Betulinic acid derived hydroxamates and betulin derived carbamates are interesting scaffolds for the synthesis of novel cytotoxic compounds

Article abstract of DOI:10.1016/j.ejmech.2015.10.043

The betulinic acid-derived hydroxamates 5-18, the amides 19-24, and betulin-derived bis-carbamates 25-28 as well as the carbamates 31-40 and 44-48 were prepared and evaluated for their antiproliferative activity in a photometric sulforhodamine B (SRB) assay against several human cancer cell lines and nonmalignant mouse fibroblasts (NIH 3T3). While for 3-O-acetyl hydroxamic acid 5 EC₅₀ values as low as EC₅₀ = 1.3 μM were found, N,O-bis-alkyl substituted hydroxamates showed lowered cytotoxicity (EC₅₀ = 16-20 μM). In general, hydroxamic acid derivatives showed only reduced selectivity for tumor cells, except for allyl substituted compound 13 (EC₅₀ = 5.9 μM for A2780 human ovarian carcinoma cells and EC₅₀ > 30 μM for nonmalignant mouse fibroblasts). The cytotoxicity of betulinic acid derived amides 19-24 and of betulin derived bis-carbamates 25-28 was low, except for N-ethyl substituted 25. Hexyl substituted 39 showed EC₅₀ = 5.6 μM (518A2 cells) while for mouse fibroblasts EC₅₀ > 30 was determined.

Full text of DOI:10.1016/j.ejmech.2015.10.043

European Journal of Medicinal Chemistry 106 (2015) 194e210
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Betulinic acid derived hydroxamates and betulin derived carbamates
are interesting scaffolds for the synthesis of novel cytotoxic
compounds
*
€
ꢀ
Jana Wiemann, Lucie Heller, Vincent Perl, Ralph Kluge, Dieter Strohl, 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:
The betulinic acid-derived hydroxamates 5e18, the amides 19e24, and betulin-derived bis-carbamates
25e28 as well as the carbamates 31e40 and 44e48 were prepared and evaluated for their anti-
proliferative activity in a photometric sulforhodamine B (SRB) assay against several human cancer cell
lines and nonmalignant mouse fibroblasts (NIH 3T3). While for 3-O-acetyl hydroxamic acid 5 EC50 values
Received 27 August 2015
Received in revised form
20 October 2015
Accepted 26 October 2015
Available online 5 November 2015
as low as EC50 ¼ 1.3
m
M were found, N,O-bis-alkyl substituted hydroxamates showed lowered cytotox-
icity (EC50 ¼ 16e20
m
M). In general, hydroxamic acid derivatives showed only reduced selectivity for
tumor cells, except for allyl substituted compound 13 (EC50 ¼ 5.9
m
M for A2780 human ovarian carci-
M for nonmalignant mouse fibroblasts). The cytotoxicity of betulinic acid
derived amides 19e24 and of betulin derived bis-carbamates 25e28 was low, except for N-ethyl
Keywords:
noma cells and EC50 > 30
m
Triterpenes
Betulinic acid
Betulin
Hydroxamates
Carbamates
Tumor cells
substituted 25. Hexyl substituted 39 showed EC50 ¼ 5.6
m
M (518A2 cells) while for mouse fibroblasts
EC50 > 30 was determined.
© 2015 Elsevier Masson SAS. All rights reserved.
1. Introduction
class of compounds bearing high potential as antitumor-active
compounds [20,21], to our knowledge, there are only two reports
describing a hydroxamic acid derived from glycyrrhetinic acid.
These compounds (Fig. 1) acted as selective inhibitors of 11b-
hydroxysteroid dehydrogenase 2; there were no data provided
whether these compounds were cytotoxic.
During our continuing search for antitumor active compounds
from natural products [20e22] we became interested in the syn-
thesis of betulinic acid (BA, 1, Fig. 1) derived hydroxamic acid and
derivatives and their cytotoxicity. Previous studies suggested
introducing a sulfamate [23,24] or carbamate [25,26] to the pen-
tacyclic skeleton of triterpenes can significantly improve the cyto-
toxicity of these compounds. Thus, we decided to prepare several
mono- and bis-carbamates derived from betulin (2, Fig. 1). Betulin
as well as betulinic acid have been shown to be interesting scaffolds
for developing analogs displaying various biological and medicinal
properties especially potent anticancer effects [27e37].
Hydroxamic acids have been known since Lossen's discovery of
oxalohydroxamic acid (Fig.1) in 1869 [1,2]. Then and for many years
to follow they were regarded as rather exotic compounds. Nowa-
days, they have been recognized [3] as a unique family of com-
pounds that hold a wide spectrum of biological activities. They act
as selective inhibitors of many enzymes, such as matrix metal-
loproteinases [4,5], hydrolases [6], ureases [7], lipoxygenase [8],
tumor necrosis factor-a converting enzyme [9,10], carbonic anhy-
drase [11,12], ribonucleotide reductase [13,14] and many others.
Their acidity is much weaker than that of structurally related car-
boxylic acids but the hydroxamic acid moiety may act as a bidentate
ligand to chelate with several metal ions. It also controls multiple
sites for potential hydrogen bond interactions with enzymes and
receptors [15].
Nonspecific hydroxamic acid derivatives have been prepared
starting inter alia from benzodiazepines [16],
a-amino-suberic
acids [17], N-alkylated amino acids [18] as well as from arylsulfo-
namides [19]. Although triterpenoic acids represent an important
2. Results and discussion
2.1. Chemistry
€
* Corresponding author. Bereich Organische Chemie, Martin-Luther Universitat
Halle-Wittenberg, Kurt-Mothes-Str. 2, D-06120 Halle (Saale), Germany.
Our synthetic approach started from BA whose acetylation gave
3-O-acetyl-BA (3, Scheme 1) while from the Jones oxidation of BA
E-mail address: rene.csuk@chemie.uni-halle.de (R. Csuk).
http://dx.doi.org/10.1016/j.ejmech.2015.10.043
0223-5234/© 2015 Elsevier Masson SAS. All rights reserved.

J. Wiemann et al. / European Journal of Medicinal Chemistry 106 (2015) 194e210
195
methylhydroxylammonium
chloride
or
O-methylhydrox-
ylammonium chloride or O-allylhydroxylammonium chloride in
the presence of triethylamine furnished products 7, 9, 11 and 13;
their deacetylation yielded compounds 8, 10, 12 or 14, respectively.
Under similar conditions betulonic acid (4) gave substituted
hydroxamic acids 15e18. Yields dropped slightly for these reactions
because of the accompanying formation of C-3-oximes.
For comparison, we prepared several betulinic acid derived
amides as well as betulin derived carbamates. Reaction of 3-O-
acetyl-betulinic acid (3) with oxalyl chloride (Scheme 2) followed
by a reaction with dry ammonia in DCM furnished amide 19 in 95%
yield. Deacetylation of 19 with potassium hydroxide in methanol
gave amide 20 [47,48]. In a similar way, from the reaction of 3 with
oxalyl chloride and benzylamine, benzylamide 21 was obtained
whose deacetylation yielded 22; the Jones oxidation of 21 gave 3-
oxo compound 23. Following the procedure given for the synthe-
sis of 12, from betulonic acid (4) with dry ammonia in DCM 3-oxo-
amide 24 [49e52] was obtained. Compound 19 is characterized in
Fig. 1. Structure of Lossen's oxalohydroxamic acid (A) and a representative glycyr-
rhetinic acid derived hydroxamic acid (B), betulinic acid (BA, 1) and betulin (2).
betulonic acid (4) was obtained [38,39]. Treatment of 3 with oxalyl
chloride in DCM for 2 h at 25 ꢀC followed by adding hydrox-
ylammonium chloride in the presence of trimethylamine [40]
provided a 68% yield of hydroxamic acid 5. Deacetylation of 5
with potassium hydroxide in methanol gave 63% of 6 [41].
While the reaction of 1 with oxalyl chloride/hydroxylammonium
chloride gave target compound 5 nicely, the reaction of 1 with
propylphosphonic anhydride (T3P) or with1,10-carbonyldiimidazole
failed togive highyields undera broad varietyof different conditions
[42e44].
its 1H NMR spectrum by the presence of a signal at
d
¼ 5.55 ppm
that was assigned to the CONH2 moiety. In addition, the signal for
H-19 was shifted to lower fields (compared to parent compound 3
showing Dd ¼ 0.08 ppm). In the 13C NMR spectrum the CONH2
moiety was detected at
d
¼ 179.3 ppm.
Hydroxamic acid 5 is characterized in its 13C NMR spectrum by a
Previous studies suggested the introduction of a sulfamate [23]
or carbamate [25] to the skeleton of a pentacyclic triterpenoid can
significantly improve its cytotoxicity. Thus, we decided to prepare
several bis-carbamates. The synthesis of these bis-carbamates
25e29 started from betulin (2, Scheme 3). While the reaction of
betulin with ethyl isocyanate in refluxing chloroform for 48 h gave
only low yields, the microwave assisted reaction of 2 with ethyl
isocyanate in dry THF worked nicely, and 3,28-bis-N-ethyl-carba-
mate 25 was obtained in 81% isolated yield. Similarly bis-
carbamates 26e28 were prepared.
signal at
d
¼ 175.1 ppm being assigned to the CONHOH moiety (C-
28) of 5. For comparison, C-28 was found in 3 at
shifting of the resonance signal of carbonyl carbon C-28 to higher
fields is typical for hydroxamic acids [45,46]. In the IR spectrum the
CeO stretch vibration was detected at
Reaction of
with N,O-dimethylhydroxylammonium
d
¼ 182.5 ppm. This
n
¼ 1643 cmꢁ1
.
3
with oxalyl chloride followed by reaction
chloride,
or
N-
For the synthesis of 3-O-acetyl-28-N-alkyl-carbamates 31e35,
3-O-acetyl-betulin (30) was used as a starting material. Compound
30 is easily accessible from betulin; thus, diacetylation of betulin
gave diacetate 29 whose selective deacetylation with KOH in
MeOH/THF at 0 ꢀC yielded 57% of monoacetate 30.
The microwave assisted reaction of 30 with alkyl isocyanates or
phenyl isocyanate allowed a quick and reliable synthesis of 3-O-
acetylated 28-N-substituted carbamates 31e35. From their deace-
tylation with potassium hydroxide in MeOH compounds 36e40
were obtained.
The synthesis of 3-oxo-28-N-alkyl-carbamates 44e48 (Scheme
4) started from 3-oxo-betulin 43, and the microwave assisted re-
action of 43 with alkyl isocyanates in THF gave target compounds
44e48 in good yields. The starting material for these reactions, 43,
was obtained from a deacetylation reaction of 42 (KOH in MeOH) in
90% isolated yield. Compound 42 was easily prepared by Jones
oxidation of 41 (72% yield); the latter was made by a selective
acetylation of betulin in 60% isolated yield.
2.2. Biology
The betulinic acid-derived hydroxamates 5e18, the amides
19e24, and betulin-derived bis-carbamates 25e28 as well as the
carbamates 31e40 and 44e48 were evaluated for their anti-
proliferative activity in a photometric sulforhodamine B (SRB) assay
[53e56] against several human cancer cell lines and nonmalignant
mouse fibroblasts (NIH 3T3). For comparison, betulinic acid (1),
betulin (2), betulonic acid (4) and acetates 3, 29 and 30 were
included into this screening (Table 1).
Scheme 1. Synthesis of betulinic acid derived hydroxamic acids 5e18: a) Ac2O, NEt3,
pyridine, DMAP, 12 h, 25 ꢀC, 75%; b) Jones oxidation (4 h, 25 ꢀC), 81%; c) oxalyl chloride,
DCM, 2 h, 25 ꢀC, then NHR1OR2, NEt3, DCM, 2e12 h, 25 ꢀC: 5 (from NH2OH$HCl, 68%), 7
(from HNMeOMe$HCl, 77%), 9 (from HNMeOH$HCl, 66%), 11 (from NH2OMe$HCl, 63%),
13 (from NH2OAll$HCl, 90%), 15 (from HNMeOMe$HCl, 52%), 16 (from HNMeOH$HCl,
81%), 17 (from NH2OMe$HCl, 45%), 18 (from NH2OAll$HCl, 68%); d) KOH in MeOH,
25 ꢀC: 6 (4 d, 63%), 8 (5 d, 89%), 10 (5 d, 60%), 12 (5 d, 95%), 14 (7 d, 94%).
Betulin (2) and its diacetate 29 displayed no (EC50 > 30 mM;
cut-off of the assay) cytotoxicity; low activity was found for the
betulin-monoacetate 30. Betulinic acid (1) is well-known for its

196
J. Wiemann et al. / European Journal of Medicinal Chemistry 106 (2015) 194e210
Scheme 2. Synthesis of betulinic or betulonic acid derived amides 19e24: a) oxalyl chloride, DCM 2 h 25 ꢀC, then NH3, 1 h, 25 ꢀC, 95% (for 19) and 84% (for 24); b) oxalyl chloride,
DCM, 2 h, 25 ꢀC, then BnNH2, 1 h, reflux, 59%; c) KOH, MeOH, THF, 1 d, 25 ꢀC, 97% (for 20) and 92% (for 22); d) Jones oxidation, 20 min, 25 ꢀC, 74%; f) a) oxalyl chloride, DCM, 2 h,
25 ꢀC, then NH3, 1 h, 25 ꢀC, 84%.
Scheme 3. Synthesis of betulin derived 3,28-bis-carbamates 25e28 and 28-mono-carbamates 31e40: a) microwave assisted (7 h, 120 ꢀC, THF, RNCO): 25 (from EtNCO, 81%), 26
(from PropNCO, 83%), 27 (from ButNCO, 81%), 28 (from HexNCO, 73%); b) Ac2O, NEt3, DMAP, DCM, 12 h, 25 ꢀC, 90%; c) KOH, MeOH, THF, 0 ꢀC, 30 min, 57%; d) KOH, MeOH, THF, 25 ꢀC:
36 (2 d, 95%), 37 (1 d, 93%), 38 (2 d, 94%), 39 (1 d, 88%, 40 (2 d, 89%).
Scheme 4. Synthesis of 3-oxo-betulin derived 28-mono-carbamates 44e48: a) Ac2O, NEt3, DMAP, 12 h, 25 ꢀC, 60%; b) Jones oxidation, 40 min, 25 ꢀC, 72%; c) KOH, MeOH, THF, 12 h,
25 ꢀC, 90%; d) miocrowave assisted (7 h, 120 ꢀC, THF, RNCO): 44 (from EtNCO, 68%), 45 (from PropNCO, 62%), 46 (from ButNCO, 72%), 47 (from HexNCO, 55%), 48 (from PhNCO, 85%).
cytotoxicity, and EC50 values between 8.8 and 17.1
determined while for betulonic acid (4) EC50 values between
EC50 ¼ 13.1e29.0 M were found. Betulinic acid 3-acetate 3
showed higher cytotoxicity, especially for 518A2 human mela-
noma cells (EC50 ¼ 4.5 M). Betulinic acid derived 3-O-acetyl
hydroxamic acid 5 showed significantly higher cytotoxicity, and
m
M were
EC50 values as low as EC50 ¼ 1.3
stitution (as in 7) in the hydroxamate moiety lowered cytotoxicity
M) significantly. The presence of at least one
acidic hydrogen substituent in the hydroxamic acid part seems
mandatory to obtain cytotoxic compounds: Compounds 9 and 11
mM were found. Bis-alkyl sub-
m
(EC50 ¼ 16e20
m
m
gave EC50 values between EC50
¼
2.0e13.5 mM. For allyl-

J. Wiemann et al. / European Journal of Medicinal Chemistry 106 (2015) 194e210
197
Table 1
19e24, and betulin-derived bis-carbamates 25e28 as well as the
carbamates 31e40 and 44e48 were prepared and evaluated for
their antiproliferative activity in a photometric sulforhodamine B
(SRB) assay against several human cancer cell lines and nonma-
lignant mouse fibroblasts (NIH 3T3). While betulin (2) and its
diacetate 29 exhibited no cytotoxicity, low activity was found for
the betulin-monoacetate 30. Betulinic acid 3-acetate 3 showed
higher cytotoxicity than its parent compound betulinic acid, but
significantly lower EC50 values were determined for 3-O-acetyl
Cytotoxicity of betulinic acid-derived hydroxamates 5e18 and starting materials
1e4, 29, 30 (EC50 values in mM from SRB assays after 96 h of treatment; the values are
averaged from three independent experiments performed each in triplicate; confi-
dence interval CI ¼ 95%). The cell lines are human cancer cell lines: 518A2 (mela-
noma), A2780 (ovarian carcinoma), A549 (alveolar basal epithelial adenocarcinoma),
MCF7 (breast adenocarcinoma) and non-malignant mouse fibroblasts (NIH 3T3).
518A2
A2780
A549
MCF7
NIH 3T3
1
2
9.4 0.7
>30
8.8 0.9
>30
17.1 1.1
>30
10.2 1.2
>30
16.1 1.4
>30
hydroxamic acid 5, and EC50 values as low as EC50 ¼ 1.3
found. Bis-alkyl substitution in the hydroxamate moiety lowered
M). The presence of at least one acidic
hydrogen substituent in the hydroxamic acid part seems manda-
tory to obtain cytotoxic compounds. Despite their low EC50 values,
hydroxamic acid derivatives showed only reduced selectivity for
tumor cells, except for allyl substituted compound 13
mM were
3
4
29
30
5
6
7
8
9
10
11
12
13
14
15
16
17
18
4.5 0.1
29.0 2.0
>30
18.3 0.5
20.4 1.9
>30
11.0 1.3
1.3 0.1
3.7 0.5
19.3 1.1
7.6 0.3
2.0 0.1
6.2 0.4
25.9 2.5
>30
14.0 2.4
1.3 0.1
5.3 0.6
20.1 1.4
19.0 3.0
4.3 0.7
11.0 0.5
25.9 2.0
>30
11.2 1.4
1.4 0.1
3.9 0.6
16.4 0.9
10.2 1.4
2.3 0.3
>30
13.1 0.9
>30
cytotoxicity (EC50 ¼ 16e20
m
15.2 1.6
1.6 0.1
2.4 0.2
17.5 0.6
7.3 0.5
3.5 0.3
Not soluble
8.0 0.6
5.8 1.2
7.6 0.1
6.7 0.1
>30
>30
1.1 0.1
2.0 0.5
17.4 1.1
7.7 1.3
2.9 0.6
(EC50 ¼ 5.9
mM for A2780 human ovarian carcinoma cells and
EC50 > 30 M for nonmalignant mouse fibroblasts). The cytotoxicity
m
8.9 0.5
12.3 0.9
5.9 0.6
7.3 0.7
20.7 1.6
3.0 0.2
12.0 1.5
19.7 2.0
11.7 2.0
6.9 0.2
6.6 1.5
4.1 1.7
14.5 1.3
6.0 0.3
6.1 1.1
25.0 2.1
13.5 0.9
21.9 0.7
12.2 2.0
8.6 1.0
18.7 1.2
10.2 0.9
13.8 1.6
18.4 1.9
9.5 0.5
15.2 0.5
>30
6.1 0.4
26.7 2.0
5.0 0.4
12.7 1.1
19.7 1.7
of betulinic acid derived amides 19e24 as well as of betulin derived
bis-carbamates 25e28 was low, except for N-ethyl substituted 25.
While almost all of these compounds showed more or less the same
cytotoxicity for tumor cell lines as well as for mouse fibroblasts,
6.3 0.2
17.2 1.6
22.4 1.5
hexyl substituted 39 showed EC50 ¼ 5.6
mM (518A2 cells) while for
mouse fibroblasts EC50 > 30 was determined.
4. Experimental section
substituted 14 EC50 values of the same magnitude as for 11 were
found (Table 2).
4.1. General e chemistry
Deacetylated compound 6 also gave low EC50 values, but this
compound was less active than parent 5. Substituted hydroxamic
acids 8, 12 and 14 were of similar cytotoxicity. Despite the low EC50
values, hydroxamic acid derivatives showed only reduced selec-
tivity for tumor cells, except for allyl substituted compound 13
Melting points are uncorrected (Leica hot stage microscope),
NMR spectra were recorded using the Varian spectrometers Gemini
2000 or Unity 500 (d given in ppm, J in Hz, internal Me4Si, MS
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%. Betulinic acid (1) and betulin (2) and
were obtained from different commercial suppliers in bulk quan-
tities. Microwave assisted reactions were performed in a Mono-
wave 300 apparatus (Anton Parr, Austria).
(EC50 ¼ 5.9
mM for A2780 human ovarian carcinoma cells and
EC50 > 30 M for nonmalignant mouse fibroblasts) (Table 3).
m
The cytotoxicity of amides 19e24, however, was moderate; the
lowest EC50 value was determined for 3-O-acetylated compounds
19 and 21. The cytotoxicity of betulin derived bis-carbamates
25e28 was also low, except for N-ethyl substituted 25; the same
was observed for their 3-O-acetylated analogues 31e35 and their 3-
oxo-analogues 44e48.
Significantly improved cytotoxicity was found for carbamates
36e40 holding a free hydroxyl group at position C-3. While almost
all of these compounds showed more or less the same cytotoxicity
for tumor cell lines as well as for mouse fibroblasts, hexyl
4.2. General e biological screening
substituted 39 showed EC50 ¼ 5.5
mM (518A2 cells) while for mouse
fibroblasts EC50 > 30 was determined.
The SRB assay was performed as previously described [53e56].
4.3. Syntheses
3. Conclusion
4.3.1. (3
b) 3-Acetyloxy-lup-20(29)-en-28-acid (3)
The betulinic acid-derived hydroxamates 5e18, the amides
Prepared by reaction (12 h, 25 ꢀC) of 1 (4.0 g, 8.78 mmol) with
Table 2
Cytotoxicity of betulinic acid derived amides 19e24 (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%). The cell lines are human cancer cell lines: 518A2 (melanoma), A2780 (ovarian carcinoma), HT29 (colorectal
adenocarcinoma), MCF7 (breast adenocarcinoma), A549 (alveolar basal epithelial adenocarcinoma), HeLa (epitheloid cervix carcinoma) and non-malignant mouse fibroblasts
(NIH 3T3).
518A2
A2780
HT-29
MCF7
A549
HeLa
NIH 3T3
19
20
21
22
23
24
13.4 1.1
9.9 0.7
6.2 1.3
>30
>30
21.5 1.9
5.7 0.6
15.7 1.4
6.2 1.1
11.8 1.4
7.4 1.1
15.6 1.4
11.5 0.7
10.2 1.3
>30
11.1 0.6
10.7 0.5
>30
18.0 2.1
>30
12.4 1.4
8.2 0.9
10.3 0.8
>30
>30
14.7 0.9
12.0 1.1
11.2 1.2
16.9 1.8
>30
>30
22.9 1.7
20.0 1.3
10.7 0.6
>30
11.1 0.9
>30
e
>30
17.9 1.4
10.3 1.1
17.7 1.1

