Esters and amides of maslinic acid trigger apoptosis in human tumor cells and alter their mode of action with respect to the substitution pattern at C-28

Article abstract of DOI:10.1016/j.ejmech.2013.10.016

Cancer is one of the most commonly diagnosed diseases worldwide; its mortality rate is high, and there is still a demand for the development of antitumor active drugs. Triterpenoic acids show many pharmacological effects, among them antitumor activity. On

Full text of DOI:10.1016/j.ejmech.2013.10.016

European Journal of Medicinal Chemistry 70 (2013) 259e272
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Esters and amides of maslinic acid trigger apoptosis in human tumor
cells and alter their mode of action with respect to the substitution
pattern at C-28
Bianka Siewert, Elke Pianowski, René Csuk*
Bereich Organische Chemie, Martin-Luther-Universität Halle-Wittenberg, Kurt-Mothes-Straße 2, D-06120 Halle (Saale), Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 10 June 2013
Received in revised form
Cancer is one of the most commonly diagnosed diseases worldwide; its mortality rate is high, and there
is still a demand for the development of antitumor active drugs. Triterpenoic acids show many phar-
macological effects, among them antitumor activity. One of these, maslinic acid-1 is of interest because of
its antitumor profile. It is not only cytotoxic but also triggers apoptosis in various human tumor cell lines.
To improve the cytotoxicity of parent 1 we set out to synthesize a series of esters and amides differing in
structure and lipophilicity. These compounds were tested in a sulforhodamine B assay for cytotoxicity,
and screened for their ability to induce apoptosis using an acridine orange/propidium iodide assay, DNA
laddering and cell cycle experiments. Esters containing small-chain, lipophilic residues increased the
cytotoxicity whereas amides as well long-chain esters led to a decrease in activity. The antitumor activity
seems to be independent from the substitution pattern at position C-28 for esters and amides but alters
their mode of action.
1
October 2013
Accepted 7 October 2013
Available online 12 October 2013
Keywords:
Maslinic acid
Antitumor activity
Apoptosis
Structureeactivity relationships
Ó 2013 Elsevier Masson SAS. All rights reserved.
1. Introduction
several groups [3,18e21]. Furthermore, compound 1 exhibits [22]
some selectivity between malignant and non-malignant cell lines,
Cancer is one of the most commonly diagnosed diseases
worldwide. Recent research results [1e5] correlate the low can-
cer mortality rate in Mediterranean countries to a diet rich in
olives. The triterpenoic compounds maslinic acid (1, Scheme 1)
and oleanolic acid seem to be the main antitumor active in-
gredients of olives (Olea europea L.) [1,3,6e8]. Thus, for 1 an
inhibitory activity for glycogen phosphorylases [9,10], protein
tyrosine phosphatase 1B [11] as well as an anti-HIV-1 activity
and selective parasitostatic activities of 1 were established in mice
[23,24].
Structural modifications of triterpenoids have a high impact
onto their biological activity [25]. As compared to other triterpe-
noids, for maslinic acid only a limited number of derivatives is
known, and even less of them have been investigated with respect
to their antitumor activity. Recent studies [26] gave evidence for an
apoptotic effect of 1. To the best of our knowledge, however, only
one report [26] has been published showing a correlation between
the ability to induce apoptosis as a function of structural modifi-
cations e but unfortunately no IC50 values have been reported. In
that study, Parra et al. [26] demonstrated that the ability for
inducing apoptosis increases by “simple” modifications of 1: Thus,
especially amide and benzyl maslinoates induce apoptosis in >90%
of the tumor cells e thus making these compounds interesting
candidates for further development. For betulinic acid several
structureeactivity relationships have been deduced but almost
nothing is known for compound 1 and derivatives thereof [27e29].
Hence, we became interested in a more systematic investigation on
maslinic acid derivatives: Several esters and amides of 1 were
prepared, characterized and their cytotoxicity (IC50 values from
[
12] were reported. Both triterpenoic acids can be extracted in
high amounts from waste [13] of the olive oil production (“olive
pomace”), hence underlining an economical interest connected
with the investigation of the pharmaceutical effects attributed to
these natural products.
Consequently, several groups investigated the effects of maslinic
acid onto cancer cell lines [1,6,8,14e16]. Among others, Reyes et al.
[
8,14,15] could demonstrate that 1 is able to induce cell death by
apoptosis in the colon cancer cell lines HT29 and in Caco-2. In 2007,
was reported to be cytotoxic for the breast cancer cell line MCF7
17], and quite recently these initial findings were confirmed by
1
[
photometric sulforhodamine
determined.
B protein (SRB) assays) were
*
Corresponding author. Tel.: þ49 (0) 345 55 25660; fax: þ49 (0) 345 55 27030.
E-mail address: rene.csuk@chemie.uni-halle.de (R. Csuk).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.10.016

260
B. Siewert et al. / European Journal of Medicinal Chemistry 70 (2013) 259e272
ꢂ
ꢂ
Scheme 1. Synthesis of the esters 2e23 from maslinic acid 1: using DMF, K
2
CO
3
, 25 C, 30 min followed by the addition of Hal-R, 25 C, 12 h.
2
. Chemistry
Maslinic acid-1, a triterpenoic acid, carries three different
Consecutively, a series of homologous amides was synthesized
(
Scheme 2) to investigate the influence of a nitrogen substituent.
Starting from 1, diacetylation furnished a diacetate 24 whose
treatment with thionylchloride followed by the reaction with
amines yielded diacetylated amides 25e29 whose de-acetylation
with potassium carbonate in MeOH gave amides 30e34 in 61e
82% isolated yield.
functional groups: two hydroxyl groups in ring A, a double bond in
ring C and a carboxylic group (C-28). Antitumor screening of de-
rivatives of betulinic acid, oleanolic or ursolic acid revealed the
presence of a C]O moiety to be essential for obtaining good anti-
tumor activity [25e29].
Firstly, we prepared several esters at position C-28. This
carboxyl group is remarkably unreactive, and, Fischer esterification
protocols failed. The esters were obtained by treating 1 in N,N-
dimethylformamide (DMF) or acetone with alkyl bromides in the
presence of freshly grounded potassium carbonate. An aqueous
work-up using 5% cold aqueous hydrochloric acid facilitated the
isolation of the products by destroying some byproducts [30]. Thus,
following this general procedure, aliphatic esters (Scheme 1) were
synthesized in good to excellent yields (75e99%), and esters con-
taining side chains of different lengths (2,3,6,13,20e21) as well as
halogenated (5, 10e12, 16e19) and hydroxylated (e.g. 4) esters were
obtained.
3. Biology
Photometric SRB assays [33] were performed, the results of
which are compiled in Table 1. Thus, IC50 values (Table 1) were
determined for six different human cancer cell lines and non-
malignant mouse fibroblasts (NIH 3T3).
By and large, esterification of parent 1 results in an increase of
cytotoxicity. As shown for three different human cancer cell lines
[A2780 (ovarian carcinoma), HT29 (colon adenocarcinoma) and
MCF7 (breast adenocarcinoma)] short alkyl esters of 1 up to a
maximum length of six carbons possess a higher cytotoxicity than
parent 1 (Fig. 1). Activity is lost, however, for long chain esters (e.g.
20e22).
The allyl ester of 2-cyano-3,12-dioxooleana-1,9(11)dien-28-acid
(CDDO) has previously been shown to possess a five-fold higher
inhibitory activity for the production of nitric oxide NO in mouse
macrophages than a homologous butyl ester, whereas a significant
drop in activity was determined for the corresponding CDDO ethyl
ester [34]. Recently, we were able to show for several derivatives of
glycyrrhetinic acid that unsaturated esters exhibit higher cytotox-
icity towards several human tumor cell lines as compared to their
saturated analogs. However, comparison of the IC50 values from the
SRB tests using the estrogen receptor positive breast cancer cell line
MCF7 showed no significant differences in the cytotoxicity for the
allyl (7), propargyl (8) or but-3-enyl (14) ester as compared to
parent 1. A drop in activity was observed for the alkynyl ester 15.
The structures of the esters 2e23 were confirmed by extensive
analysis. Thus, the mass spectrometric analysis revealed for all es-
ters the presence of a characteristic methyl oleanoate decomposi-
tion signal m/z ¼ 203 [31,32]. Also, the elimination of two
molecules of water followed by a subsequent splitting off of two
13
molecules of alcohol was observed (m/z ¼ 409). All C NMR signals
for the triterpenoic skeleton in this series of compounds were
similar with exception of the signal for C-28 (and the additional
signals for the alcohol part of the ester). For C-28 a shift to higher
field for the esters was observed as compared to parent 1
(
d
¼ 180.2 ppm for 1 to
d
¼ 177.8 ꢀ 0.47 ppm for the esters). In
addition, for the alkynic derivatives 8 and 15 in the IR spectra the
typical bands for alkynes were found at
respectively.
¼ 3314 and 3296 cmꢁ
1
,
n

B. Siewert et al. / European Journal of Medicinal Chemistry 70 (2013) 259e272
261
ꢂ
ꢂ
ꢂ
ꢂ
0
Scheme 2. a) Ac
2
O, pyridine, TEA b) DCM/THF, SOCl
2
, 0 C / 25 C, 30 min c) DCM, RNH
2
, 25 C, 12 h d) K
2
CO
3
, MeOH, 25 C, 8 h; R ¼ H (25), ethyl (26), propyl-27, propenyl (28),
propargyl-29; R ¼ H (30), ethyl (31), propyl (32), propenyl (33), propargyl (34).
Following these observations we assumed that the improve-
ments in cytotoxicity following the introduction of an ester moiety
might be explained by differences in the transport of the com-
pounds through the cell membrane. In addition, the overall lip-
ophilicity of the C-28 ester seems to be more important for activity
than individual modifications at this functional group. Thus, the
IC50 values of several halogenated analogs of the propyl 6 as well as
of the butyl ester 13 are similar; these substituents exhibited no
strong impact onto the cytotoxicity of the compounds. By and large,
these halogenated analogs showed similar cytotoxicity as their
non-halogenated analogs. Interestingly, introducing additional
polar groups (as exemplified in the 2-hydroxyethyl ester 4) resulted
in a complete loss of activity in melanoma cells (518A2), thyroid
carcinoma (8505C) and lung cancer (A549) cells. The same is true
for the butinyl analog 19. Promising results could be seen for the 4-
chloro-butyl compound 16 whereas a drop of activity was observed
for the chlorobutinyl derivative 19.
Several amides of 1 [26] are able to induce apoptosis; this par-
allels previous findings for CDDO derivatives [34]. Contrary to the
results for the esters, introduction of a lipophilic moiety resulted in
a significant loss of cytotoxicity for the amides. The propyl deriva-
tive 32 was shown to be significantly more potent than the ethyl
derivative 31. Introducing moieties containing C]C double bonds
led to a decrease in activity. It seems that esters exhibit a 2e3 fold
higher cytotoxicity than the amides. These findings may be the
result either of different transport mechanisms of the compounds
through the membrane or of a different integration into the cell
membrane [35,36]. The effects of several analogs of betulinic acid
onto the membrane of erythrocytes has been studied several years
ago [37]. Compounds possessing a hydrogen bond donor group (for
the cell cycle by FACS-analysis [38]. These experiments revealed
(Fig. 2) that the compounds induced a significant G1/G0 arrest in
the living, adherent growing cells after 24 h. After 48 h, a further
increase of cells in the G1/G0 phase with a total reduction of cells in
the S as well as the G2/M phase was observed. Hence, these analogs
of 1 are potent cell proliferation stopping agents.
Controlled dying of cells is characterized by an intact cell
membrane until the middle phases of the death process [39e42].
The combination of a cell impermeable dye (e.g. propidium iodide,
PI) and a permeable dye (e.g. acridine orange, AO) [42] in a fluo-
rescence microscopy based assay allows quantitative information
concerning the different modes of cell death. In this AO/PI assay the
cells were treated with maslinic acid derivatives possessing a three
carbon chain in the ester or amide moiety, and a green fluorescence
(Fig. 3) was observed; hence a controlled cell death had been
induced. A hallmark of apoptosis is the activation of caspase-
activated DNAse (CAD) by caspase 3 [43]. As a result, DNA is cut
into several smaller pieces of 178 bps length. Using gel
Table 1
Cytotoxicity (IC50 values in microM, from SRB assays after 96 h of treatment; the
values are averaged from at least
3 independent experiments; confidence
interval ¼ 95%, individual positive (upper value) and negative (lower value) errors
are given in the supplementary part; n.D. not detected) for maslinic acid derivatives
1
e23, 30e34 and betulinic acid (BA, as a standard) using a panel of various cancer
cell lines (518A2, 8505C, A2780, A549, HT29 and MCF7) and non-malignant mouse
fibroblasts NIH 3T3.
518A2
8505c
A2780
A549
HT29
MCF7
NIH 3T3
BA
1
2
3
4
5
6
7
8
11.9
13.7
15.6
11.1
32.5
16.9
13.9
14.2
20.4
31.6
12.4
19.4
12.8
12.2
13.3
22.7
8.4
6.7
17
11.0
19.5
17.3
12.6
13.4
10.2
14.1
n.d
14.9
23.4
17.8
13.7
32.5
19
13.2
14.2
17.3
30.3
14
20.6
13.8
17.5
12.8
18.6
12.2
12.7
27.4
9.9
14.4
135.6
104.1
10.5
>60
37.3
23.4
37
n.d.
28.8
12.8
12
14.8
24.4
16.3
8.8
10.0
16.6
21.4
12.1
>30
14
13.4
15.1
18.1
32.6
17.3
21.6
12.3
16
14.1
26.1
12.9
12.9
33.1
12.6
14.1
>120
56.9
11.9
47.8
14.2
17.6
24.8
26.1
example COOH, CONH
2
or CHOH) at position C-28 acted echino-
14.7
12.2
>30
18.9
13.8
12.4
14.9
37.3
11.6
22.2
13.5
13.7
7.1
cytogenic, whereas esters, aldehydes and lupeol resulted in the
formation of stomatocytes. The latter were formed when the
incorporation took place predominantly into the inner leaflet. The
amides 30e34, parent 1 as well as inactive 4 and 9 carry a hydrogen
bond donor group whereas all of the active compounds of our study
are devoid of this function.
The amide 30 exhibited IC50 values comparable to those of 1
employing the malignant human tumor cell lines A549 (human
lung carcinoma) and HT29. This compound showed a significantly
higher cytotoxicity towards tumor cell lines than towards the non-
malignant mouse fibroblasts. Furthermore, the introduction of a
saturated, bulky cyclohexylmethyl residue (as in 23) led to prom-
ising results; for example for the ovarian cancer cell line an IC50
24.1
14.9
12.8
14.7
12.6
30.9
11.4
16.4
14.3
11.5
10.1
9.8
24
17.8
13.5
15.1
14.3
36
13.8
21.1
13.4
13.1
12.7
19.7
8.6
7.8
9
18.1
12.1
n.d.
11.7
17.7
8
10.1
6.5
5.9
1
1
1
0
1
2
13
14
1
1
1
1
5
6
7
8
26.3
8.5
13.2
29.1
9.2
8.9
n.D
13.1
27.9
12.4
12.4
111
38.5
12.8
23.4
7.7
22.5
8.5
13
20.5
8.5
13.3
107
75.7
9.3
43.7
37.7
25.2
37.6
29.5
value of 3.6
m
M was observed.
19
20
21
22
23
7.2
13
The IC50 values from the SRB assays show the cytotoxicity of a
compound but these tests don’t indicate whether the cell death
occurs via apoptosis or necrosis. To investigate the compounds for
their ability to induce apoptosis or for a stop of cell growing,
additional cell cycle investigations (after incubation periods of 24
and 48 h) were performed.
141.3
>90
5.9
61.3
45.2
28.1
42.5
27.7
80.3
93.8
3.6
29.7
27.4
18.3
25.8
21.9
67.7
123.4
8.2
81.1
29.1
13.9
28.2
24.6
8
30
31
41.2
42.1
29.1
39.3
24.5
3
3
3
2
3
4
Treatment of a cell population with a specific and proportionally
DNA-binding fluorescence dye allows measuring the distribution of
29.8

