Small structural changes of pentacyclic lupane type triterpenoid derivatives lead to significant differences in their anticancer properties

Article abstract of DOI:10.1016/j.ejmech.2010.04.018

In the present investigations five new derivatives of betulinic and betulonic acid were synthesized and the effect of this structural variations on anticancer activity was studied and discussed. The anti-proliferative activity of betulinic and betulonic acid derivatives was studied against eight tumor cell lines of different histogenic origin. The derivatives exerted a dose dependent antiproliferative action at micromolar concentrations toward target tumor cell lines. The apoptotic mode of cell death on colon cancer cell line HT-29 was induced by the most active compounds 5,2-amino-3-hydroxy-2-(hydroxy-methyl) propyl (3-O-acetyl)betulinate,and 9,2-amino-3-hydroxy-2-(hydroxymethyl)propyl betulonate. Treatment of HT-29 cells with 5 and 9 induced apoptosis,as observed by dye exclusion test (trypan blue) and by the appearance of a typical ladder pattern in the DNA fragmentation assay and FITC annexin V assay. Cell cycle perturbations caused by compound 5 are also presented.

Full text of DOI:10.1016/j.ejmech.2010.04.018

European Journal of Medicinal Chemistry 45 (2010) 3346e3353
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Small structural changes of pentacyclic lupane type triterpenoid derivatives lead
to significant differences in their anticancer properties
a
,
b
,
,
*
ꢀa
Harish Kommera , Goran N. KaluCerovic c, Jutta Kalbitz d, Birgit Dräger e, Reinhard Paschke a
a Biozentrum, Martin-Luther-Universität Halle-Wittenberg, Weinbergweg 22, 06120 Halle, Germany
b Department of Chemistry, Institute of Chemistry, Technology and Metallurgy, University of Belgrade, Studentski trg 14, 11000 Belgrade, Serbia
c Institut für Chemie, Martin-Luther-Universität Halle-Wittenberg, 06120, Halle, Kurt-Mothes-Straße 2, Germany
d BioSolutions Halle GmbH, Weinbergweg 22, 06120 Halle, Germany
e Institut für Pharmazie, Martin-Luther-Universität Halle-Wittenberg, Hoher Weg 8, 06120 Halle/Saale, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
In the present investigations five new derivatives of betulinic and betulonic acid were synthesized and
the effect of this structural variations on anticancer activity was studied and discussed. The anti-
proliferative activity of betulinic and betulonic acid derivatives was studied against eight tumor cell lines
of different histogenic origin. The derivatives exerted a dose dependent antiproliferative action at
micromolar concentrations toward target tumor cell lines. The apoptotic mode of cell death on colon
cancer cell line HT-29 was induced by the most active compounds 5, 2-amino-3-hydroxy-2-(hydroxy-
methyl)propyl (3-O-acetyl)betulinate, and 9, 2-amino-3-hydroxy-2-(hydroxymethyl)propyl betulonate.
Treatment of HT-29 cells with 5 and 9 induced apoptosis, as observed by dye exclusion test (trypan blue)
and by the appearance of a typical ladder pattern in the DNA fragmentation assay and FITC annexin V
assay. Cell cycle perturbations caused by compound 5 are also presented.
Received 11 December 2009
Received in revised form
9 April 2010
Accepted 15 April 2010
Available online 27 April 2010
Keywords:
Betulinic acid
Betulonic acid
Antitumoral activity
Apoptosis
Ó 2010 Elsevier Masson SAS. All rights reserved.
Cell cycle
1. Introduction
cancer types characterized with highest human mortalities. Despite
different sensitivities against different cancer cell lines betulinic
The antitumor properties of lupane derived triterpenoid plant
extracts have been demonstrated for the past 25 years on various in
vivo cancer model systems [1e5]. Betulinic acid (1) is a pentacyclic
lupane type triterpenoid (see Fig. 1) which can be directly isolated
from leaves of Syzigium claviflorum or synthesized from betulin.
Betulin is present in the barks of birch tree Betula spp. (Betulaceae)
up to 30% of its dry weight [6,7] and in Ziziphus spp. (Rhamnaceae),
Syzygium spp. (Myrtaceae), Diospyros spp. (Ebenaceae) and Paeonia
spp. (Paeoniaceae) [7]. Betulinic acid was found to inhibit HIV-1
replication in vitro [8]. Furthermore, other than this betulinic acid
showed antiinflammatory [9,10] and antimalarial activities [11,12].
Betulinic acid was found to show cytotoxic activity against
a broad spectrum of cancer cell lines derived from lung, colorectal,
breast, prostrate and cervical cancers which are most prevalent
acid arrested cell growth at nearly equal concentrations [13].
Betulinic acid has been reported to induce apoptosis in a p-53 and
CD-95 independent manner [14e19] and was also identified as
a potential activator of NF-kB in a number of cancer cell lines [20].
The anticancer property is linked to its ability to induce apoptotic
cell death by triggering the mitochondrial way of apoptosis.
Induction of apoptosis was found to be through loss of mitochon-
drial membrane potential, production of reactive oxygen species
and permeability of transition pore openings. These processes lead
to the release of mitochondrial apoptogenic factors, activation of
caspases and DNA fragmentation on neurectodermal tumors and
interestingly betulinic acid was found to be selective to tumor cells
and non toxic to normal non cancerous cells [21e23].
In this paper we report the synthesis and characterization of
new derivatives of betulinic acid and one new derivative of betu-
lonic acid. Furthermore, in vitro anticancer activity as well as the
structure activity relationships and mode of cell death are
described. The drastic influence of minor structural changes on the
anticancer properties is shown in detail and could contribute to
a deeper understanding of the anticancer properties of this inter-
esting class of compounds.
