Synthesis of novel 3-amino and 29-hydroxamic acid derivatives of glycyrrhetinic acid as selective 11β-hydroxysteroid dehydrogenase 2 inhibitors

Article abstract of DOI:10.1016/j.bmc.2010.08.046

Glycyrrhetinic acid, the metabolite of the natural product glycyrrhizin, is a well known nonselective inhibitor of 11β-hydroxysteroid dehydrogenase (11β-HSD) type 1 and type 2. Whereas inhibition of 11β-HSD1 is currently under consideration for treatment of metabolic diseases, such as obesity and diabetes, 11β-HSD2 inhibitors may find therapeutic applications in chronic inflammatory diseases and certain forms of cancer. So far, no selective 11β-HSD2 inhibitor has been developed and neither animal studies nor clinical trials have been reported based on 11β-HSD2 inhibition. Starting from the lead compound glycyrrhetinic acid, novel triterpene type derivatives were synthesized and analyzed for their biological activity against overexpressed human 11β-HSD1 and 11β-HSD2 in cell lysates. Several hydroxamic acid derivatives showed high selectivity for 11β-HSD2. The most potent and selective compound is active against human 11β-HSD2 in the low nanomolar range with a 350-fold selectivity over human 11β-HSD1.

Full text of DOI:10.1016/j.bmc.2010.08.046

Bioorganic & Medicinal Chemistry 18 (2010) 7522–7541
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis of novel 3-amino and 29-hydroxamic acid derivatives of
glycyrrhetinic acid as selective 11b-hydroxysteroid dehydrogenase 2 inhibitors
Christian Stanetty a, , Laszlo Czollner b, , Iris Koller a, Priti Shah b, Rawindra Gaware b, Thierry Da Cunha c,
Alex Odermatt c, Ulrich Jordis b, Paul Kosma a, Dirk Claßen-Houben d,
⇑
a Department of Chemistry, University of Natural Resources and Applied Life Sciences, Muthgasse 18, 1190 Vienna, Austria
b Institute of Applied Synthetic Chemistry, University of Technology, Getreidemarkt 9, 1060 Vienna, Austria
c Division of Molecular and Systems Toxicology, Department of Pharmaceutical Research, University of Basel, Klingelbergstrasse 50, 4056 Basel, Switzerland
d Onepharm Research and Development GmbH, Veterinärplatz 1, 1210 Vienna, Austria
a r t i c l e i n f o
a b s t r a c t
Article history:
Glycyrrhetinic acid, the metabolite of the natural product glycyrrhizin, is a well known nonselective
inhibitor of 11b-hydroxysteroid dehydrogenase (11b-HSD) type 1 and type 2. Whereas inhibition of
11b-HSD1 is currently under consideration for treatment of metabolic diseases, such as obesity and dia-
betes, 11b-HSD2 inhibitors may find therapeutic applications in chronic inflammatory diseases and cer-
tain forms of cancer. So far, no selective 11b-HSD2 inhibitor has been developed and neither animal
studies nor clinical trials have been reported based on 11b-HSD2 inhibition. Starting from the lead com-
pound glycyrrhetinic acid, novel triterpene type derivatives were synthesized and analyzed for their bio-
logical activity against overexpressed human 11b-HSD1 and 11b-HSD2 in cell lysates. Several
hydroxamic acid derivatives showed high selectivity for 11b-HSD2. The most potent and selective com-
pound is active against human 11b-HSD2 in the low nanomolar range with a 350-fold selectivity over
human 11b-HSD1.
Received 28 May 2010
Revised 22 August 2010
Accepted 27 August 2010
Available online 28 September 2010
Keywords:
11b-Hydroxysteroid dehydrogenase
11b-HSD1
11b-HSD2
Selective inhibitor
Hydroxamic acid
Glycyrrhizin
Glycyrrhetinic acid
Carbenoxolone
Ó 2010 Elsevier Ltd. All rights reserved.
Glucocorticoid metabolism
Cortisone metabolism
Inflammation
Chronic inflammatory disease
Cancer
1. Introduction
macrophages. 11b-HSD2 is a NAD+-dependent dehydrogenase
and inactivates 11b-hydroxyglucocorticoids by oxidation in kid-
11b-Hydroxysteroid dehydrogenases (11b-HSDs) are micro-
somal enzymes belonging to the short-chain dehydrogenase/
reductase (SDR) family. In humans and rodents, two isozymes,
11b-HSD1 and 11b-HSD2 have been identified which catalyze the
interconversion of active 11b-hydroxyglucocorticoids and their
inactive 11-keto counterparts (Fig. 1).1,2 11b-HSD1 NADPH-depen-
dently activates the 11-ketosteroids cortisone (human) and 11-
dehydrocorticosterone (rodents) to cortisol and corticosterone,
respectively. 11b-HSD1 is highly expressed in many glucocorticoid
target tissues, including liver, adipose tissue, skeletal muscle and
ney, colon, placenta and inflamed tissue. In classical aldosterone
target tissues, such as renal cortical collecting ducts and distal
colon, 11b-HSD2 protects the mineralocorticoid receptor from
activation by glucocorticoids.3,4
Active glucocorticoids play a vital role in the regulation of
carbohydrate, protein, lipid, and bone metabolism, the maturation
and differentiation of cells, and the modulation of inflammatory re-
sponses and stress. These cortisol effects are mediated by the acti-
vation of glucocorticoid receptors. The local concentration of active
cortisol in specific tissues is tuned by the pre-receptor metabolism
performed by 11b-HSDs. Cortisone in plasma provides a pool of
inactive precursor that can be converted to active glucocorticoids
at sites where 11b-HSD1 reductase activity is predominant. Local
excess or deficiency of glucocorticoids due to impaired function
of 11b-HSDs has been associated with specific diseases, pointing
to potential therapeutic indications for selective inhibitors of both,
11b-HSD1 and 11b-HSD2.
⇑
Corresponding author at present address: Savira pharmaceuticals GmbH,
Veterinärplatz 1, 1210 Vienna, Austria. Tel.: +43 1 250 77 5900; fax: +43 1 250
77 5999.
E-mail address: dirk.classen-houben@savira.at (D. Claßen-Houben).
These authors contributed equally to this publication.
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.08.046

C. Stanetty et al. / Bioorg. Med. Chem. 18 (2010) 7522–7541
7523
Elevated 11b-HSD1-dependent glucocorticoid activation is
associated with multiple features of the metabolic syndrome like
insulin and leptin resistance, visceral obesity, dyslipidemia, and
type 2 diabetes.5–7 Over-expression of 11b-HSD1 within adipose
tissue in transgenic mice results in insulin resistance, hyperlipid-
emia and visceral obesity, whereas 11b-HSD1 knockout mice show
decreased triglyceride and cholesterol levels and resistance to
stress-induced hyperglycemia.8,9
Glycyrrhetinic acid (GA), the metabolite of the natural product
glycyrrhizin, inhibits both, 11b-HSD1 and 11b-HSD2.10–12 In
homogenates of rat liver and kidney, a 20- to 40-fold selectivity
for 11b-HSD1 over 11b-HSD2 was reported for 11-deoxo-glycyrrh-
etinic acid, glycyrrhetinic acid 3-hemiphthalate and glycyrrhetol,
based on IC50 values.13 Other compounds that are not triterpenes
have been identified in high throughput screening campaigns
and lead optimization programs as selective 11b-HSD1 inhibitors
by different pharmaceutical companies and are currently being
developed for the treatment of metabolic diseases.14–21
Whereas beneficial effects have been reported for the inhibition
of 11b-HSD1 in metabolic diseases such as obesity and diabetes,
the target 11b-HSD2 has been associated with (chronic) inflamma-
tory diseases and cancer. Several studies found an over-expression
of 11b-HSD2 in inflamed tissues and in many cancer cell lines. It
has been demonstrated that in rheumatoid arthritis synovial cells
have a reduced capacity of local reactivation of cortisone which
has been associated with increased activity of 11b-HSD2.22–26
Thus, the impaired immunomodulatory effect of glucocorticoids
may be improved upon pharmacological inhibition of 11b-HSD2.
However, no selective 11b-HSD2 inhibitor has been developed so
far and neither animal studies nor clinical trials have been exe-
cuted based on the selective inhibition of 11b-HSD2. To our knowl-
edge, the only compound reported to exhibit noteworthy
selectivity for 11b-HSD2 is a hydroxyethylamide derivative of glyc-
yrrhetinic acid reported by Vicker et al.27 with only 36% inhibition
of rodent 11b-HSD1 but 92% inhibition of rodent 11b-HSD2 at
hydroxamic acid motifs on the GA scaffold and finally the combina-
tion of both modifications for selected compounds.
2.2. Synthetic modification at the 3-amino moiety
Within this part of GA-based structures a set of related 3-ami-
no-GA derivatives has been synthesized varying the relative
stereochemistry at the 3-position and the NH-acidity (nucleophi-
licity), respectively, and attaching additional carboxylic acid
moieties. In order to facilitate workup and purification of the inter-
mediates, a synthetic strategy with C29-protection was envisaged
accepting the additional deprotection step of the ester protecting
group. The syntheses of the relevant starting materials, 3-oximo-
and 3-methoximo GA diphenylmethyl esters (1 and 2) have been
described previously.28 The oximes 1 and 2 were reduced to the
corresponding amines and methoxyamines (3a/3b and 4a/4b)28
with tBuNH2ꢀBH3/TiCl3 and tBuNH2ꢀBH3/HCl, respectively (Scheme
1). Both pairs of diastereomers were successfully separated by
chromatography on a multigram scale. The single diastereomeric
esters were deprotected to the corresponding free acids with
TFA/anisole or by catalytic hydrogenation and isolated as hydro-
chlorides (5a/5b36 and 6a/6b). Starting from the 3-amino-esters
3a and 3b a variety of 3-(sulfon)amido GA derivatives was pre-
pared via standard acylation or sulfonylation protocols and subse-
quent hydrogenolysis (7a–9b and 10–12). In total, a subset of
thirteen 3-amino GA derivatives was prepared varying in the ste-
reochemistry of the 3-position and the acidity of the NH group
(Scheme 1). Furthermore, compounds 10–12 carry an additional
carboxylic acid moiety and thus mimic the hemisuccinate of GA,
carbenoxolone (Fig. 1).
2.3. Synthetic modifications of the C29 hydroxamic acid motif
In addition to the ring-A modifications, the C29 carboxylic acid
moiety has been widely varied in previous publications, wherein
the C29 hydroxamic acid motif was considered as one of the most
relevant C29-modification for inhibition of 11b-HSD2.30 Hence, a
systematic variation of the substitution pattern of the hydroxamic
acid motif of GA and its 3-O-acetylated derivative 13 was per-
formed to allow a first insight into structure–activity relationships.
To come up with a reliable protocol for the conversion of GA-type
carboxylic acids to hydroxamic acids, several methods have been
evaluated with varying success. A literature protocol based on
PPAA-coupling-reagent (1-propanephosphonic acid cyclic anhy-
dride)37 describing the conversion of GA to the corresponding
hydroxamic acid 20 gave significantly lower yields in our hands
(also in the analogous attempt to prepare 20). The attempted con-
version of a readily formed mixed anhydride (ethyl carbonate)38 to
the corresponding hydroxamic acid lead only to recovery of start-
ing acid 13. It turned out that the installation of the hydroxamic
acid motif at the C29 position of GA is best accomplished via for-
mation of the corresponding acid chloride and treatment with rel-
evant hydroxylamine (as hydrochloride salt). By applying these
conditions a large set of 3-O-GA based hydroxamic acids has been
prepared (Scheme 2). While O-substituted and N-substituted
hydroxamic acids could be directly synthesized from or via the acid
chloride 13a in good yields, the unsubstituted hydroxamic acid
motif (e.g., 20) was most reliably installed in two steps via the cor-
responding O-benzyl hydroxamic acid and subsequent hydrogen-
olysis. Attempts to analogously react acid chloride 13a directly
with NH2OHꢀHCl lead to irreproducible low yields and difficult
purification. It is noteworthy that the conversion of 13a into 20
with NH2OHꢀHCl was successfully implemented under TEA/TMSCl
conditions.39 However, this latter method could not be applied to
the following cases of 3-N-derivatives of GA, which were therefore
again synthesized in two steps (vide infra).
10 lM. The reported IC50 value of 0.004 nM for 11b-HSD2 is very
low. However, using human 11b-HSD2, an IC50 value in the lower
micromolar range was obtained (own observations).28
In previous work, the selectivity of several GA derivatives has
been studied using 11b-HSD1 and 11b-HSD2 isolated from rat liver
and kidney, respectively.13,27,29,30 Some of the published com-
pounds were selective for rat 11b-HSD1 over 11b-HSD2 and also
showed inhibitory activity against human 11b-HSD1. Other studies
demonstrated significant species-specific differences in the activi-
ties of inhibitors and the conversion of substrates by 11b-HSD1,
implicating that the primary biological assay should be performed
with the human enzymes.31–33
GA, as a nonselective inhibitor of both isozymes is a valuable
starting point for the development of selective 11b-HSD2 inhibi-
tors. Here, we describe the synthesis of novel hydroxamic acid
derivatives of GA, as well as their inhibitory activity against human
recombinant 11b-HSD1 and 11b-HSD2.
2. Results and discussion
2.1. Chemistry
Within the framework of ongoing studies various modifications
of the GA A-ring, including the introduction of sulfur, halides, dou-
ble bonds and additional hydroxy groups, the installation of an epi-
sulfide and ring expansions have already been reported.28,34
Furthermore, the synthesis and inhibitory potential of a variety
of C29 amides28 and a large number of Ugi-type-products have
been published.35 In the current report we discuss the synthesis
of a variety of 3-amino-GA derivatives, the installation of C-29

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C. Stanetty et al. / Bioorg. Med. Chem. 18 (2010) 7522–7541
COODpm
COODpm
COOH
H
H
O
H
O
H
O
H
a or b
c or d
Cl
H
H
H
R
R
R
N
N
H
N+
5a R=H (α)
H
3a R=H (α)
3b R=H (β)
H2
5b R=H (β)
6a R=OMe (α)
6b R=OMe (β)
1 R=OH
2 R=OMe
4a R=OMe (α)
4b R=OMe (β)
e,f
e,f
e,f
COOH
COOH
COOH
H
H
H
O
O
O
H
H
H
R1O
R
N
H
R
Y
N
N
H
H
H
H
H
O
10 Y=CO, R1=H
7a R=Ac
8a R=Ms
9a R=Tf
7b R=Ac
8b R=Ms
9b R=Tf
11 Y=SO2, R1=Me
g
12 Y=SO2, R1=H
Scheme 1. Towards 3-(sulfon)amido-GA derivatives. Reagents and conditions: (a) 1, TiCl3, tBuNH2ꢀBH3 (58% 3b, 17% 3a); (b) tBuNH2ꢀBH3, HCl (66% 4b, 14% 4a); (c) H2, Pd/C
65–83% for 5a, 5b, 6a; (d) TFA, anisole 85% for 6b; (e) Ac2O, MsCl, Tf2O, succinic anhydride or MeOOC(CH2)2SO2Cl in DCM/TEA 0 °C to rt (42–95%); (f) H2, Pd/C (50–99%);
(g) NaOH in MeOH/H2O, 97% for 12.
hydroxamic acid derivatives via the corresponding acid chlorides
(in situ). As explained in the 3-OAc series, the N-methyl hydroxa-
mic acids (28a–29b) were prepared directly and the unsubstituted
hydroxamic acids (32a–33b) were prepared via the corresponding
O-benzyl-hydroxamic acids (30a–31b). For the modification of the
3-methoxyamino-GA 4a/4b with the hydroxamic acid motif an-
other strategy was envisaged via initial conversion of 3-keto GA
34 to the corresponding hydroxamic acid derivatives (36 via 35
and 37) and subsequent transformation to the 3-methoxyamino
compounds. This way the 3-keto- and 3-oximo GA hydroxamic
acid derivatives (35–41) could be included in this investigation.
It is noteworthy that in the purification of crude 3-keto
hydroxamic acid species 35 and 37 small amounts of remaining
3-keto-GA 34 turned out to be very difficult to separate by stan-
dard normal-phase chromatography or aqueous extraction. Grati-
fyingly, by passing contaminated 35 and 37 over a short bed of
amino-phase the contaminating amounts of 34 could be separated.
With pure 35 and 36 in hands the corresponding oximes were then
prepared by standard procedures. The preparation of targeted 3b-
methoxyamino GA-N-methyl-hydroxamic acid 42 could eventually
be accomplished by tBuNH2ꢀBH3-reduction of oxime ether 41 and
purification of diastereomers by column chromatography and rep-
recipitation. The analogous reduction of oxime ether 39 (unsubsti-
tuted hydroxamic acid) failed, however, due to the hydrolytic
lability of the free hydroxamic acid.
O
O
OH
OH
OH
OH
O
HO
11β-HSD1
11β-HSD2
H
H
H
H
H
H
O
O
cortisol
cortisone
O
OH
H
O
O
H
H
HO
O
O
glycyrrhetinic acid (GA)
carbenoxolone (CBX)
Figure 1. 11b-HSD enzymes catalyze the interconversion of cortisone and cortisol.
Glycyrrhetinic acid and carbenoxolone represent nonselective 11b-HSD inhibitors
that serve as lead molecules in the present study.
2.4. Synthesis of selected 3-amino-GA based C29 hydroxamic
acid derivatives
2.5. Biology
Based on preliminary results from the 11b-HSD screening, some
structural elements (TfNH–, AcNH– and MeONH– for C3 and
–CONHOH and –CON(Me)OH) for C29) were selected to be for-
mally combined as new compounds. Two different strategies have
been applied (Scheme 3).
Glycyrrhetinic acid (GA) is a potent and nonselective inhibitor
of both 11b-HSD isozymes. Shimoyama et al. reported a lower
IC50 value using rat hepatic 11b-HSD1 homogenate (90 2 nM)
compared to rat renal 11b-HSD2 homogenate (360 2 nM).13 Pot-
ter et al. reported 85% inhibition of rat 11b-HSD1 and complete
For the (sulfon)amide type compounds the optimized procedure
from the 3-AcO-GA series was thus used to install the hydroxamic
acid moieties as the final reaction step. The corresponding carbox-
ylic acids (7a/7b and 9a/9b) were therefore converted to the
inhibition of rat 11b-HSD2 at a concentration of 10 lM of
GA.27,29,30,40 We evaluated the inhibitory activity of GA and its
derivatives against recombinant human 11b-HSD1 and 11b-
HSD2. Using a tenfold lower inhibitor concentration (1 lM) we

C. Stanetty et al. / Bioorg. Med. Chem. 18 (2010) 7522–7541
7525
R2
O
R2
N
O
O
X
N
OR1
OR1
H
H
H
b
d
O
H
O
H
O
H
14 R1=R2=Me
21 R1=R2=Me
H
H
H
15 R1=H, R2=Me
22 R1=H, R2=Me
23 R1=H, R2=tBu
24 R1=Me, R2=H
25 R1=All, R2=H
26 R1=Bn, R2=H
27 R1=R2=H
AcO
AcO
HO
16 R1=H, R2=tBu
17 R1=Me, R2=H
18 R1=All, R2=H
19 R1=Bn, R2=H
20 R1=R2=H
13 X=OH
13a X=Cl
a
c
Scheme 2. Towards 3-hydroxy-GA hydroxamic acid derivatives. Reagents and conditions: (a) SOCl2, reflux, 99%; (b) relevant hydroxylamines as hydrochloride salts, DCM,
TEA, 40–90%; (c) H2, Pd/C, THF, 94%; (d) KOH, MeOH, 30–99%.
O
O
O
H
N
N
OH
O
H
N
OBn
OH
OH
H
H
H
a
b
c
O
H
O
H
O
H
H
H
H
H
R
R
R
N
H
N
N
H
H
28a R=Ac (α)
28b R=Ac (β)
29a R=Tf (α)
29b R=Tf (β)
7a R=Ac (α)
7b R=Ac (β)
9a R=Tf (α)
9b R=Tf (β)
30a R=Ac (α)
30b R=Ac (β)
31a R=Tf (α)
31b R=Tf (β)
32a R=Ac (α)
32b R=Ac (β)
33a R=Tf (α)
33b R=Tf (β)
R1
N
R
N
OH
O
O
O
N
OR2
OH
H
H
H
O
O
H
O
H
g
d
f
3-keto-
GA
H
H
H
X
O
O
N
N
H
H
38 R=H, X=OH
39 R=H, X=OMe
40 R=Me,X=OH
41 R=Me, X=OMe
35 R1=H, R2=Bn
36 R1=R2=H
34
42
e
37 R1=Me, R2=H
Scheme 3. Synthesis of 3-amino-GA based hydroxamic acid derivatives. Reagents and conditions: (a) SOCl2, reflux, NH(Me)OHꢀHCl DCM, TEA (61–81%); (b) SOCl2, reflux,
BnONH2, DCM, TEA (81–93%); (c) H2, Pd/C in MeOH or MeOH/EtOAc with 1% AcOH (40–64%); (d) SOCl2, reflux; BnONH2 or HN(Me)OHꢀHCl, DCM, TEA; (e) H2, Pd/C in MeOH
56%); (f) NH2OHꢀHCl in pyridine or NH(Me)OHꢀCl, NaOAc in CHCl3/MeOH; (g) tBuNH2ꢀBH3, dioxane/EtOH, 3 M HCl.
found an inhibition of 83.9% of human 11b-HSD1 and 93.6% of hu-
man 11b-HSD2 for GA. The licensed drug carbenoxolone (CBX) had
comparable potency with 87.7% inhibition of 11b-HSD1 and 98.2%
inhibition of 11b-HSD2. As the lead compounds GA and CBX
showed significant inhibition of 11b-HSD1 and 11b-HSD2 at
Val180, Tyr183, Leu217, Ala223, Ala226, and Val227), specific
interactions are formed with the carboxylic acid oxygen atom, the
11-keto function and the carbonyl oxygen of the ester between GA
and succinic acid. The carboxylic acid of GA interacts with the
hydrogen bond donor functions of Tyr183 and the 20-hydroxy-
group of the ribose of the nicotinamide nucleotide of NADP+. The
11-keto function acts as a hydrogen bond acceptor for the hydroxy
group of Ser170 and the backbone amide of Ala172. Finally the car-
bonyl oxygen atom of the ester between GA and succinic acid
forms a hydrogen bond from the amide nitrogen of Leu217 as well
as from a conserved water molecule. The free acid function of the
succinate is not well resolved in the crystal structure which might
be due to high conformational flexibility and lack of specific inter-
actions. Based on this structural information a series of different
modifications of the carboxylic acid in position 29 was synthesized
and biologically evaluated (Table 1).
1
1
l
l
M, novel derivatives were first screened at a concentration of
M. As soon as first highly active compounds were found further
derivatives were screened at 0.2 lM. The activity of the com-
pounds is summarized in Tables 1–3, showing the residual conver-
tive activity of both isoenzymes. IC50 values were determined for
selected 11b-HSD2 selective compounds that comprised a low
residual acitivty for 11b-HSD2 and a high residual activity for
11b-HSD1.
The crystal structure of 11b-HSD1 in complex with carbenoxo-
lone (CBX) and NADP+ was analyzed for necessary and potential
interactions between inhibitor and protein (PDB ID: 2BEL).41,42 Be-
sides multiple hydrophobic interactions of the triterpene core with
surrounding amino acids (Thr124, Leu126, Leu171, Ala172, Tyr177,
The dipeptide derivatives 43 and 44 synthesized using Ugi-type
multiple component reaction,35 as well as the glycine amide deriv-

