Novel acidic 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) inhibitor with reduced acyl glucuronide liability: The discovery of 4-[4-(2-adamantylcarbamoyl)-5-tert-butyl-pyrazol-1-yl]benzoic acid (AZD8329)

Article abstract of DOI:10.1021/jm301252n

Inhibition of 11β-HSD1 is viewed as a potential target for the treatment of obesity and other elements of the metabolic syndrome. We report here the optimization of a carboxylic acid class of inhibitors from AZD4017 (1) to the development candidate AZD8329 (27). A structural change from pyridine to pyrazole together with structural optimization led to an improved technical profile in terms of both solubility and pharmacokinetics. The extent of acyl glucuronidation was reduced through structural optimization of both the carboxylic acid and amide substituents, coupled with a reduction in lipophilicity leading to an overall increase in metabolic stability.

Full text of DOI:10.1021/jm301252n

Article
pubs.acs.org/jmc
Novel Acidic 11β-Hydroxysteroid Dehydrogenase Type 1 (11β-HSD1)
Inhibitor with Reduced Acyl Glucuronide Liability: The Discovery of
4‑[4-(2-Adamantylcarbamoyl)-5-tert-butyl-pyrazol-1-yl]benzoic Acid
(AZD8329)
James S. Scott,* Joanne deSchoolmeester, Elaine Kilgour, Rachel M. Mayers, Martin J. Packer,
David Hargreaves, Stefan Gerhardt, Derek J. Ogg, Amanda Rees, Nidhal Selmi, Andrew Stocker,
John G. Swales, and Paul R. O. Whittamore
Cardiovascular & Gastrointestinal Innovative Medicines Unit, AstraZeneca Mereside, Alderley Park, Macclesfield, Cheshire,
SK10 4TG, United Kingdom
S
* Supporting Information
ABSTRACT: Inhibition of 11β-HSD1 is viewed as a potential
target for the treatment of obesity and other elements of the
metabolic syndrome. We report here the optimization of a
carboxylic acid class of inhibitors from AZD4017 (1) to the
development candidate AZD8329 (27). A structural change from
pyridine to pyrazole together with structural optimization led to
an improved technical profile in terms of both solubility and
pharmacokinetics. The extent of acyl glucuronidation was
reduced through structural optimization of both the carboxylic
acid and amide substituents, coupled with a reduction in lipophilicity leading to an overall increase in metabolic stability.
compound, we had observed that the major route of elimination
was formation of an acyl glucuronide metabolite. This is a normal
metabolic fate for acids,15,16 and moreover in the case of
compound 1 we had not observed any covalent binding to
proteins that can be associated with adverse toxicological events.
Nevertheless, as part of a back-up program, we deemed it
desirable to seek to identify a candidate with a similar biological
profile but with a reduced propensity to form an acyl
glucuronide.
INTRODUCTION
■
The metabolic syndrome is a collection of abnormalities
including resistance to insulin, obesity, dyslipidemia, hyper-
glycemia and hypertension that represents a major risk factor for
cardiovascular disease and type II diabetes.1 It has been proposed
that elevated intracellular levels of the glucocorticoid hormone
cortisol in liver and adipose tissue can lead to glucose intolerance
and insulin resistance and can contribute to the development of
the metabolic syndrome.2−4
11β-Hydroxysteroid dehydrogenase type 1 (11β-HSD1) is an
NADPH dependent reductase expressed mainly in liver, adipose
and brain tissue that converts the glucocorticoid inactive
hormone cortisone to the glucocorticoid active hormone
cortisol.5 Inhibition of this enzyme has the potential to reduce
intracellular glucocorticoid concentrations and has been
proposed as an attractive therapeutic paradigm for treatment of
the metabolic syndrome.6−8 The considerable interest in this
target is reflected by the large number of publications and patent
applications as described in recent reviews.9−11
11β-Hydroxysteroid dehydrogenase type 2 (11β-HSD2) is an
NAD dependent oxidase expressed mainly in kidney and colon
tissue that catalyzes the reverse reaction and prevents cortisol
activation of mineralocorticoid receptors.12 Selectivity against
this isoform was viewed as an essential requirement in order to
avoid any risk of hypertensive effects.13
Our start point for this program of work was the pyridine
amide lead 2 previously identified from a high throughput
screening campaign.14 We believed that the thiopropyl
substituent in combination with the amide functionality was
important for binding and thus screened a diverse range of five
and six membered heterocyles that contained this fragment. This
led to the identification of pyrazole 3 that showed considerably
enhanced potency at similar lipophilicity (Table 1). The
structural change from pyridine to pyrazole resulted in an
increase in both ligand lipophilicity efficiency (LLE; pIC50-
logD)17 and ligand efficiency as measured per heavy atom count.
The pyrazole retained the high free drug levels observed with the
pyridine core but, in common with the pyridine 2, pyrazole 3
showed no oral exposure when dosed in a rat pharmacokinetic
study.
In a previous publication, we described the identification of
compound 1 (AZD4017, Figure 1)14 as a potent and selective
acidic inhibitor of 11β-HSD1. During profiling of this
Received: August 30, 2012
© XXXX American Chemical Society
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Figure 1. Interconverstion of cortisone and cortisol by 11β-HSD1 and 11β-HSD2 and the acidic inhibitor AZD4017 (1).
Table 1. Enzyme Potencies, Physical and DMPK Properties for Substituted Pyrazoles
cpd human 11β-HSD1 IC50 (μM) logD7.4 LLE (pIC50-logD)
LE
rat PPB (% free) rat Heps Clint (μL/min/106 cells) rat Cl (mL/min/kg) rat F (%)
2
3
0.350
0.018
3.2
3.0
3.3
4.7
0.46
0.55
13.0
13.2
284
120
41
21
0
0
a
Scheme 1. Synthesis of Pyrazoles 4−7
a
t
(a) (OEt)3CH, Ac2O, 140 °C, 5 h, 38%; (b) RNHNH2.HCl, (iPr)2NEt, CH(OtBu)2NMe2, EtOH, 70 °C, 1 h, 32−66%; (c) BuONO, CuCl2,
n
MeCN, 65 °C, 15 min, 30−65%; (d) PrSH, NaHMDS, DMF, 20 °C, 2 h, 65−86%; (e) NaOH, MeOH, 60 °C, 24 h, 48−98%.
In efforts to optimize the oral exposure of pyrazole 3 we sought
to introduce acidic functionality with the hope of achieving
improved pharmacokinetics.14 Our initial efforts focused on
substitution at the 1N-pyrazole position and the synthesis of
compounds made as part of this optimization effort are detailed
below (Schemes 1−5).
give thiopropylpyrazoles 34 and 35. Hydrolysis of the aryl esters
under basic conditions produced the pyrazole acids 4−7.
