Novel 18β-glycyrrhetinic acid analogues as potent and selective inhibitors of 11β-hydroxysteroid dehydrogenases

Article abstract of DOI:10.1016/j.bmc.2004.06.008

Extensive structural modifications to the 18β-glycyrrhetinic acid template are described and their effects on the SAR of the 11β- hydroxysteroid dehydrogenase isozymes type 1 and 2 from the rat are investigated. Isoform selective inhibitors have been discovered and compound 7 N-(2-hydroxyethyl)-3β-hydroxy-11-oxo-18β-olean-12-en-30-oic acid amide is highlighted as a very potent selective inhibitor of 11β-hydroxysteroid dehydrogenase 2 with an IC₅₀=4pM.

Full text of DOI:10.1016/j.bmc.2004.06.008

Bioorganic & Medicinal Chemistry 12 (2004) 4439–4457
Novel 18b-glycyrrhetinic acid analogues as potent and selective
inhibitors of 11b-hydroxysteroid dehydrogenases
Xiangdong Su,a Harshani Lawrence,a Dharshini Ganeshapillai,a Adrian Cruttenden,b
Atul Purohit,b Michael J. Reed,b Nigel Vickera and Barry V. L. Pottera,*
aMedicinal Chemistry, Department of Pharmacy and Pharmacology and Sterix Ltd, University of Bath, Bath BA2 7AY, UK
bEndocrinology and Metabolic Medicine and Sterix Ltd, Imperial College School of Medicine, St. MaryÕs Hospital,
London W2 1NY, UK
Received 27 February 2003; accepted 2 June 2004
Available online 2 July 2004
Abstract—Extensive structural modifications to the 18b-glycyrrhetinic acid template are described and their effects on the SAR of
the 11b-hydroxysteroid dehydrogenase isozymes type 1 and 2 from the rat are investigated. Isoform selective inhibitors have been
discovered and compound 7 N-(2-hydroxyethyl)-3b-hydroxy-11-oxo-18b-olean-12-en-30-oic acid amide is highlighted as a very
potent selective inhibitor of 11b-hydroxysteroid dehydrogenase 2 with an IC50 ¼ 4 pM.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
corticoid cortisol and is present in liver, muscle and
adipose tissue.3 Cortisol has a central role in regulating
carbohydrate metabolism and its main action in this
respect is to oppose the action of insulin and 11b-HSD1
has been shown to mediate glucocorticoid action and
insulin release in pancreatic islets.4 In the liver, while
insulin stimulates glucose uptake and inhibits gluco-
neogenesis, cortisol acts to inhibit glucose uptake and
stimulate gluconeogenesis. Gluconeogenesis in this
organ is reduced in knockout mice without the
11b-HSD1 gene resulting in lower blood sugar levels.5
Recently, it has been shown that selective inhibition of
11b-HSD1 decreases blood glucose concentrations in
hyperglycaemic mice.6 Inhibition of 11b-HSD1 in sub-
jects suffering from impaired glucose tolerance or dia-
betes mellitus could increase glucose uptake by the liver
and inhibit gluconeogenesis. Thus, a selective inhibitor
of 11b-HSD1 has considerable potential as an antidia-
betic agent. The effects of the non-selective 11b-HSD
inhibitor carbenoxolone (CBX 2) on insulin sensitivity
in non-obese men with type 2 diabetes has recently been
shown to support this hypothesis.7 The choice of lean
patients for this study was an important consideration
as these patients had no change in the 11b-HSD1 levels
in adipose tissue.7 Several groups have programmes
targeted at the development of selective 11b-HSD1
inhibitors for the treatment of metabolic disorders such
as insulin resistance, obesity, hyperlipidemia and arterial
hypertension.4;8–11 Although it has been known for some
The short-chain dehydrogenases/reductases (SDR) are a
large well-established family of functionally hetero-
geneous enzymes present in all forms of life.1 The
11b-hydroxysteroid dehydrogenases (11b-HSDs) are
members of this family and are microsomal enzymes
catalyzing the conversion of active glucocorticoids to
their 11-dehydro products and vice versa.2 The 11b-
hydroxysteroid dehydrogenase isozymes type 1 and 2
are responsible for the interconversion of cortisone (E)
and cortisol (F) as shown in Figure 1.
The 11b-hydroxysteroid dehydrogenase type 1 (11b-
HSD1) isoform converts cortisone to the active gluco-
OH
OH
OH
OH
O
O
HO
O
Type 1
Type 2
O
O
Cortisone (E)
Cortisol (F)
Figure 1. Interconversion of cortisone (E) and cortisol (F) by 11b-
HSD type 1 and 2.
* Corresponding author. Tel.: +44-1225-386639; fax: +44-1225-
386114; e-mail: b.v.l.potter@bath.ac.uk
0968-0896/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.06.008

4440
X. Su et al. / Bioorg. Med. Chem. 12 (2004) 4439–4457
time that 18b-glycyrrhetinic acid (18b-GA 1), a principal
active ingredient of liquorice root, and its hemisuccinate
derivative carbenoxolone (CBX 2) (Fig. 2) are potent
non-selective inhibitors of both 11b-HSD1 and 11b-
HSD2,12 only recently have selective inhibitors of 11b-
HSD1 with nanomolar potency been reported.8
stantially extend the structural modifications of the 18b-
GA template and report the effects of these novel com-
pounds on the SAR of 11b-HSD1 and 11b-HSD2
isoforms from the rat.
2. Chemistry
The 11b-HSD type 2 isoform inactivates cortisol to
cortisone and is a unidirectional enzyme and exclusively
acts as a NADþ dependent dehydrogenase of adrenal
glucocorticoids. This isoform is found primarily in
mineralocorticoid (MC) tissues such as the kidney, co-
lon, salivary glands and the sodium-transporting epi-
thelia of the lung a nonclassical MC target organ.13
Inhibition of 11b-HSD2 could be used to potentiate the
anti-inflammatory effects of glucocorticoids, leading to a
reduction in the amount of glucocorticoid required for
anti-inflammatory therapy and in the severity of very
significant side effects that can result from this type of
medication. Inhibition of 11b-HSD2 with liquorice
derivatives results in cortisol-dependent mineralocorti-
coid excess with hypertension and hypokalemic alkalo-
sis.14;15 Disruption or mutations in the 11b-HSD2 gene
result in sodium retention, hypokalemia and hyperten-
sion because of inappropriate glucocorticoid occupation
of the MC receptor in the kidney, where 11b-HSD2 is
principally expressed in the distal nephron.5;16 An
excellent recent review on the 11b-HSDs highlighted the
link between 11b-HSD2 and cancer.17 Adrenal cortical
adenomas and carcinomas synthesize 11b-HSD2,18 and
11b-HSD2 levels are high in pituitary tumours.19;20
Ductal and lobular breast epithelial cells have also been
shown to synthesize 11b-HSD2, with increased synthesis
of 11b-HSD2 seen in invasive carcinomas.21 Transfec-
tion experiments to study the role of 11b-HSDs showed
that in stably transfected cells overexpressing HSD11B2
cell proliferation was increased.22 In addition, the
observation that 11b-HSD2 potentiates the antiprolif-
erative effects of glucocorticoids in endometrial, and
some breast cancer cell lines highlights a putative role
for 11b-HSD2 activity in tumourigenesis.23 These
observations highlight the need for selective inhibitors of
11b-HSD1 and 11b-HSD2 as therapeutic agents or tools
for further mechanistic studies.
The synthesis of compounds from the 18b-GA template
1 was targeted at three key areas of the molecule where
functionality is present for further modification. The
template was modified in the A, C and E rings at posi-
tions 3, 11 and 30, respectively, as indicated in Figure 3.
CO2H
CO2H
H
H
O
O
O
HO
HO2C
O
1
2
Carbenoloxone (CBX)
18β-Glycyrrhetinic acid (18β-GA)
Figure 2. Structures of 18b-glycyrrhetinic acid and carbenoxolone.
E-ring Modifications
CO2H
C-ring Modifications
30
H
11
O
3
HO
A-ring Modifications
Figure 3. Structural modification of 18b-glycyrrhetinic acid template.
2.1. Modifications to the 30-position
2.1.1. Amides. A diverse set of amides 3–33 was syn-
thesized by activation of the carboxylic acid moiety at
the 30-position and reaction with a range of amines.
Syntheses were performed classically and in parallel
using the water soluble EDCI as the activating agent
under a variety of conditions (Scheme 1).27 The com-
pounds listed in Tables 1 and 2 were synthesized by
these methods.
A number of natural products have been reported to
inhibit the 11b-HSDs and these include xenobiotics,
flavonoids, polyphenolic compounds from tea, gossypol
and the constituents of Saiboku-To, a Chinese herbal
remedy.24;25 In general, these compounds are non-selec-
tive inhibitors of the 11b-HSDs and are much less
potent than 18b-GA 1. Some selectivity for 11b-HSD1
has been reported for weakly inhibiting synthetic
steroids and chenodeoxycholic acid; the diuretic furo-
semide has also been reported as a non-selective inhib-
itor.24;25
CO2H
CONR1R2
H
H
O
O
a
As 18b-GA 1 and CBX 2 are potent inhibitors of 11b-
HSD lacking selectivity a programme of work was ini-
tiated to evaluate the effects on SAR by modifying the
18b-GA template on 11b-HSD activity and selectivity.
The use of a number of 18b-GA derivatives was the
topic of a recent patent by our group.26 Here we sub-
RO
RO
Scheme 1. Synthesis of 18b-glycyrrhetinic acid amides. Reagents and
conditions: (a) EDCI, R1R2NH, CH2Cl2 or CHCl3, DMAP, HOBt
(optional), rt.

X. Su et al. / Bioorg. Med. Chem. 12 (2004) 4439–4457
4441
Table 1. Secondary amide derivatives of 18b-GA (1) at the 30-position
and their inhibition of 11b-HSD1 and 11b-HSD2
Table 1 (continued)
Compd
R
% Inhibition % Inhibition
of 11b-HSD1 of 11b-HSD2
CONHR
at 10 lMa
at 10 lMa
H
OCH3
OCH3
O
25
26
38
15
N
N
∗
∗
HO
58
55
93
94
Compd
R
% Inhibition % Inhibition
of 11b-HSD1 of 11b-HSD2
OCH3
N
at 10 lMa
at 10 lMa
1
2
3
85
52
13
101
100
100
27
∗
S
CH2CH2CH2CH3
ꢀIndicates point of attachment.
a Mean of at least two measurements with typically ꢁ5% variation.
4
5
90
25
103
35
∗
H
COOEt
∗
2.1.2. Ethers and esters. A number of chemical modifi-
cations to the 30-position of the 18b-GA template have
been published. However, the effects of these structural
changes on 11b-HSD activity and selectivity have not
been reported.26;28;29 Schemes 2 and 4 illustrate the
introduction of alcohol and ether functionalities into the
18b-GA template. The C-30 esters were prepared from
18b-GA 1 using the standard methods with the requisite
alkyl halides as previously described to give 35–38.26
Compound 34 had previously been prepared by the
above method,26 and also by treatment of 18b-GA 1 with
diazomethane;30 in this instance p-TSA in methanol was
used.31
H
6
58
39
COOH
∗
CH2CH2OH
7
8
9
36
88
36
92
98
CH2(CH2)4OH
CH2CH2NHCH3
101
O
O
10
71
25
∗
CH(CH2CH2OH)CO2H
11
12
50
19
6
∗
66
N
O
13
14
34
7
20
92
∗
∗
N
N
O
The C-30 hydroxymethyl analogue of 18b-GA 39 was
prepared from 18b-GA 1 by reduction with LAH fol-
lowed by oxidation of the allylic alcohol with manga-
nese dioxide (Scheme 2). Esterification of the C-30 acid
of 18b-GA 1 using MeOH/p-TSA followed by protec-
tion of the 3b-OH as its THP ether using THP/PPTS
gave 45 as an intermediate for further modification.
Etherification of the C-30 hydroxymethyl moiety using
sodium hydride and the requisite alkyl halide under
standard conditions followed by deprotection of the 3-
position using PPTS gave the ether derivatives 40–43.
The tertiary gem dimethyl alcohol 44 was one of the
minor products isolated by treatment of the C-30 me-
thyl ester of 18b-GA 34 with methyl lithium (Scheme 4).
Compounds synthesized in Schemes 2 and 4 and C-30
esters where the 11-position is unchanged are listed in
Table 3.
N
NH
15
16
10
23
38
∗
∗
N
100
NH
∗
∗
17
18
36
29
23
18
N
N
19
20
66
85
99
74
∗
N
CH2C6H5
21
22
35
21
96
44
∗
2.2. Modifications to the C-3 position
N
A number of modifications have been examined at the
3-position, these include the synthesis of ether and
ester derivatives and oxidation of the C-3 hydroxyl
group to give 3-oxocompounds. The C-3 acetate 58
was prepared as previously described,31 and the C-3
ethers 47–57 were prepared as outlined in Scheme 3
using standard procedures these compounds are listed
in Table 4.
∗
∗
N
N
23
24
52
18
77
99
N
∗

4442
X. Su et al. / Bioorg. Med. Chem. 12 (2004) 4439–4457
Table 2. Tertiary and other amide modifications of (1) coupled with changes to the 3-position
CONR1R2
H
O
RO
Compd
R
R1
R2
% Inhibition of
11b-HSD1 at 10 lMa
% Inhibition of
11b-HSD2 at 10 lMa
28
29
30
31
32
COCH3
COCH3
COCH3
CH3
H
H
H
H
CH2C6H5
OH
36
32
39
32
59
27
89
4
(CH2)2OH
(CH2)2OH
0
23
H
R1R2 ¼ –(CH2)5–
33
CO(CH2)2COOH
H
∗
40
23
N
ꢀIndicates point of attachment.
a Mean of at least two measurements with typically ꢁ5% variation.
HO
HO
O
O
O
O
O
H
H
b
a
H
c, d
O
O
H
O
O
HO
O
O
HO
O
O
1
34
45
46
e
c
R
R
O
HO
HO
O
H
H
H
g
f
H
HO
O
O
O
HO
HO
HO
O
HO
O
64
40 R = m-methoxybenzyl
41 R = CH3
d
61 R = CH3
HO
42 R = COCH2Cl
43 R = CH2CH2OH
H
O
HO
39
Scheme 2. Modifications to the 30 and 11 positions of 18b-GA. Reagents and conditions: (a) CH3OH, p-TSA, reflux; (b)THP, PPTS, DCM, rt; (c)
LiAlH4, THF, 60 ꢁC; (d) MnO2, CHCl3, rt; (e) (i) NaH, RX or ClCH2CO2C2H5 for 42, THF or toluene, 60 ꢁC; (ii) PPTS, CH3OH, 50 ꢁC; (f) succinic
anhydride, pyridine, DMAP, 90 ꢁC; (g) PtO2, H2, C2H5OH–THF, rt.
2.3. 3-Oxo-compounds and carbenoxolone (CBX)
analogues
GA compounds using succinic anhydride and DMAP in
pyridine using standard methods.
The 3-oxo analogues of 18b-GA 1 were prepared by
oxidation of the 3-position of 18b-GA 1 and the corre-
sponding C-30 methyl ester 34 using Jones reagent as
previously described to give 59 and 60 (Scheme 3).26;30
2.4. Modifications to the 11-position of the C-ring
The reduced compound 64 was prepared by treatment of
18b-GA 1 with LAH followed by reduction of the allylic
alcohol at the 11-position with hydrogen using PtO2 as
the catalyst (Scheme 2).29 Reduction of 18b-GA 1 with
hydrogen over PtO2 gave the 11-deoxo-18b-GA ana-
The succinate ester derivatives 33 and 61 related to CBX
2 were prepared from the corresponding 3b-hydroxy-

