2-Aryloxy-4-alkylaminopyridines: Discovery of novel corticotropin-releasing factor 1 antagonists

Article abstract of DOI:10.1021/jm070579c

An orally active clinical candidate of corticotropin-releasing factor 1 (CRF₁) antagonist 1 showed a significant positive food effect in dog and human after oral administration. Efforts to address the food effect issue led us to explore and dis

Full text of DOI:10.1021/jm070579c

J. Med. Chem. 2008, 51, 1385–1392
1385
2-Aryloxy-4-alkylaminopyridines: Discovery of Novel Corticotropin-Releasing Factor 1
Antagonists
Yuhpyng L. Chen,*,† R. Scott Obach,‡ John Braselton,§ Michael L. Corman,† James Forman,† Jody Freeman,§
Randall J. Gallaschun,† Robert Mansbach,§ Anne W. Schmidt,§ Jeffrey S. Sprouse,§ F. David Tingley, III,§ Elizabeth Winston,§
and David W. Schulz§
Medicinal Chemistry, Neuroscience, and Drug Metabolism Departments, PGRD, Pfizer, Inc., Groton, Connecticut 06340
ReceiVed May 17, 2007
An orally active clinical candidate of corticotropin-releasing factor 1 (CRF1) antagonist 1 showed a significant
positive food effect in dog and human after oral administration. Efforts to address the food effect issue led
us to explore and discover compounds in series 2 as orally active CRF1 receptor antagonists, in which some
compounds showed improved physicochemical properties while retaining desired pharmacological properties.
Compound 3a (CP-376395) was selected for further development, due not only to its reduced food effects
but also its greater efficacy in CNS models. Compound 3a was advanced to the clinic. The synthesis of
representative potential candidates and their in Vitro, ex ViVo, and in ViVo data are described.
Introduction
CRF, a 41 amino acid peptide, was identified as a key
regulator of the hypothalamus-pituitary-adrenal (HPA) axis.1
Published preclinical data support the hypothesis that a small-
molecule CRF1 receptor antagonist, such as 4a (CP-154526) or
4b (antalarmin), is effective in blocking neuroendocrine effects,
autonomic responses, or increased heart rate or behavioral
changes induced by exogenous or endogenous CRF.2,3 Since
the first nonpeptide small-molecule CRF1 receptor subtype
antagonist 4a was published as a research tool,2 many related
chemical series have been disclosed as selective CRF1 receptor
subtype antagonists by several laboratories over the past 13
years. We have independently discovered many CRF1 antagonist
chemotypes; for example, our published pyridine series was
found to be safe and free of liver toxicity4 which has been an
issue for some other series.5,6 An orally active CRF1 receptor
antagonist 14 was advanced to phase II. Compound 1 showed
a significant food effect in dog and human.7 It has low solubility
and basicity which is thought to account for a 10- to 20-fold
positive food effect observed in dogs and humans. It was thought
that increased basicity may lead to improved solubility and
reduced food effects. In a continuation of our efforts to improve
physicochemical properties, compounds in series 2 were de-
signed, in which the oxygen at either the alkoxy or aryloxy
portion of 1 was replaced with an aminoalkyl or arylamino
Figure 1. Structures of CRF1 selective antagonists.
group, respectively (see Figure 1). It is expected that this change
will lead to compounds with greater basicity along with
compared to 10–20-fold for 1. The synthesis, pharmacology,
improvements in solubility. Efforts to further optimize physi-
and pharmacokinetic data of the representative compounds are
cochemical properties by adding a polar group or blocking
presented.
metabolic sites have led to the discovery of several potential
candidates 3a,b and 5a,b that met the desired pharmacological
and pharmacokinetic profile and were selected for exploratory
toxicology evaluations. Among these, compound 3a demon-
strated unexpected potency in several in ViVo models and was
advanced to the clinic. Compound 3a showed reduced fed-fasted
food effects, only 2–3-fold in dogs and humans7 as predicted
Chemistry
The syntheses of compounds in series 2 are illustrated in
Schemes 1-4. Scheme 1 describes a method to control
regioselectivity via a pyridine 1-oxide 78 to provide the desired
isomer in an excellent yield. Compound 6 was prepared
according to the literature procedure9 with a small modification
in which 1 equiv of N,N-diethylaniline was added to improve
the yield from 33%9 to a maximum of 72% yield. Standard
oxidation of compound 6 with m-CPBA provided the desired
1-oxide 7 in a low yield along with recovered starting material,
which remained unreacted even after 2 days. Changing the
* To whom correspondence should be addressed. Tel: (860) 444-2438.
Fax: (860) 444-2438. E-mail: yuhpyng888@yahoo.com.
†
Medicinal Chemistry Department.
Drug Metabolism Department.
Neuroscience Department.
‡
§
10.1021/jm070579c CCC: $40.75
2008 American Chemical Society
Published on Web 02/21/2008

1386 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5
Chen et al.
Scheme 1a
a Reagents and conditions: (a) NaOMe in xylene, reflux, 62%; POCl3, diethylaniline, 95%; (b) urea-HOOH, CF3COOH, rt, 18 h; 93%; (c) (i) trimethylphenol,
a base (NaH, KH, or t-BuOK) in THF, rt, 97%, (ii) PCl3 in methylene chloride, 93%; (d) NH2CH(Et)(CH2R1) in DMSO or NMP; (e) Method A (R ) Cl):
(i) 1 N DIBALH in THF, toluene, 67%, (ii) SOCl2 in methylene chloride, 100%, (iii) 1 N BH3 in THF, room temperature, 58%. Method B (R ) Me and
Br): 1 equiv of AlCl3, 3–4 equiv of LiAlH4 in THF, reflux, 2 h (100 and 90%, respectively).
Scheme 2a
oxidizing agent to urea-HOOH in CF3COOH improved the
yield dramatically (93%). On the other hand, we have found
that oxidation of 3,6-dimethyl-2,4-dichloropyridine with m-
CPBA provided a better yield4 than urea-HOOH, indicating
urea-HOOH may only be suitable for oxidation of an electron-
deficient pyridine, where m-CPBA may be a better choice for
an electron-rich pyridine. Coupling of 7 with an aryloxy
compound followed by PCl3 reduction provided the correspond-
ing aryloxypyridine (8a–c) in excellent yield. Reaction of a
4-chloronicotinic acid methyl ester with an appropriate alkyl-
amino compound yielded the corresponding 4-alkylamino-2-
aryloxypyridine derivatives (9a-c and 5a). Two reduction
methods were used for conversion of the 3-COOMe-pyridine
9a-c to the corresponding 3-Me derivatives 3a-c. The first
method involved a three-step synthesis to give the corresponding
3-methyl analogue 3b from 9b in moderate yield as shown in
Scheme 1 (see footnote e, method A). Efforts to improve the
process led us to identify a one-step method for conversion of
a 3-methyl ester-pyridine to the corresponding 3-Me-pyridine
(e.g., 3a and 3c) using LiAlH4/AlCl3 in THF in excellent yield
(>90%). This method also worked well for conversion of
3-COOH to 3-Me as shown in Schemes 3 and 4 (conversion of
12 to 13 and 15 to 16) or in the presence of a halogen group as
seen in conversion of 9c to 3c (R4 ) Br) in Scheme 1.
a Reagents and conditions: (a) (i) LiOH H2O in p-dioxane/H2O, (ii) SOCl2
in methylene chloride, (iii) MeNH2(g) in methylene chloride; (b) (S)-2-
amino-1-butanol in N-methylpyrrolidone at 150 °C.