198
J. Wiemann et al. / European Journal of Medicinal Chemistry 106 (2015) 194e210
Table 3
Cytotoxicity of betulin derived bis-carbamates 25e28 and mono-carbamates 31e40 and 44e48 (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%). The cell lines are human cancer cell lines: 518A2 (melanoma), A2780
(ovarian carcinoma), HT29 (colorectal adenocarcinoma), MCF7 (breast adenocarcinoma), A549 (alveolar basal epithelial adenocarcinoma), HeLa (epitheloid cervix carcinoma)
and non-malignant mouse fibroblasts (NIH 3T3).
518A2
A2780
HT-29
MCF7
A549
HeLa
NIH 3T3
25
26
27, 28
31
32e35
36
37
38
39
40
12.6 1.3
>30
>30
10.2 1.3
>30
12.5 1.6
15.9 2.0
8.0 1.1
5.5 0.7
23.9 1.4
16.6 1.1
>30
5.8 0.6
19.4 2.0
>30
11.1 1.4
>30
4.6 0.7
10.6 1.5
7.0 0.9
7.7 0.9
7.2 1.4
8.3 0.9
>30
8.3 0.9
>30
>30
>30
>30
23.3 2.0
>30
>30
20.4 2.5
>30
12.1 1.1
15.5 1.4
12.5 0.8
>30
10.7 1.3
>30
>30
14.1 1.2
>30
12.0 1.6
14.2 2.1
8.6 0.5
12.7 1.4
>30
17.8 0.9
>30
>30
17.0 1.5
>30
>30
9.4 0.9
10.1 1.2
7.4 1.1
11.5 1.3
13.4 1.2
>30
>30
>30
>30
>30
>30
>30
8.8 1.0
7.3 0.8
5.7 0.7
>30
14.3 2.0
4.6 0.7
>30
16.3 1.1
7.5 1.0
>30
16.3 2.5
11.5 0.9
>30
24.6 1.9
19.7 2.5
>30
44
45e48
20.3 1.9
>30
NEt3 (2.2 mL, 15.90 mmol), acetic anhydride (1.6 mL, 16.94 mmol)
and catalytic amounts of DMAP in dry pyridine (80 mL), and 3
(3.28 g, 75%) was obtained as a colorless solid; RF ¼ 0.60 (silica gel,
toluene/ethyl acetate/heptane/formic acid, 80:20:10:3); m.p.:
3H, CH3 (25)), 0.80e0.74 (m, 1H, CH (5)) ppm; 13C NMR (100 MHz,
CDCl3):
¼ 175.1 (C]O, C28), 171.1 (C]O, C31), 150.2 (Cq, C20),
d
109.8 (CH2]C, C29), 81.0 (CH, C3), 55.4 (CH, C5), 54.3 (Cq, C17), 50.4
(CH, C9), 50.4 (CH, C18), 46.9 (CH, C19), 42.4 (Cq, C14), 40.8 (Cq, C8),
38.4 (CH2, C1), 38.3 (CH2, C22), 38.0 (CH, C13), 37.8 (Cq, C4), 37.1 (Cq,
C10), 34.3 (CH2, C7), 32.8 (CH2, C16), 30.7 (CH2, C21), 29.4 (CH2,
C15), 27.9 (CH3, C23), 25.5 (CH2, C12), 23.7 (CH2, C7), 21.3 (CH3, C32),
20.8 (CH2, C11), 19.3 (CH3, C30), 18.2 (CH2, C6), 16.5 (CH3, C26), 16.2
(CH3, C25), 16.1 (CH3, C24), 14.6 (CH3, C27) ppm; MS (ESI, MeOH):
m/z ¼ 514.4 (100%, [MþH]þ), 536.3 (30%, [MþH]þ), 1027.3 (50%,
[2M þ H]þ), 1049.4 (90%, [2M þ Na]þ); analysis calcd for C32H51NO4
(513.75): C 74.81, H 10.01, N 2.73; found: C 74.63, H 10.17, N 2.51.
275e277 ꢀC (lit.: [57] 277e278 ꢀC; [
a
]
D
¼ þ20.54ꢀ (c ¼ 0.58, CHCl3)
(lit.: [57] þ22ꢀ (c ¼ 0.49, CHCl3); MS (ESI, MeOH): m/z ¼ 497.4 (25%,
[M ꢁ H]ꢁ), 543.1 (100%, [M þ HCO2]ꢁ), 995.3 (100%, [2M ꢁ H]ꢁ),
1017.8 (8%, [2M ꢁ 2H þ Na]ꢁ).
4.3.2. Betulonic acid (4)
Prepared from 1 (13.1 g, 28.7 mmol) by Jones oxidation (4 h,
25 ꢀC) as previously reported [38], followed by chromatographic
workup (silica gel, hexanes/ethyl acetate, 9:1), and 4 (10.6 g, 81%)
was obtained as a colorless solid; RF ¼ 0.79 (silica gel, hexanes/ethyl
acetate, 7:3); m.p.: 242e244 ꢀC (lit.: [38] 245e247 ꢀC;
4.3.4. (3b) 3-Hydroxy-N-hydroxy-lup-20(29)-en-28-amide (6)
To a solution of potassium hydroxide (0.28 g, 5.00 mmol) in
methanol (25 mL) compound 5 (0.20 g, 0.39 mmol) was added, and
stirring at 25 ꢀC was continued for 4 days. Usual aqueous work-up
followed by chromatography (silica gel, hexanes/ethyl acetate, 7:3,
then 8:2, then CHCl3/MeOH 9:1 followed by 10:0) gave 6 [41]
(0.12 g, 63%) as an off-white solid; RF ¼ 0.27 (silica gel, hexanes/
[
a
]
¼ þ39.42ꢀ (c ¼ 0.64, CHCl3) (lit.: [57] [
a
]
¼ þ32ꢀ (c ¼ 0.37,
D
D
CHCl3), [
a
]
¼ þ40ꢀ (c ¼ 0.86, CHCl3) [58]; MS (ESI, MeOH): m/
D
z ¼ 455.2 (100%, [MþH]þ), 508.9 (24%, [M þ Na þ MeOH]þ), 909.3
(16%, [2M þ H]þ), 931.3 (46%, [2M þ Na]þ).
4.3.3. (3
b
) 3-Acetyloxy-N-hydroxy-lup-20(29)-en-28-amide (5)
ethyl acetate, 7:3); m.p.: 188e190 ꢀC; [
a
]
¼ ꢁ5.30ꢀ (c ¼ 0.67,
D
To an ice cold solution of 3 (0.39 g, 0.78 mmol) in dry DCM
(25 mL), oxalyl chloride (0.40 mL, 4.70 mmol) was slowly added,
and the mixture was allowed to warm to 25 ꢀC and stirred for 2 h.
The solvent was removed under diminished pressure, the residue
dissolved in dry THF (20 mL), and the solvent was again removed.
The residue was dissolved in dry DCM (25 mL) containing NEt3
(0.70 mL, 5.05 mmol), and hydroxylammonium chloride (0.20 g,
2.90 mmol) was added. Stirring at 25 ꢀC was continued for another
2 h. Usual aqueous workup gave a residue that was subjected to
chromatography (silica gel, hexanes/ethyl acetate, 7:3), and 5
(0.27 g, 68%) was obtained as a colorless solid; RF ¼ 0.36 (silica gel,
CHCl3/MeOH 5:1); IR (KBr):
n
¼ 3252br, 2946s, 2868m, 1628m,
1466m, 1450m, 1388m, 1376m, 1256w, 1156w, 1110w, 1042m, 1030m,
1010m, 886m cmꢁ1; 1H NMR (400 MHz, CDCl3/CD3OD):
¼ 4.60 (s,
d
1H, CHa (29)), 4.47 (s, 1H, CHb (29)), 3.07e3.00 (m, 1H, CH (3)), 2.96
(ddd, J ¼ 10.9, 10.9, 3.8 Hz, 1H, CH (19)), 2.35e2.25 (m, 1H, CH (13)),
1.94e1.76 (m, 2H, CHa (16) þ CHa (21)), 1.73e1.62 (m, 1H, CHa (22)),
1.62e1.57 (m, 2H, CHa (12) þ CHa (1)), 1.56 (s, 3H, CH3 (30)),
1.51e1.42 (m, 3H, CH2 (2) þ CH (18)), 1.42e1.35 (m, 3H, CHb
(16) þ CHa (15) þ CHa (6)), 1.35e1.18 (m, 6H, CH2 (7) þ CHb
(21) þ CHb (22) þ CHb (6) þ CHa (11)), 1.19e1.10 (m, 2H, CHb
(11) þ CH (9)), 1.07e0.96 (m, 1H, CHb (15)), 0.96e0.86 (m, 1H, CHb
(12)), 0.84 (s, 3H, CH3 (27)), 0.82 (s, 6H, CH3 (23) þ CH3 (25)),
0.80e0.72 (m, 1H, CHb (1)), 0.70 (s, 3H, CH3 (26)), 0.62 (s, 3H, CH3
(24)), 0.57e0.52 (m, 1H, CH (5)) ppm; 13C NMR (100 MHz, CDCl3/
hexanes/ethyl acetate, 7:3); m.p.: 207e210 ꢀC; [
a]
¼ þ9.02ꢀ
D
(c ¼ 0.58, CHCl3); IR (KBr): ¼ 3355br, 3075m, 2943s, 2870s, 1736s,
n
1716s, 1643s, 1451s, 1377s, 1317w, 1265s, 1196m, 1152w, 1107m,
1066w, 1030s, 979s, 917m, 885m cmꢁ1; 1H NMR (400 MHz, CDCl3):
CD3OD):
d
¼ 174.8 (C]O, C28), 150.7 (Cq, C20), 109.3 (CH2]C, C29),
d
¼ 8.93e7.70 (m, 2H, NH þ OH), 4.73 (s, 1H, CHa (29)), 4.60 (s, 1H,
78.7 (CH, C9), 55.4 (CH, C5), 54.2 (Cq, C17), 50.5 (CH, C9), 50.4 (CH,
C18), 46.7 (CH, C19), 42.3 (Cq, C14), 40.7 (Cq, C8), 38.7 (CH2, C1), 38.2
(CH2, C22), 37.8 (CH, C13), 37.1 (Cq, C4), 36.9 (Cq, C10), 34.3 (CH2, C7),
32.6 (CH2, C16), 30.7 (CH2, C21), 29.3 (CH2, C15), 27.8 (CH3, C23),
26.9 (CH2, C2), 25.5 (CH2, C12), 20.8 (CH2, C11), 19.2 (CH3, C30), 18.2
(CH2, C6), 16.0 (CH3, C26), 15.9 (CH3, C25), 15.2 (CH3, C24), 14.5 (CH3,
C27) ppm; MS (ESI, MeOH): m/z ¼ 472.4 (100%, [MþH]þ), 494.3 (6%,
[MþNa]þ), 943.4 [20%, [2M þ H]þ), 965.5 (60%, [2M þ Na]þ);
analysis calcd for C30H49NO3 (471.71): C 76.39, H 10.47, N 2.97;
found: C 76.21, H 10.90, N 2.76.
CHb (29)), 4.50e4.42 (m, 1H, CH (3)), 3.03 (ddd, J ¼ 11.1, 11.1, 4.2 Hz,
1H, CH (19)), 2.33 (ddd, J ¼ 12.6, 12.6, 3.2 Hz, 1H, CH (13)), 2.03 (s,
3H, CH3 (32)), 2.02e1.87 (m, 2H, CHa (21) þ CHa (16)), 1.83e1.73 (m,
1H, CHa (22)), 1.74e1.68 (m, 2H, CHa (12) þ CHa (1)), 1.68 (s, 3H, CH3
(30)), 1.66e1.28 (m, 12H, CH (18) þ CH2 (2) þ CHb (22) þ CH2
(7) þ CHb (16) þ CHb (21) þ CHa (15) þ CHa (11) þ CH2 (6)),
1.29e1.21 (m, 2H, CHb (11) þ CH (9)), 1.20e1.09 (m, 1H, CHb (15)),
1.09e0.96 (m, 2H, CHb (12) þ CHb (1)), 0.95 (s, 3H, CH3 (27)), 0.93 (s,
3H, CH3 (24)), 0.84 (s, 3H, CH3 (26)), 0.83 (s, 3H, CH3 (23)), 0.82 (s,

J. Wiemann et al. / European Journal of Medicinal Chemistry 106 (2015) 194e210
199
4.3.5. (3
amide (7)
b
) 3-Acetyloxy-N-methoxy-N-methyl-lup-20(29)-en-28-
C21), 31.1 (CH2, C16), 30.1 (CH2, C15), 28.1 (CH3, C23), 27.6 (CH2, C2),
25.9 (CH2, C12), 21.3 (CH2, C11), 19.8 (CH3, C30), 18.5 (CH2, C6), 16.4
(CH3, C26), 16.3 (CH3, C25), 15.5 (CH3, C24),14.9 (CH3, C27) ppm; MS
(ESI, MeOH): m/z ¼ 500.5 (100%, [MþH]þ), 1005.7 (28%, [2M þ Li]þ),
1021.4 (55%, [2M þ Na]þ); analysis calcd for C32H53NO3 (499.77): C
76.90, H 10.69, N 2.80; found: C 76.77, H 10.84, N 2.69.
Following the procedure given for the synthesis of 5, from the
reaction of 3 (0.30 g, 0.60 mmol) with oxalyl chloride (0.30 mL,
3.52 mmol) in dry DCM (25 mL) for 2 h, then with NEt3 (0.70 mL,
5.05 mmol) and N,O-dimethylhydroxylammonium chloride (0.12 g,
1.24 mmol) in dry DCM (25 mL) for 1 day at 25 ꢀC followed by
chromatography (silica gel, hexanes/ethyl acetate, 9:1) 7 (0.25 g,
77%) [59] was obtained as a colorless solid; RF ¼ 0.72 (silica gel,
4.3.7. (3
b) 3-Acetyloxy-N-hydroxy-N-methyl-lup-20(29)-en-28-
amide (9)
hexanes/ethyl acetate, 8:2); m.p.: 260e261 ꢀC; [
a
]
D
¼ þ8.28ꢀ
Following the procedure for the synthesis of 5, reaction of 3
(0.32 g, 0.64 mmol) with oxalyl chloride (0.30 mL, 3.52 mmol), then
with NEt3 (0.70 mL, 5.05 mmol) and N-methylhydroxylammonium
chloride (0.15 g, 1.81 mmol) in dry DCM (25 mL) for 12 h at 25 ꢀC,
followed by chromatography (silica gel, hexanes/ethyl acetate, 8:2)
gave 9 (0.22 g, 0.42 mmol, 66%) as a colorless solid; RF ¼ 0.57 (silica
(c ¼ 0.66, CHCl3); IR (KBr): ¼ 3441br, 3077w, 2936s, 2868s, 1737s,
n
1651s, 1446m, 1370m, 1327w, 1249s, 1165w, 1105w, 1029m, 994m,
976m, 887m cmꢁ1 1H NMR (400 MHz, CDCl3):
¼ 4.71 (d,
;
d
J ¼ 2.4 Hz, 1H, CHa (29)), 4.56 (d, J ¼ 2.3 Hz, 1H, CHb (29)), 4.48e4.44
(m, 1H, CH (3)), 3.64 (s, 3H, OCH3 (34)), 3.15 (s, 3H, NCH3 (33)), 2.97
(ddd, J ¼ 10.8, 10.8, 4.3 Hz, 1H, CH (19)), 2.71 (ddd, J ¼ 12.0, 12.0,
3.6 Hz, 1H, CH (13)), 2.34e2.29 (m, 1H, CHa (16)), 2.11e2.05 (m, 1H,
CHa (22)), 2.03 (s, 3H, CH3 (32)), 1.84e1.75 (m, 1H, CHb (16)),
1.73e1.56 (m, 4H, CHa (1) þ CHa (12) þ CH2 (2)), 1.67 (s, 3H, CH3
(30)), 1.56e1.50 (m, 1H, CH (9)), 1.50e1.42 (m, 1H, CHa (6)),
1.42e1.20 (m, 10H, CHb (6) þ CH2 (11) þ CHa (15) þ CH2 (7) þ CHb
(22) þ CH2 (21) þ CH (18)),1.17e1.12 (m, CHa (15)),1.01e0.92 (m, 2H,
CHb (12) þ CHb (1)), 0.95 (s, 3H, CH3 (27)), 0.93 (s, 3H, CH3 (26)), 0.84
(s, 3H, CH3 (25)), 0.83 (s, 3H, CH3 (23)), 0.82 (s, 3H, CH3 (24)),
0.80e0.76 (m, 1H, CH (5)) ppm; 13C NMR (100 MHz, CDCl3):
gel, hexanes/ethyl acetate, 7:3); m.p.: 226e228 ꢀC; [
a]
¼ þ3.52ꢀ (c
D
0.84, CHCl3); IR (KBr):
n
¼ 3302br, 3073w, 2947s, 2875m, 1736m,
1701s, 1630s, 1451m, 1390m, 1375s, 1334w, 1259s, 1271s, 1193w,
1109w, 1073w, 1029m, 980m cmꢁ1 1H NMR (400 MHz, CDCl3):
¼ 8.56 (br, 1H, OH), 4.74 (d, J ¼ 1.9 Hz, 1H, CHa (29)), 4.60 (dd,
;
d
J ¼ 1.9, 1.5 Hz, 1H, CHb (29)), 4.49e4.44 (m, 1H, CH (3)), 3.41 (s, 3H,
NCH3 (33)), 2.98 (ddd, J ¼ 11.1, 11.1, 3.9 Hz, 1H, CH (19)), 2.81 (ddd,
J ¼ 13.0, 13.0, 3.5 Hz, 1H, CH (13)), 2.11 (ddd, J ¼ 13.2, 3.1, 3.1 Hz, 1H,
CHa (16)), 2.03 (s, 3H, CH3 (32)), 1.96 (dd, J ¼ 11.0, 7.8 Hz, 1H, CHa
(22)),1.91e1.80 (m,1H, CHa (15)),1.76e1.71 (m,1H, CHa (12)),1.69 (s,
3H, CH3 (30)), 1.67e1.57 (m, 4H, CH (18) þ CH2 (2) þ CHa (1)),
1.57e1.22 (m, 10H, CHb (16) þ CH2 (6) þ CH2 (11) þ CHb (22) þ CH2
(7) þ CHb (15) þ CHa (21)), 1.16 (ddd, J ¼ 13.5, 3.2, 3.2 Hz, 1H, CHb
(21)), 1.03e0.94 (m, 2H, CHb (1) þ CHb (12)), 0.96 (s, 3H, CH3 (27)),
0.94 (s, 3H, CH3 (24)), 0.85 (s, 3H, CH3 (26)), 0.83 (s, 3H, CH3 (23)),
0.83 (s, 3H, CH3 (25)), 0.81e0.75 (m, 1H, CH (5)) ppm; 13C NMR
d
¼ 177.1 (C]O, C28), 171.1 (C]O, C31), 151.5 (Cq, C20), 109.2 (CH2]
C, C29), 81.1 (CH, C3), 60.4 (OCH3, C34), 56.2 (Cq, C17), 55.7 (CH, C5),
52.2 (CH, C9), 50.9 (CH, C18), 46.5 (CH, C19), 42.4 (Cq, C14), 40.9 (Cq,
C8), 38.6 (CH2, C1), 38.0 (Cq, C4), 37.3 (Cq, C10), 37.2 (CH, C13), 35.2
(CH2, C22), 34.5 (CH2, C7), 34.1 (NCH3, C33), 31.2 (CH2, C21), 31.0
(CH2, C16), 30.1 (CH2, C15), 28.1 (CH3, C23), 25.8 (CH2, C12), 23.9
(CH2, C2), 21.4 (CH3, C32), 21.3 (CH2, C11), 19.7 (CH3, C30), 18.4 (CH2,
C6), 16.6 (CH3, C24), 16.4 (CH3, C26), 16.3 (CH3, C25), 14.9 (CH3, C27)
ppm; MS (ESI, MeOH): m/z ¼ 542.5 (100%, [MþH]þ), 564.3 (38%,
[MþNa]þ), 580.1 (24%, [M þ Li þ MeOH]þ),1089.6 (40%, [2M þ Li]þ),
1105.5 [2M þ Na]þ); analysis calcd for C34H55NO4 (541.80): C 75.37,
H 10.37, N 2.59; found: C 75.34, H 10.51, N 2.38.
(100 MHz, CDCl3):
d
¼ 171.5 (C]O, C28),171.2 (C]O, C31),150.9 (Cq,
C20),109.7 (CH2]C, C29), 81.1 (CH, C3), 55.7 (CH, C5), 53.3 (Cq, C17),
52.4 (CH, C18), 50.9 (CH, C9), 46.1 (CH, C19), 42.2 (Cq, C14), 40.9 (Cq,
C8), 38.6 (CH2, C1), 38.4 (NCH3, C33), 38.0 (Cq, C4), 37.3 (CH, C13),
37.3 (Cq, C10), 35.9 (CH2, C22), 34.5 (CH2, C7), 32.4 (CH2, C16), 31.2
(CH2, C15), 29.8 (CH2, C21), 28.1 (CH3, C23), 25.7 (CH2, C12), 23.9
(CH2, C2), 21.5 (CH3, C32), 21.3 (CH2, C11), 19.7 (CH3, C30), 18.4 (CH2,
C6), 16.6 (CH3, C26), 16.4 (CH3, C25), 16.3 (CH3, C24), 14.8 (CH3, C27)
ppm; MS (ESI, MeOH): m/z ¼ 528.4 (70%, [MþH]þ), 550.2 (20%,
[MþNa]þ), 1077.4 (100%, [2M þ Na]þ); analysis calcd for C33H53NO4
(527.78): C 75.10, H 10.12, N 2.65; found: C 75.00, H 10.21, N 2.47.
4.3.6. (3b) 3-Hydroxy-N-methoxy-N-methyl-lup-20(29)-en-28-
amide (8)
Deacetylation of 7 (0.10 g, 0.18 mmol) with potassium hydroxide
(0.10 g, 1.79 mmol) in MeOH/CHCl3 (13 mL/3.5 mL) for 5 days at
25 ꢀC as described above, followed by chromatography (silica gel,
hexanes/ethyl acetate, 8:2) gave 8 (0.80 g, 89%) as a colorless solid;
RF ¼ 0.59 (silica gel, hexanes/ethyl acetate, 7:3); m.p.: 219e220 ꢀC;
4.3.8. (3
b) 3-Hydroxy-N-hydroxy-N-methyl-lup-20(29)-en-28-
amide (10)
[
a
]
¼ þ4.66ꢀ (c ¼ 0.64, CHCl3); IR (KBr):
n
¼ 3456s, 3078w, 2944s,
Deacetylation of 9 (0.22 g, 0.42 mmol) with potassium hy-
D
2866s, 1655s, 1621s, 1447s, 1415m, 1390m, 1357m, 1259w, 1190m,
droxide (0.28 g, 5.00 mmol) in MeOH for 5 days at 25 ꢀC, followed
by chromatography gave 10 (0.12 g, 60%) as a colorless solid (only
scarcely soluble in usual solvents); RF ¼ 0.25 (silica gel, hexanes/
1166m, 1109m, 1081w, 1044m, 1032m, 995s, 885m cmꢁ1
;
1H NMR
(400 MHz, CDCl3):
d
¼ 4.71 (d, J ¼ 2.3 Hz, 1H, CHa (29)), 4.56 (dd,
J ¼ 2.3, 1.4 Hz, 1H, CHb (29)), 3.65 (s, 3H, OCH3 (32)), 3.17 (dd,
J ¼ 11.8, 5.4 Hz,1H, CH (3)), 3.15 (s, 3H, NCH3 (31)), 2.98 (ddd, J ¼ 11.1,
11.1, 4.0 Hz,1H, CH (19)), 2.71 (ddd, J ¼ 13.0,11.8, 3.6 Hz,1H, CH (13)),
2.34e2.30 (m, 1H, CHa (16)), 2.12e2.06 (m, 1H, CHa (22)), 1.85e1.74
(m, 1H, CHb (16)), 1.73e1.64 (m, 2H, CHa (12) þ CHa (1)), 1.67 (s, 3H,
CH3 (30)), 1.64e1.43 (m, 4H, CH2 (2) þ CH (18) þ CHa (6)), 1.43e1.19
(m, 10H, CH2 (11) þ CHb (6) þ CH2 (21) þ CHa (15) þ CH2 (7) þ CHb
(22) þ CH (9)), 1.18e1.12 (m, 1H, CHb (15)), 1.00e0.94 (m, 1H, CHb
(12)), 0.96 (s, 3H, CH3 (27)), 0.95 (s, 3H, CH3 (23)), 0.94 (s, 3H, CH3
(25)), 0.93e0.85 (ddd, J ¼ 13.2, 13.2, 3.6 Hz, 1H, CHb (1)), 0.82 (s, 3H,
ethyl acetate, 8:2); m.p.: 258e260 ꢀC; IR (KBr):
n
¼ 3385s, 3198m,
3084m, 2956s, 2927s, 2868s, 1653m, 1607s, 1550w, 1452m, 1419m,
1384s, 1294w, 1256w, 1196m, 1107w, 1092w, 1023m, 982m,
884m cmꢁ1; MS (ESI, MeOH): m/z ¼ 486.5 (100%, [MþH]þ), 508.4
(10%, [MþNa]þ), 993.6 (50%, [2M þ Na]þ); analysis calcd for
C31H51NO3 (485.74): C 76.65, H 10.58, N 2.88; found: C 76.55, H
10.51, N 2.67.
4.3.9. (3 ) 3-Acetyloxy-N-methoxy-lup-20(29)-en-28-amide (11)
b
Following the procedure for the synthesis of 5, reaction of 3
(0.20 g, 0.40 mmol) with oxalyl chloride (0.20 mL, 2.35 mmol)
followed by reaction with NEt3 (0.70 mL, 5.05 mmol) and O-
methylhydroxylammonium chloride (0.15 g, 1.81 mmol) in dry
DCM (25 mL) for 5 h at 25 ꢀC followed by chromatography (silica
gel, hexanes/ethyl acetate, 7:3) gave 11 (0.13 g, 63%) as a colorless
solid; RF ¼ 0.43 (silica gel, hexanes/ethyl acetate, 7:3); m.p.:
CH3 (26)), 0.75 (s, 3H, CH3 (24)), 0.69e0.66 (m, 1H, CH (5)) ppm; 13
C
NMR (100 MHz, CDCl3):
d
¼ 177.1 (C]O, C28), 151.6 (Cq, C20), 109.2
(CH2]C, C29), 79.2 (CH, C3), 60.4 (OCH3, C32), 56.2 (Cq, C17), 55.6
(CH, C5), 52.3 (CH, C18), 51.0 (CH, C9), 46.5 (CH, C19), 42.4 (Cq, C14),
40.9 (Cq, C8), 39.0 (CH2, C1), 38.9 (Cq, C4), 37.4 (Cq, C10), 37.2 (CH,
C13), 35.2 (CH2, C22), 34.6 (CH2, C7), 34.1 (NCH3, C31), 31.2 (CH2,

