Synthesis and SAR of 2-aryloxy-4-alkoxy-pyridines as potent orally active corticotropin-releasing factor 1 receptor antagonists

Article abstract of DOI:10.1021/jm070578k

A series of 2-aryloxy-4-alkoxy-pyridines (1) was identified as novel, selective, and orally active antagonists of the corticotropin-releasing factor 1 (CRF₁) receptor. Among these, compound 2 (CP-316311) is a potent and selective CRF₁

Full text of DOI:10.1021/jm070578k

J. Med. Chem. 2008, 51, 1377–1384
1377
Synthesis and SAR of 2-Aryloxy-4-alkoxy-pyridines as Potent Orally Active
Corticotropin-Releasing Factor 1 Receptor Antagonists
Yuhpyng L. Chen,*,† John Braselton,‡ James Forman,† Randall J. Gallaschun,† Robert Mansbach,‡ Anne W. Schmidt,‡
Thomas F. Seeger,‡ Jeff S. Sprouse,‡ F. David Tingley, III,‡ Elizabeth Winston,‡ and David W. Schulz‡
Medicinal Chemistry and Neuroscience Departments, PGRD, Pfizer Inc., Groton, Connecticut 06340
ReceiVed May 17, 2007
A series of 2-aryloxy-4-alkoxy-pyridines (1) was identified as novel, selective, and orally active antagonists
of the corticotropin-releasing factor 1 (CRF1) receptor. Among these, compound 2 (CP-316311) is a potent
and selective CRF1 receptor antagonist with an IC50 value of 6.8 nM in receptor binding and demonstrates
oral efficacy in central nervous system (CNS) in vivo models. The regiochemistry of compounds in this
series was determined by an X-ray structural analysis. A method to control regioselectivity via pyridine-
N-oxides was developed. The synthesis of compounds in series 1 (Figure 1) and [3H]-2 as well as the
structure-activity relationship (SAR) are discussed. The in vitro, ex vivo, and in vivo properties of
representative compounds are described herein. Compound 2 was advanced to phase II depression trials to
test the hypothesis that CRF1 antagonists could be used clinically as antidepressant drugs.
Introduction
Corticotropin-releasing factor (CRF), a 41 amino acid peptide
neurotransmitter, coordinates the body’s response to stress
through the release of adrenocorticotropic hormone (ACTH).
CRF has been shown to mediate stress-induced changes in the
autonomic system and to cause numerous neuroendocrine
changes and behavioral effects.1 Clinical data indicate that
patients with depression and post-traumatic stress disorder show
significantly elevated concentrations of CRF in cerebrospinal
fluid when compared to normal controls.2 In addition, patients
with depression, anxiety, anorexia nervosa, and post-traumatic
stress disorder showed a blunted ACTH response to intraveneous
Figure 1. CRF1 antagonists.
CRF, indicating that their CRF receptors may be down-regulated
possibly due to chronic hypersecretion of CRF.3 Receptor
in series 1. The ex vivo and in vivo results of representative
compounds are presented.
subtypes include CRF1 and CRF2 receptors that are widely
distributed throughout the central nervous system (CNS) and
peripheral tissues.4 A selective CRF1 receptor antagonist may
represent a novel class of compounds for the treatment of
anxiety, depression, and stress-related diseases. Published pre-
clinical data support the hypothesis that a small molecule CRF1
receptor antagonist such as CP-154526 (3a) or CP-156181
(antalarmin, 3b) blocks neuroendocrine effects (e.g., increased
ACTH induced by i.v. CRF), autonomic responses (e.g.,
hypertensive effects), or increased heart rate or behavioral
changes induced by i.c.v. CRF or stress stimuli.5,6 In a
continuation of our structure-activity relationship (SAR) efforts
around 3a, structural modifications in the pyrrolo-pyrimidine
core template and peripheral side chains led to various novel
chemotypes, in which the pyridine series represents one of the
most attractive series because of its low molecular weight
coupled with excellent preclinical efficacy and safety. SAR
efforts led to the discovery of a clinical candidate 2 that was
advanced to phase II to test the ability of a CRF1 receptor
antagonist to treat depressed patients. Herein we would like to
report the regioselective synthesis and the SAR of compounds
2,4-Dichloro-3,6-dimethyl-pyridine (4) was prepared by the
method analogous to a literature report7 as shown in Scheme
1. Direct coupling of 2,4-dichloro-3,6-dimethyl-pyridine with
sodium 2,4,6-trimethylphenoxide provided a 3:1 mixture of
regioisomers 5 and 6 as white crystals, in which the minor
component 6 was found to be the desired isomer. The regio-
chemistry of 5 was determined by X-ray structural analysis.
Reaction of each isomer 5 and 6 with 3-pentanoxide in DMSO
at 120 °C provided compounds 7 and 2, respectively. Compound
2, derived from the minor isomer 6, was found to be more potent
than the major isomer 7, with IC50 values for the CRF1 receptor
of 6.8 and 3400 nM, respectively. Reversing the order of
addition by adding 3-pentoxide first in an attempt to address
the regioselectivity issue surprisingly switched the selectivity
from preferably attacking at the C4 position as seen in the case
of trimethylphenoxide to the C2 position in the pyridine ring,
thus again providing the undesired regioisomer 8 as a major
component as shown in Scheme 1.
A regioselective method was needed in order to rapidly
generate SAR and to efficiently scale up key compounds to
support toxicology studies. We thought that if we could increase
the reactivity at C2 of the pyridine ring through the formation
of the pyridine-N-oxide, 8, we might be able to control the
regioselectivity in the coupling step to provide the desired
isomer. Indeed such an approach successfully delivered the bulk
* To whom correspondence should be addressed. Phone: (860) 444-2438.
Fax: (860) 444-2438. E-mail: yuhpyng888@yahoo.com.
†
Medicinal Chemistry Department.
Neuroscience Department.
‡
10.1021/jm070578k CCC: $40.75
2008 American Chemical Society
Published on Web 02/09/2008

1378 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5
Chen et al.
Scheme 1a
The area around substituents at C5 and C6 in the pyridine
series 1 was found to have a narrow SAR; for example, changing
R5 from H to Et resulted in a change in IC50 values from 6.8
nM to >10,000 nM, respectively, whereas the area at R3 seems
to tolerate a variety of groups, including Me, Et, Br, nitro, and
amino, as shown in Table 1. The optimized substituents at R3,
R5, and R6 are Me, H, and Me (compound 2), respectively,
which also offers good metabolic stability, brain penetrability,
and oral activity, as shown in the ex vivo data. Table 2 describes
the SAR in the p-aryloxy group, in which R7 seems to tolerate
various groups and R7 as Me and CONH2 provided excellent
ex vivo inhibition in cortex after p.o. administration. Although
the methoxy group in 35 seems to increase polarity and
demonstrates excellent ex vivo inhibition in cortex after oral
dosing, it shows poor metabolic stability perhaps due to
demethylation. Table 3 describes the limited SAR on the top
alkoxide side chain, in which the branched alkoxide is required
for potency based on our SAR in series 3a.10 In order to add
basicity and polarity, the 2,4,6-trimethyl-phenoxy group of 2
was replaced with a 3-(2,4,6-trimethyl-pyridoxy) to give com-
pound 14, which showed a pKa increase from ∼3.5 to 4.97 and
improved kinetic water solubility from <1 µg/mL to 10 µg/
mL.11 Compound 14 had an IC50 value of 10 nM and
demonstrated excellent ex vivo cortex displacement (70%
inhibition at 3.2 mg/kg, s.c.) but is relatively more labile than
2 in human microsomes.
Liver toxicity had been a key issue in our early CRF program,
but compounds in this series proved to be safe and no liver
toxicity was seen in five day rat toxicology studies.10 Com-
pounds in series 1 have high affinity for the CRF1 receptor
subtype and low affinity for the CRF2 receptor (>1 µM). The
representative compound 2 fully antagonized CRF-stimulated
adenylate cyclase activity in rat cortex and at human CRF1
receptors endogenously expressed in IMR32 cells with apparent
Ki values of 7.6 and 8.5 nM, respectively. Compound 2 is highly
selective for the CRF1 receptor and has affinities of greater than
1 µM for more than 40 other receptors, channels, enzymes, and
uptake proteins. Ex vivo binding studies in rats demonstrate
that oral administration of 2 (3.2 mg/kg) inhibits 125I-oCRF
binding by >80%. Oral activity has been obtained in rats by
using models that reflect antagonism of CRF-mediated responses
in the periphery and the brain. Compound 2 significantly
attenuates activation of the hypothalamic-pituitary-adrenal
(HPA) axis, as measured by increased plasma ACTH levels in
response to exogenous CRF administration with an MED value
of 10 mg/kg, p.o. (p < 0.05 versus CRF alone, One Way
ANOVA, Dunnett’s post hoc test, N ) 12) and a calculated
ID50 value (p.o.) of 35 mg/kg (4 µg/kg oCRF, i.v.) with a
confidence interval of 26.7-45.4 mg/kg. Systemically admin-
istered compound 2 blocks the effects of both the exogenous
and endogenous CRF in the CNS. For example, it demonstrated
activity in several behavioral models, including reversal of i.c.v.
