Synthesis and optimization of novel 4,4-disubstituted cyclohexylbenzamide derivatives as potent 11β-HSD1 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2010.10.129

The synthesis and SAR of a series of 4,4-disubstituted cyclohexylbenzamide inhibitors of 11β-HSD1 are described. Optimization rapidly led to potent, highly selective, and orally bioavailable inhibitors demonstrating efficacy in both rat and non-human primate ex vivo pharmacodynamic models.

Full text of DOI:10.1016/j.bmcl.2010.10.129

Bioorganic & Medicinal Chemistry Letters 21 (2011) 405–410
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and optimization of novel 4,4-disubstituted cyclohexylbenzamide
derivatives as potent 11b-HSD1 inhibitors
Daqing Sun , Zhulun Wang , Seb Caille à, Michael DeGraffenreid , Felix Gonzalez-Lopez de Turiso ,
Randall Hungate à, Juan C. Jaen §, Ben Jiang , Lisa D. Julian , Ron Kelly , Dustin L. McMinn ,
Jacob Kaizerman , Yosup Rew , Athena Sudom , Hua Tu –, Stefania Ursu , Nigel Walker ,
Maren Willcockson , 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:
The synthesis and SAR of a series of 4,4-disubstituted cyclohexylbenzamide inhibitors of 11b-HSD1 are
described. Optimization rapidly led to potent, highly selective, and orally bioavailable inhibitors demon-
strating efficacy in both rat and non-human primate ex vivo pharmacodynamic models.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 19 September 2010
Revised 23 October 2010
Accepted 26 October 2010
Available online 31 October 2010
Keywords:
11b-HSD1
11b-HSD2
Diabetes
Metabolic syndrome
Hydroxysteroid dehydrogenase
4,4-Disubstituted cyclohexylbenzamides
11b-Hydroxysteroid dehydrogenase type 1 (11b-HSD1) is a key
enzyme that acts as an NADPH-dependent reductase capable of
converting inactive glucocorticoids such as cortisone into their
active form (e.g., cortisol) in specific tissues, such as liver, adipose,
and brain.1–4 Conversely, 11b-hydroxysteroid dehydrogenase
type 2 (11b-HSD2), a structurally related isoenzyme of 11b-
HSD1, catalyzes the conversion of cortisol to cortisone using NADP+
as a cofactor. 11b-HSD2 is expressed in cells that contain the min-
eralocorticoid receptor (MR) and protects the MR by converting
cortisol to cortisone.5 Selective inhibition of 11b-HSD1 may be a
viable therapeutic strategy for the treatment of metabolic syn-
drome and has attracted significant attention from the pharmaceu-
tical research community.6–29
significantly less potential for PXR transactivation relative to the
corresponding 4-monosubstituted analogs. In addition, 1 showed
excellent cross-species pharmacokinetics and in vivo inhibition of
11b-HSD1 in a rat ex vivo pharmacodynamic model. However, the
potency of 1 made it unsuitable for further development in its
present form. We felt that additional modifications of 1 at the 4-
position of the cyclohexane ring would offer an opportunity to
increase its potency while maintaining other properties (such as
pharmacokinetics) which would provide
suitable for development.
a compound more
Compounds were synthesized via the routes outlined in
Schemes 1–5.33 4,4-Disubstituted cyclohexyl benzamides 2, 9, 14,
15, and 16, were prepared in a straightforward manner (Scheme 1).
4-Aryl/heteroaryl, 4-cyanocyclohexanones 19 were obtained by a
We previously described the design and optimization of sub-
stituted cyclohexylbenzamide 11b-HSD1 inhibitors.30–32 During
the course of that research, we noted that 4,4-disubstituted
cyclohexylbenzamides, such as compound 1 (Fig. 1), exhibited
CF3
HO
hHSD1 IC50 =14nM
N
⇑
Corresponding author. Tel.: +1 650 244 2195.
E-mail address: daqings@amgen.com (D. Sun).
Amgen, Inc., South San Francisco, CA, United States.
Amgen, Inc., 1 Amgen Center Drive, Thousand Oaks, CA 91320, United States.
Present address: ChemoCentryx, Inc., Mountain View, CA 94043, United States.
Present address: LakePharma, Inc., 530 Harbor Blvd., Belmont, CA 94002,
h293 IC50 =52nM
OH
h293 +3% HSA IC50 = 121 nM
O
à
§
1
–
United States.
