Betulinic acid derived amides are highly cytotoxic, apoptotic and selective

Article abstract of DOI:10.1016/j.ejmech.2020.112815

Betulinic and platanic acid derived amides were prepared and screened for their cytotoxic activity. All of the compounds were shown to be cytotoxic for a panel of human tumor cell lines, and especially apoptotic betulinic acid derived compounds 6, 8 and 19 showed low EC₅₀ values. Of special interest was a 4-isoquinolinyl amide of 3-O-acetyl-betulinic acid (compound 19), being the most cytotoxic compound of this series and holding EC₅₀ values as low as EC₅₀ = 1.48 μM (A375 melanoma cells) while being significantly less cytotoxic for non-malignant fibroblasts NIH 3T3 with a selectivity index of >91.2. This finding parallels previous results obtained for SAA21, a augustic acid derived compound thus making the 4-isoquinolinyl moiety to a privileged scaffold.

Full text of DOI:10.1016/j.ejmech.2020.112815

European Journal of Medicinal Chemistry 207 (2020) 112815
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Betulinic acid derived amides are highly cytotoxic, apoptotic and
selective
,
,
1
a
b
a, *
Sophie Hoenke a 1, Niels V. Heise a , Michael Kahnt , Hans-Peter Deigner , Rene Csuk
ꢀ
a Martin-Luther-University Halle-Wittenberg, Organic Chemistry, Kurt-Mothes-Str. 2, D-06120, Halle (Saale), Germany
b Furtwangen University, Medical and Life Sciences Faculty, Jakob-Kienzle Str. 17, D-78054, Villingen-Schwenningen, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Betulinic and platanic acid derived amides were prepared and screened for their cytotoxic activity. All of
the compounds were shown to be cytotoxic for a panel of human tumor cell lines, and especially
apoptotic betulinic acid derived compounds 6, 8 and 19 showed low EC50 values. Of special interest was a
4-isoquinolinyl amide of 3-O-acetyl-betulinic acid (compound 19), being the most cytotoxic compound of
Received 27 July 2020
Received in revised form
2 September 2020
Accepted 2 September 2020
Available online 9 September 2020
this series and holding EC50 values as low as EC50 ¼ 1.48
mM (A375 melanoma cells) while being
significantly less cytotoxic for non-malignant fibroblasts NIH 3T3 with a selectivity index of >91.2. This
finding parallels previous results obtained for SAA21, a augustic acid derived compound thus making the
4-isoquinolinyl moiety to a privileged scaffold.
Keywords:
Betulinic acid
Platanic acid
Amides
© 2020 Elsevier Masson SAS. All rights reserved.
Cytotoxicity
Isoquinoline
1. Introduction
More recently, triterpenes in particular have again become a
focus of research, and especially triterpene carboxylic acids derived
Despite all efforts, diseases and deaths from cancer still repre-
sent the second most frequent type of death worldwide. In high-
income countries, however, deaths from cancer are even more
common than those resulting from a cardiovascular disease [1] (see
Scheme 1)
The number of patients with cancer has risen steadily in recent
years, and a forecast for the year 2040 predicts that the number of
patients as well as the number of deaths by cancer will double [2].
The number of newly approved drugs increases continuously; thus
in 2019 alone for Germany 25 new drugs (excluding biosimilars)
have been brought to market with ten of them to treat cancer pa-
tients [3].
The development of new cytotoxic active ingredients continues
to be focused on natural substances or on drugs derived from
natural substances. Thus, nowadays 50% of all approved drugs can
be traced back to a natural product [4]. A special role thereby is
taken by terpenes and terpenoids in as much as they represent the
largest family of natural compounds that have been investigated for
their therapeutic effects [4,5].
from, for example betulinic acid, maslinic acid, oleanolic acid or
ursolic acid (Fig. 1).
Recent investigations have shown that the presence of an intact
carboxyl function is necessary to achieve good cytotoxicity. For
example, derivatives derived from betulinic acid are usually
significantly more active than those derived from betulin [6e8].
The potential of those compounds containing an additional cationic
residue, e.g. a quaternary ammonium salt [9,10], a phosphonium
moiety [11e13], a BODIPY residue [14,15], a malachite green [16] or
e of special interest - a rhodamine B residue [17e20] being con-
nected to the basic skeleton of the triterpene with or without a
suitable amine spacer, should be particularly emphasized. Inter-
estingly, however, in the past, some triterpenoid amides also
showed good and - above all - quite selective cytotoxic effects. EM2
[21e25] - a benzylamide of diacetylated maslinic acid (Fig. 2) - is
cytotoxic in the low
mM range [26] (e.g. EC50 against A2780 ovarian
cancer cells ¼ 0.5
mM) but this compound also showed good
selectivity towards cancer cells (e.g. EC50 against non-malignant
primary human fibroblasts WW030272 ¼ 156
mM). Furthermore,
small structural differences obviously influence both cytotoxicity
and selectivity. For example, SAA9 derived from augustic acid was
* Corresponding author.
quite cytotoxic (EC50 2.2
(S ¼ [EC50 (A2780)]/[EC50 (NIH 3T3)] ¼ 0.4), whereas the analogous
mM, A2780 cells), but also quite unselective
E-mail address: rene.csuk@chemie.uni-halle.de (R. Csuk).
1
These authors contributed equally.
https://doi.org/10.1016/j.ejmech.2020.112815
0223-5234/© 2020 Elsevier Masson SAS. All rights reserved.

S. Hoenke, N.V. Heise, M. Kahnt et al.
European Journal of Medicinal Chemistry 207 (2020) 112815
Scheme 1. Reactions and conditions: a) Jones oxidation, 76% for 2, 98% for 10; b) tBuOK, tBuOH, THF, 3 h, 50 ꢀC, air, 97% for 3, 85% for 11; c) NaBH4, MeOH, 23 ꢀC, 2 h, 55% (for 4), 74%
(for 12); d) Ac2O, NEt3, DMAP (cat.), DCM, 23 ꢀC, 1 d, 70% (for 5), 48% (for 13); e) (COCl)2, DCM, 23 ꢀC, 1 h, then benzylamine or 4-amino-isoquinoline or 5-aminoquinoline, DCM,
23 ꢀC, 1 h, 62% (for 6), 55% (for 7), 42% (for 8), 84% (for 14), 49% (for 15), 82% (for 16), 73% (for 18), 60% (for 19), 64% (for 20), 76% (for 22), 80% (for 23), 57% (for 24).
SAA21 derivative showed high cytotoxicity (EC50 ¼ 1.2
m
M, A2780)
2. Results and discussion
but also high selectivity (S
¼
[EC50 (A2780)]/[EC50 (NIH
3T3)] ¼ > 50) [27].
2.1. Chemistry
In the past, many derivatives obtained from betulinic acid
proved to be more cytotoxic than those derived from 2,3-O-diacy-
lated triterpene carboxylic acids [7,8]. To get a better insight into
structure/activity relationships, we decided to synthesize de-
rivatives holding a betulinic acid (or derived from it, platanic acid)
backbone but with an extra hydroxyl group at C-2. We also wanted
to investigate whether the influence of the orientation of the qui-
nolinyl substituent, as already observed for augustic acid de-
rivatives, was also reflected in these derivatives.
The synthesis of the compounds was straightforward. Silica gel
supported Jones oxidation of betulinic acid (1) gave betulonic acid
(2) in 76% isolated yield. Its treatment with tBuOK/air in tBuOH/THF
gave 3 whose reduction with NaBH4 furnished (2b, 3b) configurated
4. Compound 4 was acetylated to yield diacetylated 5. Compound 5
served as a valuable starting material for the synthesis of the am-
ides. Thereby 5 was activated with oxalyl chloride followed by the
addition of either benzylamine, 4-aminoisoquinoline or 5-
2

S. Hoenke, N.V. Heise, M. Kahnt et al.
European Journal of Medicinal Chemistry 207 (2020) 112815
Fig. 1. Structure of betulin, betulinic acid, platanic acid, maslinic acid, augustic acid, oleanolic acid and ursolic acid.
Fig. 2. Structure (and representative EC50 values) for maslinic acid derived EM2 and augustic acid derived compounds SAA9 and SAA21.
aminoquinoline to yield amides 6e8, respectively. In a similar way,
platanic acid (9) was oxidized, and 2-oxo compound 10 was ob-
tained, whose oxidation (/ 11), reduction (/12) followed by
acetylation gave 13. As described above, from 13 amides 14e16
were prepared.
For comparison and to have an insight onto the influence of the
extra hydroxyl group in these molecules (as compared to the
starting materials), 1 and 9 were acetylated, and the acetates 17 and
21 were obtained. Reaction with oxalyl chloride followed by the
addition of either benzylamine, 4-aminoisoquinoline or 5-
aminoquinoline furnished amides 18e20 and 22e24, respectively.
derivative) being the most cytotoxic compound of this series. These
results parallel previous findings for augustic acid derived SAA21,
and is also found for analogs 8, 14 and 24.
Compound 19, the most cytotoxic compound of this study also
held the highest tumor/non-tumor cell selectivity, especially for
A375 melanoma cells (S ¼ 91.2, Table 2 and Fig. 4), A2780 ovarian
cancer cells (S ¼ 61.6) and the cell line FaDu (hypopharyngeal
carcinoma, S ¼ 59.0). These results seem to indicate that the amide
substituent is more important than the substitution pattern at ring
E.
For compounds 6, 8 and 19, further investigations were carried
out to get a deeper insight into their mode of being cytotoxic;
thereby, A375 melanoma cell were used. After having treated the
cells with the compounds for 1 day, an Annexin V assay (Fig. 5)
showed that fewer cells were vital as compared to the control.
Having been treated for 2 days (Fig. 6), an even more pro-
nounced effect was observed. The number of apoptotic cells was
increased (circa 44). Approximately 20% of the cells were late-
apoptotic, but only 1.5% showed a necrotic cell death. As far as 8
is concerned, about two thirds of the cells were still vital (67.4%).
