Platanic acid: A new scaffold for the synthesis of cytotoxic agents

Article abstract of DOI:10.1016/j.ejmech.2017.11.046

Thirty-seven different derivatives (2-38) have been prepared from platanic acid, a natural occurring triterpenoid. Main emphasis was the introduction of several N-containing functional groups such as amines, amides and oximes and their screening for cytotoxic activity employing several human tumor cell lines using SRB assays. In these SRB assays, nearly all compounds showed good cytotoxicity for these human tumor cell lines. Two compounds (17 and 38), however, were submitted to extended biological testing and investigated with respect to their mode of action using fluorescence microscopy and FACS analysis. Compound 17, a methyl (3β 20R) 3-acetyloxy-20-amino-30-norlupan-28-oate, induced apoptosis in A2780 ovarian carcinoma cells.

Full text of DOI:10.1016/j.ejmech.2017.11.046

Accepted Manuscript
Platanic acid: A new scaffold for the synthesis of cytotoxic agents
Michael Kahnt, Lucie Heller, Patricia Grabandt, Ahmed Al-Harrasi, René Csuk
PII:
S0223-5234(17)30949-2
10.1016/j.ejmech.2017.11.046
EJMECH 9921
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 22 May 2017
Revised Date: 25 October 2017
Accepted Date: 18 November 2017
Please cite this article as: M. Kahnt, L. Heller, P. Grabandt, A. Al-Harrasi, René. Csuk, Platanic acid: A
new scaffold for the synthesis of cytotoxic agents, European Journal of Medicinal Chemistry (2017), doi:
10.1016/j.ejmech.2017.11.046.
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ACCEPTED MANUSCRIPT
Graphical abstract

ACCEPTED MANUSCRIPT
Platanic acid: a new scaffold for the synthesis of cytotoxic agents
Michael Kahnt a, Lucie Heller a, Patricia Grabandt a, Ahmed Al-Harrasi b, René Csuk*,a
a
Martin-Luther-University Halle-Wittenberg, Organic Chemistry, Kurt-Mothes-Str. 2, D-
06120 Halle (Saale), Germany
b
University of Nizwa, Chair of Oman’s Medicinal Plants and Marine Natural Products, PO
Box 33, Birkat Al-Mauz, Nizwa, Sultanate of Oman
*Corresponding author:
Prof. Dr. René Csuk
Martin-Luther University Halle-Wittenberg
Organic Chemistry
Kurt-Mothes-Str. 2
D-06120 Halle (Saale)
Germany
Tel.: +49 345 5525660
Fax: +49 345 5527030
e-mail: rene.csuk@chemie.uni-halle.de
Abstract
Thirty-seven different derivatives (2-38) have been prepared from platanic acid, a natural
occurring triterpenoid. Main emphasis was the introduction of several N-containing functional
groups such as amines, amides and oximes and their screening for cytotoxic activity
employing several human tumor cell lines using SRB assays. In these SRB assays, nearly all
compounds showed good cytotoxicity for these human tumor cell lines. Two compounds (17
and 38), however, were submitted to extended biological testing and investigated with respect
to their mode of action using fluorescence microscopy and FACS analysis. Compound 17, a
methyl (3β, 20R) 3-acetyloxy-20-amino-30-norlupan-28-oate, induced apoptosis in A2780
ovarian carcinoma cells.
1

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1
Introduction
Platanic acid (PA, Fig. 1) is a norlupane type pentacyclic triterpene mainly found in plants of
the family of platanaceae. Besides its occurrence in sycamore trees (Platanus x hybrida Brot
and Platanus occidentalis) [1], PA can also be extracted from Melaleuca leucadendra [2] and
Leptospermum scoparium [3]. As the content of PA in these plants is comparatively low, this
secondary metabolite can be obtained more conveniently by partial synthesis from betulin
(BN) or betulinic acid (BA). The latter compounds can be extracted easily in larger quantities
from several plants and waste from wood industry. As early as 1940, L. Ruzicka et al. were
able to synthesise PA starting from BA by chromium trioxide oxidation [4]. Improvements of
this procedure were reported in the following years [5-7] but it was the merit of A. Vystrcil et
al. [8] to establish in 1970 the transformation of BA into PA by ozonolysis.
Up to now, little is known about the pharmacological properties of PA and its derivatives. In
1994, Fujioka et al. were able to show a selective inhibitory activity of PA and structurally
related BA against HIV-1 replication in H9 lymphocyte cells [9]. HIV maturation inhibitory
activities of BA and PA derivatives were also reported by Swidorski et al. [10]. Besides anti
HIV activity, PA and its derivatives exert further pharmacological properties. Recently,
platanic acid derivatives were reported to inhibit the enzyme butyrylcholinesterase. It is
anticipated that this enzyme and its regulation is involved in the progress of Alzheimer’s
disease [11]. Furthermore, cytotoxic activity for several human tumor cell lines has been
credited to PA and its derivatives [12-16]. Compared to other triterpenoic acids, such as
ursolic, oleanolic, maslinic or betulinic acid, the number of references regarding the cytotoxic
activity of PA and its derivatives is quite low. Nevertheless, it has been shown that several
PA derivatives act as cytotoxic agents [16]. Therefore, the further investigation of platanic
acid derivatives and their cytotoxic properties was called for.
2

