Incorporation of a Michael acceptor enhances the antitumor activity of triterpenoic acids

Article abstract of DOI:10.1016/j.ejmech.2015.07.004

Finding and developing drugs for the treatment of cancer has been challenging scientists for many decades, and using compounds of natural origin represents one of several strategies. Triterpenoic acids are a very promising class of secondary metabolites being able to induce apoptosis while their cytotoxicity is low. Therefore, derivatizations have to be conducted to improve cytotoxicity while retaining their ability to induce programmed cell death. The incorporation of a Michael acceptor into molecules resulted very often in drugs of improved cytotoxicity. Thus, in this study we synthesized and evaluated several Michael acceptor substituted compounds derived from glycyrrhetinic, ursolic, oleanolic and platanic acid. The influence of the presence of such a functional group onto the cytotoxicity was investigated in colorimetric sulforhodamine B assays employing several human cancer cell lines. EC₅₀ values in the single-digit micromolar range were measured. Thus, the incorporation of a Michael acceptor unit into triterpenoic acids enhances the cytotoxicity of these compounds significantly.

Full text of DOI:10.1016/j.ejmech.2015.07.004

Accepted Manuscript
Incorporation of a Michael acceptor enhances the antitumor activity of triterpenoic
acids
Lucie Heller, Stefan Schwarz, Vincent Perl, Alexander Köwitsch, Bianka Siewert,
René Csuk, Prof. Dr.
PII:
S0223-5234(15)30137-9
10.1016/j.ejmech.2015.07.004
EJMECH 7985
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 4 May 2015
Revised Date: 29 June 2015
Accepted Date: 2 July 2015
Please cite this article as: L. Heller, S. Schwarz, V. Perl, A. Köwitsch, B. Siewert, R. Csuk, Incorporation
of a Michael acceptor enhances the antitumor activity of triterpenoic acids, European Journal of
Medicinal Chemistry (2015), doi: 10.1016/j.ejmech.2015.07.004.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Incorporation of a Michael acceptor enhances the antitumor activity of triterpenoic
acids
Lucie Heller, Stefan Schwarz, Vincent Perl, Alexander Köwitsch, Bianka Siewert and 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
Keywords: triterpenoic acid; ursolic acid; oleanolic acid; glycyrrhetinic acid; platantic acid,
tumor cells.
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Graphical abstract
Incorporation of a Michael acceptor enhances the antitumor activity of triterpenoic acids
Lucie Heller, Stefan Schwarz, Vincent Perl, Alexander Köwitsch, Bianka Siewert and René
Csuk*
Martin-Luther-University Halle-Wittenberg, Organic Chemistry, Kurt-Mothes-Str.2, D-06120
Halle (Saale) Germany
2