The number of apoptotic cells, however, was significantly increased
(18.9%) as compared to necrotic (1.3%) or late apoptotic/secondary
necrotic (12.4%) cells. In summary, these compounds mainly act by
apoptosis. Thereby, 6 showed only a slight increase of cells being
necrotic and late apoptotic and secondary necrotic range (0.7 and
7.9%, respectively), while the largest increase was observed for
2.2. Biology
The compounds were screened for their cytotoxic activity using
photometric sulforhodamine assays (SRB) [28e30], the results of
which are compiled in Table 1 and Fig. 3.
The results from the SRB assays revealed for many compounds
cytotoxic activity. While the parent compounds 1 and 9 were not
cytotoxic within the borders of the assays (cut-off 30 mM), betulonic
acid (2) was more cytotoxic than parent 1. Low cytotoxicity was also
observed for 3e5, 9e13 and compound 21. Cytotoxicity was
significantly increased as soon as an amide was formed irrespective
whether the terpenoid skeleton was derived from 1 or 9. For all
amides 6e8, 14,15, 18e18, 23, and 24 EC50 values in the low
mM
range were found with betulinic acid derived 19 (a 4-isoquinolinyl
3

S. Hoenke, N.V. Heise, M. Kahnt et al.
European Journal of Medicinal Chemistry 207 (2020) 112815
Table 1
Cytotoxicity of selected compounds; SRB assay EC50 values [
m
M] after 72 h of treatment; averaged from three independent experiments performed each in triplicate; con-
fidence interval CI ¼ 95%. Human cancer cell lines: A375 (melanoma), HT29 (colorectal carcinoma), MCF-7 (breast adenocarcinoma), A2780 (ovarian carcinoma), FaDu
(hypopharyngeal carcinoma), NIH 3T3 (non-malignant fibroblasts); n.s. not soluble under the conditions of the assay; n.d. not determined. Doxorubicin (DR) was used as a
positive control.
A375
HT29
MCF7
A2780
FaDu
NIH 3T3
1
>30
>30
>30
>30
>30
>30
2
3
4
5
6
7
8
9
14.3 1.7
13.76 1.4
11.2 1.8
15.81 1.0
3.14 0.6
3.34 0.2
2.9 0.1
>30
18.5 1.9
26.07 2.8
17.68 2.2
17.40 1.2
20.72 6.1
>15
12.0 1.5
11.96 1.1
15.8 2.1
11.25 0.8
11.3 2.1
7.33 1.0
6.26 0.8
>30
4.7 0.6
10.49 1.2
12.28 2.2
8.68 1.2
4.42 1.0
4.76 0.6
3.24 0.5
>30
17.4 2.1
14.05 1.1
16.54 2.5
13.5 0.9
5.4 1.0
4.02 0.3
3.49 0.4
>30
19.4 1.2
12.85 0.9
17.28 2.3
14.11 0.9
>70
>30
>120
>30
>30
>30
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
DR
>30
>30
>30
>30
>30
>30
>30
7.22 4.3
18.43 4.9
n.s.
21.3 2.0
>30
>30
>30
>30
n.s.
>30
>30
>30
>30
24.8 1.7
>30
>30
20.6 1.7
>30
>30
>30
>30
>30
5.57 4.4
6.41 0.8
n.s.
7.2 1.2
9.59 0.9
2.26 0.2
5.18 0.3
>30
>30
>30
>30
9.57 3.5
>30
>30
n.s.
>30
>30
>135
>30
>30
n.s.
>30
22.54 5.7
2.9 0.5
4.88 0.6
n.s.
19.2 1.7
4.05 0.2
1.48 0.1
3.05 0.1
>30
>30
>30
5.2 2.1
6.96 2.0
n.s.
11.0 0.5
26.8 6.8
4.65 0.5
12.12 1.8
>30
2.36 1.6
6.15 0.8
n.s.
18.3 0.5
6.29 0.8
2.16 0.2
5.08 0.3
>30
n.s.
n.s.
n.s.
n.s.
5.66 0.4
4.60 0.3
n.d.
9.34 1.3
7.06 1.5
1.1 0.3
7.84 0.6
5.4 0.9
0.01 0.01
7.67 0.2
6.42 0.5
n.d.
>30
1.3 0.6
Fig. 3. Cytotoxicity of selected compounds; SRB assay EC50 values [mM] after 72 h of treatment.
apoptotic cells (9.5%). Compound 8 also gave an increased number
of apoptotic cells, but here more were found being in the late phase.
A similar behavior was observed upon treatment of the cells with
19 (8.6% apoptosis and 9.6%% late apoptosis).
Visual inspection (Fig. 7) of the cells supported the findings from
the FACS investigations (vide infra). Thus, after a 24 h incubation,
cell division had already been reduced/stopped. The number of
apoptotic cells was slightly increased resulting in a small number of
cells showing morphological differences/characteristics.
apoptosis. For example, the cells were shrunken, strangulation
occurred and also blebbing was observed.
Changes in the cell cycle (Fig. 8 and Fig. 9) were visible after 24 h
of incubation. Compound 6 no longer showed a G2/M peak (0.0%),
and the number of cells being in the S phase was significantly
(30.3%), while more cells were in the G1 phase (63.8%). In addition,
approximately 6% of the cells were in the SubG1 phase. For com-
pound 8, an increase of cells in the subG1 phase could be noted
(3.5%). Compound 19 showed a similar behavior as 6. Hereby, 61.1%
of the cells were in G1 and 36.6% in S phase. Visual inspection of the
cells supported the findings from the FACS investigations (vide
After an incubation for 48h, however, all compounds caused
dramatic changes in the number and morphology of the cells due to
4

S. Hoenke, N.V. Heise, M. Kahnt et al.
European Journal of Medicinal Chemistry 207 (2020) 112815
Table 2
3. Conclusion
Selectivity S ¼ EC50 (NIH 3T3)/EC50 (tumor cell line) for the individual compounds.
A small series of amides derived from betulinic acid (1), platanic
acid (9) and (2 , 3 )-dihydroxy-20(29)-en-28-oic acid (4) as well as
(2 , 3 )-20-oxo-30-norlupan-28-28-oic acid (12) was prepared and
screened for their cytotoxic activity. All of the compounds were
shown to be cytotoxic for a panel of human tumor cell lines.
Especially betulinic acid derived compounds 6, 8 and 19 held
excellent cytotoxicity. They act mainly by apoptosis. Compound 19,
a 4-isoquinolinyl amide of 3-O-acetyl-betulinic acid, was the most
A375
HT29
MCF7
A2780
FaDu
b
b
3
4
5
6
7
8
13
14
15
17
18
19
20
23
24
0.9
1.5
0.9
22.3
9.0
41.4
0.4
10.3
6.1
1.6
7.4
0.5
1.0
0.8
3.4
2.0
4.0
0.3
4.2
1.6
1.4
1.0
4.5
1.0
1.0
1.0
1.1
1.1
1.3
6.2
4.1
19.2
0.3
5.8
4.3
2.7
1.1
29.0
2.5
3.2
4.3
1.2
1.4
1.6
15.8
6.3
37.0
0.3
12.7
4.9
1.6
4.8
0.9
1.0
1.0
13.0
7.5
34.4
0.3
5.4
4.7
4.2
3.1
59.7
5.8
3.9
4.7
b
b
cytotoxic compound holding EC50 values as low as EC50 ¼ 1.48
mM
(A375 melanoma cells) while being significantly less cytotoxic for
non-malignant fibroblasts NIH 3T3 with a selectivity index of >91.2.
This finding parallels previous results obtained for SAA21, an
augustic acid derived compound.
91.2
9.8
5.3
62.5
5.9
3.8
6.5
5.6
4. Experimental
infra). Thus, after a 24 h incubation, cell division had already been
reduced/stopped. The number of apoptotic cells was slightly
increased resulting in a small number of cells showing morpho-
logical differences/characteristics.
NMR spectra were recorded using the Varian spectrometers
Gemini 2000 or Unity 500 (d given in ppm, J in Hz; typical exper-
iments: HeHeCOSY, HMBC, HSQC, NOESY), MS spectra were taken
on a Finnigan MAT LCQ 7000 (electrospray, voltage 4.1 kV, sheath
Fig. 4. Selectivity S ¼ EC50 (NIH 3T3)/EC50 (tumor cell line) of selected compounds.
Fig. 5. Annexin V-FITC/PI assay: treatment of A375 cells with 6, 8 and 19 (6.2 mM/4.8 mM/3.0 mM) for 24 h. Examples of density plots determined by flow cytometry (Attune®
Cytometric Software v. 1.2.5). R1: necrotic, R2: secondary necrotic/late stage apoptotic, R3: vital, R4: apoptotic.
5

S. Hoenke, N.V. Heise, M. Kahnt et al.
European Journal of Medicinal Chemistry 207 (2020) 112815
Fig. 6. Annexin V-FITC/PI assay: treatment of A375 cells with 6, 8 and 19 (6.2
mM/4.8
mM/3.0
mM) for 48 h. Examples of density plots determined by flow cytometry (Attune®
Cytometric Software v. 1.2.5). R1: necrotic, R2: secondary necrotic/late stage apoptotic.
Fig. 7. Microscopic evaluation of the A375 cells treated with compounds 6, 8, 19.
Fig. 8. Cell Cycle investigation. Examples for histogram determined by flow cytometry after 24 h, green G1 G2/M, striped S phase, blue subG1. (For interpretation of the references to
color in this figure legend, the reader is referred to the Web version of this article.)
gas nitrogen) instrument. The optical rotations were measured
either on a JASCO P-2000 or 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 microscope), and elemental analyses were performed on
a Foss-Heraeus Vario EL (CHNS) unit. IR spectra were recorded on a
Perkin Elmer FT-IR spectrometer Spectrum 1000 or a Perkin-Elmer
Spectrum Two (UATR Two Unit). UV-VIS spectra were taken on a
Perkin-Elmer Lambda 14 spectrometer or on a Perkin-Elmer
Lambda 750 S (UV/VIS/NIR) spectrometer. The solvents were
dried according to usual procedures. The purity of the compounds
was determined by HPLC and found to be >96%. Column chroma-
tography was performed on a Büchi Reveleris Prep purification
system using Chromabond Flash cartridges (SiOH, 40.63 mm) from
Macherey-Nagel or Reveleris high resolution cartridges from Büchi.