200
J. Wiemann et al. / European Journal of Medicinal Chemistry 106 (2015) 194e210
217e220 ꢀC; [
a]
¼ þ14.92ꢀ (c ¼ 0.54, CHCl3); IR (KBr):
n
¼ 3639m,
(0.40 g, 0.80 mmol) with oxalyl chloride (0.40 mL, 4.60 mmol),
followed by reaction with NEt3 (0.70 mL, 5.05 mmol) and O-allyl-
hydroxylammonium chloride (0.30 g, 2.75 mmol) in dry DCM
(25 mL) for 2 h at 25 ꢀC, followed by chromatography (silica gel,
hexanes/ethyl acetate, 9:1) gave 13 (0.40 g, 90%) as a colorless solid;
RF ¼ 0.65 (silica gel, hexanes/ethyl acetate, 7:3); m.p.: 128e131 ꢀC;
D
3385m, 3226m, 3071w, 2952s, 2866m, 1740s, 1654s, 1638m, 1511w,
1466m, 1448m, 1392m, 1377m, 1350w, 1317w, 1250s, 1151w, 1108w,
1084w, 1032m, 980m, 890m cmꢁ1 1H NMR (400 MHz, CDCl3):
;
d
¼ 8.46 (s, 1H, NH), 4.73 (s, 1H, CHa (29)), 4.60 (s, 1H, CHb (29)),
4.50e4.42 (m, 1H, CH (3)), 3.75 (s, 3H, OCH3 (33)), 3.11 (ddd, J ¼ 11.0,
11.0, 4.2 Hz, 1H, CH (19)), 2.42 (ddd, J ¼ 12.6, 12.6, 3.2 Hz, 1H, CH
(13)), 2.03 (s, 3H, CH3 (32)), 2.02e1.93 (m, 1H, CHa (16)), 1.93e1.80
(m, 1H, CHa (21)), 1.80e1.73 (m, 1H, CHa (22)), 1.73e1.58 (m, 4H, CHa
(12) þ CHa (1) þ CH2 (2)), 1.67 (s, 3H, CH3 (30)), 1.57e1.51 (m, 3H, CH
(9) þ CHb (21) þ CHa (15)), 1.50e1.44 (m, 1H, CHa (6)), 1.44e1.29 (m,
6H, CHb (6) þ CHa (11) þ CHb (16) þ CH2 (7) þ CHb (22)), 1.29e1.20
(m, 2H, CHb (11) þ CH (18)), 1.20e1.11 (m, 1H, CHb (15)), 1.01e0.90
(m, 2H, CHb (12) þ CHb (1)), 0.96 (s, 3H, CH3 (27)), 0.95 (s, 3H, CH3
(26)), 0.84 (s, 3H, CH3 (25)), 0.83 (s, 3H, CH3 (23)), 0.82 (s, 3H, CH3
(24)), 0.80e0.75 (m, 1H, CH (5)) ppm; 13C NMR (100 MHz, CDCl3):
[a
]
¼ þ22.16ꢀ (c ¼ 0.59, CHCl3); IR (KBr):
¼ 3260br, 2947s, 1737s,
n
D
1644s, 1455s, 1376s, 1317m, 1245s, 1154m, 1108m, 1030s, 979s, 927s,
883s, 754m, 544m cmꢁ1; 1H NMR (400 MHz, CDCl3):
d
¼ 8.42 (s, 1H,
NH), 5.99 (ddt, J ¼ 16.8, 10.3, 6.4 Hz, 1H, CH (34)), 5.36e5.25 (m, 2H,
CH2, C35)), 4.72 (s, 1H, CHa (29)), 4.58 (s, 1H, CHb (29)), 4.50e4.43
(m, 1H, CH (3)), 4.42e4.32 (m, 2H, CH2 (33)), 3.10 (ddd, J ¼ 11.0, 11.0,
3.9 Hz, 1H, CH (19)), 2.42 (ddd, J ¼ 12.7, 12.7, 3.3 Hz, 1H, CH (13)),
2.02 (s, 3H, CH3 (32)), 2.02e1.92 (m, 1H, CHa (16)), 1.92e1.84 (m, 1H,
CHa (21)), 1.79e1.72 (m, 1H), 1.66 (s, 3H, CH3 (30)), 1.71e1.63 (m, 2H,
CHa (12) þ CHa (1)), 1.62e1.56 (m, 2H, CH2 (2)), 1.55e1.49 (m, 3H, CH
(18) þ CHb (21) þ CHa (15)), 1.49e1.28 (m, 7H, CH2 (6) þ CHa
(11) þ CHb (16) þ CH2 (7) þ CHb (22)), 1.27e1.20 (m, 2H, CH
(9) þ CHb (11)), 1.19e1.10 (m, 1H, CHb (15)), 1.05e0.93 (m, 2H, CHb
(12) þ CHb (1)), 0.95 (s, 3H, CH3 (27)), 0.94 (s, 3H, CH3 (26)), 0.83 (s,
3H, CH3 (25)), 0.82 (s, 3H, CH3 (23)), 0.81 (s, 3H, CH3 (24)),
0.79e0.72 (m, 1H, CH (5)) ppm; 13C NMR (100 MHz, CDCl3):
d
¼ 174.7 (C]O, C28), 171.2 (C]O, C31), 150.7 (Cq, C20), 109.7
(CH2]C, C29), 81.1 (CH, C3), 64.3 (OCH3, C33), 55.6 (CH, C5), 54.8
(Cq, C17), 50.7 (CH, C9), 50.7 (CH, C18), 46.9 (CH, C19), 42.5 (Cq, C14),
41.0 (Cq, C8), 38.6 (CH2, C1), 38.2 (CH2, C22), 37.9 (Cq, C4), 37.9 (CH,
C13), 37.3 (Cq, C10), 34.5 (CH2 C7), 33.2 (CH2, C21), 30.9 (CH2, C16),
29.6 (CH2, C15), 28.1 (CH3, C23), 25.7 (CH2, C12), 23.8 (CH2, C2), 21.4
(CH3, C32), 21.0 (CH2, C11), 19.6 (CH3, C30), 18.3 (CH2, C6), 16.6 (CH3,
C24), 16.3 (CH3, C26), 16.3 (CH3, C25), 14.8 (CH3, C27) ppm; MS (ESI,
MeOH): m/z ¼ 528.5 (100%, [MþH]þ), 550.3 (24%, [MþNa]þ), 1055.3
(24%, [2M þ H]þ), 1077.40 (80%, [2M þ Na]þ); analysis calcd for
d
¼ 174.6 (C]O, C28), 171.2 (C]O, C31), 150.7 (Cq, C20), 132.7 (CH]
CH2, C34), 120.4 (CH2]CH, C35), 109.7 (CH2]C, C29), 81.1 (CH, C3),
77.4 (CH2, C33), 55.6 (CH, C5), 54.8 (Cq, C17), 50.7 (CH, C18), 50.7
(CH, C9), 46.8 (CH, C19), 42.5 (Cq, C14), 40.9 (Cq, C8), 38.5 (CH2, C1),
38.3 (CH2, C22), 37.9 (Cq, C4), 37.8 (CH, C13), 37.3 (Cq, C10), 34.5
(CH2, C7), 33.3 (CH2, C21), 30.9 (CH2, C16), 29.6 (CH2, C15), 28.1
(CH3, C23), 25.7 (CH2, C12), 23.8 (CH2, C2), 21.4 (CH3, C32), 21.0
(CH2, C11), 19.6 (CH3, C30), 18.3 (CH2, C6), 16.6 (CH3, C24), 16.4 (CH3,
C25), 16.3 (CH3, C26), 14.8 (CH3, C27) ppm; MS (ESI, MeOH): m/
z ¼ 554.4 (96%, [MþH]þ), 576.4 (20%, [MþNa]þ), 1129.5 (100%,
[2M þ Na]þ); analysis calcd for C35H55NO4 (553.82): C 75.91, H
10.01, N 11.56; found: C 75.79, H 10.14, N 11.49.
C
33H53NO4 (527.78): C 75.10, H 10.12, N 2.65; found: C 74.88, H
10.34, N 2.60.
4.3.10. (3b) 3-Hydroxy-N-methoxy-lup-20(29)-en-28-amide (12)
Deacetylation of 11 (0.12 g, 0.22 mmol) with potassium hy-
droxide (0.18 g, 3.21 mmol) in MeOH/THF (18 mL/6 mL) for 5 days at
25 ꢀC as described above, followed by chromatography (silica gel,
hexanes/ethyl acetate, 7:3) gave 12 (0.10 g, 95%) as a colorless solid;
RF ¼ 0.36 (silica gel, hexanes/ethyl acetate, 7:3); m.p.: 222e224 ꢀC;
[
a
]
¼ þ3.22ꢀ (c ¼ 0.62, CHCl3); IR (KBr):
n
¼ 3421m, 3243m, 3082w,
4.3.12. (3b) 3-Hydroxy-N-allyloxy-lup-20(29)-en-28-amide (14)
D
2940s, 2869m, 1655s, 1464m, 1438m, 1390m, 1377w, 1256w, 1193w,
Deacetylation of 13 (0.17 g, 0.31 mmol) with potassium hy-
droxide (0.17 g, 3.03 mmol) in MeOH (18 mL) and THF (7 mL) for 7
days at 25 ꢀC as described above, followed by chromatography
(silica gel, hexanes/ethyl acetate, 8:2) gave 14 (0.15 g, 94%) as a
colorless solid; RF ¼ 0.44 (silica gel, hexanes/ethyl acetate, 7:3);
1108w, 1039m, 888m cmꢁ1; 1H NMR (400 MHz, CDCl3):
d
¼ 8.57 (s,
1H, NH), 4.72 (d, J ¼ 1.9 Hz,1H, CHa (29)), 4.58 (dd, J ¼ 2.0,1.4 Hz,1H,
CHb (29)), 3.74 (s, 3H, OCH3 (31)), 3.16 (dd, J ¼ 11.3, 4.9 Hz, 1H, CH
(3)), 3.10 (ddd, J ¼ 11.1, 11.1, 4.3 Hz, 1H, CH (19)), 2.41 (ddd, J ¼ 12.8,
12.8, 3.6 Hz, 1H, CH (13)), 2.02e1.94 (m, 1H, CHa (16)), 1.92e1.87 (m,
1H, CHa (21)), 1.76 (dd, J ¼ 11.8, 7.8 Hz, 1H, CHa (22)), 1.72e1.67 (m,
1H, CHa (12)), 1.66 (s, 3H, CH3 (30)), 1.66e1.63 (m, 1H, CHa (1)),
1.63e1.47 (m, 6H, CH2 (2) þ CHa (15) þ CHb (21) þ CH (18) þ CHa
(6)), 1.47e1.30 (m, 6H, CHb (6) þ CHa (11) þ CHb (16) þ CHb
(22) þ CH2 (7)), 1.28e1.21 (m, 2H, CH (9) þ CHb (11)), 1.20e1.14 (m,
1H, CHb (15)), 1.03e0.96 (m, 1H, CHb (12)), 0.95 (s, 9H, CH3
(27) þ CH3 (25) þ CH3 (25)), 0.88 (ddd, J ¼ 12.9, 12.9, 4.1 Hz, 1H, CHb
(1)), 0.80 (s, 3H, CH3 (26)), 0.74 (s, 3H, CH3 (24)), 0.69e0.63 (m, 1H,
m.p.: 129e131 ꢀC; [
a
]
¼ þ3.36ꢀ (c ¼ 0.78, CHCl3); IR (KBr):
D
n
¼ 3423br, 3075w, 2944s, 2869s, 1655m, 1453m, 1389m, 1376m,
1279w, 1247w, 1189w, 1138w, 1107w, 1034m, 1008m, 983m, 924m,
883m cmꢁ1 1H NMR (400 MHz, CDCl3):
¼ 8.31 (s, 1H, NH), 5.99
;
d
(ddt, J ¼ 16.8, 10.3, 6.4 Hz, 1H, CH (32)), 5.37e5.27 (m, 2H, CH2 (33)),
4.73 (s, 1H, CHa (29)), 4.59 (s, 1H, CHb (29)), 4.43e4.33 (m, 2H, CH2
(31)), 3.17 (dd, J ¼ 11.3, 4.8 Hz, 1H, CH (3)), 3.11 (ddd, J ¼ 11.1, 11.1,
4.3 Hz, 1H, CH (19)), 2.42 (ddd, J ¼ 12.7, 12.7, 3.5 Hz, 1H, CH (13)),
2.03e1.93 (m, 1H, CHa (16)), 1.86 (m, 1H, CHa (21)), 1.75 (dd, J ¼ 11.8,
7.9 Hz, 1H, CHa (22)), 1.72e1.63 (m, 2H, CHa (12) þ CHa (1)), 1.67 (s,
3H, CH3 (30)), 1.63e1.46 (m, 6H, CH2 (2) þ CHa (15) þ CHb (21) þ CH
(18) þ CHa (6)), 1.45e1.30 (m, 6H, CHb (6) þ CHa (11) þ CHb
(16) þ CH2 (7) þ CHb (22)), 1.29e1.20 (m, 2H, CH (9) þ CHb (11)),
1.19e1.12 (m, 1H, CHb (15)), 1.03e0.96 (m, 1H, CHb (12)), 0.96 (s, 3H,
CH3 (23)), 0.95 (s, 6H, CH3 (25) þ CH3 (27)), 0.92e0.84 (m, 1H, CHb
(1)), 0.81 (s, 3H, CH3 (26)), 0.75 (s, 3H, CH3 (24)), 0.70e0.64 (m, 1H,
CH (5)) ppm; 13C NMR (100 MHz, CDCl3):
d
¼ 174.7 (C]O, C28),
150.7 (Cq, C20), 109.6 (CH2]C, C29), 79.1 (CH, C3), 64.3 (OCH3, C31),
55.5 (CH, C5), 54.8 (Cq, C17), 50.7 (CH, C9), 50.7 (CH, C18), 46.8 (CH,
C19), 42.5 (Cq, C14), 40.9 (Cq, C8), 39.0 (CH2, C1), 38.9 (Cq, C4), 38.2
(CH2, C22), 37.9 (CH, C13), 37.3 (Cq, C10), 34.5 (CH2, C7), 33.2 (CH2,
C21), 30.9 (CH2, C16), 29.6 (CH2, C15), 28.1 (CH3, C23), 27.5 (CH2, C2),
25.7 (CH2, C12), 21.0 (CH2, C11), 19.6 (CH3, C30), 18.4 (CH2, C6), 16.3
(CH3, C26), 16.3 (CH3, C25), 15.5 (CH3, C25), 14.8 (CH3, C24) ppm; MS
(ESI, MeOH): m/z ¼ 486.5 (60%, [MþH]þ), 508.4 (5%, [MþNa]þ),
971.4 (20%, [2M þ H]þ), 993.5 (100%, [2M þ Na]þ); analysis calcd for
CH (5)) ppm; 13C NMR (100 MHz, CDCl3):
d
¼ 174.6 (C]O, C28),
150.7 (Cq, C20), 132.7 (CH]CH2, C32), 120.5 (CH2]CH, C33), 109.7
(CH2]C, C29), 79.1 (CH, C3), 77.4 (CH2, C31), 55.5 (CH, C5), 54.9 (Cq,
C17), 50.8 (CH, C18), 50.7 (CH, C9), 46.8 (CH, C19), 42.5 (Cq, C14),
41.0 (Cq, C8), 39.0 (CH2, C1), 38.9 (Cq, C4), 38.4 (CH2, C22), 37.9 (CH,
C13), 37.4 (Cq, C10), 34.6 (CH2, C7), 33.4 (CH2, C21), 30.9 (CH2, C16),
29.6 (CH2, C15), 28.1 (CH3, C23), 27.6 (CH2, C2), 25.8 (CH2, C12), 21.0
(CH2, C11), 19.6 (CH3, C30), 18.4 (CH2, C6), 16.4 (CH3, C26), 16.3 (CH3,
C
31H51NO3 (485.74): C 76.65, H 10.58, N 2.88; found: C 76.44, H
10.67, N 2.80.
4.3.11. (3
b)-3-Acetyloxy-N-allyloxy-lup-20(29)-en-28-amide (13)
Following the procedure for the synthesis of 5, reaction of 3

J. Wiemann et al. / European Journal of Medicinal Chemistry 106 (2015) 194e210
201
C25), 15.5 (CH3, C24), 14.8 (CH3, C27) ppm; MS (ESI, MeOH): m/
z ¼ 512.5 (70%, [MþH]þ), 534.4 (12%, [MþNa]þ), 1023.3 (18%,
[2M þ H]þ), 1045.6 (100%, [2M þ Na]þ); analysis calcd for
C18), 50.3 (CH, C9), 47.5 (Cq, C4), 46.1 (CH, C19), 42.2 (Cq, C14), 40.8
(Cq, C8), 39.8 (CH2, C1), 38.3 (NCH3, C31), 37.4 (CH, C13), 37.1 (Cq,
C10), 35.9 (CH2, C22), 34.3 (CH2, C2), 33.9 (CH2, C7), 32.2 (CH2, C16),
31.2 (CH2, C21), 29.8 (CH2, C15), 26.7 (CH3, C23), 25.8 (CH2, C12),
21.8 (CH2, C11), 21.2 (CH3, C24), 19.8 (CH2, C6), 19.7 (CH3, C30), 16.1
(CH3, C26), 16.1 (CH3, C25), 14.2 (CH3, C27) ppm; MS (ESI, MeOH):
m/z ¼ 484.5 (100%, [MþH]þ), 507.5 (5%, [MþNa]þ), 537.8 (12%,
[M þ Na þ MeOH]þ), 748.5 (80%, [3Mþ2Na]2þ), 989.5 (50%,
[2M þ Na]þ); analysis calcd for C31H49NO3 (483.73): C 76.97, H
10.21, N 2.90; found: C 76.76, H 10.34, N 2.74.
C
33H53NO3 (511.78): C 77.45, H 10.44, N 2.74; found: C 77.39, H
10.51, N 2.62.
4.3.13. 3-Oxo-N-methoxy-N-methyl-lup-20(29)-en-28-amide (15)
Following the procedure for the synthesis of 5, reaction of 4
(0.25 g, 0.50 mmol) in DCM (20 mL) with oxalyl chloride (0.30 mL,
3.5 mmol), then with NEt3 (0.70 mL, 5.06 mmol) and N,O-dime-
thylhydroxylammonium chloride (0.15 g, 1.55 mmol) for 12 h at
25 ꢀC followed by chromatography (silica gel, hexanes/ethyl ace-
tate, 9:1) gave 15 (0.13 g, 52%) as a colorless solid; RF ¼ 0.70 (silica
4.3.15. 3-Oxo-N-methoxy-lup-20(29)-en-28-amide (17)
Following the procedure for the synthesis of 5, reaction of 4
(0.25 g, 0.55 mmol) dry DCM (20 mL) with oxalyl chloride (0.4 mL,
4.70 mmol), followed by reaction with NEt3 (0.75 mL, 5.42 mmol)
and O-methylhydroxylammonium chloride (0.15 g, 1.81 mmol) in
dry DCM (20 mL) for 12 h at 25 ꢀC, followed by chromatography
(silica gel, hexanes/ethyl acetate, 8:2) gave 17 (0.12 g, 45%) as a
colorless solid; RF ¼ 0.43 (silica gel, hexanes/ethyl acetate, 7:3);
gel, hexanes/ethyl acetate, 7:3); m.p.: 174e176 ꢀC; [
a
]
D
¼ þ33.12ꢀ
(c ¼ 0.60, CHCl3); IR (KBr): ¼ 3398br, 3077w, 2942s, 2867s, 1709s,
n
1651s, 1456s, 1376m, 1356s, 1345m, 1283w, 1243w, 1168m, 1137w,
1110m,1082w,1022w, 993s, 881m cmꢁ1; 1H NMR (400 MHz, CDCl3):
d
¼ 4.70 (s, 1H, CHa (29)), 4.56 (s, 1H, CHb (29)), 3.65 (s, 3H, OCH3
(32)), 3.15 (s, 3H, NCH3 (31)), 2.97 (ddd, J ¼ 11.0, 11.0, 3.5 Hz, 1H, CH
(19)), 2.75 (ddd, J ¼ 12.9, 12.9, 3.3 Hz, 1H, CH (13)), 2.47 (ddd,
J ¼ 15.8, 8.2, 7.6 Hz, 1H, CHa (2)), 2.42e2.27 (m, 2H, CHb (2) þ CHa
(16)), 2.15e2.05 (m, 1H, CHa (22)), 1.88 (ddd, J ¼ 13.1, 8.1, 4.6 Hz, 2H,
CHa (1)), 1.79 (m, 1H, CHb (16)), 1.75e1.69 (m, 1H, CHa (12)), 1.67 (s,
3H, CH3 (30)),1.54 (m,1H, CH (18)),1.50e1.21 (m,13H, CH2 (6) þ CH2
(11) þ CH2 (21) þ CH2 (7) þ CHa (15) þ CHb (1) þ CHb (22) þ CH
(5) þ CH (9)), 1.20e1.13 (m, 1H, CHb (15)), 1.05 (s, 3H, CH3 (23)), 1.00
(s, 3H, CH3 (24)), 0.97 (s, 3H, CH3 (25)), 0.96 (s, 3H, CH3 (27)),
0.96e0.93 (m, 1H, CHb (12)), 0.91 (s, 3H, CH3 (26)) ppm; 13C NMR
m.p.: 159e162 ꢀC; [
a
]
¼ þ24.32ꢀ (c ¼ 0.62, CHCl3); IR (KBr):
D
n
¼ 3173br, 2944s, 2866s, 1708s, 1637s, 1515m, 1464m, 1376m,
1318w, 1281w, 1243w, 1195w, 1140w, 1117w, 1081w, 1046m, 938m,
886m cmꢁ1; 1H NMR (400 MHz, CDCl3):
¼ 8.46 (s, 1H, NH), 4.73 (s,
d
1H, CHa (29)), 4.60 (s, 1H, CHb (29)), 3.75 (s, 3H, OCH3 (31)), 3.11
(ddd, J ¼ 10.9, 10.9, 3.9 Hz, 1H, CH (19)), 2.54e2.43 (m, 2H, CH
(13) þ CHa (2)), 2.38 (ddd, J ¼ 15.6, 7.4, 4.3 Hz, 1H, CHb (2)),
2.06e1.94 (m, 1H, CHa (16)), 1.94e1.83 (m, 2H, CHa (21) þ CHa (1)),
1.81e1.68 (m, 2H, CHa (22) þ CHa (21)), 1.67 (s, 3H, CH3 (30)),
1.63e1.52 (m, 3H, CHa (15) þ CHb (21) þ CH (18)), 1.52e1.22 (m, 11H,
CH2 (6) þ CH2 (11) þ CHb (16) þ CH2 (7) þ CHb (22) þ CHb (1) þ CH
(9) þ CH (5)), 1.21e1.15 (m, 1H, CHb (15)), 1.05 (s, 3H, CH3 (23)),
1.06e1.02 (m, 1H, CHb (12)), 1.01 (s, 3H, CH3 (24)), 1.00 (s, 3H, CH3
(27)), 0.97 (s, 3H, CH3 (25)), 0.92 (s, 3H, CH3 (26)) ppm; 13C NMR
(100 MHz, CDCl3):
d
¼ 218.3 (C]O, C3), 177.0 (C]O, C28), 151.4 (Cq,
C20), 109.2 (CH2]C, C29), 60.4 (OCH3, C32), 56.1 (Cq, C17), 55.2 (CH,
C5), 52.1 (CH, C18), 50.3 (CH, C9), 47.5 (Cq, C4), 46.4 (CH, C19), 42.4
(Cq, C14), 40.8 (Cq, C8), 39.8 (CH2, C1), 37.3 (CH, C13), 37.1 (Cq, C10),
35.1 (CH2, C22), 34.3 (CH2, C2), 34.1 (NCH3, C31), 33.8 (CH2, C7), 31.1
(CH2, C16), 31.0 (CH2, C21), 30.0 (CH2, C15), 26.7 (CH3, C23), 25.8
(CH2, C12), 21.8 (CH2, C11), 21.2 (CH3, C24), 19.8 (CH2, C6), 19.7 (CH3,
C30), 16.1 (CH3, C26), 16.1 (CH3, C25), 14.8 (CH3, C27) ppm; MS (ESI,
MeOH): m/z ¼ 498.5 (100%, [MþH]þ), 1017.6 (40%, [2M þ Na]þ);
analysis calcd for C32H51NO3 (497.75): C 77.22, H 10.33, N 2.81;
found: C 76.98, H 10.54, N 2.77.
(100 MHz, CDCl3):
d
¼ 218.3 (C]O, C3), 174.7 (C]O, C28), 150.6 (Cq,
C20), 109.7 (CH2]C, C29), 64.3 (OCH3, C31), 55.2 (CH, C5), 54.7 (Cq,
C17), 50.7 (CH, C18), 50.1 (CH, C9), 47.5 (Cq, C4), 46.8 (CH, C19), 42.6
(Cq, C14), 40.9 (Cq, C8), 39.8 (CH2, C1), 38.2 (CH2, C22), 38.0 (CH,
C13), 37.1 (Cq, C10), 34.3 (CH2, C2), 33.8 (CH2, C7), 33.2 (CH2, C21),
30.9 (CH2, C16), 29.6 (CH2, C15), 26.7 (CH3, C23), 25.8 (CH2, C12),
21.6 (CH2, C11), 21.2 (CH3, C24), 19.8 (CH2, C6), 19.6 (CH3, C30), 16.1
(CH3, C26), 16.1 (CH3, C25), 14.7 (CH3, C27) ppm; MS (ESI, MeOH):
m/z ¼ 484.5 (100%, [MþH]þ), 506.3 (4%, [MþNa]þ), 967.3 (20%,
[2M þ H]þ), 989.6 (82%, [2M þ Na]þ); analysis calcd for C31H49NO3
(483.37): C 76.97, H 10.21, N 2.90; found: C 76.88, H 10.50, N 2.81.
4.3.14. 3-Oxo-N-hydroxy-N-methyl-lup-20(29)-en-28-amide (16)
Following the procedure for the synthesis of 5, reaction of 4
(0.24 g, 0.53 mmol) with oxalyl chloride (0.30 mL, 3.52 mmol)
followed by reaction with NEt3 (0.70 mL, 5.06 mmol) and N-
methylhydroxylammonium chloride (0.10 g, 1.20 mmol) in dry
DCM (25 mL) for 12 h at 25 ꢀC, followed by chromatography (silica
gel, hexanes/ethyl acetate, 8:2) gave 16 (0.21 g, 81%) as a colorless
solid; RF ¼ 0.25 (silica gel, hexanes/ethyl acetate, 7:3); m.p.:
4.3.16. 3-Oxo-N-allyloxy-lup-20(29)-en-28-amide (18)
Following the procedure for the synthesis of 5, reaction of 4
(0.26 g, 0.57 mmol) with oxalyl chloride (0.3 mL, 3.52 mmol), fol-
lowed by reaction with NEt3 (0.75 mL, 5.42 mmol) and O-allylhy-
droxylammonium chloride (0.25 g, 2.29 mmol) in dry DCM (25 mL)
for 5 h at 25 ꢀC, followed by chromatography (silica gel, hexanes/
ethyl acetate, 8:2) gave 18 (0.20 g, 68%) as a colorless solid;
RF ¼ 0.33 (silica gel, toluene/ethyl acetate/heptane/formic acid,
214e216 ꢀC; [
a
]
¼ þ21.54ꢀ (c ¼ 0.70, CHCl3); IR (KBr):
¼ 3405br,
n
D
2942s, 2868s, 1704m, 1640m, 1595m, 1455m, 1376m, 1184w, 1104w,
1076w, 1022w cmꢁ1
;
1H NMR (400 MHz, CDCl3):
d
¼ 8.53 (br, 1H,
OH), 4.73 (s, 1H, CHa (29)), 4.59 (s, 1H, CHb (29)), 3.40 (s, 1H, NCH3
(31)), 2.98 (ddd, J ¼ 11.1, 11.1, 3.6 Hz, 1H, CH (19)), 2.85 (ddd, J ¼ 12.9,
12.9, 3.4 Hz, 1H, CH (13)), 2.53e2.44 (m, 1H, CHa (2)), 2.38 (ddd,
J ¼ 15.6, 7.5, 4.3 Hz, 1H, CHb (2)), 2.14 (ddd, J ¼ 13.4, 2.8, 2.8 Hz, 1H,
CHa (16)), 1.98 (dd, J ¼ 10.6, 7.9 Hz, 1H, CHa (22)), 1.94e1.80 (m, 2H,
CHa (1) þ CHa (21)), 1.79e1.69 (m, 1H, CHa (12)), 1.68 (s, 3H, CH3
(30)), 1.62 (dd, J ¼ 11.3 Hz, 1H, CH (18)), 1.54 (ddd, J ¼ 13.9, 13.9,
3.5 Hz, 1H, CHb (16)), 1.48e1.22 (m, 12H, CH2 (6) þ CH2 (11) þ CH2
(7) þ CHb (1) þ CHb (22) þ CHb (21) þ CHa (15) þ CH (9) þ CH (5)),
1.17 (ddd, J ¼ 13.5, 3.2, 3.2 Hz, 1H, CHb (15)), 1.05 (s, 3H, CH3 (23)),
1.01 (s, 3H, CH3 (24)), 0.97e0.89 (m, 1H, CHb (12)), 0.97 (s, 3H, CH3
(27)), 0.97 (s, 3H, CH3 (25)), 0.92 (s, 3H, CH3 (26)), ppm; 13C NMR
80:20:30:4); m.p.: 113e115 ꢀC; [
(KBr):
a
]
¼ þ22.88ꢀ (c ¼ 0.89, CHCl3); IR
D
n
¼ 3277br, 3074w, 2948s, 2868s, 1705s, 1642s, 1458m,
1376m, 1241w, 1203w, 1140w, 1115w, 1039m, 924m, 883m cmꢁ1; 1H
NMR (400 MHz, CDCl3):
¼ 8.45 (s, 1H, NH), 5.99 (ddt, J ¼ 16.8, 10.3,
d
6.4 Hz, 1H, CH (32)), 5.37e5.24 (m, 2H, CH2 (33)), 4.71 (s, 1H, CHa
(29)), 4.58 (s, 1H, CHb (29)), 4.43e4.31 (m, 2H, CH2 (31)), 3.10 (ddd,
J ¼ 11.0, 11.0, 3.9 Hz, 1H, CH (19)), 2.53e2.42 (m, 2H, CH (13) þ CHa
(2)), 2.37 (ddd, J ¼ 15.6, 7.4, 4.3 Hz, 1H, CHb (2)), 2.01e1.92 (m, 1H,
CHa (16)), 1.92e1.83 (m, 2H, CHa (21) þ CHa (1)), 1.80e1.67 (m, 2H,
CHa (22) þ CHa (12)), 1.66 (s, 3H, CH3 (30)), 1.60e1.50 (m, 3H, CH
(18) þ CHb (21) þ CHa (15)), 1.49e1.21 (m, 11H, CH2 (6) þ CH2
(11) þ CHb (16) þ CH2 (7) þ CHb (22) þ CHb (1) þ CH (9) þ CH (5)),
(100 MHz, CDCl3):
C20), 109.7 (CH2]C, C29), 55.2 (CH, C5), 53.4 (Cq, C17), 52.3 (CH,
d
¼ 218.4 (C]O, C3), 171.5 (C]O, C28), 150.9 (Cq,

