Rhodamine B conjugates of triterpenoic acids are cytotoxic mitocans even at nanomolar concentrations

Article abstract of DOI:10.1016/j.ejmech.2016.12.040

Triterpenoic acids 1–6 exhibited very low or no cytotoxicity at all, but their corresponding 2,3-di-O-acetyl-piperazinyl amides 13–18 showed low EC₅₀values for several human tumor cell lines. Their cytotoxicity, however, was also high for the non-malignant mouse fibroblasts NIH 3T3. A significant improvement was achieved by preparing the rhodamine B derivatives 19–24. While rhodamine B is not cytotoxic (up to a concentration of 30μM – cut-off of the assay), the triterpenoid piperazine-spacered rhodamine B derivatives were cytotoxic in nano-molar concentration. Compound 24 (a diacetylated maslinic acid derivative) was most toxic for several human tumor cell lines but less toxic for mouse fibroblasts NIH 3T3. Staining and double-staining experiments revealed 24 to act as a mitocan.

Full text of DOI:10.1016/j.ejmech.2016.12.040

European Journal of Medicinal Chemistry 127 (2017) 1e9
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Rhodamine B conjugates of triterpenoic acids are cytotoxic mitocans
even at nanomolar concentrations
*
ꢀ
Sven Sommerwerk, Lucie Heller, Christoph Kerzig, Annemarie E. Kramell, 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:
Triterpenoic acids 1e6 exhibited very low or no cytotoxicity at all, but their corresponding 2,3-di-O-
acetyl-piperazinyl amides 13e18 showed low EC50 values for several human tumor cell lines. Their
cytotoxicity, however, was also high for the non-malignant mouse fibroblasts NIH 3T3. A significant
improvement was achieved by preparing the rhodamine B derivatives 19e24. While rhodamine B is not
Received 23 November 2016
Received in revised form
19 December 2016
Accepted 20 December 2016
Available online 23 December 2016
cytotoxic (up to a concentration of 30mM e cut-off of the assay), the triterpenoid piperazine-spacered
rhodamine B derivatives were cytotoxic in nano-molar concentration. Compound 24 (a diacetylated
maslinic acid derivative) was most toxic for several human tumor cell lines but less toxic for mouse fi-
broblasts NIH 3T3. Staining and double-staining experiments revealed 24 to act as a mitocan.
© 2016 Elsevier Masson SAS. All rights reserved.
Keywords:
Triterpenes
Maslinic acid
Mitocan
Cytotoxicity
Rhodamine B
Betulinic acid
Oleanolic acid
Ursolic acid
Platanic acid
Glycyrrhetinic acid
1. Introduction
led to an increased accumulation of cationic compounds, and as a
result, an increased cytotoxic activity can be expected for these
As early as 1924 Nobel laureate O. H. Warburg linked impaired
mitochondrial functions to the growth of tumor cells [1e4]. This
hypothesis was neglected for many years but experienced a re-
naissance during the last decade, and mitochondria have been
identified as an emerging target for developing anti-cancer agents.
All compounds targeting mitochondria as final target are regarded
as mitocans [5]. Mitocans have a large potential to be developed
into selective and efficient anti-cancer drugs [6e11]. This is
corroborated by recent findings that cells of a tumor might differ
due to mutations within the same tumor [12e14], and mutations
have been observed for the same type of tumor in different patients
[15,16]. Mitocans can be divided into eight different categories [17],
one of them being lipophilic cations acting onto the inner mitro-
chondrial membrane [18]. Previous studies showed an increased
mitochondrial membrane potential for malignant cells compared to
nonmalignant cells [19,20]. Thus, an increased potential difference
compounds as well as a higher selectivity for tumor cells. In 1980
Johnson etal. [21] showed rhodamine-123 to accumulate in mito-
chondria of src-transformed cells, and Lampidis etal. [22] proved
these compounds to exhibit selectivity for cancer cells. In addition,
a fluorinated rhodamine-docetaxel derivative targeted mitochon-
dria and showed good cytotoxicity [23]. Several derivatives of tri-
terpenoic acids (for example, ursolic, oleanolic, betulinic, maslinic
acid) [24e33] have been shown to exhibit high cytotoxicity for
human tumor cell lines, and the presence of an extra amine func-
tion increased cytotoxicity [24,25]. As a consequence of these ob-
servations we planned to synthesize triterpenoic acid derivatives
holding an extra amine substituent (in the form of a piperazine
amide) that is covalently bonded to rhodamine B as the lipophilic
component.
2. Results and discussion
2.1. Chemistry
* Corresponding author.
E-mail address: rene.csuk@chemie.uni-halle.de (R. Csuk).
Ursolic (1, Fig.1), oleanolic (2), glycyrrhetinic (3) and platanic
http://dx.doi.org/10.1016/j.ejmech.2016.12.040
0223-5234/© 2016 Elsevier Masson SAS. All rights reserved.

2
S. Sommerwerk et al. / European Journal of Medicinal Chemistry 127 (2017) 1e9
positions of all absorption bands above
l
¼250nm are quite similar.
The characteristic visible absorption band of the rhodamine B
chromophore in 24, however, was red-shifted by about 15nm.
The fluorescence emission spectrum of 24 exhibited a maximum
at
the excitation wavelength in the range from
Moreover, excitation spectra of 24 were recorded with three
detection wavelengths (
¼600, 610 and 620nm). The observed
l
¼587nm, as shown in the inset of Fig.2, and it is independent of
l
¼250e500nm.
l
wavelength-independence of both excitation and emission spectra
clearly indicates that compound 24 does not contain any fluores-
cent impurities. These promising results prompted us to perform a
determination of the fluorescence quantum yield of 24. A meth-
anolic solution of rhodamine B was chosen as reference system, and
a fluorescence quantum yield of 0.82 has been determined (the
fluorescence spectrum of pure rhodamine B is also shown in Fig.2).
The quantum yield determination was carried out using excitation
wavelengths
l
¼355 and 500nm, respectively. For these measure-
ments very similar quantum yields were obtained with an average
value of 0.57 3%. All these results provided unambiguous addi-
tional evidence (besides NMR and MS) that the fluorescence active
rhodamine B moiety is present in 24.
2.2. Biology
The cytotoxicity of the compounds was determined using the
SRB assay. The results from these assays employing different hu-
man tumor cell lines have been compiled in Table1 using parent
maslinic acid as a reference compound. As a result, compound 24 is
approximately 1000-times more cytotoxic than parent maslinic
acid, and the selectivity FSi (defined as EC50 A2780 tumor cell line
compared to EC50 nonmalignant mouse fibroblasts NIH 3T3) was
increased by factor 50.
Fig. 1. Structures of triterpenoic acids 1e6 and a generalized representation.
acid (4) were bought from different suppliers, betulinic acid (5) was
prepared by oxidation of betulin [34,35], and maslinic acid (6) was
synthesized as previously described [36,37].
Acetylation of the triterpenoic acids gave the acetates 7e12
(Scheme 1). Reaction of 7e12 with oxalyl chloride followed by a
reaction with piperazine furnished amides 13e18. Reaction of
rhodamine B with 13e18 gave violet-colored compounds 19e24
showing strong fluorescence.
No cytotoxicity was measured for rhodamine B (EC50>30mM;
cut-off of the assay). Hence, to the best of our knowledge, com-
pound 24 is the most toxic triterpenoic acid derivative so far; its
cytotoxicity is comparable to that of commercial and well estab-
lished cytotoxic therapeutics, such as doxorubicin or paclitaxel.
At the cellular level, compound 24 probably acts as a mitocan. To
support these assumptions extra investigations were carried out.
The main plot of Fig.2 compares the absorption spectra of 24 and
rhodamine B (Fig.2). In both spectra, the shapes as well as the
Scheme 1. Synthesis of compounds 7e24: a) Ac2O, NEt3, DMF (cat.), DCM, 25 ꢀC, 1 day: yields: 7 (83%), 8 (85%), 9 (81%), 10 (87%), 11 (81%), 12 (78%); b) oxalyl chloride, NEt3, DMF
(cat.), DCM, 25 ꢀC, 5 h, then piperazine, DCM, NEt3, DMAP, 0ꢀ / 25 ꢀC, 30 min: yields: 13 (80%), 14 (85%), 15 (67%), 16 (85%), 17 (79%), 18 (86%); c) rhodamine B, NHS, DCC, DMF,
25 ꢀC, 3 days: yields: 19 (58%), 20 (76%), 21 (64%), 22 (70%), 23 (66%), 24 (70%).

S. Sommerwerk et al. / European Journal of Medicinal Chemistry 127 (2017) 1e9
3
Fig. 2. Structure (right) of 24, and photochemical properties (left) of rhodamine B and 24 in methanol: comparison of the normalized absorption spectra (main plot) and emission
spectra (insert) of 24 and rhodamine B (Rh B).
Table 1
Cytotoxicity of selected compounds (EC50 values in mM from SRB assays after 96 h of treatment; the values are averaged from three independent experiments performed each
in triplicate; confidence interval CI ¼ 95%) [27,38e41]. Human cancer cell lines: 518A2 (melanoma), HT29 (colorectal adenocarcinoma), MCF7 (breast adenocarcinoma), A549
(lung adenocarcinoma), A2780 (ovarian carcinoma), 8505C (thyroid carcinoma); NIH 3T3: nonmalignant mouse fibroblasts.
Compound
518A2
A2780
HT29
MCF7
A549
8505C
NIH 3T3
1
2
3
4
14.7 0.1
>30
>30
11.7 0.6
14.0 2.3
>30
10.6 0.7
>30
>30
12.7 0.1
>30
>30
15.5 1.7
>30
>30
13.5 1.5
>30
>30
18.7 1.6
>30
18.7 1.0
n.d.