Abbreviations: Py, pyridine; DMAP, 4-dimethylaminopyridine; DMSO, dimethyl
sulfoxide; HMPD, 2-(hydroxymethyl)propane-1,3-diol; TRIS, tris(hydroxymethyl)
aminomethane; TCA, trichloroacetic acid; SRB, sulforhodamine B.
* Corresponding author. Tel.: þ49 345 55 21 600; fax: þ49 345 55 27 230.
E-mail address: reinhard.paschke@biozentrum.uni-halle.de (R. Paschke).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.04.018

H. Kommera et al. / European Journal of Medicinal Chemistry 45 (2010) 3346e3353
3347
panel of screened derivatives. Compound 5 was found to be 4e6
fold more active than betulinic acid and the best activity was
observed on head and neck cancer cell line (A253) (IC50 ¼ 2.0).
Interestingly, compound 6 was not active also at higher concen-
trations on 518A2, A253, A549, MCF-7 and SW1736 cell lines
whereas it showed a strong cytotoxicity on A413 and A2780 cell
lines (IC50w3.5
compounds 7 and 9 were moderately active whereas compound 8
was inactive (>100 M).
mM) and moderate activity on HT-29 cell line. The
m
Structure activity relation indicated that modification of
secondary hydroxyl (C3) into acetyl group of betulinic acid (1 /3)
retained cytotoxicity. The presence of acetyl group at C3 along with
primary amine in the side group substituted at C28 (5) seems to be
necessary to yield a highly cytotoxic compound with broad spec-
trum activity, because without derivatization at C3 (6) resulted in
a highly cytotoxic derivative on some types of cancer cell lines but
also a loss of broad spectrum anticancer activity exhibited by
betulinic acid was observed. Conversion of the betulinic acid into an
amide (1 /7) resulted in a moderately active derivative. On the
other hand the presence of primary amine in the side group
substituted at C28 and a keto group at C3 (9) exhibited a similar
activity like betulinic acid.
Since the structure of a compound strongly influences the
ability of a substance to enter the cell through the membrane as
well as the capability to interact with components of the cell, it is
not surprising that structural changes lead to different values in
cytotoxicity. In case of the compounds (Table 1) small structural
variations lead to drastic changes and could be therefore exploited
for a more mechanistic approach for the explanation of the cyto-
toxic properties. The lead compound betulinic acid, 1, shows an
Fig. 1. Betulinic (1) and betulonic acid (2).
2. Results and discussion
2.1. Chemistry
Synthesis of betulinic acid derivatives 3e8 and betulonic acid
derivative 9 (Schemes 1 and 2) were prepared by appropriate
modification of known method [24]. The compounds 4, 5, 7 and 9
are not soluble in water, slightly soluble in ethanol and methanol,
and fairly soluble in dimethyl sulfoxide (DMSO), dimethyl form-
amide and in chloroform whereas compounds 6 and 8 were not
soluble in chloroform and water.
2.2. Spectroscopic measurements
The 1H and 13C NMR spectroscopy have been used for charac-
terization of new derivatives. Chemical shifts arising from lupane
moiety were found as expected [25]. In 1H NMR spectra the signals
of hydrogen atoms belonging to C10 carbon atom appear at 4.20 (4),
4.07 (5) and 4.02 ppm (6). The signals of C30 and C40 protons were
found between 3.70e3.75 ppm (4), 3.45e3.47 (5) and 3.48 ppm (6)
ppm. In case of betulinamide derivatives 7 and 8 the signals arising
from C20, C30 and C40 protons were found at 3.59 and
3.69e3.72 ppm, respectively. Methyl protons from C10 and C30/C40
protons of betulonic ester derivative (9) were found at 3.98 and
IC50 of 8e14 mM on the investigated tumor cell lines. The intro-
duction of a higher dipole moment in molecular axis in the
betulonic acid 2 resulted in a similar value of the cytotoxicity but
also a remarkable increase in A431 and A2780 cell lines, respec-
tively. The introduction of an opposite dipole 3-O-acetyl group (3)
has a similar effect, i.e. increased cytotoxicity and specificity
towards some cancer cell lines. Compounds 4e8 were prepared to
improve the poor solubility in water. Forming an ester bond to tris
(hydroxymethyl)aminomethane, with an acetyl group at C3,
compound 5, leads to highly activity on all tumor cell lines.
Unexpectedly, the broad spectrum anticancer activity was lost by
removing the acetyl group in position C3 on 5 (/6). Interestingly,
the compound 6 was found to have noticeable cell specific effects
on A431, A2780 and HT-29 cell lines. Subsequent studies may lead
to the identification of the cause of resistance on 518A2, A253,
A549, MCF-7 and SW1736 cell lines as well as sensitivity on the
other three tumor cell lines. The free amino group seems to be
13
3.90e3.97 ppm, respectively. In C NMR spectra carbon atom C10
renonates between 43 and 57 ppm for 4e6, while for 7 and 8
between 62 and 68 ppm. The chemical shifts belonging to carbon
atoms C20, C30 and C40 were found between 58 and 67 ppm. Ester
and amide carbon atom resonances were found as expected
between 170 and 179 ppm for all compounds.
2.3. Cytotoxicity
The in vitro cytotoxic activity of the betulinic and betulonic acid
derivatives was studied on eight different tumor cell lines: 518A2
(melanoma), A253 (head and neck tumor), A431 (cervical), A2780
(ovarian), A549 (lung), HT-29 (colon), MCF-7 (breast) and SW1736
(anaplastic thyroid tumor) by sulforhodmine B colorimetric assay
method [26]. Since the investigated compounds were insoluble in
water, they were initially dissolved in DMSO and further diluted
with culture medium for analysis. The amount of DMSO in working
concentrations was less than 0.5% which was found to be non-toxic
to the cells. The compounds showed dose dependent antitumoral
activity against investigated cell lines. The IC50 values are summa-
rized in Table 1.
