MSBA-S – A pentacyclic sulfamate as a new option for radiotherapy of human breast cancer cells

Article abstract of DOI:10.1016/j.ejmech.2021.113721

Many pentacyclic triterpenoids show anti-cancer and anti-inflammatory properties. Recently, we detected a pronounced cytotoxicity and radiosensitivity of two betulinyl sulfamates in human breast cancer cells. Besides betulinic acid scaffold (BSBA-S), we synthesized several new sulfamate-coupled scaffolds from oleanolic acid (OSBA-S), ursolic acid (USBA-S), platanic acid (PSBA-S) and maslinic acid (MSBA-S). Highest cytotoxicity was monitored in breast cancer cell lines after MSBA-S treatment showing in SRB assays IC₅₀ values between 3.7 μM and 5.8 μM. Other sulfamate/triterpene conjugates, however, were less cytotoxic holding IC₅₀ values between 6.6 μM and >50 μM, respectively. MSBA-S-treated breast cancer cells displayed significantly reduced clonogenic survival and an increased rate of apoptosis as compared to the other conjugates. In addition, MSBA-S in combination with irradiation resulted in effects on radiosensitivity in MDA-MB-231 cells (DMF₁₀ = 1.14). In particular, ROS formation was strongly assessed in MSBA-S-treated breast cancer cells. Our findings suggest that the sulfamate derivative of maslinic acid MSBA-S might be a new option for the radiation therapy in breast cancer cells.

Full text of DOI:10.1016/j.ejmech.2021.113721

European Journal of Medicinal Chemistry 224 (2021) 113721
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
MSBA-S e A pentacyclic sulfamate as a new option for radiotherapy of
human breast cancer cells
,
Marina Petrenko a *, Antje Güttler a, Elena Pflüger a, Immo Serbian b, Michael Kahnt b,
a
a
c
c
b
ꢀ
Yvonne Eiselt , Jacqueline Keßler , Anne Funtan , Reinhard Paschke , Rene Csuk ,
Dirk Vordermark a, Matthias Bache a
a Martin Luther University Halle-Wittenberg, Department of Radiotherapy, Ernst Grube Straße 40, D-06120, Halle, Germany
b Martin Luther University Halle-Wittenberg, Organic Chemistry, Kurt Mothes Straße 2, D-06120, Halle, Germany
c Martin Luther University Halle-Wittenberg, Biozentrum, Weinbergweg 22, D-06120, Halle, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Many pentacyclic triterpenoids show anti-cancer and anti-inflammatory properties. Recently, we
detected a pronounced cytotoxicity and radiosensitivity of two betulinyl sulfamates in human breast
cancer cells. Besides betulinic acid scaffold (BSBA-S), we synthesized several new sulfamate-coupled
scaffolds from oleanolic acid (OSBA-S), ursolic acid (USBA-S), platanic acid (PSBA-S) and maslinic acid
(MSBA-S). Highest cytotoxicity was monitored in breast cancer cell lines after MSBA-S treatment
Received 25 April 2021
Received in revised form
14 July 2021
Accepted 25 July 2021
Available online 28 July 2021
showing in SRB assays IC50 values between 3.7
m
M and 5.8
m
M. Other sulfamate/triterpene conjugates,
M and >50 mM, respectively. MSBA-S-
however, were less cytotoxic holding IC50 values between 6.6
m
Keywords:
treated breast cancer cells displayed significantly reduced clonogenic survival and an increased rate of
apoptosis as compared to the other conjugates. In addition, MSBA-S in combination with irradiation
resulted in effects on radiosensitivity in MDA-MB-231 cells (DMF10 ¼ 1.14). In particular, ROS formation
was strongly assessed in MSBA-S-treated breast cancer cells. Our findings suggest that the sulfamate
derivative of maslinic acid MSBA-S might be a new option for the radiation therapy in breast cancer cells.
© 2021 Elsevier Masson SAS. All rights reserved.
Pentacyclic triterpenoic acid
Sulfamates
Triple negative human breast cancer
Cytotoxicity
Apoptosis
Radiotherapy
1. Introduction
burdened with side-effects, it is essential that multi-targeted and
effective therapies are developed to enhance chances for poor
Breast cancer is one of the commonly diagnosed cancer types
and provides the second highest cancer-related mortality rate in
women [1]. The four classified distinct subtypes, such as luminal A
or B, Her 2-positive (Her2þ), and basal-like of breast cancer cell
lines provide different response to treatment and outcome [2].
Especially the triple-negative breast cancers (TNBC) that lack
expression of estrogen receptor (ER), progesterone receptor (PR)
and HER2 are associated with an adverse prognosis [3,4]. The
diagnosis of TNBC ensures important implications for the choice of
systemic therapies. Thus, standard breast cancer treatment consists
of surgery, radiotherapy, chemotherapy, hormonal therapy and/or
immunotherapy [5,6]. Radiotherapy is a conventional treatment
method and is systematically performed as it decreases the rate of
local recurrence and mortality. Since therapy is often limited and
prognosis especially in TNBC. To reduce side-effects and improve
the outcome of radiotherapy, combination with pharmacologically
relevant therapeutic agents is of utmost importance.
For a long time, natural drugs have comprised a wide spectrum
of medicinal and pharmacological activities. Due to its multi-
targeting advantage in drug development and noteworthy anti-
cancer properties, pentacyclic triterpenes came in the focus of
scientific interest.
Naturally occurring pentacyclic triterpenoids such as ursolic
acid (UA), betulin, betulinic acid (BA), oleanolic acid (OA), platanic
acid (PA), or maslinic acid (MA) and derivatives thereof exhibit
pharmacological benefits (Fig. 1). Notably, modification on posi-
tions C-2 and C-28 of the pentacyclic triterpenoidic skeleton
resulted in profound efficiency and enhanced cytotoxicity, whereas
the introduction of ester substituents at C-3 gave compounds of
higher bioavailability, and C-28-amides held improved the solubi-
lity and higher cytotoxicity [7]. Anti-cancer properties of several
pentacyclic triterpenes and their derivatives have been described
* Corresponding author.
E-mail address: marina.petrenko@uk-halle.de (M. Petrenko).
https://doi.org/10.1016/j.ejmech.2021.113721
0223-5234/© 2021 Elsevier Masson SAS. All rights reserved.

M. Petrenko, A. Güttler, E. Pflüger et al.
European Journal of Medicinal Chemistry 224 (2021) 113721
Fig. 1. Structures of used pentacyclic triterpenoic acids.
in vitro as well as in vivo: ursolic [8e10], betulinic [11e13], oleanoic
[14,15], platanic [16,17] or maslinic acid [18,19]. Besides induction of
apoptosis, triterpenes also modulate the tumor environment
causing anti-angiogenic, anti-inflammatory, anti-oxidative, car-
dioprotective as well as anti-viral, anti-bacterial and anti-fugal ef-
fects [20,21]. In addition, terpenoids and derivatives provide
inhibition of cell proliferation, induction of tumor cell death by
targeting different genes and multiple pathways, such as Bcl-2, NF-
syntheses to access even more effective anti-tumor active tri-
terpene derivatives [31]. Previous investigations already showed
the cytotoxicity of BA to be greatly enhanced (concerning the
proliferation of MCF-7 cells) by using a conjugate at position C-28
thus providing access to compounds holding low IC50 values [32].
Moreover, C-2/C-3-sulfamates of triterpenes have been reviewed as
effective and competetive inhibitors of CA II [25].
To access the target structures 16e20, parent triterpenoidic
oleanolic acid, ursolic acid, betulinic acid, platantic acid and maslinic
acid (Fig. 1) were acetylated to yield acetates 1e5 (Sch. 1). These
acetates were activated with oxalyl chloride followed by the addi-
tion of benzyl chloride to give amides 6e10, whose deacetylation
furnished 11e15. Reaction of these compounds with sulfamoyl
chloride finally gave target compounds 16e20, respectively (Sch. 2).
k
B, STAT3, TNF, angiogenesis, PI3K/Akt/mTOR, and TLR [22,23].
Previously, a few pentacyclic sulfamate triterpenes have been
examined as carbonic anhydrase inhibitors (CAIs) [24e26]. Based
on their structure, these compounds were capable to inhibit tumor-
associated carbonic anhydrases (CA), such as hCA II, hCA IX and hCA
XII. Thereby, sulfamates and sulfonamides bind directly to the zinc
ion in the centre in the enzymes’ active site [27]. Initial own studies
showed anti-cancer benefits after incubation of pentacyclic sulfa-
mate triterpenes, resulting in enhanced cytotoxicity and induction
of apoptosis in different tumor entities as compared to their parent
compounds [26,28,29]. Furthermore, betulinyl sulfamates revealed
inhibition of migration and enhanced radiosensitivity [29]. Further,
our very recent study suggested that combination of a betulin-
derived triterpene and a CAI resulted in additive cytotoxicity and
radiosensitization on breast cancer cells [30]. As a consequence, to
identify effective pentacyclic sulfamate triterpenes with a balance
of impairing tumor-selective drugs together with enhanced radio-
sensitization was called for.
