Discovery of a potent, selective, and orally bioavailable acidic 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) inhibitor: Discovery of 2-[(3 s)-1-[5-(cyclohexylcarbamoyl)-6-propylsulfanylpyridin-2-yl]- 3-piperidyl]acetic acid (AZD4017)

Article abstract of DOI:10.1021/jm300592r

Inhibition of 11β-HSD1 is an attractive mechanism for the treatment of obesity and other elements of the metabolic syndrome. We report here the discovery of a nicotinic amide derived carboxylic acid class of inhibitors that has good potency, selectivity, and pharmacokinetic characteristics. Compound 11i (AZD4017) is an effective inhibitor of 11β-HSD1 in human adipocytes and exhibits good druglike properties and as a consequence was selected for clinical development.

Full text of DOI:10.1021/jm300592r

Article
pubs.acs.org/jmc
Discovery of a Potent, Selective, and Orally Bioavailable Acidic 11β-
Hydroxysteroid Dehydrogenase Type 1 (11β-HSD1) Inhibitor:
Discovery of 2-[(3S)-1-[5-(Cyclohexylcarbamoyl)-6-
propylsulfanylpyridin-2-yl]-3-piperidyl]acetic Acid (AZD4017)
James S. Scott,* Suzanne S. Bowker, Joanne deSchoolmeester, Stefan Gerhardt, David Hargreaves,
Elaine Kilgour, Adele Lloyd, Rachel M. Mayers, William McCoull, Nicholas J. Newcombe, Derek Ogg,
Martin J. Packer, Amanda Rees, John Revill, Paul Schofield, Nidhal Selmi, John G. Swales,
and Paul R. O. Whittamore
Cardiovascular and Gastrointestinal Innovative Medicines Unit, AstraZeneca Mereside, Alderley Park, Macclesfield, Cheshire, SK10
4TG, U.K.
S
* Supporting Information
ABSTRACT: Inhibition of 11β-HSD1 is an attractive mechanism for the treatment of obesity
and other elements of the metabolic syndrome. We report here the discovery of a nicotinic
amide derived carboxylic acid class of inhibitors that has good potency, selectivity, and
pharmacokinetic characteristics. Compound 11i (AZD4017) is an effective inhibitor of 11β-
HSD1 in human adipocytes and exhibits good druglike properties and as a consequence was
selected for clinical development.
type 2 (11β-HSD2) is an NAD dependent oxidase expressed
mainly in kidney and colon tissue that catalyzes the reverse
reaction and prevents activation of mineralocorticoid receptors
by 2.12 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
Carbenoxolone (3a), originally developed as a treatment for
esophageal ulceration and inflammation, is an example of an
acidic compound that is a potent but nonselective inhibitor of
both 11β-HSD1 and 11β-HSD2.14 This 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 because of the associated
hypertension risk.15
Reflecting the keen interest in the therapeutic potential of
this target, over 250 patent applications from over 25
pharmaceutical companies and academic groups have now
been published and comprehensively reviewed.16−18 Several
companies have now progressed compounds into development,
and the structures of those compounds where disclosed are
shown in Figure 2.
INTRODUCTION
■
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 defined as
having the metabolic syndrome.1 The underlying causes of the
metabolic syndrome are complex; however, 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 glucocorticoid levels,5 it has been hypothesized that
elevated intracellular 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
11β-Hydroxysteroid dehydrogenase type 1 (11β-HSD1) is an
NADPH dependent enzyme that is widely expressed, notably in
liver, adipose tissue, and brain.10 It catalyzes the interconversion
of the inactive glucocorticoid hormone cortisone (1) to the
active glucocorticoid hormone cortisol (2) (Figure 1) and
therefore plays a key role in the regulation of intracellular
cortisol concentrations.11 11β-Hydroxysteroid dehydrogenase
Received: April 27, 2012
Published: June 12, 2012
© 2012 American Chemical Society
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Figure 1. Interconverstion of cortisone (1) and cortisol (2) by 11β-HSD1 and 11β-HSD2 and the unselective inhibitor carbenoxolone (3a).
Figure 2. Selective 11β-HSD1 inhibitors disclosed in the literature.
Table 1. Properties of 2-Thioalkylnicotinamide 4 and Piperazinesulfonamide 5
human 11β-HSD1 IC50
(μM)
PPB rat
(% free)
solubility
(μM)
rat heps Clint
permeability, CACO-2 Papp
compd
log D7.4
((μL/min)/106 cells)
(×10−6 cm s−1
)
4
5
0.350
0.200
3.2
3.9
13.2
0.5
82
2
284
272
27
10
Biovitrum was the first company to disclose inhibitors such
as BVT-2733 (3b) and BVT-14225 (3c) that were selective for
11β-HSD1 over 11β-HSD2.19 They entered into a collabo-
ration with Amgen, and this led to the development of the first
compound to enter clinical trials, BVT-3498 (AMG-311)20
(3d) reaching phase II in 2003. This was subsequently replaced
by BVT-83370 (AMG-221)21 which progressed into phase I
trials, but development was halted in April 2011.22 Merck has
progressed two compounds into development, MK-0916,
which entered phase II in 2004, and MK-0736, which was
originally developed as a treatment for hypertension and
entered phase II trials in 2005. Initial clinical data have now
been reported showing no significant improvement in fasting
plasma glucose at week 12 relative to placebo.23,24 However,
modest dose-dependent decreases in blood pressure and body
weight were observed over the course of the study, together
with a small but significant reduction of 0.3% in hemoglobin
A1C (hBA1c) at week 12.23,24 The structures of these
candidates have not been disclosed. Pfizer took the inhibitor
PF-915275 (3f)25 into phase I trials in 2006. In 2007 it was
announced that this compound had progressed to phase II;
however, it was subsequently stopped because of tablet
formulation issues.26 Incyte has also taken a lead compound
INCB-13739, the structure of which has not yet been disclosed,
as far as phase II trials, and data in patients with type II diabetes
have recently been reported27 and subsequently reviewed.28
The study showed that after 12 weeks as an addition to
metformin monotherapy, doses of 200 mg resulted in
significant reductions in HbA1c (−0.6%), fasting plasma
glucose (−24 mg/dl), and homeostasis model assessment−
insulin resistance (HOMA-IR) (−24%) compared with
placebo. Vitae Pharmaceticals has been in collaboration with
Boehringer Ingelheim, and a compound entered phase I trials in
2010. Inhibitors such as 3g have been reported by Vitae,29
although it is not known whether this represents the structure
of their development compound. Other companies such as
Bristol-Myers Squibb, Roche, and Lilly have also progressed
compounds into the clinic, although to date the structures have
not been disclosed.
RESULTS AND DISCUSSION
■
As part of a drug discovery program to identify a potent and
selective inhibitor of 11β-HSD1, we undertook a high
throughput screening campaign against the AstraZeneca
compound collection. A number of chemotypes were found
that inhibited 11β-HSD1, and these were profiled and
developed to generate two distinct series that we felt
represented promising leads. The series are exemplified by 2-
thioalkylnicotinamide 4 and piperazinesulfonamide 5, the
properties of which are summarized in Table 1.
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a
Nicotinamide 4 had modest potency (IC50 = 350 nM) and
was suprisingly unbound (rat plasma protein binding (PPB),
13.2% free) given the measured log D7.4 (3.2). Ligand efficiency
was high as measured per heavy atom count (0.46) and
moderate as measured by ligand lipophilicity efficiency30 (LLE
= pIC50 − logD = 3.3). Sulfonamide 5 was more potent (IC50 =
200 nM) although less impressive in terms of ligand efficiency
per heavy atom count (0.33) or ligand lipophilicity efficiency
(2.8). The permeability of both was high as measured in a
CACO-2 assay with no evidence of efflux.
Scheme 1
a
Reagents and conditions: (a) (COCl)2, DMF, CH2Cl2, amine, 25 °C,
1 h, 16−54%; (b) HOBt, EDAC, iPr2EtN, CH2Cl2, amine, 25 °C, 18 h,
16−74%.