In order to facilitate exploration of the amide functionality, the
pyrazole core could also be constructed with a tert-butyl ester
instead of an amide substituent (Scheme 2). Treatment of tert-
butyl cyanoacetate with triethylorthoformate followed by
reaction with methyl 4-hydrazinobenzoate gave the amino-
pyrazole 36. Diazotisation followed by treatment with cupric
chloride gave the chloropyrazole 37 which could be displaced
with propylthiol to give the thiopyrazole 38. Cleavage of the tert-
butyl ester with trifluoroacetic acid gave the acid 39. This could
be coupled with a range of amines under HOBt/EDCI coupling
conditions to give intermediates of type 40 that, after hydrolysis
of the aryl ester, afforded compounds 8−10, 12 and 13.
Treatment of chloropyrazole 37 with a range of thioalkyl groups
produced thioalkylpyrazoles of type 41. These could be
converted to compounds 14−17 following a similar sequence
SYNTHESIS
■
Pyrazoles were initially constructed via a route that involved the
condensation of 2-cyano-N-cyclohexylacetamide 28 with
triethylorthoformate to give the enamide 29 (Scheme 1).
Reaction with alkyl or aryl hydrazines produced the correspond-
ing aminopyrazoles 30 and 31. The amino functionality could be
converted to chloro through diazotization with tert-butylnitrite
followed by treatment with cupric chloride to give chloropyr-
azoles 32 and 33. The chlorine was subsequently displaced with
propane thiol using sodium hexamethyldisilazide as the base to
B
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a
Scheme 2. Synthesis of Thiopyrazoles 8−10, 12−17
a
Reagents: (a) (i) (OEt)3CH, Ac2O, 125 °C, 3 h; (ii) (4-COOMe)C6H4NHNH2.HCl, (iPr)2NEt, CH(OtBu)2NMe2, EtOH, Δ, 5 h, 21%; (b)
tBuONO, CuCl2, MeCN, 65 °C, 15 min, 83%; (c) PrSH, K2CO3, BuCN, Δ, 5 h, 89%; (d) TFA, CH2Cl2, 20 °C, 16 h, 89−100%; (e) NR1R2,
HOBt, EDCI, (iPr)2NEt, DMF, 20 °C, 18 h, 46−92%; (f) NaOH, MeOH, 60 °C, 18 h, 48−100%; (g) R3SH, NaHMDS, DMF, 20 °C, 5 h, 58−77%;
(h) 2-adamantylamine, HOBt, EDCI, (iPr)2NEt, DMF, 20 °C, 18 h, 57−85%.
n
n
a
Scheme 3. Synthesis of Pyrazole 11
a
Reagents: (a) Et3N, EtOH, 80 °C, 6 h, 94%; (b) tBuONO, CuCl2, CH3CN, 60 °C, 30 min, 84%; (c) nPrSH, Na2CO3, DMF, 20 °C, 16 h, 88%; (d)
KOSiMe3, THF, 18 °C, 18 h, 88%; (e) trans-5-methoxyadamantan-2-amine.HCl, HOBt, EDCI, (iPr)2NEt, DMF, 20 °C, 18 h, 52%; (f) NaOH,
MeOH, 60 °C, 16 h, 41%.
a
Scheme 4. Synthesis of Thiopyrazoles 19−24, 26, 27
a
Reagents: (a) R1COMe, LiHMDS, THF, −78 °C then 2-isocyanatoadamantane −78 °C, 1 h, 81−99%; (b) CH(OMe)2NMe2, 1,4-dioxane, 100 °C,
2 h, 80−99%; (c) (4-COOMe)C6H4NHNH2.HCl, MeCOOH, EtOH, 80 °C, 2 h, 38−83%; (d) NaOH, 1,4-dioxane, 100 °C, 7 h, 42−100%; (e) (4-
CN)C6H4NHNH2·HCl, MeCOOH, EtOH, 80 °C, 3 h, 72%.
of tert-butyl ester cleavage to 42, amide coupling with 2-
adamantylamine to 43 and aryl ester hydrolysis.
In the case of the methoxy substituted adamantyl 11, the
chemistry involved an analogous route but with 4-cyano
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a
Scheme 5. Synthesis of Trifluoropyrazole 25
a
Reagents: (a) (4-CN)C6H4NHNH2·HCl, EtOH, 20 °C, 16 h, 77%; (b) KOSiMe3, THF, 20 °C, 3 h, 73%; (c) 2-adamantylamine.HCl, HOBt,
EDCI, (iPr)2NEt, DMF, 20 °C, 16 h, 58%; (d) NaOH, MeOH, 65 °C, 45 h, 93%.
Table 2. Enzyme Potencies, Physical and DMPK Properties for 1N-Substituted Pyrazoles
substituted hydrazine as starting material (Scheme 3).
Construction of the pyrazole core 44 occurred in good yield
then conversion of the amino group to a chloride 45 followed by
displacement with propylthiol gave the thiopyrazole 46. Careful
optimization revealed that the ester could be selectively
hydrolyzed in the presence of the nitrile using potassium
trimethylsilanolate to furnish the pyrazole acid 47. Amide
coupling with trans-5-methoxyadamantan-2-amine under
HOBt/EDCI coupling conditions gave cyano 48 which was
hydrolyzed under basic conditions to give acid 11.
For compounds with a carbon linked substituent, routes were
developed that involved addition of ketones to 2-isocyanatoa-
damantane to form β-keto esters of type 49 (Scheme 4).
Reaction with N,N-dimethylformamide dimethyl acetal gave the
corresponding enamines 50. Treatment with methyl 4-
hydrazinobenzoate formed pyrazoles 51 then hydrolysis of the
ester produced final compounds 20−24, 26 and 27. In the case of
compound 19, this was constructed in an analogous fashion but
using 4-cyano phenylhydrazine to form pyrazole 52. Hydrolysis
of the cyano group gave the desired acid 19.
Trifluoropyrazole 25 was synthesized via a route reacting ethyl
2-(ethoxymethylene)-4,4,4-trifluoro-3-oxobutanoate 53 with 4-
cyano phenylhydrazine to construct the pyrazole core 54
(Scheme 5). Selective hydrolysis of the ethyl ester in the
presence of the nitrile using potassium trimethylsilanolate
furnished the pyrazole acid 55. Amide coupling with 2-
adamantylamine under HOBt/EDCI coupling conditions gave
cyano 56 which was hydrolyzed under basic conditions to give
acid 25.
