Platanic acid-derived methyl 20-amino-30-norlupan-28-oates are potent cytotoxic agents acting by apoptosis

Article abstract of DOI:10.1007/s00044-018-2189-6

A set of eight derivatives of betulinic acid (2) and 12 derivatives of platanic acid (3) has been prepared and screened for their cytotoxic activity using SRB assays. First synthetic approaches were focused on the preparation of augustic acid (4) analogs of 2 and 3 by introducing a second hydroxyl group on the A-ring of both triterpenoic acids leading to compounds 5–14. Further structural modifications were performed at the C-20 keto group of the platanic acid backbone by its transformation into an oxime moiety and subsequent reduction to 20-amino-30-norlupan derivatives 17–24. In the SRB assays low EC₅₀ values were observed especially for methyl (3β, 20 R)-3-acetyloxy-20-amino-30-norlupan-28-oate (21), methyl (3β, 20 S)-3-acetyloxy-20-amino-30-norlupan-28-oate (22), and (3β, 20 R)-3,28-diacetyloxy-20-amino-30-norlupane (24) all holding an amino function at C-20. Compound 21 was selected for extensive biological testing that showed this compound to cause cell death by inducing apoptosis. [Figure not available: see fulltext.].

Full text of DOI:10.1007/s00044-018-2189-6

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research
https://doi.org/10.1007/s00044-018-2189-6
ORIGINAL RESEARCH
Platanic acid-derived methyl 20-amino-30-norlupan-28-oates are
potent cytotoxic agents acting by apoptosis
Michael Kahnt1 Lucie Heller Ahmed Al-Harrasi Renate Schäfer Ralph Kluge Christoph Wagner
1 ●
2 ●
1 ●
1 ●
3 ●
●
4 ●
1
Choijiljav Otgonbayar René Csuk
Received: 15 February 2018 / Accepted: 4 May 2018
Springer Science+Business Media, LLC, part of Springer Nature 2018
©
Abstract
A set of eight derivatives of betulinic acid (2) and 12 derivatives of platanic acid (3) has been prepared and screened for their
cytotoxic activity using SRB assays. First synthetic approaches were focused on the preparation of augustic acid (4) analogs
of 2 and 3 by introducing a second hydroxyl group on the A-ring of both triterpenoic acids leading to compounds 5–14.
Further structural modifications were performed at the C-20 keto group of the platanic acid backbone by its transformation
into an oxime moiety and subsequent reduction to 20-amino-30-norlupan derivatives 17–24. In the SRB assays low EC50
values were observed especially for methyl (3β, 20 R)-3-acetyloxy-20-amino-30-norlupan-28-oate (21), methyl (3β, 20 S)-3-
acetyloxy-20-amino-30-norlupan-28-oate (22), and (3β, 20 R)-3,28-diacetyloxy-20-amino-30-norlupane (24) all holding an
amino function at C-20. Compound 21 was selected for extensive biological testing that showed this compound to cause cell
death by inducing apoptosis.
Graphical Abstract
●
●
●
●
Keywords Platanic acid Betulinic acid Cytotoxicity Triterpenoids Apoptosis
Introduction
Electronic supplementary material The online version of this article
Treatment of severe diseases has always been a challenge
for humankind. While our early ancestors trusted in the
use of extracts from leaves, fruits, and roots of plants,
(https://doi.org/10.1007/s00044-018-2189-6) contains supplementary
material, which is available to authorized users.
th
th
*
René Csuk
during the 18 and 19 century a dramatic change occurred.
To put it into simple language: the use of natural
products was out, and the trust in products synthesized in
pharmaceutical labs was great. Nowadays we note a coex-
istence of these two ways to treat diseases: exploiting
natural products (providing valuable synthetic backbones
and scaffolds) and their derivatization (with sophisticated
synthetic transformations).
rene.csuk@chemie.uni-halle.de
1
2
Organic Chemistry, Martin-Luther University Halle-Wittenberg,
Kurt-Mothes-Str. 2, D-06120 Halle (Saale), Germany
Chair of Oman’s Medicinal Plants and Marine Natural Products,
University of Nizwa, P.O. Box 33PC 616, Birkat Al-Mauz,
Nizwa, Sultanate of Oman
3
4
Inorganic Chemistry, Martin-Luther University Halle-Wittenberg,
Kurt-Mothes-Str. 2, D-06120 Halle (Saale), Germany
There have been large efforts in surgery and irradiation
of tumors. Furthermore, the development and the refinement
of a broad variety of chemotherapeutics has been improved
Institute of Chemistry and Chemical Technology, Mongolian
Academy of Sciences, Ulanbaatar-51, Mongolia

Medicinal Chemistry Research
but the prognosis for many types of cancers remains poor.
Our own synthetic work during recent years focused
on the use of triterpenoic acids as valuable starting points
for the synthesis of cytotoxic agents being intended to
fight cancer. These triterpenoic acids (such as ursolic,
oleanolic, tormentic, boswellic, maslinic, asiatic, or gly-
cyrrhetinic acid) can conveniently be obtained from
plants. Early in the history of humankind our ancestors
made use of different parts of birch trees (Betula platy-
phylla). The bark of birches contains huge amounts of
betulin (1, Figs. 1, 2) besides smaller amounts of betulinic
acid (2) and several other triterpenes. While 1 is not cyto-
toxic at all for many human tumor cell lines, 2 shows better
cytotoxic properties. As a result, many derivatives of
betulinic acid have been prepared and examined in detail
(Ali-Seyed et al. 2016; Csuk 2014; Gheorgheosu et al.
2014; Jonnalagadda et al. 2013; Paduch and Kandefer-
Szerszen 2014; Periasamy et al. 2014; Salvador et al. 2014;
Zhang et al. 2015). Recently, derivatives of platanic
acid (3) and augustic acid (4) came in the focus of interest.
Therefore, we decided to synthesize several analogs
derived from 1 and 2, and to investigate their potential as
antitumor agents.
Following previous findings (Sommerwerk et al. 2016a,
Sommerwerk et al. 2016b) reporting increased cytotoxicity
accompanied by tumor–nontumor selectivity of augustic
acid (4) derivatives, our objective was the introduction of a
second hydroxyl moiety at C-2 of betulinic (2) and platanic
acid (3). Data from literature (Ma et al. 2005, Ma et al.
2009) also revealed amino substituted triterpenoids, espe-
cially those derived from oleanolic or usrolic acid to be
hightly cytotxic (EC < 2 µM). Therefore, we decided to
5
0
synthesize several 20-amino derivatives of platanic and
betulinic acid.
Fig. 1 Structure of betulin (1), betulinic acid (2), platanic acid (3), and
augustic acid (4) A final proof, however, for the structure of 8 was
obtained after growing suitable crystals and performing a single crystal
X-ray structure analysis. The results of this analysis are depicted and
summarized in Fig. 2 and in the Experimental part
Results and Discussion
Soxhlet extraction of dry birch bark (Betula platyphylla)
with diethyl ether delivered betulin (1) in excellent yields.
Regioselective oxidation of 1 as previously described
(Barthel et al. 2008) gave betulinic acid (2) while from the
Jones oxidation of 1 betulonic acid (5) was obtained (Csuk
et al. 2006; Barthel et al. 2008). Esterification of 5 gave
methyl ester 6 (Xu et al. 2012). Reaction of 6 with potas-
sium tert-butanolate in dry tert-butanol in the presence of
air yielded 80% of 7. Compound 7 was reduced with
NaBH in THF at 0 °C and 82% of 8 were isolated. Com-
4
1
pound 8 is characterized in its H NMR spectrum by the
presence of a signal at δ = 4.05 ppm (assigned to 2-H); 3-H
was detected at δ = 3.15 ppm. Interpretation of the NOESY
spectra allowed to determine the absolute configuration of
this molecule Scheme 1.
Acetylation of 8 in the presence of one equivalent of
acetyl chloride in pyridine afforded a low yield of mono
acetate 9. The use of an excess of acetylating agent gave
diacetate 10, however, in excellent isolated yield.
Platanic acid derivatives 11–14 were prepared using the
same procedure as described for the synthesis of 8. Thus,
Jones oxidation of platanic acid (3) gave 3-oxo-platanic acid
Fig. 2 Molecular structure of compound 8. Thermal ellipsoids at the
5
1
0% probability level. Symmetry operator: i: x − 1/2, −y + 1/2, −z +
. Intramolecular hydrogen bridge O1-H1∙∙∙O2: d(O1∙∙∙O2) 275.6 pm,
intermolecular hydrogen bridge O2-H2∙∙∙O3′: d(O2∙∙∙O3) 289.6 pm.
Formula C31H50O4, M = 486.71, colorless crystal, 0.380 × 0.380 ×
3
0
.150 mm , a = 1113.90(7) pm, b = 1408.39(9) pm, c = 1751.92(11)
3
3
pm, α = β = γ = 90°, V = 2.7484(3) nm , ρcalc = 1.176 g cm , μ =
(
11) in almost quantitative yield. Reaction of 11 with
1
0
.075 mm− , Z = 4, orthorhombic, space group P2 2 2 , T = 220(2) K,
1 1 1
potassium tert-butanolate in dry tert-butanol in the presence
of air yielded 65% of 12. Subsequent reduction of 12 with
1
8,533 reflections, independent reflections 4827, Rint = 0.0602, Data/
2
restraints/parameters 4827/0/352, Goof on F 2.204, R1 = 0.0442(I >
sigma(I)), wR2 = 0.0798(all data), CCDC: 1577240
2
NaBH in dry THF gave 2β-hydroxy-platanic acid (13) in
4

