Urea derivates of ursolic, oleanolic and maslinic acid induce apoptosis and are selective cytotoxic for several human tumor cell lines

Article abstract of DOI:10.1016/j.ejmech.2016.04.051

2,3-Di-O-acetyl-maslinic acid benzylamide (5) has previously been shown to possess high cytotoxicity for a variety of human tumor cell lines while being of low cytotoxicity to non-malignant cells. Structural modifications performed on 5 revealed that the presence of these acetyl groups in 5 and the presence of (2β,3β)-configurated centers seems necessary for obtaining high cytotoxicity combined with best selectivity between malignant cells and non-malignant mouse fibroblasts. Compounds carrying an ursane skeleton showed weaker cytotoxicity than their oleanane derived analogs. In addition, the benzylamide function in compound 5 should be replaced by a phenylurea moiety to gain better cytotoxicity while retaining and improving the selectivity. Thus, maslinic acid derived N-[2β,3β-di-O-acetyl-17β-amino-28-norolean-12-en-17-yl]phenylurea (45) gave best results showing EC₅₀ = 0.9 μM (for A2780 ovarian cancer cells) with EC₅₀ > 120 μM for fibroblasts (NIH 3T3) and triggered apoptosis while caspase-3 was not activated by this compound.

Full text of DOI:10.1016/j.ejmech.2016.04.051

European Journal of Medicinal Chemistry 119 (2016) 1e16
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Urea derivates of ursolic, oleanolic and maslinic acid induce apoptosis
and are selective cytotoxic for several human tumor cell lines
*
ꢀ
Sven Sommerwerk, Lucie Heller, Julia Kuhfs, Rene Csuk
Martin-Luther-University Halle-Wittenberg, Organic Chemistry, 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:
2,3-Di-O-acetyl-maslinic acid benzylamide (5) has previously been shown to possess high cytotoxicity
for a variety of human tumor cell lines while being of low cytotoxicity to non-malignant cells. Structural
modifications performed on 5 revealed that the presence of these acetyl groups in 5 and the presence of
Received 25 February 2016
Received in revised form
19 April 2016
Accepted 20 April 2016
Available online 23 April 2016
(2b,3b)-configurated centers seems necessary for obtaining high cytotoxicity combined with best
selectivity between malignant cells and non-malignant mouse fibroblasts. Compounds carrying an
ursane skeleton showed weaker cytotoxicity than their oleanane derived analogs. In addition, the ben-
zylamide function in compound 5 should be replaced by a phenylurea moiety to gain better cytotoxicity
Keywords:
while retaining and improving the selectivity. Thus, maslinic acid derived N-[2
b
,3
b
-di-O-acetyl-17
b-
Ursolic acid
Oleanolic acid
Maslinic acid
Tumor cells
SRB assay
amino-28-norolean-12-en-17-yl]phenylurea (45) gave best results showing EC50 ¼ 0.9
m
M (for A2780
ovarian cancer cells) with EC50 > 120
m
M for fibroblasts (NIH 3T3) and triggered apoptosis while caspase-
© 2016 Elsevier Masson SAS. All rights reserved.
3 was not activated by this compound.
Apoptosis
1. Introduction
properties have been detected, among them antimicrobial [1,2],
antifungal [3], anti-oxidative [4] and anti-inflammatory [5] as well
as antitumor-active properties [5e10].
Recently, triterpenoids carrying hydroxylic groups at position C-
2 and C-3 of ring A, especially maslinic acid (3, MA) moved into the
focus of scientific investigations while derivatives of betulinic acid
(4, BA) are of continuing interest. Although the cytotoxic properties
of maslinic acid have been shown as early as 1988 [11], its mode of
action remained by and large unclear [11e14]. Previously, an in-
crease of anti-proliferative activity was achieved by derivatization
Animal as well as human life seems associated with the occur-
rence of cancer. One of the earliest written records describing
breast cancer is found in an egyptian Papyrus (“Edwin Smith”) from
1600 BC. At that time and for many centuries surgery was the only
treatment, later on followed by radiation, chemotherapy and now-
a-days increasingly by treatment with cancer-cell specific anti-
bodies. Although chemotherapy offers the greatest chance of cure
for several types of cancer there is still an increasing need for the
development of new, selective and effective drugs.
Natural products and derivatives thereof play a key role in
treating cancer, and (tri)-terpenoid natural products came again in
the center of scientific interest during the ever-lasting search for
new cytotoxic and selective drugs. Thus, almost 40% of all scientific
publications covering the chemistry/biology of triterpenoic acids
at position C-28, and an EC50 ¼ 0.5
mM for A2780 ovarian cancer
cells was found for a 2,3-di-O-acetyl-maslinic acid benzylamide
(EM2, 5) [17]. This compound showed high cytotoxicity for a variety
of human tumor cell lines but also rather low cytotoxicity for non-
malignant mouse fibroblasts NIH 3T3 (selectivity factor approxi-
mately F ¼ 70) [16].
(holding either a
a
-amyrin, a
b
-amyrin or a lupane skeleton) have
Thus, compound 5 seems to be an ideal starting point for the
search for highly active and tumor-cell specific compound analogs,
predominantly by performing modifications in ring A (Fig. 1), in
Ring E as well as by modifications at the carbonyl group. These
derivatives were subjected to a sulforhodamine B assay [17] for the
determination of their cytotoxic activity. Representative and most
active compounds of this screening were investigated in more
detail by annexin V/propidium iodide staining and by investigation
of their action onto the cell-cycle.
been published not until 2010 to 2015. Oleanolic acid (1, OA, Fig. 1)
and ursolic acid (2, UA) and their derivatives have been investigated
in detail by several groups, and many interesting biological
€
* Corresponding author. Bereich Organische Chemie, Martin-Luther Universitat
Halle-Wittenberg, Kurt-Mothes-Str. 2, D-06120, Halle, Saale, Germany.
E-mail address: rene.csuk@chemie.uni-halle.de (R. Csuk).
http://dx.doi.org/10.1016/j.ejmech.2016.04.051
0223-5234/© 2016 Elsevier Masson SAS. All rights reserved.

2
S. Sommerwerk et al. / European Journal of Medicinal Chemistry 119 (2016) 1e16
Fig. 1. Structure of representative triterpenoic acids 1e4 and the transformation of oleanolic acid (1) into maslinic acid (3) [20] followed by its transformation into the highly
cytotoxic derivative EM2 (5) [15,27].
2. Results and discussion
Reaction of 6 with 2,3-dimethoxypropane (in the presence of p-
TsOH), however, gave a 91% yield of 13 within just a few minutes
[20]. The epoxide 14 was synthesized following the procedure
given by GarciaeGranados et al. [21] using PPh3 and diethyl azo-
dicarboxylate in DMF. While compound 15 was easily obtained
from the reaction of 6 with chloroacetic anhydride, the formation of
dialdehyde 16 required the presence of silica supported sodium
periodate [22]. The presence of the two aldehyde moieties was
easily shown by 1H NMR-spectroscopy; the 1H NMR spectrum of 16
The presence of oxygen-carrying substituents at positions C-2
and C-3 in ring A seems important for obtaining good cytotoxicity;
these functionalities, however, have to be protected. Unprotected
hydroxyl groups at these positions led to an approximately 4-fold
decrease in cytotoxicity [15]. Our strategy to obtain C2/C3-
modified derivatives of maslinic aacid (3) started from oleanolic
acid (1). Thus, 3 was obtained from 1 by partial synthesis in an
overall yield of 41% as previously described. This approach for
obtaining large amounts of 3 seems more convenient than the
extraction of 3 from olives or olive pomace [18]. Reaction of 3 with
oxalyl chloride followed by a reaction with benzylamine gave
known compound 6 that served as a valuable starting material for
the transformations to follow (Scheme 1).
Thus, reaction of 6 with sodium hydride and methyl iodide [19]
gave the 2-monomethylether 7 together with dimethyl ether 8
while no 3-monomethyl ether was formed at all (see Scheme 2).
This selective methylation is probably due to steric hindrance at C
(3)-OH caused by the presence of the geminal dimethyl group at
position C-4. Reaction of 6 under the same conditions using 1-
bromo-2-methoxyethane as the alkylating agent gave 11. The re-
action of 6 with 2,2-dimethoxymethane in the presence of catalytic
amounts of p-TsOH led to the formation of three different products
as a function of temperature and time. Thus, reaction of 6 for 6 days
showed the presence of a triplet at
¼ 9.60 ppm.
Betulinic acid (BA, as a standard), maslinic acid (MA, for com-
d
¼ 9.80 ppm and a singlet at
d
parison) and compounds 5, 7e16 were submitted to SRB assays,
and the results of these assays are compiled in Table 1.
For compounds 7, 8, 10, 12e14 and 16 (Table 1) a significant
decrease of the cytotoxicity was observed. In addition, these com-
pounds also showed a diminished selectivity concerning tumor cell
lines and non-malignant mouse fibroblasts. Compounds 9 and 11,
however, show similar biological activity compared to EM2 (5)
concerning cytotoxicity and tumor-to-non-tumor selectivity. The
selectivity index (FSI
mately 5 for both compounds and promising but still decreased as
¼
EC50, NIH 3T3/EC50, tumor cell line) is approxi-
compared to compound 5. The most active compound of this series
is the bis-chloroacetate 15 showing an EC50 ¼ 2
mM for the human
breast cancer cell line MCF-7. This increase in cytotoxicity, however,
is accompanied by an almost total loss in selectivity (FSI ¼ 0.8). This
parallel previous results observed for derivatives of the triterpenoid
tormentic acid [23]. As a result, derivatives carrying acetyl pro-
tecting groups at OHeC (2) and OHeC (3) produce the best results
at room temperature gave
methoxymethoxy derivative
a
mixture consisting of the 2-
together with the 2,3-
9
dimethoxymethoxy derivative 10, while the microwave assisted
reaction at 120 ꢀC furnished the 2,3-methylenated compound 12.

S. Sommerwerk et al. / European Journal of Medicinal Chemistry 119 (2016) 1e16
3
Scheme 1. Synthesis of maslinic acid derived compounds 6e16: (a) MeOH/KOH, 25 ꢀC, 24 h, 66%; (b)NaH, MeI, THF, 0 ꢀC / 25 ꢀC, 5 d, 38% (for 7), 33% (for 8); (c) dimethoxy-
methane, p-TsOH, 25 ꢀC, 7 d, 35% (for 9), 40% (for 10); (d) NaH, 1-bromo-2-methoxyethane, THF, 0 ꢀC / 25 ꢀC, 5 d, 58%, (e) dimethoxymethane, p-TsOH, 120 ꢀC, 2 h, microwave
assisted, 78%; (f) DMP, p-TsOH, 25 ꢀC, 10 min, 91%; (g) PPh3, DEAD, DMF, 0 ꢀC / reflux, 2 d, 65%; (h) chloroacetic anhydride, TEA, DMAP, DCM, 25 ꢀC, 20 h, 92%; (i) NaIO4 on silica,
DCM, 25 ꢀC, 2 min, 98%.
possible diastereoisomers 3, 17e19 (with respect to positions C-2
and C-3) can be synthesized very easily from commercially avail-
able oleanolic acid (1) [20]. Acetylation of 3, 17e19 with acetic
anhydride in DCM in the presence of DMAP and trimethylamine
gave diacetates 20e23; their treatment with oxalyl chloride/ben-
zylamine under Schotten-Baumann conditions afforded EM2 (5)
and analogs 24e26. Reaction of 20 with aniline or 2-
phenylethylamine applying similar conditions gave 27 and 28,
respectively.
The (2b,3b) configurated derivative 24 showed in the SRB assays
EC50 values of similar magnitude as standard compound 5 but the
tumor-to-non-tumor selectivity was significantly smaller than that
observed for EM2. A loss of selectivity was also observed for
(2
values of all compounds in this series. Quite the opposite, for
(2 ,3 )-configurated 25, a 10-fold diminished selectivity was
observed. As a result, the presence of a 3 configurated center
a,3a)-configurated 26; in addition, 26 showed the highest EC50
b
a
b
seems important for obtaining compounds of high cytotoxicity and
good-to-excellent selectivity.
In a next step, the influence of the chain length between the
aromatic substituent and the amide at C-28 was investigated.
Compared to EM2 (5) chain elongated 28 showed a decreased
cytotoxicity, while shortening of the chain (as represented in phe-
nylamide 27) increased cytotoxicity as well as tumor-to-non-tumor
cell selectivity (Table 1).
All of the derivatives described above possess an oleanane
skeleton. Hence, we became interested in possible differences in
bioactivity comparing molecules comprising an oleanane or an
ursane skeleton. Despite all limitations results from this investi-
gation and data from literature so far suggest that the presence of
Scheme 2. Synthesis of compounds 5, 20e28: (a) Ac2O, TEA, DMAP, DCM, 25 ꢀC, 20 h,
76e78%; (b) oxalyl chloride, TEA, DMF, DCM, 25 ꢀC, 2 h; (c) RNH2, TEA, DMAP, DCM,
0
ꢀC, 5 min, 81e87%; R ¼ Bn (5, 24e26), Ph (27), phenylethyl (28).
in SRB assays, i.e. they combined highest cytotoxicity with best
selectivity between malignant cells and non-malignant mouse
fibroblasts.
In continuation of these observations, the relevance of the
relative configuration at positions C-2 and C-3 of ring A was
investigated in more detail. As already shown, all of the four

4
S. Sommerwerk et al. / European Journal of Medicinal Chemistry 119 (2016) 1e16
Table 1
Cytotoxicity of selected compounds, betulinic acid (BA, standard) and maslinic acid (MA, for comparison), oleanolic acid (OA, for comparison) and ursolic acid (UA, for
comparison): EC50 values in M from SRB assays after 96 h of treatment; the values are averaged from three independent experiments performed each in triplicate; confidence
m
interval CI ¼ 95%; n.d. not detected/not determined. Human cancer cell lines: 518A2 (melanoma), FaDu (hypopharyngeal carcinoma), HT29 (colorectal adenocarcinoma), MCF7
(breast adenocarcinoma), A549 (lung adenocarcinoma), A2780 (ovarian carcinoma), SW 1736 (thyroid carcinoma); NIH 3T3: non-malignant mouse fibroblasts.
EC50
518A2
FADU
HT29
MCF-7
A549
A2780
SW1736
NIH 3T3
BA
MA
OA
UA
5
7
8
9
10
11
12
13
14
15
16
20
21
22
23
24
25
26
27
28
9.4 0.4
13.7 0.9
64.3 4.2
14.7 0.1
1.5 0.2
12.6 1.1
18.1 2.0
3.2 0.1
22.1 2.7
3.5 0.1
>30
29.8 2.4
15.7 0.9
2.1 0.0
6.0 0.1
23.1 0.5
13.8 1.0
11.7 0.9
15.3 1.0
0.9 0.3
2.3 0.2
5.7 0.8
0.7 0.7
1.2 0.2
15.3 2.1
n.d.
14.4 1.5
28.8 0.5
38.8 3.1
10.6 0.3
4.7 0.2
10.6 0.9
14.3 1.1
5.2 0.7
15.9 0.5
4.9 0.3
20.8 0.7
25.9 3.1
26.6 3.8
4.3 0.5
8.3 0.4
25.5 2.2
18.8 1.6
16.5 0.8
21.4 0.6
3.7 0.4
5.2 3.3
7.7 0.9
2.9 0.7
6.1 1.4
10.2 0.9
37.2 1.2
80.0 3.5
12.7 0.1
7.7 0.5
11.7 2.1
14.6 2.4
5.5 0.6
15.2 3.0
6.8 1.9
16.7 1.7
14.6 1.7
8.6 2.0
2.0 0.5
5.4 0.1
16.6 1.5
15.1 1.3
14.8 0.6
17.6 0.6
3.5 1.2
7.6 1.0
7.8 0.6
3.1 0.5
5.8 1.0
17.1 1.6
23.4 0.5
72.3 1.5
15.5 1.3
2.5 0.1
21.5 3.0
15.3 0.4
12.8 1.2
17.3 0.9
4.9 0.7
>30
26.4 0.4
25.8 1.7
6.5 0.0
6.9 0.7
18.9 1.5
29.1 1.0
15.2 0.9
20.4 0.8
5.6 0.4
8.8 1.4
15.6 1.0
9.5 2.1
9.2 2.3
8.8 0.7
19.5 0.8
14.0 0.7
11.7 0.6
0.5 0.1
n.d.
n.d.
n.d.
n.d.
2.7 0.9
n.d.
n.d.
n.d.
n.d.
n.d.
16.1 1.4
21.1 0.2
76.4 0.7
18.7 1.6
33.8 3.0
11.9 1.0
14.7 1.2
16.5 1.9
15.0 1.6
15.5 2.9
13.9 3.3
21.6 2.3
20.5 4.7
1.6 0.2
12.2 0.8
12.2 0.7
8.0 1.0
10.0 0.6
14.2 1.1
9.4 1.1
75.4 3.4
14.2 2.0
3.1 0.1
11.4 1.5
17.2 1.4
3.3 0.2
17.9 2.1
n.d.
n.d.
18.1 3.1
20.5 1.4
5.4 0.7
18.2 2.7
4.6 0.6
>30
>30
>30
3.3 0.5
7.5 0.7
n.d.
>30
>30
>30
12.9 1.5
8.6 1.1
n.d.
2.8 1.0
7.0 0.1
17.8 0.8
>30
9.5 0.5
17.3 0.7
2.0 0.3
3.3 0.1
5.5 1.1
1.4 0.1
2.8 0.5
n.d.
10.5 0.9
7.2 0.6
6.7 0.7
10.5 0.7
0.5 0.2
1.1 0.1
1.9 0.3
n.d.
>30
23.4 4.4
22.0 1.1
3.4 0.3
5.2 1.9
20.7 8.2
2.9 0.5
5.2 3.0
7.7 1.0
11.4 0.6
16.0 1.9
9.5 3.1
n.d.
acetyl groups at OHeC (2) and OHeC (3) seems as mandatory for
obtaining highly active compounds as a (2 ,3 )-configuration in
ring A and the presence of a phenylamide rather than of a benzy-
lamide at C-28. Hence, 2-epi-corsolic acid (33) possessing a (2 , 3
derived 35 gave an almost quantitative yield of amine 37 at 30 ꢀC
within 2 h, for the ursane-type isocyanate 36, however, a complete
reaction occurred only at 50 ꢀC after 1 day of reaction. These dif-
ferences in reactivity are probably due to the steric hindrance of the
methyl groups in ring E in molecules of the ursane-type. Reaction of
amines 37 and 38 with benzoyl chloride gave amides 41 and 42
while from the reaction of 37 and 38 with phenyl chloroformate
carbamates 43 and 44 were obtained. Reaction of the amines with
phenyl isocyanate yielded ureas 45 and 46, respectively.
Screening of the compounds in SRB assays (Table 2) showed
amides 41 and 42 of decreased cytotoxicity (compared to 39 and
40), and for these compounds, selectivity between tumor cells and
mouse fibroblasts is almost completely lost. A similar behavior was
detected for carbamates 43 and 44. Compounds 45 and 46 showed
cytotoxic effects and selectivity similar to compounds 39 and 40. By
and large, ursane-derived compounds showed diminished cyto-
toxicity than their oleanane derived analogs.
b
b
b
b)
configuration at ring A seemed an ideal starting point for these
investigations. This compound, however, is expensive, and its
extraction from natural sources is rather tedious and time
consuming. Hence, an alternative partial synthesis of 33 was
developed starting from easily accessible and cost-efficient ursolic
acid (2). Jones oxidation [20,24] of 2 furnished 3-oxo compound 29
(Scheme 3) whose bromination with pyridinium tribromide gave a
2:1 mixture of epimeric compounds 30/31 [25]. Nucleophilic sub-
stitution of the bromine substituents in the presence of air gave an
almost quantitative yield of enol 32 whose reduction with sodium
borohydride gave (2b,3b) (33) in an excellent over-all yield of 38%
(from 2, after re-crystallization from ethyl acetate). Acetylation of
33 gave di-acetate 34.
Microwave assisted reactions of 21 or 34 with diphenylphos-
phoryl azide at 180 ꢀC gave isocyanates 35 and 36, respectively
(Scheme 4). The isocyanates are characterized in their IR spectra by
To gain a deeper insight into the mode of action of compounds
onto tumor cells, some extra experiments were performed, such as
an annexin V/propidium iodide assay (Fig. 2), cell cycle evaluations
(Fig. 3 A and B) as well as fluorescence microscopy (Fig. 3 C).
the presence of strong absorption signals at
n
¼ 2256e2270 cmꢁ1
.
And in their 13C NMR spectra the signal for the N]C]O moiety was
Thus, the cells were treated with compound 24 (10
mM, 48 h) or
detected at
d
¼ 122.0 and 122.4 ppm, respectively [26].
with compound 45 (10 M, 24 h). The results from these assays
indicate that a controlled cell death had occurred as evidenced by
m
Surprising differences in reactivity were observed for the hy-
drolysis reactions of the isocyanates 35 and 36. While oleanane
the presence of green fluorescent A2780 cells. Several cells showed
Scheme 3. Synthesis of compounds 29e34: (a) Cr3O/H2SO4, silica gel, acetone, 0 ꢀC, 30 min, 100%; (b) pyridinium tribromide, acetic acid, 25 ꢀC, 3 h, 100%; (c) air, NaOH (5 equiv),
DMF, 60 ꢀC, 1 h, 97%; (d) NaBH4, THF/MeOH, 25 ꢀC, 1 h, re-crystallized from ethyl acetate, 38%; (e) Ac2O, TEA, DMAP, DCM, 25 ꢀC, 20 h, 51%.

