Discovery and optimization of piperidyl benzamide derivatives as a novel class of 11β-HSD1 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2009.01.058

Discovery and optimization of a piperidyl benzamide series of 11β-HSD1 inhibitors is described. This series was derived from a cyclohexyl benzamide lead structures to address PXR selectivity, high non-specific protein binding, poor solubility, limited in

Full text of DOI:10.1016/j.bmcl.2009.01.058

Bioorganic & Medicinal Chemistry Letters 19 (2009) 1797–1801
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery and optimization of piperidyl benzamide derivatives
as a novel class of 11b-HSD1 inhibitors
*
Yosup Rew , Dustin L. McMinn, Zhulun Wang, Xiao He, Randall W. Hungate, Juan C. Jaen ,
Athena Sudom, Daqing Sun, Hua Tu, Stefania Ursu, Elisia Villemure, Nigel P. C. Walker,
Xuelei Yan, Qiuping Ye, Jay P. Powers
Amgen Inc., 1120 Veterans Boulevard, South San Francisco, CA 94080, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Discovery and optimization of a piperidyl benzamide series of 11b-HSD1 inhibitors is described. This ser-
ies was derived from a cyclohexyl benzamide lead structures to address PXR selectivity, high non-specific
protein binding, poor solubility, limited in vivo exposure, and in vitro cytotoxicity issues observed with
the cyclohexyl benzamide structures. These efforts led to the discovery of piperidyl benzamide 15 which
features improved properties over the cyclohexyl benzamide derivatives.
Received 16 December 2008
Revised 15 January 2009
Accepted 16 January 2009
Available online 23 January 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
11b-HSD1
Type II diabetes
Insulin sensitivity
Cyclohexyl benzamide
Piperidyl benzamide
PXR
Microsomal stability
Hepatocyte stability
One of the key regulatory roles of glucocorticoids is glucose
homeostasis. In this regard, glucocorticoids regulate gluconeogen-
esis in the liver and inhibit peripheral glucose uptake in muscle
and adipose tissue. 11b-Hydroxysteroid dehydrogenase type 1
(11b-HSD1), an NADPH-dependent reductase, is a key enzyme that
converts the inactive glucocorticoid cortisone to the active gluco-
corticoid hormone cortisol in liver, adipose, and brain tissues.
Excessive concentrations of glucocorticoids in the liver and adipose
tissue can lead to glucose intolerance and insulin resistance. There-
fore, selective inhibition of the 11b-HSD1 enzyme has been consid-
ered as a promising strategy to improve insulin sensitivity and
treat type II diabetes.1,2
Previously we reported on a series of cyclohexyl benzamide
derivatives, exemplified by compound 1, as potent 11b-HSD1
inhibitors (Fig. 1).3 Compound 1 exhibited excellent in vitro inhibi-
tion of human 11b-HSD1 enzyme and favorable pharmacokinetic
profiles in the rat, cynomolgus monkey, and dog. Furthermore,
compound 1 showed a dose-dependent decrease in adipose 11b-
HSD1 activity in a monkey ex vivo pharmacodynamic model.3
CF3
HO
hHSD1 SPA IC50 = 1.4 nM
h293 IC50 = 5.8 nM
h293 ( 3% HSA) IC50 = 124 nM
N
O
N
1
Figure 1. Representative cyclohexyl benzamide 11b-HSD1 inhibitor.3,8
However, 1 exhibited low micromolar range in vitro cytotoxicity
in HeLa cells (IC50 = 2.5 l
M).4
The X-ray co-crystal structure of 1 with the 11b-HSD1 enzyme
showed that the pyridyl ring points toward the solvent exposed re-
gion of the protein dimer interface and does not make any discern-
able specific interaction with the active site of the protein.3
Combined with our initial SAR,3 this gave some indication that
the core cyclohexyl benzamide motif was the essential binding
component of these molecules. We hypothesized that, by replacing
the pyridyl group with other functional groups, we could modulate
physicochemical properties without disrupting critical interactions
with the enzyme. Moreover, in addition to enhancing the structural
diversity of compounds at the 4-position of the cyclohexane ring,
* Corresponding author. Tel.: +1 650 244 2682.
E-mail address: yrew@amgen.com (Y. Rew).
