Synthesis of glycyrrhetinic acid derivatives for the treatment of metabolic diseases

Article abstract of DOI:10.1016/j.bmc.2009.10.036

The effect of glycyrrhetinic acid (GA) and GA-derivatives towards 11β-hydroxysteroid dehydrogenase (11β-HSD) was investigated. Novel compounds with modifications at positions C-3, C-11 and C-29 of the GA skeleton were prepared. Single crystal X-ray diffraction data of selected substances are reported and discussed.

Full text of DOI:10.1016/j.bmc.2009.10.036

Bioorganic & Medicinal Chemistry 18 (2010) 433–454
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis of glycyrrhetinic acid derivatives for the treatment
of metabolic diseases
Igor Beseda a, Laszlo Czollner a, Priti S. Shah b, Rupesh Khunt a, Rawindra Gaware a, Paul Kosma c,
Christian Stanetty c, Maria Carmen del Ruiz-Ruiz c, Hassan Amer c, Kurt Mereiter d, Thierry Da Cunha e,
Alex Odermatt e, Dirk Claßen-Houben f, Ulrich Jordis a,
*
a Inst. Applied Synthetic Chemistry, Vienna University of Technology, Getreidemarkt 9, Vienna 1060, Austria
b Saurashtra University, University Campus, Rajkot 360 005, India
c Department of Chemistry, Univeristy of Natural Resources and Life Sciences, Muthgasse 18, Vienna 1190, Austria
d Institute of Chemical Technologies and Analytics, University of Technology, Getreidemarkt 9, Vienna 1060, Austria
e Division of Molecular and Systems Toxicology, Pharmacenter, University of Basel, Klingenbergstrasse 50, Basel 4056, Switzerland
f Onepharm Research and Development, Veterinärplatz 1, Vienna 1210, Austria
a r t i c l e i n f o
a b s t r a c t
Article history:
The effect of glycyrrhetinic acid (GA) and GA-derivatives towards 11b-hydroxysteroid dehydrogenase
(11b-HSD) was investigated. Novel compounds with modifications at positions C-3, C-11 and C-29 of
the GA skeleton were prepared. Single crystal X-ray diffraction data of selected substances are reported
and discussed.
Received 25 June 2009
Revised 17 October 2009
Accepted 20 October 2009
Available online 23 October 2009
Ó 2009 Elsevier Ltd. All rights reserved.
This publication is dedicated to Professor
Peter Stanetty on the occasion of his 65th
birthday
Keywords:
Hydroxysteroid dehydrogenase (HSD)
X-ray
Metabolic diseases
11b-HSD1
11b-HSD2
Inhibitors
1. Introduction
are hormones that regulate a range of pathways involved in regu-
lation of carbohydrate, protein and lipid metabolism, regulation of
Extracts from glycyrrhiza glabra have been used as medicines for
gastro-intestinal diseases, as expectorants, and for many other
indications for more than 4800 years with references from the Chi-
nese medicine, the codex Hammurabi, papyrus Eber, writings by
Plinius, Theophrastus Paracelsus and Hildegard von Bingen. Ruzi-
cka first reported the correct molecular formula of glycyrrhetinic
acid (1) (Scheme 1) as the aglycon of glycyrrhizin in 1937.1 The
biological activities of 1 have been studied extensively and the ef-
fects include, among others, antiinflammatory, antiulcer, antialler-
gic, and antitumor promoter activity, as summarized previously.2,3
In the context of the work presented here we were interested in
the effect of glycyrrhetinic acid (GA, 1) and its derivatives towards
11b-hydroxysteroid dehydrogenase (11b-HSD). Glucocorticoids
normal growth and development, influence on cognitive function,
and resistance to stress. The principal glucocorticoid hormone in
the human body is cortisol (corticosterone in rodents), which is
synthesized in the adrenal cortex from cholesterol and secreted
in response to the adrenocorticotropic hormone (ACTH) from the
25
28
CH3
H
26
CH3
24
CH3
CH3
13
18
O
17
22
2
12
1
11
9
HO
16
15
21
10
6
8
14
5
20
19
4
OH
29
7
3
27
CH
3 H3C
H3C 23
H
30
H
O
* Corresponding author. Tel.: +43 1 58801 15460; fax: +43 1 58801 15499.
E-mail address: ujordis@pop.tuwien.ac.at (U. Jordis).
Scheme 1. The structure of glycyrrhetinic acid, shown with numbering as used
throughout the text.
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.10.036

434
I. Beseda et al. / Bioorg. Med. Chem. 18 (2010) 433–454
pituitary gland in a circadian, episodic manner, but the secretion of
this hormone can also be stimulated by stress, exercise and infec-
tion. In cells, cortisol binds primarily to the glucocorticoid receptor
(GR) that then acts as a ligand-inducible transcription factor to in-
duce the expression of glucocorticoid responsive genes. The corti-
costeroid hormones are found in the body along with their
oxidized 11-dehydro counterparts (cortisone and 11-dehydrocort-
icosterone in human and rodents, respectively), which do not have
activity at the glucocorticoid receptor. The local concentration of
active hormone can differ significantly from its circulating levels
due to the intracellular conversion by 11b-HSD enzymes.4,5 In
mammalians, two distinct isozymes of the 11b-hydroxysteroid
dehydrogenases (11b-HSD) control glucocorticoid activity at the
tissue level by modification of the oxidation state of the hormones.
Both, 11b-HSD1 and 11b-HSD2 are members of the short chain
alcohol dehydrogenase (SCAD) superfamily that have been widely
conserved throughout evolution.
the enzyme by the presence of an aldehyde group at the C-18
position. The importance of protecting the MR from occupation
by glucocorticoids is seen in patients with AME (apparent mineral-
ocorticoid excess) where the gene encoding 11b-HSD2 is
mutated.22,23 Defects or inactivity of the type 2 enzyme results in
hypertensive syndromes and research has shown that patients
with hypertensive syndrome have an increased urinary excretion
ratio of cortisol to cortisone.12,24,25 Inhibition of 11b-HSD2 results
in cortisol-dependent mineralocorticoid excess syndrome with
hypertension and hypokalemic alkalosis.26,27 11b-HSD1 inhibitors
used for the therapy of diabetes or metabolic disease should be
specific for the type 1 isozyme in order to avoid mineralocorticoid
side effects.
As starting point of a program aimed towards determining the
activity of GA-derivatives for 11b-HSD1, we have optimized the
synthesis of GA-derivatives with emphasis on full characterization
of the purity and the identity of these derivatives.
11b-HSD1, originally isolated from rat liver,6 acts in vivo pre-
dominantly as an oxoreductase using NADPH as a co-factor to
transform cortisone into cortisol in a number of tissues including
liver, adipose tissue, bone, pancreas, endothelium, ocular tissue,
skeletal muscle, vascular smooth muscle and certain parts of the
central nervous system.5,7 Thus, 11b-HSD1 serves as a local regula-
tor of glucocorticoid actions in the tissues and organs where it
is expressed. 11b-HSD1 is a reversible enzyme, and in tissue
homogenates, upon purification and under certain conditions such
as reduced co-factor availability and/or in disease states, dehydro-
genase activity prevails.8–11
By contrast, the human 11b-HSD2 isozyme is a high-affinity
NAD-dependent, unidirectional dehydrogenase that converts the
secondary alcohol group at the C-11 position of cortisol to a
secondary ketone, thereby producing the inactive metabolite corti-
sone.5 11b-HSD2 is predominantly expressed in mineralocorticoid
target tissues such as kidney, sweat glands, salivary glands, colonic
mucosa, and placenta, and protects the mineralocorticoid receptor
(MR) from illicit occupation by the higher circulating concentra-
tions of cortisol.12 Its co-localization with the MR plays an
important role in the regulation of salt and water balance.13,14
Thus, 11b-HSD1 allows certain tissues to convert cortisone to
cortisol to increase local glucocorticoid activity and potentiate
adaptive response, thereby counteracting 11b-HSD2 activity that
results in a fall in active glucocorticoids.15
Alterations in 11b-HSD activities have been associated with
several human diseases, including hypertension, diabetes, obesity
and metabolic syndrome. The role of 11b-HSD1 in the metabolic
syndrome and type 2 diabetes is supported by several lines of evi-
dence. Cortisol opposes the action of insulin meaning a stimulation
of hepatic gluconeogenesis, inhibition of peripheral glucose uptake
and increased blood glucose concentration. Inhibition of 11b-HSD1
would decrease the cortisol level and as a result increase glucose
uptake and inhibit hepatic gluconeogenesis, giving a reduction in
circulatory glucose levels. Pharmacological inhibition of 11b-
HSD1 in rat and man with carbenoxolone16,17 and transgenic
knockout in mice18–21 results in enhanced hepatic insulin sensitiv-
ity and reduced gluconeogenesis and glycogenosis, suggesting that
the development of a potent 11b-HSD1 inhibitor could therefore
have considerable therapeutic potential for the treatment of condi-
tions associated with elevated blood glucose.
2. Results and discussion
18b-Glycyrrhetinic acid 1 was used as the starting material and,
with the desire to study the effect of structural changes at the posi-
tions of C-3, C-11, C-18 and C-29 of 18b-glycyrrhetinic acid, the fol-
lowing derivatives were prepared.
The C-29 esters were prepared from 18b-GA 1 by treatment
with diazomethane or diphenyldiazomethane to give 2a or 2b in
high yields (Scheme 2). Compounds 2c–2e have previously been
prepared (2c,28,29 2d, 2e30) using standard methods, for example,
with an excess of appropriate alkyl halides in a solution of NaOH
in DMSO at room temperature. Substances were purified by col-
umn chromatography, and then crystallized to give 40–70% yield.
A number of modifications have been examined at the C-3 posi-
tion; these include the synthesis of ester and ether derivatives.
The C-3 esters 3a, 3d and 3e were prepared as previously described
by reaction of 1 with corresponding anhydrides.
Ethers 3b2 and 3c were obtained by treatment of 18b-GA 1 with
trimethylsilyl chloride or phosphorus oxychloride, respectively, in
dry pyridine. Treatment of 3a with thionyl chloride gave 4a quan-
titatively that was used directly without purification. By contrast,
4b and 4c were obtained by reaction of the corresponding C-29 es-
ters with acetic anhydride. As shown in Scheme 3, 5a–e were ob-
tained by the condensation of C-29 acid chloride 4a, and the
corresponding amines. Deacetylation of 5d gave 26d in high yield.
Additionally, the aldehyde 6 was prepared from 5a and converted
into the oxime 7.
3-Keto compounds 8a–b were obtained in high yield from the
corresponding esters 2a–b with PCC in DCM (Scheme 4). These
were reacted with hydroxylamine and O-methyl-hydroxyamine
in pyridine to give oximes 9a–d.
The reduction of the oximes 9a–c with NaCNBH3 in the pres-
ence of TiIIICl3 and NH4OAc in MeOH afforded corresponding
amines 10a–d.
A number of reducing agents for the preparation of 3a-hydroxy-
GA 12 from 11 were reported. In our hands, K-Selectride was the
most useful among them and allowed for the preparation of 1231
in 95% yield (Scheme 5). This was purified by crystallization from
MeOH. The other reducing agents were less stereoselective.
To prepare the C-29 protected esters, diazomethane and diphe-
nyldiazomethane were used.
The 11b-HSD2 isoform deactivates cortisol to cortisone, thus
11b-HSD2 inhibitors could be used to increase the locally available
amount of glucocorticoids in inflammatory diseases—allowing for a
decrease of the doses required for anti-inflammatory therapy,
resulting in a reduction of the frequency and severity of the side ef-
fects of glucocorticoid therapy.
The reduction of 1–15 has been reported previously by treat-
ment of 1 in hexamethylphosphoric triamide (HMPT) with an ex-
33
cess of lithium,32 by catalytic reduction using PtO2 or by zinc
and concd HCl34 (Scheme 6). In our hands, the last method worked
best—even on larger scales. Methylation of 15 with diazomethane
gave 16 in almost quantitative yield. Reduction of 1 with NaBH4 in
aq THF containing NaOH gave a mixture of epimers 11b-GA and
On the other hand, 11b-HSD2 protects the MR in many key reg-
ulatory tissues. Aldosterone itself is protected from metabolism by

I. Beseda et al. / Bioorg. Med. Chem. 18 (2010) 433–454
435
HOOC
Me
R'OOC
Me
S
S
Me
Me
R
R
O
O
S
S
Me
Me
H
Me
Me
H
R
R
R
R
R'
S
S
S
S
H
Me
H
Me
S
S
R
R
2a
2b
2c
2d
2e
Me
Dpm
Et
Allyl
Bn
HO
1
HO
H
H
Me
Me
Me
Me
O
HOOC
Me
Me
R'
S
S
Me
Me
R
R
O
O
S
S
Me
Me
H
Me
Me
H
R
R
R
R
S
S
S
S
H
Me
H
Me
S
S
R
R
R'O
AcO
H
H
Me
Me
Me
3a. R'= Ac
Me
R'
Cl
OMe
ODpm
3d. R'=CO(CH2)2COOH
4a
4b
4c
3b. R'= Si(CH3)3
3c. R'=PO(OH)2
3e. R'=COCH2CMe2COOH
Scheme 2. Preparations of ethers and esters in position 3 and 29 of GA.
O
O
Me
Me
R''
H
N
H
S
S
Me
Me
R
R
O
O
4a
S
S
Me
Me
H
Me
Me
H
R
R
R
R
O
S
S
S
S
H
Me
H
Me
S
R
S
R
O
R'O
6
H
H
Me
Me
Me
OH
Me
N
O
Me
NH S
O
5a. R'=OAc, R''=
S
Me
5b. R'=OAc, R''=H
R
O
5c. R'=OAc, R''=NH2
S
Me
Me
H
R
R
5d. R'=OAc, R''=CH2CH=CH2
5e. R'=OAc, R''=(CH2)2NH2
26d. R'=H, R''=CH2CH=CH2
O
S
S
H
Me
S
R
O
7
H
Me
Me
Scheme 3. C-29 modification.
11
a
-GA 17 that could be separated as their corresponding esters.
20, in THF with catalytic amounts of HCl present, followed by
methylation with diazomethane to obtain 21. The synthesis of
Pure epimers 18b and 18c were obtained in ratio 5:1 by reaction
of 17 with diphenyldiazomethane followed by purification via col-
umn chromatography. Methylation of 17 with diazomethane with
excess reagent gives 19, methylated at positions C-11 and C-29,
whereas treatment with 1 equiv gives the methylester 18a
selectively.
18a
-GA 23 from 18b-GA 1 has been reported under acid or alka-
line33 conditions. The latter were used by us and the 18
a-GA esters
were prepared by treatment with diphenyldiazomethane or diazo-
methane to give 24 or 25.
A diverse set of amides 26a–26j was synthesized by activation
of the carboxylic acid moiety at the 29-position and reaction with
a range of amines (Scheme 7). Syntheses were performed classi-
The crude mixture of 17 can be dehydrated to heteroannular
diene 22 by refluxing in a mixture of CH3COOH and HCl or, via

436
I. Beseda et al. / Bioorg. Med. Chem. 18 (2010) 433–454
ROOC
Me
S
Me
R
O
2a,b
S
Me
Me
H
R
R
S
S
H
Me
R
X
8a. R= Me, X=O
8b. R= Dpm, X=O
14. R=Me, X=-OCH2CH2O-
H
Me
Me
R'OOC
Me
R'OOC
Me
S
Me
S
Me
R
O
R
O
S
Me
Me
H
R
S
R
Me
Me
H
R
R
S
S
H
Me
S
S
R
H
Me
R''
R
R''O
N
N
H
H
Me
H
Me
Me
Me
9a. R'= Me, R''= H
9b. R'= Dpm, R''= H
9c. R'= Dpm, R''= Me
10a. R'= Me, R''= H
10b. R'= Dpm, R''= H, 3β−amino
10c. R'= Dpm, R''= H, 3α-amino
10d. R'= Dpm, R''= OMe
9d. R'= H,
R''= Me
Scheme 4. C-3 oximes and amines formation.
HOOC
Me
ROOC
Me
S
S
Me
Me
R
R
O
O
1
S
S
Me
Me
H
R
Me
Me
H
R
R
R
S
S
S
S
H
Me
H
Me
R
R
R
O
11
HO
H
H
Me
Me
Me
Me
12 R= H
13a R=Me
13b R=Dpm
Scheme 5. Inversion of configuration at C-3.
cally with DCC or using the water soluble EDAC as the activating
agents.
activity assays. The solvent concentration did not exceed 0.1%
and had no effect on enzyme activities. Reactions were stopped
by adding methanol containing 2 mM unlabeled cortisone and cor-
tisol, followed by separation of steroids by TLC and scintillation
counting. Enzyme kinetics was analyzed by non-linear regression
using four parameter logistic curve fitting (Sigmaplot, Systat Soft-
ware Inc.). Data (mean SD) were obtained from three indepen-
dent experiments.
2.1. 11b-HSD activity assays
Recombinant human 11b-HSD1 and 11b-HSD2 were expressed
in HEK-293 cells that are devoid of endogenous expression of these
enzymes.35 Activities were determined in cell lysates basically as
described before.36 Briefly, 11b-HSD1 dependent reduction of
[1,2-3H]-labelled cortisone (American Radiolabeled Chemicals, St.
Louis, MO) to cortisol was measured for 10 min at 37 °C in a vol-
2.2. Synthesis and biological results
ume of 22
l
l containing a final concentration of 200 nM cortisone
18b-GA 1 has been reported to show potent and non-selective
inhibition of both isozymes of 11b-HSD. Shimoyama et al. reported
lower IC50 value for rat hepatic 11b-HSD1 extract (90 2 nM) com-
pared to rat renal 11b-HSD2 extract (360 2 nM).37 Potter and co-
workers reported 85% inhibition of rat 11b-HSD1 and complete
and 500 M NADPH. 11b-HSD2 dependent oxidation of cortisol to
l
cortisone was measured similarly using [1,2,6,7-3H]-cortisol
(Amersham Pharmacia, Piscataway, NJ, USA) at a final concentra-
tion of 50 nM and NAD+ (500
nal concentrations between 50 nM and 20
stock solutions in dimethylsulfoxide and immediately used for
lM). 18b-GA and its derivatives at fi-
l
M were diluted from
inhibition of rat 11b-HSD2 at an inhibitor concentration of 10
lM
of 18b-GA 1.30,38,39 We evaluated the inhibitory activity of 18b-

I. Beseda et al. / Bioorg. Med. Chem. 18 (2010) 433–454
437
ROOC
Me
ROOC
Me
S
Me
S
S
Me
R
O
S
Me
Me
H
S
R
R
Me
Me
H
R
R
1
S
S
H
Me
S
S
S
R
H
Me
S
R
HO
HO
H
Me
H
Me
Me
Me
15. R= H
23. R=H
24. R=Dpm
25. R=Me
16. R= Me
Me
ROOC
ROOC
Me
HOOC
Me
S
S
S
Me
Me
R
Me
R
R'O
Me
S
S
Me
Me
H
R
Me
H
R
R
S
Me
Me
R
R
S
S
S
S
Me
H
S
R
Me
R
S
S
R
S
H
Me
S
R
HO
HO
AcO
H
H
Me
Me
Me
H
Me
R'
Me
20. R=H
Me
22
21. R=Me
17
18a
H
Me
H
H
18b,c Dpm
19
H
Me
Me
Scheme 6. Various reductions of GA.
O
R'
Me
OH OH
N
R''
R
26a. R'=H, R''=
OH
S
Me
O
OH OH
1
S
Me
Me
H
R
R
26b. R'=H, R''=(CH2)2OH
CH
26c. R'=H, R''=CH2C
S
S
H
Me
S
R
HO
26e. R'=CONHCH2CH3, R''=(CH2)3N(CH3)2
26f. R'=H, R''=CH2COOCH2CH3
H
Me
Me
26g. R'=H, R''=(CH2)2N(CH3)2
O
26h. R', R''=
CH3
O
K+
26i. R'=H, R''=CH2COO -
26j. R'=H, R''=
O
O
K+
Scheme 7. Amides formation.
GA 1 and its derivatives against recombinant human 11b-HSD1
and 11b-HSD2. Using a tenfold lower inhibitor concentration
20 l
M.40 By contrast, the natural product glycyrrhizin GL has lower
inhibitory activity for 11b-HSD1 (63.2%), and especially for 11b-
(1 lM), we observed an inhibition of 83.9% for human 11b-HSD1
HSD2 (28.1%). Because the lead compounds 1 and 3d show maxi-
and 93.6% for human 11b-HSD2 for 1. The licensed drug carbenox-
olone 3d has comparable potency with 87.7% inhibition of
11b-HSD1 and 98.2% inhibition of 11b-HSD2. Although the
mal inhibition of 11b-HSD1 at 1 lM synthesized derivatives were
also screened at a concentration of 1 lM against 11b-HSD1 and
11b-HSD2.
18
a
-derivative has a lower inhibitory potency for 11b-HSD1, it is
Based on the fact, that activity was conserved when sugar moi-
eties or succinic acid are introduced in position 3, additional deriv-
selective and does not inhibit 11b-HSD2 in concentrations up to