The compound 3 was found to be less active than betulinic acid
on HT-29 and A2780 cell lines, but similar activity was observed on
A253, A431, MCF-7 and SW1736 cell lines. (3-O-acetyl)betulinic
acid (3) exhibits nearly two times higher cytotoxicity than betulinic
acid (1) on 518A2 and A549 cell lines. The compound 4 did not
show any toxicity in the investigated concentration range
crucial for the activity, since compound
4 lost the activity
completely although the structure is similar to compound 6 in all
other parts. This proposition was also confirmed by compound 7,
where tris(hydroxymethyl)aminomethane is linked by an amide
bond where a decrease in cytotoxicity was observed. The same
effect was found, complete loss of cytotoxicity when the acetyl
group was removed to convert compound 7 to compound 8.
A comparison of cytotoxicity of betulonic acid 2 and compound 9
reveals a loss of specificity towards some types of cancer cell lines
shown by betulonic acid but retained the activity similar to
betulinic acid unlike compounds 3 and 5 where an increase in
cytotoxicity was observed.
2.4. Compounds 5 and 9 induce apoptosis
Compounds 5 and 9 show apoptotic mode of cell death on HT-29
cell line which has been confirmed from annexin V staining
experiment (Fig. 2A). The number of apoptotic cells on treatment
(>100
mM). 2-Amino-3-hydroxy-2-(hydroxymethyl)propyl (3-O-
acetyl)betulinate (5) was the most active derivative among the

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H. Kommera et al. / European Journal of Medicinal Chemistry 45 (2010) 3346e3353
Scheme 1. Reagents and conditions for the synthesis of betulinic acid derivatives: (a) acetic anhydride/DMAP/Py; (b) oxalyl chloride, HMPD; (c) oxalyl chloride, TRIS, 45 min, rt; (d)
oxalyl chloride, TRIS, 3 h, rt; (e) NaOMe, MeOH.
with IC90 concentration of 5 and 9 for 48 h showed around 20%
more apoptotic cells compared to untreated cells (control). Even-
though IC50 values of 1 is lower than 5 and equal to 9, betulinic acid
(1) seem to induce greater magnitude of apoptosis in shorter
exposure time than 5 and 9 as was observed from annexin V
experiment. The induction of apoptosis by 5 and 9 is further
confirmed by DNA fragmentation assay. Floating cells from HT-29
after 24 h treatment with the IC90 concentrations were collected
Scheme 2. Reagents and conditions for the synthesis of betulonic acid derivative: (a) oxalyl chloride, CH2Cl2, TRIS, 1 h.

H. Kommera et al. / European Journal of Medicinal Chemistry 45 (2010) 3346e3353
3349
Table 1
IC50 M] for the 96 h of activity of investigated compounds on tumor cell lines determined by SRB colorimetric assay.
[
m
Compound
518A2
A253
A431
A549
A2780
HT-29
MCF-7
SW1736
1
2
3
4
5
6
7
8
9
8.13 ꢀ 0.04
10.42 ꢀ 0.07
4.65 ꢀ 0.66
>100
9.18 ꢀ 0.04
10.05 ꢀ 0.20
11.84 ꢀ 0.45
>100
12.60 ꢀ 0.29
3.26 ꢀ 0.04
10.72 ꢀ 0.22
>100
2.54 ꢀ 0.09
3.61 ꢀ 0.20
10.34 ꢀ 0.14
>100
11.1 ꢀ 1.83
10.09 ꢀ 0.17
6.73 ꢀ 2.09
>100
11.07 ꢀ 0.44
2.98 ꢀ 0.25
18.99 ꢀ 0.10
>100
3.62 ꢀ 0.56
3.46 ꢀ 0.20
10.10 ꢀ 0.10
>100
13.93 ꢀ 0.46
10.02 ꢀ 0.27
16.14 ꢀ .20
>100
2.45 ꢀ 0.06
8.90 ꢀ 0.36
10.03 ꢀ 0.06
>100
12.27 ꢀ 0.19
10.37 ꢀ 0.15
11.06 ꢀ 0.87
>100
13.09 ꢀ 0.48
3.05 ꢀ 0.29
7.30 ꢀ 0.66
>100
2.87 ꢀ 0.03
2.02 ꢀ 0.22
3.44 ꢀ 0.02
2.76 ꢀ 0.05
3.73 ꢀ 0.03
>100
>100
>100
>100
>100
29.58 ꢀ 0.05
>100
10.78 ꢀ 0.04
29.47 ꢀ 0.06
>100
10.53 ꢀ 0.42
30.52 ꢀ 0.70
>100
10.83 ꢀ 0.08
29.40 ꢀ 0.82
>100
10.56 ꢀ 0.05
10.24 ꢀ 0.89
>100
10.12 ꢀ 0.06
10.10 ꢀ 0.05
10.01 ꢀ 0.07
9.44 ꢀ 0.09
and analysed by the DNA laddering technique and compared with
betulinic acid (1) (Fig. 2B). In the colon cancer, HT-29 cell line with
the compounds, occurrence of typical DNA ladders was observed.
Furthermore, programmed cell death was observed by trypan blue
exclusion test, in which floating cells showed the ability to exclude
the blue dye similar to betulinic acid indicating that 5 and 9 caused
cell death by the induction of apoptosis (Fig. 2B).
whereas activation of caspase 2 on 6 h treatment was observed.
Meanwhile for a 24 h exposure with compound 5 did not upregu-
late any caspases. Similarly the cells treated for 2 h with 9 seem to
show activation of caspase 2. On the other hand treatment for 6 or
24 h with 9 has not upregulated any of the caspases. These results
show that compounds 5 and 9 seem to cause no significant accu-
mumlation of caspases even after 24 h treatment on HT-29 cell line.
