Type and position of linkage govern the cytotoxicity of oleanolic acid rhodamine B hybrids

Article abstract of DOI:10.1016/j.steroids.2021.108876

Oleanolic acid/rhodamine B hybrids exhibit different cytotoxicity depending on the way these two structural elements are linked. While a hybrid holding a piperazinyl spacer at C-28 proved to be cytotoxic in the nano-molar concentration range, hybrids with a direct linkage of the Rho B residue to C-3 of the triterpenoid skeleton are cytotoxic only in the low micro-molar concentration range without any selectivity. This once again underlines the importance of selecting the right spacer and the most appropriate position on the skeleton of the triterpene to achieve the most cytotoxic hybrids possible.

Full text of DOI:10.1016/j.steroids.2021.108876

Steroids 172 (2021) 108876
Contents lists available at ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
Type and position of linkage govern the cytotoxicity of oleanolic acid
rhodamine B hybrids
Niels Heisea, Sophie Hoenkea, Vivienne Simona, Hans-Peter Deignerb, Ahmed Al-Harrasic,
a,*
´
Rene Csuk
a Martin–Luther–University Halle-Wittenberg, Organic Chemistry, Kurt–Mothes–Str. 2, D-06120 Halle, Saale, Germany
b Furtwangen University, Medical and Life Sciences Faculty, Jakob–Kienzle Str. 17, D-78054 Villingen-Schwenningen, Germany
c University of Nizwa, Chair of Oman’s Medicinal Plants and Marine Natural Products, P.O. Box 33, PC 616, Birkat Al-Mauz, Nizwa, Oman
A R T I C L E I N F O
A B S T R A C T
Keywords:
Oleanolic acid/rhodamine B hybrids exhibit different cytotoxicity depending on the way these two structural
elements are linked. While a hybrid holding a piperazinyl spacer at C-28 proved to be cytotoxic in the nano-molar
concentration range, hybrids with a direct linkage of the Rho B residue to C-3 of the triterpenoid skeleton are
cytotoxic only in the low micro-molar concentration range without any selectivity. This once again underlines
the importance of selecting the right spacer and the most appropriate position on the skeleton of the triterpene to
achieve the most cytotoxic hybrids possible.
Oleanolic acid
Rhodamine B
Hybrids
Cytotoxicity
1. Introduction
focus here is on the endoplasmic reticulum, lysosomes and especially
mitochondria.
Cancer affects the lives of numerous people every year; this disease
ends fatally for many of them. Thus, despite many advances, state-of-
the-art therapy and early diagnosis, the number of people suffering
from cancer continues to rise. Whereas in 2008 there were around 12.7
million persons affected, by 2020 the figure had already risen to 19.3
million, and a further increase to 28.4 million persons is forecast for
2040. In 2020 alone, 10 million deaths were recorded. In addition to the
personal and professional impact on the concerned persons and their
families, the costs of treating the disease are also very high. It is esti-
mated that the total cost of cancer worldwide will exceed in 2030 US$
450 billion [1,2].
In recent years, derivatives of pentacyclic triterpenoic acids have
been identified as promising and, in some cases, highly cytotoxic com-
pounds, starting with studies on the cytotoxic activity of betulinic acid
and melanoma [7]. While betulinic acid causes parallel damage in both
mitochondrial and lysosomal compartments thereby inducing auto-
phagy [8], we were able to show for triterpene-derived saphirinium
derivatives [9] that the endoplasmic reticulum is the target. Rhodamine
B derivatives [10–17], as well as several phosphonium salts [18–20] or
F-16 conjugates [21] on the other hand, act as mitocans; their targets are
the mitochondria [22–26].
Hereby we could show that EC50 values in the low micro or even in
the nano-molar concentration range for cytotoxicity on human cancer
cell lines were observed depending very strongly on the triterpene
scaffold, the spacer and the cationic residue. Thus, derivatives of mas-
linic acid were found to be more active than those derived from betulinic
acid [10,14,17]. Rhodamine B derivatives were more cytotoxic than
comparable malachite green derivatives [27], and compounds holding a
(homo)-piperazinyl spacer [16] were significantly more effective than
those with an ethylenediamine spacer [14,28]. The latter derivatives
showed EC50 values > 30 µM and thus are usually considered non-
cytotoxic.
Drug targeting by definition refers to the targeted and selective
accumulation or release of a drug at one or more desired sites of action.
In cancer therapy, this ultimately reflects the ability to distinguish be-
tween malignant and normal cells. An insufficient selectivity is the cause
of severe side effects. This inevitably leads to a poor compliance of pa-
tients because of facing a reduced quality of life due to the drugs. Asa
result, an early discontinuation of therapy takes place.
Extending the original concept of drug-targeting directed at different
tissues, a new line of research (“third level drug targeting”) [3] has
emerged in recent years that applies drug-targeting to subcellular en-
tities and compartments (“organelle specific drug targeting”) [3–6]. The
Thus, the question of the spacer and its linkage is of decisive
* Corresponding author.
E-mail address: rene.csuk@chemie.uni-halle.de (R. Csuk).
https://doi.org/10.1016/j.steroids.2021.108876
Received 1 March 2021; Received in revised form 17 May 2021; Accepted 25 May 2021
Available online 12 June 2021
0039-128X/© 2021 Elsevier Inc. All rights reserved.

N. Heise et al.
Steroids 172 (2021) 108876
importance. For this comparative study, oleanolic acid (OA) was chosen
as the basic structure to access a small library of compounds. OA de-
rivatives had previously been shown to hold good cytotoxicity [29–31].
Furthermore, OA is also readily and commercially available in larger
quantities.
acetate and sodium cyanoborohydride gave the 3β-configured amine 3
[36–40]; the corresponding 3α epimer [36–41] could be determined in
trace amounts on TLC and detected by mass spectrometry using HPTLC-
ASAP MS but could not be isolated.
Amide 4 was formed in 65% yield from the reaction of rhodamine B
(Rho B) in acetonitrile in the presence of EDC and TEA showing an UV
absorption λmax = 550 nm being characteristic for the presence of an
intact Rho B moiety.
2. Results and discussion
Reaction of 1 with succinic anhydride in the presence of TEA gave
chain elongated 5 whose reaction with oxalyl chloride followed by the
addition of piperazine furnished 6. The use of a succinyl spacer has been
Thus, OA was converted into methyl ester 1 (Scheme 1) [32–35], the
oxidation of which with freshly prepared Jones reagent afforded ketone
2 in 71% isolated yield [32]. Reductive amination of 2 with ammonium
Scheme 1. Reactions and conditions: a) K2CO3, DMF, MeI, 23 ◦C, 24 h, 77.5%; b) Jones oxidation, 71.3%; c) (NH4)2CO3, NaBH3CN, MeOH, 23 ◦C, 24 h, 65%; d) Rho
B, EDC, TEA, 23 ◦C, 3 d, 65%; e) succinyl anhydride, pyridine, DMAP (cat.), 23 ◦C, 8 h, 79%; f) (CO)2Cl2, DMF, then piperazine, DCM, TEA, DMAP, 23 ◦C, 1d, 94%; g)
Rho B, DCM, (CO)2Cl2, DMF (cat.), 23 ◦C, 1 h, then 6, TEA, DMAP (cat), 23 ◦C, 1 d, 75%; h) Ac2O, DCM, TREA, DMAP (cat), 23 ◦C, 1 d, 75%; i) (CO)2Cl2, DMF, then
piperazine, DCM, REA, DMAP (cat.), 23 ◦C, 1 d, 84%; j) Rho B, DCM, (CO)2Cl2, DMF (cat.), 23 ◦C, 1 h, the 9, TEA, DMAP (cat.), 23 ◦C, 1 d, 79%; k) Rho B, DCM,
(CO)2Cl2, DMF (cat.), 23 ◦C, 1 h, then 1, TEA, DMAP (cat.), 23 ◦C, 1 d, 41%.