CRF-induced excitation of locus coeruleus neurons (60%
inhibition at 0.3 mg/kg, i.v.), reversal of startle potentiation
induced by i.c.v. CRF (100% at 32 mg/kg, p.o.), and activity
in the defensive withdrawal model of anxiety at 10 mg/kg, i.p.
administration.10
a Reagents and conditions: (a) 2,4,6,-trimethylphenol, KOt-Bu, DMSO;
(b) 3-pentanol, NaH or KOt-Bu, THF; (c) 3-pentanol, NaH or KOt-Bu,
DMSO or 1-methyl-2-pyrrolidinone.
of 2 for pharmacology and toxicology studies.8,9 Pyridine-N-
oxide 10a was prepared by reaction of 4 with m-CPBA in
chloroform at room temperature in 77% yield as white crystals.
Coupling of pyridine-N-oxide 10a with 2,4,6-trimethylphenol
and sodium hydride in refluxing THF for 2 h provided the
desired regioisomer 11a exclusively in 89% yield as cubic
crystals. Reduction of 11a with 1 equiv of PCl3 in refluxing
methylene chloride for 1 h yielded the desired product 6 as white
crystals in 95% yield as shown in Scheme 2. Analogues in this
series were prepared for SAR by the method analogous to that
described in the synthesis of compound 2 as illustrated in
Scheme 3. [2H]-2 was synthesized as a mixture of deuterium
addition products at positions C1, C2, and C3 by reduction of
the precursor 13 with deuterium in the presence of 10% Pd/C
in ethyl acetate at atmospheric pressure. Compound 13 was
prepared as a mixture of cis/trans isomers by reacting 6 with
pent-1-en-3-ol and NaH in DMSO, where the olefin isomer-
ization occurred under basic conditions as shown in Scheme 3.
The developed reduction method was used for preparing [3H]-
2 to give a 45 Ci/mmol specific activity of the radiolabeled
material, which was used for drug metabolism studies.
Compounds in series 1 were tested in a rat cortex binding
assay by using 125I-ovine-CRF (125I-oCRF) as shown in Tables
1-3. Low nanomolar compounds were selected for ex vivo
evaluations. Because compounds in the blood can gain access
to the pituitary gland without crossing the blood-brain barrier,
the decrease in binding in the brain or pituitary gland gives an
estimate of the relative brain/plasma ratio of systemically
administered compounds via either the oral (p.o.) or subcutane-
ous (s.c.) route. Several compounds exhibit excellent ex vivo
activity in the cortex and pituitary 125I-oCRF displacement assay
at 3.2 mg/kg, indicating compounds penetrate the CNS well
via either p.o. or s.c. administration.
In summary, a regioselective synthesis was developed for
rapid SAR assessment of series 1. Compounds in the pyridine
series 1 are potent and highly selective antagonists of the CRF1
receptor subtype. A representative compound, 2, fully antago-
nizes CRF-stimulated adenylate cyclase activity and significantly
attenuates activation of the HPA axis as measured by blocking
plasma ACTH elevation induced by i.v. oCRF. Systemically

Synthesis and SAR of CRF1 Antagonists
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5 1379
Scheme 2a
a Reagents and conditions: (a) m-CPBA, CHCl3, or CH2Cl2; (b) 2,4,6,-trimethylphenol, NaH, THF; (c) 2 M PCl3 in CH2Cl2, 95%; (d) 3-pentanol, NaH,
or KOt-Bu, DMSO or NMP.
Scheme 3a
Compounds 10b–d were prepared by the method analogous to
that described in preparation of 10a, starting with an appropriate
2,4-dichloro-pyridine and an oxidizing agent.
2,4-Dichloro-6-methyl-1-oxy-nicotinic Acid Methyl Ester
(10b). White crystals, mp 90–91.5 °C; 1H NMR (CDCl3) δ 7.26 (s,
1H), 3.98 (s, 3H), 2.54 (s, 3H) ppm; Anal. (C8H7NO3Cl2) C, H, N.
2,4-Dichloro-3,5,6-trimethyl-pyridine 1-Oxide (10c). White
1
crystals, mp 146–148 °C; H NMR (CDCl3) δ 2.57 (s, 3H), 2.49
(s, 3H), 2.38 (s, 3H) ppm; Anal. (C8H9NOCl2) C, H, N.
2,4-Dichloro-6-methyl-pyridine 1-Oxide (10d). White crystals,
1
mp 100–102 °C; H NMR (CDCl3) δ 7.42 (d, 1H), 7.22 (d, 1H),
2.55 (s, 3H) ppm; Anal. (C6H5NOCl2) C, H, N.
a Reagents and conditions: (a) 3 equiv of CH2dCHCH(OH)(Et), 1.2 equiv
of NaH in DMSO, 130 °C; (b) deuterium, 10% Pd/C in EtOAc, 1 atm,
quantitative yield.
4-Chloro-2,5-dimethyl-6-(2,4,6-trimethyl-phenoxy)-pyridine
1-Oxide (11a). A solution of 2,4,6-trimethylphenol (31.166 g, 0.229
mmol) in dry THF (700 mL) was treated with 60% sodium hydride
in oil (9.16 g, 0.229 mmol) at room temperature. After all of the
H2 was evolved, 2,4-dichloro-3,6-dimethyl-pyridine 1-oxide (44.000
g, 0.229 mmol) was added, and the resulting mixture was heated
at reflux for 2 h. The reaction was quenched with saturated
ammonium chloride and extracted with ethyl acetate. The organic
layer was dried and concentrated to dryness to give a solid. The
solid was recrystallized from petroleum ether to give 59.41 g (89%)
of the title compound as white crystals, mp 106–107 °C; 1H NMR
(CDCl3) δ 7.04 (s, 1H), 6.78 (s, 2H), 2.41 (s, 3H), 2.36 (s, 3H),
2.22 (s, 3H), 2.06 (s, 6H) ppm; Anal. (C16H18NO2Cl) C, H, N.
administered 2 blocks the effects induced by the exogeneous
or endogeneous CRF in the brain in rat models. It demonstrates
oral efficacy in the i.c.v. CRF-induced and fear potentiated startle
models after oral administration. 2 was selected for clinical
investigation in a placebo-controlled study in depressed pa-
tients.10
Experimental Section
Melting points were determined on a Thomas-Hoover capillary
Compounds 11b–g were prepared by the method analogous to
that described in the preparation of 11a, starting with an appropriate
2,4-dichloro-pyridine 1-oxide and an appropriate phenol in the
presence of a base (KOt-Bu, NaH, or KH) at a temperature between
room temperature and reflux in dry THF.
1
melting point apparatus and are uncorrected. High field H NMR
spectra were recorded on a Varian XL-300, XL-400, Bruker AM
250, or Bruker AM 300 instrument. Elemental analyses were carried
out by Schwarzkopf Microanalytical, Woodside, NY.
2,4-Dichloro-3,6-dimethyl-pyridine 1-Oxide (10a).8 A mixture
of 2,4-dichloro-3,6-dimethyl-pyridine (790 mg, 4.49 mmol) and
50% m-chloro-perbenzoic acid (1.544 g, 4.49 mmol) in 10 mL of
chloroform was stirred at room temperature for 20 h. The reaction
was quenched with water, washed with saturated sodium thiosulfate,
saturated sodium carbonate, and brine, and extracted with chloro-
form. The organic layer was dried and concentrated to give 0.954
g of crude material. The material was purified through silica gel
and recrystallized from THF to give 0.662 g (77%) of the desired
2-(4-Bromo-2,6-dimethyl-phenoxy)-4-chloro-3,6-dimethyl-py-
ridine 1-Oxide (11b). White crystals, mp 137–139 °C; H NMR
(CDCl3) δ 7.12 (s, 2H), 7.08 (s, 1H), 2.42 (s, 6H), 2.09 (s, 6H)
ppm; Anal. (C15H15BrClNO2) C, H, N.