S. Sommerwerk et al. / European Journal of Medicinal Chemistry 119 (2016) 1e16
5
Scheme 4. (a) Diphenylphosphoryl azide, TEA, toluene, 180 ꢀC, 3 h, microwaves, 76% (for 35), 57% (for 36); (b) HClaq, THF, 30e50 ꢀC, 2e24 h, 76% (for 37), 46% (for 38); (c) oxalyl
chloride, TEA, DMF, DCM, 25 ꢀC, 2 h; (d) aniline, TEA, DMAP, DCM, 25 ꢀC, 5 min, 86% (for 39), 85% (for 40); (e) benzoyl chloride, TEA, DMAP, DCM, 25 ꢀC, 2 h, 78% (for 41), 68% (for
42); (f) phenyl chloroformate, TEA, DMAP, DCM 25 ꢀC, 2 h, 92% (for 43), 83% (for 44); (g) phenylisocyanate, toluene, 2 h, 25 ꢀC, 69% (for 45), 58% (for 46).
Table 2
Cytotoxicity of selected compounds (EC50 values in mM from SRB assays after 96 h of treatment; the values are averaged from three independent experiments performed each
in triplicate; confidence interval CI ¼ 95%; n.d. not detected, MA for comparison). Human cancer cell lines: 518A2 (melanoma), FaDu (hypopharyngeal carcinoma), HT29
(colorectal adenocarcinoma), MCF7 (breast adenocarcinoma), A549 (lung adenocarcinoma), A2780 (ovarian carcinoma), SW 1736 (thyroid carcinoma); NiH 3T3: nonmalignant
mouse fibroblasts.
EC50
518A2
HT29
MCF-7
A549
A2780
SW1736
NIH 3T3
MA
39
40
41
42
43
44
45
46
13.7 0.9
2.0 0.2
3.4 0.1
3.5 0.8
7.9 0.5
4.4 1.6
3.1 0.9
1.2 0.3
2.5 0.5
28.8 0.5
2.6 0.5
7.3 1.0
6.8 1.1
11.7 1.5
8.5 3.2
7.6 3.0
2.3 2.7
16.0 2.3
37.2 1.2
1.8 0.7
16.4 2.9
16.8 3.7
>30
5.8 1.0
23.3 6.1
2.8 1.8
19.6 1.6
23.4 0.5
4.9 0.4
8.9 1.2
11.1 2.0
16.4 2.6
8.5 1.2
10.1 1.8
4.4 1.7
12.4 1.6
19.5 0.8
1.3 0.0
3.3 1.3
3.1 1.2
6.7 1.2
2.4 0.2
1.6 0.4
0.9 0.0
1.5 0.5
n.d.
21.1 0.2
>60
>60
12.2 3.3
14.1 2.1
15.4 7.4
>30
2.7 0.8
4.4 0.3
6.8 2.3
14.9 2.0
7.3 0.9
5.5 1.0
2.8 1.2
6.8 1.4
>120
>120
an orange color; these cells have died by secondary necrosis.
Furthermore, an annexin V/PI assay (Fig. 2) as well as a quantitative
FACS experiment (Fig. 3) was performed. Therefore, A2780 cells
were incubated with 24e26, 43 and 45 for 24 h and 48 h, respec-
tively. Fig. 2 depicts the results of this assay. For compounds 24e26
and 43, treatment of the cells for 24 h showed only a small increase
of annexin positive cells (2e5% apoptosis relative to control). On the
contrary, the urea derivative 45 significantly induced controlled cell
death after 24 h, and relative to control almost 33% of the
A2780 cells showed apoptotic behavior. After 48 h about 65e78% of
the A2780 cells carried phosphatidylserine in the outer leaf of the
membrane, thus indicating an apoptotic death of these cells. Most
of the dead cells (approximately 50%), however, showed apoptosis;
another 20e30% of dead cell had died by a process of secondary
necrosis. For the investigation of the cell cycle, A2780 cells were
cytometry. As a result, the caspase assays showed no activation of
effector caspase 3. Thus, a caspase-independent cell death was
most likely. Based on these results we assume a cytostatic behavior
of the compounds.
3. Conclusion
In this study several derivatives of 2,3-dihydroxy-triterpenoic
acids were synthesized, and most of these showed low EC50 values
in photometric SRB assays. Systematic variation of the protecting
groups [at HOeC (2) and HOeC (3)], the absolute configuration at
these centers, modifications at the carbonyl group at C-28 as well as
comparison between analogs of the ursane vs oleanane skeleton
type revealed, revealed that the presence of these acetyl groups at
positions C-2 and C-3 and the presence of (2b,3b)-configurated
treated with 24e26, 43 and 45 (10 mM) for 24 h. Close inspection of
centers seems necessary for obtaining high cytotoxicity combined
with best selectivity between malignant cells and non-malignant
mouse fibroblasts. An oleanane derived compounds showed bet-
ter cytotoxicity than their ursane derived analogs, and the presence
of a phenylurea moiety at C-28 led to a compound with improved
cytotoxicity while retaining the selectivity between malignant cells
the results (Fig. 3) for living A2780 cells showed the presence of a
weaker G1/G0 peak combined with an increase in the S phase after
24 h. An emersion of a Sub-G1 peak occurred with simultaneous
decreases of G1/G0 phase and S phase (after 48 h, cf. suppl. part).
Furthermore, a decrease of the G2/M phase was detected after 24 h.
Caspase activation was determined after 24/48/72 h by using flow
and mouse fibroblasts. Thus, maslinic acid derived N-[2b,3b,17b)-

6
S. Sommerwerk et al. / European Journal of Medicinal Chemistry 119 (2016) 1e16
Fig. 2. Evaluation of annexin V/PI assay: treatment of A2780 cells after 24 h and 48 h with derivatives 24 (10 mM), 25 (10 mM), 26 (10 mM), 43 (10 mM), 45 (10 mM). Upper part:
representative samples after 48 h treatment. Lower part: diagram with relative changes in the distribution compared to control [%], determined by Attune® Cytometric Software vl
1.2.5.
2,3-Di-O-acetyl-17-amino-28-norolean-12-en-17-yl]phenylurea
(45) gave best results showing EC50 ¼ 0.9 M (for A2780 ovarian
cancer cells) with EC50 > 120 M for fibroblasts (NIH 3T3). This
prepared as previously reported [15,18,20,27]. Microwave assisted
reactions were performed in a Monowave 300 apparatus (Anton
Paar, Austria). The SRB assay, the annexin V/propidium iodide
staining and the FACS cell cycle investigations were performed as
previously described [12,15,23,27].
m
m
compound acts by apoptosis; its activity is caspase-3 independent.
4. Experimental part
4.2. Syntheses
4.1. General
4.2.1. 2
benzylamide (7) and 2
a
-Methoxy-3
b
-hydroxy-olean-12-en-28-oic acid
,3 -dimethoxy-olean-12-en-28-oic acid
Melting points are uncorrected (Leica hot stage microscope),
a b
NMR spectra were recorded using the Varian spectrometers Gemini
benzylamide (8)
2000 or Unity 500 (
d
given in ppm, J in Hz, internal Me4Si, MS
To an ice-cold solution of 6 (120 mg, 0.21 mmol) in dry THF
(5 mL), sodium hydride (15 mg, 0.63 mmol) was added, and the
mixture was stirred for 30 min and allowed to warm to room
spectra were taken on a Finnigan MAT LCQ 7000 (electrospray,
voltage 4.1 kV, sheath gas nitrogen) instrument. The optical rota-
tion was measured on a PerkineElmer polarimeter at 20 ꢀC; TLC
was performed on silica gel (Merck 5554); elemental analyses were
performed on a Vario EL (CHNS). The solvents were dried according
to usual procedures. The purity of the compounds was determined
by HPLC and found to be >98%. Ursolic acid (1) and oleanolic acid
(2) were obtained from different commercial suppliers in bulk
quantities; maslinic acid (3), EM2 (5), and compounds 17e20 were
temperature. Methyl iodide (50
mL, 0.80 mmol) was added, and
stirring at 25 ꢀC was continued for 5 days. Et2O (100 mL) was added,
and the organic layer was washed consecutively with diluted hy-
drochloric acid (0.1
M
, 1 ꢂ 100 mL), water (2 ꢂ 100 mL) and brine
(1 ꢂ 50 mL), dried (MgSO4), filtrated, and the filtrate was evapo-
rated to dryness. The residue was subjected to column chroma-
tography (silica gel, hexane/ethyl acetate, 8:2) to afford 7 (47 mg,

S. Sommerwerk et al. / European Journal of Medicinal Chemistry 119 (2016) 1e16
7
Fig. 3. (A): Representative examples for cell cycle evaluation of the living A2780 cells after 24 h with derivatives 24 (10
arithmetic means of the population distribution [%] of living A2780 cells treated with 24e26, 43 and 45 (10
bar ¼ 10 m): control (A2780); treatment of A2780 cells with 24 (48 h, 10 M) and 45 (24 h, 10 M).
m
M) and 45 (10
m
M) via ModFit V 4.0.5. (B): Diagram with
m
M) for 24 h. (C) Representative fluorescence microscopic images (scale
m
m
m
38%) and 8 (42 mg, 33%); 7: white solid; mp 108e110 ꢀC; RF ¼ 0.21
6.3 Hz, 1 H, He33a), 4.14 (dd, J ¼ 14.7, 4.3 Hz, 1 H, He33b), 3.57 (s,
3 H, He32), 3.42 (s, 3 H, He31), 3.27 (ddd, J ¼ 11.3, 9.6, 4.5 Hz, 1 H,
He2), 2.58 (d, J ¼ 9.6 Hz, 1 H, He3), 2.54 (dd, J ¼ 12.7, 4.2 Hz, 1 H,
He18), 2.03e1.95 (m, 2 H, He1a þ He16a), 1.94e1.80 (m, 2 H,
He11a þ He11b), 1.80e1.71 (m, 2 H, He22a þ He19a), 1.70e1.48
(m, 5 H, He16b þ He9 þ He22b þ He15a þ He6a), 1.47e1.28 (m,
(silica gel, hexane/ethyl acetate, 8:2); [
a]
¼ ꢁ6.88ꢀ (c ¼ 0.30,
D
CHCl3); IR (KBr):
n
¼ 2944vs, 2878s, 1646s, 1518s, 1454s, 1388m,
1364m, 1264m, 1194m, 1092s, 1050m, 1030m, 698 m cmꢁ1; 1H NMR
(500 MHz, CDCl3)
d
¼ 7.35e7.31 (m, 2H, He35), 7.29e7.23 (m, 3 H,
He34 þ He36), 6.17 (dd, J ¼ 6.1, 4.6 Hz, 1 H, NH), 5.31 (dd, J ¼ 3.6,
3.6 Hz, 1 H, He12), 4.62 (dd, J ¼ 14.7, 6.3 Hz, 1 H, He32a), 4.15 (dd,
J ¼ 14.7, 4.4 Hz, 1 H, He32b), 3.37 (s, 3 H, He31), 3.21 (ddd, J ¼ 11.2,
9.5, 4.3 Hz,1 H, He2), 3.04 (d, J ¼ 9.5 Hz,1 H, He3), 2.56 (dd, J ¼ 13.1,
4.4 Hz, 1 H, He18), 2.05 (dd, J ¼ 12.3, 4.3 Hz, 1 H, He1a), 2.00 (ddd,
3
H, He7a
þ
He6b
þ
He21a), 1.28e1.13 (m,
3
H,
He7b þ He21b þ He19b), 1.14 (s, 3 H, He27), 1.06e1.00 (m, 1 H,
He15b), 0.98 (s, 3 H, He24), 0.91 (s, 6 H, He29 þ He30), 0.90 (s, 3 H,
He25), 0.78 (s, 3 H, He23), 0.78e0.73 (m, 2 H, He5 þ He1b), 0.65
J
¼
13.8, 13.8, 4.0 Hz,
1
H, He16a), 1.94e1.80 (m,
2
H,
(s, 3 H, H26); 13C NMR (125 MHz, CDCl3):
d
¼ 178.2 (Ce28), 145.0
He11a þ He11b), 1.80e1.70 (m, 2 H, He22a þ He19a), 1.70e1.52
(Ce13), 138.6 (Ce34), 128.8 (Ce36), 127.9 (Ce35), 127.5 (Ce37),
122.9 (Ce12), 93.0 (Ce3), 79.7 (Ce2), 62.3 (Ce32), 57.8 (Ce31), 55.3
(Ce5), 47.7 (Ce9), 46.8 (Ce19), 46.5 (Ce17), 44.5 (Ce1), 43.7
(Ce33), 42.5 (Ce18), 42.2 (Ce14), 40.5 (Ce4), 39.6 (Ce8), 37.8
(Ce10), 34.3 (Ce21), 33.1 (Ce30), 32.8 (Ce22), 32.4 (Ce7), 30.9
(Ce20), 28.7 (Ce24), 27.5 (Ce15), 25.9 (Ce27), 24.0 (Ce16), 23.8
(Ce29), 23.7 (Ce11), 18.4 (Ce6), 17.4 (Ce23), 17.1 (Ce26), 16.7
(Ce25) ppm; MS (ESI): m/z (%) ¼ 590.3 ([M þ H]þ, 100), 1179.3
([2M þ H]þ, 70), 1201.5 ([2M þ Na]þ, 74); analysis calculated for
(m, 5 H, He16b þ He9 þ He22b þ He15a þ He6a), 1.49e1.31 (m,
3
H, He7a
þ
He6b
þ
He21a), 1.29e1.13 (m,
3
H,
He7b þ He21b þ He19b), 1.15 (s, 3 H, He27), 1.07e1.01 (m, 1 H,
He15b), 1.05 (s, 3 H, He24), 0.93 (s, 3 H, He25), 0.91 (s, 6 H,
He29 þ He30), 0.83 (s, 3 H, He23), 0.81 (dd, J ¼ 11.6, 1.8 Hz, 1 H,
He5), 0.72 (dd, J ¼ 11.8, 11.8 Hz, 1 H, He1b), 0.67 (s, 3 H, He26); 13
C
NMR (125 MHz, CDCl3):
d
¼ 178.1 (Ce28), 145.2 (Ce13), 138.6
(Ce33),128.8 (Ce35),127.9 (Ce34),127.5 (Ce36),122.7 (Ce12), 81.7
(Ce3), 78.8 (Ce2), 56.6 (Ce31), 55.2 (Ce5), 47.7 (Ce9), 46.8 (Ce19),
46.5 (Ce17), 43.7 (Ce32), 42.5 (Ce18), 42.2 (Ce14), 42.1 (Ce1), 39.6
(Ce8), 39.0 (Ce4), 38.1 (Ce10), 34.3 (Ce21), 33.1 (Ce30), 32.8
(Ce22), 32.5 (Ce7), 30.9 (Ce20), 28.8 (Ce24), 27.4 (Ce15), 25.9
(Ce27), 24.0 (Ce16), 23.8 (Ce29), 23.7 (Ce11), 18.3 (Ce6), 17.1
(Ce26), 17.0 (Ce23), 16.7 (Ce25) ppm; MS (ESI): m/z (%) ¼ 576.4
([M þ H]þ, 100), 598.6 ([M þ Na]þ, 6), 1151.3 ([2M þ H]þ, 82), 1173.5
([2M þ Na]þ, 68); analysis calculated for C38H57NO3 (575.86): C
79.26, H 9.98, N 2.43; found: C 79.01, H 10.07, N 2.31; 8: white solid;
mp 96e97 ꢀC; RF ¼ 0.42 (silica gel, hexane/ethyl acetate, 8:2);
C39H59NO3 (589.89): C 79.41, H 10.08, N 2.37; found: C 70.26, H
10.19, N 2.16.
4.2.2. 2
a
-Methoxymethoxy-3
b
-hydroxy-olean-12-en-28-oic acid
benzylamide (9) and 2
acid benzylamide (10)
a,3b-dimethoxymethoxy-olean-12-en-28-oic
To a solution of 6 (100 mg, 0.18 mmol) in dimethoxymethane
(10 mL), p-TsOH (3 mg, 0.02 mmol) was added, and the mixture was
stirred at 25 ꢀC for 7 days. Et2O (100 mL) was added, the organic
layer was washed with water (2 ꢂ 100 mL) and brine (1 ꢂ 50 mL),
dried (MgSO4), filtrated, and the filtrate was evaporated to dryness.
The residue was subjected to column chromatography (silica gel,
hexane/ethyl acetate, 8:2) to afford compounds 9 (38 mg, 35%) and
10 (46 mg, 40%); 9: white solid; mp 97e99 ꢀC; RF ¼ 0.17 (silica gel,
[
a
]
¼ ꢁ7.61ꢀ (c ¼ 0.24, CHCl3); IR (KBr):
n
¼ 2946vs, 2878s, 1644s,
D
1518s, 1454s, 1388m, 1364m, 1182m, 1114s, 1096s, 1016m,
698 m cmꢁ1
;
1H NMR (500 MHz, CDCl3):
d
¼ 7.34e7.30 (m, 2H,
He36), 7.29e7.21 (m, 3 H, He35 þ He37), 6.18 (dd, J ¼ 6.0, 4.6 Hz,
hexane/ethyl acetate, 8:2); [
a
]
D
¼ ꢁ5.38ꢀ (c ¼ 0.35, CHCl3); IR (KBr):
1 H, NH), 5.30 (dd, J ¼ 3.5, 3.5 Hz, 1 H, He12), 4.62 (dd, J ¼ 14.7,

