The presence of a cationic center is not alone decisive for the cytotoxicity of triterpene carboxylic acid amides

Article abstract of DOI:10.1016/j.steroids.2020.108713

3-O-Acetyl-ursolic acid (2) and 3-O-acetyl oleanolic acid (8) were converted into piperazinylamides holding a distal NH, NMe or a NMe₂ group. These compounds as well as the corresponding N-methyl-N-oxides were accessed. Their cytotoxicity was assessed in SRB assays employing a panel of human tumor cell lines and non-malignant fibroblasts (NIH 3T3). As a result, compounds holding a quaternary distal N-substituent were less cytotoxic that those holding a NH-moiety. Hence, the presence of a distal cationic center seems not to be a sufficient criterion for obtaining triterpenoids of high cytotoxicity and selectivity.

Full text of DOI:10.1016/j.steroids.2020.108713

Steroids163(2020)108713
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Steroids
journal homepage: www.elsevier.com/locate/steroids
The presence of a cationic center is not alone decisive for the cytotoxicity of
triterpene carboxylic acid amides
Benjamin Brandesa, Lukas Kocha, Sophie Hoenkea, Hans-Peter Deignerb, René Csuka,⁎
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
A R T I C L E I N F O
A B S T R A C T
Keywords:
Oleanolic acid
Ursolic acid
Amides
N-oxide
Cytotoxicity
3-O-Acetyl-ursolic acid (2) and 3-O-acetyl oleanolic acid (8) were converted into piperazinylamides holding a
distal NH, NMe or a NMe2 group. These compounds as well as the corresponding N-methyl-N-oxides were ac-
cessed. Their cytotoxicity was assessed in SRB assays employing a panel of human tumor cell lines and non-
malignant fibroblasts (NIH 3T3). As a result, compounds holding a quaternary distal N-substituent were less
cytotoxic that those holding a NH-moiety. Hence, the presence of a distal cationic center seems not to be a
sufficient criterion for obtaining triterpenoids of high cytotoxicity and selectivity.
1. Introduction
2. Results and discussion
Attempts to successfully treat cancer are more necessary than ever,
as 9.6 million people worldwide died in 2018, and globally about 1 in 6
deaths is due to cancer [1]. Many of the novel therapeutics are derived
from natural products, such as best-sellers vincristine [2–4], paclitaxel
[5–7], etoposide [8,9], irinotecan [10–12], docetaxel [13–16] and to-
potecan [17–20]. In recent years, terpenes, especially triterpene car-
boxylic acids [21–40], have again become the focus of scientific in-
terest. In particular, esters and amides of betulinic acid, oleanolic acid,
ursolic acid and maslinic acid showed sufficiently high cytotoxicity
2.1. Chemistry
Acetylation (Scheme 1) of ursolic acid (1) gave 3-O-acetyl-ursolic
acid (2) [55]. Reaction of 2 with oxalyl chloride in DCM in the presence
of catal. amounts DMF followed by a condensation reaction with pi-
perazine gave amide (3).[56–58] Under similar conditions from N-
methyl-piperazine compound 4 [59] was obtained in almost quantita-
tive yield. Reaction of 3 with an excess of methyl iodide for 20 h gave
82% of 5, while the oxidation of 4 with hydrogen peroxide for two days
yielded 96% of N-oxide 6. While a few ammonium salts of triterpenoids
have been investigated for their cytotoxic potential, the number of
complex molecules holding a piperazinyl derived N-oxide remained
small over the years. Thus, a N-oxide derived from the aminosteroid
RM-133 was shown of negligible low cytotoxicity against HL-60, PANC-
1 and OVCAR-3 cancer cells [60].
In a similar way as described above, acetylation of oleanolic acid (7)
gave 3-O-acetyl-oleanolic acid (8) [61]. Activation with oxalyl chloride
followed by its reaction with piperazine or N-methyl-piperazine gave
amides 9 [55] and 10 [62], respectively. Quaternisation of 9 gave
ammonium salt 11 while from the hydrogen peroxide oxidation of 10N-
oxide 12 was obtained.
towards
a
large number of human tumor cell lines
[24,25,27,28,30,41–48]. The attachment of a cationic residue to the
triterpenoid skeleton proved to be particularly advantageous, since this
improved the notoriously poor solubility of triterpene carboxylic acids
and – as a consequence – also the bioavailability. Furthermore, parti-
cularly in the case of derivatives derived from rhodamine B an in-
creased inward transport into mitochondria was observed thus trig-
gering apoptosis [47–49]. A slightly increased cytotoxicity has also
been reported for triterpenoids holding an extra ammonium [50,51] or
phosphonium group [52–54]. In extension to these findings we became
interested in the synthesis and biological evaluation of ursolic and
oleanolic acid derived amides holding a distal secondary, tertiary or
quaternary amino group.
