The chemical and biological potential of C ring modified triterpenoids

Article abstract of DOI:10.1016/j.ejmech.2013.11.025

A convenient and elegant route has been developed to separate the natural regioisomers triterpenoids ursolic acid (UA) and oleanolic acid (OA) as well as derivatives thereof. Eleven unknown derivatives of OA were designed, synthesized, and their cytotoxicity was investigated. Further sixteen compounds were prepared to correlate all compounds in a SAR study. It could be shown that C-ring modifications of OA and UA have only a moderate influence onto the cytotoxic activity of the compounds but a significant impact onto the ability to trigger apoptosis in ovarian cancer cells (cell line A2780).

Full text of DOI:10.1016/j.ejmech.2013.11.025

European Journal of Medicinal Chemistry 72 (2014) 84e101
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
The chemical and biological potential of C ring modified triterpenoids
*
Bianka Siewert, Jana Wiemann, Alexander Köwitsch, René Csuk
Bereich Organische Chemie, Martin-Luther-Universität Halle-Wittenberg, Kurt-Mothes-Str. 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:
A convenient and elegant route has been developed to separate the natural regioisomers triterpenoids
ursolic acid (UA) and oleanolic acid (OA) as well as derivatives thereof. Eleven unknown derivatives of OA
were designed, synthesized, and their cytotoxicity was investigated. Further sixteen compounds were
prepared to correlate all compounds in a SAR study. It could be shown that C-ring modifications of OA
and UA have only a moderate influence onto the cytotoxic activity of the compounds but a significant
impact onto the ability to trigger apoptosis in ovarian cancer cells (cell line A2780).
Received 15 July 2013
Received in revised form
11 November 2013
Accepted 24 November 2013
Available online 5 December 2013
Ó 2013 Elsevier Masson SAS. All rights reserved.
Keywords:
Oleanolic acid acid
Ursolic acid
Triterpenoids
Antitumor activity
Apoptosis
Structureeactivity relationships
1. Introduction
oleanolic acid (OA) on a larger scale. These compounds (Scheme 1)
are widely spread in the plant kingdom and known for their broad
Triterpenoids are promising candidates [1] for the development
of new drugs. As products of the secondary metabolism they fulfill a
function in nature in their own right. But in terms of a future use for
medicinal applications, they are far off of being optimized for this
use. For these highly complex molecules carrying several stereo-
genic centers, however, total syntheses are quite challenging but for
larger scale synthesis highly uneconomic and libraries of analogs
are difficult to obtain. Thus, semi-synthetic strategies are most
rewarding; they shorten the time-to-market period tremendously.
As a prerequisite, suitable precursors have to be gained from plant
material. However, these precursors occur only in small amounts,
and, in addition, they are most often part of complex mixtures.
Thus, their isolation and/or separation is difficult, sometimes
laborious and uneconomic. For their straightforward isolation,
extractive steps have to be combined with selective derivatization
reactions. The demand for these compounds has increased since
clinical trials for NVX-207 [2] or CDDO-Me [3,4] have already
begun.
pharmaceutical activities, e.g., for noteworthy antiviral, antibacte-
rial and anticancer activities [6e8]. However, both molecules occur
very often together in leafs or peels of plants [9]. In continuation of
our previous work looking for triterpenoids of improved cytotoxic
properties, the development of an easy protocol for their separation
was still in the focus of our scientific interest.
Several groups were able to show that the introduction of an
oxygen substituent into ring C increased the biological activity of
the compounds. Due to their ability to act as “reactive oxygen
species (ROS) producer”, the presence of a,b-unsaturated systems
seems promising. For example, for the well-known compound
CDDO (2-cyano-3,12-diooxoolean-1,9-dien-28-oic acid) several
pharmaceutical activities, e.g., anti-inflammatory, anti-diabetic
nephropathy and cytotoxic activities, have been reported [10,11].
Furthermore, clinical trial using ester derivatives of CDDO have
been started, thus increasing the demand for this class of com-
pounds [10,12,13]. CDDO esters showed a fast first-pass meta-
bolism; hence, there is still a necessity for improvement [12].
Recently, Leal et al. [14] presented several promising anticancer
active lactones possessing an UA backbone.
Many data reported for the biological activity of triterpenoids,
however, are hardly comparable, and the impact of modifications of
the C-ring onto the cytotoxicity of the compounds still remains
unclear. Thus, we became interested in studying the structuree
activity relationships (SAR) of some of these derivatives combined
with a more detailed inspection of their mode of action (MOA).
To guarantee the successful commercial exploitation and the
development of analogs, effective isolation methods are mandatory
and called for. Recently, we were able to present [5] a convenient
separation of regioisomeric triterpenoids, ursolic (UA) and
* 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.2013.11.025

B. Siewert et al. / European Journal of Medicinal Chemistry 72 (2014) 84e101
85
Scheme 1. Separation of a mixture of OA/UA (and derivatives thereof) and the regeneration of OA.
2. Chemistry
these mild reaction conditions. Moreover, this type of reaction
(Scheme 2) could be used for the separation OA/UA quite univer-
sally. Neither the presence of a second double bond in the molecule
(as exemplified for 26) nor the presence of an additional hydroxyl
group in ring A (as in 12) restricted these reactions.
Usually the separation of regio- or diastereoisomers can easily
be accomplished by chromatography, by recrystallization or by
chemo-selective reactions with/without the use of enzymes. Pre-
viously, smaller amounts of UA/OA mixtures have been separated
by chromatography [15,16] or by treating these mixtures with HBr
[17]. Recently, we presented a new chemo-selective separation of
the two constitutional isomers UA and OA by treating of a UA/OA
mixture with peracids (either performic acid or m-chlor-
operbenzoic acid) [5] under mild conditions. Thus, OA is selectively
transformed into its corresponding 12-hydroxy-lactone 1 (Scheme
1) while leaving UA unchanged. The formation of an epoxide from
UA is not observed using mild conditions and low temperatures. UA
is a very valuable starting material [18] for the synthesis of cyto-
toxic compounds. As early as in 1966, Barton et al. [19] showed that
OA can be recovered by a reductive elimination (using lithium in
ammonia) starting from the 12-acetoxy-lactone 5. Compound 5 can
be prepared either from the 3-hydroxy-lactone 1 by its acetylation
with acetic anhydride in pyridine or from the 3-acetoxy-lactone 4,
which was accessed by the separation of a mixture of 2 and 3. The
To investigate the influence of C-ring modifications onto the
biological activity an oxygen substituent at position 11 (compounds
10 and 17, Scheme 3) was inserted via an
a-allylic, chromate-
assisted oxidation. For compound 10, the characteristic IR band
for the newly created
n
a,b-unsaturated system was found at
¼ 1724 cmꢀ1, in combination with an a signal in the 13C NMR
spectrum at
d
¼ 200.3 ppm. Subsequent treatment of 8, 15 and 24
with selenium dioxide in refluxing 1,4-dioxane resulted in the
formation of unknown products. Usually, this type of reaction is
known [21e23] to establish a 9,11-ene-12-oxo moiety in steroid-
derived structures. However, due to the presence of terminal
methyl groups located at positions 26 and 27 in the backbone of the
triterpenoids, an elimination of the hydroxyl group at position C-11
is not possible any longer. Hence, a keto group is created instead
and rearranged into a thermodynamically more stable enol-system
(Scheme 3). For this structural feature,
a new signal at
presence of an additional sharp singulet at
NMR spectrum of 5 indicates e together with an IR vibration band
d
¼ 2.10 ppm in the 1H
d
¼ 142.3 ppm (C12) is observed in the 13C NMR spectrum. Addi-
tional IR vibrations at
n
¼ 1652 and 1614 cmꢀ1 confirm the structure
at
n
¼ 1766 cmꢀ1 e the formation of 5. Nevertheless, a low turn-
[24]. The configuration (Scheme 3) of ring C (compounds 9 and 25)
was ascertained by the shift of the equatorial proton H-1 to higher
fields. This shifting (which has already been observed in the past for
glycyrrhetinic acid and was also found in compounds 10 and 17) is
caused by an anisotropy tensor of the 11-oxo function [25e27].
Furthermore, cross-peaks in gHMBC NMR experiments between
C12 and C18 as well as between C9 and C11 finally confirm this
over rate of 20% devaluates this separation [19,20]. Consequently,
we were looking for a more elegant strategy. Given the potential of
C-ring modifications e as exemplified in CDDO-Me e we tried to
shorten the synthetic scheme and to optimize the process of
extraction and separation.
The treatment of mixtures of ester analogs from OA/UA mix-
tures with peracids furnished 12-oxo derivatives from OA exclu-
sively but not from UA. Thus, UA esters remained unchanged under
structure which is further ascertained by its ESI-MS spectra (Dm/
z ¼ þ16 as compared to the starting material).

86
B. Siewert et al. / European Journal of Medicinal Chemistry 72 (2014) 84e101
Scheme 2. Separation of mixtures of the OA/UA ester analogs.
3
As depicted in Scheme 4, in general, the position of H-12 was
¼ 3.9 Hz, JH12(ax)eH11(eq) ¼ 2.3 Hz for compound 30) the
H11(ax)
assumed to be axial. By comparison with data from literature and
by the presence of a cross-peak between H-12 and H-18 in the 2D
configuration of this stereogenic center is confirmed. A character-
istic IR absorption at
5 and 28e31 being typical for the presence of a lactone moiety.
n
¼ 1699 cmꢀ1 was detected for compounds 4,
3
NMR spectra as well as suiting coupling constants (e.g., JH12(ax)e
Scheme 3. Oxidation of the C-28 ester analogs.

B. Siewert et al. / European Journal of Medicinal Chemistry 72 (2014) 84e101
87
Scheme 4. Lactonization of maslinic acid (MA).
3. Biological investigations
however, has been subject to controversial discussions. Thus, on the
one side the presence of an -unsaturated moiety was regarded
a,b
Many natural occurring triterpenoids are well-known for their
less important for the ability to produce ROS; a comparison of the
cytotoxicity of a 12-oxo-9,11-ene and a 11-oxo-12,13-ene revealed
no significant differences between these two compounds [30,31].
Contrary to these findings, activity was claimed to depend crucial
on the presence of an intact Michael acceptor [31], e.g., the presence
of a 12-oxo-9,11-ene moiety. The significance of an intact C ring has
also been discussed for the explanation of interactions between
tyrosine phosphatase 1B and triterpenoids [32]. In addition, several
semicarbamates derived from 1 showed an increased cytotoxicity
as compared to parent OA [33]. The introduction of lipophilic
moieties into ring A seems to result in better cytotoxicity of these
compounds [14].
Our own data make a nucleophilic attack onto position 13 as a
key step quite improbable. If an attack at position 13 should be
important, one would expect an increasing activity with an
increasing positive polarization of C-13 e or at least the activity
should be proportional to an increasing electronegativity of the
adjacent halogen substituent. Quite on the contrary, the iodo-
lactone is the most active compound of this series. This led to the
assumption that either a nucleophilic attack at position 12 or a
stereoelectronic hindrance by this substituent must be essential.
Our results parallel previous findings of Salvador et al. [33] sug-
gesting a nucleophilic attack.
cytotoxic activity with IC50 values between 10 and 80
mM [8].
Several of them trigger apoptosis, and OA as well as MA show
promising tumor-to-control selectivity. Screening of the com-
pounds to detect their cytotoxic activity is usually performed either
by an MTT [28] or a SRB [29] assay; the first of these uses the
mitochondrial dependent reduction of a thiazolium salt whereas
the latter is based on the proportional binding of a rhodamine dye
to surface membrane proteins. Advantages of Skehan’s SRB assay
are found in the linearity between cell density and the optical
density. The comparison of cytotoxicity measured by different as-
says (using different theoretical assumptions to describe more or
less the same cellular feature) remains critical, and is even more
complicated by numerous modifications of the original assay.
Hence, we decided to include several already known compounds
into this study, and to test all compounds using the same protocol
and standardized conditions. Several triterpenoids have been
shown to exhibit some selectivity towards cancer cells and some
derivatives of OA are able to induce apoptosis [8]; hence, we set out
to investigate the influence of the C ring onto antitumor activity,
their tumor-to-control selectivity as well as onto their ability to
trigger apoptosis.
Table 1 and Fig. 1 summarize some of the results. Most of the
compounds exhibited a moderate to good IC50 value in the SRB
tests. As far as the cytotoxic activity of the lactones (4, 5, 28e32) is
concerned, the presence of a hydroxyl group seems unfavorable.
For the halide-substituted compounds activity increased with the
atomic radius or was reciprocally proportional to an increasing
electronegativity. The importance of an intact C ring for retaining
good biological activity has already been demonstrated for glycyr-
rhetinic acid derivatives. The mode of action of these compounds,
Comparison of the IC50 values of the products resulting from the
oxidation of the OA esters (Fig. 1) indicate that introducing an extra
oxygen seems less important for obtaining good cytotoxicity than
variations at e.g., position 3. Cytotoxicity of the OA derivatives can
be raised by these modifications up to ca.111 fold [33]. Inspection of
the IC50 values (listed in Table 1) shows oxidation in ring C by and
large insignificant for obtaining good cytotoxic effects. These find-
ings are in perfect agreement with results obtained by Chadalapaka

88
B. Siewert et al. / European Journal of Medicinal Chemistry 72 (2014) 84e101
Table 1
Cytotoxicity of OA, MA and derivatives (IC50 values in mM from SRB assays after 96 h of treatment; the values are averaged from at least three independent experiments
performed each in triplicate; confidence interval CI ¼ 95%; the individual errors (positive upper and negative lower values are given; n.D. not detected). Cell lines 518A2, 8505C,
A2780, A549, HT29 and MCF are human cancer cell lines, NiH 3T3 cells are a non-malignant mouse fibroblasts, STP is stauroporine.
IC50
OA
518A2
8505C
A2780
14.0
A549
72.3
HT29
38.8
MCF7
NiH 3T3
76.4
>60
0.0
0.0
1.9
1.7
0.0
0.0
0.0
>60
0.0
0.0
2.1
1.9
0.0
0.0
0.0
1.1
1.1
1.8
1.7
0.0
0.0
0.0
2.1
2.5
0.3
0.3
1.3
1.1
3.5
1.9
0.6
0.6
0.0
0.0
0.0
5.4
4.8
1.0
0.9
0.0
0.0
0.0
>60
0.0
0.0
0.9
0.8
0.0
0.0
0.0
1.5
1.4
0.7
0.7
0.0
0.0
0.0
4.5
2.3
0.1
0.1
4.1
3.7
0.3
0.3
1.3
1.2
2.6
2.4
0.7
0.7
1.6
1.5
0.3
0.3
MA
13.7
17.0
19.5
23.4
28.8
16.6
21.4
1
>30
>30
>30
>30
>30
>30
>30
4
>90
6.8
>90
7.0
63.2
5.6
>90
6.6
>90
14.1
26.7
46.8
13.5
29.4
12.8
26.3
17.6
25.4
18.5
15.3
28.7
>90
>60
92.8
509.7
>90
>90
46.9
0.2
>90
18.5
16.7
>30
9.4
85.7
7.2
5
0.0
0.0
1.9
1.8
5.5
4.8
0.1
0.1
2.0
1.9
1.3
1.2
1.8
1.5
0.0
0.0
0.6
0.6
0.1
0.1
0.2
0.2
2.1
0.0
0.0
0.8
0.8
5.8
4.9
0.6
0.5
4.6
3.6
1.3
1.2
3.0
2.5
0.1
0.1
2.2
2.0
1.8
1.6
0.5
0.5
1.5
0.0
0.0
3.2
2.8
0.0
0.0
0.4
0.4
0.0
0.0
0.5
0.4
2.1
1.7
0.9
0.8
0.0
0.0
0.2
0.2
1.3
1.2
2.6
2.3
2.0
5.9
4.9
2.9
2.8
2.7
2.2
6.0
5.0
1.5
1.3
1.1
0.7
1.2
1.1
0.4
0.4
0.3
0.3
1.4
1.3
2.5
0.3
0.3
1.0
1.0
0.0
0.0
0.4
0.4
0.0
7
34.1
40.8
7.7
20.5
31.1
8.4
8.6
26.1
>30
8.2
34.3
28.2
12.5
33.6
21.4
30.5
31.7
8
>30
4.0
9
0.7
0.6
1.7
1.6
4.3
3.4
2.3
1.7
0.9
0.8
3.1
2.6
1.2
1.0
1.2
1.1
1.7
10
11
12
13
14
17
18
20
24
25
28
29
30
31
32
STP
24.1
15.6
8.6
16.2
14.7
13.3
10.3
20.6
13.0
23.3
29.1
41.9
16.4
>90
18.0
36.5
72.1
22.2
0.2
40.0
17.8
6.8
66.1
17.8
7.8
0.0
16.3
>30
23.8
22.2
21.3
25.8
15.8
>90
>60
>90
n.D.
n.D.
n.D.
n.D.
0.4
0.9
0.8
9.6
11.6
15.3
6.6
14.1
20.2
15.1
25.6
28.0
69.1
>60
>90
33.1
29.1
34.4
16.4
0.6
1.9
1.8
2.1
1.9
0.1
0.1
1.7
1.6
0.9
17.7
15.6
21.2
28.0
73.2
>60
78.4
n.D.
n.D.
n.D.
n.D.
0.2
24.8
25.8
22.4
>90
>60
22.1
>90
43.1
80.2
29.2
0.2
0.2
0.2
0.0
0.0
3.0
17.5
23.0
19.8
8.2
2,0
1.6
1.6
1,7
1,8
2,4
3.9
3.7
2,3
0,8
2,7
6.5
5.6
1.4
0.8
5.7
4.5
3.0
2.2
1.0
0.9
3.8
3.4
4.3
3.7
3.8
3.4
0.7
0.7
0.0
0.0
0.0
0.0
0.0
0.0
0.3
0.3
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
2.3
3.9
2.5
3.8
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
39.9
34.2
25.9
31.5
15.5
0.2
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
2.7
2.5
7.0
6.4
0.4
0.4
0.0
0.0
2.2
2.7
3.6
3.2
1.9
1.7
0.0
0.0
8.5
7.1
2.1
1.9
4.4
3.8
0.0
0.0
3.2
3.0
0.0
0.0
et al. [34]; these authors were able to show that the location of an
-unsaturated system (
9,11 or 12,13) is insignificant for obtaining
good cytotoxicity.
cells with compounds 1, 9 or 24 led to a reduction of the G2/M
phase; compounds 1 or 24 were the most active compounds of this
series.
a
,b
D
D
However, a comparison of IC50 values is not sufficient to describe
the biological activity of these compounds. To get a deeper insight,
some representative molecules were selected and submitted to
several MOA investigation assays applying the same concentration
(30 mM) for all compounds. Fig. 2 shows some results from cell cycle
investigations.
To evaluate whether the activity (as shown in the SRB assay) is
caused by a cytostatic effect or rather by a cytotoxic effect, living
ovarian cancer cells were investigated after 24 h of treatment with
the compounds. A significant G1/G0 arrest (as previously found for
parent maslinic acid, unpublished data) or ursolic acid [14], was not
detected at all. A weak G0/G1 arrest (<20%) was observed for
compounds 8 and 25. Compounds 4, 5 or 7, however, showed
practically no influence onto the cell cycle. The treatment of the
Obviously at least compounds 4, 5 and 7 are cytotoxic, and hence
investigations of the dead cells seemed highly interesting. Usually an
undesired necrotic cell death (happening most accidentally) is
described by a loss of the integrity of the cell membrane [35]. The
integrity of the cell membrane can be investigated by a microscopic
fluorescence dye-exclusion assay (AO/PI; Fig. 3). In this test, acridine
orange (AO) as an un-polar green fluorescence dye permeates the cell
membrane and stains dead cells possessing an intact membrane [36].
Thus, cells having died by activating a programmed cell death show
green fluorescence. Propidium iodide (PI) [37], however, a double
positive charged dye, stains only dead cells with a broken cell
membrane; hence, a deep red color is observed.
As depicted in Fig. 4, all dead cells had died due to a pro-
grammed process. Several death mechanisms are known [38]; for