1
4-Chloro-2-(4-chloro-2,6-dimethyl-phenoxy)-3,6-dimethyl-py-
ridine 1-Oxide (11c). 1H NMR (CDCl3) δ 7.08 (s, 1H), 6.97 (s, 2H),
2.42 (s, 6H), 2.09 (s, 6H) ppm; Anal. (C15H15Cl2NO2) C, H, N.
4-Chloro-6-methyl-2-(2,4,6-trimethyl-phenoxy)-1-oxy-nico-
tinic Acid Methyl Ester (11d). 1H NMR (CDCl3) δ 7.04 (s, 1H),
6.78 (s, 2H), 3.48 (s, 3H), 2.52 (s, 3H), 2.22 (s, 3H), 2.08 (s, 6H)
ppm.
1
product as white crystals, mp 131–132 °C; H NMR (CDCl3) δ
7.22 (s, 1H), 2.51 (s, 3H), 2.47 (s, 3H) ppm; Anal. (C7H7Cl2NO)
C, H, N.

1380 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5
Chen et al.
Table 1. In Vitro CRF1 Inhibition (IC50)a and ex Vivo Datab Using a Radiolabeled CRF Displacement Assay in Rat Cortex and Pituitary for
Compounds in the Pyridine Series
in vitro,a rat cortex
pIC50 ( SEM (M) IC50 (nM)
ex vivob
compound no.
R3
Me
Me
Et
R5
R6
3.2 mg/kg route
p.o.
cortex (% inh ( SD)
pituitary (% inh ( SD)
2
H
H
H
H
H
H
H
H
H
H
Me
Et
Me
Et
8.17 ( 0.04
7.06 ( 0.34
8.04 ( 0.18
6.83 ( 0.06
6.33 ( 0.08
7.82 ( 0.05
7.91 ( 0.24
7.60 ( 0.16
8.01 ( 0.14
<5.00
6.8
86
9.1
150
470
15
12*
25
75 ( 7
NT
57 ( 8
72 ( 11
NT
66 ( 18
NT
15
16
17
18
19
20
21
22
23
24
25
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
s.c.
H
NT
NT
COOMe
NO2
NH2
NMe2
Br
COOH
Me
Me
NT
s.c.
s.c.
59 ( 10
78 ( 16
NT
35 ( 9
51 ( 5
NT
NT
NT
9.8
>10,000
180
NT
NT
NT
NT
6.74 ( 0.25
<5.00
NT
NT
>10,000
a Values reported are the geometric mean of at least three experimental runs using rat cortex (*except compound 20, n ) 2). b Compounds were administered
via oral (p.o.) or subcutaneous (s.c.) route at 3.2 mg/kg. 125I-oCRF binding to brain and pituitary tissues in rats was measured by using an ex vivo autoradiographic
assay or brain homogenate binding displacement assay. Percent inhibition values are mean ( SD. NT ) not tested.
Table 2. SAR of the 2-Aryloxy Group in the Pyridine Series in Vitroa and ex Vivo Datab
in vitro,a rat cortex
ex vivob
compound no.
R7
pIC50 ( SEM (M)
IC50 (nM)
3.2 mg/kg route
p.o.
cortex (% inhibition)
pituitary (% inhibition)
2
Me
H
Et
8.17 ( 0.04
6.96 ( 0.14
7.97 ( 0.13
8.01 ( 0.09
7.50 ( 0.16
8.26 ( 0.24
8.58 ( 0.16
7.91 ( 0.27
7.74 ( 0.12
5.87 ( 0.29
8.69 ( 0.10
7.70 ( 0.11
6.96 ( 0.18
7.40 ( 0.15
7.21 ( 0.10
8.50 ( 0.12
7.40 ( 0.14
6.8
110
11
9.8
32
5.5
2.6
12
18
1400
2
20
110
40
75 ( 7
NT
55 ( 7
42 ( 21
NT
66 ( 8
47 ( 16
27 ( 4
33 ( 22
NT
75 ( 4
43 ( 15
NT
72 ( 11
NT
56 ( 16
34 ( 19
NT
76 ( 7
84 ( 1
72 ( 11
15 ( 12
NT
55 ( 1
33 ( 1
NT
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
s.c.
s.c.
n-Pr
F
Cl
Br
I
p.o.
p.o.
s.c.
p.o.
CONH2
OH
OMe
OEt
Oi-Pr
CHO
CH2OH
CH2OMe
CMe2(OH)
p.o.
p.o.
NT
NT
61
3.2
40
p.o.
p.o.
42 ( 25
57 ( 2
NT
-20 ( 4
12 ( 1
NT
a Values reported are the geometric mean of at least three experimental runs using rat cortex. b Compounds were administered via oral (p.o.) or subcutaneous
(s.c.) route at 3.2 mg/kg. 125I-oCRF binding to brain and pituitary tissues in rats was measured using an ex vivo autoradiographic assay or brain homogenate
binding displacement assay. Percent inhibition values are mean ( SD. NT ) not tested.
4-Chloro-2,3,5-trimethyl-6-(2,4,6-trimethyl-phenoxy)-pyri-
dine 1-Oxide (11e). White crystals, mp 132–134 °C; 1H NMR
(CDCl3) δ 6.75 (s, 2H), 2.47 (s, 3H), 2.38 (s, 3H), 2.35 (s, 3H),
2.20 (s, 3H), 2.04 (s, 6H) ppm; Anal. (C17H20ClNO2) C, H, N.
4-Chloro-2-methyl-6-(2,4,6-trimethyl-phenoxy)-pyridine 1-Ox-
ide (11f). White crystals, mp 191–193 °C; 1H NMR (CDCl3) δ
6.96 (s, 1H), 6.95 (s, 2H), 2.62 (s, 3H), 2.32 (s, 3H), 2.13 (s, 6H)
ppm; Anal. (C15H16ClNO2) C, H, N.
4-Chloro-3,6-dimethyl-2-(2,4,6-trimethyl-3-pyridyl)-pyri-
dine 1-Oxide (11g). White crystals, mp 113–114 °C; 1H NMR
(CDCl3) δ 7.09 (s, 1H), 6.82 (s, 1H), 2.44 (s, 6H), 2.41 (s, 3H),
2.29 (s, 3H), 2.13 (s, 3H) ppm; Anal. (C15H17ClN2O2) C, H, N.
4-Chloro-3,6-dimethyl-2-(2,4,6-trimethyl-phenyoxy)-pyri-
dine (6).9 To a solution of 4-chloro-2,5-dimethyl-6-(2,4,6-trimethyl-
phenoxy)-pyridine 1-oxide (55.00 g, 185.5 mmol) in 130 mL of
dry methylene chloride was added 2 M PCl3 in methylene chloride

Synthesis and SAR of CRF1 Antagonists
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5 1381
Table 3. In Vitro CRF1 Inhibition (IC50)a and Ex Vivo Datab Using Radiolabeled CRF for Displacement Assays in Rat Cortex and Pituitary for
Compounds with 4-Alkoxy SAR in the Pyridine Series
in vitro,a rat cortex
ex vivob
compound no.
OR
OCH(Et)2
OCH(Et)(Me)
OCH(Et)(CH2OMe)
OC()-CH3)(Et)
O-3-THF
pIC50 ( SEM (M) IC50 (nM) 3.2 mg/kg administration route cortex (% inhibition) pituitary (% inhibition)
2
8.17 ( 0.04
7.55 ( 0.13
7.96 ( 0.06
7.79 ( 0.09
6.99 ( 0.09
7.99 ( 0.27
6.8
28
11
16
100
10
p.o.
s.c.
s.c.
s.c.
75 ( 7
74 ( 5
75 ( 9
75 ( 21
NT
72 ( 11
42 ( 1
49 ( 14
55 ( 6
NT
42
43
13
44
14
s.c.
70 ( 6
6 ( 13
a Values reported are the geometric mean of at least three experimental runs using rat cortex. b Compounds were administered via oral (p.o.) or subcutaneous
(s.c.) route at 3.2 mg/kg. 125I-oCRF binding to brain and pituitary tissues in rats was measured using an ex vivo autoradiographic assay or brain homogenate
binding displacement assay. Percent inhibition values are mean ( SD. NT ) not tested.