2

N. Heise et al.
Steroids 172 (2021) 108876
applied very successfully in the past for the synthesis of biologically
active triterpene carboxylic acid derivatives. Rho B was activated with
oxalyl chloride and allowed to react with 6 to afford 7 as a purple solid
showing λmax = 559 nm.
from the 48 h incubation of 10, 44.9% of the A375 cells have died by
apoptosis and 19.6% by late apoptosis. The number of necrotic cells
remained small (0.9%).
Acetylation of OA gave well known acetate 3 whose reaction – as
described above – yielded piperazinyl amide 9. This compound was
coupled with Rho B to afford 10, again as a pink colored solid.
To get an insight onto the influence of an oxygen substituent at po-
sition C-3 of the triterpenoid skeleton (as compared to a nitrogen sub-
stituent as in 4), compound 1 was coupled with Rho B to yield 11. To
evaluate the cytotoxic activity of these compounds, photometric sulfo-
rhodamine B assays (SRB) were performed employing several human
tumor cell lines as well as non-malignant fibroblasts (NIH 3 T3). The
results from these assays are compiled in Table 1.
3. Conclusion
In this small study, using OA as the starting material, it was shown
that OA Rho B hybrids exhibit different cytotoxicity depending on the
way these two structural elements are linked. Hybrids with a direct
linkage of the Rho B residue to C–3 of the triterpenoid skeleton (whether
as ester or as amide) are cytotoxic in the low micro-molar concentration
range but also not selective. In contrast, a hybrid of OA, Rho B and a
piperazinyl spacer at C–28 proved to be cytotoxic in the nano-molar
concentration range, whereas no increase in cytotoxicity can be
observed when binding to C–3 via a succinyl spacer. This once again
underlines the importance of selecting the right spacer and the most
appropriate position on the skeleton of the triterpene to achieve the
most cytotoxic hybrids possible. Compound 10 is significantly more
cytotoxic than parent compound OA and acts mainly by apoptosis while
the number of necrotic cells remains small.
The results from the SRB assays showed no significant difference
between compounds 4 and 11; the cytotoxic effect was independent of
whether the RhoB moiety was bound to C-3 via an ester linkage or as an
amide. Their EC50 values for all tumor cells were low, but these com-
pounds also lacked selectivity. In this series of compounds, 10 performed
best. Compounds 7 and 10 showed EC50 values in the nano-molar or low
micro-molar concentration range. This highlights the importance of the
piperazinyl moiety as well as the nature of the attachment of the Rho B
residue to the triterpenoid backbone. Compound 10 is thus 25 times
more cytotoxic (to A2780 cells) than compound 7 and >1000 times
more cytotoxic than parent compound OA.
4. Experimental part
4.1. General
Molecular modeling calculations, as they have been performed in the
past, are of limited value, only. These calculations had shown, using
similar compounds as examples, that some mitochondrial enzymes (e.g.
NAD(P)H-quinone oxidoreductase or succinate dehydrogenase) could
possibly be inhibited; however, the experimental evidence for this is still
pending [42]. The cytotoxicity of the compounds could also be caused
by changes in the potential of mitochondrial membranes or their ability
to increase the concentration of reactive oxygen species. To get a deeper
insight, most cytotoxic compound 10 was subjected to flow cytometric
measurements (Annexin V/PI assay). Thereby, A375 cells were treated
with 2 × EC50 concentrations of 10 for 48 h, and the results from these
experiments are depicted in Fig. 1.
NMR spectra were recorded using the Varian spectrometers DD2 and
VNMRS (400 and 500 MHz, respectively, δgiven in ppm, J in Hz; typical
experiments: APT 13C, HMBC, HSQC); MS spectra were taken on a Fin-
nigan MAT LCQ 7000 (electrospray, voltage 4.1 kV, sheath gas nitrogen)
or an Advion expressionL CMS. TLC was performed ion silica gel (Merck
5554, detection with cerium molybdate reagent); melting points are
uncorrected (Leica hot stage microscope). IR spectra were recorded on a
Perkin Elmer FT-IR spectrometer 1000 or on a Perkin Elmer Spectrum
Two (UATR Two unit). The solvents were dried according to usual
procedures. Chemicals were obtained from local suppliers; oleanolic
ˇ
´
acid was bought “Betulinines” (Stríbrna Skalice, Czech Republic) and
used as received. SRB assays were performed as previously described
[14,34,43].
In Fig. 1, the BL1-A signal corresponds to the FITC signal for annexin
V (x-axis); PI was detected at BL3-A (y-axis). As a vonsequence, cells
found in R1 areo necrotic cells, those in R2 are late apoptotic, while cells
in R3 are viable cells, and cells in R4 have died from apoptosis. Thus,
4.2. Biology
4.2.1. Cell lines and culture conditions
Following human cancer cell lines A375 (malignant melanoma),
HT29 (colon adenocarcinoma), MCF-7 (breast cancer), A2780 (ovarian
carcinoma), and non-malignant mouse fibroblasts NIH 3 T3 were used.
All cell lines were obtained from the Department of Oncology (Martin-
Luther-University Halle Wittenberg). Cultures were maintained as
monolayers in RPMI 1640 medium with L-glutamine (Capricorn Scien-
tific GmbH, Ebsdorfergrund, Germany) supplemented with 10% heat-
inactivated fetal bovine serum (Sigma-Aldrich GmbH, Steinheim, Ger-
many) and penicillin/streptomycin (Capricorn Scientific GmbH, Ebs-
Table 1
Cytotoxicity of selected compounds; SRB assay EC50 values [µM] after 72 h of
treatment; averaged from three independent experiments performed each in
triplicate; confidence interval CI = 95%. Human cancer cell lines: A375 (mela-
noma), HT29 (colorectal carcinoma), MCF-7 (breast adenocarcinoma), A2780
(ovarian carcinoma), NIH 3 T3 (non-malignant fibroblasts); cut-off 30 μM, n.s.
not soluble, n.d. not determined. Betulinic acid (BA), oleanolic acid (OA) and
doxorubicin (DX) have been used as positive standards.
#
A375
HT29
MCF-7
A2780
NIH 3 T3
◦
dorfergrund, Germany) at 37 C in a humidified atmosphere with 5%
OA
Rho
B
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
CO2.
1
>30
>30
>30
>30
>30
4.2.2. Cytotoxicity assay (SRB assay)
2
3.8 ± 1.0
2.9 ± 0.3
1.8 ± 0.1
n.s.
4.7 ± 0.5
4.0 ± 0.4
2.4 ± 0.3
n.s.
4.5 ± 0.5
2.9 ± 0.2
1.5 ± 0.2
n.s.
3.8 ± 0.2
2.9 ± 0.5
1.5 ± 0.1
n.s.
6.3 ± 0.4
2.8 ± 0.5
5.5 ± 0.4
n.s.