Currently at ChemoCentryx, Inc., Mountain View, CA 94043, USA.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.01.058

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Y. Rew et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1797–1801
Table 1
SAR of cyclohexyl and piperidyl benzamides with polar alkyl susbstituents
CF3
HO
N
R
O
Compounda
R
hHSD1 SPAb
IC50 (nM)
h293c (cell)
IC50 (nM)
h293 (cell, 3%
% Remaining z@ 30 min
PXRg activation %
HeLa cytotoxicity
M)
HSAd) IC50 (nM)
of controlh @ 2
lM
IC50
N/Di
N/D
>10
>10
N/D
N/D
4
(l
hLMe
rLMf
2
3
4
5
6
7
8
9
5.5
7.6
0.7
2.8
0.88
2.0
0.6
36
16
60
202
11
>90
70
73
75
74
30
56
60
N/D
60
1
CN
52
5
CN
CN
1.4
>90
64
42
2
10
130
30
<5
CN
2
1.4
>90
N/D
>90
N/D
77
CN
CN
8
129
28
N/D
>90
N/D
1.2
CN
315
1190
N/D
CONH2
(trans/cis =1/1)
10
11
12
13
14
15
2.4
11
17
109
6.1
5.0
56
77
417
97
>90
>90
>90
>90
86
>90
72
5
>10
>10
>10
8
CONH2
2
100
4
CONH2
2
3.8
1.7
11
>90
>90
28
CONH2
90
6.5
3
CONH2
CN
108
148
>10
>10
N
N
14
167
>90
86
4
CONH2
b,c,dAll potency data are reported as the average of at least two determinations.
a
All compounds gave satisfactory 1H NMR, HPLC, and MS data in full agreement with their proposed structures, and purity (>95%) was determined by HPLC analysis.
b
c
d
e
f
IC50 values determined by scintillation proximity assay.
h293 = HEK 293 cells stably transfected with full-length human 11b-HSD1.
HSA = human serum albumin.
hLM = human liver microsomes.
rLM = rat liver microsomes.
g
h
i
PXR = pregnane X receptor.
Rifampin (@ 10
lM) used as a positive control.
N/D = not determined.

Y. Rew et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1797–1801
1799
such modifications would potentially offer an opportunity to ad-
dress the off-target cytotoxicity observed in HeLa cells without
affecting the 11b-HSD1 activity.
We felt that extending polar groups from the 4-position of the
cyclohexyl ring would enhance the solubility of the compounds.
We therefore initiated our efforts by the systematic introduction
of polarity via carbon tethered nitrile and primary amide groups
(compounds 2–13 in Table 1).
of ester 22 into bromide 23 followed by alkylation of 23 with the
cyclopropanecarbonitrile anion.7 All these protected ketone inter-
mediates (17, 20, 21, and 24) were converted into amines 18a–
18d by removal of the ketal protecting group, followed by reduc-
tive amination with cyclopropylamine. Finally, the target 4-cya-
noalkyl-cyclohexyl benzamides, 2–8 were synthesized via amide
coupling reaction of these amines with chiral acid 253 followed
by separation of each geometric isomer (cis and trans) using sil-
ica-gel column chromatography. Further hydrolysis of the cyano
group to the primary amide provided carboxamide derivatives,
9–13.
All synthesized compounds were evaluated for the inhibition of
human and mouse 11b-HSD1 enzymes, as well as in cell-based as-
says.8 Consistent with our previous publication,3 trans isomers (2,
4, 6, 10) were typically more potent than cis isomers (3, 5, 7, 11) in
both biochemical and cellular assays. A 2-carbon spacer length be-
tween the polar functional group (-CN or -CONH2) and the cyclo-
hexane ring resulted in a substantial increase in potency relative
to the 1-carbon spacer (2, 3 vs 4, 5). The improvement in biochem-
ical potency for 4 and 5 may reflect a greater tolerance of polar fea-
tures distanced further towards solvent exposed regions of the
protein. Within the 2-carbon linked nitrile and primary amide mol-
ecules, both biochemical potency and cellular potency were
slightly decreased in primary amides (10, 11, 12, 13) relative to
the corresponding nitriles (4, 5, 6, 8). Also, compounds in this ser-
ies showed 5- to 20-fold shift in their IC50 values in the h293 cell-
based assays upon addition of HSA.
