Carbamate derivatives of betulinic acid and betulin with selective cytotoxic activity

Article abstract of DOI:10.1016/j.bmcl.2010.04.004

Synthesis and antiproliferative activity of eight new derivatives of betulinic acid (1) and betulin (2) are described. The compounds were tested against fifteen tumor cell lines. The toxicity against normal human fibroblasts and the mode of cell death on lung cancer cell line induced by the most active compounds 9 (bis(ethylcarbamate)betulin) and 11 (3-O-ethylcarbamate of 28-O-acetylbetulin) was investigated. Caspase 3 activity on lung cancer cell line (A549) was determined for 1, 5 (3-O-ethylcarbamate of betulinic acid), 9 and 11. All derivatives exerted a dose dependent antiproliferative action at micromolar concentrations toward target tumor cell lines. Treatment of lung cancer cells for 24 h with 9 and 11 induced apoptosis, as observed by the appearance of a typical ladder pattern in the DNA fragmentation assay.

Full text of DOI:10.1016/j.bmcl.2010.04.004

Bioorganic & Medicinal Chemistry Letters 20 (2010) 3409–3412
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Carbamate derivatives of betulinic acid and betulin with selective
cytotoxic activity
a
Harish Kommera , Goran N. Kaluderovic a,b, Sebastian Dittrich a, Jutta Kalbitz c, Birgit Dräger d,
-
´
Thomas Mueller e, Reinhard Paschke a,
*
a Biozentrum, Martin-Luther-Universität Halle-Wittenberg, Weinbergweg 22, 06120 Halle, Germany
b Institut für Chemie, Martin-Luther-Universität Halle-Wittenberg, Kurt-Mothes-Straße 2, 06120 Halle, Germany
c BioSolutions Halle GmbH, Weinbergweg 22, 06120 Halle, Germany
d Institut für Pharmazie, Martin-Luther-Universität Halle-Wittenberg, Hoher Weg 8, 06120 Halle, Germany
e Faculty of Medicine, Department of Oncology, Martin-Luther-Universität Halle-Wittenberg, Ernst-Grube-Straße 40, 06120 Halle, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Synthesis and antiproliferative activity of eight new derivatives of betulinic acid (1) and betulin (2) are
described. The compounds were tested against fifteen tumor cell lines. The toxicity against normal
human fibroblasts and the mode of cell death on lung cancer cell line induced by the most active com-
pounds 9 (bis(ethylcarbamate)betulin) and 11 (3-O-ethylcarbamate of 28-O-acetylbetulin) was investi-
gated. Caspase 3 activity on lung cancer cell line (A549) was determined for 1, 5 (3-O-ethylcarbamate
of betulinic acid), 9 and 11. All derivatives exerted a dose dependent antiproliferative action at micromo-
lar concentrations toward target tumor cell lines. Treatment of lung cancer cells for 24 h with 9 and 11
induced apoptosis, as observed by the appearance of a typical ladder pattern in the DNA fragmentation
assay.
Received 23 February 2010
Revised 30 March 2010
Accepted 5 April 2010
Available online 18 April 2010
Keywords:
Betulinic acid
Betulin
Natural products
Apoptosis
Ó 2010 Elsevier Ltd. All rights reserved.
Cytotoxicity
Caspase activity
The preclinical development of bioactive natural products and
their analogs as chemotherapeutic agents is a major objective of
anticancer research programs. Triterpenoids are one of the most
important classes of natural products occurring widely in the plant
kingdom. The derivatives of triterpenoids have been one of the
most interesting areas of research in the past few years vested to
their broad range of biological and medicinal properties.1–6 Betuli-
nic acid and betulin (Fig. 1) belong to the class of pentacyclic lu-
pane-type triterpenes. The birch tree (Betula spp., Betulaceae) is
one of the substantial source for betulin.7
Betulinic acid is one of few such compounds that have been
shown to possess several medicinal properties including antican-
cer, antimalarial, antimicrobial and anti-HIV activities.2 Initially
betulinic acid was considered to be melanoma specific8–10 but re-
cent studies suggested that it shows anticancer activity against a
broad spectrum of cancers.11 Eventhough, the anticancer mecha-
nism is not completely clear and well established, betulinic acid
was found to cause cancer cell death by induction of apoptosis
through changes in the mitochondrial membrane potential, pro-
duction 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.12–15 Also, betulinic acid inhibits topoi-
somerase I and can act as possible scaffold for the design of potent
topoisomerase inhibitors.16,17 Moreover, betulinic acid was found
to be selective to tumor cells and non toxic to normal non cancer-
ous cells.18 In combination with cholesterol, the cytotoxicity of
betulin is more enhanced, which is not observed with betulinic
acid.19
Derivatives of betulin were found to show hepatoprotective and
anti-HIV activity.20 Recently, some betulin derivatives containing
sulfur showed significant antiinflammatory and immunomodula-
tory effects.21
In the present study, the synthesis and in vitro anticancer activ-
ity of eight new derivatives of betulin and betulinic acid on several
cell lines is reported. In addition, the mode of cell death with the
interplay of caspases along with structure–activity relationship is
also investigated. The derivatives 3 and 4 are prepared according
to the methods available in literature.22,23
The synthetic chemical routes followed in producing the com-
pounds in series 3–12 are outlined in Scheme 1 and details for
the synthesis and characterization are given in Supplementary
data.
* Corresponding author. Tel.: +49 345 55 21 600; fax: +49 345 55 27 230.
E-mail address: reinhard.paschke@biozentrum.uni-halle.de (R. Paschke).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.04.004