ACCEPTED MANUSCRIPT
Fig. 1. Structure of platanic acid (PA), betulinic acid (BA) and betulin (BN).
2
Results and Discussion
2.1 Chemistry
The backbone of PA differs from other common triterpenoic acids such as oleanolic or ursolic
acid by the presence of an additional carbonyl moiety at the five-membered ring E. This
functional group at C-20 offers numerous opportunities for the derivatization and structural
modification. Therefore, this carbonyl group was used as an entry point for derivatization of
PA. Preparation of platanic acid derivatives 2-34 was performed as previously described [11].
Preceding to further modifications, C-3 and C-28 protected PA derivatives 2-5 were prepared.
Subsequent oximation of compounds 4 and 5 provided oximes 6-9; the separation of the E/Z-
isomers was easily achieved by column chromatography. Determination of their absolute
configuration at C-20 was established by investigation of the 1H NMR-spectra of compounds
8 and 9, since both compounds are known in literature.[17] In comparison to compound 8
(4.18–4.08 ppm), the 19-H signal of compound 9 (3.22 ppm) is clearly shifted, which is
consistent with data from literature. Similar NMR data were observed for compounds 6 and 7,
what allowed us to determine the configuration of these compounds respectively. A table of
chemical shifts compared to literature values can be found in the supplementary material.
Beckmann rearrangement of the oximes 8 and 9 afforded derivatives 10 (38%) and 12 (81%),
whose deacetylation gave compounds 11 and 13 (Scheme 1). Reaction of 9 with acetic
anhydride [17], propionyl chloride or benzoyl chloride provided products 14-16, respectively.
3

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Reduction of the oximes 7 or 9 [18] gave 20-amino-PA-derivatives 17, 18, 21 and 22. The
epimers were separated easily by column chromatography, and their absolute configuration at
C-20 was determined using 1H NMR spectroscopy. Amines 17 or 18 were acetylated to yield
19 and 20, which are both known in the literature [19]. Optical rotation values of compounds
19 and 20 additionally confirmed the configuration at C-20, as they were in full agreement
with previously reported. Selective deprotection of 21 at C-3 or C-28 provided compounds 23
and 24.
Scheme 1 Synthesis of platanic acid derivatives 2-24: (a) DMF, K2CO3, BnBr, 120 °C, 1 h,
98%; (b) DMF, K2CO3, MeI, 25 °C, 30 min, 89%; (c) Ac2O, pyridine, 25 °C, 4 h, for 4 (91%)
.
and 5 (96%) or 1 h for 19 and 20 (94% each); (d) HONH2 HCl, pyridine, 60 °C, 2 h, 10% (for
6), 80% (for 7) from 4; 13% (for 8) and 72% (for 9) from 5; (e) p-TosCl, DCM, pyridine,
DMAP, 25 °C, 2 days, then CHCl3, silica gel, 25 °C, 1 day, 38% (for 10) and 81% (for 12), (f)
KOH/MeOH, THF, 60 °C, 6 h, 74% (for 11) or 25 °C, 2 days, 88% (for 13), (g) Ac2O (for 13,
yield: 84%) or propionyl chloride (for 15, yield: 64%) or benzoyl chloride (for 16, yield:
99%), pyridine, 25 °C, 17 h; (h) CH3COONH4, NaBH3CN, MeOH, 25 °C, then TiCl3 (≥12%
in aq. HCl (12%)), 0–25 °C, 20 h, yielding 17 (67%) and 18 (15%) from 9 or 21 (73%) and 22
(19%) from 7; (k) MeOH/KOH, 25 °C, 75%; (l) H2, Pd/C (10%), CHCl3/MeOH 2:1, 25 °C,
24 h, 88%.
4

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Further modifications were performed by Curtius degradation of 3-O-acetyl-PA (25) (yielding
isocyanate 26) and subsequent hydrolysis, which provided the corresponding 17-amino-PA
derivative 27 (Scheme 2).
The 3-amino-PA derivatives 29 and 30 were prepared in a two-step process (Scheme 2)
starting from PA-benzyl ester 2. Silica gel supported oxidation of 2 with Jones reagent [20]
provided 28. Compounds 29 and 30 were obtained as a mixture by reductive amination of 28
with ammonium acetate and sodium cyanoborohydride [21]. Separation of the epimers was
achieved by column chromatography, and their absolute configuration at C-3 was determined
by 1H-NMR spectroscopy. According to literature values of 3-amino-betulinic acid[22], there
is a slight difference in the chemical shift of 3-H between the
α and the β-amino derivative.
1
These differences were observed in the H-NMR spectra of compounds 29 and 30. An
additional confirmation of the configuration was established using the H,H-ROESY NMR
experiment and investigating 3-H signal NOE couplings (description given in the
supplementary material).
Scheme 2. Synthesis of platanic acid derived amines 27, 29 and 30. (a) Ac2O, pyridine, 25 °C,
24 h, 94%; (b) diphenylphosphoryl azide, NEt3, toluene, 130 °C, 30 min, microwaves, 92%;
(c) aq. HCl (2M), THF, 70 °C, 6 h, 84%; (d) acetone, silica gel, 25 °C, 30 min, then Jones
reagent, 0–25 °C, 30 min, 96%; (e) CH3COONH4, MeOH, 60 °C, 15 min, then NaBH3CN,
MeOH, 25 °C, 24 h, 40% (for 29) and 44% (for 30).
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Platanic acid derivative 17 can be converted easily into amine 31 by reduction of the carbonyl
moiety at C-28 to yield the corresponding primary alcohol (Scheme 3). Acetylation of both
hydroxyl groups of 31 was performed by protecting the amino group at C-20 with di-tert-
butyldicarbonate followed by reaction with acetic anhydride. Subsequent removal of the Boc
protecting group yielded compound 32.
Scheme 3. Synthesis of betulin amines 31 and 32: (a) LiAlH4, THF, 70 °C (6 h), 25 °C (24 h),
71%; (b) Boc2O, pyridine, 25 °C, 30 min, then Ac2O, 16 h, then TFA, DCM, 0–25 °C, 3 h,
74%.
Another way to introduce a primary amino group into the platanic acid backbone was
achieved by amidation with ethylenediamine at C-28. DMF-catalyzed activation of the
carboxyl moiety with oxalyl chloride in dry dichloromethane and subsequent treatment with
ethylenediamine yielded amide 33. The ethylenediamine derived amides 35 and 37 were
prepared following the same procedure (Scheme 4). Treatment of platanic acid amides 33, 35
and 37 with a methanolic potassium hydroxide solution in methanol provided deacetylated
carboxamides 34, 36 and 38, respectively. Despite the long reaction times (up to 6 days), the
deacetylation of compounds 33, 35 and 37 was performed under mild conditions at room
temperature in order to avoid undesired side reactions. TLC reaction experiments revealed,
that reflux as well as microwave conditions result in the occurrence of side products.
6