Compounds 2–13 were synthesized via the routes outlined in
Scheme 1. Three cyclohexylamine intermediates (18a–18c) were
prepared from the monoacetal (16). Reaction of diethyl cya-
nomethylphosphonate sodium salt with 16, followed by hydroge-
nation afforded nitrile 17.5 In
a
similar manner, 16 was
transformed into ester 19, which was followed by carbon-chain
elongation of 19 to provide homologated nitrile 20.6
-Dimethyl-
a,a
nitrile 21 was prepared via sequential methylation of 20. The cor-
responding cyclopropyl analog 24 was synthesized by conversion
H
N
O
O
b
a
O
d
O
CN
CN
O
16
18a
17
c
O
O
O
O
As observed previously in mono-substituted cyclohexyl benz-
amides,3 trans isomers were found to be more metabolically stable
than cis isomers in both human liver microsomes and rat liver
microsomes (2, 4, 10 vs 3, 5, 11). Replacement of the nitrile group
with a primary amide improved the rat in vitro metabolic stability
significantly (4, 6 vs 10, 12), while installation of a,a-disubsituents
such as gem-dimethyl or cyclopropyl group further enhanced
in vitro metabolic stability in rat (4 vs 6, 8).
CO2Et
CN
20
19
e
b
H
N
O
O
CN
CN
18b
21
b
Compounds were also evaluated for their potential to activate
PXR, a liability that can potentially lead to upregulation of
CYP3A4.9 In most cases, trans isomers exhibited reduced PXR activ-
ity in vitro (4, 10 vs 5, 11), while conversion of the nitrile group to
the more polar primary amide group in trans isomers significantly
reduced PXR activation potential (2, 4, 6, 8 vs 9, 10, 12, 13).
Of significance was the observation that most compounds eval-
H
N
CN
18c
O
O
O
g
f
O
O
O
uated had marked reduction in cytotoxicity (IC50 P 10 lM) rela-
Br
CN
CO2Et
24
tive to 1. Moreover, compounds 10 and 12 showed optimal
combination of limited cytotoxicity and PXR potential, good po-
tency and microsomal stability and were therefore selected for
rat pharmacokinetic (PK) studies.10a,10b However, it was found that
the in vitro rat liver microsomal stability of 10 and 12 (>90%
remaining after 30 min for both 10 and 12) was not predictive of
the in vivo rat clearance. Compounds 10 and 12 were found to have
high and moderate clearance in rat [CL (iv) = 3.0 and 1.3 L/h/kg,
respectively] in addition to moderate oral bioavailability (%F = 22
and 32, respectively).
Metabolic studies utilizing compound 10 with human and rat
hepatocytes revealed that hydrolysis of the primary amide to the
corresponding carboxylic acid occurred, followed by glucuronida-
tion to 27 (Fig. 2). This observation provided an explanation for
the disconnect between the observed in vitro microsomal stability
22
23
b
H
N
CN
CF3
18d
HO
CF3
HO
CO2H
25
+
h
N
R
R
H
N
O
R
R
X
n
CN
2-8, X = CN
9-13, X = CONH2
n
i
18a-18d
and the rat in vivo clearance. Introduction of a,a-dialkyl substitu-
Scheme 1. Reagents and conditions: (a) i—(EtO)2P(O)CH2CN, NaH, DMPU, THF, 95%;
ii—10% Pd/C, H2, AcOH/H2O, 95%; (b) i—a 1:2 (v/v) mixture of 3 N aqueous HCl and
THF, quantitatively; (ii) cyclopropylamine, NaBH(OAc)3, AcOH, 1,2-DCE, 80À95%;
(c) i—(EtO)2P(O)CH2CO2Me, NaH, DMPU, THF, 85%; ii—10% Pd/C, H2, EtOAc, 98%; (d)
i—LiAlH4, ether, 91%; ii—MsCl, TEA, DCM, quantitatively; iii—NaCN, DMSO, 71%; (e)
i—LDA, MeI, THF, 76%; ii—LDA, MeI, THF, 70%; (f) i—NaBH4, 10% MeOH-DME, 77%;
ii—CBr4, Ph3P, benzene, 81%; (g) LDA, cyclopropanecarbonitrile, THF, À78 °C, 63%;
(h) HBTU, HOBt, DIEA, DMF, 30À60% (cis/trans = 1/1À7.8/1); i—KOH, t-BuOH, 85 °C,
48À81%.
