Selective killing of cancer cells with triterpenoic acid amides - The substantial role of an aromatic moiety alignment

Article abstract of DOI:10.1016/j.ejmech.2016.06.053

2,3-Di-O-acetyl-triterpenoic acid derived amides possessing a (2β, 3β) configuration in ring A and two acetyl groups were previously shown to possess high cytotoxicity for human tumor cell lines but to exhibit low cytotoxicity for non-malignant mouse fibroblasts. In this study, augustic acid (1) and 2-epi-corosolic acid (2) were chosen as starting points for the synthesis of analogs. While augustic acid derived 3-quinolinyl amide 9 gave low EC₅₀values in SRB assays but was cytotoxic for all lines, the isomeric 4-isoquinolinyl amide 21 was very cytotoxic for the tumor cell lines but significantly less cytotoxic for the mouse fibroblasts NIH 3T3. In addition, a triacetylated 4-isoquinolinyl derivative of asiatic acid (28) gave EC₅₀?=?80?nM (for A2780 ovarian cancer cells). As shown by additional experiments (acridine orange/propidium iodide staining, fluorescence spectroscopy and cell cycle investigations) these compounds act mainly by apoptosis.

Full text of DOI:10.1016/j.ejmech.2016.06.053

Accepted Manuscript
Selective killing of cancer cells with triterpenoic acid amides - The substantial role of
an aromatic moiety alignmentx
Sven Sommerwerk, Lucie Heller, Julia Kufs, René Csuk
PII:
S0223-5234(16)30543-8
10.1016/j.ejmech.2016.06.053
EJMECH 8714
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 18 May 2016
Revised Date: 26 June 2016
Accepted Date: 28 June 2016
Please cite this article as: S. Sommerwerk, L. Heller, J. Kufs, R. Csuk, Selective killing of cancer cells
with triterpenoic acid amides - The substantial role of an aromatic moiety alignmentx, European Journal
of Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.06.053.
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ACCEPTED MANUSCRIPT
Selective killing of cancer cells with triterpenoic acid amides – the substantial role of an
aromatic moiety alignment
Sven Sommerwerk, Lucie Heller, Julia Kufs, René Csuk*
Martin-Luther-University Halle-Wittenberg, Organic Chemistry, Kurt-Mothes-Str.2, D-06120
Halle (Saale) Germany
Corresponding author:
Prof. Dr. René Csuk
Bereich Organische Chemie
Martin-Luther Universität Halle-Wittenberg
Kurt-Mothes-Str. 2
D-06120 Halle (Saale)
Germany
Tel. +49 (0) 345 5525660
Fax: +49 (0) 345 5527030
Email: rene.csuk@chemie.uni-halle.de
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Graphical Abstract:
Selective killing of cancer cells with triterpenoic acid amides – the substantial role of an
aromatic moiety alignment
Sven Sommerwerk, Lucie Heller, Julia Kufs, René Csuk*
Martin-Luther-University Halle-Wittenberg, Organic Chemistry, Kurt-Mothes-Str.2, D-06120
Halle (Saale) Germany
A small structural difference (3-quinolinyl / 4-isoquinolinyl) has a strong impact on
cytotoxicity and malignant/non-malignant cell selectivity.
2

