2-Amino-1,3-thiazol-4(5H)-ones as potent and selective 11β- hydroxysteroid dehydrogenase type 1 inhibitors: Enzyme-ligand co-crystal structure and demonstration of pharmacodynamic effects in C57Bl/6 mice

Article abstract of DOI:10.1021/jm701551j

11β-Hydroxysteroid dehydrogenase type 1 (11β-HSD1) has attracted considerable attention during the past few years as a potential target for the treatment of diseases associated with metabolic syndrome. In our ongoing work on 11β-HSD1 inhibitors, a series of new 2-amino-1,3-thiazol-4(5H)-ones were explored. By inserting various cycloalkylamines at the 2-position and alkyl groups or spirocycloalkyl groups at the 5-position of the thiazolone, several potent 11β-HSD1 inhibitors were identified. An X-ray cocrystal structure of human 11β-HSD1 with compound 6d (K_i = 28 nM) revealed a large lipophilic pocket accessible by substitution off the 2-position of the thiazolone. To increase potency, analogues were prepared with larger lipophilic groups at this position. One of these compounds, the 3-noradamantyl analogue 8b, was a potent inhibitor of human 11β-HSD1 (K_i = 3 nM) and also inhibited 11β-HSD1 activity in lean C57Bl/6 mice when evaluated in an ex vivo adipose and liver cortisone to cortisol conversion assay.

Full text of DOI:10.1021/jm701551j

J. Med. Chem. 2008, 51, 2933–2943
2933
2-Amino-1,3-thiazol-4(5H)-ones as Potent and Selective 11ꢀ-Hydroxysteroid Dehydrogenase
Type 1 Inhibitors: Enzyme-Ligand Co-Crystal Structure and Demonstration of
Pharmacodynamic Effects in C57Bl/6 Mice
Lars Johansson,*,† Christopher Fotsch,‡ Michael D. Bartberger,‡ Victor M. Castro,† Michelle Chen,‡ Maurice Emery,‡
Sonja Gustafsson,† Clarence Hale,‡ Dean Hickman,‡ Evert Homan,† Steven R. Jordan,‡ Renee Komorowski,‡ Aiwen Li,‡
Kenneth McRae,‡ George Moniz,‡ Guy Matsumoto,‡ Carlos Orihuela,‡ Gunnar Palm,† Murielle Veniant,‡ Minghan Wang,‡
Meredith Williams,†,§ and Jiandong Zhang‡
BioVitrum AB, SE-112 76 Stockholm, Sweden, and Amgen, Inc., One Amgen Center DriVe, Thousand Oaks, California 91320
ReceiVed December 12, 2007
11ꢀ-Hydroxysteroid dehydrogenase type 1 (11ꢀ-HSD1) has attracted considerable attention during the past
few years as a potential target for the treatment of diseases associated with metabolic syndrome. In our
ongoing work on 11ꢀ-HSD1 inhibitors, a series of new 2-amino-1,3-thiazol-4(5H)-ones were explored. By
inserting various cycloalkylamines at the 2-position and alkyl groups or spirocycloalkyl groups at the 5-position
of the thiazolone, several potent 11ꢀ-HSD1 inhibitors were identified. An X-ray cocrystal structure of human
11ꢀ-HSD1 with compound 6d (Ki ) 28 nM) revealed a large lipophilic pocket accessible by substitution
off the 2-position of the thiazolone. To increase potency, analogues were prepared with larger lipophilic
groups at this position. One of these compounds, the 3-noradamantyl analogue 8b, was a potent inhibitor of
human 11ꢀ-HSD1 (Ki ) 3 nM) and also inhibited 11ꢀ-HSD1 activity in lean C57Bl/6 mice when evaluated
in an ex vivo adipose and liver cortisone to cortisol conversion assay.
Introduction
Type 2 diabetes (T2D)a constitutes a rapidly growing world-
wide health problem, and it has been estimated that over 200
million people might be affected by the disease in 2010.1 The
pathogenesis of T2D is characterized by high plasma glucose
levels, peripheral insulin resistance, and hyperinsulinemia.2–4
At present, there are a number of drugs on the market that
partially lower glucose levels and improve insulin sensitivity;
however, more efficient therapies with less side effects are
needed.5 In this respect, 11ꢀ-hydroxysteroid dehydrogenase type
1 (11ꢀ-HSD1) is a biological target that has attracted consider-
able attention over the past few years.6–8 This microsomal
membrane-bound enzyme interconverts inactive cortisone (1a)
to the receptor-active glucocorticoid cortisol (2a) in humans.
In rodents, 11ꢀ-HSD1 catalyzes the corresponding conversion
between 11-dehydrocorticosterone (1b) and corticosterone (2b)
(Figure 1).9,10
Glucocorticoid hormones, such as cortisol, are important
regulators of several intracellular transcription and expression
processes. On the basis of observations from animal experi-
ments, as well as studies with humans, it has become evident
that glucocorticoid excess in tissues such as liver, adipose, and
skeletal muscle might be contributing to the onset of the
metabolic syndrome.11,12 In the liver, gluconeogenesis, which
is abnormally high in subjects with T2D,13 is stimulated by
Figure 1. Interconversion of cortisone (1a)/11-dehydrocorticosterone
(1b) and cortisol (2a)/corticosterone (2b).
cortisol via the nuclear glucocorticoid receptor (GR), primarily
by activating the enzymes phosphoenolpyruvate carboxykinase
and glucose-6-phosphatase.14 It has been demonstrated that liver-
specific GR antagonists suppress glucose output from the liver.15
In the adipose tissue, cortisol is involved in the promotion of
adipogenesis, induction of lipolysis, and release of free fatty
acids.16 The significance of glucocorticoids and 11ꢀ-HSD1 in
T2D has been demonstrated in several rodent studies. 11ꢀ-HSD1
knockout mice showed resistance toward the development of
diet-induced metabolic syndrome and had significantly lower
fasting blood glucose levels as compared to wild-type mice.17
In contrast, transgenic mice specifically overexpressing 11ꢀ-
HSD1 in adipose tissue showed typical features of the metabolic
syndrome such as visceral obesity, glucose intolerance, and
insulin resistance.18,19 Consistent with these findings, treatment
of diabetic and obese mice with selective 11ꢀ-HSD1 inhibitors
gave significantly improved insulin sensitivity and glucose
tolerance and also reduced visceral fat deposits.20,21 Moreover,
clinical evidence in humans has been indirectly obtained with
subjects suffering from Cushing’s syndrome, where abnormal
cortisol excess is characterized by impaired glucose tolerance,
visceral obesity, and hyperglycaemia.22 These findings suggest
that inhibition of 11ꢀ-HSD1 might provide beneficial effects
against diseases associated with metabolic syndrome.23,24
There are two known isoforms of 11ꢀ-HSD. The type 1
isoform (11ꢀ-HSD1) is primarily expressed in the liver and
* To whom correspondence should be addressed. Phone: +46-(0)8-
6973804. Fax: +46-(0)8-6972320. E-mail: lars.johansson@biovitrum.com.
†
Biovitrum AB.
Amgen, Inc.
‡
§
Current address: S*Bio Pte Ltd, 1 Science Park Road, no. 05-09 The
Capricorn, Singapore Science Park II, Singapore 117528.
a Abbreviations: T2D, type 2 diabetes; 11ꢀ-HSD1, 11ꢀ-hydroxysteroid
dehydrogenase type 1; GR, glucocorticoid receptor; 11ꢀ-HSD2, 11ꢀ-
hydroxysteroid dehydrogenase type 2; MR, mineralocorticoid receptor; SAR,
structure-activity relationship; SPA, scintillation proximity assay; PK,
pharmacokinetics; PD, pharmacodynamics; HTS, high-throughput screen;
DPPA, diphenyl phosphorazidate; GA, 18ꢀ-glycyrrhetinic acid; CMC,
carboxymethylcellulose.
10.1021/jm701551j CCC: $40.75
2008 American Chemical Society
Published on Web 04/18/2008

2934 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 10
Johansson et al.
Scheme 1a
a Reagents and conditions: (a) (i) SOCl2, 60 °C, (ii) Br2, HBr, 60-70
°C, (iii) MeOH, HCl, rt; (b) (i) PCl3, Br2, 85 °C, (ii) oxalyl chloride, DMF,
CH2Cl2, rt, (iii) EtOH, EtN(iPr)2, rt; (c) NH3, dioxane, rt; (d) (i) ethoxy-
carbonyl isothiocyanate, CH2Cl2, rt or benzoyl isothiocyanate, CHCl3, rt,
(ii) basic hydrolysis; (e) dioxane, 90-105 °C or EtN(iPr)2, ethanol, and
microwave heating (155-170 °C).
Figure 2. Progression of screening hit (3) to lead (5a). 11ꢀ-HSD1
potencies are expressed as the mean ( SD of at least three independent
determinations.
followed by mono R-bromination with Br2 and catalytic HBr
and subsequent reaction of the obtained R-brominated acyl
chloride with methanol to give the desired R-bromoester (10),
or (b) direct R-bromination of the carboxylic acid (9) using the
Hell-Volhard-Zelinsky reaction,35 followed by acyl chloride
formation with oxalyl chloride and subsequent ester preparation
to 10. The cycloalkylthioureas 13 were prepared from the
corresponding cycloalkylisothiocyanate (11) by addition of
ammonia.36 Alternatively, the synthesis of 13 was carried out
by reacting the corresponding cycloalkylamine 12 with ethoxy-
carbonyl isothiocyanate or benzoyl isothiocyanate, followed by
basic hydrolysis.37 The noncommercially available cycloalky-
lamines 12 were prepared according to methodologies described
in the literature.38
adipose tissue, whereas the type 2 isoform (11ꢀ-HSD2) is
located mainly in the kidney. 11ꢀ-HSD1 is a NADPH-
dependent, bidirectional enzyme, functioning predominantly as
an oxoreductase producing active glucocorticoids. 11ꢀ-HSD2,
on the other hand, is a NAD-dependent unidirectional enzyme
that acts as a dehydrogenase to form inactive 11-keto metabolites
and protects the mineralocorticoid receptor (MR) from cortisol.
