C, 68.04; H, 7.22. Found: C, 67.79;
H, 7.30.
5.1.2.2. 10b-Allyldeoxoartemisinin 6. A solution of 5 (2.13 g,
5.5 mmol) in anhydrous 1,2-dichloroethane (25 mL) was added
dropwise via cannula to a stirring mixture of allyltrimethylsilane
(4.4 mL, 27.7 mmol), anhydrous ZnCl2 (0.90 g, 6.6 mmol) and
powdered 4 Å molecular sieves in anhydrous 1,2-dichloroethane
(25 mL) at 0 °C. After stirring at 0 °C for 3 h, the reaction mixture
was diluted with EtOAc (150 mL) and washed with 5% aq citric

1332
J. Chadwick et al. / Bioorg. Med. Chem. 17 (2009) 1325–1338
Table 2
In vitro antimalarial activity of artemisinin-dimers against chloroquine-resistant K1 or chloroquine-sensitive 3D7 strains of P. falciparum
Compound
IC50
SD
Compound
IC50 (nM)
SD
(nM)
(nM)
(nM)
H
H
H
H
O
O
O
O
O
O
O
O
O
O
O
O
H
H
2a
0.20
0.30
24ab
4.8
0.4
H
O
H
O
O
O
O
O
O
P
O
P
O
O
OMe
OMe
H
H
H
H
H
H
H
H
O
O
O
O
O
O
O
O
O
O
O
O
O
O
H
H
O
3a
0.50
0.10
0.02
0.06
24bb
6.0
0.30
0.01
0.40
H
O
H
O
O
O
H
O
O
P
O
O
P
O
OPh
OPh
H
O
O
O
O
O
O
O
O
O
O
O
H
H
8a
0.04
29b
0.03
H
H
O
O
O
O
O
O
H
N
O
P
Me
O
H
H
H
O
O
O
O
O
O
O
H
O
O
O
O
10a
>1000
30b
4.60
H
O
H
O
O
O
O
P
O
O
O
Me
H
H
H
H
O
O
O
O
O
O
O
O
O
O
O
O
H
H
H
O
H
O
O
O
O
13b
4.80
0.02
31b
>60
O
O
P
HN
NH
O
OMe
H
H
O
O
H
H
O
O
O
O
O
O
H
H
O
O
O
O
O
O
O
14b
0.21
0.07
32b
0.46
0.08
H
N
H
O
O
O
N
P
O
OPh

J. Chadwick et al. / Bioorg. Med. Chem. 17 (2009) 1325–1338
1333
Table 2 (continued)
Compound
IC50
(nM)
SD
(nM)
Compound
IC50 (nM)
SD
(nM)
H
H
O
O
O
O
O
O
H
O
H
O
O
O
15b
0.50
0.3
1b
Artemisinin
12.3
1.40
O
P
Me
Artemether
3.53a,
2.11b
1.91,
0.61
a
IC50 determined against chloroquine-resistant K1 strain of P. falciparum.
IC50 determined against chloroquine-sensitive 3D7 strain of P. falciparum.
b
acid (50 mL), saturated aq NaHCO3 (50 mL) and brine (50 mL).
The organic extracts were dried over MgSO4, filtered and concen-
trated under reduced pressure to give a clear oil. Purification by
flash column chromatography (silica gel, 10:90 EtOac/n-Hex)
gave 6 (1.55 g, 92%) as a white solid. Compound 6: Mp 76–
78 °C; 1H NMR (400 MHz, CDCl3) d 5.93 (1H, m, CH@CH2), 5.33
(1H, s, H-12), 5.12 (2H, m, CH@CH2), 4.31 (1H, m, H-10), 2.68
(1H, sex, J = 7.3 Hz, H-9), 2.45–2.17 (3H, m), 2.10–0.89 (19H,
m) including 1.41 (3H, s, 3Me), 0.96 (3H, d, J = 6.0 Hz, 6Me),
and 0.89 (3H, d, J = 7.6 Hz, 9Me); 13C NMR (100 MHz, CDCl3)
136.5, 116.0, 103.1, 89.2, 81.1, 74.6, 52.4, 44.4, 37.5, 36.7, 34.5,
34.3, 30.3, 26.1, 24.9, 24.7, 20.1, and 12.9; IR (Nujol)/cmꢀ1
2931, 1642, 1454, 1376, 1278, 1105, 1041, 879 (O–O), and 840
(O–O); HRMS (CI) C18H29O4 [M+H]+ requires 309.20660, found
309.20702; Anal. calcd C18H28O4 requires: C, 70.10; H, 9.15.
Found: C, 69.62; H, 9.32.
5.1.2.4. Methyl phosphate ester-linked dimer 2. Alcohol 7
(100 mg, 0.32 mmol) was reacted according to GP1. Purification
by column chromatography (Florisil, 70:30 EtOAc/n-Hex) gave 2
(65 mg, 58%) as a white solid. Compound 2: Mp 48–50 °C; 1H
NMR (400 MHz, CDCl3) d 5.30 (2H, s, 2 ꢂ H-12), 4.49 (4H, m,
2 ꢂ OCH2), 4.16 (2H, m, 2 ꢂ H-10), 3.76 (3H, d, J = 11.1 Hz, OMe),
2.68 (2H, sex, J = 7.3 Hz, 2 ꢂ H-9), 2.28 (2H, td, J = 13.1, 4.0 Hz,
2 ꢂ H-4 ), 2.06–0.87 (42H, m) including 1.40 (6H, s, 2 ꢂ 3Me),
a
0.96 (6H, d, J = 6.0 Hz, 2 ꢂ 6Me) and 0.87 (6H, d, J = 7.5 Hz,
2 ꢂ 9Me); 13C NMR (100 MHz, CDCl3) d 103.5, 89.3, 81.3, 71.6,
67.0, 52.7, 44.6, 37.8, 36.9, 34.8, 30.9, 30.3, 26.3, 25.1, 24.8, 20.5
and 13.2; MS (FAB) [M+H]+ 701 (100), 307 (8), 237 (8), 191 (25),
154 (72), 136 (68), 107 (46), 77 (52); Anal. calcd C35H57O12P re-
quires: C, 59.99; H 8.20. Found: C, 60.03; H, 8.56.
5.1.2.5. Phenyl phosphate ester-linked dimer 3. Alcohol
7
(100 mg, 0.32 mmol) was reacted according to GP1. Purification
by column chromatography (Florisil, 70:30 EtOAc/n-Hex) gave 3
(60 mg, 49%) as a clear oil. Compound 3: 1H NMR (400 MHz, CDCl3)
d 7.32 (2H, t, Ar–H), 7.24 (2H,t, Ar–H), 7.14 (1H, t, Ar–H), 5.28 (2H,
s, 2 ꢂ H-12), 4.38 (2H, m, 2 ꢂ H-10), 4.28 (4H, m, 2 ꢂ OCH2), 2.67
(2H, sex, J = 7.3 Hz, 2 ꢂ H-9), 2.32 (2H, td, J = 13.0, 4.0 Hz, 2 ꢂ H-
5.1.2.3. 10b-(2-Hydroxyethyl)-deoxoartemisinin 7. Ozone was
bubbled through a solution of 6 (3.00 g, 9.7 mmol) in anhydrous
CH2Cl2 (250 mL) at ꢀ78 °C for 60 min until the solution became
saturated with ozone and appeared blue. Nitrogen was then bub-
bled through the solution for 20 min to purge excess ozone. The
solvent was removed under reduced pressure and the residue ta-
ken up in anhydrous MeOH/THF (10:90, 100 mL). The solution
was cooled to 0 °C and NaBH4 (2.50 g, 66.1 mmol) added over
4 h. The mixture was stirred overnight at room temperature and
then concentrated under reduced pressure, followed by addition
of CHCl3 (150 mL) and water (50 mL). The organic layer was dried
over MgSO4, filtered and concentrated under reduced pressure to
give a clear oil. Purification by flash column chromatography (silica
gel, 40:60 EtOAc/n-Hex) gave 7 (1.60 g, 53%) as a white crystalline
solid. Compound 7: Mp 104–106 °C; 1H NMR (400 MHz, CDCl3) d
5.36 (1H, s, H-12), 4.46 (1H, m, H-10), 3.84 (2H, m, CH2OH), 2.65
4a
), 2.10–0.84 (42H, m) including 1.39 (6H, s, 2 ꢂ 3Me), 0.95
(6H, d, J = 5.9 Hz, 2 ꢂ 6Me) and 0.84 (6H, d, J = 7.4 Hz, 2 ꢂ 9Me);
13C NMR (100 MHz, CDCl3) d 150.2, 130.0, 125.0, 120.5, 103.5,
89.3, 81.0, 71.5, 67.0, 52.7, 44.6, 37.8, 36.9, 34.8, 30.9, 30.3, 26.4,
25.0, 24.6, 20.5 and 13.2; HRMS (ESI) C40H60O12P [M+H]+ requires
763.3822, found 763.3821; Anal. calcd C40H59O12P requires: C,
62.98; H, 7.80. Found: C, 62.75; H, 8.01.