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Abstract
Finding and developing drugs for the treatment of cancer has been challenging scientists for
many decades, and using compounds of natural origin represents one of several strategies.
Triterpenoic acids are a very promising class of secondary metabolites being able to induce
apoptosis while their cytotoxicity is low. Therefore, derivatizations have to be conducted to
improve cytotoxicity while retaining their ability to induce programmed cell death. The
incorporation of a Michael acceptor into molecules resulted very often in drugs of improved
cytotoxicity. Thus, in this study we synthesized and evaluated several Michael acceptor
substituted compounds derived from glycyrrhetinic, ursolic, oleanolic and platanic acid. The
influence of the presence of such a functional group onto the cytotoxicity was investigated in
colorimetric sulforhodamine B assays employing several human cancer cell lines. EC50 values
in the single-digit micromolar range were measured. Thus, the incorporation of a Michael
acceptor unit into triterpenoic acids enhances the cytotoxicity of these compounds
significantly.
1. Introduction
The search for antitumor drugs has been a task for generations of chemists, physicians and
pharmacists. One of these strategies starts from biological active substances found in nature
and tries to optimize their activity by derivatization. Triterpenoic acids represent a promising
class of antitumor compounds easily to be obtained from several natural sources. Betulinic
acid,[1-4] oleanolic acid [5-7] or glycyrrhetinic acid [8-12] have already been part of
numerous investigations. In a few cases rather potent cytotoxic compounds were obtained
showing activity in micromolar or even nanomolar concentrations. Furthermore, the ability to
trigger apoptosis could be shown for several of them, comprising the triterpenoic acids [9, 10,
13] as well as analogs derived from the parent pentacyclic triterpenoic acids (Fig. 1).[8, 13]
Insert Fig. 1
Derivatization of natural occurring compounds is usually performed by functional group
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modifications. Thus, altering the molecular scaffold is an effective strategy for optimizing
biological activities. As far as the antitumor activity is concerned, the incorporation of a
Michael-acceptor into a given molecular structure has already been shown to enhance the
cytotoxic properties of secondary natural products.[14, 15] Therefore, we decided to
synthesize a series of different triterpenoic acid bearing a Michael acceptor moiety and to
evaluate their cytotoxic potential in sulforhodamine B assays employing different human
cancer celllines.
2. Results and discussion
2.1. Chemistry
Starting from the methyl esters of ursolic (1), 11-oxo ursolic (2), oleanolic (3), 11-oxo
oleanolic (4), glycyrrhetinic (5) or platanic acid (6), a Michael acceptor group was introduced
in a two-step synthesis (Scheme 1). First, these esters were oxidized in position 3 by Jones
oxidation using chromium(VI)oxide and sulphuric acid in acetone (7-11, 22)[16]. The
Michael acceptor moiety was created by the reaction of the 3-oxo-esters 7-11 (Scheme 1) or
22 (Scheme 2) with paraformaldehyde/finely grounded potassium carbonate in DMF, and α-
methylenated compounds 12-16 [6, 17] were obtained in 44-67 % isolated yield.
In order to investigate the influence of the Michael acceptor, three types of modifications
were performed: First, the Michael-acceptor moiety was reduced. The reaction of 16 with
sodium borohydride in the presence of cerium(III) chloride gave an 81 % yield of the 3-β-
hydroxy-2-methylene derivative 21. Furthermore, compounds 7, 8 and 10 were allowed to
react with bromine in glacial acetic acid and converted into their corresponding 2,2-dibromo-
3-oxo derivatives 17-19. Finally, a 2β-methyl-3-oxo derivative 20 was synthesized starting
from 7 by its reaction with diisopropylamine and n-butyllithium in THF at -78 °C followed by
the addition of iodomethane [18]. From NOESY-NMR spectra a 2β configuration of the
methyl group was deduced.
1
Inspection of the H NMR spectra of compounds 12-16 showed the chemical shift for their
methylene protons between δ = 5.90 and 6.02 ppm as well as between δ = 5.06 and 5.23 ppm,
while the chemical shift for the alkenic carbons were detected in the 13C NMR spectra
between δ = 123.6 and 124.1 ppm and δ = 141.8 and 142.0 ppm, respectively. For
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dibrominated compounds 17-19 in their 13C NMR spectra for C-2 a chemical shift of about δ
= 63 ppm was observed. Furthermore, for these compounds the typical isotopic pattern
[M79Br+1]+:[M81Br+1]+ was observed in their ESI-MS spectra.
Insert Scheme 1
Methyl 2-oxo-platanoate (22, scheme 2), however, could not be transformed into the
corresponding bis alkene: As expected, the Michael acceptor was created in ring A (scheme
2), but also the methyl ketone attached to ring E was transformed, and products 23 (71 %) and
24 (2 %) were formed. The formation of an 8-membered ring in these reactions, however, is
not unprecedented.[19, 20]
Insert Scheme 2
2.2. Biology
Cytotoxicity of the compounds was determined using the photometric sulforhodamine B assay
[21-23] employing five different human cancer cell lines. The results from these assays are
compiled in the Table. Compounds 23 and 24 could not be measured due to their poor
solubility under the conditions of the assay.
As compared to their parent 3-oxo compounds 7-11 the cytotoxicity is considerably increased
for all analogues 12-16 bearing Michael acceptor groups. While the EC50 values for
compounds 7-11 were greater than 30 µM (cut-off of the assay), the incorporation of a
Michael acceptor group into ring A significantly improved the cytotoxicity of the compounds,
and EC50 values in the single-digit micromolar range were observed. The highest activities
could be determined for methyl glcycyrrhetinoate derived 16 showing an EC50 as low as 0.74
µM for MCF-7 breast cancer cells. Interestingly, the oleanolic acid derived compounds 14 and
15 are less active than methyl ursoate derived compounds 12 and 13, respectively.
Nevertheless, all four compounds gave EC50 values between 1.2 and 5.4 µM.
Insert Table
When the Michael-acceptor unit was destroyed by reduction of either the methylene group (ꢀ
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20) or the carbonyl group (ꢀ 21) the cytotoxicity was considerably decreased, while the
introduction of two bromo-substituents in position C-3 (ꢀ 17-19) decreased cytotoxicity only
slightly or not at all. (20, 21).
3. Conclusions
A series of 18 derivatives – derived from glycyrrhetinic, oleanolic, ursolic and platanic acid –
were synthesized and tested for their cytotoxic activity employing five different human
malignant cell lines. The incorporation of a Michael acceptor unit into ring A of the
triterpenoid skeleton considerably enhanced the cytotoxic potential as compared to their
parent compounds and derivatives devoid of the this group. Being the highlight of our
investigations, compound 16, an derivative derived from glycyrrhetinic acid, gave EC50
values as low as 0.74 µM with MCF-7 cells. Although 2-cyano-3,12-dioxooleana-1,9(11)-
dien-28-oic acid (CDDO), a common known and comparable oleanolic acid analogue, is still
a little more active (here EC50 values of 0.16 µM [24] have been reported) the enhancing
potential [6] of the Michael acceptor moiety was clearly demonstrated.
Acknowledgements
We like to thank Dr. R. Kluge for measuring the ESI-MS spectra, Dr. D. Ströhl for NMR
measurements, and Miss J. Wiese for IR spectra and optical rotations. Many thanks are due to
Dr. Th. Müller from the Dep. of Haematology/Oncology for providing the cells, and to the
“Gründerwerkstatt-Biowissenschaften” for support.
4. Experimental section
4.1. General
Melting points are uncorrected (Leica hot stage microscope), NMR spectra were recorded
using the Varian spectrometers Gemini 2000 or Unity 500 (δ given in ppm, J in Hz, internal
Me4Si), IR spectra (film or KBr pellet) on a Perkin Elmer FT-IR spectrometer Spectrum 1000,
MS spectra were taken on a Finnigan MAT LCQ 7000 (electrospray, voltage 4.5 kV, sheath
gas nitrogen) instrument. The optical rotation was measured on a Perkin Elmer polarimeter at
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20 °C; TLC was performed on silica gel (Merck 5554); elemental analyses were performed on
a Vario EL (CHNS). The solvents were dried according to usual procedures. The purity of the
compounds was determined by HPLC and found to be > 98 %.
Oleanolic, ursolic and platanic acid were obtained from Betulinines (Stribrna Skalice, Czech
Republic), glycyrrhetinic acid was bought from Orgentis (Gatersleben, Germany), and the
methyl esters as well as the 3-oxo derivatives were synthesized according to literature. [22,
23] The synthesis of 11 has already been described.[25]
4.2. General procedure for oxidation at position C(3) (method A)
The methyl triterpenoate (1 equiv.) was dissolved in acetone (10 mL per 0.1 g starting
material) and cooled to 0 °C. CrO3 (2.5 equiv.) in sulphuric acid (1
M, 1 mL per 0.1 mmol
CrO3) was added, and the mixture was stirred at room temperature for 1 hour. Ethanol (half
the amount of acetone) was added, the mixture filtered, and the filtrate was evaporated to
dryness. The residue was subjected to column chromatography (silica gel, hexane/EtOAc,
4:1) to yield the methyl 3-oxo-triterpenoate.
4.3. General procedure for the incorporation of the Michael acceptor moiety
(method B)
The methyl 3-oxo-triterpenoate (1 equiv.) was dissolved in dry DMF (10 mL per 200 mg
starting material), and powdered potassium carbonate (4 equiv.) as well as paraformaldehyde
(5 equiv.) were added. The mixture was heated to 90 °C and stirred for 1 h. After cooling to
25 °C, water was added, the reaction mixture was neutralized by adding aqueous hydrochloric
acid (2
M), and the aqueous layer was extracted with DCM (3 × 20 mL). The combined
organic layers were washed with brine (25 mL), dried (Na2SO4), filtered and evaporated to
dryness. The residue was subjected to column chromatography (silica gel, hexane-EtOAc,
9:1) to yield the product.
4.4. General procedure for the bromination reaction (method C)
The methyl 3-oxo-triterpenoate derivative (1 equiv.) was dissolved in acetic glacial acetic acid
(1 mL per 10 mg of starting material), and bromine (4.5 equiv. dissolved in 1 mL of glacial
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acetic acid per 30 mg bromine) was slowly added. Stirring was continued for 15 min, water
(10 mL), an aqueous solution of potassium bisulphite solution (satd., 10 mL) and of sodium
hydrogen carbonate (satd., 20 mL) were added. The mixture was extracted with DCM (3 × 30
mL), the combined organic layers were washed (NaCl), dried (Na2SO4), filtered and
evaporated to dryness. The residue was subjected to chromatographic purification (silica gel,
hexane-EtOAc, 4:1) to yield the dibromo compound.
4.5. Methyl 3-oxo-urs-12-en-28-oate (7)
Compound 7 (2.33 g, 88 %) was obtained as colorless crystals from methyl (3β) 3-hydroxy-
urs-12-en-28-oate (1) using method A; mp 193-195 °C (lit.:[26] 192-193 °C,[27] 191-194 °C
[28]); Rf = 0.67 (hexane/EtOAc, 8:2). [α]D = +90° (c = 5.90, CHCl3) (lit.: [27] +85° (c = 1.23,
CHCl3)); UV-vis (MeOH): λmax = 222 nm (log ε = 3.80); IR (KBr): ν = 2970s, 2947s, 2857m,
1731s, 1698s, 1461m, 1446m, 1430w, 1385w, 1378w, 1366w, 1308w, 1273w, 1226m, 1202m,
1
1169m, 1144m, 1113w, 1102w, 1080w, 1030w cm-1; H NMR (500 MHz, CDCl3): δ
= 5.20
(m, 1H, H-12), 3.54 (s, 3H, OMe), 2.47 (ddd, J = 15.9, 11.0, 7.3 Hz, 1H, H-2), 2.30 (ddd, J =
15.9, 6.9, 3.8 Hz, 1H, H-2'), 2.18 (d, J = 11.2 Hz, 1H, H-18), 1.94 (ddd, J = 13.4, 13.3, 4.6 Hz,
1H, H-16), 1.91-1.87 (m, J = 8.7, 3.4 Hz, 2H, H-11, H-11'), 1.85 (ddd, J = 13.2, 7.3, 3.8 Hz,
1H, H-1), 1.71 (ddd, J = 13.7, 13.6, 4.5 Hz, 1H, H-15), 1.64-1.58 (m, 2H, H-22, H-16'), 1.55-
1.49 (m, 2H, H-9, H-22'), 1.46-1.34 (m, 5H, H-6, H-6', H-21, H-7, H-1'), 1.32-1.20 (m, 4H,
H-19, H-5, H-7', H-21'), 1.05-1.00 (m, 1H, H-15'), 1.02 (s, 3H, H-23), 1.01 (s, 3H, H-27),
0.98 (s, 3H, H-25), 0.97 (s, 3H, H-24), 0.95-0.91 (m, 1H, H-20), 0.87 (d, J = 6.3 Hz, 3H, H-
13
30), 0.79 (d, J = 6.5 Hz, 3H, H-29), 0.73 (s, 3H, H-26) ppm; C NMR (125 MHz, CDCl3):
δ = 217.7 (C-3), 177.9 (C-28), 138.2 (C-13), 125.3 (C-12), 55.3 (C-5), 52.9 (C-18), 51.4
(OMe), 48.1 (C-17), 47.4 (C-4), 46.7 (C-9), 42.1 (C-14), 39.4 (C-8), 39.1 (C-1), 39.0 (C-19),
38.8 (C-20), 36.7 (C-10), 36.6 (C-22), 34.1 (C-2), 32.5 (C-7), 30.6 (C-21), 28.0 (C-15), 26.5
(C-23), 24.2 (C-16), 23.5 (C-27), 23.4 (C-11), 21.5 (C-24), 21.1 (C-30), 19.6 (C-6), 17.0 (C-
29), 16.8 (C-26), 15.2 (C-25) ppm; ESI-MS (MeOH): m/z (%) = 469.3 ([M + H]+, 75), 523.0
([M + Na + MeOH]+, 100); elemental anal. calcd for C31H48O3 (468.71) C 79.44, H 10.32,
found: C 79.31, H 10.47.
4.6. Methyl 3,11-dioxo-urs-12-en-28-oate (8)
Compound 8 (1.70 g, 88 %) was obtained as an amorphous solid from methyl (3β) 3-hydroxy-
11-oxo-urs-12-en-28-oate (2) using method A; Rf = 0.33 (hexane/EtOAc, 8:2); [α]D = +108°
(c = 4.80, CHCl3); UV/vis (MeOH): λmax = 268 (log ε = 4.09) nm; IR (KBr): ν = 2950s,
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2871m, 1728s, 1705s, 1661s, 1619w, 1458m, 1386w, 1321w, 1273w, 1245w, 1226w, 1200m,
1
1144w, 1110w, 1084w, 1001w cm-1; H NMR (500 MHz, CDCl3): δ = 5.62 (s, 1H, H-12),
3.60 (s, 3H, OMe), 2.94 (ddd, J = 13.5, 7.1, 4.1 Hz, 1H, H-1), 2.61 (ddd, J = 15.9, 11.2, 7.2
Hz, 1H, H-2), 2.42 (d, J = 11.0 Hz, 1H, H-18), 2.38 (s, 1H, H-9), 2.31 (ddd, J = 15.8, 10.6, 6.6
Hz, 1H, H-2'), 2.08 (ddd, J = 14.6, 14.6, 4.8 Hz, 1H, H-16), 1.83-1.73 (m, 3H, H-16', H-22,
H-15), 1.64-1.46 (m, 5H, H-7, H-22', H-6, H-6', H-21), 1.43-1.34 (m, 3H, H-19, H-7', H-1'),
1.32-1.21 (m, 3H, H-5, H-15', H-21'), 1.29 (s, 3H, H-27), 1.23 (s, 3H, H-25), 1.09-1.02 (m,
1H, H-20), 1.08 (s, 3H, H-23), 1.03 (s, 3H, H-24), 0.95 (d, J = 6.6 Hz, 3H, H-30), 0.93 (s, 3H,
13
H-26), 0.85 (d, J = 6.6 Hz, 3H, H-29) ppm; C NMR (125 MHz, CDCl3): δ = 217.0 (C-3),
199.0 (C-11), 177.1 (C-28), 163.2 (C-13), 130.6 (C-12), 60.7 (C-9), 55.4 (C-5), 52.7 (C-18),
51.8 (OMe), 47.7 (C-4), 47.6 (C-17), 44.4 (C-8), 43.8 (C-14), 39.7 (C-1), 38.6 (C-19), 38.6
(C-20), 36.7 (C-10), 35.9 (C-22), 34.2 (C-2), 32.4 (C-7), 30.3 (C-21), 28.4 (C-15), 26.4 (C-
23), 23.9 (C-16), 21.4 (C-24), 21.0 (C-27), 20.9 (C-30), 18.7 (C-6), 18.7 (C-26), 17.1 (C-29),
15.5 (C-25) ppm; ESI-MS (MeOH): m/z (%) = 483.5 ([M + H]+, 100); elemental anal. calcd
for C31H46O4 (482.69): C 77.14; H9.61; found: 76.95; H 9.82.
4.7. Methyl 3-oxo-olean-12-en-28-oate (9)
Compound 9 (2.65 g, 83 %) was obtained from methyl (3β) 3-hydroxy-olean-12-en-28-oate
(3) using method A; colorless crystals; mp 183-186 °C (lit.:[29] 184 °C); Rf = 0.66
(hexane/EtOAc, 8:2); [α]D = +86° (c = 3.60, CHCl3) (lit.:[29] +90° (c = 1.20, CHCl3));
UV/vis (MeOH) λmax = 229 (log ε = 3.70) nm; IR (KBr): ν = 2941s, 2863m, 1726s, 1703s,
1458m, 1382w, 1364w, 1304w, 1264w, 1230w, 1205m, 1178w, 1163m, 1125w, 1094w,
1040w, 1016w, 991w cm-1; 1H NMR (500 MHz, CDCl3): δ = 5.29 (m, 1H, H-12), 3.61 (s, 3H,
OMe), 2.86 (dd, J = 13.7, 4.3 Hz, 1H, H-18), 2.53 (ddd, J = 16.0, 11.2, 7.3 Hz, 1H, H-2), 2.34
(ddd, J = 15.9, 6.7, 3.6 Hz, 1H, H-2'), 1.99-1.-83 (m, 4H, H-1, H-16, H-11, H-11'), 1.67 (ddd,
J = 14.0, 13.9, 4.6 Hz, 1H, H-22), 1.64-1.56 (m, 4H, H-9, H-15, H-19, H-16'), 1.53-1.44 (m,
4H, H-22', H-7, H-6, H-6'), 1.42-1.26 (m, 4H, H-1', H-21, H-7', H-5), 1.20-1.11 (m, 2H, H-
19', H-21'), 1.12 (s, 3H, H-27), 1.09-1.04 (m, 1H, H-15'), 1.06 (s, 3H, H-23), 1.02 (s, 3H, H-
13
24), 1.02 (s, 3H, H-25), 0.91 (s, 3H, H-30), 0.87 (s, 3H, H-29), 0.76 (s, 3H, H-26) ppm; C
NMR (125 MHz, CDCl3): δ = 217.6 (C-3), 178.2 (C-28), 143.8 (C-13), 122.1 (C-12), 55.3 (C-
5), 51.5 (OMe), 47.4 (C-9), 46.9 (C-4), 46.7 (C-17), 45.8 (C-19), 41.7 (C-14), 41.4 (C-18),
39.2 (C-8), 39.1 (C-1), 36.7 (C-10), 34.1 (C-2), 33.8 (C-21), 33.1 (C-29), 32.3 (C-7), 32.2 (C-
22), 30.7 (C-20), 27.7 (C-15), 26.4 (C-23), 25.8 (C-27), 23.6 (C-30), 23.5 (C-11), 23.0 (C-16),
21.4 (C-24), 19.6 (C-6), 16.7 (C-26), 15.0 (C-25) ppm; ESI-MS (MeOH): m/z (%) = 469.4
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([M + Na]+, 90), 523.0 ([M + Na + MeOH]+, 100); elemental anal. calcd for C31H48O3
(468.71): C 79.44, H 10.32, found: C 79.28, H 10.52.
4.8. Methyl 3,11-dioxo-olean-12-en-28-oate (10)
Compound 10 (1.12 g, 75 %) was obtained as an amorphous solid from methyl (3β) 3-
hydroxy-11-oxo-olean-12-en-28-oate (4) using method A; Rf = 0.46 (hexane/EtOAc, 8:2);
[α]D = +119° (c = 3.30, CHCl3); UV/vis (MeOH): λmax = 269 (log ε = 4.08) nm; IR (KBr): ν =
3432br, 2950s, 2866m, 1718s, 1704s, 1654s, 1460m, 1386m, 1365w, 1307w, 1261m, 1234w,
1
1210m, 1169m, 1125w, 1083w, 1012w, 997w cm-1; H NMR (500 MHz, CDCl3): δ = 5.66 (s,
1H, H-12), 3.63 (s, 3H, OMe), 3.05-2.95 (m, 2H, H-18, H-1), 2.64-2.56 (m, 1H, H-2), 2.41 (s,
1H, H-9), 2.39-2.32 (m, 1H, H-2'), 2.05 (ddd, J = 13.4, 13.0, 3.8 Hz, 1H, H-16), 1.78-1.57 (m,
6H, H-19, H-16', H-7, H-15, H-22, H-22'), 1.54-1.32 (m, 5H, H-1', H-7', H-21, H-6, H-6'),
1.35 (s, 3H, H-27), 1.31-1.18 (m, 4H, H-5, H-19', H-21', H-15'), 1.21 (s, 3H, H-25), 1.08 (s,
3H, H-23), 1.04 (s, 3H, H-24), 0.94 (s, 3H, H-26), 0.93 (s, 3H, H-30), 0.92 (s, 3H, H-29) ppm;
13C NMR (125 MHz, CDCl3): δ = 217.1 (C-3), 199.5 (C-11), 177.4 (C-28), 169.1 (C-13),
127.8 (C-12), 61.0 (C-9), 55.4 (C-5), 51.9 (OMe), 47.7 (C-4), 46.2 (C-17), 44.8 (C-14), 44.3
(C-19), 43.6 (C-8), 41.6 (C-18), 39.7 (C-1), 36.8 (C-10), 34.2 (C-2), 33.7 (C-21), 32.8 (C-29),
32.2 (C-7), 31.5 (C-22), 30.6 (C-20), 27.8 (C-15), 26.5 (C-23), 23.5 (C-27), 23.4 (C-30), 22.9
(C-16), 21.3 (C-24), 18.8 (C-26), 18.7 (C-6), 15.5 (C-25) ppm; ESI-MS (MeOH): m/z (%) =
483.5 ([M + H]+, 100); elemental anal. calcd for C31H46O4 (482.69): C 77.14, H 9.61; found:
C 77.02, H 9.79.
4.9. Methyl 2-methylene-3-oxo-urs-12-en-28-oate (12)
Compound 12 (640 mg, 45 %) was obtained as an amorphous solid from 7 using method B; Rf
= 0.75 (hexane/EtOAc, 8:2); [α]D = +100° (c = 3.20, CHCl3); UV-vis (MeOH). λmax = 221
(log ε = 3.82), 254 (log ε = 3.53) nm; IR (KBr): ν = 2948s, 1726s, 1688m, 1456m, 1382m,
1
1272w, 1229m, 1200m, 1166w, 1145m, 1113w, 1057w, 1033w, 1002w cm-1; H NMR (500
MHz, CDCl3): δ = 5.93 (m, 1H, methylene), 5.23 (m, 1H, H-12), 5.07 (m, 1H, methylene),
3.55 (s, 3H, OMe), 2.60 (d, J = 15.2 Hz, 1H, H-1), 2.19 (d, J = 11.4 Hz, 1H, H-18), 2.09 (d, J
= 15.0 Hz, 1H, H-1'), 1.97-1.90 (m, 3H, H-11, H-11', H-16), 1.71 (ddd, J = 13.6, 13.2, 4.6 Hz,
1H, H-15), 1.65-1.18 (m, 12H, H-19, H-5, H-9, H-6, H-6', H-21, H-21', H-7, H-7', H-22, H-
22', H-16'), 1.08-1.02 (m, 1H, H-15'), 1.06 (s, 3H, H-23), 1.04 (s, 3H, H-27), 1.00 (s, 3H, H-
24), 0.97-0.92 (m, 1H, H-20), 0.88 (d, J = 6.3 Hz, 3H, H-30), 0.86 (s, 3H, H-25), 0.80 (d, J =
13
6.2 Hz, 3H, H-29), 0.73 (s, 3H, H-26) ppm; C NMR (125 MHz, CDCl3): δ = 207.3 (C-3),
10