3. Biology
The compounds were subjected to sulforhodamine B (SRB) assays to
assess their cytotoxicity employing several human tumor cell lines, i.e.
⁎ Corresponding author.
https://doi.org/10.1016/j.steroids.2020.108713
Received 27 June 2020; Received in revised form 26 July 2020; Accepted 8 August 2020
Availableonline12August2020
0039-128X/©2020ElsevierInc.Allrightsreserved.

B. Brandes, et al.
Steroids163(2020)108713
Scheme 1. Reactions and conditions: a) Ac2O, pyridine, 25 °C, 1 d, 77% (of 2), 82% (of 8); b) (COCl)2, catal. DMF, DCM, 0 °C → r.t., 2 h; 2) piperazine, NEt3, DCM,
17 h, r.t., 84%; c) (COCl)2, catal. DMF, DCM, 0 °C → r.t., 2 h; 2) N-methylpiperazine, NEt3, DCM, 17 h, r.t., 99%; d) K2CO3, MeI, ACN, 20 h, r.t. 82%; e) H2O2, MeOH,
50 °C, 2 d, 96%; f) (COCl)2, catal. DMF, DCM, 0 °C → r.t., 2 h; 2) piperazine, NEt3, DCM, 17 h, r.t., 80%; c) (COCl)2, catal. DMF, DCM, 0 °C → r.t., 2 h; 2) N-
methylpiperazine, NEt3, DCM, 17 h, r.t., 86%; d) K2CO3, MeI, ACN, 20 h, r.t. 53%; e) H2O2, MeOH, 50 °C, 2 d, 99%.
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%; cut-off 30 μM; n.d. not detected/not determined, n.s. not soluble under the conditions of the assay. Doxorubicin (DX) and betulinic
acid (BA) were used as a positive standard.
A375
HT29
MCF-7
A2780
FaDu
NIH 3T3
1
3
4
5
6
7
9
12.3
1.8
4.1
> 30
12.9
> 30
1.8
0.9
0.1
0.2
10.6
2.4
7.3
> 30
14.6
> 30
1.3
0.7
0.1
0.2
12.7
1.9
5.5
> 30
8.8
> 30
1.7
0.1
0.2
11.7
2.3
0.6
0.2
0.7
14.7
2.2
5.3
> 30
11.2
> 30
1.6
1.1
0.2
0.2
13.1
2.7
1.1
0.2
1.0
0.2
0.4
0.7
0.2
5.14
> 30
11.1
> 30
1.7
11.1
> 30
15.3
> 30
1.7
1.3
0.7
1.8
0.2
1.6
0.7
0.3
0.1
0.1
10
11
12
BA
DX
n.s.
n.s.
n.s.
> 30
9.6
12.0
1.1
n.s.
n.s.
n.s.
> 30
11.8
14.4
1.0
> 30
12.0
18.4
0.9
> 30
11.2
12.7
0.01
> 30
11.1
13.8
0.8
> 30
18.2
16.1
0.01
1.4
1.3
0.8
2.0
2.0
0.2
0.8
1.7
0.3
1.0
1.8
0.01
1.0
2.1
0.2
2.1
1.4
0.001
2

B. Brandes, et al.
Steroids163(2020)108713
A375 (melanoma), HT29 (colorectal carcinoma), MCF-7 (breast ade-
nocarcinoma), A2780 (ovarian carcinoma) and FaDu (hypopharyngeal
carcinoma) as well as non-malignant fibroblasts (NIH 3T3). The results
from these assays (EC50 values after an incubation time of 72 h) are
compiled in Table 1.