Betulinic acid and platanic acid were obtained from Betulinines
ꢁ
ꢀ
(Stríbrna Skalice, Czech Republic) in bulk quantities and used as
received. A description of the biological screening, additional
6

S. Hoenke, N.V. Heise, M. Kahnt et al.
European Journal of Medicinal Chemistry 207 (2020) 112815
Fig. 9. Cell Cycle investigation. Examples for histogram determined by flow cytometry after 48 h, green G1 G2/M, striped S phase, blue subG1. (For interpretation of the references to
color in this figure legend, the reader is referred to the Web version of this article.)
spectroscopic data and depictions of the 1H and 13C NMR spectra
4.4. Syntheses
are provided in the supplementary materials file.
4.4.1. 3-Oxolup-20(29)-en-28-oic acid (betulonic acid, 2)
Oxidation of betulinic acid (1, 10.0 g, 21.9 mmol) with silica gel
supported Jones reagent [31] [from acetone (500 mL), CrO3 (2.51 g,
25.1 mmol), H2SO4 (2.5 mL), H2O (10 mL), silica gel (100 mL)] at 0 ꢀC
for 1 h followed by quenching the reaction by the addition of MeOH
(5 mL), removal of the solvents and Soxhlet-extraction of the res-
idue with diethyl ether (600 mL, 3.5 h) and column chromatog-
raphy (SiO2, hexanes/EtOAc, EtOAc: 3% / 15%) gave 2 (6.23 g, 76%)
as a colorless solid; m.p. 246e248 ꢀC (lit.: [32] 245e247 ꢀC);
4.1. General procedure for acetylation (GP A)
One equivalent of each triterpene carboxylic acid (compounds 1,
4, 9, 12) was dissolved in dry dichloromethane (DCM, 100 mL), and
acetic anhydride (3 eq. per hydroxyl group), triethylamine (3 eq.)
and catalytic amounts of DMAP were added. The reaction mixture
was stirred at 23 ꢀC for 1 day. A methanolic solution of ammonia
(satd., 5 mL) was added, and the reaction mixture was stirred for
another 30 min at 23 ꢀC. For workup, aq. hydrochloric acid (10%,
20 mL) was added, the aqueous phase was extracted with DCM (3 x
10 mL), the combined organic phases were washed with brine
(20 mL) and dried (Na2SO4). The volatiles were removed under
diminished pressure, and the crude product (5, 13, 17, 21) was pu-
rified by column chromatography.
[
a
]
D
¼ þ37.4ꢀ (c 0.283, CHCl3) [lit.: [33] [
a]
¼ þ33.8ꢀ (c 0.33
D
CHCl3)]; RF ¼ 0.42 (SiO2, hexanes/EtOAc, 7:1).
4.4.2. 2-Hydroxy-3-oxolupa-1,20(29)-dien-28-oic acid (3) [173106-
22-4]
To a solution of 2 (8.55 g, 18.8 mmol) in tBuOH (500 mL) and THF
(70 mL), tBuOK (30.0 g, 0.267 mol) [34] was added, and the mixture
was stirred for 3 h at 50 ꢀC while air was bubbled through. The
solvents were removed under diminished pressure, and the residue
was dissolved in EtOAc (100 mL). Usual aqueous workup followed
by column chromatography (SiO2, hexanes/EtOAc, EtOAc: 5% /
20%) gave 3 (7.41 g, 97%) as a colorless solid; m.p. 204e205 ꢀC (lit.:
4.2. General procedure for the synthesis of benzyl amides (GP B)
[35] 204e205 ꢀC); [
a
]
¼ þ40.5ꢀ (c 0.265, CHCl3) [lit.: [35]
The corresponding acetylated compound (5, 13, 17 and 21) was
dissolved in dry DCM (10 mL) and oxalyl chloride (4 eq.) and cat-
alytic quantities of DMF were added. The reaction mixture was
stirred at 23 ꢀC for 1 h. The volatiles were removed under dimin-
ished pressure, the residue was dissolved in dry DCM (10 mL), and
the corresponding amine (3 eq.) was added. The reaction mixture
was stirred at 23 ꢀC for 1 h. Usual aqueous workup followed column
chromatography gave compounds 6, 14, 18 and 22, respectively.
D
[
a
]
D
¼ þ13ꢀ (c 0.75, CHCl3)]; RF ¼ 0.68 (SiO2, hexanes/EtOAc, 3:1).
4.4.3. 2b,3b-Dihydroxylup-20(29)-en-28-oic acid (4)
To a solution of 3 (8.525 g, 18.2 mmol) in THF (200 mL) and
MeOH (40 mL), NaBH4 (1.0 g, 26.4 mmol) [31] was added, and the
mixture was stirred for 2 days at 23 ꢀC. Usual aqueous workup
followed by column chromatography (SiO2, hexanes/EtOAc, EtOAc:
10% / 30%) gave 4 (4.03 g, 55%) as a colorless solid; m.p.
270e273 ꢀC (decomp.) (lit.: [36] 273e276 ꢀC); [
CHCl3) [lit.: [36] [
hexanes/EtOAc, 1:1).
a
]
¼ þ32.4ꢀ (c 0.21,
D
a
]
D
¼ þ31.1 (c 0.262, pyridine)]; RF ¼ 0.54 (SiO2,
4.3. General procedure for the synthesis of quinolinyl amides (GP C)
The corresponding acetylated compound (5, 13, 17 and 21) was
dissolved in dry DCM (10 mL), and activated with oxalyl chloride (4
eq.) and catalytic quantities of DMF as described above. The vola-
tiles were removed under reduced pressure, the residue was dis-
solved in dry DCM (4 mL), and dry pyridine (8 mL), and the
respective amine (3 eq.) and catalytic quantities of DMAP and
triethylamine were added. The reaction mixture was stirred with
the help of microwave irradiation (Anton Parr, Graz/Austria, Mon-
owave) at 120 ꢀC for 2 h. Usual aqueous workup followed by col-
umn chromatographic purification gave compounds 7, 8, 15, 16, 19,
20, 23 and 24, respectively.
4.4.4. 2b,3b-Diacetyloxy-lup-20(29)-en-28-oic acid (5)
Acetylation of 4 (2.38 g, 5.0 mmol) according to GPA followed by
chromatography (SiO2, hexanes/EtOAc, EtOAc: 3% / 15%) gave 5
(1.97 g, 70%) as a colorless solid; m.p. 260e265 ꢀC (decomp.) (lit.:
[36] 246e248 ꢀC); [
a
]
D
¼ þ34.6ꢀ (c 0.199, CHCl3); RF ¼ 0.63 (SiO2,
hexanes/EtOAc, 7:1); IR (ATR):
n
¼ 2944m, 1742s, 1366m, 1230s,
1189m, 1031 m cmꢁ1; 1H NMR (400 MHz, CDCl3):
d
¼ 5.32 (m, 1H, 2-
H), 4.74 (s, 1H, 29-Ha), 4.66e4.56 (m, 2H, 3-H, 29-Hb), 3.00 (m, 1H,
19-H), 2.32e2.09 (m, 2H,16-Ha,13-H), 2.03 (s, 3H, 34-H), 2.02 (s, 3H,
32-H), 2.07e1.92 (m, 1H, 1-Ha), 1.96 (m, 2H, 21-Ha, 22-Ha), 1.72 (d,
7

S. Hoenke, N.V. Heise, M. Kahnt et al.
European Journal of Medicinal Chemistry 207 (2020) 112815
J ¼ 1.6 Hz, 2H, 12-Ha, 6-Ha), 1.69 (s, 3H, 30-H), 1.65e1.14 (m, 13H, 18-
H, 6-Hb, 22-Hb, 7-Hb, 16-Hb, 21-Hb, 15-Ha, 11-H, 9-H, 1-Hb, 15-Hb),
1.11 (s, 3H, 26-H), 1.02 (s, 3H, 25-H), 0.99 (m, 1H, 12-Hb), 0.97 (s, 3H,
27-H), 0.96 (s, 3H, 24-H), 0.92 (m, 1H, 5-H), 0.88 (s, 3H, 23-H) ppm;
43), 124.76 (C-44), 120.64 (C-42), 109.65 (C-39), 77.88 (C-3), 69.49
(C-2), 56.68 (C-17), 55.57 (C-5), 51.05 (C-9), 50.19 (C-18), 46.43 (C-
19), 42.71 (C-8), 42.17 (C-4), 40.90 (C-10), 38.44 (C-1), 37.58 (C-13),
37.32 (C-22), 36.94 (C-14), 34.22 (C-7), 34.08 (C-16), 30.82 (C-21),
29.61 (C-15), 28.88 (C-23), 25.49 (C-12), 21.17 (C-32), 21.02 (C-11),
20.79 (C-34), 19.45 (C-30), 17.92 (C-6), 17.43 (C-25), 16.69 (C-26),
16.28 (C-24), 14.54 (C-27) ppm; MS (ESI, MeOH): m/z 683 (92%,
[MþH]þ), 705 (44%, [MþNa]þ), 1365 (100%, [2M þ H]þ), 1388 (36%,
[2M þ Na]þ); analysis calcd for C43H58N2O5 (682.95): C 75.62, H
8.56, N 4.10; found: C 75.48, H 8.76, N 4.24.
13C NMR (126 MHz, CDCl3):
d
¼ 181.38 (C-28), 170.90 (C-33), 170.41
(C-31), 150.39 (C-20), 109.97 (C-29), 78.11 (C-3), 69.79 (C-2), 56.49
(C-17), 55.39 (C-5), 51.09 (C-9), 49.41 (C-18), 47.08 (C-19), 42.76 (C-
1), 42.35 (C-14), 40.97 (C-8), 38.53 (C-13), 37.56 (C-4), 37.20 (C-10),
37.16 (C-22), 34.31 (C-7), 32.32 (C-16), 30.72 (C-21), 29.71 (C-15),
29.14 (C-23), 25.54 (C-12), 21.41 (C-34), 21.14 (C-11), 21.02 (C-32),
19.51 (C-30), 18.15 (C-6), 17.64 (C-25), 16.89 (C-26), 16.35 (C-24),
14.76 (C-27) ppm; MS (ESI, MeOH): m/z 437 (8%, [M þ H-2HOAc]þ),
497 (10%, [M þ H-HOAc]þ), 1135 (100%, [2M þ Na]þ); analysis calcd
for C34H52O6 (556.77): C 73.34, H 9.41; found: C 73.11, H 9.63.
