Erythrocyte membrane modifying agents and the inhibition of Plasmodium falciparum growth: Structure-activity relationships for betulinic acid analogues

Article abstract of DOI:10.1016/j.bmc.2003.10.010

The natural triterpene betulinic acid and its analogues (betulinic aldehyde, lupeol, betulin, methyl betulinate and betulinic acid amide) caused concentration-dependent alterations of erythrocyte membrane shape towards stomatocytes or echinocytes according to their hydrogen bonding properties. Thus, the analogues with a functional group having a capacity of donating a hydrogen bond (COOH, CH₂OH, CONH₂) caused formation of echinocytes, whereas those lacking this ability (CH₃, CHO, COOCH ₃) induced formation of stomatocytes. Both kinds of erythrocyte alterations were prohibitive with respect to Plasmodium falciparum invasion and growth; all compounds were inhibitory with IC₅₀ values in the range 7-28 μM, and the growth inhibition correlated well with the extent of membrane curvature changes assessed by transmission electron microscopy. Erythrocytes pre-loaded with betulinic acid or its analogues and extensively washed in order to remove excess of the chemicals could not serve as hosts for P. falciparum parasites. Betulinic acid and congeners can be responsible for in vitro antiplasmodial activity of plant extracts, as shown for Zataria multiflora Boiss. (Labiatae) and Zizyphus vulgaris Lam. (Rhamnaceae). The activity is evidently due to the incorporation of the compounds into the lipid bilayer of erythrocytes, and may be caused by modifications of cholesterol-rich membrane rafts, recently shown to play an important role in parasite vacuolization. The established link between erythrocyte membrane modifications and antiplasmodial activity may provide a novel target for potential antimalarial drugs.

Full text of DOI:10.1016/j.bmc.2003.10.010

Bioorganic & Medicinal Chemistry 12 (2004) 119–127
Erythrocyte membrane modifying agents and the inhibition of
Plasmodium falciparum growth:
structure–activity relationships for betulinic acid analogues
Hanne L. Ziegler,a Henrik Franzyk,a Majid Sairafianpour,a Mehrnoush Tabatabai,a
b
Mahboubeh D. Tehrani,a KarimBagherzadeh, Henry Hagerstrand,c Dan Stærka
and Jerzy W. Jaroszewskia,*
aDepartment of Medicinal Chemistry, The Danish University of Pharmaceutical Sciences, Universitetsparken 2,
DK-2100 Copenhagen, Denmark
bMedicinal Plants Unit, Isfahan Research Centre of Natural Resources and Animal Science, Isfahan, PO Box 81785-114,
Islamic Republic of Iran
c
˚
Department of Biology, Abo Akademi University, Biocity, FIN-20520 Abo/Turku, Finland
˚
Received 20 May 2003; accepted 10 October 2003
Abstract—The natural triterpene betulinic acid and its analogues (betulinic aldehyde, lupeol, betulin, methyl betulinate and betu-
linic acid amide) caused concentration-dependent alterations of erythrocyte membrane shape towards stomatocytes or echinocytes
according to their hydrogen bonding properties. Thus, the analogues with a functional group having a capacity of donating a
hydrogen bond (COOH, CH2OH, CONH2) caused formation of echinocytes, whereas those lacking this ability (CH3, CHO,
COOCH3) induced formation of stomatocytes. Both kinds of erythrocyte alterations were prohibitive with respect to Plasmodium
falciparum invasion and growth; all compounds were inhibitory with IC50 values in the range 7–28 mM, and the growth inhibition
correlated well with the extent of membrane curvature changes assessed by transmission electron microscopy. Erythrocytes pre-
loaded with betulinic acid or its analogues and extensively washed in order to remove excess of the chemicals could not serve as
hosts for P. falciparum parasites. Betulinic acid and congeners can be responsible for in vitro antiplasmodial activity of plant
extracts, as shown for Zataria multiflora Boiss. (Labiatae) and Zizyphus vulgaris Lam. (Rhamnaceae). The activity is evidently due
to the incorporation of the compounds into the lipid bilayer of erythrocytes, and may be caused by modifications of cholesterol-rich
membrane rafts, recently shown to play an important role in parasite vacuolization. The established link between erythrocyte
membrane modifications and antiplasmodial activity may provide a novel target for potential antimalarial drugs.
# 2003 Elsevier Ltd. All rights reserved.
1. Introduction
culated) forms.2ꢀ5 Shape-transformed human ery-
throcytes exhibit altered membrane permeability6 and
increased resistance to cell deformation.7
The erythrocyte (red blood cell) membrane is a tri-
lamellar composite consisting of a lipid bilayer and an
underlying protein network. The latter, consisting
mainly of spectrin, supports the lipid bilayer and con-
tributes heavily to the maintenance of shape and elasti-
city of the cell.1 Normally, the erythrocytes have a
discocytic shape. They are, however, highly deformable
cells capable of passing blood capillaries thinner than
their diameter. Various agents and conditions cause
changes of the erythrocyte cell shape either towards
stomatocytic (cup-shaped) or towards echinocytic (spi-
Erythrocytes function as host cells during the life cycle
of Plasmodium parasites, the unicellular protozoans
causing malaria, which is one of the most prevalent and
disabling human diseases affecting 5–10% of world’s
population. Erythrocytes are invaded by merozoites, the
final stages of the parasite hepatic cycle, leading to
the development of a parasitophorous vacuole in which
the merozoite matures to the intra-erythrocytic stage
(ring stage).8 As it grows inside the erythrocyte, the
parasite causes extensive modifications of the host cell,
including rearrangement of its macromolecules, expres-
sion of numerous own proteins, and establishing new
* Corresponding author. Fax: +45-3530-6040; e-mail: jj@dfuni.dk
0968-0896/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2003.10.010

120
H. L. Ziegler et al. / Bioorg. Med. Chem. 12 (2004) 119–127
permeation pathways.9ꢀ11 These changes result in
altered membrane properties of the infected cells, such
as changed deformability and susceptibility to haemo-
lysis, and increased cell adhesiveness.10ꢀ13 Parasitized
erythrocytes exhibit altered contents of phosphati-
dylcholine and phosphatidylethanolamine molecular
species in the membrane.14 Studies indicated that the
transport of parasite proteins in the infected ery-
throcytes is vesicle-mediated,15 and that membrane oxi-
dation is involved in activation of endogenous channels
by the parasite,16 emphasizing the importance of lipid
bilayer composition for the parasite growth.
mechanism by which membrane-active chemicals affect
parasite invasion and growth. In the present work, we
correlate membrane effects of a series of analogues of
betulinic acid, a pentacyclic triterpene having a struc-
ture reminiscent of that of cholesterol, with their in vitro
inhibition of P. falciparum growth. Such correlations
are of interest in order to shed light on the mechanism
of antiplasmodial activity of membrane modulating
compounds.