A key issue for both of these series was the lack of oral
bioavailability in rat (F = 0%). The in vitro data suggested that
metabolism may be a limiting factor, as both compounds were
unstable in rat hepatocytes. In order to test this concept, we
elected to carry out diagnostic DMPK experiments using a
nonspecific inhibitor of the cytochrome P450 (CYP) enzymes,
1-aminobenzotriazole (ABT).31 Our hypothesis was that if
metabolism was the limiting factor, dosing of ABT should
attenuate oxidative drug metabolism in vivo and improve the
pharmokinetic parameters of the compounds. We elected to
predose the ABT orally according to protocols described in the
literature32 to inhibit both gut wall and hepatic P450
metabolism. The results are shown in Table 2.
Synthesis of compounds substituted in the 6-position was
achieved from 2,6-dichloronicotinic acid 8 via formation of the
acid chloride and coupling with cyclohexylamine which
proceeded in good yield (98%) to give amide 9. In contrast
to the results obtained with the corresponding ethyl ester,33 we
found that treatment of the amide 9 with sodium propylthiolate
resulted in a completely regioselective displacement of the 2-
chloro subsituent (no evidence of 6-chloro substitution evident
in crude reaction mixtures) to afford key intermediate 10.
Subsequent treatment with amine nucleophiles with pendent
ester groups and microwave heating using butyronitrile as
solvent, followed by ester hydrolysis to the corresponding acids
using lithium hydroxide, gave acidic compounds 11a−j
(Scheme 2).
Investigation of the SAR around the 2-substituent was carried
out via treatment of intermediate 9 with propanol, propyl-
amine, and N-methylpropylamine to give intermediates 12a,
12b, and 12c, respectively, again with complete regioselectivity
for displacement of the 2-chloro position. Treatment with
methyl-(S)-3-piperidine acetate with microwave heating using
butyronitrile as solvent followed by lithium hydroxide
hydrolysis gave compounds 13a−c (Scheme 2).
Table 2. DMPK Properties of 2-Thioalkylnicotinamide 4 and
Piperazinesulfonamide 5 in the Absence and Presence of an
ABT Predose
Cmax
AUC0−t
Cl
compd
expt
no ABT
(μg/mL) (μg·h/mL) (mL min−1 kg−1
)
4
4
5
5
0.039
0.514
0
0.08
2.51
0
42
7
po predose of ABT
no ABT
20
14
po predose of ABT
0
0
Compound 14 with methylene in place of sulfur was
synthesized via a different approach involving construction of
the pyridine ring. Starting from the known enamine ester 15,34
a Michael addition with methyl propiolate gave 16. Then base
mediated cyclization afforded the oxopyridine intermediate 17
in good yield. Treatment with phosphorus oxychloride to give
18 was followed by ester hydrolysis to give the chloro-
substituted acid intermediate 19. Subsequent amide coupling
with cyclohexyalamine to form 20 and then treatment with
methyl-(S)-3-piperidine acetate with microwave heating using
butyronitrile as solvent gave compound 14 (Scheme 3).
Our efforts to address the metabolism by amide modification
involved three approaches, namely, (i) introducing metabolic
blocking groups (compound 7b), (ii) lowering log D7.4 by
introduction of polar functionality (compounds 7c−g), and
(iii) reducing the levels of free drug by introducing acidic
functionality with the aim of modulating the unbound clearance
(compound 7h). The results are summarized in Table 3.
Disappointingly, most of the putatively metabolism blocking
or lower log D7.4 compounds tested showed little or no
improvement. In many cases, the clearance remained high in
hepatocyctes and moderate to high in vivo, with no significant
oral levels observed in rat. Significant increases in the free level
of these compounds (in the cases of compounds 7c and 7d
around 50% free) may have offset any reduction in lipophilicity
driven metabolism. The introduction of a carboxylic acid
functionality onto the cyclohexyl ring (compound 7h),
however, dramatically lowered the clearance (rat hepatocytes,
<2 (μL/min)/106 cells) and when dosed in vivo showed an
excellent pharmocokinetic profile in rat (Clp = 2.5 mL min−1
Upon oral predosing with ABT, 2-thioalkylnicotinamide 4
showed an improvement in AUC of approximately 30-fold
relative to the compound with no ABT predose and a
significant reduction in the clearance of the compound. This
provided confidence that absorption was not an issue for this
compound and that if the CYP mediated metabolic clearance
could be reduced, it would lead to an improvement in oral
levels. In contrast, piperazinesulfonamide 5 showed no
observable compound levels following an ABT predose, leading
us to conclude that the metabolism was not CYP mediated or
that other factors, such as solubility, were restricting oral
exposure. On the basis of these results, we elected to focus our
optimization efforts on reducing metabolic clearance in the 2-
thioalkylnicotinamide series. We were guided by metabolism
identification (Met-ID) results that indicated that no amide
bond cleavage was observed and the majority (>80%) of the
metabolism was oxidative and occurring on the cyclohexyl ring
with only moderate amounts on the pyridine and none being
observed on the thioalkyl chain. Our medicinal chemistry
strategy was to address the metabolism using amide
modification. The compounds described in this optimization
program were synthesized as described in Schemes 1−3.
An initial set of compounds (7a−h) was synthesized via
amide couplings of the commercially available 2-thionicotinic
acid 6 using either acid chloride chemistry to give compounds
4, 7a, and 7h or HOBt/EDAC methodology to give
compounds 7b−g (Scheme 1).
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a
Scheme 2
a
Reagents and conditions: (a) (i) oxalyl chloride, DMF, CH2Cl2, 25 °C, 2 h; (ii) cyclohexylamine, CH2Cl2, 25 °C, 16 h, 98%; (b) nPrSH, NaHMDS,
DMF, 25 °C, 2 h, 76%; (c) (i) RH, K2CO3, nBuCN, 170 °C, 2 h; (ii) LiOH, THF/H2O, 25 °C, 3 h, 19−64%; (d) nPrXH, K2CO3, nBuCN, 150 °C, 1
h, 60−71%; (e) (i) methyl-(S)-3-piperidine acetate. HCl, K2CO3, BuCN, 170 °C; (ii) LiOH, THF/H2O, 25 °C, 16 h, 33−81%.
n
a
Scheme 3
a
Reagents and conditions: (a) methyl propiolate, toluene, 100 °C, 96 h, 81%; (b) NaOtBu, NMP, 180 °C, 4 h, 78%; (c) POCl3, 120 °C, 2 h, 81%;
(d) NaOH, MeOH, 25 °C, 16 h, 86%; (e) HOBt, EDAC, NEt3, CH2Cl2, cyclohexylamine, 25 °C, 16 h, 75%; (f) methyl-(S)-3-piperidine acetate.
n
HCl, K2CO3, BuCN, 150 °C, 96 h, 25%.
kg−1; F = 81%). Notably this compound had high free drug
levels for an acid (rat PPB, 41% free) with considerably reduced
lipophilicity (log D7.4 < 0.5) relative to the original lead
(log D7.4 = 3.2). Unfortunately, when tested in the 11β-HSD1
assay, the activity of the compound was below the reporting
threshold (IC50 > 30 μM).
Encouraged by the fact that 3a, a known inhibitor of 11β-
HSD1, contains acidic functionality, we sought to introduce an
acidic group in our series in a position that would be tolerated
by the enzyme. The structure of 3a in complex with 11β-HSD1
was published in 2004 (PDB code 2BEL).35 The active site was
analyzed using SiteMap probes, as implemented in the
acidic group. The results of a series of 6-substituted pyridyl
compounds are presented in Table 4.
Consistent with our hypothesis and in contrast with
compound 7h, compounds 11a−k showed inhibition of the
enzyme while retaining the favorable DMPK properties
associated with the introduction of acidic functionality.
Compounds 11a and 11b demonstrated that an NH linker
was tolerated, while efforts to incorporate an O-aryl linker (11c,
11d) were less successful. Moving the position of the acid
around a piperidine ring (11e−g) revealed that the 3- and 4-
positions were particularly favored (11f, 11g). Chain
homologation at these positions (11h−j) resulted in the
identification of compound 11i which was the most potent
acidic compound identified within this series (IC50 = 7 nM).
The observation that compound 11i was >10-fold more
potent than its enantiomer 11h suggested that a specific
interaction was being made by the acidic group with the
protein. In order to understand the nature of this protein−
ligand interaction, we obtained a crystal structure of compound
11i in complex with human 11β-HSD1. Figure 3 shows a view
of 11i bound in the active site, with associated ligand density.