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C. Stanetty et al. / Bioorg. Med. Chem. 18 (2010) 7522–7541
Table 1
Amide type derivatives (C29) of GA and their inhibition of 11b-HSD1 and 11b-HSD2
O
R1
R2
N
H
O
H
HO
H
Compound
R1
R2
Concd (nM)
% Residual conversion (mean SD)
11b-HSD2 11b-HSD1
GA
CBX
43
44
45
1000
1000
1000
1000
1000
1000
1000
1000
1000
6
2
109
89
79
59
59 19
13
2
2
1
9
3
1
5
16
12
109 15
79
97
64 19
97
9
53 14
2
4
CH2CONHCH2COOH
CH2CONHCH2COOH
CH2COOH
CH2CH2N(CH3)2
CH2CH2OH
H
CH2CH2OH
CH2COOH
4
6
H
H
H
H
H
46
47
5
5
48a
27
1
2
OH
a
Tested as 3-acetate.
ative 45 were not active, neither against 11b-HSD1 nor against
11b-HSD2. Weak activity on both enzymes was observed for the
dimethylaminoethyl amide derivative 46. The hydroxyethyl amide
derivative 47 showed a complete loss of activity against 11b-HSD1
whereas the activity against 11b-HSD2 was significantly reduced,
compared to GA. The free amide 48 had activity in a similar range
compared to GA, but with a slightly preferential inhibition of 11b-
HSD1 over 11b-HSD2. The most potent compound for 11b-HSD2
was the hydroxamic acid derivative 27, which in addition showed
reasonable selectivity over 11b-HSD1 (Table 1). Determination of
IC50 values (Table 4) confirmed the high activity and demonstrated
an approximately 10-fold selectivity for 11b-HSD1 over 11b-HSD2.
In summary, many modifications of position 29 lead to a partial
or even complete loss of activity against both enzymes at concen-
and the 3a- and 3b-methoxyamine derivatives 6a and 6b inhibited
both enzymes with a preference for 11b-HSD2. The amino-carben-
oxolone derivatives 10–12 did not enhance enzyme inhibition or
selectivity compared to the parent CBX.
In summary, activity against 11b-HSD2 can be retained with
small modifications of position 3. The hydrogen bond acceptor
seen in the crystal structure of 11b-HSD1 complexed with CBX
and NADP+ has proven to be favorable for activity. The 3b-acetate
derivative 13 was the most selective compound within this series.
The findings above with the modifications of position 3 and 29 and
the published influence of a modification of position 1130 were
combined and led to the synthesis of a series of hydroxamic acids,
which retain the native keto function in position 11 (Table 3).
Position 3 was modified with the residues found to be active
within the above series of compounds and the hydroxamic acid
was substituted on the nitrogen or oxygen atom. The 3-acetate
of the hydroxamic acid 20 was twice as active as the position 3
unsubstituted hydroxamic acid 27 (Table 4). Methylation of the
hydroxamic acid oxygen atom led to compound 24 and a signifi-
cant loss in 11b-HSD2 activity. In contrast, methylation of the
hydroxamic acid nitrogen atom resulting in compounds 15 (3-ace-
tate) and 22 had no influence on 11b-HSD1 activity but increased
the 11b-HSD2 activity significantly. Interestingly, methylation of
both nitrogen and oxygen (compound 14) as well as the substitu-
tion of the hydroxamic acid oxygen with an allyl function (18) led
to a significant decrease of inhibitory activity (Table 4). Whereas
the N-methyl-hydroxamic acid 15 was the most active and selec-
tive compound, more spacious substituents like an N-tert-butyl
group in compound 16 were not accepted in this position.
Replacement of the 3b-O-acetyl function of compound 15 with a
3b-N-acetyl group resulted in the comparably active compound
28b.
trations of 1 lM. Exceptions are the free amide 48, where the activ-
ity is retained for both enzymes, and the hydroxamic acid
derivative 27, which is the most potent and selective compound
in this series with IC50 values of 122 nM and 1157 nM for
11b-HSD2 and 11b-HSD1, respectively, resulting in an approxi-
mately 10-fold selectivity (Table 4).
The 3-acetate 13 has already been suggested as an inhibitor of
11b-HSD isozymes based on a pharmacophore model.43 The
compound does not have the second carboxylic acid function pres-
ent in CBX; however, observations from the crystal structure of
11b-HSD1 with CBX suggested that this group is not involved in
essential interactions. Compound 13 and further modifications
(O-based and N-based) of the 3-position were obtained and sub-
jected to biological testing (Table 2). Compound 13 retains the po-
tential hydrogen bond acceptor function seen in the crystal
structure of 11b-HSD1 with CBX and is weakly active on both en-
zymes, with a preference for 11b-HSD2. Based on the fact that
the ester oxygen atom does not participate in the interaction net-
work with the protein, the corresponding 3b-acetamide 7b was
In summary, modification of position 3 of the hydroxamic acid
derivatives had only a minor impact on the selectivity and activity
of the compounds. The substitution of the 3-hydroxy group of the
hydroxamic acid derivatives led in all cases to a significant loss of
activity. Introduction of a methyl substituent at the hydroxamic
acid nitrogen atom resulted in compounds 15 and 22 with signifi-
cantly increased activity and selectivity toward 11b-HSD2.
synthesized. Both 7b but also the 3a-acetamide 7a were active
on 11b-HSD1 and 11b-HSD2. Whereas the 3
a
-derivative showed
similar activity on both enzymes, the 3b-derivative preferentially
inhibited 11b-HSD2. The free 3b-amine derivative 5b, which does
not have the hydrogen bond acceptor function, was only a weak
inhibitor. The 3-keto-GA 34, the 3-methoxyimine derivative 49,

C. Stanetty et al. / Bioorg. Med. Chem. 18 (2010) 7522–7541
7527
Table 2
Derivatives of the 3 hydroxy group of GA and their inhibition of 11b-HSD1 and 11b-HSD2
O
OH
H
O
H
H
bR
aR
Compound
aR
bR
Concd (nM)
% Residual conversion (mean SD)
11b-HSD2 11b-HSD1
*
GA
H
H
1000
1000
6
2
16
2
HO
HO
O
*
CBX
2
1
12
4
O
O
*
3b
6a
H
1000
200
42
5
2
2
15
43
2
3
H2N
O
O
*
*
H3C
N
H
H
O
O
*
H3C
N
H
6b
7a
H
200
2
4
1
1
33
4
9
2
H
1000
H3C
N
H
7b
8a
8b
9a
9b
H
1000
200
200
200
200
0.2 0.4
36 15
32 29
58 45
99 19
87 21
*
*
H3C
N
H
O
O
S
H
4
4
5
7
1
1
*
H3C
N
H
O
O
S
H
H3C
N
H
O
O
S
H
50
22
*
F3C
N
H
O
O
S
H
*
F3C
N
H
O
HO
*
10
11
H
H
H
1000
1000
13
3
4
1
6
1
2
2
N
H
O
O
O
O
S
*
H3C
HO
N
H
O
O
O
S
*
12
13
1000
1000
40
14
8
1
5
4
9
N
H
O
O
H
53
*
H3C
O
*
34
49
1000
1000
1
3
2
1
36 11
37 16
O
O
*
N

7528
C. Stanetty et al. / Bioorg. Med. Chem. 18 (2010) 7522–7541
Table 3
Hydroxamic acid derivatives of GA and their inhibition of 11b-HSD1 and 11b-HSD2
O R3
O
N
R2
H
O
H
R1
H
Compound
R1
R2
R3
Concd (nM)
% residual conversion (mean SD)
11b-HSD2 11b-HSD1
*
GA
1000
1000
6
1.5
16
2
HO
HO
O
*
CBX
2
1
12
4
O
O
O
14
15
CH3
CH3
CH3
H
200
72
3
1
1
95 22
51 11
*
*
*
H3C
H3C
O
O
O
O
O
1000
16
17
18
C(CH3)3
H
200
1000
200
85 14
36 40
86
38
3
9
H3C
HO
*
H
H
CH3
O
CH2CH@CH2
84
1
2
2
99 12
*
*
H3C
O
O
O
20
H
H
1000
48
8
H3C
*
22
27
CH3
H
H
H
200
7
2
2
71 13
53 14
HO
HO
*
1000
1.5
O
28b
29a
CH3
CH3
H
H
200
200
4
3
3
72 12
*
H3C
N
H
O
O
S
82
88
8
*
F3C
N
H
O
O
S
29b
42
CH3
CH3
H
H
200
200
44 40
30 18
71 27
*
F3C
H3C
N
H
O
*
N
H
93
9
human 11b-HSD2 reported to date. As synovial cells have a re-
duced capacity of local reactivation of cortisone in rheumatoid
arthritis, which is expected to result from an increased 11b-HSD2
activity the reported compounds should prove useful as mechanis-
tic tools for further anti-inflammatory in vitro and in vivo studies.
3. Conclusions
Using glycyrrhetinic acid as a lead compound and starting point,
novel highly potent and selective inhibitors of human 11b-HSD2
have been identified. The data generated lead to a significant
extension of the knowledge about structure–activity relationship
of glycyrrhetinic acid derivatives as inhibitors of human 11b-
hydroxysteroid dehydrogenases. The selectivity of synthesized
compounds for 11b-HSD2 over 11b-HSD1 ranges from 0.4 to 350,
spanning nearly three orders of magnitude. The compounds and
information will be used for the further optimization and improve-
ment of potency of the compound class. Compound 15 is the most
potent (IC50 value of 2.9 nM) and selective (350-fold) inhibitor of
4. Experimental
4.1. General
Compounds 1, 2, 13, 13a, and 34 have been prepared as
reported before.28 Compound 27 was prepared according to a liter-
ature protocol.37 Whenever reasonable, solvents were purified and

C. Stanetty et al. / Bioorg. Med. Chem. 18 (2010) 7522–7541
7529
Table 4
IC50 values and selectivities of GA and novel selective 11b-HSD2 inhibitors
COX
H
O
H
H
R
Compound
R
COX
11b-HSD2
11b-HSD1
95% CI
Selectivity HSD2/HSD1
IC50 (nM)
257
95% CI
IC50 (nM)
778
*
O
O
O
O
HO
GA
6a
6b
186–354
631–960
49–78
3
OH
OH
OH
OH
O
*
*
N
4.9
2.8–8.6
61.9
42.7
13
3
H
O
N
H
14.5
9.0–23.4
12.7–144
O
7b
458
385–546
825
634–1076
2
*
H3C
N
H
O
O
O
O
O
O
OH
N
13
15
20
750
2.9
416–1350
0.65–13.1
48–79
7138
1012
626
2240–22750
747–1372
465–846
10
*
*
*
H3C
H3C
O
O
O
350
10
OH
H
N
OH
61.1
H3C
HO
*
*
O
O
H
N
O
24
27
2130
122
1740–2600
93–160
929
787–1097
932–1440
0.4
10
H
N
OH
HO
O
1157
O
O
34
49
329
268
219–492
193–371
619
522–735
1
6
OH
OH
O
N
1508
1120–2030
dried by standard procedures. Melting points were measured on a
Büchi B-545 melting point apparatus or a Kofler-type Reichert
Thermovar micro hot stage microscope and are uncorrected.
Regarding NMR-assignment and nomenclature the carboxylic acid
of GA was assigned as C29 and the adjacent methyl group as C30.
NMR spectra were recorded at 297 K in the solvent indicated with
a Bruker AC 200-, a Bruker DPX 300-, a Bruker AC 400-, and a Bru-
ker DPX 400 spectrometer using standard Bruker NMR software.
Spectra were referenced to tetramethylsilane via calibration with
the residual solvent peaks.44 Reactions were monitored by TLC
on Silica Gel 60 F254 plates; spots were detected by UV light exam-
ination or visualized by spraying with anisaldehyde-sulfuric acid,
molybdophosphoric acid, mixture of molybdophosphoric acid
and CeIV ammonium nitrate or ninhydrine and heating. Normal
phase column chromatography was performed on Silica Gel 60
(230–400 mesh, Merck). HPLC-HRMS analysis was carried out from
CH3CN solutions (concentration: 1–10 mg/L) using an HTC PAL sys-
tem autosampler (CTC Analytics AG, Zwingen, Switzerland), an
Agilent 1100/1200 HPLC with binary pumps, degasser and column
thermostat (Agilent Technologies, Waldbronn, Germany) and Agi-
lent 6210 ESI-TOF mass spectrometer (Agilent Technologies, Palo
Alto, United States).
4.2. (3a,18b,20b)-3-Amino-11-oxo-olean-12-en-29-oic acid,
diphenylmethyl ester (3a) and (3b,18b,20b)-3-amino-11-oxo-
olean-12-en-29-oic acid, diphenylmethyl ester (3b)
Solid NaOAc (12.35 g, 150.5 mmol, 16.3 equiv) was added por-
tionwise to a TiCl3 solution (12% in 5–10% HCl, 47.6 g, 36.9 mmol,
ꢁ4 equiv) and the solution was stripped with argon. This solution
was added dropwise (30 min) to an Ar-stripped solution of oxime 1
(6.00 g, 9.23 mmol, 1.00 equiv) and tBuNH2ꢀBH3 (2.00 g, 23.1
mmol, 2.5 equiv) in dry EtOH at ꢂ9 °C. The dark blue solution
was stirred for around 2 h before satd NH4Cl (600 mL) and DCM
(600 mL) were added and the phases were separated. The aqueous
layer was extracted with DCM (3ꢃ), the combined organic layers
were washed with satd NH4Cl, satd NaHCO3 and brine, dried over
Na2SO4 and evaporated. Column chromatography (SiO2: 680 g,
CHCl3/MeOH 8:1 + 0.1% AcOH?CHCl3/MeOH 4:1 + 0.1% AcOH)
gave (after treatment with satd NaHCO3, brine and drying over
Na2SO4) pure 3a-amine 3a (980 mg, 16.7%) and pure 3b-amine
(3.40 g, 57.9%) as white solid foams. Analytical data for 3a:
Rf = 0.33 (CHCl3/MeOH 9:1 + 0.1% AcOH), ½a D20
ꢄ
+111.6 (c 1.0, CHCl3),
1H NMR (CDCl3): d 0.67 (s, 3H, H28), 0.87–0.93 (m, 6H, H23, H24),
0.93-1.02 (m, 1H, H2b), 1.09 (s, 3H, H26), 1.15 (s, 3H, H25), 1.12-

7530
C. Stanetty et al. / Bioorg. Med. Chem. 18 (2010) 7522–7541
1.16 (m, 1H, H15b), 1.17 (s, 3H, H30), 1.21–1.27 (m, 1H, H3), 1.39
(s, 3H, H27), 1.27-1.41 (m, 6H, H1b, H6b, H16b, H21b, H22b, H22a),
1.41-1.52 (m, 2H, H7b, H7a), 1.53–1.77 (m, 3H, H1a, H16a, H19b),
1.76-1.86 (m, 1H, H15a), 1.93–2.15 (m, 4H, H2a, H18, H19a, H21a),
2.46 (s, 1H, H9), 2.47–2.56 (m, 1H, H6a), 2.62–2.96 (m, 1H, H5),
5.52 (s, 1H, H12), 6.93 (s, 1H, OCHPh2), 7.22-7.43 (m, 10H,
10 ꢃ PhCH); 13C NMR (CDCl3): d 16.5 (q, C25), 17.4 (t, C7), 18.7
(q, C26), 23.5 (q, C23/24), 23.6 (q, C27), 25.6 (t, C16), 26.4 (2 ꢃ t,
C2, C15), 28.3 (2 ꢃ q, C28, C30), 28.8 (q, C23/24), 31.2 (t, C21),
31.7 (s, C17), 32.7 (t, C6), 33.6 (t, C1), 36.8 (s, C4), 37.4 (s, C10),
37.5 (t, C22), 41.1 (t, C19), 43.2 (s, C20), 44.0 (s, C8), 45.5 (s,
C14), 48.0 (2 ꢃ d, C3, C18), 56.2 (d, C5), 61.8 (d, C9), 76.6 (d, OCH-
Ph2), 127.0 (d, PhCH), 127.3 (d, PhCH), 127.8 (d, PhCH), 128.1 (d,
PhCH), 128.4 (d, PhCH), 128.58 (d, C12), 128.63 (d, PhCH), 140.10
(s, PhC), 140.13 (s, PhC), 168.8 (s, C13), 175.2 (s, C29), 200.3 (s,
C11). Analytical data for 3b: Rf = 0.23 (CHCl3/MeOH 9:1 + 0.1%
(q, C23/24), 17.3 (t, C6), 18.7 (q, C26), 23.3 (q, C27), 23.5 (t, C2),
26.37, 26.40 (2 ꢃ t, C15, C16), 28.23, 28.27, 28.6 (3 ꢃ q, C23/24,
C28, C30), 31.1 (t, C21), 31.7 (s, C17), 32.8 (t, C7), 36.9 (s, C4),
37.1 (s, C10), 37.5 (t, C22), 39.3 (t, C1), 41.1 (t, C19), 43.1 (s,
C14), 44.0 (s, C20), 45.3 (s, C8), 48.0 (d, C18), 56.2 (d, C5), 61.6
(d, C9), 61.8 (q, OMe), 67.9 (d, C3), 76.6 (d, OCHPh2), 127.0 (d,
PhCH), 127.3 (d, PhCH), 127.8 (d, PhCH), 128.1 (d, PhCH), 128.4
(d, PhCH), 128.5 (d, C12), 128.6 (d, PhCH), 140.06 (s, PhC), 140.10
(s, PhC), 168.7 (s, C13), 175.2 (s, C29), 200.1 (s, C11).
4.4. (3a,18b,20b)-3-Amino-11-oxo-olean-12-en-29-oic acid hyd-
rochloride (5a)
Diphenylmethyl ester 3a (200 mg, 0.314 mmol, 1.00 equiv) was
dissolved in MeOH/AcOH 100:1, 5 mL). The atmosphere was ex-
changed to argon by evaporation and purging with argon (3 cy-
cles), Pd/C (10%, 300 mg) was added and the atmosphere was
exchanged to H2 (3 cycles). The reaction mixture was stirred under
an atmosphere of hydrogen and monitored by TLC. Upon complete
conversion of starting material the reaction mixture was filtered
over a bed of CeliteÒ. Purification of the crude material was done
by dropwise addition of HCl in Et2O (1 M, 0.6 mmol) to a concen-
trated solution in MeOH and completion of precipitation by addi-
tion of Et2O (10 mL). The suspension was stirred for several
hours before the target compound was collected by filtration and
washed excessively with Et2O to provide compound 5a (115 mg,
72.2%) as white solid. Rf = 0.12 (CHCl3/MeOH 9:1 + 0.5% AcOH)
AcOH), ½a 2D0
ꢄ
+129.8 (c 1.0, CHCl3) HRMS: calcd [M+H]+: 636.4411
found [M+H]+: 636.4423; 1H NMR (CDCl3): d 0.66 (s, 3H, H28),
0.69–0.77 (m, 2H, H5, H16b), 0.84 (s, 3H, H23/24), 0.91-1.07 (m,
3H, H1b, H7b, H16a), 1.04 (s, 3H, H23/24), 1.09 (s, 3H, H26), 1.13
(s, 3H, H25), 1.17 (s, 3H, H30), 1.22-1.53 (m, 4H, H6b, H21b,
H22b, H22a), 1.36 (s, 3H, H27), 1.55-1.73 (m, 5H, H2b, H2a, H6a,
H7a, C19b), 1.73-1.87 (m, 1H, H15b), 1.92-2.10 (m, 4H, H15a,
H18, H19a, H21a), 2.33 (s, 1H, H9), 2.50-2.59 (m, 1H, H3), 2.75–
2.86 (m, 1H, H1a), 5.51 (s, 1H, H12), 6.93 (s, 1H, OCHPh2), 7.22–
7.42 (m, 10H, 10 ꢃ PhCH); 13C NMR (CDCl3): d 16.05 (q, C25),
16.13 (q, C23/24), 17.7 (t, C7), 18.7 (q, C26), 23.3 (q, C27), 26.4
(3 ꢃ t, C2, C15, C16), 28.2 (2 ꢃ q, C28, C30), 28.5 (q, C23/C24),
31.2 (t, C21), 31.7 (s, C17), 32.7 (t, C6), 37.2 (s, C10), 37.5
(t, C22), 38.0 (s, C4), 39.6 (t, C1), 41.1 (t, C19), 43.2 (s, C20), 44.0
(s, C8), 45.2 (s, C14), 48.0 (d, C18), 55.4 (d, C5), 60.0 (d, C3), 61.7
(d, C9), 76.6 (d, OCHPh2), 127.0 (d, PhCH), 127.3 (d, PhCH), 127.8
(d, PhCH), 128.1 (d, PhCH), 128.4 (d, PhCH), 128.5 (d, C12), 128.6
(d, PhCH), 140.07 (s, PhC), 140.11 (s, PhC), 168.7 (s, C13), 175.2
(s, C29), 199.9 (s, C11).
mp: >350 °C (MeOH/Et2O); ½a D20
ꢄ
+152.4 (c 1.0, MeOH); HRMS: calcd
[M+H]+: 470.3629, found [M+H]+: 470.3625; 1H NMR (DMSO): d
0.78 (s, 3H, H28), 0.89 (s, 3H, H23/24), 0.91 (s, 3H, H23/24),
0.93–1.03 (m, 1H, H15b), 1.03 (s, 3H, H26), 1.05 (s, 3H, H25),
1.08 (s, 3H, H30), 1.15–1.50 (m, 9H, H1b, H5, H6a, H6b, H7b,
H16b, H21b, H22a, H22b), 1.4 (s, 3H, H27), 1.55–1.85 (m, 6H,
H2b, H7a, H16a, H19a, H19b, H21a), 1.95–2.15 (m, 3H, H2a,
H15a, H18), 2.41–2.53 (m, 1H, H1a), 2.61 (s, 1H, H9), 2.94 (br s,
1H, H3), 5.45 (s, 1H, H12); 13C NMR (DMSO): d 16.5 (q, C25), 17.3
(t, C6), 18.7 (q, C26), 21.6 (t, C2), 22.8 (q, C23/24), 23.9 (q, C27),
26.2, 26.5 (2 ꢃ t, C15, C16), 28.20, 28.28 (2 ꢃ q, C23/24, C30),
28.8 (q, C28), 30.8 (t, C21), 31.9 (s, C17), 32.1 (t, C7), 32.5 (t, C1),
35.4 (s, C8), 36.9 (s, C10), 37.9 (t, C22), 41.1 (t, C19), 43.5 (s,
C20), 43.6 (s, C8), 45.4 (s, C14), 46.7 (d, C5), 48.5 (d, C18). 56.9
(d, C3), 60.6 (d, C9), 127.5 (d, C12), 170.5 (s, C13), 178.0 (s, C29),
199.6 (s, C11).
4.3. (3a,18b,20b)-3-Methoxyamino-11-oxo-olean-12-en-29-oic
acid, diphenylmethyl ester (4a) and (3b,18b,20b)-3-methoxya-
mino-11-oxo-olean-12-en-29-oic acid, diphenylmethyl ester
(4b)
tBuNH2ꢀBH3 (196 mg, 2.26 mmol, 3.0 equiv) was added to a
solution of the methoxime 2 (664 mg, 0.753 mmol, 1.00 equiv) in
EtOH/dioxane (1:2, 5.5 mL). The reaction mixture was cooled to
0 °C and 10% HCl (2.6 mL) was added dropwise keeping the tem-
perature below 5 °C. The reaction mixture was stirred at 0 °C for
2.5 h. Upon complete conversion (TLC, Hex/EtOAc 9:1), solid
Na2CO3 (ꢁ1 g) was added portionwise and the mixture was distrib-
uted between satd NaHCO3 and DCM. The organic layer was dried
over Na2SO4, filtered and evaporated. Column chromatography of
4.5. (3b,18b,20b)-3-Amino-11-oxo-olean-12-en-29-oic acid hyd-
rochloride (5b)
Ester 3b (467 mg, 0.734 mmol, 1.00 equiv) was deprotected by
hydrogenolysis analogous to 5a in MeOH/AcOH 100:1 (8 mL) with
Pd/C (100 mg). Upon complete conversion but prior to filtration,
2 M HCl (2 equiv) was added and the reaction mixture was stirred
for 30 min. The crude material was purified by column chromatog-
raphy (SiO2, CHCl3/MeOH 10:1 + 0.1% AcOH). Precipitation from a
concentrated eluent solution was achieved with 1 M HCl in Et2O
(1.4 mmol) and additional Et2O. The suspension was stirred for
several hours, filtered and the crystals were washed with Et2O to
give pure 5b (307 mg, 82.6%) as white solid. Rf = 0.07 (CHCl3/MeOH
9:1 + 0.1% AcOH); mp: 287–290 °C (toluene, MeOH) (Lit.36: >300 °C
the residue (Hex/EtOAc 20:1?Hex/EtOAc 5:1) gave pure 3a-meth-
oxyamine 4a (70 mg, 14%) and pure 3b-methoxyamine 4b (330 mg,
65.8%) as white solid foams. Analytical data for 4a: Rf = 0.11 (Hex/
EtOAc 5:1) Due to the instability of the material, it was deprotected
and characterized as free acid only. Analytical data of 4b: Rf = 0.17
(Hex/EtOAc 5:1); ½a D20
ꢄ
+143.0 (c 1.0, CHCl3); 1H NMR (CDCl3): d 0.66
(s, 3H, H28), 0.72 (s, 3H, H23/24), 0.69–0.77 (m, 1H, H5), 0.89–1.01
(m, 2H, H1b, H16b), 1.07 (s, 3H, H23/24), 1.09 (s, 3H, H26), 1.12–
1.19 (m, 1H, H15b), 1.13 (s, 3H, H25), 1.17 (s, 3H, H30), 1.20–
1.43 (m, 6H, H2b, H6b, H7b, H21b, H22a, H22b), 1.36 (s, 3H,
H27), 1.55–1.71 (m, 3H, H6a, H7a, H19b), 1.79 (td, J = 13.5 Hz,
J = 3.8 Hz, 1H, H15a), 1.86–1.94 (m, 1H, H2a), 1.94–2.08 (m, 4H,
H16a, H18, H19a, H21a), 2.34 (s, 1H, H9), 2.51 (dd, J = 11.8 Hz,
J = 4.0 Hz, 1H, H3), 2.75–2.83 (m, 1H, H1a), 3.50 (s, 3H, OCH3),
5.34 (br s, 1H, NH), 5.51 (s, 1H, H12), 6.93 (s, 1H, OCHPh2), 7.26–
7.40 (m, 10H, 10 ꢃ PhCH); 13C NMR (CDCl3): d 16.1 (q, C25), 16.7
dec., MeOH); ½a 2D0
ꢄ
+125.9 (c 1.0, MeOH/CHCl3 1:1); HRMS: calcd
[M+H]+: 470.3629, found [M+H]+: 470.3625; 1H NMR (MeOD): d
0.84 (s, 3H, H28), 0.91 (s, 3H, H23/24), 0.90–0.99 (m, 1H, H5),
1.00–1.20 (m, 3H, H1b, H15b, H16b), 1.09 (s, 3H, H23/24), 1.12–
1.18 (br s, 9H, H25, H26, H30), 1.30–1.60 (m, 5H, H6b, H7b,
H21b, H22a, H22b), 1.60–2.02 (m, 8H, H2a, H2b, H6a, H7a, H15a,
H19a, H19b, H21a), 2.08–2.30 (m, 2H, H16a, H18), 2.52 (s, 1H,
H9), 2.76–2.89 (m, 1H, H1a), 2.90–3.00 (m, 1H, H3), 5.59 (s, 1H,

