The cytotoxicity of oleanane derived aminocarboxamides depends on their aminoalkyl substituents

Article abstract of DOI:10.1016/j.steroids.2019.05.014

Several oligo-methylene diamine derived carboxamides of oleanolic and maslinic acid have been prepared, and substitutions of the terminal primary amine as well as variations of the length of alkyl chain of the diamine moiety were made. Biological evaluation of their cytotoxic activity was performed using photometric sulforhodamin B assays employing a panel of different human cancer cell lines. These experiments showed most of the carboxamides to be cytotoxic with EC₅₀ values below 10 μM. Prolongation of the alkyl chain length initially reduced EC₅₀ values to a minimum, but a decrease in cytotoxicity was observed for longer alkyl chains. Variation of substituents at the terminal nitrogen atom, however, did not influence EC₅₀ values at all. Noteworthy results were obtained particularly for compounds 4, 6 and 23 as indicated by EC₅₀ values lower than 2 μM, and in case of a maslinic derivative 23 even an increased tumor/non-tumor cell selectivity was observed. These compounds were further investigated using fluorescence microscopy and flow cytometry analysis, which revealed 6 to show indications of apoptosis.

Full text of DOI:10.1016/j.steroids.2019.05.014

Accepted Manuscript
The cytotoxicity of oleanane derived aminocarboxamides depends on their ami-
noalkyl substituents
Michael Kahnt, Anne Loesche, Immo Serbian, Sophie Hoenke, Lucie Fischer,
Ahmed Al-Harrasi, René Csuk
PII:
S0039-128X(19)30112-6
https://doi.org/10.1016/j.steroids.2019.05.014
STE 8422
DOI:
Reference:
Steroids
To appear in:
Received Date:
Revised Date:
Accepted Date:
20 March 2019
27 May 2019
29 May 2019
Please cite this article as: Kahnt, M., Loesche, A., Serbian, I., Hoenke, S., Fischer, L., Al-Harrasi, A., Csuk, R., The
cytotoxicity of oleanane derived aminocarboxamides depends on their aminoalkyl substituents, Steroids (2019),
doi: https://doi.org/10.1016/j.steroids.2019.05.014
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

The cytotoxicity of oleanane derived aminocarboxamides depends on their aminoalkyl
substituents
Michael Kahnt a, Anne Loesche a, Immo Serbian a, Sophie Hoenke a, Lucie Fischer 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, Natural and Medical Sciences Research Center, P.O. Box 33, Birkat Al
Mauz, Nizwa 616, 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
Several oligo-methylene diamine derived carboxamides of oleanolic and maslinic acid have
been prepared, and substitutions of the terminal primary amine as well as variations of the
length of alkyl chain of the diamine moiety were made. Biological evaluation of their cytotoxic
activity was performed using photometric sulforhodamin B assays employing a panel of
different human cancer cell lines. These experiments showed most of the carboxamides to be
cytotoxic with EC50 values below 10 µM. Prolongation of the alkyl chain length initially
reduced EC50 values to a minimum, but a decrease in cytotoxicity was observed for longer alkyl
chains. Variation of substituents at the terminal nitrogen atom, however, did not influence EC50
values at all. Noteworthy results were obtained particularly for compounds 4, 6 and 23 as
indicated by EC50 values lower than 2 µM, and in case of a maslinic derivative 23 even an
increased tumor/non-tumor cell selectivity was observed. These compounds were further
1

investigated using fluorescence microscopy and flow cytometry analysis, which revealed 6 to
show indications of apoptosis.
Keywords: Oleanolic acid; maslinic acid; cytotoxicity; triterpenoids, carboxamides
1
Introduction
Medicinal herbs have been playing an important role in traditional medicine for thousands of
years. In search of rescue for their disease, people have been searching for drugs in nature ever
since. The oldest written evidence of plants’ medicinal usage is approximately 5000 years
old.[1] Nowadays nearly 25% of all plant species in the world have some kind of medicinal
use.[2] Due to their broad spectrum of biological and pharmacological properties such as anti-
inflammatory, antimalarial, antitumor and antiviral activities, natural products are considered
to be ideal candidates for modern drug discovery. A recent study revealed about 50% of all new
approved drugs in the period between 1981 and 2014 being natural products or natural product
related.[3] One of the most prevalent disease in present time remains cancer, resulting in
approximately 9.6 million cancer deaths worldwide in 2018.[4] With more than 50% of all new
anticancer drugs (1981-2014), natural products and derivatives thereof still play a key role in
treating cancer.[3] A promising class of natural products are triterpenes including (among
others) oleanolic (Fig. 1, OA) and maslinic acid (Fig. 1, MA), both of which can be found in
and isolated from plants. They show some promising biological properties such as antioxidant
[5-8], antiviral [5, 9-11] and antitumor activity [5, 12-18]. In this study, we focused on the
improvement of the antitumor activity of both triterpenes by chemical modification. Oleanolic
derivatives were previously investigated by Heller et al., showing ethylene diamine derived
carboxamides to be of an increased cytotoxicity as compared to their parent compounds.[19]
Previous studies, however, focused on the investigation of the influence of the substitution at
the primary amine of the ethylene diamine moiety.[19, 20] Also included in this study was an
ursolic carboxamide holding an extended alkyl length (derived from tetramethylene diamine),
that showed promising cytotxic activity in the range of 1.3–3.2 µM.[20] Therefore, we decided
to additionally vary the length of the ,-diamine moiety to evaluate its influence on
cytotoxicity. For successful cancer treatment, cytotoxicity towards cancer cells (as indicated by
low EC50 values) is very important, but gaining selectivity between tumor and non-tumor cells
is even more essential. Recent studies revealed maslinic acid derivatives to show increased
2

cytotoxicity being accompanied by an improved tumor–non-tumor cell selectivity - as
compared to their analogs derived from oleanolic acid.[21-23] Keeping this in mind, we
prepared a small library of carboxamides of oleanolic and maslinic acid and compared both
cytotoxicity as well as selectivity.
Fig. 1: Structures of oleanolic (OA) and maslinic acid (MA).
2
Results and Discussion
Oleanolic acid (OA) was obtained from Carbone Scientific (London, UK) in 98.6% purity and
maslinic acid (MA) was synthesized as previously described.[24, 25] Acetylation of both
triterpenoic acids afforded acetates 1 (Scheme 1) and 13 (Scheme 2) in excellent yields.
Treatment of acetate 1 with oxalyl chloride and -alkyl diamines of various chain length
provided compounds 2-12 (Scheme 1).
Scheme 1: Synthesis of oleanolic carboxamides 2-12: a) Ac2O, DCM, NEt3, DMAP, 25 °C, 2
days, 90%; b) oxalyl chloride, DCM, DMF, 0–25 °C, 15 h, then amine, 25 °C, 2 h, 36-60%.
The cytotoxicity of oleanolic carboxamides 2-12 was determined in sulforhodamine B (SRB)
assays.[26] The results of this screening are compiled in Table 1.
3

Table 1: Cytotoxicity of compounds 2-12 and oleanolic acid (OA): 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%.
A253
A2780
HT29
MCF-7
SW1736
NIH 3T3
Compound
OA
2
n.d.
>30
>30
>30
n.d.
>30
3.1 ± 0.1
3.1 ± 0.1
1.2 ± 0.9
3.8 ± 0.4
3.1 ± 0.2
2.8 ± 0.5
3.7 ± 0.8
2.4 ± 0.3
2.7 ± 0.2
2.3 ± 0.4
2.5 ± 0.6
3.1 ± 0.1
3.1 ± 0.2
1.2 ± 0.4
3.1 ± 0.2
3.1 ± 0.2
3.0 ± 0.1
2.5 ± 0.2
2.0 ± 0.2
2.7 ± 0.2
2.9 ± 0.3
4.6 ± 0.7
2.0 ± 0.2
2.2 ± 0.2
1.7 ± 0.1
1.4 ± 0.3
1.2 ± 0.1
1.2 ± 0.1
1.8 ± 0.1
1.6 ± 0.1
3.1 ± 0.2
3.2 ± 0.1
5.4 ± 0.3
1.7 ± 0.2
1.7 ± 0.1
2.0 ± 0.1
1.6 ± 0.3
3.9 ± 0.9
3.1 ± 0.2
3.1 ± 0.7
2.4 ± 0.2
3.1 ± 0.1
3.2 ± 0.3
5.2 ± 0.2
3.9 ± 0.6
3.1 ± 0.3
3.6 ± 2.1
3.5 ± 0.5
3.8 ± 0.2
3.5 ± 0.1
3.5 ± 0.4
3.8 ± 0.6
7.0 ± 0.5
3.5 ± 0.1
6.9 ± 0.1
2.1 ± 0.1
2.4 ± 0.9
1.0 ± 0.3
2.0 ± 0.2
2.7 ± 0.4
2.2 ± 0.1
2.3 ± 0.2
2.3 ± 0.2
3.0 ± 0.2
3.2 ± 0.7
5.7 ± 0.1
3
4
5
6
7
8
9
10
11
12
The influence of an altered chain length on the cytotoxicity of the compounds shall be
demonstrated comparing the EC50 values for the human colon adenocarcinoma cell line HT29
(Fig. 2). Thus, EC50 values initially decreased with increasing chain length n up to a minimum
value of 1.2 μM (n = 5 (6) or n = 6 (7)). For n greater than 6, the EC50 values increased slightly
until they reached a maximum value at n = 11 (12, EC50 = 5.4 ± 0.3 µM). Comparing the EC50
values with those obtained for non-malignant mouse fibroblasts (NIH 3T3), some information
on the selectivity [which is defined as selectivity index (SI); e.g. SI = EC50 (NIH 3T3)/EC50
(HT29)] can be obtained (indicated by red numbers in Fig. 2). As a result, the most selective
substance in the series holding a SI of 2.25 was compound 6; this compound also showed the
highest cytotoxicity (EC50 = 1.2 ± 0.1 μM). The origin of this selectivity, however, remains
unclear. Investigation of the other cancer cell lines revealed also compound 4 as highly
cytotoxic. This compound showed noteworthy cytotoxic activity on human submandibular
gland A253 cancer cells (1.2 ± 0.9 µM), HT29 (1.7 ± 0.1 µM) and on the human ovarian cancer
4