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Scheme 4. Synthesis of platanic acid carboxamides 33–38: (a) oxalyl chloride, DCM, DMF,
0–25 °C, 1 h, then ethylenediamine (for 33, yield 86%) or N,N-dimethylethylenediamine (for
35, yield 93%) or 1-(2-aminoethyl)-pyrrolidine (for 37, yield 89%), 55 °C, 30 min; (b)
MeOH/KOH, 25 °C, 4 days, 86% (for 34) or 6 days, 89% (for 36) or 6 days, 76% (for 38).
2.2 Biology
Platanic acid derivatives 2-38 were screened for their cytotoxicity employing several human
tumor cell lines using the colorimetric sulforhodamine B assay. To investigate their selectivity
between tumor/nontumor cells EC50 values were also obtained for nonmalignant mouse
fibroblasts NIH 3T3. The results of the biological screening are compiled in Table 1.
Table 1: Cytotoxicity of compounds 2-38, platanic acid (PA) and staurosporine (STS): EC50
values from SRB assays after 96 h of treatment are given in µM (n.d. not detected); the values
are averaged from three independent experiments each performed in triplicate; confidence
interval CI = 95%.
518A2
A2780
HT29
MCF-7
A549
NIH 3T3
44.8 ± 1.4
n.d.
37.0 ± 0.7
15.8 ± 1.3
> 30
36.2 ± 1.6
10.1 ± 0.4
> 30
34.2 ± 1.9
9.0 ± 0.4
> 30
47.3 ± 4.5
15.9 ± 0.5
> 30
35.4 ± 0.9
18.1 ± 0.3
> 30
PA
2
n.d.
3
n.d.
> 30
> 30
> 30
n.d.
> 30
4
n.d.
> 30
> 30
> 30
n.d.
> 30
5
16.5 ± 0.4
9.0 ± 2.0
9.7 ± 2.1
7.8 ± 2.5
n.d.
9.9 ± 1.2
6.1 ± 1.3
12.9 ± 0.7
12.2 ± 0.5
11.2 ± 1.4
> 30
20.1 ± 2.2
12.3 ± 2.7
13.0 ± 2.0
14.4 ± 1.9
> 30
7.3 ± 0.5
6.8 ± 2.2
12.1 ± 0.3
12.4 ± 0.5
13.9 ± 1.5
> 30
11.2 ± 0.7
7.9 ± 1.5
8.8 ± 0.7
8.2 ± 0.9
> 30
14.0 ± 1.1
6.1 ± 1.3
12.3 ± 1.2
14.0 ± 2.4
> 30
6
7
8
9
10
11
n.d.
> 30
> 30
> 30
7