ents improved in vitro stability in both human and rat hepatocytes
(10 vs 12). Moreover, replacement of the gem-dimethyl group with
a cyclopropyl group significantly enhanced the in vitro hepatocyte
stability in human and rat (Table 2). This superior stability of the
cyclopropyl compound 13 relative to the gem-dimethyl compound
12 can potentially be explained by a combination of steric and
electronic effects.11

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Y. Rew et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1797–1801
Scheme 2. Ethyl 1-cyanocyclopropanecarboxylate 28 was prepared
CF3
HO
according to the literature procedures12 and was converted into
tosylate 29 following ester reduction. N-Alkylation of piperidine
30 with 29 provided 31. Ketal hydrolysis followed by reductive
amination afforded amine intermediate 32. Amide coupling of 32
with chiral acid 25 yielded N-cyanoalkylpiperidyl benzamide 14,
which was transformed into the primary amide derivative 15.
The potency and biological data of compound 14 and 15 are
summarized in Table 1. Both 14 and 15 were less potent than the
corresponding cyclohexyl benzamides in both biochemical and cel-
lular assays (14, 15 vs 8, 13). However, piperidyl benzamides
showed a much lower cellular potency shift upon addition of
HSA (1- to 2-fold shift) compared to cyclohexyl benzamides and
their cellular potency in the presence of 3% HSA was comparable
to that of 13. Plasma protein binding data for compound 13 and
15 are also consistent with the cellular potency shift upon addition
of HSA (Table 3).
N
O
O
NH2
10
O
O
CF3
HO
N
OH
OH
26
CF3
HO
N
OH
O
O
HO
O
Whereas 14 showed a compromised in vitro metabolic stability
in rats, the cyclopropyl amide 15 had improved metabolic stability.
Compound 15 was evaluated in rat pharmacokinetic studies. Com-
pound 15 exhibited low plasma clearance [CL (iv) = 0.89 L/h/kg]
and excellent oral bioavailability (%F = 69).10c As with the cyclo-
hexyl benzamide derivative, the in vitro human and rat hepatocyte
stability assay results were in agreement with the in vivo plasma
clearance of compound 15 (>90% remaining after 90 min in hHep,
69% remaining after 90 min in rHep).
The structure of compound 15 bound to human 11b-HSD1 was
determined by X-ray crystallography (Fig. 3) to a resolution of
2.3 Å.13 The co-crystal structure showed that the inhibitor binds
to the substrate site in a V-shape with its trifluoromehtylcarbinol
O
27
C
O
OH
Figure 2. Metabolism of compound 10 in human and rat hepatocytes.
Table 2
In vitro hepatocyte stability of compounds 10, 12, and 13
Compound
Hepatocyte stability % remaining @ 90 min
hHepa
rHepb
10
12
13
32
84
>90
<5
10
73
a
hHep = human hepatocytes.
rHep = rat hepatocytes.
b
Table 3
Plasma protein binding for compounds 13 and 15
Compound
fu in human plasmaa
fu in rat plasmaa
O
13
15
0.12
0.48
0.048
0.20
a
CN
CN
TsO
EtO
a
Samples analyzed on API-4000 and ultracentrifugation protocol-4 h. fu = frac-
tion unbound.
28
29
O
O
O
b
c
O
NH
N
CN
30
31
H
N
CF3
HO
N
d
CN
32
N
O
N
X
14, X = CN
15, X = CONH2
e
Scheme 2. Reagents and conditions: (a) i—NaBH4, 10% MeOH–DME, 90%; ii—TsCl,
pyridine, 0 °C, 75%; (b) 29, DIEA, AcCN, 85 °C, 80%; (c) (i) 5% H2O–TFA, 88%; ii—
cyclopropylamine, NaBH(OAc)3, AcOH, 1,2-DCE, 100%; (d) 25, EDCI, HOAt, NaHCO3,
DMF, 74%; (e) KOH, t-BuOH, 85 °C, 45%.
Having identified a pendant feature that appeared well toler-
ated and stable in hepatocytes, an effort was made to further en-
hance solubility and stereosimplicity by optimization of the
cyclopropylamide in the context of a more polar piperidine ring
architecture.