cell line A2780 (1.2 ± 0.4 µM). This was the reason to select this substance together with
compound 6 for an extended biological testing (Fig. 3).
Fig. 2: Influence of chain length on EC50 values of compounds 2-12 for HT29 cancer cells
compared to non-malignant mouse fibroblasts (NIH 3T3). The selectivity index (SI) is defined
as: SI = EC50 (NIH 3T3)/EC50 (HT29).
Initial investigations were performed by using dye exclusion acridine orange (AO)/ propidium
iodide (PI) assays and A2780 cancer cells (Fig. 3, A). Microscopic images of A2780 cells
treated with 4 revealed – in addition to many vital cells (green staining) – the presence of some
secondary necrotic/late-stage apoptotic cells, as indicated by their orange stained nuclei. Close
inspection also showed protrusions of the plasma membrane in some cells (membrane blebbing,
marked by white arrows in Fig. 3). These observations were also made for A2780 cells having
been treated with 6 for 24 h, while significantly less vital cells were noticed. For a quantification
of the apoptosis-inducing activity of 4 and 6, flow cytometry analyses were performed using an
annexin V-FITC/PI staining (Fig. 3, B). Most of the A2780 cells treated with 4 remained vital
(72.2%), while 10.0% or 16.1% of the cells were apoptotic or secondary necrotic/late-stage
apoptotic, respectively. Treatment of A2780 cells with 6 resulted in 56.1% annexin V-FITC-
positive cells, with 43.4% of the cells still being considered vital. Close inspection of the density
plots showed most of the cells having died by apoptosis (49.9%) while 6.2% of the cells were
secondary necrotic/late-stage apoptotic.
5

Fig. 3: (A) Fluorescence microscopic images: treatment of A2780 cells with 4 (2.4 µM) and 6
(6.2 µM) for 24 h. Scale bar = 20 µm, AO and PI were used. (B) Annexin V-FITC/PI assay:
treatment of A2780 cells with 4 and 6 (2.4 µM/6.2 µM) for 24 h. Examples of density plots
determined by flow cytometry (Attune® Cytometric Software v. 1.2.5). R1: necrotic, R2:
secondary necrotic/late stage apoptotic, R3: vital, R4: apoptotic.
Syntheses of oleanolic and maslinic carboxamides 14-28 derived from ethylene diamine were
analogous to compounds 2-12 and obtained from the acetylated triterpenoic acids by reaction
with oxalyl chloride followed by adding substituted ethylenediamines (Scheme 2). Finally, the
acetoxy groups were removed with an methanolic solution of potassium hydroxide.[27]
6

Scheme 2: Synthesis of oleanolic and maslinic carboxamides 14-28: a) Ac2O, DCM, NEt3,
DMAP, 25 °C, 2 days, 91%; b) oxalyl chloride, DCM, DMF, 0–25 °C, 1 h, then amine, 25 °C,
2 h, 71-96%; c) MeOH/KOH, 25 °C, 2-3 days, 60-93 %.
The cytotoxicity of oleanolic and maslinic carboxamides 14-28 was determined in
sulforhodamine B (SRB) assays.[26] The results of these screenings are compiled in Table 2.
Table 2: Cytotoxicity of compounds 14-28, oleanolic acid (OA) and maslinic acid (MA): 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%.
R1
H
H
H
R2
H
R3
518A2
>30
A2780
>30
HT29
>30
MCF-7
>30
A375
n.d.
NIH 3T3
>30
OA
MA
17
28-COOH
OH 28-COOH 13.7 ± 0.9
19.5 ± 0.8
n.d.
28.8 ± 0.5
3.1 ± 0.3
1.7 ± 0.2
5.0 ± 0.3
3.9 ± 0.2
3.7 ± 0.1
2.3 ± 0.3
5.1 ± 0.6
2.8 ± 0.2
3.4 ± 0.8
1.7 ± 0.2
4.6 ± 0.7
>30
n.d.
21.1 ± 0.2
3.8 ± 0.6
3.4 ± 0.5
7.4 ± 0.8
2.8 ± 0.3
3.5 ± 0.6
4.4 ± 0.4
7.6 ± 0.7
2.7 ± 0.2
2.6 ± 0.7
4.6 ± 0.3
6.2 ± 2.9
H
3.7 ± 0.1
3.3 ± 0.2
8.7 ± 1.6
3.4 ± 0.6
3.4 ± 0.3
n.d.
1.5 ± 0.1
1.8 ± 0.2
6.9 ± 1.0
2.9 ± 0.3
2.8 ± 0.6
2.4 ± 0.2
4.7 ± 0.7
2.7 ± 0.3
1.3 ± 0.1
2.0 ± 0.2
3.0 ± 0.5
3.2 ± 0.2
1.6 ± 0.1
6.5 ± 0.6
3.5 ± 0.7
2.7 ± 0.7
n.d.
21 Ac OAc
1.8 ± 0.1
7.0 ± 0.8
3.2 ± 0.1
2.9 ± 0.4
1.6 ± 0.1
4.8 ± 0.3
3.2 ± 0.1
2.4 ± 0.1
1.5 ± 0.3
3.6 ± 0.4
25
14 Ac
18
H
OH
H
H
H
22 Ac OAc
26
15 Ac
19
H
OH
H
14.5 ± 0.7
3.3 ± 0.3
3.6 ± 0.3
n.d.
7.0 ± 0.4
3.5 ± 0.3
1.6 ± 0.1
n.d.
H
H
23 Ac OAc
27 OH
H
10.7 ± 0.5
4.6 ± 1.2
7

16 Ac
20
H
H
4.6 ± 0.5
1.5 ± 0.1
1.5 ± 0.2
8.2 ± 0.4
3.5 ± 0.8
1.6 ± 0.1
3.5 ± 0.1
7.0 ± 0.3
3.5 ± 0.4
3.4 ± 0.4
2.0 ± 0.1
5.9 ± 0.2
3.5 ± 0.5
2.0 ± 0.1
1.7 ± 0.2
4.9 ± 0.7
4.9 ± 0.1
1.9 ± 0.1
0.9 ± 0.1
7.1 ± 0.4
4.4 ± 0.6
2.9 ± 0.1
2.6 ± 0.3
2.0 ± 0.6
H
24 Ac OAc
28 OH
H
Evaluation of the EC50 values in Table 2 reveals the impact of the presence of acetyloxy groups
on the cytotoxicity of the compounds. As for oleanolic carboxamides (14-20), the presence or
absence of an acetyl moiety at position 3 had no influence on the EC50 values of the compounds
at all. This is illustrated in Fig. 4 for HT29 cancer cells. For maslinic acid derivatives 21-28,
however, the impact of the acetoxy moieties on the cytotoxic properties of the compounds was
significant. Bisacetyloxy derivatives 21-24 show low EC50 values in the range of 1.7 µM to 2.3
µM. Removal of these acetoxy groups (as in compounds 25-28) more than doubled the EC50
values up to 5.9 µM. Contrary to the effect of the acetoxy groups, a substitution of the primary
amino function did not affect cytotoxicities at all. This finding is in perfect agreement with the
results from a previous investigation.[20] Furthermore, most maslinic carboxamides are
significantly higher selective than those derived from oleanolic acid. That can be illustrated by
comparing compounds 15 and 23 (Fig. 4). Both compounds share the same carbon skeleton but
15 is derived from oleanolic while 23 is a derivative of maslinic acid. This small structural
difference led to a significant increase in selectivity from SI = 0.96 (15) to SI = 2.7 (23). This
observation is consistent with previous studies [21-23], that showed maslinic acid derived
derivatives to be of higher selectivity than their oleanolic acid analogs. The presence of an
additional hydroxyl moiety at ring A of the triterpenoid backbone has been assumed as a
possible reason for the increased selectivity. The highest selectivity of the carboxamides 14-28
was observed for maslinic acid derivative 23 and A2780 tumor cells (SI = 3.1) together with a
low EC50 value of 1.5 ± 0.3 µM. Therefore, compound 23 was chosen for some extra biological
investigations. Selectivity indices of all compounds and human tumor cell lines can be found
in the supplementary material (Table 1 and Table 2).
8