2.2. Effects of the sulfamates 16e20 on cytotoxicity
Betulinyl sulfamates have been found to enhance cytotoxicity in
comparison to BA in cancer cells [26,29]. To determine the drug-
induced cytotoxicity, SRB screenings were performed with breast
cancer cell lines (Table 1). Treatment with OSBA-S (16) and PSBA-S
(19) caused lowest cytotoxic effects with IC50 values varying from
9.3
resulted in moderate cytotoxicity of the IC50 values, varying from
6.6 M to 26.9 M in the breast cancer cell lines.
However, IC50 values of BA varied from 13 M in MCF-7 breast
M in MDA-MB-231 cells in our recently
mM to ꢀ 50
mM. Incubation of BSBA-S (18) and USBA-S (17)
m
m
m
In this light, we synthesized and investigated cell- and radio-
biological effects of new pentacyclic sulfamate triterpenes in breast
cancer cell lines.
cancer cell line and 18
m
Table 1
2. Results and discussion
Cytotoxicity of the compounds shown by IC50 values (
incubation period of 96 h in one basal-like breast cancer cell line (MCF-7) and three
TNBC cells (HS578T, MDA-MB-231, BT-20). Cut-off was set to 50 M. The data
represent the average values ( SD) of at least three independent experiments.
mM) from SRB assays after an
m
2.1. Synthesis of the triterpenoid sulfamates
We aimed to investigate the anti-cancer effect and radiosensi-
tivity of the a small series of newly synthesized pentacyclic sulfa-
mate triterpenes. As the presence of a benzylamide moiety at
position C-28 was described to provide higher cytotoxic effects as
well as increased tumor-selectivity compared to esters, benzyla-
mides were chosen as cytotoxic parent compounds for further
compound
HS578T
MDA-MB-231
MCF-7
BT-20
BSBA-S
OSBA-S
USBA-S
PSBA-S
MSBA-S
15.6 2.5
15.8 2.2
18.8 6.4
16.9 4.5
3.8 2.4
23.3 2.8
ꢀ50
6.6 1.6
ꢀ50
26.9 6.9
16.2 6.4
16.2 4.0
32.0 4.5
5.8 1.6
15.2 7.1
ꢀ50
7.5 3.2
9.3 2.5
4.9 1.9
3.7 0.8
2

M. Petrenko, A. Güttler, E. Pflüger et al.
European Journal of Medicinal Chemistry 224 (2021) 113721
submitted work (under review). Derivatization at position C-28 for
drug targets BSBA-S (18), USBA-S (17) and PSBA-S (19) brought
about enhanced cytotoxicity in MCF-7 cells, whereas these modi-
fications did not result in additive cytotoxic effects on the TNBC cell
lines. Notably, the MA derivative was found to be toxic for tumor
cell lines holding the lowest IC50 values as compared to other tri-
terpenoids [25,28]. In particular, MSBA-S (20) caused the strongest
effects on all cell lines in our study. Only MSBA-S (20) assessed
strong cytotoxic effect on all breast cancer cell lines, with IC50
2.4. Effects of the sulfamates 16e20 on cell death
On account of the strong effect of MSBA-S (20) on the clonogenic
survival in TNBC cells, further investigations were focused on cell
death analyses. Amides derived from BA, OA, UA, MA as well as
from PA have been shown in several studies to exhibit anti-cancer
activities by evoking apoptosis in human tumor cells [12,17,32,33].
Significant increase of secondary necrosis inducted by triterpenes
was perceived in human tumor cells as well [31].
values from 3.7
m
M to 5.8
m
M.
DAPI staining (Fig. 3 A and B) showed that apoptosis of the breast
cancer cells was elevated after treatment with the cells with the
sulfamates showing the typical morphologic shrinkage hallmark of
the cell nuclei (Fig. 3C). Incubation with OSBA-S (16), PSBA-S (19),
USBA-S (17) and BSBA-S (18) had moderate effects on cell death in
the HS578T cell line with 3.5%e7.1% apoptotic cells. MSBA-S (20)
assessed strong significant effect with 22.1% (p ¼ 0.03) apoptotic
cells. MDA-MB-231 cells showed lower effects after application of
PSBA-S (19), USBA-S (17) and BSBA-S (18) with 2.1%e6.7% apoptotic
cells. Moderate effects were gained after incubation with OSBA-S
(16) and MSBA-S (20) with 10.5% and 8.9% apoptotic cells.
This results parallel previous findings for triterpenoids struc-
turally modified at positions C-2/C-3/C-28 [28]. Moreover, this
study revealed strong cytotoxicity of sulfamate derived from mas-
linic acid with IC50 values from 2.2
cells.
In conclusion, BSBA-S (18), USBA-S (17) and PSBA-S (19) exerted
enhanced cytotoxicity, especially in MCF-7 breast cancer cells. In-
cubation of all four cell lines with MSBA-S (20) resulted in strong
cytotoxicity, with rather low IC50 values.
mM to 4.0 mM for different cancer
Quantification of vital (Q3), necrotic (Q1), late apoptotic (Q2)
and apoptotic (Q4) cells was gained by flow-cytometry using
Annexin V and propidium iodide (PI) staining (Fig. 4 A). Annexin V
analysis (Fig. 4 B and C) was performed. BSBA-S (18) triggered
moderate effects in HS57T cells, with 0.8% necrotic cells, 2.8%
apoptotic and 12.1% late apoptotic cells. MSBA-S (20) caused strong
cell death with 2.6% necrotic cells, 8.7% apoptotic and 44.4% late
apoptotic cells, respectively. However, in MDA-MB-231 cells, the
compounds had moderate effects (BSBA-S: 0.5% necrotic cells, 1.1%
apoptotic and 4.8% late apoptotic cells; MSBA-S: 1.6% necrosis, 7.4%
apoptosis and 18.4% late apoptosis).
Besides slight effects after incubation with the other pentacyclic
sulfamate triterpenes, BSBA-S (18) and especially MSBA-S (20)
treatment resulted in strongest impact on the TNBC cells. Previ-
ously, it could be shown that betulinyl sulfamates caused apoptosis,
mediated by activation of caspases [26,29]. This parallels our re-
sults, showing induced cell death (Fig. 3) after incubation of the
pentacyclic triterpene derivatives and late apoptosis after applica-
tion of MSBA-S (20) in the Annexin V-PI assay (Fig. 4). In accordance
with our results, betulinyl triterpenes caused necrosis and
apoptosis, respectively [26,34].
2.3. Effects of the sulfamates 16e20 on clonogenic cell survival
In agreement with the SRB assay, highly significant impact on the
reduction of the clonogenic survival was monitored after incubation
of MSBA-S (20) in TNBC HS578Tcells up to 16.5% (p < 0.0001) and in
MDA-MB-231 cells up to 42.1% (Fig. 2). For the other sulfamates
decreased effects were observed showing reduction levels of the
clonogenic survival levels of 73.1%e81.3% in HS578T cells and of
80.6%e84.5% in MDA-MB-231 cells, respectively. For comparison,
incubation of the betulinyl CAIs on breast cancer cells resulted in
similar inhibition of the clonogenic survival and cytotoxic results
within the SRB-Assay in our previous work [29].
We also observed an inhibition of cell migration of 21% after
incubation with BSBA-S (18) with the wound healing assay in MDA-
MB-231 cells (data not shown). In agreement with these findings,
BA and betulinyl sulfamates caused strong inhibition of migration
in MDA-MB-231 and MCF-7 cells as well [29]. Interestingly, OA has
been found to increase MDA-MB-231 human breast cancer cell
migration and hence to show promising properties for wound
healing [32]. The inhibitory effect on cell migration might be
induced in a cell-specific pattern.
Fig. 2. Clonogenic survival after 24 h treatment with the sulfamates BSBA-S OSBA-S, USBA-S, PSBA-S and MSBA-S in TNBC cells (A) MDA-MB-231 and (B) HS578T. The data represent
the average values (þSD) of three independent experiments; ***p < 0.0001.
3

M. Petrenko, A. Güttler, E. Pflüger et al.
European Journal of Medicinal Chemistry 224 (2021) 113721
Fig. 3. Apoptotic cell death analysis (DAPI) in TNBC cells (A) HS578T and (B) MDA-MB-231 after 72 h treatment with BSBA-S OSBA-S, USBA-S, PSBA-S and MSBA-S. C displays typical
apoptotic cell nuclei in HS578T cells after 72 h treatment with MSBA-S. The data represent the average values (þSD) of three independent experiments; *p < 0.05.
2.5. Effects of the sulfamates 16e20 on reactive oxygen species
(ROS) generation
[32]. Radiosensitization was also observed in a rat model after
combined application of OA [15]. There have been studies with BA
and betulinyl triterpene derivatives, displaying enhanced radio-
sensitivity after incubation of the drugs [34,39,40]. Furthermore,
radiosensitization was detected after incubation with betulinyl
sulfamates and irradiation in human breast cancer cells under
hypoxic conditions [29].
Another triterpene derivative was found to significantly sup-
press cell proliferation, invasion and migration in human tumor
cells. Highlighting, it induced ROS-mediated ER-stress dependent
apoptosis in human tumor cells [32].
ROS formation was detected after treatment of cells with BSBA-S
(18) and MSBA-S (20). This effect was measured by the conversion
of CM-H2DCFDA to 5,6-chloromethyl-2,7-dichlorofluorescein. The
drugs caused increased ROS formation in the two TNBC cell lines
HS578T and MDA-MB-231 (Fig. 5). Compared to DMSO treatment,
treatment with BSBA-S (18) resulted in a 2.7-fold (p ¼ 0.005) sig-
nificant increase in ROS levels in MDA-MB-231 cells and a 2.8-fold
increase in ROS levels in HS578T cells. Significant 3.1-fold (p ¼ 0.01)
increase in ROS induction was detected after treatment with MSBA-
S (20) in HS578T cells and a 5.7-fold increase in MDA-MB-231 cells
was observed in comparison to DMSO-treated cells.
However, there is a lack of MA or MA derivatives studies of
radiosensitivity so far. In our analysis, in addition to treatment with
MSBA-S (20), cells were irradiated (Fig. 6). Incubation with MSBA-S
(20) had impact on radiosensitivity in MDA-MB-231 cells with the
DMF10 ¼ 1.14 (p ¼ 0.13). The association of ROS formation and
radiosensitivity was observed.
Eventually, our findings suggest that application of MSBA-S (20)
resulted in increased apoptosis and ROS formation and slight
radiosensitization under hypoxic conditions in particular in TNBC
cells. There is a need for further investigations of the effect of
MSBA-S (20) on tumor behaviour in vivo.
Highest ROS formation was achieved after treatment with
MSBA-S (20). Studies revealed ROS induction after incubation of BA
on tumor cells [35,36]. In addition, betulinyl triterpene derivatives
contributed to the ROS formation in tumor cells as well [30,37,38].