Scheme 3a
Scheme 2 illustrates the method for the synthesis of com-
pound 5b in which the final 2-aminobutanol coupling with 10
was achieved by direct heating at 150 °C in good yield.
Scheme 3 describes the synthesis of compound 13. Direct
coupling of the 2,4-dichloropyridine 6 with an alkoxide provides
a 2:1 mixture of regioisomers in favor of the desired 4-alkyl
ether product 11. Introduction of the trimethylanilino group to
the 2-Cl-pyridine proved to be challenging. Coupling of the
aniline via a pyridine N-oxide failed to provide the desired
product due to self-dimerization/oxidation of the trimethyl-
aniline. The best method we have identified after exploring
various conditions for generating 12 afforded the desired product
in about 30% yield by coupling 11 with trimethylaniline in the
presence of Cu and K2CO3 in DMF. Final reduction with AlCl3/
LiAlH4 gave the desired compound 13 for biological assay.
Compound 16 was prepared in a similar way in which the
a Reagents and conditions: (a) (i) 3-pentanol, NaH in THF, 22% isolated
yield, (ii) NaOH in MeOH/H2O, reflux, 92%; (b) 1 equiv of trimethylaniline,
1 equiv of K2CO3, 0.2 equiv of Cu in DMF, 29.4%; (c) 3 equiv of LiAlH4,
1 equiv of AlCl3 in THF, reflux, 78%.
4-alkylamino group was introduced first, followed by trimethyl-
aniline coupling and reduction as shown in Scheme 4.
Results and Discussion
Initial SAR exploration by replacing the oxygen in the alkoxy
or aryloxy substituents of 1 provided compounds in series 2 as
shown in Table 1. Compound 1 has a low pKa (∼3.6) where
the mesylate was found to be the only appropriate salt that can
form a crystalline solid; however, it dissociates easily in aqueous
solution. The mesylate salt of 1 is quite insoluble at pH 7 and

2-Aryloxy-4-alkylaminopyridines
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5 1387
Scheme 4a
Table 2. Pharmacokinetics of CRF1 Antagonists 1 and 3a in Beagle
Dogs
pharmacokinetic parameter
1
3a
intravenous dose (mg/kg)
CL (mL/min/kg)a
VDss (L/kg)a
1.0
1.0
4.1 ( 0.4
1.1 ( 0.3
3.1 ( 0.8
8.3
17 ( 5
5.3 ( 2.2
12 ( 7
8.8
VDγ (L/kg)a
t1/2γ (hr)b
oral dose (mg/kg) fasted state
Cmax (ng/mL)a
Tmax (h)a
5.0
1.0
187 ( 170
1.5 ( 0.6
3.6 ( 2.6
4.4
46.2 ( 25.3
1.8 ( 1.3
22 ( 19
73
F (%)a
a Reagents and conditions: (a) (i) 1-ethylpropylamine in DMSO, 110
°C, 42%, (ii) LiOH in dioxane/H2O, (iii) 1 N HCl, 75%; (b) 1 equiv of
trimethylaniline, 1 equiv of K2CO3, 0.2 equiv of Cu in DMF, 32.5%; (c) 3
equiv of LiAlH4, 1 equiv of AlCl3 in THF, reflux, 100%.
estimated Fa (%)b
oral dose (mg/kg) fed state
Cmax (ng/mL)a
Tmax (h)a
5.0
1.0
1740 ( 290
1.9 ( 1.0
37 ( 19
46
131 ( 62
2.0 ( 1.4
64 ( 10
100
F (%)a
estimated Fa (%)b
Table 1. In Vitro Binding Affinity and Ex ViVo Data of Compounds in
Series 2
a Values reported are mean + standard deviation. b Values reported are
harmonic mean.
p-methyl group of the trimethylphenoxy of 3a to help reduce
metabolic clearance with a chloro resulted in compound 3b with
no improvement of clearance; the in Vitro t1/2 values in human
liver microsomes were 38 and 30 min for 3a and 3b,
respectively. Introduction of a polar group such as OH at the
C4-side chain and COOMe of R3 of 3b resulted in compound
5a with increased polarity and decreased protein binding
(increased unbound fraction from 0.22% to 1.3%). Compound
5a seemed to have good stability when incubated with human
liver microsomes (intrinsic clearance ) 0.047 mL/min/mg
protein); however, the possibility existed that the 3-methyl ester
could be hydrolyzed by esterases in liver and other tissues.
Replacing the 3-COOMe with a more polar 3-CONHMe led to
compound 5b with slightly weaker in Vitro potency but reduced
protein binding (4.5% free), which may account for good ex
ViVo activity. All tested compounds in Table 1 demonstrated
good brain penetrability and oral activity measured by ex ViVo
binding in animals treated at 3.2 mg/kg. In general, compounds
that exhibit good brain penetration as measured by ex ViVo
binding also demonstrate efficacy in animal behavior models
as seen with these compounds.
Most compounds demonstrated similar in ViVo potency
compared to 1,4 with the exception of compound 3a. Compound
3a was unexpectedly more efficacious than others in the fear-
potentiated startle and locus coeruleus excitation models induced
by icv CRF, despite having a similar intrinsic affinity for the
CRF1 receptor. Several potential candidates (3a,b, 5a,b) met
the desired pharmacological and pharmacokinetic profile and
were selected for exploratory toxicology evaluation. Among
these, compound 3a passed exploratory toxicology studies and
was selected for further development.
Pharmacology Results of 3a. Compound 3a fully antago-
nizes oCRF-stimulated adenylate cyclase activity in rat cerebral
cortex and at human CRF1 receptors with an apparent Ki value
of 12 nM, indicating antagonist functional activity. It is highly
selective for the human CRF1 receptor subtype; affinity for the
CRF2 receptor is >10000 nM. It showed affinities greater than
1 µM against 40 neurotransmitter receptor and ion channels
(Nova Screen). Compound 3a significantly attenuated activation
of the HPA axis (MED ) 10 mg/kg, p.o., p < 0.05 versus CRF
alone, One-Way ANOVA, Dunnett’s post hoc test, N ) 10), as
measured by increased plasma adrenocorticotropin hormone
(ACTH) levels, in response to i.v. CRF (4 µg/kg) administration.