262
B. Siewert et al. / European Journal of Medicinal Chemistry 70 (2013) 259e272
pattern at position C-28 for esters and amides; modifications at this
position determine their mode of action. Of special interest is ethyl
ester 3 due to its pronounced cytotoxicity for HT29 cells whereas
compounds 8 and 14 exhibited good cytotoxicity against the human
ovarian cancer cell line A2780.
5. Experimental
5.1. General
Reagents were bought from commercial suppliers without any
further purification. Melting points were measured with a LEICA
hot stage microscope and were not corrected, NMR spectra were
recorded using the Varian spectrometers Gemini 2000 or Unity 500
4
(d given in ppm, J in Hz, internal Me Si), IR spectra (film or KBr
pellet) on a PerkineElmer FT-IR spectrometer Spectrum 1000. MS
spectra were taken on a Finnigan MAT TSQ 7000 (electrospray,
voltage 4.5 kV, sheath gas nitrogen) instrument. Optical rotations
were taken on a PerkineElmer 341 polarimeter (1 cm micro cell,
ꢂ
20
C) and UVevis spectra on a PerkineElmer unit, Lambda 14.
Fig. 1. Comparison of IC50 values (from SRB assays after 96 h of incubation with several
human tumor cell lines, confidence interval ¼ 95%) for compounds 1e3, 6, 13, 20e22.
Elemental analyses were measured on a FosseHeraeus Vario EL
unit and correct elemental analyses were obtained for all com-
pounds (C, H, and N where appropriate, max. deviation from
theoretical value < 0.3%). TLC was performed on silica gel (Merck
5554, detection by UV absorption). The solvents were dried ac-
cording to usual procedures. The purity of the compounds was
>95% as checked by HPLC. Atoms were numbered according to
usual IUPAC numbering for triterpenoids. The assignments of the
NMR signals attribution was realized using a combination of several
2D NMR techniques (H, H-COSY, H, H-NOESY, gHMBC and gHSQC).
Carbon shifts for all compounds are summarized in Tables 2e4.
electrophoresis and staining, the typical “DNA-ladders” are found.
The results from these DNA-laddering experiments are depicted in
Fig. 3, too. These assays, however, do not allow quantifications.
Hence, an annexin-V/PI assay was performed [44]. In these exper-
iments, the whole cell population was treated with fluorescence
labeled phosphatidyl-serine specific binding protein annexin-V and
PI. Quantitative FACS analysis allowed to distinguish vital from
necrotic or apoptotic or secondary necrotic cells. As a result, most of
the dead cells showed secondary necrosis after having been incu-
bated for 6 h. Hence, the outer leaf of the membrane carries the
phosphatidyl-serine (as expected for an apoptosis), and the integ-
rity of the membrane is lost (as typical for late apoptotic events).
These results suggest that either apoptosis is induced slowly or the
integrity of the cell membrane might be altered upon incubation of
the cells with the compounds.
To investigate whether the apoptotic process is caspase-
dependent an extra colorimetric assay [42] was applied. The cell
lysate collected from treated as well as untreated cell populations
was reacted with p-nitroaniline-containing tetra-peptides. The
changes in the optical density (Fig. 4) were measured after 12 h of
incubation. In this experiment, a high concentration of an aspartate
specific cutting caspase correlates with a high optical density. For 1
a low optical density was found after an incubation period of 12 h.
For compounds 1 and 8 caspases 3, 8 and 9 were shown to be
activated.
5.2. Biological assays
5.2.1. SRB-assay
SRB-Assays were prepared according to ref [33]. The cell lines
518A2, 8505C, A549, A2780, HT29, MCF7 and NIH 3T3 were
amplified with a starting cell number of 2000, 2500, 1500, 1000,
2000, 2000 and 2500, respectively and counted with an automatic
Ò
cell counter (InvitrogeneÔ countess ). IC values were calculated
50
from semi logarithmic dose response curves by non-linear regres-
sion applying a two parametrical Hill-slope equation [45]. Values
are given with a confidence interval CI ¼ 95% (cf. Supplementary
material).
5.2.2. Acridine orange/propidium iodide dye exclusion assay
Approximately 500,000 cells (HT29) were seeded in cell culture
flasks and were allowed to grow for 1 day. After removing of the
medium, the substance loaded medium was added, and the flasks
were incubated for about 24e48 h. The supernatant medium was
collected and centrifuged; the cell pellet was suspended in PBS and
Triterpenoic acids are known to intercalate into isolated mem-
branes [35,36,38]. These intercalation processes take place either in
the upper or deeper fatty acid regions of the cell membrane
depending on the log P value of the intercalants. Hence, the entering
of PI into cells might occur via gaps in the membrane being formed
by an intercalation between compounds and the membrane.
centrifuged again. Equal amounts of a PI/AO solution (1:1, 100 mg/
2
þ
2þ
ml) and a suspension of the cell pellet in PBS (w/o Ca and Mg )
were softly mixed. Visual analysis was performed under a fluo-
rescence microscope (Axioskop II (Carl Zeiss, Jena). While green
fluorescence shows apoptosis, a deep colored nucleus indicates
necrotic cells; weak orange dots mark lysosomes [39,40].
4
. Summary
Starting from industrial waste a series of different esters and
amides of maslinic acid was synthesized. Biological screening of
these compounds showed for esters containing small-chain, lipo-
philic residues an increase in cytotoxicity whereas for amides as
well as for long-chain esters a decrease was observed. Results from
a dye exclusion assay, from DNA laddering experiments, from a
caspase assay as well from annexin-V binding studies indicate that
the cytotoxicity seems to be independent from the substitution
5.2.3. Cell cycle investigations
Approximately 1,000,000 cells (HT29) were seeded in cell cul-
ture flasks and were allowed to grow for 1 day. After removing of
the medium, the substance loaded medium was added, and the
flasks were incubated for about 24e48 h. The living cells were
collected by centrifugation (1200 rpm, 5 min) and washed twice
(PBS, w/o). After careful fixation with ice cold ethanol (70%, min. 2

B. Siewert et al. / European Journal of Medicinal Chemistry 70 (2013) 259e272
263
Fig. 2. Upper part: Cell cycle distribution after 24 h treatment of HT29 cells with maslinic acid 1 or derivatives 6, 8 and 32. Lower part: Relative changes in the cell cycle of HT29
colon cancer cells after treatment for 24 h and 48 h respectively with MA (1, 30 M), propyl maslinoate (6, 15 M), propargyl (8, 30 M), 3-chloropropyl (10, 15 M), maslinic amide
30, 60 M), propyl amide (32, 60 M) and propargyl amide (34, 60 M).
m
m
m
m
(
m
m
m
h), the cells were centrifuged (1200 rpm, 5 min) again, washed
5.2.6. General procedure for the synthesis of the diacetylated
amides (method B)
twice with staining buffer (PBS (w/w), fetal bovine, sodium azide)
ꢂ
and treated with RNAse (100
mL, 100
m
g/mL, 30 min) at 37 C. Then,
To a solution of 24 (1 equiv) in dry DCM/THF (50 mL, 1:1, v/v),
thionyl chloride (1.1 equiv) and triethylamine (1.3 equiv) were
at room temperature PI (1 mg/ml, 30 min) was added in the dark.
Analysis was performed with using an ABÔ Attune FACS machine
and calculated with FCS express using the method described by
Dean [46].
ꢂ
ꢂ
added at 0 C, stirring at 0 C was continued for 20 min. After stirring
ꢂ
at 25 C for several hours (until TLC showed completion of the re-
action), the solvents were removed under reduced pressure, the
residue was dissolved in DCM, and the corresponding amine (1.3
equiv) and triethylamine (1.3 equiv) were added. After completion
of the reaction (as checked by TLC), the mixture was poured into ice
water, the precipitate was filtered off, washed with cold water and
purified by chromatography (silica gel, n-hexane/ethyl acetate).
5.2.4. Annexin V/PI assay
Approximately 1,000,000 cells (HT29) were seeded in cell cul-
ture flasks and were allowed to grow for 1 day. After removing of
the medium, the substance loaded medium was added, and the
flasks were incubated for about 6 h. All cells were harvested,
centrifuged (1200 rpm, 5 min) and washed twice (PBS, w/w).
5.2.7. General procedure for deacylation (method C)
Approximately 100,000 cells were washed with annexin
bounding buffer (BD) and treated in the dark with propidium io-
dide (5 L, life technologiesÔ) for 15 min at room temperature.
After adding of the annexin V bounding buffer (400 L), the sus-
V
The diacetylated amides were dissolved in methanol, and KOH
(1.2 equiv) was added; the mixture was stirred at room tempera-
ture until TLC showed completion of the reaction. Usual workup
followed by chromatography (silica gel, n-hexane/ethyl acetate
mixtures) yielded the amides.
m
m
pension was subjected to a FACS measurement. Calculation of the
Ò
data was performed as described in the suppliers (BD Biosciences )
manual.
5.2.7.1. Maslinic acid (1). Maslinic acid was isolated from olive
pomace as reported in Refs. [47]; after recrystallization from ethyl
ꢂ
5
.2.5. General procedure for the synthesis of the esters (method A)
Compound 1 (1 equiv) was dissolved in dry DMF (5 mL), and
acetate 1 was obtained as a colorless solid; m.p. 263e267 C, (lit.:
ꢂ
266e269
C
[9]);
R
F
¼
0.22 (n-hexane/ethyl acetate, 6:4);
ꢂ
ꢂ
finely grounded potassium carbonate (5 equiv) was added. After
stirring for 1 h at room temperature, the alkyl bromide (2 equiv)
was added, and stirring was continued for another 18 h. The
mixture was poured into an ice cold solution of aq. hydrochloric
acid (5%, 50 mL), and the white precipitate was filtered off. Its
chromatographic purification (silica gel, n-hexane/ethyl acetate,
[
a
]
D
¼ þ55 (c 0.42, CHCl
3
) (lit.: [
a
]
D
¼ þ60 (c 0.1, CHCl
3
) [48]). MS
H] ), 517.0 (100%,
ꢁ
(ESI, MeOH): m/z
¼
471.5 (43%, [M
ꢁ
ꢁ
ꢁ
[M þ HCO
2
] ), 943.1 (62%, [2M ꢁ H] ).
5.2.7.2. Methyl (2
a, 3b) 2,3-dihydroxy-olean-12-en-28-oate (2).
Obtained from 1 (100 mg, 0.21 mmol) by method A as fine colorless
needles; yield: 88%; m.p. 229e231 C (lit.: 214e216 C [49]);
ꢂ
ꢂ
7:3) and recrystallization (ethanol) yielded the product in an
ꢂ
analytically pure form.
R
F
¼ 0.4 (n-hexane/ethyl acetate, 6:4); [
a]
D
¼ 60.9 (c 6.5, CHCl
3
); IR

264
B. Siewert et al. / European Journal of Medicinal Chemistry 70 (2013) 259e272
Fig. 3. Left part: Dye exclusion assay and DNA-ladder (HT29, colon cancer cell line): Cells were treated with 1 (30
mM), 6 (15
mM), 7 (30
mM), 8 (30
m
M), 10 (15
mM), 32 (60
m
M), 33
(
60 M), 34 (60 M). Right part: Annexin V assay (HT29, colon cancer cells): Cells were treated for 6 h with 1 (30
m
m
m
M), 6 (15
mM), 8 (30
mM) and 32 (60
mM).
þ
þ
þ
2þ
(
KBr)
n
¼ 3571, 3300, 2947, 1739, 1461, 1386, 1363, 1262, 1229, 1190,
[M þ H] ), 504.5 (53.4%, [M þ NH
4
] ), 509.5 (100%, [M þ Na] ),
ꢁ1
1
þ
1
162, 1124, 1052, 1037, 984, 958, 921 cm
;
H NMR (500 MHz,
¼ 5.28 (dd, J ¼ 3.4, 3.4 Hz, 1H, H-12), 3.72e3.65 (m, 1H, H-
-31), 3.00 (d, J ¼ 9.4 Hz, 1H, H-3), 2.85 (dd,
J ¼ 13.8, 4.1 Hz,1H, H-18), 2.21 (br, 2H, OH),1.97 (dd, J ¼ 10.4, 3.9 Hz,
H, H -1), 1.96e1.83 (m, 3H, H
-16 þ Ha,b-11), 1.68 (ddd, J ¼ 13.9,
3.9, 4.4 Hz, 1H, H -19 þ H -15 þ H
-7), 1.64e1.56 (m, 4H, H-9 þ H
6), 1.55e1.52 (m, 1H, H -22),
-6), 1.50 (ddd, J ¼ 14.0, 3.4, 3.4 Hz, H
.43 (m, 1H, H -6), 1.29 (ddd,
-7), 1.38 (ddd, J ¼ 12.4, 12.4, 2.5 Hz, H
J ¼ 13.8, 9.6, 2.7 Hz, 1H, H -21), 1.29e1.27 (m, 1H, H -22), 1.16 (ddd,
J ¼ 14.0, 4.2, 4.2 Hz, 1H, H -21), 1.15e1.11 (m, 1H, H -19), 1.12 (s, 3H,
CH eC27), 1.06e1.02 (m, 1H, H -15), 1.02 (s, 3H, CH -23), 0.97 (s,
H, CH -25), 0.92 (s, 3H, CH -30), 0.92e0.87 (m, 1H, H -1), 0.89 (s,
H, CH -29), 0.84e0.82 (m, 1H, H-5), 0.82 (s, 3H, CH -24), 0.71 (s,
H, CH
-26) ppm; MS (ESI, MeOH, source CID): m/z ¼ 487.4 (48.8%,
541.2 (22.0%, [M þ Na þ MeOH] ), 741.5 (14.6%, [3M þ Na þ H] ),
2
þ
2þ
CDCl
2
3
):
d
749.6 (73.2%, [3M þ K þ H] ), 992.8 (48.8%, [4M þ K þ H] ).
), 3.61 (s, 3H, CH
3
5.2.7.3. Ethyl (2 2,3-dihydroxy-olean-12-en-28-oate (3).
a,
3b)
1
1
1
1
a
a
Obtained from 1 by method A as a colorless solid; yield: 86%; m.p.
ꢂ
ꢂ
a
a
a
b
-
223e224 C; R
(c 6.2, CHCl ); IR (KBr)
1320, 1302, 1258, 1180, 1161, 1126, 1098, 1081, 1050, 1037 cm ; H
NMR (500 MHz, CDCl ):
¼ 5.26 (dd, J ¼ 3.4, 3.4 Hz, 1H, H-12), 4.06
(m, 2H, CH
-31), 3.67 (m, 1H, H-2), 2.98 (d, J ¼ 9.3 Hz, 1H, H-3), 2.84
(dd, J ¼ 13.8, 4.1 Hz, 1H, H-18), 2.10e2.00 (br, 2H, OH), 1.95 (dd, 1H,
J ¼ 14.8, 3.2 Hz, H -1), 1.95e1.82 (m, 3H, H -16 þ Ha,b-11), 1.68 (ddd,
J ¼ 13.7, 13.7, 4.3 Hz, 1H, H -7), 1.64e1.54 (m, 4H, H-9 þ H -19 þ H
15 þ H -16), 1.54e1.47 (m, 1H, H -6), 1.50e1.43 (m, 1H, H -7), 1.44e
.40 (m, 1H, H -22), 1.38e1.33 (m, 1H, H -6), 1.32e1.24 (m, 2H, H
F
¼ 0.4 (n-hexane/ethyl acetate, 6:4); [
a
]
D
¼ þ201.5
a
a
3
n
¼ 3609, 3363, 2948, 1723, 1463, 1387, 1365,
ꢁ
1 1
b
b
a
b
3
d
b
b
2
3
b
3
3
3
3
3
3
b
a
a
3
3
a
a
a
-
3
b
a
b
1
a
b
a
-
-
2
3
1 þ H
b
-22), 1.24e1.15 (m, 1H, H
b
-21), 1.20 (t, J ¼ 7.1 Hz, 3H, CH
3
2), 1.16e1.10 (m, 1H, H
-15), 1.01 (s, 3H, CH
0), 0.92e0.86 (m, 1H, H
H, H-5), 0.80 (s, 3H, CH
b
-19), 1.11 (s, 3H, CH
3
-27), 1.06e1.00 (m, 1H,
-25), 0.90 (s, 3H, CH
-1), 0.88 (s, 3H, CH -29), 0.84e0.82 (m,
-24), 0.72 (s, 3H, CH
H
b
3
-23), 0.96 (s, 3H, CH
3
3
-
3
1
b
3
3
3
-26) ppm; MS (ESI,
þ
MeOH, source CID): m/z ¼ 523.6 (100%, [M þ Na] ), 555.1 (19.5%,
þ
2þ
2þ
[
[
M þ Na þ MeOH] ), 770.5 (75%, [3M þ K þ H] ), 1020.8 (38.7%,
2
þ
4M þ K þ H] ), 1024.2 (36.4%, [4Mþ2Na þ H] )).
5
.2.7.4. 2-Hydroxyethyl (2 , 3 ) 2,3-dihydroxy-olean-12-en-28-oate
a
b
(
4). Obtained from 1 by method A as a colorless solid; yield: 84%;
ꢂ
m.p. 224e226 C;
R
F
¼ 0.08 (n-hexane/ethyl acetate, 6:4);
ꢂ
[a
]
D
¼ 57.1 (c 3.5, CHCl
3
); IR (KBr)
n
¼ 3422, 2948, 2866,1706,1048,
ꢁ
1 1
1
458, 1034, 1176, 1718, 754,1262, 1162, 1388, 1080, 1364 cm
; H
NMR (500 MHz, CDCl
3
):
d
¼ 5.30 (dd, J ¼ 3.4, 3.4 Hz, 1H, H-12), 4.19
-31), 4.13 (ddd, J ¼ 11.8, 5.4, 3.6 Hz,
-31), 3.79 (dd, J ¼ 5.3, 3.8 Hz, 2H, CH -32), 3.68 (ddd,
J ¼ 10.7, 10.7, 3.6 Hz, 1H, H-2), 2.99 (d, J ¼ 9.3 Hz, 1H, H-3), 2.87 (dd,
(ddd, J ¼ 11.7, 5.8, 3.8 Hz, 1H, H
a
1
H, CH
b
2
Fig. 4. Relative activity of caspases 3, 8 and 9 after treatment of HT29 colon-cancer
cells with 30 M of MA(1) or propargyl maslinoate (8) for 6, 12 and 24 h, respectively.
m