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Abstract
2,3-Di-O-acetyl-triterpenoic acid derived amides possessing a (2β, 3β) configuration in ring A
and two acetyl groups were previously shown to possess high cytotoxicity for human tumor
cell lines but to exhibit low cytotoxicity for non-malignant mouse fibroblasts. In this study,
augustic acid (1) and 2-epi-corosolic acid (2) were chosen as starting points for the synthesis
of analogs. While augustic acid derived 3-quinolinyl amide 9 gave low EC50 values in SRB
assays but was cytotoxic for all lines, the isomeric 4-isoquinolinyl amide 21 was very
cytotoxic for the tumor cell lines but significantly less cytotoxic for the mouse fibroblasts
NIH 3T3. In addition, a triacetylated 4-isoquinolinyl derivative of asiatic acid (28) gave EC50
= 80 nM (for A2780 ovarian cancer cells). As shown by additional experiments (acridine
orange/propidium iodide staining, fluorescence spectroscopy and cell cycle investigations)
these compounds act mainly by apoptosis.
Keywords: maslinic acid, oleanolic acid, asiatic acid, tumor cells, SRB assay, apoptosis
1. Introduction
Despite of improved technology and medical progress for the last decades, cancer remains
one of the world’s leading causes of death finally resulting in approximately 8 million deaths
globally per year.[1] Options to treat this family of diseases include surgery, radiotherapy,
chemotherapy or a combination thereof. Many of the chemotherapeutics commonly applied
for the treatment of cancer are characterized altogether by a high degree of non-selective
toxicity for the cells. For example, cis-platinum (one of the most common used
chemotherapeutics accounting to annually sales of approximately 3 billion Euro) is highly
active for many types of human tumors [2] (e.g. ovarian cancer as well as cancer of the head,
neck, testes but also for treating small cell lung cancer), but this drug also exhibits strong
nephro- and neurotoxic effects.[3] Thus, a main goal of an effective treatment of cancer
pursues to kill cancer cells selectively without any (or negligible) damage to non-malignant
cells. Modern therapeutic schemes to achieve these goals include, for example, prodrug
targeting,[4] advanced drug delivery systems [5, 6] or by applying light [7, 8] or heat [9, 10]
or by a selective drug targeting using magnetic triggering.[11, 12]
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In the course of investigating a small library of maslinic acid derivative compounds, we
discovered an interesting molecule named EM2 (Fig. 1), and this di-O-acetylated
benzylamide of maslinic acid exhibited rather low EC50 values (sulforhodamine B assays [13]
(SRB), EC50 = 0.5 µM for human ovarian cancer cells) while being significantly less toxic for
non-malignant mouse fibroblasts (NIH 3T3, EC50 = 33.8 µM).[14] We considered EM2 as an
ideal basis to start additional investigations concerning the biological activity of pentacyclic
triterpenoid analogs carrying an additional nitrogen containing heterocycle attached to ring E
by a suitable spacer. In addition, we became also interested in possible effects of the
presence/absence of acetyl groups in ring A onto cytotoxicity and/or selectivity of the
compounds. Hence, a panel of analogs was synthesized, and their cytotoxicity measured using
SRB assays. For selected compounds (showing low EC50 values for the cancer cells and high
EC50 values for the mouse fibroblasts), cell cycle investigations, annexin V/propidium iodide
staining experiments as well as visual inspections of the cells by fluorescence microscopy
were carried out.
Fig. 1. Structures of EM2, augustic acid (1) and 2-epi-corosolic acid (2).
2. Results and discussion
Augustic acid (1, Fig. 1) and 2-epi-corosolic acid (2) were chosen as starting points for the
synthesis of analogs. Both of these compounds carry two hydroxyl functions in ring A
showing a (2β, 3β)-configuration, and they are easily accessed by synthesis.[15-18]
Acetylation of 1 or 2 gave diacetates 3 and 4 (Scheme 1) whose reactions under Schotten-
Baumann conditions with oxalyl chloride followed by the addition of an amine furnished
amides 5–21 in moderate to excellent isolated yields between 38 and 90%. Amides 5–21 were
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characterized by NMR, IR, UV-vis spectroscopy and ESI-MS. Thus, in the IR spectra of these
amides a strong absorption band at v = 1506–1534 cm-1 was detected being characteristic for
the presence of a NHCO moiety. In their UV-vis spectra the aromatic chromophore gave
characteristic absorption maxima between λ = 244–279 nm for the pyridinyl substituted
compounds while for the quinolinyl amides absorption maxima λ = 241-335 nm were
1
detected. As far as their H NMR spectra are concerned, the signal for the NH moiety was
detected between δ = 10.30–10.37 ppm for the 8-quinolinyl amides while all of the other
compounds this signal was found between δ = 7.77–8.10 ppm, respectively.[19, 20]
Scheme 1. Synthesis of compounds 3–21: (a) Ac2O, TEA, DMAP, DCM, 25°C, 20h, 65–
79%; (b) oxalyl choride, TEA, DMF, DCM, 25°C, 2h; then: R3-NH2, TEA, DMAP, DCM,
25°C, 2d, 38–90%.
The compounds were subjected to SRB assays,[13] the results of which are summarized in
Table 1.
Table 1. Cytotoxicity of selected compounds (EC50 values in µM from SRB assays after 96
hours of treatment; the values are averaged from three independent experiments performed
each in triplicate; confidence interval CI = 95 %; n.d. not detected). Human cancer cell lines:
518A2 (melanoma), HT29 (colorectal adenocarcinoma), MCF7 (breast adenocarcinoma),
A549 (lung adenocarcinoma), A2780 (ovarian carcinoma), 8505C (thyroid carcinoma); NIH
3T3: nonmalignant mouse fibroblasts.
EC50 R1 R2 R3
518A2
25.7±0.7 >30
>30 >30
HT29
MCF7
A549
A2780
8505C
NIH 3T3
>30
H
Me -
Me H
Me -
25.1±2.2 n.d.
29.1±0.7 >30
n.d.
n.d.
1
2
3
-
24.9±2.7 >30
n.d. n.d.
>30
H
13.8±1.0 23.1± 0.6 16.1± 3.8 n.d.
>30
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Me H
Me 3-pyridinyl
Me H 3-pyridinyl
Me 4-pyridinyl
Me H 4-pyridinyl
Me 3-quinolinyl
Me H 3-quinolinyl
Me 4-quinolinyl
Me H 4-quinolinyl
Me 5-quinolinyl
Me H 5-quinolinyl
Me 6-quinolinyl
Me H 6-quinolinyl
Me 7-quinolinyl
Me H 7-quinolinyl
Me 8-quinolinyl
Me H 8-quinolinyl
-
17.9±0.6 16.2±1.2 10.4±2.9 15.6±0.4 17.9±0.6 15.6±0.5 12.7± 0.7
4
H
1.7±0.5 2.2±0.2
2.0±0.1 3.2±0.1
4.2±0.5 2.7±0.2
8.3±0.2 9.4±0.6
2.9±0.1 6.3±0.7
2.5±0.2 6.5±1.4
2.4±0.2 4.1±0.3
6.4±0.3 5.5±0.6
1.2±0.2 2.4±0.2
2.0±0.8 3.6±0.5
2.9±0.1 4.4±0.4
4.2±0.2 9.2±1.0
1.6±0.2 1.3±0.1
2.4±0.2 2.0±0.2
6.2±1.0 >30
2.0±0.2
3.5±0.3
3.1±0.7
9.0±1.3
9.3±1.3
3.2±0.4
4.3±0.4
4.0±1.0
2.5±0.1
5.5±1.0
5.8±0.8
6.5±1.3
1.4±0.2
1.6±0.4
2.8±0.4 0.9±0.1 2.3±0.4 2.4±0.2
3.5±0.2 2.0±0.1 2.7±0.3 4.2±0.3
4.0±0.3 1.6±0.7 4.1±0.3 1.8±0.2
10.1±0.9 8.3±0.2 8.5±0.1 5.7±0.9
7.4±0.7 2.2±0.6 5.0±0.4 0.9±0.3
3.4±0.4 2.8±0.4 3.1±0.2 2.2±0.2
4.2±0.3 2.0±0.9 3.4±0.2 2.9±0.8
8.2±0.7 3.5±0.1 6.6±0.8 5.2±0.9
3.0±0.4 0.7±0.1 2.3±0.2 8.6±1.8
3.7±0.5 2.7±0.5 3.2±0.3 >60
4.9±0.5 2.0±0.5 3.8±0.2 0.6±0.1
5.3±0.4 4.2±0.1 4.3±0.1 0.9±0.1
2.6±0.1 1.0±0.1 1.6±0.1 0.7±0.1
3.1±0.0 1.8±0.0 2.5±0.5 1.8±0.1
5
6
H
7
8
H
9
10
11
12
13
14
15
16
17
18
19
20
21
H
H
H
H
H
24.6±9.7 >30
5.1±0.9 16.1±3.5 >30
6.7±0.9 >30 >30
3.3±0.3 1.2±0.6 2.6±0.2 >60
13.3±1.4 >30
>30
>30
H
Me 4-isoquinolinyl 1.6±0.2 2.3±0.4
2.4±0.4
Compounds 5–8 carrying a pyridine substituent showed an improved cytotoxicity as
compared to acetylated acids 3 and 4. Thus, compound 5 gave an EC50 = 0.9 µM for the
human carcinoma cell line A2780 but selectivity between the human tumor cell lines and non-
malignant mouse fibroblasts NIH 3T3 remained low for these compounds. A different
performance, however, was observed during the biological evaluation of quinolinyl
substituted compounds – depending on their substitution pattern. While the cytotoxicity of
compounds 9–12 and 15–18 was similar to that of the pyridinyl substituted amides, 8-
quinolinyl derivatives (19 and 20) showed selective cytotoxicity towards different human
tumor cell lines. However, their overall cytotoxicity was low, and their solubility in water was
quite poor. As a consequence, we discarded these compounds from additional investigations.
5-Quinolinyl substituted compounds 13 and 14, however, performed better because they gave
rather low EC50 values, they were cytotoxic to the human tumor cell lines but of significantly
lowered cytotoxicity for the non-malignant mouse fibroblasts. From these findings we
presumed a potentially existing connection between cytotoxicity and the orientation of the
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aromatic system, and – as a consequence – we synthesized 4-isoquinolinyl compound 21.
Compounds 9 (a 3-quinolinyl derivative) and 21 are formally distinct from each other by a re-
orientation of the aromatic substituent. (Fig. 2)
Fig. 2. Comparison of cytotoxicity of 3-quinolinyl amide 9 and 4-isoquinolinyl amide 21
(EC50 values from SRB assays in µM) for different human tumor cell lines and nonmalignant
mouse fibroblasts (NIH 3T3).
While this re-orientation had only a slight but significant effect in gaining high cytotoxicity,
its impact on the selectivity for tumor cells was dramatically enhanced, and selectivity factors
greater/equal 50 were observed.
To get a first impression of the impact of the presence of the hydroxyl groups of ring A, some
derivatives of ursolic and asiatic acid were synthesized (Scheme 2).
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Scheme 2. Synthesis of compounds 4, 14, 24–27: (a) Ac2O, TEA, DMAP, DCM, 25 °C, 20 h,
65–89%; (b) oxalyl choride, TEA, DMF, DCM, 25°C, 2h; then 5-aminoquinoline, TEA,
DMAP, DCM, 25 °C, 2 d, 52–87%.
The cytotoxic activities of acetylated amides of ursolic acid [21-26] has already been
investigated by several groups in detail while for asiatic acid mainly amides without
additional acetyl groups in ring A have been investigated so far. 27, 28 Screening of our analogs
for their cytotoxic activity showed (cf. Table 2) gave for compound 26 (an ursolic acid
derived 5-quinolinyl derivative) an almost complete loss of selectivity between cancer cells
and nonmalignant mouse fibroblasts. For analog 27 (derived from asiatic acid), however,
selectivity was maintained, and an increased cytotoxicity was gained.
Table 2. Cytotoxicity of selected compounds (EC50 values in µM from SRB assays after 96
hours of treatment; the values are averaged from three independent experiments performed
each in triplicate; confidence interval CI = 95 %). Human cancer cell lines: 518A2
(melanoma), HT29 (colorectal adenocarcinoma), MCF7 (breast adenocarcinoma), A549 (lung
adenocarcinoma), A2780 (ovarian carcinoma), 8505C (thyroid carcinoma); NIH 3T3:
nonmalignant mouse fibroblasts.
EC50
14
518A2
2.0±0.8
3.5±0.9
HT29
MCF7
A549
A2780
2.7±0.5
4.4±0.6
8505C
3.2±0.3
3.5±0.0
NIH 3T3
> 60
3.6±0.5
5.1±0.4
5.5±1.0
3.9±0.1
3.7±0.5
5.1±0.6
3.5±0.2
26
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0.5±0.1
0.8±0.1
1.4±0.1
1.1±0.1
0.4±0.2
0.9±0.0
10.2±4.0
27
Within the panel of compounds investigated so far, two factors appear particularly critical for
obtaining cytotoxic as well as tumor-selective compounds, viz. the presence of an
isoquinolinyl moiety at position 28 as well as a minimum of three O-acetyl groups attached to
ring A. Combining these factors led to the synthesis of compound 28, an 4-isoquinolinyl
derivative of asiatic acid whose EC50 values are depicted in Figure 3.
Fig. 3. Cytotoxicity of compound 28 [EC50 values (in µM from SRB assays)] for different
human tumor cell lines and non-malignant mouse fibroblasts (NIH 3T3).
Hence, we managed the synthesis of a triterpenoid derivative showing both low EC50 values
(for example, EC50 = 80 nM for A2780 human ovarian cancer) but also high
tumor/nonmalignant cell selectivity (EC50 = 3.23 µM for NIH 3T3, thus a selectivity index of
40).
To have a better understanding of the cytotoxicity of these compounds (and their mode of
action) some additional experiments were carried out (Fig. 4). A2780 Ovarian cancer cells
were incubated with compounds 9, 11, 13, 15, 21, 27 and 28, and annexin V/propidiuum
iodide staining experiments as well as quantitative FACS experiments were performed.
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Incubation of the cells with each of the compounds for two days showed a significant increase
in the number of cells having died by apoptosis; even more cells had died by secondary
necrosis. (Fig. 4 B)
Fig. 4. (A) Representative samples of FACS based annexin V/PI assay of A2780 cells after
24h and 48h treatment with 21 and 28. (B) Evaluation of annexin V/PI assay: treatment of
A2780 cells with derivatives 9, 11, 13, 15, 21, 27 and 28 after 24h and 48h. Diagram with
arithmetic population distribution [%], determined by Attune® Cytometric Software vl 1.2.5.
(C) Fluorescence microscopic and bright-field microscopic images (scale bar = 20 µm):
control (A2780, 40h); treatment of A2780 cells with 21.
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Treatment of the cells with compound 28 for 24 h, however, showed the number of apoptotic
cells significantly higher than that of cells having died of secondary necrosis. For example,
treatment of A2780 cells with compound 28 (concentration as low as 1.5 µM, treatment for 24
h) gave an increase in the number of apoptotic cells (as compared to control) of 20% while the
number of cells having died of necrosis or secondary necrosis remained unchanged.
Prolongation of the time of incubation (up to 48 hours) led to a decrease of apoptotic cells by
10% while the number of secondary necrotic cells increased by 45% (compared to control).
Furthermore, inspection of dead A2780 cells (after having been incubated with 21, 6 µM, 40
h) by fluorescence microscopy and application of an acridine orange/propidium iodide
staining (AO/PI, Fig. 4C) allowed the identification of apoptose induced modifications of the
cell membrane. Comparison of cells (treated either with 21 or untreated) showed for the
untreated cells the complete absence of apoptosis while the cells that were treated with 21
showed an unsymmetrical distribution of AO (typically crescent and granular) [27] and
membrane blebbing – both findings are characteristic hallmarks for the presence of apoptosis.
Some of the cells were shining in orange – these cells were at a secondary necrotic stage.
For the investigation of the cell cycle, A2780 cells were incubated with compounds 9, 11, 13,
15, 21, 27 and 28 for 24h and 48h, respectively. Inspection of the results showed after an
incubation for 24 h a decrease of the G1 peak together with a simultaneous increase of cells in
the S phase and a complete loss of the G2 peak (Fig. 5). After incubation of the cells for 48 h,
a decrease of the S phase was observed, and a subG1 peak appeared. This hypodiploidic peak
can be explained by a cleavage of DNA; the presence of this peak is usually regarded as one
of the typical hallmarks of apoptotic cells. The results from some additional investigations of
the cell cycle as well as quantitative FACS measurements have been compiled in the
supplementary material.
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Fig. 5. (A): Representative examples for cell cycle evaluation of A2780 cells after 24h and
48h treatment with derivatives 21 and 28 via ModFit V 4.0.5. (B): Diagram with arithmetic
means of the population distribution [%] of A2780 cells treated with 9, 11, 13, 15, 21, 27 and
28 for 24h and 48h.
3. Conclusion
In this study amides of 2,3-O-acetyl-augustic acid and -2-epi-corosolic acid were synthesized,
and their cytotoxicity was determined photometric SRB assays. While augustic acid derived
3-quinolinyl amide 9 gave low EC50 values in SRB assays (EC50 = 2.2 to 9.3 µM) but was
cytotoxic for all lines including non-malignant mouse fibroblasts (EC50 = 0.9 µM), the
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isomeric 4-isoquinolinyl amide 21 was even higher cytotoxic for the tumor cell lines (EC50 =
1.2 to 2.6 µM) but significantly less cytotoxic for the mouse fibroblasts NIH 3T3 (EC50 > 60
mM). In addition, a triacetylated 4-isoquinolinyl derivative of asiatic acid (28) gave rather
low EC50 = 80 nM (for A2780 ovarian cancer cells) values. As shown by additional
experiments (acridine orange/propidium iodide staining, fluorescence spectroscopy and cell
cycle investigations) these compounds act mainly by apoptosis. These derivatives can
function as ideal starting points for further screening and testing.
4. Experimental Part
4.1.General
Information about equipment and general techniques has been compiled in the supplementary
materials section.
4.2.Syntheses
4.2.1. 2β, 3β-Diacetyloxy-olean-12-en-28-oic acid (3)
To a solution of 1 (950 mg, 2.01 mmol) in dry DCM (100 mL), acetic anhydride (820 mg,
8.04 mmol), triethylamine (915 mg, 9.05 mmol) and DMAP (25 mg, 0.20 mmol) were added,
and the mixture was stirred at room temperature for 20 hours. Et2O (500 mL) was added, and
the organic layer was washed with diluted HCl (0.1 M, 1 × 500 mL), water (2 × 500 mL) and
brine (1 × 250 mL), dried (MgSO4), filtrated and concentrated in vacuum. The residue was
subjected to column chromatography (silica gel, hexane/ethyl acetate, 8:2) to provide 3
(884 mg, 79%) as a white solid; m.p. 322 °C (decomp.) (lit.[28]: 148–150 °C); RF = 0.24
(silica gel, hexane/ethyl acetate, 8:2); [α]D = +83.81° (c = 0.32, CHCl3); IR (KBr): ν = 2944s,
1744vs, 1698s, 1464w, 1436w, 1366m, 1252s, 1196m, 1160w, 1058m, 1030m cm−1; 1H NMR
(500 MHz, CDCl3): δ = 5.33–5.30 (m, 1 H, H–2), 5.27 (dd, J = 3.6, 3.6 Hz, 1 H, H–12), 4.62
(d, J = 3.9 Hz, 1 H, H–3), 2.82 (dd, J = 13.9, 4.5 Hz, 1 H, H–18), 2.08 (s, 3 H, H–32), 2.02 (s,
3 H, H–34), 2.01–1.91 (m, 3 H, H–1a + H–16a + H–11a), 1.89–1.69 (m, 3 H, H–11b + H–22a
+ H–15a), 1.64–1.41 (m, 7 H, H–6a + H–6b + H–16b + H–19a + H–22b + H–9 + H–7a),
1.38–1.28 (m, 3 H, H–7b + H–21a + H–1b), 1.24–1.11 (m, 2 H, H–21b + H–19b), 1.20 (s, 3
H, H–25), 1.12 (s, 3 H, H–27), 1.09–1.03 (m, 1 H, H–15b), 1.04 (s, 3 H, H–23), 1.00–0.96 (m,
1 H, H–5), 0.92 (s, 3 H, H–29), 0.90 (s, 3 H, H–30), 0.90 (s, 3 H, H–24), 0.76 (s, 3 H, H–26)
ppm; 13C NMR (125 MHz, CDCl3): δ = 184.3 (C–28), 170.9 (C–33), 170.4 (C–31), 143.9 (C–
13), 122.6 (C–12), 78.1 (C–3), 69.8 (C–2), 55.4 (C–5), 48.2 (C–9), 46.7 (C–17), 45.9 (C–19),
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42.0 (C–1), 41.8 (C–14), 41.1 (C–18), 39.5 (C–8), 37.5 (C–4), 37.0 (C–10), 33.9 (C–21), 33.2
(C–30), 32.6 (C–22), 32.6 (C–7), 30.8 (C–20), 29.3 (C–24), 27.7 (C–15), 26.2 (C–27), 23.7
(C–29), 23.6 (C–11), 22.9 (C–16), 21.4 (C–32), 21.0 (C–34), 18.1 (C–6), 17.8 (C–23), 17.5
(C–26), 16.1 (C–25) ppm; MS (ESI): m/z (%) = 557.2 ([M+H]+, 13), 574.3 ([M+NH4]+, 100),
579.4 ([M+Na]+, 21), 1249.1 ([2M+Na]+, 87); analysis calculated for C34H52O6 (556.77): C
73.34, H 9.41; found: C 73.18, H 9.57.
4.2.2. 2β, 3β-Diacetyloxy-urs-12-en-28-oic acid (4)
Following the procedure given for 1, from 2 compound 4 (728 mg, 65%) was obtained as a
white solid; m.p. 247–252 °C; RF = 0.48 (silica gel, hexane/ethyl acetate, 7:3); [α]D = +75.00°
(c = 0.39, CHCl3); IR (KBr): ν = 3410m, 2950s, 2872m, 1746vs, 1698s, 1458m, 1398m,
1
1370s, 1254vs, 1234s, 1194m, 1160m, 1056m, 1030s, 972m cm−1; H NMR (500 MHz,
CDCl3): δ = 5.32 (ddd, J = 3.8, 3.8, 3.5 Hz, 1 H, H–2), 5.24 (dd, J = 3.6, 3.6 Hz, 1 H, H–12),
4.63 (d, J = 3.9 Hz, 1 H, H–3), 2.18 (d, J = 11.2 Hz, 1 H, H–18), 2.08–1.84 (m, 4 H, H–11a +
H–11b + H–15a + H–16a), 2.04 (s, 3 H, H–32), 2.02 (s, 3 H, H–34), 1.97 (dd, J = 6.8, 3.9 Hz,
1 H, H–1a), 1.75–1.63 (m, 3 H, H–16b + H–22a + H–22b), 1.61–1.43 (m, 5 H, H–6a + H–6b
+ H–7a + H–9 + H–21a), 1.39–1.24 (m, 4 H, H–1b + H–7b + H–19 + H–21b), 1.22 (s, 3 H,
H–25), 1.14–1.03 (m, 1 H, H–15b), 1.07 (s, 3 H, H–27), 1.04 (s, 3 H, H–23), 1.03–0.96 (m, 2
H, H–5 + H–20), 0.95 (d, J = 6.3 Hz, 3 H, H–30), 0.90 (s, 3 H, H–24), 0.86 (d, J = 6.4 Hz, 3
13
H, H–29), 0.79 (s, 3 H, H–26) ppm; C NMR (125 MHz, CDCl3): δ = 183.7 (C–28), 170.9
(C–33), 170.4 (C–31), 138.3 (C–13), 125.7 (C–12), 78.1 (C–3), 69.8 (C–2), 55.3 (C–5), 52.6
(C–18), 48.1 (C–17), 48.1 (C–9), 42.2 (C–14), 42.1 (C–1), 39.8 (C–8), 39.1 (C–19), 39.0 (C–
20), 37.5 (C–4), 36.9 (C–22), 36.9 (C–10), 32.9 (C–7), 30.7 (C–21), 29.3 (C–24), 28.0 (C–
15), 24.1 (C–16), 23.8 (C–27), 23.5 (C–11), 21.4 (C–32), 21.3 (C–30), 21.0 (C–34), 18.1 (C–
6), 17.8 (C–23), 17.3 (C–26), 17.2 (C–29), 16.3 (C–25) ppm; MS (ESI): m/z (%) = 555.5 ([M-
H]-, 63), 1111.2 ([2M-H]-, 100), 1133.5 ([2M-2H+Na]-, 28); analysis calculated for C34H52O6
(556.77): C 73.34, H 9.41; found: C 73.09, H 9.61.
4.2.3. 2β, 3β-Diacetyloxy-olean-12-en-28-oic acid 3-pyridinyl amide (5)
To a solution of 3 (100 mg, 0.18 mmol) in dry DCM (10 mL), oxalyl chloride (70 mg,
0.55 mmol), triethylamine (3 mg, 0.03 mmol) and DMF (2 mg, 0.03 mmol) were added, and
the mixture was stirred at room temperature for 2 hours. The solvent was removed under
reduced pressure, the residue was dissolved in dry THF (1 × 10 mL), the solvent was
removed, and the residue was immediately dissolved in dry DCM (10 mL). The solution was
14

