Further studies with the 2-amino-1,3-thiazol-4(5H)-one class of 11β-hydroxysteroid dehydrogenase type 1 inhibitors: Reducing pregnane X receptor activity and exploring activity in a monkey pharmacodynamic model

Article abstract of DOI:10.1021/jm801073z

A series of compounds containing the 2-amino-1,3-thiazol-4(5H)-one core were found to be potent inhibitors of the enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1). One of our lead compounds from this series activated the human nuclear xenobiotic

Full text of DOI:10.1021/jm801073z

J. Med. Chem. 2008, 51, 7953–7967
7953
Further Studies with the 2-Amino-1,3-thiazol-4(5H)-one Class of 11ꢀ-Hydroxysteroid
Dehydrogenase Type 1 Inhibitors: Reducing Pregnane X Receptor Activity and Exploring
Activity in a Monkey Pharmacodynamic Model
Christopher Fotsch,*,† Michael D. Bartberger,† Eric A. Bercot,† Michelle Chen,‡ Rod Cupples,‡ Maury Emery,§ Jenne Fretland,§
Anil Guram,† Clarence Hale,‡ Nianhe Han,† Dean Hickman,§ Randall W. Hungate,† Michael Hayashi,§ Renee Komorowski,‡
Qingyian Liu,† Guy Matsumoto,§ David J. St. Jean, Jr.,† Stefania Ursu,‡ Murielle Ve´niant,‡ Guifen Xu,§ Qiuping Ye,§
Chester Yuan,† Jiandong Zhang,† Xiping Zhang,§ Hua Tu,‡ and Minghan Wang‡
Departments of Chemical Research and DeVelopment, Metabolic Disorders, and Pharmacokinetics and Drug Metabolism, Amgen, Inc., One
Amgen Center DriVe, Thousand Oaks, California 91320, and Amgen Inc., 1120 Veterans BouleVard, South San Francisco, California 94080
ReceiVed August 28, 2008
A series of compounds containing the 2-amino-1,3-thiazol-4(5H)-one core were found to be potent inhibitors
of the enzyme 11ꢀ-hydroxysteroid dehydrogenase type 1 (11ꢀ-HSD1). One of our lead compounds from
this series activated the human nuclear xenobiotic receptor, pregnane X receptor (PXR). To try and mitigate
the PXR activity, we prepared analogues of our lead series that contained polar groups on the right-hand
side of the thiazolone. Several analogues containing amides or alcohols appended to the C-5 position of the
thiazolone showed a significant reduction in PXR activity. Through these structure-activity efforts, a
compound containing a tert-alcohol group off the C-5 position, analogue (S)-33a, was found to have an
11ꢀ-HSD1 Ki ) 35 nM and negligible PXR activity. Compound (S)-33a was advanced into a pharmaco-
dynamic model in cynomolgus monkeys, where it inhibited adipose 11ꢀ-HSD1 activity after being orally
administered.
Scheme 1. Interconversion of Cortisone, 1a
(Dehydrocorticosterone, 1b), to Cortisol, 2a (Corticosterone, 2b)
Introduction
An estimated 171 million people worldwide suffered from
diabetes in the year 2000.1 By the year 2030, the number of
people afflicted with diabetes could more than double.1 The
majority of these cases are defined as type II diabetes mellitus
(T2DM), a condition characterized by an abnormally high
fasting blood glucose (>126 mg/dL) and insulin resistance. Left
untreated, T2DM can lead to debilitating sequelae such as
retinopathy, neuropathy, coronary artery disease, and ischemic
stroke. While many therapies are currently available for manag-
ing the symptoms associated with T2DM (e.g., insulin secre-
tagogues, insulin sensitizers, and modulators of glucose uptake
and production), improved therapies are being sought that target
novel biological pathways involved in this disease. One target
that has piqued the interest of many pharmaceutical companies
is the enzyme 11ꢀ-hydroxysteroid dehydrogenase type 1
(11ꢀ-HSD1a).2-5
Expressed mainly in liver, adipose, and brain, 11ꢀ-HSD1 is
an NADPH-dependent enzyme that primarily acts as a reductase
to convert the inactive glucocorticoid cortisone (1a) to the active
form, cortisol (2a) (Scheme 1). In rodents, 11ꢀ-HSD1 converts
dehydrocorticosterone (1b) to the active form, corticosterone
(2b). The isozyme of 11ꢀ-HSD1, 11ꢀ-HSD2, is mainly ex-
pressed in the kidney and acts as a dehydrogenase to convert
cortisol (2a) to cortisone (1a) (Scheme 1). In adipose and liver,
cortisol activates the glucocorticoid receptor (GR), which
upregulates the expression of genes involved in energy homeo-
stasis, that is, genes that regulate gluconeogenesis,6 adipogenesis,
and lipolysis.7 Because 11ꢀ-HSD1 is the enzyme that controls
the tissue levels of the GR ligand, cortisol, excessive activity
of 11ꢀ-HSD1 is thought to play a role in the development of
hyperglycemia, insulin resistance, and lipid disorders. Evidence
supporting this hypothesis was obtained in mice that have
genetically altered 11ꢀ-HSD1 expression. Mice that overexpress
11ꢀ-HSD1 in adipose tissue developed central obesity, insulin
resistance, and dyslipidemia.8 Conversely, mice deficient in 11ꢀ-
HSD1 were resistant to diet-induced obesity and insulin
resistance.9-11 In addition, orally available 11ꢀ-HSD1 inhibitors
have been shown to improve glucose tolerance and insulin
12,13
resistance in mouse models of type II diabetes
(see Chart
1 for examples of 11ꢀ-HSD1 inhibitors14-18). Although there
is no concrete evidence that patients with diabetes exhibit
increased circulating glucocorticoid levels, elevated adipose 11ꢀ-
HSD1 activity has been correlated with obesity in humans,
suggesting that there is increased tissue-specific GR activity in
obesity.19,20 The weight of evidence suggests that inhibition of
11ꢀ-HSD1 is an attractive strategy to lower GR activity, which
could be useful for treating type II diabetes.
* To whom correspondence should be addressed. Tel: 805-447-7746.
Fax: 805-480-1337. E-mail: cfotsch@amgen.com.
†
Department of Chemical Research and Development.
Department of Metabolic Disorders.
Department of Pharmacokinetics and Drug Metabolism.
‡
We have previously described a class of 11ꢀ-HSD1 inhibitors
containing the 1,3-thiazol-4(5H)-one core (referred to as
thiazolone).21,22 In our publications,21,23 compound 3 was shown
to be a potent and selective inhibitor of 11ꢀ-HSD1, which
§
a Abbreviations: 11ꢀ-HSD1, 11ꢀ-hydroxysteroid dehydrogenase; CYP,
cytochrome P450; PXR, pregnane X receptor; thiazolone, 1,3-thiazol-4(5H)-
one; AUC, area under the curve; POC, percent of control.
10.1021/jm801073z CCC: $40.75
2008 American Chemical Society
Published on Web 11/19/2008

7954 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24
Chart 1. Examples of 11ꢀ-HSD1 Inhibitors
Fotsch et al.
which were then methylated at the 5-position of the thiazolone
ring to give compounds 8 and 9. The aryl bromide on compound
9 was converted to a carboxymethyl group through a palladium-
mediated carbonylation reaction to provide intermediate 10. This
intermediate was converted to the carboxylic acid 11, which
was then converted to amide derivatives 12a-e and 13.
Alternatively, the ester 10 was converted to alcohol 14, which
was then converted in a three-step process to amide 15.
The thiourea 5 was also treated with methyl 2-bromopro-
panoate to give 5-methyl thiazolone 16 as a key intermediate,
which was used for the preparation of the final compounds
shown in Scheme 4. Amide 18 was prepared from thiazolone
16 by first alkylating the 5-position with 4-(bromomethyl)ben-
zonitrile and then hydrolyzing the nitrile with KOH. To
introduce an aromatic ring directly onto the 5-position of the
thiazolone, aryl bromide 19 was cross-coupled with intermediate
16 using LiHMDS, Pd2(dba)3, and BINAP. The nitrile was then
hydrolyzed as before to provide amide compound 20. The cross-
coupling procedure used to prepare 20 was also used in the
preparation of analogues 23, 26, and 29. Amide analogue 30
was prepared from alcohol 29 through commonly used func-
tional group interconversions.
Compounds containing tertiary alcohols off the 5-position of
the thiazolone were prepared as outlined in Scheme 5. The
thioureas for these compounds were prepared from commercially
available benzyl amines using the procedure described above.
To prepare 5-methyl thiazolones 32, thioureas 31 were heated
with 2-bromoproprionic acid and NaOAc (Compound 16 was
prepared by this alternate procedure in addition to being prepared
by the method illustrated in Scheme 4. See the Experimental
Section). Tertiary alcohol derivatives 33 were prepared from
thiazolone 32 using LDA and the appropriate ketone. Analogue
35 was prepared from 5-methyl thiazolone 16 by alkylating the
5-position with 2-methyloxirane to give intermediate 34. Oxida-
tion of 34 and subsequent addition of methyl Grignard afforded
the desired tertiary alcohol 35.
a Ref 14. b Ref 15. c Ref 16. d Ref 17. e Ref 18.
inhibited adipose 11ꢀ-HSD1 activity in vivo and was efficacious
in rodent models of diabetes.24 After further profiling in drug
metabolism studies, compound 3 was found to activate the
human pregnane X receptor (PXR), a nuclear receptor that
upregulates genes involved in drug metabolism [e.g., cytochrome
P450 (CYP) 3A4 and multidrug resistance proteins]. By
activating PXR, the efficacy of a compound may be reduced
since PXR upregulates proteins that may increase the clearance
of a drug.25 Our strategy to reduce the PXR activation of
compound 3 was to replace the CF3 group at the 5-position with
polar groups to decrease the logP, a strategy that is known to
lower PXR activity.26 Also, the X-ray data27 and a homology
model28 of PXR suggest that its binding site is very hydrophobic,
so adding polar groups to our compounds might make binding
to this site unfavorable. Because we knew from our previous
work that the left-hand R-methyl benzylamine group of 3 was
important for 11ꢀ-HSD1 activity and metabolic stability,21 we
kept this group in the analogues prepared in this paper. Herein,
we describe the structure-activity relationship (SAR) of 2-ami-
nobenzyl thiazolone analogues that contain amides, carboxylic
acids, and alcohols appended to the 5-position, and we describe
how some of these changes affected PXR activity.
Results and Discussion
Inhibition of 11ꢀ-HSD1 and 11ꢀ-HSD2 enzymatic activity
was measured using a scintillation proximity assay (SPA).21,29
Cell-based activity was measured by monitoring the conversion
of cortisone to cortisol in Chinese hamster ovary (CHO) cells
stably transfected with human 11ꢀ-HSD1.29 Results are reported
as the average of at least two independent experiments with at
least two replicates at each concentration. The variance in the
measurements is expressed as the standard error of the mean
(SEM).
During the expansion of the SAR for lead compound 3,
analogue 8, containing a 4-fluoro phenyl group at the 5-position
of the thiazolone, was found to have 11ꢀ-HSD1 activity similar
to our lead compound 3 (both have Ki values ) 22 nM, see
Table 1). Rather than trying to substitute polar groups off the
CF3 group of 3, we felt that the C-5 phenyl group on 8 was an
easier platform to append polar groups. Table 1 summarizes
the analogues of 8 that we prepared that contained polar groups
on the C-5 aromatic ring. The first attempt at incorporating a
polar group led to carboxylate analogue 11, which had a Ki )
3.5 µM. We were able to quickly improve the activity of this
compound with ester and primary amide derivatives, 10 and
12a, which were g80-fold more potent than carboxylate
analogue 11. Keeping the primary amide in place, and extending
the aromatic ring by one methylene group, gave compound 18,
which was ∼40-fold less potent than the amide analogue 12a.
Because activity decreased with the addition of a methylene
Chemistry
Compounds containing an aryl group at the 5-position of the
thiazolone were prepared using the methods outlined in Schemes
3 and 4. Thiourea 5 was prepared in two steps from the
commercially available amine 4 by first treating the amine with
benzoyl isothiocyanate followed by basic hydrolysis. Thiourea
5 was treated with R-bromo carboxylates 7 to give thiazolones,

11ꢀ-Hydroxysteroid Dehydrogenase Type 1 Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24 7955
Scheme 2. Strategy To Address the Issue of PXR Activation
a PXR activity with a 20 µM concentration of 3 (100% of control is equivalent to the activity achieved with 12.5 µM rifampin).
Scheme 3a
a Reagents and conditions: (a) Benzoyl isothiocyanate, CH2Cl2. (b) K2CO3, THF/methanol/H2O, 98% for two steps. (c) NBS, AIBN, CCl4, 80 °C (X )
F, R ) H, 86%; X ) Br, R ) Et, 96%). (d) X ) F, NaOAc, ethanol, reflux, 48%; X ) Br, NEt(i-Pr)2, ethanol, reflux, 71%. (e) LDA, CH3I, THF, -78 °C,
(X ) F, 68%, X ) Br, 97%). (f) CO, PPh3, Pd(OAc)2, KOAc, methanol, DMF, 100 °C, 99%. (g) LiOH, H2O, THF, methanol, reflux, 62%. (h) LiAlH4, THF,
0 °C, 70%. (i) SOCl2, CH2Cl2, room temperature, 100%. (j) HNR1R2, THF, 0 °C, 8-79%. (k) Morpholine, HOBT, dicyclohexanecarbodiimide, CH2Cl2,
room temperature, 91%. (l) SOCl2, dioxane, room temperature, 100%. (m) Bu4NCN, CH2Cl2, reflux, 63%. (n) KOH, t-BuOH, 100 °C, 17%.
spacer, subsequent analogues in this series contained an aromatic
ring directly attached to 5-position of the thiazolone ring.
Tertiary and secondary amide analogues, 12b-12d and 13, were
all less potent (Ki values ) 76-330 nM) than the primary amide
analogue 12a, but the pyrrolidine amide analogue 12e was
slightly more potent than 12a. Four other primary amide
analogues were also prepared. Analogue 15, containing a
methylene spacer between the amide and the aryl ring, was ∼2-
fold less potent than amide analogue 12a. An analogue
containing geminal methyl groups at the benzylic position
(analog 23) and an analogue with a two-carbon spacer (30) were
also less potent than primary amide 12a. However, the analogue
containing a cyclopropyl ring at the benzylic position, compound
20, was slightly more potent than the primary amide 12a (cf.
Ki ) 30 vs 44 nM). Analogues containing alcohols were also
tested in this series of compounds. As observed in the SAR of
the primary amides, the alcohol containing geminal methyl
groups at the benzylic position (compound 26) and the alcohol
with a two-carbon spacer (compound 29) were less potent that
analogue containing the primary alcohol (compound 14, Ki )
46 nM).
The most potent compounds from Table 1 were then tested
in the PXR reporter gene assay. Analogue 8 had PXR activity
of ca. 65% of control when tested at both 2 and 20 µM. At a
test concentration of 2 µM, however, analogues with polar
groups off the right-hand phenyl ring, compounds 12a,e, 20,
and 14, showed little activation of PXR (Table 2). Raising the
test concentration to 20 µM, pyrrolidine amide 12e and
cyclopropyl analogue 20 activated PXR at 35 and 97% of
control, respectively. On the other hand, the primary amide 12a
and alcohol 14, at a test concentration of 20 µM, activated PXR
by only 9.3 and 25%, respectively, which was significantly lower
than our lead compound 3 (PXR ) 45% of control). While these
results were encouraging, amide analogue 12a was found to be

