Ursolic and oleanolic acid derivatives with cholinesterase inhibiting potential

Article abstract of DOI:10.1016/j.bioorg.2018.12.013

Triterpenoids are in the focus of scientific interest, and they were evaluated for many pharmacological applications among them their ability to act as inhibitors of cholinesterases. These inhibitors are still of interest as drugs that improve the life quality of patients suffering from age-related dementia illnesses especially of Alzheimer's disease. Herein, we prepared several derivatives of ursolic and oleanolic acid and screened them in Ellman's assays for their ability to inhibit acetylcholinesterase and/or butyrylcholinesterase, and for each of the active compounds the type of inhibition was determined. As a result, several compounds were shown as good inhibitors for acetylcholinesterase and butyrylcholinesterase even in a micromolar range. An ursolic acid derived hydroxyl-propinyl derivative 10 was a competitive inhibitor for butyrylcholinesterase with an inhibition constant of K_i = 4.29 μM, and therefore being twice as active as gold standard galantamine hydrobromide. The best inhibitor for acetylcholinesterase, however, was 2-methyl-3-oxo-methyl-ursoloate (18), acting as a mixed-type inhibitor showing K_i = 1.72 μM and K_i′ = 1.28 μM, respectively.

Full text of DOI:10.1016/j.bioorg.2018.12.013

Accepted Manuscript
Ursolic and oleanolic acid derivatives with cholinesterase inhibiting potential
Anne Loesche, Alexander Köwitsch, Susana D. Lucas, Zayan Al-Halabi,
Wolfgang Sippl, Ahmed Al-Harrasi, René Csuk
PII:
S0045-2068(18)31228-8
DOI:
https://doi.org/10.1016/j.bioorg.2018.12.013
YBIOO 2677
Reference:
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
26 October 2018
3 December 2018
10 December 2018
Please cite this article as: A. Loesche, A. Köwitsch, S.D. Lucas, Z. Al-Halabi, W. Sippl, A. Al-Harrasi, R. Csuk,
Ursolic and oleanolic acid derivatives with cholinesterase inhibiting potential, Bioorganic Chemistry (2018), doi:
https://doi.org/10.1016/j.bioorg.2018.12.013
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Ursolic and oleanolic acid derivatives with cholinesterase inhibiting potential
Anne Loesche1, Alexander Köwitsch1, Susana D. Lucas2, Zayan Al-Halabi 3, Wolfgang Sippl
3, Ahmed Al-Harrasi4, René Csuk*1
1
Martin-Luther-University Halle-Wittenberg, Organic Chemistry, Kurt-Mothes-Str. 2, D-
06120 Halle (Saale), Germany;
2
Universidade de Lisboa, Faculdade de Farmácio, Instituto de Investigacao do Medicamento
(iMed.ULisboa), Av. Prof. Gama Pinto, 1649-003 Lisboa, Portugal;
3
Martin-Luther-University Halle-Wittenberg, Institute of Pharmacy, Wolfgang-Langenbeck-
Str. 4, D-06120 Halle (Saale), Germany;
4
University of Nizwa, Chair of Oman’s Medicinal Plants and Marine Natural Products, PO
Box 33, Birkat Al-Mauz, Nizwa, Sultanate of Oman.
* Corresponding author
Graphical abstract:
Ursolic and oleanolic acid derivatives with cholinesterase inhibiting potential
1

Abstract
Triterpenoids are in the focus of scientific interest, and they were evaluated for many
pharmacological applications among them their ability to act as inhibitors of cholinesterases.
These inhibitors are still of interest as drugs that improve the life quality of patients suffering
from age-related dementia illnesses especially of Alzheimer’s disease. Herein, we prepared
several derivatives of ursolic and oleanolic acid and screened them in Ellman’s assays for
their ability to inhibit acetylcholinesterase and/or butyrylcholinesterase, and for each of the
active compounds the type of inhibition was determined. As a result, several compounds were
shown as good inhibitors for acetylcholinesterase and butyrylcholinesterase even in a
micromolar range. An ursolic acid derived hydroxyl-propinyl derivative 10 was a competitive
inhibitor for butyrylcholinesterase with an inhibition constant of Ki = 4.29 M, and therefore
being twice as active as gold standard galantamine hydrobromide. The best inhibitor for
acetylcholinesterase, however, was 2-methyl-3-oxo-methyl-ursoloate (18), acting as a mixed-
type inhibitor showing Ki = 1.72 µM and Ki’ = 1.28 M, respectively.
1. Introduction
Spending their last days on earth having already lost many cognitive functions remains a
major fear of elderly people. Although during the last century their lifespans increased, the so-
called “old-age diseases” became more and more important. Alzheimer's disease (AD) besides
cancer, strokes, heart attacks and dementia diseases became the most important diseases of the
21st century; these diseases cause the highest death rates. Nowadays, one person in 200 suffers
from any kind of dementia, and this number is expected to double within the next 30 years
[1,2]. Therefore, the scientific and economic interest remains unbroken, and the research for a
better understanding of dementia and possible treatments especially of AD have been in
progress for decades.
The large number of patients suffering from AD makes this disease of special interest within
the field of dementia diseases. Many theories regarding the causes of these diseases were
postulated, resulting in some therapeutic concepts. One of the most often followed ideas is the
-amyloid hypothesis basing essentially on the neurotoxic effect of -amyloid plaques inside
the human brain having been formed by the action of -,  and -secretases [3,4]. However,
about 30 percent of middle-aged people have AD-equal amounts of these plaques but without
2

suffering from AD [5,6]. In addition, several therapies basing on this theory failed to increase
the cognitive abilities [7-10] by decreasing the -amyloid plaque's concentration. Other
therapeutic targets refer, for example, to inflammatory processes or mitochondrial disorder of
the -protein [11-13].
Another concept relies on the neurotransmitter acetylcholine (ACh) since its concentration
seems reduced during AD; AD typical symptoms such as amnesia or behavioral disorders [14-
17] have been credited to a lowered concentration of ACh. Usually, the cleavage of this
neurotransmitter is performed by the hydrolase acetylcholinesterase (AChE, E.C. 3.1.1.7) but
another enzyme, butyrylcholinesterase (BChE, E.C. 3.1.1.8), seems also important for
controlling the concentration of ACh in different tissues of an organism including the brain. It
was assumed that BChE is able to compensate a lack of AChE activity [18,19]. Furthermore,
the AChE/BChE ratio in the brain alters from 0.2 in healthy brains to 11 during advanced AD
[14,20,21]. Thus, both enzymes represent interesting targets for potential AD cures or – at
least - as tools for a deeper insight into AD's pathology.
Triterpenes represent a group of pharmacologically active substances having already been
tested for cholinergic activities.[22-24] Triterpenes holding hopyl [25], lanostyl [26] or lupyl
[27] skeletons have been targets in recent studies. Also, pentacyclic triterpenoic acids and
compounds derived thereof showed an inhibitory potential for AChE in micromolar range
[28]. Especially several subgroups of - and -amyrins were most effective inhibitors for this
cholinesterase. Furthermore, AChE inhibition has been found for derivatives of oleanolic acid
[29-32], ursolic acid [33] as well as of glycyrrhetinic acid [34] or platanic acid derived
compounds [35]. Therefore, we decided to synthesize and to test several ursolic and oleanolic
acid (Fig. 1) derived compounds in Ellman’s assays for their ability to act as inhibitors of
AChE as well as of BChE and to obtain inhibitory constants (Ki and Ki′).
Fig. 1. Structure of ursolic acid (1) and oleanolic acid (20).
3