B. Siewert et al. / European Journal of Medicinal Chemistry 70 (2013) 259e272
265
Table 2
1
3C NMR shifts (100 MHz, CDCl
3
) for compounds 1e12.
1
2
3
4
5
6
7
8
9
10
46.5
11
46.5
12
46.5
C-1
C-2
C-3
C-4
C-5
C-6
C-7
C-8
46.6
68.6
83.8
39.8
55.9
18.8
33.2
39.8
48.1
38.5
23.9
46.3
68.9
84.0
39.1
55.3
18.3
32.5
39.3
47.6
38.3
23.0
122.1
143.8
41.6
27.6
23.4
46.7
41.2
45.8
3.7
33.8
32.3
28.6
16.7
16.5
16.9
25.9
178.2
33.1
23.6
51.5
46.3
68.9
84.0
39.1
55.3
18.3
32.3
39.3
47.6
38.3
23.0
122.1
143.8
41.6
27.6
23.4
46.7
41.2
45.8
30.7
33.8
32.5
28.6
16.7
16.6
16.9
25.9
178.2
33.1
23.6
51.5
46.5
69.1
84.0
39.3
55.4
18.5
32.7
39.5
47.7
38.4
23.8
122.3
144.3
42.0
27.7
23.2
47.1
41.6
45.9
30.8
34.0
32.6
28.8
17.2
16.8
16.9
26.0
178.3
33.2
23.6
66.2
61.8
47.0
69.1
84.2
39.3
55.5
18.5
32.6
39.6
47.8
38.5
23.8
122.6
143.7
42.0
27.8
26.7
47.0
41.4
46.0
30.9
33.2
32.6
28.8
17.2
16.8
16.9
26.0
177.6
33.3
23.8
64.0
41.7
46.5
69.1
84.1
39.3
55.4
18.5
32.6
39.6
47.7
38.4
23.1
122.3
144.0
41.9
27.7
23.6
46.8
41.4
46.0
30.9
34.1
32.8
28.8
16.9
16.8
17.2
26.0
177.9
33.2
23.6
66.0
22.2
10.8
46.6
69.1
84.1
39.3
55.4
18.5
32.4
39.6
47.8
38.5
23.8
122.6
143.6
41.9
27.8
23.1
46.9
41.4
46.0
30.8
34.0
32.8
28.8
17.3
16.9
16.8
26.0
176.8
33.1
23.6
51.8
78.3
74.5
46.3
68.9
84.0
39.1
55.3
18.3
32.4
39.4
47.6
38.3
23.0
122.2
143.8
41.7
27.6
23.4
46.7
41.3
45.8
30.7
33.9
32.6
28.6
16.7
16.6
17.0
25.9
177.3
33.2
23.7
64.8
132.5
177.7
48.4
69.5
84.5
40.5
55.7
19.5
33.5
40.7
48.6
39.3
24.6
123.7
145.1
42.9
28.7
24.0
47.1
42.8
48.1
31.6
33.7
34.8
29.3
17.8
17.1
17.4
26.4
179.5
33.5
23.9
62.6
32.7
59.6
69.1
84.1
39.3
55.4
18.5
32.6
39.6
47.7
38.5
23.8
122.5
144.0
41.5
27.8
23.2
47.0
41.9
45.9
30.9
34.0
31.9
28.8
17.3
16.8
16.9
26.1
177.6
33.2
23.7
62.0
31.9
29.7
69.0
84.0
39.3
55.4
18.5
32.6
39.5
47.7
38.4
23.6
122.4
143.9
41.5
27.7
23.1
46.9
41.9
45.9
30.8
34.0
32.8
28.4
16.9
16.7
17.2
26.0
177.7
33.2
23.8
61.0
31.8
41.4
69.1
84.1
39.3
55.4
18.5
32.6
39.6
47.8
38.5
23.7
122.4
143.9
41.5
27.8
23.2
47.0
41.9
45.9
30.9
34.0
32.8
28.4
16.9
16.8
17.4
26.0
177.7
33.1
23.6
63.8
32.6
1.9
C-9
C-10
C-11
C-12
C-13
C-14
C-15
C-16
C-17
C-18
C-19
C-20
C-21
C-22
C-23
C-24
C-25
C-26
C-27
C-28
C-29
C-30
C-31
C-32
C-33
122.4
144.8
42.4
28.3
23.7
47.7
42.0
46.4
30.9
34.2
33.2
29.3
17.6
17.4
16.8
26.2
180.2
33.3
23.8
ꢂ
ꢂ
J ¼ 13.6, 3.9 Hz,1H, H-18), 2.16 (brs, 3H, OH),1.97 (ddd, J ¼ 13.9,13.9,
.3 Hz, 1H, H -16), 1.96e1.93 (m, 1H, H -1), 1.93e1.87 (m, 2H, Ha,b
1), 1.72 (ddd, J ¼ 13.8, 13.8, 4.4 Hz, 1H, H -7), 1.65e1.59 (m, 4H, H-
þ H -15 þ H -19 þ H -16), 1.53 (m, 2H, H -6 þ H -7), 1.44 (ddd,
J ¼ 10.1, 10.1, 2.7 Hz, 1H, H -22), 1.39e1.27 (m, 3H, H -6 þ CH -21),
.22e1.14 (m, 2H, H -19), 1.14 (s, 3H, CH -27), 1.10e1.05 (m,
-22 þ H
H, H -15), 1.02 (s, 3H, CH -23), 0.97 (s, 3H, CH -25), 0.92 (s, 3H,
CH3ꢁ30), 0.90 (s, 3H, CH -29), 0.85e0.81 (m, 1H, H-5), 0.81 (s, 3H,
CH -24), 0.74 (s, 3H, CH -26) ppm; MS (ESI, MeOH, source CID): m/
z ¼ 539.6 (100%, [M þ Na] ), 1055.2 (36%, [2M þ Na] ).
167e169 C; R
F
¼ 0.52 (n-hexane/ethyl acetate, 6:4); [
a
]
D
¼ þ53.8
4
1
9
a
a
-
(c 3.0, CHCl
1162, 1125, 1052, 1032, 958 cm
3
); IR (KBr):
n
¼ 3384, 2946, 1721, 1462, 1386, 1364, 1262,
ꢁ
1 1
a
;
H NMR (400 MHz, CDCl
3
):
-31),
a
a
b
a
b
d
¼ 5.28 (dd, J ¼ 3.3, 3.3 Hz, 1H, H-12), 4.05e3.88 (m, 2H, CH
2
a
b
2
3.68 (ddd, J ¼ 11.1, 11.1, 4.4 Hz, 1H, H-2), 2.99 (d, J ¼ 9.5 Hz, 1H, H-3),
2.88 (dd, J ¼ 13.8, 3.9 Hz,1H, H-18), 2.21 (brs, 2H, OH), 1.97e1.83 (m,
1
1
b
b
3
b
3
3
4H, CH
a
-1 þ H
a
-16 þ Ha,b-11), 1.71 (dd, J ¼ 13.5, 13.5, 4.3 Hz, 1H, H
a
-
3
7),1.71e1.55 (m, 6H, H-9 þ H
1.48 (m, 2H, H -7), 1.42 (ddd, J ¼ 12.2, 12.2, 2.7 Hz, 1H, H
-6 þ H
22), 1.38e1.34 (m, 1H, H -6), 1.32e1.24 (m, 2H, H
-21 þ H
.17e1.13 (m, 2H, H -21), 1.13 (s, 3H, CH -27), 1.10e1.01 (m,
-19 þ H
1H, H -15), 1.02 (s, 3H, CH -23), 0.97 (s, 3H, CH -25), 0.95 (t,
J ¼ 7.4 Hz, CH -30), 0.92e0.86 (m, 1H, H -1),
0.90 (s, 3H, CH -24), 0.73 (s,
a
-19 þ H
a
-15 þ H
b
-16 þ CH
2
-32),1.52e
3
3
a
b
a
-
þ
þ
b
a
b
-22),
1
b
b
3
5
.2.7.5. 2-Chloroethyl (2 , 3 ) 2,3-dihydroxy-olean-12-en-28-oate
a
b
b
3
3
(
5). Obtained from 1 by method A as a colorless solid; yield: 56%;
3
(33)), 0.92 (s, 3H, CH
3
b
ꢂ
m.p. 169e170 C;
R
F
¼
0.37 (n-hexane/ethyl acetate, 6:4);
3 3
-29), 0.86 (m, 1H, H-5), 0.82 (s, 3H, CH
ꢂ
[
a
]
D
¼ þ34 (c 1.3, CHCl
3
); IR (KBr):
n
¼ 3430, 2944, 2926, 2852,
3H, CH
[M
3
-26) ppm; MS (ESI, MeOH, source CID): m/z ¼ 537.6 (100%,
þ
2þ
H] ), 1049.3 (49%,
1
730, 1634, 1462, 1434, 1384, 1366, 1302, 1262, 1238, 1196, 1174,
þ
Na] ), 792.0 (61%, [3M
þ
K
þ
ꢁ
1
1
2
1
160, 1124, 1108, 1094, 1082, 1050, 1034 cm ; H NMR (500 MHz,
CDCl ):
¼ 5.32 (dd, J ¼ 3.4, 3.4 Hz, 1H, H-12), 4.31 (ddd, J ¼ 11.6,
.7, 5.7 Hz, 1H, H -31),
-31), 4.24 (ddd, J ¼ 11.6, 5.6, 5.6 Hz, 1H, CH
.68 (ddd, J ¼ 10.2, 9.4, 4.7 Hz, 1H, H-2), 3.66 (dd, J ¼ 5.7, 5.7 Hz, 2H,
-32), 3.01 (d, J ¼ 9.4 Hz,1H, H-3), 2.88 (dd, J ¼ 14.1, 4.4 Hz,1H, H-
8), 2.03e1.88 (m, 4H, H -16 þ Ha,b-11), 1.74 (dd, J ¼ 13.7,
-1 þ H
3.7, 4.2 Hz, 1H, H -15 þ H -19 þ H
-7), 1.68e1.50 (m, 6H, H-9 þ H
6 þ H -6 þ H -7), 1.49e1.44 (m, 1H, H -22), 1.43e1.20 (m, 5H, CH
-6 þ H -19), 1.23e1.15 (m, 1H, H
-27), 1.07 (m, 1H, H
[4M þ K þ H] ).
3
d
5
3
CH
1
1
1
(
CH
a
b
5.2.7.7. 2-Propen-1-yl (2a, 3b) 2,3-dihydroxy-olean-12-en-28-oate
(7). Obtained from 1 by method A as a colorless solid; yield: 90%;
m.p. 203e205 C;
ꢂ
2
R
F
¼ 0.49 (n-hexane/ethyl acetate, 6:4);
ꢂ
a
a
[a
]
D
¼ þ55.4 (c 3.6, CHCl
3
); IR (KBr):
n
¼ 3406, 2947, 1727, 1648,
ꢁ1 1
a
a
a
b
-
1464, 1386, 1363, 1261, 1199, 1178, 1159, 1123, 1051, 1034 cm ; H
NMR (500 MHz, CDCl ):
¼ 5.87 (ddd, 1H, J ¼ 22.7, 10.8, 5.6 Hz, CH-
32), 5.32e5.25 (m, 1H, CH (33)), 5.27 (m, 1H, H-12), 5.18 (dd,
J ¼ 10.4, 1.2 Hz, 1H, CH (33)), 4.55e4.44 (m, 2H, CH -31), 3.65 (ddd,
J ¼ 11.1, 9.7, 4.5 Hz, 1H, H-2), 2.98 (d, J ¼ 9.5 Hz, 1H, H-3), 2.86 (dd,
J ¼ 13.8, 4.1 Hz, 1H, H-18), 2.03 (br, 2H, OH), 2.00e1.84 (m, 4H, Ha,b
11 þ H -16 þ H -1), 1.69 (ddd, J ¼ 13.7, 13.7, 4.3 Hz, 1H, H -7), 1.66e
.46 (m, 4H, H -15 þ H -19 þ H-9), 1.55e1.49 (m, 2H, H
-16 þ H
7 þ H -6), 1.44 (ddd, J ¼ 15.4, 15.4, 3.8 Hz, 1H, H
J ¼ 12.6, 12.6, 2.1 Hz, 1H, H -6), 1.33 (m, 1H, H -21), 1.28 (m, 1H, H
a
b
a
2
3
d
21) þ H
b
b
-22 þ H
b
b
-15), 1.14 (s, 3H,
a
3
b
-1), 1.03 (s, 3H, CH
3
-23), 0.98 (s, 3H, CH
3
-
b
2
2
(
5), 0.93 (s, 3H, CH
s, 3H, CH
CID): m/z ¼ 557.3 (100%, [M þ Na] ), 1091.3 (90%, [2M þ Na] ).
3
3
-30), 0.91 (s, 3H, CH -29, 0.84 (m, 1H, H-5), 0.83
3
-24), 0.74 (s, 3H, CH
3
-26) ppm; MS (ESI, MeOH, source
-
þ
þ
a
a
a
1
b
a
a
b
-
-
5
.2.7.6. Propyl (2 2,3-dihydroxy-olean-12-en-28-oate (6).
a
,
3
b
)
a
a
-22), 1.37 (ddd,
Obtained from 1 by method A as a colorless solid; yield: 78%; m.p.
b
a
b

266
B. Siewert et al. / European Journal of Medicinal Chemistry 70 (2013) 259e272
Table 3
1
3C NMR shifts (100 MHz, CDCl
3
) for compounds 13-22.
1
3
14
46.4
15
46.5
16
45.9
17
45.8
18
46.5
19
46.6
20
46.5
21
46.5
22
46.6
C-1
C-2
C-3
C-4
C-5
C-6
C-7
C-8
46.4
68.9
84.0
39.2
55.3
18.3
32.7
39.4
47.5
38.2
23.5
68.9
83.9
39.2
55.3
18.3
32.4
39.4
47.6
38.3
22.9
122.2
143.8
41.7
27.6
23.4
46.6
41.2
45.8
30.7
33.9
32.6
28.1
16.7
16.6
17.0
25.9
177.6
33.1
23.6
63.3
33.1
134.3
117.0
69.1
84.1
39.3
55.4
18.5
32.6
39.6
47.8
38.5
23.7
122.4
143.8
41.9
27.8
23.1
46.9
41.4
46.0
30.9
34.0
32.8
28.3
17.2
16.8
16.9
26.0
177.6
33.2
23.6
62.1
19.1
69.8
28.8
69.0
84.0
39.4
55.3
18.4
32.5
39.2
47.6
38.3
23.0
122.3
143.9
41.8
27.6
23.5
46.7
41.3
46.4
30.7
33.9
32.6
28.3
17.1
16.6
16.7
25.9
177.7
33.2
23.6
63.3
26.1
29.4
44.7
68.9
83.9
39.2
55.3
18.3
32.3
41.2
47.6
38.3
23.0
122.3
143.8
41.7
27.6
23.5
46.7
39.4
46.4
30.9
33.8
32.6
28.3
17.1
16.6
16.7
25.9
177.7
33.3
23.6
63.2
27.3
29.5
33.0
69.1
84.1
39.3
55.4
18.5
32.5
39.6
47.7
38.4
23.6
122.4
143.8
41.9
27.8
23.2
46.9
41.4
46.0
30.8
34.0
32.8
28.8
17.3
16.8
16.9
26.0
177.5
33.2
23.8
59.2
128.4
129.9
39.0
69.0
84.1
39.3
55.4
18.5
32.8
39.6
47.7
38.4
23.6
122.6
143.6
41.9
27.8
23.2
46.9
41.4
46.0
30.8
34.0
32.4
28.8
17.2
16.8
16.9
26.0
177.0
33.2
23.8
51.8
80.9
81.3
30.6
69.1
84.1
39.3
55.4
18.5
32.6
39.6
47.8
38.4
23.1
122.3
144.0
41.9
27.7
23.6
46.8
41.4
46.0
30.9
34.1
32.8
28.8
16.9
16.7
17.2
26.0
177.9
33.3
23.8
64.5
28.8
25.9
31.6
14.0
69.1
84.1
39.3
55.4
18.5
32.6
39.6
47.8
38.5
23.6
122.3
144.1
41.9
27.8
23.1
46.8
41.0
46.0
30.9
34.1
32.8
28.8
17.2
16.8
16.9
26.0
177.9
32.1
29.4
64.4
28.8
26.2
29.5
69.1
84.1
39.3
55.4
18.5
32.6
39.6
47.7
38.5
23.6
122.3
144.0
41.9
27.8
23.1
46.8
41.4
46.0
30.9
34.1
32.8
29.4
17.2
16.7
16.9
26.0
177.9
33.3
23.8
66.0
29.4
26.2
C-9
C-10
C-11
C-12
C-13
C-14
C-15
C-16
C-17
C-18
C-19
C-20
C-21
C-22
C-23
C-24
C-25
C-26
C-27
C-28
C-29
C-30
C-31
C-32
C-33
C-34
C-36
Chain
C-42
C-35 þ C-45
C-46
C-48
122.3
144.0
41.7
27.6
23.0
46.6
41.3
45.8
30.7
33.9
32.6
28.8
16.8
16.6
17.0
25.9
177.9
33.1
23.6
64.0
30.8
19.2
13.7
29.8
14.3
29.8
29.5
32.1
14.3
2
1
CH
CH
2), 1.22e1.07 (m, 2H, H
b
-21 þ H
b
-19), 1.13 (s, 3H, CH
3
-27), 1.05 (m,
26) ppm; MS (ESI, MeOH, source CID): m/z ¼ 533.6 (63%,
þ
2þ
H, H
b
-15), 1.02 (s, 3H, CH
3
-23), 0.97 (s, 3H, CH
3
-25), 0.92 (s, 3H,
-1), 0.81 (s, 3H,
[M þ Na] ), 785.4 (46%, [3M þ K þ H] ), 1040.8 (100%,
þ
3
-30), 0.90 (s, 3H, CH
3
-29), 0.91e0.80 (m, 1H, H
b
[4M þ K þ H] ).
3
-24), 0.81e0.79 (m, 1H, H-5), 0.72 (s, 3H, CH
3
-26) ppm; MS (ESI,
þ
MeOH, source CID): m/z ¼ 495.5 (7.3%, [M þ H] ), 530.4 (6.1%,
5.2.7.9. 3-Hydroxy-prop-1-yl (2a, 3b) 2,3-dihydroxy-olean-12-en-
28-oate (9). Obtained from 1 by method A as a colorless solid; yield:
þ
þ
[
[
M
þ
NH
4
] ), 535.5 (100%, [M
þ
Na] ), 788.5 (36.6%,
2
þ
þ
ꢂ
3M þ K þ H] ), 1047.2 (48.8%, [2M þ Na] ).
80%; m.p. 119e121 C; R
F
¼ 0.17 (n-hexane/ethyl acetate, 6:4);
ꢂ
[a
]
D
¼ þ51.7 (c 1.4, CHCl
3
); IR (KBr):
n
¼ 3446, 2948, 2878, 2864,
ꢁ
1
5
.2.7.8. 2-Propyn-1-yl (2 , 3 ) 2,3-dihydroxy-olean-12-en-28-oate
a
b
1724,1706,1646,1636,1458,1384,1364,1262,1178,1162,1050 cm
;
1
(8). Obtained from 1 by method A as a colorless solid; yield: 78%;
H NMR (400 MHz, CD
4.19 (ddd, J ¼ 12.7, 6.3, 6.3 Hz, 1H, CH
6.3Hz, CH
-31), 3.68(ddd, J¼ 6.5,1.4Hz, 2H, CH
1H, H-2), 2.96 (d, J ¼ 9.6 Hz,1H, H-3), 2.95 (dd, J ¼ 13.8, 4.5 Hz,1H, H-
18), 2.10 (ddd, J ¼ 12.8, 12.8, 3.5 Hz, 1H, H -16), 2.03e1.96 (m, 3H,
CH -32), 1.82e1.67 (m, 2H, H
-1 þ Ha,b-11), 1.95e1.85 (m, 2H, CH
19 þ H-7),1.70e1.60 (m, 3H, H-9 þ H -15 þ H -16),1.63e1.38 (m, 5H,
CH -21),1.38e1.33 (m,1H, H -22),1.25e1.20
(m,1H, H
1.07 (s, 3H, CH
3H, CH
3
OH):
d
¼ 5.33 (dd, J ¼ 3.5, 3.5 Hz, 1H, H-12),
-31), 4.15 (ddd, J ¼ 12.7, 6.3,
(33)), 3.72e3.64 (m,
ꢂ
ꢂ
m.p. 225e227 C (lit.: 233e234 C [50]); R
acetate, 6:4); [
F
¼ 0.23 (n-hexane/ethyl
a
ꢂ
a
]
D
¼ þ58.6 (c 3.0, CHCl
3
); IR (KBr):
n
¼ 3568, 3406,
b
2
3
310, 3296, 2946, 2862, 1736, 1466, 1442, 1386, 1364, 1260, 1228,
ꢁ
1 1
1
196, 1176, 1156, 1118, 1066, 1052, 1034 cm ; H NMR (400 MHz,
CDCl ):
¼ 5.31 (dd, J ¼ 3.5, 3.5 Hz, 1H, H-12), 4.68 (dd, J ¼ 15.6,
.4 Hz,1H, CH -31), 3.69 (ddd,
a
3
d
a
2
a
-
2
a
-31), 4.57 (dd, J ¼ 15.6, 2.5 Hz,1H, CH
b
a
b
J ¼ 11.2, 9.6, 4.5 Hz, 1H, H-2), 3.00 (d, J ¼ 9.5 Hz, 1H, H-3), 2.87 (dd,
J ¼ 13.7, 3.9 Hz, 1H, H-18), 2.41 (dd, J ¼ 2.5, 2.5 Hz, 1H, CH (33)), 2.10
2
-6 þ H
b
-7 þ H
a
-22 þ H
a
b
b
-21),1.22 (s, 3H, CH
3
-27),1.21e1.08 (m, 2H, H
b
-15 þ H
b
-19),
-30), 0.97 (s,
(
brs, 2H, OH), 2.02 (ddd, J ¼ 12.6, 12.6, 3.5 Hz, 1H, H
a
-16), 1.95 (m,
3
-23),1.06 (s, 3H, CH
3
-25),1.00 (s, 3H, CH
3
1H, H
1H, H
7 þ H
1), 1.29 (m, 1H, H
H, CH
-27), 1.07 (ddd, J ¼ 9.5, 9.5, 5.3 Hz, 1H, H
CH -23), 0.98 (s, 3H, CH -25), 0.93 (s, 3H, CH -30), 0.90 (s, 3H, CH
9), 0.88e0.78 (m, 1H, H-5), 0.82 (s, 3H, CH -24), 0.76 (s, 3H, CH
a
-1), 1.94e1.87 (m, 2H, Ha,b-11), 1.70 (ddd, J ¼ 13.7, 13.7, 4.3 Hz,
3
-29), 0.95e0.88 (m, 2H, H-5 þ H
b
-1), 0.87 (s, 3H, CH
3
-24),
a
-7), 1.68e1.49 (m, 6H, H
-6), 1.49e1.39 (m, 1H, H
-22), 1.24e1.17 (m, 2H, H
b
-16 þ H
-22), 1.39e1.32 (m, 2H, H
-21 þ H
-1), 1.03 (s, 3H,
b
-19 þ H
a
-15 þ H-9 þ H
b
-
0.82 (s, 3H, CH
3
-26) ppm; MS (ESI, MeOH, source CID): m/z ¼ 531.1
þ
þ
2þ
a
a
b
-6 þ H
a
-
(8%, [M þ H] ), 553.3 (92%, [M þ Na] ), 815.9 (70%, [3M þ K þ H] ),
þ
2
3
b
b
b
-19), 1.14 (s,
1083.3 (100%, [2M þ Na] ).
3
b
3
3
3
3
3
-
-
5.2.7.10. 3-Chloro-prop-1-yl (2a, 3b) 2,3-dihydroxy-olean-12-en-28-
2
3
oate (10). Obtained from 1 by method A as a colorless solid; yield