X. Su et al. / Bioorg. Med. Chem. 12 (2004) 4439–4457
Table 3. Ester, ether and alcohol modifications of the 30-position of 18b-GA (1)
4443
R
H
O
HO
Compd
R
% Inhibition of
11b-HSD1 at 10 lMa
% Inhibition of
11b-HSD2 at 10 lMa
34
35
36
COOCH3
COOCH2CH3
COOC(CH3)3
85
81
51
105
98
23
COO
37
38
34
38
39
COOCH2C6H5
CH2OH
53
90
60
100
OCH3
H2CO
40
6
14
41
42
43
44
CH2OCH3
CH2OCOCH2Cl
43
6
8
92
19
9
CH2OCH2CH2OH
C(CH3)2OH
88
31
a Mean of at least two measurements with typically ꢁ5% variation.
HO
HO
O
O
O
O
O
O
H
H
O
H
H
O
a
b
O
c
O
HO
RO
HO
RO
34
1
d
47 R = CH3
48 R = CH2CH3
52 R = CH3
53 R = CH2CH3
54 R = CH2CH(CH3)2
d
49 R = CH2CH(CH3)2
50 R = CH2CH2OCH3
51 R = CH2CH2OH
55 R = p-t-butylbenzyl
56 R = m-methoxybenzyl
57 R = benzyl
HO
O
O
O
H
H
O
O
O
O
60
59
Scheme 3. Modifications to the 3-position of 18b-GA. Reagents and conditions: (a) CH3OH, p-TSA, reflux; (b) NaH, RX, THF, reflux; (c) NaOH,
CH3OH–THF, reflux; (d) Jones reagent.19
logue 62, the corresponding C-30 methyl ester 63 was
prepared in the same manner from 18b-GA methyl ester
34 formed from 18b-GA 1 by treatment with p-TSA in
methanol (Scheme 4).26;31 Treatment of 18b-GA methyl
ester 34 with methyl lithium gave three products as
indicated in Scheme 4 with the major products being the
1,2-addition product 65 and the exo-methylene deriva-
tive 66 resulting from the dehydration of 65. The corre-
sponding C-30 acid derivatives were formed by
hydrolysis of the esters with potassium hydroxide to yield
67 and 68. Alternatively, 68 was also synthesized in high
yield from 18b-GA 1 by treatment with methyl lithium
followed by dehydration under acidic conditions with
hydrochloric acid during work up (Scheme 4, Table 5).
3. Results and discussion
On the rat 11b-HSD type 1 and 2 isozymes 18b-GA 1
and CBX 2 show potent and non-selective inhibition at
10 lM. Both 1 and 2, although being non-selective, in-
hibit the type 2 enzyme to a higher degree as evidenced
by the greater inhibition of 11b-HSD2 over 11b-HSD1
at different concentrations.26 As 1 and 2 show maximal

4444
X. Su et al. / Bioorg. Med. Chem. 12 (2004) 4439–4457
Table 4. 3-Ether and ester 18b-GA analogues and their inhibitions for 11b-HSD
COOR1
H
O
RO
Compd
R
R1
% Inhibition of
11b-HSD1 at 10 lMa
% Inhibition of
11b-HSD2 at 10 lMa
47
48
49
50
51
52
53
54
CH3
CH2CH3
CH3
CH3
CH3
CH3
CH3
H
8
32
33
11
38
28
74
61
6
18
19
10
5
CH2CH(CH3)2
CH2CH2OCH3
CH2CH2OH
CH3
99
89
43
CH2CH3
CH2CH(CH3)2
H
H
55
CH3
CH3
15
26
H2C
OCH3
56
2
4
H2C
57
58
CH2C6H5
COCH3
CH3
H
18
24
17
99
a Mean of at least two measurements with typically ꢁ5% variation.
HO
H
O
HO
44
O
HO
HO
O
O
O
O
+
O
H
H
H
HO
H
HO
O
a
b
HO
HO
HO
HO
34
65
67
62
+
HO
HO
c
O
c
O
O
O
O
O
H
H
O
H
H2C
H
H2C
b
a
HO
HO
HO
HO
66
68
1
63
Scheme 4. Modifications to the 11 and 30 positions of 18b-GA. Reagents and conditions: (a) CH3Li–ether, THF, )78 to 25 ꢁC; (b) KOH, CH3OH–
THF–H2O; (c) PtO2, H2, C2H5OH–THF, rt.
inhibition of 11b-HSD2 at 10 lM, compounds synthe-
sized by template modifications were screened at 10 lM
against both isozymes. Selected analogues were exam-
ined at lower concentrations and IC50 values determined
for a number of highly potent compounds. In general
terms compounds have been classed as showing some
selectivity if there is greater than 2.5-fold difference in %
inhibition, provided >50% inhibition is observed for one
isozyme. For the more potent selective inhibitors high-
lighted the percentage inhibition for one isozyme is
>90%.
3.1. Amide modifications
The secondary amide modifications at the 30-position
(Table 1) gave a wide range of activities on both 11b-
HSD1 and 11b-HSD2. In general, most compounds

X. Su et al. / Bioorg. Med. Chem. 12 (2004) 4439–4457
4445
Table 5. 11-Modified 18b-GA analogues and their inhibition for 11b-HSD1 and 11b-HSD2
R1
H
R
HO
Compd
R
R1
% Inhibition of
11b-HSD1 at 10 lMa
% Inhibition of
11b-HSD2 at 10 lMa
62
63
64
65
66
67
68
H
COOH
93
20
13
30
40
88
57
109
20
60
5
H
COOCH3
CH2OH
COOCH3
COOCH3
COOH
H
b-OH-a-CH3
@CH2
b-OH-a-CH3
20
14
22
@CH2
COOH
a Mean of at least two measurements with typically ꢁ5% variation.
showed a greater inhibition of 11b-HSD2 over 11b-
HSD1 and were non-selective. A number of compounds
showed >90% inhibition of 11b-HSD2 with >2.5-fold %
inhibition over 11b-HSD1, indicating selectivity. These
compounds included the neutral species 7 and 3 with
hydroxyethyl and n-butyl side chains, respectively. Most
compounds in this series that displayed the above cri-
teria contained basic side chains. These derivatives
included the 2-pyridinylethyl and 2-pyridinylmethyl
analogues 24 and 21, the 4-ethylpiperidinyl and piper-
azinylmethyl analogues 16 and 14, and the ethylami-
nomethyl derivative 9.
18 and 12 are weakly active compared to 16 and 14 the
importance of the basic interaction in this region is
highlighted. As the 2-pyridinyl compounds 24 and 21 are
both potent and selective the position of the basic
nitrogen is further highlighted as both the 3-pyridinyl
and 4-pyridinyl compounds 22 and 23 are less potent and
non-selective. The ethylaminomethyl analogue 9 is po-
tent and selective and has a secondary amine in the same
region as the pyridyl nitrogen in 21. As both pyridyl and
secondary amine derivatives have been shown to be po-
tent selective inhibitors of 11b-HSD2 this suggests that a
hydrogen bond acceptor interaction may be important in
this region. Although the position of the basic group in
the side chain is important for activity and selectivity it is
likely that different interactions are being picked up by
the sets 16, 14, 9, and 21, 24 as the basic group in these
sets occupies a different spatial region.
The 2-hydroxyethyl analogue 7 was designed to mimic
the C-17 side chain of cortisol/cortisone and probe the
hydrogen bonding characteristics in this region. Al-
though being longer by two atoms than the side chain in
cortisone the amide carbonyl in 7 is isosteric with the
ketone at the 20-position of cortisone and the hydroxyl
is in a similar spatial region to the C-21 hydroxyl in
cortisone. This compound has an IC50 of 4 pM against
11b-HSD2 with only 36% inhibition against 11b-HSD1
at 10 lM, and is the most potent 11b-HSD2 inhibitor
reported against the rat isozyme. The position of the
hydroxyl group in 7 is important for selectivity as the
5-hydroxypentyl analogue 8 is potent on 11b-HSD1
and 11b-HSD2.
The 4-methoxypyridin-3-yl analogue 25 was weakly
active on both isozymes, however the 2,4-dimethoxy-
pyridin-3-yl compound 26, the thiazol-2-yl analogue 27
and the pyrrolidinyl hydrazide 19, although non-selec-
tive, were potent 11b-HSD2 inhibitors, highlighting the
importance of electronic effects in this region.
A number of compounds in this series were weakly ac-
tive and non-selective, these include the ethyl ester 5 and
its corresponding carboxylic acid analogue 6; the N-
methylpiperazinyl, morpholinyl and derivatives 15, and
13 also show low activity and poor selectivity. These
results indicate that the steric and electronic effects of
these groups in this region are not providing favourable
interactions with either of the 11b-HSD isozymes.
The n-butyl analogue 3 is a hydrophobic derivative with
a linear alkyl chain. The other hydrophobic derivatives 4
and 20 with cyclopropyl and benzyl side chains did not
show selectivity for 11b-HSD2. This observation indi-
cates that the hydrophobic interactions in this region of
11b-HSD1 are more restricted than in 11b-HSD2 as 3,
with a hydrophobic side chain with more degrees of
freedom, is poorly active against 11b-HSD1.
The cyclic lactone 10, although not potent, does show
some selectivity for 11b-HSD1 as does the hydroxy acid
11, which is the ring opened version of 10. The possibility,
although unlikely, that 10 is hydrolytically converted to
11 under the assay conditions cannot be ruled out.
The active and selective basic amides indicate that the
position of the basic moiety is important for activity and
selectivity on 11b-HSD2. Both 16 and 14 have a sec-
ondary amine moiety in a ring on the basic side chain in a
similar region of space, and as the N-ethylpiperidinyl, N-
ethylpyrrolidinyl and N-ethylmorpholinyl analogues 17–
Other secondary amide modifications (Table 2) include
compounds with O-acetyl 28, 30, O-methyl 31 and
succinate 33 groups at the 3-position. The 3-O-acetyl

4446
X. Su et al. / Bioorg. Med. Chem. 12 (2004) 4439–4457
benzyl amide 28 was weakly active and non-selective.
The 3-acetyl hydroxamate analogue 29 showed selec-
tivity for 11b-HSD2 similar to the close structural ana-
logue 58, 3-O-acetyl 18b-GA (Table 4). Interestingly, 30
the 3-O-acetyl analogue of the potent selective 11b-
HSD2 inhibitor 7 (Table 1) was inactive on 11b-HSD2,
indicating that the combination of these groups is not
tolerated by the active site of the type 2 isozyme. This
observation was also noted for the 3-O-methyl analogue
31, which was also inactive on 11b-HSD2 (Table 2)
compared to 52 the 3-O-methyl analogue of 18b-GA
(Table 4), which was a potent inhibitor of 11b-HSD2.
This indicates that changes to the 3 and 30 positions to
give 50 were not additive and probably modifications at
both ends of the template are resulting in unfavourable
steric effects. The tertiary amide 32 was a weak non-
selective inhibitor of both isozymes, possibly indicating
that a tertiary amide in this region does not have the
required electronic effect, but as the similar secondary
amide analogues 17 and 18 discussed above were also
weakly active, an unfavourable hydrophobic interaction
in the region is also possible.
sponding carbenoxolone analogue (with 75% and 19%
inhibition of 11b-HSD1 and 11b-HSD2, respectively at
10 lM, not shown in tables). Conversely, 42 is a potent
and selective 11b-HSD2 inhibitor; clearly, interactions
in this area of space are key to determine selectivity with
unfavourable hydrophobic effects being limiting for
both isozymes.
The 3-ether analogues of the 18b-GA template are listed
in Table 4. The direct analogues of 18b-GA 1 itself show
that the 3-methoxy ether 52 is potent and selective for
11b-HSD2. By increasing the steric bulk to ethyl and
isopropyl selectivity for 11b-HSD2 is lost, although
moderate activity, decreasing with hydrophobicity is
maintained. The 3-ether analogues of the C-30 methyl
ester of 18b-GA (47–51, 55–57) are essentially inactive
on both isozymes irrespective of the size and hydro-
phobicity of the ether. Clearly a combination of C-30
ester and C-3 ether moieties is sufficient to block key
interactions on both 11b-HSD1 and 11b-HSD2.
The acetyl ester derivative of 18b-GA, 58 was a potent
selective inhibitor of 11b-HSD2. The potency of 58 on
11b-HSD2 is similar to that of the succinate ester CBX
2, however hydrolytic instability of ester groups any-
where on the GA template cannot be ruled out and 58
may be slowly converted to 18b-GA 1.
In summary, amide modifications at the 30-position are
generally non-selective but show greater inhibition of
11b-HSD2 than 11b-HSD1. The position of the basic
nitrogen in amide side chains is important for activity
and selectivity on 11b-HSD2. The most potent com-
pound in this series is the hydroxyethyl analogue 7 and
elongation of the alkyl chain decreases activity.
The 3-oxo analogue of 18b-GA, 60 is a non-selective and
potent inhibitor of 11b-HSD1 and 11b-HSD2 and the
corresponding C-30 methyl ester 59, although losing
some potency, is also non-selective (activities not shown,
Scheme 3).26 This indicates that a hydrogen bond donor
at the 3-position is not essential for activity and a
hydrogen bond acceptor interaction may be taking place
in this region. This hypothesis is in agreement with the
results observed above for the 3-ethers 52–53 and acetyl
analogue of 18b-GA 58 and also for CBX 2 where a
hydrogen bond donor interaction close to C-3 may be
important. The succinate ester side chain present in CBX
2 was incorporated into selected active compounds that
had indicated selectivity for either 11b-HSD1 or 11b-
HSD2; the synthetic ease of introduction of the succinate
side chain was also taken into account when choosing the
analogues. In the secondary amide series 21 was chosen
as a selective 11b-HSD2 inhibitor and, although only
moderately active, 41 with a methoxymethyl ether at C-
30 was chosen as a selective 11b-HSD1 inhibitor. Activity
and selectivity for 11b-HSD1 was retained with 61;
however, for 33 selectivity was lost and only weak activity
observed indicating the combination of structural chan-
ges at C-3 and C-30 was not tolerated in this case (ac-
tivities not shown).26
3.2. Ester and ether modifications
Further modifications to the 30-position including
esters, ethers and alcohols are listed in Table 3. Esters
with small alkyl groups such as methyl and ethyl, 34 and
35, are potent non-selective inhibitors of 11b-HSD1 and
11b-HSD2. The more bulky hydrophobic esters such as
its t-butyl, cyclohexylmethyl and benzyl derivatives, 36–
38 are less potent and non-selective, indicating that large
hydrophobic groups in this region are not beneficial for
activity.
The hydroxymethyl derivative 39 is potent on both 11b-
HSD1 and 11b-HSD2 and has comparable activity to
18b-GA 1. This indicates that a carbonyl group at the
30-position is not a requirement for activity. However,
the more bulky gem-dimethyl derivative 44 shows weak
activity, indicating the poor tolerance of steric bulk and
the importance of an interaction in this region. Inter-
estingly, the 2-hydroxyethyloxymethyl compound 43 is
selective for 11b-HSD1 and this compound has the
hydroxyl moiety in a similar region to 11 indicating an
important interaction in this region for 11b-HSD1
activity.
In summary, small alkyl esters such as methyl or ethyl at
the 30-position are potent but not selective 11b-HSD
inhibitors and larger groups result in loss of potency. At
the 3-position the acetyl compound is a potent and
selective 11b-HSD2 inhibitor however potency is lost
with additional substitution at the 30-position. In the
ether series the 3-methoxy compound is a potent and
selective inhibitor of 11b-HSD2 and a loss of selectivity
is observed with larger substituents.
The bulky ether 40 is inactive on both 11b-HSD1 and
11b-HSD2, indicating that the tolerance for steric bulk
for activity has been exceeded. The smaller methoxy-
methyl ether 41 although weakly active, shows some
selectivity for 11b-HSD1 and it is interesting to note this
selectivity is also observed in 61 (Scheme 2), the corre-