ACCEPTED MANUSCRIPT
177.9 (C-28), 141.9 (C-2), 138.2 (C-13), 125.3 (C-12), 123.6 (C-31), 54.2 (C-5), 53.0 (C-18),
51.4 (OMe), 48.1 (C-17), 47.0 (C-1), 45.8 (C-4), 45.2 (C-9), 42.2 (C-14), 39.4 (C-8), 39.1 (C-
19), 38.8 (C-20), 36.6 (C-10), 36.6 (C-22), 32.3 (C-7), 30.6 (C-21), 28.3 (C-23), 28.0 (C-15),
24.2 (C-16), 23.4 (C-27), 23.4 (C-11), 22.7 (C-24), 21.1 (C-30), 20.1 (C-6), 17.0 (C-29), 16.8
(C-26), 15.1 (C-25) ppm; ESI-MS (MeOH): m/z (%) = 481.1 ([M + H]+, 100); elemental anal.
calcd for C32H48O3 (480.72): C 79.95, H 10.06; found: C 79.93, H 10.19.
4.10. Methyl 2-methylene-3,11-dioxo-urs-12-en-28-oate (13)
Compound 13 (435 mg, 44 %) was obtained from 8 using method B; colorless crystals; mp
219-222 °C; Rf = 0.5 (hexane/EtOAc, 8:2); [α]D = +126° (c = 4.40, CHCl3), UV/vis (MeOH):
λmax = 266 (log ε = 4.13) nm; IR KBr): ν = 2950s, 2870m, 1728s, 1687m, 1661s, 1618w,
1458m, 1384m, 1320w, 1273w, 1225w, 1200m, 1147w, 1113w, 1085w, 1057w, 1004w cm-1;
1H NMR (500 MHz, CDCl3): δ = 5.95 (m, 1H, methylene), 5.59 (s, 1H, H-12), 5.18 (m, 1H,
methylene), 3.69 (d, J = 15.6 Hz, 1H, H-1), 3.56 (s, 3H, OMe), 2.39 (d, J = 11.4 Hz, 1H, H-
18), 2.35 (s, 1H, H-9), 2.10 (d, J = 15.3 Hz, 1H, H-1'), 2.03 (ddd, J = 14.7, 14.4, 4.7 Hz, 1H,
H-16), 1.78-1.68 (m, 3H, H-16', H-22, H-15), 1.61-1.46 (m, 3H, H-7, H-22', H-21), 1.44-1.20
(m, 7H, H-19, H-7', H-6, H-6', H-5, H-15', H-21'), 1.25 (s, 3H, H-27), 1.08 (s, 3H, H-25), 1.06
(s, 3H, H-23), 1.04-1.02 (m, 1H, H-20), 1.01 (s, 3H, H-24), 0.91 (d, J = 6.4 Hz, 3H, H-30),
13
0.89 (s, 3H, H-26), 0.81 (d, J = 6.4 Hz, 3H, H-29) ppm; C NMR (125 MHz, CDCl3): δ =
206.6 (C-3), 198.9 (C-11), 177.1 (C-28), 163.3 (C-13), 141.8 (C-2), 130.6 (C-12), 124.1
(methylene), 58.9 (C-9), 54.4 (C-5), 52.8 (C-18), 51.8 (OMe), 47.6 (C-17), 47.2 (C-1), 46.0
(C-4), 44.3 (C-8), 43.8 (C-14), 38.7 (C-19), 38.6 (C-20), 36.7 (C-10), 35.9 (C-22), 32.1 (C-7),
30.3 (C-21), 28.4 (C-15), 28.3 (C-23), 23.9 (C-16), 22.7 (C-24), 20.9 (C-27), 20.9 (C-30),
19.3 (C-6), 18.5 (C-26), 17.0 (C-29), 15.0 (C-25) ppm; ESI-MS (MeOH): m/z (%) = 495.5
([M + H]+, 100); elemental anal. calcd for C32H46O4 (494.71): C 77.69, H 9.37; found C
77.50, H 9.49.
4.11. Methyl 2-methylene-3-oxo-olean-12-en-28-oate (14)
Compound 14 (720 mg, 56 %) was obtained as an amorphous solid from 9 using method B; Rf
= 0.76 (hexane/EtOAc, 8:2); [α]D = +102° (c = 3.60, CHCl3); UV/vis (MeOH): λmax = 217
(log ε = 4.06) nm; IR (KBr): ν = 2949s, 1729s, 1669w, 1461m, 1385m, 1364w, 1303w,
1
1262m, 1231w, 1191m, 1163m, 1124w, 1077w, 1060w, 1035w, 1016w cm-1; H NMR (500
MHz, CDCl3): δ = 5.92 (m, 1H, methylene), 5.26 (m, 1H, H-12), 5.07 (m, 1H, methylene),
3.56 (s, 3H, OMe), 2.82 (dd, J = 13.9, 4.4 Hz, 1H, H-18), 2.57 (d, J = 15.0 Hz, 1H, H-1), 2.06
11