2. Results and discussion
2.1. Chemistry
Reaction of ursolic acid (1, Scheme 1) or of its 11-oxo analogue 2 [36] with methyl iodide in
the presence of potassium carbonate gave esters 3 and 4 in good yields. These esters served as
valuable starting materials for the syntheses to follow. Acetylation of 3 or 4 with acetic
anhydride and triethylamine in dry DCM furnished acetates 5 and 6, respectively. Jones
oxidation of 3 or 4 gave 3-oxo compounds 7 and 8. Their reaction with lithium
diisopropylamide in THF followed by adding 2-propyn-1-ol yielded 3-hydroxyprop-1-ynyl
13
derivatives 9 and 10, respectively. Compound 9 is characterized in its C NMR spectrum by
the presence of the alkynyl carbons detected at  = 88.9 and 83.7 ppm. The 1H NMR chemical
shift of 33-H was determined at  = 4.26 ppm.
Scheme 1. Synthesis of ursolic acid (1) derived compounds 2-10: a) K2CO3, MeI, DMF, 12
h, 25 °C, 78%; b) K2CO3, MeI, DMF, 12 h, 25 °C, 82%; c) Ac2O, TEA, DCM, 12 h, 25 °C,
87%; d) Na2Cr2O7 AcOH, NHS, acetone, 2 d, 40 °C, 65%; e) CrO3, H2SO4, acetone, 1 h, 25
°C, 88%; f) CrO3, H2SO4, acetone, 1 h, 25 °C, 88%; g) LDA, THF, -78 °C→25 °C, 53%; h)
LDA, THF, -78 °C→25 °C, 63%.
4

The 3-O-acryloyl derivative 11 (Scheme 2) was obtained from the reaction of 3 with acryloyl
chloride in the presence of triethylamine and catalytic amounts of DMAP. Reaction of 3 with
sodium hydride and sulfamoyl chloride in THF gave 12 which was converted into the
corresponding carbamoylsulfamate 13 using sodium hydride, 1,1’-carbonyldiimidazole and a
saturated solution of ammonia. Products dibrominated at position C-2 were obtained from the
3-oxo esters 7 and 8. Thus, 7 or 8 was allowed to react with bromine in glacial acetic acid,
and compounds 14 and 15 were obtained.
Reaction of 7 and 8 with paraformaldehyde in DMF under basic conditions gave the 2-
1
methylenated derivatives 16 and 17, respectively. Compound 16 is characterized in its H
NMR spectrum by the presence of a methylene group resulting in signals at  = 5.93 ppm and
13
5.07 ppm (for 17: H = 5.95 and 5.18 ppm, respectively). In the C NMR spectrum signals at
 = 141.9 and 123.6 ppm (for 17: C= 141.8 and 124.1 ppm) were assigned to this group.
Treatment of 7 with LDA and iodomethane in dry THF yielded compound 18. Compound 19
holding a double bond in ring A was synthesized as previously reported.[37]
Scheme 2. Synthesis of compounds 11-19: a) TEA, acryloyl chloride, DMAP, CHCl3, 30
min, 25 °C, 45%; b) NaH, THF, 0°C→25 °C, 30 min then sulfamoyl chloride, THF, 5 d, 25
°C, 69%; c) NaH, THF, 0°C→25 °C, 30 min then CDI, NH3, THF, 3 h, 25 °C,95%; d) Br2,
AcOH, 15 min, 25 °C, 80%; e) Br2, AcOH, 15 min, 25 °C, 93%; f) K2CO3, paraformaldehyde,
5

DMF, 1 h, 90 °C, 45%; g) K2CO3, paraformaldehyde, DMF, 1 h, 90 °C, 44%; h) LDA, MeI,
THF, -78°C→25 °C, 71%; i) 3,3-dimethylglutarimide, PPh3, DEAD, THF, 12 h, 0 °C, 93%.
Following the procedures given for the synthesis of the derivatives of ursolic acid, starting
from oleanolic acid (20) and 11-oxo-oleanolic acid 21 [38] the corresponding methyl esters 22
and 23 were prepared (Scheme 3). Acetylation of 22 afforded 24; from the reaction of 22 with
acryloyl chloride/trimethylamine/DMAP acryloylated 25 was obtained in 63% isolated yield.
1
The olefinic protons of this compound were detected in the H NMR spectrum at  = 6.30,
6.04 and 5.73 ppm, respectively.
Scheme 3. Synthesis of 21-28: a) K2CO3, MeBr, DMF, 18 h, 25 °C, 88%; b) K2CO3, MeBr,
DMF, 18 h, 25 °C, 90%; c) Ac2O, TEA, DCM, 12 h, 25 °C, 95%; d) TEA, acryloyl chloride,
DMAP, CHCl3, 30 min, 25 °C, 63%; e) CrO3, H2SO4, acetone, 1 h, 25 °C, 75%; f) LDA,
THF, -78 °C→25 °C, 52%; g) 3,3-dimethylglutarimide, PPh3, DEAD, THF, 12 h, 0 °C, 96%.
6

Oxidation of 21 gave a 3,11-dioxo-oleanoate 26 whose reaction with LDA and 2-propyn-1-ol
afforded 27 in 52% yield. Reaction of 23 with triphenylphosphane, 3,3-dimethylglutarmide
and diethyl azodicarboxylate in THF gave 87% of a 2,12-diene 28. Sulfamoylated 29 was
prepared from 22 (Scheme 4) according to our previously published procedure;[39] this
compound was further transformed into carbamoylsulfamated 30. The diene 28 served as a
starting material for the synthesis of the 1,9-endoperoxide 31 and epoxide 32, respectively.
[34] Bromination of 32 gave 33 whose Jones-oxidation furnished 34.
Scheme 4. Synthesis of 29-34: a) NaH, THF, 0°C→25 °C, 30 min then sulfamoyl chloride,
THF, 5 d, 25 °C, 70%; c) NaH, THF, 0°C→25 °C, 30 min then CDI, NH3, THF, 3 h, 25 °C,
96%; c) Na2Cr2O7, AcOH, NHS, acetone, 2 d, 40 °C, 46%; d) m-CPBA, MeCl, 2 d, 25 °C,
55%; e) HBr, THF, 10 min, 10 °C, 77%; f) CrO3, H2SO4, acetone, 30 min, 25 °C, 53%.
2.2. Biology
Compounds 1–34 and galantamine hydrobromide (GH, as a standard) were subjected to
Ellman’s assays to measure their ability to inhibit the enzymes acetylcholinesterase (from
Electrophorus electricus, electric eel) and butyrylcholinesterase (from equine serum).
7

Cholinesterases from different species are very similar. Due to these large and functional
conservations, AChE from the electric eel, and BChE from equine serum can be used as
suitable models for the corresponding enzymes from humans. As a result, while most of the
compounds inhibited AChE, only two compounds displayed a significant activity for BChE;
several compounds were insoluble under the conditions of the assay. The results from these
assays are compiled in Table 1.
Table 1. Inhibitory constants for galantamine hydrobromide (Ki = AChE: 0.54 ± 0.01 µM;
BChE: Ki = 9.37 ± 0.67 µM) as a standard and compounds 1‒34 (except insoluble and
inactive ones) using Ellman’s assay employing AChE (electric eel) and BChE (equine serum);
four different substrate concentrations and four different inhibitor concentrations were used;
Ki and Ki’ are reported in µM; mean ± SE; inactive means less than 30% inhibition at 50 µM
concentration.
AChE
BChE
AChE
BChE
Compound
Ki (µM) [Ki’(µM)]
(Type of inhibition)
8.54 ± 1.33 [38.16 ± 3.28]
(Mixed type)
Ki (µM) [Ki’(µM)]
(Type of inhibition)
Compound
Ki (µM) [Ki’(µM)]
(Type of inhibition)
11.62 ± 2.82
Ki (µM) [Ki’(µM)]
(Type of inhibition)
1
20
inactive
inactive
inactive
inactive
inactive
inactive
(UA)
(OA)
(competitive)
7.48 ± 0.53 [17.40 ± 0.63]
(Mixed type)
4.22 ± 0.68[12.24 ± 0.85]
(Mixed type)
2
21
22
27
28
29
31
32
34
9.48 ± 0.98 [45.90 ± 0.81]
(Mixed type)
3.46 ± 0.56 [49.11 ± 7.28]
(Mixed type)
3
4.29 ± 0.29
24.35 ± 9.07
(competitive)
inactive
inactive
10
12
14
16
18
19
(competitive)
7.39 ± 1.51 [11.1 ± 1.37]
(Mixed type)
6.26 ± 2.71 [15.06 ± 2.00]
(Mixed type)
inactive
inactive
inactive
inactive
inactive
inactive
inactive
inactive
inactive
inactive
11.50 ± 1.75 [35.83 ± 1.21]
(Mixed type)
6.37 ± 0.26 [26.69 ± 0.18]
(Mixed type)
20.73 ± 0.17 [12.42 ± 1.33]
(Mixed type)
17.01 ± 1.02 [36.51 ± 4.25]
(Mixed type)
1.72 ± 0.24 [1.28 ± 0.05]
(Mixed type)
21.89 ± 5.93 [21.53 ± 3.57]
(Mixed type)
4.43 ± 0.46 [5.26 ± 1.04]
(Mixed type)
12,88 ± 0.77 [14.58 ± 0.92]
(Mixed type)
For the parent triterpenoic acids inhibition constants for AChE in low micromolar magnitude
were measured. Thus, ursolic acid (1) gave a mixed-type inhibition (Ki = 8.54 ± 1.33 M, Ki’
= 38.16 ± 3.28 M) while for oleanolic acid (20) a competitive inhibition (Ki = 11.62 ± 2.82
M) was found. Inhibition of both compounds, however, was lower than the inhibition
measured for standard galantamine hydrobromide (GH, Ki = 0.54 ± 0.01 M). Esterification
of 1 had no significant impact on the ability to act as an inhibitor, and the inhibition constants
of 3 were similar to those of parent ursolic acid. Interestingly, the methyl ester of oleanolic
8