B. Siewert et al. / European Journal of Medicinal Chemistry 70 (2013) 259e272
267
Table 4
1
3C NMR shifts (100 MHz, CDCl
3
) for compounds 23-34.
2
3
24
44.0
25
44.1
26
44.1
27
44.1
28
44.1
29
44.1
30
46.5
31
48.1
32
48.1
33
45.5
34
48.1
C-1
C-2
C-3
C-4
C-5
C-6
C-7
C-8
46.6
69.1
84.1
39.3
55.5
18.5
32.8
39.6
47.8
38.5
23.6
70.2
80.8
39.5
55.0
18.3
32.5
39.5
47.7
38.3
23.6
122.4
143.8
41.0
27.8
23.0
46.7
41.7
46.0
30.8
33.9
32.5
28.6
17.3
16.6
17.8
26.0
183.8
33.2
23.7
70.1
80.7
39.4
55.0
18.4
32.7
39.5
47.6
38.3
23.9
122.6
145.1
42.3
27.4
23.8
46.6
42.7
46.8
30.9
34.2
32.4
28.6
17.2
16.6
17.8
25.9
181.2
33.1
23.7
70.1
80.7
39.5
55.0
18.3
32.6
39.6
47.6
38.2
23.9
122.3
145.4
42.3
27.4
23.7
46.3
42.4
47.0
30.9
34.3
32.3
28.6
17.0
16.6
17.8
25.9
178.1
33.2
23.7
34.5
14.8
70.0
80.7
39.5
55.0
18.3
32.7
39.6
47.6
38.2
23.9
122.3
145.4
42.3
27.4
23.7
46.4
42.4
47.0
30.9
34.3
32.3
28.6
17.0
16.6
17.8
25.9
178.1
33.1
23.7
41.3
22.8
11.6
70.1
80.7
39.5
55.0
18.4
32.7
39.6
47.6
38.2
24.0
122.6
145.3
42.1
27.4
23.7
46.5
42.3
46.9
30.9
34.3
32.3
28.6
17.0
16.6
17.8
25.9
178.1
33.1
23.7
42.3
134.6
116.4
70.1
80.7
39.5
55.0
18.3
32.4
39.6
47.6
38.2
24.0
122.9
145.0
42.2
27.3
23.8
46.5
42.3
46.8
30.9
34.2
32.3
28.5
17.1
16.6
17.7
25.9
178.0
33.1
23.8
29.5
71.8
70.0
69.0
84.7
39.3
55.4
18.5
32.6
39.4
47.7
38.4
24.0
122.8
145.1
42.3
27.5
23.7
46.5
42.7
46.8
30.9
34.1
32.5
28.8
16.9
16.8
17.3
25.9
181.4
33.1
23.7
69.5
84.5
40.5
56.6
19.5
34.3
40.7
48.6
39.2
24.6
123.8
145.5
43.0
28.5
24.0
47.4
42.6
47.7
31.6
35.1
33.8
29.3
17.4
17.1
17.9
26.5
180.2
33.5
24.0
35.6
14.9
69.5
84.4
40.5
56.6
19.5
34.4
40.7
48.8
39.2
24.0
123.8
145.5
43.0
28.5
23.6
47.5
42.6
47.7
31.6
35.1
33.8
29.3
17.5
17.1
17.9
26.5
180.2
33.6
24.0
42.5
24.6
11.9
69.0
84.1
39.3
55.3
18.4
32.6
39.6
47.7
38.3
23.9
122.8
145.2
42.1
27.4
23.7
46.5
42.4
46.9
30.9
34.3
32.4
28.7
16.9
16.8
17.1
25.9
178.2
33.1
23.7
42.3
134.5
116.5
69.5
85.5
40.5
56.7
19.5
34.1
40.8
48.4
39.3
24.7
123.9
145.3
42.9
28.4
24.0
47.5
42.5
47.6
31.6
35.1
33.8
29.3
17.4
17.1
18.1
26.5
180.0
33.5
24.0
29.7
81.0
71.8
C-9
C-10
C-11
C-12
C-13
C-14
C-15
C-16
C-17
C-18
C-19
C-20
C-21
C-22
C-23
C-24
C-25
C-26
C-27
C-28
C-29
C-30
C-31
C-32
C-33
C-34
C-35
Ac
122.2
144.0
41.9
27.8
23.1
46.9
41.5
46.0
30.9
34.1
32.7
28.8
16.9
16.8
17.3
26.1
177.9
33.3
23.8
69.6
32.3
26.6
25.9
30.0
21.1
21.3
21.1
21.3
21.1
21.3
21.1
21.3
21.1
21.3
21.0
21.3
Ac
AcC ¼ O
107.7
171.0
170.7
171.0
170.7
171.0
170.7
171.0
170.7
171.0
170.7
171.0
AcC ¼ O
ꢂ
8
4%; m.p. 146e147 C; R
F
¼ 0.3 (n-hexane/ethyl acetate, 5:3);
18), 2.17 (dtt, J ¼ 12.5, 6.3, 6.3 Hz, 2H, CH
1 þ H -16), 1.85e1.79 (m, 2H, Ha,b-11), 1.70 (ddd, J ¼ 13.8, 13.8,
4.4 Hz, 1H, H -19 þ H -15 þ H -16),
-7), 1.68e1.59 (m, 4H, H-9 þ H
1.58e1.53 (m, 1H, H -22),
-6), 1.45 (ddd, J ¼ 13.8, 13.8, 4.4 Hz, 1H, H
-7), 1.38 (ddd, J ¼ 12.5, 12.5, 2.5 Hz, 1H, H -6),
-21), 1.20e1.15 (m, 2H, H -21),
-15), 1.03 (s, 3H, CH
-25), 0.93e0.89 (m, 1H, H -1), 0.92 (s, 3H, CH
-29), 0.79e0.75 (m, 1H, H-5), 0.75 (s, 3H, CH
2 a
-32), 1.98 (m, 2H, CH -
ꢂ
[
a
]
D
¼ þ41.2 (c 2.1, CHCl
3
); IR (KBr):
n
¼ 3440, 2948, 2864, 1726,
a
1
1
3
4
1
2
1
1
3
1
654, 1648, 1636, 1628, 1618, 1458, 1438, 1386, 1260, 1176, 1162,
a
a
a
b
ꢁ
1 1
050, 1034 cm ; H NMR (500 MHz, CDCl
.3 Hz, 1H, H-12), 4.17e4.13 (m, 2H, CH
-31), 3.67 (ddd, J ¼ 11.1, 9.5,
.4 Hz, 1H, H-2), 3.60 (t, J ¼ 6.4 Hz, 2H, CH (33)), 2.98 (d, J ¼ 9.5 Hz,
3
):
d
¼ 5.27 (dd, J ¼ 3.3,
a
a
2
1.41e1.35 (m, 1H, H
1.37e1.28 (m, 2H, H
1.13 (s, 3H, CH
0.99 (s, 3H, CH
0.90 (s, 3H, CH
0.74 (s, 3H, CH
b
b
2
b
-22 þ H
a
b
-1 þ H
b
H, H-3), 2.85 (dd, J ¼ 13.8, 4.0 Hz, 1H, H-18), 2.49 (brs, 2H, OH),
3
-27), 1.10e1.04 (m, 1H, H
b
3
-23),
3
-30),
3
-24),
.09e2.00 (m, 2H, CH
6), 1.70 (ddd, J ¼ 13.9, 13.9, 4.3 Hz, 1H, H
9 þ H-9 þ H -16 þ H -15), 1.55e1.48 (m, 1H, H
H, H -22 þ H -7), 1.36e1.32 (m, 2H, H -21 þ H
-6 þ H
.16 (m, 2H, H -19), 1.12 (s, 3H, CH -27), 1.08e1.02 (m, 1H,
-21 þ H
-15), 1.01 (s, 3H, CH -23), 0.96 (s, 3H, CH -25), 0.91 (s, 3H, CH
0), 0.89 (s, 3H, CH -29), 0.91e0.84 (m, 2H, H
-1 þ H-5), 0.81 (s, 3H,
CH -23), 0.72 (s, 3H, CH -26) ppm; MS (ESI, MeOH, source CID): m/
z ¼ 549.1 (11%, [M þ H] ), 571.4 (100%, [M þ Na] ), 844.6 (10%,
2
-32), 1.98e1.87 (m, 4H, Ha,b-11 þ CH
a
-1 þ H
a
-
-
3
b
a
-7), 1.66e1.56 (m, 4H, H
a
3
b
a
a
-6), 1.43e1.35 (m,
-22), 1.22e
3
-26) ppm; MS (ESI, MeOH, source CID): m/z ¼ 615.7
7
9
þ
81
þ
b
a
b
a
b
(75%, [ M þ Na] ), 617.5 (100%, [ M þ Na] ), 1209.1 (60%,
79 81
þ
þ
b
b
3
[2 M þ Na] ), 1211.1 (38%, [2 M þ Na] ).
H
3
b
3
3
3
-
3
b
5.2.7.12. 3-Iodo-prop-1-yl (2 , 3 ) 2,3-dihydroxy-olean-12-en-28-
a
b
3
3
oate (12). Obtained from 1 by method A as a colorless solid;
þ
þ
ꢂ
yield: 92%; m.p. 145e147 C; R
F
¼ 0.29 (n-hexane/ethyl acetate,
2
þ
þ
ꢂ
[
3M þ Na] ), 1119.5 (67%, [2M þ Na] ).
5:3); [
a
]
D
¼ þ47.3 (c 3.1, CHCl
3
); IR (KBr):
n
¼ 3424, 2948, 1726,
2
864, 1162, 1034, 1050, 1460, 1170, 1384, 1260, 1630, 1124, 1364,
ꢁ1 1
5
.2.7.11. 3-Bromo-prop-1-yl (2 , 3 )-2,3-dihydroxy-olean-12-en-28-
a
b
1302 cm ; H NMR (500 MHz, CDCl
1H, H-12), 4.14e4.10 (m, 2H, CH
-31), 3.69 (ddd, J ¼ 11.3, 9.7, 4.5 Hz,
1H, H-2), 3.23 (ddd, J ¼ 6.8, 6.7, 1.0 Hz, 2H, CH (33)), 3.00 (d,
J ¼ 9.5 Hz, 1H, H-3), 2.85 (dd, J ¼ 13.8, 4.2 Hz, 1H, H-18), 2.20e2.01
(br, 2H, OH), 2.15e2.07 (m, 2H, CH -32), 2.00e1.93 (m, 2H, CH
1 þ H -16), 1.95e1.83 (m, 2H, Ha,b-11), 1.71 (ddd, J ¼ 13.8, 13.8,
4.4 Hz, 1H, H -19 þ H -15 þ H -16),
-7), 1.64e1.58 (m, 4H, H-9 þ H
1.60e1.55 (m, 1H, H -6), 1.53e1.40 (m, 3H, H -7 þ H -22),
-6 þ H
1.39e1.30 (m, 2H, H -21), 1.22e1.14 (m, 2H, H -21 þ H
-22 þ H
3
):
d
¼ 5.28 (dd, J ¼ 3.3, 3.3 Hz,
oate (11). Obtained from 1 by method A as a colorless solid; yield:
2
ꢂ
9
2%; m.p. 117e119 C; R
F
¼ 0.30 (n-hexane/ethyl acetate, 5:3);
2
ꢂ
[
a
]
D
¼ þ44.4 (c 4.0, CHCl
3
); IR (KBr):
n
¼ 3442, 2948, 2864, 1726,
1
1
(
3
628, 1458, 1386, 1364, 1302, 1260, 1176, 1162, 1124, 1050,
2
a
-
ꢁ
1 1
034 cm ; H NMR (500 MHz, CDCl
dt, J ¼ 11.1, 6.9 Hz,1H, CH -31), 4.15 (dt, J ¼ 11.1, 6.9 Hz,1H, CH
.70 (ddd, J ¼ 11.0, 9.5, 4.2 Hz, 1H, H-2), 3.46 (t, J ¼ 6.6 Hz, 2H, CH
33)), 3.01 (d, J ¼ 9.4 Hz, 1H, H-3), 2.86 (dd, J ¼ 13.8, 4.0 Hz, 1H, H-
3
):
d
¼ 5.29 (brs, 1H, H-12), 4.17
a
a
b
-31),
a
a
a
b
2
a
b
b
a
(
b
a
b
b
-