5.1.2.6. Methylphosphonate-linked
dimer
8. Alcohol
7
(260 mg, 0.83 mmol) was reacted according to GP2. Purification
by column chromatography (silica gel, EtOAc) gave 8 (90 mg,
31%) as a clear oil. Compound 8: 1H NMR (400 MHz, CDCl3) d
5.30 (2H, s, 2 ꢂ H-12), 4.37–4.03 (6H, m, 2 ꢂ OCH2 and 2 ꢂ H-10),
2.70 (2H, sex, J = 7.3 Hz, 2 ꢂ H-9), 2.33 (2H, td, J = 4.0, 14.5 Hz,
(1H, sex, J = 7.3 Hz, H-9), 2.33 (1H, td, J = 13.4, 4.0 Hz, H-4a),
3.07–0.87 (21H, m) including 1.42 (3H, s, 3Me), 0.96 (3H, d,
J = 6.0 Hz, 6Me) and 0.87 (3H, d, J = 7.6 Hz, 9Me); 13C NMR
(100 MHz, CDCl3) d 103.2, 89.3, 81.1, 75.0, 62.8, 52.3, 44.2, 37.5,
36.6, 34.5, 31.7, 30.9, 30.4, 26.1, 24.8, 20.2 and 12.9; IR (Nujol)/
cmꢀ1 3512 (O–H), 2928, 1462, 1379, 1273, 1093, 1048, 883 (O–
O) and 838 (O–O); MS (CI) [M+H]+ 313 (11), 296 (20), 295 (100),
267 (19) and 253 (27); HRMS (CI) C17H29O5 [M+H]+ requires
313.20151, found 313.20185; Anal. calcd C17H28O5 requires: C,
65.36; H, 9.03. Found: C, 65.64; H, 9.29.
2 ꢂ H-4
a), 2.05–0.87 (45H, m) including 1.48 (3H, d, J = 17.5 Hz,
P-Me), 1.40 (6H, s, 2 ꢂ 3Me), 0.96 (6H, d, J = 6.0 Hz, 2 ꢂ 6Me) and
0.87 (6H, d, J = 7.5 Hz, 2 ꢂ 9Me); 13C NMR (100 MHz, CDCl3) d
103.6, 92.4, 81.4, 71.8, 64.1, 44.7, 37.8, 36.9, 34.8, 31.1, 30.3,
30.1, 26.4, 25.1, 20.5, 13.3, 12.0 and 10.6; HRMS (ESI)
C35H57NaO11P [M+Na]+ requires 707.3536, found 707.3513; Anal.

1334
J. Chadwick et al. / Bioorg. Med. Chem. 17 (2009) 1325–1338
calcd C35H57O11P requires: C, 62.63; H, 8.59. Found: C, 62.17; H,
8.77.
C, 65.65; H, 9.28. Compound 12: Mp 131–132 °C; 1H NMR
(400 MHz, CDCl3) d 5.22 (1H, s, H-12), 3.74 and 3.61 (2H, AB quar-
tet, J = 11.6, 1.0 Hz, H-10), 2.32 (1H, td, J = 14.1, 4.0 Hz, H-4a), 2.11–
5.1.2.7. 10b-Deoxy-(2-hydroxyethyl)-deoxoartemisinin 9. Zinc
dust (1 g) was activated by washing with 5% aq HCl (3 ꢂ 10 mL),
water (3 ꢂ 10 mL), EtOH (3 ꢂ 10 mL) and diethyl ether
(3 ꢂ 10 mL), then thoroughly dried in vacuo. Activated zinc dust
(50 mg) was added to a stirring solution of 7 (322 mg, 1.06 mmol)
in glacial acetic acid (35 mL). The reaction mixture was stirred at
room temperature for 72 h, with more zinc dust (50 mg) being
added at 24 and 48 h, respectively. After 72 h CHCl3 (50 mL) was
added, the mixture filtered through a sintered glass funnel and
the zinc dust washed with CHCl3 (3 ꢂ 25 mL). The filtrate and
washings were combined and neutralized with saturated aq NaH-
CO3. The organic layer was separated, washed with saturated aq
NaHCO3 (25 mL), brine (25 mL) and water (25 mL), dried over
MgSO4, filtered and concentrated under reduced pressure to give
a white solid. Purification by flash column chromatography (silica
gel, 1:2 EtOAc/n-Hex) gave 9 (255 mg, 81%) as a white solid. Com-
pound 9: Mp 90–92 °C; 1H NMR (400 MHz, CDCl3) d 5.27 (1H, s, H-
12), 4.36 (1H, m, H-10), 3.57 (2H, m, CH2OH), 2.50 (1H, br s, OH),
2.23 (1H, sex, J = 7.6 Hz, H-9), 1.99–0.89 (21H, m) including 1.53
(3H, s, 3Me), 0.89 (6H, m, 6Me and 9Me); 13C NMR (100 MHz,
CDCl3) d 107.6 (C-3), 97.4, 82.9, 68.8, 62.1, 45.7, 40.6, 36.0, 34.9,
33.6, 30.1, 25.6, 25.1, 24.1, 22.6, 19.2 and 12.8; IR (Nujol)/cmꢀ1
3340 (O–H), 2925, 2855, 2359, 1458, 1385, 1377, 1266, 1208,
1144, 1097, 1053, 1011, 996, 984, 953, 905 and 877; HRMS (CI)
C17H29O4 [M+H]+ requires 297.20660, found 297.20670; Anal. calcd
C17H28O4 requires: C, 68.89; H, 9.52. Found: C, 68.73; H, 9.42.
0.96 (19H, m) including 1.57 (3H, s, 9Me), 1.41 (3H, s, 3Me) and
0.96 (3H, d, J = 5.9 Hz, 6Me); 13C NMR (100 MHz, CDCl3) d 104.0,
92.3, 81.9, 70.3, 52.6, 38.1, 37.9, 37.7, 36.7, 34.2, 29.4, 26.1, 24.8,
24.2 and 20.5; IR (Nujol)/cmꢀ1 3438 (O–H), 2929, 2858, 1461,
1378, 1277, 1252, 1136, 1091, 1065, 1040, 996, 934, 911, 874
(O–O), 830 (O–O); HRMS (ESI) C15H24NaO5 [M+Na]+ requires
307.1521, found 307.1508; C15H24O5 requires C 63.36; H 8.51.
Found C, 62.89; H, 8.37.