7956 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24
Fotsch et al.
Scheme 4a
a Reagents and conditions: (a) Methyl 2-bromopropanoate, Et3N, ethanol, 100 °C, microwave, 88%. (b) Compound 16, LDA, THF, -78 to 0 °C, 87%.
(c) KOH, t-BuOH, 85-100 °C, 66-86%. (d) Compound 16, LiHMDS, Pd2(dba)3, BINAP, toluene, 95 °C, 26-37%. (e) t-BuOK, 18-crown-6, CH3I, THF,
-78 °C to room temperature, 99%. (f) CH3MgBr, THF, 0 °C to room temperature, 74%. (g) TMSCl, NEt(i-Pr)2, K2CO3, CH2Cl2, 0 °C to room temperature,
84%. (h) K2CO3, methanol, room temperature, 93%. (i) Imidazole, iodine, PPh3, CH2Cl2, room temperature, 82%. (j) Bu4NCN, CH2Cl2, reflux, 65%.
Scheme 5a
a Reagents and conditions: (a) 2-Bromopropanoic acid, NaOAc, ethanol, reflux, 88-100%. (b) LDA, THF, -78 °C. (c) R1R2CdO, THF, -78-0 °C,
17-100%. (d) Lithium bis(trimethylsilyl)amide, THF, 0 °C. (e) 2-Methyloxirane, LiClO4, THF, 0 °C to room temperature, 71%. (f) Dess-Martin periodinane,
CH2Cl2, 83%. (g) CH3MgBr, THF, 0 °C, 61%.
an efflux substrate in CACO-2 cells (efflux ratio ) 10).
Therefore, we focused on expanding the SAR of derivatives
containing alcohols to maintain low PXR activation.
In the next set of analogues, we added hydroxyl groups to
aliphatic chains on the C-5 position (Table 3). An analogue
containing the 2-methyl-propan-2-ol side chain, compound 35,

11ꢀ-Hydroxysteroid Dehydrogenase Type 1 Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24 7957
Table 2. Transactivation of PXRa
Table 1. SAR of Thiazolones Substituted with Polar Aryl Rings
a PXR luciferase reporter gene assay. b Data were expressed as a percent
of control (POC) where 100% of control is equivalent to the activity
achieved with 12.5 µM rifampin. Compounds were run in triplicate and
have been reported with the SEM.
containing groups larger than a fluorine atom at the para-
position, analogues 33d-f, or the analogue without a substituent
on the phenyl ring, analogue 33h, were less potent than analogue
33a. Moving the fluoro to the meta-position gave analogue 33i,
which was ∼3-fold less potent than para-fluoro analogue 33a.
The ortho-fluoro analogue 33j, on the other hand, was 2-fold
more potent than 33a. Other analogues with electron-withdraw-
ing groups at the ortho-position, for example, 33k and 33l, were
also more potent than para-fluoro analogue 33a. However, all
three of these ortho-substituted analogues, 33j-l, were also
more potent in the PXR assay than the para-fluoro substituted
analogue 33a. From this last set of results, PXR transactivation
seems to be affected by subtle changes made to the phenyl ring
on 33. For example, the slight change of moving the fluoro from
the para-position to the ortho-position led to a statistically
significant increase in PXR transactivation (cf. the PXR values
for 33a and 33j). Even more surprising is the PXR activity of
the unsubstituted analogue 33h, which had a PXR activity nearly
7-fold higher than the para-fluoro analogue 33a. Because the
goal of this work was to mitigate PXR transactivation, we only
measured PXR functional activity and not PXR binding. We
cannot, therefore, exclude the possibility that 33a may bind to
PXR and not activate the receptor.30 Nevertheless, compound
33a seemed to strike the right balance between 11ꢀ-HSD1
potency and low PXR activity, so we subjected compound 33a
to further in vitro and in vivo testing.
a Ki was derived from a SPA using human 11ꢀ-HSD1 expressed in E.
3
coli cells and H-cortisone as the substrate. b IC50 was determined from a
whole cell assay using CHO cells overexpressing human 11ꢀ-HSD1. c SEM
for at least two independent determinations. d n ) 1.
was 6-fold less potent than the benzyl alcohol analogue 14 but
had similar PXR activity. Shortening the side chain further gave
tertiary alcohol 33a, which was nearly as potent as benzyl
alcohol analogue 14 in the biochemical assay, but showed a
significant decrease in PXR transactivation [10% (not signifi-
cant) of control at a test concentration of 20 µM]. Encouraged
by the lower PXR activity, more analogues were prepared that
contained the tertiary alcohol directly attached to the C-5
position of the thiazolone. The biochemical and cell-based
potency of such analogues improved with tertiary alcohol
analogues containing the cyclobutyl and cylcopentyl groups
(analogues 33b and 33c). However, in both cases, PXR activity
increased (26 and 61% of control). Therefore, we kept the
dimethyl tertiary alcohol in place and tried to improve potency
by modifying the left-hand side of the molecule. Analogues
Before running additional studies with compound 33a, the
two diastereomers were separated using supercritical fluid
chromatography. The C-5 (S)-isomer,31 compound (S)-33a, had
a Ki and cell IC50 of ∼35 nM, while the corresponding (R)-
isomer was 6-8-fold less potent (Table 4). Compound (S)-33a
was cocrystallized with human 11ꢀ-HSD1 (Figure 1) and
occupied the catalytic binding pocket of the enzyme. As we
observed with a related thiazolone inhibitor,32 the OH of Tyr183

7958 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24
Table 3. SAR of Thiazolones Substituted with Aliphatic Alcohols
Fotsch et al.
Figure 1. X-ray structure of human 11ꢀ-HSD1-(S)-33a complex.
presence of the para-fluoro substituent.33 The fluorophenyl ring
also formed hydrophobic contacts with the side chain of Leu126
and other nearby residues. The “rearward” methyl group at the
chiral C-5 position of (S)-33a formed a van der Waals contact
with the backbone nitrogen atom of Leu217. The proximity of
the C-5 substituent to Leu217 may explain why (R)-33a is less
potent than (S)-33a in the human 11ꢀ-HSD1 assay, since the
bulkier tertiary alcohol group at the C-5 of (R)-33a would suffer
a steric clash with Leu217. The tertiary alcohol group of (S)-
33a formed a hydrophobic contact with the side chain of Tyr177,
but the hydroxyl group did not appear to form a direct H-bond
contact with the protein. Instead, the hydroxyl may have
interacted with solvent molecules. From prior crystallographic
studies, water molecules have been observed in the 11ꢀ-HSD1
active site.22,32
In the PXR assay, the POCs at 20 µM of compound (S)-33a
and its diastereomer (R)-33a were 6 and 11, respectively. The
result for (S)-33a represented a greater than 7-fold improvement
over the PXR value obtained for compound 3. To determine
how PXR activation correlated with induction of CYP3A4, we
measured the mRNA expression and enzyme activity of
CYP3A4 in cryopreserved human hepatocytes.34-36 Using
hepatocytes from two different donors, compound 3 at a test
concentration of 20 µM showed an increase in both CYP3A4
mRNA and enzyme activity (67-110 and 56-64% of control,
respectively). Compound (S)-33a, on the other hand, only
showed a modest increase in CYP3A4 mRNA expression and
enzyme activity (13-45 and 10-17% of control, respectively).
The CYP3A4 activities for compounds 3 and (S)-33a are
consistent with the results from the PXR reporter gene assay;
that is, increased PXR transactivation led to higher levels of
CYP3A4 mRNA expression and enzyme activity.
a Ki was derived from a SPA using human 11ꢀ-HSD1 expressed in E.
3
coli cells and H-cortisone as the substrate. b IC50 was determined from a
whole cell assay using CHO cells overexpressing human 11ꢀ-HSD1. c SEM
for at least two independent determinations. d n ) 1. e PXR luciferase
reporter gene assay. f Data were expressed as a POC where 100% of control
is equivalent to the activity achieved with 12.5 µM rifampin. Compounds
were run in triplicate and have been reported with the SEM. g Compounds
33a-c and 33j-l had IC50 values > 10000 nM in the human 11ꢀ-HSD2
assay.
Table 4
hu11ꢀ-HSD1 activity
SPAa
cellb
#
Ki ( SEMc
IC50 ( SEMc
(R)-33a
(S)-33a
210 ( 38
35 ( 1
300 ( 80
34 ( 3
Encouraged by the PXR results, we next assessed (S)-33a
for nonhuman 11ꢀ-HSD1 activity and in vivo pharmacokinetics
(PK) to determine which species might be most appropriate for
in vivo assessment (Table 6). In mouse, rat, dog, and monkey,
the in vivo pharmacokinetics showed that compound (S)-33a
had moderate to low clearance and oral bioavailabilities between
56 and 100%. In the 11ꢀ-HSD1 biochemical and cell-based
assays, (S)-33a was less potent in the rodent assays (Ki ) 100
and 300 nM for mouse and rat, respectively) than in the
nonrodent assays (Ki ) 12 and 48 nM for dog and monkey,
respectively). Because compound (S)-33a was less potent in the
rat assay than in the monkey assay, we chose to run the in vivo
a Ki was derived from a SPA using the human 11ꢀ-HSD1 expressed in
E. coli cells and 3H-cortisone as the substrate. b IC50 was determined from
a whole cell assay using CHO cells overexpressing the human 11ꢀ-HSD1.
c SEM for at least two independent determinations.
formed a hydrogen bond with the C-2 NH of (S)-33a, and the
backbone NH of Ala172 formed a hydrogen bond with the
carbonyl group of thiazolone (S)-33a. The Ser170 OH formed
a hydrogen bond with the side chain oxygen of Tyr183, which
was also observed in our previously reported structure.32 The
phenyl ring of (S)-33a formed an edge-to-face π-stacking
interaction with Tyr183, which was likely enhanced by the