8
S. Sommerwerk et al. / European Journal of Medicinal Chemistry 119 (2016) 1e16
n
¼ 2946vs, 1644s, 1520m, 1454m, 1388m, 1364m, 1212m, 1186w,
1H NMR (500 MHz, CDCl3):
¼ 7.35e7.31 (m, 2H, He36), 7.29e7.23 (m, 3 H, He35 þ He37), 6.17
for 5 days. Work-up as described above followed by column chro-
1146m, 1104s, 1040s, 698 m cmꢁ1
;
matography (silica gel, hexane/ethyl acetate, 8:2) gave 11 (84 mg,
d
58%) as white solid; mp 74e76 ꢀC; RF ¼ 0.12 (silica gel, hexane/ethyl
(dd, J ¼ 6.1, 4.7 Hz,1 H, NH), 5.30 (dd, J ¼ 3.6, 3.6 Hz, 1 H, He12), 4.73
(d, J ¼ 6.8 Hz, 1 H, He31a), 4.69 (d, J ¼ 6.8 Hz, 1 H, He31b), 4.62 (dd,
J ¼ 14.7, 6.3 Hz, 1 H, He33a), 4.14 (dd, J ¼ 14.7, 4.4 Hz, 1 H, He33b),
3.53 (ddd, J ¼ 11.5, 9.4, 4.3 Hz, 1 H, He2), 3.40 (s, 3 H, He32), 3.07 (d,
J ¼ 9.5 Hz,1 H, He3), 2.55 (dd, J ¼ 13.2, 4.2 Hz,1 H, He18), 2.03e1.95
(m, 2 H, He1a þ He16a), 1.92e1.81 (m, 2 H, He11a þ He11b),
acetate, 8:2); [
a
]
¼ 7.51ꢀ (c ¼ 0.38, CHCl3); IR (KBr):
n
¼ 2944vs,
D
2874s, 1654s, 1518m, 1454s, 1388m, 1362m, 1198m, 1114vs, 1030m,
698 m cmꢁ1
;
1H NMR (500 MHz, CDCl3):
d
¼ 7.34e7.30 (m, 2H,
He40), 7.28e7.23 (m, 3 H, He39 þ He41), 6.17 (dd, J ¼ 6.2, 4.6 Hz,
1 H, NH), 5.30 (dd, J ¼ 3.5, 3.5 Hz, 1 H, He12), 4.62 (dd, J ¼ 14.7,
6.3 Hz, 1 H, He37a), 4.14 (dd, J ¼ 14.7, 4.3 Hz, 1 H, He37b),
1.80e1.71 (m,
2
H, He22a
þ
He19a), 1.70e1.52 (m,
5
H,
4.09e4.05
He31b
(m,
1H,
He31a),
He34b),
3.74e3.64
3.56e3.47
(m,
(m,
3H,
4H,
He16b þ He9 þ He22b þ He15a þ He6a), 1.49e1.32 (m, 3 H,
þ
He34a
þ
He7a He6b He21a), 1.30e1.13 (m, H,
þ
þ
3
He32a þ He32b þ He35a þ He35b), 3.43 (ddd, J ¼ 11.4, 9.6, 4.5 Hz,
1 H, He2), 3.37 (s, 3 H, He33), 3.36 (s, 3 H, He36), 2.75 (d, J ¼ 9.5 Hz,
1 H, He3), 2.54 (dd, J ¼ 13.1, 4.2 Hz, 1 H, He18), 2.03e1.94 (m, 2 H,
He7b þ He21b þ He19b), 1.15 (s, 3 H, He27), 1.07e1.02 (m, 1 H,
He15b), 1.06 (s, 3 H, He24), 0.95e0.87 (m, 1 H, He1b), 0.93 (s, 3 H,
He25), 0.90 (s, 6 H, He29 þ He30), 0.83 (s, 3 H, He23), 0.81 (dd,
J ¼ 11.7, 1.9 Hz, 1 H, He5), 0.66 (s, 3 H, H26); 13C NMR (125 MHz,
He1a
He11a
þ
He16a),
He22a
1.92e1.71
(m,
4
H,
H,
þ
He11b
þ
þ
He19a), 1.70e1.48 (m, 5
CDCl3):
d
¼ 178.1 (Ce28), 145.1 (Ce13), 138.6 (Ce34), 128.8 (Ce36),
He16b þ He9 þ He22b þ He15a þ He6a), 1.47e1.28 (m, 3 H,
127.9 (Ce35), 127.5 (Ce37), 122.8 (Ce12), 96.8 (Ce31), 81.5 (Ce3),
78.7 (Ce2), 55.8 (Ce32), 55.0 (Ce5), 47.6 (Ce9), 46.8 (Ce19), 46.5
(Ce17), 44.7 (Ce1), 43.7 (Ce33), 42.5 (Ce18), 42.3 (Ce14), 39.6
(Ce8), 39.3 (Ce4), 38.2 (Ce10), 34.3 (Ce21), 33.1 (Ce30), 32.8
(Ce22), 32.4 (Ce7), 30.9 (Ce20), 29.0 (Ce24), 27.4 (Ce15), 25.9
(Ce27), 24.0 (Ce16), 23.8 (Ce29), 23.7 (Ce11), 18.3 (Ce6), 17.1
(Ce26), 17.1 (Ce23), 16.7 (Ce25) ppm; MS (ESI): m/z (%) ¼ 606.4
([M þ H]þ,100), 628.5 ([M þ Na]þ, 4),1211.3 ([2M þ H]þ, 94),1233.5
([2M þ Na]þ, 63); analysis calculated for C39H59NO4 (605.89): C
77.31, H 9.82, N 2.31; found: C 77.08, H 9.97, N 2.03; 10: white solid;
mp 90e92 ꢀC; RF ¼ 0.32 (silica gel, hexane/ethyl acetate, 8:2);
He7a He6b He21a), 1.28e1.14 (m, H,
þ
þ
3
He7b þ He21b þ He19b), 1.13 (s, 3 H, He27), 1.06e1.00 (m, 1 H,
He15b), 0.99 (s, 3 H, He24), 0.91 (s, 3 H, He30), 0.90 (s, 3 H, He29),
0.89 (s, 3 H, He25), 0.88e0.82 (m, 1 H, He1b), 0.81 (s, 3 H, He23),
0.76 (dd, J ¼ 11.5, 1.6 Hz, 1 H, He5), 0.64 (s, 3 H, He26); 13C NMR
(125 MHz, CDCl3):
d
¼ 178.1 (Ce28), 145.0 (Ce13), 138.6 (Ce38),
128.8 (Ce40), 127.9 (Ce39), 127.5 (Ce41), 122.9 (Ce12), 91.4 (Ce3),
78.6 (Ce2), 73.5 (Ce31), 72.7 (Ce32), 72.7 (Ce35), 69.5 (Ce34), 59.1
(Ce33), 59.1 (Ce36), 55.3 (Ce5), 47.6 (Ce9), 46.8 (Ce19), 46.5
(Ce17), 44.9 (Ce1), 43.7 (Ce37), 42.5 (Ce18), 42.2 (Ce14), 40.5
(Ce4), 39.5 (Ce8), 37.9 (Ce10), 34.3 (Ce21), 33.1 (Ce30), 32.8
(Ce22), 32.4 (Ce7), 30.9 (Ce20), 28.8 (Ce24), 27.4 (Ce15), 25.9
(Ce27), 24.0 (Ce16), 23.8 (Ce29), 23.7 (Ce11), 18.4 (Ce6), 17.7
(Ce23), 17.1 (Ce26), 16.6 (Ce25) ppm; MS (ESI): m/z (%) ¼ 678.3
([M þ H]þ, 100), 1355.3 ([2M þ H]þ, 20); analysis calculated for
[
a
]
¼ 53.31ꢀ (c ¼ 0.41, CHCl3); IR (KBr):
n
¼ 2946vs, 1646s, 1518s,
D
1454s, 1390m, 1364m, 1212m, 1198m, 1148s, 1106s, 1030vs, 918s,
698 m cmꢁ1
;
1H NMR (500 MHz, CDCl3):
d
¼ 7.34e7.30 (m, 2H,
He38), 7.29e7.23 (m, 3 H, He37 þ He39), 6.17 (dd, J ¼ 6.1, 4.6 Hz,
1 H, NH), 5.30 (dd, J ¼ 3.6, 3.6 Hz, 1 H, He12), 4.89 (d, J ¼ 6.5 Hz, 1 H,
He33a), 4.72 (d, J ¼ 6.4 Hz, 1 H, He33b), 4.71 (d, J ¼ 6.7 Hz, 1 H,
He31a), 4.67 (d, J ¼ 6.7 Hz, 1 H, He31b), 4.61 (dd, J ¼ 14.7, 6.3 Hz,
1 H, He35a), 4.14 (dd, J ¼ 14.7, 4.4 Hz, 1 H, He35b), 3.68 (ddd,
J ¼ 11.3, 9.6, 4.5 Hz, 1 H, He2), 3.42 (s, 3 H, He34), 3.36 (s, 3 H,
He32), 2.98 (d, J ¼ 9.6 Hz, 1 H, He3), 2.54 (dd, J ¼ 13.0, 4.1 Hz, 1 H,
He18), 2.03e1.95 (m, 2 H, He1a þ He16a), 1.92e1.80 (m, 2 H,
He11a þ He11b), 1.80e1.71 (m, 2 H, He22a þ He19a), 1.70e1.51
(m, 5 H, He16b þ He9 þ He22b þ He15a þ He6a), 1.49e1.30 (m,
C43H67NO5 (678.00): C 76.17, H 9.96, N 2.07; found: C 75.88, H 10.11,
N 1.93.
4.2.4. 2a,3b-Methylenedioxy-olean-12-en-28-oic acid benzylamide
(12)
To a solution of 6 (50 mg, 0.09 mmol) in dimethoxymethane
(10 mL) p-TsOH (3 mg, 0.02 mmol) was added followed by micro-
wave irradiation (120 ꢀC, 2 h). The mixture was diluted with Et2O
(100 mL), and the organic layer was washed with water
(2 ꢂ 100 mL) and brine (1 ꢂ 50 mL), dried (MgSO4), filtrated, and
the filtrate was concentrated in vacuo. The residue was subjected to
column chromatography (silica gel, hexane/ethyl acetate, 8:2) to
provide 12 (40 mg, 78%) as a white solid; mp 113e116 ꢀC; RF ¼ 0.41
3
H, He7a
þ
He6b
þ
He21a), 1.29e1.13 (m,
3
H,
He7b þ He21b þ He19b), 1.14 (s, 3 H, He27), 1.07e1.01 (m, 1 H,
He15b), 1.03 (s, 3 H, He24), 0.93e0.85 (m, 1 H, He1b), 0.93 (s, 3 H,
He25), 0.90 (s, 6 H, He29 þ He30), 0.84 (s, 3 H, He23), 0.80 (dd,
J ¼ 11.6, 1.7 Hz, 1 H, He5), 0.65 (s, 3 H, He26); 13C NMR (125 MHz,
(silica gel, hexane/ethyl acetate, 8:2); [
CHCl3); IR (KBr):
1364m, 1102s, 1018
¼ 7.35e7.31 (m, 2H, He35), 7.29e7.23 (m, 3 H, He34 þ He36),
a
]
¼ 15.08ꢀ (c ¼ 0.21,
D
CDCl3):
d
¼ 178.1 (Ce28), 145.1 (Ce13), 138.5 (Ce36), 128.8 (Ce38),
n
¼ 2944vs, 2868s, 1644s, 1518m, 1454m, 1386m,
127.9 (Ce37), 127.5 (Ce39), 122.8 (Ce12), 99.4 (Ce33), 96.4 (Ce31),
89.5 (Ce3), 76.2 (Ce2), 56.3 (Ce34), 55.5 (Ce32), 55.3 (Ce5), 47.6
(Ce9), 46.8 (Ce19), 46.5 (Ce17), 45.6 (Ce1), 43.7 (Ce35), 42.5
(Ce18), 42.2 (Ce14), 39.8 (Ce4), 39.5 (Ce8), 37.9 (Ce10), 34.3
(Ce21), 33.1 (Ce30), 32.8 (Ce22), 32.5 (Ce7), 30.9 (Ce20), 28.9
(Ce24), 27.4 (Ce15), 25.9 (Ce27), 24.0 (Ce16), 23.8 (Ce29), 23.7
(Ce11), 18.5 (Ce6), 17.8 (Ce23), 17.1 (Ce26), 16.6 (Ce25) ppm; MS
(ESI): m/z (%) ¼ 650.3 ([M þ H]þ, 100), 672.4 ([M þ Na]þ, 8), 1299.3
([2M þ H]þ, 42), 1321.3 ([2M þ Na]þ, 38); analysis calculated for
m ;
cmꢁ1 1H NMR (500 MHz, CDCl3):
d
6.16 (dd, J ¼ 6.1, 4.6 Hz, 1 H, NH), 5.31 (dd, J ¼ 3.6, 3.6 Hz, 1 H, He12),
5.08 (dd, J ¼ 10.3, 1.0 Hz, 2 H, He31a þ He31b), 4.63 (dd, J ¼ 14.7,
6.4 Hz,1 H, He32a), 4.14 (dd, J ¼ 14.7, 4.4 Hz,1 H, He32b), 3.54 (ddd,
J ¼ 11.7, 9.4, 4.0 Hz, 1 H, He2), 2.89 (d, J ¼ 9.4 Hz, 1 H, He3), 2.55 (dd,
J ¼ 13.1, 3.9 Hz,1 H, He18), 2.10 (dd, J ¼ 11.5, 4.0 Hz,1 H, He1a), 2.00
(ddd, J ¼ 13.7, 13.7, 3.8 Hz, 1 H, He16a), 1.91e1.85 (m, 2 H,
He11a þ He11b), 1.80e1.72 (m, 2 H, He22a þ He19a), 1.72e1.55
(m, 5 H, He16b þ He9 þ He22b þ He15a þ He6a), 1.48e1.32 (m,
C
41H63NO5 (649.94): C 75.77, H 9.77, N 2.16; found: C 75.52, H 9.91,
N 2.04.
3
H, He7a
þ
He6b
þ
He21a), 1.32e1.14 (m,
3
H,
He7b þ He21b þ He19b), 1.16 (s, 3 H, He27), 1.08 (s, 3 H, He24),
1.07e1.01 (m, 2 H, He15b þ He1b), 0.97 (s, 3 H, He25), 0.91 (s, 3 H,
He30), 0.91 (s, 3 H, He29), 0.89 (s, 3 H, He23), 0.83e0.79 (m, 1 H,
4.2.3. 2a,3b-Dimethoxyethoxy-olean-12-en-28-oic acid
benzylamide (11)
To an ice-cold solution of 6 (120 mg, 0.21 mmol) in dry THF
(5 mL) sodium hydride (15 mg, 0.63 mmol) was added. The mixture
was stirred for 30 min at 25 ꢀC, and 1-bromo-2-methoxyethane
He5), 0.68 (s, 3 H, He26); 13C NMR (125 MHz, CDCl3):
d
¼ 178.1
(Ce28), 145.2 (Ce13), 138.6 (Ce33), 128.8 (Ce35), 127.9 (Ce34),
127.5 (Ce36), 122.7 (Ce12), 95.5 (Ce31), 89.1 (Ce3), 73.4 (Ce2),
56.2 (Ce5), 48.0 (Ce9), 46.8 (Ce19), 46.5 (Ce17), 43.7 (Ce34), 42.4
(80
m
L, 0.85 mmol) was added. Stirring at 25 ꢀC was continued

