Modulation of 11β-hydroxysteroid dehydrogenase type 1 activity by 1,5-substituted 1H-tetrazoles

Article abstract of DOI:10.1016/j.bmcl.2010.04.055

Inhibitors of 11β-hydroxysteroid dehydrogenase (11β-HSD1) show promise as drugs to treat metabolic disease and CNS disorders such as cognitive impairment. A series of 1,5-substituted 1H-tetrazole 11β-HSD1 inhibitors has been discovered and chemically modified. Compounds are selective for 11β-HSD1 over 11β-HSD2 and possess good cellular potency in human and murine 11β-HSD1 assays. A range of in vitro stabilities are observed in human liver microsome assays.

Full text of DOI:10.1016/j.bmcl.2010.04.055

Bioorganic & Medicinal Chemistry Letters 20 (2010) 3265–3271
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Modulation of 11b-hydroxysteroid dehydrogenase type 1 activity
by 1,5-substituted 1H-tetrazoles
a
a
a
a
Scott P. Webster a, , Margaret Binnie , Kirsty M. M. McConnell , Karen Sooy , Peter Ward ,
Michael F. Greaney b, Andy Vinter c, T. David Pallin d, Hazel J. Dyke d, Matthew I. A. Gill d,
Ines Warner d, Jonathan R. Seckl a, Brian R. Walker a
*
a Endocrinology Unit, Centre for Cardiovascular Science, The Queen’s Medical Research Institute, University of Edinburgh, 47 Little France Crescent, Edinburgh EH16 4TJ, UK
b School of Chemistry, University of Edinburgh, Joseph Black Building, King’s Buildings, West Mains Road, Edinburgh EH9 3JJ, UK
c Cresset Biomolecular Discovery Ltd, Biopark Hertfordshire, Broadwater Road, Welwyn Garden City, Herts AL7 3AX, UK
d Argenta, 8/9 Spire Green Centre, Flex Meadow, Harlow, Essex CM19 5TR, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
Inhibitors of 11b-hydroxysteroid dehydrogenase (11b-HSD1) show promise as drugs to treat metabolic
disease and CNS disorders such as cognitive impairment. A series of 1,5-substituted 1H-tetrazole 11b-
HSD1 inhibitors has been discovered and chemically modified. Compounds are selective for 11b-HSD1
over 11b-HSD2 and possess good cellular potency in human and murine 11b-HSD1 assays. A range of
in vitro stabilities are observed in human liver microsome assays.
Received 22 March 2010
Revised 12 April 2010
Accepted 13 April 2010
Available online 18 April 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
11b-Hydroxysteroid dehydrogenase type 1
11b-HSD1
Drug discovery
Metabolic syndrome
Increased circulating levels of the glucocorticoid hormone corti-
sol (Cushing’s syndrome) causes central obesity, hyperglycaemia,
dyslipidaemia and hypertension by acting in key metabolic tissues,
including liver and adipose; and causes depression and cognitive
impairments by acting in the CNS.1 Elevated cortisol is thought
to be important in metabolic syndrome and in age-related cogni-
tive impairment.2–5
Glucocorticoid activity at the tissue level is modulated by the
11b-hydroxysteroid dehydrogenase (11b-HSD) enzymes, which
interconvert inactive cortisone and cortisol (or dehydrocorticoster-
one and corticosterone in rodents), and thus control access of glu-
cocorticoids to intra-cellular receptors.6,7 Two isoforms of 11b-HSD
are known. In the periphery, 11b-HSD1 is present predominantly
in the liver and adipose tissue, while in the brain it is found mainly
in the hippocampus and cortex, where it catalyses predominant
reductase activity, regenerating glucocorticoids. 11b-HSD2, which
catalyses the reverse reaction, is expressed mainly in the kidney
where it protects mineralocorticoid receptors from glucocorticoid
activation. Dysregulation of the 11b-HSDs is also thought impor-
tant in metabolic syndrome.2
sis, suggesting that inhibitors of 11b-HSD1 may be useful for the
treatment of type 2 diabetes.8 These mice also have low serum tri-
glycerides and increased HDL cholesterol and apo-lipoprotein A1
levels, suggesting that 11b-HSD1 inhibition may prevent athero-
sclerosis.9 11b-HSD1 knockout mice are also protected against
age-related cognitive impairment, suggesting that inhibitors may
be useful in the treatment of diseases characterised by cognitive
dysfunction, such as Alzheimer’s disease.10
Substantial activity in the pharmaceutical industry has led to
the discovery of numerous classes of non-steroidal 11b-HSD1
inhibitors.11–13 These include triazoles such as compound 544
(1), sulphonamides like PF-915275 (2), adamantyl carboxamides
(3) and thiazolones such as BVT116429 (4) (Fig. 1).14–17 Compound
1 has been shown to produce beneficial effects in animal models of
atherosclerosis and of type 2 diabetes, with lowering of plasma tri-
glycerides, glucose, rate of body weight gain and food intake.18 At
high doses compound 4 lowers fasting plasma glucose in Ldlr 3KO
mice, while compound 2 has been shown to alter plasma insulin
levels in cynomolgus monkeys.19,20
We have discovered and modified a series of 1,5-substituted
1H-tetrazoles that display selective inhibition of 11b-HSD1
in vitro. Compounds were tested for inhibition in mammalian cells
stably transfected with human 11b-HSD1 or human 11b-HSD2
using a scintillation proximity assay (SPA) at a 20 nM substrate
concentration.21 Selected compounds were also tested in cells
expressing murine 11b-HSD1.
In mice, global knockout of 11b-HSD1 leads to enhanced hepatic
insulin sensitivity and reduced gluconeogenesis and glycogenoly-
* Corresponding author. Tel.: +44 1312426738; fax: +44 1312426779.
E-mail address: scott.webster@ed.ac.uk (S.P. Webster).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.04.055