ACCEPTED MANUSCRIPT
(ddd, J = 15.0 Hz, 1H, H-1'), 1.94-1.87 (m, 3H, H-16, H-11, H-11'), 1.66-1.51 (m, 5H, H-22,
H-9, H-15, H-19, H-16'), 1.49-1.24 (m, 7H, H-21, H-22', H-7, H-7', H-6, H-6', H-5), 1.15-1.02
(m, 3H, H-19', H-21', H-15'), 1.09 (s, 3H, H-27), 1.06 (s, 3H, H-23), 0.99 (s, 3H, H-24), 0.87
(s, 3H, H-25), 0.86 (s, 3H, H-30), 0.84 (s, 3H, H-29), 0.72 (s, 3H, H-26) ppm; 13C NMR (125
MHz, CDCl3): δ = 207.3 (C-3), 178.2 (C-28), 143.8 (C-13), 142.0 (C-2), 123.6 (C-31), 122.1
(C-12), 54.1 (C-5), 51.5 (OMe), 46.7 (C-1), 46.0 (C-4), 45.9 (C-17), 45.8 (C-19), 45.4 (C-9),
41.8 (C-14), 41.4 (C-18), 39.1 (C-8), 36.8 (C-10), 33.8 (C-21), 33.1 (C-29), 32.3 (C-22), 32.0
(C-7), 30.7 (C-20), 28.3 (C-23), 27.7 (C-15), 25.7 (C-27), 23.6 (C-30), 23.4 (C-11), 23.0 (C-
16), 22.7 (C-24), 20.1 (C-6), 16.6 (C-26), 14.9 (C-25) ppm; ESI-MS (MeOH): m/z (%) =
481.3 ([M + H]+, 80), 535.0 ([M + Na + MeOH]+, 100); elemental anal. calcd for C32H48O3
(468.71): C 79.95, H 10.06; found: C 79.83, H 10.25.
4.12. Methyl 2-methylene-3,11-dioxo-olean-12-en-28-oate (15)
Compound 15 (295 mg, 67 %) was obtained from 10 using method B; colorless crystals; mp
210-212 °C; Rf = 0.52 (hexane/EtOAc, 8:2); [α]D = +129° (c = 4.30, CHCl3); UV/vis
(MeOH): λmax = 266 (log ε = 4.14) nm; IR (KBr): ν = 3432br, 2949s, 2867m, 1728s, 1687m,
1659s, 1622w, 1463m, 1385m, 1363w, 1328w, 1263w, 1190m, 1167m, 1126w, 1081w, 1059w,
1014w, 978w cm-1; 1H NMR (500 MHz, CDCl3): δ = 5.99 (m, 1H, methylene), 5.67 (s, 1H, H-
12), 5.23 (m, 1H, methylene), 3.78 (d, J = 15.8 Hz, 1H, H-1), 3.62 (s, 3H, OMe), 3.02 (dd, J =
13.8, 4.0 Hz, 1H, H-18), 2.43 (s, 1H, H-9), 2.13 (d, J = 15.6 Hz, 1H, H-1'), 2.04 (ddd, J =
14.0, 13.8, 4.0 Hz, 1H, H-16), 1.78-1.57 (m, 6H, H-19, H-16', H-7, H-15, H-22, H-22'), 1.51-
1.44 (ddd, J = 8.6, 7.4, 3.1 Hz, 1H, H-6), 1.41-1.30 (m, 4H, H-5, H-7, H-21, H-6'), 1.36 (s,
3H, H-27), 1.28-1.17 (m, 3H, H-19', H-21', H-15'), 1.11 (s, 3H, H-25), 1.10 (s, 3H, H-23),
1.06 (s, 3H, H-24), 0.93 (s, 3H, H-26), 0.92 (s, 3H, H-30), 0.91 (s, 3H, H-29) ppm; 13C NMR
(125 MHz, CDCl3): δ = 206.7 (C-3), 199.4 (C-11), 177.4 (C-28), 169.2 (C-13), 141.8 (C-2),
127.8 (C-12), 124.0 (methylene), 59.3 (C-9), 54.4 (C-5), 51.9 (OMe), 47.1 (C-1), 46.2 (C-17),
46.1 (C-4), 44.6 (C-14), 44.3 (C-19), 43.6 (C-8), 41.6 (C-18), 36.8 (C-10), 33.7 (C-21), 32.8
(C-29), 31.9 (C-7), 31.5 (C-22), 30.7 (C-20), 28.3 (C-23), 27.8 (C-15), 23.4 (C-27), 23.4 (C-
30), 22.9 (C-16), 22.6 (C-24), 19.3 (C-6), 18.5 (C-26), 15.0 (C-25) ppm; ESI-MS (MeOH):
m/z (%) = 495.5 ([M + H]+, 100); elemental anal. calcd for C32H46O4 (494.71): C 77.69, H
9.37; found: C 77.49, H 9.50.
4.13. Methyl 2-methylene-3,11-dioxo-olean-12-en-30-oate (16)
Compound 16 (450 mg, 59 %) was obtained from methyl 3,11-dioxo-olean-12-en-30-oate
12

