Targeting cancer cells with oleanolic and ursolic acid derived hydroxamates

Article abstract of DOI:10.1016/j.bmcl.2015.12.064

Oleanolic and ursolic acid derived hydroxamates were easily obtained from their parent compounds; they were screened for their cytotoxicity applying SRB assays employing several human tumor cell lines. Low EC₅₀values were determined for compounds in which the nitrogen as well as the oxygen in the hydroxamic acid part still holds acidic hydrogens. Thus, ursolic acid derived compounds having at least an OH and/or NH moiety in the hydroxamate part of the molecule showed good cytotoxicity but they are significantly less selective for the tumor cells than oleanolic acid derived compounds. Good results were determined for oleanolic acid derived 7 for tumor cell lines 518A2 (melanoma, EC₅₀= 3.3 μM), A2780 (ovarian carcinoma, EC₅₀= 3.4 μM) and HT29 (colon adenocarcinoma, EC₅₀= 5.6 μM) while being significantly less cytotoxic for fibroblasts (EC₅₀= 20.4 μM).

Full text of DOI:10.1016/j.bmcl.2015.12.064

Accepted Manuscript
Targeting cancer cells with oleanolic and ursolic acid derived hydroxamates
Jana Wiemann, Lucie Heller, René Csuk
PII:
S0960-894X(15)30377-2
DOI:
http://dx.doi.org/10.1016/j.bmcl.2015.12.064
BMCL 23425
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
16 November 2015
16 December 2015
19 December 2015
Please cite this article as: Wiemann, J., Heller, L., Csuk, R., Targeting cancer cells with oleanolic and ursolic acid
derived hydroxamates, Bioorganic & Medicinal Chemistry Letters (2015), doi: http://dx.doi.org/10.1016/j.bmcl.
2
015.12.064
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Targeting cancer cells with oleanolic and ursolic acid derived hydroxamates
Jana Wiemann, Lucie Heller and René Csuk*
Martin-Luther Universität Halle-Wittenberg, Bereich Organische Chemie, Kurt-Mothes-Str.
2
, D-06120 Halle (Saale), Germany; rene.csuk@chemie.uni-halle.de
Keywords: triterpenes; oleanolic acid; ursolic acid; hydroxamates; SRB assay; cancer
Abstract
Oleanolic and ursolic acid derived hydroxamates were easily obtained from their parent
compounds; they were screened for their cytotoxicity applying SRB assays employing several
human tumor cell lines. Low EC50 values were determined for compounds in which the
nitrogen as well as the oxygen in the hydroxamic acid part still holds acidic hydrogens. Thus,
ursolic acid derived compounds having at least an OH and/or NH moiety in the hydroxamate
part of the molecule showed good cytotoxicity but they are significantly less selective for the
tumor cells than oleanolic acid derived compounds. Good results were determined for
oleanolic acid derived 7 for tumor cell lines 518A2 (melanoma, EC50 = 3.3 µM), A2780
(ovarian carcinoma, EC50 = 3.4 µM) and HT29 (colon adenocarcinoma, EC50 = 5.6 µM)
while being significantly less cytotoxic for fibroblasts (EC50 = 20.4 µM).
Cancer remains one of the most threatening diseases in the world. Worldwide, 8.2 million
people died of cancer in 2012, with lung cancers (with bronchus and trachea cancers) rising to
th
become the 5 leading cause of death in 2012 killing 1.1 million men and 0.5 million women
[
1]
.
Expenses for cancer-related drugs increased from 80.8 billion US$ in 2010 to 100.2 billion
[
2]
US$ in 2014 . Although over the past decades notable progress has been achieved on
developing new antitumor active drugs, there is still an increasing demand to develop new and
innovative drugs to target tumor cell specific mechanisms. Recently, the importance of
[
3-8]
molecular pathways regulating the cellular epigenome has been pointed out
, and their
[
9-11]
dysregulation has been related to cancer
.
[
12, 13]
Among others, histone deacetylases play a key-role in these processes
, and hydroxamate
substituted inhibitors have been recognized as potent antitumor active drugs. Several of them
1