Inappropriate cortisol-induced activation of the MR might lead
to sodium retention, hypokalemia, and hypertension,25 so 11ꢀ-
HSD2 inhibition should be avoided for any potential drug aimed
at inhibiting 11ꢀ-HSD1.
In contributions from our laboratories26 and others,27–33 the
discovery of selective, nonsteroidal, small-molecule 11ꢀ-HSD1
inhibitors has been reported during the past few years. As part
of our ongoing efforts to develop 11ꢀ-HSD1 inhibitors for the
treatment of T2D, the 1,3-thiazol-4(5H)-one (henceforth ab-
breviated as thiazolone) compound 3 was identified as a hit in
a high-throughput screen of our compound collection (Figure
2). The compound was a moderate 11ꢀ-HSD1 inhibitor (Ki )
189 nM), selective versus 11ꢀ-HSD2, possessed good aqueous
solubility, and had a relatively low molecular weight. Further
optimization of this compound eventually led to compound 5a
(Ki ) 126 nM), which no longer contained the aniline func-
tionality at the 2-position (Figure 2). In this paper, we describe
the structure-activity relationships (SAR) of a series of
analogues to 5a that led to the identification of a potent inhibitor
of 11ꢀ-HSD1 with in vivo activity.
Results and Discussion
We initially focused our efforts on probing the importance
of the size of the group at the thiazolone 2-position (Table 1,
compounds 5a-d). The inhibitory properties of the molecules
were evaluated in a scintillation proximity assay (SPA) with
human 11ꢀ-HSD1, and selected compounds were counter-
screened in a human 11ꢀ-HSD2 assay. Potency in the 11ꢀ-
HSD1 enzymatic binding assay improved about 2-fold, when
compared to 5a, by either extending the cyclohexyl ring at the
2-position by one methylene (5b) or by increasing the ring size
(5c and 5d). The enzymatic potency could be improved even
further by substituting an n-propyl group for the ethyl group at
the 5-position (6a-d). The most potent compound from this
series, the cyclooctyl analogue 6d, was a selective inhibitor for
11ꢀ-HSD1 with a Ki value of 28 nM and exhibited no ap-
preciable activity against 11ꢀ-HSD2.
Chemistry
Compound 6d was cocrystallized with human 11ꢀ-HSD1, and
the X-ray structure was determined (Figure 3) (PDB ID for
X-ray cocrystal structure of human 11ꢀ-HSD1 with 6d: 3BYZ).
The overall fold of the 11ꢀ-HSD1 protein in the structure is
very similar to those previously published.39–41 Even though
racemic 6d was used for the cocrystallization with human 11ꢀ-
HSD1, only the C-5 (S)-stereoisomer ((S)-6d) cocrystallized with
the enzyme. The inhibitor occupies the substrate binding site
and thus competes with the substrate steroid. However, the
aminothiazolone core of (S)-6d is in a reversed binding mode
compared to our previously published structure of the N-(2-
fluorophenyl) derivative 14.26e Furthermore, it can be inferred
from the hydrogen bonding pattern of the active site residues
that (S)-6d exists as the endo-double bond tautomer, which is
different from compound 14 where the bound tautomer contains
the exo-double bond. In the cocrystal structure of 14,26e the
backbone NH proton of Ala172 forms a hydrogen bond with
Synthesis. The synthetic routes toward the 5,5-dialkyl and
5-spirocycloalkyl substituted thiazolone end products 5-8 are
outlined in Scheme 1. The key step in the synthesis, the
construction of the thiazolone ring system, was accomplished
by a substitution/cyclization reaction between a N-cycloalky-
lthiourea (13) and an R-bromoester (10).34 The rate of this
reaction step was found to be sensitive to the steric properties
of the R-bromoesters 10, and the process required long reaction
times (3-8 days) by conventional heating at 90-105 °C.
However, the reaction time could be substantially reduced by
microwave-assisted heating at 155-170 °C in the presence of
N,N′-diisopropylethylamine, and under these conditions the
desired 5,5-dialkyl and 5-spirocycloalkyl analogues were ob-
tained within 2.5 h. The noncommercially available R-bro-
moesters were either synthesized by (a) converting carboxylic
acid (9) to the corresponding acyl chloride with thionyl chloride

2-Amino-1,3-thiazol-4(5H)-ones as 11ꢀ-HSD1 Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 10 2935
Table 1. Human 11ꢀ-HSD1 and 11ꢀ-HSD2 Activities of Compounds 5
and 6
various methylene groups of the N-cyclooctyl function of (S)-
6d to the cofactor and nearby residues in the active site (not
shown), it was envisaged that even larger groups might be
accommodated in the hydrophobic pocket occupied by the
cyclooctyl group.
Armed with this structural information, we prepared ana-
logues with bulkier cycloalkyl substituents at the 2-position of
the thiazolone ring (Table 1, compounds 5e-i and 6e-i). Three
of the compounds from this set, the 1-norbornyl analogue 6f
and the 1-adamantyl analogues 5i and 6i, showed comparable
potency to the cycloctyl analogue 6d. However, the most potent
compounds in the series were the 3-noradamantyl species 5h
and 6h with 11ꢀ-HSD1 Ki values of 13 and 12 nM, respectively.
Noteworthy is also that the 7-norbornyl analogues 5g and 6g
were significantly less potent when compared to the correspond-
ing 1-norbornyl species 5f and 6f. These results indicate that
both the size and the shape of the cycloalkyl moiety in the
thiazolone 2-position are of importance when optimizing the
interactions in this hydrophobic region of the enzyme.
To exploit the second hydrophobic pocket close to the
5-position of the thiazolone, the diethyl analogues 7 and the
spirocyclopentyl analogues 8 were prepared (Table 2). These
structural changes not only increased the steric bulk at the thia-
zolone 5-position when compared to compounds 5 and 6 but
they also had the advantage of giving achiral molecules. Of the
six analogues prepared, the cycloctyl substituted thiazolone 8a
and the 3-noradamantyl analogue 8b, both with the spirocyclo-
pentyl group on the 5-position, showed improved potency over
their 5-methyl-5-propyl congeners 6d and 6h. The most potent
of these, 8b, had an 11ꢀ-HSD1 Ki value of 3 nM, exhibited
high selectivity versus 11ꢀ-HSD2, and was selected for further
in vitro and in vivo profiling.
Before assessing the potency of compound 8b in our rodent
pharmacodynamic (PD) model, the binding affinity in the murine
11ꢀ-HSD1 assays and rat pharmacokinetics of 8b were evalu-
ated. As seen in Table 3, compound 8b was 40-60-fold less
potent against murine 11ꢀ-HSD1 when compared to human 11ꢀ-
HSD1. This result is consistent with our previous observation
that compounds that are active in the human 11ꢀ-HSD1 assay
are not necessarily potent in the murine 11ꢀ-HSD1 assays. Such
discrepancies in 11ꢀ-HSD1 affinity between species might
be anticipated, e.g., the mouse and human 11ꢀ-HSD1 enzymes
only share a 79% amino acid identity.26a Nonetheless, we
deemed compound 8b sufficiently potent against murine 11ꢀ-
HSD1 to further investigate its pharmacokinetic profile in rats
(Table 3). While the intravenous clearance was high (6.25 L
h-1 kg-1), the oral bioavailability and half-life (45% and 2 h,
respectively) were both considered acceptable for an in vivo
evaluation of 8b.
In our rodent PD model, the inhibition of 11ꢀ-HSD1 was
measured in adipose and liver, the two tissues where 11ꢀ-HSD1
is believed to play a role in metabolic diseases. Compound 8b
was administered to lean C57Bl/6 mice in a single 30 mg/kg
oral dose, and after 2 and 6 h, the inhibition of 11ꢀ-HSD1 was
measured ex vivo by incubating epididymal fat and liver in
media containing 3H-cortisone. Enzyme activity of 11ꢀ-HSD1
was significantly lower when compared to vehicle controls after
2 h (86% and 89% reduction in adipose and liver, respectively)
and 6 h (87% and 79% reduction in adipose and liver,
respectively) (Figure 4). To achieve this level of inhibition, the
plasma concentrations of compound 8b were 20 ( 1 µM at 2 h
and 7.3 ( 0.5 µM at 6 h, which, after accounting for protein
binding (mouse fu ) 0.63%), corresponds to fraction unbound
concentrations of 120 and 44 nM, respectively. Considering the
a Racemic mixtures. b Potencies are expressed as the mean ( SD of at
least three independent determinations. c Single-point determinations at 10
µM concentration.
the oxygen lone pair supplied by the nearby side chain oxygen
of Ser170 and the OH proton of Ser170 interacts with the
acceptor lone pair on the exocyclic nitrogen of the inhibitor. In
the cocrystal structure of (S)-6d, the backbone NH proton of
Ala172 is in direct contact with the thiazolone carbonyl oxygen
atom, and the side chain OH proton of Ser170 hydrogen bonds
with the side chain oxygen of Tyr183. The NH protons on both
inhibitors, exocyclic NH for analogue (S)-6d and endocyclic
NH for analogue 14, form hydrogen bonds with the side chain
oxygen of Tyr183. The OH proton of Tyr183 in each cocrystal
structure interacts with a ribose OH group of the adjacent
NADPH cofactor, which in turn appears to be exposed to bulk
solvent.