ACCEPTED MANUSCRIPT
cooled to 0 °C and triethylamine (24 mg, 0.24 mmol), DMAP (2 mg, 0.02 mmol) as well as 3-
aminopyridine (52 mg, 0.55 mmol) were added. After 2 days of stirring, Et2O (100 mL) was
added, the organic was washed with diluted HCl (0.1 M, 1 × 100 mL), water (2 × 100 mL) and
brine (1 × 50 mL), dried (MgSO4), filtrated and evaporated to dryness. Column
chromatography (silica gel, hexane/ethyl acetate, 7:3) afforded 5 (85 mg, 75%) as a white
solid; m.p. 156–160 °C; RF = 0.60 (silica gel, chloroform/ethyl acetate, 1:1); [α]D = +70.84°
(c = 0.34, CHCl3); UV-vis (CHCl3): λmax (log ε) = 244 nm (3.81), 279 nm (3.30) ; IR (KBr): ν
= 3388m, 2950s, 2878m, 1746vs, 1684m, 1586m, 1524s, 1480s, 1418m, 1366s, 1252vs, 1234s,
1192m, 1158m, 1056m, 1030m, 708m cm−1; 1H NMR (500 MHz, CDCl3): δ = 8.40 (d, J = 2.5
Hz, 1 H, H–36), 8.31 (dd, J = 4.7, 1.4 Hz, 1 H, H–37), 8.22 (ddd, J = 8.4, 2.5, 1.5 Hz, 1 H, H–
39), 7.77 (s, 1 H, NH), 7.24 (dd, J = 8.3, 4.7 Hz, 1 H, H–38), 5.57 (dd, J = 3.4, 3.4 Hz, 1 H,
H–12), 5.31 (ddd, J = 3.7, 3.7, 3.7 Hz, 1 H, H–2), 4.60 (d, J = 3.9 Hz, 1 H, H–3), 2.67 (dd, J =
12.7, 3.2 Hz, 1 H, H–18), 2.12–1.90 (m, 4 H, H–16a + H–11a + H–11b + H–1a), 2.04 (s, 3 H,
H–32), 2.02 (s, 3 H, H–34), 1.87–1.65 (m, 4 H, H–19a + H–22a + H–22b + H–16b), 1.64–
1.52 (m, 3 H, H–15a + H–6a + H–9), 1.52–1.22 (m, 7 H, H–7a + H–7b + H–6b + H–21a + H–
21b + H–1b + H–19b), 1.19 (s, 3 H, H–27), 1.14 (s, 3 H, H–25), 1.09 (ddd, J = 14.1, 2.8, 2.8
Hz, 1 H, H–15b), 1.02 (s, 3 H, H–23), 0.98–0.92 (m, 1 H, H–5), 0.93 (s, 6 H, H–29 + H–30),
13
0.88 (s, 3 H, H–24), 0.71 (s, 3 H, H–26) ppm; C NMR (125 MHz, CDCl3): δ = 177.3 (C–
28), 170.8 (C–33), 170.4 (C–31), 145.3 (C–37), 145.2 (C–13), 141.1 (C–36), 134.9 (C–35),
127.2 (C–39), 123.8 (C–38), 123.5 (C–12), 78.0 (C–3), 69.6 (C–2), 55.2 (C–5), 48.1 (C–9),
47.5 (C–17), 46.7 (C–19), 42.7 (C–18), 42.5 (C–14), 42.1 (C–1), 39.7 (C–8), 37.4 (C–4), 36.8
(C–10), 34.2 (C–21), 33.0 (C–30), 32.5 (C–22), 32.4 (C–7), 30.9 (C–20), 29.2 (C–24), 27.4
(C–15), 25.9 (C–27), 24.2 (C–16), 23.9 (C–11), 23.7 (C–29), 21.4 (C–32), 21.0 (C–34), 18.0
(C–6), 17.8 (C–23), 17.2 (C–26), 16.1 (C–25) ppm; MS (ESI): m/z (%) = 633.5 ([M+H]+,
100), 1265.1 ([2M+H]+, 16); analysis calculated for C39H56N2O5 (632.87): C 74.01, H 8.92, N
4.43; found: C 73.82, H 9.03, N 4.19.
4.2.4. 2β, 3β-Diacetyloxy-ursan-12-en-28-oic acid 3-pyridinyl amide (6)
As described for 5, compound 6 (120 mg, 56%) was obtained from 4 and 3-aminopyridine as
a white solid; m.p. 146–149 °C; RF = 0.42 (silica gel, hexane/ethyl acetate, 4:6); [α]D =
+51.20° (c = 0.35, CHCl3); UV-vis (CHCl3): λmax (log ε) = 244 nm (4.02), 279 nm (3.57); IR
(KBr): ν = 3394m, 2928s, 2872m, 1746vs, 1684m, 1618w, 1586m, 1526s, 1480s, 1485m,
1418s, 1398m, 1370s, 1326m, 1252vs, 1234vs, 1192m, 1156m, 1106w, 1078m, 1056m, 1030s,
1
972m, 944m, 754m, 706m cm−1; H NMR (500 MHz, CDCl3): δ = 8.39 (d, J = 2.2 Hz, 1 H,
15

ACCEPTED MANUSCRIPT
H–36), 8.31 (dd, J = 4.7, 1.1 Hz, 1 H, H–37), 8.19 (ddd, J = 8.4, 2.4, 1.4 Hz, 1 H, H–39), 7.76
(s, 1 H, NH), 7.24 (dd, J = 8.3, 4.7 Hz, 1 H, H–38), 5.51 (dd, J = 3.5, 3.5 Hz, 1 H, H–12), 5.32
(ddd, J = 3.8, 3.8, 3.5 Hz, 1 H, H–2), 4.61 (d, J = 3.9 Hz, 1 H, H–3), 2.12–1.93 (m, 6 H, H–1a
+ H–11a + H–11b + H–16a + H–18 + H–22a), 2.04 (s, 3 H, H–32), 2.02 (s, 3 H, H–34), 1.87–
1.81 (m, 1 H, H–16b), 1.75–1.67 (m, 1 H, H–15a), 1.62–1.45 (m, 6 H, H–6a + H–7a + H–9 +
H–19 + H–21a + H–22b), 1.42 (d, J = 11.8, 2.9 Hz, 1 H, H–6b), 1.39–1.26 (m, 3 H, H–1b +
H–7b + H–21b), 1.15 (s, 3 H, H–25), 1.13 (s, 3 H, H–27), 1.11–1.08 (m, 1 H, H–15b), 1.04–
0.90 (m, 2 H, H–5 + H–20), 1.02 (s, 3 H, H–23), 0.98 (d, J = 6.0 Hz, 3 H, H–30), 0.92 (d,
13
J = 6.5 Hz, 3 H, H–29), 0.89 (s, 3 H, H–24), 0.70 (s, 3 H, H–26) ppm; C NMR (125 MHz,
CDCl3): δ = 177.1 (C–28), 170.8 (C–33), 170.4 (C–31), 145.2 (C–37), 141.0 (C–36), 140.5
(C–13), 135.0 (C–35), 127.2 (C–39), 126.3 (C–12), 123.8 (C–38), 78.0 (C–3), 69.6 (C–2),
55.2 (C–5), 54.4 (C–18), 49.0 (C–17), 48.1 (C–9), 43.0 (C–14), 42.2 (C–1), 40.0 (C–19), 39.8
(C–8), 39.2 (C–20), 37.4 (C–4), 37.2 (C–22), 36.8 (C–10), 32.7 (C–7), 31.0 (C–21), 29.3 (C–
24), 27.9 (C–15), 25.2 (C–16), 23.8 (C–11), 23.5 (C–27), 21.4 (C–32), 21.3 (C–30), 21.0 (C–
34), 18.0 (C–6), 17.8 (C–23), 17.4 (C–29), 17.1 (C–26), 16.3 (C–25) ppm; MS (ESI): m/z (%)
= 633.7 ([M+H]+, 100), 1265.5 ([2M+H]+, 46); analysis calculated for C39H56N2O5 (632.87):
C 74.01, H 8.92, N 4.43; found: C 73.84, H 9.09, N 4.35.
4.2.5. 2β, 3β-Diacetyloxy-olean-12-en-28-oic acid 4-pyridinyl amide (7)
As described for 5, compound 7 (62 mg, 55%) was obtained from 3 and 4-aminopyridine as a
white solid; m.p. 171–174 °C; RF = 0.31 (silica gel, chloroform/ethyl acetate, 1:1); [α]D =
+32.92° (c = 0.31, MeOH); UV-vis (MeOH): λmax (log ε) = 248 nm (4.21); IR (KBr): ν =
3386m, 2950s, 2878m, 1746vs, 1696m, 1588s, 1506s, 1474m, 1412m, 1366s, 1326m, 1252vs,
1
1236s, 1200m, 1158m, 1056m, 1030m, 1012m, 992m, 822m cm−1; H NMR (500 MHz,
CD3OD): δ = 8.39–8.36 (m, 2 H, H–37), 7.70–7.67 (m, 2 H, H–36), 5.42 (dd, J = 3.5, 3.5 Hz,
1 H, H–12), 5.32 (ddd, J = 3.7, 3.7, 3.7 Hz, 1 H, H–2), 4.58 (d, J = 3.9 Hz, 1 H, H–3), 2.99
(dd, J = 13.2, 3.4 Hz, 1 H, H–18), 2.20 (ddd, J = 14.0, 14.0, 3.8 Hz, 1 H, H–16a), 2.07–1.90
(m, 3 H, H–11a + H–11b + H–1a), 2.02 (s, 3 H, H–32), 1.99 (s, 3 H, H–34), 1.86–1.75 (m, 2
H, H–19a + H–16b), 1.73–1.61 (m, 4 H, H–22a + H–22b + H–15a + H–9), 1.61–1.42 (m, 4 H,
H–6a + H–6b + H–7a + H–21a), 1.38 (dd, J = 15.0, 3.7 Hz, 1 H, H–1b), 1.32–1.18 (m, 3 H,
H–7b + H–21b + H–19b), 1.21 (s, 3 H, H–27), 1.21 (s, 3 H, H–25), 1.12 (ddd, J = 13.9, 3.0,
3.0 Hz, 1 H, H–15b), 1.06 (s, 3 H, H–23), 1.05–1.01 (m, 1 H, H–5), 0.98 (s, 3 H, H–29), 0.94
(s, 3 H, H–30), 0.90 (s, 3 H, H–24), 0.72 (s, 3 H, H–26) ppm; 13C NMR (125 MHz, CD3OD):
δ = 179.4 (C–28), 172.5 (C–33), 172.1 (C–31), 150.5 (C–37), 148.4 (C–35), 145.3 (C–13),
16