X. Su et al. / Bioorg. Med. Chem. 12 (2004) 4439–4457
4447
3.3. Modification of the 11-position
identification of novel nonsteroidal templates as 11b-
HSD inhibitors and as templates for further design.
A number of modifications were made at the 11-position
of the 18b-GA 1 template, the region where the 11b-
HSD isozymes invoke their catalysis. The 11-exo-
methylene compounds 66 and 68 were weakly active and
non-selective showing that this carbonyl isostere, al-
though having a similar size to a carbonyl, does not have
the electronic and hydrogen bonding properties in this
region likely to be required for inhibition of 11b-HSD
catalysis. The tertiary alcohol analogue 65 showed
poor activity against both 11b-HSD1 and 11b-HSD2
although some selectivity for 11b-HSD1 was indicated.
This observation was strengthened by the corresponding
C-30 acid derivative 67 showing reasonable potency and
selectivity for the type 1 isozyme. Removal of the car-
bonyl at the 11-position to give the desoxo analogues
62–64 showed that with this change only the C-30 acid
derivative 62 retained potency but was non-selective.
The ester analogue 63 was weakly active and non-se-
lective. The C-30 hydroxymethyl derivative 64, although
weakly active, demonstrated selectivity for 11b-HSD2,
an observation previously noted for small nonhindered
C-30 side chain alcohols such as 7. In summary, with
hydrogen at the 11-position and the 30-position
unsubstituted potency is maintained with lack of selec-
tivity. Introduction of a methylene group at the 11-po-
sition gives weakly active non-selective compounds.
Introduction of a tertiary alcohol giving 67 results in
selectivity for 11b-HSD1.
4. Conclusions
Novel highly potent and selective inhibitors of rat 11b-
HSD2 have been identified and the knowledge of the
SAR of glycyrrhetinic acid analogues as inhibitors of the
11b-hydroxysteroid dehydrogenases has been greatly
extended. The selective 11b-HSD2 described herein have
potencies ranging from low picomolar to hundred
nanomolar levels. Compound 7 is the most potent
selective 11b-HSD2 inhibitor reported to date with an
IC50 ¼ 4 pM on the rat isozyme. Compounds such as 7
should prove useful as mechanistic tools for in vivo
studies in rodent models to evaluate the biological effects
of potent and selective 11b-HSD2 inhibitors. In addition,
they will serve as templates for further design of selective
nonsteroidal inhibitors and the recent publication of a
homology model of 11b-HSD2 will be used in structure
based drug design to accelerate this process.35
5. Experimental
5.1. General
All chemicals were either purchased from the Aldrich
Chemical Co. (Gillingham, UK), Lancaster Synthesis
(Morecambe, UK) or ACROS Organics (Loughbor-
ough, UK). All organic solvents of A.R. grade were
supplied by Fisher Scientific (Loughborough, UK).
As the 11b-HSD isozymes exert their catalytic action at
the 11-position, it is not surprising to see dramatic
changes in potency when this region of the molecule is
modified.
Rat livers and rat kidneys from normal Wistar rats were
obtained from Harlan Olac, Bicester, Oxon, UK. The
PBS–sucrose buffer assay medium, DulbeccoÕs Phos-
phate Buffered Saline, 1 tablet/100 mL with 0.25 M su-
crose, pH 7.4 was purchased from BDH Laboratory
supplies, UK. Ecoscint A scintillation fluid was obtained
from National Diagnostics, US. Radioactive cortisol
solutions, [1,2,6,7-3H]-cortisol was obtained from
American Radiolabelled Chemical Inc., (ARC) MD,
USA with 2 · 105 dpm and [4-14C]-cortisol was obtained
from Perkin Elmer, MS, USA with 10,000 dpm.
Radiolabelled cortisone was obtained from American
Radiolabelled Chemical Inc., (ARC) MD, USA with
2 · 105 dpm and [4-14C]-cortisone was bought from ARC
(10,000 dpm). Cortisol, cortisone and cofactors
NADPH and NADP were purchased from Sigma
Chemical Co., Poole, UK.
From the extensive number of structural changes to the
18b-GA template discussed a number of potent and
selective inhibitors of 11b-HSD2 have been identified
and the IC50 values of a selection these compounds are
shown in Table 6 with 18b-GA 1 included for compar-
ison.
From the extensive modifications on the 18b-GA tem-
plate 1 discussed a number of potent and selective
inhibitors of 11b-HSD2 have been identified. A pre-
liminary communication relating to some of this work
has been published.34 These compounds are currently
being used in the construction of a pharmacophore for
11b-HSD2, which will not only be used in the design of
further selective inhibitors of 11b-HSD2 but also in the
Thin layer chromatography (TLC) was performed on
pre-coated plates (Merck TLC aluminium sheets silica
gel 60 F254, Art. No 5554). Compounds were visualized
by either viewing under UV light or treating with an
ethanolic solution of phosphomolybdic acid (PMA)
followed by heating. Flash chromatography was carried
out using Sorbsil C60 silica gel or Isoluteꢂ pre-packed
Flash Si columns from Argonaut Technologies. Parallel
synthesis was performed on either Radleys Carousel
reaction stations or Radleys GreenHouse parallel syn-
thesizers. Solvent removal from parallel syntheses was
Table 6. IC50 values for selective inhibitors of 11b-HSD2
Compd
IC50 (nM) on 11b-HSD2a
1
360b
0.004
164
7
42
52
58
184
0.02
a Mean of at least two measurements with typically ꢁ5% variation.
b Reported value.32

4448
X. Su et al. / Bioorg. Med. Chem. 12 (2004) 4439–4457
performed on a GeneVac DD4 evaporation system.
NMR spectra were recorded with a JEOL GX-270 or
Varian-Mercury-400 spectrometer, and chemical shifts
are reported in parts per million (ppm, d) relative to
tetramethylsilane (TMS) as an internal standard. Only
diagnostic proton signals are listed. Mass spectra were
recorded at the Mass Spectrometry Service Centre,
University of Bath. FAB-MS were carried out using m-
nitrobenzyl alcohol (NBA) as the matrix. High perfor-
mance liquid chromatography (HPLC) analysis was
performed with a Waters Delta 600 liquid chromato-
graph with a Waters 996 photodiode Array Detector
using a Waters Radialpack C18, 8 · 100 mm column.
Melting points (mp) were measured with a Reichert-
Jung ThermoGalen Kofler block or a Sanyo Gallenk-
amp melting point apparatus and are uncorrected.
product steroids were extracted with diethyl ether
(4 mL). The organic phase was removed and taken to
dryness under air. The resulting residue was dissolved in
diethyl ether (50–100 lL), transferred to a thin-layer
chromatography plate, which contained a fluorescent
dye, and developed using the system chloroform:
methanol (9:1, v/v). Product steroids were visualized
under UV light. In this system the Rf values for cortisol
and cortisone were 0.5 and 0.8, respectively. The areas
containing cortisol or cortisone were cut from the TLC
plate and placed in a 20 mL scintillation vials. Steroids
were eluted from the plate by the addition of methanol
(0.5 mL) before the addition of scintillation fluid
(10 mL) and determining the radioactivity using scintil-
lation spectrometry. Enzyme activity was determined as
the amount of product formed/mg protein/h after cor-
rection for procedural losses. Assays were carried out in
the absence or presence of inhibitors. Initially, all com-
pounds were tested at 10 lM with IC50 values being
determined for compounds showing >90% inhibition
when tested at this concentration.
Incubations were performed in a mechanically shaken
water bath, SW 20, Germany. Evaporations were car-
ried out using a Techne Driblock DB 3A, UK. Scintil-
lation vials used were 20 mL polypropylene vials with
caps, SARSTEDT, Germany, and scintillation coun-
tering was performed on a Beckman LS 6000 SC,
Beckman Instruments Inc., USA.
5.3. Chemistry
5.3.1. General synthetic procedures
5.2. Biology
5.3.1.1. Method A: Synthesis of 18b-GA 30-amides (4,
5, 7, 8, 10, 12, 30–32). To a solution of 18b-GA (1)
(0.5 mmol) in DCM (15 mL) were added the amine
(1.0 mmol), HOBt (0.26 mmol), EDCI (0.55 mmol),
DMAP (0.55 mmol) and triethylamine (0.55 mmol). The
mixture was stirred under nitrogen at room temperature
for 16–24 h. When TLC showed the completion of the
reaction, the reaction mixture was poured into water
and extracted with DCM. The organic phase was wa-
shed with 2% HCl and water, dried over MgSO4.
Evaporation of the solvent gave a residue that was
purified by flash chromatography (SiO2, ethyl acetate–
hexane, gradient elution) to give the amide product.
Assays were performed essentially as described previ-
ously.26 Kidneys and livers from normal Wistar rats
were homogenized on ice in PBS–sucrose buffer (1 g/
10 mL) using an Ultra-Turrax. After the livers and
kidneys were homogenized the homogenate was centri-
fuged for 5 min at 4000 rpm. The supernatant obtained
was removed and stored in glass vials at )20 ꢁC. The
amount of protein per lL of rat liver and kidney cen-
trifuged supernatants was determined using the Brad-
ford method.33
For the assay of 11b-HSD type 1 activity rat centrifuged
supernatant (76 lg protein) was incubated for 90 min
with [3H] cortisone and adjusted to a final substrate
concentration of 0.5 lM with cortisone. The assay was
carried out in phosphate-buffered saline (PBS) with
NADPH (1 mM) as the co-factor. At the end of the
incubation period [4-14C] cortisol was added to monitor
procedural losses together with 50 lg of unlabelled
cortisol to assist in the location of product steroid.
Using this assay, conversion of cortisone to cortisol was
linear with respect to time (0–120 min) and protein
concentration (10–100 lg) (data not shown).
5.3.1.2. Method B: Synthesis of 18b-GA 30-amides (20,
28). To a solution of 18b-GA (1) (0.2 mmol) in chloro-
form (5 mL) the requisite amine (0.4 mmol) and EDCI
(0.4 mmol) were added. The mixture was stirred under
nitrogen at room temperature for 6–16 h. When TLC
showed the completion of the reaction, the reaction
mixture was poured into water, extracted with DCM.
The organic phase was washed with 2% HCl and water,
dried over MgSO4 and concentrated in vacuo to give a
residue that was purified by flash chromatography
(SiO2, ethyl acetate–hexane, gradient elution) to give the
amide products.
The assay for 11b-HSD type 2 activity employed rat
kidney centrifuged supernatant (136 lg protein) and [3H]
cortisol adjusted to a final substrate concentration of
0.5 lM with cortisol. PBS–sucrose was used as the buffer
with NADP (1 mM) as the co-factor. [4-14C] Cortisone
was added at the end of the incubation period (60 min)
to monitor procedural losses with 50 lg unlabelled cor-
tisone being added to assist product location,
5.3.1.3. Method C: Parallel synthesis of 18b-GA 30-
amides using Radleys GreenHouse Parallel Synthesizer
(3, 9, 13–19, 22–27). To a suspension of 18b-GA (1)
(50 mg, 0.106 mmol) in anhydrous DCM (4 mL, 10 mL
tubes) under an argon atmosphere at room temperature,
EDCI (1.1 M equiv), DMAP (cat.) were added followed
by Et3N (1.1 M equiv). The reaction mixture was stirred
for 0.5 h at room temperature, added the amine
(2 M equiv) and stirred overnight. The reactions were
At the end of the incubation period and addition of
[4-14C] and unlabelled product steroids, precursor and

X. Su et al. / Bioorg. Med. Chem. 12 (2004) 4439–4457
4449
monitored using TLC (DCM–MeOH, 95:5) and quen-
ched by addition of water (1 mL). The resulting mixtures
were stirred at rt for 10 min and the aqueous layer was
separated. The organic phase was then washed subse-
quently with 1 M HCl (1 mL) and water (1 mL). The
aqueous phase was removed and the bi-phasic mixture
was evaporated using the GeneVac. The crude products
were purified in a parallel manner using Argonaut pre-
packed columns (5 g) on the Vacuum Flash Master using
a gradient elution with hexane–ethyl acetate–methanol
mixtures to give the products as white solids.
(dt, J ¼ 13:3, 3.4 Hz, 1H, 1b-H), 2.71 (m, 1H, –NHCH),
2.34 (s, 1H, 9a-H), 2.15 (t, J ¼ 8:2 Hz, 1H, 18b-H), 1.37
(s, 3H, CH3), 1.14 (s, 3H, CH3), 1.13 (s, 3H, CH3), 1.11
(s, 3H, CH3), 1.01 (s, 3H, CH3), 0.82 (s, 3H, CH3), 0.81
(s, 3H, CH3); FAB-MS m=z: 510 (100, MHþ); FAB-
HRMS calcd for C33H53NO3 (MHþ) 510.3947, found
510.3956.
5.3.1.8. N-(3b-Hydroxy-11,30-dioxo-18b-olean-12-en-
30-yl)-L-alanine ethyl ester (5). 5 was synthesized with
general method A starting from 1 (1.0 mmol). White
amorphous powder (430 mg, 75%) was obtained. HPLC
purity 99% (tR 4.2 min in 6% water–methanol); 1H
NMR (400 MHz, CDCl3) d 6.14 (d, J ¼ 8 Hz, 1H, NH),
5.76 (s, 1H, 12-H), 4.60 (m, 1H, NHCH), 4.23 (q,
J ¼ 7:3 Hz, 2H, –OCH2CH3), 3.23 (m, 1H, 3a-H), 2.82
(dt, J ¼ 13:3, 3.5 Hz, 1H, 1b-H), 2.34 (s, 1H, 9a-H), 2.28
(t, J ¼ 8:5 Hz, 1H, 18b-H), 1.40 (d, J ¼ 7 Hz, 3H, CH3),
1.39 (s, 3H, CH3), 1.30 (t, J ¼ 7:3 Hz, 3H, CH3), 1.15 (s,
3H, CH3), 1.14 (s, 3H, CH3), 1.13 (s, 3H, CH3), 1.01 (s,
3H, CH3), 0.82 (s, 3H, CH3), 0.81 (s, 3H, CH3); FAB-
MS m=z: 570 (100, MHþ); FAB-HRMS calcd for
C35H56NO5 (MHþ) 570.4158, found 570.4150.
5.3.1.4. Method D: Synthesis of 3-alkylated 18b-GA
30-methyl ester derivatives (47–51, 55–57). The inter-
mediate methyl ester 34 was suspended in anhydrous
THF under an argon atmosphere, NaH (60% dispersed
in oil, 2 equiv) was added at rt over 5 min, followed by
the requisite alkyl or aryl halide (2 equiv). The crude
mixtures were heated at reflux under inert atmosphere.
The reactions were monitored by TLC (EtOAc–hexane,
2:8). Once completed, the reactions were quenched by
addition of water (dropwise) at rt. The crude mixtures
were extracted with DCM, and the organic phase was
washed with saturated NaCl solution. The organic ex-
tracts were dried (MgSO4) and concentrated in vacuo to
obtain the product as a solid. The crude product was
then purified using flash chromatography (SiO2, hex-
ane–ethyl acetate, gradient elution) to obtain in general
a colourless solid.
5.3.1.9. N-(3b-Hydroxy-11,30-dioxo-18b-olean-12-en-
30-yl)-L-alanine (6). To a solution of 5 (300 mg,
0.53 mmol) in methanol–THF (8 mL:4 mL) was added 1
N NaOH solution (1 mL). The mixture was stirred un-
der nitrogen at rt for 2 h, adjusted to pH 3 with 10% HCl
and extracted with DCM. The organic phase was wa-
shed with water, dried over MgSO4 and concentrated to
give a residue, which was purified with flash chroma-
tography and recrystallized from ethyl ether. White
crystalline solid (180 mg, 63%) was obtained. Mp 272–
5.3.1.5. Method E: Synthesis of 3-alkylated 18b-GA
derivatives (52–54). The starting ester was suspended in
THF–water (3:1, 4 mL), LiOH (5 M equiv) were added,
and the mixture heated at reflux for 24 h. The crude
reaction was cooled to rt, acidified with 2 M HCl
(pH ¼ 2) and extracted with DCM. The organic phase
was dried (MgSO4) and concentrated to obtain the
crude product as a pale yellow solid. The crude product
was then purified using flash chromatography eluting
with ethyl acetate–methanol or DCM–methanol.
1
275 ꢁC; HPLC purity 97% (tR 2.6 min in methanol); H
NMR (400 MHz, CDCl3) d 6.67 (d, J ¼ 7:8 Hz, 1H,
NH), 5.64 (s, 1H, 12-H), 4.65 (m, 1H, –NHCH–), 3.18
(m, 1H, 3a-H), 2.70 (dt, J ¼ 13:3, 3.2 Hz, 1H, 1b-H),
2.27 (s, 1H, 9a-H), 1.33 (d, J ¼ 7 Hz, 3H, CH3), 1.28 (s,
3H, CH3), 1.09 (s, 3H, CH3), 1.05 (s, 3H, CH3), 1.04 (s,
3H, CH3), 0.94 (s, 3H, CH3), 0.74 (s, 3H, CH3), 0.73 (s,
3H, CH3); FAB-MS m=z: 542 (100, MHþ); FAB-HRMS
calcd for C35H52NO5 (MHþ) 542.3845, found 542.3853.
5.3.1.6. Method F: Synthesis of 18b-GA 30-esters (35–
38). A solution of 1 (1.0 g; 2.12 mmol) in anhydrous
DMF (50 mL) containing anhydrous K2CO3 (2.9 g;
21.2 mmol) was stirred at rt under N2 for 1 h. The req-
uisite alkyl halide (11 equiv) was introduced to the
reaction mixture followed by tetra-butyl ammonium
iodide (200 mg). The mixture was stirred for 24 h at rt,
poured into brine and extracted with ethyl acetate
(3 · 200 mL). The organic phase was washed with water
(3 · 100 mL), dried over MgSO4, filtered and evaporated
in vacuo to give a white solid. Purification by flash
chromatography (SiO2, EtOAc–Hexane gradient elu-
tion) and recrystallization from absolute ethanol gave
the required product.
5.3.1.10. N-(2-Hydroxyethyl)-3b-hydroxy-11-oxo-18b-
olean-12-en-30-oic acid amide (7). 7 was synthesized with
general method A. White crystalline solid (110 mg, 43%)
was obtained. Mp 160–163 ꢁC; HPLC purity 96% (tR
1
6.2 min in 55% water–methanol); H NMR (400 MHz,
CDCl3) d 6.06 (t, J ¼ 5:9 Hz, 1H, NH), 5.61 (s, 1H, 12-
H), 3.67 (t, J ¼ 5:1 Hz, 2H, CH2), 3.39 (m, 2H,
–NHCH2–), 3.16 (m, 1H, 3a-H), 2.72 (dt, J ¼ 13:6,
3.6 Hz, 1H, 1b-H), 2.27 (s, 1H, 9a-H), 2.12 (t,
J ¼ 8:6 Hz, 1H, 18b-H), 1.30 (s, 3H, CH3), 1.08 (s, 3H,
CH3), 1.06 (s, 3H, CH3), 1.05 (s, 3H, CH3), 0.94(s, 3H,
CH3), 0.75 (s, 3H, CH3), 0.74 (s, 3H, CH3); FAB-MS
m=z: 542 (100, MHþ); FAB-HRMS calcd for
C35H52NO5 (MHþ) 542.3845, found 542.3853.
5.3.1.7. N-Cyclopropyl-3b-hydroxy-11-oxo-18b-olean-
12-en-30-oic acid amide (4). 4 was synthesized with
general method A. White crystalline solid (150 mg, 59%)
was obtained. Mp 253–256 ꢁC; HPLC purity 99% (tR
5.3.1.11.
N-(5-Hydroxypentyl)-3b-hydroxy-11-oxo-
1
3.9 min in 6% water–methanol); H NMR (400 MHz,
18b-olean-12-en-30-oic acid amide (8). 8 was synthe-
sized with general method A. White crystalline solid
CDCl3) d 5.64 (s, 1H, 12-H), 3.23 (m, 1H, 3a-H), 2.80

