Discovery and structure-activity relationships of ent-Kaurene diterpenoids as potent and selective 11β-HSD1 inhibitors: Potential impact in diabetes

Article abstract of DOI:10.1016/j.ejmech.2013.05.010

The biological screening of a collection of nature occurring diterpenoids against 11β-HSD1 resulted in the discovery of the lead compound 1b, which pointed to the therapeutic potential for type 2 diabetes. Subsequently, an optimization project was initiated. Starting from compound 1b and its counterpart 2, the hemi-synthesis was performed on kaurenic acid scaffolds yielding 36 derivatives. Further evaluations on both human and mouse 11β-HSD revealed that seven urea derivatives exhibited significant improved potency and selectivity. Especially, the urea 19a has an IC₅₀ (human 11β-HSD1) = 9.4 nM and selectivity index (human 11β-HSD) > 10,649. The 2D and 3D binding models of the complex 19a/11β-HSD1 were generated using docking simulations. Based on the results, the structural-activity relationships (SARs) of compounds 1b and 2 were also discussed.

Full text of DOI:10.1016/j.ejmech.2013.05.010

European Journal of Medicinal Chemistry 65 (2013) 403e414
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Discovery and structureeactivity relationships of ent-Kaurene
diterpenoids as potent and selective 11
b-HSD1 inhibitors: Potential
impact in diabetes
,
,
Xu Deng a, Yu Shen b, Jing Yang a b, Juan He a, Yu Zhao a, Li-Yan Peng a, Ying Leng b
*
,
,
Qin-Shi Zhao a
*
a State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650204,
China
b Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China
a r t i c l e i n f o
a b s t r a c t
Article history:
The biological screening of a collection of nature occurring diterpenoids against 11b-HSD1 resulted in the
Received 26 December 2012
Received in revised form
8 May 2013
Accepted 9 May 2013
Available online 18 May 2013
discovery of the lead compound 1b, which pointed to the therapeutic potential for type 2 diabetes.
Subsequently, an optimization project was initiated. Starting from compound 1b and its counterpart 2,
the hemi-synthesis was performed on kaurenic acid scaffolds yielding 36 derivatives. Further evaluations
on both human and mouse 11
potency and selectivity. Especially, the urea 19a has an IC50 (human 11
index (human 11 -HSD) > 10,649. The 2D and 3D binding models of the complex 19a/11
b
-HSD revealed that seven urea derivatives exhibited significant improved
-HSD1) ¼ 9.4 nM and selectivity
-HSD1 were
b
b
b
Keywords:
ent-Kaurene diterpenoids
Structureeactivity relationship studies
generated using docking simulations. Based on the results, the structuraleactivity relationships (SARs) of
compounds 1b and 2 were also discussed.
Ó 2013 Elsevier Masson SAS. All rights reserved.
Selective 11b-HSD1 inhibitors
Urea derivatives
Diabetes
1. Introduction
important regulators of glucose and lipid homeostasis [4]. Elevated
level of glucocorticoids can lead to insulin resistance by decreasing
The prevalence of type 2 diabetes and obesity is one of the main
public health threats in 21st century. It is estimated that more than
150 million people worldwide suffer from diabetes. And the num-
ber is expected to rise to 300 million by the year of 2025 [1].
However, the medicines for treatment of type 2 diabetes are far
from sufficient. So the development of novel agents with good
therapeutic index and low side effects is still in urgent demand [2].
Metabolic syndrome is a pre-diabetic state, which features with
abdominal obesity, impaired glucose tolerance, dyslipidemia, low
levels of high density lipoprotein (HDL), and hypertension [3].
When metabolic syndrome progresses to diabetes, complications
associating with this disorder, including cardiovascular disease,
kidney failure and diabetic retinopathy, become prominent. Recent
investigations revealed that aberrant glucocorticoid receptor (GR)
signaling was closely associated with metabolic syndrome. Gluco-
corticoid hormones, including cortisone and cortisol in human, are
insulin dependent glucose uptake, enhancing hepatic gluconeo-
genesis, and inhibiting insulin secretion from pancreatic cells. Pa-
tients with sustained glucocorticoid excess will develop
dyslipidemia, visceral obesity, and other metabolic syndromes [5].
11
inter-conversion of the glucocorticoids, cortisone and cortisol, in
human (Fig. 1) [6]. 11 -HSD has two isoforms, 11 -HSD1 and 11
HSD2. 11 -HSD1, which is primarily found in liver, adipose and
brain, converses the inactive cortisone to the active cortisol. Its
counterpart 11 -HSD2, which is mainly expressed in kidney, cata-
lyzes the reverse conversion. Both 11 -HSD1 and 11 -HSD2 are
involved in maintenance of the balance of glucocorticoid hor-
mones. Evidences from homozygous 11 -HSD1 knock-out mice
model revealed that impaired function of 11 -HSD1 could result in
reducing of gluconeogenesis and lipophilia in liver, and increasing
of insulin sensitivity. However, inhibition of 11 -HSD2 would lead
to sodium retention, hypokalemia and hypertension [7,8]. There-
fore, selective inhibition of 11 -HSD1 will be a therapeutic strategy
to combat type 2 diabetes and obesity [9e11].
b-hydroxysteroid dehydrogenase (11b-HSD) catalyzes the
b
b
b-
b
b
b
b
b
b
b
b
* Corresponding authors.
E-mail address: qinshizhao@mail.kib.ac.cn (Q.-S. Zhao).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.05.010

404
X. Deng et al. / European Journal of Medicinal Chemistry 65 (2013) 403e414
compounds 1b and 2 were chosen as the starting materials for
structural modifications (Fig. 3).
3. Chemistry
Starting from compounds 1b and 2, thirty-six structurally
diverse derivatives varying on the positions C-11, C-15, C-16(17),
and C-19 were designed and synthesized for the SARs studies.
Generally, the derivatives were designed on basis of bioisosteric
replacement, functionality “knock-out” strategies combining with
chemical accessibility. In medicinal chemistry, bioisosteric
replacement is a frequently used strategy in lead optimization in
order to gain the desired biological or physical properties of a
compound. On the other hand, functionality “knock-out” is also a
common protocol to clearly state the contributions of the structural
motifs to the biological activities.
Fig. 1. Inter-conversion of cortisone and cortisol.
As a result,11
medicinal chemistry community over the past few years [12e14]. A
number of potent and selective 11 -HSD1 inhibitors was reported up
b-HSD1 has attracted considerable attention among
b
to now, some of which are progressing to clinical trial (Fig. 2) [15].
However, most of the inhibitors are synthetic chemicals [12]. Nature-
derived inhibitors were still scarce in literatures. So unearthing the
selective11b-HSD1inhibitorsfromthe inexhaustible naturalproduct
3.1. Modifications on C-11 position
reservoir will be a promising project.
Herein, we presented the discovery of the ent-kaurene diter-
penoids 1b and 2 as selective 11b-HSD1 inhibitors along with their
The investigations on C-11 concerned modifications of the hy-
droxyl group into ester, ketone, alkene, and so on (Scheme 1). The
C-11 hydroxyl group of compound 2 was selectively esterified by
deprotonation with NaH and subsequent nucleophilic addition
with CS2 followed by trapping with MeI, affording compound 3 in
moderate yield. Mitsunobu reaction of compound 2 with diethyl
azodicarboxylate (DEAD) in THF led to the alkene 4 in good yield
[16]. Treatment of compound 2 with the acyl isothiocyanate in the
presence of base delivered a series of C-11 mono-esters, 5ae5d.
Moreover, two C-11 carbamic acid esters, 6a and 6b, were also
prepared in good yield by reacting with triphosgene and amines
(Scheme 1) [17,18].
structureeactivity relationship studies. Starting from the leads 1b
and 2, thirty-six derivatives were designed and synthesized. The
representatives were C-4 urea substituted analogs, which were
accessible through Curtius rearrangement and subsequent nucle-
ophilic addition. Enzymatic evaluations revealed that seven urea
derivatives exhibited potent human 11
b
-HSD1 inhibitory activity
and improved selectivity over human 11
b-HSD2.
2. Discovery of the lead compounds
As a part of our efforts to identify novel drug candidates for type
2 diabetes, we discovered a novel selective 11 -HSD1 inhibitor,
b
3.2. Modifications on C-15, C-16,17 positions
kaur-16-en-19-oic acid (1b), by screening a collection of natural
products (Table 1). As shown in Table 1, compound 1b, with IC50
(mouse) ¼ 357 nM and selectivity index (mouse) ¼ 1400, has su-
perior activity and selectivity over the other counterparts.
To demonstrate the contributions of C-15 alcohol and C-16,17
alkene to the activity, a class of regio-selective reactions was also
performed on these motifs (Scheme 2).
However, several obstacles, including poor water solubility, lay
ahead of compound 1b on its way to a drug candidate. Therefore, it
is important to initiate an optimization project to circumvent these
problems. The limited amount of compound 1b in plant had once
Using compound 1b as the substrate, the C-16,17 alkene was
oxidized to its corresponding bioisostere ketone 7, under K2OsO4e
NaIO4 reaction system [19]. Reaction of compound 1b with SeO2
provided the allylic alcohol 8a in moderate yield [20,21]. The
structure of compound 8a was assigned by comparing the spectra
data with the literature [22]. The alcohol 8a was further trans-
formed to its fluoride derivative 8b by treatment with dieth-
ylaminosulfur trifluoride (DAST). Based on the SN2 mechanism of
the reaction concerning DAST, the C-15 stereochemistry of com-
pound 8b was opposite to that of C-15 in compound 8a.
bothered us a lot. Gratifyingly, the easily available 11b,15b-dihy-
droxy-kaur-16-en-19-oic acid (2) can serve as alternative. It is
relatively abundant in Nouelia insignis Franch. Moreover, compound
2 has more functional groups, which facilitates structure modifi-
cations and structureeactivity relationship studies. So both
We also started our efforts toward the modifications of com-
pound 2 on C-15 and C-16,17. The C-16,17 alkene group was hy-
drogenated enantioselectively with Pd/C under H2 to afford the
alkane 9 in excellent yield [23]. The epoxidation reaction of com-
pound 2 with meta-chloroperoxybenzoic acid (m-CPBA) gave the
target 10 as a single diastereoisomer. The high stereo-selectivity of
compounds 9 and 10 is benefited from the directing effect of the
hydroxyl group at position C-15 [23,24]. Treatment of compound 2
with the (2,2,6,6-tetramethylpiperidin-1-yl)oxyl (TEMPO) and
phenyliodine diacetate (PIDA) in the presence of HOAc delivered
the a,b unsaturated ketone 11 in good yield (Scheme 2) [25].
3.3. Modifications on C-19 position
We then turned our hemi-synthesis explorations to the C-19
carboxyl acid, which was a top priority in our schedule.
Again, both compounds 1b and 2 were used as the substrates.
We first attempted to convert the C-19 carboxyl acid to the
Fig. 2. Representative 11b-HSD1 inhibitors.