11ꢀ-Hydroxysteroid Dehydrogenase Type 1 Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24 7959
Table 5. Human PXR Activity and CYP3A4 Activity in Cryopreserved Hepatocytes
activity in cryopreserved human hepatocytes
CYP3A4 mRNA (% of control)b ( SD
CYP3A4 activityc (% of control)b ( SD
PXR activity (% of control) ( SEMa
donor DJV
donor NPV
donor DJV
donor NPV
rifampin
3
(S)-33a
100 ( 10
45 ( 5
6 ( 2
100 ( 9
110 ( 10
45 ( 4
100 ( 5
67 ( 4
13 ( 1
100 ( 2
64 ( 10
17 ( 1
100 ( 3
56 ( 5
10 ( 1
a PXR luciferase reporter gene assay. Data are expressed as a POC where 100% of control is equivalent to the activity achieved with 12.5 µM rifampin.
Compounds were run in triplicate and have been reported with the SEM. b Data are expressed as a POC where 100% of control is equivalent to the activity
achieved with 20 µM rifampin. Compounds were run twice and have been reported with the SD. c Enzyme activity was determined by measuring the
conversion of midazolam to 1′-hydroxy midazolam. For details, see the Experimental Section.
Table 6. Potency and Pharmacokinetics of Compound (S)-33a in Four Different Species
11ꢀ-HSD1 activity
in vivo pharmacokineticsd
SPAa Ki ( SEMc
cellb IC50 ( SEMc
CL (L h-1kg-1
)
Vss (L kg-1
)
t1/2 (i.v., h)
AUCoral (ng h mL-1
)
Foral (%)
e
species
mouse
rat
dog
100 ( 18
300 ( 70
12 ( 2
140 ( 60
340 ( 80
45 ( 22
42 ( 6
1.6
0.31
0.085
1.2
1.4
2.5
0.88
1.9
3.3
5.6
7.3
2.2
6500
9200
54000
5700
100
56
91
monkey
48 ( 13
65
a Ki was derived from a SPA using the species 11ꢀ-HSD1 expressed in E. coli cells and 3H-cortisone as the substrate. b IC50 was determined from a whole
cell assay using CHO cells overexpressing the species 11ꢀ-HSD1. c SEM for at least two independent determinations. d Mouse dosed i.v., 2 mg kg-1 in 10%
dimethylacetamide, 23% water, 67% PEG 400; p.o. 10 mg kg-1 in 0.1% Tween 80, 0.5% CMC, 99.4% water. Rat dosed i.v., 2 mg kg-1 in DMSO; dosed
p.o., 5 mg kg-1 in 0.1% Tween 80, 0.5% CMC, 99.4% water. Dog dosed i.v., 2 mg kg-1 in 10% dimethylacetamide, 23% water, 67% PEG 400; dosed p.o.,
5 mg kg-1 in 1% Tween 80, 47.5% OraPlus, 51.5% water. Monkey dosed i.v., 2 mg kg-1 in 10% dimethylacetamide, 40% water, 50% PEG 400; dosed p.o.,
10 mg kg-1 in 10% dimethylacetamide, 40% water, 50% PEG 400. e AUCoral ) oral area under the curve (AUC) for time 0-∞.
In summary, while our strategy to lower PXR activity by
adding polar groups worked for some analogues, in other cases,
the functionality on other parts of the inhibitors also affected
PXR activity. Reducing steric bulk on the right-hand side of
the molecule often helped reduce PXR activity. In addition, the
substituents on the left-hand aromatic ring also influenced the
PXR activity. The right balance of low PXR activity, biochemi-
cal potency, and cell-based activity was achieved with a
compound that had a 4-fluorophenyl group on the left-hand side
of the molecule and a dimethyl tertiary alcohol group at the
C-5 position (compound 33a). The C-5 (S)-enantiomer of this
compound had biochemical and cell-based hu11ꢀ-HSD1 activity
of ∼35 nM. In cryopreserved human hepatocytes, the CYP3A4
Figure 2. Inhibition of 11ꢀ-HSD1 activity with (S)-33a (dosed PO)
mRNA and enzyme activity for (S)-33a was significantly lower
in the mesenteric fat of cynomolgus monkeys. *p ) 0.0048, **p )
than the lead compound 3. The X-ray cocrystal structure of (S)-
0.0007, and n g 4. Statistical analyses were carried out using a two-
33a with human 11ꢀ-HSD1 indicated that the compound formed
tailed Student’s t-test.
hydrogen bonds and hydrophobic interactions with the amino
pharmacodynamic assay in monkeys. In addition, performing
such a study in monkeys would lay the groundwork for
additional studies in diabetic models with nonhuman primates.
To measure the pharmacodynamic effects of (S)-33a in
cynomolgus monkeys, we measured the activity of 11ꢀ-HSD1
in a fat tissue explant 2 h after the compound was given orally
to the animals. The enzyme activity was measured by incubating
acid residues in the catalytic pocket of the enzyme. Compound
(S)-33a also had good pharmacokinetics across four different
species. In a monkey pharmacodynamic model, compound (S)-
33a showed significant inhibition of 11ꢀ-HSD1 activity in the
mesenteric fat of monkeys at both 3 and 30 mg kg-1. With
these results, compound (S)-33a may be used to further
understand the biological effects of inhibiting 11ꢀ-HSD1 in
nonhuman primates.
3
the mesenteric fat with H-cortisone and monitoring for the
conversion to 3H-cortisol. At the same time, blood was collected
from the animals so that the plasma concentrations of (S)-33a
could be determined. Statistically significant inhibition of 11ꢀ-
HSD1 activity was observed at 3 and 30 mg kg-1 (Figure 2).
Plasma concentrations of (S)-33a at 3 and 30 mg kg-1 were
4.9 and 26 µM, respectively. Running a linear regression
analysis on the data from the plasma concentrations vs the
percent enzyme inhibition gave a plasma IC50 ) 910 nM.
Adjusting for plasma protein binding (monkey funbound ) 10.8%),
the unbound IC50 was calculated as 98 nM, which was slightly
greater than the monkey cell IC50 (42 nM). These data indicate
that, when given orally, compound (S)-33a is able to penetrate
the fat and inhibit 11ꢀ-HSD1 in the target tissue at plasma
concentrations that are near the cellular IC50, when corrected
for fraction unbound.
Experimental Section
General. Unless otherwise noted, all materials were obtained
from commercial sources and used without further purification.
Preparative flash chromatography was carried out on Merck silica
gel 60 (230-400 mesh) or prepacked silica gel cartridges (Biotage
or Isco). 1H NMR spectra were recorded on Bruker 400 MHz
instrument. All spectra were recorded using the residual solvent
proton resonance or tetramethylsilane (TMS) as an internal standard.
Chemical shifts are reported in parts per million (ppm, δ units).
1
For products containing a thiazolone, the H NMR spectra were
complex due to the presence of two diastereomers and the two
tautomeric forms of the thiazolone. In many instances, the 1H NMR
spectra could be simplified by adding one drop of trifluoroacetic
acid, which shifted the equilibrium of thiazolone tautomers to one
form. In instances where the compound was a mixture of diaster-

7960 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24
Fotsch et al.
eomers, a pair of signals was observed for many of the protons in
the spectra. The chemical shifts for the other diastereomer are
reported parenthetically. Mass spectrometry (MS) was performed
using an electrospray Agilent 1100 Series liquid chromatograph/
mass selective detector (MSD) to give the molecular [M + H]+
ion of the target molecules. The purity of final compounds was
determined by either elemental analysis (performed by Atlantic
Microlab, Inc., Norcross, GA) or by analytical reverse-phase high-
performance liquid chromatography (HPLC) using two separate
signals (215 and 254 nm) on two separate instruments (see the
Supporting Information for details).
(2.04) (s, 3H), 1.77 (1.75) (d, 3H, J ) 6 Hz). MS (ESI, pos. ion)
m/z: 347 (M + H). Anal. (C18H16F2N2OS) C, H, N.
Ethyl 2-Bromo-2-(4-bromophenyl)acetate (7, X ) Br). Accord-
ing to the procedure described for 7, X ) F, the title compound
was prepared from ethyl 2-(4-bromophenyl)acetate (6, X ) Br)
(3.0 g, 12 mmol), N-bromosuccinimide (2.4 g, 14 mmol), and AIBN
(0.20 g, 1.2 mmol). After purification by silica gel chromatography
(7% EtOAc in hexane), the desired product was isolated as a
colorless oil (3.8 g, 96%). 1H NMR (CDCl3, 400 MHz): δ 7.50 (d,
2H, J ) 8 Hz), 7.43 (d, 2H, J ) 8 Hz), 5.28 (s, 1H), 4.24 (m, 2H),
1.28 (m, 3H).
(S)-1-[1-(4-Fluorophenyl)ethyl]thiourea (5). A solution of (S)-
1-(4-fluorophenyl)ethanamine (4) (5.0 g, 36 mmol) and benzoyl
isothiocyanate (4.8 mL, 36 mmol) in CH2Cl2 was stirred at ambient
temperature in a round-bottomed flask overnight. The solvent was
removed in vacuo, and the crude product was dissolved in THF/
MeOH/H2O (30, 30, and 30 mL, respectively). K2CO3 (20 g, 140
mmol) was added, and the mixture was stirred at ambient
temperature in a round-bottomed flask. After 4 h, the solvent was
removed under reduced pressure, and the crude reaction mixture
was diluted with water (60 mL) and extracted with EtOAc (3 ×
100 mL). The combined organic layers were washed with brine,
filtered, and concentrated in vacuo. Purification by flash column
chromatography (silica gel, 0-5% MeOH in CH2Cl2) afforded the
5-(4-Bromophenyl)-2-[(S)-1-(4-fluorophenyl)ethylamino]thiazol-
4(5H)-one. According to the procedure described for 5-(4-fluo-
rophenyl)-2-[(S)-1-(4-fluorophenyl)ethylamino]thiazol-4(5H)-one,
the title compound was prepared from (S)-1-[1-(4-fluorophenyl)-
ethyl]thiourea (5) (1.0 g, 5.0 mmol), ethyl 2-bromo-2-(4-bromophe-
nyl)acetate (7, X ) Br) (2.0 g, 6.1 mmol), and di-iso-propylethy-
lamine (1.05 mL, 6.05 mmol). After purification (silica gel
chromatography: 34% EtOAc in hexane and then 40% EtOAc in
hexane), the desired product was isolated as a white solid (1.4 g,
71%). 1H NMR (CDCl3, 400 MHz): δ 7.53-7.34 (m, 4H),
7.24-6.99 (m, 4H), 5.17 (5.09) (s, 1H), 4.62 (q, 1H, J ) 8 Hz),
1.80 (1.77) (d, 3H, J ) 8 Hz). MS (ESI, pos. ion) m/z: 393, 395
(M + H).
1
title compound 5 (7 g, 98%) as a white solid. H NMR (CDCl3,
5-(4-Bromophenyl)-2-[(S)-1-(4-fluorophenyl)ethylamino]-5-me-
thylthiazol-4(5H)-one (9). According to the procedure described for
8, the title compound was prepared from 5-(4-bromophenyl)-2-[(S)-
1-(4-fluorophenyl)ethylamino]thiazol-4(5H)-one (1.3 g, 3.2 mmol),
LDA (9.6 mL of a 2.0 M solution in heptane/THF/ethylbenzene,
19 mmol), and iodomethane (1.2 mL, 19 mmol). After purification
(silica gel chromatography, 1.5% MeOH in CH2Cl2, and then 3%
MeOH in CH2Cl2), the product was isolated as a light yellow solid
(1.27 g, 97%). 1H NMR (CDCl3, 400 MHz): δ 7.50-7.35 (m, 5H),
7.19 (d, 1H, J ) 8 Hz), 7.10-6.96 (m, 3H), 4.57 (m, 1H), 2.06
(1.98) (s, 3H), 1.78 (1.76) (d, 3H, J ) 8 Hz). MS (ESI, pos. ion)
m/z: 407, 409 (M + H).
Methyl 4-{2-[(S)-1-(4-fluorophenyl)ethylamino]-5-methyl-4-oxo-
4,5-dihydrothiazol-5-yl}benzoate (10). A round-bottomed flask with
a mixture of 5-(4-bromophenyl)-2-[(S)-1-(4-fluorophenyl)ethy-
lamino]-5-methylthiazol-4(5H)-one (9) (0.79 g, 1.9 mmol), triph-
enylphosphine (0.15 g, 0.58 mmol), and palladium acetate (0.11 g,
0.48 mmol) in MeOH/DMF (3 mL/3 mL) was pressurized with
CO(g), purged twice with CO(g), and then heated to 100 °C
overnight at 40-50 psi of CO(g). The reaction mixture was cooled
to room temperature, and the mixture was filtered through Celite.
The Celite pad was washed with CH2Cl2, and the filtrate was washed
with water (3×). The organic phase was dried over Na2SO4, filtered,
and concentrated in vacuo. Purification by flash column chroma-
tography (silica gel, 2-3.6% MeOH in CH2Cl2) afforded the title
compound as a light yellow solid (0.74 g, 99%). 1H NMR (CDCl3,
400 MHz): δ 11.42 (br s, 1H), 7.94-8.04 (m, 2H), 7.39-7.67 (m,
4H), 6.63-7.01 (m, 2H), 4.56 (q, 1H, J ) 8 Hz), 3.92 (s, 3H),
2.11 (2.02) (s, 3H), 1.82 (1.80) (d, 3H, J ) 8 Hz). MS (ESI, pos.
ion) m/z: 387 (M + H).
400 MHz): δ 7.33-7.20 (m, 2H), 7.10-7.05 (m, 2H), 6.48 (br s,
1H), 5.60 (br s, 2H), 4.56 (m, 1H), 1.52 (d, 3H, J ) 4 Hz). MS
(ESI, pos. ion) m/z: 199 (M + H).
2-Bromo-2-(4-fluorophenyl)acetic Acid (7, X ) F). To a round-
bottomed flask containing a solution of 2-(4-fluorophenyl)acetic acid
(6, X ) F) (2.0 g, 13 mmol) in CCl4 (30 mL) were added
N-bromosuccinimide (2.5 g, 14 mmol) and AIBN (0.32 g, 2.0
mmol). The mixture was gradually heated to reflux, and after 2.5 h,
the heating was stopped and the mixture was stirred overnight at
room temperature. The liquid was filtered and concentrated in
vacuo. Purification by flash column chromatography (silica gel,
20-50% EtOAc in hexane) afforded the title compound as yellow
1
oil (2.6 g, 86%). H NMR (CDCl3, 400 MHz): δ 7.56 (dd, 2H, J
) 4, 8 Hz), 7.08 (t, 2H, J ) 8 Hz), 5.35 (s, 1H). MS (ESI, pos.
ion) m/z: 231, 233 (M + H).
5-(4-Fluorophenyl)-2-[(S)-1-(4-fluorophenyl)ethylamino]thiazol-
4(5H)-one. To a round-bottomed flask with (S)-1-[1-(4-fluorophe-
nyl)ethyl]thiourea (5) (0.12 g, 0.62 mmol) and 2-bromo-2-(4-
fluorophenyl)acetic acid (7, X ) F) (0.16 g, 0.68 mmol) in ethanol
(2 mL) was added sodium acetate (0.10 g, 1.2 mmol), and the
mixture was heated to reflux overnight. A 10% solution of Na2CO3
was then added to the reaction mixture, and the mixture was
extracted with EtOAc. The organic layer was dried over Na2SO4,
filtered, and concentrated in vacuo. Purification by flash column
chromatography (silica gel, 0-5% MeOH in CH2Cl2) afforded the
1
title compound as a white solid (0.10 g, 48%). H NMR (CDCl3,
400 MHz): δ 7.42-7.48 (m, 2H), 7.32-7.36 (m, 1H), 7.18-7.22
(m, 1H), 6.96-7.09 (m, 4H), 5.20 (tautomer 5.12) (s, 1H), 4.63
(q, 1H, J ) 8 Hz), 1.83 (tautomer 1.80) (d, 3H, J ) 8 Hz). MS
(ESI, pos. ion) m/z: 333 (M + H).
4-{2-[(S)-1-(4-Fluorophenyl)ethylamino]-5-methyl-4-oxo-4,5-di-
hydrothiazol-5-yl}benzoic Acid (11). In a round-bottomed flask was
added a mixture of methyl 4-{2-[(S)-1-(4-fluorophenyl)ethylamino]-
5-methyl-4-oxo-4,5-dihydrothiazol-5-yl}benzoate (10) (1.2 g, 3.1
mmol), THF (6 mL), MeOH (2 mL), H2O (2 mL), and lithium
hydroxide hydrate (0.26 g, 6.2 mmol). The mixture was heated to
reflux for 2 h, then neutralized with 1 N HCl, and extracted with
CH2Cl2 (5×). The organic solution was dried over Na2SO4, filtered,
and concentrated in vacuo. Purification by flash column chroma-
tography (silica gel, 3.6-10% MeOH in CH2Cl2) afforded the title
compound as a light brown solid (0.72 g, 62%). 1H NMR (DMSO-
d6, 400 MHz): δ 9.85 (d, 1H, J ) 8 Hz), 7.83-7.93 (m, 2H),
7.40-7.65 (m, 4H), 7.17-7.24 (m, 2H), 5.28 (qn, 1H, J ) 8 Hz),
1.90 (1.95) (s, 3H), 1.50 (d, 3H, J ) 8 Hz). MS (ESI, pos. ion)
m/z: 373 (M + H).
5-(4-Fluorophenyl)-2-[(S)-1-(4-fluorophenyl)ethylamino]-5-me-
thylthiazol-4(5H)-one (8). To a round-bottomed flask, cooled to -78
°C, containing a solution of 5-(4-fluorophenyl)-2-[(S)-1-(4-fluo-
rophenyl)ethylamino]thiazol-4(5H)-one (0.10 g, 0.30 mmol) in 2
mL of THF, was added LDA (0.90 mL of a 2.0 M solution in
heptane/THF/ethylbenzene, 1.8 mmol). After 1 h, iodomethane (0.11
mL, 1.8 mmol) was added, and the reaction mixture was gradually
warmed to -40 °C and quenched with 10 mL of saturated NH4Cl.
The aqueous phase was extracted with ethyl acetate (3×), and the
combined organic layers were dried over Na2SO4, filtered, and
concentrated in vacuo. Purification of the crude material by flash
column chromatography (silica gel, 0-5% MeOH-CH2Cl2) af-
forded the title compound as a white solid (71 mg, 68%). 1H NMR
(CDCl3 + one drop of TFA, 400 MHz): δ 7.39-7.42 (m, 1H),
7.27-7.33 (m, 3H), 7.02-7.14 (m, 4H), 4.54-4.61 (m, 1H), 2.14