D
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Table 3. Enzyme Potencies, Physical and DMPK Properties for Amide Variation
In efforts to increase potency, we investigated the SAR of the
amide substituent (Table 3). In contrast to the results observed
in the previous pyridine series,14 N-methylcyclohexyl amide 8 did
not improve potency and led to a decrease in stability in rat
hepatocytes and a reduction in oral bioavailability. 2-Adamantyl
9 increased 10-fold in potency relative to 6 but at the expense of a
log unit increase in lipophilicity leading to an overall similar value
for LLE (6.7). Despite this, the in vitro and in vivo clearance
remained low and the compound had moderate bioavailability.
Attempts to introduce a hydroxyl substituent 10 onto the
adamantyl ring resulted in retention of potency despite a
lowering of lipophilicity and a corresponding increase in LLE
(8.6). Despite high stability in hepatocytes, the compound
showed increased in vivo clearance and poor bioavailability,
potentially as a result of high free drug levels. The corresponding
methyl 11 and difluoromethyl 12 ethers retained good potency
and showed improved bioavailability over the corresponding
hydroxyl 10. Sulphone 13 showed a drop in potency and very
RESULTS AND DISCUSSION
■
Both cis 4 and trans 5 cyclohexylacids showed modest potency
with the trans isomer 5 being slightly more potent and with a
higher LLE (5.5). The introduction of acidic functionality
resulted in improved oral exposure of these compounds with the
trans isomer 5 in particular showing high stability in hepatocytes
that translated into low in vivo clearance and complete
bioavailability (100%). As both stereoisomers retained potency
we felt this indicated some degree of spatial tolerance for acidic
functionality. We explored alternative linking groups and as a
consequence made the benzoic acids 6 and 7. The para-
substituted acid resulted in an increase in potency together with a
lowering of lipophilicity that resulted in an increase in LLE (6.9).
Compound 6 has reasonable free levels for an acid and showed
low turnover in rat hepatocytes that corresponded to low in vivo
clearance and excellent bioavailability (Table 2).
E
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Table 4. Enzyme Potencies, Physical and DMPK Properties for Thioalkyl Pyrazoles
high in vivo clearance leading to poor bioavailability for this
compound.
more stable both in vitro and in vivo leading to an excellent
pharmacokinetic profile for this compound.
On balance we concluded that adamantyl 9 represented the
most promising point for further optimization and looked to
optimize the thio-alkyl substituent (Table 4). Truncation of the
thiopropyl-substituent to ethyl 14 and methyl 15 resulted in
small reductions in potency and an increase in free drug levels, as
expected based on the reduction in lipophilicity. Expansion to the
five-membered ring 16 increased lipophilicity but did not bring a
corresponding increase in potency, leading to a reduction in LLE
(5.7). Further expansion to the six-membered ring 17 resulted in
a further drop in potency and a lower LLE (4.9).
Although compound 15 looked attractive in terms of overall
profile, we elected to reinvestigate the SAR with respect to
whether the thio-group could be replaced (Table 5). Simple
deletion of the thio-substituent (18) resulted in a significant loss
in potency. Replacement with methyl 19 failed to increase
potency but extension to ethyl 20 resulted in a significant
improvement in LLE (5.5). Branching at this position, as
exemplified by isopropyl 21, resulted in a further improvement in
LLE (6.6). The 3 and 4-membered cyclic variants 22 and 23
maintained similar LLE levels although this deteriorated upon
expansion to the 5-membered ring 24. Notably, cyclopropyl 22
was less stable in rat hepatocytes than either the isopropyl 21 or
cyclobutyl 23 and this translated to higher in vivo clearance in rat.
Replacement with trifluoromethyl 25 resulted in a drop in
potency and a reduction in LLE. The methyl cyclopropyl 26 and
tert-butyl 27 analogues showed a further increase in LLE (7.1 and
7.0 respectively) surpassing the thiopropyl compound 9 (LLE
6.7) in terms of efficiency. In a similar manner to 22, cyclopropyl
26 had moderate turnover in rat hepatocytes and moderate in
vivo clearance in rat. In contrast, tert-butyl 27 was significantly
We obtained a crystal structure of compound 27 bound in the
active site of 11β-HSD1, using conditions described previously.14
Figure 2A shows the binding mode of compound 27 highlighting
the key interactions of the amide carbonyl with Ser170 and of the
acid which has a strong hydrogen bond contact to Tyr284. Figure
2B shows the structure of 27 overlaid with that of 1. The
cyclohexyl ring of 1 and the NH group overlay very closely with a
cyclohexyl ring of the adamantane portion of compound 27. The
amide carbonyl groups are at different angles; this is
accommodated by a change in position of the Ser170 side
chain, which makes a contact with the carbonyl in both
complexes. There is no equivalent to the propyl chain of 1, but
a methyl group from the tert-butyl substituent is overlaid on the
sulfur atom. The acid groups occupy very different positions in
the active site. In 1, we noted a key interaction with the backbone
at Leu217; this interaction is absent for compound 27 and
instead we observe a strong hydrogen bond contact to Tyr284.
This residue is not resolved in the structure with 1 and it appears
that compound 27 is able to stabilize the C-terminal loop of the
protein via this interaction, with the effect that it is now clearly
visible in the electron density. Comparing the ribbon trace for the
structure of 1 (yellow) with that of compound 27 (orange), we
see that the loop comprising residues Gly229 to His232 is pushed
outward (on the right of the Figure 2B) to accommodate the
tyrosine side chain. The C-terminal loop in the AZD4017
structure is not resolved beyond Ser283.
On the basis of this program of work, compound 27 was
profiled further (Table 6). The compound showed no significant
activity (<25% inhibition at 10 μM) against five isoforms of the
cytochrome P450 enzymes (CYP1A2, CYP2C9, CYP2C19,
F
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Table 5. Enzyme Potencies, Physical and DMPK Properties for Nonthioalkyl Pyrazoles
CYP2D6 and CYP3A4). Plasma protein binding showed good
free levels for an acid (1.3−3.5% free across species) in line with
the low lipophilicity of the compound (logD7.4 1.0). The
measured pKA of the carboxylic acid was 3.9. The solubility as
measured on crystalline material was >1900 μM and when taken
together with moderate cellular permeability, as measured in an
MDCK assay (Papp(A-B) 7.3 × 10−6 cm.s−1; efflux ratio 0.5), it
was predicted that this would lead to good absorption in vivo.
Four stable, nonsolvated crystalline forms were identified and no
photostability issues or chemical stability concerns across the pH
range 1−10 were highlighted with this compound.
