Antidiabetic activity of passive nonsteroidal glucocorticoid receptor modulators

Article abstract of DOI:10.1021/jm050205o

Much has been learned about the consequences of glucocorticoid receptor antagonism by studying steroidal active antagonists such as RU-38486 (1). In the liver glucocorticoid receptor antagonism suppresses hepatic glucose production decreasing plasma glucose levels; however, extrahepatic antagonism produces several undesirable side effects including activation of the hypothalamic pituitary adrenal axis. A series of nonsteroidal passive N-(3-dibenzylamino-2- alkyl-phenyl)-methanesulfonamide glucocorticoid receptor modulators was discovered. Liver selective and systemically available members of this series were found and characterized in diabetes and side effect rodent models. A highly liver selective member of this series, acid 14, shows efficacy in the ob/ob model of diabetes. It lowers plasma glucose, cholesterol, and free fatty acid concentrations and reduces the rate of body weight gain. The structurally related systemically available passive modulator 12 lowers glucose, HbA _1c, triglyceride, free fatty acid, and cholesterol levels. Interestingly, it did not acutely activate the hypothalamic pituitary adrenal axis in unstressed CD-1 mice or have the abortive effects observed with 1. These results indicate that passive GR antagonists may have utility as antidiabetic agents.

Full text of DOI:10.1021/jm050205o

J. Med. Chem. 2005, 48, 5295-5304
5295
Antidiabetic Activity of Passive Nonsteroidal Glucocorticoid Receptor
Modulators
J. T. Link,*,† Bryan Sorensen,† Jyoti Patel,§ Marlena Grynfarb,‡ Annika Goos-Nilsson,‡ Jiahong Wang,†
Steven Fung,† Denise Wilcox,† Brad Zinker,† Phong Nguyen,† Bach Hickman,§ James M. Schmidt,§
Sue Swanson,§ Zhenping Tian,§ Thomas J. Reisch,§ Gary Rotert,§ Jia Du,§ Benjamin Lane,†
Thomas W. von Geldern,† and Peer B. Jacobson†
Metabolic Disease Research, Abbott Laboratories, 100 Abbott Park Road, Department 4CB, Building AP-10, Room L-14, Abbott
Park, Illinois 60064-6098, Abbott Laboratories, 100 Abbott Park Road, Abbott Park, Illinois 60064, and Karo Bio AB, Novum,
SE-141 57 Huddinge, Sweden
Received March 7, 2005
Much has been learned about the consequences of glucocorticoid receptor antagonism by
studying steroidal active antagonists such as RU-38486 (1). In the liver glucocorticoid receptor
antagonism suppresses hepatic glucose production decreasing plasma glucose levels; however,
extrahepatic antagonism produces several undesirable side effects including activation of the
hypothalamic pituitary adrenal axis. A series of nonsteroidal passive N-(3-dibenzylamino-2-
alkyl-phenyl)-methanesulfonamide glucocorticoid receptor modulators was discovered. Liver
selective and systemically available members of this series were found and characterized in
diabetes and side effect rodent models. A highly liver selective member of this series, acid 14,
shows efficacy in the ob/ob model of diabetes. It lowers plasma glucose, cholesterol, and free
fatty acid concentrations and reduces the rate of body weight gain. The structurally related
systemically available passive modulator 12 lowers glucose, HbA1c, triglyceride, free fatty acid,
and cholesterol levels. Interestingly, it did not acutely activate the hypothalamic pituitary
adrenal axis in unstressed CD-1 mice or have the abortive effects observed with 1. These results
indicate that passive GR antagonists may have utility as antidiabetic agents.
Glucocorticoids are steroid hormones that mediate
immune function, glucose homeostasis, fat distribution,
normal growth and development, stress responses, and
a multitude of other processes.1 They exert their action
via the glucocorticoid receptor (GR), which is a popular
target for pharmacologic intervention.2 Agonists, an-
tagonists, and modulators are studied as potential
therapies for multiple diseases. GR agonists have been
explored primarily as antiinflammatory agents and
immunosuppressants while antagonists have histori-
cally received less attention. The potent steroidal GR
antagonist 1 is known for its abortifacient effects due
to potent progesterone receptor (PR) antagonism (Figure
1).3 Treatment of humans, or rodent models of diabetes,
with the antagonist 1 lowers hepatic glucose pro-
duction (HGP) by reducing the expression of the key
gluconeogenic enzymes (PEPCK) and glucose-6-phos-
phatase (G6Pase).4,5 Extrahepatic GR antagonism, how-
Figure 1. The GR ligands 1, 2, cyproterone acetate (3), and
ever, has both desirable and undesirable consequences.
Potential benefits include increased insulin sensitivity,
fat redistribution, and effects on bone while detriments
include activation of the hypothalamic pituitary adrenal
(HPA) axis, diminution of immune response, and a
decreased stress response. Recently, mice with a selec-
tive inactivation of the GR gene in hepatocytes have
sulfonamide 4.
been created to determine the effect of hepatospecific
GR antagonism.6a Also, improvements in hyperglycemia
and hyperlipidemia after treatment with antisense
oligonucleotides that cause selective reduction of the GR
in liver and white adipose tissue of diabetic rodents have
been reported.6b We have also explored a number of
strategies to develop a treatment for type II diabetes
by hepatoselective GR antagonism. One is the liver
targeting of steroidal GR antagonists related to 1.7 In
particular, a steroidal bile acid conjugate was discovered
that gave a desirable profile in several pharmacology
* Author to whom correspondence should be addressed. Mailing
address: Metabolic Disease Research, Abbott Laboratories, 100 Abbott
Park Road, Dept 4CB, Bldg AP-10, Rm. L-14, Abbott Park, IL 60064-
6098. Phone: (847) 935-6806. Fax: (847) 938-1674. E-mail: james.link@
abbott.com.
† Metabolic Disease Research, Abbott Laboratories.
§ Abbott Laboratories.
‡ Karo Bio AB.
10.1021/jm050205o CCC: $30.25 © 2005 American Chemical Society
Published on Web 07/13/2005

5296 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 16
Scheme 1. GR Sulfonamide Modulator Synthesisa
Link et al.
a (a) SELECTFLUOR, CH3CN, reflux, 12 h; (b) TosCl, K2CO3, acetone, 60 °C, 12 h; allyl iodide, 60 °C, 24 h, 24% (2 steps); (c) KOH,
EtOH, H2O, 90 °C, 2 h, 68%; (d) 4-fluorobenzaldehyde, K2CO3, DMF, 100 °C, 14 h, 73%; (e) 2-methyl-3-nitroaniline, AcOH, DCE, rt, 4 h,
then Na(OAc)3BH, rt, 12 h, 88%; (f) 2,4-difluorobenzyl bromide, i-Pr2NEt, DMF, 100 °C, 18 h, 100%; (g) Pd(PPh3)4, PhSiH3, CH2Cl2, rt,
2 h, 92%; (h) ethyl glycolate, PPh3, di-tert-butyl azodicarboxylate, THF, rt, 12 h, 72%; (i) Fe, NH4Cl, EtOH, H2O, 80 °C, 1 h; (j) MsCl,
pyridine, rt, 12 h; 100% (2 steps); (k) NaOH, H2O, THF, EtOH, rt, 12 h, 97%.
Table 1. GR Sulfonamide Modulators 12-15
models.7d,e We have also reported the development of
several new classes of nonsteroidal modulators.8
A
groundbreaking novel class of modulators has also been
reported for the treatment of obesity.9 The results
obtained from the in vivo evaluation of key members of
a series of N-(3-dibenzylamino-2-alkyl-phenyl)-methane-
sulfonamides8a-e are summarized herein.
The GR receptor is a cytoplasmic nuclear hormone
receptor that is normally complexed to heat shock
proteins.10 Upon ligand binding, the GR-ligand complex
dissociates from these chaperones, dimerizes, and trans-
locates to the nucleus. There it interacts with DNA
sequences (termed glucocorticoid response elements
[GREs]) and transcription factors to initiate, or repress,
gene transcription.11 This complex signaling pathway
allows for the possibility that ligands might be dif-
ferentiated on the basis of binding potency, facility/
extent of dimerization, translocation, and DNA inter-
action of the modulator-GR complex. Ligands that
compete for agonist GR binding and agonist receptor
complex DNA response element binding are called
“active” antagonists, while ligands that only compete
for agonist GR binding have been termed “passive”
antagonists.12 Steroid 1 is an example of an active
antagonist. A number of passive antagonists have been
reported, including steroid (2)13 and cyproterone acetate
(3).14 Our efforts to convert a series of GR selective
nonsteroidal agonists into GR selective antagonists
resulted in the discovery of subnanomolar passive
modulators such as 4.8b,c We sought to discover GR
selective systemically available and liver selective pas-
sive GR modulators within this series to determine the
potential of these types of compounds as antidiabetic
agents.
compd
R1
R2
R3
12
13
14
15
F
F
H
F
F
OCH2CO2H
OCH2CO2H
H
Br
OCH2CO2H
Cl
OCH2CO2H
tosylate 6.16 Basic hydrolysis removes the tosylate, and
a nucleophilic aromatic substitution reaction of the
resultant phenol with 4-fluorobenzaldehyde yields al-
dehyde 8. Reductive amination with 2-methyl-3-nitro-
aniline provides aniline 9.17 Alkylation with 2,4-difluoro-
benzoldehyde yields allyl ether 10. Palladium mediated
removal of the allyl group18 followed by Mitsunobu
reaction19 with ethyl glycolate provides nitroarene 11.
Iron mediated reduction of the nitro group, aniline
mesylation, and ester hydrolysis then yields the acid 12.
In Vitro Assays. Nuclear hormone receptor binding
activity is assessed using radioligand binding assays.
In the case of the GR, the radioligand is the potent
agonist [3H]-dexamethasone. Receptor binding selectivi-
ties against progesterone (PR), androgen (AR), estrogen
(ERR and ERâ), mineralocorticoid (MR), and thyroid
hormone (TRR and TRâ) are determined similarly.