438
I. Beseda et al. / Bioorg. Med. Chem. 18 (2010) 433–454
atives with a modification in that position were synthesized and
tested for 11b-HSD1 inhibition. The 3-acetate 3a has already been
suggested as an inhibitor for 11b-HSD isozymes based on a phar-
macophore model41 and was found to be weakly active on 11b-
HSD1. The compound does not have the second carboxylic acid
function of the carbenoxolone derivative but retains the hydrogen
bond acceptor function. The 3-phosphate 3c, however, is as active
as the carbenoxolone derivative, even though the acidic functional-
ity is not attached with a spacer of three carbon atoms. Due to the
good activity of 3c, we also tested its 11b-HSD2 activity of that
derivative but could not find a better selectivity compared to car-
benoxolone 3d. On the other hand, 3a had a weak selectivity for
11b-HSD2.
HSD1. However, the reduction of ketone 11 to the diastereomeric
-hydroxy derivative 12 of the lead compound 1 resulted in
excellent inhibition of 11b-HSD1. Therefore, we also tested upon
inhibition of the other isozyme and found a comparable inhibition
of both enzymes.
Not only the modification in position 3 but also the reduction of
position 11 to the deoxo derivate 15 and the inversion of configu-
ration at position 18 to 18a-glycyrrhetinic acid 23 led to a moder-
ate loss in activity. Modification of position 29 to the free amide 5c
let to a compound with comparable activity as 1, whereas the
oxime 7 had significantly reduced activity. Also, secondary amide
modifications of the carboxylic acid function in position 29 result-
ing in compounds 26a–e, 26g, 26i,j led to a moderate to significant
reduction in inhibitory potency against human 11b-HSD1 and also
11b-HSD2.
3a
The oxidation of the 3b-hydroxy group to the ketone 11 and the
conversion to the O-methyloxime 9d reduced the activity on 11b-
Compound
% Inhibition of human
% Inhibition of human
11b-HSD1 at 1
lM ( SD)
11b-HSD2 at 1 lM ( SD)
CH3
CH3
H
O
CH3
CH3
HO
1
91.2 ( 3.1)
99.9 ( 1.2)
OH
H3C
H3C
H3C
H
H
O
CH3
H
O
CH3
CH3
O
CH3
H3C
O
3a
47.1 ( 9.2)
85.7 ( 1.3)
OH
CH3
H3C
H3C
H
H
O
CH3
H
O
CH3
CH3
O
CH3
HO
O
3c
86.0 ( 1.7)
94.6 ( 1.0)
P
OH
CH3
H3C
H3C
HO
H
H
O
CH3
H
O
CH3
CH3
O
CH3
O
3d
87.7 ( 4.4)
98.2 ( 0.9)
OH
HO
CH3
H3C
H
H
O
H3C
O
CH3
H
CH3
O
H
CH3
CH3
H3C
O
5c
90.9 ( 5.3)
86.9 ( 0.7)
NH2
H3C
H3C
H
H3C
O
O

I. Beseda et al. / Bioorg. Med. Chem. 18 (2010) 433–454
439
Compound
% Inhibition of human
11b-HSD1 at 1 M ( SD)
% Inhibition of human
11b-HSD2 at 1 lM ( SD)
l
CH3
H
O
CH3
CH3
O
CH3
7
43.3 ( 17.4)
59.8 ( 7.3)
O
N
CH3
H3C
H3C
OH
H3C
H
H
CH3
H
O
CH3
CH3
CH3
O
9d
63.2 ( 16.2)
97.5 ( 0.7)
OH
H3C
N
CH3
H3C
H3C
H
H
O
CH3
H
O
H
CH3
CH3
CH3
11
63.8 ( 11.5)
98.7 ( 1.6)
OH
O
CH3
H3C
H3C
H
O
CH3
H
CH3
CH3
CH3
O
12
97.4 ( 1.9)
97.0 ( 1.6)
OH
CH3
H3C
CH3
H
OH
H
O
O
CH3
OH
CH3
O
CH3
CH3
23
49.7 ( 15.1)
49.3 ( 1.4)
HO
CH3
H
CH3
H3C
H
H
CH3
CH3
H
O
CH3
OH
OH
CH3
OH
HO
26a
ꢀ12.9 ( 17.9)
3.4 ( 9.1)
NH
CH3
H3C
OH OH
H3C
H
H
O
CH3
H
O
CH3
CH3
CH3
HO
H
N
26b
2.9 ( 4.6)
40.7 ( 19.4)
CH3
OH
H3C
H3C
H
H
O
(continued on next page)

440
I. Beseda et al. / Bioorg. Med. Chem. 18 (2010) 433–454
Compound
% Inhibition of human
11b-HSD1 at 1 M ( SD)
% Inhibition of human
l
11b-HSD2 at 1
lM ( SD)
CH3
H
CH3
O
CH3
CH3
HO
H
N
26c
10.1 ( 5.6)
ꢀ13.1 ( 15.6)
H3C
CH
H3C
H3C
H
H
O
CH3
H
CH3
O
H
CH3
CH3
H
N
26d
26e
26g
HO
HO
HO
6.2 ( 0.8)
ꢀ22.5 ( 4.7)
14.8 ( 11.8)
41.1 ( 4.8)
CH3
CH2
H3C
H
H3C
H
O
CH3
CH3
O
CH3
CH3
N
CH3
CH3
CH3
32.5 ( 9.8)
H
N
N
H3C
H3C
H3C
H
H
O
O
CH3
H
O CH3
CH3
CH3
CH3
H
N
36.0 ( 19.0)
N
CH3
H3C
H3C
H
H
CH3
O
CH3
H
O
CH3
CH3
CH3
K+
O
HO
H
N
26i
2.7 ( 6)
21.4 ( 0.7)
O
CH3
H3C
H3C
H
H
O
O
CH3
H
CH3
K+
O
CH3
CH3
O
HO
26j
19.5 ( 4.9)
ꢀ28.6 ( 2.8)
N
CH3
H3C
H
H
H3C
O
2.3. X-ray crystal structure analyses
duced by altering the backbone of the parent compound. Technical
details of this work are given at the end of the experimental section
and in Table 1, selected structural diagrams are shown in Figures
1–4. Glycyrrhetinic acid consists of five six-membered rings with
two trans-configured, one cis-configured and one singly unsatu-
rated ring–ring-fusion. Four of the five rings (A, B, D, and E) adopt
chair conformations, whereas ring C with one C@C and one C@O
double bond adopts a half chair conformation. The molecule is rel-
atively rigid, but shows a limited flexibility that finds expression in
some bending along the long axis of the molecule, which is in part
Seven crystal structures of compounds of the present study
were investigated with X-ray single crystal diffraction, namely
glycyrrhetinic acid (1) in the form of the solvate 1ꢁDMSO and
two esters of 1 (2aꢁsolv where solv is disordered acetone/ethanol;
2b), the 3-methoxyamino derivative 10d, and three derivatives
(15ꢁDMSO, 19, and 20) with modifications in the unsaturated ring
C (see below). This was done partly to provide support for their
chemical structures and partly to learn about the changes intro-

I. Beseda et al. / Bioorg. Med. Chem. 18 (2010) 433–454
441
Table 1
Details for the crystal structure determinations of 1ꢁDMSO, 2aꢁsolv, 2b, 10d, 15ꢁDMSO, 19, and 20
1ꢁDMSO
C32H52O5S
548.80
Monoclinic
P21 (no. 4)
13.0687(9)
6.4997(4)
18.2299(12)
105.808(1)
1489.93(17)
100
2aꢁSolv
2b
10d
15ꢁDMSO
C31H48O4S
534.81
Monoclinic
P21 (no. 4)
12.9717(5)
6.5050(3)
18.3846(7)
106.131(1)
1490.23(11)
100
19
20
Formula
fw
Crystal system
Space group
a (Å)
C32H54O4Sa
484.69a
Hexagonal
P65 (no. 170)
26.8604(7)
26.8604(7)
7.0894(4)
90
C43H56O4
636.88
Monoclinic
P21 (no. 4)
13.0784(7)
7.1969(4)
20.3287(12)
108.404(1)
1815.55(18)
173
C44H59NO4
665.92
C32H52O4
500.74
Orthorhombic
P212121 (no. 19)
7.1475(3)
12.4992(5)
31.3422(13)
90
C30H46O3
454.67
Orthorhombic
P212121 (no. 19)
7.4426(3)
20.4774(9)
24.0381(11)
90
Monoclinic
C2 (no. 5)
36.205(4)
6.7089(7)
25.815(3)
123.593(2)
5223.2(9)
100
b (Å)
c (Å)
b (°)
V (Å3)
T (K)
4429.6(3)
100
3663.5(3)
100
2800.1(2)
100
Z
2
6
2
4
2
4
8
qcalcd (g cmꢀ3
)
1.223
0.147
600
1.090a
1.165
0.073
692
1.207
0.076
1448
1.192
0.143
588
1.188
0.076
1104
1.156
0.072
2000
l
(mmꢀ1) (Mo K
a
)
0.070a
F(000)
1596a
h range (°)
No. of rflns measd
Rint
2.4–30.0
21,935
0.014
2.3–27.0
53,231
0.043
3.0–30.0
26,753
0.025
2.6–30.0
29,332
0.024
2.3–30.0
21,999
0.013
2.5–30.0
41,155
0.025
2.9–30.5
22,121
0.033
No. of rflns unique
8500
8292
352/1
0.0351
0.0358
0.0902
0.0908
0.00(4)
ꢀ0.17/0.50
6380
5584
324/1
0.1014
0.1104
0.2660
0.2761
1(2)
5664
5312
433/1
0.0449
10,669
9810
453/0
0.0492
0.0537
0.1173
0.1213
8415
8279
343/1
0.0337
0.0342
0.0863
0.0869
0.02(3)
ꢀ0.25/0.45
8128
7823
334/0
0.0446
0.0463
0.1111
0.1123
0.3(7)
14,314
11,180
613/1
0.0513
0.0748
0.1184
0.1314
ꢀ0.1(8)
ꢀ0.26/0.53
No. of rflns I > 2
No. of params/constr.
R1 (F2 > 2 (F2))b
R1 (all data)
wR2 (F2 > 2
r
(I)
r
0.0477
r
(F2))
0.1113
0.1139
0.2(9)
ꢀ0.16/0.38
wR2 (all data)
Flack abs.str. param.
ꢀ0.2(7)
Diff. four peaks min/max (e Åꢀ3
)
ꢀ0.32/0.70
ꢀ0.15/0.45
ꢀ0.17/0.47
a
Disordered solvate of acetone/ethanol disregarding the unknown solvent content.
b
2
R1
=
R
||Fo| ꢀ |Fc||/
R
||Fo|; wR2 = {
R
[w(Fo ꢀFc2)2]/
R .
[w(Fo2)2]}1/2
Figure 1. Molecular structure of 1ꢁDMSO in the crystalline state showing the hydrogen bond to the DMSO molecule, O4ꢁ ꢁ ꢁO5 = 2.62 Å.
attributable to distance mismatches between the carbon backbone
and the three neighbouring methyl groups C24, C25, and C26.
These three methyl groups show C24–C25 and C25–C26 contact
distances of 3.2 Å, whereas the corresponding C–C distances of
their carrier atoms in the ring system, C4–C10 and C10–C8, are dis-
tinctly shorter (ca. 2.65 Å). As the result, the molecule shows some
kind of bending in the region of the A+B+C rings. A further result of
steric congestion between the three methyl groups of C24, C25,
and C26 is that the orientation of the methyl group C25H3 relative
to the quaternary carbon C10 is not eclipsed but halfway between
staggered and eclipsed, showing for instance in 1ꢁDMSO a C–C–C–
H torsion angle of 28°. The molecular structure of glycyrrhetinic
acid in the form of its DMSO solvate is shown in Figure 1. Glycyrrh-
etinic acid is known to crystallize in the form of a variety of sol-
vates of which by now the crystal structures of the solvates with
acetone+H2O and MeOH were reported.42,43 Characteristically, in
the solvates of glycyrrhetinic acid both the COOH and the alcoholic
OH group are involved in hydrogen bonds, for example, in the
DMSO solvate, where the O1–H1 group donates a hydogen bond
to the C@O oxygen O3, the O4–H4 group donates a hydrogen bond
to the DMSO oxygen O5, and the keto oxygen O2 is inactive. In or-
der to achieve this, the lipophilic part of glycyrrhetinic forms
stacks of molecules with a head to tail arrangement of the OH
and the COOH group (Fig. 2). This arrangement is favourable be-
cause it separates polar and apolar parts of the molecule and is
in a related fashion also found in the acetone–H2O solvate.42
Remarkably, the crystals of 1ꢁDMSO and 15ꢁDMSO are practically
isostructural, where 15ꢁDMSO having in 11-position a CH2 group
instead of a C@O group in 1ꢁDMSO (Table 1). Returning in this con-
text to the conformation of glycyrrhetinic acid derivatives studied
in the present work, it is shown in Figure 4 that their cores exhibit
only modest variations in shape. This indicates that changed

442
I. Beseda et al. / Bioorg. Med. Chem. 18 (2010) 433–454
Figure 2. Packing diagram of 1ꢁDMSO viewed along b-axis. Broken red lines are the hydrogen bonds O1 ? O3 and O4 ? O5. 1ꢁDMSO is in principle isostructural with
15ꢁDMSO (C11@O2 group replaced by C11H2).
Figure 3. Molecular structure of 2b in the crystalline state.
Figure 4. Superposition plots of the mutually least-squares fitted glycerrhetinic acid moieties of the crystalline compounds 1ꢁDMSO, 2aꢁsolv, 2b, 10d, 15ꢁDMSO, 19, and 20
(hydrogen atoms and substituting groups omitted for clarity). Note the outlier compound 20, which mismatches clearly the rest of the compounds for carbon atoms C9, C8,
C7, C6, C3, and C2.
hybridization of the C-ring atoms, from sp2 for C11 (1, 2a, 2b, 10d)
3. Experimental
to sp3 (15, 19), has little effects on the molecular conformation of
the glycyrrhetinic acid backbone, and that intermolecular forces
of crystal packing have comparable effects. The only outlier in this
respect is compound 20, which has a cyclohexadiene structure for
the C-ring, which causes by a sp3 to sp2 transition of C10 a more
pronounced conformational effect to be seen particularly well at
C9, Figure 4.
All solvents were purified and dried by standard procedures.
Melting points were measured on a Büchi B-545 melting point
apparatus. 1H and 13C NMR spectra were recorded on a Bruker
AC 200 (200 MHz) and AC 400 (400 MHz) pulse Fourier-transform
NMR spectrometer in CDCl3 or DMSO-d6. Chemical shifts are
reported relative to the resonance of tetramethylsilane (TMS) or