Fig. 4: Cytotoxicity of oleanolic (2, 14-20) and maslinic carboxamides (21-28) for HT29 tumor
cells vs. non-malignant mouse fibroblasts (EC50 from SRB assays). The selectivity index (SI)
is defined as: SI = EC50 (NIH 3T3)/EC50 (HT29).
Microscopic images of A2780 cells treated with 23 for 48h showed the presence of some vital
cells, and ruptures in the plasma membrane were noticed (membrane blebbing, marked by white
arrows in Fig. 5). Additionally, some orange stained nuclei could be observed, thus indicating
late-stage apoptotic cells. For quantification of the apoptosis-inducing activity of 23, a flow
cytometry analysis was performed using an annexin V-FITC/PI staining (Fig. 5). The density
plots for the treatment of A2780 cells with 23 for 24 h and 48 h showed significant differences.
After 24 h, 73.8% of the tumor cells were still vital, 12.2% were secondary necrotic/late stage
apoptotic and 12.6% of the cells having died by apoptosis. After 48 h the quantity of vital cells
decreased to 67.7%, while 30.5% of the cells were annexin V-FITC-positive. In addition, the
number of apoptotic cells increased to 18.6 %.
9

Fig. 5: Fluorescence microscopic images: treatment of A2780 cells with 23 (3.0 µM) for 48 h.
Scale bar = 20 µm, AO and PI were used. Flow cytometry analysis: Annexin V-FITC/PI assay,
treatment of A2780 cells with 23 (3.0 µM) for 24 h and 48 h, respectively. Examples of density
plots determined by flow cytometry (Attune® Cytometric Software v. 1.2.5). R1: necrotic, R2:
secondary necrotic/late stage apoptotic, R3: vital, R4: apoptotic.
3
Conclusion
In this study, several carboxamides derived from oleanolic and maslinic acid have been
prepared using various -diamines and N-substituted ethylenediamines. The cytotoxicity of
the compounds was determined employing photometric sulforhodamine B assays, which
showed most of the carboxamides to be cytotoxic with EC50 values below 10 µM. Increasing
chain lengths of the -alkyldiamines seems to reduce EC50 values until a minimum value of
1.2 ± 0.1 µM (6, HT29 tumor cells). While variation of the terminal substituent of ethylene
diamines did not affect the cytotoxicity at all, the presence or absence of an acetyloxy moiety
at the A ring of the triterpenoic backbone had a significantly higher influence, and especially
for maslinic derivatives 21-28 increased cytotoxicity was observed. Carboxamides derived
from maslinic acid were higher selective than their oleanolic acid derived analogs. Compounds
4, 6 and 23 were selected for further investigations regarding their mode of action using dye
10

exclusion acridine orange (AO)/ propidium iodide (PI) assays and flow cytometry analysis.
Incubation of A2780 cells with 6 triggered apoptosis.
4
Experimental Part
Information about technical equipment, experimental procedures and full analytical data of
these compounds can be found in the supplementary material. Oleanolic and maslinic acid
derivatives 14-28 have been synthesized as previously reported.[27]
4.1.1 General procedure A for the acetylation of triterpenoic acids (1, 13)
To a solution of the triterpenoic acid (11 mmol) in dry DCM (150 mL), triethylamine (4.6 mL,
33 mmol), acetic anhydride (3.1 mL, 33 mmol) and DMAP (cat.) were added. After stirring for
two days at 25 °C a saturated solution of NH3 in MeOH was added (3 mL), and the mixture was
stirred for another 30 minutes. Dilution with DCM and subsequent aqueous work-up provided
the crude acetates. Recrystallization from EtOH yielded acetates 1 (90%) and 13 (91%) as
colorless solids; their spectroscopic data were in full agreement with data from the literature.
4.1.2 General procedure B for the synthesis of oleanolic carboxamides (2-12)
To an ice-cold solution of 1 (0.5 mmol) in dry DCM (10 mL), oxalyl chloride (3.2 mmol) and
dry DMF (2 drops) were added. After warming to 25 °C, the mixture was stirred for 15 h. The
solvent was removed under reduced pressure, re-evaporated with dry THF (4 x 15 mL), and the
residue was immediately resolved in dry DCM (10 mL). This mixture was added dropwise to a
solution of the amine (3.0 mmol) in dry DCM (2 mL) and stirred at 25 °C for 2 h. After usual
aqueous work-up, the solvent was removed under reduced pressure, and the crude products
were subjected to column chromatography (silica gel, chloroform/methanol, 9:1). Compounds
2-12 were each obtained as colorless solids.
(3-3-Acetyloxy-olean-12-en-28-oic acid (1)
Compound 1 was prepared according to general procedure A from oleanolic acid. Yield: 90%;
m.p. 259–261 °C (Lit.: 255–257 °C[28]).
(3)-N-(2-Aminoethyl)-3-acetyloxy-olean-12-en-28-amide (2)
11

Compound 2 was prepared from 1 according to general procedure B using ethylenediamine.
Column chromatography (SiO2, CHCl3/MeOH, 9:1) gave 2 (yield: 60%); m.p. 202–205 °C
(decomp.); []D = +54.7° (c 0.310, CHCl3); Rf = 0.20 (CHCl3/MeOH, 9:1); IR (KBr):  =
3406br, 2946s, 2878s, 1734s, 1624m, 1524m, 1466m, 1432m, 1384vs, 1340m, 1314m, 1248s,
1
1214w, 1148w, 1174w, 1148w, 1096w, 1074w, 1028m, 986m cm-1; H NMR (500 MHz,
DMSO-d6):  = 7.49 (dd, J = 5.6, 5.6 Hz, 1H, NH), 5.21 (dd, J = 3.4, 3.4 Hz, 1H, 12-H), 4.38
(dd, J = 11.7, 4.3 Hz, 1H, 3-H), 3.27 (ddd, J = 13.1, 13.1, 6.6 Hz, 1H, 31-Ha), 3.16 (ddd, J =
13.1, 13.1, 6.9 Hz, 1H, 31-Hb), 2.78 (t, J = 7.0 Hz, 2H, 32-H), 2.76 (m, 1H, 18-H), 1.99 (s, 3H,
Ac), 1.93 (ddd, J = 13.3, 13.3, 4.4 Hz 1H, 16-Ha), 1.86 – 1.75 (m, 2H, 2-Ha, 11-Ha), 1.66 (dd, J
= 13.5, 13.5 Hz, 1H, 19-Ha), 1.62 – 1.26 (m, 12H, 1-Ha, 2-Hb, 6-Ha, 6-Hb, 7-Ha, 9-H, 11-Hb, 15-
Ha, 16-Hb, 21-Ha, 22-Ha, 22-Hb), 1.25 – 1.16 (m, 1H, 7-Hb), 1.10 (s, 3H, 27-H), 1.14 – 0.90 (m,
4H, 1-Hb, 15-Hb, 19-Hb, 21-Hb), 0.88 (s, 6H, 25-H, 30-H), 0.87 (s, 3H, 29-H), 0.86 – 0.81 (m,
1H, 5-H), 0.81 (s, 6H, 23-H, 24-H), 0.66 (s, 3H, 26-H) ppm; 13C NMR (126 MHz, DMSO-d6):
 = 177.4 (C-28), 170.1 (Ac), 143.8 (C-13), 121.5 (C-12), 79.9 (C-3), 54.5 (C-5), 46.8 (C-9),
45.9 (C-19), 45.3 (C-17), 41.2 (C-14), 40.4 (C-18), 38.9 (C-8), 38.7 (C-32), 37.5 (C-1), 37.2
(C-4), 36.9 (C-31), 36.4 (C-10), 33.5 (C-21), 32.8 (C-29), 32.6 (C-22), 32.1 (C-7), 30.4 (C-20),
27.7 (C-23), 26.9 (C-15), 25.6 (C-27), 23.6 (C-30), 23.2 (C-2), 22.9 (C-11), 22.2 (C-16), 20.9
(Ac), 17.7 (C-6), 16.7 (C-26), 16.6 (C-24), 15.0 (C-25) ppm; MS (ESI, MeOH): m/z = 541.3
(100%, [M+H]+), 1081.3 (4%, [2M+H]+); analysis calcd for C34H56N2O3 (540.83): C 75.51, H
10.44, N 5.18; found: C75.31, H 10.63, N 5.01.
(3)-N-(3-Aminopropyl)-3-acetyloxy-olean-12-en-28-amide (3)
Compound 3 was prepared from 1 according to general procedure B using 1,3-diaminopropane.
Column chromatography (SiO2, CHCl3/MeOH, 9:1) gave 3 (yield: 50%); m.p. 188–190 °C
(decomp.); []D = +30.5° (c 0.310, CHCl3); Rf = 0.19 (CHCl3/MeOH, 9:1); IR (KBr):  =
3424br, 2948m, 2876w, 1734w, 1628w, 1534w, 1464w, 1432w, 1384vs, 1248w, 1148vw, 1028w
cm-1; 1H NMR (500 MHz, DMSO-d6):  = 7.45 (dd, J = 5.8, 5.8 Hz, 1H, NH), 5.21 (dd, J = 3.4,
3.4 Hz, 1H, 12-H), 4.38 (dd, J = 11.6, 4.3 Hz, 1H, 3-H), 3.14 – 3.00 (m, 2H, 33-Ha, 31-Ha, 31-
Hb), 2.79 (dd, J = 13.3, 3.6 Hz, 1H, 18-H), 2.73 (t, J = 7.4 Hz, 2H, 33-Ha, 33-Hb), 1.99 (s, 3H,
Ac), 1.96 – 1.88 (m, 1H, 16-Ha), 1.84 – 1.73 (m, 2H, 2-Ha, 11-Ha), 1.73 – 1.27 (m, 15H, 1-Ha,
2-Hb, 6-Ha, 6-Hb, 7-Ha, 9-H, 11-Hb, 15-Ha, 16-Hb, 19-Ha, 21-Ha, 22-Ha, 22-Hb, 32-Ha, 32-Hb),
1.25 – 0.89 (m, 5H, 1-Ha, 7-Hb, 15-Hb, 19-Hb, 21-Hb), 1.10 (s, 3H, 27-H), 0.88 (s, 3H, 25-H),
0.88 (s, 3H, 30-H), 0.87 (s, 3H, 29-H), 0.81 (s, 6H, 23-H, 24-H), 0.87 – 0.80 (m, 1H, 5-H), 0.66
(s, 3H, 26-H) ppm; 13C NMR (126 MHz, DMSO-d6):  = 177.1 (C-28), 170.1 (Ac), 144.0 (C-
12