>30
>30
>30
>30
>30
>30
5
6
13
14
15
16
17
18
19
20
21
22
23
24
9.4 0.7
13.7 0.9
2.6 0.1
2.7 0.2
9.4 0.2
2.7 0.1
1.5 0.1
n.d.
0.047 0.007
0.043 0.004
0.147 0.016
0.100 0.031
0.046 0.007
0.011 0.000
>30
8.8 0.9
19.5 0.8
2.1 0.1
1.7 0.1
8.2 0.5
3.1 0.1
1.9 0.1
1.9 0.1
0.038 0.002
0.032 0.001
0.083 0.009
0.036 0.001
0.050 0.004
0.007 0.000
>30
14.4 2.3
28.8 0.5
1.9 0.3
1.3 0.1
4.5 0.6
2.4 0.3
1.0 0.0
1.6 0.1
0.083 0.007
0.091 0.010
0.108 0.005
0.099 0.016
0.151 0.022
0.057 0.015
>30
10.2 1.2
>30
17.1 1.1
23.4 0.5
3.2 0.3
2.3 0.3
10.5 0.1
3.9 0.9
2.2 0.1
1.5 0.1
0.140 0.003
0.120 0.006
0.175 0.014
0.131 0.005
0.176 0.022
0.186 0.028
>30
n.d.
16.1 1.4
21.1 0.2
2.1 0.1
1.7 0.1
8.7 0.7
17.0 0.5
3.5 0.5
3.8 0.3
10.0 0.3
3.7 0.2
2.2 0.1
n.d.
0.047 0.019
0.045 0.017
0.167 0.018
0.145 0.011
0.062 0.013
n.d.
2.0 0.1
1.7 0.2
8.4 0.8
2.8 0.1
1.4 0.1
1.0 0.1
0.075 0.005
0.062 0.004
0.086 0.012
0.070 0.002
0.081 0.005
0.028 0.005
>30
0.7 0.1
0.9 0.0
3.2 0.02
0.137 0.009
0.137 0.006
0.139 0.009
0.171 0.006
0.208 0.034
0.334 0.061
>30
rhodamine B
>30
Firstly, A2780 tumor cells were treated with compound 24 in the
presence of fluorescence dyes Hoechst 33,342 and rhodamine 123
(Fig.3). Excitation- and emission spectra of these compounds differ
significantly to accomplish a distinction which areas within the
cells show an accumulation of the dyes. Fluorescence dye Hoechst
33,342 binds selectively to DNA; this dye shows a maximum exci-
Cell death of A2780cells upon treatment with 24 didn't occur via
apoptosis (concentration of 0.1 M). The typical morphological
m
hallmarks of apoptosis could not be detected by microscopy. To
support these findings, A2780cells were treated with 24 followed
by dying with annexin V/propidium iodide and quantitative FACS
measurements. The results show (cf. supplementary material) that
the amount of annexin V positive cells has not been altered upon
treatment with compound 24 (as compared to control). Apoptosis
was not induced in the cells even upon increasing the concentra-
tation wavelength of
¼461nm. In contrast, dye rhodamine 123 is usually used for dying
of mitochondria; this dye shows an absorption at
¼505nm, and a
maximum of emission at
¼560nm. The results of these in-
l
¼350nm and emits at a maximum at
l
l
l
tion of 24 to a concentration of 0.4mM; as a result of an increased
vestigations are depicted in Fig.3. As a result, compound 24 is
localized within the cells exactly in the same area as rhodamine
123. In addition, a localization of 24 in the nucleus can be ruled out
by comparison with the staining experiment using Hoechst 33,342.
concentration of 24, the population of the cells is shifted towards PI
positive cells. This reflects also a limited applicability of this assay
for compounds of significant fluorescence. PI shows a maximum of
emission at
l¼590nm (not intercalated into DNA), and its spectrum

4
S. Sommerwerk et al. / European Journal of Medicinal Chemistry 127 (2017) 1e9
Fig. 3. Fluorescence microscopic investigation of A2780 ovarian carcinoma cells. The cells were incubated with compound 24 for 24 h (concentration of 0.1
mM), and after double-
staining using rhodamine 123 and Hoechst 33,342 the cells were inspected using fluorescence and light microscope (scale bar ¼ 20
mm). The overlay indicates a mitochondrial
localization of compound 24.
is superimposed by the emission of compound 24 (
l¼561nm).
fluorescence/absorption were smaller than 0.1. All emission spectra
were corrected using the spectrometer's sensitivity function. 2-
Amino-pyridine and N,N-dimethylamino-m-nitrobenzene were
used for the calibration of the instrument [42].
3. Conclusions
While triterpenoic acids 1e6 exhibit very low or no cytotoxicity
at all, their corresponding 2,3-di-O-acetyl-piperazinyl amides
13e18 showed low EC50 values for several human tumor cell lines
but their cytotoxicity was significant for the non-malignant mouse
fibroblasts NIH 3T3, too. A significant improvement, however, was
achieved by preparing the rhodamine B derivatives 19e24. While
4.4. SRB assay, and annexin V/propidium iodide assay
These assays were carried out as previously described (see
supplementary material).
rhodamine B is not cytotoxic (up to a concentration of 30mM e cut-
4.5. Double-staining using Hoechst 33,342 and rhodamine 123
off of the assay), the triterpenoid piperazine-spacered rhodamine B
derivatives were cytotoxic in nano-molar concentration, with
compound 24 (a diacetylated maslinic acid derivative) being most
toxic at all. This compound was significantly less toxic for the
mouse fibroblasts. Staining and double-staining experiments
revealed 24 to act as a mitocan.
The morphological changes of the cells were analyzed using
fluorescence microscopy employing the human cancer cell line
A2780. Approx. 800,000cells were seeded in cell culture flasks
(25cm2), and the cells were allowed to grow up for 24h. After
removing of the used medium, the substance loaded fresh medium
(concentration of 0.1mM) was reloaded (or a blank new medium as a
4. Experimental part
control). After 24h, the content of the flask was collected and
centrifuged (1200rpm, 4ꢀC), the pellet was gently suspended in
phosphate-buffered saline (PBS (w/w), 1mL) and centrifuged again.
The PBS was removed, and the pellet gently re-suspended in PBS
4.1. General - chemistry
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; typical
(50
tion of Hoechst 33,342/rhodamine 123 (10
were incubated for 30minat 37ꢀC in the dark and washed with PBS
L). The analysis of the cells was performed by fluo-
rescence microscope after re-suspending the pellet in the super-
natant. The Hoechst 33,342 dye was used for live-cell fluorescent
staining of DNA, and mitochondria were stained by rhodamine 123.
m
L). After having mixed the cell suspension (10
m
L) with a solu-
mg/mL, 20
mL) the cells
experiments: H-H-COSY, HMBC, HSQC, NOESY, DQF-COSY), MS
spectra were taken on a Finnigan MAT LCQ 7000 (electrospray,
voltage 4.1kV, sheath gas nitrogen) instrument. The optical rota-
tions were measured on a Perkin-Elmer polarimeter at 20ꢀC; TLC
was performed on silica gel (Merck 5554, detection with cer-
iummolybdate reagent); elemental analyses were performed on a
Vario EL (CHNS). IR spectra were recorded on a Perkin Elmer FT-IR
spectrometer Spectrum 1000. Fluorescence microscopic images
were recorded on an Axioskop 20 with an AxioCam MR3 (Carl Zeiss
AG). The solvents were dried according to usual procedures. The
purity of the compounds was determined by HPLC and found to be
>98%.
(w/w, 2ꢁ500
m
4.6. Syntheses
4.6.1. General procedure A for the acetylation of triterpenoic acids
7e12
A solution of the triterpenoic acid 1e6 (2.56mmol) in dry DCM
(100mL) was treated with acetic anhydride (1.045g, 10.24mmol),
triethylamine (1.167g, 11.53mmol) and DMAP (31mg, 0.25mmol).
The mixture was stirred at room temperature for one day, diluted
with Et2O (500mL), and the mixture was subsequently washed
with aqueous HCl (0.1M, 1ꢁ500mL), water (2ꢁ500mL) and brine
(1ꢁ250mL), dried (MgSO4), and the filtrate was concentrated under
diminished pressure. The residue was subjected to column chro-
matography (silica gel, hexane/ethyl acetate, mixtures) to provide
acetates 7e12 each as a colorless solid. Their spectroscopic data
were in full agreement with data from the literature.
4.2. Absorption spectroscopy
The spectra were taken in quartz cuvettes (thickness 1cm) using
a Shimadzu UV-1800 spectrometer; the baseline was measured
using methanol (Aldrich, >99.9%).
4.3. Fluorescence spectrometry
The spectra were measured using a Perkin Elmer LS 50B in-
strument. All solutions were diluted (to avoid filter effects), and
absorptions at the excitation wavelength and in the overlap area
4.6.2. General procedure B for the synthesis of the piperazine
amides 13e18
Compounds 7e12 (0.90mmol) were each dissolved in dry DCM

S. Sommerwerk et al. / European Journal of Medicinal Chemistry 127 (2017) 1e9
5
(30mL), oxalyl chloride (570mg, 4.5mmol), triethylamine (20mg,
0.2mmol) and DMF (15mg, 0.2mmol) were added. The mixture was
stirred at room temperature for 5h. The solvent was removed under
reduced pressure, re-evaporated with dry THF (1ꢁ10mL), and the
residue was immediately dissolved in dry DCM (30mL). The solu-
tion was cooled to 0ꢀC, and triethylamine (100mg, 0.99mmol),
DMAP (12mg, 0.1mmol) as well as piperazine (233mg, 2.70mmol)
were added. After 30min of stirring, the solvent was removed, and
the residue was re-dissolved in MeOH (5mL) and precipitated in
water (20mL). Filtration and drying (CaCl2, 24h) afforded com-
pounds 13e18 each as a colorless solid.