We observed an interaction between the amidecarbonyl
group and the hydroxyl side chain of Y183. There is a hydrogen
bond between the carboxylate group and the backbone NH of
L217. The carboxylate is also in proximity to the side chain of
Schrodinger software package. This identified two primary
̈
positions for hydrogen bond acceptors: at the active site
residues S170 and Y183 and near the backbone NH of L217.
The site analysis suggested a slight concavity near L217, which
would promote a hydrogen bond interaction, and indeed, 3a
utilizes this hydrogen bond position. Similarly, there is a
significant region of the pocket that is predicted to favor
hydrophobic groups and that is occupied by the steroid scaffold
of 3a. We speculated that a potential binding mode for our
compound could involve the amidecarbonyl interacting with
Y183. We therefore chose to explore substitution of the
pyridine core as a potential way to access this hydrophobic
pocket with the hope of finding a suitable way to position an
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Table 3. Enzyme Potencies and Physicochemical and DMPK Properties for Pyridineamides
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Table 4. Enzyme Potencies and Physical and DMPK Properties for Acidic Compounds
a
Bioavailabilities of >100% are believed to reflect enterohepatic recirculation of acylglucuronide metabolite.
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Figure 3. (a) Binding mode of compound 11i in human 11β-HSD1, with associated 2Fo − Fc density contoured at 1σ. The ligand was cocrystallized
along with the NADP cofactor, but there are no contacts between the two molecules. (b) Interaction map of compound 11i in the 11β-HSD1 active
site. Blue arrows indicate a backbone hydrogen bond. Green arrows indicate a side chain hydrogen bond. L217 and A172 show hydrophobic packing
against the pyridyl group. Labels D, E, and F refer to the protein chain indices. The dotted line marks the protein surface. Purple patches indicate
solvent exposure. The interaction map was generated using the MOE software package.
D259, although the O···O separation is slightly longer than
expected for a hydrogen bond interaction. The pyridyl ring is
sandwiched between the hydrophobic side chains of L217 and
A172. The S-propyl and cyclohexyl groups are packed within
the hydrophobic region of the pocket, with some limited
exposure to solvent for the cyclohexyl. There is no contact with
the NADP+ cofactor, which remains in situ on binding the
inhibitor. Overall, the high affinity of compound 11i may be
explained both from a complementary shape match and burial
of hydrophobic groups and from two hydrogen bond
interactions: first, an essential interaction with the active site
residue Y183 and second, an optional, but energetically
favorable, interaction with the backbone at L217.
The nature of the 2-substituent was examined to determine if
the thioalkyl subsituent was optimal by varying the atom
directly attached to the pyridyl ring while keeping the rest of
the molecule constant (Table 5). The oxo analogue 13a was
less potent and more lipophilic, perhaps because of the
potential for formation of an internal H-bond with the amide
NH. Amino analogues 13b and 13c were isolipophilic but had
reduced potency. Switching to the carbon analogue 14 that has
no potential to form an internal H-bond resulted in a reduction
in lipophilicity, a drop in potency, and an overall less ligand
efficient compound. All compounds showed good oral
bioavailability in rat. On the basis of these results, the thioalkyl
substituent was determined to be optimal and compound 11i
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Table 5. Enzyme Potencies and Physical and DMPK Properties for Acidic Compounds
a
Bioavailabilities of >100% are believed to reflect enterohepatic recirculation of acylglucuronide metabolite.
Table 6. Physical Properties of Compound 11i
a
b
b
b
b
c
aqueous solubility, pH 7.4
mouse PPB
(% free)
rat PPB
dog PPB
human PPB
(% free)
MDCK permeability Papp
(×10−6 cm·s−1
(μM)
(% free)
(% free)
)
144 (n = 2)
2.9 (n = 2)
1.8 (n = 4)
2.3 (n = 2)
0.8 (n = 3)
b
29 (A−B), 22 (B−A) (n = 2)
a
Solubility of compounds in aqueous phosphate buffer at pH 7.4 after 24 h at 25 °C (μM). PPB was assessed by equilibrium dialysis in the
appropriate species plasma at 37 °C. Free and bound concentrations were quantified by LC−UV−MS. Compounds were incubated at 10 μM in
cultured MDCK cells. Permeability was measured in both the A to B and B to A direction.
c
was selected for further profiling. The LLE for 11i is 6.0,
significantly higher than those of all of the other acidic
analogues and a considerable improvement over the initial lead
4.
an MDCK assay (Papp(A−B) = 29 × 10−6 cm·s−1; efflux ratio of
0.8), it was predicted that this would lead to good absorption in
vivo.
The pharmacokinetic profile of compound 11i was examined
in other species (mouse, rat, dog), and the results are shown in
Table 7. The compound showed consistently good properties
with moderate clearances and high bioavailabilities (F > 60%)
across all three species. The compound had notably high
volumes of distribution across species (mouse, 4.2 L/kg; rat, 2.6
L/kg; dog, 0.9 L/kg), which was viewed as surprising for an
acidic compound. Additionally in mouse and dog (but not rat),
secondary peaks were observed in the pharmacokinetic profiles,
leading to the hypothesis that the high volumes could be
resulting from enterohepatic recirculation (EHC) whereby an
Further profiling of compound 11i indicated that the
properties of this compound made it suitable for further
development (Table 6). The compound was inactive (<25%
inhibition at 10 μM) against five isoforms of the cytochrome
P450 enzymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6, and
CYP3A4). Plasma protein binding showed good free levels for
an acid (0.8−2.9% free across species), perhaps structurally
related to the high free levels of the initial lead. The aqueous
solubility as measured on crystalline material was 144 μM, and
when coupled with good cellular permeability as measured in
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a
following a polymorph screen, providing confidence that solid-
state issues should not hinder development.
Despite having high potency for the human form of 11β-
HSD1, compound 11i showed much reduced activity across
species as shown in Table 9 with the exception of cynomolgous
Table 7. Pharmacokinetic Parameters of Compound 11i
iv
oral
half-life
(h)
Clp
Vdss
(L/kg)
half-life
(h)
bioavailability
(%)
species (mL min−1 kg−1
)
b
mouse
rat
16
9
4.2
2.6
0.9
4.2
4.4
6.2
3.2
4.7
4.9
>100
b
>100
Table 9. In Vitro Potencies for Compound 11i
dog
7
60
a
Compounds were dosed at 1−3 mg/kg in either solution (DMSO/
IC50 (μM)
b
hydroxy-β-cyclodextrin) or suspension (HPMC/Tween). Bioavail-
abilities of >100% are believed to reflect enterohepatic recirculation of
acylglucuronide metabolite.
human
enzyme
mouse
enzyme
rat
enzyme
dog
enzyme
cyno
enzyme
human
adipocyte
0.007
0.750
4.06
3.77
0.029
0.002
acylglucuronide of the acid was being formed and then
subsequently cleaved leading to release of the parent
compound. In mouse and dog, species that possess a gall
bladder, it was postulated that the secondary peaks observed
were a result of parent compound being released into the GI
tract, whereas these were not evident in the rat, a species that
lacks a gall bladder.36 It was hypothesized that this was the
effect responsible for the high volumes of distribution observed.
Met-ID results in hepatocytes (rat, dog, human) revealed
that conversion of the acid to the acylglucuronide was indeed
the major route of metabolism in all three species (Table 8).
monkey (IC50 = 0.029 μM). Additionally, as we believed
adipose is a key target organ, inhibition of 11β-HSD1 activity
was measured in isolated human adipocytes from nondiabetic
volunteers. The compound was shown to be a potent inhibitor
in this key target tissue (IC50 = 0.002 μM) in good agreement
with the enzyme potency, thus providing some confidence that
the compound was not restricted from adipose tissue by the
fact that it was acidic.
Since 11i had lower potency against the mouse enzyme, only
a limited number of preclinical pharmacodynamic measure-
ments were performed. An ex vivo assay measuring the
3
3
inhibition of conversion of H-cortisone to H-cortisol was
conducted in epididymal adipose tissue. Figure 4 shows the
Table 8. Metabolism-ID Summaries for Selected
Compounds 11i
species
parent (%)
[+Glu] (%)
[+O] (%)
[+Glu]/[O]
rat
66
34
93
23
53
6
11
13
1
2.1
4.1
6
dog
human
While this is a normal metabolic fate for acids, acylglucuronides
have been associated in some cases with idiosyncratic toxicity.37
It is postulated that the initially formed 1β-isomer of the
acylglucuronide metabolite may lead directly to drug−protein
conjugates while Amadori rearrangement can result in the
formation the 2-O- and 4-O-acylated products which can in
turn lead to protein adducts through Schiff base formation with
the amino groups of proteins.38 To assess the potential risk
associated with this, 14C radiolabeled compound 11i was
prepared and dosed orally to a rat to assess the degree of
covalent binding. Pleasingly this compound did not show any
significant covalent binding in plasma or liver tissue when
dosed to male rats at 20 mg/kg (40 μCi/kg). Less than 25
pmol/mg protein was detected after 2 h, and no detectable
binding was observed after 24 h. This suggested that this
compound would be unlikely to give rise to idiosyncratic
toxicity as a result of acylglucuronide formation.