C. Stanetty et al. / Bioorg. Med. Chem. 18 (2010) 7522–7541
7531
H12); 13C NMR (MeOD): d 17.33, 17.43 (2 ꢃ q, C23/24, C25), 19.3 (t,
C6), 20.1 (q, C26), 24.7 (q, C27), 25.0 (t, C2), 28.19, 28.42 (2 ꢃ t,
C15, C16), 29.2, 29.6, 30.1 (3 ꢃ q, C23/24, C28, C30), 32.8 (t, C21),
33.8 (s, C17), 34.4 (t, C7), 38.6 (s, C4), 39.0 (s, C10), 39.9 (t, C22),
40.3 (t, C1), 43.2 (t, C19), 45.5, 45.8, 47.5 (3 ꢃ s, C8, C14, C20),
50.8 (d, C18), 56.7 (d, C5), 62.1 (d, C3), 63.5 (d, C9), 129.7 (d,
C12), 174.0 (s, C13), 181.2 (s, C29), 203.0 (s, C11).
18.4 (q, C26), 19.4 (t, C2), 23.0 (q, C27), 26.1, 26.2 (2 ꢃ t, C15,
C16), 28.1, 28.16, 28.22 (3 ꢃ q, C23/24, C28, C30), 30.7 (t, C21),
31.6 (s, C17), 32.2 (t, C7), 36.3, 36.6 (2 ꢃ s, C4, C10), 37.4 (t, C22),
38.0 (t, C1), 40.9 (t, C19), 43.1 (s, C14), 43.5 (s, C20), 45.2 (s, C8),
48.2 (d, C18), 55.5 (d, C5), 61.1 (d, C9), 61.6 (q, OCH3), 70.0 (d,
C3), 127.8 (d, C12), 171.0 (s, C13), 179.0 (s, C29), 200.2 (s, C11).
4.8. (3a,18b,20b)-3-(Acetylamino)-11-oxo-olean-12-en-29-oic
4.6. (3a,18b,20b)-3-Methoxyamino-11-oxo-olean-12-en-29-oic
acid (7a)
acid hydrochloride (6a)
Amino ester 3a (500 mg, 0.79 mmol, 1.00 equiv) was dissolved
in dry DCM (10 mL) before TEA was added and the reaction mix-
ture was cooled to 0 °C. Acetic anhydride (370 L, 3.93 mmol,
Ester 4a (200 mg, 0.3 mmol, 1.0 equiv) was deprotected by
hydrogenolysis analogous to 5a in MeOH/EtOAc 1:1 + 1% AcOH
(30 mL). The crude material was purified by precipitation as hydro-
chloride by dissolution in THF and addition of 1 M HCl in Et2O
(2 equiv). Filtration and washing with Et2O and collection of solids
by centrifugation gave pure 6a (104 mg, 64.6%) as white solid.
l
5.00 equiv) was added slowly under temperature control and the
reaction mixture was allowed to warm to rt and was stirred until
complete conversion of starting material. The reaction mixture
was diluted with EtOAc or DCM, washed with diluted HCl, with
satd NaHCO3 and brine, dried over Na2SO4 and evaporated (aque-
ous workup). The crude material was submitted to chromatogra-
phy (SiO2, Hex/EtOAc 1:3) to give pure ester intermediate
(509 mg, 95.5%) as white solid. Analytical data for intermediate es-
Rf = 0.29 (Hex/EtOAc 1:1 + 0.1% AcOH); ½a D20
ꢄ
+115.0 (c 0.3, MeOH)
HRMS: calcd [M+H]+: 500.3734, found [M+H]+: 500.3734; 1H
NMR (CDCl3/MeOD 1:5): d 0.85 (s, 3H, H28), 0.98–1.08 (m, 1H,
H16b), 1.09 (s, 3H, H23/24), 1.12 (s, 3H, H23/24), 1.15–1.25 (m,
1H, H15b), 1.16 (s, 3H, H26), 1.18 (s, 3H, H30), 1.22 (s, 3H, H25),
1.17–1.27 (m, 1H, H1b), 1.35–1.50 (m, 5H, H5, H7b, H21b, H22a,
H22b), 1.45–1.65 (m, 2H, H6a, H6b), 1.49 (s, 3H, H27), 1.65–1.75
(m, 1H, H19b), 1.75–2.05 (m, 5H, H2b, H7a, H15a, H19a, H21a),
2.05–2.30 (m, 1H, H2a, H16a, H18), 2.61–2.70 (m, 1H, H1a), 2.69
(s, 1H, H9), 3.36–3.40 (m, 1H, H3), 4.01 (s, 3H, OCH3), 5.64 (s, 1H,
H12); 13C NMR (CDCl3/MeOD 1:5): d 17.4 (q, C25), 18.5 (t, C6),
18.9 (t, C2), 19.2 (q, C26), 23.7 (q, C23/24), 24.1 (q, 27), 27.2 (t,
C16/15), 27.2 (q, C23/24), 27.4 (t, C15/16), 28.8 (q, C30), 29.1 (q,
C28), 31.9 (t, C21), 32.8 (s, C17), 33.1 (t, C7), 34.9 (t, C1), 36.7 (s,
C4), 37.9 (s, C10), 38.8 (t, C22), 42.3 (t, C19), 44.66, 44.69 (2 ꢃ s,
C14, C20), 46.6 (s, C8), 49.6 (d, C18), 50.0 (d, C5), 62.0 (q, OCH3),
62.1 (d, C9), 67.8 (d, C3), 128.7 (d, C12), 173.1 (s, C13), 180.2 (s,
C29), 202.0 (s, C11).
ter: Rf = 0.20 (Hex/EtOAc 1:2) ½a D20
ꢄ
+99.0 (c 1.0, CHCl3); HRMS:
calcd [M+H]+: 678.4517, found [M+H]+: 678.4523. An aliquot of
the intermediate ester (170 mg, 0.251 mmol, 1.00 equiv) was
deprotected by hydrogenolysis analogous to 5a in MeOH/EtOAc
50:50 + 1% AcOH (6 mL) with Pd/C (17 mg, 10%). Trituration of
the crude material with hot EtOAc gave pure 7a (105 mg, 81.8%)
as white solid. Rf = 0.27 (DCM/MeOH 8:1); ½a D20
ꢄ
+95.0 (c 1.0,
CHCl3/MeOH); HRMS: calcd [M+H]+: 512.3734, found [M+H]+:
512.3737; 1H NMR (CDCl3): d 0.84 (s, 3H, H28), 0.86 (s, 3H, H23/
24), 0.93–1.29 (m, 4H, H1b, H5, H15b, H16b), 0.98 (s, 3H, H23/
24), 1.15 (s, 3H, H26), 1.16 (s, 3H, H25), 1.19 (s, 3H, H30), 1.28–
1.59 (m, 8H, H2a, H2b, H6b, H7a, H7b, H21b, H22a, H22b), 1.46
(s, 3H, H27), 1.58–1.76 (m, 2H, H6a, H19b), 1.84–2.15 (m, 4H,
H10a, H15a, H19a, H21a), 2.02 (s, 3H, NHAc), 2.15–2.27 (m, 1H,
H18), 2.56 (s, 1H, H9), 2.62 (br s, 1H, H1a), 3.79 (s, 1H, H9), 5.66
(s, 1H, H12); 13C NMR (CDCl3): d 16.1 (q, C25), 17.0 (t, C7), 18.3
(q, C26), 22.2 (q, NHCOCH3), 22.8 (q, C27), 22.9 (t, C2), 23.0 (q,
C23/24), 26.0 (t, C15), 26.1 (t, C16), 27.8 (q, C23/24), 28.1 (q,
C30), 28.2 (q, C28), 30.7 (t, C21), 31.5 (s, C17), 32.1 (t, C6), 33.9
(t, C1), 36.0 (s, C4), 36.9 (s, C10), 37.4 (t, C22), 40.9 (t, C19), 43.1
(s, C20), 43.4 (s, C8), 45.4 (s, C14), 48.1 (d, C18), 49.6 (d, C5), 53.6
(d, C3), 61.2 (d, C9), 127.7 (d, C12), 170.6 (s, NHCOCH3), 171.0 (s,
C13), 179.2 (s, C29), 201.2 (s, C11).
4.7. (3b,18b,20b)-3-Methoxyamino-11-oxo-olean-12-en-29-oic
acid hydrochloride (6b)
To a solution of ester 4b in dry DCM (13 mL), first anisole
(1.60 g, 15.02 mol, 50 equiv) and then TFA (4.5 mL) was added
dropwise at ꢂ10 °C and the reaction mixture was stirred at 0 °C un-
til all starting material was consumed (TLC, Hex/EtOAc 5:2). After
1 h the reaction mixture was diluted with DCM and treated with
satd NaHCO3 (55 mL) until the pH was slightly basic. The solution
was buffered by addition of AcOH, the phases were separated and
the aqueous layer was extracted with DCM (3ꢃ). The combined or-
ganic layers were washed with brine, dried over Na2SO4 and evap-
orated to give a crude material, which was submitted to column
chromatography (SiO2: 25 g, stepwise gradient from toluene/EtOAc
4:1?1:1 with 0.1% AcOH) to give pure 6b (137 mg, 85.1%) as white
solid. Part of the material was converted to the hydrochloride by
dissolving in MeOH and precipitation with 2 M HCl in Et2O (2–
3 equiv) and Et2O. Filtration and washing with Et2O gave 6b as
hydrochloride as white solid. Rf = 0.29 (CHCl3/MeOH 30:1); mp:
4.9. (3b,18b,20b)-3-(Acetylamino)-11-oxo-olean-12-en-29-oic
acid (7b)
Amino ester 3b (440 mg, 0.692 mmol, 1.00 equiv) was acyl-
ated analogous to 7a in dry DCM (10 mL) with TEA (0.490 g,
4.8 mmol, 7.00 equiv) and Ac2O (350 mg, 3.46 mmol, 5.00 equiv).
Aqueous workup and recrystallization from DCM/EtOAc gave
intermediate ester (395 mg, 84.2%) as white crystals. Analytical
data of intermediate ester: Rf = 0.22 (Hex/EtOAc 1:2); mp: 162–
165 °C (EtOAc/DCM); ½a D20
ꢄ
+101.2 (c 1.0, CHCl3); HRMS: calcd
249–251 °C (Et2O); ½a D20
ꢄ
+160.3 (c 0.8, MeOH); HRMS: calcd
[M+H]+: 678.4517, found [M+H]+: 678.4522. An aliquot of the
intermediate ester (200 mg, 0.295 mmol, 1.00 equiv) was depro-
tected by hydrogenolysis analogous to 5a in MeOH/EtOAc
50:50 + 1% AcOH (10 mL) with Pd/C (20 mg, 10%). The crude
material was purified by separation between 0.5 M NaOH and
Et2O, reacidification and subsequent trituration with hot EtOAc
to give pure 7b (104 mg, 68.9%) as white solid. Rf = 0.48 (Hex/
[M+H]+: 500.3735, found [M+H]+: 500.3734; 1H NMR (CDCl3/MeOD
2:5) d 0.78–0.86 (m, 1H, H5), 0.83 (s, 3H, H28), 0.96–1.09 (m, 2H,
H1b, H16b), 1.00 (s, 3H, H23/24), 1.15 (s, 3H, H26), 1.17 (s, 3H,
H25), 1.19–1.26 (m, 1H, H15a), 1.19 (s, 3H, H30), 1.27 (s, 3H,
H23/24), 1.29–1.45 (m, 3H, H21b, H22a, H22b), 1.39 (s, 3H, H27),
1.41–1.52 (m, 1H, H6b, H7b), 1.56–1.66 (m, 3H, H6a, H7a, H19b),
1.80–2.11 (m, 6H, H2a, H2b, H15b, H16a, H19a, H21a), 2.18–2.25
(m, 1H, H18), 2.39 (s, 1H, H9), 2.89–2.99 (m, 1H, H1a), 3.03–3.11
(m, 1H, H3), 4.06 (s, 3H, OCH3), 5.67 (s, 1H, H12); 13C NMR
(CDCl3/MeOD 2:5): d 15.5 (q, 25), 16.5 (q, C23/24), 16.9 (t, C6),
EtOAc 1:5) ½a 2D0
ꢄ
+113.0 (c 1.0, CHCl3/MeOH 3:1); HRMS: calcd
[M+H]+: 512.3734, found [M+H]+: 512.3739; 1H NMR (CDCl3): d
0.82 (s, 3H, H25), 0.83 (s, 3H, H28), 0.85–0.94 (m, 1H, H5),
0.89 (s, 3H, H23/24), 0.99–1.17 (m, 2H, H1b, H16b), 1.14

