Antimalarial and Antitumor Evaluation of Novel C-10 Non-Acetal Dimers of 10β-(2-Hydroxyethyl)deoxoartemisinin

Article abstract of DOI:10.1021/jm030974c

Four series of C-10 non-acetal dimers were prepared from key trioxane alcohol 10β-(2-hydroxyethyl)deoxoartemisinin (9b). All of the dimers prepared displayed potent low nanomolar antimalarial activity versus the K1 and HB3 strains of Plasmodium falciparum

Full text of DOI:10.1021/jm030974c

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1290
J . Med. Chem. 2004, 47, 1290-1298
An tim a la r ia l a n d An titu m or Eva lu a tion of Novel C-10 Non -Aceta l Dim er s of
10â-(2-Hyd r oxyeth yl)d eoxoa r tem isin in
J . Prince J eyadevan,† Patrick G. Bray,‡ J ames Chadwick,† Amy E. Mercer,† Aoife Byrne,† Stephen A. Ward,‡
B. Kevin Park,§ Dominic P. Williams,§ Rick Cosstick,† J ill Davies,‡ Adrian P. Higson,# Ed Irving,#
Gary H. Posner,| and Paul M. O’Neill*,†,§
Department of Chemistry, The Robert Robinson Laboratories, University of Liverpool, Liverpool L69 7ZD, U.K.,
Department of Pharmacology and Therapeutics, University of Liverpool, Liverpool L69 3GE, U.K.,
Liverpool School of Tropical Medicine, University of Liverpool, Liverpool L69 3GE, U.K., UFC Ltd., Synergy House,
Guildhall Close, Manchester Science Park, Manchester M15 6SY, U.K., and Department of Chemistry,
School of Arts and Sciences, The J ohns Hopkins University, Baltimore, Maryland 21218
Received J uly 28, 2003
Four series of C-10 non-acetal dimers were prepared from key trioxane alcohol 10â-(2-
hydroxyethyl)deoxoartemisinin (9b). All of the dimers prepared displayed potent low nanomolar
antimalarial activity versus the K1 and HB3 strains of Plasmodium falciparum. The most
potent compound assayed was phosphate dimer 14a , which was greater than 50 times more
potent than the parent drug artemisinin and about 15 times more potent than the clinically
used acetal artemether. In contrast to their potent activity versus malaria parasites, virtually
all of the dimers expressed poor anticancer activity apart from the trioxane phosphate ester
dimers 14a and 14b, which expressed nanomolar growth inhibitory (GI50) values versus a range
of cancer cell lines in the NCI 60 human cell line screen. Further detailed studies on these
dimers in vitro in HL60 cells demonstrate that both phosphate ester dimers (14a and 14b) are
more potent than the anticancer agent doxorubicin. Interestingly, phosphate ester monomers
9c and 9d , antimalarially active in the low nanomolar region versus P. falciparum, are inactive
as anticancer agents even at concentrations in the millimolar region. This observation
emphasizes the importance of two trioxane units for high antiproliferative activity, and we
propose that the nature of the linker in dimers of this type plays a crucial role in imparting
potent anticancer activity.
In tr od u ction
The compound artemisinin, also known as qinghaosu
(1), is a tetracyclic 1,2,4-trioxane occurring in Artemisia
annua. Artemisinin and its derivatives dihydroarte-
misinin (2), artemether (3), and sodium artesunate (4)
are routinely used for the treatment of malaria and are
particularly effective against cerebral malaria.1
Different modes of action have been proposed by
various groups to account for the action of artemisinin
and its derivatives in treating malaria.2 While the mode
of action of artemisinin as an antimalarial has not been
unequivocally established, it has been demonstrated
that the peroxide linkage is essential for expression of
activity.
Certain artemisinin derivatives, which contain a
peroxide moiety, have also been tested for biological
activity other than antimalarial activity. For instance,
the cytoxicity of artemisinin, dihydroartemisinin, and
an ether dimer of artemisinin to Ehrlich ascites tumor
3
cells has been reported.
Haynes and co-workers
disclosed in a patent the cytotoxicity of a range of
* To whom correspondence should be addressed. Address: Depart-
ment of Chemistry, Robert Robinson Laboratories, University of
Liverpool, Liverpool L69 7ZD, U.K. Phone: 0151-794-3553. Fax: 0151-
794-3588. E-mail: P. M.oneill01@liv.ac.uk.
† The Robert Robinson Laboratories, University of Liverpool.
‡ Liverpool School of Tropical Medicine, University of Liverpool.
§ Department of Pharmacology and Therapeutics, University of
Liverpool.
#
UFC Ltd.
| The J ohns Hopkins University.
10.1021/jm030974c CCC: $27.50 © 2004 American Chemical Society
Published on Web 01/31/2004