oxalyl chloride (0.385 mL, 2.0 mmol) and a catal. amount of DMF was
added. The mixture was stirred for 2 h at room temperature. The vo-
latiles were removed under diminished pressure, dry THF (25 mL) was
added, and evaporated. The residue was dissolved in dry DCM (15 mL)
and added within 30 min to a solution of dry piperazine (330 mg,
3.8 mmol), triethylamine (142 mL, 1.9 mmol) in dry DCM (30 mL) at
−15 °C. After stirring at room temperature for 17 h followed by usual
aqueous workup and chromatography (silica gel, CHCl3/MeOH, gra-
dient 0% MeOH → 5% MeOH), 3 (418 mg, 84%) was obtained as a
colorless solid; m.p. 162 °C (decomp.) (lit.: [64] 158–161 °C);
[α]D = +38.2° (c 0.191, CHCl3); RF = 0.6 (CHCl3/MeOH, 9:1); IR
(KBr): ν = 698 m, 751 m, 979 m, 1027 m, 1080w, 1109w, 1139w,
1162w, 1193 m, 1244 s, 1367 m, 1454 m, 1518 m, 1646 m, 1709 m,
1732 m, 2868w, 2943 m cm−1; UV/Vis (CHCl3): λmax (log ε) = 226
(3.66) nm; 1H NMR (400 MHz, CDCl3): δ = 5.23–5.18 (m, 1H, 12-H),
4.52–4.45 (m, 1H, 3-H), 3.79–3.75 (m, 4H, 33-H2 + 33′-H2), 2.95–2.89
(m, 4H, 34-H2 + 34′-H2), 2.41 (d, J = 10.9, 1H, 18-H1), 2.26–2.09 (m,
1H, 16-Ha), 2.04 (s, 3H, 32-H3), 1.95–1.86 (m, 3H, 11-H2 + 16-Hb),
1.78–1.65 (m, 2H, 22-H2), 1.66–1.56 (m, 3H, 1-Ha + 2-H2), 1.56–1.20
(m, 8H, 6-H2 + 7-H2 + 9-H + 19-H + 21-H2), 1.12–0.98 (m, 5H, 1-
H2 + 15-Hb + 27-H3), 0.97–0.91 (m, 7H, 20-H + 25-H3 + 30-H3),
0.90–0.78 (m, 11H, 5-H + 15-Hb + 23-H3 + 24-H3 + 29-H3), 0.73 (s,
3H, 26-H3) ppm; 13C NMR (101 MHz, CDCl3): δ = 175.6 (C-28), 171.2
(C-31), b138.7 (C-13), 125.4 (C-12), 81.1 (C-3), 55.5 (C-5), 55.0 (C-18),
48.7 (C-17), 47.7 (C-9), 45.5 (C-33 + C-33′ + C-34 + C-34′), 42.3 (C-
14), 39.6 (C-8), 39.6 (C-19), 38.9 (C-20), 38.4 (C-1), 37.8 (C-4), 37.1
(C-10), 34.5 (C-22), 33.1 (C-7), 30.6 (C-21), 28.2 (C-15), 28.2 (C-23),
23.7 (C-2), 23.7 (C-27), 23.5 (C-11), 21.5 (C-32), 21.5 (C-16) 21.4 (C-
30), 18.3 (C-6), 17.6 (C-29), 17.3 (C-26), 16.9 (C-24), 15.6 (C-25) ppm;
As a result, cytotoxicity decreases for compounds holding a qua-
ternary nitrogen substituent (EC50 > 30 μM). Slightly better results
were obtained for the N-oxides 6 and 12. Furthermore, the results from
these assays show the substitution pattern of the distal piperazinyl ni-
trogen of significant influence onto the cytotoxicity of the compounds.
Thus, derivatives holding a remote NH moiety were more cytotoxic
than their N-methylated analogs. While the exact mechanism still re-
mains unclear it appears that the presence of a lipophilic cation in the
conjugates is not a sufficient for achieving good cytotoxicity. The
acetylated piperazinylamides 3 and 9 showed cytotoxic effects com-
parable to those of doxorubicin but with a slightly worse selectivity.
However, they represent a promising starting point for the further de-
velopment of cytostatics derived from triterpene carboxylic acids.
4. Conclusion
Ursolic acid (1) and oleanolic acid (7) were converted into their
respective 3-O-acetates. The latter compounds were transformed into
piperazinylamides. These compounds as well as their N- and N,N-di-
methylated analogs and N-methyl-N-oxides were accessed in good
yields and subjected to SRB assays to assess their cytotoxicity em-
ploying a panel of human tumor cell lines and non-malignant fibro-
blasts (NIH 3T3). As a result, and contrary to some expectations,
compounds holding a quaternary distal N-substituent were less cyto-
toxic that those holding a NH-moiety. Hence, the presence of a distal
cationic center seems not to be a sufficient criterion for assessing tri-
terpenoids of high cytotoxicity. Furthermore the selectivity was di-
minished for these compounds; the best result was obtained for com-
pound, a N-methyl-piperazinyl derivative of ursolic acid holding a
selectivity factor of 2.7 for A375 melanoma cells/non-malignant fi-
broblasts NIH 3T3.
MS (ESI, MeOH): m/z
36H58N2O3 (566.87): C 76.28, H 10.31, N 4.94; found: C 75.97, H
10.51, N 4.76.