Figure 1. An early 4,4-disubstituted cyclohexylbenzamide 11b-HSD1 inhibitor (1).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.10.129

406
D. Sun et al. / Bioorg. Med. Chem. Lett. 21 (2011) 405–410
O
c
O
O
O
O
a, b
d
a, b
c
CN
O
O
COOH
O
19
O
CN
OH
CHO
Ar
Ar
22 Ar
20
19
Ar
Ar
34
d, e
33
e
CF3
O
O
HO
d, e
O
OH
OH
f,g
O
COOMe
CN
Ar
N
23
f
Ar
Ar
35
X
O
21 (Ar = 2-Py)
CF3
Ar
HO
5, X = CN
7, 11, 12, 13, X = CONH2
HN
h
g
N
O
Ar
Scheme 3. Reagents and conditions: (a) ethylene glycol, p-toluenesulfonic acid,
benzene, reflux, 45–92%; (b) LiAlH4 (0.5 equiv), THF, 80–98%; (c) NaH, diethyl
(cyanomethyl) phosphonate, THF, 63–80%; (d) H2, 10% Pd/C, EtOAc, 89–99%; (e) 3 N
HCl, THF, 63–76%; (f) cyclopropylamine, NaBH(OAc)3, acetic acid, dichloroethane,
33%; (g) EDC, HOAt, (S)-4-(1,1,1-trifluoro-2-hydroxypropan-2-yl)benzoic acid, DMF,
52%; (h) KOH, t-BuOH, 90 °C, 77%.
X
24
Ar
2, Ar = Ph,X = OH
25, X = OH
h
9, 14, 15, 16, X = OCONH2
Scheme 1. Reagents and conditions: (a) ethylene glycol, p-toluenesulfonic acid,
benzene, reflux, 45–92%; (b) ethylene glycol, KOH, 170 °C, 54–88%; (c) K2CO3, MeI,
THF, 52–74%; (d) LiAlH4, THF, reflux, 54–92%; (e) 3 N HCl, THF, 60–95%; (f)
cyclopropylamine, NaBH(OAc)3, acetic acid, dichloroethane, 8–24%; (g) EDC, HOAt,
(S)-4-(1,1,1-trifluoro-2-hydroxypropan-2-yl)benzoic acid, DMF, 48–57%; (h) tri-
chloroacetyl isocyanate, neutral Al2O3, CHCl3, 84–95%.
CF3
HO
a, b
19
N
CN
Ph
O
36
O
O
O
CO2Me
CN
a
c
b
O
O
O
CO2Me
CN
CF3
HO
O
Ph
28
26
27
37, X =NH2
c
d
e
N
8, X = NHSO2Me
O
2
37, X =NH
O
O
O
X
10, X =NHCOMe
OH
d
e, f
O
O
37
Ph
O
CN
CO2Me
Ph
Ph
Scheme 4. Reagents and conditions: (a) cyclopropylamine, NaBH(OAc)3, acetic acid,
dichloroethane, 75–78%; (b) EDC, HOAt, (S)-4-(1,1,1-trifluoro-2-hydroxypropan-2-
yl)benzoic acid, DMF, 49–57%; (c) H2, Raney Ni, 28% NH4OH, MeOH, 98%; (d) MsCl,
Et3N, 75%; (e) Hunig’s base, acetyl chloride, DMF, 79%.
Ph
31
29
30
f
g, h
CF3
CN
HO
Ph
32
O
N
HN
a
g, h
CO2Me
OH
HO
O
CO2Me
CF3
Ar
N
Ph
Ar
38
3
39
N
b
O
CF3
X
CF3
HO
HO
Ph
c
4, X = CN
6, X = CONH2
i
N
CO2Me
O
40
O
X
Scheme 2. Reagents and conditions: (a) ethyl cyanoacetate, acetic acid, NH4OAc,
toluene, reflux, 70%; (b) 2-bromobenzene, n-BuLi, CuI, n-Bu2S, Et2O, 49%; (c) DMSO,
NaCl, H2O, 80%; (d) (i) ethylene glycol, KOH, 170 °C, 54–92%; (ii) K2CO3, MeI, THF, 52–
74%; (e) LiAlH4, THF, 65–84%; (f) 3 N HCl, THF, 60–75%; (g) cyclopropylamine,
NaBH(OAc)3, acetic acid, dichloroethane, 21–35%; (h) EDC, HOAt, (S)-4-(1,1,1-
trifluoro-2-hydroxypropan-2-yl)benzoic acid, DMF, 58–62%; (i) KOH, t-BuOH,
90 °C, 81%.