According to the X-ray structure with compound 14, Tyr177
forms an edge-to-face aromatic stacking interaction with the
2-fluorophenyl moiety.26e In the X-ray with (S)-6d, Tyr177
adopted a change in conformation, accompanied by a rotation
of the Cꢀ-Cγ bond of nearby residue Leu171, to form a van
der Waals contact with the C-5 propyl substituent of (S)-6d.
The C-5 methyl was only 3.6 Å from the backbone NH of
Leu217. The proximity of the C-5 methyl group to the protein
might explain why (R)-6d does not cocrystallize with human
11ꢀ-HSD1 because the propyl group on (R)-6d would sterically
clash with Leu217. From analysis of the distances between the

2936 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 10
Johansson et al.
Figure 3. (a) Cocrystal structures of previously reported N-(2-fluorophenyl) analogue 14 (left) and N-cyclooctyl inhibitor (S)-6d (right) in the
active site of human 11ꢀ-HSD1, depicting the reversal of binding mode of the thiazolone core (red ) oxygen, blue ) nitrogen, yellow ) sulfur,
orange ) phosphorus, carbon: green ) human 11ꢀ-HSD1, pink ) NADPH, light blue ) 14, grey ) (S)-6d). Distances are in Å; (b) schematic
picture of the key interactions of 14 and (S)-6d with human 11ꢀ-HSD1.
Table 2. Human 11ꢀ-HSD1 and 11ꢀ-HSD2 Activities of Compounds 7
and 8
the cyclooctyl analogue (S)-6d with 11ꢀ-HSD1, we observed
that the hydrophobic pockets near the catalytic site could
accommodate larger groups, which might be exploited to
improve potency. To that end, we prepared analogues with larger
lipophilic groups on both sides of the thiazolone core and
identified the achiral 3-noradamantyl analogue 8b, which had
an 11ꢀ-HSD1 Ki value of 3 nM. Compound 8b was also orally
active in mice, where it inhibited 11ꢀ-HSD1 activity in both
the epididymal fat and liver. Our findings suggest that this type
of thiazolones might provide suitable 11ꢀ-HSD1 inhibitors for
further evaluation in preclinical models of diseases associated
with metabolic syndrome.
Experimental Section
Chemistry. 1H NMR spectra were recorded on Varian Inova
(400 MHz), Bruker DRX (500 MHz), Bruker 300-Avance (300
MHz), or on Avance DMX-400 (400 MHz) instruments. All spectra
were recorded using the residual solvent proton resonance or
tetramethylsilane (TMS) as internal standard. Chemical shifts are
reported in parts per million (ppm, δ units). Elemental analysis
was performed by Atlantic Microlab, Inc., Norcross, GA. The purity
of compounds was determined by analytical reverse-phase HPLC.
The retention time (tR) was determined on Agilent Series 1100
spectrometers using either ACE 3 C8 (3 µm, 3.0 mm × 50 mm)
column (system A), Chromolith SpeedROD RP-18e (4.6 mm ×
50 mm) column (system B), Phenomenex Synergi MAX-RP (2.0
mm × 50 mm) column (systems C and F), Phenomenex Luna C8(2)
column (4.6 mm × 100 mm) (system D), or Agilent Zorbax SB-
C18 (3.0 mm × 50 mm) column (system E). For systems A-C
and F, 0.1% trifluoroacetic acid in acetonitrile (M1) and 0.1%
trifluoroacetic acid in H2O (M2) were used as mobile phases.
Gradient system A: 0-3.0 min, 10-97% M1; 3.0-3.1 min,
97-100% M1; 3.1-4.1 min, 100% M1; 4.1-4.2 min, 100-10%
M1; 4.2-6.2 min, 10% M1. Flow rate 0.7 mL/min. Injection
volume 3.0 µL. Gradient system B: 0-2.0 min, 2-95% M1;
2.0-2.1 min, 95-100% M1; 2.1-3.0 min, 100% M1; 3.0-3.1 min,
100-2% M1; 3.1-3.6 min, 2% M1. Flow rate 5.0 mL/min.
Injection volume 5.0 µL. Gradient system C: 0-0.2 min, 10% M1;
0.2-3.0 min, 10-100% M1; 3.0-4.5 min, 10% M1; 4.5-5.0 min,
100-10% M1. Flow rate 0.8 mL/min. Injection volume 4.0 µL.
a Potencies are expressed as the mean ( SD of at least three independent
determinations. b Single-point determinations at 10 µM concentration.
moderate inhibitory potency against mouse 11ꢀ-HSD1 for 8b
(Ki ) 120 nM), the great pharmacodynamic effects on 11ꢀ-
HSD1 observed at these relatively low plasma concentrations
might indicate that the distribution of 8b into adipose and liver
tissues is highly efficient. Furthermore, it cannot be excluded
that hypothetical 11ꢀ-HSD1 active metabolites of 8b are
contributing to the observed inhibitory effects as well.
Conclusion
Optimization of the HTS hit compound 3 led to a series of
potent and selective thiazolones with mono-, bi-, and tricy-
cloalkylamines at the 2-position and short-chain alkyl groups
or spirocycloalkyl groups at the 5-position. Many of the
compounds showed low nanomolar potencies in the human 11ꢀ-
HSD1 SPA binding assay and exhibited high selectivity for 11ꢀ-
HSD1 versus 11ꢀ-HSD2. In the X-ray cocrystal structure of

2-Amino-1,3-thiazol-4(5H)-ones as 11ꢀ-HSD1 Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 10 2937
Table 3. Mouse and Rat 11ꢀ-HSD1 Activities and Rat Pharmacokinetics of Compound 8b
b
c
c
c
c
mouse 11ꢀ-HSD1 Kia (nM) rat 11ꢀ-HSD1 Kia (nM) CLb (L h-1 kg-1
)
Vss (L kg-1
)
tmax (h) AUC0-inf (ng h-1 mL-1
)
t1/2 (h) %Foral
120 ( 5 180 ( 33 6.25
1.84
2.5 175
2
45
a n ) 2, mean ( SD reported. b Dosed iv (0.5 mg kg-1) in DMSO. c Dosed po (2 mg kg-1 in 0.5% CMC/0.1% Tween 80 in water).
to the residue was added diethyl ether (50 mL) and H2O (25 mL).
The phases were separated, and the organic phase was washed with
brine (10 mL) and dried over MgSO4. After filtration, the solvent
was carefully removed to give 10a as a colorless liquid (3.33 g,
1
58%). H NMR (400 MHz, CDCl3) δ: 0.99 (t, J ) 7.3 Hz, 3 H),
1.87 (s, 3 H), 2.14 (m, 2 H), 3.78 (s, 3 H).
Ethyl 2-Bromo-2-methylbutanoate (10b). This compound was
prepared according to the method described for 10h. A reaction
between 2-methylbutyric acid (3.64 g, 35.6 mmol), bromine (2.10
mL, 41.0 mmol), phosphorus trichloride (0.15 mL, 1.78 mmol),
and oxalyl chloride (3.50 mL, 38.9 mmol) gave 10b as a colorless
oil (4.90 g, 66%). 1H NMR (300 MHz, CDCl3) δ: 0.99 (t, J ) 7.5
Hz, 3 H), 1.31 (t, J ) 7.5 Hz, 3 H), 1.88 (s, 3 H), 2.15 (q, J ) 8.0
Hz, 2 H), 4.24 (q, J ) 7.0 Hz, 2 H).
Methyl 2-Bromo-2-methylpentanoate (10c). This compound
was prepared according to the method described for 10a. Starting
from 2-methylpentanoic acid (2.10 g, 18.1 mmol), the synthesis
gave 10c as a pale-yellow liquid (1.98 g, 53%). 1H NMR (400
MHz, CDCl3) δ: 0.95 (t, J ) 7.3 Hz, 3 H), 1.26-1.52 (m, 2 H),
1.88 (s, 3 H), 2.08 (m, 2 H), 3.77 (s, 3 H).
Figure 4. Ex vivo data for 8b. Acute effects of 8b as assessed by ex
vivo cortisone to cortisol turnover. The compound was administered
orally to lean C57Bl/6 mice (vehicle ) 0.5% CMC/0.1% Tween 80 in
water; n ) 6/group). Animals were sacrificed at 2 and 6 h post dose,
at which time inguinal fat pads and liver were removed to measure
11ꢀ-HSD1 activity. Values are expressed as mean ( SEM of n ) 6.
Gradient system F: 0-0.2 min, 10% M1; 0.2-3.0 min, 10-95%
M1; 3.5-3.7 min, 10% M1; 3.7-5.0 min, 100-10% M1. Flow
rate 1.5 mL/min. Injection volume ) 1.0 µL. For systems D and
E, 0.1% formic acid in acetonitrile (M3) and 0.1% formic acid in
H2O (M4) were employed as mobile phases. Gradient system D:
0-0.5 min, 10% M3; 0.5-7.0 min, 10-100% M3; 7.0-9.5 min,
100% M3; 9.5-10.0 min, 100-10% M3. Flow rate 1.0 mL/min.
Injection volume 3.0 µL. Gradient system E: 0-3.0 min, 10-95%
M3; 3.0-3.5 min, 95% M3; 3.5-3.51 min, 95-10% M3. Flow
rate 1.5 mL/min. Flow rate 1.5 mL/min. Injection volume 2.0 µL.