7532
C. Stanetty et al. / Bioorg. Med. Chem. 18 (2010) 7522–7541
(2 ꢃ s, 6H, H23/24, H26), 1.17–1.28 (m, 1H, H15b), 1.20 (s, 3H,
H30), 1.30–1.54 (m, 6H, H2b, H6b, H7b, H21b, H22a, H22b),
1.41 (s, 3H, H27), 1.55–1.74 (m, 4H, H2a, H6a, H7a, H19b),
1.79–2.15 (m, 4H, H15a, H16a, H19a, H21a); 1.99 (s, 3H, NHAc),
2.15–2.26 (m, 1H, H18), 2.44 (s, 1H, H9), 2.69–2.80 (m, 1H, H1a),
3.58–3.70 (m, 1H, H3), 5.65 (s, 1H, H12); 13C NMR (CDCl3): d
15.8 (q, C23/24), 16.0 (q, C25), 17.3 (t, C7), 18.2 (q, C26), 22.3
(q, NHCOCH3), 22.8 (q, C27), 24.6 (t, C2), 26.0 (2 ꢃ t, C15, C16),
27.9–28.1 (3 ꢃ q, C23/24, C28, C30), 30.6 (t, C21), 31.5 (s, C17),
32.2 (t, C6), 36.6 (s, C10), 37.3 (t, C22), 37.9 (s, C4), 39.4 (t,
C1), 40.7 (t, C19), 43.0 (s, C20), 43.3 (s, C8), 45.1 (s, C14), 48.6
(d, C18), 55.0 (d, C5), 56.3 (d, C3), 61.4 (d, C9), 127.6 (d, C12),
170.8 (2 ꢃ s, NHCOCH3, C13), 179.0 (s, C29), 201.0 (s, C11).
[M+H]+: 548.3404, found [M+H]+: 548.3405; 1H NMR (CDCl3/MeOD
5:1): d 0.80 (s, 3H, H23/24), 0.80–0.87 (m, 1H, H5), 0.83 (s, 3H,
H28), 1.00–1.12 (m, 2H, H1b, H16b), 1.04 (s, 3H, H23/24), 1.14
(br s, 6H, H25, H26), 1.19–1.24 (m, 1H, H15b), 1.19 (s, 3H, H30),
1.30–1.54 (m, 5H, H6b, H7b, H21b, H22a, H22b), 1.40 (s, 3H,
H27), 1.57–1.82 (m, 5H, H2a, H2b, H6a, H7a, H19b), 1.83–2.03
(m, 3H, H15a, H19a, H21a), 2.00–2.13 (m, 1H, H16a), 2.20 (dd,
J = 13.4 Hz, J = 3.3 Hz, 1H, H18), 2.40 (s, 1H, H9), 2.79 (dt,
J = 13.0 Hz, J = 4.0 Hz, 1H, H1a), 2.94–3.02 (m, 1H, H3), 2.97 (s,
3H, SO2CH3), 5.66 (s, 1H, H12); 13C NMR (CDCl3/MeOD 5:1): d
15.8 (q, C25), 15.9 (q, C23/24), 17.7 (t, C6), 18.3 (q, C26), 22.9 (q,
C27), 26.04 (t, C15/16), 26.15 (t, C2 + C15/16), 28.02, 28.09, 28.15
(3 ꢃ q, C23/24, C28, C30), 30.7 (t, C21), 31.6 (s, C17), 32.4 (t, C7),
36.6 (s, C10), 37.4 (t, C22), 38.3 (s, C4), 39.6 (t, C1), 40.8 (t, C19),
41.1 (q, SO2CH3), 43.1 (s, C14), 43.4 (s, C20), 45.2 (s, C8), 48.1 (d,
C18), 55.6 (d, C5), 61.5 (d, C9), 61.9 (d, C3), 127.7 (d, C12), 170.8
(s, C13), 179.0 (s, C29), 200.8 (s, C11).
4.10. (3a,18b,20b)-3-Methylsulfonylamino-11-oxo-olean-12-en-
29-oic acid (8a)
Amino ester 3a (250 mg, 0.393 mmol, 1.00 equiv) was sulfony-
lated analogous to 7a in dry DCM (8 mL) with TEA (0.164 mL,
4.12. (3a,18b,20b)-11-Oxo-3-trifluoromethylsulfonylamino-
1.18 mmol, 3.0 equiv) and mesyl chloride (520
lL, 0.67 mmol,
olean-12-en-29-oic acid (9a)
1.7 equiv in 2 mL DCM) at ꢂ10 °C. The reaction mixture was directly
evaporated onto SiO2 (700 mg) and purified by column chromatog-
raphy (SiO2: 30 g, Hex/EtOAc 2:1) to give pure diphenylmethyl ester
(208 mg, 74.0%) as white solid which was submitted to the hydro-
genolysis step. Intermediate ester (187 mg, 0.262 mmol, 1.00 equiv)
was deprotected by hydrogenolysis analogous to 5a in MeOH/EtOAc
2:1 + 1% AcOH. Crude material was evaporated onto SiO2 (650 mg)
and submitted to column chromatography (SiO2: 10 g, Hex/EtOAc
1:1 + 0.1% AcOH) to give pure compound 8a. Rf = 0.42 (Hex/EtOAc
Amino ester 3a (250 mg, 0.393 mmol, 1.00 equiv) was sulfony-
lated analogous to 7a in dry DCM (25 mL) with TEA (119 mg,
1.18 mmol, 3.00 equiv) and Tf2O (133 mg, 0.427 mmol, 1.20 equiv,
precooled in 1 mL dry DCM) at ꢂ10 °C and stirred at ꢂ10 °C for 1 h.
The reaction mixture was directly evaporated onto SiO2 and puri-
fied by column chromatography (SiO2: 40 g, Hex/EtOAc 10:1) to
give pure diphenylmethyl ester (243 mg, 80.5%) as white solid
which was submitted to hydrogenolysis. Analytical data of ester:
3:4 + 0.5% AcOH); ½a D20
ꢄ
+96.3 (c 1.0, MeOH/CHCl3 5:1); HRMS: calcd
Rf = 0.51 (Hex/EtOAc 2:1); ½a D20
ꢄ
+86.8 (c 1.0, CHCl3/MeOH 3:1);
[M+H]+: 548.3404, found [M+H]+: 548.3415; 1H NMR (CDCl3): d
0.77–0.83 (m, 1H, H5), 0.80 (s, 3H, H28), 0.97 (s, 3H, H23/24),
0.97–1.05 (m, 1H, H16b), 1.01 (s, 3H, H23/24), 1.10 (s, 3H, H26),
1.10–1.20 (m, 1H, H15b), 1.19, 1.20 (2 ꢃ s, 2 ꢃ 3H, H25, H30),
1.24–1.45 (m, 6H, H1b, H6b, H7b, H21b, H22a, H22b), 1.40 (s, 3H,
H27), 1.41–1.51 (m, 4H, H2b, H6a, H7a, H19b), 1.76–1.86 (m, 2H,
H15a, H19a), 1.94–2.06 (m, 4H, H2a, H18, H21a, H16a), 2.50
(s, 1H, H9), 2.72–2.81 (m, 1H, H1a), 2.96 (s, 3H, SO2CH3), 3.16–
3.23 (m, 1H, H3), 5.45 (s, 1H, H12), 6.21 (d, 10.0 Hz, 1H, NH);
13C NMR (CDCl3): d 16.4 (q, C25), 17.2 (t, C6), 18.5 (q, C26), 23.1
(q, C23/24), 23.9 (t, C2), 24.4 (q, C27), 26.25, 26.38 (2 ꢃ t, C15,
C16), 28.4 (q, C30), 28.6 (q, C28), 29.4 (q, C23/24), 31.0 (t, C21),
31.9 (s, C17), 32.6 (t, C7), 33.2 (t, C1), 36.8, 37.1 (2 ꢃ s, C4, C10),
38.1 (t, C22), 41.1 (t, C19), 41.9 (q, SO2CH3), 43.3 (s, C20), 43.8
(s, C14), 45.6 (s, C8), 48.1 (d, C18), 49.9 (d, C5), 59.6 (d, C3),
61.2 (d, C9), 128.1 (d, C12), 168.0 (s, C13), 180.4 (s, C29), 198.7
(s, C11).
HRMS: calcd [M+H]+: 768.3904, found [M+H]+: 768.3904. Interme-
diate ester (140 mg, 1.82 mmol, 1.00 equiv) was deprotected by
hydrogenolysis analogous to 5a in MeOH/AcOH 100:1 (12 mL) with
Pd/C (14 mg, 10%). The crude material was purified by column
chromatography (SiO2: 30 g, Hex/EtOAc 3:1?Hex/EtOAc 2:1 +
0.1% AcOH) to give pure 9a (101 mg, 92.1%) as white solid.
Rf = 0.41 (CHCl3/MeOH 9:1 + 0.5% AcOH); ½a D20
ꢄ
+82.0 (c 1.0, CHCl3/
MeOH 3:1); HRMS: calcd [M+H]+: 602.3122, found [M+H]+:
602.3121; 1H NMR (CDCl3/MeOD 5:1): d 0.83 (s, 3H, H28), 0.98
(s, 3H, H23/24), 0.99 (s, 3H, H23/24), 0.95–1.10 (m, 2H, H16b,
H5), 1.14 (s, 3H, H26), 1.16 (s, 3H, H25), 1.19 (s, 3H, H30), 1.10–
1.28 (m, 2H, H1b, H15b), 1.42 (s, 3H, H27), 1.28–1.47 (m, 5H,
H6b, H7b, H21b, H22a, H22b), 1.50–1.75 (m, 4H, H2b, H6a, H7a,
H19b), 1.75–1.94 (m, 2H, H15a, H19a), 1.94–2.16 (m, 3H, H2a,
H16a, H21a), 2.20 (dd, 1H, J = 13.6 Hz, J = 3.6 Hz, H18), 2.58 (s,
1H, H9), 2.62 (dt, 1H, J = 14.2 Hz, J = 3.7 Hz, H1a), 3.29–3.36 (m,
1H, H3), 5.66 (s, 1H, 12); 13C NMR (CDCl3/MeOD 5:1): d 16.3 (q,
C25), 17.0 (t, C6), 18.4 (q, C26), 22.7 (q, C23/24), 22.9 (q, C27),
23.9 (t, C2), 26.1 (t, C15), 26.2 (t, C16), 28.1 (q, C30), 28.2 (q,
C28), 28.5 (q, C23/24), 30.8 (t, C21), 31.6 (s, C17), 32.2 (t, C7),
33.3 (t, C1), 36.67 (s, C10), 36.74 (s, C4), 37.5 (t, C22), 41.0 (t,
C19), 43.2 (s, C20), 43.5 (s, C8), 45.4 (s, C14), 48.2 (d, C18), 48.9
(d, C5), 61.0 (d, C9), 61.4 (d, C3), 127.8 (d, C12), 170.8 (s, C13),
179.1 (s, C29), 201.1 (s, C11).
4.11. (3b,18b,20b)-3-Methylsulfonylamino-11-oxo-olean-12-en-
29-oic acid (8b)
Amino ester 3b (252 mg, 0.396 mmol, 1.00 equiv) was sulfony-
lated analogous to 7a in dry DCM (8 mL) with TEA (0.165 mL,
1.19 mmol, 3.00 equiv) and mesyl chloride (670 lL, 0.859 mmol,
2.17 equiv in 2 mL DCM) at ꢂ10 °C (EtOH/ice bath). The reaction
mixture was diluted with DCM (50 mL), evaporated onto SiO2
(600 mg) and purified by column chromatography (SiO2: 30 g,
Hex/EtOAc 2:1?Hex/EtOAc 3:2) to give pure diphenylmethyl ester
(224 mg, 79.2%) as white solid which was submitted to the hydrog-
enolysis step. Intermediate ester (196 mg, 0.274 mmol, 1.00 equiv)
was deprotected by hydrogenolysis analogous to 5a in MeOH/
EtOAc 2:1 + 1% AcOH (55 mL). The reaction mixture was evapo-
rated onto SiO2 (600 mg) and submitted to column chromatogra-
phy (SiO2: 23 g, Hex/EtOAc 3:4 + 0.1% AcOH) to give pure 8b
(148 mg, 98.6%) as white solid. Rf = 0.35 (Hex/EtOAc 3:4 + 0.5%
4.13. (3b,18b,20b)-11-Oxo-3-trifluoromethylsulfonyl-amino-
olean-12-en-29-oic acid (9b)
Amino ester 3b (250 mg, 0.393 mmol, 1.00 equiv) was sulfony-
lated analogous to 7a in dry DCM (7 mL) with TEA (119 mg,
1.18 mmol, 3.00 equiv) and Tf2O (122 mg, 0.432 mmol, 1.10 equiv;
precooled in 1 mL DCM) at -10 °C. The reaction mixture was di-
rectly evaporated onto SiO2 (1 g) and purified by column chroma-
tography (Hex/EtOAc 7:1) to give pure diphenylmethyl ester
(250 mg, 82.8%) as white solid foam which was submitted to
hydrogenolysis. Analytical data of ester: Rf = 0.46 (Hex/EtOAc
AcOH);
½
a 2D0
+122.6 (c 1.0, MeOH/CHCl3 5:1); HRMS: calcd
ꢄ

C. Stanetty et al. / Bioorg. Med. Chem. 18 (2010) 7522–7541
7533
2:1); ½a 2D0
ꢄ
+82.8 (c 1.0, CHCl3/MeOH 3:1); HRMS: calcd [M+H]+:
4.15. (3b,18b,20b)-3-(2-Methoxycarbonyl-ethylsulfonylamino)-
11-oxo-olean-12-en-29-oic acid (11)
768.3904, found [M+H]+: 768.3907. Diphenylmethyl ester
(140 mg, 0.182 mmol, 1.00 equiv) was deprotected by hydrogenol-
ysis analogous to 5a in MeOH/AcOH 100:1 with Pd/C (12 mg, 10%).
The crude material was purified by column chromatography (SiO2:
23 g, Hex/EtOAc 2:1?1:1 + 0.1% AcOH) and subsequent trituration
with Et2O to give pure 9b (100 mg, 91.2%) as white solid. R = 0.40
Amino ester 3b (400 mg, 0.629 mmol, 1.00 equiv) was sulfony-
lated analogously to 7a in dry DCM (14 mL) with TEA (0.26 mL,
1.89 mmol, 3.0 equiv) and 3-chlorosulfonyl propionic acid methyl-
ester (141 mg, 0.755 mmol, 1.2 equiv; in <2 mL dry DCM). The
temperature was kept below 0 °C during addition and the reaction
mixture was stirred for 1 h at rt. The reaction mixture was diluted
with DCM, evaporated onto SiO2 (1.5 g) and purified by column
chromatography (SiO2: 40 g, Hex/EtOAc 2:1) to give pure diphenyl-
methyl ester (381 mg, 77.1%) as white solid which was submitted
to hydrogenolysis. Analytical data for ester: Rf = 0.31 (Hex/EtOAc
(CHCl3/MeOH 9:1 + 0.5% AcOH); ½a D20
ꢄ
+96.4 (c 1.0, CHCl3/MeOH
3:1); HRMS: calcd [M+H]+: 602.3122, found [M+H]+: 602.3130;
1H NMR (CDCl3/MeOD 5:1): d 0.83 (s, 6H, H23/24, H28), 0.76–
0.87 (m, 1H, H5), 1.03 (s, 3H, H23/24), 0.97–1.11 (m, 2H, H1b,
H16b), 1.14 (s, 6H, H25, H26), 1.19 (s, 3H, H30), 1.22–1.30 (m,
1H, H15b), 1.39 (s, 3H, H27), 1.30–1.56 (m, 5H, H6b, H7b, H21b,
H22a, H22b), 1.56–1.73 (m, 5H, H2a, H2b, H6a, H7a, H19b), 1.74–
2.14 (m, 4H, H15a, H16a, H19a, H21a), 2.21 (dd, 1H, J = 13.5 Hz,
J = 3.8 Hz, H18), 2.41 (s, 1H, H9), 2.79 (dt, 1H, J = 13.4 Hz,
J = 3.3 Hz, H1a), 3.13 (dd, 1H, J = 12.5 Hz, J = 4.2 Hz, H3), 5.66
(s, 1H, H12); 13C NMR (CDCl3/MeOD 5:1): d 15.8 (q, C25), 15.9
(q, C23/24), 17.7 (t, C6), 18.2 (q, C26), 22.9 (q, C27), 25.3 (t, C2),
26.0 (t, C15), 26.1 (t, C16), 28.0 (q, C30), 27.9 (q, C28), 28.1 (q,
C23/24), 30.7 (t, C21), 31.5 (s, C17), 32.3 (t, C7), 36.5 (s, C10),
37.4 (t, C22), 38.4 (s, C4), 39.6 (t, C1), 40.8 (t, C19), 43.1 (s, C20),
43.4 (s, C8), 45.1 (s, C14), 48.1 (d, C18), 55.3 (d, C5), 61.4 (d, C9),
63.8 (d, C3), 127.7 (d, C12), 171.0 (s, C13), 179.0 (s, C29), 200.8
(s, C11).
2:1) ½a 2D0
ꢄ
+100.9 (c 0.8, CHCl3); HRMS: calcd [M+H]+: 786.4398,
found [M+H]+: 786.4403. Intermediate ester (350 mg, 0.445 mmol,
1.00 equiv) was deprotected by hydrogenolysis analogous to 5a in
MeOH/AcOH 100:1 (22 mL) with Pd/C at rt for 3 h. The crude mate-
rial was evaporated onto SiO2 (1 g) and submitted to column chro-
matography (SiO2: 25 g, Hex/EtOAc 2:1 + 0.1% AcOH to Hex/EtOAc
1:1 + 0.1% AcOH). The product fractions were dissolved in EtOAc
and washed twice with water, once with brine, dried over Na2SO4,
evaporated and co-evaporated from DCM to afford pure 11
(270 mg, 97.8%) as solid white foam. Rf = 0.25 (Hex/EtOAc 1:1 +
0.5% AcOH); ½a 2D0
ꢄ
+100.4 (c 1.0, MeOH/CHCl3 5:1); HRMS: calcd
[M+H]+: 620.3616, found [M+H]+: 620.3626; 1H NMR (CDCl3): d
0.79–0.87 (m, 1H, H5), 0.79 (s, 3H, H23/24), 0.84 (s, 3H, H28),
0.98–1.11 (m, 2H, H1b, H16b), 1.04 (s, 3H, H23/24), 1.13 (br s,
6H, H25, H26), 1.15–1.23 (m, 1H, H15b), 1.23 (s, 3H, H30), 1.37
(s, 3H, H27), 1.38–1.53 (m, 5H, H6b, H7b, H21b, H22a, H22b),
1.56–1.77 (m, 5H, H2a, H2b, H6a, H7a, H19b), 1.81 (td,
J = 13.7 Hz, J = 3.8 Hz, 1H, H15a), 1.88–2.09 (m, 3H, H16a, H19a,
H21a), 2.19 (dd, J = 13.2 Hz, J = 3.4 Hz, 1H, H18), 2.36 (s, 1H, H9),
2.78–2.87 (m, 1H, H1a), 2.85 (app. t, J = 7.6 Hz, 2H, COCH2), 2.99–
3.09 (m, 1H, H3), 3.29–3.40 (m, 2H, SO2CH2), 3.73 (s, 3H, OCH3),
4.31 (d, J = 9.9 Hz, 1H, NH), 5.71 (s, 1H, H12); 13C NMR (CDCl3): d
16.2 (q, C25), 16.4 (q, C23/24), 18.0 (t, C6), 18.6 (q, C26), 23.3 (q,
C27), 26.37, 26.43 (2 ꢃ t, C15, C16), 27.2 (t, C2), 28.42 (3 ꢃ q,
C23/24, C28, C30), 28.8 (t, COCH2), 30.9 (t, C21), 31.9 (s, C17),
32.7 (t, C7), 36.8 (s, C10), 37.7 (t, C22), 38.5 (s, C4), 39.8 (t, C1),
40.8 (t, C19), 43.2 (s, C14), 43.8 (s, C20), 45.4 (s, C8), 48.2 (d,
C18), 49.3 (t, SO2CH2), 52.3 (q, OCH3), 55.9 (d, C5), 61.6 (d, C9),
62.4 (d, C3), 128.4 (d, C12), 169.5 (s, C13), 171.0 (s, COOCH3),
181.5 (s, C29), 200.2 (s, C11).
4.14. (3b,18b,20b)-11-Oxo-3-succinylamino-olean-12-en-29-oic
acid (10)
Amino ester 3b (400 mg, 0.629 mmol, 1.00 equiv) was acylated
analogous to 7a in dry DCM (9.5 mL) with TEA (610 lL, 4.40 mmol,
7.00 equiv) and succinic anhydride (315 mg, 3.15 mmol, 5.00 equiv)
at 0 °C to rt for 3d. Aqueous workup and purification by column
chromatography (SiO2: 20 g, Hex/EtOAc 1:1 + 0.1% AcOH to Hex/
EtOAc 1:3 + 0.1% AcOH) and (SiO2: 7 g, DCM/MeOH 70:1 + 0.1%
AcOH) gave pure diphenylmethyl ester (192 mg, 41.5%) as white so-
lid foam which was submitted to hydrogenolysis. Analytical data for
ester: Rf = 0.15 (Hex/EtOAc 3:1 + 0.5% AcOH); ½a D20
ꢄ
+97.6 (c 0.3,
CHCl3); HRMS: calcd [M+H]+: 736.4572, found [M+H]+: 736.4568.
Intermediate ester (260 mg, 0.253 mmol, 1.00 equiv) was deprotec-
ted by hydrogenolysis analogous to 5a on Pd/C in MeOH with 1%
AcOH. The crude material was distributed between Et2O and
0.3 M NaOH. The aqueous layer was acidified with 2 M HCl and ex-
tracted with DCM. The combined organic layers were washed twice
with water, once with brine, dried over Na2SO4 and evaporated. Trit-
uration with hot EtOAc and washing with EtOAc and Et2O gave pure
10 (100 mg, 49.7%) as white solid. Rf = 0.32 (CHCl3/MeOH 9:1 + 0.5%
4.16. (3b,18b,20b)-3-(2-Carboxy-ethylsulfonylamino)-11-oxo-
olean-12-en-29-oic acid (12)
Ester 11 (210 mg, 0.339 mmol, 1.00 equiv) was dissolved in
MeOH (5 mL), 0.2 M NaOH in MeOH (5 mL, 3 equiv) was added at
0 °C and the reaction mixture was stirred at rt overnight. Another
5 mL of 0.2 M NaOH in MeOH, then water (1 mL) and finally 2 M
NaOH (0.5 mL) were added and the reaction mixture was stirred
at 6 °C for another night to complete conversion. The reaction mix-
ture was diluted with DCM, acidified with diluted AcOH (10%) and
extracted with EtOAc. The combined organic layers were washed
once with diluted AcOH, with water, with brine, dried over Na2SO4
and evaporated. The crude material was evaporated onto SiO2 (1 g)
and submitted to flash column chromatography (SiO2: 10 g, DCM/
MeOH 30:1 + 0.1% AcOH to DCM/MeOH 15:1 + 0.1% AcOH) to give
pure acid 12 (199 mg, 97%) as white solid. Rf = 0.18 (Hex/EtOAc
AcOH); ½a 2D0
ꢄ
+106.6 (c 1.0, CHCl3/MeOH 5:1); HRMS: calcd [M+H]+:
570.3789, found [M+H]+: 570.3799; 1H NMR (CDCl3/MeOD 5:1): d
0.73 (s, 3H, H23/24), 0.74 (s, 3H, H28), 0.76–0.83 (m, 1H, H5), 0.79
(s, 3H, H23/24), 0.90–1.05 (m, 2H, H1b, H16b), 1.05 (s, 6H, H25,
H26), 1.11 (s, 3H, H30), 1.09–1.43 (m, 7H, H2b, H6b, H7b, H15b,
H21b, H22a, H22b), 1.32 (s, 3H, H27), 1.48–1.58 (m, 4H, H2a, H6a,
H7a, H19b), 1.70–1.90 (m, 3H, H15a, H19a, H21a), 1.91–2.05 (m,
1H, H16a), 2.07–2.17 (m, 1H, H18), 2.35 (s, 1H, H9), 2.36–2.46 (m,
2H, CH2), 2.51–2.61 (m, 2H, CH2), 2.60–2.71 (m, 1H, H1a), 3.50–
3.58 (m, 1H, H3), 5.57 (s, 1H, H12), 6.78 (d, J = 9.8 Hz, 1H, NH); 13
C
NMR (CDCl3/MeOD 5:1): d 15.8 (q, C25), 16.0 (q, C23/24), 17.3 (t,
C6), 18.2 (q, C26), 22.9 (q, C27), 24.6 (t, C2), 25.97, 26.07 (2 ꢃ t,
C15, C16), 27.97, 28.01 (2 ꢃ q, C23/24, C30), 28.11 (q, C28), 29.5 (t,
CH2), 30.62, 30.70 (2 ꢃ t, CH2, C21), 31.5 (s, C17), 32.3 (t, C7), 36.6
(s, C10), 37.4 (t, C22), 37.9 (s, C4), 39.4 (t, C1), 40.8 (t, C19), 43.0
(s, C20), 43.4 (s, C14), 45.2 (s, C8), 48.1 (s, C18), 55.1 (s, C5), 56.3
(s, C3), 61.4 (s, C9), 127.7 (s, C12), 170.9 (s, C13), 172.1 (s, NHCO),
175.0 (s, COOH), 179.0 (s, C29), 201.1 (s, C11).
1:3 + 0.5% AcOH); ½a D20
ꢄ
+95.9 (c 1.0, MeOH/CHCl3 5:1); HRMS: calcd
[M+H]+: 606.3459, found [M+H]+: 606.3467; 1H NMR (CDCl3 + 3dr
MeOD): d 0.79 (s, 3H, H23/24), 0.80–0.87 (m, 1H, H5), 0.82 (s, 3H,
H28), 0.98–1.09 (m, 2H, H1b, H16b), 1.03 (s, 3H, H23/24), 1.12 (br
s, 6H, H25, H26), 1.19 (s, 3H, H30), 1.19–1.22 (m, 1H, H15b), 1.25–
1.51 (m, 5H, H6b, H7b, H21b, H22a, H22b), 1.37 (s, 3H, H27), 1.56–

