Identification and optimisation of 3,3-dimethyl-azetidin-2-ones as potent and selective inhibitors of 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1)

Article abstract of DOI:10.1039/c3md00234a

3,3-Di-methyl-azetidin-2-ones were identified as potent and selective 11β-HSD1 inhibitors against the human and mouse forms of the enzyme. Structure guided optimisation of LLE was conducted, utilising a key polar interaction and identifying stereochemical preference for the 4S isomer. Metabolic stability was improved to afford oral exposure, providing tool compounds suitable for pre-clinical evaluation.

Full text of DOI:10.1039/c3md00234a

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Identification and optimisation of 3,3-dimethyl-
azetidin-2-ones as potent and selective inhibitors
of 11b-hydroxysteroid dehydrogenase type 1 (11b-
Cite this: Med. Chem. Commun., 2014,
5, 57
HSD1)†
a
William McCoull, Martin Augustin,c Caroline Blake,a Anne Ertan,b Elaine Kilgour,a
*
Stephan Krapp,c Jane E. Moore,a Nicholas J. Newcombe,a Martin J. Packer,a
Amanda Rees,a John Revill,a James S. Scott,a Nidhal Selmi,a Stefan Gerhardt,a
Derek J. Ogg,a Stefan Steinbacherc and Paul R. O. Whittamorea
Received 21st August 2013
Accepted 4th November 2013
3,3-Di-methyl-azetidin-2-ones were identified as potent and selective 11b-HSD1 inhibitors against the
human and mouse forms of the enzyme. Structure guided optimisation of LLE was conducted, utilising a
key polar interaction and identifying stereochemical preference for the 4S isomer. Metabolic stability was
improved to afford oral exposure, providing tool compounds suitable for pre-clinical evaluation.
DOI: 10.1039/c3md00234a
www.rsc.org/medchemcomm
liver, adipose tissue, and brain.10 It catalyses the interconver-
sion of the inactive glucocorticoid hormone cortisone 1 to the
Introduction
active glucocorticoid hormone cortisol 2 and therefore plays a
key role in the regulation of intracellular cortisol concentrations
(Fig. 1).11 11b-Hydroxysteroid dehydrogenase type 2 (11b-HSD2)
is an NAD dependent oxidase expressed mainly in kidney and
colon tissue that catalyses the reverse reaction and prevents
cortisol activation of mineralocorticoid receptors.12 Inhibition
of this enzyme has been associated with hypertension and other
complications and therefore selectivity over this isoform is a key
requirement for any potential therapeutic agent.13 Carbenox-
olone (CBX) 3, originally developed as a treatment for oeso-
The principal strategy in the management or prevention of type
II diabetes and cardiovascular disease involves treatment of a
collection of risk factors. When a patient exhibits central obesity
in combination with two of the following risk factors; elevated
fasting plasma glucose, dyslipidemia, hypertension, reduced
high density lipoprotein (HDL) cholesterol they are dened as
having the metabolic syndrome.1 The underlying causes of the
metabolic syndrome are complex and the synthesis and
metabolism of glucocorticoids have been proposed to play a key
role.2,3 Human patients with Cushing’s syndrome, who exhibit
elevated circulating glucocorticoid levels, display a phenotype
similar to the metabolic syndrome.4 Although patients with the
metabolic syndrome do not exhibit elevated plasma glucocor-
ticoid levels,5 it has been hypothesised that elevated intracel-
lular concentrations may play a crucial role. Therefore, the
ability to reduce intracellular glucocorticoid concentrations has
been proposed as an attractive therapeutic paradigm for the
metabolic syndrome.6–9
phageal ulceration and inammation, is
a potent but
unselective inhibitor of both 11b-HSD1 & 11b-HSD2.14 It has
been shown to improve insulin sensitivity and reduce glucose
production in patients with type II diabetes but is not used
clinically for the treatment of the metabolic syndrome due to
the associated hypertension risk.15
There has been signicant interest in the therapeutic
potential of selective inhibitors of 11b-HSD1. Over 250
patent applications from over 25 pharmaceutical companies
and academic groups have now been published and
11b-Hydroxysteroid dehydrogenase type 1 (11b-HSD1) is a
NADPH dependent enzyme that is widely expressed, notably in
aCardiovascular and Gastrointestinal Innovative Medicines Unit, AstraZeneca,
Alderley Park, Maccleseld, Cheshire, SK10 4TG, UK. E-mail: william.mccoull@
astrazeneca.com; Tel: +44 (0)1625 519444
bMedicines Evaluation Pharmaceutical Development, AstraZeneca R&D, 151 85
¨
¨
Sodertalje, Sweden
cProteros biostructures GmbH, Am Klopferspitz 19, Martinsried, Germany
† Electronic supplementary information (ESI) available: Synthetic details for the
preparation of compounds; experimental protocols and X-ray coordinates for
the crystal structure of 30 (CCDC: 882283) bound to 11b-HSD1; 11b-HSD1 & 2
assay protocols. See DOI: 10.1039/c3md00234a
Fig. 1 Interconversion of cortisone 1 to cortisol 2 by 11b-HSD1 and 2.