Electrospray mass spectrometry (MS) was performed using an
Agilent 1100 series liquid chromatograph/mass selective detector
(MSD) to obtain the pseudomolecular [M + H]+ ion of the target
molecules. Accurate mass determinations were performed on the
following two systems. (A) Agilent MSD-TOF (positive electro-
spray ionization) connected to an Agilent 1100 HPLC system with
a diode array detector. The instrument was calibrated by Agilent
ES-TOF tuning mix. (B) Bruker FTMS (positive electrospray
ionization) with an external calibration using PEG 600 as the
calibrant. Preparative HPLC was performed on a Gilson 305 HPLC
system equipped with an ACE 5 C8 (5 µm, 21.2 mm × 100 mm)
column, a Varian PrepStar HPLC system with a Phenomenex C18
(5 µm, 30 mm × 150 mm) column, or on a Waters 2700/Micromass
ZQ system equipped with an ACE 5 C8 (5 µm, 21.2 mm × 100
mm) column. Acetonitrile and H2O containing 0.1% TFA, respec-
tively, were used as mobile phases. Preparative flash chromatog-
raphy was carried out on Merck silica gel 60 (230-400 mesh) or
prepacked silica gel cartridges (Biotage). Microwave reactions were
performed with a Personal Chemistry Smith creator or synthesizer
using 0.5-2 mL or 2-5 mL Smith process vials fitted with
aluminum caps and septa. Unless otherwise noted, all materials were
obtained from commercial sources and used without further
purification. Norbornane-1-carboxylic acid was prepared from
norbornane-2-carboxylic acid as described in the literature.38 For
a majority of the products 5-8, 1H NMR spectra with dual signal
set were observed. The explanation to this feature is the existence
of two rotameric forms due to restricted rotation around the
thiazolone C2-NH bond.
Ethyl 2-Bromo-2-methylpentanoate (10d). This compound was
prepared according to the method described for 10h. A reaction
between 2-methylvaleric acid (5.88 g, 50.6 mmol), bromine (3.00
mL, 58.2 mmol), phosphorus trichloride (0.22 mL, 2.5 mmol), and
oxalyl chloride (4.73 mL, 53.1 mmol) gave 10d as a pale oil (8.80
g, 78%). 1H NMR (300 MHz, CDCl3) δ: 0.97 (t, J ) 7.5 Hz, 3 H),
1.13-1.60 (m, 5 H), 1.89 (s, 3 H), 2.11 (q, J ) 7.5 Hz, 2 H), 4.25
(q, J ) 7.5 Hz, 2 H).
Methyl 2-Bromo-2-ethylbutanoate (10e). This compound was
prepared according to the method described for 10a. Starting from
2-ethylbutanoic acid (3.00 g, 25.8 mmol), the synthesis gave 10e
1
as a colorless liquid (4.08 g, 75%). H NMR (400 MHz, CDCl3)
δ: 0.97 (t, J ) 7.3 Hz, 6 H), 2.12 (m, 4 H), 3.77 (s, 3 H).
Ethyl 2-Bromo-2-ethylbutanoate (10f). This compound was
prepared according to the method described for 10h. A reaction
between 2-ethylbutanoic acid (6.09 g, 51.9 mmol), bromine (3.10
mL, 60.0 mmol), phosphorus trichloride (0.23 mL, 2.6 mmol), and
oxalyl chloride (4.90 mL, 54.6 mmol) gave 10f as a pale oil (9.14
g, 79%). 1H NMR (300 MHz, CDCl3) δ: 0.99 (t, J ) 7.2 Hz, 6 H),
1.31 (t, J ) 7.2 Hz, 3 H), 2.09-2.17 (m, 4 H), 4.25 (q, J ) 7.2
Hz, 2 H).
Methyl 1-Bromocyclopentanecarboxylate (10g). This com-
pound was prepared according to the method described for 10a.
Starting from cyclopentanecarboxylic acid (1.06 g, 9.26 mmol),
the synthesis gave 10g as a pale-yellow liquid (591 mg, 31%). 1H
NMR (400 MHz, CDCl3) δ: 1.77 (m, 2 H), 1.97 (m, 2 H), 2.29
(m, 4 H), 3.79 (s, 3 H).
Ethyl 1-Bromocyclopentanecarboxylate (10h). Phosphorus
trichloride (0.54 mL, 6.2 mmol) was added dropwise to the mixture
of cyclopentanecarboxylic acid (14.2 g, 124 mmol) and bromine
(7.35 mL, 143 mmol). The reaction was then gradually heated to
85 °C and stirred at this temperature in a sealed vessel for 12 h.
After cooling to rt, the mixture was partitioned between ethyl acetate
and H2O. The combined organic portions were washed with H2O
and brine and concentrated in vacuo to give 23.9 g of the
R-brominated carboxylic acid intermediate. The intermediate (23.9
g, 124 mmol) was dissolved in CH2Cl2 (250 mL), mixed with oxalyl
chloride (11.6 mL, 130 mmol), and two drops of DMF were added.
After stirring at rt for 2 h, the solvent was removed in vacuo and
then ethanol (50 mL) was added to the residue followed by the
addition of N,N-diisopropylethylamine (22.7 mL, 130 mmol). The
mixture was stirred at rt for 20 min. The solvent was removed in
vacuo, and the residue was partitioned between diethyl ether and
H2O. The organic portion was washed with H2O and brine and
concentrated in vacuo. The crude product was filtered through a
Methyl 2-Bromo-2-methylbutanoate (10a). A solution of
2-methylbutanoic acid (3.00 g, 29.4 mmol) in thionyl chloride (20
mL) was stirred at 60 °C for 1 h. The solution was cooled to rt,
and Br2 (3.00 mL, 58.7 mmol, 2 equiv) and one drop of 48%
aqueous HBr were added. The mixture was heated at 60 °C for 1 h
and at 70 °C for 2 h. The solvent was removed, and the residual
R-brominated acyl chloride intermediate was dissolved in methanol
(40 mL) and 1.25 M HCl in methanol (1 mL) was added. The
mixture was stirred at rt for 22 h. The solvent was removed, and

2938 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 10
Johansson et al.
plug of silica gel eluting with 10% ethyl acetate in hexanes.
Removal of the solvent gave 10h as a pale oil (21.2 g, 77%). H
as a white solid (325 mg). The crude material was suspended in a
mixture of CH2Cl2 (10 mL) and triethylamine (137 µL, 0.99 mmol),
and after addition of ethoxycarbonyl isothiocyanate (117 µL, 0.99
mmol), the reaction was stirred at rt for 35 min. To the resulting
pale-yellow solution was added 5 M NaOH (15 mL), and the
reaction was heated at 50 °C for 3.5 h and at 65 °C for 1.5 h. The
mixture was cooled, and CH2Cl2 (30 mL) was added. The phases
were separated, and the aqueous layer was extracted with CH2Cl2
(20 mL). The combined organic phases were washed with saturated
aqueous NaHCO3 (20 mL), H2O (15 mL), and brine (10 mL).
Evaporation of the solvent and drying in vacuo yielded 13f as an
1
NMR (300 MHz, CDCl3) δ: 1.31 (t, J ) 7.1 Hz, 3 H), 1.70-1.87
(m, 2 H), 1.90-2.08 (m, 2 H), 2.28-2.35 (m, 4 H), 4.25 (q, J )
7.1 Hz, 2 H).
N-Cyclohexylthiourea (13a). A mixture of isothiocyanatocy-
clohexane (6.74 g, 46.8 mmol) and 0.5 M ammonia in 1,4-dioxane
(150 mL) in a 500 mL round-bottomed flask was stirred at rt under
nitrogen overnight. The solvent was removed in vacuo to afford
13a as a white solid (5.10 g, 69%). 1H NMR (300 MHz, CD3OD)
δ: 1.12-1.45 (m, 5 H), 1.57-1.82 (m, 3 H), 1.83-2.00 (m, 2 H),
3.90-4.11 (m, 1 H).
1
off-white solid (139 mg, 11%). H NMR (400 MHz, CDCl3) δ:
N-(Cyclohexylmethyl)thiourea (13b). This compound was
prepared according to the method described for 13a. A reaction
between (isothiocyanatomethyl)cyclohexane (3.88 g, 25.0 mmol)
and 0.5 M ammonia in 1,4-dioxane (100 mL) gave 13b as a white
solid (4.21 g, 98%). H NMR (300 MHz, CD3OD) δ: 0.80-1.07
(m, 2 H), 1.11-1.38 (m, 3 H), 1.47-1.83 (m, 6 H), 2.88-3.25(m,
2 H).
1.43-1.49 (m, 2 H), 1.64 (m, 2 H), 1.71-1.87 (m, 6 H), 2.24 (m,
1 H), 5.87 (br, 2 H), 6.70 (br, 1 H). MS (ESI+) for C8H14N2S m/z:
171 [M + H]+.