2. Results
It may thus be expected that changes in composition,
properties and form of the erythrocyte membrane,
manifested as rheological changes or as microscopically
observable alterations of the cell curvature, can affect
the Plasmodium parasite invasion and growth. There are
indeed various reports to support this.17 Ovalocytic
erythrocytes typical for the Melanesians, which have an
altered cytoskeleton, are more resistant to the deforma-
tion than normal erythrocytes and less prone to the
invasion by merozoites.18,19 Merozoites preferentially
invade young red blood cells, which are more deform-
able and have a different membrane composition than
mature erythrocytes.20 The inhibition of invasion was
observed in resealed erythrocyte ghosts upon chemical
or immunochemical cross-linking of spectrin.21 More-
over, inhibition of invasion has been observed upon
treatment of erythrocytes with membrane-active drugs
such as colchicine and vinblastine, but very high (milli-
molar) concentrations were used.22 Very recently, com-
pounds possibly acting as cholesterol mimics were
shown to inhibit proliferation of malaria parasites in
vitro in a low-micromolar concentration range.23,24 This
shows that there is a link between the erythrocyte
membrane properties and the ability of erythrocytes to
function as hosts for the Plasmodium parasites, which
could possibly be exploited for a novel antimalarial
therapy.
2.1. Betulinic acid as an in vitro antiplasmodial plant
constituent
Ethanol extracts of Iranian medicinal plants Zataria
multiflora Boiss. (Labiatae) and Zizyphus vulgaris Lam.
(Rhamnaceae) showed in vitro antiplasmodial activity
against P. falciparum strain 3D7 with IC50<12.5 mg
mLꢀ1. Bioactivity-directed fractionation of the extracts
led to isolation of betulinic acid (1, Fig. 1) fromboth
plants. In addition, Z. vulgaris yielded betulinic alde-
hyde (2). The compounds were identified by comparison
1
of their H and 13C NMR spectroscopic data and opti-
cal rotations with those of authentic compounds. IC50
values for antiplasmodial aꢀct1ivity of purified isolates 1
and 2 were about 6 mg m L (14 mM). After the com-
pounds were identified as antiplasmodial constituents of
the plants, all subsequent experiments were carried out
using a commercial sample of 1 and a synthetic sample
of 2.
2.2. Synthesis of betulinic acid analogues
In order to investigate structure–activity relationships
for the antiplasmodial effect, several synthetic analogues
of 1 were included in the investigation along with lupeol
(3).23 The aldehyde 2, only a small amount of which was
isolated from Z. vulgaris, was synthesized by oxidation
of the commercially available betulin (4) with pyr-
33
Already early studies linked the erythrocyte membrane
cholesterol content with the susceptibility to parasitic
invasion in vitro.25 Plasmodium falciparum cannot syn-
thesize cholesterol, and malaria infection results in
hypocholesterolaemic conditions.26 Recent studies
demonstrated the presence of lipid membrane micro-
domains or rafts, which can be isolated as detergent-
resistant membranes and which have an elevated
content of cholesterol.27ꢀ29 It was recently found, that
raft-anchored proteins of the erythrocyte host are inter-
nalised by Plasmodium vacuoles.30 When cholesterol is
idiniumdichromate.
The oxidation of the primary
hydroxy group in 4 proceeded with enough selectivity to
afford the required product (2) in 20% yield, but con-
comitant oxidation of the secondary hydroxy group was
observed.33 The ester 5 and the amide 6 were obtained
by reaction of betulinic acid pentafluorophenyl ester 3-
O-trifluoroacetate (7) with methoxide ion and ammonia,
depleted fromthe infected erythrocytes, the parasite
31
is expelled fromthe vacuole,
and Plasmodium infec-
tion is blocked by cholesterol depletion fromthe rafts. 32
Since lipid rafts apparently play a role in endovacuola-
tion of the parasites and macromolecular trafficking in
the infected erythrocytes, and because of the cholesterol
demand for production of new generations of mer-
ozoites, the infection may be generally affected by com-
pounds altering the cholesterol content or properties of
the erythrocyte membrane, for example, by structural
analogues of cholesterol. This may provide a general
Figure 1. Structures of compounds 1–7.

H. L. Ziegler et al. / Bioorg. Med. Chem. 12 (2004) 119–127
121
respectively. The active ester 7 was prepared from 1 and
pentafluorophenyl trifluoroacetate.34 Deprotection of
the 3-hydroxy group in 7 proceeded simultaneously with
the reaction of the pentafluorophenyl ester group. High
reactivity usually observed with pentaflurophenyl esters
was not shared by 7 and its conversion to 5 and 6
required rather harsh conditions, reflecting a consider-
able steric hindrance around C-28. Physical and spec-
troscopic data for 2 and 5 were identical with those
reported35ꢀ37 (the 13C NMR spectrumof a compound
formulated as 2, published by Zhong et al.,38 is different
particularly for the E-ring carbons, and thus this com-
pound cannot have the structure assigned to it, being
probably a stereoisomer of 2). Compounds 6 and 7 are
new.
with glutaric dialdehyde, dehydrated and embedded in
Epon similarly as previously described.23 Alterations of
the cell shape were assessed using transmission electron
microscopy. Control cells showed the normal discocytic
shape, and DMSO used for preparation of the stock
solutions and present in the medium did not affect the
cells at concentrations up to 2% (Fig. 2).23 Erythrocytes
incubated with 1–6 showed concentration-dependent
changes of the erythrocyte shape. Betulinic acid (1),
betulin, (4) and betulinic acid amide (6) induced forma-
tion of echinocytes, whereas betulinic aldehyde (2) and
methyl betulinate (5) caused formation of stomatocytes,
similarly as lupeol (3).23 A summary of the cell shape
changes is given in Table 2. Figure 2 shows concen-
tration dependence of the cell shape alterations caused
by 1, and examples of the effects of 2, 4 and 6 are given
in Figure 3. Increased concentration led to increasingly
altered cell shapes, with all cells transformed and some
lysis taking place at the highest concentrations used.