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S. P. Webster et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3265–3271
5-thioethanone moiety. A number of compounds were synthesised
according to Scheme 1. A selection of 1-substituted 1H-tetrazole-5-
thiols were reacted with a range of acylbromides in the presence of
sodium methoxide to yield compounds 6a–w.
N
N
O
O
S
H2N
N
N
N
H
Compound 5 was treated with oxone in aqueous methanol to
yield the sulphoxide 7 and the sulphone 8 (Scheme 1).
1
2
CN
Cl
O
A number of analogues were generated by forming the tetrazole
moiety from a series of 4-oxo-butyramide derivatives as shown in
Scheme 2A. A series of isomeric tetrazoles was also generated by
forming the ‘reverse’ amide from benzoyl chloride and 3-amino-
1-phenylpropan-1-one, followed by reaction with azidotrimethyl
silane (Scheme 2B).
H
N
N
O
O
CF3
S
N
H
O
H2N
F
3
4
A 1-phenyl-1H-tetrazol-5-oxy linked compound was prepared
by reacting 5-chloro-1-phenyl-1H-tetrazole with 2-hydroxyaceto-
phenone (Scheme 2C).
Figure 1. Selected non-steroidal 11b-HSD1 inhibitors.
Our next goal was to explore SAR in the sulphanyl tetrazole ser-
ies. The results indicated that a hydrophobic group directly linked
to the ketone, such as tert-butyl, benzyl and phenyl, conferred good
potency suggesting that this part of the scaffold occupies a lipo-
philic binding site within the enzyme. Substitution on the phenyl
group with fluorine or methoxy groups (compounds 6a–g) was
generally detrimental to good cellular potency (Table 1). However,
in contrast to mono substitution, 3,4-dimethoxy substitution con-
ferred approximately 10-fold greater potency (compound 6h). In
general compounds were highly selective for 11b-HSD1 over
11b-HSD2, although the heterocyclic analogues, 6j and 6k, inhib-
ited 11b-HSD2 substantially and were not progressed further.
Since the 3,4-dimethoxy phenyl group conferred good potency
when combined with phenyl at the 1-position of the central tetra-
zole, a series of 3,4-dimethoxy phenyl analogues was prepared
according to Scheme 1 in an effort to improve potency.
N
N
N
N
S
O
hHSD1 IC50 = 173nM
hHSD2 %inh @ 10µM = 0%
5
Figure 2. Initial hit from the primary in vitro screen.
An 11b-HSD1 inhibitor containing a 5-sulphanyl 1H-tetrazole
moiety was identified following a primary in vitro compound
screen on a selection of putative 11b-HSD1 inhibitors identified
from a pharmacophore field-based in silico screen.22,23 Compound
5 displays sub-micromolar potency in cellular assays and is selec-
tive for 11b-HSD1 (Fig. 2).
Only marginal increases in potency relative to compound 6h
were achieved within the group of compounds tested. Replace-
ment of the 1-phenyl group with alkyl groups such as tert-butyl
We initially sought to explore SAR at the 1-position of the tet-
razole and at the position adjacent to the carbonyl group of the
R
N
N
N N
N
R2
a
2
N
Br
+
S
SH
N
N
O
O
R1
R1
N
5, 6a - 6w
b
N
N
N
R2
N
N
R2
S
S
+
N
N
R1
O
O
O
O
O
R1
8
7
Scheme 1. Reagents and conditions: (a) MeOH, NaOH, 60 °C, 4 h; (b) MeOH, KHSO5, rt, 18 h.
A
O
N
O
N
R2
R2
R2
N
a
a
NH2
b
b
HO
HN
R1
N
R1
+
R1
O
O
O
O
O
O
B
N
N
N
H2N
R2
H
N
O
Cl
R2
R2
O
N
+
R1
R1
R1
C
N
N
N
N
N
R2
R2
N
HO
+
Cl
N
O
N
R1
O
R1
O
Scheme 2. Reagents and conditions: (A) a—DCM, HATU, DIPEA, rt, 20 min; b—DCM, PCl5, N2, rt, 15 min then TMS-N3, rt, 18 h; (B) a—DCM, DIPEA, rt, 1 h; b—DCM, PCl5, N2, rt,
15 min then TMS-N3, rt, 48 h; (C) DMF, NaH, rt, N2, rt, 1 h.