Medicinal Chemistry Research
Scheme 1 Synthesis of betulin-derived compounds 5–10 and platanic
acid-derived compounds 11–14: a Jones oxidation, 4 h, 25 °C, 81% for
from 11); d NaBH4, THF, EtOH, 25 °C, 60 min, 81% (for 8 from 7) or
NaBH4, THF, MeOH, 25 °C, 22 h, 63% (for 13 from 12); e AcCl
(1 equiv.), pyridine, 25 °C, 2 days, 42% (9) or AcCl (3 equiv.), pyridine,
25 °C, 2 days, 52% (10)
5
1
8
from 1 or 1 h, 25 °C, 98% for 11 from 3; b MeI, K2CO3, DMF, 25 °C,
t
t
h, 70% (6) or 30 min 79% (14); c BuOK, BuOH, air, 40 °C, 60 min,
t
t
0% (for 7 from 6) or BuOK, BuOH, THF, air, 50 °C, 4 h, 65% (for 12
Scheme 2 Synthesis of
compounds 15–20: a O , DCM,
3
–
60 °C, 60 min then Zn
powder) AcOH, 25 °C, 2 h, 73
; b Ac2O, pyridine, 25 °C, 4 h,
(
%
.
9
6
6%; c HONH2 HCl, pyridine,
0 °C, 3 h, 13 % (for 19) and 72
%
(for 20)
6
3% isolated yield. For comparison, compound 13 was
yielded 18. Reaction of 18 with hydroxylammonium
chloride in dry pyridine at 60 °C for 3 h gave a mixture of
oximes (20 Z)-19 (13%) and (20 E)-20 (72 %) that were
easily separated by column chromatography. Their absolute
configuration was deduced from the interpretation of
NOESY spectra (L. Heller et al. 2017).
transformed into its methyl ester 14 by esterification with
MeI in DMF (L. Heller et al. 2017).
From the ozonolysis of betulin-3,28-diacetate (15) in dry
DCM at −60 °C diacetate 16 was obtained (Scheme 2).
Esterification of 3 gave methyl ester 17 whose acetylation

Medicinal Chemistry Research
Scheme 3 Synthesis of amines
1–24: a CH3COONH4,
2
NaBH3CN, MeOH, 25 °C then
TiCl (≥12% in aq. HCl (12%)),
3
0
(
–25 °C, 20 h, yielding 21
67%) and 22 (15%); b LiAlH4,
THF, 70 °C (6 h), 25 °C (24 h),
7
3
1%; c Boc2O, pyridine, 25 °C,
0 min then Ac2O, 16 h then
TFA, DCM, 0–25 °C, 3 h, 74%
As previously described, reduction of the oximes 19 or
Table 1 Cytotoxicity of compounds 1–24
2
0 (L. Heller et al. 2017; Sun et al. 1998) with NaBH CN/
3
#
518A2
A549
A2780
MCF-7
>30
NIH 3T3
>30
TiCl /NH OAc in methanol gave the amines 21 and 22 in
good yields (Scheme 3); these compounds were separated
3
4
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
>30
>30
>30
18.9 ± 1.7 14.6 ± 1.6 11.0 ± 1.9 14.8 ± 1.9 13.1 ± 1.1
>30 >30 >30 >30 >30
25.7 ± 0.7 >30 18.9 ± 1.8 25.1 ± 2.2 >30
14.2 ± 1.1 13.1 ± 0.9 4.7 ± 0.6 12.0 ± 1.5 19.4 ± 1.2
by chromatography, and their absolute configuration at C-
1
2
0 was determined using H NMR spectroscopy. Reaction
of 21 with lithium aluminiumhydride in dry THF at 70 °C
yielded 23. Acetylation of both hydroxyl groups of 23 was
performed by protecting the amino group at C-20 with di-
tert-butyldicarbonate followed by a reaction with acetic
anhydride in dry pyridine. Subsequent removal of the Boc
protecting group with trifluoroacetic acid in DCM furnished
>30
>30
>30
>30
21.0 ± 1.7 >30
>30 >30
>30
>30
20.4 ± 2.3 17.0 ± 1.1 8.9 ± 0.6
17.8 ± 1.3 25.2 ± 2.6
21.4 ± 1.9 20.9 ± 0.9 11.3 ± 1.5 21.0 ± 2.4 27.3 ± 2.5
24.3 ± 1.7 21.3 ± 1.1 10.7 ± 3.0 29.2 ± 2.9 17.1 ± 1.5
2
4 (Heller et al. 2017; Kahnt et al. 2018).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
The cytotoxicity of all compounds was determined by
sulforhodamine B (SRB) assays; the results are compiled in
Table 1.
The betulinic acid derivatives 5–7 were found to be of
moderate cytotoxicity or to be not cytotoxic at all. These
results agree with results previously reported (Urban et al.
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
20.6 ± 1.7 24.8 ± 1.7 >30
>30
>30
>30
>30
>30
>30
10.2 ± 1.3 >30
11.1 ± 1.4 20.4 ± 2.6 >30
>30
>30
21.0 ± 1.7 >30
>30
>30
>30
2
004; Urban et al. 2005. The cytotoxicity of augustic acid
>30
>30
>30
>30
>30
>30
analogs 8–10 was of the same order of magnitude as
compound 5. Thus, they show similar EC50 values as
augustic acid (4). Parent platanic acid (3) and its analogs
were not cytotoxic (EC > 30 μM) for the human tumor cell
>30
>30
9.7 ± 2.1
7.8 ± 2.5
4.1 ± 0.3
6.3 ± 0.2
8.8 ± 0.7
8.2 ± 0.9
5.3 ± 0.7
6.6 ± 0.8
12.9 ± 0.7 12.1 ± 0.3 12.3 ± 1.2
12.2 ± 0.5 12.4 ± 0.5 14.0 ± 2.4
50
4.7 ± 0.1
6.0 ± 0.1
>30
5.1 ± 0.1
4.6 ± 0.2
>30
5.6 ± 0.2
4.4 ± 0.6
16.0 ± 1.1
4.0 ± 0.7
lines. This indicates that the presence of a carbonyl function
at position C-20 has no influence onto cytotoxic properties
of this compound. This finding was also deduced by com-
paring the results obtained for compounds 15 and 16. For
example, for betulin-3,28-diacetate (15) a EC = 11.1 μM
23.7 ± 1.8 >30
2.2 ± 0.1 2.3 ± 0.1
2.5 ± 0.1
2.1 ± 0.2
50
EC50 values from SRB assays after 96 h of treatment are given in µM;
the values are averaged from three independent experiments each
performed in triplicate; confidence interval CI = 95%; positive control:
betulinic acid (2)
was measured for ovarian carcinoma cells A2780,
and diacetate 16 showed an EC50 value of 21.0 μM. The
platanic acid derivatives 17 and 18 show no cytotoxic
properties, too.

Medicinal Chemistry Research
Fig. 3 a Annexin V-FITC/ PI assay: treatment of A2780 cells with 21
microscopic images (scale bar = 10 µm), AO and PI were used. c
(
8 µM) for 24 h. Examples of density plots determined by flow cyto-
Representative examples for cell cycle evaluation via ModFit V 4.0.5
®
metry (Attune Cytometric Software vl 1.2.5). b Fluorescence
The idea of achieving enhanced activities by modifying
C-20 was realized by synthesizing oximes 19 and 20; in
their SRB screening both compounds gave EC50 values in
the range of 7.8–14.0 μM. The 20-amino derivatives 21 and
quantification of the apoptosis-inducing activity of com-
pound 21. Thus, treatment of A2780 cells with 21 resulted
in about 23.7% apoptotic cells and 16.6% secondary
necrotic cells, with 58.1% of the cells still being con-
sidered vital. Representative examples of the density plots
determined by flow cytometry and the distributions are
shown in Fig. 3a.
From these results, we assume that compound 21 is able
to trigger programmed cell death, including apoptotic
mechanisms.
2
2
2, however, were even more cytotoxic. In comparison, the
0-amino betulin derivative 23 showed low cytotoxicity.
Thus, the most active compound of this series was (3β,
0 R)-3,28-diacetyloxy-20-amino-30-norlupane (24) with
2
an EC50 value of 2.1 μM for MCF-7 cells.
For compound 21 extra experiments were performed by
treating A2780 cells with 21 (8 µM) for one day. A dye
exclusion test using acridine orange and propidium iodide
(
AO/PI) revealed that in some cells ruptures of the plasma
Conclusion
membrane occurred, while the red-green color indicates a
controlled cell death (Fig. 3b). To elucidate whether the
growth inhibitory effects of 21 were due to cell cycle
arrest, a flow cytometry analysis was performed. As
shown in Fig. 3c, treatment of the cells with 21 resulted in
a decrease in the S phase from 34.0 to 22.7% compared to
control. However, there were no cell cycle arrests to
identify. Furthermore, an accumulation (9.7% of the cell
population) in sub-G1 phase, indicator of cell apoptosis,
was detected after treating the cells with 21 exposure for
one day. An annexin V-FITC/PI staining allowed a
Augustic acid analogs of betulinic and platanic acid have
been prepared and screened for their cytotoxic activity using
SRB assays. As a result, the compounds derived from
betulinic acid revealed to be more potent than those from
platanic acid. Therefore, further structural modifications
were performed, which showed that the replacement of the
keto group at position C-20 improves the cytotoxicity of the
compounds. The best results were observed for the 20-
amino derivatives 21–24. All of these compounds showed
EC50 values < 7 µM. The highest cytotoxic activity was