4.4.7. Quinolin-5-yl 2b,3b-diacetyloxy-lup-20(29)-en-28-amide (8)
According to GPC from 5 (250 mg, 0.45 mmol) and 5-amino-
quinoline (250 mg, 1.7 mmol) followed by chromatography (SiO2,
hexanes/EtOAc, EtOAc: 20% / 55%) 8 (130 mg, 42%) was obtained
4.4.5. Benzyl 2
b
,3
b-Diacetyloxy-lup-20(29)-en-28-amide (6)
as an off-white solid; m.p. 249e252 ꢀC (decomp.); [
a]
¼ þ2.9ꢀ (c
D
Reaction of 5 (250 mg, 0.45 mmol) with benzyl amine (0.3 mL,
2.8 mmol) according to GPB followed by chromatography (SiO2,
EtOAc/hexanes, EtOAc 8% / 20%) gave 6 (180 mg, 62%) as a
0.162, CHCl3); RF ¼ 0.53 (SiO2, CHCl3/MeOH, 95:5); UVeVis (CHCl3):
lmax (log ε) ¼ 305 nm (4.47), 317 nm (4.45); IR (ATR):
1741s, 1474m, 1366m, 1232s, 1187m, 1030m, 798m, 730 m cmꢁ1; 1H
NMR (400 MHz, CDCl3):
¼ 8.84 (d, J ¼ 3.1 Hz, 1H, 41-H), 8.29 (d,
n
¼ 2942m,
colorless solid; m.p. 138e141 ꢀC; [
a
]
¼ þ39.0ꢀ (c 0.222, CHCl3);
d
D
RF ¼ 0.64 (SiO2, toluene/EtOAc/heptane/HCOOH, 80:26:10:5); IR
J ¼ 8.4 Hz, 1H, 36-H), 8.04e7.94 (m, 1H, 37-H), 7.77e7.66 (m, 2H, 38-
H, 43-H), 7.46 (dd, J ¼ 8.6, 4.4 Hz, 1H, 42-H), 5.31 (m, 1H, 2-H), 4.74
(s, 1H, 29-Ha), 4.65e4.58 (m, 2H, 29-Hb, 3-H), 3.16 (td, 1H, 19-H),
2.58 (td,1H,13-H), 2.32 (m,1H,16-Ha), 2.02 (s, 3H, 32-H), 2.02 (s, 3H,
34-H), 2.20e1.95 (m, 3H, 1-Ha, 21-Ha, 22-Ha), 1.72 (s, 3H, 30-H),
1.88e1.21 (m, 16H, 16-Hb, 12-H, 15-Ha, 22-Hb, 18-H, 6-H, 21-Hb, 7-H,
11-H, 15-Hb, 9-H, 1-Hb), 1.10 (s, 3H, 26-H), 1.03 (s, 3H, 27-H), 1.01 (m,
6H, 24-H, 25-H), 0.93 (m, 1H, 5-H), 0.88 (s, 3H, 23-H) ppm; 13C NMR
(ATR):
n
¼ 2943w, 1741s, 1620w, 1365m, 1231s, 1188m, 1029m,
¼ 7.38e7.24 (m, 5H, 37-
698 m cmꢁ1; 1H NMR (400 MHz, CDCl3):
d
H, 38-H, 39-H, 40-H, 41-H), 5.88 (t, J ¼ 5.7 Hz, 1H, NH) 5.36e5.26
(m, 1H, 2-Ha), 4.74 (d, J ¼ 2.0 Hz, 1H, 29-Ha), 4.60 (m, 2H, 29-Hb, 3-
H), 4.48 (dd, J ¼ 15.1, 5.8 Hz, 1H, 35-Ha), 4.37 (dd, J ¼ 14.6, 5.6 Hz, 1H,
35-Hb), 3.16 (td, J ¼ 11.0, 4.1 Hz, 1H, 19-H), 2.55e2.44 (td, 1H, 13-H),
2.03 (s, 3H, 32-H), 2.01 (s, 3H, 34-H), 2.06e1.87 (m, 3H, 1-H, 16-Ha,
21-Ha), 1.68 (s, 3H, 30-H), 1.81e1.21 (m, 16H, 22-Ha, 12-H, 16-Hb, 18-
H, 6-H, 22-Hb, 21-Hb, 7-H, 11-H, 1-Hb, 9-H), 1.12 (s, 1H, 15-H), 1.10 (s,
3H, 26-H), 1.02 (s, 3H, 25-H), 0.95 (s, 3H, 27-H), 0.93 (s, 3H, 24-H),
0.90 (m, 1H, 5-H), 0.88 (s, 3H, 23-H) ppm; 13C NMR (101 MHz,
(101 MHz, CDCl3):
d
¼ 175.66 (C-28), 170.91 (C-33), 170.41 (C-31),
150.54 (C-20), 148.81 (C-41), 148.39 (C-39), 133.54 (C-44), 132.84
(C-36), 130.38 (C-43), 125.85 (C-37), 124.09 (C-35), 123.17 (C-38),
120.89 (C-42), 109.92 (C-29), 78.10 (C-3), 69.71 (C-2), 56.92 (C-17),
55.44 (C-5), 51.27 (C-9), 50.43 (C-18), 46.68 (C-19), 42.93 (C-8),
42.40 (C-1), 41.14 (C-10), 38.62 (C-22), 37.83 (C-13), 37.55 (C-14),
37.17 (C-4), 34.46 (C-7), 34.15 (C-16), 31.05 (C-21), 29.86 (C-15),
29.10 (C-23), 25.71 (C-12), 21.40 (C-32), 21.27 (C-11), 21.02 (C-34),
19.66 (C-30), 18.15 (C-6), 17.66 (C-25), 16.92 (C-26), 16.54 (C-24),
14.78 (C-27) ppm; MS (ESI, MeOH): m/z 683 (100%, [MþH]þ), 705
(22%, [MþNa]þ), 1365 (44%, [2M þ H]þ), 1387 (58%, [2M þ Na]þ);
analysis calcd for C43H58N2O5 (682.95): C 75.62, H 8.56, N 4.10;
found: C 75.41, H 8.74, N 4.29.
CDCl3):
d
¼ 176.01 (C-28), 170.88 (C-33), 170.40 (C-31), 150.97 (C-
20), 139.32 (C-36), 128.80 (C-38, C-40), 127.93 (C-37, C-41), 127.47
(C-39), 109.61 (C-29), 78.13 (C-3), 69.76 (C-2), 55.78 (C-17), 55.42
(C-5), 51.27 (C-9), 50.32 (C-18), 46.83 (C-19), 43.94 (C-8), 43.44 (C-
35), 42.82 (C-4), 42.38 (C-1), 41.05 (C-10), 38.56 (C-22), 37.77 (C-13),
34.43 (C-7), 33.94 (C-16), 31.02 (C-21), 29.46 (C-15), 29.11 (C-23),
25.72 (C-12), 21.41 (C-32), 21.25 (C-11), 21.02 (C-34), 19.63 (C-30),
18.18 (C-6), 17.67 (C-25), 16.92 (C-26), 16.42 (C-24), 14.69 (C-27)
ppm; MS (ESI, MeOH): m/z 646 (100%, [MþH]þ), 668 (76%,
[MþNa]þ), 1291 (52%, [2M þ H]þ), 1313 (84%, [2M þ Na]þ); analysis
calcd for C41H59NO4 (645.93): C 76.24, H 9.21, N 2.17; found: C
76.02, H 9.37, N 2.30.
4.4.8. 3,20-Dioxo-30-norlupan-28-oic acid (10)
Silica gel assisted Jones oxidation [31] of platanic acid (9, 5.0 g,
10.9 mmol) followed by column chromatography (SiO2, hexanes/
EtOAc, EtOAc: 10% / 30%) gave 10 (4.90 g, 98%) as a colorless solid;
4.4.6. Isoquinolin-4-yl 2
(7)
b
,3
b
-diacetyloxy-lup-20(29)-en-28-amide
(150 mg, 0.27 mmol) and 4-
m.p. 230 ꢀC (decomp.) (lit.: [37] 232e233 ꢀC); [
a
]
D
¼ þ4.2ꢀ (c 0.308,
According to GPC from
5
CHCl3) (lit.: [37] [
EtOAc, 1:1).
a
]
D
¼ ꢁ11ꢀ (c 1.0, C6H6)]; RF ¼ 0.64 (SiO2, hexanes/
aminoisoquinoline (150 mg, 1.0 mmol) followed by chromatog-
raphy (SiO2, hexanes/EtOAc, EtOAc: 20% / 55%) 7 (76 mg, 42%) was
obtained as an off-white solid; m.p. 186e190 ꢀC (decomp.);
4.4.9. 2-Hydroxy-3,20-dioxo-30-norlupan-1-en ꢁ28-oic acid (11)
Following the procedure [34] given for the synthesis of 3, from
10 (4.846 g, 10.6 mmol) 11 (4.30 g, 85%) was obtained and directly
used in the next step; an analytical sample showed m.p.