S. Sommerwerk et al. / European Journal of Medicinal Chemistry 119 (2016) 1e16
9
(Ce18), 42.3 (Ce14), 41.8 (Ce1), 40.3 (Ce4), 39.9 (Ce8), 37.8 (Ce10),
34.3 (Ce21), 33.1 (Ce30), 32.8 (Ce22), 32.7 (Ce7), 30.9 (Ce20), 28.8
(Ce24), 27.5 (Ce15), 26.0 (Ce27), 23.9 (Ce16), 23.9 (Ce11), 23.8
(Ce29), 17.9 (Ce6), 17.4 (Ce26), 17.1 (Ce25), 16.4 (Ce23) ppm; MS
(ESI): m/z (%) ¼ 574.4 ([M þ H]þ, 100), 596.5 ([M þ Na]þ, 7), 1147.3
([2M þ H]þ, 72), 1169.5 ([2M þ Na]þ, 82); analysis calculated for
1.9 Hz, 1 H, He1a), 1.98 (ddd, J ¼ 13.7, 13.7, 3.8 Hz, 1 H, He16a),
1.89e1.84 (m, H, He11a He11b), 1.81e1.72 (m, H,
2
þ
2
He22a þ He19a), 1.71e1.51 (m, 3 H, He16b þ He22b þ He15a),
1.47e1.32 (m, 5 H, He9 þ He6a þ He7a þ He6b þ He21a),
1.28e1.14 (m, 4 H, He7b þ He21b þ He1b þ He19b), 1.13 (s, 3 H,
He27), 1.08 (s, 3 H, He24), 1.07e1.01 (m, 1 H, He15b), 1.03 (s, 3 H,
He25), 1.00 (s, 3 H, He23), 0.91 (s, 6 H, He30 þ He29), 0.73e0.69
(m, 1 H, He5), 0.67 (s, 3 H, He26); 13C NMR (125 MHz, CDCl3):
C
38H55NO3 (573.85): C 79.53, H 9.66, N 2.44; found: C 79.37, H 9.82,
H 2.29.
d
¼ 178.1 (Ce28), 144.9 (Ce13), 138.6 (Ce32), 128.8 (Ce34), 127.9
4.2.5. 2
benzylamide (13)
a
,3
b
-(Isopropylidenedioxy)-olean-12-en-28-oic acid
(Ce33), 127.5 (Ce35), 123.1 (Ce12), 61.1 (Ce3), 54.4 (Ce2), 52.2
(Ce5), 47.8 (Ce9), 46.9 (Ce19), 46.6 (Ce17), 43.7 (Ce31), 42.6
(Ce18), 42.4 (Ce14), 39.7 (Ce8), 38.5 (Ce1), 36.9 (Ce10), 34.3
(Ce21), 33.2 (Ce30), 32.8 (Ce22), 32.5 (Ce4), 32.1 (Ce7), 30.9
(Ce20), 30.4 (Ce24), 27.4 (Ce15), 25.8 (Ce27), 24.0 (Ce16), 23.8
(Ce29), 23.6 (Ce11), 20.7 (Ce23), 19.8 (Ce6), 16.9 (Ce26), 16.8
(Ce25) ppm; MS (ESI): m/z (%) ¼ 544.3 ([M þ H]þ, 98), 566.5
([M þ Na]þ, 14), 1087.3 ([2M þ H]þ, 94), 1109.3 ([2M þ Na]þ, 100);
analysis calculated for C37H53NO2 (543.82): C 81.72, H 9.82, N 2.58;
found: C 81.56, H 9.93, N 2.41.
p-TsOH (3 mg, 0.02 mmol) was added to a solution of compound
6 (100 mg, 0.18 mmol) in 2,2-dimethoxypropane (10 mL). After
10 min of stirring at room temperature, Et2O (100 mL) was added,
and the organic layer was washed with water (2 ꢂ 100 mL) and
brine (1 ꢂ 50 mL), dried (MgSO4), filtrated, and the filtrate was
evaporated to dryness followed by column chromatography (silica
gel, hexane/ethyl acetate, 8:2) of the residue to afford 13 (98 mg,
91%) as a white solid; mp 122e123 ꢀC; RF ¼ 0.45 (silica gel, hexane/
ethyl acetate, 8:2); [
a]
¼ 29.29ꢀ (c ¼ 0.38, CHCl3); IR (KBr):
D
n
¼ 2944vs, 2866s, 1660s, 1514s, 1454s, 1368s, 1230s, 1170m, 1004m,
4.2.7. 2a,3b-Bis(chloroacetyloxy)-olean-12-en-28-oic acid
benzylamide (15) (general procedure A, GP A)
1070m, 1054s, 854m, 698 m cmꢁ1
;
1H NMR (500 MHz, CDCl3):
d
¼ 7.35e7.31 (m, 2H, He37), 7.29e7.23 (m, 3 H, He36 þ He38), 6.16
To a solution of compound 6 (200 mg, 0.36 mmol) in dry DCM
(20 mL), chloroacetic anhydride (246 mg,1.44 mmol), triethylamine
(164 mg, 1.62 mmol) and DMAP (5 mg, 0.04 mmol) were added, and
the mixture was stirred at room temperature for 20 h. Et2O
(100 mL) was added, and the organic layer was washed with diluted
(dd, J ¼ 6.2, 4.6 Hz,1 H, NH), 5.30 (dd, J ¼ 3.6, 3.6 Hz,1 H, He12), 4.63
(dd, J ¼ 14.7, 6.4 Hz, 1 H, He34a), 4.13 (dd, J ¼ 14.7, 4.3 Hz, 1 H,
He34b), 3.69 (ddd, J ¼ 11.7, 9.4, 4.0 Hz, 1 H, He2), 3.03 (d, J ¼ 9.4 Hz,
1 H, He3), 2.54 (dd, J ¼ 12.6, 4.2 Hz, 1 H, He18), 2.04e1.96 (m, 2 H,
He1a þ He16a), 1.89e1.83 (m, 2 H, He11a þ He11b), 1.79e1.72 (m,
HCl (0.1
M
, 1 ꢂ 100 mL), water (2 ꢂ 100 mL) and brine (1 ꢂ 50 mL),
2
H,
He22a
þ
He19a),
1.72e1.54
(m,
5
H,
dried (MgSO4), filtrated, and the filtrate was concentrated in vacuo.
The residue was subjected to column chromatography (silica gel,
hexane/ethyl acetate, 8:2) to provide 15 (234 mg, 92%) as a white
solid; mp 116e119 ꢀC; RF ¼ 0.38 (silica gel, hexane/ethyl acetate
He16b þ He9 þ He22b þ He15a þ He6a), 1.48e1.33 (m, 3 H,
He7a þ He6b þ He21a), 1.42 (s, 3 H, He32), 1.40 (s, 3 H, He33),
1.31e1.14 (m, 3 H, He7b þ He21b þ He19b), 1.15 (s, 3 H, He27),
1.06e0.98 (m, 2 H, He15b þ He1b), 1.04 (s, 3 H, He24), 0.97 (s, 3 H,
He25), 0.91 (s, 3 H, He30), 0.91 (s, 3 H, He29), 0.88 (s, 3 H, He23),
0.82e0.78 (m, 1 H, He5), 0.68 (s, 3 H, He26); 13C NMR (125 MHz,
8:2); [
a
]
¼ ꢁ5.04ꢀ (c ¼ 0.28, CHCl3); IR (KBr):
n
¼ 3432m, 2948s,
D
1744s, 1650m, 1517m, 1454s, 1396w, 1309s, 1264s, 1185s, 1025m,
1001s, 751s, 699 s cmꢁ1; 1H NMR (500 MHz, CDCl3):
d
¼ 7.40e7.20
CDCl3):
d
¼ 178.1 (Ce28), 145.2 (Ce13), 138.6 (Ce35), 128.8 (Ce37),
(m, 5 H, He37 þ He38 þ He39), 6.16 (dd, J ¼ 5.2, 5.2 Hz, 1 H, NH),
5.31 (dd, J ¼ 3.2, 3.2 Hz, 1 H, He12), 5.18 (ddd, J ¼ 11.2, 11.0, 4.5 Hz,
1 H, He2), 4.86 (d, J ¼ 10.3 Hz, 1 H, He3), 4.61 (dd, J ¼ 14.6, 6.1 Hz,
1 H, He35a), 4.17 (dd, J ¼ 14.6, 4.4 Hz, 1 H, He35b), 4.05 (s, 1 H,
He32a), 4.05 (s, 1 H, He32b), 3.96 (s, 2 H, He34a þ He34b), 2.57
(dd, J ¼ 12.7, 3.3 Hz, 1 H, He18), 2.12e2.03 (m, 1 H, He1a),
2.03e1.93 (m, 1 H, He16a), 1.87e1.80 (m, 2 H, He11a þ He11b),
127.9 (Ce36), 127.5 (Ce38), 122.8 (Ce12), 108.4 (Ce31), 88.8 (Ce3),
72.6 (Ce2), 56.2 (Ce5), 48.0 (Ce9), 46.8 (Ce19), 46.5 (Ce17), 43.7
(Ce34), 42.5 (Ce18), 42.3 (Ce14), 42.0 (Ce1), 40.1 (Ce4), 39.9
(Ce8), 37.5 (Ce10), 34.3 (Ce21), 33.1 (Ce30), 32.8 (Ce22), 32.7
(Ce7), 30.9 (Ce20), 28.7 (Ce24), 27.5 (Ce15), 27.3 (Ce32), 27.0
(Ce33), 26.0 (Ce27), 23.9 (Ce16), 23.9 (Ce11), 23.8 (Ce29), 17.9
(Ce6), 17.4 (Ce26), 17.2 (Ce25), 16.3 (Ce23) ppm; MS (ESI): m/z
(%) ¼ 602.4 ([M þ H]þ, 68), 624.5 ([M þ Na]þ, 9), 1203.3 ([2M þ H]þ,
50), 1225.5 ([2M þ Na]þ, 100); analysis calculated for C40H59NO3
(601.90): C 79.82, H 9.88, N 2.33; found: C 79.77, H 9.93, N 2.18.
1.81e1.53
(m,
7
H,
He19a þ He22a þ He16b þ He22b þ He9 þ He15a þ He6a),
1.53e1.18 (m, 5 H, He7a þ He6b þ He21a þ He7b þ He21b),
1.17e1.12 (m,
4
H, He19b
þ
He27), 1.12e1.05 (m, 2 H,
He1b þ He15b), 1.03 (s, 3 H, He25), 1.01e0.96 (m, 1 H, He5), 0.94
4.2.6. 2b,3b-Epoxy-olean-12-en-28-oic acid benzylamide (14)
(s, 3 H, He23), 0.94 (s, 3 H, He24), 0.92 (s, 3 H, He30), 0.92 (s, 3 H,
Compound 6 (100 mg, 0.18 mmol) was dissolved in DMF (1 mL),
and PPh3 (140 mg, 0.53 mmol) was added. The reaction mixture
was stirred at 0 ꢀC for 1 h, and a solution of diethyl azodicarboxylate
(93 mg, 0.53 mmol) in DMF (0.2 mL) was slowly added. This
mixture was heated under reflux for 2 h, and the solvent was
removed under reduced pressure. Et2O (100 mL) was added, and
aqueous work-up as described above followed by chromatography
(silica gel, hexane/ethyl acetate, 8:2) gave 14 (63 mg, 65%) as a
white solid; mp 105e106 ꢀC; RF ¼ 0.47 (silica gel, hexane/ethyl
He29), 0.66 (s, 3 H, He26); 13C NMR (125 MHz, CDCl3):
d
¼ 177.9
(Ce28), 167.3 (Ce31), 167.0 (Ce33), 145.1 (Ce13), 138.5 (Ce36),
128.8 (Ce38), 127.9 (Ce37), 127.5 (Ce39), 122.1 (Ce12), 82.3 (Ce3),
72.2 (Ce2), 54.9 (Ce5), 47.5 (Ce9), 46.7 (Ce19), 46.4 (Ce17), 43.7
(Ce1), 43.7 (Ce35), 42.4 (Ce18), 42.2 (Ce14), 41.0 (Ce32), 40.9
(Ce34), 39.7 (Ce4), 39.5 (Ce8), 38.3 (Ce10), 34.2 (Ce21), 33.1
(Ce30), 32.8 (Ce22), 32.2 (Ce7), 30.9 (Ce20), 28.4 (Ce24), 27.4
(Ce15), 25.8 (Ce27), 23.8 (Ce16), 23.7 (Ce29), 23.6 (Ce11), 18.2
(Ce6), 17.6 (Ce23), 17.0 (Ce26), 16.5 (Ce25) ppm; MS (ESI): m/z
(%) ¼ 714.3 ([M þ H]þ, 100), 736.1 ([M þ Na]þ, 26), 1429.9
([2M þ H]þ, 32), 1451.3 ([2M þ Na]þ, 52); analysis calculated for
acetate, 8:2); [
a
]
D
¼ 56.30ꢀ (c ¼ 0.32, CHCl3); IR (KBr):
n
¼ 2950vs,
2868s, 1642s, 1520s, 1454m, 1386m, 1364m, 1280w, 1286w, 1240w,
1216w, 1184w, 840m, 698 m cmꢁ1
;
1H NMR (500 MHz, CDCl3):
C41H57Cl2NO5 (714.80): C 68.89, H 8.04, N 1.96; found: C 68.64, H
d
¼ 7.35e7.31 (m, 2H, He34), 7.29e7.23 (m, 3 H, He33 þ He35), 6.17
7.87, N 1.79.
(dd, J ¼ 6.1, 4.7 Hz,1 H, NH), 5.32 (dd, J ¼ 3.6, 3.6 Hz,1 H, He12), 4.62
(dd, J ¼ 14.7, 6.4 Hz, 1 H, He31a), 4.12 (dd, J ¼ 14.7, 4.3 Hz, 1 H,
He31b), 3.23 (ddd, J ¼ 3.9, 1.9, 1.9 Hz, 1 H, He2), 2.82 (d, J ¼ 4.0 Hz,
1 H, He3), 2.53 (dd, J ¼ 13.1, 4.0 Hz, 1 H, He18), 2.12 (dd, J ¼ 14.9,
4.2.8. 2,3-Seco-olean-12-en-2,3-dial-28-oic acid benzylamide (16)
Compound 6 (100 mg, 0.18 mmol) was dissolved in DCM (30 mL)
and silica supported sodium periodate (silica gel, 320 mg; sodium

10
S. Sommerwerk et al. / European Journal of Medicinal Chemistry 119 (2016) 1e16
periodate, 80 mg, 0.37 mmol) was added. The suspension was
stirred for 2 min at 25 ꢀC. Water (30 mL) was added, and the organic
layer was washed with water (2 ꢂ 30 mL) and brine (1 ꢂ 20 mL),
dried (MgSO4), filtrated, and the filtrate evaporated to dryness to
yield 16 (98 mg, 98%) as white solid; mp 127e129 ꢀC; RF ¼ 0.24
[
a
]
¼ þ75.98ꢀ (c ¼ 0.50, CHCl3); IR (KBr):
n
¼ 2950s, 2876m,
D
1746vs, 1696s, 1462m, 1434m, 1368s, 1252s, 1244vs, 1166m, 1122w,
1030 s cmꢁ1; 1H NMR (500 MHz, CDCl3):
d
¼ 5.27 (dd, J ¼ 3.5, 3.5 Hz,
1 H, He12), 4.99 (d, J ¼ 6.5 Hz, 1 H, He3), 4.94 (ddd, J ¼ 6.2, 6.2,
5.1 Hz, 1 H, He2), 2.82 (dd, J ¼ 13.6, 4.1 Hz, 1 H, He18), 2.06 (s, 3 H,
(silica gel, hexane/ethyl acetate, 8:2); [
a
]
¼ 51.38ꢀ (c ¼ 0.33,
He32), 2.01 (s,
3
H, He34), 2.02e1.65 (m,
6
H,
D
CHCl3); IR (KBr):
n
¼ 3418s, 2950vs, 2872s, 2712w, 1716s, 1642s,
He12a þ He11a þ He11b þ He22a þ He15a þ He1a), 1.65e1.51
(m, 5 H, He19a þ He16b þ He1b þ He22b þ He9), 1.51e1.25 (m,
7 H, He6a þ He6b þ He7a þ He7b þ He21a þ He21b þ He5),
1.17e1.03 (m, 2 H, He19b þ He15b), 1.15 (s, 3 H, He27), 1.13 (s, 3 H,
He25), 1.00 (s, 3 H, He23), 0.92 (s, 3 H, He29), 0.90 (s, 6 H,
He24 þ He30), 0.74 (s, 3 H, He26) ppm; 13C NMR (125 MHz,
1518s, 1454m, 1386m, 1364m, 1030m, 698 m cmꢁ1ꢁ1
;
1H NMR
(500 MHz, CDCl3)
d
¼ 9.80 (t, J ¼ 2.2 Hz, 1 H, He2), 9.60 (s, 1 H,
He3), 7.34e7.30 (m, 2H, He34), 7.28e7.22 (m, 3 H, He33 þ He35),
6.12 (dd, J ¼ 5.8, 5.0 Hz,1 H, NH), 5.29 (dd, J ¼ 3.6, 3.6 Hz,1 H, He12),
4.57 (dd, J ¼ 14.7, 6.2 Hz, 1 H, He31a), 4.17 (dd, J ¼ 14.7, 4.6 Hz, 1 H,
He31b), 2.58 (dd, J ¼ 13.1, 4.0 Hz, 1 H, He18), 2.50 (dd, J ¼ 17.8,
2.4 Hz, 1 H, He1a), 2.30 (dd, J ¼ 17.8, 2.1 Hz, 1 H, He1b), 2.21 (dd,
J ¼ 10.4, 7.2 Hz, 1 H, He9), 2.10 (dd, J ¼ 8.4, 5.3 Hz, 1 H, He5), 1.99
(ddd, J ¼ 13.7, 13.7, 3.9 Hz, 1 H, He16a), 1.91e1.81 (m, 2 H,
CDCl3):
d
¼ 184.2 (Ce28), 170.4 (Ce31), 170.1 (Ce33), 143.7 (Ce13),
122.6 (Ce12), 76.2 (Ce3), 70.7 (Ce2), 50.6 (Ce5), 48.1 (Ce9), 46.7
(Ce17), 46.0 (Ce19), 41.9 (Ce14), 41.2 (Ce1), 41.1 (Ce18), 39.6
(Ce8), 37.1 (Ce4), 36.7 (Ce10), 34.0 (Ce21), 33.2 (Ce30), 32.6
(Ce22), 32.3 (Ce7), 30.8 (Ce20), 27.7 (Ce15), 26.9 (Ce24), 26.2
(Ce27), 23.7 (Ce29), 23.5 (Ce11), 23.0 (Ce16), 22.2 (Ce23), 21.4
(Ce34), 21.1 (Ce32), 18.8 (Ce6), 18.2 (Ce25), 17.1 (Ce26) ppm; MS
(ESI): m/z (%) ¼ 555.4 ([MꢁH]-, 100), 1111.3 ([2MꢁH]-, 100), 1133.6
([2M-2H þ Na]-, 34); analysis calculated for C34H52O6 (556.77): C
73.34, H 9.41; found: C 73.25, H 9.53.
He11a
He19a
He15a
þ
He11b),
1.77e1.59
(m,
4
H,
H,
H,
þ
þ
He22a
He6a
þ
He22b
He6b
þ
He16b), 1.58e1.47 (m,
He7a), 1.39e1.14 (m,
4
4
þ
þ
He21a þ He21b þ He7b þ He19b), 1.14 (s, 3 H, He27), 1.12 (s, 3 H,
He24), 1.10e1.05 (m, 1 H, He15b), 1.09 (s, 3 H, He23), 0.95 (s, 3 H,
He25), 0.90 (s, 6 H, He29 þ He30), 0.68 (s, 3 H, He26); 13C NMR
(125 MHz, CDCl3):
d
¼ 206.9 (Ce3), 202.5 (Ce2), 177.9 (Ce28), 144.8
(Ce13), 138.5 (Ce32), 128.8 (Ce34), 127.9 (Ce33), 127.5 (Ce35),
122.3 (Ce12), 52.2 (Ce1), 50.9 (Ce4), 49.2 (Ce5), 46.6 (Ce19), 46.5
(Ce17), 43.7 (Ce31), 43.2 (Ce10), 42.6 (Ce14), 42.3 (Ce18), 41.2
(Ce9), 39.5 (Ce8), 34.2 (Ce21), 33.1 (Ce30), 32.9 (Ce22), 31.9
(Ce7), 30.8 (Ce20), 27.4 (Ce15), 25.4 (Ce27), 24.2 (Ce24), 24.0
(Ce16), 23.9 (Ce11), 23.7 (Ce29), 20.3 (Ce6), 19.9 (Ce23), 19.5
(Ce25), 17.0 (Ce26) ppm; MS (ESI): m/z (%) ¼ 560.3 ([M þ H]þ, 100),
1119.3 ([2M þ H]þ, 46); analysis calculated for C37H53NO3 (559.82):
C 79.38, H 9.54, N 2.50; found: C 79.21, H 9.66, N 2.36.
4.2.11. 2a,3a-Diacetyloxy-olean-12-en-28-oic acid (23)
Compound 23 (180 mg, 76%) was obtained from 19 and acetic
anhydride according to GP A as a white solid; mp ¼ 151e155 ꢀC;
RF ¼ 0.18 (silica gel, hexane/ethyl acetate, 8:2); [
a]
¼ þ28.78ꢀ
D
(c ¼ 0.36, CHCl3); IR (KBr): ¼ 2958s, 2876m, 1748vs, 1698s, 1462m,
n
1434w, 1366m, 1250s, 1234s, 1158m, 1036m, 1018 m cmꢁ1; 1H NMR
(500 MHz, CDCl3):
d
¼ 5.28 (dd, J ¼ 3.6, 3.5 Hz,1 H, He12), 5.23 (ddd,
J ¼ 12.4, 4.5, 2.9 Hz,1 H, He2), 4.96 (d, J ¼ 2.5 Hz,1 H, He3), 2.83 (dd,
J ¼ 13.9, 4.5 Hz, 1 H, He18), 2.11 (s, 3 H, He32), 2.03e1.84 (m, 3 H,
He11a þ He11b þ He16a), 1.96 (s, 3 H, He34), 1.81e1.67 (m, 3 H,
4.2.9. 2
b
,3b
-Diacetyloxy-olean-12-en-28-oic acid (21)
He15a
He1a
He6a
þ
He22a
þ
He9), 1.66e1.53
He22b), 1.52e1.42 (m,
H, He1b
(m,
4
H,
H,
Compound 21 (185 mg, 78%) was obtained from 17 and acetic
anhydride according to GP A as a white solid; mp ¼ 322 ꢀC
(decomp.) (lit [28].: 148e150 ꢀC); RF ¼ 0.24 (silica gel, hexane/ethyl
þ
He19a
þ
He16b
þ
2
þ
He7a), 1.39e1.05 (m,
8
þ
He21a
þ He21b þ He6b þ He7b þ He5 þ He19b þ He15b), 1.17 (s, 3 H,
acetate, 8:2); [
a
]
¼ þ83.81ꢀ (c ¼ 0.32, CHCl3); IR (KBr):
n
¼ 2944s,
He27), 1.03 (s, 3 H, He25), 0.97 (s, 3 H, He23), 0.93 (s, 3 H, He29),
D
1744vs, 1698s, 1464w, 1436w, 1366m, 1252s, 1196m, 1160w, 1058m,
0.91 (s, 3 H, He30), 0.87 (s, 3 H, He24), 0.75 (s, 3 H, He26) ppm; 13
C
1030 m cmꢁ1
;
1H NMR (500 MHz, CDCl3):
d
¼ 5.33e5.30 (m, 1 H,
NMR (125 MHz, CDCl3):
d
¼ 183.7 (Ce28), 170.8 (Ce31), 170.6
He2), 5.27 (dd, J ¼ 3.6, 3.6 Hz, 1 H, He12), 4.62 (d, J ¼ 3.9 Hz, 1 H,
He3), 2.82 (dd, J ¼ 13.9, 4.5 Hz, 1 H, He18), 2.08 (s, 3 H, He32), 2.02
(s, 3 H, He34), 2.01e1.91 (m, 3 H, He1a þ He16a þ He11a),
1.89e1.69 (m, 3 H, He11b þ He22a þ He15a), 1.64e1.41 (m, 7 H,
He6a þ He6b þ He16b þ He19a þ He22b þ He9 þ He7a),
1.38e1.28 (m, 3 H, He7b þ He21a þ He1b), 1.24e1.11 (m, 2 H,
He21b þ He19b), 1.20 (s, 3 H, He25), 1.12 (s, 3 H, He27), 1.09e1.03
(m,1 H, He15b),1.04 (s, 3 H, He23),1.00e0.96 (m,1 H, He5), 0.92 (s,
3 H, He29), 0.90 (s, 3 H, He30), 0.90 (s, 3 H, He24), 0.76 (s, 3 H,
(Ce33), 143.8 (Ce13), 122.5 (Ce12), 77.3 (Ce3), 68.4 (Ce2), 49.8
(Ce5), 47.6 (Ce9), 46.7 (Ce17), 46.1 (Ce19), 41.8 (Ce14), 41.1
(Ce18), 39.6 (Ce8), 38.9 (Ce1), 38.7 (Ce4), 38.3 (Ce10), 34.0
(Ce21), 33.2 (Ce30), 32.6 (Ce22), 32.5 (Ce7), 30.8 (Ce20), 27.9
(Ce24), 27.8 (Ce15), 26.2 (Ce27), 23.7 (Ce29), 23.6 (Ce11), 23.0
(Ce16), 21.7 (Ce23), 21.3 (Ce34), 21.2 (Ce32), 18.0 (Ce6), 17.4
(Ce26), 16.3 (Ce25) ppm; MS (ESI): m/z (%) ¼ 557.1 ([M þ H]þ, 26),
574.2 ([M þ NH4]þ, 72), 579.2 ([M þ Na]þ, 28), 1135.3 ([2M þ Na]þ,
100); analysis calculated for C34H52O6 (556.77): C 73.34, H 9.41;
found: C 73.19, H 9.49.
He26) ppm; 13C NMR (125 MHz, CDCl3):
d
¼ 184.3 (Ce28), 170.9
(Ce33), 170.4 (Ce31), 143.9 (Ce13), 122.6 (Ce12), 78.1 (Ce3), 69.8
(Ce2), 55.4 (Ce5), 48.2 (Ce9), 46.7 (Ce17), 45.9 (Ce19), 42.0 (Ce1),
41.8 (Ce14), 41.1 (Ce18), 39.5 (Ce8), 37.5 (Ce4), 37.0 (Ce10), 33.9
(Ce21), 33.2 (Ce30), 32.6 (Ce22), 32.6 (Ce7), 30.8 (Ce20), 29.3
(Ce24), 27.7 (Ce15), 26.2 (Ce27), 23.7 (Ce29), 23.6 (Ce11), 22.9
(Ce16), 21.4 (Ce32), 21.0 (Ce34), 18.1 (Ce6), 17.8 (Ce23), 17.5
(Ce26), 16.1 (Ce25) ppm; MS (ESI): m/z (%) ¼ 557.2 ([M þH ]þ, 13),
574.3 ([M þ NH4]þ, 100), 579.4 ([M þ Na]þ, 21), 1249.1 ([2M þ Na]þ,
87); analysis calculated for C34H52O6 (556.77): C 73.34, H 9.41;
found: C 73.21, H 9.57.
4.2.12. 2b,3b-Diacetyloxy-olean-12-en-28-oic acid benzylamide
(24) (general procedure B, GP B)
To a solution of 21 (90 mg, 0.16 mmol) in dry DCM (10 mL),
oxalyl chloride (62 mg, 0.48 mmol), triethylamine (3 mg,
0.03 mmol) and DMF (2 mg, 0.03 mmol) were added. The mixture
was stirred at room temperature for 2 h. The solvent was removed
under reduced pressure, re-evaporated with dry THF (1 ꢂ 10 mL),
and the residue was immediately dissolved in dry DCM (10 mL). The
solution was cooled to 0 ꢀC, and triethylamine (21 mg, 0.21 mmol),
DMAP (2 mg, 0.02 mmol) and benzylamine (52 mg, 0.48 mmol)
were added. After 5 min of stirring, Et2O (100 mL) was added, and
usual aq. work-up followed by column chromatography (silica gel,
hexane/ethyl acetate, 8:2) afforded 24 (89 mg, 85%) as a white solid;
mp ¼ 120e122 ꢀC; RF ¼ 0.28 (silica gel, hexane/ethyl acetate, 8:2);
4.2.10. 2b,3a-Diacetyloxy-olean-12-en-28-oic acid (22)
Compound 22 (180 mg, 76%) was obtained from 18 and acetic
anhydride according to GP A as a white solid; mp ¼ 163e166 ꢀC (lit
[29].: 206e210 ꢀC); RF ¼ 0.23 (silica gel, hexane/ethyl acetate, 8:2);