ACCEPTED MANUSCRIPT
(11) using method B; colorless crystals; mp 186-190 °C; Rf = 0.67 (hexane/EtOAc, 8:2); [α]D
= +160° (c = 0.33, CHCl3); UV/vis (MeOH): λmax = 247 (log ε = 4.10) nm; IR (KBr) ν =
3427br, 2926s, 1724s, 1686m, 1651s, 1468m, 1385m, 1324w, 1279w, 1247w, 1221m, 1160m,
1090w, 1050m cm-1; 1H NMR (500 MHz, CDCl3): δ = 6.02 (dd, J = 2.2 Hz, 1H, methylene),
5.72 (s, 1H, H-12), 5.26 (dd, J = 2.2 Hz, 1H, methylene), 3.76 (d, J = 15.3 Hz, 1H, H-1), 3.70
(s, 3H, OMe), 2.47 (s, 1H, H-9), 2.14 (m, 1H, H-1'), 2.14 (m, 1H, H-18), 2.04 (ddd, J = 13.7,
13.7, J = 4.4 Hz, 1H, H-15), 2.00 (m, 1H, H-21), 1.93 (ddd, J = 13.6, 3.7, 2.8 Hz, 1H, H-19),
1.85 (ddd, J = 13.7, 13.7, 4.6 Hz, 1H, H-16), 1.71 (m, 1H, H-7), 1.62 (dd, J = 13.5, 13.5 Hz,
1H, H-19'), 1.54 (m, 1H, H-6), 1.53 (m, 1H, H-6'), 1.48 (ddd, J = 12.8, 3.1, 3.1 Hz, 1H, H-7'),
1.41 (m, 1H, H-5), 1.40 (m, 1H, H-22), 1.39 (s, 3H, H-27), 1.32 (m, 2H, H-22', H-21'), 1.23
(m, 1H, H-16'), 1.17 (s, 3H, H-26), 1.17 (s, 3H, H-25), 1.15 (s, 3H, H-29), 1.15 (s, 3H, H-23),
1.09 (s, 3H, H-24), 1.04 (m, 1H, H-15'), 0.83 (s, 3H, H-28) ppm; 13C NMR (125 MHz,
CDCl3): δ = 206.7 (C-3), 199.3 (C-11), 176.9 (C-30), 169.7 (C-13), 141.9 (C-2), 128.5 (C-12),
124.1 (methylene), 59.3 (C-9), 54.4 (C-5), 51.8 (OMe), 48.4 (C-18), 47.2 (C-1), 46.1 (C-4),
45.0 (C-8), 44.0 (C-20), 43.3 (C-14), 41.2 (C-19), 37.7 (C-22), 36.7 (C-10), 31.8 (C-7), 31.8
(C-17), 31.1 (C-21), 28.6 (C-28), 28.3 (C-29), 28.2 (C-23), 26.5 (C-16), 26.4 (C-15), 23.3 (C-
27), 22.7 (C-24), 19.4 (C-6), 18.2 (C-26), 15.1 (C-25) ppm; ESI-MS (MeOH): m/z (%) =
495.5 ([M + H]+, 100), 517.4 ([M + Na]+, 7), 548.9 ([M + Na + MeOH]+, 61), 989.1 ([2M +
H]+, 16), 1011.2 ([2M + Na]+, 18); elemental anal. calcd for C32H46O4 (494.71): C 77.69, H
9.37; found: 77.45, H 9.52.
4.14. Methyl 2,2-dibromo-3-oxo-urs-12-en-28-oate (17)
Compound 17 (195 mg, 80 %) was obtained as an amorphous solid from 7 using method C; Rf
= 0.57 (hexane/EtOAc, 9:1); [α]D = +43° (c = 5.70, CHCl3); UV/vis (MeOH): λmax = 217 (log
ε = 3.98) nm; IR (KBr): ν = 3424br, 2952s, 1725s, 1637w, 1457m, 1387w, 1203m, 1112w cm-
1; 1H NMR (500 MHz, CDCl3): δ = 5.28 (m, 1H, H-12), 3.60 (d, J = 15.9 Hz, 1H, H-1), 3.59
(s, 3H, OMe), 3.17 (d, J = 16.2 Hz, 1H, H-1'), 2.25 (d, J = 11.3 Hz, 1H, H-18), 2.02-1.90 (m,
3H, H-16, H-11, H-11'), 1.78-1.73 (m, 2H, H-5, H-15), 1.70-1.63 (m, 3H, H-22, H-22', H-16'),
1.57-1.45 (m, 4H, H-9, H-6, H-21, H-7), 1.51 (s, 3H, H-23), 1.42-1.35 (m, 3H, H-19, H-7', H-
6'), 1.30-1.25 (m, 1H, H-21'), 1.23 (s, 3H, H-24), 1.13-1.09 (m, 1H, H-15'), 1.10 (s, 3H, H-
27), 1.03 (s, 1H, H-20), 1.00 (s, 3H, H-25), 0.94 (d, J = 6.1 Hz, 3H, H-30), 0.86 (d, J = 6.4
Hz, 3H, H-29), 0.78 (s, 3H, H-26) ppm; 13C NMR (125 MHz, CDCl3): δ = 204.4 (C-3), 177.9
(C-28), 138.4 (C-13), 124.9 (C-12), 65.7 (C-1), 63.2 (C-2), 53.0 (C-18), 52.8 (C-5), 51.5
(OMe), 48.1 (C-17), 47.9 (C-4), 45.0 (C-9), 42.3 (C-14), 39.3 (C-8), 39.1 (C-19), 38.8 (C-20),
13

ACCEPTED MANUSCRIPT
38.7 (C-10), 36.5 (C-22), 33.2 (C-23), 31.9 (C-7), 30.6 (C-21), 27.9 (C-15), 24.2 (C-24), 24.1
(C-16), 23.5 (C-11), 23.4 (C-27), 21.1 (C-30), 20.0 (C-6), 17.0 (C-29), 16.7 (C-26), 15.5 (C-
25) ppm; ESI-MS (MeOH): m/z (%) = 647.1 ([M(2×79Br) + Na]+, 56), 649.2 ([M(79Br, 81Br) +
Na]+, 100), 651.2 ([M(2×81Br) + Na]+, 46); elemental anal. calcd for C31H46Br2O3 (626.50): C
59.43, H 7.40; found: C 59.11, H. 7.53.
4.15. Methyl 2,2-dibromo-3,11-dioxo-urs-12-en-28-oate (18)
Compound 18 (123 mg, 93 %)was obtained from 8 using method C; slightly yellowish
crystals; mp 225-227 °C; Rf = 0.5 (hexane/EtOAc, 8:2); [α]D = +48° (c = 5.90, CHCl3);
UV/vis (MeOH): λmax = 269 (log ε = 4.11); IR (KBr): ν = 3436br, 2951s, 1728s, 1684w,
1655s, 1612w, 1458m, 1387m, 1318w, 1287w, 1273w, 1248w, 1223m, 1204m, 1167w, 1150w,
1137w, 1110w, 1080w, 1036w, 1012w cm-1; 1H NMR (500 MHz, CDCl3): δ = 5.66 (s, 1H, H-
12), 4.51 (d, J = 16.8 Hz, 1H, H-1), 3.60 (s, 3H, OMe), 3.20 (d, J = 16.5Hz, 1H, H-1'), 2.44
(d, J = 11.4 Hz, 1H, H-18), 2.37 (s, 1H, H-9), 2.09 (ddd, J = 13.8, 13.4, 4.7 Hz, 1H, H-16),
1.89 (dd, J = 11.8, 3.0 Hz, 1H, H-5), 1.84-1.73 (m, 3H, H-16', H-22, H-15), 1.65-1.47 (m, 5H,
H-7, H-22', H-6, H-21), 1.53 (s, 3H, H-23), 1.43-1.35 (m, 3H, H-19, H-7', H-6'), 1.33-1.26 (m,
2H, H-15', H-21'), 1.31 (s, 3H, H-27), 1.24 (s, 3H, H-24), 1.21 (s, 3H, H-25), 1.09-1.01 (m,
1H, H-20), 0.96 (d, J = 6.4 Hz, 3H, H-30), 0.90 (s, 3H, H-26), 0.87 (d, J = 6.4 Hz, 3H, H-29)
13
ppm; C NMR (125 MHz, CDCl3): δ = 203.5 (C-3), 197.7 (C-11), 177.0 (C-28), 163.8 (C-
13), 130.3 (C-12), 65.1 (C-1), 63.0 (C-2), 59.2 (C-9), 52.8 (C-18), 51.9 (C-5), 51.8 (OMe),
47.7 (C-4), 46.8 (C-17), 44.2 (C-8), 44.0 (C-14), 38.7 (C-19), 38.6 (C-20), 38.5 (C-10), 35.9
(C-22), 33.2 (C-23), 31.6 (C-7), 30.3 (C-21), 28.5 (C-15), 24.1 (C-24), 23.9 (C-16), 21.1 (C-
27), 21.0 (C-30), 19.2 (C-6), 18.5 (C-26), 17.1 (C-29), 16.1 (C-25) ppm; ESI-MS (MeOH):
m/z (%) = 639.3 ([M(2×79Br) + H]+, 23), 641.3 ([M(79Br, 81Br) + H]+, 51), 643.3 ([M(2×81Br)
+ H]+, 27), 661.1 ([M(2×79Br) + Na]+, 46), 663.1 ([M(79Br, 81Br) + Na]+, 100), 665.0
([M(2×81Br) + Na]+, 52); elemental anal. calcd for C31H44Br2O4 (624.49): C 58.13, H 6.92;
found: C57.84, H 7.14.
4.16. Methyl 2,2-dibromo-3,11-dioxo-olean-12-en-28-oate (19)
Compound 19 (95 mg, 71 %) was obtained from 10 using method C; slightly yellowish
crystals; mp 223-226 °C; Rf = 0.55 (hexane/EtOAc, 8:2); [α]D = +82° (c = 3.40, CHCl3),
UV/vis (MeOH): λmax = 269 (log ε = 4.18) nm; IR (KBr): ν = 3434br, 2948s, 1727s, 1684w,
1658s, 1464m, 1387m, 1364w, 1330w, 1264w, 1199m, 1162m, 1138w, 1083w, 1039w, 1014w
1
cm-1; H NMR (500 MHz, CDCl3): δ = 5.70 (s, 1H, H-12), 4.55 (d, J = 16.5 Hz, 1H, H-1),
14