=
567.5 ([M+H]+); analysis calcd for
C
8. 28-(4-Methyl-piperazin-1-yl)-28-oxours-12-en-3 β-yl acetate (4)
Following the procedure given for the synthesis of 3, from 2 (1.0 g,
2.0 mmol) and N-methylpiperazine (450 mL, 5.0 mmol), 4 (1.1 g, 99%)
5. Experimental
was obtained as a colorless solid; m.p. 139–141 °C (decomp.);
[α]D = 34.6° (c 0.16, CHCl3); RF = 0.59 (CHCl3/MeOH, 9:1); IR (KBr):
ν = 698 m, 751 m, 979 m, 1027 m, 1080w, 1109w, 1139w, 1162w,
1193 m 1244 s, 1367 m, 1454 m, 1518 m, 1646 m, 1709 m, 1732 m,
2868w, 2943 m cm−1; UV/Vis (CHCl3): λmax (log ε) = 226 (3.60) nm;
1H NMR (400 MHz, CDCl3): δ = 5.23–5.18 (m, 1H, 12-H), 4.52–4.45
(m, 1H, 3-H), 3.85–3.65 (m, 4H, 33-H2 + 33′-H2), 2.57 (s, 4H, 33-
H2 + 33′-H2), 2.44–2.39 (m, 4H, 18-H + 35-H3), 2.20–2.07 (m, 1H, 16-
Ha), 2.05–2.02 (s, 3H, 32-H3), 1.94–1.88 (m, 3H, 11-H2 + 16-Hb),
1.75–1.68 (m, 1H, 22-Ha), 1.68–1.57 (m, 5H, 1-H2 + 2H2 + 22-Hb),
1.55–1.42 (m, 4H, 6-Ha + 7-Ha + 9-H + 21-Ha), 1.42–1.33 (m, 2H, 6-
Hb + 19-H), 1.33–1.25 (m, 2H, 7-Hb + 21-Hb), 1.06 (s, 3H, 27-H3),
NMR spectra were recorded using the Varian spectrometers Gemini
2000 or Unity 500 (δ given in ppm, J in Hz; typical experiments:
HeHeCOSY, HMBC, HSQC, NOESY), MS spectra were taken on a
Finnigan MAT LCQ (electrospray, voltage 4.1 kV, sheath gas nitrogen)
instrument. The optical rotations were measured on a Jasco P-200 po-
larimeter at 20 °C; TLC was performed on silica gel (Merck 5554, de-
tection with cerium molybdate reagent); melting points are uncorrected
(Büchi Melting Point M-565), and elemental analyses were performed
on a Foss-Heraeus Vario EL (C-HNS) unit. IR spectra were recorded on a
Perkin Elmer FT-IR spectrometer Spectrum Two; for UV/Vis a Perkin
Elmer Lambda 750S instrument was used. The solvents were dried ac-
cording to usual procedures. The purity of the compounds was de-
termined by HPLC and found to be > 96%. Ursolic acid (1) and olea-
nolic acid (7) and were obtained from Betulinines (Stříbrná Skalice,
Czech Republic) in bulk quantities and used as received.
1.09–1.03 (m, 1H, 15-Hb), 1.02–0.96 (m, 2H, 15-Hb
+ 20-H),
0.96–0.92 (m, 6H, 25-H3 + 30-H3), 0.90–0.79 (m, 9H, 23-H3 + 24-
H3 + 29-H3), 0.84–0.79 (m, 1H, 5-H) 0.73 (s, 3H, 26-H3) ppm; 13C
NMR (101 MHz, CDCl3): δ = 175.6 (C-28), 171.2 (C-31), 138.6 (C-13),
125.4 (C-12), 81.1 (C-3), 55.5 (C-5), 54.7 (C-18 + C-34 + C-34′), 48.7
(C-17), 47.7 (C-9), 45.3 (C-35), 44.5 (C-33 + C-33′), 42.3 (C-14), 39.6
(C-19), 39.6 (C-8), 38.9 (C-20), 38.4 (C-1), 37.8 (C-4), 37.1 (C-10), 34.5
(C-22), 33.1 (C-7), 30.6 (C-21), 28.3 (C-15), 28.2 (C-23), 23.8 (C-27),
23.7 (C-2), 23.4 (C-11), 23.3 (C-32), 21.4 (C-16), 21.4 (C-30), 18.3 (C-
6), 17.6 (C-29), 17.0 (C-26), 16.9 (C-24), 15.6 (C-25) ppm; MS (ESI,
MeOH): m/z = 580.3 ([M+H]+); analysis calcd for C37H60N2O3
(580.90): C 76.50, H 10.41, N 4.82; found: C 76.39, H 10.63, N 4.61.
6. 3β-Acetyloxy-urs-12-en-28-oic acid (2)
This compound was obtained from the acetylation of ursolic acid (1,
10.01 g, 17.6 mmol) with acetic anhydride in dry pyridine followed by
recrystallization from MeOH; 2 (8.43 g, 77%) was obtained as a col-
orless solid. An analytical sample showed m.p. 279–281 °C (lit.: [63]
279–280 °C).
