Synthesis and cytotoxic evaluation of malachite green derived oleanolic and ursolic acid piperazineamides

Article abstract of DOI:10.1007/s00044-020-02536-1

The coupling of acetylated piperazinylamide spacered triterpenoic oleanolic acid and ursolic acid with meta or para substituted carboxylated malachite green analogs gave conjugates 10, 11, 15, and 16 that were cytotoxic for several human tumor cell lines. Especially, an oleanolic acid-derived compound 10 was cytotoxic for MCF-7 human breast carcinoma cells (EC₅₀ = 0.7 μM). These derivatives represent first examples of triterpenoic acid derivatives holding a cationic scaffold derived from malachite green. [Figure not available: see fulltext.]

Full text of DOI:10.1007/s00044-020-02536-1

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research
https://doi.org/10.1007/s00044-020-02536-1
ORIGINAL RESEARCH
Synthesis and cytotoxic evaluation of malachite green derived
oleanolic and ursolic acid piperazineamides
Sander Friedrich1 Immo Serbian Sophie Hoenke Ratna Kancana Wolfram René Csuk1
1 ●
1 ●
1 ●
●
Received: 23 January 2020 / Accepted: 24 March 2020
©
The Author(s) 2020
Abstract
The coupling of acetylated piperazinylamide spacered triterpenoic oleanolic acid and ursolic acid with meta or para
substituted carboxylated malachite green analogs gave conjugates 10, 11, 15, and 16 that were cytotoxic for several human
tumor cell lines. Especially, an oleanolic acid-derived compound 10 was cytotoxic for MCF-7 human breast carcinoma cells
(
EC = 0.7 μM). These derivatives represent first examples of triterpenoic acid derivatives holding a cationic scaffold
50
derived from malachite green.
Graphical Abstract
●
●
●
●
Keywords Oleanolic acid Ursolic acid Cytotoxicity Terpenoids Malachite green
Introduction
difficult to be handled, and the cure rate (Holzel et al. 2017;
Laffman-Johnson 2012; Vliek et al. 2018) remained low
over all the years despite intensive research.
Recently the potential of scaffolds holding a cationic
functional group came into the focus of renewed interest
inasmuch as their transport into cells might be facilitated by
cation-transporters (Cai et al. 2016; Everett et al. 2013; Qian
et al. 2016). Some of these transporters were reported to be
overexpressed in malignant cells (Cai et al. 2014; Cai et al.
Chemotherapeutic treatment of cancer represents still a
scientific challenge. Although nowadays many patients
suffering from cancer can be cured or—at least—their life
span can be increased. Several types of cancer, however, are
Supplementary information The online version of this article (https://
doi.org/10.1007/s00044-020-02536-1) contains supplementary
material, which is available to authorized users.
2016; Everett et al. 2013). Several molecules holding a
cationic residue are cytotoxic and seem to target the mito-
chondria, such as ammonium (Biedermann et al. 2010;
Kataev et al. 2014) or phosphonium salts (Spivak et al.
*
René Csuk
rene.csuk@chemie.uni-halle.de
2013, 2017) of more complex molecules. Thus, these
1
compounds are mitocans (“mitochondrially targeted antic-
ancer drugs”).
Organic Chemistry, Martin-Luther-University Halle-Wittenberg,
Kurt-Mothes-Str. 2, D-06120 Halle (Saale), Germany

Medicinal Chemistry Research
Some conjugates of pentacyclic triterpenes are mitocans,
too. Thus, hybrids holding an extra cationic functional
group have shown promising to excellent cytotoxic results.
While “simple” quaternary ammonium salts (Biedermann
et al. 2010; Kataev, Strobykina, and Zakharova 2014) were
only of moderate cytotoxicity with their EC50 values being
in the same potency range as phosphonium salts (Spivak
et al. 2017, 2013), hybrids consisting of a suitable penta-
cyclic triterpene, an amine spacer and a BODIPY-FL group
biological evaluation of conjugates holding a cationic tri-
phenylmethane moiety especially of scaffolds of the
“malachite green type”, i.e., 1 and 2; these scaffolds differ
from malachite green by the presence of an additional car-
boxyl group; the latter is necessary for the attachment to the
triterpene-spacer adduct.
Recently conjugates holding this type of a malachite
green moiety (Müller et al. 1997; Rassow and Gruber 1915;
Yang et al. 2007) have been used for the production of
antibodies to be used in environmental analyses (Yang et al.
2007), as part of a fluorophore to label antimicrobial pep-
tides (Zhao et al. 2016) and for the detection of nerve gas
simulants by chromogenic chemodosimeters (Costero et al.
2012).
The synthesis of 1 (Scheme 1) started with the reaction
of m-formyl-benzoic acid (3) with N, N-dimethylaniline in
the presence of zinc chloride to yield 4 (Rassow and
Gruber 1915; Sinev et al. 1978). Similarly, from p-for-
mylbenzoic acid (5) compound 6 was obtained in 89%
yield as a blue-greenish compound. Reaction of 4 with
tetrachloro-p-benzoquinone afforded 41% of 1 while from
6 (Harle et al. 2018; Mueller et al. 1981; Rassow and
Gruber 1915) under the same conditions 2 was obtained;
both compounds are dark green solids. Compounds of this
type are also known as “Mordant Green” or “Mordant
Blue”.