202
J. Wiemann et al. / European Journal of Medicinal Chemistry 106 (2015) 194e210
1.19e1.14 (m, 1H, CHb (15)), 1.04 (s, 3H, CH3 (23)), 1.00 (s, 3H, CH3
(24)), 0.99 (s, 3H, CH3 (27)), 0.95 (s, 3H, CH3 (25)), 0.91 (s, 3H, CH3
(lit.: [61] [
a
]
¼ þ6.0ꢀ, CHCl3); IR (KBr):
n
¼ 3422br, 2943s, 2869m,
1H NMR
D
1656m, 1451m, 1376m, 1044w, 883m, 543w cmꢁ1
;
(26)) ppm; 13C NMR (100 MHz, CDCl3):
d
¼ 218.3 (C]O, C3), 174.6
(400 MHz, CDCl3):
d
¼ 5.53 (br, 1H, NHa), 5.43 (br, 1H, NHb), 4.73 (s,
(C]O, C28), 150.6 (Cq, C20), 132.7 (CH]CH2, C32), 120.5 (CH2]C,
C33), 109.7 (CH2]C, C29), 77.4 (CH2, C31), 55.1 (CH, C5), 54.8 (Cq,
C17), 50.7 (CH, C18), 50.1 (CH, C9), 47.5 (Cq, C4), 46.7 (CH, C19), 42.6
(Cq, C14), 40.9 (Cq, C8), 39.8 (CH2, C1), 38.3 (CH2, C22), 37.9 (CH,
C13), 37.1 (Cq, C10), 34.3 (CH2, C2), 33.8 (CH2, C7), 33.3 (CH2, C21),
30.9 (CH2, C16), 29.6 (CH2, C15), 26.7 (CH3, C23), 25.7 (CH2, C12),
21.6 (CH2, C11), 21.1 (CH3, C24), 19.8 (CH2, C6), 19.6 (CH3, C30), 16.2
(CH3, C26), 16.1 (CH3, C25), 14.7 (CH3, C27) ppm; MS (ESI, MeOH):
m/z ¼ 510.5 (100%, [MþH]þ), 532.4 (14%, [MþNa]þ), 784.9 (20%,
[3M þ K þ H]2þ),1019.3 (24%, [2M þ H]þ),1041.6 (84%, [2M þ Na]þ);
analysis calcd for C33H51NO3 (509.76): C 77.75, H 10.08, N 2.75;
found: C 77.59, H 10.19, N 2.63.
1H, CHa (29)), 4.59 (s, 1H, CHb (29)), 3.17 (dd, J ¼ 11.1, 5.1 Hz, 1H, CH
(3)), 3.07 (dt, J ¼ 11.0, 4.2 Hz, 1H, CH (19)), 2.45 (dt, J ¼ 12.7, 3.3 Hz,
1H, CH (13)), 2.03e1.89 (m, 2H, CHa (16) þ CHa (21)), 1.80 (dd,
J ¼ 11.9, 7.8 Hz, 1H, CHa (22)), 1.72e1.63 (m, 2H, CHa (1) þ CHa (12)),
1.68 (s, 3H, CH3 (30)), 1.63e1.52 (m, 5H, CH2 (2) þ CH (18) þ CHa
(15) þ CHb (16)), 1.52e1.49 (m, 1H, CHa (6)), 1.45e1.32 (m, 6H, CH2
(7) þ CHb (6) þ CHa (11) þ CHb (21) þ CHb (22)), 1.30e1.21 (m, 2H,
CH (9) þ CHb (11)), 1.20e1.13 (m, 1H, CHb (15)), 1.05e0.94 (m, 1H,
CHb (12)), 0.96 (s, 3H, CH3 (27)), 0.95 (s, 3H, CH3 (23)), 0.95 (s, 3H,
CH3 (25)), 0.93e0.84 (m, 1H, CHb (1)), 0.82 (s, 3H, CH3 (26)), 0.75 (s,
3H, CH3 (24)), 0.67 (m, 1H, CH (5)) ppm; 13C NMR (100 MHz, CDCl3):
d
¼ 178.9 (C]O, C28),150.8 (C]CH2, C20),109.4 (H2C]C, C29), 79.0
(CH, C3), 55.8 (Cq, C17), 55.4 (CH, C5), 50.6 (CH, C9), 49.9 (CH, C18),
46.6 (CH, C19), 42.5 (Cq, C14), 40.7 (Cq, C8), 38.8 (CH2, C1), 38.7 (CH2,
C22), 38.4 (Cq, C4), 37.7 (CH, C13), 37.2 (Cq, C10), 34.4 (CH2, C7), 34.0
(CH2, C16), 30.7 (CH2, C21), 29.5 (CH2, C15), 28.0 (CH3, C23), 27.4
(CH2, C2), 25.6 (CH2, C12), 20.9 (CH2, C11), 19.5 (CH3, C30),18.3 (CH2,
C6), 16.2 (CH3, C25), 16.1 (CH3, C26), 15.3 (CH3, C24), 14.6 (CH3, C27)
ppm; MS (ESI, MeOH): m/z ¼ 456.3 (84%, [M þ H]þ), 911.4 (78%,
[2M þ H]þ), 933.4 (100%, [2M þ Na]þ).
4.3.17. (3b) 3-Acetyloxylup-20(29)-en-28-amide (19)
Compound 3 (4.0 g, 8.0 mmol) was allowed to react with oxalyl
chloride (3.7 g, 2.5 mL, 29.2 mmol) in dry DCM (50 mL) as described
above. The volatiles were removed, and the residue was dissolved
in dry DCM (50 mL). A solution of dry ammonia in DCM (satd.,
50 mL) was added, and the mixture was stirred at 25 ꢀC for 1 h.
Water (50 mL) and diluted aqueous hydrochloric acid (1 N) were
added until the aqueous layer showed pH ¼ 7. The layers were
separated, the aqueous layer was extracted with DCM (3 ꢂ 50 mL),
the combined organic phases were dried (Na2SO4), and the solvent
was distilled off under reduced pressure. The crude product was
purified by chromatography (silica gel, hexanes/ethyl acetate,
7:3) to yield 19 (3.8 g, 95%) as a colorless solid; RF ¼ 0.12 (silica
gel, hexanes/ethyl acetate, 8:2); m.p.: 336e338 ꢀC (lit.: [60]
4.3.19. (3b) 3-Acetyloxylup-N-benzyl-20(29)-en-28-amide (21)
Following the procedure given for the synthesis of 19, the re-
action of 3 (0.35 g, 0.7 mmol) with oxalyl chloride (0.22 g, 0.15 mL,
1.8 mmol) in dry DCM (15 mL), followed by the reaction with
benzylamine (0.15 g, 0.15 mL) (1 h, reflux) and chromatographic
work-up (silica gel, hexanes/ethyl acetate, 8:2) gave 21 (0.24 g, 59%)
as a colorless solid; RF ¼ 0.87 (silica gel, hexanes/ethyl acetate, 7:3);
340e341 ꢀC);
[a
]
¼
þ17.0ꢀ (c
¼
0.48, CHCl3); IR (KBr):
D
n
¼ 3471m, 3325m, 3202w, 3074w, 2940s, 2868m, 1742s, 1686s,
m.p.: 124e127 ꢀC; [
a
]
D
¼ þ23.2ꢀ (c ¼ 0.35, CHCl3); IR (KBr):
1642s, 1610m, 1449w, 1392m, 1367s, 1301w, 1242s, 1139w, 1108w,
1082w, 1025m, 1009m, 979m, 946w, 899w, 875m, 738w, 655w,
n
¼ 3425br, 2946s, 1736m, 1638m, 1517w, 1454w, 1384m, 1246m,
1028w, 979w, 882m, 698w cmꢁ1
;
UVevis (CHCl3): lmax
608w, 579w, 558w, 541w, 516w cmꢁ1
;
1H NMR (400 MHz, CDCl3):
(log ε) ¼ 253.8 (3.49) nm; 1H NMR (400 MHz, CDCl3):
¼ 7.35e7.29
d
d
¼ 5.53 (br, 2H, NH2), 4.73 (s, 1H, CHa (29)), 4.60 (s, 1H, CHb (29)),
(m, 2H, CH (36/360)), 7.29e7.22 (m, 3H, CH (35/350) þ CH (37)), 5.89
(br, 1H, NH)), 4.74 (s, 1H, CHa (29)), 4.59 (s, 1H, CHb (29)), 4.53e4.42
(m, 2H, CH (3) þ CHa (33)), 4.36 (dd, J ¼ 14.7, 5.6 Hz, 1H, CHb (33)),
3.17 (dt, J ¼ 11.0, 4.0 Hz,1H, CH (19)), 2.49 (dt, J ¼ 12.8, 3.5 Hz,1H, CH
(13)), 2.03 (s, 3H, CH3 (32)), 2.02e1.94 (m, 2H, CH2 (21)), 1.94e1.87
(m, 2H, CH2 (16)), 1.80e1.70 (m, 2H, CHa (22) þ CHa (12)), 1.68 (s, 3H,
CH3 (30)), 1.67e1.62 (m, 1H, CHa (1)), 1.62e1.51 (m, 3H, CH
(18) þ CH2 (2)), 1.50e1.41 (m, 3H, CHb (22) þ CHa (15) þ CHa (6)),
1.40e1.28 (m, 7H, CH2 (7) þ CHa (11) þ CHb (6)), 1.28e1.21 (m, 2H,
CH (9) þ CHb (11)), 1.15e1.08 (m, 1H, CHb (15)), 1.04e0.93 (m, 2H,
CHb (1) þ CHb (12)), 0.95 (s, 3H, CH3 (27)), 0.91 (s, 3H, CH3 (25)), 0.84
(s, 3H, CH3 (26)), 0.83 (s, 3H, CH3 (23)), 0.83 (s, 3H, CH3 (24)), 0.77
4.46 (dd, J ¼ 9.7, 6.6 Hz, 1H, CH (3)), 3.07 (dt, J ¼ 11.1, 4.4 Hz, 1H, CH
(19)), 2.44 (dt, J ¼ 12.8, 3.5 Hz, 1H, CH (13)), 2.03 (s, 3H, CH3 (32)),
2.02e1.89 (m, 2H, CHa (16) þ CHa (21)), 1.81 (dd, J ¼ 11.9, 7.8 Hz, 1H,
CHa (22)), 1.73e1.64 (m, 2H, CHa (1) þ CHa (12)), 1.68 (s, 3H, CH3
(30)), 1.63e1.52 (m, 5H, CH2 (2) þ CH (18) þ CHa (15) þ CHb (16)),
1.52e1.44 (m, 2H, CHa (6) þ CHa (11)), 1.44e1.34 (m, 5H, CH2
(7) þ CHb (6) þ CHb (21) þ CHb (22)), 1.30e1.21 (m, 2H, CH (9) þ CHb
(11)), 1.21e1.14 (m, 1H, CHb (15)), 1.03e0.92 (m, 2H, CHb (1) þ CHb
(12)), 0.96 (s, 3H, CH3 (27)), 0.95 (s, 3H, CH3 (25)), 0.84 (s, 3H, CH3
(23)), 0.83 (s, 3H, CH3 (26)), 0.82 (s, 3H, CH3 (24)), 0.78 (m, 1H, CH
(5)) ppm; 13C NMR (100 MHz, CDCl3):
d
¼ 179.3 (C]O, C28), 171.0
(C]O, C31), 150.7 (C]CH2, C20), 109.5 (H2C]C, C29), 80.9 (CH, C3),
55.8 (Cq, C17), 55.5 (CH, C5), 50.5 (CH, C9), 49.9 (CH, C18), 46.6 (CH,
C19), 42.5 (Cq, C14), 40.8 (Cq, C8), 38.4 (CH, C13), 38.4 (CH2, C1), 37.8
(Cq, C4), 37.7 (CH2, C22), 37.1 (Cq, C10), 34.3 (CH2, C7), 33.9 (CH2,
C16), 30.7 (CH2, C21), 29.5 (CH2, C15), 27.9 (CH3, C23), 25.6 (CH2,
C12), 23.7 (CH2, C2), 21.3 (CH2, C32), 20.9 (CH2, C11),19.4 (CH3, C30),
18.2 (CH2, C6), 16.5 (CH3, C24), 16.2 (CH3, C26), 16.2 (CH3, C25), 14.6
(CH3, C27) ppm; MS (ESI, MeOH): m/z ¼ 498.5 (34%, [M þ H]þ),
520.4 (11%, [M þ Na]þ), 552.0 (5%, [M þ Na þ MeOH]þ), 995.4
(100%, [2M þ H]þ), 1017.4 (46%, [2M þ Na]þ).
(m, 1H, CH (5)) ppm; 13C NMR (100 MHz, CDCl3):
d
¼ 175.9 (C]O,
C28), 171.0 (C]O, C31), 150.8 (C]CH2, C20), 139.2 (Cq, C34), 128.6
(CH, C36/360), 127.8 (CH, C35/350), 127.3 (CH, C37), 109.4 (H2C]C,
C29), 81.0 (CH, C3), 55.7 (Cq, C17), 55.5 (CH, C5), 50.6 (CH, C9), 50.2
(CH, C18), 46.7 (CH, C19), 43.3 (CH2, C33), 42.5 (Cq, C14), 40.8 (Cq,
C8), 38.4 (CH2, C1), 38.4 (CH2, C22), 37.8 (Cq, C4), 37.7 (CH, C13), 37.1
(Cq, C10), 34.4 (CH2, C7), 33.8 (CH2, C16), 30.9 (CH2, C21), 29.4 (CH2,
C15), 27.9 (CH3, C23), 25.6 (CH2, C12), 23.7 (CH2, C2), 21.3 (CH3, C32),
21.0 (CH2, C11), 19.5 (CH3, C30), 18.2 (CH2, C6), 16.5 (CH3, C24), 16.2
(CH3, C26), 16.1 (CH3, C25), 14.6 (CH3, C27) ppm; MS (ESI, MeOH):
m/z ¼ 588.5 (58%, [M þ H]þ), 610.5 (26%, [M þ Na]þ), 1175.3 (90%,
[2M þ H]þ), 1197.4 (100%, [2M þ Na]þ); analysis calcd for
4.3.18. (3b) 3-Hydroxylup-20(29)-en-28-amide (20)
Deacetylation of 19 (1.78 g, 3.58 mmol) with potassium hy-
droxide (0.4 g, 7.16 mmol) in THF (30 mL)/MeOH (20 mL) for 1 day
at 25 ꢀC as described above, followed by chromatography (silica gel,
hexanes/ethyl acetate, 7:3) gave 20 (1.57 g, 96.5%) as a colorless
solid; RF ¼ 0.1 (silica gel, hexanes/ethyl acetate, 8:2); m.p.:
C39H57NO3 (587.87): C 79.68, H 9.77, N 2.38; found: C 79.52, H 9.85,
N 2.21.
4.3.20. (3b) N-Benzyl-3-hydroxylup-20(29)-en-28-amide (22)
Deacetylation of 21 (210 mg, 0.36 mmol) with potassium hy-
droxide (0.1 g, 1.8 mmol) in THF (15 mL)/MeOH (5 mL) for 1 day at
256e257 ꢀC (lit.: [47] 254e257 ꢀC); [
a
]
D
¼ þ2.0ꢀ (c ¼ 0.3, CHCl3),

J. Wiemann et al. / European Journal of Medicinal Chemistry 106 (2015) 194e210
203
25 ꢀC as described above, followed by chromatography (silica gel,
hexanes/ethyl acetate, 7:3) gave 22 (180 mg, 91.8%) as a colorless
solid; RF ¼ 0.6 (silica gel, hexanes/ethyl acetate, 7:3); m.p.:
C2), 33.8 (CH2, C16), 33.7 (CH2, C7), 30.9 (CH2, C21), 29.4 (CH2, C15),
26.6 (CH3, C23), 25.7 (CH2, C12), 21.5 (CH2, C11), 21.0 (CH3, C24),19.7
(CH2, C6), 19.5 (CH3, C30), 16.0 (CH3, C26), 16.0 (CH3, C25), 14.6 (CH3,
C27) ppm; MS (ESI, MeOH): m/z ¼ 544.5 (66%, [M þ H]þ), 566.2 (9%,
[M þ Na]þ), 1087.3 (70%, [2M þ H]þ), 1109.4 (100%, [2M þ Na]þ);
analysis calcd for C37H53NO2 (543.82): C 81.72, H 9.82, N 2.58;
found: C 81.54, H 9.70, N 2.64.
246e248 ꢀC; [
a
]
¼ þ14.3ꢀ (c ¼ 0.31, CHCl3); IR (KBr):
n
¼ 3496s,
D
3448s, 2941s, 2868m, 1634s, 1496s, 1458m, 1376w, 1319w, 1280w,
1223w, 1190w, 1105w, 1077w, 1045w, 892w, 761w, 702w, 596w,
496w cmꢁ1; UVevis (CHCl3): lmax (log ε) ¼ 258.8 (3.43) nm; 1H
NMR (400 MHz, CDCl3):
d
¼ 7.35e7.29 (m, 2H, CH (34/340)),
7.29e7.23 (m, 3H, CH (33/330) þ CH (35)), 5.88 (br, 1H, NH)), 4.74 (s,
1H, CHa (29)), 4.59 (s,1H, CHb (29)), 4.48 (dd, J ¼ 14.7, 5.8 Hz,1H, CHa
(31)), 4.36 (dd, J ¼ 14.7, 5.6 Hz, 1H, CHb (31)), 3.17 (m, 2H, CH
(19) þ CH (3)), 2.49 (dt, J ¼ 12.8, 3.5 Hz, 1H, CH (13)), 2.03e1.94 (m,
2H, CH2 (21)), 1.94e1.87 (m, 2H, CH2 (16)), 1.80e1.69 (m, 2H, CHa
(22) þ CHa (12)), 1.68 (s, 3H, CH3 (30)), 1.68e1.63 (m, 1H, CHa (1)),
1.62e1.54 (m, 3H, CH (18) þ CH2 (2)), 1.53e1.44 (m, 2H, CHa
(15) þ CHa (6)), 1.43e1.36 (m, 2H, CHb (22) þ CHa (11)), 1.36e1.28
(m, 3H, CH2 (7) þ CHb (6)), 1.29e1.20 (m, 2H, CH (9) þ CHb (11)),
1.16e1.08 (m, 1H, CHb (15)), 1.05e0.97 (m, 1H, CHb (12)), 0.96 (s, 3H,
CH3 (27)), 0.96 (s, 3H, CH3 (23)), 0.91 (s, 3H, CH3 (25)), 0.91e0.87 (m,
1H, CHb (1)), 0.82 (s, 3H, CH3 (26)), 0.75 (s, 3H, CH3 (24)), 0.66 (m,
4.3.22. 3-Oxolup-20(29)-en-28-amide (24)
Reaction of 4 (3.0 g, 6.61 mmol) with oxalyl chloride (3.0 g, 2 mL,
23.3 mmol) in abs. DCM (50 mL) followed by a reaction with
ammonia in DCM as described above and chromatographic workup
(silica gel, hexanes/ethyl acetate, 7:3) gave 24 (2.52, 84.1%) as a
colorless solid; RF ¼ 0.21 (silica gel, hexanes/ethyl acetate, 8:2);
m.p.: 155e1157 ꢀC (lit.: [50] 156e157 ꢀC); [
a
]
¼ þ32.9ꢀ (c ¼ 0.37,
D
CHCl3); IR (KBr):
n
¼ 3446br, 2947s, 2868m, 1703s, 1670s, 1458m,
1375m, 1204w, 1116w, 882w, 755w, 543w cmꢁ1; 1H NMR (400 MHz,
CDCl3):
¼ 5.55 (br, 1H, NHa), 5.32 (br, 1H, NHb), 4.74 (s, 1H, CHa
d
(29)), 4.59 (s, 1H, CHb (29)), 3.08 (dt, J ¼ 11.1, 4.4 Hz, 1H, CH (19)),
2.56e2.43 (m, 2H, CH (13) þ CHa (2)), 2.42e2.35 (m, 1H, CHb (2)),
2.03e1.94 (m, 1H, CHa (21)), 1.93e1.85 (m, 2H, CHa (16) þ CHa (1)),
1.84e1.77 (m, 1H, CHa (22)), 1.76e1.69 (m, 1H, CHa (12)), 1.68 (s, 3H,
CH3 (30)),1.62e1.53 (m, 3H, CH (9) þ CHb (16) þ CHa (15)),1.53e1.47
(m, 2H, CHa (6) þ CHb (22)), 1.46e1.34 (m, 7H, CH (18) þ CHb
(1) þ CH2 (7) þ CHb (21) þ CHa (11) þ CHb (6)), 1.34e1.28 (m, 2H, CH
(5) þ CHb (11)), 1.23e1.17 (m, 1H, CHb (15)), 1.06 (s, 3H, CH3 (23)),
1.03e0.99 (m, 1H, CHb (12)), 1.01 (s, 3H, CH3 (24)), 1.00 (s, 3H, CH3
(25)), 0.98 (s, 3H, CH3 (27)), 0.92 (s, 3H, CH3 (26)) ppm; 13C NMR
1H, CH (5)) ppm; 13C NMR (100 MHz, CDCl3):
d
¼ 175.9 (C]O, C28),
151.0 (C]CH2, C20), 139.2 (Cq, C32), 128.6 (CH, C34/340), 127.8 (CH,
C33/330), 127.3 (CH, C35), 109.4 (H2C]C, C29), 79.0 (CH, C3), 55.7
(Cq, C17), 55.4 (CH, C5), 50.7 (CH, C9), 50.2 (CH, C18), 46.7 (CH, C19),
43.3 (CH2, C31), 42.5 (Cq, C14), 40.8 (Cq, C8), 38.9 (CH2, C1), 38.7
(CH2, C22), 38.4 (Cq, C4), 37.7 (CH, C13), 37.2 (Cq, C10), 34.5 (CH2,
C7), 33.8 (CH2, C16), 30.9 (CH2, C21), 29.5 (CH2, C15), 28.0 (CH3,
C23), 27.4 (CH2, C2), 25.7 (CH2, C12), 21.0 (CH2, C11), 19.5 (CH3, C30),
18.3 (CH2, C6), 16.2 (CH3, C26), 16.2 (CH3, C25), 15.4 (CH3, C24), 14.7
(CH3, C27) ppm; MS (ESI, MeOH): m/z ¼ 546.5 (100%, [M þ H]þ),
568.4 (9%, [M þ Na]þ), 1091.4 (28%, [2M þ H]þ), 1113.4 (52%,
[2M þ Na]þ); analysis calcd for C37H55NO2 (545.84): C 81.42, H
10.16, N 2.57; found: C 81.39, H 10.29, N 2.43.
(100 MHz, CDCl3):
d
¼ 218.1 (C]O, C3),178.9 (C]O, C28),150.7 (C]
CH2, C20), 109.4 (H2C]C, C29), 55.8 (Cq, C17), 55.0 (CH, C5), 50.0
(CH, C18), 49.8 (CH, C9), 47.3 (Cq, C4), 46.5 (CH, C19), 42.6 (Cq, C14),
40.7 (Cq, C8), 39.6 (CH2, C1), 38.3 (CH2, C22), 37.7 (CH, C13), 36.9 (Cq,
C10), 34.1 (CH2, C2), 33.9 (CH2, C16), 33.7 (CH2, C7), 30.7 (CH2, C21),
29.5 (CH2, C15), 26.6 (CH3, C23), 25.6 (CH2, C12), 21.4 (CH2, C11),
21.0 (CH3, C24), 19.6 (CH2, C6), 19.5 (CH3, C30), 16.0 (CH3, C26), 15.9
(CH3, C25), 14.5 (CH3, C27) ppm; MS (ESI, MeOH): m/z ¼ 454.5 (59%,
[M þ H]þ), 907.3 (100%, [2M þ H]þ), 929.5 (71%, [2M þ Na]þ).
4.3.21. N-Benzyl-3-oxolup-20(29)-en-28-amide (23)
Jones oxidation of 22 (137 mg, 0.25 mmol) in acetone (25 mL)
using freshly prepared reagent [from CrO3 (63 mg, 0.63 mmol),
sulfuric acid (62 mg, 34 mL, 0.63 mmol, 96%) and water (0.5 mL)] for
20 min, followed by usual aqueous work-up and chromatography
(silica gel, hexanes/ethyl acetate, 7:3) gave 23 (100 mg, 73.5%) as a
colorless solid; RF ¼ 0.68 (silica gel, hexanes/ethyl acetate, 7:3);
4.3.23. (3b) Lup-20(29)-en-3,28-bis-N-ethylcarbamate (25)
Microwave assisted reaction (7 h, 120 ꢀC) of 2 (0.3 g, 0.68 mmol)
in dry THF (5 mL) with ethyl isocyanate (0.29 g, 0.32 mL, 4.1 mmol),
followed by chromatographic work-up (silica gel, hexanes/ethyl
acetate, 8:2) gave 25 (0.32 g, 80.8%) as a colorless solid; RF ¼ 0.29
(silica gel, hexanes/ethyl acetate, 8:2); m.p.: 233e235 ꢀC;
m.p.: 108e111 ꢀC; [
a
]
¼ þ38.3ꢀ (c ¼ 0.37, CHCl3), (lit.: [61]
D
[
a
]
¼ þ43.0ꢀ, CHCl3); IR (KBr):
n
¼ 3396br, 2947s, 2867m, 1705s,
D
1640s, 1519m, 1454m, 1384m, 1241w, 1185w, 1080w, 986w, 882w,
724w, 698w, 583w cmꢁ1; UVevis (CHCl3): lmax (log ε) ¼ 259.0
[
a
]
¼ þ24.9ꢀ (c ¼ 0.44, CHCl3); IR (KBr):
¼ 3330br, 2945s, 2870m,
n
D
(3.42) nm; 1H NMR (500 MHz, CDCl3):
d
¼ 7.35e7.30 (m, 2H, CH (34/
2361w, 1687s, 1536s, 1449m, 1376w, 1306m, 1264s, 1142w, 1107w,
340)), 7.30e7.24 (m, 3H, CH (33/330) þ CH (35)), 5.89 (br, 1H, NH)),
4.74 (s, 1H, CHa (29)), 4.60 (s, 1H, CHb (29)), 4.49 (dd, J ¼ 14.7, 5.8 Hz,
1H, CHa (31)), 4.36 (dd, J ¼ 14.7, 5.6 Hz, 1H, CHb (31)), 3.18 (dt,
J ¼ 11.1, 4.3 Hz, 1H, CH (19)), 2.54 (dt, J ¼ 12.8, 3.5 Hz, 1H, CH (13)),
2.51e2.44 (m, 1H, CHa (2)), 2.48 (ddd, J ¼ 14.7, 7.5, 2.2 Hz, 1H, CHa
(2)), 2.38 (ddd, J ¼ 15.6, 7.5, 4.3 Hz, 1H, CHb (2)), 2.03e1.95 (m, 1H,
CHa (21)), 1.94e1.85 (m, 2H, CHa (1) þ CHa (16)), 1.80e1.70 (m, 2H,
CHa (22) þ CHa (12)), 1.68 (s, 3H, CH3 (30)), 1.64e1.52 (m, 2H, CH
(18) þ CHb (16)), 1.51e1.39 (m, 5H, CHb (22) þ CHa (15) þ CHa
(11) þ CH2 (6)), 1.39e1.33 (m, 5H, CH (9) þ CHb (1) þ CH2 (7) þ CHb
(21)), 1.32e1.23 (m, 1H, CHb (15) þ CHb (11)), 1.18e1.11 (m, 1H, CH
(5)), 1.06 (s, 3H, CH3 (23)), 1.02 (s, 3H, CH3 (24)), 1.02e0.98 (m, 1H,
CHb (12)), 0.97 (s, 3H, CH3 (27)), 0.94 (s, 3H, CH3 (25)), 0.92 (s, 3H,
1083w, 1020m, 986w, 894w, 781w, 636w cmꢁ1; 1H NMR (400 MHz,
CDCl3):
d
¼ 4.68 (s, 1H, CHa (29)), 4.65e4.51 (br, 2H, 2ꢂ NH), 4.58 (s,
1H, CHb (29)), 4.34 (dd, J ¼ 11.2, 4.0 Hz, 1H, CH (3)), 4.25 (d,
J ¼ 10.8 Hz, 1H, CHa (28)), 3.83 (d, J ¼ 10.3 Hz, 1H, CHb (28)), 3.21 (br,
4H, CH2 (32) þ CH2 (320)), 2.4 (dt, J ¼ 11.0, 5.8 Hz, 1H, CH (19)),
2.02e1.91 (m, 1H, CHa (21)), 1.86e1.79 (m, 1H, CHa (16)), 1.78e1.72
(m, 1H, CHa (22)) 1.72e1.68 (m, 1H, CHa (15)), 1.68 (s, 3H, CH3 (30)),
1.68e1.64 (m, 1H, CH (13)), 1.63e1.53 (m, 4H, CHa (2) þ CHa
(1) þ CH2 (12)), 1.52e1.45 (m, 2H, CHb (2) þ CH (18)), 1.45e1.41 (m,
1H, CHa (6)), 1.40e1.33 (m, 6H, CHb (6) þ CH2 (11) þ CHb (21) þ CH2
(7)), 1.32e1.26 (m, 1H, CH (9)), 1.25e1.17 (m, 1H, CHb (16)), 1.14 (t,
J ¼ 7.3 Hz, 3H, CH3 (33)), 1.13 (t, J ¼ 7.2 Hz, 3H, CH3 (330)), 1.09e0.95
(m, 3H, CHb (15) þ CHb (22) þ CHb (1)), 1.03 (s, 3H, CH3 (25)), 0.96 (s,
3H, CH3 (27)), 0.88 (s, 3H, CH3 (23)), 0.83 (s, 3H, CH3 (26)), 0.81e0.76
(m, 1H, CH (5)), 0.80 (s, 3H, CH3 (24)) ppm; 13C NMR (100 MHz,
CH3 (26)) ppm; 13C NMR (125 MHz, CDCl3):
d
¼ 218.1 (C]O, C3),
175.8 (C]O, C28), 150.8 (C]CH2, C20), 139.2 (Cq, C32), 128.6 (CH,
C34/340), 127.8 (CH, C33/330), 127.3 (CH, C35), 109.4 (H2C]C, C29),
55.6 (Cq, C17), 55.0 (CH, C5), 50.1 (CH, C9), 50.1 (CH, C18), 47.3 (Cq,
C4), 46.6 (CH, C19), 43.3 (CH2, C31), 42.6 (Cq, C14), 40.7 (Cq, C8), 39.7
(CH2, C1), 38.4 (CH2, C22), 37.8 (CH, C13), 36.9 (Cq, C10), 34.1 (CH2,
CDCl3):
d
¼ 157.2 (C]O, C31), 156.9 (C]O, C310), 150.4 (C]CH2,
C20), 109.7 (H2C]C, C29), 80.9 (CH, C3), 62.8 (CH2, C28), 55.4 (CH,
C5), 50.3 (CH, C9), 48.8 (CH, C18), 47.7 (CH, C19), 46.6 (Cq, C17), 42.7
(Cq, C14), 40.9 (Cq, C8), 38.4 (CH2, C1), 38.0 (Cq, C4), 37.5 (CH, C13),