268
B. Siewert et al. / European Journal of Medicinal Chemistry 70 (2013) 259e272
1
9), 1.13 (s, 3H, CH
3
-27), 1.09e1.03 (m, 1H, H
-25), 0.92 (s, 3H, CH -30), 0.90 (s, 3H, CH
.92e0.85 (m, 2H, H-5 þ H -1), 0.82 (s, 3H, CH
b
-15), 1.03 (s, 3H, CH
3
-
1.21e1.15 (m, 2H, H
1H, H -15), 1.03 (s, 3H, CH
CH -30), 0.90 (s, 3H, CH -29), 0.91e0.81 (m, 2H, H
(s, 3H, CH
b
-21 þ H
b
-19), 1.13 (s, 3H, CH
-23), 0.98 (s, 3H, CH
3
-27), 1.11e1.00 (m,
2
3), 0.98 (s, 3H, CH
3
3
3
-29),
b
3
3
-25), 0.93 (s, 3H,
0
CH
b
3
-24), 0.75 (s, 3H,
3
3
b
-1 þ H-5), 0.82
3
-26) ppm; MS (ESI, MeOH, source CID): m/z ¼ 641.1 (20%,
3
-24), 0.74 (s, 3H, CH
3
-26) ppm; MS (ESI, MeOH): m/
þ
þ
þ
þ
þ
[
(
M þ H] ), 658.1 (10%, [M þ NH
4
] ), 663.3 (62%, [M þ Na] ), 1303.2
z ¼ 525.2 (25%, [M þ H] ), 547.4 (81%, [M þ Na] ), 806.7 (84%,
þ
2þ
þ
100%, [2M þ Na] ).
[3M þ K þ H] ), 1071.4 (100%, [2M þ Na] ).
5.2.7.13. Butyl (2
a,
3
b)
2,3-dihydroxy-olean-12-en-28-oate (13).
5.2.7.16. 4-Chloro-but-1-yl (2 , 3 ) 2,3-dihydroxy-olean-12-en-28-
a
b
Obtained from 1 by method A as a colorless solid; yield: 95%; m.p.
oate (16). Obtained from 1 by method A as a colorless solid;
ꢂ
ꢂ
ꢂ
1
(
1
49e152 C; R
c 4.0, CHCl ); IR (KBr):
434, 1386, 1380, 1364, 1304, 1260, 1242, 1176, 1164, 1064, 1052,
F
¼ 0.51 (n-hexane/ethyl acetate 1:1); [
a
]
D
¼ þ51.4
yield: 72%; m.p. 134e136 C; R
F
¼ 0.41 (n-hexane/ethyl acetate,
); IR (KBr):
¼ 3396, 2945, 1723,
):
ꢂ
3
n
¼ 3368, 2950, 2870, 2864, 1720, 1460,
1:1); [
a]
D
¼ þ49.0 (c 3.3, CHCl
3
n
ꢁ
1 1
1462, 1387, 1261, 1161, 1124, 1049 cm ; H NMR (500 MHz, CDCl
¼ 5.22 (dd, J ¼ 3.5, 3.5 Hz, 1H, H-12), 3.98 (t, J ¼ 6.2 Hz, 2H, CH
31), 3.62 (ddd, J ¼ 11.0, 9.8, 4.6 Hz, 1H, H-2), 3.49 (t, J ¼ 6.5 Hz, 2H,
CH
H-18), 1.91 (dd, J ¼ 12.5, 4.4 Hz, 1H, H
16 þ Ha,b-11), 1.80e1.75 (m, 2H, CH (33)), 1.74e1.67 (m, 2H, CH
32), 1.64 (ddd, J ¼ 13.9, 13.9, 4.4 Hz, 1H, H
19 þ CH -9 þ CH -11 þ H -7 þ H -16 þ H
J ¼ 14.0, 11.1, 3.4 Hz, 1H, H -21), 1.30 (ddd, J ¼ 11.3, 9.6, 2.9 Hz, 1H,
-21), 1.27e1.22 (m, 1H, H -21), 1.16e1.02 (m, 2H, H -19),
-21 þ H
1.07 (s, 3H, CH -27), 1.03e0.97 (m, 1H, H -15), 0.96 (s, 3H, CH -23),
-25), 0.86 (s, 3H, CH -30), 0.84 (s, 3H, CH -29), 0.83e
3
ꢁ
1 1
1
032 cm ; H NMR (500 MHz, CDCl ): ¼ 5.27 (dd, J ¼ 3.4, 3.4 Hz,
3
d
d
2
-
1
H, H-12), 4.00 (m, 2H, CH -31), 3.67 (ddd, J ¼ 11.0, 9.5, 4.3 Hz, 1H,
H-2), 2.99 (d, J ¼ 9.5 Hz, 1H, H-3), 2.94 (br, 2H, OH), 2.86 (dd,
J ¼ 14.0, 4.2 Hz, 1H, H-18), 1.96 (dd, 1H, J ¼ 12.6, 3.6 Hz, H -1), 1.96e
-16),1.69 (ddd, J ¼ 13.8,13.8, 4.3 Hz,1H, H
-19 þ H -15 þ CH (11)), 1.54e1.47 (m, 3H,
-32), 1.52e1.47 (m, 1H, H -6), 1.45e1.40 (m, 1H, H -7),
-22), 1.38e1.33 (m, 1H, H -6), 1.37 (m, 2H, CH
-21), 1.32e1.24 (m, 1H, H -22), 1.20e1.13
-19), 1.12 (s, 3H, CH -27), 1.05e1.00 (m, 1H, H
-23), 0.96 (s, 3H, CH
-25), 0.90 (t, J ¼ 7.4 Hz, 3H,
(36)), 0.91 (s, 3H, CH -30), 0.93e0.86 (m, 1H, H -1), 0.88 (s, 3H,
-29), 0.83e0.77 (m, 1H, H-5), 0.80 (s, 3H, CH -24), 0.72 (s, 3H,
2
2
(34)), 2.98 (d, J ¼ 9.4 Hz, 1H, H-3), 2.80 (dd, J ¼ 13.5, 3.9 Hz, 1H,
a
a
-1), 1.89e1.81 (m, 3H, H
a
-
-
-
1
7
.83 (m, 3H, Ha,b-11 þ H
a
a
-
2
2
), 1.64e1.54 (m, 3H, H
a
a
b
a
-7), 1.61e1.44 (m, 6H, H
-15 þ H
a
H-9 þ CH
2
a
b
2
b
b
b
a
a
-22), 1.38 (ddd,
1.43e1.38 (m, 1H, H
a
b
2
a
(
(
33)), 1.35e1.31 (m, 1H, H
m, 2H, H
a
b
H
a
b
b
b
b
-21 þ H
b
3
b
-
3
b
3
15), 1.01 (s, 3H, CH
3
3
0.91 (s, 3H, CH
3
3
3
CH
CH
CH
3
3
3
3
b
0.77 (m, 2H, H
b
-1 þ H-5), 0.76 (s, 3H, CH
3
-24), 0.66 (s, 3H, CH
3
-26)
þ
3
ppm; MS (ESI, MeOH): m/z ¼ 585.5 (21%, [M þ Na] ), 1147.2 (100%,
þ
-26) ppm; MS (ESI, MeOH, source CID): m/z ¼ 551.6 (100%,
[2M þ Na] ).
þ
2þ
[
[
M þ Na] ), 812.5 (20%, [3M þ K þ H] ), 1079.3 (52.4%,
þ
2M þ Na] ).
5.2.7.17. 4-Bromo-but-1-yl (2a, 3b) 2,3-dihydroxy-olean-12-en-28-
oate (17). Obtained from 1 by method A as a colorless solid;
ꢂ
5
.2.7.14. 3-Buten-1-yl (2
a
, 3
b
) 2,3-dihydroxy-olean-12-en-28-oate
yield: 55%; m.p. 151e155 C; R
F
¼ 0.25 (n-hexane/ethyl acetate,
ꢂ
(
8
14). Obtained from 1 by method A as a colorless solid; yield:
1:1); [
a
]
D
¼ þ45.0 (c 4.1, CHCl
3
); IR (KBr):
n
¼ 3385, 2947, 1725,
ꢂ
ꢁ1 1
7%; m.p. 149e152 C; R
F
¼ 0.56 (n-hexane/ethyl acetate, 1:1);
1464, 1386, 1260, 1178, 1161, 1051, 1035 cm ; H NMR (400 MHz,
CDCl ):
¼ 5.26 (dd, J ¼ 3.5, 3.5 Hz, 1H, H-12), 4.02 (t, J ¼ 6.2 Hz, 2H,
CH
-31), 3.66 (ddd, J ¼ 11.2, 9.6, 4.5 Hz, 1H, H-2), 3.40 (t, J ¼ 6.7 Hz,
2H, CH
(34)), 2.97 (d, J ¼ 9.5 Hz, 1H, H-3), 2.84 (dd, J ¼ 12.9, 3.2 Hz,
1H, H-18), 2.03e1.95 (m,1H, H -1),1.95e1.86 (m, 5H, CH
(33) þ H
16 þ Ha,b-11), 1.79e1.72 (m, 2H, CH -32), 1.72e1.69 (m, 2H, H-
7 þ H-19)), 1.63e1.48 (m, 5H, H-9 þ H -16 þ H -15 þ H -6 þ H -7),
1.48e1.33 (m, 2H, H -6), 1.32e1.25 (m, 2H, H -22 þ H -21),
-22 þ H
1.20e1.09 (m, 2H, H -21),1.11 (s, 3H, CH -27),1.07e1.02 (m,
-19 þ H
1H, H -15), 1.00 (s, 3H, CH -23), 0.96 (s, 3H, CH -25), 0.90 (s, 3H,
CH -30), 0.88 (s, 3H, CH -29), 0.83e0.77 (m, 2H, H
-1 þ H-5), 0.80
(s, 3H, CH -24), 0.70 (s, 3H, CH3ꢁ26) ppm; MS (ESI, MeOH): m/
z ¼ 629.3 (15%, [M þ Na] ), 1237.1 (100%, [2M þ Na] ).
ꢂ
[
a
]
D
¼ þ55.4 (c 3.2, CHCl
3
); IR (KBr):
n
¼ 3392, 2946, 2864, 1720,
3
d
1
9
6
642, 1460, 1434, 1386, 1364, 1260, 1242, 1176, 1162, 1052, 1032,
2
ꢁ
1 1
92 cm ; H NMR (500 MHz, CDCl
.7 Hz, 1H, CH (33)), 5.27 (dd, J ¼ 3.4, 3.4 Hz, 1H, H-12), 5.10 (dd,
(34)), 5.06 (dd, J ¼ 10.2, 1.2 Hz, 1H, CH (34)),
3
):
d
¼ 5.78 (ddd, J ¼ 17.0, 10.3,
2
a
2
a
-
J ¼ 17.2, 1.5 Hz, 1H, CH
a
b
2
4
4
4
.07 (ddd, J ¼ 13.2, 10.8, 5.4 Hz, 2H, CH
2
-31), 3.69 (ddd, J ¼ 11.3, 9.7,
b
a
a
b
.5 Hz, 1H, H-2), 3.00 (d, J ¼ 9.5 Hz, 1H, H-3), 2.86 (dd, J ¼ 13.8,
.1 Hz,1H, H-18), 2.47 (br, 2H, OH), 2.35 (ddd, J ¼ 6.6, 6.6, 6.6 Hz, 2H,
a
b
b
a
b
b
3
CH
2
-32), 1.99e1.91 (m, 4H, Ha,b-11 þ H
a
-16 þ H
a
-1), 1.69 (ddd,
-16 þ H
-6), 1.47 (dd, J ¼ 17.3,
-6), 1.38e1.27 (m, 2H, H
b
3
3
J ¼ 13.8, 13.8, 4.3 Hz, 1H, H
5 þ H -19 þ H-9), 1.56e1.47 (m, 2H, H
.4 Hz, 1H, H -22), 1.43e1.38 (m, 1H, H
1 þ H -22), 1.20e1.14 (m, 2H, H -21 þ H
.07e1.03 (m, 1H, H -15), 1.02 (s, 3H, CH -23), 0.97 (s, 3H, CH
-30), 0.89 (s, 3H, CH
-24), 0.85e0.79 (m, 1H, H-5), 0.72 (s, 3H, CH
a
-7), 1.66e1.46 (m, 4H, H
b
a
-
3
3
b
1
3
2
1
0
0
a
b
-7 þ H
a
3
þ
þ
a
b
a
-
b
b
b
-19), 1.12 (s, 3H, CH
3
-27),
-25),
b
3
3
5.2.7.18. (2Z) 4-Chlorobut-2-en-1-yl (2 , 3 ) 2,3-dihydroxy-olean-
a
b
.91 (s, 3H, CH
.82 (s, 3H, CH
3
3
-29), 0.91e0.80 (m, 1H, H
b
-1),
12-en-28-oate (18). Obtained from 1 by method A as a colorless
ꢂ
3
3
-26)
solid; yield: 78%; m.p. 148e149 C; R
F
¼ 0.27 (n-hexane/ethyl ac-
þ
ꢂ
ppm; MS (ESI, MeOH, source CID): m/z ¼ 549.6 (83.0%, [M þ Na] ),
etate, 5:3); [
a
]
D
¼ þ46.9 (c 3.2, CHCl
3
); IR (KBr):
n
¼ 3422, 2948,
þ
2þ
5
8
80.9 (9.8%, [M þ Na þ MeOH] ), 801.6 (12.2%, [3M þ Na þ H] ),
2864, 1726, 1636, 1460, 1386, 1364, 1260, 1230, 1196, 1176, 1160,
2
þ
þ
ꢁ1 1
10.0 (34.1%, [3M þ K þ H] ), 1075.3 (100%, [2M þ Na] ).
1122, 1050, 1034 cm ; H NMR (400 MHz, CDCl
3
):
d
¼ 5.82 (dddd,
J ¼ 10.8, 7.7, 7.7, 1.3 Hz, 1H, CH (33)), 5.71 (ddd, J ¼ 10.8, 6.8, 6.8 Hz,
CH-32), 5.29 (dd, J ¼ 3.5, 3.5 Hz, 1H, H-12), 4.66 (ddd, J ¼ 13.1, 6.9,
5
.2.7.15. 3-Butyn-1-yl (2 , 3 ) 2,3-dihydroxy-olean-12-en-28-oate
a
b
(
8
15). Obtained from 1 by method A as a colorless solid; yield:
1.2 Hz, 1H, CH
a
-31), 4.59 (ddd, J ¼ 13.1, 6.7, 1.2 Hz, 1H, CH
b
-31), 4.15
ꢂ
9%; m.p. 168e169 C; R
F
¼ 0.22 (n-hexane/ethyl acetate 1:1);
(ddd, J ¼ 11.9, 7.7, 0.9 Hz, 1H, CH
0.9 Hz, 1H, CH
(34)), 3.69 (ddd, J ¼ 11.4, 9.5, 4.5 Hz, 1H, H-2), 3.0 (d,
J ¼ 9.5 Hz, 1H, H-3), 2.85 (dd, J ¼ 13.7, 3.7 Hz, 1H, H-18), 2.11 (brs,
2H, OH), 2.03e1.87 (m, 4H, H -1 þ Ha,b-11), 1.70 (dd,
-16 þ CH
J ¼ 13.7, 13.7, 4.4 Hz, 1H, H -7), 1.68e1.50 (m, 6H, H-9 þ H -19 þ H
15 þ H -16 þ H -6 þ H -7), 1.47e1.40 (m, 2H, H -6 þ H -21), 1.39e
1.27 (m, 2H, H -21), 1.23e1.15 (m, 2H, H -21 þ H -19), 1.13
-22 þ H
(s, 3H, CH -27), 1.09e1.01 (m, 1H, H -15), 1.02 (s, 3H, CH -23), 0.98
(s, 3H, CH -25), 0.92 (s, 3H, CH -30), 0.90 (s, 3H, CH -29), 0.89e0.84
(m, 2H, H-5 þ H -1), 0.82 (s, 3H, CH -24), 0.75 (s, 3H, CH
a
(34)), 4.25 (ddd, J ¼ 12.0, 7.7,
ꢂ
[
a
]
D
¼ þ48.3 (c 3.7, CHCl
3
); IR (KBr):
n
¼ 3422, 2942, 2864, 1724,
b
1
636, 1466, 1458, 1386, 1364, 1304, 1260, 1228, 1198, 1178, 1160,
ꢁ
1 1
1
124, 1088, 1050, 1040 cm ; H NMR (400 MHz, CDCl
dd, J ¼ 3.5, 3.5 Hz, 1H, H-12), 4.19e4.05 (m, 2H, CH -31), 3.72 (dd,
J ¼ 14.0, 7.4 Hz, 2H, CH -32), 3.68 (ddd, J ¼ 11.3, 9.6, 4.5 Hz,1H, H-2),
.00 (d, J ¼ 9.5 Hz,1H, H-3), 2.88 (dd, J ¼ 13.8, 4.1 Hz, 1H, H-18), 2.50
-32), 2.01e1.95 (m, 2H, H -16),
.95e1.88 (m, 2H, Ha,b-11), 1.71 (ddd, J ¼ 13.8, 13.8, 4.4 Hz,1H, H
.71e1.50 (m, 6H, H -15 þ H -16 þ H -6 þ H
-19 þ H-9 þ H
.47e1.35 (m, 2H, H -22), 1.39e1.26 (m, 2H, H -21 þ H
-6 þ H
3
):
d
¼ 5.30
a
a
(
2
a
a
a
-
2
b
a
b
b
a
3
b
b
b
b
(
1
dt, J ¼ 6.8, 2.7 Hz, 2H, CH
2
a
-1 þ H
a
3
b
3
a
-7),
-7),
3
3
3
1
1
a
a
b
a
b
b
3
3
-26) ppm;
þ
b
a
a
b
-22),
MS (ESI, MeOH, source CID): m/z ¼ 583.3 (100%, [M þ Na] ), 584.3