Med. Chem. Commun., 2014, 5, 57–63 | 57
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comprehensively reviewed.16–18 Our approach to identifying 11b-
HSD1 inhibitors was to run a high throughput screen (HTS) and
progress multiple chemical series in parallel. A 2-thioalkyl
nicotinamide chemotype was progressed but initially exhibited
modest rodent potency as recently reported elsewhere.19–23 We
wished to progress another novel, structurally distinct series
that, while selective for 11b-HSD1 over 11b-HSD2, was also
potent against a rodent species to allow pre-clinical evaluation.
In this communication we describe the optimisation of an
azetidin-2-one series to deliver human and mouse potent 11b-
HSD1 inhibitors that are selective over 11b-HSD2 with sufficient
oral exposure to be used as preclinical tool compounds.
Fig. 2 Key interaction pharmacophore defined by the crystal structure
of carbenoxolone 3 bound to human 11b-HSD1, showing a 6 A˚
separation between acceptor groups bound in the active site. See
Fig. 5 for a detailed view of the active site as compared with the 2-
azetidinone series.
Results and discussion
The lead compound 4 was obtained direct from HTS and had
several attractive features. It was potent against human and
murine 11b-HSD1 (although not rat), selective against 11b-
HSD2 and hERG and displayed sub-optimal physicochemical
properties (Table 1). High log D7.4 and very high in vitro
metabolism translated into zero oral exposure in rat and this
was a key focus of the optimisation campaign. The low molec-
ular weight and synthetic accessibility of the azetidin-2-one core
allowed for SAR exploration around all three positions.
Our initial focus was to establish SAR to achieve reduction of
lipophilicity while maintaining potency. In doing so we expected
that the two electron-rich aryl rings (likely metabolic so spots)
would be replaced leading to modications that would suggest a
way to reduce metabolism as well. Our tactic was to keep at least
one of these aryl groups and use ligand lipophilicity efficiency
(LLE)24 as a key optimisation parameter. At this time, a crystal
structure with carbenoxolone (CBX) bound to human 11b-HSD1
was available (PDB code 2bel)25 and compound design was
guided by docking virtual ligands into this structure.
Fig. 3 Docking of 43 into 11b-HSD1 binding site, which was modelled
from the 2bel crystal structure.
We believed that the azetidin-2-one carbonyl would mimic the
ketone of CBX and postulated that adding a polar interaction
˚
about 6 A distant from this into our ligands could realise an
additional binding interaction. Docking azetidin-2-ones into
this structure using Glide26 oriented the carbonyl as expected
and the gem-di-Me group into shallow lipophilic pockets as
shown in Fig. 3.27 The aryl ring on the azetidin-2-one nitrogen
was orientated in the direction of Tyr183, which suggested the
incorporation of a polar substituent, capable of hydrogen
bonding, at the meta-position, as a way to increase LLE.
Furthermore, only the (4S)-enantiomer docked well suggesting
that it would be more potent than the (4R)-enantiomer.