For the evaluation of the cytotoxicity of the compounds the
sulforhodamine-B (Kiton-Red S, ABCR) micro-culture colorimetric assay
was used as previously reported. The EC50 values were averaged from
three independent experiments performed each in triplicate calculated
from semi-logarithmic dose–response curves applying a non-linear 4P
Hills-slope equation.
3
4
5
6
9.2 ± 0.6
1.1 ± 0.1
13.0 ± 1.1
1.9 ± 0.2
0.06 ±
0.004
11.3 ± 0.6
0.9 ± 0.1
20.5 ± 1.7
1.3 ± 0.1
0.09 ±
10.7 ± 0.4
0.8 ± 0.1
12.9 ± 1.9
2.0 ± 0.1
0.06 ±
12.7 ± 1.3
0.8 ± 0.1
9.4 ± 0.5
2.1 ± 0.1
0.032 ±
0.001
7.9 ± 0.3
1.4 ± 0.1
17.5 ± 1.5
2.1 ± 0.1
0.137 ±
0.006
7
8
9
10
4.2.3. Annexin V/PI assay
0.01
0.004
11
1.6 ± 0.2
n.d.
2.8 ± 0.4
12.7 ± 1.8
0.9 ± 0.2
2.3 ± 0.1
18.4 ± 2.0
1.1 ± 0.3
1.1 ± 0.2
12.0 ± 1.7
0.02 ± 0.01
2.6 ± 0.2
16.1 ± 1.4
0.06 ± 0.03
Approx. 600,000 cells (A375) were seeded in cell culture flasks; they
were allowed to grow for 1 day. The medium was removed, and the
substance loaded medium was added; incubation lasted for for 48 h. All
BA
DX
n.d.
3

N. Heise et al.
Steroids 172 (2021) 108876
Fig. 1. Annexin V/PI flow cytometry of 10 employing A375 cells (48 h of incubation, 2 × EC50 concentration); control experiment (left), incubation with 10 (right).
cells were harvested, centrifuged (1200 rpm, 5 min) and washed twice
(PBS (w/w)). Approx. 100,000 cells were washed with annexin V
bounding buffer (BD Biosciences®) and treated with a propidium iodide
removed under diminished pressure, water was added, and the aqueous
phase was extracted with DCM (3 × 25 mL). After evaporation of the
DCM, column chromatography (silica gel, hexane/EtOAc, 9:1) furnished
solution (3
μ
L, 1 mg/mL) and annexin V (5
μL, BD Biosciences®) for 15
2 as a white solid (5.18 g, 71.3%); m.p. 186 ◦C (lit.:[45] 184 ◦C) ; [
α] =
min at room temperature in the dark. After adding annexin V bounding
+93.2◦ (c 0.320, CHCl3) [lit.:[46] [
α
]
= 90◦ (c 1.2, CHCl3); RF = 0D.66
buffer (400
μ
L) the suspension was submitted to a FACS measurement.
(SiO2, hexanes/EtOAc, 8:2)]; UV–VisD(CHCl3): λmax (log
ε) = 310 nm
Calculation was performed as suggested from the supplier (BD
(3.09); IR (ATR): ν = 3432 s, 2941 s, 1726vs, 1703 s, 1458 m, 1382w,
Biosciences®).
1364w, 1264w, 1205 m, 1163 m, 1125w, 1040w, 1016 m cmꢀ 1; 1H NMR
(500 MHz, CDCl3): δ = 5.29 (m, 1H, 12-H), 3.61 (s, 3H, OMe), 2.86 (dd,
1H, J = 13.7, 4.3 Hz, 18-H), 2.53 (ddd, 1H, J = 16.0, 11.2, 7.3 Hz, 2-Ha),
2.34 (ddd, 1H, J = 15.9, 6.7, 3.6 Hz, 2-Hb), 1.99–1.-83 (m, 4H, 1-Ha, 16-
Ha, 11-H), 1.67 (ddd, 1H, J = 14.0, 13.9, 4.6 Hz, 22-Ha), 1.64–1.56 (m,
4H, 9-H, 15-Ha, 19-Ha, 16-Hb), 1.53–1.44 (m, 4H, 22-Hb, 7-Ha, 6-H),
1.42–1.26 (m, 4H, 1-Hb, 21-Ha, 7-Hb, 5-H), 1.20–1.11 (m, 2H, 19-Hb,
21-Hb), 1.12 (s, 3H, 27-H), 1.09–1.04 (m, 1H, 15-Hb), 1.06 (s, 3H, 23-H),
1.02 (s, 3H, 24-H), 1.02 (s, 3H, 25-H), 0.91 (s, 3H, 30-H), 0.87 (s, 3H, 29-
H), 0.76 (s, 3H, 26-H) ppm; 13C NMR (126 MHz, CDCl3): δ = 217.6 (C-
3), 178.2 (C-28), 143.8 (C-13), 122.1 (C-12), 55.3 (C-5), 51.5 (OMe),
47.4 (C-9), 46.9 (C-4), 46.7 (C-17), 45.8 (C-19), 41.7 (C-14), 41.4 (C-
18), 39.2 (C-8), 39.1 (C-1), 36.7 (C-10), 34.1 (C-2), 33.8 (C-21), 33.1 (C-
29), 32.3 (C-7), 32.2 (C-22), 30.7 (C-20), 27.7 (C-15), 26.4 (C-23), 25.8
(C-27), 23.6 (C-30), 23.5 (C-11), 23.0 (C-16), 21.4 (C-24), 19.6 (C-6),
16.7 (C-26), 15.0 (C-25) ppm; MS (ESI, MeOH): m/z 469.2 (100%, [M +
H]+), 491.0 (32%, [M + Na]+), 408.4 (12%, [M + Na + H2O]+); analysis
calcd for C30H46O3 (454.69): C 79.25, H 10.20; found: C 78.98, H 10.32.