11ꢀ-Hydroxysteroid Dehydrogenase Type 1 Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24 7961
4-{2-[(S)-1-(4-Fluorophenyl)ethylamino]-5-methyl-4-oxo-4,5-di-
hydrothiazol-5-yl}benzamide (12a). To a solution of 4-{2-[(S)-1-
(4-fluorophenyl)ethylamino]-5-methyl-4-oxo-4,5-dihydrothiazol-5-
yl}benzoic acid (11) (0.25 g, 0.67 mmol) in CH2Cl2 (2 mL) were
added thionyl chloride (0.10 mL, 1.3 mmol) and two drops of DMF.
After the reaction mixture was stirred overnight at room temper-
ature, the mixture was concentrated in vacuo. The crude product
was dissolved in THF (2.5 mL), and then, ammonium hydroxide
(37% solution in H2O, 0.6 mL) was added to the solution at 0 °C.
After 1 h, the mixture was diluted with water (5 mL) and extracted
with CH2Cl2 (3×). The organic phase was dried over Na2SO4,
filtered, and concentrated in vacuo. Purification by flash column
chromatography (silica gel, 4-10% MeOH in CH2Cl2) gave the
product as a white solid (0.20 g, 79%). Analytical data for single
MS (ESI, pos. ion) m/z: 426 (M + H). Anal. (C23H24FN3O2S ·
0.4H2O) C, H, N.
2-[(S)-1-(4-Fluorophenyl)ethylamino]-5-methyl-5-[4-(morpholine-
4-carbonyl)phenyl]thiazol-4(5H)-one (13). Into a round-bottomed
flask was added 4-{2-[(S)-1-(4-fluorophenyl)ethylamino]-5-methyl-
4-oxo-4,5-dihydrothiazol-5-yl}benzoic acid (89 mg, 0.24 mmol),
dicyclohexanecarbodiimide (74 mg, 0.36 mmol), 1-hydroxybenzo-
triazole (29 mg, 0.22 mmol), and morpholine (0.042 mL, 0.48
mmol) in CH2Cl2 (2 mL). After the mixture was stirred overnight
at room temperature, the reaction mixture was diluted with 10%
Na2CO3 (15 mL) and extracted with CH2Cl2 (3×). The organic
phase was dried over Na2SO4, filtered, and concentrated in vacuo.
After purification (preparative TLC, 10% MeOH in CH2Cl2), the
1
desired product was isolated as a white solid (97 mg, 91%). H
1
diastereomer: H NMR (CDCl3 + 1 drop of TFA, 400 MHz): δ
NMR (CDCl3 + 1 drop of TFA, 400 MHz): δ 7.53-7.30 (m, 6H),
7.15-7.08 (m, 2H), 4.64-4.57 (m, 1H), 3.87 (m, 4H), 3.69 (m,
2H), 3.46 (m, 2H), 2.18 (2.08) (s, 3H), 1.78 (1.76) (d, 3H, J ) 8
Hz). MS (ESI, pos. ion) m/z: 442 (M + H). Anal. (C23H24FN3O3S)
C, H, N.
7.87 (br s, 1H), 7.82 (d, 2H, J ) 8 Hz), 7.45 (d, 2H, J ) 8 Hz),
7.30-7.33 (m, 2H), 7.08-7.12 (m, 2H), 6.51 (br s, 1H), 4.62 (q,
1H, J ) 8 Hz), 2.19 (s, 3H), 1.78 (d, 3H, J ) 8 Hz). MS (ESI, pos.
ion) m/z: 372 (M + H). Anal. (C19H18FN3O2S·0.3H2O) C, H, N.
N-Cyclopropyl-4-{2-[(S)-1-(4-fluorophenyl)ethylamino]-5-meth-
yl-4-oxo-4,5-dihydrothiazol-5-yl}benzamide (12b). The title com-
pound was prepared according to the procedure of example 12a
using 4-{2-[(S)-1-(4-fluorophenyl)ethylamino]-5-methyl-4-oxo-4,5-
dihydrothiazol-5-yl}benzoyl chloride (0.080 g, 0.20 mmol) and
cyclopropylamine (0.20 mL, 2.9 mmol). After purification (prepara-
tive TLC, 5% MeOH in CH2Cl2), the product was obtained as a
white solid (6.7 mg, 8%). 1H NMR (CDCl3, 400 MHz): δ
7.72-7.63 (m, 2H), 7.51-7.34 (m, 4H), 7.06-6.90 (m, 2H), 6.49
(6.63) (br s, 1H), 4.56 (q, 1H, J ) 8 Hz), 2.88 (m, 1H), 2.09 (2.00)
(s, 3H), 1.79 (1.77) (d, 3H, J ) 8 Hz), 0.89-0.80 (m, 2H), 0.61
(m, 2H). MS (ESI, pos. ion) m/z: 412 (M + H). HPLC purity at
215 and 254 nm: 98 and 100% in system C; 96 and 99% in system
A.
2-[(S)-1-(4-Fluorophenyl)ethylamino]-5-[4-(hydroxymethyl)phe-
nyl]-5-methylthiazol-4(5H)-one (14). Into a round-bottomed flask
was added methyl 4-{2-[(S)-1-(4-fluorophenyl)ethylamino]-5-meth-
yl-4-oxo-4,5-dihydrothiazol-5-yl}benzoate (23 mg, 0.060 mmol) in
THF (1 mL) and lithium aluminum hydride (1.0 M solution in THF,
0.089 mL, 0.089 mmol) at 0 °C. The reaction was quenched by
adding sodium sulfate decahydrate. The solid was filtered and
washed with CH2Cl2 (3×). The filtrate was dried over Na2SO4,
filtered, and concentrated in vacuo. After purification (preparative
TLC 8% MeOH in CH2Cl2), the desired product was isolated as a
white solid (15 mg, 70%). 1H NMR (CDCl3, 400 MHz): δ
7.47-7.31 (m, 6H), 7.00-6.91 (m, 2H), 4.67 (m, 2H), 4.55 (m,
1H), 2.09 (2.00) (s, 3H), 1.81 (1.79) (d, 3H, J ) 8 Hz). MS (ESI,
pos. ion) m/z: 359 (M + H). HPLC purity at 215 and 254 nm: 99
and 100% in system C; 98 and 96% in system A.
N-Cyclopentyl-4-{2-[(S)-1-(4-fluorophenyl)ethylamino]-5-methyl-
4-oxo-4,5-dihydrothiazol-5-yl}benzamide (12c). The title compound
was prepared according to the procedure described for example
12a using 4-{2-[(S)-1-(4-fluorophenyl)ethylamino]-5-methyl-4-oxo-
4,5-dihydrothiazol-5-yl}benzoyl chloride (0.10 g, 0.30 mmol) and
cyclopentylamine (0.2 mL, 2.0 mmol). After purification (prepara-
tive TLC, 5% MeOH in CH2Cl2, and then 50% EtOAc in hexane),
5-[4-(Chloromethyl)phenyl]-2-[(S)-1-(4-fluorophenyl)ethylamino]-
5-methylthiazol-4(5H)-one. Into a round-bottomed flask was added
2-[(S)-1-(4-fluorophenyl)ethylamino]-5-[4-(hydroxymethyl)phenyl]-
5-methylthiazol-4(5H)-one (0.17 g, 0.47 mmol) in 1,4-dioxane (5
mL) and thionyl chloride (0.14 mL, 1.9 mmol). After 45 min of
stirring at room temperature, the reaction mixture was concentrated
in vacuo. The white solid was used in the next step without
purification. MS (ESI, pos. ion) m/z: 377 (M + H).
1
the product was isolated as a white solid (44 mg, 49%). H NMR
(CDCl3 + 1 drop of TFA, 400 MHz): δ 7.76 (7.69) (d, 2H, J ) 8
Hz), 7.51 (7.39) (d, 2H, J ) 8 Hz), 7.29-7.34 (m, 2H), 7.07-7.14
(m, 2H), 6.255 (6.21) (d, 1H, J ) 8 Hz), 4.55-4.62 (m, 1H),
4.36-4.43 (m, 1H), 2.16 (2.05) (s, 3H), 2.08-2.13 (m, 2H), 1.75
(1.77) (d, 3H, J ) 8 Hz), 1.46-1.73 (m, 6H). MS (ESI, pos. ion)
m/z: 440 (M + H). Anal. (C24H26FN3O2S·0.2H2O) C, H, N.
4-{2-[(S)-1-(4-Fluorophenyl)ethylamino]-5-methyl-4-oxo-4,5-di-
hydrothiazol-5-yl}-N-methylbenzamide (12d). The title compound
was prepared according to the procedure of example 12a using 4-{2-
[(S)-1-(4-fluorophenyl)ethylamino]-5-methyl-4-oxo-4,5-dihydrothia-
zol-5-yl}benzoyl chloride (0.10 g, 0.26 mmol) and methylamine
(1.0 mL of a 2 M solution in THF, 2.0 mmol). After purification
(preparative TLC, 5% MeOH in CH2Cl2), the product was obtained
2-(4-{2-[(S)-1-(4-Fluorophenyl)ethylamino]-5-methyl-4-oxo-4,5-
dihydrothiazol-5-yl}phenyl)acetonitrile (Intermediate B). Into a
round-bottomed flask was added a solution of 5-[4-(chlorometh-
yl)phenyl]-2-[(S)-1-(4-fluorophenyl)ethylamino]-5-methylthiazol-
4(5H)-one (0.18 g, 0.48 mmol) in CH2Cl2 (3.5 mL) and tetrabu-
tylammonium cyanide (0.26 g, 0.95 mmol). The reaction mixture
was heated to reflux for 4 h, and then, the mixture was concentrated
in vacuo. After purification by flash column chromatography (silica
gel, 30-50% EtOAc in hexane), the desired product was isolated
1
as a white solid (0.11 g, 63%). H NMR (CDCl3, 400 MHz): δ
11.33 (br s, 1H), 7.50-7.28 (m, 6H), 6.99-6.92 (m, 2H), 4.56 (m,
1H), 3.73 (m, 2H), 2.09 (2.00) (s, 3H), 1.82 (1.80) (d, 3H, J ) 8
Hz). MS (ESI, pos. ion) m/z: 368 (M + H).
1
as a white solid (10 mg, 10%). H NMR (CDCl3, 400 MHz): δ
7.75-7.66 (m, 2H), 7.53-7.36 (m, 4H), 7.09-6.92 (m, 2H), 6.63
(6.18) (m, 1H), 4.56 (q, 1H, J ) 8 Hz), 3.02-2.96 (m, 3H), 2.09
(2.05) (s, 3H), 1.79 (1.77) (d, 3H, J ) 8 Hz). MS (ESI, pos. ion)
m/z: 386 (M + H). HPLC purity at 215 and 254 nm: 97 and 100%
in system C; 98 and 99% in system A.
2-(4-{2-[(S)-1-(4-Fluorophenyl)ethylamino]-5-methyl-4-oxo-4,5-
dihydrothiazol-5-yl}phenyl)acetamide (15). Into a round-bottomed
flask were added 2-(4-{2-[(S)-1-(4-fluorophenyl)ethylamino]-5-
methyl-4-oxo-4,5-dihydrothiazol-5-yl}phenyl)acetonitrile (60 mg,
0.16 mmol), potassium hydroxide (0.12 g, 2.1 mmol), and t-BuOH
(2 mL). The mixture was gradually heated to 100 °C, and after
2 h, the reaction mixture was cooled to room temperature and
concentrated in vacuo. The residue was neutralized with 2 N HCl
until the pH was 5-6, and then, the aqueous layer was extracted
with CH2Cl2 (3×). The organic phase was dried over Na2SO4,
filtered, and concentrated in vacuo. After purification (preparative
TLC, 10% MeOH in CH2Cl2), the title compound was isolated as
a white solid (11 mg, 17%). 1H NMR (CD3OD, 400 MHz): δ
7.42-7.23 (m, 6H), 7.12-7.07 (m, 2H), 5.34 (q, 1H, J ) 8 Hz),
3.47-3.51 (m, 2H), 2.02-1.92 (m, 3H), 1.62-1.57 (m, 3H). MS
2-[(S)-1-(4-Fluorophenyl)ethylamino]-5-methyl-5-[4-(pyrrolidine-
1-carbonyl)phenyl]thiazol-4(5H)-one (12e). The title compound was
prepared according to the procedure of example 12a using 4-{2-
[(S)-1-(4-fluorophenyl)ethylamino]-5-methyl-4-oxo-4,5-dihydrothia-
zol-5-yl}benzoyl chloride (0.10 g, 0.26 mmol) and pyrrolidine
(0.063 mL, 0.77 mmol). After purification (preparative TLC, 5%
MeOH in CH2Cl2, and then 100% EtOAc), the product was obtained
1
as a white solid (67 mg, 61%). H NMR (CDCl3 + one drop of
TFA, 400 MHz): δ 7.58-7.30 (m, 6H), 7.15-7.08 (m, 2H),
4.64-4.57 (m, 1H), 3.73-3.67 (m, 2H), 3.46-3.40 (m, 2H), 2.17
(2.07) (s, 3H), 2.06-1.92 (m, 4H), 1.76 (1.78) (d, 3H, J ) 8 Hz).