Medicinal Chemistry Research
observed for (3β, 20 R)-3,28-diacetyloxy-20-amino-30-nor-
lupane (24, EC = 2.1 ± 0.2, MCF-7). Compound 21 was
[α] = +8.7 (c = 0.4, CHCl ), lit.: [α] = +9.0 (c = 0.5,
D
3
D
CHCl ) (Csuk et al. 2006).
5
0
3
selected for extensive biological testing, that showed this
compound mainly acts by apoptosis.
Platanic acid (3)
This compound was obtained commercially from betuli-
nines (Stříbrná Skalice, Czech Republic); as an alternative,
it was obtained by ozonolysis of betulinic acid (2) (Vystrčil
and Buděšínský 1970).
Experimental
General
NMR spectra were recorded using the Varian spectrometers
Gemini 2000 or Unity 500 (δ given in ppm, J in Hz; typical
experiments: H-H-COSY, HMBC, HSQC, and NOESY),
MS spectra were taken on a Finnigan MAT LCQ 7000
Augustic acid (4)
This compound was prepared from oleanolic acid as pre-
viously described (Sommerwerk et al. 2015a, 2015b); col-
orless solid; m.p. 309–311 °C, lit.: 308–310 °C (Wen et al.
(
electrospray, voltage 4.1 kV, sheath gas nitrogen) instru-
ment. The optical rotations were measured on
a
2008), [α] = +88.1° (c = 0.31, THF), lit.:+93.5 (c = 0.17,
D
Perkin–Elmer polarimeter at 20 °C; TLC was performed on
silica gel (Merck 5554, detection with cerium molybdate
reagent); melting points are uncorrected (Leica hot stage
microscope), and elemental analyses were performed on a
Foss-Heraeus Vario EL (C-HNS) unit. IR spectra were
recorded on a Perkin–Elmer FT-IR spectrometer Spectrum
pyridine) (Cheng et al. 2008).
Betulonic acid (5)
This compound was prepared from 1 by Jones oxi-
dation as previously described ((Barthel et al. 2008) and
obtained as a colorless solid; m.p. 249–252 °C, lit.:
1
000. The solvents were dried according to usual proce-
1
5
dures. The purity of the compounds was determined by
HPLC and found to be >96%. Platanic acid (3) was
obtained from Betulinines (Stříbrná Skalice, Czech
Republic) in bulk quantities. Fluorescence microscopic
images were recorded on an Axioskop 20 with an AxioCam
MR3 (Carl Zeiss AG). Flow cytometric experiments were
performed on an Attune acoustic focusing cytometer (Life
Technologies GmbH). Data sets for the crystal structure
were collected with a STOE-IPDS2 diffractometer. Pro-
grams used: structure solution SHELXS-97 (Sheldrick
250–254 °C (Urban et al. 2004) [α] = +31.1° (c = 0.3,
D
CHCl ), lit.: [α] = +32.0 (c = 0.4, CHCl ) (Urban et al.
3
D
3
2004).
Methyl 3-oxo-lup-20(29)-en-28-oate (6)
Esterification of 5 with MeI/K CO in dry DMF as pre-
2
3
viously described (Xu et al. 2012) gave 6 (85%) as a col-
orless solid; m.p. 162–165 °C, lit.: 161–165 °C (Urban et al.
2004); [α] = +27.2° (c = 0.56, MeOH), lit.: [α] =
D
D
2
008); structure refinement SHELXL-97 (Sheldrick 2008),
+28.0° (c = 0.4, CHCl ) (Urban et al. 2004).
3
and graphics Diamond (Diamond 1997).
Methyl 2-hydroxy-3-oxolupa-1,20(29)-dien-28-oate (7)
Synthesis
To a suspension of potassium tert-butanolate (18.5 g,
164,9 mmol) in tert-butanol (175 ml) at 40 °C 6 (2.0 g,
Isolation of betulin (1)
4.3 mmol) was added, and stirring was continued for an
Soxhlet extraction of dry birch bark (Betula platyphylla,
additional 60 min. During this period, a stream of air was
bubbled through the reaction mixture. Usual aqu. work-up
followed by chromatography (silica gel, toluene/diethyl
ether, 15:1) gave 7 (1.67 g, 80%) as a colorless solid; m.p.
122–126 °C, lit.: 122–124 °C (Urban et al. 2004; Urban
et al. 2005); [α] = +26.1° (c = 0.54, MeOH), lit.: [α] =
1
80 g) with diethyl ether for two days followed by chro-
matography (silica gel, toluene/EtOAc/heptane/HCOOH,
8
2
0/20/10/3) gave 1 (39.6 g, 22%) as a colorless solid; m.p.
53–255 °C, lit.: 254–255 °C (Jarolim et al. 1961) [α] =
D
+
26.5° (c = 0.9, CHCl ), lit.: [α] = +22.6° (c = 0.1,
3
D
D
D
CHCl ) (Gu et al. 2004).
+3° (c = 0.67, CHCl ) (Urban et al. 2005); R = 0.77
3
3
F
(toluene/diethyl ether, 5:1); IR (KBr): ν = 2948s, 1727s,
Betulinic acid (2)
1668m, 1646m, 1457m, 1406m, 1234m, 1156m, and
−
1
1
133m cm ; UV/Vis (MeOH): λ
(log ε) = 288 nm
max
1
This compound was prepared as previously described
Barthel et al. 2008) from 1 and obtained as a colorless
solid; m.p. 309–312 °C, lit.: 310–313 °C (Csuk et al. 2006)
(4.09); H NMR (400 MHz, CDCl ): δ = 6.41 (s, 1H, 1-H),
3
(
5.86 (s, 1H, OH), 4.73 (m, 1H, 29-H ), 4.59 (m, 1H, 29-H ),
a
b
3.65 (s, 3H, 31-H), 2.98 (ddd, 1H, J = 11.1, 10.8, 4.5 Hz,

Medicinal Chemistry Research
1
9-H), 2.29–2.19 (m, 2H, 13-H, 16-H ), 1.93–1.85 (m, 2H,
Methyl (2β, 3β) 3-acetyloxy-2-hydroxylup-20(29)-en-28-
a
2
2-H , 21-H ), 1.81–1.72 (m, 1H, 12-H ), 1.67 (s, 3H, 30-
oate (9)
a
a
a
H), 1.63–1.30 (m, 15H, 22-H , 18-H, 16-H , 15-H , 6-H ,
b
b
a
a
1
1
2
1-H , 6-H , 21-H , 7-H , 7-H , 9-H, 11-H , 5-H, 15-H ,
Acetylation of 8 (0.08 g, 0.16 mmol) with acetyl chloride
(0.013 g, 0.014 mL, 0.19 mmol) in dry pyridine (3 mL) at
room temperature for two days followed by usual work-up
and chromatography (silica gel, hexanes/EtOAc, 8:2) gave
a
b
b
a
b
b
b
2-H ), 1.17 (s, 3H, 23-H), 1.10 (s, 3H, 25-H), 1.08 (s, 3H,
b
1
3
4-H), 0.96 (s, 3H, 27-H), 0.94 (s, 3H, 26-H) ppm;
C
NMR (125 MHz, CDCl ): δ = 200.9 (C-3), 176.4 (C-28),
3
1
50.1 (C-20), 143.8 (C-2), 128.7 (C-1), 109.7 (C-29), 56.6
9 (0.036 g, 42%) as a colorless solid; m.p. 217 °C; [α] =
D
(
C-17), 54.1 (C-5), 51.3 (C-31), 49.5 (C-9), 47.0 (C-18),
+25.3° (c = 0.5, MeOH); R = 0.28 (hexanes/EtOAc, 8:2);
F
4
8
5.9 (C-19), 44.0 (C-4), 42.8 (C-10), 41.6 (C-14), 38.7 (C-
), 38.4 (C-13), 37.0 (C-22), 34.1 (C-7), 32.2 (C-16), 30.7
IR (KBr): ν = 3496s, 3073m, 2947s, 2869s, 1728s, 1643m,
1451s, 1433m, 1376s, 1318m, 1247s, 1189s, 1156s, 1134s,
−
1 1
(
C-21), 29.7 (C-15), 27.2 (C-23), 25.5 (C-12), 21.7 (C-24),
1107m, 1070m, 1054m, 1030s, 980s, 883m cm ; H NMR
2
2
1.3 (C-6), 20.2 (C-25), 19.5 (C-30), 18.9 (C-11), 16.6 (C-
(500 MHz, CDCl ): δ = 4.72 (s, 1H, 29-H ), 4.60 (s, 1H,
3
a
6), and 14.7 (C-27) ppm; MS (ESI, MeOH): m/z = 1003
29-H ), 4.59 (d, J = 4.1 Hz, 1H, 3-H), 4.09 (m, 1H, 2-H),
b
+
+
(
44% [2M+K] ), 987 (100% [2M+Na] ), 505 (36% [M
3.64 (s, 3H, 31-H), 2.97 (dt, 1H, J = 10.7, 4.4 Hz, 19-H),
2.24–2.08 (m, 3H, 1-H , 13-H, 16-H ), 2.12 (s, 3H, 33-H),
+
+
+
Na] ), and 483 (25% [M+H] ); analysis calcd for
a
a
C H O (482.71): C 77.14, H 9.61; found: C 76.89, H
1.92–1.82 (m, 2H, 21-H , 22-H ), 1.73–1.69 (m, 2H, 6-H ,
a a a
3
1
46
4
9
.83.
12-H ), 1.67 (s, 3H, 30-H), 1.60–1.18 (m, 12H, 6-H , 7-H,
a b
9
-H, 11-H, 15-H, 16-H , 18-H, 21-H , 22-H ), 1.16 (s, 3H,
b b b
Methyl (2β, 3β) 2,3-dihydroxylup-20(29)-en-28-oate (8)
To a solution of 7 (2.28 g, 4.8 mmol) in dry THF (40 mL)
26-H), 1.14–1.07 (m, 1H, 1-H ), 1.05 (s, 3H, 25-H),
b
1
.03–0.96 (m, 1H, 12-H ), 0.95–0.79 (m, 1H, 5-H),
b
0.93 (s, 3H, 27-H), 0.91 (s, 3H, 24-H), 0.84 (s, 3H, 23-H)
1
3
and EtOH (7 mL) at 0 °C NaBH (0.72 g, 19.2 mmol) was
ppm; C NMR (100 MHz, CDCl ): δ = 176.5 (C-28),
4
3
added. The mixture was allowed to warm to room tem-
perature, and stirring was continued for another 60 min.
Usual aq. work-up followed by chromatography (silica gel,
hexanes/EtOAc, 8:2) gave 8 as a colorless solid (1.90 g,
170.4 (C-32), 150.5 (C-20), 109.6 (C-29), 80.9 (C-3),
69.6 (C-2), 56.7 (C-5), 55.6 (C-17), 51.3 (C-31), 51.2
(C-9), 49.6 (C-18), 47.1 (C-19), 44.0 (C-1), 42.7
(C-14), 41.0 (C-8), 38.4 (C-13), 37.7 (C-10), 37.2 (C-22),
37.1 (C-4), 34.4 (C-7), 32.4 (C-16), 30.8 (C-21), 29.7 (C-
15), 29.3 (C-23), 25.7 (C-12), 21.3 (C-33), 21.3 (C-11),
19.6 (C-30), 18.2 (C-6), 18.1 (C-25), 17.3 (C-26), 16.2 (C-
24), 14.8 (C-27) ppm; MS (ESI, MeOH): m/z = 1079.1
8
1%); m.p. 219–221 °C, lit.: 115 °C (Krainova et al. 2016);
[
0
α] = +14.0° (c = 0.70, MeOH), lit.: [α] = +24.0° (c =
.4, CHCl3) ; R = 0.33 (toluene/EtOAc, 3:1); IR (KBr):
F
D
D
2
5
ν = 3558m, 3510s, 2952s, 2867m, 1709s, 1646w, 1454w,
−
1
1
+
+
1
193m, 1173m, 1159m cm ; H NMR (400 MHz, CDCl ):
(100% [2M+Na] ), 551.5 (78% [M+Na] ); analysis calcd
3
δ = 4.71 (d, 1H, J = 1.8 Hz, 29-H ), 4.58 (s, 1H, 29-H ),
for C H O (528.77): C 74.96, H 9.91; found: C 74.71,
a
b
33 52
5
4
3
2
2
3
1
1
2
.05 (m, 1H, 2-H), 3.64 (s, 3H, 31-H), 3.15 (d, 1H, 4.1 Hz,
-H), 2.96 (ddd, 1H, J = 11.0, 10.6, 4.4 Hz, 19-H),
.30–2.10 (m, 3H, 16-H , 13-H, 1-H ), 1.95–1.80 (m, 2H,
H 10.13.
Methyl (2β, 3β) 2,3-bis(acetyloxy)lup-20(29)-en-28-oate (10)
a
a
1-H , 22-H ), 1.70–1.65 (m, 2H, 12-H , 6-H ), 1.66 (s, 3H,
a
a
a
a
0-H), 1.60–1.27 (m, 10H, 18-H, 6-H , 11-H, 22-H , 15-H ,
Acetylation of 8 (0.55 g, 1.1 mmol) in dry pyridine (16 mL)
with acetyl chloride (0.26 g, 0.24 mL, 3.3 mmol) at 25 °C
for two days, followed by usual work-up and chromato-
graphy (silica gel, toluene/EtOAc, 5:1) gave 10 (0.33 g,
52%) as an amorphous solid; [α] = +21.3° (c = 0.4,
MeOH); R = 0.73 (toluene/EtOAc, 3:1); IR (KBr): ν =
b
b
a
6-H , 21-H , 7-H), 1.24–1.00 (m, 4H, 9-H, 15-H , 12-H ,
b
b
b
b
-H ), 1.11 (s, 3H, 26-H), 0.96 (s, 3H, 25-H), 0.95 (s, 3H,
b
3-H), 0.93 (s, 3H, 27-H), 0.90 (s, 3H, 24-H), 0.73 (dd, 1H,
13
J = 10.8, 2.8 Hz, 5-H) ppm; C NMR (125 MHz, CDCl3):
δ = 176.5 (C-28), 150.4 (C-20), 109.5 (C-29), 78.5 (C-3),
D
F
7
1
1.2 (C-2), 56.6 (C-17), 55.4 (C-5), 51.3 (C-31), 51.1 (C-
9), 49.6 (C-18), 47.0 (C-9), 44.6 (C-1), 42.6 (C-14), 40.9
2949s, 1745s, 1643w, 1368m, 1252s, 1189m, 1155m,
−
1
1
1134m, 1031m cm ; H NMR (400 MHz, CDCl ): δ =
3
(
C-8), 38.4 (C-13), 38.3 (C-4), 37.0 (C-10), 37.0 (C-22),
5.29 (m, 1H, 2-H), 4.71 (s, 1H, 29-H ), 4.58 (d, J = 4.1 Hz,
a
3
1
4.4 (C-7), 32.3 (C-16), 30.7 (C-21), 29.7 (C-23), 29.7 (C-
5), 25.7 (C-12), 21.2 (C-11), 19.5 (C-30), 18.2 (C-6), 17.2
1H, 3-H), 4.57 (m, 1H, 29-H ), 3.64 (s, 3H, 31-H), 2.96 (dt,
b
1H, J = 10.9, 4.5 Hz, 19-H), 2.24–2.12 (m, 2H, 16-H , 13-
a
(
(
[
C-25), 17.2 (C-26), 16.1 (C-24), 14.8 (C-27) ppm; MS
H), 2.03–1.92 (m, 1H, 1-H ), 2.00 (s, 3H, 35-H), 1.99 (s,
a
+
ESI, MeOH): m/z = 487.5 (12.5% [M+H] ), 509.6 (100%
3H, 33-H), 1.91–1.80 (m, 2H, 22-H , 21-H ), 1.73–1.60 (m,
a
a
+
+
M+Na] ), 996.1 (55% [2M+Na] ); analysis calcd for
2H, 12-H , 6-H ), 1.66 (s, 3H, 30-H), 1.59–1.17 (m, 12H,
a a
C H O (486.74): C 76.50, H 10.35; found: C 74.31, H
18-H, 6-H , 22-H , 7-H, 16-H , 21-H , 15-H , 11-H, 9-H, 1-
b b b b a
3
1
50
4
1
0.37.
H ), 1.16–1.05 (m, 1H, 15-H )), 1.07 (s, 3H, 26-H),
b b