[
17, 18]
are already applied in clinical trials for cancer treatment
. In addition, hydroxamic acids
have again entered the focus of scientific interest reflecting the fact that they show various
[
16, 19-23]
biological activities
.
Although many derivatives of pentacyclic triterpenoic acids have been prepared and screened
[24-29]
for their cytotoxic activity
hydroxamate substituted triterpenoids have only scarcely
been described so far. There are only two reports on glycyrrhetinic acid derived
hydroxamates, and they acted as inhibitors of 11β hydroxysteroid-dehydrogenase 2 but these
[
30, 31]
[32]
compounds have not been screened for any cytotoxic activity
. Recently, we
described the synthesis and antiproliferative properties of betulinic acid derived
hydroxamates.
Scheme 1. Synthesis of hydroxamates 2–9 from 3-O-acetyl-oleanolic acid (1): (a) Ac
pyridine, NEt , DMAP, 25 °C, 12 h, 86 %; (b) (COCl) , DCM, 25 °C, 2 h then NH OH.HCl,
NEt , DCM, 25 °C, 3 h, 74 %; (c) KOH, MeOH, 25 °C, 4 d, 98 %; (d) (COCl) , DCM, 25 °C,
h then HNMeOMe.HCl, NEt , DCM, 25 °C, 12 h, 84 %; (e) KOH, MeOH, 25 °C, 3 d, 99
; (f) (COCl) , DCM, 25 °C, 2 h then HNMeOH.HCl, NEt , DCM, 25 °C, 1 d, 48 %; (g)
, DCM, 25 °C, 2 h then NH OMe.HCl, NEt
DCM, 25 °C, 30 min, 75 %; (i) KOH, MeOH, 25 °C, 4 d, 93 %.
2
O,
3
2
2
3
2
2
3
%
2
3
KOH, MeOH, 25 °C, 5 d, 62 %; (h) (COCl)
2
2
3
,
2

To gain a deeper insight into this class of neglected compounds we decided to synthesize
representative examples of hydroxamates derived from oleanolic acid (OA) and ursolic acid
(
UA) and to investigate their cytotoxic activity employing several human tumor cell lines and
nonmalignant mouse fibroblasts (NIH 3T3) in photometric sulforhodamine B (SRB) assays
[
33]
.
Commercially available oleanolic acid (OA) was acetylated, and 3-O-acetyl-oleanolic acid (1)
was obtained in 86 % yield (Scheme 1). Reaction of 1 with oxalyl chloride followed by
reaction with hydroxylammonium chloride/triethylamine for 3 h furnished a hydroxamate 2
whose deacetylation with potassium hydroxide in methanol for 4 days gave an almost
1
quantitative yield of 3. Hydroxamic acid 2 is characterized in its C NMR spectrum by a
3
signal at δ = 176.7 ppm being assigned to the CONHOH moiety (C28) of 2. In the IR
-1
spectrum the C-O stretch vibration was detected at ν = 1646 cm .
Reaction of
1
with oxalyl chloride followed by reaction with N,O-
dimethylhydroxylammonium chloride or N-methylhydroxylammonium chloride or O-
methylhydroxylammonium chloride in the presence of triethylamine gave products 4–6 whose
deacetylation yielded compounds 7– 9, respectively. Under similar conditions, 3-oxo-
oleanolic acid (10, obtained by Jones-oxidation from OA in 81 % yield) gave substituted
hydroxamic acids 11–13 (Scheme 2).
Scheme 2. Synthesis of hydroxamates 11–13 from 3-keto-oleanolic acid (10): (a) CrO
3
,
,
H
2
SO
4
, H
2
O, acetone, 25 °C, 2 h, 81 %; (b) (COCl)
2
, DCM, 25 °C, 2 h then HNMeOMe.HCl,
NEt , DCM, 25 °C, 12 h, 43 %; (c) (COCl)
3
2
, DCM, 25 °C, 2 h then HNMeOH.HCl, NEt
3
3

DCM, 25 °C, 12 h, 44 %; (d) (COCl)
C, 2 h, 80 %.
2 2 3
, DCM, 25 °C, 2 h then NH OMe.HCl, NEt , DCM, 25
°
Similarly, commercially available ursolic acid (UA) was acetylated to yield 3-O-acetate 14
Scheme 3) that was transformed into acetylated hydroxamates 15–18; deacetylation as
described above gave compounds 19–22. Jones oxidation of UA yielded 3-oxo-usolic acid
23, Scheme 4) that was converted into hydroxamic acids 24–26.
(
(
Scheme 3. Synthesis of hydroxamates 15–22 from 3-O-acetyl-ursolic acid (14): (a) Ac
pyridine, NEt , DMAP, 25 °C, 12 h, 97 %; (b) (COCl) , DCM, 25 °C, 2 h then NH OH.HCl,
NEt , DCM, 25 °C, 3 h, 68 %; (c) KOH, MeOH, 25 °C, 6 d, 89 %; (d) (COCl) , DCM, 25 °C,
h then HNMeOMe.HCl, NEt , DCM, 25 °C, 12 h, 43 %; (e) KOH, MeOH, 25 °C, 6 d, 92
; (f) (COCl) , DCM, 25 °C, 2 h then HNMeOH.HCl, NEt , DCM, 25 °C, 12 h, 78 %; (g)
, DCM, 25 °C, 2 h then NH OMe.HCl, NEt
DCM, 25 °C, 1 h, 73 %; (i) KOH, MeOH, 25 °C, 4 d, 93 %.
2
O,
3
2
2
3
2
2
3
%
2
3
KOH, MeOH, 25 °C, 5 d, 89 %; (h) (COCl)
2
2
3
,
The oleanolic acid derived hydroxamates 2–9/11–13 and ursolic acid derived compounds 15–
2/24–26 were evaluated for their cytotoxic activity using photometric sulforhodamine B
assays employing several human tumor cell lines (518A2, A2780, A549, FaDu, HT29, MCF-
2
[
33]
7
) and nonmalignant mouse fibroblasts (NIH 3T3) . For comparison, OA, UA as well as
standard compound betulinic acid (BA) were included into this screening. The results of these
assays are summarized in Table 1.
4