(Ce14), 39.6 (Ce19), 39.6 (Ce8), 38.9 (Ce20), 38.4 (Ce1), 37.8
(Ce4), 37.1 (Ce10), 34.5 (Ce22), 33.1 (Ce7), 30.6 (Ce21), 28.3
(Ce15), 28.2 (Ce24), 23.7 (Ce16), 23.7 (Ce11), 23.7 (Ce27), 23.5
(Ce2), 21.4 (Ce30), 21.4 (Ce32), 18.3 (Ce6), 17.6 (Ce29), 17.0
(Ce26), 16.9 (Ce23), 15.6 (Ce25) ppm; MS (ESI): m/z (%)¼567.4
([MþH]þ, 57); analysis calculated for C36H58N2O3 (566.86): C 76.28,
H 10.31, N 4.94; found: C 76.01, H 10.53, N 4.79.
4.6.11. 3b-Acetyloxy-olean-12-en-28-oyl piperazine (14) [48,49]
General procedure B; from 8 (513mg, 1.00mmol), yield: 482mg,
85%; m.p. 170e176ꢀC; RF¼0.46 (silica gel, chloroform/methanol,
9:1); IR (KBr):
n
¼3446m, 2946vs, 2876s, 2490w, 1734s, 1636s, 1464s,
4.6.3. General procedure C for the synthesis of the rhodamine B
derivatives 19e24
1384vs, 1248vs, 1144m, 1026s, 1006scmꢂ1
CDCl3):
;
1H NMR (500MHz,
d
¼5.25 (dd, J¼3.4, 3.4Hz, 1H, He12), 4.51e4.45 (m, 1H,
A mixture of rhodamine B (170mg, 0.35mmol), NHS (44mg,
0.38mmol) and DCC (78mg, 0.38mmol) in DMF (5mL) was stirred at
ambient temperature for 20h. The precipitated dicyclohexylurea
was removed by filtration and compounds 13e18 (0.32mmol, dis-
solved in dry DMF (3mL)) were added to the filtrate. After stirring
for two days at room temperature, the solvent was removed under
reduced pressure, and the resulting solid was subjected to column
chromatography (silica gel, chloroform/methanol, 9:1) to yield
compounds 19e24 each as a purple solid.
He3), 3.85e3.72 (m, 4H, He33), 2.88e2.75 (m, 5H, He34þHe18),
2.18e2.09 (m, 1H, He16a), 2.03 (s, 3H, He32), 1.96e1.81 (m, 2H,
He11),
1.75e1.48
(m,
10H,
He6aþHe16bþHe2þHe15aþHe7þHe1aþHe19aþHe9),
1.48e1.30 (m, 3H, He6bþHe22aþHe21a), 1.29e1.23 (m, 2H,
He21bþHe1b), 1.13 (s, 3H, He27), 1.11e0.99 (m, 2H,
He15bþHe1b), 0.92 (s, 6H, He25þHe29), 0.90 (s, 3H, He30), 0.85
(s, 3H, He24), 0.84 (s, 3H, He23), 0.84e0.81 (m, 1H, He5), 0.71 (s,
3H, He26) ppm; 13C NMR (126MHz, CDCl3):
d¼175.5 (Ce28), 171.1
(Ce31), 144.6 (Ce13), 121.8 (Ce12), 81.1 (Ce3), 55.5 (Ce5), 47.8
(Ce9), 47.7 (Ce17), 46.5 (Ce19), 45.1 (Ce34), 44.5 (Ce33), 43.7
(Ce18), 42.0 (Ce14), 39.3 (Ce8), 38.2 (Ce1), 37.8 (Ce4), 37.1 (Ce10),
34.1 (Ce21), 33.1 (Ce30), 32.9 (Ce22), 30.5 (Ce20), 30.1 (Ce7), 28.2
(Ce24), 28.1 (Ce15), 26.1 (Ce27), 24.2 (Ce29), 23.7 (Ce2), 23.5
(Ce11), 23.0 (Ce16), 21.4 (Ce32), 18.3 (Ce6), 17.0 (Ce26), 16.8
(Ce23), 15.5 (Ce25) ppm; MS (ESI): m/z (%)¼567.4 ([MþH]þ, 47);
analysis calculated for C36H58N2O3 (566.86): C 76.28, H 10.31, N
4.94; found: C 76.13, H 10.49, N 4.69.
4.6.4. 3b-Acetyloxy-ursan-12-en-28-oic acid (7)
General procedure A; yield: 83%; m.p. 292e295ꢀC (lit.:
293e294ꢀC [43]).
4.6.5. 3b-Acetyloxy-olean-12-en-28-oic acid (8)
General procedure A; yield: 85%; m.p. 264e265ꢀC (lit.:
264e265ꢀC [44]).
4.6.6. 3b-Acetyloxy-11-oxo-olean-12-en-29-oic acid (9)
General procedure A; yield: 81%; m.p. 309e312ꢀC (lit.:
310e313ꢀC [45]).
4.6.12. 3b-Acetyloxy-11-oxo-olean-12-en-29-oyl piperazine (15)
General procedure B; from 9 (513mg, 1.00mmol); yield: 389mg,
67%; m.p. 160ꢀC (decomp.); RF¼0.30 (silica gel, chloroform/meth-
anol, 9:1); UV-Vis (chloroform): lmax (logε)¼249 (4.42) nm; IR
4.6.7. 3b-Acetyloxy-20-oxo-30-norlupan-28-oic acid (10)
General procedure A; yield: 87%; m.p. 251e254ꢀC (lit.:
252e255ꢀC [46]).
(KBr):
n
¼3442m, 2968s, 2950s, 2874m, 1730s, 1654s, 1464m, 1384vs,
1248s, 1140m, 1028mcmꢂ1
;
1H NMR (500MHz, CDCl3):
d
¼5.67 (s,
1H, He12), 4.50 (dd, J¼11.7, 4.7Hz, 1H, He3), 3.81e3.71 (m, 4H,
He33), 3.08e3.03 (m, 4H, He34), 2.76 (ddd, J¼13.2, 3.2, 3.2Hz, 1H,
He1a), 2.35 (s, 1H, He9), 2.28 (dd, J¼13.0, 2.5Hz, 1H, He18),
2.08e2.20 (m, 2H, He16aþHe19a), 2.03 (s, 3H, He32), 1.96 (ddd,
J¼13.4, 3.1, 3.1Hz, 1H, He21a), 1.82 (ddd, J¼13.4, 13.4, 4.1Hz, 1H,
He15a), 1.75e1.53 (m, 5H, He6aþHe2þHe7aþHe21b), 1.50e1.32
(m, 5H, He6bþHe7bþHe19bþHe22), 1.34 (s, 3H, He27),
1.23e0.97 (m, 3H, He15bþHe16bþHe1b), 1.21 (s, 3H, He29), 1.14
(s, 3H, He25), 1.10 (s, 3H, He26), 0.86 (s, 6H, He23þHe24), 0.80 (s,
3H, He28), 0.79e0.76 (m, 1H, He5) ppm; 13C NMR (126MHz,
4.6.8. 3b-Acetyloxy-lup-20(29)en-28-oic acid (11)
General procedure A; yield: 81%; m.p. 280e283ꢀC (lit.:
287e289ꢀC [47]).
4.6.9. 2a, 3b-Bis(acetyloxy)-olean-12-en-28-oic acid (12)
General procedure A; yield: 78%; m.p. 280e283ꢀC (lit.:
287e289ꢀC [33]).
4.6.10. 3b-Acetyloxy-urs-12-en-28-oyl piperazine (13) [48,49]
General procedure B; from 7 (513mg, 1.00mmol), yield: 453mg,
CDCl3):
d
¼200.2 (Ce11), 174.2 (Ce30), 171.1 (Ce31), 169.8 (Ce13),
80%; m.p. 158e161ꢀC; RF¼0.44 (silica gel, chloroform/methanol,
128.7 (Ce12), 80.7 (Ce3), 61.8 (Ce9), 55.2 (Ce5), 48.3 (Ce18), 45.5
(Ce8), 45.4 (Ce34), 44.7 (Ce33), 44.0 (Ce20), 44.0 (Ce21), 43.5
(Ce14), 39.0 (Ce1), 38.2 (Ce4), 37.9 (Ce22), 37.1 (Ce10), 33.2
(Ce19), 32.9 (Ce7), 31.9 (Ce17), 28.6 (Ce28), 28.2 (Ce24), 27.1
(Ce29), 26.9 (Ce16), 26.5 (Ce15), 23.7 (Ce2), 23.2 (Ce27), 21.4
(Ce32), 18.9 (Ce26), 17.5 (Ce6), 16.8 (Ce23), 16.6 (Ce25) ppm; MS
(ESI): m/z (%)¼581.4 ([MþH]þ, 42); analysis calculated for
9:1); IR (KBr):
n
¼3442m, 2968s, 2946s, 2928s, 2872m, 1734s,
1636m, 1456m, 1384vs, 1248s, 1138w, 1026mcmꢂ1
(500MHz, CDCl3):
;
1H NMR
d¼5.21 (dd, J¼3.5, 3.5Hz, 1H, He12), 4.51e4.46
(m, 1H, He3), 3.84e3.70 (m, 4H, He33), 3.11e3.01 (m, 4H, He34),
2.34 (d, J¼9.8Hz, 1H, He18), 2.21e2.13 (m, 1H, He16a), 2.04 (s, 3H,
He32),
1.95e1.24
(m,
18H,
He2aþHe22aþHe2bþHe16bþHe15aþHe22bþHe11aþHe11b-
þHe1aþHe21aþHe21bþHe9þHe19þHe7aþHe7bþHe19þH-
e6aþHe6b), 1.12e0.79 (m, 4H, He15bþHe1bþHe20þHe5), 1.06
(s, 3H, He27), 0.95 (d, J¼6.3Hz, 3H, He30), 0.93 (s, 3H, He25), 0.88
(d, J¼6.4Hz, 3H, He29), 0.86 (s, 3H, He24), 0.85 (s, 3H, He23), 0.73
C36H56N2O4 (580.84): C 74.44, H 9.72, N 4.82; found: C 74.22, H
9.95, N 4.59.