Ar
Ar
41, X = OH
d
17, 18, X = OCONH2
Scheme 5. Reagents and conditions: (a) cyclopropylamine, NaBH(OAc)3, acetic acid,
dichloroethane, 94%; (b) EDC, HOAt, (S)-4-(1,1,1-trifluoro-2-hydroxypropan-2-
yl)benzoic acid, DMF, 74%; (c) LiAlH4, THF, 21–31%; (d) trichloroacetyl isocyanate,
neutral Al2O3, CHCl3, 54%.
tandem one-pot double Michael addition–Dieckman condensation
between methyl acrylate and the appropriate benzylic nitrile,
followed by Krapcho decarboxylation.34 Protection of 19 as its
monoethylene ketal was followed by hydrolysis of the nitrile to pro-
duce acid 20. Reduction of 20 with LiAlH4 followed by removal of
the ketal protecting group gave disubstituted cyclohexanones 23.
In a similar fashion, 2-pyridyl cyclohexanone 23 was prepared by
conversion of 20 into ester 21 followed by reduction and deprotec-
tion. Reductive amination of 23 with cyclopropylamine resulted in a
mixture of cis/trans cyclopropylamine products, and the desired

D. Sun et al. / Bioorg. Med. Chem. Lett. 21 (2011) 405–410
407
trans isomer 24 was separated by silica gel chromatography and
coupled with (S)-4-(1,1,1-trifluoro-2-hydroxypropan-2-yl)benzoic
acid30 to afford primary alcohol 25 (2, Ar = Ph, X = OH), which was
transformed into the corresponding carbamates 9, 14, 15, and 16
using trichloroacetyl isocyanate.
Compounds 3 and 4 were synthesized via reductive amination
of cyclohexanones 31 and 32 with cyclopropylamine followed by
an amide coupling reaction (Scheme 2). Preparation of 31 and 32
proceeded through the monoethylene ketal of cyclohexadione 26.
Reaction of ketal 26 with ethyl cyanoacetate, followed by conju-
gate addition afforded intermediate 28. Krapcho decarboxylation
of 28 was followed by deprotection of the ketal gave cyclohexa-
none 32. Intermediate 31 was prepared from 29 using a series of
common transformations. Finally, hydrolysis of the cyano group
in 4 afforded to the primary amide 6.
The synthesis of compounds 5, 7, 11, and 13 was completed from
the corresponding disubstituted cyclohexanones 19 (Scheme 3).
Protection of 19 with ethylene glycol, followed by partial reduction
of the cyano group with LiAlH4 gave aldehyde 33. Reaction of 33 with
diethyl cyanomethylphosphonate and sodium hydride produced
unsaturated nitrile 34. Hydrogenation of 34, followed by removal
of the ketal protecting group afforded ketone 35. Reductive amina-
tion of 35 with cyclopropylamine followed by standard amide bond
formation produced the nitrile 5. Finally, hydrolysis of the cyano
group to the primary amide afforded carboxamide derivatives 5, 7,
11, and 13.
Syntheses of compounds 8 and 10 were accomplished in a
straightforward manner, starting from the appropriate 4,4-disub-
stituted cyclohexanone 19 (Scheme 4). Primary amine intermedi-
ate 37 was prepared from 19. Reductive amination of 19 with
cyclopropylamine, followed by amide bond formation produced
4-phenyl-4-cyano cyclohexylbenzamide 36, which was reduced
to amine 37 in the presence of Raney nickel. The targeted com-
pounds 8 and 10 were then synthesized via a coupling reaction
of 37 with either methylsulfonyl or acetyl chloride, respectively.
Heteroarylcarbamates 17 and 18 were prepared from cyclohexa-
nones 38 as shown in Scheme 5. Reductive amination followed by
standard amide bond formation gave intermediate 40, which was
reduced to primary alcohols. The desired trans alcohol 41 was sepa-
rated by silica gel chromatography and treated with trichloroacetyl
isocyanate as before to produce carbamates 17 and 18.