The pharmacokinetic profile of compound 27 was examined in
other species and the results are summarized in Table 7. The
compound showed good properties (mouse, rat, dog) with low in
vivo clearance in mouse and very low in vivo clearance in both rat
and dog. Volumes of distribution were low as expected of an
acidic compound and good bioavailabilities (F > 40%) were
observed across all three species. The renal component of the in
vivo clearance was found to be minimal, comprising <1% of the
total clearance in rat and <5% in dog. Compound 27 tested
negative for reactive metabolites from oxidative metabolism in an
in vitro trapping assay in human liver microsomes using reduced
glutathione as a trapping agent.
In order to understand the mechanism of clearance,
compounds 1 and 27 were incubated in hepatocytes and the
proportion of glucuronidation relative to oxidative metabolism,
as measured by peak area of metabolites, was calculated as shown
in Table 8. In the case of compound 1, acyl glucuonidation was
the predominant metabolic pathway for this compound in all
three species with ratios relative to oxidative metabolism of
approximately 2, 4, and 6 in rat, dog and human respectively. In
stark contrast, compound 27 showed significantly greater
metabolic stability (greater % parent remaining) and minimal
amounts of acyl glucuronide formation (0.5% or less) in all three
species. A switch to oxidative metabolism as the predominant
metabolic pathway had occurred such that the ratios of acyl
G
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Figure 2. (A) Crystal structure of compound 27 bound to human 11β-HSD1 highlighting key residues Ser170 and Tyr284. (B) Crystal structure of
compound 27 bound to human 11β-HSD1, overlaid with 1. The protein constructs are identical, but compound 27 induces a loop motion, as seen to the
right, due to ordering of Tyr284 via a strong hydrogen bond contact.
Table 6. Physical Properties of Compound 27
A metabolism-ID study was carried out in bile duct canulated
rats with the results for the 0−6 h samples shown in Table 9. For
c
MDCK
aqueous
mouse
b
rat
dog
b
human
b
permeability
Papp
a
b
solubility
PPB
PPB
PPB
PPB
Table 9. Metabolism-ID Summaries in Rat Bile and Urine for
Selected Compounds 1 and 27
(μM)
(% free) (% free) (% free) (% free) (× 10−6 cm s−1
)
>1900
(n = 2)
3.5
(n = 1)
1.3
(n = 3)
2.0
(n = 2)
1.4
(n = 2)
7.3 (A-B); 3.9 (B-
A) (n = 3)
cpd
excreta
parent (%) [+Glu] (%) [+O] (%) ratio [+Glu]:[O]
a
Solubility of compounds in aqueous phosphate buffer at pH 7.4 after
1
rat bile
15
75
68
10
7.5
2.4
0.1
2.0
b
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-UVMS. Compounds were incubated at 10 μM
in cultured MDCK cells. Permeability was measured in both the A to B
and B to A direction.
1
rat urine
rat bile
4
28
27
27
10.8
99.4
9.1
0.4
80.1
0.2
c
rat urine
compound 1, acyl glucuronidation was the predominant
metabolic pathway. By contrast, oxidation was the main route
of metabolism observed for compound 27 in rat bile, with lower
amounts of glucuronide detected (9% vs 75% for 1). In rat urine,
the majority of the analyte was parent (99.4%), with only low
levels of the glucuronide (0.4%) and oxidized products (0.2%)
being detected, consistent with the lower clearance of this
compound. Three isomers of the glucuronide of 27 were
detected in rat bile compared to five glucuronide isomers of
compound 1.
Table 7. Pharmacokinetic Parameters for Selected
Compound 27
a
Clp
Vdss
i.v. half-
life (h)
oral half- bioavailability
life (h)
species (mL/min/kg) (L/kg)
(%)
mouse
rat
9
0.4
0.3
0.1
0.8
5.8
5.5
2.2
4.9
5.1
100
95
0.6
0.3
dog
45
a
Dosed at between 1 and 3 mg/kg in either solution (DMSO/
hydroxy-beta-cyclodextrin) or suspension (HPMC/Tween).
Compound 27 displayed excellent selectivity versus the related
enzymes 11β-HSD2 (IC50 >30 μM), 17β-HSD1 (IC50 >30 μM)
and 17β-HSD3 (<5% inhibition at 10 μM) and showed no
measurable activity against the glucocorticoid or mineralocorti-
coid receptors. Further profiling revealed that compound 27 was
nonmutagenic in a two-strain Ames test and negative in a mouse
lymphoma assay. It had an IC50 > 100 μM in the hERG IonWorks
assay and was assessed in a guinea pig MAP assay with no
changes observed in any parameters. A good selectivity profile
(>2500 fold selective) was observed versus a diverse panel of 140
targets.
Table 8. Metabolism-ID Summaries for Selected Compounds
1 and 27
cpd
species parent (%) [+Glu] (%) [+O] (%) ratio [+Glu]:[O]
1
rat
66
23
53
6
11
13
1
2.1
1
dog
34
4.1
1
human
rat
93
6
27
27
27
96
0.1
3.9
0.02
0.08
0.10
dog
93.7
95
0.5
0.4
5.8
4.6
human
Profiling of 11-βHSD1 activity across species revealed that,
although the potency of compound 27 was similar to that of 1 in
human and cynomolgous monkey, significant improvements
were observed in the rat (>50 fold) and dog (>450 fold) activity.
The potency as measured in human adipocytes (IC50 0.002 μM)
was in good agreement with the enzyme potency (Table 10).
Because of the improved potency of 27 in rat relative to 1 it was
feasible to carry out pharmacodynamic measurements in an ex
vivo assay measuring the inhibition of conversion of 3H-cortisone
glucuronidation to oxidative metabolism were now 0.1 or less.
We rationalized these observations as being a combination of
several factors; in the nature of the carboxylic acid (aryl vs alkyl)
dis-favoring acyl glucuronidation; the higher local lipophilicity of
the amide (adamantyl vs cyclohexyl) favoring metabolism; and
the overall reduction in lipophilicity leading to an overall increase
in metabolic stability.
H
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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, art.
5715) were used for TLC analysis. The purity of compounds submitted
for screening was >95% as determined by UV analysis of liquid
chromatography−mass spectroscopy (LC−MS) chromatograms at 254
nM and substantiated using the TAC (Total Absorption Chromato-
gram). Further support for the purity statement was provided using the
MS TIC (Total Ion Current) trace in ESI +ve and −ve ion modes,
HRMS and NMR analysis. Solutions were dried over anhydrous
magnesium sulfate and the solvent was removed by rotary evaporation
under reduced pressure.