(
Brandes et al. 2020; Krajcovicova et al. 2018) held lower
EC50 values against a variety of different human cancer cell
lines. Superior cytotoxicity, however, was found for those
triterpenoids holding one or two O-acetyl groups on ring A,
an amide spacer at C-28 (preferentially a piperazinyl resi-
due), and a rhodamine B moiety attached to this spacer
(
Sommerwerk et al. 2017). EC50 values in the low micro-
molar (Kahnt et al. 2018; Wolfram et al. 2018a, 2018b) and
even nano-molar range (Sommerwerk et al. 2017) were
reported for these conjugates.
Results and discussion
In extension of these findings and due to the close structural
similarity between malachite green (A) and rhodamine B
(
B, Fig. 1) we became interested in the synthesis and
Fig. 1 Structure of malachite
green (a) and rhodamine B (b)
as well as of scaffolds 1 and 2.
The lower section displays the
close structural similarity
between malachite green and
rhodamine B

Medicinal Chemistry Research
Scheme 1 Synthesis of
malachite green analogs 1 and 2:
reactions and conditions:
a ZnCl , EtOH, N, N-
2
dimethylaniline, reflux, 1 day,
8
9% (of 4), 69% (of 6);
b tetrachloro-p-benzoquinone,
CHCl3, 35 °C, 2 h, 41% (of 1),
6
5% (of 2)
Oleanolic acid (7) was acetylated (Sommerwerk et al.
Conclusion
2
017) (Scheme 2) to yield acetate 8; the latter compound
was coupled with piperazine as previously reported to yield
(Sommerwerk et al. 2017). Compound 1 was activated
Coupling of acetylated triterpenoic oleanolic acid and
ursolic acid holding a piperazinylamide spacer with meta or
para substituted carboxylated malachite green analogs gave
conjugates 10, 11, 15, and 16, respectively. These com-
pounds were cytotoxic for several human tumor cell lines.
Thereby, oleanolic acid-derived compound 10 was espe-
cially cytotoxic for MCF-7 human breast carcinoma cells
(EC = 0.7 μM).
9
in situ with oxalyl chloride and coupled with 9 to afford
target compound 10. Reaction of 9 with 2 gave 11.
Acetylation of ursolic acid (12) gave acetate 13 followed by
its reaction with piperazine to yield amide 14 (Sommerwerk
et al. 2017). Coupling of the latter compound with 1 or 2 as
described above afforded the hybrids 15 and 16, respectively.
The compounds were screened for their cytotoxic activity
using sulforhodamine B assays; the results from these assays
are summarized in Table 1. Betulinic acid and doxorubicin
were used as standards. While ursolic acid (12) is of similar
cytotoxicity as standard betulinic acid, no significant cyto-
toxicity was found for oleanolic acid (7). Cytotoxicity
increased about tenfold for the actylated piperazinylamides 9
and 14. Except for the HT29 human adenocarcinoma cells,
the EC50 values determined for the carboxylated malachite
green derivatives 1 and 2 were found in the low one-digit
μM range. As far as the coupling products 10, 11, 15, and 16
are concerned, oleanolic acid-derived compounds 10 and 11
were of significantly higher cytotoxicity than ursolic acid-
derived 15 and 16. The selectivity between tumor cells and
the nonmalignant fibroblasts is—by and large—the same as
in betulinic acid but significantly better than that of doxor-
ubicin. Furthermore, meta substituted malachite green car-
boxylates 10 and 15 were about twice as cytotoxic than para
substituted analogs 11 and 16, respectively. Thus, this makes
oleanolic acid-derived 10 to the most cytotoxic compound of
this series holding EC50 values between 0.7 and 0.9 μM.
Furthermore, 10 is ~17 times more cytotoxic than standard
triterpenoic acid betulinic acid and circa 50 times more
cytotoxic than starting material oleanolic acid. Interestingly,
while the lowest cytotoxicity was observed for HT29 cells,
for an analogous rhodamine B derivative especially for this
cell line the highest cytotoxicity has been noted. This seems
to prove that both the type of triterpenoic acid, the type of
amide linkage, the type of cationic residue, and its sub-
stitution pattern are of great importance both with respect to
a tumor cell line-specific cytotoxicity and to cytotoxicity in
general. Ongoing studies in our laboratory try to gain a
deeper insight into these observations.
5
0
Experimental
NMR spectra were recorded using the Varian spectrometers
Gemini 2000 or Unity 500 (δ given in ppm, J in Hz; typical
experiments: H-H-COSY, HMBC, HSQC, NOESY), MS
spectra were taken on a Finnigan MAT LCQ 7000 (elec-
trospray, voltage 4.1 kV, sheath gas nitrogen) instrument.
The optical rotations were measured on a Perkin-Elmer
polarimeter at 20 °C; TLC was performed on silica gel
(Merck 5554, detection with cerium molybdate reagent);
melting points are uncorrected (Leica hot stage micro-
scope), 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 1000. The
solvents were dried according to usual procedures. The
purity of the compounds was determined by HPLC and
found to be >96%. Oleanolic acid (7) and ursolic acid (12)
were obtained from Betulinines (Stříbrná Skalice, Czech
Republic) in bulk quantities.