5.1.2.10. Methyl phosphate ester-linked dimer 13. Alcohol 11
(120 mg, 0.36 mmol) was reacted according to GP1. Purification
by column chromatography (Florisil, 30:70 EtOAc/n-Hex) gave 13
(32 mg, 25%) as a colorless oil. Compound 13: 1H NMR (400 MHz,
CDCl3) d 5.33 (2H, s, 2 ꢂ H-12), 4.28–4.05 (6H, m, 2 ꢂ OCH2 and
2 ꢂ H-10), 3.78–3.64 (3H, m, OMe), 2.66 (2H, sex, J = 7.5 Hz,
2 ꢂ H-9), 2.32 (2H, td, J = 14.0, 4.0 Hz, 2 ꢂ H-4
a), 2.06–0.87 (46H,
m) including 1.41 (6H, s, 2 ꢂ 3Me), 0.96 (6H, d, J = 6.0 Hz,
2 ꢂ 6Me) and 0.87 (6H, d, J = 7.6 Hz, 2 ꢂ 9Me); 13C NMR
(100 MHz, CDCl3) d 103.2, 89.3, 81.1, 75.3, 62.8, 52.3, 44.3, 37.5,
36.6, 34.5, 31.2, 30.6, 29.7, 26.5, 26.0, 24.9, 24.8, 20.2 and 12.9;
HRMS (ESI) C37H61NaO12
P
[M+Na]+ requires 751.3798, found
751.3782; Anal. calcd C37H61O12P requires: C, 60.97; H, 8.44.
Found: C, 60.43; H, 8.67.
5.1.2.11. Phenyl phosphate ester-linked dimer 14. Alcohol 11
(99 mg, 0.30 mmol) was reacted according to GP1. Purification by
column chromatography (Florisil, 60:40 EtOAc/n-Hex) gave 14
(24 mg, 20%) as a colorless oil. Compound 14: 1H NMR (400 MHz,
CDCl3) d 7.32 (2H, t, Ar–H), 7.22 (2H, d, Ar–H), 7.15 (1H, t, Ar–H),
5.27 (2H, s, 2 ꢂ H-12), 4.29–4.11 (6H, m, 2 ꢂ OCH2 and 2 ꢂ H-10),
2.64 (2H, sex, J = 7.2 Hz, 2 ꢂ H-9), 2.32 (2H, td, J = 14.3, 3.8 Hz,
5.1.2.8. Deoxy methylphosphonate-linked dimer 10. Alcohol 9
(128 mg, 0.43 mmol) was reacted according to GP2. Purification
by column chromatography (silica gel, EtOAc) gave 10 (66 mg,
47%) as a clear oil. Compound 10: 1H NMR (400 MHz, CDCl3) d
5.25 (2H, s, 2 ꢂ H-12), 4.30–3.97 (6H, m, 2 ꢂ OCH2 and 2 ꢂ H-10),
2.22 (2H, sex, J = 7.3 Hz, 2 ꢂ H-9), 2.08–0.88 (47H, m) including
1.50 (9H, m, P-Me and 2 ꢂ 3Me) and 0.88 (12H, m, 2 ꢂ 6Me and
2 ꢂ 9Me); 13C NMR (100 MHz, CDCl3) d 107.4, 97.4, 82.7, 64.9,
63.6, 45.7, 40.7, 35.9, 34.9, 32.9, 29.7, 25.5, 24.1, 22.5, 19.2, 12.5
an 10.6; HRMS (ESI) C35H57NaO9P [M+Na]+ requires 657.3638,
found 657.3629; Anal. calcd C35H57O9P requires: C, 64.40; H,
8.80. Found: C, 64.27; H, 8.85.
2 ꢂ H-4 ), 2.06–0.83 (46H, m) including 1.40 (6H, s, 2 ꢂ 3Me),
a
0.95 (3H, d, J = 5.9 Hz, 2 ꢂ 6Me), 0.95 (3H, d, J = 5.9 Hz, 2 ꢂ 6Me),
0.83 (3H, d, J = 7.5 Hz, 9Me) and 0.83 (3H, d, J = 7.5 Hz, 9Me); 13C
NMR (100 MHz, CDCl3) d 130.0, 125.3, 120.4, 120.4, 103.5, 89.5,
81.5, 75.1, 68.9, 52.7, 44.7, 37.8, 37.0, 34.8, 30.7, 26.5, 25.9, 25.3,
25.1, 20.5 and 13.2; HRMS (ESI) C42H63NaO12P+ [M+Na]+ requires
813.3955, found 813.3948; Anal. calcd C42H63O12P requires: C,
63.78; H, 8.03, Found: C, 63.42; H, 8.19.
5.1.2.9. 10b-(3-Hydroxypropyl)-deoxoartemisinin
5.1.2.12. Methylphosphonate-linked dimer 15. Alcohol 11
(100 mg, 0.31 mmol) was reacted according to GP3. Purification
by flash column chromatography (silica gel, EtOAc) gave 15
(31 mg, 28%) as a colorless oil. Compound 15: 1H NMR (400 MHz,
CDCl3) d 5.30 (2H, s, 2 ꢂ H-12), 4.25–3.90 (6H, m, 2 ꢂ OCH2 and
2 ꢂ H-10), 2.67 (2H, sex, J = 7.0 Hz, 2 ꢂ H-9), 2.32 (2H, td, J = 14.1,
11. BH3ꢁSMe2 [2.0 M in diethyl ether] (2.00 mL, 4.00 mmol) was
added to a stirring solution of 6 (1.04 g, 3.36 mmol) in anhydrous
THF (20 mL) at 0 °C. The reaction mixture was allowed to warm
up to room temperature and stirred for 24 h. After 24 h, a suspen-
sion of NaBO3ꢁ4H2O (2.50 g, 16.25 mmol) in water (25 mL) was
slowly added and the resulting suspension stirred for a further
24 h. After 24 h, water (25 mL) was added and the mixture ex-
tracted with CH2Cl2 (4 ꢂ 25 mL). The organic extracts were dried
over Na2SO4, filtered and concentrated under reduced pressure to
give a colorless oil. Purification by gradient flash column chroma-
tography (silica gel, 20:80?30:70 EtOAc/n-Hex) gave 11 (0.66 g,
62%) as a white solid along with a byproduct 12 (387 mg, 30%) as
a white solid. Compound 11: 1H NMR (400 MHz, CDCl3) d 5.33
(1H, s, H-12), 4.24 (1H, m, H-10), 3.70 (2H, CH2OH), 2.65 (1H,
4.0 Hz, 2 ꢂ H-4
a), 2.04–0.87 (49H, m) including 1.48 (3H, d,
J = 17.5 Hz, P-Me), 1.41 (6H, s, 2 ꢂ 3Me), 0.96 (6H, d, J = 5.8 Hz,
2 ꢂ 6Me) and 0.87 (6H, d, J = 7.5 Hz, 2 ꢂ 9Me); 13C NMR
(100 MHz, CDCl3) d 103.5, 89.5, 81.5, 75.2, 65.7, 44.6, 37.8, 36.9,
34.8, 30.7, 28.9, 26.5, 25.9, 25.3, 25.0, 20.6, 13.3 and 10.5; HRMS
(ESI) C37H61NaO11P+ [M+Na]+ requires 735.3849, found 735.3839;
Anal. calcd C37H61O11P requires: C, 62.34; H, 8.63. Found: C,
61.95; H, 8.19.