2.5. Effect of 5 and 9 on the kinetics of caspases 2, 3, 8 and 9
2.6. Cell cycle perturbations
activities
For analysis of cell cycle perturbations induced by 5, exponen-
tially growing HT-29 cells were treated with IC90 concentration for
24 and 48 h (Table 2). When compared to control, the compound
(IC90; 24 h) caused no changes in G1 and S phases but a decrease in
the number of cells in G2-M-phase with an increase in number of
apoptotic cells (SubG1-peak). In contrast compound 9 (IC90; 48 h)
did not induce significant cell cycle arrest in any cell cycle phase but
rather directly provoked an increase of number of apoptotic cells
To gain insight into the molecular changes of the new deriva-
tives-induced apoptosis, it was analyzed whether caspases are
involved as effectors in the induced cell death. The upstream cas-
pases 2, 8 and 9, and the downstream caspase 3 were used for the
present investigation (Fig. 3). Colon cancer HT-29 cell line has been
chosen for the investigation of the caspase activity. When treated
with compound 5, for 2 h, none of the caspases were activated
Fig. 2. (A) Representative FACS dot plots show control cells and treated cells stained with FITC annexin V. (B) Dye exclusion test and DNA laddering of 1, 5 and 9 on HT-29 cell line.

3350
H. Kommera et al. / European Journal of Medicinal Chemistry 45 (2010) 3346e3353
Fig. 3. Activity of caspases 2 (C2), 3 (C3), 8 (C8) and 9 (C9) on colon cancer HT-29 cell line treated for 2, 6 and 24 h with 5 and 9.
with concomitant decline of all other cell cycle phases in HT-29
cells. It was observed that there is a translation of cells from G2M
phase to apoptotic phase (SubG1) after 24 h treatment with
compound 5, it indicated that apoptosis caused by 5 on HT-29 cells
may be due to disturbances caused in G2M phase in the cell cycle.
With respect to the size of the betulinic acid core, the variations
in the C3 and C28 positions are marginal. Present investigation
shows, eventhough not clear, small polar groups in these positions
can lead to significant differences in cytotoxicity. More systematic
structural variations must be carried out in order to get more clarity
about the reasons for these findings.
3. Conclusion
4. Experimental
The modification with acetyl group at C3 and a primary amine
containing group at C28 in betulinic acid yields potentially cytotoxic
derivatives. The modification of C28 carboxylic acid group of betu-
linic acid into an ester of hydroxymethylpropanediol has resulted in
a loss ofcytotoxicity, but modification ofC28 carboxylic acidgroupof
(3-O-acetyl)betulinic acid into ester of tris(hydroxymethyl)amino-
methane yielded a highly cytotoxic derivative and removal of acetyl
group from C3 in this compund has shown cytotoxicity only on few
cell lines indicating a loss in broad spectrum anticancer activity. On
the other hand modifying C28 of (3-O-acetyl)betulinic acid into
amide of tris(hydroxymethyl)aminomethane resulted in a moder-
ately toxic compound. Deacetylation of (3-O-acetyl)betulinamide
derivate 7 yielded an inactive compound. Compounds 5 and 9 cause
cancer cell death by the induction of apoptosis without significant
caspase accumulation. The compound 5 seems to cause apoptosis by
perturbations in G2M phase of cell cycle.
4.1. Materials and methods
Synthesis of betulinic acid derivatives 4e8 and betulonic acid
derivative 9 has been described in Schemes 1 and 2, respectively.
3-O-acetylbetulinic acid (3) was synthesized as described in liter-
ature [25]. Solvents were obtained commercially and used without
further purification. The 1H and 13C NMR spectra were recorded on
a Varian Unity 500 NMR spectrometer in CDCl3 or DMF-d7. MS
spectra were recorded on LCQ e Thermo Finnigen with both posi-
tive and negative ionization modes as mentioned below. Betulinic
acid (1) was obtained as a kind gift from BioSolutions Halle GmbH
(Halle, Germany) and betulonic acid (2) was purchased from
Betulinines (Stribrna Skalice, Czech Republic). Numbering system
of the carbon skeleton is given in the Fig. 4 for compounds 5 and 7
as an example.
4.2. Chemical synthesis
Table 2
Cell cycle analysis of HT-29 cells untreated (control) and treated with the IC90
concentration of 5 for 24 and 48 h.
4.2.1. Synthesis of 3-hydroxy-2-(hydroxymethyl)propyl betulinate (4)
To the solution of (3-O-acetyl)betulinic acid (3) (5.0 g, 10 mmol)
in 25 ml dry dichloromethane 4 ml (45 mmol) of freshly distilled
oxalyl chloride was added. The mixture was stirred until the CO2
bubbling stopped; approximately 20 min. Dichloromethane and the
excess of oxalyl chloride were removed in vacuum. The synthesis of
(3-O-acetyl)betulinyl chloride has to be carried out under moisture
free conditions. Aftercooling down to room temperature the residue
was dissolved in a mixture of 40 ml pyridine and 10 ml of
Phase
24 h (%)a
Control
48 h (%)a
Control
5
5
G2/M
S
G1
30.12
21.62
45.40
2.95
16.39
19.83
38.41
25.11
14.62
37.24
46.22
2.33
7.60
13.85
18.07
60.66
SubG1
a
Cells in different cell cycle phases.

H. Kommera et al. / European Journal of Medicinal Chemistry 45 (2010) 3346e3353
3351
bubbling stopped, approximately 20 min. Dichloromethane and the
excess of oxalyl chloride were removed in vacuum. The synthesis of
(3-O-acetyl)betulinyl chloride has to be carried out under moisture
free conditions. After cooling down to room temperature the
residue was dissolved in a mixture of 40 ml pyridine and 10 ml of
dichloromethane and 0.8 g (6.5 mmol) of DMAP was added. After
complete dissolution of DMAP,
a solution containing 2.4 g
(20 mmol) of TRIS (tris(hydroxymethyl)aminomethane) in 2 ml of
pyridine was added. The mixture was stirred for 45 min at room
temperature and the solvent was removed rapidly under vacuum.