7534
C. Stanetty et al. / Bioorg. Med. Chem. 18 (2010) 7522–7541
1.77 (m, 5H, H2a, H2b, H6a, H7a, H19b), 1.83 (td, J = 13.9 Hz,
J = 4.2 Hz, 1H, H15a), 1.88–2.08 (m, 3H, H19a, H21a, H16a), 2.19
(dd, J = 13.3 Hz, J = 3.9 Hz, 1H, H18), 2.37 (s, 1H, H9), 2.75–2.83
(m, 1H, H1a), 2.82 (app. t, J = 7.6 Hz, 2H, HOOCCH2), 3.00 (dd,
J = 12.0 Hz, J = 4.6 Hz, 1H, H3), 3.27–3.39 (m, 2H, SO2CH2), 5.20
(d, J = 9.8 Hz, 1H, NH), 5.66 (s, 1H, H12); 13C NMR (CDCl3 + 3dr
MeOD): d 16.1 (q, C25), 16.2 (q, C23/24), 17.9 (t, C6), 18.5 (q,
C26), 23.1 (q, C27), 26.23, 26.31 (2 ꢃ t, C15, C16) 26.7 (t, C2),
28.28, 28.32, 28.36 (3 ꢃ q, C23/24, C28, C30), 28.6 (t, HOOCCH2),
30.9 (t, C21), 31.7 (s, C17), 32.5 (t, C7), 36.7 (s, C10), 37.6 (t,
C22), 38.4 (s, C4), 39.7 (t, C1), 40.9 (t, C19), 43.2 (s, C14), 43.6 (s,
C20), 45.3 (s, C8), 48.2 (d, C18), 49.1 (t, SO2CH2), 55.7 (d, C5),
61.5 (d, C9), 62.0 (d, C3), 128.0 (d, C12), 170.5 (s, C13), 172.9 (s,
COOH), 179.4 (s, C29), 200.7 (s, C11).
16.0 (q, C25), 16.3 (q, C23/24), 17.0 (t, C6), 18.3 (q, C26), 20.8 (q,
COCH3), 22.7 (q, C27), 23.2 (t, C2), 25.5 (q, C30), 26.2 (t, C15),
26.4 (t, C16), 27.6 (q, C23/24), 28.2 (q, C28), 31.5 (t, C21), 31.7 (s,
C17), 32.4 (t, C7), 36.7 (s, C10), 37.59 (t, C22), 37.61 (q, N-Me),
37.7 (s, C4), 38.5 (t, C1), 42.0 (t, C19), 43.2 (s, C20), 44.0 (s, C8),
45.2 (s, C14), 48.4 (d, C18), 54.7 (d, C5), 61.4 (d, C9), 80.7 (d, C3),
127.6 (d, C12), 171.4 (s, COCH3), 171.6 (s, C13), 175.6 (s, C29),
201.1 (s, C11).
4.19. (3b,18b,20b)-3-Acetoxy-N-tertbutyl-N-hydroxy-11-oxo-
olean-12-en-29-amide (16)
Acid chloride 13a (2.0 g, 3.7 mmol, 1.00 equiv) was reacted
analogously to 14 with TEA (1 mL, 10.0 mmol, 2.7 equiv) and N-
tert-butyl-hydroxylamine hydrochloride (500 mg, 4.0 mmol,
1.08 equiv) to give compound 16 (1.95 g, 88%) as solid foam.
Rf = 0.3 (DCM/MeOH 98:2); HRMS: calcd [M+H]+: 584.4310, found
[M+H]+: 584.4300; 1H NMR (200 MHz, CDCl3): d 0.77 (s, 3H), 0.84
(s, 6H), 1.09 (s, 3H), 1.12 (s, 3H), 1.14 (s, 9H), 1.18 (s, 3H), 1.33
(s, 3H), 2.37–0.72 (m, 19H), 2.01 (s, 3H), 2.91 (m, 1H), 4.32 (m,
1H), 5.20 (s, 1H), 5.92 (s, 1H), 6.95 (br, 1H); 13C NMR (200 MHz,
CDCl3): d 26.7 (3C), 26.3 (2C), 4.7, 8.5, 16.4, 16.6, 17.3, 18.6, 21.3,
23.3, 23.5, 28.0, 28.4, 28.7, 31.1, 31.8, 32.6, 36.9, 37.6, 38.7, 39.0,
43.1, 44.0, 48.1, 54.3, 54.9, 55.7, 61.7, 128.6, 168.8, 170.9, 176.1,
199.9.
4.17. (3b,18b,20b)-3-Acetoxy-N-methyl-N-methoxy-11-oxo-
olean-12-en-29-amide (14)
To
1.00 equiv) and TEA (1.011 g, 10.0 mmol, 10.0 equiv) in dry DCM
(5% solution), N,O-dimethylhydroxylamine hydrochloride
a solution of acid chloride 13a (531 mg, 1.00 mmol,
(945 mg, 10.0 mmol, 10.0 equiv) was added portionwise. The reac-
tion mixture was stirred at rt under TLC-monitoring. Upon com-
plete conversion of starting material the reaction mixture was
washed with 2 M HCl, water and brine, dried with MgSO4 and
evaporated. The crude material was purified by column chroma-
tography (DCM/MeOH gradients) to give pure 14 (300 mg, 54%)
as yellowish solid. Rf = 0.38 (CHCl3/MeOH = 96:4); HRMS: calcd
[M+H]+: 556.3997, found [M+H]+: 556.4045; 1H NMR (200 MHz,
CDCl3): d 0.75 (s, 3H), 0.81 (s, 6H), 1.06 (s, 3H), 1.1 (s, 3H), 1.12
(s, 3H), 1.30 (s, 3H), 0.66–2.32 (m, 20H), 1.98 (s, 3H), 2.72 (dt,
1H), 3.11 (s, 3H), 3.6 (s, 3H), 4.44 (dd, 1H), 5.63 (s, 1H); 13C NMR
(200 MHz, CDCl3): d 16.4., 16.6, 17.3, 18.6, 21.3, 23.1, 23.5, 26.2,
26.5, 26.7, 28.0, 28.4, 31.9, 31.9, 32.7, 33.8, 36.9, 37.9, 38.0, 38.8,
42.3, 43.2, 44.7, 45.3, 48.3, 55.0, 60.5, 61.6, 80.6, 128.4, 169.7,
170.9, 176.8, 200.0.
4.20. (3b,18b,20b)-3-Acetoxy-N-methoxy-11-oxo-olean-12-en-
29-amide (17)
Acid chloride 13a (531 mg, 1.00 mmol, 1.00 equiv) was reacted
analogously to 14 with TEA (1.011 g, 10.0 mmol, 10.0 equiv) and
O-methyl-hydroxylamine hydrochloride (835 mg, 10.0 mmol,
10.0 equiv) to give compound 17 (250 mg, 46%) as solid foam.
Rf = 0.38 (DCM/MeOH 96:4); HRMS: calcd [M+H]+: 542.3840, found
[M+H]+: 542.3826; 1H NMR (200 MHz, CDCl3): d 0.40–2.70 (m,
19H), 0. 79 (s, 3H), 0.85 (s, 6H), 1.09 (s, 3H), 1.12 (s, 6H), 1.32 (s,
3H), 2.02 (s, 3H), 2.75 (m, 1H), 3.72 (s, 3H), 4.47 (dd, 1H), 5.67 (s,
1H), 9.80 (br, 1H); 13C NMR (200 MHz, CDCl3): d 16.3, 16.6, 17.3,
18.6, 21.3, 23.3, 23.5, 26.3, 26.4, 28.0, 28.4, 29.2, 31.1, 31.8, 32.6,
36.9, 37.3, 38.0, 38.8, 41.0, 42.6, 43.2, 45.4, 48.0, 55.0, 61.7, 63.9,
80.6, 128.4, 169.6, 171.1, 173.6, 200.3.
4.18. (3b,18b,20b)-3-Acetoxy-N-methyl-N-hydroxy-11-oxo-
olean-12-en-29-amide (15)
Carboxylic acid 13 (500 mg, 0.98 mmol, 1.00 equiv) was stirred
in SOCl2/toluene 1:1 (14 mL each) for 2 h. Upon complete conver-
sion according to TLC (SiO2, DCM/MeOH) the excess SOCl2 was
evaporated and the residue was coevaporated from toluene twice.
4.21. (3b,18b,20b)-3-Acetoxy-N-allyloxy-11-oxo-olean-12-en-29-
amide (18)
The residue was dissolved in DCM (20 mL) and first TEA (540 lL,
3.90 mmol, 4.00 equiv) and then N-methyl-hydroxylamine hydro-
chloride (122 mg, 1.46 mmol, 1.50 equiv) were added at rt. The
reaction mixture was stirred at rt monitored by TLC (SiO2: DCM/
Et2O 1:1) for given periods of time. Upon complete conversion, sol-
vents were evaporated, the residue was taken up in DCM and
washed with water, NaHCO3 and brine, dried over Na2SO4 and
evaporated (aqueous workup). The crude material was purified
by flash column chromatography (SiO2: 20 g, DCM/MeOH 94:6)
to give pure 15 (486 mg, 92%) as white solid. Rf = 0.20 (DCM/MeOH
Acid chloride 13a (531 mg, 1.00 mmol, 1.00 equiv) was reacted
analogous to 14 with TEA (1.011 g, 10.0 mmol, 10.0 equiv) and O-
allyl-hydroxylamine
hydrochloride
(1.096 g,
10.0 mmol,
10.0 equiv) to give compound 18 (250 mg, 44%) as solid foam.
Rf = 0.4 (DCM/MeOH 97:3); HRMS: calcd [M+H]+: 568.3997, found
[M+H]+: 568.3993; 1H NMR (200 MHz, CDCl3): d 0.76 (s, 3H), 0.82
(s, 6H), 1.07 (s, 3H), 1.1 (s, 6H), 1.30 (s, 3H), 0.65–2.40 (m, 20H),
2.00 (s, 3H), 2.70 (m, 1H), 4.34 (m, 2H), 4.43 (m, 1H), 5.26 (m,
2H), 5.65 (m, 1H), 5.93 (m, 1H), 9.48 (s, 1H); 13C NMR (200 MHz,
CDCl3): d 16.3, 16.6, 17.3, 18.6, 21.2, 23.2, 23.4, 26.4, 28.0, 28.4,
29.2, 31.2, 31.7, 32.6, 36.8, 37.3, 38.0, 38.8, 40.9, 42.8, 43.2, 45.4,
48.0, 53.4, 54.9, 61.7, 77.1, 80.6, 120.3, 128.3, 132.9, 169.8, 171.0,
173.5, 200.3.
94:6); ½a 2D0
ꢄ
+121.7 (c 1.0, CHCl3/MeOH 3:1); HRMS: calcd [M+H]+:
540.3689, found [M+H]+: 540.3635; 1H NMR (CDCl3/MeOD 5:1): d
0.78–0.87 (m, 1H, H5), 0.82 (s, 3H, H28), 0.90 (s, 6H, H23/24),
0.99–1.12 (m, 2H, H1b, H16b), 1.14 (s, 3H, H26), 1.17 (s, 3H,
H25), 1.21 (s, 3H, H30), 1.26–1.36 (m, 1H, H21b), 1.36–1.55 (m,
4H, H6b, H7b, H22a, H22b), 1.39 (s, 3H, H27), 1.55–1.77 (m, 5H,
H2a, H7a, H2b, H6a, H19b), 1.77–1.92 (dt, 1H, J = 13.9 Hz,
J = 4.7 Hz, H15a), 2.042.18 (m, 1H, H16a), 2.06 (s, 3H, OAc), 2.18–
2.33 (m, 3H, H19a, H21a, H18), 2.42 (s, 1H, H9), 1.18–1.26 (m,
1H, H15b), 2.72–2.82 (td, 1H, J = 13.6 Hz, J = 3.4 Hz, H1a), 3.25 (s,
3H, NHCH3), 4.13 (s, 1H, NH/OH), 4.46–4.55 (dd, 1H, J = 11.5 Hz,
J = 4.9 Hz, H3), 5.71 (s, 1H, H12); 13C NMR (CDCl3/MeOD 5:1): d
4.22. (3b,18b,20b)-3-Acetoxy-N-benzyloxy-11-oxo-olean-12-en-
29-amide (19)
Carboxylic acid 13 (500 mg, 0.98 mmol, 1.00 equiv) was - anal-
ogous to 15 - first converted to the acid chloride in SOCl2/toluene
(14 mL each) within 2 h which was then reacted with a solution
of NH2OBn (144 mg 1.17 mmol, 1.20 equiv) and TEA (540 lL,

C. Stanetty et al. / Bioorg. Med. Chem. 18 (2010) 7522–7541
7535
3.90 mmol, 4.00 equiv) in DCM (30 mL). Aqueous workup gave
crude material which was purified by flash column chromatogra-
phy (SiO2: 25 g, DCM/Et2O 5:1?DCM/Et2O 1:1) to give pure 19
(554 mg, 90.3%) as orange solid. Rf = 0.46 (DCM/MeOH 20:1);
HRMS: calcd [M+H]+: 618.4153, found [M+H]+: 618.4145; 1H
NMR (CDCl3): d 0.78–0.80 (m, 1H, H5), 0.80 (s, 3H, H28), 0.88 (s,
6H, H23/24, H25), 0.96–1.09 (m, 2H, H1b, H16b), 1.11 (s, 6H,
H26, H30), 1.16 (s, 3H, H23/24), 1.32 (s, 3H, H27), 1.24–1.50 (m,
5H, H6b, H7b, H22a, H22b, H21b), 2.05 (s, 3H, OAc), 2.32 (s, 1H,
H9), 1.13–1.20 (m, 1H, H15b), 1.54–1.72 (m, 6H, H2a, H19a,
H19b, H7a, H2b, H6a), 1.73–1.85 (m, 1H, H15a), 1.89–2.11 (m,
3H, H18, H16a, H21a), 2.74–2.83 (td, 1H, J = 13.4 Hz, J = 3.3 Hz,
H1a), 4.46–4.55 (dd, 1H, J = 11.6 Hz, J = 4.8 Hz, H3), 4.94 (s, 2H,
Bn), 5.44 (s, 1H, H12), 7.29–7.45 (m, 5H, Bn), 8.42 (s, 1H, NH);
13C NMR (CDCl3): d 16.4 (q, C23/24), 16.6 (q, C25), 17.3 (t, C6),
18.6 (q, C26), 21.3 (q, COCH3), 23.3 (q, C27), 23.5 (t, C2), 26.3 (t,
C15), 26.4 (t, C16), 28.0 (q, C23/24), 28.3 (q, C28), 29.5 (q, C30),
31.2 (t, C21), 31.8 (s, C17), 32.6 (t, C7), 36.9 (s, C10), 37.3 (t,
C22), 38.0 (s, C4), 38.8 (t, C1), 41.1 (t, C19), 43.0 (s, C20), 43.1 (s,
C8), 45.3 (s, C14), 47.9 (d, C18), 55.0 (d, C5), 61.7 (d, C9), 77.9
(2 ꢃ d, Bn), 80.6 (d, C3), 128.5 (d, C12), 128.7 (2 ꢃ d, Bn), 128.9
(d, Bn), 129.2 (2 ꢃ d, Bn), 135.3 (s, Bn), 168.8 (s, C13), 171.0 (s,
COCH3), 173.5 (s, C29), 200.0 (s, C11).
purified by column chromatography (SiO2, gradient of 0–5% meth-
anol in DCM) to give pure compound 21 (50 mg, 30%) as white so-
lid. Rf = 0.30 (DCM/MeOH 96:4); HRMS: calcd [M+H]+: 514.3891,
found [M+H]+: 514.3900; 1H NMR (200 MHz, CDCl3): d 0.58–2.31
(m, 26H), 0.74 (s, 3H), 0.94 (s, 3H), 1.05 (s, 3H), 1.06 (s, 3H), 1.12
(s, 3H), 1.30 (s, 3H), 2.70 (m, 1H), 3.12 (s, 3H), 3.35 (m, 1H), 3.61
(s, 3H), 5.64 (s, 1H); 13C NMR (50 MHz, CDCl3): d 15.6, 16.4, 17.5,
18.7, 23.1, 26.2, 26.5, 26.7, 27.2, 28.1, 28.4, 32.8, 33.9, 37.0, 37.9,
31.9 (2C), 39.1 (2C), 42.4, 43.3, 44.8, 45.3, 48.3, 54.9, 60.6, 61.7,
78.8, 128.4, 169.9, 176.9, 200.4.
4.25. (3b,18b,20b)-3,N-Dihydroxy-N-methyl-11-oxo-olean-12-
en-29-amide (22)
Ester 15 (300 mg, 0.55 mmol, 1.00 equiv) was reacted analo-
gous to 21 with KOH (310 mg, 5.5 mmol, 10.0 equiv) in MeOH to
give compound 22 (141 mg, 51%) as white solid. Rf = 0.45 (DCM/
MeOH 96:4) HRMS: calcd [M+H]+: 500.3734, found [M+H]+:
500.3729; 1H NMR (200 MHz, pyridine-d5): d 0.70–3.56 (m, 23H),
0.79 (s, 3H), 1.05 (s, 3H), 1.11 (s, 3H), 1.24 (s, 3H), 1.31 (s, 3H),
1.38 (s, 3H), 1.45 (s, 3H), 3.46 (s, 3H), 5.67–5.77 (m, 1H), 6.05 (s,
1H); 13C NMR (200 MHz, pyridine-d5) d 17.06, 17.30, 18.41,
19.32, 23.71, 26.75, 27.25, 27.43, 28.60, 29.22, 32.28, 32.59,
33.51, 38.09, 39.03, 39.07, 40.25, 40.26, 43.78, 44.03, 45.08,
45.95, 49.12, 50.12, 55.80, 62.62, 78.37, 129.14, 170.61, 176.46,
200.17.
4.23. (3b,18b,20b)-3-Acetoxy-N-hydroxy-11-oxo-olean-12-en-
29-amide (20)
O-Benzyl-hydroxamic acid 19 (544 mg, 0.88 mmol, 1.00 equiv)
was dissolved in THF (11 mL). The atmosphere was exchanged to
argon by evaporation and purging with argon (3 cycles), Pd/C
(110 mg, 20%) was added and the atmosphere was exchanged to
H2 (3 cycles). The reaction mixture was stirred under an atmo-
sphere of hydrogen under TLC-monitoring. Upon complete conver-
sion of starting material the reaction mixture was filtered over a
bed of CeliteÒ. The reaction mixture was evaporated onto SiO2
(600 mg) and submitted to column chromatography (SiO2: 25 g,
DCM/Et2O 1:1?DCM/MeOH 5:1) to give pure 20 (465 mg, 94.7%)
as white solid. Rf = 0.19 (DCM/Et2O), HRMS: calcd [M+H]+:
528.3689, found [M+H]+: 528.3867; 1H NMR (CDCl3): d 0.77–0.83
(m, 1H, H5), 0.81 (s, 3H, H28), 0.88 (s, 6H, H23/24), 0.98–1.06 (m,
2H, H1b, H16b), 1.12 (s, 3H, H26), 1.15 (s, 3H, H25), 1.18 (s, 3H,
H30), 1.21–1.16 (m, 1H, H15b), 1.33–1.48 (m, 5H, H6b, H22a,
H22b, H7b, H21b), 1.36 (s, 3H, H27), 1.54–1.75 (m, 5H, H19b,
H2a, H7a, H2b H6a), 1.77–1.93 (m, 2H, H19a, H15a), 1.97–2.07
(m, 2H, H21a, H16a), 2.05 (s, 3H, OAc), 2.36 (s, 1H, H9), 2.20–
2.27 (m, 1H, H18), 2.73–2.80 (td, 1H, J = 13.3 Hz, J = 3.4 Hz, H1a),
4.47–4.54 (dd, 1H, J = 11.6 Hz, J = 4.8 Hz, H3), 5.79 (s, 1H, H12),
7.66 (br s, NH/OH); 13C NMR (CDCl3): d 16.4 (q, C25), 16.6 (q,
C23/24), 17.3 (t, C6), 18.6 (q, C26), 21.3 (q, COCH3), 23.3 (q, C27),
23.5 (t, C2), 26.4 (2 ꢃ t, C15, C16), 28.0 (q, C23/24), 28.4 (q, C28),
29.3 (q, C30), 31.0 (t, C21), 31.8 (s, C17), 32.6 (t, C7), 36.9 (s,
C10), 37.2 (t, C22), 38.0 (s, C4), 38.8 (t, C1), 40.7 (t, C19), 42.3 (s,
C20), 43.2 (s, C8), 45.4 (s, C14), 47.7 (d, C18), 55.0 (d, C5), 61.7
(d, C9), 80.6 (d, C3), 128.5 (d, C12), 169.5 (s, C13), 171.0 (s, COCH3),
173.9 (s, C29), 200.6 (s, C11).
4.26. (3b,18b,20b)-3,N-Dihydroxy-N-tertbutyl-11-oxo-olean-12-
en-29-amide (23)
Ester 16 (1.60 g, 2.70 mmol, 1.00 equiv) was reacted analogous
to 21 with KOH (0.70 g, 10.6 mmol, 3.9 equiv) in MeOH/DCM
(50 mL + 10 mL) to give compound 23 (990 mg, 67.7%) as white so-
lid. Rf = 0.35 (DCM/MeOH 95:5); HRMS: calcd [M+H]+: 542.4204,
found [M+H]+: 542.4202; 1H NMR (200 MHz, CDCl3): d 0.75–2.40
(m, 19H), 0.79 (s, 3H), 0.80 (s, 3H), 0.99 (s, 3H), 1.11 (s, 6H), 1.16
(s, 9H), 1.20 (s, 3H), 1.36 (s, 3H), 2.76 (m, 1H), 3.20 (m, 1H), 5.65
(s, 1H), 7.00 (br, 1H); 13C NMR (200 MHz, CDCl3): d 15.58, 16.33,
17.47, 176.15, 18.65, 23.43, 26.35, 26.39, 26.40, 26.67 (3C), 27.28,
28.09, 28.42, 28.78, 31.08, 31.80, 32.73, 37.06, 37.67, 39.11,
40.76, 43.18, 44.01, 45.36, 48.11, 54.90, 55.77, 61.80, 78.67,
128.67, 168.78, 200.09.
4.27. (3b,18b,20b)-3-Hydroxy-N-methoxy-11-oxo-olean-12-en-
29-amide (24)
Ester 17 (148 g, 0.27 mmol, 1.00 equiv) was reacted analogous
to 21 with KOH (179 mg, 2.7 mmol, 10 equiv) in MeOH (13 mL)
to give compound 24 (135 mg, 99%) as white solid. Rf = 0.25
(DCM/MeOH 96:4); HRMS: calcd [M+H]+: 500.3734, found
[M+H]+: 500.3725; 1H NMR (200 MHz, CDCl3): d 0.60–2.40 (m,
21H), 0.78 (s, 3H), 0.80 (s, 3H), 0.98 (s, 3H), 1.10 (s, 6H), 1.14 (s,
3H), 1.34 (s, 3H), 2.74 (d, 1H), 3.20 (m, 1H), 3.73 (s, 3H), 5.67 (s,
1H), 9.33 (br, 1H); 13C NMR (50 MHz, CDCl3): d 15.6, 16.3, 169.6,
17.4, 18.6, 23.3, 26.4 (2C), 27.2, 28.1, 28.5, 29.2, 31.1, 31.8, 32.7,
37.1, 37.3, 39.1, 39.2, 41.1, 42.5, 43.2, 45.4, 48.1, 55.0, 61.8, 64.0,
78.8, 128.4, 173.6, 200.4.
4.24. (3b,18b,20b)-3-Hydroxy-N-methyl-N-methoxy-11-oxo-
olean-12-en-29-amide (21)
4.28. (3b,18b,20b)-N-Allyloxy-3-hydroxy-11-oxo-olean-12-en-
To
a
stirred solution of the ester (180 mg, 0.324 mmol,
29-amide (25)
1.00 equiv) in methanol (ꢁ1% solution) KOH pellets (85%;
211 mg, 3.20 mmol, 10.0 equiv) were added and the reaction mix-
ture was stirred at rt under TLC-monitoring. After 48 h the solvent
was evaporated and the residue was separated between water and
DCM. The combined organic layers were washed with 2 M HCl and
water, dried over Na2SO4 and evaporated. The crude material was
Ester 18 (243 mg, 0.43 mmol, 1.00 equiv) was reacted analo-
gous to 21 with KOH (283 mg, 4.3 mmol, 10 equiv) in MeOH
(20 mL) to give compound 25 (90 mg, 40%) as yellow solid.
Rf = 0.25 (DCM/MeOH 96:4); HRMS: calcd [M+H]+: 526.3891, found
[M+H]+: 526.3882; 1H NMR (200 MHz, CDCl3): d 0.65–2.33 (m,