9. 3 β-Acetyloxy-28-(4,4-dimethylpiperazin-4-ium-1-yl)-28-
oxours-12-ene iodide (5)
7. 28-Oxo-28-(1-piperazinyl)urs-12-en-3 β-yl acetate (3)
To a solution of 2 (0.5 g, 1.0 mmol) in dry DCM (50 mL) at 0 °C
To a solution of 3 (130 mg, 0.23 mmol) in dry ACN (5 mL) at 25 °C
3

B. Brandes, et al.
Steroids163(2020)108713
finely ground K2CO3 (63 mg, 0.46 mmol), methyl iodide (260 μL,
0.8 mmol) were added, and the mixture was stirred at room tempera-
ture for 20 h. The volatiles were removed under diminished pressure,
and the residue was subjected to chromatography (silica gel, CHCl3/
MeOH, gradient 0% MeOH → 5% MeOH) to yield 5 (136 mg, 82%) as a
colorless solid; m.p. 303 °C (decomp.); [α]D = +17.6° (c 0.29, CHCl3);
RF = 0.37 (CHCl3/MeOH, 9:1); IR (KBr): ν = 961 m, 1014 m, 1025 s,
1090w, 1106w, 1147 m, 1153w, 1206 m, 1260 s, 1301w, 1362 s,
1387 m, 1466 m, 1639 s, 1691w, 1733 s, 2910 m, 2941 m, 2953 m,
2966 m cm−1; UV/Vis (CHCl3): λmax (log ε) = 229 (3.99) nm; 1H NMR
(500 MHz, CD3OD): 1H NMR (500 MHz, CD3OD) δ 5.27 (t, J = 3.9 Hz,
1H, 12-H), 4.51 (dd, J = 11.4, 5.0 Hz, 1H, 3-H), 4.11–4.08 (m, 2H, 33-
H2), 4.02–3.99 (m, 2H, 33′-H2), 3.50–3.44 (m, 4H, 34-H2 + 34′-H2),
3.32–3.28 (m, 6H, 35-H3 + 36-H3), 2.43 (d, J = 11.1 Hz, 1H, 18-H),
2.35–2.26 (m, 1H, 16-Ha), 2.05 (s, 3H, 32-H3), 2.02–1.96 (m, 2H, 11-
H2), 1.96–1.83 (m, 4H, 1-H2 + 16-Hb + 21-Ha), 1.78–1.55 (m, 8H, 2-
H2 + 6-Ha + 7-H2 + 9-H + 20-H + 21-Hb), 1.55–1.33 (m, 4H, 6-
Hb + 19-H + 22-H2), 1.14 (s, 3H, 27-H3), 1.23–1.07 (m, 2H, 15-H2),
1.0–0.97 (m, 6H. 25-H3 + 30-H3), 0.98–0.95 (m, 3H, 29-H3), 0.90–0.87
(m, 6H, 23-H3 + 24-H3), 0.87–0.85 (m, 1H, 5-H), 0.84–0.80 (m, 3H,
26-H3) ppm; 13C NMR (125 MHz, CD3OD): δ = 182.5 (C-28), 171.5 (C-
31), 125.4 (C-13), 125.3 (C-12), 81.1 (C-3), 61.1 (C-34 + C-34′), 55.3
(C-5), 55.0 (C-18), 50.7 (C-35 + C-36), 48.7 (C-17), 47.4 (C-9), 41.9 (C-
14), 39.4 (C-8), 39.2 (C-33 + C-33′), 39.2 (C-19), 38.0 (C-20), 37.3 (C-
4), 36.7 (C-10), 33.9 (C-1), 32.6 (C-22), 30.1 (C-7), 28.4 (C-21), 28.0
(C-15), 27.2 (C-23), 23.2 (C-16), 23.0 (C-2), 22.9 (C-11), 22.8 (C-27),
20.0 (C-30), 19.7 (C-32), 17.9 (C-6), 16.5 (C-29), 16.0 (C-26), 15.8 (C-
24), 25.3 (C-25) ppm; MS (ESI, MeOH): m/z = 595.5 ([M]+); analysis
calcd for C38H63N2O3I (722.84): C 63.14, H 8.79, N 3.88; found: C
62.84, H 8.99, N 3.59.
11. 3 β-Acetyloxy-olean-12-en-28-oic acid (8)
This product (8, 9.0 g, 82%) was obtained from the acetylation of
oleanolic acid (7, 10.01 g, 17.6 mmol) with acetic anhydride in dry
pyridine followed by re-crystallization from MeOH as a colorless solid;
an analytical sample showed m.p. 258–260 °C, lit.: [65] 264–265 °C.
12. 28-Oxo-28-(1-piperazinyl)-olean-12-en-3 β-yl acetate (9)
Following the synthesis of 3, from 8 (500 mg, 1.0 mmol), 9 (400 mg,
80%) was obtained as a colorless solid; m.p. 171 °C (decomp.), lit.
170–176 [64].