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n.d.
22.1 ± 1.3
> 30
> 30
10.0 ± 1.0
> 30
27.7 ± 0.9
> 30
24.1 ± 2.0
> 30
12
13
14
15
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
STS
n.d.
> 30
16.5 ± 0.7
17.9 ± 2.1
4.1 ± 0.3
6.3 ± 0.2
27.0 ± 0.7
30.1 ± 0.8
2.2 ± 0.1
2.5 ± 0.1
7.5 ± 0.1
23.2 ± 1.0
> 30
n.d.
23.8 ± 3.1
28.7 ± 2.9
6.3 ± 0.4
2.0 ± 0.3
27.6 ± 1.0
24.7 ± 1.1
2.3 ± 0.2
1.3 ± 0.3
5.0 ± 0.2
20.7 ± 0.6
> 30
13.1 ± 0.7
13.4 ± 0.8
5.1 ± 0.1
4.6 ± 0.2
21.4 ± 0.2
17.9 ± 1.1
3.0 ± 0.1
1.3 ± 0.3
5.6 ± 0.2
> 30
13.5 ± 1.4
13.2 ± 1.3
5.3 ± 0.7
6.6 ± 0.8
22.9 ± 2.1
25.5 ± 1.1
3.1 ± 0.1
3.3 ± 0.2
10.1 ± 1.6
> 30
15.0 ± 1.3
15.3 ± 2.6
5.6 ± 0.2
4.4 ± 0.6
16.0 ± 1.1
19.4 ± 0.1
4.1 ± 0.3
2.0 ± 0.9
7.3 ± 0.1
27.2 ± 3.0
> 30
n.d.
4.7 ± 0.1
6.0 ± 0.1
27.3 ± 0.7
25.3 ± 1.6
4.0 ± 0.1
2.3 ± 0.8
7.5 ± 0.1
> 30
> 30
> 30
> 30
n.d.
> 30
> 30
> 30
n.d.
> 30
> 30
> 30
18.7 ± 1.2
> 30
> 30
> 30
27.5 ± 0.7
> 30
> 30
> 30
> 30
> 30
2.3 ± 0.3
1.9 ± 0.1
23.7 ± 1.8
2.2 ± 0.1
3.2 ± 0.2
2.5 ± 0.1
3.7 ± 0.7
3.6 ± 0.6
3.3 ± 0.3
2.5 ± 1.2
0.03 ± 0.01
2.8 ± 0.7
2.2 ± 0.1
> 30
2.4 ± 0.2
1.5 ± 0.1
20.3 ± 0.5
1.2 ± 0.1
3.3 ± 1.2
2.3 ± 0.1
4.5 ± 0.2
3.9 ± 0.1
2.2 ± 0.8
2.8 ± 0.6
1.9 ± 0.3
1.8 ± 0.1
> 30
3.6 ± 0.1
2.1 ± 0.2
> 30
2.5 ± 0.5
2.3 ± 0.2
16.0 ± 1.1
4.0 ± 0.7
2.1 ± 0.1
2.5 ± 0.1
1.4 ± 0.1
3.5 ± 1.4
0.6 ± 0.6
2.8 ± 1.0
0.008 ± 0.001
2.5 ± 0.1
3.2 ± 0.2
2.5 ± 0.1
3.8 ± 0.6
4.0 ± 0.7
4.3 ± 0.5
2.2 ± 0.2
0.12 ± 0.01
2.1 ± 0.2
3.1 ± 0.1
2.4 ± 0.2
2.5 ± 0.3
3.2 ± 0.2
2.0 ± 0.5
2.1 ± 0.2
2.3 ± 0.1
3.2 ± 0.4
2.6 ± 0.2
2.9 ± 0.5
5.1 ± 0.3
2.9 ± 0.6
2.3 ± 0.1
0.15 ± 0.02 0.10 ± 0.02 0.04 ± 0.01
The introduction of various protecting groups (compounds 2-5) had, except for compound 2
(PA-benzyl ester), no influence onto the EC50 values; compound 2 showed an increased
cytotoxicity compared to parent PA. Modification of the carbonyl moiety at C-20 [such as
oximes (compounds 6-9)] resulted in moderate antitumor activity (6.1 – 16.5 µM) whereby
the configuration of the oxime (E or Z) did not have any influence onto the EC50 values.
Furthermore, no significant differences in cytotoxicity were detected for compounds differing
in the substituents at C-28 (Bn or Me). Beckmann-rearranged products exhibited decreased
cytotoxicity for most cell lines. The lowest EC50 values observed for compounds 10 (11.2
µM, A2780) and 12 (10.0 µM, MCF-7) are approximately in the same range as for the parent
compound. Deacetylation of the Beckmann products resulted in a lowered cytotoxicity.
8

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Compounds 11 and 13 are regarded non-cytotoxic since EC50 values greater than 30 µM were
observed for all tumor cell lines.
Since the products resulting from the Beckmann rearrangements did not show desirable
cytotoxicity, we turned our attention to investigate alkyl substituted oximes in more detail.
Thus (E) configurated oxime 9 served as a starting material for accessing O-alkyl substituted
oximes. Two of these O-substituted oximes (14 and 15) showed EC50 values similar to the
cytotoxicity observed for the Beckmann products but no significant improvements were
observed. EC50 values of 16, however, could not be determined due to its low solubility in
solvents compatible with the SRB assay.
Cytotoxicity of 17 and 18, each holding an amino moiety at C-20 was significantly improved
(2.0 – 6.6 µM) as compared to parent 9. Compound 18 displayed an EC50 of 2.0 µM (± 0.3
µM) for HT29 cancer cells.
Acetylation gave 19 and 20, which both showed decreased cytotoxicity. Similar to compound
9, oxime 7 (holding a benzyl moiety at C-28) was reduced; this provided the even more active
amines 21 and 22. For compound 22 the lowest EC50 of 1.3 µ
M was observed for the colon
adenocarcinoma cell line HT29. Deacetylation (yielding 23) resulted in a slight increase in
EC50 values. Removal of the benzyl moiety at C-28 (yielding compound 24), however, led to
an almost complete loss of cytotoxicity. Compound 27 (holding an amino group at C-17) is
significantly less cytotoxic than 17 or 18 (carrying an amino group at C-20). Following
previous findings [23-26] reporting increased cytotoxicity of 3-amino substituted
triterpenoids, amines 29 and 30 were prepared, and both compounds were highly cytotoxic
(e.g. 29: EC50 = 1.9 µM for breast adenocarcinoma cells MCF-7 and 30: EC50 = 1.5 µM for
colon adenocarcinoma HT29).
Transformation of 17 into 31 resulted in a dramatic loss of cytotoxic activity. As shown for
21, the presence of an O-acetyl moiety might have a positive influence onto cytotoxicity. EC50
values of 21 were lowered by a factor of two as compared to deacetylated 23. Keeping that in
mind, 31 was acetylated at HO-C(3) and HO-C(28) to yield the most active compound of this
study (32) exhibiting an EC50 = 1.2 µM for HT29 cancer cells.
9