I. Beseda et al. / Bioorg. Med. Chem. 18 (2010) 433–454
443
corresponding solvent peak. Infrared spectra were recorded on a
BIORAD ATR-FT-IR spectrometer as solutions in DCM or MeOH.
(m, 3H, 1-H, 16-H, 21-H), 1.87–0.89 (m, 15H, 1-H, 2-H, 6-H, 7-H,
15-H, 16-H, 19-H, 21-H, 22-H), 1.35 (s, 3H, 30-H), 1.17 (s, 3H, 27-
H), 1.13 (s, 3H, 25-H), 1.09 (s, 3H, 26-H), 1.00 (s, 3H, 23-H), 0.81–
0.65 (m, 1H, 5-H), 0.80 (s, 3H, 24-H), 0.66 (s, 3H, 28-H); 13C NMR
(100 MHz, CDCl3) d 200.1 (C-11), 175.2 (C-29), 168.9 (C-13),
140.1 (C-Ar), 140.1 (C-Ar), 128.6 (C-Ar), 128.6 (C-Ar), 128.5 (C-
Ar), 128.5 (C-Ar), 128.1 (C-12), 127.8 (C-Ar), 127.3 (C-Ar), 127.3
(C-Ar), 127.0 (C-Ar), 127.0 (C-Ar), 78.7 (C-3), 76.6 (C-31), 61.8 (C-
9), 54.9 (C-5), 48.1 (C-18), 45.3 (C-14), 44.0 (C-8), 43.1 (C-20),
41.1 (C-19), 39.2 (C-1), 39.1 (C-4), 37.5 (C-22), 37.1 (C-10), 32.7
(C-7), 31.7 (C-17), 31.2 (C-21), 28.3 (C-30), 28.3 (C-28), 28.1 (C-
23), 27.3 (C-2), 26.4 (C-16), 26.4 (C-15), 23.3 (C-27), 18.7 (C-26),
Absorbance peak maxima are given in m
(cmꢀ1). For thin layer chro-
matography (TLC), Merck TLC aluminum sheets (Silica 60 F254,
0.25 mm) were used. Visualization was by UV light at 254 and
366 nm or spray reagents (molybdophosphoric acid, mixture of
molybdophosphoric acid and CeIV ammonium nitrate or ninhy-
drine and heating). Compounds were purified by MPLC (medium
pressure liquid chromatography) using preparative silica gel (40–
63 mm) columns. HPLC was performed using a Waters 2695
instrument with Merck Chromolith RP18 columns and a gradient
of 3–60% acetonitrile and water containing HCOOH 0.1% at a flow
of 1.0–3.0 mL/min. Preparative LC was performed using a Waters
instrument with Merck Geminy RP18 columns and a gradient of
3–60% acetonitrile and water containing HCOOH 0.1% at a flow of
25 mL/min. The HPLC reported purity is the number generated
for the peak area as calculated using the Waters Millennium soft-
ware with the Maxplot option for the UV maximum of the corre-
sponding peak. Mass spectra were measured on a JEOL DX-300.
The HRMS positive ionisation mode displayed a [M+1]+ ion corre-
sponding to the elementar formula.
17.5 (C-6), 16.4 (C-25), 15.6 (C-24). IR (DCM)
m: 3744, 3624,
3057, 2980, 2941, 2871, 1725, 1665, 1459, 1424, 1389, 1312,
1212, 1166, 1158, 1034, 992, 961, 911, 895. MS-CI, m/z (Irel. (%)):
167.04 (100); 637.01 [M+1] (34); 638.05 (16); 168.05 (13);
181.92 (11); 183.86 (7); 427.18 (6); 182.99 (5); 639.06 (4);
425.11 (4).
3.3. (3b,18b,20b)-3-Hydroxy-11-oxo-olean-12-en-29-oic acid
ethyl ester (2c)
3.1. (3b,18b,20b)-3-Hydroxy-11-oxo-olean-12-en-29-oic acid
methyl ester (2a)
A solution of 1 (1.00 g, 2.1 mmol) in anhydrous DMF (50 mL)
containing anhydrous K2CO3 (2.90 g, 21.2 mmol) was stirred for
24 h at rt, then poured into brine and extracted with EtOAc
(3 ꢃ 200 mL). The organic phase was washed with water
(3 ꢃ 100 mL), dried over MgSO4, filtered and evaporated in vacuo
to give a white solid. The crude product was subjected to SiO2 chro-
matography eluting with PE–EtOAc (5–30% gradient elution) to
give 2c as a white solid (0.45 g, 42.5%, HPLC >99%). Mp 104–
105 °C (Ref. 51: 93–94 °C); 1H NMR (400 MHz, CDCl3): d 5.63 (s,
1H), 4.18–4.10 (m, 2H), 3.22 (dd, J = 10.8, 5.4 Hz, 1H), 2.72 (dd,
J = 13.3, 3.5 Hz, 1H), 2.33 (s, 1H), 2.13–1.78 (m, 5H), 1.70–1.58
(m, 6H), 1.52–1.24 (m, 8H), 1.35 (s, 3H), 1.23–1.13 (m, 1H), 1.13
(s, 3H), 1.13 (s, 3H), 1.11 (s, 3H), 1.05–0.98 (m, 2H), 0.99 (s, 3H),
0.81 (s, 3H), 0.79 (s, 3H), 0.74–0.67 (m, 1H); 13C NMR (100 MHz,
CDCl3) d 200.2, 176.4, 169.3, 128.5, 78.7, 61.8, 60.3, 54.9, 48.4,
45.4, 43.8, 43.1, 41.0, 39.1, 37.6, 37.6, 37.1, 32.7, 31.8, 31.1, 28.5,
28.3, 28.1, 27.3, 26.5, 26.4, 23.4, 18.6, 17.5, 16.3, 15.6, 14.3. IR
Compound 1 (20.0 g, 42.5 mmol) was placed into DCM (100 mL)
and Et2O (100 mL) and transferred into a diazomethane apparatus.
Diazomethane was added slowly until complete esterification was
apparent on TLC. When diazomethane was gone, the solvent was
evaporated. The crude product was crystallized from MeOH
(500 mL) to give 2a as colorless crystals (17.1 g, 83%, HPLC >99%).
Mp 250–251 °C (Ref. 44: 245–248.5 °C); ½a D20
ꢂ
+161.8 (c 1, CHCl3)
(Ref. 44: +161.1°). 1H NMR (400 MHz, CDCl3): d 5.65 (s, 1H, 12-
H), 3.70 (s, 3H, 31-H), 3.24 (dd, J = 10.7, 5.7 Hz, 1H, 3-H), 2.81 (d,
J = 13.7 Hz, 1H, 1-H), 2.35 (s, 1H, 9-H), 2.13–1.78 (m, 5H, 15-H,
16-H, 18-H, 19-H, 21-H), 1.70–1.58 (m, 5H, 2-H, 6-H, 7-H, 19-H),
1.52–1.03 (m, 6H, 6-H, 7-H, 15-H, 16-H, 21-H, 22-H), 1.38 (s, 3H,
27-H), 1.23–1.13 (m, 1H), 1.16 (s, 3H, 30-H), 1.15 (s, 3H, 25-H),
1.14 (s, 3H, 26-H), 1.07–0.99 (m, 2H, 1-H), 1.02 (s, 3H, 23-H),
0.81 (s, 3H, 24-H), 0.80 (s, 3H, 28-H), 0.74–0.69 (m, 1H, H-5); 13C
NMR (100 MHz, CDCl3) d 200.3 (C-11), 177.0 (C-29), 169.2 (C-13),
128.6 (C-12), 78.7 (C-3), 61.8 (C-9), 54.9 (C-5), 51.8 (C-31), 48.4
(C-18), 45.4 (C-8), 44.1 (C-20), 43.2 (C-14), 41.1 (C-19), 39.1 (C-
4), 39.1 (C-1), 37.7 (C-22), 37.0 (C-10), 32.7 (C-7), 31.8 (C-17),
31.1 (C-21), 28.5 (C-28), 28.4 (C-30), 28.1 (C-23), 27.3 (C-2), 26.4
(C-16), 26.4 (C-15), 23.4 (C-27), 18.7 (C-26), 17.5 (C-6), 16.4 (C-
(DCM) m: 3385, 3053, 2972, 2941, 2871, 1721, 1656, 1621, 1459,
1389, 1362, 1324, 1216, 1173, 1154, 1088, 1042, 996, 949, 922,
899, 880.
3.4. (3b,18b,20b)-3-Hydroxy-11-oxo-olean-12-en-29-oic acid
allyl ester (2d)
25), 15.6 (C-24). IR (DCM)
m
: 3350, 2949, 2871, 1725, 1656, 1455,
To a solution of 1 (10.0 g, 21.3 mmol) in DMSO (150 mL) at 20 °C
was added powdered KOH (2.07 g, 27.6 mmol) and during 10 min
allyl bromide (3.6 g, 29.7 mmol). The reaction mixture was stirred
for 24 h at rt (monitored by TLC), poured into ice-water (250 mL)
and extracted with CHCl3 (3 ꢃ 250 mL). The combined organic
phases was washed with water (2 ꢃ 100 mL), dried (MgSO4), fil-
tered, evaporated and purified by MPLC (solvent system: DCM–
MeOH gradient elution 0–3%) to get 8.4 g of product. The thus ob-
tained crude product was finally purified by recrystallization from
hot methanol (400 mL) to give 2d as a white crystalline compound
(7.5 g, 69%, HPLC >99%). Mp 208–210 °C. 1H NMR (400 MHz, CDCl3)
d 6.00–5.80 (m, 1H), 5.62 (s, 1H), 5.37–5.19 (m, 2H), 4.61–4.52 (m,
2H), 3.26–3.14 (m, 1H), 2.77 (dd, J = 13.4, 3.4 Hz, 1H), 2.32 (s, 1H),
2.08–1.86 (m, 4H), 1.73–1.50 (m, 7H), 1.46–1.26 (m, 5H), 1.35 (s,
3H), 1.22–1.08 (m, 1H), 1.15 (s, 3H), 1.11 (s, 3H), 1.10 (s, 3H),
1.06–0.86 (m, 2H), 0.98 (s, 3H), 0.79 (s, 6H), 0.73–0.63 (m, 1H);
13C NMR (100 MHz, CDCl3) d 200.2, 176.0, 169.2, 132.2, 128.5,
118.4, 78.7, 65.0, 61.8, 54.9, 48.3, 45.3, 44.0, 43.2, 41.0, 39.1,
37.7, 37.0, 32.7, 31.8, 31.1, 28.5, 28.3, 28.1, 27.4, 26.4, 26.4, 23.3,
1389, 1220, 1193, 1158, 1088, 1042, 996. MS-CI, m/z (Irel. (%)):
484.98 [M] (100); 485.99 (36); 135.05 (15); 177.99 (11); 292.98
(11); 188.98 (10); 174.98 (9); 482.97 (8); 316.88 (7); 483.96 (7).
3.2. (3b,18b,20b)-3-Hydroxy-11-oxo-olean-12-en-29-oic acid
diphenylmethyl ester (2b)
Glycyrrhetinic acid 1 (90.0 g, 192 mmol) in MeOH (500 mL) was
heated to 45 °C and a solution of diphenyldiazomethane (113.0 g,
573 mmol) in Et2O (600 mL) was added slowly until complete
esterification was apparent on TLC. The solvent was evaporated
at reduced pressure and the crude product was filtered through a
short pad of silica, the solvent was evaporated and the crude com-
pound crystallized from a mixture of Et2O (250 mL)–pentane
(800 mL) to yield diphenylmethyl ester 2b as colorless crystals
(108.0 g, 87%, HPLC >99%). Mp 182.5–183.5 °C; ½a D20
ꢂ
+140 (c 0.2,
CHCl3). 1H NMR (400 MHz, CDCl3): d 7.41–7.21 (m, 10H, arom),
6.95 (s, 1H, 31-H), 5.54 (s, 1H, 12-H), 3.28–3.18 (m, 1H, 3-H),
2.80 (dd, J = 13.5, 3.5 Hz, 1H, 18-H), 2.33 (s, 1H, 9-H), 2.08–1.88
18.6, 17.4, 16.3, 15.5. IR (DCM) m: 3740, 3501, 2937, 2871, 1729,

444
I. Beseda et al. / Bioorg. Med. Chem. 18 (2010) 433–454
1656, 1621, 1540, 1459, 1389, 1362, 1328, 1316, 1212, 1154, 1085,
1042, 984, 934, 922, 880, 830. MS-CI, m/z (Irel. (%)): 511.20 [M+1]
(100); 512.17 (35); 513.16 (8); 510.25 (4); 135.22 (3); 514.18
(2); 175.13 (2); 189.14 (2); 319.13 (2); 343.09 (2).
tion and dried to give 3b as a white solid (0.41 g, 54%, HPLC >97%).
Mp 260–261 °C (Ref. 2: 244–246 °C). 1H NMR (400 MHz, CDCl3) d
5.73 (s, 1H), 3.22 (dd, J = 11.5, 4.5 Hz, 1H), 2.76 (d, J = 13.3 Hz,
1H), 2.36 (s, 1H), 2.20 (d, J = 10.6 Hz, 1H), 2.07–1.97 (m, 2H),
1.90–1.81 (m, 1H), 1.68–1.59 (m, 3H), 1.49–1.38 (m, 5H), 1.39 (s,
3H), 1.31–1.21 (m, 2H), 1.25 (s, 3H), 1.19–1.13 (m, 1H), 1.16 (s,
3H), 1.14 (s, 3H), 1.08–1.01 (m, 2H), 0.91 (s, 3H), 0.85 (s, 3H),
0.79 (s, 3H), 0.73–0.67 (m, 1H), 0.12 (s, 9H); 13C NMR (100 MHz,
CDCl3) d 200.7, 181.9, 169.5, 128.5, 79.5, 61.9, 55.1, 48.3, 45.5,
43.8, 40.9, 39.5, 39.2, 39.1, 37.7, 37.1, 32.8, 31.9, 30.9, 28.6, 28.5,
28.1, 27.6, 26.5, 26.4, 23.4, 18.7, 17.7, 16.5, 16.1, 0.51. +1, m/z (Irel.
(%)): 543.27 [M] (100); 544.18 (35); 453.41 (12); 545.14 (10);
615.02 (7); 454.45 (5); 90.04 (5); 452.61 (4); 191.19 (4); 471.48
(4).
3.5. (3b,18b,20b)-3-Hydroxy-11-oxo-olean-12-en-29-oic acid
benzyl ester (2e)
A solution of glycyrrhetinic acid (10.0 g, 21.0 mmol) in anhy-
drous DMF (250 mL) containing anhydrous K2CO3 (29.0 g,
212.0 mmol) was stirred at rt under N2 for 1 h. Benzyl bromide
(40.0 g, 234.0 mmol) was added to the reaction mixture followed
by tetrabutylammonium iodide (2.0 g, 5.5 mmol). The mixture
was stirred for 24 h at room temperature, poured into brine and
extracted with EtOAc (3 ꢃ 200 mL). The combined organic phases
was washed with water (3 ꢃ 100 mL), dried over Na2SO4, filtered
and evaporated in vacuo to give a yellow resin. Purification by flash
chromatography (PE–EtOAc gradient elution, 5–35%) yielded ben-
zyl ester 2e as a white solid (5.5 g, 46%, HPLC >99%). Mp 129–
3.8. (3b,18b,20b)-3-[(Phosphono)oxy]-11-oxo-olean-12-en-29-
oic acid (3c)
A mixture of glycyrrhetinic acid (2.0 g, 4.3 mmol) in dry pyri-
dine (3.4 mL) and dry THF (20 mL) was added dropwise to a stir-
red solution of phosphorus oxychloride (1.36 mL, 14.6 mmol) in
dry THF (40 mL), during 20 min. at 0 °C. The ice bath was re-
moved and the reaction mixture was stirred additional 1.5 h at
rt. The reaction mixture was then concentrated under reduced
pressure. The residual oil was slowly poured into acidified
cracked ice from 0.5 N HCl (20 mL). The solid part was separated
by filtration and washed with cold water (2 ꢃ 50 mL). The crude
product was dried 2 h under 7 mbar vacuum to yield crude dihy-
drogen phosphate 3c as a white solid (2.5 g, 75%, HPLC ꢄ60%). An
analytical sample was purified by preparative LC (HPLC >99%). Mp
242–243 °C; 1H NMR (400 MHz, DMSO-d6) d 5.41 (s,1H), 3.75 (m,
1H), 2.61 (d, 1H), 2.36 (s, 1H), 2.08–2.04 (m, 2H), 1.87–1.54 (m,
2H),1.51 (m, 1H), 1.40–1.20 (m, 5H), 1.35 (s, 3H), 1.16–0.84 (m,
3H), 1.09 (s, 3H), 1.04 (s, 3H), 1.03 (s, 3H), 0.94 (s, 3H), 0.83–
0.66 (m, 4H), 0.75 (s, 3H); 13C NMR (100 MHz, DMSO-d6) d
199.41, 178.12, 170.27, 127.69, 83.21, 83.15, 61.36, 54.41, 48.49,
45.29, 43.51, 43.36, 41.07, 38.97, 38.91, 38.49, 37.95, 36.85,
32.45, 31.96, 30.80, 28.82, 28.27, 26.51, 26.23, 25.13, 23.42,
130 °C (Ref. 45: 131–133); ½a D20
ꢂ
+141.5 (c 0.018, CHCl3) (Ref. 45:
137.5°). 1H NMR (400 MHz, CDCl3) d 7.42–7.33 (m, 5H), 5.56 (s,
1H), 5.22 (d, J = 13.3 Hz, 1H), 5.10 (d, J = 13.2 Hz, 1H), 3.24 (dd,
J = 10.7, 5.7 Hz, 1H), 2.80 (dd, J = 13.4, 3.2 Hz, 1H), 2.33 (s, 1H),
2.07–1.96 (m, 3H), 1.82 (d, J = 4.1 Hz, 1H), 1.69–1.59 (m, 5H),
1.56–1.49 (m, 1H), 1.40–1.29 (m, 3H), 1.36 (s, 3H), 1.20–1.12 (m,
1H), 1.18 (s, 3H), 1.15 (s, 3H), 1.12 (s, 3H), 1.04–0.96 (m, 2H),
1.02 (s, 3H), 0.91–0.84 (m, 1H), 0.82 (s, 3H), 0.75 (s, 3H), 0.73–
0.69 (d, J = 10.3 Hz, 1H); 13C NMR (100 MHz, CDCl3) d 200.18,
176.22, 169.05, 136.12, 128.62, 128.52, 128.31, 128.25, 78.75,
66.23, 61.78, 54.92, 48.20, 45.35, 43.99, 43.17, 41.07, 39.14,
37.65, 37.06, 32.75, 31.78, 31.17, 28.43, 28.30, 28.11, 27.30,
26.47, 26.39, 23.36, 18.66, 17.49, 16.38, 15.60. IR (DCM) m: 3744,
3605, 3057, 2983, 2941, 2871, 1721, 1656, 1459, 1424, 1389,
1212, 1166, 1154, 1085, 1038, 992, 895. MS-CI, m/z (Irel. (%)):
561.12 [M+1] (100); 562.12 (42); 563.12 (10); 107.93 (9); 560.13
(7); 427.20 (7); 425.20 (2); 106.16 (2); 574.45 (2); 566.73 (2).
3.6. (3b,18b,20b)-3-(Acetyloxy)-11-oxo-olean-12-en-29-oic acid
(3a)
22.53, 18.77, 17.55, 16.69, 16.61. IR (MeOH) m: 3365, 2950,
2875, 1702, 1656, 1563, 1544, 1463, 1389, 1328, 1212, 1177,
Glycyrrhetinic acid (10.0 g, 21.3 mmol) was heated under reflux
with Ac2O (160 mL, 1.7 mol) for 1.5 h. AcOH (20 mL) and H2O
(40 mL) were then added to the still hot solution. The product,
which crystallized upon cooling was filtered off and washed with
cold H2O (4 ꢃ 50 mL) and Et2O (15 mL) and dried in vacuo to give
O-acetyl 3a as a white solid (10.5 g, 96.4%, HPLC >99%). Mp 310–
1019, 1000, 880, 841.
3.9. (3b,18b,20b)-3-(3-Carboxy-1-oxopropoxy)-11-oxo-olean-
12-en-29-oic acid (3d)
A mixture of acid 1 (2.00 g, 4.25 mmol), succinic anhydride
(2.13 g, 21.25 mmol) and DMAP (1.04 g, 8.50 mmol) in dry pyridine
(50 mL) was refluxed for 24 h. After cooling, the mixture was acid-
ified with 2 N HCl (300 mL) to pH ꢄ3 and extracted with EtOAc
(300 mL), the organic phase washed with cold water (150 mL)
and dried over Na2SO4, filtered and the mixture was evaporated
in vacuo to give a white powder. The crude product was crystal-
lized from MeOH (20 mL) to give 3d as a white solid (2.04 g,
83.8%, HPLC >99%). Mp 302–303 °C (Ref. 31: 313–315 °C); 1H
NMR (400 MHz, DMSO-d6) d 5.15, 4.23–4.11 (m, 1H), 2.26–2.09
(m, 6H), 1.83–1.75 (m, 1H), 1.61–0.87 (m, 10H), 1.10 (s, 3H),
0.85–0.61 (m, 4H), 0.83 (s, 3H), 0.80 (s, 3H), 0.77 (s, 3H), 0.57–
0.41 (m, 6H), 0.56 (s, 3H), 0.55 (s, 3H), 0.49 (s, 3H); 13C NMR
(100 MHz, DMSO-d6) d 200.6, 179.3, 175.1, 173.3, 171.5, 128.9,
81.4, 62.5, 55.4, 49.7, 46.5, 44.7, 44.6, 39.6, 39.3, 39.2, 38.2, 33.6,
33.2, 32.0, 30.9, 30.5, 30.1, 29.5, 29.3, 27.8, 27.5, 24.9, 24.7, 24.1,
313 °C (Ref. 46: 319–321 °C); ½a D20
ꢂ
+163.3 (c 1, CHCl3). 1H NMR
(400 MHz, CDCl3) d 5.73 (s, 1H), 4.53 (dd, J = 11.5, 5.2 Hz, 1H),
2.80 (d, J = 13.9 Hz, 1H, 2.38 (s, 1H), 2.19 (d, J = 13.1 Hz, 1H), 2.07
(s, 3H), 2.06–1.55 (m, 12H), 1.48–1.02 (m, 7H), 1.38 (s, 3H), 1.24
(s, 3H), 1.18 (s, 3H), 1.14 (s, 3H), 0.89 (s, 6H), 0.85 (s, 3H); 13C
NMR (100 MHz, CDCl3) d 200.4, 181.8, 171.1, 169.6, 128.4, 80.6,
61.7, 55.0, 48.2, 45.4, 43.8, 43.1, 40.8, 38.7, 38.0, 37.7, 36.9, 32.7,
31.8, 30.8, 28.5, 28.4, 28.0, 26.4, 26.3, 23.5, 23.3, 21.3, 18.6, 17.3,
16.6, 16.3. MS-CI, m/z (Irel. (%)): 513.12 [M+1] (100); 514.15 (34);
469.22 (7); 515.14 (7); 191.14 (5); 189.08 (4); 135.00 (4); 175.09
(3); 262.04 (3); 303.07 (3).
3.7. (3b,18b,20b)-3-[(Trimethylsilyl)oxy]-11-oxo-olean-12-en-
29-oic acid (3b)
To a solution of glycyrrhetinic acid (0.65 g, 1.38 mmol) in dry
pyridine (25 mL) were added hexamethyldisilazane (1.11 g,
6.91 mmol) and trimethylsilyl chloride (0.75 g, 6.91 mmol). The
mixture was allowed to stand for 3 h under nitrogen atmosphere.
After the mixture had been cooled, the solid was collected by filtra-
20.0, 18.6, 18.2, 17.8. IR (DCM) m: 3740, 2956, 2871, 1729, 1660,
1497, 1455, 1386, 1362, 1328, 1312, 1208, 1154, 1081, 1030,
980, 965, 888. MS-CI, m/z (Irel. (%)): 453.00 (100); 167.03 (52);
454.00 (33); 469.73 (14); 135.01 (11); 189.04 (10); 135.96 (8);
234.92 (8); 168.01 (8); 409.07 (7).