4.2.4. Synthesess
4.2.4.1. Methyl 3 β -hydroxyolean-12-en-28-oate (1). Oleanolic acid
(15.0 g, 32.8 mmol) was dissolved in DMF (200 mL) and potassium
carbonate (4.5 g, 32.8 mmol) was added. The reaction mixture was
stirred at 23 ◦C for 30 min. Iodomethane (2.5 mL, 39.7 mmol) was added
dropwise, and the mixture was stirred at 23 ◦C for 24 h. HCl (0.1 m, 65
mL) and water (0.5 L) were added, the precipitate was collected, washed
with water (2 × 250 mL) and dried. Recrystallization from ethanol gave
1 as a white solid (11.63 g, 77.5%); m.p. 203 ◦C (lit.:[44] 200–202 ◦C);
[
α
]
D = +66.5◦ (c 0.34, CHCl3) [lit.:[44] [
α]
D = 70◦ (c 1.0, CHCl3)]; RF =
0.78 (SiO2, hexanes/EtOAc, 7:3); UV–Vis (CHCl3): λmax (logε) = 257 nm
(3.74) IR (ATR):
ν = 3441 s, 2947 m, 1728 m, 1636w, 1464w, 1386w,
1163w, 1032w cmꢀ 1; 1H NMR (500 MHz, CDCl3): δ = 5.26 (m,1H, 12-H)
3.60 (s, 3H, OMe), 3.19 (dd, 1H, J = 11.0, 4.4 Hz, 3-H), 2.84 (dd, 1H, J
= 13.9, 4.2 Hz, 18-H), 1.95 (ddd, 1H, J = 14.5, 14.4, 4.1 Hz, 16-Ha),
1.88–1.82 (m, 2H,11-Ha,b), 1.67 (ddd, 1H, J = 13.9, 13.9, 4.4 Hz, 22-Ha),
1.63–1.47 (m, 9H, 9-H, 1-Ha, 19-Ha, 6-Ha, 15-Ha, 22-Hb, 16-Hb, 2-H),
1.43–1.22 (m, 4H, 21-Ha, 7-H, 6-Hb), 1.19–1.12 (m, 2H, 19-Hb, 21-
Hb), 1.10 (s, 3H, 27-H), 1.03(dd, 1H, J = 14.9, 4.0 Hz, 15-Hb) 0.97–0.92
(m, 1H, 1-Hb), 0.96 (s, 3H, 23-H), 0.90 (s, 3H, 30-H), 0.88 (s, 3H, 25-H),
0.87 (s, 3H, 29-H), 0.76 (s, 3H, 24-H), 0.73–0.68 (m, 1H, 5-H), 0.70 (s,
3H, 26-H) ppm; 13C NMR (126 MHz, CDCl3): δ = 178.2 (C-28), 143.7 (C-
13), 122.3 (C-12), 79.0 (C-3), 55.2 (C-5), 51.5 (OMe), 47.6 (C-9), 46.7
(C-17), 45.8 (C-19), 41.6 (C-14), 41.3 (C-18), 39.2 (C-8), 38.7 (C-4.),
38.4 (C-1), 37.0 (C-10), 33.8 (C-21), 33.1 (C-29), 32.6 (C-7), 32.3 (C-
22), 30.6 (C-20), 28.1 (C-23), 27.7 (C-15), 27.1 (C-2), 25.9 (C-27), 23.6
(C-30), 23.4 (C-11), 23.0 (C-16), 18.3 (C-6), 16.8 (C-26), 15.5 (C-24),
15.3 (C-25) ppm; MS (ESI, MeOH): m/z 453.1 (68%, [M + H-H2O]+),
471.3 (16%, [M + H]+) 493.2 (100%, [M + H + H2O]+); analysis calcd
for C31H50O3 (470.73): C 79.10, H 10.71; found: C 78.85, H 10.97.
4.2.4.3. Methyl 3 β -aminoolean-12-en-28-oate (3). A suspension of 2
(580 mg, 1.24 mmol)) and ammonium acetate (950 mg, 12.4 mmol) in
◦
MeOH (50 mL) stirred at 23 C for 10 min. A 1 m solution of sodium
cyanoborohydride in THF (0.54 mL) was added, and the reaction
mixture was stirred at 23 ◦C for 24 h. Usual aqueous workup and puri-
fication by column chromatography (SiO2, CHCl3/MeOH, 9:1) gave 3 as
a white solid (196 mg, 71.4%); m.p. 216–220 ◦C; [
α
]
= +39.0◦ (c
D
0.134, CHCl3); RF = 0.36 (SiO2, CHCl3/MeOH, 9:1); IR (ATR):
ν
= 3413
m, 2946w, 1726w, 1635 m, 1328 s, 1191w, 1040w, 824 m cmꢀ 1
;
1H
NMR (400 MHz, CDCl3): δ = 5.33–5.23 (m, 1H, 12-H), 3.62 (s, 3H, 31-
H), 3.20–3.08 (m, 1H, 3-H), 2.86 (dd, J = 13.7, 4.6 Hz, 1H, 18-H),
2.20–1.17 (m, 19H, 16-H, 11-Ha, 7-H, 22-H, 2-H, 11-Hb, 19-Ha, 15-Ha,
6-H, 1-H, 21-H, 9-H), 1.13 (s, 3H, 30-H), 1.09 (s, 3H, 23-H), 1.08–1.03
(m, 2H, 15-Hb, 19-Hb), 0.96 (s, 3H, 24-H), 0.93 (s, 6H, 25-H, 26-H), 0.88
(s, 3H, 29-H), 0.81–0.76 (m, 1H, 5-H), 0.72 (s, 3H; 27-H) ppm; 13C NMR
(101 MHz, CDCl3) δ = 178.5 (C-28), 144.1 (C-13), 122.3 (C-12), 58.5 (C-
3), 56.3 (C-5), 48.3 (C-9), 46.9 (C-19), 46.7 (C-17), 42.0 (C-14), 41.6 (C-
18), 39.5 (C-8), 37.0 (C-4), 35.5 (C-10), 34.1 (C-1), 34.1 (C-21), 33.2 (C-
29), 32.6 (C-2), 32.5 (C-7), 32.4 (C-22), 30.9 (C-20), 27.9 (C-15), 27.7
(C-23), 25.9 (C-30), 23.8 (C-26), 23.3 (C-16), 22.9 (C-24), 17.0 (C-27),
15.4 (C-25) ppm; MS (ESI, MeOH): m/z 470.1 (100%, [M + H]+);
analysis calcd for C30H49NO2 (455.72): C 79.07, H 10.84, N 3.07; found:
4.2.4.2. Methyl 3-oxoolean-12-en-28-oate (2). A solution of 1 (5.61 g,
12.28 mmol) in acetone (300 mL) was heated under reflux for 30 min.
After cooling to 0 ◦C, Jones reagent [prepared from CrO3 (6.27 g, 62.7
mmol), water (26 mL), and conc. H2SO4 (6.23 mL)] was slowly added,
◦
and the mixture was stirred for 1 h at 0 C. Then MeOH (10 mL) was
added, and stirring was continued for another 30 min. The solvents were
4

N. Heise et al.
Steroids 172 (2021) 108876
C 78.83, H 11.03, N 2.86.
4.2.4.6. Methyl (3 β)-3-{[(4-oxo-4-(piperazin-1-yl)butanoyl]oxy}olean-
12-en-28-oate (6). Compound 5 (300 mg, 0.526 mmol) was dissolved in
dry DCM (20 mL), and oxalyl chloride (4 eq.) and catalytic quantities of
DMF were added. The reaction mixture was stirred at 23 ◦C for one hour.
The volatiles were removed under diminished pressure, the residue was
dissolved in dry DCM (15 mL), and piperazine (4 eq.), TEA (1 eq.) and
DMAP (cat.) were added. The reaction mixture was stirred at 23 ◦C for
one day. Usual aqueous workup followed column chromatography
(SiO2, hexanes/EtOAc, 7:3) gave 6 as a white solid (295 mg, 94%); m.p.
4.2.4.4. 6-(Diethylamino)-N,N-diethyl-9-(2-{[(3 β)-28-methoxy-28-oxoo-
lean-12-en-3-yl]carbamoyl}phenyl)3H-xanthen-3-iminium chloride (4).
To a solution of rhodamine B (184.9 mg, 0.386 mmol) in acetonitrile
(60 mL), EDC (74.0 mg, 0.386 mmol) and TEA (0.06 mL, 0.386 mmol)
were added, and the mixture was stirred at 23 ◦C for 90 min. Compound
3 (165 mg, 0.351 mmol) was added, and stirring at 23 ◦C was continued
for 3 days. Usual aqueous workup followed by column chromatography
(SiO2, CHCl3/MeOH, 9:1) gave 4 as a purple solid (160 mg, 65%); m.p.