The stomatocytogenic compounds (2 and 5) induced
endovesiculation, and the number as well as the size of
the vesicles increased with increasing concentration. The
vesicles were located mainly along the erythrocyte
membrane. The echinocytogenic compounds (1, 4 and
6) caused formation of exovesicles, the amide 6 being
the most active. Interestingly, betulin (4) caused a pro-
gressive echinocyte formation with concomitant forma-
tion of small exovesicles at concentrations up to 50 mg
2.3. In vitro antiplasmodial activity of betulinic acid
analogues
Antiplasmodial activity of 1–6 was determined by inhi-
bition of incorporation of [3H]phenylalanine into a
chloroquine-sensitive 3D7 strain of P. falciparum simi-
larly as previously described.23,39 The results are shown
in Table 1. The IC50 values observed for the analogues
were in a range of 7–28 mM, the parent compound
lupeol (3) being the least active. Betulin (4) exhibited a
non-sigmoid (biphasic) dependence of the growth inhi-
bition with concentration (data not shown). Limited
solubility of 4 possibly contributed to this effect, but
various procedures for stock dilution (sequential dilu-
tion of a DMSO stock with growth medium or a paral-
lel addition of the stock solution to obtain the final
concentrations in the growth medium directly) had not
affected the final result. However, more than 50%
growth inꢀhi1bition was observed at concentrations below
12 mg m L (27 mM).
ꢀ1
mLꢀ1, but the cells incubated with 200 mg m L of 4
showed almost normal cell shape and a smaller number
of exovesicles (Fig. 4). Consistent findings were
observed using various methods of sample fixation, and
thus the observed changes are not artefacts induced by
sample preparation. Regardless whether the compounds
caused stomatocyte or echinocyte formation, there was
a good correlation between the extent of the micro-
scopically observed cell shape changes (Table 2, Figs 2
and 3) and the IC50 values for the antiplasmodial effect
(Table 1). The most potent analogue 5 showed detect-
able cell shape changes at the lowest concentrations.
2.4. Effects of betulinic acid and analogues on ery-
throcyte membrane
In order to assess effects of 1–6 on the erythrocyte
membrane, various concentrations of the compounds
were incubated with non-parasitized erythrocytes as in
the assay for the antiplasmodial activity. The concen-
tration ranges used were chosen to cover the regions in
which the compounds exhibited the antiplasmodial
effects (Table 1). After incubation the cells were fixed
Table 2. Erythrocyte shape changes caused by incubation with
betulinic acid and analogues determined by transmission electron
microscopya
ꢀ1
Concentration mg m L
Compd
3b
1
2
4
5
6
200
100
50
25
12.5
6.25
3.13
1.56
0.78
0.39
0 (control)
—
—
—
—
SS E1-D
—
—
—
—
S2
—
S1
D–S1
D–S1
—
—
—
E3
—
E3
E1
E1
—
D
Table 1. In vitro antiplasmodial activity of betulinic acid and
analogues against Plasmodium falciparum strain 3D7
—
E2
E2 S2–SS SS
—
E2
E1
E1–D
—
E1–D
—
a
—
S2
S1
S1
—
D
S1–S2 E2
Compd
IC50
S1–S2
—
D–S1
—
—
—
E1
D
D
—
D
ꢀ1
mg m L
mM
1
2
3
4
6.3ꢁ1.3
6.2ꢁ1.5
<12c
13.9ꢁ2.9
14.0ꢁ3.5
27.7ꢁ0.5b
<27c
11.8ꢁ0.2b
—
D
—
D
D–S1–E1
D
—
D
D
D
5
3.3ꢁ0.9
7.0ꢁ2.0
a Only the dominating erythrocyte shapes are listed: D, discocyte; E1,
E2, E3, echinocyte type 1, 2 and 3; S1, S2, stomatocyte type 1 and 2;
SS, spherostomatocyte (according to the nomenclature of Bessis2);
the compounds were not tested at the concentrations for which no
shape symbols are given.
6
Chloroquine
6.4ꢁ0.6
14.1ꢁ1.3
0.048ꢁ0.013
0.025ꢁ0.007
a Concentration necessary to inhibit growth by 50%.
b From ref 23.
c Biphasic growth inhibition curve (see text).
b Used as positive control, cf. ref 23.

122
H. L. Ziegler et al. / Bioorg. Med. Chem. 12 (2004) 119–127
Figure 2. Effecꢀt 1of betulinic acid (1) on human erythrocyte shape: (a) Control erythrocytes incubated with 2% DMSO; (b)–(f) erythrocytes treated
ꢀ1
ꢀ1
ꢀ1
ꢀ1
with 50 mg m L (109.5 mM), 12.5 mg m L (27.4 mM), 6.25 mg m L (13.7 mM), 3.13 mg m L (6.9 mM) and 0.78 mg m L (1.7 mM) of 1, respec-
tively. Micrographs were obtained with a transmission electron microscope at 1250 times magnification except for (b), which was obtained at 2500
times magnification.
Figure 3. Effect of betulinic aldehyde (2), lupeol (3), methyl betulinate (5), and betulinic acꢀid1 amide (6) on human erythrocyte shape: (a) erythrocytes
ꢀ1
incubated with 6.25 mg m L (14.2 mM) of 2; (b) erythrocytes incubated with 12.5 mg m L (29.3 mM) of 3; (c) erythrocytes incubated with 3.13 mg
mLꢀ1 (6.7 mM) of 5; (d) erythrocytes incubated with 6.25 mg m L (13.7 mM) of 6. Micrographs were obtained with a transmission electron micro-
scope at 1250 times magnification.
ꢀ1

H. L. Ziegler et al. / Bioorg. Med. Chem. 12 (2004) 119–127
123
Figure 4. Effecꢀt 1of betulin (4) on human erythrocyte shape: (a) erythrocytes incubated with 200 mg m Lꢀ1 (451.7 mM) of 4; (b) erythrocytes incubated
with 50 mg m L (112.9 mM) of 4. Micrographs were obtained with a transmission electron microscope at 1250 times magnification.