Synthesis
N-(4-((3-carboxyphenyl)(4-(dimethylamino)phenyl)
methylene)cyclohexa-2,5-dien-1-yliden)-N-
methylmethanaminium chloride (1)
Following the procedure given for the synthesis of 2 from
4
(1.34 mmol, 500 mg), tetrachloro-p-benzoquinone
(393 mg, 1.6 mmol) and glacial acetic acid (1 mL;
0.1 mmol) followed by crystallization of the solid from
water (100 mL) 1 (224 mg, 41%) was obtained as a purple

Medicinal Chemistry Research
Scheme 2 Synthesis of
compounds 7–16: reactions and
conditions: a Ac2O, NEt3,
DMAP, DCM, 88% (of 8), 64%
(
of 13); b oxalyl chloride, THF;
DCM, piperazine 49%; c oxalyl
chloride, THF; NEt3, DMAP,
2
5 °C, 2 h, 50%; d oxalyl
chloride, THF; NEt3, DMAP,
5 °C, 2 h, 50%; e oxalyl
chloride, THF; DCM, piperazine
7%; f oxalyl chloride, THF;
2
5
NEt3, DMAP, 25 °C, 2 h, 50%;
g oxalyl chloride, THF; NEt3,
DMAP, 25 °C, 2 h, 41%
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%
#
FaDu
A2780
HT29
MCF-7
SW1736
NIH 3T3
1
2
7
9
1
1
1
1
1
1
6.1 ± 1.0
8.2 ± 0.9
>30
9.7 ± 1.1
4.6 ± 0.7
>30
18.2 ± 1.4
12.4 ± 1.2
>30
6.4 ± 0.8
6.5 ± 1.1
>30
8.1 ± 1.5
9.7 ± 0.8
>30
6.3 ± 1.7
5.2 ± 1.1
>30
n.d.
1.7 ± 0.1
0.9 ± 0.2
2.1 ± 0.3
11.7 ± 0.6
2.1 ± 0.1
1.4 ± 0.3
4.9 ± 0.6
12.7 ± 1.8
0.01 ± 0.01
1.3 ± 0.1
4.3 ± 0.5
4.6 ± 0.2
10.6 ± 0.7
1.9 ± 0.3
2.4 ± 0.2
7.9 ± 0.5
18.4 ± 2.0
0.9 ± 0.2
1.7 ± 0.1
0.7 ± 0.1
1.4 ± 0.3
12.7 ± 0.1
2.0 ± 0.1
1.2 ± 0.1
2.9 ± 0.7
12.0 ± 1.7
1.1 ± 0.3
1.8 ± 0.9
0.9 ± 0.1
1.7 ± 0.1
14.3 ± 0.4
3.1 ± 0.8
1.7 ± 0.4
4.0 ± 0.5
16.4 ± 1.9
1.7 ± 0.3
2.2 ± 1.0
0.6 ± 0.2
1.5 ± 0.3
5.2 ± 1.4
9.7 ± 0.8
1.2 ± 0.3
2.6 ± 0.7
16.1 ± 1.4
0.008 ± 0.001
0
1
2
4
5
6
0.8 ± 0.2
1.5 ± 0.1
n.d.
n.d.
1.5 ± 0.1
2.3 ± 0.2
n.d.
Betulinic acid
Doxorubicin
n.d.
Human cancer cell lines: FaDu (hypopharyngeal carcinoma), A2780 (ovarian carcinoma), HT29 (colorectal
carcinoma), MCF-7 (breast adenocarcinoma), SW1736 (thyroid carcinoma) NIH 3T3 (nonmalignant
fibroblasts); cutoff 30 μM
n.d. not determined

Medicinal Chemistry Research
solid; R = (ACN/H O, 85:15) = 0.44; m.p. 207–210 °C;
DMSO-d ): δ = 7.92 (d, J = 13.8 Hz, 2H, 2′-H, 4′-H), 7.29
F
2
6
1
H NMR (400 MHz, DMSO-d ): δ = 10.23 (s, 1H,
(d, J = 19.0 Hz, 2H, 6′-H, 3′-H), 6.94 (d, J = 13.8 Hz, 4H,
3-H), 6.67 (d, J = 13.8 Hz, 4H, 2-H), 5.4 (s, 1H, 5-H), 2.88
6
COOH), 8.25 (d, J = 7.8 Hz, 1H, 4′-H), 7.80 (t, J =
1
3
1
7
.8 Hz, 1H, 2′-H), 7.73 (t, J = 7.7 Hz, 1H, 5′-H), 7.56 (dt,
.7, 1.5 Hz, 1H, 6′-H), 7.34–7.29 (m, 4H, 2-H, 7-H), 7.08
(s, 12H, 1–NMe ) ppm; C-NMR (400 MHz, DMSO-d6):
2
δ = 148.8 (C-1), 145.5 (C-1′), 134.1 (C-2′), 132.8 (C-3′),
131.1 (C-5′), 129.9 (C-3, C-4), 129.1 (C-6′), 128.1 (C-4′),
1
3
(
d, J = 9.3 Hz, 4H, 3-H, 8-H) ppm; C NMR (400 MHz,
DMSO-d ): δ = 174.0 (C-5), 172.4 (C-OOH), 167.0
113.0 (C-2), 54.9 (C-5), 40.9 (NMe ) ppm; IR (KBr): ν =
6
2
−
1
(
C-9), 156.9 (C-1), 140.5 (C-7), 138.5 (C-6′), 134.9 (C-
′), 133.5 (C-4′), 131.6 (C-1′), 129.6 (C-3′), 126.9 (C-6,
C4), 121.1 (C-5′), 114.7 (C-3, C8), 41.0 (1-NMe ), 21.5
3448s, 1614m, 1519m, 1351w cm ; UV–vis (CHCl ): λ
3 max
2
(log ε) = 289.65 (4.3 nm); MS (ESI, MeOH): m/z (%) =
+
375.27 (100%, [M + H] ); analysis calcd for C H N O
2
24 24
2
2
(
9-NMe ) ppm; IR (KBr): ν = 3447sbr, 1617w, 1584m,
369m, 1168m cm ; UV–Vis (CHCl3): λmax (log ε) =
20 (3.4), 345.17 (2.82), 475.22 (2.72), 682.72 (3.06),
(374.48): C 76.98, H 7.00, N 7.48; found: C 76.83, H 7.15,
N 7.31.