Medicinal Chemistry Research
1
.04–0.96 (m, 1H, 12-H ), 0.99 (s, 3H, 25-H), 0.95–0.83
2-Hydroxy-3,20-dioxo-1-en-30-norlupan-28-oic acid (12)
b
(
(
(
m, 1H, 5-H), 0.93 (s, 3H, 27-H), 0.91 (s, 3H, 24-H), 0.85
s, 3H, 23-H) ppm; 13C NMR (125 MHz, CDCl ): δ = 176.4
To a suspension of potassium tert-butanolate (1.5 g,
13.37 mmol) in tert-butanol (26 ml) and tetrahydrofuran
(3.5 mL) 11 (0.5 g, 1.09 mmol) was added, and stirring was
continued for 4 h at 50 °C. During this period, a stream of
air was bubbled through the reaction mixture. Usual aq.
work-up followed by chromatography (silica gel, hexanes/
EtOAc, 3:2) gave 12 (334 mg, 65%) as a colorless solid; m.
p. 224–228 °C; [α] = +9.0° (c = 0.66, MeOH), R = 0.46
3
C-28), 170.5 (C-34), 170.0 (C-32), 150.3 (C-20), 109.6 (C-
2
(
9), 78.0 (C-3), 69.6 (C-2), 56.6 (C-17), 55.3 (C-5), 51.2
C-31), 51.1 (C-9), 49.5 (C-18), 47.1 (C-19), 42.6 (C-1),
4
1
2.3 (C-14), 40.9 (C-8), 38.3 (C-13), 37.5 (C-4), 37.1 (C-
0), 37.0 (C-22), 34.3 (C-7), 32.3 (C-16), 30.7 (C-21), 29.6
(
C-15), 29.1 (C-23), 25.5 (C-12), 21.3 (C-35), 21.1 (C-11),
2
1.0 (C-33), 19.4 (C-30), 18.1 (C-6), 17.6 (C-25), 16.8 (C-
D
F
2
=
6), 16.2 (C-24), 14.7 (C-27) ppm; MS (ESI, MeOH): m/z
1163.2 (100% [2M+Na] ), 593.5 (20% [M+Na] );
(toluene/EtOAc/heptane/HCOOH, 80:26:10:5); IR (KBr): ν
+
+
= 3433s, 2947s, 2871m, 1709s, 1668s, 1455m, 1406m,
−
1 1
analysis calcd for C H O (570.81): C 73.65, H 9.54;
1382m, 1359m, 1236m, 1166m, 1132m, 1055m cm ; H
35
54
6
found: C 85.42, H 9.72.
NMR (500 MHz, CDCl ): δ = 6.41 (s, 1H, 1-H), 3.25 (ddd,
3
J = 11.2, 11.2, 4.8 Hz, 1H, 19-H), 2.34–2.27 (m, 1H, 16-
H ), 2.19 (s, 3H, 29-H), 2.19–1.97 (m, 4H, 18-H, 21-H , 13-
a
a
3
-Oxo-platanic acid (11)
H, 22-H ), 1.68–1.37 (m, 12H, 11-H , 9-H, 22-H , 5-H, 21-
a a b
H , 15-H , 6-H , 6-H , 16-H , 7-H , 7-H , 11-H ),
b
a
a
b
b
a
b
b
A suspension of 3 (10.5 g, 21.8 mmol) and silica gel
100 mL) in acetone (500 mL) was stirred for 30 min. After
cooling to 0 °C Jones reagent (freshly prepared from CrO3
1.28–1.12 (m, 3H, 15-H , 12-H , 12-H ), 1.20 (s, 3H, 23-
b a b
(
H), 1.12 (s, 3H, 25-H), 1.09 (s, 3H, 24-H), 1.01 (s, 3H, 27-
1
3
H), 0.98 (s, 3H, 26-H) ppm; C NMR (125 MHz, CDCl3):
δ = 212.1 (C-20), 201.3 (C-3), 181.8 (C-28), 144.1 (C-2),
128.9 (C-1), 56.4 (C-17), 54.1 (C-5), 51.3 (C-19), 49.1 (C-
18), 45.7 (C-9), 44.1 (C-4), 42.7 (C-14), 41.6 (C-8), 38.8
(C-10), 37.8 (C-13), 36.8 (C-22), 34.0 (C-7), 31.5 (C-16),
30.2 (C-29), 29.7 (C-15), 28.4 (C-21), 27.3 (C-23), 27.1 (C-
12), 21.7 (C-24), 21.2 (C-11), 20.4 (C-25), 18.9 (C-6), 16.5
(C-26), 14.8 (C-27) ppm; MS (ESI, MeOH): m/z = 471
(
(
3
2.5 g, 24.9 mmol), water (10 mL), and concd. H SO
2 4
2.5 mL)) was slowly added, and the mixture was stirred for
0 min at 25 °C. Then MeOH (5 mL) was added, and stir-
ring was continued for another 30 min. The solvents were
removed under diminished pressure, and the resulting solid
was extracted with diethyl ether (Soxhlet apparatus, 6 h).
Evaporation of the ether furnished 11 (9.74 g, 98%) as a
white solid; m.p. 231–233 °C, lit.: 230–233 °C (Baratto
et al. 2013); [α] = +5.4° (c = 0.39, CHCl ), lit.: [α] =
+
+
(100%, [M+H] ), 493 (14%, [M+Na] ); analysis calcd for
C H O (470.65): C 74.01, H 9.00; found: C 73.72, H
D
3
D
29 42
5
+
31° (c = 0.1, CHCl ) (Samoshina et al. 2003); R = 0.5
9.19.
3
F
(
=
toluene/EtOAc/heptane/HCOOH, 80:26:10:5); IR (KBr): ν
32946s, 2869s, 1706s, 1683s, 1455m, 1379m, 1354m,
321w, 1278m, 1243m, 1203m, 1166m, 1140m, 1082w cm
(2β, 3β) 2,3-Dihydroxy-20-oxo-30-norlupan-28-oic acid (13)
1
−
1 1
;
H NMR (400 MHz, CDCl ): δ = 3.24 (ddd, J = 11.3,
To a solution of 12 (217 mg, 0.46 mmol) in dry THF
(10 mL) and MeOH (2 mL) at 0 °C NaBH4 (32 mg,
0.85 mmol) was added. The mixture was allowed to warm
to room temperature, and stirring was continued for another
22 h. Usual aq. work-up followed by chromatography
(silica gel, chloroform/acetone, 4:1) gave 13 as a colorless
3
1
2
1.3, 4.9 Hz, 1H, 19-H), 2.51–2.37 (m, 2H, 2-H , 2-H ),
a b
.32–2.26 (m, 1H, 16-H ), 2.18 (s, 3H, 29-H), 2.19–1.96
a
(
m, 4H, 21-H , 18-H, 13-H, 22-H ), 1.91–1.84 (m, 1H,
a a
1
-H ), 1.64–1.21 (m, 14H, 22-H , 21-H , 15-H , 16-H , 6-
a b b a b
H , 11-H , 7-H , 7-H , 9-H, 1-H , 11-H , 6-H , 5-H,
a
a
a
b
b
b
b
1
5-H ), 1.16–1.07 (m, 2H, 12-H , 12-H ), 1.06 (s, 3H, 23-
solid (138 mg, 63%); m.p. 266–270 °C; [α] = –6.2° (c =
b
a
b
D
H), 1.01 (s, 3H, 27-H), 1.00 (s, 3H, 24-H), 0.94 (s, 3H, 26-
0.215, CHCl3); RF = 0.43 (toluene/EtOAc/heptane/
HCOOH, 80:26:10:5); IR (KBr): ν = 3436br s, 2944s,
2870m, 1698s, 1450m, 1378m, 1358m, 1322w, 1278w,
13
H), 0.90 (s, 3H, 25-H) ppm;
C NMR (100 MHz,
CDCl ): δ = 218.6 (C-3), 212.4 (C-20), 181.9 (C-28), 56.4
3
−
1
1
(
C-17), 54.9 (C-5), 51.3 (C-19), 49.8 (C-9), 49.2 (C-18),
1242m, 1190m, 1170m, 1134m, 1044m cm ; H NMR
4
3
1
2
2
=
7.4 (C-4), 42.4 (C-14), 40.7 (C-8), 39.7 (C-1), 37.8 (C-13),
7.0 (C-10), 36.8 (C-22), 34.2 (C-2), 33.6 (C-7), 31.5 (C-
6), 30.2 (C-29), 29.8 (C-15), 28.4 (C-21), 27.3 (C-12),
6.9 (C-23), 21.5 (C-11), 21.1 (C-24), 19.8 (C-6), 16.1 (C-
5), 15.8 (C-26), 14.8 (C-27) ppm; MS (ESI, MeOH): m/z
(400 MHz, THF-d ): δ = 3.91 (ddd, J = 4.0, 4.0, 2.9 Hz,
8
1H, 2-H), 3.22 (ddd, J = 11.2, 11.2, 4.3 Hz, 1H, 19-H), 3.02
(d, J = 4.0 Hz, 1H, 3-H), 2.24 (ddd, J = 12.6, 3.1, 3.1 Hz,
1H, 16-H ), 2.19–1.96 (m, 4H, 13-H, 1-H , 18-H, 21-H ),
a
a
a
2.08 (s, 3H, 29-H), 1.92–1.86 (m, 1H, 22-H ), 1.59–1.23
a
−
−
455 (100% [M-H] ), 911 (77% [2M-H] ); analysis
(m, 11H, 15-H , 6-H , 6-H , 11-H , 22-H , 21-H , 7-H , 7-
a
a
b
a
b
b
a
calcd for C H O (456.67): C 76.27, H 9.71; found:
H , 16-H , 11-H , 9-H), 1.16 (s, 3H, 25-H), 1.18–1.00 (m,
b b b
2
9
44
4
C 76.07, H 9.88.
4H, 15-H , 12-H , 12-H , 1-H ), 0.99 (s, 3H, 27-H), 0.95 (s,
b a b b

