Discovery of a potent, orally active 11β-hydroxysteroid dehydrogenase type 1 inhibitor for clinical study: Identification of (S)-2-((1 S,2 S,4 R)-Bicyclo[2.2.1]heptan-2-ylamino)-5-isopropyl-5-methylthiazol-4(5 H)-one (AMG 221)

Article abstract of DOI:10.1021/jm100242d

Thiazolones with an exo-norbornylamine at the 2-position and an isopropyl group on the 5-position are potent 11β-HSD1 inhibitors. However, the C-5 center was prone to epimerization in vitro and in vivo, forming a less potent diastereomer. A methyl group was added to the C-5 position to eliminate epimerization, leading to the discovery of (S)-2-((1S,2S,4R)-bicyclo[2.2.1] heptan-2-ylamino)-5-isopropyl-5-methylthiazol-4(5H)-one (AMG 221). This compound decreased fed blood glucose and insulin levels and reduced body weight in diet-induced obesity mice.

Full text of DOI:10.1021/jm100242d

J. Med. Chem. 2010, 53, 4481–4487 4481
DOI: 10.1021/jm100242d
Discovery of a Potent, Orally Active 11β-Hydroxysteroid Dehydrogenase Type 1 Inhibitor for
Clinical Study: Identification of (S)-2-((1S,2S,4R)-Bicyclo[2.2.1]heptan-2-ylamino)-5-isopropyl-
5-methylthiazol-4(5H)-one (AMG 221)
†
Murielle M. Veniant, Clarence Hale, Randall W. Hungate, Kyung Gahm, Maurice G. Emery, Janan Jona, Smriti Joseph,
†
‡
‡
§
ꢀ
Jeffrey Adams,‡,2 Andrew Hague,‡,3 George Moniz,‡,4 Jiandong Zhang,‡ Michael D. Bartberger,‡ Vivian Li,‡ Rashid Syed,‡,[
‡
†
†
†
†
‡
ꢀ
Steven Jordan, Renee Komorowski, Michelle M. Chen, Rod Cupples, Ki Won Kim, David J. St. Jean Jr.,
Lars Johansson,^,# Martin A. Henriksson,^ Meredith Williams,^,1 Jerk Vallgarda,^ Christopher Fotsch,‡ and
Minghan Wang*,†
˚
Departments of †Metabolic Disorders, ‡Chemistry Research and Development, §PKDM, and Pharmaceutics, Amgen Inc., One Amgen Center
Drive, Thousand Oaks, California 91320, and ^Biovitrum AB, SE-112 76 Stockholm, Sweden. 3Current address: Infinity Pharmaceuticals,
780 Memorial Drive, Cambridge, Massachusetts 02139. 4Current address: Process Research, Eisai, Inc., 4 Corporate Drive, Andover,
Massachusetts 01810. 2Current address: Process Chemistry, Ricerca Biosciences, LLC, 7528 Auburn Road, Concord Twp., Ohio 44077.
[Current address: Department of Chemistry and Biochemistry, California State University Northridge, 18111 Nordhoff Street, Northridge,
California 91330-8262. 1Current address: S*BIO, #05-09 The Capricorn, Science Park II, 117528 Singapore. #Current address:
Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-171 77 Stockholm, Sweden.
Received February 23, 2010
Thiazolones with an exo-norbornylamine at the 2-position and an isopropyl group on the 5-position
are potent 11β-HSD1 inhibitors. However, the C-5 center was prone to epimerization in vitro and in
vivo, forming a less potent diastereomer. A methyl group was added to the C-5 position to eliminate
epimerization, leading to the discovery of (S)-2-((1S,2S,4R)-bicyclo[2.2.1]heptan-2-ylamino)-5-isopro-
pyl-5-methylthiazol-4(5H)-one (AMG 221). This compound decreased fed blood glucose and insulin
levels and reduced body weight in diet-induced obesity mice.
Scheme 1
Introduction
Glucocorticoids have many physiological functions, such as
augmentation of hepatic glucose production and suppression of
glucose utilization in skeletal muscle and adipose tissue.1 Gluco-
corticoid excess in Cushing’s syndrome causes metabolic ab-
normalities similar to the features of the metabolic syndrome,2
suggesting that glucocorticoid action may be involved in the
development of these disorders. Thus, blockade of glucocorticoid
action is considered a viable strategy to treat the metabolic
syndrome.3,4 11β-Hydroxysteroid dehydrogenase (11β-HSD1a)
is a bidirectional enzyme in vitro with both dehydrogenase and
reductase activities.5,6 However, in tissues and intact cells, it acts
predominately as an oxoreductase, converting inert cortisone to
active cortisol in humans or 11-dehydrocorticosterone (11-DHC)
to corticosterone in rodents.7-10 The isozyme of 11β-HSD1, 11β-
HSD2, is a dehydrogenase and catalyzes the opposite reaction.