3,3-Dimethyl-azetidin-2-ones were synthesised through
routes summarised in Scheme 1. The Staudinger cycloaddi-
tion28 was used to directly prepare di-aryl azetidin-2-ones 6 from
imines 5 usually in good yield. Azetidin-2-ones with other 3-
alkyl substituents were prepared similarly. To prepare N-alkyl
analogues 9, the para-methoxyphenyl (PMP) protecting group
was oxidatively cleaved by treatment with ceric ammonium
nitrate,29 followed by alkylation of 8 at low temperature.30
Alternatively, N-arylation could be achieved from 8 via Buch-
wald–Hartwig amination.31,32 Alkyl groups were incorporated
into the C4 position by conversion of the easily accessible
aldehyde 11 into the corresponding vinylazetidine, which was
hydrogenated in excellent yield to give 12, or reduction and
methylation to give ether 13.
Our analysis of the CBX/11b-HSD1 structure identied that
the active site Tyr183 and Ser170 formed polar interactions with
˚
the tertiary acid and ketone groups, spaced about 6 A apart
(Fig. 2); there is also a hydrogen bond interaction between the
tertiary acid and a ribose hydroxyl group of the NAD co-factor.
Table 1 Properties of 2-azetidinone lead compound 4
h 11b-HSD1 IC50 (mM)a
mu 11b-HSD1 IC50 (mM)a
Rat 11b-HSD1 IC50 (mM)
h 11b-HSD2 IC50 (mM)
hERG IC50 (mM)
0.098
0.25
5.9
>30
13
MW
log D7.4
Solubility (mM)
Rat ppb %free
Rat heps Clint
(mL per min per 106 cells)
311
>4.2
7.9
0.63
>300
a
Mean value of at least 3 experiments.
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Table 3 Potency, log D7.4 and LLE of selected 1-substituted azetidin-
2-ones
R
h 11b-HSD1 IC50a (mM)
log D7.4
LLE
4
4-MeO-Ph
Et
PhCH2
Ph
2-Pyridyl
2-F-Ph
0.098
2.8
0.20
0.089
2.5
0.20
0.30
0.15
0.11
0.029
0.057
13
0.076
0.66
0.40
0.057
>4.2
2.7
3.7
>4.3
3.2
4.2
—
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33b
2.9
3.0
—
2.4
2.5
—
—
—
—
—
2.1
4.2
3
2.6
4.5
Scheme 1 Representative synthetic route to 3,3-dimethylazetidine-2-
i
ones. Reagents and conditions: PMP ¼ para-MeO-phenyl. (a) Pr-
CO2Et, LiHMDS, PhMe–THF, 0 ꢀC – RT, 8–90%; (b) CeIV(NH4)2(NO3)6,
MeCN–H2O, 0 ꢀC, 50%; (c) LiHMDS, R–X, ꢁ78 ꢀC, THF, 29–45% or
Pd2(dba)3, xantphos, Cs2CO3, dioxane, PhBr, 150 ꢀC 30%; (d) iPrCO2Et,
LDA, THF, 0 ꢀC – RT, 55%; (e) HCl, MeOH, 65%; (f) tBuOK, THF,
Ph3P+MeIꢁ, 86%. (g) H2, 10% Pd/C, EtOAc, RT, 3 h, 98%; (h) NaBH4,
MeOH, 75%; (i) NaH, MeI, THF, 55%.
3-F-Ph
4-F-Ph
>4
>4.3
>4.3
>4.3
>4.1
2.8
2.9
3.2
3.8
2.8
2-Me-Ph
3-Me-Ph
4-Me-Ph
2-MeSO2-Ph
3-MeSO2-Ph
4-MeSO2-Ph
3-MeS-Ph
3-MeSO-Ph
Table 2 Potency of selected 3-substituted azetidin-2-ones
a
b
Mean value of at least 3 experiments. Diastereomers unresolved.
(Table 3). Alkyl substituents such as ethyl (19) were less potent,
although benzyl (20) did maintain potency. Introduction of
nitrogen in pyridyl (22) did lower lipophilicity but lost potency
relative to phenyl (21). A scan of uorine and methyl groups at
all three positions was conducted (23–28) which suggested that
R1
R2
h 11b-HSD1 IC50a (mM)
Table 4 Potency, log D7.4 and LLE of selected 4-substituted azetidin-
2-ones
4
Me
Me
Et
H
H
Me
Et
Et
c-Pr
n-Pr
0.098
2.1
>30
0.90
>30
0.83
14b
15
16c
17c
18
–(CH2)4–
a
Mean value of at least 3 experiments. b 2 : 1 trans : cis. c trans-Racemic.