acid 22 was a mixed-type inhibitor, and elimination of HO-C(3) provided compounds with
rather lower Ki values. Furthermore, introduction of a 2-methyl-3-oxo-moiety increased the
inhibition potential, and inhibition constants of Ki = 1.72 ± 0.24 M and Ki’ = 1.28 ± 0.05 M
were determined for 18. Finally, introduction of a 3-hydroxyprop-1-ynyl moiety provided a
selective BChE inhibitor; thus, ursolic acid derived 10 (Ki = 4.29 ± 0.29 M, competitive
inhibitor) was twice as active as galantamine hydrochloride.
1500000
1000000
500000
0
750000
500000
250000
0
500
250
0
[S]
[I]
[S]
-500000
-1000000
-1500000
Ki' = 1.28 ± 0.05 µM
-250
Ki = 1.72 ± 0.24 µM
-250000
-30000-25000-20000-15000-10000 -5000
0
5000 10000 15000 20000
-10
-5
0
5
10
-5
0
5
10
1/[S] in M-1
[I] in µM
[I] in µM
Fig. 2. Lineweaver-Burk (left), Dixon (middle) and Cornish-Bowden (right) plots for mixed-
type inhibitor 18.
3.3. Docking
Molecular modeling is a valuable tool to rationalize different biological outcomes from
closely related compounds. Therefore, we used GOLD 5.2 docking software [40] to evaluate
the preferred poses of some of the synthesized compounds in the active site of the targeted
enzymes AChE and BChE. Compound 18 being the most active compound towards AChE
showed a preferential U-shape pose facing the active site Ser203. This led to an acceptable
positioning for subsequent ester hydrolysis and hence to an inactivation of the enzyme.
Docking of several UA derived compounds revealed that their preferential pose did not allow
the entrance into the AChE gorge and thus there was no inhibition of the targeted esterase.
From the results of the biological assay it was known that the selectivity of the inhibitors to
AChE is higher than to BChE. This can probably be explained by the fact that the inhibitors
interact with the residues Phe 330, Trp 279, Tyr 121 in the AChE binding pocket via pi-pi
interactions whereas the corresponding residues in BChE (Ala 328, Val 277, Gln 119 cannot.
The active compounds could fit reasonably into the AChE hydrophobic binding pocket. In
addition, the carboxyl groups of the compounds fit very well into the hydrophilic part of the
pocket.
9

Fig. 3. Docked poses at the AChE and BChE active sites calculated with GOLD 5.2 [37]. A)
AChE: compounds 18 (green), 17 (cyano) and 10 (pink); B) BChE, compound 10 (pink).
Pictures made with MOE2014 software [41].ꢀ
4. Conclusions
Several derivatives of ursolic and oleanolic acid varying in their substitution pattern were
synthesized in this study and screened for their inhibitory potential for the enzymes AChE and
BChE applying Ellman’s assays. The inhibition constants and the type of inhibition were
determined for each compound. While parent ursolic acid gave inhibition constants of about
10 µM for AChE, no inhibition for BChE was detected. However, several compounds showed
a higher inhibition for AChE and two compounds inhibited BChE even in micromolar
magnitude. The results obtained in the biological assay can be explained by appropriate
molecular modelling calculations.
5. Experimental
5.1. Chemistry
5.1.1 General
Reagents were bought from commercial suppliers without any further purification.
Instrumental analysis was performed as previously reported. [42] The purity of the
compounds was checked by HPLC, and found to > 96%.
10

5.1.2. Syntheses
5.2.1. 3-Hydroxy-urs-12-en-28-oic acid (ursolic acid, 1)
This compound was commercially obtained from Betulinines (Stříbrná Skalice, Czech
Republic) and used as received.
5.2.3. Methyl 3-hydroxy-urs-12-en-28-oate (3)
This compound was synthesized according to literature from 1;[39] m.p. 165‒167 °C; []D =
+68° (c = 0.53, CHCl3); MS (ESI): m/z (%) = 471.2 ([M + H]+, 80), 493.4 ([M + Na]+, 100).
5.2.4. Methyl 3-hydroxy-11-oxo-urs-12-en-28-oate (4)
Esterification [39] of 2 [36] gave compound 4; m.p. 132‒135 °C; []D = +87° (c = 3.0,
CHCl3); MS (ESI): m/z (%) = 485.5 ([M + H]+, 100), 507.5 ([M + Na]+, 50).
5.2.5. Methyl 3-acetyloxy-urs-12-en-28-oate (5)
This compound was synthesized by acetylation of 3; [42] m.p. 243‒246 °C; []D = 64.7° (c =
0.30, CHCl3); MS (ESI): m/z (%) = 513.2 ([M + H]+, 40), 535.5 ([M + Na]+, 100).
5.2.6. Methyl 3-acetyloxy-11-oxo-urs-12-en-28-oate (6)
Compound 5 (480 mg, 0.94 mmol) was dissolved in acetone (50 mL) and glacial acetic acid
(5 mL). N-hydroxysuccinimide (1.20 g, 10.43 mmol) and potassium dichromate (1.05 g, 3.56
mmol) were added. After 2 days of continuous stirring at 40 °C, solutions of saturated
potassium bisulfite solution and saturated sodium bicarbonate were added. The aqueous
solution was extracted with dichloromethane (4 × 50 mL); the combined organic layers were
washed with water (20 mL) and brine (20 mL), dried (Na2SO4) and evaporated to dryness.
The residue was subjected to column chromatography (silica gel, hexanes/ethyl acetate, 8:2)
to afford 6 (0.32 g, 65%) as an colorless solid; m.p. 245–250 °C (lit.: 235 °C [43]; RF = 0.66
(chloroform/diethyl ether, 95:5); []D = +75° (c = 3.3, CHCl3); UV–Vis (methanol): max (log
) = 274 nm (4.21); IR (KBr):  = 3441br, 2971s, 2875m, 1731s, 1655s, 1620w, 1459m,
1427w, 1396m, 1371m, 1319m, 1307m, 1272m, 1239s, 1202m, 1142m, 1116m, 1101w,
1088w, 1028m, 1002m, 983m cm-1; 1H NMR (500 MHz, CDCl3):  = 5.58 (s, 1H, H-12), 4.51
(dd, 1H, J = 11.6, 4.6 Hz, H-3), 3.59 (s, 3H, OMe), 2.77 (d, 1H, J = 13.8 Hz, H-1), 2.40 (d,
1H, J = 11.3 Hz, H-18), 2.30 (s, 1H, H-9), 2.11-2.01 (m, 1H, H-16), 2.03 (s, 3H, Ac)), 1.81-
11