Medicinal Chemistry Research
6
1
H, 23-H, 24-H), 0.94 (s, 3H, 26-H), 0.82–0.77 (dd, J =
22-H ), 1.12–0.89 (m, 4H, 1-H , 2-H, 21-H), 0.99 (s, 3H,
b b
0.3, 3.0 Hz, 1H, 5-H) ppm; 13C NMR (100 MHz, THF-d8):
26-H), 0.97 (s, 3H, 27-H), 0.82 (s, 6H, 24-H, 25-H), 0.81 (s,
1
3
δ = 210.4 (C-20), 177.6 (C-28), 78.7 (C-3), 72.0 (C-2), 56.7
C-17), 56.6 (C-5), 52.1 (C-19), 52.0 (C-9), 49.8 (C-18),
3H, 23-H), 0.76 (d, 1H, J = 9.9 Hz, 5-H) ppm; C NMR
(
(125 MHz, CDCl ): δ = 211.6 (C-20), 171.5 (C-32), 170.9
3
4
3
1
2
2
5.6 (C-1), 43.2 (C-14), 41.7 (C-8), 39.2 (C-4), 38.3 (C-13),
8.0 (C-10), 37.6 (C-22), 35.3 (C-7), 32.6 (C-16), 30.7 (C-
5), 30.1 (C-23), 29.6 (C-29), 29.0 (C-21), 28.2 (C-12),
2.1 (C-11), 19.1 (C-6), 17.8 (C-24), 17.6 (C-25), 16.5 (C-
6), and 15.1 (C-27) ppm; MS (ESI, MeOH): m/z = 475
(C-30), 80.8 (C-3), 62.5 (C-28), 55.3 (C-5), 51.7 (C-19),
50.1 (C-9), 49.4 (C-18), 46.3 (C-17), 42.5 (C-14), 40.8 (C-
8), 38.3 (C-1), 37.8 (C-4), 37.0 (C-10), 36.4 (C-13), 34.4
(C-22), 34.0 (C-7), 29.6 (C-29), 29.3 (C-16), 27.9 (C-23),
27.5 (C-21), 27.2 (C-12), 27.0 (C-15), 23.6 (C-2), 21.3 (C-
33), 21.0 (C-31), 20.8 (C-11), 18.1 (C-6), 16.5 (C-24), 16.1
(C-25), 16.0 (C-26), 14.6 (C-27) ppm; MS (ESI, MeOH): m/
+
+
(
12%, [M+H] ), 492 (100%, [M+NH ] ), 971 (86%, [2M
4
+
+
9
Na] ); analysis calcd for C H O (474.68): C 73.38, H
29
46
5
+
+
.77; found: C 73.05, H 9.90.
z = 546.5 (16% [M+NH ] ), 551.5 (26% [M+Na] ), 815.9
4
2
+
+
(
63% [3M+2Na] ), and 1079.2 (100% [2M+Na] );
Methyl (2β, 3β)-2,3-dihydroxy-20-oxo-30-norlupan-28-oate
14)
analysis calcd for C H O (528.77): C 74.96, H 9.91;
found: C 74.65, H 10.16.
3
3
52
5
(
Esterification of 13 (98 mg, 0.21 mmol) in dry DMF (2 mL)
Methyl (3β) 3-hydroxy-20-oxo-30-norlupan-28-oate (17)
with K CO (120 mg, 0.87 mmol) and MeI (0.03 mL,
2
3
0
.48 mmol) as previously described (Xu et al. 2012; Heller
et al. 2017) gave 14 as a colorless solid (80 mg, 79%); m.p.
27–229 °C; [α] = –10.7° (c = 0.38, CHCl ); R = 0.31
This compound was prepared according to (Sommerwerk
et al. 2015a, 2015b) from 3; colorless solid; m.p. 242–246 °
2
C, lit.: 240–244 °C (Mayer 1996); R = 0.38 (toluene/ethyl
D
3
F
F
(
toluene/EtOAc/heptane/HCOOH, 80:26:10:5); MS (ESI,
acetate/n-heptane/HCOOH, 80:26:10:5); [α] = −28.2° (c
D
+
MeOH): m/z = 506 (100%, [M+NH ] ), 999 (20%, [2M
+
= 0.52, CHCl ), lit.: [α] = –30.0° (c = 0.40, CHCl )
4
3
D
3
+
Na] ).
(Mayer 1996); MS (ESI, MeOH): m/z = 473 (60%, [M+H]
+
+
+
)
, 490 (98%, [M+NH ] ), 495 (74%, [M+Na] , 505
4
+
2+
(
3β)-3,28-Diacetyloxylup-20(29)-ene (15)
(20%, [M+H+MeOH] ), 731 (70%, [M+2Na] ), and 967
+
(98%, [2M+Na] ).
This compound was prepared according to (Gauthier et al.
2
006) from 1; colorless solid; m.p. 219–221 °C, lit.:
Methyl (3β)-3-acetyloxy-20-oxo-30-norlupan-28-oate (18)
2
23–224 °C (Gauthier et al. 2006); [α] = +22.1° (c = 0.8,
D
CHCl ), lit.: 19.7° (c = 1.7, CHCl ) (Gauthier et al. 2006).
This compound was prepared according to (Vystrčil and
Buděšínský 1970) from 17; colorless solid m.p. 201–204 °
3
3
(
3β) 3,28-Diacetyloxy-30-norlup-20-one (16)
C, lit.: 205–207 °C (Vystrčil and Buděšínský 1970); R =
F
0.66 (toluene/ethyl acetate/n-heptane/HCOOH, 80:25:30:4);
Ozone was bubbled through a solution of 15 (0.9 g,
.71 mmol) in dry DCM (50 mL) at −60 °C for 60 min.
After warming to room temperature powdered Zn (1.0 g,
.015 mol) and glacial acetic acid (10 mL) were added
[α] = −8.2° (c = 0.35, CHCl ), lit.: [α] = –14.6° (c =
D
3
D
2
1
1
0.80, CHCl ) ; MS (ESI, MeOH): m/z = 455 (70%, [M
3
+
+
+H-HOAc] ), 537 (26%, [M+Na] ), 749 (100%, [3M
2
+
+
0
+2Na] ), and 1051 (78%, [2M+Na] ).
within 1 h. The suspension was stirred for another hour,
followed by usual aq. work-up and chromatography (silica
gel, hexanes/EtOAc, 9:1) to afford 16 (0.66 g, 73%) as a
colorless solid; m.p. 185–187 °C, lit.: 188–190 °C (Flekhter
et al. 2005); [α] = –10.9° (c = 0.50, CHCl ), lit.: [α]
Methyl (3β, 20Z) 3-acetyloxy-20-hydroxyimino-30-
norlupan-28-oate (19) and methyl (3β, 20E) 3-acetyloxy-20-
hydroxyimino-30-norlupan-28-oate (20)
D
3
D
=
–9.8° (c = 0.75, CHCl ) (Vystrčil and Buděšínský 1970);
A solution of 18 (1.2 g, 2.34 mmol) and hydro-
3
RF = 0.42 (hexanes/EtOAc, 8:2); IR (KBr): ν = 2948s,
xylammonium chloride (1.0 g, 14.95 mmol) in pyridine
(12 mL) was stirred at 60 °C for 3 h. The solvent was
removed under reduced pressure by re-evaporating with
toluene (3 × 20 mL). The residue was dissolved in dichlor-
omethane (20 mL) and washed successively with diluted
HCl (0.1 M, 1 × 50 mL) and water (3 × 30 mL). The aqueous
layer was extracted with dichloromethane (3 × 20 mL), and
the combined organic layers were dried with Na SO , and
2
1
871s, 1737s, 1457m, 1392s, 1368s, 1316w, 1247s, 1169m,
−
1 1
106w, 1032s, 979s cm ; H NMR (400 MHz, CDCl ): δ
3
=
4.44 (dd, 1H, J = 10.9, 5.2 Hz, 3-H), 4.18 (d, 1H, J =
1
1.1 Hz, 28-H ), 3.75 (d, 1H, J = 11.1 Hz, 28-H ), 2.63 (dt,
a
b
1
H, J = 11.3, 5.8 Hz, 19-H), 2.13 (s, 3H, 29-H), 2.11–1.95
(
m, 2H, 15-H , 18-H), 2.05 (s, 3H, 31-H), 2.00 (s, 3H, 33-
a
H), 1.88–1.75 (m, 2H, 16-H , 22-H ), 1.70–1.13 (m, 15H, 1-
a
a
2
4
H , 2-H, 6-H, 7-H, 9-H, 11-H, 12-H , 13-H, 15-H ), 16-H ,
the solvent was evaporated. The residue was fractionated by
a
a
b
b