I. Beseda et al. / Bioorg. Med. Chem. 18 (2010) 433–454
445
3.10. (3b,18b,20b)-3-(4-Hydroxy-3,3-dimethyl-1,4-
dioxobutoxy)-11-oxo-olean-12-en-29-oic acid (3e)
rated in vacuo and the crude product was crystallized from MeOH
(20 mL) to give O-acetyl 4c as a white solid (1.01 g, 94.7%, HPLC
>99%). Mp 130–132 °C; 1H NMR (300 MHz, CDCl3) d 7.40–7.25
(m, 10H), 6.93 (s, 1H), 5.51 (s, 1H), 4.52 (dd, J = 11.5, 5.2 Hz, 1H),
2.80 (dd, J = 13.5, 3.4 Hz, 1H), 2.34 (s, 1H), 2.05 (s, 3H), 2.04–1.94
(m, 4H), 1.75–1.55 (m, 7H), 1.38–1.24 (m, 4H), 1.36 (s, 3H), 1.17
(s, 3H), 1.16 (s, 3H), 1.09 (s, 3H), 1.07–0.94 (m, 3H), 0.88 (s, 3H),
0.88 (s, 3H), 0.80 (d, J = 9.5 Hz, 1H), 0.66 (s, 3H); 13C NMR
(75 MHz, CDCl3) d 199.9, 175.2, 171.0, 168.9, 140.1, 140.0, 128.6,
128.6, 128.5, 128.5, 128.1, 128.1, 127.8, 127.3, 127.3, 127.0,
127.0, 80.6, 76.6, 61.7, 55.0, 48.1, 45.3, 44.0, 43.2, 41.1, 38.8, 38.0,
37.5, 36.9, 32.7, 31.7, 31.2, 28.3, 28.2, 28.0, 26.4, 26.3, 23.6, 23.3,
Compound 1 (1.00 g, 2.13 mmol) was heated with 2,2-dim-
ethylsuccinic anhydride (1.09 g, 8.50 mmol) and DMAP (0.52 g,
4.25 mmol) in pyridine (60 mL) to reflux overnight. After cooling,
the mixture was acidified with 2 N HCl (300 mL) to pH ꢄ3 and ex-
tracted with EtOAc (300 mL), the organic phase washed with cold
water (150 mL) and dried over Na2SO4, filtered and the mixture
was evaporated in vacuo to give a white powder. Purification by
flash chromatography (DCM/MeOH, gradient elution 0–5%) gave
3e as a white solid (0.65 g, 50.9%, HPLC >98%). Mp 301–304 °C;
1H NMR (400 MHz, DMSO-d6) d 5.41 (s, 1H), 4.42 (dd, J = 11.5,
4.5 Hz, 1H), 2.64 (m, 1H), 2.57–2.47 (m, 2H), 2.13–2.04 (m, 2H),
1.83–1.77 (m, 1H), 1.73–1.62 (m, 4H), 1.52–1.43 (m, 2H), 1.41–
1.32 (m, 5H), 1.36 (s, 3H), 1.30–1.21 (m, 2H), 1.19–1.13 (m, 1H),
1.18 (s, 3H), 1.17 (s, 3H), 1.10 (s, 3H), 1.08–1.02 (m, 2H), 1.06 (s,
3H), 1.04 (s, 3H), 0.96 (d, J = 10.7 Hz, 1H), 0.91–0.84 (m, 2H), 0.82
(s, 3H), 0.82 (s, 3H), 0.76 (s, 3H); 13C NMR (100 MHz, DMSO-d6) d
199.3, 178.2, 178.1, 171.0, 170.2, 127.7, 80.3, 61.3, 54.2, 48.5,
45.3, 44.4, 43.5, 43.4, 41.1, 40.6, 40.1, 39.3, 38.4, 38.0, 36.9, 32.4,
32.0, 30.8, 28.8, 28.3, 28.1, 26.5, 26.3, 25.8, 25.7, 23.6, 23.5, 18.8,
21.3, 18.7, 17.4, 16.7, 16.4. IR (DCM) m: 3740, 3057, 2956, 2871,
1729, 1683, 1656, 1621, 1559, 1540, 1455, 1389, 1366, 1320,
1212, 1150, 1085, 1030, 984, 899, 865. MS-CI, m/z (Irel. (%)):
167.02 (100); 181.96 (38); 168.00 (16); 178.04 (14); 678.79 [M]
(12); 513.00 (10); 183.02 (10); 679.83 (6); 183.90 (6); 165.03 (5).
Anal. Calcd for C45H58O5: C, 79.61; H, 8.61; O, 11.78. Found: C,
79.57; H, 8.57; O, 11.75.
3.14. (3b,18b,20b)-2-[(4-Methylphenyl)sulfonyl]hydrazide-3-
(acetyloxy)-11-oxo-olean-12-en-29-oic acid (5a)
17.1, 16.6. IR (DCM)
m: 3057, 2976, 2941, 2875, 1702, 1656,
1459, 1389, 1324, 1212, 1158, 1139, 992, 946, 895, 880. MS-CI,
m/z (Irel. (%)): 453.13 (100); 282.12 (68); 454.09 (33); 298.22
(16); 300.28 (14). 283.12 (12); 471.14 (12); 272.37 (10); 299.05
(10); 244.88 (9).
To a ice cold solution of tosylhydrazine (160 mg, 0.86 mmol) in
dry pyridine (0.25 mL) there was slowly added a solution of 4a
(300 mg, 0.57 mmol) in dry THF (2 mL). The mixture was left
standing for 24 h at rt, then poured into 5% HCl (4 mL). The aque-
ous solution was extracted with DCM (3 ꢃ 20 mL) and the organic
layers were washed with water (10 mL), dried over Na2SO4, filtered
and concentrated to dryness to obtain the crude product as white
solid. Purification by flash chromatography (PE–EtOAc gradient
elution, 10–50%) gave 5a as a white solid (120 mg, 31%, HPLC
>97%). Mp 245–246 °C (Ref. 48: 245 °C). 1H NMR (400 MHz, CDCl3)
d 8.62 (d, J = 8.2 Hz, 2H), 7.31 (d, J = 8.2 Hz, 2H), 5.62 (s, 1H), 4.52
(dd, J = 11.6, 4.8 Hz, 1H), 2.79 (d, J = 13.5 Hz, 1H), 2.41 (s, 3H),
2.35 (s, 1H), 2.07 (s, 3H), 2.00–1.89 (m, 1H), 1.87–1.55 (m, 9H),
1.49–1.38 (m, 2H), 1.34–1.28 (m, 1H), 1.32 (s, 3H), 1.27–1.18 (m,
2H), 1.18 (s, 3H), 1.14 (s, 3H), 1.15–1.05 (m, 1H), 1.04 (s, 3H),
0.96 (d, J = 14.0 Hz, 2H), 0.89 (s, 3H), 0.89 (s, 3H), 0.81 (d,
J = 11.1 Hz, 1H), 0.69 (s, 3H); 13C NMR (100 MHz, CDCl3) d 200.3,
174.8, 171.1, 169.0, 144.8, 133.6, 129.7, 129.0, 128.6, 80.6, 61.7,
55.0, 47.5, 45.4, 43.1, 42.9, 40.5, 38.8, 38.0, 37.0, 36.9, 32.6, 31.6,
30.7, 29.2, 28.0, 28.0, 26.4, 26.3, 23.5, 23.4, 21.7, 21.3, 18.7, 17.4,
3.11. (3b,18b,20b)-3-(Acetyloxy)-11-oxo-olean-12-en-29-oyl
chloride (4a)
Compound 3a (3.0 g, 5.9 mmol) was added to thionyl chloride
(7.0 g, 59 mmol) and the mixture was refluxed for 1 h. After evap-
oration of excess thionyl chloride, there was added dry toluene
(30 mL). The mixture was evaporated in vacuo to give 4a as a yel-
low powder (3.1 g, 99%). Mp 289–292 °C (Ref. 47: 297–303 °C). The
crude product was used as such.
3.12. (3b,18b,20b)-3-(Acetyloxy)-11-oxo-olean-12-en-29-oic
acid methyl ester (4b)
Compound 2a (1.00 g, 2.06 mmol) was heated under reflux with
Ac2O (20 mL, 211.60 mmol) for 1.5 h. The solvent was evaporated
in vacuo and the crude product was purified by flash chromatogra-
phy (PE–EtOAc gradient elution, 5–20%) to yield 4b as a white solid
(0.95 g, 87.4%, HPLC >99%). Mp 282.5–283.5 °C (Ref. 1: 300–
301 °C); 1H NMR (300 MHz, CDCl3) d 5.63 (s, 1H), 4.48 (dd,
J = 10.9, 5.6 Hz, 1H), 3.65 (s, 3H), 2.76 (dd, J = 13.5, 3.4 Hz, 1H),
2.33 (s, 1H), 2.10–1.91 (m, 4H), 2.01 (s, 3H), 1.88–1.83 (m, 1H),
1.72–1.49 (m, 6H), 1.44–1.24 (m, 5H), 1.33 (s, 3H), 1.20–1.13 (m,
1H), 1.12 (s, 3H), 1.11 (s, 3H), 1.09 (s, 3H), 1.04–0.91 (m, 2H),
0.84 (s, 6H), 0.77 (s, 3H); 13C NMR (75 MHz, CDCl3) d 200.0,
176.9, 170.9, 169.2, 128.4, 80.6, 61.7, 55.0, 51.7, 48.4, 45.4, 44.0,
43.1, 41.0, 38.7, 38.0, 37.7, 36.9, 32.7, 31.8, 31.1, 28.5, 28.3, 28.0,
16.7, 16.4. IR (DCM)
m: 3740, 3616, 3057, 2987, 2879, 1737,
1652, 1459, 1424, 1397, 1343, 1169, 1092, 1030, 984, 895. MS-CI,
m/z (Irel. (%)): 537.67 (100); 291.17 (67); 84.06 (46); 59.13 (41);
377.13 (39); 100.94 (20); 840.27 (17); 97.99 (9); 128.04 (9);
416.94 (9).
3.15. (3b,18b,20b)-29-Amino-11,29-dioxo-olean-12-en-3-yl
acetate (5b)
A cold solution of 4a (531 mg, 1.0 mmol) in dry DCM (20 mL)
was slowly bubbled with gaseous NH3. The mixture was stirred
for 15 min and monitored by TLC, till completed. The reaction mix-
ture was stirred additional 30 min. The organic layer was washed
with water (20 mL), dried with Na2SO4 and the solvent was re-
moved in vacuo to obtain 5b as a white solid (490 mg, 95%, HPLC
>96%). Mp 312–314 °C. 1H NMR (400 MHz, CDCl3) d 6.12 (br, 2H),
5.68 (s, 1H), 4.52 (dd, J = 11.6, 4.8 Hz, 1H), 2.78 (dd, J = 13.5,
3.4 Hz, 1H), 2.35 (s, 1H), 2.23–2.17 (m, 1H), 2.05 (s, 3H), 2.00–
0.99 (m, 17H), 1.36 (s, 3H), 1.19 (s, 3H), 1.15 (s, 3H), 1.12 (s, 3H),
0.91–0.78 (m, 1H), 0.87 (s, 3H), 0.86 (s, 3H), 0.83 (s, 3H); 13C
NMR (100 MHz, CDCl3) d 200.1, 179.3, 171.0, 169.3, 128.5, 80.6,
61.7, 55.0, 48.1, 45.4, 43.2, 41.7, 38.8, 38.0, 37.4, 36.9, 36.9, 32.7,
31.9, 31.3, 29.5, 28.4, 28.0, 26.4, 26.4, 23.5, 23.3, 21.3, 18.7, 17.3,
26.4, 26.4, 23.5, 23.3, 21.3, 18.6, 17.3, 16.7, 16.4. IR (DCM)
m:
3740, 3057, 2956, 2875, 1729, 1656, 1621, 1459, 1436, 1389,
1366, 1320, 1250, 1216, 1193, 1158, 1088, 1030, 988, 899. MS-CI,
m/z (Irel. (%)): 526.98 [M+1] (100); 527.99 [M+1] (30); 467.02
(25); 189.01 (20); 135.01 (17); 191.04 (15); 499.02 (15); 292.94
(11); 135.93 (11); 175.01 (11).
3.13. (3b,18b,20b)-3-(Acetyloxy)-11-oxo-olean-12-en-29-oic
acid diphenylmethyl ester (4c)
Compound 2b (1.00 g, 1.57 mmol) was heated under reflux with
Ac2O (25 mL, 264.50 mmol) for 50 min. The solvent was evapo-

446
I. Beseda et al. / Bioorg. Med. Chem. 18 (2010) 433–454
16.7, 16.4. IR (DCM)
m
: 3362, 3203, 2366, 2335, 1725, 1656, 1617,
(100 MHz, CDCl3) d 200.8, 180.2, 177.7, 165.8, 123.9, 78.6, 60.6,
54.9, 50.3, 45.3, 44.8, 44.2, 43.8, 43.5, 43.1, 42.6, 42.3, 40.5, 37.7,
39.0, 37.5, 37.0, 36.8, 35.4, 34.6, 31.9, 29.5, 28.2, 24.8, 23.7, 21.2,
19.2, 16.7, 16.3.
1459, 1389, 1366, 1328, 1208, 1142, 1088, 1030, 1000, 984, 903,
884, 865. MS-CI, m/z (Irel. (%)): 512.18 [M+1] (100); 527.54 (91);
528.54 (69); 526.36 (38); 513.21 (37); 511.22 (32); 523.98 (25);
521.93 (21); 529.21 (17); 510.2 (17).
3.19. (3b,18b,20b)-3-(Acetyloxy)-11-oxo-olean-12-en-29-al (6)
3.16. (3b,18b,20b)-29-Hydrazino-11,29-dioxo-olean-12-en-3-yl
acetate (5c)
Compound 5a (1.00 g, 1.45 mmol) was heated in ethylene gly-
col (15 mL) to 160 °C. To this solution was quickly added Na2CO3
(0.47 g, 4.40 mmol). After a minute, the mixture was poured on
ice (40 g), extracted with chloroform (3 ꢃ 60 mL), the combined
organic layers dried over Na2SO4, filtered and evaporated in va-
cuo. The crude material was crystallized from MeOH (15 mL) to
give 6 as a white solid (0.25 g, 36.4%). Mp 232–235 °C. 1H NMR
(200 MHz, CDCl3) d 9.35 (s, 1H), 5.60 (s, 1H), 4.45 (dd, J = 11.6,
4.8 Hz, 1H), 2.73 (dd, J = 13.4, 3.2 Hz, 1H), 2.29 (s, 1H), 2.06–
1.87 (2H), 1.98 (s, 3H), 1.86–1.64 (m, 4H), 1.63–1.45 (m, 5H),
1.40–1.22 (m, 4H), 1.32 (s, 3H), 1.19–1.02 (m, 2H), 1.09 (s,
3H), 1.05 (s, 3H), 1.03–0.94 (m, 1H), 0.91 (s, 3H), 0.81 (s, 3H),
0.81 (s, 3H), 0.79–0.72 (m, 1H), 0.73 (s, 3H); 13C NMR
(50 MHz, CDCl3) d 205.6, 199.9, 170.9, 168.6, 128.5, 80.6, 61.7,
55.0, 47.6, 46.8, 45.4, 43.2, 38.8, 38.3, 38.0, 37.1, 36.9, 32.6,
31.9, 28.4, 28.3, 28.0, 26.3, 26.1, 24.0, 23.6, 23.5, 21.3, 18.6,
17.3, 16.6, 16.4.
To a cold solution of NH2NH2ꢁH2O (250 mg, 5.0 mmol) in dry
THF (20 mL) was dropwise added 4a (0.531 mg, 1 mmol) in dry
THF (5 mL). The reaction mixture was stirred at rt for hour. The or-
ganic layer was washed with water (20 mL), dried with Na2SO4 and
the solvent was removed in vacuo to obtain crude product. This
was crystallizes from toluene–petrolether mixture to give 5c as a
white solid (250 mg, 47%, HPLC ꢄ94%). Mp >175 °C. 1H NMR
(200 MHz, CDCl3) d 5.60 (s, 1H), 4.49 (dd, J = 10.8, 5.3 Hz, 1H),
2.76 (d, J = 13.4 Hz, 1H), 2.32 (s, 1H), 2.10–0.98 (m, 20H), 2.03 (s,
3H), 1.35 (s, 3H), 1.20 (s, 3H), 1.13 (s, 3H), 1.09 (s, 3H), 0.85 (s,
6H), 0.78 (s, 3H); 13C NMR (50 MHz, CDCl3) d 200.0, 178.8, 171.0,
169.1, 128.4, 80.6, 61.7, 55.0, 48.0, 46.7, 45.3, 43.2, 41.9, 41.1,
38.8, 38.0, 36.9, 32.7, 31.8, 29.6, 29.5, 28.4, 28.0, 26.4, 25.5, 23.5,
23.3, 21.3, 18.6, 17.3, 16.6, 16.4. IR (DCM) m: 3300, 2366, 2343,
1729, 1656, 1544, 1525, 1509, 1459, 1366, 1324, 1212, 1169,
1142, 1088, 1030, 984, 903, 880, 861. MS-CI, m/z (Irel. (%)):
567.17 (100); 568.18 (41); 469.15 (36); 527.14 (32); 467.14 (16);
528.14 (16); 113.08 (15); 543.30 (13); 566.20 (12); 58.06 (11).
3.20. (3b,18b,20b)-3-Hydroxy-11-oxo-olean-12-en-29-al oxime
(7)
3.17. (3b,18b,20b)-29-(Allylamino)-11,29-dioxo-olean-12-en-3-
yl acetate (5d)
To a cold solution of 6 (697 mg, 1.0 mmol) in dry pyridine
(7 mL) was slowly added a solution of NH2OHꢁHCl in dry pyridine
(3 mL) and stirred for 12 h at rt. The reaction mixture was diluted
with DCM (100 mL), washed with 10% HCl (3 ꢃ 50 mL) and water
(50 mL). The combined organic layers were dried over Na2SO4, fil-
tered and roto-evaporated to give 7 as a white solid (160 mg, 31%,
HPLC ꢄ95%). 1H NMR (200 MHz, CDCl3) d 7.19 (s, 1H), 5.57 (s, 1H),
4.49 (m, 1H), 2.70 (m, 1H), 2.35–0.65 (m, 21H), 1.98 (s, 3H), 1.30 (s,
3H), 1.15 (s, 3H), 1.09 (s, 3H), 0.98 (s, 3H), 0.80 (s, 6H), 0.75 (s, 3H);
13C NMR (50 MHz, CDCl3) d 200.0, 171.1, 169.4, 156.7, 128.4, 80.6,
61.6, 55.0, 47.1, 45.4, 43.2, 41.3, 38.8, 38.0, 37.6, 36.9, 36.6, 32.6,
32.1, 31.9, 29.2, 28.4, 28.0, 26.4, 26.3, 23.5, 23.4, 21.3, 18.6, 17.3,
16.7, 16.4. Anal. Calcd for C32H49NO4: C, 75.11; H, 9.65; N, 2.74.
Found: C, 74.95; H, 9.82; N, 2.40.
To a stirred solution of allylamine (90 mg, 1.6 mmol) and TEA
(300 mg, 3 mmol) in DCM (25 mL) were added 4a (531 mg,
1 mmol) in small portions. After stirring for 12 h at rt, the solution
was extracted with 1 N HCl (3 ꢃ 10 mL) and water (1 ꢃ 10 mL) and
sat. NaHCO3 (1 ꢃ 10 mL). The organic layer was dried with Na2SO4
and the solvent was removed in vacuo to obtain crude product.
This was crystallizes from EtOH (3 mL)–petrolether (24 mL) mix-
ture to give 5d as a white solid (230 mg, 42%, HPLC >97%). Mp
230–231 °C. 1H NMR (200 MHz, CDCl3) d 5.90 (br, 1H), 5.80 (m,
1H), 5.62 (s, 1H), 5.13 (m, 1H), 4.5 (m, 1H), 3.87 (m, 1H), 2.7 (m,
1H), 2.03 (m, 1H), 2.38–0.70 (m, 22H), 2.08 (s, 3H), 1.34 (s, 3H),
1.13 (s, 6H), 1.10 (s, 3H), 0.86 (s, 6H), 0.80 (s, 3H); 13C NMR
(50 MHz, CDCl3) d 199.9, 175.5, 170.9, 169.3, 134.5, 128.4, 116.3,
80.6, 61.7, 54.9, 48.1, 45.3, 43.6, 43.2, 41.9, 41.7, 38.7, 38.0, 37.4,
36.9, 32.6, 31.9, 31.4, 29.6, 28.4, 28.0, 26.4, 26.3, 23.4, 23.3, 21.2,
3.21. (18b,20b)-3,11-Dioxo-olean-12-en-29-oic acid methyl
ester (8a)
18.6, 17.3, 16.6, 163. IR (DCM)
m
: 3358, 2362, 2339, 1725, 1656,
To a solution of 2a (6.0 g, 12.4 mmol) in DCM (0.5 L) was added
PCC (4.0 g, 18.6 mmol). The mixture was stirred for 2 h at rt, mon-
itored by TLC. The mixture was diluted with Et2O (0.5 L) and fil-
tered through a short pad (100 g) of silica. The pad was eluted
once with Et2O (0.5 L), the combined eluates concentrated under
vacuum and dried to give 8a as colorless crystals (4.9 g, 82%). Mp
1525, 1459, 1432, 1424, 1389, 1366, 1324, 1173, 1169, 1142,
1088, 1030, 984, 919, 880, 865. HRMS, m/z: 552.4044 [M+1] (calcd
for C35H54O4N, 552.4044).
3.18. (3b,18b,20b)-29-[(2-Aminoethyl)amino]-11,29-dioxo-
olean-12-en-3-yl acetate (5e)
242–243 °C; (Ref. 49: 242 °C); ½a D20
ꢂ
+182.1 (c 0.2, CHCl3) (Ref. 49:
181.5°). 1H NMR (200 MHz, CDCl3) d 5.63 (s, 1H), 3.63 (s, 3H),
2.97–2.82 (m, 1H), 2.66–2.48 (m, 1H), 2.41–2.21 (m, 1H), 2.37 (s,
1H), 2.11–1.14 (m, 16H), 1.30 (s, 3H), 1.20 (s, 3H), 1.10 (s, 3H),
1.08 (s, 3H), 1.03 (s, 3H), 1.00 (s, 3H), 0.95–0.89 (m, 1H), 0.75 (s,
3H); 13C NMR (50 MHz, CDCl3) d 217.1, 199.4, 176.9, 169.7,
128.4, 61.0, 55.4, 51.8, 48.4, 47.7, 45.2, 44.0, 43.3, 41.1, 39.7,
37.7, 36.7, 34.2, 32.1, 31.8, 31.1, 28.5, 28.3, 26.5, 26.4, 26.3, 23.3,
To a cold solution of 4a (531 mg, 1.0 mmol) in dry DCM (20 mL)
was slowly added a solution of ethane-1,2-diamine (600 mg,
10 mmol) in dry DCM (10 mL). The mixture was left for 24 h at
rt. The organic layer was washed with water (10 mL), dried with
Na2SO4 and the solvent was removed in vacuo to obtain crude
product as white solid. Purification by flash chromatography
(DCM–MeOH gradient elution, 1–5%) give 5e as a white solid
(250 mg, 45%, HPLC >98%). Mp >210 °C. 1H NMR (400 MHz, CDCl3)
d 6.92 (br, 1H), 5.57 (s, 1H), 3.47–3.34 (m, 4H), 3.23–3.18 (m, 1H),
2.64 (dd, J = 13.5, 3.4 Hz, 1H), 2.30–2.17 (m, 6H), 2.07–0.93 (m,
18H), 1.33 (s, 3H), 1.19 (s, 3H), 1.17 (s, 3H), 1.10 (s, 3H), 0.98 (s,
3H), 0.90–0.64 (m, 1H), 0.79 (s, 3H), 0.68 (s, 3H); 13C NMR
21.4, 18.8, 18.5, 15.6. IR (DCM) m: 3740, 3682, 3624, 3057, 2976,
2945, 2875, 1721, 1702, 1656, 1459, 1436, 1389, 1366, 1320,
1220, 1208, 1193, 1158, 1112, 1088, 1000, 980, 895. +1, m/z (Irel.
(%)): 483.01 [M] (100); 484.02 (34); 485.04 (21); 499.11 (14).
97.15 (10); 498.16 (9); 135.03 (7); 482.09 (6); 486.05 (6); 189.07
(6).