R
h 11b-HSD1 IC50a (mM)
log D7.4
LLE
Firstly, substitution was investigated at the 3-position with
gem-di-Me found to be optimal for potency (Table 2). According
to our postulated docking pose, both methyl groups t snugly
into lipophilic pockets and even small changes were not toler-
ated thus we accepted that this was not a position where lip-
ophilicity could be reduced. It is notable that a butyrolactam
series of 11b-HSD1 inhibitors33 has been reported where gem-di-
methyl substitution next to the lactam carbonyl was found to be
optimal for human and murine potency, consistent with our
results. All compounds in Table 2 had log D7.4 that were too
high to measure accurately (>3.8) and rat hepatocytes gave high
Clint values (>300 mL per min per 106 cells).
12
13
34
35
4
36
37
38
39
40
41
42
43
Et
6.3
>30
0.24
6.0
0.098
0.28
0.13
0.086
0.18
3.0
0.11
0.079
0.033
3.0
2.2
3.7
2.6
>4.2
3.9
3.8
3.9
3.4
>4.3
>4.1
>4.3
3.7
2.2
—
2.9
2.6
—
2.7
3.1
3.1
3.3
—
MeOCH2
Ph
2-Pyridyl
2-MeO-Ph
3-MeO-Ph
4-MeO-Ph
2-HO-Ph
4-F-Ph
(R)-4-Br-Ph
(S)-4-Br-Ph
1-Naphthyl
5-Quinoxalyl
—
—
3.8
The N1 position of the azetidin-2-one was more tolerant to
structural modication and allowed lipophilicity to be lowered
a
Mean value of at least 3 experiments.
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substitution at all positions could be tolerated. We envisaged enantiomer 41 was more potent than (4R)-enantiomer 40 in
˚
that a sulfone at the meta-position, about 6 A distant from the accord with our docking hypothesis. The absolute stereo-
carbonyl would satisfy the favourable polar interaction with chemistry of (4R)-40 was unambiguously determined by X-ray
Tyr183 as suggested by our docking studies. This was conrmed crystallographic analysis (Fig. 4).34 Naphthyl 42 was potent
when all three sulfones were tested, the meta-sulfone 30 being 9- and was evolved into quinoxaline 43 which, in line with our
fold more potent than the para-sulfone 31, and 170-fold more docking studies, realised increased potency and the highest LLE
potent than ortho-sulfone 29. Furthermore, the meta-sulde 32 from this set.
lacking the hydrogen bonding acceptor was less potent while
The crystal structure of sulfone 30 bound to murine 11b-
the meta-sulfoxide 33 which does provide an acceptor was HSD1 was obtained to explore our binding pharmacophore
˚
equipotent. Consequently, 30 and 33 represent signicant hypothesis of two polar interactions spaced about 6 A apart
progress in terms of reducing log D7.4 and improving LLE (Fig. 5). The potency of 30 against the murine form of 11b-HSD1
relative to the initial lead 4.
(IC50 ¼ 0.15 mM) is similar to that in human, thus we expected
In parallel, we explored the 4-position of the azetidin-2-one similar binding characteristics in both species. The tertiary acid
with the N1 position xed as 4-methoxyphenyl (Table 4). of CBX makes a hydrogen bond contact to a ribose hydroxyl
Although non-polar alkyl groups were tolerated, ethyl (12) group and to Tyr183; we therefore expected the sulfone group of
resulted in lower potency relative to phenyl (34). Attempts to 30 to mimic this interaction. However, it appears from the
introduce polarity such as pyridine (35) resulted in lower contact network that Tyr183 is forced to act as proton donor to
potency albeit with similar LLE. Aryl substitution was tolerated both the sulfone and to the azetidin-2-one carbonyl for ligand
at all positions (4, 36–39) and it was conrmed that the (4S)- 30. The hydroxyl proton could be located between the two
acceptor oxygen atoms to create a favourable polar environ-
ment, which is however not optimal for either interaction. This
motif is not present for CBX, where the ketone group is
deployed towards Ser170 only, leaving Tyr183 to act as donor for
the acid. The azetidin-2-one carbonyl, is oriented between
Ser170 and Tyr183, rather than only towards Ser170 as for the
CBX ketone group. There is also a short contact to the serine,
suggesting a strong hydrogen bond. This denes a favourable
network of contacts, with the azetidin-2-one carbonyl posi-
tioned more optimally than the ketone of CBX. It is a feature of
inhibitors of this target that contacts are observed to one or
both of the active site residues.23
We obtained the murine structure because human struc-
tures were not accessible at the time of this work, although they
did subsequently become available for a range of ligands.19,20,23
Given our restriction to a murine construct, we compared the
Fig. 4 X-ray crystallographic structure of (4R)-40.