In the CNS, systemically administered 3a blocks the effects of
both exogenous and endogenous CRF. Pretreatment with 3a
reversed the excitation of locus coeruleus neurons induced by
in Vitroa
pIC50
compd SEM (M) (nM) (% inhibition ( SD) (% inhibition ( SD)
ex ViVob at 3.2 mg/kg (oral)
(
IC50
cortex pituitary
1
8.17 ( 0.04
8.29 ( 0.05
8.14 ( 0.06
8.25 ( 0.19
8.19 ( 0.08
6.8
5.1
7.2
5.6
6.4
75 ( 7
81 ( 17
79 ( 23
86 ( 4
111 ( 17
86 ( 2
NT
72 ( 11
49 ( 16
72 ( 4
81 ( 4
86 ( 4
63 ( 2
NT
3a
3b
3c
5a
5b
13
16
7.51 ( 0.05 31
8.43 ( 0.02
6.13 ( 0.08 750
3.7
NT
NT
a Values reported are the geometric mean of at least three experimental
runs using rat cortex.2a b Compounds were administered orally. 125I-
ovineCRF (125I-oCRF) binding to brain and pituitary tissues in rats was
measured using an ex ViVo brain homogenate binding assay. NT: not tested.
2 with solubility of 0.2 and 3 µg/mL, respectively. Replacement
of the 3-alkoxy group with a 3-alkylamino group resulted in
compound 3a with increased basicity (pKa of 6.9) and solubility
in simulated gastrointestinal fluid or at low pH (0.1 µg/mL at
pH 7 and 5.4 mg/mL at pH 2.4), which also made a significant
impact on reduction of food effects from 10- to 20-fold seen
with 1 down to 2–3 fold in dogs and humans as expected. A
very basic 2,4-diaminopyridine 16 (pKa of 9.3) reduced CRF1
binding affinity dramatically (IC50 value of 750 nM). During
the course of our CRF program, a CRF1 pharmacophore was
developed based on the SAR from several chemotypes, in which
the lone pair electron of the “N” in the core template (e.g., pyrido
in series 2) is essential for a compound to exhibit potent CRF1
binding affinity. The basic nitrogen in 16 exists as a protonated
form in the pyrido ring rather than amino side chain (X-ray
structural analysis) at physiological pH; thus, the interaction of
the “N” with the CRF receptor is diminished, which may
account for the poor CRF1 binding affinity. The polar 3-COOH
intermediate 12 was found to be inactive as expected. Except
for the very basic 2,4-diaminopyridine 16, all compounds in
Table 1 showed low nanomolar binding affinity. Replacing the

1388 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5
Chen et al.
1
melting point apparatus and are uncorrected. High-field H NMR
icv CRF (3 µg) with an ID50 of <0.01 mg/kg, i.v. Compound
3a completely blocked the enhanced startle response induced
by icv CRF (1 µg) at 17.8 mg/kg, p.o. and partially blocked at
10 mg/kg, p.o. without significantly altering baseline startle. The
attenuation of fear-potentiated startle was statistically significant
at lower doses (0.32–3.2 mg/kg, p.o., with 62–83% blockade)
and completely reversed by 3a at 10 mg/kg, p.o. In conclusion,
3a has been shown to behave as a CRF1 receptor antagonist in
several different in ViVo paradigms.
Pharmacokinetics of CRF1 Receptor Antagonists (1 and
3a) in Dogs. Both compounds 1 and 3a were studied for their
pharmacokinetic behavior in beagle dogs after intravenous and
oral administration. Following i.v. administration, the clearance
of 1 was low relative to hepatic blood flow (4.1 vs 35 mL/min/
kg), while the clearance of 3a was moderate (17 vs 35 mL/
min/kg). The kinetics were multiphasic for both compounds and
demonstrated volumes of distribution during the terminal
elimination phase that were considerably greater than the steady-
state volumes of distribution. Such an observation can be
explained by partitioning into a deep pharmacokinetic compart-
ment (e.g., adipose tissue) from which the compound slowly
leeches back into the circulation, and is a common phenomenon
for lipophilic compounds. (In fact, in a tissue distribution study
conducted in rats, sequestration of 1 into adipose tissue was
observed; data not shown.)
Following oral administration, the bioavailability of 1 was
lower than 3a, despite the lower systemic clearance. This is
consistent with 3a being better absorbed than 1. Back calcula-
tions of estimates of the fraction absorbed suggest that 3a is
73% absorbed while 1 is only 4% absorbed in animals that have
been fasted. When these compounds were administered to
animals 1 h following feeding, the oral bioavailability of 1 was
markedly increased (10-fold), while the oral bioavailability
increased 3-fold for 3a. Positive food effects can arise by several
different mechanisms, including possible inhibition of intestinal
efflux transporters by food constituents or enhanced dissolution
of the compound by food. In the case of 1 and 3a, the latter
explanation is more plausible because administration of these
compounds in triglyceride formulations also resulted in increased
oral bioavailability in the fasted state (data not shown). These
results clearly show that in progressing from compound 1 to
compound 3a, increasing the basicity in this series improved
the oral pharmacokinetics and reduced the magnitude of the
positive food effect.
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-6-methyl-1-oxynicotinic Acid Methyl Ester (7).
Urea hydrogen peroxide addition compound (98% pure) (3.860 g,
40.9 mmol) was added to a stirring solution of 2,4-dichloro-6-
methylnicotinic acid methyl ester (3.000 g, 13.6 mmol) in trifluo-
roacetic acid (15 mL) under nitrogen. The reaction mixture was
stirred at room temperature for 18 h. Additional urea-hydrogen
peroxide (0.640 g, 6.8 mmol) was added and the reaction stirred
for an additional 3 h. The reaction mixture was poured over
ice–water and stirred rapidly. The mixture was treated with sodium
thiosulfate, neutralized with 2 N aqueous NaOH to pH 11, and
extracted three times with chloroform. The combined chloroform
extracts were washed with water andbrine, dried over sodium
sulfate, and concentrated to give 2.98 g (93%) of the desired product
1
as a white crystal: mp 90–91.5 °C; H NMR (CDCl3) δ 7.26 (s,
1H), 3.98 (s, 3H), 2.54 (s, 3H). Anal. (C8H7NO2Cl2) C, H, N.
