Design and synthesis of tricyclic corticotropin-releasing factor-1 antagonists

Article abstract of DOI:10.1021/jm049085v

Antagonists of the corticotropin-releasing factor (CRF) neuropeptide should prove to be effective in treating stress and anxiety-related disorders. In an effort to identify antagonists with improved physicochemical properties, new tricyclic CRF₁

Full text of DOI:10.1021/jm049085v

5780
J. Med. Chem. 2005, 48, 5780-5793
Design and Synthesis of Tricyclic Corticotropin-Releasing Factor-1 Antagonists
Raymond S. Gross,# Zhiqiang Guo,# Brian Dyck,# Tim Coon,# Charles Q. Huang,# Richard F. Lowe,#
Dragan Marinkovic,# Manisha Moorjani,# Jodene Nelson,# Said Zamani-Kord,# Dimitri E. Grigoriadis,‡
Sam R. J. Hoare,‡ Paul D. Crowe,‡ Jane Han Bu,# Mustapha Haddach,# James McCarthy,# John Saunders,#
Robert Sullivan,# TaKung Chen,† and John P. Williams*,#
Departments of Medicinal Chemistry, Pharmacology and Lead Discovery, and Preclinical Development, Neurocrine Biosciences,
12790 El Camino Real, San Diego, California 92130
Received November 12, 2004
Antagonists of the corticotropin-releasing factor (CRF) neuropeptide should prove to be effective
in treating stress and anxiety-related disorders. In an effort to identify antagonists with
improved physicochemical properties, new tricyclic CRF1 antagonists were designed, synthe-
sized, and tested for biological activity. As a result of studies aimed at establishing a relationship
between structure and CRF1 binding affinity, NBI 35965 (12a) was identified as a high-affinity
antagonist with a pKi value of 8.5. Compound 12a proved to be a functional CRF1 antagonist
with pIC50 values of 7.1 and 6.9 in the in vitro CRF-stimulated cAMP accumulation and ACTH
production assays, respectively, and 12a also reduced CRF or stress induced ACTH production
in vivo.
Introduction
Hypersecretion of hypothalamic CRF manifests itself in
a down-regulation of CRF receptors in the anterior
pituitary, as demonstrated by the blunted ACTH re-
sponse to peripherally administered CRF.8,9 In de-
pressed patients CRF has also been shown to be
elevated extrahypothalamically and is found in high
concentrations in the cerebrospinal fluid.10 This, in turn,
has been shown to result in decreased expression of the
CRF1 receptor when measured in the frontal cortex of
suicide victims.11 Furthermore, a significant positive
correlation exists between the elevated CRF levels in
the cerebrospinal fluid and the degree of insensitivity
to dexamethasone suppression of plasma cortisol in
depressed individuals.7 These studies suggest that in
these disorders the CRF system is attempting to at-
tenuate the effects of elevated CRF levels by reducing
the number of sites for this hormone’s action.
Over the past decade, several non-peptide CRF1
receptor antagonists have been reported and several
representative examples are illustrated in Figure 1.12
Compounds such as antalarmin (1),13 R121919 (2),14,15
compound 3,16 and DMP 696 (4)17 have proven to be
functional antagonists both in vitro and in vivo with
exquisite selectivity for the CRF1 receptor. The com-
pound R121919 (2) has demonstrated preliminary phar-
macologic effects in a small open label phase IIA clinical
trial in major depressive disorder patients. Compound
2 showed that while effective in reducing the Hamilton
depression and anxiety rating scale (HAM-D and HAM-
A) scores of depressed individuals, there was little effect
on the basal functioning of the HPA axis, suggesting
that non-peptide CRF1 antagonists may prove to be
useful therapeutics in the treatment of anxiety and
depression.18,19
Corticotropin-releasing factor (CRF) is a neuropeptide
that has generated a great deal of interest during the
past 2 decades, and it is now commonly understood to
be the primary mediator of the hypothalamic-pituitary-
adrenal (HPA) stress response.1 Since its purification
and molecular identification in the early 1980s, a role
for this hormone has been proposed in a variety of
endocrine, neuropsychiatric, and neurodegenerative
disorders. Primarily CRF is produced in and secreted
from parvocellular neurons of the paraventricular hy-
pothalamus and is the primary regulator of the release
of adrenocorticotropic hormone (ACTH) as well as other
proopiomelanocortin (POMC) derived peptides from the
anterior pituitary gland.2 In addition to its distribution
in the hypothalamus, it has been found that CRF is
widely expressed extrahypothalamically in many re-
gions of the central nervous system where it mediates
the central processes of the stress response including
the autonomic, electrophysiological, and behavioral
responses to stress.3-6 It is now well-known that CRF
exerts its action through a specific interaction with two
distinct transmembrane receptors that belong to the
class B subfamily of G-protein-coupled receptors. These
receptors, encoded by two distinct genes, are termed
CRF1 and CRF2 and have been extensively character-
ized and quantified in a number of species.