4450
X. Su et al. / Bioorg. Med. Chem. 12 (2004) 4439–4457
(165 mg, 59%) was obtained. Mp 136–139 ꢁC; HPLC
purity 98% (tR 5.1 min in 8% water–methanol); 1H
NMR (400 MHz, CDCl3) d 5.67 (s, 1H, 12-H), 5.63 (m,
1H, NH), 3.68 (m, 2H, -CH2OH), 3.42 (m, 1H,
–NHCH–), 3.24 (m, 1H, –NHCH–), 3.23 (m, 1H, 3a-H),
2.80 (dt, J ¼ 13:6, 3.5 Hz, 1H, 1b-H), 2.38 (s, 1H, 9a-H),
1.34 (s, 3H, CH3), 1.14 (s, 6H, 2 · CH3), 1.13 (s, 3H,
CH3), 1.01 (s, 3H, CH3), 0.82 (s, 3H, CH3), 0.81 (s, 3H,
CH3); FAB-MS m=z: 556 (100, MHþ); FAB-HRMS
calcd for C35H58NO4 (MHþ) 556.4366, found 556.4376.
5.3.1.15. N-Benzyl-3b-hydroxy-11-oxo-18b-olean-12-
en-30-oic acid amide (20). 20 was synthesized with gen-
eral method B. Off-white crystalline powder (40 mg,
33%) was obtained. Mp 233–235 ꢁC; HPLC purity 98%
(tR 2.6 min in 4% water–methanol); 1H NMR (400 MHz,
CDCl3) d 7.32–7.36 (m, 2H, aromatic protons), 7.25–
7.30 (m, 3H, aromatic protons), 5.83 (broad, 1H, NH),
5.57 (s, 1H, 12-H), 4.47 (m, 2H, NHCH2Ph), 3.21 (dd,
J ¼ 10:5, 6.2 Hz, 1H, 3a-H), 2.78 (dt, J ¼ 13:3, 4.5 Hz,
1H, 1b-H), 2.31 (s, 1H, 9a-H), 2.18 (dd, J ¼ 9:8, 3.5 Hz,
1H, 18b-H), 1.36 (s, 3H, CH3), 1.16 (s, 3H, CH3), 1.13
(s, 3H, CH3), 1.12 (s, 3H, CH3), 1.00 (s, 3H, CH3), 0.81
(s, 3H, CH3), 0.80 (s, 3H, CH3); FAB-MS m=z: 560 (100,
MHþ); FAB-HRMS calcd for C37H54NO3 (MHþ)
560.4104, found 560.4090.
5.3.1.12. N-(2-Oxo-tetrahydrofuran-3-yl)-3b-hydroxy-
11-oxo-18b-olean-12-en-30-oic acid amide (10). The dia-
stereoisomers 10 were synthesized with general method
A starting from 1 (0.75 mmol). White powder (305 mg,
74%) was obtained. HPLC purity 99% (as diastereoiso-
mers, tR ¼ 3:9 min in 4% water–methanol); 1H NMR
(400 MHz, CDCl3) d 6.37 (d, J ¼ 6:2 Hz, 1H, NH), 6.32
(d, J ¼ 6:2 Hz, 1H, NH), 5.75 (s, 1H, 12-H), 5.66 (s, 1H,
12-H), 4.58 (m, 2H, 2 · NHCH), 4.49 (m, 2H, –OCH2),
4.30 (m, 2H, –OCH2), 3.23 (m, 2H, 2 · 3a-H), 2.80 (m,
2H, 2 · 1b-H), 2.32 (s, 2H, 2 · 9a-H), 1.35 (s, 6H,
2 · CH3), 1.16 (s, 6H, 2 · CH3), 1.11 (s, 12H, 4 · CH3),
0.98 (s, 6H, 2 · CH3), 0.81 (s, 6H, 2 · CH3), 0.79 (s,
6H, 2 · CH3); FAB-MS m=z: 554 (100, MHþ); FAB-
HRMS calcd for C34H52NO5 (MHþ) 554.3845, found
554.3832.
5.3.1.16. N-Benzyl-3b-acetyloxy-11-oxo-18b-olean-12-
en-30-oic acid amide (28). 28 was synthesized with gen-
eral method B starting from 3b-acetyloxy glycyrrhetinic
acid (58).31 White crystalline solid (55 mg, 48%) was
obtained. Mp 150–152 ꢁC; HPLC purity 97.7% (tR
3.6 min in 4% water–methanol); 1H NMR (400 MHz,
CDCl3) d 7.32–7.37 (m, 2H, aromatic protons), 7.24–
7.30 (m, 3H, aromatic protons), 5.83 (t, J ¼ 5:5 Hz, 1H,
NH), 5.56 (s, 1H, 12-H), 4.41–4.53 (m, 3H, 3a-H and
NHCH2Ph), 2.78 (dt, J ¼ 13:6, 4.5 Hz, 1H, 1b-H), 2.33
(s, 1H, 9a-H), 2.16 (dd, J ¼ 9:7, 3.2 Hz,1H, 18b-H), 2.05
(s, 3H, 3-CH3CO–), 1.35 (s, 3H, CH3), 1.16 (s, 6H,
2 · CH3), 1.15 (s, 3H, CH3), 1.12 (s, 3H, CH3), 0.88 (s,
3H, CH3), 0.81 (s, 3H, CH3); FAB-MS m=z: 602 (100,
MHþ); FAB-HRMS calcd for C39H56 NO (MHþ)
602.4209, found 602.4211.
5.3.1.13. N-(3b-Hydroxy-11,30-dioxo-18b-olean-12-
en-30-yl)-2-amino-4-hydroxy-butyric acid (11). To a
solution of 10 (110 mg, 0.20 mmol) in methanol (4 mL)
was added 1N KOH solution (1 mL). The mixture was
stirred under nitrogen at rt for 24 h, then adjusted to
pH 2 with 10% HCl and extracted with DCM. The or-
ganic phase was washed with water, dried over MgSO4
and concentrated to give a residue, which was purified
with flash chromatography. White solid (65 mg, 57%)
was obtained. Mp 220–229 ꢁC; HPLC purity 95% (as
5.3.1.17. N-Hydroxy-3b-acetyloxy-11-oxo-18b-olean-
12-en-30-oic acid amide (29). To a solution of 58
(235 mg, 0.45 mmol) in anhydrous THF (8 mL) was
added DMAP (11 mg, 0.09 mmol), followed by carbon-
yldiimidazole (89 mg, 0.55 mmol). The mixture was
stirred at rt under nitrogen for 18 h. A solution of
hydroxylamine hydrochloride (64 mg, 0.92 mmol) in
DMF (2 mL) was added. The mixture was stirred at
60 ꢁC for 5 h, and poured into water, extracted with
DCM. The organic phase was washed brine, dried over
MgSO4 and concentrated in vacuo to afford a residue
that was purified with flash chromatography (EtOAc–
hexane gradient elution). White crystalline solid (90 mg,
38%) was obtained. Mp 294–299 ꢁC; HPLC purity 99%
(tR 7.2 min in 10% water–methanol); 1H NMR
(400 MHz, CDCl3) d 6.37 (s, 1H, NH), 5.69 (s, 1H, 12-
H), 4.52 (dd, J ¼ 11:7, 7.1 Hz, 1H, 3a-H), 2.81 (dt,
J ¼ 13:6, 3.5 Hz, 1H, 1b-H), 2.28 (s, 1H, 9a-H), 2.08 (s,
3H, CH3CO–), 1.36 (s, 3H, CH3), 1.20 (s, 3H, CH3), 1.16
(s, 3H, CH3), 1.13 (s, 3H, CH3), 0.88 (s, 6H, 2 · CH3),
0.81 (s, 3H, CH3); FAB-MS m=z: 528 (100, MHþ); FAB-
HRMS calcd for C32H50NO5 (MHþ) 528.3689, found
528.3698.
1
diastereoisomers, tR 2.0 min in 4% water–methanol); H
NMR (400 MHz, CDCl3) d 7.0 (m, 1H, NH), 5.75 (s,
1H, 12-H), 4.40 (m, 1H, –NHCH–), 3.65 (m, 2H,
–OCH2), 3.18 (m, 1H, 3a-H), 1.31 (s, 3H, CH3), 1.19 (s,
3H, CH3), 1.05 (s, 3H, CH3), 1.02 (s, 3H, CH3), 0.92 (s,
3H, CH3), 0.75 (s, 6H, 2 · CH3); FAB-MS m=z: 570 (30,
M)Hþ); FAB-HRMS calcd for C34H52NO6 (M)Hþ)
570.3795, found 570.3797.
5.3.1.14. N-[2-(Morpholin-4-yl)ethyl]-3b-hydroxy-11-
oxo-18b-olean-12-en-30-oic acid amide (12). 12 was syn-
thesized with general method A. White crystalline solid
(200 mg, 69%) was obtained. Mp 135–138 ꢁC; HPLC
purity 97% (tR 3.9 min in 6% water–methanol); 1H
NMR (400 MHz, CDCl3) : d 6.21 (m, 1H, NH), 5.71 (s,
1H, 12-H), 3.72 (m, 4H, –(CH2)2O of morpholine), 3.38
(m, 2H, –CONHCH2), 3.23 (m, 1H, 3a-H), 2.82 (dt,
J ¼ 13:5, 3.2 Hz, 1H, 1b-H), 2.52 (t, J ¼ 5:9 Hz, 2H,
–CH2N), 2.47 (m, 4H, –N(CH2)2), 2.37 (s, 1H, 9-H),
1.40 (s, 3H, CH3), 1.14 (s, 9H, 3 · CH3), 1.01 (s, 3H,
CH3), 0.82 (s, 3H, CH3), 0.81 (s, 3H, CH3); FAB-MS
m=z: 583 (100, MHþ); FAB-HRMS calcd for
C36H59N2O4 (MHþ) 583.4475, found 583.4492.
5.3.1.18.
N-(2-Hydroxyethyl)-3b-acetyloxy-11-oxo-
18b-olean-12-en-30-oic acid amide (30). 30 was synthe-
sized with general method A starting from 58 (872 mg,
1.70 mmol). White crystalline solid (688 mg, 73%) was
obtained. Mp 236–238 ꢁC; HPLC purity 99% (tR 2.2 min