Table 10. In Vitro Potencies for Compounds 1 and 27
rat
dog
enzyme
IC50
cyno
human
human
mouse
enzyme
IC50
enzyme adipocyte
enzyme
enzyme
IC50
(μM)
IC50
cpd IC50 (μM) IC50 (μM)
(μM)
(μM)
(μM)
1
0.007
0.009
0.750
6.1
4.06
3.77
0.029
0.024
0.002
0.002
27
0.086
0.008
3
to H-cortisol in this preclinical species. The effects in adipose
and liver tissue of dosing compound 27 orally to lean rats (1−30
mg/kg) are shown in Figure 3. At 3 h post dose at 3 mg/kg, 50%
cis-4-[4-(Cyclohexylcarbamoyl)-5-propylsulfanyl-pyrazol-1-
yl]cyclohexanecarboxylic Acid (4). cis-Ethyl 4-[4-(cyclohexylcarba-
moyl)-5-propylsulfanyl-pyrazol-1-yl]cyclohexane-1-carboxylate 34a
(116 mg, 0.28 mmol) was dissolved in methanol (5 mL) and treated
with 2 M sodium hydroxide (1.4 mL, 2.80 mmol). The mixture was
stirred at ambient temperature for 24 h. The reaction mixture was
concentrated in vacuo to remove the methanol and diluted with water
(25 mL). The aqueous solution was washed with ether (20 mL),
acidified to pH 3 with 2 M HCl and extracted with EtOAc (3 × 25 mL).
The combined extracts were washed with water (2 × 20 mL) and brine
(10 mL), dried (MgSO4) and evaporated to leave a white solid.
Trituration with ether gave a white solid (52 mg, 48%); 1H NMR (400
MHz, DMSO-d6) δ 0.91 (3H, t, J = 7.3 Hz), 1.10−1.47 (7H, m), 1.52−
1.74 (7H, m), 1.77−1.84 (2H, m), 1.85−1.99 (2H, m), 2.10−2.22 (2H,
m), 2.59−2.66 (1H, m), 2.87 (2H, t, J = 7.2 Hz), 3.66−3.79 (1H, m),
4.61−4.72 (1H, m), 7.77 (1H, d, J = 8.0 Hz), 7.92 (1H, s), 12.32 (1H, s);
HRMS (EI) for C20H32O3N3S (MH+); calcd, 394.2159; found,
394.2158.
4-[4-(N-Cyclohexyl-N-methyl-carbamoyl)-5-propylsulfanyl-
pyrazol-1-yl]benzoic Acid (8). Methyl 4-[4-(cyclohexyl-methyl-
carbamoyl)-5-propylsulfanyl-pyrazol-1-yl]benzoate 40a (162 mg, 0.39
mmol) was dissolved in methanol (10 mL) and treated at ambient
temperature with 2 M aqueous sodium hydroxide solution (0.96 mL,
1.95 mmol). The mixture was stirred at ambient temperature for 18 h.
Methanol was removed by evaporation under reduced pressure and the
clear solution was diluted with water (25 mL). 2 M HCl was added until
pH 4 and the mixture was extracted with ethyl acetate (2 × 25 mL). The
combined extracts were washed with water (2 × 10 mL) and brine (10
mL), dried (MgSO4) and evaporated to give the title compound as a
white solid (150 mg, 96%); 1H NMR (300 MHz, DMSO-d6) δ 0.68 (3H,
t, J = 7.3 Hz), 1.10−1.41 (4H, m), 1.42−1.85 (8H, m), 2.58 (2H, t, J =
7.0 Hz), 2.86 (3H, s), 3.45−3.60 (0.5H.m), 4.21−4.38 (0.5H,m), 7.74
(2H, d, J = 8.2 Hz), 7.89 (1H, s), 8.09 (2H, d, J = 8.4 Hz), 13.20 (1H, s);
HRMS (EI) for C21H28O3N3S (MH+); calcd, 402.1846; found,
402.1844.
Figure 3. Inhibition of 11β-HSD1 in mouse ex vivo model in adipose
and liver for compound 27.
inhibition was observed in liver tissue, approximately equal to the
in vitro rat IC50. In adipose tissue, a similar observation was made
at a dose of 10 mg/kg, approximating to five times the rat IC50
value.
In summary, we have discovered novel acidic inhibitors of 11β-
HSD1 that show excellent physicochemical and pharmacokinetic
profiles. Re-engineering of the initial structure involving a
structural change from pyridine to pyrazole, a switch from an
alkyl to an aryl acid and replacement of a thioalkyl group with a
tert-butyl substituent resulted in compound 27 that had
significantly reduced acyl glucuronidation liability. On the basis
of a strong technical profile and the ability to inhibit 11β-HSD1
in both rat liver and adipose tissue, compound 27 was selected for
development as AZD8329 and progressed into toxicity studies.
The results of further studies with this compound will be
reported in due course.
4-[4-(2-Adamantylcarbamoyl)-5-propylsulfanyl-pyrazol-1-
yl]benzoic Acid (9). Prepared according to the procedure of 8 from
methyl 4-[4-(2-adamantylcarbamoyl)-5-propylsulfanyl-pyrazol-1-yl]-
1
benzoate 40b in 94% yield; H NMR (300 MHz, DMSO-d6) δ 0.65
(3H, t, J = 7.3 Hz), 1.17−1.29 (2H, m), 1.54−1.66 (2H, m), 1.68−2.06
(12H, m), 2.62 (2H, t, J = 7.0 Hz), 4.05−4.14 (1H, m), 7.75 (2H, d, J =
8.5 Hz), 8.03 (1H, d, J = 7.5 Hz), 8.13 (2H, d, J = 8.5 Hz), 8.20 (1H, s),
13.19 (1H, s); HRMS (EI) for C24H30O3N3S (MH+); calcd, 440.2002;
found, 440.2002.
4-[4-[((trans)-5-Methoxy-2-adamantyl)carbamoyl]-5-propyl-
sulfanyl-pyrazol-1-yl]benzoic Acid (11). A solution of sodium
hydroxide (1.05 mL, 2.11 mmol) was added in one portion to a stirred
suspension of 1-(4-cyanophenyl)-N-(5-methoxy-2-adamantyl)-5-pro-
pylsulfanyl-pyrazole-4-carboxamide 48 (190 mg, 0.42 mmol), in MeOH
(4 mL) warmed to 60 °C. The resulting solution was stirred at 60 °C for
16 h. The reaction mixture was concentrated and diluted with 1,4-
dioxane (6 mL), and a further 2 mL sodium hydroxide solution and 3
mL water were added then stirred at 100 °C for 16 h. The reaction
mixture was evaporated to remove the organic solvents, and washed with
CH2Cl2 (10 mL), the aqueous layer was acidified with 2 N HCl, the
precipitate was collected by filtration, washed with water (10 mL) and
EXPERIMENTAL SECTION
■
All solvents and chemicals used were reagent grade. Anhydrous solvents
tetrahydrofuran (THF), dimethylformamide (DMF) and dimethylace-
tamide (DMA) 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 eluted
using an Isco Companion system. 1H NMR were recorded on a Varian
Gemini 2000 (300 MHz) or a Bruker Avance DPX400 (400 MHz) and
were determined in CDCl3 or DMSO-d6. Chemical shifts are reported in
I
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Journal of Medicinal Chemistry
Article
dried under vacuum to afford a cream solid that was purified by
preparative HPLC (Phenomenex Gemini C18 110A (axia) column, 5
μm silica, 30 mm diameter, 100 mm length), using decreasingly polar
mixtures of water (containing 0.5% NH3) and MeCN as eluents.