204
J. Wiemann et al. / European Journal of Medicinal Chemistry 106 (2015) 194e210
37.0 (Cq, C10), 35.9 (CH2, C32/C320), 34.5 (CH2, C22), 34.1 (CH2, C7),
29.8 (CH2, C16), 29.6 (CH2, C21), 27.9 (CH3, C23), 27.1 (CH2, C15),
25.2 (CH2, C12), 24.1 (CH2, C2), 20.8 (CH2, C11), 19.1 (CH3, C30), 18.1
(CH2, C6), 16.1 (CH3, C24), 16.0 (CH3, C26), 15.7 (CH3, C25), 15.3 (CH3,
C33/C330), 14.7 (CH3, C27) ppm; MS (ESI, MeOH): m/z ¼ 607.4 (37%,
[M þ Na]þ), 1169.5 (100%, [2M þ H]þ), 1191.5 (71%, [2M þ Na]þ);
analysis calcd for C36H60N2O4 (584.87): C 73.93, H 10.34, N 4.79;
found: C 73.77, H 10.51, N 4.69.
(15) þ CHb (12) þ CHb (22)), 1.04 (s, 3H, CH3 (25)), 0.99e0.94 (m, 1H,
CHb (1)), 0.97 (s, 3H, CH3 (27)), 0.92 (t, J ¼ 7.3 Hz, 3H, CH3 (35)), 0.92
(t, J ¼ 7.3 Hz, 3H, CH3 (350)), 0.88 (s, 3H, CH3 (23)), 0.84 (s, 3H, CH3
(26)), 0.81e0.76 (m,1H, CH (5)), 0.80 (s, 3H, CH3 (24)) ppm; 13C NMR
(125 MHz, CDCl3):
d
¼ 157.2 (C]O, C31), 156.8 (C]O, C310), 150.3
(C]CH2, C20), 109.7 (H2C]C, C29), 81.0 (CH, C3), 62.9 (CH2, C28),
55.4 (CH, C5), 50.3 (CH, C9), 48.8 (CH, C18), 47.7 (CH, C19), 46.6 (Cq,
C17), 42.7 (Cq, C14), 40.9 (Cq, C8), 40.7 (CH2, C32/C320), 38.4 (CH2,
C1), 38.0 (Cq, C4), 37.5 (CH, C13), 37.0 (Cq, C10), 34.6 (CH2, C22), 34.1
(CH2, C7), 32.1 (CH2, C33/C330), 29.8 (CH2, C16), 29.7 (CH2, C21), 27.9
(CH3, C23), 27.1 (CH2, C15), 25.2 (CH2, C12), 24.1 (CH2, C2), 20.8 (CH2,
C11), 19.9 (C34/C340), 19.1 (CH3, C30), 18.2 (CH2, C6), 16.5 (CH3, C24),
16.1 (CH3, C26), 16.0 (CH3, C25), 14.7 (CH3, C27), 13.7 (CH3, C35/350)
ppm; MS (ESI, MeOH): m/z ¼ 641.1 (7%, [M þ H]þ), 663.3 (30%,
[M þ Na]þ), 1281.4 (100%, [2M þ H]þ), 1303.5 (24%, [2M þ Na]þ);
analysis calcd for C40H68N2O4 (640.98): C 74.95, H 10.69, N 4.37;
found: C 74.87, H 10.82, N 4.19.
4.3.24. (3b) Lup-20(29)-en-3,28-bis-N-propylcarbamate (26)
Microwave assisted reaction (7 h, 120 ꢀC) of 2 (0.3 g, 0.68 mmol)
in dry THF (5 mL) with propyl isocyanate (0.35 g, 0.39 mL,
4.1 mmol) followed by chromatographic work-up (silica gel, hex-
anes/ethyl acetate, 8:2) gave 26 (0.362 g, 83.4%) as a colorless solid;
RF ¼ 0.4 (silica gel, hexanes/ethyl acetate, 8:2); m.p.: 123e126 ꢀC
[
a
]
¼ þ25.5ꢀ (c ¼ 0.32, CHCl3); IR (KBr):
n
¼ 3358s, 2963s, 2873s,
D
1717s, 1520s, 1458s, 1389m, 1265s, 1138m, 1106m, 1042m, 1008m,
976m, 882w, 774w, 638w cmꢁ1
;
1H NMR (500 MHz, CDCl3):
d
¼ 4.64e4.45 (br, 2H, 2ꢂ NH), 4.61 (s, 1H, CHa (29)), 4.57 (s, 1H, CHb
4.3.26. (3b) Lup-20(29)-en-3,28-bis-N-hexylcarbamate (28)
(29)), 4.27 (dd, J ¼ 7.7, 2.9 Hz,1H, CH (3)), 4.19 (d, J ¼ 10.6 Hz,1H, CHa
(28)), 3.76 (d, J ¼ 10.1 Hz, 1H, CHb (28)), 3.07 (br, 4H, CH2 (32) þ CH2
(320)), 2.38 (dt, J ¼ 11.1, 5.7 Hz, 1H, CH (19)), 1.92 (dq, J ¼ 13.8,
10.5 Hz, 1H, CHa (21)), 1.80e1.72 (m, 1H, CHa (16)), 1.72e1.63 (m, 2H,
CHa (22) þ CHa (15)), 1.61 (s, 3H, CH3 (30)), 1.62e1.54 (m, 4H, CHa
(2) þ CH (13) þ CHa (1) þ CHa (12)), 1.54e1.47 (m, 2H, CHb (2) þ CH
(18)), 1.47e1.40 (m, 5H, CH2 (33) þ CH2 (330) þ CHa (6)), 1.37e1.28
(m, 5H, CH2 (7) þ CHb (21) þ CHa (11) þ CHb (6)), 1.25e1.20 (m, 1H,
CH (9)), 1.20e1.11 (m, 2H, CHb (16) þ CHb (11)), 1.03e0.92 (m, 3H,
CHb (22) þ CHb (15) þ CHb (12)), 0.97 (s, 3H, CH3 (25)), 0.92e0.88
(m, 1H, CHb (1)), 0.90 (s, 3H, CH3 (27)), 0.86 (t, J ¼ 7.7 Hz, 3H, CH3
(34)), 0.85 (t, J ¼ 7.7 Hz, 3H, CH3 (340)), 0.81 (s, 3H, CH3 (23)), 0.77 (s,
3H, CH3 (26)), 0.74e0.70 (m, 1H, CH (5)), 0.73 (s, 3H, CH3 (24)) ppm;
Microwave assisted reaction (7 h, 120 ꢀC) of 2 (0.3 g, 0.68 mmol)
in dry THF (5 mL) with hexyl isocyanate (0.52 g, 0.58 mL, 4.1 mmol)
followed by chromatography (silica gel, hexanes/ethyl acetate, 9:1)
gave 28 (0.35 g, 73.4%) as a colorless solid; RF ¼ 0.49 (silica gel,
hexanes/ethyl acetate, 8:2); m.p.: 85e88 ꢀC;
[
a
]
D
¼
þ20.5ꢀ
(c ¼ 0.53, CHCl3); IR (KBr): ¼ 3347br, 2954s, 2870m, 2361w, 1700s,
n
1540m, 1458m, 1376w, 1247s, 1140w, 1040w, 1012w, 982w, 883w,
775w, 668w cmꢁ1; 1H NMR (500 MHz, CDCl3):
¼ 4.67 (s, 1H, CHa
d
(29)), 4.64e4.59 (br, 2H, 2ꢂ NH), 4.57 (s, 1H, CHb (29)), 4.34 (dd,
J ¼ 10.9, 3.0 Hz, 1H, CH (3)), 4.25 (d, J ¼ 10.7 Hz, 1H, CHa (28)), 3.82
(d, J ¼ 10.4 Hz, 1H, CHb (28)), 3.15 (br, 4H, CH2 (32) þ CH2 (320)), 2.44
(dt, J ¼ 11.0, 5.9 Hz, 1H, CH (19)), 2.04e1.92 (m, 1H, CHa (21)),
1.85e1.78 (m, 1H, CHa (16)), 1.77e1.69 (m, 2H, CHa (15) þ CHa (22)),
1.69e1.60 (m, 4H, CH (13) þ CHa (1) þ CHa (2) þ CHa (12)), 1.67 (s,
3H, CH3 (30)), 1.60e1.53 (m, 2H, CH (18) þ CHb (2)), 1.52e1.43 (m,
5H, CH2 (33) þ CH2 (330) þ CHa (6)), 1.43e1.34 (m, 5H, CH2 (7) þ CHb
(6) þ CHa (11) þ CHb (21)), 1.34e1.25 (m, 13H, CH2 (34) þ CH2
(340) þ CH2 (35) þ CH2 (350) þ CH2 (36) þ CH2 (360) þ CH (9)),
1.23e1.13 (m, 2H, CHb (11) þ CHb (16)), 1.10e0.99 (m, 3H, CHb
(12) þ CHb (15) þ CHb (22)), 1.03 (s, 3H, CH3 (25)), 0.98e0.92 (m, 1H,
CHb (1)), 0.96 (s, 3H, CH3 (27)), 0.91e0.85 (m, 9H, CH3 (23) þ CH3
(37) þ CH3 (370)), 0.83 (s, 3H, CH3 (26)), 0.79 (s, 3H, CH3 (24)),
0.78e0.75 (m, 1H, CH (5)) ppm; 13C NMR (125 MHz, CDCl3):
13C NMR (125 MHz, CDCl3):
d
¼ 157.2 (C]O, C31), 156.9 (C]O,
C310), 150.3 (C]CH2, C20), 109.8 (H2C]C, C29), 81.0 (CH, C3), 62.9
(CH2, C28), 55.4 (CH, C5), 50.3(CH, C9), 48.8 (CH, C18), 47.7 (CH,
C19), 46.6 (Cq, C17), 42.8 (CH2, C32/C320), 42.7 (Cq, C14), 40.9 (Cq,
C8), 38.4 (CH2, C1), 38.0 (Cq, C4), 37.5 (CH, C13), 37.0 (Cq, C10), 34.6
(CH2, C22), 34.1 (CH2, C7), 29.8 (CH2, C16), 29.7 (CH2, C21), 27.9
(CH3, C23), 27.1 (CH2, C15), 25.2 (CH2, C12), 24.1 (CH2, C2), 23.3 (CH2,
C33/C330), 20.8 (CH2, C11), 19.1 (CH3, C30), 18.2 (CH2, C6), 16.5 (CH3,
C24), 16.1 (CH3, C26), 16.0 (CH3, C25), 14.7 (CH3, C27), 11.2 (CH3, C34/
C340) ppm; MS (ESI, MeOH): m/z ¼ 612.8 (6%, [M þ H]þ), 635.5 (26%,
[M þ Na]þ), 1125.5 (100%, [2M þ H]þ), 1247.6 (32%, [2M þ Na]þ);
analysis calcd for C38H64N2O4 (612.93): C 74.46, H 10.52, N 4.57;
found: C 74.39, H 10.69, N 4.50.
d
¼ 157.1 (C]O, C31), 156.8 (C]O, C310), 150.3 (C]CH2, C20), 109.7
(H2C]C, C29), 81.0 (CH, C3), 62.9 (CH2, C28), 55.4 (CH, C5), 50.3 (CH,
C9), 48.8 (CH, C18), 47.7 (CH, C19), 46.6 (Cq, C17), 42.7 (Cq, C14), 41.0
(CH2, C32/C320), 40.9 (Cq, C8), 38.4 (CH2, C1), 38.0 (Cq, C4), 37.5 (CH,
C13), 37.0 (Cq, C10), 34.6 (CH2, C22), 34.1 (CH2, C7), 31.5 (CH2, C35/
350), 30.0 (CH2, C33/C330), 29.8 (CH2, C16), 29.7 (CH2, C21), 27.9
(CH3, C23), 27.1 (CH2, C15), 26.4 (CH2, C34/340), 25.2 (CH2, C12), 24.1
(CH2, C2), 22.5 (CH2, C36/360), 20.8 (CH2, C11), 19.1 (CH3, C30), 18.2
(CH2, C6), 16.5 (CH3, C24), 16.1 (CH3, C26), 16.0 (CH3, C25), 14.7 (CH3,
C27), 14.0 (CH3, C37/370) ppm; MS (ESI, MeOH): m/z ¼ 697.0 (12%,
[M þ H]þ), 719.5 (34%, [M þ Na]þ), 1394.6 (100%, [2M þ H]þ), 1416.4
(20%, [2M þ Na]þ); analysis calcd for C44H76N2O4 (697.09): C 75.81,
H 10.99, N 4.02; found: C 75.69, H 11.14, N 3.94.
4.3.25. (3b) Lup-20(29)-en-3,28-bis-N-butylcarbamate (27)
Microwave assisted reaction (7 h, 120 ꢀC) of 2 (0.3 g, 0.68 mmol)
in dry THF (5 mL) with butyl isocyanate (0.4 g, 0.45 mL, 4.1 mmol)
followed by chromatography (silica gel, hexanes/ethyl acetate, 8:2)
gave 27 (0.35 g, 80.7%) as a colorless solid; RF ¼ 0.41 (silica gel,
hexanes/ethyl acetate, 8:2); m.p.: 106e109 ꢀC; [
a
]
¼ þ31.8ꢀ
D
(c ¼ 0.32, CHCl3); IR (KBr): ¼ 3364br, 2957s, 2872m, 2361w, 1709s,
n
1528m, 1465m, 1376w, 1247m, 1138w, 1018w, 883w, 775w cmꢁ1; 1H
NMR (400 MHz, CDCl3):
¼ 4.68 (s,1H, CHa (29)), 4.66e4.50 (br, 2H,
d
2ꢂ NH), 4.58 (s, 1H, CHb (29)), 4.34 (dd, J ¼ 11.0, 4.0 Hz, 1H, CH (3)),
4.26 (d, J ¼ 10.9 Hz, 1H, CHa (28)), 3.83 (d, J ¼ 10.2 Hz, 1H, CHb (28)),
3.17 (br, 4H, CH2 (32) þ CH2 (320)), 2.45 (dt, J ¼ 10.9, 5.6 Hz, 1H, CH
(19)), 2.04e1.93 (m, 1H, CHa (21)), 1.86e1.79 (m, 1H, CHa (16)),
1.78e1.70 (m, 2H, CHa (22) þ CHa (15)), 1.69e1.61 (m, 4H, CHa
(1) þ CH (13) þ CHa (12) þ CHa (2)), 1.68 (s, 3H, CH3 (30)), 1.61e1.54
(m, 2H, CH (18) þ CHb (2)), 1.52e1.44 (m, 5H, CH2 (33) þ CH2
(330) þ CHa (6)), 1.44e1.38 (m, 6H, CH2 (7) þ CH2 (11) þ CHb
(21) þ CHb (6)), 1.38e1.32 (m, 4H, CH2 (34) þ CH2 (340)), 1.29e1.24
(m, 1H, CH (9)), 1.23e1.17 (m, 1H, CHb (16)), 1.10e0.99 (m, 3H, CHb
4.3.27. 3,28-Di-O-acetyl-betulin (29)
This compound was prepared from 1 (4.5 g, 10.16 mmol) by
acetylation (12 h, 25 ꢀC) with acetic anhydride (7.5 mL, 79.2 mmol),
NEt3 (15 mL, 108.2 mmol) and DMAP (0.12 g, 0.95 mmol) as pre-
viously described, followed by one re-crystallization from EtOH
(30 mL) to yield 29 (4.78 g, 89.5%) as a colorless solid; RF ¼ 0.73
(silica gel, hexanes/ethyl acetate, 8:2); m.p.: 219e222 ꢀC (lit.: [62]
216e218 ꢀC);
[
a
]
¼
þ20.7ꢀ (c
¼
0.36, CHCl3), (lit.: [62]
D
[a
]
¼ þ19.7ꢀ (CHCl3)); MS (ESI, MeOH): m/z ¼ 467.3 (91%,
D