Functional activity is determined in three different
assays. GRAF cells are a genetically engineered mam-
malian cell line. They express h-GR and a transcrip-
tional unit composed of a GRE and core promoter
Chemistry. A typical modulator synthesis is detailed
in Scheme 1. Resorcinol 5 is fluorinated utilizing [1-
(chloromethyl)-4-fluoro-1,4-diazoniabicyclo[2.2.2]octane-
bis(tetrafluoroborate)] (SELECTFLUOR).15 Regio-
selective tosylation and allylation then provide aryl

Passive Glucocorticoid Receptor Modulators
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 16 5297
Table 2. Nuclear Hormone Assay Results for Active and Passive Antagonists 1-4 and 12-15
IC50 (nM)a
IC50 (nM)a
rat
VP16
EC50
VP16
agonism
(%)
h-GR
hepatocyte
r-GR
binding
h-PR
h-AR
binding
h-MR
binding
compd
binding
GRAF
TAT (µM)a
(nM)a
binding
1
1.1
2.4
0.6
4.8
1.8
5.4
2.6
4.8
43
0.27
7.3
>30
1.9
4.3
>30
2.3
0.7
7
59
1.4
2.4
1.0
0.2
ND
2.1
1.9
2.9
8.8
6000
6300
120
2
51
2400
550
180
140
1700
150
6500
1600
NDc
ND
4
86
NEb
NE
NE
NE
NE
NE
NE
NE
NE
NE
12
13
14
15
440
330
255
500
ND
ND
11700
3600
690
1000
a The data are the geometric mean of at least two experiments. b NE ) no effect up to 10-100 µM. c ND ) not determined.
Table 3. Metabolic Stability for Steroids 1 and 2 and
sequences linked to a reporter gene. The reporter gene
Sulfonamide Modulators 12 and 14
encodes a secreted form of alkaline phosphatase (ALP).
Antagonists are evaluated by their inhibition of dexa-
t1/2 (h)
rat
human
methasone-induced ALP expression in these cells. Com-
pounds are also tested in freshly isolated rat hepatocytes
for their effects on dexamethasone induced expression
of the GR regulated enzyme tyrosine aminotransferase
(TAT). A third functional assay is used to assess the
“active” or “passive” nature of a compound. VP16 is a
transcriptional activation domain that, if present in the
nucleus, activates GREs. Human liver hepatoma (HuH7)
cells were transiently transfected with VP16-GR fusion
protein expression plasmid and a reporter (GRE-Luc).
In this assay “passive” antagonists show little, or no,
response while “active” antagonists robustly stimulate
luciferase expression.
RLM
hepatocyte
HLM
hepatocyte
compd
stabilitya,b
stabilityc
stabilityb,d
stabilityc
1
<0.33
0.33
4.1
<0.1
<0.2
6.5
NDe
ND
2.3
ND
ND
9.1
2
12
14
4.6
9.9
8.9
31.1
a RLM ) rat liver microsomes. b 0.25 mg/mL of protein and 10
µM compound. c 5 x 105 cells/mL and 10 µM compound. d HLM )
human liver microsomes. e ND ) not determined.
Results and Discussion
These assays were used to profile the GR modulators
4, 12-15 (Table 1), antagonist 1, and steroid 2. Pub-
lished data for 3 indicates a GR Kd ) 45 nM and a PR
Kd ) 15 nM.14 The GR modulators 4 and 12-15 potently
inhibit GR binding (0.6-5.4 nM) and show good to
excellent selectivity over other nuclear hormone recep-
tors (Table 2). The steroids 1 and 3 have similar GR
binding potency but are less selective for PR in particu-
lar. Modulators 12, 13, and 15, where R3 ) OCH2CO2H,
are modestly PR selective (IC50 ) 140-180 nM) while
modulator 14, in which R2 ) OCH2CO2H, is more
selective. The steroid 2 is both potent and selective.
Modulator 4, 12-15, and the steroid 2 are less potent
in the GRAF and rat hepatocyte TAT assays than 1.
The VP16-GR assay indicates that the steroids 1 and 2
are “active” antagonists while all of the sulfonamide
modulators are “passive”.
Figure 2. Sprague-Dawley rat pharmacokinetics at 5 mpk
of GR modulators 12 and 14. 3 animals per time point.
Table 4. Liver and Plasma Levels for Modulators 12 and 14 (5
mpk Dose in Sprague-Dawley Rats)a
Since long duration blockade of hepatic GR is sought,
robust metabolic stability is required. The metabolic
profile of 1 has been thoroughly studied.20 It undergoes
rapid N-demethylation and other transformations. How-
ever, these metabolites also inhibit parent metabolism.
The primary metabolites also have potent GR activity.
The steroidal GR ligand (2) also does not have robust
metabolic stability. Modulators 12 and 14 have better
metabolic stability as assessed by incubation with rat
and human liver microsomes and hepatocytes (Table 3).
Identification of metabolites from 12 revealed that the
primary metabolic event was N-debenzylation, with
removal of the smaller benzyl group being the major
pathway.
1 h liver
1 h plasma
7 h liver
7 h plasma
compd level (µg/g) level (µg/mL) level (µg/g) level (µg/mL)
12 (iv)
14 (oral)
74
20
1.6
0.03
40
3.8
0.65
0.01
a 3 animals per time point for 12 and 14.
intravenous arm of a Sprague-Dawley rat 5 mpk PK
experiment reveals that the compound has a short half-
life (0.4 h), low plasma AUC, and high clearance (14.0
L/h‚kg). After 5 h, plasma levels were undetectable.
Liver levels at 1 h postdose were high (20 µg/g) and
decreased after 7 h (3.8 µg/g) (Table 4). The correspond-
ing oral dose had very low plasma levels and liver levels
similar to those for the intravenous route of administra-
tion. Throughout the pharmacokinetic study the com-
The pharmacokinetic profile of acid 14 suggests a high
degree of liver selectivity (Figure 2). Examination of the

5298 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 16
Link et al.
Table 5. Drug Level Data from 2 Week ob/ob Studies for
Modulators 12 and 14
as antidiabetic agents, acid modulators 12 and 14 were
tested in a diabetic animal model. The ob/ob mouse
model of diabetes was chosen since it is sensitive to GR
antagonists and allows for comparison to agents with
other mechanisms of action. The liver selective acid 14
was given to ob/ob mice orally b.i.d. for 13 days at 10
and 100 mpk. It dose dependently lowered postprandial
blood glucose levels, with a 46% decrease observed at
the high dose (Table 6). Body weight gain was also
reduced at 100 mpk, as were circulating aspartate
aminotransferase (AST)/alanine aminotransferase (ALT)
enzyme levels, liver weights, cholesterol concentrations
(89%), and free fatty acid (FFA) levels (39%). The
decreases in AST and ALT levels are reassuring as the
amount of both can increase in the presence of a
hepatotoxic agent. Epididymal poststudy fat pad weights
were unaltered by compound treatment. The end of
study drug levels (2 h past final dose) also supported a
highly liver selective profile with high liver and low
plasma drug levels (Table 5). Systemically available
modulator 12 also demonstrates dose dependent glucose
lowering without affecting insulin levels in a 14 day
study (Table 7). Decreases in liver glycogen levels and
in liver TAT levels were observed. This is consistent
with the proposed mechanism of action. A decrease in
the rate of weight gain relative to control animals was
seen. At the high dose, triglycerides and FFAs were
lowered (although FFA levels were similar at both 30
and 100 mpk). End of study drug levels indicate hepato-
selective distribution; however, plasma drug levels are
also significant.
dose
(mpk)
liver level
plasma level
compd
(µg/g)a
(µg/mL)a
12
12
14
14
30
20
0.82
6
100
10
157
NDb
207
0
100
1.6
a 2 h after the final study dose. b ND ) not determined.
pound maintains a high liver to plasma distribution
consistent with a hepatoselective profile. Although liver
selective, modulator 12 also achieves high plasma levels
following oral dosing at 5 mpk in Sprague-Dawley rats.
Liver levels 7 h postdose were high (40 µg/g) as was the
plasma AUC (3.85 µg‚h/mL) from a 5 mpk oral dose.
Oral bioavailability of 12 was moderate (F ) 40%).
In Vivo Results. Fasted Sprague-Dawley rats are
used to determine whether the modulators are able to
block hepatic GR in vivo. An oral challenge with 10 mpk
of the potent GR agonist prednisolone induces expres-
sion of hepatic tyrosine aminotransferase (TAT) and
increases hepatic glycogen levels (as assessed in liver
biopsy punches 6 h postdose). It also induces severe
lymphopenia. Oral administration of 1 (100 mpk) 1 h
prior to dosing completely blocks both the hepatic (TAT
and glycogen) and peripheral (lymphopenic) responses.
Similar administration of the passive antagonist 12 at
100 mpk does not antagonize either response. Interest-
ingly, the passive antagonist cyproterone acetate (3) has
been shown to inhibit transactivation of liver GR.14 In
the reported experiment, fasted male Sprague-Dawley
rats were given compound (80 mpk), dexamethasone 10
min later (0.01 mg/kg), and 4 h later TAT activity was
measured. It was active in this less stringent challenge
model (dose of the agonist dexamethasone is signifi-
cantly lower) but less active than 1 at one-half of the
dose. No drug levels were reported from the study. In
combination, these two experiments suggest that a
passive antagonist can block hepatic GR. However, the
blockade is not as strong as is observed with an active
antagonist.
Due to the efficacy observed with modulator 12, a
longer 30 day study in ob/ob mice was conducted. At
the end of the study glucose levels were lowered by 60-
70% relative to vehicle leading to a 1.7% reduction in
HbA1c levels. In addition, triglyceride levels dropped
55%, cholesterol was lowered 33%, and free fatty acid
levels decreased 25% relative to vehicle (see Table 9
for details). A reduction in ALT and AST was also
observed, which suggests that 12 is not highly hepato-
toxic. This is the first demonstration of antidiabetic
activity in a rodent model of diabetes with a passive GR
antagonist.
To determine if passive antagonists provided suf-
ficient blockade of hepatic GR to be of potential utility
Table 6. 13 Day ob/ob Study for Vehicle, Steroid 1, and Modulator 14 Treated ob/ob Micea
body wt (g)
glucose
(mg/dL)
insulin
(ng/mL)
FFA
(mequiv/L)
ALT
(units/mL)
AST
(units/mL)
animal
compd
po dose
b.i.d.