7962 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24
Fotsch et al.
(ESI, pos. ion) m/z: 386 (M + H). HPLC purity at 215 and 254
nm: 98 and 99% in system C; 99 and 100% in system A.
0.79 mmol), 1-(4-bromophenyl)cyclopropanecarbonitrile (19) (0.35
g, 1.6 mmol), Pd2(dba)3 (51 mg, 0.055 mmol), BINAP (0.099 g,
0.16 mmol), and LiHMDS (0.33 g, 2.0 mmol) in toluene (8 mL).
The mixture was heated to 95 °C and stirred overnight. Saturated
NH4Cl (5 mL) was then added, and the mixture was extracted with
EtOAc (30 mL). The organic phase was dried over Na2SO4, filtered,
and concentrated in vacuo. After purification (preparative TLC, 50%
EtOAc in hexane), the desired product was isolated as a light yellow
2-[(S)-1-(4-Fluorophenyl)ethylamino]-5-methylthiazol-4(5H)-
one (16). A mixture of (S)-1-[1-(4-fluorophenyl)ethyl]thiourea (5)
(3.5 g, 18 mmol), methyl 2-bromopropanoate (2.2 mL, 19 mmol),
and triethylamine (2.7 mL, 19 mmol) in ethanol (10 mL) was heated
in a sealed vessel at 100 °C in a microwave reactor (Personal
Chemistry). After 1 h, the reaction vessel was cooled to room
temperature, and the solvent was removed in vacuo. The crude
mixture was diluted with water (100 mL) and extracted with EtOAc
(3 × 100 mL). The combined organic layers were washed with
brine, dried over Na2SO4, filtered, and concentrated in vacuo.
Purification by flash column chromatography (silica gel, 0-50%
EtOAc in hexane) afforded the title compound 16 (4.0 g, 88%) as
1
solid (0.12 g, 37%). H NMR (CD3OD, 400 MHz): δ 7.44-7.26
(m, 6H), 7.10 (7.08) (d, 2H, J ) 8 Hz), 5.33 (q, 1H, J ) 8 Hz),
2.02-1.91 (m, 3H), 1.73-1.67 (m, 2H), 1.61-1.56 (m, 3H),
1.49-1.43 (m, 2H). MS (ESI, pos. ion) m/z: 394 (M + H).
1-(4-{2-[(S)-1-(4-Fluorophenyl)ethylamino]-5-methyl-4-oxo-4,5-
dihydrothiazol-5-yl}phenyl)cyclopropanecarboxamide (20). The
title compound was prepared according to the procedure of example
18 using 1-(4-{2-[(S)-1-(4-fluorophenyl)ethylamino]-5-methyl-4-
oxo-4,5-dihydrothiazol-5-yl}phenyl)cyclopropanecarbonitrile (0.073
g, 0.19 mmol), KOH (0.13 g, 2.4 mmol), and t-BuOH (2 mL). After
purification (preparative TLC, 10% MeOH in CH2Cl2, then 50%
EtOAc in hexane), the title compound was isolated as a white solid
1
a white solid. H NMR (CDCl3 + 1 drop of TFA, 400 MHz): δ
7.36-7.32 (m, 2H), 7.13-7.08 (m, 2 H), 4.57 (4.566) (q, 1H, J )
8 Hz), 4.21 (4.13) (q, 1H, J ) 8 Hz), 1.76 (1.75) (d, 3H, J ) 8
Hz), 1.72 (1.66) (d, 3H, J ) 8 Hz)). MS (ESI, pos. ion) m/z: 253
(M + H).
2-[(S)-1-(4-Fluorophenyl)ethylamino]-5-methylthiazol-4(5H)-
one (16) (Alternative Procedure). Into a round-bottomed flask
equipped with stirring was added a mixture of (S)-1-[1-(4-
fluorophenyl)ethyl]thiourea (5) (60 g, 30 mmol), 2-bromopropanoic
acid (30 mL, 340 mmol), and sodium acetate (36 g, 440 mmol) in
ethanol (220 mL), which was heated overnight at reflux. The
reaction mixture was then cooled to room temperature, and then,
water (500 mL) and saturated Na2CO3 (50 mL) were added. The
mixture was extracted with ethyl acetate (3 × 500 mL), and the
combined organic layers were washed with saturated Na2CO3, water
(3 × 400 mL), and brine, dried over Na2SO4, filtered, and
concentrated in vacuo to give 70 g of the desired product (91%
yield).
1
(50 mg, 66%). H NMR (CDCl3, 400 MHz): δ 10.91 (br s, 1H),
7.47-7.31 (m, 6H), 7.03-6.97 (m, 2H), 5.57 (5.32) (m, 2H), 4.57
(m, 1H), 2.10 (2.00) (s, 3H), 1.79 (1.77) (d, 3H, J ) 8 Hz),
1.64-1.60 (m, 2H), 1.08-1.04 (m, 2H). MS (ESI, pos. ion) m/z:
412 (M + H). HPLC purity at 215 and 254 nm: 97 and 99% in
system C; 100 and 100% in system A.
2-(4-Bromophenyl)-2-methylpropanenitrile (22). Into a round-
bottomed flask was added 2-(4-bromophenyl)acetonitrile (21) (1.0
g, 5.1 mmol), 18-crown-6 (0.34 g, 1.3 mmol), iodomethane (0.70
mL, 11 mmol), and THF (50 mL). After the reaction flask was
cooled to -78 °C, potassium tert-butoxide (1.3 g, 11 mmol) was
added. The mixture was stirred at -78 °C for 2 h, then warmed to
room temperature, and stirred overnight. Saturated NH4Cl was then
added, and the reaction mixture was extracted with EtOAc (3×).
The combined organic phases were dried over Na2SO4, filtered,
and concentrated in vacuo. After purification by flash column
chromatography (silica gel, 0-50% EtOAc in hexane), the desired
product was isolated as an orange oil (1.1 g, 99%). 1H NMR
(CDCl3, 400 MHz): δ 7.52 (d, 2H, J ) 8 Hz), 7.35 (d, 2H, J ) 8
Hz), 1.71 (s, 6H).
2-(4-{2-[(S)-1-(4-Fluorophenyl)ethylamino]-5-methyl-4-oxo-4,5-
dihydrothiazol-5-yl}phenyl)-2-methylpropanamide (23). This com-
pound was prepared according to the procedure described for 20.
1H NMR (CDCl3 + 1 drop of TFA, 400 MHz): δ 7.46-7.29 (m,
6H), 7.15-7.09 (m, 2H), 4.62 (m, 1H), 2.17 (2.07) (s, 3H), 1.77
(1.75) (d, 3H, J ) 8 Hz), 1.63 (1.60) (s, 6H). MS (ESI, pos. ion)
m/z: 414 (M + H). Anal. (C22H24FN3O2S·0.3H2O) C, H, N.
2-(4-Bromophenyl)propan-2-ol (25). To a round-bottomed flask
containing a solution of methyl 4-bromobenzoate (24) (1.0 g, 4.7
mmol) in THF (30 mL) cooled to 0 °C was added methylmagnesium
bromide (4.7 mL of a 3.0 M solution in diethyl ether, 14 mmol).
After the addition, the reaction mixture was warmed to room
temperature and stirred for 2 h. Saturated NH4Cl (10 mL) was
added, and the reaction mixture was extracted with EtOAc (3×).
The combined organic phases were dried over Na2SO4, filtered,
and concentrated in vacuo. After purification by flash column
chromatography (silica gel, 9-14% EtOAc in hexane), the title
compound was isolated as a colorless oil (0.74 g, 74%). 1H NMR
(CDCl3, 400 MHz): δ 7.45 (d, 2H, J ) 8 Hz), 7.36 (d, 2H, J ) 8
Hz), 1.56 (s, 6H). MS (ESI, pos. ion) m/z: 197, 199 [(M + H) -
H2O].
4-({2-[(S)-1-(4-Fluorophenyl)ethylamino]-5-methyl-4-oxo-4,5-di-
hydrothiazol-5-yl}methyl)benzonitrile. Into a round-bottomed flask
was added a solution of 2-[(S)-1-(4-fluorophenyl)ethylamino]-5-
methylthiazol-4(5H)-one (16) (0.30 g, 1.2 mmol) in THF (3 mL),
and LDA (2.0 M solution in heptane/tetrahydrofuran/ethylbenzene,
2.4 mL, 4.8 mmol) was added at -78 °C. After 1 h, a solution of
4-(bromomethyl)benzonitrile (17) (0.70 g, 3.6 mmol) in THF (3
mL) was added at -78 °C. The mixture was gradually warmed to
0 °C, and then, saturated NH4Cl (4 mL) was added to quench the
reaction. The mixture was extracted with EtOAc (3×), and the
combined organic fractions were dried over Na2SO4, filtered, and
concentrated in vacuo. After purification by flash column chroma-
tography (silica gel, 17-50% EtOAc in hexane), the desired product
was isolated as a white solid (0.38 g, 87%). 1H NMR (CDCl3, 400
MHz): δ 7.48-7.62 (m, 1H), 7.27-7.39 (m, 3H), 7.00-7.20 (m,
4H), 4.44 (m, 1H), 2.32-3.36 (m, 1H), 2.94-3.10 (m, 1H),
1.61-1.74 (m, 6H). MS (ESI, pos. ion) m/z: 368 (M + H).
4-({2-[(S)-1-(4-Fluorophenyl)ethylamino]-5-methyl-4-oxo-4,5-di-
hydrothiazol-5-yl}methyl)benzamide (18). Into a round-bottomed
flask was added 4-({2-[(S)-1-(4-fluorophenyl)ethylamino]-5-methyl-
4-oxo-4,5-dihydrothiazol-5-yl}methyl)benzonitrile (70 mg, 0.19
mmol), potassium hydroxide (0.14 g, 2.5 mmol), and t-BuOH (2
mL). The mixture was gradually heated to 85 °C, and after 2 h, the
reaction was concentrated in vacuo. A 2 N solution of HCl was
added until the pH was 7-8. The mixture was then extracted with
CH2Cl2 (3×), and the organic phases were dried over Na2SO4,
filtered, and concentrated in vacuo. After purification by flash
column chromatography (silica gel, 0-10% MeOH in CH2Cl2), the
title compound was isolated as a white solid (54 mg, 74%). 1H
NMR (CDCl3 + 1 drop of TFA, 400 MHz): δ 7.83 (d, 1H, J ) 8
Hz), 7.77 (7.71) (br s, 1H), 7.61 (d, 1H, J ) 8 Hz), 7.33 (d, 1H, J
) 8 Hz), 7.23-7.26 (m, 1H), 7.04-7.11 (m, 4H), 6.64 (6.57) (br
s, 1H), 4.46 (q, 1H, J ) 8 Hz), 3.01-3.45 (m, 2H), 1.82 (1.73) (s,
3H), 1.66 (1.63) (d, 3H, J ) 8 Hz). MS (ESI, pos. ion) m/z: 386
(M + H). Anal. (C20H20FN3O2S·0.2H2O) C, H, N.
2-[(S)-1-(4-Fluorophenyl)ethylamino]-5-[4-(2-hydroxypropan-2-
yl)phenyl]-5-methylthiazol-4(5H)-one (26). Using the cross-coupling
procedure used for the preparation of intermediate A, the title
compound was prepared from 2-[(S)-1-(4-fluorophenyl)ethylamino]-
5-methylthiazol-4(5H)-one (16) (0.10 g, 0.40 mmol), 2-(4-bro-
mophenyl)propan-2-ol (25) (0.17 g, 0.79 mmol), Pd2(dba)3 (0.025
g, 0.028 mmol), (S)-BINAP (0.049 g, 0.079 mmol), and LiN(TMS)2
(0.33 g, 2.0 mmol). After purification (preparative TLC, 50% EtOAc
in hexane), the desired product was isolated as an off-white solid
1-(4-{2-[(S)-1-(4-Fluorophenyl)ethylamino]-5-methyl-4-oxo-4,5-
dihydrothiazol-5-yl}phenyl)cyclopropanecarbonitrile (Intermedi-
ate A). To a round-bottomed flask was added 2-[(S)-1-(4-
fluorophenyl)ethylamino]-5-methylthiazol-4(5H)-one (16) (0.20 g,
1
(45 mg, 30%). H NMR (CDCl3 + 1 drop of TFA, 400 MHz): δ