X. Su et al. / Bioorg. Med. Chem. 12 (2004) 4439–4457
4451
1
in methanol); H NMR (400 MHz, CDCl3) d 6.22 (t,
7.15–7.23 (m. 1H, Ar–H), 5.71 (s, 1H, 12-H), 4.40–4.57
(m, 3H, 3a-H and –CH2Py), 2.71 (dt, J ¼ 13:3, 3.5 Hz,
1H, 1b-H), 2.58–2.64 (m, 4H, –COCH2CH2CO–), 2.34
(s, 1H, 9a-H), 1.34 (s, 3H, CH3), 1.19 (s, 3H, CH3), 1.04
(s, 6H, 2 · CH3), 0.82 (s, 3H, CH3), 0.81 (s, 3H, CH3),
0.66 (s, 3H, CH3); FAB-MS m=z: 661 (60, MHþ); FAB-
HRMS calcd for C40H57N2O6 (MHþ) 661.4217, found
661.4231.
J ¼ 5:5 Hz, 1H, NH), 5.68 (s, 1H, 12-H), 4.51 (dd,
J ¼ 12:7, 5.1 Hz, 1H, 3a-H) 3.73 (m, 2H, –CH2OH),
3.54 (m, 1H, –NHCH–), 3.39 (m, 1H, –NHCH–), 2.86
(broad, 1H, OH), 2.77 (dt, J ¼ 13:7, 3.5 Hz, 1H, 1b-H),
2.36 (s, 1H, 9a-H), 2.18 (t, J ¼ 8:6 Hz, 1H, 18b-H), 2.06
(s, 3H, CH3CO–), 1.37 (s, 3H, CH3), 1.15 (s, 6H,
2 · CH3), 1.12 (s, 3H, CH3), 0.88 (s, 6H, 2 · CH3), 0.81
(s, 3H, CH3); 13C NMR (270 MHz, CDCl3) d 200.2,
176.9, 171.0, 169.5, 128.4, 80.6, 62.5, 61.7, 55.0, 48.2,
45.4, 43.7, 43.2, 42.0, 41.9, 38.8, 38.0, 37.4, 36.9, 32.7,
31.9, 31.5, 29.5, 28.5, 28.0, 26.4, 26.3, 23.5, 23.3, 21.3,
18.7, 17.3, 16.7, 16.4; FAB-MS m=z: 556 (100, MHþ);
FAB-HRMS calcd for C34H54NO5 (MHþ) 556.4002,
found 556.3991.
The following amides (3, 9, 13–19, 21–27) were synthe-
sized with general method C.
5.3.1.22. N-Butyl-3b-hydroxy-18b-olean-12-en-11-one-
30-oic acid amide (3). 3: HPLC purity 90% (tR 2.38 min
in 100% MeOH); 1H NMR (CDCl3, 400 MHz) d 5.69 (s,
1H, olefinic), 3.50–3.21 (m, 3H), 2.34 (s, 1H, CH), 1.64–
1.43 (broad m, 4H), 1.37, 1.25, 1.23, 1.14, 1.01, 0.83 and
0.81 (7 · s, 3H each, CH3); APCI-MS m=z: 526 (50,
MHþ), 567 (50, M+CH3CNþ); FAB-HRMS calcd for
C35H55NO3 (MHþ) 526.4260, found 526.4267.
5.3.1.19. N-(2-Hydroxyethyl)-3b-methoxy-11-oxo-18b-
olean-12-en-30-oic acid amide (31). 31 was synthesized
with general method A starting from 52 (500 mg,
1.03 mmol). White crystalline solid (396 mg, 75%) was
obtained. Mp 146–148 ꢁC; HPLC purity 99% (tR 2.6 min
1
in methanol); H NMR (400 MHz, CDCl3) d 6.18 (t,
5.3.1.23. N-(2-Amino-N-methylethyl)-3b-hydroxy-18b-
olean-12-en-11-one-30-oic acid amide (9). 9: mp 153–
156 ꢁC; HPLC purity 95% (tR 2.38 min in 100% MeOH);
1H NMR (CDCl3, 400 MHz) d 5.70 (s, 1H, olefinic H),
3.25–3.21 (m, 2H), 3.13–2.78 (broad dt, 1H, 1b-CH),
2.18 (s, 3H N–CH3), 1.37, 1.35, 1.12, 1.10, 1.00, 0.80 and
0.79 (7 · s, 3H each, 7 · CH3); APCI-MS m=z: 526 (100,
Mþ); FAB-HRMS calcd for C33H54N2O3 (Mþ)
526.4089, found 526.4049.
J ¼ 5:4 Hz, 1H, NH), 5.68 (s, 1H, 12-H), 3.74 (m, 2H,
–CH2OH), 3.38–3.48 (m, 2H, –NHCH2–), 3.28 (s, 3H,
CH3O–), 2.80 (dt, J ¼ 13:7, 3.5 Hz, 1H, 1b-H), 2.68 (dd,
J ¼ 12:6, 5.0 Hz, 1H, 3a-H), 2.33 (s, 1H, 9a-H), 2.17 (t,
J ¼ 8:5 Hz, 1H, 18b-H), 1.37 (s, 3H, CH3), 1.15 (s, 3H,
CH3), 1.13 (s, 3H, CH3), 1.12 (s, 3H, CH3), 0.99 (s, 3H,
CH3), 0.82 (s, 3H, CH3), 0.79 (s, 3H, CH3); FAB-MS
m=z: 528 (100, MHþ); FAB-HRMS calcd for
C33H54NO4 (MHþ) 528.4053, found 528.4041.
5.3.1.24.
olean-12-en-11-one-30-oic acid amide (13). 13: mp 183–
N-(Morpholin-4-amino)-3b-hydroxy-18b-
5.3.1.20. N-(3b-Hydroxy-11,30-dioxo-18b-olean-12-
en-30-yl)-piperidine (32). 32 was synthesized with gen-
eral method A. White crystalline solid (110 mg, 41%)
was obtained. Mp 276–280 ꢁC; HPLC purity 98% (tR
186 ꢁC; HPLC purity >99% (tR 1.53 min in 100%
1
MeOH); H NMR (CDCl3, 270 MHz) d 6.30 (broad s,
1H, NH), 5.60 (s, 1H, olefinic), 3.82–3.78 (broad t, 4H,
CH2), 3.22–3.20 (broad m, 1H, CH), 2.80–2.77 (broad
m, 5H, CH2), 2.34 (s, 1H, CH), 1.37, 1.25, 1.10, 1.12,
1.01, 0.81 and 0.75 (7 · s, 3H each, CH3); APCI-MS m=z:
555 (40, MHþ), 577 (30, M+Naþ); FAB-HRMS calcd
for C37H54N2O4 (MHþ) 555.4525, found 555.4512.
1
4.8 min in 4% water–methanol); H NMR (400 MHz,
CDCl3) d 5.71 (s, 1H, 12-H), 3.55 (m, 4H, –N(CH2)2 of
piperidine), 3.23 (m, 1H, 3a-H), 2.82 (dt, J ¼ 13:3,
3.5 Hz, 1H, 1b-H), 2.35 (s, 1H, 9a-H), 2.30 (t,
J ¼ 8:2 Hz, 1H, 18b-H), 1.37 (s, 3H, CH3), 1.22 (s, 3H,
CH3), 1.14 (s, 3H, CH3), 1.13 (s, 3H, CH3), 1.01 (s, 3H,
CH3), 0.82 (s, 3H, CH3), 0.81 (s, 3H, CH3); FAB-MS
m=z: 538 (100, MHþ); FAB-HRMS calcd for
C35H56NO3 (MHþ) 538.4260, found 538.4239.
5.3.1.25.
olean-12-en-11-one-30-oic acid amide (14). 14: HPLC
N-(1-Piperazinylethyl)-3b-hydroxy-18b-
1
purity >90% (tR 2.45 min in 100% MeOH); H NMR
(CDCl3, 270 MHz) d 5.71 (s, 1H, olefinic), 3.64 (broad t,
4H, CH2), 3.20–3.10 (broad m, CH), 2.81–2.71 (broad
dt, 1H, 1b-CH), 2.38–2.32 (broad t, 4H, CH2), 2.30 (s,
1H, CH), 1.35, 1.25, 1.13, 1.12, 1.00, 0.81 and 0.80 (7 · s,
3H each, CH3); APCI-MS m=z: 582 (100, MHþ), 553
(40, M)CHþ3 ).
5.3.1.21. N-(Pyridin-2-yl-methyl)-3b-hydroxy-11-oxo-
18b-olean-12-en-30-oic acid amide 3b-succinic semiester
(33). To a solution of 21 (122 mg, 0.218 mmol) in pyr-
idine (5 mL) were added succinic anhydride (304 mg,
3.04 mmol) and DMAP (106 mg, 0.88 mmol). The mix-
ture was stirred at 90 ꢁC for 72 h, and poured over 10%
HCl solution, extracted with DCM. The organic phase
was washed brine, dried over MgSO4 and concentrated
in vacuo to afford a residue, which was purified with
flash chromatography. Yellow solid (65 mg, 45%) was
obtained. Mp 152–154 ꢁC; HPLC purity 99% (tR 3.4 min
5.3.1.26.
N-(4-Methylpiperazinyl-1-amino)-3b-hydr-
oxy-18b-olean-12-en-11-one-30-oic acid amide (15). 15:
mp 190–191 ꢁC; HPLC purity >99% (tR 2.45 min in
1
100% MeOH); H NMR (CDCl3, 270 MHz) d 5.71 (s,
1H, olefinic), 3.64 (broad t, 4H, CH2), 3.20–3.10 (broad
m, CH), 2.81–2.71 (broad dt, 1H, 1b-CH), 2.38–2.32
(broad t, 4H, CH2), 2.30 (s, 1H, CH), 1.35, 1.25, 1.13,
1.12, 1.00, 0.81 and 0.80 (7 · s, 3H each, CH3); APCI-
MS m=z: 568 (30, MHþ), 553 (100, M)CHþ3 ).
1
in methanol); H NMR (400 MHz, CDCl3) d 8.50 (dd,
J ¼ 7:9, 2.0 Hz 1H, Ar–H), 7.66 (dt, J ¼ 7:8, 2.0 Hz, 1H,
Ar–H), 7.53 (m, 1H, NH), 7.31 (d, J ¼ 7:8, 1H, Ar–H),

4452
X. Su et al. / Bioorg. Med. Chem. 12 (2004) 4439–4457
5.3.1.27. N-(4-Piperidinylmethyl)-3b-hydroxy-11-oxo-
3.23–3.22 (broad m, 1H, CH), 2.79–2.76 (dt, J ¼ 13:2,
3.1 Hz, 1H, 1b-CH), 2.32 (s, 1H, CH), 1.36, 1.15, 1.13,
1.12 and 1.00 (5 · s, 3H each, 5 · CH3), 0.80 (s, 6H,
2 · CH3); APCI-MS m=z: 561 (100, MHþ), 583 (50,
M+Naþ); FAB-HRMS calcd for C36H52N2O3 (MHþ)
561.4056, found 561.4035.
18b-olean-12-en-30-oic acid amide (16). 16: mp 198–
200 ꢁC HPLC purity >99% (tR 1.68 min in 100%
1
MeOH); H NMR (CDCl3, 400 MHz) d 5.67 (s, 1H,
olefinic), 3.47–3.16 (m, 3H), 2.80–2.72 (broad dt, 1H,
1b-CH), 2.32 (s, 1H, CH), 1.34, 1.20, 1.11, 1.10, 0.98,
0.81 and 0.78 (7 · s, 3H each, CH3); APCI-MS m=z: 547
(70, MHþ).
5.3.1.33. N-(Pyridin-3-yl-methyl)-3b-hydroxy-11-oxo-
18b-olean-12-en-30-oic acid amide (23). 23: mp 250–
251 ꢁC; HPLC purity >99% (tR 1.48 min in 100%
5.3.1.28. N-(Piperidinylethyl)-3b-hydroxy-11-oxo-18b-
olean-12-en-30-oic acid amide (17). 17: HPLC purity 90%
(tR 2.46 min in 100% MeOH); 1H NMR (CDCl3,
400 MHz) d 5.66 (s, 1H, olefinic H), 3.97–3.54 (m, 2H,
CH2), 3.30–3.20 (m, 3H), 2.84–2.73 (m, 1H, CH), 2.34
(s, 1H, CH), 1.39, 1.35, 1.13, 1.12, 1.00 (5 · s, 3H each,
5 · CH3), 0.80 (s, 6H, 2 · CH3); APCI-MS m=z: 581 (85,
MHþ); FAB-HRMS calcd for C37H60N2O3 (MHþ)
581.4682, found 581.4674.
1
MeOH); H NMR (CDCl3, 400 MHz) d 8.57–8.55 and
7.18–7.17 (2 · d, J ¼ 5:8 Hz each, 4H, aromatic); 6.03
(broad t, 1H, NH), 5.62 (s, 1H, olefinic), 4.50–4.46 (m,
2H, benzylic CH2), 3.23 (broad m, 1H, CH), 2.80–2.70
(broad dt, 1H, 1b-CH), 2.33 (s, 1H, CH), 1.37, 1.19,
1.13, 1.00, 0.88, 0.82 and 0.81 (7 · s, 3H each, 7 · CH3);
APCI-MS m=z: 561 (100, MHþ), 583 (30, M+Naþ).
5.3.1.34. N-(Pyridin-2-yl-ethyl)-3b-hydroxy-11-oxo-
18b-olean-12-en-30-oic acid amide (24). 24: mp 150–
153 ꢁC; HPLC purity >99% (tR 1.59 min in 100%
5.3.1.29. N-(Pyrrolindinylethyl)-3b-hydroxy-11-oxo-
18b-olean-12-en-30-oic acid amide (18). 18: mp 156–
159 ꢁC; HPLC purity 95% (tR 2.37 min in 100% MeOH);
1H NMR (CDCl3, 400 MHz) 5.67 (s, 1H, olefinic H),
3.31–3.28 (m, 2H, CH2), 3.13–2.78 (broad s, 1H, b-CH),
2.77–2.73 (m, 1H, CH), 2.34–2.18 (m, 5H), 1.38, 1.21
1.12, 1.11 and 1.00 (5 · s, 3H each, 5 · CH3), 0.80 (s, 6H,
2 · CH3); FAB-MS m=z: 567 (100, Mþ); APCI-MS m=z:
567 (70, MHþ); FAB-HRMS calcd for C36H59N2O3
(MHþ) 567.4525, found 567.4533.
1
MeOH); H NMR (CDCl3, 400 MHz) d 8.57–8.56 (m,
1H, aromatic), 7.65–7.61 (ddd, J ¼ 7:8, 1.5 Hz, 1H,
aromatic), 7.21–7.19 (m, 2H, aromatic), 7.05 (broad t,
1H, NH), 5.63 (s, 1H, oefinic), 3.67–3.73 (m, 2H, CH2),
3.21–3.25 (dd, J ¼ 10:9, 5.8 Hz, CH), 3.02–2.99 (t,
J ¼ 11:7 Hz, 2H, CH2), 2.83–2.79 (dt, J ¼ 10:1, 3.5 Hz,
1H, 1b-CH), 2.34 (s, 1H, CH), 1.37, 1.13, 1.10, 1.07,
1.01, 0.81 and 0.75 (7 · s, 3H each, CH3), APCI-MS m=z:
575 (100, MHþ); FAB-HRMS calcd for C37H54N2O3
(MHþ) 575.4167, found 575.4199.
5.3.1.30.
N-(Pyrrolidinyl)-3b-hydroxy-11-oxo-18b-
olean-12-en-30-oic acid amide (19). 19: mp 191–195 ꢁC;
1
HPLC purity >99% (tR 1.79 min in 100% MeOH); H
5.3.1.35. N-(6-Methoxypyridin-3yl-methyl)-3b-hydr-
oxy-11-oxo-18b-olean-12-en-30-oic acid amide (25). 25:
mp 180–183 ꢁC; HPLC purity >99% (tR 1.63 min in
100% MeOH); 1H NMR (CDCl3, 400 MHz) d 8.11–8.10
(s, 1H, aromatic), 7.88–7.85 (d, J ¼ 8:5 Hz, 1H, aro-
matic), 7.24 (s, 1H, NH), 6.75–6.73 (d, J ¼ 8:5 Hz, 1H,
aromatic), 5.67 (s, 1H, olefinic), 3.91 (s, 3H, OCH3),
3.24–3.04 (broad m, 1H, CH), 2.81–2.76 (dt, J ¼ 13:6,
3.9 Hz, 1H, 1b-CH), 2.34 (s, 1H, CH), 1.61, 1.39, 1.26,
1.12, 1.01, 0.83 and 0.80 (7 · s, 3H each, CH3); APCI-
MS m=z: 577 (40, M)CHþ3 ); FAB-HRMS calcd for
C37H54N2O4 (MHþ) 591.3627, found 591.3606.
NMR (CDCl3, 400 MHz) d 5.68 (s, 1H, olefinic H),
3.25–3.21 (dd, J ¼ 10:5, and 5.4 Hz, 1H, CH–OH),
2.94–2.81 (broad m, 4H), 2.26 (s, 1H, CH), 1.37, 1.23
1.14, 1.13, 1.01, 0.83 and 0.81 (7 · s, 3H each, 7 · CH3);
APCI-MS m=z: 539 (75, MHþ), 580 (100, M+41þ);
FAB-HRMS calcd for C37H60N2O3 (MHþ) 539.4212,
found 539.4209.
5.3.1.31. N-(Pyridin-2-yl-methyl)-3b-hydroxy-11-oxo-
18b-olean-12-en-30-oic acid amide (21). 21: mp 150–
153 ꢁC; HPLC purity >99% (tR 1.48 min in 100%
1
MeOH); H NMR (CDCl3, 400 MHz) d 8.66–8.67 (d,
J ¼ 4:6 Hz, 1H, aromatic), 7.69–7.65 (m, 1H, aromatic),
7.38 (broad t, 1H, NH), 7.25–7.22 (m, 2H, aromatic),
5.89 (s, 1H olefinic), 4.68–4.47 (m, J ¼ 16:7, 5.0 Hz, 1H,
benzylic CH), 4.48–4.43 (m, J ¼ 16:7, 5.0 Hz, 1H, benz-
ylic CH), 3.26–3.22 (dd, J ¼ 10:5, 5.4 Hz, 1H, CH),
2.84–2.79 (dt, J ¼ 13:2, 3.9 Hz, 1H, 1b-CH), 2.37 (s, 1H,
CH), 1.41, 1.19, 1.14, 1.12, 0.88, 0.81 and 0.77 (7 · s, 3H
each, CH3); APCI-MS m=z: 561 (MHþ); FAB-HRMS
calcd for C36H52N2O3 (MHþ) 561.4056, found 561.4045.
5.3.1.36. N-(2,6-Dimethoxypyridin-3yl-methyl)-3b-hy-
droxy-11-oxo-18b-olean-12-en-30-oic acid amide (26). 26:
mp 210–215 ꢁC; HPLC purity >99% (tR 1.63 min in
100% MeOH); 1H NMR (CDCl3, 400 MHz) d 8.46–8.43
(d, J ¼ 8:5 Hz, 1H, aromatic), 7.68 (broad s, 1H, NH),
6.33–6.32 (d, J ¼ 8:5 Hz, 1H, aromatic), 5.78 (s, 1H,
aromatic), 4.08–3.89 (s, 3H, OCH3), 3.50–3.49 (s, 3H,
OCH3), 3.23–3.21 (broad m, 1H, CH), 2.80–2.37 (dt,
J ¼ 13:6, 3.5 Hz, 1H, 1b-CH), 2.32 (s, 1H, CH), 2.20–
2.16 (dd, J ¼ 12:8, 3.5 Hz, 1H, CH), 1.37, 1.26, 1.00,
0.86 and 0.81 (5 · s, 3H each, CH3), 1.13 (s, 6H,
2 · CH3); FAB-MS m=z: 607 (100, MHþ); FAB-HRMS
calcd for C37H54N2O5 (MHþ) 607.4110, found 607.4090.
5.3.1.32. N-(Pyridin-3-yl-methyl)-3b-hydroxy-11-oxo-
18b-olean-12-en-30-oic acid amide (22). 22: mp 236–
237 ꢁC; HPLC purity >99% (tR 1.48 min in 100%
1
MeOH); H NMR (CDCl3, 400 MHz) d 8.54–8.52 (m,
2H, aromatic), 7.63–7.61 (d, J ¼ 7:8 Hz, 1H, aromatic),
7.29–7.28 (m, 1H, aromatic), 6.01 (broad t, 1H, NH),
5.59 (s, 1H,olefinic), 4.55–4.47 (m, 2H, benzylic CH2),
5.3.1.37.
N-(2-Thiazolyl)-3b-hydroxy-11-oxo-18b-
olean-12-en-30-oic acid amide (27). 27: mp 224–227 ꢁC;
HPLC purity >99% (tR 1.91 min in 100% MeOH); H
1