4.6.13. 3b-Acetyloxy-20-oxo-30-norlupan-28-oyl piperazine (16)
General procedure B; from 10 (513mg,1.00mmol); yield: 481mg,
(s, 3H, He26) ppm; 13C NMR (126MHz, CDCl3):
171.1 (Ce31), 138.7 (Ce13), 125.5 (Ce12), 81.1 (Ce3), 55.5 (Ce5),
55.2 (Ce18), 48.8 (Ce17), 47.7 (Ce9), 45.0 (Ce34), 45.0 (Ce33), 42.3
d
¼175.7 (Ce28),
85%; m.p. 115e125ꢀC; RF¼0.33 (silica gel, chloroform/methanol,
9:1); IR (KBr):
n
¼3440m, 2944vs, 2868s, 2742w, 1734s, 1710s, 1632s,
1452m, 1414s, 1370s, 1246vs, 1192s, 1142m, 1030scmꢂ1 1H NMR
;

6
S. Sommerwerk et al. / European Journal of Medicinal Chemistry 127 (2017) 1e9
(500MHz, CDCl3):
d
¼4.47e4.42 (m, 1H, He3), 3.60e3.50 (m, 4H,
(Ce18), 42.0 (Ce14), 39.5 (Ce4), 39.3 (Ce8), 38.4 (Ce10), 34.2
(Ce21), 33.2 (Ce30), 32.8 (Ce22), 30.5 (Ce20), 30.0 (Ce7), 28.6
(Ce24), 28.0 (Ce15), 26.1 (Ce27), 24.2 (Ce29), 23.6 (Ce11), 22.8
(Ce16), 21.3 (Ce32), 21.0 (Ce34), 18.4 (Ce6), 17.8 (Ce23), 17.0
(Ce26), 16.6 (C25) ppm; MS (ESI): m/z (%)¼625.4 ([MþH]þ, 100);
analysis calculated for C38H60N2O5 (624.89): C 73.04, H 9.68, N 4.48;
found: C 72.85, H 9.90, N 4.31.
He32), 3.26e3.20 (m, 1H, He19), 2.86e2.76 (m, 4H, He33), 2.69
(ddd, J¼12.6, 12.6, 3.8Hz, 1H, He13), 2.14 (s, 3H, He29), 2.13e2.06
(m, 2H, He16aþHe18), 2.02 (s, 3H, He31), 2.00e1.94 (m, 1H,
He22a), 1.94e1.86 (m, 1H, He21a), 1.69e1.20 (m, 16H,
He6þHe11þHe2þHe12aþHe15þHe21bþHe16bþHe7þHe22-
bþHe1aþHe9), 1.19e1.11 (m, 1H, He15b), 1.07e0.89 (m, 2H,
He12bþHe1b), 0.96 (s, 3H, He27), 0.91 (s, 3H, He26), 0.83 (s, 3H,
He25), 0.82 (s, 3H, He24), 0.81 (s, 3H, He23), 0.79e0.75 (m, 1H,
4.6.16. 9-[2-[[4-(3b-Acetyloxy-ursan-12-en-28-oyl)-piperazinyl]
He5) ppm; 13C NMR (126MHz, CDCl3):
d¼213.2 (Ce20),173.5
carbonyl]phenyl]- 3,6-bis(diethylamino]-xanthylium chloride (19)
General procedure C: from 13 (181mg, 0.32mmol); yield:
190mg, 58%; m.p. 243e245ꢀC; RF¼0.39 (silica gel, chloroform/
methanol, 9:1); UV-Vis (methanol): lmax (logε)¼257 (4.10), 525
(Ce28), 171.0 (Ce30), 81.1 (Ce3), 55.6 (Ce5), 54.6 (Ce17), 52.8
(Ce18), 50.8 (Ce9), 50.2 (Ce19), 46.5 (Ce32), 46.5 (Ce33), 41.9
(Ce14), 40.7 (Ce8), 38.5 (Ce1), 37.9 (Ce4), 37.3 (Ce10), 36.1 (Ce13),
35.8 (Ce22), 34.4 (Ce7), 32.2 (Ce16), 30.5 (Ce29), 30.0 (Ce15), 28.9
(Ce21), 28.1 (Ce24), 27.6 (Ce12), 23.8 (Ce2), 21.4 (Ce31), 21.3
(Ce11), 18.3 (Ce6), 16.6 (Ce23), 16.4 (Ce25), 16.2 (Ce26), 14.8
(Ce27) ppm; MS (ESI): m/z (%)¼569.3 ([MþH]þ, 32); analysis
calculated for C35H56N2O4 (568.83): C 73.90, H 9.92, N 4.92; found:
C 73.77, H 10.12, N 4.78.
(3.95), 561 (4.42) nm; IR (KBr):
n
¼3448vs, 1636s, 1590s, 1412s,
1384vs, 1338s, 1248s, 1182scmꢂ1
;
1H NMR (500MHz, CDCl3):
d¼7.69e7.63 (m, 2H, He38þHe39), 7.59e7.54 (m, 1H, He37),
7.34e7.26 (m, 3H, He40þHe45’þHe45), 7.08e6.97 (m, 2H,
He44’þHe44), 6.78e6.74 (m, 2H, He47’þHe47), 5.18e5.12 (m, 1H,
He12), 4.49e4.43 (m, 1H, He3), 3.68e3.53 (m, 8H, He49þHe49’),
3.52e3.10 (m, 8H, He33þHe34), 2.39e2.26 (m, 1H, He18),
2.14e2.04 (m, 1H, He16a), 2.02 (s, 3H, He32), 1.90e1.20 (m, 26H,
He2aþHe22aþHe2bþHe16bþHe15aþHe22bþHe11aþHe11b-
þHe1aþHe21aþHe21bþHe9þHe19þHe7aþHe7bþHe6aþH-
4.6.14. 3b-Acetyloxy-lup-20(29)en-28-oyl piperazine (17)
General procedure B; from 11 (500mg, 1.00mmol); yield:
449mg, 79%; m.p. 177e181ꢀC; RF¼0.41 (silica gel, chloroform/
methanol, 9:1); IR (KBr):
n
¼3348s, 3070m, 2944vs, 2868s, 2488w,
e6bþHe19þHe50þHe50’),
1.08e0.76
(m,
4H,
1734vs, 1636vs, 1454s, 1384vs, 1248vs, 1192s, 1128m, 1030s,
He15bþHe1bþHe20þHe5), 1.01 (s, 3H, He27), 0.91e0.87 (m, 6H,
980scmꢂ1 1H NMR (500MHz, CDCl3):
;
d
¼4.71 (s, 1H, He29a), 4.58
He25þHe30), 0.85e0.81 (m, 9H, He29þHe24þHe23), 0.64 (s, 3H,
(s, 1H, He29b), 4.48e4.43 (m, 1H, He3), 3.76e3.70 (m, 4H, He33),
3.08e2.99 (m, 4H, He34), 2.97e2.91 (m, 1H, He19), 2.82 (ddd,
J¼13.1, 13.1, 3.6Hz,1H, He13), 2.07e2.01 (m,1H, He16a), 2.03 (s, 3H,
He32), 1.94e1.98 (m, 1H, He22a), 1.87e1.78 (m, 1H, He21a),
He26) ppm; 13C NMR (126MHz, CDCl3):
d
¼176.4 (Ce28), 171.1
(Ce31), 168.0 (Ce35), 157.8 (Ce48), 157.8 (Ce480), 155.9 (Ce42),
155.8 (Ce46), 155.6 (Ce460), 138.5 (Ce13), 135.1 (Ce41), 132.3
(Ce45), 132.2 (Ce45’), 130.5 (Ce36), 130.4 (Ce40), 130.4 (Ce38),
130.2 (Ce39), 128.0 (Ce37), 125.3 (Ce12), 114.7 (Ce44), 114.5
(Ce440), 113.8 (Ce43), 113.7 (Ce430), 96.4 (Ce47), 96.4 (Ce470), 81.1
(Ce3), 55.4 (Ce5), 55.1 (Ce18), 48.8 (Ce17), 47.6 (Ce9), 46.2
(Ce49), 46.2 (Ce490), 45.0 (Ce34), 45.0 (Ce33), 42.2 (Ce14), 39.5
(Ce19), 39.5 (Ce8), 38.7 (Ce20), 38.3 (Ce1), 37.8 (Ce4), 37.0
(Ce10), 34.2 (Ce22), 33.0 (Ce7), 30.5 (Ce21), 28.2 (Ce24), 28.1
(Ce15), 23.7 (Ce16), 23.7 (Ce11), 23.7 (Ce27), 23.5 (Ce2), 21.4
(Ce30), 21.3 (Ce32), 18.3 (Ce6), 17.6 (Ce29), 17.0 (Ce26), 16.9
(Ce23), 15.6 (Ce25), 12.8 (Ce50), 12.8 (Ce500) ppm; MS (ESI): m/z
(%)¼991.7 ([MꢂCl]þ, 100); analysis calculated for C64H87ClN4O5
(1027.85): C 74.79, H 8.53, N 5.45; found: C 74.57, H 8.73, N 5.30.