X. Deng et al. / European Journal of Medicinal Chemistry 65 (2013) 403e414
405
Table 1
Discovery of 11b-HSD1 inhibitors..
Entry
Mouse (IC50
11 -HSD1
1.553
)
Human (IC50
11 -HSD1
)
b
11
b
-HSD2
HSD2/HSD1
b
11
10
b
-HSD2
HSD2/HSD1
1a
1b
1c
m
M
>1 mM
500
>1 mM
>1 mM
ND
>640
1400
>311
>357
ND
1.417
ND
m
M
m
M
7.0
357 nM
m
M
ND
ND
3.216
2.805
m
m
M
M
1.236
m
M
>1 mM
>1 mM
1.22 nM
>809
>1095
0.42
1d
0.91
mM
GAa
40.21 nM
29.1 nM
a
GA: glycyrrhetinic acid was used as a positive control; ND: not determined.
hydroxyl group. Refluxing of compounds 1b and 2 with LiAlH4 in
THF furnished the desired alcohols 12 and 16 in excellent yields.
Further treatment of the alcohol 12 with DAST afforded the fluoride
13 in moderate yield. Reaction of compound 2 with diazomethane
in ether gave the methyl ester 17 quantitatively (Scheme 3).
Inspiring by the fact that the urea constitutes a family of potent
(indicated by dashed lines) between compound 19a and human
11 -HSD1 is stronger than between compound 19a and mouse 11
HSD1, from the 2D interaction scheme (Fig. 4A and B). Besides,
there are two hydrogen bonds between C-15 hydroxyl group and
b
b-
Tyr183 and Ser170 in the complex 19a/mouse 11b-HSD1. And there
ꢀ
is a strong hydrogen interaction with the O/O distance of 2.9 A
selective 11
b
-HSD1 inhibitors [14,26,27], we envisaged to incor-
between C-15 hydroxyl group and the residue Tyr183 in complex
porate the urea scaffold into the core structure. The carboxyl group
was converted to urea moiety in a two-step procedure. Upon
heating compounds 1b and 2 with diphenylphosphoryl azide
(DPPA) in anisole, the Curtius rearrangement proceeded smoothly
to afford intermediates 14 and 18 in excellent yields [28,29]. The C-
4 stereochemistry of derivatives 14 and 18 is still R because Curtius
reaction doesn’t affect the stereochemistry of carbon adjacent to
the carboxyl acid [28]. Subsequent nucleophilic addition of the
isocyanates 14 and 18 with various reagents delivered a diverse set
of urea derivatives 15ae15c in moderate to excellent yields
(Scheme 3).
19a/human 11b-HSD1 (Fig. 4).
4.2. Biological effects of the derivatives
With derivatives secured, we then determined the enzymatic
potency on inhibition of human and mouse 11
HSD2 by scintillation proximity assay (SPA) using microsomes
containing 11 -HSD1 or 11 -HSD2 [30]. Results are reported as the
b-HSD1 and 11b-
b
b
average of at least two independent experiments with at least two
replicates at each concentration.
The preliminary evaluations of the derivatives were performed
at the concentration of 1 mM. Glycyrrhetinic acid was used as the
4. Results and discussions
positive control [31] (Table 2).
The derivatives with inhibitory ratio more than 50% in pre-
liminary tests were further evaluated to calculate the IC50 value and
4.1. Protein based modeling
the selectivity ratio against 11
At the discovery stage, several ent-kaurene diterpenoids were
assessed for their 11 -HSD inhibitory activity and selectivity
b-HSD2 (Table 3).
To gain insight into the interactions between the ligand and the
receptor, the 2D and 3D binding models of the compound 19a to
b
mouse and human 11
the molecular docking (Fig. 4). It’s found that the top ranking
docking score for compound 19a with mouse 11 -HSD1
is ꢀ6.98 kcal/mol, while with human 11 -HSD1 the score
is ꢀ8.60 kcal/mol. These indicate that the binding affinity of com-
pound 19a with human 11 -HSD1 is stronger than that with mouse
11 -HSD1. The results are also in good agreement with the exper-
imental results (IC50 (human 11
-HSD1) ¼ 9.3 nM and IC50 (mouse
11 -HSD1) > 1 M). Additionally, the hydrophobic interaction
b
-HSD1 were generated respectively based on
(Table 1). Structural comparisons between the active and the
inactive demonstrated that the carboxyl group at C-18 (see
compounds 1a and 1b) and the double bond at C-16,17 (see
compounds 1c and 1d) were preferential to the hydroxyl group and
the vicinal diol group respectively for the activity and selectivity.
On basis of the biological results illustrated in Tables 2 and 3, the
structureeactivity relationships of the leads 1b and 2 were dis-
cussed herein.
b
b
b
b
b
b
m
The effects of structural variations at C-11 on the activity and
selectivity are first examined. Eliminating of the C-11 hydroxyl
group to the alkene 4 exerted negligible effects on the inhibition of
both mouse and human 11b-HSD1, indicating that the C-11 hy-
droxyl group is not necessary for the binding. The C-11 ester ana-
logs 3 and 5a showed an impressive improvement of the potency
and selectivity on human 11
that the proper non-polar functionality at C-11 was beneficial to the
activity and selectivity on human 11 -HSD instead of mouse 11
HSD. However, the potency on both mouse and human 11 -HSD1
b-HSD. These observations suggested
b
b-
b
was decreasing with increase in the steric bulk of C-11 (see esters
Fig. 3. Structure of compounds 1b and 2.
5be5d, 6a and 6b).

406
X. Deng et al. / European Journal of Medicinal Chemistry 65 (2013) 403e414
Scheme 1. Modifications on the hydroxyl group at position C-11. Reagents and Conditions: a) NaH, CS2, then MeI, THF, RT; b) DEAD, PPh3, THF, RT; c) RCOSCN, Et3N, THF, 0 ꢁC-RT; d)
Triphosgene, Et3N, R1R2NH, DCM, 0 ꢁC-RT.
Next, the contributions of C-15 scaffold to the biological ac-
tivities were discussed. The C-15 hydroxyl derivative 8a was less
Oxidation of the C-15 hydroxyl group into ketone 11 produced
negligible effects on the activity. These results demonstrated
that C-15 hydroxyl group played a minor role for the binding,
which was in agreement with the molecular docking
predictions.
The ketone analog 7, alkane analog 9, the epoxide analog 10, and
the diol analog 1c were designed to mimic the C-16,17 double bond
and probe the binding characteristics in this region. However,
active than compound 1b on both mouse and human 11
but partially maintained the activity and selectivity on mouse
11 -HSD. Interestingly, the fluoride analog 8b was also poorly
active on both mouse and human 11 -HSD1. These observations
revealed that the functionality combination of these groups was
not tolerated by active site of both mouse and human 11 -HSD1.
b-HSD1,
b
b
b
Scheme 2. Modifications at positions C-15, C-16 and C-17 Reagents and Conditions: a) K2OsO4, NaIO4, 2,6-lutidine, t-BuOH/THF/H2O, RT; b) SeO2, 1,4-dioxane, RT; c) DAST,
DCM, ꢀ78 ꢁC; d) Pd/C, H2, MeOH/EtOAc, RT; e) m-CPBA, NaHCO3, DCM, RT; f) TEMPO, PIDA, HOAc, CH3CN, RT.

X. Deng et al. / European Journal of Medicinal Chemistry 65 (2013) 403e414
407
Scheme 3. Modifications at positions C-18 Reagents and Conditions: a) LiAlH4, THF, reflux; b) DAST, DCM, ꢀ78 ꢁC; c) DPPA, Et3N, anisole, 90 ꢁC; d) R1R2NH, Et3N, THF, RT-reflux; e)
CH2N2, Et2O, 0 ꢁC.
replacements of the substitutes at the C-16,17 double bond by these
bioisosteric manipulations resulted in a drop of potency on both
derivative 19a was 100 folds more active than the parent com-
pound 2 on human 11 -HSD1, and was with excellent selectivity
over human 11 -HSD2 (Table 3), indicating that compound 19a
b
mouse and human 11
b-HSD1, which implied that the double bond
b
was the optimum choice for this region. These results were also
supported by the evidence that the C-16,17 alkene had hydrophobic
interactions with residues Thr124, Thr222, Ile121, Ala223 and
Tyr183, from the 2D binding schemes of the complex 19a/human
contained the optimal functional group combination for the bind-
ing. These superior features merit compound 19a for further
investigations.
We also noticed that mouse 11
responded quite differently to the functionality changes. Human
11 -HSD1 was more sensitive to the even subtle structural varia-
tion, whereas, mouse 11 -HSD1 was rather obtuse. It was reasoned
that there was significant structural variability between the active
b-HSD1 and human 11b-HSD1
11
b
-HSD1.
We then focused on the effects of the variations at the C-19
carboxyl group. The C-19 hydroxyl analogs 12 and 16, the C-19
fluoride analog 13 were weakly active on both human and mouse
b
b
11
b
-HSD1, comparing to compound 1b and 2. The ester derivative
pockets of mouse 11b-HSD1 and human 11b-HSD1 [32].
17 had comparable activity to compound 2 on both species 11
b
-
HSD1. All these results highlighted the importance of electronic
5. Conclusions
effects in this region. The urea modifications at C-19 gave a wide
range of activities on human 11
15c, 19a, 19b, 19e, 19h and 19i showed potent inhibitory activity on
human 11 -HSD1, and were with modest to excellent selectivity
over human 11 -HSD2 (Table 3). Especially, the most potent
b
-HSD1. Seven urea derivatives 15b,
In summary, we reported the discovery of an ent-kaurene diter-
penoids, compound 1b, as a novel selective 11 -HSD1 inhibitor. Using
compound 1b and its counterpart 2 as the starting materials, thirty-
b
b
b
six structurally diverse derivatives were designed and synthesized.

408
X. Deng et al. / European Journal of Medicinal Chemistry 65 (2013) 403e414
Fig. 4. Two-dimensional (2D) and three-dimensional (3D) interaction schemes between compound 19a and mouse/human 11
(http://www.inteligand.com/ligandscout/) and PyMOL (http://pymol.sourceforge.net/). The ligands and the residues near the pocket which are different in mouse and human 11
HSD1 are shown as sticks (receptor carbon in green and ligand carbon in cyan). The left two figures belong to mouse 11 -HSD1 and the right two figures belong to human 11 -HSD1.
b-HSD1. The figures were prepared using LigandScout
b-
b
b
Critical different residues of the binding pocket are labeled in the 3D interaction views. Hydrogen bonds are shown as dotted lines in both 2D and 3D views. (For interpretation of
the references to colour in this figure legend, the reader is referred to the web version of this article.)
The derivatives were further evaluated on SPA for their 11
inhibitory potency and selectivity. It’s found that the acetyl ester 5a
and several urea derivatives, 19a, 19b, 19e, 19h, exhibited significant
b
-HSD
spectrometer at ambient temperature. The residual solvent protons
(1H) or the solvent carbons (13C) were used as internal standards.
1H-NMR data are presented as follows: chemical shift in ppm
downfield from tetramethylsilane (multiplicity, coupling constant,
integration). The following abbreviations are used in reporting
NMR data: s, singlet; d, doublet; t, triplet; dd, doublet of doublets;
dt, doublet of triplets; m, multiplet. EI-MS and HR-EI-MS were
taken on a VG Auto Spec-3000 or on a Finnigan MAT 90 instrument.
improved potency and selectivity on human 11
2D and 3D interaction schemes of compound 19a and human/mouse
11 -HSD1 were generated by docking simulation. The present work
b-HSD1. Moreover, the
b
enriched the structural diversity of selective 11b-HSD1 inhibitors.
6. Experimental sections
6.2. Chemistry
6.1. General information
6.2.1. Isolation of ent-kaur-16-en-19-ol (1a), ent-kaur-16-en-19-oic
All reactions were performed under argon atmosphere using
flame-dried glassware unless otherwise noted. CH2Cl2, CH3CN and
1,4-dioxane were distilled over CaH2. THF was distilled over so-
dium/benzophenone ketyl. All reagents were commercially avail-
able and used without further purification unless indicated
otherwise. Thin layer chromatographies were carried out on Merck
silica plates (0.25 mm layer thickness). Flash chromatography was
performed with 300e400 mesh silica gels. Yields reported were for
isolated, spectroscopically pure compounds. 1H- and 13C-NMR ex-
periments were performed on a Bruker AM-400 and DRX-500 NMR
acid (1b), ent-16
16 ,17,19-kaurenetriol (1d)
Dried samples of aerial part of Diplopterygium gigantea (Wall.)
Nakai (5.4 kg) were ground and extracted with 95% ethanol. The
extraction was subjected to macroporous resin eluting with 95%
methanol. The crude product was then purified by silica gel chro-
matography with petrol ether/acetone mixture to give five frac-
tions. The fraction of the general chromatography eluted with
petrol ether/acetone (20:80) was further chromatographed over
silica gel columns, Sephadex LH column and RP-18 reverse column
b, 17-dihydroxy-19-kauranoic acid (1c), ent-
b