11β-HSD1 is primarily expressed in liver, adipose tissue, and
brain, while 11β-HSD2 is mainly expressed in kidney. Since
glucocorticoid activation of the mineralocorticoid receptor
(MR) in kidney leads to hypertension, 11β-HSD2 activity pro-
tects MR from activation by inactivating cortisol. The adipose
expression of 11β-HSD1 in obese human subjects is elevated,11,12
suggesting that there is glucocorticoid excess in adipose tissues
of these subjects, which could be linked to the development
of obesity and insulin resistance. Transgenic overexpression of
11β-HSD1 in mouse adipose tissue resulted in the development of
obesity, insulin resistance, dyslipidemia, and hypertension.13,14
Moreover, mice with genetic ablation of 11β-HSD1 became
resistant to diet-induced obesity (DIO), hyperglycemia, and
dyslipidemia.15,16 These data suggest that selective inhibition of
11β-HSD1 could have therapeutic value for the treatment of
metabolic syndrome. In addition, liver-selective overexpression
of 11β-HSD1 only led to mild insulin resistance and dyslipidemia
but without obesity.17 These findings also indicate that adipose is
the primary target tissue for 11β-HSD1 inhibition.1 A number of
pharmaceutical and biotechnology companies have developed
11β-HSD1 inhibitors for the treatment of type 2 diabetes,
including Biovitrum,18 Abbott,19,20 Merck,21,22 Pfizer,23-25 and
Incyte.26 Recently, clinical studies with an 11β-HSD1 inhibitor in
patients with type 2 diabetes have provided additional evidence in
support of the therapeutic value of this target.26
Discussion
*To whom correspondence should be addressed. Phone: 805-447-
5598. Fax: 805-499-0953. E-mail: mwang@amgen.com.
Through SAR work on our thiazolone series of compounds
described in a previous report,27 a lead compound, 1, was
identified. Compound 1, prepared by the method outlined in
Scheme 1, was a mixture of eight diastereomers with a
a Abbreviations: 11β-HSD1, 11β-hydroxysteroid dehydrogenase
type 1; 11-DHC, 11-dehydrocorticosterone; DIO, diet-induced obesity;
GR, glucocorticoid receptor; GTT, glucose tolerance test; SGF, simu-
lated gastric fluid ; SIF, simulated intestinal fluid.
r
2010 American Chemical Society
Published on Web 05/13/2010
pubs.acs.org/jmc

4482 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 11
Veꢀniant et al.
calculated Ki of 58 nM. We prepared all of the (() exo-
norbornyl and (() endo-norbornyl analogues separately to
identify the most potent diastereomer (see Supporting In-
formation). The four (() endo-norbornyl analogues had
calculated Ki values in the range of 400-5000 nM and,
therefore, were not further pursued. One of the exo-norbornyl
diastereomers had better potency compared with the other
three diastereomers (Table 1). The most potent exo-diaster-
eomer was the (S,S) analogue, 2, where the stereochemical
assignmentreferstothe C-2 position of the norbornylringand
the C-5 position of the thiazolone ring, respectively (see
Table 1). We suspected that the C-5 position might be prone
to epimerization. Incubation of compound 2 with either
simulated gastric fluid (SGF) or simulated intestinal fluid
(SIF) led to the formation of a mixture of compound 2 and its
less active epimer, 3 (Figure 1). The formation of 3 in SGF is
more pronounced thanin SIF(Figure1). Thisfindingsuggests
that 2 may undergo epimerization within the gastrointestinal
tract after oral administration.
Table 1. Human 11β-HSD1 Activities of Compoundsa
To evaluate if compound 2 undergoes epimerization in
plasma, it was incubated with human or rat plasma and the
sample mixture was then analyzed for the presence of 2 and its
less active isomer, 3. In fact, at the end of the incubation, the
isomer was found in both human and rat plasma samples,
indicating that the C-5 position on the thiazolone ring was
susceptible to epimerization (Figure 2). In vitro epimerization
of compound 2 readily occurred in both human and rat
plasma (Figure 2). Incubation of compound 3 in rat and
human plasma showed similar results with the formation of
compound2 (datanotshown). The epimerization of2 to3 was
also confirmed in vivo in the rat. Following intravenous
administration of 2 mg/kg 2 to rats, small but measurable
concentrations of 3 were observed (Figure 3). In contrast,
there was a relatively greater amount of 3 formed following
oral administration of 10 mg/kg compound 2 to rats
(Figure 3). The mean AUC ratio of 3 to 2 following the iv
dose was 8.5%, whereas following the oral dose a mean AUC
ratio of 39% was observed. We hypothesized that oral
administration might have resulted in greater epimerization
of 2, since it was exposed to acidic conditions in the stomach,
which is consistent with the level of epimerization found in
SGF (Figure 2).