4-Chloro-6-methyl-2-(2,4,6-trimethylphenoxy)-1-nicotinic Acid
Methyl Ester (8a). A mixture of 2,4-dichloro-6-methyl-1-oxyni-
cotinic acid methyl ester (2.240 g, 9.49 mmol) and trimethylphenol
(1.290 g, 9.49 mmol) in dry THF was cooled in an ice bath, and
60% NaH in oil (380 mg, 9.49 mmol) was added portionwise during
a period of 5 min. The reaction mixture was stirred at room
temperature for 6 h. The mixture was quenched with water and
saturated ammonium chloride and extracted twice with chloroform.
The organic layer was dried and concentrated to give 3.120 g (97%)
of the title compound, which was used directly in the next step:
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).
To a solution of crude 4-chloro-6-methyl-2-(2,4,6-trimethylphe-
noxy)-1-oxynicotinic acid methyl ester (3.100 g) in 30 mL of dry
methylene chloride was added 2 M PCl3 in methylene chloride (5.3
mL, 10.6 mmol) at room temperature. The resulting mixture was
heated at reflux for 1 h. The mixture was concentrated to dryness,
and the residue was poured over ice–water and extracted with
CHCl3. The organic layer was dried and concentrated to give 2.820
g (93%) of the crude title compound. The crude material was
purified through silica gel column chromatography using CHCl3/
hexane (1:1) as eluent to give 1.452 g of yellow crystals: mp
127–128.5 °C; 1H 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). Anal. C17H18ClNO3
(C,H,N).
4-Chloro-6-methyl-2-(2,6-dimethyl-4-chlorophenoxy)nicotin-
ic Acid Methyl Ester (8b). The title compound was prepared by
the method analogous to that described for compound 8a as a white
crystal: mp 125–128 °C; 1H NMR (CDCl3) δ 7.03 (s, 2H), 6.86 (s,
1H), 3.96 (s, 3H), 2.25 (s, 3H), 2.05 (s, 6H).
Conclusions
4-Chloro-6-methyl-2-(2,6-dimethyl-4-bromophenoxy)nicotin-
ic Acid Methyl Ester (8c). The title compound was prepared by
the method analogous to that described for compound 8a to give
white crystals: mp 108–110 °C. Anal. (C16H15BrClNO3) C, H, N.
4-(1-Ethylpropylamino)-6-methyl-2-(2,4,6-trimethylphenoxy)
nicotinic Acid Methyl Ester (9a). A mixture of 4-chloro-6-methyl-
2-(2,4,6-trimethylphenoxy)nicotinic acid methyl ester (3.434 g,
10.74 mmol) and 1-ethylpropylamine (10 mL) in 10 mL of DMSO
was heated at 120 °C for 15 h. The mixture was quenched with
water and extracted with ethyl acetate. The organic layer was dried
and concentrated to give a yellow solid. The yellow solid was
recrystallized with hexane to give 2.519 g (63%) of the title
compound as white crystals: mp 106–107.5 °C; 1H NMR (CDCl3)
δ 8.07 (d, 1H), 6.87 (s, 2H), 6.08 (s, 1H), 3.87 (s, 3H), 3.34 (m,
1H), 2.30 (s, 3H), 2.12 (s, 3H), 2.09 (s, 6H), 1.64 (m, 4H), 0.97 (t,
6H). Anal. (C22H30N2O3) C, H, N.
Compounds in series 2 were designed to address physico-
chemical properties. All compounds exhibited potent CRF1
binding affinity except 16 because of its high basicity. These
low nanomolar compounds in series 2 also demonstrated good
brain ex ViVo displacement activity after oral administration,
indicating good oral activity and brain penetrability. Several
potential candidates 3a,b and 5a,b met the desired pharmaco-
logical and pharmacokinetic profile. Compound 3a is clearly
more potent than 1 in relevant in ViVo models, despite having
a similar intrinsic affinity for the CRF1receptor subtype. In dogs,
a moderate food effect was observed; oral bioavailability was
22% in fasted animals and 64% in fed animals. As a result, 3a
was selected to be a back up candidate and advanced to the
clinic, where it may be useful in evaluating the utility of CRF1
receptor antagonists in stress-related disorders in the clinic.
4-(1-Ethylpropylamino)-6-methyl-2-(2,6-dimethyl-4-chlorophe-
noxy)nicotinic Acid Methyl Ester (9b). A mixture of 4-chloro-
6-methyl-2-(4-chloro-2,6-dimethylphenoxy)nicotinic acid methyl
ester (77 mg, 0.226 mmol) and 1-ethylpropylamine in DMSO was
heated at 120 °C for 4 h. The mixture was quenched with saturated
ammonium chloride, water, and brine and extracted with ethyl
Experimental Section
The syntheses of compounds in series 2 are described below.
Melting points were determined on a Thomas-Hoover capillary

2-Aryloxy-4-alkylaminopyridines
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5 1389
acetate. The organic layer was dried and concentrated to give 140
mg of yellow solid: 1H NMR (CDCl3) δ 8.10 (d, 1H), 7.03 (s,
2H), 6.09 (s, 1H), 3.88 (s, 3H), 3.35 (m, 1H), 2.10 (s, 3H), 2.08 (s,
6H), 1.5–1.7 (m, 4H), 0.96 (t, 6H).
2-(4-Chloro-2,6-dimethylphenoxy)-4-(1-hydroxymethylpropyl-
amino)-6-methylnicotinic acid methyl Ester (5a). A mixture of
4-chloro-2-(4-chloro-2,6-dimethylphenoxy)-6-methylnicotinic acid
methyl ester (9.0 g, 26.45 mmol) and (S)-2-amino-1-butanol (12.7
mL) in 1-methyl-2-pyrrolidinone was heated at 130 °C for 2 h and
then at 100 °C overnight. The mixture cooled to room temperature,
poured into ice–water, and diluted with ethyl acetate. The organic
layer was separated, washed with water, dried over anhydrous
sodium sulfate, filtered, and concentrated to dryness to give 13.6 g
of crude product as a light yellow oil. The oil was purified by silica
gel column chromatography using chloroform to 2% MeOH in
chloroform as eluent to give 6.684 g (64%) of the title compound
as white glass foam. The glass foam was triturated with hexane to
give a white solid. The solid was recrystallized from diisopropyl
ether to give white crystals, mp 122.5–124 °C. Anal. (C20H25-
ClN2O4) C, H, N.