Compounds were evaluated for the in vitro inhibition of human
and mouse 11b-HSD1 enzymes, as well as in cell-based assays.
11b-HSD1 enzyme activity was determined by measuring the con-
version of [3H]-cortisone to [3H]-cortisol. The 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 enzyme assays were
performed with baculovirus-produced recombinant full-length
human 11b-HSD1 as the enzyme source and NADPH as cofactor
(h-HSD1 IC50). Cell-based enzyme assays (h-293 IC50) utilized
HEK293 cells stably expressing recombinant human full-length
11b-HSD1 as the enzyme source without supplementation of
NADPH. Inhibition of 11b-HSD1 in adipocytes was evaluated by a
cell-based [3H]-cortisone conversion assay with differentiated
human fat cells and media containing 3% of human serum albumin
(HSA). IC50 values for enzyme inhibition were calculated with a
dose response curve fitting algorithm with at least duplicate sets
of samples. These compounds were also tested against the 11b-
HSD2 enzyme in the SPA assay to determine 11b-HSD selectivity.
All analogs had IC50 >10 lM against 11b-HSD2, highlighting the
excellent inhibitor selectivity between the two 11b-HSD isoforms.
Our initial optimization efforts began with the goal of improv-
ing the potency of 1 by replacement of the cyclopropyl with a phe-
nyl group and the replacement of the hydroxyl moiety with carbon
tethered polar groups such as alcohols, nitriles, and primary
amides (Table 1). Compounds with a one-carbon spacer between
the polar group and the cyclohexane ring (2, 4, and 6) were more
potent than 1 in both biochemical and cellular assays. Increasing
the spacer length from one to two carbons resulted in a further
potency improvement (3, 5, and 7). These observations indicated
that spacer length and polarity in the features pendant from the
4-position on the cyclohexane ring were important for 11b-
HSD1 inhibitory activity. Within the two-atom length sub-series,
Table 1
4,4-Disubstituted cyclohexylbenzamide 11b-HSD1 inhibitors
CF3
HO
N
R
O
Ph
Compound
R
h-HSD1a SPA
IC50 (nM)
h-293a
IC50 (nM)
h-293a + 3% HSA
IC50 (nM)
Adipocytea + 3% HSA
IC50 (nM)
% TO at 30 min
HLM
% TO at
30 min RLM
PXR% of control
at 2
lM
1
2
14
52
41
121
—
50
—
<10
10
<10
32
<5
2.2
<5
OH
OH
3
4
5
6
7
1.9
1.4
3.9
1.4
0.8
12
35
—
15
—
<10
19
<10
17
6
9
10.4
—
CN
CN
—
—
10
51
—
CONH2
CONH2
16
92
37
—
<10
<10
<10
<10
<5
<5
3.9
17
SO2Me
N
H
8
9
1.7
1
86
259
20
20
8.8
17
<10
<10
<10
21
<10
<10
<5
<5
<5
CONH2
COMe
4.6
6.7
O
N
H
10
2.2
129
a
IC50 values determined by scintillation proximity assay (SPA), and all potency data are reported as the average of at least two determinations; h-293 = HEK 293 cells
stably transfected with full-length human 11b-HSD1; HSA = human serum albumin; adipocyte (3% HSA) IC50 values determined by cell-based [3H]-cortisone conversion assay
with differentiated human fat cells and media containing 3% human serum albumin; HLM and RLM = human and rat liver microsomes, respectively; %TO = % turnover.

408
D. Sun et al. / Bioorg. Med. Chem. Lett. 21 (2011) 405–410
Table 2
4,4-Disubstituted cyclohexylbenzamide 11b-HSD1 inhibitors
CF3
HO
N
R
O
Ar
Compound Ar
R
h-HSD1a SPA
IC50 (nM)
h-293a IC50
(nM)
h-293a + 3% HSA
IC50 (nM)
Adipocytea + 3% HSA
IC50 (nM)
% TO at 30 min % TO at 30 min
HLM
RLM
CONH2
CONH2
11
1.3
7
109
36
<10
<10
F
12
13
1.8
9.6
0.6
12
—
55
—
24
>100
14
<10
<10
<10
<10
<10
<10
F
CONH2
CONH2
N
O
O
14
5.7
135
F
CONH2
15
16
17
18
0.5
3.5
2.4
1.8
15
19
30
16
34
74
93
63
12
25
31
18
<10
<10
<10
<10
<10
<10
<10
<10
F
F
CONH2
CONH2
O
O
N
N
CONH2
O
S
N
a
IC50 values determined by scintillation proximity assay (SPA), and all potency data are reported as the average of at least two determinations; h-293 = HEK 293 cells
stably transfected with full-length human 11b-HSD1; HSA = human serum albumin; adipocyte (3% HSA). IC50 values determined by cell-based [3H]-cortisone conversion
assay with differentiated human fat cells and media containing 3% of human serum albumin; HLM and RLM = human and rat liver microsomes, respectively; %TO = %
turnover.