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Compound 33, holding another primary amino group attached to the platanic acid backbone,
showed EC50 values in the range of 2.1 – 3.3 µM. Transformation of 33 into 34 had no
significant influence onto the cytotoxic activity. Data from literature revealed
ethylenediamine derived carboxamides of oleanolic acid [27] to be highly cytotoxic (EC50 =
1.3 µM –3.0 µM). Therefore, substituents at the terminal amino group of the ethylenediamine
moiety were varied and compounds 35-38 were obtained. All of them showed EC50 values
similar to those observed for 33. As already observed for 33 and 34, there were no significant
differences in cytotoxicity between acetylated (35 and 37) and deacetylated derivatives (36
and 38). The highest cytotoxic activity was determined for 37 for MCF-7 cancer cells (EC50 =
2.0 µM). Unfortunately, these compounds did not show any significant selectivity between
tumor cells and nonmalignant mouse fibroblasts (NIH 3T3).
Compounds 17 and 38 were selected for a pro-apoptotic screening. Initial investigations were
performed by using dye exclusion acridine orange (AO)/ propidium iodide (PI) test (Fig. 2,
B). Treating A2780 cells with 17 (8 µM) and 38 (5 µM) for 24 hours caused cell death to a
high degree. Close inspection by fluorescence microscopy revealed the presence of some vital
cells (green staining) after treatment with 17, while cells treated with 38 featured green and
orange colors thus indicating late-stage apoptosis. A quantification of the apoptosis-inducing
activity of the compounds was determined by an annexin V-FITC/PI staining (Fig. 2, A).
Treatment with 17 resulted in 40.3% annexin V-FITC-positive A2780 cells, while upon
treatment with 38 54.5% of the cells were annexin V-FITC positive. Cells incubated with 17
showed most of the cells having died by apoptosis (23.7%), while 47.4% of cells treated with
compound 38 were secondary necrotic/late-stage apoptotic. An extra investigation of the cell
cycle revealed also some differences in the mechanism of cell death (Fig. 2, C). Thus, no cell
arrest was detected for both compounds; investigations of the adherent cells treated with
compound 38 showed an almost uniform distribution of the DNA content over a relatively
broad range of the DNA histogram. The activation of caspase 3, an important mediator of
apoptosis, was further investigated. The expression level of caspase 3 protein was
significantly enhanced by a treatment of the cells for 24 hours with compounds 17 and 38. A
comparison of the two compounds showed that 17 slightly more potent than 38 (cf.
Supplementary Material Figure 1). In summary, the externalization of phosphatidylserine and
the release of caspase 3 protein could be identified as a significant feature of the cell death for
the A2780 ovarian carcinoma cells treated with compound 17. Similar results were observed
for compound 38, however an additional permeabilization of the cell membrane can be noted.
10

ACCEPTED MANUSCRIPT
Fig. 2: (A) Annexin V-FITC/ PI assay: treatment of A2780 cells with 17 and 38 (8 µM/ 5
µM) for 24h. Examples of density plots determined by flow cytometry (Attune® Cytometric
Software vl 1.2.5). (B) Fluorescence microscopic images (scale bar = 10 µm), AO and PI
were used a) control, b) 17 (8 µM), c) 38 (5 µM). (C) Representative examples for cell cycle
evaluation via ModFit V 4.0.5 a) control, b) 17 (8 µM), c) 38 (5 µM).
3.
Conclusions
To sum up, a set of thirty-seven derivatives of platanic acid (2-38) has been prepared and
screened for their cytotoxic activity against several human tumor cell lines and non-malignant
mouse fibroblasts using SRB assays. Nearly all of them displayed good cytotoxicity for
11

ACCEPTED MANUSCRIPT
several tumor cells. Low EC50 values were obtained for ethylenediamine derived
carboxamides 33-38, for platanic acid amines 17, 18, 21, 22, 29 and 30 as well as for the
amine 32. Compounds 17 and 38 were subjected to extended biological testing showing 17 to
trigger cell death by apoptosis.
4
Experimental Part
4.1 General
NMR spectra were recorded using the Varian spectrometers Gemini 2000 or Unity 500 (δ
given in ppm, J in Hz; typical experiments: H-H-COSY, HMBC, HSQC, NOESY), MS
spectra were taken on a Finnigan MAT LCQ 7000 (electrospray, voltage 4.1 kV, sheath gas
nitrogen) instrument. The optical rotations were measured on a Perkin-Elmer polarimeter at
20 °C; TLC was performed on silica gel (Merck 5554, detection with cerium molybdate
reagent); melting points are uncorrected (Leica hot stage microscope), and elemental analyses
were performed on a Foss-Heraeus Vario EL (CHNS) unit. IR spectra were recorded on a
Perkin Elmer FT-IR spectrometer Spectrum 1000. Microwave assisted reactions were
performed in a Monowave 300 apparatus (Anton Parr, Austria). The solvents were dried
according to usual procedures. The purity of the compounds was determined by HPLC and
found to be >96%. Platanic acid (PA) was obtained from different commercial suppliers in
bulk quantities. Fluorescence microscopic images were recorded on an Axioskop 20 with an
AxioCam MR3 (Carl Zeiss AG). Flow cytometric experiments were performed on an Attune
acoustic focusing cytometer (Life Technologies GmbH).
4.2 Biology
4.2.1 Cell lines and culture conditions
The cell lines used are human cancer cell lines: 518A2 (melanoma), A2780 (ovarian
carcinoma), HT29 (colon adenocarcinoma), MCF-7 (breast adenocarcinoma), A549 (alveolar
basal epithelial adenocarcinoma) and non-malignant mouse fibroblasts NIH 3T3. Cultures
were maintained as monolayers in RPMI 1640 medium with L-glutamine (Capricorn
Scientific GmbH, Ebsdorfergrund, Germany) supplemented with 10% heat inactivated fetal
bovineserum
(Sigma-Aldrich
Chemie
GmbH,
Steinheim,
Germany)
and
penicillin/streptomycin (Capricorn Scientific GmbH, Ebsdorfergrund, Germany) at 37 °C in a
humidified atmosphere with 5% CO2.
12