247–250 ◦C; RF = 0.48 (SiO2, CHCl3/MeOH, 9:1); UV–Vis (CHCl3): λmax
78–82 ◦C; [
α]
= +11.4◦ (c 0.163, CHCl3); RF = 0.21 (SiO2, hexanes/
D
EtOAc, 7:3); IR (ATR): ν = 3442 m, 2946 s, 1725 s, 1628 m, 1328 s, 1173
m, 1036w, 822 m cmꢀ 1; 1H NMR (400 MHz, CDCl3): δ = 5.27 (t, J = 3.6
Hz, 1H, 12-H), 4.50 (dd, J = 8.8, 7.1 Hz, 1H, 3-H), 3.68–3.44 (m, 8H, 36-
H, 37-H, 38-H, 39-H), 3.62 (s, 3H, 31-H), 2.85 (dd, J = 13.8, 4.5 Hz, 1H,
18-H), 2.71–2.59 (m, 4H, 33-H, 34-H), 2.02–1.91 (m, 1H, 16-Ha),
1.91–1.84 (m, 2H, 11-H), 1.73–1.14 (m, 16H, 22-Ha, 16-Hb, 2-H, 19-Ha,
1-Ha, 15-Ha, 9-H, 6-Ha, 7-H, 6-Hb, 21-Ha, 22-Hb, 21-Hb, 19-Hb), 1.12 (s,
3H, 27-H), 1.08–0.98 (m, 2H, 1-Hb, 15-Hb), 0.92 (d, J = 1.3 Hz, 6H, 25-
H, 30-H), 0.89 (s, 3H, 29-H), 0.86 (s, 3H, 23-H), 0.85 (s, 3H, 24-H),
0.82–0.79 (m, 1H, 5-H), 0.72 (s, 3H, 26-H) ppm; 13C NMR (101 MHz,
CDCl3): δ = 178.7 (C-28), 173.1 (C-32), 170.6 (C-35), 144.3 (C-13),
122.7 (C-12), 81.7 (C-3), 55.8 (C-5), 52.0 (C-31), 48.0 (C-9) 47.2 (C-17),
46.3 (C-19), 45.5 (C-37, C-38), 42.1 (C-14), 42.1 (C-36, C-39), 41.8 (C-
18), 39.8 (C-8), 38.6 (C-1), 38.2 (C-4), 37.4 (C-10), 34.3 (C-21), 33.6 (C-
29), 33.1 (C-7), 32.8 (C-22), 31.2 (C-20), 30.0 (C-34), 28.5 (C-23), 28.4
(C-33), 28.2 (C-15), 26.4 (C-27), 24.1 (C-30), 24.0 (C-16), 23.9 (C-11),
23.5 (C-2), 18.7 (C-6), 17.3 (C-26), 17.2 (C-24), 15.8 (C-25) ppm; MS
(ESI, MeOH): m/z 639.3 (100%, [M + H]+), 1277.2 (12%, [2 M + H]+),
1299.3 (100%, [2 M + Na]+); analysis calcd for C39H62N2O5 (638.92): C
73.31, H 9.78, N 4.38; found: C 73.05, H 9.98, N 4.18.
(log
ε
) = 550 nm (4.97); IR (ATR):
ν
= 2938w, 1645 m, 1587 s, 1409 m,
1332 s, 1178 s, 1130 m, 1073 m, 823 m, 683 m cmꢀ 1; H NMR (500
MHz, CDCl3): δ = 7.93 (d, J = 7.7 Hz, 1H, 37-H), 7.67–7.62 (m, 1H, 35-
H), 7.61–7.51 (m, 2H, 38-H, 41-H), 7.47 (d, J = 9.4 Hz, 1H, 41′-H), 7.22
(dd, J = 7.5, 1.1 Hz, 1H, 36-H), 6.99 (d, J = 9.4 Hz, 1H, 42-H), 6.91 (d, J
= 9.4 Hz, 1H, 42′-H), 6.70–6.65 (m, 2H, 44-H, 44′-H), 5.23 (t, J = 3.7 Hz,
1H, 12-H), 3.60 (s, 3H, 31-H), 3.64–3.51 (m, 8H, 46-H, 46-H’),
3.47–3.37 (m, 1H, 18-H), 2.82 (dd, J = 13.7, 4.6 Hz, 1H), 1.97–0.93 (m,
20H, 16-H, 2-Ha, 22-Ha, 15-Ha, 19-Ha, 22-Hb, 1-Ha, 6-H, 9-H, 7-Ha, 21-
Ha, 7-Hb, 21-Hb, 2-Hb, 19-Hb, 15-Hb), 1.35–1.24 (m, 12H, 47-H, 47′-H),
1.03 (s, 3H, 30-H), 0.90 (s, 3H, 26-H), 0.86 (d, J = 1.6 Hz, 6H, 25-H, 29-
H), 0.82 (s, 1H, 1-Hb), 0.81 (s, 3H, 27-H), 0.70 (m, 1H, 5-H), 0.67 (s, 3H,
24-H), 0.58 (s, 3H, 23-H) ppm; 13C NMR (126 MHz, CDCl3): δ = 178.4
(C-28), 168.0 (C-32), 160.2 (C-39), 158.0 (C-45′), 157.9 (C-45), 155.7
(C-43′), 155.4 (C-43), 143.8 (C-13), 138.3 (C-34), 133.2 (C-41′), 132.9
(C-41), 130.8 (C-33), 130.5 (C-35), 130.1 (C-38), 129.3 (C-36), 128.6
(C-37), 122.6 (C-12), 114.6 (C-40′), 114.3 (C-42′), 114.2 (C-40), 114.2
(C-42), 96.0 (C-44′), 96.0 (C-44), 57.8 (C-3), 56.3 (C-5), 51.6 (C-31),
47.7 (C-9), 46.9 (C-17), 46.1 (C-46, C-46′), 46.1 (C-19), 41.7 (C-14),
41.5 (C-18), 39.4 (C-1), 39.3 (C-8), 38.3 (C-4), 37.0 (C-10), 34.0 (C-21),
33.2 (C-29), 32.8 (C-7), 32.5 (C-22), 30.8 (C-20), 28.6 (C-23), 27.8 (C-
15), 25.9 (C-30), 24.6 (C-2), 23.8 (C-26), 23.5 (C-11), 23.2 (C-16), 18.6
(C-6), 16.9 (C-24), 16.7 (C-27), 15.3 (C-25), 12.8 (C-47, C-47′) ppm; MS
(ESI, MeOH): m/z 895.2 (100%, [M + H]+); analysis calcd for
1
4.2.4.7. 6-(Diethylamino)-N,N-diethyl-9-(2-{[(3 β)-2-methoxy-28-oxoo-
lean-12-en3-yl]carbamoyl}phenyl)–3H-xanthen-3-iminium chloride (7).
Rhodamine B (277 mg, 0.6 mmol) was dissolved in dry DCM (15 mL)
and oxalyl chloride (4 eq.) and catalytic quantities of DMF were added.