The crude product was dried, purified using column chromatog-
raphy (dichloromethane:methanol in 10:1). Yield 40%. MS [M þ H]þ
602.2. 1H NMR (500 MHz, CDCl3):
d 0.81 (m, 1H, C5H), 0.82 (s, 3H,
C24H3), 0.83(s, 3H, C23H3), 0.90 (s, 3H, C25H3), 0.93 and 1.85 (m,
2H, C1H2), 0.96 (s, 3H, C26H3),1.00 and 1.85 (m, 2H, C15H2),1.16 and
1.88 (m, 2H, C12H2), 1.31 and 1.59 (m, 2H, C11H2), 1.34 and 1.49 (m,
2H, C7H2), 1.36 and 1.57 (m, 2H, C6H2), 1.38 (m, 1H, C9H), 1.40 and
2.18 (m, 2H, C16H2), 1.54 and 2.20 (m, 2H, C21H2), 1.62 and 2.23 (m,
2H, C22H2), 1.65 (s, 3H, C30H3), 1.67 (m, 2H, C2H2), 1.69 (m, 1H,
C18H), 2.02 (s, 3H, C200H3), 2.17 (m, 1H, C13H), 2.96 (m, 1H, C19H),
3.45e3.47 (m, 2H, C30H2 and m, 2H, C40H2), 4.07 (m, 2H, C10H2), 4.44
(m, 1H, C3H), 4.60 and 4.72 (s, 2H, C29H2). 13C NMR (125 MHz,
CDCl3): d 14.6 (C27), 16.0 (C24), 16.2 (C25), 16.4 (C26), 18.1 (C6), 19.3
(C29), 20.9 (C11), 21.2 (C200), 23.6 (C2), 25.4 (C12), 27.9 (C23), 29.7
(C21), 30.5 (C15), 32.1 (C16), 34.2 (C7), 37.1 (C22), 37.2 (C4), 37.7
(C10), 38.2 (C13), 38.4 (C1), 40.7 (C8), 42.4 (C14), 56.8 (C10), 46.8
(C18), 49.3 (C19), 50.4 (C9), 55.4 (C5), 56.6 (C17), 64.7 (C30 and C40),
65.2 (C20), 109.9 (C30), 150.0 (C20), 171.0 (C100), 176.8 (C28).
Fig. 4. Numbering system of the carbon skeleton for compounds 5 and 7.
dichloromethane and 0.8 g (6.5 mmol) of DMAP (4-dimethylami-
nopyridine) was added. After complete dissolution of DMAP,
a solution containing 2.12 g of HMPD (2-(hydroxymethyl)propane-
1,3-diol), in 5 ml of pyridine was added. The mixture was stirred at
roomtemperature and after45 minthe solventwasremoved rapidly
under vacuum. The crude product was dried, purified using column
chromatography (dichloromethane:methanol in 10:1). To the iso-
lated substance 4 ml of a 0.5 M solution of sodium methoxide
(NaOMe) was added and refluxed overnight. Further 2 ml of 0.5 M
NaOMe solution were added. The reaction was stopped by neutral-
ization with Dowex 50 W-X8 (Hþ) exchange resin. The mixture was
refluxed again and filtered hot to remove the ion exchange resin. The
residuewas washed twicewith hotmethanol. The crudeproductof 4
was dried, purified using column chromatography (dichloro-
methane:methanol in 10:1). Yield 45%. MS [M þ Na]þ 567.7. 1H NMR
4.2.3. Synthesis of 2-amino-3-hydroxy-2-(hydroxymethyl)propyl
betulinate (6)
To the solution of 5 (1.0 g) dissolved in 90 ml of dry methanol
4 ml of a 0.5 M NaOMe was added and refluxed overnight. Further
2 ml of 0.5 M NaOMe solution were added. The reaction was
stopped by neutralization with Dowex 50 W-X8 (Hþ) exchange
resin. The mixture was refluxed again and filtered hot to remove
the ion exchange resin. The residue was washed twice with hot
methanol. The crude product of 5 was dried, purified using column
chromatography (dichloromethane:methanol in 10:1). Yield 90%.