S. Sommerwerk et al. / European Journal of Medicinal Chemistry 119 (2016) 1e16
11
[
a
]
¼ þ44.41ꢀ (c ¼ 0.35, CHCl3); IR (KBr):
n
¼ 2948s, 2878m,
1252vs, 1240s, 1036m, 1018 m cmꢁ1
;
1H NMR (500 MHz, CDCl3):
D
1746vs, 1654m, 1518m, 1454m, 1434m, 1364s, 1252vs, 1192m, 1160w,
d
¼ 7.35e7.31 (m, 2H, He38), 7.29e7.23 (m, 3 H, He37 þ He39), 6.17
1056m, 1030 s cmꢁ1; 1H NMR (500 MHz, CDCl3):
d
¼ 7.34e7.30 (m,
(dd, J ¼ 6.3, 4.5 Hz,1 H, NH), 5.31 (dd, J ¼ 3.6, 3.6 Hz,1 H, He12), 5.22
(ddd, J ¼ 12.4, 4.5, 2.8 Hz, 1 H, He2), 4.96 (d, J ¼ 2.3 Hz, 1 H, He3),
4.63 (dd, J ¼ 14.7, 6.3 Hz, 1 H, He35a), 4.14 (dd, J ¼ 14.7, 4.4 Hz, 1 H,
He35b), 2.55 (dd, J ¼ 13.1, 4.0 Hz, 1 H, He18), 2.10 (s, 3 H, He32),
2.00 (ddd, J ¼ 13.5, 13.5, 3.7 Hz, 1 H, He16a), 1.96 (s, 3 H, He34),
1.93e1.54 (m, 9 H, He11a þ He11b þ He19a þ He22a þ He22b þ
He9 þ He16b þ He1a þ He15a), 1.52e1.12 (m, 9 H, H e7a þ He7b
þ He6a þ He6b þ He21a þ He21b þ He1b þ He19b þ He5), 1.20
2H, He38), 7.28e7.23 (m, 3 H, He37 þ He39), 6.17 (dd, J ¼ 6.2,
4.7 Hz, 1 H, NH), 5.31e5.28 (m, 2 H, He2 þ He12), 4.62e4.57 (m,
2 H, He3 þ He35a), 4.15 (dd, J ¼ 14.7, 4.4 Hz, 1 H, He35b), 2.54 (dd,
J ¼ 13.0, 3.8 Hz, 1 H, He18), 2.04 (s, 3 H, He32), 2.03e1.92 (m, 2 H,
He1a
þ
He16a), 2.02 (s, 3 H, He34), 1.89e1.79 (m, 2 H,
He11a þ He11b), 1.79e1.71 (m, 2 H, He22a þ He19a), 1.70e1.44
(m, H,
7
He6a þ He6b þ He16b þ He15a þ He22b þ He9 þ He7a),
1.40e1.12 (m, 5 H, He1b þ He21a þ He21b þ He19b þ He7b), 1.14
(s, 6 H, He27 þ He25), 1.06e1.00 (m, 1 H, He15b), 1.05 (s, 3 H,
He23), 0.97e0.93 (m, 1 H, He5), 0.90 (s, 3 H, He30), 0.90 (s, 3 H,
He29), 0.89 (s, 3 H, He24), 0.68 (s, 3 H, He26) ppm; 13C NMR
(s,
He25 þ He23), 0.91 (s, 6 H, He29 þ He30), 0.87 (s, 3 H, He24),
0.67 (s, 3 H, He26) ppm; 13C NMR (125 MHz, CDCl3):
¼ 178.1
3 H, He27), 1.08e1.02 (m, 1 H, He15b), 0.99 (s, 6 H,
d
(Ce28), 170.7 (Ce31), 170.6 (Ce33), 145.1 (Ce13), 138.6 (Ce36),
128.8 (Ce38), 127.9 (Ce37), 127.5 (Ce39), 122.8 (Ce12), 77.3 (Ce3),
68.3 (Ce2), 49.7 (Ce5), 47.5 (Ce9), 46.9 (Ce19), 46.5 (Ce17), 43.8
(Ce35), 42.5 (Ce18), 42.3 (Ce14), 39.7 (Ce8), 38.9 (Ce1), 38.5
(Ce4), 38.3 (Ce10), 34.3 (Ce21), 33.2 (Ce30), 32.8 (Ce22), 32.3
(Ce7), 30.9 (Ce20), 27.9 (Ce24), 27.5 (Ce15), 26.1 (Ce27), 24.0
(Ce16), 23.8 (Ce29), 23.6 (Ce11), 21.7 (Ce23), 21.3 (Ce34), 21.1
(Ce32), 18.0 (Ce6), 17.2 (Ce26), 16.4 (Ce25) ppm; MS (ESI): m/z
(%) ¼ 646.4 ([M þ H]þ, 100), 668.3 ([M þ Na]þ, 10), 1291.2
([2M þ H]þ, 14), 1313.5 ([2M þ Na]þ, 26); analysis calculated for
(125 MHz, CDCl3):
d
¼ 178.1 (Ce28), 170.8 (Ce33), 170.3 (Ce31),
145.1 (Ce13), 138.5 (Ce36), 128.8 (Ce38), 127.9 (Ce37), 127.5
(Ce39), 122.7 (Ce12), 78.0 (Ce3), 69.7 (Ce2), 55.3 (Ce5), 48.1
(Ce9), 46.7 (Ce19), 46.5 (Ce17), 43.7 (Ce35), 42.4 (Ce18), 42.3
(Ce14), 42.0 (Ce1), 39.6 (Ce8), 37.4 (Ce4), 36.8 (Ce10), 34.3
(Ce21), 33.1 (Ce30), 32.8 (Ce22), 32.5 (Ce7), 30.8 (Ce20), 29.2
(Ce24), 27.3 (Ce15), 25.9 (Ce27), 23.9 (Ce16), 23.7 (Ce29), 23.6
(Ce11), 21.4 (Ce32), 21.0 (Ce34), 18.1 (Ce6), 17.8 (Ce23), 17.2
(Ce26),16.0 (Ce25) ppm; MS (ESI): m/z (%) ¼ 646.5 ([M þ H]þ,100),
668.5 ([M þ Na]þ, 12), 1291.3 ([2M þ H]þ, 45), 1313.6 ([2M þ Na]þ,
28); analysis calculated for C41H59NO5 (645.91): C 76.24, H 9.21, N
2.17; found: C 76.03, H 9.39, N 2.00.
C41H59NO5 (645.91): C 76.24, H 9.21, N 2.17; found: C 76.11, H 9.17, N
2.29.
4.2.15. 2a,3b-Diacetyloxy-olean-12-en-28-oic acid phenylamide
(27)
4.2.13. 2
b
,3
a-Diacetyloxy-olean-12-en-28-oic acid benzylamide
Compound 27 (95 mg, 84%) was obtained from 20 and aniline
(25)
(50 ml, 0.55 mmol) according to GP B as a white solid;
Compound 25 (85 mg, 81%) was obtained from 22 according to
mp ¼ 185e186 ꢀC; RF ¼ 0.40 (silica gel, hexane/ethyl acetate, 8:2);
GP B as a white solid; mp ¼ 114e116 ꢀC; RF ¼ 0.25 (silica gel,
[
a
]
¼
þ26.41ꢀ (c
¼
0.31, CHCl3); UVevis (CHCl3): lmax
D
hexane/ethyl acetate, 8:2); [
a
]
¼ þ36.43ꢀ (c ¼ 0.46, CHCl3); IR
(log ε) ¼ 247 nm (4.07); IR (KBr):
n
¼ 2948s, 2866m, 1744vs, 1680m,
D
(KBr):
n
¼ 2948s, 2874m,1744vs,1654s, 1518s, 1454m, 1434m, 1368s,
1598m, 1526m, 1500m, 1438s, 1368s, 1310m, 1252vs, 1176w, 1158w,
1246vs, 1230vs, 1030s, 698 m cmꢁ1
;
1H NMR (500 MHz, CDCl3):
¼ 7.35e7.30 (m, 2H, He38), 7.29e7.22 (m, 3 H, He37 þ He39),
1042m, 756 m cmꢁ1 1H NMR (500 MHz, CDCl3):
;
d
¼ 7.65 (s, 1 H,
d
NH), 7.48e7.45 (m, 2H, He36), 7.32e7.27 (m, 2H, He37), 7.09e7.06
(m, 1 H, He38), 5.53 (dd, J ¼ 3.5, 3.5 Hz, 1 H, He12), 5.08 (ddd,
J ¼ 11.5, 10.5, 4.7 Hz, 1 H, He2), 4.74 (d, J ¼ 10.3 Hz, 1 H, He3), 2.67
6.17 (dd, J ¼ 5.9, 4.8 Hz,1 H, NH), 5.30 (dd, J ¼ 3.5, 3.5 Hz, 1 H, He12),
4.98 (d, J ¼ 6.4 Hz, 1 H, He3), 4.92 (ddd, J ¼ 6.1, 6.1, 4.9 Hz, 1 H, He2),
4.61 (dd, J ¼ 14.6, 6.2 Hz, 1 H, He35a), 4.15 (dd, J ¼ 14.7, 4.4 Hz, 1 H,
He35b), 2.54 (dd, J ¼ 13.0, 3.7 Hz, 1 H, He18), 2.06 (s, 3 H, He32),
2.05e1.94 (m, 1 H, He16a), 2.02 (s, 3 H, He34),1.87e1.52 (m, 10 H, H
11a þ H 11b þ H 19a þ He22a þ He22b þ He1a þ He1b þ He16b
(dd,
J
¼
12.5, 3.7 Hz, 1 H, He18), 2.12e1.90 (m, 4 H,
He16a þ He11a þ He11b þ He1a), 2.05 (s, 3 H, He34), 1.98 (s, 3 H,
He32), 1.86e1.59 (m, H,
6
He19a þ He22a þ He22b þ He16b þ He15a þ He9),1.55e1.21 (m,
7 H, He6a þ He6b þ He7a þ He7b þ He21a þ He21b þ He19b),
1.19 (s, 3 H, He27), 1.12e1.02 (m, 2 H, He15b þ He1b), 1.03 (s, 3 H,
He25), 0.98e0.92 (m,1 H, He5), 0.94 (s, 6 H, He29 þ He30), 0.88 (s,
6 H, He23 þ He24), 0.71 (s, 3 H, He26) ppm; 13C NMR (125 MHz,
þ
He9
þ
He15a),
1.51e1.12
(m,
8
H,
He7a
þ He7b þ He6a þ He6b þ He21a þ He21b þ He19b þ He5), 1.17
(s, 3 H, He27),1.09 (s, 3 H, He25),1.08e1.00 (m, 1 H, He15b),1.02 (s,
3 H, He23), 0.90 (s, 9 H, He30 þ He24 þ He29), 0.67 (s, 3 H, He26)
ppm; 13C NMR (125 MHz, CDCl3):
d
¼ 178.1 (Ce28), 170.3 (Ce31),
CDCl3):
d
¼ 176.5 (Ce28), 171.0 (Ce33), 170.7 (Ce31), 145.3 (Ce13),
170.1 (Ce33), 145.0 (Ce13), 138.5 (Ce36), 128.8 (Ce38), 127.9
(Ce37), 127.5 (eC39), 122.8 (Ce12), 76.2 (Ce3), 70.7 (Ce2), 50.5
(Ce5), 48.0 (Ce9), 46.8 (Ce19), 46.5 (Ce17), 43.7 (Ce35), 42.6
(Ce18), 42.4 (Ce14), 41.2 (Ce1), 39.6 (Ce8), 37.0 (Ce4), 36.7 (Ce10),
34.3 (Ce21), 33.1 (Ce30), 32.8 (Ce22), 32.1 (Ce7), 30.9 (Ce20), 27.4
(Ce15), 27.0 (Ce24), 26.0 (Ce27), 24.0 (Ce16), 23.7 (Ce29), 23.5
(Ce11), 22.2 (Ce23), 21.4 (Ce34), 21.1 (Ce32), 18.7 (Ce6), 18.2
(Ce25),16.9 (Ce26) ppm; MS (ESI): m/z (%) ¼ 646.3 ([M þ H]þ,100),
668.5 ([M þ Na]þ, 17), 1291.4 ([2M þ H]þ, 36), 1313.5 ([2M þ Na]þ,
55); analysis calculated for C41H59NO5 (645.91): C 76.24, H 9.21, N
2.17; found: C 76.04, H 9.41, N 2.05.
138.5 (Ce35), 129.1 (Ce37), 124.2 (Ce38), 123.0 (Ce12), 119.9
(Ce36), 80.7 (Ce3), 70.1 (Ce2), 54.9 (Ce5), 47.6 (Ce9), 47.3 (Ce17),
46.9 (Ce19), 44.1 (Ce1), 42.7 (Ce18), 42.4 (Ce14), 39.6 (Ce8), 39.5
(Ce4), 38.2 (Ce10), 34.3 (Ce21), 33.1 (Ce30), 32.6 (Ce22), 32.3
(Ce7), 30.9 (Ce20), 28.6 (Ce24), 27.5 (Ce15), 25.9 (Ce27), 24.2
(Ce16), 23.9 (Ce11), 23.7 (Ce29), 21.3 (Ce32), 21.0 (Ce34), 18.3
(Ce6), 17.7 (Ce23), 17.0 (Ce26), 16.6 (Ce25) ppm; MS (ESI): m/z
(%) ¼ 632.4 ([M þ H]þ, 100), 654.5 ([M þ Na]þ, 14), 1263.3
([2M þ H]þ, 30); analysis calculated for C40H57NO5 (631.88): C
76.03, H 9.09, N 2.22; found: C 75.97, H 9.23, N 1.84.
4.2.16. 2a,3b-Diacetyloxy-olean-12-en-28-oic acid
4.2.14. 2
a
,3a-Diacetyloxy-olean-12-en-28-oic acid benzylamide
ethylphenylamide (28)
(26)
Compound 28 (102 mg, 86%) was obtained from 20 and 2-
Compound 26 (91 mg, 87%) was obtained from 23 according to
phenylethylamine (50 ml, 0.40 mmol) according to GP B as a
GP B as a white solid; mp ¼ 117e119 ꢀC; RF ¼ 0.25 (silica gel,
white solid; mp ¼ 117e120 ꢀC; RF ¼ 0.21 (silica gel, hexane/ethyl
hexane/ethyl acetate, 8:2); [
a
]
D
¼ ꢁ5.47ꢀ (c ¼ 0.35, CHCl3); IR (KBr):
acetate, 8:2); [
a
]
D
¼ þ25.13ꢀ (c ¼ 0.33, CHCl3); IR (KBr):
n
¼ 2946s,
n
¼ 2946s, 2876m, 1746vs, 1658m, 1516m, 1454m, 1432w, 1364m,
2864m, 1744vs, 1660s, 1518m, 1454m, 1368s, 1252vs, 1232s, 1042s,