2
−
1
1
2
7
(
(
14.92 (3.27) nm; MS (ESI, MeOH): m/z (%) = 373.33
4-[Bis(4-dimethylamino)phenyl]methyl-benzoic acid (6)
−
100%, [M–Cl] ); analysis calcd for C H N O Cl
2
4
25
2
2
408.93): C 70.49, H 6.16, N 6.85; found: C 70.35, H
.38, N 6.73.
To a solution of 5 (1.44 g, 9.6 mmol) and ZnCl (3.9 g,
2
6
28.8 mmol) in ethanol (100 mL) N, N-dimethylaniline
(3.6 mL, 28.8 mmol) was slowly added, and the mixture
N-(4-((4-carboxyphenyl)(4-(dimethylamino)phenyl)
methylene)cyclohexa-2,5-dien-1-yliden)-N-
methylmethanaminium chloride (2)
was stirred under reflux for 1 day. The mixture was diluted
with methanol (40 mL), the pH value was adjusted to 5 by
adding aq. HCl, and the precipitate was filtered off. Com-
pound 6 (2.45 g, 69%) was obtained as light green/blue
To a solution of 6 (500 mg 1.34 mmol) and tetrachloro-p-
solid; R = 0.95 (n-hexane/EtOAc, 3:1); m.p. 252 °C
(decomp.) (lit: 250 °C, decomp. (Yang et al. 2007)); H
F
1
benzoquinone (393 mg, 1.6 mmol) in CHCl (50 mL) gla-
3
cial acetic acid (1 mL, 0.1 mmol) was added, and stirring at
NMR (400 MHz, DMSO-d ): δ = 12.76 (s, 1H, 5′-H),
6
3
5 °C was continued for 2 h. The volatiles were removed
7.88–7.82 (m, 2H, 3′-H), 7.21–7.16 (m, 2H, 2′-H),
under reduced pressure, and the residue was crystallized
from water (100 mL) to yield 2 (970 mg, 65%) as a dark
green solid; R = 0.3 (ACN/H O, 85:15); m.p. 230–232 °C;
6.93–6.85 (m, 4H, 2-H), 5.38 (s, 1H, 5-H), 2.83 (s, 12H,
1
3
NMe ) ppm; C NMR (400 MHz, DMSO-d ): δ = 167.8
2
6
(C-5′), 151.1 (C-1′), 149.3 (C-1), 131.9 (C-4), 129.8 (C-2),
129.7 (C-3′), 129.4 (C-2′), 112.8 (C-3′), 54.59 (C-5), 40.7
(NMe ) ppm; IR (KBr): ν = 3447s , 2883m, 1683s, 1610s,
F
2
1
H NMR (400 MHz, DMSO-d ): δ = 10.21 (s, 1H, COOH),
6
8
.12 (d, J = 8.7 Hz, 2H, 3′-H), 7.42 (d, J = 8.5 Hz, 2H, 2′-
2
br
H), 7.32 (d, J = 9.2 Hz, 2H, 3-H), 7.08 (d, J = 9.2 Hz, 2H,
1571w, 1519s, 1479w, 1443w, 1428w, 1349w, 1314w,
1293w, 1227w, 1202w, 1177w, 1164w, 1061w, 1017w cm ;
−1
2
-H), 6.97 (d, J = 9.2 Hz, 2H, 7-H), 6.61 (d, J = 9.2 Hz, 2H,
-H), 3.31 (s, 12H, 1–NMe ), 2.85 (s, 6H, 9–NMe ) ppm;
8
UV–vis (CHCl ): λ (log ε) = 270.37 (4.16) nm; MS (ESI,
2
2
3
max
1
3
+
C NMR (100 MHz, DMSO-d ): δ = 173.6 (C-5), 166.7
MeOH): m/z (%) = 375.33 (100%, [M + H] ); anlysis calcd
6
(
C-OOH), 156.7 (C-1), 154.3 (C-9), 140.3 (C-3), 136.0 (C-
for C H N O (374.48): C 76.98, H 7.00, N 7.48; found:
2
4
24
2
2
1
1
′), 134.9 (C-2′), 134.3 (C-4′), 129.7 (C-3′), 128.9 (C-7),
21.0 (C-6), 111.9 (C-8), 41.1 (1–NMe ), 40.6 (9–NMe )
C 76.77, H 7.19, N 7.29.