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4.2.2 Cytotoxic assay (SRB assay), AO/PI dye exclusion test, cell cycle investigations,
annexin V-FITC/PI assay and caspase 3 activity assay
Experimental details can be found in the supplementary material.
4.3 Syntheses
Platanic acid derivatives 2-34 were prepared as described previously [11]. Procedures for their
synthesis and full analytical data can be found in the supplementary material.
4.3.1 (3β)-N-[2-(Dimethylamino)ethyl]-3-acetyloxy-20-oxo-30-norlupan-28-amide (35)
Compound 25 (0.4 g, 0.8 mmol) was dissolved in dry DCM (15 mL), cooled to 0 °C and
treated with oxalyl chloride (0.3 mL, 3.52 mmol) and dry DMF (3 drops). The mixture was
allowed to warm to 25 °C and stirred for one hour. The solution was concentrated under
reduced pressure and re-evaporated with dry THF (3 x 15 mL). The residue was dissolved in
dry DCM (15 mL), cooled to 0 °C and NEt3 (3 drops) 2-(dimethylamino)-ethylamine (0.26
mL, 2.4 mmol) and DMAP (cat.) were added. The mixture was then stirred at 55 °C for 30
minutes. After completion of the reaction (as indicated by TLC) the solvent was removed
under reduced pressure. The crude product was purified by column chromatography (silica
gel, CHCl3/MeOH 9:1) to yield 35 (425 mg, 93%) as a white solid; m.p. 143 – 147 °C; RF =
0.46 (silica gel, chloroform/methanol 9:1); [α]D = –6.2° (c 0.395, CHCl3); IR (KBr): ν =
3409br m, 2946s, 2871m, 1733m, 1654m, 1522m, 1450m, 1384s, 1248s, 1196m, 1162w,
1029m, 979m cm-1; 1H NMR (500 MHz, CDCl3): δ = 6.98 (br s, 1H, NH), 4.45 (dd, J = 11.0,
5.0 Hz, 1H, H-3), 3.56–3.48 (m, 2H, H-32), 3.34 (td, J = 11.5, 3.9 Hz, 1H, H-19), 3.02–2.94
(m, 2H, H-33), 2.71 (s, 6H, Me-34 + Me-35), 2.21 (td, J = 12.1, 4.0 Hz, 1H, H-13), 2.14 (s,
3H, Me-29), 2.08 – 2.03 (m, 2H, H-18 + H-16a), 2.02 (s, 3H, Me-31), 2.00 – 1.91 (m, 1H, H-
21a), 1.84 – 1.78 (m, 1H, H-22a), 1.67 – 1.13 (m, 15H, H-1a + H-2ab + H-16b + H-22b + H-
6a + H-21b + H-11a + H-7ab + H-15a + H-6b + H-9 + H-11b + H-15b), 1.10 – 0.91 (m, 3H,
H-12ab +H-1b), 0.97 (s, 3H, Me-27), 0.89 (s, 3H, Me-26), 0.82 (s, 6H, Me-23 + Me-25), 0.81
13
(s, 3H, Me-24), 0.80 – 0.74 (m, 1H, H-5) ppm; C NMR (126 MHz, CDCl3): δ = 212.8 (C-
20), 177.7 (C-28), 171.0 (C-30), 81.0 (C-3), 58.2 (C-33), 55.8 (C-17), 55.5 (C-5), 51.3 (C-19),
50.5 (C-9), 50.2 (C-18), 44.6 (C-34 + C-35), 42.4 (C-14), 40.8 (C-8), 38.5 (C-1), 37.9 (C-22),
37.9 (C-4), 37.3 (C-10), 37.0 (C-13), 35.7 (C-32), 34.4 (C-7), 32.6 (C-16), 30.2 (C-29), 29.6
(C-15), 28.6 (C-21), 28.1 (C-23), 27.3 (C-12), 23.8 (C-2), 21.4 (C-31), 21.1 (C-11), 18.3 (C-
13