Figure 3. X-ray co-crystal structure of compound 15 in human 11b-HSD1. The key
hydrogen bonding interaction of 15 to the enzyme is shown with magenta dashed
line. The color scheme is red for oxygen atoms, blue for nitrogen atoms, orange for
sulfur atoms, magenta for fluorine atoms. Carbon atoms are in green for protein 1,
yellow for protein 2, gray for NADP+, and beige for inhibitor. The water molecules
are shown in red spheres.
Piperidyl benzamide derivatives, 14 and 15 were synthesized in
a straightforward manner, through a sequence illustrated in

Y. Rew et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1797–1801
1801
D.; Shuai, Q.; Wang, J.; Fung, S.; Monzon, K. M.; Chiou, W. J.; Pan, L.; Deng, X.;
Chovan, L. E.; Ramaiya, A.; Mullally, M.; Henry, R. F.; Stolarik, D. F.; Imade, H.
M.; Marsh, K. C.; Beno, D. W. A.; Fey, T. A.; Droz, B. A.; Brune, M. E.; Camp, H. S.;
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(g) Yuan, C.; St. Jean, D. J., Jr.; Liu, Q.; Cai, L.; Li, A.; Han, N.; Moniz, G.; Askew, B.;
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group pointing towards the cofactor NADP + side. The central
amide carbonyl group makes a hydrogen bond interaction with
the hydroxyl group of S170, one of the three residues defined as
the catalytic triad for 11b-HSD1 activity.14 The cyclopropanecarb-
oxamide group locates in the dimer interface and forms a weak
water-mediated hydrogen bond with C-terminal region of adjacent
protein.
In summary, we have described our efforts towards the expan-
sion of structural diversity and optimization of a structurally novel
cyclohexyl benzamide series of 11b-HSD1 inhibitors. Improve-
ments in metabolic stability and reduction in cytotoxicity within
the cyclohexyl benzamide series transferred well to the piperidyl
subclass culminating in compound 15 which represents a potent
and promising benzamide subclass. Compound 15 effectively ad-
dresses PXR activation, high non-specific protein binding, poor
pharmacokinetic properties, and in vitro cytotoxicity issues previ-
ously observed with the cyclohexyl benzamide structures. This
represents a promising step forward in our continued development
of our benzamide class of 11b-HSD1 inhibitors.
3. Julian, L. D.; Wang, Z.; Bostick, T.; Caille, S.; Choi, R.; DeGraffenreid, M.; Di, Y.;
He, X.; Hungate, R. W.; Jaen, J. C.; Liu, J.; Monshouwer, M.; McMinn, D.; Rew, Y.;
Sudom, A.; Sun, D.; Tu, H.; Ursu, S.; Walker, N.; Yan, X.; Ye, Q.; Powers, J. P. J.
Med. Chem. 2008, 51, 3953.
4. Hela cytotoxicity assay: IC50 for inhibition of cell growth of Hela cells at 72
hours was measured using Alamar blue assay.
5. Yamamoto, T.; Eki, T.; Nagumo, S.; Suemune, H.; Saikai, K. Tetrahedron 1992, 48,
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[3H]-cortisone to [3H]-cortisol. Product [3H]-cortisol, captured by an anti-
cortisol monoclonal antibody conjugated to scintillation proximity assay (SPA)
beads, was quantified with
a microscintillation plate reader. Biochemical
References and notes
enzyme assays were performed with Baculovirus-produced recombinant full-
length human or mouse 11b-HSD1 as the enzyme source and NADPH as
cofactor. Cell-based enzyme assays (h293) utilized HEK293 cells stably
expressing recombinant human full-length 11b-HSD1 as the enzyme source
without supplementation of NADPH. IC50 values for enzyme inhibition were
calculated with a dose response curve fitting algorithm with at least duplicate
sets of samples. In the cellular assay, selected compounds were tested in the
presence of human serum albumin (HSA) to measure the impact of protein
binding.
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9. Pregnane X Receptor (PXR) assay: HepG2 cells transfected with a luciferase
reporter construct driven by CYP3A4 gene and human PXR cDNA. Cells were
exposed to test article and the luciferase activity determined by chemilum.
Rifampin is used as control.
10. a Compound 10: dosed iv 0.51 mg/kg, po 2.0 mg/kg.; b Compound 12: dosed iv
0.39 mg/kg, po 2.0 mg/kg.; c Compound 15: dosed iv 0.50 mg/kg, po 2.0 mg/kg.
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Accession Code 3FRJ.
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