N-(7-Norbornyl)thiourea (13g). A 100 mL round-bottomed
flask was charged with magnesium (639 mg, 26.3 mmol), and dry
diethyl ether (6 mL) was added. A small portion of a solution of
7-bromonorbornane (4.18 g, 23.9 mmol) in dry diethyl ether (10
mL), and a few I2 crystals were then added to the flask. The
Grignard reaction was initiated by heating the mixture at reflux
temperature under stirring. The remaining diethyl ether solution of
7-bromonorbornane was then added dropwise via a syringe over
10 min. The reaction was heated under reflux for a further 30 min
and then cooled to rt. A rubber balloon was charged with CO2(g),
and via a syringe, the gas was slowly bubbled through the solution
under stirring for 3.5 h. To the resulting suspension, diethyl ether
(50 mL) and 2 M aqueous HCl (50 mL) were added. The phases
were separated, and the aqueous layer was extracted with diethyl
ether (2 × 50 mL). The combined organic phases were washed
with brine (15 mL) and dried over MgSO4. Removal of the solvent
yielded 2.09 g of a white residue containing norbornane-7-
carboxylic acid intermediate. To the crude material dissolved in
acetonitrile (25 mL) was added triethylamine (2.27 mL, 16.4 mmol)
and DPPA (3.54 mL, 16.4 mmol). After stirring the mixture at 50
°C for 2 h, tert-butanol (80 mL) was added and the reaction was
heated at 80 °C overnight. The resulting mixture was concentrated
under vacuum, and CH2Cl2 (100 mL) and saturated aqueous
NaHCO3 (50 mL) were added. The phases were separated, and the
organic phase was washed with saturated aqueous NaHCO3 (50
mL), H2O (50 mL), brine (25 mL), and dried (MgSO4). Evaporation
of the solvent gave a sticky transparent solid that was dissolved in
CH2Cl2 (10 mL). After addition of concentrated trifluoroacetic acid
(5 mL), the solution was stirred under reflux overnight. The resulting
solution was concentrated in vacuo to give the amine intermediate
as an off-white sticky solid. The crude material was suspended in
CH2Cl2 (10 mL) and triethylamine (2.3 mL), and after addition of
ethoxycarbonyl isothiocyanate (1.94 mL, 16.4 mmol), the mixture
was stirred at rt for 1 h. To the resulting solution was added 5 M
NaOH (15 mL), and the reaction was heated at 65 °C for 3 h. The
mixture was cooled, and CH2Cl2 (30 mL) was added. The phases
were separated, and the aqueous layer was extracted with CH2Cl2
(20 mL). The combined organic phases were washed with saturated
aqueous NaHCO3 (20 mL), H2O (15 mL), and brine (15 mL).
Evaporation of the solvent and drying in vacuo gave a sticky solid
residue. Further purification of the material by silica gel flash
chromatography (pentane/ethyl acetate, 4:6) yielded the title
compound 13g as an off-white solid (382 mg, 15%, 78% pure by
LCMS). MS (ESI+) for C8H14N2S m/z: 171 [M + H]+. The
material was used in subsequent reactions without any further
purification.
1
N\-Cycloheptylthiourea (13c). This compound was prepared
according to the method described for 13a. A reaction between
isothiocyanatocycloheptane (8.47 g, 54.6 mmol) and 0.5 M am-
monia in 1,4-dioxane (220 mL) gave 13c as a white solid (7.03 g,
75%). 1H NMR (300 MHz, CD3OD) δ: 1.40-1.80 (m, 10 H),
1.85-2.09 (m, 2 H), 4.15-4.35 (m, 1 H).
N-Cyclooctylthiourea (13d). Benzoyl isothiocyanate (7.40 mL,
54.0 mmol) was added to a solution of cyclooctanamine (6.25 g,
49.1 mmol) in chloroform (200 mL) in a 500 mL round-bottomed
flask containing a magnetic stirring bar. The mixture was stirred at
rt overnight, after which the solvents were removed in vacuo. The
residue was stirred with potassium carbonate (34.6 g, 250 mmol)
in methanol (200 mL), H2O (100 mL) and THF (100 mL) at rt
overnight. The low boiling solvents were removed in vacuo, and
the residue was partitioned between ethyl acetate and H2O. The
organic portion was washed with brine and concentrated in vacuo.
The residue was purified by silica gel flash chromatography
(0-100% of ethyl acetate in hexanes) to give 13d as a white solid
(8.50 g, 93%). 1H NMR (300 MHz, CD3OD) δ: 1.47-1.77 (m, 12
H), 1.78-1.94 (m, 2 H), 4.22-4.35 (m, 1 H).
1-(2,2,3,3-Tetramethylcyclopropyl)thiourea (13e). To a solu-
tion of 2,2,3,3-tetramethylcyclopropanecarboxylic acid (681 mg,
4.79 mmol) in dry acetonitrile (10 mL) was added triethylamine
(668 µL, 4.79 mmol) and diphenyl phosphorazidate (DPPA) (1.03
mL, 4.79 mmol). The reaction mixture was stirred at 50 °C for 2 h
and then cooled to rt. After addition of 1 M aqueous HCl (10 mL)
the mixture was heated at reflux for 5 h. The organic solvent was
then evaporated and the remaining aqueous solution was neutralized
with solid NaHCO3 and extracted with chloroform. Ethoxycarbonyl
isothiocyanate (565 µL, 4.79 mmol) was added to the combined
chloroform phases, and the reaction mixture was stirred at rt for
24 h. The solvent was evaporated in vacuo, and the crude material
was purified by silica gel flash chromathography (cyclohexane/ethyl
acetate, 95:5). The residue was suspended in 5 M aqueous NaOH
(7 mL), and the reaction mixture was stirred at 50 °C for 3 h. The
precipitate was collected by filtration, yielding 380 mg (46%) of
13e as a white solid. 1H NMR (400 MHz, CDCl3) δ: ppm 1.03 (s,
6 H) 1.14 (s, 6 H) 1.91 (s, 1 H) 5.82 (s, 2 H) 6.12 (s, 1 H). MS
(ESI+) for C8H16N2S m/z: 173 [M + H]+.
N-(1-Norbornyl)thiourea (13f). To a solution of norbornane-
1-carboxylic acid (1.05 g, 7.48 mmol) and triethylamine (1.14 mL,
8.22 mmol) in dry acetonitrile (20 mL) was added DPPA (1.77
mL, 8.22 mmol), and the mixture was heated at 50 °C for 3 h.
After cooling to rt, concentrated HCl (15 mL) was added and the
mixture was heated under reflux (90 °C) for 3.5 h. The temperature
was decreased to 85 °C, and the mixture was stirred for an additional
17 h. After cooling, solid NaHCO3 was added until the pH was
basic. H2O and CH2Cl2 were added, and the mixture was vigorously
stirred until everything dissolved. The phases were separated, and
the aqueous layer was extracted with several portions of CH2Cl2
(10 × 100 mL). The combined organic phases were dried (Na2SO4),
and the solvent was evaporated to give the crude intermediate amine
N-(3-Noradamantyl)thiourea (13h). To 3-noradamantyl amine
(367 mg, 2.67 mmol) in CH2Cl2 (1 mL) was added dropwise
ethoxycarbonyl isothiocyanate (316 µL, 2.67 mmol), and the
mixture was stirred for 5 min. After addition of 5 M aqueous NaOH
(8 mL), the reaction was stirred at 70 °C for 6 h. H2O (20 mL) and
ethyl acetate (25 mL) were added, and the layers were separated.
The aqueous phase was extracted with ethyl acetate (5 mL), and
the combined organic phases were dried over MgSO4. After
evaporation of the solvent and drying in vacuo, 13h was obtained
1
as a white solid (481 mg, 92%). H NMR (400 MHz, CDCl3) δ:

2-Amino-1,3-thiazol-4(5H)-ones as 11ꢀ-HSD1 Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 10 2939
1.48-1.66 (m, 4 H), 1.81-1.96 (m, 4 H), 2.00-2.12 (m, 2 H),
2.33 (s, 2 H), 2.50 (m, 1 H), 6.04 (br, 2 H), 6.77 (br, 1 H).
N-(1-Adamantyl)thiourea (13i). This compound was prepared
according to the method described for 13a. A reaction between
1-adamantyl isothiocyanate (4.83 g, 25.0 mmol) and 0.5 M ammonia
in 1,4-dioxane (100 mL, 50 mmol) gave 13i as a white solid (4.22
NMR (400 MHz, DMSO-d6) δ: 0.77-0.82 (m, 3 H), 1.34-1.64
(m, 13 H), 1.71 (q, J ) 7.3 Hz, 2 H), 1.83-1.91 (m, 2 H),
3.35-3.96 (m, 1 H), 9.10-9.67 (m, 1 H, NH). HRMS m/z calcd
for C13H22N2OS: 254.1453; found: 254.1448. HPLC 100%, tR
2.81 min (system A), 100%, tR ) 1.30 min (system B).
)
(()-2-(Cycloheptylamino)-5-methyl-5-propyl-1,3-thiazol-4(5H)-
one (6c). This compound was prepared according to the method
described for 5c. A reaction between N-cycloheptylthiourea (13c)
(76.5 mg, 0.444 mmol) and methyl 2-bromo-2-methylpentanoate
(10c) (92.8 mg, 0.444 mmol) at 90 °C for 6 days gave 6c as a
white solid (47 mg, 40%). 1H NMR (400 MHz, DMSO-d6) δ:
0.82-0.86 (m, 3 H), 0.96-1.11 (m, 1 H), 1.33-1.73 (m, 16 H),
1.83-1.91 (m, 2 H), 3.36-3.96 (m, 1 H), 9.08-9.65 (m, 1 H, NH).
HRMS m/z calcd for C14H24N2OS: 268.1609; found: 268.1618.
HPLC 100%, tR ) 3.00 min (system A), 100%, tR ) 1.43 min
(system B).