1.73e1.19
(m,
17H,
He6þHe11þHe2þHe12aþHe15þHe21bþHe16bþHe7þHe22-
bþHe1aþHe9þHe18), 1.67 (s, 3H, He30), 1.19e1.13 (m, 1H,
He15b), 1.10e0.90 (m, 2H, He12bþHe1b), 0.95 (s, 3H, He27), 0.92
(s, 3H, He26), 0.84 (s, 3H, He25), 0.83 (s, 3H, He24), 0.82 (s, 3H,
He23), 0.80e0.75 (m, 1H, He5) ppm; 13C NMR (126MHz, CDCl3):
d¼173.9 (Ce28), 171.1 (Ce31), 151.2 (Ce20), 109.5 (Ce29), 81.1
(Ce3), 55.7 (Ce5), 54.7 (Ce17), 52.8 (Ce18), 50.9 (Ce9), 45.8
(Ce19), 45.4 (Ce33), 45.4 (Ce34), 42.0 (Ce14), 40.9 (Ce8), 38.6
(Ce1), 38.0 (Ce4), 37.3 (Ce10), 37.1 (Ce13), 36.0 (Ce22), 34.5 (Ce7),
32.6 (Ce16), 31.4 (Ce21), 30.0 (Ce15), 28.1 (Ce24), 25.7 (Ce12),
23.9 (Ce2), 21.5 (Ce32), 21.3 (Ce11), 19.8 (Ce30), 18.4 (Ce6), 16.6
(Ce23), 16.4 (Ce25), 16.3 (Ce26), 14.8 (Ce27) ppm; MS (ESI): m/z
(%)¼567.3 ([MþH]þ, 36); analysis calculated for C36H58N2O3
(566.86): C 76.28, H 10.31, N 4.94; found: C 76.13, H 10.52, N 5.09.
4.6.17. 9-[2-[[4-(3b-Acetyloxy-olean-12-en-28-oyl)-1-piperazinyl]
carbonyl] phenyl]-3,6-bis(diethylamino]-xanthylium chloride (20)
General procedure C: from 14 (181mg, 0.32mmol); yield:
250mg, 76%; m.p. 245e248C; RF¼0.39 (silica gel, chloroform/
methanol, 9:1); UV-Vis (methanol): lmax (logε)¼257 (4.70), 524
4.6.15. 2a, 3b-Bis(acetyloxy)-olean-12-en-28-oyl piperazine (18)
General procedure B; from 12 (500mg, 0.90mmol); yield:
(4.69), 561 (5.19) nm; IR (KBr):
n
¼3448s, 2942m, 1630m, 1590s,
483mg, 86%; m.p. 157e160ꢀC; RF¼0.36 (silica gel, chloroform/
1468m, 1414m, 1384vs, 1338m, 1274m, 1248m, 1182m, 1134m,
methanol, 9:1); IR (KBr):
n
¼3446m, 2946s, 2864m, 1744vs, 1632s,
1004mcmꢂ1 1H NMR (500MHz, CDCl3):
;
d
¼7.69e7.63 (m, 2H,
1458m, 1438m, 1396m, 1368s, 1252vs, 1232s, 1186m, 1044scmꢂ1; 1H
NMR (400MHz, CDCl3):
He38þHe39), 7.55e7.52 (m, 1H, He37), 7.35e7.26 (m, 3H,
He40þHe45’þHe45), 7.11e7.03 (m, 1H, He440), 7.00e6.94 (m, 1H,
He44), 6.81e6.74 (m, 2H, He470þHe47), 5.20 (dd, J¼3.5, 3.5Hz, 1H,
He12), 4.50e4.44 (m, 1H, He3), 3.65e3.58 (m, 8H, He49þHe490),
3.58e3.46 (m, 2H, He33a), 3.41e3.20 (m, 6H, He33bþHe34),
3.01e2.95 (m, 1H, He18), 2.11e1.98 (m, 1H, He16a), 2.02 (s, 3H,
He32), 1.92e1.75 (m, 2H, He11), 1.69e1.24 (m, 23H,
He6þHe16bþHe2þHe15aþHe7þHe22aþHe21aþHe1aþH-
d
¼5.24 (dd, J¼3.4, 3.4Hz, 1H, He12), 5.09
(ddd, J¼11.3, 11.3, 4.6Hz, 1H, He2), 4.73 (d, J¼10.3Hz, 1H, He3),
3.67e3.52 (m, 4H, He35), 3.07 (dd, J¼13.7, 3.7Hz, 1H, He18),
2.88e2.75
(m,
4H,
He36),
2.16e1.90
(m,
3H,
He16aþHe1aþHe11a), 2.04 (s, 3H, He34), 1.96 (s, 3H, He32),
1.88e1.77
(m,
1H,
He11b),
1.76e1.56
1.56e1.23
(m,
(m,
6H,
5H,
He16bþHe15aþHe7þHe9þHe19a),
He6þHe22þHe21a), 1.21e1.10 (m, 2H, He21bþHe19b), 1.12 (s,
3H, He27), 1.10e1.00 (m, 2H, He15bþHe1b), 1.04 (s, 3H, He25),
0.98e0.93 (m, 1H, He5), 0.92 (s, 3H, He29), 0.89 (s, 3H, He23), 0.88
(s, 6H, He24þHe30), 0.73 (s, 3H, He26) ppm; 13C NMR (100MHz,
e19aþHe9þHe50þHe500),
1.23e0.95
(m,
5H,
He15bþHe22bþHe21bþHe1bþHe19b), 1.08 (s, 3H, He27), 0.88
(s, 3H, He25), 0.86 (s, 3H, He29), 0.85 (s, 6H, He30þHe24), 0.83 (s,
3H, He23), 0.82e0.79 (m, 1H, He5), 0.64 (s, 3H, He26) ppm; 13C
CDCl3):
d
¼175.1 (Ce28), 171.0 (Ce33), 170.6 (Ce31), 145.1 (Ce13),
NMR (126MHz, CDCl3):
d
¼175.9 (Ce28), 171.1 (Ce31), 167.9 (Ce35),
121.2 (Ce12), 80.8 (Ce3), 70.2 (Ce2), 55.1 (Ce5), 47.9 (Ce9), 47.5
(Ce17), 46.9 (Ce35), 46.6 (Ce19), 46.4 (Ce36), 44.0 (Ce1), 43.7
157.9 (Ce48), 157.8 (Ce480), 155.9 (Ce42), 155.9 (Ce46), 155.7
(Ce460), 144.9 (Ce13), 135.2 (Ce41), 132.4 (Ce45), 132.4 (Ce450),

S. Sommerwerk et al. / European Journal of Medicinal Chemistry 127 (2017) 1e9
7
130.7 (Ce36), 130.4 (Ce40), 130.4 (Ce38), 130.2 (Ce39), 127.8
(Ce37), 121.5 (Ce12), 114.7 (Ce44), 114.3 (Ce440), 113.9 (Ce43),
113.9 (Ce430), 96.5 (Ce47), 96.4 (Ce470), 81.0 (Ce3), 55.5 (Ce5),
47.8 (Ce34), 47.8 (Ce9), 47.6 (Ce17), 46.4 (Ce19), 46.2 (Ce49), 46.2
(Ce490), 43.7 (Ce18), 42.2 (Ce33), 42.0 (Ce14), 39.2 (Ce8), 38.2
(Ce1), 37.8 (Ce4), 37.1 (Ce10), 34.0 (Ce21), 33.1 (Ce30), 32.9
(Ce22), 30.4 (Ce20), 29.9 (Ce7), 28.2 (Ce24), 27.9 (Ce15), 25.9
(Ce27), 24.1 (Ce29), 23.6 (Ce2), 23.5 (Ce11), 22.4 (Ce16), 21.4
(Ce32), 18.3 (Ce6), 17.0 (Ce26), 16.8 (Ce23), 15.5 (Ce25), 12.8
(Ce50), 12.8 (Ce500) ppm; MS (ESI): m/z (%)¼991.8 ([MꢂCl]þ, 100);
analysis calculated for C64H87ClN4O5 (1027.85): C 74.79, H 8.53, N
5.45; found: C 74.52, H 8.74, N 5.29.
He6þHe11þHe2þHe12aþHe15þHe21bþHe16bþHe7þHe22-
bþHe1aþHe9þHe49þHe490), 1.03e0.97 (m, 1H, He15b),
0.97e0.89 (m, 2H, He12bþHe1b), 0.91 (s, 3H, He27), 0.83 (s, 3H,
He26), 0.81 (s, 3H, He24), 0.80 (s, 6H, He25þHe23), 0.77e0.72 (m,
1H, He5) ppm; 13C NMR (126MHz, CDCl3):
d¼213.1 (Ce20), 173.3
(Ce28), 171.1 (Ce30), 168.2 (Ce34), 157.8 (Ce47), 157.8 (Ce470),
155.9 (Ce41), 155.8 (Ce45), 155.8 (Ce450), 134.9 (Ce40), 132.4
(Ce44), 132.4 (Ce440), 130.6 (Ce35), 130.4 (Ce39), 130.3 (Ce37),
130.3 (Ce38), 128.0 (Ce36), 114.6 (Ce43), 114.5 (Ce430), 113.8
(Ce42), 113.8 (Ce420), 96.4 (Ce46), 96.4 (Ce460),81.0 (Ce3), 55.6
(Ce5), 54.6 (Ce17), 52.6 (Ce18), 50.7 (Ce9), 50.2 (Ce19), 47.8
(Ce33), 46.2 (Ce48), 46.2 (Ce480), 42.3 (Ce32), 41.8 (Ce14), 40.7
(Ce8), 38.5 (Ce1), 37.9 (Ce4), 37.2 (Ce10), 36.1 (Ce13), 35.7 (Ce22),
34.3 (Ce7), 32.0 (Ce16), 30.1 (Ce29), 29.9 (Ce15), 28.8 (Ce21), 28.1
(Ce24), 27.5 (Ce12), 23.8 (Ce2), 21.4 (Ce31), 21.2 (Ce11), 18.3
(Ce6), 16.6 (Ce23), 16.3 (Ce25), 16.1 (Ce26), 14.7 (Ce27), 12.8
(Ce50), 12.8 (Ce500) ppm; MS (ESI): m/z (%)¼993.8 ([MꢂCl]þ, 100);
analysis calculated for C63H85ClN4O6 (1029.83): C 73.48, H 8.32, N
5.44; found: C 73.20, H 8.41, N 5.23.