A number of clinical investigators have described
a clear and significant correlation between hyper-
secretion of central CRF and the severity of affective or
anxiety disorders. Given the primary role of CRF in
stimulating pituitary-adrenocortical secretion, it is
plausible that hypersecretion of central CRF may play
a fundamental role in the etiology of major depression.7
It has been suggested that the clinical development
of the early CRF1 antagonists was limited because of
the highly lipophilic nature of compounds such as
antalarmin (1).20 The overall objective of this study was
to reduce lipophilicity of small-molecule CRF1 antago-
nists. It was hoped that restricting the conformational
* To whom correspondence should be addressed. Phone: 858-617-
7662. Fax: 858-617-7619. E-mail: jpwilliams@neurocrine.com.
# Department of Medicinal Chemistry.
‡ Department of Pharmacology and Lead Discovery.
† Department of Preclinical Development.
10.1021/jm049085v CCC: $30.25 © 2005 American Chemical Society
Published on Web 08/10/2005

Design and Synthesis of CRF1 Antagonists
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 18 5781
Attempts to prepare compounds 12v and 12w via this
route failed and, as a consequence, a modified route
illustrated in Scheme 2. The R1 side chains were
installed first by condensing 10 with the amines (cyclo-
hexylamine and 2-amino-1-methoxybutane), and the
final cyclization with 1,2-dibromoethane provided 12v
and 12w.
For the bottom region SAR studies, a divergent
intermediate strategy was employed. It was presumed
that treatment of intermediate 19 with the requisite
amino alcohol, cyclization, and alkylation would yield
the intermediate 22 that would be then be poised to
undergo palladium-mediated Suzuki coupling reactions
to rapidly introduce aromatic side chains (Scheme 3).
Preparation of 19 first required nitration of pyrazole
followed by palladium-catalyzed hydrogenation of the
nitro group to afford 4-aminopyrazole 15. Analogous to
the sequence in Scheme 1, 15 was condensed with
ethylacetoacetate and subjected to a thermal cyclization
to provide 17. Conversion of 17 to the 7-chloro inter-
mediate 18 was accomplished with POCl3. Owing to the
inductively withdrawing nature of the 7-chloro group,
18 was selectively brominated to afford 19 in 13%
overall yield over six steps. This synthesis was success-
fully performed on a 100 g scale, and 19 proved to be a
versatile and important intermediate for the subsequent
SAR studies. Preparation of the final compounds re-
quired the condensation of 19 with (S)-2-aminobutanol
analogous to the route illustrated in Scheme 1. Cycliza-
tion of 20 using 48% HBr at 130 °C followed by
alkylation of 21 with cyclopropylmethyl bromide and
sodium hydride gave the penultimate intermediate 22.
The final aryl side chains were incorporated by a
palladium-catalyzed cross coupling with a variety of aryl
boronic acids.
The synthesis of 23n required a modified route that
is outlined in Scheme 4 because the necessary boronic
acid was not available commercially. First, the 2-chloro-
5-bromobenzotrifluoride (24) was acylated in a two-step
process that involved cross-coupling with tributyl-(1-
ethoxyvinyl)tin to yield 25 after hydrolysis of the enol
ether. Intermediate 26 was prepared by a palladium-
catalyzed boronation, which was then used as the
coupling partner with 22 in a Suzuki cross-coupling
reaction to afford 27. Conversion of the acyl group
involved condensation of 27 with methylmagnesium
bromide followed by dehydration to provide 28, which
was then hydrogenated to give the final compound 23n.
Figure 1. Examples of non-peptide CRF1 antagonists.
Figure 2. Design of tricyclic CRF1 antagonists.
freedom would result in compounds that would populate
a small subset of the biologically relevant conformations
of compound 1, reducing the potential entropic penalty
incurred when flexible small-molecule ligands bind to
large proteins.21 Covalent attachment of the N-1 methyl
group present in compound 3 to the top region alkyl side
chain would restrict the conformational freedom of the
flexible “top region” (Figure 2). It was hypothesized that
this reduced flexibility would result in reduction of the
size of the alkyl chains required for optimal biological
activity.