The results from the SRB assays showed ursolic acid (UA) as cytotoxic as betulinic acid
BA); oleanolic acid (OA) was significantly less cytotoxic. This displays the potential of OA
(
and UA as starting materials for the development of cytotoxic/antitumor active drugs. All
hydroxamates derived from OA or UA were more cytotoxic than their parent compounds but
most of the compounds were as cytotoxic for cancer cells as for non-malignant mouse
fibroblasts. Low EC50 values were determined for compounds OA derived 2 and 3 – in these
compounds the nitrogen as well as the oxygen in the hydroxamic acid part still holds acidic
hydrogens.
Scheme 4. Synthesis of hydroxamates 24–26 from 3-keto-ursolic acid (23): a) CrO
3
, H
2
SO
4
3
,
,
H
2
O, acetone, 25 °C, 2 h, 96 %; b) (COCl)
2
, DCM, 25 °C, 2 h then HNMeOMe.HCl, NEt
DCM, 25 °C, 5 h, 65 %; c) (COCl)
2
3
, DCM, 25 °C, 2 h then HNMeOH.HCl, NEt , DCM, 25
°C, 1 d, 66 %; d) (COCl) , DCM, 25 °C, 2 h then NH OMe.HCl, NEt , DCM, 25 °C, 2 h, 74
2 2 3
%.
However, these compounds were of equal cytotoxicity for fibroblasts and for A2780 ovarian
cancer cells with EC50 values as low as 2.6 µM (for 2) and 3.4 µM (3). Low EC50 values were
also found for compounds 7 (EC50 = 3.4 µM), 17 (EC50 = 6.5 µM), 19 (EC50 = 4.6 µM), 21
(
EC50 = 4.5 µM), 24 (EC50 = 4.5 µM) and 25 (EC50 = 4.0 µM) for A2780 cells. In addition,
compound 24 showed a noteworthy low EC50 = 8.3 µM for the multidrug resistant human
tumor cell FaDu while for nonmalignant fibroblasts EC50 = 18.5 µM was determined.
5