sex, J = 7.2 Hz, H-9), 2.33 (1H, td, J = 14.0, 4.0 Hz, H-4
a), 2.07–
5.1.2.13. 10b-(Propa-1,2-dienyl)deoxoartemisinin 18. A solu-
0.87 (23H, m) including 1.41 (3H, s, 3Me), 0.96 (3H, d, J = 6.0 Hz,
6Me) and 0.87 (3H, d, J = 7.6 Hz, 9Me); 13C NMR (100 MHz, CDCl3)
d 103.5, 89.7, 81.5, 75.7, 63.1, 52.7, 44.6, 40.0, 37.9, 34.8, 31.6, 30.9,
26.9, 26.4, 25.3, 25.1, 20.5 and 13.2; IR (film)/cmꢀ1 3424 (O–H),
2940, 2875, 1715, 1452, 1377, 1100 (C–OH), 877 (O–O), 825 (O–
O) and 735; HRMS (CI) C18H31O5 [M+H]+ requires 327.21713, found
327.21772; Anal. calcd C18H30O5 requires: C, 66.13; H, 9.26. Found:
tion of 5 (980 mg, 2.52 mmol) in anhydrous 1,2-dichloroethane
(10 mL) was added dropwise via cannula to a stirring mixture
of propargyl trimethylsilane (1.00 g, 8.91 mmol), anhydrous
ZnCl2 (0.45 g, 3.30 mmol) and powdered 4 Å molecular sieves
in anhydrous 1,2-dichloroethane (10 mL) at 0 °C. After 2, 5 and
8 h more ZnCl2 (0.45 g, 3.30 mmol) was added to the reaction
mixture. After stirring overnight, the reaction mixture was

J. Chadwick et al. / Bioorg. Med. Chem. 17 (2009) 1325–1338
1335
diluted with EtOAc (50 mL) and washed with 5% aq citric acid
(25 mL), saturated aq NaHCO3 (25 mL) and brine (25 mL). The
organic extracts were dried over MgSO4, filtered and
concentrated under reduced pressure to give a clear oil. Purifica-
tion by flash column chromatography (silica gel, 5:95 EtOAc/n-
Hex) gave 18 (328 mg, 42%) as a pale yellow oil. 18: 1H NMR
(400 MHz, CDCl3) d 5.49 (1H, s, H-12), 5.32 (1H, ddd, J = 7.5,
6.8, 4.3 Hz CH@C@CH2), 4.86 (2H, dd, J = 6.8, 4.3 Hz), 4.67 (1H,
m, H-10), 2.89 (1H, m, H-9), 2.37 (1H, td, J = 14.1, 4.0 Hz, H-
C
21H28NaO6S [M+Na]+ requires 431.1504, found 431.1500; Anal.
calcd C21H28O6S requires: C, 61.74; H, 6.91. Found: C, 61.61; H,
7.03.
5.1.2.16. 10b-Vinyl-9-epi-deoxoartemisinin
22. Vinylmagne-
sium bromide (1.0 M in THF, 8.0 mL, 8.00 mmol) was added to a
stirring solution of anhydrous zinc chloride (0.63 g, 4.64 mmol)
in anhydrous THF (25 mL). After stirring for 30 min at room tem-
perature, 21 (1.58 g, 3.87 mmol) in anhydrous THF (16 mL) was
added dropwise via cannula. After stirring for 48 h, 5% aq citric acid
solution (50 mL) was added and the mixture extracted with EtOAc
(3 ꢂ 30 mL). The organic extracts were washed with saturated
NaHCO3 (25 mL) and brine (25 mL), dried over MgSO4, filtered
and concentrated under reduced pressure to give a pale yellow
oil. Purification by flash column chromatography (silica gel, 8:92
EtOAc/n-Hex) gave an inseparable mixture of 22 and 17 (600 mg)
as a colorless oil. Compound 22: 1H NMR (400 MHz, CDCl3) d
5.78 (1H, m, CH@CH2), 5.55 (1H, s, H-12), 5.33 (1H, dm,
4a), 2.06–0.83 (19H, m) including 1.44 (3H, s, 3Me), 0.96 (3H,
d, J = 6.0 Hz, 6Me) and 0.83 (3H, d, J = 7.5 Hz, 9Me); 13C NMR
(100 MHz, CDCl3) d 207.8 (CH@C@CH2), 104.4, 89.7, 88.5, 81.3,
77.6, 74.2, 53.0, 45.6, 37.9, 36.8, 34.9, 30.8, 26.5, 25.0, 24.2,
20.7 and 14.1; IR (neat)/cmꢀ1 2239, 2875, 2245, 1956 (C@C@C),
1738, 1717, 1451, 1375, 1242, 1188, 1126, 1090, 1052, 993, 937,
876 (O–O), 845 (O–O) and 732; MS (CI) [M+NH4]+ 324 (31), 261
(100) and 247 (57); HRMS (CI) C18H30NO4 [M+NH4]+ requires
324.21750, found 324.21808; Anal. calcd C18H26O4 requires: C,
70.56; H, 8.55. Found: C, 70.07; H, 8.79.
J = 17.2 Hz,
CH@C(H)Hcis), 4.52 (1H, dd, J = 10.3, 7.0 Hz, H-10), 2.29 (1H, td,
J = 13.9, 4.0 Hz, H-4 ), 2.10–0.96 (20H, m) including 1.41 (3H, s,
3Me), 1.02 (3H, d, J = 6.8 Hz, 9Me) and 0.96 (3H, d, J = 6.0 Hz,
6Me); 13C NMR (100 MHz, CDCl3)
138.4 (CH@CH2), 117.1
CH@C(H)Htrans),
5.19
(1H,
dm,
J = 10.3 Hz,
5.1.2.14. Anhydroartemisinin 17.
A
solution of BF3ꢁOEt2
a
(8.4 mmol) was added to a stirring solution of dihydroartemisinin
4 (2.00 g, 7 mmol) in anhydrous THF (50 mL) at room temperature.
The reaction mixture was stirred at 66 °C for 2.5 h, after which
time it was allowed to cool to room temperature, diluted with
EtOAc (50 mL) and washed with saturated aq NaHCO3 (25 mL)
and brine (25 mL). The organic extracts were dried over MgSO4, fil-
tered and concentrated under reduced pressure to give a pale yel-
low solid. Purification by flash column chromatography (silica gel,
10:90 EtOAc/n-Hex) gave 17 (1.47 g, 79%) as a white solid. Com-
pound 17: Mp 95–97 °C; 1H NMR (400 MHz, CDCl3) d 6.18 (1H, q,
J = 1.3 Hz, H-10), 5.54 (1H, s, H-12), 2.45–2.35 (1H, m), 2.10–2.00
(2H, m), 1.95–0.98 (17H, m) including 1.59 (3H, d, J = 1.1 Hz,
9Me), 1.42 (3H, s, 3Me) and 0.98 (3H, d, J = 5.8 Hz, 6Me); 13C
NMR (100 MHz, CDCl3) d 135.6, 108.8, 105.2, 90.3, 79.6, 52.1,
45.1, 38.1, 36.9, 34.8, 30.6, 26.5, 25.1, 20.9 and 16.8; IR (Nujol)/
cmꢀ1 2931, 2857, 2358, 1686, 1652, 1461, 1376, 1280, 1251,
1198, 1177, 1158, 1141, 1112, 1079, 1029, 1016, 992, 954, 904,
879 (O–O), 848 (O–O), 828 and 722; HRMS (CI) C15H23O4 [M+H]+
requires 267.15964, found 267.16045; Anal. calcd C15H22O4 re-
quires C, 67.64; H, 8.33. Found: C, 67.48; H, 8.35.
d
(CH@CH2), 102.2, 90.4, 82.1, 77.4, 51.4, 46.7, 40.8, 37.3, 36.6,
34.1, 32.0, 24.8, 19.9 and 19.4; HRMS (CI) C17H27O4 [M+H]+ re-
quires 295.1909, found 295.1907.
5.1.2.17. 10b-(Hydroxymethyl)-9-epi-deoxoartemisinin 23. Ozone
was bubbled through a solution of 22 (600 mg) in anhydrous
CH2Cl2 (50 mL) at ꢀ78 °C for 60 min until the solution became
saturated with ozone and appeared blue. Nitrogen was then bub-
bled through the solution for 20 min to purge excess ozone. The
solvent was removed under reduced pressure to give a white
foam which taken up in anhydrous MeOH/THF (10:90, 100 mL).