ACCEPTED MANUSCRIPT
3.63 (s, 3H, OMe), 3.18 (d, J = 16.8 Hz, 1H, H-1'), 3.04 (dd, J = 13.7, 4.8 Hz, 1H, H-18), 2.40
(s, 1H, H-9), 2.06 (ddd, J = 13.7, 13.7, 4.0 Hz, 1H, H-16), 1.91 (dd, J = 11.6, 3.4 Hz, 1H, H-
5), 1.78-1.68 (m, 2H, H-16', H-22), 1.66-1.57 (m, 4H, H-7, H-19, H-22', H-15), 1.55-1.47 (m,
2H, H-6, H-6'), 1.53 (s, 3H, H-23), 1.42-1.33 (m, 2H, H-21, H-7'), 1.38 (s, 3H, H-27), 1.30-
1.19 (m, 3H, H-19', H-21', H-15'), 1.25 (s, 3H, H-24), 1.20 (s, 3H, H-25), 0.94 (s, 3H, H-29),
13
0.94 (s, 3H, H-30), 0.91 (s, 3H, H-26) ppm; C NMR (125 MHz, CDCl3): δ = 203.7 (C-3),
198.4 (C-11), 177.4 (C-28), 169.9 (C-13), 127.5 (C-12), 65.0 (C-1), 63.0 (C-2), 59.5 (C-9),
51.9 (C-5), 51.9 (OMe), 46.8 (C-4), 46.1 (C-17), 44.6 (C-14), 44.3 (C-19), 43.8 (C-8), 41.7
(C-18), 38.5 (C-10), 33.7 (C-21), 33.2 (C-23), 32.8 (C-29), 31.5 (C-22), 31.4 (C-7), 30.7 (C-
20), 27.8 (C-15), 24.0 (C-24), 23.5 (C-27), 23.4 (C-30), 22.8 (C-16), 19.1 (C-6), 18.5 (C-26),
16.0 (C-25) ppm; ESI-MS (MeOH): m/z (%) = 639.3 ([M(2×79Br) + H]+, 30), 641.3 ([M(79Br,
81Br) + H]+, 61), 643.2 ([M(2×81Br) + H]+, 35), 661.1 ([M(2×79Br) + Na]+, 49), 663.1
([M(79Br, 81Br) + Na]+, 100), 665.1 ([M(2×81Br) + Na]+, 50); elemental anal. calcd for
C31H44Br2O4 (640.49): C 58.13, H 6.92; found: C 57.81, H 6.99.
4.17. Methyl (2
To a solution of diisopropylamine (85 mg, 0.84 mmol) in dry THF (10 mL) at -78 °C, n-
butyllithium (1.6 in hexane, 0.60 mL, 0.96 mmol) was added, and the mixture was stirred
β) 2-methyl-3-oxo-urs-12-en-28-oate (20)
M
for 15 min. A solution of 7 (300 mg, 0.64 mmol) in dry THF (3 mL) iodomethane (200 mg,
1.41 mmol, dissolved in dry THF (3 mL)) were added dropwise. The solution was allowed to
warm to room temperature and water (30 mL) was added. The mixture was extracted with
DCM (3 × 20 mL), the combined organic layers were dried (Na2SO4), filtered and evaporated
to dryness. The residue was subjected to column chromatography (silica gel, hexane/EtOAc,
9:1) to yield 20 (180 mg, 58 %); amorphous solid; Rf = 0.4 (hexane/EtOAc, 9:1); [α]D = +49°
(c = 2.00, CHCl3); UV/vis: λmax (MeOH): 216 (log ε = 3.80) nm; IR (KBr): ν = 3440br,
1
2928s, 1726m, 1704m, 1653w, 1456w, 1387w, 1229w, 1199w, 1145w cm-1; H NMR (500
MHz, CDCl3): δ = 5.22 (m, 1H, H-12), 3.59 (s, 3H, OMe), 2.78-2.70 (m, 1H, H-2), 2.21 (d, J
= 11.3 Hz, 1H, H-18), 2.02-1.89 (m, 4H, H-11, H-11', H-16, H-1), 1.75 (ddd, J = 13.8, 13.8,
4.6 Hz, 1H, H-15), 1.69-1.62 (m, 2H, H-22, H-16'), 1.56 (ddd, J = 13.4, 13.4, 4.0 Hz, 1H, H-
22'), 1.52-1.41 (m, 5H, H-6, H-6', H-21, H-7, H-9), 1.35-1.22 (m, 3H, H-19, H-7', H-21'), 1.19
(s, 3H, H-31), 1.12 (d, J = 12.2 Hz, 1H, H-5), 1.08-1.01 (m, 2H, H-15', H-1'), 1.05 (s, 3H, H-
23), 1.04 (s, 3H, H-27), 1.03 (s, 3H, H-24), 1.00-0.95 (m, 1H, H-20), 0.99 (s, 3H, H-25), 0.91
(d, J = 6.1 Hz, 3H, H-30), 0.83 (d, J = 6.4 Hz, 3H, H-29), 0.78 (s, 3H, H-26) ppm; 13C NMR
(125 MHz, CDCl3): δ = 218.0 (C-3), 177.9 (C-28), 138.3 (C-13), 125.2 (C-12), 57.2 (C-5),
15