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Dimers of Deoxoartemisinin
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 5 1291
Ch a r t 1
directed alkylation or protein alkylation, after reductive
activation. Other antitumor agents, such as FR-900482
(7), upon reductive N-O scission, rearrange to produce
cytotoxic mitosene-like intermediates, 8, which can
efficiently cross-link DNA.13 In a similar manner, we
have recently shown that C-10 carba trioxane analogues
of dihydroartemisinin (9a ), upon reductive activation,
14
generate potentially toxic dicarbonyl products (9e).
In terms of the antimalarial mechanism of action, it was
suggested that selective intraparasitic generation of 9e
and subsequent protein alkylation in tandem with free
radical mediated damage may have a role to play in the
mechanism of action of these analogues.
semisynthetic monomers of dihydroartemisinin.4 The
most potent derivatives were shown to be capable of
causing extensive DNA damage, as assessed by the
DNA comet assay. It was proposed that the observed
antitumor effect of these peroxide-based monomers was
due to nuclear DNA damage.4 More recently, Wu and
co-workers have described the cytotoxicity of some
cyanoarylmethyl analogues of dihydroartmisinin. Some
of these peroxides had activities in the nanomolar range
against P388 murine leukaemia cell lines.5
The aim of the present study was to examine the
antimalarial and antitumor activities of a new series
of non-acetal C-10 carba dimers (Chart 1). 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.14-17 Consequently,
several groups have developed synthetic and semisyn-
thetic approaches to C-10 carba analogues. In the
approach described here, we have employed trioxane
alcohol (9b) as a key intermediate for the preparation
of new dimeric artemisinin analogues.
Alkylating agents remain an effective class of anti-
cancer drugs whose cytotoxic and therapeutic effects are
derived primarily from their ability to form intra- and
interstrand DNA cross-links. Some dimeric chemical
structures capable of DNA cross-linking have especially
high biological activities. These include bismustard
lexitropsins,6 dimeric cephalostatin analogues,7 bis-
enediynes,8 and dimers of the saponin antitumor agent
OSW-1.9 Recently, there have been several reports of
the potent antimalarial and antitumor activities of a
series of synthetic (5)10 and semisynthetic artemisinin-
derived dimers (6a ).11 Dimers of this type (6a ) were
found to possess potent antitumor activity in the NCI
60 cell line assay with activities comparable to that of
paclitaxel. The potential drawback in these derivatives
is the presence of the metabolically susceptible C-10
acetal linkage, and Posner and co-workers subsequently
prepared a set of metabolically more robust dimers
(6b).12a These compounds were found to possess both
potent antimalarial and tumor growth inhibitory prop-
erties. More recently Posner has reported on the concise
synthesis and antimalarial and antitumor activity of
highly stable three-carbon-linked dimers.12b In parallel,
J ung and co-workers disclosed data on amide- and
sulfone-linked C-10 carba dimers. Some of these com-
pounds demonstrated comparable activity to taxol and
adriamycin against murine P388 cell lines and human
breast cancer MCF7 cell lines.12c
Ch em istr y
Our optimized procedure was used for the synthesis
of 9b.17 Thus, the benzoate 10 was prepared in high
yield by treatment of DHA with benzoyl chloride in
pyridine. NMR studies on the white crystalline solid
indicated R stereochemistry with respect to the C-O
linkage at C-10 (C-10 H appears as a doublet with a
J 9-10 value of 9.9 Hz, indicating a dihedral angle of
about 180° between itself and C-9 H). Reaction of the
benzoate with allyltrimethylsilane (5 equiv) in the
presence of ZnCl2 catalyst (1.2 equiv) at 0 °C for 4 h
gave the desired product, 10a , in 90% yield (Scheme 1).
Ozonolysis of 10a and in situ reduction of the ozonide
provided the key alcohol in 55% yield required for
dimerization.
Trioxane dimers 11a -c and 12a ,b were prepared by
treating 2 equiv of vacuum-dried alcohol 9b and 1 equiv
of the requisite acid chloride in the presence of a
catalytic quantity of 4-(dimethylamino)pyridine. Reac-
The mechanism of antitumor and antimalarial activ-
ity of these trioxane dimers may involve either DNA-