X. Deng et al. / European Journal of Medicinal Chemistry 65 (2013) 403e414
409
Table 2
6.2.2. Isolation of 11b,15b-dihydroxy-kaur-16-en-19-oic acid (2)
Enzymatic assay.a
The pulverized branch and leaf of N. insignis Franch (22 kg) was
extracted with 95% ethanol and concentrated to give a residue
(750 g). The residue was subjected to silica gel chromatography
eluting with petrol ether/acetone (from 90:10 to 30:70) to separate
into fractions. The polar fraction eluted with petrol ether/acetone
(30:70) was further purified over silica gel columns, Sephadex LH
column and RP-18 reverse column to give compound 2 (65 g).
Compound 2 was characterized by comparing the spectral data
with the references indicated [35].
Entry
Inhibitory ratio
Mouse 11
b-HSD1 (%)
Human 11b-HSD1 (%)
1b
2
3
4
5a
5b
5c
5d
6a
6b
7
8a
8b
9
10
11
12
13
14
15a
15b
15c
16
17
18
19a
19b
19c
19d
19e
19f
19g
19h
19i
19j
19k
19l
19m
GAb
82.39
16.54
11.06
19.58
12.40
21.28
32.67
33.98
35.23
11.60
19.58
55.54
55.97
11.60
16.78
25.27
20.43
37.67
55.52
18.24
25.89
18.17
27.26
25.06
16.83
24.40
7.92
81.79
39.72
59.99
43.57
74.91
29.33
24.82
32.31
30.07
15.49
56.31
35.17
47.13
15.49
27.32
19.59
28.58
31.20
28.35
19.74
63.98
55.66
17.42
42.26
6.14
94.96
64.54
44.71
9.70
68.81
2.12
11.81
75.95
65.69
0.24
13.89
23.15
44.60
94.53
6.2.3. 11
b-((Methylthio)-thioxomethoxy)-15b-hydroxy-kaur-16-
en-19-oic acid (3)
To a suspension of NaH (32 mg, 60%, 1 mmol) in THF (1.0 mL) at
0
ꢁC under N2 was added a solution of compound 2 (33 mg,
0.1 mmol) in THF (0.5 mL) dropwise. The mixture was stirred at 0 ꢁC
for 0.5 h. Then CS2 (39 mg, 0.5 mmol) was added dropwise. The
mixture was warmed to the room temperature and continued
stirring for 2 h. Then MeI (35 mg, 0.25 mmol) was added dropwise
and stirred for another 4 h until no starting material was detected
by TLC judgment. A saturated aqueous NH4Cl solution was added to
quench the reaction. The mixture was extracted with dichloro-
methane (10 mL ꢂ 3). The combined organic layers were washed
with brine, dried over anhydrous Na2SO4 and concentrated. The
crude product was subjected to flash chromatography on silica gel
(ethyl acetate/petrol ether ¼ 1:3) to afford compound 3 as white
foams (20 mg, 47%). 1H-NMR (CDCl3, 400 MHz)
d 6.30 (s,1H), 5.18 (s,
3.69
5.39
1H), 5.18 (s, 1H), 3.86 (d, J ¼ 4 Hz, 1H), 2.76 (s, 1H), 2.63 (s, 3H), 2.18
(d, J ¼ 8 Hz, 1H), 2.04e2.09 (m, 3H), 2.01 (s, 1H), 1.82e1.89 (m, 2H),
1.79 (d, J ¼ 16 Hz, 1H), 1.69 (s, 1H), 1.63 (d, J ¼ 12 Hz, 1H), 1.53 (dd,
J ¼ 20 Hz, 8 Hz, 1H), 1.51 (d, J ¼ 20 Hz, 1H), 1.34 (dd, J ¼ 12 Hz, 8 Hz,
1H), 1.25 (s, 1H), 1.24 (s, 3H), 1.19 (dd, J ¼ 20 Hz, 8 Hz, 1H), 1.09 (d,
J ¼ 4 Hz, 1H), 1.07 (s, 1H), 0.85 (s, 3H); 13C-NMR (CDCl3, 100 MHz)
52.23
19.44
11.32
16.34
20.77
16.07
21.39
12.71
9.21
d
216.7,183.8, 151.4, 109.3, 89.9, 66.5, 57.0, 56.1, 45.4, 43.7, 42.6, 40.2,
38.6, 38.6, 38.4, 37.4, 35.9, 29.6, 28.8, 20.9, 19.2, 18.9, 15.0; HR-EI-MS
(m/z): calcd. for C22H32O4S2 [M]þ, 424.1742, found 424.1744.
96.68
a
The value was determined at the concentration of 1 mM. And the values are
6.2.4. 15b-Hydroxy-kaur-11(12),16-dien-19-oic acid (4) [16]
To a solution of DEAD (87 mg, 0.5 mmol) in THF (1.5 mL) at 0 ꢁC
were added compound 2 (33 mg, 0.1 mmol) and PPh3 (65 mg,
0.25 mmol) successively. The mixture was warmed to the room
temperature and was stirred for 8 h until no starting material was
detected. Then the reaction was quenched with water. The aqueous
phase was extracted with EtOAc (8 mL ꢂ 3). The combined organic
layers were washed with brine, dried over anhydrous Na2SO4 and
concentrated. The crude product was subjected to flash chroma-
tography on silica gel (ethyl acetate/petrol ether ¼ 1:4) to afford
compound 4 as white foams (28 mg, 89%). 1H-NMR (CDCl3,
averages of two determinations and deviation from the average is 10% of the
average value.
b
GA: glycyrrhetinic acid, which was used as a positive control.
to give compound 1a (450 mg), compound 1b (3.0 g), compound 1c
(8 mg), and compound 1d (8 mg). Compounds 1a [33], 1b [33], 1c
[34], and 1d [34] were characterized by comparing the spectral data
with the references indicated.
Table 3
Enzymatic assay.a
400 MHz)
d
6.02 (t, J ¼ 8 Hz, 8 Hz, 1H), 5.48 (dd, J ¼ 8 Hz, 8 Hz, 1H),
Entry Mouse (IC50
11 -HSD1 11
64.2 nM
)
Human (IC50
-HSD2 HSD2/HSD1 11 -HSD1 11
131.4 nM 19.0
)
4.86 (s, 1H), 4.81 (d, J ¼ 4 Hz, 1H), 3.88 (s, 1H), 2.83 (dd, J ¼ 8 Hz,
4 Hz,1H), 2.13 (m, 2H),1.97 (s,1H),1.84 (m, 3H),1.76 (m,1H),1.54 (d,
J ¼ 12 Hz, 1H), 1.45 (m, 1H), 1.25 (s, 3H), 1.18 (d, J ¼ 12 Hz, 2H), 1.07
b
b
b
b-HSD2 HSD2/HSD1
1b
3
5a
8a
m
M
144.8
(m, 3H), 0.86 (s, 3H); 13C-NMR (CDCl3, 100 MHz)
d 183.8, 157.6,
>1
>1
m
m
M
M
ND
ND
ND
ND
>386.7
ND
ND
ND
ND
ND
ND
770.6 nM >100
116.4 nM 228.7
m
m
M
M
>129.7
1965.4
ND
>302.3
>245.7
>10649.0
>401.6
6.2
134.2, 125.4, 103.1, 83.4, 55.1, 48.8, 44.9, 43.7, 42.3, 39.7, 38.8, 38.4,
37.5, 34.1, 28.8, 21.3, 18.9, 15.4; HR-EI-MS (m/z): calcd. for C20H28O3
[M]þ, 316.2042, found 316.2038.
258.6 nM >100
mM
>1
m
M
ND
15b >1
15c >1
19a >1
19b >1
19e >1
19h >1
mM
mM
mM
mM
mM
mM
mM
ND
ND
ND
ND
ND
ND
ND
ND
330.8 nM >100
407.3 nM >100
m
m
M
M
9.4 nM
249.0 nM >100
266.1 nM 1.6
165.4 nM >100
403.7 nM 0.7
0.83 nM 1.22 nM
>1 mM
m
M
M
6.2.5. Typical procedures for compounds 5ae5d (preparation of
compound 5b)
mM
m
> 604.6
1.7
0.4012
To a solution of compound 2 (17 mg, 0.05 mmol) in THF (1.0 mL)
at 0 ꢁC were added Et3N (15 mg, 0.15 mmol) and benzoyl isothio-
cyanate (16 mg, 0.1 mmol) successively. The mixture was warmed
to the room temperature and was stirred for 1.5 h until no starting
material was detected. Then the reaction was quenched with water.
The aqueous phase was extracted with EtOAc (8 mL ꢂ 3). The
19i
>1
ND
ND
mM
GAb 2.0 nM
a
Values are averages of two determinations and deviation from the average is
10% of the average value.
b
GA: glycyrrhetinic acid, which was used as
a positive control; ND: not
determined.