1.50 (m, 10H, H-2, H-6, H-7, H-15, H-16’, H-21, H-22), 1.44-1.20 (m, 4H, H-7’, H-19, H-15’,
H-21’), 1.28 (s, 3H, H-27), 1.13 (s, 3H, H-25), 1.10-1.01 (m, 2H, H-1’, H-20), 0.95 (d, 3H, J
= 6.4 Hz, H-30), 0.89 (s, 3H, H-26), 0.86 (d, 3H, J = 6.1 Hz, H-29), 0.85 (s, 3H, H-24), 0.85
13
(s, 3H, H-23), 0.76 (d, 1H, J = 11.4 Hz, H-5) ppm; C NMR (125 MHz, CDCl3):  = 199.6
(C-11), 177.1 (C-28), 170.9 (Ac), 162.8 (C-13), 130.6 (C-12), 80.6 (C-3), 61.3 (C-9), 55.0 (C-
5), 52.7 (C-18), 51.8 (OMe), 47.6 (C-17), 44.6 (C-8), 43.7 (C-14), 38.8 (C-4), 38.6 (C-19),
38.5 (C-20), 38.0 (C-1), 37.0 (C-10), 35.9 (C-22), 32.9 (C-7), 30.3 (C-21), 28.3 (C-15), 28.0
(C-23), 23.9 (C-16), 23.5 (C-2), 21.3 (Ac), 21.0 (C-27), 20.9 (C-30), 18.8 (C-26), 17.3 (C-
6),17.1 (C-29), 16.7 (C-24), 16.2 (C-25) ppm; MS (ESI): m/z (%) = 527.5 ([M + H]+, 85),
549.5 ([M + Na]+, 100); anal. calcd. for C33H50O5 (526.75): C, 75.25; H, 9.57; found: C,
75.07; H, 9.74.
5.2.7. Methyl 3-oxo-urs-12-en-28-oate (7)
This compound was synthesized according to literature from 3;[44] m.p. 193‒195°C; []D =
+90° (c = 5.90, CHCl3); MS (ESI): m/z (%) = 496.3 ([M + H]+, 75), 523.0 ([M + Na +
MeOH]+,100).
5.2.8. Methyl 3,11-dioxo-urs-12-en-28-oate (8)
This amorphous compound was synthesized according to literature from 4;[44] []D = +108°
(c = 4.80, CHCl3); MS (ESI): m/z (%) = 483.5 ([M + H]+, 100).
5.2.9. Methyl 3-hydroxy-3-hydroxy-1-propynyl)-urs-12-en-28-oate (9)
To a freshly prepared solution of lithium diisopropylamide [made from diisopropylamine (605
mg, 5.99 mmol) and n-butyllithium (1.6 M in hexane, 3.78 mL, 6.05 mmol) in dry THF (20
mL)] at -78°C, 2-propyn-1-ol (223 mg, 4 mmol) in dry THF (4 mL) was added, and the
mixture was stirred for 30 min. Compound 7 (360 mg, 0.74 mmol) in dry THF (5 mL) was
added dropwise, and the mixture was allowed to warm to room temperature. Usual aqueous
workup followed by column chromatography (silica gel, hexanes/ethyl acetate, 7:3) gave 9
(207 mg, 53%) as an amorphous solid; RF = 0.35 (hexanes/ethyl acetate, 7:3); []D = 32° (c =
3.0, CHCl3); UV–Vis (methanol): max (log ) = 213 nm (3.86); IR (KBr):  = 3433br, 2948s,
2870m, 1725m, 1455m, 1388m, 1308w, 1231m, 1200w, 1144w, 1114w, 1076w, 1033m, 994m
cm-1; 1H NMR (500 MHz, CDCl3):  = 5.19 (m, 1H, H-12), 4.26 (s, 2H), 3.54 (s, 3H, OMe),
2.17 (d, 1H, H-18, J = 11.0 Hz), 1.97–1.83 (m, 4H, H-11, H-11′, H-16, H-2), 1.70 (ddd, 1H,
H-15, J = 13.8, 13.8, 4.6 Hz), 1.63–1.57 (m, 3H, H-22, H-16′, H-2′), 1.55–1.47 (m, 3H, H-9,
12

H-22′, H-1), 1.45–1.39 (m, 3H, H-6, H-21, H-7), 1.32–1.18 (m, 5H, H-19, H-6′, H-1′, H-7′, H-
21′), 1.06 (d, 1H, H-5, J = 11.0 Hz), 1.03–0.92 (m, 2H, H-15′, H-20), 1.02 (s, 3H, H-27), 0.98
(s, 3H, H-23), 0.87 (d, 3H, H-30, J = 5.6 Hz), 0.86 (s, 3H, H-25), 0.81 (s, 3H, H-24), 0.80 (d,
3H, H-29, J = 6.0 Hz), 0.67 (s, 3H, H-26) ppm; 13C NMR (125 MHz, CDCl3):  = 178.0 (C-
28), 138.1 (C-13), 125.5 (C-12), 88.9 (C-31, C≡C), 83.7 (C-32, C≡C), 75.8 (C-3), 53.1 (C-5),
52.8 (C-18), 51.4 (OMe), 51.2 (C-33, H2C-O), 48.1 (C-17), 47.6 (C-9), 41.9 (C-14), 41.4 (C-
4), 39.4 (C8), 39.0 (C-19), 38.8 (C-20), 37.8 (C-1), 36.9 (C-10), 36.6 (C-22), 32.8 (C-7), 32.4
(C-2), 30.6 (C-21), 28.0 (C-15), 25.8 (C-23), 24.2 (C-16), 23.7 (C-27), 23.3 (C-11), 21.1 (C-
30), 18.5 (C-6), 17.8 (C-24), 17.0 (C-29), 16.8 (C-26), 15.7 (C-25) ppm; MS (ESI): m/z (%) =
547.5 ([M + Na]+, 100); anal. calcd. for C34H52O4 (524.77): C, 77.82; H, 9.99; found: C,
77.52; H 10.19.
5.2.10. Methyl 3-hydroxy-3-hydroxy-1-propynyl)-11-oxo-urs-12-en-28-oate (10)
Following procedure described for the synthesis of 9, compound 10 (282 mg, 63%) was
obtained as an amorphous solid from the reaction of 8 (400 mg, 0.83 mmol) followed by
column chromatography (silica gel, hexanes/ethyl acetate, 7:3); RF = 0.54 (hexanes/ethyl
acetate, 1:1); []D = 66° (c = 3.5, CHCl3); UV–Vis (MeOH): max (log ) = 269 nm (4.04); IR
(KBr):  = 3432br, 2950s, 2871m, 1728s, 1661s, 1457m, 1388w, 1323w, 1272w, 1231w,
1202m, 1167w, 1145w, 1112w, 1083w, 1036m, 993m cm-1; 1H NMR (500 MHz, CDCl3):  =
5.59 (s, 1H, H-12), 4.29 (s, 2H), 3.59 (s, 3H, OMe), 2.75 (dd, 1H, H-1, J = 14.0, 2.8 Hz), 2.40
(d, 1H, H-18, J = 11.9 Hz), 2.34 (s, 1H, H-9), 2.09–1.95 (m, 2H, H-2, H-16), 1.81–1.71 (m,
3H, H-16′, H-22, H-15), 1.68–1.49 (m, 5H, H-2′, H-7, H-22′, H-6, H-21), 1.43–1.34 (m, 5H,
H-19, H-7′, H-16′, H-1′, H-15′, H-21′), 1.30 (s, 3H, H-27), 1.11 (s, 3H, H-25), 1.08–1.00 (m,
2H, H-5, H-20), 1.03 (s, 3H, H-23), 0.95 (d, 3H, H-30, J = 6.4 Hz), 0.89 (s, 3H, H-26), 0.87
13
(s, 3H, H-24), 0.85 (d, 3H, H-29, J = 6.7 Hz) ppm; C NMR (125 MHz, CDCl3):  = 199.8
(C-11), 177.2 (C-28), 162.9 (C-13), 130.6 (C-12), 88.9 (C-31, C≡C), 83.7 (C-32, C≡C), 75.5
(C-3), 61.5 (C-9), 52.8 (C-5), 52.7 (C-18), 51.8 (OMe), 51.1 (C-33, H2C-O), 47.6 (C-17), 44.6
(C-8), 43.6 (C-14), 41.5 (C-4), 38.6 (C-19), 38.5 (C-20), 38.3 (C-1), 37.1 (C-10), 36.0 (C-22),
32.9 (C-7), 32.5 (C-2), 30.3 (C-21), 28.4 (C-15), 25.9 (C-23), 23.9 (C-16), 21.0 (C-27), 20.9
(C-30), 18.8 (C-26), 17.8 (C-24), 17.7 (C-6), 17.0 (C-29), 16.5 (C-25) ppm; MS (ESI): m/z
(%) = 539.5 ([M + H]+, 100); anal. calcd. for C34H50O5 (538.76): C, 75.80; H, 9.35; found: C,
75.63; H, 9.51.
5.2.11. Methyl 3-acryloyloxy-urs-12-en-28-oate (11)
13