MS [M þ H]þ 560.5. 1H NMR (500 MHz, DMF-d7):
d 0.73 (m, 1H,
C5H), 0.82 (s, 3H,C24H3), 0.92 (s, 3H, C23H3), 0.94 and 1.65 (m, 2H,
C1H2), 0.99 (s, 3H, C25H3), 1.00 and 1.71 (m, 2H, C15H2), 1.29 and
1.62 (m, 2H, C11H2), 1.30 (s, 3H, C26H3), 1.33 and 1.52 (m, 2H, C6H2),
1.36 and 1.45 (m, 2H, C7H2), 1.46 and 2.73 (m, 2H, C16H2), 1.54 and
2.26 (m, 2H, C21H2), 1.65 (s, 3H, C30H3), 1.68 (m, 1H, C18H), 1.70 (m,
1H, C9H), 1.87 and 2.27 (m, 2H, C22H2), 1.89 (m, 2H, C12H2), 1.90
and 2.25 (m, 2H, C2H2), 2.26 (m, 1H, C13H), 3.06 (m, 1H, C19H), 3.48
(m, 2H, C30H2 and m, 2H, C40H2), 4. 02 (m, 2H, C10H2), 4.04 (m, 1H,
C3H), 4.60 and 4.73 (s, 2H, C29H2). 13C NMR (125 MHz, DMF-d7):
(500 MHz, DMF-d7): d 0.76 (m, 1H, C5H), 0.80 (s, 3H, C25H3), 0.81 (s,
3H, C27H3), 0.88 (s, 3H, C26H3), 0.93 and 1.62 (m, 2H, C1H2), 1.11 (m,
2H, C15H2), 1.12 and 1.84 (m, 2H, C12H2), 1.15 (s, 3H, C24H3), 1.24 (s,
3H, C23H3),1.29 and 1.60 (m, 2H, C11H2),1.36 and 1.53 (m, 2H, C6H2),
1.37 and 1.43(m, 2H, C7H2), 1.44 and 2.21 (m, 2H, C16H2), 1.55 and
2.18 (m, 2H, C21H2),1.59 and 2.20 (m, 2H, C22H2),1.65 (s, 3H, C30H3),
1.67 (m, 1H, C9H), 1.68 and 1.83 (m, 2H, C2H2), 1.81 (m, 2H, C15H2),
1.82 (m, 1H, C18H), 2.03 (m, 3H, C13H), 2.70 (m, 1H, C20H), 3.70-3.75
(m, 2H, C30H2 and m, 2H, C40H2), 4.20 (d, 2H, 3J ¼ 5.8 Hz, C10H2), 4.44
(m,1H, C3H), 4.57 and 4.69 (s, 2H, C29H2). 13C NMR (125 MHz, DMF-
d
15.0 (C27), 16.2 (C24), 16.5 (C25), 16.6 (C26), 19.0 (C6), 19.5 (C29),
21.6 (C11), 28.6 (C23), 29.6 (C2), 29.8 (C12), 30.2 (C21), 30.6 (C15),
32.7 (C16), 34.9 (C7), 37.8 (C22), 39.5 (C4), 37.3 (C10), 39.0 (C13),
39.4 (C1), 41.4 (C8), 43.1 (C14), 48.0 (C18), 49.9(C19), 51.2 (C9), 56.2
(C5), 57.1 (C17), 57.3 (C10), 64.7 (C30 and C40), 66.6 (C20), 109.9 (C30),
151.4 (C20), 176.1 (C28).
d7): d 15.2 (C27),16.4 (C24),16.6 (C25),16.7 (C26),19.1 (C6),19.6 (C29),
21.7 (C11), 26.4 (C2), 28.5 (C12), 28.7 (C23), 31.4 (C21), 31.4 (C15), 35.1
(C16), 37.4 (C7), 37.9 (C10), 39.2 (C13), 39.5 (C1), 39.6 (C22), 39.7 (C4),
41.5 (C8), 44.8 (C14), 43.1 (C20), 48.1 (C18), 50.1 (C9), 51.3 (C19), 56.2
(C5), 57.3 (C17), 60.8 (C30 and C40), 63.2 (C10), 110 (C30), 151.4 (C20)
170.3 (C28).
4.2.4. Synthesis of N-(2,3-hydroxy-2-(hydroxymethyl)propyl)
(3-O-acetyl)betulinamide (7)
The compound 7 was synthesized using method described for
compound 5 with except that the mixture was stirred for 3 h at
room temperature and the solvent was removed rapidly under
vacuum. The crude product was dried, purified using column
chromatography (dichloromethane:methanol in 10:1). Yield 40%.
4.2.2. Synthesis of 2-amino-3-hydroxy-2-(hydroxymethyl)propyl
(3-O-acetyl)betulinate (5)
To the solution of (3-O-acetyl)betulinic acid (3) (5.0 g, 10 mmol)
in 25 ml dry dichloromethane 4 ml (45 mmol) freshly distilled
oxalyl chloride was added. The mixture was stirred until the CO2
MS [M þ H]þ 602.4. 1H NMR (500 MHz, CDCl3):
d 0.82 (m, 1H, C5H),
0.83 (s, 3H, C24H3), 0.84 and 1.93 (m, 2H, C1H2), 0.90 (s, 3H, C23H3),

3352
H. Kommera et al. / European Journal of Medicinal Chemistry 45 (2010) 3346e3353
0.94 (s, 3H, C25H3), 0.96 (s, 3H, C26H3), 0.99 (s, 3H, C27H3), 1.02 (m,
2H, C16H2), 1.30 and 1.96 (m, 2H, C21H2), 1.35 and 1.99 (m, 2H,
C15H2), 1.38 and 1.57 (m, 2H, C6H2), 1.39 (m, 2H, C7H2), 1.38 and
1.54 (m, 2H, C11H2), 1.40 (m, 1H, C9H), 1.50 (m, 1H, C18H), 1.62 (m,
2H, C2H2), 1.65 (s, 3H, C30H3), 1.75 (m, 2H, C22H2), 2.03 (s, 3H,
C200H3), 2.34 (m, 2H, C12H2), 2.36 (m, 1H, C13H), 3.01 (m, 1H, C19H),
3.59 (s, 2H, C20H2, s, 2H, C30H2 and s, 2H, C40H2), 4.45 (m, 1H, C3H),
(C1), 47,4 (C4), 41.5 (C8), 41.4 (C14), 47.8 (C18), 50.0 (C9), 48.4 (C19),
55.2 (C5), 56.4 (C17), 58.1 (C20), 64.5 (C30 and C40), 68.0 (C10), 109.6
(C30), 150.9 (C20), 178.5(C28), 218 (C3).
4.3. Cytotoxicity assay
4.3.1. Preparation of solutions of drugs
4.60 and 4.73 (s, 2H, C29H2). 13C NMR (125 MHz, CDCl3):
d
14.6
Stock solutions of investigated compounds were prepared in
a dimethyl sulfoxide (DMSO) at concentrations of 20 mM and
afterwards they were diluted with complete nutrient medium
(C27), 16.0 (C24), 16.2 (C25), 16.5 (C26), 18.1 (C6), 19.4 (C29), 20.9
(C11), 21.4 (C200), 23.7 (C2), 25.5 (C12), 27.9 (C23), 29.4 (C21), 30.8
(C15), 33.9 (C16), 34.2 (C7), 37.1 (C4), 37.7 (C10), 37.8 (C13), 38.4 (C1),
38.7 (C22), 40.8 (C8), 42.5 (C14), 46.7 (C18), 49.9 (C19), 50.4 (C9),
55.4 (C5), 56.3 (C17), 61.1 (C20, C30 and C40), 61.7 (C10), 109.6 (C30),
150.5 (C20), 171.0 (C100), 178.8 (C28).