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52.8 (C-18), 51.4 (OMe), 49.5 (C-1), 48.0 (C-17), 48.0 (C-4), 47.0 (C-9), 42.0 (C-14), 39.5
(C-8), 38.9 (C-19), 38.8 (C-20), 37.2 (C-10), 36.6 (C-22), 36.4 (C-2), 32.7 (C-7), 30.6 (C-21),
28.0 (C-15), 25.4 (C-23), 24.1 (C-16), 23.5 (C-27), 23.4 (C-11), 22.0 (C-24), 21.1 (C-30),
19.3 (C-6), 17.0 (C-29), 16.9 (C-26), 15.5 (C-25), 15.5 (C-31) ppm; ESI-MS (MeOH): m/z
(%) = 483.3 ([M + H]+, 65), 537.0 ([M + Na + MeOH]+, 100); elemental anal. calcd for
C32H50O3 (482.74): C 79.62, H 10.44; found: 79.45, H 10.61.
4.18. Methyl (3β) 3-hydroxy-2-methylene-11-oxo-olean-12-en-30-oate (21)
Compound 16 (390 mg, 0.79 mmol) was dissolved in 100 mL dry methanol and cerium
trichloride heptahydrate (630 mg, 1.69 mmol) was added. Sodium borohydride (50 mg, 1.32
mmol) was added, and the mixture was stirred at room temperature for 48 hours. The solution
was concentrated to half volume and poured into water. The aqueous layer was extracted with
DCM (3 x 20 mL), the combined organic layers were washed with brine (25 mL), dried
(Na2SO4), filtered and evaporated to dryness. Chromatographic purification (silica gel,
hexane/EtOAc, 4:1) yielded 21 (320 mg, 81 %); colorless crystals; mp 111-114 °C; Rf = 0.52
(hexane/EtOAc, 8:2); [α]D = +121° (c = 0.51, CHCl3); UV/vis (MeOH): λmax = 249 (log ε =
4.09) nm; IR (KBr): ν = 3529br, 2950s, 2868s, 1731s, 1653s, 1621m, 1455s, 1388s, 1365m,
1315m, 1280m, 1249m, 1219s, 1190s, 1155s, 1112m, 1081m, 1055s, 1030m, 977m, 965m,
1
897m, 733m cm-1; H NMR (500 MHz, CDCl3): δ = 5.67 (s, 1H, H-12), 5.04 (m, 1H,
methylene), 4.97 (m, 1H, methylene), 3.74 (m, 1H, H-3), 3.70 (s, 3H, OMe), 3.44 (d, J = 12.9
Hz, 1H, H-1), 2.44 (s, 1H, H-9), 2.10 (dd, J = 13.9, 3.8 Hz, 1H, H-18), 2.03 (ddd, J = 13.5,
13.5, 4.6 Hz, 1H, H-15), 2.00 (m, 1H, H-21), 1.93 (ddd, J = 13.4, 4.2, 2.7 Hz, 1H, H-19), 1.84
(ddd, J = 13.5, 13.5, 4.5 Hz, 1H, H-16), 1.70 (m, 1H, H-7), 1.68 (m, 1H, H-1'), 1.62 (dd, J =
13.5, 13.5 Hz, 1H, H-19'), 1.57 (m, 1H, H-6), 1.54 (m, 1H, H-6'), 1.45 (m, 1H, H-7'), 1.42 (m,
1H, H-22), 1.39 (s, 3H, H-27), 1.35 (m, 2H, H-22'), 1.32 (m, 1H, H-21'), 1.20 (m, 1H, H-16'),
1.15 (s, 3H, H-29), 1.13 (s, 3H, H-26), 1.11 (s, 3H, H-23), 1.06 (s, 3H, H-25), 1.02 (m, 1H, H-
13
15'), 0.96 (m, 1H, H-5), 0.81 (s, 3H, H-28), 0.70 (s, 3H, H-24) ppm; C NMR (125 MHz,
CDCl3): δ = 199.8 (C-11), 176.9 (C-30), 169.3 (C-13), 146.9 (C-2), 128.6 (C-12), 108.4
(methylene), 80.3 (C-3), 61.1 (C-9), 55.0 (C-5), 51.8 (OMe), 49.2 (C-1), 48.4 (C-18), 45.5 (C-
8), 44.0 (C-20), 43.2 (C-14), 41.4 (C-4), 41.1 (C-19), 39.2 (C-10), 37.8 (C-22), 32.7 (C-7),
31.8 (C-17), 31.1 (C-21), 28.5 (C-28), 28.3 (C-29), 28.2 (C-23), 26.5 (C-16), 26.4 (C-15),
23.4 (C-27), 18.6 (C-26), 17.9 (C-6), 16.1 (C-25), 15.4 (C-24) ppm; ESI-MS (MeOH): m/z
(%) = 497.6 ([M + H]+, 16), 519.5 ([M + Na]+, 6), 537.3 ([M + Na + H2O]+, 5), 551.1 ([M +
Na + MeOH]+, 100), 993.2 ([2M + H]+, 18), 1015.3 ([2M + Na]+, 74); elemental anal. calcd
16

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for C32H48O4 (496.72): C 77.38, H 9.74; found C 77.19, H 9.81.
4.19. Methyl 3,20-dioxo-30-norlupan-28-oate (22)
Compound 22 (3.4 g, 97 %) was obtained from methyl (3β) 3-hydroxy-20-oxo-30-norlupan-
28-oate (6) using method A; colorless crystals; mp 155-157 °C; Rf = 0.38
(toluene/EtOAc/formic acid/heptane, 80:26:5:10); [α]D = + 3° (c = 0.55, CHCl3), IR (KBr): ν
= 3446w, 2941s, 2866s, 2362w, 1733s, 1703s, 1455s, 1430m, 1385m, 1352m, 1319m, 1274m,
1
1241m, 1188s, 1161s, 1129s, 1079m, 1046w, 1005m, 978m cm-1; H NMR (400 MHz,
CDCl3): δ = 3.61 (s, 3H, H-30), 3.19 (dt, J = 11.0, 4.4 Hz, 1H, H-19), 2.46-2.29 (m, 2H, H-2),
2.19 (dt, J = 11.9, 3.0 Hz, 1H, H-16), 2.11 (s, 3H, H-29), 2.04 (dd, J = 21.4, 10.6 Hz, 1H, H-
18), 1.99-1.89 (m, 2H, H-13, H-21), 1.86-1.76 (m, 2H, H-1, H-22), 1.51-1.20 (m, 13H, H-1',
H-5, H-6, H-7, H-9, H-11, H-15, H-16', H-21', H-22'), 1.16-1.09 (m, 1H, H-15'), 1.06-1.01 (m,
2H, H-12), 1.00 (s, 3H, H-23), 0.95 (s, 3H, H-24), 0.94 (s, 3H, H-27), 0.86 (s, 3H, H-26), 0.84
13
(s, 3H, H-25) ppm; C NMR (100 MHz, CDCl3): δ = 218.0 (C-3), 212.1 (C-20), 176.4 (C-
28), 56.3 (C-17), 54.8 (C-5), 51.4 (C-30), 51.2 (C-19), 49.7 (C-9), 49.2 (C-18), 47.2 (C-4),
42.2 (C-14), 40.5 (C-8), 39.5 (C-1), 37.4 (C-13), 36.9 (C-10), 36.5 (C-22), 34.0 (C-2), 33.5
(C-7), 31.6 (C-16), 30.1 (C-29), 29.7 (C-15), 28.2 (C-21), 27.2 (C-12), 26.7(C-23), 21.4 (C-
11), 21.0 (C-24), 19.6 (C-6), 15.9 (C-25), 15.6 (C-26), 14.6 (C-27) ppm; ESI-MS (MeOH):
m/z (%) = 471.3 ([M + H]+, 24), 488.3 ([M + NH4]+, 12), 493.5 ([M + Na]+, 5), 728.8 ([3M +
2Na]2+, 100), 963.3 ([2M + Na]+, 64); elemental anal. calcd for C30H46O4 (494.71): C 76.55,
H 9.85; found: C 76.41, H 9,97.
4.20. Methyl 19-(hydroxymethylacryloyl)-2-methylene-3-oxo-20,29,30-trinorlupan-28-oate
(23) and methyl 29-hydroxymethyl-2-methylene-3,20-oxo-29-(1,3,5-trioxocane)-20,29,34-
trinorlupan-28-oate (24)
Compounds 23 (1.45 g, 71 %) and 24 (30 mg, 2 %) were obtained from 22 using method B.
Data for compound 23: colorless crystals; mp 204-206 °C; Rf = 0.19 (toluene/EtOAc/formic
acid/heptane, 80:26:5:10); [α]D = +10° (c = 0.57, CHCl3); IR (KBr): ν = 3470m, 2947s,
1
1728s, 1690s, 1456m, 1379m, 1270w, 1138s, 1049m cm-1; H NMR (400 MHz, CDCl3): δ =
6.10 (br t, J = 4.3 Hz, 1H, methylene-31), 5.95 (br t, J = 4.3 Hz, 1H, methylene-31), 5.90 (t, J
= 2.1 Hz, 1H, methylene-34), 5.06 (t, J = 2.1 Hz, 1H, methylene-34), 4.78-4.68 (m, 2H, H-
30), 3.62 (s, 3H, H-32), 3.73 (dt, 1H, J = 11.2, 4.7 Hz, H-19), 2.63 (d, J = 15.3 Hz, 1H, H-1),
2.33-2.18 (m, 1H, H-18), 2.21 (dt, J = 12.5, 3.0 Hz, 1H, H-16), 2.02-1.96 (m, 2H, H-1', H-13),
1.96-1.88 (m, 1H, H-21), 1.88-1.78 (m, 1H, H-22), 1.51-1.11 (m, 13H, H-5, H-6, H-7, H-9, H-
17