4.6.18. 9-[2-[[4-(3b-Acetyloxy-11-oxo-olean-12-en-29-oyl)-
piperazinyl]carbonyl]phenyl]-3,6-bis(diethylamino]-xanthylium
chloride (21)
General procedure C: from 15 (186mg, 0.32mmol); yield:
214mg, 64%; m.p. 237e240ꢀC; RF¼0.32 (silica gel, chloroform/
methanol, 9:1); UV-Vis (methanol): lmax (logε)¼257 (4.68), 524
(4.58), 561 (5.08) nm; IR (KBr):
n
¼3448vs, 2972m, 1636m, 1590m,
1H NMR
¼7.70e7.63 (m, 2H, He38þHe39), 7.55e7.51
1414m, 1384s, 1338m, 1248m, 1182m, 1074mcmꢂ1
(500MHz, CDCl3):
;
4.6.20. 9-[2-[[4-(3b-Acetyloxy-lup-20(29)en-28-oyl)-1-
piperazinyl]carbonyl]phenyl]-3,6-bis(diethylamino]-xanthylium
chloride (23)
General procedure C: from 17 (181mg, 0.32mmol); yield:
218mg, 66%; m.p. 246e250ꢀC; RF¼0.38 (silica gel, chloroform/
methanol, 9:1); UV-Vis (methanol): lmax (logε)¼257 (4.37), 524
d
(m, 1H, He37), 7.33e7.27 (m, 3H, He40þHe450þHe45), 7.10e7.01
(m, 2H, He440þ He44), 6.79e6.76 (m, 2H, He470þHe47), 5.62 (s,
1H, He12), 4.50 (dd, J¼11.6, 4.8Hz, 1H, He3), 3.65e3.56 (m, 8H,
He49þHe490), 3.54e3.35 (m, 8H, He33aþHe33bþHe34), 2.77
(ddd, J¼13.0, 3.1, 3.1Hz, 1H, He1a), 2.34 (s, 1H, He9), 2.27e2.22 (m,
1H, He18), 2.06e1.97 (m, 2H, He16aþHe19a), 2.03 (s, 3H, He32),
1.93e1.88 (m,1H, He21a),1.78 (ddd, J¼13.5,13.5, 3.7Hz,1H, He15a),
1.73e1.52 (m, 5H, He6aþHe2þHe7aþHe21b), 1.46e1.24 (m, 17H,
He6bþHe7bþHe19bþHe22þHe50þHe500), 1.32 (s, 3H, He27),
1.18e0.94 (m, 3H, He15bþHe16bþHe1b), 1.16 (s, 3H, He29), 1.14
(s, 3H, He25), 1.09 (s, 3H, He26), 0.86 (s, 6H, He23þHe24),
0.81e0.76 (m, 1H, He5), 0.75 (s, 3H, He28), ppm; 13C NMR
(4.40), 561 (4.89) nm; IR (KBr):
n
¼3448vs, 2940s, 1636s, 1590m,
1H NMR
¼7.70e7.64 (m, 2H, He38þHe39), 7.54e7.49
1414m, 1384s, 1340m, 1248m, 1182m, 1074mcmꢂ1
(500MHz, CDCl3):
;
d
(m, 1H, He37), 7.36e7.27 (m, 3H, He40þHe45’þHe45), 7.17e6.94
(m, 2H, He440þHe44), 6.88e6.75 (m, 2H, He470þHe47), 4.67 (s,
1H, He29a), 4.54 (s, 1H, He29b), 4.48e4.42 (m, 1H, He3),
3.66e3.57 (m, 8H, He49þHe490), 3.54e3.22 (m, 8H, He33þHe34),
2.89 (ddd, J¼11.1, 11.1, 4.2Hz, 1H, He19), 2.78e2.71 (m, 1H, He13),
2.05e1.97 (m, 1H, He16a), 2.02 (s, 3H, He32), 1.87e1.81 (m, 1H,
(126MHz, CDCl3):
d¼200.1 (Ce11), 174.8 (Ce30), 171.0 (Ce31), 169.9
(Ce13), 167.7 (Ce35), 157.8 (Ce48), 157.8 (Ce480), 156.1 (Ce42),
155.8 (Ce46), 155.8 (Ce460), 135.1 (Ce41), 132.4 (Ce45), 132.3
(Ce450), 130.9 (Ce36), 130.3 (Ce40), 130.3 (Ce38), 130.2 (Ce39),
128.5 (Ce12), 127.8 (Ce37), 114.6 (Ce44), 114.5 (Ce440), 114.0
(Ce43), 113.9 (Ce430), 96.4 (Ce47), 96.4 (Ce470), 80.7 (Ce3), 61.8
(Ce9), 55.1 (Ce5), 48.2 (Ce18), 47.9 (Ce34), 46.2 (Ce49), 46.2
(Ce490), 45.4 (Ce8), 44.1 (Ce21), 44.0 (Ce20), 43.4 (Ce14), 42.2
(Ce33), 38.9 (Ce1), 38.1 (Ce4), 37.8 (Ce22), 37.1 (Ce10), 32.8
(Ce19), 32.6 (Ce7), 31.9 (Ce17), 28.5 (Ce28), 28.2 (Ce24), 26.9
(Ce29), 26.7 (Ce16), 26.5 (Ce15), 23.7 (Ce2), 23.1 (Ce27), 21.4
(Ce32), 18.8 (Ce26), 17.5 (Ce6), 16.8 (Ce23), 16.5 (Ce25), 12þ.8
(Ce50), 12.8 (Ce500) ppm; MS (ESI): m/z (%)¼1005.8 ([MꢂCl] ,
100); analysis calculated for C64H85ClN4O6 (1041.84): C 73.78, H
8.22, N 5.38; found: C 73.50, H 8.46, N 5.16.
He22a),
1.73e1.19
(m,
30H,
He6þHe11þHe2þHe12aþHe15þHe21þHe16bþHe7þHe22b-
þHe1aþHe9þHe18þHe50þHe500), 1.64 (s, 3H, He30), 1.13e1.07
(m, 1H, He15b), 0.99e0.89 (m, 2H, He12bþHe1b), 0.91 (s, 3H,
He27), 0.86 (s, 3H, He26), 0.82 (s, 3H, He24), 0.81 (s, 6H,
He25þHe23), 0.78e0.74 (m, 1H, He5) ppm; 13C NMR (126MHz,
CDCl3):
d¼174.4 (Ce28), 171.1 (Ce31), 167.8 (Ce35), 157.9 (Ce48),
157.8 (Ce480), 156.0 (Ce42), 155.9 (Ce46), 155.8 (Ce460), 151.3
(Ce20), 135.3 (Ce41), 132.4 (Ce45), 132.4 (Ce450), 130.8 (Ce36),
130.4 (Ce40), 130.3 (Ce38), 130.2 (Ce39), 127.7 (Ce37), 114.7
(Ce44), 114.3 (Ce440), 114.0 (Ce43), 114.0 (Ce430), 109.4 (Ce29),
96.5 (Ce47), 96.5 (Ce470), 81.1 (Ce3), 55.6 (Ce5), 54.8 (Ce17), 52.6
(Ce18), 50.8 (Ce9), 47.8 (Ce34), 46.2 (Ce49), 46.2 (Ce490), 45.9
(Ce19), 42.3 (Ce33), 42.0 (Ce14), 40.8 (Ce8), 38.5 (Ce1), 37.9
(Ce4), 37.2 (Ce10), 37.0 (Ce13), 35.9 (Ce22), 34.4 (Ce7), 32.5
(Ce16), 31.4 (Ce21), 29.9 (Ce15), 28.1 (Ce24), 25.6 (Ce12), 23.8
(Ce2), 21.4 (Ce32), 21.2 (Ce11), 19.6 (Ce30), 18.3 (Ce6), 16.6
(Ce23), 16.3 (Ce25), 16.2 (Ce26), 14.7 (Ce27), 12.8 (Ce50), 12.8
(Ce500) ppm; MS (ESI): m/z (%)¼991.7 ([MꢂCl]þ, 100); analysis
calculated for C64H87ClN4O5 (1027.85): C 74.79, H 8.53, N 5.45;
found: C 74.55, H 9.03, N 5.27.