100 mpk b.i.d.
10 mpk b.i.d.
100 mpk b.i.d.
b.i.d.
initial
final
ob/ob mice
ob/ob mice
ob/ob mice
ob/ob mice
lean mice
vehicle
14
14
1
vehicle
377 ( 24
290 ( 19
333 ( 34
141 ( 11
190 ( 7
19.7 ( 2.2
28.1 ( 4.5
20.0 ( 2.6
7.5 ( 0.7
0.8 ( 0.1
45.8 ( 1.2
46.0 ( 0.9
45.4 ( 0.8
46.1 ( 1.3
26.3 ( 0.7
49.9 ( 1.3
46.6 ( 0.5
49.3 ( 0.7
48.2 ( 1.3
27.0 ( 0.8
1.3 ( 0.3
1.0 ( 0.3
1.3 ( 0.7
1.5 ( 0.2
0.52 ( 0.02
840 ( 74
282 ( 40
786 ( 200
1110 ( 80
63 ( 11
474 ( 35
330 ( 54
420 ( 80
310 ( 30
132 ( 11
a 8-10 animals per time point with results reported with standard error.
Table 7. 14 Day ob/ob Data for Vehicle, Steroid 1, and Modulator 12a
liver TAT
(nmol of
glycogen
body wt (g)
glucose
(mg/dL)
insulin
(ng/mL)
(mg/g
cholesterol pHB/min/mg
FFA
(mequiv/L)
animal
compd
po dose
initial
final
of liver)
(mg/dL)
of protein)
ob/ob mice vehicle b.i.d.
342 ( 21 38.2 ( 0.6.9 40.0 ( 0.9 49.5 ( 1.1 64.3 ( 3.2 223 ( 21
100 mpk b.i.d. 233 ( 15 32.8 ( 6.3 40.0 ( 0.7 46.9 ( 0.7 44.2 ( 4.0 134 ( 21
2.7 ( 0.3
2.4 ( 0.3
1.7 ( 0.3
1.8 ( 0.2
2.7 ( 0.3
1.26 ( 0.15
1.01 ( 0.07
0.99 ( 0.11
0.84 ( 0.09
0.84 ( 0.09
ob/ob mice 12
ob/ob mice 12
30 mpk b.i.d. 314 ( 23 25.0 ( 5.6
100 mpk b.i.d. 170 ( 6 14.0 ( 2.8
lean mice vehicle b.i.d. 170 ( 9 1.3 ( 0.3
41.5 ( 0.5 49.2 ( 0.8 54.6 ( 1.7 225 ( 16
41.7 ( 0.5 51.2 ( 0.4 30.5 ( 1.8 366 ( 25
23.0 ( 0.9 26.2 ( 1.2 74.2 ( 3.0 151 ( 6
ob/ob mice
1
a 8-10 animals per time point with results reported with standard error.

Passive Glucocorticoid Receptor Modulators
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 16 5299
Table 8. Plasma Protein Binding of [3H]-12a by Equilibrium
Dialysis
Experimental Section
Compound Preparation. Unless otherwise specified, all
solvents and reagents were obtained from commercial suppli-
ers and used without further purification. All reactions were
performed under nitrogen atmosphere unless specifically
noted. Normal-phase flash chromatography was done using
Merck silica gel 60 (230-400 mesh) from E.M. Science, or was
performed on a hybrid system employing Gilson components
and Biotage prepacked columns. Reversed-phase chromatog-
raphy was performed using a Gilson 215 solvent handler-
driven HPLC system (CH3CN:0.1% TFA in H2O or CH3CN:
NH4OAc in H2O) on a YMC ODS Guardpak column. Analytical
LC-MS was performed on a Shimadzu HPLC system with a
Zorbax SB-C8, 5 µm 2.1 × 50 mm (Agilent technologies)
column, with a PE Sciex, API 150EX single quadropole mass
spectrometer, at a flow rate of 1 mL/min (0.05% NH4OAc-
buffer:MeCN and 0.05% HCOOH in H2O:MeCN). 1H NMR
spectra were recorded at 300 and 500 MHz; all values are
referenced to tetramethylsilane as internal standard and are
reported as shift (multiplicity, coupling constants, proton
count). Mass spectral analysis is accomplished using fast atom
bombardment (FAB-MS), electrospray (ESI-MS), or direct
chemical ionization (DCI-MS) techniques. All elemental analy-
ses are consistent with theoretical values to within +0.4%
unless indicated.
4-Fluorobenzene-1,3-diol. [1-(Chloromethyl)-4-fluoro-1,4-
diazoniabicyclo[2.2.2]octane bis(tetrafluoroborate)] (SELECT-
FLUOR) (28.4 g., 80.1 mmol) in anhydrous acetonitrile (100
mL) was treated with resorcinol (8.82 g., 80.1 mmol) and
heated at 100 °C overnight. The reaction was diluted with
diethyl ether, washed twice with H2O, washed twice with
saturated NaHCO3, rinsed with brine, dried (Na2SO4), and
filtered, and the filtrate was concentrated under reduced
pressure to provide a mixture of isomers: 1H NMR (300 MHz,
CDCl3) δ 6.94 (dd, J ) 14, 10 Hz,1 H), 6.51 (dd, J ) 8, 3 Hz,1
H), 6.29 (m, 1 H), 5.12 (s, 1 H), 4.64 (s, 1 H); MS (DCI) m/z
129 (M + H)+.
concentration
species
(µg/mL)
% bound
rat
mouse
human
2
2
2
99.9
99.7
99.9
a For the synthesis of [3H]-12 see the Experimental Section.
One of the primary side effect concerns associated
with GR antagonism is activation of the HPA axis. To
determine the potential of a passive GR modulator to
impact the HPA axis, acid modulator 12 (which achieves
significant plasma levels) was given to male CD-1 mice
at 100 mpk. Two hours later, plasma levels of adreno-
corticotropic hormone (ACTH) and corticosterone levels
were analyzed. No change relative to vehicle was
observed, indicating minimal acute impact upon the
HPA axis under unstressed conditions. In contrast, the
steroidal active antagonist 1 causes a dramatic increase
in both plasma ACTH and corticosterone levels. Al-
though this experiment does not demonstrate that
modulator 12 has no effect on the HPA axis, it does show
that passive GR modulators and antagonists can have
significantly different profiles.
Steroid 1 is also an abortive agent whose effect is
mediated through its PR activity. Modulator 12 was
checked for in vivo effects on PR, for which it has greater
in vitro selectivity relative to 1 (Table 2). At 30 and 100
mpk q.d. oral dosing for 5 days in pregnant rats, no
effects on uterine weight or progesterone levels were
observed. In the same experiment, 1 reduced both by
50% at 3 mpk.
3-(Allyloxy)-4-fluorophenyl 4-methylbenzenesulfonate
(6). p-Toluenesulfonyl chloride (15.2 g, 80.1 mmol) was added
to a solution of 4-fluorobenzene-1,3-diol (10.2 g, 80.1 mmol)
and K2CO3 (33.2 g, 240 mmol) in acetone (160 mL). The
reaction mixture was heated at 60 °C overnight and treated
with allyl iodide (7.30 mL, 80.1 mmol), and heating was
continued at 60 °C for 24 h.16
Determination of the protein binding of one of the
lipophilic acids using a radiolabeled variant (12, c log
P ) 6.17) indicates a low free drug fraction in rodent
and human plasma (Table 8). This property may con-
tribute to the lack of peripheral effects on the HPA axis
or PR relative to 1.
The reaction mixture was cooled
to room temperature, quenched with aqueous NH4Cl, and
concentrated under reduced pressure. The crude products were
diluted with ethyl acetate, extracted with H2O, washed with
brine, dried (Na2SO4), and concentrated under reduced pres-
sure. The residue was purified by flash chromatography on a
prepacked Biotage column (hexane f 9:1 hexane:ethyl acetate)
on silica gel to provide benzenesulfonate (6) (6.24 g, 24%) as a
clear colorless oil: 1H NMR (300 MHz, CDCl3) δ 7.70 (d, J )
12 Hz, 2 H), 7.33 (d, J ) 12 Hz, 2 H), 6.95 (dd, J ) 14, 12 Hz,
1 H), 6.66 (dd, J ) 12, 3 Hz, 1 H), 6.45 (m, 1 H), 5.98 (m, 1 H),
5.33 (m, 2 H), 4.48 (m, 2 H), 2.44 (s, 3 H); MS (ESI) m/z 323
(M + H)+.
Conclusion. Starting from a nonsteroidal GR agonist
a series of passive GR modulators was discovered.8a-c
Analogues were found that are GR selective, metaboli-
cally stable, bioavailable, and hepatoselectively distrib-
uted. These passive modulators lower blood glucose
levels in ob/ob mice. Furthermore, improvement in the
lipid profile was observed. Surprisingly, even a com-
pound such as 12 that achieves significant plasma
exposure fails to acutely activate the HPA axis in
unstressed CD-1 mice, whereas the active antagonist
steroid 1 strongly affects it. Overall, these results
suggest that passive GR antagonists may be of greater
potential interest as antidiabetic agents than is cur-
rently suspected. It remains to be seen whether a GR
ligand can become an antidiabetic therapy for modulat-
ing hepatic glucose production.21
3-(Allyloxy)-4-fluorophenol (7). Potassium hydroxide
(10.9 g, 194 mmol) was added to a solution of 3-(allyloxy)-4-
fluorophenyl 4-methylbenzenesulfonate (6.24 g, 19.4 mmol) in
EtOH (20 mL) and H2O (20 mL). The reaction mixture was
heated to 90 °C for 2 h, cooled to room temperature, and
concentrated under reduced pressure. The residue was purified
by flash chromatography (9:1 f 4:1 hexane:ethyl acetate) on
silica gel to provide the phenol (7) (2.20 g, 68%): 1H NMR (300
Table 9. 30 Day ob/ob Data for Vehicle and Modulator 12a
HbA1c
triglycerides
(mg/dL)
FFA
cholesterol
(mg/dL)
AST ALT
animal
compd
po dose
b.i.d.
100 mpk b.i.d.
30 mpk b.i.d.
b.i.d.