X. Su et al. / Bioorg. Med. Chem. 12 (2004) 4439–4457
4453
NMR (CDCl3, 400 MHz) d 9.24 (1H, s, NH), 7.36 (d,
J ¼ 3:5 Hz, 1H, aromatic), 6.91–6.90 (d, J ¼ 3:5 Hz, 1H,
aromatic), 5.67 (s, 1H, olefinic), 3.16–3.13 (broad m, 1H,
CH), 2.72–2.69 (dt, J ¼ 13:6, 3.9 Hz, 1H, 1b-CH), 2.26
(s, 1H, CH), 1.18, 1.06, 1.05, 1.04, 0.94, 0.74 and 0.73
(7 · s, 3H each, CH3); APCI-MS m=z 553 (25, MHþ),
594 (100, M+CH3CNþ); FAB-HRMS calcd for
C33H48N2O3S (MHþ) 553.3430, found 553.3455.
3,4-dihydro-2H-pyran (250 lL, 2.74 mmol), followed by
p-toluenesulfonic acid (0.1 g). The mixture was stirred at
rt for 6.5 h, poured into 5% sodium bicarbonate, ex-
tracted with DCM. The organic phase was washed with
brine, dried over magnesium sulfate and concentrated to
afford white solid (1.0 g, 95%). HPLC showed the purity
of diastereoisomers 51% and 48%, respectively (tR 23
and 24 min in 10% water–methanol); 1H NMR
(400 MHz, CDCl3) d 5.66 (s, 2H, 2 · 12-H), 4.73 (broad
t, 1H, 20-H of tetrahydropyranyl), 4.58 (dd, J ¼ 4:7,
3.5 Hz, 1H, 20-H of tetrahydropyranyl), 3.95 (m, 2H, 60-
H of tetrahydropyranyl), 3.68 (s, 3H, –OCH3), 3.47 (m,
2H, 60-H of tetrahydropyranyl), 3.25 (dd, J ¼ 11:7,
4.7 Hz, 1H, 3a-H), 3.06 (m, 1H, 3a-H), 2.78 (m, 2H, 1b-
H), 2.33 (s, 2H, 9a-H), 1.36 (s, 3H, CH3), 1.35 (s, 3H,
CH3), 1.15 (s, 12H, 4 · CH3), 1.12 (s, 6H, 2 · CH3), 1.06
(s, 3H, CH3), 0.92 (s, 3H, CH3), 0.83 (s, 3H, CH3), 0.82
(s, 3H, CH3), 0.80 (s, 6H, 2 · CH3); FAB-MS m=z: 569
(80, MHþ); FAB-HRMS calcd for C36H57O5 (MHþ)
569.4206, found 569.4215.
5.3.1.38. 3b-Hydroxy-11-oxo-18b-olean-12-en-30-oic
acid methyl ester (34). To a solution of 1 (15 g,
31.9 mmol) in methanol (200 mL) was added p-toluene-
sulfonic acid (2 g). The mixture was refluxed for 72 h,
concentrated in vacuo, poured into water, extracted with
DCM. The organic phase was washed with 5% sodium
bicarbonate and brine, dried over magnesium sulfate
and concentrated to yield white solid, which was re-
crystallized from methanol. White crystals (14 g, 91%)
1
were obtained. Mp 254–258 ꢁC (lit.25 248–256 ꢁC); H
NMR (400 MHz, CDCl3) d 5.67 (s, 1H, 12-H), 3.70 (s,
3H, –OCH3), 3.23 (m, 1H, 3a-H), 2.80 (dt, J ¼ 13:6,
4.5 Hz, 1H, 1b-H), 2.37 (s, 1H, 9a-H), 1.37 (s, 3H, CH3),
1.15 (s, 3H, CH3), 1.14 (s, 3H, CH3), 1.13 (s, 3H, CH3),
1.01 (s, 3H, CH3), 0.81 (s, 6H, 2 · CH3); FAB-MS m=z:
485 (100, MHþ).
5.3.1.45.
3b-Tetrahydropyran-2-yloxy-11-oxo-18b-
olean-12-en-30-ol (46). To a solution of 45 (900 mg,
1.58 mmol) in THF (50 mL) was added LiAlH4 (500 mg,
13 mmol) in portions. The mixture was stirred at 60 ꢁC
for 20 h, quenched with saturated NH4Cl solution and
extracted with DCM. The organic phase was washed
with brine, dried over magnesium sulfate and concen-
trated to afford an oily residue, which was dissolved in
CHCl3 (100 mL). MnO2 (8 g, 92 mmol) was added. The
suspension was stirred at rt for 8 h, filtered. Evaporation
of the solvent in vacuo gave a brown residue that was
purified with flash chromatography (EtOAc–hexane
gradient). White crystalline solid (500 mg, 58%) was
obtained. HPLC purity 99% (as diastereoisomers, tR
5.3.1.39. 3b-Hydroxy-11-oxo-18b-olean-12-en-30-oic
acid ethyl ester (35). 35 was synthesized with general
method F. Pale yellow crystals (592 mg; 56%) were ob-
tained. Mp 93–94 ꢁC; FAB-MS m=z: 499 (100, MHþ);
FAB-HRMS calcd for C32H51O4 (MHþ) 499.3787,
found 499.3786.
5.3.1.40. 3b-Hydroxy-11-oxo-18b-olean-12-en-30-oic
acid t-butyl ester (36). 36 was synthesized with general
method F. Yellow crystals (201 mg; 18%) were obtained.
Mp 171–174 ꢁC; FAB-MS m=z: 527 (100, MHþ); FAB-
HRMS calcd for C34H55O4 (MHþ) 527.4100, found
527.4105.
1
7.7 min in 4% water–methanol); HNMR showed the
diastereoisomers in 2:1 ratio (400 MHz, CDCl3) d 5.58
(s, 2H, 2 · 12-H), 4.73 and 4.58 (m, 2H, 20-H of tetra-
hydropyranyl), 3.95 and 3.45 (m, 4H, 60-H of tetrahy-
dropyranyl), 3.50 (m, 4H, –OCH2–), 3.22 and 3.05 (m,
2H, 3a-H), 2.78 (m, 2H, 1b-H), 2.35 (s, 2H, 9a-H), 1.36
and 1.35 (s, 6H, 2 · CH3), 1.15 (s, 6H, 2 · CH3), 1.13 (s,
3H, CH3), 1.06 (s, 3H, CH3), 0.93 (s, 12H, 4 · CH3), 0.92
(s, 6H, 2 · CH3), 0.83 and 0.82 (s, 6H, 2 · CH3); FAB-
MS m=z: 541 (100, MHþ); FAB-HRMS calcd for
C35H57O4 (MHþ) 541.4257, found 541.4279.
5.3.1.41. 3b-Hydroxy-11-oxo-18b-olean-12-en-30-oic
acid cyclohexylmethyl ester (37). 37 was synthesized
with general method F. White crystals (703 mg; 59%)
were obtained. Mp 98–99 ꢁC; FAB-MS m=z: 567 (100,
MHþ); FAB-HRMS calcd for C37H59O4 (MHþ)
567.4413, found 567.4409.
5.3.1.42. 3b-Hydroxy-11-oxo-18b-olean-12-en-30-oic
acid benzyl ester (38). 38 was synthesized with general
method F. White solid (680 mg; 57%) were obtained.
Mp 125–126 ꢁC; FAB-MS m=z: 561 (100, MHþ); FAB-
HRMS calcd for C37H53O4 (MHþ) 561.3944, found.
561.3933.
5.3.1.46.
3b,30-Dihydroxy-18b-olean-12-en-11-one
30-m-methoxybenzyl ether (40). To a solution of 46
(60 mg, 0.11 mmol) in THF (5 mL) was added 3-meth-
oxybenzyl chloride (87 mg, 0.56 mmol), followed by
NaH (60% dispersion, 22 mg, 0.56 mmol). The mixture
was stirred at 60 ꢁC under nitrogen for 3 h, quenched
with water and extracted with DCM. The organic phase
was washed with brine, dried over magnesium sulfate
and concentrated to afford white solid, which was dis-
solved in CH3OH (6 mL). Pyridium p-toluenesulfonate
(50 mg) was added. The mixture was stirred at 50 ꢁC for
7 h, concentrated in vacuo gave white solid that was
purified with flash chromatography (EtOAc–hexane
gradient). White powder (28 mg, 44%) was obtained;
HPLC purity 99% (tR 8.1 min in 4% water–methanol);
5.3.1.43. 11-Oxo-18b-olean-12-ene-3b,30-diol (39).
39 was synthesized by the literature28 method. Mp
275–277 ꢁC (lit.28 274–277 ꢁC); HPLC purity 99% (tR
7.6 min in 15% water–methanol).
5.3.1.44.
3b-Tetrahydropyran-2-yloxy-11-oxo-18b-
olean-12-en-30-oic acid methyl ester (45). To a solution
of 34 (900 mg, 1.86 mmol) in DCM (10 mL) was added