Compound 3 (233 mg, 0.49 mmol) was dissolved in dry chloroform (30 mL), triethylamine
(184 mg, 1.81 mmol), acryloyl chloride (113 mg, 1.25 mmol) and catalytic amounts of 4-
dimethylaminopyridine were added successively. After 30 min of continuous stirring, water
(40 mL) was added, and the layers were separated. The aqueous solution was extracted with
chloroform (2 × 30 mL); the combined organic layers were washed with water (20 mL) and
brine (20 mL), dried (Na2SO4) and evaporated to dryness. The residue was subjected to
column chromatography (silica gel, hexanes/ethyl acetate, 9:1) to afford 11 (115 mg, 45%) as
an amorphous solid; RF = 0.78 (hexanes/ethyl acetate, 8:2); []D = 67° (c = 5.0, CHCl3); UV–
Vis (methanol): max (log ) = 221 nm (4.19); IR (KBr):  = 2935s, 2872s, 1728s, 1634m,
1456m, 1409m, 1390w, 1370w, 1295m, 1272w, 1225m, 1204s, 1187s, 1146m, 1114w, 1034w,
1012w cm-1; 1H NMR (500 MHz, CDCl3):  = 6.36 (dd, 1H, acryloyl, J = 17.2, 1.5 Hz), 6.10
(dd, 1H, acryloyl, J = 17.2, 10.4 Hz), 5.79 (dd, 1H, acryloyl, J = 10.6, 1.5 Hz), 5.23 (m, 1H,
H-12), 4.56 (dd, 1H, H-3, J = 9.8, 7.3 Hz), 3.59 (s, 3H, OMe), 2.22 (d, 1H, H-18, J = 11.0
Hz), 1.99 (ddd, 1H, H-16, J = 13.6, 13.4, 4.8 Hz), 1.91 (d, 1H, H-11, J = 3.7 Hz), 1.89 (d, 1H,
H-11′, J = 3.7 Hz), 1.76 (ddd, 1H, H-15, J = 13.9, 13.6, 4.8 Hz), 1.69–1.56 (m, 6H, H-22, H-
22′, H-1, H-16′, H-2, H-2′), 1.54–1.44 (m, 4H, H-9, H-6, H-21, H-7), 1.37–1.23 (m, 4H, H-19,
H-6′, H-7′, H-21), 1.12–1.03 (m, 2H, H-15′, H-1′), 1.06 (s, 3H, H-27), 0.97–0.91 (m, 1H, H-
20), 0.94 (s, 3H, H-25), 0.93 (d, 3H, H-30, J = 6.2 Hz), 0.89–0.83 (m, 1H, H-5), 0.88 (s, 3H,
H-23), 0.87 (s, 3H, H-26), 0.85 (d, 3H, H-29, J = 6.6 Hz), 0.74 (s, 3H, H-24) ppm; 13C NMR
(125 MHz, CDCl3):  = 178.0 (C-28), 166.0 (C-31), 138.1 (C-13), 130.0 (C-33, H2C=CH),
129.1 (C-32, HC=CH2), 125.4 (C-12), 81.0 (C-3, HC-O), 55.3 (C-5), 52.9 (C-18), 51.4
(OMe), 48.0 (C-17), 47.5 (C-9), 42.0 (C-14), 39.5 (C-8), 39.0 (C-19), 38.8 (C-20), 38.2 (C-1),
37.8 (C-4), 36.8 (C-22), 36.6 (C-10), 32.9 (C-7), 30.6 (C-21), 28.1 (C-23), 28.0 (C-15), 24.2
(C-16), 23.6 (C-27), 23.5 (C-2), 23.3 (C-11), 21.1 (C-30), 18.2 (C-6), 17.0 (C-29), 16.9 (C-
24), 16.7 (C-26), 15.4 (C-25) ppm; MS (ESI): m/z (%) = 525.3 ([M + H]+, 60), 547.3 ([M +
Na]+, 100); anal. calcd. for C34H52O4 (524.77): C, 77.82; H, 9.99; found: C, 77.66; H, 10.15.
5.2.12. Methyl 3-[(aminosulfonyl)oxy]-urs-12-en-28-oate (12)
This compound was synthesized from 3 as previously reported;[39] m.p. 115‒118 °C; []D =
+62.10° (c = 0.32, CHCl3); MS (ESI): m/z (%) = 548.3 ([M - H]-, 90), 1097.1 ([2M - H]-,
100).
5.2.13. Methyl 3-{[[(aminocarbonyl)amino]sulfonyl]oxy}-urs-12-en-28-oate (13)
14

This compound was synthesized from 12 as previously reported; [45] m.p. 49‒53 °C; []D =
+81.4 (c = 0.3, CHCl3); MS (ESI): m/z (%)) = 591.5 ([M - H]-, 32), 1183.1 ([2M - H]-, 31).
5.2.14. Methyl 2,2-dibromo-3-oxo-urs-12-en-28-oate (14)
This amorphous compound was synthesized from 7 as previously reported;[44] []D = +43° (c
= 5.70, CHCl3); MS (ESI): m/z (%) = 647.1 ([M (2 x 79Br) + Na]+, 56), 649.2 ([M (79Br, 81Br)
+ Na]+, 100), 651.2 ([M (2 x 81Br) + Na]+,46).
5.2.15. Methyl 2,2-dibromo-3,11-dioxo-urs-12-en-28-oate (15)
This compound was synthesized from 8 as previously reported;[41] m.p. 225‒227 °C; []D =
79
+48° (c = 5.90, CHCl3); MS (ESI): m/z (%) = 639.3 ([M (2 x Br) + H]+, 23), 641.3 ([M
(79Br, 81Br) + H]+, 51), 643.3 ([M (2 x 81Br) + H]+,27), 661.1 ([M (2 x 79Br) + Na]+, 46), 663.1
([M (79Br, 81Br) + Na]+, 100), 665.0 ([M (2 x 81Br) + Na]+,52).
5.2.16. Methyl 2-methylene-3-oxo-urs-12-en-28-oate (16)
This amorphous compound was synthesized from 7 as previously reported;[44] []D = +100°
(c = 3.20, CHCl3); MS (ESI): m/z (%) = 481.1 ([M + H]+, 100).
5.2.17. Methyl 2-methylene-3,11-dioxo-urs-12-en-28-oate (17)
This compound was synthesized from 8 as previously reported;[44] m.p. 219‒222 °C; []D =
+126° (c = 4.40, CHCl3); MS (ESI): m/z (%) = 495.5 ([M + H]+, 100).
5.2.18. Methyl 2-methyl-3-oxo-urs-12-en-28-oate (18)
To a solution of lithium diisopropylamide [prepared from diisopropylamine (85 mg, 0.84
mmol) and n-butyllithium (1.6 M in hexane, 0.60 mL, 0.96 mmol) in dry THF (10 mL)] at -
78°C methyl 3-oxo-urs-12-en-28-oate (300 mg, 0.64 mmol) in dry THF (5 mL) was added
followed by iodomethane (200 mg, 1.41 mmol) in dry THF (3 mL) after additional 30 min.
The mixture was allowed to reach room temperature, and water (30 mL) was added. Usual
aqueous work-up and extraction with dichloromethane (3 × 20 mL) followed by column
chromatography (silica gel, hexanes/ethyl acetate, 9:1) afforded 18 (220 mg, 71 %) as an
amorphous solid; RF = 0.4 (hexanes/ethyl acetate, 9:1); []D = 49° (c = 2.0; CHCl3); UV–Vis
15