410
X. Deng et al. / European Journal of Medicinal Chemistry 65 (2013) 403e414
combined organic layers were washed with brine, dried over
anhydrous Na2SO4 and concentrated. The crude product was sub-
jected to flash chromatography on silica gel (ethyl acetate/petrol
ether ¼ 1:3) to afford compound 5b as white foams (22 mg, 100%).
(15 mg, 0.15 mmol) dropwise. The mixture was stirred at 0 ꢁC for
0.5 h, and then morpholine (9 mg, 0.1 mmol) was added dropwise.
The mixture was stirred for another 2.5 h until no starting material
was detected by TLC. Then the reaction was quenched with water.
The aqueous phase was extracted with EtOAc (8 mL ꢂ 3). The
combined organic layers were washed with brine, dried over
anhydrous Na2SO4 and concentrated. The crude product was sub-
jected to flash chromatography on silica gel (ethyl acetate/petrol
ether ¼ 1:1.5) to afford compound 6a as white foam (20 mg, 89%).
6.2.5.1. 11b-Acetyloxy-15b-hydroxy-kaur-16-en-19-oic acid (5a).
Compound 5a (10 mg, 53%) was prepared from compound 2 (17 mg,
0.05 mmol) as white foam according to the synthetic procedure
described for 5b. 1H-NMR (CDCl3, 400 MHz)
d 5.21 (s, 1H), 5.14 (s,
1H), 4.93 (s, 1H), 4.83 (s, 1H), 2.68 (s, 1H), 2.17 (s, 3H), 1.99 (d,
J ¼ 10 Hz, 1H), 1.95 (d, J ¼ 15 Hz, 2H), 1.90 (s, 3H), 1.83e1.85 (m, 3H),
1.58 (s, 1H), 1.43e1.52 (m, 2H), 1.24 (s, 3H), 1.03e1.04 (m, 3H), 0.90
6.2.6.1. 11
b
-((Morphilin-4-yl)-carbonyl-oxy)-15
b
-hydroxy-kaur-16-
5.19 (d, J ¼ 4 Hz,
en-18-oic acid (6a). 1H-NMR (CDCl3, 400 MHz)
d
(s, 3H); 13C-NMR (CDCl3, 100 MHz)
d
183.6, 170.4, 152.6, 108.8, 81.9,
1H), 5.07 (s, 1H), 4.99 (s, 1H), 3.78 (s, 1H), 3.61e3.69 (m, 6H), 3.50e
3.56 (m, 2H), 2.64 (d, J ¼ 8 Hz, 1H), 2.30 (d, J ¼ 16 Hz, 1H), 2.16 (s,
1H), 2.09 (d, J ¼ 12 Hz, 1H), 1.87e1.93 (m, 2H), 1.78 (t, J ¼ 12 Hz,
12 Hz, 2H), 1.64 (t, J ¼ 12 Hz, 12 Hz, 1H), 1.53e1.55 (m, 1H), 1.50 (s,
1H), 1.46 (d, J ¼ 12 Hz, 2H), 1.27 (s, 3H), 1.24 (d, J ¼ 4 Hz, 2H), 1.06e
66.4, 56.4, 56.2, 44.4, 43.7, 42.5, 40.1, 38.6, 38.3, 37.4, 36.0, 28.8, 21.2,
20.9,18.9, 15.0; HR-EI-MS (m/z): calcd. for C22H32O5 [M]þ, 376.2250,
found 376.2247.
6.2.5.2. 11
b
-Benzoyloxy-15
b
-hydroxy-kaur-16-en-19-oic acid (5b).
1.16 (m, 4H), 0.97 (s, 3H); 13C-NMR (CDCl3, 100 MHz)
d 176.7, 157.1,
Compound 5b (22 mg,100%) was prepared from compound 2 (17 mg,
0.05 mmol) as white foam according to the synthetic procedure
154.5, 106.8, 83.2, 77.6, 71.4, 67.3, 61.1, 54.0, 47.0, 46.6, 45.2, 44.6,
41.6, 40.1, 40.0, 39.6, 39.4, 39.0, 38.0, 36.0, 28.3, 22.4, 20.3,18.9,15.8;
HR-EI-MS (m/z): calcd. for C25H37NO6 [M]þ, 447.2621, found
447.2640.
described above. 1H-NMR (CDCl3, 400 MHz)
d
8.05 (d, J ¼ 8 Hz, 2H),
7.66 (t, J ¼ 8 Hz,1H), 7.51 (t, J ¼ 8 Hz, 2H), 5.11 (s,1H), 5.02 (s,1H), 4.04
(d, J ¼ 4 Hz,1H), 3.76 (s, 2H), 2.63 (s,1H), 2.33 (d, J ¼ 16 Hz,1H),1.92e
2.05 (m, 5H),1.69e1.80 (m, 4H),1.60 (s,1H),1.55 (d, J ¼ 16 Hz,1H),1.47
(dd, J ¼ 8 Hz, 4 Hz, 1H), 1.43 (s, 3H), 1.31 (d, J ¼ 12 Hz, 1H), 1.25 (d,
J ¼ 12 Hz, 1H), 1.18 (dd, J ¼ 12 Hz, 4 Hz, 1H), 1.09 (dd, J ¼ 12 Hz, 4 Hz,
6.2.6.2. 11b-((N-2-thiazolyl)-carbonyl-oxy)-15b-hydroxy-kaur-16-
en-18-oic acid (6b). Compound 6b (11 mg, 69% base on the recovery
starting material (brsm)) was prepared from compound 2 (17 mg,
0.05 mmol) as white solid according to the synthetic procedure
described for 6a, with 5 mg starting material recovered. 1H-NMR
1H), 0.99 (s, 3H); 13C-NMR (CDCl3, 100 MHz)
d 172.8, 162.2, 158.2,
134.3,130.3, 129.1, 128.8, 106.0, 82.3, 66.7, 56.3, 53.4, 45.5, 44.4, 42.9,
40.0, 38.8, 38.7, 37.8, 37.7, 35.6, 28.2, 21.3, 18.8, 16.1; HR-EI-MS (m/z):
calcd. for C27H34O5 [M]þ, 438.2406, found 438.2414.
(CDCl3, 400 MHz)
d
7.27 (d, J ¼ 4 Hz, 1H), 6.83 (d, J ¼ 4 Hz, 1H), 5.10
(s, 1H), 4.91 (s, 1H), 3.79 (s, 1H), 2.58 (s, 1H), 2.24 (d, J ¼ 12 Hz, 1H),
1.98 (dd, J ¼ 12 Hz, 8 Hz, 1H), 1.79e1.97 (m, 5H), 1.72 (d, J ¼ 16 Hz,
1H),1.64 (s, 1H),1.45 (d, J ¼ 12 Hz, 1H), 1.42 (d, J ¼ 12 Hz,1H), 1.22 (s,
3H), 1.18 (s, 3H), 1.13 (s, 1H), 1.08 (d, J ¼ 16 Hz, 2H), 1.04 (d, J ¼ 8 Hz,
6.2.5.3. 11b-(4-Bromo-benzoyloxy)-15b-hydroxy-kaur-16-en-19-oic
acid (5c). Compound 5c (23 mg, 89%) was prepared from com-
pound 2 (17 mg, 0.05 mmol) as white solid according to the syn-
thetic procedure described for 5b. 1H-NMR (CD3OD, 400 MHz)
2H), 0.89 (s, 3H); 13C-NMR (CDCl3, 100 MHz)
d 183.7, 162.4, 157.2,
152.6, 136.5, 112.9, 106.6, 83.2, 72.7, 56.3, 51.8, 44.9, 44.3, 40.5, 40.3,
38.9, 38.7, 38.6, 38.1, 36.0, 30.1, 29.5, 21.7, 19.5, 15.9; HR-EI-MS (m/
z): calcd. for C24H30N2O5S [M]þ, 458.1875, found 458.1883.
d
7.83 (d, J ¼ 4 Hz, 2H), 7.51 (d, J ¼ 4 Hz, 2H), 4.96 (s,1H), 4.93 (s, 1H),
3.84 (d, J ¼ 4 Hz, 1H), 3.67 (s, 1H), 2.50 (s, 1H), 2.09 (d, J ¼ 12 Hz,1H),
1.94 (d, J ¼ 12 Hz,1H),1.91 (d, J ¼ 8 Hz,1H),1.79 (t, J ¼ 8 Hz, 4H),1.73
(d, J ¼ 12 Hz, 1H), 1.57 (dt, J ¼ 8 Hz, 4 Hz, 2H), 1.45 (s, 1H), 1.34e1.38
(m, 3H), 1.13 (s, 3H), 1.09 (d, J ¼ 4 Hz, 1H), 1.03e1.06 (m, 3H), 0.99 (d,
J ¼ 4 Hz,1H), 0.97 (d, J ¼ 4 Hz,1H), 0.81 (s, 3H), 0.79 (d, J ¼ 4 Hz, 2H);
6.2.7. 17-Norkaur-16-oxo-19-oic acid (7) [19]
To a solution of compound 1b (30 mg, 0.1 mmol) in t-BuOH/
THF/H2O (0.75 mL/0.5 mL/0.25 mL) were added 2,6-lutidine
(20 mg, 0.2 mmol) and K2OsO4 (2 mg, 0.004 mmol) succes-
sively. The mixture was stirred at room temperature for 10 min,
then NaIO4 (87 mg, 0.4 mmol) was added in one portion. The
mixture was stirred for another 3 h until no starting material was
detected by TLC. Then the reaction was quenched with a satu-
rated Na2SO3 aqueous solution. The aqueous phase was extracted
with EtOAc (8 mL ꢂ 3). The combined organic layers were washed
with brine, dried over anhydrous Na2SO4 and concentrated. The
crude product was subjected to flash chromatography on silica gel
(ethyl acetate/petrol ether ¼ 1:4) to afford compound 7 as white
foam (28 mg, 93%). The structure of compound 7 was assigned by
comparing the spectra data with that reported in the literature
[36].
13C-NMR (125 MHz, MeOD)
d 180.9, 168.5, 158.0, 132.0, 131.8, 130.1,
129.7, 128.2, 106.5, 82.6, 66.3, 56.8, 54.9, 45.3, 44.0, 42.2, 40.7, 39.7,
39.6, 38.3, 38.3, 36.4, 30.0, 29.2, 21.8, 19.5, 15.7; HR-EI-MS (m/z):
calcd. for C27H33O5Br [M] þ, 516.1511, found 516.1533.
6.2.5.4. 11b-(4-Dimethylamimo-benzoyloxy)-15b-hydroxy-kaur-16-
en-19-oic acid (5d). Compound 5d (24 mg, 99%) was prepared from
compound 2 (17 mg, 0.05 mmol) as white solid according to the
synthetic procedure described for 5b. 1H-NMR (CDCl3, 500 MHz)
d
7.73 (d, J ¼ 10 Hz, 2H), 6.55 (d, J ¼ 10 Hz, 2H), 4.92 (s, 1H), 4.88 (s,
1H), 3.91 (s, 3H), 3.82 (dd, J ¼ 15 Hz, 5 Hz, 1H), 3.62 (s, 1H), 2.91 (s,
6H), 2.46 (s, 1H), 2.17 (d, J ¼ 15 Hz, 1H), 1.91 (s, 1H), 1.82e1.89 (m,
4H), 1.79 (d, J ¼ 15 Hz, 2H), 1.70 (d, J ¼ 15 Hz, 2H), 1.54 (t, J ¼ 15 Hz,
1H), 1.43 (d, J ¼ 20 Hz, 2H), 1.34 (d, J ¼ 15 Hz,1H), 1.27 (s, 3H),1.12 (s,
3H), 1.08 (s, 2H), 1.04 (d, J ¼ 5 Hz, 1H), 1.00 (s, 1H), 0.90e0.96 (m,
6.2.8. 15a-Hydroxy-kaur-16-en-19-oic acid (8a) [20,21]
2H), 0.86 (s, 3H); 13C-NMR (CDCl3, 125 MHz)
d
173.6, 162.6, 157.3,
To a solution of compound 1b (30 mg, 0.1 mmol) in 1,4-dioxane
(1.0 mL) was added SeO2 (17 mg, 0.15 mmol) in one portion. The
mixture was stirred at room temperature under N2 for 2 h until no
starting material was detected by TLC. Then the solvent was
removed under vacuo. The crude product was subjected to flash
chromatography on silica gel (ethyl acetate/petrol ether ¼ 1:4) to
afford compound 8a (18 mg, 57%). The structure of compound 8a
was characterized by comparing with the spectra data in the
literature [22].
154.1, 132.2, 114.6, 110.6, 105.8, 81.9, 65.7, 56.2, 53.9, 45.1, 43.2, 41.6,
39.9, 39.6, 38.8, 38.7, 37.5, 35.6, 29.3, 28.6, 28.0, 21.1, 18.8, 15.8; HR-
EI-MS (m/z): calcd. for C29H39NO5 [M]þ, 481.2828, found 481.2821.
6.2.6. Typical procedure for compounds 6ae6b (preparation of
compound 6a) [17,18]
To a solution of compound 2 (17 mg, 0.05 mmol) and tri-
phosgene (15 mg, 0.05 mmol) in DCM (1.0 mL) was added Et3N