◦
C
59H80N3O4Cl (930.74): C 76.14, H 8.66, N 4.52; found: C 75.95, H 8.90,
The reaction mixture was stirred at 23 C for one hour. The volatiles
N 4.31.
were removed under diminished pressure, the residue was dissolved in
dry DCM (15 mL), and compound 6 (200 mg, 0.469 mmol), TEA (1
equiv.) and DMAP (cat.) were added. The reaction mixture was stirred at
23 ◦C for one day. Usual aqueous workup followed column chroma-
tography (SiO2, CHCl3/MeOH, 9:1) gave 7 as a purple solid (151 mg,
75.5%); m.p. 165–175 ◦C; RF = 0.44 (SiO2, CHCl3/MeOH, 9:1); UV–Vis
4.2.4.5. 4-{[(3 β)-28-Methoxy-28-oxoolean-12-en-3-yl]oxy}-4-oxobuta-
noic acid (5). Compound 1 (2 g, 4.23 mmol) and catalytic amounts of
DMAP were added to a solution of succinic anhydride (2.1 g, 21.15
mmol) in dry pyridine (50 mL), and the mixture was stirred under reflux
for 8 h. Usual aqueous workup followed by column chromatography
(SiO2, hexanes/EtOAc, 7:3) gave 5 as a white solid (1.9 g, 79%); m.p.
(CHCl3): λmax (log ε) = 559 nm (4.92); IR (ATR): ν = 3411 m, 2932w,
1722w, 1633 m, 1588 m, 1411 m, 1334 s, 1245 m, 1179 m, 1074w,
1007w, 979w, 823w, 683w cmꢀ 1
;
1H NMR (500 MHz, CDCl3): δ =
210–212 ◦C; [
α
]
D = +60.8◦ (c 0.201, CHCl3); RF = 0.29 (SiO2, hexanes/
7.78–6.64 (m, 10H, 45-H, 43-H, 46-H, 49-H, 49′-H, 44-H, 50-H, 50′-H,
44-H, 44′-H), 5.29–5.22 (m, 1H, 12-H), 4.45 (t, J = 7.9 Hz, 1H, 3-H),
3.61 (s, 3H, 31-H), 3.76–3.26 (m, 16H, 54-H, 54′-H, 36-H, 37-H, 38-H,
39-H), 2.85 (dd, J = 14.4, 4.8 Hz, 1H, 18-H), 2.73–2.52 (m, 4H, 33-H,
34-H), 2.01–1.90 (m, 1H, 16-Ha), 1.86 (dd, J = 9.5, 4.0 Hz, 2H, 11-H),
1.74–1.12 (m, 16H, 22-Ha, 16-Hb, 2-H, 19-Ha, 1-Ha, 15-Ha, 9-H, 6-Ha,
7-H, 6-Hb, 21-Ha, 22-Hb, 21-Hb, 19-Hb), 1.31 (s, 12H, 55-H, 55′-H), 1.11
(s, 3H, 27-H), 1.08–0.95 (m, 2H, 15-Hb, 1-Hb), 0.91 (s, 3H, 30-H), 0.90
(s, 3H, 25-H), 0.89 (s, 3H, 29-H), 0.83 (s, 3H, 23-H), 0.81 (s, 3H, 24-H),
0.78–0.76 (m, 1H, 5-H), 0.71 (s, 3H, 26-H) ppm; 13C NMR (126 MHz,
CDCl3): δ = 178.4 (C-28), 173.1 (C-32), 170.5 (C-35), 168.0 (C-40),
163.1 (C-47), 157.9 (C-53, C-53′), 155.9 (C-51, C-51′), 143.9 (C-13),
137.2 (C-42), 132.6 (C-49, C-49′), 131.0 (C-41), 130.1, 127.8 (C-45),
122.4 (C-12), 115.3 (C-48, C-48′, C-50, C-50′), 96.1 (C-52, C-52′), 81.2
(C-3), 55.5 (C-5), 51.6 (C-31), 47.7 (C-9), 46.9 (C-17), 46.2 (C-54, C-
54′), 46.0 (C-19), 45.5 (C-37, C-38), 42.4 (C-36′, C-39), 41.8 (C-14), 41.4
(C-18), 39.4 (C-8), 38.2 (C-1), 37.9 (C-4), 37.0 (C-10), 34.0 (C-21), 33.2
(C-29), 32.7 (C-7), 32.5 (C-22), 32.3 (C-20), 30.8 (C-34), 28.2 (C-23),
28.0 (C-33), 27.8 (C-15), 26.0 (C-27), 23.8 (C-30), 23.6 (C-16), 23.5 (C-
11), 23.2 (C-2), 18.3 (C-6), 17.0 (C-26), 16.9 (C-24), 15.5 (C-25), 12.7
EtOAc, 7:3); IR (ATR): ν = 2935 m, 1728 s, 1710 s, 1440w, 1381w, 1317
m, 1175 s, 1148 m, 1036w, 1013w, 985 m, 801w, 645 m cmꢀ 1; 1H NMR
(400 MHz, CDCl3): δ = 5.27 (t, J = 3.7 Hz, 1H, 12-H), 4.52 (dd, J = 9.0,
6.9 Hz, 1H, 3-H), 3.62 (s, 3H, 31-H), 2.86 (dd, J = 13.9, 4.5 Hz, 1H, 18-
H), 2.73–2.56 (m, 4H, 33-H, 34-H), 2.02–1.91 (m, 1H, 16-Ha), 1.90–1.83
(m, 2H, 11-H), 1.74–1.14 (m, 16H, 22-Ha, 16-Hb, 2-H, 19-Ha, 1-Ha, 15-
Ha, 9-H, 6-Ha, 7-H, 6-Hb, 21-Ha, 22-Hb, 21-Hb, 19-Hb), 1.12 (s, 3H, 27-
H), 1.09–0.99 (m, 2H, 15-Hb, 1-Hb), 0.92 (s, 3H, 25-H), 0.92 (s, 3H,
30-H), 0.89 (s, 3H, 29-H), 0.85 (s, 3H, 23-H), 0.85 (s, 3H, 24-H),
0.82–0.76 (m, 1H, 5-H), 0.72 (s, 3H, 26-H) ppm; 13C NMR (101 MHz,
CDCl3): δ = 178.5 (C-28), 177.8 (C-32), 171.9 (C-35), 144.0 (C-13),
122.4 (C-12), 81.7 (C-3), 55.5 (C-5), 51.7 (C-31), 47.7 (C-9), 46.9 (C-
17), 46.0 (C-19), 41.8 (C-14), 41.5 (C-18), 39.4 (C-8), 38.2 (C-1), 37.9
(C-4), 37.1 (C-10), 34.0 (C-21), 33.2 (C-29), 32.8 (C-7), 32.5 (C-22),
30.8 (C-20), 29.5 (C-33), 29.1 (C-34), 28.1 (C-23), 27.8 (C-15), 26.1 (C-
27), 23.8 (C-30), 23.6 (C-16), 23.2 (C-2), 18.4 (C-6), 17.0 (C-26), 16.8
(C-24), 15.5 (C-25) ppm; MS (ESI, MeOH): m/z 493.1 (38%, [M + Na]+),
1163.3 (100%, [2 M + Na]+); analysis calcd for C35H54O6 (570.80): C
73.65, H 9.54.
5

N. Heise et al.
Steroids 172 (2021) 108876
(C-55, C-55′) ppm; MS (ESI, MeOH): m/z 1064.1 (100%, [M + H]+);
analysis calcd for C67H91N4O7Cl (1099.92): C 73.16, H 8.34, N 5.09;
found: C 72.96, H 8.51, N 4.87.
[10] S. Hoenke, I. Serbian, H.-P. Deigner, R. Csuk, Mitocanic Di- and triterpenoid
rhodamine B conjugates, Molecules 25 (2020) 5443.