I. Beseda et al. / Bioorg. Med. Chem. 18 (2010) 433–454
447
3.22. (18b,20b)-3,11-Dioxo-olean-12-en-29-oic acid
diphenylmethyl ester (8b)
128.6, 128.6, 128.5, 128.5, 128.1, 127.8, 127.3, 127.0, 127.0,
126.8, 126.8, 76.6, 61.2, 55.6, 48.1, 45.3, 44.0, 43.2, 41.1, 40.5,
39.0, 37.5, 37.1, 32.4, 31.7, 31.1, 28.3, 28.2, 27.1, 26.4, 26.3, 23.3,
To a solution of 2b (8.0 g, 10 mmol) in DCM (600 mL) was added
PCC (4.0 g, 15 mmol). After stirring for 3.5 h at 40–45 °C (conver-
sion was checked by TLC for completeness of turnover), the mix-
ture was filtered through a short pad (100 g) of silica. The pad
was eluted once with Et2O (0.8 L)). The combined eluates were
concentrated on a rotavapor to give 8b as a white solid (7.7 g,
96%, HPLC >97%). Mp 103–104 °C. 1H NMR (400 MHz, CDCl3) d
7.45–7.22 (m, 10H), 6.95 (s, 1H), 5.57 (s, 1H), 3.03–2.95 (m, 1H),
2.71–2.61 (m, 1H), 2.44 (s, 1H), 2.42–2.35 (m, 1H), 2.11–2.01 (m,
3H), 1.88–1.78 (m, 1H), 1.75–1.53 (m, 4H), 1.48–1.26 (m, 7H),
1.39 (s, 3H), 1.29 (s, 3H), 1.25–1.18 (m, 1H), 1.20 (s, 3H), 1.15 (s,
3H), 1.12 (s, 3H), 1.09 (s, 3H), 1.05–0.98 (m, 1H), 0.70 (s, 3H); 13C
NMR (100 MHz, CDCl3) d 217.3, 199.4, 175.2, 169.4, 140.1, 140.0,
128.7, 128.7, 128.5, 128.5, 128.4, 128.2, 127.9, 127.2, 127.2,
127.0, 127.0, 76.7, 61.0, 55.4, 48.1, 47.8, 45.2, 44.0, 43.3, 41.2,
39.8, 37.5, 36.7, 34.3, 32.1, 31.8, 31.2, 28.4, 28.2, 26.5, 26.4, 26.4,
23.2, 18.6, 18.2, 17.4, 15.5. IR (DCM) m: 3740, 3620, 3061, 2983,
2941, 2875, 1729, 1656, 1459, 1424, 1389, 1316, 1212, 1154,
1085, 1015, 984, 922, 895, 733. MS-CI, m/z (Irel. (%)): 167.04
(100); 650.01 (24). 182.01 (18); 168.04 (14); 651.02 (14); 440.09
(13); 648.83 (11); 183.81 (9); 183.03 (8); 441.05 (7). Anal. Calcd
for C43H55NO4: C, 79.47; H, 8.53; N, 2.16. Found: C, 79.28; H,
8.34; N, 1.88.
3.25. (18b,20b)-3-(Methoxyimino)-11-oxo-olean-12-en-29-oic
acid diphenylmethyl ester (9c)
A solution of 8b (4.0 g, 6.3 mmol) and O-methylhydroxylamine
hydrochloride (2.63 g, 31.5 mmol) in dry pyridine (30 mL) was
heated for 2.5 h at 50 °C. After cooling to room temperature the
reaction mixture was diluted DCM (400 mL), washed with 10%
HCl (3 ꢃ 100 mL) and water (50 mL). The combined organic layers
were dried over Na2SO4, filtered and roto-evaporated to give 9c as
a white solid (4.0 g, 95.6%, HPLC >98%). Mp 105–110 °C. 1H NMR
(200 MHz, CDCl3): d 7.33–7.15 (m, 10H), 6.86 (s, 1H), 5.44 (s,
1H), 3.73 (s, 3H), 2.93–2.69 (m, 2H), 2.27 (s, 1H), 2.23–2.07 (m,
1H), 2.06–1.85 (m, 4H), 1.66–1.47 (m, 4H), 1.37–1.20 (m, 4H),
1.26 (s, 3H), 1.17–1.02 (m, 2H), 1.15 (s, 3H), 1.09 (s, 3H), 1.09 (s,
3H), 1.04 (s, 3H), 1.01–0.85 (m, 3H), 0.98 (s, 3H), 0.59 (s, 3H); 13C
NMR (50 MHz, CDCl3) d 199.7, 175.2, 169.0, 165.7, 140.1, 140.0,
128.6, 128.6, 128.5, 128.5, 128.1, 127.8, 127.3, 127.3, 127.0,
127.0, 76.6, 61.3, 61.0, 55.6, 48.1, 45.3, 44.0, 43.2, 41.1, 40.1,
39.1, 37.5, 36.9, 32.4, 31.7, 31.2, 28.3, 28.2, 27.3, 26.4, 26.4, 23.5,
23.3, 21.5, 18.8, 18.5, 15.7. IR (DCM) m: 3740, 2980, 2945, 2871,
1729, 1702, 1656, 1621, 1494, 1459, 1389, 1362, 1212, 1154,
1112, 1085, 1030, 1000, 980, 915, 749. MS-CI, m/z (Irel. (%)):
167.00 (100); 181.97 (94); 79.98 (84); 177.98 (47); 199.56 (35);
194.52 (20); 182.98 (19); 168.09 (16); 468.99 (13); 183.90 (10);
200.80 (10); 180.03 (8); 105.01 (8); 634.81 [M] (7). Anal. Calcd
for C43H54O4: C, 81.35; H, 8.57. Found: C, 81.08; H, 8.25.
3.23. (18b,20b)-3-(Hydroxyimino)-11-oxo-olean-12-en-29-oic
acid methyl ester (9a)
A solution of 8a (4.0 g, 8.3 mmol) and NH2OH.HCl (2.9 g,
41.4 mmol) in dry pyridine (30 mL) were heated for 2.5 h at
50 °C. After cooling to rt, the reaction mixture was diluted with
DCM (300 mL) and washed with 10% HCl (3 ꢃ 150 mL). The com-
bined organic layers were dried and concentrated on a rotavapor
to give 9a as colorless crystals (3.8 g, 92%, HPLC >99%). Mp 284–
285 °C. 1H NMR (200 MHz, CDCl3) d 5.61 (s, 1H), 3.62 (s, 3H),
3.07–2.94 (m, 1H), 2.88–2.75 (m, 1H), 2.31 (s, 1H), 2.30–2.22 (m,
1H), 2.15–1.69 (m, 6H), 1.67–1.31 (m, 8H), 1.27 (s, 3H), 1.24–
1.16 (m, 2H), 1.19 (s, 3H), 1.11 (s, 3H), 1.08 (s, 3H), 1.08 (s, 3H),
1.15–0.89 (m, 2H), 1.02 (s, 3H), 0.74 (s, 3H); 13C NMR (50 MHz,
CDCl3) d 199.8, 176.9, 169.4, 166.9, 128.4, 61.3, 55.6, 51.8, 48.4,
45.3, 44.0, 43.2, 41.1, 40.5, 39.1, 37.7, 37.0, 32.4, 31.8, 31.1, 28.5,
28.3, 27.1, 26.4, 26.4, 23.3, 23.2, 18.6, 18.2, 17.2, 15.7. IR (DCM)
23.3, 18.6, 18.2, 17.7, 15.6. IR (DCM) m: 3740, 3057, 2980, 2945,
2871, 2821, 1729, 1656, 1621, 1494, 1459, 1389, 1366, 1328,
1312, 1212, 1162, 1150, 1085, 1050, 984, 915, 880, 826. MS-CI,
m/z (Irel. (%)): 664.17 [M+1] (100); 167.05 (88); 665.15 (44);
454.18 (29); 182.02 (29); 168.13 (15); 452.21 (13); 183.13 (11);
666.15 (11); 199.53 (9). HRMS, m/z: 664.4352 [M+1] calcd for
C44H58O4N, 664.4366.
3.26. (18b,20b)-3-(Methoxyimino)-11-oxo-olean-12-en-29-oic
acid (9d)
Preparation as described for 9c. Crystallization from MeOH
gives product 9d as a white solid (93.2%, HPLC >98%). 1H NMR
(200 MHz, CDCl3): d 5.73 (s, 1H), 3.80 (s, 3H), 3.02–2.70 (m, 2H),
2.38 (s, 1H), 2.30–2.08 (m, 2H), 2.07–1.73 (m, 4H), 1.71–1.28 (m,
8H), 1.35 (s, 3H), 1.25–0.93 (m, 5H), 1.23 (s, 3H), 1.22 (s, 3H),
1.17 (s, 3H), 1.15 (s, 3H), 1.05 (s, 3H), 0.84 (s, 3H); 13C NMR
(50 MHz, CDCl3) d 200.2, 182.1, 169.8, 165.8, 128.3, 61.3, 61.0,
55.6, 48.2, 45.4, 43.8, 43.2, 40.8, 40.1, 39.0, 37.7, 36.9, 32.4, 31.8,
30.8, 28.5, 28.4, 27.2, 26.4, 26.4, 23.4, 23.3, 18.6, 18.2, 17.7, 15.6.
MS-CI, m/z (Irel. (%)): 498.16 [M+1] (100); 499.15 (33); 454.26
(17); 500.21 (7); 452.24 (6); 455.29 (5); 466.23 (3); 453.26 (2);
482.23 (2); 122.01 (2).
m
: 3300, 2362, 1729, 1660, 1459, 1389, 1366, 1316, 1277, 1216,
1189, 1158, 1115, 1088, 1050, 1027, 992, 946, 930, 880, 834,
803. MS-CI, m/z (Irel. (%)): 498.16 [M+1] (100); 499.13 (47);
482.26 (27); 485.18 (16); 83.23 (12). 500.12 (12); 497.07 (8);
486.31 (6); 484.23 (6); 480.26 (6).
3.24. (18b,20b)-3-(Hydroxyimino)-11-oxo-olean-12-en-29-oic
acid diphenylmethyl ester (9b)
A solution of 8b (4.0 g, 6.3 mmol) and NH2OH.HCl (2.2 g,
31.5 mmol) in dry pyridine (30 mL) was heated for 2.5 h at 50 °C.
After cooling to rt, the reaction mixture was diluted with DCM
(400 mL), washed with 10% HCl (3 ꢃ 150 mL) and water (50 mL).
The combined organic layers were dried over Na2SO4, filtered
and roto-evaporated to give 9b as a white solid (4.0 g, 98%, HPLC
ꢄ99%). Mp 215–216 °C. 1H NMR (200 MHz, CDCl3): d 7.40–7.27
(m, 10H), 6.94 (s, 1H), 5.54 (s, 1H), 3.08 (dd, J = 15.8, 4.3 Hz, 1H),
2.96–2.86 (m, 1H), 2.37 (s, 1H), 2.34–2.23 (m, 1H), 2.11–1.93 (m,
4H), 1.88–1.72 (m, 2H), 1.68–1.52 (m, 4H), 1.42–1.50 (m, 2H),
1.39–1.24 (m, 3H), 1.35 (s, 3H), 1.26 (s, 3H), 1.20 (s, 3H), 1.18 (s,
3H), 1.14–1.03 (m, 3H), 1.12 (s, 3H), 1.10 (s, 3H), 0.68 (s, 3H); 13C
NMR (50 MHz, CDCl3) d 199.7, 175.2, 169.2, 167.2, 140.1, 140.0,
3.27. (3b,18b,20b)-3-Amino-11-oxo-olean-12-en-29-oic acid
methyl ester (10a)
NaOAc (2.64 g, 32.2 mmol) was added portion wise to a TiCl3
solution (10.30 g of 12% in 10% HCl) and allowed to stand for 1 h.
The solution was diluted with water (2 mL) and bubbled with ar-
gon for 5 min. The solution was added dropwise to a solution of
oxime 9a (1.00 g, 2.0 mmol) and BH3ꢁtBuNH2 (0.43 g, 5.0 mmol)
in EtOH (20 mL) at 0 °C. The reaction mixture was allowed to come
to rt and stirred for 12 h. The reaction was monitored by TLC. The
reaction mixture was poured to a brine solution (150 mL) and
stirred for 10 min. This mixture was extracted with EtOAc