The solution was cooled to 0 °C and NaBH4 (2.50 g, 66.1 mmol)
added over 4 h. The mixture was stirred overnight at room tem-
perature and then concentrated under reduced pressure, followed
by addition of EtOAc (150 mL) and water (50 mL). The organic
layer was dried over MgSO4, filtered and concentrated under re-
duced pressure to give a colorless oil. Purification by flash column
chromatography (silica gel, 40:60 EtOac/n-Hex) gave 23 (372 mg,
32% from 21) as a colorless oil. Compound 23: 1H NMR (400 MHz,
CDCl3) d 5.45 (1H, s, H-12), 4.22 (1H, ddd, J = 10.7, 5.5, 2.6 Hz, H-
10), 3.84 (1H, dd, J = 11.8, 2.6 Hz, CHOH), 3.53 (1H, dd, J = 11.8,
5.1.2.15. 10b-Benzenesulfonyl-9-epi-dihydroartemisinin 21. Ben-
zene sulfinic acid was prepared from its commercially available
sodium salt by treatment with 1.0 M HCl (10 mL) and extrac-
tion with CH2Cl2 (2 ꢂ 10 mL). The organic extracts were dried
over MgSO4, filtered and concentrated under reduced pressure
to give a white solid which was used immediately without fur-
ther purification. Benzene sulfinic acid (1.00 g, 7.0 mmol) was
added to a stirring solution of anhydroartemisinin 17 (0.93 g,
3.5 mmol) in anhydrous CH2Cl2 (60 mL). After stirring for
30 min at room temperature, the reaction mixture was diluted
with CH2Cl2 (100 mL) and washed with saturated NaHCO3
(50 mL) and brine (50 mL). The organic layer was dried over
MgSO4, filtered and concentrated under reduced pressure to
give an off-white solid. Purification by flash column chromatog-
raphy (silica gel, 20:80 EtOAc/n-Hex) gave 21 (0.95 g, 66%) as a
white solid. Compound 21: Mp 106–108 °C; 1H NMR (400 MHz,
CDCl3) d 7.98 (2H, t, J = 7.2 Hz, Ar–H), 7.66 (1H, t, J = 7.2 Hz, Ar–
H), 7.55 (2H, t, J = 7.8 Hz, Ar–H), 5.41 (1H, s, H-12), 5.14 (1H, d,
J = 10.3 Hz, H-10), 2.28–0.92 (21H, m) including 1.43 (3H, d,
J = 6.9 Hz, 9Me), 1.06 (3H, s, 3Me) and 0.92 (3H, d, J = 5.7 Hz,
6Me); 13C NMR (100 MHz, CDCl3) d 137.8, 133.9, 129.6, 129.2,
128.9, 102.7, 91.0, 90.1, 82.3, 51.1, 48.9, 37.5, 36.3, 35.3, 34.4,
34.1, 31.4, 25.5, 25.0, 21.1 and 20.1; IR (film)/cmꢀ1 2926,
1551 (Ar), 1384, 1250, 1210, 1000 and 1020; HRMS (ESI)
5.5 Hz, CHOH), 2.30 (1H, td, J = 14.0, 4.0 Hz, H-4a), 2.10–0.96
(20H, m) including 1.41 (3H, s, 3Me), 1.04 (3H, d, J = 7.0 Hz,
9Me) and 0.96 (3H, d, J = 6.0 Hz, 6Me); 13C NMR (100 MHz, CDCl3)
d 102.3, 90.5, 82.1, 75.7, 64.1, 51.4, 47.0, 37.3, 36.5, 35.6, 34.1,
31.8, 25.8, 24.7, 20.3 and 19.9; IR (neat)/cmꢀ1 3493 (O–H),
2927, 2875, 1740, 1457, 1375, 1280, 1239, 122, 1200, 1158,
1140, 1114, 1072, 1052, 1011, 991, 964, 944, 931, 911, 889 (O–
O), 866, 836 (O–O), 818, 763, 732 and 701; HRMS (CI)
C16H30NO5 [M+NH4]+ requires 316.21240, found 316.21204; Anal.
calcd C16H26O5 requires: C, 64.41; H, 8.78. Found: C, 64.35; H,
8.91.
5.1.2.18. Methyl phosphate ester-linked dimer 24a. Alcohol 23
(104 mg, 0.35 mmol) was reacted according to GP1. Purification by
column chromatography (Florisil, 50:50 EtOAc/n-Hex) gave 24a
(38 mg, 32%) as
a
colorless oil. Compound 24a: 1H NMR
(400 MHz, CDCl3) d 5.45 (2H, s, 2 ꢂ H-12), 4.37–4.07 (6H, m,
2 ꢂ OCH2 and 2 ꢂ H-10), 3.82 (3H, d, J = 11.2 Hz, OMe), 2.28 (2H,
td, J = 13.8, 3.8 Hz, 2 ꢂ H-4
a), 2.07–1.89 (6H, m), 1.70–0.96 (34H,
m) including 1.38 (6H, s, 2 ꢂ 3Me), 1.08 (6H, d, J = 6.9 Hz,
2 ꢂ 9Me) and 0.96 (6H, d, J = 5.8 Hz, 2 ꢂ 6Me); 13C NMR
(100 MHz, CDCl3) d 102.2, 90.5, 82.1, 74.1, 68.6, 51.4, 47.0, 37.3,

1336
J. Chadwick et al. / Bioorg. Med. Chem. 17 (2009) 1325–1338
36.5, 35.8, 34.1, 31.7, 30.2, 29.7, 25.8, 24.8 and 19.9; HRMS (ESI)
C33H53NaO12P [M+Na]+ requires 695.3172, found 695.3147; Anal.
calcd C33H53O12P requires: C, 58.92; H, 7.94. Found: C, 59.32; H,
8.33.
to a stirring solution of 7 (0.19 g, 0.61 mmol) in anhydrous CH2Cl2
(10 mL). After stirring for 30 min at room temperature, the reaction
mixture was diluted with diethyl ether (50 mL) and poured into a
stirring solution of sodium thiosulfate (2.5 g) in saturated aq NaH-
CO3 (50 mL). After stirring for 15 min at room temperature, the or-
ganic layer was separated, washed with saturated aq NaHCO3
(20 mL) and water (20 mL), dried over Na2SO4, filtered and concen-
trated under reduced pressure to give a colorless oil. Purification
by flash column chromatography (silica gel, 30:70 EtOAc/n-Hex)
gave 27 (0.13 g, 68%) as a white solid.
5.1.2.19. Phenyl phosphate ester-linked dimer 24b. Alcohol 23
(104 mg, 0.35 mmol) was reacted according to GP1. Purification by
column chromatography (Florisil, 50:50 EtOAc/n-Hex) gave 24b
(38 mg, 32%) as
a
colorless oil. Compound 24b: 1H NMR
(400 MHz, CDCl3) d 7.38–7.27 (4H, m, Ar–H), 7.14 (2H, m, Ar–H),
5.40 (2H, s, 2 ꢂ H-12), 4.4.46–4.18 (6H, m, 2 ꢂ OCH2 and 2 ꢂ H-
10), 2.28 (2H, td, J = 13.6, 3.6Hz, 2 ꢂ 4
a
), 2.04–1.89 (6H, m),
5.1.2.23. 10b-(2-Oxoethyl)deoxoartemisinin 27. Ozone was
bubbled through a solution of 6 (2.44 g, 7.9 mmol) in anhydrous
MeOH (100 mL) at ꢀ78 °C for 60 min until the solution became sat-
urated with ozone and appeared blue. Nitrogen was then bubbled
through the solution for 20 min to purge excess ozone. PPh3
(4.15 g, 15.8 mmol) was added to the stirring solution at ꢀ78 °C.
The mixture was allowed to warm up to room temperature and
stirred for 18 h. The solvent was then removed under reduced
pressure and the residue purified by flash column chromatography
(silica gel, 15:85 EtOAc/n-Hex) to give 27 (1.86 g, 76%) as a white
solid.
1.66–0.95 (34H, m) including 1.37 (6H, s, 2 ꢂ 3Me), 1.05 (6H, d,
J = 7.0 Hz, 2 ꢂ 9Me) and 0.95 (6H, d, J = 5.7 Hz, 2 ꢂ 6Me); 13C
NMR (100 MHz, CDCl3) d 129.9, 125.1, 120.6, 114.3, 102.5, 90.8,
82.5, 74.2, 51.8, 47.4, 37.6, 36.9, 36.1, 35.9, 34.5, 32.0, 30.1, 26.2,
25.2 and 20.2; HRMS (ESI) C38H55NaO12
P
[M+Na]+ requires
757.3329, found 757.3318; Anal. calcd C38H55O12P requires: C,
62.11; H, 7.54. Found: C, 61.79; H, 7.76.