(95 mL, 190 mmol) dropwise during a period of 1 h. After addition,
the mixture was heated at reflux for 0.5 h, cooled, and concentrated
to dryness. The residue was poured into ice–water and extracted
with methylene chloride. The organic layer was washed with brine,
neutralized with saturated sodium carbonate, dried, and concentrated
to give 56.75 g of the crude material. The crude material was
purified through a 150 g silica gel column by using chloroform as
eluent to give 49.27 g (95%) of the desired product as white crystals,
mp 57–62 °C; HRMS for C16H18ClNO: calc 275.1072, found
mixture was heated at 130 °C for 3 h. The reaction was quenched
with water and extracted with ethyl acetate. The organic layer was
separated, dried, and concentrated to give 68.21 g of yellow solid.
The solid was purified through silica gel column chromatography using
10% chloroform in hexane grading to chloroform as eluent to give
52.20 g (92%) of the title compound as white crystals, mp 72.5–74
°C; 1H NMR (CDCl3) δ 6.84 (s, 2H), 6.26 (s, 1H), 4.16 (m, 1H), 2.27
(s, 3H), 2.17 (s, 6H), 2.04 (s, 6H), 1.69 (m, 4H), 0.95 (t, 6H) ppm;
Anal. (C21H29NO2) C, H, N. The mesylate salt of 4-(1-ethyl-propoxy)-
3,6-dimethyl-2-(2,4,6-trimethyl-phenoxy)-pyridine was prepared by
addition of 1 equiv of methanesulfonic acid in ethyl acetate. The white
crystals formed from ethyl acetate, mp 117–119 °C; Anal.
(C22H33NO5S) C, H, N, S.
275.70172; IR (KBr) 2951, 2920, 1592, 1564 cm-1 1H NMR
;
(CDCl3) δ 6.87 (s, 2H), 6.77 (s, 1H), 2.39 (s, 3H), 2.29 (s, 3H),
2.18 (s, 3H), 2.03 (s, 6H) ppm.
Compounds 12b–g were prepared by the method analogous to
that described in the preparation of 6, starting with an appropriate
4-chloro-6-substituted-phenoxy/pyridoxy-pyridine 1-oxide and phos-
phorus trichloride.
2-(4-Bromo-2,6-dimethyl-phenoxy)-4-chloro-3,6-dimethyl-py-
ridine (12b). White crystals; 1H NMR (CDCl3) δ 7.22 (s, 2H),
6.81 (s, 1H), 2.40 (s, 3H), 2.20 (s, 3H), 2.05 (s, 6H) ppm; Anal.
(C15H15BrClNO) C, H, N.
4-Chloro-2-(4-chloro-2,6-dimethyl-phenoxy)-3,6-dimethyl-py-
ridine (12c). White crystals; 1H NMR (CDCl3) δ 7.07 (s, 2H), 6.81
(s, 1H), 2.41 (s, 3H), 2.20 (s, 3H), 2.06 (s, 6H) ppm; Anal.
(C15H15Cl2NO) C, H, N.
The following compounds were prepared by the method analo-
gous to that described for compound 2, starting from an appropriate
4-chloro-2-methyl-6-substituted-phenoxy-pyridine with an appropri-
ate alcohol and base.
cis/trans-4-(1-Ethyl-propenyloxy)-3,6-dimethyl-2-(2,4,6-tri-
methyl-phenoxy)-pyridine (13). 1H NMR (CDCl3) δ 6.85 (s, 2H),
6.30 (s, 0.3H), 6.21 (s, 0.7H), 5.10 (m, 0.7H), 4.95 (m, 0.3H), 2.27
(s, 3H), 2.24 (s, 2.1H), 2.19 (s, 0.9H), 2.14 (s, 3H), 2.05 (s, 6H),
1.65 (d, 0.9H), 1.50 (d, 2.1H), 1.08 (t, 1.8H), 1.05 (t, 3.2H) ppm.
6-Ethyl-4-(1-ethyl-propoxy)-3-methyl-2-(2,4,6-trimethyl-phe-
1
noxy)-pyridine (15). H NMR (CDCl3) δ 6.87 (s, 2H), 6.28 (s,
4-Chloro-6-methyl-2-(2,4,6-trimethyl-phenoxy)-nicotinic Acid
1
1H), 4.20 (m, 1H), 2.46 (q, 2H), 2.30 (s, 3H), 2.20 (s, 3H), 2.07 (s,
6H), 1.72 (m, 4H), 1.05 (t, 3H), 0.99 (t, 6H) ppm; Anal.
(C22H31NO2) C, H, N.
Methyl Ester (12d). Yellow crystals; mp 122–125 °C; H NMR
(CDCl3) δ 6.84 (s, 2H), 6.82 (s, 1H), 3.94 (s, 3H), 2.27 (s, 3H),
2.25 (s, 3H), 2.04 (s, 6H) ppm; Anal. (C17H18ClNO3) C, H, N.
4-Chloro-2,3,5-trimethyl-6-(2,4,6-trimethyl-phenoxy)-pyri-
3-Ethyl-4-(1-ethyl-propoxy)-6-methyl-2-(2,4,6-trimethyl-phe-
noxy)-pyridine (16). 1H NMR(CDCl3) δ 6.85 (s, 2H), 6.26 (s, 1H),
4.18 (m, 1H), 2.73 (q, 2H), 2.28 (s, 3H), 2.17 (s, 3H), 2.05 (s, 6H),
(m, 4H), 1.18 (t, 3H), 0.96 (t, 6H) ppm.
1
dine (12e). White crystals; mp 101–103 °C; H NMR (CDCl3) δ
6.85 (s, 2H), 2.39 (s, 3H), 2.28 (s, 3H), 2.22 (s, 3H), 2.20 (s, 3H),
2.01 (s, 6H) ppm; Anal. (C17H20ClNO) C, H, N.
4-Chloro-2-methyl-6-(2,4,6-trimethyl-phenoxy)-pyridine (12f).
4-(1-Ethyl-propoxy)-2-methyl-6-(2,4,6-trimethyl-phenoxy)-py-
1
White crystals; mp 46–48 °C; H NMR (CDCl3) δ 6.92 (s, 2H),
1
ridine (17). H NMR (CDCl3) δ 6.90 (s, 2H), 6.34 (d, J ) 2 Hz,
6.83 (d, 1H), 6.30 (d, 1H), 2.44 (s, 3H), 2.31 (s, 3H), 2.09 (s, 6H)
ppm; Anal. (C15H16ClNO) C, H, N.
1H), 5.70 (d, J ) 2 Hz, 1H), 4.05 (m, 1H), 2.40 (s, 3H), 2.30 (s,
3H), 2.11 (s, 6H), 1.62 (m, 4H), 0.89 (t, 6H) ppm; Anal.
(C20H27NO2) C, H, N.
4-Chloro-3,6-dimethyl-2-(2,4,6-trimethyl-3-pyridyl)-pyri-
dine (12g). White crystals; mp 72–73 °C; 1H NMR (CDCl3) δ 6.89
(s, 1H), 6.81 (s, 1H), 2.50 (s, 3H), 2.40 (s, 3H), 2.27 (s, 3H), 2.18 (s,
3H), 2.04 (s, 3H) ppm; Anal. (C15H17ClN2O·0.25H2O) C, H, N.
4-(1-Ethyl-propoxy)-3,6-dimethyl-2-(2,4,6-trimethyl-phenoxy)-
pyridine (2). To a solution of 3-pentanol (56 mL, 0.5205 mol) in
DMSO (760 mL) was added 60% sodium hydride in oil (7.64 g, 0.191
mol) portionwise. After stirring at room temperature for 30 min, a
solution of 4-chloro-2,5-dimethyl-6-(2,4,6-trimethyl-phenoxy)-pyridine
(47.80 g, 0.174 mol) in 50 mL of dry THF was added, and the resulting
4-(1-Ethyl-propoxy)-6-methyl-2-(2,4,6-trimethyl-phenoxy)-
nicotinic Acid Methyl Ester (18). Clear oil; 1H NMR (CDCl3) δ
6.84 (s, 2H), 6.39 (s, 1H), 5.04 (m, 1H), 3.85 (s, 3H), 2.27 (s, 3H),
2.23 (s, 3H), 2.05 (s, 6H), 1.5–1.7 (m, 4H), 0.95 (s, 6H) ppm; Exact
mass (C22H29NO4) calc 371.21, found 371.2097.
