Amino(oxo)acetate moiety: A new functional group to improve the cytotoxicity of betulin derived carbamates

Article abstract of DOI:10.1016/j.bmcl.2016.04.055

While 3-O-acetylated betulin derivatives carrying a carbamate moiety at position C-28 are of rather low cytotoxicity for human tumor cell lines, the corresponding C-3 amino(oxo) acetates show good cytotoxicity. For example, an EC₅₀ as low as 2.0 μM was found for (3β) 28-{[(hexylamino)carbonyl]oxy}lup-20(29)-en-3-yl amino(oxo)acetate (16) employing the ovarian cancer cell line A2780.

Full text of DOI:10.1016/j.bmcl.2016.04.055

Bioorganic & Medicinal Chemistry Letters xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Amino(oxo)acetate moiety: A new functional group to improve the
cytotoxicity of betulin derived carbamates
a
a
a
b
a,
⇑
Lucie Heller , Vincent Perl , Jana Wiemann , Ahmed Al-Harrasi , René Csuk
a
Martin-Luther-University Halle-Wittenberg, Organic Chemistry, Kurt-Mothes-Str. 2, D-06120 Halle (Saale), Germany
University of Nizwa, Chair of Oman’s Medicinal Plants and Marine Natural Products, PO Box 33, Birkat Al-Mauz, Nizwa, Oman
b
a r t i c l e i n f o
a b s t r a c t
Article history:
While 3-O-acetylated betulin derivatives carrying a carbamate moiety at position C-28 are of rather low
Received 4 March 2016
Revised 17 April 2016
Accepted 20 April 2016
Available online xxxx
cytotoxicity for human tumor cell lines, the corresponding C-3 amino(oxo) acetates show good cytotox-
icity. For example, an EC50 as low as 2.0
29)-en-3-yl amino(oxo)acetate (16) employing the ovarian cancer cell line A2780.
Ó 2016 Elsevier Ltd. All rights reserved.
lM was found for (3b) 28-{[(hexylamino)carbonyl]oxy}lup-20
(
Keywords:
Betulin
Cytotoxicity
Triterpene
Cancers are a family of diseases, and one of the hallmarks of
cancer is a continuous and unregulated growth of cells leading
either to a solid tumor or to diffusely spread cells. In Europe, North
America and parts of Asia nearly half of people diagnosed with
cancer and receiving a treatment die of cancer or because of the
treatment. Cancer can be treated and even cured either by surgery,
application of radiation, immunotherapy or by chemotherapy.
Although it is indisputable that notable progress and major
achievements in the therapy of all kinds of cancer have been made
by chemotherapy, but there remains the need to search for new
chemotherapeutics. Starting several decades ago, natural product
derived compounds made their way into modern anticancer
chemotherapy, and nowadays they play a major role in developing
new cytotoxic agents. Among these natural products, many
Figure 1. Structure of betulin (BN) and betulinic acid (BA).
1
–8
terpenoids
finding new therapeutic drugs
preventive agents
have shown to act as excellent starting points for
2,3,5,6,8
and even as potential chemo-
During our search for new cytotoxic
1
,4–7
obtain in huge amounts from extracting birch bark, and it is an
interesting scaffold for accessing derivatives displaying various
biological properties. For example, in HeLa cancer cells BN, unlike
BA, seems to trigger apoptosis through an intrinsic pathway by a
sequential activation of caspase-9 and caspase 3/-7 and the cleav-
compounds derived from secondary natural products, we became
9
interested in triterpenoic acids (cf. betulinic acid (BA), boswellic
1
0,11
12
13
14
acid,
glycyrrhetinic acid, maslinic acid, oleanolic acid,
1
4
15
ursolic acid, tormentic acid and others) and other triterpenes
2
6
age of poly-(ADP-ribose)-polymerase. Recently, BN derived car-
(
cf. betulin, BN, Fig. 1). Interestingly enough, while there are many
16–22
bamates came into the focus of scientific interest because of their
reports on the cytotoxicity of BA or derivatives,
BN is of
2
7–31
increased cytotoxic activity
and an improved therapeutic
reduced cytotoxicity, and the number of betulin derived cytotoxic
3
0,32
derivatives remained limited.2
3–25
index. Hence, we decided to have a closer look onto this class
of compounds, and to explore their cytotoxic properties.
3
2
Apart from the fact that BN is a cheap, renewable resource usu-
ally obtained from agro-industrial waste. Thus, BN is easily to
Following well known procedures, BN was acetylated and diac-
etate 1 was obtained in excellent yields (Scheme 1). A regioselective
and partial deacetylation of 1 with KOH in a mixture of THF and
⇑
Corresponding author.
http://dx.doi.org/10.1016/j.bmcl.2016.04.055
960-894X/Ó 2016 Elsevier Ltd. All rights reserved.
0
Please cite this article in press as: Heller, L.; et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.04.055