ACCEPTED MANUSCRIPT
124.0 (C–12), 115.8 (C–36), 79.6 (C–3), 70.8 (C–2), 56.4 (C–5), 49.3 (C–9), 48.9 (C–17),
47.4 (C–19), 43.1 (C–14), 42.8 (C–1), 42.3 (C–18), 40.7 (C–8), 38.3 (C–4), 37.9 (C–10), 35.0
(C–21), 33.6 (C–7), 33.5 (C–30), 33.4 (C–22), 31.6 (C–20), 29.5 (C–24), 28.5 (C–15), 26.6
(C–27), 24.6 (C–11), 24.1 (C–29), 23.8 (C–16), 21.2 (C–32), 20.8 (C–34), 19.0 (C–6), 18.2
(C–23), 17.7 (C–26), 16.6 (C–25) ppm; MS (ESI): m/z (%) = 633.4 ([M+H]+, 100); analysis
calculated for C39H56N2O5 (632.87): C 74.01, H 8.92, N 4.43; found: C 73.79, H 9.13, N 4.22.
4.2.6. 2β, 3β-Diacetyloxy-ursan-12-en-28-oic acid 4-pyridinyl amide (8)
As described for 5, compound 8 (41 mg, 56%) was obtained from 4 and 4-aminopyridine as a
white solid; m.p. 160–163 °C; RF = 0.23 (silica gel, hexane/ethyl acetate, 3:7); [α]D = +41.60°
(c = 0.86, CHCl3); UV-vis (CHCl3): λmax (log ε) = 248 nm (3.90); IR (KBr): ν = 3422s,
2950m, 2872w, 1744m, 1690m, 1636m, 1588m, 1506m, 1432s, 1414s, 1384vs, 1328s, 1252m,
1234m, 1196w, 1156w, 1052w, 1032w, 1010w cm−1; 1H NMR (500 MHz, CD3OD): δ = 8.36
(dd, J = 5.0, 1.5 Hz, 2 H, H–37), 7.67 (dd, J = 4.9, 1.6 Hz, 2 H, H–36), 5.42 (dd, J = 3.6,
3.6 Hz, 1 H, H–12), 5.32 (ddd, J = 3.9, 3.9, 3.2 Hz, 1 H, H–2), 4.59 (d, J = 4.0 Hz, 1 H, H–3),
2.37 (d, J = 10.4 Hz, 1 H, H–18), 2.23–2.15 (m, 1 H, H–16a), 2.03–1.95 (m, 3 H, H–1a + H–
11a + H–11b), 2.01 (s, 3 H, H–32), 1.99 (s, 3 H, H–34), 1.91–1.82 (m, 2 H, H–15a + H–16b),
1.82–1.76 (m, 1 H, H–22a), 1.70–1.46 (m, 7 H, H–6a + H–6b + H–7a + H–9 + H–19 + H–21a
+ H–22b), 1.44–1.36 (m, 2 H, H–1b + H–21b), 1.35–1.30 (m, 1 H, H–7b), 1.19 (s, 3 H, H–
25), 1.17 (s, 3 H, H–27), 1.14–1.08 (m, 2 H, H–15b + H–20), 1.07–1.01 (m, 1 H, H–5), 1.06
(s, 3 H, H–23), 1.00 (d, J = 6.4 Hz, 3 H, H–30), 0.95 (d, J = 6.5 Hz, 3 H, H–29), 0.90 (s, 3 H,
H–24), 0.70 (s, 3 H, H–26) ppm; 13C NMR (125 MHz, CD3OD): δ = 179.4 (C–28), 172.5 (C–
33), 172.1 (C–31), 150.5 (C–37), 148.4 (C–35), 140.1 (C–13), 127.1 (C–12), 115.7 (C–36),
79.6 (C–3), 70.7 (C–2), 56.3 (C–5), 53.8 (C–18), 50.2 (C–17), 49.1 (C–9), 43.4 (C–14), 43.0
(C–1), 40.9 (C–8), 40.7 (C–19), 40.1 (C–20), 38.3 (C–4), 37.8 (C–10), 37.8 (C–22), 33.8 (C–
7), 31.7 (C–21), 29.5 (C–24), 28.9 (C–15), 25.0 (C–16), 24.5 (C–11), 24.3 (C–27), 21.5 (C–
30), 21.1 (C–32), 20.8 (C–34), 19.0 (C–6), 18.2 (C–23), 17.7 (C–29), 17.6 (C–26), 16.6 (C–
25) ppm; MS (ESI): m/z (%) = 633.5 ([M+H]+, 100), 1287.5 ([2M+Na]+, 1); analysis
calculated for C39H56N2O5 (632.87): C 74.01, H 8.92, N 4.43; found: C 73.75, H 8.83, N 4.31.
4.2.7. 2β, 3β-Diacetyloxy-olean-12-en-28-oic acid 3-quinolinyl amide (9)
As described for 5, compound 9 (88 mg, 72%) was obtained from 3 and 3-aminoquinoline as
a white solid; m.p. 174–177 °C; RF = 0.69 (silica gel, chloroform/ethyl acetate, 1:1); [α]D =
+110.93° (c = 0.32, CHCl3); UV-vis (CHCl3): λmax (log ε) = 254 nm (4.60), 322 nm (3.69),
17

ACCEPTED MANUSCRIPT
335 nm (3.70); IR (KBr): ν = 3388w, 2948s, 2876m, 1746vs, 1684m, 1608w, 1534s, 1486s,
1
1468m, 1420m, 1364s, 1252vs, 1196m, 1158m, 1056m, 1030m cm−1; H NMR (500 MHz,
CDCl3): δ = 8.91 (d, J = 2.4 Hz, 1 H, H–43), 8.55 (d, J = 2.6 Hz, 1 H, H–36), 8.03–8.00 (m, 2
H, H–38 + NH), 7.78 (d, J = 8.0 Hz, 1 H, H–39), 7.61 (ddd, J = 8.4, 6.9, 1.4 Hz, 1 H, H–40),
7.52 (ddd, J = 8.1, 6.9, 1.2 Hz, 1 H, H–41), 5.65 (dd, J = 3.5, 3.5 Hz, 1 H, H–12), 5.32 (ddd, J
= 3.8, 3.8, 3.8 Hz, 1 H, H–2), 4.61 (d, J = 3.9 Hz, 1 H, H–3), 2.75 (dd, J = 12.9, 3.6 Hz, 1 H,
H–18), 2.18–1.95 (m, 4 H, H–16a + H–11a + H–11b + H–1a), 2.04 (s, 3 H, H–32), 2.02 (s, 3
H, H–34), 1.90–1.71 (m, 4 H, H–19a + H–22a + H–22b + H–16b), 1.68–1.52 (m, 3 H, H–15a
+ H–6a + H–9), 1.52–1.23 (m, 7 H, H–7a + H–7b + H–6b + H–21a + H–21b + H–1b + H–
19b), 1.22 (s, 3 H, H–27), 1.15–1.09 (m, 1 H, H–15b), 1.12 (s, 3 H, H–25), 1.01 (s, 3 H, H–
23), 0.98–0.94 (m, 1 H, H–5), 0.97 (s, 3 H, H–29), 0.96 (s, 3 H, H–30), 0.88 (s, 3 H, H–24),
13
0.70 (s, 3 H, H–26) ppm; C NMR (125 MHz, CDCl3): δ = 177.6 (C–28), 170.9 (C–33),
170.4 (C–31), 145.4 (C–37), 145.3 (C–13), 143.8 (C–36), 131.6 (C–35), 129.1 (C–38), 128.5
(C–42), 128.3 (C–40), 128.0 (C–39), 127.4 (C–41), 123.8 (C–43), 123.7 (C–12), 78.0 (C–3),
69.6 (C–2), 55.2 (C–5), 48.1 (C–9), 47.7 (C–17), 46.8 (C–19), 42.8 (C–18), 42.6 (C–14), 42.1
(C–1), 39.7 (C–8), 37.4 (C–4), 36.8 (C–10), 34.3 (C–21), 33.1 (C–30), 32.5 (C–22), 32.4 (C–
7), 30.9 (C–20), 29.2 (C–24), 27.4 (C–15), 26.0 (C–27), 24.4 (C–16), 23.9 (C–11), 23.7 (C–
29), 21.4 (C–32), 21.0 (C–34), 18.0 (C–6), 17.8 (C–23), 17.2 (C–26), 16.1 (C–25) ppm; MS
(ESI): m/z (%) = 683.5 ([M+H]+, 100); analysis calculated for C43H58N2O5 (682.93): C 75.62,
H 8.56, N 4.10; found: C 75.47, H 8.77, N 3.98.
4.2.8. 2β, 3β-Diacetyloxy-ursan-12-en-28-oic acid 3-quinolinyl amide (10)
As described for 5, compound 10 (60 mg, 65%) was obtained from 4 and 3-aminoquinoline as
a white solid; m.p. 141–143 °C; RF = 0.38 (silica gel, hexane/ethyl acetate, 6:4); [α]D =
+84.40° (c = 0.31, CHCl3); UV-vis (CHCl3): λmax (log ε) = 254 nm (4.60), 322 nm (3.66), 336
nm (3.66); IR (KBr): ν = 3396m, 2924s, 2870m, 2852m, 1744vs, 1684m, 1608w, 1576w,
1534s, 1486m, 1468m, 1458m, 1420m, 1396m, 1364s, 1252vs, 1234s, 1196m, 1158w, 1144w,
1
1116w, 1102w, 1078w, 1056m, 1030m cm−1; H NMR (500 MHz, CDCl3): δ = 8.86 (d,
J = 2.5 Hz, 1 H, H–43), 8.55 (d, J = 2.6 Hz, 1 H, H–36), 8.02 (d, J = 9.1 Hz, 1 H, H–38),
7.79–7.77 (m, 1 H, H–41), 7.63–7.59 (m, 1 H, H–39), 7.54–7.50 (m, 1 H, H–40), 5.59 (dd,
J = 3.6, 3.6 Hz, 1 H, H–12), 5.32 (ddd, J = 3.8, 3.8, 3.5 Hz, 1 H, H–2), 4.61 (d, J = 3.9 Hz, 1
H, H–3), 2.17–1.97 (m, 6 H, H–1a + H–11a + H–11b + H–16a + H–18 + H–22a), 2.04 (s, 3 H,
H–32), 2.02 (s, 3 H, H–34), 1.92–1.86 (m, 1 H, H–16b), 1.79–1.70 (m, 1 H, H–15a), 1.68–
1.46 (m, 6 H, H–6a + H–7a + H–9 + H–19 + H–21a + H–22b), 1.44–1.34 (m, 3 H, H–1b + H–
18

ACCEPTED MANUSCRIPT
6b + H–21b), 1.32–1.26 (m, 1 H, H–7b), 1.17–1.10 (m, 1 H, H–15b), 1.15 (s, 3 H, H–27), 1.12
(s, 3 H, H–25), 1.08–0.98 (m, 4 H, H–20 + H–30), 1.00 (s, 3 H, H–23), 0.97–0.93 (m, 1 H, H–
13
5), 0.95 (d, J = 6.5 Hz, 3 H, H–29), 0.88 (s, 3 H, H–24), 0.68 (s, 3 H, H–26) ppm; C NMR
(125 MHz, CDCl3): δ = 177.4 (C–28), 170.9 (C–33), 170.4 (C–31); 145.3 (C–42), 143.8 (C–
43), 140.7 (C–13), 131.7 (C–35), 129.1 (C–41), 128.5 (C–37), 128.2 (C–39), 128.0 (C–40),
127.3 (C–38), 126.4 (C–12), 123.7 (C–36), 78.0 (C–3), 69.6 (C–2), 55.2 (C–5), 54.4 (C–18),
49.1 (C–17), 48.1 (C–9), 43.0 (C–14), 42.2 (C–1), 40.0 (C–19), 39.8 (C–8), 39.2 (C–20), 37.4
(C–22), 37.2 (C–4), 36.8 (C–10), 32.7 (C–7), 31.0 (C–21), 29.3 (C–24), 28.0 (C–15), 25.4
(C–16), 23.8 (C–11), 23.5 (C–27), 21.4 (C–32), 21.3 (C–30), 21.0 (C–34), 18.0 (C–6), 17.8
(C–23), 17.4 (C–29), 17.2 (C–26), 16.3 (C–25) ppm; MS (ESI): m/z (%) = 683.5 ([M+H]+,
100), 1365.3 ([2M+H]+, 38); analysis calculated for C43H58N2O5 (682.93): C 75.62, H 8.56, N
4.10; found: C 75.51, H 8.89, N 3.99.
4.2.9. 2β, 3β-Diacetyloxy-olean-12-en-28-oic acid 4-quinolinyl amide (11)
As described for 5, compound 11 (49 mg, 40%) was obtained from 3 and 4-aminoquinoline as
a white solid; m.p. 226–227 °C; RF = 0.72 (silica gel, chloroform/ethyl acetate, 1:1); [α]D =
+17.60° (c = 0.29, CHCl3); UV-vis (CHCl3): λmax (log ε) = 299 nm (4.03), 315 nm (3.88); IR
(KBr): ν = 2946s, 2870m, 1744vs, 1692m, 1624m, 1570m, 1522s, 1494s, 1458s, 1432s,
1
1396m, 1366m, 1314m, 1252vs, 1236s, 1162m, 1056m, 1030m, 1012m cm−1; H NMR (500
MHz, CDCl3): δ = 8.83 (d, J = 5.1 Hz, 1 H, H–37), 8.58 (s, 1 H, NH), 8.49 (d, J = 5.1 Hz, 1
H, H–36), 8.13 (d, J = 8.3 Hz, 1 H, H–39), 7.76–7.71 (m, 2 H, H–41 + H–42), 7.59 (ddd, J =
8.3, 7.0, 1.2 Hz, 1 H, H–40), 5.76 (dd, J = 3.5, 3.5 Hz, 1 H, H–12), 5.30 (ddd, J = 3.8, 3.8, 3.8
Hz, 1 H, H–2), 4.60 (d, J = 3.9 Hz, 1 H, H–3), 2.85 (dd, J = 12.9, 3.8 Hz, 1 H, H–18), 2.16
(ddd, J = 14.0, 14.0, 3.8 Hz, 1 H, H–16a), 2.03–1.82 (m, 6 H, H–11a + H–11b + H–1a + H–
19a + H–22a + H–16b), 2.02 (s, 3 H, H–32), 2.02 (s, 3 H, H–34), 1.78 (ddd, J = 14.0, 14.0,
4.3 Hz, 1 H, H–22b), 1.66 (ddd, J = 13.9, 13.9, 4.3 Hz, 1 H, H–15a), 1.61–1.41 (m, 4 H, H–9
+ H–6a + H–21a + H–7a), 1.41–1.21 (m, 5 H, H–6b + H–19b + H–1b + H–21b + H–7b), 1.23
(s, 3 H, H–27), 1.14 (ddd, J = 13.7, 3.1, 3.1 Hz, 1 H, H–15b), 0.99 (s, 3 H, H–25), 0.98 (s, 3
H, H–29), 0.98 (s, 6 H, H–30 + H–23), 0.96–0.91 (m, 1 H, H–5), 0.87 (s, 3 H, H–24), 0.51 (s,
13
3 H, H–26) ppm; C NMR (125 MHz, CDCl3): δ = 177.5 (C–28), 170.9 (C–33), 170.4 (C–
31), 151.6 (C–37), 149.0 (C–35), 145.3 (C–13), 140.7 (C–38), 130.9 (C–39), 129.4 (C–41),
126.3 (C–40), 124.1 (C–12), 120.2 (C–43), 118.9 (C–42), 110.3 (C–36), 78.0 (C–3), 69.6 (C–
2), 55.3 (C–5), 48.8 (C–17), 48.1 (C–9), 46.9 (C–19), 43.1 (C–18), 42.5 (C–14), 42.0 (C–1),
39.6 (C–8), 37.4 (C–4), 36.7 (C–10), 34.2 (C–21), 33.1 (C–30), 32.8 (C–22), 32.4 (C–7), 30.9
19