Fractions containing the desired compound were evaporated to dryness
to afford a white solid (82 mg, 41%). 1H NMR (400 MHz, DMSO-d6) δ
0.66 (3H, t, J = 7.3 Hz), 1.18−1.30 (2H, m), 1.41−1.51 (2H, m), 1.66−
1.86 (6H, m), 1.88−1.98 (2H, m), 2.07−2.18 (3H, m), 2.63 (2H, t, J =
7.1 Hz), 3.14 (3H, s), 3.96−4.10 (1H, m), 7.71−7.77 (1H, m), 8.01 (1H,
d, J = 7.3 Hz), 8.08−8.15 (1H, m), 8.18 (1H, s), 13.26 (1H, s); HRMS
(EI) for C25H32O4N3S (MH+); calcd, 470.2108; found, 470.2108.
4-[4-(2-Adamantylcarbamoyl)pyrazol-1-yl]benzoic Acid (18).
Prepared according to the procedure of 8 from methyl 4-[4-(2-
adamantylcarbamoyl)pyrazol-1-yl]benzoate in 28% yield; 1H NMR
(300 MHz, DMSO-d6) δ 1.48−1.61 (2H, m), 1.66−1.89 (8H, m), 1.90−
1.99 (2H, m), 2.04−2.18 (2H, m), 4.01−4.06 (1H, m), 7.58 (1H, d, J =
7.0 Hz), 7.98 (2H, d, J = 8.6 Hz), 8.08 (2H, d, J = 8.6 Hz), 8.23 (1H, s),
9.17 (1H, s), 13.00 (1H, s); HRMS (EI) for C21H24O3N3 (MH+); calcd,
366.1812; found, 366.1812.
4-[4-(2-Adamantylcarbamoyl)-5-methyl-pyrazol-1-yl]-
benzoic Acid (19). A solution of 1 M sodium hydroxide (24.28 mL,
24.28 mmol) was added to a stirred suspension of N-(2-adamantyl)-1-
(4-cyanophenyl)-5-methyl-pyrazole-4-carboxamide 52 (1.25 g, 3.47
mmol) in dioxane (25 mL). The resulting suspension was stirred at 100
°C for 7 h. The reaction mixture was concentrated, diluted with water
(40 mL) and filtered through Celite. The filtrates were acidified with 1
M citric acid. The precipitate was recovered by filtration, washed with
water (3 × 20 mL) and dried under vacuum at 50 °C. The crude product
was purified by preparative HPLC (Phenomenex Gemini C18 110A
(axia) column, 5 μm silica, 30 mm diameter, 100 mm length), using
decreasingly polar mixtures of water (containing 1% formic acid) and
MeCN as eluents. Fractions containing the desired compound were
evaporated to dryness to afford a pale yellow powder (550 mg, 42%); 1H
NMR (400 MHz, DMSO-d6) δ 1.45−1.54 (2H, m), 1.70−1.88 (8H, m),
1.90−2.00 (2H, m), 2.05−2.18 (2H, m), 2.56 (3H, s), 4.00−4.10 (1H,
m), 7.55 (1H, d, J = 6.8 Hz), 7.63−7.68 (2H, m), 8.05−8.10 (2H, m),
8.29 (1H, s), 13.25 (1H, s); HRMS (EI) for C22H26O3N3 (MH+); calcd,
380.1969; found, 380.1969.
7.55 (2H, m), 7.80 (1H, d, J = 7.1 Hz), 7.93 (1H, s), 8.04−8.10 (2H, m),
13.40 (1H, s); HRMS (EI) for C25H30O3N3 (MH+); calcd, 406.2125;
found, 406.2123.
4-[4-(2-Adamantylcarbamoyl)-5-cyclopentyl-pyrazol-1-yl]-
benzoic Acid (24). Prepared according to the procedure of 8 from
methyl 4-[4-(2-adamantylcarbamoyl)-5-cyclopentyl-pyrazol-1-yl]-
1
benzoate 51g in 97% yield; H NMR (400 MHz, DMSO-d6) δ 1.43
−1.55 (4H, m), 1.74 - 1.85 (12H, m), 1.89 - 1.96 (2H, m), 2.03 - 2.12
(4H, m), 2.99 - 3.08 (1H, m), 3.98 - 4.03 (1H, m), 7.53 - 7.55 (2H, m),
7.74 (1H, d, J = 6.9 Hz), 8.09 (1H, s), 8.10 - 8.12 (2H, m), 13.30 (1H, s);
HRMS (EI) for C26H32O3N3 (MH+); calcd, 434.2438; found, 434.2438.
4-[4-(Adamantan-2-ylcarbamoyl)-5-(trifluoromethyl)-1H-
pyrazol-1-yl]benzoic Acid (25). A solution of sodium hydroxide
(1.056 mL, 2.11 mmol) was added in one portion to a stirred solution of
N-adamantan-2-yl-1-(4-cyanophenyl)-5-(trifluoromethyl)-1H-pyra-
zole-4-carboxamide 56 (250 mg, 0.60 mmol), in methanol (10 mL)
under air. The resulting solution was stirred at 65 °C for 45 h. The
resulting mixture was evaporated to dryness and the residue dissolved in
ice/water (25 mL) and the mixture was acidified with 2 M HCl. The
precipitate was collected by filtration, washed with water (25 mL) and
1
dried under vacuum to afford a white solid (243 mg, 93%). H NMR
(400 MHz, DMSO-d6) δ 1.46−1.56 (2H, m), 1.66−1.73 (2H, m), 1.75−
1.88 (5H, m), 1.89−1.97 (3H, m), 2.00−2.10 (2H, m), 3.98−4.05 (1H,
m), 7.63 (2H, d, J = 8.5 Hz), 8.11−8.14 (3H, m), 8.34 (1H, d, J = 7.1
Hz), 13.30 (1H, s); HRMS (EI) for C22H23O3N3F3 (MH+); calcd,
434.1686; found, 434.1684.