B. Siewert et al. / European Journal of Medicinal Chemistry 70 (2013) 259e272
269
þ
þ
þ
þ
(
[
41%, [M
þ
Na] ), 585.3 (41%, [M
þ
Na] ), 861.9 (6%,
z ¼ 641.7 (20%, [M þ H] ), 663.7 (100%, [M þ Na] ), 1303.3 (50%,
2
þ
2þ
þ
3M þ K þ H] ), 1038.9 (32%, [4M þ K þ H] ).
[2M þ Na] ).
5
.2.7.19. 4-Chloro-but-3-yn-1-yl (2 , 3 ) 2,3-dihydroxy-olean-12-
a
b
5.2.7.22. Octadecyl (2a, 3b) 2,3-dihydroxy-olean-12-en-28-oate
en-28-oate (19). Obtained from 1 by method A as a colorless
(22). Obtained from 1 by method A as a colorless solid; yield:
ꢂ
ꢂ
solid; yield: 72%; m.p. 184e185 C; R
F
¼ 0.36 (n-hexane/ethyl ace-
82%; m.p. 119e120 C; R
F
¼ 0.5 (n-hexane/ethyl acetate, 1:1);
ꢂ
ꢂ
tate, 1:1); [
a
]
D
¼ þ53.6 (c 3.1, CHCl
3
); IR (KBr):
n
¼ 3430, 2944,
[
a
]
D
¼ 37.2 (c 3.4, CHCl
3
); IR (KBr):
n
¼ 3572, 3314, 3150, 2922,
2
862, 1734, 1466, 1458, 1388, 1364, 1262, 1174, 1158, 1118, 1052,
2852, 1726, 1468, 1386, 1362, 1262, 1228, 1200, 1180, 1160, 1124,
ꢁ
1
1
ꢁ1 1
1034 cm ; H NMR (400 MHz, CDCl
3
):
d
¼ 5.30 (dd, J ¼ 3.3, 3.3 Hz,
-31), 4.64 (ddd,
-31), 4.14 (dd, J ¼ 1.9 Hz, 2H, CH (34)),
1052, 1036 cm ; H NMR (400 MHz, CDCl
3.3 Hz, 1H, H-12), 4.06e3.93 (m, 2H, CH -31), 3.72e3.64 (m, 1H,
H-2), 3.00 (d, J ¼ 9.3 Hz, 1H, H-3), 2.87 (dd, J ¼ 13.8, 4.1 Hz, 1H, H-
18), 2.14 (brs, 2H, OH), 2.00e1.87 (m, 4H, Ha,b-11 þ H -16 þ H -1),
1.69 (ddd, J ¼ 13.8, 13.8, 4.4 Hz, 1H, H -7), 1.65e1.50 (m, 6H, H-
9 þ H -19 þ H -15 þ CH -32 þ H -16), 1.50e1.43 (m, 1H, H -6),
1.42e1.30 (m, 3H, H -22 þ H -6), 1.37e1.22 (m, 27H, H
-7 þ H
21 þ CH -33 þ CH -47 þ H -22 þ CH -46 þ CH -(36e44)), 1.22e
1.11 (m, 2H, H -21), 1.13 (s, 3H, CH -27), 1.08e1.00 (m,
-19 þ H
1H, H -15), 1.02 (s, 3H, CH -23), 0.97 (s, 3H, CH -25), 0.91 (s, 3H,
CH -30), 0.90 (s, 3H, CH -48),
0.86e0.75 (m, 2H, H-5 þ H
CH
3
):
d
¼ 5.28 (dd, J ¼ 3.3,
1
H, H-12), 4.71 (ddd, J ¼ 15.7, 1.9, 1.9 Hz, 1H, CH
a
2
J ¼ 15.7, 1.9, 1.9 Hz, 1H, CH
b
2
3
2
.70 (ddd, J ¼ 11.2, 9.5, 4.4 Hz, 1H, H-2), 2.99 (d, J ¼ 9.5 Hz, 1H, H-3),
a
a
.86 (dd, J ¼ 13.7, 3.7 Hz, 1H, H-18), 2.14 (brs, 2H, OH), 2.01 (ddd,
-16), 2.00e1.87 (m, 3H, CH
-1 þ Ha,b-11),
.71 (ddd, J ¼ 13.8, 13.8, 4.2 Hz, 1H, H -7), 1.68e1.50 (m, 6H, H-
a
J ¼ 13.3, 13.2, 3.3 Hz, 1H, H
a
a
a
a
2
b
a
1
9
a
b
a
b
a
-
þ H
þ H
1 þ H
-15), 1.02 (s, 3H, CH
0), 0.90 (s, 3H, CH
-29), 0.89e0.84 (m, 2H, H-5 þ H
CH -24), 0.75 (s, 3H, CH -26) ppm; MS (ESI, MeOH, source CID): m/
z ¼ 581.5 (100%, [M þ Na] ), 858.4 (24%, [3M þ K þ H] ), 1038.9
a
-19 þ H
-21), 1.39e1.27 (m, 2H, H
-19), 1.13 (s, 3H, CH
-23), 0.98 (s, 3H, CH
a
-15 þ H
b
-16 þ H
a
-6 þ H
b
-7), 1.47e1.40 (m, 2H, H
b
-
2
2
b
2
2
6
2
H
a
b
-22 þ H
a
-21), 1.23e1.15 (m, 2H, H
b
-
b
b
3
b
3
-27), 1.06 (ddd, J ¼ 14.6, 3.9, 3.9 Hz, 1H,
-25), 0.92 (s, 3H, CH
-1), 0.82 (s, 3H,
b
3
3
b
3
3
3
-
3
3
-29), 0.88 (t, J ¼ 7.0 Hz, 3H, CH
3
3
3
b
b
-1), 0.82 (s, 3H, CH
3
-23), 0.73 (s, 3H,
3
3
3
-26) ppm; MS (ESI, MeOH, source CID): m/z ¼ 725.5 (36%,
þ
2þ
þ
þ
2þ
[M þ H] ), 747.6 (100%, [M þ Na] ), 1107.2 (32%, [3M þ K þ H] ),
2
þ
þ
(
54%, [4M þ K þ H] ).
1471.5 ( 85%, [2M þ Na] ).
5
.2.7.20. Hexyl (2 , 3
a
b
) 2,3-dihydroxy-olean-12-en-28-oate (20).
5.2.7.23. (Cyclohexyl)-methyl (2a, 3b) 2,3-dihydroxy-olean-12-en-
Obtained from 1 by method A as a colorless solid; yield: 81%;
28-oate (23). Obtained from 1 by method A as a colorless solid;
ꢂ
ꢂ
m.p. 161e163 C;
R
F
¼ 0.24 (n-hexane/ethyl acetate, 1:1);
yield: 74%; m.p. 198e200 C; R
F
¼ 0.43 (n-hexane/ethyl acetate,
ꢂ
ꢂ
[
a
]
D
¼ þ53.2 (c 6.2, CHCl
3
); IR (KBr):
n
¼ 3418, 2950, 2861, 1725,
1:1); [
a]
D
¼ þ46 (c 3.2, CHCl
3
); IR (KBr): n
¼ 3424, 2928,1725, 1615,
ꢁ1 1
1
9
3
466, 1386, 1364, 1302, 1262, 1200, 1178, 1161, 1124, 1051, 1034,
1451, 1385, 1261, 1161, 1051, 1034 cm ; H NMR (500 MHz, CDCl
¼ 5.21 (dd, J ¼ 3.5, 3.5 Hz, 1H, H-12), 3.78e3.70 (m, 2H, CH -31),
3.62 (ddd, J ¼ 11.1, 11.0, 4.5 Hz, 1H, H-2), 2.93 (d, 1H, J ¼ 9.5 Hz, H-3),
2.81 (dd, J ¼ 13.8, 4.2 Hz, 1H, H-18), 1.91 (dd, J ¼ 12.5, 4.7 Hz, 1H, H
1), 1.89e1.81 (m, 3H, H
-16 þ Ha,b-11), 1.69e1.50 (m, 10H, CH-
32 þ CH -33 þ CH -34 þ CH -35 þ H -7 þ H -19 þ H -16 þ H
15 þ H-9), 1.50e1.35 (m, 4H, H -22 þ CH -6 þ H-7), 1.30 (ddd,
J ¼ 14.6, 10.8, 3.2 Hz, 1H, H -21), 1.25e1.07 (m, 3H, H
3
):
ꢁ1
1
96 cm
;
H NMR (500 MHz, CDCl
3
):
d
¼ 5.27 (dd, J ¼ 3.5,
d
2
.5 Hz, 1H, H-12), 4.00 (dt, J ¼ 6.5, 3.0 Hz, 2H, CH
a
-31), 3.68 (ddd,
J ¼ 11.2, 9.5, 4.4 Hz, 1H, H-2), 2.99 (d, J ¼ 9.5 Hz, 1H, H-3), 2.87
dd, J ¼ 13.8, 4.1 Hz, 1H, H-18), 2.25 (brs, 1H, OH), 2.21 (brs, 1H,
OH), 2.00e1.87 (m, 4H, CH -16 þ Ha,b-11), 1.70 (ddd,
-1 þ H
J ¼ 13.8, 13.8, 4.4 Hz, 1H, H-7), 1.68e1.55 (m, 6H, H-9 þ H
-16), 1.58e1.50 (m, 2H, H -6),
-6), 1.34e1.26 (m, 8H, H -35 þ CH
-21 þ CH
-22), 1.16e1.08 (m, 2H, H -19), 1.12 (s,
-21 þ H
-27), 1.08e1.00 (m, 1H, H -15), 1.02 (s, 3H, CH -23), 0.97
-25), 0.92 (s, 3H, CH -30), 0.90 (s, 3H, CH -29), 0.92e
.85 (m, 1H, H -36), 0.85e0.79 (m,
-1), 0.89 (t, J ¼ 6.8 Hz, 3H, CH
H, H-5), 0.82 (s, 3H, CH -24), 0.73 (s, 3H, CH
a
-
(
a
a
a
a
a
2
a
a
b
a
-
a
-
a
2
1
9 þ CH
2
-32 þ H
a
-15 þ H
b
b
-7 þ H
a
a
b
-21 þ H
-19), 1.06 (s,
-1 þ H -15),
-29), 0.82e
-26) ppm; MS
b
-
1
3
3
.41e1.35 (m, 1H, H
b
a
2
2
-
22 þ H
3H, CH
0.91 (s, 3H, CH
b
-34 þ H
b
-33), 1.12 (dd, J ¼ 14.9, 4.8 Hz, 1H, H
b
b
3 þ CH
H, CH
2
-34 þ H
a
b
b
3
-27), 0.96 (s, 3H, CH
3
-23), 1.01e0.89 (m, 2H, H
b
3
b
3
3
3 3
-25), 0.86 (s, 3H, CH -30), 0.83 (s, 3H, CH
(
s, 3H, CH
3
3
3
0.77 (m,1H, H-5), 0.76 (s, 3H, CH
3
-24), 0.66 (s, 3H, CH
3
þ
0
1
b
3
(ESI, MeOH): m/z ¼ 569.5 (18%, [2M þ H] ), 1159.3 (100%,
þ
3
3
-26) ppm; MS (ESI,
[2M þ Na] ).
þ
MeOH, source CID): m/z ¼ 557.3 (14.6%, [M þ H] ), 579.6 (100.0%,
þ
þ
[
[
M þ Na] ), 607.5 (34.1%, [M þ NH
4
þMeOH þ H] ), 855.0 (26.9%,
5.2.7.24. (2
a,
3b
)
2,3-Di-O-acetyl-maslinic
acid
(24).
2
þ
þ
3M þ K þ H] ), 1135.3 (85.3%, [2M þ Na] ).
Acetylation of 1 (1.2 g; 2.54 mmol) in dry dichloromethane (50 mL)
with acetic anhydride (0.96 mL, 10.16 mmol) and triethylamine
ꢂ
5.2.7.21. Dodecyl (2
a, 3b
) 2,3-dihydroxy-olean-12-en-28-oate (21).
(0.96 mL, 10.16 mmol) for 12 h at 24 C followed by aqueous work-
Obtained from 1 by method A as a colorless solid; yield: 99%; M.p.
up and recrystallization from ethanol gave 24 as a colorless solid;
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
1
41e142 C; R
F
¼ 0.5 (n-hexane/ethyl acetate, 1:1); [
a
]
D
¼ þ45.7 (c
yield: 88%; m.p. 170e173 C, (lit.: 175e180 C [51]); [
8.3, CHCl ); R
80:25:30:4); IR (KBr):
a
]
D
¼ þ30 (c
2
.9, CHCl ); IR (KBr):
3
n
¼ 3326, 2926, 2854, 1726, 1704, 1466, 1460,
3
F
¼ 0.61 (toluene/ethyl acetate/n-heptane/formic acid,
1438, 1386, 1382, 1362, 1262, 1200, 1180, 1160, 1124, 1052,
n
¼ 2949, 1748, 1694, 1464, 1369, 1252, 1183,
ꢁ
1
1
ꢁ1 1
1034 cm ; H NMR (400 MHz, CDCl
3
):
d
¼ 5.28 (dd, J ¼ 3.4, 3.4 Hz,
1156, 1045 cm ; H NMR (500 MHz, CDCl ):
3
d
¼ 5.18 (dd, J ¼ 3.4,
1
4
4
1
H, H-12), 4.06e3.94 (m, 2H, CH
2
-31), 3.68 (ddd, J ¼ 10.8, 9.5,
3.4 Hz, 1H, H-12), 5.01 (ddd, J ¼ 11.4, 9.8, 4.7 Hz, 1H, H-2), 4.66 (d,
J ¼ 10.4 Hz, 1H, H-3), 2.73 (dd, J ¼ 13.8, 4.1 Hz, 1H, H-18), 1.96 (s, 3H,
.1 Hz, 1H, H-2), 3.00 (d, J ¼ 9.4 Hz, 1H, H-3), 2.87 (dd, J ¼ 13.7,
.1 Hz, 1H, H-18), 2.20e2.10 (br, 2H, OH), 2.98 (dd, J ¼ 12.2, 4.0 Hz,
CH
CH
3
a
-32), 1.92 (dd, J ¼ 11.3 Hz, 4.3 Hz, 1H, H
a
-1), 1.91e1.90 (m, 1H,
H, H
3.8, 4.4 Hz, H
32) þ H -19), 1.54e1.48 (m, 1H, H
2), 1.37e1.33 (m, 1H, H -6), 1.33e1.20 (m, 15H, H
6 þ H -22 þ CH -42 þ CH -40) þ CH -35 þ CH -36 þ CH
7 þ CH -38),1.21e1.15 (m,1H, H -21),1.17e1.11 (m,1H, H
s, 3H, CH -27), 1.08e1.04 (m, 1H, H -15), 1.03 (s, 3H, CH
s, 3H, CH -25), 0.92 (s, 3H, CH -30), 0.90 (s, 3H, CH -29), 0.91e0.85
m, 1H, H -1), 0.86e0.82 (m, 1H, H-5), 0.82 (s, 3H, CH -24), 0.73 (s,
H, CH -26) ppm; MS (ESI, MeOH, source CID): m/
(42) þ CH
a
-1), 1.97e1.88 (m, 3H, H
a
-16 þ Ha,b-11), 1.71 (ddd, J ¼ 13.8,
(11)), 1.89 (s, 3H, CH
3
(33)), 1.84 (ddd, J ¼ 12.5, 9.9, 3.5 Hz, 1H,
-16 þ CH (11)), 1.68 (ddd, 1H, J ¼ 14.1,
-7), 1.65e1.62 (m, 1H, H
-15), 1.60 (dd, J ¼ 11.8,
4.1 Hz, 1H, H-9), 1.56e1.48 (m, 2H, H -19), 1.48e1.45 (m, 1H,
-7 þ H
-6), 1.41e1.32 (m, 2H, H -6), 1.27 (ddd, J ¼ 11.7, 11.7,
-22 þ H
1.9 Hz, 1H, H -21), 1.19e1.09 (m, 2H, H -22), 1.06 (ddd,
-21 þ H
J ¼ 13.9, 4.5, 2.0 Hz, 1H, H -19), 1.03 (s, 3H, CH3ꢁ27), 1.01e0.86 (m,
4H, CH -1 þ H-5), 0.96 (s, 3H, CH -23), 0.84 (s, 3H, CH -25),
-15 þ H
0.83 (s, 3H, CH -30), 0.82 (s, 3H, CH -29), 0.81 (s, 3H, CH
(s, 3H, CH
1
(
2
1
a
-7), 1.66e1.57 (m, 6H, H-9 þ H
-6), 1.47e1.42 (m, 2H, H
-21 þ H
b
-16 þ H
a
-15 þ CH
2
H
a
-16), 1.80e1.72 (m, 2H, H
b
b
a
a
b
-7 þ H
a
-
-
-
10.8, 4.5 Hz, H
a
a
b
a
b
2
b
a
b
2
2
2
2
H
a
a
b
3
2
b
b
-19),1.13
-23), 0.97
a
b
b
(
(
(
3
b
3
b
3
3
3
2
b
3
3
b
3
3
3
3
-24), 0.65
þ
6
3
3
3
-26) ppm; MS (ESI, MeOH): m/z ¼ 557.4 (49%, [M þ H] ),