11ꢀ-Hydroxysteroid Dehydrogenase Type 1 Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24 7963
7.53-7.28 (m, 6H), 7.14-7.08 (m, 2H), 4.60 (m, 1H), 2.16 (2.06)
(s, 3H), 1.77 (1.75) (d, 3H, J ) 8 Hz), 1.63 (1.59) (s, 6H). MS
(ESI, pos. ion) m/z: 387 (M + H). Anal. (C21H23FN2O2S·H2O) C,
H, N.
3-(4-{2-[(S)-1-(4-Fluorophenyl)ethylamino]-5-methyl-4-oxo-4,5-
dihydrothiazol-5-yl}phenyl)propanamide (30). According to the
procedure of example 18, the title compound was prepared from
3-(4-{2-[(S)-1-(4-fluorophenyl)ethylamino]-5-methyl-4-oxo-4,5-di-
hydrothiazol-5-yl}phenyl)propanenitrile (70 mg, 0.18 mmol) and
KOH (51 mg, 0.92 mmol). After purification (preparative TLC,
10% MeOH in CH2Cl2), the title compound was isolated as a white
solid (63 mg, 86%). 1H NMR (CDCl3 + 1 drop of TFA, 400 MHz):
δ 7.35-7.29 (m, 2H), 7.27-7.18 (m, 4H), 7.14-7.07 (m, 2H), 4.60
(m, 1H), 2.98 (m, 2H), 2.60 (m, 2H), 2.15 (2.05) (s, 3H), 1.77
(1.75) (d, 3H, J ) 8 Hz). MS (ESI, pos. ion) m/z: 400 (M + H).
Anal. (C21H22FN3O2S·0.2H2O) C, H, N.
(S)-2-[1-(4-Fluorophenyl)ethylamino]-5-(2-hydroxypropan-2-yl)-
5-methylthiazol-4(5H)-one [(S)-33a, (R)-33a]. According to the
general procedure described for 33d, 33a was synthesized from
2-[(S)-1-(4-fluorophenyl)ethylamino]-5-methylthiazol-4(5H)-one
(16) (330 mg, 1.3 mmol), LDA (3.5 mL of a 1.5 M solution in
THF/cyclohexane, 5.3 mmol), and acetone (0.29 mL, 3.9 mmol).
After purification of the crude reaction mixture (silica gel, 0-50%
EtOAc in hexanes), a colorless film was obtained as the mixture
of diastereomers (300 mg, 73%). The isomers were separated using
a Berger Multigram II supercritical fluid chromatography system.
The column was Chiralpak ASH (21 mm × 250 mm, 5 µm) using
CO2(l) with 25% methanol as the eluant with a flow rate of 60
mL/min. The column temperature was 40 °C, and the outlet pressure
was 100 bar.
(S)-2-[(S)-1-(4-Fluorophenyl)ethylamino]-5-(2-hydroxypropan-
2-yl)-5-methylthiazol-4(5H)-one [(S)-33a]. Yield: 57 mg, white solid
(14%). 1H NMR (CDCl3 + 1 drop of TFA, 400 MHz): δ 7.35-7.30
(m, 2H), 7.14-7.08 (m, 2 H), 4.59 (q, 1H, J ) 8 Hz), 1.81 (s, 3H),
1.75 (d, 3H, J ) 8 Hz), 1.31 (s, 3 H), 1.26 (s, 3H). MS (ESI, pos.
ion) m/z: 311 (M + H). Anal. (C15H19FN2O2S) C, H, N.
(R)-2-[(S)-1-(4-Fluorophenyl)ethylamino]-5-(2-hydroxypropan-
2-yl)-5-methylthiazol-4(5H)-one [(R)-33a]. Yield: 50 mg, white solid
(12%). 1H NMR (CDCl3 + 1 drop of TFA, 400 MHz): δ 7.35-7.30
(m, 2H), 7.14-7.09 (m, 2H), 4.62 (q, 1H, J ) 8 Hz), 1.76 (d, 3H,
J ) 7 Hz), 1.72 (s, 3H), 1.46 (s, 3H), 1.43 (s, 3H). MS (ESI, pos.
ion) m/z: 311 (M + H). Anal. (C15H19FN2O2S) C, H, N.
2-[(S)-1-(4-Fluorophenyl)ethylamino]-5-(1-hydroxycyclobutyl)-
5-methylthiazol-4(5H)-one (33b). According to the procedure
described 33d, this compound was prepared from 2-[(S)-1-(4-
fluorophenyl)ethylamino]-5-methylthiazol-4(5H)-one (16) (0.20,
0.79 mmol), LDA (1.6 mL of a 2 M solution in THF, 0.32 mmol),
and cyclobutanone (0.33 g, 4.8 mmol). After purification (silica
gel, 2-5% methanol/CH2Cl2), the title compound was isolated as
a white solid (65 mg, 25%). 1H NMR (CDCl3, 400 MHz): δ
7.67-7.47 (m, 2H), 7.11-6.97 (m, 2H), 6.01-5.96 (tautomer
3.95-3.79) (br s, 1H), 5.53-5.42 (tautomer 4.66-4.52) (m, 1H),
2.45-1.39 (m, 13H). MS (ESI, pos. ion) m/z: 323.2 (M + H). HPLC
purity at 215 and 254 nm: 99 and 100% in systems B and C.
2-[(S)-1-(4-Fluorophenyl)ethylamino]-5-(1-hydroxycyclopentyl)-
5-methylthiazol-4(5H)-one (33c). According to the general procedure
described for 33d, this compound was synthesized from 2-[(S)-1-
(4-fluorophenyl)ethylamino]-5-methylthiazol-4(5H)-one (16) (0.25
g, 1.0 mmol), LDA (2.5 mL of a 2 M solution in THF, 2.5 mL, 5
mmol), and cyclopentanone (0.27 mL, 3.0 mmol). After purification
of the crude product (silica gel, 4:1 hexanes/ethyl acetate to 1:1
hexanes/ethyl acetate), the title compound was isolated as a white
solid (230 mg, 67%). 1H NMR (CDCl3 + 1 drop of TFA, 400
MHz): δ 7.31 (7.30) (d, 2H, J ) 8 Hz); 7.11 (app t, 2H, J ) 8
Hz), 4.69 (q, 1H, J ) 8 Hz), 1.93-1.58 (m, 14H). MS (ESI, pos.
ion) m/z: 337 (M + H). Anal. (C17H21FN2O2S) C, H, N.
(4-Bromophenethoxy)trimethylsilane (28). To a round-bottomed
flask with a solution of 2-(4-bromophenyl)ethanol (27) (3.0 g, 15
mmol) and diisopropylethylamine (3.9 mL, 22 mmol) in CH2Cl2
(30 mL) cooled to 0 °C was added chlorotrimethylsilane (2.8 mL,
22 mmol). The mixture was warmed to room temperature and stirred
overnight. K2CO3 (1.1 g, 8.0 mmol) was then added, and the
reaction mixture was stirred for an additional 3 h at room
temperature. The solids were then filtered off, and the filtrate was
concentrated in vacuo. After purification by flash column chroma-
tography (silica gel, 15% EtOAc in hexane), the title compound
was isolated as a colorless oil (3.4 g, 84%). 1H NMR (CDCl3, 400
MHz): δ 7.40 (d, 2H, J ) 8 Hz), 7.08 (d, 2H, J ) 8 Hz), 3.75 (t,
2H, J ) 8 Hz), 2.77 (t, 2H, J ) 8 Hz), 0.06 (s, 9H).
2-[(S)-1-(4-Fluorophenyl)ethylamino]-5-methyl-5-{4-[2-
(trimethylsilyloxy)ethyl]phenyl}thiazol-4(5H)-one. Using the cross-
coupling procedure used for the preparation of intermediate A, the
title compound was prepared using 2-[(S)-1-(4-fluorophenyl)ethy-
lamino]-5-methylthiazol-4(5H)-one (16) (0.80 g, 3.2 mmol), (4-
bromophenethoxy)trimethylsilane (28) (1.7 g, 6.3 mmol), Pd2(dba)3
(0.20 g, 0.22 mmol), (S)-BINAP (0.40 g, 0.63 mmol), and
LiN(TMS)2 (1.6 g, 9.5 mmol). After purification (silica gel, 23%
EtOAc in hexane, then 39% EtOAc in hexane), the title compound
was isolated as a light yellow solid (0.43 g, 31%). 1H NMR (CDCl3,
400 MHz): δ 7.44-7.13 (m, 6H), 7.02-6.94 (m, 2H), 4.56 (m,
1H), 3.75 (m, 2H), 2.82 (m, 2H), 2.07 (1.98) (s, 3H), 1.79 (1.76)
(d, 3H, J ) 8 Hz), 0.08 (0.07) (s, 9H). MS (ESI, pos. ion) m/z: 373
[(M + H) - Si(CH3)3].
2-{(S)-[1-(4-fluorophenyl)ethyl]amino}-5-[4-(2-hydroxyethyl)phe-
nyl]-5-methyl-1,3-thiazol-4(5H)-one (29). Into a round-bottomed
flask was added 2-[(S)-1-(4-fluorophenyl)ethylamino]-5-methyl-5-
{4-[2-(trimethylsilyloxy)ethyl]phenyl}thiazol-4(5H)-one (0.41 g,
0.92 mmol), K2CO3 (0.64 g, 4.6 mmol), and MeOH (1.5 mL). After
the mixture was stirred at room temperature for 1 h, the solids were
filtered off, and the filtrate was concentrated in vacuo. Purification
by flash column chromatography (silica gel, 0-6% MeOH in
CH2Cl2) gave the desired product as a white solid (0.32 g, 93%).
1H NMR (CDCl3, 400 MHz): δ 7.42-7.17 (m, 6H), 7.04-6.96
(m, 2H), 4.57 (m, 1H), 3.85 (m, 2H), 2.86 (m, 2H), 2.09 (2.00) (s,
3H), 1.78 (1.75) (d, 3H, J ) 8 Hz). MS (ESI, pos. ion) m/z: 373
(M + H). HPLC purity at 215 and 254 nm: 100 and 100% in
systems C and A.
2-[(S)-1-(4-Fluorophenyl)ethylamino]-5-[4-(2-iodoethyl)phenyl]-
5-methylthiazol-4(5H)-one. Into a round bottomed flask was added
2-[(S)-1-(4-fluorophenyl)ethylamino]-5-[4-(2-hydroxyethyl)phenyl]-
5-methylthiazol-4(5H)-one (29) (0.29 g, 0.79 mmol), triphenylphos-
phine (0.25 g, 0.94 mmol), imidazole (64 mg, 0.94 mmol), and
iodine (0.22 g, 0.86 mmol) in CH2Cl2 (2 mL). After the mixture
was stirred at room temperature for 2 h, the mixture was filtered,
and the filtrate was concentrated in vacuo. Purification of the crude
product by flash column chromatography (silica gel, 43% EtOAc
in hexane) afforded the title compound as a white solid (0.31 g,
82%). 1H NMR (CDCl3, 400 MHz): δ 7.43-7.26 (m, 4H),
7.20-6.95 (m, 4H), 4.57 (m, 1H), 3.32 (m, 2H), 3.17 (m, 2H),
2.09 (2.00) (s, 3H), 1.78 (1.76) (d, 3H, J ) 8 Hz). MS (ESI, pos.
ion) m/z: 483 (M + H).
3-(4-{2-[(S)-1-(4-Fluorophenyl)ethylamino]-5-methyl-4-oxo-4,5-
dihydrothiazol-5-yl}phenyl)propanenitrile. Using the cyanide dis-
placement reaction used in the synthesis intermediate B, the title
compound was prepared from 2-[(S)-1-(4-fluorophenyl)ethylamino]-
5-[4-(2-iodoethyl)phenyl]-5-methylthiazol-4(5H)-one (0.31 g, 0.64
mmol) and tetrabutylammonium cyanide (0.86 g, 3.2 mmol). After
purification (silica gel, 55% EtOAc in hexane), the desired product
was isolated as a white solid (0.16 g, 65%). 1H NMR (CDCl3, 400
MHz): δ 7.45-7.40 (m, 2H), 7.31-7.17 (m, 4H), 7.02-6.95 (m,
2H), 4.57 (m, 1H), 2.94 (m, 2H), 2.61 (m, 2H), 2.08 (2.00) (s,
3H), 1.80 (1.77) (d, 3H, J ) 8 Hz). MS (ESI, pos. ion) m/z: 382
(M + H).
4-{(S)-1-[5-(2-Hydroxypropan-2-yl)-5-methyl-4-oxo-4,5-dihy-
drothiazol-2-ylamino]ethyl}benzonitrile (33d). To a round-bottomed
flask equipped with magnetic stirring and nitrogen purge was added
a solution of 4-[(S)-1-(5-methyl-4-oxo-4,5-dihydrothiazol-2-ylami-
no)ethyl]benzonitrile37 (0.21 g, 0.81 mmol) in THF (10 mL). After
the reaction flask was cooled to -78 °C, LDA (1.8 mL of a 1.8 M
solution in heptane/THF/ethylbenzene, 3.2 mmol) was added. The
reaction mixture was stirred at -78 °C for 30 min, and then, acetone
(0.24 g, 4.0 mmol) was added. After the mixture was stirred for