(MeOH): max (log ) = 216 nm (3.80); IR (KBr):  = 3440br, 2929s, 1725m, 1704m, 1654w,
1456w, 1388w, 1230w, 1200w, 1146w cm-1; 1H NMR (500 MHz, CDCl3):  = 5.24 (m, 1H, H-
12), 3.60 (s, 3H, OMe), 2.77–2.71 (m, 1H, H-2), 2.21 (d, 1H, H-18, J = 11.3 Hz), 2.02–1.89
(m, 4H, H-11, H-11′, H-16, H-1), 1.75 (ddd, 1H, H-15, J = 13.8, 13.8, 4.6 Hz), 1.69–1.62 (m,
2H, H-22, H-16′), 1.56 (ddd, 1H, H-22′, J = 13.4, 13.4, 4.0 Hz), 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, 1H, H-5,
J = 12.2 Hz), 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, 3H, H-30, J = 6.1 Hz), 0.83
13
(d, 3H, H-29, J = 6.4 Hz), 0.78 (s, 3H, H-26) ppm; C NMR (125 MHz, CDCl3):  = 218.0
(C-3), 177.9 (C-28), 138.3 (C-13), 125.2 (C-12), 57.2 (C-5), 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; MS (ESI): m/z (%) = 483.3 ([M + H]+, 65), 537.0 ([M +
Na + MeOH]+, 100); anal. calcd. for C32H50O3 (482.74): C, 79.62; H, 10.44; found: C 79.51;
H, 10.58.
5.2.19. Methyl 11-oxo-urs-2,12-dien-28-oate (19)
This compound was synthesized from 4 as previously reported;[37] m.p. 179‒187 °C; []D =
+141° (c = 1.8, CHCl3); MS (ESI): m/z (%) = 467.5 ([M + H]+, 100), 489.5 [M + Na]+, 30).
5.2.20. 3-Hydroxy-olean-12-en-28-oic acid (oleanolic acid, 20)
This compound was commercially obtained from Betulinines (Stříbrná Skalice, Czech
Republic) and used as received.
5.2.22. Methyl 3-hydroxy-olean-12-en-28-oate (22)
This compound was synthesized from 20 as previously reported;[42] m.p. 198‒200 °C; []D =
+70° (c = 0.43, CHCl3); MS (ESI): m/z (%) = 493.5 ([M + Na]+, 100).
5.2.23. Methyl 3-hydroxy-11-oxo-olean-12-en-28-oate (23)
Esterification of 21 as previously reported [42] gave 21 as a colorless solid; m.p. 184‒188 °C;
[]D = +82 (c = 0.14, CHCl3); MS (ESI): m/z (%) = 485.6 ([M + H]+, 100), 507.5 ([M + Na]+,
35).
16

5.2.24. Methyl 3-acetyloxy-olean-12-en-28-oate (24)
The acetylation of 22 using acetic anhydride gave 24;[37] m.p. 219-221 °C 224-225 °C; []D
= +69.3 (c = 3.8, CHCl3); MS (ESI): m/z (%) = 535.5 ([M + Na]+, 100).
5.2.25. Methyl 3-acryloyloxy-olean-12-en-28-oate (25)
Following the procedure given for the synthesis of 11, from 22 (198 mg, 0.42 mmol) and
acryloyl chloride followed by chromatography (silica gel, chloroform/diethyl ether, 98:2) 25
(138 mg, 63%) was obtained as a colorless solid; m.p. 227–233 °C; RF = 0.62 (hexanes/ethyl
acetate, 9:1); []D = 70° (c = 4.2, CHCl3); UV–Vis (MeOH): max (log ) = 219 nm (4.28); IR
(KBr):  = 3425br, 2940s, 2862m, 1722s, 1471m, 1404m, 1388w, 1363w, 1272m, 1240w,
1204m, 1191s, 1163m, 1124w, 1096w, 1046m, 1014w cm-1; 1H NMR (500 MHz, CDCl3):  =
6.30 (dd, 1H, acryloyl, J = 17.4, 1.5 Hz), 6.04 (dd, 1H, acryloyl, J = 17.2, 10.6 Hz), 5.73 (dd,
1H, acryloyl, J = 10.7, 1.5 Hz), 5.21 (m, 1H, H-12), 4.50 (dd, 1H, H-3, J = 8.6, 7.3 Hz), 3.56
(s, 3H, OMe), 2.82 (dd, 1H, H-18, J = 13.8, 4.2 Hz), 1.90 (ddd, 1H, H-16, J = 14.4, 14.5, 4.0
Hz), 1.85–1.80 (m, 2H, H-11, H-11′), 1.65–1.42 (m, 11H, H-9, H-1, H-19, H-6, H-7, H-15, H-
22, H-22′, H-16′, H-2, H-2′), 1.39–1.09 (m, 4H, H-6′, H-21, H-21′, H-7′, H-19′), 1.07 (s, 3H,
H-27), 1.02–0.96 (m, 2H, H-15′, H-1′), 0.88 (s, 3H, H-25), 0.86 (s, 3H, H-30), 0.84–0.77 (m,
1H, H-5), 0.83 (s, 3H, H-29), 0.83 (s, 3H, H-26), 0.81 (s, 3H, H-23), 0.66 (s, 3H, H-24) ppm;
13C NMR (125 MHz, CDCl3):  = 178.2 (C-28), 166.0 (C-31), 143.8 (C-13), 130.0 (C-33,
H2C=C), 129.9 (C-32), 122.2 (C-12), 81.1 (C-3, HC-O), 55.3 (C-5), 51.5 (OMe), 47.4 (C-9),
46.7 (C-17), 45.8 (C-19), 41.6 (C-14), 41.2 (C-18), 39.3 (C-8), 38.1 (C-1), 37.8 (C-4), 36.9
(C-10), 33.8 (C-21), 33.1 (C-29), 32.6 (C-7), 32.3 (C-22), 30.7 (C-20), 28.0 (C-23), 27.6 (C-
15), 25.8 (C-27), 23.5 (C-30), 23.4 (C-11), 23.4 (C-2), 23.0 (C-16), 18.2 (C-6), 16.7 (C-24),
16.6 (C-26), 15.3 (C-25) ppm; MS (ESI): m/z (%) = 547.3 ([M + Na]+, 100); anal. calcd. for
C34H52O4 (524.77): C, 77.82; H, 9.99; found: C, 77.64; H, 10.13.
5.2.26. Methyl 3,11-dioxo-olean-12-en-28-oate (26)
This amorphous compound was synthesized from 21 as previously reported;[44] []D = +119°
(c = 3.30, CHCl3); MS (ESI): m/z (%) = 483.5 ([M + H]+, 100).
5.2.27. Methyl 3-hydroxy-3-(3-hydroxy-1-propynyl)-11-oxo-olean-12-en-28-oate (27)
As described for 10, compound 27 was obtained from methyl 3,11-dioxo-olean-12-en-28-oate
(26, 300 mg, 0.62 mmol) as an amorphous solid (175 mg, 52%); RF = 0.54 (hexanes/ethyl
acetate, 1:1); []D = 42° (c = 4.4, CHCl3); UV–Vis (MeOH): max (log ) = 269 nm (4.02); IR
17