2
L. Heller et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
Scheme 1. Synthesis of target compounds 13–17: (a) Ac
2
O, NEt
3
, DMAP, DCM, 12 h, 25 °C, 90%; (b) KOH, MeOH, THF, 0 °C, 30 min, 57%; (c) microwave-assisted, 7 h, 120 °C,
, DCM, 25 °C,
THF, RNCO, 3 (81%), 4 (83%), 5 (81%), 6 (73%), 7 (87%); (d) KOH, MeOH, THF, 25 °C, 8 (2 d, 95%), 9 (1 d, 93%), 10 (2 d, 94%), 11 (1 d, 88%), 12 (2 d, 89%); (e) (COCl
h; (f) NH , DCM, 25 °C, 12 h, 13 (72%), 14 (63%), 15 (78%), 16 (76%), 17 (60%).
2 2
)
1
3
methanol advanced smoothly at 0 °C and provided monoacetate 2 in
7% yield. Microwave assisted reaction of 2 with alkyl isocyanates or
ingly enough, on deacetylation of 3–7, for compounds 8–12
cytotoxicity was restored, and EC50 values in the same magnitude
as observed for BA were measured.
5
phenyl isocyanate gave 3-O-acetylated carbamates 3–7. They were
deacetylated with KOH in methanol32 to yield carbamates 8–12.
Previous screening of compounds 3–7 in photometric sulforho-
The reaction of secondary alcohols with oxalyl chloride is
3
4–38
known to yield alkyl chloro(oxo) acetates,
but their reactions
3
3
damine B (SRB) assays showed these compounds of low cytotox-
icity (EC50 >30 mol = cut-off of the assay)—these compounds
with ammonia or amines leading to alkyl amino(oxo)acetates have
3
9–44
l
only scarcely been applied.
As an alternative, decomposing
4
5
were of the same low cytotoxicity as parent BN (Table 1). Interest-
halonitro-acetic acid derivatives has been suggested.
Table 1
Cytotoxicity of selected compounds, betulin (BN) and betulinic acid (BA, standard) (EC50 values in
independent experiments performed each in triplicate; confidence interval CI = 95 %; cut-off of the assay 30
l
M from SRB assays after 96 h of treatment; the values are averaged from three
M)
l
EC50
518A2
A2780
HT29
MCF-7
A549
HeLa
BA
BN
1
2
3
11.9 ± 1.7
>30
>30
>30
10.2 ± 1.3
>30
12.5 ± 3.1
15.9 ± 2.3
8.0 ± 1.1
5.6 ± 0.7
23.9 ± 3.0
11.4 ± 1.1
9.9 ± 0.9
7.9 ± 1.2
6.5 ± 1.1
15.3 ± 1.0
11.0 ± 1.9
>30
20.3 ± 2.4
28.5 ± 1.9
11.1 ± 1.4
>30
4.6 ± 1.0
10.6 ± 1.1
7.0 ± 0.9
7.6 ± 1.3
7.2 ± 0.7
10.0 ± 0.9
7.0 ± 0.9
6.4 ± 1.0
2.0 ± 0.4
17.3 ± 0.7
14.4 ± 1.5
>30
25.7 ± 2.9
>30
>30
>30
14.8 ± 1.9
>30
>30
20.2 ± 2.0
20.4 ± 2.6
>30
12.1 ± 2.1
15.5 ± 3.0
12.5 ± 1.4
>30
24.6 ± 2.1
11.2 ± 0.9
12.5 ± 2.5
10.0 ± 1.5
5.6 ± 1.1
17.6 ± 0.6
14.9 ± 1.6
>30
21.5 ± 3.0
>30
14.1 ± 1.2
>30
12.0 ± 2.6
14.2 ± 1.7
8.6 ± 1.4
12.7 ± 2.6
>30
13.7 ± 1.1
10.5 ± 1.7
9.1 ± 1.8
7.4 ± 1.3
20.4 ± 0.8
8.8 ± 0.7
>30
>30
>30
17.0 ± 1.6
>30
4
8
9
1
1
1
1
1
1
1
1
–7
>30
>30
16.3 ± 2.0
7.5 ± 1.3
>30
16.3 ± 1.8
15.3 ± 2.0
12.8 ± 2.7
10.1 ± 1.2
7.3 ± 1.6
21.4 ± 0.6
9.4 ± 0.6
10.1 ± 1.7
7.4 ± 1.9
11.5 ± 1.5
14.1 ± 2.4
12.8 ± 1.9
11.2 ± 1.8
10.1 ± 1.5
10.5 ± 0.7
0
1
2
3
4
5
6
7
Human cancer cell lines: 518A2 (melanoma), A2780 (ovarian carcinoma), HT29 (colorectal adenocarcinoma), MCF-7 (breast adenocarcinoma), A549 (lung adenocarcinoma),
and HeLa (epitheloid cervix carcinoma).
Please cite this article in press as: Heller, L.; et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.04.055

L. Heller et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
3
As a result, the influence of the presence of an amino(oxo)-acet-
ate onto the biological activity of a molecule has never been exam-
ined before. Thus, compounds 8–12 were allowed to react with
oxalyl chloride followed by reacting the intermediary (and not iso-
lated) chloro(oxo)-acetates with ammonia, and products 13–17
were obtained in good to moderate yields. Screening of these com-
pounds in photometric SRB assays gave surprising results: While
acetates 3–7 were of rather low cytotoxicity for a variety of differ-
ent human tumor cell lines, the amino(oxo) acetates 13–17
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Please cite this article in press as: Heller, L.; et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.04.055