5.1.2.20. 10b-(2-Methanesulfonylethyl)deoxoartemisinin 25. NEt3
(0.4 mL, 2.9 mmol) and MsCl (0.2 mL, 2.6 mmol) were added to a
stirring solution of 7 (0.45 g, 1.4 mmol) in anhydrous CH2Cl2
(20 mL) at 0 °C. After stirring at 0 °C for 2 h, water (20 mL) was
added and the aqueous phase extracted with CH2Cl2 (3 ꢂ 10 mL).
The combined organic layers were dried over MgSO4, filtered and
the solvent removed under reduced pressure. Purification by flash
column chromatography (silica gel, 40:60 EtOAc/n-Hex) gave 25
(0.56 g, 100%) as a white crystalline solid. Compound 25: Mp
123–125 °C; 1H NMR (400 MHz, CDCl3) d 5.31 (1H, s, H-12),
4.48–4.32 (3H, m, H-10 and CH2OMs), 3.03 (3H, s, OMs), 2.69
5.1.2.24. 10b-(2-Oxoethyl)deoxoartemisinin
27. 2,6-Lutidine
(0.45 mL, 3.87 mmol), OsO4 (2.5% w/v in 2-methyl-2-propanol)
and NaIO4 (1.66 g, 7.74 mmol) were added to a stirring solution
of 6 (597 mg, 1.94 mmol) in dioxane-water (3:1, 20 mL). After stir-
ring at room temperature for 24 h, water (20 mL) and CH2Cl2
(20 mL) were added. The organic layer was separated and the
aqueous phase extracted with CH2Cl2 (3 ꢂ 20 mL). The combined
organic extracts were washed with brine (20 mL), dried over
Na2SO4, filtered and concentrated under reduced pressure to give
a colorless oil. Purification by flash column chromatography (silica
gel, 15:85 EtOAc/n-Hex) gave 27 (380 mg, 63%) as a white solid.
Compound 27: 1H NMR (400 MHz, CDCl3) d 9.80 (1H, dd, J = 3.2,
1.6 Hz, CHO), 5.32 (1H, s, H-12), 4.96 (1H, m, H-10), 2.79–2.64
(2H, m, CH2CHO), 2.47–2.41 (1H, m), 2.33 (1H, td, J = 14.1, 4.1 Hz,
(1H, sex, J = 7.3 Hz, H-9), 2.33 (1H, td, J = 13.8, 4.1 Hz, H-4a),
2.10–0.88 (21H, m) including 1.41 (3H, s, 3Me), 0.97 (3H, d,
J = 6.0 Hz, 6Me) and 0.88 (3H, d, J = 7.6 Hz, 9Me); 13C NMR
(100 MHz, CDCl3) d 103.2, 89.2, 81.0, 70.9, 68.4, 52.2, 44.1, 37.3,
36.5, 34.4, 29.9, 29.6, 26.0, 24.8, 24.7, 20.1 and 12.7; IR (Nujol)/
cmꢀ1 2926, 1462, 1378, 1350, 1279, 1167, 1100, 1037, 876 (O–O)
and 824 (O–O); HRMS (ESI) C18H30O7S [M]+ requires 390.17123,
found 390.17127; Anal. calcd C18H30O7S requires: C, 55.39; H,
7.74. Found: C, 55.11; H, 7.81.
H-4a), 2.07–2.00 (1H, m), 1.97–1.89 (1H, m), 1.84–1.76 (1H, m),
1.73–1.65 (2H, m), 1.48–0.86 (14H, m) including 1.40 (3H, s,
3Me), 0.97 (3H, d, J = 6.0 Hz, 6Me) and 0.86 (3H, d, J = 7.6 Hz); 13C
NMR (100 MHz, CDCl3) d 201.7 (CHO), 103.2, 89.4, 80.9, 69.4,
52.2, 44.5, 44.0, 37.5, 36.5, 34.4, 29.8, 26.0, 24.8, 24.7, 20.1 and
13.0; HRMS (CI) C17H30NO5 [M+NH4]+ requires 328.21240, found
328.21203; Anal. calcd C17H26O5 requires: C, 65.78; H, 8.44. Found:
C, 65.62; H, 8.51.
5.1.2.21. 10b-(2-Aminoethyl)-deoxoartemisinin 26. A solution
of 25 (0.78 g, 2.0 mmol) in NH4OH/EtOH (1:1, 40 mL) was stirred
at room temperature for five days. The reaction mixture was then
concentrated under reduced pressure, treated with 5% aq HCl until
acidic and washed with CH2Cl2 (2 ꢂ 50 mL). The aqueous phase
was then treated with 2.0 M NaOH until basic and extracted with
CH2Cl2 (2 ꢂ 50 mL). The organic extracts were dried over MgSO4,
filtered and concentrated under reduced pressure to give an off-
white solid. Purification by flash column chromatography (silica
gel, 20:80 MeOH/CH2Cl2) gave 26 (0.61 g, 98%) as a white solid.
Compound 26: Mp 101–103 °C; 1H NMR (400 MHz, CDCl3) d 5.35
(1H, s, H-12), 4.29 (1H, m, H-10), 2.99–2.82 (2H, m, CH2NH2),
2.67 (1H, 1H, sex, J = 7.5 Hz, H-9), 2.33 (1H, td, J = 13.5, 4.0 Hz, H-
5.1.2.25. 10b-(2-Carboxyethyl)deoxoartemisinin 28. NaH2PO4
(203 mg, 1.69 mmol) was added to a stirring solution of 27
(2.02 g, 6.51 mmol) in t-BuOH (90 mL) and water (18 mL). 2-
Methyl-2-butene (2.0 M in THF, 38 mL, 76 mmol) was then added,
followed by NaClO2 (1.15 g, 9.58 mmol). The resulting pale yellow
solution was stirred at room temperature for 2 h and then concen-
trated under reduced pressure. 1.0 M aq NaOH (50 mL) was added
and the resulting solution washed with CH2Cl2 (3 ꢂ 50 mL). The
aqueous phase was acidified with 1.0 M aq HCl and extracted with
CH2Cl2 (3 ꢂ 50 mL). The combined organic extracts were dried over
MgSO4, filtered and concentrated under reduced pressure to give
28 (2.12 g, 100%) as a white brittle foam. Compound 28: 1H NMR
(400 MHz, CDCl3) d 9.70 (1H, br s, CO2H), 5.37 (1H, s, H-12), 4.88
(1H, ddd, J = 9.9, 6.2, 3.3 Hz, H-10), 2.78–2.61 (2H, H-9 and
CHHCO2H), 2.50 (1H, dd, J = 15.7, 3.3 Hz, CHHCO2H), 2.33 (1H, td,
4a), 2.09 (2H, br s, NH2), 2.06–0.87 (21H, m) including 1.41 (3H,
s, 3Me), 0.96 (3H, d, J = 6.1 Hz, 6Me) and 0.87 (3H, d, J = 7.6 Hz,
9Me); 13C NMR (100 MHz, CDCl3) d 103.5, 92.3, 81.5, 74.2, 52.8,
44.7, 41.1, 37.8, 37.0, 34.9, 32.9, 30.7, 30.0, 26.5, 25.2, 20.5 and
13.3; IR (Nujol)/cmꢀ1 3365 (N–H), 2925, 2875, 1664, 1570, 1455,
1377, 1114, 1054, 1011, 944, 877 (O–O) and 839 (O–O); HRMS
(CI) C17H30NO4 [M+H]+ requires 312.21747, found 312.21776;
Anal. calcd C17H29NO4 requires: C, 65.57; H, 9.39; N 4.50. Found:
C, 65.38; H, 9.37; N, 4.00.