ACCEPTED MANUSCRIPT
(C–20), 29.2 (C–24), 27.4 (C–15), 26.2 (C–27), 24.5 (C–16), 23.8 (C–29), 23.7 (C–11), 21.4
(C–32), 21.0 (C–34), 17.9 (C–6), 17.7 (C–23), 16.8 (C–26), 16.0 (C–25) ppm; MS (ESI): m/z
(%) = 683.4 ([M+H]+, 100); analysis calculated for C43H58N2O5 (682.93): C 75.62, H 8.56, N
4.10; found: C 75.41, H 8.81, N 4.00.
4.2.10. 2β, 3β-Diacetyloxy-ursan-12-en-28-oic acid 4-quinolinyl amide (12)
As described for 5, compound 12 (47 mg, 38%) was obtained from 4 and 4-aminoquinoline as
a white solid; m.p. 162–164 °C; RF = 0.56 (silica gel, chloroform/ethyl acetate, 1:1); [α]D = -
9.12° (c = 0.27, CHCl3); UV-vis (CHCl3): λmax (log ε) = 300 nm (4.03), 315 nm (3.90); IR
(KBr): ν = 2928s, 2872m, 1744vs, 1692m, 1570m, 1518s, 1494s, 1458s, 1394m, 1370m,
1310m, 1252vs, 1234s, 1196m, 1158w, 1056m, 1030m cm−1; 1H NMR (500 MHz, CDCl3): δ =
8.82 (d, J = 5.2 Hz, 1 H, H–37), 8.56 (s, 1 H, NH), 8.46 (d, J = 5.2 Hz, 1 H, H–36), 8.18 (d, J
= 8.5 Hz, 1 H, H–39), 7.77–7.73 (m, 2 H, H–41 + H–42), 7.63–7.58 (m, 1 H, H–40), 5.69–
5.66 (m, 1 H, H–12), 5.32–5.28 (m, 1 H, H–2), 4.59 (d, J = 3.6 Hz, 1 H, H–3), 2.22–1.91 (m, 7
H, H–18 + H–16a + H–16b + H–11a + H–11b + H–22a + H–1a), 2.01 (s, 6 H, H–32 + H–34),
1.81–1.31 (m, 10 H, H–15a + H–22b + H–21a + H–21b + H–19 + H–9 + H–7a + H–6a + H–
6b + H–1b), 1.31–1.24 (m, 1 H, H–7b), 1.18–1.12 (m, 1 H, H–15b), 1.16 (s, 3 H, H–27), 1.11–
0.91 (m, 2 H, H–20 + H–5), 1.03–0.98 (m, 12 H, H–23 + H–25 + H–29 + H–30), 0.87 (s, 3 H,
H–24), 0.54 (s, 3 H, H–26) ppm; 13C NMR (125 MHz, CDCl3): δ = 177.4 (C–28), 170.9 (C–
33), 170.4 (C–31), 151.1 (C–37), 148.4 (C–35), 141.3 (C–38), 140.5 (C–13), 130.4 (C–39),
129.6 (C–41), 126.7 (C–12), 126.5 (C–40), 120.2 (C–43), 118.9 (C–42), 110.3 (C–36), 78.0
(C–3), 69.6 (C–2), 55.3 (C–5), 54.6 (C–18), 50.3 (C–17), 48.1 (C–9), 42.9 (C–14), 42.1 (C–
1), 40.1 (C–19), 39.8 (C–8), 39.2 (C–20), 37.4 (C–22), 37.4 (C–4), 36.7 (C–10), 32.6 (C–7),
31.0 (C–21), 29.2 (C–24), 27.9 (C–15), 25.5 (C–16), 23.8 (C–11), 23.5 (C–27), 21.4 (C–32),
21.3 (C–30), 21.0 (C–34), 18.0 (C–6), 17.7 (C–23), 17.5 (C–29), 16.9 (C–26), 16.1 (C–25)
ppm; MS (ESI): m/z (%) = 683.5 ([M+H]+, 100); analysis calculated for C43H58N2O5 (682.93):
C 75.62, H 8.56, N 4.10; found: C 75.33, H 8.69, N 3.85.
4.2.11. 2β, 3β-Diacetyloxy-olean-12-en-28-oic acid 5-quinolinyl amide (13)
As described for 5, compound 13 (82 mg, 67%) was obtained from 3 and 5-aminoquinoline as
a white solid; m.p. 167–168 °C; RF = 0.60 (silica gel, chloroform/ethyl acetate, 1:1); [α]D =
+38.91° (c = 0.34, CHCl3); UV-vis (CHCl3): λmax (log ε) = 242 nm (4.49), 307 nm (3.77), 317
nm (3.77); IR (KBr): ν = 3386w, 2948s, 2866m, 1746vs, 1684m, 1594w, 1572w, 1522m,
1
1488s, 1396m, 1364s, 1252vs, 1196m, 1156m, 1056m, 1030m, 798m cm−1; H NMR (500
20

ACCEPTED MANUSCRIPT
MHz, CDCl3): δ = 8.93 (dd, J = 4.2, 1.5 Hz, 1 H, H–40), 8.14–8.12 (m, 2 H, H–42 + H–36),
8.10 (s, 1 H, NH), 7.92 (ddd, J = 8.5, 0.9, 0.9 Hz, 1 H, H–38), 7.69 (dd, J = 8.4, 7.7 Hz, 1 H,
H–37), 7.43 (dd, J = 8.6, 4.2 Hz, 1 H, H–41), 5.63 (dd, J = 3.5, 3.5 Hz, 1 H, H–12), 5.30 (ddd,
J = 3.9, 3.9, 3.9 Hz, 1 H, H–2), 4.60 (d, J = 3.9 Hz, 1 H, H–3), 2.86 (dd, J = 12.8, 4.1 Hz, 1 H,
H–18), 2.14 (ddd, J = 14.0, 14.0, 3.9 Hz, 1H, H–16a), 2.02 (s, 6 H, H–32 + H–34), 1.99–1.94
(m, 3 H, H–11a + H–11b + H–1a), 1.92–1.79 (m, 4 H, H–19a + H–22a + H–22b + H–16b),
1.74 (ddd, J = 14.1, 14.1, 4.4 Hz, 1H, H–15a), 1.61–1.36 (m, 5 H, H–9 + H–6a + H–6b + H–
7a + H–21a), 1.36–1.24 (m, 4 H, H–19b + H–1b + H–21b + H–7b), 1.23 (s, 3 H, H–27), 1.15
(ddd, J = 13.8, 3.1, 3.1 Hz, 1H, H–15b), 1.06 (s, 3 H, H–25), 1.00 (s, 3 H, H–23), 1.00 (s, 3 H,
H–29), 0.98–0.94 (m, 1 H, H–5), 0.97 (s, 3 H, H–30), 0.88 (s, 3 H, H–24), 0.67 (s, 3 H, H–26)
ppm; 13C NMR (125 MHz, CDCl3): δ = 177.0 (C–28), 170.8 (C–33), 170.4 (C–31), 150.3 (C–
40), 148.8 (C–39), 145.4 (C–13), 132.9 (C–35), 129.7 (C–37), 129.3 (C–42), 126.7 (C–38),
123.5 (C–12), 122.2 (C–43), 120.8 (C–41), 120.2 (C–36), 78.0 (C–3), 69.6 (C–2), 55.3 (C–5),
48.1 (C–9), 48.1 (C–17), 46.9 (C–19), 43.0 (C–18), 42.6 (C–14), 42.0 (C–1), 39.7 (C–8), 37.4
(C–4), 36.8 (C–10), 34.3 (C–21), 33.1 (C–30), 33.1 (C–22), 32.5 (C–7), 30.9 (C–20), 29.2
(C–24), 27.4 (C–15), 26.1 (C–27), 24.2 (C–16), 23.8 (C–29), 23.7 (C–11), 21.4 (C–32), 21.0
(C–34), 18.0 (C–6), 17.7 (C–23), 17.4 (C–26), 16.1 (C–25) ppm; MS (ESI): m/z (%) = 683.6
([M+H]+, 100); analysis calculated for C43H58N2O5 (682.93): C 75.62, H 8.56, N 4.10; found:
C 75.53, H 8.71, N 3.76.
4.2.12. 2β, 3β-Diacetyloxy-ursan-12-en-28-oic acid 5-quinolinyl amide (14)
As described for 5, compound 14 (43 mg, 58%) was obtained from 4 and 5-aminoquinoline as
a white solid; m.p. 144–146 °C; RF = 0.35 (silica gel, hexane/ethyl acetate, 4:6); [α]D =
+18.90° (c = 1.02, CHCl3); UV-vis (CHCl3): λmax (log ε) = 241 nm (4.42), 308 nm (3.71), 317
nm (3.71); IR (KBr): ν = 3346m, 2948s, 2872m, 1744vs, 1658s, 1620m, 1596m, 1572m,
1518s, 1488s, 1458s, 1398s, 1366s, 1320m, 1252vs, 1234vs, 1194s, 1156m, 1114m, 1078m,
1
1056s, 1030s, 996m, 972m, 944m, 798s, 754s cm−1; H NMR (500 MHz, CDCl3): δ = 8.92
(dd, J = 4.2, 1.5 Hz, 1 H, H–40), 8.13 (d, J = 8.6 Hz, 1 H, H–42), 8.03 (s, 1 H, NH), 8.02 (d,
J = 6.8 Hz, 1 H, H–36), 7.92 (d, J = 8.5 Hz, 1 H, H–38), 7.68 (dd, J = 8.1, 8.1 Hz, 1 H, H–37),
7.41 (dd, J = 8.6, 4.2 Hz, 1 H, H–41), 5.55 (dd, J = 3.3, 3.3 Hz, 1 H, H–12), 5.30 (ddd,
J = 3.7, 3.7, 3.5 Hz, 1 H, H–2), 4.60 (d, J = 3.9 Hz, 1 H, H–3), 2.19 (d, J = 10.5 Hz, 1 H, H–
18), 2.17–2.10 (m, 1 H, H–16a), 2.06–1.95 (m, 4 H, H–1a + H–11a + H–11b + H–22a), 2.02
(s, 3 H, H–32), 2.01 (s, 3 H, H–34), 1.95–1.80 (m, 2 H, H–15a + H–16b), 1.77–1.69 (m, 1 H,
H–22b), 1.63–1.47 (m, 5 H, H–6a + H–7a + H–9 + H–19 + H–21a), 1.46–1.29 (m, 4 H, H–1b
21