(KBr):  = 3423br, 2950s, 2867m, 1727s, 1655s, 1464m, 1385s, 1366w, 1328w, 1264m,
1206m, 1163w, 1081w, 1036m, 994w cm-1; 1H NMR (500 MHz, CDCl3):  = 5.62 (s, 1H, H-
12), 4.27 (s, 2H), 3.61 (s, 3H, OMe), 2.98 (dd, 2H, H-18, J = 13.4, 3.7 Hz), 2.77 (dd, 1H, H-1,
J = 13.4, 3.7 Hz), 2.36 (s, 1H, H-9), 2.07–1.94 (m, 2H, H-2, H-16), 1.75–1.56 (m, 7H, H-19,
H-16′, H-2, H-7, H-15, H-22, H-22′), 1.54–1.49 (m, 1H, H-6), 1.41–1.27 (m, 4H, H-1′, H-7′,
H-21, H-6′), 1.37 (s, 3H, H-27), 1.26–1.15 (m, 4H, H-5, H-19′, H-21′, H-15′), 1.09 (s, 3H, H-
25), 1.03 (s, 3H, H-23), 0.91 (s, 3H, H-30), 0.91 (s, 3H, H-29), 0.88 (s, 3H, H-26), 0.86 (s,
3H, H-24) ppm; 13C NMR (125 MHz, CDCl3):  = 200.4 (C-11), 177.5 (C-28), 168.8 (C-13),
127.7 (C-12), 88.9 (C-31, C≡C), 83.7 (C-32, C≡C), 75.5 (C-3), 61.9 (C-9), 52.7 (C-5), 51.9
(OMe), 51.0 (C-33), 46.2 (C-17), 45.0 (C-14), 44.3 (C-19), 43.4 (C-8), 41.6 (C-18), 41.6 (C-
4), 38.2 (C-1), 37.2 (C-10), 33.7 (C-21), 32.8 (C-29), 32.7 (C-7), 32.5 (C-2), 31.6 (C-22), 30.6
(C-20), 27.7 (C-15), 25.9 (C-23), 23.6 (C-27), 23.4 (C-30), 22.9 (C-16), 18.9 (C-26), 17.7 (C-
24), 17.6 (C-6), 16.5 (C-25) ppm; MS (ESI): m/z (%) = 539.5 ([M + H]+, 100); anal. calcd. for
C34H50O5 (538.76): C, 75.80; H, 9.35; found: C, 75.56; H, 9.51.
5.2.28. Methyl 11-oxo-olean-2,12-dien-28-oate (28)
This compound was synthesized according to literature from 23;[37]; m.p. 177‒183 °C; []D
= +147° (c = 1.5, CHCl3); MS (ESI): m/z (%) = 467.5 ([M + H]+, 100).
5.2.29. Methyl 3-[(aminosulfonyl)oxy]-olean-12-en-28-oate (29)
As described for 12, compound 29 was obtained from 22 [39]; m.p. 128‒129 °C; []D =
73.69° (c = 0.3, CHCl3); MS (ESI): m/z (%) = 548.3 ([M - H]-, 96), 1098.2 ([2M – H]-, 100).
5.2.30. Methyl 3-{[[(aminocarbonyl)amino]sulfonyl]oxy}-olean-12-en-28-oate (30)
As described for 13, this compound was obtained from 29;[45] m.p. 77‒82 °C; []D = +79.2°
(c = 0.28, CHCl3); MS (ESI): m/z (%) = 91.5 ([M - H]-, 95), 1183.1 ([2M – H]-, 97).
5.2.31. Methyl 2,3-dihydro-1,9-peroxo-11-oxo-olean-12-en-28-oate (31)
This compound was synthesized according to literature from 28; [37]; m.p. 160‒165 °C; []D
= -19° (c = 1.7, CHCl3); MS (ESI): m/z (%) = 497.4 ([M + H]+, 25), 519.6 ([M + Na]+, 100).
5.2.32. Methyl 3-epoxy-11-oxo-olean-12-en-28-oate (32)
To a mixture of 28 (391 mg, 0.84 mmol) in DCM (40 mL) m-CPBA (550 mg, 3,19 mmol)
was added, and the mixture was stirred at 25 °C for 2 h. Cold saturated solution of sodium
18

bicarbonate (100 mL) was added, and the reaction mixture was extracted with DCM (2 x 100
mL). The solvent was evaporated under diminished pressure. The residue was subjected to
chromatography (silica gel, hexane/ethyl acetate, 9:1) to afford 32 (227 mg, 55%) as an
colorless solid; m.p. 205–207 °C; RF = 0.60 (hexanes/ethyl acetate, 4:1); []D = +70° (c = 3.1,
CHCl3); UV–Vis (methanol): max (log ) = 269 nm (4.00); IR (KBr):  = 3424br, 2948s,
1720s, 1653s, 1576m, 1461m, 1438m, 1387m, 1366m, 1329m, 1308m, 1259m, 1236m, 1214m,
1
1169m, 1129w, 1064w, 1012w cm-1; H NMR (500 MHz, CDCl3):  = 5.64 (s, 1H, H-12),
3.62 (s, 3H, OMe), 3.21-3.14 (m, 2H, H-2, H-1), 2.99 (dd, 1H, J = 13.8, 3.7 Hz, H-18), 3.05
(dd, 1H, J = 17.8, 5.9 Hz, H-1’), 2.80 (d, 1H, J = 3.7 Hz, H-3), 2.28 (s, 1H, H-9), 2.02 (ddd,
1H, J = 13.8, 13.8, 4.3 Hz, H-16), 1.74-1.66 (m, 2H, H-16’, H-22), 1.64-1.51 (m, 5H, H-6 H-
7, H-15, H-19, H-22’), 1.49- 1.20 (m, 6H, H-6’, H-7’ H-15’, H-19’, H-21), 1.29 (s, 3H, H-27),
1.10 (s, 3H, H-25), 1.08 (s, 3H, H-23), 1.01 (s, 3H, H-26), 0.96-0.90 (m, 1H, H-5), 0.92 (s,
13
3H, H-30), 0.91 (s, 3H, H-29), 0.87 (s, 3H, H-24) ppm; C NMR (125MHz, CDCl3):  =
199.9 (C11), 177.5 (C-28), 169.1 (C-13), 127.8 (C-12), 61.3 (C-3), 60.4 (C-9), 52.6 (C-2, HC-
O), 51.8 (OMe), 46.6 (C-5), 46.2 (C-17), 44.8 (C-14), 44.2 (C-19), 43.5 (C-8), 41.6 (C-18),
40.6 (C-1), 36.1 (C-4), 33.7 (C-21), 32.8 (C-29), 32.6 (C-10), 32.0 (C-7), 31.5 (C-22), 30.6
(C-20), 28.2 (C-23), 27.7 (C-15), 23.4 (C-27), 23.4 (C-26), 22.9 (C-16), 22.0 (C-30), 18.2 (C-
25), 18.1 (C-24), 17.8 (C-6) ppm; MS (ESI): m/z (%) = 483.5 ([M + H]+, 100); anal. calcd. for
C31H46O4 (482.69): C, 77.14; H, 9.61; found: C, 76.91; H, 9.72.
5.2.33. Methyl 2-bromo-3-hydroxy-11-oxo-olean-12-en-28-oate (33)
To a solution of 32 (95 mg, 0.20 mmol) in THF (10 mL), HBr (47 %, 100 mg) was added
dropwise at 10 °C. After stirring for 10 min, the solvent was removed in vacuo, water (10 mL)
was added, and the reaction mixture was extracted with DCM (2 x 20 mL). Chromatographic
purification (silica gel, hexane/ethyl acetate, 4:1) gave 33 (85 mg, 77%) as an amorphous
solid; RF = 0.50 (hexanes/ethyl acetate, 4:1); []D = +102° (c = 4.2, CHCl3); UV–Vis
(MeOH): max (log ) = 268 nm (4.08); IR (KBr):  = 3447br, 2949s, 2869m, 1727s, 1657s,
1463m, 1388m, 1367m, 1329w, 1304w, 1262m, 1210m, 1164m, 1104w, 1080w, 1052w,
1032w, 1009w, 757m cm-1; 1H NMR (500 MHz, CDCl3):  = 5.61 (s, 1H, H-12), 4.23 (ddd,
1H, J = 11.9, 8.9, 7.9 Hz, H-2), 3.80 (d, 1H, J = 12.2 Hz, H-3), 3.56 (s, 3H, OMe), 2.96 (dd,
1H, J = 13.7, 4.3 Hz, H-18), 2.79 (dd, 1H, J = 15.3, 11.0 Hz, H-1), 2.43 (s, 1H, H-9), 2.19
(dd, 1H, J = 15.3, 7.9 Hz, H-1’), 1.97 (ddd, 1H, J = 13.7, 13.7, 4.3 Hz, H-16), 1.70-1.61 (m,
2H, H-16’, H-22), 1.60-1.50 (m, 4H, H-7, H-15, H-19, H-22’), 1.45-1.39 (m, 2H, H-6), 1.31-
1.11 (m, 5H, H-7’, H-15’, H-19’H-21), 1.28 (s, 3H, H-27), 1.21 (s, 3H, H-25), 1.02-0.96 (m,
19