270
B. Siewert et al. / European Journal of Medicinal Chemistry 70 (2013) 259e272
þ
þ
ꢂ
5
74.5 (100%, [M þ NH
4
] ), 579.5 (51%, [M þ Na] ), 1135.2 (100%,
n-heptane/formic acid, 80:25:30:4); [
(KBr):
NMR (500 MHz, CDCl
5.88e5.78 (m, 2H, CH
(dd, J ¼ 17.2, 1.4 Hz,1H, H
(dd, J ¼ 11.2, 10.9, 4.5 Hz,1H, H-2), 4.75 (d, J ¼ 10.3 Hz, 1H, H-3), 4.01
(dddd, J ¼ 11.9, 6.0, 5.9, 1.2 Hz, 1H, CH -32), 3.61 (ddd, J ¼ 15.6, 5.8,
4.5 Hz, 1H, H
-32), 2.54 (dd, J ¼ 12.8, 3.4 Hz, 1H, H-18), 2.06e2.00
(m, 1H, H -1), 2.05 (s, 3H, CH
(Ac)), 2.03 (ddd, J ¼ 12.4, 12.4, 4.7 Hz,
1H, H -16), 1.98 (s, 3H, CH (Ac)), 1.96e1.82 (m, 2H, Ha,b-11), 1.77
(dd, J ¼ 13.3 Hz, 1H, H -19), 1.74e1.69 (m, 1H, H -7), 1.69e1.61 (m,
2H, H-9 þ H -16), 1.61e1.41 (m, 5H, H -22 þ H -7 þ H -15 þ CH -6),
1.41e1.24 (m, 3H, H -21), 1.24e1.16 (m, 1H, H -19), 1.15 (s,
-22 þ CH
3H, CH -27), 1.09e0.96 (m, 2H, H -15), 1.05 (s, 3H, CH -25),
-1 þ H
a
]
D
¼ þ11 (c 4.2, CHCl
3
); IR
þ
ꢁ1 1
[2M þ Na] ).
n
¼ 3428, 2948, 1744, 1662, 1517, 1368, 1252, 1043 cm ; H
3
):
-31), 5.37 (dd, J ¼ 3.3, 3.3 Hz, 1H, H-12), 5.16
-33), 5.12 (dd, J ¼ 10.3,1.3 Hz, H -33), 5.09
d
¼ 5.97e5.91 (dd, J ¼ 5.3, 5.2 Hz, 1H, NH),
5.2.7.25. (2
a
, 3
b
) 2,3-Dihydroxy-28-oxo-olean-12-en-28-amide (25).
Obtained from 24 by method B as a colorless solid; yield: 61%; m.p.
92e295 C, (lit.: 160e162 C [25]); R
tate/n-heptane/formic acid, 80:25:30:4); [
2
a
b
ꢂ
ꢂ
2
F
¼ 0.21 (toluene/ethyl ace-
ꢂ
a]
D
¼ þ52 (c 3.7, CHCl
3
),
a
ꢂ
(
1
lit.: þ60 , c 1.0, CHCl
3
/MeOH 2:1 [25]); IR (KBr):
n
¼ 3456, 2946,
):
), 5.31 (dd, J ¼ 3.5, 3.5 Hz,1H, H-12), 3.63 (ddd,
J ¼ 11.3, 9.9, 4.5 Hz, 1H, H-2), 2.94 (d, J ¼ 9.5 Hz, 1H, H-3), 2.46 (dd,
J ¼ 13.1, 3.9 Hz, 1H, H-18), 2.02e1.92 (m, 2H, H -16 þ H -1), 1.90e
.81 (m, 2H, Ha,b-11),1.80e1.71 (m, 2H, H -7), 1.65e1.52 (m,
-19 þ H
-6), 1.52e1.30 (m, 3H, H
-22 þ H -19 þ H -21), 1.11
-15), 0.97 (s, 3H, CH -30), 0.92
-1), 0.85 (s, 3H, CH -29), 0.85
b
ꢁ1 1
660, 1596, 1463, 1364, 1191, 1050 cm ; H NMR (500 MHz, CDCl
3
a
3
d
¼ 5.81 (brs, 2H, NH
2
a
3
a
a
a
a
b
a
b
a
2
1
5
6
a
a
b
2
b
H, H-9 þ H
þ H -21 þ H
-27), 1.07e0.98 (m, 1H, H
-23), 0.85e0.77 (m, 1H, H
-24), 0.84e0.82 (m, 1H, H-5), 0.76 (s, 3H, CH
b
-7 þ H
a
-15 þ H
b
-16 þ H
a
b
-
3
b
b
3
a
a
-22), 1.24e1.12 (m, 5H, H
b
b
b
0.96 (dd, J ¼ 11.9, 1.5 Hz, 1H, H-5), 0.90 (s, 12H, CH
3
-23 þ CH
3
-
(s, 3H, CH
(s, 3H, CH
(s, 3H, CH
3
b
3
30 þ CH
3
-24 þ CH
3
-29), 0.75 (s, 3H, CH
3
-26) ppm; MS (ESI, MeOH):
þ
þ
3
b
3
m/z ¼ 596.5 (100%, [M þ H] ), 618.5 (36%, [M þ Na] ), 1191.0 (28%,
þ
þ
3
3
-25), 0.76 (s,
[2M þ H] ), 1213.2 (74%, [2M þ Na] ).
þ
3
H, CH
3
-26) ppm; MS (ESI, MeOH): m/z ¼ 943.5 (22%, [2M þ H] ),
þ
9
65.4 (100%, [2M þ Na] ).
5.2.7.29. 2-Propyn-1-yl (2 , 3 ) 2,3-di-O-acetyl-28-oxo-olean-12-
en-28-amide (29). Obtained from 24 by method B as a colorless
a
b
ꢂ
5
.2.7.26. Ethyl (2
a
, 3
b
) 2,3-di-O-acetyl-28-oxo-olean-12-en-28-
amide (26). Obtained from 24 by method B as a colorless solid;
yield: 81%; m.p. 146e149 C; R
heptane/HCOOH, 80:25:30:4); [
solid; yield: 56%; m.p. 226e227 C; R
F
¼ 0.51 (toluene/ethyl
ꢂ
acetate/n-heptane/formic acid, 80:25:30:4); [
CHCl ); IR (KBr):
1251, 1044 cm
a
]
D
¼ þ1 (c 3.8,
ꢂ
F
¼ 0.41 (toluene/ethyl acetate/n-
3
n
¼ 3432, 3250, 2947, 2362, 1744, 1654, 1508, 1369,
ꢂ
ꢁ1
1
a]
D
¼ þ16 (c 3.1, CHCl
3
); IR (KBr):
;
H NMR (500 MHz, CDCl
3
):
d
¼ 6.04 (dd,
ꢁ
1 1
n
¼ 3432, 2947, 1745, 1662, 1517, 1368, 1252, 1044 cm
400 MHz, CDCl ):
¼ 5.82 (dd, J ¼ 5.1, 5.1 Hz, 1H, NH), 5.36 (dd,
J ¼ 3.4, 3.4 Hz, 1H, H-12), 5.09 (ddd, J ¼ 11.4, 11.4, 4.6 Hz, 1H, H-2),
-31), 3.19e3.03
;
H NMR
J ¼ 4.8 Hz, 1H, NH), 5.40 (dd, J ¼ 3.4, 3.4 Hz, 1H, H-12), 5.08 (ddd,
(
3
d
J ¼ 11.4, 10.7, 4.6 Hz, 1H, H-2), 4.74 (d, J ¼ 10.3 Hz, 1H, H-3), 4.03
(ddd, J ¼ 17.6, 5.4, 2.6 Hz,1H, CH -31), 3.90 (ddd, J ¼ 17.6, 4.4, 2.6 Hz,
a
4
.74 (d, J ¼ 10.3 Hz, 1H, H-3), 3.38e3.30 (m, 1H, CH
m, 1H, CH2ꢁ31), 2.50 (dd, J ¼ 12.8, 3.4 Hz, 1H, H-18), 2.05 (s, 3H, CH
Ac)), 2.02 (ddd, J ¼ 12.5, 12.5, 4.7 Hz, 1H, H -16), 1.98 (s, 3H, CH
Ac)), 1.96e1.87 (m, 3H, Ha,b-11 þ H
3.4 Hz, H
-19), 1.70e1.61 (m, 3H, H-9 þ H
m, 4H, H -22 þ H -15 þ H -6), 1.44 (ddd, J ¼ 11.9, 11.9,
-7 þ H
.3 Hz, 1H, H -6), 1.38e1.25 (m, 2H, H -21), 1.22e1.16 (m,
-22 þ H
H, H -21), 1.17e1.08 (m, 1H, H -19), 1.15 (s, 3H, CH -27), 1.10 (t,
-32), 1.06 (s, 3H, CH -25), 1.05e0.98 (m, 2H, H
-1), 0.90 (m, 13H, H-5 þ CH -23 þ CH -30 þ CH -24 þ CH
9), 0.77 (s, 3H, CH
a
1H, H
1H, C^CH (33)), 2.05 (s, 3H, CH
4.7 Hz, 1H, H -16), 1.97 (s, 3H, CH
11 þ H -1), 1.75 (dd, J ¼ 13.3, 13.3 Hz, 1H, H
b
-31), 2.53 (dd, J ¼ 12.9, 3.4 Hz, 1H, H-18), 2.20 (dd, J ¼ 2.6 Hz,
(Ac)), 2.02 (ddd, J ¼ 12.4, 12.4,
(Ac)), 1.99e1.84 (m, 3H, Ha,b
-19), 1.72e1.67 (m, 1H,
-16 þ H -6),1.51e1.41 (m,
-6 þ H -21 þ H -22), 1.22e
-27), 1.14e1.00 (m, 1H, H -19),
-15), 0.99e0.94
(24) þ CH -29),
(
(
(
1
(
3
3
a
3
a
3
-
a
-1), 1.75 (m, 1H, J ¼ 13.4,
-16 þ H -7), 1.60e1.47
a
a
a
b
a
H
a
-7), 1.66e1.51 (m, 4H, H-9 þ H
a
-22 þ H
-7), 1.41e1.26 (m, 3H, H
-21), 1.15 (s, 3H, CH
-25), 1.08e0.99 (m, 2H, H
(m,1H, H-5), 0.90 (s,12H, CH (30) þ CH
(23) þ CH
b
a
b
a
a
a
2H, H
a
-15 þ H
b
b
a
b
2
1
b
b
a
1.16 (m, 1H, H
b
3
b
b
b
3
1.06 (s, 3H, CH
3
b
-1 þ H
b
J ¼ 7.3 Hz, CH
3
3
b
3
-
-
3
3
3
3
1
2
5 þ H
b
3
3
3
0.79 (s, 3H, CH
3
-26) ppm; MS (ESI, MeOH): m/z ¼ 594.5 (91%,
þ
þ
þ
3
-26) ppm; MS (ESI, MeOH): m/z ¼ 584.5 (100%,
[M þ H] ), 616.5 (39%, [M þ Na] ), 1186.9 (24%, [2M þ H] ), 1209.2
þ
þ
þ
þ
[
M þ H] ), 606.4 (34%, [M þ Na] ), 1166.9 (14%, [2M þ H] ), 1189.2
(100%, [2M þ Na] ).
þ
(
48%, [2M þ Na] ).
5
.2.7.30. (2
a, 3b) 2,3-Dihydroxy-28-oxo-olean-12-en-28-amide (30).
5
.2.7.27. Propyl (2a, 3b) 2,3-di-O-acetyl-28-oxo-olean-12-en-28-
Obtained from 25 by method C as a colorless solid; yield: 61%; m.p.
ꢂ
ꢂ
amide (27). Obtained from 24 by method B as an off-white solid;
yield: 61%; m.p. 129e130 C; R
292e295 C (lit.: 160e162 C [26]); R
tate/n-heptane/formic acid, 80:25:30:4); [
F
¼ 0.21 (toluene/ethyl ace-
ꢂ
ꢂ
F
¼ 0.54 (toluene/ethyl acetate/n-
a
]
D
¼ þ52 (c 3.7, CHCl
3
),
ꢂ
ꢂ
heptane/formic acid, 80:25:30:4); [
KBr):
NMR (400 MHz, CDCl
a]
D
¼ þ21 (c 2.6, CHCl
3
); IR
(lit.: þ60 , c 1.0, CHCl
3
/MeOH 2:1 [26]); IR (KBr):
n
¼ 3456, 2946,
):
), 5.31 (dd, J ¼ 3.5, 3.5 Hz,1H, H-12), 3.63 (ddd,
J ¼ 11.3, 9.9, 4.5 Hz, 1H, H-2), 2.94 (d, J ¼ 9.5 Hz, 1H, H-3), 2.46 (dd,
J ¼ 13.1, 3.9 Hz, 1H, H-18), 2.02e1.92 (m, 2H, H -16 þ H -1), 1.90e
1.81 (m, 2H, Ha,b-11), 1.80e1.71 (m, 2H, H -7), 1.65e1.52 (m,
-19 þ H
5H, H-9 þ H -7 þ H -15 þ H -16 þ H -6), 1.52e1.30 (m, 3H, H
6 þ H -21 þ H -22), 1.24e1.12 (m, 5H, H -22 þ H -19 þ H -21), 1.11
(s, 3H, CH -27), 1.07e0.98 (m, 1H, H -15), 0.97 (s, 3H, CH -30), 0.92
(s, 3H, CH -23), 0.85e0.77 (m, 1H, H -1), 0.85 (s, 3H, CH -29), 0.85
(s, 3H, CH -24), 0.84e0.82 (m, 1H, H-5), 0.76 (s, 3H, CH
ꢁ1
1
ꢁ1 1
(
n
¼ 3432, 2948, 1744, 1661, 1517, 1368, 1251, 1043 cm ; H
1660, 1596, 1463, 1364,1191, 1050 cm ; H NMR (500 MHz, CDCl
¼ 5.81 (brs, 2H, NH
3
3
):
d
¼ 5.88 (dd, J ¼ 5.9, 4.9 Hz, 1H, NH), 5.36
d
2
(
dd, J ¼ 3.4, 3.4 Hz, 1H, H-12), 5.09 (ddd, J ¼ 11.4, 10.3, 4.6 Hz, 1H, H-
2
), 4.75 (d, J ¼ 10.3 Hz, 1H, H-3), 3.34 (dt, J ¼ 13.6, 7.1 Hz, 1H, CH
-31), 2.51 (dd, J ¼ 13.4, 3.3 Hz,
(Ac)), 2.02 (ddd, J ¼ 12.5, 12.5, 4.8 Hz, 1H,
(Ac)), 1.91e1.86 (m, 3H, H
-16 þ Ha,b-11), 1.77
dd, J ¼ 13.4,13.4 Hz, H -19),1.74e1.59 (m, 3H, H-9 þ H -7 þ H -16),
.55e1.36 (m, 7H, H -7 þ H -15 þ CH (32) þ CH -6), 1.36e
-22 þ H
.24 (m, 2H, H -22), 1.24e1.16 (m, 1H, C (19)), 1.15 (s, 3H,
-21 þ H
CH (17)), 1.11e1.00 (m, 2H, H -15), 1.06 (s, 3H, CH -25),
-1 þ H
.00e0.94 (m, 1H, H-5), 0.93e0.89 (m, 12H, CH
a
-
a
a
3
1
H
(
1
1
1), 2.95 (dt, J ¼ 13.6, 7.2 Hz, 1H, CH
b
a
a
H, H-18), 2.05 (s, 3H, CH
-1), 1.98 (s, 3H, CH
3
b
a
b
a
b
-
a
3
a
a
a
b
b
b
a
a
b
3
b
3
a
b
a
2
2
3
b
3
a
b
3
3
-25), 0.76 (s,
þ
3
b
b
3
3H, CH3ꢁ26) ppm; MS (ESI, MeOH): m/z ¼ 943.5 (22%, [2M þ H] ),
þ
1
3
-23 þ CH
3
-
965.4 (100%, [2M þ Na] ).
3
0 þ CH
3
-24 þ CH
3
-29), 0.77 (s, 3H, CH
3
-26) ppm; MS (ESI, MeOH):
þ
þ
m/z ¼ 598.4 (100%, [M þ H] ), 620.5 (39%, [M þ Na] ), 1195.0 (28%,
5.2.7.31. Ethyl (2a, 3b) 2,3-dihydroxy-28-oxo-olean-12-en-28-amide
(31). Obtained from 26 by method C as a colorless solid; yield: 82%;
þ
þ
[2M þ H] ), 1217.3 (68%, [2M þ Na] ).
ꢂ
ꢂ
m.p. 170e173 C; [
a
]
D
¼ þ33 (3.0, CHCl
3
); R
F
¼ 0.15 (toluene/ethyl
¼ 3422,
1 1
5
.2.7.28. 2-Propen-1-yl (2 , 3 ) 2,3-di-O-acetyl-28-oxo-olean-12-
a
b
acetate/n-heptane/formic acid, 80:25:30:4); IR (KBr): n
2945, 1637, 1525, 1384, 1259, 1050, 1031 cm ; H NMR (500 MHz,
CD OD):
¼ 7.25 (dd, J ¼ 5.5, 5.5 Hz, 1H, NH), 5.40 (dd, J ¼ 3.4,
ꢁ
en-28-amide (28). Obtained from 24 by method B as a colorless
solid; yield: 65%; m.p.158e159 C; R
ꢂ
F
¼ 0.48 (toluene/ethyl acetate/
3
d