448
I. Beseda et al. / Bioorg. Med. Chem. 18 (2010) 433–454
(3 ꢃ 100 mL), washed with NaHCO3, brine, dried with Na2SO4, fil-
tered and evaporated under vacuum to obtain the white solid.
The crude product was subjected to SiO2 chromatography eluting
with CHCl3/MeOH+1%AcOH (0–20%) to give 10a as a white solid
(0.85 g, 87.5%, HPLC >95%). 1H NMR (200 MHz, CDCl3): d 5.65 (s,
1H), 3.70 (s, 3H), 3.24 (dd, J = 10.7, 5.7 Hz, 1H), 2.81 (d,
J = 13.7 Hz, 1H), 2.35 (s, 1H), 2.13–1.78 (m, 5H), 1.70–1.58 (m,
6H), 1.52–1.03 (m, 5H), 1.38 (s, 3H), 1.23–1.13 (m, 1H), 1.16 (s,
3H), 1.15 (s, 3H), 1.14 (s, 3H), 1.07–0.99 (m, 2H), 1.02 (s, 3H),
0.81 (s, 3H), 0.80 (s, 3H), 0.74–0.69 (m, 1H); 13C NMR (200 MHz,
CDCl3) d 200.3, 177.0, 169.2, 128.6, 78.6, 61.8, 54.9, 51.8, 48.4,
45.4, 44.1, 43.2, 41.1, 39.1, 39.1, 37.7, 37.1, 32.7, 31.8, 31.1, 28.5,
28.4, 28.1, 27.3, 26.5, 26.4, 23.4, 18.7, 17.5, 16.4, 15.6.
(50 MHz, CDCl3) d 200.3 (C-11), 175.2 (C-29), 168.8 (C-13), 140.1
(C-Ar), 140.1 (C-Ar), 128.6 (C-Ar), 128.6 (C-Ar), 128.6 (C-12), 128.4
(C-Ar), 128.4 (C-Ar), 128.1 (C-Ar), 127.8 (C-Ar), 127.3 (C-Ar), 127.3
(C-Ar), 127.0 (C-Ar), 127.0 (C-Ar), 76.6 (C-31), 61.8 (C-9), 56.2 (C-
5), 48.0 (C-3), 48.0 (C-18), 45.5 (C-14), 44.0 (C-8), 43.2 (C-20), 41.1
(C19), 37.5 (C-22), 37.4 (C-10), 36.8 (C-4), 33.6 (C-1), 32.7 (C-6),
31.7 (C-17), 31.2 (C-21), 28.8 (C-23), 28.3 (C-28), 28.3 (C-30), 26.4
(C-2), 26.4 (C-15), 25.6 (C-16), 23.6 (C-27), 23.5 (C-24), 18.7 (C-26),
17.4 (C-7), 16.5 (C-25). Anal. Calcd for C43H57NO3: C, 81.22; H,
9.03; N, 2.20. Found: C, 80.94; H, 8.77; N, 1.88.
3.30. (3b,18b,20b)-3-(Methoxyamino)-11-oxo-olean-12-en-29-
oic acid diphenylmethyl ester (10d)
3.28. (3b,18b,20b)-3-Amino-11-oxo-olean-12-en-29-oic acid
diphenylmethyl ester (10b)
Compound 9c (2.16 g, 3.3 mmol) was suspended in EtOH (7 mL)
and dioxane (15 mL), and then cooled to 0 °C. Borane tert-butyl-
amine complex (0.93 g, 10.7 mmol) was added, followed by the
dropwise addition of 10% HCl (10 mL). The reaction mixture was
stirred at 0 °C for 2.5 h. After this time, Na2CO3 was added until
gas evolution ceased, and the mixture was partitioned between
saturated NaHCO3 and DCM. The organic layer was dried over
Na2SO4, filtered and concentrated. The crude product was applied
on SiO2 column and eluted with PE/EtOAc (2–20%) to give 10d as
a white powder (0.90 g, 42%, HPLC >98%). Mp 198–200 °C. 1H
NMR (400 MHz, CDCl3) d 7.44–7.22 (m, 10H), 6.94 (s, 1H), 5.52
(s, 1H), 5.34 (br, 1H), 3.50 (s, 3H), 2.80 (dd, J = 13.5, 3.5 Hz, 1H),
2.59–2.48 (m, 1H), 2.35 (s, 1H), 2.13–1.48 (m, 9H), 1.46–1.25 (m,
6H), 1.37 (s, 3H), 1.23–0.79 (m, 4H), 1.18 (s, 3H), 1.14 (s, 3H),
1.09 (s, 3H), 1.08 (s, 3H), 0.72 (s, 3H), 0.67 (s, 3H); 13C NMR
(100 MHz, CDCl3) d 200.2, 175.2, 168.7, 140.1, 140.1, 128.6,
128.6, 128.5, 128.4, 128.4, 128.1, 127.8, 127.3, 127.2, 126.9,
126.9, 76.6, 67.9, 61.8, 61.6, 56.1, 48.0, 45.3, 44.0, 43.1, 41.1,
39.3, 37.5, 37.1, 36.9, 32.7, 31.7, 31.1, 28.6, 28.3, 28.2, 26.4, 23.5,
23.3, 18.7, 17.3, 16.7, 16.1. Anal. Calcd for C44H459NO4: C, 79.36;
H, 8.93; N, 2.10. Found: C, 79.04; H, 8.72; N, 1.82.
NaBH3CN (8.32 g, 132.0 mmol) was added to a mixture of 9b
(4.0 g, 6,2 mmol) and NH4OAc (7.73 g, 100.0 mmol) in MeOH
(400 ml) under argon atmosphere. The solution was chilled in
ice-water and TiCl3 (23 mL, 15% in H2O) was added dropwise over
20 min. The mixture was stirred at room temperature for 12 h and
then adjusted with 2 N NaOH to pH 10. The aqueous solution was
extracted with DCM (3 ꢃ 300 mL) and the combined organic layers
were washed with water, dried over Na2SO4, filtered and concen-
trated to dryness to obtain the crude product as white solid. The
crude product was subjected to SiO2 chromatography eluting with
CHCl3/MeOH+1%AcOH (0–20%) to give 10b as a white solid (2.1 g,
54%, HPLC >99%). Mp 232–235 °C; ½a D20
ꢂ
+129.8 (c 1.0, CHCl3). 1H
NMR (400 MHz, CDCl3) d 7.40–7.28 (m, 10H, Arom.), 6.95 (s, 1H,
31-H), 5.53 (s, 1H, 12-H), 2.78 (dd, J = 13.5, 3.4 Hz, 1H, 1-H),
2.42–2.31 (m, 1H, 3-H), 2.36 (s, 1H, 9-H), 2.10–1.94 (m, 4H, 16-H,
18-H, 19-H, 21-H), 1.87–1.75 (m, 1H, 15-H), 1.74–1.00 (m, 13H,
1-H, 2-H, 6-H, 7-H, 15-H, 16-H, 19-H, 21-H, 22-H), 1.38 (s, 3H,
27-H), 1.19 (s, 3H, 30-H), 1.14 (s, 3H, 25-H), 1.11 (s, 3H, 26-H),
0.98 (s, 3H, 23-H), 0.77 (s, 3H, 24-H), 0.75–0.71 (m, 1H, 5-H),
0.68 (s, 3H, 28-H); 13C NMR (100 MHz, CDCl3) d 200.2 (C-11),
175.2 (C-29), 168.8 (C-13), 140.1 (C-Ar), 140.1 (C-Ar), 128.6 (C-
Ar), 128.6 (C-Ar), 128.5 (C-12), 128.1 (C-Ar), 127.8 (C-Ar), 127.3
(C-Ar), 127.0 (C-Ar), 76.6 (C-31), 61.9 (C-9), 59.6 (C-3), 55.5 (C-5),
48.1 (C-18), 45.3 (C-8), 44.0 (C-20), 43.2 (C-14), 41.1 (C-19), 40.0
(C-1), 38.5 (C-4), 37.5 (C-22), 37.3 (C-10), 32.8 (C-7), 31.7 (C-17),
31.2 (C-21), 28.4 (C-23), 28.3 (C-30), 28.3 (C-28), 28.2 (C-2), 26.4
(C-15), 26.4 (C-16), 23.4 (C-27), 18.7 (C-26), 17.9 (C-6), 16.3 (C-
3.31. (18b,20b)-3,11-Dioxo-olean-12-en-29-oic acid (11)
Jones reagent (20 mL) was added to a solution of 1 (10.0 g,
21 mmol) in acetone (500 mL). The reaction mixture was stirred
for 30 min at 0 °C (monitored by TLC), poured into ice-water and ex-
tracted with CHCl3 (500 mL). The organic phase was washed with
water (3 x 200 mL), dried (MgSO4), filtered and evaporated to give
a white solid. The crude product was purified by recrystallization
from MeOH (150 mL) to give 11 as white crystals (8.2 g, 82%, HPLC
25), 15.7 (C-24). IR (DCM) m: 3755, 3686, 2972, 2941, 2871, 1775,
1729, 1656, 1621, 1582, 1563, 1544, 1525, 1494, 1459, 1389,
1366, 1328, 1308, 1212, 1150, 1085, 1050, 1030, 980, 915, 880,
868, 822, 737. MS-CI, m/z (Irel. (%)): 166.97 (100); 635.89 [M]
(20); 181.94 (18); 167.99 (17); 425.99 (10). 36.89 (9); 165.01 (8);
424.02 (7); 183.78 (6); 182.95 (5). Anal. Calcd for C43H57NO3: C,
81.22; H, 9.03; N, 2.200. Found: C, 80.87; H, 8.71; N, 1.91.
>99%). Mp 311–313 °C; (Ref. 31: 298–302 °C); ½a D20
ꢂ
+184.5 (c 0.4,
CHCl3) (Ref. 50: +184°). 1H NMR (400 MHz, CDCl3) d 5.77 (s, 1H),
3.03–2.94 (m, 1H), 2.71–2.60 (m, 1H), 2.47 (s, 1H), 2.43–2.34 (m,
1H), 2.24 (dd, J = 13.5, 3.2 Hz, 1H), 2.10–2.00 (m, 2H), 1.99–1.84
(m, 2H), 1.74–1.67 (m, 1H), 1.66–1.54 (m, 3H), 1.48–1.37 (m, 4H),
1.40 (s, 3H), 1.34–1.22 (m, 1H), 1.30 (s, 3H), 1.27 (s, 3H), 1.25 (s,
3H), 1.19 (s, 3H), 1.13 (s, 3H), 1.09 (s, 3H), 1.07–1.02 (m, 1H), 0.88
(s, 3H). 13C NMR (100 MHz, CDCl3) d 217.3, 199.7, 181.7, 169.9,
128.4, 61.1, 55.5, 48.3, 47.8, 45.3, 43.8, 43.3, 40.9, 39.7, 37.7, 36.7,
34.2, 32.1, 31.9, 30.9, 29.7, 28.6, 28.4, 26.5, 26.4, 23.4, 21.4, 18.8,
3.29. (3a,18b,20b)-3-Amino-11-oxo-olean-12-en-29-oic acid
diphenylmethyl ester (10c)
Another elution gives (3a,18b,20b)-3-Amino-11-oxo-olean-12-
18.5, 15.6. IR (DCM) m: 3736, 3061, 2937, 2875, 1702, 1656, 1555,
en-29-oic acid diphenylmethyl ester (10c) as a white solid (0.60 g,
1540, 1459, 1386, 1366, 1324, 1208, 1162, 1112, 1088, 1000, 980,
946, 922, 880, 818. MS-CI, m/z (Irel. (%)): 468.98 [M] (100); 423.03
(98); 425.04 (95); 469.98 (44); 441.82 (38); 424.08 (31); 407.06
(31); 426.07 (27); 440.96 (23); 439.95 (23).
15%, HPLC >96%). ½a D20
ꢂ
+111.6 (c 1.0, CHCl3). 1H NMR (200 MHz,
CDCl3) d 7.43–7.22 (m, 10H, arom.), 6.93 (s, 1H, 31-H), 5.52 (s, 1H,
12-H), 2.96–62 (m, 1H, 5-H), 2.56–2.47 (m, 1H, 6-H), 2.46 (s, 1H, 9-
H), 2.15–1.93 (m, 4H, 2-H, 18-H, 19-H, 21-H), 1.86–1.76 (m, 1H,
15-H), 1.77–1.53 (m, 3H, 1-H, 16-H, 19-H), 1.52–1.41 (m, 2H, 7-H),
1.41–1.27 (m, 6H, 1-H, 6-H, 16-H, 21-H, 22-H), 1.39 (s, 3H, 27-H),
1.27–1.21 (m, 1H, 3-H), 1.17 (s, 3H, 30-H), 1.16–1.12 (m, 1H, 15-
H), 1.15 (s, 3H, 25-H), 1.09 (s, 3H, 26-H), 1.02–0.93 (m, 1H, 2-H),
0.93–0.87 (d, 6H, 23-H, 24-H), 0.67 (s, 3H, 28-H); 13C NMR
3.32. (3a,18b,20b)-3-Hydroxy-11-oxo-olean-12-en-29-oic acid
(12)
A solution of 11 (3.0 g, 6.5 mmol) in dry THF (200 mL) under
nitrogen atmosphere was cooled in methanol slush bath
a

I. Beseda et al. / Bioorg. Med. Chem. 18 (2010) 433–454
449
(ꢀ74 °C), and a 1.0 M solution of K-selectride in THF (20 mL,
(C-24). IR (DCM)
m
: 3543, 2937, 2871, 1729, 1656, 1621, 1559,
20 mmol) was added dropwise by syringe. After two more hours
at this temperature, the reaction mixture was warmed to and left
to stand overnight at rt. The reaction was quenched with 1 N HCl
to pH 2.0 under ice-cooling and extracted with CHCl3 (750 mL).
The extract was washed with brine, dried over anhydrous MgSO4,
filtered and evaporated in vacuo. The product was crystallized
from MeOH to afford 12 as a white solid (1.2 g, 39.8%, HPLC
>99%). Mp 335–338 °C. 1H NMR (200 MHz, DMSO-d6) d 5.39 (s,
1H), 3.16 (br, 1H), 2.49 (m, 1H), 2.38 (s, 1H), 2.37–2.20 (m, 1H),
2.19–1.95 (m, 2H), 1.92–1.50 (m, 6H), 1.49–1.15 (m, 10H), 1.35
(s, 3H), 1.13–0.94 (m, 1H), 1.09 (s, 3H), 1.02 (s, 6H), 0.92–0.64
(m, 1H), 0.83 (s, 3H), 0.76 (s, 3H), 0.75 (s, 3H); 13C NMR (50 MHz,
DMSO-d6) d 199.6, 178.1, 170.0, 127.8, 74.0, 61.6, 48.5, 47.9, 45.5,
43.5, 43.4, 41.1, 38.0, 37.6, 37.2, 33.5, 32.6, 32.0, 30.8, 29.3, 28.8,
1540, 1497, 1455, 1386, 1362, 1328, 1312, 1212, 1189, 1162,
1077, 1030, 980, 969, 938, 915, 880. MS-CI, m/z (Irel. (%)): 167.05
(100); 637.02 [M+1] (45); 182.01 (27); 183.10 (20); 298.91 (13);
638.04 (12); 282.09 (12); 272.37 (8); 283.16 (8); 199.84 (7).
3.35. (18b,20b)-3,3-Ethane-1,2-diyldioxy-11-oxo-olean-12-en-
29-oic acid methyl ester (14)
A suspension of 8a (1.0 g, 2.1 mmol), toluene (50 mL), ethylene-
glycol (0.6 mL) and pyridinium p-toluenesulfonate (20.7 mg,
0.09 mmol) was refluxed for 12 h with azeotropic removal of water
by a Dean-Stark apparatus filled with molecular sieves in the recei-
ver. The resulting mixture was cooled and washed with water
(5 mL) and the aqueous layer was extracted with DCM. The organic
layers were combined and concentrated to give crude 14 as a white
powder (0.7 g, 64%, HPLC >96%). Mp 188–189 °C. 1H NMR
(200 MHz, CDCl3) d 5.59 (s, 1H), 3.89 (m, 4H), 3.62 (s, 3H), 2.63
(dd, J = 13.3, 3.2 Hz, 1H), 2.36 (s, 1H), 2.10–1.61 (m, 5H), 1.50–
1.12 (m, 12H), 1.30 (s, 3H), 1.10 (s, 3H), 1.08 (s, 3H), 1.06 (s, 3H),
1.02–0.93 (m, 1H), 0.89 (s, 3H), 0.85–0.75 (m, 1H), 0.79 (s, 3H),
0.73 (s, 3H); 13C NMR (50 MHz, CDCl3) d 200.4, 176.9, 169.3,
128.5, 112.3, 64.8, 64.6, 61.7, 52.9, 51.7, 48.3, 45.4, 44.0, 43.2,
42.3, 41.1, 37.7, 37.3, 37.0, 32.6, 31.8, 31.1, 28.5, 28.3, 26.7, 26.4,
28.3, 26.4, 26.3, 25.6, 23.6, 22.7, 18.8, 17.4, 16.6. IR (DCM)
m,
3736, 2956, 2918, 2879, 2852, 1717, 1648, 1617, 1455, 1386,
1362, 1208, 1162, 1088, 1061, 1034, 976, 930, 884. MS-CI, m/z (Irel.
(%)): 470.99 [M] (100); 471.99 (30); 178.00 (28); 194.78 (11);
469.01 (10); 427.03 (9); 175.03 (8); 135.02 (8); 303.01 (8);
261.99 (8).
3.33. (3a,18b,20b)-3-Hydroxy-11-oxo-olean-12-en-29-oic acid
methyl ester (13a)
26.4, 23.6, 23.0, 20.1, 18.6, 17.6, 16.3. IR (DCM)
m: 2956, 2875,
Preparation as described for 2a. Purification by flash chroma-
tography (ethylacetate–petrolether) gives methylester 13a as col-
orless needles (81%, HPLC >98%). Mp 250–252 °C. 1H NMR
(400 MHz, CDCl3) d 5.56 (s, 1H, 12-H), 3.68 (s, 3H, 31-H), 3.41 (t,
J = 3.2 Hz, 1H, 3-H), 2.53 (dd, J = 13.4, 3.2 Hz, 1H, 1-H), 2.46 (s,
1H, 9-H), 2.09–1.97 (m, 4H, 2-H, 18-H, 19-H, 21-H), 1.94–0.91
(m, 15H, 1-H, 2-H, 5-H, 6-H, 7-H, 15-H, 16-H, 19-H, 21-H, 22-H),
1.37 (s, 3H, 27-H), 1.14 (s, 3H, 25-H), 1.14 (s, 3H, 30-H), 1.12 (s,
3H, 26-H), 0.96 (s, 3H, 24-H), 0.86 (s, 3H, 23-H), 0.80 (s, 3H, 28-
H); 13C NMR (100 MHz, CDCl3) d 200.5 (C-11), 177.0 (C-29), 169.2
(C-13), 128.5 (C-12), 75.7 (C-3), 61.7 (C-9), 51.7 (C-31), 48.4 (C-
18), 48.4 (C-5), 45.6 (C-8), 44.0 (C-20), 43.3 (C-14), 41.1 (C-19),
37.8 (C-22), 37.5 (C-4), 37.1 (C-10), 33.4 (C-1), 32.6 (C-7), 31.8
(C-17), 31.1 (C-21), 28.5 (C-28), 28.5 (C-24), 28.3 (C-30), 26.4 (C-
15), 26.4 (C-16), 25.4 (C-2), 23.6 (C-27), 22.4 (C-23), 18.7 (C-26),
1729, 1660, 1621, 1459, 1389, 1366, 1320, 1208, 1154, 1127,
1108, 1088, 1058, 1027, 949, 919, 903, 880, 826. MS-CI, m/z (Irel.
(%)): 526.99 [M] (100); 528.00 (39); 483.00 (35). 99.03 (19);
427.06 (15); 465.09 (13); 484.02 (12); 543.77 (9); 498.95 (7);
189.09 (7); 529.04 (6); 134.97 (6); 173.06 (5); 135.92 (5); 512.83
(5).
3.36. (3b,18b,20b)-3-Hydroxy-olean-12-en-29-oic acid (15)
Zn (6.7 g, 102 mmol) was added to a solution of dioxane
(180 mL) and 1 (6.0 g, 14 mmol). The mixture was cooled to 5–
10 °C and HCl (35% aq, 27 mL) was added over 30 min. The result-
ing mixture was stirred for 30 min, the unreacted Zn was removed
by decantation and the dioxane was distilled off. The residue was
purified by SiO2 column chromatography, eluting with 97:3
DCM/THF to obtain 15 as a white powder (4.0 g, 69%). Mp 324–
326 °C; (Ref. 31: 320–325). 1H NMR (400 MHz, DMSO-d6) d 5.17
(t, J = 3.2 Hz, 1H), 3.01 (dd, J = 9.7, 6.1 Hz, 1H), 2.02–1.94 (m, 1H),
1.91–1.81 (m, 3H), 1.79–1.67 (m, 3H), 1.63–1.57 (d, J = 13.2 Hz,
1H), 1.55–1.43 (m, 6H), 1.33–1.23 (m, 5H), 1.12 (s, 3H), 1.06 (s,
3H), 0.99–0.79 (m, 4H), 0.91 (s, 0.91), 0.91 (s, 3H), 0.88 (s, 3H),
0.74 (s, 3H), 0.73–0.62 (m, 1H), 0.69 (s, 3H); 13C NMR (100 MHz,
DMSO-d6) d 178.5, 144.8, 122.3, 77.2, 55.2, 48.3, 47.5, 43.6, 42.9,
41.6, 40.6, 38.6, 38.6, 38.5, 37.0, 32.6, 32.1, 31.1, 28.6, 28.6, 28.6,
27.4, 26.8, 26.2, 26.1, 23.5, 18.5, 17.0, 16.5, 15.7. MS-CI, m/z (Irel.
(%)): 439.02 (100); 191.14 (74); 411.09 (42); 203.05 (40); 248.99
(32); 247.99 (29); 189.10 (26); 440.03 (24); 205.08 (24); 265.83
(22); 456.14 [M] (20); 207.09 (19); 393.10 (18); 471.90 (17);
95.12 (17).
17.4 (C-6), 16.3 (C-25). IR (DCM)
m: 3736, 3057, 2953, 2871,
2157, 2134, 1729, 1652, 1555, 1540, 1513, 1459, 1389, 1370,
1316, 1220, 1193, 1158, 1088, 1069, 1034, 992, 976, 946, 919,
880, 826. MS-CI, m/z (Irel. (%)): 485.06 [M+1] (100); 486.04 (29);
316.93 (8); 135.03 (7); 292.97 (7); 487.03 (6); 484.07 (5); 175.04
(5); 189.04 (5); 135.94 (4).
3.34. (3a,18b,20b)-3-Hydroxy-11-oxo-olean-12-en-29-oic acid
diphenylmethyl ester (13b)
Preparation as described for 2b. Purification by flash chomatog-
raphy (DCM–MeOH, 0–0.5% gradient elution) gives 13b as a white
solid (85%, HPLC >98%). Mp 131–132 °C. 1H NMR (200 MHz, CDCl3)
d 7.44–7.21 (m, 10H), 6.94 (s, 1H, 31-H), 5.52 (s, 1H, 12-H), 3.42 (t,
J = 3.1, 1H, 3-H), 2.51 (dd, J = 13.5, 3.1 Hz, 1H, 18-H), 2.45 (s, 1H, 9-
H), 2.17–1.85 (m, 5H, 2-H, 16-H, 19-H, 21-H), 1.84–1.01 (m, 13H, 1-
H, 2-H, 6-H, 7-H, 15-H, 16-H, 21-H, 22-H), 1.38 (s, 3H, 30-H), 1.18
(s, 3H, 27-H), 1.15 (s, 3H, 25-H), 1.10 (s, 3H, 26-H), 1.00–0.90 (m,
1H, 5-H), 0.97 (s, 3H, 23-H), 0.87 (s, 3H, 24-H), 0.67 (s, 3H, 28-
H); 13C NMR (50 MHz, CDCl3) d 200.3 (C-11), 175.2 (C-29), 168.9
(C-13), 140.1 (C-Ar), 140.0 (C-Ar), 128.6 (C-Ar), 128.4 (C-12),
127.8 (C-Ar), 127.3 (C-Ar), 127.0 (C-Ar), 76.6 (C-3), 75.8 (C-31),
61.6 (C-9), 48.3 (C-5), 45.5 (C-14), 44.0 (C-8), 43.2 (C-20), 41.1
(C-19), 37.5 (C-4), 37.1 (C-22), 33.5 (C-10), 32.6 (C-7), 31.7 (C-
17), 31.1 (C-21), 28.5 (C-30), 28.3 (C-28), 28.2 (C-23), 26.4 (C-2),
25.4 (C-15), 23.5 (C-27), 22.3 (C-26), 18.6 (C-25), 17.4 (C-6), 16.3
3.37. (3b,18b,20b)-3-Hydroxy-olean-12-en-29-oic acid methyl
ester (16)
Preparation as described for 2a. Purification by crystallization
from MeOH gives 16 as colorless crystals (96.1%). Mp 247–248 °C
(Ref. 51: 248–249°); ½a D20
ꢂ
+110 (c 1, CHCl3) (Ref. 47: ½a D20
ꢂ
+122 (c
0.12, CHCl3)). 1H NMR (400 MHz, CDCl3) d 5.29 (t, J = 3.5 Hz, 1H,
12-H), 3.69 (s, 3H, 31-H), 3.23 (dd, J = 10.8, 4.7 Hz, 1H, 3-H),
1.99–1.83 (m, 6H, 11-H,16-H, 18-H, 19-H, 21-H), 1.80–0.90 (m,
15H, 1-H, 2-H, 6-H, 7-H, 15-H, 16-H, 19-H, 21-H, 22-H), 1.15 (s,
3H, 27-H), 1.14 (s, 3H, 30-H), 1.01 (s, 3H, 24-H), 0.97 (s, 3H, 26-