To circumvent the epimerization problem, a methyl group
was introduced at the C-5 position on analogue 6, which
produced a mixture of four diastereomers (7) (Scheme 2). This
mixture had a Ki of 17.3 nM, and after separation of the four
stereoisomers by chiral chromatography, the most potent
compound was 8 (AMG 221) (Ki = 12.8 nM; see Table 1).28
Compound 8 contained the (S,S) configuration at the C-2
position of the norbornyl and the C-5 position of the thiazo-
lone, which was the configuration found in the most potent
des-methyl analogue, compound 2. Compound 8 was also
potent in our cell-based assay and showed selectivity over
a The absolute configuration of compounds 2, 8, and 9 were con-
firmed by X-ray analysis. Data are expressed as the mean ( SEM.
Figure 1. In vitro epimerization of 2 to 3 in simulated gastric fluid (SGF) and simulated intestinal fluid (SIF).

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 11 4483
Figure 2. In vitro epimerization of 2 to 3 in rat and human plasmas.
Figure 4. Key binding interactions of 8 with human 11β-HSD1.
Carbon atoms of 11β-HSD1 are in green, NADPH in magenta, and
8 in cyan. Relevant hydrogen bonding and van der Waals intera-
€
˚
tomic distances are reported in angstroms.
Scheme 2
Figure 3. In vivo conversion of 2 to 3 in the rat following an
intravenous (2 mg/kg) or oral dose (10 mg/kg) of 2.
Table 2. Preclinical Pharmacokinetic Properties of Compound 8 in
Animalsa
11β-HSD2, 17β-HSD1, and glucocorticoid receptor (GR)
(IC50 values for all assays were >10 μM). This compound
was also selective over 61 other human targets (less than 25%
binding at 10 μM, data not shown). The cocrystal structure of
8 with the human enzyme was resolved. The overall fold of the
enzyme was virtually the same as that previously published.27
The binding mode of the compound and the arrangement of
the active site residues were almost identical to an analogue
complex structure reported previously27 (PDB code 3BYZ)
(Figure 4). The tautomerization state of the aminothiazolone
core of 8, possessing an endocyclic CdN double bond and
exocyclic NH, was the same as in this previous cocrystal
structure27 as was its hydrogen bonding pattern with nearby
residues. A hydrogen bonding interaction was observed be-
tween the thiazolone carbonyl oxygen atom and the backbone
NH of Ala172. This was augmented by an additional hydro-
gen bond between the exocyclic NH of the inhibitor and the
CD1 mouse SD rat beagle dog cynomolgus monkey
CL ((L/h)/kg)
ss (L/kg)
1/2 (h)
F (%)
3.16
1.00
15.6 b
54
1.36
1.73
41.5 b
34
0.94
1.02
8.1 b
50
1.89
0.98
0.6
7
V
t
a iv dose and formulation: mouse (5 mg/kg), rat (2 mg/kg), dog (3 mg/
kg), and monkey (2 mg/kg) in 10% dimethylacetamide, 23% water, and
67% PEG 400. po dose and formulation: mouse (30 mg/kg), rat (10 mg/
kg), and monkey (10 mg/kg) in 0.1% Tween-80 and 0.5% CMC in water;
dog (10 mg/kg) in 10% dimethylacetamide, 23% water, and 67% PEG
400. b Terminal t1/2
.
side chain oxygen of Tyr183, the latter forming a second
hydrogen bonding interaction with the Ser170 side chain. An
alternative binding mode involving the exocyclic double bond
tautomer (endocyclic NH) accompanied by a 180ꢀ reversal of
the aminothiazolone core in the binding site has been obser-
ved29 (PDB: 2RBE). The 2-norbonyl moiety of 8 occupied

4484 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 11
Veꢀniant et al.
Figure 5. Effects of 8 on glucose and insulin levels, glucose tolerance test (GTT), and body weight in DIO mice: (A) blood glucose levels
measured after 13 days of treatment (/, P < 0.05 vs vehicle); (B) plasma insulin levels measured after 13 days of treatment (/, P < 0.05 vs
vehicle); (C) intraperitoneal GTT performed in DIO mice treated with vehicle, rosiglitazone (5 (mg/kg)/day, q.d.), or 8 at 25 or 50 mg/kg b.i.d.;
(D) body weight measured at the end of the treatment period (//, P e 0.01 vs vehicle; ///, P < 0.001 vs vehicle).
a similar area of space as the cycloalkyl moiety in our
previously published inhibitor complex 3BYZ.27
8in a disease animal model, we administered this compound at 25
and 50 mg/kg, b.i.d., in DIO mice for 13-14 days. Administering
8b.i.d. was necessary to inhibit 11β-HSD1 activity in the fat for at
least 8 h per dose so that the total inhibition period within a day
was more than 16 h. At the end of the study, fed blood glucose
showed statistically significant reduction in comparison to the
vehicle group (Figure 5A). On day 13, there were statistically
significant decreases in insulin levels in all treated groups when
compared with the vehicle control group (Figure 5B). On day 14
and after a 12 h fast, glucose tolerance was slightly improved in
the compound 8 treatment groups compared with the vehicle
group (Figure 5C). As expected, mice treated for only 13 days
with rosiglitazone did not show improved glucose tolerance.