the methysulfonamide 8 exhibited the biggest potency shift in cell-
based assays and the acetyl amide 10 suffered a significant potency
decrease upon addition of human serum albumin (HSA). The carba-
mate 9 showed an optimal cellular potency within this set of com-
pounds. Overall, compounds 7 and 9 showed a significant increase
in potency compared with 1 (ꢀ17-fold in the biochemical assay;
ꢀ13-fold in the h-293 cell-based assay; and ꢀthreefold in adipo-
cyte cell-based assay, respectively). Moreover, the two compounds
exhibited an optimal combination of limited PXR risk and excellent
metabolic stability in human and rat liver microsomes (HLM and
RLM).
Having identified the two-atom tethered primary amide and
carbamate as the best functional groups with respect to activity,
we then turned our attention to the modification of the phenyl
group on the 4-position of the cyclohexane ring (Table 2). Within
the primary amide sub-series, the 4-(2-fluorophenyl) substituted
cyclohexylbenzamide 11 appeared to be potent in the biochemical
assay, but showed a considerable potency shift in the cellular assay
upon addition of HSA (ꢀ15-fold). On the other hand, pyridine ana-
logue 13 exhibited a significant potency decrease in the adipocyte
cell-based assay with 3% HSA. The 4-(4-fluorophenyl) 12 showed
minimal potency shifts in the cellular assays with and without
the addition of HSA. Compounds in the carbamate sub-series that
had a heteroaryl ring in the 4-position showed a greater potency
shift in the cellular assays (16, 17, and 18), and 15 constituted
the optimal carbamate containing compound. All of the amides
and carbamates proved to have excellent in vitro metabolic
stability in both human and rat liver microsomes (HLM and RLM
turnover <10%), as well as limited PXR activation.
Since compounds 9, 14, 16, and 18 exhibited satisfactory potency
in the adipocyte assay and in vitro metabolic stability, these
compounds were examined in rat pharmacokinetic experiments
(Table 3). It was found that 9, 14, and 16 had a low clearance
(CL = 0.15–0.21 L/h/kg) and oral bioavailability (%F) between =
20–64%. Compound 18 had moderate clearance (CL = 0.82) and oral
bioavailability (%F = 20). In addition, these compounds also inhib-
ited the rat 11b-HSD1 enzyme (see Table 4, IC50 = 30–220 nM). We
decided to test these molecules in a pharmacodynamic model in rats
and assess the effects of oral administration towards inhibition of
11b-HSD1. Compounds 9, 14, 16, and 18 were dosed orally in
Sprague–Dawley rats at 4, 20, and 100 mg/kg, respectively. Two
Table 3
Rat pharmacokinetic data for compounds 9, 14, 16, and 18
Compound
CL (iv,
L/kg/h)
Vdss
(L/kg)
T1/2
(iv, h)
F
AUC
(po,
(po, %)
lg h/L)
9
14
16
18
0.16
0.15
0.21
0.82
1.0
3.9
5.2
4.2
4.5
20
48
64
25
2770
6790
6180
618
1.28
1.24
3.7
Dosed iv 0.5 mg/kg, po 2.0 mg/kg.