4.6.19. 9-[2-[[4-(3b-Acetyloxy-20-oxo-30-norlupan-28-oyl)-1-
piperazinyl]carbonyl]phenyl]-3,6-bis(diethylamino]-xanthylium
chloride (22)
General procedure C: from 16 (182mg, 0.32mmol); yield:
230mg, 70%; m.p. 235e243ꢀC; RF¼0.35 (silica gel, chloroform/
methanol, 9:1); UV-Vis (methanol): lmax (logε)¼260 (4.53), 525
(4.50), 651 (5.00) nm; IR (KBr):
1248m, 1182mcmꢂ1
n
¼3450vs, 1630m, 1590m, 1384s,
¼7.70e7.63 (m,
;
1H NMR (500MHz, CDCl3):
d
4.6.21. 9-[2-[[4-(2a,3b-Bis(acetyloxy)-olean-12-en-28-oyl)-1-
piperazinyl]carbonyl]phenyl]-3,6-bis(diethylamino]-xanthylium
chloride (24)
General procedure C: from 18 (200mg, 0.32mmol); yield:
244mg, 70%; m.p. 247e249ꢀC; RF¼0.31 (silica gel, chloroform/
methanol, 9:1); UV-vis (CHCl3): lmax (logε)¼260nm (4.51), 306nm
(4.15), 357nm (3.84), 405nm (3.48), 562nm (5.01); IR (KBr):
2H, He37þHe38), 7.60e7.54 (m, 1H, He36), 7.36e7.25 (m, 3H,
He40þHe440þHe44), 7.09e6.93 (m, 2H, He430þHe43),
6.86e6.71 (m, 2H, He460þHe46), 4.46e4.41 (m, 1H, He3),
3.69e3.54 (m, 8H, He48þHe480), 3.52e3.17 (m, 8H, He32þHe33),
3.12 (ddd, J¼11.4, 11.4, 4.0Hz, 1H, He19), 2.59e2.50 (m, 1H, He13),
2.11 (s, 3H, He29), 2.02e1.95 (m, 2H, He16aþHe18), 2.01 (s, 3H,
He31), 1.89e1.73 (m, 2H, He22aþHe21a), 1.65e1.04 (m, 28H,
n
¼2968w, 2926w, 2870w, 1636m, 1464m, 1436m, 1384m, 1248w,

8
S. Sommerwerk et al. / European Journal of Medicinal Chemistry 127 (2017) 1e9
1182w,1076w,1044wcmꢂ1; 1H NMR (500MHz, CDCl3):
d
¼7.69e7.36
drug discovery, Nat. Rev. Drug Discov. 10 (2011) 221e237.
[12] M. Gerlinger, A. Rowan, S. Horswell, J. Larkin, D. Endesfelder, E. Gronroos,
P. Martinez, N. Matthews, A. Stewart, P. Tarpey, I. Varela, B. Phillimore,
S. Begum, N. McDonald, A. Butler, D. Jones, K. Raine, C. Latimer, C. Santos,
M. Nohadani, A. Eklund, B. Spencer-Dene, G. Clarke, L. Pickering, G. Stamp,
M. Gore, Z. Szallasi, J. Downward, P.A. Futreal, C. Swanton, Intra-tumor het-
erogeneity and Darwinian selection revealed by multi-region exome
sequencing of renal cell carcinomas, abstract 964, Cancer Res. 72 (2012),
http://dx.doi.org/10.1158/1538-7445.AM2012-964.
[13] T.A. Yap, M. Gerlinger, P.A. Futreal, L. Pusztai, C. Swanton, Intratumor het-
erogeneity: seeing the wood for the trees, Sci. Transl. Med. 4 (2012).
[14] M. Gerlinger, A.J. Rowan, S. Horswell, J. Larkin, D. Endesfelder, E. Gronroos,
P. Martinez, N. Matthews, A. Stewart, P. Tarpey, I. Varela, B. Phillimore,
S. Begum, N.Q. McDonald, A. Butler, D. Jones, K. Raine, C. Latimer, C.R. Santos,
M. Nohadani, A.C. Eklund, B. Spencer-Dene, G. Clark, L. Pickering, G. Stamp,
M. Gore, Z. Szallasi, J. Downward, P.A. Futreal, C. Swanton, Intratumor het-
erogeneity and branched evolution revealed by multiregion sequencing, New
Engl, J.Med. 366 (2012) 883e892.
[15] S. Jones, X.S. Zhang, D.W. Parsons, J.C.H. Lin, R.J. Leary, P. Angenendt,
P. Mankoo, H. Carter, H. Kamiyama, A. Jimeno, S.M. Hong, B.J. Fu, M.T. Lin,
E.S. Calhoun, M. Kamiyama, K. Walter, T. Nikolskaya, Y. Nikolsky, J. Hartigan,
D.R. Smith, M. Hidalgo, S.D. Leach, A.P. Klein, E.M. Jaffee, M. Goggins, A. Maitra,
C. Iacobuzio-Donahue, J.R. Eshleman, S.E. Kern, R.H. Hruban, R. Karchin,
N. Papadopoulos, G. Parmigiani, B. Vogelstein, V.E. Velculescu, K.W. Kinzler,
Core signaling pathways in human pancreatic cancers revealed by global
genomic analyses, Science 321 (2008) 1801e1806.
[16] D.W. Parsons, S. Jones, X.S. Zhang, J.C.H. Lin, R.J. Leary, P. Angenendt,
P. Mankoo, H. Carter, I.M. Siu, G.L. Gallia, A. Olivi, R. McLendon, B.A. Rasheed,
S. Keir, T. Nikolskaya, Y. Nikolsky, D.A. Busam, H. Tekleab, L.A. Diaz, J. Hartigan,
D.R. Smith, R.L. Strausberg, S.K.N. Marie, S.M.O. Shinjo, H. Yan, G.J. Riggins,
D.D. Bigner, R. Karchin, N. Papadopoulos, G. Parmigiani, B. Vogelstein,
V.E. Velculescu, K.W. Kinzler, An integrated genomic analysis of human glio-
blastoma Multiforme, Science 321 (2008) 1807e1812.
(m, 2H, He40þHe41), 7.58e7.54 (m, 1H, He39), 7.34e7.26 (m, 3H,
He42þHe470þHe47), 7.10e7.03 (m, 1H, He460), 6.99e6.94 (m, 1H,
He46), 6.79e6.76 (m, 2H, He490þHe49), 5.18 (dd, J¼3.6, 3.6Hz, 1H,
He12), 5.07 (ddd, J¼10.0, 11.0, 4.6Hz, 1H, He2), 4.72 (d, J¼10.3Hz,
1H, He3), 3.72e3.47 (m, 10H, He51þHe510þHe35a), 3.36e3.18 (m,
6H, He35bþHe36), 3.00e2.93 (m, 1H, He18), 2.10e1.73 (m, 4H,
He16aþHe1aþHe11), 2.03 (s, 3H, He34), 1.95 (s, 3H, He32),
1.69e1.26
(m,
22H,
He16aþHe19aþHe9þHe7þHe6þHe15aþHe22aþHe21aþH-
e52þHe520), 1.26e1.20 (m, 1H, He22b), 1.15e0.98 (m, 4H,
He15bþHe21bþHe19bþHe1b), 1.07 (s, 3H, He27), 1.10 (s, 3H,
He25), 0.96e0.92 (m, 1H, He5), 0.88 (s, 6H, He23þHe24), 0.86 (s,
3H, He29), 0.85 (s, 3H, He30), 0.62 (s, 3H, He26) ppm; 13C NMR
(126MHz, CDCl3):
d
¼176.0 (Ce28),170.9 (Ce33),170.6 (Ce31),168.1
(Ce37), 157.8 (Ce50), 157.8 (Ce500), 155.9 (Ce44), 155.9 (Ce48),
155.7 (Ce480), 144.9 (Ce13), 135.1 (Ce43), 132.5 (Ce47), 132.5
(Ce470), 130.5 (Ce38), 130.4 (Ce42), 130.4 (Ce40), 130.2 (Ce41),
128.0 (Ce39), 121.2 (Ce12), 114.8 (Ce46), 114.3 (Ce460), 113.9
(Ce45), 113.8 (Ce450), 96.5 (Ce49), 96.4 (Ce490), 80.8 (Ce3), 70.2
(Ce2), 55.0 (Ce5), 47.8 (Ce17), 47.8 (Ce36), 47.6 (Ce9), 46.4 (Ce19),
46.2 (Ce51), 46.2 (Ce510), 44.0 (Ce1), 43.6 (Ce18), 42.3 (Ce35),
42.0 (Ce14), 39.5 (Ce4), 39.2 (Ce8), 38.3 (Ce10), 34.0 (Ce21), 33.1
(Ce30), 32.7 (Ce22), 30.4 (Ce20), 29.8 (Ce7), 28.5 (Ce24), 27.8
(Ce15), 26.0 (Ce27), 24.1 (Ce29), 23.5 (Ce11), 22.4 (Ce16), 21.2
(Ce32), 21.0 (Ce34), 18.3 (Ce6), 17.7 (Ce23), 16.9 (Ce26), 16.6
(Ce25), 12.8 (Ce52), 12.8 (Ce520) ppm; MS (ESI): m/z (%)¼1049.7
([MꢂCl-]þ, 100); analysis calculated for C66H89ClN4O7 (1085.89): C
73.00, H 8.26, N 5.16; found: C 72.73, H 8.43, N 5.00.
[17] J. Neuzil, L.-F. Dong, J. Rohlena, J. Truksa, S.J. Ralph, Classification of mitocans,
anti-cancer drugs acting on mitochondria, Mitochondrion 13 (2013) 199e208.
[18] J.S. Modica-Napolitano, J.R. Aprille, Delocalized lipophilic cations selectively
target the mitochondria of carcinoma cells, Adv. Drug Deliv. Rev. 49 (2001)
63e70.