Fig. 5 Binding modes for carbenoxolone in the active site of human 11b-HSD1 (red ribbon) and for azetidin-2-one 30 in the active site of murine
11b-HSD1 (yellow ribbon), as revealed by X-ray crystal structures. Carbenoxolone interacts with Ser170 via its ketone group (2.9 A˚) and with
Tyr183 via the tertiary acid (2.6 A˚); there is also a contact between the acid and a ribose hydroxyl group of NADP+ (2.76 A˚). By contrast, the amide
group of compound 30 acts as an isostere of the ketone from CBX, but interacts with both Ser170 (2.5 A˚); and Tyr183 (2.8 A˚). The sulfone acts as
an isostere for the tertiary acid, but does not make an optimum contact with either Tyr183 (3.3 A˚); or the ribose group. This is despite the similar
pharmacophore pattern that we observe in these two ligands, with a 6 A˚ separation between the two acceptor groups in 30, similar to CBX
(Fig. 2).
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the active site which would increase human affinity at the
expense of mouse, and this has been conrmed in other dis-
closed series.19
Furthermore, the crystal structure conrmed that the gem-di-
methyl group ts into a hydrophobic depression in the enzyme
surface that would not accommodate larger groups (anked by
Leu171 and Tyr177) and it is the (4S)-enantiomer that exists in
the crystal structure in line with higher potency than the (4R)-
enantiomer.
The substituents at the 1 and 4 positions which gave the
highest LLE were combined and it was found that the 1-meta-
sulfone-4-quinoxaline combination in 45 gave the most advan-
tageous potency/lipophilicity balance with an LLE of 4.7 (Table
5). The enantiomer pair 46 and 47 conrmed the dependence of
potency on stereochemistry at the 4-position, although in this
case the absolute stereochemistry was not unambiguously
determined. Selectivity against 11b-HSD2 (>30 mM) was main-
tained throughout this work and 45 and 48 were conrmed as
potent against murine 11b-HSD1 (0.35 mM and 0.68 mM
respectively) and inactive against rat 11b-HSD1 (>10 mM). All
Fig. 6 Overlay of active site for human 11b-HSD1 (red ribbon) and
murine 11b-HSD1 (yellow ribbon) demonstrating close homology
between side-chains in the region contacted by ligand 30 (shown in
yellow). Contacts available to this ligand do not favour binding to the
human enzyme over the murine form.
available human and murine structures to explore side-chain compounds containing a methoxy-phenyl had high in vitro
variations within the active site. Fig. 6 shows an overlay of the metabolism (Clint > 200 mL per min per 106 cells), except 31
2bel human binding site (red ribbon) and our murine structure (Clint ¼ 82 mL per min per 106 cells), 12 (Clint ¼ 86 mL per min
(yellow ribbon). It is clear that variations are relatively minor in per 106 cells), and 13 (Clint ¼ 130 mL per min per 106 cells).
the active site, especially in regions that ligand 30 contacts. This However, all combination compounds 44–48 showed lower in
suggests that the limited size of the scaffold facilitates binding vitro metabolism (Table 5) which translated into low to
to both enzymes; the two active sites differ very little within the moderate clearance in vivo (Table 6). Two compounds (44 and
region that this ligand can access. There were clearly regions of 48) were progressed to oral dosing in rat and this conrmed that
Table 5 Selected properties for combinations of substituents at 1 and 4-position of azetidin-2-one
Rat/mouse hep Clint
R1
R4
h 11b-HSD1 IC50a (mM)
log D7.4
LLE
(mL per min per 106 cells)
44
45
46
47
48
3-MeSO2-Ph
3-MeSO2-Ph
4-F-Ph
4-F-Ph
4-F-Ph
4-F-Ph
0.91
0.074
>30
0.28
0.091
2.8
2.4
4.2
4
3.2
4.7
—
2.6
3.3
16/46
32/<3
30/18
25/NT
117/125
5-Quinoxalyl
(R)-4-F-Ph
(S)-4-F-Ph
5-Quinoxalyl
3.7
a
Mean value of at least 3 experiments; NT ¼ not tested.