4-(1-Ethylpropylamino)-6-methyl-2-(2,6-trimethyl-4-bromophe-
noxy)nicotinic Acid Methyl Ester (9c). A mixture of 4-chloro-
6-methyl-2-(4-bromo-2,6-dimethylphenoxy)nicotinic acid methyl
ester and 1-ethylpropylamine in DMSO was heated at 120 °C for
16 h. The mixture was quenched with water and brine and extracted
with ethyl acetate. The organic layer was dried and concentrated
to dryness. The residue was purified by silica gel column chroma-
tography using hexane to 3% ethyl acetate in hexane as eluent to
1
give the title compound as a white solid: H NMR (CDCl3) δ 8.1
(d, 1H), 7.18 (s, 2H), 6.08 (s, 1H), 3.87 (s, 3H), 3.35 (m, 1H), 2.10
(s, 3H), 2.08 (s, 6H), 1.4–1.7 (m, 4H), 0.96 (t, 6H).
[3,6-Dimethyl-2-(2,4,6-trimethylphenoxy)pyridin-4-yl](1-eth-
ylpropyl)amine (3a). To a mixture of 4-(1-ethylpropylamino)-6-
methyl-2-(2,4,6-trimethylphenoxy)nicotinic acid methyl ester (29
mg, 0.078 mmol) and AlCl3 (10 mg, 0.078 mmol) in dry THF was
added 1 M LiAlH4 in diethyl ether (0.31 mL, 0.31 mmol) at room
temperature. After being sitrred for 10 min, the mixture was heated
at reflux for 2 h. The mixture was quenched with 0.2 mL of water,
0.2 mL of 1 N NaOH, 0.4 mL of water, and 10 mL of dry THF
and stirred for 15 min. The mixture was filtered through Celite
and washed with chloroform. The filtrate was dried over dry sodium
sulfate, filtered and concentrated to dryness to give 26 mg (93%)
of white crystalline solid: mp 64–68 °C; 1H NMR (free base, CDCl3,
300 MHz) δ 6.82 (s, 2H), 6.05 (s, 1H), 3.70 (d, 1H, NH), 3.32 (m,
1H), 2.29 (s, 3H), 2.15 (s, 3H), 2.11 (s, 3H), 2.08 (s, 6H), 1.45–1.70
(m, 4H), 0.93 (t, 6H). The corresponding HCl salt was prepared
and recrystallized from diethyl ether to give a white solid: mp
4-Chloro-2-(2,4,6-trimethylphenoxy)-6,N-dimethylnicotina-
mide (10). To a solution of 4-chloro-6-methyl-2-(2,6-dimethyl-4-
chlorophenoxy)nicotinic acid methyl ester (10.00 g, 29.4 mmol)
in 60 mL of p-dioxane was added a solution of LiOH ·H2O (2.50
g, 103.4 mmol) in 60 mL of water. The resulting mixture was heated
at reflux for 2 h and then stirred at room temperature overnight.
The mixture was cooled in an ice bath and quenched with 11 mL
of concentrated HCl. The resulting mixture was extracted twice
with methylene chloride. The organic layer was separated, dried
over Na2SO4, and filtered. The filtrate was concentrated to give a
crude 4-chloro-6-methyl-2-(2,4,6-trimethylphenoxy)nicotinic acid
as a yellow oil (9.87 g) that was used as is.
To a solution of 4-chloro-2-(4-chloro-2,6-dimethylphenoxy)-6-
methylnicotinic acid (3.002 g, 9.2 mmol) in 10 mL of methylene
chloride was added thionyl chloride (4 mL, 54.8 mmol) at room
temperature, and the mixture was stirred for 1 h. The resulting
mixture was concentrated to dryness to give 3.112 g of 4-chloro-
2-(4-chloro-2,6-dimethy-phenoxy)-6-methylnicotinic acyl chloride
as a light yellow solid. An excess of methylamine was bubbled
into a solution of the crude acyl chloride (0.841 g, 5 mmol) in 6
mL of methylene chloride until a precipitate formed. The mixture
was stirred at room temperature for 1 h, quenched with water, and
extracted with chloroform. The organic layer was separated, dried
over Na2SO4, and filtered. The filtrate was concentrated to dryness
to give 0.721 g of the desired product as an off-white solid. The
solid was recrystallized from EtOAc/Et2O to give off-white crystals:
mp 243.1 °C; 1H NMR (CDCl3) δ 7.04 (s, 2H), 6.87 (s, 1H), 5.87
(brs,1H), 3.06 (d, 3H), 2.25 (s, 3H), 2.07 (s, 6H).
1
212–215 °C; H NMR (hydrochloride salt, CDCl3, 300 MHz) δ
6.89 (s, 2H), 6.20 (s, 1H), 5.18 (d, 1H,NH), 3.46 (m, 1H), 2.66 (s,
3H), 2.27 (s, 3H), 2.11 (s, 6H), 2.04 (s, 3H), 1.68 (m, 4H), 0.95 (t,
6H). Anal. (C21H30N2O·HCl) C, H, N.
3,6-Dimethyl-2-(2,6-dimethyl-4-bromophenoxy)pyridin-4-yl](1-
ethylpropyl)amine (3c). The title compound was prepared by the
1
method analogous to that for 3a to give a white solid: H NMR
(CDCl3) δ 7.19 (s, 2H), 6.09 (s, 1H), 3.36 (d, 1H), 3.33 (m, 1H),
2.15 (s, 3H), 2.12 (s, 3H), 2.09 (s, 6H), 1.4–1.8 (m, 4H), 0.97 (t,
6H).
[3,6-Dimethyl-2-(2,6-dimethyl-4-chlorophenoxy)pyridin-4-
yl](1-ethylpropyl)amine (3b). To a 0 °C solution of 4-(1-ethylpropyl-
amino)-6-methyl-2-(2,6-dimethyl-4-chlorophenoxy)nicotinic acid
methyl ester (145 mg, 0.37 mmol) in toluene (1 mL) was added 1
M diisobutylaluminum hydride in THF (1.2 mL, 1.2 mmol). The
mixture was then stirred at room temperature. The mixture was
quenched with methanol, stirred for 1 h, filtered through Celite,
and washed with methanol. The filtrate was concentrated to dryness
to give 140 mg of crude product that was purified by silca gel
column chromatography using 1:1 hexane/chloroform to give the
desired 3-hydroxymethyl product as a white solid (90 mg, 67%).
2-(4-Chloro-2,6-dimethylphenoxy)-4(S)-(1-hydroxymethylpro-
pylamino)-6,N-dimethylnicotinamide (5b). A mixture of 4-chloro-
2-(2,4,6-trimethylphenoxy)-6,N-dimethylnicotinamide (22.36 g, 65.9
mmol) and (S)-2-amino-1-butanol (17.8 mL, 189 mmol) in N-
methylpyrrolidinone (50 mL) was heated at 150 °C overnight. The
mixture was quenched with water and extracted twice with ethyl
acetate. The organic layer was washed with brine and separated.