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11, H-15, H-16', H-21', H-22'), 1.10-1.00 (m, 1H, H-12), 1.00-0.91 (m, 1H, H-12'), 1.04 (s,
3H, H-23), 0.97 (s, 3H, H-24), 0.96 (s, 3H, H-27), 0.87 (s, 3H, H-26), 0.76 (s, 3H, H-25) ppm;
13C NMR (100 MHz, CDCl3): δ = 207.6 (C-3), 205.8 (C-20), 176.6 (C-28), 146.8 (C-31),
142.2 (C-33), 125.3 (C-29), 123.7 (C-2), 63.0 (C-30), 56.3 (C-17), 53.9 (C-5), 51.5 (OMe),
49.5 (C-9), 48.3 (C-18), 47.0 (C-1), 45.8 (C-4), 45.0 (C-19), 42.3 (C-14), 40.5 (C-8), 37.4 (C-
13), 37.0 (C-10), 36.8 (C-22), 33.3 (C-7), 31.4 (C-16), 29.7 (C-15), 29.8 (C-21), 28.1 (C-23),
27.4 (C-12), 22.4 (C-24), 21.5 (C-11), 19.7 (C-6), 15.5 (C-26), 15.1 (C-25), 14.6 (C-27) ppm;
ESI-MS (MeOH): m/z (%) = 525.5 ([M + H]+, 21), 547.4 ([M + Na]+, 7); elemental anal.
calcd for C33H48O5 (524.73): C 75.53, H 9.22; found: C 75.38, H 9.37.
Data for compound 24: colorless crystals; mp 190-194 °C; Rf = 0.20 (toluene/EtOAc/formic
acid/heptane, 80:26:5:10); [α]D = +5° (c = 0.53, CHCl3); IR (KBr): ν = 3473s, 2948s, 1728s,
1
1690s, 1445m, 1377m, 1260w, 1168s, 1138s, 1047m cm-1; H NMR (500 MHz, CDCl3): δ =
5.90 (t, J = 2.2 Hz, 1H, methylene), 5.06 (t, J = 2.2 Hz, 1H, methylene), 4.76-4.69 (m, 4H, H-
31, H-32), 4.10 (d, J = 11.9 Hz, 1H, H-30), 3.99 (d, J = 11.9 Hz, 1H, H-33), 3.88-3.84 (m, 1H,
H-30'), 3.83-3.79 (m, 1H, H-33'), 3.71 (d, J = 10.9 Hz, 1H, H-34), 3.67 (d, J = 10.9 Hz, 1H,
H-34'), 3.62 (s, 3H, H-35), 3.53 (dt, J = 11.3, 4.9 Hz, 1H, H-19), 2.62 (d, J = 15.3 Hz, 1H, H-
1), 2.27 (t, J = 11.2 Hz, 1H, H-18), 2.21 (dt, J = 12.5, 3.0 Hz, 1H, H-16), 2.02-1.97 (m, 1H,
H-1'), 1.97 (s, 1H, H-13), 1.96-1.88 (m, 1H, H-21), 1.85-1.79 (m, 1H, H-22), 1.51-1.11 (m,
13H, H-5, H-6, H-7, H-9, H-11, H-15, H-16', H-21', H-22'), 1.10-1.00 (m, 1H, H-12), 1.00-
0.91 (m, 1H, H-12'), 1.04 (s, 3H, H-23), 0.97 (s, 3H, H-24), 0.96 (s, 3H, H-27), 0.87 (s, 3H,
H-26), 0.76 (s, 3H, H-25) ppm; 13C NMR (125 MHz, CDCl3): δ = 215.8 (C-20), 207.6 (C-3),
176.4 (C-28), 142.2 (C-2), 123.6 (methylene), 95.5 (C-31 and C-32), 68.1 (C-33), 67.6 (C-
30), 64.3 (C-34), 57.7 (C-29), 56.0 (C-17), 53.9 (C-5), 51.5 (C-36), 48.6 (C-18), 48.2 (C-9),
47.8 (C-19), 47.0 (C-1), 45.8 (C-4), 42.3 (C-14)), 40.4 (C-8), 37.4 (C-13), 36.9 (C-10), 36.6
(C-22), 33.3 (C-7), 31.4 (C-16), 29.6 (C-15), 28.1 (C-23), 28.0 (C-21), 27.2 (C-12), 22.4 (C-
24), 21.5 (C-11), 20.1 (C-6), 15.5 (C-26), 15.4 (C-25), 14.6 (C-27) ppm; ESI-MS (MeOH):
m/z (%) = 615.4 ([M + H]+, 49), 637.7 ([M + Na]+, 17), 1251.3 ([2M + Na]+, 100); elemental
anal. calcd for C36H54O8 (614.81): C 70.33, H 8.85; found: C 70.17, H 8.98.
4.21. Cytotoxicity studies
4.21.1. Cell lines and culture conditions
The human cell lines 518A2 (melanoma), 8505C (thyroid carcinoma), A549 (alveolar basal
epithelial adenocarcinoma), HT29 (colorectal adenocarcinoma), MCF-7 (breast
adenocarcinoma) were included in this study. Cultures were maintained as monolayer in
18

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RPMI 1640 (PAA Laboratories, Pasching, Germany) supplemented with 10 % heat
inactivated fetal bovine serum (Biochrom AG, Berlin, Germany) and penicillin / streptomycin
(PAA Laboratories) at 37 °C in a humidified atmosphere of 5 % CO2/ 95 % air.
4.21.2. Cytotoxicity assay [22, 30, 31]
The cytotoxicity of the compounds was evaluated using the sulforhodamine-B (SRB) (Sigma
Aldrich) microculture colorimetric assay. In short, exponentially growing cells were seeded
into 96-well plates on day 0 at the appropriate cell densities to prevent confluence of the cells
during the period of experiment. After 24 h, the cells were treated with serial dilutions of the
compounds (0-100 µM) for 96 h. The final concentration of DMSO or DMF solvent never
exceeded 0.5 %, which was non-toxic to the cells. The percentages of surviving cells relative
to untreated controls were determined 96 h after the beginning of drug exposure. After a 96 h
treatment, the supernatant medium from the 96 well plates was discarded, and the cells were
fixed with 10 % TCA. For a thorough fixation, the plates were allowed to rest at 4 °C. After
fixation, the cells were washed in a strip washer. The washing was done four times with water
using alternate dispensing and aspiration procedures. The plates were then dyed with 100 µl
of 0.4 % SRB (sulforhodamine B) for about 20 min. After dying, the plates were washed with
1 % acetic acid to remove the excess of the dye and allowed to air dry overnight. 100 µl of 10
mM Tris base solution were added to each well, and absorbance was measured at λ = 570 nm
(using a 96 well plate reader, Tecan Spectra, Crailsheim, Germany). The EC50 value was
determined from three independent measurements (each in triplicate) applying the two-
parametric Hill slope equation.
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Chem., 74 (2014) 1-6.
[31] B. Siewert, E. Pianowski, A. Obernauer, R. Csuk, Towards cytotoxic and selective
derivatives of maslinic acid, Bioorgan. Med. Chem., 22 (2014) 594-615.
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Figure Captions
Figure 1. Structure of triterpenoic acids: ursolic acid (UA), oleanolic acid (OA),
glycyrrhetinic acid (GA), and platanic acid (PA).
Scheme 1. Synthesis of the 2-methylene-3-oxo (12-16) and the 2,2-dibromo-3-oxo (17-19)
derivatives starting from the corresponding 3-oxo compounds as well as of 20 and 21:
Reagents and conditions: a) CrO3, acetone, H2SO4, r.t., 1 hour; b) K2CO3, paraformaldehyde,
DMF, 90 °C, 1 hour; c) Br2, CH3COOH, r.t., 15 min; d) diisopropylamine, n-BuLi, THF, -78
°C, 30 min; then: iodomethane, THF, -78 °C → r.t.; e) CeCl3, NaBH4, MeOH, r.t., 48 hours.
Scheme 2. Formation of 23 (71 %) and 24 (2 %) from methyl platanoate (22): Reagents and
conditions: a) K2CO3, paraformaldehyde, DMF, 90 °C, 1 hour.
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Table 1. Cytotoxicity (EC50 in µM, 96 hours of treatment, from SRB assay) of compounds 1-
16 as well as of parent compounds glycyrrhetinic acid (GA), ursolic acid (UA), oleanolic acid
(OA) and platanic acid (PA). EC50 values represent mean values of three independent
measurements (each performed in triplicate) and were calculated applying the two-parametric
Hill slope equation (confidence interval CI = 95 %). The cell lines are human cancer cell
lines: 518A2 (melanoma), 8505C (thyroid carcinoma), A549 (alveolar basal epithelial
adenocarcinoma), HT29 (colorectal adenocarcinoma), MCF-7 (breast adenocarcinoma); n.d. =
not detected.
EC50 518A2
8505C
A549
HT-29
MCF-7
84.70 ± 4.36
>30
83.92 ± 4.20
86.50 ± 4.33
>30
82.76 ± 4.14
>30
80.09 ± 4.05
>30
GA
5
>30
1.19 ± 0.06
16.78 ± 0.84
14.7 ± 0.13
>30
1.58 ± 0.08
15.45 ± 0.77
13.5 ± 1.54
n.d.
1.23 ± 0.06
17.90 ± 0.90
15.5 ± 1.72
n.d.
1.34 ± 0.13
n.d.a
0.74 ± 0.08
n.d.
10
15
UA
1
10.6 ± 0.71
>30
12.7 ± 0.13
>30
>30
n.d.
n.d.
>30
>30
2
1.50 ± 0.15
1.24 ± 0.12
>30
1.57 ± 0.16
1.56 ± 0.16
20.5 ± 0.82
6.10 ± 0.61
>30
1.54 ± 0.15
1.64 ± 0.16
26.1 ± 3.04
2.93 ± 0.29
>30
1.69 ± 0.17
1.15 ± 0.12
26.7 ± 5.42
1.35 ± 0.14
>30
1.55 ± 0.16
1.30 ± 0.13
16.7 ± 1.03
4.43 ± 0.44
>30
6
7
11
12
14
OA
3
2.16 ± 0.22
>30
>30
>30
>30
>30
>30
>30
n.d.
n.d.
>30
>30
>30
n.d.
n.d.
27.62
28.69
4
3.71 ± 0.21
4.11 ± 0.41
1.51 ± 0.15
4.32 ± 0.24
n.d.
3.83 ± 0.20
n.d.
3.71 ± 0.21
4.47 ± 0.45
3.16 ± 0.31
4.24 ± 0.23
5.44 ± 0.54
4.68 ± 0.46
8
9
1.62 ± 0.16
1.57 ± 0.15
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>30
>30
>30
>30
>30
>30
>30
>30
>30
PA
16
24.11 ± 2.12

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