J. Wiemann et al. / European Journal of Medicinal Chemistry 106 (2015) 194e210
205
[M þ H-HOAc]þ), 544.2 (16%, [M þ NH4]þ), 549.4 (12%, [MþNa]þ),
1075.6 (10%, [2M þ Na]þ).
1.67e1.58 (m, 4H, CHa (1) þ CH (13) þ CH2 (12)), 1.58e1.48 (m, 3H,
CH2 (2) þ CH (18)), 1.44e1.34 (m, 3H, CHa (6) þ CH2 (35)), 1.32e1.20
(m, 6H, CH2 (7) þ CHb (21) þ CH2 (11) þ CHb (6)), 1.25e1.16 (m, 2H,
CHb (16) þ CH (9)), 1.10e0.99 (m, 2H, CHb (22) þ CHb (15)), 1.04 (s,
3H, CH3 (25)), 0.99e0.95 (m, 1H, CHb (1)), 0.96 (s, 3H, CH3 (27)), 0.92
(t, J ¼ 4.0 Hz, 3H, CH3 (36)), 0.84 (s, 3H, CH3 (23)), 0.84 (s, 3H, CH3
(26)), 0.83 (s, 3H, CH3 (24)), 0.79e0.76 (m, 1H, CH (5)) ppm; 13C
4.3.28. 3-O-Acetyl-betulin (30)
Deacetylation of 29 (8.2 g, 15.6 mmol) with KOH (0.87 g,
15.6 mmol) in THF (50 mL) and MeOH (20 mL) for 30 min at 0 ꢀC
followed by usual aqueous workup and chromatography (silica gel,
hexanes/ethyl acetate, 8:2) gave 30 (4.33 g, 57.4%) as a colorless
solid; RF ¼ 0.40 (silica gel, hexanes/ethyl acetate, 8:2); m.p.:
NMR (100 MHz, CDCl3):
d
¼ 171.0 (C]O, C31), 157.2 (C]O, C33),
150.3 (C]CH2, C20), 109.8 (H2C]C, C29), 80.9 (CH, C3), 62.9 (CH2,
C28), 55.4 (CH, C5), 50.3 (CH, C9), 48.8 (CH, C18), 47.7 (CH, C19), 46.6
(Cq, C17), 42.8 (CH2, C34), 42.7 (Cq, C14), 40.9 (Cq, C8), 38.4 (CH2, C1),
37.8 (Cq, C4), 37.5 (CH, C13), 37.1 (Cq, C10), 34.6 (CH2, C22), 34.1 (CH2,
C7), 29.8 (CH2, C16), 29.7 (CH2, C21), 27.9 (CH3, C23), 27.1 (CH2, C15),
25.2 (CH2, C12), 23.7 (CH2, C2), 23.2 (CH2, C35), 21.3 (CH3, C32), 20.8
(CH2, C11), 19.1 (CH3, C30), 18.2 (CH2, C6), 16.5 (CH3, C24), 16.1 (CH3,
C26), 16.0 (CH3, C25), 14.7 (CH3, C27), 11.2 (CH3, C36) ppm; MS (ESI,
MeOH): m/z ¼ 570.1 (8%, [M þ H]þ), 592.4 (9%, [M þ Na]þ), 643.3
(13%, [M þ H þ DMF]þ), 665.2 (14%, [M þ Na þ DMF]þ), 1139.5
(100%, [2M þ H]þ), 1161.6 (66%, [2M þ Na]þ); analysis calcd for
255e258 ꢀC (lit.: [62] 258e260 ꢀC); [
a]
¼ þ34.1ꢀ (c ¼ 0.37, CHCl3),
D
(lit.: [62] [
a
]
D
¼ þ25.7ꢀ (CHCl3)); MS (ESI, MeOH): m/z ¼ 485.3 (8%,
[MþH]þ), 538.9 (25%, [M þ Na þ MeOH]þ), 969.5 (18%, [2M þ H]þ),
991.4 (100%, [2M þ Na]þ).
4.3.29. (3b) 3-Acetyloxylup-20(29)-en-28-N-ethylcarbamate (31)
Microwave assisted reaction (7 h, 120 ꢀC) of 30 (1.0 g, 2.1 mmol)
in dry THF (5 mL) with ethyl isocyanate (0.44 g, 0.49 mL, 6.2 mmol)
as described above, followed by chromatography (silica gel, hex-
anes/ethyl acetate, 9:1) gave 31 (1.05 g, 91.2%) as a colorless solid;
RF ¼ 0.44 (silica gel, hexanes/ethyl acetate, 8:2); m.p.: 116e119 ꢀC;
C36H59NO4 (569.86): C 75.88, H 10.44, N 2.46; found: C 75.69, H
[
a
]
¼ þ21.0ꢀ (c ¼ 0.32, CHCl3); IR (KBr):
¼ 3424br, 2946s, 2872m,
n
10.53, N 2.31.
D
1731s, 1641w, 1521m, 1456m, 1369m, 1245s, 1146w, 1106w, 1081w,
1026m, 980w, 883w, 775w, 548w cmꢁ1; 1H NMR (400 MHz, CDCl3):
4.3.31. (3b) 3-Acetyloxylup-20(29)-en-28-N-butylcarbamate (33)
d
¼ 4.68 (s, 1H, CHa (29)), 4.61 (br, 1H, NH), 4.58 (s, 1H, CHb (29)),
Microwave assisted reaction (7 h, 120 ꢀC) of 30 (1 g, 2.1 mmol) in
dry THF (5 mL) with butyl isocyanate (0.61 g, 0.7 mL, 6.2 mol) as
described above, followed by chromatography (silica gel, hexanes/
ethyl acetate, 9:1) gave 33 (1.0 g, 83.3%) as a colorless solid;
RF ¼ 0.57 (silica gel, hexanes/ethyl acetate, 8:2); m.p.: 94e97 ꢀC;
4.46 (dd, J ¼ 9.9, 6.2 Hz, 1H, CH (3)), 4.25 (d, J ¼ 10.7 Hz, 1H, CHa
(28)), 3.82 (d, J ¼ 10.7 Hz, 1H, CHb (28)), 3.22 (br, 2H, CH2 (34)), 2.44
(dt, J ¼ 11.0, 5.7 Hz,1H, CH (19)), 2.03 (s, 3H, CH3 (32)), 2.02e1.92 (m,
1H, CHa (21)), 1.86e1.76 (m, 1H, CHa (16)), 1.75e1.69 (m, 2H, CHa
(22) þ CHa (15)), 1.68 (s, 3H, CH3 (30)), 1.67e1.61 (m, 4H, CHa
(1) þ CH (13) þ CH2 (12)), 1.60e1.52 (m, 3H, CH (18) þ CH2 (2)),
1.52e1.47 (m, 1H, CHa (6)), 1.44e1.33 (m, 6H, CH2 (7) þ CHb
(21) þ CH2 (11) þ CHb (6)), 1.32e1.24 (m, 1H, CH (9)), 1.20 (dd,
J ¼ 12.6, 3.6 Hz, 1H, CHb (16)), 1.14 (t, J ¼ 7.2 Hz, 3H, CH3 (35)),
1.10e0.99 (m, 2H, CHb (22) þ CHb (15)), 1.03 (s, 3H, CH3 (25)),
0.98e0.92 (m, 1H, CHb (1)), 0.96 (s, 3H, CH3 (27)), 0.84 (s, 3H, CH3
(23)), 0.84 (s, 3H, CH3 (26)), 0.83 (s, 3H, CH3 (24)), 0.78 (m, 1H, CH
[a
]
¼ þ18.6ꢀ (c ¼ 0.29, CHCl3); IR (KBr):
¼ 3421br, 2951s, 2872m,
n
D
2366w, 1734m, 1636w, 1534w, 1458w, 1374w, 1245m, 1134w, 1106w,
1025w, 980w, 882w, 776w cmꢁ1
;
1H NMR (400 MHz, CDCl3):
d
¼ 4.68 (s, 1H, CHa (29)), 4.62 (br, 1H, NH), 4.58 (s, 1H, CHb (29)),
4.46 (dd, J ¼ 10.3, 5.8 Hz, 1H, CH (3)), 4.26 (d, J ¼ 10.7 Hz, 1H, CHa
(28)), 3.82 (d, J ¼ 10.5 Hz, 1H, CHb (28)), 3.17 (br, 2H, CH2 (34)), 2.44
(dt, J ¼ 11.1, 5.8 Hz, 1H. CH (19)), 2.03 (s, 3H, CH3 (32)), 2.02e1.92 (m,
1H, CHa (21)), 1.87e1.76 (m, 1H, CHa (16)), 1.75e1.69 (m, 2H, CHa
(22) þ CHa (15)), 1.68 (s, 3H, CH3 (30)), 1.67e1.61 (m, 4H, CHa
(1) þ CH (13) þ CH2 (12)), 1.61e1.53 (m, 3H, CH2 (2) þ CH (18)),
1.52e1.44 (m, 3H, CHa (6) þ CH2 (35)), 1.43e1.29 (m, 6H, CH2
(7) þ CHb (21) þ CH2 (11) þ CHb (6)), 1.29e1.24 (m, 2H, CH2 (36)),
1.23e1.15 (m, 2H, CHb (16) þ CH (9)), 1.10e0.99 (m, 2H, CHb
(22) þ CHb (15)), 1.04 (s, 3H, CH3 (25)), 0.98e0.95 (m, 1H, CHb (1)),
0.96 (s, 3H, CH3 (27)), 0.92 (t, J ¼ 4.0 Hz, 3H, CH3 (37)), 0.84 (s, 3H,
CH3 (23)), 0.84 (s, 3H, CH3 (26)), 0.83 (s, 3H, CH3 (24)), 0.79e0.76 (m,
(5)) ppm; 13C NMR (100 MHz, CDCl3):
d
¼ 171.0 (C]O, C31), 157.0
(C]O, C33), 150.3 (C]CH2, C20), 109.8 (H2C]C, C29), 80.9 (CH, C3),
63.0 (CH2, C28), 55.4 (CH, C5), 50.3 (CH, C9), 48.8 (CH, C18), 47.7 (CH,
C19), 46.6 (Cq, C17), 42.7 (Cq, C14), 40.9 (Cq, C8), 38.4 (CH2, C1), 37.8
(Cq, C4), 37.5 (CH, C13), 37.1 (Cq, C10), 35.1 (CH2, C34), 34.5 (CH2,
C22), 34.1 (CH2, C7), 29.8 (CH2, C16), 29.6 (CH2, C21), 27.9 (CH3,
C23), 27.1 (CH2, C15), 25.2 (CH2, C12), 23.7 (CH2, C2), 21.3 (CH3, C32),
20.8 (CH2, C11), 19.1 (CH3, C30), 18.2 (CH2, C6), 16.5 (CH3, C24), 16.1
(CH3, C26), 16.0 (CH3, C25), 15.3 (CH3, C35), 14.7 (CH3, C27) ppm; MS
(ESI, MeOH): m/z ¼ 555.9 (18%, [M þ H]þ), 578.3 (15%, [M þ Na]þ),
1111.3 (100%, [2M þ H]þ), 1133.3 (53%, [2M þ Na]þ); analysis calcd
for C35H57NO4 (555.83): C 75.63, H 10.34, N 2.52; found: C 75.41, H
10.47, N 2.39.
1H, CH (5)) ppm; 13C NMR (100 MHz, CDCl3):
d
¼ 171.0 (C]O, C31),
157.1 (C]O, C33), 150.3 (C]CH2, C20), 109.8 (H2C]C, C29), 80.9
(CH, C3), 62.9 (CH2, C28), 55.4 (CH, C5), 50.3 (CH, C9), 48.8 (CH, C18),
47.7 (CH, C19), 46.6 (Cq, C17), 42.7 (Cq, C14), 40.9 (Cq, C8), 40.8 (CH2,
C34), 38.4 (CH2, C1), 37.8 (Cq, C4), 37.5 (CH, C13), 37.1 (Cq, C10), 34.6
(CH2, C22), 34.1 (CH2, C7), 32.1 (CH2, C35), 29.8 (CH2, C16), 29.7
(CH2, C21), 27.9 (CH3, C23), 27.1 (CH2, C15), 25.2 (CH2, C12), 23.7
(CH2, C2), 21.3 (CH3, C32), 20.8 (CH2, C11), 19.9 (CH2, C36), 19.1 (CH3,
C30), 18.2 (CH2, C6), 16.5 (CH3, C24), 16.1 (CH3, C26), 16.0 (CH3, C25),
14.7 (CH3, C27), 13.7 (CH3, C37) ppm; MS (ESI, MeOH): m/z ¼ 584.1
(25%, [M þ H]þ), 606.5 (10%, [M þ Na]þ), 1167.5 (100%, [2M þ H]þ),
1189.5 (22%, [2M þ Na]þ); analysis calcd for C37H61NO4 (583.88): C
76.11, H 10.53, N 2.40; found: C 76.00, H 10.63, N 2.29.
4.3.30. (3b) 3-Acetyloxylup-20(29)-en-28-N-propylcarbamate (32)
Microwave assisted reaction (7 h,120 ꢀC) of 30 (0.78 g,1.6 mmol)
in dry THF (5 mL) with propyl isocyanate (0.41 g, 0.45 mL,
4.8 mmol) as described above, followed by chromatography (silica
gel, hexanes/ethyl acetate, 9:1) gave 32 (0.68 g, 73.6%) as a colorless
solid; RF ¼ 0.52 (silica gel, hexanes/ethyl acetate, 8:2); m.p.:
108e111 ꢀC; [
a
]
¼ þ29.9ꢀ (c ¼ 0.34, CHCl3); IR (KBr):
n
¼ 3418br,
D
2949s, 2872m, 2362w, 1732m, 1642w, 1530w, 1461w, 1372w, 1246m,
1136w, 1106w, 1031w, 980w, 882w, 776w, 668w cmꢁ1
;
1H NMR
¼ 4.68 (s, 1H, CHa (29)), 4.65 (br, 1H, NH), 4.58
4.3.32. (3b) 3-Acetyloxylup-20(29)-en-28-N-hexylcarbamate (34)
(400 MHz, CDCl3):
d
Microwave assisted reaction (7 h, 120 ꢀC) of 30 (1 g, 2.1 mmol) in
dry THF (5 mL) with hexyl isocyanate (0.79 g, 0.9 mL, 6.2 mmol) as
described above, followed by chromatography (silica gel, hexanes/
ethyl acetate, 9:1) gave 34 (1.09 g, 86.5%) as a colorless solid;
RF ¼ 0.82 (silica gel, hexanes/ethyl acetate, 8:2); m.p.: 80e82 ꢀC;
(s, 1H, CHb (29)), 4.46 (dd, J ¼ 9.9, 6.1 Hz, 1H, CH (3)), 4.26 (d,
J ¼ 10.6 Hz, 1H, CHa (28)), 3.83 (d, J ¼ 10.6 Hz, 1H, CHb (28)), 3.14 (br,
2H, CH2 (34)), 2.44 (dt, J ¼ 11.1, 5.8 Hz, 1H, CH (19)), 2.03 (s, 3H, CH3
(32)), 2.02e1.92 (m, 1H, CHa (21)), 1.85e1.76 (m, 1H, CHa (16)),
1.76e1.69 (m, 2H, CHa (22) þ CHa (15)), 1.68 (s, 3H, CH3 (30)),
[a
]
¼ þ19.6ꢀ (c ¼ 0.43, CHCl3); IR (KBr):
¼ 3420br, 2950s, 2870m,
n
D

206
J. Wiemann et al. / European Journal of Medicinal Chemistry 106 (2015) 194e210
2362w, 1734m, 1646w, 1534w, 1457w, 1347w, 1245m, 1134w, 1106w,
1030w, 980w, 882w, 776w, 668w, 610w cmꢁ1 1H NMR (400 MHz,
CDCl3):
¼ 4.68 (s, 1H, CHa (29)), 4.62 (br, 1H, NH), 4.58 (s, 1H, CHb
[M þ Na]þ), 1207.3 (48%, [2M þ H]þ), 1229.3 (100%, [2M þ Na]þ);
analysis calcd for C39H57NO4 (603.87): C 77.57, H 9.51, N 2.32;
found: C 77.36, H 9.69, N 2.18.
;
d
(29)), 4.46 (dd, J ¼ 9.4, 6.5 Hz, 1H, CH (3)), 4.26 (d, J ¼ 10.8 Hz, 1H,
CHa (28)), 3.82 (d, J ¼ 10.3 Hz, 1H, CHb (28)), 3.16 (br, 2H, CH2 (34)),
2.44 (dt, J ¼ 11.2, 5.9 Hz, 1H, CH (19)), 2.03 (s, 3H, CH3 (32)),
2.02e1.92 (m, 1H, CHa (21)), 1.85e1.76 (m, 1H, CHa (16)), 1.76e1.70
(m, 2H, CHa (22) þ CHa (15)), 1.68 (s, 3H, CH3 (30)), 1.66e1.62 (m, 2H,
CHa (1) þ CH (13)), 1.61e1.53 (m, 5H, CH (18) þ CH2 (12) þ CH2 (2)),
1.53e1.44 (m, 3H, CH2 (35) þ CHa (6)), 1.44e1.35 (m, 6H, CH2
(7) þ CHb (21) þ CH2 (11) þ CHb (6)), 1.33e1.24 (m, 7H, CH (9) þ CH2
(36) þ CH2 (37) þ CH2 (38)), 1.24e1.15 (m, 1H, CHb (16)), 1.08e0.99
(m, 2H, CHb (22) þ CHb (15)), 1.04 (s, 3H, CH3 (25)), 0.98e0.93 (m,
1H, CHb (1)), 0.96 (s, 3H, CH3 (27)), 0.88 (t, J ¼ 6.4 Hz, 3H, CH3 (39)),
0.84 (s, 3H, CH3 (23)), 0.84 (s, 3H, CH3 (26)), 0.83 (s, 3H, CH3 (24)),
0.79e0.76 (m, 1H, CH (5)) ppm; 13C NMR (100 MHz, CDCl3):
4.3.34. (3b) 3-Hydroxylup-20(29)-en-28-N-ethylcarbamate (36)
Deacetylation of 31 (0.98 g, 1.77 mmol) with potassium hy-
droxide (0.25 g, 5.3 mmol) in THF (30 mL) and MeOH (10 mL) for 2
days at 25 ꢀC as described above, followed by chromatography
(silica gel, hexanes/ethyl acetate, 7:3) gave 36 (0.86 g, 94.5%) as a
colorless solid; RF ¼ 0.13 (silica gel, hexanes/ethyl acetate, 8:2);
m.p.: 223e225 ꢀC; [
a]
¼ þ10.1ꢀ (c ¼ 0.29, CHCl3); IR (KBr):
D
n
¼ 3432br, 2938s, 1689m, 1640w, 1536w, 1458w, 1375w, 1248m,
1024w, 883m, 543w cmꢁ1
;
1H NMR (400 MHz, CDCl3):
d
¼ 4.67 (s,
1H, CHa (29)), 4.62 (br, 1H, NH), 4.57 (s, 1H, CHb (29)), 4.24 (d,
J ¼ 10.8 Hz, 1H, CHa (28)), 3.83 (d, J ¼ 10.5 Hz, 1H, CHb (28)), 3.21 (br,
2H, CH2 (32)), 3.17 (dd, J ¼ 11.2, 5.0 Hz, 1H, CH (3)), 2.44 (dt, J ¼ 11.1,
5.8 Hz, 1H, CH (19)), 1.98 (ddd, J ¼ 19.5, 11.4, 7.0 Hz, 1H, CHa (21)),
1.86e1.72 (m, 3H, CHa (15) þ CHa (16) þ CHa (22)), 1.72e1.54 (m, 5H,
CH (18) þ CH (13) þ CHa (1) þ CHa (2) þ CHa (12)), 1.67 (s, 3H, CH3
(30)), 1.54e1.48 (m, 2H, CHb (2) þ CHa (6)), 1.44e1.33 (m, 5H, CH2
(7) þ CHb (6) þ CHa (11) þ CHb (21)), 1.29e1.17 (m, 3H, CH (9) þ CHb
(11) þ CHb (16)), 1.13 (t, J ¼ 7.2 Hz, 3H, CH3 (33)), 1.10e0.99 (m, 3H,
CHb (12) þ CHb (15) þ CHb (22)), 1.03 (s, 3H, CH3 (25)), 0.96 (s, 3H,
CH3 (27)), 0.96 (s, 3H, CH3 (23)), 0.89 (dt, J ¼ 12.8, 4.4 Hz, 1H, CHb
(1)), 0.81 (s, 3H, CH3 (26)), 0.75 (s, 3H, CH3 (24)), 0.67e0.65 (m, 1H,
d
¼ 171.0 (C]O, C31), 157.2 (C]O, C33), 150.3 (C]CH2, C20), 109.8
(H2C]C, C29), 80.9 (CH, C3), 62.9 (CH2, C28), 55.4 (CH, C5), 50.3
(CH, C9), 48.8 (CH, C18), 47.7 (CH, C19), 46.6 (Cq, C17), 42.7 (Cq, C14),
41.0 (CH2, C34), 40.9 (Cq, C8), 38.4 (CH2, C1), 37.8 (Cq, C4), 37.5 (CH,
C13), 37.1 (Cq, C10), 34.6 (CH2, C22), 34.1 (CH2, C7), 31.5 (CH2, C37),
30.0 (CH2, C35), 29.8 (CH2, C16), 29.7 (CH2, C21), 27.9 (CH3, C23),
27.1 (CH2, C15), 26.4 (CH2, C36), 25.2 (CH2, C12), 23.7 (CH2, C2), 22.5
(CH2, C38), 21.3 (CH3, C32), 20.8 (CH2, C11), 19.1 (CH3, C30), 18.2
(CH2, C6), 16.5 (CH3, C24), 16.1 (CH3, C26), 16.0 (CH3, C25), 14.7 (CH3,
C27), 14.0 (CH3, C39) ppm; MS (ESI, MeOH): m/z ¼ 612.2 (60%,
[M þ H]þ), 634.5 (19%, [M þ Na]þ),1223.6 (100%, [2M þ H]þ),1245.6
(26%, [2M þ Na]þ); analysis calcd for C39H65NO4 (611.94): C 76.55, H
10.71, N 2.29; found: C 76.37, H 10.84, N 2.19.
CH (5)) ppm; 13C NMR (100 MHz, CDCl3):
d
¼ 157.0 (C]O, C31),
150.3 (C]CH2, C20), 109.7 (H2C]C, C29), 79.0 (CH, C3), 62.9 (CH2,
C28), 55.3 (CH, C5), 50.4 (CH, C9), 48.8 (CH, C18), 47.7 (CH, C19), 46.6
(Cq, C17), 42.7 (Cq, C14), 40.9 (Cq, C8), 38.8 (Cq, C4), 38.7 (CH2, C1),
37.5 (CH, C13), 37.1 (Cq, C10), 35.9 (CH2, C32), 34.6 (CH2, C22), 34.2
(CH2, C7), 29.8 (CH2, C16), 29.7 (CH2, C21), 28.0 (CH3, C23), 27.4
(CH2, C2), 27.1 (CH2, C15), 25.2 (CH2, C12), 20.8 (CH2, C11), 19.1 (CH3,
C30), 18.3 (CH2, C6), 16.1 (CH3, C26), 16.0 (CH3, C25), 15.3 (CH3, C24),
15.3 (CH3, C33), 14.7 (CH3, C27) ppm; MS (ESI, MeOH): m/z ¼ 514.1
(14%, [M þ H]þ), 536.4 (6%, [M þ Na]þ), 1027.5 (100%, [2M þ H]þ),
1049.4 (96%, [2M þ Na]þ); analysis calcd for C33H55NO3 (513.79): C
77.14, H 10.79, N 2.73; found: C 77.04, H 10.90, N 2.59.
4.3.33. (3b) 3-Acetyloxylup-20(29)-en-28-N-phenylcarbamate (35)
Microwave assisted reaction (7 h, 120 ꢀC) of 30 (1 g, 2.1 mmol) in
dry THF (5 mL) with phenyl isocyanate (0.74 g, 0.67 mL, 6.2 mmol)
as described above, followed by chromatography (silica gel, hex-
anes/ethyl acetate, 9:1) gave 35 (1.08 g, 87.1%) as a colorless solid;
RF ¼ 0.84 (silica gel, hexanes/ethyl acetate, 7:3); m.p.: 221e223 ꢀC;
[
a
]
¼ þ13.2ꢀ (c ¼ 0.44, CHCl3); IR (KBr):
¼ 3431m, 3080w, 2948s,
n
D
2873m, 1737s, 1647w, 1602m, 1523s, 1501w, 1442s, 1391w, 1374m,
1311m, 1246m, 1204s, 1178w, 1105w, 1084w, 1054m, 1030m, 978m,
898w, 872w, 848w, 757m, 693w, 545w, 510w cmꢁ1; UVevis (CHCl3):
4.3.35. (3b) 3-Hydroxylup-20(29)-en-28-N-propylcarbamate (37)
Deacetylation of 32 (0.53 g, 0.93 mmol) with potassium hy-
droxide (0.10 g, 1.8 mmol) in THF (5 mL)/MeOH (5 mL) for 1 day as
decribed above, followed by chromatography (silica gel, hexanes/
ethyl acetate, 8:2) gave 37 (0.46 g, 93.0%) as a colorless solid;
RF ¼ 0.26 (silica gel, hexanes/ethyl acetate, 8:2); m.p.: 111e114 ꢀC;
lmax (log e) ¼ 236.1 (4.17) nm; 1H NMR (400 MHz, CDCl3):
d
¼ 7.39
(d, J ¼ 7.9 Hz, 2H, HC]C (35/350)), 7.31 (t, J ¼ 7.9 Hz, 2H, HC]C (36/
360)), 7.06 (t, J ¼ 7.3 Hz,1H, HC]C (37)), 6.61 (br,1H, NH), 4.70 (s,1H,
CHa (29)), 4.60 (s,1H, CHb (29)), 4.47 (dd, J ¼ 10.2, 5.9 Hz,1H, CH (3)),
4.37 (d, J ¼ 10.8 Hz, 1H, CHa (28)), 3.95 (d, J ¼ 10.8 Hz, 1H, CHb (28)),
2.47 (dt, J ¼ 11.0, 5.8 Hz, 1H, CH (19)), 2.04 (s, 3H, CH3 (32)),
2.03e1.96 (m, 1H, CHa (21)), 1.92e1.82 (m, 1H, CHa (16)), 1.81e1.71
(m, 2H, CHa (22) þ CHa (15)), 1.69 (s, 3H, CH3 (30)), 1.68e1.66 (m, 1H,
CH (13)),1.66e1.59 (m, 3H, CHa (1) þ CH2 (12)),1.60e1.55 (m, 3H, CH
(18) þ CH2 (2)), 1.53e1.48 (m, 1H, CHa (6)), 1.46e1.36 (m, 6H, CH2
(7) þ CHb (21) þ CH2 (11) þ CHb (6)), 1.33e1.19 (m, 2H, CH (9) þ CHb
(16)), 1.15e1.02 (m, 2H, CHb (22) þ CHb (15)), 1.05 (s, 3H, CH3 (25)),
1.02e0.93 (m, 1H, CHb (1)), 0.98 (s, 3H, CH3 (27)), 0.85 (s, 3H, CH3
(23)), 0.84 (s, 3H, CH3 (26)), 0.84 (s, 3H, CH3 (24)), 0.79e0.76 (m, 1H,
[
a
]
¼ þ10.5ꢀ (c ¼ 0.31, CHCl3); IR (KBr):
¼ 3456br, 2941s, 2869s,
n
D
2362w, 1702s, 1528m, 1460m, 1375w, 1244m, 1106w, 1045m, 883w,
668w cmꢁ1
;
1H NMR (400 MHz, CDCl3):
d
¼ 4.68 (s, 1H, CHa (29)),
4.64 (br, 1H, NH), 4.58 (s, 1H, CHb (29)), 4.25 (d, J ¼ 10.7 Hz, 1H, CHa
(28)), 3.84 (d, J ¼ 10.6 Hz, 1H, CHb (28)), 3.18 (dd, J ¼ 11.2, 5.0 Hz, 1H,
CH (3)), 3.14 (br, 2H, CH2 (32)), 2.44 (dt, J ¼ 11.1, 5.7 Hz, 1H, CH (19)),
2.03e1.92 (m, 1H, CHa (21)), 1.87e1.79 (m, 1H, CHa (16)), 1.79e1.69
(m, 2H, CHa (22) þ CHa (15)), 1.67 (s, 3H, CH3 (30)), 1.66e1.61 (m, 3H,
CHa (1) þ CH (13) þ CHa (12)), 1.61e1.55 (m, 3H, CH (18) þ CH2 (2)),
1.55e1.47 (m, 3H, CH2 (33) þ CHa (6)), 1.44e1.33 (m, 5H, CH2
(7) þ CHb (21) þ CHa (11) þ CHb (6)),1.29e1.17 (m, 3H, CH (19) þ CHb
(16) þ CHb (11)), 1.10e0.99 (m, 3H, CHb (22) þ CHb (15) þ CHb (12)),
1.04 (s, 3H, CH3 (25)), 0.97 (s, 3H, CH3 (23)), 0.96 (s, 3H, CH3 (27)),
0.92 (t, J ¼ 7.3 Hz, 3H, CH3 (34)), 0.89e0.84 (m, 1H, CHb (1)), 0.82 (s,
3H, CH3 (26)), 0.75 (s, 3H, CH3 (24)), 0.69e0.66 (m, 1H, CH (5)) ppm;
CH (5)) ppm; 13C NMR (100 MHz, CDCl3):
d
¼ 171.0 (C]O, C31),
154.0 (C]O, C33), 150.1 (C]CH2, C20), 137.9 (Cq, C34), 129.0 (CH,
C36/360), 123.4 (CH, C37), 118.6 (CH, C35/350), 109.9 (H2C]C, C29),
80.9 (CH, C3), 63.6 (CH2, C28), 55.4 (CH, C5), 50.3 (CH, C9), 48.8 (CH,
C18), 47.7 (CH, C19), 46.6 (Cq, C17), 42.7 (Cq, C14), 40.9 (Cq, C8), 38.4
(CH2, C1), 37.8 (Cq, C4), 37.6 (CH, C13), 37.1 (Cq, C10), 34.5 (CH2, C22),
34.1 (CH2, C7), 29.8 (CH2, C16), 29.6 (CH2, C21), 27.9 (CH3, C23), 27.1
(CH2, C15), 25.2 (CH2, C12), 23.7 (CH2, C2), 21.3 (CH3, C32), 20.8
(CH2, C11), 19.1 (CH3, C30), 18.2 (CH2, C6), 16.5 (CH3, C24), 16.1 (CH3,
C26), 16.1 (CH3, C25), 14.7 (CH3, C27) ppm; MS (ESI, MeOH): m/
z ¼ 604.1 (6%, [M þ H]þ), 621.0 (10%, [M þ NH4]þ), 626.3 (24%,
13C NMR (100 MHz, CDCl3):
d
¼ 157.2 (C]O, C31), 150.3 (C]CH2,
C20), 109.7 (H2C]C, C29), 79.0 (CH, C3), 63.0 (CH2, C28), 55.3 (CH,
C5), 50.4 (CH, C9), 48.8 (CH, C18), 47.7 (CH, C19), 46.6 (Cq, C17), 42.8
(CH2, C32), 42.7 (Cq, C14), 40.9 (Cq, C8), 38.8 (Cq, C4), 38.7 (CH2, C1),
37.5 (CH, C13), 37.1 (Cq, C10), 34.6 (CH2, C22), 34.2 (CH2, C7), 29.9
(CH2, C16), 29.7 (CH2, C21), 28.0 (CH3, C23), 27.4 (CH2, C2), 27.1 (CH2,