(%)
(mequiv/L)
(units/mL)
ob/ob mice
ob/ob mice
ob/ob mice
lean mice
vehicle
12
12
vehicle
9.5 ( 0.4
7.7 ( 0.3
8.9 ( 0.4
4.8 ( 0.2
272 ( 33
163 ( 22
214 ( 82
73 ( 12
1.32 ( 0.09
1.27 ( 0.05
0.99 ( 0.13
0.56 ( 0.04
291 ( 17
239 ( 11
266 ( 18
133 ( 5
322 ( 30
303 ( 45
395 ( 53
33 ( 4
a 9-12 animals per time point with results reported with standard error.

5300 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 16
Link et al.
MHz, CDCl3) δ 6.93 (dd, J ) 16, 10 Hz, 1 H), 6.49 (dd, J ) 10,
3 Hz, 1 H), 6.36 (m, 1 H), 6.06 (m, 1 H), 5.37 (m, 2 H), 4.61 (s,
1 H), 4.57 (m, 2 H); MS (DCI) m/z 169 (M + H)+.
methylene chloride:hexanes) to provide the ester (11) (3.25 g,
72%): 1H NMR (300 MHz, CDCl3) δ 7.53 (dd, J ) 8, 2 Hz, 1
H), 7.15 (m, 5 H), 7.03 (dd, J ) 16, 12 Hz, 1 H), 6.88 (d, J )
12 Hz, 2 H), 6.75 (m, 2 H), 6.61 (dd, J ) 10, 3 Hz, 1 H), 6.55
(m, 1 H), 4.62 (s, 2 H), 4.22 (q, J ) 9 Hz, 2 H), 4.14 (s, 2 H),
4.08 (s, 2 H), 2.54 (s, 3 H), 1.26 (t, J ) 10 Hz, 3 H); MS (ESI+)
m/z 581 (M + H)+.
4-(3-(Allyloxy)-4-fluorophenoxy)benzaldehyde (8). 4-
Fluorobenzaldehyde (1.41 mL, 13.1 mmol) was added to a
solution of 3-(allyloxy)-4-fluorophenol (2.20 g, 13.1 mmol) and
K2CO3 (5.40 g, 39.3 mmol) in DMF (13 mL). The reaction
mixture was heated to 100 °C for 14 h and was cooled to room
temperature. The crude products were diluted with ethyl
acetate, washed with H2O (2x) and brine, dried (Na2SO4), and
concentrated under reduced pressure. The residue was purified
by flash chromatography (40:1 f 9:1 hexane:ethyl acetate) on
silica gel to provide aldehyde (8) (2.59 g, 73%): MS (DCI) m/z
273 (M + H)+.
N-(4-(3-(Allyloxy)-4-fluorophenoxy)benzyl)-N-(2-meth-
yl-3-nitrophenyl)amine (9). 2-Methyl-3-nitroaniline (1.45 g,
9.52 mmol) and 4-(3-(allyloxy)-4-fluorophenoxy)benzaldehyde
(2.59 g, 9.52 mmol) in dichloroethane (10 mL) were treated
with glacial acetic acid (2.18 mL, 38.1 mmol). The yellow
reaction mixture was stirred for 4 h at room temperature,
treated with sodium triacetoxyborohydride (4.04 g, 19.0 mmol),
and stirred overnight at room temperature. The mixture was
poured into saturated aqueous NaHCO3 (50 mL) and extracted
with ethyl acetate (50 mL). The organic phase was washed
with brine (50 mL), dried (Na2SO4), and filtered, and the
filtrate was concentrated under reduced pressure. The residue
was purified by flash chromatography (40:1 f 6:1 hexane:ethyl
acetate) on silica gel to provide nitroarene (9) (3.41 g, 88%):
1H NMR (300 MHz, CDCl3) δ 7.32 (d, J ) 10 Hz, 2 H), 7.15
(m, 2 H), 7.02 (m, 2 H), 6.94 (d, J ) 10 Hz, 2 H), 6.78 (m, 1 H),
6.67 (dd, J ) 9, 3 Hz, 1 H), 6.52 (m, 1 H), 6.03 (m, 1 H), 5.34
(m, 2 H), 4.56 (m, 2 H), 4.39 (s, 2 H), 2.22 (s, 3 H); MS (ESI-)
m/z 407 (M-H)-.
Ethyl (5-(4-(((2,4-Difluorobenzyl)(2-methyl-3-((methyl-
sulfonyl)amino)phenyl)amino)methyl)phenoxy)-2-fluoro-
phenoxy)acetate. Ethyl (5-(4-(((2,4-difluorobenzyl)(2-methyl-
3-nitrophenyl)amino)methyl)phenoxy)-2-fluorophenoxy)ace-
tate (3.25 g, 5.60 mmol) and NH4Cl (208 mg, 3.92 mmol) in
ethanol (10 mL) and water (3 mL) were treated with iron
powder (2.19 g, 39.2 mmol) and heated at 80 °C for 1 h. The
reaction mixture was cooled to room temperature, diluted with
CH2Cl2 (100 mL), and filtered through Celite with CH2Cl2
washes, and the filtrate was concentrated under reduced
pressure to provide the aniline compound, which was used
without further purification. The residue was dissolved in
anhydrous pyridine (10 mL), and to this was added methane-
sulfonyl chloride (0.52 mL, 6.72 mmol). The reaction mixture
was stirred overnight at room temperature. The mixture was
diluted with ethyl acetate, washed with H2O, 1 N H3PO4, and
brine, dried (Na2SO4), and filtered, and the filtrate was
concentrated under reduced pressure. The residue was purified
by flash chromatography (9:1 f 6:4 hexane:ethyl acetate) on
silica gel to provide the sulfonamide (3.52 g, 100%): 1H NMR
(300 MHz, CDCl3) δ 7.22-7.00 (m, 6 H), 6.92 (m, 1 H), 6.86
(d, J ) 10 Hz, 2 H), 6.73 (m, 2 H), 6.56 (m, 2 H), 6.19 (s, 1 H),
4.62 (s, 2 H), 4.22 (q, J ) 9 Hz, 2 H), 4.07 (s, 2 H), 4.02 (s, 2
H), 2.95 (s, 3 H), 2.30 (s, 3 H), 1.25 (t, J ) 9 Hz, 3 H); MS
(ESI+) m/z 629 (M + H)+.
(5-(4-(((2,4-Difluorobenzyl)(2-methyl-3-((methylsulfon-
yl)amino)phenyl)amino)methyl)phenoxy)-2-fluorophe-
noxy)acetic Acid (12). A solution of ethyl (5-(4-(((2,4-difluoro-
benzyl)(2-methyl-3-((methylsulfonyl)amino)phenyl)amino)-
methyl)phenoxy)-2-fluorophenoxy)acetate (3.52 g, 5.60 mmol)
in THF (10 mL) was treated with aqueous 4 N sodium
hydroxide (0.70 mL, 28.0 mmol) and ethanol (2 mL) and stirred
overnight at room temperature. The mixture was concentrated
under reduced pressure, diluted with water, and extracted
with diethyl ether. The aqueous phase was acidified with 1 N
aqueous H3PO4 and extracted with chloroform. The chloroform
phase was dried (Na2SO4) and filtered, and the filtrate was
concentrated under reduced pressure. The residue was purified
by flash chromatography (9:1 methylene chloride:methanol)
on silica gel to provide the acid (12) (3.25 g, 97%) as a light
brown solid: 1H NMR (500 MHz, DMSO) δ 8.95 (s, 1 H), 7.26
(d, J ) 8.4 Hz, 2 H), 7.21 (m, 2 H), 7.11 (m, 1 H), 7.05 (d, J )
7.8 Hz, 1 H), 7.00 (d, J ) 7.5 Hz, 1 H), 6.95 (m, 2 H), 6.88 (d,
J ) 8.4 Hz, 2 H), 6.82 (m, 1 H), 6.48 (m, 1 H), 4.75 (s, 2 H),
4.07 (s, 2 H), 4.05 (s, 2 H), 2.90 (s, 3 H), 2.33 (s, 3 H); MS
(ESI+) m/z 601 (M + H)+. Anal. Calcd for C30H27F3N2O6S‚
1H2O: C, 58.25; H, 4.73; N, 4.53. Found: C, 58.47; H, 4.78; N,
4.30. Analytical HPLC using a YMC CombiScreen ODS-A
analytical column and 20% acetonitrile/0.1% TFA aqueous
buffer to 100% acetonitrile gradient showed >95% purity.
N-(4-(3-(Allyloxy)-4-fluorophenoxy)benzyl)-N-(2,4-di-
fluorobenzyl)-N-(2-methyl-3-nitrophenyl)amine (10). N-
(4-(3-(Allyloxy)-4-fluorophenoxy)benzyl)-N-(2-methyl-3-nitrophen-
yl)amine (3.41 g, 8.36 mmol) and diisopropylethylamine (2.91
mL, 16.7 mmol) in DMF (10 mL) were treated with 2,4-
difluorobenzyl bromide (1.29 mL, 10.0 mmol) and heated for
18 h at 100 °C. After cooling to room temperature, the mixture
was diluted with ethyl acetate (50 mL). The mixture was
washed with saturated ammonium chloride (50 mL), water (2
× 25 mL), and brine (25 mL), dried (Na2SO4), and filtered,
and the filtrate was concentrated under reduced pressure. The
residue was purified by flash chromatography (silica gel, 19:1
f 9:1 hexane:ethyl acetate) to provide dibenzylaniline (10) as
a yellow solid (4.46 g, 100%): 1H NMR (300 MHz, CDCl3) δ
7.52 (dd, J ) 10, 2 Hz, 1 H), 7.15 (m, 5 H), 7.02 (dd, J ) 16, 12
Hz, 1 H), 6.89 (d, J ) 12 Hz, 2 H), 6.75 (m, 2 H), 6.65 (dd, J )
10, 3 Hz, 1 H), 6.48 (m, 1 H), 6.01 (m, 1 H), 5.31 (m, 2 H), 4.53
(m, 2 H), 4.14 (s, 2 H), 4.09 (s, 2 H), 2.53 (s, 3 H); MS (ESI+)
m/z 535 (M + H)+.