Table 6 Pharmacokinetic parameters for selected azetidin-2-onesa
Species
Clp (mL minꢁ1 kgꢁ1
)
Vdss (L kgꢁ1
)
IV half-life (h)
PO Cmax (mM)
Bioavailability (%)
44
45
47
48
45
Rat
Rat
Rat
Rat
2.9
52
19
57
42
1.2
3.2
9.7
2.6
1.0
5.4
0.77
7.2
0.76
0.3
0.95
NT
NT
0.11
0.7
27
NT
NT
39
Mouse
22
a
Compound was dosed to rats IV at 0.4 mg kgꢁ1 and PO at 2 mg kgꢁ1, to mouse IV at 2 mg kgꢁ1 and PO at 2.5 mg kgꢁ1, in 5% DMSO : 95%
hydroxypropyl beta cyclodextrin; NT ¼ not tested.
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acceptable oral exposure could be obtained for this series. 12 R. C. Wilson, S. Dave-Sharma, J. Wei, V. R. Obeyesekere,
Mouse hepatocyte data (Table 5) showed a similar trend to rat
and in vivo pharmacokinetic data (Table 6) conrmed that 45
had acceptable oral exposure in mouse suitable for use in pre-
clinical models.
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Conclusions
In summary, we identied a 3,3-di-methyl-azetidin-2-one series
of 11b-HSD1 inhibitors which are potent against both the
human and mouse enzymes and selective against 11b-HSD2.
Optimisation of LLE was conducted, using the CBX/11b-HSD1
crystal structure to guide incorporation of a sulfone to pick up a
polar interaction with the enzyme that was conrmed by a
ligand/protein crystal structure. The preference for the (4S)
stereoisomer being more potent was predicted by docking and
conrmed in practice. Incorporation of less metabolically labile
functional groups allowed oral exposure in rat and mouse to be
realised. The potential of the 3,3-di-methyl-azetidin-2-ones
series to deliver pre-clinical tools has been demonstrated.
Acknowledgements
This research was sponsored by AstraZeneca. The authors thank
Steve Bloor, Rob Garcia and Sally Johnson for the testing of
compounds in biological assays and colleagues in physical
chemistry, DMPK and safety assessment for the provision of
secondary ADMET data.
21 J. S. Scott, A. L. Gill, L. Godfrey, S. D. Groombridge, A. Rees,
¨
J. Revill, P. Schoeld, P. Sorme, A. Stocker, J. G. Swales and
P. R. O. Whittamore, Bioorg. Med. Chem. Lett., 2012, 22,
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22 J. S. Scott, P. Barton, S. N. L. Bennett, J. deSchoolmeester,
L. Godfrey, E. Kilgour, R. M. Mayers, M. J. Packer, A. Rees,
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template for a docking study of compound 43. We used
the Maestro protein preparation wizard from Schrodinger
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position of hydroxyl protons; protonations and tautomers
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34 Crystal structure of (4R)-40: molecular formula
¼
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C
18H18BrNO2, formula weight ¼ 360.23, crystal system ¼
˚
orthorhombic, space group ¼ P212121, a ¼ 5.9750(2) A,
3
˚
˚
˚
b ¼ 11.6900(3) A, c ¼ 23.7850(5) A, V ¼ 1661.33(8) A , T ¼
200 K, Z ¼ 4, Dc ¼ 1.440 mg mꢁ3, (Mo-Ka) ¼ 2.481 mmꢁ1
,
3815 reections measured, 3788 independent reections,
2938 observed reections [I > 2.0s(I)], R[F2 > 2s(F2)] ¼
0.037, goodness of t ¼ 1.054. The absolute conguration
around carbon C4 was determined to (R). Flack’s x ¼
0.007(9).
This journal is © The Royal Society of Chemistry 2014
Med. Chem. Commun., 2014, 5, 57–63 | 63