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1292 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 5
J eyadevan et al.
Sch em e 1. Synthesis of Trioxane Alcohol 9b
Sch em e 2. Synthesis of Aliphatic and Aromatic Diester
Sch em e 4. Synthesis of Phosphate Ester Dimers
Dimers
Ta ble 1. In Vitro Antimalarial Activity of Dimers versus the
K1 and HB3 Strains of Plasmodium falciparum in Vitroa
K1 strain
IC50 (nM)
HB3 strain
IC50 (nM)
analogue
9c
9d
11a
11b
11c
12a
12b
13a
13b
13c
14a
14b
artemisinin
chloroquine
SD
SD
2.2
3.1
2.6
42.2
4.6
1.8
2.4
2.9
2.7
2.4
(0.4
(0.9
(0.9
(1.9
(1.4
(1.4
(0.8
(1.9
(1.6
(0.8
(0.3
(0.1
(1.4
(8
ND
ND
1.6
ND
ND
1.4
1.1
1.3
2.1
ND
0.09
0.18
14.5
20.
Sch em e 3. Synthesis of Ether-Linked Dimers by
Reductive Etherification
(0.4
(1.1
(0.8
(1.1
(1.3
0.2
0.5
12.3
190
0.1
0.20
1.5
3.1
a
ND: not determined.
addition of the appropriate phosphate dichloride (Scheme
4). For comparison, two phosphate monomers 9c and
9d were prepared in a similar manner using trioxane
alcohol 9b and the appropriate chlorophosphate.
An tim a la r ia l Assessm en t of New C-10 Ca r ba
Dim er s
The antimalarial activities for the new dimers, against
the chloroquine-resistant K1 strain of Plasmodium
falciparum, are recorded in Table 1. Several of these
dimers have remarkable activity against this strain. For
example, phosphate dimer 14a is greater than 50× more
potent than the parent drug artemisinin and about 15×
more potent than the clinically used acetal artemether.
Even more dramatic is the comparison with the 4-ami-
noquinoline chloroquine in this strain, where trioxanes
14a and 14b are between 400× and 900× more potent.
The table clearly demonstrates that trioxane dimers in
this class are among the most potent antimalarials to
have been tested against the K1 strain of Plasmodium
falciparum. Against the chloroquine-sensitive HB3 strain,
the phosphate dimers were again the most potent of the
dimers tested with subnanomolar antimalarial activity.
tions were complete after 16 h, and the products were
isolated by standard workup and silica gel chromatog-
raphy (Scheme 2).
For the preparation of ether derivatives, we decided
to employ the mild reductive etherification procedure
developed by Hatakeyama and co-workers.18 The triox-
ane alcohol was converted into the corresponding TMS
ether, which was then coupled with an aromatic bis-
aldehyde at -78 °C in the presence of TMSOTf. After 1
h at -78 °C, the reducing agent Et3SiH was added and
the reaction was allowed to warm to -30 °C over 3 h.
Standard workup gave the required bis-ether dimers
13a -c in good yield (Scheme 3). The phosphate ester
dimers were prepared from 9b by deprotonation with
sodium hexamethyldisilazide (NaHMDS) followed by