Also, as shown from the baseline of the glucose tolerance test,
fasted blood glucose levels of compound 8 treated mice were not
changed compared to the vehicle treated mice. Body weight was
also decreased in a dose-dependent fashion in mice treated with 8
compared to the control group (Figure 5D). Also, as expected,
body weight of mice treated with rosiglitazone was statistically
significantly increased.
The PK profiles of 8wereevaluatedinseveral animal species,
including mouse, rat, dog, and monkey. The in vivo pharma-
cokinetic properties are summarized in Table 2. The data from
a 5 mg/kg iv study in mice are reported for ease of comparison
with those in other species, since the same vehicle was used
(Table 2). Mouse iv PK was also carried out at 2 mg/kg, and
similar CL and Vss values were obtained (data not shown). The
plasma clearance of 8 was moderate to high in mouse, rat,
monkey, and dog relative to hepatic blood flow (Table 2).
Disposition was biphasic where the compound exhibited a
rapid initial phase of decline which accounted for the majority
of the area under the concentration-time profile followed by
the appearance of a “deep” compartment characterized by a
much longer terminal half-life in all the species tested. Com-
pound 8 had a good bioavailability in mouse, rat, and dog.
However, the bioavailability in monkey was low, which could
be attributed to first-pass hepatic metabolism consistent with
low liver microsomal stability. This compound did not inhibit
any of the five main cytochrome P450 enzymes, 3A4, 2D6,
1A2, 2C9, and 2C19 (IC50 > 15 μM). The in vitro Km for
metabolism was greater than 100 μM and there was no
evidence for transport by P-glycoprotein.
Compound 8 was administered in a mouse pharmacodynamic
model measuring 11β-HSD1 activity in an adipose tissue explant
as described previously.27 11β-HSD1 activity was inhibited by
33%, 55%, and 47% in the inguinal fat at 4 h after 8 was orally
gavaged at 5, 15, and 50 mg/kg, respectively. At 8 h, the 11β-
HSD1 activity in the inguinal fat of the 5 mg/kg group had
returned to a level (∼10% inhibition) close to that in the control
animals treated with vehicle, but there was still significant
inhibition in the 15 and 50 mg/kg groups (36% and 39%
inhibition, respectively). To test the pharmacological effects of
Conclusion
We identified a potent, selective 11β-HSD1 inhibitor com-
pound 2. However, since this compound underwent both in vitro
and in vivo epimerization, it was not suitable for further advance-
ment. Through structural modification of this compound to pre-
vent epimerization, 8was discovered. This compound had accep-
table potency, selectivity, PK properties, and pharmacological
efficacy. The favorable profile of compound 8justified preclinical
safety studies, after which it was advanced to clinical trials.
Experimental section
Preparation of Compounds. (R,S)-2-((()-exo-Bicyclo[2.2.1]-
heptan-2-ylamino)-5-isopropylthiazol-4(5H)-one. A mixture of

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 11 4485
(()-exo-1-(bicyclo[2.2.1]heptan-2-yl)thiourea (530 mg, 3.1 mmol),
(S)-2-bromo-3-methylbutanoic acid (560 mg, 3.1 mmol), and so-
dium acetate (310 mg, 3.7 mmol) in anhydrous ethanol (10 mL) was
heated in a round-bottomed flask to reflux under nitrogen. After
4.5 h, the reaction mixture was cooled to room temperature, and the
mixture was concentrated in vacuo. To the residue was added ethyl
acetate (20 mL), which was washed with water (20 mL) and brine
(20 mL). The organic layer was then dried over Na2SO4, filtered, and
concentrated in vacuo. The crude product was purified by flash
chromatography (4:1 hexanes/acetone) to yield the desired product
as a colorless oil (355 mg, 45%). The mixture was further purified by
SFC (supercritical fluid chromatography method, column OD-H
(4.6 mm ꢀ 15 cm, 5 μm), flow rate of 2.8 mL/min, 89% CO2 (l) and
11% ethanol, T = 35 ꢀC, back pressure of 120 bar) to give the four
diastereomers described below.