[11] M. Kahnt, J. Wiemann, L. Fischer, S. Sommerwerk, R. Csuk, Transformation of
asiatic acid into a mitocanic, bimodal-acting rhodamine B conjugate of nanomolar
cytotoxicity, Eur. J. Med. Chem. 159 (2018) 143–148.
[12] I. Serbian, S. Hoenke, R. Csuk, Synthesis of some steroidal mitocans of nanomolar
cytotoxicity acting by apoptosis, Eur. J. Med. Chem. 199 (2020), 112425.
[13] I. Serbian, S. Hoenke, O. Kraft, R. Csuk, Ester and amide derivatives of rhodamine B
exert cytotoxic effects on different human tumor cell lines, Med. Chem. Res. 29
(2020) 1655–1661.
4.2.4.8. 3 β -Acetyloxy-olean-12-en-28-oic acid (8). This compound was
prepared by acetylation of OA as previously reported, and 8 (10 g, 75%)
was obtained as
a
colorless solid; m.p. 265–267 ◦C (lit.:[47]
264–265 ◦C); MS (ESI, MeOH): m/z 499.2 (13%, [M + H]+), 521.2 (35%,
[M + Na]+, 1019.4 [2 M + Na]+).
[14] S. Sommerwerk, L. Heller, C. Kerzig, A.E. Kramell, R. Csuk, Rhodamine B
conjugates of triterpenoic acids are cytotoxic mitocans even at nanomolar
concentrations, Eur. J. Med. Chem. 127 (2017) 1–9.
¨
[15] J. Wiemann, L. Fischer, J. Kessler, D. Strohl, R. Csuk, Ugi multicomponent-
4.2.4.9. (3 β) 28-Oxo-28-(piperazine-1-yl)-olean-12-en-3-yl acetate (9).
As described for the synthesis of 6, from 8 and piperazine, compound 9
(0.42 g, 84%) was obtained as a colorless solid; m.p. 171–175 ◦C; (lit.:
[48,49] m.p. 170–176 ◦C); MS (ESI, MeOH): m/z 567.2 (52%, [M +
H]+).
reaction: Syntheses of cytotoxic dehydroabietylamine derivatives, Bioorg. Chem.
81 (2018) 567–576.
¨
[16] R.K. Wolfram, L. Fischer, R. Kluge, D. Strohl, A. Al-Harrasi, R. Csuk,
Homopiperazine-rhodamine B adducts of triterpenoic acids are strong mitocans,
Eur. J. Med. Chem. 155 (2018) 869–879.
[17] R.K. Wolfram, L. Heller, R. Csuk, Targeting mitochondria: esters of rhodamine B
with triterpenoids are mitocanic triggers of apoptosis, Eur. J. Med. Chem. 152
(2018) 21–30.
4.2.4.10. 9-(2-{4[(3
β)-3-Acetyloxy-28-oxoolean-12-en-28-yl]-pipera-
[18] A.Y. Spivak, D.A. Nedopekina, R.R. Khalitova, R.R. Gubaidullin, V.N. Odinokov, Y.
P. Bel’skii, et al., Triphenylphosphonium cations of betulinic acid derivatives:
synthesis and antitumor activity, Med. Chem. Res. 26 (2017) 518–531.
[19] A.Y. Spivak, D.A. Nedopekina, E.R. Shakurova, R.R. Khalitova, R.R. Gubaidullin, V.
N. Odinokov, et al., Synthesis of lupane triterpenoids with triphenylphosphonium
substituents and studies of their antitumor activity, Russ. Chem. Bull. 62 (2013)
188–198.
zine-1-carbonyl}phenyl)-6-(diethylamino)-N,N-diethyl-3H-xanthen-3-imi-
nium chloride (10). As described for the synthesis of 7, from 9 and
piperazine, compound 10 (0.67 g, 79%) was obtained as a colorless
◦
◦
solid; m.p. 244–247 C, (lit.:[14] 245–248 C); MS (ESI, MeOH): m/z
991.7 (100%, [Mꢀ Cl]+).
[20] O.V. Tsepaeva, A.V. Nemtarev, T.I. Abdullin, L.R. Grigor’eva, E.V. Kuznetsova, R.
A. Akhmadishina, et al., Design, synthesis, and cancer cell growth inhibitory
activity of triphenylphosphonium derivatives of the triterpenoid betulin, J. Nat.
Prod. 80 (2017) 2232–2239.
4.2.4.11. 6-(Diethylamino)-N,N-diethyl-9-[2-({[(3
β)-28-methoxy-28-
oxoolean-12-en-3-yl]oxy}carbonyl)phenyl]–3H-xanthen-3-iminium chlo-
ride (11). This compound was prepared as previously described; 11 was
obtained as a pink solid (0.68 g, 41%); m.p. 237–240 ◦C (lit.:[17]
235–240 ◦C); MS (ESI, MeOH): m/z 896.1 (100%, [Mꢀ Cl]+).
[21] Spivak AY, Nedopekina DA, Gubaidullin RR, Davletshin EV, Tukhbatullin AA,
D’Yakonov VA, et al. Pentacyclic triterpene acid conjugated with mitochondria-
targeting cation F16: Synthesis and evaluation of cytotoxic activities. Med Chem
Res. 2021:Ahead of Print.
[22] L. Dong, V. Gopalan, O. Holland, J. Neuzil, Mitocans revisited: mitochondrial
targeting as efficient anti-cancer therapy, Int. J. Mol. Sci. 21 (2020) 7941.
[23] D. Guzman-Villanueva, V. Weissig, Mitochondria-targeted agents:
mitochondriotropics, mitochondriotoxics, and mitocans, Handb. Exp. Pharmacol.
240 (2017) 423–438.
CRediT authorship contribution statement
Niels Heise: Investigation, Writing - review & editing. Sophie
Hoenke: Investigation, Writing - review & editing. Vivienne Simon:
Investigation, Writing - review & editing. Hans-Peter Deigner:
Conceptualization, Writing - original draft, Writing - review & editing.
Ahmed Al-Harrasi: Conceptualization, Writing - original draft, Writing
[24] S. Mani, G. Swargiary, K.K. Singh, Natural agents targeting mitochondria in cancer,
Int. J. Mol. Sci. 21 (2020) 6992.
[25] J. Neuzil, L.-F. Dong, J. Rohlena, J. Truksa, S.J. Ralph, Classification of mitocans,
anti-cancer drugs acting on mitochondria, Mitochondrion 13 (2013) 199–208.
[26] V. Panda, P. Khambat, S. Patil, Mitocans as novel agents for anticancer therapy: an
overview, Int. J. Clin. Med. 2 (2011) 515–529.
´
- review & editing. Rene Csuk: Conceptualization, Writing - original
[27] S. Friedrich, I. Serbian, S. Hoenke, R.K. Wolfram, R. Csuk, Synthesis and cytotoxic
evaluation of malachite green derived oleanolic and ursolic acid piperazineamides,
Med. Chem. Res. 29 (2020) 926–933.
draft, Writing - review & editing.
[28] M. Kahnt, L. Fischer, A. Al-Harrasi, R. Csuk, Ethylenediamine derived
carboxamides of betulinic and ursolic acid as potential cytotoxic agents, Molecules
23 (2018) 1–19.