X. Deng et al. / European Journal of Medicinal Chemistry 65 (2013) 403e414
411
6.2.9. 11
b
,15
b
-dihydroxy-kaur-19-oic acid (9)
was detected by TLC judgment. A saturated aqueous NH4Cl solution
was added to quench the reaction and the mixture was stirred for
0.5 h. The aqueous phase was extracted with EtOAc (10 mL ꢂ 3). The
combined organic layers were washed with H2O and brine suc-
cessively, dried over anhydrous Na2SO4 and concentrated. The
crude product was subjected to flash chromatography on silica gel
(ethyl acetate/petrol ether ¼ 1:10) to afford compound 12 as white
foams (24 mg, 84%).
To a solution of compound 2 (17 mg, 0.05 mmol) in MeOH
(4.0 mL) was added Pd/C (3.0 mg, 17%wt) in one portion. The
mixture was stirred at room temperature under H2 for 1 h until no
starting material was detected by TLC. Then the insoluble substance
was removed. The filtrate was concentrated under vacuo, and the
crude product was subjected to flash chromatography on silica gel
(ethyl acetate/petrol ether ¼ 1:2) to afford compound 9 as white
foams (16 mg, 94%). 1H-NMR (CDCl3:CD3OD ¼ 1:1, 500 MHz)
d 3.8
(d, J ¼ 5 Hz,1H), 3.28 (s, 2H), 3.24 (s, 1H), 2.33 (d, J ¼ 10 Hz, 2H), 2.17
(t, J ¼ 10 Hz, 1H), 2.07 (d, J ¼ 15 Hz, 1H), 1.73e1.86 (m, 8H), 1.64 (d,
J ¼ 10 Hz, 1H), 1.35 (d, J ¼ 10 Hz, 1H), 1.29 (d, J ¼ 10 Hz, 2H), 1.17 (s,
1H), 1.15 (d, J ¼ 5 Hz, 3H), 1.10 (s, 3H), 1.07 (d, J ¼ 15 Hz, 1H), 0.96 (t,
J ¼ 10 Hz, 1H), 0.87 (s, 3H); 13C-NMR (CDCl3:CD3OD ¼ 1:1, 125 MHz)
6.2.12.1. 19-Hydroxy-kaur-16-ene (12). The structure of compound
12 was assigned by comparing the spectra data with the literature
[37].
6.2.12.2. 11b,15b,19-Trihydroxy-kaur-16-ene (16). Compound 16
d
182.4, 65.2, 61.8, 57.1, 52.1, 50.3, 44.6, 40.2, 39.3, 39.0, 37.9, 35.6,
(30 mg, 94%) was prepared from compound 2 (33 mg, 0.1 mmol) as
white solid according to the synthetic procedure described for 12.
35.4, 34.0, 29.5, 20.9, 19.7, 15.9, 11.3; HR-EI-MS (m/z): calcd. for
C
20H32O4 [M]þ, 336.2144, found 336.2155.
1H-NMR (CDCl3, 400 MHz)
d 4.76 (s, 1H), 4.74 (s, 1H), 3.65 (d,
J ¼ 4 Hz, 1H), 3.45 (s, 1H), 3.44 (d, J ¼ 12 Hz, 1H), 3.06 (s, 5H), 2.31 (s,
1H),1.70 (d, J ¼ 12 Hz, 2H), 1.64 (m, 1H), 1.55 (d, J ¼ 16 Hz,1H), 1.35e
1.43 (m, 1H), 1.27 (s, 1H), 1.17e1.20 (m, 2H), 1.02e1.06 (m, 2H), 0.98
(s, 3H), 0.85 (t, J ¼ 12 Hz, 1H), 0.73e0.78 (m, 2H), 0.68 (s, 3H), 0.67
6.2.10. 11b,15b-dihydroxy-16b(17)-epoxy-kaur-19-oic acid (10)
To a suspension of compound 2 (33 mg, 0.1 mmol) and NaHCO3
(17 mg, 0.2 mmol) in DCM (1.0 mL) was added m-CPBA (26 mg, 70%,
0.1 mmol) in one portion. The mixture was stirred at room tem-
perature for 1.5 h until no starting material was detected by TLC.
Then the reaction was quenched with a saturated Na2SO3 aqueous
solution. The aqueous phase was extracted with EtOAc (10 mL ꢂ 3).
The combined organic layers were washed with H2O and brine
successively, dried over anhydrous Na2SO4 and concentrated. The
crude product was subjected to flash chromatography on silica gel
(ethyl acetate/petrol ether ¼ 1:1) to afford compound 10 as white
(s, 1H); 13C-NMR (CDCl3, 100 MHz)
d 155.8, 102.9, 65.4, 56.7, 56.1,
49.0, 44.1, 43.9, 41.5, 40.4, 39.6, 39.1, 38.6, 35.5, 33.1, 27.0, 20.4, 18.2,
18.1, 18.0; HR-EI-MS (m/z): calcd. for C20H32O3 [M]þ, 320.2351,
found 320.2353.
6.2.13. Typical procedure for compounds 8b and 13 (preparation of
compound 13)
To a solution of compound 12 (15 mg, 0.05 mmol) in DCM
(1.0 mL) at ꢀ78 ꢁC under N2 was added a solution of DAST (16 mg,
0.1 mmol) in DCM (0.5 mL) dropwise. The mixture was stirred for
0.5 h until no starting material was detected by TLC judgment. A
saturated aqueous NH4Cl solution was added to quench the reac-
tion. The aqueous phase was extracted with EtOAc (10 mL ꢂ 3). The
combined organic layers were washed with H2O and brine suc-
cessively, dried over anhydrous Na2SO4 and concentrated. The
crude product was subjected to flash chromatography on silica gel
(chloroform/petrol ether ¼ 1:2) to afford compound 13 as white
foams (7 mg, 48%).
foams (32 mg, 91%). 1H-NMR (CD3OD, 400 MHz)
d 4.37 (s, 1H), 3.67
(d, J ¼ 12 Hz,1H), 3.63 (d, J ¼ 12 Hz,1H), 3.08 (s,1H), 2.48 (t, J ¼ 8 Hz,
1H), 2.20 (d, J ¼ 16 Hz,1H), 2.01 (t, J ¼ 12 Hz, 2H), 1.79e1.89 (m, 4H),
1.73 (d, J ¼ 12 Hz, 1H), 1.64 (s, 1H), 1.56 (dt, J ¼ 12 Hz, 4 Hz, 1H), 1.27
(s, 1H), 1.21 (s, 3H), 1.12e1.16 (m, 2H), 1.05 (d, J ¼ 12 Hz, 2H), 1.02 (s,
3H); 13C-NMR (CD3OD, 100 MHz)
d 180.4, 86.9, 78.0, 76.6, 62.3, 56.5,
51.3, 45.4, 43.1, 41.2, 38.4, 38.3, 37.7, 37.6, 36.6, 34.3, 28.7, 20.9, 18.8,
17.4; HR-EI-MS (m/z): calcd. for C20H30O5 [M]þ, 350.2093, found
350.2100.
6.2.11. 11b-Hydroxy-kaur-15-oxo-16-en-19-oic acid (11) [25]
To a suspension of compound 2 (17 mg, 0.05 mmol) in CH3CN
(1.0 mL) was added TEMPO (2 mg, 0.013 mmol), iodobenzene
diacetate (25 mg, 0.077 mmol) and HOAc (10 mg, 0.17 mmol) suc-
cessively. The mixture was stirred at room temperature for 6 h until
most starting material was conversed by TLC. Then the reaction was
quenched with a saturated Na2SO3 aqueous solution. The aqueous
phase was extracted with EtOAc (10 mL ꢂ 3). The combined organic
layers were washed with brine, dried over anhydrous Na2SO4 and
concentrated. The crude product was subjected to flash chroma-
tography on silica gel (ethyl acetate/petrol ether ¼ 1:2) to afford
compound 11 as white foams (5 mg, 72% brsm). 1H-NMR (CD3OD,
6.2.13.1. 19-Fluoro-kaur-16-ene (13). 1H-NMR (CDCl3, 400 MHz)
d
4.80 (s, 1H), 4.74 (s, 1H), 4.15 (dd, J ¼ 60 Hz, 12 Hz, 1H), 3.70 (dd,
J ¼ 48 Hz, 12 Hz, 1H), 3.64 (s, 2H), 2.65 (s, 1H), 2.06 (m, 2H), 1.94 (d,
J ¼ 12 Hz, 1H), 1.88 (d, J ¼ 12 Hz, 1H), 1.76 (m, 1H), 1.65 (m, 1H), 1.57
(s, 3H), 1.48 (m, 4H), 1.35 (m, 1H), 1.26 (s, 1H), 1.09 (d, J ¼ 4 Hz, 1H),
1.03 (d, J ¼ 4 Hz, 3H), 0.99 (s, 4H); 13C-NMR (CDCl3, 100 MHz)
d
155.6, 103.0, 65.1, 64.9, 56.7, 56.0, 48.9, 47.9, 43.8, 41.3, 40.1, 39.5,
39.0, 37.4, 35.8, 35.7, 33.1, 30.9, 29.6, 27.5, 27.4, 20.3, 18.1, 18.0; HR-
EI-MS (m/z): calcd. for C20H31F [M]þ, 290.2410, found 290.2447.
6.2.13.2. 15b-Fluoro-kaur-16-en-19-oic acid (8b). Compound 8b
400 MHz)
d
6.30 (s, 1H), 5.76 (s, 1H), 4.54 (s, 1H), 3.87 (s, 1H), 3.56 (s,
(6 mg, 49%) was prepared from compound 8a (12 mg, 0.038 mmol)
1H), 2.93 (d, J ¼ 12 Hz, 1H), 2.69 (d, J ¼ 12 Hz, 1H), 2.56 (m, 1H),
2.46e2.50 (m, 3H), 2.32e2.34 (m, 3H), 1.85e2.00 (m, 5H), 1.75 (s,
3H), 1.69e1.77 (m, 4H), 1.53e1.62 (m, 3H), 1.47 (s, 3H); 13C-NMR
as white solid according to the synthetic procedure described for
13. 1H-NMR (CDCl3, 400 MHz)
d
5.51 (d, J ¼ 4 Hz, 1H), 5.33 (d,
J ¼ 8 Hz, 1H), 5.23 (d, J ¼ 4 Hz, 1H), 4.97 (s, 1H), 4.86 (s, 1H), 4.62 (d,
J ¼ 56 Hz, 1H), 2.83 (s, 1H), 2.65 (d, J ¼ 4 Hz, 2H), 2.18 (s, 1H), 2.14
(dd, J ¼ 8 Hz, 4 Hz, 1H), 2.10 (d, J ¼ 8 Hz, 1H), 1.92 (d, J ¼ 16 Hz, 4H),
1.82 (dd, J ¼ 12 Hz, 4 Hz, 4H), 1.65 (d, J ¼ 8 Hz, 2H), 1.62 (d, J ¼ 4 Hz,
4H), 1.55 (s, 2H), 1.45e1.52 (m, 6H), 1.37 (dd, J ¼ 16 Hz, 8 Hz, 1H),
1.32 (s, 3H), 1.09e1.18 (m, 5H), 1.03 (d, J ¼ 8 Hz, 2H), 0.80e0.88 (m,
(CD3OD, 100 MHz)
d 210.9, 180.0, 150.1, 112.1, 65.2, 62.3, 55.7, 50.4,
43.1, 40.0, 39.2, 38.5, 37.4, 36.6, 36.2, 33.5, 28.5, 19.5, 18.5, 15.1; HR-
EI-MS (m/z): calcd. for C20H28O4 [M]þ, 332.1988, found 332.1992.
6.2.12. Typical procedure for compounds 12 and 16 (preparation of
compound 12)
5H); 13C-NMR (CDCl3, 100 MHz)
d 169.8, 139.3, 139.2, 111.7, 111.7,
To a suspension of LiAlH4 (14 mg, 0.37 mmol) in THF (1.0 mL) at
102.6, 100.8, 81.7, 80.1, 56.3, 56.2, 51.7, 48.9, 47.0, 43.5, 41.9, 40.8,
40.1, 40.0, 38.5, 37.7, 37.7, 36.2, 32.4, 29.6, 27.2, 25.2, 20.3, 18.7, 18.5,
15.2; HR-EI-MS (m/z): calcd. for C20H29FO2 [M]þ, 320.2152, found
320.2144.
0
ꢁC under N2 was added a solution of compound 1b (30 mg,
0.1 mmol) in THF (0.5 mL) dropwise. The mixture was warmed to
the room temperature and stirred for 2 h until no starting material