Table 1. Cytotoxicity of oleanolic (2–9/11–13) or ursolic acid (15–22/24–26) derived
hydroxamates and starting materials OA and UA as well as betulinic acid (BA) as standard
(
EC50 values in µM from SRB assays after 96 hours of treatment; confidence interval CI =
5%). The cell lines are human cancer cell lines: 518A2, A2780, A549, FaDu, HT29, MCF-7
and nonmalignant mouse fibroblasts (NIH 3T3).
9
5
18A2
A2780
A549
FaDu
HT29
MCF-7
NIH 3T3
16.1±1.4
76.4±0.7
18.7±1.6
2.6±0.3
BA
OA
UA
2
9.4±0.7
64.3±4.2
14.7±0.1
4.2±0.1
5.5±0.6
8.4±0.2
9.4±0.3
14.2±0.4
3.3±0.5
11.6±0.2
12.8±1.1
20.9±2.1
12.9±0.6
17.7±0.4
3.6±0.1
12.7±0.5
8.4±0.2
15.5±0.6
9.4±0.1
22.9±0.8
12.5±0.9
14.3±1.2
15.5±1.8
8.8±0.9
14.0±0.7
11.7±0.6
2.6±0.5
3.4±0.6
6.8±1.3
7.5±1.1
10.8±2.0
3.4±0.1
3.9±0.2
12.3±1.5
8.8±0.7
4.8±0.7
6.1±1.4
2.7±0.1
9.8±0.9
6.5±0.2
10.6±1.5
4.6±0.7
21.8±3.1
4.5±1.3
12.1±1.7
4.5±1.5
17.1±1.1
72.3±1.5
15.5±1.3
3.1±0.3
4.7±0.3
17.6±2.0
10.8±1.9
18.6±1.4
11.8±2.0
9.8±0.6
20.9±1.1
5.6±0.8
8.1±0.4
4.8±0.4
3.9±0.1
9.6±0.8
5.7±1.0
6.6±0.5
6.7±0.4
11.6±2.0
9.7±0.6
8.3±0.9
8.7±1.1
15.3±2.1
75.4±3.4
14.2±2.0
10.6±0.4
8.2±0.1
14.4±2.3
38.8±3.1
10.6±0.3
4.9±0.1
10.2±1.2
80.0±3.5
12.7±0.1
9.5±0.1
8.2±0.2
7.5±0.8
6.2±1.2
9.1±0.6
16.2±1.2
8.3±1.1
17.0±1.8
16.4±1.5
16.4±1.1
20.0±1.8
3.3±0.2
15.4±0.6
7.9±0.8
6.8±0.7
13.1±0.2
18.7±1.7
15.4±0.3
10.0±2.1
10.7±0.3
3
4.7±0.1
4.3±0.2
4
9.4±1.1
13.1±1.0
10.6±0.4
17.5±0.4
5.6±1.0
13.3±0.7
12.3±0.7
13.2±1.9
20.4±2.1
11.1±0.4
18.2±0.6
13.7±0.9
9.2±0.3
5
12.1±0.2
12.3±0.6
29.8±3.1
5.8±0.3
6
7
8
8.7±0.4
9
14.3±1.1
24.3±2.1
14.1±0.9
23.1±0.7
6.4±0.4
18.8±0.8
15.6±1.3
11.9±0.4
18.6±0.9
3.5±0.3
1
1
1
1
1
1
1
1
2
2
2
2
1
2
3
5
6
7
8
9
0
1
2
4
12.2±1.8
2.5±0.6
15.0±1.1
11.3±0.7
12.1±0.3
18.1±1.1
26.8±2.0
15.1±0.3
11.2±0.8
8.3±1.2
17.6±1.0
8.7±0.3
23.0±1.7
6.6±0.4
13.2±1.0
7.5±0.2
14.8±0.7
5.5±0.5
18.4±0.7
12.3±1.4
15.4±2.1
18.7±1.3
23.3±1.5
10.1±0.2
16.2±1.5
18.5±2.3
6

2
2
5
6
12.4±1.4
12.9±1.1
4.0±0.8
9.3±1.1
9.2±1.1
17.6±0.6
12.0±1.3
12.8±0.8
12.9±1.1
17.6±1.2
11.2±2.0
10.5±0.1
14.3±1.5
14.3±2.0
Best results, however, were determined for oleanolic acid derived 7 for tumor cell lines
18A2 (EC50 = 3.3 µM), A2780 (EC50 = 3.4 µM) and HT29 (EC50 = 5.6 µM) while being
5
significantly less cytotoxic for fibroblasts (EC50 = 20.4 µM). Interestingly, the ursolic acid
derived analogue, 20 was significantly less cytotoxic [518A2 (EC50 = 22.9 µM), A2780 (EC50
=
21.8 µM) and HT29 (EC50 = 18.4 µM)] but also less selective for the tumor cells [EC50 for
NIH 3T3: 7 (20.4 µM), 20 (23.3 µM)]. Ursolic acid derived compounds possessing at least an
OH and/or NH moiety in the hydroxamate part of the molecule (as in 15, 19, 20) showed good
cytotoxicity (as good as OA derived 7) but they are significantly less selective for the tumor
cells.
To sum up, oleanolic and ursolic acid derived hydroxamates were easily obtained from their
parent compounds. Low EC50 values were determined in SRB assays for those compounds in
which the nitrogen as well as the oxygen in the hydroxamic acid part still holds acidic
hydrogens. Ursolic acid derived compounds showed good cytotoxicity but they were
significantly less selective for the tumor cells compared to oleanolic acid derived compounds.
Thus, oleanolic acid derived hydroxamate 7 showed low EC50 values for several tumor cell
lines while being significantly less cytotoxic for fibroblasts.
Acknowledgments
Thanks are due to Mrs J. Wiese for measuring the IR spectra and optical rotations. We like to
thank Dr. D. Ströhl and his team for many NMR spectra and Dr. R. Kluge for measuring the
ESI-MS spectra. The cell lines were kindly provided by Dr. Th. Müller (Dept. of
Haematology/Oncology, Martin-Luther Universität Halle-Wittenberg). Support by the
“
Gründerwerkstatt–Biowissenschaften” is gratefully acknowledged
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Graphical abstract
1
0