13. 28-(4-Methyl-piperazin-1-yl)-28-oxoolean-12-en-3 β -yl
acetate 10)
Following the synthesis of 4, from 8 (470 mg, 0.94 mmol) and 4-
methylpiperazine (220 μL, 2.4 mmol) followed by chromatography
(silica gel, CHCl3/MeOH, gradient 0% MeOH → 5% MeOH), 10
(472 mg, 86%) was obtained as a colorless solid; m.p. 216–218 °C
(decomp.) (lit.: [62] 218–220 °C); [α]D = +44.0° (c 0.305, CHCl3);
RF = 0.56 (CHCl3/MeOH, 9:1); IR (KBr): ν = 1004 s, 1023 m, 1076w,
1143 m, 1167w, 1205 m, 1244 s, 1286w, 1370 m, 1392 m, 1398w,
1411 m, 1457 m, 1616 s, 1734 s, 2746w, 2791w, 2848w, 2941 m cm−1
;
UV/Vis (CHCl3): λmax (log ε) = 227 (3.65) nm; 1H NMR (400 MHz,
CDCl3): δ = 5.39–5.13 (m, 1H, 12-H), 4.65–4.32 (m, 1H, 3-H),
3.75–3.70 (m, 4H, 33-H2
+ 33′-H2), 3.23–2.98 (m, 1H, 18-H),
2.66–2.26 (m, 7H, 34-H2 + 34′-H2 + 35-H3), 2.20–2.00 (m, 5H, 2-
Ha + 16-Ha + 32-H3), 1.99–1.78 (m, 2H, 11-H2), 1.76–1.48 (m, 8H, 1-
Ha + 2-Hb + 6-Ha, 9-H, 16-Hb, 19-Ha, 22-H2), 1.50–1.22 (m, 4H, 6-
Hb + 7-H2 + 21-Ha), 1.22–1.12 (m, 5H, 19-Hb + 21-Hb + 27-H3),
1.11–0.97 (m, 3H, 1-Hb, 15-H2), 0.95–0.80 (m, 16H, 5-H + 23-
10. 28-(4-Methyl-4-oxido-piperazin-1-yl)-28-oxours-12-en-3 β-yl
acetate (6)
H C
3 + 24-H3, 25-H3, 29-H3, 30-H3), 0.74–0.70 (m, 3H, 26-H3) ppm; 13
NMR (101 MHz, CDCl3): δ = 175.0 (C-28), 171.0 (C-31), 144.7 (C-13),
121.5 (C-12), 80.9 (C-3), 55.4 (C-5), 55.0 (C-34 + C-34′), 47.7 (C-9),
47.5 (C-17), 46.4 (C-19), 45.8 (C-35), 44.9 (C-33 + C-33′), 43.6 (C-18),
41.8 (C-14), 39.1 (C-8), 38.1 (C-1), 37.7 (C-4), 37.0 (C-10), 34.0 (C-21),
33.0 (C-29), 32.8 (C-7), 30.4 (C-20), 30.1 (C-22), 28.0 (C-23), 27.9 (C-
15), 25.9 (C-27), 24.1 (C-30), 23.5 (C-11), 23.4 (C-16), 22.8 (C-2), 21.3
(C-32), 18.2 (C-6), 16.9 (C-26), 16.7 (C-24), 15.4 (C-25) ppm; MS (ESI,
MeOH): m/z = 581.5 ([M+H]+), 1161.6 ([2M+H]+), 1183.8 ([2M
+Na]+); analysis calcd for C37H60N2O3 (580.90): C 76.50, H 10.41, N
4.82; found: C 76.32, H 10.60, N 4.67.
To a solution of 4 (500 mg, 086 mmol) in MeOH (15 mL) at room
temperature an aqueous solution of H2O2 (35%, 370 μL, 4.3 mmol) was
added. The mixture was stirred at 50 °C for 24 h, another H2O2 (35%,
370 μL, 4.3 mmol) was added, and stirring was continued for another
24 h. The volatiles were removed under reduced pressure and the re-
sidue was subjected to chromatography (silica gel, CHCl3/MeOH, gra-
dient 0% MeOH → 30% MeOH) to yield 6 (492 mg, 96%) as a colorless
solid; m.p. 203 °C (decomp.); [α]D
= +29.9° (c 0.29, CHCl3);
RF = 0.25 (CHCl3/MeOH, 9:1); IR (KBr): ν = 984 m, 1005w, 1026 m,
1105w, 1132w, 1207w, 1245 s, 1370 m, 1391 m, 1453 m, 1630 m,
1731 m, 2872 m, 2926 m, 2945 m cm−1; UV/Vis (CHCl3): λmax (log
ε) = 227 (3.36) nm; 1H NMR (500 MHz, CDCl3): δ = 5.21–5.16 (m, 1H,
12-H), 4.50–4.43 (m, 1H, 3-H), 4.35–4.28 (m, 1H, 34-Ha), 4.27–4.20
(m, 1H, 35-Ha), 3.88–3.64 (m, 2H, 34-Hb + 34-Hb), 3.62–3.35 (m, 2H,
33-Ha + 36-Ha), 3.34 (s, 3H, 37-H3), 3.26–3.08 (m, 2H, 33-Hb + 36-
Hb), 2.39–2.34 (m, 1H, 18-H), 2.21–2.11 (m, 1H, 16-Ha), 2.02 (s, 3H,