Medicinal Chemistry Research
column chromatography (silica gel, hexanes/ethyl acetate,
31.6 (C-16), 29.8 (C-15), 28.1 (C-23), 27.1 (C-12), 23.8 (C-
2), 22.3 (C-21), 21.7 (C-29), 21.4 (C-32), 21.0 (C-11), 18.3
(C-6), 16.6 (C-24), 16.2 (C-25), 16.1 (C-26), 14.5 (C-27)
4
:1) to afford compound 19 (164 mg, 13%) and compound
2
0 (892 mg, 72%).
+
Data for 19: white solid; m.p. 244–247 °C, lit.:
ppm; MS (ESI, MeOH): m/z = 516 (100%, [M+H] );
2
45–248 °C (Vystrčil et al. 1986); R = 0.48 (toluene/ethyl
analysis calcd for C H NO (515.78): C 74.52, H 10.36,
F
32 53
4
acetate/n-heptane/HCOOH, 80:26:10:5); [α] = +45.0° (c
N 2.72; found: C 74.39, H 10.50, N 2.51.
D
=
0.32, CHCl ), lit.: [α] = +30° (c = 0.4, CHCl ) (Vystr-
Data for 22: white solid, m.p. 218–220 °C; R = 0.31
3
D
3
F
čil et al. 1986); MS (ESI, MeOH): m/z = 530 (94%, [M+H]
(CHCl /MeOH/NH OH, 90:10:0.1); [α] = −10.7° (c =
3
4
D
+
2+
2+
)
, 795 (34%, [3M+2H] ), 806 (10%, [3M+H+Na] ),
0.285, CHCl ); IR (KBr): ν = 2948s, 2871m, 1732s,
3
+
and 1059 (100%, [2M+H] .
1456w, 1368m, 1246s, 1156m, 1135m, 1029w, 980m cm
−
1
1
Data for 20: white solid; m.p. 245–247 °C, lit.:
; H NMR (400 MHz, CDCl ): δ = 4.46 (dd, J = 10.4,
3
2
44–246 °C (Vystrčil et al. 1986); R = 0.56 (toluene/ethyl
5.9 Hz, 1H, 3-H), 3.65 (s, 3H, 30-H), 3.09 (dd, J = 12.4,
F
acetate/n-heptane/HCOOH, 80:26:10:5); [α] = +6.1° (c =
5.9 Hz, 1H, 20-H), 2.37–2.16 (m, 3H, 19-H, 16-H , 13-H),
D
a
0
.36, CHCl ), lit.: [α] = +8.0° (c = 0.4, CHCl ) (Vystrčil
2.03 (s 3H, 32-H), 1.85–1.77 (m, 1H, 22-H ), 1.73–1.45 (m,
3
D
3
a
et al. 1986); MS (ESI, MeOH): m/z = 530 (100%, [M+H]
8H, 1-H , 12-H , 2-H, 21-H, 6-H , 11-H ), 1.43–1.25 (m,
9H, H-18, 6-H , H-16 , 15-H , 7-H, 11-H , 9-H, 22-H ),
b b a b b
a
a
a
a
+
+
)
, 1059 (44%, [2M+H] ).
1
.26–1.05 (m, 2H, 12-H , 15-H ), 1.06 (d, J = 6.6 Hz, 3H,
b b
Methyl (3β, 20 R)-3-acetyloxy-20-amino-30-norlupan-28-
oate (21) and methyl (3β, 20 S)-3-acetyloxy-20-amino-30-
norlupan-28-oate (22)
29-H), 1.03–0.94 (m, 1H, 1-H ), 0.95 (s, 3H, 27-H), 0.90 (s,
b
3H, 26-H), 0.85 (s, 3H, 25-H), 0.83 (s, 3H, 23-H), 0.83 (s,
1
3
3H, 24-H), 0.80–0.74 (m, 1H, 5-H) ppm; C NMR
(
CDCl , 125 MHz): δ = 176.9 (C-28), 171.1 (C-31), 81.1
3
To a solution of 20 (1.15 g, 2.17 mmol) and ammonium
acetate (1.0 g, 13.0 mmol) in MeOH (120 mL), sodium
cyanoborohydride (0.4 g, 6.4 mmol) was added under N2
atmosphere. The reaction was cooled to 0 °C, and titanium
trichloride (≥12% in HCl (12%), 4 mL, 3.9 mmol) was
added dropwise during 30 min. The mixture was stirred at
room temperature for 20 h and then treated with 2 N sodium
hydroxid until pH = 10. The aqueous solution was extracted
with DCM (3 × 200 mL), and the organic layer was dried
over anhydrous Na SO ; the solvent was concentrated in
(C-3), 57.1 (C-17), 55.6 (C-5), 51.4 (C-30), 50.3 (C-9), 48.6
(C-18), 48.5 (C-20), 45.2 (C-19), 42.6 (C-14), 40.8
(C-8), 38.5 (C-1), 38.2 (C-13), 37.9 (C-4), 37.2 (C-10), 37.2
(C-22), 34.4 (C-7), 32.0 (C-16), 29.8 (C-15), 28.1 (C-23),
27.1 (C-12), 23.8 (C-2), 23.5 (C-29), 22.3 (C-21), 21.5
(C-32), 21.0 (C-11), 18.3 (C-6), 16.6 (C-24), 16.3 (C-25),
16.1 (C-26), 14.7 (C-27) ppm; MS (ESI, MeOH): m/z =
+
516 (100%, [M+H] ); analysis calcd for C H NO
3
2
53
4
(515.78): C 74.52, H 10.36, N 2.72; found: C 74.34, H
10.47, N 2.43.
2
4
vacuo. The residue was fractionated by column chromato-
graphy (silica gel, chloroform/methanol/NH OH, 90:10:0.1)
4
to afford 21 (751 mg, 67%) and 22 (168 mg, 15%).
(3β, 20 R) 3-Hydroxy-20-amino-30-norlupan-28-ol (23)
Compound 23 (245 mg, 71%) was obtained from 21
Data for 21: white solid; m.p. 208–211 °C; R = 0.37
F
(
CHCl /MeOH/NH OH, 90:10:0.1); [α] = −18.4° (c =
3
4
D
0
1
1
.46, CHCl ); IR (KBr): ν = 3440sbr, 2948s, 2872m,
(400 mg, 0.78 mmol) by reduction within LiAlH in THF
(1 M, 6 mL) as previously described (Heller et al. 2017);
3
4
729s, 1636w, 1457m, 1384s, 1246s, 1166m, 1135m,
−
1 1
028w, 980w cm ; H NMR (400 MHz, CDCl ): δ = 4.46
white solid, m.p. 240–246 °C; R = 0.37 (CHCl /MeOH/
3
F
3
(
(
dd, J = 10.6, 5.7 Hz, 1H, 3-H), 3.66 (s, 3H, 30-H), 3.31
dd, J = 12.6, 6.0 Hz, 1H, 20-H), 2.41 (dd, J = 16.2, 8.6 Hz,
NH OH, 90:10:0.1); [α] = –47.8° (c = 0.29, pyridine); MS
(ESI, MeOH): m/z = 446 (100%, [M+H] ).
4
D
+
1
3
2
2
H, 19-H), 2.30–2.16 (m, 2H, 16-H , 13-H), 2.03 (s, 3H,
a
2-H), 1.84–1.74 (m, 1H, 22-H ), 1.73–1.56 (m, 8H, 1-H ,
(3β, 20 R) 3,28-Diacetyloxy-20-amino-30-norlupane (24)
a
a
1-H , 2-H, 12-H ), 1.55–1.23 (m, 9H, H-18, 11-H , 6-H ,
a
a
a
a
2-H , 16-H , 15-H , 6-Hb, 7-H, 11-H , 9-H, 12-H ), 1.22
Di-tert-butyldicarbonate (70 mg, 0.32 mmol) was added to a
solution of 23 (150 mg, 0.33 mmol) in pyridine (2 mL)
(Kahnt et al. 2018), and stirring at 25 °C was continued for
30 min. Without additional work-up, acetic anhydride
(0.1 mL, 1.06 mmol) was added, and the mixture was stirred
at room temperature for 16 h. The reaction mixture was then
diluted with toluene (3 × 20 mL), and all organic solvents
were removed under reduced pressure to yield a crude solid
(170 mg) which was used without further purification. After
b
a
a
b
b
(
d, J = 6.6 Hz, 3H, 29-H), 1.18–1.05 (m, 1H, 15-Hb),
1
2
0
.04–0.94 (m, 1H, 1-H ), 0.97 (s, 3H, 27-H), 0.90 (s, 3H,
b
6-H), 0.85 (s, 3H, 25-H), 0.83 (s, 6H, 23-H, 24-H),
.80–0.72 (m, 1H, 5-H) ppm; 13C NMR (CDCl , 125 MHz):
3
δ = 176.7 (C-28), 171.0 (C-31), 81.0 (C-3), 57.1 (C-17),
5
5.6 (C-5), 51.5 (C-30), 50.2 (C-9), 49.9 (C-20), 48.5 (C-
1
8), 44.4 (C-19), 42.6 (C-14), 40.8 (C-8), 38.5 (C-1), 38.2
(
C-13), 37.9 (C-4), 37.2 (C-10), 36.6 (C-22), 34.4 (C-7),