[
a
]
¼ ꢁ3.2ꢀ (c 0.216, CHCl3); RF ¼ 0.56 (SiO2, CHCl3/MeOH, 95:5);
D
UVeVis (CHCl3): lmax (log ε) ¼ 282 nm (4.20), 323 nm (4.24); IR
(ATR):
n
¼ 2942m, 1741s, 1367m, 1251s, 1187m, 1030m, 577w cmꢁ1
;
1H NMR (400 MHz, CDCl3):
d
¼ 9.12 (s, 1H, 38-H), 8.75 (s, 1H, 36-H),
225e228 ꢀC (lit.: [29] 224e228 ꢀC); [
a
]
¼ þ9.2ꢀ (c 0.71, MeOH)
D
8.01 (d, J ¼ 8.2 Hz, 1H, 40-H), 7.83e7.70 (m, 2H, 42-H, 41-H),
7.68e7.59 (m, 1H, 43-H), 5.32 (td, 1H, 2-H), 4.75 (s, 1H, 29-Ha),
4.66e4.56 (m, 2H, 29-Hb, 3-H), 3.19 (s, 1H, 19-H), 2.59 (td, 1H, 13-H),
2.18e1.92 (m, 3H, 1-Ha, 12-Ha, 21-Ha), 2.02 (s, 3H, 32-H), 2.02 (s, 3H,
34-H),1.72 (s, 3H, 30-H),1.91e1.19 (m,17H,12-Hb,18-H,1-Hb, 21-Hb,
22-H,16-H, 7-H,11-H,15-H, 9-H,17-H),1.10 (s, 3H, 26-H),1.03 (s, 3H,
27-H), 1.02 (m, 6H, 24-H, 25-H), 0.98e0.91 (m, 1H, 5-H), 0.88 (s, 3H,
[lit.: [29] [
a]
¼ þ9.0ꢀ (c 0.66, MeOH); MS (ESI, MeOH): m/z 472
D
(14%, [MþH]þ), 493 (100%, [MþNa]þ), 509 (32%, [MþK]þ).
4.4.10. 2b,3b-dihydroxy-20-oxo-30-norlupan-28-oic acid (12)
To a solution of 11 (4.9 g, 10.4 mmol) in THF (100 mL) and MeOH
(20 mL), NaBH4 (0.5 g, 13.2 mmol) [31] was added in several por-
tions, and the mixture was stirred for 1 d at 23 ꢀC. Usual aqueous
work-up followed by chromatography (SiO2, CHCl3/acetone,
acetone: 5% / 20%) gave 12 (3.7 g, 74%) as a colorless solid; m.p.
23-H) ppm; 13C NMR (101 MHz, CDCl3):
(C-33), 170.19 (C-31), 150.33 (C-20), 149.89 (C-38), 138.61 (C-36),
131.07 (C-35), 130.56 (C-41), 128.75 (C-39), 128.11 (C-40), 127.31 (C-
d
¼ 175.20 (C-28), 170.68
265e269 ꢀC (lit.: [29] 266e270 ꢀC) [
a]
¼ ꢁ11.2ꢀ (c 0.130, CHCl3)
D
8

S. Hoenke, N.V. Heise, M. Kahnt et al.
European Journal of Medicinal Chemistry 207 (2020) 112815
(lit.: [
1:1).
a
]
¼ ꢁ6.2ꢀ (c 0.215, CHCl3)]; RF ¼ 0.31 (SiO2, hexanes/EtOAc,
[
a
]
¼ ꢁ14.2ꢀ (c 0.258, CHCl3); RF ¼ 0.20 (SiO2, CHCl3/MeOH, 95:5);
D
D
UVeVis (CHCl3): lmax (log ε) ¼ 228 nm (3.78); IR (ATR): ¼ 2943w,
n
1741m, 1666w, 1588w, 1449w, 1396m, 1368m, 1253s, 1188m, 1032m,
4.4.11. 2b,3b-Diacetyloxy-20-oxo-30-norlupan-28-oic acid (13)
947w, 873w, 780w, 750w, 653w, 622w, 576w, 497w cmꢁ1; 1H NMR
According to GPA from 12 (1.75 g, 7.4 mmol) followed by chro-
matography (SiO2, hexanes/EtOAc, EtOAc: 5% / 50%), 13 (993 mg,
(500 MHz, CDCl3):
d
¼ 9.13 (s, 1H, 37-H), 8.71 (s, 1H, 35-H), 8.02 (d,
J ¼ 8.2 Hz,1H, 39-H), 7.82e7.72 (m, 2H, 42-H, 40-H), 7.65 (m, 1H, 41-
H), 5.31 (m, 1H, 2-H), 4.60 (d, J ¼ 3.9 Hz, 1H, 3-H), 3.45 (td, J ¼ 11.3,
5.7 Hz, 1H, 19-H), 2.41e2.32 (m, 1H, 13-H), 2.29e2.20 (m, 2H, 16-Ha,
18-H), 2.18 (m, 1H, 21-Ha), 2.17 (s, 3H, 29-H), 2.09 (m, 1H, 22-Ha),
2.02 (s, 3H, 33-H), 2.01 (s, 3H, 31-H), 1.97 (m, 1H, 1-Hb), 1.88 (m, 1H,
16-Hb), 1.74 (m, 2H, 15-H, 22-Hb), 1.62e1.21 (m, 10H, 21-Hb, 6-H, 7-
H, 11-H, 15-Hb, 9-H, 1-Hb), 1.09 (s, 3H, 25-H), 1.07 (m, 2H, 12-H), 1.06
(s, 3H, 27-H), 1.02 (s, 3H, 24-H), 1.00 (s, 3H, 25-H), 0.97e0.91 (m, 1H,
48%) was obtained as a colorless solid; m.p. 150 ꢀC; [
a
]
¼ þ6.9ꢀ (c
D
0.146, CHCl3); RF ¼ 0.33 (SiO2, hexanes/EtOAc, 3:1); IR (ATR):
n
¼ 2944w, 1740m, 1365m, 1231s, 1189w, 1134w, 1031m, 946w,
752m, 667w, 605w, 510w cmꢁ1; 1H NMR (400 MHz, CDCl3):
d
¼ 5.31
(td, 2H, 2-H), 4.60 (d, J ¼ 3.9 Hz, 1H, 3-H), 3.24 (td, J ¼ 11.3, 4.7 Hz,
2H, 19-H), 2.29 (m, 1H, 16-Ha), 2.18 (s, 3H, 29-H), 2.15e2.05 (m, 1H,
18-H), 2.02 (s, 3H, 33-H), 2.01 (s, 3H, 31-H), 2.06e1.92 (m, 3H, 1-Ha,
13-H, 22-Ha),1.67e1.15 (m,13H, 22-Hb, 6-H,15-Ha, 21-H,16-Hb, 7-H,
11-H, 1-Hb, 15-Hb), 1.10 (s, 3H, 25-H), 1.08 (s, 2H, 12-H), 1.01 (s, 3H,
24-H), 1.00 (s, 3H, 27-H), 0.95 (m, 1H, 5-H), 0.93 (s, 3H, 26-H), 0.88
5-H), 0.88 (s, 3H, 23-H) ppm; 13C NMR (126 MHz, CDCl3):
d
¼ 212.60
(C-20), 175.39 (C-28), 170.86 (C-32), 170.39 (C-30), 150.25 (C-37),
138.78 (C-35), 131.38 (C-34), 130.94 (C-40), 128.97 (C-38), 128.44
(C-39), 128.28 (C-43), 127.66 (C-41), 120.82 (C-42), 78.04 (C-3),
69.73 (C-2), 56.77 (C-17), 55.40 (C-5), 51.11 (C-19), 50.86 (C-9), 50.11
(C-18), 42.72 (C-14), 42.34 (C-1), 41.03 (C-8), 38.18 (C-22), 37.55 (C-
4), 37.16 (C-10), 36.89 (C-13), 34.34 (C-7), 33.53 (C-16), 30.51 (C-29),
29.91 (C-15), 29.10 (C-23), 28.66 (C-21), 27.29 (C-12), 21.39 (C-33),
21.22 (C-11), 21.01 (C-31), 18.12 (C-6), 17.65 (C-24), 16.89 (C-25),
16.44 (C-26), 14.84 (C-27) ppm; MS (ESI, MeOH): m/z 685 (100%,
[MþH]þ); analysis calcd for C42H56N2O6 (684.92): C 73.65, H 8.24, N
4.09; found: C 73.40, H 8.42, N 4.17.
(s, 3H, 23-H) ppm; 13C NMR (101 MHz, CDCl3):
d
¼ 212.12 (C-20),
181.60 (C-28), 170.84 (C-32), 170.37 (C-30), 78.02 (C-3), 69.79 (C-2),
56.34 (C-17), 55.32 (C-5), 51.35 (C-19), 50.90 (C-9), 49.26 (C-18),
42.55 (C-14), 42.27 (C-1), 40.85 (C-8), 37.62 (C-13), 37.55 (C-4), 37.15
(C-10), 36.84 (C-22), 34.18 (C-7), 31.59 (C-16), 30.21 (C-29), 29.71
(C-15), 29.12 (C-23), 28.41 (C-21), 27.19 (C-12), 21.39 (C-33), 21.13
(C-11), 21.00 (C-31), 18.10 (C-6), 17.61 (C-24), 16.87 (C-25), 16.29 (C-
26), 14.79 (C-27) ppm; MS (ESI, MeOH): m/z 439 (28%, [M þ H-
HOAc]þ), 499 (32%, [M þ H-2HOAc]þ), 576 (100%, [M þ NH4]þ), 581
(48%, [MþNa]þ); analysis calcd for C33H50O7 (558.76): C 70.94, H
9.02; found: C 70.73, H 9.24.