B. Siewert et al. / European Journal of Medicinal Chemistry 72 (2014) 84e101
89
Fig. 1. IC50 values (in
mM from SRB with a confidence interval (95%) for OA, MA and derivatives utilizing the human ovarian cancer cell line A2780. The colors of the bars indicate the
type of modification at positions C-3 and C-28. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
triterpenoids most commonly apoptosis [8] as well as autophagy
[39] was reported.
cells with 5, 9, 17 or 18 the typical DNA ladders were found.
Furthermore, the ability of 9, 17 and 18 to trigger apoptosis affirms
Microscopic inspection of the dead cells allowed a preliminary
evaluation: membrane blebbing and condensed nuclei as well as
cell shrinking indicated apoptosis [40]. Staining the cells with AO/PI
gave red dots e a sign for at least partial autophagy [41]. Fig. 3
depicts representative microscopic views of the cells, and signifi-
cant morphologic changes have been marked using different
colored arrows. One unique feature of apoptosis is the activation of
ICAD [42]; this results in the degradation of DNA into several 178 bp
long pieces (Fig. 3). After 24 h of treatment only compounds 5 and
17 showed significant DNA ladders as indicated by electrophoresis
and staining of the DNA. In the electrophoretic gels for compounds
1, 9 and 10 only weak ladders were found.
that the presence of an a,b-unsaturated system in ring C seems
necessary for inducing apoptosis very quickly.
The ability to trigger apoptosis is independent from the IC50
values; although compound 9 exhibited a lower IC50 value in the
SRB tests than compound 18, the latter of which showed a signifi-
cantly higher ability to trigger apoptosis. The drop down of cyto-
toxicity found for compounds 24 and 25 might be explained by the
low polarity of these compounds. The presence of an increased
necrotic population after having treated the cells with 9 might be
due to a concentration dependent toxic effect. Hence, OA methyl
esters possessing a modified ring C are promising compounds
possessing the ability to induce programmed cell death very
quickly.
Membrane integrity and DNA-laddering are typical hallmarks
for an apoptotic death; another hallmark of apoptosis is the
translocation of phosphatidylserine. This compound is located on
the inner side of the cell and translocates to the outer cell mem-
brane during apoptosis thus acting as a signal for macrophages [43].
During apoptosis several executing proteinases, also known as
caspases, are activated in a highly controlled manner. During this
lethal caspase-cascade process other enzymes get activated and/or
essential parts of the cell skeleton are broken down leading to a
subsequent exposition of the phosphatidylserine. This process can
be measured using an annexin V assay. By double staining and
submitting the stained cell population to a FACS measurement the
distribution of vital versus dead cells can be determined. Fig. 4
shows the result of one of these assays. The experimental data
were evaluated by plotting the green fluorescence (belonging to the
annexin V-FITC) versus the red one (belonging to propidium iodide)
and splitting these plots into four quadrants. While PI stained only
cells possessing a leak membrane, red labeled cells (upper left) are
usually regarded as necrotic cells. Cells carrying both signals (upper
right) are assumed to be secondary necrotic; this phenomenon is
known to occur for cells held in cell cultures.
4. Conclusion
A convenient method for separating a mixture of regioisomeric
triterpenoids UA/OA was found. This separation technique using
per-acids was shown to be quite universal. UA could be recovered
very easily from the mixtures, and the intermediates were trans-
formed into compounds showing promising antitumor activity. In
addition, different MOAs were found for compounds differing only
slightly in their structure. As far as the OA derived lactone 1 is
concerned, the introduction of an additional hydroxy substituent at
position C-2 (as in 28) as well as of a lipophilic moiety at position C-
12 (as in 5) decreases the IC50 values significantly. An additional
oxygen substituent in ring C increases the cytotoxicity, too. Thus,
the highest activity was determined for the previously unknown
11-oxo-12-hydroxy-olean-12-enes 17 and 18, respectively.
5. Experimental part
5.1. General e chemistry
As indicated in Fig. 4, the treatment of the cells with 17 or 18
triggered apoptosis after incubating for 24 h in >50% of the cell
population. In addition, 3,12-diacetylated 5 as well as compound 9
triggered apoptosis. These results are in excellent agreement to the
results from the DNA ladder assays; therein, after treatment of the
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 on VARIAN Gemini 2000 or Unity 500 spectrometers at

90
B. Siewert et al. / European Journal of Medicinal Chemistry 72 (2014) 84e101
Fig. 2. Cell cycle investigations. Ovarian cancer cells (A2780) were treated for 24 h with a 30 mM solution of compounds 1, 4, 5, 7e10, 18, 24 or 25, respectively. The living cells were
harvested, fixed, the population was aligned and submitted to a FACS supported measurement. The DNA was dyed with PI after treatment with RNAse. In the upper part of the figure
(A) the relative changes to control have been depicted; the lower part (B) shows typical graphs for the cell cycle distribution.
27 ꢁC (chemical shifts
d
are given in ppm and J in Hz. Mass spectra
aq soln. of sodium thiosulfate, and usual work up, the solvents were
evaporated under diminished pressure. The residue was subjected
to chromatography (silica gel, hexane/ethyl acetate).
were taken on a FINNIGAN MAT TSQ 7000 (electrospray, voltage
4.5 kV, sheath gas nitrogen) instrument. Elemental analyses were
measured on a Foss-Heraeus Vario EL unit. IR spectra were
recorded on a Perkin Elmer FT-IR spectrometer Spectrum 1000,
optical rotations on a Perkin Elmer 341 polarimeter (1 cm micro
cell, 25 ꢁC) and UVevis spectra on a Perkin Elmer unit, Lambda 14.
Elemental analysis was performed for all new compounds and
correct values (ꢂ0.3 for C, H, N) were obtained. TLC was performed
on silica gel (Merck 5554, detection with ceriummolybdate spray
reagent). Solvents were dried according to usual procedures. The
purity of the compounds was checked by HPLC/DAD (Prontosil
C18, MeOH/H2O 95/5, 1% H3PO4) and found to be >98% for each
compound.
5.2.2. Oxidation with mCPBA (method B)
To a mixture of ursolic acid and oleanolic acid (1 equiv., 2:1) in
DCM mCPBA (1.5 equiv.) was added, and the mixture was stirred at
25 ꢁC for 24 h. After washing with an aq soln. of sodium thiosulfate
and usual workup, the solvents were evaporated under diminished
pressure. The residue was subjected to chromatography (silica gel,
hexane/ethyl acetate).
5.2.3. Acetylation (method C)
Acetylation was performed in dry DCM (100 mL) with acetic
anhydride (2 equiv.) and triethylamine (0.3 equiv.) for 12 h at 24 ꢁC
and gave, after usual work-up and recrystallization from ethanol,
the product.
5.2. General procedures
5.2.1. Oxidation with hydrogen peroxide and formic acid
(method A)
5.2.4. Esterfication (method D)
To a mixture of ursolic acid and oleanolic acid (1 equiv., 2:1) in
DCM, formic acid (concd, 2 equiv.)/H2O2 (30%, 2 equiv.) was added,
and the mixture was stirred at 25 ꢁC for 24 h. After washing with an
The starting material (1 equiv.) was dissolved in dry DMF (5 mL),
and finely grounded potassium carbonate (5 equiv.) was added.
After 60 min of stirring at room temperature, the alkylbromide (2

B. Siewert et al. / European Journal of Medicinal Chemistry 72 (2014) 84e101
91
Fig. 3. Dye exclusion (AO/PI) assay and representative DNA laddering experiments. Ovarian cancer cells (A2780) were treated with the compounds 1, 4, 5, 7e10, 17, 18, 24 or 25
(30 M each, 24 h). The floating (dead) cell cells were collected, washed with PBS and submitted to a fluorescence microscopic investigation. Green dyed cells are typical for a
m
programmed, non-necrotic death. Light green colored arrows indicate typical membrane blebbing; light purple arrows indicate the typical membrane blebbing of the nucleus
membrane, and the blue arrows point to orange colored dots which could be autophagosomes and indicate an autophagy mediated apoptosis. In addition, the results from the DNA
laddering experiment are shown for a control experiment as well as for the experiments using compounds 5 and 17, respectively. (For interpretation of the references to colour in
this figure legend, the reader is referred to the web version of this article.)
Fig. 4. Annexin V/PI assay: Ovarian cancer cells (A2780) were treated with compounds 1, 4, 5, 7e10, 17, 18, 24 or 25 (30 mM each), respectively. After 24 h, all cells were harvested
and submitted to a FACS based assay. Diagram A (upper part) shows the relative changes in the distribution compared to control. Measurements were performed as technical
replicates. Typical samples of these experiments are depicted in the lower part (B). (For interpretation of the references to colour in this figure legend, the reader is referred to the
web version of this article.)
equiv.) was added, and stirring was continued for an additional
18 h. The mixture was poured into an ice cold solution of aq HCl
(3.7%, 50 mL), and the white precipitate was filtered off. Chro-
matographic purification (silica gel, hexane/ethyl acetate, 7:3) and
recrystallization (ethanol) afforded the product.
5.2.5. Oxidation with selenium dioxide (method E)
Equal molar amounts of selenium dioxide and the 12-oxo
compound were stirred in refluxing 1,4-dioxane for 24 h. After
filtration, an aq solution of sodium thiosulfate was added, and the
reaction mixture was extracted with DCM. After following usual

92
B. Siewert et al. / European Journal of Medicinal Chemistry 72 (2014) 84e101
workup, the crude product was subjected to a chromatographic
purification followed by recrystallization from ethanol.
hexane/ethyl acetate, 5:3); 1H NMR (400 MHz, d6 e pyridine):
¼ 5.45 (dd, J ¼ 3.5, 3.5 Hz, 1H, CH (12)), 3.43 (dd, J ¼ 9.6, 6.5 Hz, 1H,
d
CH (3)), 2.60 (d, J ¼ 11.2 Hz, 1H, CH (18)), 2.29 (ddd, J ¼ 13.3, 13.3,
4.5 Hz, 1H, CHa (15)), 2.09 (ddd, J ¼ 13.3, 13.3, 4.1 Hz, 1H, CHa (16)),
1.98e1.90 (m, 5H, CHb (16) þ CH2 (7) þ CH2 (11)), 1.88e1.77 (m, 2H,
CH2 (2)), 1.62e1.51 (m, 4H, CH (9) þ CHa (6) þ CHa (22) þ CHb (1)),
1.46e1.39 (m, 3H, CH (19) þ CH2 (21)), 1.38e1.25 (m, 2H, CHb
(6) þ CHb (22)),1.21 (s, 3H, CH3 (27)),1.20 (s, 3H, CH3 (23)),1.23e1.11
(m, 1H, CHb (15)), 1.02 (s, 3H, CH3 (30)), 0.99 (s, 3H, CH3 (25)), 0.97
(d, 3H, 3J ¼ 6.8 Hz, CH3 (29)), 0.92 (d, 3H, 3J ¼ 6.1 Hz, CH3 (24)), 0.86
(s, 3H, CH3 (26)), 0.82 (m, 1H, CH (5)) ppm; 13C NMR (400 MHz, d6 e
5.2.6. Deacetylation (method F)
The acetylated starting material (1 equiv.) was dissolved in
methanol. Finally grounded KOH (1.2 equiv.) was added, and the
mixture was stirred at room temperature until completed as
checked by TLC. The mixture was poured into a cold aq solution of
HCl (3.7%), and the precipitate was collected. Usual work up fur-
nished the product.
5.3. Biological material and procedures
pyridine):
d
¼ 180.1 (C]O, C28), 139.5 (C]CH, C13), 125.9 (CH]C,
C12), 78.4 (CHOH, C3), 56.1 (CH, C5), 53.8 (CH, C18), 48.3 (CH, C9),
48.3 (Cquart, C17), 42.7 (Cquart, C14), 40.2 (Cquart, C8), 39.7 (CH, C19),
39.7 (CH, C20), 39.6 (Cquart, C4), 39.3 (CH2, C1), 37.7 (CH2, C22), 37.5
(Cquart, C10), 33.8 (CH2, C7), 31.3 (CH2, C21), 29.1 (CH3, C23), 28.9
(CH2, C15), 28.4 (CH2, C2), 25.2 (CH2, C16), 24.2 (CH3, C27), 23.8
(CH2, C11), 21.7 (CH3, C30), 19.0 (CH2, C6), 17.7 (CH3, C29), 17.7 (CH3,
C26), 16.8 (CH3, C24), 15.9 (CH3, C25) ppm; ESI-MS (MeOH): m/
z ¼ 456.6 (44%, [M ꢀ H]ꢀ), 501.2 (100%, [M þ HCO2]ꢀ), 911.0 (44%,
[2M ꢀ H]ꢀ).
5.3.1. Cell lines and culture conditions
The cell lines 518A2, 8505C, A2780, A549, HT29, MCF-7, NiH 3T3
and WW030272 were included in this study. Cultures were main-
tained as monolayer in RPMI 1640 (PAA Laboratories, Pasching,
Germany) supplemented with 10% heat inactivated fetal bovine
serum (Biochrom AG, Berlin, Germany) and penicillin/streptomycin
(PAA Laboratories) at 37 ꢁC in a humidified atmosphere of 5% CO2/
95% air.
5.3.2. Cytotoxicity assay, dye-exclusion assay, DNA-laddering and
apoptosis assay
5.4.2. (3
lactone (1)
b, 12a) 3,12 Dihydroxy-18b-olean-28-oic acid 28,13-
Instrumentation, cell lines and culture conditions, cytotoxicity
assay, dye exclusion tests and DNA fragmentation was performed as
previously described [44,45].
By using method A or B, compound 2 was obtained from a
mixture of UA/OA (ca. 2:1); yield: 10%; m.p.: 249e252 ꢁC (lit.: [32]:
247e249 ꢁC); RF ¼ 0.25 (hexane/ethyl acetate, 5:3); [
a]
¼ þ32.4ꢁ (c
D
0.35, CHCl3); IR (KBr):
n
¼ 3668w, 3524s, 2947s, 2867s, 1736s,
5.3.3. Cell cycle investigation
1470m,1396m, 1362m,1306w,1255m, 1226m,1176w,1146m,1092w,
Approximately 1*106 cells (HT29, A2780 or NIH 3T3) were
seeded in cell culture flasks (25 cm2), and the cells were allowed to
grow for 24 h. After removing of the used medium, the substance
loaded medium was reloaded (or a blank fresh medium as a con-
trol). After 24 or 48 h, the living cells were harvested, washed with
PBS (with Mg2þ and Ca2þ) twice and fixed with ethanol (70%, 4 ꢁC,
1 h). After removing of the fixation and permeabilization agent, the
cells were washed with PBS buffer (with Mg2þ and Ca2þ, containing
1% BSA and 0.1% NaN3, 3 ꢃ 1 mL, 1000 rpm) and adjusted to 1*105
million cells. The pellet was gently suspended in staining buffer
(PBS buffer containing BSA, RNAase, NaN3 and PI following the
procedure of Darzynkiewicz et al. [41]) and incubated for 30 min at
37 ꢁC. Analyses were performed using the AttuneÒ FACS machine;
collecting data from the BL-2A channel. Doublet cells were
excluded from the measurements by plotting BL-2A against BL-2H.
For each cell cycle distribution 20.000 events were collected. Dis-
tribution was calculated by the method of Dean et al. [46].
1077m, 1060m, 1037m cmꢀ1; 1H NMR (500 MHz, CDCl3):
d
¼ 3.83e
3.80 (dd, J ¼ 2.8, 2.8 Hz,1H, CH (12)), 3.15 (dd, J ¼ 11.4, 4.4 Hz,1H, CH
(3)), 2.07 (ddd, J ¼ 13.3, 5.8, 5.8 Hz, 1H, CHa (16)), 2.01e1.88 (m, 3H,
CH (18) þ CHa (19) þ CHa (11)), 1.86e1.75 (m, 2H, CHb (19) þ CHa
(15)), 1.66 (ddd, J ¼ 12.8, 12.8, 3.5 Hz, 1H, CHa (1)), 1.59e1.51 (m, 3H,
CH2 (2) þ CHa (22)), 1.53e1.48 (m, 1H, CH (9)), 1.49e1.44 (m, 2H, CHa
(6) þ CHa (7)), 1.42e1.37 (m, 2H, CHb (11) þ CHb (16)), 1.35e1.30 (m,
1H, CHb (21)),1.24 (s, 3H, CH3 (27)),1.22e1.15 (m, 4H, CH2 (15) þ CHb
(7) þ CHb (6)), 1.12e1.09 (m, 1H, CHb (22)), 1.08 (s, 3H, CH3 (26)),
0.92 (s, 3H, CH (23)), 0.91 (s, 3H, CH (29)), 0.92e0.87 (m, 1H, CHb
(21)), 0.83 (s, 3H, CH (30)), 0.81 (s, 3H, CH (25)), 0.71 (s, 3H, CH (24)),
0.68 (dd, J ¼ 11.8, 1.9 Hz, 1H, CH (5)) ppm; 13C NMR (125 MHz,
CDCl3):
d
¼ 179.9 (C]O, C28), 90.6 (Cquart, C13), 78.9 (CHOH, C3),
76.4 (CH, C12), 55.2 (CH, C5), 51.1 (CH, C18), 44.7 (Cquart, C17), 44.6
(CH, C9), 42.3 (Cquart, C14), 42.1 (Cquart, C8), 39.4 (CH2, C19), 38.9
(CH2, C1), 38.9 (Cquart, C4), 36.5 (Cquart, C10), 34.2 (CH2, C21), 34.0
(CH2, C7), 33.3 (CH3, C29), 31.6 (Cquart, C20), 28.8 (CH2, C11), 28.1
(CH3, C23), 28.1 (CH2, C15), 28.0 (CH2, C22), 27.5 (CH2, C2), 23.9
(CH3, C30), 21.2 (CH2, C16), 18.6 (CH3, C27), 18.5 (CH3, C26), 17.8
(CH2, C6), 16.3 (CH3, C25), 15.4 (CH3, C24) ppm; MS (ESI, MeOH): m/
z ¼ 473.5 (27%, [M þ H]þ), 495.6 (15%, [M þ Na]þ), 527.1 (26%,
[M þ Na þ MeOH]þ), 967.3 (100%, [2M þ Na]þ).
5.3.4. Annexin V/PI assay
Approx. 500,000 cells (A2780) 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 24 h. All cells were harvested, centrifuged
(1200 rpm, 5 min) and washed twice (PBS (w/w). Approx. 100,000
cells were washed with annexin V bounding buffer (BD) and treated
5.4.3. (3b) 3-Acetoxy-urs-11-en-28-acid (2)
Compound 2 was obtained as a colorless solid using method A
from a mixture of acetylated 2 and 3. The latter compounds were
obtained either using method C or directly from UA applying
method C; yield: 88%; m.p.: 257e259 ꢁC (lit.: 258 ꢁC [47]);
with a propidium iodide solution (3
(5 L, life technologiesÔ) for 15 min at room temperature in the
dark. After adding Annexin V bounding buffer (400 L), the sus-
mL, 50 mg/mL) and Annexin V
m
m
pension was submitted to a FACS measurement. Calculation was
[
a
]
¼ þ69ꢁ (c 0.19, CHCl3); RF ¼ 0.64 (n-hexane/ethyl acetate, 8:2);
D
performed as suggested from the supplier (BD BiosciencesÒ).
UVeVis (MeOH): lmax (log
3
) ¼ 224 nm (3.71); IR (KBr): ¼ 3423br,
n
2947s, 1736s, 1696s, 1464m, 1366m, 1245s, 1180w, 1148w, 1097w,
5.4. Syntheses
1076w, 1028m, 1009 m cmꢀ1 1H NMR (500 MHz, CDCl3):
;
d
¼ 5.26
(m,1H, CH (12)), 4.48 (dd, J ¼ 9.4, 7.0 Hz,1H, CH (3)), 2.80 (dd, J ¼ 3.8,
4.3 Hz, 1H, CH (18)), 2.08 (s, 3H, Ac), 1.97 (ddd, J ¼ 13.7, 13.5, 4.0 Hz,
1H, CHa (16)), 1.90e1.84 (m, 2H, CH2 (11)), 1.76 (ddd, J ¼ 14.0, 13.8,
4.3 Hz, 1H, CHa (22)), 1.69 (ddd, J ¼ 13.8, 13.8, 3.7 Hz, 1H, CHa (15)),
5.4.1. Ursolic acid (UA)
By method A or B, UA was obtained from a mixture of UA/OA (ca.
2:1); yield: 57%; m.p.: 257e259 ꢁC (lit.: [47]: 258ꢁ); RF ¼ 0.62 (n-