32-H3), 1.92–1.86 (m, 2H, 11-H2), 1.76–1.66 (m, 3H, 15-Ha + 22-H2),
1.65–1.54 (m, 4H, 2-H2 + 20-H + 21-Ha), 1.54–1.41 (m, 6H, 1-Ha + 6-
Ha + 9-H + 16-Hb + 19-H + 21-Hb), 1.41–1.23 (m, 3H, 7-H2 + 6-Hb),
1.12–1.07 (m, 1H, 1-Hb), 1.05 (s, 3H, 27-H3), 1.03–0.95 (m, 1H, 15-Hb),
1.0–0.89 (m, 6H, 30-H3 + 25-H3), 0.88–0.77 (m, 9H, 23-H3 + 24-
H3 + 29-H3), 0.82–0.78 (m, 1H, 5-Hb), 0.69 (s, 3H, 26-H3) ppm; 13C
NMR (125 MHz, CDCl3): δ = 175.6 (C-28), 170.9 (C-31), 138.2 (C-13),
125.2 (C-12), 80.8 (C-3), 65.6 (C-36 + C-33), 59.8 (C-37), 55.2 (C-5),
54.9 (C-18), 48.5 (C-17), 47.4 (C-9), 42.0 (C-14), 40.2 (C-35 + C-34),
39.3 (C-19), 39.3 (C-8), 38.6 (C-20), 38.1 (C-1), 37.5 (C-4), 36.7 (C-10),
34.4 (C-22), 32.7 (C-7), 30.2 (C-21), 28.0 (C-15), 27.9 (C-23), 25.2 (C-
27), 23.4 (C-16), 23.2 (C-11), 21.1 (C-32), 21.0 (C-30), 18.3 (C-2), 18.3
(C-6), 17.2 (C-29), 16.7 (C-26), 16.6 (C-24), 15.3 (C-25) ppm; MS (ESI,
MeOH): m/z = 597.4 ([M+H]+), 1194.6 ([2M+H]+); analysis calcd
for C37H60N2O4 (596.90): C 74.45, H 10.13, N 4.69; found: C 74.32, H
10.36, N 4.44.
14. 3 β-Acetyloxy-28-(4,4-dimethylpiperazin-4-ium-1-yl)-28-
oxoolean-12-ene iodide (11)
Following the procedure given for the synthesis of 5, from 9
(500 mg, 0.88 mmol), 11 (337 mg, 53%) was obtained as a colorless
solid; m.p. 241 °C (decomp.); [α]D = +17.82° (c 0.19, CHCl3);
RF = 0.4 (CHCl3/MeOH, 9:1); IR (KBr): ν = 950 m, 960 m, 984 m,
1003 m, 1025 m, 1085w, 1102w, 1152 m, 1165w, 1206 m, 1257 s,
1302w 1364 m, 1388 m, 1468 m, 1637 s, 1731 s, 2871w, 2941 m cm−1
;
UV/Vis (CHCl3): λmax (log ε) = 229 (4.01) nm; 1H NMR (500 MHz,
CD3OD): δ = 5.29–5.24 (m, 1H, 12-H), 4.53–4.46 (m, 1H, 3-H),
4.22–4.11 (m, 2H, 33-H2), 4.07–3.92 (m, 2H, 36-H2), 3.55–3.45 (m, 4H,
34-H2 + 35-H2), 3.31 (s, 6H, 37-H3 + 38-H3), 3.11–3.04 (m, 1H, 18-
H), 2.32–2.22 (m, 1H, 16-Ha), 2.06 (s, 3H, 32-H3), 1.99–1.92 (m, 2H,
11-H2), 1.88–1.44 (m, 13H, 1-Ha + 2-H2 + 6-H2 + 7-H2 + 9-H + 15-
Ha + 16-Ha + 19-Ha + 21-Ha + 22-Ha), 1.42–1.33 (m, 1H, 22-Hb),
1.31–1.26 (m, 1H, 21-Hb), 1.25–1.19 (m, 5H, 15-Hb + 19-Hb + 27-H3),
1.15–1.06 (m, 1H, 1-Hb), 1.02 (s, 3H, 25-H3), 0.99 (s, 3H, 29-H3), 0.97
(s, 3H, 30-H3), 0.95–0.89 (m, 7H, 5-H + 29-H3 + 30-H3), 0.80 (s, 3H,
26-H3) ppm; 13C NMR (125 MHz, CD3OD): δ = 177.7 (C-28), 172.9 (C-
31), 145.7 (C-13), 123.1 (C-12), 82.5 (C-3), 62.6 (C-34), 61.5 (C-35),
56.8 (C-5), 52.1 (C-37 + C-38), 47.7 (C-9), 48.0 (C-17), 47.5 (C-19),
4

B. Brandes, et al.
Steroids163(2020)108713
45.0 (C-18), 43.1 (C-14), 40.5 (C-36 + C-33), 40.5 (C-8), 39.3 (C-1),
38.7 (C-4), 38.2 (C-10), 35.0 (C-21), 33.8 (C-22), 33.4 (C-30), 31.2 (C-
20), 30.8 (C-7), 29.1 (C-15), 28.6 (C-24), 26.4 (C-27), 24.5 (C-2), 24.5
(C-11), 24.3 (C-29), 23.3 (C-16), 21.1 (C-32), 19.3 (C-6), 17.5 (C-26),
17.1 (C-23), 16.0 (C-25) ppm; MS (ESI, MeOH): m/z = 595.5 ([M]+);
analysis calcd for C38H63N2O3I (722.84): C 63.14, H 8.79, N 3.88;
found: C 62.96, H 8.95, N 3.62.