J. Wiemann et al. / European Journal of Medicinal Chemistry 106 (2015) 194e210
207
C15), 25.2 (CH2, C12), 23.3 (CH2, C33), 20.8 (CH2, C11), 19.1 (CH3,
C30), 18.3 (CH2, C6), 16.1 (CH3, C26), 16.0 (CH3, C25), 15.3 (CH3, C24),
14.7 (CH3, C27), 11.2 (CH3, C34) ppm; MS (ESI, MeOH): m/z ¼ 528.1
(H2C]C, C29), 79.0 (CH, C3), 62.9 (CH2, C28), 55.3 (CH, C5), 50.4
(CH, C9), 48.8 (CH, C18), 47.7 (CH, C19), 46.6 (Cq, C17), 42.7 (Cq, C14),
41.1 (CH2, C32), 40.9 (Cq, C8), 38.8 (Cq, C4), 38.7 (CH2, C1), 37.5 (CH,
C13), 37.1 (Cq, C10), 34.6 (CH2, C22), 34.2 (CH2, C7), 31.5 (CH2, C35),
30.0 (CH2, C33), 29.8 (CH2, C16), 29.7 (CH2, C21), 28.0 (CH3, C23),
27.4 (CH2, C2), 27.1 (CH2, C15), 26.4 (CH2, C34), 25.2 (CH2, C12), 22.5
(CH2, C36), 20.8 (CH2, C11), 19.1 (CH3, C30), 18.3 (CH2, C6), 16.1 (CH3,
C26), 16.0 (CH3, C25), 15.3 (CH3, C24), 14.7 (CH3, C27), 14.0 (CH3,
C37) ppm; MS (ESI, MeOH): m/z ¼ 570.1 (12%, [M þ H]þ), 592.3 (4%,
[M þ Na]þ), 1139.5 (100%, [2M þ H]þ), 1161.5 (60%, [2M þ Na]þ);
analysis calcd for C37H63NO3 (569.90): C 77.98, H 11.14, N 2.46;
found: C 77.77, H 11.29, N 2.27.
(14%, [M
þ
H]þ), 1055.4 (100%, [2M
þ
H]þ), 1077.5 (72%,
[2M þ Na]þ); analysis calcd for C34H57NO3 (527.82): C 77.37, H
10.88, N 2.65; found: C 77.15, H 10.91, N 2.52.
4.3.36. (3b) 3-Hydroxylup-20(29)-en-28-N-butylcarbamate (38)
Deacetylation of 33 (0.23 g, 0.39 mmol) with potassium hy-
droxide (0.11 g, 2.0 mmol) in THF (10 mL)/MeOH (5 mL) for 2 days at
25 ꢀC as described above, followed by chromatography (silica gel,
hexanes/ethyl acetate, 8:2) gave 38 (0.2 g, 94.0%) as a colorless
solid; RF ¼ 0.39 (silica gel, hexanes/ethyl acetate, 8:2); m.p.:
100e102 ꢀC; [
a]
¼ þ11.6ꢀ (c ¼ 0.43, CHCl3); IR (KBr):
n
¼ 3456br,
4.3.38. (3b) 3-Hydroxylup-20(29)-en-28-N-phenylcarbamate (40)
D
2941s, 2870s, 2362w, 1700m, 1638w, 1522w, 1458m, 1374w, 1248w,
Deacetylation of 35 (1 g, 1.8 mmol) with potassium hydroxide
(0.4 g, 7.16 mmol) in THF (30 mL)/MeOH (20 mL) for 2 days at 25 ꢀC
as described above, followed by chromatography (silica gel, hex-
anes/ethyl acetate, 7:3) gave 40 (0.83 g, 89.2%) as a colorless solid;
RF ¼ 0.28 (silica gel, hexanes/ethyl acetate, 8:2); m.p.: 233e234 ꢀC;
1136w, 1106w, 1047w, 984w, 882w, 774w, 638w cmꢁ1
;
1H NMR
(400 MHz, CDCl3):
d
¼ 4.68 (s, 1H, CHa (29)), 4.62 (br, 1H, NH), 4.58
(s, 1H, CHb (29)), 4.25 (d, J ¼ 10.9 Hz, 1H, CHa (28)), 3.82 (d,
J ¼ 10.5 Hz, 1H, CHb (28)), 3.18 (m, 3H, CH2 (32) þ CH (3)), 2.45 (dt,
J ¼ 11.0, 5.7 Hz, 1H, CH (19)), 2.03e1.92 (m, 1H, CHa (21)), 1.86e1.76
(m, 1H, CHa (16)), 1.76e1.69 (m, 2H, CHa (22) þ CHa (15)), 1.67 (s, 3H,
CH3 (30)), 1.66e1.61 (m, 3H, CHa (1) þ CH (13) þ CHa (12)), 1.61e1.55
(m, 3H, CH2 (2) þ CH (18)), 1.54e1.44 (m, 3H, CHa (6) þ CH2 (33)),
1.43e1.36 (m, 5H, CH2 (7) þ CHb (21) þ CHa (11) þ CHb (6)),
1.36e1.30 (m, 2H, CH2 (34)), 1.29e1.17 (m, 3H, CHb (16) þ CH
(9) þ CHb (11)), 1.10e1.00 (m, 3H, CHb (22) þ CHb (15) þ CHb (12)),
1.04 (s, 3H, CH3 (25)), 0.96 (s, 3H, CH3 (23)), 0.96 (s, 3H, CH3 (27)),
0.92 (t, J ¼ 7.3 Hz, 3H, CH3 (35)), 0.89e0.84 (m, 1H, CHb (1)), 0.82 (s,
3H, CH3 (26)), 0.76 (s, 3H, CH3 (24)), 0.69e0.66 (m, 1H, CH (5)) ppm;
[
a
]
¼ þ7.2ꢀ (c ¼ 0.27, CHCl3); IR (KBr):
n
¼ 3511br, 2945s, 2867m,
D
1721s, 1602m, 1550s, 1503w, 1446s, 1385w, 1316w, 1228s, 1086m,
1070m, 1031w, 984w, 886w, 751m, 692w 506w cmꢁ1; UVevis
(CHCl3): lmax (log ε) ¼ 235.8 (4.06) nm; 1H NMR (400 MHz, CDCl3):
d
¼ 7.39 (d, J ¼ 7.9 Hz, 2H, HC]C (33/330)), 7.31 (t, J ¼ 7.9 Hz, 2H,
HC]C (34/340)), 7.06 (t, J ¼ 7.3 Hz,1H, HC]C (35)), 6.63 (br,1H, NH),
4.70 (s, 1H, CHa (29)), 4.60 (s, 1H, CHb (29)), 4.37 (d, J ¼ 10.7 Hz, 1H,
CHa (28)), 3.96 (d, J ¼ 10.8 Hz, 1H, CHb (28)), 3.18 (dd, J ¼ 11.1, 5.0 Hz,
1H, CH (3)), 2.47 (dt, J ¼ 11.0, 5.8 Hz, 1H, CH (19)), 2.02 (ddd, J ¼ 19.2,
13.9, 10.5 Hz, 1H, CHa (21)), 1.93e1.82 (m, 1H, CHa (16)), 1.81e1.72
(m, 2H, CHa (22) þ CHa (15)), 1.69 (s, 3H, CH3 (30)), 1.68e1.55 (m, 4H,
CH (18) þ CHa (1) þ CH (13) þ CHa (12)), 1.55e1.44 (m, 3H, CH2
(2) þ CHa (6)),1.44e1.36 (m, 5H, CH2 (7) þ CHb (21) þ CHa (11) þ CHb
(6)), 1.32e1.22 (m, 3H, CH (9) þ CHb (16) þ CHb (11)), 1.15e1.02 (m,
2H, CHb (22) þ CHb (15)), 1.05 (s, 3H, CH3 (25)), 1.01e0.95 (m, 1H,
CHb (12)), 0.98 (s, 3H, CH3 (27)), 0.97 (s, 3H, CH3 (23)), 0.90 (dt,
J ¼ 12.9, 4.3 Hz, 1H, CHb (1)), 0.83 (s, 3H, CH3 (26)), 0.76 (s, 3H, CH3
(24)), 0.69e0.66 (m, 1H, CH (5)) ppm; 13C NMR (100 MHz, CDCl3):
13C NMR (100 MHz, CDCl3):
d
¼ 157.1 (C]O, C31), 150.3 (C]CH2,
C20), 109.7 (H2C]C, C29), 79.0 (CH, C3), 62.9 (CH2, C28), 55.3 (CH,
C5), 50.4 (CH, C9), 48.8 (CH, C18), 47.7 (CH, C19), 46.6 (Cq, C17), 42.7
(Cq, C14), 40.9 (Cq, C8), 40.8 (CH2, C32), 38.8 (Cq, C4), 38.7 (CH2, C1),
37.5 (CH, C13), 37.1 (Cq, C10), 34.6 (CH2, C22), 34.2 (CH2, C7), 32.1
(CH2, C33), 29.8 (CH2, C16), 29.7 (CH2, C21), 28.0 (CH3, C23), 27.4
(CH2, C2), 27.1 (CH2, C15), 25.2 (CH2, C12), 20.8 (CH2, C11), 19.9 (CH2,
C34), 19.1 (CH3, C30), 18.3 (CH2, C6), 16.1 (CH3, C26), 16.0 (CH3, C25),
15.3 (CH3, C24), 14.7 (CH3, C27), 13.7 (CH3, C35) ppm; MS (ESI,
MeOH): m/z ¼ 542.1 (14%, [M þ H]þ), 1083.5 (100%, [2M þ H]þ),
1105.5 (22%, [2M þ Na]þ); analysis calcd for C35H59NO3 (541.85): C
77.58, H 10.98, N 2.58; found: C 77.40, H 11.14, N 2.41.
d
¼ 154.1 (C]O, C31),150.1 (C]CH2, C20),137.9 (Cq, C32),129.0 (CH,
C34/340), 123.4 (CH, C35), 118.6 (CH, C33/330), 109.8 (H2C]C, C29),
79.0 (CH, C3), 63.5 (CH2, C28), 55.3 (CH, C5), 50.4 (CH, C9), 48.9 (CH,
C18), 47.7 (CH, C19), 46.6 (Cq, C17), 42.7 (Cq, C14), 40.9 (Cq, C8), 38.9
(Cq, C4), 38.7 (CH2, C1), 37.6 (CH, C13), 37.2 (Cq, C10), 34.6 (CH2,
C22), 34.2 (CH2, C7), 29.8 (CH2, C16), 29.6 (CH2, C21), 28.0 (CH3,
C23), 27.4 (CH2, C2), 27.1 (CH2, C15), 25.2 (CH2, C12), 20.8 (CH2, C11),
19.2 (CH3, C30), 18.3 (CH2, C6), 16.1 (CH3, C26), 16.1 (CH3, C25), 15.3
(CH3, C24), 14.8 (CH3, C27) ppm; MS (ESI, MeOH): m/z ¼ 579.1 (6%,
[M þ NH4]þ), 584.3 (6%, [M þ Na]þ), 1123.4 (48%, [2M þ H]þ), 1146.5
(44%, [2M þ Na]þ); analysis calcd for C37H55NO3 (561.84): C 79.10, H
9.87, N 2.49; found: C 79.03, H 10.02, N 2.33.
4.3.37. (3b) 3-Hydroxylup-20(29)-en-28-N-hexylcarbamate (39)
Deacetylation of 34 (0.27 g, 0.56 mmol) with potassium hy-
droxide (0.1 g, 1.8 mmol) in THF (10 mL)/MeOH (5 mL) for 1 day as
described above, followed by chromatography (silica gel, hexanes/
ethyl acetate, 8:2) gave 39 (0.22 g, 87.5%) as a colorless solid;
RF ¼ 0.32 (silica gel, hexanes/ethyl acetate, 8:2); m.p.: 95e97 ꢀC;
[
a
]
¼ þ11.7ꢀ (c ¼ 0.41, CHCl3); IR (KBr):
n
¼ 3456br, 2940s, 2869s,
D
1702s, 1642w, 1523m, 1456m, 1375w, 1248m, 1106w, 1045m, 883w,
637w cmꢁ1
;
1H NMR (400 MHz, CDCl3):
d
¼ 4.68 (s, 1H, CHa (29)),
4.3.39. 28-O-Acetyl-betulin (41)
4.63 (br, 1H, NH), 4.57 (s, 1H, CHb (29)), 4.25 (d, J ¼ 10.4 Hz, 1H, CHa
(28)), 3.83 (d, J ¼ 10.4 Hz, 1H, CHb (28)), 3.17 (m, 3H, CH2 (32) þ CH
(3)), 2.44 (dt, J ¼ 11.0, 5.7 Hz, 1H, CH (19)), 2.06e1.92 (m, 1H, CHa
(21)), 1.79 (dd, J ¼ 17.5, 9.3 Hz, 1H, CHa (16)), 1.76e1.69 (m, 2H, CHa
(22) þ CHa (15)), 1.67 (s, 3H, CH3 (30)), 1.66e1.61 (m, 3H, CHa
(1) þ CH (13) þ CHa (12)), 1.61e1.51 (m, 4H, CH (18) þ CH2 (2) þ CHa
(6)), 1.51e1.44 (m, 2H, CH2 (33)), 1.44e1.36 (m, 5H, CH2 (7) þ CHb
(21) þ CHa (11) þ CHb (6)), 1.34e1.23 (m, 7H, CH (9) þ CH2
(34) þ CH2 (35) þ CH2 (36)), 1.23e1.14 (m, 2H, CHb (11) þ CHb (16)),
1.09e1.01 (m, 3H, CHb (22) þ CHb (15) þ CHb (12)), 1.03 (s, 3H, CH3
(25)), 0.96 (s, 3H, CH3 (23)), 0.96 (s, 3H, CH3 (27)), 0.93e0.87 (m, 1H,
CHb (1)), 0.88 (t, J ¼ 6.7 Hz, 3H, CH3 (37)), 0.82 (s, 3H, CH3 (26)), 0.75
(s, 3H, CH3 (24)), 0.69e0.66 (m, 1H, CH (5)) ppm; 13C NMR
Acetylation of 2 (15.0 g, 33.88 mmol) in DCM (100 mL) with
acetic anhydride (3.2 mL, 33.88 mmol) in the presence of NEt3
(4.3 mL) and DMAP (0.24 g, 1.96 mmol) as previously described,
followed by usual workup and chromatography (silica gel, hexanes/
ethyl acetate, 8:2) gave 41 (9.87 g, 60.4%) as a colorless solid;
RF ¼ 0.40 (silica gel, hexanes/ethyl acetate, 8:2); m.p.: 216e217 ꢀC
(lit.: [63] 214e215 ꢀC); [
a
]
D
¼ þ11.1ꢀ (c ¼ 0.39, CHCl3), (lit.: [64]
[a
]
¼ þ10.3ꢀ (CHCl3)); MS (ESI, MeOH): m/z ¼ 467.3 (100%,
D
[M þ HeH2O]þ), 485.1 (4%, [MþH]þ), 991.4 (16%, [2M þ Na]þ).
4.3.40. 28-Acetyloxy-lup-20(29)-en-3-one (42)
Jones oxidation of 41 (3.0 g, 6.22 mmol) for 40 min at 25 ꢀC as
previously described, followed by usual aqueous workup and
chromatography (silica gel, hexanes/ethyl acetate, 8:2) gave 42
(100 MHz, CDCl3):
d
¼ 157.2 (C]O, C31), 150.3 (C]CH2, C20), 109.7

208
J. Wiemann et al. / European Journal of Medicinal Chemistry 106 (2015) 194e210
(2.14 g, 71.6%) as a colorless solid; RF ¼ 0.73 (silica gel, hexanes/
ethyl acetate, 8:2); m.p.: 91e94 ꢀC (lit.: [65] 77e79 ꢀC);
CHb (28)), 3.13 (br, 2H, CH2 (32)), 2.53e2.42 (m, 2H, CH (19) þ CHa
(2)), 2.37 (ddd, J ¼ 15.6, 7.5, 4.4 Hz, 1H, CHb (2)), 2.07e1.93 (m, 1H,
CHa (21)), 1.92e1.82 (m, 1H, CHa (1)), 1.82e1.69 (m, 3H, CHa
(16) þ CHa (22) þ CHa (15)), 1.67 (s, 3H, CH3 (30)), 1.69e1.55 (m, 4H,
CH (13) þ CH2 (12) þ CH (18)), 1.54e1.47 (m, 2H, CH2 (33)),
1.47e1.33 (m, 8H, CH2 (6) þ CH2 (7) þ CHa (11) þ CHb (21) þ CHb
(1) þ CH (9)), 1.25 (m, 3H, CH (5) þ CHb (11) þ CHb (16)), 1.10e0.99
(m, 2H, CHb (22) þ CHb (15)), 1.07 (s, 3H, CH3 (25)) 1.05 (s, 3H, CH3,
(23)), 1.01 (s, 3H, CH3 (24)), 0.97 (s, 3H, CH3, (27)), 0.9 (s, 3H, CH3
(26)), 0.91 (t, J ¼ 2.0 Hz, 3H, CH3 (34)) ppm; 13C NMR (100 MHz,
[
a]
¼ þ37.1ꢀ (c ¼ 0.35, CHCl3), (lit.: [66] [
a
]
D
¼ þ38.0ꢀ (CHCl3)); MS
D
(ESI, MeOH): m/z ¼ 483.2 (26%, [MþH]þ), 505.3 (8%, [MþNa]þ),
536.8 (10%, [M þ Na þ MeOH]þ), 965.1 (12%, [2M þ H]þ), 987.3 (70%,
[2M þ Na]þ).
4.3.41. 28-Hydroxy-lup-20(29)en-3-one (43)
Deacetylation of 42 (2.1 g, 4.36 mmol) with KOH (0.61 g,
10.9 mmol) in THF (20 mL) and MeOH (10 mL) for 12 h at 25 ꢀC as
described above, followed by usual workup and chromatography
(silica gel, hexanes/ethyl acetate, 8:2) gave 43 (1.73 g, 90.4%) as a
colorless solid; RF ¼ 0.45 (silica gel, hexanes/ethyl acetate, 8:2);
CDCl3):
d
¼ 218.0 (C]O, C3), 157.1 (C]O, C31), 150.2 (C]CH2, C20),
109.8 (H2C]C, C29), 62.8 (CH2, C28), 55.0 (CH, C5), 49.7 (CH, C9),
48.7 (CH, C18), 47.6 (CH, C19), 47.3 (Cq, C4), 46.6 (Cq, C17), 43.3 (CH2,
C32), 42.7 (Cq, C14), 40.8 (Cq, C8), 39.6 (CH2, C1), 37.6 (CH, C13), 36.9
(Cq, C10), 34.5 (CH2, C22), 34.1 (CH2, C2), 33.5 (CH2, C7), 29.8 (CH2,
C16), 29.6 (CH2, C21), 27.1 (CH2, C15), 26.5 (CH3, C23), 25.2 (CH2, C2),
23.2 (CH2, C33), 21.3 (CH2, C11), 21.0 (CH3, C24), 19.6 (CH2, C6), 19.1
(CH3, C30), 15.9 (CH3, 25), 15.8 (CH3, C26), 14.7 (CH3, C27), 11.2 (CH3,
C34) ppm; MS (ESI, MeOH): m/z ¼ 526.2 (40%, [M þ H]þ), 548.4 (6%,
[M þ Na]þ), 1051.6 (100%, [2M þ H]þ], 1073.6 (46%, [2M þ Na]þ);
analysis calcd for C34H55NO3 (525.81): C 77.66, H 10.54, N 2.66;
found: C 77.51, H 10.69, N 2.50.
m.p.: 103e105 ꢀC (lit.: [67] 94e96 ꢀC); [
a
]
D
¼ þ45.0ꢀ (c ¼ 0.40,
CHCl3), (lit.: [67] [
a
]
¼ þ52.7ꢀ (CHCl3)); MS (ESI, MeOH): m/
z ¼ 423.1 (31%, [M þ DHeH2O]þ), 441.2 (46%, [MþH]þ), 881.4 (29%,
[2M þ H]þ), 903.4 (100%, [2M þ Na]þ).
4.3.42. 3-Oxo-lup-20(29)-en-28-N-ethylcarbamate (44)
Microwave assisted reaction (7 h, 120 ꢀC) of 43 (0.3 g,
0.682 mmol) in dry THF (5 mL) with ethyl isocyanate (0.15 mg,
0.16 mL, 2.05 mmol) as described above, followed by chroma-
tography (silica gel, hexanes/ethyl acetate, 8:2) gave 44 (0.237 g,
68.0%) as a colorless solid; RF ¼ 0.32 (silica gel, hexanes/ethyl
4.3.44. 3-Oxo-lup-20(29)-en-28-N-butylcarbamate (46)
acetate, 8:2); m.p.: 83e86 ꢀC; [
a
]
¼ þ34.7ꢀ (c ¼ 0.32, CHCl3); IR
Microwave assisted reaction (7 h, 120 ꢀC) of 43 (0.28 g,
0.682 mmol) in dry THF (5 mL) with butyl isocyanate (0.2 g,
0.23 mL, 2.05 mmol) as described above, followed by chroma-
tography (silica gel, hexanes/ethyl acetate, 8:2) gave 46 (0.246 g,
72.0%) as a colorless solid; RF ¼ 0.51 (silica gel, hexanes/ethyl
D
(KBr):
n
¼ 3441s, 2945s, 2870m, 1706s, 1639m, 1523w, 1458m,
1384w, 1243m, 1138w, 1112w, 1080w, 1023m, 882w, 774w cmꢁ1; 1H
NMR (400 MHz, CDCl3):
¼ 4.68 (s, 1H, CHa (29)), 4.60 (br, 1H, NH),
d
4.58 (s, 1H, CHb (29)), 4.26 (d, J ¼ 10.7 Hz, 1H, CHa (28)), 3.83 (d,
J ¼ 10.9 Hz, 1H, CHb (28)), 3.22 (br, 2H, CH2 (32)), 2.53e2.43 (m, 2H,
CH (19) þ CHa (2)), 2.37 (ddd, J ¼ 15.7, 7.4, 4.5 Hz, 1H, CHb (2)),
2.05e1.94 (m, 1H, CHa (21)), 1.92e1.86 (m, 1H, CHa (1)), 1.81e1.71
(m, 3H, CHa (22) þ CHa (16) þ CHa (15)), 1.68 (s, 3H, CH3 (30)),
1.68e1.64 (m, 2H, CH (13) þ CHa (12)), 1.62e1.56 (m, 1H, CH (18)),
1.49e1.39 (m, 4H, CH2 (6) þ CH2 (7)), 1.36e1.30 (m, 4H, CH
(9) þ CHb (1) þ CHb (21) þ CHa (11)), 1.28e1.26 (m, 1H, CH (5)),
1.25e1.20 (m, 2H, CHb (11) þ CHb (16)), 1.14 (t, J ¼ 7.2 Hz, 3H, CH3
(33)), 1.08 (s, 3H, CH3 (25)), 1.06 (s, 3H, CH3, (23)), 1.05e1.00 (m,
3H, CHb (22) þ CHb (15) þ CHb (12)), 1.02 (s, 3H, CH3 (24)), 0.98 (s,
3H, CH3 (27)), 0.93 (s, 3H, CH3 (26)) ppm; 13C NMR (100 MHz,
acetate, 8:2); m.p.: 84e86 ꢀC; [
a
]
¼ þ32.4ꢀ (c ¼ 3.65, CHCl3); IR
D
(KBr):
n
¼ 3416s, 2956s, 2870m, 1706s, 1642w, 1530m, 1460m,
1384w, 1245m, 1136w, 1112w, 1052w, 1021m, 882w, 776w cmꢁ1; 1H
NMR (400 MHz, CDCl3):
¼ 4.68 (s, 1H, CHa (29)), 4.64 (br, 1H, NH),
d
4.58 (s, 1H, CHb (29)), 4.26 (d, J ¼ 10.5 Hz, 1H, CHa (28)), 3.83 (d,
J ¼ 10.5 Hz, 1H, CHb (28)), 3.17 (br, 2H, CH2 (32)), 2.55e2.42 (m, 2H,
CH (19) þ CHa (2)), 2.38 (ddd, J ¼ 15.5, 7.4, 4.3 Hz, 1H, CHb (2)),
2.07e1.93 (m, 1H, CHa (21)), 1.92e1.83 (m, 1H, CHa (1)), 1.82e1.69
(m, 3H, CHa (16) þ CHa (22) þ CHa (15)), 1.69e1.54 (m, 4H, CH
(13) þ CH2 (12) þ CH (18)), 1.67 (s, 3H, CH3 (30)), 1.53e1.41 (m, 6H,
CH2 (33) þ CH2 (6) þ CH2 (7)), 1.41e1.33 (m, 5H, CH2 (11) þ CHb
(21) þ CHb (1) þ CH (9)), 1.32e1.17 (m, 4H, CH (5) þ CHb (16) þ CH2
(34)), 1.11e1.05 (m, 2H, CHb (22) þ CHb (15)), 1.07 (s, 3H, CH3 (25)),
1.06 (s, 3H, CH3 (23)), 1.02 (s, 3H, CH3 (24)), 0.97 (s, 3H, CH3 (27)),
0.94 (s, 3H, CH3 (26)), 0.92 (t, J ¼ 7.7 Hz, 3H, CH3 (35)) ppm; 13C
CDCl3):
d
¼ 218.0 (C]O, C3), 157.0 (C]O, C31), 150.2 (C]CH2,
C20), 109.8 (H2C]C, C29), 62.8 (CH2, C28), 55.0 (CH, C5), 49.8 (CH,
C9), 48.7 (CH, C18), 47.6 (CH, C19), 47.3 (Cq, C4), 46.6 (Cq, C17), 42.8
(Cq, C14), 40.8 (Cq, C8), 39.6 (CH2, C1), 37.6 (CH, C13), 36.9 (Cq, C10),
36.1 (CH2, C32), 34.5 (CH2, C22), 34.1 (CH2, C2), 33.5 (CH2, C7), 29.8
(CH2, C16), 29.6 (CH2, C21), 27.1 (CH2, C15), 26.5 (CH3, C23), 25.2
(CH2, C12), 21.3 (CH2, C11), 21.0 (CH3, C24), 19.6 (CH2, C6), 19.1
(CH3, C30), 15.9 (CH3, 25), 15.8 (CH3, C26), 15.3 (CH3, C33), 14.7
(CH3, C27) ppm; MS (ESI, MeOH): m/z ¼ 512.1 (100%, [M þ H]þ),
529.1 (7%, [M þ NH4]þ), 534.3 (17%, [M þ Na]þ), 1023.5 (100%,
[2M þ H]þ), 1025.5 (64%, [2M þ Na]þ); analysis calcd for
NMR (100 MHz, CDCl3):
d
¼ 218.0 (C]O, C3), 157.2 (C]O, C31),
150.2 (C]CH2, C20), 109.8 (H2C]O, C29), 62.8 (CH2, C28), 55.0
(CH, C5), 49.7 (CH, C9), 48.8 (CH, C18), 47.6 (CH, C19), 47.3 (Cq, C4),
46.6 (Cq, C17), 42.8 (Cq, C14), 40.8 (Cq, C8), 40.8 (CH2, C32), 39.1
(CH2, C1), 37.6 (CH, C13), 36.9 (Cq, C10), 34.5 (CH2, C22), 34.1 (CH2,
C2), 33.5 (CH2, C7), 32.1 (CH2, C33), 29.8 (CH2, C16), 29.6 (CH2,
C21), 27.1 (CH2, C15), 26.5 (CH3, C23), 25.2 (CH2, C12), 21.3 (CH2,
C11), 21.0 (CH3, C24), 19.9 (CH2, C34), 19.6 (CH2, C6), 19.1 (CH3,
C30), 15.9 (CH3, C25), 15.9 (CH3, C26), 14.7 (CH3, C27), 13.7 (CH3,
C35) ppm; MS (ESI, MeOH): m/z ¼ 540.2 (72%, [M þ H]þ), 562.5
C
33H53NO3 (511.78): C 77.45, H 10.44, N 2.74; found: C 77.39, H
10.51, N 2.57.
4.3.43. 3-Oxo-lup-20(29)-en-28-N-propylcarbamate (45)
(8%, [M
þ
Na]þ), 1079.5 (100%, [2M
þ
H]þ), 1101.5 (49%,
Microwave assisted reaction (7 h, 120 ꢀC) of 43 (0.29 g,
0.659 mmol) in dry THF (5 mL) with propyl isocyanate (0.17 g,
0.19 mL, 1.98 mmol) as described above, followed by chromatog-
raphy (silica gel, hexanes/ethyl acetate, 8:2) gave 45 (0.216 g, 62.0%)
as a colorless solid; RF ¼ 0.35 (silica gel, hexanes/ethyl acetate, 8:2);
[2M þ Na]þ); analysis calcd for C35H57NO3 (539.83): C 77.87, H
10.64, N 2.59; found: C 77.61, H 10.83, N 2.41.
4.3.45. 3-Oxo-lup-20(29)-en-28-N-hexylcarbamate (47)
Microwave assisted reaction (7 h, 120 ꢀC) of 43 (0.29 g,
0.66 mmol) in dry THF (5 mL) with hexyl isocyanate (0.25 g,
0.29 mL, 1.98 mmol) as described above, followed by chromatog-
raphy (silica gel, hexanes/ethyl acetate, 8:2) gave 47 (0.205 g, 55.1%)
as a colorless solid; RF ¼ 0.57 (silica gel, hexanes/ethyl acetate, 8:2);
m.p.: 87e91 ꢀC; [
a
]
D
¼ þ34.5ꢀ (c ¼ 3.4, CHCl3); IR (KBr):
¼ 3406s,
n
2958s, 2870m, 1706s, 1642w, 1530w, 1459m, 1384w, 1236m, 1138w,
1112w, 1044w, 1004w, 882w, 776w cmꢁ1
CDCl3):
¼ 4.72e4.64 (br, 1H, NH), 4.67 (s, 1H, CHa (29)), 4.57 (s, 1H,
CHb (29)), 4.26 (d, J ¼ 10.8 Hz, 1H, CHa (28)), 3.82 (d, J ¼ 10.7 Hz, 1H,
;
1H NMR (400 MHz,
d
m.p.: 76e79 ꢀC; [
a
]
D
¼ þ29.8ꢀ (c ¼ 0.52, CHCl3); IR (KBr):