4.4.14. Quinolin-5-yl 2
b,3b -diacetyloxy-20-oxo-30-norlupan-28-
amide (16)
4.4.12. Benzyl 2
(14)
b
,3
b
-diacetyloxy-20-oxo-30-norlupan-28-amide
According to GPC fron 13 (244 mg, 0.44 mmol) and 5-amino-
quinoline (25 mg, 1.8 mmol) followed by chromatography (SiO2,
hexanes/EtOAc, EtOAc: 10% / 60%) 16 (244 mg, 82%) was isolated
According to GPB from 13 (12.41 g, 2.22 mmol) and benzylamine
(0.49 mL, 4.5 mmol) followed by chromatography (SiO2, hexanes/
EtOAc, 3:2), 14 (1.21 g, 84%) was obtained as a colorless solid; m.p.
as an off-white solid; m.p. 290e292 ꢀC (decomp.); [
a
]
D
¼ ꢁ28.6ꢀ (c
0.213, CHCl3); RF ¼ 0.26 (SiO2, CHCl3:MeOH, 95:5); UVeVis (CHCl3):
150e154 ꢀC; [
a]
¼ þ21.5ꢀ (c 0.355, CHCl3); RF ¼ 0.49 (SiO2, hex-
lmax (log ε) ¼ 304 nm (4.34), 316 nm (4.31); IR (ATR):
1741m, 1364m, 1249s, 1027w, 809 s cmꢁ1 1H NMR (400 MHz,
CDCl3):
¼ 8.93 (d, J ¼ 2.8 Hz, 1H, 40-H), 8.17 (d, J ¼ 8.5 Hz, 1H, 42-
n
¼ 2939w,
D
anes/EtOAc, 3:1); UVeVis (CHCl3): lmax (log ε) ¼ 259 nm (3.22); IR
;
(ATR):
n
¼ 2943m, 1741s, 1660m, 1516m, 1231s, 1188m, 1030m,
d
699m, 603w cmꢁ1
;
1H NMR (400 MHz, CDCl3):
d
¼ 7.36e7.23 (m,
H), 8.02 (dd, J ¼ 6.9, 2.5 Hz, 1H, 41-H), 7.78e7.68 (m, 2H, 37-H, 35-
H), 7.45 (dd, J ¼ 8.6, 4.2 Hz, 1H, 36-H), 5.34e5.28 (m, 1H, 2-H), 4.60
(d, J ¼ 3.9 Hz, 1H, 3-H), 3.51e3.41 (m, 1H, 19-H), 2.41e2.31 (m, 1H,
13–H), 2.23 (m, 3H,16-Ha,18-H, 21-Ha), 2.17 (s, 3H, 29-H), 2.14e2.04
(m, 1H, 22-Ha), 2.02 (s, 3H, 33-H), 2.01 (s, 3H, 31-H), 1.97 (s, 1H, 1-
Ha), 1.89 (m, 1H, 16-Hb), 1.80e1.67 (m, 1H, 15-Ha, 22-Hb),
1.64e1.22 (m, 10H, 21-Hb, 6-H, 7-H, 11-H, 15-Hb, 9-H, 1-Hb), 1.10 (s,
3H, 25-H), 1.13e1.03 (m, 2H, 12-H), 1.06 (s, 3H, 27-H), 1.02 (s, 3H, 24-
H), 1.00 (s, 3H, 26-H), 0.97e0.91 (m, 1H, 5-H), 0.88 (s, 3H, 23-H)
5H, 36-H, 37-H, 38-H, 39-H, 40-H), 5.97 (t, J ¼ 5.7 Hz, 1H, NH) 5.30
(td, J ¼ 3.6 Hz, 1H, 2-H), 4.59 (d, J ¼ 3.9 Hz, 1H, 3-H), 4.51e4.44 (m,
1H, 34-Ha), 4.38e4.30 (m, 1H, 34-Hb), 3.46 (td, J ¼ 11.2, 4.1 Hz, 1H,
19-H), 2.27 (td, J ¼ 12.1, 4.1 Hz, 1H, 13-H), 2.16 (s, 3H, 29-H),
2.12e2.03 (m, 2H, 18-H, 21-Ha), 2.02 (s, 3H, 33-H), 2.01 (s, 3H, 31-H),
1.98e1.93 (m, 1H, 1-Hb), 1.93e1.85 (m, 1H, 16-Ha), 1.81e1.73 (m, 1H,
22-Ha), 1.71e1.19 (m, 12H, 16-Hb, 22-Hb, 6-H, 21-Hb, 15-Ha, 7-H, 11-
H, 1-Hb, 9-H), 1.14 (m, 1H, 15-Hb), 1.09 (s, 3H, 25-H), 1.13e1.05 (m,
2H, 12-H), 1.01 (s, 3H, 24-H), 0.97 (s, 3H, 27-H), 0.95e0.92 (m, 1H, 5-
H), 0.89 (s, 3H, 26-H), 0.87 (s, 3H, 23-H) ppm; 13C NMR (101 MHz,
ppm; 13C NMR (101 MHz, CDCl3):
d
¼ 212.51 (C-20), 175.35 (C-28),
170.84 (C-32), 170.36 (C-30), 150.19 (C-40), 147.96 (C-38), 132.88 (C-
34), 130.79 (C-42), 129.57 (C-35), 127.46 (C-41), 123.84 (C-43),
122.61 (C-37), 121.14 (C-36), 78.02 (C-3), 69.72 (C-2), 56.75 (C-17),
55.41 (C-5), 51.11 (C-19), 50.91 (C-9), 50.13 (C-18), 42.73 (C-14),
42.35 (C-1), 41.05 (C-8), 38.18 (C-22), 37.56 (C-4), 37.16 (C-10), 36.94
(C-13), 34.35 (C-7), 33.53 (C-16), 30.49 (C-29), 29.92 (C-15), 29.10
(C-23), 28.67 (C-21), 27.28 (C-12), 21.40 (C-33), 21.24 (C-11), 21.01
(C-31), 18.12 (C-6), 17.66 (C-24), 16.89 (C-25), 16.47 (C-26), 14.85 (C-
27) ppm; MS (ESI, MeOH): m/z 685 (100%, [MþH]þ), 707 (8%,
[MþNa]þ), 1370 (18%, [2M þ H]þ), 1391 (10%, [2M þ Na]þ); analysis
calcd for C42H56N2O6 (684.92): C 73.65, H 8.24, N 4.09; found: C
73.47, H 8.39, N 4.20.
CDCl3):
d
¼ 212.76 (C-20), 175.70 (C-28), 170.65 (C-32), 170.19 (C-
30), 138.96 (C-35), 128.68 (C-38, C-37), 127.73 (C-40, C-36), 127.39
(C-38), 77.89 (C-3), 69.60 (C-2), 55.48 (C-17), 55.22 (C-5), 50.97 (C-
19), 50.93 (C-9), 49.92 (C-18), 43.30 (C-34), 42.42 (C-14), 42.17 (C-
1), 40.79 (C-8), 37.95 (C-22), 37.39 (C-4), 36.99 (C-10), 36.68 (C-13),
34.15 (C-7), 32.98 (C-16), 30.30 (C-29), 29.33 (C-15), 28.94 (C-23),
28.54 (C-21), 27.13 (C-12), 21.24 (C-33), 21.06 (C-11), 20.84 (C-31),
17.97 (C-6), 17.50 (C-24), 16.73 (C-25), 16.19 (C-26), 14.60 (C-27)
ppm; MS (ESI, MeOH): m/z 648 (72%, [MþH]þ), 994 (52%,
[3Mþ2Na]2þ), 1295 (34%, [2M þ H]þ), 1317 (72%, [2M þ Na]þ);
analysis calcd for C40H57NO6 (647.90): C 74.15, H 8.87, N 2.16; found:
C 73.97, H 9.03, N 2.31.
4.4.15. 3b-Acetyloxy-lup-20(29)-en-28-oic acid (17) [10376-50-8]
4.4.13. Isoquinolin-4-yl 2
28-amide (15)
According to GPC from 13 (137 mg, 0.24 mmol) and 4-
aminoisoquinoline (150 mg, 1.0 mmol) 15 (83 mg, 49%) was ob-
tained as an off-white solid; m.p. 205e208 ꢀC (decomp.);
b
,3
b
-diacetyloxy-20-oxo-30-norlupan-
According to GPA from 1 (0.5 g, 1.10 mmol) followed by crys-
tallization from EtOH, 17 (489 mg, 89%) was obtained as a colorless
solid; m.p. 275e278 ꢀC (lit.: [35] 277e278 ꢀC); [
a
]
¼ þ20.6ꢀ (c
D
0.350, CHCl3) [lit.: [35] [
hexanes/EtOAc, 4:1).
a
]
¼ þ22ꢀ (c 0.49, CHCl3)]; RF ¼ 0.58 (SiO2,
D
9

S. Hoenke, N.V. Heise, M. Kahnt et al.
European Journal of Medicinal Chemistry 207 (2020) 112815
4.4.16. Benzyl 3
b
-acetyloxy-lup-20(29)-en-28-amide (18)
21.12 (C-11), 19.66 (C-30), 18.32 (C-6), 16.62 (C-24), 16.38 (C-25, C-
26), 14.83 (C-27) ppm; MS (ESI, MeOH): m/z 625 (100%, [MþH]þ),
1249 (44%, [2M þ H]þ); C41H56N2O3 (624.91): C 78.80, H 9.03, N
4.48; found: C 78.53, H 9.23, N 4.57.
According to GPB from 17 (407 mg, 0.82 mmol) and benzyl-
amine (0.3 mL, 2.8 mmol) followed by chromatography (SiO2,
hexanes/EtOAc, 9:1), 18 (350 mg, 73%) was obtained as a colorless
solid; m.p. 135 ꢀC, 210 ꢀC (decomp.) (lit.: [30,38] 124e127 ꢀC);
[
a
]
¼ 20.9ꢀ (c 0.348, CHCl3) [lit.: [30,38] [
a]
¼ þ23.2ꢀ (c 0.35,
4.4.19. 3b-Acetyloxy-20-oxo-30-norlupan-28-oic acid (21)
D
D
CHCl3)]; RF ¼ 0.65 (SiO2, hexanes/EtOAc, 7:1).
According to GPA from 9 (15.02 g, 32.8 mmol) followed by
crystallization from EtOH, 21 (13.8 g, 84%) was obtained as a
colorless solid; m.p. 268e270 ꢀC (decomp.) (lit.: [39] 252e255 ꢀC);
4.4.17. Isoquinolin-4-yl 3b-acetyloxy-lup-20(29)-en-28-amide (19)
According to GPC from 17 (300 mg, 0.60 mmol) and 4-amino-
isoquinoline (300 mg, 2.1 mmol) followed by chromatography
(SiO2, hexanes/EtOAc, EtOAc: 15% / 60%), 19 (214 mg, 56%) was
obtained as an off-white solid; m.p. 235 ꢀC (decomp.);
[
a
]
¼ ꢁ9.1ꢀ (c 0.34, CHCl3) [lit.: [39] [
a
]
¼ ꢁ9.5ꢀ (c 0.80, CHCl3)];
D
D
RF ¼ 0.50 (SiO2, toluene/EtOAc/heptane/HCOOH, 80:26:10:5).