Chemistry
The synthesis of tricyclic analogues of 3 was ac-
complished by first assembling the pyrazole ring (in-
termediate 8) by treating hydrazine hydrochloride with
the 1,3-dicarbonyl equivalent 7 (Scheme 1). Intermedi-
ate 7 was prepared by condensation of the R-amino-
acetophenone 6 with N,N-dimethylformamide dimethyl-
acetal, and alkylating potassium phthalamide with
commercially available chloroketone 5 provided ketone
6. Formation of an enamine derived from 8 and ethyl-
acetoacetate followed by thermal cyclization in diphenyl
ether led to the production of compound 9 in 77%
isolated yield over two steps. Conversion of 9 to 10 was
carried out in the presence of POCl3. A two-step, one-
pot procedure involving a reaction with intermediate 10
and a variety of amino alcohols followed by aqueous
HBr-mediated cyclization afforded compounds 11a-g.
Final top region side chains were installed via base-
mediated alkylation of intermediates 11a-g to yield the
final compounds 12a-u. The tritiated analogue of 12a
was prepared bromination of 12a followed by palladium-
catalyzed tritiation.
Results
The compounds prepared in this study were screened
for their ability to inhibit [125I]sauvagine binding to
h-CRF1 LtK transfectant cells, and the pKi values are
reported in Tables 1 and 2.22 Antalarmin (1) and
compound 3 were prepared as a positive control, and
the pKi values determined were consistent with previ-
ously reported values (Table 1).13,16 Covalent attachment
of the N-1 methyl group to the C-7 branched alkyl side
chain of 3 resulted in compound 12a, which is a high-
affinity CRF1 antagonist with a pKi value of 8.5. The
activity of the enantiomer 12b was significantly reduced
(pKi ) 6.9), providing evidence that the pocket within
the receptor that interacts with top region side chain
has a defined asymmetric shape. Once it was estab-

5782 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 18
Gross et al.
Scheme 1a
a (a) Potassium phthalimide, DMF, 25 °C, 71%; (b) DMFDMA, ∆ 74%; (c) NH2NH2‚HCl, aqueous EtOH, ∆; (d) NH2NH2‚H2O aqueous
EtOH, ∆, 91% (two steps); (e) ethyl acetoacetate, p-TsOH, toluene, ∆; (f) Ph2O, 250 °C, 77% (two steps); (g) POCl3, MeCN, ∆, 95%; (h)
amino alcohols, p-TsOH, 120 °C; (i) 48% aqueous HBr, 140 °C, 76% (two steps); (j) alkyl halides, NaH, DMF, 25 °C; (l) Br2, CH2Cl2, 25C,
75%; (m) 3H2, Pd/C, EtNiPr2.
Scheme 2a
a (a) R1NH2, TsOH, 130 °C; (b) 1,2-dibromoethane, K2CO3, 85 °C.
lished that the tricyclic core structure allows the side
chains to adopt biologically relevant conformations, a
study was launched to test the hypothesis of whether
restricting the conformational freedom of the top region
side chains would ultimately result in the introduction
of smaller less lipophilic side chains at positions R1 and
R2.
the receptor may tolerate larger substituents. The
phenyl derivative 12g was prepared to further probe the
shape of this putative binding pocket. Filling the space
with the larger phenyl ring did not increase the affinity
for this compound; if anything, the activity was slightly
reduced (pKi ) 8.0), leaving the original ethyl substitu-
ent as the optimal R2 side chain.
Also contained within Table 1 are the results of a
study of the R1 position in this series of CRF1 antago-
nists. Analogous to the R2 site, the R1 side chains also
appear to be sensitive to the size and shape of the alkyl
substituent. Removal of the cyclopropylmethyl side
chain resulted in significant loss of biological activity
(11c, pKi ) 6.1). From the rebuilding of the alkyl chain
at R1, it was concluded that four carbon atoms are
Complete removal of the R2 ethyl group (12c) resulted
in a 25-fold loss of affinity at the CRF1 receptor, but
much of the lost binding affinity was regained by the
introduction of a methyl group at position R2 (12d, pKi
) 8.1). Replacing the (S)-ethyl group with a (S)-propyl
group (12e, pKi ) 8.4) or the branched (S)-isopropyl
group (12f, pKi ) 8.3) had no effect on binding affinity
within this series, suggesting that this region within

Design and Synthesis of CRF1 Antagonists
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 18 5783
Scheme 3a
a (a) HNO3, H2SO4, 55 °C, 33%; (b) H2, Pd/C 95%; (c) ethyl acetoacetate, toluene, ∆; (d) Ph2O, 200 °C, 46% (two steps); (e) POCl3,
MeCN, ∆, 91%; (f) Br2, MeOH, 98%; (g) (S)-(+)-2-amino-1-butanol, p-TsOH, 140 °C; (h) 48% aqueous HBr, 130 °C, 43% (two steps); (i)
alkyl halides, NaH, DMF, 25 °C, 94%; (j) boronic Acids, Pd(Ph3P)4, aqueous Na2CO3, Ba(OH)2, EtOH, toluene, 60%.