(RPMI-1640 with L-glutamine (PAA Labs, Pasching, Austria)) con-
taining 10% heat inactivated fetal bovine serum (FBS) (Biochrom
AG, Berlin, Germany) and 1% penicillin/streptomycin (100ꢁ) (PAA
Labs, Pasching, Austria).
4.2.5. Synthesis of N-(2,3-hydroxy-2-(hydroxymethyl)propyl)
betulinamide (8)
4.3.2. Cell culture
The cell lines 518A2 (melanoma), A253 (head and neck
tumor), A431 (cervical), A2780 (ovarian), A549 (lung), HT-29
(colon), MCF-7 (breast) and SW1736 (anaplastic thyroid tumor)
were included in this study. All these cell lines were kindly
provided by Dr Thomas Mueller, Department of Hematology/
Oncology, Martin Luther University of Halle-Wittenberg, Halle
(Saale), Germany. Cultures were maintained as monolayers in
RPMI 1640 supplemented with 10% heat inactivated fetal bovine
serum and 1% penicillin/streptomycin at 37 ꢂC in a humidified
atmosphere with 5% CO2.
The compound 8 was synthesized and isolated using method
described for compound 6, using 7 instead of 5. Yield 90%. MS
[M þ H]þ 560.8. 1H NMR (500 MHz, DMF-d7):
d 0.73 (m, 1H, C5H),
0.83 (s, 3H, C24H3), 0.94 (s, 3H, C23H3), 0.96 (s, 3H, C25H3), 0.99
and 1.63 (m, 2H, C1H2), 1.02 (C26H3), 1.03 and 1.94 (m, 2H,
C16H2), 1.24 (s, 3H, C27H3), 1.33 and 2.13 (m, 2H, C21H2), 1.34 and
2.10 (m, 2H, C15H2), 1.35 and 1.43 (m, 2H, C7H2), 1.36 and 1.51
(m, 2H, C11H2), 1.37 (m, 1H, C9H), 1.36 and 1.57 (m, 2H, C6H2),
1.53 (m, 1H, C18H), 1.63 (m, 2H, C16H2), 1.65 (s, 3H, C30H3), 1.84
and 1.90 (m, 2H, C2H2), 1.88 and 2.13 (m, 2H, C22H2), 2.56 (m, 2H,
C12H2), 2.57 (m, 1H, C13H), 3.08 (m, 1H, C19H), 3.69e3.72 (s, 2H,
C20H2, s, 2H, C30H2 and s, 2H, C40H2), 4.25 (m, 1H, C3H), 4.58 and
4.3.3. Cytotoxicity assay
The cytotoxic activities of all the compounds were evaluated
using the sulforhodamine-B (SRB) (Sigma Aldrich) microculture
colorimetric assay [26]. In short, exponentially growing cells were
seeded into 96 well plates on day 0 at the appropriate cell densities
to prevent confluence of the cells during the period of experiment.
After 24 h, the cells were treated with serial dilutions of the
4.70 (s, 2H, C29H2). 13C NMR (125 MHz, DMF-d7):
d 14.9 (C27),
16.2 (C24), 16.5 (C25), 16.6 (C26), 19.0 (C6), 19.6 (C29), 21.7 (C11),
26.4 (C2), 28.1 (C12), 28.6 (C23), 30.0 (C21), 30.8 (C15), 31.5 (C16),
34.2 (C7), 34.3 (C10), 37.8 (C22), 38.2 (C13), 39.4 (C1), 39.5 (C4),
41.5 (C8), 43.1 (C14), 47.5 (C18), 50.7 (C9), 51.3 (C19), 56.2 (C5),
56.9 (C17), 62.5 (C10), 62.6 (C20, C30 and C40), 109.6 (C30), 151.9
(C20), 178.5 (C28).
compounds (0e100 mM) for 96 h. The percentages of surviving cells
relative to untreated controls were determined 96 h after the
beginning of drug exposure. After 96 h treatment, the supernatant
medium from the 96 well plates was thrown away and the cells
were fixed with 10% TCA. For a thorough fixation plates are now
allowed to stand at 4 ꢂC for at least 2 h. After fixation the cells are
washed in a strip washer. The washing is done four times with
water using alternate dispensing and aspiration procedures. The
4.2.6. Synthesis of 2-amino-3-hydroxy-2-(hydroxymethyl)propyl
betulonate (9)
To the solution of betulonic acid (2) (0.455 g, 1.0 mmol) in 5 ml
dry dichloromethane 0.5 ml freshly distilled oxalyl chloride was
added. The mixture was stirred until the CO2 bubbling stopped,
approximately 30 min. Dichloromethane and the excess of oxalyl
chloride were removed in vacuum. After cooling down to room
temperature the residue was dissolved in a mixture of 8 ml pyri-
dine and 3 ml of dichloromethane and 10 mg (0.08 mmol) of
DMAP was added. To the reaction mixture a solution containing
0.24 g (2.0 mmol) of TRIS in 2 ml of pyridine was added. The
mixture was stirred for 1 h at room temperature and the solvent
was removed rapidly under vacuum. The crude product was dried,
purified using column chromatography (dichloromethane:meth-
anol in 8:2). Yield 42%. [M þ H]þ 559.1. 1H NMR (500 MHz, CDCl3:
plates are then dyed with 100 mL of 0.4% SRB for about 45 min. After
dying the plates are again washed to remove the dye with 1% acetic
acid and allowed to air dry overnight. Hundred microliters of
10 mM Tris base solution were added to each well of the plate the
next day and absorbance was measured at 570 nm using a 96 well
plate reader (Tecan Spectra, Crailsheim, Germany). The IC50 and
IC90 values, defined as the concentrations of the compound at
which 50 and 90% cell inhibition is observed, was estimated from
the semi-logarithmic dose-response curves.