B. Siewert et al. / European Journal of Medicinal Chemistry 72 (2014) 84e101
93
1.64e1.49 (m, 8H, CH (9) þ CHa (1) þ CHa (19) þ CHa (6) þ CHb
(22) þ CHa (16) þ CH2 (2)), 1.45e1.22 (m, 5H, CHb (6)þ CH2
(21) þ CH2 (7)), 1.20e1.02 (m, 3H, CHa (19) þ CHb (1) þ CHb (15)),
1.12 (s, 3H, CH3 (27)), 0.93 (s, 3H, CH3 (23)), 0.91 (s, 3H, CH3 (30)),
0.89 (s, 3H, CH3 (29)), 0.86e0.81 (m, 1H, CH (5)), 0.85 (s, 3H, CH3
(25)), 0.84 (s, 3H, CH3 (26)), 0.74 (s, 3H, CH3 (24)) ppm; 13C NMR
(Cquart, C13), 81.0 (CHOH, C3), 76.3 (CHOH, C12), 55.4 (CH, C5), 51.2
(CH, C18), 44.8 (Cquart, C17), 44.6 (CH, C9), 42.4 (Cquart, C14), 42.2
(Cquart, C8), 39.5 (CH2, C19), 38.6 (CH2, C1), 38.0 (Cquart, C4), 36.5
(Cquart, C10), 34.3 (CH2, C7), 34.1 (CH2, C21), 33.4 (CH3, C30), 31.7
(Cquart, C20), 28.9 (CH2, C22), 28.2 (CH2, C15), 28.3 (CH3, C23), 27.6
(CH2, C11), 24.0 (CH3, C27), 23.7 (CH2, C2), 21.4 (CH3, Ac), 21.4 (CH2,
16), 18.7 (CH3, C29), 18.7 (CH3, C26), 17.8 (CH2, C6), 16.6 (CH3, C24),
16.5 (CH3, C25) ppm; MS (ESI): m/z ¼ 527.3 (100%, [M þ H]þ), 549.3
(125 MHz, CDCl3):
d
¼ 184.4 (C]O, C28), 171.1 (C]O, Ac),143.5 (C]
CH, C13), 122.5 (CH]C, C12), 80.9 (CHOH, C3), 55.3 (CH, C5), 47.5
(CH, C9), 46.5 (Cquart, C17), 45.8 (CH2, C19), 41.5 (Cquart, C14), 40.9
(CH, C18), 39.3 (Cquart, C8), 38.0 (CH2, C1), 37.6 (Cquart, C4), 36.9
(Cquart, C10), 33.7 (CH2, C21), 33.0 (CH3, C29), 32.5 (CH2, C7), 32.4
(CH2, C22), 30.6 (Cquart, C20), 28.0 (CH3, C23), 27.6 (CH2, C15), 25.9
(CH3, C27), 23.6 (CH3, C30), 23.5 (CH2, C11), 23.4 (CH2, C2), 22.8
(CH2, C16), 21.3 (CH3, Ac), 18.1 (CH2, C6), 17.0 (CH3, C24), 16.6 (CH3,
C26), 15.3 (CH3, C25) ppm; MS (ESI, MeOH): m/z ¼ 497.7 (100%,
[M ꢀ H]-), 543.3 (50%, [M þ HCOꢀ2 ]ꢀ).
(10%, [M
þ
Na]þ), 1053.2 (25%, [2M
þ
H]þ), 1075.2 (25%,
[2M þ Na]þ).
5.4.6. (3b, 12a) 3,12-Diacetoxy-olean-28-oic acid 28,13-lactone (5)
From compound 4 following method C, 5 was obtained as fine
colorless needles (from ethanol); yield: 76%; m.p.: 236e239 ꢁC (lit.:
241e243 ꢁC [50]); RF
¼
0.80 (n-hexane/ethyl acetate, 6:4);
[a
]
D
¼ þ60.6ꢁ (c 0.27, CHCl3); IR (KBr):
n
¼ 3448brs, 2938s, 1766s,
1746m, 1724m, 1474m, 1466m, 1458m, 1438m, 1394m, 1376m,
5.4.4. (3
b
) 3-Acetoxy-olean-11-en-28-acid (3)
was obtained as colorless needles applying
1246m, 1232m, 1044m, 1032m, 1012 m cmꢀ1
;
1H NMR (400 MHz,
Compound
3
CDCl3):
d
¼ 5.7 (brs, 1H, CH (12)), 4.48 (dd, J ¼ 9.8, 6.3 Hz, 1H, CH
method C starting from OA; yield: 95%; m.p.: 257e261 ꢁC, (lit.:
(12)), 2.10e2.03 (m, 1H, CH (16)), 2.10 (s, 3H, CH3 (Ac)), 2.04 (s, 3H,
CH3 (Ac)), 1.96e1.86 (m, 4H, CH (18) þ CHa (19) þ CHa (7) þ CHa
(15)), 1.66e1.48 (m, 9H, CHa (6) þ CHa (1) þ CHa (16) þ CH (9) þ CHa
(22) þ CH2 (11) þ CH2 (2)), 1.45e1.28 (m, 6H, CHb (6) þ CHb
(19) þ CHb (7) þ CH2 (21) þ CHb (16)), 1.27 (s, 3H, CH3 (27)), 1.21 (dd,
J ¼ 14.5, 8.1 Hz, 1H, CHb (22)), 1.16 (s, 3H, CH3 (26)), 1.19e1.15 (m, 2H,
CHb (1) þ CHb (15)), 0.96 (s, 3H, CH3 (30)), 0.88 (s, 3H, CH3 (25)), 0.87
(s, 3H, CH3 (29)), 0.84 (s, 3H, CH3 (23)), 0.82 (s, 3H, CH3 (24)), 0.91e
257e259 ꢁC [48]); [
a]
¼ þ65ꢁ (c 0.33, CHCl3) [lit.: þ74ꢁ (c 1.0;
D
CHCl3 [49])]; RF ¼ 0.60 (toluene/ethyl acetate/n-heptane/formic
acid, 80:20:30:4); IR (KBr):
1370m, 1252s, 1179m, 1161m, 1127w, 1026m, 1010 m cmꢀ1; 1H NMR
(400 MHz, CDCl3):
¼ 5.20 (dd, J ¼ 3.5, 3.5 Hz,1H, CH (12)), 4.47 (dd,
n
¼ 3219br, 2942s, 1731s, 1680m, 1466m,
d
J ¼ 8.5, 7.3 Hz,1H, CH (3)), 2.80 (dd, J ¼ 13.7, 4.1 Hz,1H, CH (18)), 2.02
(s, 3H, Ac), 1.95 (ddd, J ¼ 13.4, 13.4, 3.9 Hz, 1H, CHa (16)), 1.90e1.77
(m, 2H, CH2 (11)), 1.73 (ddd, J ¼ 14.9, 14.6, 4.4 Hz, 1H, CHa (7)), 1.67
(ddd, J ¼ 13.3, 13.3, 4.1 Hz, 1H, CHa (15)), 1.54e1.34 (m, 8H, CHa
(1) þ CHb (16) þ CHa (19) þ CH (9) þ CHa (6) þ CHa (22) þ CH2 (2)),
1.34e1.11 (m, 5H, CHb (7) þ CHb (6) þ CH2 (21) þ CHb (22)), 1.10 (s,
3H, CH3 (27)),1.08e0.93 (m, 3H, CHb (19) þ CHb (15) þ CHb (1)), 0.92
(s, 3H, CH3 (25)), 0.90 (s, 3H, CH3 (30)), 0.88 (s, 3H, CH3 (29)), 0.84 (s,
3H, CH3 (23)), 0.86e0.74 (m, 1H, CH (5)), 0.83 (s, 3H, CH3 (26)), 0.72
0.88 (m, 1H, CH (5)) ppm; 13C NMR (125 MHz, CDCl3):
d
¼ 179.0 (C]
O, C28), 171.1 (C]O, Ac), 169.4 (C]O, Ac), 89.4 (Cquart, C13), 80.7
(CHOH, C3), 55.3 (CH, C5), 50.2 (CH, C18), 45.3 (Cquart, C17), 44.6 (CH,
C9), 42.3 (Cquart, C14), 42.2 (Cquart, C8), 39.5 (CH2, C19), 38.4 (CH2,
C1), 37.8 (Cquart, C4), 36.4 (Cquart, C10), 33.9 (CH2, C7), 34.1 (CH2,
C21), 33.3 (CH3, C29), 31.5 (Cquart, C20), 27.9 (CH2, C22), 27.8 (CH2,
C15), 27.4 (CH3, C23), 25.2 (CH2, C11), 23.8 (CH3, C27), 23.5 (CH2,
C2), 21.4 (CH3, Ac), 21.3 (CH3, Ac), 21.1 (CH2, 16), 18.5 (CH3, C30), 18.4
(CH3, C26), 17.6 (CH2, C6), 16.4 (CH3, C24), 16.3 (CH3, C25) ppm; MS
(ESI, MeOH): m/z ¼ 579.3 (100%, [M þ Na]þ), 1135.3 (100%,
[2M þ Na]þ).
(s, 3H, CH3 (24)) ppm; 13C NMR (100 MHz, CDCl3):
d
¼ 184.3 (C]O,
C28), 171.0 (C]O, C31), 143.6 (C]CH, C13), 122.5 (CH]C, C12), 80.9
(CHOAc, C3), 55.3 (CH, C5), 47.5 (CH, C9), 46.5 (Cquart, C17), 45.8
(CH2, C19), 41.5 (Cquart, C14), 40.8 (CH, C18), 39.2 (Cquart, C8), 38.0
(CH2, C1), 37.6 (Cquart, C4), 36.9 (Cquart, C10), 33.7 (CH2, C21), 33.0
(CH3, C29), 32.5 (CH2, C7), 32.4 (CH2, C22), 30.6 (Cquart, C20), 28.0
(CH3, C23), 27.6 (CH2, C15), 26.9 (CH2, C2), 25.8 (CH3, C27), 23.6
(CH3, C30), 23.5 (CH2, C16), 23.3 (CH2, C11), 21.2 (CH3, Ac), 18.1 (CH2,
C6), 17.1 (CH3, C24), 16.6 (CH3, C26), 15.3 (CH3, C25) ppm; MS (ESI,
MeOH): m/z ¼ 497.5 (100%, [M ꢀ H]ꢀ), 543.1 (66%, [M þ HCO2]ꢀ),
995.1 (64%, [2M ꢀ H]ꢀ), 1017.5 (16%, [2M ꢀ 2H þ Na]ꢀ).
5.4.7. (3b) Methyl 3-acetoxy-urs-11-en-28-oate (6)
Compound 6 was obtained as colorless needles either by using
method A from a mixture of the methyl triterpenoates 6 and 7 or
directly using method D starting from 2; yield: 91%; m.p.: 243e
246 ꢁC (lit.: 243e245 ꢁC [45,52]); RF ¼ 0.75 (hexane/ethyl acetate,
8:2); [
(KBr):
a
]
¼ þ64.7ꢁ (c 0.30, CHCl3) [lit [45,52].: þ61 ꢁC (CHCl3)]; IR
D
n
¼ 3433br, 2942s, 2868m, 1734s, 1458m, 1385m, 1371m,
5.4.5. (3
b
, 12
a
) 3-Acetyl-12-hydroxy-18
b
-olean-28-oic acid 28,13-
1312w, 1244s, 1202m, 1187m, 1167w, 1150w, 1114w, 1073w, 1027m,
lactone (4)
1006w cmꢀ1; 1H NMR (500 MHz, CDCl3):
d
¼ 5.23 (m, 1H, CH (12)),
Compound 4 was obtained from a mixture of 2 and 3 or directly
from 1 using method C; yield: 54.4% (from 1); m.p.: 295e298 ꢁC
(lit.: [50]: 261e263 ꢁC; lit.: [51]: 285e287 ꢁC); RF ¼ 0.36 (toluene/
4.48 (dd, J ¼ 10.6, 5.5 Hz, 1H, CH (3)), 3.59 (s, 3H, CH3), 2.17 (d,
J ¼ 11.3 Hz, 1H, CH (18)), 2.03 (s, 3H, Ac), 1.98 (ddd, J ¼ 13.5, 13.4,
4.3 Hz,1H, CHa (16)),1.89 (dd, J ¼ 8.7, 3.4 Hz, 2H, CH2 (11)), 1.85 (ddd,
J ¼ 13.6, 13.5, 4.3 Hz, 1H, CHa (15)), 1.69e1.55 (m, 6H, CH2 (7) þ CHa
(1) þ CHb (16) þ CH2 (2)), 1.54e1.43 (m, 4H, CH (9) þ CHa (6) þ CHa
(21) þ CHa (22)), 1.38e1.26 (m, 4H, CH (19) þ CHb (6) þ CHb
(22) þ CHb (21)), 1.10e1.02 (m, 2H, CHb (15) þ CHb (1)), 1.06 (s, 3H,
CH3 (27)), 0.99e0.95 (m, 1H, CH (20)), 0.93 (s, 3H, CH3 (25)), 0.93 (d,
J ¼ 5.0 Hz, 3H, CH3 (30)), 0.87e0.83 (m, 1H, CH (5)), 0.86 (s, 3H, CH3
(23)), 0.85 (d, J ¼ 5.4 Hz, 3H, CH3 (29)), 0.84 (s, 3H, CH3 (26)), 0.73 (s,
ethyl acetate/n-heptane/formic acid, 80:20:30:4); [
a
]
¼ þ44.4ꢁ (c
D
0.34, CHCl3), IR (KBr):
1384s, 1248s, 1060m, 1028 m cmꢀ1
¼ 4.48 (dd, J ¼ 10.1, 6.2 Hz, 1H, CH (3)), 3.87 (dd, J ¼ 2.7, 2.7 Hz, 1H,
n
¼ 3528 brs, 3448vs, 2948s, 1736vs, 1468m,
;
1H NMR (400 MHz, CDCl3):
d
CH (12)), 2.13 (ddd, J ¼ 13.3, 13.3, 5.8 Hz, 1H, CH (16)), 2.03 (s, 3H,
CH3 (Ac)), 2.06e1.84 (m, 4H, CH (18) þ CH2 (19) þ CHa (7)),1.84 (ddd,
J ¼ 13.5, 13.5, 6.1 Hz, 1H, CHa (15)), 1.76e1.66 (m, 1H, CHa (1)), 1.69e
1.1.52 (m, 6H, CH (9) þ CHa (22) þ CH2 (11) þ CH2 (2)),1.52e1.40 (m,
3H, CH2 (6) þ CHb (7)), 1.39e1.32 (m, 1H, CHb (22)), 1.30 (s, 3H, CH3
(27)), 1.29e1.22 (m, 3H, CHb (16) þ CH2 (21)), 1.19e1.12 (m, 1H, CHb
(15)), 1.14 (s, 3H, CH3 (26)), 1.13e1.09 (m, 1H, CHb (1)), 0.97 (s, 3H,
CH3 (30)), 0.89 (s, 6H, CH3 (25) þ CH3 (29)), 0.86 (s, 3H, CH3 (23)),
0.85 (s, 3H, CH3 (24)), 0.85e0.82 (m, 1H, CH (5)) ppm; 13C NMR
3H, CH3 (24)) ppm; 13C NMR (125 MHz, CDCl3):
d
¼ 178.0 (C]O,
C28), 170.9 (C]O, Ac), 138.1 (C]CH, C13), 125.4 (CH]C, C12), 80.9
(CHOH, C3), 55.3 (CH, C5), 52.8 (CH, C18), 51.4 (CH3, C31), 48.0
(Cquart, C17), 47.5 (CH, C9), 41.9 (Cquart, C14), 39.5 (Cquart, C8), 39.0
(CH, C19), 38.8 (CH, C20), 38.3 (CH2, C1), 37.6 (Cquart, C4), 36.8 (CH2,
C21), 36.6 (Cquart, C10), 32.9 (CH2, C7), 30.6 (CH2, C22), 28.0 (CH3,
C23), 28.0 (CH2, C15), 24.2 (CH2, C16), 23.5 (CH3, C27), 23.5 (CH2,
(100 MHz, CDCl3):
d
¼ 180.2 (C]O, C28), 171.3 (C]O, Ac), 90.8