7964 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24
Fotsch et al.
1.5 h at -78 °C, the reaction mixture was quenched with a saturated
solution of NH4Cl. The reaction mixture was then extracted with
EtOAc (3 × 100 mL), and the combined organic extracts were
washed with brine, dried over Na2SO4, filtered, and concentrated
in vacuo. Purification by flash column chromatography (silica gel,
0-3% MeOH-CH2Cl2) afforded the title compound (116 mg, 45%)
ethyl acetate/hexanes) to give the title compound as a white foam
(0.55 g, 68%). 1H NMR (CDCl3, 400 MHz): δ 7.35-6.94 (m, 4H),
4.63-4.53 (m, 1H), 2.64 (s, 1H), 1.85-1.58 (m, 6H), 1.36-1.13
(m, 6H). MS (ESI, pos. ion) m/z: 311.2 (M + H). HPLC purity at
215 and 254 nm: 98 and 98% in system B; 98 and 96% in system
C.
1
as a tan solid. H NMR (CDCl3 + 1 drop of TFA, 400 MHz): δ
2-[(S)-1-(2-Fluorophenyl)ethylamino]-5-(2-hydroxypropan-2-yl)-
5-methylthiazol-4(5H)-one (33j). According to the general procedure
described for 33d, this compound was synthesized from 2-[(S)-1-
(2-fluorophenyl)ethylamino]-5-methylthiazol-4(5H)-one37 (0.75 g,
3.0 mmol), LDA (6.0 mL of a 2 M solution in THF, 12 mmol),
and acetone (0.47 mL, 6.0 mmol). After purification of the crude
product (silica gel, 4:1 hexanes/ethyl acetate), the title compound
7.74 (d, 2H, J ) 8 Hz), 7.48 (d, 2H, J ) 8 Hz), 4.66 (4.65) (q, 1H,
J ) 8 Hz), 1.78 (d, 3H, J ) 8 Hz), 1.71 (1.82) (s, 1H), 1.46 (1.34)
(s, 3H), 1.42 (1.26) (s, 3H). MS (ESI, pos. ion) m/z: 318 (M + H).
Anal. (C16H19N3O2S·0.6H2O) C, H, N.
2-[(S)-1-(4-Bromophenyl)ethylamino]-5-(2-hydroxypropan-2-yl)-
5-methylthiazol-4(5H)-one (33e). Compound 33e was prepared
according to the general procedure described 33d using 2-[(S)-1-
(4-bromophenyl)ethylamino]-5-methylthiazol-4(5H)-one37 (0.70 g,
2.2 mmol), LDA (5 mL of a 1.8 M solution in heptane/THF/
ethylbenzene, 8.9 mmol), and acetone (0.65 g, 11 mmol). After
purification of the crude product (silica gel, 0-3% CH3OH in
CH2Cl2), the product was isolated as a white solid (700 mg, 84%).
1H NMR (CDCl3 + 1 drop of TFA, 400 MHz): δ 7.57 (d, 2H, J )
8 Hz), 7.22 (d, 2H, J ) 8 Hz), 4.615 (q, 1H, J ) 8 Hz), 1.83
(1.73) (s, 3H), 1.75 (1.46) (d, 3H, J ) 8 Hz), 1.37 (1.35) (s, 3H),
1.31 (1.27) (s, 3H). MS (ESI, pos. ion) m/z: 371, 373 (M + H).
Anal. (C15H19BrN2O2S) C, H, N.
1
was isolated as a white solid (160 mg, 17%). H NMR (CDCl3 +
1 drop of TFA, 400 MHz): δ 7.40-7.33 (m, 2H); 7.23 (t, 1H, J )
8 Hz), 7.13 (dd, 1H, J ) 8 Hz, 12 Hz), 5.01 (m, 1H), 1.84 (1.73)
(3H, s), 1.78 (d, 3H, J ) 4 Hz), 1.48(1.38) (s, 3H), 1.48(1.34) (s,
3H). MS (ESI, pos. ion) m/z: 311 (M + H). Anal. (C15H19FN2O2S)
C, H, N.
2-[(S)-1-(2-Chlorophenyl)ethylamino]-5-(2-hydroxypropan-2-yl)-
5-methylthiazol-4(5H)-one (33k). According to the procedure
described 33d, this compound was prepared from 2-[(S)-1-(2-
chlorophenyl)ethylamino]-5-methylthiazol-4(5H)-one37 (1.3 g, 4.8
mmol), LDA (9.7 mL of a 2 M solution in THF, 19 mmol), and
acetone (2.2 g, 39 mmol). After purification (silica gel, 10-50%
ethyl acetate/hexane), the title compound was isolated as a light
yellow foam (1.2 g, 76%). 1H NMR (CDCl3, 400 MHz): δ
7.62-7.16 (m, 4H), 5.15-5.01 (m, 1H), 3.99 (tautomer 3.48) (s,
1H), 2.62 (s, 1H), 1.80-1.58 (m, 6H), 1.36-1.07 (m, 6H). MS
(ESI, pos. ion) m/z: 327.1 (M + H). HPLC purity at 215 and 254
nm: 100 and 100% in system B; 98 and 97% in system C.
(S)-5-(2-Hydroxypropan-2-yl)-5-methyl-2-{1-[2-(trifluoromethyl)-
phenyl]ethylamino}thiazol-4(5H)-one (33l). According to the pro-
cedure described for 33d, this compound was prepared from
5-methyl-2-[(S)-1-(2-(trifluoromethyl)phenyl]ethylamino)thiazol-
4(5H)-one37 (0.50 g, 1.7 mmol), LDA ·THF (4.4 mL of a 1.5 M
solution in cyclohexane, 6.6 mmol), and acetone (0.49 mL, 6.6
mmol). After purification (silica gel, 0-4% MeOH in CH2Cl2), the
5-(2-Hydroxypropan-2-yl)-5-methyl-2-[(S)-1-p-tolylethylami-
no]thiazol-4(5H)-one (33f). Compound 33f was prepared according
to the general procedure described 33d using 5-methyl-2-[(S)-1-
p-tolylethylamino]thiazol-4(5H)-one37 (0.60 g, 2.4 mmol), LDA (5.4
mL of a 1.8 M solution in heptane/THF/ethylbenzene, 9.7 mmol),
and acetone (0.70 g, 12 mmol). After purification of the crude
product (silica gel, 0-3% CH3OH in CH2Cl2), the product was
1
isolated as a colorless thin film (670 mg, 90%). H NMR (CDCl3
+ 1 drop of TFA, 400 MHz): δ 7.21 (m, 4H), 4.615 (m, 1H), 2.38
(s, 3H), 1.82 (1.76) (s, 3H), 1.445 (1.75) (d, 3H, J ) 8 Hz), 1.28
(1.35) (s, 3H). MS (ESI, pos. ion) m/z: 307 (M + H). Anal.
(C16H22N2O2S·0.2H2O) C, H, N.
2-[(S)-1-(4-Chlorophenyl)ethylamino]-5-(3-hydroxypropan-2-yl)-
5-methylthiazol-4(5H)-one (33g). According to the procedure
described for 33d, this compound was synthesized from 2-[(S)-1-
(4-chlorophenyl)ethylamino]-5-methylthiazol-4(5H)-one37 (0.30 g,
1.1 mmol), LDA (2.2 mL of a 2.0 M solution in THF, 4.4 mmol),
and acetone (0.52 g, 8.9 mmol). After purification (silica gel,
30-70% ethyl acetate/hexane), the title compound was isolated as
a white foam (0.17 g, 47%). 1H NMR (CDCl3, 400 MHz): δ
7.41-7.28 (m, 4H), 4.62-4.51 (m, 1H), 3.96-3.83 (br s, 1H), 2.61
(s, 1H), 1.83-1.58 (m, 5H), 1.37-1.14 (m, 7H). MS (ESI, pos.
ion) m/z: 327.1 (M + H). HPLC purity at 215 and 254 nm: 100
and 100% in system B; 99 and 97% in system C.
1
desired product was isolated as a white solid (530 mg, 89%). H
NMR (CDCl3, 400 MHz): δ 7.81 (dd, 1H, J ) 8.0, 16.5 Hz), 7.67
(d, 1H, J ) 8.0 Hz), 7.60-7.53 (m, 1H), 7.40 (m, 1H), 5.04-5.00
(m, 1H), 3.91 (m, 1H), 1.81-1.64 (m, 6 H), 1.34-1.12 (m, 6H).
MS (ESI, pos. ion) m/z: 361 (M + H). HPLC purity at 215 and
254 nm: 100 and 100% in system A; 98 and 100% in system D.
2-[(S)-1-(4-Fluorophenyl)ethylamino]-5-(2-hydroxypropyl)-5-me-
thylthiazol-4(5H)-one (34). To a round-bottomed flask equipped with
stirring was added 2-[(S)-1-(4-fluorophenyl)ethylamino]-5-meth-
ylthiazol-4(5H)-one 16 (0.34 g, 1.4 mmol) in THF (3 mL) under
nitrogen at 0 °C. Lithium bis(trimethylsilyl)amide (5.4 mL of a 1
M solution in THF, 5.4 mmol) was added, and the reaction mixture
was stirred at 0 °C for 1 h. A solution of 2-methyloxirane (0.38
mL, 5.4 mmol) and lithium perchlorate (0.29 g, 2.7 mmol) in THF
(4 mL) was added, and the reaction mixture was stirred at 0 °C for
2 h and then warmed to ambient temperature overnight. The reaction
mixture was quenched with a saturated solution of NH4Cl and
extracted with EtOAc (3×). The combined organic layers were
washed with brine, dried over Na2SO4, filtered, and concentrated
in vacuo. Purification by flash column chromatography (silica gel,
0-7% MeOH in CH2Cl2) afforded the title compound 34 (310 mg,
71%) as a white solid. 1H NMR (CDCl3, 400 MHz): δ 7.45-7.30
(m, 2H), 7.03-6.98 (m, 2H), 4.59-4.51 (m, 1 H), 4.06-3.91 (m,
1H), 2.15-1.98 (m, 2 H), 1.82-1.48 (m, 6H), 1.27-1.14 (m, 3H).
MS (ESI, pos. ion) m/z: 311 (M + H).
5-(2-Hydroxypropan-2-yl)-5-methyl-2-[(S)-1-phenylethylami-
no]thiazol-4(5H)-one (33h). Compound 33h was prepared according
to procedure described for 33d using 5-methyl-2-[(S)-1-phenyl-
ethylamino]thiazol-4(5H)-one37 (0.51 g, 2.2 mmol), LDA (4.8 mL
of a 1.8 M solution in heptane/THF/ethylbenzene, 8.7 mmol), and
acetone (0.63 g, 11 mmol). The crude material was purified by
flash column chromatography (silica gel, 0-3% MeOH-CH2Cl2)
1
to afford the title compound as a white solid (600 mg, 94%). H
NMR (CDCl3 + 1 drop of TFA, 400 MHz,): δ 7.45-7.37 (m, 3H),
7.34-7.31 (m, 2H) 4.645 (q, 1H, J ) 7 Hz), 1.82 (1.71) (s, 3H),
1.775 (1.45) (d, 3H, J ) 7 Hz), 1.27 (1.33) (s, 6H). MS (ESI, pos.
ion) m/z: 293(M + H). Anal. (C15H20N2O2S·0.2H2O) C, H, N.
2-[(S)-1-(3-Fluorophenyl)ethylamino]-5-(2-hydroxypropan-2-yl)-
5-methylthiazol-4(5H)-one (33i). A 100 mL round-bottomed flask
was charged with 2-[(S)-1-(3-fluorophenyl)ethylamino]-5-meth-
ylthiazol-4(5H)-one37 (0.66 g, 2.6 mmol) and 10 mL of THF. After
the mixture was cooled to -78 °C, LDA (6.5 mL of a 2 M solution
in THF, 13 mmol) was added, and the solution was stirred for 1 h
at -78 °C. After that time, acetone (1.7 mL, 24 mmol) was added,
and the mixture was stirred at -78 °C for 2.5 h. The reaction was
quenched with a saturated solution of NH4Cl. The mixture was
extracted with EtOAc, dried with MgSO4, and concentrated to give
an oil, which was purified by silica gel chromatography (30-70%
2-[(S)-1-(4-Fluorophenyl)ethylamino]-5-methyl-5-(2-oxopropyl)-
thiazol-4(5H)-one. To a round-bottomed flask equipped with stirring
was added 2-[(S)-1-(4-fluorophenyl)ethylamino]-5-(2-hydroxypro-
pyl)-5-methylthiazol-4(5H)-one (34) (0.28 g, 0.90 mmol) and
Dess-Martin periodinane (0.54 g, 1.3 mmol) in CH2Cl2 (8 mL).
After the mixture was stirred at ambient temperature overnight,
Na2S2O3 (1.5 g) and a saturated solution of NaHCO3 (5 mL) were
added, and the mixture was stirred at ambient temperature for 30