450
I. Beseda et al. / Bioorg. Med. Chem. 18 (2010) 433–454
H), 0.95 (s, 3H, 25-H), 0.92–0.75 (m, 1H, 5-H), 0.80 (s, 3H, 23-H),
0.79 (s, 3H, 28-H); 13C NMR (100 MHz, CDCl3) d 177.7 (C-29),
144.4 (C-13), 122.6 (C-12), 79.0 (C-3), 55.2 (C-5), 51.6 (C-31),
48.2 (C-18), 47.6 (C-9), 44.3 (C-20), 42.9 (C-19), 41.5 (C-14), 39.8
(C-8), 38.8 (C-4), 38.6 (C-1), 38.4 (C-22), 36.9 (C-10), 32.7 (C-7),
31.9 (C-17), 31.3 (C-21), 28.6 (C-30), 28.2 (C-28), 28.1 (C-24),
27.2 (C-2), 27.0 (C-16), 26.2 (C-15), 26.0 (C-27), 23.5 (C-11), 18.4
(C-6), 16.8 (C-26), 15.6 (C-23), 15.5 (C-25).
1.10 (s, 3H), 1.04–0.97 (m, 1H), 1.01 (s, 3H), 0.96–0.76 (m, 2H),
0.83 (s, 3H), 0.76–0.66 (m, 1H), 0.71 (s, 3H). 13C NMR (100 MHz,
CDCl3) d 175.7, 147.5, 140.4, 140.2, 128.5, 128.5, 128.4, 128.4,
127.9, 127.8, 127.4, 127.4, 127.0,127.0, 126.3, 79.0, 77.8, 66.6,
55.8, 52.4, 47.6, 44.2, 42.8, 42.2, 39.5, 38.8, 38.7, 38.3, 38.0, 33.2,
31.7, 31.3, 28.5, 28.3, 28.2, 27.0, 26.8, 26.5, 25.3, 19.3, 18.7, 18.1,
15.8. IR (DCM)
m: 3612, 3057, 2976, 2937, 2871, 1725, 1455,
1420, 1378, 1216, 1153, 1082, 1031, 992, 916, 895, 835. MS-CI,
m/z (Irel. (%)): 167.03 (100); 168.05 (14); 183.77 (8); 225.07 (7);
182.98 (5); 181.92 (5); 453.05 (4); 206.90 (2); 180.17 (2); 152.13
(2). Anal. Calcd for C43H58O4: C, 80.83; H, 9.15. Found: C, 81.21;
H, 8.81.
3.38. (3b,18b,20b)-3,11-Dihydroxy-olean-12-en-29-oic acid (17)
Compound 1 (5.0 g, 10.6 mmol), NaBH4 (24.0 g, 634.5 mmol)
and NaOH (2.5 g, 62.5 mmol) were suspended in a mixture of
THF (200 mL) and water (200 mL). The heterogeneous mixture
was refluxed for 4 h. The solvent THF was evaporated under
reduced pressure and the residue was poured into a 5% aqueous
solution of NaH2PO4 (1 L) and extracted with ethylacetate (2 ꢃ
250 mL). The combined organic layers were washed with water
(2 ꢃ 100 mL), dried (Na2SO4), filtered and evaporated in vacuo to
give 17 as white crystals (4.8 g, 95.6%). Mp 265–270 °C. 1H NMR
3.41. (3b,11a,18b,20b)-3,11-Dihydroxy-olean-12-en-29-oic acid
diphenylmethyl ester (18c)
Another elution gives (3b,11a,18b,20b)-3,11-dihydroxy-olean-
12-en-29-oic acid diphenylmethyl ester (18c) as a white solid
(60 mg, 9%, HPLC >96%). Mp 115–118 °C. 1H NMR (200 MHz,
CDCl3) d 7.34–7.16 (m, 10H), 6.82 (s, 1H), 5.07 (d, J = 3.9 Hz,1H),
4.08 (dd, J = 8.3, 3.6 Hz, 1H), 3.16 (dd, J = 9.9, 6.0 Hz,1H), 2.00–
1.80 (m, 5H), 1.75–1.48 (m, 7H), 1.40–1.20 (m, 6H), 1.14 (s, 3H),
1.11–1.05 (m, 1H), 1.09 (s, 3H), 1.04–0.98 (m, 1H), 0.97 (s, 3H),
0.96–0.78 (m, 2H), 0.93 (s, 3H), 0.89 (s, 3H), 0.76–0.68 (m, 1H),
0.73 (s 3H), 0.60 (s, 3H). 13C NMR (100 MHz, CDCl3) d 175.7,
147.5, 140.4, 140.2, 128.5, 128.5, 128.4, 128.4, 127.9, 127.8,
127.4, 127.4, 127.0,127.0, 126.3, 79.0, 77.8, 66.6, 55.8, 52.4,
47.6, 44.2, 42.8, 42.2, 39.5, 38.8, 38.7, 38.3, 38.0, 33.2, 31.7,
31.3, 28.5, 28.3, 28.2, 27.0, 26.8, 26.5, 25.3, 19.3, 18.7, 18.1,
(300 MHz, pyridine-d5)
d 5.86 (d, J = 3.5 Hz, 1H), 4.67 (d,
J = 3.6 Hz, 1H), 3.50 (dd, J = 11.28, 4.16 Hz, 1H), 2.58–2.36 (m,
2H), 2.33–2.11 (m, 4H), 2.00–1.81 (m, 4H), 1.80–1.54 (m, 6H),
1.76 (s, 3H), 1.53–1.21 (m, 4H), 1.49 (s, 3H), 1.35 (s, 3H), 1.27 (s,
3H), 1.27 (s, 3H), 1.18–1.00 (m, 2H), 1.12 (s, 3H), 0.98–0.82 (m,
2H), 0.90 (s, 3H); 13C NMR (75 MHz, pyridine-d5) d (ppm) 179.9,
146.6, 128.9, 78.8, 66.2, 56.9, 53.7, 48.8, 47.5, 44.8, 42.9, 40.4,
39.9, 39.5, 39.3, 39.1, 38.2, 32.6, 32.3, 31.4, 29.6, 29.5, 29.2, 28.2,
26.3, 25.0, 21.3, 19.6, 19.1, 17.2. IR (MeOH) m: 3342, 2366, 2347,
1702, 1656, 1640, 1629, 1563, 1536, 1459, 1389, 1366, 1339,
1316, 1277, 1262, 1223, 1177, 1139, 1104, 1027, 976, 949, 922,
892, 857, 814. MS-CI, m/z (Irel. (%)): 314.51 (100); 212.45 (78);
270.50 (68); 57.28 (55); 323.50 (54); 67.36 (53); 311.28 (36);
70.08 (34); 103.22 (33); 463.44 (31).
15.8. IR (DCM) m: 3612, 3056, 2980, 2936, 2871, 1724, 1455,
1420, 1378, 1215, 1154, 1081, 1030, 992, 915, 895, 835. MS-CI,
m/z (Irel. (%)): 167.32 (100); 167.67 (12); 183.87 (9); 225.07 (7);
183.05 (6); 181.92 (5); 452.74 (5); 207.05 (2); 180.11 (2);
152.69 (2). Anal. Calcd for C43H58O4: C, 80.83; H, 9.15. Found: C,
81.20; H, 8.76.
3.39. (3b,11b,18b,20b)-3,11-Dihydroxy-olean-12-en-29-oic acid
methyl ester (18a)
3.42. (3b,11b,18b,20b)-3-Hydroxy-11-methoxyolean-12-en-29-
oic acid methyl ester (19)
Preparation as described for 2a. Purification by flash chroma-
tography (DCM–MeOH, gradient elution, 0–1%) yields 18a as a
white solid (51%). Mp 206–207 °C. 1H NMR (400 MHz, CDCl3) d
5.41 (d, J = 5.4 Hz, 1H), 4.35 (dd, J = 7.9, 2.9 Hz, 1H), 3.71 (s, 3H),
3.26 (dd, J = 10.8, 5.3 Hz, 1H), 2.08 (dd, J = 13.0, 3.4 Hz, 1H), 2.03–
1.93 (m, 3H), 1.88–1.72 (m, 5H), 1.64–1.54 (m, 3H), 1.42 (s, 3H),
1.41–1.29 (m, 5H), 1.27–1.19 (m, 2H), 1.25 (s, 3H), 1.15 (s, 3H),
1.12–0.99 (m, 2H), 1.11 (s, 3H), 1.01 (s, 3H), 0.93 (d, J = 13.5 Hz,
1H), 0.84 (s, 3H), 0.83 (s, 3H), 0.72 (dd, J = 10.1, 2.8 Hz, 1H); 13C
NMR (100 MHz, CDCl3) d 177.5, 147.7, 126.3, 79.1, 66.7, 55.8,
52.5, 51.7, 47.9, 44.3, 42.8, 42.3, 39.5, 38.8, 38.7, 38.3, 38.2, 33.2,
31.8, 31.3, 28.5, 28.5, 28.3, 27.1, 26.8, 26.5, 25.3, 19.3, 18.7, 18.1,
Compound 17 (1.0 g, 2.1 mmol) was placed into DCM (100 mL)
and Et2O (100 mL) and transferred into a diazomethane apparatus.
Diazomethane was added slowly and the mixture was kept at 40 °C
for 12 h. When diazomethane was gone, the solvent was evapo-
rated. The crude product was crystallized from MeOH (500 mL)
to give 19 as colorless crystals (0.9 g, 85%, HPLC >99%). Mp 185–
187 °C.
3.43. (3b,18b,20b)-3-Hydroxy-oleana-(9(11),12-dien-29-oic acid
(20)
15.7. IR (DCM)
m: 3744, 3620, 3057, 2983, 2945, 2871, 2165,
To a solution of 17 (473 mg, 1.0 mmol) in dry THF (20 mL) was
1721, 1459, 1424, 1389, 1223, 1162, 1081, 1038, 1027, 919, 895.
MS-CI, m/z (Irel. (%)): 206.93 (100); 189.07 (64); 469.03 (37);
451.12 (29); 470.08 (15); 207.95 (14); 249.00 (12); 452.19 (12);
468.03 (11); 205.01 (10).
added HCl (100 lL) and refluxed for 8 h. The reaction was moni-
tored by TLC. The solvent was evaporated to dryness. The crude
product was crystallized from MeOH (10 mL) to give acid 20 as a
white solid (395 mg, 87%, HPLC >98%). Mp 301–302 °C. 1H NMR
(200 MHz, pyridine-d5)
d 5.85 (d, J = 5.6 Hz, 1H), 5.71 (d,
3.40. (3b,11b,18b,20b)-3,11-Dihydroxy-olean-12-en-29-oic acid
diphenylmethyl ester (18b)
J = 5.6 Hz, 1H), 3.45 (m, 1H), 2.59 (dd, J = 12.6, 3.0 Hz, 1H), 2.28
(d, J = 12.8 Hz, 2H), 2.16–1.59 (m, 6H), 1.57–1.15 (m, 5H), 1.33 (s,
3H), 1.26 (s, 3H), 1.22 (s, 3H), 1.21 (s, 3H), 1.17 (s, 3H), 1.12–0.85
(m, 2H), 1.06 (s, 3H), 0.91 (s, 3H); 13C NMR (50 MHz, pyridine-d5)
d 180.0, 155.7, 147.2, 122.2, 116.5, 78.2, 52.2, 47.4, 44.7, 44.0,
43.5, 42.3, 41.3, 40.0, 39.6, 39.6, 39.4, 38.2, 33.0, 32.5, 32.1, 29.4,
Preparation as described for 2b. Purification by flash chroma-
tography (DCM–MeOH gradient elution, 0–1%) gives 11b isomer
18b as a white solid (240 mg, 35.5%, HPLC >97%). Mp 101–102 °C.
1H NMR (200 MHz, CDCl3) d 7.42–7.24 (m, 10H), 6.95 (s, 1H),
5.16 (d, J = 3.9 Hz,1H), 4.29 (t, J = 4.1 Hz, 1H), 3.24 (dd, J = 9.6,
6.2 Hz,1H), 2.08–1.84 (m, 5H), 1.77–1.53 (m, 7H), 1.41 (s, 3H),
1.38–1.22 (m, 6H), 1.21 (s, 3H), 1.18 (s, 3H), 1.13–1.05 (m, 1H),
29.3, 28.0, 26.5, 26.0, 21.6, 20.8, 19.2, 17.1. IR (MeOH) m: 3439,
2366, 2343, 1706, 1656, 1555, 1536, 1525, 1459, 1378, 1366,
1343, 1316, 1277, 1262, 1223, 1177, 1146, 1104, 1077, 1034,
992, 922, 834, 818. MS-CI, m/z (Irel. (%)): 206.94 (100); 437.10

I. Beseda et al. / Bioorg. Med. Chem. 18 (2010) 433–454
451
(65); 189.04 (61); 455.09 [M] (46); 454.17 (29); 438.10 (25);
409.14 (18); 207.97 (15); 456.09 (12); 203.10 (12).
1H, 15-H), 1.79 (dd, J = 13.8, 3.6 Hz, 1H, 21-H), 1.69–1.18 (m,
17H, 2-H, 6-H, 7-H, 15-H, 16-H, 19-H, 21-H, 22-H, 27-H), 1.17 (s,
3H, 30-H), 1.09 (s, 3H, 25-H), 1.05 (s, 3H, 26-H), 1.05–0.92 (m,
1H, 1-H), 0.90 (s, 3H, 23-H), 0.72–0.67 (m, 1H, 5-H), 0.70 (s, 3H,
24-H), 0.67 (s, 3H, 28-H); 13C NMR (100 MHz, DMSO-d6) d 199.2
(C-11), 179.9 (C-29), 166.5 (C-13), 123.5 (C-12), 77.0 (C-3), 60.4
(C-9), 54.6 (C-5), 45.1 (C-14), 43.7 (C-8), 42.0 (C-20), 40.6 (C-18),
39.3 (C-4), 38.9 (C-1), 37.1 (C-16), 36.8 (C-10), 35.7 (C-22), 35.5
(C-17), 33.6 (C-7), 31.8 (C-19), 28.8 (C-21), 28.6 (C-23), 27.4 (C-
2), 26.7 (C-15), 21.0 (C-30), 20.8 (C-27), 18.7 (C-26), 17.7 (C-6),
3.44. (3b,18b,20b)-3-Hydroxy-oleana-(9(11),12-dien-29-oic acid
methyl ester (21)
Preparation as described for 2a. Purification by flash chroma-
tography (DCM–MeOH, gradient elution, 0–1%) yields 21 as a white
solid (90%, HPLC >99%). Mp 240–243 °C. 1H NMR (400 MHz, CDCl3)
d 5.74–5.63 (m, 2H, 11-H, 12-H), 3.78 (s, 3H, 31-H), 3.26–3.18 (m,
1H, 3-H), 2.13–0.92 (m, 19H, 1-H, 2-H, 6-H, 7-H, 15-H, 16-H, 18-H,
19-H, 21-H, 22-H), 1.20 (s, 3H, 25-H), 1.13 (s, 3H, 30-H), 1.13 (s, 3H,
26-H), 1.04 (s, 3H, 23-H), 1.00 (s, 3H, 27-H), 0.87–0.76 (m, 1H, 5-H),
0.85 (s, 3H, 28-H), 0.82 (s, 3H, 24-H); 13C NMR (100 MHz, CDCl3) d
177.7 (C-29), 154.6 (C-9), 146.0 (C-13), 121.4 (C-12), 115.7 (C-11),
78.7 (C-3), 51.6 (C-31), 51.1 (C-5), 46.4 (C-18), 44.2 (C-20), 42.8 (C-
19), 42.7 (C-8), 40.4 (C-14), 38.9 (C-4), 38.7 (C-10), 38.3 (C-22),
37.1 (C-1), 32.1 (C-7), 31.6 (C-17), 31.2 (C-21), 28.4 (C-28), 28.4
(C-30), 28.2 (C-23), 27.9 (C-2), 27.3 (C-16), 25.6 (C-15), 25.3 (C-
16.9 (C-25), 16.5 (C-24), 16.1 (C-28). IR (DCM)
m: 3496, 2969,
2926, 2867, 1704, 1661, 1297, 1200, 1112, 1028, 989, 658. MS-CI,
m/z (Irel. (%)): 425.07 (100); 427.08 (68); 471.04 [M+1] (37);
426.07 (32); 428.12 (24); 423.06 (19); 472.08 (17); 191.00 (16);
424.11 (15); 175.02 (15).
3.47. (3b,18a,20b)-3-Hydroxy-11-oxo-olean-12-en-29-oic acid
diphenylmethyl ester (24)
25), 20.9 (C-26), 20.1 (C-27), 18.3 (C-6), 15.7 (C-24). IR (DCM)
m
,
Preparation as described for 2b. Purification by crystallization
from MeOH gives diphenylmethyl ester 24 as a white powder
3736, 3350, 3034, 2980, 2949, 2914, 2875, 2157, 1721, 1648,
1555, 1540, 1470, 1455, 1378, 1316, 1277, 1262, 1220, 1189,
1162, 1088, 1065, 1038, 992, 822, 749.
(84%, HPLC >99%). Mp 128–131 °C; ½a D20
ꢂ
+58 (c 0.061, DCM) (Ref.
47: +57.6°). 1H NMR (200 MHz, CDCl3) d 7.39–7.28 (m, 10H), 6.88
(s, 1H), 5.56 (s, 1H), 3.24 (dd, J = 11.2, 4.9 Hz, 1H), 2.70 (m, 1H),
2.28–2.19 (m, 2H), 2.01–1.84 (m, 2H), 1.83–1.30 (m, 13H), 1.29
(s, 3H), 1.26 (s, 3H), 1.27–1.19 (m, 1H), 1.17 (s, 3H), 1.11 (s, 3H),
0.99 (s, 3H), 0.98–0.86 (m, 1H), 0.79 (s, 3H), 0.69–0.63 (m, 4H);
13C NMR (50 MHz, CDCl3) d 199.8, 175.0, 165.5, 140.4, 140.4,
128.6, 128.6, 128.5, 128.5, 128.1, 127.8, 127.3, 127.3, 127.0,
127.0, 78.7, 76.6, 76.5, 60.7, 55.0, 48.7, 45.3, 44.2, 43.1, 41.5,
39.4, 39.1, 37.5, 37.2, 32.7, 31.8, 31.2, 28.3, 28.3, 28.0, 27.3, 26.5,
3.45. (3b,20b)-3-(Acetyloxy)-oleana-11,13(18)-dien-29-oic acid
(22)
The mixture of crude 17 (1.00 g, 2.1 mmol) was added to a mix-
ture of acetic acid (40 mL) and concd hydrochloric acid (10 mL).
The mixture was refluxed for 2 h, poured into ice-water (200 mL)
and filtered. Purification by flash chromatography (DCM–MeOH
gradient elution, 0–5%) gave 22 as a white solid (0.65 g, 62%). Mp
274–275 °C. 1H NMR (400 MHz, DMSO-d6) d 6.30 (d, J = 11.0 Hz,
1H, 12-H), 5.58 (d, J = 11.4 Hz, 1H, 11-H), 4.45 (dd, J = 10.8,
3.8 Hz, 1H, 3-H), 2.7 (d, J = 14.5 Hz, 1H, 19-H), 2.20 (d, J = 14.6 Hz,
1H, 19-H), 2.08–1.04 (m, 18H, 1-H, 2-H, 5-H, 6-H, 7-H, 9-H, 15-H,
16-H, 21-H, 22-H), 1.13 (s, 3H, 30-H), 1.12 (s, 3H, 28-H), 0.98 (s,
3H, 27-H), 0.94 (s, 3H, 25-H), 0.89 (s, 3H, 23-H), 0.87 (s, 3H, 24-
H), 0.73 (s, 3H, 26-H); 13C NMR (100 MHz, DMSO-d6) d 184.5 (C-
29), 171.1 (C-31), 135.4 (C-13), 135.0 (C-18), 126.3 (C-11), 125.4
(C-12), 80.9 (C-3), 54.9 (C-5), 54.1 (C-9), 43.8 (C-20), 42.5 (C-14),
40.3 (C-8), 37.8 (C-4), 37.6 (C-1), 37.5 (C-22), 36.6 (C-10), 35.8
(C-16), 34.7 (C-17), 32.2 (C-7), 32.0 (C-19), 29.8 (C-21), 27.8 (C-
23), 25.2 (C-28), 24.3 (C-15), 23.4 (C-2), 21.3 (C-32), 20.1 (C-27),
19.5 (C-30), 18.2 (C-6), 18.0 (C-25), 16.5 (C-26), 16.2 (C-24). IR
26.4, 23.3, 18.7, 17.5, 16.1, 15.7. IR (DCM) m: 3057, 2980, 2945,
2871, 1729, 1656, 1459, 1389, 1227, 1193, 1158, 1104, 1034,
992, 895.
3.48. (3b,18a,20b)-3-Hydroxy-11-oxo-olean-12-en-29-oic acid
methyl ester (25)
Preparation as described for 2a. Purification by flash chroma-
tography (DCM–MeOH gradient elution, 0–1%) gives 25 as a white
solid (76.3%, HPLC >99%). Mp 264–266 °C. 1H NMR (200 MHz,
CDCl3) d 5.55 (s, 1H), 3.67 (s, 3H), 3.21 (dd, J = 11.1, 3.8 Hz), 2.67
(dd, J = 13.3, 3.2 Hz, 1H), 2.25 (s, 1H), 2.22–2.15 (m, 1H), 2.08–
1.78 (m, 3H), 1.71–1.43 (m, 12H), 1.41–1.26 (m, 2H), 1.33 (s, 3H),
1.25–1.16 (m, 1H), 1.21 (s, 3H), 1.18 (s, 3H), 1.12 (s, 3H), 0.99 (s,
3H), 0.95–0.90 (m, 1H), 0.79 (s, 3H), 0.70 (s, 3H); 13C NMR
(50 MHz, CDCl3) d 199.8, 178.8, 165.6, 124.1, 78.7, 60.7, 55.0,
51.9, 44.9, 43.8, 42.5, 40.3, 39.1, 39.0, 37.6, 36.8, 35.9, 35.5, 33.7,
31.8, 28.4, 28.1, 27.2, 26.7, 20.8, 20.8, 18.5, 17.5, 16.5, 16.0, 15.7.
(DCM) m: 3041, 2941, 2868, 1733, 1698, 1470, 1378, 1370, 1193,
1150, 1115, 1085, 1030, 988, 930, 899, 822, 741. MS-CI, m/z (Irel.
(%)): 436.96 (100); 189.06 (86); 248.79 (51); 437.98 (32); 190.04
(13); 203.04 (12); 496.93 [M] (10); 213.02 (10); 496.09 (10);
135.92 (8).
IR (DCM)
m: 3732, 3501, 2976, 2945, 2871, 1729, 1663, 1625,
1459, 1389, 1362, 1328, 1277, 1231, 1193, 1158, 1115, 1042,
996, 942, 880, 753, 706. MS-CI, m/z (Irel. (%)): 485.04 [M] (100);
486.05 [M+1] (32); 316.90 (15); 483.04 (14); 191.04 (12); 487.08
(10); 175.05 (9); 135.01 (8); 501.14 (8); 311.85 (8).
3.46. (3b,18a,20b)-3-Hydroxy-11-oxo-olean-12-en-29-oic acid
(23)
KOH (16.0 g, 285 mmol) was added to diethylene glycol
(100 mL) and heated to 100 °C. Compound 1 (40 g, 85 mmol) was
added, and the solution was stirred at 220 °C for 2 h. Afterwards
the mixture was cooled rt. Water (500 mL) and concentrated HCl
were added to the cooled solution, adjusting it to pH ꢄ5. The solid
was filtered, washed with water (200 mL), and then extracted with
hot acetone (200 mL). The remaining white solid was filtered and
recrystallized from EtOH (2 ꢃ 1.5 L) to give 23 as colorless needles
(18.0 g, 43.7%, HPLC >99%). Mp 338–340 °C; (Ref. 52: 320–325 °C);
3.49. 1-Deoxy-1-{[(3b,18
a,20b)-3-hydroxy-11,29-dioxo-olean-
12-en-29-yl]amino}- -glucitol (26a)
D
Compound
1 (470 mg, 1.0 mmol) and D-glucitol (200 mg,
1.1 mmol) were transferred into a cold mixture of DCC (210 mg,
1.0 mmol) in DCM (50 mL) and the solution was stirred rapidly.
After 15 h DCM was evaporated in vacuo and the residue was dis-
solved in EtOAc (50 mL). After 24 h in the fridge the precipitate was
filtered, the mother liquor evaporated in vacuo and the residue was
purified by flash chromatography (DCM–MeOH gradient elution,
0–20%) to give 26a as a white powder (200 mg, 31%). Mp 193–
½
a 2D0 +74.4° (c 0.081, EtOH). 1H NMR (400 MHz, DMSO-d6) d 5.32 (s,
ꢂ
1H, 12-H), 3.00 (dd, J = 11.3, 4.3 Hz, 1H, 3-H), 2.46 (d, J = 13.3 Hz,
1H, 1-H), 2.30–2.25 (m, 2H, 9-H, 18-H), 1.89 (dd, J = 13.2, 3.8 Hz,