However, few studies have been examined ROS formation after
treatment with triterpenes. Our own study indicate enhancement
of radiosensitivity in association with ROS formation [30].
3. Conclusion
In conclusion, our findings suggest that application of a sub-
stantial lower concentration of MSBA-S (20) in comparison to the
other pentacyclic sulfamate triterpenes results in an increased
cytotoxicity, significant reduction of clonogenic survival, apoptosis
induction and increased ROS formation in TNBC cell lines. Addi-
tionally, enhanced radiosensitivity was shown in MDA-MB-
231 cells. The other potential natural compounds OSBA-S (16),
USBA-S (17), PSBA-S (19) and BSBA-S (18) have been simulta-
neously well been characterized but showed only marginal effects
2.6. Effects of the sulfamates 16e20 on radiosensitivity
Sensitivity of radiotherapy to carcinoma cells was previously
found to be increased by a several dammarane-type triterpenes
4

M. Petrenko, A. Güttler, E. Pflüger et al.
European Journal of Medicinal Chemistry 224 (2021) 113721
Fig. 4. Analysis of Annexin V-PI staining in TNBC cells (B) HS578T and (C) MDA-MB-231 after 24 h incubation of BSBA-S or MSBA-S. A displays flow cytometry analysis of Annexin V-
PI staining and the classification of vital (Q3), necrotic (Q1), late apoptotic (Q2) and apoptotic (Q4) cells.
Fig. 5. ROS formation in TNBC cells (A) HS578T and (B) MDA-MB-231 after 24 h treatment with BSBA-S and MSBA-S. The data represent the average values (þSD) of three in-
dependent experiments; *p < 0.05.
5

M. Petrenko, A. Güttler, E. Pflüger et al.
European Journal of Medicinal Chemistry 224 (2021) 113721
Fig. 6. The effects of MSBA-S on radiosensitivity in TNBC cells MDA-MB-231 after irradiation with 6 Gy, 10 Gy and 14 Gy under hypoxia (0.1% O2) (A and B). The data represent the
average values (þSD) of three independent experiments.
on cellular parameters. MSBA-S (20) might be considered as a po-
tential anti-cancer drug in breast cancer cell lines due to its pa-
rameters being analyzed in this study.
4.1.4. 3b-Acetyloxy-urs-12-en-oic acid (2)
Following GPA, compound 2 (2.41 g, 89 %) was obtained from UA
as a colorless solid; Rf ¼ 0.55 (n-hexane/ethyl acetate, 3:1); m. p.
280e283 ꢂC (lit:[6] 280e285 ꢂC); [
a
]
¼ þ66.1ꢂ (c 0.35, CHCl3)
D
[lit:[6] [
a
]D ¼ þ65.59ꢂ (c 0.33, CHCl3)]; MS (ESI, MeOH): m/z 499.2
4. Experimental
(11 %, [MþH]þ), 521.3 (35 %, [MþH]þ), 1019.2 (100 %, [2 M þ Na]þ).
4.1. Syntheses
4.1.5. 3b-Acetyloxy-lup-20(29)-en-28-oic acid (3)
Following GPA, compound 3 (4.90 g, 90 %) was obtained from BA
as a colorless solid; Rf ¼ 0.59 (n-hexane/ethyl acetate, 4:1); m. p.
4.1.1. General
Melting points are uncorrected (Leica hot stage microscope or
Büchi M ꢁ 565), NMR spectra were recorded using the Agilent
276e278 ꢂC (lit:[6] 282e285 ꢂC); [
a]
¼ þ20.4ꢂ (c 0.30, CHCl3)
D
[(lit:[6] [
a
]
¼ þ20.98ꢂ (c 0.33, CHCl3)]; MS (ESI, MeOH): m/z 487.0
spectrometers VNMRS or DD2 (d given in ppm, J in Hz, internal
D
(30 %, [M ꢁ H]-, 995.1 (100 %, [2M ꢁ H]-; 1018.4 (31 % [2M-
Me4Si or residual solvent peaks; typical experiments: HeHeCOSY,
HMBC, HSQC, NOESY, DQF-COSY), MS spectra were taken on a
Finnigan MAT LCQ 7000 (electrospray, voltage 4.1 kV, sheath gas
nitrogen) instrument; ASAP-MS spectra were taken on an Advion
expressionL CMS-L mass spectrometer (source voltage: 77 V, APCI
2H þ Na]-).
4.1.6. 3b-Acetyloxy-20-oxo-30-norlupan-28-oic acid (4)
Following GPA, compound 4 (6.9 g, 84 %) was obtained from PA
as a colorless solid; Rf ¼ 0.51 (toluene/ethyl acetate/heptane/formic
acid, 80:26:10.5); m. p. 267e269 ꢂC (lit:[7] 252e255 ꢂC);
corona discharge: 4.2 m
A, capillary temperature: 250 ꢂC, capillary
voltage: 180 V, sheath gas: N2). Optical rotations were measured on
a JASCO P-2000 instrument. TLC was performed on silica gel (Merck
5554, detection with cerium molybdate reagent); IR spectra were
recorded on a PerkinElmer Spectrum Two (UATR Two Unit). The
solvents were dried according to usual procedures. Triterpenoic
[
a
]
D
¼ ꢁ9.3ꢂ (c 0.30, CHCl3) [(lit: [
a
]
D
¼ ꢁ9.5ꢂ (c 0.5, CHCl3)]; MS
(ESI, MeOH): m/z 999.4 (100 %, [2M ꢁ H]-).
4.1.7. (2a, 3b) 2,3-Bis(acetyloxy)-olean-12-en-28-oic acid (5)
ꢁ
ꢀ
acids were obtained from “Betulinines” (Stríbrna Skalice, Czech
Republic) and used as received.
Following GPA, compound 5 (1.1 g, 92 %) was obtained from MA
as a colorless solid; Rf ¼ 0.32 (n-hexane/ethyl acetate, 3:1); m. p.
223e226 ꢂC (lit: 221e223 ꢂC); [
a]
¼ þ30.5ꢂ (c 0.5, CHCl3) [(lit:
D
[
a
]
D
¼ þ29.3ꢂ (c 0.3, CHCl3)]; MS (ESI, MeOH): m/z 557.4 (52 %,
4.1.2. General procedure of acetylation (GPA)
[MþH]þ), 1135.1 (100 %, [2 M þ Na]þ).
To a solution of the triterpenoic acid (OA, UA, BA, PA, MA, 1
equiv.) in dry DCM, acetic anhydride (3 equiv.), triethylamine (3
equiv.) and DMAP (C-at.) were added, and the mixture was stirred
at 20 ꢂC for 1 day. Usual aqueous work-up followed by re-
crystallization from ethanol furnished products 1e5.
4.1.8. General procedure for the synthesis of acetylated benzyl
amides (GPB)
To a solution of the acetylated triterpenoic acid 1e5 (1 equiv.) in
dry DCM, DMF (cat.) and oxalyl chloride (4 equiv.) were added
followed by the addition of benzyl amine (1.5 equiv.) After stirring
for 1 h at 20 ꢂC followed by usual aqueous work-up and column
chromatography, products 6e10 were obtained.
4.1.3. 3b-Acetyloxy-olean-12-en-oic acid (1)
Following GPA, compound 1 (2.45 g, 89 %) was obtained from OA
as a colorless solid; Rf ¼ 0.70 (toluene/ethyl acetate/heptane/formic
acid, 80:26:10:5); m. p. 287e290 ꢂC (lit:[4, 5] 281e283 ꢂC);
[
a
]
¼ þ68.7ꢂ (c 0.25, CHCl3) [(lit:[4, 5] [
a]
¼ þ74.1ꢂ (c 0.4, CHCl3)];
4.1.9. 3b-Acetyloxy-N-benzyl-olean-12-en-28-oic amide (6)
Following GPC, compound 6 (1.1 g, 95 %) was obtained from 1 as
a colorless solid; Rf ¼ 0.35 (n-hexane/ethyl acetate, 7:1); m. p.
D
D
MS (ESI, MeOH): m/z 499.1 ([MþH]þ), 516.2 (34 %, [M þ NH4]þ,
521.4 [30 %, [MþNa]þ.
6

M. Petrenko, A. Güttler, E. Pflüger et al.
European Journal of Medicinal Chemistry 224 (2021) 113721
243e244 ꢂC (lit:[6] 247e249 ꢂC); [
a
]
D
¼ þ29.8ꢂ (c 0.33, CHCl3),
[
a
]
¼ þ4.1ꢂ (c 0.40, CHCl3), [(lit:[11] [
a]
D
¼ þ2.0ꢂ (c 0.3, CHCl3)];
D
[(lit:[6] [
a
]
¼ þ30.66ꢂ (c 0.35, CHCl3)]; MS (ESI, MeOH): m/z 588.2
MS (ESI, MeOH): m/z 546.2 (30 %, [MþH]þ), 568.4 (10 %, [MþNa]þ),
1091.5 (30 %, [2 M þ H]þ), 1113.1 (100 %, [2 M þ Na]þ).
D
(90 %, [MþH]þ), 610.1 (22 %, [MþNa]þ), 1175.0 (46 %, [2 M þ H]þ),
1197.3 (100 %, [2 M þ Na]þ).
4.1.18. 3b-Hydroxy-N-benzyl-20-oxo-30-norlupan-28-oic amide
4.1.10. 3
b-Acetyloxy-N-benzyl-urs-12-en-28-oic amide (7)
(14)
Following GPC, compound 7 (1.4 g, 94 %) was obtained from 2 as
a colorless solid; Rf ¼ 0.69 (n-hexane/ethyl acetate, 7:1); m. p.