94
B. Siewert et al. / European Journal of Medicinal Chemistry 72 (2014) 84e101
C2), 23.3 (CH2, C11), 21.3 (CH3, Ac), 21.1 (CH3, C29), 18.2 (CH2, C6),
17.0 (CH3, C30), 17.0 (CH3, C24), 16.8 (CH3, C26), 15.5 (CH3, C25)
ppm; MS (ESI, MeOH): m/z ¼ 513.2 (40%, [M þ H]þ), 535.5 (100%,
[M þ Na]þ).
MeOH): m/z ¼ 529.1 (20%, [M þ H]þ), 551.3 (100%, [M þ Na]þ),
583.2 (17%, [M þ Na þ MeOH]þ).
5.4.10. (3
en-28-oate (9)
By using method E compound 9 was obtained from 8; yield 83%;
b) Methyl 3-acetoxy-12-hydroxy-11-oxo-olean-D-12,13-
5.4.8. (3b) Methyl 3-acetoxy-olean-11-en-28-oate (7)
Compound 7 was obtained from 3 as a colorless solid using
RF
¼
0.5 (n-hexane/ethyl acetate, 8:2); m.p.: 199e201 ꢁC;
method D; yield: 85%; m.p.: 220e221 ꢁC (lit.: 219e220 ꢁC [53]);
[a]
¼ þ112.7ꢁ (c 0.34, CHCl3); IR (KBr):
¼ 3454br, 2948s, 2868m,
n
D
[
a
]
¼ þ66ꢁ (c 0.32, CHCl3) [lit.: þ69ꢁ (CHCl3 [53])]; RF ¼ 0.70
1730vs, 1664m, 1636s, 1466m, 1390m, 1366s, 1306m, 1244vs, 1202m,
D
(toluene/ethyl acetate/n-heptane/formic acid, 80:20:30:4); IR
1164m, 1140w, 1120w, 1034m cmꢀ1; UVeVis (CHCl3): lmax (log
(KBr):
1240s, 1162m, 1123w, 1038m, 1022 m cmꢀ1
CDCl3):
¼ 5.28 (dd, J ¼ 3.5, 3.5 Hz, 1H, CH (12)), 4.49 (dd, J ¼ 8.9,
n
¼ 3428br, 2938s, 2861m, 1731s, 1451m, 1364m, 1266m,
3
) ¼ 280.0 nm (4.51); 1H NMR (500 MHz, CDCl3):
d
¼ 6.21 (s, 1H,
;
1H NMR (400 MHz,
OH), 4.50 (dd, J ¼ 11.6, 4.9 Hz, 1H, CH (3)), 3.71e3.64 (ddd, J ¼ 9.2,
9.2, 1.6, 1H, CH (18)), 3.63 (s, 3H, CH3 (31)), 2.80 (ddd, J ¼ 13.6, 13.6,
3.6 Hz, 1H, CHa (1)), 2.46 (s, 1H, CH (9)), 2.04 (ddd, J ¼ 13.8, 13.8,
3.0 Hz, 1H, CHa (16)), 2.05 (s, 3H, CH3 (Ac)), 1.76 (ddd, J ¼ 13.9, 13.9,
4.4 Hz, 1H, CHa (7)), 1.73e1.67 (m, 2H, CH2 (2)), 1.67e1.60 (m, 2H,
CHb (7) þ CHb (16)), 1.60e1.55 (m, 3H, CHa (21) þ CHa (15) þ CHa
(6)), 1.43e1.36 (m, 5H, CH2 (19) þ CHa (22) þ CHb (21) þ CHb (6)),
1.36 (s, 3H, CH3 (27)), 1.29e1.23 (m, 1H, CHb (22)), 1.20 (ddd, J ¼ 14.0,
14.0, 3.4 Hz, 1H, CHb (15)), 1.13 (s, 3H, CH3 (25)), 1.10 (ddd, J ¼ 13.3,
13.3, 4.1 Hz, CHb (1)), 1.00 (s, 3H, CH3 (30)), 0.93 (s, 3H, CH3 (29)),
0.92 (s, 3H, CH3 (26)), 0.87 (s, 6H, CH3 (23) þ CH3 (24)), 0.85e0.77
d
7.1 Hz, 1H, CH (3)), 3.62 (s, 3H, CH3 (31)), 2.86 (dd, J ¼ 13.9, 4.1 Hz,
1H, CH (18)), 2.04 (s, 3H, CH3 (Ac)),1.97 (ddd, J ¼ 14.3, 5.6, 3.8 Hz,1H,
CHa (16)), 1.92e1.82 (m, 2H, CHa (2) þ CHa (11)), 1.70 (dd, J ¼ 13.8,
4.5 Hz, 1H, CHa (19)), 1.67e1.57 (m, 6H, CHa (1) þ CHb (11) þ CHb
(2) þ CHb (16) þ CHa (15) þ CHa (7)), 1.57e1.47 (m, 2H, CHa
(22) þ CHa (6)), 1.51 (dd, J ¼ 10.9, 3.8 Hz, 1H, CH (9)), 1.47e1.40 (m,
2H, CHb (7) þ CHb (6)), 1.34 (ddd, J ¼ 14.7, 10.5, 5.9 Hz, 1H, CHa (21)),
1.28e1.14 (m, 3H, CHb (21) þ CHb (19) þ CHb (22)), 1.12 (s, 3H, CH3
(27)), 1.10e0.94 (m, 2H, CHb (15) þ CHb (1)), 0.93 (s, 3H, CH3 (30)),
0.92 (s, 3H, CH3 (25)), 0.90 (s, 3H, CH3 (29)), 0.86 (s, 3H, CH3 (23)),
0.85 (s, 3H, CH3 (24)), 0.79 (dd, J ¼ 12.9, 7.2 Hz, 1H, CH (5)), 0.72 (s,
(m, 1H, CH (5)) ppm; 13C NMR (125 MHz, CDCl3):
d
¼ 195.5 (C]O,
C11), 178.0 (C]O, C28), 171.1 (C]O, Ac), 142.3 (COH]C, C12), 136.9
(C]CHOH, C13), 80.6 (CHOH, C3), 60.5 (CH, C9), 55.2 (CH, C5), 52.0
(CH3, C31), 46.2 (Cquart, C1), 45.6 (Cquart, C17), 41.7 (Cquart, C8), 40.4
(CH2, C19), 38.8 (CH2, C1), 38.2 (Cquart, C4), 37.5 (Cquart, C10), 34.2
(CH2, C22), 33.4 (CH, C18), 33.2 (CH2, C21), 33.0 (CH3, C29), 32.0
(CH2, C7), 30.7 (Cquart, C20), 28.2 (CH3, C23), 28.0 (CH2, C15), 23.7
(CH3, C27), 23.4 (CH3, C30), 23.3 (CH2, C2), 21.4 (CH3, Ac), 19.0 (CH3,
C26), 17.4 (CH2, C6), 16.8 (CH3, C24), 16.5 (CH3, C25) ppm; MS (ESI,
MeOH): m/z ¼ 543.2 (38%, [M þ H]þ), 565.4.5 (50%, [M þ Na]þ),
1107.3 (100%, [2M þ Na]þ).
3H, CH3 (26)) ppm; 13C NMR (125 MHz, CDCl3):
d
¼ 178.3 (C]O,
C28), 170.9 (C]O, Ac), 143.8 (C]CH, C13), 122.2 (CH]C, C12), 80.9
(CHOAc, C3), 55.3 (CH, C5), 51.5 (CH3, C31), 47.6 (CH, C9), 46.7
(Cquart, C17), 45.8 (CH2, C19), 41.6 (Cquart, C4), 41.3 (CH, C18), 41.2
(Cquart, C8), 38.1 (CH2, C1), 37.7 (Cquart, C14), 36.9 (Cquart, C10), 33.4
(CH2, C21), 33.1 (CH3, C29), 32.6 (CH2, C7), 32.4 (CH2, C22), 30.7
(Cquart, C20), 28.0 (CH3, C23), 27.6 (CH2, C15), 25.9 (CH3, C27), 23.6
(CH3, C30), 23.5 (CH2, C2), 23.4 (CH2, C16), 23.1 (CH2, C11), 21.2 (CH3,
Ac), 18.2 (CH2, C6), 16.8 (CH3, C26), 16.7 (CH3, C24), 15.3 (CH3, C25)
ppm; MS (ESI, MeOH): m/z ¼ 535.5 (22%, [M þ Na]þ), 1047.3 (100%,
[2M þ Na]þ).
5.4.11. (3b) Methyl 3-acetoxy-11-oxo-olean-12-en-28-oate (10)
N-Hydroxysuccinimide (1.2 g, 10.4 mmol) and potassium di-
chromate (1.1 g, 3.6 mmol) were added to a solution of 7 (0.5 g,
0.94 mmol) in acetone (50 mL) containing glacial acetic acid (5 mL);
the mixture was stirred at 40ꢁ for 48 h. The reaction was quenched
with potassium disulfite solution and after usual work up 10 was
obtained as a colorless solid; yield: 65%; m.p.: 216e220 ꢁC (lit.:
5.4.9. (3b) Methyl 3-acetoxy-12-oxo-olean-28-oate (8)
Compound 7 was treated according to method A to yield 8; yield
75%; RF ¼ 0.5 (n-hexane/ethyl acetate, 8:2); m.p.: 189e191 ꢁC (lit.:
194 ꢁC [54]; [
a]
¼ ꢀ13.3ꢁ (c 0.54, CHCl3; lit.: ꢀ12ꢁ (c 1.4, CHCl3
¼ 3426br, 2948s, 2866m, 1728vs, 1700s, 1466m,
D
n
[54]); IR (KBr):
1388m, 1368m, 1244vs, 1192m, 1162m, 1082w, 1030m, 1004 m cmꢀ1
;
[55]: 245e247 ꢁC); [
ethyl acetate, 7:3); UVeVis (MeOH): lmax (log
a
]
D
¼ þ82ꢁ (c 0.14, CHCl3); RF ¼ 0.62 (hexane/
UVeVis (CHCl3): lmax (log
CDCl3):
3
) ¼ 278 nm (4.31); 1H NMR (500 MHz,
3
) ¼ 269 nm (4.03); IR
d
¼ 4.47 (dd, J ¼ 11.5, 4.9 Hz, 1H, CH (3)), 3.67 (s, 3H, CH3
(KBr):
1330w, 1304w, 1261m, 1227w, 1209m, 1189m, 1162m, 1125w, 1089w,
1039m, 1013w cmꢀ1; 1H NMR (500 MHz, CDCl3):
¼ 5.61 (s, 1H, CH
n
¼ 3339br, 2949s, 2866s,1724s,1661s,1466m,1387m,1365w,
(31)), 2.81e2.76 (ddd, J ¼ 13.4, 4.3, 3.5, 1H, CH (18)), 2.61 (d,
J ¼ 4.3 Hz, 1H, CH (13)), 2.23 (dd, J ¼ 16.8, 5.1 Hz, 1H, CHa (11)), 2.14
(dd, J ¼ 16.8, 3.5 Hz, 1H, CHb (11)), 2.04 (s, 3H, CH3 (Ac)), 1.93 (ddd,
J ¼ 13.5, 3.6, 2.5 Hz,1H, CHa (19)), 1.89 (ddd, J ¼ 14.8, 14.8, 4.2 Hz,1H,
CHa (16)), 1.79 (ddd, J ¼ 13.7, 13.7, 4.6 Hz, 1H, CHa (7)), 1.68e1.58 (m,
6H, CH (18) þ CHa (15) þ CH2 (2) þ CHa (6) þ CHb (16)), 1.55 (ddd,
J ¼ 12.9, 7.5, 3.8 Hz, 1H, CHa (1)), 1.49e1.43 (m, 3H, CHb (6) þ CHb
(7) þ CHa (22)),1.36e1.28 (m, 2H, CHb (22) þ CHa (21)),1.27e1.17 (m,
2H, CHb (21) þ CHb (19)),1.06 (ddd, J ¼ 13.3, 4.1, 1.8 Hz,1H, CHb (15)),
1.04e0.98 (m, 1H, CHb (1)), 0.97 (s, 3H, CH3 (30)), 0.96 (s, 3H, CH3
(26)), 0.93 (s, 3H, CH3 (27)), 0.90 (s, 3H, CH3 (29)), 0.87 (s, 3H, CH3
(25)), 0.86 (s, 3H, CH3 (23)), 0.85 (s, 3H, CH3 (24)), 0.84e0.80 (m, 1H,
d
(12)), 3.61 (s, 3H, CH3 (31)), 3.20 (dd, J ¼ 10.8, 5.5 Hz, 1H, CH (3)),
2.98 (dd, J ¼ 13.8, 3.7 Hz, 1H, CH (18)), 2.85e2.75 (m, 1H, CHa (1)),
2.30 (s, 1H, CH (9)), 2.02 (ddd, J ¼ 13.8, 13.8, 4.0 Hz, 1H, CHa (16)),
1.75e1.52 (m, 9H, CHa (19) þ CHb (16) þ CH2 (7) þ CHa (15) þ CHa
(22) þ CH2 (2) þ CHa (6)), 1.42e1.13 (m, 6H, CHb (19) þ CHb
(22) þ CH2 (21) þ CHb (15) þ CHb (6)), 1.34 (s, 3H, CH3 (27)), 1.08 (s,
3H, CH3 (25)), 1.00e0.95 (m, 1H, CHb (1)), 0.97 (s, 3H, CH3 (23)), 0.92
(s, 3H, CH3 (30)), 0.91 (s, 3H, CH3 (29)), 0.89 (s, 3H, CH3 (26)), 0.78 (s,
3H, CH3 (24)), 0.66 (brd, J ¼ 11.3 Hz, 1H, CH (5)) ppm; 13C NMR
(125 MHz, CDCl3):
d
¼ 200.3 (C]O, C11), 177.4 (C]O, C28), 168.6
CH (5)) ppm; 13C NMR (125 MHz, CDCl3):
d
¼ 211.7 (C]O, C12),
(C]CH, C13), 127.9 (CH]C, C12), 78.7 (CHOH, C3), 61.7 (CH, C9),
55.0 (CH, C5), 51.8 (CH3, C31), 46.2 (Cquart, C17), 45.0 (Cquart, C8), 44.2
(CH2, C19), 43.4 (Cquart, C14), 41.5 (CH, C18), 39.1 (CH2, C1), 38.8
(Cquart, C4), 37.2 (Cquart, C10), 33.7 (CH2, C21), 32.9 (CH2, C7), 32.8
(CH3, C29), 31.6 (CH2, C22), 30.6 (Cquart, C20), 28.1 (CH3, C23), 27.7
(CH2, C15), 27.3 (CH2, C2), 23.5 (CH3, C27), 23.4 (CH3, C30), 22.9
(CH2, C16), 18.9 (CH3, C26), 17.4 (CH2, C6), 16.1 (CH3, C25), 15.5 (CH3,
C24) ppm; MS (ESI, MeOH): m/z ¼ 485.6 (100%, [M þ H]þ), 507.5
(35%, [M þ Na]þ).
178.5 (C]O, C28), 171.0 (C]O, Ac), 80.6 (CHOAc, C3), 55.3 (CH, C5),
52.0 (CH3, C13), 51.9 (CH, C31), 49.8 (CH, C9), 47.5 (Cquart, C17), 42.0
(Cquart, C14), 41.4 (Cquart, C8), 38.6 (CH2, C11), 37.9 (CH2, C1), 37.8
(Cquart, C10), 37.0 (Cquart, C4), 36.4 (CH2, C19), 34.6 (CH2, 21), 33.5
(CH3, C29), 33.1 (CH2, C22), 32.1 (CH, C18), 31.9 (CH2, C7), 30.8
(Cquart, C20), 28.1 (CH3, C23), 27.7 (CH2, C15), 23.6 (CH2, C2), 23.3
(CH3, C30), 22.9 (CH2, C16), 21.4 (CH3, Ac), 20.7 (CH3, C27),18.3 (CH2,
C6), 16.6 (CH3, C24), 16.3 (CH3, C26), 15.4 (CH3, C25) ppm; MS (ESI,