2
2
−
1
ppm; IR (KBr): ν = 3447s, 1589m, 1363m, 1169m cm ;
UV–vis (CHCl3): λmax (log ε) = 345 (4.38), 480 (4.14), 675
3β-Acetyloxy-olean-12-en-28-oic acid (8)
Acetylation of 7 as previously described gave 8 (88%) as
(
(
(
4.43), 725 (4.65) nm; MS (ESI, MeOH): m/z (%) = 373.33
100, [M − Cl] ); analysis calcd for C H N O Cl
408.93): C 70.49, H 6.16, N 6.85; found: C 70.30, H 6.32,
−
a colorless solid; R = 0.7 (silica gel, toluene/ethyl acet-
24
25
2
2
F
ate/formic acid/heptane, 80:26:5:1); m.p.: 263–265 °C
N 6.69.
-[Bis(4-dimethylamino)phenyl]methyl-benzoic acid (4)
To a solution of 3 (1 g, 6.67 mmol) and ZnCl (2.7 g,
(lit.: 260–261 °C) (Sommerwerk et al. 2017); [α] =
D
+
117.7° (c 0.37, CHCl ), (lit.: +119° (c 0.1, CHCl )
3
3
3
(Sommerwerk et al. 2017); MS (ESI, MeOH): m/z = 497.5
−
−
(
75%, [M − H] ), 995.2 (100%, [2M – H] ), 1017.7
−
(29%, [2M − 2H + Na] ).
2
2
(
0.01 mmol) in ethanol (50 mL) N, N-dimethylaniline
2.5 g, 20.01 mmol) was added, and stirring under reflux
was continued for 1 day. Work-up as described for 6 gave 4
2.23 g, 89%) as a greenish/blue solid; R = 0.89 (n-hexane/
3β-Acetyloxy-olean-12-en-28-oyl piperazine (9)
(
Reaction of 8 with oxalyl chloride and piperazine as pre-
F
1
EtOAc, 8:1); m.p. 149–151 °C; H NMR (400 MHz,
viously described gave 9 (49%); R = 0.46 (silica gel,
F

Medicinal Chemistry Research
CHCl /MeOH, 9:1); m.p. 172–176 °C (lit.: 170–176 °C)
solvents were evaporated DMF. The solid was dissolved in
dry DCM (50 mL), triethylamine (0.2 g, 0.3 mL, 10 mmol),
DMAP (4-dimethylaminopyridine, 15 mg, 0.1 mmol), and a
solution of 9 (849 mg, 1.5 mmol) in dry DCM (20 mL) were
added. The mixture was stirred at 25 °C for 2 h; usual work-
3
(
(
Sommerwerk et al. 2017); MS (ESI, MeOH): m/z = 567.4
45%, [M + H] ).
+
N-(4-((4-dimethylamino)phenyl)-3-((4-(3β-acetyloxyolean-
2-en-28-carboxy)piperazin)-1-yl)oxy)-
1
up followed by chromatography (silica gel, CHCl /MeOH,
3
carbonylphenylmethylencyclohexa-2,5-dien-1-yliden)-N-
methylmethanaminium chloride (10)
9:1) gave 11 (594 mg, 50%) as a dark green solid; RF
1
(CHCl /MeOH, 9:1) = 0.35; m.p. 192–195 °C; H NMR
3
(
400 MHz, CDCl ): δ = 7.99 (s, 2H, 37-H), 7.56 (d, J =
3
Following the procedure given for the synthesis of 11, from
8.2 Hz, 2H, 38-H), 7.37 (dd, J = 8.73, 3.5 Hz, 4H, 2′-H, 8′-
H), 6.97 (d, J = 9.2 Hz, 4H, 3′-H, 7′-H), 5.21 (m, 1H, 12-
H), 4.47 (t, J = 8.3 Hz, 1H, 3-H), 3.69 (m, 4H, 33-H), 3.36
1
(200 mg, 0.49 mmol) compound 10 (240 mg, 49.8%) was
obtained as a dark green solid; R (CHCl /MeOH, 9:1) =
F
3
1
0
8
4
.35; m.p. 212–215 °C; H NMR (400 MHz, CDCl ): δ =
(s, 6H, N–Me ), 3.1 (q, J = 14.0, 7.3 Hz, 4H, 34-H), 2.94 (s,
3
2