Following GPC, compound 14 (0.82 g, 92 %) was obtained from 9
as a colorless solid; Rf ¼ 0.40 (toluene/ethyl acetate/heptane/formic
acid, 80:26:10:5); m. p. 266e268 ꢂC (lit:[8] 266e267 ꢂC),
143e145 ꢂC (lit:[6] 145 ꢂC), [
a]
¼ þ18.4ꢂ (c 0.4, CHCl3), [(lit:
D
[
a
]
¼ þ17.89ꢂ (c 0.35, CHCl3)]; MS (ESI, MeOH): m/z 588.0(43 %,
[
a
]
D
¼ ꢁ6.1ꢂ (c 0.3, CHCl3), [(lit:[8] [
a]
D
¼ ꢁ5.9ꢂ (c 0.33, CHCl3)]; MS
D
[MþH]þ), 610.3 (20 %, [MþNa]þ), 1175.2 (66 %, [2 M þ H]þ), 1197.1
(ESI, MeOH): m/z 548.3 (100 %, [MþH]þ), 1095.1 (50 %, [2 M þ H]þ),
1117.2 (70 %, [2 M þ Na]þ).
(74 %, [2 M þ Na]þ).
4.1.11. 3
b
-Acetyloxy-N-benzyl-lup-20(29)-en-28-oic amide (8)
4.1.19. (2a, 3b) Dihydroxy-N-benzyl-olean-12-en-28-oic amide (15)
Following GPC, compound 8 (2.1 g, 84 %) was obtained from 3 as
a colorless solid; Rf ¼ 0.24 (n-hexane/ethyl acetate, 7:1); m. p.
Following GPC, compound 15 (0.34 g, 89 %) was obtained from
10 as a colorless solid; Rf ¼ 0.15 (toluene/ethyl acetate/heptane/
formic acid, 80:25:30:4); m. p. 147e149 ꢂC (lit:[9] 148e151 ꢂC);
143e146 ꢂC (lit:[6] 138e144 ꢂC), [
a]
¼ þ22.5ꢂ (c 0.37, CHCl3),
D
[(lit:[6] [
a
]
¼ þ22.19ꢂ (c 0.36, CHCl3)]; MS (ESI, MeOH): m/z 588.1
[
a
]
D
¼ þ28.2ꢂ (c 0.20, CHCl3), [(lit:[9] [
a]
¼ þ29.0ꢂ (c 0.29, CHCl3)];
(40 %, [MþDH]þ), 610.1 (23 %, [MþNa]þ), 1197.2 (100 %, [2 M þ Na]þ).
MS (ESI, MeOH): m/z 562.3 (40 %, [MþH]þ, 1145.3 (100 %,
D
[2 M þ Na]þ).
4.1.12. 3b-Acetyloxy-N-benzyl-20-oxo-30-norlupan-28-oic amide
(9)
4.1.20. General procedure for the synthesis of the sulfamates (GPD)
4.1.20.1. Sulfamoyl chloride. At 0 ꢂC to vigorously stirred chlor-
osulfonyl isocyanate (24.6 mmol, 3.48 g, 2.14 mL), formic acid
(24.6 mmol, 0.93 mL) was added, and stirring was continued until
the evolution of gases had ceased. The precipitate was filtered off
and dissolved in benzene and filtered again. The filtrate was
evaporated to dryness, and the product was obtained as a colorless
solid (2.45 g, 86 %) being used for all subsequent reactions without
any further purification. It can be kept under argon in the dark at
20 ꢂC for at least 2 months without deterioration.
To an ice-cold solution of the triterpenoic amides 11e15
(0.64 mmol) in THF (30 mL), NaH (0.90 mmol) was added, and the
suspension was stirred at 25 ꢂC for 30 min. A freshly prepared so-
lution of sulfamoyl chloride (1.02 mmol) in THF (2 mL) was added,
and the mixture was stirred for 5 days at 25 ꢂC. The solvent was
removed under diminished pressure, and the residue portioned
between ether (100 mL) and H2O (100 mL). The organic layer was
separated, washed with brine (100 mL), dried (MgSO4), concen-
trated under diminished pressure, and the residue was purified by
column chromatography (silica gel).
Following GPC, compound 9 (0.95 g, 88 %) was obtained from 4
as a colorless solid; Rf ¼ 0.38 (n-hexane/ethyl acetate, 7:1); m. p.
291e293 ꢂC (lit:[8] 290 ꢂC), [
a
]
¼ þ2.2ꢂ (c 0.32, CHCl3), [(lit:[8]
D
[
a
]
¼ þ0.5ꢂ (c 0.159, CHCl3)]; MS (ESI, MeOH): m/z 590.4 (100 %,
D
[MþH]þ), 612.4 (28 %, [MþNa]þ).
4.1.13. (2a, 3b) Bisacetyloxy-N-benzyl-olean-12-en-28-oic amide
(10)
Following GPB compound 10 (0.8 g, 84 %) was obtained from 5 as
a colorless solid; Rf ¼ 0.70 (n-hexane/ethyl acetate, 7:3); m. p.
143e145 ꢂC (lit:[9] 143e145 ꢂC); [
a
]
¼ ꢁ7.3ꢂ (c 0.25, CHCl3)
D
[(lit:[9] [
a]
¼ ꢁ7.0ꢂ (c 0.32, CHCl3)]; MS (ESI, MeOH): m/z 646.5
D
(100 %, [MþH]þ), 668.4 (50 %, [MþNa]þ).
4.1.14. General procedure for deacetylation (GPC)
To a solution of compounds 6e10 (1.0 equiv.) in methanol,
methanolic KOH (1.2 equiv, freshly prepared) was added; the
mixture was stirred at 20 ꢂC until completion of the reaction (as
indicated by TLC). The mixture was poured into ice cold aq. HCl
(3.7 %), the precipitate was collected, dried, and subjected to col-
umn chromatography.
4.1.21. 3b-Aminosulfonyloxy-N-benzyl-olean-12-en-28-oic amide
(16)
4.1.15. 3
b
-Hydroxy-N-benzyl-olean-12-en-28-oic amide (11)
Following GPD, compound 16 (0.43 g, 83 %) was obtained from
11 as a colorless amorphous solid; Rf ¼ 0.17 (0.17, n-hexane/ethyl
Following GPC, compound 11 (0.62 g, 87 %) was obtained from 6
as a colorless solid; Rf ¼ 0.82 (toluene/ethyl acetate/heptane/formic
acid, 80:26:10:5); Rf ¼ 0.28 (toluene/EtOAc/n-heptane/HCOOH,
acetate, 7:3); [
a
]
D
¼ þ28.1ꢂ (c 0.385, CHCl3); IR (ATR): v ¼ 3406w,
2946 m, 2879w, 1639 m, 1635 m, 1520 m, 1497 m, 1464 m, 1455 m,
1434w, 1364s, 1266w, 1210w, 1203w, 1178s, 1010w, 961 m, 910vs,
80:26:10:5); m. p. 248e250 ꢂC (lit: 249e251 ꢂC), [
a
]
¼ þ30.3ꢂ (c
D
0.32, CHCl3), [(lit: [
a
]
D
¼ þ30.42ꢂ (c 0.33, CHCl3)]; MS (ESI, MeOH):
871 m, 836 m, 698 m cmꢁ1 1H NMR (400 MHz, CDCl3):
;
m/z 546.1 (54 %, [MþH]þ), 568.3 (15 %, [MþNa]þ), 1091.0 (45 %,
d
¼ 7.36e7.22 (m, 5H, 33-H þ 33‘-H þ 34-H þ 34‘-H þ H-35), 6.18
[2 M þ H]þ), 1113.5 (100 %, [2 M þ Na]þ).
(dd, J ¼ 6.4, 4.4 Hz, 1H, H-amide), 5.30 (dd, J ¼ 3.6 Hz, 1H, H-12),
4.87 (s, 2H, NH2-sulfamate), 4.62 (dd, J ¼ 14.7, 6.3 Hz, 1H, 31-Ha),
4.22 (dd, J ¼ 12.0, 4.6 Hz, 1H, 3-H), 4.14 (dd, J ¼ 14.7, 4.4 Hz, 1H, 31-
Hb), 2.54 (dd, J ¼ 13.0, 4.4 Hz, 1H, 18-H), 2.09e1.93 (m, 2H, 2-Ha þ
22-Ha), 1.92e1.71 (m, 5H, 2-Hb þ 11-Ha þ 11-Hb þ 15-Ha þ 19-Ha),
1.71e1.49 (m, 6H, 22-Hb þ 15-Hb þ 1-Ha þ 16-Ha þ 6-Ha þ 9-H),
1.49e1.31 (m, 3H, 7-Ha þ 6-Hb þ 21-Ha), 1.31e1.13 (m, 3H, 7-
Hb þ 21-Hb þ 19-Hb), 1.14 (s, 3H, 27-H), 1.03 (s, 3H, 24-H),
1.07e0.98 (m, 2H, 16-Hb þ 1-Hb), 0.90 (s, 3H, 30-H), 0.90 (s, 3H, 29-
H), 0.90 (s, 3H, 26-H), 0.86 (s, 3H, 23-H), 0.84e0.79 (m, 1H, 5-H),
4.1.16. 3b-Hydroxy-N-benzyl-urs-12-en-28-oic amide (12)
Following GPC, compound 12 (0.72 g, 87 %) was obtained from 7
as a colorless solid; Rf ¼ 0.70 (toluene/ethyl acetate/heptane/formic
acid, 80:26:10:5); m. p. 266e269 ꢂC (lit:[10] 267e268 ꢂC),
[
a
]
¼ þ32.0ꢂ (c 0.33, CHCl3), [(lit:[6] [
a
]
¼ þ32.33ꢂ (c 0.34,
D
D
CHCl3)]; MS (ESI, MeOH): m/z ¼ 546.1 (100 %, [MþH]þ), 568.5 (12 %,
[MþNa]þ), 1091.2 (72 %, [2 M þ H]þ), 1114.4 (94 %, [2 M þ Na]þ).