S. P. Webster et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3265–3271
3267
Table 1
Table 2
Human 11b-HSD1 and 11b-HSD2 inhibition for compounds 6a–k
Human 11b-HSD1 and 11b-HSD2 inhibition for compounds 6l–w
N
N
OMe
R2
N
OMe
N
N
N
S
N
O
N
R1
S
O
Compound
R1
hHSD1 IC50, nM
hHSD2 %inh at 10
0
lM
Compound
R2
hHSD1 IC50, nM
14,500
hHSD2 %inh at 10
nd
lM
*
Me
6l
2180
954
6a
*
Me
*
6m
6n
nd
0
6b
6c
252
257
9
7
*
*
*
*
1660
183
F
6o
6p
6q
0
6d
6e
6f
1300
1340
1050
1600
nd
0
*
*
*
*
F
1450
93
28
nd
*
*
OMe
38
nd
OMe
OMe
6r
2450
0
6g
*
*
OMe
6h
116
nd
6s
6t
1000
1600
3
*
*
*
*
6i
289
485
18
OMe
nd
6j
69
O
S
*
*
6k
1700
100
6u
1040
nd
nd, not determined.
Cl
*
*
6v
860
0
0
and isopropyl led to a loss in potency, while substitution at the
ortho- and para-positions of the 1-phenyl group was generally det-
rimental to potency (Table 2). Only methyl substitution at the
meta-position led to a minor increase in potency suggesting that
the 1-phenyl group occupies a confined binding pocket within
the enzyme.
S
6w
1700
nd, not determined.
The microsomal stability of several 5-sulphanyl-tetrazole ana-
logues was assessed in vitro using human liver microsomes (data
not shown). The results suggested that microsomal stability was
poor in this series. To investigate whether the sulphanyl group rep-
resented the main metabolically labile group, we oxidised the sul-
phanyl group and isolated both the sulphoxide and sulphone
derivatives of compound 5. We also targeted oxygen, nitrogen
and carbon replacements of the sulphur atom linked to the 5-posi-
tion of the tetrazole (Scheme 2).
microsomal stability was lower for these compounds. In contrast,
replacement of the 5-sulphanyl group with methylene led to only
a small drop in potency and a large increase in microsomal stabil-
ity. These results suggested that carbon-linked tetrazoles offered
an improved profile for in vivo dosing and further optimization.
Compound 11 possessed both moderate potency and high
microsomal stability. Replacement of the 1-phenyl group with
cycloalkyl groups such as cyclohexyl or adamantyl resulted in a
loss of potency (Table 4). Substitution on the 1-phenyl ring at the
ortho- and para-positions generally lowered potency. However,
The results from our initial studies showed that the ether, sul-
phone and sulphoxide analogues possessed lower potency than
the corresponding sulphanyl analogues (Table 3). In addition,