B. Siewert et al. / European Journal of Medicinal Chemistry 72 (2014) 84e101
95
5.4.12. (2a,3b
) Methyl 2,3-dihydroxy-olean-12-en-28-oate (11)
5.4.14. Methyl (2a,3b)2,3edi-O-acetyl-12-oxo-olean-28-oate (13)
Compound 11 [56] was obtained from MA (100 mg,
0.21 mmol) using method D as fine colorless needles; yield: 88%;
m.p. 229e231 ꢁC (214e216 ꢁC [56]); RF ¼ 0.4 (n-hexane/ethyl
Compound 13 obtained as colorless solid from 12 using method
B; yield: 84%; m.p.: 144e147 ꢁC; RF ¼ 0.21 (n-hexane/ethyl acetate,
8:2); [
a
]
¼ ꢀ46.7ꢁ (c 0.40, CHCl3); IR (KBr):
n
¼ 3588w, 3442s,
D
acetate, 6:4); [
a
]
¼ þ60.9ꢁ (c 0.65, CHCl3); IR (KBr)
n
¼ 3571br,
3430s, 2949m, 2867w, 1742vs, 1727s, 1700m, 1472m, 1458m, 1440w,
1395w, 1387m, 1369m, 1250vs, 1192m, 1160m, 1154m, 1119w, 1086w,
D
3300s, 2947s, 1739s, 1461s, 1386m, 1363m, 1262m, 1229m, 1190s,
1162s, 1124m, 1052s, 1037s, 984m, 958s, 921s cmꢀ1
;
1H NMR
1042m cmꢀ1
;
1H NMR (400 MHz, CDCl3):
d
¼ 5.06 (ddd, J ¼ 11.4,
(500 MHz, CDCl3):
d
¼ 5.28 (dd, J ¼ 3.4, 3.4 Hz, 1H, CH (12)), 3.72e
10.4, 4.7 Hz, 1H, CH (2)), 4.74 (d, J ¼ 10.4 Hz, 1H, CH (3)), 3.65 (s, 3H,
CH3 (31)), 2.80 (ddd, J ¼ 13.6, 4.0, 4.0 Hz, 1H, CH (18)), 2.60 (d,
J ¼ 4.0 Hz, 1H, CH (13)), 2.18 (m, 1H, CHa (11)), 2.16 (m, 1H, CHb (11)),
2.05 þ 1.98 (s, 6H, 2ꢃ CH3 (Ac)), 2.00e1.88 (m, 3H, CHa (19) þ CHa
(1) þ CHa (16)), 1.85e1.78 (m, 1H, CHa (7)), 1.79e1.71 (m, 1H, CH (9)),
1.70e1.60 (m, 3H, CHa (15) þ CHb (16) þ CHa (6)), 1.52e1.42 (m, 3H,
CHb (6) þ CHb (7) þ CHa (22)), 1.39e1.28 (m, 2H, CHb (22) þ CHa
(21)), 1.24e1.18 (m, 1H, CHb (19) þ CHb (21)), 1.12e1.08 (m, 1H, CHb
(15)), 1.06e0.88 (m, 1H, CHb (1)), 1.02 (s, 3H, CH3 (27)), 0.98 (s, 3H,
CH3 (25)), 0.97 (s, 3H, CH3 (30)), 0.96 (m,1H, CH (5)), 0.94 (s, 3H, CH3
(23)), 0.90 (s, 3H, CH3 (24)), 0.89 (s, 3H, CH3 (29)), 0.89 (s, 3H, CH3
3.65 (m, 1H, CH (2)), 3.61 (s, 3H, CH3 (31)), 3.00 (d, J ¼ 9.4 Hz, 1H,
CH (3)), 2.85 (dd, J ¼ 13.8, 4.1 Hz, 1H, CH (18)), 2.21 (br, 2H, OH),
1.97 (dd, J ¼ 10.4, 3.9 Hz, 1H, CHa (1)), 1.96e1.83 (m, 3H, CHa
(16) þ CH2 (11)), 1.68 (ddd, J ¼ 13.9, 13.9, 4.4 Hz, 1H, CHa (7)),
1.64e1.56 (m, 4H, CH (9) þ CHa (19) þ CHa (15) þ CHb (16)), 1.55e
1.52 (m, 1H, CHa (6)), 1.50 (ddd, J ¼ 14.0, 3.4, 3.4 Hz, CHa (22)), 1.43
(m, 1H, CHb (7)), 1.38 (ddd, J ¼ 12.4, 12.4, 2.5 Hz, CHb (6)), 1.29
(ddd, J ¼ 13.8, 9.6, 2.7 Hz, 1H, CHa (21)), 1.29e1.27 (m, 1H, CHb
(22)), 1.16 (ddd, J ¼ 14.0, 4.2, 4.2 Hz, 1H, CHb (21)), 1.15e1.11 (m,
1H, CHb (19)), 1.12 (s, 3H, CH3 (C27)), 1.06e1.02 (m, 1H, CHb (15)),
1.02 (s, 3H, CH3 (C23)), 0.97 (s, 3H, CH3 (C25)), 0.92 (s, 3H, CH3
(C30)), 0.92e0.87 (m, 1H, CHb (1)), 0.89 (s, 3H, CH3 (C29)), 0.84e
0.82 (m, 1H, CH (5)), 0.82 (s, 3H, CH3 (C24)), 0.71 (s, 3H, CH3 (C26))
(26)); 13C NMR (100 MHz, CDCl3):
O, C28), 170.4 (C]O, Ac), 170.5 (C]O, Ac), 80.1 (CHOAc, C3), 69.5
d
¼ 210.6 (C]O, C12), 178.0 (C]
(CHOAc, C2), 54.8 (CH, C5), 51.6 (CH, C13), 49.6 (CH, C9), 47.4 (Cquart
C17), 43.3 (CH2, C1), 41.7 (Cquart, C14), 41.2 (Cquart, C8), 39.0 (Cquart
,
,
ppm; 13C NMR (125 Hz, CDCl3):
d
¼ 178.2 (C]O, C28), 143.8 (C]
CH, C13), 122.1 (CH]C, C12), 84.0 (CHOH, C3), 68.9 (CHOH, C2),
55.3 (CH, C5), 51.5 (CH3, C31), 47.6 (CH, C9), 46.7 (Cquart, C17), 46.3
(CH2, C1), 45.8 (CH2, C19), 41.6 (Cquart, C14), 41.2 (CH, C18), 39.3
(Cquart, C8), 39.1 (Cquart, C4), 38.3 (Cquart, C10), 33.8 (CH2, C21), 33.1
(CH3, C29), 32.5 (CH2, C22), 32.3 (CH2, C7), 30.7 (Cquart, C20), 28.6
(CH3, C23), 27.6 (CH2, C15), 25.9 (CH3, C27), 23.6 (CH3, C30), 23.4
(CH2, C16), 23.0 (CH2, C11), 18.3 (CH2, C6), 16.9 (CH3, C26), 16.7
(CH3, C24), 16.6 (CH3, C25) ppm; MS (ESI, MeOH, source CID): m/
z ¼ 487.4 (48.8%, [M þ H]þ), 504.5 (53.4%, [M þ NH4]þ), 509.5
(100%, [M þ Na]þ), 541.2 (22.0%, [M þ Na þ MeOH]þ), 741.5
(14.6%, [3M þ Na þ H]2þ), 749.6 (73.2%, [3M þ K þ H]2þ), 992.8
(48.8%, [4M þ K þ H]2þ).
C4), 38.5 (CH2, C11), 38.1 (Cquart, C10), 36.4 (CH2, C19), 34.5 (CH2,
C21), 33.5 (CH3, C29), 32.8 (CH2, C7), 32.0 (CH, C18), 31.6 (CH2, C22),
30.8 (Cquart, C20), 28.2 (CH3, C23), 27.7 (CH2, C15), 23.0 (CH3, C30),
22.9 (CH2, C16), 21.0 (CH3, Ac), 21.1 (CH3, Ac), 20.4 (CH3, C27), 18.3
(CH2, C6), 17.5 (CH3, C26), 16.5 (CH3, C24), 16.0 (CH3, C25) ppm; MS
(ESI, MeOH): m/z ¼ 587.3 (37%, [M þ H]þ), 604.4 (26%, [M þ NH4]þ),
609.5 (100%, [M þ Na]þ), 1190.2 (29%, [2M þ NH4]þ), 1195.1 (85%,
[2M þ Na]þ). C35H54O7
5.4.15. (3b) Methyl 3-hydroxy-urs-11-en-28 oate (14)
Compound 14 was obtained from UA as fine colorless needles
using method D; yield: 95%; m.p.: 165e167 ꢁC (lit [59].: 166e
168 ꢁC); RF ¼ 0.47 (n-hexane/ethyl acetate, 8:2); [
a]
¼ þ68ꢁ (c
D
5.4.13. (2
a,3b
) Methyl 2,3-diacetyl-olean-12-en-28-oate (12)
0.53; CHCl3) [lit.: 49.8ꢁ (c 1.0; CHCl3) [52]; UVeVis (MeOH): lmax
Obtained from 11 using method C as a colorless solid; yield:
(log
1457m, 1386m, 1378m, 1308w, 1270w, 1232m, 1200m, 1187m,
1168w, 1142m, 1113w, 1093w, 1031m cmꢀ1 1H NMR (500 MHz,
CDCl3):
¼ 5.22 (m, 1H, CH (12)), 3.59 (s, 3H, CH3 (31)), 3.20 (dd,
3
) ¼ 224 nm (3.62); IR (KBr):
n
¼ 3374br, 2927s, 2871m, 1728s,
92%; m.p.: 173e175 ꢁC (lit.: 165e168 ꢁC [57], 176e178 ꢁC [58];
RF ¼ 0.48 (n-hexane/ethylacetate, 8:2); [
CHCl3); IR (KBr):
a
]
¼ þ25.1ꢁ (c 0.37,
;
D
n
¼ 3430br, 2972s, 2949s, 2882m, 2866m, 1745vs,
d
1722s, 1460m, 1433m, 1393m, 1369m, 1250vs, 1190m, 1165m,
J ¼ 11.0, 4.9 Hz, 1H, CH (3)), 2.21 (d, J ¼ 11.3 Hz, 1H, CH (18)), 1.98
(ddd, J ¼ 13.4, 13.4, 4.6 Hz, 1H, CHa (2)), 1.90 (dd, J ¼ 8.9, 3.7 Hz, 2H,
CH2 (11)), 1.85 (ddd, J ¼ 13.8, 13.8, 4.8 Hz, 1H, CHa (15)), 1.69e1.56
(m, 5H, CH2 (7) þ CHa (1) þ CHa (16) þ CHb (2)),1.55e1.44 (m, 5H, CH
(9) þ CHa (6) þ CHa (21) þ CHa (22) þ CHb (16)), 1.36e1.24 (m, 4H,
CH (19) þ CHb (6) þ CHb (22) þ CHb (21)), 1.08e1.03 (m, 1H, CHb
(15)), 1.06 (s, 3H, CH3 (27)), 1.01e0.95 (m, 2H, CH (20) þ CHb (1)),
0.97 (s, 3H, CH3 (23)), 0.93 (d, J ¼ 6.0 Hz, 3H, CH3 (30)), 0.91 (s, 3H,
CH3 (25)), 0.85 (d, J ¼ 6.4 Hz, 3H, CH3 (29)), 0.77 (s, 3H, CH3 (24)),
0.73 (s, 3H, CH3 (26)), 0.71 (brd, J ¼ 10.4 Hz, 1H, CH (5)) ppm; 13C
1042m, 1034m cmꢀ1 1H NMR (400 MHz, CDCl3):
;
d
¼ 5.28 (dd,
J ¼ 3.6, 3.6 Hz, 1H, CH (12)), 5.11 (ddd, J ¼ 11.7, 10.4, 4.8 Hz, 1H, CH
(2)), 4.73 (d, J ¼ 10.4 Hz, 1H, CH (3)), 3.60 (s, 3H, CH3 (31)), 2.85 (dd,
J ¼ 13.8, 4.4 Hz,1H, CH (18)), 2.04 þ1.97 (s, 6H, CH3 (Ac)), 2.08e1.93
(m, 3H, CHa (1) þ CHa (16) þ CHa (11)), 1.93e1.88 (m, 1H, CHb (11)),
1.73e1.68 (m, 1H, CHa (7)), 1.66e1.58 (m, 4H, CHa (15) þ CHa
(19) þ CHb (16) þ CH (9)), 1.55e1.48 (m, 2H, CHa (6) þ CHb (7)),
1.48e1.38 (m, 2H, CHa (22) þ CHb (6)), 1.35e1.28 (m, 2H, CHb
(22) þ CHa (21)), 1.20e1.12 (m, 2H, CHb (21) þ CHb (19)), 1.11 (s, 3H,
CH3 (27)), 1.10 (m, 2H, CHb (1) þ CHb (15)), 1.06 (s, 3H, CH3 (23)),
1.00e0.92 (m, 1H, CH (5)), 0.94 (s, 3H, CH3 (25)), 0.90 (s, 3H, CH3
(30)), 0.89 (s, 3H, CH3 (29)), 0.89 (s, 3H, CH3 (24)), 0.74 (s, 3H, CH3
NMR (125 MHz, CDCl3):
d
¼ 177.9 (C]O, C28), 138.1 (C]CH, C13),
125.5 (HC]C, C12), 79.0 (CHOH, C3), 55.2 (CH, C5), 52.9 (CH, C18),
51.4 (CH3, C31), 48.0 (Cquart, C17), 47.5 (CH, C9), 41.9 (Cquart, C14),
39.5 (Cquart, C8), 39.0 (CH, C19), 38.8 (CH, C20), 38.7 (Cquart, C4), 38.6
(CH2, C1), 36.9 (Cquart, C10), 36.7 (CH2, C22), 32.9 (CH2, C7), 30.6
(CH2, C21), 28.1 (CH3, C23), 28.0 (CH2, C15), 27.2 (CH2, C16), 24.2
(CH2, C2), 23.6 (CH3, C27), 23.3 (CH2, C11), 21.1 (CH3, C30), 18.3 (CH2,
C6), 17.0 (CH3, C29), 16.9 (CH3, C26), 15.6 (CH3, C24), 15.4 (CH3, C25)
ppm; MS (ESI, MeOH): m/z ¼ 471.2 (80%, [M þ H]þ), 493.4 (100%,
[M þ Na]þ).
(26)); 13C NMR (100 MHz, CDCl3):
d
¼ 178.3 (C]O, C28), 170.7 (C]
O, Ac), 170.3 (C]O, Ac), 143.8 (C]CH, C13), 122.2 (CH]C, C12),
80.6 (CHOAc, C3), 70.0 (CHOAc, C2), 54.9 (CH, C5), 47.6 (CH, C9),
46.7 (Cquart, C17), 45.9 (CH2, C19), 43.8 (CH2, C1), 41.6 (Cquart, C14),
41.2 (CH, C18), 39.5 (Cquart, C4), 39.3 (Cquart, C8), 38.3 (Cquart, C10),
33.8 (CH2, C21), 33.0 (CH3, C29), 32.4 (CH2, C22), 32.3 (CH2, C7),
30.6 (Cquart, C20), 28.5 (CH3, C23), 27.6 (CH2, C15), 25.8 (CH3, C27),
23.8 (CH3, C30), 23.4 (CH2, C11), 23.1 (CH2, C16), 21.0 (CH3, Ac), 20.9
(CH3, Ac), 18.3 (CH2, C6), 17.7 (CH3, C26), 16.9 (CH3, C24), 16.5 (CH3,
C25) ppm; MS (MeOH): m/z ¼ 571.1 (41%, [M þ H]þ), 588.2 (71%,
[M þ NH4]þ), 593.5 (16%, [M þ Na]þ, 1163.4 (100%, [2M þ Na]þ).
5.4.16. (3b) Methyl 3-hydroxy-olean-11-en-28 oate (15)
OA (1 equiv.) was treated with iodomethane following method
D. Compound 16 was obtained as fine colorless needles; yield:
88.3%; m.p.: 198e200 ꢁC (lit [60].: 198e200 ꢁC); RF ¼ 0.50 (n-
C
35H54O6

96
B. Siewert et al. / European Journal of Medicinal Chemistry 72 (2014) 84e101
hexane/ethyl acetat, 8:2);
[
a
]
¼
þ70ꢁ (c 0.43; CHCl3) [lit
CDCl3):
d
¼ 6.22 (s, 1H, OH), 3.66 (ddd, J ¼ 9.0, 9.0, 1.8 Hz, 1H, CH
D
[61].: þ70ꢁ (c 1.0; CHCl3)]; IR (KBr):
n
¼ 3334br, 2947s, 2865m,
(18)), 3.62 (s, 3H, CH3 (31)), 3.22 (dd, J ¼ 10.4, 5.9 Hz, 1H, CH (3)),
2.79 (ddd, J ¼ 13.4, 3.4, 3.4 Hz, 1H, CHa (1)), 2.44 (s, 1H, CH (9)), 2.04
(ddd, J ¼ 13.7, 13.7, 3.9 Hz, 1H, CHa (16)), 1.76 (ddd, J ¼ 13.9, 13.9,
4.4 Hz, 1H, CHa (7)), 1.70 (brd, J ¼ 13.7 Hz, 1H, CHb (6)), 1.67e1.52 (m,
6H, CHb (7) þ CHa (21) þ CH2 (11) þ CHa (15) þ CHa (6)), 1.38e1.26
(m, 5H, CH2 (19) þ CHb (21) þ CHa (22) þ CHb (6)), 1.36 (s, 3H, CH3
(27)), 1.26e1.22 (m, 1H, CHb (22)), 1.19 (ddd, J ¼ 14.0, 3.2, 3.2 Hz, 1H,
CHb (15)), 1.10 (s, 3H, CH3 (25)), 1.04e0.95 (m, 1H, CHb (1)), 0.99 (s,
6H, CH3 (30) þ CH3 (23)), 0.92 (s, 3H, CH3 (29)), 0.91 (s, 3H, CH3
(26)), 0.79 (s, 3H, CH3 (24)), 0.70 (brd, J ¼ 11.4 Hz, 1H, CH (5)) ppm;
1725s, 1662w, 1464m, 1386m, 1363m, 1304w, 1263m, 1241m, 1202m,
1190m, 1162m, 1125m, 1094w, 1065w, 1032m cmꢀ1
; UVeVis
(MeOH): lmax (log
3
) ¼ 223 nm (3.66); 1H NMR (500 MHz, CDCl3):
d
¼ 5.26 (brs, 1H, CH (12)), 3.60 (s, 3H, CH3 (31)), 3.19 (dd, J ¼ 11.0,
4.4 Hz, 1H, CH (3)), 2.84 (dd, J ¼ 13.9, 4.2 Hz, 1H, CH (18)), 1.95 (ddd,
J ¼ 14.5, 14.4, 4.1 Hz, 1H, CHa (16)), 1.88e1.82 (m, 2H, CH2 (11)), 1.67
(ddd, J ¼ 13.9, 13.9, 4.4 Hz, 1H, CHa (7)), 1.63e1.47 (m, 9H, CH
(9) þ CHa (1) þ CHa (19) þ CHa (6) þ CHa (15) þ CHb (7) þ CHb
(16) þ CH2 (2)), 1.43e1.22 (m, 4H, CHa (21) þ CH2 (22) þ CHb (6)),
1.19e1.12 (m, 2H, CHb (19) þ CHb (21)), 1.10 (s, 3H, CH3 (27)), 1.08e
0.98 (m, 1H, CHb (15)), 0.97e0.92 (m, 1H, CHb (1)), 0.96 (s, 3H, CH3
(23)), 0.90 (s, 3H, CH3 (30)), 0.88 (s, 3H, CH3 (29)), 0.87 (s, 3H, CH3
(25)), 0.76 (s, 3H, CH3 (24)), 0.73e0.68 (m, 1H, CH (5)), 0.70 (s, 3H,
13C NMR (125 MHz, CDCl3):
d
¼ 195.6 (C]O, C11), 178.0 (C]O, C28),
142.3 (COH]C, C12), 136.8 (C]COH, C13), 78.8 (CHOH, C3), 60.6
(CH, C9), 55.1 (CH, C5), 52.0 (CH3, C31), 46.2 (Cquart, C14), 45.6
(Cquart, C17), 41.7 (Cquart, C8), 40.4 (CH2, C1), 39.3(CH2, C19), 39.2
(Cquart, C4), 37.6 (Cquart, C10), 34.2 (CH2, C22), 33.4 (CH, C18), 33.2
(CH2, C21), 33.0 (CH3, C29), 32.0 (CH2, C7), 30.7 (Cquart, C20), 28.2
(CH3, C23), 28.0 (CH2, C15), 27.4 (CH2, C11), 23.5 (CH3, C27), 23.4
(CH3, C30), 23.3 (CH2, C2), 18.9 (CH3, C26), 17.5 (CH2, C6), 16.5 (CH3,
C25), 15.7 (CH3, C24) ppm; MS (ESI): m/z ¼ 501.3 (100%, [M þ H]þ),
1023.4 (64%, [2M þ Na]þ).
CH3 (26)) ppm; 13C NMR (125 MHz, CDCl3):
d
¼ 178.2 (C]O, C28),
143.7 (C]CH, C13), 122.3 (CH]C, C12), 79.0 (C3, CHOH), 55.2 (CH,
C5), 51.5 (CH3, C31), 47.6 (CH, C9), 46.7 (Cquart, C17), 45.8 (CH2, C19),
41.6 (Cquart, C14), 41.3 (CH, C18), 39.2 (Cquart, C8), 38.7 (Cquart, C4),
38.4 (CH2, C1), 37.0 (Cquart, C10), 33.8 (CH2, C21), 33.1 (CH3, C29),
32.6 (CH2, C7), 32.3 (CH2, C22), 30.6 (Cquart, C20), 28.1 (CH3, C23),
27.7 (CH2, C15), 27.1 (CH2, C2), 25.9 (CH3, C27), 23.6 (CH3, C30), 23.4
(CH2, C11), 23.0 (CH2, C16), 18.3 (CH2, C6), 16.8 (CH3, C26), 15.5 (CH3,
C24), 15.3 (CH3, C25) ppm; MS (ESI, MeOH): m/z ¼ 493.5 (100%,
[M þ Na]þ).
5.4.19. (3b) Methyl 3-hydroxy-11-oxo-olean-12-en-28-oate (18)
To a mixture of 16 (450 mg, 0.88 mmol) in acetone (50 mL) and
glacial acetic acid (5 mL) N-hydroxysuccinimide (950 mg,
8.25 mmol) and potassium dichromate (780 mg, 2.64 mmol) were
added, and the mixture was stirred at 45 ꢁC for 48 h. The mixture
was quenched with an aq potassium disulfide solution and washed
with an aq. sodium hydrogen carbonate solution. After extracting
with DCM, the organic phases were combined, washed, dried and
evaporated. Chromatographic purification gave compound 18;
yield 71%; m.p.: 184e188 ꢁC (lit.: [36]: 181e188 ꢁC); RF ¼ 0.62 (n-
5.4.17. (3b) Methyl 3-hydroxy-12-oxo-olean-28-oate (16)
Compound 16 was obtained using method A; yield: 42%;
RF ¼ 0.20 (hexane/ethyl acetate, 8:2); m.p.: 194e196 ꢁC (lit [62].:
132e133 ꢁC); [
a
]
D
¼ ꢀ26.0ꢁ (c0.35; CHCl3); IR (KBr):
n
¼ 3492vs,
3446vs, 3432s, 2994w, 2970m, 2946m, 2926m, 2860w, 1718m,
1688s, 1654w, 1636w, 1628w, 1470w, 1458w, 1438w, 1240m, 1192w,
1178w, 1162w, 1048w, 1038w, 994w cmꢀ1
;
1H NMR (500 MHz,
hexane/ethyl acetate, 7:3; [
(MeOH): lmax (log
2866s, 1724s, 1661s, 1466m, 1387m, 1365w, 1330w, 1304w, 1261m,
1227w, 1209m, 1189m, 1162m, 1125w, 1089w, 1039m, 1013w cmꢀ1
1H NMR (500 MHz, CDCl3):
¼ 5.61 (s, 1H, CH (12)), 3.61 (s, 3H, CH3
a
]
¼ þ82ꢁ (c 0.14; CHCl3); UVeVis
D
CDCl3):
d
¼ 3.67 (s, 3H, CH3 (31)), 3.19 (dd, J ¼ 11.4, 4.4 Hz, 1H, CH
3
) ¼ 269 nm (4.03); IR (KBr):
n
¼ 3339br, 2949s,
(3)), 2.81e2.74 (m, 1H, CH (18)), 2.61 (d, J ¼ 3.9 Hz, 1H, CH (13)), 2.23
(dd, J ¼ 16.7, 4.8 Hz, 1H, CHa (11)), 2.12 (dd, J ¼ 16.1, 13.8 Hz, 1H, CHb
(11)), 1.94 (m, 1H, CHa (19)), 1.87 (ddd, J ¼ 14.6, 14.6 Hz, 4.1 Hz, 1H,
CHa (16)), 1.79 (ddd, J ¼ 13.7, 13.7, 4.3 Hz, 1H, CHa (7)), 1.69e1.56 (m,
4H, CH (9) þ CH2 (16) þ CHa (2)), 1.54 (m, 1H, CHa (1)), 1.50e1.39 (m,
2H, CHa (22) þ CHa (21)), 1.35e1.24 (m, 2H, CHb (22) þ CHb (21)),
1.20 (dd, J ¼ 13.3, 13.3 Hz, 1H, CHb (19)), 1.07 (d, 1H, CHb (2)), 0.98 (s,
3H, CH3 (30)), 0.98e0.95 (m, 1H, CHb (16)), 0.96 (s, 3H, CH3 (27)),
0.95 (s, 3H, CH3 (23)), 0.93 (s, 3H, CH3 (28)), 0.89 (s, 3H, CH3 (29)),
0.84 (s, 3H, CH3 (26)), 0.77 (s, 3H, CH3 (25)), 0.74e0.67 (m, 1H, CH
;
d
(31)), 3.20 (dd, J ¼ 10.8, 5.5 Hz, 1H, CH (3)), 2.98 (dd, J ¼ 13.8, 3.7 Hz,
1H, CH (18)), 2.80 (d, J ¼ 13.4 Hz, 1H, CHa (1)), 2.30 (s, 1H, CH (9)),
2.02 (ddd, J ¼ 13.8, 13.8, 4.0 Hz, 1H, CHa (16)), 1.75e1.52 (m, 9H, CHa
(19) þ CHb (16) þ CHa (7) þ CHa (15) þ CH2 (22) þ CH2 (2) þ CHa (6)),
1.42e1.13 (m, 6H, CHb (19) þ CHb (7) þ CH2 (21) þ CHb (15) þ CHb
(6)), 1.34 (s, 3H, CH3 (27)), 1.08 (s, 3H, CH3 (23)), 1.00e0.95 (m, 1H,
CHb (1)), 0.97 (s, 3H, CH3 (30)), 0.92 (s, 3H, CH3 (29)), 0.91 (s, 3H, CH3
(25)), 0.89 (s, 3H, CH3 (24)), 0.78 (s, 3H, CH3 (26)), 0.66 (brd,
(5)) ppm; 13C NMR (125 MHz, CDCl3):
d
¼ 211.7 (C]O, C12), 178.2
(C]O, C28), 78.6 (CHOH, C3), 55.1 (CH, C5), 51.8 (CH, C13), 51.9
(CH3, C31), 49.7 (CH, C9), 47.3 (Cquart, C17), 41.9 (Cquart, C14), 41.2
(Cquart, C8), 38.8 (CH2, C11), 38.5 (CH2, C1), 37.9 (Cquart, C10), 36.9
(Cquart, C4), 36.2 (CH2, C19), 34.4 (CH2, C1), 33.4 (CH3, C29), 32.9
(CH2, C7 þ C22), 32.0 (CH2, C21), 31.8 (CH, C18), 30.6 (Cquart, C20),
27.9 (CH3, C29), 27.5 (CH2, C2), 27.04 (CH3, C23), 23.1 (CH3, C27),
22.8 (CH2, C16), 20.5 (CH3, C30), 18.3 (CH2, C6), 16.0 (CH3, C24), 15.3
(CH3, C26), 15.2 (CH3, C25) ppm; MS (ESI, MeOH): m/z ¼ 487.5
(54.8%, [M þ H]þ), 509.6 (79.6%, [M þ Na]þ), 995.3 (100%,
[2M þ Na]þ).
J ¼ 11.3 Hz, 1H, CH (5)) ppm; 13C NMR (125 MHz, CDCl3):
¼ 200.3
d
(C]O, C11), 177.4 (C]O, C28), 168.6 (C ¼ CH, C13), 127.9 (CH ¼ C,
C12), 78.7 (CHOH, C3), 61.7 (CH, C9), 55.0 (CH, C5), 51.8 (CH3, C31),
46.2 (Cquart, C17), 45.0 (Cquart, C8), 44.2 (CH2, C19), 43.4 (Cquart, C14),
41.5 (CH, C18), 39.1 (CH2, C1), 38.8 (Cquart, C4), 37.2 (Cquart, C10), 33.7
(CH2, C21), 32.9 (CH2, C7), 32.8 (CH3, C29), 31.6 (CH2, C22), 30.6
(Cquart, C20), 28.1 (CH3, C23), 27.7 (CH2, C15), 27.3 (CH2, C2), 23.5
(CH3, C27), 23.4 (CH3, C30), 22.9 (CH2, C16), 18.9 (CH3, C26), 17.4
(CH2, C6), 16.1 (CH3, C24), 15.5 (CH3, C25) ppm; MS (ESI, MeOH): m/
z ¼ 485.6 (100%, [M þ H]þ), 507.5 (35%, [M þ Na]þ).
5.4.18. (2
a, 3b
) Methyl 3,12-dihydroxy-11-oxo-olean-12-en-28-
5.4.20. (3b) Benzyl 3-hydroxy-urs-12-en-28-oate (19)
oate (17)
UA (1 equiv.) was dissolved in DMF and treated with benzyl-
chloride following method D. Compound 19 was obtained as a
colorless solid; yield: 82%; m.p.: 182 ꢁC (lit.: 180e182 ꢁC [63]);
Compound 17 was obtained using method E starting from
compound 16 as colorless crystals; yield: 96%; m.p.: 206e210 ꢁC;
RF ¼ 0.43 (n-hexane/ethyl acetate, 7:3); [
IR (KBr):
a
]
¼ þ70ꢁ (c 0.43; CHCl3);
RF ¼ 0.25 (n-hexane/ethyl actetate 8:2); [
a
]
¼ þ45.6ꢁ (c 0.30,
D
D
n
¼ 3530s, 3472m, 3008m, 2986s, 2948s, 2928s, 2864s,
CHCl3); IR (KBr):
n
¼ 3518vs, 3474br, 3266m, 3034w, 2988s, 2970s,
1726vs, 1652m, 1614s, 1470m, 1454m, 1390s, 1366s, 1306m, 1276m,
2942s, 2922vs, 2868s, 2854s, 2836m, 2798w, 1714vs, 1498w, 1464m,
1452m, 1376m, 1308w, 1288m, 1272m, 1248m, 1230m, 1198m,
1180m, 1164m, 1140m, 1114m, 1096w, 1050m, 1028 m cmꢀ1; 1H NMR
1262s, 1242m, 1204m, 1192s, 1162m, 1046s, 758s, 736 s cmꢀ1; UVe
Vis (CHCl3): lmax (log
3
) ¼ 281.2 nm (4.48); 1H NMR (500 MHz,