11ꢀ-Hydroxysteroid Dehydrogenase Type 1 Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24 7965
min. The reaction mixture was extracted with CH2Cl2 (3 × 60 mL),
and the combined organic layers were washed with brine, dried
over Na2SO4, filtered, and concentrated in vacuo. Purification by
flash column (silica gel, 0-3% MeOH in CH2Cl2) afforded the title
compound (230 mg, 83%) as a white solid. 1H NMR (CDCl3 + 1
drop of TFA, 400 MHz): δ 7.35-7.29 (m, 2H), 7.14-7.05 (m,
2H), 4.58 (4.53) (q, 1H, J ) 8 Hz), 3.31 (3.30) (d, 1H, J ) 20
Hz), 3.05 (2.91) (d, 1H, J ) 20 Hz), 2.25 (2.19) (s, 3H), 1.74,
1.725 (2d, 3H, J ) 8 Hz), 1.72 (1.63) (s, 3H). MS (ESI, pos. ion)
m/z: 309 (M + H).
nonessential amino acids, and sodium pyruvate. Assay plates were
prepared 1 day prior to the addition of the compound. Cells were
seeded at a density of 30000 cells per well in 100 mL of
maintenance medium. The assay was initiated by removing the
medium and rinsing cells twice with glucose buffer (50 mM HEPES
containing 120 mM NaCl, 1.85 mM CaCl2, 1.3 mM MgSO4, and
4.8 mM KCl, pH 7.4), followed by the addition of 80 mL of glucose
buffer containing 0.1% DMSO. The compound was added to
achieve a final concentration of 0.1 nM to 10 µM. The plates were
incubated for 60 min at 37 °C. Following the incubation, cortisone
was added to each well to achieve a final concentration of 100
nM. The plates were then incubated at 37 °C. The incubation time
for human 11ꢀ-HSD1 was 40 min; for mouse 11ꢀ-HSD1, 45 min;
for dog 11ꢀ-HSD1, 1.5 h; and for rat and monkey 11ꢀ-HSD1, 2 h.
Cortisol formed from cortisone was quantitated using a cortisol
enzyme-linked immunosorbent assay kit.
2-[(S)-1-(4-Fluorophenyl)ethylamino]-5-(2-hydroxy-2-methylpro-
pyl)-5-methylthiazol-4(5H)-one (35). In a round-bottomed flask
equipped with stirring was added methylmagnesium bromide (0.42
mL of a 3 M solution in diethyl ether, 1.3 mmol) to a solution of
2-[(S)-1-(4-fluorophenyl)ethylamino]-5-methyl-5-(2-oxopropyl)thi-
azol-4(5H)-one (98 mg, 0.32 mmol) in THF at 0 °C under nitrogen.
The resulting reaction mixture was stirred at 0 °C for 5 h, and then,
additional methylmagnesium bromide (0.42 mL of a 3 M solution
in diethyl ether, 1.3 mmol) was added. The reaction mixture was
stirred at 0 °C for 1 h, and again, methylmagnesium bromide (0.42
mL of a 3 M solution in diethyl ether, 1.3 mmol) was added. After
the reaction mixture was stirred to ambient temperature for 1 h,
the mixture was quenched with a saturated solution of NH4Cl and
extracted with EtOAc (3 × 60 mL). The combined organic layers
were washed with brine, dried over Na2SO4, filtered, and concen-
trated in vacuo. Purification by flash column (silica gel, 0-3%
MeOH in CH2Cl2) afforded the title compound (63 mg, 61%) as a
PXR Reporter Gene Assay. This assay was conducted at Indigo
Biosciences (State College, PA).38
Human Hepatocyte Culture and Experimental Procedure. Fresh
human hepatocytes from two donors were purchased from Cellz-
Direct. Donor 1 was a 60 year old Caucasian male (lot DJV) with
no adverse medical history or history of substance or alcohol abuse,
and donor 2 was a 40 year old Caucasian female (lot NPV) with a
history of tobacco usage and substance usage (cocaine and
marijuana). On day 1, fresh hepatocytes were received and
suspended in plating medium (0.8 × 106 cells mL-1). Hepatocytes
were counted and plated in collagen-coated 24 well plates (BD
Biosciences, Bedford, MA) with 0.4 × 106 cells per well. The
hepatocytes were placed in a 37 °C incubator (Steri-Cult CO2
Incubator, model 3310, Thermo Electron Corp., Waltham, MA)
under an atmosphere of 95% air/5% CO2 and 90% relative humidity
and allowed a 3-5 h attachment period. Following the attachment
period, the plating medium and unattached cells were aspirated,
sandwich medium was applied (0.5 mL per well), and the cells
were incubated overnight. On day 2, the sandwich medium was
aspirated, and culture medium (0.5 mL per well) was applied for
an overnight acclimation period. On days 3 and 4, culture medium
containing either DMSO (0.1%) or rifampin (0.05-5 µM) was
applied on each day (0.5 mL per well). The test articles were
prepared in DMSO stock solutions resulting in final incubation
concentrations of 0.1% DMSO. Compound treatment was main-
tained for a total of 48 h. On day 5, hepatocytes were gently washed
with Krebs-Henseleit buffer (KHB) (2 × 0.5 mL per well; ∼37
°C) and allowed to acclimate for an additional 10 min. Subse-
quently, CYP enzyme activities were determined by the addition
of the marker substrate midazolam (10 µM; CYP3A4) dissolved
in KHB (0.5 mL per well; ∼37 °C). Following a 10 min incubation
time period, the supernatant was removed and stored at -80 °C
until analyzed. Hepatocytes designated for mRNA analysis were
washed once with PBS (0.5 mL per well) containing calcium and
magnesium and aspirated. To each well was added 0.5 mL of 33%
lysis mixture (Panomics, Inc., Fremont, CA) and then stored at -80
°C until assay.
mRNA Analysis. The CYP3A4 mRNA content was determined
with branched DNA (bDNA) signal amplification technology using
the Panomics Discover XL Kit (Panomics, Inc.) with assays
performed according to the manufacturer’s instructions. bDNA
probe sets, containing capture extender, label extender, and blocking
probes for human CYP3A4 (catalog no. PA-10909) and GAPDH
(catalog no. PA-10382) were also purchased from Panomics, Inc.
Plate washing steps were performed on an Elx405 automated
microplate washer (BIO-TEK, Winooski, VT), and luminescence
was analyzed on a Luminoskan Ascent microplate luminometer
(Thermo Labsystems, Helsinki, Finland). CYP mRNA levels were
normalized to the mRNA levels of the housekeeping gene GAPDH.
Enzyme Activity. Following the induction treatment period, cell
cultures were assayed for the metabolism of the CYP3A4 marker
substrate, midazolam. Analysis and quantification of 1′-OH-
midazolam, the major midazolam metabolite in hepatocyte cultures,
were performed by LC-MS/MS on a system comprising a reverse
phase HPLC (Shimadzu, Kyoto, Japan) and a triple quadrupole mass
1
white solid. H NMR (CDCl3 400 MHz): δ 7.47-7.30 (m, 2 H),
7.07-6.95 (m, 2H), 4.62-4.60 (m, 1H), 2.28-2.11 (m, 2H, m),
1.77 (m, 3H), 1.70-1.63 (m, 3H), 1.33-1.12 (m, 6H). MS (ESI,
pos. ion) m/z: 325 (M + H). Anal. (C16H21FN2O2S·0.3H2O) C, H,
N.
11ꢀ-HSD1 SPA Assay. Enzyme assays were performed using
purified recombinant 11ꢀ-HSD1 that was expressed in E. coli.
Assays were run in a total volume of 100 µL, including 40 µL of
purified enzyme, 10 µL of compound dilutions, 10 µL of [3H]cor-
tisone (100 nM final), 10 µL of NADPH (200 µM final), and 30
µL of assay buffer (50 mM Tris-HCl, 1 mM EDTA, pH 7.2). The
assay was initiated by the addition of purified enzyme preparation.
The final concentrations of enzyme varied for different species.
For human, rat, and dog 11ꢀ-HSD1, the final concentration of
enzyme was 20 nM. For mouse and monkey 11ꢀ-HSD1, the final
concentration of enzyme was 5 and 10 nM, respectively. Assay
plates were incubated on an orbital shaker for 1 h at room
temperature. The reaction was stopped by the addition of 10 µL of
buffer containing 100 µM 18 ꢀ-glycyrrhetinic acid (GE). At the
same time, 10 µL of a 1:50 dilution of anticortisol and 100 µL of
15 mg mL-1 antimouse SPA beads were added to the wells. Plates
were incubated on an orbital shaker for another 30 min at room
temperature. Radiometric quantitation was determined on a Top-
Count NXT instrument (Perkin-Elmer, Downers Grove, IL).
11ꢀ-HSD2 SPA Assay. Enzyme assays were performed using
human 11ꢀ-HSD2 enzyme expressed in CHO cells. Assays were
run in a total volume of 100 µL, including 40 µL of enzyme, 10
µL of compound dilutions, 10 µL of [3H]cortisol (3 nM final), 10
µL of NAD+ (300 µM final), and 30 µL of assay buffer (0.1 M
sodium phosphate buffer, pH 7.6). The assay was initiated by the
addition of 40 µL of enzyme preparation. Assay plates were
incubated on an orbital shaker for 1 h at room temperature. The
reaction was stopped by the addition of 10 µL of buffer containing
100 µM 18 ꢀ-glycyrrhetinic acid (GE). At the same time, 10 µL of
a 1:70 dilution of anticortisol and 100 µL of 10 mg mL-1 antimouse
SPA beads were added to the wells. Plates were incubated on an
orbital shaker for another 30 min at room temperature. Radiometric
quantitation was determined on a TopCount NXT instrument
(Perkin-Elmer).
Measurement of 11ꢀ-HSD1 Activity in Whole Cells. Cell-based
activity was measured by monitoring the conversion of cortisone
to cortisol in CHO cell lines stably overexpressing 11ꢀ-HSD1. Cells
were maintained in Dulbecco’s modified Eagle’s medium supple-
mented with 10% dialyzed serum, penicillin/streptomycin/glutamine,

7966 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24
Fotsch et al.
(8) Masuzaki, H.; Paterson, J.; Shinyama, H.; Morton, N. M.; Mullins,
J. J.; Seckl, J. R.; Flier, J. S. A transgenic model of visceral obesity
and the metabolic syndrome. Science 2001, 294, 2166–2170.
(9) Kotelevtsev, Y.; Holmes, M. C.; Burchell, A.; Houston, P. M.; Schmoll,
D.; Jamieson, P.; Best, R.; Brown, R.; Edwards, C. R.; Seckl, J. R.;
Mullins, J. J. 11beta-Hydroxysteroid dehydrogenase type 1 knockout
mice show attenuated glucocorticoid-inducible responses and resist
hyperglycemia on obesity or stress. Proc. Natl. Acad. Sci. U.S.A. 1997,
94, 14924–9.
(10) Morton, N. M.; Paterson, J. M.; Masuzaki, H.; Holmes, M. C.; Staels,
B.; Fievet, C.; Walker, B. R.; Flier, J. S.; Mullins, J. J.; Seckl, J. R.
Novel adipose tissue-mediated resistance to diet-induced visceral
obesity in 11ꢀ-hydroxysteroid dehydrogenase type 1-deficient mice.
Diabetes 2004, 53, 931–938.
spectrometer (Applied Biosystems API 5000, Foster City, CA) using
Turbo Ionspray (Applied Biosystems) via multiple reaction moni-
toring. Samples (25 µL) were loaded on a C18 column (Phenom-
enex Onyx Monolithic C18, 100 mm × 3.0 mm, P/no. CHO-8158)
eluted with a linear gradient of mobile phase A (H2O with 0.1%
acetic acid and 5% methanol) to B (H2O with 0.1% acetic acid
and 95% methanol). The flow rate was 1 mL/min. Metabolites were
quantitated by a peak area ratio of metabolite to internal standard
(prazosin).
X-ray Structure Determination. The X-ray cocrystal structure
of (S)-33a with human 11ꢀ-HSD1 was collected using the procedure
described in ref 32.
(11) Morton, N. M.; Holmes, M. C.; Fievet, C.; Staels, B.; Tailleux, A.;
Mullins, J. J.; Seckl, J. R. Improved lipid and lipoprotein profile,
hepatic insulin sensitivity, and glucose tolerance in 11ꢀ-hydroxysteroid
dehydrogenase type 1 null mice. J. Biol. Chem. 2001, 276, 41293–
41300.
(12) Alberts, P.; Nilsson, C.; Selen, G.; Engblom, L. O. M.; Edling,
N. H. M.; Norling, S.; Klingstro¨m, G.; Larsson, C.; Forsgren, M.;
Ashkzari, M.; Nilsson, C. E.; Fiedler, M.; Bergqvist, E.; Oehman, B.;
Bjo¨rkstrand, E.; Abrahmse´n, L. B. Selective inhibition of 11ꢀ-
hydroxysteroid dehydrogenase type 1 improves hepatic insulin sen-
sitivity in hyperglycemic mice strains. Endocrinology 2003, 144, 4755–
4762.
(13) Hermanowski-Vosatka, A.; Balkovec, J. M.; Cheng, K.; Chen, H. Y.;
Hernandez, M.; Koo, G. C.; Le Grand, C. B.; Li, Z.; Metzger, J. M.;
Mundt, S. S.; Noonan, H.; Nunes, C. N.; Olson, S. H.; Pikounis, B.;
Ren, N.; Robertson, N.; Schaeffer, J. M.; Shah, K.; Springer, M. S.;
Strack, A. M.; Strowski, M.; Wu, K.; Wu, T.; Xiao, J.; Zhang, B. B.;
Wright, S. D.; Thieringer, R. 11ꢀ-HSD1 inhibition ameliorates
metabolic syndrome and prevents progression of atherosclerosis in
mice. J. Exp. Med. 2005, 202, 517–527.
(14) Gu, X.; Dragovic, J.; Koo, G. C.; Koprak, S. L.; LeGrand, C.; Mundt,
S. S.; Shah, K.; Springer, M. S.; Tan, E. Y.; Thieringer, R.;
Hermanowski-Vosatka, A.; Zokian, H. J.; Balkovec, J. M.; Waddell,
S. T. Discovery of 4-heteroarylbicyclo[2.2.2]octyltriazoles as potent
and selective inhibitors of 11beta -HSD1: Novel therapeutic agents
for the treatment of metabolic syndrome. Bioorg. Med. Chem. Lett.
2005, 15, 5266–5269.
In Vivo Pharmacodynamics in Monkeys. Male cynomolgus
monkeys were assigned based on their body weight to treatment
groups [0, 0.3, 1, 3, and 30 mg kg-1 of compound (S)-31a] with
at least four animals per group. The animals were dosed via the
nasal-gastric route once with compound of target dosage in dosing
vehicle (1% Tween 80, 47.5% Ora-Plus, and 51.5% water). At 2 h
postdosing, plasma and tissue samples were collected, snap-frozen
in liquid nitrogen, and stored either in dry ice or at -80 °C.
Approximately 50 mg of mesenteric fat tissues was cut in triplicate
into a 24 well plate on dry ice. The tissues were then incubated
with 600 µL of warm Krebs Ringer Phosphate (KRP) buffer (0.9%
NaCl, 1.15% KCl, 1.8% MgSO4, and 0.1 M Na2HPO4 at pH 7.4)
3
containing H-cortisone (Amersham, GE Healthcare) at 37 °C on
a shaker for 1 h. After the incubation, 150 µL of media was
collected into a 96 well plate. To measure 3H-cortisol by SPA, 40
µL of SPA PVT beads (Amersham, GE Healthcare) mix containing
cortisol monoclonal antibody (5.4 µg mL-1) (East Coast Biologics,
MA) was added to all wells. The plates were incubated at room
temperature on a shaker for 15 min, followed by centrifugation at
2500 RPM for 5 min. After the supernatant was removed, 100 µL
of PBS/0.1% BSA was added for further incubation at room
temperature on a shaker, followed by another round of centrifuga-
tion. The beads were resuspended in 150 µL of PBS/0.1% BSA
and transferred to a 96 well Opti-Plate white assay plates (Perki-
nElmer, Waltham, MA) to be read on a TopCount plate reader
(PerkinElmer). The DPM readout from TopCount was converted
to the absolute amount of cortisol produced, which was then
normalized by the amount of fat tissue used and the assay incubation
time. The unit of ex vivo activity was expressed as fmol cortisol
produced per mg of fat tissue used per hour (fmol cortisol/mg tissue/
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Acknowledgment. We thank Kyung Gahm and Wes Barn-
hart for their assistance in separating the enantiomers of 33a.
Supporting Information Available: Analytical data for final
compounds and X-ray structure coordinates for (S)-33a and (S)-
33a with human 11ꢀ-HSD1 (PDB ID: 3EY4). This material is
available free of charge via the Internet at http://pubs.acs.org.
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