d, 0.96 (s, 3H, C25H3), 0.98 (s, 3H, C27H3), 1.01 (s, 3H, C24H3), 1.02
4.3.4. Apoptosis tests
(s, 3H, C26H3), 1.08 (s, 3H, C23H3), 1.10 and 1.97 (m, 2H, C12H2),
1.31 and 1.80 (m, 2H, C1H2), 1.36 and 1.59 (m, 2H, C11H2), 1.37 (m,
1H, C9H), 1.39 and 1.73 (m, 2H, C7H2), 1.40 and 2.34 (m, 2H,
C16H2), 1.43 and 2.44 (m, 2H, C16H2), 1.45 (m, 2H, C15H2), 1.46 (m,
1H, C5H), 1.50 and 1.66 (m, 2H, C6H2), 1.53 and 2.23 (m, 2H,
C21H2), 1.58 and 2.33 (m, 2H, C22H2),1.65 (s, 3H, C30H3), 1.71 (m,
1H, C18H), 2.17 (m, 1H, C13H), 2.30 and 2.41 (m, 2H, C2H2), 3.02
(m, 1H, C19H), 3.90-3.97 (m, 2H, C30H2 and m, 2H, C40H2), 3.98 (m,
2H, C10H2), 4.58 and 4.68 (s, 2H, C29H2). 13C NMR (125 MHz,
4.3.4.1. Trypan blue exclusion test. Apoptotic cell death was
analyzed by trypan blue dye (Sigma Aldrich, Germany) on HT-29
cell line. The cell culture flasks with 70e80% confluence were
treated with IC90 doses of 1, 5 and 9 for 24 h. The supernatant
medium with floating cells was collected after treatment and
centrifuged to collect the dead and apoptotic cells. The cell pellet
was re-suspended in serum free media. Equal amounts of cell
suspension and trypan blue were mixed and was analysed under
a microscope. The cells, which were apoptotic, excluded the dye
and were colorless and the ones whose cell membrane was
destroyed (necrotic cells) were turning into blue. Control cells were
obtained harvesting untreated HT-29 cells by trypsinization.
CDCl3): d 14.6 (C27), 15.8 (C24), 16.2 (C25), 16.4 (C26), 19.3 (C6), 19.6
(C29), 21.2 (C11), 34.0 (C2), 25.8 (C12), 26.6 (C23), 30.0 (C21), 30.4
(C15), 31.9 (C16), 33.6 (C7), 36.9 (C10), 37.0 (C22), 38.2 (C13), 39.4

H. Kommera et al. / European Journal of Medicinal Chemistry 45 (2010) 3346e3353
3353
4.3.4.2. DNA fragmentation assay. Determination of apoptotic cell
death was performed by DNA gel electrophoresis. Briefly, HT-29
cells were treated with respective IC90 doses of 1, 5 and 9for 24 h,
respectively. Floating cells induced by drug exposure were
collected, washed with PBS and lysed with lysis buffer (100 mM
TriseHCl Ph 8.0; 20 mM EDTA; 0.8% SDS; all from Sigma Aldrich).
Then, cells were treated with RNAse A at 37 ꢂC for 2 h and
proteinase K at 50 ꢂC (both from Roche Diagnostics chemical
company, Mannheim, Germany). DNA laddering was observed by
running the samples on 2% agarose gel followed by ethidium
bromide (Sigma Aldrich) staining.
samples are then centrifuged, the cell pellet is washed with 2 ml of
staining buffer (PBS þ 2% FCS þ 0.01% NaN3) and again centrifuged.
The cell pellet is resuspended in 100 ml of RNase A (1 mg/ml) and
incubated for 30 min at 37 ꢂC. At the end of incubation the samples
are treated with propidium iodide (20 g/1 ml of staining buffer)
m
and allowed to stand in dark at least for 30 min before analysis. The
fluorescence intensity was determined by a Facscalibur (Becton
Dickinson, Heidelberg, Germany). Each analysis was done using
about 1 ꢁ104 events.
Acknowledgement
4.3.4.3. Annexin V staining assay. Apoptotic cells were detected
using the FITC Annexin V Apoptosis Detection Kit (BD PharMingen,
BD Biosciences, Heidelberg, Germany). FITC Annexin V is a fluores-
cent probe which binds to phosphatidylserine. At the onset of
apoptosis, phosphatidylserine which is normally found on the
internal part of the plasma membrane becomes translocated to the
external portion of the membrane and becomes available to bind to
the FITC annexin V. After 48 h of treatment with compounds 1, 5
and 9 at IC90 concentrations HT-29 cells (0.5 ꢁ106/ml) were
collected, washed in PBS and resuspended in ice cold binding
buffer. AnnexinVeFITC and PI were added to each sample and
incubated in the dark for 15 min. The fluorescence intensity was
The authors are grateful to the Wirtschaftsministerium Sachsen-
Anhalt for financial support (Antitumor drugs on the basis of
natural compounds FuE 62/05) and BioSolutions Halle GmbH,
Germany.
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4.3.6. Cell cycle analysis
Cell cycle was assessed by flow cytometry using a fluorescence-
activated cell sorter (FACS). For this assay, 1 ꢁ106 HT-29 cells were
seeded in 25 sq cm cell culture flasks, with 10 ml of medium. After
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