412
X. Deng et al. / European Journal of Medicinal Chemistry 65 (2013) 403e414
6.2.14. Typical procedure for compounds 14 and 18 (preparation of
compound 18) [28,29]
0.97 (s, 3H), 0.88 (d, J ¼ 12 Hz, 1H), 0.73 (t, J ¼ 12 Hz, 1H); 13C-NMR
(CDCl3, 100 MHz)
d 159.7, 157.3, 155.6, 155.4, 135.3, 121.7, 121.6,
To a solution of compound 2 (33 mg, 0.3 mmol) in anisole
(1.0 mL) at room temperature was added DPPA (28 mg, 0.1 mmol)
and Et3N (15 mg, 0.15 mmol), successively. The mixture was
warmed to 90 ꢁC and stirred for 1.5 h until no starting material was
detected by TLC. The mixture was cooled to the room temperature,
and then was subjected to flash chromatography on silica gel (ethyl
acetate/petrol ether ¼ 1:10) directly to afford compound 18 as
white foams (34 mg, 100%).
115.4, 115.2, 103.0, 56.4, 55.4, 55.0, 48.5, 43.8, 43.7, 40.7, 39.9, 39.7,
38.9, 36.4, 33.0, 27.5, 19.2, 17.8, 17.1; HR-EI-MS (m/z): calcd. for
C
26H35N2OF [M]þ, 410.2733, found 410.2717.
6.2.15.3. N-(tricyclo(3.3.1.13,7)-dec-1-yl)-N-(kaur-16-ene-4
a-yl) urea
(15c). Compound 15b (21 mg, 88%) was prepared from compound
14 (15 mg, 0.05 mmol) as white solid according to the synthetic
procedure described for 19h. 1H-NMR (CDCl3, 400 MHz)
d 4.80 (s,
1H), 4.75 (s, 1H), 3.91 (s, 1H), 2.74 (d, J ¼ 16 Hz,1H), 2.65 (s,1H), 2.06
(d, J ¼ 4 Hz, 5H), 1.94 (s, 3H), 1.93 (s, 3H), 1.86 (d, J ¼ 12 Hz, 1H), 1.78
(d, J ¼ 12 Hz, 1H), 1.65 (s, 7H), 1.60 (s, 4H), 1.56 (d, J ¼ 8 Hz, 3H),
1.38e1.42 (m, 2H), 1.40 (s, 1H), 1.38 (s, 3H), 1.26 (s, 1H), 1.15 (s, 3H),
1.09 (m, 1H), 0.92 (d, J ¼ 8 Hz, 1H), 0.80 (dt, J ¼ 12 Hz, 4 Hz, 1H); 13C-
6.2.14.1. 4
a
-Isocyanato-11
b
,15
d
b
-dihydroxy-kaur-16-ene
5.09 (s, 1H), 5.01 (s, 1H), 4.01 (d,
(18).
1H-NMR (CDCl3, 400 MHz)
J ¼ 4 Hz, 1H), 3.77 (dd, J ¼ 16 Hz, 12 Hz, 2H), 2.63 (s, 1H), 2.03 (d,
J ¼ 16 Hz, 1H), 1.95 (d, J ¼ 16 Hz, 2H), 1.76 (t, J ¼ 12 Hz, 12 Hz, 5H),
1.53 (s, 2H), 1.44 (m, 2H), 1.39 (d, J ¼ 4 Hz, 1H), 1.38 (s, 3H), 1.12 (m,
2H), 1.06 (s, 3H), 1.03 (d, J ¼ 12 Hz, 1H); 13C-NMR (CDCl3, 100 MHz)
NMR (CDCl3, 100 MHz)
d 156.4, 155.4, 103.1, 56.6, 55.6, 55.0, 50.8,
48.9, 44.0, 43.8, 42.5, 40.8, 40.2, 39.9, 39.1, 36.7, 36.4, 33.2, 29.5,
27.9, 19.6, 18.0, 17.9; HR-EI-MS (m/z): calcd. for C30H46N2O [M]þ,
450.3610, found 450.3613.
d
158.0, 121.6, 106.1, 82.2, 66.2, 59.1, 54.2, 53.7, 44.3, 43.0, 41.3, 38.9,
38.8, 37.8, 37.2, 36.0, 31.8, 19.4, 18.0, 16.5; HR-EI-MS (m/z): calcd. for
20H29NO3 [M]þ, 331.2147, found 331.2144.
C
6.2.15.4. N-(phenyl)-N-(11b,15b-dihydroxy-kaur-16-ene-4a-yl) urea
6.2.14.2. 4
a
-Isocyanato -kaur-16-ene (14). Compound 14 (26 mg,
(19a). Compound 19a (32 mg, 84%) was prepared from compound
18 (33 mg, 0.1 mmol) as white solid according to the synthetic
88%) was prepared from compound 1b (30 mg, 0.1 mmol) as white
solid according to the synthetic procedure described for 18. 1H-
procedure described for 19h. 1H-NMR (CDCl3, 400 MHz)
d 7.77 (d,
NMR (CDCl3, 400 MHz)
d
4.81 (s, 1H), 4.75 (s, 1H), 2.66 (s, 1H), 2.07
J ¼ 8 Hz, 1H), 7.70 (t, J ¼ 8 Hz, 1H), 7.43 (t, J ¼ 8 Hz, 1H), 5.52 (s, 1H),
5.49 (s, 1H), 4.39 (s, 1H), 4.21 (s, 1H), 3.25 (t, J ¼ 16 Hz, 1H), 3.06 (s,
1H), 2.43 (m, 2H), 2.31 (s, 2H), 2.25 (d, J ¼ 16 Hz, 1H), 2.19 (m, 2H),
2.01 (s, 1H), 1.92 (d, J ¼ 16 Hz, 1H), 1.86 (s, 3H), 1.61 (m, 3H), 1.47 (s,
(s, 2H), 2.04 (s, 1H), 1.87 (d, J ¼ 12 Hz, 1H), 1.68e1.78 (m, 3H), 1.43e
1.59 (m, 9H), 1.36 (s, 3H), 1.26 (s, 2H), 1.15 (s, 3H), 1.06 (d, J ¼ 4 Hz,
1H), 0.95 (d, J ¼ 8 Hz, 1H), 0.77 (td, J ¼ 12 Hz, 4 Hz, 4 Hz, 1H); 13C-
NMR (CDCl3,100 MHz)
d
155.5,103.1, 59.1, 55.2, 55.1, 49.1, 43.9, 43.8,
3H); 13C-NMR (CDCl3, 100 MHz)
d 156.6, 155.1, 138.9, 128.2, 121.8,
41.5, 40.1, 39.9, 39.4, 39.0, 33.1, 31.8, 29.6, 19.9, 18.2, 18.0, 16.6; HR-
118.9, 105.6, 81.3, 64.7, 55.2, 54.4, 54.2, 44.0, 41.2, 38.9, 38.5, 38.0,
36.6, 36.1, 35.4, 28.2, 28.2, 26.9, 18.3, 17.2, 16.4; HR-EI-MS (m/z):
calcd. for C26H36N2O3 [M]þ, 424.2726, found 424.2733.
EI-MS (m/z): calcd. for C20H29NO [M]þ, 299.2249, found 299.2242.
6.2.15. Typical procedure for compounds 15ae15c and 19ae19m
(preparation of compound 19h)
6.2.15.5. N-(4-Fluoro-phenyl)-N-(11
b,15b-dihydroxy-kaur-16-ene-
To a solution of compound 18 (33 mg, 0.1 mmol) in THF (1.0 mL)
at room temperature was added Et3N (30 mg, 0.3 mmol) and
cyclohexyl amino (30 mg, 0.3 mmol) successively. The mixture was
stirred at room temperature for 2.5 h until no starting material was
detected by TLC. The solvent was removed under vacuo. The crude
product was subjected to flash chromatography on silica gel (ethyl
acetate/petrol ether ¼ 1:1.5) directly to afford compound 19h as
white foams (41 mg, 95%).
4a-yl) urea (19b). Compound 19b (19 mg, 86%) was prepared from
compound 18 (17 mg, 0.05 mmol) as white solid according to the
synthetic procedure described for 19h. 1H-NMR (CD3OD, 400 MHz)
d
7.17 (dd, J ¼ 8 Hz, 4 Hz, 2H), 6.87 (t, J ¼ 8 Hz, 2H), 4.98 (s, 1H), 4.94
(s, 1H), 3.85 (d, J ¼ 4 Hz,1H), 3.32 (s, 1H), 2.67 (d, J ¼ 12 Hz,1H), 2.52
(d, J ¼ 4 Hz, 1H), 1.85 (t, J ¼ 12 Hz, 2H), 1.76 (dd, J ¼ 12 Hz, 4 Hz, 2H),
1.69 (s, 1H), 1.64 (dd, J ¼ 16 Hz, 4 Hz, 1H), 1.55 (d, J ¼ 12 Hz, 1H), 1.46
(s, 1H), 1.35 (d, J ¼ 12 Hz, 4 Hz, 2H), 1.31 (s, 3H), 1.27 (d, J ¼ 4 Hz, 1H),
1.22 (m, 1H), 1.17 (s, 2H), 1.05 (m, 4H), 0.93 (d, J ¼ 12 Hz, 1H), 0.89 (s,
6.2.15.1. N-(2-thiazolyl)-N-(kaur-16-ene-4
a
-yl)
urea
(15a).
3H); 13C-NMR (MeOD, 100 MHz)
d 159.7, 157.2, 155.6, 135.3, 121.7,
Compound 15a (12 mg, 84%brsm) was prepared from compound 14
(15 mg, 0.05 mmol) as white solid according to the synthetic pro-
cedure described for 19h. Another 6 mg starting material was
115.4, 115.2, 106.2, 81.9, 65.4, 55.6, 55.1, 54.6, 44.4, 42.0, 39.4, 39.0,
38.5, 37.1, 36.6, 36.0, 29.5, 27.5, 18.8, 17.7, 17.0; HR-EI-MS (m/z):
calcd. for C26H35N2O3F [M]þ, 442.2632, found 442.2629.
recovered. 1H-NMR (CDCl3, 400 MHz)
d
7.37 (d, J ¼ 4 Hz, 1H), 6.79
(d, J ¼ 4 Hz, 1H), 4.82 (s, 1H), 4.76 (s, 1H), 2.95 (d, J ¼ 16 Hz, 1H), 2.67
(s, 1H), 2.09 (s, 2H), 1.86 (dd, J ¼ 16 Hz, 8 Hz, 1H), 1.70 (d, J ¼ 16 Hz,
1H), 1.61 (d, J ¼ 8 Hz, 1H), 1.47 (s, 3H), 1.21 (s, 2H), 1.13e1.18 (d,
J ¼ 4 Hz, 3H), 1.01 (d, J ¼ 8 Hz, 1H), 0.83e0.87 (m, 1H); 13C-NMR
6.2.15.6. N-(4-Trifluoronmethyl-phenyl)-N-(11
b,15b-dihydroxy-kaur-
16-ene-4 -yl) urea (19c). Compound 19c (13 mg, 53%) was pre-
a
pared from compound 18 (17 mg, 0.05 mmol) as white solid ac-
cording to the synthetic procedure described for 19h. 1H-NMR
(CDCl3, 100 MHz)
d
162.8, 155.6, 153.7, 136.7, 110.3, 103.1, 56.5, 55.6,
(CDCl3, 400 MHz) d 7.40 (s, 4H), 5.02 (s, 1H), 4.98 (s, 1H), 3.91 (d,
48.9, 44.0, 43.8, 40.8, 40.1, 40.0, 39.0, 36.5, 33.2, 27.6, 19.7, 17.9, 17.9;
HR-EI-MS (m/z): calcd. for C23H33N3OS [M]þ, 399.2344, found
399.2341.
J ¼ 4 Hz, 1H), 3.71 (s, 2H), 2.73 (d, J ¼ 12 Hz, 1H), 2.58 (d, J ¼ 8 Hz,
1H), 2.13 (s, 1H), 1.91 (m, 3H), 1.67 (m, 5H), 1.51 (s, 1H), 1.42 (d,
J ¼ 16 Hz, 2H), 1.35 (s, 3H), 1.20 (s, 3H), 1.02 (s, 3H); 13C-NMR (CDCl3,
100 MHz)
d 157.5, 154.5, 143.2, 125.9, 125.9, 117.8, 106.2, 81.9, 65.4,
6.2.15.2. N-(4-Fluoro-phenyl)-N-(kaur-16-ene-4
a
-yl) urea (15b).
55.6, 55.1, 54.6, 45.0, 41.1, 40.7, 38.3, 37.9, 36.6, 36.2, 36.0, 29.6, 27.6,
18.6, 18.5, 17.6; HR-EI-MS (m/z): calcd. for C27H35N2O3F3 [M]þ,
492.2600, found 492.2599.
Compound 15b (11 mg, 87%brsm) was prepared from compound 14
(15 mg, 0.05 mmol) as white solid according to the synthetic pro-
cedure described for 19h. Another 5 mg starting material was
recovered. 1H-NMR (CDCl3, 400 MHz)
d
7.17e7.21 (m, 2H), 6.88 (t,
6.2.15.7. N-(Thiazol-2-yl)-N-(11b,15b-dihydroxy-kaur-16-ene-4a-yl)
J ¼ 8 Hz, 1H), 4.73 (s, 1H), 4.67 (s, 1H), 2.71 (d, J ¼ 12 Hz, 1H), 2.56 (s,
1H),1.99 (s, 2H),1.88 (d, J ¼ 12 Hz,1H),1.77 (d, J ¼ 12 Hz,1H),1.67 (d,
J ¼ 12 Hz, 1H), 1.40e1.57 (m, 7H), 1.31 (s, 3H), 1.01e1.04 (m, 2H),
urea (19d). Compound 19d (11 mg, 42%) was prepared from com-
pound 18 (17 mg, 0.05 mmol) as white solid according to the syn-
thetic procedure described for 19h. 1H-NMR (CDCl3, 400 MHz)
d 7.2