B. Siewert et al. / European Journal of Medicinal Chemistry 70 (2013) 259e272
271
3
(
.4 Hz,1H, H-12), 3.66 (ddd, J ¼ 11.4, 9.8, 4.5 Hz,1H, H-2), 3.30e3.20
m, 1H, CH -31), 3.20e3.12 (m, 1H, CH
-31), 2.95 (dd, J ¼ 9.6 Hz, 1H,
H-3), 2.83 (dd, J ¼ 13.7, 4.1 Hz, 1H, H-18), 2.10 (ddd, J ¼ 13.8, 13.8,
.0 Hz, 1H, H -16), 1.99 (m, 3H, CH
-1 þ Ha,b-11), 1.82 (dd, J ¼ 13.5,
3.5 Hz, 1H, H -15 þ H -16 þ H
-19), 1.70e1.52 (m, 7H, H-9 þ H
þ CH -7 þ H -22), 1.49 (ddd, J ¼ 11.4, 11.4, 3.3 Hz, 1H, H -6), 1.43
ddd, J ¼ 13.9, 13.9, 4.1 Hz, H -21), 1.35 (m, 1H, H -22), 1.24e1.19 (m,
-21), 1.21 (s, 3H, CH
-27), 1.11 (t, J ¼ 7.2 Hz, 3H, CH
2), 1.10e1.01 (m, 1H, H -15), 1.05 (s, 3H, CH -23), 1.05 (s, 3H, CH
5), 0.99 (s, 3H, CH -30), 0.95 (s, 3H, CH -29), 0.97e0.90 (m, 1H, H
), 0.88 (dd, J ¼ 11.3, 0.7 Hz, 1H, H-5), 0.85 (s, 3H, CH
H, CH
J ¼ 17.3, 2.5 Hz, 1H, CH
2.95 (d, J ¼ 9.6 Hz,1H, H-3), 2.84 (dd, J ¼ 13.2, 3.5 Hz,1H, H-18), 2.54
(d, J ¼ 2.5 Hz, 1H, CH (33)), 2.14 (ddd, J ¼ 13.3, 13.3, 3.5 Hz, 1H, H
16), 2.06e1.92 (m, 3H, Ha,b-11 þ H -1), 1.81 (dd, J ¼ 13.5,13.5 Hz,1H,
-19), 1.77e1.39 (m, 9H, H-9 þ H -15 þ H -16 þ CH -6 þ CH
7 þ H -22 þ H -21), 1.37e1.25 (m, 2H, H -21 þ H -22), 1.22 (s, 3H,
CH -27), 1.29e1.16 (m, 1H, H -19), 1.14e1.06 (m, 1H, H -15), 1.05 (s,
3H, CH -23), 1.05 (s, 3H, CH -25), 0.99 (s, 6H, CH -29),
(30) þ CH
0.95 (s, 3H, CH -23), 0.91 (s, 3H, CH -24), 0.89e0.81 (m, 2H, H
b
-31), 3.66 (ddd, J ¼ 11.3, 9.7, 4.5 Hz, 1H, H-2),
a
b
a
-
4
1
6
(
a
a
a
a
a
b
a
-
H
a
a
b
2
2
-
2
a
a
a
a
b
b
a
b
3
b
b
2
3
2
1
3
5
H, H
b
-19 þ H
b
3
3
3
b
-
-
-
3
3
3
3
b
3
3
3
b
-
3
3
1 þ H-5), 0.85 (s, 3H, CH
3
-26) ppm; MS (ESI, MeOH): m/z ¼ 510.6
þ
þ
3
-24), 0.83 (s,
(40%, [M
þ
H] ), 1019.2 (10%, [2M
þ
H] ), 1041.3 (100%,
þ
þ
3
-26) ppm; MS (ESI, MeOH): m/z ¼ 500.6 (88%, [M þ H] ),
[2M þ Na] ).
þ
þ
22.7 (20%, [M þ Na] ), 999.3 (22%, [2M þ H] ), 1021.3 (100%,
þ
[2M þ Na] ).
Acknowledgments
5
.2.7.32. Propyl (2
a
, 3
b
) 2,3-dihydroxy-28-oxo-olean-12-en-28-
We like to thank Dr. D. Ströhl for the NMR spectra, Dr. R. Kluge
for the measurement of the ESIeMS spectra and Ms J. Wiese for the
recording of the IR und UVevis spectra as well as for the mea-
surement of the optical rotations. The cell lines were kindly pro-
vided by Dr. T. Müller (Dept. of Haematology/Oncology, Univ. Halle).
Support by the “Gründerwerkstatt e Biowissenschaften” is grate-
fully acknowledged. We also like to thank Dr. G. Kaluderovics and
Prof. Dr. R. Paschke for their help with the cells; B.S. would like to
thank Dr. A. Stojanovic for many fruitful discussions.
amide (32). Obtained from 27 by method C as a colorless solid;
yield: 80%; m.p. 152e154 C; R
ꢂ
F
¼ 0.19 (toluene/ethyl acetate/n-
ꢂ
heptane/formic acid, 80:25:30:4); [
KBr):
a
]
D
¼ þ47 (c 4.4, CHCl
3
); IR
ꢁ1
(
n
¼ 3417, 2945,1639,1525,1463,1384,1266,1186,1050 cm
H NMR (500 MHz, CD OD):
¼ 5.40 (dd, J ¼ 3.5, 3.5 Hz, 1H, H-12),
.65 (ddd, J ¼ 11.3, 9.8, 4.5 Hz, 1H, H-2), 3.24e3.16 (m, 1H, CH -31),
.09e3.02 (m, 1H, CH
;
1
3
d
3
3
a
b
-31), 2.94 (d, J ¼ 9.6 Hz, 1H, H-3), 2.83 (dd,
J ¼ 13.3, 3.8 Hz, 1H, H-18), 2.10 (ddd, J ¼ 13.1, 13.1, 4.4 Hz, 1H, H
.03e1.94 (m, 3H, CH
-1 þ Ha,b-11), 1.82 (dd, J ¼ 13.5 Hz, 1H, H
.73e1.50 (m, 9H, H-9 þ CH -7 þ H -22 þ H -15 þ CH
6 þ H -6), 1.49 (ddd J ¼ 11.8, 11.8, 3.5 Hz, 1H, H -6), 1.43 (ddd,
J ¼ 13.5, 13.5, 3.9 Hz, 1H, H -22), 1.37e1.30 (m, 1H, H -21), 1.22 (s,
H, CH -27),1.25e1.17 (m, 2H, H -19),1.11e1.03 (m,1H, H
-21 þ H
5), 1.05 (s, 3H, CH -23), 1.04 (s, 3H, CH -25), 0.99 (s, 3H, CH -30),
.95 (s, 3H, CH (33)), 1.00e0.87 (m,
-29), 0.94 (t, J ¼ 7.4 Hz, 3H, CH
H, H -1), 0.85 (s, 3H, CH -24),
-26) ppm; MS (ESI, MeOH): m/z ¼ 514.7 (54%,
a
-16),
2
1
1
a
a
-19),
2
a
a
2
(32) þ H
b
-
Appendix A. Supplementary data
a
b
a
b
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2013.10.016.
3
1
0
1
0
3
b
b
b
-
3
3
3
3
3
References
b
-15), 0.88e0.82 (m, 2H, H-5 þ H
b
3
.83 (s, 3H, CH
3
[1] M.E. Juan, U. Wenzel, V. Ruiz-Gutierrez, H. Daniel, J.M. Planas, Olive fruit ex-
tracts inhibit proliferation and induce apoptosis in HT-29 human colon cancer
cells, J. Nutr. 136 (2006) 2553e2557.
þ
þ
þ
[M þ H] ), 1027.3 (28%, [2M þ H] ), 1049.4 (100%, [2M þ Na] ).
[
2] Y. Allouche, G. Beltran, J.J. Gaforio, M. Uceda, M.D. Mesa, Antioxidant and
antiatherogenic activities of pentacyclic triterpenic diols and acids, Food
Chem. Toxicol. 48 (2010) 2885e2890.
5
.2.7.33. N-Prop-2-en-1-yl (2a, 3b) 2,3-dihydroxy-28-oxo-olean-12-
en-28-amide (33). Obtained from 28 by method C as white solid;
yield: 62%; m.p. 152e154 C; R
[
3] Y. Allouche, F. Warleta, M. Campos, C. Sanchez-Quesada, M. Uceda, G. Beltran,
J.J. Gaforio, Antioxidant, antiproliferative, and pro-apoptotic capacities of
pentacyclic triterpenes found in the skin of olives on MCF-7 human breast
cancer cells and their effects on DNA damage, J. Agric. Food Chem. 59 (2011)
121e130.
ꢂ
F
¼ 0.12 (toluene/ethyl acetate/n-
ꢂ
heptane/formic acid, 80:25:30:4); [
KBr):
a]
D
¼ þ42 (c 3.6, CHCl
3
); IR
(
n
¼ 3527, 3416, 2945, 1637, 1540, 1469, 1386, 1259, 1050,
ꢁ1 1
1031 cm ; H NMR (500 MHz, CDCl
3
):
H, NH), 5.81 (dddd, J ¼ 16.2, 10.6, 5.8, 5.8 Hz, 1H, CH-32), 5.38 (dd,
(33)),
d
¼ 5.97 (dd, J ¼ 5.0, 5.2 Hz,
[
[
4] Y. Allouche, M. Uceda, A. Jimenez, M.P. Aguilera, J.J. Gaforio, G. Beltran, Fruit
quality and olive leaf and stone addition affect Picual virgin olive oil tri-
terpenic content, J. Agric. Food Chem. 57 (2009) 8998e9001.
5] Y. Allouche, A. Jimenez, M. Uceda, M.P. Aguilera, J.J. Gaforio, G. Beltran, Tri-
terpenic content and chemometric analysis of virgin olive oils from forty olive
cultivars, J. Agric. Food Chem. 57 (2009) 3604e3610.
1
J ¼ 3.5, 3.5 Hz, 1H, H-12), 5.16 (dd, J ¼ 17.3, 3.1, 1.5 Hz, 1H, CH
.12 (ddd, J ¼ 10.3, 2.7, 1.4 Hz, 1H, CH (33)), 4.05e3.97 (m, 1H, CH
1), 3.69 (ddd, J ¼ 11.1, 11.1, 4.5 Hz, 1H, H-2), 3.64e3.57 (m, 1H, CH
a
5
3
b
a
-
-
b
3
2
1
1
1), 3.00 (d, J ¼ 9.3 Hz, 1H, H-3), 2.54 (dd, J ¼ 13.0, 3.6 Hz, 1H, H-18),
.18 (brs, 2H, OH), 2.00 (ddd, J ¼ 12.6, 12.6, 3.9 Hz, 1H, H -16), 1.98e
.91 (m, 3H, CH -19),
-1 þ Ha,b-11), 1.76 (dd, J ¼ 13.5, 13.5 Hz, 1H, H
.73 (ddd, J ¼ 14.0, 3.6, 3.6 Hz, 1H, H -7), 1.68e1.52 (m, 3H, H
6 þ H -7 þ H-9), 1.53e1.48 (m, 1H, H -6), 1.48e1.42 (m, 1H, H -15),
.42e1.32 (m, 2H, H -22), 1.29e1.17 (m, 4H, CH (21) þ H
-6 þ H
-19), 1.16 (s, 3H, CH -27), 1.03 (s, 3H, CH -23), 1.02e0.95 (m,
-15), 0.98 (s, 3H, CH -25), 0.94e0.87 (m, 1H, H -1), 0.91 (s,
H, CH -29), 0.89e0.81 (m, 1H, H-5), 0.83 (s, 3H, CH
[6] M.E. Juan, J.M. Planas, V. Ruiz-Gutierrez, H. Daniel, U. Wenzel, Antiproliferative
and apoptosis-inducing effects of maslinic and oleanolic acids, two pentacyclic
triterpenes from olives, on HT-29 colon cancer cells, Br. J. Nutr. 100 (2008)
a
a
a
36e43.
a
b
-
[7] 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 Ca2þ-evoked
p38 signaling pathway, Naunyn-Schmiedeberg’s Arch. Pharmacol. 383
1
b
a
a
1
2
1
6
2
b
a
2
b
-
(2011) 321e330.
2 þ H
b
3
3
[8] F.J. Reyes-Zurita, E.E. Rufino-Palomares, J.A. Lupianez, M. Cascante, Maslinic
acid, a natural triterpene from Olea europaea L., induces apoptosis in HT29
human colon-cancer cells via the mitochondrial apoptotic pathway, Cancer
Lett. 273 (2009) 44e54.
[9] X. Wen, H. Sun, J. Liu, G. Wu, L. Zhang, X. Wu, P. Ni, Pentacyclic triterpenes.
Part 1: the first examples of naturally occurring pentacyclic triterpenes as a
new class of inhibitors of glycogen phosphorylases, Bioorg. Med. Chem. Lett.
H, H
b
3
b
3
(30) þ CH
3
3
-
4), 0.75 (s, 3H, CH
3
-26) ppm; MS (ESI, MeOH): m/z ¼ 512.5 (65%,
þ
þ
þ
[
(
M þ H] ), 534.5 (15%, [M þ Na] ), 1023.3 (18%, [2M þ H] ), 1045.5
þ
100%, [2M þ Na] ).
15 (2005) 4944e4948.
[
[
[
10] X. Wen, P. Zhang, J. Liu, L. Zhang, X. Wu, P. Ni, H. Sun, Pentacyclic triterpenes.
Part 2: synthesis and biological evaluation of maslinic acid derivatives as
glycogen phosphorylase inhibitors, Bioorg. Med. Chem. Lett. 16 (2006) 722e
5.2.7.34. N-Prop-2-yn-1-yl (2a, 3b)-2,3-dihydroxy-28-oxo-olean-12-
en-28-amide (34). Obtained from 29 by method C as a bright yel-
low solid; yield: 62%; m.p. 181e183 C; R
acetate/n-heptane/formic acid, 80:25:30:4); [
CHCl ); IR (KBr):
726.
ꢂ
F
¼ 0.22 (toluene/ethyl
11] W.W. Qiu, Q. Shen, F. Yang, B. Wang, H. Zou, J.Y. Li, J. Li, J. Tang, Synthesis and
biological evaluation of heterocyclic ring-substituted maslinic acid derivatives
as novel inhibitors of protein tyrosine phosphatase 1B, Bioorg. Med. Chem.
Lett. 19 (2009) 6618e6622.
12] A. Parra, F. Rivas, P.E. Lopez, A. Garcia-Granados, A. Martinez, F. Albericio,
N. Marquez, E. Munoz, Solution- and solid-phase synthesis and anti-HIV
ꢂ
a
]
D
¼ þ34 (c 0.35,
3
n
1
¼ 3418, 2946, 2360, 1646, 1515, 1464, 1387, 1256,
ꢁ1
1186, 1050 cm ; H NMR (400 MHz, CD
3
OD):
d
¼ 5.40 (dd, J ¼ 3.5,
-31), 3.91 (dd,
3
.5 Hz, 1H, H-12), 4.0 (dd, J ¼ 17.3, 2.5 Hz, 1H, CH
a

272
B. Siewert et al. / European Journal of Medicinal Chemistry 70 (2013) 259e272
activity of maslinic acid derivatives containing amino acids and peptides,
Bioorg. Med. Chem. 17 (2009) 1139e1145.
[32] M. Burnouf-Radosevich, N.E. Delfel, R. England, Gas chromatography-mass
spectrometry of oleanane- and ursane-type triterpenes application to
e
[
[
13] A. Parra, P.E. Lopez, A. Garcia-Granados, Different pathways for the deoxy-
genation of the A-ring of natural triterpene compounds, Nat. Prod. Res. 24
Chenopodium quinoa triterpenes, Phytochemistry 24 (1985) 2063e2066.
[33] 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, J. Natl. Cancer Inst. 82 (1990) 1107e1112.
[34] T. Honda, Y. Honda, F.G. Favaloro Jr., G.W. Gribble, N. Suh, A.E. Place,
(
2010) 177e196.
14] F.J. Reyes, J.J. Centelles, J.A. Lupianez, M. Cascante, (2Alpha,3beta)-2,3-
dihydroxyolean-12-en-28-oic acid, new natural triterpene from Olea
a
europea, induces caspase dependent apoptosis selectively in colon adeno-
carcinoma cells, FEBS Lett. 580 (2006) 6302e6310.
15] F.J. Reyes-Zurita, G. Pachon-Pena, D. Lizarraga, E.E. Rufino-Palomares,
M. Cascante, J.A. Lupianez, The natural triterpene maslinic acid induces
apoptosis in HT29 colon cancer cells by a JNK-p53-dependent mechanism,
BMC Cancer 11 (2011) 154.
M.H. Rendi, M.B. Sporn, A novel dicyanotriterpenoid, 2-cyano-3,12-
dioxooleana-1,9(11)-dien-28-onitrile, active at picomolar concentrations for
inhibition of nitric oxide production, Bioorg. Med. Chem. Lett. 12 (2002)
1027e1030.
[
[35] M. Broniatowski, M. Flasi nꢀ ski, P. Wydro, Investigation of the interactions of
lupane type pentacyclic triterpenes with outer leaflet membrane phospho-
[
16] R. Martin, J. Carvalho-Tavares, E. Ibeas, M. Hernandez, V. Ruiz-Gutierrez,
M.L. Nieto, Acidic triterpenes compromise growth and survival of astrocytoma
cell lines by regulating reactive oxygen species accumulation, Cancer Res. 67
lipids e Langmuir monolayer and synchrotron X-ray scattering study,
J. Colloid Interface Sci. 381 (2012) 116e124.
[36] J. Prades, O. Vogler, R. Alemany, M. Gomez-Florit, S.S. Funari, V. Ruiz-Gutier-
rez, F. Barcelo, Plant pentacyclic triterpenic acids as modulators of lipid
membrane physical properties, Biochim. Biophys. Acta 1808 (2011) 752e760.
[37] H.L. Ziegler, H. Franzyk, M. Sairafianpour, M. Tabatabai, M.D. Tehrani,
K. Bagherzadeh, H. Hagerstrand, D. Staerk, J.W. Jaroszewski, Erythrocyte
membrane modifying agents and the inhibition of Plasmodium falciparum
growth: structure-activity relationships for betulinic acid analogues, Bioorg.
Med. Chem. 12 (2004) 119e127.
[38] Z. Darzynkiewicz, H.D. Halicka, H. Zhao, Analysis of cellular DNA content by
flow and laser scanning cytometry, Adv. Exp. Med. Biol. 676 (2010) 137e147.
[39] Z. Darzynkiewicz, X. Huang, M. Okafuji, M.A. King, Cytometric methods to
detect apoptosis, Methods Cell. Biol. 75 (2004) 307e341.
[40] Z. Darzynkiewicz, G. Juan, X. Li, W. Gorczyca, T. Murakami, F. Traganos,
Cytometry in cell necrobiology: analysis of apoptosis and accidental cell death
(necrosis), Cytometry 27 (1997) 1e20.
[41] D. Wlodkowic, J. Skommer, Z. Darzynkiewicz, Cytometry in cell necrobiology
revisited. Recent advances and new vistas, Cytometry Part A 77A (2010) 591e
606.
(
2007) 3741e3751.
[
[
[
[
[
17] X. He, R.H. Liu, Triterpenoids isolated from apple peels have potent anti-
proliferative activity and may be partially responsible for apple’s anticancer
activity, J. Agric. Food Chem. 55 (2007) 4366e4370.
18] V. Amico, V. Barresi, D. Condorelli, C. Spatafora, C. Tringali, Antiproliferative
terpenoids from almond hulls (Prunus dulcis): identification and structure-
activity relationships, J. Agric. Food Chem. 54 (2006) 810e814.
19] B. Bednarczyk-Cwynar, L. Zaprutko, P. Ruszkowski, B. Hladon, Anticancer ef-
fect of A-ring or/and C-ring modified oleanolic acid derivatives on KB, MCF-7
and HeLa cell lines, Org. Biomol. Chem. 10 (2012) 2201e2205.
20] A. Ortiz, A. Cardoso-Taketa, M.R. Monroy, J. Arellano, G. Hernandez,
M.L. Villarreal, Transformed cell suspension culture of Galphimia glauca pro-
ducing sedative nor-friedelanes, Planta Med. 76 (2010) 386e392.
21] S.H. Youn, J.S. Lee, M.S. Lee, E.Y. Cha, P.T. Thuong, J.R. Kim, E.S. Chang, Anti-
cancer properties of pomolic acid-induced AMP-activated protein kinase
activation in MCF7 human breast cancer cells, Biol. Pharm. Bull. 35 (2012)
105e110.
[
[
[
22] R. Rodriguez-Rodriguez, J.S. Perona, M.D. Herrera, V. Ruiz-Gutierrez, Tri-
terpenic compounds from “orujo” olive oil elicit vasorelaxation in aorta from
spontaneously hypertensive rats, J. Agric. Food Chem. 54 (2006) 2096e2102.
23] C. Moneriz, P. Marin-Garcia, A. Garcia-Granados, J.M. Bautista, A. Diez,
A. Puyet, Parasitostatic effect of maslinic acid. I. Growth arrest of Plasmodium
falciparum intraerythrocytic stages, Malar. J. 10 (2011) 82.
24] C. Moneriz, P. Marin-Garcia, J.M. Bautista, A. Diez, A. Puyet, Parasitostatic ef-
fect of maslinic acid. II. Survival increase and immune protection in lethal
Plasmodium yoelii-infected mice, Malar. J. 10 (2011) 103.
[42] A.V. Zelenin, Chapter nine e acridine orange as a probe for cell and molecular
biology, in: W.T. Mason (Ed.), Fluorescent and Luminescent Probes for Biological
Activity, second ed., Academic Press, London, 1999, pp. 117e135.
[43] H. Sakahira, M. Enari, S. Nagata, Cleavage of CAD inhibitor in CAD activation
and DNA degradation during apoptosis, Nature 391 (1998) 96e99.
[44] I. Vermes, C. Haanen, H. Steffens-Nakken, C. Reutellingsperger, A novel assay
for apoptosis flow cytometric detection of phosphatidylserine expression on
early apoptotic cells using fluorescein labelled Annexin V, J. Immunol.
Methods 184 (1995) 39e51.
[
25] J.A.R. Salvador, Pentacyclic Triterpenes as Promising Agents in Cancer, Nova
Science Pub Inc, 2010.
[45] H.J. Motulsky, A. Christopoulos, Fitting Models to Biological Data Using Linear
and Nonlinear Regression. A Practical Guide to Curve Fitting, Oxford Univer-
sity Press, New York, 2004.
[46] P.N. Dean, J.H. Jett, Mathematical analysis of DNA distributions derived from
flow microfluorometry, J. Cell Biol. 60 (1974) 523e527.
[
26] 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) 5991e6001.
[
[
[
27] S. Fulda, Betulinic acid for cancer treatment and prevention, Int. J. Mol. Sci. 9
[47] A. Garcia-Granados, Process for the industrial recovery of oleanolicand mas-
linic acids contained in the olive milling by products. P.C.T. Int. Appl, WO
9804331, Chem. Abstr. 128 (1998) 179706.
[48] M.S. Zheng, Y.-K. Lee, Y. Li, K. Hwangbo, C.-S. Lee, J.-R. Kim, S.K.-S. Lee, H.-
W. Chang, J.-K. Son, Inhibition of DNA topoisomerases I and II and cytotoxicity
of compounds from Ulmus davidiana var. japonica, Arch. Pharmacal. Res. 33
(2010) 1307e1315.
(
2008) 1096e1107.
28] S. Fulda, Modulation of apoptosis by natural products for cancer therapy,
Planta Med. 76 (2010) 1075e1079.
29] J. Sarek, M. Kvasnica, M. Urban, J. Klinot, M. Hajduch, Correlation of cytotoxic
activity of betulinines and their hydroxy analogues, Bioorg. Med. Chem. Lett.
15 (2005) 4196e4200.
[
[
30] R. Csuk, S. Schwarz, B. Siewert, R. Kluge, D. Ströhl, Conversions at C-30 of
glycyrrhetinic acid and their impact on antitumor activity, Arch. Pharm. 345
[49] K. Majumdar, M. Biswas, U.K. Som, S. Das, Chemical constituents of the bark of
Terminalia myriocarpa, Indian J. Chem. Soc. 82 (2005) 673e674.
[50] K. Cheng, J. Liu, H. Sun, E. Bokor, K. Czifrak, B. Konya, M. Toth, T. Docsa,
P. Gergely, L. Somsak, Tethered derivatives of d-glucose and pentacyclic tri-
terpenes for homo/heterobivalent inhibition of glycogen phosphorylase, New
J. Chem. 34 (2010) 1450e1464.
(
2012) 223e230.
31] H. Budzikiewicz, J.M. Wilson, C. Djerassi, Mass spectrometry in structural and
stereochemical problems. XXXII.1 pentacyclic triterpenes, J. Am. Chem. Soc.
85 (1963) 3688e3699.