(S)-2-((1S,2S,4R)-Bicyclo[2.2.1]heptan-2-ylamino)-5-isopro-
pylthiazol-4(5H)-one (2). Yield = 41 mg, 5.2%. 1HNMR(CDCl3
þ 1 drop of TFA, 400 MHz): δ 4.35 (d, J = 4.02 Hz, 1 H), 3.42
(m, 1 H), 2.68 (m, 1 H), 2.48 (br s, 2 H), 1.90 (ddd, J = 2.26, 8.03,
13.3 Hz, 1 H), 1.69 (m, 2 H), 1.585 (br d, J=12Hz, 2H), 1.38(brd,
J = 10.5 Hz, 1 H), 1.23 (m, 2 H), 1.16 (d, J = 7.03 Hz, 3 H), 1.05
(d, J = 6.52 Hz, 3 H). MS (ESI) m/z: 253.2 (M þ 1). Anal. Calcd
for C13H20N2OS: C, 61.87; H, 7.99; N, 11.10. Found: C, 62.01; H,
7.96; N, 11.08.
was isolated as a white solid (2.37 g). The mixture was further
purified by SFC (supercritical fluid chromatography method,
two columns run in series: AD-H (4.6 mm ꢀ15 cm, 5 μm) and
OD-H (4.6 mm ꢀ 15 cm, 5 μm) (flow rate of 3 mL/min, 90%
CO2 (l) and 10% ethanol, T = 40 ꢀC, back pressure of 120 bar).
(S)-2-((1S,2S,4R)-Bicyclo[2.2.1]heptan-2-ylamino)-5-isopropyl-
1
5-methylthiazol-4(5H)-one (8). Yield = 210 mg, 9%. H NMR
(CDCl3 þ 1 drop of TFA, 400 MHz): δ 3.40 (br d, J = 5 Hz, 1 H),
2.46 (m, 2 H), 2.34 (dq, J = 6.61, 6.78 Hz, 1 H), 1.89 (ddd, J =
2.01, 8.03, 13.5 Hz, 1 H), 1.78 (s, 3 H), 1.69 (m, 2 H), 1.58 (m, 2 H),
1.38 (br d, J = 11.0 Hz, 1 H), 1.22 (m, 2 H), 1.12 (d, J = 7.03 Hz,
3 H), 1.025 (d, J = 6.52 Hz, 3 H). MS (ESI) m/z: 267.2 (M þ 1).
Anal. Calcd for C14H22N2OS: C, 63.12; H, 8.32; N, 10.52. Found:
C, 63.31; H, 8.29; N, 10.59.
(S)-2-((1R,2R,4S)-Bicyclo[2.2.1]heptan-2-ylamino)-5-isopropyl-
5-methylthiazol-4(5H)-one (9). Yield = 240 mg, 10%. 1H NMR
(CDCl3 þ 1 drop of TFA, 400 MHz): δ 3.39 (dd, J = 2.26,
7.78 Hz, 1 H), 2.47 (m, 2 H), 2.33 (sept, J = 6.69 Hz, 1 H), 1.90
(ddd, J = 2.26, 8.03, 13.3 Hz, 1 H), 1.78 (s, 3 H), 1.71-1.55 (m,
4 H), 1.37 (br d, J = 10.0 Hz, 1 H), 1.22 (m, 2 H), 1.10 (d, J =
6.52 Hz, 3 H), 1.00 (d, J = 6.52 Hz, 3 H). MS (ESI) m/z: 267.2
(M þ 1). Anal.Calcdfor C14H22N2OS: C, 63.12; H, 8.32;N, 10.52.
Found: C, 63.34; H, 8.39; N, 10.54.
(R)-2-((1S,2S,4R)-Bicyclo[2.2.1]heptan-2-ylamino)-5-isopropyl-
5-methylthiazol-4(5H)-one (10). Yield = 260 mg, 11%. 1H NMR
(CDCl3 þ 1 drop of TFA, 400 MHz): δ 3.41 (dd, J = 2.26,
8.28 Hz, 1 H), 2.47 (m, 2 H), 2.33 (dq, J = 6.61, 6.78 Hz, 1 H), 1.91
(ddd, J = 2.01, 7.53, 13.6 Hz, 1 H), 1.79 (s, 3 H), 1.69-1.56 (m,
4 H), 1.37 (br d, J = 10.5 Hz, 1 H), 1.21 (m, 2 H), 1.11 (d, J =
7.03 Hz, 3 H), 1.01 (d, J = 6.52 Hz, 3 H). MS (ESI) m/z: 267.2
(M þ 1). HPLC (method A (5 min)) Rf = 1.94 min, 100, 96%
(215, 254 nm); (method B (15 min)) Rf = 10.3 min, 99, 96%
(215, 254 nm).