J = 14.1, 4.0 Hz, H-4a), 2.08–2.00 (1H, m), 1.99–1.90 (1H, m),
1.84–1.75 (1H, m), 1.73–1.64 (2H, m), 1.48–0.88 (14H, m) includ-
ing 1.41 (3H, s, 3Me), 0.97 (3H, d, J = 5.9 Hz, 6Me) and 0.88 (3H,
d, J = 7.5 Hz, 9Me); 13C NMR (100 MHz, CDCl3) d 176.1 (CO2H),
103.3, 89.4, 80.8, 70.9, 52.1, 43.9, 37.5, 37.4, 36.5, 35.9, 34.4,
5.1.2.22. 10b-(2-Oxoethyl)deoxoartemisinin 27. Dess–Martin
periodinane (15% w/v in CH2Cl2, 1.8 mL, 0.64 mmol) was added

J. Chadwick et al. / Bioorg. Med. Chem. 17 (2009) 1325–1338
1337
29.8, 25.8, 24.7, 20.1 and 12.7; IR (neat)/cmꢀ1 3500 (O–H), 2941,
2877, 2360, 1714 (C@O), 1452, 1378, 1281, 1192, 1126, 1092,
1055, 1014, 943, 877 (O–O), 845 (O–O), 824 and 736; HRMS (CI)
C17H30NO6 [M+NH4]+ requires 344.20734, found 344.20673; Anal.
calcd C17H26O6 requires: C, 62.56; H, 8.03. Found: C, 62.11; H, 8.53.
was dried over MgSO4, filtered and concentrated under reduced
pressure to give a pale yellow oil. Purification by flash column
chromatography (silica gel, 25:75 EtOAc/n-Hex) gave 31
(49 mg, 18%) as colorless oil. Compound 31: 1H NMR
(400 MHz, CDCl3)
d
5.29 (2H, s, 2 ꢂ H-12), 4.12 (2H, ddd,
J = 11.3, 6.4, 2.2 Hz, 2 ꢂ H-10), 3.58–3.41 (4H, m, 2 ꢂ CH2N),
5.1.2.26. Amide-linked dimer 29. Oxalyl chloride (2.0 M in
CH2Cl2, 0.30 mL, 0.60 mmol) was slowly added to a stirring solu-
tion of 28 (133 mg, 0.41 mmol) in anhydrous CH2Cl2 (5 mL) at
0 °C. The reaction mixture was allowed to warm up to room tem-
perature and stirred for 90 min. After 90 min, the solvent was re-
moved under reduced pressure and the residue dried under high
vacuum for 30 min. The residue was taken up in anhydrous CH2Cl2
(5 mL) and added via cannula to a stirring solution of 26 (146 mg,
0.41 mmol) and NEt3 (0.1 mL, 0.71 mmol) in anhydrous CH2Cl2
(5 mL) at 0 °C. The reaction mixture was allowed to warm up to
room temperature and stirred overnight. The solvent was removed
under reduced pressure and the residue purified by flash column
chromatography (silica gel, 2:1 EtOAc/n-Hex) to give 29 (161 mg,
74%) as a white solid. Compound 29: 1H NMR (400 MHz, CDCl3) d
7.08 (1H, br s, NH), 5.37 (1H, s, H-12), 5.33 (1H, s, H-12), 4.75
(1H, ddd, J = 11.0, 6.3, 2.1 Hz, H-10), 4.21 (1H, ddd, J = 10.8, 5.9,
2.3 Hz, H-10), 3.52–3.34 (2H, m, CH2N), 2.74–2.60 (3H, m), 2.54–
2.44 (2H, m), 2.41–2.37 (4H, m), 2.13–0.85 (37H, m) including
1.40 (3H, s, 3Me), 1.40 (3H, s, 3Me), 0.97 (3H, d, J = 6.1 Hz, 6Me),
0.96 (3H, d, J = 6.0 Hz, 6Me) and 0.85 (6Me, pseudo t, 2 ꢂ 9Me);
13C NMR (100 MHz, CDCl3) d 171.8 (C@O), 103.7, 103.4, 90.0,
89.2, 81.5, 81.2, 75.3, 71.1, 52.8, 52.4, 44.9, 44.2, 39.0, 38.0, 37.8,
37.0, 34.9, 34.7, 30.7, 30.6, 29.2, 26.5, 26.3, 25.2, 25.2, 20.6, 20.4, 13.7
and 13.6; IR (Nujol)/cmꢀ1 3315 (O–H), 2919, 2727, 2359, 2340,
1717, 1665 (C@O), 1542 (C@O), 1457, 1377, 1324, 1279, 1251, 1229,
1207, 1188, 1152, 1126, 1101, 1054, 1040, 1013, 959, 934, 877
(O–O), 850, 826 (O–O), 721 and 668; HRMS (ESI) C34H54NO9 [M+H]+
requires 620.3799, found 620.3792; Anal. calcd C34H53NO9 requires:
C, 65.89; H, 8.62; N, 2.26. Found: C, 65.42; H, 9.14; N, 1.89.
2.67 (2H, sex, J = 7.5 Hz, 2 ꢂ H-9), 2.33 (2H, td, J = 14.0, 4.0 Hz,
2 ꢂ H-4
a), 2.07–2.00 (2H, m), 1.98–0.87 (40H, m) including
1.42 (6H, s, 2 ꢂ 3Me), 0.97 (6H, d, J = 6.0 Hz, 2 ꢂ 6Me) and 0.87
(6H, d, J = 7.6 Hz, 2 ꢂ 9Me); 13C NMR (100 MHz, CDCl3) d 144.7
(C@O), 103.2, 89.3, 81.0, 71.6, 52.2, 44.1, 40.9, 37.3, 36.5, 34.4,
31.5, 29.7, 26.0, 24.8, 20.1 and 12.8; IR (neat)/cmꢀ1 3390,
2921, 1716 (C@O), 1519, 1455, 1377, 1263, 1190, 1100, 1039,
1013, 947, 879 (O–O), 843 (O–O), 824, 778, 738, 703 and 600;
HRMS (ESI) C35H57N2O9 [M+H]+ requires 649.4064, found
649.4037; Anal. calcd C35H56N2O9 requires: C, 64.79; H, 8.70;
N, 4.32. Found: C, 64.53; H, 8.51; N, 3.91.
5.1.2.29. 4,40-bipiperidine-linked dimer 32. 4,40-Bipiperidine
was prepared from its commercially available dihydrochloride by
treatment with 1.0 M NaOH and extraction with EtOAc. The organ-
ic extracts were dried over Na2SO4, filtered and concentrated under
reduced pressure to give a white solid (mp 172 °C, lit. 172–173 °C)