7536
C. Stanetty et al. / Bioorg. Med. Chem. 18 (2010) 7522–7541
20H), 0.79 (s, 3H), 0.80 (s, 3H), 0.98 (s, 3H), 1.11 (s, 6H), 1.14 (s, 3H),
1.34 (s, 3H), 2.72 (m, 1H), 2.75 (m, 1H), 3.21 (m, 1H), 4.38 (d, 2H),
(s, 3H, H28), 0.90 (s, 3H, H23/24), 0.83–0.93 (m, 1H, H5), 0.96–
1.07 (m, 1H, H16b), 1.11 (s, 3H, H26), 1.13 (s, 3H, H23/24), 1.21
(s, 3H, H30), 1.36 (s, 3H, H27), 1.23–1.45 (m, 5H, H7b, H6b,
H22b, H21b, H15b), 1.45–1.75 (m, 7H, H7a, H19b, H2a, H6a,
H22a, H1b, H2b), 1.81 (dt, 1H, J = 13.6 Hz, J = 4.0 Hz, H15a), 2.03
(s, 3H, OAc), 2.05–2.26 (m, 4H, H21a, H19a, H18, H16a), 2.39 (s,
1H, H9), 2.80–2.70 (m, 1H, H1a), 3.35 (s, 3H, N-Me), 3.62–3.74
(m, 1H, H3), 5.68 (s, 1H, H12), 8.34 (br s, NH/OH); 13C NMR
(CDCl3/MeOD 5:1): d 16.3 (q, C23/24), 16.6 (q, C25), 17.7 (t, C6),
18.6 (q, C26), 23.0 (q, C27), 23.6 (q, COCH3), 25.3 (t, C2), 26.3 (q,
C30), 26.4 (t, C15), 26.7 (t, C16), 28.5 (q(2C), C23/24, C28), 31.8
(t, C21), 32.5 (s, C17), 32.7 (t, C7), 36.9 (s, C10), 37.7 (t, C22),
38.0 (s, C4), 38.3 (q, N-Me), 39.7 (t, C1), 42.3 (t, C19), 43.3 (s,
C20), 43.6 (s, C8), 45.3 (s, C14), 48.5 (d, C18), 55.5 (d, C5), 56.7
(d, C3), 61.7 (d, C9), 128.2 (d, C12), 170.0 (s, COCH3), 170.5 (s,
C13), 174.0 (s, C29), 200.6 (s, C11).
5.28 (s, 1H), 5.34 (d, 1H), 5.67 (s, 1H), 6.00 (m, 1H), 9.05 (s, 1H); 13
C
NMR (50 MHz, CDCl3): d 15.6, 16.3, 17.5, 18.7, 23.3, 26.3, 26.4, 27.2,
28.1, 28.4, 29.4, 31.2, 31.4, 32.7, 37.1, 37.3, 39.2 (2C), 41.1, 42.9,
43.2, 45.4, 47.9, 55.0, 61.8, 64.5, 78.8, 120.6, 128.5, 132.7, 169.4,
173.8, 200.4.
4.29. (3b,18b,20b)-N-Benzyloxy-3-hydroxy-11-oxo-olean-12-en-
29-amide (26)
Ester 19 (483 mg, 0.78 mmol, 1.00 equiv) was reacted analo-
gous to 21 with KOH (514 mg, 7.8 mmol, 10 equiv) in MeOH
(35 mL) to give compound 26 (180 mg, 40%) as yellow solid.
Rf = 0.27 (DCM/MeOH=96:4); HRMS: calcd [M+H]+: 576.4047,
found [M+H]+: 576.4050; 1H NMR (200 MHz, CDCl3): d 0.59–2.30
(m, 21H), 0.76 (s, 3H), 0.78 (s, 3H), 0.96 (s, 3H), 1.11 (s, 9H), 1.31
(s, 3H), 2.72 (m, 1H), 3.15 (m, 1H), 4.92 (s, 2H), 5.44 (s, 1H), 7.37
(m, 5H), 8.98 (m, 1H); 13C NMR (200 MHz, CDCl3): d 15.5, 16.3,
17.4, 18.6, 23.3, 26.3, 26.4, 27.1, 28.1, 28.3, 29.4, 31.1, 31.8, 32.7,
37.0, 37.3, 39.1, 39.4, 40.9, 42.8, 43.1, 45.4, 47.9, 55.0, 61.8, 77.7,
78.8, 128.6, 128.8 (5C), 135.6, 169.1, 175.2, 200.1.
4.32. (3a,18b,20b)-N-Hydroxy-N-methyl-11-oxo-3-[(trifluoro-
methylsulfonyl)amino]-olean-12-en-29-amide (29a)
Carboxylic acid 9a (150 mg, 0.25 mmol, 1.00 equiv) was—analo-
gous to 15—first converted to the acid chloride in SOCl2/toluene
(3.6 mL each) within 2 h and then reacted with MeNHOHꢀHCl
4.30. (3a,18b,20b)-3-Acetylamino-N-hydroxy-N-methyl-11-oxo-
(31.0 mg, 0.37 mmol, 1.50 equiv) and TEA (138 lL, 1.00 mmol,
olean-12-en-29-amide (28a)
4.00 equiv) in DCM (10 mL). Aqueous workup gave crude material
which was purified by flash column chromatography (SiO2: 20 g,
DCM/MeOH 50:1?DCM/MeOH 30:1) to give pure 29a (96 mg,
Carboxylic acid 7a (50 mg, 0.098 mmol, 1.00 equiv) was—analo-
gous to 15—first converted to the acid chloride in SOCl2/toluene
(1.4 mL each) within 2 h and was then reacted with MeNHOHꢀHCl
61.0%) as orange solid. Rf = 0.61 (CHCl3/MeOH 9:1 + AcOH); ½a D20
ꢄ
+81.9 (c 1.0, CHCl3/MeOH 3:1); HRMS: calcd [MꢂH]ꢂ: 629.3236,
found [MꢂH]ꢂ: 629.2848; 1H NMR (CDCl3/MeOD 5:1): d 0.82 (s,
3H, H28), 0.98 (s, 3H, H23/24), 0.99 (s, 3H, H23/24), 0.96–1.08
(m, 2H, H16b, H5), 1.13 (s, 3H, H26), 1.16 (s, 3H, H25), 1.21 (s,
3H, H30), 1.08–1.25 (m, 2H, H15b, H1b), 1.25–1.37 (m, 1H,
H21b), 1.41 (s, 3H, H27), 1.37–1.50 (m, 4H, H22a, H7b, H6b,
H22b), 1.61–1.91 (m, 3H, H15a, H7a, H19b), 1.61 1.50 (m, 2H,
H6a, H2b), 2.03–2.31 (m, 5H, H19a, H21a, H18, H2a, H16a), 2.58
(s, 1H, H9), 2.57–2.67 (m, 1H, H1a), 3.25 (s, N-Me), 3.28–3.38 (m,
1H, H3), 3.67 (br s, NH/OH), 5.70 (s, 1H, H12); 13C NMR (CDCl3/
MeOD 5:1): d 16.3 (q, C25), 17.1 (t, C6), 18.5 (q, C26), 22.7 (q,
C23/24), 22.8 (q, C27), 23.9 (t, C2), 25.7 (q, C30), 26.3 (t, C15),
26.5 (t, C16), 28.3 (q, C28), 28.5 (q, C23/24), 31.6 (t, C21), 31.9 (s,
C17), 32.3 (t, C7), 33.3 (t, C1), 36.7 (s, C10), 36.8 (s, C4), 37.7 (t,
C22), 37.8 (q, N-Me), 42.1 (t, C19), 43.4 (s, C20), 44.1 (s, C8), 45.4
(s, C14), 48.5 (d, C18), 48.9 (d, C5), 61.0 (d, C9), 61.4 (d, C3),
127.6 (d, C12), 171.9 (s, C13), 175.6 (s, C29), 201.6 (s, C11).
(12.0 mg, 0.15 mmol, 1.50 equiv) and TEA (54 lL, 0.39 mmol,
4.00 equiv) in DCM (10 mL). Aqueous workup gave crude material
which was purified by flash column chromatography (SiO2: 5 g,
DCM/MeOH 30:1?DCM/MeOH 10:1) to give pure 28a (38 mg,
71.2%) as orange solid. Rf = 0.74 (DCM/MeOH 5:1); ½a D20
ꢄ
+96.4 (c
1.0, CHCl3/MeOH 3:1); HRMS: calcd [MꢂH]ꢂ: 539.3849, found
[MꢂH]ꢂ: 539.3737; 1H NMR (CDCl3): d 0.75–0.84 (m, 1H, H5),
0.82 (s, 3H, H28), 0.87 (s, 3H, H23/24), 0.94–1.09 (m, 2H, H16b,
H1b), 0.98 (s, 3H, H23/24), 1.13 (s, 3H, H26), 1.16 (s, 3H, H25),
1.21 (s, 3H, H30), 1.25–1.76 (m, 11H, H7a, H19b, H6a, H22a, H7b,
H6b, H22b, H21b, H15b, H2a, H2b), 1.41 (s, 3H, H27), 1.77–1.92
(m, 1H, H15a), 1.96–2.27 (m, 4H, H18, H21a, H19a, H16a), 2.04
(s, 3H, NHAc), 2.41 (s, 1H, H9), 2.62–2.73 (d, 1H, J = 13.1 Hz,
H1a), 3.36 (s, 3H, N-Me), 3.79–3.88 (d, 1H, J = 7.6 Hz, H3), 5.68 (s,
1H, H12), 6.02 (br s, NH), 8.59 (br s, OH); 13C NMR (CDCl3): d
16.5 (q, C25), 17.3 (t, C6), 18.7 (q, C26), 23.2 (q, C23/24), 23.4 (q,
C27), 23.6 (q, COCH3), 26.3 (q, C30), 26.4 (2 ꢃ t, C2, C15), 26.7 (t,
C16), 28.4 (q, C23/24), 28.5 (q, C28), 31.8 (t, C21), 32.5 (s, C17),
32.6 (t, C7), 34.9 (t, C1), 36.5 (s, C10), 37.3 (s, C4), 37.7 (t, C22),
38.3 (q, N-Me), 42.6 (t, C19), 43.4 (s, C20), 43.5 (s, C8), 45.5 (s,
C14), 48.4 (d, C18), 51.1 (d, C5), 53.8 (d, C3), 61.8 (d, C9), 128.3
(d, C12), 169.6 (s, C13), 170.5 (s, COCH3), 173.8 (s, C29), 200.4 (s,
C11).
4.33. (3b,18b,20b)-N-Hydroxy-N-methyl-11-oxo-3-[(trifluoro-
methylsulfonyl)amino]-olean-12-en-29-amide (29b)
Carboxylic acid 9b (300 mg, 0.50 mmol, 1.00 equiv) was—analo-
gous to 15—first converted to the acid chloride in SOCl2/toluene
(7.2 mL each) within 2 h and then reacted with MeNHOHꢀHCl
(62.0 mg, 0.75 mmol, 1.50 equiv) and TEA (276 lL, 2.00 mmol,
4.31. (3b,18b,20b)-3-Acetylamino-N-hydroxy-N-methyl-11-oxo-
olean-12-en-29-amide (28b)
4.00 equiv) in DCM (25 mL). Aqueous workup gave crude material
which was purified by flash column chromatography (SiO2: 30 g,
DCM/MeOH 40:1?DCM/MeOH 30:1) to give pure 29b (257 mg,
Carboxylic acid 7b (300 mg, 0.59 mmol, 1.00 equiv) was—analo-
gous to 15–first converted to the acid chloride in SOCl2/toluene
(8.5 mL each) within 3 h and was then reacted with MeNHOHꢀHCl
81.4%) as white solid. Rf = 0.30 (CHCl3/MeOH 9:1 + AcOH); ½a D20
ꢄ
+97.1 (c 1.0, CHCl3/MeOH 3:1); HRMS: calcd [MꢂH]ꢂ: 629.3236,
found [MꢂH]ꢂ: 629.2745; 1H NMR (CDCl3): d 0.75 (s, 3H, H23/
24), 0.82 (s, 3H, H28), 0.80–0.87 (m, 1H, H5), 1.04 (s, 3H, H23/
24), 1.08 (s, 3H, H26), 1.10 (s, 3H, H25), 0.99–1.13 (m, 2H, H1b,
H16b), 1.21 (s, 3H, H30), 1.16–1.29 (m, 2H, H21b, H15b), 1.37 (s,
3H, H27), 1.34–1.48 (m, 3H, H7b, H6b, H22b), 1.57–1.87 (m, 6H,
H2b, H15a, H19b, H7a, H6a, H22a), 1.88–1.98 (m, 1H, H2a), 2.04–
2.17 (m, 3H, H21a, H18, H16a), 2.22–2.30 (d, 1H, J = 13.6 Hz,
H19a), 2.40 (s, 1H, H9), 2.80–2.88 (m, 1H, H1a), 3.14–3.24 (m,
(73.0 mg, 0.88 mmol, 1.50 equiv) and TEA (325 lL, 2.35 mmol,
4.00 equiv) in DCM (10 mL). Aqueous workup gave crude material
which was purified by flash column chromatography (SiO2: 85 g,
DCM/MeOH 20:1) to give pure 28b (255 mg, 80.4%) as white solid.
Rf = 0.49 (CHCl3/MeOH 9:1 + AcOH); ½a D20
ꢄ
+115.2 (c 1.0, CHCl3/
MeOH 3:1); HRMS: calcd [MꢂH]ꢂ: 539.3849, found [MꢂH]ꢂ:
539.3854; 1H NMR (CDCl3/MeOD 5:1): d 0.80 (s, 3H, H25), 0.81

C. Stanetty et al. / Bioorg. Med. Chem. 18 (2010) 7522–7541
7537
1H, H3), 3.33 (s, 3H, N-Me), 3.70 (br s, 1H, NH/OH), 5.81 (s, 1H,
H12), 7.21 (br s, NH/OH); 13C NMR (CDCl3): d 16.0 (q, C25), 16.5
(q, C23/24), 18.0 (t, C6), 18.5 (q, C26), 23.0 (q, C27), 25.7 (t, C2),
25.9 (q, C30), 26.5 (t, C15), 26.7 (t, C16), 28.48 (q, C28), 28.52 (q,
C23/24), 31.8 (s, C17), 32.6 (t, C21), 33.3 (t, C7), 36.9 (s, C10),
37.5 (t, C22), 38.3 (q, N-Me), 38.8 (s, C4), 39.7 (t, C1), 42.0 (t,
C19), 43.4 (s, C20), 43.8 (s, C8), 45.5 (s, C14), 49.1 (d, C18), 55.5
(d, C5), 61.5 (d, C9), 64.4 (d, C3), 127.4 (d, C12), 172.8 (s, C13),
174.6 (s, C29), 201.7 (s, C11).
C6), 36.9 (s, C10), 37.3 (t, C22), 38.1 (s, C4), 39.8 (t, C1), 40.8 (t,
C19), 42.8 (s, C20), 43.1 (s, C8), 45.3 (s, C14), 47.7 (d, C18), 55.5
(d, C5), 56.7 (d, C3), 61.7 (d, C9), 77.7 (t, OCH2Ph), 128.5 (d, C12),
128.6 (2 ꢃ d, PhC3, PhC5), 128.8 (d, PhC4), 129.2 (d, PhC2, PhC6),
135.4 (s, PhC1), 169.0 (s, C13), 170.0 (s, COCH3), 173.5 (s, C29),
200.0 (s, C11).
4.36. (3a,18b,20b)-N-Benzyloxy-11-oxo-3-[(trifluoromethyl-
sulfonyl)amino]-olean-12-en-29-amide (31a)
4.34. (3
12-en-29-amide (30a)
a
,18b,20b)-3-(Acetylamino)-N-benzyloxy-11-oxo-olean-
Carboxylic acid 9a (150 mg, 0.25 mmol, 1.00 equiv) was—analo-
gous to 15—first converted to the acid chloride in SOCl2/toluene
(4 mL each) within 2 h and was then reacted with NH2OBn
Carboxylic acid 7a (100 mg, 0.20 mmol, 1.00 equiv) was—analo-
gous to 15—first converted to the acid chloride in SOCl2/toluene
(2.8 mL each) overnight, and was then reacted with a pre-stirred
solution of NH2OBn (29 mg 0.23 mmol, 1.20 equiv) and TEA
(37.0 mg, 0.30 mmol, 1.20 equiv) and TEA (69.0 lL, 0.50 mmol,
2.00 equiv) in DCM (20 mL). Aqueous workup gave crude material
which was purified by flash column chromatography (SiO2: 20 g,
Hex/EtOAc 3:1?Hex/EtOAc 1:1 + 0.1% AcOH) to give pure 31a
(157 mg, 89.2%) as orange solid. Rf = 0.68 (CHCl3/MeOH 9:1 +
(54 lL, 0.40 mmol, 2.00 equiv) in acetonitrile (4 mL). Aqueous
workup gave crude material which was purified by flash column
chromatography (SiO2: 10 g, DCM/Et2O 5:1?DCM/MeOH 10:1) to
give pure 30a (102 mg, 84.6%) as orange solid. Rf = 0.41 (DCM/
AcOH); ½a 2D0
ꢄ
+94.2 (c 1.0, CHCl3/MeOH 3:1) HRMS: calcd [MꢂH]ꢂ:
705.3549, found [MꢂH]ꢂ: 705.4057; 1H NMR (CDCl3/MeOD 5:1):
d 0.77 (s, 3H, H28), 0.98 (s, 3H, H23/24), 0.99 (s, 3H, H23/24),
1.01–1.10 (m, 2H, H5, H16b), 1.11 (s, 3H, H30), 1.12 (s, 3H, H26),
1.16 (s, 3H, H25), 1.15–1.25 (m, 2H, H15b, H1b), 1.27–1.34 (m,
3H, H22a, H22b, H21b), 1.39 (s, 3H, H27), 1.38–1.42 (m, 2H, H6b,
H7b), 1.49–1.69 (m, 3H, H19b, H2b, H6a), 1.69–1.88 (m, 3H, H15a,
H19a, H7a), 1.88–1.96 (m, 1H, H21a), 1.96–2.08 (m, 2H, H18,
H16a), 2.08–2.16 (m, 1H, H2a), 2.59 (s, 1H, H9), 2.57–2.66 (m, 1H,
H1a), 3.31–3.36 (m, 1H, H3), 4.17 (br s, NH/OH), 4.91 (s, 2H, Bn),
5.48 (s, 1H, H12), 7.32–7.46 (m, 5H, Bn); 13C NMR (CDCl3/MeOD
5:1): d 16.2 (q, C25), 16.9 (t, C6), 18.3 (q, C26), 22.6 (q, C23/24),
22.8 (q, C27), 23.8 (t, C2), 25.98 (t, C15), 26.02 (t, C16), 28.0 (q,
C28), 28.3 (q, C23/24), 28.7 (q, C30), 30.5 (t, C21), 31.3 (s, C17),
32.0 (t, C7), 33.1 (t, C1), 36.5 (s, C10), 36.7 (s, C4), 37.0 (t, C22), 40.5
(t, C19), 42.4 (s, C20), 43.1 (s, C8), 45.2 (s, C14), 47.5 (d, C18), 48.5
(d, C5), 60.9 (d, C9), 61.3 (d, C3), 77.3 (t, OCH2Ph), 127.7 (d, C12),
128.2 (2 ꢃ d, PhCH), 128.4 (d, PhCH), 128.9 (2 ꢃ d, 2 ꢃ PhCH),
135.3 (s, PhC), 170.8 (s, C13), 173.7 (s, C29), 201.4 (s, C11).
MeOH 5:1); ½a 2D0
ꢄ
+89.6 (c 1.0, CHCl3/MeOH 3:1); HRMS: calcd
[MꢂH]ꢂ: 615.4162, found [MꢂH]ꢂ: 615.4016; 1H NMR (CDCl3): d
0.68–0.77 (m, 1H, H5), 0.72 (s, 3H, H28), 0.80 (s, 3H, H23/24),
0.86–1.00 (m, H, H2b, H16b), 0.90 (s, 3H, H25), 1.00–1.16 (m, 1H,
H15b), 1.06 (s, 6H, H26, H30), 1.09 (s, 3H, H23/24), 1.16–1.50 (m,
7H, H2a, H2b, H6b, H7a, H7b, H22a, H22b), 1.31 (s, 3H, H27),
1.50–1.82 (m, 5H, H6a, H16a, H19a, H19b, H21b), 1.82–2.08 (m,
3H, H15a, H18, H21a), 1.97 (s, 3H, NHAc), 2.32 (s, 1H, H9), 2.54–
2.65 (td, 1H, H2a), 3.72–3.81 (m, 1H, H3), 4.88 (s, 2H, CH2-Ph),
5.43 (s, 1H, H12), 7.22 7.38 (m, 5H, Aromat), 8.71 (s, 1H, NH); 13C
NMR (CDCl3): d 16.5 (q, C25), 17.2 (t, C7), 18.6 (q, C26), 23.1 (q,
C23/24), 23.6 (t, 2 ꢃ q, C2, COCH3, C27), 26.3 (2 ꢃ t, C15, C16),
28.4 (2 ꢃ q, C23/24, C28), 29.4 (q, C30), 31.2 (t, C21), 31.7 (s,
C17), 32.4 (t, C6), 34.8 (t, C1), 36.5 (s, C4), 37.2 (s, C10), 37.3 (t,
C22), 41.1 (t, C19), 42.9 (s, C20), 43.2 (s, C8), 45.5 (s, C14), 47.8
(d, C18), 51.0 (d, C5), 53.8 (d, C3), 61.7 (d, C9), 77.9 (t, OCH2Ph),
128.4 (d, C12), 128.6 (2 ꢃ d, PhC3, PhC5), 128.8 (d, PhC4), 129.2
(2 ꢃ d, PhC2, PhC6), 135.3 (s, PhC1), 169.2 (s, COCH3), 169.5 (s,
C13), 173.5 (s, C29), 200.0 (s, C11).
4.37. (3b,18b,20b)-N-Benzyloxy-11-oxo-3-[(trifluoromethyl-
sulfonyl)amino]-olean-12-en-29-amide (31b)
4.35. (3b,18b,20b)-3-(Acetylamino)-N-benzyloxy-11-oxo-olean-
12-en-29-amide (30b)
Carboxylic acid 9b (500 mg, 0.83 mmol, 1.00 equiv) was—analo-
gous to 15—first converted to the acid chloride in SOCl2/toluene
(12 mL each) (within 2 h) and was then reacted with a pre-stirred
solution of NH2OBn (123 mg, 1.00 mmol, 1.20 equiv) and TEA
Carboxylic acid 7b (200 mg, 0.39 mmol, 1.00 equiv) was—analo-
gous to 15—first converted to the acid chloride in SOCl2/toluene
(5.7 mL each) overnight and was then reacted with a pre-stirred
solution of NH2OBn (58 mg, 0.47 mmol, 1.20 equiv) and TEA
(230 lL, 1.66 mmol, 2.00 equiv) in DCM (20 mL). Aqueous workup
gave crude material which was purified by flash column chroma-
tography (SiO2: 30 g, Hex/EtOAc 3:1 + 0.1% AcOH to Hex/EtOAc
1:1 + 0.1% AcOH to CHCl3/MeOH 9:1 + 0.1% AcOH) to give pure
31b (476 mg, 81.0%) as white solid. Rf = 0.67 (CHCl3/MeOH
(108 lL, 0.78 mmol, 2.00 equiv) in acetonitrile (3 mL). Aqueous
workup gave crude material which was purified by flash column
chromatography (SiO2: 25 g, DCM/Et2O 5:1?DCM/MeOH 20:1) to
give pure 30b (224 mg, 92.9%) as orange solid. Rf = 0.45 (DCM/
9:1 + AcOH); ½a 2D0
ꢄ
+106.4 (c 1.0, CHCl3/MeOH 3:1); HRMS: calcd
[MꢂH]ꢂ: 705.3549, found [MꢂH]ꢂ: 705.4055; 1H NMR (CDCl3/
MeOD 5:1): d 0.76 (s, 3H, H28), 0.83 (s, 3H, H23/24), 0.73–0.87
(m, 1H, H5), 1.02 (s, 3H, H23/24), 0.96–1.06 (m, 2H, H1b, H16b),
1.11 (s, 3H, H30), 1.12 (s, 3H, H26), 1.14 (s, 3H, H25), 1.06–1.25
(m, 1H, H15b), 1.35 (s, 3H, H27), 1.25–1.38 (m, 3H, H22a, H22b,
H21b), 1.38–1.55 (m, 2H, H6b, H7b), 1.55–1.71 (m, 5H, H7a, H6a,
H2a, H2b, H19b), 1.71–1.93 (m, 2H, H15a, H19a), 1.93–2.09 (m,
3H, H18, H16a, H21a), 2.38 (s, 1H, H9), 2.75–2.86 (m, 1H, H1a),
3.08–3.17 (m, 1H, H3), 4.09 (s, 1H, NH), 4.91 (s, 2H, OCH2Ph),
5.49 (s, 1H, H12), 7.29–7.48 (m, 5H, Bn); 13C NMR (CDCl3/MeOD
5:1): d 15.9 (q, C25), 16.0 (q, C23/24), 17.7 (t, C6), 18.3 (q, C26),
23.0 (q, C27), 25.3 (t, C2), 26.1 (2 ꢃ t, C15, C16), 28.0 (q, C28),
28.1 (q, C30), 28.7 (q, C23/24), 30.6 (t, C21), 31.3 (s, C17), 32.3 (t,
C7), 36.6 (s, C10), 37.0 (t, C22), 38.4 (s, C4), 39.7 (t, C1), 40.5 (t,
C19), 42.4 (s, C20), 43.0 (s, C8), 45.1 (s, C14), 47.5 (d, C18), 55.3
MeOH 5:1); ½a 2D0
ꢄ
+108.9 (c 1.0, CHCl3/MeOH 3:1); HRMS: calcd
[MꢂH]ꢂ: 615.4162, found [MꢂH]ꢂ: 615.4016; 1H NMR (CDCl3): d
0.70 (s, 3H, H28), 0.72 (s, 3H, C23/24), 0.73–0.79 (m, 1H, H5),
0.81 (s, 3H, H23/24), 0.86–0.99 (m, 2H, H16b, H1b), 1.04 (s, 6H,
H26, H30), 1.05 (s, 3H, H25), 1.07–1.14 (m, 1H, H15b), 1.14–1.39
(m, 6H, H6b, H7b, H16a, H21b, H22a, H22b), 1.26 (s, 3H, H27),
1.39–1.60 (m, 5H, H2a, H2b, H6a, H7a, H19b), 1.63–1.79 (m, 2H,
H15a, H19a), 1.83–1.93 (m, 1H, H21a), 1.94 (s, 3H, NHAc), 1.97–
2.06 (m, 1H, H18), 2.26 (s, 1H, H9), 2.63–2.73 (m, 1H, H1a), 2.64–
2.74 (m, 1H, H3), 4.89 (s, 2H, CH2Ph), 5.43 (s, 1H, H12), 7.24–7.38
(m, 5H, Aromat), 8.83 (s, 1H, NH); 13C NMR (CDCl3): d 16.2 (q,
C25), 16.6 (q, C23/24), 17.6 (t, C7), 18.6 (q, C26), 23.3 (q, C27),
23.4 (q, COCH3), 25.3 (t, C2), 26.3 (2 ꢃ t, C15, C16), 28.3 (q, C28),
28.6 (q, C23/24), 29.4 (q, C30), 31.2 (t, C21), 31.7 (s, C17), 32.6 (t,