Acknowledgments
[29] J.-H. Feng, W. Chen, Y. Zhao, X.-L. Ju, Anti-tumor activity of oleanolic, ursolic and
glycyrrhetinic acid, Open Nat Prod J. 2 (2009) 48–52.
¨
We like to thank Th. Schmidt for the MS spectra, and Dr. D. Strohl, Y.
Schiller and S. Ludwig for numerous NMR spectra. IR and UV/vis
spectra, optical rotations and micro-analyses were performed by M.
Schneider. The cell lines were provided by Dr. Th. Müller (Dep. of
Oncology, MLU).
[30] R. Paduch, M. Kandefer-Szerszen, Antitumor and antiviral activity of pentacyclic
triterpenes, Mini-Rev. Org. Chem. 11 (2014) 262–268.
[31] A. Paszel-Jaworska, A. Romaniuk, M. Rybczynska, Molecular mechanisms of
biological activity of oleanolic acid - a source of inspiration for a new drugs design,
Mini-Rev. Org. Chem. 11 (2014) 330–342.
¨
[32] L. Heller, S. Schwarz, V. Perl, A. Kowitsch, B. Siewert, R. Csuk, Incorporation of a
Michael acceptor enhances the antitumor activity of triterpenoic acids, Eur. J. Med.
References
Chem. 101 (2015) 391–399.
¨
[33] A. Loesche, A. Kowitsch, S.D. Lucas, Z. Al-Halabi, W. Sippl, A. Al-Harrasi, et al.,
[1] https://de.statista.com/statistik/daten/studie/204615/umfrage/kosten-neuer-kre
bserkrankungen-weltweit-nach-krebs-und-kostenart/; last accessed: 01.03.2021.
[2] E. Dolgin, Cancer’s cost conundrum, Nature 555 (2018) S26–S29.
[3] A.A. Khan, K.S. Allemailem, A. Almatroudi, S.A. Almatroodi, M.A. Alsahli, A.
H. Rahmani, Novel strategies of third level (Organelle-specific) drug targeting: an
innovative approach of modern therapeutics, J. Drug Delivery Sci. Technol. 61
(2021), 102315.
Ursolic and oleanolic acid derivatives with cholinesterase inhibiting potential,
Bioorg. Chem. 85 (2019) 23–32.
¨
[34] B. Siewert, J. Wiemann, A. Kowitsch, R. Csuk, The chemical and biological
potential of C ring modified triterpenoids, Eur. J. Med. Chem. 72 (2014) 84–101.
[35] S. Sommerwerk, L. Heller, R. Csuk, Synthesis and cytotoxic activity of pentacyclic
triterpenoid sulfamates, Arch. Pharm. (Weinheim, Ger.) 348 (2015) 46–54.
[36] C.-M. Ma, N. Nakamura, M. Hattori, Chemical modification of oleanene type
triterpenes and their inhibitory activity against HIV-1 protease dimerization,
Chem. Pharm. Bull. 48 (2000) 1681–1688.
[4] U. Joshi, V.V. Nimbalkar, Ligand based drug targeting system and their
application, Int. J. Pharm. Sci. Rev. Res. 65 (2020) 233–240.
[5] F. Shakeel, Recent advances in liposomal drug delivery system for drug targeting,
Curr. Drug Deliv. 17 (2020) 824–825.
[37] C.-M. Ma, N. Nakamura, M. Hattori, T. Kawahata, T. Otake, Inhibitory effects of
triterpene-azidothymidine conjugates on proliferation of human
immunodeficiency virus type 1 and its protease, Chem. Pharm. Bull. 50 (2002)
877–880.
[6] S. Tabassum, S.A. Farheen, A review on liposomes as novel drug delivery system,
Eur. J. Biomed. Pharm. Sci. 7 (2020) 411–416.
[7] E. Pisha, H. Chai, I.S. Lee, T.E. Chagwedera, N.R. Farnsworth, G.A. Cordell, et al.,
Discovery of betulinic acid as a selective inhibitor of human-melanoma that
functions by induction of apoptosis, Nat. Med. 1 (1995) 1046–1051.
[8] S. Fulda, Autophagy and cell death, Autophagy 8 (2012) 1250–1251.
[9] O. Kraft, M. Kozubek, S. Hoenke, I. Serbian, D. Major, R. Csuk, Cytotoxic
triterpenoid-safirinium conjugates target the endoplasmic reticulum, Eur. J. Med.
Chem. 209 (2021), 112920.
[38] C.-M. Ma, X.-H. Wu, H. Masao, X.-J. Wang, Y. Kano, HCV protease inhibitory,
cytotoxic and apoptosis-inducing effects of oleanolic acid derivatives, J. Pharm.
Pharm. Sci. 12 (2009) 243–248.
[39] U. Wrzeciono, L. Gorczynska, Nitrogen derivatives of triterpenes. VIII. Sulfonamide
derivatives of oleanane, Rocz Chem. 47 (1973) 1185–1190.
[40] U. Wrzeciono, W. Turowska, L. Gorczynska, Nitrogen derivatives of triterpenes.
VII. Products of the reduction of oleanonic acid oxime and its methyl ester, Rocz
Chem. 47 (1973) 955–962.
6

N. Heise et al.
Steroids 172 (2021) 108876
[41] G. Drefahl, S. Huneck, The reduction products of various triterpene oximes and
triterpenic acid amides, Chem. Ber. 93 (1960) 1967–1975.
[42] I. Macasoi, M. Mioc, D. Berceanu Vaduva, R. Ghiulai, A. Mioc, D. Muntean,
V. Dumitrascu, In silico ecaluation of the antiproliferative mitochondrial targeted
mechanism of action of some pentacyclic triterpene derivatives, Rev. Chim. 69
(2018) 3361–3363.
[45] A. Garcia-Granados, P.E. Lopez, E. Melguizo, A. Parra, Y. Simeo, Remote
hydroxylation of methyl groups by regioselective cyclopalladation. Partial
synthesis of hyptatic acid-A, J. Org. Chem. 72 (2007) 3500–3509.
¨
[46] H.S. Triterpene, 4. Die Triterpensauren des Balsams von Liquidambar Orientalis
Miller, Tetrahedron 19 (1963) 479–482.
[47] Ruzicka L, Hofmann K. Polyterpenes and polyterpenoids. C. Transpositions in the
rings A and E of oleanolic acid. Carbon skeleton of pentacyclic triterpenes. Helv
Chim Acta. 1936;19:114-28.
[43] S. Hoenke, N.V. Heise, M. Kahnt, H.-P. Deigner, R. Csuk, Betulinic acid derived
amides are highly cytotoxic, apoptotic and selective, Eur. J. Med. Chem. 207
(2020), 112815.
[48] B. Brandes, S. Hoenke, L. Fischer, R. Csuk, Design, synthesis and cytotoxicity of
BODIPY FL labelled triterpenoids, Eur. J. Med. Chem. 185 (2020), 111858.
[49] B. Brandes, L. Koch, S. Hoenke, H.-P. Deigner, R. Csuk, The presence of a cationic
center is not alone decisive for the cytotoxicity of triterpene carboxylic acid
amides, Steroids 163 (2020), 108713.
[44] L.R. Row, S.R.S. Rao, Chemistry of Terminalia species. VI. Constitution of
tomentosic acid, a new triterpene carboxylic acid from T. tomentosa, Tetrahedron
18 (1962) 827–838.
7