Figure 4. Inhibition of 11-βHSD1 in mouse epididymal adipose tissue
ex vivo for compound 11i.
effects of compound 11i dosed orally to lean mice (10−1500
mg/kg). At 1 h postdose, at a dose of 50 mg/kg compound 11i,
50% inhibition was observed. This corresponded to a plasma
exposure equivalent to the IC50 in the mouse. Increasing the
dose further led to a maximal effect of approximately 70%
inhibition at 1500 mg/kg, equivalent to 10 × IC50 in the mouse,
demonstrating the dose dependent inhibition of 11β-HSD1 by
compound 11i in this model.
In summary, we have discovered novel acidic inhibitors of
11β-HSD1 that show excellent pharmacokinetic profiles. On
the basis of these encouraging results and the overall profile,
compound 11i was selected for development as the clinical
candidate AZD4017. Further details of these investigations will
be reported in due course.
Compound 11i displayed excellent selectivity versus the
related enzymes 11-βHSD2, 17β-HSD1, 17β-HSD3 (all IC50
>
30 μM) and showed no measurable activity against the
glucocorticoid and mineralocorticoid receptors. Further profil-
ing revealed that 11i was nonmutagenic in a two-strain Ames
test and negative in a mouse lymphoma assay. It had IC50 > 100
μM in the hERG IonWorks assay and was assessed in a guinea
pig MAP assay with no changes observed in any of the
parameters. No significant induction of CYP450 enzymes was
observed following administration to rats for 1 month. A good
selectivity profile was observed versus a panel of 119 unrelated
enzymes and receptors with the only two weak hits being
angiotensin (AT2) receptor and choleocystokinin (CCK2)
receptor. One stable anhydrous crystalline form was identified
EXPERIMENTAL SECTION
All solvents and chemicals used were reagent grade. Anhydrous
solvents tetrahydrofuran (THF), benzene, dimethoxyethane (DME)
■
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were purchased from Aldrich. Flash column chromatography was
carried out using prepacked silica cartridges (from 4 g up to 330 g)
from Redisep, Biotage, or Crawford and elution was with an Isco
Companion system. 1H NMR data were recorded on a Varian Gemini
2000 (300 MHz) or a Bruker Avance DPX400 (400 MHz) instrument
and were determined in CDCl3 or DMSO-d6. Chemical shifts are
reported in ppm relative to tetramethylsilane (TMS) (0.00 ppm) or
solvent peaks as the internal reference, and coupling constant (J)
values are reported in hertz (Hz). Splitting patterns are indicated as
follows: s, singlet; d, doublet; t, triplet; m, multiplet; br, broad peak.
Merck precoated thin layer chromatography (TLC) plates (silica gel
60 F254, 0.25 mm, no. 5715) were used for TLC analysis. The purity of
compounds submitted for screening was >95% as determined by UV
analysis of liquid chromatography−mass spectrometry (LC−MS)
chromatograms at 254 nm and substantiated using the TAC (total
absorption chromatogram). Further support for the purity statement
was provided using the MS TIC (total ion current) trace in ESI +ve
and −ve ion modes and HRMS and NMR analysis. Solutions were
dried over anhydrous magnesium sulfate, and the solvent was removed
by rotary evaporation under reduced pressure.
1.59−1.70 (4H, m), 1.78−1.89 (2H, m), 2.78 (3H, s), 3.18 (2H, t, J =
7.5), 3.21−3.33 (3H, m), 3.86−3.96 (2H, m), 7.15 (1H, dd, J = 7.5,
4.8), 7.49 (1H, dd, J = 7.5, 1.7), 8.46 (1H, dd, J = 4.8, 1.7). HRMS
(EI) for C15H23O2N2S(MH+): calcd, 295.1475; found, 295.1474.
N-[(1R,3S)-5-Hydroxy-2-adamantyl]-2-propylsulfanylpyri-
dine-3-carboxamide (7d). 7d was prepared according to the
procedure of 7b from (1R,3S)-5-hydroxy-2-adamantylamine in 45%
1
yield. H NMR (300 MHz, DMSO-d6) δ 0.95 (3H, t, J = 7.8), 1.27−
1.37 (2H, m), 1.53−1.76 (8H, m), 1.90−2.11 (5H, m), 3.08 (2H, t, J =
7.8), 3.87−3.95 (1H, m), 4.39 (1H, s), 7.10−7.17 (1H, m), 7.59−7.66
(1H, m), 8.20 (1H, d, J = 6.9), 8.43−8.50 (1H, m). HRMS (EI) for
C19H27O2N2S(MH+): calcd, 347.1788; found, 347.1785.
N-[(1R,3S)-5-Hydroxy-2-adamantyl]-N-methyl-2-propylsulfa-
nylpyridine-3-carboxamide (7e). 7e was prepared according to the
procedure of 7b from (1R,3S)-N-methyl-5-hydroxy-2-adamantylamine
in 20% yield. 1H NMR (300 MHz, DMSO-d6, 373 K) δ 0.98 (3H, t, J
= 7.2), 1.46−1.56 (2H, m), 1.59−1.73 (8H, m), 1.93−2.04 (2H, m),
2.09−2.18 (1H, m), 2.32−2.40 (2H, m), 3.00 (3H, s), 3.18 (2H, t, J =
7.8), 3.98 (1H, s), 4.00−4.04 (1H, m), 7.14 (1H, dd, J = 7.5, 4.9), 7.49
(1H, dd, J = 7.5, 1.8), 8.45 (1H, dd, J = 4.9, 1.8). HRMS (EI) for
C20H29O2N2S(MH+): calcd, 361.1944; found, 361.1943.
N-Cyclohexyl-2-propylsulfanylpyridine-3-carboxamide (4).
Oxalyl chloride (0.332 mL, 3.81 mmol) was added dropwise to 2-
(propylthio)nicotinic acid (0.751 g, 3.81 mmol) and N,N-dimethyl-
formamide (0.014 g, 0.19 mmol) in CH2Cl2 (50 mL) over a period of
2 min. The resulting solution was stirred for 2 h at ambient
temperature and then added dropwise to cyclohexylamine (0.755 g,
7.61 mmol) in CH2Cl2 (20 mL) and stirred for 1 h. The reaction
mixture was diluted with CH2Cl2 (75 mL) and washed sequentially
with saturated NaHCO3 (25 mL), water (25 mL), and saturated brine
(25 mL). The organic layer was dried over Na2SO4, filtered, and
evaporated to afford crude product that was purified by flash silica
chromatography, with elution gradient from 0% to 50% EtOAc in
isohexane. Pure fractions were evaporated to dryness to give a yellow
solid. This was triturated with Et2O (10 mL) and then the suspension
filtered to afford N-cyclohexyl-2-(propylthio)nicotinamide (0.435 g,
41%) as a white solid. 1H NMR (300 MHz, DMSO-d6) δ 0.95 (3H, t, J
= 7.2), 1.02−1.38 (5H, m), 1.50−1.90 (6H, m), 3.06 (2H, t, J = 7.2),
3.60−3.76 (1H, m), 7.13 (1H, dd, J = 7.5, 4.9), 7.63 (1H, dd, J = 7.5,
1.8), 8.24 (1H, d, J = 7.8), 8.46 (1H, dd, J = 4.9, 1.8). HRMS (EI) for
C15H23ON2S (MH+): calcd, 279.1526; found, 279.1525.