[19] S. Davis, M.J. Weiss, J.R. Wong, T.J. Lampidis, L.B. Chen, Mitochondrial and
plasma-membrane potentials cause unusual accumulation and retention of
Rhodamine-123 by human-breast adenocarcinoma-derived Mcf-7 cells, J.Biol.
Chem. 260 (1985) 3844e3850.
Acknowledgments
[20] J.S. Modicanapolitano, J.R. Aprille, Basis for the selective cytotoxicity of
Rhodamine-123, Cancer Res. 47 (1987) 4361e4365.
[21] L.V. Johnson, M.L. Walsh, L.B. Chen, Localization of mitochondria in living cells
with rhodamine 123, Proc. Nat. Acad. Sci. 77 (1980) 990e994.
[22] T.J. Lampidis, Y. Hasin, M.J. Weiss, L.B. Chen, Selective killing of carcinoma cells
“invitro” by lipophilic-cationic compounds: a cellular basis, Biomed. Phar-
macother. 39 (1985) 220e226.
[23] C. Xie, J. Chang, X.-D. Hao, J.-M. Yu, H.-R. Liu, X. Sun, Mitochondrial-targeted
prodrug cancer therapy using a rhodamine B labeled fluorinated docetaxel,
Eur. J. Pharm. Biopharm. 85 (2013) 541e549.
[24] S. Sommerwerk, L. Heller, J. Kuhfs, R. Csuk, Selective killing of cancer cells with
triterpenoic acid amides - the substantial role of an aromatic moiety align-
ment, Eur. J. Med. Chem. 122 (2016) 452e464.
[25] S. Sommerwerk, L. Heller, J. Kuhfs, R. Csuk, 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 (2016) 1e16.
[26] L. Heller, V. Perl, J. Wiemann, A. Al-Harrasi, R. Csuk, Amino(oxo)acetate moi-
ety: a new functional group to improve the cytotoxicity of betulin derived
carbamates, Bioorg. Med. Chem. Lett. 26 (2016) 2852e2854.
[27] J. Wiemann, L. Heller, R. Csuk, Targeting cancer cells with oleanolic and ursolic
acid derived hydroxamates, Bioorg. Med. Chem. Lett. 26 (2016) 907e909.
[28] L. Heller, A. Obernauer, R. Csuk, Simple structural modifications confer cyto-
toxicity to allobetulin, Bioorgan. Med. Chem. 23 (2015) 3002e3012.
[29] S. Sommerwerk, L. Heller, R. Csuk, Synthesis and cytotoxic activity of penta-
cyclic triterpenoid sulfamates, Arch. Pharm 348 (2015) 46e54.
€
We like to thank Dr. D. Strohl and his team for the NMR spectra,
and Dr. R. Kluge for measuring the MS spectra. The IR spectra were
recorded by Mrs V. Simon and Mrs J. Wiese, M.Sc.; the micro-
analyses were measured by Mrs J. Pech. The cell lines were kindly
provided by Dr Th. Müller (Dep. of Haematology/Oncology, Martin-
Luther Universitat Halle-Wittenberg). Support by the “Wissen-
schaftsCampus Halle WCH” (W13004216) 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.2016.12.040.
References
[1] O. Warburg, K. Posener, E. Negelein, Metabolism of carcinoma cells, Biochem.
Z. 152 (1924) 309e344.
[2] O. Warburg, On respiratory impairment in cancer cells, Science 124 (1956)
269e270.
[30] R. Csuk, Betulinic acid and its derivatives: a patent review (2008-2013), Expert
Opin. Ther. Pat. 24 (2014) 913e923.
[3] O. Warburg, Origin of cancer cells, Oncologia 9 (1956) 75e83.
[4] O. Warburg, On the origin of cancer cells, Science 123 (1956) 309e314.
[5] V. Panda, P. Khambat, S. Patil, Int. J. Clin. Med. 2 (2011) 515e529.
[6] S. Fulda, G. Kroemer, Mitochondria as therapeutic targets for the treatment of
malignant disease, Antioxid, Redox Sign 15 (2011) 2937e2949.
[7] S. Fulda, L. Galluzzi, G. Kroemer, Targeting mitochondria for cancer therapy,
Nat. Rev. Drug Discov. 9 (2010) 447e464.
[8] S.J. Ralph, S. Rodriguez-Enriquez, J. Neuzil, E. Saavedra, R. Moreno-Sanchez,
The causes of cancer revisited: “Mitochondrial malignancy” and ROS-induced
oncogenic transformation - why mitochondria are targets for cancer therapy,
Mol. Asp. Med. 31 (2010) 145e170.
[9] S.J. Ralph, S. Rodriguez-Enriquez, J. Neuzil, R. Moreno-Sanchez, Bioenergetic
pathways in tumor mitochondria as targets for cancer therapy and the
importance of the ROS-induced apoptotic trigger, Mol. Asp. Med. 31 (2010)
29e59.
[10] A. Lemarie, S. Grimm, Mitochondrial respiratory chain complexes: apoptosis
sensors mutated in cancer, Oncogene 30 (2011) 3985e4003.
[11] O. Kepp, L. Galluzzi, M. Lipinski, J.Y. Yuan, G. Kroemer, Cell death assays for
[31] R. Csuk, Recent developments in the synthesis of antitumor-active glycyr-
rhetinic acid derivatives, Mini-Rev. Org. Chem. 11 (2014) 253e261.
[32] B. Siewert, E. Pianowski, A. Obernauer, R. Csuk, Towards cytotoxic and se-
lective derivatives of maslinic acid, Bioorg. Med. Chem. 22 (2014) 594e615.
[33] 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.
[34] A. Barthel, S. Stark, R. Csuk, Oxidative transformations of betulinol, Tetrahe-
dron 64 (2008) 9225e9229.
€
[35] R. Csuk, K. Schmuck, R. Schafer, Apractical synthesis of betulinic acid, Tetra-
hedron Lett. 47 (2006) 8769e8770.
[36] S. Sommerwerk, R. Csuk, Convenient and chromatography-free partial syn-
theses of maslinic acid and augustic acid, Tetrahedron Lett. 55 (2014)
5156e5158.
[37] S. Sommerwerk, L. Heller, I. Serbian, R. Csuk, Straightforward partial synthesis
of four diastereomeric 2,3-dihydroxy-olean-12-en-28-oic acids from oleanolic

S. Sommerwerk et al. / European Journal of Medicinal Chemistry 127 (2017) 1e9
9
acid, Tetrahedron 71 (2015) 8528e8534.
[38] L. Heller, S. Schwarz, V. Per, A. Kowitsch, B. Siewert, R. Csuk, Incorporation of a
isolated from Ilex paraguariensis and derivatives on aromatase inhibition, Eur.
J. Med. Chem. 43 (2008) 1865e1877.
€
Michael acceptor enhances the antitumor activity of triterpenoic acids, Eur. J.
Med. Chem. 101 (2015) 391e399.
[44] L. Ruzicka, K. Hofmann, Polyterpenes and polyterpenoids. C. Transpositions in
the rings A and E of oleanolic acid. Carbon skeleton of pentacyclic triterpenes,
Helv. Chim. Acta 19 (1936) 114e128.
[45] G. Drefahl, S. Huneck, The preparation of acetylglycyrrhetinic acid and its
Curtius degradation, Chem. Ber. 94 (1961) 2015e2018.
[46] A. Vystrcil, M. Budesinsky, Triterpenes. XVI. Unusual epimerization of the C-19
acetyl group in 20-oxo-30-norlupane derivatives, Collect. Czech. Chem.
Commun. 35 (1970) 295e311.
[47] 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.
[39] B. Siewert, E. Pianowski, A. Obernauer, R. Csuk, Towards cytotoxic and se-
lective derivatives of maslinic acid, Bioorg. Med. Chem. 22 (2014) 594e615.
[40] R. Csuk, S. Schwarz, R. Kluge, D. Strohl, Does one Keto group Matter?
Structure-Activity relationships of glycyrrhetinic acid derivatives modified at
position C-11, Arch. Pharm. 345 (2012) 28e32.
[41] L.C. Baratto, M.V. Porsani, I.C. Pimentel, A.B. Pereira Netto, R. Paschke,
B.H. Oliveira, Preparation of betulinic acid derivatives by chemical and
biotransformation methods and determination of cytotoxicity against selected
cancer cell lines, Eur. J. Med. Chem. 68 (2013) 121e131.
[42] K. Suzuki, A. Kobayashi, S. Kaneko, K. Takehira, T. Yoshihara, H. Ishida,
Y. Shiina, S. Oishi, S. Tobita, Reevaluation of absolute luminescence quantum
yields of standard solutions using a spectrometer with an integrating sphere
[48] X. Yang, Y. Li, W. Jiang, M. Ou, Y. Chen, Y. Xu, Q. Wu, Q. Zheng, F. Wu, L. Wang,
W. Zou, Y.J. Zhang, J. Shao, Synthesis and biological evaluation of novel ursolic
acid derivatives as potential anticancer prodrugs, Chem. Biol. Drug Des. 86
(2015) 1397e1404.
and
a back-thinned CCD detector, Phys. Chem. Chem. Phys. 11 (2009)
9850e9860.
[49] S.-X. Hua, R.-Z. Huang, M.-Y. Ye, Y.-M. Pan, G.-Y. Yao, Y. Zhang, H.-S. Wang,
Design, synthesis and invitro evaluation of novel ursolic acid derivatives as
potential anticancer agents, Eur. J. Med. Chem. 95 (2015) 435e452.
[43] S.C.B. Gnoatto, A. Dassonville-Klimpt, S. Da Nascimento, P. Galera,
K. Boumediene, G. Gosmann, P. Sonnet, S. Moslemi, Evaluation of ursolic acid