5-(4-(((2,4-Difluorobenzyl)(2-methyl-3-nitrophenyl)-
amino)methyl)phenoxy)-2-fluorophenol. N-(4-(3-(Allyloxy)-
4-fluorophenoxy)benzyl)-N-(2,4-difluorobenzyl)-N-(2-methyl-3-
nitrophenyl)amine (4.46 g, 8.36 mmol) and Pd(PPh3)4 (1.00 g,
0.836 mmol) in CH2Cl2 (10 mL) were treated with phenylsilane
(2.08 mL, 16.7 mmol) and stirred for 2 h at room temperature.
The reaction mixture was concentrated under reduced pres-
sure, and the residue was purified by flash chromatography
(silica gel, 19:1 f 6:1 hexane:ethyl acetate) to provide the
phenol as a yellow oil (3.87 g, 92%).
Ethyl (5-(4-(((2,4-Difluorobenzyl)(2-methyl-3-nitrophen-
yl)amino)methyl)phenoxy)-2-fluorophenoxy)acetate (11).
A solution of 5-(4-(((2,4-difluorobenzyl)(2-methyl-3-nitrophenyl)-
amino)methyl)phenoxy)-2-fluorophenol (3.87 g, 7.73 mmol),
triphenylphosphine (4.05 g, 15.5 mmol), and di-tert-butyl
azodicarboxylate (2.67 g, 11.6 mmol) in anhydrous THF (16
mL) was treated with ethyl glycolate (0.92 mL, 9.67 mmol).
The reaction mixture was stirred overnight at room temper-
ature. The mixture was diluted with ethyl acetate, washed
with H2O and brine, dried (Na2SO4), and filtered, and the
filtrate was concentrated under reduced pressure. The residue
was purified by flash chromatography (silica gel, 1:1 f 7:3
Compounds 13-15 and the [H3]-12 precursor 16 were
prepared using similar methods and conditions.
(2-Bromo-5-(4-(((2,4-difluorobenzyl)(2-methyl-3-((meth-
ylsulfonyl)amino)phenyl)amino)methyl)phenoxy)phe-
noxy)acetic Acid (13). Light brown solid: 1H NMR (500
MHz, CDCl3) δ 7.48 (d, J ) 8.7 Hz, 1 H), 7.35 (dd, J ) 15, 8.4
Hz, 1 H), 7.28 (dd, J ) 7.8, 1.2 Hz, 1 H), 7.21 (m, 2 H), 7.09 (s,
1 H), 7.03 (d, J ) 8.4 Hz, 2 H), 6.78 (dd, J ) 8.4, 2.2 Hz, 1 H),
6.73 (m, 1 H), 6.63 (dd, J ) 8.6, 2.7 Hz, 1 H), 6.55 (d, J ) 2.5
Hz, 1 H), 4.64 (s, 2 H), 4.35 (s, 2 H), 4.20 (s, 2 H), 2.89 (s, 3 H),
2.01 (s, 3 H); MS (ESI+) m/z 663 (M + H)+; HRMS calcd for
C30H27N2O6S1Br1F2 660.0741, found 660.0762. Analytical HPLC
using a YMC CombiScreen ODS-A analytical column and 20%
acetonitrile/0.1% TFA aqueous buffer to 100% acetonitrile
gradient showed >95% purity.
(4-{4-[(Benzyl{2-methyl-3-[(methylsulfonyl)amino]-
phenyl}amino)methyl]phenoxy}phenoxy)acetic Acid (14).

Passive Glucocorticoid Receptor Modulators
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 16 5301
Scheme 2. Synthesis of [3H]-12
Light brown solid: 1H NMR (400 MHz, CDCl3) δ 7.25 (m, 6
H), 7.15 (m, 3 H), 6.95 (m, 5 H), 6.83 (d, J ) 8.8 Hz, 2 H), 6.29
(s, 1 H), 4.66 (s, 2 H), 4.05 (s, 2 H), 4.01 (s, 2 H), 2.94 (s, 3 H),
2.33 (s, 3 H); MS (ESI+) m/z 547 (M + H)+; HRMS calcd for
C30H31N2O6S1 547.1897, found 547.1899. Anal. Calcd for
C30H30N2O6S‚1.5 H2O: C, 62.71; H, 5.81; N, 4.88. Found: C,
62.91; H, 5.77; N, 4.63. Analytical HPLC using a YMC
CombiScreen ODS-A analytical column and 20% acetonitrile/
0.1% TFA aqueous buffer to 100% acetonitrile gradient showed
>95% purity.
(2-Chloro-5-{4-[((2,4-difluorobenzyl){2-methyl-3-
[(methylsulfonyl)amino]phenyl}amino)methyl]phenoxy}-
phenoxy)acetic Acid (15). Light brown solid: 1H NMR (300
MHz, CDCl3) δ 7.32 (d, J ) 8.8 Hz, 2 H), 7.25 (m, 3 H), 7.20
(m, 2 H), 6.96 (d, J ) 8.5 Hz, 2 H), 6.81 (d, J ) 8.8 Hz, 2 H),
6.77 (s, 1 H), 6.75 (m, 1 H), 6.62 (d, J ) 2.7 Hz, 1 H), 4.65 (s,
2 H), 4.17 (s, 2 H), 3.98 (s, 2 H), 2.91 (s, 3 H), 1.97 (s, 3 H); MS
(ESI+) m/z 617 (M + H)+. Anal. Calcd for C30H27Cl1F2N2O6S‚
1.5H2O: C, 55.94; H, 4.69; N, 4.35. Found: C, 56.02; H, 4.35;
N, 4.25. Analytical HPLC using a YMC CombiScreen ODS-A
analytical column and 20% acetonitrile/0.1% TFA aqueous
buffer to 100% acetonitrile gradient showed >95% purity.
(total activity ) 69.5 mCi). Radio-HPLC analysis (from 0.1%
TFA:60% CH3CN to 75% CH3CN in 15 min on Phenomenex
Luna C18(2), 5 µm, 4.6 × 250 mm at 1 mL/min, 220 nM UV)
showed ∼89% radiochemical purity. The UV trace showed
about equal amounts of starting material and product. About
6 mL of [3H]-12 was evaporated to dryness, and the residue
was dissolved in 10 mL of acetonitrile:water:TFA (52:48:0.1).
Approximately 2 mL per injection was made onto a Phenom-
enex Synergi Max-RP column (4 µm, 250 mm × 21.2 mm i.d.)
using a Shimadzu HPLC system. [3H]-12 was eluted at a flow
rate of approximately 20 mL/min by an isocratic mobile phase
consisting of acetonitrile:water:TFA (56:44:0.1). Peak detection
and chromatograms were obtained using a Shimadzu variable
wavelength UV detector set at 220 nm and Shimadzu Class-
VP software. The fractions containing [3H]-12 were collected
at approximately 24 min using a Shimadzu fraction collector.
Fractions were combined and the solvents were evaporated
in vacuo. The residue was dissolved in 200 proof ethanol (20
mL).
The synthesis of compound 4 has been reported previously
(by methods similar to those for the preparation of sulfonamide
12).8c A procedure that is not reported herein and character-
ization information are included below.
Ethyl 4-[3-(4-{[Benzyl(2-methyl-3-nitrophenyl)amino]-
methyl}phenoxy)phenoxy]butanoate. A solution of N-(3-
{benzyl[4-(3-hydroxyphenoxy)benzyl]amino}-2-methyl-phenyl)-
methanesulfonamide (0.100 g, 0.227 mmol) in DMF (0.57 mL)
was treated with NaH (0.011 g, 0.27 mmol, 60% dispersion).
After 15 min, ethyl 4-bromobutyrate (0.039 mL, 0.27 mmol)
was added and the reaction mixture was stirred overnight. The
crude products were diluted with diethyl ether, washed with
saturated NH4Cl, extracted twice with H2O, washed with
brine, dried (Na2SO4), and concentrated under reduced pres-
sure. The residue was purified by flash chromatography
on silica gel (hexane f 7:1 hexane:ethyl acetate) to provide
ethyl 4-[3-(4-{[benzyl(2-methyl-3-nitrophenyl)amino]methyl}-
phenoxy)phenoxy]butanoate as a yellow oil.
4-(3-{4-[(Benzyl{2-methyl-3-[(methylsulfonyl)amino]-
phenyl}amino)methyl]phenoxy}phenoxy)butanoic Acid.
1H NMR (300 MHz, DMSO-d6): δ 8.97 (s, 1 H), 7.23 (m, 8 H),
6.88-7.08 (m, 5 H), 6.68 (dd, J ) 8.3, 2.5 Hz, 1 H), 6.49 (m, 2
H), 4.06 (s, 2 H), 4.02 (s, 2 H), 3.93 (t, J ) 6.4 Hz, 2 H), 3.33-
3.61 (br s, 1 H), 2.91 (s, 3 H), 2.40 (s, 3 H), 2.35 (t, J ) 7.3 Hz,
2 H), 1.90 (m, 2 H). MS (ESI): m/z 575 (M + H+).
Determination of Purity and Specific Activity. [3H]-
12 was assayed using an Agilent 1100 series HPLC system
consisting of a quaternary pump, an autosampler, and a
photodiode array UV detector. A Packard Radiomatic A 500
radioactivity detector was connected to the HPLC system. For
radiodetection, a 500 µL flow cell and a 3:1 ratio of Ultima-
Flo M scintillation cocktail to HPLC mobile phase were used.
The analyses were performed using a Phenomenex Synergi,
Max-RP column (4 µm, 250 mm × 4.6 mm i.d.). The mobile
phase was isocratic at 52% B for 25 min followed by ramping
to 90% B in 1 min and holding at 90% B for 9 min, where
mobile phase A ) 0.1% TFA/water and mobile phase B ) 0.1%
TFA/acetonitrile. The flow rate was set at approximately 1 mL/
min, and the UV detection was set at 220 nm.
The radiochemical purity of [3H]-12 was found to be >99%.
Confirmation of the identity of [3H]-12 was obtained when 12
had an identical retention time. The specific activity was
determined to be 17.7 Ci/mmol by measuring the mass and
radioactivity concentrations of [3H]-12 dissolved in the mobile
phase. The concentration of radioactivity was determined by
liquid scintillation counting of an accurately measured aliquot.