Mesylate Salt of 4-(1-Ethyl-propoxy)-2,3,5-trimethyl-6-(2,4,6-
trimethyl-phenoxy)-pyridine (24). White crystals, mp 58–60 °C;
1H NMR (CDCl3) δ 6.90 (s, 2H), 4.20 (m, 1H), 2.70 (s, 3H), 2.61

1382 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5
Chen et al.
(s, 3H), 2.28 (s, 3H), 2.16 (s, 3H), 2.08 (s, 6H), 1.5–1.8 (m, 4H),
0.96 (t, 6H) ppm; Exact mass (C22H31NO2) calc 341.2355, found
342.2440.
4-(1-Ethyl-propoxy)-3,6-dimethyl-5-ethyl-2-(2,4,6-trimethyl-
phenoxy)-pyridine (25). Clear oil; Anal. (C23H33NO2) C, H, N.
2-(2,6-Dimethyl-phenoxy)-4-(1-ethyl-propoxy)-3,6-dimethyl-
pyridine (26). 1H NMR (CDCl3) δ 7.06 (m, 3H), 6.30 (s, 1H),
4.20 (m, 1H), 2.21 (s, 6H), 2.11 (s, 6H), 1.73 (m, 4H), 0.99 (t, 6H)
ppm; APCI (C21H28N2O3) M + 1 ) 357.3.
2-(4-Chloro-2,6-dimethyl-phenoxy)-4-(1-ethyl-propoxy)-3,6-
dimethyl-pyridine (30). 1H NMR (CDCl3) δ 7.05 (s, 2H), 6.31 (s,
1H), 4.20 (m, 1H), 2.20 (s, 6H), 2.08 (s, 6H), 1.73 (m, 4H), 0.99
(t, 6H) ppm; Anal. (C20H26NO2Cl) C, H, N.
2-(4-Bromo-2,6-dimethyl-phenoxy)-4-(1-ethyl-propoxy)-3,6-
dimethyl-pyridine (31). 1H NMR (CDCl3) δ 7.18 (s, 2H), 6.30 (s,
1H), 4.22 (m, 1H), 2.20 (s, 6H), 2.05 (s, 6H), 1.73 (m, 4H), 1.00
(t, 6H) ppm; Anal. (C20H26BrNO2) C, H, N.
(brs, 2H), 2.30 (s, 3H), 2.18 (s, 3H), 2.10 (s, 6H), 1.74 (m, 4H),
1.00 (t, 6H) ppm.
[4-(1-Ethyl-propoxy)-6-methyl-2-(2,4,6-trimethyl-phenoxy)-
pyridin-3-yl]-dimethyl-amine (21). To a solution of 4-(1-ethyl-
propoxy)-6-methyl-2-(2,4,6-trimethyl-phenoxy)-pyridin-3-
ylamine in dry THF was added lithium bis(trimethylsilyl)amide at
–78 °C. After stirring at –78 °C for 10 min, an excess of methyl
iodide was added. The title compound was isolated after quenching
with water and extracting with ethyl acetate. The crude material
was purified by silica gel column chromatography using CHCl3 as
eluent to give 31 mg (70%) of the desired product as a tan foam;
1H NMR (CDCl3) δ 6.86 (s, 2H), 6.31 (s, 1H), 4.25 (m, 1H), 2.89
(s, 6H), 2.29 (s, 3H), 2.17 (s, 3H), 2.08 (s, 6H), 1.76 (m, 4H), 1.00
(t, 6H) ppm.
3-Bromo-4-(1-ethyl-propoxy)-6-methyl-2-(2,4,6-trimethyl-
phenoxy)-pyridine (22). A mixture of 3-amino-4-(1-ethyl-pro-
poxy)-6-methyl-2-(2,4,6-trimethyl-phenoxy)-pyridine with NaNO2,
48% HBr, CuBr, and water was stirred at 0 °C, heated at reflux for
10 min, and then cooled to room temperature. The resulting mixture
was adjusted to pH 7.0 and extracted with ethyl acetate. The organic
layer was separated, dried, and concentrated to give the title
compound as a golden oil. The oil was purified by silica gel column
chromatography using CHCl3 as eluent to give a white solid, mp
85–87 °C; 1H NMR (CDCl3) δ 6.86 (s, 2H), 6.3 (s, 1H), 4.28 (m,
1H), 2.28 (s, 3H), 2.18 (s, 3H), 2.02 (s, 6H), 1.7 (m, 4H), 1.0 (t,
6H) ppm; Anal. (C20H26BrNO2) C, H, N.
4-s-Butoxy-3,6-dimethyl-2-(2,4,6-trimethyl-phenoxy)-pyri-
1
dine (42). H NMR (CDCl3) δ 6.88 (s, 2H), 6.31 (s, 1H), 4.35 (m,
1H), 2.30 (s, 3H), 2.21 (s, 3H), 2.19 (s, 3H), 2.07 (s, 6H), 1.7–1.9 (m,
2H), 1.34 (d, 3H), 1.01 (t, 3H) ppm; Anal. (C20H27NO2) C, H, N.
4-(1-Methoxymethyl-propoxy)-3,6-dimethyl-2-(2,4,6-trimeth-
yl-phenoxy)-pyridine (43). 1H NMR (CDCl3) δ 6.88 (s, 2H), 6.38
(s, 1H), 4.42 (m, 1H), 3.5–3.7 (m, 2H), 3.42 (s, 3H), 2.31 (s, 3H),
2.21 (s, 6H), 2.07 (s, 6H), 1.7–1.85 (m, 2H), 1.02 (t, 3H) ppm;
Anal. (C21H29NO3) C, H, N.
3,6-Dimethyl-4-(tetrahydro-furan-3-yloxy)-2-(2,4,6-trimethyl-
1
4-(1-Ethyl-propoxy)-6-methyl-2-(2,4,6-trimethyl-phenoxy)-
nicotinic Acid (23). To a -78 °C solution of 3-bromo-4-(1-ethyl-
propoxy)-6-methyl-2-(2,4,6-trimethyl-phenoxy)-pyridine (50 mg,
0.127 mmol) in 1 mL of dry THF was added 0.061 mL of 2.5 M
n-BuLi in hexanes at -78 °C. The resulting mixture was stirred at
-78 °C for 5 min. An excess of dry ice was added, and the resulting
mixture was gradually warmed to room temperature. The reaction
was quenched with saturated NH4Cl and extracted with ethyl acetate
to give 50 mg of a light yellow oil that crystallized upon standing.
The material was purified by silica gel column chromatography
using CHCl3 as eluent to give 41 mg of the desired product that
was then dissolved in Et2O and extracted with 1 N NaOH. The
aqueous layer was acidified with 1 N HCl to pH 4 and extracted
with Et2O. The organic layer was separated, dried, and concentrated
to give 31 mg of white crystals, mp 145–147 °C; Anal. (C21H27NO4)
C, H, N.
phenoxy)-pyridine (44). H NMR (CDCl3) δ 6.88 (s, 2H), 6.25
(s, 1H), 4.99 (m, 1H), 3.9–4.1 (m, 4H), 2.31 (s, 3H), 2.23 (s, 3H),
2.20 (s, 3H), 2.1–2.3 (m, 2H), 2.07 (s, 6H) ppm; Anal. (C20H25NO3)
C, H, N.
4-(1-Ethyl-propoxy)-3,6-dimethyl-2-[3-(2,4,6-trimethyl-pyri-
dinoxy)]-pyridine (14). To a solution of 3-pentanol (0.11 mL) in
dry THF was added sodium hydride (60% in oil, 20 mg). After
stirring for 5 min, a solution of 4-chloro-2,5-dimethyl-6-[3-(2,4,6-
trimethyl-pyridinoxy)]-pyridine (92 mg, 0.33 mmol) in THF was
added. DMSO was added, and the resulting mixture was heated at
130 °C in an oil bath overnight. The reaction was quenched with
water and brine and extracted 3 times with ethyl acetate. The organic
layer was separated, dried (MgSO4), and concentrated to dryness.
After silica gel column chromatography purification, 47 mg (43%)
of the desired product was obtained as a clear oil; 1H NMR (CDCl3)
δ 6.88 (s, 1H), 6.37 (s, 1H), 4.21 (m, 1H), 2.5 (s, 3H), 2.29 (s,
3H), 2.19 (s, 3H), 2.18 (s, 3H), 2.07 (s, 3H), 1.70 (m, 4H), 0.98 (t,
6H) ppm. The oil was converted to the corresponding HCl salt to
give a white solid (63 mg).