Medicinal Chemistry Research
dissolving the residue in dichloromethane (50 mL), the
Cytotoxic assay
mixture was cooled to 0 °C, and trifluoroacetic acid
(
0.08 mL, 1 mmol) was added. Cooling was removed, and
The cytotoxicity of the compounds was evaluated using the
sulforhodamine-B (Kiton-Red S, ABCR) micro culture
colorimetric assay. Cells were seeded into 96-well plates on
day 0 at appropriate cell densities to prevent confluence of
the cells during the period of experiment. After 24 h, the
cells were treated with six different concentrations (1, 3, 7,
12, 20, and 30 µM) minimum. The final concentration of
DMSO/DMF never exceeded 0.5 %, which was non-toxic
to the cells. After a 96 h treatment, the supernatant medium
from the 96-well plates was discarded, the cells were fixed
with 10% trichloroacetic acid (TCA) and allowed
to rest at 4 °C. After 24 h fixation, the cells were washed in
a strip washer and dyed with SRB solution (100 µL, 0.4 %
in 1 % acetic acid) for about 20 min. After dying,
the plates were washed four times with 1% acetic acid to
remove the excess of the dye and allowed to air-dry over-
night. Tris base solution (200 µL, 10 mM) was added to
each well and absorbance was measured at λ = 570 nm
using a 96-well plate reader (Tecan Spectra, Crailsheim,
Germany). The EC50 values were averaged from three
independent experiments performed each in triplicate cal-
culated from semi logarithmic dose response curves
applying a non-linear 4 P Hills-slope equation (GraphPad
Prism5; variables top and bottom were set to 100 and 0,
respectively).
the reaction mixture was allowed to warm up to room
temperature. Stirring was continued for additional 3 h, then
the reaction mixture was concentrated to dryness under
reduced pressure, re-dissolved in dichloromethane and re-
evaporated. The residue was purified by column chroma-
tography (silica gel, CHCl /MeOH/NH OH, 90:10:0.1) to
afford 24 (130 mg, 74%) as a white solid; m.p. 204–206 °C;
RF = 0.49 (CHCl /MeOH/NH OH, 90:10:0.1); [α] =
−
3
4
3
4
D
24.4° (c = 0.33, CHCl ), IR (KBr): ν = 3442br s, 2946m,
3
1
736m, 1636m, 1524w, 1458w, 1384vs, 1245m, 1031m cm
−
1
1
;
H NMR (400 MHz, CDCl ): δ = 4.44 (dd, J = 10.4,
3
5
.9 Hz, 1H, 3-H), 4.21 (d, J = 11.1 Hz, 1H, 28-H ), 3.82 (d,
a
J = 11.1 Hz, 1H, 28-H ), 3.60 (q, J = 6.1 Hz, 1H, 20-H),
b
2
3
2
2
.44 (ddd, J = 11.1, 11.1, 5.3 Hz, 1H, 19-H), 2.05 (s, 3H,
3-H), 2.03 (s, 3H, 31-H), 1.98 (td, J = 11.4, 3.7 Hz, 1H,
2-H ), 1.92–1.83 (m, 1H, 16-H ), 1.82–1.54 (m, 9H, H-18,
a
a
1-H, 13-H, 1-H , 2-H, 12-H , 15-H ), 1.50–1.44 (m, 1H, 6-
a
a
a
H ), 1.43–1.31 (m, 5H, 6-Hb, 11-H, 7-H), 1.37 (d, J =
6
1
H), 1.00–0.93 (m, 1H, 1-H ), 0.99 (s, 3H, 25-H), 0.85 (s,
3
0
a
.6 Hz, 3H, 29-H), 1.30–1.20 (m, 2H, 9-H, H-16b),
.09–1.01 (m, 3H, 22-H , 15-H , 12-H ), 1.04 (s, 3H, 27-
b
b
b
b
H, 23-H), 0.83 (s, 3H, 24-H), 0.82 (s, 3H, 26-H),
13
.79–0.70 (m, 1H, 5-H) ppm; C NMR (100.5 MHz,
CDCl ): δ = 171.6 (C-32), 171.0 (C-30), 80.9 (C-3), 62.3
3
(
C-28), 55.6 (C-5), 51.0 (C-20), 49.8 (C-9), 47.8 (C-18),
4
1
3
6.7 (C-17), 43.6 (C-19), 43.0 (C-14), 41.0 (C-8), 38.4 (C-
), 37.9 (C-4), 37.2 (C-13), 37.1 (C-10), 34.2 (C-7),
3.5 (C-22), 29.3 (C-16), 28.1 (C-23), 27.2 (C-15), 27.0
AO/PI dye exclusion test
Morphological characteristics of cell death were analyzed
employing an AO/PI assay using human cancer cell line
(
C-12), 23.8 (C-2), 21.6 (C-21), 21.4 (C-31), 21.1 (C-33),
5
2
2
=
0.8 (C-11), 19.6 (C-29), 18.3 (C-6), 16.6 (C-24), 16.2 (C-
A2780. Approximately 8∙10 cells were seeded in cell
2
5), 16.2 (C-26), 14.3 (C-27) ppm; MS (ESI, MeOH): m/z
culture flasks (25 cm ), and the cells were allowed to grow
+
530 (100%, [M+H] ); analysis calcd for C H NO
up for 24 h. After removing of the used medium, the
substance loaded fresh medium was reloaded (or a blank
new medium as a control). After 24 h, the content of the
flask was collected and centrifuged (1200 rpm, 4 °C), the
pellet was gently suspended in phosphate-buffered saline
33
55
4
(
529.81): C 74.81, H 10.46, N 2.64; found: C 74.77, H
1
0.63, N 2.37.
Biological assays
(
PBS (w/w), 1 mL) and centrifuged again. The PBS
was removed, and the pellet gently suspended in PBS
50 µL) again. The analysis of the cells was performed
Cell lines and culture conditions
(
The cell lines used are human cancer cell lines: 518A2
using a fluorescence microscope after having mixed
the cell suspension (10 µL) with a solution of AO/PI
(1 µg/mL, 10 µL).
(
melanoma), A549 (alveolar basal epithelial adenocarci-
noma), A2780 (ovarian carcinoma), MCF-7 (breast adeno-
carcinoma), and non-malignant mouse fibroblasts NIH 3T3.
Cultures were maintained as monolayers in RPMI 1640
medium with L-glutamine (Capricorn Scientific GmbH,
Ebsdorfergrund, Germany) supplemented with 10% heat
inactivated fetal bovineserum (Sigma-Aldrich Chemie
GmbH, Steinheim, Germany) and penicillin/streptomycin
Cell cycle investigations
5
Approximately 8∙10 cells (A2780) were seeded in cell
2
culture flasks (25 cm ), and the cells were allowed to grow
up for 24 h. After removing of the used medium, the sub-
stance loaded fresh medium was reloaded (or a blank fresh
medium as a control). After 24 h, only the adherent cells
(
3
Capricorn Scientific GmbH, Ebsdorfergrund, Germany) at
7 °C in a humidified atmosphere with 5% CO2.