The mass concentration was measured by comparing the
HPLC-UV peak area of an accurately measured aliquot to a
standard solution.
4-{[4-(3-{4-[(Benzyl{2-methyl-3-[(methylsulfonyl)amino]-
phenyl}amino)methyl]phenoxy}phenoxy)butanoyl]-
amino}butanoic Acid (4). 1H NMR (300 MHz, DMSO-d6):
δ 8.97 (s, 1 H), 7.83 (t, J ) 5.8 Hz, 1 H), 7.13-7.34 (m, 8 H),
6.87-7.09 (m, 5 H), 6.67 (m, 1 H), 6.47 (m, 2 H), 4.27-4.62
(br s, 1 H), 4.06 (s, 2 H), 4.03 (s, 2 H), 3.91 (t, J ) 6.4 Hz, 2 H),
3.03 (dd, J ) 12.5, 6.8 Hz, 2 H), 2.91 (s, 3 H), 2.40 (s, 3 H),
2.19 (t, J ) 7.5 Hz, 4 H), 1.88 (m, 2 H), 1.59 (m, 2 H). MS
(APCI): m/z 660 (M + H+). HRMS calcd for C36H42N3O7S1
660.2738, found 660.2739. Analytical HPLC using a YMC
CombiScreen ODS-A analytical column and 20% acetonitrile/
0.1% TFA aqueous buffer to 100% acetonitrile gradient showed
>95% purity.
Assay Protocols. GR Binding Assay. 3H-Dexamethasone
(TRK 645, “3H-dex”) was purchased from Pharmacia Amer-
sham, Uppsala, Sweden. Dexamethasone (“dex”) was pur-
chased from Sigma. The Costar 96-well polypropylene plates
(3794 or 3365) were purchased from Life Technologies AB,
Ta¨by, Sweden. The GF/B filter (1450-521), filter cassette (1450-
104), MeltiLex scintillating wax (1450-441), sample bag (1450-
42), Microbeta 1450-PLUS, and Microsealer 1495-021 were all
purchased from Wallac Oy, Turkku, Finland. Human gluco-
corticoid receptors were extracted from Sf9 cells infected with
a recombinant baculovirus transfer vector containing the
cloned h-GR gene. Recombinant baculovirus was generated
utilizing the BAC-TO-BAC expression system (Life Technolo-
gies) in accordance with instruction from the supplier. The
h-GR coding sequences were cloned into a baculovirus transfer
vector by standard techniques. The recombinant baculoviruses
expressing h-GR were amplified and used to infect Sf9 cells.
[3H]-12 (Scheme 2). The brominated precursor, 16 (4.0 mg,
0.00589 mmol), was taken up in methanol (1.0 mL) in a 5 mL
flask. Triethylamine (0.010 mL, 0.072 mmol) and 10% Pd/C
(8 mg) were added. The flask was attached to the apparatus
for tritium gas reactions, frozen in liquid nitrogen, and
evacuated. It was thawed, frozen in liquid nitrogen, and
evacuated again. Tritium gas (1.3 Ci, 0.0069 mmol, ∼58 Ci/
mmol) was generated and admitted to the reaction flask. It
was warmed to room temperature and the reaction mixture
stirred for 1.5 h. The volatiles were removed under vacuum,
and then the reaction mixture was filtered. It was evaporated
to dryness in vacuo, taken up in methanol (∼1 mL), and then
evaporated again. This process was repeated two times and
the residue again taken up in methanol (1 mL) and assayed

5302 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 16
Link et al.
Infected cells were harvested 48 h postinfection. The receptors
were extracted from the cell pellet with a phosphate buffer (1
mM EDTA, 20 mM KPO4 (pH 8), 8.6% glycerol, 12 mM MTG,
20 mM Na2MoO4). The concentration of h-GR in the extract
was measured via specific binding of 3H-dexamethasone (“3H-
dex”) with the G25-assay22 and estimated to approximately
25 nM. The extract was aliquoted and stored at -70 °C.
Dilution series of the test compounds and dexamethasone
(“dex”) as reference were made from 10 mM (1 mM dex) stock
solutions in DMSO. Ten microliters of each dilution was added
in duplicate to the wells. The cell extracts were diluted 10-
fold in EPMo + MTG buffer (1 mM EDTA, HPO4 20 mM (pH
8), 6 mM MTG). The diluted extract was added to the wells
(110 µL). 3H-Dex was diluted from the stock solution to 10 nM
in EPMo + MTG buffer. Then 110 µL aliquots of the diluted
3H-dex were added to the wells. The final concentration of
h-GR in the experiment was estimated to 1 nM. All prepara-
tions were made in ambient temperature (20-25 °C) on ice
and with buffers at +4 °C. The plates were incubated overnight
at +4 °C (15-20 h).
for 4 h. Cells were then washed with PBS and lysed with lysis
buffer (50 mM Tris-HCl, pH 7.5, 0.1 mM EDTA, 10 µg/mL
PMSF and 0.1% Lubrol PX). The tyrosine aminotransferase
assay was performed according to standard protocols.23 Basi-
cally, the substrate mixture containing 6mM L-tyrosine, 11 µM
R-ketoglutarate and 56 µM pyridoxal-5′ phosphate in potas-
sium phosphate buffer was added to the lysates and incubated
for 30 min at 37 °C. The reaction was stopped by 10N KOH
and plates were read on a Spectrophotometer at 340 nm. Wells
that contained substrate without compound or Dex were used
as the background, while the wells that contained substrate
and Dex without any compound were considered as maximal
signal. Percent inhibition of each compound was calculated
relative to the maximal signal and IC50 curves were generated.
In Vivo Pharmacology. Rat Prednisolone Challenge
Model. Overnight fasted 150 g male Sprague-Dawley rats
are orally dosed with vehicle, 1 (30-100 mg/kg), or selected
glucocorticoid receptor antagonists (30-100 mg/kg), 60 min
prior to an oral challenge with prednisolone at 10 mg/kg. Six
hours following the prednisolone challenge, rats are euthanized
with CO2, and bled via cardiac puncture for evaluation of blood
lymphocytes and plasma drug levels. Seven millimeter liver
biopsy punches are harvested for evaluation of tyrosine
aminotransferase (TAT), hepatic glycogen, and GR antagonist
levels. Additional liver tissue, retroperitoneal fat, skeletal
muscle, kidney, and skin from an ear biopsy are also removed
for isolation an evaluation of mRNA. Prednisolone increases
hepatic TAT and glycogen levels, and induces severe lym-
phopenia during the 6 h challenge interval. At doses of 100
mpk, 1 completely antagonizes these hepatic and peripheral
responses.
The incubation was stopped by filtration through a GF/B
filter on a Tomtec CellHarvester. The GF/B filters were dried
for at least 1 h at 65 °C. A MeltiLex scintillation wax was
melted onto filters with the Microsealer. Filters were placed
in a sample bag, which was thereafter trimmed with scissors
to fit the filter cassette. The cassettes were placed in the
Microbeta and measured for 1 min/position, returning ccpm
(corrected counts per minute).
For compounds able to displace the 3(H)-dexamethasone
from the receptor an IC50 value (the concentration required to
3
inhibit 50% of the binding of (H)-dex) was determined by a
nonlinear four parameter logistic model:
Mouse Hypothalamic-Pituitary-Adrenal (HPA) Ac-
tivation Model. Nonfasted CD-1 male mice, weighing ap-
proximately 25 g, are dosed with vehicle, 1 (30-100 mg/kg),
or GR antagonist (30-100 mg/kg) at 0800 h when cortico-
sterone levels are low. Two hours later, mice are euthanized
with CO2 and bled by cardiac puncture, and the plasma is
analyzed for corticosterone by mass spectroscopy and for
adrenocorticotropic hormone (ACTH) levels by ELISA. Brains
and plasma are also removed for analysis of GR antagonist
levels. At doses of 100 mpk, 1 significantly increases ACTH
and corticosterone levels compared to vehicle controls.
b ) ((bmax - bmin)/(1 + (I/IC50)S)) + bmin
I
where I is added concentration of binding inhibitor, IC50 is the
concentration for inhibitor at half-maximal binding, and S is
3
a slope factor. For determinations of the concentration of H-
dex in the solutions, regular scintillation counting in a Wallac
Rackbeta 1214 was performed using the scintillation cocktail
Supermix (Wallac).
The Microbeta instrument generates the mean cpm (counts
per minute) value/minute and corrects for individual variations
between the detectors thus generating corrected cpm values.
Counting efficiency between detectors differed by less than 5%.
Similar protocols were employed to measure affinity of the
compounds for progesterone receptor (PR), mineralocorticoid
receptor (MR), androgen receptor (AR), estrogen receptors (ERR
and ERâ), and thyroid hormone receptors (TRR and TRâ).
GRAF Assay. Chinese hamster ovarian cells (CHO-K1)
were stably transfected with an expression plasmid encoding
hGR and a reporter construct containing a glucocorticoid
response element driving expression of alkaline phosphatase
(ALP) to produce the GRAF cell line. GRAF cells were grown
in HAM’s F12 medium supplemented with 10% FCS and 1%
L-glutamine. Induction medium was Opti-MEM with 1%
L-glutamine, 50 µg/mL gentamycin. GRAF cells were seeded
in growth medium in 96-well plates at ∼50 000 cells per well.
After 24 h of incubation, the cells were induced with test
compounds as well as dexamethasone, as a positive control,
at serial dilutions, to measure agonist response. In order to
examine antagonist effects GRAF cells were treated with
increasing amounts of test compounds in the presence of 5 nM
dexamethasone. After 48 h of induction, disodium 3-(4-meth-
oxyspiro{1,2-dioxetane-,2′-(5′-chloro)tricyclo[3.3.1.13.7]decan}-
4-yl)phenylphosphate (CSPD; Applied Biosystems) was added
to the medium, and levels of secreted alkaline phos-
phatase were analyzed by chemiluminescence on a MicroBeta
Trilux.
ob/ob Mouse Efficacy Model. Male B6.VLepob(-/-) (ob/ob)
mice and their lean littermates (Jackson Laboratory) were
group housed and allowed free access to food (Purina 5015)
and water. Mice were 6-7 weeks old at the start of each study.