4-(1-Ethyl-propoxy)-6-methyl-3-nitro-2-(2,4,6-trimethyl-phe-
noxy)-pyridine (19). To a mixture of 2-chloro-4-(1-ethyl-propoxy)-
6-methyl-3-nitro-pyridine (500 mg, 1.93 mmol) and 2,4,6-trimeth-
ylphenol (289 mg, 2.13 mmol) in dry THF was added KOt-Bu. The
resulting mixture was stirred at room temperature overnight. The
reaction was quenched with water and brine and extracted 3 times
with ethyl acetate. The organic layer was separated, dried (MgSO4),
and concentrated to dryness. After silica gel column chromatography
purification, the title compound was obtained as a light yellow crystal,
mp 106–109 °C; 1H NMR (CDCl3) δ 6.87 (s, 2H), 6.43 (s, 1H), 4.30
(m, 1H), 2.29 (s, 3H), 2.28 (s, 3H), 2.09 (s, 6H), 1.74 (m, 4H), 0.97
(t, 6H) ppm; Anal. (C20H26N2O4) C, H, N.
2-(4-Ethyl-2,6-dimethyl-phenoxy)-4-(1-ethyl-propoxy)-3,6-di-
methyl-pyridine (27). To a solution of 2.5 N n-BuLi in hexane
(0.47 mL, 1.18 mmol) in 5 mL of dry THF was added a solution
of 2-(4-bromo-2,6-dimethyl-phenoxy)-4-(1-ethyl-propoxy)-3,6-di-
methyl-pyridine (465 mg, 1.18 mmol) in 5 mL of dry THF at -78
°C. After stirring at that temperature for 5 min, an excess of ethyl
iodide (0.4 mL) was added, and the resulting mixture was stirred
at -78 °C for 30 min and then at 0 °C for 15 min. The reaction
was quenched with saturated ammonium chloride and extracted with
ethyl acetate. The organic layer was dried and concentrated to give
a brown oil. The oil was purified by silica gel column chromatog-
raphy using chloroform as eluent to give 260 mg of the title
compound as a white solid; 1H NMR (CDCl3) δ 6.90 (s, 2H), 6.38
(s, 1H), 4.20 (m, 1H), 2.61 (q, 2H), 2.24 (s, 3H), 2.21 (s, 3H), 2.10
(s, 6H), 1.70 (m, 4H), 1.30 (t, 3H), 0.98 (t, 6H) ppm; Anal.
(C22H31NO2) C, H, N.
4-(1-Ethyl-propoxy)-6-methyl-2-(2,4,6-trimethyl-phenoxy)-py-
ridin-3-ylamine (20). A mixture of 4-(1-ethyl-propoxy)-6-methyl-
3-nitro-2-(2,4,6-trimethyl-phenoxy)-pyridine (150 mg, 0.418 mmol)
and 10% Pd/C (23 mg) in ethanol was hydrogenated at 50 psi for
15 h. An additional 10% Pd/C was added, and the resulting mixture
was hydrogenated for an additional 24 h. The mixture was filtered
through Celite, and the filtrate was concentrated to dryness to give
200 mg of crude material. The crude material was purified by silica
gel column chromatography using 1:1 ethyl acetate/hexane as eluent
to give 61 mg of the desired product with 94% purity by GC/MS.
A portion of the free base (10 mg) was converted to the
The following compounds were prepared by the method analo-
gous to that described for compound 27, starting from n-BuLi and
2-(4-bromo-2,6-dimethyl-phenoxy)-4-(1-ethyl-propoxy)-3,6-dimeth-
yl-pyridine, followed by quenching with an appropriate electrophile.
2-(2,6-Dimethyl-4-propyl-phenoxy)-4-(1-ethyl-propoxy)-3,6-
dimethyl-pyridine (28). 1H NMR (CDCl3) δ 6.88 (s, 2H), 6.30 (s,
1H), 4.20 (m, 1H), 2.54 (dd, 2H), 2.22 (s, 3H), 2.20 (s, 3H), 2.09
(s, 6H), 1.6–1.8 (m, 6H), 0.9–1.1 (m, 9H) ppm; Anal. (C23H33NO2)
C, H, N.
1
corresponding HCl salt to give a white solid, mp 96–98 °C; H
4-(1-Ethyl-propoxy)-2-(4-fluoro-2,6-dimethyl-phenoxy)-3,6-di-
NMR (CDCl3) δ 6.88 (s, 2H), 6.03 (s, 1H), 4.20 (m, 1H), 3.72
methyl-pyridine (29). Colorless oil; Anal. (C20H26FNO2) C, H, N.

Synthesis and SAR of CRF1 Antagonists
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5 1383
4-(1-Ethyl-propoxy)-2-(4-iodo-2,6-dimethyl-phenoxy)-3,6-di-
methyl-pyridine (32). H NMR (CDCl3) δ 7.39 (s, 2H), 6.30 (s,
2.30 (brs, 1H), 2.22 (s, 3H), 2.21 (s, 3H), 2.12 (s, 6H), 1.73 (m,
4H), 0.91 (t, 6H) ppm; Anal. (C21H29NO3) C, H, N.
1
1H), 4.19 (m, 1H), 2.20 (s, 3H), 2.18 (s, 3H), 2.05 (s, 6H), 1.72
(m, 4H), 0.98 (t, 6H) ppm; Anal. (C20H26INO2) C, H, N.
4-[4-(1-Ethyl-propoxy)-3,6-dimethyl-pyridin-2-yloxy]-3,5-di-
methyl-benzaldehyde (38). 1H NMR (CDCl3) δ 9.94 (s, 1H), 7.61
(s, 2H), 6.32 (s, 1H), 4.20 (m, 1H), 2.21 (s, 3H), 2.16 (s, 9H), 1.70
(m, 4H), 0.98 (t, 6H) ppm; Anal. (C21H27NO3) C, H, N.
2-{4-[4-(1-Ethyl-propoxy)-3,6-dimethyl-pyridin-2-yloxy]-3,5-
dimethyl-phenyl}-propan-2-ol (41). 1H NMR (CDCl3) δ 7.15 (s,
2H), 6.25 (s, 1H), 4.20 (m, 1H), 2.20 (s, 3H), 2.19 (s, 3H), 2.10 (s,
6H), 1.85 (brs, 1H), 1.70 (m, 4H), 1.60 (s, 6H), 0.95 (t, 6H) ppm;
Anal. (C23H33NO3) C, H, N.
The following compounds were prepared by the method analo-
gous to that described for compound 35, starting with an appropriate
pyridine-3,5-dimethylphenol or pyridine-3,5-dimethyl-phenyl metha-
nol with a base, followed by quenching with an appropriate alkyl
halide.
2-(4-Ethoxy-2,6-dimethyl-phenoxy)-4-(1-ethyl-propoxy)-3,6-
dimethyl-pyridine (36). 1H NMR (CDCl3) δ 6.60 (s, 2H), 6.28 (s,
1H), 4.19 (m, 1H), 3.99 (q, 2H), 2.19 (s, 3H), 2.18 (s, 3H), 2.07 (s,
6H), 1.74 (m, 4H), 1.40 (t, 3H), 0.97 (t, 6H) ppm.
4-(1-Ethyl-propoxy)-2-(4-methoxymethyl-2,6-dimethyl-phe-
noxy)-3,6-dimethyl-pyridine (40). mp 58–60 °C; 1H NMR (CDCl3)
δ 7.05 (s, 2H), 6.30 (s, 1H), 4.41 (s, 2H), 4.19 (m, 1H), 3.42 (s,
3H), 2.21 (s, 3H), 2.18 (s, 3H), 2.11 (s, 6H), 1.72 (m, 4H), 0.98 (s,
6H) ppm; Anal. (C22H31NO3) C, H, N.
4-[4-(1-Ethyl-propoxy)-3,6-dimethyl-pyridin-2-yloxy]-3,5-di-
methyl-benzamide (33). To a solution of 4-[4-(1-ethyl-propoxy)-
3,6-dimethyl-pyridin-2-yloxy]-3,5-dimethyl-benzoic acid in anhy-
drous methylene chloride was added SOCl2, and the mixture was
stirred at room temperature for 1 h. The mixture was concentrated
to dryness to provide the corresponding acyl chloride. The acyl
chloride was quenched with an excess of NH3 and stirred at room
Biological Evaluation. Experimental details for the CRF receptor
binding assays, in vitro adenylate cyclase functional assay, effect
on elevations in plasma ACTH levels, CRF-induced increases in
locus coeruleus firing in the rat, acoustic startle response induced
by i.c.v. CRF, and fear-potentiated startle induced by electric foot
shock have been reported previously.5b
1
temperature to provide the desired product; H NMR (CDCl3) δ
7.51 (s, 2H), 6.32 (s, 1H), 6.2 (brs, 1H), 5.7 (brs, 1H), 4.20 (m,
1H), 2.22 (s, 3H), 2.19 (s, 3H), 2.12 (s, 6H), 1.72 (m, 4H), 0.97 (t,
6H) ppm.