13), 121.4 (C-12), 79.9 (C-3), 54.5 (C-5), 46.9 (C-9), 46.0 (C-19), 45.4 (C-17), 41.3 (C-14),
40.4 (C-18), 38.9 (C-8), 37.6 (C-1), 37.3 (C-4), 36.9 (C-33), 36.5 (C-10), 35.8 (C-31), 33.6 (C-
21), 32.9 (C-22), 32.9 (C-29), 32.2 (C-7), 30.4 (C-20), 27.8 (C-23), 27.5 (C-32), 27.0 (C-15),
25.6 (C-27), 23.6 (C-30), 23.2 (C-2), 22.9 (C-11), 22.2 (C-16), 21.0 (Ac), 17.8 (C-6), 16.9 (C-
26), 16.6 (C-24), 15.1 (C-25) ppm; MS (ESI, MeOH): m/z = 555.4 (100%, [M+H]+), 1109.2
(4%, [2M+H]+) ; analysis calcd for C35H58N2O3 (554.86): C 75.76, H 10.54, N 5.05; found: C
75.50, H 10-74, N 4.87.
(3)-N-(4-Aminobutyl)-3-acetyloxy-olean-12-en-28-amide (4)
Compound 4 was prepared from 1 according to general procedure B using 1,4-diaminobutane.
Column chromatography (SiO2, CHCl3/MeOH, 9:1) gave 4 (yield: 46%); m.p. 149–152 °C;
[]D = +31.6° (c 0.190, CHCl3); Rf = 0.16 (CHCl3/MeOH, 9:1); IR (KBr):  = 3428br, 2946m,
1
2876w, 1736w, 1630m, 1534w, 1452m, 1384m, 1246m, 1092w, 1028m cm-1; H NMR (500
MHz, DMSO-d6):  = 7.32 (dd, J = 5.6, 5.6 Hz, 1H, NH), 5.21 (dd, J = 3.2, 3.2 Hz, 1H, 12-H),
4.38 (dd, J = 11.7, 4.3 Hz, 1H, 3-H), 3.04 (ddd, J =12.7, 12.7, 6.5 Hz, 1H, 31-Ha), 2.96 (ddd, J
=12.8, 12.8, 6.6 Hz, 1H, 31-Hb), 2.79 (dd, J = 13.3, 3.8 Hz, 1H, 18-H), 2.72 (t, J = 7.4 Hz, 2H,
34-Ha, 34-Hb), 1.99 (s, 3H, Ac), 1.95 – 1.85 (m, 1H, 16-Ha), 1.84 – 1.77 (m, 2H, 2-Ha, 11-Ha),
1.66 (dd, J = 13.5, 13.5 Hz, 1H, 19-Ha), 1.66 – 1.16 (m, 17H, 1-Ha, 2-Hb, 6-Ha, 6-Hb, 7-Ha, 7-
Hb, 9-H, 11-Hb, 15-Ha, 16-Hb, 21-Ha, 22-Ha, 22-Hb, 32-Ha, 32-Hb, 33-Ha, 33-Hb), 1.14 – 0.87
(m, 4H, 1-Ha, 15-Hb, 19-Hb, 21-Hb), 1.10 (s, 3H, 27-H), 0.89 (s, 3H, 25-H), 0.88 (s, 3H, 30-H),
0.87 (s, 3H, 29-H), 0.85 – 0.82 (m, 1H, 5-H), 0.81 (s, 6H, 23-H, 24-H), 0.67 (s, 3H, 26-H) ppm;
13C NMR (126 MHz, DMSO-d6):  = 176.2 (C-28), 170.1 (Ac), 144.1 (C-13), 121.2 (C-12),
79.9 (C-3), 54.5 (C-5), 46.9 (C-9), 46.0 (C-19), 45.2 (C-17), 41.3 (C-14), 40.4 (C-18), 38.9 (C-
8), 38.7 (C-34), 38.1 (C-31), 37.5 (C-1), 37.2 (C-4), 36.5 (C-10), 33.6 (C-21), 32.9 (C-29), 32.8
(C-22), 32.2 (C-7), 30.4 (C-20), 27.8 (C-23), 27.0 (C-15), 26.2 (C-33), 25.6 (C-27), 25.0 (C-
32), 23.6 (C-30), 23.2 (C-2), 22.9 (C-11), 22.2 (C-16), 21.0 (Ac), 17.8 (C-6), 16.8 (C-26), 16.6
(C-24), 15.0 (C-25) ppm; MS (ESI, MeOH): m/z = 569.4 (100%, [M+H]+), 1137.5 (10%,
[2M+H]+), 1159,6 (4%, [2M+Na]+); analysis calcd for C36H60N2O3 (568.89): C 76.01, H 10.63,
N 4.92; found: C 75.88, H 10.87, N 4.67.
(3)-N-(5-Aminopentyl)-3-acetyloxy-olean-12-en-28-amide (5)
Compound 5 was prepared from 1 according to general procedure B using 1,5-diaminopentane.
Column chromatography (SiO2, CHCl3/MeOH, 9:1) gave 5 (yield: 49%); m.p. 161–164 °C;
[]D = +39.3° (c 0.330, CHCl3); Rf = 0.20 (CHCl3/MeOH, 9:1); IR (KBr):  = 3424br, 2946s,
13