On day 0 animals were weighed and postprandial glucose
levels determined (Precision X glucometer, Abbott Laborato-
ries). Mean glucose levels did not differ significantly from
group to group (n ) 10) at the start of the studies. Animals
were weighed and postprandial glucose measurements were
taken weekly throughout the study. On the last day of the
study 16 h postdose the mice were euthanized and blood
samples (EDTA) were taken by cardiac puncture and im-
mediately placed on ice. Whole blood measurements for HbA1c
were taken with hand held meters (A1c NOW, Metrika Inc.,
Sunnyvale, CA). Blood samples were then spun, and plasma
was removed and frozen until further analysis. Plasma
parameters were run according to instructions by the manu-
facturer and consisted of ALT/AST for hepatic enzymes (GO-&
GP-transaminase kit, Sigma Diagnostics, St. Louis, MO), free
fatty acids (NEFA C kit, Wako Chemicals, Neuss, Germany),
triglyceride and cholesterol lipid measurements (Infinity kits,
Sigma Diagnostics, St. Louis, MO), and insulin and ACTH
hormones by ELISA (American Laboratory Products Co.,
Windham, NH). Tissue samples were collected, weighed, and
snap frozen.
Liver biopsies were taken for hepatic tyrosine aminotrans-
ferase activity. The TAT activity was determined by the
spectrophotometric method of Diamondstone and normalized
for hepatic protein content by the method of Lowry.24,25
Glycogen content was measured by known methods.26
13 Day ob/ob Study with Modulator 14, Mice were dosed
orally twice a day for 13 consecutive days with compound and
Hepatocyte TAT Assay. Primary rat hepatocytes were
plated in 96-well collagen-coated plates at 50 000 cells per well.
Cells were incubated in DMEM with 10% charcoal-stripped
serum at 37 °C for 4 h. Cells were pretreated with serial dilu-
ted compounds for 30 min, followed by 100 nM of prednisolone

Passive Glucocorticoid Receptor Modulators
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 16 5303
Group: Conjugate of a Glucocorticoid Antagonist Bioorg. Med.
Chem. Lett. 2003, 13, 2307-2310. (c) von Geldern, T. W.; Link,
J. T.; Tu, N.; Kym, P. R.; Lai, C.; Richards, S. J.; Jacobson, P.
B.; Bishop, R. D.; Gates, B. D. WO 04/000869. (d) von Geldern,
T. W.; Tu, N.; Kym, P. R.; Link, J. T.; Jae, H.; Lai, C.; Apelqvist,
T.; Rhonnstadt, P.; Hagberg, L.; Koehler, K.; Grynfarb, M.; Goos-
Nilsson, A.; Sandberg, J.; O¨ sterlund, M.; Wang, J.; Fung, S.;
Wilcox, D.; Nguyen, P.; Jakob, C.; Hutchins, C.; Fa¨rnegårdh, M.;
Kauppi, B.; O¨ hman, L.; Jacobsen, P. B. Liver-Selective Gluco-
corticoid Antagonists. 1. Bile Acid Conjugates. The Identification
of A-348441. J. Med. Chem. 2004, 47, 4213-4230. (e) Jacobson,
P. B.; von Geldern, T. W.; O˜ hman, L.; O˜ sterland, M.; Wang, J.;
Zinker, B.; Wilcox, D.; Nguyen, P. T.; Mika, A.; Fung, S.; Fey,
T.; Goos-Nilsson, A.; Grynfarb, M.; Barkhem, T.; Marsh, K.;
Beno, D. W. A.; Nga-Nguyen, B.; Kym, P. R.; Link, J. T.; Tu, N.;
Edgerton, D. S.; Cherrington, A.; Efendic, S.; Lane, B. C.;
Opgenorth, T. J. J. Pharmacol. Exp. Ther. 2005, 314, 191-200.
(8) (a) Link, J. T.; Sorensen, B. K.; Patel, J. R.; Arendsen, D. L.; Li,
G. WO 02/064550, 2002. (b) Link, J. T.; Sorensen, B. K.; Patel,
J.; Emery, M.; Grynfarb, M.; Goos-Nilsson, A. Discovery of Novel
Nonsteroidal Glucocorticoid Receptor Modulators. Bioorg. Med.
Chem. Lett. 2004, 14, 2209-2212. (c) Link, J. T.; Sorensen, B.
K.; Patel, J.; Arendsen, D.; Li, G.; Swanson, S.; Nguyen, B.;
Emery, M.; Grynfarb, M.; Goos-Nilsson, A. Optimization and
Metabolic Stabilization of a Class of Nonsteroidal Glucocorticoid
Modulators. Bioorg. Med. Chem. Lett. 2004, 14, 4169-4172. (d)
Link, J. T.; Sorensen, B.; Lai, C.; Wang, J.; Fung, S.; Deng, D.;
Emery, M.; Carroll, S.; Grynfarb, M.; Goos-Nilsson, A.; von
Geldern, T. Synthesis, Activity, and Liver Targeting of Gluco-
corticoid Receptor Modulator-Statin Hybrids. Bioorg. Med.
Chem. Lett. 2004, 14, 4173-4178. (e) Tu, N.; Link, J. T.;
Sorensen, B. K.; Emery, M.; Grynfarb, M.; Goos-Nilsson, A.;
Nguyen, B. Bile Acid Coinjugates of a Nonsteroidal Glucocorti-
coid Receptor Modulator. Bioorg. Med. Chem. Lett. 2004, 14,
4179-4183. (f) Apelqvist, T.; Efendic, S. WO 99/63976, 1999. (g)
Aqelqvist, T.; Goede, P.; Holmgren, E. WO 00/07972, 2000. (h)
Kym, P. R.; Lane, B. C.; Pratt, J. K.; von Geldern, T.; Winn, M.;
Brenneman, J.; Patel, J. R.; Arendsen, D. L.; Akritopoulou-
Zanze, I. US 6,329,534, 2001. (i) Akritopoulou-Zanze, I., Patel,
J. R.; Hartandi, K.; Brenneman, J.; Winn, M.; Pratt, J. K.;
Grynfarb, M.; Goos-Nisson, A.; von Geldern, T. W.; Kym, P. R.
Synthesis and Biological Evaluation of Novel, Selective, Non-
steroidal Glucocorticoid Receptor Antagonists. Bioorg. Med.
Chem. Lett. 2004, 14, 2079-2082.
(9) (a) Morgan, B. P.; Swick, A. G.; Hargrove, D. M.; LaFlamme, J.
A.; Moynihan, M. S.; Carroll, R. S.; Martin, K. A.; Lee, E.;
Decosta, D.; Bordner, J. Discovery of Potent, Nonsteroidal, and
Highly Selective Glucocorticoid Receptor Antagonists. J. Med.
Chem. 2002, 45, 2417-2424. (b) Morgan, B. P.; Liu, K. K.-C.;
Dalvie, D. K.; Swick, A. G.; Hargrove, D. M.; Wilson, T. C.;
LaFlamme, J. A.; Moynihan, M. S.; Rushing, M. A.; Woodworth,
G. F.; Li, J.; Trilles, R. V.; Yang, X.; Harper, K. W.; Carroll, R.
S.; Martin, K. A.; Nardone, N. A.; O’Donnell, J. P.; Faletto, M.
B.; Vage, C.; Soliman, V. Discovery of Potent, Non-Steroidal and
Highly Selective Glucocorticoid Receptor Antagonists with Anti-
Obesity Activity. Lett. Drug Des. Discovery 2004, 1, 1-5.
(10) Bamberger, C. M.; Schulte, H. M.; Chrousos, G. P. Molecular
Determinants of Glucocorticoid Receptor Function and Tissue
Sensitivity to Glucocorticoids. Endocr. Rev. 1996, 17, 245-261.
(11) Truss, M.; Beato, M. Steroid Hormone Receptors: Interaction
with Deoxyribonucleic acid and Transcription Factors. Endocr.
Rev. 1993, 14, 459-479.
vehicle controls. Control ob/ob mice and lean littermates were
dosed with vehicle (1 equiv of NaOH, 5% ethanol, 10% PEG
400, 85% saline) that was also used to formulate 14.
14 Day ob/ob Study with Modulator 12. Mice were dosed
orally twice a day for 14 consecutive days with compound and
vehicle controls. Control ob/ob mice and lean littermates were
dosed with vehicle (0.2% hydroxypropyl methylcellulose) that
was also used to formulate 12.
30 Day ob/ob Study with Modulator 12. Mice were dosed
orally twice a day for 30 consecutive days with compound and
vehicle controls. Control ob/ob mice and lean littermates were
dosed with vehicle (0.2% hydroxypropyl methylcellulose) that
was also used to formulate 12.
Acknowledgment. We thank Dr. Kennan Marsh
and Dr. Dan Plata for assistance with the pharmaco-
kinetic experiments and compound scale-up, respec-
tively.
References
(1) (a) Munck, A.; Guyre, P. M.; Holbrook, J. J. Physiological
Functions of Glucocorticoids in Stress and their Relationship to
Pharmacological Actions. Endocr. Rev. 1984, 5, 25-44. (b)
Sapolsky, R. M.; Romero, M.; Munck, A. U. How do Glucocorti-
coids Influence Stress Responses? Integrating Permissive, Sup-
pressive, Stimulatory, and Preparative Actions. Endocr. Rev.
2000, 21, 55-89.
(2) (a) Coghlan, M. J.; Elmore, S. W.; Kym, P. R.; Kort, M. E.
Selective Glucocorticoid Receptor ModulatorsAnn. Reports Med.
Chem. 2002, 37, 167-176, (b) Coghlan, M. J.; Elmore, S. W.;
Kym, P. R.; Kort, M. E. The Pursuit of Differentiated Ligands
for the Glucocorticoid Receptor. Curr. Topics Med. Chem. 2003,
3, 1617-1635. (c) Ali, A.; Thompson, C. F.; Balkovec, J. M.;
Graham, D. W.; Hammond, M. L.; Quraishi, N.; Tata, J. R.;
Einstein, M.; Ge, L.; Harris, G.; Kelly, T. M.; Mazur, P.; Pandit,
S.; Santoro, J.; Sitlani, A.; Wang, C.; Williamson, J.; Miller, D.