2.5. Effect of pre-treatment of erythrocytes with betulinic
acid, betulinic aldehyde and betulin on P. falciparum
growth
After the erythrocytes were pre-treated with betulinic
acid (1), betulinic aldehyde (2) or betulin (4), extensively
washed in order to remove the chemicals from the
medium, and inoculated with a P. falciparum culture,
strong growth impairment was observed. Thus, para-
sitized erythrocytes (parasitemia 5%) were mixed with
erythrocytes pre-incubated for 24 h with 1 (6.25ꢀ1 or 50
ꢀ1
ꢀ1
ꢀ1
mg m L ), 2 (6.25 or 50 mg m L ) or 4 (6.25, 50 or 200
mg m L ), and growth of the parasites in the cultures
was followed as a function of time (Fig. 5). During the
first 4 days of the experiment, parasites of all stages
could be observed in the cultures. After additional 2
days most of the parasites were extracellular, with an
excess of ruptured schizonts and merozoites. At day 8,
virtually no live parasites were observed in any of the
cultures employing pre-treated erythrocytes. At the
same time, the control culture exhibited a normal
growth pattern, reaching 5% parasitemia on day 4,
when the parasites were sub-cultured and then con-
tinued to grow normally. Erythrocytes used for the
control culture were incubated and washed in the same
way as the test cultures, but without the chemicals pre-
sent in the incubation medium.
Figure 5. Growth of Plasmodium falciparum 3D7 in erythrocytes loa-
ded with betulinic acid (1), betulinic aldehyde (2) and betulin (4). For
each day, the columns represent frꢀo1m left to right: erythrocytes pre-
ꢀ1
incubated for 24 h with 50 mg m L (109.5 mM) of 1, 6ꢀ.215 mg m L
ꢀ1
(13.7 mM) of 1, 50 mg m L (113.5 mM) of 2, 6.25 mg m L (14.2 mM)
ꢀ1
ꢀ1
of 2, 200 mg m L (451.7 mM) of 4, 50 mg m L (112.9 mM) of 4, 6.25
ꢀ1
mg m L (14.1 mM) of 4 (white columns), and control (black column).
The control was sub-cultured on day 4.
no clues about a possible mechanism of the in vitro
antiplasmodial effect of 1. We have now found, that 1
and its analogues 2 and 4–6 are potent membrane-active
agents exerting their stomatocytogenic or echinocyto-
genic action in the concentration region, where the in
vitro antiplasmodial effect is observed. This is similar to
what was very recently reported for 323 and the diter-
pene dehydroabietinol.24
3. Discussion
Bioassay-guided fractionation of plant extracts is a
standard method for isolation of bioactive natural pro-
ducts. Especially in the area of antiparasitic agents,
natural products have played a pivotal role in the past,
and there is a continued interest in antimalarial activity
of plants and plant isolates.40,41 Bioactivity-guided
fractionation of extracts of Z. multiflora42 and Z. vul-
garis43 using P. falciparum growth inhibition assay led
to isolation of the pentacyclic triterpenes 1 and 2, which
exhibited antiplasmodial activity as shown in Table 1.
There are only minor variations in the in vitro anti-
plasmodial activity of 1–6 (Table 1). The observed ery-
throcyte membrane alterations correlated well with the
antiplasmodial effect (Table 2). However, the effects on
the membrane shape differed conspicuously in the series
1–6. Thus, in spite of the closely related structures,
compounds 2, 3 and 5 caused formation of stomato-
cytes, whereas the formation of echinocytes was
observed with 1, 4 and 6 (Table 2, Figs 2–4). As expec-
ted, endo- or exovesiculation was observed with com-
pounds that induce formation of stomatocytes or
echinocytes, respectively (Figs 2–4). To our knowledge,
this is the first demonstration of varying membrane
In vitro antiplasmodial activity of 1 has been reported
previously44,45 with IC50 values similar to those found in
the present work (Table 1). The disclosure of the in vitro
antiplasmodial activity of 1 has led to an evaluation of
its in vivo effects using Plasmodium berghei murine
malaria model.45 However, no in vivo activity was
found.45 Very recently, the antiplasmodial activity of 1
was sought improved using a combinatorial synthesis
approach.46 However, these previous studies provided

124
H. L. Ziegler et al. / Bioorg. Med. Chem. 12 (2004) 119–127
effects in a series of closely related compounds where a
single functional group is being modified while keeping
a large hydrocarbon skeleton unchanged. There is a
clear structure–activity relationship in the observed
membrane effects (Table 2). Thus, compounds 1, 4 and
6 having a functional group at C-28 that is capable of
acting as a hydrogen bond donor (COOH, CONH2,
CH2OH) are echinocytogenic, while the presence at this
position of a group that cannot donate a hydrogen
bond (CH3, CHO, COOCH3) results in stomatocyto-
genic compounds 2, 3 and 5 (Table 2). According to the
classical bilayer couple theory,5,47,48 echinocytes are
formed when a compound incorporates into and
expands the outer leaflet of the membrane lipid bilayer,
whereas stomatocytes are formed when the incor-
poration takes place predominantly into the inner leaf-
let. Thus, 1, 4 and 6 are apparently incorporated mainly
into the outer leaflet, and 2, 3 and 5 into the inner leaf-
let. Previous studies have highlighted the importance of
amphiphile charge for the type of membrane effect they
induce.49ꢀ51 The present results emphasize the impor-
tance of hydrogen bonds in the interaction with the
erythrocyte membrane. Since hydrogen bonding
appears to be important for the interactions of choles-
terol with membrane phospholipids,52 mainly with
sphingomyelin present predominantly in the outer
membrane leaflet, the triterpenoids may also interact
with sphingomyelin. The biphasic behavior of 4 (Fig. 4)
may be due to its redistribution into the inner leaflet at
high concentrations, similarly as previously reported
with some amphiphiles.50
membrane, and (2) that incorporation of the triterpenes
into erythrocyte membrane is prohibitive with respect to
malaria parasite invasion and growth irrespectively of
the type of transformation they induce (formation of
stomatocytes or echinocytes). Although the membrane
changes observed by electron microscopy (Figs 2–4) are
difficult to quantify, there is a very good qualitative
correlation between the extent of membrane shape
alterations and the observed IC50 values for the inhibi-
tion of proliferation of malaria parasites. Erythrocyte
membrane and the parasite vacuolization process might
thus be a possible novel target53 for antimalarial ther-
apy, especially if a way of selective targeting of the
infected erythrocytes can be found. The link between
the in vitro antiplasmodial effect and the erythrocyte
membrane modification has also an immediate sig-
nificance with respect to interpretation of Plasmodium
drug sensitivity assays employing intra-erythrocytic
parasite stages. Moreover, compounds 1–6 provide a
potentially useful model for studies of interaction of
cholesterol analogues with lipid bilayers.54
5. Experimental
5.1. General
NMR spectra were obtained on a Bruker AMX 400 or a
Varian Gemini 2000 instrument (proton frequency
400.13 or 300.06 MHz, respectively), using tetra-
methylsilane (1H and 13C) or trichlorofluoromethane
(19F) as internal standard. Optical rotations were mea-
sured using a Perkin-Elmer 241 polarimeter. Merck
silica gel 60 F254 plates were used for thin layer chro-
matography (TLC), the spots being visualised by
spraying with anisaldehyde-sulfuric acid reagent fol-
lowed by heating. Vacuumliquid chromatographic
(VLC) separations were performed on Merck silica gel
60H for TLC. Preparative HPLC separations were
achieved on a chromatograph consisting of a Gynkotek
P 580 pump, Rheodyne 7725 injector, Shimadzu SPD-
10AV spectrophotometric detector operating at 205 nm,
and a recorder, using a 25 cmꢂ1.6 cmI. D. column
packed with Lichrosorb Si-60 (7 mm) and eluted with
heptane–EtOH 9:1 at 6 mL minꢀ1. Melting points were
determined in capillaries and are uncorrected. Betulinic
acid (1), lupeol (3) and betulin (4) were purchased from
Sigma-Aldrich. All chemicals and materials were used
as received. Elemental analyses were performed at the
Department of Chemistry, H. C. Ørsted Institute, Uni-
versity of Copenhagen.