(R)-2-((1S,2S,4R)-Bicyclo[2.2.1]heptan-2-ylamino)-5-isopro-
pylthiazol-4(5H)-one (3). Yield = 24 mg, 3.1%. 1HNMR(CDCl3
þ 1 drop of TFA, 400 MHz): δ 4.36 (d, J = 3.51 Hz, 1 H), 3.44 (br
d, J = 5.52 Hz, 1 H), 2.67 (m, 1 H), 2.48 (m, 2 H), 1.92 (ddd, J =
2.01, 8.03, 13.6 Hz, 1 H), 1.71-1.54 (m, 4 H), 1.375 (br d, J =
12 Hz, 1 H), 1.22 (m, 2 H), 1.16 (d, J = 6.52 Hz, 3 H), 1.04 (d, J =
6.52 Hz, 3 H). MS (ESI) m/z: 253.1 (M þ 1). Anal. Calcd for
C13H20N2OS: C, 61.87; H, 7.99; N, 11.10. Found: C, 61.94; H,
7.88; N, 11.09.
(R)-2-((1R,2R,4S)-Bicyclo[2.2.1]heptan-2-ylamino)-5-isopro-
pylthiazol-4(5H)-one (4). Yield = 34 mg, 4.3%. 1HNMR(CDCl3
þ 1 drop of TFA, 400 MHz): δ 4.33 (t, J = 3.76 Hz, 1 H), 3.42 (d,
J = 7.53, 1 H), 2.68 (m, 1 H), 2.48 (br s, 2 H), 1.90 (dd, J = 8.03,
13.1 Hz, 1 H), 1.73-1.58 (m, 4 H), 1.38 (d, J = 10.5 Hz, 1 H), 1.22
(m, 2 H), 1.16 (d, J = 6.52 Hz, 3 H), 1.05 (d, J = 6.52 Hz, 3 H). MS
(ESI) m/z: 253.2 (M þ 1). HPLC (method A (5 min)) Rf = 1.81
min, 100, 100% (215, 254 nm); (method B (15 min)) Rf = 8.72 min,
100, 100% (215, 254 nm).
(R)-2-((1R,2R,4S)-Bicyclo[2.2.1]heptan-2-ylamino)-5-isopropyl-
5-methylthiazol-4(5H)-one (11). Yield = 233 mg, 10%. 1H NMR
(CDCl3 þ 1 drop of TFA, 400 MHz): δ3.39 (dd, J= 3.40, 5.02 Hz,
1 H), 2.46 (m, 2 H), 2.34 (dq, J = 6.61, 6.78 Hz, 1 H), 1.89 (ddd,
J= 2.26, 7.91, 13.4 Hz, 1 H), 1.77 (s, 3 H), 1.72-1.55(m, 4H), 1.38
(br d, J = 10.5 Hz, 1 H), 1.21 (m, 2 H), 1.11 (d, J = 6.52 Hz, 3 H),
1.02 (d, J = 6.52 Hz, 3 H). MS (ESI) m/z: 267.2 (M þ 1). Anal.
Calcd for C14H22N2OS: C, 63.12; H, 8.32; N, 10.52. Found: C,
63.06; H, 8.27; N, 10.58.
(S)-2-((1R,2R,4S)-Bicyclo[2.2.1]heptan-2-ylamino)-5-isopro-
pylthiazol-4(5H)-one (5). Yield = 24 mg, 3.1%. 1HNMR(CDCl3
þ 1 drop of TFA, 400 MHz): δ 4.34 (d, J = 3.51 Hz, 1 H), 3.43 (d,
J = 5.02, 1 H), 2.66 (m, 1 H), 2.47 (br s, 2 H), 1.91 (ddd, J = 2.26,
7.91, 13.4 Hz, 1 H), 1.71-1.56 (m, 4 H), 1.37 (d, J = 10.6 Hz, 1 H),
1.24 (m, 2 H), 1.15 (d, J= 7.03 Hz, 3H), 1.04(d, J=6.52Hz, 3H).
MS(ESI) m/z: 253.2 (M þ 1). HPLC (method A (5 min)) Rf =1.82
min, 100, 100% (215, 254 nm); (method B (15 min)) Rf = 8.73 min,
100, 100% (215, 254 nm).
(R,S)-2-((()-exo-Bicyclo[2.2.1]heptan-2-ylamino)-5-isopropyl-
5-methylthiazol-4(5H)-one. To a 250 mL round-bottomed flask
equipped with magnetic stirring was added diisopropylamine
(8.0 mL, 57 mmol) in 100 mL of anhydrous THF. After the
reaction mixture was cooled to -78 ꢀC under an inert atmo-
sphere, n-butyllithium (34 mL of a 1.6 M solution in hexanes,
54 mmol) was added dropwise over 15 min. Following the complete
addition of n-butyllithium, the reaction mixture was stirred for an
additional 40 min, and then (R,S)-2-((()-exo-bicyclo[2.2.1]heptan-
2-ylamino)-5-isopropylthiazol-4(5H)-one (2.2 g, 8.7 mmol) in
20 mL of anhydrous THF was added over 5 min. After ∼1.5 h,
methyl iodide (2.2 mL, 35 mmol) was added, and the reaction
mixture was stirred for another 5.5 h at -78 ꢀC. Brine (60 mL)
was then added, and the reaction flask was allowed to warm to
room temperature. The organic layer was then separated from
the aqueous layer, and the aqueous layer was extracted with
THF (2ꢀ). The organic layers were combined and dried over
MgSO4, filtered, and concentrated in vacuo. Following flash
chromatography (hexanes/ethyl acetate) the desired product
Purity of all final compounds were >95% as determined by
LC-MS and/or elemental analysis.