B. Siewert et al. / European Journal of Medicinal Chemistry 72 (2014) 84e101
97
(500 MHz, CDCl3):
d
¼ 7.53e7.29 (m, 5H, CHaromat), 5.37 (br, 1H, OH),
2.45 (d, J ¼ 4.2 Hz, 1H, CH (13)), 2.18 (dd, J ¼ 16.7, 16.7, 4.8 Hz, 1H,
CHa (16)), 2.03 (s, 3H, CH3 (37)), 2.04e1.98 (m, 1H, CHa (15)), 1.95e
1.90 (m, 1H, CHa (19)), 1.89e1.80 (m, 2H, CHa (7) þ CHb (16)), 1.69e
1.52 (m, 5H, CHb (15) þ CHa (6) þ CH2 (11) þ CHa (2)), 1.51e1.47 (m,
3H, CH (9) þ CHa (21) þ CHb (7)), 1.44e1.30 (m, 3H, CHa (1) þ CHb
(6) þ CHa (22)), 1.29e1.15 (m, 3H, CHb (1) þ CHb (22) þ CHb (19)),
1.05e0.94 (m, 1H, CHb (2)), 0.99 (s, 3H, CH3 (30)), 0.97 (s, 3H, CH3
(23)), 0.90 (s, 6H, CH3 (27) þ CH3 (29)), 0.87e0.84 (m, 1H, CHb
(15)), 0.85 (s, 3H, CH3 (25)), 0.81 (s, 3H, CH3 (24)), 0.79 (m, 1H, CH
(5)), 0.61 (s, 3H, CH3 (26)) ppm; 13C NMR (100 MHz, CDCl3):
5.24 (dd, J ¼ 3.4, 3.4 Hz, 1H, CH (12)), 5.10 (d, J ¼ 12.5 Hz, 1H, CHa
(31)), 4.98 (d, J ¼ 12.5 Hz, 1H, CHb (31)), 3.21 (dd, J ¼ 11.0, 4.5 Hz, 1H,
CH (3)), 2.27 (d, J ¼ 11.3 Hz, 1H, CH (18)), 2.01 (ddd, J ¼ 13.3, 13.3,
4.4 Hz, 1H, CHa (16)), 1.94e1.84 (m, 2H, CH2 (2)), 1.84e1.75 (m, 1H,
CHa (15)), 1.75e1.67 (m, 1H, CHb (16)), 1.67e1.57 (m, 5H, CH2
(22) þ CHa (1), CH2 (11)), 1.52e1.40 (m, 4H, CHa (6) þ CHa (7) þ CHa
(21) þ CH (9)), 1.41e1.20 (m, 5H, CH (19) þ CH (20) þ CHb (7) þ CHb
(21) þ CHb (6)), 1.07 (s, 3H, CH3 (27)), 1.02e0.95 (m, 2H, CHb
(15) þ CHb (1)), 0.99 (s, 3H, CH3 (23)), 0.94 (d, J ¼ 6.2 Hz, 3H, CH3
(30)), 0.89 (s, 3H, CH3 (25)), 0.85 (d, J ¼ 6.4 Hz, 3H, CH3 (29)), 0.78 (s,
3H, CH3 (24)), 0.71 (brd, J ¼ 11.7 Hz, 1H, CH (5)), 0.65 (s, 3H, CH3
d
¼ 211.6 (C]O, C12), 177.6 (C]O, C28), 171.0 (C]O, C36), 136.5
(Cquart, C32), 128.7 (Caromat, C33), 128.5 (Caromat, C35), 128.3 (Caromat
,
(26)) ppm; 13C NMR (125 MHz, CDCl3):
d
¼ 177.4 (C]O, C28), 138.2
C34), 80.6 (CH, C3), 66.1 (CH2, C31), 55.3 (CH, C5), 52.0 (CH, C13),
49.8 (CH, C9), 47.4 (Cquart, C17), 42.0 (Cquart, C14), 41.3 (Cquart, C8),
38.6 (Cquart, C4), 37.9 (CH2, C21), 37.8 (CH2, C11), 36.9 (Cquart, C10),
36.4 (CH2, C19), 34.6 (CH2, C7), 33.5 (CH3, C29), 33.0 (CH2, C22),
32.2 (CH, C18), 31.8 (CH2, C1), 30.8 (Cquart, C20), 28.0 (CH3, C23),
27.5 (CH2, C15), 23.5 (CH2, C2), 23.3 (CH3, C30), 22.9 (CH2, C16),
21.4 (CH3, C37), 20.6 (CH3, C27), 18.3 (CH2, C6), 16.6 (CH3, C26), 15.9
(CH3, C24), 15.3 (CH3, C25) ppm; MS (ESI, MeOH): m/z ¼ 605.1
(56%, [M þ H]þ), 622.1 (26%, [M þ NH4]þ), 585.3 (32%, [M þ Na]þ),
929.9 (26%, [3Mþ2Na]2þ), 1209.2 (26%, [2M þ H]þ), 1231.3 (100%,
[2M þ Na]þ).
(C]CH, C13), 136.5 (Caromat, C32), 128.5 (CHaromat, C33), 128.3
(CHaromat, C34), 128.1 (CHaromat, C35), 125.9 (CH]C, C12), 79.2 (CH,
C3), 66.1 (CH2, C31), 55.4 (CH, C5), 53.0 (CH, C18), 48.3 (Cquart, C17),
47.7 (CH, C9), 42.2 (Cquart, C14), 39.7 (Cquart, C8), 39.2 (CH, C19), 39.0
(CH, C20), 38.9 (Cquart, C4), 38.8 (CH2, C1), 37.1 (Cquart, C10), 36.8
(CH2, C22), 33.2 (CH2, C7), 30.8 (CH2, C21), 28.3 (CH3, C23), 28.1
(CH2, C15), 27.4 (CH2, C11), 24.4 (CH2, C16), 23.7 (CH3, C27), 23.4
(CH2, C2), 21.3 (CH3, C30), 18.5 (CH2, C6), 17.2 (CH3, C29), 17.1 (CH3,
C26), 15.8 (CH3, C24), 15.6 (CH3, C25) ppm; MS (ESI, MeOH): m/
z ¼ 547.3 (10%, [M þ H]þ), 569.5 (100%, [M þ Na]þ), 1115.2 (82%,
2M þ Na]þ).
5.4.23. (3b) Benzyl 3-acetoxy-urs-11-en-28-oate (22)
5.4.21. (3
b
) Benzyl 3-hydroxy-olean-12-en-28-oate (20)
Compound 22 [66] was obtained from a mixture of 22 and 23
by using method A; yield: 56%; RF ¼ 0.7 (n-hexane/ethyl acetate,
OA (1 equiv.) was treated with benzylchloride following method
D, and 21 was obtained as fine colorless needles; yield: 92%; m.p.:
85:15); m.p.: 173e174 ꢁC; [
a]
¼ þ45.1ꢁ (c 0.66, CHCl3); IR (KBr):
D
185e187 ꢁC (lit [64].: 189 ꢁC); [
a]
¼ þ58.4ꢁ (c 0.45; CHCl3) [lit.:
n
¼ 3433w, 3069w, 2926m, 2926s, 2874s, 1727s, 1499w, 1465s,
D
[65]: 50.1ꢁ (c 2.44; CHCl3)]; RF ¼ 0.42 (n-hexane/ethyl acetate, 8:2);
1388m, 1379s, 1039m, 1283m, 1270m, 1237s, 1197s, 1182s, 1140s,
1110s, 1028s, 1005m, 986s, 970s, 697s cmꢀ1 1H NMR (500 MHz,
CDCl3):
¼ 7.37e7.27 (m, 5H, CH (33) þ CH (35) þ CH (36)), 5.23
IR (KBr):
n
¼ 3583s, 2938s, 1726s, 1498w, 1463m, 1386m, 1363w,
;
1323w, 1305w, 1264w, 1234w, 1201m, 1182m, 1162m, 1129m, 1095w,
1044m, 1016 m cmꢀ1; 1H NMR (500 MHz, CDCl3):
¼ 7.37e7.27 (m,
d
d
(dd, J ¼ 3.5, 3.5 Hz, 1H, CH (12)), 5.10 (d, J ¼ 12.5 Hz, 1H, CHa (31)),
4.98 (d, J ¼ 12.5 Hz, 1H, CHb (31)), 4.49 (dd, J ¼ 9.7, 6.3 Hz, 1H, CH
(3)), 2.26 (d, J ¼ 11.2 Hz, 1H, CH (18)), 2.04 (s, 3H, CH3 (37)), 2.00
(dd, J ¼ 13.2, 13.2, 4.6 Hz, 1H, CHá (16)), 1.88 (m, 2H, CHa (2)), 1.85
(ddd, J ¼ 10.5, 10.5, 4.5 Hz, 1H, CHa (11)), 1.80 (ddd, J ¼ 13.5, 13.5,
4.6 Hz, 1H, CHa (15)), 1.69 (ddd, 1H, J ¼ 10.5, 3.2, 3.2 Hz, CHb (11)),
1.66e1.60 (m, 4H, CH2 (22) þ CHa (1) þ CHb (2)), 1.52e1.44 (m, 4H,
CH (9) þ CHa (6) þ CHa (21) þ CHb (16)), 1.33e1.30 (m, 1H, CH
(20)), 1.30e1.27 (m, 1H, CHb (21)), 1.28e1.25 (m, 1H, CHb (7)), 1.07
(s, 3H, CH3 (27)), 1.11e1.03 (m, 2H, CHb (1) þ CHb (15)), 0.99 (dd,
J ¼ 11.6, 5.7 Hz, 1H, CH (19)), 0.94 (s, 3H, CH3 (30)), 0.91 (s, 3H, CH3
(25)), 0.86 (s, 6H, CH3 (23) þ CH3 (29)), 0.85 (s, 3H, CH3 (24)),
0.87e0.80 (m, 1H, CH (5)), 0.64 (s, 3H, CH3 (26)) ppm; 13C NMR
5H, CHaromat), 5.29 (dd, J ¼ 3.6, 3.6 Hz, 1H, CH (12)), 5.09 (d,
J ¼ 12.6 Hz,1H, CHa (31)), 5.05 (d, J ¼ 12.5 Hz,1H, CHb (31)), 3.20 (dd,
J ¼ 11.3 Hz, 4.5 Hz, 1H, CHOH (3)), 2.91 (dd, J ¼ 13.8, 4.2 Hz, 1H, CH
(18)), 1.98 (ddd, J ¼ 13.4, 13.0, 4.1 Hz, 1H, CHa (16)), 1.85 (dd, J ¼ 8.9,
3.6 Hz, 1H, CH2 (11)), 1.76e1.14 (m, 16H, CH (9) þ CH2 (19) þ CHa
(1) þ CH2 (21)þ CH2 (7) þ CH2 (22) þ CHa (15)þ CH2 (2)þ CHb (16)þ
CH2 (6)), 1.13 (s, 3H, CH3 (27)), 1.09e1.00 (m, 2H, CHb (1) þ CHb (15)),
0.98 (s, 3H, CH3 (23)), 0.92 (s, 3H, CH3 (30)), 0.90 (s, 3H, CH3 (29)),
0.88 (s, 3H, CH3 (25)), 0.77 (s, 3H, CH3 (24)), 0.70 (brd, J ¼ 11.5 Hz,1H,
CH (5)), 0.61 (s, 3H, CH3 (26)) ppm; 13C NMR (100 MHz, CDCl3):
d
¼ 177.4 (C]O, C28), 143.7 (C]CH, C13), 136.4 (Cquart, C32), 128.4
(CHaromat, C35), 128.0 (CHaromat, C34), 127.9 (CHaromat, C33), 122.5
(CH]C, C12), 80.0 (CHOH, C3), 65.9 (CH2, C31), 55.2 (CH, C5), 47.6
(CH, C9), 46.7 (Cquart, C17), 45.9 (CH2, C19), 41.7 (Cquart, C14), 41.4
(CH, C18), 39.3 (Cquart, C8), 38.7 (Cquart, C4), 38.4 (CH2, C1), 37.0
(Cquart, C10), 33.8 (CH2, C21), 33.1 (CH3, C29), 32.7 (CH2, C7), 32.4
(CH2, C22), 30.7 (Cquart, C20), 28.1 (CH3, C23), 27.6 (CH2, C15), 27.2
(CH2, C2), 25.9 (CH3, C27), 23.6 (CH3, C30), 23.4 (CH2, C11), 23.0
(CH2, C16), 18.3 (CH2, C6), 16.9 (CH3, C26), 15.6 (CH3, C24), 15.3 (CH3,
C25) ppm; MS (ESI, MeOH): m/z ¼ 569.5 (100%, [M þ Na]þ).
(500 MHz, CDCl3):
d
¼ 177.2 (C]O, C28), 170.9 (C]O, C36), 138.1
(C]CH, C13), 136.4 (Cquart, C32), 128.4 (CHaromat, C34), 128.1
(CHaromat, C33), 127.9 (CHaromat, C35), 125.6 (CH]C, C12), 80.9
(CHOAc, C3), 65.9 (CH2, C31), 55.3 (CH, C5), 52.9 (CH, C18), 48.1
(Cquart, C17), 47.5 (CH, C9), 42.0 (Cquart, C14), 39.5 (Cquart, C8), 39.1
(CH, C19), 38.8 (Cquart, C20), 38.3 (CH2, C1), 37.7 (Cquart, C4), 36.8
(Cquart, C10), 36.6 (CH2, C21), 32.9 (CH2, C7), 30.6 (CH2, C22), 28.1
(CH3, C29), 27.9 (CH2, C15), 24.2 (CH2, C16), 23.5 (CH2, C11), 23.5
(CH3, C27), 23.2 (CH2, C2), 21.3 (CH3, Ac), 21.1 (CH3, C30), 18.2 (CH2,
C6), 17.0 (CH3, C26), 17.0 (CH3, C24), 16.7 (CH3, C23), 15.5 (CH3, C25)
ppm; MS (ESI, MeOH): m/z ¼ 589.2 (30%, [M þ H]þ), 611.5 (30%,
[M þ Na]þ), 1199.2 (100%, [2M þ Na]þ).
5.4.22. (3b) Benzyl 3-hydroxy-12-oxo-olean-28-oate (21)
Compound 21 was obtained following method A as a colorless
solid; yield: 83%; m.p.: 198e201 ꢁC (lit.: 196e198 ꢁC [4]); RF ¼ 0.74
(silica
80:20:3:20); [
(log
gel,
toluene/ethyl
acetate/formic
acid/n-heptane,
a
]
¼ þ54.1ꢁ (c 0.5, CHCl3); UVeVis (CHCl3): lmax
5.4.24. (3b) Benzyl 3-acetoxy-olean-11-en-28-oate (23)
D
3
) ¼ 258.17 (1.35) nm; IR (KBr):
n
¼ 2942s, 1728s, 1711s, 1472m,
Compound 23 [67] was obtained using method D as a colorless
solid; yield: 83%; RF ¼ 0.7 (n-hexane/ethyl acetate, 85:15); m.p.:
1456m, 1387w, 1372m, 1240s, 1204m, 1180m, 1161m, 1140m, 1119m,
1086w, 1037m, 1005w cmꢀ1; 1H NMR (400 MHz, CDCl3):
d
¼ 7.36e
228e230 ꢁC; [
a]
¼ þ55.8ꢁ (c 0.33, CHCl3); IR (KBr):
n
¼ 2940brs,
D
7.29 (m, 5H, CH (33) þ CH (34) þ CH (35)), 5.19 (d, J ¼ 11.6 Hz, 1H,
CHa (31)), 5.07 (d, J ¼ 11.6 Hz, 1H, CHb (31)), 4.45 (dd, J ¼ 11.3,
5.0 Hz, 1H, CH (3)), 2.83 (ddd, J ¼ 10.2, 3.3, 3.3 Hz, 1H, CH (18)),
1726vs, 1468w, 1456w, 1388w, 1378w, 1364w, 1264m, 1242s, 1200w,
1178m, 1162m, 1122w, 1030m, 1012m cmꢀ1
;
1H NMR (500 MHz,
CDCl3):
d
¼ 7.37e7.27 (m, 5H, CHaromat), 5.28 (dd, J ¼ 3.5, 3.5 Hz, 1H,