Loading the triterpenes 1, 2 and 4 into the erythrocyte
membrane, followed by removal of excess of the chemi-
cals by washing, rendered the cells unsuitable for para-
site growth, as illustrated in Figure 5. Thus, the presence
of the compounds in the erythrocyte membrane and not
in the growth medium is important for the inhibition of
the parasite growth. It is evident that both the stoma-
tocytogenic and the echinocytogenic compounds exhibit
this effect. The observation of excess of extracellular
merozoites in the cultures employing the membrane-
modified erythrocytes suggests that the invasion was
inhibited. The presence of extra-erythrocytic schizonts
suggests that the parasites may have been expelled from
the host cells, similarly as recently reported upon
depletion of cholesterol.31
Pentacyclic triterpenes are ubiquitous dietary con-
stituents and their interactions with cellular membranes
may be responsible for various physiological and in
vitro effects. Further studies are necessary to investigate
the possible influence of pentacyclic triterpenes on the
composition and function of lipid rafts, which were
recently shown to play a key role in the erythrocyte
invasion by merozoites.31,32
5.2. P. falciparum drug sensitivity assay
The assay was performed with P. falciparum strain 3D7
(initial parasitemia 1.5%) essentially as previously
described.23,39 Briefly, the parasites were exposed to test
compounds for 48 h in microtiter plates, measuring
uptake of [3H]phenylalanine during the second half of
this period. Extracts and fractions were tested at 12.5,
4. Conclusion
ꢀ1
The key observations reported here are: (1) that hydro-
gen bonding properties of pentacyclic triterpenes deter-
mine their mode of incorporation into the erythrocyte
25, 50 and 100 mg m L . Inhibition curves for pure
compounds were constructed using 8–15 different con-
centrations, each tested in duplicate. IC50 values were

H. L. Ziegler et al. / Bioorg. Med. Chem. 12 (2004) 119–127
125
derived fromthe radioactivity uptake curves by non-
linear curve fitting with GraFit ver. 4.06 programfrom
Erithacus Software Ltd., using four-parameter logistic
equation. The reported values are averages of three
independent determinations. Chloroquine diphosphate
was used as a reference in all determinations.
jujuba Mill., Zizyphus sativa Gaertn.), were collected in
Ardestan, Isfahan, Iran. Voucher specimens (Alt. 899
and Alt. 1058) were deposited in the herbariumof the
Isfahan Research Centre of Natural Resources and
Animal Science, Isfahan, Iran.
5.6. Isolation of betulinic acid (1) from Z. multiflora
Boiss.
5.3. Parasite growth in erythrocytes pre-treated with bet-
ulinic acid (1), betulinic aldehyde (2) and betulin (4)
Dried and pulverised plant material (aerial parts, 100 g)
was extracted with 3ꢂ1.5 L of 96% EtOH. The extract
was evaporated, and the residue (9.4 g) fractionated on
a VLC column, eluting with toluene followed by tolu-
ene–EtOAc 4:1, EtOAc, and MeOH, and testing the
fractions for antiplasmodial activity. Active material
Solutions of the test compounds (1, 2 and 4) in 0.4 mL
of DMSO were mixed with 38.6 mL of RPMI 1640
medium (containing all additives), 1 mL of packed
human erythrocytes (1010 cells) was added, and the sus-
pensions were incubated at 37 ꢃC in an atmosphere
containing 93% N2, 5% CO2 and 2% O2. The final
concentration of the compounds was 6.25 or 50 mg
ꢀ1
(IC50<12.5 mg m L ) eluted with EtOAc (1.8 g) was
further fractionated by VLC using toluene, toluene–
EtOAc 95:5, and toluene–EtOAc 1:1. Crystallisation
fromEtOAc of a fraction eluted with toluene–EtOAc
95:5 gave 24 mg (0.024%) of betulinic acid (1);
ꢀ1
mLꢀ1, with an additional concentration of 200 mg m L
for 4. Reference flasks contained the same amount of
DMSO but no test compounds. After a 24 h incubation
period, the erythrocytes were separated (2000 rpm, 10
min) and washed three times with RPMI 1640 medium
and once with the growth medium. The washing process
was monitored by microscopy in order to exclude the
possibility that cell shape changes occurred during this
procedure. The washed cells were then used for sub-
cultivation of P. falciparum 3D7 in 25 cm2 flasks. Thus,
200 mL of the pre-incubated erythrocytes were mixed
with 20 mL of parasitized erythrocytes (parasitemia 5%)
and incubated with 5 mL of the growth medium. The
medium was replaced with a fresh portion every 48 h.