ACCEPTED MANUSCRIPT
6), 16.6 (C-24), 16.3 (C-25), 16.2 (C-26), 14.8 (C-27) ppm; MS (ESI, MeOH): m/z = 571
(100%, [M+H]+); analysis calculated for C35H58N2O4 (570.44): C 73.64, H 10.24, N 4.91;
found: C 73.51, H 10.39, N 4.80.
4.3.2 (3β)-N-[2-(Dimethylamino)ethyl]-3-hydroxy-20-oxo-30-norlupan-28-amide (36)
To a solution of compound 35 (200 mg, 0.35 mmol) in MeOH (10 mL) a solution of
potassium hydroxide (2 equiv) in MeOH (2 mL) was added. The mixture was stirred at room
temperature. After 6 days, aq. HCl was added until pH = 7. After usual workup, the crude
product was subjected to column chromatography (silica gel, CHCl3/MeOH 9:1) to yield 36
(164 mg, 89%) as a white solid; m.p. 173 – 176 °C; RF = 0.39 (silica gel, CHCl3/MeOH 9:1);
[α]D = –23.8° (c 0.630, MeOH); IR (KBr): ν = 3424br s, 2942m, 2868m, 1694m, 1642m,
1526m, 1384s, 1198w, 1170w, 1044w cm-1; 1H NMR (500 MHz, CDCl3): δ = 7.20 (t, J = 5.4
Hz, 1H, NH), 3.66 – 3.60 (m, 2H, H-30ab), 3.34 (ddd, J = 11.2, 11.2, 4.1 Hz, 1H, H-19), 3.21
– 3.14 (m, 3H, H-3 + H-31ab), 2.85 (s, 6H, Me-32 + Me-33), 2.20 (td, J = 11.4, 4.1 Hz, 1H,
H-13), 2.15 (s, 3H, Me-29), 2.10 – 2.02 (m, 2H, H-18 + H-16a), 2.01 – 1.90 (m, 1H, H-21a),
1.87 – 1.77 (m, 1H, H-22a), 1.68 – 0.99 (m, 17H, H-1a + H-2ab + H-16b + H-22b + H-6a +
H-21b + H-11a + H-6b +H-7ab + H-15a + H-9 + H-11b +H-15b + H-12ab), 0.98 (s, 3H, Me-
27), 0.95 (s, 3H, Me-23), 0.89 (s, 3H, Me-26), 0.93 – 0.82 (m, 1H, H-1b), 0.80 (s, 3H, Me-
25), 0.74 (s, 3H, Me-24), 0.70 – 0.65 (m, 1H, H-5) ppm; 13C NMR (126 MHz, CDCl3):
δ = 212.7 (C-20), 177.8 (C-28), 79.0 (C-3), 58.3 (C-31), 55.9 (C-17), 55.4 (C-5), 51.3 (C-19),
50.6 (C-9), 50.2 (C-18), 44.3 (C-32 + C-33), 42.4 (C-14), 40.8 (C-8), 39.0 (C-4), 38.8 (C-1),
37.8 (C-22), 37.4 (C-10), 37.0 (C-13), 35.1 (C-30), 34.4 (C-7), 32.5 (C-16), 30.2 (C-29), 29.7
(C-15), 28.7 (C-21), 28.1 (C-23), 27.5 (C-12), 27.4 (C-2), 21.1 (C-11), 18.4 (C-6), 16.3 (C-
25), 16.3 (C-26), 15.5 (C-24), 14.9 (C-27) ppm; MS (ESI, MeOH): m/z = 529 (100%,
[M+H]+); analysis calculated for C33H56N2O3 (528.43): C 74.95, H 10.67, N 5.30; found: C
74.78, H 10.92, N 5.17.
4.3.3. (3β)-N-[2-Pyrrolidin-1-yl)-ethyl]-3-acetyloxy-20-oxo-30-norlupan-28-amide (37)
Compound 37 was synthesized following the procedure given for 35 from 25 (0.4 g, 0.8
mmol), oxalyl chloride (0.3 mL, 3.52 mmol), DMF (3 drops) in dry DCM (15 mL), NEt3 (3
drops) 1-(2-aminoethyl)-pyrrolidine (0.3 mL, 2.4 mmol) in dry DCM (15 mL). Column
chromatography (silica gel, CHCl3/MeOH 9:1) afforded 37 (426 mg, 89%) as a white solid;
m.p. 162 – 165 °C; RF = 0.35 (silica gel, chloroform/methanol 9:1); [α]D = –7.8° (c 0.330,
CHCl3); IR (KBr): ν = 3404br s, 2947s, 1733s, 1652s, 1525s, 1383s, 1248s, 1196m, 1029m,
14