Epimerization of Compounds in Vitro and in Vivo. Epimeriza-
tion study was conducted in both simulated gastric fluid (SGF,
which contains 2 g of NaCl, 3.2 g of pepsin, and 7 mL of HCl QS
to 1000 mL of water, pH 1.12) and simulated intestinal fluid
(SIF, which contains 6.8 g of potassium phosphate monobasic,
190 mL of 0.2 N NaOH, 10 g of pancreatin QS to 1000 mL of
water, pH 7.5 at 23 ꢀC). The study was conducted by preparing a
3 mL solution of compound 2 at 0.1 mg/mL in SIF and SGF.
The solution was maintained in a water bath at 37 ꢀC. Samples
of 0.25 mL were withdrawn at each time point, and the reaction
was quenched by adding acetonitrile. Samples were centrifuged,
and the supernatant was analyzed by LC-MS.
Compounds 3 and 2 were incubated in rat and human plasma
at 37 ꢀC for 24 h. Aliquots of plasma were taken at 0, 6, and 24 h
for measurement of the corresponding 2 or its diastereomer 3 for
assessment of epimerization at the C-5 position on the thiazo-
lone ring. To determine the extent of epimerization in vivo,
Sprague-Dawley rats (n = 2/group) were administered either
an intravenous (2 mg/kg) or oral (10 mg/kg) dose of 2. Blood was
collected for the measurement of 3 and 2 predose and at intervals
up to 24 h to characterize the pharmacokinetics. Compounds 3
and 2 were analyzed in plasma using HPLC to separate the
diastereomers and then detected using electrospray ionization
(positive ion) MS/MS (API 4000, Applied Biosystems) and
single ion monitoring (SIM). The assay quantitation limit was
2 ng/mL for 2 and 1 ng/mL for 3.

4486 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 11
Crystal Structure. The X-ray structure of the complex of 8 and
Veꢀniant et al.
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˚
human 11β-HSD1 was determined to a resolution of 2.6 A using
a published protocol.27 The final R and Rfree are 21.7% and
30.0%, respectively.
(4) Walker, B. R.; Seckl, J. R. 11Beta-hydroxysteroid dehydrogenase
type 1 as a novel therapeutic target in metabolic and neurodegen-
erative disease. Expert Opin. Ther. Targets 2003, 7, 771–783.
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Expression of 11 beta-hydroxysteroid dehydrogenase using recom-
binant vaccinia virus. Mol. Endocrinol. 1990, 4, 1827–1832.
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length recombinant human and rat type 1 11beta-hydroxysteroid
dehydrogenases with retained oxidoreductase activities. Protein
Expression Purif. 2002, 26, 349–356.
PK Studies. Compound 8 was administered via po or iv to
mouse, rat, or monkey. Blood samples were collected at various
time points, and compound concentrations were measured.
Ex Vivo Assay. The assay was carried out as described
previously.27
Animals for Pharmacology Studies. Three-week-old male
C57BL/6 mice were fed high fat (HF) diet chow (D12492 high
fat diet, Research Diets Inc., New Brunswick, NJ) for 12 weeks
(age of mice at start of experiment was 15-16 weeks) to induce
insulin resistance and obesity. Animals were maintained at a
constant temperature and had free access to food and drinking
water in a light-dark cycle with lights on from 6:30 a.m. to 6:30 p.m.
Pharmacology Study of 8 in Diet-Induced Obesity (DIO) Mice.
Mice were sorted into groups of n = 11-12 to provide equal
distribution of pretreatment glucose levels and body weight
between groups. Fed blood glucose and insulin levels were
measured before starting the treatment and at the end of the
treatment. Mice were orally gavaged with 8 at doses of 50 or 100
(mg/kg)/day twice a day (i.e., 25 or 50 mg/kg twice a day with
half dose in the morning and half in the afternoon) or with
vehicle (0.1% Tween-80 and 0.5% CMC in water) for 13 or
14 days. Rosiglitazone was included in the study and delivered
at ∼5 mg/kg/day in HF diet chow for 13 or 14 days.