ACCEPTED MANUSCRIPT
+ H–6b + H–7b + H–21b), 1.18–1.12 (m, 1 H, H–15b), 1.16 (s, 3 H, H–27), 1.11–1.03 (m, 1
H, H–20), 1.08 (s, 3 H, H–25), 1.01 (d, J = 3.9 Hz, 3 H, H–30), 1.01 (s, 3 H, H–23), 0.99–
0.93 (m, 1 H, H–5), 0.97 (d, J = 6.4 Hz, 3 H, H–29), 0.88 (s, 3 H, H–24), 0.71 (s, 3 H, H–26)
ppm; 13C NMR (125 MHz, CDCl3): δ = 176.8 (C–28), 170.8 (C–33), 170.4 (C–31), 150.3 (C–
40), 148.8 (C–39), 140.4 (C–13), 133.0 (C–35), 129.6 (C–37), 129.5 (C–42), 126.7 (C–38),
126.2 (C–12), 122.3 (C–43), 120.7 (C–41), 120.5 (C–36), 78.0 (C–3), 69.6 (C–2), 55.3 (C–5),
54.6 (C–18), 49.5 (C–17), 48.1 (C–9), 43.0 (C–14), 42.1 (C–1), 40.1 (C–19), 39.9 (C–8), 39.3
(C–20), 37.8 (C–22), 37.4 (C–4), 36.8 (C–10), 32.9 (C–7), 31.0 (C–21), 29.2 (C–24), 27.9
(C–15), 25.2 (C–16), 23.7 (C–27), 23.6 (C–11), 21.4 (C–32), 21.3 (C–30), 21.0 (C–34),
18.0 (C–6), 17.8 (C–23), 17.5 (C–26), 17.5 (C–29), 16.2 (C–25) ppm; MS (ESI): m/z (%) =
683.5 ([M+H]+, 100), 705.3 ([M+Na]+, 3), 1365.3 ([2M+H]+, 15), 1387.6 ([2M+Na]+, 10),
1403.1 ([2M+K]+, 3); analysis calculated for C43H58N2O5 (682.93): C 75.62, H 8.56, N 4.10;
found: C 75.39, H 8.82, N 4.03.
4.2.13. 2β, 3β-Diacetyloxy-olean-12-en-28-oic acid 6-quinolinyl amide (15)
As described for 5, compound 15 (109 mg, 89%) was obtained from 3 and 6-aminoquinoline
as a white solid; m.p. 167–169 °C; RF = 0.63 (silica gel, chloroform/ethyl acetate, 1:1); [α]D =
+101.63° (c = 0.37, CHCl3); UV-vis (CHCl3): λmax (log ε) = 247 nm (4.70), 274 nm (3.86),
282 nm (3.84), 322 nm (3.70), 335 nm (3.68); IR (KBr): ν = 3396w, 2948s, 2878m, 1744vs,
1684m, 1626w, 1600w, 1532s, 1496m, 1464m, 1430m, 1364s, 1252vs, 1194m, 1156w, 1118w,
1056m, 1030m, 1012m cm−1; 1H NMR (500 MHz, CDCl3): δ = 8.81 (dd, J = 4.2, 1.7 Hz, 1 H,
H–39), 8.49 (d, J = 2.4 Hz, 1 H, H–43), 8.09 (dd, J = 8.1, 1.6 Hz, 1 H, H–41), 8.01 (d,
J = 9.0 Hz, 1 H, H–37), 7.94 (s, 1 H, NH), 7.39–7.34 (m, 2 H, H–40 + H–36), 5.62 (dd, J =
3.6, 3.6 Hz, 1 H, H–12), 5.31 (ddd, J = 3.8, 3.8, 3.8 Hz, 1 H, H–2), 4.61 (d, J = 3.9 Hz, 1 H,
H–3), 2.73 (dd, J = 12.7, 3.5 Hz, 1 H, H–18), 2.17–1.94 (m, 4 H, H–16a + H–11a + H–11b +
H–1a), 2.03 (s, 3 H, H–32), 2.02 (s, 3 H, H–34), 1.90–1.70 (m, 4 H, H–19a + H–22a + H–22b
+ H–16b), 1.70–1.52 (m, 3 H, H–15a + H–9 + H–6a), 1.52–1.23 (m, 7 H, H–7a + H–7b + H–
21a + H–21b + H–6b + H–1b + H–19b), 1.21 (s, 3 H, H–27), 1.15–1.08 (m, 1 H, H–15b), 1.14
(s, 3 H, H–25), 1.01 (s, 3 H, H–23), 0.98–0.94 (m, 1 H, H–5), 0.96 (s, 3 H, H–29), 0.95 (s, 3
13
H, H–30), 0.88 (s, 3 H, H–24), 0.70 (s, 3 H, H–26) ppm; C NMR (125 MHz, CDCl3): δ =
177.1 (C–28), 170.9 (C–33), 170.4 (C–31), 149.4 (C–40), 145.7 (C–41), 145.4 (C–13), 136.0
(C–35), 136.0 (C–38), 130.2 (C–42), 129.1 (C–37), 123.4 (C–12), 123.1 (C–43), 121.7 (C–
39), 116.0 (C–36), 78.0 (C–3), 69.7 (C–2), 55.3 (C–5), 48.1 (C–9), 47.5 (C–17), 46.8 (C–19),
42.8 (C–18), 42.5 (C–14), 42.1 (C–1), 39.7 (C–8), 37.5 (C–4), 36.8 (C–10), 34.3 (C–21), 33.1
22

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(C–30), 32.6 (C–22), 32.4 (C–7), 30.9 (C–20), 29.2 (C–24), 27.4 (C–15), 26.0 (C–27), 24.4
(C–16), 23.9 (C–11), 23.7 (C–29), 21.4 (C–32), 21.0 (C–34), 18.0 (C–6), 17.7 (C–23), 17.1
(C–26), 16.2 (C–25) ppm; MS (ESI): m/z (%) = 683.5 ([M+H]+, 100); analysis calculated for
C43H58N2O5 (682.93): C 75.62, H 8.56, N 4.10; found: C 75.59, H 8.80, N 3.85.
4.2.14. 2β, 3β-Diacetyloxy-ursan-12-en-28-oic acid 6-quinolinyl amide (16)
As described for 5, compound 16 (200 mg, 87%) was obtained from 4 and 6-aminoquinoline
as a white solid; m.p. 156–159 °C; RF = 0.39 (silica gel, hexane/ethyl acetate, 4:6); [α]D =
+76.70° (c = 0.31, CHCl3); UV-vis (CHCl3): λmax (log ε) = 248 nm (4.62), 284 nm (3.77), 322
nm (3.60), 335 nm (3.60); IR (KBr): ν = 3394m, 2928s, 2872m, 1744vs, 1682s, 1626m,
1600m, 1576m, 1534s, 1496s, 1458s, 1430s, 1398m, 1378s, 1364s, 1252vs, 1234vs, 1194s,
1
1156m, 1138m, 1118m, 1078m, 1056m, 1030s, 966m, 944m, 934m, 828m, 752m cm−1; H
NMR (500 MHz, CDCl3): δ = 8.81 (dd, J = 4.2, 1.7 Hz, 1 H, H–39), 8.46 (d, J = 2.4 Hz, 1 H,
H–43), 8.09 (d, J = 6.8 Hz, 1 H, H–41), 8.01 (d, J = 9.0 Hz, 1 H, H–37), 7.95 (s, 1 H, NH),
7.37–7.34 (m, 2 H, H–40 + H–36), 5.57 (dd, J = 3.5, 3.5 Hz, 1 H, H–12), 5.31 (ddd, J = 3.8,
3.8, 3.8 Hz, 1 H, H–2), 4.61 (d, J = 3.9 Hz, 1 H, H–3), 2.16–2.03 (m, 4 H, H–11a + H–11b +
H–16a + H–18), 2.03 (s, 3 H, H–32), 2.02 (s, 3 H, H–34), 2.03–1.97 (m, 2 H, H–1a + H–22a),
1.88 (dd, J = 8.3, 6.3 Hz, 1 H, H–16b), 1.80–1.72 (m, 1 H, H–15a), 1.67–1.46 (m, 6 H, H–6a
+ H–7a + H–9 + H–19 + H–21a + H–22b), 1.44–1.34 (m, 3 H, H–1b + H–6b + H–21b), 1.32–
1.26 (m, 1 H, H–7b), 1.16–1.10 (m, 1 H, H–15b), 1.15 (s, 3 H, H–27), 1.13 (s, 3 H, H–25),
1.06–0.98 (m, 1 H, H–20), 1.00 (d, J = 2.3 Hz, 3 H, H–30), 0.99 (s, 3 H, H–23), 0.97–0.93 (m,
1 H, H–5), 0.95 (d, J = 6.4 Hz, 3 H, H–29), 0.88 (s, 3 H, H–24), 0.68 (s, 3 H, H–26) ppm; 13C
NMR (125 MHz, CDCl3): δ = 176.9 (C–28), 170.8 (C–33), 170.4 (C–31), 149.4 (C–39),
145.6 (C–38), 140.6 (C–13), 136.1 (C–35), 136.1 (C–41), 130.2 (C–37), 129.1 (C–42), 126.2
(C–12), 123.0 (C–36), 121.7 (C–40), 115.9 (C–43), 78.0 (C–3), 69.6 (C–2), 55.2 (C–5), 54.4
(C–18), 48.9 (C–17), 48.1 (C–9), 43.0 (C–14), 42.2 (C–1), 40.0 (C–19), 39.8 (C–8), 39.3 (C–
20), 37.4 (C–4), 37.2 (C–22), 36.8 (C–10), 32.7 (C–7), 31.0 (C–21), 29.2 (C–24), 28.0 (C–
15), 25.3 (C–16), 23.8 (C–11), 23.5 (C–27), 21.4 (C–32), 21.3 (C–30), 21.0 (C–34), 18.0 (C–
6), 17.8 (C–23), 17.5 (C–29), 17.1 (C–26), 16.3 (C–25) ppm; MS (ESI): m/z (%) = 683.7
([M+H]+, 100), 1365.3 ([2M+H]+, 34), 1388.3 ([2M+Na]+, 4); analysis calculated for
C43H58N2O5 (682.93): C 75.62, H 8.56, N 4.10; found: C 75.41, H 8.74, N 3.91.
23

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4.2.15. 2
β
, 3β
-Diacetyloxy-olean-12-en-28-oic acid 7-quinolinyl amide (17)
As described for 5, compound 17 (104 mg, 85%) was obtained from 3 and 7-aminoquinoline
as a white solid; m.p. 167–170 °C; RF = 0.67 (silica gel, chloroform/ethyl acetate, 1:1); [α]D =
+48.44° (c = 0.27, CHCl3); UV-vis (CHCl3): λmax (log ε) = 247 nm (4.85), 275 nm (4.14), 324
nm (3.99), 335 nm (3.95); IR (KBr): ν = 2948s, 2866m, 1744vs, 1684m, 1624m, 1526m,
1
1496s, 1458s, 1430s, 1364m, 1252vs, 1192w, 1156m, 1056m, 1030m cm−1; H NMR (500
MHz, CDCl3): δ = 8.86 (dd, J = 4.3, 1.7 Hz, 1 H, H–38), 8.10 (dd, J = 8.3, 1.1 Hz, 1 H, H–
42), 8.07 (dd, J = 8.9, 2.1 Hz, 1 H, H–43), 7.98 (s, 1 H, NH), 7.90 (d, J = 2.0 Hz, 1 H, H–36),
7.77 (d, J = 8.9 Hz, 1 H, H–40), 7.32 (dd, J = 8.2, 4.3 Hz, 1 H, H–39), 5.61 (dd, J = 3.4, 3.4
Hz, 1 H, H–12), 5.32 (ddd, J = 3.7, 3.7, 3.7 Hz, 1 H, H–2), 4.62 (d, J = 3.9 Hz, 1 H, H–3),
2.73 (dd, J = 12.3, 3.4 Hz, 1 H, H–18), 2.16–1.92 (m, 4 H, H–16a + H–11a + H–11b + H–1a),
2.04 (s, 3 H, H–32), 2.02 (s, 3 H, H–34), 1.90–1.63 (m, 5 H, H–19a + H–22a + H–22b + H–
16b + H–15a), 1.62–1.36 (m, 5 H, H–9 + H–21a + H–7a + H–6a + H–6b), 1.36–1.24 (m, 4 H,
H–1b + H–19b + H–21b + H–7b), 1.21 (s, 3 H, H–27), 1.15–1.08 (m, 1 H, H–15b), 1.14 (s, 3
H, H–25), 1.01 (s, 3 H, H–23), 0.99–0.94 (m, 1 H, H–5), 0.96 (s, 3 H, H–29), 0.95 (s, 3 H, H–
13
30), 0.88 (s, 3 H, H–24), 0.75 (s, 3 H, H–26) ppm; C NMR (125 MHz, CDCl3): δ = 177.0
(C–28), 170.9 (C–33), 170.4 (C–31), 150.9 (C–38), 148.8 (C–37), 145.2 (C–13), 139.6 (C–
35), 135.9 (C–42), 128.7 (C–40), 125.5 (C–41), 123.6 (C–12), 121.1 (C–43), 120.1 (C–39),
117.1 (C–36), 78.0 (C–3), 69.7 (C–2), 55.3 (C–5), 48.2 (C–9), 47.6 (C–17), 46.8 (C–19), 42.8
(C–18), 42.5 (C–14), 42.1 (C–1), 39.7 (C–8), 37.5 (C–4), 36.8 (C–10), 34.3 (C–21), 33.1 (C–
30), 32.6 (C–22), 32.4 (C–7), 30.9 (C–20), 29.2 (C–24), 27.4 (C–15), 26.0 (C–27), 24.3 (C–
16), 23.9 (C–11), 23.8 (C–29), 21.4 (C–32), 21.0 (C–34), 18.0 (C–6), 17.8 (C–23), 17.2 (C–
26), 16.2 (C–25) ppm; MS (ESI): m/z (%) = 683.6 ([M+H]+, 100), 1365.4 ([2M+H]+, 15);
analysis calculated for C43H58N2O5 (682.93): C 75.62, H 8.56, N 4.10; found: C 75.43, H
8.69, N 3.86.
4.2.16. 2β, 3β-Diacetyloxy-ursan-12-en-28-oic acid 7-quinolinyl amide (18)
As described for 5, compound 18 (83 mg, 68%) was obtained from 4 and 7-aminoquinoline as
a white solid; m.p. 157–162 °C; RF = 0.51 (silica gel, chloroform/ethyl acetate, 1:1); [α]D =
+24.50° (c = 0.28, CHCl3); UV-vis (CHCl3): λmax (log ε) = 248 nm (4.90), 275 nm (4.24), 325
nm (4.06), 335 nm (4.02); IR (KBr): ν = 2948s, 2872m, 1744vs, 1684m, 1624m, 1582w,
1528m, 1496s, 1456s, 1430s, 1364m, 1252vs, 1236s, 1194m, 1156w, 1056m, 1032m cm−1; 1H
NMR (500 MHz, CDCl3): δ = 8.86 (d, J = 3.9 Hz, 1 H, H–38), 8.09 (d, J = 8.2 Hz, 1 H, H–
42), 8.04 (d, J = 9.2 Hz, 1 H, H–43), 7.99 (s, 1 H, NH), 7.87 (s, 1 H, H–36), 7.76 (d,
24