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S. P. Webster et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3265–3271
Table 3
Table 4 (continued)
Human 11b-HSD1 and liver microsomal stability for compounds 6c and 7–11
Compound
R1
hHSD1 IC50, nM
HLMa % parent
N
N
*
N
R2
N
R1
X
12j
686
37
O
Compound
X
R1
R2
hHSD1 IC50, nM
HLMa % parent
CN
*
6c
7
8
9
10
11
S
Ph
Ph
Ph
Ph
Ph
Ph
Ph
257
1230
943
971
Inactive
359
34
25
4
4
nd
88
S(@O)
S(@O)2
O
NMe
CH2
t-Bu
t-Bu
Ph
Ph
Ph
12k
12l
3900
1500
1100
81
1
N
*
*
a
Percentage parent remaining after 30 min incubation with human liver
microsomes (HLM).
12m
7
Table 4
Human 11b-HSD1 and liver microsomal stability for compounds 12a–r
*
N
N
N
N
12n
12o
8000
987
73
nd
*
*
R1
O
Compound
R1
hHSD1 IC50, nM
HLMa % parent
33
*
12p
12q
915
14
nd
12a
2100
*
*
*
*
1500
12b
12c
683
948
22
2
*
12r
2400
nd
O
nd, not determined.
a
Percentage parent remaining after 30 min incubation with human liver
microsomes (HLM).
12d
12e
588
114
2
the introduction of a meta-chloro group increased cellular potency
approximately 3-fold relative to compound 11. This compound
(12e) also possessed moderate microsomal stability.
The SAR adjacent to the carbonyl group of compound 11 was
also investigated. The majority of substitutions on the phenyl ring
failed to improve either the potency or the microsomal stability
relative to the unsubstituted compound, with only the meta-meth-
oxy analogue (compound 13g) showing a slight increase in potency
(Table 5). Replacement of the phenyl moiety with alkyl and cyclo-
alkyl groups also tended to reduce potency.
Since compound 12e displayed acceptable levels of potency ver-
sus human and murine 11b-HSD1 (IC50 = 114 nM and IC50 = 302 nM,
respectively) we investigated its inhibition profile in vivo in C57/Bl6
mice.24 The compound displayed good levels of inhibition in liver,
subcutaneous adipose tissue and brain following an oral dose sug-
gesting that 12e was orally bioavailable (Table 6).
Since ketones are potentially reactive in vivo a suitable replace-
ment for the carbonyl group was desirable. A wide array of ana-
logues containing amido, hydroxyl, hydroxyamino and sulphanyl
groups were prepared. In addition, a variety of non-conventional
heterocyclic ketone isosteres were also synthesised. However,
these analogues were either inactive or displayed low inhibition
and no suitable replacement for the ketone was identified.
A number of isomeric 1,5-substituted tetrazoles were produced
to explore SAR around the central tetrazole group (Table 7). In
*
*
52
Cl
12f
575
34
Cl
*
OMe
OMe
12g
12h
2200
304
42
6
*
*
12i
426
12
OMe

S. P. Webster et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3265–3271
3269
Table 5
Table 7
Human 11b-HSD1 and microsomal stability for compounds 13a–i
Human 11b-HSD1 and microsomal stability for compounds 14a–k
N
N
N
N
R2
N
N
N
N
RX
O
O
Compound
RX
hHSD1 IC50, nM
HLMa % parent
*
*
*
Compound
R2
hHSD1 IC50, nM
820
HLMa % parent
nd
14a
843
29
9
*
13a
OMe
13b
13c
987
597
nd
21
14b
14c
751
493
*
F
18
11
*
*
F
OMe
*
13d
13e
13f
13g
4600
310
539
215
nd
8
Cl
14d
722
O
O
*
*
*
Cl
Cl
14e
14f
1800
1100
5
9
*
*
OMe
20
14
Cl
*
*
13h
13i
>10,000
506
nd
11
14g
>10,000
nd
Cl
*
*
14h
14i
3900
1900
4500
1100
81
31
53
6
nd, not determined.
a
N
Percentage parent remaining after 30 min incubation with human liver
microsomes (HLM).
*
*
N
N
N
N
Table 6
Ex vivo pharmacodynamic inhibition of 11b-HSD1 for compound 12e
14j
Route
% inhibition
in liver 2 h
% inhibition in
adipose 2 h
% inhibition
in brain 2 h
*
ip
po
61 19
38 11
52
35
4
8
25
1
22 13
14k
Compound was administered at 10 mg/kg.
S
nd, not determined.
a
Percentage parent remaining after 30 min incubation with human liver
microsomes (HLM).
general, the regioisomeric 5-phenyl tetrazole analogues were less
potent than the 1-phenyl tetrazoles and SAR was not transferable
between each series.
An in silico model, generated using Glide and based on the crystal
structure of human 11b-HSD1 (PDB: 2BEL) was prepared (Fig. 3). The
model places the tetrazole group in approximately the same orien-
tation as the A ring of the steroid substrate.25 The phenyl-tetrazole
ring stacks close to Y177 and is orientated towards the mouth of
the pocket. The key hydrogen-bonding interaction is made by the
ketone moiety and the catalytic residues S170 and Y183. The phe-
nyl-ketone ring is orientated towards the back of the binding pocket
where there is only limited space for further substitution. The mod-
elling is in keeping with our in vitro results, which indicate limited
scope for significant modification of the phenyl groups.
The majority of the compounds prepared in the carbon-linked
tetrazole series possessed only low to moderate microsomal stabil-
ity. To investigate this further metabolite identification studies on
compound 12e were undertaken. The compound was incubated