4.4.20. Benzyl 3b-acetyloxy-20-oxo-30-norlupan-28-amide (22)
[
a
]
¼ ꢁ10.7ꢀ (c 0.124, CHCl3); RF ¼ 0.5 (SiO2, CHCl3/MeOH, 95:5);
According to GPB from 21 (250 mg, 0.5 mmol) and benzylamine
(0.2 mL, 1.9 mmol) followed by chromatography (SiO2, hexanes/
EtOAc, EtOAc: 5% / 30%) 22 (244 mg, 76%) [40] was obtained as a
D
UVeVis (CHCl3): lmax (log ε) ¼ 229 nm (3.87); IR (ATR): ¼ 2943m,
n
2870w, 1731m, 1656w, 1585w, 1516w, 1468m, 1411w, 1391m, 1373m,
1244s,1182w,1135w,1106w,1026m, 979m, 884m, 797w, 778w, 749s,
colorless solid; m.p. 290 ꢀC (decomp.); [
a
]
¼ þ0.5ꢀ (c 0.159, CHCl3);
D
559w cmꢁ1; 1H NMR (500 MHz, CDCl3):
d
¼ 9.08 (s, 1H, 36-H), 8.69
RF ¼ 0.35 (SiO2, hexanes/EtOAc, 3:1); IR (ATR):
n
¼ 2943m, 1733m,
(s, 1H, 34-H), 7.99 (d, J ¼ 8.2 Hz, 1H, 38-H), 7.82e7.70 (m, 2H, 40-H,
39-H), 7.63 (t, J ¼ 7.1 Hz, 1H, 41-H), 4.74 (s, 1H, 29-Ha), 4.62 (s, 1H,
19-Hb), 4.50e4.42 (m, 1H, 3-H), 3.23e3.14 (m, 1H, 19-H), 2.58 (m,
1H, 13-H), 2.33e2.24 (m, 1H, 16-Ha), 2.12e2.05 (m, 2H, 22-Ha, 21-
Ha), 2.03 (s, 3H, 32-H), 1.71 (s, 3H, 30-H), 1.86e1.17 (m, 16H, 16-
Hb, 12-Ha, 15-Ha, 1-Ha, 18-H, 22-Hb, 2-H, 21-Hb, 11-Ha, 7-H, 6-Hb,
15-Hb, 9-H, 11-Hb), 1.03 (s, 3H, 27-H), 1.00 (s, 3H, 25-H), 0.97 (m, 2H,
12-Hb, 1-Hb), 0.84 (m, 6H, 23-H, 25-H), 0.83 (s, 3H, 24-H), 0.79 (m,
1654m, 1519m, 1453w, 1367m, 1244s, 1027m, 979m, 753m, 698m,
609w cmꢁ1; 1H NMR (500 MHz, CDCl3):
¼ 7.37e7.27 (m, 5H, 35-H,
d
37-H, 38-H, 34-H, 36-H), 5.95 (t, J ¼ 5.7 Hz, 1H, NH), 4.48 (m, 2H, 3-
H, 32-Ha), 4.37e4.27 (m, 1H, 32-Hb), 3.48 (m, 1H, 19-H), 2.26 (m, 1H,
13-H), 2.17 (s, 3H, 29-H), 2.13e2.04 (m, 1H, 18-H), 2.03 (s, 3H, 31-H),
1.88 (m,1H,16-Ha),1.77 (m,1H, 21-Ha),1.68e1.20 (m,17H,1-Ha, 2-H,
6-H, 7-H, 9-H, 11-H, 12-H, 15-H, 16-Hb, 21-Hb, 22-Hb), 1.19e1.02 (m,
1H, 1-Hb), 0.98 (s, 3H, 27-H), 0.88 (s, 3H, 26-H), 0.84e0.80 (m, 9H,
23-H, 26-H, 27-H), 0.78 (m, 1H, 5-H) ppm; 13C NMR (126 MHz,
1H, 5-H) ppm; 13C NMR (126 MHz, CDCl3):
d
¼ 175.58 (C-28), 171.18
(C-31), 150.68 (C-20), 149.77 (C-36), 138.49 (C-34), 131.49 (C-33),
130.90 (C-39), 128.88 (C-37), 128.68 (C-42), 128.35 (C-38), 127.62
(C-41), 121.15 (C-40), 109.80 (C-29), 81.08 (C-3), 56.93 (C-17), 55.65
(C-5), 50.76 (C-9), 50.45 (C-18), 46.66 (C-19), 42.77 (C-14), 41.03 (C-
8), 38.64 (C-1), 38.59 (C-4), 37.95 (C-10), 37.89 (C-13), 37.29 (C-22),
34.56 (C-7), 34.21 (C-16), 31.06 (C-21), 30.00 (C-15), 28.08 (C-23),
25.79 (C-12), 23.84 (C-2), 21.45 (C-32), 21.12 (C-11), 19.68 (C-30),
18.34 (C-6), 16.62 (C-24), 16.38 (C-25, C-26), 14.83 (C-27) ppm; MS
(ESI, MeOH): m/z 626 (76%, [MþH]þ), 1249 (100%, [2M þ H]þ);
analysis calcd for C41H56N2O3 (624.91): C 78.80, H 9.03, N 4.48;
found: C 78.61, H 9.18, N 4.61.
CDCl3):
d
¼ 213.01 (C-20), 175.88 (C-28), 171.09 (C-30), 139.12 (C-
33),128.84 (C-35, C-37),127.89 (C-34, C-38),127.55 (C-36), 81.01 (C-
3), 55.66 (C-17), 55.55 (C-5), 51.17 (C-19), 50.57 (C-9), 50.19 (C-18),
43.46 (C-32), 42.42 (C-14), 40.84 (C-8), 38.52 (C-1), 38.11 (C-4),
37.94 (C-22), 37.27 (C-10), 36.94 (C-13), 34.41 (C-7), 33.13 (C-16),
30.47 (C-29), 29.62 (C-15), 28.72 (C-21), 28.08 (C-23), 27.39 (C-12),
23.81 (C-2), 21.44 (C-31), 21.09 (C-11), 18.32 (C-6), 16.63 (C-24),
16.36 (C-25), 16.22 (C-26), 14.82 (C-27) ppm; MS (ESI, MeOH): m/z
590 (100%, [MþH]þ), 1179 (66%, [2M
þ
H]þ), 1201 (54%,
[2M þ Na]þ); analysis calcd for C38H55NO4 (589.86): C 77.38, H 9.40,
N 2.37; found: C 77.19, H 9.65, N 2.50.
4.4.18. Quinolin-5-yl 3
b
-acetoxy-lup-20(29)-en-28-amide (20)
4.4.21. Isoquinolin-4-yl 3
amide (23)
According to GPC from 21 (250 mg, 0.5 mmol) and 4-
aminoisoquinoline (250 mg, 1.7 m mol) followed by chromatog-
raphy SiO2, hexanes/EtOAc, EtOAc: 20% / 80%) 23 (228 mg, 72%)
was obtained as an off-white solid; m.p. 235 ꢀC (decomp.);
b-acetyloxy-20-oxo-30-norlupan-28-
According to GPC from 17 (200 mg, 0.4 mmol) and 5-
aminoquinoline (200 mg, 1.4 mmol) followed by chromatography
(SiO2: hexanes/EtOAc, EtOAc: 25% / 50%), 20 (160 mg, 64%) was
obtained as an off-white solid; m.p. 277e280 ꢀC (decomp.);
[
a
]
¼ þ21.2ꢀ (c 0.261, CHCl3); RF ¼ 0.54 (SiO2, CHCl3/MeOH, 95:5);
D
UVeVis (CHCl3): lmax (log ε) ¼ 236 nm (3.93); IR (ATR):
n
¼ 2943m,
[
a
]
¼ ꢁ20.3ꢀ (c 0.211, CHCl3); RF ¼ 0.26 (SiO2, CHCl3/MeOH, 95:5);
D
1730m, 1649w, 1594w, 1477m, 1392w, 1367m, 1244s, 1181w, 1028m,
979m, 883m, 797s, 755m, 651w cmꢁ1 1H NMR (400 MHz, CDCl3):
¼ 8.91e8.86 (m, 1H, 39-H), 8.15 (d, J ¼ 8.5 Hz, 1H, 41-H), 7.97 (dd,
UVeVis (CHCl3): lmax (log ε) ¼ 228 nm (3.74); IR (ATR): ¼ 2943m,
n
;
1710m, 1585w, 1516w, 1467m, 1368m, 1245s, 1183w, 1024m, 979m,
893w, 749s, 574w cmꢁ1; 1H NMR (500 MHz, CDCl3):
¼ 9.12 (s, 1H,
d
d
J ¼ 6.8, 2.6 Hz,1H, 34-H), 7.71e7.63 (m, 2H, 36-H, 35-H), 7.40 (dd, J ¼
8.5, 4.2 Hz, 1H, 40-H), 4.74 (s, 1H, 29-Ha), 4.62 (s, 1H, 29-Hb),
4.50e4.41 (m, 1H, 3-H), 3.19 (td, J ¼ 10.9, 4.7 Hz, 1H, 19-H), 2.57 (td,
J ¼ 12.6, 3.4 Hz, 1H, 13-H), 2.30e2.20 (m, 1H, 16-Ha), 2.11e1.99 (m,
2H, 21-Ha, 22-Ha), 2.03 (s, 3H, 32-H),1.71 (s, 3H, 30-H),1.89e1.15 (m,
17H, 16-Hb, 12-Ha, 15-Ha, 9-H, 1-Ha, 22-Hb, 2-H, 6-Ha, 21-Hb, 11-Ha,
7-H, 6-Hb, 15-Hb, 18-H, 11-Hb), 1.03 (s, 3H, 27-H), 1.11e0.91 (m, 2H,
12-Hb,1-Hb), 0.99 (s, 3H, 24-H), 0.84 (s, 3H. 23-H), 0.83 (s, 3H, 25-H),
0.82 (s, 3H, 26-H), 0.79 (m, 1H, 5-H) ppm; 13C NMR (101 MHz,
35-H), 8.72 (s, 1H, 33-H), 8.02 (d, J ¼ 8.1 Hz, 1H, 37-H), 7.78 (m, 2H,
38-H, 39-H), 7.65 (t, J ¼ 7.3 Hz, 1H, 40-H), 4.47 (dd, J ¼ 10.9, 5.3 Hz,
1H, 3-H), 3.50e3.42 (m, 1H, 19-H), 2.36 (m, 1H, 13-H), 2.30e2.20 (m,
2H,16-Ha,18-H), 2.17 (s, 3H, 29-H), 2.15e2.06 (m,1H, 22-Ha), 2.03 (s,
3H, 31-H),1.92e1.82 (m,1H,16-Hb),1.78e1.14 (m,15H, 22-Hb,15-Ha,
1-Ha, 2-H, 21-H, 6-Ha, 11-Ha, 7-H, 15-Hb, 6-Hb, 9-H, 11-Hb), 1.08 (s,
2H, 12-H), 1.06 (s, 3H, 27-H), 0.99 (s, 3H, 25-H), 0.96 (s, 1H, 1-Hb),
0.83 (d, J ¼ 7.1 Hz, 9H, 23-H, 24-H, 26-H), 0.80 (m, 1H, 5-H) ppm; 13
C
NMR (126 MHz, CDCl3):
d
¼ 212.90 (C-20), 175.69 (C-28), 171.38 (C-
CDCl3):
d
¼ 175.51 (C-28),171.17 (C-31),150.66 (C-20),150.13 (C-39),
30), 150.31 (C-35), 138.85 (C-33), 131.70 (C-32), 131.26 (C-38),