X. Deng et al. / European Journal of Medicinal Chemistry 65 (2013) 403e414
413
(d, J ¼ 4 Hz, 1H), 6.79 (d, J ¼ 4 Hz, 1H), 4.99 (s, 1H), 4.96 (s, 1H), 3.89
(d, J ¼ 4 Hz,1H), 3.70 (s, 1H), 3.28 (s, 1H), 2.72 (d, J ¼ 12 Hz,1H), 2.56
(d, J ¼ 8 Hz, 1H), 2.12 (s, 1H),1.97 (d, J ¼ 12 Hz,1H), 1.90 (d, J ¼ 12 Hz,
1H), 1.78 (m, 2H), 1.69 (d, J ¼ 12 Hz, 1H), 1.62 (d, J ¼ 16 Hz, 1H), 1.50
(s, 2H), 1.43 (d, J ¼ 12 Hz, 2H), 1.36 (s, 3H), 1.18 (s, 3H), 1.03 (s, 3H);
39.4, 38.9, 38.4, 37.1, 36.8, 35.9, 33.6, 27.6, 25.3, 24.8, 18.9, 17.6, 17.1;
HR-EI-MS (m/z): calcd. for C26H42N2O3 [M]þ, 430.3195, found
430.3185.
6.2.15.12. N-(Tricyclo(3.3.1.13,7)-dec-1-yl)-N-(11
kaur-16-ene-4 -yl) urea (19i). Compound 19i (24 mg, 93%) was
b,15b-dihydroxy-
13C-NMR (CDCl3, 100 MHz)
d
157.5, 152.8, 134.1, 111.8, 106.4, 82.1,
a
65.6, 55.9, 54.8, 44.8, 42.1, 39.6, 39.3, 38.7, 37.4, 36.7, 36.2, 29.0, 27.5,
19.2, 17.9, 17.47; HR-EI-MS (m/z): calcd. for C23H33N3O3S [M]þ,
431.2243, found 431.2223.
prepared from compound 18 (17 mg, 0.05 mmol) as white solid
according to the synthetic procedure described for 19h. 1H-NMR
(CDCl3, 400 MHz)
d
5.10 (s, 1H), 5.01 (s, 1H), 4.01 (d, J ¼ 4 Hz, 1H),
3.93 (s, 1H), 3.83 (d, J ¼ 12 Hz, 2H), 3.75 (d, J ¼ 12 Hz, 1H), 2.74 (d,
J ¼ 16 Hz,1H), 2.62 (s,1H), 2.03 (s, 3H),1.95 (d, J ¼ 12 Hz, 4H),1.91 (s,
6H), 1.79 (m, 4H), 1.64 (s, 6H), 1.57 (s, 1H), 1.47 (m, 2H), 1.35 (s, 3H),
1.24 (s, 1H), 1.12 (m, 1H), 1.06 (s, 3H); 13C-NMR (CDCl3, 100 MHz)
6.2.15.8. N-(Naphthalen-1-yl)-N-(11
b,15b-dihydroxy-kaur-16-ene-
4
a-yl) urea (19e). Compound 19e (9 mg, 75%brsm) was prepared
from compound 18 (17 mg, 0.05 mmol) as white solid according to
the synthetic procedure described for 19h. Another 8 mg starting
d
157.9, 156.9, 106.1, 82.2, 66.1, 55.7, 55.0, 54.2, 50.8, 44.4, 43.0, 42.5,
material was recovered. 1H-NMR (CDCl3, 400 MHz)
d
8.14 (d,
39.7, 38.8, 38.4, 37.3, 36.7, 36.3, 36.0, 29.5, 29.2, 27.9, 19.2, 17.8, 17.8;
HR-EI-MS (m/z): calcd. for C30H46N2O3 [M]þ, 482.3508, found
482.3503.
J ¼ 8 Hz, 1H), 7.92 (d, J ¼ 8 Hz, 1H), 7.85 (d, J ¼ 8 Hz, 1H), 7.54e7.61
(m, 2H), 7.45e7.52 (m, 2H), 6.51 (s, 1H), 5.07 (s, 1H), 4.98 (s, 1H),
4.34 (s, 1H), 3.80 (d, J ¼ 4 Hz, 1H), 3.64 (d, J ¼ 12 Hz, 1H), 3.59 (d,
J ¼ 12 Hz, 1H), 3.23 (dd, J ¼ 8 Hz, 16 Hz, 1H), 2.73 (d, J ¼ 12 Hz, 1H),
2.52 (s, 1H), 2.19 (s, 1H), 1.58e1.77 (m, 8H), 1.52 (d, J ¼ 12 Hz, 1H),
1.49 (s, 3H), 1.39 (s, 2H), 1.36e1.38 (m, 2H), 1.27 (s, 6H), 1.16 (d,
J ¼ 8 Hz, 1H), 1.14 (s, 1H), 1.11 (d, J ¼ 8 Hz, 1H), 1.04e1.09 (m, 2H),
6.2.15.13. N-(Methyl, methoxy)-N-(11
b,15b-dihydroxy-kaur-16-ene-
4a-yl) urea (19j). Compound 19j (19 mg, 96%) was prepared from
compound 18 (17 mg, 0.05 mmol) as white solid according to the
synthetic procedure described for 19h. 1H-NMR (MeOD, 400 MHz)
0.85e0.89 (m, 4H); 13C-NMR (CDCl3, 100 MHz)
d
158.0, 134.7, 133.0,
d 5.16 (s, 1H), 5.15 (s, 1H), 4.05 (s, 1H), 3.87 (s, 1H), 3.79 (s, 3H), 3.44
131.1,130.6,129.0,128.5,128.1,127.4,127.0,125.8,125.2,122.7,106.3,
84.4, 81.5, 76.4, 55.7, 55.6, 51.3, 44.9, 41.6, 40.3, 38.4, 37.9, 36.1, 35.8,
33.9, 27.7, 20.3, 20.3, 19.3, 18.2, 17.7, 17.3; HR-EI-MS (m/z): calcd. for
(s, 1H), 3.12 (s, 3H), 2.74 (m, 2H), 2.14 (m, 2H), 2.00 (m, 3H), 1.88 (dd,
J ¼ 12 Hz, 4 Hz, 1H), 1.71 (s, 2H), 1.65 (d, J ¼ 12 Hz, 4 Hz, 2H), 1.51 (s,
3H), 1.37 (m, 4H), 1.26 (dd, J ¼ 12 Hz, 4 Hz, 2H), 1.23 (s, 3H), 0.98 (d,
C
30H38N2O3 [M]þ, 474.2882, found 474.2888.
J ¼ 8 Hz, 2H); 13C-NMR (MeOD, 100 MHz)
d 161.2, 158.3, 107.5, 83.2,
66.4, 62. 5, 56.9, 56.4, 56.2, 43.0, 40.6, 40.6, 39.8, 38.5, 37.6, 37.2,
6.2.15.9. N-(Methyl)-N-(11
b
,15
b
-dihydroxy-kaur-16-ene-4
a
-yl) urea
36.2, 29.9, 28.4, 20.3, 19.0, 18.5; HR-EI-MS (m/z): calcd. for
(19f). Compound 19f (17 mg, 97%brsm) was prepared from com-
pound 18 (17 mg, 0.05 mmol) as white solid according to the syn-
thetic procedure described for 19h. 1H-NMR (CD3OD, 500 MHz)
C
22H36N2O4 [M]þ, 392.2675, found 392.2675.
6.2.15.14. 4-Morpholine-carboxamide,N-(11
b,15b-dihydroxy-kaur-
d
5.10 (s, 1H), 5.02 (s, 1H), 4.33 (d, J ¼ 4 Hz, 1H), 4.12 (s, 1H), 4.01 (d,
16-ene-4 -yl) urea (19k). Compound 19k (19 mg, 99%) was pre-
a
J ¼ 4 Hz, 1H), 3.86 (d, J ¼ 8 Hz, 1H), 3.76 (d, J ¼ 8 Hz, 1H), 2.74 (d,
J ¼ 12 Hz, 1H), 2.70 (d, J ¼ 4 Hz, 1H), 2.63 (s, 1H), 2.06 (s, 1H), 1.93e
1.96 (m, 3H), 1.77e1.86 (m, 4H), 1.62 (d, J ¼ 12 Hz, 1H), 1.58 (s, 1H),
1.43e1.48 (m, 1H), 1.38 (s, 3H), 1.32 (d, J ¼ 8 Hz, 1H), 1.24 (s, 1H),
pared from compound 18 (17 mg, 0.05 mmol) as white solid ac-
cording to the synthetic procedure described for 19h. 1H-NMR
(CDCl3, 400 MHz)
d 5.10 (s, 1H), 5.02 (s, 1H), 4.39 (s, 1H), 4.00 (d,
J ¼ 4 Hz, 1H), 3.77 (s, 1H), 3.68 (t, J ¼ 4 Hz, 2H), 3.24 (t, J ¼ 4 Hz, 2H),
2.77 (d, J ¼ 16 Hz, 1H), 2.63 (d, J ¼ 4 Hz, 1H), 1.94 (d, J ¼ 12 Hz, 3H),
1.82 (m, 3H), 1.60 (s, 1H), 1.53 (m, 3H), 1.39 (s, 3H), 1.28 (dd, J ¼ 8 Hz,
4 Hz, 1H), 1.24 (s, 1H), 1.17 (dd, J ¼ 16 Hz, 4 Hz, 1H), 1.10 (s, 1H), 1.07
1.06e1.17 (m, 4H), 1.04 (s, 3H); 13C-NMR (CD3OD, 125 MHz)
d 157.9,
157.9, 106.2, 82.2, 66.1, 55.6, 55.1, 54.3, 44.4, 43.0, 39.6, 38.9, 38.4,
37.3, 36.6, 36.1, 27.8, 27.1, 19.1, 17.8, 17.6; HR-EI-MS (m/z): calcd. for
C
21H34N2O3 [M]þ, 362.2569, found 362.2575.
(s, 3H); 13C-NMR (CDCl3, 100 MHz)
d 157.7, 156.6106.2, 82.1, 66.3,
65.9, 55.6, 54.3, 44.1, 43.0, 39.4, 38.8, 38.3, 37.2, 36.1, 35.9, 27.7, 27.7,
19.1, 18.0, 17.6; HR-EI-MS (m/z): calcd. for C24H38N2O4 [M]þ,
418.2832, found 418.2838.
6.2.15.10. N-(Tert-butyl)-N-(11b,15b-dihydroxy-kaur-16-ene-4a-yl)
urea (19g). Compound 19g (19 mg, 99%) was prepared from com-
pound 18 (17 mg, 0.05 mmol) as white solid according to the syn-
thetic procedure described for 19h. 1H-NMR (CDCl3, 400 MHz)
6.2.15.15. 1-Piperzaine-carboxamide,N-(11
b,15b-dihydroxy-kaur-16-
d
5.06 (s, 1H), 4.97 (s, 1H), 3.97 (d, J ¼ 4 Hz, 1H), 3.71 (s, 1H), 2.58 (d,
ene-4 -yl) urea (19l). Compound 19l (16 mg, 86%) was prepared
a
J ¼ 4 Hz, 2H), 2.11 (s, 1H), 1.88e1.95 (m, 3H), 1.69e1.73 (m, 3H), 1.57
(d, J ¼ 20 Hz, 1H), 1.52 (s, 1H), 1.40e1.45 (m, 3H), 1.33 (s, 3H), 1.32 (s,
9H), 1.21 (s, 1H), 1.19 (s, 2H), 1.09e1.13 (m, 2H), 1.04 (s, 3H), 1.01 (s,
from compound 18 (17 mg, 0.05 mmol) as white solid according to
the synthetic procedure described for 19h. 1H-NMR (MeOD,
500 MHz) d 5.02 (s, 1H), 5.01 (s, 1H), 3.92 (s, 1H), 3.74 (s, 1H), 3.34 (t,
1H); 13C-NMR (CDCl3,100 MHz)
d
157.2,157.0,106.0, 82.5, 76.4, 56.0,
J ¼ 5 Hz, 4H), 3.30e3.31 (m, 2H), 3.14 (dd, J ¼ 10 Hz, 5 Hz, 4H), 2.91
(dd, J ¼ 10 Hz, 5 Hz, 3H), 2.74 (d, J ¼ 15 Hz, 1H), 2.59 (s, 1H), 2.03 (d,
J ¼ 10 Hz, 1H), 1.97 (d, J ¼ 10 Hz, 1H), 1.86 (s, 2H), 1.83 (d, J ¼ 10 Hz,
1H), 1.74 (t, J ¼ 5 Hz, 1H), 1.57 (s, 2H), 1.48 (d, J ¼ 10 Hz, 1H), 1.42 (d,
J ¼ 10 Hz, 1H), 1.37 (s, 3H), 1.15 (td, J ¼ 10 Hz, 5 Hz, 2H), 1.10 (s, 3H),
51.3, 45.1, 41.7, 40.5, 38.3, 38.1, 36.6, 36.3, 34.0, 29.2, 29.0, 27.7, 20.2,
19.2, 19.0, 17.7; HR-EI-MS (m/z): calcd. for C24H40N2O3 [M]þ,
404.3039, found 404.3039.
6.2.15.11. N-(Cyclohexyl)-N-(11
b
,15
b
-dihydroxy-kaur-16-ene-4
a
-yl)
1.08 (d, J ¼ 15 Hz, 2H); 13C-NMR (MeOD, 125 MHz)
d 157.7, 156.7,
urea (19h). Compound 19h (41 mg, 95%) was prepared from
compound 18 (33 mg, 0.1 mmol) as white solid according to the
synthetic procedure described for 19h. 1H-NMR (MeOD, 500 MHz)
106.8, 82.6, 65.9, 56.7, 55.8, 47.3, 45.4, 45.2, 44.0, 42.5, 40.2, 40.0,
39.3, 38.0, 37.0, 28.1, 19.7, 18.5, 18.4; HR-EI-MS (m/z): calcd. for
C
24H40N4O3 [M]þ, 432.3100, found 432.3123.
d
4.97 (s,1H), 4.93 (s,1H), 3.86 (d, J ¼ 5 Hz,1H), 3.72 (m, 6H), 3.25 (d,
J ¼ 15 Hz, 1H), 2.61 (d, J ¼ 15 Hz, 1H), 2.51 (s, 1H), 1.90 (d, J ¼ 10 Hz,
1H), 1.84 (d, J ¼ 15 Hz, 1H),1.79 (s, 2H), 1.75 (s, 2H), 1.67 (m, 4H),1.51
(m,1H),1.46 (s,1H),1.35 (t, J ¼ 15 Hz,1H),1.21 (d, J ¼ 10 Hz, 2H),1.01
(m, 4H), 0.97 (s, 3H), 0.92 (d, J ¼ 15 Hz, 1H); 13C-NMR (MeOD,
6.2.15.16. 1-Pyrrolidine-carboxamide,N-(11
b,15b-dihydroxy-kaur-16-
ene-4 -yl) urea (19m). Compound 19m (19 mg, 98%) was prepared
a
from compound 18 (17 mg, 0.05 mmol) as white solid according to
the synthetic procedure described for 19h. 1H-NMR (MeOD,
125 MHz)
d
157.4, 157.2, 106.0, 81.8, 65.3, 55.7, 54.7, 54.6, 44.4, 42.0,
500 MHz) d 4.56 (s,1H), 4.55 (s, 1H), 3.46 (s, 1H), 3.27 (s, 1H), 2.88 (s,