4454
X. Su et al. / Bioorg. Med. Chem. 12 (2004) 4439–4457
1
1H NMR (400 MHz, CDCl3) d 6.92 (broad, 1H, Ar–H),
6.91 (broad, 1H, Ar–H), 6.89 (broad, 1H, Ar–H), 6.82
(dd, J ¼ 7:5, 2.8 Hz, 1H, Ar–H), 5.56 (s, 1H, 12-H), 4.49
(s, 2H, –CH2Ph), 3.82 (s, 3H, –OCH3), 3.28(AB,
J ¼ 25 Hz, 2H, 30-OCH2–), 3.23 (m, 1H, 3a-H), 2.79
(dt, J ¼ 13:6, 3.6 Hz, 1H, 1b-H), 2.34 (s, 1H, 9a-H), 1.37
(s, 3H, CH3), 1.14 (s, 3H, CH3), 1.12 (s, 3H, CH3), 1.01
(s, 3H, CH3), 0.97 (s, 3H, CH3), 0.81 (s, 6H, 2 · CH3);
FAB-MS m=z: 577 (85, MHþ); FAB-HRMS calcd for
C38H57O4 (MHþ) 577.4257, found 577.4275.
methanol); H NMR (400 MHz, CDCl3) d 5.57 (s, 1H,
12-H), 3.73 (m, 2H, –OCH2–), 3.56 (m, 2H, –CH2O–),
3.40 (m, 2H, 30-OCH2–), 3.23 (m, 1H, 3a-H), 2.79 (dt,
J ¼ 13:6, 3.5 Hz, 1H, 1b-H), 2.34 (s, 1H, 9a-H), 1.38 (s,
3H, CH3), 1.14 (s, 3H, CH3), 1.13 (s, 3H, CH3), 1.01 (s,
3H, CH3), 0.95 (s, 3H, CH3), 0.87 (s, 3H, CH3), 0.81 (s,
3H, CH3); FAB-MS m=z: 471 (100, MHþ); FAB-HRMS
calcd for C32H53O4 (MHþ) 501.3944, found 501.3928.
The following compounds (47–51, 55–57) were synthe-
sized with general method D.
5.3.1.47.
3b,30-Dihydroxy-18b-olean-12-en-11-one
30-methyl ether (41). Starting from 46 (95 mg,
0.18 mmol) and CH3I (50 lL, 0.80 mmol), 41 was syn-
thesized as described for 40. White crystalline solid
(65 mg, 78%) was obtained. Mp 224–227 ꢁC; HPLC
purity 99% (tR 4.3 min in methanol); 1H NMR
(400 MHz, CDCl3) d 5.59 (s, 1H, 12-H), 3.34 (s, 3H, –
OCH3), 3.23 (m, 1H, 3a-H), 3.20 (AB, J ¼ 25 Hz, 2H,
30-OCH2–), 2.79 (dt, J ¼ 13:7, 3.5 Hz, 1H, 1b-H), 2.34
(s, 1H, 9a-H), 1.37 (s, 3H, CH3), 1.14 (s, 3H, CH3), 1.13
(s, 3H, CH3), 1.01 (s, 3H, CH3), 0.92 (s, 3H, CH3), 0.86
(s, 3H, CH3), 0.81 (s, 3H, CH3); FAB-MS m=z: 471 (100,
MHþ); FAB-HRMS calcd for C31H51O3 (MHþ)
471.3838, found 471.3826.
5.3.1.50. 3b-Methoxy-18b-olean-12-en-11-one-30-oic
acid methyl ester (47). 47: mp P 300 ꢁC; HPLC purity
1
>95% (tR 5.63 min in 100% MeOH); H NMR (CDCl3,
400 MHz) d 5.67 (s, 1H, olefinic), 3.69 (s, 3H, CH3), 3.36
(s, 3H, CH3), 2.95–2.81 (dt, J ¼ 13:2, 3.1 Hz, 1H, 1b-
CH), 2.70–2.66 (dd, J ¼ 11:7, 4.6 Hz, 1H, CH), 2.33 (s,
1H, CH), 2.09–1.66 (m, 6H), 1.36, 1.15, 1.14, 1.12, 0.99,
0.81 and 0.79 (7 · s, 3H each, CH3); FAB-MS m=z: 499
(100, MHþ); FAB-HRMS calcd for C32H50O2 (MHþ)
499.3787, found 499.3801.
5.3.1.51.
3b-Ethoxy-18b-olean-12-en-11-one-30-oic
acid methyl ester (48). 48: HPLC purity >90% (tR
2.59 min in 100% MeOH); H NMR (CDCl3, 400 MHz)
1
d 5.66 (s, 1H, olefinic), 4.47–4.17 (m, 2H, CH2), 3.68 (s,
3H, CH3), 2.84–2.81 (dt, J ¼ 13:6, 3.5 Hz, 1H, 1b-CH),
2.35 (s, 1H, CH), 2.09–2.01 (m, 1H, 18b-CH), 1.36,
1.157, 1.150 and 1.12, 0.95, 0.90 and 0.80 (7 · s, 3H each,
CH3); FAB-MS m=z: 513 (30, MHþ), 557 (80,
M+CH3CN)þ ; FAB-HRMS calcd for C33H52O4 (MHþ)
513.3943, found 513.3941.
5.3.1.48.
3b,30-Dihydroxy-18b-olean-12-en-11-one
30-chloroacetyl ester (42). Starting from 46 (60 mg,
0.11 mmol) and chloroacetic acid ethyl ester (60 lL,
0.56 mmol), 42 was synthesized as described for 40.
White solid (30 mg, 52%) was obtained. Mp 178–179 ꢁC;
HPLC purity 99% (tR 5.2 min in 4% water–methanol);
1H NMR (400 MHz, CDCl3) 5.58 (s, 1H, 12-H), 4.12 (s,
2H, –CH2O–), 4.08 (s, 2H, –CH2Cl–), 3.23 (m, 1H, 3a-
H), 2.79 (dt, J ¼ 13:2, 3.4 Hz, 1H, 1b-H), 2.33 (s, 1H,
9a-H), 1.38 (s, 3H, CH3), 1.14 (s, 3H, CH3), 1.13 (s, 3H,
CH3), 1.01 (s, 3H, CH3), 0.96 (s, 3H, CH3), 0.88 (s, 3H,
CH3), 0.81 (s, 3H, CH3); FAB-MS m=z: 533 (100, MHþ);
FAB-HRMS calcd for C32H50ClO4 (MHþ) 533.3398,
found 533.3389.
5.3.1.52. 3b-Isobutyloxy-18b-olean-12-en-11-one-30-
oic acid methyl ester (49). 49: mp 298–300 ꢁC; HPLC
1
purity >99% (tR 4.97 min in 100% MeOH); H NMR
(CDCl3, 400 MHz) d 5.59 (s, 1H, olefinic), 3.61 (s, 3H,
CH3), 3.35–3.32 (dd, J ¼ 8:9, 6.2 Hz, CH), 2.90–2.86
(dd, J ¼ 8:9, 6.2 Hz, CH), 2.75–2.69 (dt, J ¼ 13:2,
3.5 Hz, 1H, 1b-CH), 2.68–2.64 (dd, J ¼ 12:1, 4.6 Hz, 1H,
CH), 2.25 (s, 1H, CH), 1.48 (s, 6H, 2 · CH3), 1.29, 1.071,
1.05,0.91 (4 · s, 3H each, CH3), 0.86–0.84 (d, J ¼ 5:8 Hz,
3H, CH3), 0.82–0.80 (d, J ¼ 6:6 Hz, 3H, CH3), 0.73 (s,
6H, 2 · CH3); FAB-MS m=z: 527 (100, MHþ); FAB-
HRMS calcd for C37H52O4 (MHþ) 527.3736, found
527.3734.
5.3.1.49.
3b,30-Dihydroxy-18b-olean-12-en-11-one
30-(2-hydroxyethyl) ether (43). To a solution of 46
(100 mg, 0.19 mmol) in toluene (6 mL) was added NaH
(60% dispersion, 42 mg, 1.05 mmol). After stirring at
50 ꢁC for 10 min, 2-(2-bromoethoxy)-tetrahydro-pyran
(200 lL, 1.32 mmol) was added. The mixture was stirred
at 90 ꢁC under nitrogen for 8 h. More NaH (30 mg,
0.75 mmol) and 2-(2-bromo-ethoxy)-tetrahydro-pyran
(100 lL, 0.66 mmol) were added. The solution was stir-
red at 100 ꢁC for 24 h, quenched with water and ex-
tracted with DCM. The organic phase was washed with
brine, dried over magnesium sulfate and concentrated in
vacuo to afford an oily residue that was dissolved in
CH3OH (10 mL). Pyridium p-toluenesulfonate (50 mg)
was added to the solution. The mixture was stirred at
50 ꢁC for 7 h, diluted with 5% NaHCO3 solution, ex-
tracted with DCM. The organic phase was washed with
brine, dried and concentrated in vacuo gave a yellow
residue that was purified with flash chromatography.
Off-white solid (37 mg, 39%) was obtained. Mp 208–
211 ꢁC; HPLC purity 98% (tR 3.5 min in 4% water–
5.3.1.53. 3b-(2-Methoxyethoxy)-18b-olean-12-en-11-
one-30-oic acid methyl ester (50). 50: HPLC purity
1
>90% (tR 4.82 min in 100% MeOH); H NMR (CDCl3,
400 MHz) d 5.66 (s, 1H, olefinic), 4.23–4.09 (m, 1H,
CH), 3.78–3.73 (m, 1H, CH), 3.69 (s, 3H, OCH3), 3.58–
3.44 (m, 2H, CH2), 3.38 (s, 3H, OCH3), 2.83–2.78 (m,
2H, 2 · CH), 2.33 (s, 1H, CH), 1.36, 1.15, 1.14, 1.12,
1.00, 0.81 and 0.80 (7 · s, 3H each, CH3); FAB-MS m=z:
543 (90, MHþ); FAB-HRMS calcd for C34H54O5 (MHþ)
543.4049, found 543.4035.
5.3.1.54. 3b-(2-Hydroxyethoxy)-18b-olean-12-en-11-
one-30-oic acid methyl ester (51). 51: mp P 300 ꢁC;
1
HPLC purity >99% (tR 4.82 min in 100% MeOH); H

X. Su et al. / Bioorg. Med. Chem. 12 (2004) 4439–4457
4455
NMR (CDCl3, 400 MHz) d 5.66 (s, 1H, olefinic), 3.74–
3.70 (broad m, 2H, CH2), 3.69 (s, 3H, OCH3), 3.43–3.38
(m, 2H, CH2), 2.87–2.80 (m, 2H, 2 · CH), 2.33 (s, 3H,
CH), 1.61, 1.36, 1.15, 1.12, 1.00, 0.81 and 0.80 (7 · s, 3H
each, CH3); FAB-MS m=z: 529 (55, MHþ), 467 (30, [M-
61þ]); FAB-HRMS calcd for C33H52O5 (MHþ)
529.3893, found 529.3873.
5.3.1.59. 3b-Ethyloxy-18b-olean-12-en 11-one 30-oic
acid (53). 53: mp 284 ꢁC (dec.); HPLC purity >95% (tR
1
2.59 min in 100% MeOH); H NMR (CDCl3) d 5.70 (s,
1H, olefinic), 3.70–3.64 (m, 1H, CH), 3.38–3.31 (m, 1H,
CH), 2.82–2.75 (m, 2H, 2 · CH), 2.34 (s, 1H, CH), 2.19–
2.03 (m, 1H, 18b-CH), 1.37, 1.23, 1.14, 1.13, 0.98, 0.84
and 0.80 (7 · s, 3H each, CH3), 1.19–1.65 (t, J ¼ 13:6,
3H, CH3); FAB-MS m=z: 499 (80, MHþ), 453 (40, M-
45)þ, FAB-HRMS calcd for C32H50O4 (MHþ) 499.3787,
found 499.3772.
5.3.1.55. 3b-(4-t-Butylbenzyloxy)-18b-olean-12-en-11-
one-30-oic acid methyl ester (55). 55: mp 234–236 ꢁC;
1
HPLC purity >95% (tR 4.97 min in 100% MeOH); H
5.3.1.60. 3b-Isobutyloxy-18b-olean-12-en-11-one-30-
oic acid (54). 54: mp 238 ꢁC (dec.); HPLC purity >95%
(tR 5.14 min in 100% MeOH); 1H NMR (CDCl3,
400 MHz) d 5.70 (s, 1H, olefinic), 3.43–3.39 (dd, J ¼ 8:9,
6.2 Hz, 1H, CH), 2.98–2.94 (dd, J ¼ 8:9, 7.4 Hz, 1H),
2.81–2.75 (dt, J ¼ 13:7, 3.4 Hz, 1H, 1b-CH), 2.75–2.71
(dd, J ¼ 12:1, 4.6 Hz, 1H, CH), 2.33 (s, 1H, CH), 1.37,
1.23, 1.14, 1.13, 0.99, 0.83, 0.81 (7 · s, 3H each, CH3),
0.93–0.88 (2 · d, J ¼ 6:6 Hz each, 2 · CH3); FAB-MS
m=z: 527 (100, MHþ); FAB-HRMS calcd for C34H54O4
[MHþ] 527.4100, found 527.4090.
NMR (CDCl3, 400 MHz) d 7.41–7.28 (2 · d, J ¼ 8:5 Hz
each), 5.66 (s, 1H, olefinic), 4.66–4.63 (d, 1H,
J ¼ 11:7 Hz, benzylic CH), 4.40–4.37 (d, 1H,
J ¼ 11:7 Hz, benzylic CH) 3.69 (s, 3H, CH3), 2.96–2.92
(dd, J ¼ 11:3, 4.2 Hz, 1H, CH), 2.84–2.80 (dt, J ¼ 13:2,
3.5 Hz, 1H, 1b-CH), 2.33 (s, 1H, CH), 2.09–2.08 (m, 18-
bCH,), 1.315 (s, 9H, tert-butyl), 1.360, 1.154, 1.150,
1.12, 1.01, 0.86 and 0.80 (7 · s, 3H each, CH3); FAB-MS
m=z: 631 (40, MHþ); FAB-HRMS calcd for C42H62O4
(MHþ) 632.4759, found 632.4771.
5.3.1.56. 3b-(3-Methoxybenzyloxy)-18b-olean-12-en-
11-one-30-oic acid methyl ester (56). 56: mp P 300 ꢁC;
5.3.1.61.
3,11-Dioxo-18b-olean-12-en-30-oic
acid
(59). Jones reagent (1 mL) was added to a solution of
1 (100 mg; 0.21 mmol) in acetone (5 mL). The reaction
mixture was stirred for 0.5 h at 0 ꢁC, poured into ice-
water and extracted with CHCl3 (50 mL). The organic
phase was washed with water (3 · 50 mL), dried
(MgSO4) and evaporated to get a white solid (108 mg).
The crude product was purified by recrystallization from
hot absolute ethanol to give 59 as white crystals (82 mg;
82%). Mp 302–303 ꢁC (lit.23 mp 298–302 ꢁC); FAB-MS
m=z: 469 (100, MHþ); FAB-HRMS calcd for C30H45O4
(MHþ) 469.3318, found 469.3322.
1
HPLC purity >99% (tR 5.03 min in 100% MeOH); H
NMR (CDCl3, 400 MHz) d 7.24–7.22 (m, 1H, aromatic),
6.94 (appd, 1H, aromatic), 6.92 (s, 1H, aromatic), 6.82–
6.80 (m, 1H, aromatic), 5.66 (s, 1H, olefinic), 4.68–4.65
(d, J ¼ 12:1 Hz, 1H, benzylic CH), 4.42–4.39 (d,
J ¼ 12:1, 1H, benzylic CH), 3.81 (s, 3H, CH3), 3.69 (s,
3H, CH3), 2.96–2.92 (dd, J ¼ 11:7, 4.2 Hz, 1H, CH),
2.84–2.80 (dt, J ¼ 13:2, 3.1 Hz, 1H, 1b-CH), 2.33 (s, 1H,
CH), 1.36, 1.16, 1.15, 1.13, 1.01, 0.87 and 0.81 (7 · s, 3H
each, CH3), FAB-MS m=z: 605 (100, MHþ); FAB-
HRMS calcd for C39H56O5 (MHþ) 605.4222, found
605.4206.
5.3.1.62.
3,11-Dioxo-18b-olean-12-en-30-oic
acid
methyl ester (60). Starting from 34, 60 was synthesized
as described for the preparation of 59. White crystals
(59 mg; 59%) was obtained. Mp 249–250 ꢁC (lit.30 mp
254–256 ꢁC); FAB-MS m=z: 483 (100, MHþ); FAB-
HRMS calcd for C31H47O4 (MHþ) 483.3474, found
483.3472.
5.3.1.57.
3b-Benzyloxy-18b-olean-12-en
11-one
30-oic acid methyl ester (57). 57: mp 302–304 ꢁC;
HPLC purity >95% (tR 4.97 min in 100% MeOH); H
1
NMR (CDCl3, 400 MHz) d 7.36–7.30 (m, 5H, aromatic),
5.66 (s, 1H, olefinic), 4.70–4.67 (d, J ¼ 12:1 Hz, 1H,
benzylic CH), 4.42–4.39 (d, J ¼ 11:7 Hz, 1H, benzylic
CH), 3.69 (s, 3H, CH3), 2.95–2.91 (dd, J ¼ 11:7, 4.2 Hz,
1H, CH), 2.84–2.80 (dt, J ¼ 13:6, 3.5 Hz, 1H, 1b-CH),
2.32 (s, 1H, CH), 2.09–1.66 (m, 6H), 1.35, 1.15, 1.14,
1.12, 1.00, 0.86 and 0.80 (7 · s, 3H each, CH3); FAB-MS
m=z: 575 (30, MHþ); FAB-HRMS calcd for C37H52O4
(MHþ) 575.4055, found 575.4106.
5.3.1.63.
3b,30-Dihydroxy-18b-olean-12-en-11-one
30-methyl ether 3b-succinic semiester (61). Starting
from 41 (25 mg, 0.05 mmol) and succinic anhydride
(27 mg, 0.27 mmol), 61 was synthesized as described for
33. Off-white solid (17 mg, 60%) was obtained. Mp 237–
239 ꢁC; HPLC purity 99% (tR 2.5 min in 2% water–
1
methanol); H NMR (400 MHz, CDCl3) d 5.58 (s, 1H,
The following compounds (52–53) were synthesized with
general method E.
12-H), 4.55 (dd, J ¼ 11:7, 4.7 Hz, 1H, 3a-H), 3.33 (s,
3H, –OCH3), 3.22 (AB, J ¼ 25 Hz, 2H, 30-OCH2–), 2.78
(dt, J ¼ 13:3, 3.5 Hz, 1H, 1b-H), 2.62–2.72 (m, 4H, –
COCH2CH2CO–), 2.35 (s, 1H, 9a-H), 1.36 (s, 3H, CH3),
1.16 (s, 3H, CH3), 1.13 (s, 6H, 2 · CH3), 0.92 (s, 3H,
CH3), 0.88 (s, 6H, 2 · CH3), 0.86 (s, 3H, CH3); FAB-MS
m=z: 571 (100, MHþ); FAB-HRMS calcd for C35H55O6
(MHþ) 571.3999, found 571.3999.
5.3.1.58.
3b-Methyloxy-18b-olean-12-en
11-one
30-oic acid (52). 52: mp P 300 ꢁC; HPLC purity >95%
(tR 5.63 min in 100% MeOH); 1H NMR (CDCl3,
400 MHz) d 5.62 (s, 1H, olefinic), 3.29 (s, 3H, CH3),
2.95–2.81 (dt, J ¼ 13:2, 3.5 Hz, 1H, 1b-CH), 2.70–2.66
(dd, J ¼ 11:7, 4.2 Hz, 1H, CH), 2.26 (s, 1H, CH), 2.09–
1.66 (m, 6H), 1.29, 1.15, 1.07, 1.05, 0.91, 0.76 and 0.71
(7 · s, 3H each, CH3), FAB-MS 485 (100, MHþ).
5.3.1.64.
18b-Olean-12-ene-3b,30-diol (64).
64
was synthesized with literature29 method. Mp 245–247