128.70 (C-37),127.98 (C-40),121.22 (C-39), 81.25 (C-3), 57.06 (C-17),
55.83 (C-5), 51.14 (C-19), 50.86 (C-9), 50.47 (C-18), 42.82 (C-14),
41.19 (C-8), 38.80 (C-1), 38.43 (C-4), 38.21 (C-22), 37.54 (C-10), 37.24
(C-13), 34.71 (C-7), 33.76 (C-16), 30.77 (C-29), 30.32 (C-15), 28.93
(C-21), 28.33 (C-23), 27.65 (C-12), 24.07 (C-2), 21.70 (C-31), 21.36
(C-11), 18.57 (C-6), 16.88 (C-24), 16.62 (C-25), 16.57 (C-26), 15.15 (C-
148.40 (C-37), 133.14 (C-42), 130.88 (C-41), 129.47 (C-35), 127.29 (C-
34), 123.89 (C-33), 122.56 (C-36), 121.00 (C-40), 109.81 (C-29), 81.06
(C-3), 56.88 (C-17), 55.64 (C-5), 50.75 (C-18), 50.46 (C-9), 46.71 (C-
19), 42.76 (C-14), 41.03 (C-8), 38.64 (C-4), 38.64 (C-1), 37.94 (C-22),
37.91 (C-13), 37.28 (C-10), 34.56 (C-7), 34.19 (C-16), 31.06 (C-21),
29.99 (C-15), 28.07 (C-23), 25.77 (C-12), 23.84 (C-2), 21.45 (C-32),
10

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27) ppm; MS (ESI, MeOH): m/z 627 (100%, [MþH]þ), 1253 (58%,
[2M þ H]þ); analysis calcd for C40H54N2O4 (626.88): C 76.64, H 8.68,
N 4.47; found: C 76.51, H 8.96, N 4.62.
4.4.22. Quinolin-5-yl 3
(24)
b -acetyloxy-20-oxo-30-norlupan-28-amide
According to GPC from 21 (250 mg, 0.5 mmol) and 5-
aminoquinoline (250 mg, 1.7 mmol) followed by chromatography
(SiO2, hexanes/EtOAc, EtOAc: 25% / 80%) 24 (177 mg, 57%) was
obtained as an off-white solid; m.p. 277e280 ꢀC (decomp.);
[
a
]
¼ ꢁ35.9ꢀ (c 0.106, CHCl3); RF ¼ 0.36 (SiO2, CHCl3/MeOH, 95:5);
D
UVeVis (CHCl3): lmax (log ε) ¼ 235 nm (3.95); IR (ATR): ¼ 2944m,
n
1732m, 1711m, 1595w, 1515w, 1476m, 1367m, 1244s, 1182w, 1133w,
1024m, 979m, 798s, 752s, 665w, 610w, 496w cmꢁ1 1H NMR
(400 MHz, CDCl3):
¼ 8.90 (d, J ¼ 3.0 Hz, 1H, 38-H), 8.21 (d,
;
d
J ¼ 8.4 Hz,1H, 40-H), 8.03 (d, J ¼ 7.7 Hz,1H, 33-H), 7.71 (m, 2H, 35-H,
34-H), 7.45 (dd, J ¼ 8.5, 4.3 Hz, 1H, 39-H), 4.51e4.42 (m, 1H, 3-H),
3.47 (m, 1H, 19-H), 2.36 (s, 1H, 13-H), 2.25 (m, 2H, 16-Ha, 18-H), 2.17
(s, 3H, 29-H), 2.14e2.05 (m, 2H, 21-Ha, 22-Ha), 2.03 (s, 3H, 31-H),
1.88 (m, 1H, 16-Hb), 1.80e1.16 (m, 14H, 22-Hb, 15-Ha, 1-Ha, 2-H, 6-Ha,
11-Ha, 7-H,15-Hb, 6-Hb, 9-H,11-Hb, 21-Hb),1.09 (s, 2H,12-H), 1.07 (s,
3H, 27-H), 0.99 (s, 3H 25-H), 0.97 (s, 1H, 1-Hb), 0.83 (m, 9H, 23-H,
24-H, 26-H), 0.79 (s, 1H, 5-H) ppm; 13C NMR (101 MHz, CDCl3):
d
¼ 212.59 (C-20), 175.45 (C-28),171.12 (C-30),149.77 (C-38),133.06
(C-36), 131.51 (C-40), 129.82 (C-34), 127.05 (C-33), 123.97 (C-32),
122.86 (C-35), 121.06 (C-39), 107.47 (C-41), 80.97 (C-3), 56.79 (C-
17), 55.57 (C-5), 50.94 (C-19), 50.59 (C-9), 50.24 (C-18), 42.57 (C-
14), 40.95 (C-8), 38.55 (C-1), 38.17 (C-4), 37.95 (C-22), 37.29 (C-10),
37.04 (C-13), 34.46 (C-7), 33.48 (C-16), 30.49 (C-29), 30.07 (C-15),
28.69 (C-21), 28.08 (C-23), 27.38 (C-12), 23.81 (C-2), 21.45 (C-31),
21.12 (C-11), 18.31 (C-6), 16.63 (C-24), 16.37 (C-25), 16.35 (C-26),
14.91 (C-27) ppm; MS (ESI, MeOH): m/z 628 (100%, [MþH]þ), 1253
(24%, [2M þ H]þ); analysis calcd for C40H54N2O4 (626.88): C 76.64,
H 8.68, N 4.47; found: C 76.46, H 8.87, N 4.65.
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ꢁ
ꢁ
ꢀ
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Synthetic approach for rapid preparation of BODIPY conjugates and their use
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asiatic acid into
a mitocanic, bimodal-acting rhodamine B conjugate of
Declaration of competing interest
nanomolar cytotoxicity, Eur. J. Med. Chem. 159 (2018) 143e148.
[18] 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.
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.
€
[19] R.K. Wolfram, L. Fischer, R. Kluge, D. Strohl, A. Al-Harrasi, R. Csuk, Homo-
piperazine-rhodamine B adducts of triterpenoic acids are strong mitocans,
Eur. J. Med. Chem. 155 (2018) 869e879.
[20] R.K. Wolfram, L. Heller, R. Csuk, Targeting mitochondria: esters of rhodamine
B with triterpenoids are mitocanic triggers of apoptosis, Eur. J. Med. Chem.
152 (2018) 21e30.
Acknowledgements
[21] I.Z. Pavel, R. Csuk, C. Danciu, S. Avram, F. Baderca, A. Cioca, E.A. Moaca,
C.V. Mihali, I. Pinzaru, D.M. Muntean, C.A. Dehelean, Assessment of the anti-
angiogenic and anti-inflammatory properties of a maslinic acid derivative and
its potentiation using zinc chloride, Int. J. Mol. Sci. 20 (2019).
[22] I.Z. Pavel, C. Danciu, C. Oprean, C.A. Dehelean, D. Muntean, R. Csuk,
D.M. Muntean, In vitro evaluation of the antimicrobial ability and cytotoxicity
on two melanoma cell lines of a benzylamide derivative of maslinic acid, Anal.
Cell Pathol. (2016), 2787623.
[23] I.Z. Pavel, C.A. Dehelean, L. Farczadi, D.M. Muntean, L. Vlase, C. Danciu, R. Csuk,
F. Birsasteanu, D.M. Muntean, Assessment of a maslinic acid derivative and its
metabolite in rat blood by liquid chromatography coupled with mass spec-
trometry, Rev. Chim.-Bucharest 68 (2017) 1089e1094.
€
We like to thank Dr. D. Strohl, S. Ludwig and Y. Schiller for the
NMR spectra and the late Dr. R. Kluge for numerous ESI-MS mea-
surements. IR, UVeVis spectra and optical rotations were recorded
by V. Simon, micro-analyses by S. Ludwig and M. Schneider. Cordial
thanks are due to E. Merkel for her help in the synthesis of some
starting materials as well as Dr. L. Fischer for performing pre-
liminary cytotoxicity tests. The cell lines were provided by Dr. T.
Müller (Dept. Oncology).
[24] I.Z. Pavel, O.M. Duicu, I. Scurtu, D. Coricovac, C. Dehelean, R. Csuk,
D.M. Muntean, Assessment of the effects of a maslinic acid derivative in iso-
lated mouse liver mitochondria, Eur. J. Clin. Invest. 45 (2015), 29-29.
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ment of the antioxidant effect of a maslinic acid derivative in an experimental
model of acute inflammation, FARMACIA 65 (2017) 390e395.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2020.112815.
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triterpenoic acid amides - the substantial role of an aromatic moiety align-
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