(7) Apostolova, G.; Schweizer, R. A.; Balazs, Z.; Kostadinova, R. M.;
Odermatt, A. Dehydroepiandrosterone inhibits the amplification
of glucocorticoid action in adipose tissue. Am. J. Physiol.: Endo-
crinol. Metab. 2005, 288, E957–E964.
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Chalder, S. M.; Arlt, W.; Lavery, G. G.; Bedendo, O.; Ray,
D. W.; Laing, I.; Malunowicz, E.; White, P. C.; Hewison, M.;
Mason, P. J.; Connell, J. M.; Shackleton, C. H.; Stewart, P. M.
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genase type 1 and hexose-6-phosphate dehydrogenase interact to
cause cortisone reductase deficiency. Nat. Genet. 2003, 34, 434–439.
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type 1 is a predominant 11 beta-reductase in the intact perfused rat
liver. J. Endocrinol. 2000, 165, 685–692.
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Kaprio, J.; Hamsten, A.; Yki-Jarvinen, H. Overexpression of
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studies in young adult monozygotic twins. J. Clin. Endocrinol.
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After 13 days of treatment, each treatment group was split
into two post-treatment subgroups, subgroup A with n = 5 and
subgroup B with n = 6-7. From subgroup A, plasma was
collected for measurements of fed blood glucose and insulin
levels (blood was also collected from all animals at baseline
[day - 1] for the same measurements). For subgroups B, 14 days
after starting treatments, glucose tolerance testing (GTT, 2 g/kg
of body weight intraperitoneal glucose injection) was performed
after a 12 h fast.
Intraperitoneal GTT was carried out in DIO mice fasted for
12 h (9 p.m. to 9 a.m.). Fasted blood glucose was measured prior
to an ip injection of glucose (0.5 g/mL dextrose) at 2 g/kg body
weight. Blood samples were collected from the retro-orbital
sinuses of nonanesthetized mice. In order to minimize stress
levels in the tested animals, only 30 and 90 min blood glucose
levels were measured using the OneTouch profile meter.
Blood Chemistry Measurements. Blood glucose levels were
measured immediately following blood collection on a glucose
monitor (OneTouch profile meter, Lifescan, Inc., Milpitas, CA).
Plasma insulin was measured by radioimmunoassay using a kit
from Linco Research (St. Charles, MO).
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J. J.; Seckl, J. R.; Flier, J. S. A transgenic model of visceral obesity
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Morton, N. M.; Paterson, J. M.; Shinyama, H.; Sharp, M. G.;
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blood pressure in mice. J. Clin. Invest. 2003, 112, 83–90.
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Schmoll, D.; Jamieson, P.; Best, R.; Brown, R.; Edwards, C. R.;
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11β-HSD1 Enzymatic Assays. In vitro biochemical scintillation
proximity assay (SPA) and cell-based assays are as described
previously.27
Statistical Analyses. All statistical analyses were carried out
using a two-tailed Student’s t test assuming unequal variances
(GraphPad Prism software; San Diego, CA).
(17) Paterson, J. M.; Morton, N. M.; Fievet, C.; Kenyon, C. J.; Holmes,
M. C.; Staels, B.; Seckl, J. R.; Mullins, J. J. Metabolic syndrome
without obesity: hepatic overexpression of 11beta-hydroxysteroid
dehydrogenase type 1 in transgenic mice. Proc. Natl. Acad. Sci.
U.S.A. 2004, 101, 7088–7093.
(18) Barf, T.; Vallgarda, J.; Emond, R.; Haggstrom, C.; Kurz, G.;
Nygren, A.; Larwood, V.; Mosialou, E.; Axelsson, K.; Olsson,
R.; Engblom, L.; Edling, N.; Ronquist-Nii, Y.; Ohman, B.;
Alberts, P.; Abrahmsen, L. Arylsulfonamidothiazoles as a new
class of potential antidiabetic drugs. Discovery of potent and
selective inhibitors of the 11beta-hydroxysteroid dehydrogenase
type 1. J. Med. Chem. 2002, 45, 3813–3815.
(19) Yeh, V. S.; Kurukulasuriya, R.; Madar, D.; Patel, J. R.; Fung, S.;
Monzon, K.; Chiou, W.; Wang, J.; Jacobson, P.; Sham, H. L.;
Link, J. T. Synthesis and structural activity relationship of 11beta-
HSD1 inhibitors with novel adamantane replacements. Bioorg.
Med. Chem. Lett. 2006, 16, 5408–5413.
Supporting Information Available: Preparation of (S)-2-bro-
mo-3-methylbutanoic acid and (()-exo-1-(bicyclo[2.2.1]heptan-
2-yl)thiourea, details of the HPLC conditions used, details of the
stereochemical determination, and X-ray data. This material is
available free of charge via the Internet at http://pubs.acs.org.
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