2876m, 1734m, 1628m, 1532w, 1466w, 1432w, 1384vs, 1318w, 1246m, 1214w, 1188w,
1176vw, 1148w, 1096w, 1074w, 1028w cm-1; 1H NMR (500 MHz, DMSO-d6):  = 7.23 (dd, J
= 5.5, 5.5 Hz, 1H, NH), 5.20 (dd, J = 3.4, 3.4 Hz, 1H, 12-H), 4.39 (dd, J = 11.6, 4.3 Hz, 1H, 3-
H), 3.03 (ddd, J =13.0, 13.0, 7.0 Hz, 1H, 31-Ha), 2.94 (ddd, J =13.0, 13.0, 6.9 Hz, 1H, 31-Hb),
2.81 – 2.76 (m, 1H, 18-H), 2.75 (t, J = 7.7 Hz, 2H, 35-Ha, 35-Hb), 1.99 (s, 3H, Ac), 1.91 (ddd,
J = 14.3, 14.3, 3.2 Hz, 1H, 16-Ha), 1.84 – 1.77 (m, 2H, 2-Ha, 11-Ha), 1.66 (dd, J = 13.4, 13.4
Hz, 1H, 19-Ha), 1.62 – 1.16 (m, 19H, 1-Ha, 2-Hb, 6-Ha, 6-Hb, 7-Ha, 7-Hb, 9-H, 11-Hb, 15-Ha,
16-Hb, 21-Ha, 22-Ha, 22-Hb, 32-Ha, 32-Hb, 33-Ha, 33-Hb, 34-Ha, 34-Hb), 1.10 (s, 3H, 27-H),
1.11 – 0.89 (m, 4H, 1-Ha, 15-Hb, 19-Hb, 21-Hb), 0.89 (s, 3H, 25-H), 0.88 (s, 3H, 30-H), 0.87 (s,
3H, 29-H), 0.86 – 0.83 (m, 1H, 5-H), 0.81 (s, 6H, 23-H, 24-H), 0.67 (s, 3H, 26-H) ppm;
13C NMR (126 MHz, DMSO-d6):  = 176.1 (C-28), 170.1 (Ac), 144.1 (C-13), 121.2 (C-12),
79.9 (C-3), 54.5 (C-5), 46.8 (C-9), 46.0 (C-19), 45.2 (C-17), 41.3 (C-14), 40.4 (C-18), 38.9 (C-
8), 38.8 (C-35), 38.5 (C-31), 37.5 (C-1), 37.2 (C-4), 36.5 (C-10), 33.6 (C-21), 32.9 (C-29), 32.8
(C-22), 32.3 (C-7), 30.4 (C-20), 28.6 (C-32), 27.8 (C-23), 27.0 (C-15), 26.7 (C-34), 25.6 (C-
27), 23.6 (C-30), 23.3 (C-33), 23.2 (C-2), 22.9 (C-11), 22.3 (C-16), 20.9 (Ac), 17.8 (C-6), 16.8
(C-26), 16.6 (C-24), 15.0 (C-25) ppm; MS (ESI, MeOH): m/z = m/z = 583.4 (100%, [M+H]+),
1165.4 (15%, [2M+H]+); analysis calcd for C37H62N2O3 (582.91): C 76.24, H 10.72, N 4.81;
found: C 76.02, H 10.96, N 4.63.
(3)-N-(6-Aminohexyl)-3-acetyloxy-olean-12-en-28-amide (6)
Compound 6 was prepared from 1 according to general procedure B using 1,6-diaminohexane.
Column chromatography (SiO2, CHCl3/MeOH, 9:1) gave 3 (yield: 50%); m.p. 125–129 °C;
[]D = +34.4° (c 0.335, CHCl3); Rf = 0.21 (CHCl3/MeOH, 9:1); IR (KBr):  = 3444br, 2942m,
2864vw, 1734vw, 1634m, 1538vw, 1462vw, 1432vw, 1384vs, 1248w, 1089w, 1028w cm-1;
1H NMR (500 MHz, DMSO-d6):  = 7.22 (dd, J = 5.6, 5.6 Hz, 1H, NH), 5.20 (dd, J = 3.4, 3.4
Hz, 1H, 12-H), 4.38 (dd, J = 11.5, 4.4 Hz, 1H, 3-H), 3.02 (ddd, J = 12.8, 12.8, 6.8 Hz, 1H, 31-
Ha), 2.95 (ddd, J = 12.8, 12.8, 6.9 Hz, 1H, 31-Hb), 2.81 – 2.75 (m, 1H, 18-H), 2.76 (t, J = 7.6
Hz, 2H, 36-Ha, 36-Hb), 1.99 (s, 3H, Ac), 1.89 (ddd, J = 14.9, 14.9, 4.0 Hz 1H, 16-Ha), 1.83 –
1.76 (m, 2H, 2-Ha, 11-Ha), 1.65 (dd, J = 13.4, 13.4 Hz, 1H, 19-Ha), 1.60 – 1.17 (m, 21H, 1-Ha,
2-Hb, 6-Ha, 6-Hb, 7-Ha, 7-Hb, 9-H, 11-Hb, 15-Ha, 16-Hb, 21-Ha, 22-Ha, 22-Hb, 32-Ha, 32-Hb, 33-
Ha, 33-Hb, 34-Ha, 34-Hb, 35-Ha, 35-Hb), 1.09 (s, 3H, 27-H), 1.11 – 0.88 (m, 4H, 1-Hb, 15-Hb,
19-Hb, 21-Hb), 0.88 (s, 3H, 25-H), 0.87 (s, 3H, 30-H), 0.86 (s, 3H, 29-H), 0.86 – 0.80 (m, 1H,
5-H), 0.81 (s, 6H, 23-H, 24-H), 0.67 (s, 3H, 26-H) ppm; 13C NMR (126 MHz, DMSO-d6):  =
14

176.1 (C-28), 170.1 (Ac), 144.2 (C-13), 121.2 (C-12), 79.9 (C-3), 54.6 (C-5), 46.9 (C-9), 46.0
(C-19), 45.2 (C-17), 41.3 (C-14), 40.5 (C-18), 38.9 (C-8), 38.9 (C-36), 38.7 (C-31), 37.6 (C-1),
37.3 (C-4), 36.5 (C-10), 33.7 (C-21), 32.9 (C-29), 32.8 (C-22), 32.3 (C-7), 30.4 (C-20), 29.0
(C-33), 27.8 (C-23), 27.1 (C-34), 27.0 (C-15), 26.1 (C-32), 25.6 (C-27), 25.6 (C-35), 23.6 (C-
30), 23.2 (C-2), 22.9 (C-11), 22.3 (C-16), 21.0 (Ac), 17.8 (C-6), 16.8 (C-26), 16.7 (C-24), 15.0
(C-25) ppm; MS (ESI, MeOH): m/z = 597.5 (100%, [M+H]+), 1193.5 (5%, [2M+H]+); analysis
calcd for C38H64N2O3 (596.94): C 76.46, H 10.81, N 4.69; found: C 76.20, H 11.04, N 4.42.
(3)-N-(7-Aminoheptyl)-3-acetyloxy-olean-12-en-28-amide (7)
Compound 7 was prepared from 1 according to general procedure B using 1,7-diaminoheptane.
Column chromatography (SiO2, CHCl3/MeOH, 9:1) gave 7 (yield: 52%); m.p. 120–124 °C;
[]D = +35.4° (c 0.075, CHCl3); Rf = 0.30 (CHCl3/MeOH, 9:1); IR (KBr):  = 3440br, 2940s,
1
2860m, 1734m, 1628m, 1524m, 1438m, 1384s, 1246m, 1080m, 1028m cm-1; H NMR (500
MHz, DMSO-d6):  = 7.24 (dd, J = 5.5, 5.5 Hz, 1H, NH), 5.21 (dd, J = 3.3, 3.3 Hz, 1H, 12-H),
4.38 (dd, J = 11.5, 4.4 Hz, 1H, 3-H), 3.02 (ddd, J = 13.3, 13.3, 7.2 Hz, 1H, 31-Ha), 2.95 (ddd,
J = 12.8, 12.8, 7.5 Hz, 1H, 31-Hb), 2.78 (dd, J = 13.4, 3.6 Hz, 1H, 18-H), 2.72 (t, J = 7.6 Hz,
2H, 37-Ha, 37-Hb), 1.99 (s, 3H, Ac), 1.94 – 1.84 (m, 1H, 16-Ha), 1.83 – 1.76 (m, 2H, 2-Ha, 11-
Ha), 1.65 (dd, J = 13.5, 13.5 Hz, 1H, 19-Ha), 1.61 – 1.15 (m, 23H, 1-Ha, 2-Hb, 6-Ha, 6-Hb, 7-Ha,
7-Hb, 9-H, 11-Hb, 15-Ha, 16-Hb, 21-Ha, 22-Ha, 22-Hb, 32-Ha, 32-Hb, 33-Ha, 33-Hb, 34-Ha, 34-
Hb, 35-Ha, 35-Hb, 36-Ha, 36-Hb), 1.09 (s, 3H, 27-H), 1.11 – 0.88 (m, 4H, 1-Hb, 15-Hb, 19-Hb,
21-Hb), 0.88 (s, 3H, 25-H), 0.87 (s, 3H, 30-H), 0.86 (s, 3H, 29-H), 0.85 – 0.80 (m, 1H, 5-H),
0.81 (s, 6H, 23-H, 24-H), 0.67 (s, 3H, 26-H) ppm; 13C NMR (126 MHz, DMSO-d6):  = 176.0
(C-28), 170.1 (Ac), 144.2 (C-13), 121.2 (C-12), 79.9 (C-3), 54.6 (C-5), 46.9 (C-9), 46.0 (C-19),
45.2 (C-17), 41.3 (C-14), 40.4 (C-18), 38.9 (C-8), 38.8 (C-37), 38.7 (C-31), 37.6 (C-1), 37.2
(C-4), 36.5 (C-10), 33.7 (C-21), 32.9 (C-29), 32.7 (C-22), 32.3 (C-7), 30.4 (C-20), 29.0 (C-33),
28.3 (C-34), 27.8 (C-23), 27.1 (C-35), 26.9 (C-15), 26.4 (C-32), 25.9 (C-36), 25.6 (C-27), 23.6
(C-30), 23.2 (C-2), 22.9 (C-11), 22.3 (C-16), 21.0 (Ac), 17.8 (C-6), 16.8 (C-26), 16.6 (C-24),
15.0 (C-25) ppm; MS (ESI, MeOH): m/z = 611.1 (100%, [M+H]+), 1222.5 (6%, [2M+H]+);
analysis calcd for C39H66N2O3 (610.97): C 76.67, H 10.89, N 4.59; found: C 76.31, H 11.00, N
4.29.
(3)-N-(8-Aminooctyl)-3-acetyloxy-olean-12-en-28-amide (8)
Compound 8 was prepared from 1 according to general procedure B using 1,8-diaminooctane.
Column chromatography (SiO2, CHCl3/MeOH, 9:1) gave 8 (yield: 38%); m.p. 143–146 °C;
15