3410
H. Kommera et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3409–3412
Figure 1. Betulinic acid (1) and betulin (2).
Scheme 1. Reagent and reaction conditions: (a) CH2N2, ether, rt; (b) ethyl/phenyl isocyanate, chloroform, 60 °C, 48 h; (c) acetic anhydride, dichloromethane, rt.
The in vitro cytotoxic activity of the betulin and betulinic acid
to the ethyl carbamates, thereby with a lower ability to penetrate
the cell membrane, as observed by Drag-Zalesinska et al.25
Interestingly, modification of C28 carboxylic acid group of bet-
ulinic acid (1) into a methyl ester (3) lead to a fall in activity but a
modification of C3 hydroxy group of 1 or 3 into ethylcarbamate has
yielded derivatives 5 and 7, respectively, where compound 5 was
found to be less cytotoxic than betulinic acid in most of the cases
(except on lung cancer A549, ovarian cancer A2780, anaplastic thy-
roid cancer SW1736) and compound 7 was twofold more toxic
than 5 on most of the investigated cell lines.
The derivatization of C3 secondary hydroxyl group of 28-O-
acetylbetulin (4) and both primary and secondary hydroxyl groups
of betulin (2) into ethylcarbamate has yielded highly cytotoxic
derivatives 11 and 9, respectively. Introduction of imidazole scaf-
fold into the lupane skeleton has yielded similar toxicities26 as
the active new carbamate derivatives reported in this work.
Another important parameter to evaluate for an anticancer drug
is its stability in solution. The in vitro stability was determined by
HPLC after incubation at 37 °C of compound 9 in phosphate buffer
(pH 7.2). It was found that compound was quite stable, and no
decomposition products were observed even after 96 h incubation
time in phosphate buffer.
derivatives on fifteen different tumor cell lines: 8505C and
SW1736 (anaplastic thyroid), A253 and FaDu (head and neck),
A431 (cervical), A2780 (ovarian), DLD-1, HCT-8, HCT-116, HT-29
and SW480 (colon), MCF-7 (breast), 518A2 (melanoma), A549
(lung) and liposarcoma (connective tissue) was studied by sulfo-
rhodamine B colorimetric assay24 in triplicate. The compounds
showed dose dependent antitumoral activity against investigated
cell lines. The IC50 values are summarized in Table 1.
The most active compound in the panel of derivatives synthe-
sized is compound 11, and was found to be two to eight times
more cytotoxic than betulinic acid on the tumor cell lines used in
the study. The best activity of 3-O-ethylcarbamate of 28-O-acetyl-
betulin (11) was observed on lung cancer cell line (A549)
(IC50 = 1.69
1.77 M).
lM) and on colon cancer cell line (SW480) (IC50 =
l
Interestingly, the phenylcarbamate derivatives 6, 8, 10 and 12
were found to be inactive or seem to show activity only at higher
concentrations whereas in most of the cases the ethylcarbamate
derivatives 5, 7, 9 and 11 show higher activity than betulinic acid
on the investigated tumor cell lines. A modification of secondary
hydroxyl group at C3 into ethylcarbamate seems to be the reason
for higher activity. On the other hand, modification into a phenyl
carbamate yields less cytotoxic derivatives. One reason for low
activity of the phenyl carbamates can be due the straight and rigid
The most active derivatives, 9 and 11, were screened for their
selectivity towards tumor cells and summarized in Table 2. The
compounds were tested on human fibroblasts (WWO70327). The
phenyl groups and the possible interactions with the
p-system of
IC50 of betulinic acid, 1, on fibroblasts was found to be 20.81
lM
the phenyl ring lead to a much more bulky molecule compared
and for 9 and 11, >100 and 24.35 M, respectively. Thus the
l