4-[4-(2-Adamantylcarbamoyl)-5-(1-methylcyclopropyl)-
pyrazol-1-yl]benzoic Acid (26). Prepared according to the procedure
of
8 from methyl 4-[4-(2-adamantylcarbamoyl)-5-(1-
1
methylcyclopropyl)pyrazol-1-yl]benzoate 51h in 89% yield; H NMR
(400 MHz, DMSO-d6) δ 0.51−0.53 (2H, m), 0.68−0.69 (2H, m), 1.54−
1.58 (5H, m), 1.69−1.74 (2H, m), 1.84−1.87 (6H, m), 1.92−2.08 (4H,
m), 4.03−4.09 (1H, m), 7.44 (1H, d, J = 7.1 Hz), 7.62−7.68 (2H, m),
8.06 (1H, s), 8.07−8.12 (2H, m), 13.16 (1H, s); HRMS (EI) for
C25H30O3N3 (MH+); calcd, 420.2282; found, 420.2281.
4-[4-(2-Adamantylcarbamoyl)-5-tert-butyl-pyrazol-1-yl]-
benzoic Acid (27). 2 M aqueous sodium hydroxide solution (51.7 mL,
103.32 mmol) was added to methyl 4-[4-(2-adamantylcarbamoyl)-5-
tert-butyl-pyrazol-1-yl]benzoate 51a (4.5 g, 10.33 mmol) in methanol
(100 mL). The mixture was stirred at 70 °C for 1 h and then cooled to
ambient temperature, concentrated under reduced pressure and diluted
with water (100 mL). The reaction mixture was adjusted to pH 3 with 2
M HCl. The reaction mixture was extracted with EtOAc (500 mL) and
washed sequentially with water (2 × 100 mL), and saturated brine (50
mL). The organic layer was dried over MgSO4, filtered and evaporated
to give a pale yellow solid. The solid was washed with EtOAc (20 mL),
collected by filtration and dried under vacuum to give a cream crystalline
solid (3.89 g, 89%); mp 310−312 °C; 1H NMR (400 MHz, DMSO-d6) δ
1.18 (9H, s), 1.44−1.54 (2H, m), 1.65−1.87 (8H, m), 1.90−1.92 (2H,
m), 2.03−2.12 (2H, m), 3.97−4.01 (1H, m), 7.48−7.51 (2H, m), 7.60
(1H, s), 8.05−8.08 (2H, m), 8.12 (1H, d, J = 7.2 Hz), 13.23 (1H, s);
HRMS (EI) for C25H32O3N3 (MH+); calcd, 422.2438; found, 422.2437.
N-(2-Adamantyl)-4,4-dimethyl-3-oxo-pentanamide (49a). A 1
M solution of solution of lithium bis(trimethylsilyl)amide in THF
(22.84 mL, 22.84 mmol) was added to THF (25 mL) and cooled under
nitrogen to −78 °C. A solution of 3,3-dimethyl-2-butanone (2.287 g,
22.84 mmol) in THF (25 mL) was added dropwise over a period of 5
min. The resulting solution was stirred at −78 °C under nitrogen for 15
min. A solution of 2-isocyanatoadamantane (prepared from 2-
adamantylamine hydrochloride by the method of R. Reck and C.
Jochims18) (3.68 g, 20.76 mmol) in THF (20 mL) was added over a
period of 5 min. The resulting solution was stirred at −78 °C for 1 h and
then allowed to warm to 20 °C over 1 h. The reaction mixture was
poured into saturated NH4Cl (150 mL) and extracted with EtOAc (2 ×
100 mL), the organic layer was washed with water (50 mL) and brine
(50 mL) dried over MgSO4, filtered and evaporated to afford a yellow
oil.The crude product was purified by flash silica chromatography,
elution gradient 0 to 50% EtOAc in isohexane to afford a white solid
(4.64 g, 81%); 1H NMR (400 MHz, DMSO-d6) δ 1.08−1.09 (9H, m),
1.50 (2H, d), 1.66−1.89 (10H, m), 1.95−2.00 (2H, m), 3.53 (1.4H, s),
4-[4-(2-Adamantylcarbamoyl)-5-ethylpyrazol-1-yl]benzoic
Acid (20). Prepared according to the procedure of 8 from methyl 4-[4-
(2-adamantylcarbamoyl)-5-ethylpyrazol-1-yl]benzoate 51c in 59%
1
yield; H NMR (400 MHz, DMSO-d6) δ 1.03 (3H, t, J = 7.4 Hz),
1.46−1.57 (2H, m), 1.66−1.87 (8H, m), 1.90−1.98 (2H, s), 2.04−2.14
(2H, m), 2.97 (2H, q, J = 7.4 Hz), 3.99−4.06 (1H, m), 7.58−7.63 (3H,
m), 8.09−8.13 (2H, m), 8.28 (1H, s); HRMS (EI) for C23H28O3N3
(MH+); calcd, 394.2125; found, 394.2124.
4-[4-(2-Adamantylcarbamoyl)-5-propan-2-ylpyrazol-1-yl]-
benzoic Acid (21). Prepared according to the procedure of 8 from
methyl 4-[4-(2-adamantylcarbamoyl)-5-propan-2-ylpyrazol-1-yl]-
1
benzoate 51d in 100% yield; H NMR (400 MHz, DMSO-d6) δ 1.29
(6H, d, J = 7.0 Hz), 1.46−1.56 (2H, m), 1.66−1.88 (8H, m), 1.92−1.99
(2H, m), 2.04−2.14 (2H, m), 3.12 (1H, hept, J = 7.0 Hz), 4.02−4.05
(1H, m), 7.50−7.55 (2H, m), 7.67 (1H, d, J = 6.8 Hz), 8.05 (1H, s),
8.07−8.12 (2H, m), 13.20 (1H, s); HRMS (EI) for C24H30O3N3
(MH+); calcd, 408.2281; found, 408.2281.
4-[4-(2-Adamantylcarbamoyl)-5-cyclopropyl-pyrazol-1-yl]-
benzoic Acid (22). Prepared according to the procedure of 8 from
methyl 4-[4-(2-adamantylcarbamoyl)-5-cyclopropylpyrazol-1-yl]-
1
benzoate 51e in 95% yield; H NMR (300 MHz, DMSO-d6) δ 0.41−
0.46 (2H, m), 0.85−0.91 (2H, m), 1.48−1.60 (2H, m), 1.67−1.89 (8H,
m), 1.90−1.99 (2H, m), 2.00−2.11 (2H, m), 2.21−2.30 (1H, m), 4.00−
4.09 (1H, m), 7.58 (1H, d, J = 7.3 Hz), 7.77 (2H, d, J = 8.5 Hz), 7.94
(1H, s), 8.09 (2H, d, J = 8.5 Hz), 13.11 (1H, s); HRMS (EI) for
C24H28O3N3 (MH+); calcd, 406.2125; found, 406.2123.