12
S. Sommerwerk et al. / European Journal of Medicinal Chemistry 119 (2016) 1e16
1032s, 700 m cmꢁ1
;
1H NMR (500 MHz, CDCl3):
d
¼ 7.34e7.30 (m,
(Ce13), 128.5 (Ce1), 125.3 (Ce12), 54.0 (Ce5), 52.8 (Ce18), 48.2
(Ce17), 44.0 (Ce4), 43.1 (Ce9), 42.5 (Ce14), 40.3 (Ce8), 39.1
(Ce19), 39.0 (Ce20), 38.5 (Ce10), 36.8 (Ce22), 32.9 (Ce7), 30.7
(Ce21), 28.1 (Ce15), 27.4 (Ce24), 24.1 (Ce16), 23.7 (Ce27), 23.5
(Ce11), 21.9 (Ce23), 21.3 (Ce30), 19.9 (Ce25), 18.8 (Ce6), 17.7
(Ce26), 17.1 (Ce29) ppm; MS (ESI): m/z (%) ¼ 469.1 ([M þ H]þ, 100),
486.3 ([M þ NH4]þ, 16), 491.1 ([M þ Na]þ, 33); analysis calculated
for C30H44O4 (468.67): C 76.88, H 9.46; found: C 76.77, H 9.52.
2 H, He39), 7.27e7.23 (m, 1 H, He40), 7.20e7.16 (m, 2 H, He38),
5.81 (dd, J ¼ 7.5, 3.3 Hz, 1 H, NH), 5.08 (ddd, J ¼ 11.5, 10.4, 4.6 Hz, 1 H,
He2), 4.93 (dd, J ¼ 3.5, 3.5 Hz, 1 H, He12), 4.72 (d, J ¼ 10.4 Hz, 1 H,
He3), 3.85 (dddd, J ¼ 13.2, 7.5, 6.4, 5.4 Hz, 1 H, He35a), 3.09 (dddd,
J ¼ 13.2, 9.2, 5.8, 3.4 Hz, 1 H, He35b), 2.85 (ddd, J ¼ 13.6, 5.6, 5.6 Hz,
1 H, He36a), 2.70 (ddd, J ¼ 13.7, 9.0, 6.4 Hz, 1 H, He36b), 2.29 (dd,
J ¼ 13.1, 4.2 Hz, 1 H, He18), 2.05 (s, 3 H, He34), 2.00e1.84 (m, 2 H,
He16a
He11a þ He11b þ He16b þ He22a þ He19a), 1.57e1.13 (m, 9 H,
He9 He22b
þ
He1a), 1.98 (s, 3 H, He32), 1.76e1.57 (m, 5 H,
4.2.20. 2
acid (33)
b
,3b
-Dihydroxyurs-12-en-28-oic acid ¼ 2-epi-corosolic
þ
þ He6a þ He6b þ He15a þ He7a þ He7b þ He21a þ He21b),
1.10e0.89 (m, 4 H, He19b þ He1b þ He15b þ He5), 1.08 (s, 3 H,
He27), 0.99 (s, 3 H, He25), 0.90 (s, 3 H, He23), 0.88 (s, 3 H, He30),
0.88 (s, 6 H, He29 þ He24), 0.55 (s, 3 H, He26) ppm; 13C NMR
Compound 33 (3.70 g, 39%) was prepared from 32 as previously
described for 3 [15], and it was obtained as a white solid;
mp ¼ 282e285 ꢀC (lit [31].: 284 ꢀC); RF ¼ 0.28 (silica gel, hexane/
ethyl acetate, 6:4); [
a
]
¼ þ82.50ꢀ (c ¼ 0.32, C5H5N); IR (KBr):
D
(125 MHz, CDCl3):
d
¼ 178.1 (Ce28), 170.9 (Ce33), 170.7 (Ce31),
n
¼ 3496s, 3436s, 3024s, 2922vs, 2854s, 2612w, 1706vs, 1458s,
144.7 (Ce13), 139.2 (Ce37), 128.9 (Ce38), 128.8 (Ce39), 126.8
(Ce40), 122.5 (Ce12), 80.7 (Ce3), 70.1 (Ce2), 54.9 (Ce5), 47.6
(Ce9), 46.9 (Ce19), 46.4 (Ce17), 44.0 (Ce1), 42.2 (Ce18), 42.0
(Ce14), 44.0 (Ce35), 39.5 (Ce8), 39.5 (Ce4), 38.1 (Ce10), 35.6
(Ce36), 34.3 (Ce21), 33.1 (Ce30), 32.5 (Ce22), 32.1 (Ce7), 30.8
(Ce20), 28.5 (Ce24), 27.3 (Ce15), 25.8 (Ce27), 23.9 (Ce16), 23.6
(Ce29), 23.6 (Ce11), 21.3 (Ce32), 21.0 (Ce34), 18.3 (Ce6), 17.7
(Ce23), 17.0 (Ce26), 16.6 (Ce25) ppm; MS (ESI): m/z (%) ¼ 660.5
([M þ H]þ, 100), 682.5 ([M þ Na]þ, 12), 1319.5 ([2M þ H]þ, 86),
1341.6 ([2M þ Na]þ, 72); analysis calculated for C42H61NO5
(659.94): C 76.44, H 9.32, N 2.12; found: C 76.30, H 9.41, N 2.05.
1448m, 1410m, 1376s, 1362s, 1344w, 1320m, 1308m, 1286m, 1264m,
1250m, 1226s, 1200s, 1182s, 1164m, 1146m, 1114m, 1102m, 1088m,
1026s, 998s, 974m, 964m, 946m, 928s, 768 m cmꢁ1 1H NMR
;
(500 MHz, pyridine-d5):
4.43 (ddd, J ¼ 3.8, 3.8, 3.3 Hz,1 H, He2), 3.47 (d, J ¼ 4.0 Hz,1 H, He3),
2.67 (d, J ¼ 11.3 Hz, 1 H, He18), 2.41e2.33 (m, 1 H, He15a), 2.31 (dd,
d
¼ 5.52 (dd, J ¼ 3.6, 3.6 Hz, 1 H, He12),
J
¼
14.1, 2.8 Hz,
1
H, He1a), 2.20e1.93 (m,
6
H,
He11a þ He11b þ He16a þ He16b þ He22a þ He22b), 1.69e1.53
(m, 4 H, He6a þ He6b þ He7a þ He9), 1.54e1.47 (m, 2 H,
He19
He7
þ
He21a), 1.53 (s,
He21b), 1.39 (s,
3
H, He25), 1.45e1.39 (m,
2
2
H,
H,
þ
3 H, He23), 1.32e1.17 (m,
He1b þ He15b), 1.29 (s, 3 H, He24), 1.27 (s, 3 H, He27), 1.13 (s, 3 H,
He26), 1.11e1.00 (m, 2 H, He5 þ He20), 1.04 (d, J ¼ 6.5 Hz, 3 H,
He29), 0.98 (d, J ¼ 6.3 Hz, 3 H, He30) ppm; 13C NMR (125 MHz,
4.2.17. 3-Oxours-12-en-28-oic acid (29)
Compound 29 (5.0 g, 100%) was prepared as previously
described (for 3-oxoolean-12-en-28-oic acid [15]), and it was ob-
tained as a white solid; mp ¼ 255e258 ꢀC (lit [30].: 250e268 ꢀC);
pyridine-d5):
d
¼ 180.3 (Ce28), 139.7 (Ce13), 126.2 (Ce12), 78.8
(Ce3), 71.8 (Ce2), 56.4 (Ce5), 54.0 (Ce18), 48.9 (Ce9), 48.5 (Ce17),
45.6 (Ce1), 43.1 (Ce14), 40.6 (Ce8), 39.9 (Ce19), 39.8 (Ce20), 39.2
(Ce4), 37.9 (Ce22), 37.7 (Ce10), 34.1 (Ce7), 31.5 (Ce21), 30.7
(Ce24), 29.1 (Ce15), 25.4 (Ce16), 24.4 (Ce27), 24.2 (Ce11), 21.8
(Ce30), 19.1 (Ce6), 18.6 (Ce23), 17.9 (Ce29), 17.9 (Ce26), 17.2
(Ce25) ppm; MS (ESI): m/z (%) ¼ 471.4 ([MꢁH]-, 64), 943.3
([2MꢁH]-, 100), 965.5 ([2M-2H þ Na]-, 44); analysis calculated for
RF ¼ 0.50 (silica gel, hexane/ethyl acetate, 7:3); [
a
]
¼ þ80.30ꢀ
D
(c ¼ 0.41, CHCl3); MS (ESI): m/z (%) ¼ 453.5 ([MꢁH]-, 100), 485.4
([MꢁH þ MeOH]-, 20), 907.4 ([2MꢁH]-, 76), 930.6 ([2Mꢁ2H þ Na]-,
60); analysis calculated for C30H46O3 (454.68): C 79.25, H 10.20;
found: C 79.00, H 10.38.
4.2.18. 2
oxours-12-en-28-oic acid (31)
A mixture (2:1, 11.0 g, 100%) of 30 and 31 was prepared from 29
as previously described (for 2 -bromo-3-oxoolean-12-en-28-oic
acid and 2 -bromo-3-oxoolean-12-en-28-oic acid [18]), and this
b-Bromo-3-oxours-12-en-28-oic acid (30), 2
a-bromo-3-
C30H48O4 (472.70): C 76.23, H 10.24; found: C 76.05, H 10.38.
4.2.21. 2 ,3 -Diacetyloxyurs-12-en-28-oic acid (34)
b
b
b
Compound 34 (120 mg, 51%) was obtained from 33 and acetic
anhydride according to GP A as a white solid; mp ¼ 247e252 ꢀC;
a
mixture was used without any further purification.
RF ¼ 0.48 (silica gel, hexane/ethyl acetate, 7:3); [
a
]
¼ þ75.00ꢀ
D
(c ¼ 0.39, CHCl3); IR (KBr): ¼ 3410m, 2950s, 2872m, 1746vs, 1698s,
n
4.2.19. 2-Hydroxy-3-oxours-1,12-dien-28-oic acid (32)
1458m, 1398m, 1370s, 1254vs, 1234s, 1194m, 1160m, 1056m, 1030s,
Compound 32 (9.80 g, 97%) was prepared from 30/31 as previ-
ously described (for 2-hydroxy-3-oxoolean-1,12-dien-28-oic acid
[18]), and it was obtained as white solid; mp ¼ 143e145 ꢀC;
972 m cmꢁ1; 1H NMR (500 MHz, CDCl3):
d
¼ 5.32 (ddd, J ¼ 3.8, 3.8,
3.5 Hz, 1 H, He2), 5.24 (dd, J ¼ 3.6, 3.6 Hz, 1 H, He12), 4.63 (d,
J ¼ 3.9 Hz, 1 H, He3), 2.18 (d, J ¼ 11.2 Hz, 1 H, He18), 2.08e1.84 (m,
4 H, He11a þ He11b þ He15a þ He16a), 2.04 (s, 3 H, He33), 2.02
(s, 3 H, He32), 1.97 (dd, J ¼ 6.8, 3.9 Hz,1 H, He1a),1.75e1.63 (m, 3 H,
RF ¼ 0.66 (silica gel, hexane/ethyl acetate, 6:4); [
a]
¼ þ96.70ꢀ
D
(c ¼ 0.31, CHCl3); UVevis (CHCl3): lmax (log ε) ¼ 272 nm (3.87); IR
(KBr):
n
¼ 3566w, 3438m, 2974vs, 2928vs, 2870s, 2644w, 1792w,
He16b
þ
He22a
þ
He22b), 1.61e1.43 (m,
5
H,
1694vs, 1670vs, 1650s, 1570w, 1456s, 1408s, 1382s, 1362m, 1316m,
1288m, 1238s, 1212m, 1190m, 1164w, 1146w, 1120w, 1108w, 1086m,
He6a þ He6b þ He7a þ He9 þ He21a), 1.39e1.24 (m, 4 H,
He1b þ He7b þ He19 þ He21b), 1.22 (s, 3 H, He25), 1.14e1.03 (m,
1 H, He15b), 1.07 (s, 3 H, He27), 1.04 (s, 3 H, He23), 1.03e0.96 (m,
2 H, He5 þ He20), 0.95 (d, J ¼ 6.3 Hz, 3 H, He30), 0.90 (s, 3 H,
1054s, 1030m, 1018w, 968 m cmꢁ1 1H NMR (500 MHz, CDCl3):
;
d
¼ 6.36 (s, 1 H, H-e1), 5.95 (s, 1 H, OHe2), 5.30 (dd, J ¼ 3.6, 3.6 Hz,
1 H, He12), 2.22 (d, J ¼ 11.1 Hz, 1 H, He18), 2.18e1.97 (m, 3 H,
He11a þ He11b þ He16a), 1.91e1.81 (m, 2 H, He9 þ He15a),
1.76e1.63 (m, 3 H, He16b þ He22a þ He22b), 1.63e1.44 (m, 5 H,
He5 þ He6a þ He6b þ He7a þ He21a), 1.43e1.37 (m, 1 H, He7b),
1.36e1.24 (m, 2 H, He21b þ He19), 1.23 (s, 3 H, He25), 1.21 (s, 3 H,
He24), 1.14e1.08 (m, 1 H, He15b), 1.10 (s, 3 H, He23), 1.09 (s, 3 H,
He27), 1.05e0.98 (m, 1 H, He20), 0.95 (d, J ¼ 6.3 Hz, 3 H, He30),
0.85 (s, 3 H, He26), 0.85 (d, J ¼ 8.0 Hz, 3 H, He29) ppm; 13C NMR
He24), 0.86 (d, J ¼ 6.4 Hz, 3 H, He29), 0.79 (s, 3 H, He26) ppm; 13
C
NMR (125 MHz, CDCl3):
d
¼ 183.7 (Ce28), 170.9 (Ce31), 170.4
(Ce34), 138.3 (Ce13), 125.7 (Ce12), 78.1 (Ce3), 69.8 (Ce2), 55.3
(Ce5), 52.6 (Ce18), 48.1 (Ce17), 48.1 (Ce9), 42.2 (Ce14), 42.1 (Ce1),
39.8 (Ce8), 39.1 (Ce19), 39.0 (Ce20), 37.5 (Ce4), 36.9 (Ce22), 36.9
(Ce10), 32.9 (Ce7), 30.7 (Ce21), 29.3 (Ce24), 28.0 (Ce15), 24.1
(Ce16), 23.8 (Ce27), 23.5 (Ce11), 21.4 (Ce33), 21.3 (Ce30), 21.0
(Ce32), 18.1 (Ce6), 17.8 (Ce23), 17.3 (Ce26), 17.2 (Ce29), 16.3
(Ce25) ppm; MS (ESI): m/z (%) ¼ 555.5 ([MꢁH]ꢁ, 63), 1111.2
(125 MHz, CDCl3):
d
¼ 201.2 (Ce3), 183.9 (Ce28), 143.9 (Ce2), 138.6

S. Sommerwerk et al. / European Journal of Medicinal Chemistry 119 (2016) 1e16
13
([2MꢁH]-,100),1133.5 ([2Mꢁ2H þ Na]ꢁ, 28); analysis calculated for
(Ce21), 29.3 (Ce24), 28.3 (Ce16), 27.3 (Ce15), 23.7 (Ce11), 23.4
(Ce27), 21.4 (Ce32), 21.0 (Ce34), 20.9 (Ce30), 18.2 (Ce6), 17.9
(Ce23), 17.5 (Ce29), 17.2 (Ce26), 16.4 (Ce25) ppm; MS (ESI): m/z
(%) ¼ 494.2 ([M þ H-HOAc]þ, 18), 554.1 ([M þ H]þ, 1), 571.3
([M þ NH4]þ, 52), 576.4 ([M þ Na]þ, 29), 1129.3 ([2M þ Na]þ, 100);
analysis calculated for C34H51NO5 (553.77): C 73.74, H 9.28, N 2.53;
found: C 73.51, H 9.40, N 2.32.
C
34H52O6 (556.77): C 73.34, H 9.41; found: C 73.09, H 9.56.
4.2.22. 2b,3b-Diacetyloxy-17b-isocyanato-28-norolean-12-ene
(35)
Compound 21 (370 mg, 0.66 mmol) was dissolved in a mixture
of toluene (9 mL) and triethylamine (100 mg, 0.99 mmol), and the
mixture was stirred for 15 min at room temperature. Diphenyl-
phosphoryl azide (218 mg, 0.79 mmol) was added, and stirring was
continued in a microwave reactor at 180 ꢀC for 3 h. Et2O (100 mL)
4.2.24. 2
To a solution of compound 35 (280 mg, 0.51 mmol) in THF
(19 mL), aqueous HCl (2.53 mmol, 2 ) was added, and the mixture
was stirred for 2 h at 30 ꢀC. Et2O (100 mL) and aqueous NaOH
(2.53 mmol, 2 ) were added; usual aq. work-up followed by col-
b,3b-Diacetyloxy-17b-amino-28-norolean-12-ene (37)
was added, the organic layer was washed with diluted HCl (0.1
M
,
M
1 ꢂ 100 mL), water (2 ꢂ 100 mL) and brine (1 ꢂ 50 mL), dried
(MgSO4), filtrated, and the filtrate was evaporated to dryness. Col-
umn chromatography (silica gel, hexane/ethyl acetate, 9:1) of the
M
umn chromatography (silica gel, chloroform/methanol, 9:1) pro-
residue afforded 35 (280 mg, 76%) as
a
white solid;
vided 37 (280 mg, 76%) as a white solid; mp ¼ 190e193 ꢀC;
mp ¼ 255e260 ꢀC; RF ¼ 0.70 (silica gel, hexane/ethyl acetate, 7:3);
RF ¼ 0.51 (silica gel, chloroform/methanol, 9:1); [
a
]
¼ þ79.27ꢀ
D
[
a
]
¼ þ81.66ꢀ (c ¼ 0.40, CHCl3); IR (KBr):
n
¼ 2950m, 2916m,
(c ¼ 0.39, CHCl3); IR (KBr): ¼ 3290w, 2950s, 2868m,1746vs,1616w,
n
D
2866w, 2256vs, 1738vs, 1728s, 1458w, 1434w, 1400w, 1368m,
1518m, 1464m, 1432m, 1372s, 1300m, 1248vs, 1196m, 1056m,
1256vs, 1242s, 1054m, 1028m, 1012 m cmꢁ1
CDCl3):
;
1H NMR (500 MHz,
1032 m cmꢁ1; 1H NMR (500 MHz, CDCl3):
d
¼ 7.28 (s, 2 H, NH2), 5.54
d
¼ 5.33e5.29 (m, 2 H, He2 þ He12), 4.63 (d, J ¼ 3.9 Hz, 1 H,
(dd, 1 H, J ¼ 3.5, 3.5 Hz, He12), 5.32 (ddd, 1 H, J ¼ 3.7, 3.7, 3.7 Hz,
He2), 4.62 (d, J ¼ 3.9 Hz, 1 H, He3), 2.62 (dd, J ¼ 13.3, 3.8 Hz, 1 H,
He18), 2.20 (ddd, 1 H, J ¼ 14.4, 14.4, 5.0 Hz, He16a), 2.07e1.95 (m,
3 H, He11a þ He22a þ He1a), 2.04 (s, 3 H, He32), 2.03 (s, 3 H,
He34), 1.92e1.79 (m, 2 H, He11b þ He15a), 1.79e1.46 (m, 7 H,
He22b þ He19a þ He16b þ He6a þ He6b þ He7a þ He9),
He3), 2.36 (dd, J ¼ 13.9, 4.6 Hz, 1 H, He18), 2.10e1.81 (m, 6 H,
He11a þ He11b þ He15a þ He16a þ He1a þ He22a), 2.05 (s, 3 H,
He32), 2.03 (s, 3 H, He34), 1.70 (ddd, J ¼ 13.6, 3.5, 3.5 Hz, 1 H,
He22b),
1.65e1.48
(m,
5
H,
He6a þ He6b þ He19a þ He9 þ He7a), 1.45e1.38 (m, 1 H, He7b),
1.36e1.17 (m, 5 H, He1b þ He21a þ He21b þ He16b þ He19b),
1.22 (s, 3 H, He25), 1.14e1.05 (m, 1 H, He15b), 1.12 (s, 3 H, He27),
1.07 (s, 3 H, He23), 1.01 (s, 3 H, He26), 1.01e0.97 (m, 1 H, He5), 0.96
(s, 3 H, He29), 0.91 (s, 3 H, He24), 0.90 (s, 3 H, He30) ppm; 13C NMR
1.43e1.20
(m,
6
H,
He7b þ He21a þ He21b þ He1b þ He19b þ He15b), 1.21 (s, 3 H,
He25), 1.18 (s, 3 H, He27), 1.06 (s, 3 H, He23), 1.00 (s, 3 H, He26),
1.00e0.95 (m, 1 H, He5), 0.98 (s, 3 H, He29), 0.92 (s, 3 H, He30),
(125 MHz, CDCl3):
d
¼ 170.9 (Ce33), 170.4 (Ce31), 142.9 (Ce13),
0.91 (s, 3 H, He24) ppm; 13C NMR (125 MHz, CDCl3):
d
¼ 170.9
124.1 (Ce12), 122.0 (Ce35), 78.1 (Ce3), 69.7 (Ce2), 62.3 (Ce17),
55.4 (Ce5), 49.1 (Ce18), 48.2 (Ce9), 47.3 (Ce19), 42.1 (Ce1), 41.9
(Ce14), 39.9 (Ce8), 37.8 (Ce22), 37.5 (Ce4), 36.9 (Ce10), 35.6
(Ce21), 32.9 (Ce7), 32.8 (Ce30), 30.9 (Ce20), 29.3 (Ce24), 27.2
(Ce16), 26.8 (Ce15), 25.9 (Ce27), 23.9 (Ce29), 23.8 (Ce11), 21.4
(Ce32), 21.0 (Ce34), 18.2 (Ce6), 17.8 (Ce23), 17.2 (Ce26), 16.2
(Ce25) ppm; MS (ESI): m/z (%) ¼ 576.3 ([M þ Na]þ, 94), 1129.3
([2M þ Na]þ, 100); analysis calculated for C34H51NO5 (553.77): C
73.74, H 9.28, N 2.53; found: C 73.55, H 9.40, N 2.44.
(Ce33), 170.3 (Ce31), 139.1 (Ce13), 128.2 (Ce12), 77.9 (Ce3), 69.5
(Ce2), 58.3 (Ce17), 55.3 (Ce5), 47.8 (Ce9), 46.6 (Ce19), 44.7
(Ce18), 42.2 (Ce1), 41.6 (Ce14), 40.1 (Ce8), 37.5 (Ce4), 36.7 (Ce10),
34.8 (Ce21), 32.7 (Ce30), 32.4 (Ce7), 32.2 (Ce22), 31.0 (Ce20), 29.2
(Ce24), 25.9 (Ce27), 24.7 (Ce15), 24.1 (Ce16), 24.0 (Ce11), 23.7
(Ce29), 21.4 (Ce32), 21.0 (Ce34), 18.1 (Ce6), 17.8 (Ce23), 17.1
(Ce26),16.2 (Ce25) ppm; MS (ESI): m/z (%) ¼ 528.1 ([M þ H]þ,100);
analysis calculated for C33H53NO4 (527.78): C 75.10, H 10.12, N 2.65;
found: C 75.02, H 10.31, N 2.55.
4.2.23. (2
b
,3b
,17
b)-2,3-Diacetyloxy-17-isocyanato-28-norurs-12-
4.2.25. 2b,3b-Diacetyloxy-17b-amino-28-norurs-12-ene (38)
ene (36)
To a solution of compound 36 (280 mg, 0.51 mmol) in THF
Following the procedure given for the preparation of 35, com-
pound 36 (210 mg, 57%) was synthesized starting from 34 and
obtained as a white solid; mp ¼ 92e95 ꢀC; RF ¼ 0.64 (silica gel,
(19 mL), aqueous HCl (2.53 mmol, 2 M) was added, and the mixture
was stirred for 24 h at 50 ꢀC. Work-up as described above followed
by chromatography (silica gel, chloroform/methanol, 9:1) gave 38
(123 mg, 46%) as a white solid; mp ¼ 174e176 ꢀC; RF ¼ 0.78 (silica
hexane/ethyl acetate, 8:2); [
a
]
¼ þ71.00ꢀ (c ¼ 0.29, CHCl3); IR
D
(KBr):
n
¼ 3438w, 3036w, 2972s, 2934s, 2878m, 2844m, 2270vs,
gel, chloroform/methanol, 9:1); [
a
]
¼ þ69.30ꢀ (c ¼ 0.32, CHCl3); IR
D
1738vs, 1660w, 1654w, 1490w, 1458m, 1434m, 1400m, 1390m, 1370s,
1364s, 1342w, 1306w, 1254vs, 1238vs, 1194m, 1188m, 1158m, 1134w,
(KBr):
n
¼ 3418m, 3288m, 2954s, 2872s, 2540m, 2362w, 2342w,
1982w, 1746vs, 1654w, 1636w, 1600m, 1558w, 1500s, 1456s, 1432m,
1392s, 1374s, 1338m, 1248vs, 1194s, 1158m, 1116w, 1090m, 1056s,
1116w, 1090w, 1056m, 1026m, 1014m, 976m, 946 m cmꢁ1 1H NMR
;
(500 MHz, CDCl3):
d
¼ 5.32 (ddd, J ¼ 4.0, 4.0, 3.4 Hz, 1 H, He2),
1030s, 990m, 970m, 946m, 752s, 662 m cmꢁ1 1H NMR (500 MHz,
;
5.29e5.26 (m, 1 H, He12), 4.63 (d, J ¼ 3.9 Hz, 1 H, He3), 2.08e1.98
(m, 4 H, He1a þ He11a þ He15a þ He16a), 2.05 (s, 3 H, He32),
2.03 (s, 3 H, He34),1.94e1.87 (m, 2 H, He11b, He22a),1.81e1.73 (m,
CDCl3):
3.8, 3.8 Hz, 1 H, He2), 4.62 (d, J ¼ 3.9 Hz, 1 H, He3), 2.23e2.08 (m,
H, He16a He22a He22b), 2.07e1.90 (m, H,
d
¼ 5.53 (dd, J ¼ 3.5, 3.5 Hz, 1 H, He12), 5.33 (ddd, J ¼ 3.8,
3
þ
þ
4
2
H,
He22b
þ
He18),
1.63e1.47
(m,
5
H,
He1a þ He11a þ He11b þ He18), 2.05 (s, 3 H, He32), 2.03 (s, 3 H,
He34), 1.87e1.78 (m, 2 H, He15b þ He16b), 1.66e1.47 (m, 5 H,
He6a þ He6b þ He7a þ He9 þ He21a), 1.44e1.30 (m, 3 H,
He1b þ He7b þ He19),1.29e1.17 (m, 2 H, He15b þ He21b),1.23 (s,
3 H, He25), 1.16e1.09 (m, 1 H, He20), 1.12 (s, 3 H, He27), 1.09e0.95
(m, 1 H, He5), 1.06 (s, 3 H, He23), 1.02 (s, 3 H, He26), 0.94 (d,
J ¼ 6.3 Hz, 3 H, He30), 0.91 (s, 3 H, He24), 0.84 (d, J ¼ 6.5 Hz, 3 H,
He6a þ He6b þ He7a þ He9 þ He21a), 1.44e1.40 (m, 1 H, He7b),
1.39e1.34 (m, 1 H, He1b), 1.32e1.25 (m, 2 H, He19 þ He16b), 1.24
(s, 3 H, He25),1.22e1.18 (m, 1 H, He21b), 1.13e1.07 (m, 1 H, He15b),
1.06 (s, 3 H, He23),1.06 (s, 3 H, He26),1.06 (s, 3 H, He27),1.04e0.95
(m, 2 H, He5 þ He20), 0.93 (d, J ¼ 6.3 Hz, 3 H, He30), 0.91 (s, 3 H,
He24), 0.83 (d, J ¼ 6.5 Hz, 3 H, He29) ppm; 13C NMR (125 MHz,
CDCl3):
d
¼ 170.9 (Ce33), 170.4 (Ce31), 138.0 (Ce13), 126.8 (Ce12),
He29) ppm; 13C NMR (125 MHz, CDCl3):
(Ce31), 134.9 (Ce13), 131.2 (Ce12), 77.9 (Ce3), 69.5 (Ce2), 58.8
(Ce17), 56.7 (Ce18), 55.3 (Ce5), 47.9 (Ce9), 42.3 (Ce1), 41.9 (Ce14),
40.5 (Ce19), 40.3 (Ce8), 38.4 (Ce20), 37.5 (Ce4), 36.7 (Ce10), 36.1
d
¼ 170.9 (Ce33), 170.3
122.4 (Ce35), 78.1 (Ce3), 69.7 (Ce2), 63.0 (Ce17), 60.7 (Ce18), 55.4
(Ce5), 48.3 (Ce9), 42.3 (Ce1), 42.3 (Ce14), 41.9 (Ce22), 41.0 (Ce19),
40.1 (Ce8), 39.1 (Ce20), 37.5 (Ce4), 36.9 (Ce10), 33.2 (Ce7), 31.9