Every second day a small sample of the erythrocytes
was withdrawn and parasite counts were determined
microscopically, counting at least three microscopic
fields with an average of 100 erythrocytes in each field.
When parasitemia reached 5% (day 4, control culture),
the cells were sub-cultured similarly as described above.
[a]D25=+8ꢃ (c 0.11, pyridine); H and 13C NMR data
1
identical with those of authentic material.
5.7. Isolation of betulinic acid (1) and betulinic aldehyde
(2) from Z. vulgaris Lam.
Dried and pulverised plant material (root, 100 g) was
extracted with 3ꢂ1 L of 96% EtOH, the extract was
evaporated, and the residue (12.8 g) fractionated simi-
larly as above, to give 27 mg (0.027%) of betulinic acid
(1), [a]2D5=+7ꢃ (c 0.14, pyridine), and 6 mg (0.006%) of
betulinic aldehyde (2); [a]2D5=+16ꢃ (c 0.14, CHCl3); H
1
and 13C NMR spectra identical with those obtained
with authentic samples (commercial 1 or synthetic 2, see
below).
5.8. Synthesis of betulinic aldehyde (2)
Betulin (4) (100 mg) was added to a vigorously stirred
suspension of pyridiniumdichromate (79 mg) in dry
CH2Cl2 (8 mL).33 After 0.5 h, an additional portion of 4
(100 mg) was added to the reaction mixture, which was
then stirred for an additional 21.5 h. The mixture was
filtered through a layer of silica gel (Merck 60H) on a 2-
cmBu chner funnel, washing with EtOAc (75 mL). The
filtrate was concentrated, the residue (0.19 g) was dis-
solved in hexane–CH2Cl2 (1:2, 8 mL), and the solution
was applied to a VLC column (3ꢂ3 cm). Elution with
hexane, and then with hexane–EtOAc (50:1 to 5:1),
5.4. Erythrocyte membrane shape changes
Non-parasitized erythrocytes were incubated in 96-well
microtiter plates in medium containing test compounds
1–6 as in the assay for antiplasmodial activity,23,39 but
omitting the addition of [3H]phenylalanine. Each com-
pound was tested in at least five concentrations around
its IC50 value (50, 12.5, 6.25, 3.13 and 0.78ꢀm1g m Lꢀ1 for
1 and 2; 200, 50, 25, 12.5 and 3.13 mg m L for 3; 200,
50, 25, 6.25, 3.13, 1.56 and ꢀ0.139 mg m Lꢀ1 for 4; 25, 6.25,
3.13, 1.56 and 0.39 mg m L for 5; 50, 12.5, 6.25, 3.13
ꢀ1
1
and 0.78 mg m L for 6). Reference wells contained
monitored by TLC and H NMR, gave 2 (40 mg, 20%)
erythrocytes in the medium alone or in the medium
containing 2% of DMSO. After 48 h of incubation, 20
mL samples were spread on microscope slides, allowed
to dry, fixed with methanol, and stained with Giemsa
for phase-contrast light microscopy. Parallel samples
were prepared for transmission electron microscopy as
previously described.23
in addition to unreacted 4 (82 mg, 41%) and products
with 3-oxo group.33 The aldehyde 2 was recrystallized
fromMeOH; white crystals, mp 183–187 ꢃC, lit.35 mp
192–193 ꢃC; [a]2D5=+18ꢃ (c 0.38, CHCl3), lit.35
[a]2D5=+19ꢃ; H and 13C NMR data as reported.35,36
1
5.9. Synthesis of betulinic acid methyl ester (5)
The ester 7 (104 mg) was treated with 1 M methanolic
NaOMe (25 mL) in a closed steel vessel at 70 ꢃC for 16
h. The mixture was neutralized with HOAc (about 1.2
mL), concentrated, and the residue partitioned between
EtOAc and H2O. The organic layer was separated,
5.5. Plant material
Aerial parts of Zataria multiflora Boiss., Lamiaceae
(syn. Zataria bracteata Boiss.), and roots of Zizyphus
(Ziziphus) vulgaris Lam., Rhamnaceae (syn. Zizyphus

126
H. L. Ziegler et al. / Bioorg. Med. Chem. 12 (2004) 119–127
2JC,F=14.5 Hz, C-10), 114.7 (q, JC,F=286 Hz, CF3),
1
washed with water, dried (Na2SO4) and evaporated.
The residue was dissolved in CH2Cl2 and loaded onto a
VLC column (3ꢂ3 cm), which was eluted with hexane
and then with hexane–EtOAc (50:1 to 10:1), to yield the
methyl ester 5 (56 mg, 82%). The ester 5 was recrys-
tallized fromMeOH; white needles, mp 217–220 ꢃC,
lit.37 mp 224–225 ꢃC; [a]D25=+5ꢃ (c 0.17, CHCl3), lit.37
[a]2D5=+4ꢃ; 1H and 13C NMR data as reported.37
110.2 (C-29), 86.3 (C-3), remaining carbons: 57.7, 55.4,
50.5, 49.5, 46.7, 42.5, 40.7, 38.4, 38.3, 38.11, 37.13, 37.0,
34.2, 31.8, 30.3, 29.7, 27.8, 25.5, 23.3, 20.9, 19.4, 18.1,
16.2 (two carbons), 15.8, 14.7; 19F NMR (CDCl3):
d=ꢀ75.8 (s, 3 F, CF3), ꢀ153.7 (m, 2 F, F-20), ꢀ158.8 (t,
1 F, F-40), ꢀ163.0 (m, 2 F, F-30); elemental analysis:
calcd (%) for C38H46F8O4 C 63.50, H 6.45, found C
63.43, H 6.41.