.02 (s, 1H, 37-H), 7.78-7.69 (m, 1H, 39-H), 7.49-7.3 (m,
H, 2′-H, 7′-H), 7.05–6.91 (d, J = 8.9 Hz, 1H, 40-H), 6.99
1H, 18-H), 2.12 (m, 1H, 16-H ), 2.02 (s, 3H, 32-H), 1.89
a
(m, 2H, 11-H), 1.79–1.54 (m, 8H, 6-H, 16-H , 2-H, 15-H ,
b
a
(
5
=
1
d, J = 8.8 Hz, 4H, 3′-H, 8′-H), 6.72–6.58 (m, 1H, 38-H),
.21 (t, J = 3.6 Hz, 1H, 12-H), 4.49 (m, 1H, 3-H), 3.72 (q, J
7.0 Hz, 4H, 33-H), 3.39 (s, 6H, N-Me ), 3.23 (d, J =
7-H), 1.53–1.25 (m, 8H, 22-H, 21-H, 1-H , 19-H, 9-H),
a
1.22 (s, 3H, 27-H), 1.06 (m, 2H, 1-H , 15-H ), 0.92 (m, 6H,
b
b
25-H, 29-H), 0.79 (m, 1H, 5-H), 0.88–0.77 (dd, J = 10.9,
2
5.5 Hz, 4H, 34-H), 2.96 (s, 1H, 18-H), 2.17 (m, 1H, 16-
6.2 Hz, 9H, 23-H, 24-H, 30-H), 0.72 (s, 3H, 26-H) ppm; 13
C
H ), 2.04 (s, 3H, 32-H), 1.94–1.86 (m, 2H, 11-H),
a
NMR (100 MHz, CDCl ): δ = 171.0 (C-28), 169.3 (C-35),
3
1
.63–1.54 (m, 8H, 6-H, 16-H , 2-H, 15-H , 7-H), 1.53–1.20
162.5 (C-31), 157.0 (C-1′, C9′), 143.6 (C-7′), 140.9 (C-8′),
140.8 (C-2′), 140.8 (C-13), 139.3 (C-39), 134.7 (C-36),
127.3 (C-38), 127.2 (C-37), 125.2 (C-12), 119.4 (C-6′),
114.1 (C-3′), 80.9 (C-3), 55.3 (C-30), 47.5 (C-9), 45.9 (C-
b
a
(
m, 11H, 22-H, 15-H , 21-H, 1-H, 19-H, 9-H), 0.79 (m, 1H,
b
5
3
-H), 0.89–0.80 (m, 12H, 23-H, 24-H, 25-H, 27-H), 0.72 (s,
H, 30-H), 0.70 (s, 6H, 26-H, 29-H) ppm; 13C NMR
(
(
(
100 MHz, CDCl ): δ = 171.0 (C-28), 171.0 (C-31), 169.3
C-35), 157.0 (C-1′, C-9′), 143.6 (C-7′), 140.9 (C-8′), 140.8
C-2′), 138.5 (C-41), 134.9 (C-39), 133.4 (C-37), 131.5 (C-
0), 129.5 (C-38), 125.0 (C-12), 121.9 (C-36), 120.4 (C-6′),
34), 42.1 (C-33), 41.2 (1-NMe ), 39.4 (C-8), 38.8 (C-5),
3
2
38.7 (C-17), 38.2 (C-1), 37.7 (C-4), 36.9 (C-10), 36.5 (C-
18), 34.4 (C-21), 33.0 (C-22), 31.4 (C-14), 30.4 (C-20),
29.7 (C-27), 28.1 (C-24), 24.1 (C-2), 23.5 (C-15), 23.3 (C-
19), 23.0 (C-16), 21.3 (C-32), 21.2 (C-29), 18.2 (C-6), 16.9
(C-23), 16.7 (C-26), 15.5 (C-25), 8.6 (C-7), 0.98 (C-5′)
4
1
3
3
1
3
1
1
0
1
14.3 (C-3′), 81.3 (C-3), 56.0 (C-30), 47.4 (C-9), 46.6 (C-
4), 43.4 (C-33), 41.2 (1-NCH ), 39.4 (C-8), 39.0 (C-5),
3
8.8 (C-17), 38.6 (C-1), 37.5 (C-4), 36.5 (C-10), 36.0 (C-
8), 34.9 (C-21), 33.3 (C-22), 31.9 (C-14), 30.4 (C-20),
0.3 (C-27), 28.3 (C-24), 23.7 (C-15), 23.6 (C-2), 23.5 (C-
1), 23.4 (C-19), 23.0 (C-16), 21.6 (C-32), 21.2 (C-29),
8.4 (C-6), 17.0 (C-23), 16.1 (C-26), 15.9 (C-25), 8.4 (C-7),
ppm; IR (KBr): ν = 3445s , 2956w, 1636m, 1584m,
br
−
1
1371m, 1170w cm ; UV–vis (CHCl3): λ
(log ε) = 242
max
(3.16), 345 (3.83), 475 (3.61), 617.15 (1.79), 732.12 nm
(3.22) nm; MS (ESI, MeOH): m/z (%) = 921.80 (100%, [M
−
− Cl] ); analysis calcd for C H N O Cl (957.78): C
2
6
81
4
4
.89 (C-5′) ppm; IR (KBr): ν = 3447s , 2925m, 1618m,
75.24, H 8.52, N 5.85; found: C 75.02, H 8.76, N 5.39.