Medicinal Chemistry Research
were harvested, centrifuged (1200 rpm, 4 °C), and washed
twice with PBS (w/w), 1 mL). The cells were counted and
approximately 1∙10 cells were fixed with ethanol (70%, 4 °
C, 24 h). After centrifugation (1200 rpm, 4 °C) the cells
were washed with staining buffer (PBS (w/w), containing
References
6
Ali-Seyed M, Jantan I, Vijayaraghavan K, Bukhari SNA (2016)
Betulinic acid: recent advances in chemical modifications,
effective delivery, and molecular mechanisms of a promising
anticancer therapy. Chem Biol Drug Des 87:517–536
2
% FCS and 0.01% NaN (2% in H O), 1 mL) and cen-
Baratto LC, Porsani MV, Pimentel IC, Pereira Netto AB, Paschke R,
Oliveira BH (2013) Preparation of betulinic acid derivatives by
chemical and biotransformation methods and determination of
cytotoxicity against selected cancer cell lines. Eur J Med Chem
3
2
trifuged. The pellet was gently suspended in RNAase A
100 µL, 100 mg/mL) and incubated for 30 min at 37 °C.
(
After resuspending cells in 1 mL PI buffer (20 µL PI solu-
tion (1 mg/mL) in staining buffer) and incubating for 30 min
at room temperature in the dark, the cells were analyzed
using the Attune® FACS machine; collecting data from the
BL-2A channel. Doublet cells were excluded from the
measurements by plotting BL-2A against BL-2H. For each
cell cycle distribution 20,000 events were collected in
technical triplicates, each sample was measured in dupli-
cates. Cell cycle distribution was calculated using Mod-
FitLT™ (Verity Software House, Topsham,US).
6
8:21–131
Barthel A, Stark S, Csuk R (2008) Oxidative transformations of
betulinol. Tetrahedron 64:9225–9229
Cheng K, Zhang P, Liu J, Xie J, Sun H (2008) Practical synthesis of
bredemolic acid, a natural inhibitor of glycogen phosphorylase. J
Nat Prod 71:1877–1880
Csuk R (2014) Betulinic acid and its derivatives: a patent review
(2008–2013). Expert Opin Ther Pat 24:913–923
Csuk R, Schmuck K, Schäfer R (2006) A practical synthesis of
betulinic acid. Tetrahedron Lett 47:8769–8770
Diamond (1997) Visual Crystal Structure Information System, Crystal
Impact GbR, Bonn, K. Brandenburg (1997-2017), version 440
Flekhter OB, Giniyatullina GV, Galin FZ, Baschenko NZ, Makara NS,
Zarudii FS, Boreko EI, Savinova OV, Pavlova NI, Starikova ZA,
Tolstikov GA (2005) Synthesis and pharmacological activity of
Annexin V-FITC/ PI assay
2
0-Keto-29-norlupane Derivatives. Chem Nat Compd
5
Approximately 8∙10 cells (A2780) were seeded in cell
41:706–709
2
Gauthier C, Legault J, Lebrun M, Dufour P, Pichette A (2006) Gly-
cosidation of lupane-type triterpenoids as potent in vitro cytotoxic
agents. Bioorg Med Chem 14:6713–6725
Gheorgheosu D, Duicu O, Dehelean C, Soica C, Muntean D (2014)
Betulinic acid as a potent and complex antitumor phytochemical:
a minireview. Anti-Cancer Agents Med Chem 14:936–945
Gu J-Q, Wang Y, Franzblau SG, Montenegro G, Yang D, Timmer-
mann BN (2004) Antitubercular constituents of Valeriana laxi-
flora. Planta Med 70:509–514
culture flasks (25 cm ), and the cells were allowed to grow
up for 24 h. After removing of the used medium, the sub-
stance loaded fresh medium was reloaded (or a blank fresh
medium as a control). After 24 h, the cells were harvested,
centrifuged (1200 rpm, 4 °C), and washed twice with PBS
w/w), 1 mL). The cells were counted and approximately
∙10 cells were washed with Annexin V binding buffer
(
6
1
(
BioLegend®, San Diego,US) and treated with propidium
Heller L, Kahnt M, Loesche A, Grabandt P, Schwarz S, Brandt W,
Csuk R (2017) Amino derivatives of platanic acid act as selective
and potent inhibitors of butyrylcholinesterase. Eur J Med Chem
iodide solution (3 µL, 1 mg/mL) and Annexin V-FITC (5
µL, BioLegend®, San Diego, US) for 15 min in the dark at
room temperature. After adding Annexin V binding
buffer (400 µL) the suspension was analyzed using Attune®
FACS machine. After gating for living cells, the data from
detectors BL-1A and BL-3A were collected (20,000 events)
in technical triplicates. The assay was performed in dupli-
cates; cell distribution was calculated using Attune®
Software.
1
26:652–668
Jarolim V, Hejno K, Streibl M, Horak M, Sorm F (1961) Composition
of brown coal II Further constituents of Montan wax. Collect
Czech Chem Commun 26:459–465
Jonnalagadda SC, Corsello MA, Sleet CE (2013) Betulin-betulinic
acid natural product based analogs as anti-cancer agents. Anti
Cancer Agents Med Chem 13:1477–1499
Kahnt M, Heller L, Grabandt P, Al-Harrasi A, Csuk R (2018) Platanic
acid: a new scaffold for the synthesis of cytotoxic agents. Eur J
Med Chem 143:259–265
Krainova GF, Tolmacheva IA, El’tsov OS, Gorbunova MN, Grishko
VV (2016) Synthesis of vinyl-containing lupane- and A-
Secolupane-type triterpene esters. Chem Nat Compd 52:256–261
Ma C-M, Cai S-Q, Cui J-R, Wang R-Q, Tu P-F, Hattori M, Danesh M
Acknowledgements We would like to thank Dr. D. Ströhl and his
team for the NMR spectra, and H. Rost, MSc, for preparing some of
the starting materials and his help in the lab. Thank is also due to Mrs.
U. Lammel, Mrs. J. Wiese, MSc, and Mrs. V. Simon, B. Sc., for
measuring the IR spectra and optical rotations. Elemental analyses
were performed by Mrs. U. Lammel. The cell lines were kindly pro-
vided by Dr. Th. Müller (Dept. of Haematology/Oncology, Martin-
Luther Universität Halle-Wittenberg). Support by the “Wis-
senschaftsCampus Halle WCH” (W13004216 to R.C.) and the
(2005) The cytotoxic activity of ursolic acid derivatives. Eur J
Med Chem 40:582–589
Ma C-M, Wu X-H, Hattori M, Wang X-J, Kano Y (2009) HCV
protease inhibitory, cytotoxic and apoptosis-inducing effects of
oleanolic acid derivatives. J Pharm Pharm Sci 12:243–248
Mayer R (1996) Three lupane derivatives from Leptospermum sco-
parium. Arch Pharm 329:447–450
“International Office” (Martin-Luther Universität Halle-Wittenberg to
C.O.) is gratefully recognized.
Paduch R, Kandefer-Szerszen M (2014) Antitumor and antiviral
activity of pentacyclic triterpenes. Mini-Rev Org Chem
Compliance with ethical standards
1
1:262–268
Periasamy G, Teketelew G, Gebrelibanos M, Sintayehu B, Gebrehiwot
M, Karim A, Geremedhin G (2014) Betulinic acid and its
Conflict of interest The authors declare that they have no conflict of
interest.

Medicinal Chemistry Research
derivatives as anti-cancer agent: a review. Arch Appl Sci Res
Urban M, Sarek J, Klinot J, Korinkova G, Hajduch M (2004) Synth-
esis of A-Seco derivatives of betulinic acid with cytotoxic
activity. J Nat Prod 67:1100–1105
6
:47–58
Salvador JAR, Leal AS, Alho DPS, Goncalves BMF, Valdeira AS,
Mendes VIS, Jing Y (2014) Highlights of pentacyclic triterpe-
noids in the cancer settings. Stud Nat Prod Chem 41:33–73
Samoshina NF, Denisenko MV, Denisenko VA, Uvarova NI (2003)
Synthesis of glycosides of lupane-type triterpene acids. Chem Nat
Comp 39:575–582
Sheldrick GM (2008) A short history of SHELX. Acta Crystallogr
A64:112–122
Sommerwerk S, Heller L, Csuk R (2015a) Synthesis and cytotoxic
activity of pentacyclic triterpenoid sulfamates. Arch Pharm
Urban M, Sarek J, Tislerova I, Dzubak P, Hajduch M (2005) Influence
of esterification and modification of A-ring in a group of lupane
acids on their cytotoxicity. Bioorg Med Chem 13:5527–5535
Vystrčil A, Buděšínský M (1970) Triterpenes XVI Unuusual epi-
merisation of the C (19)-acetyl group of 20-oxo-30-norlupane
derivatives. Collect Czech Chem Commun 35:295–311
Vystrčil A, Křeček V, Buděšínský M, Protiva J (1986) Isomeric oxi-
mes of 30-norlupan-20-one and its derivatives. Collect Czech
Chem Commun 51:581–592
3
48:46–54
Wen X, Sun H, Liu J, Cheng K, Zhang P, Zhang L, Hao J, Zhang L, Ni
P, Zographos SE, Leonidas DD, Alexacou K-M, Gimisis T,
Hayes JM, Oikonomakos NG (2008) Naturally occurring penta-
cyclic triterpenes as inhibitors of glycogen phosphorylase:
synthesis, structure-activity relationships, and X-ray Crystal-
lographic studies. J Med Chem 51:3540–3554
Xu Jun, Li Zhenxi, Luo Jian, Yang Fan, Liu Ting, Liu Mingyao, Qiu
Wen-Wei, Tang Jie (2012) Synthesis and Biological Evaluation
of Heterocyclic Ring-Fused Betulinic Acid Derivatives as Novel
Inhibitors of Osteoclast Differentiation and Bone Resorption. J
Med Chem 55(7):3122–3134
Sommerwerk S, Heller L, Serbian I, Csuk R (2015b) Straightforward
partial synthesis of four diastereomeric 2,3-dihydroxy-olean-12-
en-28-oic acids from oleanolic acid. Tetrahedron 71:8528–8534
Sommerwerk S, Heller L, Kuhfs J, Csuk R (2016a) Urea derivates of
ursolic, oleanolic and maslinic acid induce apoptosis and are
selective cytotoxic for several human tumor cell lines. Eur J Med
Chem 119:1–16
Sommerwerk S, Heller L, Kuhfs J, Csuk R (2016b) Selective killing of
cancer cells with triterpenoic acid amides-the substantial role of
an aromatic moiety alignment. Eur J Med Chem 122:452–464
Sun I-C, Wang H-K, Kashiwada Y, Shen J-K, Cosentino LM, Chen C-
H, Yang L-M, Lee K-H (1998) Synthesis and biological eva-
luation of heterocyclic ring-fused betulinic acid derivatives as
novel inhibitors of osteoclast differentiation and bone resorption.
J Med Chem 41:4648–4657
Zhang Dong-Mei, Xu Hong-Gui, Wang Lei, Li Ying-Jie, Sun Ping-
Hua, Wu Xiao-Ming, Wang Guang-Ji, Chen Wei-Min, Ye Wen-
Cai (2015) Betulinic Acid and its Derivatives as Potential Anti-
tumor Agents. Med Res Rev 35(6):1127–1155