[]D = +28.4° (c 0.320, CHCl3); Rf = 0.22 (CHCl3/MeOH, 9:1); IR (KBr):  = 3426br, 2940w,
2860w, 1734w, 1636w, 1532w, 1466w, 1432w, 1384vs, 1246w, 1214vw, 1148vw, 1028w cm-1;
1H NMR (500 MHz, DMSO-d6):  = 7.20 (dd, J = 5.5, 5.5 Hz, 1H, NH), 5.21 (dd, J = 3.2, 3.2
Hz, 1H, 12-H), 4.38 (dd, J = 11.6, 4.3 Hz, 1H, 3-H), 3.02 (ddd, J = 13.1, 13.1, 7.0 Hz, 1H, 31-
Ha), 2.95 (ddd, J = 12.9, 12.9, 7.0 Hz, 1H, 31-Hb), 2.82 – 2.73 (m,3H, 18-H, 38-Ha, 38-Hb), 1.99
(s, 3H, Ac), 1.89 (ddd, J = 14.6, 14.6, 3.6Hz, 1H 16-Ha), 1.83 – 1.77 (m, 2H, 2-Ha, 11-Ha), 1.66
(dd, J = 13.4, 13.4 Hz, 1H, 19-Ha), 1.64 – 1.16 (m, 25H, 1-Ha, 2-Ha, 2-Hb, 6-Ha, 6-Hb, 7-Ha, 7-
Hb, 9-H, 11-Hb, 15-Ha, 16-Hb, 21-Ha, 22-Ha, 22-Hb, 32-Ha, 32-Hb, 33-Ha, 33-Hb, 34-Ha, 34-Hb,
35-Ha, 35-Hb, 36-Ha, 36-Hb, 37-Ha, 37-Hb), 1.10 (s, 3H, 27-H), 1.15 – 0.87 (m, 4H, 1-Hb, 15-
Hb, 19-Hb, 21-Hb), 0.88 (s, 3H, 25-H), 0.87 (s, 3H, 30-H), 0.86 (s, 3H, 29-H), 0.85 – 0.82 (m,
1H, 5-H), 0.81 (s, 6H, 23-H, 24-H), 0.67 (s, 3H, 26-H) ppm; 13C NMR (126 MHz, DMSO-d6):
 = 176.0 (C-28), 170.1 (Ac), 144.2 (C-13), 121.2 (C-12), 79.9 (C-3), 54.5 (C-5), 46.9 (C-9),
46.0 (C-19), 45.2 (C-17), 41.3 (C-14), 40.5 (C-18), 38.9 (C-8), 38.9 (C-38), 38.8 (C-31), 37.6
(C-1), 37.2 (C-4), 36.5 (C-10), 33.7 (C-21), 32.9 (C-29), 32.8 (C-22), 32.3 (C-7), 30.4 (C-20),
29.1 (C-32), 28.7 (CH2), 28.6 (CH2), 27.8 (C-23), 27.0 (C-36), 27.0 (C-15), 26.5 (C-33), 25.8
(C-37), 25.6 (C-27), 23.6 (C-30), 23.2 (C-2), 22.9 (C-11), 22.3 (C-16), 21.0 (Ac), 17.8 (C-6),
16.8 (C-26), 16.6 (C-24), 15.0 (C-25) ppm; MS (ESI, MeOH): m/z = 625.5 (100%, [M+H]+),
1249.5 (8%, [2M+H]+); analysis calcd for C40H68N2O3 (625.00): C 76.87, H 10.97, N 4.48;
found: C 76.58, H 11.18, N 4.17.
(3)-N-(9-Aminononyl)-3-acetyloxy-olean-12-en-28-amide (9)
Compound 9 was prepared from 1 according to general procedure B using 1,9-diaminononane.
Column chromatography (SiO2, CHCl3/MeOH, 9:1) gave 9 (yield: 50%); m.p. 143–146 °C;
[]D = +27.5° (c 0.370, CHCl3); Rf = 0.21 (CHCl3/MeOH, 9:1); IR (KBr):  = 3426br, 2930m,
2858m, 1734w, 1630m, 1530w, 1464w, 1432w, 1384vs, 1246m, 1214w, 1186w, 1148w, 1096w,
1074w, 1028w cm-1; 1H NMR (500 MHz, DMSO-d6):  = 7.20 (dd, J = 5.4, 5.4 Hz, 1H, NH),
5.20 (dd, J = 3.4, 3.4 Hz, 1H, 12-H), 4.38 (dd, J = 11.6, 4.4 Hz, 1H, 3-H), 3.02 (ddd, J = 12.8,
12.8, 6.9 Hz, 1H, 31-Ha), 2.94 (ddd, J = 12.7, 12.7, 6.7 Hz, 1H, 31-Hb), 2.81 – 1.77 (m, 1H, 18-
H), 2.76 (t, J = 7.6 Hz, 2H, 39-Ha, 39-Hb), 1.99 (s, 3H, Ac), 1.89 (ddd, J = 14.3, 14.3, 3.2 Hz,
1H, 16-Ha), 1.83 – 1.77 (m, 2H, 2-Ha, 11-Ha), 1.66 (dd, J = 13.5, 13.5 Hz, 1H, 19-Ha), 1.61 –
1.14 (m, 27H, 1-Ha, 2-Hb, 6-Ha, 6-Hb, 7-Ha, 7-Hb, 9-H, 11-Hb, 15-Ha, 16-Hb, 21-Ha, 22-Ha, 22-
Hb, 32-Ha, 32-Hb, 33-Ha, 33-Hb, 34-Ha, 34-Hb, 35-Ha, 35-Hb, 36-Ha, 36-Hb, 37-Ha, 37-Hb, 38-
Ha, 38-Hb), 1.10 (s, 3H, 27-H), 1.10 – 0.87 (m, 4H, 1-Hb, 15-Hb, 19-Hb, 21-Hb), 0.88 (s, 3H, 25-
H), 0.87 (s, 3H, 30-H), 0.86 (s, 3H, 29-H), 0.86 – 0.82 (m, 1H, 5-H), 0.81 (s, 6H, 23-H, 24-H),
16

0.67 (s, 3H, 26-H) ppm; 13C NMR (126 MHz, DMSO-d6):  = 176.0 (C-28), 170.1 (Ac), 144.2
(C-13), 121.2 (C-12), 79.9 (C-3), 54.5 (C-5), 46.9 (C-9), 46.0 (C-19), 45.2 (C-17), 41.3 (C-14),
40.5 (C-18), 38.9 (C-8, C-39) 38.8 (C-31), 37.5 (C-1), 37.2 (C-4), 36.5 (C-10), 33.7 (C-21),
32.9 (C-29), 32.8 (C-22), 32.3 (C-7), 30.4 (C-20), 29.1 (C-33), 28.9 (CH2), 28.7 (CH2), 28.5
(CH2), 27.8 (C-23), 27.0 (C-37), 26.9 (C-15), 26.6 (C-32), 25.8 (C-38), 25.6 (C-27), 23.6 (C-
30), 23.2 (C-2), 22.9 (C-11), 22.3 (C-16), 21.0 (Ac), 17.8 (C-6), 16.8 (C-26), 16.6 (C-24), 15.0
(C-25) ppm; MS (ESI, MeOH): m/z = 639.5 (100%, [M+H]+), 1277.4 (9%, [2M+H]+); analysis
calcd for C41H70N2O3 (639.02): C 77.06, H 11.04, N 4.38; found: C 76.86, H 11.17, N 4.07.
(3)-N-(10-Aminodecyl)-3-acetyloxy-olean-12-en-28-amide (10)
Compound 10 was prepared from 1 according to general procedure B using 1,10-
diaminodecane. Column chromatography (SiO2, CHCl3/MeOH, 9:1) gave 10 (yield: 46%); m.p.
108–110 °C; []D = +32.5° (c 0.320, CHCl3); Rf = 0.32 (CHCl3/MeOH, 9:1); IR (KBr):  =
3442br, 2928s, 2854m, 1734m, 1636m, 1526m, 1464m, 1370m, 1246s, 1028m cm-1; 1H NMR
(500 MHz, DMSO-d6):  = 7.21 (dd, J = 5.5, 5.5 Hz, 1H, NH), 5.21 (dd, J = 3.2, 3.2 Hz, 1H,
12-H), 4.39 (dd, J = 11.6, 4.4 Hz, 1H, 3-H), 3.03 (ddd, J = 12.6, 12.6, 6.5 Hz, 1H, 31-Ha), 2.94
(ddd, J = 12.7, 12.7, 6.7 Hz, 1H, 31-Hb), 2.78 (dd, J = 13.1, 3.4 Hz, 1H, 18-H), 2.69 (t, J = 7.5
Hz, 2H, 40-Ha, 40-Hb), 1.99 (s, 3H, Ac), 1.89 (ddd, J = 14.2, 14.2, 3.1 Hz, 1H, 16-Ha), 1.83 –
1.77 (m, 2H, 2-Ha, 11-Ha), 1.66 (dd, J = 13.5, 13.5 Hz, 1H, 19-Ha), 1.74 – 1.17 (m, 29H, 1-Ha,
2-Hb, 6-Ha, 6-Hb, 7-Ha, 7-Hb, 9-H, 11-Hb, 15-Ha, 16-Hb, 21-Ha, 22-Ha, 22-Hb, 32-Ha, 32-Hb, 33-
Ha, 33-Hb, 34-Ha, 34-Hb, 35-Ha, 35-Hb, 36-Ha, 36-Hb, 37-Ha, 37-Hb, 38-Ha, 38-Hb, 39-Ha, 39-
Hb), 1.10 (s, 3H, 27-H), 1.12 – 0.88 (m, 4H, 1-Hb, 15-Hb, 19-Hb, 21-Hb), 0.88 (s, 3H, 25-H),
0.88 (s, 3H, 30-H), 0.86 (s, 3H, 29-H), 0.86 – 0.81 (m, 1H, 5-H), 0.81 (s, 6H, 23-H, 24-H), 0.67
(s, 3H, 26-H) ppm; 13C NMR (126 MHz, DMSO-d6):  = 176.0 (C-28), 170.1 (Ac), 144.2 (C-
13), 121.2 (C-12), 79.9 (C-3), 54.5 (C-5), 46.9 (C-9), 46.0 (C-19), 45.2 (C-17), 41.3 (C-14),
40.5 (C-18), 39.4 (C-40), 38.9 (C-8), 38.8 (C-31), 37.5 (C-1), 37.2 (C-4), 36.5 (C-10), 33.7 (C-
21), 32.9 (C-29), 32.7 (C-22), 32.3 (C-7), 30.4 (C-20), 29.1 (C-33), 29.0 (CH2), 28.9 (CH2),
28.9 (CH2), 28.7 (CH2), 28.3 (C-38), 27.8 (C-23), 26.9 (C-15), 26.6 (C-32), 26.0 (C-39), 25.6
(C-27), 23.6 (C-30), 23.2 (C-2), 22.9 (C-11), 22.3 (C-16), 21.0 (Ac), 17.8 (C-6), 16.8 (C-26),
16.6 (C-24), 15.0 (C-25) ppm; MS (ESI, MeOH): m/z = 653.5 (100%, [M+H]+), 1305.5 (5%,
[2M+H]+); analysis calcd for C42H72N2O3 (653.05): C 77.25, H 11.11, N 4.29; found: C 77.04,
H 11.39, N 4.09.
(3)-N-(11-Aminoundecyl)-3-acetyloxy-olean-12-en-28-amide (11)
17