414
X. Deng et al. / European Journal of Medicinal Chemistry 65 (2013) 403e414
1H), 2.81 (m, 5H), 2.29 (d, J ¼ 8 Hz, 1H), 2.13 (d, J ¼ 4 Hz, 1H), 1.58 (d,
J ¼ 8 Hz, 1H), 1.52 (d, J ¼ 8 Hz, 1H), 1.46 (t, J ¼ 8 Hz, 1H), 1.39 (m, 3H),
1.30 (dt, J ¼ 12 Hz, 4 Hz, 1H), 1.18 (d, J ¼ 12 Hz, 1H), 1.12 (s, 1H), 1.04
(d, J ¼ 12 Hz, 1H), 0.99 (m, 1H), 0.93 (s, 3H), 0.69 (s, 3H), 0.63 (dd,
J ¼ 8 Hz, 4 Hz,1H), 0.60 (d, J ¼ 8 Hz,1H); 13C-NMR (MeOD,125 MHz)
Acknowledgments
We thanked professor Wei-Liang Zhu for constructive advices.
We also thanked the National Natural Science Foundation of China
(Nos. U0932602, 2011CB915503, 90813004, and 2009CB522300)
for financial support.
d
158.0, 157.6, 106.9, 82.8, 66.0, 56.9, 56.3, 56.1, 46.2, 45.5, 42.6, 40.3,
40.2, 39.5, 38.1, 37.1, 37.1, 28.4, 26.2, 19.8, 18.6, 18.4; HR-EI-MS (m/z):
calcd. for C24H38N2O3 [M]þ, 425.2780, found 425.2782.
Appendix A. Supplementary data
6.2.16. 11b,15b-dihydroxy-kaur-16-en-19-oic acid methyl ester (17)
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2013.05.010.
To a solution of compound 2 (17 mg, 0.05 mmol) in Et2O (1.0 mL)
at 0 ꢁC under N2 was added a freshly prepared solution of CH2N2 in
Et2O (1 N, 1.0 mL) dropwise. The mixture was stirred for 15 min
until no starting material was detected by TLC judgment. Then
HOAc (0.5 mL) was added to quench the reaction, and H2O was
added to dilute the mixture. The aqueous phase was extracted with
EtOAc (10 mL ꢂ 3). The combined organic layers were washed with
H2O and brine successively, dried over anhydrous Na2SO4 and
concentrated. The crude product was subjected to flash chroma-
tography on silica gel (ethyl acetate/petrol ether ¼ 1:4) to afford
compound 17 as white foams (18 mg, 99%). The structure of com-
pound 17 was characterized by comparing the spectra data with
that reported in the literature [38].
References
[1] P. Zimmet, K.G.M.M. Alberti, J. Shaw, Nature 414 (2001) 782e787.
[2] J.S. Skyler, J. Med. Chem. 47 (2004) 4113e4117.
[3] P. Malhotra, R. Kumar, S. Kumari, M.M. Singh, Lancet 347 (1996) 1833e1835.
[4] J.W. Tomlinson, E.A. Walker, I.J. Bujalska, N. Draper, G.G. Lavery, M.S. Cooper,
M. Hewison, P.M. Stewart, Endocr. Rev. 25 (2004) 831e866.
[5] G. Arnaldi, A. Angeli, A.B. Atkinson, X. Bertagna, F. Cavagnini, G.P. Chrousos,
G.A. Fava, J.W. Findling, R.C. Gaillard, A.B. Grossman, B. Kola, A. Lacroix,
T. Mancini, F. Mantero, J. Newell-Price, L.K. Nieman, N. Sonino, M.L. Vance,
A. Giustina, M. Boscaro, J. Clin. Endocrinol. Metab. 88 (2003) 5593e5602.
[6] P.M. Stewart, C.B. Worwood, B.R. Walker, Steroids 58 (1993) 614e620.
[7] B.R. Walker, A.A. Connacher, R.M. Lindsay, D.J. Webb, C.R.W. Edwards, J. Clin.
Endocrinol. Metab. 80 (1995) 3155e3159.
[8] Y. Kotelevtsev, R.W. Brown, S. Fleming, C. Kenyon, C.R.W. Edwards, J.R. Seckl,
J.J. Mullins, J. Clin. Invest. 103 (1999) 683e689.
6.3. Biological assay
[9] H. Masuzaki, J. Paterson, H. Shinyama, N.M. Morton, J.J. Mullins, J.R. Seckl,
J.S. Flier, Science 294 (2001) 2166e2170.
6.3.1. Scintillation proximity assay (SPA) for identification of 11b-
HSD1 inhibitors [29]
[10] B. Davani, A. Khan, M. Hult, E. Mårtensson, S. Okret, S. Efendic, H. Jörnvall,
U.C.T. Oppermann, J. Biol. Chem. 275 (2000) 34841e34844.
[11] T.M. Stulnig, W. Waldhaeusl, Diabetologia 47 (2004) 1e11.
[12] B.R. Walker, J.R. Seckl, Expert Opin. Ther. Targets 7 (2003) 771e783.
[13] C. Fotsch, B.C. Askew, J.J. Chen, Expert Opin. Ther. Pat. 15 (2005) 289e303.
[14] C.D. Boyle, T.J. Kowalski, L. Zhang, Annu. Rep. Med. Chem. 41 (2006) 127e140.
[15] R. Ge, Y. Huang, G. Liang, X. Li, Curr. Med. Chem. 17 (2010) 412e422.
[16] K.C.K. Swamy, N.N.B. Kumar, E. Balaraman, K.V.P.P. Kumar, Chem. Rev. 109
(2009) 2551e2651.
[17] O. Illa, M. Arshad, A. Ros, E.M. McGarrigle, V.K. Aggarwal, J. Am. Chem. Soc. 132
(2010) 1828e1830.
[18] D. Mormeneo, A. Llebaria, A. Delgado, Tetrahedron Lett. 45 (2004) 6831e6834.
[19] J.J. Topczewski, J.D. Neighbors, D.F. Wiemer, J. Org. Chem. 74 (2009) 6965e
6972.
[20] N. Rabjohn, Org. React. 5 (1949) 331e386.
[21] Y. Zou, C.H. Chen, C.D. Taylor, B.M. Foxman, B.B. Snider, Org. Lett. 9 (2007)
1825e1828.
[22] G.O. Lobitz, G. Tamayo-Castillo, L. Poveda, I. Merfort, Phytochemistry 49
(1998) 805e809.
[23] R. Fujimoto, Y. Kishi, J.F. Blount, J. Am. Chem. Soc. 102 (1980) 7154e7156.
[24] J.E. McMurry, Tetrahedron Lett. 11 (1970) 3735e3738.
[25] A. De Mico, R. Margarita, L. Parlanti, A. Vescovi, G. Piancatelli, J. Org. Chem. 62
(1997) 6974e6977.
Inhibition of compounds on human 11
HSD1 enzymatic activities was determined by the scintillation
proximity assay (SPA) using microsomes containing 11 -HSD1 ac-
cording to previous studies [29]. Briefly, the full-length cDNAs of
human 11 -HSD1 and mouse 11 -HSD1 were isolated from the
b-HSD1 and mouse 11b-
b
b
b
cDNA libraries provided by NIH Mammalian Gene Collection and
cloned into pcDNA3 expression vector. HEK-293 cells were trans-
fected with the pcDNA3-derived expression plasmid and selected
by cultivation in the presence of 700
somal fraction overexpressing 11 -HSD1 was prepared from the
HEK-293 cells stable transfected with 11 -HSD1 and used as the
enzyme source for SPA. Microsomes containing 11 -HSD1 was
mg/mL of G418. The micro-
b
b
b
incubated with NADPH and [3H] cortisone (Amersham), then the
product, [3H] cortisol was specifically captured by a monoclonal
antibody coupled to protein A-coated SPA beads (GE). IC50 values
were calculated by using Prism Version 4 (GRAPHPAD Software,
San Diego, CA).
[26] C.D. Boyle, Curr. Opin. Drug Discov. Dev. 11 (2008) 495e511.
[27] C.D. Boyle, T.J. Kowalski, Expert Opin. Ther. Pat. 19 (2009) 801e825.
[28] E.F.V. Scriven, K. Turnbull, Chem. Rev. 88 (1988) 297e368.
[29] T. Curtius, J. Prakt. Chem. 50 (1894) 275e294.
6.4. Molecular modeling of compound 19a with 11b-HSD1
[30] S. Mundt, K. Solly, R. Thieringer, A. Hermanowski-Vosatka, Assay Drug Dev.
Tech. 3 (2005) 367e375.
[31] Y.L. Ye, Z. Zhou, H.J. Zou, Y. Shen, T.F. Xu, J. Tang, H.Z. Yin, M.L. Chen, Y. Leng,
J.H. Shen, Bioorg. Med. Chem. 17 (2009) 5722e5732.
[32] M. Hult, N. Shafqat, B. Elleby, D. Mitschke, S. Svensson, M. Forsgren, T. Barf,
J. Vallgårda, L. Abrahmsen, U. Oppermann, Mol. Cell. Endocrinol. 248 (2006)
26e33.
[33] Y.-C. Wu, Y.-C. Hung, F.-R. Chang, M. Cosentino, H.-K. Wang, K.-H. Lee, J. Nat.
Prod. 59 (1996) 635e637.
[34] M.O. Fatope, O.T. Audu, Y. Takeda, L. Zeng, G. Shi, H. Shimada, J.L. McLaughlin,
J. Nat. Prod. 59 (1996) 301e303.
[35] W. Herz, R.P. Sharma, J. Org. Chem. 41 (1976) 1021e1026.
[36] E.L. Ghisalberti, P.R. Jefferies, M.A. Sefton, P.N. Sheppard, Tetrahedron 33
(1977) 2451e2456.
[37] I. Hueso-Falcón, I. Cuadrado, F. Cidre, J.M. Amaro-Luis, Á.G. Ravelo, A. Estevez-
Braun, B. de las Heras, S. Hortelano, Eur. J. Med. Chem. 46 (2011) 1291e1305.
[38] P.C. Cheng, C.D. Hufford, N.J. Doorenbos, J. Nat. Prod. 42 (1979) 183e186.
[39] R.A. Friesner, J.L. Banks, R.B. Murphy, T.A. Halgren, J.J. Klicic, D.T. Mainz,
M.P. Repasky, E.H. Knoll, M. Shelley, J.K. Perry, D.E. Shaw, P. Francis,
P.S. Shenkin, J. Med. Chem. 47 (2004) 1739e1749.
The compound 19a was prepared (generating stereoisomers and
valid single 3D conformers) by means of the Ligand Preparation
module in Maestro. The crystal structures of mouse and human
11b-HSD1 were retrieved from the Protein Data Bank (PDB entry:
1Y5R and 2IRW). All crystallographic water molecules were
removed from the coordinate set. Glide was used for docking. In the
docking process, the standard docking score was used to rank the
docking conformations. All the parameters were set as the default
values. The grid-enclosing box was centered on the centroid of
cocomplexed 19a in 11b-HSD1 and defined so as to enclose residues
ꢀ
located within 14.0 A around the 19a binding site, and default van
der Waal scaling was used (1.0 for the receptor and 0.8 for the
ligand). The RMSD between the top-scoring ligand orientation and
the crystal ligand orientation was less than 2.0 A. The binding poses
of compound 19a were modeled by Glide (Schrödinger, Inc.) in SP
mode with the same parameter settings [39,40].
[40] T.A. Halgren, R.B. Murphy, R.A. Friesner, H.S. Beard, L.L. Frye, W.T. Pollard,
J.L. Banks, J. Med. Chem. 47 (2004) 1750e1759.