J. Wiemann et al. / European Journal of Medicinal Chemistry 106 (2015) 194e210
209
n
¼ 3397m, 2951s, 2869s, 1706s, 1640w, 1522m, 1458m, 1384w,
Acknowledgments
1243m, 1138w, 1112w, 1041w, 1018w, 882w, 776w, 580w cmꢁ1
;
1H
NMR (400 MHz, CDCl3):
d
¼ 4.68 (s, 1H, CHa (29)), 4.63 (br, 1H, NH),
Thanks are due to Mrs J. Wiese for measuring the IR spectra and
optical rotations. The cell lines were kindly provided by Dr. Th. Müller
4.58 (s, 1H, CHb (29)), 4.26 (d, J ¼ 10.7 Hz, 1H, CHa (28)), 3.83 (d,
J ¼ 10.8 Hz, 1H, CHb (28)), 3.17 (br, 2H, CH2 (32)), 2.54e2.42 (m, 2H,
CH (19) þ CHa (2)) 2.38 (ddd, J ¼ 15.6, 7.5, 4.3 Hz, 1H, CHb (2)), 2.00
(m, 1H, CHa (21)), 1.93e1.83 (m, 1H, CHa (1)), 1.83e1.69 (m, 3H, CHa
(16) þ CHa (22) þ CHa (15)), 1.67 (s, 3H, CH3 (30)), 1.69e1.54 (m, 4H,
CH (13) þ CH2 (12) þ CH (18)), 1.54e1.40 (m, 6H, CH2 (33) þ CH2
(6) þ CH2 (7)), 1.39e1.31 (m, 4H, CH (9) þ CHb (1) þ CHb (21) þ CHa
(11)), 1.30e1.26 (m, 7H, CH2 (34) þ CH2 (36) þ CH2 (35) þ CH (5)),
1.26e1.16 (m, 2H, CHb (11) þ CHb (16)), 1.07 (s, 3H, CH3 (25)), 1.06 (s,
3H, CH3 (23)) 1.12e0.95 (m, 2H, CHb (22) þ CHb (15)), 1.02 (s, 3H,
CH3 (24)), 0.97 (s, 3H, CH3 (27)), 0.92 (s, 3H, CH3 (26)), 0.88 (t,
€
(Dept. of Haematology/Oncology, Martin-Luther Universitat Halle-
Wittenberg). Support by the “GründerwerkstatteBiowissenschaften”
is gratefully acknowledged.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2015.10.043.
References
J ¼ 6.5 Hz, 3H, CH3 (37)) ppm; 13C NMR (100 MHz, CDCl3):
¼ 218.0
d
(C]O, C3), 157.1 (C]O, C31), 150.2 (C]CH2, C20), 109.8 (H2C]C,
C29), 62.8 (CH2, C28), 55.0 (CH, C5), 49.8 (CH, C9), 48.8 (CH, C18),
47.6 (CH, C19), 47.3 (Cq, C4), 46.6 (Cq, C17), 42.8 (Cq, C14), 41.1 (CH2,
C32), 40.8 (Cq, C8), 39.6 (CH2, C1), 37.6 (CH, C13), 36.9 (Cq, C10), 34.5
(CH2, C22), 34.1 (CH2, C2), 33.5 (CH2, C7), 31.5 (CH2, C35), 30.0 (CH2,
C33), 29.8 (CH2, C6), 29.7 (CH2, C21), 27.1 (CH2, C15), 26.5 (CH3,
C23), 26.4 (CH2, C34), 25.2 (CH2, C12), 22.5 (CH2, C36), 21.3 (CH2,
C11), 21.0 (CH3, C24), 19.6 (CH2, C6), 19.1 (CH3, C30), 15.9 (CH3, C25),
15.8 (CH3, C26), 14.7 (CH3, C27), 14.0 (CH3, C37) ppm; MS (ESI,
MeOH): m/z ¼ 568.1 (30%, [M þ H]þ), 590.5 (4%, [M þ Na]þ), 1135.6
(100%, [2M þ H]þ), 1157.8 (32%, [2M þ Na]þ); analysis calcd for
€
[1] H. Lossen, Über die Oxalohydroxamsaure, Liebigs Ann. Chem. 150 (1869)
314e316.
[2] A.S. Begum, V.K. Jain, C.L. Khetrapal, N.C. Shivaprakash, Structural studies of
oxalohydroxamic acid by single-crystal X-ray-diffraction and spectroscopic
methods, J. Cryst. Spectrosc. 17 (1987) 545e555.
[3] S.P.E. Gupta, Hydroxamic Acids e a Unique Family of Chemicals with Multiple
Biological Activties, Springer, Berlin, 2013.
[4] E.M.F. Muri, M.J. Nieto, R.D. Sindelar, J.S. Williamson, Hydroxamic acids as
pharmacological agents, Curr. Med. Chem. 9 (2002) 1631e1653.
[5] M. Sani, D. Belotti, R. Giavazzi, W. Panzeri, A. Volonterio, M. Zanda, Synthesis
and evaluation of stereopure alpha-trifluoromethyl-malic hydroxamates as
inhibitors of matrix metalloproteinases, Tetrahedron Lett. 45 (2004)
1611e1615.
[6] D.A. Brown, W.K. Glass, N.J. Fitzpatrick, T.J. Kemp, W. Errington, G.J. Clarkson,
W. Haase, F. Karsten, A.H. Mahdy, Structural variations in dinuclear model
hydrolases and hydroxamate inhibitor models: synthetic, spectroscopic and
structural studies, Inorg. Chim. Acta 357 (2004) 1411e1436.
[7] Z. Amtul, Atta-ur-Rahman, R.A. Siddiqui, M.I. Choudhary, Chemistry and
mechanism of urease inhibition, Curr. Med. Chem. 9 (2002) 1323e1348.
C
37H61NO3 (567.89): C 78.25, H 10.83, N 2.47; found: C 78.01, H
10.97, N 2.38.
4.3.46. 3-Oxo-lup-20(29)-en-28-N-phenylcarbamate (48)
[8] C. Pergola, O. Werz, 5-Lipoxygenase inhibitors:
a review of recent de-
Microwave assisted reaction (7 h, 120 ꢀC) of 43 (0.3 g,
0.682 mmol) in dry THF (5 mL) with phenyl isocyanate (0.24 g,
0.22 mL, 2.05 mmol) as described above, followed by chromatog-
raphy (silica gel, hexanes/ethyl acetate, 8:2) gave 48 (0.325 g,
85.3%) as a colorless solid; RF ¼ 0.53 (silica gel, hexanes/ethyl ac-
velopments and patents, Expert Opin. Ther. Pat. 20 (2010) 355e375.
[9] G.R. Ott, N. Asakawa, R.Q. Liu, M.B. Covington, M.X. Qian, K. Vaddi,
R.C. Newton, J.M. Trzaskos, D.D. Christ, L. Galya, T. Scholz, W. Marshall,
J.J.W. Duan, alpha,beta-Cyclic-beta-benzamido hydroxamic acids: novel
oxaspiro[4.4] nonane templates for the discovery of potent, selective, orally
bioavailable inhibitors of tumor necrosis factor-alpha converting enzyme
(TACE), Bioorg. Med. Chem. Lett. 18 (2008) 1288e1292.
[10] G.R. Ott, N. Asakawa, Z. Lu, R.Q. Liu, M.B. Covington, K. Vaddi, M. Qian,
R.C. Newton, D.D. Christ, J.M. Traskos, C.P.D. James, alpha,beta-Cyclic-beta-
benzamido hydroxamic acids: novel templates for the design, synthesis, and
evaluation of selective inhibitors of TNF-alpha converting enzyme (TACE),
Bioorg. Med. Chem. Lett. 18 (2008) 694e699.
[11] A. Scozzafava, A. Mastrolorenzo, C.T. Supuran, Modulation of carbonic anhy-
drase activity and its applications in therapy, Expert Opin. Ther. Pat. 14 (2004)
667e702.
[12] J.Y. Winum, A. Scozzafava, J.L. Montero, C.T. Supuran, Therapeutic applications
of sulfamates, Expert Opin. Ther. Pat. 14 (2004) 1273e1308.
[13] B. Stearns, K.A. Losee, J. Bernstein, Hydroxyurea e a new type of potential
antitumor agent, J. Med. Chem. 6 (1963) 201.
[14] B. Chetan, M. Bunha, M. Jagrat, B.N. Sinha, P. Saiko, G. Graser, T. Szekeres,
G. Raman, P. Rajendran, D. Moorthy, A. Basu, V. Jayaprakash, Design, synthesis
and anticancer activity of piperazine hydroxamates and their histone deace-
tylase (HDAC) inhibitory activity, Bioorg. Med. Chem. Lett. 20 (2010)
3906e3910.
[15] C.J. Marmion, D. Griffith, K.B. Nolan, Hydroxamic acids e an intriguing family
of enzyme inhibitors and biomedical ligands, Eur. J. Inorg. Chem. (2004)
3003e3016.
etate, 8:2); m.p.: 200e202 ꢀC; [
a
]
¼ þ23.3ꢀ (c ¼ 0.46, CHCl3); IR
D
(KBr):
n
¼ 3443s, 2945s, 2868m, 2362w, 2344w, 1734s, 1700s,
1602m, 1540m, 1442m, 1386w, 1374w, 1216m, 1057w, 1030w, 882w,
752w, 692w cmꢁ1; UVevis (CHCl3): lmax (log ε) ¼ 234.4 (4.23) nm;
1H NMR (400 MHz, CDCl3):
d
¼ 7.39 (d, J ¼ 7.9 Hz, 2H, HC]C (33/
330)), 7.31 (t, J ¼ 7.8 Hz, 2H, HC]C (34/340)), 7.06 (t, J ¼ 7.3 Hz, 1H,
HC]C (35)), 6.63 (br, 1H, NH), 4.70 (s, 1H, CHa (29)), 4.60 (s, 1H, CHb
(29)), 4.38 (d, J ¼ 10.8 Hz, 1H, CHa (28)), 3.96 (d, J ¼ 10.7 Hz, 1H, CHb
(28)), 2.55e2.43 (m, 2H, CH (19) þ CHa (2)), 2.39 (ddd, J ¼ 15.6, 7.4,
4.2 Hz, 1H, CHb (2)), 2.10e1.95 (m, 1H, CHa (21)), 1.95e1.80 (m, 3H,
CHa (1) þ CHa (22) þ CHa (16)), 1.78e1.57 (m, 4H, CHa (15) þ CHa
(12) þ CH 13 þ CH (18)), 1.69 (s, 3H, CH3 (30)), 1.55e1.35 (m, 8H, CH2
(7) þ CH2 (6) þ CHb (21) þ CHa (11) þ CHb (1) þ CH (9)), 1.35e1.22
(m, 3H, CH (5) þ CHb (11) þ CHb (16)), 1.14e1.04 (m, 3H, CHb
(22) þ CHb (15) þ CHb (12)) 1.09 (s, 3H, CH3 (25)), 1.07 (s, 3H, CH3
(23)), 1.03 (s, 3H, CH3 (24)), 0.99 (s, 3H, CH3 (27)), 0.94 (s, 3H, CH3
(26)) ppm; 13C NMR (100 MHz, CDCl3):
d
¼ 218.5 (C]O, C3), 154.0
[16] L.P. Tardibono, M.J. Miller, Synthesis and anticancer activity of new
hydroxamic acid containing 1,4-benzodiazepines, Org. Lett. 11 (2009)
1575e1578.
(C]O, C31), 150.0 (C]CH2, C20), 137.9 (Cq, C32), 129.1 (CH, C34/
340), 123.4 (CH, C35), 118.6 (CH, C33/330), 109.9 (H2C]C, C29), 63.5
(CH2, C28), 55.0 (CH, C5), 49.7 (CH, C9), 48.8 (CH, C18), 47.6 (CH,
C19), 47.4 (Cq, C4), 46.6 (Cq, C17), 42.8 (Cq, C14), 40.8 (Cq, C8), 39.6
(CH2, C1), 37.7 (CH, C13), 36.9 (Cq, C10), 34.5 (CH2, C22), 34.2 (CH2,
C2), 33.5 (CH2, C7), 29.8 (CH2, C16), 29.6 (CH2, C21), 27.1 (CH2, C15),
26.6 (CH3, C23), 25.2 (CH2, C12), 21.3 (CH2, C11), 21.1 (CH3, C24), 19.6
(CH2, C6), 19.2 (CH3, C30), 15.9 (CH3, C25), 15.9 (CH3, C26), 14.7 (CH3,
C27) ppm; MS (ESI, MeOH): m/z ¼ 560.0 (12%, [M þ H]þ), 582.3
(34%, [M þ Na]þ), 613.3 (10%, [M þ Na þ MeOH]þ), 1119.3 (58%,
[2M þ H]þ), 1141.5 (100%, [2M þ Na]þ); analysis calcd for
[17] P. Kahnberg, A.J. Lucke, M.P. Glenn, G.M. Boyle, J.D.A. Tyndall, P.G. Parsons,
D.P. Fairlie, Design, synthesis, potency, and cytoselectivity of anticancer
agents derived by parallel synthesis from alpha-aminosuberic acid, J. Med.
Chem. 49 (2006) 7611e7622.
ꢁꢀ
[18] J. Jiang, A. Thyagarajan-Sahu, V. Krchnak, A. Jedinak, G.E. Sandusky, D. Sliva,
NAHA, a novel hydroxamic acid-derivative, inhibits growth and angiogenesis
of breast cancer in vitro and in vivo, PLoS ONE 7 (2012) e34283.
[19] S. Mochizuki, Y. Okada, ADAMs in cancer cell proliferation and progression,
Cancer Sci. 98 (2007) 621e628.
[20] R. Csuk, Recent developments in the synthesis of antitumor-active glycyr-
rhetinic acid derivatives, Mini-Rev. Org. Chem. 11 (2014) 253e261.
[21] B. Siewert, E. Pianowski, A. Obernauer, R. Csuk, Towards cytotoxic and se-
lective derivatives of maslinic acid, Bioorg. Med. Chem. 22 (2014) 594e615.
[22] R. Csuk, Betulinic acid and its derivatives: a patent review (2008e2013),
Expert Opin. Ther. Pat. 24 (2014) 913e923.
C
37H53NO3 (559.82): C 79.38, H 9.54, N 2.50; found: C 79.13, H 9.61,
N 2.43.

210
J. Wiemann et al. / European Journal of Medicinal Chemistry 106 (2015) 194e210
[23] S. Sommerwerk, L. Heller, R. Csuk, Synthesis and cytotoxic activity of penta-
cyclic triterpenoid sulfamates, Arch. Pharm. 348 (2015) 46e54.
[24] S. Schwarz, S. Sommerwerk, S.D. Lucas, L. Heller, R. Csuk, Sulfamates of methyl
triterpenoates are effective and competitive inhibitors of carbonic anhydrase
II, Eur. J. Med. Chem. 86 (2014) 95e102.
[47] H.L. Ziegler, H. Franzyk, M. Sairafianpour, M. Tabatabai, M.D. Tehrani,
K. Bagherzadeh, H. Hagerstrand, D. Staerk, J.W. Jaroszewski, Erythrocyte
membrane modifying agents and the inhibition of Plasmodium falciparum
growth: structureeactivity relationships for betulinic acid analogues, Bioorg.
Med. Chem. 12 (2004) 119e127.
[48] Shakeel-u-Rehman, S.N. Sofi, M.A. Khuroo, S.C. Taneja, K.A. Bhat,
R. Vishwakarma, New natural compounds from Rhododendron lepidotum,
Nat. Prod. Res. 27 (2013) 2033e2038.
[49] L.R. Mikhailova, L.A. Baltina Jr., L.A. Baltina, R.M. Kondratenko,
S.A. Nepogodiev, R.A. Field, O. Kunert, M.C. Yin, A simple method of synthesis
of triterpene glycosides similar to glycyrrhizic acid and their hepatoprotective
activity in vitro, Russ. J. Bioorg. Chem. 35 (2009) 619e627.
[50] O.B. Flekhter, E.I. Boreko, L.R. Nigmatullina, E.V. Tret'yakova, N.I. Pavlova,
L.A. Baltina, S.N. Nikolaeva, O.V. Savinova, V.F. Eremin, F.Z. Galin,
G.A. Tolstikov, Synthesis and antiviral activity of betulonic acid amides and
conjugates with amino acids, Russ. J. Bioorg. Chem. 30 (2004) 80e88.
[51] L.A. Baltina, Chemical modification of glycyrrhizic acid as a route to new
bioactive compounds for medicine, Curr. Med. Chem. 10 (2003) 155e171.
[52] L.A. Baltina, O.B. Flekhter, L.R. Nigmatullina, E.I. Boreko, N.I. Pavlova,
S.N. Nikolaeva, O.V. Savinova, G.A. Tolstikov, Lupane triterpenes and de-
rivatives with antiviral activity, Bioorg. Med. Chem. Lett. 13 (2003)
3549e3552.
€
[25] H. Kommera, G.N. Kaluderovic, S. Dittrich, J. Kalbitz, B. Drager, T. Müller,
R. Paschke, Carbamate derivatives of betulinic acid and betulin with selective
cytotoxic activity, Bioorg. Med. Chem. Lett. 20 (2010) 3409e3412.
[26] D. Chaturvedi, Role of organic carbamates in anticancer drug design, Chem.
Pharmacol. Nat. Occurr. Bioactive Comp. (2013) 117e139.
[27] E.E. Rufino-Palomares, A. Perez-Jimenez, F.J. Reyes-Zurita, L. Garcia-Salguero,
K. Mokhtari, A. Herrera-Merchan, P.P. Medina, J. Peragon, J.A. Lupianez, Anti-
cancer and anti-angiogenic properties of various natural tri-terpenoids and
some of their chemical derivatives, Curr. Org. Chem. 19 (2015) 919e947.
[28] G. Periasamy, G. Teketelew, M. Gebrelibanos, B. Sintayehu, M. Gebrehiwot,
A. Karim, G. Geremedhin, Betulinic acid and ist derivatives as anti-cancer
agent: a review, Arch. Appl. Sci. Res. 6 (2014) 47e58.
[29] D. Gheorgheosu, O. Duicu, C. Dehelean, C. Soica, D. Muntean, Betulinic acid as
a potent and complex antitumor phytochemical: a minireview, Anti-Cancer
Agents Med. Chem. 14 (2014) 936e945.
[30] J.A.R. Salvador, A.S. Leal, D.P.S. Alho, B.M.F. Goncalves, A.S. Valdeira,
V.I.S. Mendes, Y. Jing, Highlights of pentacyclic triterpenoids in the cancer
settings, Stud. Nat. Prod. Chem. 41 (2014) 33e73.
[53] P. Skehan, R. Storeng, D. Scudiero, A. Monks, J. Mcmahon, D. Vistica,
J.T. Warren, H. Bokesch, S. Kenney, M.R. Boyd, New colorimetric cytotoxicity
assay for anticancer-drug screening, J. Natl. Cancer Inst. 82 (1990) 1107e1112.
[31] S.C. Jonnalagadda, M.A. Corsello, C.E. Sleet, Betulin-betulinic acid natural
product based analogs as anti-cancer agents, Anti-Cancer Agents Med. Chem.
13 (2013) 1477e1499.
€
[54] B. Siewert, J. Wiemann, A. Kowitsch, R. Csuk, The chemical and biological po-
[32] M.G. Moghaddam, F.B.H. Ahmad, A. Samzadeh-Kermani, Biological activity of
betulinic acid: a review, Pharmacol. Pharm. 3 (2012) 119e123.
[33] F.B. Mullauer, J.H. Kessler, J.P. Medema, Betulinic acid, a natural compound
with potent anticancer effects, Anti-Cancer Drugs 21 (2010) 215e227.
[34] L. Tripathi, P. Kumar, R. Singh, A review on extraction, synthesis and anti-
cancer activity of betulinic acid, Curr. Bioactive Compd. 5 (2009) 160e168.
[35] S. Fulda, Betulinic acid: a natural product with anticancer activity, Mol. Nutr.
Food Res. 53 (2009) 140e146.
[36] S. Fulda, Betulinic acid for cancer treatment and prevention, Int. J. Mol. Sci. 9
(2008) 1096e1107.
[37] R. Csuk, Betulinic acid and its derivatives: a patent review (2008e2013),
Expert Opin. Ther. Pat. 24 (2014) 913e923.
tential of C ring modified triterpenoids, Eur. J. Med. Chem. 72 (2014) 84e101.
[55] B. Siewert, E. Pianowski, R. Csuk, Esters and amides of maslinic acid trigger
apoptosis in human tumor cells and alter their mode of action with respect to
the substitution pattern at C-28, Eur. J. Med. Chem. 70 (2013) 259e272.
[56] B. Siewert, E. Pianowski, A. Obernauer, R. Csuk, Towards cytotoxic and se-
lective derivatives of maslinic acid, Bioorg. Med. Chem. 22 (2014) 594e615.
[57] M. Urban, J. Sarek, J. Klinot, G. Korinkova, M. Hajduch, Synthesis of A-seco
derivatives of betulinic acid with cytotoxic activity, J. Nat. Prod. 67 (2004)
1100e1105.
[58] J. Ito, F.R. Chang, H.K. Wang, Y.K. Park, M. Ikegaki, N. Kilgore, K.H. Lee, Anti-
AIDS agents. 48. Anti-HIV activity of moronic acid derivatives and the new
melliferone-related triterpenoid isolated from Brazilian propolis, J. Nat. Prod.
64 (2001) 1278e1281.
[38] A. Barthel, S. Stark, R. Csuk, Oxidative transformations of betulinol, Tetrahe-
dron 64 (2008) 9225e9229.
[59] T.J. Nitz, C. Montalbetti, R. Mears, X. Gai, E. Glenn, Extended triterpene de-
rivatives as antiretroviral agents, WO2008057420A2, 2008; Chem. Abstr.
(2008) 585712.
€
[39] R. Csuk, K. Schmuck, R. Schafer, A practical synthesis of betulinic acid, Tet-
rahedron Lett. 47 (2006) 8769e8770.
[40] C. Stanetty, L. Czollner, I. Koller, P. Shah, R. Gaware, T. Da Cunha, A. Odermatt,
U. Jordis, P. Kosma, D. Claßen-Houben, Synthesis of novel 3-amino and 29-
hydroxamic acid derivatives of glycyrrhetinic acid as selective 11 beta-
hydroxysteroid dehydrogenase 2 inhibitors, Bioorg. Med. Chem. 18 (2010)
7522e7541.
[41] H.H. Sun, P.V. Kaplita, D.R. Houck, M.B. Stawicki, R. McGarry, R.C. Wahl,
A.M. Gillum, R. Cooper, A metalloproteinase inhibitor from Doliocarpus ver-
ruculosus, Phytother. Res. 10 (1996) 194e197.
[42] B. Vasantha, H.P. Hemantha, V.V. Sureshbabu, 1-Propanephosphonic acid cy-
clic anhydride (T3P) as an efficient promoter for the lossen rearrangement:
application to the synthesis of urea and carbamate derivatives, Synthesis
(2010) 2990e2996.
[43] A. Ech-Chahad, A. Minassi, L. Berton, G. Appendino, An expeditious hydrox-
yamidation of carboxylic acids, Tetrahedron Lett. 46 (2005) 5113e5115.
[44] H.A. Staab, M. Lueking, F.H. Dürr, The preparation of imidazolides. Synthesis of
amides, hydrazides, and hydroxamic acids by the imidazolide method, Chem.
Ber. 95 (1962) 1275e1283.
[45] R.C. Santos, J.A.R. Salvador, S. Marin, M. Cascante, J.N. Moreira, T.C.P. Dinis,
Synthesis and structure-activity relationship study of novel cytotoxic carba-
mate and N-acylheterocyclic bearing derivatives of betulin and betulinic acid,
Bioorg. Med. Chem. 18 (2010) 4385e4396.
[46] R.C. Santos, J.A.R. Salvador, S. Marin, M. Cascante, Novel semisynthetic de-
rivatives of betulin and betulinic acid with cytotoxic activity, Bioorg. Med.
Chem. 17 (2009) 6241e6250.
[60] C.R. Dorr, S. Yemets, O. Kolomitsyna, P. Krasutsky, L.M. Mansky, Triterpene
derivatives that inhibit human immunodeficiency virus type 1 replication,
Bioorg. Med. Chem. Lett. 21 (2011) 542e545.
[61] A.N. Antimonova, N.V. Uzenkova, N.I. Petrenko, M.M. Shakirov, E.E. Shul'ts,
G.A. Tolstikov, Synthesis of betulonic acid amides, Chem. Nat. Compd. 44
(2008) 327e333.
[62] D. Thibeault, C. Gauthier, J. Legault, J. Bouchard, P. Dufour, A. Pichette, Syn-
thesis and structure-activity relationship study of cytotoxic germanicane- and
lupane-type 3 beta-O-monodesmosidic saponins starting from betulin, Bioorg.
Med. Chem. 15 (2007) 6144e6157.
[63] S. Boryczka, E. Bebenek, J. Wietrzyk, K. Kempinska, M. Jastrzebska, J. Kusz,
M. Nowak, Synthesis, structure and cytotoxic activity of new acetylenic de-
rivatives of betulin, Molecules 18 (2013) 4526e4543.
[64] C. Horwedel, S.B. Tsogoeva, S.W. Wei, T. Efferth, Cytotoxicity of artesunic acid
homo- and heterodimer molecules toward sensitive and multidrug-resistant
CCRF-CEM leukemia cells, J. Med. Chem. 53 (2010) 4842e4848.
[65] L. Pohjala, S. Alakurtti, T. Ahola, J. Yli-Kauhaluoma, P. Tammela, Betulin-
derived compounds as inhibitors of alphavirus replication, J. Nat. Prod. 72
(2009) 1917e1926.
[66] A.V. Korovin, A.V. Tkachev, Synthesis of quinoxalines fused with triterpenes,
ursolic acid and betulin derivatives, Russ. Chem. B 50 (2001) 304e310.
[67] K. Hata, K. Hori, S. Takahashi, Differentiation- and apoptosis-inducing activ-
ities by pentacyclic triterpenes on a mouse melanoma cell line, J. Nat. Prod. 65
(2002) 645e648.