14
S. Sommerwerk et al. / European Journal of Medicinal Chemistry 119 (2016) 1e16
(Ce22), 32.5 (Ce7), 30.8 (Ce21), 29.2 (Ce24), 25.3 (Ce15), 25.2
(Ce16), 23.9 (Ce11), 23.5 (Ce27), 21.4 (Ce32), 21.0 (Ce34), 20.7
(Ce30), 18.0 (Ce6), 17.8 (Ce23), 17.4 (Ce26), 17.1 (Ce29), 16.4
(Ce25) ppm; MS (ESI): m/z (%) ¼ 528.2 ([M þ H]þ, 100); analysis
calculated for C33H53NO4 (527.78): C 75.10, H 10.12, N 2.65; found: C
75.03, H 10.34, N 2.51.
(Ce1), 40.0 (Ce19), 39.8 (Ce8), 39.3 (Ce20), 37.5 (Ce4), 37.2
(Ce22), 36.8 (Ce10), 32.8 (Ce7), 31.0 (Ce21), 29.3 (Ce24), 28.0
(Ce15), 25.2 (Ce16), 23.8 (Ce11), 23.5 (Ce27), 21.4 (Ce32), 21.3
(Ce30), 21.0 (Ce34), 18.1 (Ce6), 17.8 (Ce23), 17.5 (Ce29), 17.1
(Ce26),16.3 (Ce25) ppm; MS (ESI): m/z (%) ¼ 632.3 ([M þ H]þ,100),
654.5 ([M þ Na]þ, 27), 970.3 ([3M þ 2Na]2þ, 26), 1263.1 ([2M þ H]þ,
23), 1285.5 ([2M þ Na]þ, 86); analysis calculated for C40H57NO5
(631.88): C 76.03, H 9.09, N 2.22; found: C 75.86, H 9.23, N 2.14.
4.2.26. 2b,3b-Diacetyloxy-olean-12-en-28-oic acid phenylamide
(39)
Compound 39 (98 mg, 86%) was obtained from 21 and aniline
4.2.28. N-[2b,3b-Diacetyloxy-17b-amino-28-norolean-12-en-17-
(50
ml, 0.55 mmol) according to GP B as a white solid;
yl]-benzamide (41)
mp ¼ 153e154 ꢀC; RF ¼ 0.58 (silica gel, hexane/ethyl acetate, 7:3);
Compound 37 (120 mg, 0.23 mmol) was dissolved in dry DCM
(12 mL). After sequential addition of benzoyl chloride (120 mg,
0.86 mmol), triethylamine (42 mg, 0.42 mmol), and DMAP (2 mg,
0.02 mmol), the reaction mixture was stirred at room temperature
for 2 h. Et2O (50 mL) was added, the organic layer was washed with
diluted HCl (0.1 M, 1 ꢂ 50 mL), water (2 ꢂ 50 mL) and brine
(1 ꢂ 25 mL), dried (MgSO4), filtrated, and the filtrate was evapo-
rated to dryness. Column chromatography (silica gel, hexane/ethyl
[
a]
¼
þ70.58ꢀ (c
¼
0.27, CHCl3); UVevis (CHCl3): lmax
D
(log ε) ¼ 246 nm (4.25); IR (KBr):
n
¼ 2948s, 2878m, 1744vs, 1678m,
1598m, 1526m, 1500m, 1438s, 1366m, 1310m, 1250vs, 1194m, 1158w,
1056m, 1030m, 756 m cmꢁ1; 1H NMR (500 MHz, CDCl3):
d
¼ 7.67 (s,
1 H, NH), 7.49e7.46 (m, 2H, He36), 7.32e7.27 (m, 2H, He37),
7.10e7.05 (m, 1 H, He38), 5.54 (dd, J ¼ 3.4, 3.4 Hz, 1 H, He12), 5.31
(ddd, J ¼ 3.7, 3.7, 3.7 Hz,1 H, He2), 4.62 (d, J ¼ 3.9 Hz,1 H, He3), 2.67
(dd,
J
¼
12.8, 3.0 Hz,
1
H, He18), 2.12e1.90 (m,
4
H,
acetate, 8:2) gave 41 (112 mg, 78%) as a white solid;
He16a þ He11a þ He11b þ He1a), 2.04 (s, 3 H, He32), 2.03 (s, 3 H,
He34), 1.86e1.61 (m, H,
He19a þ He22a þ He22b þ He16b þ He15a), 1.61e1.53 (m, 2 H,
He6a He9), 1.52e1.22 (m, H,
mp ¼ 140e143 ꢀC; RF ¼ 0.54 (silica gel, hexane/ethyl acetate, 7:3);
5
[
a
]
¼
þ69.56ꢀ (c
¼
0.34, CHCl3); UVevis (CHCl3): lmax
D
(log ε) ¼ 234 nm (3.98); IR (KBr):
n
¼ 3428w, 2950s, 2870m, 1746vs,
þ
7
1670m, 1516m, 1484m, 1458m, 1436m, 1366m, 1318m, 1250vs,
He7a þ He7b þ He6b þ He21a þ He21b þ He1b þ He19b), 1.19
(s, 3 H, He27), 1.16 (s, 3 H, He25), 1.09 (ddd, J ¼ 14.1, 3.1, 3.1 Hz, 1 H,
He15b), 1.03 (s, 3 H, He23), 0.99e0.95 (m, 1 H, He5), 0.94 (s, 6 H,
He29 þ He30), 0.89 (s, 3 H, He24), 0.74 (s, 3 H, He26) ppm; 13C
1192m, 1056m, 1030
m
cmꢁ1 1H NMR (500 MHz, CDCl3):
;
d
¼ 7.73e7.69 (m, 2 H, He37), 7.49e7.38 (m, 3 H, He39 þ He38),
5.87 (s, 1 H, NH), 5.40 (dd, J ¼ 3.5, 3.5 Hz, 1 H, He12), 5.32 (ddd,
J ¼ 3.7, 3.7, 3.7 Hz,1 H, He2), 4.62 (d, J ¼ 3.9 Hz,1 H, He3), 2.56 (ddd,
NMR (125 MHz, CDCl3):
d
¼ 176.5 (Ce28), 170.9 (Ce33), 170.4
J
¼
13.9, 3.2, 3.2 Hz, 1 H, He22a), 2.38e2.29 (m, 2 H,
(Ce31), 145.3 (Ce13), 138.2 (Ce35), 129.0 (Ce37), 124.2 (Ce38),
123.2 (Ce12), 119.9 (Ce36), 78.0 (Ce3), 69.7 (Ce2), 55.3 (Ce5), 48.2
(Ce9), 47.3 (Ce17), 46.8 (Ce19), 42.8 (Ce18), 42.5 (Ce14), 42.1
(Ce1), 39.7 (Ce8), 37.5 (Ce4), 36.8 (Ce10), 34.3 (Ce21), 33.1
(Ce30), 32.6 (Ce22), 32.4 (Ce7), 30.9 (Ce20), 29.2 (Ce24), 27.4
(Ce15), 26.0 (Ce27), 24.2 (Ce16), 23.9 (Ce11), 23.7 (Ce29), 21.4
(Ce32), 21.0 (Ce34), 18.1 (Ce6), 17.8 (Ce23), 17.2 (Ce26), 16.2
(Ce25) ppm; MS (ESI): m/z (%) ¼ 632.3 ([M þ H]þ, 100), 1263.1
([2M þ H]þ, 40); analysis calculated for C40H57NO5 (631.88): C
76.03, H 9.09, N 2.22; found: C 75.87, H 9.17, N 2.07.
He18 þ He16a), 2.05e1.94 (m, 3 H, He16b þ He11a þ He1a), 2.04
(s, 3 H, He32), 2.03 (s, 3 H, He34), 1.92e1.85 (m, 1 H, He11b),
1.85e1.69 (m, 3 H, He19a þ He22b þ He15a), 1.60e1.54 (m, 2 H,
He6a
þ
He9),
1.53e1.19
(m,
7
H,
He6b þ He7a þ He7b þ He21a þ He21b þ He1b þ He19b), 1.17
(s, 3 H, He27), 1.16 (s, 3 H, He25), 1.09e1.02 (m, 1 H, He15b), 1.03 (s,
3 H, He23), 0.99e0.94 (m, 1 H, He5), 0.98 (s, 3 H, He29), 0.93 (s,
3 H, He30), 0.89 (s, 3 H, He24), 0.80 (s, 3 H, He26) ppm; 13C NMR
(125 MHz, CDCl3):
d
¼ 170.9 (Ce33), 170.4 (Ce31), 166.2 (Ce35),
143.5 (Ce13), 135.5 (Ce36), 131.2 (Ce39), 128.6 (Ce38), 126.7
(Ce37), 124.6 (Ce12), 78.1 (Ce3), 69.7 (Ce2), 57.1 (Ce17), 55.3
(Ce5), 48.1 (Ce9), 47.5 (Ce18), 46.7 (Ce19), 42.1 (Ce1), 42.0 (Ce14),
39.8 (Ce8), 37.5 (Ce4), 36.8 (Ce10), 35.3 (Ce21), 33.0 (Ce30), 32.5
(Ce22), 32.4 (Ce7), 30.9 (Ce20), 29.2 (Ce24), 26.2 (Ce15), 26.0
(Ce27), 24.1 (Ce29), 23.8 (Ce11), 22.1 (Ce16), 21.4 (Ce32), 21.0
(Ce34), 18.1 (Ce6), 17.8 (Ce23), 17.2 (Ce26), 16.1 (Ce25) ppm; MS
(ESI): m/z (%) ¼ 632.2 ([M þ H]þ, 100), 1285.4 ([2M þ Na]þ, 14);
analysis calculated for C40H57NO5 (631.88): C 76.03, H 9.09, N 2.22;
found: C 75.92, H 9.23, N 2.03.
4.2.27. 2
b,3b-Diacetyloxy-urs-12-en-28-oic acid phenylamide (40)
Compound 40 (180 mg, 85%) was obtained from 34 and aniline
(100
ml, 1.1 mmol) according to GP B as a white solid;
mp ¼ 142e146 ꢀC; RF ¼ 0.61 (silica gel, chloroform/hexane/ethyl
acetate, 10:9:1); [
a]
¼ þ47.20ꢀ (c ¼ 0.34, CHCl3); UVevis (CHCl3):
D
lmax (log ε) ¼ 238 nm (4.21); IR (KBr):
n
¼ 3404m, 2950s, 2872m,
1746vs, 1678s, 1600s, 1528s, 1500s, 1456s, 1438s, 1398m, 1366s,
1314m, 1292m, 1250vs, 1234vs, 1194s, 1156m, 1116w, 1078m, 1056m,
1030s, 972m, 944m, 754s, 692 m cmꢁ1; 1H NMR (500 MHz, CDCl3):
d
¼ 7.67 (s, 1 H, NH), 7.45 (d, J ¼ 7.5 Hz, 2 H, He36), 7.32e7.27 (m,
4.2.29. N-[2
benzamide (42)
b,3b-Diacetyloxy-17b-amino-28-norurs-12-en-17-yl]-
2 H, He37), 7.09e7.05 (m, 1 H, He38), 5.49 (dd, J ¼ 3.5, 3.5 Hz, 1 H,
He12), 5.32 (ddd, J ¼ 3.7, 3.7, 3.5 Hz, 1 H, He2), 4.62 (d, J ¼ 3.9 Hz,
Following the procedure for the preparation of 41, compound 42
(97 mg, 68%) was synthesized from 38; white solid;
mp ¼ 114e117 ꢀC; RF ¼ 0.37 (silica gel, hexane/ethyl acetate, 8:2);
1
H,
He3),
2.08e1.94
(m,
6
H,
He1a þ He11a þ He11b þ He16a þ He18 þ He22a), 2.04 (s, 3 H,
He32), 2.03 (s, 3 H, He34), 1.87e1.82 (m, 1 H, He16b), 1.80e1.72 (m,
[
a
]
¼
þ42.30ꢀ (c
¼
0.68, CHCl3); UVevis (CHCl3): lmax
D
1
H,
He15a),
1.64e1.41
(m,
7
H,
(log ε) ¼ 234 nm (3.98); IR (KBr): ¼ 3424m, 2926s, 2870m, 1744vs,
n
He6a þ He6b þ He7a þ He9 þ He19 þ He21a þ He22b),
1.40e1.28 (m, 3 H, He1b þ He7b þ He21b), 1.16 (s, 3 H, He25), 1.13
(s, 3 H, He27), 1.12e1.07 (m, 1 H, He15b), 1.03 (s, 3 H, He23),
1.01e0.93 (m, 2 H, He5 þ He20), 0.98 (d, J ¼ 5.2 Hz, 3 H, He30),
0.93 (d, J ¼ 6.5 Hz, 3 H, He29), 0.89 (s, 3 H, He24), 0.73 (s, 3 H,
1668m, 1602w, 1580w, 1560w, 1514m, 1484s, 1456m, 1398m, 1366m,
1342w, 1318m, 1250vs, 1234s, 1194m, 1160w, 1090w, 1058m, 1030m,
710 m cmꢁ1
;
1H NMR (500 MHz, CDCl3):
d
¼ 7.70e7.68 (m, 2 H,
He37), 7.47e7.43 (m, 1 H, He39), 7.41e7.37 (m, 2 H, He38), 5.88 (s,
1 H, NH), 5.38 (dd, J ¼ 3.6, 3.6 Hz, 1 H, He12), 5.32 (ddd, J ¼ 3.8, 3.8,
3.4 Hz, 1 H, He2), 4.61 (d, J ¼ 3.9 Hz, 1 H, He3), 2.85e2.79 (m, 1 H,
He26) ppm; 13C NMR (125 MHz, CDCl3):
d
¼ 176.3 (Ce28), 170.8
(Ce33), 170.4 (Ce31), 140.4 (Ce13), 138.3 (Ce35), 129.0 (Ce37),
126.0 (Ce12), 124.1 (Ce38), 119.8 (Ce36), 78.0 (Ce3), 69.7 (Ce2),
55.3 (Ce5), 54.4 (Ce18), 48.7 (Ce17), 48.1 (Ce9), 42.9 (Ce14), 42.2
He22a), 2.47e2.41 (m, 1 H, He16a), 2.08e1.90 (m, 4 H,
He1a þ He11a þ He11b þ He16b), 2.04 (s, 3 H, He32), 2.02 (s, 3 H,
He34), 1.81e1.73 (m, 1 H, He15a), 1.64 (d, J ¼ 10.8 Hz, 1 H, He18),