Compound 11 was prepared from 1 according to general procedure B using 1,11-
diaminoundecane. Column chromatography (SiO2, CHCl3/MeOH, 9:1) gave 11 (yield: 36%);
m.p. 162–165 °C; []D = +24.3° (c 0.315, CHCl3); Rf = 0.18 (CHCl3/MeOH, 9:1); IR
(KBr):  = 3426br, 2928m, 2854m, 1734w, 1718w, 1636m, 1526w, 1466w, 1432w, 1384vs,
1246m, 1148 vw, 1028m cm-1; 1H NMR (500 MHz, DMSO-d6):  = 7.20 (dd, J = 5.5, 5.5 Hz,
1H, NH), 5.20 (dd, J = 3.3, 3.3 Hz, 1H, 12-H), 4.38 (dd, J = 11.6, 4.3 Hz, 1H, 3-H), 3.03 (ddd,
J = 12.9, 12.9, 6.8 Hz, 1H, 31-Ha), 2.93 (ddd, J = 12.6, 12.6, 6.7 Hz, 1H, 31-Hb), 2.81 – 2.73
(m, 3H, 18-H, 41-Ha, 41-Hb), 1.99 (s, 3H, Ac), 1.89 (ddd, J = 14.1, 14.1, 2.8 Hz, 1H, 16-Ha),
1.82 – 1.77 (m, 2H, 2-Ha, 11-Ha), 1.65 (dd, J = 13.5, 13.5 Hz, 1H, 19-Ha), 1.61 – 1.16 (m, 31H,
1-Ha, 2-Hb, 6-Ha, 6-Hb, 7-Ha, 7-Hb, 9-H, 11-Hb, 15-Ha, 16-Hb, 21-Ha, 22-Ha, 22-Hb, 32-Ha, 32-
Hb, 33-Ha, 33-Hb, 34-Ha, 34-Hb, 35-Ha, 35-Hb, 36-Ha, 36-Hb, 37-Ha, 37-Hb, 38-Ha, 38-Hb, 39-
Ha, 39-Hb, 40-Ha, 40-Hb), 1.45 – 0.86 (m, 4H, 1-Hb, 15-Hb, 19-Hb, 21-Hb), 1.09 (s, 3H, 27-H,
0.87 (s, 3H, 25-H), 0.87 (s, 3H, 30-H), 0.86 (s, 3H, 29-H), 0.85 – 0.81 (m, 1H, 5-H), 0.80 (s,
3H, 23-H), 0.80 (s, 3H, 24-H), 0.67 (s, 3H, 26-H) ppm; 13C NMR (126 MHz, DMSO-d6):  =
176.1 (C-28), 170.1 (Ac), 144.3 (C-13), 121.2 (C-12), 79.9 (C-3), 54.6 (C-5), 46.9 (C-9), 46.1
(C-19), 45.2 (C-17), 41.3 (C-14), 40.5 (C-18), 38.9 (C-41), 38.9 (C-8), 38.8 (C-31), 37.6 (C-1),
37.3 (C-4), 36.5 (C-10), 33.7 (C-21), 33.0 (C-29), 32.8 (C-22), 32.3 (C-7), 30.4 (C-20), 29.1
(CH2), 29.1 (CH2), 29.0 (CH2), 28.9 (CH2), 29.0 (CH2), 28.6 (CH2), 27.8 (C-23), 27.0 (CH2),
*
*
27.0 (C-15), 26.7 (CK ), 25.9 (CK ), 25.6 (C-27), 23.6 (C-30), 23.2 (C-2), 22.9 (C-11), 22.3 (C-
16), 21.0 (Ac), 17.8 (C-6), 16.9 (C-26), 16.6 (C-24), 15.0 (C-25) ppm; MS (ESI, MeOH): m/z
= 667.5 (100%, [M+H]+), 1333.6 (5%, [2M+H]+); analysis calcd for C43H74N2O3 (667.08): C
77.42, H 11.18, N 4.20; found: C 77.21, H 11.42, N 4.12.
(3)-N-(12-Aminododecyl)-3-acetyloxy-olean-12-en-28-amide (12)
Compound 12 was prepared from 1 according to general procedure B using 1,12-
diaminododecane. Column chromatography (SiO2, CHCl3/MeOH, 9:1) gave 12 (yield: 42%);
m.p. 151–155 °C; []D = +35.9° (c 0.330, CHCl3); Rf = 0.38 (CHCl3/MeOH, 9:1); IR
(KBr):  = 3422br, 2928s, 2854s, 1736m, 1632s, 1528m, 1464s, 1434s, 1384s, 1318s, 1246s,
1214w, 1188w, 1148w, 1096w, 1074w, 1028m, 1008m cm-1; 1H NMR (500 MHz, CDCl3):  =
6.05 (dd, J = 4.8, 4.8 Hz, 1H, NH), 5.37 (dd, J = 3.4, 3.4 Hz, 1H, 12-H), 4.48 (dd, J = 9.9, 6.0
Hz, 1H, 3-H), 3.34 (ddd, J = 13.2, 13.2, 6.9 Hz, 1H, 31-Ha), 3.09 – 3.01 (m, 2H, 42-Ha, 42-Hb),
2.97 (ddd, J = 12.0, 12.0, 6.6 Hz, 1H, 31-Hb), 2.49 (dd, J = 12.7, 2.9 Hz, 1H, 18-H), 2.04 (s,
3H, Ac), 2.00 – 1.85 (m, 3H, 2-Ha, 11-Ha, 16-Ha), 1.80 – 1.14 (m, 36H, 1-Ha, 2-Hb, 6-Ha, 6-Hb,
7-Ha, 7-Hb, 9-H, 11-Hb, 15-Ha, 16-Hb, 19-Ha, 19-Hb, 21-Ha, 21-Hb, 22-Ha, 22-Hb, 32-Ha, 32-Hb,
18

33-Ha, 33-Hb, 34-Ha, 34-Hb, 35-Ha, 35-Hb, 36-Ha, 36-Hb, 37-Ha, 37-Hb, 38-Ha, 38-Hb, 39-Ha,
39-Hb, 40-Ha, 40-Hb, 41-Ha, 41-Hb), 1.15 (s, 3H, 27-H), 1.10 – 0.98 (m, 2H, 1-Hb, 15-Hb), 0.93
(s, 3H, 25-H), 0.90 (s, 3H, 30-H), 0.89 (s, 3H, 29-H), 0.86 (s, 3H, 23-H), 0.85 (s, 3H, 24-H),
0.86 – 0.81 (m, 1H, 5-H), 0.76 (s, 3H, 26-H) ppm; 13C NMR (126 MHz, CDCl3):  = 178.5 (C-
28), 171.2 (Ac), 145.2 (C-13), 122.9 (C-12), 81.0 (C-3), 55.4 (C-5), 47.6 (C-9), 46.9 (C-19),
46.4 (C-17), 42.5 (C-18), 42.2 (C-14), 40.7 (C-42), 39.7 (C-31), 39.5 (C-8), 38.3 (C-1), 37.8
(C-4), 37.0 (C-10), 34.3 (C-21), 33.1 (C-29), 32.5 (C-22), 32.5 (C-7), 30.9 (C-20), 29.6 (CH2),
29.6 (CH2), 29.5 (CH2), 29.5 (CH2), 29.4 (CH2), 29.4 (C-35), 29.2 (CH2), 28.2 (C-23), 27.6
(CH2), 27.4 (C-15), 27.2 (C-34), 26.6 (CH2), 25.8 (C-27), 23.9 (C-16), 23.8 (C-30), 23.7 (C-2),
23.7 (C-11), 21.4 (Ac), 18.3 (C-6), 17.0 (C-26), 16.8 (C-24), 15.6 (C-25) ppm; MS (ESI,
MeOH): m/z = 681.5 (100%, [M+H]+), 1361.6 (5%, [2M+H]+); analysis calcd for C44H76N2O3
(681.10): C 77.59, H 11.25, N 4.11; found: C 77.28, H 11.49, N 3.97.
(2,3-2,3-Diacetyloxy-olean-12-en-28-oic acid (13)
Compound 13 was prepared according to general procedure A from maslinic acid. Yield: 91%;
m.p. 172–175 °C (Lit.: 170–173 °C[23]).
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, but also 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). Financial support by
the ‘‘WissenschaftsCampus Halle WCH” (W13004216 to R.C.) and the Oman Research
Council (ORG/HSS/14/004 to A. A.-H.) is gratefully recognized.
Appendix A. Supplementary material
Supplementary data related to this article can be found, in the online version, at …
References
[1] B.B. Petrovska, Historical review of medicinal plants' usage, Pharmacogn. Rev. 6 (2012) 1-5.
19