4456
X. Su et al. / Bioorg. Med. Chem. 12 (2004) 4439–4457
ꢁC (lit.29 250–251 ꢁC, lit.28 246–250 ꢁC); 1H NMR
(400 MHz, CDCl3) d 5.18 (t, 1H,, J ¼ 3:9 Hz, 12-H),
3.52 (m, 2H, 30-CH2–), 3.22 (dd, J ¼ 11:3, 5.9 Hz, 1H,
3a-H), 1.15 (s, 3H, CH3), 1.00 (s, 3H, CH3), 0.96 (s, 3H,
CH3), 0.94 (s, 3H, CH3), 0.90 (s, 3H, CH3), 0.83 (s, 3H,
CH3), 0.81 (s, 3H, CH3).
tallized from CH3OH–DCM. White solid (80 mg, 82%)
was obtained. Mp 287–290 ꢁC; HPLC purity 95% (tR
1
2.6 min in methanol); H NMR (400 MHz, CDCl3) d
5.10 (s, 1H, 12-H), 3.23 (m, 1H, 3a-H), 2.20 (dt,
J ¼ 12:5, 3.5 Hz, 1H, 1b-H), 1.43 (s, 3H, CH3), 1.35 (s,
3H, CH3), 1.22 (s, 3H, CH3), 1.16 (s, 3H, CH3), 1.07 (s,
3H, CH3), 1.00 (s, 3H, CH3), 0.86 (s, 3H, CH3), 0.83 (s,
3H, CH3); FAB-MS m=z: 486 (10, Mþ), 469
(MHþ)H2O, 100); FAB-HRMS calcd for C31H51O4
(MHþ) 487.3787, found 487.3730.
5.3.1.65. 3b,30-Dihydroxy-30,30-dimethyl-18b-olean-
12-en-11-one (44), 3b, 11b-dihydroxy-11a-methyl-18b-
olean-12-en-30-oic acid methyl ester (65), 3b-hydroxy-
11-methylene-18b-olean-12-en-30-oic acid methyl ester
(66). To a solution of 34 (2.37 g, 4.90 mmol) in THF
(25 mL) was added CH3Li–ether solution (1.6 M,
6.2 mL, 9.92 mmol) dropwise at )78 ꢁC under nitrogen.
The mixture was stirred at )78 ꢁC for 30 min, and then
brought to rt gradually. After stirring at rt for 1 h, the
reaction was quenched with 1% HCl and extracted with
DCM. The organic phase was washed with 5% NaHCO3
solution, brine, dried over magnesium sulfate and con-
centrated to give a white solid that was subjected to flash
chromatography (EtOAc–hexane gradient elution).
Starting material 34 (820 mg) was recovered. 44 was
obtained as white crystals (130 mg, 8%). Mp 241–246 ꢁC;
5.3.1.67. 3b-Hydroxy-11-methylene-18b-olean-12-en-
30-oic acid (68). Method 1: Starting from 66 (150 mg,
0.31 mmol), 68 was synthesized as described for the
preparation of 67. White crystals (122 mg, 84%) were
obtained. Mp 295–296 ꢁC; HPLC purity 99% (tR 3.1 min
in methanol); 1H NMR (400 MHz, CDCl3) d 5.72 (s, 1H,
12-H), 5.06 and 4.98 (s, 2H, 11-CH2), 3.27 (m, 1H, 3a-
H), 2.50 (dt, J ¼ 13:7, 3.5 Hz, 1H, 1b-H), 2.31 (s, 1H,
9a-H), 1.23 (s, 3H, CH3), 1.22 (s, 3H, CH3), 1.21 (s, 3H,
CH3), 1.02 (s, 3H, CH3), 0.96 (s, 3H, CH3), 0.83 (s, 3H,
CH3), 0.82 (s, 3H, CH3); FAB-MS m=z: 469 (100, MHþ);
FAB-HRMS calcd for C31H49O3 (MHþ) 469.3682,
found 469.3629.
1
HPLC purity 99% (tR 2.4 min in methanol); H NMR
(400 MHz, CDCl3) d 5.59 (s, 1H, 12-H), 3.23 (m, 1H, 3a-
H), 2.70 (dt, J ¼ 13:7, 3.5 Hz, 1H, 1b-H), 2.27 (s, 1H,
9a-H), 1.35 (s, 3H, CH3), 1.22 (s, 3H, CH3), 1.21 (s, 3H,
CH3), 1.20 (s, 3H, CH3), 1.14 (s, 3H, CH3), 1.00 (s, 3H,
CH3), 0.95 (s, 3H, CH3), 0.81 (s, 3H, CH3), 0.68 (s, H,
CH3); FAB-MS m=z: 500 (15, Mþ), 483 (MHþ)H2O,
100); FAB-HRMS calcd for C32H53O3 (MHþ) 485.3955,
found 485.4000.
Method 2: To a solution of 1 (0.8 g, 1.7 mmol) in THF
(50 mL) was added CH3Li–ether solution (1.6 M,
6.2 mL, 9.92 mmol) dropwise at rt under nitrogen. The
mixture was stirred at rt for 1 h, and then quenched with
1% HCl and extracted with ethyl acetate. The organic
phase was washed with 5% NaHCO3, brine, dried over
magnesium sulfate and concentrated to give a white
solid that was re-crystallized from CH3OH–DCM.
White crystals (760 mg, 95%) were obtained. Mp 293–
296 ꢁC; 1H NMR was identical with the compound from
method 1.
65 was obtained as white crystals (455 mg, 28%). Mp
251–257 ꢁC; HPLC purity 91% (tR 2.6 min in methanol);
1H NMR (400 MHz, CDCl3) d 5.07 (s, 1H, 12-H), 3.70
(s, 3H, –OCH3), 3.25 (broad, 1H, 3a-H), 2.20 (dt,
J ¼ 12:5, 3.5 Hz, 1H, 1b-H), 1.44 (s, 3H, CH3), 1.35 (s,
3H, CH3), 1.16 (s, 3H, CH3), 1.14 (s, 3H, CH3), 1.06 (s,
3H, CH3), 1.00 (s, 3H, CH3), 0.82 (s, 6H, 2 · CH3);
FAB-MS m=z: 500 (15, Mþ), 483 (MHþ-H2O, 100);
FAB-HRMS calcd for C32H53O4 (MHþ) 501.3944,
found 501.3905.
Acknowledgements
We thank Sterix Ltd for financial support of this work,
Alison Smith for expert technical assistance and Del-
phine Fischer for supplying compound 58.
66 was obtained as white crystals (350 mg, 22%). Mp
168–170 ꢁC; HPLC purity 99% (tR 4.2 min in methanol);
1H NMR (270 MHz, CDCl3) d 5.67 (s, 1H, 12-H), 5.03
and 4.95 (s, 2H, 11-CH2), 3.68 (s, 3H, –OCH3), 3.24 (m,
1H, 3a-H), 2.45 (dt, J ¼ 13:3, 3.5 Hz, 1H, 1b-H), 2.35 (s,
1H, 9a-H), 1.18 (s, 3H, CH3), 1.17 (s, 3H, CH3), 1.11 (s,
3H, CH3), 0.99 (s, 3H, CH3), 0.92 (s, 3H, CH3), 0.80 (s,
3H, CH3), 0.75 (s, 3H, CH3); FAB-MS m=z: 483 (100,
MHþ); FAB-HRMS calcd for C32H51O3 (MHþ)
483.3838, found 483.3838.
References and notes
€
1. Jornvall, H.; Persson, B.; Krook, M.; Atrian, S.; Gonz-
alez-Duarte, R.; Jeffery, J.; Ghosh, D. Biochemistry 1995,
34, 6003–6013.
2. Edwards, C. R.; Stewart, P. M.; Burt, D.; Brett, L.;
McIntyre, M. A.; Sutano, W. S. Lancet 1988, 2, 986–989.
3. Seckl, J. R.; Walker, B. R. Endocrinology 2001, 142, 1371–
1376.
ꢀ
4. Davani, B.; Kahn, A.; Maartensson, E.; Efendic, S.;
Jornvall, H.; Oppermann, U. C. T. J. Biol. Chem. 2000,
€
275, 34841–34844.
5.3.1.66. 3b, 11b-Dihydroxy-11a-methyl-18b-olean-
12-en-30-oic acid (67). To a solution of 65 (100 mg,
0.2 mmol) in methanol–THF–water (5:2:1 mL) was ad-
ded KOH (88 mg, 1.56 mmol). The mixture was stirred
under nitrogen at 70 ꢁC for 48 h, poured into 2% HCl,
extracted with DCM. The organic phase was washed
with brine, dried over magnesium sulfate and concen-
trated in vacuo to give a white solid that was recrys-
5. Kotelevtsev, Y. V.; Brown, R. W.; Fleming, S.; Kenyon,
C.; Edwards, C. R. W.; Seckl, J. R.; Mullins, J. J. J. Clin.
Invest. 1999, 103, 683–689.
6. Alberts, P.; Engblom, L.; Edling, N.; Forsgren, M.;
€
Kilngstrom, G.; Larsson, C.; Ronquist-Nii, Y.; Ohman,
€
B.; Abrahmsen, L. Diabetalogica 2002, 45, 1528–1532.
€
ꢁ
7. Andrews, R. C.; Rooyackers, O.; Walker, B. R. J. Clin.
Endocrinol. Metab. 2003, 88, 285–291.

X. Su et al. / Bioorg. Med. Chem. 12 (2004) 4439–4457
4457
ꢀ
8. Barf, T.; Vallgarda, J.; Emond, R.; Haggstrom, G. K.;
Nygren, A.; Larwood, V.; Mosialou, E.; Axelsson, K.;
€
20. Rabbitt, E. H.; Ayuk, J.; Boelaert, K.; Sheppard, M. C.;
Hewison, M.; Stewart, P. M.; Gittoes, N. J. L. Oncogene
2003, 22, 1663–1667.
21. Sasano, H.; Frost, A. R.; Saitoh, R.; Matsunaga, G.;
Nagura, H.; Krozowski, Z. S.; Silverberg, S. G. Anticancer
Res. 1997, 17, 2001–2007.
22. Rabbitt, E. H.; Lavery, G. C.; Walker, E. A.; Cooper, M.
S.; Stewart, P. M.; Hewison, M. FASEB J. 2002, 16, 36–
44.
23. Rabbitt, E. H.; Gittoes, N. J. L.; Stewart, P. M.; Hewison,
M. J. Steroid. Mol. Biol. 2003, 85, 415–421.
€
Olsson, R.; Engblom, L.; Edling, N.; Ronquist-Nii, Y.;
€
ꢁ
Ohman, B.; Alberts, P.; Abrahmsen, L. J. Med. Chem.
2002, 45, 3813–3815.
9. Sandeep, T. C.; Walker, B. R. Trends Endocrinol. Metab.
2001, 12, 446–453.
10. Kotelevtsev, Y. V.; Holmes, M. C.; Burchell, A.; Houston,
P. M.; Schmoll, D.; Jamieson, P.; Best, R.; Brown, R.;
Edwards, C. R. W.; Seckl, J. R.; Mullins, J. J. Proc. Natl.
Acad. Sci. U.S.A. 1997, 94, 14924–14929.
€
11. Masusaki, H.; Paterson, J.; Shinyama, H.; Morton, N. M.;
Mullins, J. J.; Seckl, J. R.; Flier, J. S. Science 2001, 294,
2166–2170.
12. Monder, C.; Stewart, P. M.; Laksmi, V.; Valentino, R.;
Burt, D.; Edwards, C. R. W. Endocrinology 1989, 125,
1046–1053.
24. Hult, M.; Jornvall, H.; Oppermann, U. C. T. FEBS Lett.
1998, 441, 25–28.
25. Guo, J.; Reidenberg, M. M. J. Lab. Clin. Med. 1998, 132,
32–38.
26. Potter, B. V. L.; Purohit, A.; Reed, M. J.; Vicker, N. WO
02072084, 2002.
13. Diederich, S.; Grossmann, C.; Hanke, B.; Quinkler, M.;
27. Sheehan, J. C.; Ledis, S. L. J. Am. Chem. Soc. 1973, 95,
875–879.
28. Terasawa, T.; Okada, T.; Nishino, H. Eur. J. Med. Chem.
1992, 27, 689–692.
29. Shibata, S.; Takahashi, K.; Yano, S.; Harada, M.; Saito,
H.; Tamura, Y.; Kumagai, A.; Hirabayashi, K.; Yama-
moto, M.; Nagata, N. Chem. Pharm. Bull. 1987, 35, 1910–
1918.
30. Terasawa, T.; Okada, T.; Hara, T.; Itoh, I. Eur. J. Med.
Chem. 1992, 27, 351–354.
31. Sakano, K.; Ohshima, M. Agric. Biol. Chem. 1986, 50,
763–766.
32. Shimoyama, Y.; Hirabayashi, K.; Matsumoto, H.; Sato,
T.; Shibata, S.; Inoue, H. J. Pharm. Pharmacol. 2003, 55,
811–817.
33. Bradford, M. M. Anal. Biochem. 1976, 72, 248–
254.
34. Vicker, N.; Su, X.; Lawrence, H.; Cruttenden, A.; Purohit,
A.; Reed, M. J.; Potter, B. V. L. Bioorg. Med. Chem. Lett.,
2004, 14, 3263–3267.
€
Herrmann, M.; Bahr, V.; Oelkers, W. Eur. J. Endocrinol.
2000, 142, 200–207.
14. Stewart, P. M.; Valentino, R.; Wallace, A. M.; Burt, D.;
Shackleton, C. H. L.; Edwards, C. R. W. Lancet 1987, 2,
281–284.
15. Stewart, P. M.; Wallace, A. M.; Atherton, S. M.; Shearing,
C. H.; Edwards, C. R. W. Clin. Sci. 1990, 78, 49–54.
16. Dave-Sharma, S.; Wilson, R. C.; Harbison, M. D.;
Newfield, R.; Azar, M. R.; Krozowski, M. R.; Funder,
J. W.; Shackleton, C. L.; Bradlow, H. L.; Wei, J.-Q.;
Hertecant, J.; Moran, A.; Neiberger, R. E.; Balfe, J. W.;
Fattah, A.; Daneman, D.; Akkurt, H. I.; de Santis, C. J.
Clin. Endocrinol. Metab. 1998, 83, 2244–2254.
17. Walker, E. A.; Stewart, P. M. Trends Endocrinol. Metab.
2003, 14, 334–339.
18. Coulter, C. L.; Smith, R. E.; Stowaser, M.; Sasano, H.;
Krozowski, Z. S.; Gordon, R. D. Endocr. Res. 1998, 24,
875–876.
19. Korbonits, M.; Bujalska, I.; Shimojo, M.; Nobes, J.;
Kordan, S.; Grossman, A. B.; Stewart, P. M. J. Clin.
Endocrinol. Metab. 2001, 86, 2728–2733.
35. Arnold, P.; Tam, S.; Yan, L.; Baker, M. E.; Frey, J. F.;
Odermatt, A. Mol. Cell. Endocrinol. 2003, 201, 177–187.