ACCEPTED MANUSCRIPT
J = 8.9 Hz, 1 H, H–40), 7.31 (dd, J = 8.2, 4.2 Hz, 1 H, H–39), 5.57–5.54 (m, 1 H, H–12),
5.33–5.30 (m, 1 H, H–2), 4.61 (d, J = 3.8 Hz, 1 H, H–3), 2.16–1.96 (m, 6 H, H–11a + H–11b
+ H–16a + H–22a + H–1a + H–18), 2.03 (s, 3 H, H–32), 2.02 (s, 3 H, H–34), 1.92–1.85 (m, 1
H, H–16b), 1.78 (ddd, J = 13.6, 13.6, 3.7 Hz, 1 H, H–15a), 1.69–1.46 (m, 6 H, H–6a + H–7a +
H–9 + H–19 + H–21a + H–22b), 1.44–1.25 (m, 4 H, H–6b + H–21b + H–7b + H–1b), 1.16–
1.09 (m, 1 H, H–15b), 1.15 (s, 3 H, H–27), 1.13 (s, 3 H, H–25), 1.06–0.98 (m, 7 H, H–20 +
H–30 + H–23), 0.98–0.92 (m, 1 H, H–5), 0.95 (d, J = 6.3 Hz, 3 H, H–29), 0.88 (s, 3 H, H–24),
13
0.73 (s, 3 H, H–26) ppm; C NMR (125 MHz, CDCl3): δ = 176.8 (C–28), 170.9 (C–33),
170.4 (C–31), 150.9 (C–38), 148.8 (C–37), 140.4 (C–13), 139.7 (C–35), 135.9 (C–42), 128.6
(C–40), 126.3 (C–12), 125.5 (C–41), 121.1 (C–43), 120.1 (C–39), 116.9 (C–36), 78.0 (C–3),
69.6 (C–2), 55.2 (C–5), 54.5 (C–18), 49.0 (C–17), 48.2 (C–9), 43.0 (C–14), 42.2 (C–1), 40.0
(C–19), 39.9 (C–8), 39.3 (C–20), 37.4 (C–4), 37.2 (C–22), 36.8 (C–10), 32.8 (C–7), 31.0 (C–
21), 29.3 (C–24), 28.0 (C–15), 25.3 (C–16), 23.8 (C–11), 23.5 (C–27), 21.4 (C–32), 21.3 (C–
30), 21.0 (C–34), 18.0 (C–6), 17.8 (C–23), 17.5 (C–29), 17.1 (C–26), 16.4 (C–25) ppm; MS
(ESI): m/z (%) = 683.6 ([M+H]+, 100), 1365.5 ([2M+H]+, 20); analysis calculated for
C43H58N2O5 (682.93): C 75.62, H 8.56, N 4.10; found: C 75.47, H 8.73, N 3.92.
4.2.17. 2β, 3β-Diacetyloxy-olean-12-en-28-oic acid 8-quinolinyl amide (19)
As described for 5, compound 19 (136 mg, 90%) was obtained from 3 and 8-aminoquinoline
as a white solid; m.p. 140–145 °C; RF = 0.86 (silica gel, chloroform/ethyl acetate, 1:1); [α]D =
+24.59° (c = 0.36, CHCl3); UV-vis (CHCl3): λmax (log ε) = 247 nm (4.52), 324 nm (3.70); IR
(KBr): ν = 3332m, 2950vs, 2866s, 1744vs, 1672s, 1528vs, 1486vs, 1464s, 1424s, 1364s,
1326s, 1250vs, 1192s, 1162s, 1056s, 1030s, 1010s, 944m, 826s, 792s, 756m, 684m, 602m
cm−1; 1H NMR (500 MHz, CDCl3): δ = 10.37 (s, 1 H, NH), 8.85 (dd, J = 7.5, 1.4 Hz, 1 H, H–
43), 8.80 (dd, J = 4.2, 1.6 Hz, 1 H, H–37), 8.14 (dd, J = 8.3, 1.6 Hz, 1 H, H–39), 7.53–7.43
(m, 3 H, H–42 + H–41 + H–38), 5.71 (dd, J = 3.5, 3.5 Hz, 1 H, H–12), 5.30 (ddd, J = 3.7, 3.7,
3.7 Hz, 1 H, H–2), 4.60 (d, J = 3.9 Hz, 1 H, H–3), 2.99 (dd, J = 13.0, 3.7 Hz, 1 H, H–18), 2.16
(ddd, J = 13.8, 13.8, 3.9 Hz, 1 H, H–16a), 2.01 (s, 6 H, H–32 + H–34), 2.00–1.68 (m, 8 H, H–
1a + H–11a + H–11b + H–16b + H–19a + H–22a + H–22b + H–15a), 1.59–1.39 (m, 4 H, H–9
+ H–6a + H–7a + H–21a), 1.38–1.19 (m, 5 H, H–6b + H–19b + H–1b + H–21b + H–7b), 1.20
(s, 3 H, H–27), 1.10 (ddd, J = 13.6, 3.3, 3.3 Hz, 1H, H–15b), 0.98 (s, 6 H, H–25 + H–29),
0.97 (s, 3 H, H–23), 0.96 (s, 3 H, H–30), 0.95–0.91 (m, 1 H, H–5), 0.86 (s, 3 H, H–24), 0.51
13
(s, 3 H, H–26) ppm; C NMR (125 MHz, CDCl3): δ = 177.1 (C–28), 170.8 (C–33), 170.4
(C–31), 148.0 (C–37), 143.4 (C–13), 139.2 (C–36), 136.3 (C–39), 135.1 (C–35), 128.1 (C–
25

ACCEPTED MANUSCRIPT
40), 127.7 (C–42), 124.0 (C–12), 121.6 (C–38), 121.2 (C–41), 116.6 (C–43), 78.1 (C–3), 69.8
(C–2), 55.3 (C–5), 48.2 (C–17), 48.2 (C–9), 46.8 (C–19), 42.3 (C–18), 42.2 (C–14), 42.1 (C–
1), 39.7 (C–8), 37.4 (C–4), 36.7 (C–10), 34.5 (C–21), 33.2 (C–30), 33.1 (C–22), 32.5 (C–7),
30.9 (C–20), 29.2 (C–24), 27.6 (C–15), 26.1 (C–27), 24.3 (C–16), 23.8 (C–29), 23.7 (C–11),
21.4 (C–32), 21.0 (C–34), 18.0 (C–6), 17.7 (C–23), 16.6 (C–26), 16.0 (C–25) ppm; MS (ESI):
m/z (%) = 683.3 ([M+H]+, 100); analysis calculated for C43H58N2O5 (682.93): C 75.62, H
8.56, N 4.10; found: C 75.52, H 8.68, N 3.94.
4.2.18. 2β, 3β-Diacetyloxy-ursan-12-en-28-oic acid 8-quinolinyl amide (20)
As described for 5, compound 20 (83 mg, 90%) was obtained from 4 and 8-aminoquinoline as
a white solid; m.p. 145–147 °C; RF = 0.58 (silica gel, hexane/ethyl acetate, 7:3); [α]D = -5.00°
(c = 0.29, CHCl3); UV-vis (CHCl3): λmax (log ε) = 247 nm (4.51), 324 nm (3.71); IR (KBr): ν
= 3440m, 3364m, 2948s, 2870m, 1744vs, 1672m, 1596w, 1578w, 1526vs, 1486s, 1456m,
1424m, 1378m, 1324m, 1252vs, 1234s, 1194m, 1156m, 1132w, 1116w, 1102w, 1084w,
1
1058m, 1030m, 826m, 792m, 756m cm−1; H NMR (500 MHz, CDCl3): δ = 10.30 (s, 1 H,
NH), 8.84–8.82 (m, 1 H, H–43), 8.81–8.79 (m, 1 H, H–37), 8.15 (dd, J = 8.3, 1.6 Hz, 1 H, H–
39), 7.53–7.49 (m, 1 H, H–42), 7.48–7.44 (m, 2 H, H–41 + H–38), 5.69 (dd, J = 3.6, 3.6 Hz, 1
H, H–12), 5.29 (ddd, J = 3.8, 3.8, 3.5 Hz, 1 H, H–2), 4.59 (d, J = 3.9 Hz, 1 H, H–3), 2.33 (d,
J = 10.8 Hz, 1 H, H–18), 2.20–2.12 (m, 1 H, H–16a), 2.04–1.80 (m, 6 H, H–1a + H–11a + H–
11b + H–15a + H–16b + H–22a), 2.01 (s, 3 H, H–32), 2.00 (s, 3 H, H–36), 1.73–1.65 (m, 1 H,
H–22b), 1.62–1.38 (m, 6 H, H–6a + H–7a + H–9 + H–19 + H–21a + H–21b), 1.38–1.28 (m, 2
H, H–1b + H–6b), 1.28–1.22 (m, 1 H, H–7b), 1.15–1.03 (m, 2 H, H–15b + H–20), 1.14 (s, 3
H, H–27), 1.01–0.98 (m, 6 H, H–29 + H–30), 0.96 (s, 6 H, H–23) + H–25), 0.95–0.90 (m, 1 H,
H–5), 0.86 (s, 3 H, H–24), 0.51 (s, 3 H, H–26) ppm; 13C NMR (125 MHz, CDCl3): δ = 177.0
(C–28), 170.8 (C–33), 170.4 (C–31), 147.9 (C–37), 139.2 (C–36), 138.0 (C–13), 136.4 (C–
39), 135.2 (C–35), 128.1 (C–40), 127.7 (C–42), 127.2 (C–12), 121.5 (C–38), 121.2 (C–41),
116.6 (C–43), 78.1 (C–3), 69.8 (C–2), 55.3 (C–5), 53.9 (C–18), 49.6 (C–17), 48.2 (C–9), 42.5
(C–14), 42.2 (C–1), 40.0 (C–19), 39.9 (C–8), 39.2 (C–20), 37.6 (C–22), 37.4 (C–4), 36.7 (C–
10), 32.7 (C–7), 31.2 (C–21), 29.2 (C–24), 28.0 (C–15), 25.3 (C–16), 23.8 (C–27), 23.5 (C–
11), 21.4 (C–30 + C–32), 21.0 (C–34), 18.0 (C–6), 17.8 (C–23), 17.5 (C–29), 16.6 (C–26),
16.2 (C–25) ppm; MS (ESI): m/z (%) = 683.7 ([M+H]+, 100); analysis calculated for
C43H58N2O5 (682.93): C 75.62, H 8.56, N 4.10; found: C 75.46, H 8.65, N 3.81.
26