4-[4-(2-Adamantylcarbamoyl)-5-cyclobutylpyrazol-1-yl]-
benzoic Acid (23). Prepared according to the procedure of 8 from
methyl 4-[4-(2-adamantylcarbamoyl)-5-cyclobutylpyrazol-1-yl]-
1
benzoate 51f in 99% yield; H NMR (400 MHz, DMSO-d6) δ 1.47−
1.58 (2H, m), 1.60−1.89 (10H, m), 1.92−2.00 (2H, m), 2.03−2.13 (4H,
m), 2.20−2.28 (2H, m), 3.76−3.85 (1H, m), 4.01−4.06 (1H, m), 7.48−
J
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Journal of Medicinal Chemistry
Article
3.80−3.94 (1H, m), 5.30 (0.3H, s), 7.77−7.87 (1H, m), 14.43 (0.3H, s)
(2:1 mixture of keto and enol forms); LRMS m/z (M+ + H) = 278.
(2)-N-(2-Adamantyl)-2-(dimethylaminomethylidene)-4,4-di-
methyl-3-oxo-pentanamide (50a). N,N-Dimethylformamide di-
methyl acetal (3.02 mL, 22.71 mmol) was added to a stirred suspension
of N-(2-adamantyl)-4,4-dimethyl-3-oxo-pentanamide 49a (5.25 g,
18.93 mmol) in 1,4-dioxane (50 mL) under nitrogen. The resulting
mixture was stirred at 100 °C for 2 h. The reaction mixture was
evaporated to dryness and the resulting pale cream solid was dried under
ASSOCIATED CONTENT
* Supporting Information
Experimental details for the syntheses of compounds 5−7, 10,
12−17 and intermediates are described together with biological,
analytical and crystallographic information. This material is
available free of charge via the Internet at http://pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Author
*Tel: +44 (0)1625 232567. Fax: +44 (0)1625 516667. E-mail:
jamie.scott@astrazeneca.com.
■
1
vacuum to afford the title compound (5.83 g, 93%); H NMR (400
MHz, DMSO-d6) δ 1.13 (9H, s), 1.47 (2H, d), 1.69−1.83 (10H, m),
2.03 (2H, d), 2.92 (6H, s), 3.90 (1H, d), 7.24 (1H, s), 7.94 (1H, d);
LRMS m/z (M+ + H) = 333.
Notes
Methyl 4-[4-(2-Adamantylcarbamoyl)-5-tert-butyl-pyrazol-
1-yl]benzoate (51a). 2-(4-(Methoxycarbonyl)phenyl)hydrazinium
chloride (3.04 g, 15.00 mmol) was added in one portion to (2)-N-(2-
adamantyl)-2-(dimethylaminomethylidene)-4,4-dimethyl-3-oxo-penta-
namide 50a (4.99 g, 15 mmol) in ethanol (100 mL). Five drops of acetic
acid were added and the resulting solution was stirred at 80 °C for 2 h.
The reaction mixture was concentrated and diluted with EtOAc (500
mL), and washed sequentially with water (200 mL), and saturated brine
(200 mL). The organic layer was dried over MgSO4, filtered and
evaporated to afford crude product that was purified by flash silica
chromatography, elution gradient 0 to 50% EtOAc in isohexane. Pure
fractions were evaporated to dryness to afford title compound (4.66 g,
71%) as a yellow solid; 1H NMR (400 MHz, DMSO-d6) δ 1.19 (9H, s),
1.50 (2H, d), 1.69−1.95 (10H, m), 2.09 (2H, d), 3.91 (3H, s), 3.99 (1H,
d), 7.53−7.56 (2H, m), 7.62 (1H, s), 8.09−8.12 (2H, m), 8.20 (1H, d);
LRMS m/z (M+ + H) = 436.
LogD7.4. Measurements were made using a shake-flask method
where the extent of partitioning between pH 7.4 buffer and octanol was
measured.19 Compounds were dissolved in a known volume buffer, and
following the addition of a known amount of octanol, the solutions were
shaken for 30 min. Following centrifugation, analysis of the aqueous
layer was performed by LC−UV to quantify the amount of compound in
solution and then compared to analysis of the compound in solution
before the addition of octanol to calculate the partitioning coefficient,
D7.4.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
William McCoull is thanked for initial work leading to the
identification of the pyrazole scaffold. Gemma Convey, Clare
Stacey, Ruth Brownlie and Julie Cook are thanked for the
generation of biological data. Peter Barton, Stuart Bennett, Linda
Godfrey and John Revill are thanked for synthetic chemistry
contributions. Paul Davey is thanked for analytical support.
ABBREVIATIONS USED
■
11β-HSD1, 11β-hydroxysteroid dehydrogenase type 1; 11β-
HSD2, 11β-hydroxysteroid dehydrogenase type 2; Clp, plasma
clearance; CYP, cytochrome P450; DMPK, drug metabolism and
pharmacokinetics; EDCI, 1-(3-dimethylaminopropyl)-3-ethyl-
carbodiimide; HMDS, hexamethyldisilazide; HOBT, N-hydrox-
ybenzotriazole; LLE, ligand-lipophilicity efficiency; MAP, mono-
phasic action potential; MDCK, Madin-Darby canine kidney cell
line; NAD, nicotinamide adenine dinucleotide; NADPH,
nicotinamide adenine dinucleotide phosphate; PPB, plasma
protein binding; SAR, structure activity relationship; TFA,
trifluoroacetic acid
11β-HSD1 Activity in Liver and Adipose Tissue. Han Wistar rats
were maintained on a chow diet were dosed by oral gavage with
compound suspended in 0.5% w/v hydroxypropropyl methylcellulose,
HPMC (4000−15 000 cPs) and 0.1% w/v Tween 80. Animals were
euthanased 3 h post dose using a rising concentration of CO2 at a time
postdose as determined by PK. Blood samples were taken via cardiac
puncture for compound determination. Activity of the 11β-HSD1
enzyme in liver and epididymal adipose tissue was measured as the
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3
or 90 min (rat adipose tissue) in the presence of H-Cortisone (20
nmol/L, 1 μCi/mL, specific activity 1.97GBq/mmol, Perkin-Elmer)
containing DMEM Ham F12 media (Sigma Aldrich, Poole, UK)
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3
medium samples were collected for analysis of H-cortisone to H-
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resuspended in mobile phase for HPLC analysis (methanol/H2O,
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Kromasil C18 5 μm column, 4.6 mm × 250 mm (Crawford Scientific,
Lanarkshire, UK) with methanol/H2O (50:50) at flow rate of 1.5 mL/
min. Radioactivity measured using a flow scintillation analyzer
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FLO-ONE software.
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