Ex Vivo Receptor Binding Assay with 125I-oCRF. 125I-oCRF
binding to brain and pituitary tissues in rats was measured at 1 h
after dosing compounds at 3.2 mg/kg, p.o., by using either an ex
vivo brain homogenate or autoradiographic binding techniques as
described below.
4-[4-(1-Ethyl-propoxy)-3,6-dimethyl-pyridin-2-yloxy]-3,5-di-
1
methyl-phenol (34). H NMR (CDCl3) δ 7.85 (brs, 1H), 6.36 (s,
1H), 6.24 (s, 2H), 4.24 (m, 1H), 2.39 (s, 3H), 2.20 (s, 3H), 2.02 (s,
6H), 1.74 (m, 4H), 1.00 (t, 6H) ppm.
A. Homogenate ex Vivo Binding Assay. Male Sprague–Dawley
rats (weight of 200–300 g) were treated with a compound or vehicle
at the doses indicated. After a specified treatment period, they were
sacrificed via decapitation, and the brains and pituitary glands were
rapidly removed and frozen immediately on dry ice. The tissues
were suspended at 25 mg wet weight/mL in buffer (20 mM PIPES
pH 7.0, 10 mM MgCl2, 2 mM EGTA, 0.015% bacitracin, and 100
U/mL aprotinin). 100 µL aliquots of tissue homogenate were added
to assay samples containing 40 pM 125I-oCRF, yielding a final
volume of 200 µL. Nonspecific binding was determined using 1
µM rat/human CRF. After a 2 h incubation at room temperature,
assay samples were centrifuged for 10 min at 1000g. The
supernatant was discarded. Samples were rinsed with 100 µL of
ice-cold assay buffer and recentrifuged. Pellets were filtered onto
betaplate filtermats A by using a Skatron cell harvester (setting
222). Radioactivity was quantified using a betaplate scintillation
counter (Wallac).
B. Autoradiographic ex Vivo Binding Assay. Male Spra-
gue–Dawley rats (N ) 4, weight of 200–300 g) were sacrificed,
and the brains and pituitary glands were rapidly removed. The
pituitary was apposed to the underside of the brain close to its
original location, and the brain was frozen in isopentane maintained
at -30 °C. The brains were sectioned (16 µm) at the level
containing the pituitary, mounted onto microscope slides, and dried
at 5 °C under vacuum. After storage at -70 °C for no more than
one week, midbrain sections were thawed and incubated in 20 mM
PIPES buffer (pH 7.0), containing 10 mM MgCl2, 2 mM EGTA,
0.05% bovine serum albumin, 0.01% bacitracin, and 60 pM 125I-
oCRF, for one hour at room temperature. Successive serial sections
were incubated in the above mixture plus no inclusion (total
binding) or 1 µM cold rat/human CRF (nonspecific binding). All
sections were then washed for 2 × 5 min in ice cold wash buffer
(50 mM Tris-HCl, 5 mM MgCl2, 2 mM EGTA, pH 7.4), dipped
briefly in distilled water, and dried in a stream of compressed air.
The resulting slides were placed in standard X-ray cassettes and
apposed to tritium-sensitive LKB Ultrofilm for a period of 7–10
days before developing.
4-(1-Ethyl-propoxy)-2-(4-methoxy-2,6-dimethyl-phenoxy)-3,6-
dimethyl-pyridine (35). To a solution of 4-[4-(1-ethyl-propoxy)-
3,6-dimethyl-pyridin-2-yloxy]-3,5-dimethyl-phenol (40 mg, 0.12
mmol) in 3 mL of dry THF was added 10 mg of 60% sodium
hydride in oil at room temperature. After stirring for 5 min, 0.3
mL of methyl iodide was added, and the resulting mixture was
stirred at room temperature overnight. The reaction was quenched
with water and extracted with ethyl acetate. The organic layer was
dried and concentrated to give a yellow solid. The solid was purified
by silica gel column chromatography using hexane to 1:1 chloroform/
hexane as eluent to yield 20 mg of the title compound as a yellow
solid; 1H NMR (CDCl3) δ 6.66 (s, 2H), 6.28 (s, 1H), 4.20 (m, 1H),
3.79 (s, 3H), 2.20 (s, 3H), 2.19 (s, 3H), 2.08 (s, 6H), 1.71 (m, 4H),
0.97 (t, 6H) ppm.
4-(1-Ethyl-propoxy)-2-(4-isopropoxy-2,6-dimethyl-phenoxy)-
3,6-dimethyl-pyridine (37). To a solution of 4-[4-(1-ethyl-pro-
poxy)-3,6-dimethyl-pyridin-2-yloxy]-3,5-dimethyl-phenol (58 mg,
0.18 mmol) in 3 mL of dry THF was added triphenylphosphine
(70 mg, 0.264 mmol) and isopropanol (60 mg, 0.22 mmol). The
resulting mixture was stirred at room temperature for 5 min; diethyl
azodicarboxylate (46 mg, 0.26 mmol) was added. The mixture was
stirred at room temperature overnight. An additional 20 mg of
diethyl azodicarboxylate was added, and the mixture was stirred
for an additional 4 h. The reaction was quenched with water and
extracted with methylene chloride. The organic layer was dried and
concentrated to give an oil. The oil residue was purified by silica
gel column chromatography using 1:1 hexane/chloroform to 1:2
hexane/chloroform as eluent to give 38 mg (58%) of the title
compound as a colorless oil; 1H NMR (CDCl3) δ 6.60 (s, 2H),
6.28 (s, 1H), 4.50 (m, 1H), 4.18 (m, 1H), 2.20 (s, 3H), 2.19 (s,
3H), 2.07 (s, 6H), 1.71 (m, 4H), 1.34 (d, 6H), 0.98 (t, 6H) ppm;
Anal. (C23H33NO3) C, H, N.
{4-[4-(1-Ethyl-propoxy)-3,6-dimethyl-pyridin-2-yloxy]-3,5-di-
methyl-phenyl}-methanol (39). A mixture of 4-[4-(1-ethyl-pro-
poxy)-3,6-dimethyl-pyridin-2-yloxy]-3,5-dimethyl-benzaldehyde (114
mg, 0.41 mmol) and sodium borohydride (63 mg, 1.6 mmol) in 3
mL of methanol was stirred at room temperature for 2 h. The
reaction was quenched with water and extracted with ethyl acetate.
The organic layer was dried and concentrated to give a yellow oil.
The oil was purified through silica gel using chloroform as eluent
Acknowledgment. We thank Dr. Jon Bordner for the X-ray
structure analysis, David J am Ende for conducting DSC
analysis, Franco Lombardo, Christopher Lipinski, and the Pfizer
ADME group for pKa and solubility determination, and Chem-
syn Science Laboratories, Lenexa, Kansas, for conducting the
1
to give 70 mg of the title compound as a colorless oil; H NMR
(CDCl3) δ 7.04 (s, 2H), 6.32 (s, 1H), 4.55 (s, 2H), 4.21 (m, 1H),

1384 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5
Chen et al.