br
−
1
584m, 1370m, 1247m, 1170m cm ; UV–vis (CHCl3):
λmax (log ε) = 229.01 (3.56), 244.02 (4.26), 347.2 (3.87),
3β-Acetyloxy-urs-12-en-28-oic acid (13)
4
9
7
85.3 (3.71), 731.09 (4.35) nm; MS (ESI, MeOH): m/z (%):
−
21.73 (100%, [M − Cl] ); C H N O Cl (957.78): C
Acetylation of 12 as previously described (Sommerwerk
26
81
4
4
5.24, H 8.52, N 5.85; found: C 75.13, H 8.81, N 5.43.
et al. 2017) gave 13 (64%) as a colorless solid; R = 0.7
F
(silica gel, toluene/ethyl acetate/formic acid/heptane,
N-(4-((4-dimethylamino)phenyl)-4-((4-(3β-acetyloxyolean-
2-en-28-carboxy)piperazin)-1-yl)oxy)-
carbonylphenylmethylencyclohexa-2,5-dien-1-yliden)-N-
80:26:5:1); m.p. 242–244 °C (lit.: 242.7–244.1 °C); [α] =
D
1
+69.89° (c 0.86, CHCl ), (lit.: +71.2° (c 1.0, CHCl ); MS
3
3
−
(ESI, MeOH): m/z = 497.5 (64%, [M − H] ), 542.9 (30%,
[M + HCO ] ), 995.1 (68%, [2M − H] ), 1017.5 (100%,
−
−
methylmethanaminium chloride (11)
2
−
[
2M − 2H + Na] ).
To a solution of 2 (500 mg, 1.23 mmol) in dry dichlor-
omethane (DCM, 50 mL), oxalyl chloride (0.62 mg,
3β-Acetyloxy-olean-12-en-28-oyl piperazine (14)
4
.92 mmol) and three drops of dry dimethylformamide
(
DMF) were added. After stirring for 30 min, the volatiles
Reaction of 13 with oxalyl chloride and piperazine as pre-
viously (Sommerwerk et al. 2017) described gave 14 (57%);
R = 0.44 (silica gel, CHCl /MeOH, 9:1); m.p. 158–161 °C
were removed under reduced pressure, the residue was
dissolved in dry tetrahydrofuran (THF, 3 × 20 mL), and the
F
3

Medicinal Chemistry Research
(
[
lit.: 158–161 °C); MS (ESI, MeOH): m/z = 567.4 (61%,
M + H] ).
(t, J = 8.0 Hz, 1H, 3-H), 3.74 (m, 4H, 33-H), 3.38 (s, 6H,
+
1′–NMe ), 3.10 (q, 7.5 Hz, 5H, 34-H, 18-H), 2.15 (s, 1H,
2
1
6-H ), 2.02 (s, 3H, 32-H), 1.96–1.46 (m, 4H, 11-H, 6-H ,
a
a
N-(4-((4-dimethylamino)phenyl)-3-((4-(3β-acetyloxyurs-12-
en-28-carboxy)piperazin1-1-yl)oxy)-
16-H ), 1.39 (m, 8H, 2-H, 15-H , 19-H, 7-H, 9-H, 21-H ),
b a a
1.28–1.14 (m, 9H, 20-H, 6-H , 22-H 21-H , 1-H, 15-H , 5-
b
,
b
b
carbonylphenylmethylencyclohexa-2,5-dien-1-yliden)-N-
methylmethanaminium chloride (15)
H), 1.12 (s, 3H, 27-H), 0.90 (s, 3H, 30-H), 0.89 (s, 3H, 25-
H), 0.85 (s, 3H, 29-H), 0.83 (s, 3H, 24-H), 0.71 (s, 3H, 23-
1
3
H), 0.65 (s, 3H, 26-H) ppm; C NMR (100 MHz, CDCl3):
δ = 175.3 (C-28), 171.0 (C-31), 169.2 (C-35), 157.0 (C-1′,
C-9′), 144.7 (C-13), 144.5 (C-7′), 140.9 (C-8′), 140.8 (C-
2′), 139.4 (C-36), 134.7 (C-37), 127.2 (C-38), 121.6 (C-12),
114.1 (C-3′), 80.9 (C-3), 55.3 (C-30), 52.5 (C-18), 47.7 (C-
Following the procedure given for the synthesis of 11 from
1
(200 mg, 0.49 mmol) and 14 (333 mg, 0.59 mmol) com-
pound 15 (200 mg, 50%) was obtained as a dark green
1
solid; R (CHCl /MeOH, 9:1) = 0.35; m.p. 214–218 °C; H
F
3
NMR (400 MHz, CDCl ): δ = 8.00 (s, 1H, 37-H), 7.70 (d, J
17), 47.6 (C-9), 47.6 (C-34), 45.9 (NMe ), 43.6 (C-33),
3
2
=
7
3.8 Hz, 1H, 39-H), 7.62 (t, J = 3.7 Hz, 1H, 38-H), 7.44-
.28 (m, 5H, 2′-H, 7′-H, 40-H), 7.01 (d, J = 5.4 Hz, 4H, 3′-
H, 8′-H), 5.23 (t, J = 3.7 Hz, 1H, 12-H), 4.47 (t, J = 7.9 Hz,
H, 3-H), 3.70 (m, 4H, 33-H), 3.38 (s, 12H, NMe ), 3.14