K.; Thompson, C. M.; Zaller, D. M.; Forrest, M. J.; Carballo-
Jane, E.; Luell, S. Novel N-Arylpyrazolo[3,2-c]-Based Ligands
for the Glucocorticoid Receptor Binding and in Vivo Activity. J.
Med. Chem. 2004, 47, 2441-2452, (d) Thompson, C. F.; Quraishi,
N.; Ali, A.; Tata, J. R.; Hammond, M. L.; Balkovec, J. M.;
Einstein, M.; Ge, L.; Harris, G.; Kelly, T. M.; Mazur, P.; Pandit,
S.; Santoro, J.; Sitlani, A.; Wang, C.; Williamson, J.; Miller, D.
K.; Yamin, T. D.; Thompson, C. M.; O’Neill, E. A.; Zaller, D.;
Forrest, M. J.; Carballo-Jane, E.; Luell, S. Novel Heterocyclic
Glucocorticoids: in Vitro Profile and in Vivo Efficacy. Bioorg.
Med. Chem. Lett. 2005, 15, 2163-2167, (e) Einstein, M.; Green-
lee, M.; Rouen, G.; Sitlani, A.; Santoro, J.; Wang, C.; Pandit, S.;
Mazur, P.; Smalera, I.; Weaver, A. P. M.; Zeng, Y. Y.; Ge, L.;
Kelly, T.; Paiva, T.; Geissler, W.; Mosley, R. T. Williamson, J.;
Ali, A.; Balkovec, J.; Harris, G. Selective Glucocorticoid Receptor
Nonsteroidal Ligands Completely Antagonize the Dexametha-
sone Mediated Induction of Enzymes Involved in Gluconeogen-
esis and Glutamine Metabolism. J. Steroid Biochem. Mol. Biol.
2004, 92, 345-356.
(3) Spitz, I. M.; Bardin, C. W. Drug Therapy: Mifepristone (RU
486)sA Modulator of Progestin and Glucocorticoid Action. N.
Engl. J. Med. 1993, 329, 404-412.
(4) Garrel, D. R.; Moussali, R.; De Oliveira, A.; Lesie`ge, D.; Larivie`re,
F. RU 486 Prevents the Acute Effects of Cortisol on Glucose and
Leucine Metabolism. J. Clin. Endocrinol. Metab. 1995, 80, 379-
185.
(5) Friedman, J. E.; Sun, Y.; Ishizuka, T.; Farrell, C. J.; McCormack,
S. E.; Herron, L. M.; Hakimi, P.; Lechner, P.; Yun, J. S.
Phosphoenolpyruvate Carboxykinase (GTP) Gene Transcription
and Hyperglycemia are Regulated by Glucocorticoids in Geneti-
cally Obese db/db Transgenic Mice. J. Biol. Chem. 1997, 272,
31475-31481.
(6) (a) Opherk, C.; Tronche, F.; Kellendonk, C.; Kohlmu¨ller, D.;
Schulze, A.; Schmid, W.; Schu¨tz, G. Inactivation of the Gluco-
corticoid Receptor in Hepatocytes Leads to Fasting Hypoglyce-
mia and Ameliorates Hyperglycemia in Streptozotocin-Induced
Diabetes Mellitus. Mol. Endocrinol. 2003, 16, 1346-1353. (b)
Watts, L. M.; Manchem, V. P.; Leedom, T. A.; Rivard, A. L.;
McKay, R. A.; Bao, D.; Neroladakis, T.; Monia, B. P.; Boden-
miller, D. M.; Cao, J. X.-C.; Zhang, H. Y.; Cox, A. L.; Jacobs, S.
J.; Michael, M. D.; Sloop, K. W.; Bhanot, S. Reduction of Hepatic
and Adipose Tissue Glucocorticoid Receptor Expression with
Antisense Oligonucleotides Improves Hyperglycemia and Hy-
perlipidemia in Diabetic Rodents without causing Systemic
Glucocorticoid Antagonism. Diabetes 2005, 54, 1846-1853.
(7) (a) Apelqvist, T.; Wu, J.; Hoehler, K. F. WO 00/058337, 2000.
(b) Sorensen, B. K.; Link, J. T.; von Geldern, T.; Emery, M.;
Wang, J.; Hickman, B.; Grynfarb, M.; Goos-Nilsson, A.; Carroll,
S. An Evaluation of a C-Glucuronide as a Liver Targeting
(12) Wagner, B. L.; Pollio, G.; Giangrande, P.; Webster, J. C.; Breslin,
M.; Mais, D. E.; Cook, C. E.; Vedeckis, W. V.; Cidlowski, J. A.;
McDonnell, D. P. The Novel Progesterone Receptor Antagonists
RTI 3021-012 and RTI 3021-022 Exhibit Complex Glucocorticoid
Receptor Antagonists Activities: Implications for Development
of Dissociated Antiprogestins. Endocrinology 1999, 140, 1449-
1458.
(13) (a) Philibert, D.; Costerousse, G.; Gaillard-Moguilewsky, M.;
Nedelec, L.; Nique, F.; Toiurnemine, C.; Teutsch, G. From RU
38486 towards Dissociated Antiglucocorticoid and Antiproges-
terone. Front. Horm. Res. 1991, 19, 1-17. (b) Belikov, S.; Gelius,
B.; Wrange, O¨ . Hormone-induced Nucleosome Positioning in the
MMTV Promoter is Reversible. EMBO J. 2001, 20, 2802-2811.
(14) Honer, C.; Nam, K.; Fink, C.; Marshall, P.; Ksander G.; Chat-
elain, R. E.; Cornell, W.; Steele, R.; Schweitzer, R.; Schumacher,
C. Glucocorticoid Receptor Antagonism by Cypterone Acetate
and RU486. Mol. Pharmacol. 2003, 63, 1012-1020.
(15) Lal, G. S., Site-Selective Fluorination of Organic Compounds
Using 1-Alkyl-4-fluoro-1,4-diazabicyclo[2.2.2]octane Salts (Select-
fluor Reagents). J. Org. Chem. 1993, 58, 2791-2796.
(16) Bos, M. E.; Wulff, W. D.; Miller, R. A.; Chamberlin, S.; Brand-
void, T. A. Substrate Regulation of Product Distribution in the
Reactions of Aryl Chromium Carbene Complexes with Alkynes.
J. Am. Chem. Soc. 1991, 113, 9293.

5304 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 16
Link et al.
(17) Abdel-Magid, A. F.; Carson, K. G.; Harris, B. D.; Maryanoff, C.
A.; Shah, R. D. Reductive Amination of Aldehydes and Ketones
with Sodium Triacetoxyborohydride. Studies on Direct and
Indirect Reductive Amination Procedures. J. Org. Chem. 1996,
61, 3849-3862.
(18) Dessolin, M.; Guillerez, M.-G.; Thieriet, N.; Guibe´, F.; Loffet, A.
New Allyl Group Acceptors for Palladium Catalyzed Removal
of Allylic Protections and Transacylation of Allyl Carbamates.
Tetrahedron Lett. 1995, 36, 5741-5744.
(19) Mitsunobu, O. The Use of Diethyl Azodicarboxylate and Tri-
phenylphosphine in Synthesis and Transformation of Natural
Products. Synthesis 1981, 1-28.
(20) (a) Heikinheimo, O.; Kontula, K.; Croxatto, H.; Spitz, I.; Luuk-
kainen, T.; La¨hteenma¨ki, P. Plasma Concentrations and Recep-
tor Binding of RU 486 and its Metabolites in Humans. J. Steroid
Biochem. 1987, 26, 279-284. (b) Heikinheimo, O.; Haukkamaa,
M.; La¨hteenma¨ki, P. Distribution of RU 486 and its Demeth-
ylated Metabolites in Humans. J. Clin. Endocrinol. Metab. 1989,
68, 270-275. (c) Chasserot-Golaz, S.; Parcollet, P.; Beck, G.
Interrelationship between RU38486 and the P450 Activities in
Rat Liver. J. Steroid Biochem. 1989, 34, 473-478.
(21) (a) Kurukulasuriya, R.; Link, J. T.; Madar, D. J.; Pei, Z.; Rohde,
J. J.; Richards, S. J.; Souers, A. J.; Szczepankiewicz, B. G.
Prospect for Pharmacologic Inhibition of Hepatic Glucose Pro-
duction. Curr. Med. Chem. 2003, 10, 99-121. (b) Kurukulasur-
iya, R.; Link, J. T.; Madar, D. J.; Pei, Z.; Richards, S. J.; Rohde,
J. J.; Souers, A. J.; Szczepankiewicz, B. G. Potential Drug
Targets and Progress Towards Pharmacologic Inhibition of
Hepatic Glucose Production. Curr. Med. Chem. 2003, 10, 123-
154. (c) Link, J. T. Pharmacological Regulation of Hepatic
Glucose Production. Curr. Opin. Invest. Drugs 2003, 4, 421-
429.
(22) Salomonsson, M.; Carlsson, B.; Ha¨ggblad, J. Equilibrium Hor-
mone Binding to Human Estrogen Receptors in Highly Diluted
Cell Extracts is Non-cooperative and has a Km of Approximately
10 pM. J. Steroid Biochem. Mol. Biol.1994, 50 (5/6), 313-318.
(23) Granner, D. K.; Tomkins, G. M.; Tyrosine Aminotransferase (Rat
Liver). Methodology 1970, 17A, 633-637.
(24) Diamondstone, T. I. Assay of Tyrosine Aminotransferase Activity
by Conversion of p-hydroxyphenylpyruvate to p-hydroxybenz-
aldehyde. Anal. Biochem. 1966, 16, 395-401.
(25) Lowry, O. M., Rosebrough, N. J., Farr, A. L.; Randall, R. J.
Protein measurement with the Folin phenol reagent. J. Biol.
Chem. 1951, 173, 265-272.
(26) Keppler, D. and Decker, K. In Glycogen: Determination with
Amyloglucosidase. Methods of Enzymatic Analysis; Bergmeyer,
H. U., Ed.; John Wiley & Sons: New York, 1994; Vol. 3,
1127-1131.
JM050205O