4.1.17. 3
b
-Hydroxy-N-benzyl-lup-20(29)-en-28-oic amide (13)
0.67 (s, 3H, 25-H) ppm; 13C NMR (101 MHz, CDCl3):
28),144.9 (C-13),138.4 (C-32),128.7 (C-33 þ C-33‘),127.7 (C-34 þ C-
34‘), 127.4 (C-35), 122.7 (C-12), 91.5 (C-3), 55.4 (C-5), 47.4 (C-9), 46.7
(C-19), 46.3 (C-17), 43.6 (C-31), 42.3 (C-18), 42.0 (C-14), 39.3 (C-8),
d
¼ 178.0 (C-
Following GPC, compound 13 (0.82 g, 71 %) was obtained from 8
as a colorless solid; Rf ¼ 0.57 (toluene/ethyl acetate/heptane/formic
acid, 80:26:10:5); m. p. 254e256 ꢂC (lit:[11] 254e256 ꢂC),
7

M. Petrenko, A. Güttler, E. Pflüger et al.
European Journal of Medicinal Chemistry 224 (2021) 113721
38.6 (C-4), 38.3 (C-1), 36.7 (C-10), 34.1 (C-21), 33.0 (C-30), 32.6 (C-
15), 32.3 (C-7), 30.7 (C-20), 28.1 (C-24), 27.3 (C-16), 25.7 (C-27), 24.2
(C-2), 23.8 (C-22), 23.6 (C-29), 23.4 (C-11), 18.3 (C-6), 16.9 (C-25),
16.4 (C-23), 15.3 (C-26) ppm; MS (ESI, MeOH): m/z 528.4 (5 %,
[M þ HeH2NSO3H]þ), 625.3 (100 %, [MþH]þ), 647.3 (90 %,
21), 29.4 (C-16), 28.0 (C-24), 25.6 (C-12), 24.4 (C-2), 21.0 (C-11), 19.5
(C-30), 18.3 (C-6), 16.2 (C-25), 16.2 (C-23), 16.1 (C-26), 14.6 (C-27)
ppm; MS (ESI, MeOH): m/z 528.3 (10 %, [M þ HeH2NSO3H]þ), 625.3
(88 %, [MþH]þ), 647.2 (100 %, [MþNa]þ), 1271.3 (28 %, [2 M þ Na]þ);
analysis calcd for C37H56N2SO4 (624.93): C 71.11, H 9.03, N 4.48; S
5.13; found: C 70.85, H 9.24, N 4.32, S 4.95.
[MþNa]þ), 1271.3 (48 %, [2
M
þ
Na]þ); analysis calcd for
C
37H56N2SO4 (624.93): C 71.11, H 9.03, N 4.48; S 5.13; found: C
70.86, H 9.21, N 4.20, S 4.98.
4.1.24. 3
amide (19)
Following GPD, compound 19 (0.40 g, 75 %) was obtained from
b-AminosulfonyloxyN-benzyl-20-oxo-30-norlupan-28-
4.1.22. 3 -Aminosulfonyloxy-N-benzyl-urs-12-en-28-amide (17)
b
Following GPD, compound 17 (0.62 g, 79 %) was obtained from
12 as a colorless amorphous solid; Rf ¼ 0.20 (n-hexane/ethyl ace-
14 as a colorless amorphous solid; Rf ¼ 0.28 (n-hexane/ethyl ace-
tate, 7:3); [
a]
¼ ꢁ2.9ꢂ (c 0.340, CHCl3); IR (ATR): v ¼ 3365w,
D
tate, 7:3); [
a]
¼ þ14.5ꢂ (c 0.355, CHCl3); IR (ATR): v ¼ 3398w,
2946 m, 2866w, 1694 m, 1658 m, 1651 m, 1635 m, 1525 m, 1520 m,
1496w, 1469w, 1454 m, 1362s, 1248w, 1177s, 940s, 920vs, 909vs,
D
2973 m, 2921 m, 2872 m, 1646 m, 1628 m, 1524s, 1506 m, 1489 m,
1469 m, 1453 m, 1367s, 1180s, 1142 m, 1080 m, 908s, 871s, 837 m,
872 m, 837 m, 700
d
s ; 1H NMR (400 MHz, CDCl3):
cmꢁ1
748 m, 724 m, 696
m
cmꢁ1
;
1H NMR (500 MHz, CDCl3):
¼ 7.38e7.24 (m, 5H, 32-H þ 320-H þ 33-H þ 330-H þ 34-H), 5.98 (t,
d
¼ 7.35e7.21 (m, 5H, 35-H þ 34-H þ 34‘-H þ 33-H þ 33‘-H),
J ¼ 5.8 Hz, 1H, NH-amide), 4.48 (dd, J ¼ 14.7, 5.7 Hz, 1H, 30-Ha), 4.33
(dd, J ¼ 14.7, 5.4 Hz, 1H, 30-Hb), 4.19 (dd, J ¼ 12.0, 4.7 Hz, 1H, 3-H),
3.47 (td, J ¼ 11.2, 4.2 Hz, 1H, 19-H), 2.26 (td, J ¼ 12.0, 4.1 Hz, 1H, 13-
H), 2.17 (s, 3H, 29-H), 2.12e1.99 (m, 3H, 2-Ha þ 2-Hb þ 18-H),
1.94e1.56 (m, 5H, 15-Ha þ 22-Ha þ 1-Ha þ 15-Hb þ 22-Hb),
1.56e1.20 (m, 10H, 21-Ha þ 7-Ha þ 6-Ha þ 7-Hb þ 16-Ha þ 11-Ha þ
11-Hb þ 9-H þ 6-Hb þ 16-Hb), 1.17e1.02 (m, 3H, 21-Hb þ 12-Ha þ
12-Hb), 1.01 (s, 3H, 24-H), 0.98 (s, 3H, 27-H), 0.95e0.89 (m, 1H, 1-
Hb), 0.87 (s, 3H, 26-H), 0.84 (s, 3H, 23-H), 0.83 (s, 3H, 25-H),
6.16e6.12 (m, 1H, NH-amide), 5.21 (dd, J ¼ 3.5 Hz, 3.5 Hz, 1H, 12-H),
4.87 (s, 2H, NH2-sulfamate), 4.54 (dd, J ¼ 14.5, 6.0 Hz, 1H, 31-Ha),
4.22 (dd, J ¼ 12.0, 4.6 Hz, 1H, 3-H), 4.16 (dd, J ¼ 14.6, 4.5 Hz, 1H, 31-
Hb), 2.08e2.00 (m, 1H, 2-Ha), 2.00e1.95 (m, 1H, 15-Ha), 1.94e1.84
(m, 4H, 11-Ha þ 2-Hb þ 7-Ha þ 18-H), 1.84e1.73 (m, 2H, 11-Hb þ 15-
Hb), 1.73e1.62 (m, 2H, 16-Ha þ 1-Ha), 1.61e1.28 (m, 9H, 6-Ha þ 21-
Ha þ 9-H þ 22-Ha þ 7-Hb þ19-H þ6-Hb þ 21-Hb þ 22-Hb), 1.08 (s,
3H, H-27), 1.09e1.04 (m, 2H, 16-Hb þ 1-Hb), 1.04 (s, 3H, H-24),
0.97e0.91 (m, 1H, H-20),0.95 (s, 3H, H-30), 0.91 (s, 3H, H-26), 0.86
(s, 3H, H-24), 0.85 (d, J ¼ 6.4 Hz, 3H, H-29), 0.83e0.78 (m, 1H, H-5),
0.80e0.73 (m, 1H, 5-H) ppm; 13C NMR (101 MHz, CDCl3):
d
¼ 213.0
(C-20), 175.7 (C-28), 138.9 (C-31), 128.7 (C-33 þ C-330), 127.7 (C-
32 þ C-320), 127.4 (C-34), 91.7 (C-3), 55.6 (C-5), 55.5 (C-17), 51.0 (C-
19), 50.4 (C-9), 49.9 (C-18), 43.3 (C-30), 42.3 (C-14), 40.7 (C-8), 38.7
(C-4), 38.5 (C-1), 38.0 (C-22), 37.0 (C-10), 36.7 (C-13), 34.2 (C-7),
33.0 (C-15), 30.3 (C-29), 29.4 (C-21), 28.6 (C-16), 28.0 (C-24), 27.2
(C-12), 24.4 (C-2), 21.0 (C-11), 18.3 (C-6), 16.2 (C-25), 16.2 (C-23);
16.1 (C-26), 14.7 (C-27) ppm; MS (ESI, MeOH): m/z 530.3 (18 %,
[M þ HeH2NSO3H]þ), 627.4 (100 %, [MþH]þ), 649.2 (90 %,
0.70 (s, 3H, H-25) ppm; 13C NMR (126 MHz, CDCl3):
d
¼ 177.9 (C-28),
139.9 (C-13), 138.3 (C-32), 128.7 (C-34 þ C-34’), 127.9 (C-33 þ C-
330), 127.4 (C-35), 125.5 (C-12), 91.5 (C-3), 55.5 (C-5), 54.0 (C-18),
47.8 (C-17), 47.4 (C-9), 43.7 (C-31), 42.5 (C-14), 39.7 (C-19), 39.5 (C-
8), 39.1 (C-20), 38.6 (C-4), 38.4 (C-1), 37.2 (C-7), 36.7 (C-10), 32.7 (C-
22), 30.9 (C-21), 28.1 (C-24), 27.9 (C-16), 24.8 (C-15), 24.3 (C-2), 23.3
(C-11), 23.2 (C-27), 21.2 (C-30), 18.3 (C-6), 17.2 (C-29), 17.0 (C-25),
16.4 (C-23), 15.5 (C-26) ppm; MS (ESI, MeOH): m/z 528.5 (4 %,
[M þ HeH2NSO3H]þ), 625.3 (100 %, [MþH]þ), 647.3 (62 %,
[MþNa]þ), 1249.2 (18 %, [2 M þ H]þ), 1271.4 (24 %, [2 M þ Na]þ);
analysis calcd for C37H56N2SO4 (624.93): C 71.11, H 9.03, N 4.48; S
5.13; found: C 71.01, H 9.28, N 4.29, S 4.96.