43.5 (C-14), 41.9 (C-8), 41.3 (9′-NMe ), 39.1 (C-5), 38.1
2
(C-20), 37.6 (C-1), 36.9 (C-4), 33.9 (C-10), 33.0 (C-22),
30.4 (C-21), 29.9 (C-15), 28.0 (C-24), 27.8 (C-16), 25.9 (C-
11), 24.1 (C-27), 23.5 (C-2), 23.4 (C-19), 21.3 (C-32), 18.2
(C-29), 16.9 (C-23), 16.6 (C-26), 15.4 (C-25), 8.7 (C-7), 0.9
1
2
(
m, 4H, 34-H), 2.94 (m, 1H, 18-H), 2.12 (m, 1H, 16-Ha),
2
1
1
3
2
.02 (s, 3H, 32-H), 1.97–1.79 (m, 2H, 11-H), 1.78–1.45 (m,
(C-5′) nm; IR (KBr): ν = 3442s , 2940m, 2678m, 1618m,
br
−1
6H, 19-H, 6-H, 16-H , 2-H, 15-H, 7-H, 22-H, 21-H, 1-H),
1584m, 1371m, 1248w, 1171m cm ; UV–vis (CHCl ):
b
3
.44–1.28 (m, 1H, 20-H); 1.19 (m, 2H, 5-H, 9-H), 1.11 (s,
H, 23-H), 0.89 (s, 12H, 27-H, 25-H, 24-H), 0.84 (s, 6H,
6-H, 30-H), 0.69 (s, 3H, 29-H) ppm; 13C NMR (100 MHz,
λmax (log ε) = 226.9 (3.9), 256.0 (4.1), 486.2 (3.98), 682
(4.21), 737.0 (4.6) nm; MS (ESI, MeOH): m/z (%) = 921.73
−
(100%, [M − Cl] ); C H N O Cl (957.78): C 75.24, H
2
6
81
4
4
CDCl ): δ = 175.1 (C-28), 171.0 (C-31), 169.1 (C-35),
8.52, N 5.85; found: C 74.99, H 8.68, N 5.44.
3
1
57.0 (C-1′, C-9′), 144.6 (C-13), 144.5 (C-36), 140.7 (C-2′,
C7′), 140.0 (C-5′), 135.8 (C-41),132.8 (C-40), 131.2 (C-
9), 129.0 (C-38), 127.3 (C-4′, C6′), 121.6 (C-12), 114.2
C-3′, C8′), 80.9 (C-3), 55.3 (C-5), 47.7 (C-9), 47.6 (C-17),
Acknowledgements We like to thank Dr D. Ströhl and his team for the
NMR spectra and the late Dr R. Kluge for numerous ESI-MS mea-
surements. IR and UV–vis spectra were recorded by V. Simon. The
cell lines were provided by Dr T. Müller. Open Access funding pro-
vided by Projekt DEAL.
3
(
4
1
7.6 (C-34), 46.3 (C-33), 43.7 (C-18), 43.6 (C-14), 41.9 (C-
9), 41.3 (C-10), 39.3 (C-8), 39.1 (C-22), 38.2 (C-1), 38.1
(
3
3
(
1
C-7), 37.8 (C-4), 37.7 (C-21), 36.9 (C-15), 33.0 (C-20),
0.3 (C-16), 28.0 (C-11), 27.9 (C-27), 24.0 (C-2), 23.5 (C-
0), 23.4 (C-32), 21.3 (C-6), 18.2 (C-29), 16.9 (26C), 16.7
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
C-23), 15.4 (C-25) ppm; IR (KBr): ν = 3447s , 2990w,
br
−
1
636w, 1584w, 1371w, 1169w cm ; UV–vis (CHCl ): λ
3 max
Publisher’s note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
(
6
log ε) = 220.98 (3.54), 346.19 (3.87), 484.15 (3.76),
82.69 (4.11), 746.26 (4.32) nm; MS (ESI, MeOH): m/z
−
(
%) = 921.73 (100%, [M − Cl] ); C H N O Cl (957.78):
26 81 4 4
Open Access This article is licensed under a Creative Commons
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long as you give appropriate credit to the original author(s) and the
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org/licenses/by/4.0/.
C 75.24, H 8.52, N 5.85; found: C 75.11, H 8.69, N 5.49.
N-(4-((4-dimethylamino)phenyl)-4-((4-(3β-acetyloxyurs-12-
en-28-carboxy)piperazin)-1-yl)oxy)-
carbonylphenylmethylencyclohexa-2,5-dien-1-yliden)-N-
methylmethanaminium chloride (16)
Following the procedure given for the synthesis of 11, from
2
(200 mg, 0.49 mmol) and 14 (333 mg, 0.59 mmol) 16
(
164 mg, 41%) was obtained as a green solid; R (CHCl /
F
3
1
MeOH, 9:1) = 0.35; m.p. 212–215 °C; H NMR (400 MHz,
CDCl ): δ = 8.01 (d, J = 7.3 Hz, 2H, 37-H), 7.56 (d, J =
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