7538
C. Stanetty et al. / Bioorg. Med. Chem. 18 (2010) 7522–7541
(d, C5), 61.4 (d, C9), 63.8 (d, C3), 77.4 (t, OCH2Ph), 127.8 (d, C12),
128.2 (2 ꢃ d, 2 ꢃ PhCH), 128.4 (d, PhCH), 128.9 (2 ꢃ d, PhCH),
135.3 (s, PhC), 170.7 (s, C13), 173.7 (s, C29), 200.9 (s, C11).
(2.8 mL) with Pd/C (28 mg, 20%). The crude material was purified
by flash column chromatography (SiO2: 20 g, Hex/EtOAc 3:1,
240 mL to DCM/MeOH 20:1?DCM/MeOH 5:1) to give pure 33a
(57 mg, 64%) as orange solid. Rf = 0.43 (CHCl3/MeOH 9:1 + AcOH);
4.38. (3a,18b,20b)-3-(Acetylamino)-N-hydroxy-11-oxo-olean-
½
a 2D0
ꢄ
+89.9 (c 1.0, CHCl3/MeOH 3:1); HRMS: calcd [MꢂH]ꢂ:
12-en-29-amide (32a)
615.3080, found [MꢂH]ꢂ: 615.2673; 1H NMR (CDCl3/MeOD 5:1):
d 0.83 (s, 3H, H28), 0.98 (s, 3H, H23/24), 0.99 (s, 3H, H23/24),
1.13 (s, 3H, H26), 1.15 (s, 6H, H25, H30), 1.01–1.27 (m, 5H, H15b,
H1b, H21b, H16b, H5), 1.42 (s, 3H, H27), 1.37–1.47 (m, 4H, H7b,
H6b, H22a, H22b), 1.50–1.61 (m, 2H, H2b, H6a), 1.62–1.75 (m,
2H, H7a, H19b), 1.78–1.99 (m, 3H, H21a, H15a, H19a), 2.00–2.34
(m, 3H, H18, H2a, H16a), 2.61 (s, 1H, H9), 2.56–2.67 (m, 1H,
H1a), 3.30–3.35 (m, 1H, H3), 4.02 (br s, NH/OH), 5.68 (s, 1H,
H12); 13C NMR (CDCl3/MeOD 5:1): d 16.2 (q, C25), 17.0 (t, C6),
18.4 (q, C26), 22.6 (q, C23/24), 22.8 (q, C27), 23.9 (t, C2), 26.1
(2 ꢃ t, C15, C16), 28.1 (q, C28), 28.4 (q, C23/24), 28.9 (q, C30),
30.5 (t, C21), 31.4 (s, C17), 32.2 (t, C7), 33.2 (t, C1), 36.6 (s, C10),
36.7 (s, C4), 37.0 (t, C22), 40.7 (t, C19), 42.1 (s, C20), 43.2 (s, C8),
45.3 (s, C14), 47.8 (d, C18), 48.7 (d, C5), 60.9 (d, C9), 61.4 (d, C3),
127.7 (d, C12), 170.8 (s, C13), 173.5 (s, C29), 201.4 (s, C11).
O-Bn-hydroxamic acid 30a (90 mg, 0.15 mmol, 1.00 equiv) was
debenzylated by hydrogenolysis analogous to 20 in THF (2 mL)
with Pd/C (18 mg, 22%). The crude material was purified by flash
column chromatography (SiO2: 6 g, DCM/Et2O 1:1 + 0.1% AcOH to
DCM/MeOH 20:1 and DCM/MeOH 5:1). Target compound contain-
ing fractions were pooled and washed with NaHCO3 and brine,
dried over Na2SO4 and evaporated to give pure 32a (31 mg,
40.0%) as orange solid. Rf = 0.29 (DCM/MeOH 5:1); ½a D20
ꢄ
+87.8 (c
1.0, CHCl3/MeOH 3:1); HRMS: calcd [MꢂH]ꢂ: 525.3693, found
[MꢂH]ꢂ: 525.3559; 1H NMR (CDCl3): d 0.71–0.93 (m, 2H, H5,
H16b), 0.82 (s, 3H, H28), 0.87 (s, 3H, H23/24), 0.93–1.09 (m, 2H,
H1b, H15b), 0.97 (s, 3H, H23/24), 1.09–1.30 (m, 2H, H16a, H21b),
1.13 (s, 3H, H26), 1.15 (s, 3H, H25), 1.19 (s, 3H, H30), 1.30–1.58
(m, 7H, H2a, H2b, H6b, H7a, H7b, H22a, H22b), 1.41 (s, 3H, H27),
1.58–1.76 (m, 2H, H6a, H19b), 1.77–1.95 (m, 2H, H15a, H19a),
1.94–2.13 (m, 1H, H21a), 2.03 (s, 3H, NHAc), 2.15–2.32 (m, 1H,
H18), 2.41 (s, 1H, H9), 2.57–2.71 (m, 1H, H1a), 3.78–3.89 (m, 1H,
H3), 5.78 (s, 1H, H12); 13C NMR (CDCl3): d 16.5 (q, C25), 17.2 (t,
C7), 18.6 (q, C26), 23.1 (q, C23/24), 23.6 (2 ꢃ q, COCH3, C27), 26.4
(3 ꢃ t, C2, C15, C16), 28.4 (2 ꢃ q, C23/24, C28), 29.5 (q, C30), 30.9
(t, C21), 31.8 (s, C17), 32.4 (t, C6), 34.8 (t, C1), 36.5 (s, C4), 37.2 (s,
C10, t, C22), 40.8 (t, C19), 42.3 (s, C20), 43.3 (s, C8), 45.6 (s, C14),
47.7 (d, C18), 51.0 (d, C5), 53.7 (d, C3), 61.7 (d, C9), 128.5 (d, C12),
169.7 (s, COCH3), 169.9 (s, C13), 173.7 (s, C29), 200.7 (s, C11).
4.41. (3b,18b,20b)-N-Hydroxy-11-oxo-3-[(trifluoro-methylsul-
fonyl)amino]-olean-12-en-29-amide (33b)
O-Bn-hydroxamic acid 31b (375 mg, 0.53 mmol, 1.00 equiv)
was debenzylated by hydrogenolysis analogous to 20 in THF
(7.5 mL) with Pd/C (75 mg, 20%). The crude material was purified
by flash column chromatography (SiO2: 85 g, DCM/MeOH
9:1 + 0.1% AcOH to DCM/MeOH 5:1 + 0.1% AcOH) to give pure
33b (149 mg, 45.4%) as orange solid. Rf = 0.43 (CHCl3/MeOH
9:1 + AcOH); ½a 2D0
ꢄ
+96.2 (c 1.0, CHCl3/MeOH 3:1); HRMS: calcd
[MꢂH]ꢂ: 615.3080, found [MꢂH]ꢂ: 615.2993; 1H NMR (CDCl3/
MeOD 5:1): d 0.74 (s, 3H, H28), 0.75 (s, 3H, H23/24), 0.69–0.78
(m, 1H, H5), 0.94 (s, 3H, H23/24), 1.05 (s, 6H, H25, H26), 1.07 (s,
3H, H30), 0.89–1.10 (m, 2H, H16b, H1b), 1.10–1.17 (m, 1H,
H15b), 1.30 (s, 3H, H27), 1.17–1.42 (m, 5H, H6b, H7b, H22a,
H22b, H21b), 1.50–1.65 (m, 4H, H19b, H7a, H6a, H15a), 1.69–
2.16 (m, 6H, H18, H16a, H21a, H19a, H2a, H2b), 2.31 (s, 1H, H9),
2.66–2.77 (m, 1H, H1a), 3.09–3.00 (m, 1H, H3), 3.93 (br s, NH/
OH), 5.60 (s, 1H, H12); 13C NMR (CDCl3/MeOD 5:1): d 16.1 (q,
C25), 16.2 (q, C23/24), 17.9 (t, C6), 18.5 (q, C26), 23.2 (q, C27),
25.0 (t, C2), 25.5 (t, C15), 26.3 (t, C16), 28.2 (q, C23/24), 28.3 (q,
C28), 29.1 (q, C30), 30.7 (t, C21), 31.6 (s, C17), 32.5 (t, C7), 36.8
(s, C10), 37.2 (t, C22), 38.6 (s, C4), 39.9 (t, C1), 40.8 (t, C19), 42.3
(s, C20), 43.3 (s, C8), 45.4 (s, C14), 47.9 (d, C18), 55.5 (d, C5), 61.7
(d, C9), 64.0 (d, C3), 128.0 (d, C12), 171.0 (s, C13), 173.5 (s, C29),
201.2 (s, C11).
4.39. (3b,18b,20b)-3-(Acetylamino)-N-hydroxy-11-oxo-olean-12-
en-29-amide (32b)
O-Bn-hydroxamic acid 30b (90 mg, 0.15 mmol, 1.00 equiv) was
debenzylated by hydrogenolysis analogous to 20 in THF (2 mL)
with Pd/C (18 mg, 22%). The crude material was purified by column
chromatography (SiO2: 6 g, DCM/MeOH 20:1, DCM/MeOH 10:1).
Target compound containing fractions were pooled and washed
with NaHCO3 and brine, dried over Na2SO4 and evaporated to give
pure 30b (37 mg, 47.5%) as orange solid. Rf = 0.37 (DCM/MeOH
5:1); ½a 2D0
ꢄ
+112.1 (c 1.0, CHCl3/MeOH 3:1); HRMS: calcd [MꢂH]ꢂ:
525.3693, found [MꢂH]ꢂ: 525.3465; 1H NMR (CDCl3): d 0.67–
0.77 (m, 1H, H5), 0.72 (s, 3H, H23/24), 0.75 (s, 3H, H23/24), 0.81
(s, 3H, H23/24), 0.82–0.92 (m, 1H, H1b), 0.92–0.99 (m, 1H,
H16b), 1.03 (s, 3H, H26), 1.04 (s, 3H, H25), 1.05–1.17 (m, 1H,
H15b), 1.08 (s, 3H, H30), 1.16–1.41 (m, 5H, H6b, H7b, H21b,
H22a, H22b), 1.31 (s, 3H, H27), 1.41–1.68 (m, 5H, H2a, H2b, H6a,
H7a, H19b), 1.68–1.81 (m, 1H, H15a), 1.86–2.10 (m, 3H, H16a,
H19a, H21a), 2.00 (s, 3H, NHAc), 2.12–2.23 (m, 1H, H18), 2.28 (s,
1H, H9), 2.64–2.74 (m, 1H, H1a), 3.50–3.61 (m, 1H, H3), 5.76 (s,
1H, H12), 10.37 (s, 1H, NH); 13C NMR (CDCl3) d 16.2 (q, C25),
16.6 (q, C23/24), 17.7 (t, C7), 18.6 (q, C26), 23.5 (2 ꢃ q, C27,
COCH3), 25.3 (t, C2), 26.4 (2 ꢃ t, C15, C16), 28.4 (q, C28), 28.7 (q,
C23/24), 29.4 (q, C30), 31.1 (t, C21), 31.7 (s, C17), 32.7 (t, C6),
37.0 (s, C10), 37.4 (t, C22), 38.2 (s, C4), 40.2 (2 ꢃ t, C1, C19), 42.3
(s, C20), 43.2 (s, C8), 45.4 (s, C14), 47.6 (d, C18), 55.8 (d, C5), 56.8
(d, C3), 61.8 (d, C9), 129.1 (d, C12), 168.9, 169.8 (2 ꢃ s, C13,
COCH3), 173.2 (s, C29), 200.1 (s, C11).
4.42. (18b,20b)-N-Benzyloxy-3,11-dioxo-olean-12-en-29-amide
(35)
Carboxylic acid 34 (1.00 g, 2.13 mmol, 1.00 equiv) was sus-
pended in dry DCM (20 mL, 5 drops DMF added as catalyst) and
oxalyl chloride (240 lL, 2.55 mmol, 1.2 equiv) was added at 0 °C.
The reaction mixture was allowed to reach rt and was stirred under
TLC-monitoring (sample from MeOH solution, SiO2: Hex/EtOAc
2:1) until complete conversion was obtained. The reaction mixture
was evaporated at rt, co-evaporated from DCM once and re-dis-
solved in DCM. The clear solution was cooled to 0 °C and first
TEA (648 mg, 6.40 mmol, 3.0 equiv) and then a concentrated solu-
tion of BnONH2 (289 mg, 2.35 mmol, 1.1 equiv) in DCM was added
at 0 °C. The formation of hydroxamic acid was followed via TLC
(SiO2: Hex/EtOAc 2:1 and amino-phase: DCM/MeOH 20:1). Upon
complete conversion, the reaction mixture was diluted with DCM
and washed with 1 M HCl, satd NaHCO3 and brine, dried over
Na2SO4 and evaporated to give crude material which was purified
4.40. (3a,18b,20b)-N-Hydroxy-11-oxo-3-[(trifluoromethylsul-
fonyl)amino]-olean-12-en-29-amide (33a)
O-Bn-hydroxamic acid 31a (142 mg, 0.20 mmol, 1.00 equiv)
was debenzylated by hydrogenolysis analogous to 20 in THF