98
B. Siewert et al. / European Journal of Medicinal Chemistry 72 (2014) 84e101
CH (12)), 5.09 (d, J ¼ 12.6 Hz, 1H, CHa (31)), 5.04 (d, J ¼ 12.5 Hz, 1H,
CHb (31)), 4.48 (m, 1H, CH (3)), 2.90 (dd, J ¼ 13.7, 4.1 Hz, 1H, CH (18)),
2.04 (s, 3H, CH3 (Ac)), 1.98 (ddd, J ¼ 13.5, 13.5, 4.1 Hz, 1H, CHa (16)),
1.85 (dd, J ¼ 8.9, 3.5 Hz, 2H, CH2 (11)), 1.75e1.68 (ddd, J ¼ 13.8, 13.8,
4.3 Hz, 1H, CHa (7)), 1.68e1.58 (m, 7H, CHa (19) þ CHa (1) þ CHb
(7) þ CHa (15) þ CH2 (2) þ CHb (16)), 1.60e1.48 (m, 1H, CHa (6)), 1.42
(ddd, J ¼ 12.6, 12.6, 3.4 Hz, 1H, CHa (21)), 1.38e1.29 (m, 2H, CHb
(6) þ CHb (22)), 1.27e1.20 (ddd, J ¼ 9.7, 3.5, 2.5 Hz, 1H, CHb (21)),
1.22e1.16 (ddd, J ¼ 11.7, 4.1, 2.4 Hz, 1H, CHb (22)), 1.20e1.13 (ddd,
J ¼ 11.5, 4.5, 2.3 Hz, 1H, CHb (19)), 1.12 (s, 3H, CH3 (27)), 1.08e0.98
(m, 2H, CHb (1) þ CHb (15)), 0.92 (s, 3H, CH3 (30)), 0.90 (s, 3H, CH3
(25)), 0.90 (s, 3H, CH3 (29)), 0.86 (s, 3H, CH3 (23)), 0.85 (s, 3H, CH3
(24)), 0.84e0.78 (dd, J ¼ 11.8, 1.7 Hz, 1H, CH (5)), 0.61 (s, 3H, CH3
formic acid/n-heptane, 80:20:3:20); [
a
]
¼ þ98.9ꢁ (c 0.34, CHCl3);
D
IR (KBr):
n
¼ 3461m, 3033w, 2948s, 2868m, 1720s, 1667m, 1640s,
1498s, 1463m, 1372s, 1322w, 1306m, 1286m, 1263s, 1238m, 1205m,
1182m, 1163s, 1140m, 1118m, 1085m, 1085w, 1070w, 1035s,
1012m cmꢀ1; UVeVis (CHCl3): lmax (log
3
) ¼ 289.93 (4.09) nm; 1
H
NMR (500 MHz, CDCl3):
d
¼ 7.41e7.27 (m, 5H, CH (33) þ CH
(34) þ CH (35)), 6.23 (s, 1H, COH]C (12)), 5.12e5.05 (m, 2H, CH2
(31)), 4.50 (dd, J ¼ 11.5, 4.9 Hz, 1H, CH (3)), 3.75e3.71 (m, 1H, CH
(18)), 2.78 (ddd, J ¼ 13.4, 3.4, 3.4 Hz, 1H, CHa (1)), 2.42 (s, 1H, CH
(9)), 2.09e2.00 (m, 1H, CHa (16)), 2.04 (s, 3H, CH3 (Ac)), 1.80 (ddd,
J ¼ 13.9, 13.9, 4.5 Hz, 1H, CHa (7)), 1.76e1.71 (m, 3H, CHb (16) þ CH2
(2)), 1.71e1.62 (m, 1H, CHb (7)), 1.58e1.50 (m, 3H, CHa (6) þ CHa
(15) þ CHa (22)), 1.43e1.34 (m, 2H, CH2 (19) þ CHa (21)), 1.34 (s,
3H, CH3 (27)), 1.33e1.24 (m, 3H, CHb (21) þ CHb (6) þ CHb (22)),
1.18e1.13 (ddd, J ¼ 14.2, 3.3, 3.3 Hz, 1H, CHb (15)), 1.07 (s, 3H, CH3
(25)), 1.09e1.03 (m, 1H, CHb (1)), 0.99 (s, 3H, CH3 (30)), 0.92 (s, 3H,
CH3 (29)), 0.86 (s, 6H, CH3 (23) þ CH3 (24)), 0.82e0.75 (m, 1H, CH
(5)), 0.69 (s, 3H, CH3 (26)) ppm; 13C NMR (100 MHz, CDCl3):
(26)) ppm; 13C NMR (125 MHz, CDCl3):
(C]O, Ac), 143.9 (C]CH, C13), 136.6 (Caromat, C32), 128.6 (CHaromat
d
¼ 177.6 (C]O, C28), 171.1
,
C34), 128.1 (CHaromat, C33), 128.0 (CHaromat, C35), 122.6 (CH]C,
C12), 81.1 (CHOH, C3), 66.1 (CH2, C31), 55.5 (CH, C5), 47.7 (CH, C9),
46.9 (Cquart, C17), 46.0 (CH2, C19), 41.8 (Cquart, C8), 41.6 (CH, C18),
39.5 (Cquart, C20), 38.3 (CH2, C1), 37.8 (Cquart, C4), 37.1 (Cquart, C14),
34.0 (CH2, C22), 33.2 (CH3, C29), 32.8 (CH2, C21), 32.5 (CH2, C7), 30.9
(Cquart, C10), 28.2 (CH3, C23), 27.8 (CH2, C15), 26.0 (CH3, C27), 23.8
(CH3, C30), 23.7 (CH2, C2), 23.6 (CH2, C11), 23.2 (CH2, C16), 21.5
(CH3, Ac), 18.4 (CH2, C6), 17.0 (CH3, C26), 16.8 (CH3, C24), 15.5 (CH3,
C25) ppm; MS (ESI, MeOH): m/z ¼ 589.2 (42.9%, [M þ H]þ), 611.3
(82.5%, [M þ Na]þ), 1199.4 (100%, [2M þ Na]þ).
d
¼ 195.4 (C]O, C11), 177.1 (C]O, C28), 171.1 (C]O, CAc), 142.2
(COH]C, C12), 136.7 (C]COH, C13), 136.3 (Cquart, C32), 128.6
(Caromat, C34), 128.1 (Caromat, C33), 128.1 (Caromat, C35), 80.6 (CH,
C3), 66.2 (CH2, C31), 60.4 (CH, C9), 55.2 (CH, C5), 46.0 (Cquart, C17),
45.6 (Cquart, C14), 41.7 (Cquart, C8), 40.4 (CH2, C19), 38.8 (CH2, C1),
38.2 (Cquart, C4), 37.4 (Cquart, C10), 34.2 (CH2, C21), 33.4 (CH, C18),
33.2 (CH2, C7), 33.0 (CH3, C29), 32.0 (CH2, C22), 30.7 (Cquart, C20),
28.2 (CH3, C23), 27.9 (CH2, C15), 23.6 (CH2, C2), 23.4 (CH3, C27),
23.3 (CH3, C30), 23.2 (CH2, C16), 21.4 (CH3, C37), 18.8 (CH3, C26),
17.4 (CH2, C6), 16.8 (CH3, C24), 16.5 (CH3, C25) ppm; MS (ESI,
MeOH): m/z ¼ 619.1 (48%, [M þ H]þ), 641.3 (29%, [M þ Na]þ),
1259.3 (100%, [2M þ Na]þ).
5.4.25. (3b) Benzyl 3-acetoxy-12-oxo-olean-28-oate (24)
Treatment of 23 following method A gave 24 as a colorless solid;
yield: 83%; m.p.: 196e198 ꢁC (lit [68].: 196e198 ꢁC); RF ¼ 0.38 (n-
hexane/ethyl acetate, 8:2); [
a
]
¼ þ5.2ꢁ (c 0.63, CHCl3); IR
D
(KBr):
n
¼ 3404w, 2943s, 1728s, 1711s, 1472m, 1456m, 1420w, 1387m,
1366m, 1332w, 1308w, 1241s, 1204m, 1180m, 1161m, 1140m, 1119m,
1086w, 1038w, 1006w cmꢀ1; 1H NMR (500 MHz, CDCl3):
¼ 7.39e
d
5.4.27. (3b) But-3-enyl 3ehydroxy-olean-12-en-28-oate (26)
7.27 (m, 5H, CHaromat), 5.21 (d, J ¼ 12.2 Hz, 1H, CHa (31)), 5.08 (d,
J ¼ 12.2 Hz, 1H, CHb (31)), 4.47 (dd, J ¼ 11.5, 4.8 Hz, 1H, CH (3)), 2.85
(ddd, J ¼ 10.3, 3.2, 3.8 Hz, 1H, CH (18)), 2.46 (d, J ¼ 4.2 Hz, 1H, CH
(13)), 2.19 (dd, J ¼ 16.6, 4.8 Hz, 1H, CHa (11)), 2.05 (s, 3H, CH3 (Ac)),
2.02 (dd, J ¼ 16.5, 3.1 Hz,1H, CHb (11)),1.94 (ddd, J ¼ 12.8, 2.4, 2.2 Hz,
1H, CHa (1)), 1.90e1.85 (m, 1H, CHa (16)), 1.88 (ddd, J ¼ 13.8, 4.4,
4.4 Hz, 1H, CHa (22)), 1.70e1.64 (m, 1H, CHb (16)), 1.64e1.55 (m, 5H,
CH (9) þ CH2 (2) þ CHa (21) þ CHa (6)), 1.56e1.48 (m, 1H, CHb (19)),
1.47 (ddd, 1H, J ¼ 14.0, 3.4, 3.4 Hz, CHb (7)), 1.41e1.33 (m, 3H, CHb
(22) þ CHb (6) þ CHa (15)), 1.33e1.26 (m, 3H, CHb (22) þ CHb
(15) þ CHb (1)), 1.06e0.99 (m, 1H, CHb (21)), 1.00 (s, 3H, CH3 (27)),
0.99e0.95 (m, 1H, CHb (19)), 0.91 (s, 6H, CH3 (30) þ CH3 (28)), 0.86
(s, 3H, CH3 (29)), 0.86 (s, 3H, CH3 (24)), 0.83 (s, 3H, CH3 (25)), 0.80
(dd, J ¼ 11.1, 1.1 Hz, 1H, CH (5)), 0.68 (s, 3H, CH3 (26)) ppm; 13C NMR
Compound 26 was prepared according to method D and ob-
tained as colorless, fine needles (recrystallization from ethanol);
yield 84%; m.p.: 80e81 ꢁC; RF ¼ 0.52 (n-hexane/ethyl acetate,
6:4); [
a
]
¼ þ110.8 (c 0.31, CHCl3); IR (KBr):
n
¼ 3442br, 2944m,
D
1636m, 1620w, 1458s, 1446m, 1432s, 1174w, 1106m, 1032m,
1010m cmꢀ1 1H NMR (500 MHz, CDCl3):
¼ 5.79 (dddd, J ¼ 17.0,
;
d
10.2, 6.7, 6.7 Hz, 1H, CH (33)), 5.27 (dd, J ¼ 3.5 Hz, 1H, CH (12)),
5.10 (ddd, J ¼ 17.2, 3.2, 1.5 Hz, 1H, CHa (34)), 5.06 (ddd, J ¼ 10.3,
3.1, 1.6 Hz, 1H, CHb (34)), 4.12e4.01 (m, 2H, CH2 (31), AA ‘XX’-
system), 3.21 (dd, J ¼ 11.4, 4.6 Hz, 1H, CH (3)), 2.86 (dd, J ¼ 13.9,
4.3 Hz, 1H, CH (18)), 2.39e2.32 (m, 2H, CH2 (32), AA’XX’ system),
1.96 (ddd, J ¼ 11.6, 11.6, 4.5 Hz, 1H, CHa (16)), 1.89e1.84 (m, 2H,
CH2 (11)), 1.69 (ddd, J ¼ 13.8, 13.8, 4.4 Hz, 1H, CHa (7)), 1.66e1.50
(m, 9H, CHa (19) þ CH (9) þ CHa (1) þ CHb (7) þ CH2 (2) þ CHa
(15) þ CHb (16) þ CHa (6)), 1.50e1.45 (m, 1H, CHa (22)), 1.45e1.38
(m, 1H, CHb (6)), 1.38e1.30 (m, 1H, CHa (21)), 1.31e1.25 (m, 1H,
CHb (22)), 1.20e1.10 (m, 2H, CHb (19) þ CHb (21)), 1.12 (s, 3H, CH3
(27)), 1.07e1.00 (m, 1H, CHb (15)), 0.98 (s, 3H, CH3 (23)), 0.97e
0.92 (m, 1H, CHb (1)), 0.91 (s, 3H, CH3 (30)), 0.90 (s, 3H, CH3 (25)),
0.89 (s, 3H, CH3 (29)), 0.77 (s, 3H, CH3 (24)), 0.73 (s, 3H, CH3 (26)),
0.72 (dd, J ¼ 9.6, 1.4 Hz, 1H, CH (5)) ppm; 13C NMR (125 MHz,
(125 MHz, CDCl3):
d
¼ 211.4 (C]O, C12), 177.4 (C]O, C28), 170.8
(C]O, Ac), 136.3 (Caromat, C32), 128.5 (CHaromat, C34), 128.4 (CHar-
omat, C33), 128.1 (CHaromat, C35), 80.4 (CHOH, C3), 65.9 (CH3, C31),
55.1 (CH, C5), 51.8 (CH, C13), 49.6 (CH, C9), 47.2 (Cquart, C17), 41.8
(Cquart, C14), 41.2 (Cquart, C8), 38.4 (CH2, C11), 37.7 (Cquart, C10), 37.6
(CH2, C19), 36.8 (Cquart, C4), 36.2 (CH2, C1), 34.5 (CH2, C21), 33.4
(CH3, C29), 32.9 (CH2, C7), 32.1 (CH, C18), 31.7 (CH2, C22), 30.6
(Cquart, C20), 27.9 (CH3, C30), 27.4 (CH2, C16), 23.4 (CH2, C2), 23.2
(CH3, C27), 22.7 (CH2, C15), 21.2 (CH3, Ac), 20.5 (CH3, C23), 18.1 (CH2,
C6), 16.4 (CH3, C24), 15.7 (CH3, C26), 15.2 (CH3, C25) ppm; (ESI,
MeOH): m/z ¼ 605.5 (100%, [M þ H]þ), 622.4 (54.8%, [M þ NH4]þ),
627.5 (32.6%, [M þ Na]þ), 929.8 (20%, [3Mþ2Na]2þ), 1209.2 (27.9%,
[2M þ H]þ), 1231.2 (76.0%, [2M þ Na]þ).
CDCl3):
d
¼ 177.8 (C]O, C28), 143.9 (C]CH, C13), 134.5 (CH]
CH2, C33), 122.5 (CH]C, C12), 117.1 (CH2]CH, C34), 79.2 (CHOH,
C3), 63.4 (CH2, C1), 55.4 (CH, C5), 47.8 (CH, C9), 46.8 (Cquart, C17),
46.1 (CH2, C19), 41.9 (Cquart, C8), 41.4 (CH, C18), 39.5 (Cquart, C20),
38.9 (Cquart, C14), 38.6 (CH2, C1), 37.2 (Cquart, C4), 34.1 (CH2, C22),
33.3 (CH3, C29), 33.3 (CH2, C32), 32.9 (CH2, C21), 32.6 (CH2, C7),
30.9 (Cquart, C10), 28.3 (CH3, C23), 27.8 (CH2, C15), 27.4 (CH2, C2),
26.0 (CH3, C27), 23.7 (CH3, C30), 23.6 (CH2, C11), 23.2 (CH2, C16),
18.5 (CH2, C6), 17.2 (CH3, C26), 15.7 (CH3, C24), 15.5 (CH3, C25)
ppm; MS (ESI): m/z ¼ 511.3 (30%, [M þ H]þ), 1043.4 (100%,
[2M þ Na]þ).
5.4.26. (3
b) Benzyl 3-acetoxy-11-oxo-12-hydroxy-olean-D-12,13-
en-28-oate (25)
Obtained from 24 as a colorless solid following method E;
yield: 75%; m.p.: 237e240 ꢁC; RF ¼ 0.69 (toluene/ethyl acetate/