N-[(1R,3R)-5-Hydroxy-2-adamantyl]-N-methyl-2-propylsulfa-
nylpyridine-3-carboxamide (7f). 7f was prepared according to the
procedure of 7b from (1R,3R)-N-methyl-5-hydroxy-2-adamantylamine
in 16% yield. 1H NMR (300 MHz, DMSO-d6, 373 K) δ 0.98 (3H, t, J
= 7.2), 1.51−1.73 (10H, m), 1.92−2.09 (3H, m), 2.39−2.48 (2H, m),
2.94 (3H, s), 3.18 (2H, t, J = 7.8), 3.95 (1H, s), 4.02−4.06 (1H, m),
7.14 (1H, dd, J = 7.5, 4.9), 7.49 (1H, dd, J = 7.5, 1.8), 8.45 (1H, dd, J =
4.9, 1.8). HRMS (EI) for C20H29O2N2S(MH+): calcd, 361.1944;
found, 361.1943.
(2-Propylsulfanyl-3-pyridyl)-[2-(4-pyridyl)pyrrolidin-1-yl]-
methanone (7g). 7g was prepared according to the procedure of 7b
from 4-pyrrolidin-2-ylpyridine in 62% yield. 1H NMR (400 MHz,
DMSO-d6, mixture of rotamers in a 2:1 ratio) δ 0.97 (3H, dt), 1.51−
1.99 (5H, m), 2.32−2.57 (1H, m), 2.98−3.11 (0.67H, m), 3.19
(1.34H, t), 3.27−3.41 (0.67H, m), 3.48−3.56 (0.67H, m), 3.77
(0.67H, t), 4.72 (0.34H, dd), 5.14 (0.67H, dd), 6.86 (0.34H, dd), 6.98
(0.67H, d), 7.17−7.26 (1H, m), 7.41 (1.34H, d), 7.74 (0.67H, dd),
8.31 (0.34H, dd), 8.33−8.38 (0.67H, m), 8.50−8.56 (2H, m). HRMS
(EI) for C18H22ON3S(MH+): calcd, 328.1478; found, 328.1477.
4-[(2-Propylsulfanylpyridine-3-carbonyl)amino]-
cyclohexanecarboxylic Acid (7h). Oxalyl chloride (0.496 g, 3.91
mmol) was added dropwise to 2-(propylthio)nicotinic acid (0.154 g,
0.78 mmol) and N,N-dimethylformamide (6.05 μL, 0.08 mmol) in
CH2Cl2 (5 mL). The resulting solution was stirred for 2 h at ambient
temperature and then concentrated under reduced pressure and
azeotroped with toluene (10 mL). The residue was suspended in THF
(1 mL) and added to a solution of (1S,4S)-4-aminocyclohexanecar-
boxylic acid (0.112 g, 0.78 mmol) in 1 M NaOH in water (1.56 mL,
1.56 mmol) simultaneously with 1 M NaOH in water (0.78 mL, 0.78
mmol). The reaction mixture was stirred at ambient temperature
overnight. The pH was adjusted to 5 with 1 M hydrochloric acid, and
all volatiles were removed under reduced pressure. The resultant solid
was triturated with water (10 mL), filtered and the solid washed with
water (5 mL), Et2O (5 mL), then isohexane (5 mL) to afford (1S,4S)-
[(2-propylsulfanylpyridine-3-carbonyl)amino]cyclohexanecarboxylic
acid (0.137 g, 54%) as an off white solid. 1H NMR (300 MHz, DMSO-
d6, 373 K) δ 0.98 (3H, t, J = 7.3), 1.55−1.75 (8H, m), 1.84−2.04 (2H,
m), 2.35−2.45 (1H, m), 3.13 (2H, t, J = 7.2), 3.80−3.96 (1H, m), 7.10
(1H, dd, J = 7.5, 4.8), 7.62 (1H, dd, J = 7.5, 1.7), 7.82−7.98 (1H, m),
8.44 (1H, dd, J = 4.8, 1.7), 11.56 (1H, s). HRMS (EI) for
C16H23O3N2S(MH+): calcd, 323.1424; found, 323.1423.
N-Cyclohexyl-N-methyl-2-propylsulfanylpyridine-3-carbox-
amide (7a). 7a was prepared according to the procedure of 4 from N-
1
methylcyclohexylamine in 16% yield. H NMR (300 MHz, CDCl3) δ
0.85−1.15 (4H, m), 1.30−1.85 (11H, m), 2.65 (1.5H, s, rotamers),
2.93 (1.5H, s), 3.01−3.21 (2.5H, m), 4.44−4.58 (0.5H, m), 6.90−6.98
(1H, m), 7.24−7.35 (1H, m), 8.33−8.40 (1H, m). HRMS (EI) for
C16H25ON2S (MH+): calcd, 293.1682; found, 293.1681.
N-(4,4-Difluorocyclohexyl)-2-propylsulfanylpyridine-3-car-
boxamide (7b). 2-(Propylthio)nicotinic acid (0.130 g, 0.66 mmol),
1-hydroxybenzotriazole (0.105 g, 0.78 mmol), and N,N-diisopropyle-
thylamine (0.314 mL, 1.80 mmol) was added to 4,4-difluorocyclohex-
anamine hydrochloride (0.103 g, 0.60 mmol) in CH2Cl2 (5 mL)
followed by N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydro-
chloride (0.150 g, 0.78 mmol). The resulting suspension was stirred at
ambient temperature for 18 h. The reaction mixture was diluted with
CH2Cl2 (50 mL) and washed with water (5 mL). The organic layer
was dried over Na2SO4, filtered, and evaporated to afford crude
product that was purified by flash silica chromatography, with elution
gradient from 0% to 10% EtOAc in CH2Cl2. Pure fractions were
evaporated to dryness to afford N-(4,4-difluorocyclohexyl)-2-
1
(propylthio)nicotinamide (0.139 g, 74%) as a white solid. H NMR
(300 MHz, DMSO-d6) δ 0.96 (3H, t, J = 7.5), 1.52−1.68 (4H, m),
1.81−2.16 (6H, m),, 3.07 (2H, t, J = 7.8), 3.85−4.00 (1H, m), 7.15
(1H, dd, J = 7.5, 4.8), 7.66 (1H, dd, J = 7.5, 1.7), 8.38 (1H, d, J = 7.5),
8.48 (1H, dd, J = 4.8, 1.7). HRMS (EI) for C15H21ON2F2S (MH+):
calcd, 315.1337; found, 315.1337.
N-Methyl-2-propylsulfanyl-N-tetrahydropyran-4-yl-pyri-
dine-3-carboxamide (7c). 7c was prepared according to the
procedure of 7b from N-methyltetrahydropyran-4-amine in 66%
yield. 1H NMR (500 MHz, DMSO-d6, 373 K) δ 0.97 (3H, t, J = 7.5),
2-[(3R)-1-[5-(Cyclohexylcarbamoyl)-6-propylsulfanylpyridin-
2-yl]-3-piperidyl]acetic Acid (11h) [General Procedure for SNAr
and Ester Hydrolysis]. 6-Chloro-N-cyclohexyl-2-propylsulfanylpyr-
idine-3-carboxamide 10 (1.88 g, 6.03 mmol), methyl-(R)-3-piperidine
acetate hydrochloride (1.17 g, 6.03 mmol), K2CO3 (2.50 g, 18.08
mmol), and butyronitrile were mixed in a microwave tube and stirred
at 170 °C for 2 h. The solvent was evaporated under reduced pressure.
Then water (20 mL) was added and the product was extracted with
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EtOAc (2 × 40 mL), washed with brine (10 mL), dried over MgSO4,
filtered, and evaporated under reduced pressure to give an orange oil.
Purification by flash column chromatography (SiO2, eluent gradient
from 0% to 50%, hexane/EtOAc) afforded a slightly yellow oil which
crystallized to give a white solid (1.44 g, 55%). The solid was dissolved
in THF (20 mL), and water was added (10 mL) followed by LiOH
(281 mg). The mixture was stirred at room temperature for 3 h. The
solution was acidified with 2 N HCl between pH 4 and pH 5 and the
product extracted in EtOAc (2 × 40 mL). The solution was washed
with brine (10 mL), dried over MgSO4 and the organic phase
evaporated under reduced pressure to give a white solid (1.38 g, 99%).