5.10. Synthesis of betulinic acid amide (6)
The ester 7 (108 mg) was treated with liquid NH3–THF
(1:1, 30 mL) in a closed steel vessel at 55–60 ꢃC for 3
days. After cooling to ꢀ78 ꢃC the vessel was opened,
ammonia was allowed to evaporate, and the solvent was
removed in vacuo. The residue was dissolved in CH2Cl2
and loaded onto a VLC column (3ꢂ3 cm), which was
eluted with hexane and then with hexane–EtOAc (5:1 to
5:2) to yield the amide 6 (66 mg, 93%), recrystallized
Acknowledgements
We thank Ms. Dorte Brix and Ms. Katja Maj Nielsen
(The Danish University of Pharmaceutical Sciences) for
performing the in vitro Plasmodium assay, Ms. Anne
Corfitz (Centre of Medical Parasitology, Copenhagen)
for growing the parasites, Ms. Gunilla Henriksson and
Mr. Esa Nummelin (Abo Akademi University) for help
with electron microscopy, and Ms. Uraiwan Ngamra-
biab Adamsen (The Danish University of Pharmaceu-
tical Sciences) for help with the synthetic work.
fromMeOH; white prisms, mp 254–257
ꢃC (decom-
position); [a]2D5=+6ꢃ (c 0.47, CHCl3); 1H NMR
(CDCl3): d=5.53 and 5.35 (each 1H, br s, amide pro-
tons), 4.73 and 4.59 (each 1H, br s, olefinic protons),
3
3
3.18 (dd, 1H, J3ax,2ax=11.1 Hz and J3ax,2eq=5.1 Hz,
3
H-3ax), 3.08 (dt, 1H, J19ax,18ax=3J19ax,21ax=11.1 Hz,
References and notes
3J19ax,21eq=4.4 Hz, H-19ax) 1.68 (s, 3H, H-30), 0.97,
0.96, 0.96, 0.81, 0.75 (each 3H, s, H-23, H-24, H-25, H-
26 and H-27), 2.50–0.66 (m, remaining protons); 13C
NMR (CDCl3): d=178.9 (C-28), 150.8 (C-20), 109.4 (C-
29), 79.0 (C-3), remaining carbons: 55.9, 55.4, 50.7,
49.9, 46.6, 42.6, 40.8, 38.9, 38.8, 38.4, 37.7, 37.2, 34.4,
34.0, 30.8, 29.5, 28.0, 27.4, 25.6, 20.9, 19.5, 18.3, 16.2
(two carbons), 15.4, 14.6; elemental analysis: calcd (%)
for C30H49NO3ꢂ0.5H2O C 76.53, H 10.84, N 3.01,
found C 77.20, H 10.75, N 3.02.
1. Discher, D. E.; Carl, P. Cell. Mol. Biol. Lett. 2001, 6, 593.
2. Bessis, M. In Red Cell Shape: Physiology, Pathology,
Ultrastructure; Bessis, M., Weed, I., Leblond, P. F., Eds.;
Springer: Heidelberg, 1973; p 1.
3. Reinhart, W. H.; Chien, S. Am. J. Hematol. 1987, 24, 1.
4. Deuticke, B.; Grebe, R.; Haest, C. W. M. In Blood Cell
Biochemistry; Harris, J. R., Ed.; Plenum: New York,
1990; Vol. 1, p 475.
5. Fischer, T. M. Biophys. J. 1993, 65, 687.
6. Isomaa, B.; Hagerstrand, H.; Paatero, G.; Engblom, A. C.
Biochim. Biophys. Acta 1986, 860, 510.
7. Chabanel, A.; Reinhart, W.; Chien, S. Blood 1987, 69,
739.
8. Mitchell, G. H.; Bannister, L.-H. C. R. C. Crit. Rev.
Oncol./Hematol. 1988, 8, 255.
9. Desai, A. S.; Bezrukov, S. M.; Zimmerberg, J. Nature
2000, 406, 1001.
10. Kirk, K. Physiol. Rev. 2001, 81, 495.
11. Cooke, B. M.; Mohandas, N.; Coppel, R. L. Adv. Parasitol.
2001, 50, 1.
12. Deguercy, A.; Schrevel, J.; Duportail, G.; Laustriat, G.;
Kuhry, J. G. Biochem. Int. 1986, 12, 21.
13. Glenister, F. K.; Coppel, R. L.; Cowman, A. F.; Mohandas,
N.; Cooke, B. M. Blood 2002, 99, 1060.
14. Simoes, A. P.; Moll, G. N.; Beaumelle, B.; Vial, H. J.;
Roelofsen, B.; Op den Kamp, J. A. F. Biochim. Biophys.
Acta 1990, 1022, 135.
15. Taraschi, T. F.; Trelka, D.; Martinez, S.; Schneider, T.;
O’Donnell, M. E. Int. J. Parasitol. 2001, 31, 1381.
16. Huber, S. M.; Uhlemann, A.-C.; Gamper, N. L.;
Duranton, C.; Kremser, P. G.; Lang, F. EMBO J. 2002,
21, 22.
5.11. Synthesis of betulinic acid pentafluorophenyl ester
3-O-trifluoroacetate (7)
Betulinic acid (1) (519 mg, 1.14 mmol) was dissolved in
DMF (6 mL), and pentafluorophenyl trifluoroacetate
(588 mL, 3ꢂ1.14 mmol) and pyridine (201 mL, 2.2ꢂ1.14
mmol) were added. The mixture was stirred for 3.5 h,
when it was diluted with EtOAc (100 mL), and washed
successively with 0.1 M HCl, saturated aqueous
NaHCO3 and water (each with 2ꢂ80 mL). The organic
layer was dried (Na2SO4) and concentrated. The residue
was dissolved in CH2Cl2 and purified repeatedly on a
VLC column (5ꢂ5 cm), eluting with hexane and hex-
ane–EtOAc (100:1), which afforded 7 (743 mg, 76%) as
a white foam, recrystallized from ether–pentane; mp
176–177 ꢃC; [a]D25=ꢀ4.5ꢃ (c 0.37, CHCl3); 1H NMR
(CDCl3): d=4.74 and 4.63 (each 1H, br s, olefinic pro-
tons), 4.67 (dd, 1H, 3J3ax,2ax=11.0 Hz and 3J3ax,2eq=5.2
3
Hz, H-3ax), 2.96 (dt, 1H, J19ax,18ax=3J19ax,21ax=11.1
3
Hz, J19ax,21eq=4.9 Hz, H-19ax) 1.71 (s, 3H, H-30),
17. Pasvol, G.; Wilson, R. J. M. Parasitol. Today 1989, 5,
218.
18. Kidson, C.; Lamont, G.; Saul, A.; Nurse, G. T. Proc.
Natl. Acad. Sci. U.S.A. 1981, 78, 5829.