1H, H-5), 1.00 (s, 3H, H-24), 0.88 (s, 3H, H-23), 0.87 (s, 3H, H-30), 0.87 (s, 3H, H-29), 0.80
13
(s, 3H, H-26) ppm; C NMR (125 MHz, CDCl3):  = 198.7 (C-11), 177.4 (C-28), 169.6 (C-
13), 127.6 (C-12), 76.2 (C-2), 62.9 (C-9), 58.7 (C-3), 52.4 (C-1), 51.9 (OMe), 50.5 (C-5),
46.1 (C-17), 44.8 (C-14), 44.3 (C-19), 43.6 (C-8), 41.6 (C-18), 39.2 (C-4), 39.0 (C-10), 33.7
(C-21), 32.8 (C-29), 31.7 (C-7), 31.6 (C-22), 30.7 (C 20), 27.7 (C-15), 23.6 (C 27), 23.4 (C-
23), 23.4 (C-24), 23.0 (C-30), 22.8 (C-16), 21.9 (C 25), 19.1 (C-6), 18.5 (C-26) ppm; MS
(ESI): m/z (%) = 585.2 ([M (79Br) + Na]+, 94), 587.2 ([M (81Br) + Na]+, 100); anal. calcd. for
C31H47BrO4 (563.61): C, 66.06; H, 8.41; found: C, 65.83; H, 8.62.
5.2.34. Methyl 2-bromo-311-dioxo-olean-12-en-28-oate (34)
To a solution of 33 (84 mg, 0.15 mmol) in acetone (10 mL) at 0 °C CrO3 (40 mg, 0.40 mmol)
in sulfuric acid (2 M, 0.28 mL) was added. The mixture was stirred for 30 min and then
evaporated to dryness. Saturated potassium disulfite solution (10 mL) and saturated sodium
bicarbonate solution (10 mL) were added, and the mixture was extracted with DCM (2 x 20
mL). The combined organic layers were washed with brine (10 mL), dried (Na2SO4, 10 mL),
filtered and evaporated to dryness. The residue was subjected to column chromatography
(silica gel, hexane/ethyl acetate, 4:1) to yield 34 (45 mg, 53%) as a colorless solid; m.p. 221–
223 °C; RF = 0.36 (hexane/ ethyl acetate, 4:1); []D = +121° (c = 5.1, CHCl3); UV–Vis
(methanol): max (log ) = 269 nm (4.08); IR (KBr):  = 2950s, 2868m, 1728s, 1659s, 1461m,
1
1388m, 1366w, 1329w, 1262w, 1190w, 1163m, 1125w, 1062w, 1012w cm-1; H NMR (500
MHz, CDCl3):  = 5.70 (s, 1H, H-12), 5.05 (dd, 1H, J = 10.4, 10.4 Hz, H-2), 3.62 (s, 3H,
OMe), 3.04 (dd, 1H, J = 13.4, 4.3 Hz, H-18), 2.89 (dd, 1H, J = 14.7, 10.4 Hz, H-1), 2.59 (s,
1H, H-9), 2.53 (dd, 1H, J = 15.0, 11.0 Hz, H-1’), 2.06 (ddd, 1H, J = 14.4, 13.6, 4.8 Hz, H-16),
1.74-1.47 (m, 9H, H-5, H-6, H-7, H-15, H-16’, H-19, H-22), 1.41-1.33 (m, 2H, H-7’, H-21),
1.38 (s, 3H, H-27), 1.25-1.09 (m, 3H, H-15’, H-19’, H-21’), 1.15 (s, 3H, H-25), 1.13 (s, 3H,
H-23), 1.04 (s, 3H, H-26), 0.93 (s, 3H, H-30), 0.93 (s, 3H, H-29), 0.87 (s, 3H, H-24) ppm;
13C NMR (125 MHz, CDCl3):  = 208.6 (C-3), 198.1 (C-11), 177.3 (C-28), 169.8 (C-13),
127.5 (C-12), 65.8 (C-9), 54.2 (C-1,CH2), 52.2 (C-5), 51.9 (OMe), 51.0 (C-2, HC-Br), 47.5
(C-4), 46.1 (C-17), 44.6 (C-14), 44.3 (C-19), 43.7 (C-8), 41.6 (C-18), 38.5 (C-10), 33.6 (C-
21), 32.3 (C-29), 31.5 (C-7), 31.5 (C-22), 30.7 (C-20), 29.3 (C-23), 27.8 (C-15), 23.6 (C-27),
23.4 (C-30), 22.8 (C-16), 20.0 (C-25), 19.1 (C-6), 19.0 (C-26), 18.1 (C-24) ppm; MS (ESI):
m/z (%) = 561.4 ([M (79Br) + H]+, 93), 563.4 ([M (81Br) + H]+, 100), 583.3 ([M (79Br) + Na]+,
34), 585.2 ([M (81Br) + Na]+, 40); anal. calcd. for C31H45BrO4 (561.59): C, 66.30; H, 8.08;
found: C, 66.11; H, 8.26.
20

5.2 Enzymatic studies
5.2.1 Spectrometer and chemicals
The Ellman assays were performed as previously described [35] using a TECAN
SpectraFluorPlus working in the kinetic mode and measuring the absorbance at  = 415 nm
was used for the enzymatic studies. Acetylcholinesterase (from Electrophorus electricus),
5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) and acetylthiocholine iodide were purchased
from Fluka. Butyrylcholinesterase (from equine serum) was purchased from Sigma, and
butyrylthiocholine iodide was bought from Aldrich. Details can be found in the
Supplementary Material.
6. Modelling
A detailed description can be found in the Supplementary Material.
Acknowledgements
The authors like to thank Dr. Ralph Kluge for ESI-MS and Dr. Dieter Ströhl and his team for
the NMR measurements. The IR and optical rotations were measured by Mrs. U. Lammel,
MSc. J. Wiese and B.Sc. V. Simon. We like to thank Dr. St. Schwarz for his help in the
screening. Support by the “ScienceCampus Halle WCH (W13004216 to RC)” is gratefully
recognized.
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25

Highlights





Derivatives of ursolic and oleanolic acid were prepared and tested in Ellman’s assays
Several compounds acted as good inhibitors for acetyl- or butyrylcholinesterase
2-Methyl-3-oxo-methyl-ursolate 18 acted as a mixed type inhibitor for AChE
Ursolic acid derived hydroxy-propinyl 10 was a competitive inhibitor for BChE
This compound was twice as active as standard galantamine hydrobromide
26