1H NMR (400 MHz, CDCl3) δ 1.03 (3H, t, J = 7.6), 1.18−1.48 (6H,
m), 1.51−1.65 (2H, m), 1.68−1.78 (5H, m), 1.89−2.13 (4H, m),
2.25−2.37 (2H, m), 2.81 (1H, dd, J = 10.0, 13.1), 2.95−3.21 (3H, m),
3.92−4.05 (1H, m), 4.16−4.32 (2H, m), 6.36 (1H, d, J = 8.8), 6.57
(1H, d, J = 7.7), 7.80 (1H, d, J = 8.8). HRMS (EI) for C22H34O3N3S
(MH+): calcd, 420.2314; found, 420.2317.
HRMS (EI) for C21H32O3N3S(MH+): calcd, 406.2159; found,
406.2157.
1-[5-(Cyclohexylcarbamoyl)-6-propylsulfanylpyridin-2-yl]-
piperidine-3-carboxylic Acid (11f). 11f was prepared according to
the procedure of 11h from 6-chloro-N-cyclohexyl-2-propylsulfanylpyr-
1
idine-3-carboxamide 10 and ethyl nipecotate in 64% yield. H NMR
(400 MHz, CDCl3) δ 0.95 (3H, t, J = 7.2), 1.11−1.37 (6H, m), 1.46−
1.57 (2H, m), 1.65−1.78 (5H, m), 1.92−1.97 (2H, m), 2.03−2.07
(1H, m), 2.50−2.57 (1H, m), 3.02−3.12 (3H, m), 3.42 (1H, q, J =
7.2), 3.88−3.95 (1H, m), 4.03 (1H, m), 4.36−4.41 (1H, m), 6.33 (1H,
d, J = 8.8), 6.49 (1H, d, J = 7.6), 7.73 (1H, d, J = 8.8). HRMS (EI) for
C21H32O3N3S(MH+): calcd, 406.2159; found, 406.2158.
1-[5-(Cyclohexylcarbamoyl)-6-propylsulfanylpyridin-2-yl]-
piperidine-4-carboxylic Acid (11g). 11g was prepared according to
the procedure of 11h from 6-chloro-N-cyclohexyl-2-propylsulfanylpyr-
1
idine-3-carboxamide 10 and methyl isonipecotate in 19% yield. H
NMR (400 MHz, DMSO-d6) δ 0.96 (3H, t, J = 7.6), 1.05−1.35 (5H,
m), 1.43−1.93 (10H, m), 2.44−2.60 (2H, m), 2.94 (2H, t, J = 7.2),
2.99−3.10 (2H, m), 3.58−3.71 (1H, m), 4.21−4.31 (2H, m), 6.53
(1H, d, J = 8.8), 7.62 (1H, d, J = 8.8), 7.81 (1H, d, J = 7.6), 12.28 (1H,
s). HRMS (EI) for C21H32O3N3S (MH+): calcd, 406.2159; found,
406.2157.
4-[[5-(Cyclohexylcarbamoyl)-6-propylsulfanylpyridin-2-yl]-
amino]cyclohexane-1-carboxylic Acid (11a). 11a was prepared
according to the procedure of 11h from 6-chloro-N-cyclohexyl-2-
propylsulfanylpyridine-3-carboxamide 10 and E-4-aminocyclohexylcar-
1
boxylic acid methyl ester hydrochloride in 48% yield. H NMR (400
2-[(3S)-1-[5-(Cyclohexylcarbamoyl)-6-propylsulfanylpyridin-
2-yl]-3-piperidyl]acetic Acid (11i). 11i was prepared according to
the procedure of 11h from 6-chloro-N-cyclohexyl-2-propylsulfanylpyr-
idine-3-carboxamide 10 and methyl-(S)-3-piperidine acetate hydro-
chloride in 50% yield. 1H NMR (400 MHz, DMSO-d6) δ 0.95 (3H, t, J
= 7.6), 1.05−1.18 (1H, m), 1.15−1.35 (5H, m), 1.41−1.49 (1H, m),
1.55−1.90 (10H, m), 2.12−2.26 (2H, m), 2.71−2.77 (1H, m), 2.85−
3.01 (3H, m), 3.63−3.66 (1H, m), 4.15−4.34 (2H, m), 6.47 (1H, d, J
= 8.8), 7.61 (1H, d, J = 8.8), 7.79 (1H, d, J = 7.9), 12.15 (1H, s).
HRMS (EI) for C22H34O3N3S (MH+): calcd, 420.2314; found,
420.2317.
2-[1-[5-(Cyclohexylcarbamoyl)-6-propylsulfanylpyridin-2-
yl]-4-piperidyl]acetic Acid (11j). 11j was prepared according to the
procedure of 11h from 6-chloro-N-cyclohexyl-2-propylsulfanylpyr-
idine-3-carboxamide 10 and 2-(piperidin-4-yl)acetic acid ethyl ester in
61% yield. 1H NMR (300 MHz, CDCl3) δ 0.95 (3H, t, J = 7.6), 1.11−
1.42 (7H, m), 1.49−1.59 (1H, m), 1.62−1.71 (4H, m), 1.77−1.81
(2H, m), 1.91−2.08 (3H, m), 2.19−2.29 (2H, m), 2.83−2.90 (2H, m),
3.06 (2H, t, J = 7.6), 3.92 (1H, m), 4.24−4.33 (2H, m), 6.29 (1H, d, J
= 8.8), 6.47 (1H, d, J = 7.6), 7.72 (1H, d, J = 8.8). HRMS (EI) for
C22H34O3N3S(MH+): calcd, 420.2314; found, 420.2312.
2-[(3S)-1-[5-(Cyclohexylcarbamoyl)-6-propoxypyridin-2-yl]-
3-piperidyl]acetic Acid (13a). 6-Chloro-N-cyclohexyl-2-propoxyni-
cotinamide 12a (211 mg, 0.71 mmol), methyl-(S)-3-piperidine acetate
hydrochloride (276 mg,1.42 mmol), and potassium carbonate (393
mg, 2.84 mmol) were stirred in butyronitrile (5 mL). The mixture was
sealed and heated in a microwave (Biotage initiator, 300 W) at 170 °C
for 4 h. The mixture was diluted with dichloromethane (25 mL),
washed with water (20 mL), brine (20 mL), and dried over MgSO4,
then filtered and the solvent removed in vacuo. Chromatography SiO2,
eluting with ethyl acetate/isohexane 20−60%, gave a clear gum (215
mg). The gum was dissolved in MeOH (15 mL). Then 2 M NaOH
(540 μL, 1.07 mmol) was added and the mixture was stirred for 32 h.
Solvent was removed in vacuo and the residue was taken up in water
(10 mL) and pH adjusted to ∼3 to give 2-[(3S)-1-[5-(cyclo-
hexylcarbamoyl)-6-propoxypyridin-2-yl]-3-piperidyl]acetic acid (167
mg, 81%) as a white powder after trituration with ether/isohexane.
1H NMR (400 MHz, DMSO-d6) δ 1.02 (3H, t, J = 7.6), 1.15−1.90
MHz, DMSO-d6) δ 0.97 (3H, t, J = 7.2), 1.15 (2H, m), 1.24 −1.30
(6H, m), 1.37−1.44 (2H, m), 1.57−1.66 (3H, m), 1.70−1.78 (4H, m),
1.95−2.03 (4H, m), 2.15−2.25 (1H, m), 2.97 (2H, t, J = 7.2), 3.64
(1H, s), 3.71−3.77 (1H, m), 6.14 (1H, d, J = 8.8), 7.48 (1H, d, J =
8.8), 7.63 (1H, d, J = 7.2). HRMS (EI) for C22H34O3N3S (MH+):
calcd, 420.2315; found, 420.2314.
4-[[[5-(Cyclohexylcarbamoyl)-6-propylsulfanylpyridin-2-yl]-
amino]methyl]cyclohexane-1-carboxylic Acid (11b). 11b was
prepared according to the procedure of 11h from 6-chloro-N-
cyclohexyl-2-propylsulfanylpyridine-3-carboxamide 10 and aminome-
thylcyclohexane carboxylic acid methyl ester hydrochloride in 33%
1
yield. H NMR (400 MHz, DMSO-d6) δ 0.94−0.99 (5H, m), 1.10−
1.22 (1H, m), 1.23−1.40 (6H, m), 1.53−1.69 (4H, m), 1.71−1.81
(6H, m), 1.93−2.01 (2H, m), 2.10−2.16 (1H, m), 2.94−2.96 (2H, t, J
= 7.2), 3.16 (2H, d, J = 6.4), 3.55−3.65 (1H, m), 6.14 (1H, d, J = 8.4),
7.15 (1H, s), 7.47 (1H, d, J = 8.4), 7.69 (1H, d, J = 8.0), 12.20 (1H, s).