Sci. U.S.A. 1996, 93, 10477. (c) Mansbach, R. S.; Winston, E. N.;
Chen, Y. L. Antidepressant-like effects of CP-154,526, a selective
CRF1 receptor antagonist. Eur. J. Pharmacol. 1997, 323, 21. (d)
Shaham, Y.; Erb, S.; Leung, S.; Buczek, Y.; Stewart, J. CP-154 526:
a selective, nonpeptide antagonist of the corticotropin-releasing factor
1 receptor attenuates stress-induced relapse to drug seeking in cocaine-
and heroin-trained rats. Psychopharmacology 1998, 137, 184. (e)
Iredale, P. A.; Alvaro, J. D.; Lee, Y.; Terwilliger, R.; Chen, Y. L.;
Duman, R. S. Role of corticotropin-releasing factor receptor-1 in opiate
withdrawal. J. Neurochem. 2000, 74, 199. (f) Arborelius, L.; Skelton,
K. H.; Thrivikraman, K. V.; Plotsky, P. M.; Schulz, D. W.; Owens,
M. J. Chronic administration of the selective corticotropin-releasing
factor 1 receptor antagonist CP-154 526: behavioral, endocrine, and
neurochemical effects in the rat. J. Pharmacol. Exp. Ther. 2000, 294,
588. (g) Hikichi, T.; Akiyoshi, J.; Yamamoto, Y.; Tsutsumi, T.;
Isogawa, K.; Nagayama, H. Suppression of conditioned fear by
administration of CRF receptor antagonist CP-154 526. Pharmaco-
psychiatry 2000, 33, 189. (h) Le, A. D.; Harding, S.; Juzytsch, W.;
Watchus, J.; Shalev, U.; Shaham, Y. The role of corticotropin-releasing
factor in stress-induced relapse to alcohol-seeking behavior in rats.
Psychopharmacology 2000, 150, 317. (i) Maillot, C.; Million, M.; Wei,
J. Y.; Gauthier, A.; Tache, Y. Peripheral corticotropin-releasing factor
and stress-stimulated colonic motor activity involve type 1 receptor
in rats. Gastroenterology 2000, 119, 1569.
radiolabeled synthesis. Additional thanks is extended to Dr.
David Raunig for statistical assistance and calculations pertain-
ing to the ACTH data.
Supporting Information Available: Tables of elemental analy-
sis data for compounds 2, 15, 17, 19, 22, 23, 25–32, 34, and 37–44
and intermediates 10a–d, 11a–c, 11e–g, and 12b–g, as well as X-ray
data for 5 and atomic coordinates, bond lengths and angles, and
displacement parameters for 3. This material is available free of
charge via the Internet at http://pubs.acs.org.
References
(1) (a) Vale, W.; Spiess, J.; Rivier, C.; Rivier, J. Characterization of a 41
residue ovine hypothalamic peptide that stimulates secretion of
corticotropin and beta endorphin. Science 1981, 213, 1394. (b) Owens,
M. J.; Nemeroff, C. B. Physiology and pharmacology of corticotropin-
releasing factor. Pharmacol. ReV. 1991, 43, 425. (c) Black, P. H.
Psychoneuroimmunology: brain and immunity. Sci. Am. Sci. Med.
1996, 16.
(2) (a) Nemeroff, C. B.; Widerlov, E.; Bissette, G.; Wallens, H.; Karlsson,
I.; Eklund, K.; Kilts, C. D.; Loosen, P. T.; Vale, W. Elevated
concentrations of CSF corticotropin-releasing factor-like immunore-
activity in depressed patients. Science 1984, 226, 1342. (b) Darnell,
A.; Bremner, J. D.; Licinio, J.; Krystal, J.; Nemeroff, C. B.; Owens,
M.; Erdos, J.; Charnev, D. S. CSF levels of corticotropin releasing
factor in chronic post-traumatic stress disorder. Soc. Neurosci. Abstr.
1994, 20, 17.
(6) (a) Webster, E. L.; Lewis, D. B.; Torpy, D. J.; Zachman, E. K.; Rice,
K. C.; Chrousos, G. P. In vivo and in vitro characterization of
antalarmin, a nonpeptide corticotropin-releasing hormone (CRF)
receptor antagonist: suppression of pituitary ACTH release and
peripheral inflammation. Endocrinology 1996, 137, 5747. (b) Born-
stein, S. R.; Webster, E. L.; Torpy, D. J.; Richman, S. J.; Mitsiades,
N.; Igel, M.; Lewis, D. B.; Rice, K. C.; Joost, H. G.; Tsokos, M.;
Chrousos, G. P. Chronic effects of a nonpeptide corticotropin-releasing
hormone type 1 receptor antagonist on pituitary-adrenal function, body
weight, and metabolic regulation. Endocrinology 199, 139, 1546. (c)
Deak, T.; Nguyen, K. T.; Ehrlich, A. L.; Watkins, L. R.; Spencer,
R. L.; Maier, S. F.; Licinio, J.; Wong, M. L.; Chrousos, G. P.; Webster,
E.; Gold, P. W. The impact of the nonpeptide corticotropin-releasing
hormone antagonist antalarmin on behavioral and endocrine responses
to stress. Endocrinology 1999, 140, 79.
(3) (a) Holsboer, F.; Von Bardeleben, U.; Gerken, A.; Stalla, G. K.; Muller,
O. A. Blunted corticotropin and normal cortisol response to human
corticotropin-releasing-factor in depression. N. Engl. J. Med. 1984,
311, 1127. (b) Taylor, A. L.; Fishman, L. M. Corticotropin-releasing
hormone. N. Engl. J. Med. 1988, 319, 213.
(4) (a) Chen, R.; Lewis, K. A.; Perrin, M. H.; Vale, W. W. Expression
cloning of a human corticotropin-releasing-factor receptor. Proc. Natl.
Acad. Sci. U.S.A. 1993, 90, 8967. (b) Perrin, M. H.; Donaldson, C. J.;
Chen, R.; Lewis, K. A.; Vale, W. W. Cloning and functional expression
of a rat brain corticotropin releasing factor (CRF) receptor. Endocri-
nology 1993, 133, 3058. (c) Perrin, M.; Donaldson, C.; Chen, R.;
Blount, A.; Berggren, T.; Bilezikjian, L.; Sawchenko, P.; Vale, W.
Identification of a second corticotropin-releasing factor receptor gene
and characterization of a cDNA expressed in heart. Proc. Natl. Acad.
Sci. U.S.A. 1995, 92, 2969. (d) Liaw, C. W.; Lovenberg, T. W.; Barry,
G.; Oltersdorf, T.; Grigoriadis, D. E.; De Souza, E. B. Cloning and
characterization of the human corticotropin-releasing factor-2 receptor
complementary deoxyribonucleic acid. Endocrinology 1996, 137, 72.
(e) Chalmers, D. T.; Lovenberg, T. W.; Grigoriadis, D. E.; Behan,
D. P.; De Souza, E. B. Corticotropin-releasing factor receptors: from
molecular biology to drug design. Trends Pharmacol. Sci. 1996, 17,
166.
(5) (a) Chen, Y. L.; Mansbach, R. S.; Winter, S. M.; Brooks, E.; Collins,
J.; Corman, M. L.; Dunaiskis, A. R.; Faraci, W. S.; Gallaschun, R. J.;
Schmidt, A.; Schulz, D. W. Synthesis and oral efficacy of a
4-(butylethylamino)pyrrolo[2,3-d]pyrimidine: a centrally active cor-
ticotropin-releasing factor 1 receptor antagonist. J. Med. Chem. 1997,
40, 1749. (b) Schulz, W. D.; Mansbach, R. S.; Sprouse, J.; Braselton,
J. P.; Collins, J.; Corman, M.; Dunaiskis, A.; Faraci, S.; Schmidt, A.;
Seeger, T.; Seymour, P.; Tingley, F. D., III; Winston, E. N.; Chen,
Y. L.; Heym, J. CP-154 526: a potent and selective nonpeptide
antagonist of corticotropin releasing factor receptors. Proc. Natl. Acad.
(7) Prelog, V.; Szpilfogel, S. Steroids and sex hormones. LXXIX. 2-Ethyl-
5-methylpyridine, a dehydrogenation product of solanidine. HelV.
Chim. Acta 1942, 25, 1306.
(8) Because the pyridine-N-oxide intermediates may show exothermic
decomposition, it is recommended to test the differential scanning
calorimeter (DSC) prior to scale-up synthesis. Pyridine-N-oxide
intermediates 10 and 11 are not recommended for large scale
preparation, and caution is required to ensure safety in the laboratory.
In general, acceptable decomposition energy depends in part on the
conditions of the chemistry, for example dilution and operating
temperature, and so forth. In general, a 100 °C difference between
process temperature and decomposition is considered to be acceptable.
(9) An alternative method for regioselective synthesis of compound 6 was
developed for kilogram bulk preparation by the Process Department
and will be described in a separate paper.
(10) The detailed information will be described in a separate publication.
(11) Kinetic water solubility data was obtained from an in-house high
throughput assay. Compound 2 has water solubility of 0.2 and 3 µg/
mL at pH 7.0 and pH 2.0, respectively. pKa was determined by a UV
method and after extrapolation to 0% organic solvent.
JM070578K