ACCEPTED MANUSCRIPT
979m, 752m cm-1; 1H NMR (500 MHz, CDCl3): δ = 7.39 (t, J = 5.7 Hz, 1H, NH), 4.44 (dd, J
= 11.2, 4.9 Hz, 1H, H-3), 4.02 – 3.69 (m, 2H, H-34a + H-37a), 3.69 – 3.56 (m, 2H, H-32ab),
3.36 – 3.22 (m, 3H, H-19 + H-33ab), 3.19 – 2.76 (m, 2H, H-34b + H-37b), 2.19 (td, J = 12.0,
4.1 Hz, 1H, H-13), 2.14 (s, 3H, Me-29), 2.14 – 2.03 (m, 6H, H-35ab + H-36ab + H-16a + H-
18), 2.02 (s, 3H, Me-31), 2.00–1.87 (m, 1H, H-21a), 1.87 – 1.77 (m, 1H, H-22a), 1.67 – 1.11
(m, 15H, H-1a + H-2ab + H-16b + H-22b +H-6a + H-21b +H-11a +H-6b + H-7ab + H-15a +
H-9 + H-11b + H-15b), 1.10 – 0.91 (m, 3H, H-12ab + H-1b), 0.96 (s, 3H, Me-27), 0.87 (s, 3H,
Me-26), 0.82 (s, 6H, Me-23 + Me-25), 0.81 (s, 3H, Me-24), 0.79 – 0.75 (m, 1H, H-5) ppm;
13C NMR (126 MHz, CDCl3): δ = 212.6 (C-20), 177.9 (C-28), 171.0 (C-30), 81.0 (C-3), 55.8
(C-17), 55.6 (C-33), 55.5 (C-5), 55.0 (C-34 + C-37), 51.3 (C-19), 50.5 (C-9), 50.2 (C-18),
42.3 (C-14), 40.8 (C-8), 38.5 (C-1), 37.9 (C-4), 37.7 (C-22), 37.3 (C-10), 37.0 (C-13), 36.0
(C-32), 34.3 (C-7), 32.4 (C-16), 30.1 (C-29), 29.6 (C-15), 28.6 (C-21), 28.1 (C-23), 27.3 (C-
12), 23.8 (C-2), 23.4 (C-35 + C-36), 21.4 (C-31), 21.1 (C-11), 18.3 (C-6), 16.6 (C-24), 16.3
(C-25), 16.2 (C-26), 14.8 (C-27) ppm; MS (ESI, MeOH): m/z = 597 (100%, [M+H]+);
analysis calculated for C35H58N2O4 (570.44): C 74.45, H 10.13, N 4.69; found: C 74.30, H
10.41, N 4.47
4.3.4. (3β)-N-[2-Pyrrolidin-1-yl)-ethyl]-3-hydroxy-20-oxo-30-norlupan-28-amide (38)
Compound 38 was prepared according to the procedure given for 36 from 37 (0.2 g, 0.34
mmol), potassium hydroxide (2 equiv.) in methanol (10 mL). After 6 days of reaction usual
workup and column chromatography (silica gel, CHCl3/MeOH 9:1) afforded 38 (143 mg,
76%) as a white solid; m.p. 170–173 °C; RF = 0.27 (silica gel, CHCl3/MeOH 9:1); [α]D = –
29.3° (c 0.415, MeOH); IR (KBr): ν = 3422br s, 2944s, 2868m, 1704m, 1646s, 1530m, 1354s,
1
1246m, 1198m, 1082w, 1034m, 752m cm-1; H NMR (500 MHz, CDCl3): δ = 7.43 (t, J = 5.5
Hz, 1H, NH), 3.95 – 3.74 (m, 2H, H-32a + H-35a), 3.67 – 3.59 (m, 2H, H-30ab), 3.35 – 3.25
(m, 3H, H-19 + H-31ab), 3.16 (dd, J = 11.3, 4.7 Hz, 1H, H-3), 3.05 – 2.86 (m, 2H, H-32b + H-
35b), 2.19 (td, J = 12.0, 4.1 Hz, 1H, H-13), 2.13 (s, 3H, Me-29), 2.14 – 1.87 (m, 7H, H-33ab +
H-34ab + H-16a + H-18 + H-21a), 1.85 – 1.78 (m, 1H, H-22a), 1.68 – 1.12 (m, 15H, H-1a +
H-12ab + H-16b + H-22b + H-6a + H-21b + H-11a + H-6b + H-7ab + H-15a + H-9 + H-11b +
H-15b), 0.96 (s, 3H, Me-27), 1.09 – 0.82 (m, 3H, H-2ab + H-1b), 0.94 (s, 3H, Me-23), 0.86 (s,
3H, Me-26), 0.78 (s, 3H, Me-25), 0.72 (s, 3H, Me-24), 0.68–0.64 (m, 1H, H-5) ppm;
13C NMR (126 MHz, CDCl3): δ = 212.7 (C-20), 178.0 (C-28), 78.9 (C-3), 55.8 (C-17), 55.5
(C-31), 55.4 (C-5), 55.0 (C-32 + C-35), 51.3 (C-19), 50.6 (C-9), 50.2 (C-18), 42.3 (C-14),
40.8 (C-8), 39.0 (C-4), 38.8 (C-1), 37.7 (C-22), 37.3 (C-10), 37.0 (C-13), 36.0 (C-30), 34.4
15

ACCEPTED MANUSCRIPT
(C-7), 32.4 (C-16), 30.2 (C-29), 29.6 (C-15), 28.6 (C-21), 28.1 (C-23), 27.5 (C-12), 27.3 (C-
2), 23.4 (C-33 + C-34), 21.1 (C-11), 18.4 (C-6), 16.3 (C-25), 16.2 (C-26), 15.5 (C-24), 14.8
(C-27) ppm; MS (ESI, MeOH): m/z = 555 (100%, [M+H]+); analysis calculated for
C35H58N2O3 (554.44): C 75.76, H 10.54, N 5.05; found: C 75.51, H 10.70, N 4.81.
Acknowledgments
We’d like to thank Dr. R. Kluge for measuring the ESI-MS spectra and Dr. D. Ströhl and his
team for the NMR spectra. Thanks are also due to Mrs. J. Wiese, MSc and Mrs. V. Simon for
measuring the IR spectra and optical rotations. The cell lines were kindly provided by Dr. Th.
Müller (Dept. of Haematology/Oncology, Martin-Luther Universität Halle-Wittenberg).
Support by the ‘‘WissenschaftsCampus Halle WCH” (W13004216 to R.C.) and the Oman
Research Council is gratefully recognized.
Appendix A. Supplementary material
Supplementary data related to this article can be found, in the online version, at …
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Highlights
* Thirty seven derivatives of platanic acid have been prepared
* The compounds were tested for cytotoxicity employing human tumor cell lines
* SRB assays revealed most these compounds cytotoxic in low µM concentration
* Extra screening showed a methyl 3-acetyloxy-20-amino-norlupanoate to act by apoptosis