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4.2.19. 2
β
, 3β
-Diacetyloxy-olean-12-en-28-oic acid 4-isoquinolinyl amide (21)
As described for 5, compound 21 (71 mg, 58%) was obtained from 3 and 4-aminoisoquinoline
as a white solid; m.p. 176–180 °C; RF = 0.69 (silica gel, chloroform/ethyl acetate, 1:1); [α]D =
+39.41° (c = 0.35, CHCl3); UV-vis (CHCl3): λmax (log ε) = 290 nm (3.75), 302 nm (3.75), 324
nm (3.79); IR (KBr): ν = 2948s, 2878m, 1744vs, 1684m, 1648w, 1522m, 1490m, 1474m,
1458m, 1398m, 1366m, 1328w, 1252vs, 1236s, 1194m, 1162w, 1056m, 1030m cm−1; 1H NMR
(500 MHz, CDCl3): δ = 9.13 (s, 1 H, H–36), 9.08 (s, 1 H, H–37), 8.02–7.99 (m, 2 H, H–39 +
NH), 7.79–7.73 (m, 2 H, H–42 + H–40), 7.64 (ddd, J = 8.0, 5.8, 2.1 Hz, 1 H, H–41), 5.63 (dd,
J = 3.4, 3.4 Hz, 1 H, H–12), 5.30 (ddd, J = 3.8, 3.8, 3.8 Hz, 1 H, H–2), 4.61 (d, J = 3.9 Hz, 1
H, H–3), 2.88 (dd, J = 12.4, 3.4 Hz, 1 H, H–18), 2.15 (ddd, J = 13.7, 13.7, 3.9 Hz, 1 H, H–
16a), 2.02 (s, 3 H, H–32), 2.02 (s, 3 H, H–34), 2.01–1.81 (m, 7 H, H–11a + H–11b + H–16b +
H–22a + H–22b + H–1a + H–19a), 1.76 (ddd, J = 13.8, 13.8, 3.9 Hz, 1 H, H–15a), 1.61–1.37
(m, 5 H, H–6a + H–6b + H–7a + H–21a + H–9), 1.37–1.27 (m, 4 H, H–7b + H–21b + H–1b +
H–19b), 1.23 (s, 3 H, H–27), 1.19–1.13 (m, 1 H, H–15b), 1.07 (s, 3 H, H–25), 1.01 (s, 6 H,
H–23 + H–29), 0.99–0.94 (m, 1 H, H–5), 0.98 (s, 3 H, H–30), 0.88 (s, 3 H, H–24), 0.71 (s, 3
H, H–26) ppm; 13C NMR (125 MHz, CDCl3): δ = 177.0 (C–28), 170.9 (C–33), 170.4 (C–31),
149.4 (C–37), 145.2 (C–13), 137.2 (C–36), 130.6 (C–40), 129.8 (C–35), 128.8 (C–38), 128.5
(C–39), 128.4 (C–43), 127.4 (C–41), 123.6 (C–12), 120.0 (C–42), 78.0 (C–3), 69.7 (C–2),
55.3 (C–5), 48.1 (C–9), 48.1 (C–17), 46.8 (C–19), 43.1 (C–18), 42.6 (C–14), 42.0 (C–1), 39.7
(C–8), 37.5 (C–4), 36.8 (C–10), 34.3 (C–21), 33.1 (C–30), 33.1 (C–22), 32.5 (C–7), 30.9 (C–
20), 29.2 (C–24), 27.5 (C–15), 26.1 (C–27), 24.2 (C–16), 23.8 (C–29), 23.7 (C–11), 21.4 (C–
32), 21.0 (C–34), 18.0 (C–6), 17.7 (C–23), 17.4 (C–26), 16.1 (C–25) ppm; MS (ESI): m/z (%)
= 683.5 ([M+H]+, 100), 1365.2 ([2M+H]+, 46); analysis calculated for C43H58N2O5 (682.93):
C 75.62, H 8.56, N 4.10; found: C 75.43, H 8.65, N 3.97.
4.2.20. 3β-Acetyloxy-urs-12-en-28-oic acid (24)
Compound 24 (192 mg, 88%) was synthesized as described for compound 3 starting from 22.
The spectroscopic data were in accordance with the data from the literature.
4.2.21. 2α, 3β, 24-Triacetyloxy-urs-12-en-28-oic acid (25)
Compound 25 (209 mg, 83%) was synthesized as described for 3 starting from 23 and
obtained as a white solid; m.p. 153–159 °C (lit.[29]: 151.2–154.6 °C); RF = 0.23 (silica gel,
hexane/ethyl acetate, 7:3); [α]D = +34.71° (c = 0.35, CHCl3); IR (KBr): ν = 3298m, 2950s,
1
2872m, 1748vs, 1698s, 1456s, 1370s, 1234vs, 1156m, 1144s, 964m, 918m cm−1; H NMR
27

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(500 MHz, CDCl3): δ = 5.24 (dd, J = 3.5, 3.5 Hz, 1 H, H–12), 5.16 (ddd, J = 10.9, 10.9,
4.6 Hz, 1 H, H–2), 5.08 (d, J = 10.3 Hz, 1 H, H–3), 3.85 (d, J = 11.7 Hz, 1 H, H–24a), 3.58 (d,
J = 11.8 Hz, 1 H, H–24b), 2.19 (d, J = 11.3 Hz, 1 H, H–18), 2.11–1.90 (m, 4 H, H–11a + H–
11b + H–1a + H–16a), 2.08 (s, 3 H, H–36), 2.02 (s, 3 H, H–34), 1.98 (s, 3 H, H–32), 1.84
(ddd, J = 13.4, 13.4, 4.1 Hz, 1 H, H–15a), 1.75–1.59 (m, 4 H, H–16b + H–22a + H–22b + H–
9), 1.54–1.25 (m, 8 H, H–6a + H–6b + H–21a + H–21b + H–7a + H–7b + H–5 + H–19), 1.17–
0.96 (m, 3 H, H–15b + H–1b + H–20), 1.10 (s, 3 H, H–25), 1.07 (s, 3 H, H–27), 0.94 (d,
J = 6.3 Hz, 3 H, H–30), 0.87 (s, 3 H, H–23), 0.84 (d, J = 6.7 Hz, 3 H, H–29), 0.76 (s, 3 H, H–
26) ppm; 13C NMR (125 MHz, CDCl3): δ = 183.6 (C–28), 171.0 (C–35), 170.6 (C–31), 170.5
(C–33), 138.2 (C–13), 125.4 (C–12), 75.0 (C–3), 70.1 (C–2), 65.4 (C–24), 52.7 (C–18), 48.1
(C–17), 47.8 (C–9), 47.7 (C–5), 43.9 (C–1), 42.1 (C–14), 42.1 (C–4), 39.7 (C–8), 39.2 (C–
19), 38.9 (C–20), 38.0 (C–10), 36.8 (C–22), 32.6 (C–7), 30.7 (C–21), 28.0 (C–15), 24.1 (C–
16), 23.6 (C–27), 23.5 (C–11), 21.3 (C–30), 21.2 (C–32), 21.0 (C–36), 20.9 (C–34), 18.0 (C–
6), 17.2 (C–25), 17.1 (C–29), 17.1 (C–26), 14.1 (C–23) ppm; MS (ESI): m/z (%) = 615.1
([M+H]+, 14), 632.2 ([M+NH4]+, 40), 637.4 ([M+Na]+, 100); analysis calculated for C36H54O8
(614.81): C 70.33, H 8.85; found: C 70.02, H 8.97.
4.2.22. 3β-Acetyloxy-ursan-12-en-28-oic acid 5-quinolinyl amide (26)
As described for 5, compound 26 (109 mg, 87%) was obtained from 24 and 5-aminoquinoline
as a white solid; m.p. 163–165 °C; RF = 0.53 (silica gel, chloroform/ethyl acetate, 1:1); [α]D =
+2.98° (c = 0.38, CHCl3); UV-vis (CHCl3): λmax (log ε) = 241 nm (4.44), 308 nm (3.72), 317
nm (3.72); IR (KBr): ν = 3260s, 2968vs, 2926vs, 2870s, 1736vs, 1684s, 1650vs, 1596s, 1520s,
1490vs, 1370s, 1320s, 1246vs, 1204s, 1146m, 1026s, 1006s, 986s, 968s, 802s cm−1; 1H NMR
(500 MHz, CDCl3): 8.91 (dd, J = 4.2, 1.5 Hz, 1 H, H–38), 8.14 (d, J = 8.4 Hz, 1 H, H–40),
8.08 (s, 1 H, NH), 7.99 (d, J = 7.4 Hz, 1 H, H–34), 7.92 (d, J = 8.5 Hz, 1 H, H–36), 7.67 (dd,
J = 8.5, 7.7 Hz, 1 H, H–35), 7.41 (dd, J = 8.6, 4.2 Hz, 1 H, H–39), 5.54 (dd, J = 3.4, 3.4 Hz, 1
H, H–12), 4.47 (dd, J = 11.0, 4.8 Hz, 1 H, H–3), 2.19 (d, J = 10.4 Hz, 1 H, H–18), 2.13 (ddd,
J = 13.6, 13.6, 4.1 Hz, 1 H, H–16a), 2.06–1.98 (m, 2 H, H–2a + H–22a), 2.02 (s, 3 H, H–32),
1.95–1.79 (m, 3 H, H–2b + H–16b + H–15a), 1.72 (ddd, J = 13.6, 13.6, 4.2 Hz, 1 H, H–22b),
1.65–1.23 (m, 11 H, H–11a + H–11b + H–1a + H–21a + H–21b + H–9 + H–19 + H–7a + H–
7b + H–6a + H–6b), 1.17–0.98 (m, 1 H, H–15b + H–1b + H–19 + H–20), 1.15 (s, 3 H, H–27),
1.01 (d, J = 6.2 Hz, 3 H, H–30), 0.96 (d, J = 6.5 Hz, 3 H, H–29), 0.84 (s, 3 H, H–24), 0.83 (s,
3 H, H–25), 0.83–0.78 (m, 1 H, H–5), 0.81 (s, 3 H, H–23), 0.69 (s, 3 H, H–26) ppm; 13C NMR
(125 MHz, CDCl3): δ = 176.9 (C–28), 171.1 (C–31), 150.1 (C–38), 148.5 (C–37), 140.2 (C–
28

ACCEPTED MANUSCRIPT
13), 133.1 (C–33), 130.0 (C–40), 129.7 (C–35), 126.5 (C–36), 126.4 (C–12), 122.6 (C–41),
120.8 (C–34), 120.7 (C–39), 80.9 (C–3), 55.4 (C–5), 54.6 (C–18), 49.5 (C–17), 47.6 (C–9),
42.8 (C–14), 40.1 (C–19), 39.7 (C–8), 39.3 (C–20), 38.4 (C–1), 37.8 (C–4), 37.7 (C–22), 36.9
(C–10), 32.9 (C–7), 31.0 (C–21), 28.2 (C–24), 28.0 (C–15), 25.2 (C–16), 23.6 (C–11),
23.6 (C–27), 23.5 (C–2), 21.4 (C–30), 21.3 (C–32), 18.2 (C–6), 17.4 (C–29), 17.4 (C–26),
16.8 (C–23), 15.6 (C–25) ppm; MS (ESI): m/z (%) = 625.5 ([M+H]+, 100), 1249.3 ([2M+H]+,
26); analysis calculated for C41H56N2O3 (624.89): C 78.80, H 9.03, N 4.48; found: C 78.64, H
8.75, N 4.29.
4.2.23. 2α, 3β, 24-Triacetyloxy-ursan-12-en-28-oic acid 5-quinolinyl amide (27)
As described for 5, compound 27 (63 mg, 52%) was obtained from 25 and 5-aminoquinoline
as a white solid; m.p. 168–172 °C; RF = 0.49 (silica gel, chloroform/ethyl acetate, 1:1); [α]D =
+6.08° (c = 0.31, CHCl3); UV-vis (CHCl3): λmax (log ε) = 241 nm (4.26), 306 nm (3.53), 317
nm (3.52); IR (KBr): ν = 3388w, 2950s, 2926s, 2870m, 1746vs, 1682m, 1596m, 1572w,
1
1522m, 1486s, 1370s, 1234vs, 1044s, 800 cm−1; H NMR (500 MHz, CDCl3): δ = 8.93 (dd,
J = 4.2, 1.5 Hz, 1 H, H–42), 8.12 (d, J = 8.3 Hz, 1 H, H–44), 8.02 (d, J = 7.1 Hz, 1 H, H–38),
7.98 (s, 1 H, NH), 7.93 (d, J = 8.5 Hz, 1 H, H–40), 7.68 (dd, J = 8.5, 7.7 Hz, 1 H, H–39), 7.42
(dd, J = 8.6, 4.2 Hz, 1 H, H–43), 5.55 (dd, J = 3.4, 3.4 Hz, 1 H, H–12), 5.13 (ddd, J = 10.7,
10.7, 4.5 Hz, 1 H, H–2), 5.07 (d, J = 10.3 Hz, 1 H, H–3), 3.82 (d, J = 11.8 Hz, 1 H, H–24a),
3.57 (d, J = 11.9 Hz, 1 H, H–24b), 2.21 (d, J = 10.4 Hz, 1 H, H–18), 2.14 (ddd, J = 13.7, 13.7,
4.2 Hz, 1 H, H–16a), 2.10–1.89 (m, 5 H, H–1a + H–11a + H–11b + H–22a + H–16b), 2.08 (s,
3 H, H–36), 2.01 (s, 3 H, H–34), 1.98 (s, 3 H, H–32), 1.85 (ddd, J = 14.5, 14.5, 4.4 Hz, 1 H,
H–15a), 1.79–1.66 (m, 2 H, H–22b + H–9), 1.65–1.51 (m, 2 H, H–19 + H–21a), 1.50–1.28 (m,
6 H, H–7a + H–7b + H–21b + H–6a + H–6b + H–5), 1.18–1.12 (m, 3 H, H–15b + H–1b + H–
20), 1.17 (s, 3 H, H–27), 1.01 (d, J = 6.3 Hz, 3 H, H–30), 1.00 (s, 3 H, H–25), 0.96 (d,
13
J = 6.5 Hz, 3 H, H–29), 0.84 (s, 3 H, H–23), 0.70 (s, 3 H, H–26) ppm; C NMR (125 MHz,
CDCl3): δ = 176.7 (C–28), 170.9 (C–35), 170.6 (C–31), 170.5 (C–33), 150.4 (C–42), 148.8
(C–31), 140.3 (C–13), 132.9 (C–37), 129.6 (C–39), 129.4 (C–44), 126.8 (C–40), 125.9 (C–
12), 122.5 (C–45), 120.8 (C–43), 120.6 (C–38), 74.8 (C–3), 70.0 (C–2), 65.4 (C–24), 54.6
(C–18), 49.5 (C–17), 47.7 (C–5), 47.7 (C–9), 43.9 (C–1), 42.8 (C–14), 42.0 (C–4), 40.1 (C–
19), 39.8 (C–8), 39.3 (C–20), 37.9 (C–10), 37.8 (C–22), 32.5 (C–7), 31.0 (C–21), 28.0 (C–
15), 25.2 (C–16), 23.6 (C–11), 23.5 (C–27), 21.3 (C–30), 21.2 (C–32), 21.0 (C–36), 20.9 (C–
34), 17.9 (C–6), 17.4 (C–26), 17.3 (C–29), 17.2 (C–25), 14.0 (C–23) ppm; MS (ESI): m/z (%)
29