C. Stanetty et al. / Bioorg. Med. Chem. 18 (2010) 7522–7541
7539
by column chromatography (SiO2: 60 g DCM/MeOH 50:1) and
passing over a short bed of amino-phase gel (7 g) to give pure 35
(1.05 g, 85.8%) as white solid foam. Rf = 0.29 (Hex/EtOAc 1:1);
HRMS: calcd [M+H]+: 498.3578, found [M+H]+: 498.3572; 1H NMR
(CDCl3): d 0.83 (s, 3H, H28), 1.00–1.08 (m, 1H, H16b), 1.07 (s, 3H,
H23/24), 1.10 (s, 3H, H23/24), 1.16 (s, 3H, H26), 1.2 (s, 3H, H30),
1.21–1.36 (m, 3H, H5, H15b, H21b), 1.26 (s, 3H, H25), 1.33–1.46
(m, 2H, H1b, H22b), 1.37 (s, 3H, H27), 1.41–1.64 (m, 6H, H6a,
H6b, H7a, H7b, H19b, H22a), 1.86 (td, J = 13.6 Hz, J = 4.3 Hz, 1H,
H15a), 2.09 (td, J = 13.6 Hz, J = 4.3 Hz, 1H, H16a), 2.18–2.25 (m,
3H, H18, H19a, H21a), 2.37 (ddd, J = 15.8 Hz, J = 6.5 Hz, J = 4.1 Hz,
1H, H2b), 2.45 (s, 1H, H9), 2.62 (ddd, J = 15.8 Hz, J = 11.1 Hz,
J = 7.1 Hz, 1H, H2a), 2.93 (ddd, J = 13.5 Hz, J = 7.1 Hz, J = 4.1 Hz,
1H, H1a), 3.36 (s, 3H, NCH3), 5.69 (s, 1H, H12); 13C NMR (CDCl3):
d 15.7 (q, C25), 18.5 (q, C26), 18.7 (t, C7), 21.4 (q, C23/24), 23.1
(q, C27), 26.3 (q, C30), 26.4 (q, C23/24), 26.5 (t, C15), 26.7 (t,
C16), 28.6 (q, C28), 31.9 (s, C17), 32.1 (t, C6), 32.5 (t, C21), 34.2
(t, C2), 36.7 (s, C10), 37.7 (t, C22), 38.3 (q, NCH3), 39.7 (t, C1),
42.5 (t, C19), 43.5 (s, C8/20), 43.6 (s, C8/20), 45.3 (s, C14), 47.8 (s,
C4), 48.5 (d, C18), 55.4 (d, 5), 61.1 (d, C9), 128.1 (d, C12), 171.4
(s, C13), 174.0 (s, C29), 200.2 (s, C11), 217.4 (s, C3).
Rf = 0.45 (DCM/MeOH 30:1 + TEA); ½a D20
ꢄ
+138.8 (c 1.0, CHCl3);
HRMS: calcd [M+H]+: 574.3891, found [M+H]+: 574.3886; 1H
NMR (CDCl3): d 0.81 (s, 3H, H28), 0.97–1.05 (m, 1H, H16b), 1.07
(s, 3H, H23/24), 1.1 (s, 3H, H23/24), 1.12 (s, 3H, H30), 1.16 (s, 3H,
H26), 1.15–1.23 (m, 1H, H15b), 1.27 (s, 3H, H25), 1.25–1.32 (m,
1H, H5), 1.31 (s, 3H, H27), 1.32–1.42 (m, 4H, H1b, H21b, H22a,
H22b), 1.4–1.48 (m, 1H, H6b), 1.49–1.60 (m, 2H, H7a, H7b), 1.60–
1.69 (m, 3H, H6a, H19a, H19b), 1.78–1.87 (m, 1H, H15a), 1.84–
1.92 (m, 1H, H21a), 2.00 (dt, J = 13.6 Hz, J = 4.3 Hz, 1H, H16a),
2.05–2.13 (m, 1H, H18), 2.3–2.40 (m, 1H, H2b), 2.39 (s, 1H, H9),
2.57–2.68 (m, 1H, H2a), 2.90–3.00 (m, 1H, H1a), 4.94 (br s, 2H,
OCH2Ph), 5.50 (s, 1H, H12), 7.35–7.45 (m, 5H, 5 ꢃ Ph-H), 8.31 (s,
1H, NH); 13C NMR (CDCl3): d 15.6 (q, C25), 18.5 (q, C26), 18.7 (t,
C7), 21.4 (q, C23/24), 23.3 (q, C27), 26.31 (t, C15/16), 26.35 (q,
C23/24), 26.4 (t, C15/16), 28.4 (q, C28), 29.5 (q, C30), 31.2 (t,
C21), 31.8 (s, C17), 32.1 (t, C6), 34.2 (t, C2), 36.7 (s, C10), 37.3 (t,
C22), 39.8 (t, C1), 41.3 (t, C19), 43.0 (s, C20), 43.2 (s, C14), 45.1
(s, C8), 47.78 (s, C4), 47.84 (d, C18), 55.4 (d, C5), 61.0 (d, C9),
78.0 (t, OCH2Ph), 128.4 (d, PhCH), 128.7 (d, PhCH), 128.9 (d, C12),
129.3 (d, PhCH), 135.3 (s, PhC), 169.2 (s, C13), 173.6 (s, C29),
199.3 (s, C11), 217.2 (s, C3).
4.45. (18b,20b)-N-Hydroxy-3-hydroxyimino-11-oxo-olean-12-
en-29-amide (38)
Ketone 36 (50 mg, 0.103 mmol, 1.00 equiv), NaOAc (30 mg,
0.36 mmol, 3.5 equiv) and NH2OHꢀHCl (25 mg, 0.36 mmol, 3.5
equiv) were dissolved in CHCl3/MeOH 2:1 (2.5 mL). In a closed
reaction tube (15 mL-volume) the reaction mixture was stirred at
60 °C in a sand-bath under HPLC monitoring for 90 min and for
2 h at rt. The reaction mixture was diluted with CHCl3, washed
three times with half satd NaCl. The aqueous layers were extracted
with CHCl3 (some MeOH added), the combined organic layers were
washed with brine, dried over Na2SO4, and evaporated (a drop of
TEA was added for stability). The residue was taken up in distilled
dioxane (5 mL) and lyophilized to give pure 38 (45 mg, 87%) as
white lyophilisate, only contaminated with small amounts of TEA
and dioxane according to 1H NMR and 13C NMR. Rf = 0.15 (DCM/
4.43. (18b,20b)-N-Hydroxy-3,11-dioxo-olean-12-en-29-amide
(36)
O-Benzyl-hydroxamic acid 35 (500 mg, 0.871 mmol, 1.00 equiv)
was debenzylated by hydrogenolysis analogous to 20 in MeOH
(50 mL) with Pd/C (50 mg, 10%). The crude material was purified
by column chromatography (SiO2: 40 g, DCM/MeOH 50:1 + 0.1%
TEA to DCM/MeOH 30:1 + 0.1% TEA) pure 35 (236 mg, 56%) as
white lyophilisate after lyophilization from dioxane/water solu-
tion. Rf = 0.09 (DCM/MeOH 30:1 + TEA); ½a D20
ꢄ
+168.9 (c 1.1, CHCl3);
HRMS: calcd [M+H]+: 484.3421, found [M+H]+: 484.3423; 1H NMR
(CDCl3): d 0.83 (s, 3H, H28), 1.00–1.05 (m, 1H, H16b), 1.07 (s, 3H,
H23/24), 1.10 (s, 3H, H23/24), 1.15–1.24 (m, 1H, H15b), 1.16 (s,
3H, H26), 1.17 (s, 3H, H30), 1.25–1.34 (m, 1H, H5), 1.26 (s, 3H,
H25), 1.34–1.49 (m, 5H, H1b, H7b, H21b, H22a, H22b), 1.37 (s,
3H, H27), 1.47–1.61 (m, 2H, H6a, H6b), 1.61–1.73 (m, 2H, H7a,
H19b), 1.77–2.00 (m, 3H, H15a, H19a, H21a), 1.97–2.08 (m, 1H,
H16a), 2.16–2.27 (m, 1H, H18), 2.30–2.40 (m, 1H, H2b), 2.43 (s,
1H, H9), 2.55–2.66 (m, 1H, H2a), 2.88–2.98 (m, 1H, H1a), 5.78 (s,
1H, H12); 13C NMR (CDCl3): d 15.7 (q, C25), 18.5 (q, C26), 18.8 (t,
C6), 21.4 (q, C23/24), 23.3 (q, C27), 26.4 (q, C23/24), 26.4 (t, C15/
16), 26.5 (t, C15/16), 28.4 (q, C28), 29.5 (q, C30), 31.0 (t, C21),
31.8 (s, C17), 32.1 (t, C7), 34.2 (t, C2), 36.7 (s, C10), 37.3 (t, C22),
39.8 (t, C1), 40.9 (t, C19), 42.3 (s, C20), 43.3 (s, C14), 45.2 (s, C8),
47.77 (s, C4), 47.82 (d, C18), 55.4 (d, C5), 61.1 (d, C9), 128.5 (d,
C12), 169.9 (s, C13), 173.8 (s, C29), 199.8 (s, C11), 217.3 (s, C3).
MeOH 20:1 + 0.5% TEA); ½a D20
ꢄ
+93.9 (c 0.5, CHCl3); HRMS: calcd
[M+H]+: 499.3530, found [M+H]+: 499.3531; 1H NMR (CDCl3): d
0.83 (s, 3H, H28), 0.98–1.10 (m, 3H, H1b, H5, H16b), 1.07 (s, 3H,
H23/24), 1.15 (s, 3H, H26), 1.16 (s, 3H, H23/24), 1.17–1.25 (m,
1H, H15b), 1.18 (s, 3H, H30), 1.23 (s, 3H, H25), 1.34 (s, 3H, H27),
1.36–1.52 (m, 5H, H6b, H7b, H21b, H22a, H22b), 1.57–1.75 (m,
3H, H6a, H7a, H19b), 1.78–1.90 (m, 2H, H15a, H19a), 1.92–2.04
(m, 2H, H16a, H21a), 2.15–2.29 (m, 2H, H2b, H18), 2.37 (s, 1H,
H9), 2.83 (app. dt, J = 13.4 Hz, J = 4.6 Hz, 1H, H1a), 3.04 (app. dt,
J = 15.5 Hz, J = 4.3 Hz, 1H, H2a), 5.74 (s, 1H, H12); 13C NMR (CDCl3):
d 15.7 (q, C25), 17.2 (t, C2), 18.2 (t, C6), 18.7 (q, C26), 23.2 (q, C23/
24), 23.3 (q, C27), 26.38, 26.42 (2 ꢃ t, C15, C16), 27.1 (q, C23/24),
28.4 (q, C28), 29.5 (q, C30), 31.0 (t, C21), 31.8 (s, C17), 32.4 (t,
C7), 37.0 (s, C10), 37.3 (t, C22), 39.1 (t, C1), 40.4 (s, C4), 40.9 (t,
C19), 42.3 (s, C17), 43.3, 45.4 (2 ꢃ s, C8, C20), 47.9 (d, C18), 55.6
(d, C5), 61.4 (d, C9), 128.5 (d, C12), 167.0 (s, C3), 169.5 (s, C13),
173.8 (s, C29), 200.1 (s, C11).
4.44. (18b,20b)-N-Hydroxy-N-methyl-3,11-dioxo-olean-12-en-
29-amide (37)
4.46. (18b,20b)-N-Hydroxy-3-methoxyimino-11-oxo-olean-12-
en-29-amide (39)
Carboxylic acid 34 (2.00 g, 4.27 mmol, 1.00 equiv) was con-
verted to the hydroxamic acid derivative analogous to 35 by first
converting to the acid chloride in dry DCM (40 mL + 20 drops of
Ketone 36 (96 mg, 0.198 mmol, 1.00 equiv), NaOAc (65 mg,
0.79 mmol, 4.0 equiv) and NH2OMeꢀHCl (50 mg, 0.60 mmol,
3.0 equiv) were dissolved in CHCl3/MeOH 2:1 (4.5 mL) and stirred
at 60 °C in a sand-bath under an atmosphere of argon for 7 h and at
rt overnight. The reaction mixture was diluted with DCM, washed
twice with half saturated NaCl, diluted AcOH, satd NaHCO3 and
brine (always with subsequent back-extraction with DCM/MeOH
10:1). The organic layer was dried over Na2SO4, one drop of TEA
was added and evaporated, taken up in distilled dioxane (5 mL)
and lyophilized to give pure 39 (97 mg, 95.3%) as white lyophili-
DMF) with oxalyl chloride (490 lL, 5.12 mmol, 1.2 equiv) and sub-
sequent treatment with TEA (2.38 mL, 17.1 mmol, 4.0 equiv) and
N-methyl-hydroxylamine hydrochloride (392 mg, 4.69 mmol,
1.1 equiv) which was added in one portion. Aqueous workup and
column chromatography (SiO2: 115 g, DCM/MeOH 45:1?35:1)
and column chromatography on amino-phase (15 g, DCM to
DCM/MeOH 50:1) gave pure 37 (1.48 g, 69.7%) as white solid.
Rf = 0.14 (Hex/EtOAc 1:1 + 0.1% AcOH); ½a D20
ꢄ
+164.4 (c 0.55, CHCl3);

7540
C. Stanetty et al. / Bioorg. Med. Chem. 18 (2010) 7522–7541
sate. Rf = 0.19 (DCM/MeOH 20:1 + 0.5% TEA); ½a D20
ꢄ
+102.0 (c 0.5,
H19b, H22a), 1.83 (td, J = 13.5 Hz, J = 4.1 Hz, 1H, H15a), 1.99–2.14
(m, 1H, H16a), 2.11–2.24 (m, 4H, H2b, H18, H19a, H21a), 2.38 (s,
1H, H9), 2.72–2.82 (m, 1H, H1a), 2.86–2.96 (m, 1H, H2a), 3.36 (s,
3H, NCH3), 3.82 (s, 3H, OCH3), 5.66 (s, 1H, H12); 13C NMR (CDCl3):
d 15.7 (q, C25), 17.7 (t, C2), 18.2 (t, C7), 18.6 (q, C26), 23.1 (q, C27),
23.4 (q, C23/24), 26.3 (q, C30), 26.5 (t, C15), 26.7 (t, C16), 27.3 (q,
C23/24), 28.5 (q, C28), 31.9 (s, C17), 32.5, 32.7 (2 ꢃ t, C6, C21),
37.0 (s, C10), 37.7 (t, 22), 38.3 (q, NCH3), 39.1 (t, C1), 40.1 (s, C4),
42.4 (t, C19), 43.4, 43.5 (2 ꢃ s, C8, C20), 45.4 (s, C14), 48.5 (d,
C18), 55.6 (d, C5), 61 (q, OCH3), 61.4 (d, C9), 128.2 (d, C12), 165.6
(s, C3), 170.9 (s, C13), 173.7 (s, C29), 200.5 (s, C11).
CHCl3); HRMS: calcd [M+H]+: 513.3687, found [M+H]+: 513.3688;
1H NMR (CDCl3): d 0.82 (s, 3H, H28), 0.99–1.10 (m, 3H, H1b, H5,
H16b), 1.06 (s, 3H, H23/24), 1.14 (s, 3H, H26), 1.17 (br s, 6H,
H23/24, H30), 1.15–1.30 (m, 2H, H15b, H21b), 1.22 (s, 3H, H25),
1.34 (s, 3H, H27), 1.34–1.55 (m, 4H, H6b, H7b, H22a, H22b),
1.56–1.76 (m, 3H, H6a, H7a, H19a), 1.76–1.90 (m, 2H, H15a,
H19b), 1.90–2.10 (m, 2H, H16a, H21a), 2.12–2.26 (m, 2H, H2b,
H18), 2.37 (s, 1H, H9), 2.75–2.85 (m, 1H, H1a), 2.85–2.98 (m, 1H,
H2a), 3.81 (s, 3H, OCH3), 5.75 (br s, 1H, H12); 13C NMR (CDCl3): d
15.6 (q, C25), 17.7 (t, C2), 18.2 (t, C6), 18.6 (q, C26), 23.28, 23.43
(2 ꢃ q, C23/24, C27), 26.4 (t, C16), 26.4 (t, C15), 27.2 (q, C23/24),
28.4 (q, C28), 29.5 (q, C30), 31.0 (t, C21), 31.8 (s, C17), 32.4 (t,
C7), 37.0 (s, C10), 37.3 (t, C22), 39.1 (t, C1), 40.1 (s, C4), 40.8 (t,
C19), 42.3, 43.3, 45.4 (s, C8, C14, C20), 47.8 (d, C18), 55.6 (d, C5),
61.0 (q, OCH3), 61.4 (d, C9), 128.6 (d, C12), 165.6 (s, C3), 169.4 (s,
C13), 173.8 (s, C29), 200.2 (s, C11).
4.49. (3b,18b,20b)-N-Hydroxy-N-methyl-3-methoxyamino-11-
oxo-olean-12-en-29-amide (42)
Oxime 41 (275 mg, 0.522 mmol, 1.0 equiv) was dissolved in
dioxane/EtOH 2:1 (12 mL). This solution was cooled to 0 °C before
tBuNH2ꢀBH3 (91 mg, 1.04 mmol, 2.00 equiv) was added at 0 °C and
5 min later 3 M HCl (1.2 mL) was added dropwise via a syringe. The
reaction mixture was stirred at 0 °C for several hours. After some
time the reaction mixture became milky turbid but well stirrable.
According to TLC almost all starting material was converted to
the target compound. Addition of another equivalent of
tBuNH2ꢀBH3 and HCl did not lead to further conversion. The reac-
tion mixture was worked up by pouring onto satd NaHCO3 and
extraction with EtOAc, washing of the organic layers with brine,
drying over Na2SO4 and evaporation to give crude material which
was purified by column chromatography (SiO2: 30 g, DCM/Et2O
2:1) and subsequent precipitation from DCM/MeOH to give pure
target compound 42 (50 mg, 18.1%) as white solid. For better solu-
bility part of the material was transformed to the corresponding
hydrochloride by taking up in THF, precipitating with HCl in Et2O
(1 M, 2 equiv equiv) and washing with Et2O. Rf = 0.31 (toluene/
4.47. (18b,20b)-N-Hydroxy-3-hydroxyimino-N-methyl-11-oxo-
olean-12-en-29-amide (40)
A solution of ketone 37 (320 mg, 0.643 mmol, 1.00 equiv) and
hydroxylamine hydrochloride (223 mg, 3.22 mmol, 5.00 equiv) in
dry pyridine (4 mL) was stirred at rt under TLC-monitoring
(DCM/MeOH 40:1). Upon complete conversion (3 h), the reaction
mixture was diluted with DCM and washed with cooled 10% HCl,
satd NaHCO3, and brine, dried over Na2SO4 and evaporated. The
residual white solid was dissolved in MeOH and evaporated to give
a fine white solid, which was triturated with DCM to give pure 40
(320 mg, 97%) as white solid. Rf = 0.14 (Hex/EtOAc 1:1 + 0.1%
AcOH);
½
a 2D0 +110.8 (c 1.0, CHCl3); HRMS: calcd [M+H]+:
ꢄ
513.3687, found [M+H]+: 513.3681; 1H NMR (CDCl3): d 0.82 (s,
3H, H28), 0.97–1.16 (m, 3H, H1b, H5, H16b), 1.07 (s, 3H, H23/24),
1.15–1.27 (m, 2H, H15b, H21b), 1.15 (s, 3H, H26), 1.17 (s, 3H,
H23/24), 1.2 (s, 3H, H30), 1.23 (s, 3H, H25), 1.31–1.40 (m, 2H,
H6b, H22b), 1.35 (s, 3H, H27), 1.44–1.73 (m, 5H, H6a, H7a, H7b,
H19b, H22a), 1.85 (td, J = 13.6 Hz, J = 4.4 Hz, 1H, H15a), 2.02–2.15
(m, 1H, H16a), 2.13–2.33 (m, 1H, H2b, H18, H19a, H21a), 2.41 (s,
1H, H9), 2.75–2.85 (m, 1H, H1a), 3 (dt, J = 15.6 Hz, J = 4.6 Hz, 1H,
H2a), 3.26 (s, 3H, NCH3), 5.70 (s, 1H, H12); 13C NMR (CDCl3): d
15.6 (q, C25), 17.1 (t, C2), 18.1 (t, C7), 18.5 (q, C26), 22.9 (q, C27),
23.2 (q, C23/24), 25.9 (q, C30), 26.4 (t, C16), 26.6 (t, C15), 27.2 (q,
C23/24), 28.5 (q, C28), 31.8 (s + t, C17, C21), 32.3 (t, C6), 37 (s,
C10), 37.7 (t, C22), 38 (q, NCH3), 38.9 (t, C1), 40.2 (s, C4), 42.1 (t,
C19), 43.5 (s, C20), 44.1, 45.3 (2 ꢃ s, C8, C14), 48.7 (d, C18), 55.4
(d, C5), 61.3 (d, C9), 127.7 (d, C12), 166.8 (s, C3), 171.9 (s, C13),
175.5 (s, C29), 201.1 (s, C11).
EtOAc 2:1 + 0.1% AcOH); ½a D20
ꢄ
+152.6 (c 0.7, CHCl3/MeOH 5:1);
HRMS: calcd [M+H]+: 529.4000, found [M+H]+: 529.3993; 1H
NMR (CDCl3/MeOD 5:1): d 0.73 (s, 3H, H23/24), 0.73–0.79 (m,
1H, H5), 0.82 (s, 3H, H28), 0.90–0.99 (m, 1H, H1b), 0.98–1.05 (m,
1H, H16b), 1.07 (s, 3H, H23/24), 1.13 (s, 3H, H26), 1.14 (s, 3H,
H25), 1.16–1.32 (m, 2H, H15b, H21b), 1.21 (s, 3H, H30), 1.32–
1.52 (m, 5H, H2b, H6b, H7b, H22a, H22b), 1.39 (s, 3H, H27),
1.54–1.74 (m, 3H, H6a, H7a, H19b) 1.79–1.92 (m, 2H, H2a, H15a),
2.05 (td, J = 13.6 Hz, J = 4.4 Hz, 1H, H16a), 2.16–2.32 (m, 1H, H18,
H19a, H21a), 2.40 (s, 1H, H9), 2.52 (dd, J = 11.8 Hz, J = 4.1 Hz, 1H,
H3), 2.72 (dt, J = 13.4 Hz, J = 3.4 Hz, 1H, H1a), 3.25 (s, 3H, NCH3),
3.52 (s, 3H, OMe), 5.69 (s, 1H, H12); 13C NMR (CDCl3/MeOD 5:1):
d 15.8 (q, C25), 16.4 (q, C23/24), 17.0 (t, C6), 18.4 (q, C26), 22.8
(q, C27), 23.0 (t, C2), 25.6 (q, C30), 26.2 (t, C15), 26.4 (t, C16),
28.2, 28.3 (2 ꢃ q, C28, C23/24), 31.6 (s, C17), 31.9 (t, C21), 32.5 (t,
C7), 36.6 (s, C4), 37.0 (s, C10), 37.6 (t, C22), 37.7 (q, NCH3), 38.9
(t, C1), 41.9 (t, C19), 43.2 (s, C20), 44.1 (s, C14), 45.2 (s, C8), 48.5
(d, C18), 55.9 (d, C5), 61.2 (q, OCH3), 61.6 (d, C9), 67.5 (d, C3),
127.6 (d, C12), 171.6 (s, C13), 175.6 (s, C29), 201.5 (s, C11).
4.48. (18b,20b)-N-Hydroxy-N-methyl-3-methoxyimino-11-oxo-
olean-12-en-29-amide (41)
A solution of ketone 37 (500 mg, 1.01 mmol, 1.00 equiv) and
methoxylamine hydrochloride (252 mg, 3.01 mmol, 3.00 equiv) in
dry pyridine (5 mL) was stirred at rt under TLC-monitoring
(DCM/MeOH 40:1). Upon complete conversion (3 h), the reaction
mixture was diluted with DCM and washed with chilled 10% HCl,
satd NaHCO3, and brine, dried over Na2SO4 and evaporated. The
crude material was purified by column chromatography (SiO2:
25 g DCM/MeOH 60:1?DCM/MeOH 50:1) to give pure 41
(536 mg, quantitative) as white solid foam. Rf = 0.23 (Hex/EtOAc
4.50. Biology
The activity of 11b-HSD1 and 11b-HSD2 was measured as de-
scribed previously.45 Briefly, HEK-293 cells were transfected with
pcDNA3 plasmids containing either human 11b-HSD1 or 11b-
HSD2 with a C-terminal FLAG epitope. 11b-HSD1 dependent reduc-
tion of [1,2-3H]-labeled cortisone (American Radiolabeled Chemi-
cals, St. Louis, MO) to cortisol was measured in cell lysates for
1:1 + 0.1% AcOH); ½a D20
ꢄ
+96.7 (c 0.5. CHCl3); HRMS: calcd [M+H]+:
527.3843, found [M+H]+: 527.3833; 1H NMR (CDCl3): d 0.82 (s,
3H, H28), 0.96–1.05 (m, 3H, H1b, H5, H16b), 1.07 (s, 3H, H23/24),
1.12–1.24 (m, 1H, H15b), 1.14 (s, 3H, H26), 1.18 (s, 3H, H23/24),
1.19 (s, 3H, H30), 1.22 (s, 3H, H25), 1.23–1.40 (m, 2H, H21b,
H22b), 1.34 (s, 3H, H27), 1.38–1.68 (m, 6H, H6a, H6b, H7a, H7b,
10 min at 37 °C in a volume of 22
tion of 200 nM cortisone and 500
l
l
L containing a final concentra-
M NADPH. 11b-HSD2 depen-
dent oxidation of cortisol to cortisone was measured similarly
using [1,2,6,7-3H]-cortisol (Amersham Pharmacia, Piscataway, NJ,
USA) at a final concentration of 50 nM and NAD+ (500
lM). In a

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Acknowledgments
28. Beseda, I.; Czollner, L.; Shah, P. S.; Khunt, R.; Gaware, R.; Kosma, P.; Stanetty, C.;
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The work was supported by the ZIT Zentrum für Innovation und
Technologie GmbH (Vienna Spot of Excellence: 182081). A.O. has a
chair in Molecular and Systems Toxicology by the Novartis
Research Foundation and was supported by the Swiss National
Science Foundation, Grant 31003A-124912.
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