HRMS (EI) for C23H36O3N3S (MH+): calcd, 434.2471; found,
434.2468.
2-[4-[5-(Cyclohexylcarbamoyl)-6-propylsulfanylpyridin-2-
yl]oxyphenyl]acetic Acid (11c). 11c was prepared according to the
procedure of 11h from 6-chloro-N-cyclohexyl-2-propylsulfanylpyr-
idine-3-carboxamide 10 and methyl 4-hydroxyphenylacetate in 38%
yield. 1H NMR (300 MHz, CDCl3) δ 0.73 (t, 3H, J = 7.5), 1.14−1.50
(m, 7H), 1.55−1.81 (m, 3H), 1.95−2.07 (m, 2H), 2.75 (t, 2H, J =
7.5), 3.66 (s, 2H), 3.91−4.07 (m, 1H), 6.33 (d, 1H, J = 7.8), 6.62 (d,
1H, J = 8.4), 7.08 (d, 2H, J = 8.4), 7.31 (d, 2H, J = 8.4), 7.92 (d, 1H, J
= 8.1). MS m/z (M+ + H) 429. HRMS (EI) for C23H29O4N2S (MH+):
calcd, 429.1842; found, 429.1841.
2-[3-[5-(Cyclohexylcarbamoyl)-6-propylsulfanylpyridin-2-
yl]oxyphenyl]acetic Acid (11d). 11d was prepared according to the
procedure of 11h from 6-chloro-N-cyclohexyl-2-propylsulfanylpyr-
idine-3-carboxamide 10 and methyl 3-hydroxyphenylacetate in 45%
yield. 1H NMR (400 MHz, CDCl3) δ 0.71 (t, 3H, J = 7.2), 1.14−1.49
(m, 7H), 1.56−1.66 (m, 1H), 1.68−1.80 (m, 2H), 1.95−2.06 (m, 2H),
2.74 (t, 2H, J = 7.2), 3.65 (s, 2H), 3.92−4.06 (m, 1H), 6.35 (d, 1H, J =
7.2), 6.62 (d, 1H, J = 8.4), 7.01−7.09 (m, 2H), 7.14 (d, 1H, J = 7.6),
7.35 (t, 1H, J = 8.0), 7.91 (d, 1H, J = 8.0). HRMS (EI) for
C23H29O4N2S(MH+): calcd, 429.1842; found, 429.1841.
(17H, m), 2.13−2.26 (2H, m), 2.77−2.83 (1H, m), 2.93−3.05 (1H,
m), 3.71−3.83 (1H, m), 4.07−4.18 (1H, m), 4.23−4.35 (3H, m), 6.43
(1H, d, J = 8.8), 7.70 (1H, d, J = 7.6), 8.00 (1H, d, J = 8.8), 12.17 (1H,
s). HRMS (EI) for C22H34O4N3 (MH+): calcd, 404.2544; found,
404.2542.
2-[(3S)-1-[5-(Cyclohexylcarbamoyl)-6-propylaminopyridin-
2-yl]-3-piperidyl]acetic Acid (13b). 6-Chloro-N-cyclohexyl-2-
(propylamino)nicotinamide 12b (180 mg, 0.61 mmol), methyl-(S)-
3-piperidine acetate hydrochloride (148 mg, 0.76 mmol), potassium
carbonate (210 mg, 1.52 mmol) in butyronitrile (4 mL) were sealed in
1-[5-(Cyclohexylcarbamoyl)-6-propylsulfanylpyridin-2-yl]-
piperidine-2-carboxylic Acid (11e). 11e was prepared according to
the procedure of 11h from 6-chloro-N-cyclohexyl-2-propylsulfanylpyr-
idine-3-carboxamide 10 and methyl 3-hydroxyphenylacetate in 45%
1
yield. H NMR (400 MHz, DMSO-d6) δ 0.94 (3H, t, J = 7.2), 1.08−
1.13 (1H, m), 1.21−1.33 (4H, m), 1.41−1.47 (1H, m), 1.52−1.67
(4H, m), 1.68−1.80 (4H, m), 2.19−2.28 (2H, m), 2.91 (2H, t, J =
7.2), 3.09−3.17 (2H, m), 3.60−3.66 (2H, m), 4.11 (1H, s), 5.02 (1H,
s), 6.42 (1H, d, J = 8.8), 7.59 (1H, d, J = 8.8), 7.78 (1H, d, J = 7.6).
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a microwave tube and heated (Biotage Initiator 300 W) at 150 °C for a
total of 8 h. The mixture was diluted with water (20 mL) and extracted
with dichloromethane (2 × 20 mL). The combined extracts were
washed with brine (20 mL), dried over MgSO4, then filtered, and the
solvent was removed in vacuo. Chromatography (SiO2), eluting with
ethyl acetate/isohexane 0−40%, gave a clear gum (158 mg). The gum
was stirred in methanol (3 mL), and 2 M sodium hydroxide (475 μL,
0.95 mmol) was added. The mixture was stirred at room temperature
for 16 h. The solvent was removed in vacuo and the residue taken up
in water (10 mL) and acidified to pH ≈ 3 with 2 M HCl. The resulting
precipitate was filtered, washed with water, and dried to give 2-[(3S)-
1-[5-(cyclohexylcarbamoyl)-6-propylaminopyridin-2-yl]-3-piperidyl]-
were incubated for 2 h at room temperature. Following excitation at
320 nm, fluorescence at 665 and 620 nm was measured and the (665
nm)/(620 nm) ratio calculated using an Envision plate reader. This
data were then used to calculate IC50 values for each compound
(Origin 7.5, Microcal software, Northampton, MA, U.S.).
Measurement of 11-βHSD1 Activity in Isolated Human
Adipocytes. Subcutanesous (sc) adipose tissue was obtained through
a needle biopsy from the lower part of the abdomen after dermal local
anesthesia with lidocaine (Xylocain; AstraZeneca, Sodertalje, Sweden)
̈
̈
from nondiabetic volunteers. Adipocytes were isolated from sc adipose
tissue by collagenase digestion. In brief, adipocytes were isolated from
the adipose tissue following shaking in medium 199 (Invitrogen, U.K.)
supplemented with 5.6 mmol/L glucose, 4% BSA (Sigma-Aldrich,
Poole, U.K.), and 0.6 mg/mL collagenase (Roche, Burgess Hill, U.K.)
at 37 °C for ∼60 min. After filtration through a 250 μm nylon mesh
the adipocytes were washed 4 times with medium 199 supplemented
with 5.6 mmol/L glucose and 1% BSA. Isolated adipocytes were
diluted to approximately 3−5% lipocrit containing DMEM (6 mmol/L
glucose, 10% FCS (Invitrogen, UK), 1% penicillin/streptomycin
(Invitrogen, U.K.)) and incubated in six-well plates in duplicate for 6 h
1
acetic acid as a white solid (126 mg, 51%). H NMR (400 MHz,
DMSO-d6) δ 0.90 (3H, t, J = 7.6), 1.03−1.95 (17H, m), 2.10−2.24
(2H, m), 2.65−2.75 (1H, m), 2.84−2.96 (1H, m), 3.27 (2H, t, J =
6.7), 3.60−3.76 (1H, m), 4.10−4.28 (2H, m), 5.92 (1H, d, J = 8.8),
7.58 (1H, d, J = 7.6), 7.75 (1H, d, J = 8.8), 8.76 (1H, bs), 12.12 (1H,
bs); LRMS m/z (M+ + H) 403.
2-[(3S)-1-[5-(Cyclohexylcarbamoyl)-6-(methylpropylamino)-
pyridin-2-yl]-3-piperidyl]acetic Acid (13c). 13c was prepared
according to the procedure of 13b from 6-chloro-N-cyclohexyl-2-
[methyl(propyl)amino]nicotinamide 12c and methyl-(S)-3-piperidine
acetate hydrochloride in 33% yield. 1H NMR (400 MHz, DMSO-d6) δ
0.82 (3H, t, J = 7.6), 1.08−1.81 (17H, m), 2.10−2.22 (2H, m), 2.64−
2.71 (1H, m), 2.76 (3H, s), 2.83−2.93 (1H, m), 3.14−3.25 (2H, m),
3.63−3.68 (1H, m), 4.05−4.25 (2H, m), 6.18 (1H, d, J = 8.4), 7.49
(1H, d, J = 8.4), 8.22 (1H, d, J = 8.4), 12.16 (1H, s). HRMS (EI) for
C23H37O3N4(MH+): calcd, 417.2860; found, 417.2858.