B. Siewert et al. / European Journal of Medicinal Chemistry 72 (2014) 84e101
99
5.4.28. (3
b
) But-3-enyl 3ehydroxy-12-oxo-olean-28-oate (27)
5.4.30. (2
a,3b) 2,3eDiydroxy-12-chloro-olean-28-oic acid 28,13-
Compound was prepared using method B followed by chro-
matography (silica gel, hexane/ethyl acetate, 7:3) and recrystal-
lization from ethanol as a colorless solid; yield 76%; m.p.: 72e
lactone (29)
MA (0.3 g, 0.6 mmol) was solved in a DCM/water mixture (1:1,
40 mL) and sodium hypochlorite (3 mL; 13% Cl2) was added. After
stirring for 5 h at 25 ꢁC, the mixture was quenched with sodium
sulfide and stirred for one additional hour. Usual workup afforded
29 as a colorless solid; yield: 84%; m.p.: 306e309 ꢁC; RF ¼ 0.40 (n-
73 ꢁC; RF ¼ 0.36 (n-hexane/ethyl acetate, 6:4); [
a
]
¼ ꢀ19.2 (c
D
0.31, CHCl3); IR (KBr):
n
¼ 3440br, 2972s, 2940s, 2862m, 2362m,
2344m, 1700vs, 1654m, 1646m, 1636m, 1466m, 1458m, 1438m,
1418m, 1388m, 1304m, 1256m, 1242m, 1188m, 1176m, 1088m,
hexane/ethyl acetate, 1:2); [
a
]
¼ þ34.5ꢁ (c 0.5, CHCl3); IR (KBr):
D
1048m, 1036m cmꢀ1
;
1H NMR (500 MHz, CDCl3):
d
¼ 5.82e5.73
n
¼ 3395m, 2950s, 1767s, 1464m, 1395m, 1361m, 1254m, 1216m,
(m, 1H, CH (33)), 5.11 (ddd, J ¼ 17.2, 3.2, 1.5 Hz, 1H, CHa (34)),
5.06 (ddd, J ¼ 10.2, 1.6, 1.1 Hz, 1H, CHb (34)), 4.19e4.08 (m, 2H,
CH (31)), 3.20 (dd, J ¼ 11.4, 4.7 Hz, 1H, CH (3)), 2.79 (dd, J ¼ 10.1,
3.4, 3.4 Hz, 1H, CH (18)), 2.62 (d, J ¼ 4.3 Hz, 1H, CH (13)), 2.38
(brdd, J ¼ 6.6, 6.6 Hz, 2H, CH2 (32)), 2.23 (dd, J ¼ 16.7, 5.0 Hz, 1H,
CHa (11)), 2.13 (dd, J ¼ 16.6, 13.2 Hz, 1H, CHb (11)), 1.97e1.90 (m,
1H, CHa (19)), 1.91e1.84 (m, 1H, CHa (16)), 1.79 (ddd, J ¼ 13.8,
13.8, 4.7 Hz, 1H, CHa (7)), 1.70e1.51 (m, 6H, CHb (16) þ CHa
(6) þ CH2 (2) þ CH (9) þ CHa (1)), 1.48e1.37 (m, 3H, CHb
(7) þ CHb (6) þ CHa (22)), 1.36e1.29 (m, 2H, CHa (21) þ CHb
(22)), 1.25e1.18 (m, 1H, CHb (19)), 1.11e1.02 (m, 1H, CHb (15)),
0.99 (s, 3H, CH3 (30)), 0.97 (s, 3H, CH3 (23)), 0.97 (s, 3H, CH3
(25)), 0.94 (s, 3H, CH3 (27)), 0.90 (s, 3H, CH3 (29)), 0.92e0.87 (m,
1H, CHb (1)), 0.85 (s, 3H, CH3 (24)), 0.78 (s, 3H, CH3 (26)), 0.74e
1134m, 1050m cmꢀ1
;
1H NMR (500 MHz, CDCl3):
d
¼ 4.16 (dd,
J ¼ 3.9, 2.3 Hz, 1H, CH (12)), 3.72 (ddd, J ¼ 11.5, 9.7, 4.7 Hz, 1H, CH
(2)), 3.03 (d, J ¼ 9.6 Hz, 1H, CH (3)), 2.28 (ddd, J ¼ 15.0, 12.9, 4.0 Hz,
1H, CHa (11)), 2.18e2.10 (m, 2 H, CHa (19) þ CHa (16)), 2.08e2.05 (m,
1H, CHa (1)), 2.04e1.87 (m, 3H, CHb (19) þ CH (18) þ CHa (15)),1.77e
1.71 (m, 2H, CH (9) þ CHb (11)),1.68e1.59 (m, 2H, CH2 (7)),1.57e1.49
(m, 2H, CHa (22) þ CHa (6)), 1.47e1.42 (m, 1H, CHb (6)), 1.37 (s, 3H,
CH3 (27)), 1.36e1.26 (m, 2H, CHa (21) þ CHb (22)), 1.27e1.10 (m, 3H,
CHa (16) þ CHb (21) þ CHb (15)), 1.15 (s, 3H, CH3 (26)), 1.00 (s, 3H,
CH3 (23)), 1.03e0.96 (m, 1H, CHb (1)), 0.99 (s, 3H, CH3 (30)), 0.95 (s,
3H, CH3 (25)), 0.89 (s, 3H, CH3 (29)), 0.90e0.84 (m, 1H, CH (5)), 0.84
(s, 3H, CH3 (24)) ppm; 13C NMR (125 MHz, CDCl3):
C28), 91.7 (Cquart, C13), 83.4 (CHOH, C3), 69.1 (CHOH, C2), 64.6
(CHCl, C12), 55.5 (CH, C5), 51.8 (CH, C18), 46.6 (CH2, C1), 45.1 (Cquart
d
¼ 179.2 (C]O,
,
0.70 (m, 1H, CH (5)) ppm; 13C NMR (125 MHz, CDCl3):
d
¼ 211.9
C17), 44.8 (CH, C9), 43.2 (Cquart, C14), 42.4 (Cquart, C8), 39.6 (CH2,
C19), 39.2 (Cquart, C4), 37.9 (Cquart, C10), 34.2 (CH2, C7), 33.7 (CH2,
C21), 33.5 (CH3, C30), 31.7 (Cquart, C20), 29.4 (CH2, C11), 29.3 (CH2,
C15), 28.4 (CH3, C23), 27.3 (CH2, C22), 23.5 (CH3, C29), 21.2 (CH2,
C16), 20.2 (CH3, C27), 18.7 (CH3, C26), 18.1 (CH3, C25), 17.9 (CH2, C6),
16.5 (CH3, C24) ppm; MS (ESI, MeOH): m/z ¼ 529.5 (85%,
[M þ Na]þ), 561.1 (37%, [M þ Na þ MeOH]þ), 1035.3 (100%,
[2M þ Na]þ).
(C]O, C12), 177.9 (C]O, C28), 134.2 (CH, C13), 117.4 (CH2]CH,
C34), 78.8 (CHOH, C3), 63.6 (CH2, C31), 55.3 (CH, C5), 52.0 (CH,
C13) 49.9 (CH, C9), 47.5 (Cquart, C17), 42.1 (Cquart, C14), 41.5
(Cquart, C8), 39.0 (CH2, C11), 38.7 (Cquart, C20), 38.1 (CH2, C1), 37.1
(Cquart, C4), 36.4 (CH2, C19), 34.7 (CH2, C32), 33.5 (CH3, C29), 33.3
(CH2, C21), 33.2 (CH2, C7), 32.1 (CH, C18), 32.0 (CH2, C32), 30.8
(Cquart, C10), 28.1 (CH3, C23), 27.7 (CH2, C15), 27.2 (CH2, C2), 23.3
(CH3, C30), 22.9 (CH3, C21), 20.7 (CH3, C30), 18.5 (CH2, C6), 16.4
(CH3, C24), 15.5 (CH3, C26), 15.4 (CH3, C25) ppm; MS (ESI): m/
z ¼ 527.2 (100%, [M þ H]þ), 544.2 (20%, [M þ NH4]þ), 1053.3
(51%, [2M þ Na]þ), 1075.3 (42%, [2M þ Na]þ).
5.4.31. (2
lactone (30)
MA (300 mg, 0.63 mmol) in acetic acid (20 mL, 90%) was treated
with sodium acetate (0.5 g, 6.1 mmol) and bromine (50 L,1 mmol).
a,3b) 2,3eDihydroxy-12-bromo-olean-28-oic acid 28,13-
m
5.4.29. (2
lactone (28)
a
, 3
b
, 12
a
) 2,3,12eTrihydroxy-olean-28-oic acid 28,13-
After 10 min stirring at room temperature the reaction was
quenched with water and an aq. solution of sodium sulfide. The
white precipitate were filtered off and washed with water to yield
30 as a colorless solid; yield: 72%; m.p.: 260e262 ꢁC (lit. 261e
Compound 28 obtained from MA following method A; yield:
40%; m.p.: 229e231 ꢁC; RF ¼ 0.21 (n-hexane/ethyl acetate, 1:1);
[
a
]
¼ 18.8ꢁ (c 0.36, CHCl3); IR (KBr):
n
¼ 3442vs, 3432vs, 2950s,
263 ꢁC [69]); RF ¼ 0.44 (n-hexane/ethyl acetate, 1:2); [
a]
¼ þ33.0ꢁ
¼ 3384s,
D
D
n
2936s, 2866m, 1754s, 1466m, 1456m, 1386m, 1366m, 1252m,
1222m, 1144m, 1134m, 1084m, 1046m, 1032m, 974m, 942m,
(c0.50, CHCl3) [lit. 31.6ꢁ (c 3.0, CHCl3 [69])]; IR (KBr):
2950s, 1768s, 1462m, 1394m, 1360m, 1301w, 1246w, 1210m, 1130m,
732m cmꢀ1; 1H NMR (400 MHz, CDCl3):
d
¼ 3.89 (brs, 1H, CH (12)),
1051 m cmꢀ1; 1H NMR (400 MHz, CDCl3):
d
¼ 4.26 (dd, J ¼ 3.7, 2.3,
3.85e3.68 (m, 1H, CH (2)), 3.06 (d, J ¼ 8.6 Hz, 1H, CH (3)), 3.15e2.80
(brs, 2H, OH), 2.15 (ddd, J ¼ 13.5, 13.5, 5.6 Hz, 1H, CHa (16)), 2.10e
1.91 (m, 5H, CH (18) þ CHa (1) þ CHa (19) þ CH2 (11)), 1.90e1.79 (m,
1H, CHa (15)), 1.72e1.59 (m, 3H, CH (9) þ CH2 (7)), 1.58e1.45 (m,
4H, CHa (22) þ CH2 (6) þ CHb (16)), 1.46e1.34 (m, 2H, CHb
(22) þ CHa (21)), 1.31 (s, 3H, CH3 (27)), 1.29e1.20 (m, 2H, CHb
(19) þ CHb (21)), 1.15e1.09 (m, 1H, CHb (15)), 1.13 (s, 3H, CH3 (26)),
1.03 (s, 3H, CH3 (23)), 0.98 (s, 3H, CH3 (30)), 1.08e0.89 (m, 1H, CHb
(1)), 0.94 (s, 3H, CH3 (25)), 0.90 (s, 3H, CH3 (29)), 0.88e0.83 (m, 1H,
CH (5)), 0.82 (s, 3H, CH3 (24)) ppm; 13C NMR (100 MHz, CDCl3):
1H, CH (12)), 3.70 (ddd, J ¼ 11.3, 9.6, 4.6 Hz, 1H, CHa (2)), 3.05 (d,
J ¼ 9.6 Hz, 1H, CH (3)), 2.39 (ddd, J ¼ 15.0, 12.4, 3.9 Hz, 1H, CHa (11)),
2.34e2.28 (m, 1H, CHa (19)), 2.18e2.00 (m, 2H, CHa (16) þ CHa (1)),
2.01e1.90 (m, 3H, CH (18) þ CHb (19) þ CHa (15)), 1.89e1.83 (m, 1H,
CHb (11)), 1.82e1.75 (m, 1H, CH (9)), 1.69e1.52 (m, 2H, CH2 (7)),
1.60e1.50 (m, 1H, CHa (22)), 1.55e1.48 (m, 1H, CHa (6)), 1.47e1.44
(m, 1H, CHb (6)), 1.44 (s, 3H, CH3 (27)), 1.40e1.37 (m, 1H, CHa (21)),
1.33e1.38 (m, 1H, CHb (22)), 1.29e1.20 (m, 2H, CHb (16) þ CHb (21)),
1.17 (s, 3H, CH3 (26)), 1.19e1.10 (m, 1H, CHb (15)), 1.01 (s, 3H, CH3
(23)), 1.03e0.95 (m, 1 H, CHb (1)), 0.99 (s, 3H, CH3 (30)), 0.94 (s, 3H,
CH3 (25)), 0.92e0.89 (m, 1H, CH (5)), 0.89 (s, 3H, CH3 (29)), 0.82 (s,
d
¼ 180.2 (C]O, C28), 90.8 (CeO, C13), 84.0 (CHOH, C3), 76.1
(CHOH, C12), 69.2 (CHOH, C2), 55.4 (CH, C5), 51.2 (CH, C18), 46.7
(CH2, C1), 44.9 (Cquart, C17), 44.7 (CH, C9), 42.5 (Cquart, C14), 42.3
(Cquart, C8), 39.5 (CH2, C19), 39.4 (Cquart, C4), 37.9 (Cquart, C10), 34.3
(CH2, C21), 34.0 (CH2, C7), 33.4 (CH3, C30), 31.7 (Cquart, C20), 29.0
(CH2, C11), 28.6 (CH3, C23), 28.1 (CH2, C15), 27.6 (CH2, C22), 24.0
(CH3, C29), 21.3 (CH2, C16), 18.8 (CH3, C27), 18.7 (CH3, C26), 17.9
(CH2, C6), 17.7 (CH3, C25), 16.7 (CH3, C24) ppm; MS (ESI, MeOH):
m/z ¼ 489.2 (60%, [M þ H]þ), 977.4 (12%, [2M þ H]þ), 944.4 (100%,
[2M þ Na]þ).
3H, CH3 (24)) ppm; 13C NMR (100 MHz, CDCl3):
d
¼ 178.7 (C]O,
C28), 91.5 (CeO, C13), 83.8 (CHOH C3), 69.3 (CHOH, C2), 56.0 (CHBr,
C12), 55.5 (CH, C5), 52.1 (CH, C18), 46.1 (CH2, C1), 45.6 (CH, C9), 45.6
(Cquart, C17), 43.7 (Cquart, C14), 42.4 (Cquart, C8), 39.8 (CH2, C19), 39.2
(Cquart, C4), 37.9 (Cquart, C10), 34.3 (CH2, C7), 33.8 (CH2, C21), 33.5
(CH3, C30), 31.7 (Cquart, C20), 30.5 (CH2, C11), 29.3 (CH2, C15), 28.4
(CH3, C23), 27.4 (CH2, C22), 23.6 (CH3, C29), 21.2 (CH2, C16), 21.1
(CH3, C27), 19.0 (CH3, C26), 18.0 (CH3, C25), 17.8 (CH2, C6), 16.8 (CH3,
C24) ppm; MS (ESI, MeOH): m/z ¼ 551.3 (11%, [M þ H]þ), 568.3 (19%,

100
B. Siewert et al. / European Journal of Medicinal Chemistry 72 (2014) 84e101
NH4]þ), 573.5 (62%, [M
þ
Na]þ), 605.1 (20%,
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thetic triterpenoids: potent agents for prevention and treatment of tissue
injury caused by inflammatory and oxidative stress, J. Nat. Prod. 74 (2011)
537e545.
[M
þ
[M þ Na þ MeOH]þ), 1123.1 (100%, [2M þ Na]þ).
[5] R. Csuk, B. Siewert, A convenient separation of ursolic and oleanolic acid,
Tetrahedron Lett. 52 (2011) 6616e6618.
[6] J. Liu, Pharmacology of oleanolic acid and ursolic acid, J. Ethnopharmacol. 49
(1995) 57e68.
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with a broad range of applications for prevention and treatment of chronic
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[11] N. Suh, Y. Wang, T. Honda, G.W. Gribble, E. Dmitrovsky, W.F. Hickey,
R.A. Maue, A.E. Place, D.M. Porter, M.J. Spinella, C.R. Williams, G. Wu,
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5.4.32. (2a,3b
) O,O0-2,3eDiydroxy-12-iodo-olean-28-oic acid
28,13-lactone (31)
MA (0.3 g, 0.6 mmol)was solved in DCM/water (1:1, 40 mL) and
treated with sodium hydrocarbonate (250 mg, 3 mmol), potassium
iodide (1 g, 6 mmol) and iodine (500 mg, 2 mmol). After 48 h
stirring at 25 ꢁC and work up as described above, compound 31 was
obtained as a colorless solid; yield: 46%; m.p.: 230e233 ꢁC;
RF ¼ 0.44 (n-hexane/ethyl acetate, 1:2); [
a
]
¼ þ69.1ꢁ (c 0.40,
D
CHCl3); IR (KBr):
n
¼ 3424s, 2954s, 1775s, 1637w, 1468m, 1386m,
1368w, 1301w, 1210w, 1198w, 1179m, 1130m, 1103w, 1052m cmꢀ1
;
1H NMR (400 MHz, CDCl3):
d
¼ 4.45 (dd, J ¼ 4.5, 2.3 Hz, 1H, CH (12)),
3.76 (ddd, J ¼ 11.7, 9.2, 4.5 Hz, 1H, CHa (2)), 3.07 (d, J ¼ 9.2 Hz, 1H, CH
(3)), 2.60e2.51 (m, 1H, CHa (19)), 2.42e2.36 (m, 1H, CHa (11)), 2.14e
2.08 (m, 1H, CHa (16)), 2.10e2.06 (m, 1H, CHa (1)), 1.98e1.83 (m, 4H,
CHb (11) þ CHb (15) þ CHb (19) þ CH (18)), 1.79e1.72 (m, 1H, CH (9)),
1.68e1.40 (m, 4H, CH2 (22) þ CHa (7) þ CHa (6)), 1.50 (s, 3H, CH3
(27)), 1.50e1.40 (m, 1H, CHb (6)), 1.33e1.20 (m, 3H, CHb (7) þ CHa
(21) þ CHb (16)), 1.23 (s, 3H, CH3 (26)), 1.21e1.13 (m, 2H, CHb
(21) þ CHa (15)), 1.12e1.08 (m, 1H, CHb (1)), 1.04 (s, 3H, CH3 (23)),
0.99 (s, 3H, CH3 (30)), 0.94 (s, 3H, CH3 (25)), 0.95e0.85 (m, 1H, CH
(5)), 0.88 (s, 3H, CH3 (29)), 0.80 (s, 3H, CH3 (24)) ppm; 13C NMR
M.B. Sporn,
A novel synthetic oleanane triterpenoid, 2-cyano-3,12-
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(100 MHz, CDCl3):
(CHOH, C3), 69.2 (CHOH, C2), 55.2 (CH, C5), 52.7 (CH, C18), 47.1 (CH,
C9), 45.8 (Cquart, C17), 45.9 (CH2, C1), 44.1 (Cquart, C14), 42.6 (Cquart
C8), 40.0 (CH2, C19), 39.4 (Cquart, C4), 38.0 (Cquart, C10), 34.6 (CH2,
C7), 33.7 (CH2, C21), 33.6 (CH3, C30), 33.1 (CH2, C11), 32.0 (CHI, C12),
32.1 (Cquart, C20), 29.6 (CH2, C15), 28.3 (CH3, C23), 27.6 (CH2, C22),
23.4 (CH3, C29), 22.9 (CH3, C27), 21.5 (CH2, C16), 19.4 (CH3, C26),
18.5 (CH3, C25), 17.6 (CH2, C6), 16.8 (CH3, C24) ppm; MS (ESI,
MeOH): m/z ¼ 599.1 (16%, [M þ H]þ), 621.3 (100%, [M þ Na]þ), 652.9
(49%, [M þ Na þ MeOH]þ), 1218.9 (87%, [2M þ Na]þ).
d
¼ 178.3 (C]O, C28), 91.4 (Cquart, C13), 83.6
,
high-performance chromatography, J. Chromatogr.
6670.
A 1216 (2009) 6662e
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We like to thank Dr. R. Kluge for the measurement of the ESIeMS
spectra, Dr. D. Ströhl for the NMR spectra, and Dr. R. Schäfer, A.
Obernauer, V. Perl, S. Ludwig, W. Rebel and S. Oschatz for their help
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were recorded by J. Wiese. The cell lines were kindly provided by
Dr. Thomas Müller (Dept. of Haematology/Oncology, Universität
Halle-Wittenberg). Special thanks are due to L. Baratto and to Prof.
Dr. R. Paschke for helpful discussions. Support by “Gründerwerk-
statt e Biowissenschaften” is gratefully acknowledged. Special
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