[MþNa]þ), 1275.5 (28 %, [2
M
þ
Na]þ); analysis calcd for
C
36H54N2SO5 (628.90): C 68.97, H 8.68, N 4.47, S 5.11; found: C
68.70, H 8.83, N 4.26, S 4.96.
4.1.25. (2
a, 3b) Bis(aminosulfionyloxy)-N-benzyl-olean-12-en-28-
amide (20)
4.1.23. 3
amide (18)
Following GPD, compound 18 (0.51 g, 80 %) was obtained from
b
-Aminosulfonyloxy-N-benzyl-lup-20(29)-en-28-oic
Following GPD, compound 20 (0.52 g, 78 %) was obtained from
15 as a colorless amorphous solid; Rf ¼ 0.15 (n-hexane/ethyl ace-
tate, 7:3); [
a]
¼ þ7.3ꢂ (c 0.30, CHCl3); IR (ATR): v ¼ 3338 m,
D
13 as a colorless amorphous solid; Rf ¼ 0.20 (n-hexane/ethyl ace-
2949 m, 1626 m, 1524w, 1454 m, 1434 m, 1389s, 1367s, 1343vs,
tate, 7:3); [
a
]
¼ þ17.2ꢂ (c 0.345, CHCl3); IR (ATR): v ¼ 3347w,
1172s, 987 m, 950 m, 937 m, 830 m, 819s, 697 m cmꢁ1; 1H NMR
D
2944 m, 2868 m, 1639 m, 1514 m, 1497 m, 1467 m, 1454 m, 1374s,
1362s, 1318w, 1246w, 1211w, 1178vs, 956 m, 930s, 911vs, 881s,
(400 MHz, CD3OD):
d
¼ 7.29e7.26 (m, 5H, 35-H þ 34-H þ 340-
H þ 33-H þ 330-H), 5.32 (dd, J ¼ 3.6 Hz,1H,12-H), 4.72 (ddd, J ¼ 11.6,
9.9, 4.7 Hz, 1H, 2-H), 4.37 (d, J ¼ 14.7 Hz, 1H, 31-Ha), 4.26 (d,
J ¼ 14.4 Hz, 1H, 31-Hb), 4.22 (d, J ¼ 10.0 Hz, 1H, 3-H), 2.83 (dd,
J ¼ 13.3, 4.3 Hz, 1H, 18-H), 2.36 (dd, J ¼ 12.6, 4.7 Hz, 1H, 1-Ha),
2.13e2.02 (m, 1H, 22-Ha), 1.95e1.86 (m, 2H, 11-Ha þ 11-Hb),
1.82e1.73 (m,1H,19-Ha),1.72e1.30 (m, 9H, 9-H þ 7-Ha þ 7-Hb þ 16-
Ha þ 22-Hb þ 6-Ha þ 6-Hb þ 15-Ha þ 21-Ha), 1.30e1.13 (m, 4H, 15-
Hb þ 21-Hb þ 1-Hb þ 19-Hb), 1.17 (s, 3H, H-27), 1.10 (s, 3H, H-23),
1.04e1.00 (m, 1H, 16-Hb), 1.02 (s, 3H, 25-H), 1.01e0.95 (m, 1H, 5-H),
0.95 (s, 3H, 30-H), 0.93 (s, 3H, 24-H), 0.91 (s, 3H, 29-H), 0.58 (s, 3H,
837 m, 698 m cmꢁ1; 1H NMR (400 MHz, CDCl3):
d
¼ 7.36e7.23 (m,
5H, 33-H þ 33‘-H þ 34-H þ 34‘-H þ 35-H), 5.89 (t, J ¼ 5.8 Hz, 1H,
NH-amide), 4.79 (s, 2H, NH2-sulfamate), 4.74 (d, J ¼ 2.4 Hz, 1H, 29-
Ha), 4.60 (dd, J ¼ 2.4, 1.4 Hz,1H, 29-Hb), 4.48 (dd, J ¼ 14.7, 5.7 Hz, 1H,
31-Ha), 4.36 (dd, J ¼ 14.7, 5.6 Hz, 1H, 31-Hb), 4.21 (dd, J ¼ 12.0,
4.7 Hz, 1H, 3-H), 3.16 (td, J ¼ 11.1, 4.1 Hz, 1H, 19-H), 2.50 (ddd,
J ¼ 12.9, 11.3, 3.6 Hz, 1H, 13-H), 2.07e1.96 (m, 2H, 2-Ha þ 21-Ha),
1.97e1.82 (m, 2H, 15-Ha þ 2-Hb), 1.80e1.67 (m, 3H, 22-Ha þ 22-
Hb þ 12-Ha), 1.69 (s, 3H, 30-H), 1.62e1.21 (m, 12H, 18-H þ 15-
Hb þ 1-Ha þ 7-Ha þ 7-Hb þ 21-Hb þ 11-Ha þ 11-Hb þ 16-Ha þ 6-
Ha þ 6-Hb þ 9-H), 1.15e1.08 (m, 1H, 16-Hb), 1.01 (s, 3H, 24-H),
0.99e0.93 (m, 1H, 1-Hb), 0.95 (s, 3H, 27-H), 0.91 (s, 3H, 26-H), 0.85
(s, 3H, 23-H), 0.84 (s, 3H, 25-H), 0.79e0.74 (m, 1H, 5-H) ppm; 13C
26-H) ppm; 13C NMR (101 MHz, CD3OD):
d
¼ 178.6 (C-28), 144.0 (C-
13), 139.0 (C-32), 128.0 (C-34 þ C-340), 127.4 (C-33 þ C-330), 126.7
(C-35), 122.1 (C-12), 90.2 (C-3), 77.3 (C-2), 54.7 (C-5), 47.3 (C-9),
46.2 (C-19), 46.1 (C-17), 44.6 (C-1), 42.9 (C-31), 41.6 (C-14), 41.2 (C-
18), 40.1 (C-4), 39.2 (C-8), 37.9 (C-10), 33.7 (C-21), 32.9 (C-15), 32.1
(C-29), 32.1 (C-7), 30.2 (C-20), 27.8 (C-23), 27.1 (C-16), 25.0 (C-27);
23.2 (C-11), 22.6 (C-30), 22.6 (C-22), 18.0 (C-6), 16.5 (C-24), 16.3 (C-
26), 15.3 (C-25) ppm; MS (ESI, MeOH): m/z 720.3 (100 %, [MþH]þ),
742.2 (84 %, [MþNa]þ), 1102.3 (10 %, [3 Mþ2Na]2þ, 1461.3 (20 %,
NMR (101 MHz, CDCl3):
d
¼ 175.9 (C-28), 150.9 (C-20), 139.1 (C-32),
128.7 (C-34 þ C-34’), 127.8 (C-33 þ C-330), 127.3 (C-35), 109.4 (C-
29), 91.9 (C-3), 55.7 (C-5), 55.6 (C-17), 50.6 (C-9), 50.2 (C-18), 46.7
(C-19), 43.3 (C-31), 42.5 (C-14), 40.8 (C-8), 38.7 (C-4), 38.6 (C-1),
38.4 (C-22), 37.7 (C-13), 37.0 (C-10), 34.3 (C-7), 33.7 (C-15), 30.9 (C-
8

M. Petrenko, A. Güttler, E. Pflüger et al.
European Journal of Medicinal Chemistry 224 (2021) 113721
[2 M þ Na]þ); analysis calcd for C37H57N3S2O7 (720.00): C 61.72, H
7.98, N 5.84; S 8.91; found: C 61.50, H 8.13, N 5.62, S 8.67.
4.2. Cell lines, treatment with pentacyclic triterpenoic acid
derivatives
The human breast cancer cell lines MDA-MB-231, HS578T, MCF-
7 and BT-20 were grown under standard conditions under 37 ꢂC in
a humidified atmosphere containing 5% CO2 in RPMI 1640 medium
(Lonza, Walkersville, MD, USA), enriched with L-Glutamine and
25 mM HEPES with additives of 10 % fetal bovine serum (Capricorn
Scientific, Ebsdorfergrund, Germany), 1% pyruvate (Gibco, Thermo
Fisher Scientific, Waltham, MA, USA), and 2 % penicillin/strepto-
mycin (Sigma-Aldrich, St. Louis, MO, USA). The pentacyclic sulfa-
mate triterpene structures are shown in Fig. 1 and Schemes 1 and 2.
All of the compounds were dissolved in dimethyl sulfoxide (DMSO)
to achieve a 20 mM stock solution. Cells were seeded in cell culture
flasks 24 h before treatment. The treatment was performed for 24 h.
4.3. SRB assay
The cytotoxicity of the drugs was determined by Sulforhod-
amine B (SRB) assay. Treatment started 24 h after seeding the cells
in 96-well plates. BSBA-S (18), OSBA-S (16), USBA-S (17), PSBA-S
(19), and MSBA-S (20) in concentrations between 1 mM and
100 mM were applied for treatment lasting 96 h under standard
conditions. Cells were fixed after incubation by use of 10 % tri-
chloroacetic acid (Carl Roth GmbH, Karlsruhe, Germany). Following
a wash step using iced water, a 0.4 % SRB solution (Sigma-Aldrich)
was applied for staining. Another washing step with 1 % acetic acid
(Carl Roth GmbH) was performed before drying. For extinction
measurement at
l
¼ 540 nm on TECAN GENios FL TWT (Tecan
€
Treading Ag, Mannedorf, Switzerland), fixed cells were dissolved in
300 ml 20 mM Trizma base (Sigma-Aldrich). The IC50 values were
defined as the half-maximal inhibitory concentrations of the
compounds.
Scheme 2. Synthesis of sulfamates 16e20; reactions and conditions: a) NaH, THF
25 ꢂC, 30 min, then ClSO2NH2, 25 ꢂC, 5 d: 16 (83 %), 17 (79 %), 18 (80 %), 19 (75 %), 20
(78 %).