[2] R. Verpoorte, Medicinal Plants: A Renewable Resource for Novel Leads and Drugs, in: K.G.
Ramawat (Ed.), Herbal Drugs: Ethnomedicine to Modern Medicine, Springer, Berlin, Heidelberg,
2009, pp. 1-5.
[3] D.J. Newman, G.M. Cragg, Natural Products as Sources of New Drugs from 1981 to 2014, J. Nat.
Prod. 79 (2016) 629-661.
[4] F. Bray, J. Ferlay, I. Soerjomataram, R.L. Siegel, L.A. Torre, A. Jemal, Global cancer statistics
2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries,
Ca-Cancer J. Clin. 68 (2018) 394-424.
[5] P.-J. Anna, R. Aleksandra, R. Maria, Molecular Mechanisms of Biological Activity of Oleanolic
Acid - A Source of Inspiration for A New Drugs Design, Mini-Rev. Org. Chem. 11 (2014) 330-342.
[6] M.P. Montilla, A. Agil, M.C. Navarro, M.I. Jiménez, A. García-Granados, A. Parra, M.M. Cabo,
Antioxidant activity of maslinic acid, a triterpene derivative obtained from Olea europaea, Planta Med.
69 (2003) 472-474.
[7] Y. Allouche, G. Beltrán, J.J. Gaforio, M. Uceda, M.D. Mesa, Antioxidant and antiatherogenic
activities of pentacyclic triterpenic diols and acids, Food Chem. Toxicol. 48 (2010) 2885-2890.
[8] L. Huang, T. Guan, Y. Qian, M. Huang, X. Tang, Y. Li, H. Sun, Anti-inflammatory effects of
maslinic acid, a natural triterpene, in cultured cortical astrocytes via suppression of nuclear factor-
kappa B, Eur. J. Pharmacol. 672 (2011) 169-174.
[9] S. Taniguchi, Y. Imayoshi, E. Kobayashi, Y. Takamatsu, H. Ito, T. Hatano, H. Sakagami, H.
Tokuda, H. Nishino, D. Sugita, Production of bioactive triterpenes by Eriobotrya japonica calli,
Phytochemistry 59 (2002) 315-323.
[10] A. Parra, F. Rivas, P.E. Lopez, A. Garcia-Granados, A. Martinez, F. Albericio, N. Marquez, E.
Muñoz, Solution-and solid-phase synthesis and anti-HIV activity of maslinic acid derivatives
containing amino acids and peptides, Bioorg. Med. Chem. 17 (2009) 1139-1145.
[11] L.Y. Mooi, N.A. Wahab, N.H. Lajis, A.M. Ali, Chemopreventive properties of phytosterols and
maslinic acid extracted from Coleus tuberosus in inhibiting the expression of EBV early‐antigen in
Raji cells, Chem. Biodiversity 7 (2010) 1267-1275.
[12] D.-W. Mu, H.-Q. Guo, G.-B. Zhou, J.-Y. Li, B. Su, Oleanolic acid suppresses the proliferation of
human bladder cancer by Akt/mTOR/S6K and ERK1/2 signaling, Int. J. Clin. Exp. Pathol. 8 (2015)
13864-13870.
[13] Y.Y. Zhu, H.Y. Huang, Y.L. Wu, Anticancer and apoptotic activities of oleanolic acid are
mediated through cell cycle arrest and disruption of mitochondrial membrane potential in HepG2
human hepatocellular carcinoma cells, Mol. Med. Rep. 12 (2015) 5012-5018.
[14] F.J. Reyes, J.J. Centelles, J.A. Lupiáñez, M. Cascante, (2α,3β)-2,3-Dihydroxyolean-12-en-28-oic
acid, a new natural triterpene from Olea europea, induces caspase dependent apoptosis selectively in
colon adenocarcinoma cells, FEBS Lett. 580(27) (2006) 6302-6310.
20

[15] F.J. Reyes-Zurita, G. Pachón-Peña, D. Lizárraga, E.E. Rufino-Palomares, M. Cascante, J.A.
Lupiáñez, The natural triterpene maslinic acid induces apoptosis in HT29 colon cancer cells by a JNK-
p53-dependent mechanism, BMC cancer 11 (2011) 154.
[16] A. Parra, F. Rivas, S. Martin-Fonseca, A. Garcia-Granados, A. Martinez, Maslinic acid
derivatives induce significant apoptosis in b16f10 murine melanoma cells, Eur. J. Med. Chem. 46
(2011) 5991-6001.
[17] C. Li, Z. Yang, C. Zhai, W. Qiu, D. Li, Z. Yi, L. Wang, J. Tang, M. Qian, J. Luo, Maslinic acid
potentiates the anti-tumor activity of tumor necrosis factor α by inhibiting NF-κB signaling pathway,
Mol. Cancer 9 (2010) 73.
[18] D.-M. Wu, D. Zhao, D.-Z. Li, D.-Y. Xu, W.-F. Chu, X.-F. Wang, Maslinic acid induces apoptosis
in salivary gland adenoid cystic carcinoma cells by Ca 2+-evoked p38 signaling pathway, Naunyn-
Schmiedeberg's Arch. Pharmacol. 383 (2011) 321-330.
[19] L. Heller, A. Knorrscheidt, F. Flemming, J. Wiemann, S. Sommerwerk, I.Z. Pavel, A. Al-Harrasi,
R. Csuk, Synthesis and proapoptotic activity of oleanolic acid derived amides, Bioorg. Chem. 68
(2016) 137-151.
[20] M. Kahnt, L. Fischer , A. Al-Harrasi, R. Csuk, Ethylenediamine Derived Carboxamides of
Betulinic and Ursolic Acid as Potential Cytotoxic Agents, Molecules 23 (2018) 2558.
[21] S. Sommerwerk, L. Heller, J. Kuhfs, R. Csuk, Urea derivates of ursolic, oleanolic and maslinic
acid induce apoptosis and are selective cytotoxic for several human tumor cell lines, Eur. J. Med.
Chem. 119 (2016) 1-16.
[22] S. Sommerwerk, L. Heller, J. Kuhfs, R. Csuk, Selective killing of cancer cells with triterpenoic
acid amides - The substantial role of an aromatic moiety alignment, Eur. J. Med. Chem. 122 (2016)
452-464.
[23] B. Siewert, E. Pianowski, A. Obernauer, R. Csuk, Towards cytotoxic and selective derivatives of
maslinic acid, Bioorg. Med. Chem. 22 (2014) 594-615.
[24] S. Sommerwerk, R. Csuk, Convenient and chromatography-free partial syntheses of maslinic acid
and augustic acid, Tetrahedron Lett. 55 (2014) 5156-5158.
[25] S. Sommerwerk, L. Heller, I. Serbian, R. Csuk, Straightforward partial synthesis of four
diastereomeric 2,3-dihydroxy-olean-12-en-28-oic acids from oleanolic acid, Tetrahedron 71 (2015)
8528-8534.
[26] P. Skehan, R. Storeng, D. Scudiero, A. Monks, J. McMahon, D. Vistica, J.T. Warren, H.
Bokesch, S. Kenney, M.R. Boyd, New colorimetric cytotoxicity assay for anticancer-drug screening,
JNCI, J. Natl. Cancer Inst. 82 (1990) 1107-1112.
[27] A. Loesche, M. Kahnt, I. Serbian, W. Brandt, R. Csuk, Triterpene-Based Carboxamides Act as
Good Inhibitors of Butyrylcholinesterase, Molecules 24 (2019) 948.
[28] M.H.L. Wong, H.K. Bryan, I.M. Copple, R.E. Jenkins, P.H. Chiu, J. Bibby, N.G. Berry, N.R.
Kitteringham, C.E. Goldring, P.M. O’Neill, B.K. Park, Design and Synthesis of Irreversible
21

Analogues of Bardoxolone Methyl for the Identification of Pharmacologically Relevant Targets and
Interaction Sites, J. Med. Chem. 59 (2016) 2396-2409.
Highlights
* Oleanolic acid and maslinic acid occur in many plants
* These triterpenoids show promising biological activities
* Their substituted ethylene diamine derived carboxamides are highly cytotoxic
* The alkyl chain length of the diamine units influences the cytotoxicity
* Low EC50 values were observed for these compounds and human tumor cells
* Maslinic acid derivatives are higher selective than oleanolic analogs
* Cell death in the human tumor cells is triggered by apoptosis
Graphical abstract
22

23