D. Sun et al. / Bioorg. Med. Chem. Lett. 21 (2011) 405–410
409
Table 4
Table 5
Dose dependent inhibition of 11b-HSD1 by 9, 14, 16, and 18 in the rat ex vivo PD
Plasma exposure in cynomolgus monkeys after oral dosing with 7
model
PO dose
N
[7] (ng/mL) plasma
a
a
Compound h-HSD1
r-HSD1
Inhibition over vehicle (%)
Vehicle
6
5
6
5
BQL
IC50 (nM) IC50 (nM)
4 (mg/kg) 20 (mg/kg) 100 (mg/kg)
4 mg/kg
20 mg/kg
100 mg/kg
151 93
1999 2144
1709 779
9
14
16
18
1.0
86
144
220
30
23
27
14
6
43
43
44
38
71
70
72
75
0.63
3.5
1.8
a
Values are means of at least two determinations.
hours after receiving the dose, animals were sacrificed, epididymal
fat was isolated and incubated in media containing [3H]-cortisone.
Rat 11b-HSD1 activity was then measured by detection of [3H]-
cortisol. Although the inhibition of the rat enzyme was less potent
than the inhibition of the human enzyme, a dose-dependent reduc-
tion in cortisol production was observed for all of four compounds
(Table 4).
Furthermore, a non-human primate pharmacodynamic model
was used to evaluate the effects of the compound toward
inhibition of 11b-HSD1. Since compound 7 had suitable pharmaco-
kinetic profile in cynomologous monkey (CL = 0.06 L/h/kg,
%F = 81%) and activity against cynomologous monkey 11b-HSD1
enzyme (IC50 = 22 nM), this molecule was selected for evaluation
in our non-human primate ex vivo pharmacodynamic model. In
this study, non-human primates were dosed orally with 4, 20,
and 100 mg/kg of inhibitor 7. At 2 h post-dose, mesenteric fat tis-
sues were collected. Following 1 h incubation of these tissues in
media containing [3H]-cortisone, 11b-HSD1 activity was measured
through detection of tritiated cortisol levels using a scintillation
proximity assay. Relative to controls, all dose groups showed a de-
crease in [3H]-cortisol production in mesenteric fat (Fig. 2). These
results demonstrate that 7 is effective in lowering 11b-HSD1 adi-
pose activity in cynomolgus monkeys when administered orally
and has a reasonable potential to serve as an effective orally bio-
availability inhibitor of 11b-HSD1 in humans (Table 5).
Finally, the co-crystal structure of 7 with human 11b-HSD1
containing NADP cofactor was obtained by X-ray crystallography
(Fig. 3).35 As was seen with our earlier published structures from
this and closely related compound classes,30–32 7 binds to the sub-
strate site in a V-shape with its trifluoromethylcarbinol group
pointing toward the cofactor NADP+ side. Both the central and
the primary amide carbonyl groups have hydrogen bond interac-
tions with the adjacent protein molecules.
Figure 3. Co-crystal structure of compound 7 in human 11b-HSD1. The protein is
shown in both stick and molecular surface representations which are color coded
(red for oxygen atoms, blue for nitrogen, orange for sulfur, and pink and cyan for
carbon). The inhibitor and the cofactor NADP+ are shown in sticks and color coded
gray for carbon atoms in NADP+ and yellow for the inhibitor, the fluorine atoms of
the inhibitor are colored in magenta.
In summary, modification of compound 1 at the 4-position of
the cyclohexane ring led to the discovery of the two-carbon teth-
ered primary amide and carbamate groups, producing significant
improvements in biochemical and cellular potency. Furthermore,
these modifications resulted in a group of potent, selective, and
orally bioavailable compounds that demonstrated 11b-HSD1 inhi-
bition in both rat and cynomolgus monkey ex vivo models. Finally,
X-ray co-crystallographic data of 7 with 11b-HSD1 revealed the
key small molecule–protein complex interactions for the binding
of this class of 11b-HSD1 inhibitor.
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Figure 2. Ex vivo 11b-HSD1 enzyme activity in intact mesenteric fat collected from
cynomolgus monkeys dosed orally with compound 7. Plasma and mesenteric fat
samples were collected 2 h after compound was administered orally in dosing
vehicle (1% methylcellulose and 1% Tween 80 in sterile water). 11b-HSD1 enzyme
activity was measured as the [3H]-cortisol formed after 1 h incubation of fat
samples in reaction buffer containing [3H]-cortisone at 37 °C. Compound concen-
tration in plasma was determined by LC/MS/MS.

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with their proposed structures and purity (>95%) was determined by HPLC
analysis.
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35. The atomic coordinate has been deposited in the Protein Data Bank under an
accession code 3PDJ.