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Dimers of Deoxoartemisinin
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 5 1293
Ta ble 2. Growth Inhibitory Activities of Trioxane Dimers 14a
and 14b
For purposes of comparison, two phosphate ester
monomers 9c and 9d were prepared. Although these
compounds were active in the subnanomolar region
versus K1 Plasmodium falciparum, both compounds
proved to have no antiproliferative activity in the cell
lines examined, even at concentrations approaching 1
mM. This result is important because it rules out any
role of the phosphate ester function in mediating the
observed cytotoxic effects of the dimers and emphasizes
the necessity for a bivalent unit.
dimer 14a
dimer 14b
a
b
c
a
b
c
trioxane panel
and cell line
GI50
TGI
LC50
GI50
TGI
LC50
(µM)
(nM)
(µM)
(µM)
(nM)
(µM)
leukemia
CCRF-CEM
HL-60(TB)
K-562
MOLT-4
RPMI-8226
SR
769
<10
<10
<10
<10
<10
30
1.0
1.17
9.80
13.7 >100
2.84 >100
100
20
34.2 <10
33.5 <10
25.9
11.5
56
>100
2.23 >100
100
11.9
0.01 >100
0.263 >100
>100
>100
<10
<10
Having obtained the NCI data shown in Table 2, the
two phosphate dimers 14a and 14b were studied in
more detail in HL60 leukaemia and J urkat cell lines
using dihydroartemisinin and doxorubicin as a positive
control. Doxorubicin provides a useful comparison of
therapeutic utility because it is used to treat acute
leukemias and clinically the maximal initial plasma
concentration achieved with this drug after bolus ad-
ministration is 5 µM, with the lowest concentration
achieved at 0.3 µM. Generally, initial plasma concentra-
tions are in the range of 1-3 µM, declining rapidly
within 1 h to 25-250 µM. This illustrates that the GI50
value obtained in this in vitro study for DOX is
comparable to the plasma concentrations achieved
clinically.20a
colon cancer
COLO 205
HCC-2998
HCT-116
HCT-15
HT29
KM12
SW-620
melanoma
LOX IMVI
MALME-3M
SK-MEL-5
UACC-62
prostate
PC-3
breast cancer
MCF7
NCI/ ADR-RES 994
<10
1250
1850
<10
<10
<10
<10
0.21
16.4
32.6 >100
2.59 <10
0.17 >100
2.85 >100
52.9 64.8
>100
38.7 <10 >100
>100
>100
>100
>100
>100
14.4
12.6
12.6
12.6
40
40
40
<10
<10
<10
23.2
17.9
100
<10
16.4
52.9
19.1
194
13.0
75.3
15.6
30.8
2.25 >100
21.8
>100
>100
47.4 32.6 >100
199
486
15.2
18.4
38.9
42.9
>100
>100
<10
18.8
70.3 <10
65.5
50.3 18.3
22.1
<10 <10
72.2 106 >100
75.5 500
25.6 <10
>100
>100
>100
45.2
74.6
T-47D
a
Figure 1 summarizes typical curves obtained for these
drugs against the two cell lines studied, and the results
are recorded in Table 3. Against the HL60 cell line, both
phosphate esters demonstrated excellent activity with
IC50 values superior to that of doxorubicin. The J urkat
cell line in contrast was more resistant to the peroxides
studied, and doxorubicin proved to be superior in this
cell line. Both phosphate dimers outperformed DHA
by almost 10 orders of magnitude. In terms of general
toxicity to normal cell lines, we observed that these
trioxane dimers were not toxic to lymphocytes at doses
approaching 100 µM.
It is fascinating to note that the most potent com-
pound prepared in this study has a similar linker chain
length and hydrogen bond acceptor in the side chain to
two of the lead compounds prepared by J ung and co-
workers. As in this study, J ung has noted that longer
dimer linkers result in poor antiproliferative activity
(Figure 2).
GI50 ) concentration of drug required to achieve 50% growth
b
inhibition. TGI ) concentration of drug required to achieve total
growth inhibition. c LC50 ) concentration of drug required to
achieve 50% cell kill (i.e., cytotoxicity).
An titu m or Activities
To determine the growth inhibitory effects of the new
dimers, screening assays were performed by the Na-
tional Cancer Institute (NCI) using a 60 cell line panel
using concentrations of drug from the micromolar to
nanomolar range.19 The two most potent compounds in
these tests were the phosphate dimers 14a and 14b.
Data for phosphate ester dimers are recorded in Table
2. GI50 is the concentration of drug that inhibits
percentage growth by 50%, and a drug effect of this
intensity represents primary growth inhibition. In many
cases, for 14a , this value is lower than 10 nM. TGI (total
growth inhibition) is the concentration of drug required
to achieve cytostasis. TGI values are in the range of
1-10 µΜ against leukaemia, colon, and certain mela-
noma and breast cancer cell lines, indicating that this
compound has activity in the region recorded for pacli-
taxel in the recent patent disclosed by Hauser.11 Out of
the different panels of cell lines examined, dimer 14b
is also particularly effective against leukaemia, colon,
and melanoma cell lines with TGI values from nano-
molar to micromolar concentrations. Notably for both
dimers, there was little activity against lung, central
nervous system (CNS), and renal cancer cell lines (data
not shown). In contrast to the phosphate ester dimers,
the rest of the dimeric compounds tested had poor
antitumor activity in NCI assays, suggesting that the
presence of two trioxane units alone is not sufficient to
impart potent growth inhibitory activity. More impor-
tantly, it appears that the separation between the two
trioxane units is crucial. The dimers 14a and 14b have
a single phosphorus atom between the two representa-
tive trioxane carba alcohols 9b. The other dimers have
significicantly larger linker groups, and this observation
suggests a specific cellular target for 14a and 14b.
It is also noteworthy that the cell lines that are
particularly sensitive to trioxane dimers 14a and 14b
all express transferrin.20-25 Transferrin is an endog-
enous protein that transports iron from the circulation
into cells. Most cancer cell lines express higher surface
cell concentrations of transferrin receptors than normal
cells and have higher rates of iron influx.26a For
example, human hepatoma cells can express 800 000
transferrin receptors per cell on the cell surface.26b We
have noted that all the cell lines that are sensitive to
the peroxides 14a and 14b overexpress transferrin
receptors. On the basis of this knowledge, we propose
that the tumoricidal mechanism of action of these
peroxides may involve nuclear iron-dependent activation
to free radical species that damage DNA.
In preliminary studies using a J urkat (human leu-
kemic T-cell lymphoblast) cell line, we observed induc-
tion of apoptotic cell death on exposure to 14b (g30 µM,
Table 4).27 The mechanism underlying the apoptosis