(()-2-(Cyclooctylamino)-5-ethyl-5-methyl-1,3-thiazol-4(5H)-
one (5d). This compound was prepared according to the method
described for 5c. A reaction between N-cyclooctylthiourea (13d)
(114 mg, 0.610 mmol) and methyl 2-bromo-2-methylbutanoate
(10a) (119 mg, 0.610 mmol) at 95 °C for 5 days gave 5d as a
white solid (68 mg, 42%). 1H NMR (400 MHz, DMSO-d6) δ:
0.77-0.86 (m, 3 H), 1.41-1.82 (m, 19 H), 3.42-4.01 (m, 1 H),
9.09-9.66 (m, 1 H, NH). HRMS m/z calcd for C14H24N2OS:
268.1609; found: 268.1601. HPLC 100%, tR ) 3.01 min (system
A), 100%, tR ) 1.41 min (system B).
1
g, 80%). H NMR (400 MHz, CD3OD) δ: 1.70-1.75 (m, 6 H),
2.04-2.29 (m, 9 H).
(()-2-(Cyclohexylamino)-5-ethyl-5-methyl-1,3-thiazol-4(5H)-
one (5a). A 5 mL microwave vial containing a magnetic stirring
bar was charged with N-cyclohexylthiourea (13a) (234 mg, 1.48
mmol), ethyl 2-bromo-2-methylbutanoate (10b) (310 mg, 1.48
mmol), N,N-diisopropylethylamine (0.30 mL), and ethanol (0.50
mL). The sealed vial was heated in a microwave oven at 155 °C
for 2 h. After cooling to rt, the solid was collected by filtration,
washed with ethanol, and dried under vacuum to give 5a as a white
1
solid (81 mg, 23%). H NMR (300 MHz, CDCl3) δ: 0.95 (t, J )
6 Hz, 3 H), 1.12-2.14 (m, 15 H), 3.16-4.19 (m, 1 H), 5.60 (br, 1
H, NH). Anal. (C12H20N2OS) C, H, N.
(()-2-(Cyclohexylamino)-5-methyl-5-propyl-1,3-thiazol-4(5H)-
one (6a). A 5 mL microwave vial containing a magnetic stirring
bar was charged with N-cyclohexylthiourea (13a) (340 mg, 2.15
mmol), ethyl 2-bromo-2-methylpentanoate (10d) (480 mg, 2.15
mmol), N,N-diisopropylethylamine (0.50 mL), and ethanol (1.0 mL).
The sealed vial was heated in a microwave oven at 155 °C for 2 h,
then at 170 °C for 30 min. After cooling to rt, the solvents were
removed in vacuo. The residue was partitioned between ethyl acetate
and H2O. The combined organic portions were washed with brine
and dried over Na2SO4. After removing the solvent, the residue
was purified by preparative reverse-phase HPLC. The fractions
containing the product were combined, and acetonitrile was
removed in vacuo. The aqueous solution was then neutralized with
saturated aqueous NaHCO3 and extracted with ethyl acetate. The
organic portion was washed with brine and dried over Na2SO4. The
solvent was evaporated to give 6a as a tan solid (130 mg, 23%).
1H NMR (400 MHz, CDCl3 + 1 drop of TFA) δ: 0.99 (t, 3 H, J
) 7.5 Hz), 1.24-1.37 (m, 4 H), 1.52-1.61 (m, 3 H), 1.68-1.71
(m, 1 H), 1.77 (s, 3H), 1.88-1.94 (m, 3 H), 1.98-2.05 (m, 3 H)
3.32 (m, 1 H). Anal. (C13H22N2OS) C, H, N.
(()-2-((Cyclohexylmethyl)amino)-5-ethyl-5-methyl-1,3-thia-
zol-4(5H)-one (5b). This compound was prepared according to the
method described for 6a. A reaction between N-(cyclohexylmeth-
yl)thiourea (13b) (264 mg, 1.53 mmol) and ethyl 2-bromo-2-
methylbutanoate (10b) (320 mg, 1.53 mmol) at 155 °C for 2 h
gave 5b as a white solid after extraction with CH2Cl2 (83 mg, 21%).
1H NMR (400 MHz, CDCl3 + 1 drop of TFA) δ: 0.97-1.02 (m,
2 H), 1.05 (t, 3 H, J ) 7.0 Hz), 1.14-1.34 (m, 3 H), 1.70-1.79
(m, 9 H), 1.90-1.99 (m, 1 H), 2.07-2.16 (m, 1 H), 3.32 (d, 2 H,
J ) 6 Hz). Anal. (C13H22N2OS·0.1H2O) C, H, N.
(()-2-(Cyclooctylamino)-5-methyl-5-propyl-1,3-thiazol-4(5H)-
one (6d). This compound was prepared according to the method
described for 5c. A reaction between N-cyclooctylthiourea (13d)
(82.7 mg, 0.444 mmol) and methyl 2-bromo-2-methylpentanoate
(10c) (92.8 mg, 0.444 mmol) at 90 °C for 6 days gave 6d as a
white solid (57 mg, 46%). 1H NMR (400 MHz, DMSO-d6) δ:
0.82-0.86 (m, 3 H), 0.96-1.09 (m, 1 H), 1.31-1.81 (m, 20 H),
3.40-4.01 (m, 1 H), 9.07-9.65 (m, 1 H, NH). HRMS m/z calcd
for C15H26N2OS: 282.1766; found: 282.1774. HPLC 100%, tR
3.14 min (system A), 100%, tR ) 1.53 min (system B).
)
(()-5-Ethyl-5-methyl-2-[(2,2,3,3-tetramethylcyclopropyl)amino]-
1,3-thiazol-4(5H)-one (5e). This compound was prepared according
to the method described for 5c. A reaction between N-(2,2,3,3-
tetramethylcyclopropyl)thiourea (13e) (70.4 mg, 0.409 mmol) and
methyl 2-bromo-2-methylbutanoate (10a) (79.7 mg, 0.533 mmol)
gave 5e as a white solid (16 mg, 16%). 1H NMR (400 MHz,
DMSO-d6) δ: 0.76-0.81 (m, 3 H), 0.92 (s, 1 H), 0.94 (s, 1 H),
0.96 (s, 2 H), 0.96 (s, 2 H), 1.06 (s, 1 H), 1.07 (s, 1 H), 1.08 (s, 2
H), 1.09 (s, 2 H), 1.46 (s, 1 H), 1.48 (s, 2 H), 1.69-1.78 (m, 2 H),
2.06-2.38 (m, 1 H), 8.81-9.68 (m, 1 H, NH). HRMS m/z calcd
for C13H22N2OS: 254.1453; found: 254.1443. HPLC 100%, tR
2.94 min (system A), 100%, tR ) 1.36 min (system B).
)
(()-5-Methyl-5-propyl-2-[(2,2,3,3-tetramethylcyclopropyl)ami-
no]-1,3-thiazol-4(5H)-one (6e). This compound was prepared
according to the method described for 5c. A reaction between
N-(2,2,3,3-tetramethylcyclopropyl)thiourea (13e) (83.0 mg, 0.482
mmol) and methyl 2-bromo-2-methylpentanoate (10c) (101 mg,
0.482 mmol) at 90 °C for 6 days gave 6e as a white solid (15 mg,
11%). 1H NMR (400 MHz, DMSO-d6) δ: 0.81-0.86 (m, 3 H),
0.92 (s, 1 H), 0.94 (s, 1 H), 0.95 (s, 2 H), 0.96 (s, 2 H), 0.98-1.06
(m, 1 H), 1.06 (s, 1 H), 1.06 (s, 1 H), 1.08 (s, 2 H), 1.09 (s, 2 H),
1.31-1.42 (m, 1 H), 1.45 (s, 1 H), 1.47 (s, 2 H), 1.61-1.77 (m, 2
H), 2.05-2.38 (m, 1 H), 8.80-9.61 (m, 1 H, NH). HRMS m/z
calcd for C14H24N2OS: 268.1609; found: 268.1609. HPLC 100%,
tR ) 3.12 min (system A), 100%, tR ) 1.49 min (system B).
(()-2-((Cyclohexylmethyl)amino)-5-methyl-5-propyl-1,3-thia-
zol-4(5H)-one (6b). This compound was prepared according to the
method described for 6a. A reaction between N-(cyclohexylmeth-
yl)thiourea (13b) (320 mg, 1.84 mmol) and ethyl 2-bromo-2-
methylpentanoate (10d) (410 mg, 1.84 mmol) gave 6b as a tan
solid (82 mg, 17%). 1H NMR (400 MHz, CDCl3 + 1 drop of TFA)
δ: 0.99 (t, 3 H, J ) 7.5 Hz), 1.02-1.05 (m, 2 H), 1.13-1.36 (m,
4 H), 1.49-1.57 (m, 1 H), 1.70-1.79 (m, 9 H), 1.85-1.93 (m, 1
H), 1.98-2.05 (m, 1 H), 3.26 (d, 2 H, J ) 6 Hz). Anal. (C14H24
N2OS·0.1H2O) C, H, N.