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15. 28-(4-Methyl-4-oxido-piperazin-1-yl)-28-oxoolean-12-en-3 β
-yl acetate (12)
Following the procedure given for the synthesis of 6, from 10
(473 mg, 0.82 mmol), 12 (480 mg, 99%) was obtained as a colorless
solid; m.p. 191 °C (decomp.); [α]D = +28.9° (c 0.25, CHCl3); RF = 0.3
(CHCl3/MeOH, 9:1); IR (KBr): ν = 973 m, 1007 m, 1027 m, 1136w,
1193w, 1200 m, 1245 s, 1365 m, 1391 m, 1447 m, 1633 m, 1731 m,
2876w, 2944 m cm−1; UV/Vis (CHCl3): λmax (log ε) = 226 (334) nm;
1H NMR (500 MHz, CDCl3): = 5.29–5.22 (m, 1H, 12-H), 4.52–4.43 (m,
1H, 3-H), 4.43–4.35 (m, 1H, 33-Ha) 4.30–4.22 (m, 1H, 36-Ha),
3.99–3.88 (m, 1H, 36-Hb), 3.80–3.67 (m, 1H, 33-Hb), 3.43–3.29 (m, 5H,
37-H3 + 34-Ha + 35-Ha), 3.28–3.09 (m, 2H, 34-Hb + 35-Hb),
3.09–3.00 (m, 1H, 18-H), 2.21–2.08 (m, 1H, 16-Ha), 2.05–2.02 (m, 3H,
32-H3), 1.95–1.78 (m, 2H, 11-H2), 1.78–1.49 (m, 10H, 1-Ha + 2-
H2 + 6-Ha + 7-H2 + 9-H + 15-Ha + 16-Hb + 19-Ha), 1.49–1.30 (m,
2H, 6-Hb + 22-Ha), 1.30–1.16 (m, 4H, 19-Hb + 21-H2 + 22-Hb), 1.12
(s, 3H, 27-H3), 1.09–0.95 (m, 2H, 1-Hb + 15-Hb), 0.94–0.91 (m, 6H,
25-H3 + 30-H3), 0.90 (s, 3H, 29-H3), 0.85 (s, 3H, 23-H3), 0.84 (s, 3H,
24-H3), 0.83–0.79 (m, 1H, 5-H), 0.69 (s, 3H, 26-H3) ppm; 13C NMR
(125 MHz, CDCl3): δ = 175.4 (C-28), 171.0 (C-31), 144.3 (C-13), 121.8
(C-12), 80.9 (C-3), 66.0 (C-35), 65.8 (C-34), 60.3 (C-37), 55.4 (C-5),
47.6 (C-9), 47.6 (C-17), 46.2 (C-19), 43.5 (C-18), 41.8 (C-14), 40.5 (C-
33), 39.5 (C-36), 39.1 (C-8), 38.1 (C-1), 37.7 (C-4), 37.0 (C-10), 33.9
(C-21), 33.0 (C-29), 32.7 (C-22), 30.4 (C-20), 30.2 (C-7), 28.0 (C-23),
27.9 (C-15), 25.9 (C-27), 24.0 (C-30), 23.5 (C-2), 23.4 (C-11), 22.8 (C-
16), 21.3 (C-32), 18.2 (C-6), 16.9 (C-26), 16.6 (C-24), 15.4 (C-25) ppm;
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MS (ESI, MeOH): m/z
37H60N2O4 (596.90): C 74.45, H 10.13, N 4.69; found: C 74.27, H
10.31, N 4.47.
=
597.5 ([M+H]+); analysis calcd for
C
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Benjamin Brandes: Investigation, Writing - review & editing.
Lukas Koch: Investigation. Sophie Hoenke: Investigation. Hans-Peter
Deigner: Conceptualization, Writing - review & editing. René Csuk:
Conceptualization, Writing - original draft, Writing - review & editing.
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Acknowledgment
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apoptosis in human tumor cells and alter their mode of action with respect to the
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We like to thank Dr. D. Ströhl and his team for the NMR spectra and
the late Dr. R. Kluge for numerous ESI-MS measurements; additional MS
spectra were recorded by Th. Schmidt. IR and UV-Vis spectra as well as
optical rotations were measured by V. Simon and M. Schneider; mi-
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Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
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