which was used without further purification. A solution of 4,40-
bipiperidine (44 mg, 0.26 mmol) and 25 (203 mg, 0.52 mmol) in
anhydrous benzene (5 mL) was stirred at 75 °C for 48 h. After
48 h, saturated aq NaHCO3 (10 mL) was added and the mixture ex-
tracted with diethyl ether (3 ꢂ 25 mL). The organic extracts were
dried over Na2SO4, filtered and concentrated under reduced pres-
sure to give a colorless oil. Purification by reversed-phase column
chromatography (octadecyl-functionalized silica gel, MeOH) gave
32 (124 mg, 63%) as a colorless oil. Compound 32: 1H NMR
(400 MHz, CDCl3) d 5.31 (2H, s, 2 ꢂ H-12), 4.11 (2H, m, 2 ꢂ H-10),
3.00 (4H, m), 2.72 (4H, m), 2.32 (4H, m), 2.07–1.18 (34H, m),
1.41 (6H, s, 2 ꢂ 3Me), 1.13 (4H, m), 0.95 (6H, d, J = 6.0 Hz,
2 ꢂ 6Me) and 0.87 (6H, d, J = 7.6 Hz, 2 ꢂ 6Me); 13C NMR
(100 MHz, CDCl3) d 103.8, 89.1, 81.5, 75.4, 58.1, 54.9, 52.9, 44.9,
41.3, 37.8, 36.9, 34.9, 30.6, 29.8, 29.7, 27.2, 26.6, 25.1, 20.7 and
13.7; HRMS (ESI) C44H73N2O8 [M+H]+ requires 757.5367, found
757.5354; Anal. calcd C44H72N2O8 requires: C, 69.81; H, 9.59; N,
3.70. Found: C, 69.42; H, 9.83; N, 3.24.
5.1.2.27. Carbonate-linked dimer 30. Trichloromethyl chloro-
formate (41
l
L, 0.33 mmol) was added to a stirring solution of 7
L, 0.70 mmol)
(206 mg, 0.66 mmol) and anhydrous pyridine (37
l
in anhydrous diethyl ether (25 mL) at 0 °C. The reaction mixture
was stirred at 0 °C for 1 h before being allowed to warm up to room
temperature and stirred for 48 h. Water (10 mL) was then added
and the mixture extracted with EtOAc (2 ꢂ 25 mL). The organic ex-
tracts were dried over MgSO4, filtered and concentrated under re-
duced pressure to give a clear oil. Purification by flash column
chromatography (silica gel, 20:80 EtOAc/n-Hex) gave 30 (167 mg,
73%) as white crystalline solid. Compound 30: 1H NMR
(400 MHz, CDCl3) d 5.30 (2H, s, 2 ꢂ H-12), 4.57–4.35 (6H, m,
2 ꢂ CH2O and 2 ꢂ H-10), 2.65 (2H, sex, J = 7.0 Hz, 2 ꢂ H-9), 2.32
5.2. Biology
5.2.1. Cytotoxicity studies
5.2.1.1. Materials. RPMI-1640 culture media,
cillin/streptomycin solution, Hanks balanced salt solution (HBSS),
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
L-glutamine, peni-
(MTT), N-(2-hydroxyethyl)piperazine-N0-(2-ethanesulfonic acid)
hemisodium salt (HEPES), dimethylsulfoxide (DMSO), sodium
dodecyl sulfate (SDS), n-dimethylformamide (DMF) and trypan
blue (0.4%) solution were all purchased from Sigma (Poole, UK).
Foetal bovine serum (FBS) was purchased from Bio Whittaker Eur-
ope (Verviers, Belgium). Human promyelocytic leukemia HL-60
cells were obtained from the European Collection of Cell Cultures
(Salisbury, UK).
(2H, td, J = 13.9, 4.0 Hz, 2 ꢂ H-4a), 2.07–0.88 (42H, m) including
1.40 (6H, s, 2 ꢂ 3Me), 0.97 (6H, d, J = 5.9 Hz, 2 ꢂ 6Me) and 0.88
(6H, d, J = 7.5 Hz, 2 ꢂ 9Me); 13C NMR (100 MHz, CDCl3) d 150.6
(C@O), 103.1, 89.4, 81.0, 70.3, 52.2, 44.0, 37.5, 36.6, 34.4, 30.0,
28.9, 26.0, 24.8, 20.1 and 12.6; IR (Nujol)/cmꢀ1 2923, 1778
(C@O), 1466, 1455, 1378, 1152, 1017, 943, 872 (O–O), 845 (O–O)
and 690; HRMS (ES) C35H54NaO11 [M+Na]+ requires 673.3564,
found 673.3585; Anal. calcd C35H54O11 requires: C, 64.59; H,
8.36. Found: C, 64.51; H, 8.41.
5.2.1.2. Cell culture and experimental preparation. HL-60 cells
were maintained in RPMI-1640 media supplemented with 10% v/v
FBS and 1% v/v L-glutamine. On reaching confluency (1 ꢂ 106 cells/
mL in a 75 mL flask), 2 ꢂ 106 cells were seeded in 30 mL of fresh
supplemented media. The cells were incubated under humidified
air containing 5% CO2 at 37 °C. Cell density was kept below
1 ꢂ 106 cells/mL to ensure exponential growth and to avoid differ-
entiation. Cells were only used between passages 5 and 15 to pre-
vent cell differentiation.
5.1.2.28. Urea-linked dimer 31. Trichloromethyl chloroformate
(50 lL, 0.42 mmol) was added to a stirring solution of NEt3
(0.12 mL, 0.83 mmol) and 26 (260 mg, 0.83 mmol) in anhydrous
diethyl ether (10 mL) at 0 °C. The solution was allowed to warm
up to room temperature and stirred overnight. The reaction mix-
ture was then diluted with EtOAc (20 mL) and washed with sat-
urated aq CuSO4 (10 mL) and brine (10 mL). The organic layer

1338
J. Chadwick et al. / Bioorg. Med. Chem. 17 (2009) 1325–1338
Cell viability was above 95% for all experiments. The viable cell
count was based on trypan blue exclusion from the cells and was
were calculated by interpolation of the probit transformation of
the log dose–response curve.
performed in a hemocytometer using a light microscope (ꢂ10;
Zeiss Axioskop, Welwyn Garden City, UK). To 100
l
L of cells was
Acknowledgments
added 20 lL of trypan blue 0.4% solution and an aliquot was
counted. During experiments cells were exposed to drug stock
solutions which were made up in 100% DMSO and the final solvent
concentration was below 0.5% v/v in each incubation. Each concen-
tration in every experiment was carried out in quadruplicate and
the experiments were repeated on at least three separate occasions.
We would like to thank Ultrafine Chemicals (now SAFC, Synergy
House, Guildhall Close, Manchester UK) for an industrial case
award to JC and Dr. Ed Irving, Dr. Feo Scheinmann and Dr. Adrian
Higson for helpful discussion concerning the synthetic chemistry.
References and notes
5.2.1.3. MTT assay. The MTT assay is based upon the ability of
dehydrogenase enzymes within viable cells to reduce the soluble
MTT solution to an insoluble formazan salt.25 The amount of for-
mazan present is directly proportional to the number of viable
cells. HL-60 cells (2.5 ꢂ 104/well) were plated in flat-bottomed
96-well plates in triplicate, and exposed to concentrations of each
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compound ranging from 0.01
incubation, 20 L of MTT solution (5 mg/mL in HBSS) was added
to each well. After 4 h of incubation at 37 °C, 100 L of a lysing buf-
lM to 100 lM for 72 h. Following
l
l
fer (20% w/v sodium dodecyl sulfate; 50% v/v n-dimethylformam-
ide) was added to each well to dissolve the formazan crystals,
and incubated for a further 4 h. The absorbance of the wells was
read using a test wavelength of 570 nm and a reference wave-
length of 590 nm with a plate reader (MRX, Dynatech Laborato-
ries). The results are expressed as a percent of vehicle only cells.
IC50 values were estimated from individual inhibition curves plot-
ted using GraFit software.
10. O’Neill, P. M.; Pugh, M.; Stachulski, A. V.; Ward, S. A.; Davies, J.; Park, B. K. J.
Chem. Soc., Perkin Trans. 1 2001, 2682.
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5.2.2. Antimalarial activity
For in vitro antimalarial assessment versus the 3D7 and K1
strains of P. falciparum the following protocol was employed. Para-
sites were maintained in continuous culture using the method of
Jensen and Trager.26 Cultures were grown in flasks containing hu-
man erythrocytes (2–5%), with parasitemia in the range of 1–10%,
suspended in RPMI-1640 medium supplemented with 25 mM
HEPES, 32 mM NaHCO3 and 10% human serum (complete med-
ium). Cultures were gassed with a mixture of 3% O2, 4% CO2 and
93% N2. Antimalarial activity was assessed with an adaption of
the 48 h sensitivity assay of Desjardins et al. using [3H]-hypoxan-
thine incorporation as an assessment of parasite growth.27 Stock
drug solutions were prepared in 100% DMSO and diluted to the
appropriate concentration using complete medium. Assays were
performed in sterile 96-well microtitre plates, each plate contained
200
or without 10
and parasite growth compared to control wells (which constituted
100% parasite growth). After 24 h incubation at 37 °C, 0.5 Ci
lL of parasite culture (2% parasitemia, 0.5% hematocrit) with
l
L drug dilutions. Each drug was tested in triplicate
l
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
24. Still, W. C.; Kahn, M.; Mitra, A. J. Org. Chem. 1978, 43, 2923.
25. Mosmann, T. J. Immunol. Methods 1983, 65, 55.
26. Jensen, J. B.; Trager, W. J. Parasitol. 1977, 63, 883.
27. Desjardins, R. E.; Canfield, C. J.; Haynes, D.; Chulay, D. J. Antimicrob. Agents
Chemother. 1979, 16, 710.