(()-2-(Cycloheptylamino)-5-ethyl-5-methyl-1,3-thiazol-4(5H)-
one (5c). A solution of N-cycloheptylthiourea (13c) (91.8 mg, 0.533
mmol) and methyl 2-bromo-2-methylbutanoate (10a) (104 mg,
0.533 mmol) in 1,4-dioxane (600 µL) was stirred at 95 °C in a
sealed tube for 3 days. The solvent was removed, and the residue
was purified by preparative reverse-phase HPLC. The fractions
containing pure product were combined, and ethyl acetate (50 mL)
and saturated aqueous NaHCO3 (50 mL) were added. The phases
were separated, and the aqueous layer was extracted with ethyl
acetate (25 mL). The combined organic phases were washed with
brine (25 mL) and dried over MgSO4. Evaporation of the solvent,
(()-5-Ethyl-5-methyl-2-(1-norbornylamino)-1,3-thiazol-4(5H)-
one (5f). This compound was prepared according to the method
described for 5c. A reaction between N-(1-norbornyl)thiourea (13f)
(61.3 mg, 0.360 mmol) and methyl 2-bromo-2-methylbutanoate
(10a) (70.2 mg, 0.360 mmol) at 95 °C for 3 days and 100 °C for
1
1 day yielded 5f as a white solid (49 mg, 38%). H NMR (400
MHz, DMSO-d6) δ: 0.79 (t, J ) 7.3 Hz, 3 H), 1.27-1.40 (m, 2
H), 1.45-1.47 (m, 3 H), 1.62-1.86 (m, 10 H), 2.11-2.16 (m, 1
H), 9.33-9.93 (m, 1 H, NH). HRMS m/z calcd for C13H20N2OS:
1
and drying in vacuo gave 5c as a white solid (56 mg, 41%). H

2940 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 10
Johansson et al.
252.1296; found: 252.1296. HPLC 100%, tR ) 2.84 min (system
A), 96%, tR ) 1.22 min (system B).
2-(Cyclooctylamino)-5,5-diethyl-1,3-thiazol-4(5H)-one (7a).
This compound was prepared according to the method described
for 5c. A reaction between N-cyclooctylthiourea (13d) (109 mg,
0.583 mmol) and methyl 2-bromo-2-ethylbutanoate (10e) (122 mg,
0.583 mmol) at 95 °C for 5 days and 105 °C for 3 days gave 7a as
a white solid (15 mg, 9%). 1H NMR (400 MHz, CDCl3) δ:
0.85-0.95 (m, 6 H), 1.45-2.05 (m, 18 H), 3.55 and 4.28 (m, 1
H), 5.85 and 10.08 (m, 1 H, NH). HRMS m/z calcd for C15H26N2OS:
282.1766; found: 282.1758. HPLC 98%, tR ) 3.11 min (system
A), 100%, tR ) 1.52 min (system B).
2-(Cyclooctylamino)-1-thia-3-azaspiro[4.4]non-2-en-4-
one (8a). This compound was prepared according to the method
described for 6c. A reaction between N-cyclooctylthiourea (13d)
(77.3 mg, 0.415 mmol) and methyl 1-bromocyclopentanecarboxy-
late (10g) (85.9 mg, 0.415 mmol) at 90 °C for 3 days yielded 8a
as a white solid (24 mg, 20%). 1H NMR (400 MHz, DMSO-d6) δ:
1.41-1.70 (m, 20 H), 2.08-2.17 (m, 2 H), 4.04 (m, 1 H), 9.10 (d,
J ) 7.6 Hz, 1 H). HRMS m/z calcd for C15H24N2OS: 280.1609;
found: 280.1611. HPLC 99%, tR ) 3.11 min (system A), 100%, tR
) 1.46 min (system B).
(()-5-Methyl-2-(1-norbornylamino)-5-propyl-1,3-thiazol-4(5H)-
one (6f). This compound was prepared according to the method
described for 5c. A reaction between N-(1-norbornyl)thiourea (13f)
(99.4 mg, 0.583 mmol) and methyl 2-bromo-2-methylpentanoate
(10c) (122 mg, 0.583 mmol) at 110 °C for 4 days yielded 6f as a
white solid (42 mg, 27%). 1H NMR (400 MHz, CDCl3) δ:
0.84-0.96 (m, 3 H), 1.14-1.27 (m, 1 H), 1.33-1.50 (m, 3 H),
1.58-1.60 (m, 3 H), 1.66-2.03 (m, 10 H), 2.15-2.28 (m, 1 H),
6.52 and 10.11 (br, 1 H, NH). HRMS m/z calcd for C14H22N2OS:
266.1453; found: 266.1445. HPLC 100%, tR ) 3.15 min (system
A), 100%, tR ) 1.34 min (system B).
(()-5-Ethyl-5-methyl-2-(7-norbornylamino)-1,3-thiazol-4(5H)-
one (5g). This compound was prepared according to the method
described for 5c. A reaction between N-(7-norbornyl)thiourea (13
g) (86.1 mg, 0.505 mmol) and methyl 2-bromo-2-methylbutanoate
(10a) (98.6 mg, 0.505 mmol) at 100 °C for 4 days gave 5g as a
white solid (19 mg, 15%). 1H NMR (400 MHz, CDCl3) δ:
0.91-0.95 (m, 3 H), 1.21-1.42 (m, 5 H), 1.60-1.98 (m, 9 H),
2.30-2.47 (m, 2 H), 3.46-4.12 (m, 1 H). HRMS m/z calcd for
C13H20N2OS: 252.1296; found: 252.1293. HPLC 97%, tR ) 2.80
min (system A), 93%, tR ) 1.22 min (system B).
(()-5-Methyl-2-(7-norbornylamino)-5-propyl-1,3-thiazol-4(5H)-
one (6g). This compound was prepared according to the method
described for 5c. A reaction between N-(7-norbornyl)thiourea (13
g) (87.2 mg, 0.512 mmol) and methyl 2-bromo-2-methylpentanoate
(10c) (107 mg, 0.512 mmol) at 100 °C for 4 days gave 6g as a
white solid (19 mg, 14%). 1H NMR (400 MHz, CDCl3) δ:
0.85-0.93 (m, 3 H), 1.16-1.91 (m, 15 H), 2.29-2.47 (m, 2 H),
3.44-4.12 (m, 1 H). HRMS m/z calcd for C14H22N2OS: 266.1453;
found: 266.1443. HPLC 100%, tR ) 2.96 min (system A), 100%,
tR ) 1.35 min (system B).
5,5-Diethyl-2-(3-noradamantylamino)-1,3-thiazol-4(5H)-one
(7b). This compound was prepared according to the method
described for 5c. A reaction between N-(3-noradamantyl)thiourea
(13h) (115 mg, 0.588 mmol) and methyl 2-bromo-2-ethylbutanoate
(10e) (123 mg, 0.588 mmol) at 95 °C for 5 days and 110 °C for 3
1
days gave 7b as a white solid (12 mg, 7%). H NMR (400 MHz,
CDCl3) δ: 0.88-1.00 (m, 6 H), 1.45-2.35 (m, 16 H), 2.48 and
2.67 (m, 1 H), 6.08 and 8.96 (m, 1 H, NH). HRMS m/z calcd for
C16H24N2OS: 292.1609; found: 292.1622. HPLC 99%, tR ) 3.17
min (system A), 100%, tR ) 1.54 min (system B).
2-(3-Noradamantylamino)-1-thia-3-azaspiro[4.4]non-2-en-4-
one (8b). This compound was prepared according to the method
described for 5a. A reaction between N-(3-noradamantyl)thiourea
(13h) (320 mg, 1.63 mmol) and ethyl 1-bromocyclopentanecar-
boxylate (10h) (360 mg, 1.63 mmol) yielded 8b as a white solid
(()-5-Ethyl-5-methyl-2-(3-noradamantylamino)-1,3-thiazol-
4(5H)-one (5h). This compound was prepared according to the
method described for 5c. A reaction between N-(3-noradaman-
tyl)thiourea (13h) (103 mg, 0.522 mmol) and methyl 2-bromo-2-
methylbutanoate (10a) (102 mg, 0.522 mmol) gave 5h as a white
1
(165 mg, 35%). H NMR (300 MHz, CD3OD) δ: 1.52-2.39 (m,
20 H), 2.50-2.68 (m, 1 H). HRMS m/z calcd for C16H22N2OS [M
+ H]+: 291.1526; found: 291.1533. HPLC 98%, tR ) 3.06 min
(system C), 99%, tR ) 7.01 min (system D).
1
solid (47 mg, 32%). H NMR (400 MHz, DMSO-d6) δ: 0.79 (t, J
) 7.3 Hz, 3 H), 1.46 (s, 3 H), 1.47-1.57 (m, 4 H), 1.71 (q, J )
7.3 Hz, 2 H), 1.89-2.10 (m, 6 H), 2.21-2.29 (m, 2 H), 2.45 (m,
1 H), 9.23 (s, 1 H). HRMS m/z calcd for C15H22N2OS: 278.1453;
found: 278.1459. HPLC 100%, tR ) 2.98 min (system A), 100%,
tR ) 1.36 min (system B).
(()-5-Methyl-2-(3-noradamantylamino)-5-propyl-1,3-thiazol-
4(5H)-one (6h). This compound was prepared according to the
method described for 5c. A reaction between N-(3-noradaman-
tyl)thiourea (13h) (117 mg, 0.596 mmol) and methyl 2-bromo-2-
methylpentanoate (10c) (125 mg, 0.596 mmol) at 95 °C for 4 days
gave 6h as a white solid (65 mg, 37%). 1H NMR (400 MHz,
DMSO-d6) δ: 0.82-0.91 (m, 3 H), 1.03 (m, 1 H), 1.33-1.73 (m,
10 H), 1.88-2.10 (m, 6 H), 2.21-2.29 (m, 2 H), 2.45 (m, 1 H),
9.22-9.78 (m, 1 H, NH). HRMS m/z calcd for C16H24N2OS:
292.1609; found: 292.1614. HPLC 92%, tR ) 3.16 min (system
A), 97%, tR ) 1.49 min (system B).