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S. P. Webster et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3265–3271
In summary, we have discovered moderately potent and selec-
tive inhibitors of 11b-HSD1 that display inhibition in vivo. How-
ever, certain compounds have been shown to act as substrates
and inhibit the enzyme by competing with the substrate.
Acknowledgments
We thank Enamine Ltd for the provision of some synthetic
chemistry services and the Wellcome Trust for funding.
References and notes
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Figure 3. In silico model of compound 11 bound to human 11b-HSD1.
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microsomal stability did not serve as substrates for the enzyme
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a scintillation
proximity assay (SPA). Human 11b-HSD1 enzyme inhibition was assessed in
HEK293 cells stably transfected with the full length human hsd11b1 gene.
HEK293 cells were plated in 96-well poly-
D-Lys coated flat-bottomed
microplates in DMEM containing 1% glutamine, 1% penicillin and
streptomycin. Compounds were added to plates such that the final
concentration of DMSO was 1%. Tritiated cortisone was added at
a final
concentration of 20 nM and the cells incubated at 37 °C in 5% CO2, 95% O2 for
2 h. The assay solutions were transferred to a scintillation microplate and
mixed with a solution of anti-mouse YSi SPA beads and anti-cortisol antibody
in assay buffer (50 mM Tris–HCl, pH 7.0; 300 mM NaCl, 1 mM EDTA, 5%
glycerol). The plate was incubated for 2 h at room temperature and read on a
scintillation counter. The percentage inhibition was determined relative to a
Table 8
Metabolic stability data for selected tetrazoles
Compound
hHSD1
IC50, nM
HLMa
parent
%
% ketone reduction
by 11b-HSD1b
non-inhibited control and the median inhibitory concentration (IC50
)
12e
12f
12j
13e
13f
13g
13h
14c
114
575
686
310
539
215
934
493
52
34
37
8
19
14
4
31
0
0
0
5
0
1
0
determined by plotting fractional inhibition against log compound
concentration. Data were fitted to the four parameter logistic equation.
Murine 11b-HSD1 enzyme inhibition was assessed in CHO cells stably
transfected with the full length murine hsd11b1 gene. Enzyme inhibition
was determined as described for human 11b-HSD1 following a 4-h incubation
of cells, compound and substrate.
22. Cheeseright, T.; Mackey, M.; Rose, S.; Vinter, A. J. Chem. Inf. Model. 2006, 46,
665.
23. Cheeseright, T.; Mackey, M.; Rose, S.; Vinter, A. Expert Opin. Drug Discov. 2007,
2, 131.
18
a
Percentage parent remaining after 30 min incubation with human liver
24. Male C57BL/6 mice (25–30 g in weight) were group housed and allowed
free access to food and water. Compounds was dissolved in 5% DMSO,
3% ethanol, 4 mM cyclodextrin. Animals (n = 3 per group) were dosed
microsomes (HLM).
b
Percentage of ketone reduced in the presence of recombinant 11b-HSD1
enzyme after 30 min at 37 °C.

S. P. Webster et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3265–3271
3271
intraperitoneally or orally with vehicle or compound at 12-hourly
intervals. At 2 h following the third dose mice were killed by cervical
dislocation. Liver, adipose and brain samples were removed and frozen
until analysis was performed. Inhibition of 11b-HSD1 in each tissue was
determined by incubating homogenates with 20 nM tritiated cortisone,
2 mM NADPH and 0.2% glucose in Krebs buffer, pH 7.4. Cortisone and
cortisol levels were measured by HPLC and the percentage inhibition
determined relative to vehicle treated tissue.
25. Wu, X.; Kavanagh, K.; Svensson, S., Elleby, B., Hult, M., von Delft, F.; Marsden,
B.; Jornvall, H.; Abrahamsen, L.; Oppermann, U. Unpublished. http://
www.rcsb.org/pdb/explore/explore.do?structureId=2BEL.
26. Xiang, J.; Ipek, M.; Suri, V.; Tam, M.; Xing, Y.; Huang, N.; Zhang, Y.; Tobin, J.;
Mansour, T. S.; McKew, J. Bioorg. Med. Chem. 2007, 15, 4396.