452
I. Beseda et al. / Bioorg. Med. Chem. 18 (2010) 433–454
195 °C. 1H NMR (200 MHz, DMSO-d6) d 7.22 (m, 1H), 5.28 (s, 1H),
4.48 (d, 1H), 4.19 (m, 1H), 4.09 (m, 1H), 3.39–0.20 (m, 33H), 1.08
(s, 3H), 1.02 (s, 3H), 0.76 (s, 6H), 0.64 (s, 3H), 0.46 (s, 3H), 0.42 (s,
3H); 13C NMR (50 MHz, DMSO-d6) d 199.1, 175.5, 169.7, 127.4,
76.5, 72.2, 71.9, 71.4, 69.4, 63.2, 61.1, 54.0, 47.4, 44.7, 42.9, 42.8,
42.1, 40.8, 38.7, 38.7, 37.2, 36.6, 32.1, 31.3, 30.4, 28.6, 28.3, 28.1,
2.30 (s, 1H), 2.22–0.65 (m, 21H), 1.35 (s, 3H), 1.12 (s, 3H), 1.10
(s, 6H), 0.98 (s, 3H), 0.79 (s, 3H), 0.78 (s, 3H); 13C NMR (50 MHz,
CDCl3) d 200.1, 175.5, 169.3, 134.5, 128.4, 116.3, 78.7, 61.8, 54.9,
48.1, 45.3, 43.6, 43.2, 41.8, 41.7, 39.1 (2C), 37.4, 37.0, 32.7, 31.9,
31.4, 29.6, 28.4, 28.1, 27.2, 26.4, 26.3, 23.3, 18.6, 17.4, 16.3, 15.6.
IR (DCM)
m: 3362, 2933, 2871, 1648, 1528, 1459, 1424, 1389,
26.9, 25.8 (2C), 22.9, 18.3, 17.1, 16.1, 15.9. IR (DCM)
m: 3319,
1362, 1328, 1277, 1208, 1189, 1088, 1034, 996, 919.
2929, 2868, 1656, 1640, 1532, 1459, 1386, 1366, 1328, 1308,
1208, 1193, 1135, 1077, 1038, 996, 922, 880.
3.53. (3b,18a,20b)-N-[3-(Dimethylamino)propyl]-N-
[(ethylamino)carbonyl]-3-hydroxy-11-oxo-olean-12-en-29-
3.50. (3b,18a,20b)-3-Hydroxy-N-(2-hydroxyethyl)-11-oxo-
amide (26e)
olean-12-en-29-amide (26b)
To a stirred suspension of 1 (470 mg, 1 mmol) in dry DCM
(10 ml) was added EDAC (230 mg, 1.2 mmol) and DMAP (150 mg,
1.2 mmol). The reaction mixture was stirred at rt for 30 min. Barbi-
turic acid (160 mg, 1.2 mmol) was added to the clear solution and
the stirring was continued at rt for 5 days. The reaction mixture
poured into 1 N HCl (40 ml) and extracted with mixture of DCM
(32 mL) and MeOH (8 mL). The organic layers were dried and the
solvent was evaporated in vacuo to give 26e as a white powder
(53 mg, 8.4%). 1H NMR (200 MHz, pyridine-d5) d 9.26 (m, 1H),
8.33 (s, 1H), 5.77 (s, 1H), 3.75 (m, 2H), 3.54 (m, 2H), 3.15 (m,
1H), 2.89 (m, 1H), 2.59–0.49 (m, 27H), 2.08 (s, 6H), 1.38 (s, 3H),
1.06 (s, 3H), 1,05 (s, 3H), 0.99 (s, 3H), 0.86 (s, 3H), 0.80 (s, 3H),
0.54 (s, 3H); 13C NMR (50 MHz, pyridine-d5) d 200.2, 178.7,
170.0, 158.5, 129.2, 78.3, 62.6, 55.7, 55.6, 49.5, 48.1, 46.5, 46.0,
44.0, 43.9, 43.9, 43.1, 40.2, 39.2, 38.1, 36.7, 33.9, 33.4, 32.5, 29.2,
29.2, 29.2, 28.6, 27.3, 27.1, 25.2, 25.2, 23.7, 19.3, 18.4, 17.3, 17.0,
15.0. HRMS, m/z: 626.4895 [M+1] (calcd for C38H64O4N3,
626.4887).
DCC (1.30 g, 6.0 mmol) and HOBt (1.00 g, 6 mmol) were dis-
solved in DCM (150 mL) and stirred for 30 min. Compound 1
(2.40 g, 5.0 mmol) was added to this solution and stirred for
20 min. 2-Aminoethanol (370 mg, 6.0 mmol was added and stirred
at rt for 12 h. The solvent was evaporated in vacuo and the residue
was dissolved in EtOAc (150 mL). After standing in the fridge for
12 h the solid material was filtered, the solvent evaporated in va-
cuo and the residue was purified by flash chromatography
(DCM–MeOH gradient elution, 0–10%) to give 26b as a white pow-
der (0.82 g, 32%). Mp 262–265 °C. 1H NMR (200 MHz, DMSO-d6) d
7.25 (s, 1H), 5.25 (s, 1H), 4.37 (m, 1H), 4.06 (m, 1H), 3.14 (s, 6H),
3.10 (s, 3H), 3.05–0.40 (m, 17H), 1.08 (s, 3H), 1.02 (s, 3H), 0.77 (s,
3H), 0.75 (s, 3H), 0.65 (s, 3H), 0.46 (s, 3H), 0.43 (s, 3H); 13C NMR
(50 MHz, DMSO-d6) d 199.0, 175.2, 169.6, 127.4, 76.5, 61.1, 59.8,
54.0, 47.5, 44,7, 42.8, 41.4, 40.8, 38.7, 38.5, 38.5, 37.2, 36.6, 32.1,
31.3, 30.5, 28.5, 28.4, 28.1, 26.9, 26.0, 25.9, 22.9, 18.2, 17.1, 16.2,
15.9. IR (DCM)
m: 3373, 2937, 2871, 1652, 1544, 1528, 1463,
1389, 1366, 1277, 1208, 1050, 1038, 996.
3.54. Ethyl N-[(3b,18a,20b)-3-hydroxy-11,29-dioxo-olean-12-
en-29-yl]glycinate (26f)
3.51. (3b,18
a,20b)-3-Hydroxy-11-N-prop-2-yn-1-ylolean-12-en-
29-amide (26c)
DCC (2.5 g, 12 mmol) and HOBt (1.8 g, 12 mmol) were dissolved
in DCM (150 mL) CH2Cl2 and stirred for 30 min. Glycyrrhetinic
acid 1 (4.7 g, 10 mmol) was added to the clear solution and stirred
for another 20 min. Glycine ethylester hydrochloride (1.7 g,
12 mmol) and TEA (3.0 g, 30 mmol) were added and stirred at rt
for 24 h. The solvent was evaporated and the residue was heated
with EtOAc (200 mL) to 50 °C and than put into the fridge. After
24 h the solid material was removed by filtration and solvent
was evaporated to give crude material. The residue was purified
by flash chromatography (DCM–MeOH gradient elution, 0–5%) to
give 26f as a white powder (5.2 g, 93%). Mp 87–91 °C. 1H NMR
(200 MHz, CDCl3) d 6.28 (m, 1H), 4.22 (q, 2H), 4.00 (m, 2H), 3.22
(m, 1H), 2.77 (m, 1H), 2.40–0.72 (m, 22H), 1.37 (s, 3H), 1.28 (t,
3H), 1.15 (s, 3H), 1.11 (s, 6H), 0.99 (s, 3H), 0.81 (s, 3H), 0.79 (s,
3H); 13C NMR (50 MHz, CDCl3) d 200.2, 176.2, 170.2, 169.2,
128.5, 78.7, 61.9, 61.5, 55.0, 47.8, 45.3, 43.6, 43.1, 41.7, 41.3,
39.1, 39.0, 37.3, 37.0, 32.7, 31.9, 31.4, 29.5, 28.4, 28.0, 27.2, 26.5,
Compound 1 (705 mg, 1.5 mmol), EDAC (350 mL, 1.8 mmol) and
TEA (355 mg, 3.5 mmol) were stirred in DCM (40 mL) at rt. HOBt
(275 mg, 1.8 mmol) was added to this mixture and stirred for
30 min, propargylamine (165 mg, 1.8 mmol) was then added and
the solution was stirred for 48 h at rt. The solvent was evaporated
in vacuo and the residue was purified by flash chromatography
(DCM–MeOH gradient elution, 0–15%) to give 26c as a white pow-
der (600 mg, 78%). Mp >260 °C. 1H NMR (200 MHz, CDCl3) d 6.21
(m, 1H), 5.67 (s, 1H), 4.06 (m, 2H), 3.20 (m, 1H), 2.75 (m, 1H),
2.32 (s, 1H), 2.23 (m, 1H), 2.20–0.70 (m, 19H), 1.35 (s, 3H), 1.12
(s, 3H), 1.10 (s, 6H), 0.98 (s, 3H), 0.79 (s, 3H), 0.78 (s, 3H), 0.67
(d, 1H); 13C NMR (50 MHz, CDCl3) d 200.2, 175.6, 169.3, 128.4,
79.9, 78.7, 71.5, 61.8, 54.9, 48.0, 45.4, 43.5, 43.2, 41.6, 39.2, 39.1,
37.3, 37.0, 32.7, 31.8, 31.4, 29.2, 29.1, 28.4, 28.1, 27.2, 26.4, 26.3,
23.3, 18.6, 17.4, 16.3, 15.6. IR (DCM) m: 3315, 2933, 2871, 1652,
1525, 1459, 1424, 1389, 1362, 1347, 1332, 1208, 1189, 1135,
1092, 1034, 996, 949, 922, 880. Anal. Calcd for C33H49NO3: C,
78.06; H, 9.73; N, 2.76; O. Found: C, 79.94; H, 9.66; N, 2.39.
26.3, 23.4, 18.7, 17.4, 16.4, 15.6, 14.2. IR (DCM) m: 3365, 2968,
1744, 1656, 1544, 1528, 1463, 1424, 1389, 1332, 1196, 1100,
1050, 1034, 1019, 996, 884, 868.
3.52. (3b,18
a,20b)-N-Allyl-3-hydroxy-11-oxo-olean-12-en-29-
amide (26d)
3.55. (3b18a,20b)-N-[2-(Dimethylamino)ethyl]-3-hydroxy-11-
oxoolean-12-en-29-amide (26g)
A
solution of 5d (380 mg, 0.7 mmol) and KOH (100 mg,
1.5 mmol) in MeOH (30 mL) was stirred at rt for 12 h. The solvent
was evaporated and the residue diluted with water and 2 N HCl
and extracted with DCM (3 ꢃ 30 mL). The organic layers were dried
and the solvent was evaporated in vacuo. The residue was purified
by flash chromatography (DCM–MeOH gradient elution, 5–20%) to
give 26d as a white powder (156 mg, 44%). Mp >260 °C. 1H NMR
(200 MHz, CDCl3) d 5.93 (m, 1H), 5.80 (m, 1H), 5.62 (s, 1H), 5.19
(m, 1H), 5.13 (m, 1H), 3.87 (m, 1H), 3.20 (m, 1H), 2.75 (m, 1H),
Preparation as described for 26b. Purification by flash chroma-
tography (DCM–MeOH gradient elution, 0–20%) gives 26g as a
white solid (89%). 1H NMR (200 MHz, CDCl3) d 7.12 (m, 1H), 5.57
(s, 1H), 3.76 (s, 1H), 3.35 (m, 1H), 3.21 (m, 1H), 3.03 (m, 1H),
2.73–0.55 (m, 23H), 2.20 (s, 6H), 1.35 (s, 3H), 1.10 (s, 3H), 1.00 (s,
6H), 0.93 (s, 3H), 0.76 (s, 3H), 0.73 (s, 3H); 13C NMR (50 MHz,
CDCl3) d 199.6, 176.0, 169.4, 128.0, 78.3, 62.0, 58.2, 55.3, 48.7,
45.5, 44.9 (2C), 43.5, 43.1, 41.4, 40.0, 39.2, 37.4, 37.0, 36.4, 32.8,

I. Beseda et al. / Bioorg. Med. Chem. 18 (2010) 433–454
453
31.9, 31.3, 29.4, 28.4, 28.1, 27.0, 26.6, 26.2, 23.3, 18.5, 17.4, 16.4,
15.7. IR (DCM) : 3342, 2833, 2787, 1656, 1544, 1528, 1463,
Acknowledgements
m
1389, 1362, 1328, 1208, 1193, 1166, 1131, 1092, 1046, 996, 957,
922, 880, 841. HRMS, m/z: 541.4374 [M+1] (calcd for C34H57O3N2,
514.4369).
The work was supported by the ZIT Zentrum für Innovation und
Technologie GmbH (Vienna Spot of Excellence: 182081). A.O. has a
Novartis Chair of Molecular and Systems Toxicology and was sup-
ported by the Swiss National Science Foundation (No. 31003A-
124912).
3.56. Ethyl 1-[(3b,18a,20b)-3-hydroxy-11,29-dioxoolean-12-en-
29-yl]piperidine-4-carboxylate (26h)
Supplementary data
Preparation as described for 26e. Purification by flash chroma-
tography (DCM–MeOH gradient elution, 0–20%) gives 26h as a
white solid (33%). 1H NMR (200 MHz, CDCl3) d 5.65 (s, 1H), 4.29–
4.05 (m, 4H), 3.15 (m, 1H), 3.10–0.63 (m, 29H), 1.34 (s, 3H), 1.24
(s, 3H), 1.20 (s, 6H), 1.11 (s, 3H), 1.09 (s, 3H), 0.98 (s, 3H), 0.78 (s,
3H); 13C NMR (50 MHz, CDCl3) d 200.1, 174.2, 173.8, 169.6,
128.5, 78.7, 61.7, 60.6, 54.9, 48.0, 45.2, 45.0, 44.4, 44.0, 43.8,
43.2, 41.0, 39.1, 39.0, 37.8, 37.0, 33.0, 32.8, 31.7, 28.4, 28.3, 28.2,
28.1, 27.2, 27.0, 26.7, 26.4, 23.1, 18.6, 17.5, 16.3, 15.6, 14.2. IR
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2009.10.036.
References and notes
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A
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X-ray diffraction data of crystals of 1ꢁDMSO, 2aꢁsolv (a disor-
dered solvate from acetone/ethanol), 2b, 10d, 15ꢁDMSO, 19, and
20 were collected on Bruker Smart APEX CCD diffractometers using
graphite-monochromated Mo K
-scan frames covering complete spheres of the reciprocal space
with hmax = 27–30.5°. After data integration with program SAINT
corrections for absorption, k/2 effects, and crystal decay were ap-
a radiation (k = 0.71073 Å) and 0.3°
x
+
53
plied with program SADABS
.
The structures were solved by direct
methods (program SHELXS97) and refined on F2 with program SHEL-
XL97. All non-hydrogen atoms were refined anisotropically. Most
H atoms were placed in calculated positions and thereafter treated
as riding. A torsional parameter was refined for each methyl group.
For compound 2aꢁsolv the disordered solvent (acetone/ethanol)
54
was squeezed with program PLATON prior to final refinement The
crystal structures of 1ꢁDMSO and 15ꢁDMSO are practically isostruc-
tural with 15ꢁDMSO having in 11-position a CH2 group instead of in
1ꢁDMSO a C@O group. Crystal data and experimental are given in
Table 1. A complete set of structural drawings is given in the Sup-
plementary data (Figs. S1–S7). For a brief discussion of selected
structural aspects see chapter ‘X-ray crystal structure analyses’
above. CCDC 699828–699834 contain the supplementary crystallo-
graphic data for this paper. These data can be obtained free of
charge from The Cambridge Crystallographic Data Centre via
www.ccdc.cam.ac.uk/data_request/cif.

454
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