(()-2-(1-Adamantylamino)-5-ethyl-5-methyl-1,3-thiazol-4(5H)-
one (5i). This compound was prepared according to the method
described for 6a. A reaction between N-(1-adamantyl)thiourea (13i)
(270 mg, 1.29 mmol) and ethyl 2-bromo-2-methylbutanoate (10b)
(270 mg, 1.29 mmol) at 155 °C for 2 h gave 5i as a white solid (26
mg, 7%). 1H NMR (300 MHz, CDCl3) δ: 0.90-1.02 (m, 3 H),
1.57-2.28 (m, 20 H), 5.45 (br, 1 H, NH). Anal. (C16H24-
N2OS·0.5H2O) C, H, N.
(()-2-(1-Adamantylamino)-5-methyl-5-propyl-1,3-thiazol-
4(5H)-one (6i). This compound was prepared according to the
method described for 6a. A reaction between N-(1-adamantyl)th-
iourea (13i) (460 mg, 2.20 mmol) and ethyl 2-bromo-2-methyl-
pentanoate (10d) (490 mg, 2.20 mmol) gave 6i as a tan solid after
extraction with CH2Cl2 (41 mg, 6%). 1H NMR (300 MHz, CDCl3)
δ: 0.90-1.00 (m, 3 H), 1.18-1.60 (m, 3 H), 1.61-2.20 (m, 19 H),
5.48 (br, 1 H, NH). Anal. (C17H26N2OS·0.14CH2Cl2) C, H, N.
2-(1-Adamantylamino)-5,5-diethyl-1,3-thiazol-4(5H)-one (7c).
This compound was prepared according to the method described
for 6a. A reaction between N-(1-adamantyl)thiourea (13i) (370 mg,
1.75 mmol) and ethyl 2-bromo-2-ethylbutanoate (10f) (399 mg, 1.75
mmol) gave 7c as an off-white solid (55 mg, 10%). 1H NMR (300
MHz, DMSO-d6) δ: 0.73-0.85 (m, 6 H), 1.52-1.89 (m, 11 H),
1.98-2.11 (m, 8 H), 8.82 (br, 1 H, NH). HRMS m/z calcd for
C17H26N2OS [M + H]+: 307.1844; found: 307.1828. HPLC 100%,
tR ) 2.92 min (system E), 100%, tR ) 7.38 min (system D).
2-(1-Adamantylamino)-1-thia-3-azaspiro[4.4]non-2-en-4-
one (8c). This compound was prepared according to the method
described for 6a. A reaction between N-(1-adamantyl)thiourea (13i)
(248 mg, 1.18 mmol) and ethyl 1-bromocyclopentanecarboxylate
(10h) (260 mg, 1.18 mmol) at 170 °C for 1.5 h gave 8c as a white
1
solid (93 mg, 28%). H NMR (300 MHz, CDCl3) δ: 1.63-1.90
(m, 8 H), 1.96-2.27 (m, 13 H), 2.36-2.55 (m, 2 H), 5.38 (br, 1
H, NH). HRMS m/z calcd for C17H24N2OS [M + H]+: 305.1682;
found: 305.1684. HPLC 97%, tR ) 2.32 min (system F), 100%, tR
) 7.26 min (system D).
Biology. Materials. 1,2(n)-3H]-Cortisone and [1,2,6,7-3H]-
hydrocortisone were purchased from Amersham. NADPH (tetra-
sodium salt) and NAD+ (lithium salt, 10 mM stock in Tris buffer)
were obtained from Sigma-Aldrich. Glucose-6-phosphate (G-6-P),
18ꢀ-glycyrrhetinic acid (GA), and carbenoxolone (disodium salt)
were supplied by Sigma-Aldrich. Anticortisol monoclonal mouse
antibody, clone 6D6.7, was obtained from Beckman Coulter
(Marseille, France), and yttrium silicate (YSi) scintillation proximity
assay (SPA) beads coated with monoclonal antimouse antibodies
were from Amersham. The human 11ꢀ-HSD1 enzyme used was
expressed in Escherichia coli.42 The 11ꢀ-HSD2 enzyme was
produced in HEK-293 cells transfected with a pcDNA3-derived
plasmid. The 3-fold serial dilutions of test compounds in assay

2-Amino-1,3-thiazol-4(5H)-ones as 11ꢀ-HSD1 Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 10 2941
buffer (30 mM Tris-HCl, pH 7.2 containing 1 mM EDTA) were
performed on a Tecan Genesis RSP 150. The final concentration
of the compounds spanned from 18 µM to 308 pM. Triplicates of
the serial dilutions were performed on a Tomtec Quadra 96
instrument using 96-well microtiter plates (Perkin-Elmer, white
Optiplate-96). The amount of the product [3H]-cortisol bound to
the beads was determined in a Packard Top Count NXT microplate
liquid scintillation counter. Dilutions of stock solutions were made
in assay buffer unless otherwise indicated.
and sectioned into three 30-40 mg samples and placed into 24-
well Falcon plates containing prewarmed assay media that consisted
of 1 µM cortisone in DMEM. Plates were then transferred to a 37
°C/CO2 incubator and incubated for 2 h. Then cortisol product in
the media was quantitated using a cortisol ELISA kit (Assay
Designs Inc., Ann Arbor, MI). Enzyme activity is expressed as pg/
mL of product formed per mg wet tissue weight. Data were analyzed
for statistical significance (two-tailed t test) using GraphPad Prism 4.
X-ray Structure Determination. Human 11ꢀ-HSD1 (24-292)
C272S mutant was expressed and purified as described previously.39
The protein sample was concentrated to 5 mg/mL and passed
through a Pierce Extracti-Gel D column. The inhibitor was dissolved
in DMSO at 100 mM concentration and then added to the protein
sample by 1:100 dilution. The enzyme was crystallized by vapor
diffusion using hanging drops. The protein sample was mixed with
an equal volume of well solution of 25% PEG 3350, 0.2 M
ammonium acetate, and 0.1 M sodium citrate buffer (pH 5.6) at rt.
The crystals belong to space group P212121 with cell dimensions
of a ) 49.03, b ) 138.49, and c ) 155.03 Å, with four molecules
in the asymmetric unit. Data were collected at beamline 5.0.2 at
the Advanced Light Source using an ADSCQ315 detector and
processed using HKL2000.43 The phases were obtained by mo-
lecular replacement method using Molrep in CCP4 suite.44 The
model was built using the visualization program QUANTA (Ac-
celrys). The model was refined using CNX45 to a resolution of 3
Å with a crystallographic R ) 20.45% and Rfree ) 29.67%.
Human 11ꢀ-HSD1 Scintillation Proximity Assay (SPA). The
11ꢀ-HSD1 enzyme assay was carried out in the replica plates of
the compounds in a total well volume of 110 µL. Each reaction
replica contained 10 µL of diluted compound, 50 µL of assay buffer,
and 25 µL of substrate mixture [3H]-cortisone/NADPH (175 nM/
200 µM). Reactions were initiated by the addition of 25 µL of 11ꢀ-
HSD1 enzyme (40-60 nM final concentration, depending on the
batch). Following mixing, the plates were incubated on a shaker at
rt for 30-60 min depending on batch. The reactions were terminated
by addition of 10 µL of stop solution (1 mM GA in ethanol). Mouse
monoclonal anticortisol antibody was then added (10 µL of 1.92
µM working solution) followed by 50 µL of YSi SPA beads
(suspended according to the manufacturer instructions). Appropriate
controls were set up, i.e., determinations of nonspecific binding
(NSB) and of total activity (TOT), by adding or omitting 10 µL of
1 mM GA, respectively, before starting the reaction. As reference
substance carbenoxolone was run in each plate. The plates were
sealed with plastic film (Perkin-Elmer, Top Seal-A) and incubated
on a shaker for 30 min at rt before counting. The amount of the
product, [3H]-cortisol, captured on the beads was determined in a
microplate liquid scintillation counter. Kinetic constants were
calculated employing the Microsoft Excel integrated application
XLfit (Version 5.3.0.19, ID Business Solutions Ltd.) using the
sigmoidal dose-response model 205, which is based on the
nonlinear curve fitting based on Levenberg-Marquardts algorithm.
Human 11ꢀ-HSD2 Assay. The assay was performed at rt in a
96-well microtiter plate (Costar 96 well V-bottom polypropylene,
catalogue no. 3363) containing 160 µL of substrate mixture [3H]-
cortisol/NAD+ (200 nM/200 µM) in assay buffer, 20 µL of a 10
µM solution of inhibitors or control substance, and 20 µL of 11ꢀ-
HSD2 (crude extract from sonicated HEK-293 cells diluted 10-20
fold in assay buffer). When a dose response was desirable, the
inhibitor was diluted in a 3-fold manner from 10 µM to 169 pM.
Pure DMSO was diluted in parallel with inhibitors and used to
assess the solvent effect on the enzyme. Following incubation for
10 min, the reaction was terminated with perchloric acid (6 M, 50
µL). Samples were centrifuged for 5 min at 1800g, and tritiated
substrate and product were separated by HPLC (HP1100, Agilent
Technologies). The injection volume was 15 µL and the flow rate
0.8 mL/min on a Hichrom Ltd. C18 (5 µm, 4.6 mm × 150 mm)
column using acetonitrile/H2O (72:28) as the eluant. Detection was
performed with a flow scintillation detector using an Ultima-Flo
M (Packard) scintillator. Enzyme activity was quantified as area
percentage of the product peak compared to the total area (substrate
peak + product peak).
Supporting Information Available: Elemental analysis data,
spectroscopic data, and X-ray crystallographic data. This material
is available free of charge via the Internet at http://pubs.acs.org.
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