Scheme 4a
a (a) Bu3Sn(CHCHOEt), PdCl2(PP3)2, PhMe, 100 °C; (b) 1 N HCl, THF, 100%; (c) bis(pinacolato)diboron, PdCl2(dppf), KOAc, PhMe, 100
°C, 62%; (d) 22, Pd(Ph3)4, Na2CO3(aq), Ba(OH)2(aq), PhMe, EtOH, 95%; (e) MeMgBr, THF, -78 °C; (f) TFA, MgSO4, TsOH(cat.), PhMe, 100
°C, 45%; (g) H2, PtO, EtOH, 40 atm; (h) HCl, Et2O, 70%.
necessary for high affinity. The methyl (12h, pKi ) 5.3),
ethyl (12i, pKi ) 7.2), propyl (12j, pKi ) 7.5), and
isopropyl (12l, pKi ) 7.1) derivatives were all less active
than the cyclopropylmethyl (12a, pKi ) 8.5), n-butyl
(12k, pKi ) 8.2), or isobutyl (12m, pKi ) 7.8) analogues.
Branching of the alkyl substituent results in a moderate
loss of affinity when comparing the isobutyl derivative
12m versus the more compact cyclopropylmethyl ana-
logue 12a. This observation is further substantiated by
the decrease of affinity observed when the ring size is
increased to cyclobutyl (12n, pKi ) 7.7) or benzyl (12o,
pKi ) 6.8). Another strategy for reducing lipophilicity
in the alkyl side chains involves replacing carbon atoms
with oxygen atoms for carbon, and despite the success
of this strategy in previous reports,17 the 2-methoxyethyl
derivative 12p was 17-fold less active in the binding
assay.
than the isobutyl derivative 12q (pKi ) 6.4), suggesting
a distinct SAR in the R2 ) H series. As a consequence,
additional analogues with increased branching were
prepared and evaluated for CRF1 binding. Addition of
two (12s, pKi ) 8.4), four (12t, pKi ) 8.3), and six (12u,
pKi ) 8.3) methylene units to the branched alkyl side
chain increased the affinity for the receptor. Tying the
branched alkyl group back into a cyclohexyl ring (12v,
pKi ) 6.9) system led to a significant loss of activity,
supporting the contention that the hydrophobic pocket
that these side chains interact with has a distinct shape
that is long (five to seven atoms) and narrow. Analogous
to 12p, exchanging methylene units for oxygen also led
to a decrease of binding affinity, as illustrated by
compound 12w (pKi ) 7.2). More polar side chains may
adopt a completely different conformation upon receptor
binding, and it is possible that adopting the necessary
conformation is prevented by the more rigid analogues
12p and 12w in contrast to the more flexible DMP 696
(4).
An alternative top region SAR study was performed
on the basis of the observation that when R2 ) H, the
trend of branching in the R1 region and binding was
reversed. Unlike the previous example (with R2 ) ethyl),
the isopropyl analogue (12r, pKi ) 7.1) was more active
Table 2 summarizes the SAR studies completed in
regards to the bottom region of the tricyclic series. Goals

5784 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 18
Table 1. Top Region SAR
Gross et al.