The organic layer was dried over Na2SO4 and filtered. The filtrate
was purified by silica gel column chromatography using 1%
methanol in methylene chloride as eluent to give 25.87 g (∼100%)
of the desired product as a light yellow foam form that was
recrystallized from Et2O/hexane to give white crystals: mp 110.2
To a solution of the 3-hydroxymethyl solid (85 mg, 0.23 mmol)
in methylene chloride (0.3 mL) was added SOCl2 (0.3 mL). The
mixture was stirred at room temperature for 1 h and then
concentrated to dryness to give a light golden oil that was pumped
in vacuo to give crude 3-chloromethyl product. To a solution of
3-chloromethyl derivative (75 mg, 0.196 mmol) in dry THF was
added 1.0 M BH3 in THF (0.59 mL, 0.59 mmol) and the mixture
stirred for 2 h. The mixture was quenched with dilute HCl and
stirred for 5 min. The reaction mixture was neutralized with 2 N
NaOH and water and extracted with ethyl acetate. The organic layer
was separated, dried, and concentrated to dryness. The residue was
purified by silica gel column chromatography to give the title
1
°C; H NMR(CDCl3) δ 9.80 (d, 1H), 8.12 (s, 1H), 7.04 (s, 2H),
6.22 (s, 1H), 3.5–3.8 (m, 3H), 2.93 (d, 3H), 2.06 (s, 9H),
1.8(brs,1H), 1.5–1.7 (m, 2H), 0.99 (t, 3H) ppm. Anal. (C20H26-
ClN3O3) C, H, N.
2-Chloro-4-(1-ethylpropoxy)-6-methylnicotinic Acid (11). To
a solution of 3-pentanol (11.7 mL, 108.25 mmol) in 200 mL of
THF was added 60% NaH in oil (2.60 g, 108.25 mmol) at 0 °C,
and stirring was continued for 30 min. 2,4-Dichloro-6-methylni-
cotinic methyl ester (22.75 g, 103.10 mmol) was added at 0 °C,
and the mixture was stirred at room temperature overnight. The
mixture was quenched with water (400 mL) and extracted three
times with ethyl acetate (400 mL). The organic layer was separated,
dried, and concentrated to give 30.59 g of yellow oil as a mixture
of isomers with at least four components. The desired product was
isolated from silica gel column chromatography using 8% ethyl
1
compound as a colorless oil (36 mg): H NMR(CDCl3) δ 7.03 (s,
2H), 6.08 (s, 1H), 3.73 (d, 1H), 3.3 (m, 1H), 2.15 (s, 3H), 2.12 (s,
3H), 2.08 (s, 6H), 1.4–1.6 (m, 4H), 0.96 (t, 6H). The HCl salt was
prepared as a light yellow crystal, mp 194–197 °C. Anal.
(C20H27ClN2O·HCl) C, H, N.

1390 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5
Chen et al.
acetate in hexane as eluent to give 7.72 g (27.5%) as a clear oil.
Anal. (C13H18ClNO3) C, H, N.
purified by silica gel column chromatography using 4% methanol
in chloroform as eluent to give the desired product (1.8 g, 32.5%)
as a solid: mp 130–135 °C; 1H NMR (CDCl3) δ 13.54 (brd s, 1H),
11.90 (brd s, 1H), 8.20 (s, 2H), 5.88 (s, 1H), 3.34 (m, 1H), 2.24 (s,
3H), 2.19 (s, 6H), 2.16 (s, 3H), 1.54–1.67 (m, 4H), 0.96 (t, 6H).
4-(1-Ethylpropylamino)-3,6-dimethyl-2-(2,4,6-trimethylphe-
nylamino)pyridine (16). To a solution of 4-(1-ethylpropylamino)-
6-methyl-2-(2,4,6-trimethylphenylamino)nicotinic acid (100 mg,
0.28 mmol) in 4 mL of dry THF were added 1 M lithium aluminum
hydride in diethyl ether (1.12 mL, 1.12 mmol) and aluminum
trichloride (38 mg, 0.28 mmol) at room temperature. The resulting
mixture was heated at reflux and then cooled to room temperature.
The mixture was quenched with 0.8 mL of water, 0.8 mL of 1 N
NaOH, and then 1.6 mL of water. The mixture was stirred for 5
min and then filtered through Celite and washed with ethyl acetate.
The filtrate was diluted with water, and the organic layer was
separated, dried over MgSO4, and concentrated to give the desired
product as a golden oil (100 mg, >100%) which was purified by
silica gel column chromatography using 0.1 mL of NH4OH in 500
mL of 20% ethyl acetate in hexane as eluent to give the desired
product (98 mg, 100%) as a tan solid: mp 77–79 °C; 1H NMR
(CDCl3) δ 6.88 (s, 2H), 5.94 (s, 1H), 5.4 (brd s, 1H), 3.60 (d, 1H),
3.31 (m, 1H), 2.28 (s, 3H), 2.22 (s, 3H), 2.15 (s, 6H) 1.4–1.7 (m,
4H), 0.94 (t, 6H) ppm. Anal. (C21H31N3 ·0.5H2O) C, H, N.
Biological Evaluation. Experimental details for the CRF receptor
binding assay, 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 icv CRF, and fear-potentiated startle induced by electric foot
shock have been reported previously.2a,b
A mixture of 2-chloro-4-(1-ethylpropoxy)-6-methylnicotinic acid
methyl ester (7.70 g, 28.34 mmol) and NaOH (11.33 g, 283.36
mmol) in 130 mL of MeOH and 75 mL of water was heated at
reflux for 3 h. The mixture was concentrated to dryness and diluted
with water, adjusted to pH 3 with 1 N HCl, and then extracted
with EtOAc. The organic layer was separated, dried, and concen-
trated to give the desired product which was triturated with EtOAc
to give 6.3 g (86%) of white crystals, mp 170–172 °C. Anal.
(C12H16ClNO3) C, H, N.
4-(1-Ethylpropoxy)-6-methyl-2-(2,4,6-trimethylphenylami-
no)nicotinic Acid (12). A mixture of 2-chloro-4-(1-ethylpropoxy)-
6-methylnicotinic acid (372 mg, 1.44 mmol), trimethylaniline (195
mg, 1.44 mmol), potassium carbonate (199 mg, 1.44 mmol), and
copper (18 mg, 0.29 mmol) in 6 mL of DMF was heated at 110 °C
for 24 h. The mixture was quenched with water (200 mL) and
extracted with ethyl acetate (3 × 150 mL). The organic layer was
separated, dried, and concentrated to give a brown oil (750 mg).