S. Sommerwerk et al. / European Journal of Medicinal Chemistry 119 (2016) 1e16
15
1.60e1.39
(m,
7
H,
2.54e2.50 (m,
1
H, He22a), 2.15e1.90 (m,
5
H,
He6a þ He6b þ He7a þ He9 þ He19 þ He21a þ He22b),
1.38e1.22 (m, 3 H, He1b þ He7b þ He21b), 1.16 (s, 3 H, He25), 1.11
(s, 3 H, He27), 1.09e0.98 (m, 2 H, He15b þ He20), 1.02 (s, 3 H,
He23), 0.97 (d, J ¼ 6.2 Hz, 3 H, He30), 0.92e0.83 (m, 1 H, He5), 0.89
(s, 3 H, He24), 0.87 (d, J ¼ 6.4 Hz, 3 H, He29), 0.75 (s, 3 H,
He1a þ He11a þ He11b þ He16a þ He16b), 2.06 (s, 3 H, He32),
2.04 (s, 3 H, He34), 1.90e1.81 (m, 1 H, He15a), 1.65e1.52 (m, 7 H,
He6a þ He6b þ He7a þ He9 þ He18 þ He21a þ He22b),
1.48e1.41 (m, 2 H, He7b þ He19), 1.41e1.35 (m, 1 H, He1b), 1.24 (s,
3 H, He25), 1.23e1.18 (m, 1 H, He21b), 1.13e1.04 (m, 1 H, He15b),
1.11 (s, 3 H, He27), 1.08 (s, 3 H, He26), 1.07 (s, 3 H, He23), 1.04e0.97
(m, 2 H, He5 þ He20), 0.96 (d, J ¼ 6.2 Hz, 3 H, He30), 0.93 (s, 3 H,
He24), 0.86 (d, J ¼ 6.4 Hz, 3 H, He29) ppm; 13C NMR (125 MHz,
He26 ppm; 13C NMR (125 MHz, CDCl3):
d
¼ 170.8 (Ce33), 170.3
(Ce31), 165.9 (Ce35), 138.8 (Ce13), 135.5 (Ce36), 131.1 (Ce39),
128.5 (Ce38), 127.2 (Ce12), 126.7 (Ce37), 78.0 (Ce3), 69.7 (Ce2),
59.2 (Ce18), 57.5 (Ce17), 55.2 (Ce5), 48.1 (Ce9), 42.4 (Ce14), 42.2
(Ce1), 40.0 (Ce19), 39.9 (Ce8), 39.4 (Ce20), 37.4 (Ce4), 36.7
(Ce22), 36.7 (Ce10), 32.6 (Ce7), 31.4 (Ce21), 29.2 (Ce24), 26.8
(Ce15), 23.7 (Ce16), 23.4 (Ce27), 23.3 (Ce11), 21.4 (Ce32), 21.0
(Ce30), 21.0 (Ce34), 18.0 (Ce6), 17.8 (Ce23), 17.6 (Ce29), 17.1
(Ce26),16.3 (Ce25) ppm; MS (ESI): m/z (%) ¼ 632.2 ([M þ H]þ,100),
654.3 ([M þ Na]þ, 19), 685.7 ([M þ Na þ MeOH]þ, 8), 970.3
([3M þ 2Na]2þ,17),1263.3 ([2M þ H]þ,14),1285.4 ([2M þ Na]þ, 71);
analysis calculated for C40H57NO5 (631.88): C 76.03, H 9.09, N 2.22;
found: C 75.81, H 9.27, N 2.00.
CDCl3):
d
¼ 170.9 (Ce33), 170.5 (Ce31), 155.9 (Ce35), 151.1 (Ce36),
138.4 (Ce13), 129.3 (Ce38), 127.5 (Ce12), 125.2 (Ce39), 122.0
(Ce37), 78.1 (Ce3), 69.7 (Ce2), 58.8 (Ce18), 56.4 (Ce17), 55.3
(Ce5), 48.2 (Ce9), 42.3 (Ce14), 42.2 (Ce1), 40.1 (Ce8), 40.0 (Ce19),
39.3 (Ce20), 37.5 (Ce4), 37.1 (Ce22), 36.8 (Ce10), 32.9 (Ce7), 31.4
(Ce21), 29.3 (Ce24), 26.7 (Ce15), 23.7 (Ce16), 23.5 (Ce27), 23.3
(Ce11), 21.4 (Ce32), 21.0 (Ce34), 20.9 (Ce30), 18.1 (Ce6), 17.9
(Ce29), 17.6 (Ce23), 17.1 (Ce26), 16.3 (Ce25) ppm; MS (ESI): m/z
(%) ¼ 648.1 ([M þ H]þ, 100), 670.3 ([M þ Na]þ, 23), 1296.2
([2M þ H]þ, 18), 1317.3 ([2M þ Na]þ, 34); analysis calculated for
C40H57NO6 (647.88): C 74.15, H 8.87, N 2.16; found: C 73.96, H 9.01,
4.2.30. N-[2
b,3
b-Diacetyloxy-17
b-amino-28-norolean-12-en-17-
N. 2.05.
yl]-phenylcarbamate (43)
Following the procedure for the synthesis of 41, compound 43
(90 mg, 92%) was obtained from 37 and phenyl chloroformate
(89 mg, 0.57 mmol) as a white solid; mp ¼ 123e125 ꢀC; RF ¼ 0.62
4.2.32. N-[2b,3b-Diacetyloxy-17b-amino-28-norolean-12-en-17-
yl]-phenylurea (45)
A solution of compound 37 (130 mg, 0.25 mmol) in dry toluene
(10 mL) was treated with phenylisocyanate (44 mg, 0.37 mmol),
and the mixture was stirred 2 h at room temperature. Et2O (50 mL)
was added, and usual aq. work-up followed by column chroma-
tography (silica gel, hexane/ethyl acetate, 8:2) gave 45 (110 mg,
69%) as a white solid; mp ¼ 169e172 ꢀC; RF ¼ 0.46 (silica gel,
(silica gel, hexane/ethyl acetate, 7:3); [
a
]
¼ þ109.64ꢀ (c ¼ 0.30,
D
CHCl3); UVevis (CHCl3): lmax (log ε) ¼ 261 nm (2.57); IR (KBr):
n
¼ 3426w, 2950s, 2876m, 1746vs, 1486s, 1366m, 1252s, 1234s,
1200s, 1164m, 1030m, 986 m cmꢁ1 1H NMR (500 MHz, CDCl3):
;
d
¼ 7.36e7.31 (m, 2 H, He38), 7.19e7.15 (m, 1 H, He39), 7.10e7.05
(m,1 H, He37), 5.36 (dd, J ¼ 3.5, 3.5 Hz,1 H, He12), 5.33 (ddd, J ¼ 3.7,
3.7, 3.7 Hz, 1 H, He2), 4.89 (s, 1 H, NH), 4.64 (d, J ¼ 3.9 Hz, 1 H, He3),
2.28e2.22 (m, 2 H, He18 þ He22a), 2.05 (s, 3 H, He32), 2.04 (s, 3 H,
hexane/ethyl acetate, 7:3); [
a
]
¼ þ88.50ꢀ (c ¼ 0.34, CHCl3);
D
UVevis (CHCl3): lmax (log ε) ¼ 243 nm (4.15); IR (KBr):
n
¼ 3402m,
2950s, 2870m, 1746vs, 1700m, 1664m, 1598m, 1540s, 1498s, 1438m,
1364m, 1310m, 1252vs, 1232vs, 1056m, 1030m, 1012 m cmꢁ1 1H
NMR (500 MHz, CDCl3):
¼ 7.30e7.23 (m, 4 H, He37 þ He38),
He34),
2.02e1.72
(m,
8
H,
He16a
;
þ He16b þ He11a þ He11b þ He15a þ He1a þ He19a þ He22b),
1.66e1.50 (m, 4 H, He6a þ He6b þ He7a þ He9),1.46e1.18 (m, 5 H,
He7b þ He1b þ He21a þ He21b þ He19b), 1.22 (s, 3 H, He25),
1.16 (s, 3 H, He27), 1.10e0.99 (m, 2 H, He15b þ He5), 1.07 (s, 3 H,
He23), 1.03 (s, 3 H, He26), 0.98 (s, 3 H, He29), 0.92 (s, 6 H,
d
7.06e7.01 (m, 1 H, He39), 6.10 (s, 1 H, NH), 5.32 (ddd, J ¼ 3.7, 3.7,
3.7 Hz, 1 H, He2), 5.27 (dd, J ¼ 3.4, 3.4 Hz, 1 H, He12), 4.62 (d,
J ¼ 3.9 Hz, 1 H, He3), 4.51 (s, 1 H, NH), 2.44 (ddd, J ¼ 13.8, 3.3, 3.3 Hz,
1 H, He22a), 2.19e2.10 (m, 2 H, He18 þ He16a), 2.05 (s, 3 H, He32),
He24 þ He30) ppm; 13C NMR (125 MHz, CDCl3):
d
¼ 170.9 (Ce33),
2.04
(s,
3
H,
He34),
2.00e1.67
(m,
7
H,
170.4 (Ce31), 152.6 (Ce35), 151.1 (Ce36), 143.1 (Ce13), 129.3
(Ce38), 125.2 (Ce39), 124.8 (Ce12), 121.9 (Ce37), 78.1 (Ce3), 69.7
(Ce2), 55.9 (Ce17), 55.4 (Ce5), 48.2 (Ce9), 47.0 (Ce18), 46.4
(Ce19), 42.1 (Ce1), 41.8 (Ce14), 39.9 (Ce8), 37.5 (Ce4), 36.9 (Ce10),
35.3 (Ce21), 33.0 (Ce30), 32.9 (Ce22), 32.6 (Ce7), 30.9 (Ce20), 29.3
(Ce24), 26.2 (Ce15), 26.1 (Ce27), 24.1 (Ce29), 23.8 (Ce11), 22.1
(Ce16), 21.4 (Ce32), 21.0 (Ce34), 18.1 (Ce6), 17.8 (Ce23), 17.1
(Ce26), 16.1 (Ce25) ppm; MS (ESI): m/z (%) ¼ 648.1 ([M þ H]þ, 22),
670.3 ([M þ Na]þ, 28), 1317.5 ([2M þ Na]þ, 100); analysis calculated
for C40H57NO6 (647.88): C 74.15, H 8.87, N 2.16; found: C 73.87, H
8.97, N 1.96.
He16b þ He11a þ He11b þ He15a þ He22b þ He1a þ He19a),
1.63e1.44 (m, 4 H, He6a þ He6b þ He7a þ He9),1.38e1.14 (m, 5 H,
He7b þ He21a þ He21b þ He1b þ He19b),1.17 (s, 3 H, He25),1.13
(s,
3 H, He27), 1.06 (s, 3 H, He23), 1.05e0.94 (m, 2 H,
He15b þ He5), 0.96 (s, 3 H, He29), 0.91 (s, 3 H, He30), 0.91 (s, 3 H,
He24), 0.80 (s, 3 H, He26) ppm; 13C NMR (125 MHz, CDCl3):
d
¼ 170.9 (Ce33), 170.4 (Ce31), 154.3 (Ce35), 143.5 (Ce13), 139.1
(Ce36), 129.2 (Ce38), 124.4 (Ce12), 124.8 (Ce39), 120.8 (Ce37),
78.1 (Ce3), 69.8 (Ce2), 56.1 (Ce17), 55.3 (Ce5), 48.1 (Ce9), 47.8
(Ce18), 46.4 (Ce19), 42.0 (Ce1), 41.9 (Ce14), 39.8 (Ce8), 37.5 (Ce4),
36.8 (Ce10), 35.4 (Ce21), 33.3 (Ce22), 33.0 (Ce30), 32.4 (Ce7), 30.9
(Ce20), 29.2 (Ce24), 26.3 (Ce15), 26.0 (Ce27), 24.2 (Ce29), 23.8
(Ce11), 22.4 (Ce16), 21.4 (Ce32), 21.0 (Ce34), 18.1 (Ce6), 17.8
(Ce23), 16.9 (Ce26), 16.1 (Ce25) ppm; MS (ESI): m/z (%) ¼ 647.2
([M þ H]þ, 100), 669.3 ([M þ Na]þ, 10), 993.7 ([3M þ 2Na]2þ, 12),
1293.2 ([2M þ H]þ, 48); analysis calculated for C40H58N2O5
(646.90): C 74.27, H 9.04, N 4.33; found: C 73.97, H 9.28, N 4.09.
4.2.31. N-[2b,3b-Diacetyloxy-17b-amino-28-norurs-12-en-17-yl]-
phenylcarbamate (44)
Compound 44 (82 mg, 83%) was obtained as described above for
41 from 38 and phenyl chloroformate (89 mg, 0.57 mmol) as a
white solid; mp ¼ 102e104 ꢀC; RF ¼ 0.54 (silica gel, chloroform/
hexane/ethyl acetate, 10:9:1); [
a
]
¼ þ95.00ꢀ (c ¼ 0.77, CHCl3);
D
UVevis (CHCl3): lmax (log ε) ¼ 271 nm (3.06); IR (KBr):
n
¼ 3420m,
4.2.33. N-[2b,3b-Diacetyloxy-17b-amino-28-norurs-12-en-17-yl]-
2950s, 2870m, 1746vs, 1606w, 1596m, 1504m, 1484s, 1456m, 1432m,
1398m, 1364s, 1344w, 1314m, 1252s, 1234s, 1198vs, 1162m, 1116w,
1090w, 1056m, 1042m, 1026m, 986m, 966m, 944m, 690 m cmꢁ1; 1H
phenylurea (46)
As described for 45, compound 46 (93 mg, 58%) was synthesized
from 38 and obtained as a white solid; mp ¼ 173e174 ꢀC; RF ¼ 0.44
NMR (500 MHz, CDCl3):
d
¼ 7.36e7.31 (m, 2 H, He38), 7.17 (t,
(silica gel, hexane/ethyl acetate, 7:3); [
a
]
¼ þ69.20ꢀ (c ¼ 1.20,
D
J ¼ 7.4 Hz, 1 H, He39), 7.07 (d, J ¼ 7.7 Hz, 2 H, He37), 5.36e5.31 (m,
CHCl3); UVevis (CHCl3): lmax (log ε) ¼ 243 nm (4.17); IR (KBr):
2 H, He2 þ He12), 4.90 (s, 1 H, NH), 4.64 (d, J ¼ 3.9 Hz, 1 H, He3),
n
¼ 3388m, 3136w, 2950s, 2870m, 1746vs, 1700s, 1660s, 1600s,

16
S. Sommerwerk et al. / European Journal of Medicinal Chemistry 119 (2016) 1e16
1507e1514.
1542s, 1498vs, 1456s, 1440s, 1398m, 1366s, 1342m, 1314s, 1290m,
1252vs, 1232vs, 1194s, 1176m, 1160m, 1116w, 1078m, 1058s, 1030s,
[7] R. Hiramatsu, S. Fukuhara, S. Mitsuda, T. Yokomichi, T. Kataoka, Betulinic acid
and oleanolic acid, natural pentacyclic triterpenoids, interfere with N-linked
glycan modifications to intercellular adhesion molecule-1, but not its intra-
cellular transport to the cell surface, Eur. J. Pharmacol. 767 (2015) 126e134.
[8] Y. Bi, X. Yang, T. Zhang, Z. Liu, X. Zhang, J. Lu, K. Cheng, J. Xu, H. Wang, G. Lv,
P.J. Lewis, Q. Meng, C. Ma, Design, synthesis, nitric oxide release and anti-
bacterial evaluation of novel nitrated ocotillol-type derivatives, Eur. J. Med.
Chem. 101 (2015) 71e80.
996m, 972m, 946m, 750s, 692m, 666m, 604 m cmꢁ1 1H NMR
;
(500 MHz, CDCl3):
d
¼ 7.27e7.21 (m, 4 H, He37 þ He38), 7.04e7.00
(m, 1 H, He39), 6.20 (s, 1 H, NH), 5.31 (ddd, J ¼ 3.7, 3.7, 3.7 Hz, 1 H,
He2), 5.20 (dd, J ¼ 3.5, 3.5 Hz, 1 H, He12), 4.61 (d, J ¼ 5.4 Hz, 1 H,
He3), 4.60 (s, 1 H, NH), 2.76e2.66 (m, 1 H, He22a), 2.26e2.20 (m,
1 H, He16a), 2.05 (s, 3 H, He32), 2.03 (s, 3 H, He34), 2.00e1.76 (m,
5 H, He1a þ He11a þ He11b þ He15a þ He16b), 1.60e1.38 (m,
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Design, synthesis and antitumor activity of triterpenoid pyrazine derivatives
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8
H,
He6a þ He6b þ He7a þ He9 þ He18 þ He19 þ He21a þ He22b),
1.37e1.30 (m, 2 H, He1b þ He7b),1.23e1.18 (m, 1 H, He21b),1.17 (s,
3 H, He25), 1.06 (s, 3 H, He27), 1.05 (s, 3 H, He23), 1.04e0.99 (m,
1 H, He15b), 0.99e0.92 (m, 2 H, He5 þ He20), 0.94 (d, J ¼ 5.9 Hz,
3 H, He30), 0.90 (s, 3 H, He24), 0.82 (d, J ¼ 5.8 Hz, 3 H, He29), 0.79
€
[12] B. Siewert, J. Wiemann, A. Kowitsch, R. Csuk, The chemical and biological
(s, 3 H, He26) ppm; 13C NMR (125 MHz, CDCl3):
d
¼ 170.9 (Ce33),
potential of C ring modified triterpenoids, Eur. J. Med. Chem. 72 (2014)
84e101.
170.4 (Ce31), 154.2 (Ce35), 139.2 (Ce36), 138.8 (Ce13), 129.2
(Ce38), 127.1 (Ce12), 123.4 (Ce39), 120.9 (Ce37), 78.1 (Ce3), 69.7
(Ce2), 59.4 (Ce18), 56.5 (Ce17), 55.3 (Ce5), 48.1 (Ce9), 42.3
(Ce14), 42.2 (Ce1), 40.0 (Ce8), 39.9 (Ce19), 39.4 (Ce20), 37.5
(Ce4), 37.5 (Ce22), 36.7 (Ce10), 32.7 (Ce7), 31.5 (Ce21), 29.2
(Ce24), 26.8 (Ce15), 23.7 (Ce11 þ Ce16), 23.4 (Ce27), 21.4 (Ce32),
21.0 (Ce34), 20.9 (Ce30), 18.1 (Ce6), 17.8 (Ce23), 17.6 (Ce29), 16.9
(Ce26), 16.3 (Ce25) ppm; MS (ESI): m/z (%) ¼ 647.3 ([M þ H]þ, 86),
669.3 ([M þ Na]þ, 31), 992.3 ([3M þ 2Na]2þ, 20), 1293.3 ([2M þ H]þ,
94), 1315.4 ([2M þ Na]þ, 100); analysis calculated for C40H58N2O5
(646.90): C 74.27, H 9.04, N 4.33; found: C 74.03, H 9.27, N 4.15.
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cells in culture and their development into established lines, J. Cell Biol. 17
(1963) 299e313.
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Acknowledgments
Thanks are due to Dr. R. Kluge for measuring the ESI-MS spectra,
€
and to Dr. D. Strohl and his team for numerous NMR spectra. We
like to thank J. Wiese, MSc, for measuring optical rotations, UVeVis
and IR spectra, and Ms J. Pech for the micro-analyses. The cell lines
were kindly provided by Dr. Thomas Müller (Dept. of Haematology/
Oncology, Martin-Luther Universitat Halle-Wittenberg). Support by
the “Gründerwerkstatt e Biowissenschaften” and the “Wissen-
schaftsCampus Halle WCH” is gratefully acknowledged.
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€
Appendix A. Supplementary data
€
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and apoptotic activity, Eur. J. Med. Chem. 56 (2012) 237e245.
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