4.4. Clonogenic survival assay and radiosensitivity
Incubation started 24 h after seeding cells in 6-well plates with
Aldrich) and stained with 10% Giemsa solution (Sigma-Aldrich).
Exclusively colonies counting more than around 50 cells were
scored to determine the surviving fraction. The ratio of the number
of colonies formed after irradiation with 6 Gy, 10 Gy or 14 Gy and
concentrations of 40
m
M BSBA-S (18), 40
mM OSBA-S (16), 40 mM
USBA-S (17), 40 M PSBA-S (19), and 10
m
mM MSBA-S (20). Following
24 h of incubation, trypsinization and counting of cells was per-
formed for seeding of cells in small quantities (300 or 600 cells) in
75 cm2 cell culture flasks. Allowing the cells to form colonies for
about 12e14 days, they were fixed with paraformaldehyde (Sigma-
treatment with 5 mM MSBA-S (20) was compared to the number of
colonies formed in unirradiated controls. The dose modification
factor (DMF) was defined as the ratio of the surviving fraction of
DMSO-treated control cells to that of pentacyclic triterpenoic acid
derivatives-treated cells and was dependent on the dose of
irradiation.
4.5. ROS
For measurement of intracellular ROS levels, cells were plated in
6-well plates for 24 h. After treatment with 5
20 M BSBA-S (18) for 24 h, the cells were incubated with the in-
dicator CM-H2DCFDA (Thermo Fisher, Schwerte, Germany) at a
concentration of 0.5 M in PBS (complemented with CaCl2 and
mM MSBA-S (20) or
m
m
MgCl2) for 30 min at 37 ꢂC. Following a washing step with PBS, cells
were incubated with trypsin-EDTA (0.5%) (Life Technologies,
Carlsbad, USA) for 3 min at 37 ꢂC. After decanting Trypsin, cells
were detached with PBS. Fluorescent ROS signals were measured
with an LSRFortessa™ flow cytometer (BD Biosciences, Heidelberg,
Germany).
Scheme 1. Synthesis of compounds 1e15; reactions and conditions: a) Ac2O, NEt3,
DCM, DMAP (cat.), 20 ꢂC, 1 d: 1 (89 %), 2 (89 %), 3 (90 %), 4 (84 %), 5 (92 %); b) (COCl)2,
DCM, DMF (cat.), 20 ꢂC, 1 h: 6 (95 %), 7 (84 %), 8 (84 %), 9 (88 %), 10 (84 %); c) KOH,
MeOH, 20 ꢂC: 11 (87 %), 12 (87 %), 13 (71 %), 14 (92 %), 15 (89 %).
9

M. Petrenko, A. Güttler, E. Pflüger et al.
European Journal of Medicinal Chemistry 224 (2021) 113721
4.6. DAPI analysis
vis spectra, optical rotations as well elemental analysis was per-
formed by M. Schneider. Many thanks are due to Ms E. Merkel and
Dr. S. Sommerwerk for their valuable help in the preparation of
some starting materials.
After treatment with 20
m
M OSBA-S (16), 40
mM BSBA-S (18),
40
m
M USBA-S (17), 40 M PSBA-S (19) and 10 m
m
M MSBA-S (20) for
72 h under standard conditions, cells were trypsinized and
centrifuged at 800 rpm, 4 min with the cell culture supernatant.
The suspension was re-suspended in PBS and cells were fixed with
70% ethanol. Fixed cells were then centrifuged at 1500 rpm, 5 min
with use of a cytospin chamber and fixed on a slide. For analysis of
cell nuclei the fluorescent dye 40,6-diamidino-2-phenylindole-
dihydrochloride (DAPI) (Serva, Heidelberg, Germany) was incu-
bated for 5 min. Further the slide was washed three times with PBS,
cells were covered with ProLong® Gold Antifade Reagent (Life
technologies, Darmstadt, Deutschland) and a cover slip. The mi-
croscopy was performed with BZ-8000 (Keyence, Osaka, Japan), the
apoptotic cell nuclei were counted.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2021.113721.
References
[1] C.E. DeSantis, J. Ma, M.M. Gaudet, L.A. Newman, K.D. Miller, A. Goding Sauer,
A. Jemal, R.L. Siegel, Breast cancer statistics, 2019, Ca - Cancer J. Clin. 69 (2019)
438e451, https://doi.org/10.3322/caac.21583.
[2] R. Thomas, G. Al-Khadairi, J. Decock, Immune checkpoint inhibitors in triple
negative breast cancer treatment: promising future prospects, Front. Oncol. 10
(2020) 600573, https://doi.org/10.3389/fonc.2020.600573.
[3] W.D. Foulkes, I.E. Smith, J.S. Reis-Filho, Triple-negative breast cancer, N. Engl. J.
Med. 363 (2010) 1938e1948, https://doi.org/10.1056/NEJMra1001389.
[4] S. Cleator, W. Heller, R.C. Coombes, Triple-negative breast cancer: therapeutic
4.7. Annexin V-PI assay
Cells were seeded in 6-wells and after growth time of 24 h, cells
options, Lancet Oncol.
2045(07)70074-8.
8 (2007) 235e244, https://doi.org/10.1016/S1470-
were treated with 40 mM BSBA-S (18) or 10 mM MSBA-S (20) for
[5] B. Fisher, S. Anderson, J. Bryant, R.G. Margolese, M. Deutsch, E.R. Fisher, J.-
H. Jeong, N. Wolmark, Twenty-year follow-up of a randomized trial comparing
total mastectomy, lumpectomy, and lumpectomy plus irradiation for the
treatment of invasive breast cancer, N. Engl. J. Med. 347 (2002) 1233e1241,
https://doi.org/10.1056/NEJMoa022152.
24 h. Cell culture supernatant was collected and 1.0 ꢃ 106 cells were
detached with accutase. After medium was detached, cells were re-
suspended in 1 mL 1x binding buffer (10 mM HEPES/NaOH,140 mM
NaCl, 5 mM CaCl2 x H2O, pH ¼ 7.4), 125
incubated for 15 min with the staining solution propidium iodide
(PI) (50 g/mL (Invitrogen, California, US) and/or Annexin V (Invi-
trogen, California, US). Before measurement with flow cytometry
(FACS), 400 l binding buffer was added.
ml of the suspension was
[6] J. Parsons, C. Francavilla, 'Omics approaches to explore the breast cancer
landscape, Front. Cell Dev. Biol.
fcell.2019.00395.
7 (2019) 395, https://doi.org/10.3389/
m
[7] R. Mukherjee, V. Kumar, S.K. Srivastava, S.K. Agarwal, A.C. Burman, Betulinic
acid derivatives as anticancer agents: structure activity relationship, Anti-
m
cancer. Agents Med. Chem.
187152006776930846.
6 (2006) 271e279, https://doi.org/10.2174/
[8] V. Khwaza, O.O. Oyedeji, B.A. Aderibigbe, Ursolic acid-based derivatives as
potential anti-cancer agents: an update, Int. J. Mol. Sci. 21 (2020), https://
doi.org/10.3390/ijms21165920.
4.8. Statistical analysis
Statistical analysis was performed by two-way ANOVA (Dun-
net's test). The significant results were based on a p-value of <0.05
in comparison with DMSO-treated cells.
[9] H. Chen, Y. Gao, A. Wang, X. Zhou, Y. Zheng, J. Zhou, Evolution in medicinal
chemistry of ursolic acid derivatives as anticancer agents, Eur. J. Med. Chem.
92 (2015) 648e655, https://doi.org/10.1016/j.ejmech.2015.01.031.
[10] Z. Zhu, Z. Qian, Z. Yan, C. Zhao, H. Wang, G. Ying, A phase I pharmacokinetic
study of ursolic acid nanoliposomes in healthy volunteers and patients with
advanced solid tumors, Int. J. Nanomed. 8 (2013) 129e136, https://doi.org/
10.2147/IJN.S38271.
Author contributions
[11] M. Ali-Seyed, I. Jantan, K. Vijayaraghavan, S.N.A. Bukhari, Betulinic acid: recent
advances in chemical modifications, effective delivery, and molecular mech-
anisms of a promising anticancer therapy, Chem. Biol. Drug Des. 87 (2016)
517e536, https://doi.org/10.1111/cbdd.12682.
[12] D.-M. Zhang, H.-G. Xu, L. Wang, Y.-J. Li, P.-H. Sun, X.-M. Wu, G.-J. Wang, W.-
M. Chen, W.-C. Ye, Betulinic acid and its derivatives as potential antitumor
agents, Med. Res. Rev. 35 (2015) 1127e1155, https://doi.org/10.1002/
med.21353.
[13] S. Amiri, S. Dastghaib, M. Ahmadi, P. Mehrbod, F. Khadem, H. Behrouj, M.-
R. Aghanoori, F. Machaj, M. Ghamsari, J. Rosik, A. Hudecki, A. Afkhami,
M. Hashemi, M.J. Los, P. Mokarram, T. Madrakian, S. Ghavami, Betulin and its
derivatives as novel compounds with different pharmacological effects, Bio-
M.B, D.V, R.P. and R.C. designed the study; E.P, M.P, A.G. per-
formed the experimental procedures; J.K, Y.E. and A.F. aided in
methods; M.B, A.G, and M.P. analyzed the data and reviewed the
manuscript; R.C, I$S, and M.K. synthesized the chemical drugs and
contributed to the chemical study; M.P. wrote the paper. All authors
read and approved the final manuscript.
Declaration of competing interest
technol.
Adv.
38
(2020)
107409,
https://doi.org/10.1016/
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
j.biotechadv.2019.06.008.