The oil was purified by silica gel column chromatography using
3.5% MeOH in chloroform as solvent to give the less polar spot
first (210 mg) as an undesired material, and the spot collected next
was found to be the desired product (151 mg, 29.4%), mp 195–200
°C. Anal. (C21H28N2O3) C, H. N.
[4-(1-Ethylpropoxy)-3,6-dimethylpyridin-2-yl](2,4,6-trimeth-
ylphenyl)amine (13). To a solution of 4-(1-ethylpropoxy)-6-methyl-
2-(2,4,6-trimethylphenylamino)nicotinic acid (240 mg, 0.673 mmol)
in dry THF (8 mL) were added aluminum chloride (90 mg, 0.673
mmol) and 1.0 M of lithium aluminum hydride in THF (2.7 mL,
2.7 mmol). The resulting mixture was heated at reflux for 3 h. The
mixture was quenched with 0.1 mL of water and 0.1 mL of 2 N
NaOH and then quenched with water and ethyl acetate. The organic
layer was separated, dried, and concentrated to give 250 mg of
brown oil. After silica gel column chromatography, 170 mg (78%)
of the title compound was obtained which was prepared as an HCl
Ex ViWo Receptor Binding Assay with 125I-oCRF. 125I-oCRF
binding to brain and pituitary tissues in rats was measured using
brain homogenate assay. Male Sprague–Dawley rats (200–300 g)
were treated with a compound or vehicle at the doses indicated.
After a specified treatment period (usually 1 h), they were sacrificed
via decapitation and the brains and pituitary glands rapidly removed
and frozen immediately on dry ice. 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, 100 U/mL aprotinin).
Aliquots of tissue homogenate (100 µL) 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)
using a Skatron cell harvester (setting 222). Radioactivity was
quantified using a betaplate scintillation counter (Wallac).
Pharmacokinetic Studies in Dogs. All animal studies were
conducted using protocols approved by a local Animal Care and
Usage Procedure Committee in facilities accredited by IACUC. Four
beagle dogs (two male and two female) weighing between 8 and
15 kg were administered 1 mesylate salt or 3a hydrochloride salt
(1.0 mg/kg) into the cephalic vein of the foreleg. The dosing solution
for 1 was neat ethanol (15.4 mg/mL) and for 3a was sterile saline
at pH 2 (10 mg/mL). Oral dosing was conducted by gavage with
drugs suspended in 0.1% methyl cellulose at pH 2. For the
intravenous and fasted oral legs of the study, animals had last eaten
approximately 21 h prior to drug administration and were permitted
food and water approximately 3.5 h postdose. In the fed oral leg
of the study, the animals were given one can of wet dog food 1 h
before dosing. Blood samples were collected by venipuncture of
the jugular vein prior to drug administration and at time points of
0.083 (i.v. only), 0.25, 0.5, 1.0, 2.0, 3.0, 4.0, 6.0, 8.0, 12, and 24 h
postdose, processed to obtain serum and stored frozen until the day
of analysis.
1
salt to give a white solid: mp 132–133 °C; H NMR (CDCl3) δ
6.87 (s, 2H), 6.09 (s, 1H), 5.39 (brs, 1H), 4.13 (m, 1H), 2.27 (s,
3H), 2.22 (s, 3H), 2.15 (s, 6H), 1.98 (s, 3H), 1.67 (m, 4H), 0.94 (t,
6H).
2-Chloro-4-(1-ethylpropylamino)-6-methylnicotinic Acid (14).
A mixture of 2,4-dichloro-6-methylnicotinic acid methyl ester
(2.228 g, 10.13 mmol) and 1-ethylpropylamine (1.762 g, 20.26
mmol) in DMSO (4 mL) was heated at 110 °C for 5 h and then at
room temperature overnight. The mixture was quenched with water
and extracted with ethyl acetate. The organic layer was dried and
concentrated to give 1.796 g of crude material. The crude material
was purified by silica gel column chromatography using 1:1 hexane/
chloroform, then chloroform to 5% methanol in chloroform gradient
as eluent to give 0.309 g of the undesired regioisomer, then 1.167
g (43%) of the desired product as a colorless oil, and then 0.259 g
of the bis-1-ethylpropylamino adduct. The desired product: 1H NMR
(CDCl3) δ 7.14 (brs, 1H), 6.27 (s, 1H), 3.86 (s, 3H), 3.27 (m, 1H),
2.33 (s, 3H), 1.3–1.6 (m, 4H), 0.88 (t, 6H).
A mixture of 2-chloro-4-(1-ethylpropylamino)-6-methylnicotinic
acid methyl ester (1.45 g, 5.36 mmol) and LiOH ·H2O (0.45 g, 10.72
mmol) in 28 mL of a 1:1 mixture of p-dioxane/water was stirred at
room temperature overnight. The mixture was quenched with 10.7
mL of 1 N HCl and concentrated to dryness. The residue was diluted
with chloroform, filtered, and washed with chloroform. The filtrate
was dried over anhydrous Na2SO4, concentrated, and pumped in
vacuo to give a white foam (1.03 g, 75%) of 2-chloro-4-(1-
ethylpropylamino)-6-methylnicotinic acid that was used as is.
4-(1-Ethylpropylamino)-6-methyl-2-(2,4,6-trimethylphenyl-
amino)nicotinic Acid (15). A mixture of 2-chloro-4-(1-ethylpro-
pylamino)-6-methylnicotinic acid (4.00 g, 15.58 mmol), trimeth-
ylaniline (2.11 g, 15.58 mmol), potassium carbonate (2.15 g, 15.58
mmol), and copper (0.20 g, 3.12 mmol) in DMF was heated at
reflux. The mixture was quenched with ammonium chloride, stirred
for 20 min, filtered through Celite, and washed with ethyl acetate.
The filtrate was extracted with ethyl acetate. The organic layer was
separated, dried, and concentrated to dryness. The residue was
Serum samples (0.9 mL) were delivered to silanized test tubes
and fortified with internal standard. Samples were alkalinized with
NaOH (0.4 mL; 1 M) and extracted with methyl tert-butyl ether (4
mL), the aqueous layer was frozen in a dry ice/acetone bath, and
the organic layer was decanted into a silanized tube and evaporated

2-Aryloxy-4-alkylaminopyridines
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5 1391
under nitrogen at 35 °C. Sample residues were reconstituted in 20
µL of acetonitrile and analyzed by GC-MS.
Acknowledgment. We thank Dr. Jon Bordner for the X-ray
structure analysis and David J. am Ende for conducting DSC.
We extend our gratitude to Ms. Jeannine Margolis and Mr. Erik
Kuja for their excellent technical assistance in gathering the
pharmacokinetic and drug metabolism data. We acknowledge
Professor E. J. Corey for his interest and valuable discussions.