19. Liu, S.-C.; Palek, J.; Yi, S. J.; Nichols, P. E.; Derick,
L. H.; Chiou, S.-S.; Amato, D.; Corbett, J. D.; Cho,
M. R.; Golan, D. E. Blood 1995, 86, 349.
1.01, 0.96, 0.89, 0.89, 0.78 (each 3H, s, H-23, H-24, H-
25, H-26 and H-27), 2.51–0.81 (m, remaining protons);
13C NMR (CDCl3): d=172.1 (C-28), 175.4 (q,
2JC,F=41.5 Hz, CO of the trifluoroacetyl group), 149.7
(C-20), 141.3 (dd, 1JC,F=251 Hz, 2JC,F=12.5 Hz, C-20),
139.3 (dt, 1JC,F=252 Hz, 2JC,F=14 Hz, C-40), 137.9 (dt,
1JC,F=252 Hz, JC,F=14 Hz, C-30), 125.3 (t,
2
20. Mons, B. Blood Cells 1990, 16, 299.

H. L. Ziegler et al. / Bioorg. Med. Chem. 12 (2004) 119–127
127
21. McColm, A. A.; Hommel, M.; Trigg, P. I. Mol. Biochem.
Parasitol. 1980, 1, 119.
B. A.; Kharazmi, A.; Bagherzadeh, K.; Jaroszewski, J. W.
J. Nat. Prod. 2001, 64, 1398.
22. Dluzewski, A. R.; Rangachari, K.; Gratzer, W. B.; Wilson,
R. J. M. Br. J. Haematol. 1983, 55, 629.
23. Ziegler, H. L.; Stærk, D.; Christensen, J.; Hviid, L.;
Hagerstrand, H.; Jaroszewski, J. W. Antimicrob. Agents
Chemother. 2002, 46, 1441.
40. Tagboto, S.; Townson, S. Adv. Parasitol. 2001, 50, 199.
41. Christensen, S. B.; Kharazmi, A. In Bioactive Compounds
from Natural Sources, Isolation, Characterisation and
Biological Properties; Trigala, C., Ed.; Taylor & Francis:
London, 2001; p 380.
24. Ziegler, H. L.; Jensen, T. H.; Christensen, J.; Stærk, D.;
Hagerstrand, H.; Sittie, A. A.; Olsen, C. E.; Staalsø, T.;
Ekpe, P.; Jaroszewski, J. Planta Med. 2002, 68, 547.
25. Dluzewski, A. R.; Rangachari, K.; Wilson, R. J. M.;
Gratzer, W. B. Parasitology 1985, 91, 273.
26. Sein, K. K.; Aikawa, M. Med. Hypoth. 1998, 51, 105.
27. Brown, D. A.; London, E. Annu. Rev. Cell Dev. Biol.
1998, 14, 111.
28. Varma, R.; Mayor, S. Nature 1998, 394, 798.
29. Friedrichson, T.; Kurzchalia, T. V. Nature 1998, 394, 802.
30. Haldar, K.; Samuel, B. U.; Mohandas, N.; Harrison, T.;
Hiller, N. L. Int. J. Parasitol. 2001, 31, 1393.
31. Lauer, S.; Van Wye, J.; Harrison, T.; McManus, H.;
Samuel, B. U.; Hiller, N. L.; Mohandas, N.; Haldar, K.
EBMO J. 2000, 19, 3556.
32. Samuel, B. U.; Mohandas, N.; Harrison, T.; McManus,
H.; Rosse, W.; Reid, M.; Haldar, K. J. Biol. Chem. 2001,
276, 29319.
42. Ali, M. S.; Saleem, M.; Ali, Z.; Ahmad, V. U. Phyto-
chemistry 2000, 55, 933.
43. Lee, S.-S.; Lin, B.-F.; Liu, K. C. Phytochemistry 1996, 43,
847.
44. Bringmann, G.; Saeb, W.; Assi, L. A.; Francois, G.;
Narayanan, S. S.; Peters, K.; Peters, E.-M. Planta Med.
1997, 63, 255.
45. Steele, J. C. P.; Warhurst, D. C.; Kirby, G. C.; Simmonds,
M. S. J. Phytother. Res. 1999, 13, 115.
46. Pathak, A.; Singh, S. K.; Biabani, M. A. F.; Kulsh-
reshtha, D. K.; Puri, S. K.; Srivastava, S.; Kundu, B.
Comb. Chem. High Throughput Screen. 2002, 5, 241.
47. Sheetz, M. P.; Singer, S. J. Proc. Nat. Acad. Sci. U.S.A.
1974, 71, 4457.
48. Sheetz, M. P.; Alhanaty, E. Ann. N. Y. Acad. Sci. 1983,
416, 58.
49. Nwafor, A.; Coakley, W. T. Biochem. Pharmacol. 1986,
35, 953.
33. Kim, D. S. H. L.; Pezzuto, J. M.; Pisha, E. Bioorg. Med.
Chem. Lett. 1998, 8, 1707.
50. Isomaa, B.; Hagerstrand, H.; Paatero, G. Biochim. Biophys.
Acta 1987, 899, 93.
34. Green, M.; Berman, J. Tetrahedron Lett. 1990, 31, 5851.
35. Monaco, P.; Previtera, L. J. Nat. Prod. 1984, 47, 673.
36. Macias, F. A.; Simonet, A. M.; Esteban, M. D. Phyto-
chemistry 1994, 36, 1369.
37. Kojima, H.; Tominaga, H.; Sato, S.; Ogura, H. Phyto-
chemistry 1987, 26, 1107.
38. Zhong, S.-M.; Waterman, P. G.; Jeffreys, J. A. D. Phyto-
chemistry 1984, 23, 1067.
39. Sairafianpour, M.; Christensen, J.; Stærk, D.; Budnik,
51. Hagerstrand, H.; Bobacka, J.; Bobrowska-Hagerstrand,
M.; Kralj-Iglic, V.; Fosnaric, M.; Iglic, A. Cell. Mol. Biol.
Lett. 2001, 6, 161.
52. Ohvo-Rekila, H.; Ramstedt, B.; Leppimaki, P.; Slotte,
J. P. Progr. Lipid Res. 2002, 41, 66.
53. Olliaro, P. L.; Yuthavong, Y. Pharmacol. Ther. 1999, 81,
91.
54. Xu, X.; Bittman, R.; Duportail, G.; Heissler, D.; Vilcheze,
C.; London, E. J. Biol. Chem. 2001, 276, 33540.