2-[(3S)-1-[6-Butyl-5-(cyclohexylcarbamoyl)pyridin-2-yl]-3-
piperidyl]acetic Acid (14). 2-Butyl-6-chloro-N-cyclohexylnicotina-
mide 20 (310 mg, 1.05 mmol), methyl-(S)-3-piperidine acetate
hydrochloride (500 mg, 2.58 mmol), and potassium carbonate (436
mg, 3.15 mmol) were stirred in butyronitrile (20 mL) under nitrogen
at 150 °C for 96 h. Solvent was removed. The residue was dissolved in
CH2Cl2 (20 mL), and water (20 mL) was added. The mixture was
acidified, and the organic layer was separated. The aqueous layer was
extracted with further dichloromethane (2 × 20 mL). The combined
extracts were washed with brine, dried over MgSO4, filtered, and the
solvent was removed in vacuo. Chromatography (SiO2), eluting with
5% MeOH/EtOAc 0−100%, gave 2-[(3S)-1-[6-butyl-5-
(cyclohexylcarbamoyl)pyridin-2-yl]-3-piperidyl]acetic acid (106 mg,
25%) as a white foam. 1H NMR (400 MHz, DMSO-d6) δ 0.87 (3H, t,
J = 7.6), 1.02−1.48 (10H, m), 1.55−1.88 (9H, m), 2.11−2.27 (2H,
m), 2.65−2.79 (3H, m), 2.84−2.94 (1H, m), 3.61−3.73 (1H, m),
4.13−4.26 (2H, m), 6.57 (1H, d, J = 8.8), 7.42 (1H, d, J = 8.8), 7.84
(1H, d, J = 8.0), 12.07 (1H, s). HRMS (EI) for C23H36O3N3(MH+):
calcd, 402.2751; found, 402.2749.
3
with H-cortisone (20 nmol/L, 1 μCi/mL, Amersham, Chalfont St.
Giles, U.K.) containing DMEM (6 mmol/L glucose, 100 nmol/L
cortisone (Sigma-Aldrich, Poole, U.K.), 10% FCS, 1% PEST) at 37 °C,
5% CO2. Following incubations, media samples were collected and
cortisone to cortisol conversion was analyzed. Radiolabeled steroids
were extracted using ethyl acetate. Samples were evaporated to dryness
under nitrogen and resuspended in mobile phase for HPLC analysis
(methanol/H2O, 50:50), adapted from Napolitano et al.39 Radio-
labeled steroids were separated using reversed phase HPLC, Agilent
1200 HPLC instrument using a Kromasil C18 5 μm column, 4.6 mm
× 25 0 mm (Crawford Scientific, Lanarkshire, U.K.) with methanol/
H2O (50:50) at a flow rate of 1.5 mL/min. Radioactivity was measured
using a flow scintillation analyzer (Radiomatic series 500TR, Perkin-
Elmer Analytical Instruments) with FLO-ONE software.
Measurement of Inhibition of 11β-HSD1 Activity in the
Mouse. Male C57Bl6J mice maintained on a chow diet were dosed by
oral gavage with compound suspended in HPLC/Tween. At 1 h after
dose, animals were euthanized using a rising concentration of CO2.
Blood samples were taken via cardiac puncture for compound
determination, and epididymal fat pads were removed and snap-
frozen in liquid N2. Adipose tissue (50−100 mg) was cut into 2−3
mm3 pieces using scissors and incubated in 24-well plate with medium
(DMEM Ham F12 medium supplemented with 1% penicillin/
streptomycin/ampicillin and 10% FCS) containing 20 nM 3H-
cortisone for 1 h at 37 °C. Following incubation, medium was
removed and steroid extracted and HPLC performed as per adipocyte
assay.
Measurement of 11β-HSD1 Activity. The conversion of
cortisone to the active steroid cortisol by 11β-HSD1 oxo-reductase
can be measured using a cortisol competitive homogeneous time-
resolved fluorescence assay (HTRF) assay (CisBio International,
R&D, Administration and Europe Office, In Vitro Technologies,
ASSOCIATED CONTENT
* Supporting Information
Experimental details for the syntheses of intermediates and
crystallographic information. This material is available free of
charge via the Internet at http://pubs.acs.org.
■
S
̀
HTRF/Bioassays BP 84175, 30204 Bagnols/Ceze Cedex, France).
The evaluation of compounds was carried out using a baculovirus
expressed N terminal 6-His tagged full length human, rat, dog, or
cynomolgous monkey 11β-HSD1 enzyme. The enzyme was purified
from a detergent solubilized cell lysate, using a copper chelate column.
Inhibitors of 11β-HSD1 reduce the conversion of cortisone to cortisol,
which is identified by an increase in signal, in the above assay. The
assay incubation was carried out in black 384-well plates (Matrix,
Hudson, NH, U.S.), consisting of cortisone (Sigma, Poole, Dorset,
U.K., 160 nM), glucose 6-phosphate (Roche Diagnostics, 1 mM),
NADPH (Roche Diagnostics, 100 μM), glucose 6-phosphate
dehydrogenase (Roche Diagnostics, 12.5 μg/mL), EDTA (Sigma,
Poole, Dorset, U.K., 1 mM), assay buffer (K2HPO4/KH2PO4, 100
mM), pH 7.5, recombinant 11β-HSD1 (1.5 μg/mL) plus test
compound in a total volume of 20 μL. The assay plates were
incubated for 25 min at 37 °C, and the reaction was stopped by the
addition of 10 μL of 0.5 mM glycerrhetinic acid (Sigma) plus cortisol-
XL665. Then 10 μL of anti-cortisol cryptate was added, and the plates
AUTHOR INFORMATION
Corresponding Author
*Phone: +44 (0)1625 232567. Fax: +44 (0)1625 516667. E-
mail: jamie.scott@astrazeneca.com.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
Paul Kemmit, Linda Godfrey, and Pernilla Sorme are thanked
for their synthetic chemistry contributions, and Roger Butlin
and Clive Green are thanked for useful medicinal chemistry
discussions. Gemma Convey, Sally Johnson, Clare Stacey, and
Alice Yu are thanked for the generation of biological data.
■
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(16) Webster, S. P.; Pallin, T. D. 11β-Hydroxysteroid dehydrogenase
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ABBREVIATIONS USED
■
11β-HSD1, 11β-hydroxysteroid dehydrogenase type 1; 11β-
HSD2, 11β-hydroxysteroid dehydrogenase type 2; ABT, 1-
aminobenzotriazole; AG, acylglucuronide; Clp, plasma clear-
ance; CYP, cytochrome P450; DMPK, drug metabolism and
pharmacokinetics; EDAC, 1-(3-dimethylaminopropyl)-3-ethyl-
carbodiimide; HDL, high density lipoprotein; HOBT, N-
hydroxybenzotriazole; LLE, ligand lipophilicity efficiency;
MDCK, Madin−Darby canine kidney cell line; NAD,
nicotinamide adenine dinucleotide; NADPH, nicotinamide
adenine dinucleotide phosphate; PPB, plasma protein binding;
SAR, structure−activity relationship
(17) Boyle, C. D.; Kowalski, T. J. 11β-Hydroxysteroid dehydrogenase
type 1 inhibitors: a review of recent patents. Expert Opin. Ther. Pat.
2009, 19, 801−825.
(18) Morgan, S. A.; Tomlinson, J. W. 11β-Hydroxysteroid
dehydrogenase type 1 inhibitors for the treatment of type 2 diabetes.
Expert Opin. Invest. Drugs 2010, 19, 1067−1076.
(19) Barf, T.; Vallgårda, J.; Emond, R.; Haggstrom, C.; Kurz, G.;
̈
̈
Nygren, A.; Larwood, V.; Mosialou, E.; Axelsson, K.; Olsson, R.;
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Engblom, L.; Edling, N.; Ronquist-Nii, Y.; Ohman, B.; Alberts, P.;
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Abrahmsen, L. Arylsulfonamidothiazoles as a new class of potential
antidiabetic drugs. Discovery of potent and selective inhibitors of the
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