The GC-MS consisted of a Hewlett-Packard 5890 model
equipped with an HP-5 (5% phenyl methylpolysiloxane) column
(length ) 12 m; i.d. ) 0.25 mm; film thickness ) 0.25 µm). Carrier
gas (He) was at a flow rate of 1.5 mL/min. The initial oven
temperature was at 150 °C for 2 min, followed by an increase of
40 °C/min to 220 °C for 0.20 min, and followed by a second
increase of 25 °C/min to 290 °C for 1.5 min. Injection port and
transfer line temperatures were 250 and 280 °C, respectively. The
MS was operated in SIM mode for the ion current of the base peaks
for analytes and internal standards. The sample volume injected
was 3 µL. The lower limit of quantitation for the assay was 1.0
ng/mL.
Supporting Information Available: Elemental analysis for final
testing compounds 3a,b, 5a,b, and 16 and intermediates 7, 8a,c,
9a, 11, and 12, as well as the X-ray structural analysis of the HCl
salt of 16. This material is available free of charge via the Internet
at http://pubs.acs.org.
References
AUC(0ftlast) and AUMC(0ftlast) were calculated using the linear
trapezoid approximation. The elimination rate constant (kel) was
determined by the slope of linear regression of elimination phase
observed on the plot of the log of the serum concentration vs time.
AUC(0f∞) was comprised of the sum of AUC(0ftlast) and
AUC(tlastf∞). The latter value was calculated by dividing Ctlast
estimated from the aforementioned regression by kel. With i.v.
(1) (a) Vale, W.; Spiess, J.; Rivier, C.; Rivier; J. Characterization of a 41
residue ovine hypothalamic peptide that stimulates secretion of corti-
cotropin 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.
(2) (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-(butyl-
ethylamino)pyrrolo[2,3-d]pyrimidine: a centrally active corticotropin-
releasing factor1 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 corti-
cotropin releasing factor receptors. Proc. Natl. Acad. 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, 21–26. (d) Shaham, Y.; Erb,
S.; Leung, S.; Buczek, Y.; Stewart, J. CP-154,526, a selective, non-
peptide antagonist of the corticotropin-releasing factor1 receptor at-
tenuates 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 neurochemi-
cal 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. Pharmacopsychiatry 2000, 33,
189. (h) Le, A. D.; Harding, S.; Juzytsch, W.; Watchus, J.; Shalev, U.;
Shaham, Y. The role of corticotrophin-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.
(3) 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) Bornstein, 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 I receptor
antagonist on pituitary-adrenal function, body weight, and metabolic
regulation. Endocrinology 1998, 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.
administration, Ct
was estimated by extrapolation of the log-
)
0
linear regression of the data to t ) 0 and used in the determination
of AUC(tlastf∞). Elimination half-life (t1/2) was calculated as 0.693/
kel. Values of clearance (CL), volume of distribution at steady state
(VDss), volume of distribution during the terminal elimination phase
(VDγ), and oral bioavailability were calculated by
dose
AUC(0 f ∞)
CL )
(1)
(2)
(3)
dose · AUMC(0 f ∞)
AUC(0 f ∞)
VDss )
CL
VDγ )
kel
dosei.v. · AUC(0 f ∞)p.o.
dosep.o. · AUC(0 f ∞)i.v.
F )
(4)
Estimates of the fraction absorbed were done by calculating the
fraction extracted by the liver during first pass. A value of 35 mL/
min/kg was used for hepatic blood flow (Qh) in the dog. Serum
clearance values were corrected to blood values using blood/plasma
(B/P) values of 0.61 and 0.69 for 1 and 3a, respectively.
F
CL
B
P
Fa ) 100
(5)
1 -
(
)
(
· Qh
)
(
)
Protein Binding. Protein binding was determined using the
technique of equilibrium dialysis. Test compounds were added to
fresh serum and dialyzed against 67 mM KH2PO4, pH 7.4 with
0.9% (w/v) NaCl in a 20-cell Spectrum equilibrium dialysis
apparatus using Teflon cells and #4 membranes with a molecular
weight cutoff of 12–14 kDa. The samples were rotated at 20 rpm
for 5 h at 37 °C. Individual experiments were conducted in replicates
of three. When using radiolabeled compounds, the concentrations
were determined by liquid scintillation counting of portions of serum
and buffer fractions and when using nonlabeled compounds,
analysis was conducted by GC-MS. The fraction bound to protein
(fb) was calculated using the following formula
(4) Chen, Y. L.; Braselton, J.; Forman, J.; Gallaschun, R. J.; Mansbach,
R.; Schmidt, A.; Seeger, T. F.; Sprouse, J.; Tingley, F. D.; Winston,
E.; Schulz, D. W. Synthesis and SAR of 2-aryloxy-4-alkoxy-pyridines
as potent orally active corticotrophin-releasing factor 1 antagonists.
J. Med. Chem. 2008, ASAP Article, DOI: 10.021/jm070578k.
(5) Unpublished data. A representative compound in pyrrolo[2,3-d]pyri-
midine series, e.g., 2-[7-(4-bromo-2,6-dimethylphenyl)-2,5-dimethyl-
7H-pyrrolo[2,3-d]pyrimidin-4-ylamino]-(S)-butan-1-ol, was found to
produce liver toxicity in 90 day toxicology studies in rats and dogs.
(6) Chen, C.; Grigoriadis, D. E. NBI 30775 (R121919), an orally active
antagonist of the corticotropin-releasing factor (CRF) type-1 receptor
(Cs - Cb) · volume correction factor
(Cs - Cb) · volume correction factor + Cb
fb )
(6)
where Cs and Cb represent drug concentrations in the serum and
buffer fractions, respectively, and the volume correction factor
represents the quotient of the final serum volume/initial serum
volume. (This difference is due to osmotic flow.) Unbound fractions
were calculated as 1 - fb. Bound and unbound fractions were
multiplied by 100 and reported as percentages.

1392 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5
Chen et al.
for the treatment of anxiety and depression. Drug DeV. Res. 2005, 65,
216.
between process temperature and decomposition temperature is con-
sidered to be acceptable.
(7) Unpublished phase I data.
(9) Chlorination yield was improved in the presence of 1 equiv of N,N-
diethylaniline. Croisy-Delcey, M.; Bisagni, E. Aza analogs of lucan-
thone: synthesis and antitumor and bactericidal properties. J. Med.
Chem. 1983, 26, 1329.
(8) Because the pyridine N-oxide intermediates may show exothermic
decomposition, it is recommended to test the differential scanning
calorimeter prior to scale-up synthesis. Pyridine N-oxide intermediates
are not recommended for large-scale preparation, and caution is required
to ensure safety in the laboratory. In general, a 100 °C difference
JM070579C