H. Kommera et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3409–3412
3411
Table 1
IC50
M)a for the 96 h of activity of investigated compounds on tumor cell lines determined by SRB colorimetric assay
(l
1
3
4
5
6
7
8
9
10
11
12
518A2
8505C
A253
A431
A549
A2780
DLD-1
FaDu
HCT-8
HCT-116
HT-29
Liposarcoma
MCF-7
SW480
SW1736
8.13
7.26
9.18
28.75
23.67
18.51
26.99
20.63
29.31
45.86
20.33
17.25
20.47
30.72
23.26
24.39
17.92
32.10
15.84
14.74
12.87
12.19
14.37
11.74
13.12
13.95
17.98
10.71
10.96
15.54
11.38
13.68
11.95
17.84
17.56
17.28
15.20
5.55
51.49
39.55
34.66
26.71
22.36
24.82
26.32
24.25
25.18
21.40
31.63
51.85
47.88
41.08
18.76
16.72
7.22
16.69
14.80
2.32
8.35
18.94
5.53
14.98
13.46
7.22
6.85
7.03
>100
>100
>100
91.45
>100
69.52
>100
>100
>100
>100
68.96
>100
>100
>100
>100
8.18
9.39
9.71
6.08
2.91
4.37
8.32
7.88
3.91
3.84
6.20
>100
>100
>100
>100
>100
80.62
>100
>100
>100
>100
69.24
>100
>100
>100
>100
5.12
5.05
4.88
5.00
1.69
4.28
6.93
4.62
3.99
4.30
5.07
5.35
5.98
1.77
3.15
>100
>100
>100
>100
>100
84.23
>100
>100
>100
>100
76.44
>100
>100
>100
>100
12.60
11.10
11.07
11.87
10.19
13.10
10.80
13.93
12.07
12.27
6.48
7.41
17.00
13.82
16.74
15.20
16.74
17.93
17.37
16.56
5.24
12.03
17.70
2.77
6.67
2.35
13.09
3.60
a
Values are mean of three experiments, standard deviation values are less than 10% in all cases.
Table 2
Selectivity indexa of 1, 9 and 11 towards tumor cells
1
9
11
4.76
518A2
8505C
A253
A431
A549
A2780
DLD-1
FaDu
HCT-8
HCT-116
HT-29
Liposarcoma
MCF-7
SW480
SW1736
2.56
2.87
2.27
1.65
1.87
1.88
1.75
2.04
1.59
1.93
1.49
1.72
1.70
3.21
1.59
>12.22
>10.65
>10.30
>16.45
>34.36
>22.88
>12.02
>12.69
>25.58
>26.04
>16.13
>8.31
4.82
4.99
4.87
14.41
5.69
3.52
5.39
6.20
5.67
4.80
4.55
4.07
13.76
7.73
>5.65
>36.10
>26.32
a
Selectivity index (IC50 on human fibrobalsts/IC50 on corresponding tumor cell
line).
Figure 2. Activation of caspase 3 on A549 lung cancer cell line treated for 2 and 6 h
with 1, 5, 9 and 11. Values are mean of three experiments.
compounds, 9 and 11 were less toxic on normal fibroblasts than on
the investigated tumor cell lines and more selective to cancer cells
than betulinic acid. It was observed that 11 was 3–14 times more
toxic to tumor cells, for example, 14 times more selective towards
lung cancer cell line (A549) and colon cancer cell line (SW480).
Furthermore, the compound 9 did not show any cytotoxic effect
A549 treated with the IC90 concentrations of 9 and 11 were col-
lected and extracted DNA was analyzed by DNA gel electrophoresis
(see Supplementary data Fig. S1). Occurrence of typical DNA lad-
ders was observed in both cases.
on human fibroblasts even at 100
l
M concentration (Table 2). Sim-
In conclusion, we have described the synthesis of new carba-
mate derivatives of betulinic acid and their in vitro anticancer
activity on fifteen tumor cell lines. Compounds 9 and 11 were
found to be most cytotoxic among the derivatives synthesized
and they seem to induce apoptosis with the involvement of casp-
ases on lung tumor cell line. Moreover compounds 9 and 11 exhibit
a good degree of selectivity towards tumor cells than non tumor
cells. The present investigation demonstrates that simple modifica-
tions of the parent structure of betulinic acid or betulin can pro-
duce a number of highly potent derivatives, which may improve
the selective toxicity profile or introduce general toxic effects.
However, results from a more extensive investigation using a
greater number of derivatives are needed for structure–activity
relationship (SAR) study for the design and ultimate synthesis of
a more effective betulinic acid and betulin derived antitumor
agents.
ilar to compound 11, compound 9 was found to be more than 34
and 36 times more selective towards A549 and SW480 cell lines.
In addition, the human fibroblasts (WWO70327) showed better
tolerance to compound 9 and 11 in comparison with betulinic acid
in all cases.
Furthermore, cell death induced by the new compounds was
mediated by apoptosis. One critical hallmark of apoptosis is activa-
tion of caspases, in particular effector-caspases-3/-7, which are
responsible for the degradation process that eventually leads to
the typical features of apoptosis. We have chosen lung cancer cell
line A549 to investigate general activation of caspases using sub-
strate cleavage assay. After exposure to equitoxic IC90 concentra-
tions of 1, 5, 9 and 11, cells were sampled 2 and 6 h for cleavage
of typical effector-caspase substrate DEVD. When treated with
compounds 5, 9, 11 and betulinic acid, 1, for 2 h it was observed
that caspase activation was seen for compound 11 only. After 6 h
greater caspase activation was observed for compounds 5 and 11
in contrast to 1 and 9 (Fig. 2).
Acknowledgments
In addition, apoptotic cell death was confirmed by analysis of
typical DNA fragmentation. Floating cells from lung cancer cell line
The authors are grateful to the Wirtschaftsministerium Sach-
sen-Anhalt for financial support (Antitumor drugs on the basis of

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H. Kommera et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3409–3412
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