ꢁ
ꢁ
[14] L. Ziberna, D. Samec, A. Mocan, S.F. Nabavi, A. Bishayee, A.A. Farooqi, A. Sureda,
S.M. Nabavi, Oleanolic acid alters multiple cell signaling pathways: implica-
tion in cancer prevention and therapy, Int. J. Mol. Sci. 18 (2017), https://
doi.org/10.3390/ijms18030643.
Acknowledgements
[15] H. Wang, Y. Zhang, W. Yu, Y. Xue, L. Xiao, H. Xu, Hypoxia imaging and bio-
logical evaluation of the radiosensitizing effect of oleanolic acid, BioMed Res.
Int. 2018 (2018) 2694679, https://doi.org/10.1155/2018/2694679.
[16] S. Sommerwerk, L. Heller, C. Kerzig, A.E. Kramell, R. Csuk, Rhodamine B con-
jugates of triterpenoic acids are cytotoxic mitocans even at nanomolar con-
centrations, Eur. J. Med. Chem. 127 (2017) 1e9, https://doi.org/10.1016/
j.ejmech.2016.12.040.
[17] M. Kahnt, L. Heller, P. Grabandt, A. Al-Harrasi, R. Csuk, Platanic acid: a new
scaffold for the synthesis of cytotoxic agents, Eur. J. Med. Chem. 143 (2018)
259e265, https://doi.org/10.1016/j.ejmech.2017.11.046.
[18] S. Zhang, D. Ding, X. Zhang, L. Shan, Z. Liu, Maslinic acid induced apoptosis in
bladder cancer cells through activating p38 MAPK signaling pathway, Mol.
Cell. Biochem. 392 (2014) 281e287, https://doi.org/10.1007/s11010-014-
2038-y.
[19] Y. Tian, H. Xu, A.A. Farooq, B. Nie, X. Chen, S. Su, R. Yuan, G. Qiao, C. Li, X. Li,
X. Liu, X. Lin, Maslinic acid induces autophagy by down-regulating HSPA8 in
pancreatic cancer cells, Phytother, Res. 32 (2018) 1320e1331, https://doi.org/
10.1002/ptr.6064.
This work was supported by Deutsche Forschungsgemeinschaft
(DFG, German Research Foundation) e 410899006. We would like
to thank the colleagues from the Department of Radiotherapy for
their work and support. We are thankful to colleagues from the
Organic Chemistry for their great contribution and provision of the
pentacyclic triterpenoic acid derivatives. Colleagues from the Bio-
zentrum are acknowledged for their advice and collaboration.
Moreover, we would like to thank Dr. Alexander Navarette-Santos
from ZMG, UKH Halle for his support in flow cytometry. We
€
further like to thank Dr. D. Strohl, Y. Schiller and S. Ludwig for taking
the NMR spectra; the MS spectra were recorded by the late Dr. R.
Kluge; additional MS spectra were measured by T. Schmidt. IR, UV/
10

M. Petrenko, A. Güttler, E. Pflüger et al.
European Journal of Medicinal Chemistry 224 (2021) 113721
[20] M.-C. Yin, Anti-glycative potential of triterpenes: a mini-review, Biomedicine
2 (2012) 2e9, https://doi.org/10.1016/j.biomed.2011.12.001.
[21] M.N. Laszczyk, Pentacyclic triterpenes of the lupane, oleanane and ursane
group as tools in cancer therapy, Planta Med. 75 (2009) 1549e1560, https://
doi.org/10.1055/s-0029-1186102.
[22] V.R. Yadav, S. Prasad, B. Sung, R. Kannappan, B.B. Aggarwal, Targeting in-
flammatory pathways by triterpenoids for prevention and treatment of can-
cer, Toxins 2 (2010) 2428e2466, https://doi.org/10.3390/toxins2102428.
[23] B.S. Gill, S. Kumar, Navgeet, Triterpenes in cancer: significance and their in-
fluence, Mol. Biol. Rep. 43 (2016) 881e896, https://doi.org/10.1007/s11033-
016-4032-9.
triterpenoic acid amides - the substantial role of an aromatic moiety align-
ment, Eur. J. Med. Chem. 122 (2016) 452e464, https://doi.org/10.1016/
j.ejmech.2016.06.053.
[32] Y. Ren, A.D. Kinghorn, Natural product triterpenoids and their semi-synthetic
derivatives with potential anticancer activity, Planta Med. 85 (2019) 802e814,
https://doi.org/10.1055/a-0832-2383.
[33] R. Yin, T. Li, J.X. Tian, P. Xi, R.H. Liu, Ursolic acid, a potential anticancer com-
pound for breast cancer therapy, Crit. Rev. Food Sci. Nutr. 58 (2018) 568e574,
https://doi.org/10.1080/10408398.2016.1203755.
[34] M. Bache, M.P. Zschornak, S. Passin, J. Kessler, H. Wichmann, M. Kappler,
ꢀ
R. Paschke, G.N. KaluCerovic, H. Kommera, H. Taubert, D. Vordermark,
[24] J.-Y. Winum, S. Pastorekova, L. Jakubickova, J.-L. Montero, A. Scozzafava,
J. Pastorek, D. Vullo, A. Innocenti, C.T. Supuran, Carbonic anhydrase inhibitors:
synthesis and inhibition of cytosolic/tumor-associated carbonic anhydrase
isozymes I, II, and IX with bis-sulfamates, Bioorg. Med. Chem. Lett 15 (2005)
579e584, https://doi.org/10.1016/j.bmcl.2004.11.058.
[25] S. Schwarz, S. Sommerwerk, S.D. Lucas, L. Heller, R. Csuk, Sulfamates of methyl
triterpenoates are effective and competitive inhibitors of carbonic anhydrase
II, Eur. J. Med. Chem. 86 (2014) 95e102, https://doi.org/10.1016/
j.ejmech.2014.08.051.
[26] K. Vanchanagiri, D. Emmerich, M. Bruschke, M. Bache, F. Seifert, R. Csuk,
D. Vordermark, R. Paschke, Synthesis and biological investigation of new
carbonic anhydrase IX (CAIX) inhibitors, Chem. Biol. Interact. 284 (2018)
12e23, https://doi.org/10.1016/j.cbi.2018.02.014.
[27] R. McKenna, C.T. Supuran, Carbonic anhydrase inhibitors drug design, Subcell.
Biochem. 75 (2014) 291e323, https://doi.org/10.1007/978-94-007-7359-2_
15.
Increased betulinic acid induced cytotoxicity and radiosensitivity in glioma
cells under hypoxic conditions, Radiat. Oncol. 6 (2011) 111, https://doi.org/
10.1186/1748-717X-6-111.
[35] Y. Tan, R. Yu, J.M. Pezzuto, Betulinic acid-induced programmed cell death in
human melanoma cells involves mitogen-activated protein kinase activation,
Clin. Canc. Res. 9 (2003) 2866e2875.
[36] H. Yu, H. Zhang, Z. Chu, Q. Ruan, X. Chen, D. Kong, X. Huang, H. Li, H. Tang,
H. Wu, Y. Wang, W. Xie, Y. Ding, P. Yao, Combination of betulinic acid and
chidamide synergistically inhibits Epstein-Barr virus replication through over-
generation of reactive oxygen species, OncoTargets 8 (2017) 61646e61661,
https://doi.org/10.18632/oncotarget.18661.
[37] B. Chakraborty, D. Dutta, S. Mukherjee, S. Das, N.C. Maiti, P. Das,
C. Chowdhury, Synthesis and biological evaluation of a novel betulinic acid
derivative as an inducer of apoptosis in human colon carcinoma cells (HT-29),
Eur. J. Med. Chem. 102 (2015) 93e105, https://doi.org/10.1016/
j.ejmech.2015.07.035.
[28] S. Sommerwerk, L. Heller, R. Csuk, Synthesis and cytotoxic activity of penta-
cyclic triterpenoid sulfamates, Arch. Pharm. (Weinheim) 348 (2015) 46e54,
https://doi.org/10.1002/ardp.201400297.
[29] M. Bache, C. Münch, A. Güttler, H. Wichmann, K. Theuerkorn, D. Emmerich,
R. Paschke, D. Vordermark, Betulinyl sulfamates as anticancer agents and
radiosensitizers in human breast cancer cells, Int. J. Mol. Sci. 16 (2015)
26249e26262, https://doi.org/10.3390/ijms161125953.
[30] M. Petrenko, A. Güttler, A. Funtan, J. Keßler, D. Emmerich, R. Paschke,
D. Vordermark, M. Bache, Combined 3-O-acetylbetulin treatment and car-
bonic anhydrase IX inhibition results in additive effects on human breast
cancer cells, Chem. Biol. Interact. 333 (2021) 109326, https://doi.org/10.1016/
j.cbi.2020.109326.
[38] N. Yao, Y.-J. Li, Y.-H. Lei, N. Hu, W.-M. Chen, Z. Yao, M. Yu, J.-S. Liu, W.-C. Ye, D.-
M. Zhang, A piperazidine derivative of 23-hydroxy betulinic acid induces a
mitochondria-derived ROS burst to trigger apoptotic cell death in hepato-
cellular carcinoma cells, J. Exp. Clin. Canc. Res. 35 (2016) 192, https://doi.org/
10.1186/s13046-016-0457-1.
[39] E. Selzer, E. Pimentel, V. Wacheck, W. Schlegel, H. Pehamberger, B. Jansen,
R. Kodym, Effects of betulinic acid alone and in combination with irradiation
in human melanoma cells, J. Invest. Dermatol. 114 (2000) 935e940, https://
doi.org/10.1046/j.1523-1747.2000.00972.x.
€
[40] C. Eder-Czembirek, B.M. Erovic, C. Czembirek, M. Brunner, E. Selzer, R. Potter,
D. Thurnher, Betulinic acid a radiosensitizer in head and neck squamous cell
carcinoma cell lines, Strahlenther. Onkol. 186 (2010) 143e148, https://
doi.org/10.1007/s00066-010-2069-6.
[31] S. Sommerwerk, L. Heller, J. Kuhfs, R. Csuk, Selective killing of cancer cells with
11