Table 2. Bottom Region SAR Summary
a
compd
R1
R2
pKi
a
compd
X
Y
pKi
1
3
8.6 ( 0.3
8.8 ( 0.2
8.5 ( 0.1
6.9 ( 0.1
7.1 ( 0.1
8.1 ( 0.1
8.4 ( 0.3
8.3 ( 0.2
8.0 ( 0.3
6.1 ( 0.1
5.3 ( 0.2
7.2 ( 0.1
7.5 ( 0.1
8.2 ( 0.1
7.1 ( 0.1
7.8 ( 0.2
7.7 ( 0.1
6.8 ( 0.3
7.3 + 0.1
6.4 ( 0.2
7.1 ( 0.1
8.4 ( 0.1
8.3 ( 0.1
8.3 ( 0.1
6.8 ( 0.1
7.2 ( 0.2
12a
23a
23b
23c
23d
23e
23f
23g
23h
23i
23j
23k
23l
23m
23n
Cl
Cl
8.5 ( 0.1
7.7 ( 0.4
8.1 ( 0.3
7.6 ( 0.1
7.3 ( 0.1
8.4 ( 0.2
8.2 ( 0.2
8.4 ( 0.1
7.9 ( 0.1
6.8 ( 0.1
7.9 ( 0.1
8.0 ( 0.1
8.1 ( 0.4
8.1 ( 0.1
8.1 ( 0.3
CF3
CH3
CH3O
F
Cl
12a
12b
12c
12d
12e
12f
12g
11c
12h
12i
12j
c-propylmethyl
c-propylmethyl
c-propylmethyl
c-propylmethyl
c-propylmethyl
c-propylmethyl
c-propylmethyl
H
(S)-ethyl
(R)-ethyl
H
Cl
Cl
Cl
(S)-methyl
(S)-propyl
(S)-isopropyl
(S)-phenyl
(S)-ethyl
(S)-ethyl
(S)-ethyl
(S)-ethyl
(S)-ethyl
(S)-ethyl
(S)-ethyl
(S)-ethyl
(S)-ethyl
(S)-ethyl
H
Cl
CF3
CH3
CH3O
F
COOMe
CH3
CH3O
CF3
CH3O
isopropyl
Cl
Cl
Cl
Cl
methyl
CH3
CH3
CF3
CF3
CF3
ethyl
propyl
12k
12l
butyl
isopropyl
isobutyl
c-butylmethyl
Bn
12m
12n
12o
12p
12q
12r
12s
12t
12u
12v
12w
a Mean pKi ( SEM, h-CRF1.
2-methoxyethyl
isobutyl
highest affinity analogues were combined. The dimethyl
(23j, pKi ) 7.9), bis-trifluoromethyl (23l, pKi ) 8.1), and
p-methoxy substituted 23k (pKi ) 8.0) and 23m (pKi )
8.1) did not increase CRF1 binding. The 4-isopropyl
derivative 23n (pKi ) 8.1) also did not increase binding,
suggesting that increasing the size of the 4-substituent
would not further increase antagonist binding.
The tactic of restricting the flexibility of the top region
side chains resulted in 12a, which was the highest
affinity CRF1 antagonist identified in this study. Sur-
prisingly compound 12a was significantly less lipophilic
than compound 3 with experimentally determined
log D7.4 values of 4.4 and 5.3, respectively. The tricyclic
core structure appears to be more basic (pKa value of
7.93 was measured for the conjugate acid of 12a) than
the starting bicyclic heterocycle (pKa ) 7.5 for the
conjugate acid of 3) presumably because of restriction
of rotation of the nitrogen lone pair of electrons at C-7.
As a consequence, compound 12a (NBI 35965) was
further evaluated for functional CRF1 antagonism both
in vitro and in vivo.
Measurement of inhibition of CRF-induced cAMP
production was performed in the same cell line as that
used in the binding studies above but using a live whole-
cell preparation to determine intracellular cAMP ac-
cumulation. To further elucidate the functional antago-
nism of these compounds, CRF-stimulated ACTH release
was assessed in cultured rat anterior pituitary cells. The
IC50 values were essentially identical for either the
second messenger cAMP (pIC50 ) 7.1) or the release of
ACTH (pIC50 ) 6.9), demonstrating that this compound
acts as a functional antagonist. The equivalent potency
determined for compounds in both the human receptor
(cAMP) or rat receptor (ACTH) assay demonstrated that
these compounds do not exhibit any species selectivity
for the CRF1 receptor. In the absence of agonist in these
preparations, there was no cAMP accumulation or
ACTH release, demonstrating that 12a exhibited no
intrinsic agonist activity. In addition, in cells expressing
isopropyl
H
3-pentyl
H
4-heptyl
H
5-nonyl
H
c-hexyl
H
1-MeO-2-butyl
H
a Mean pKi ( SEM, h-CRF1.
of the study were twofold: (1) to find the optimal aryl
substituents that would allow for decreasing carbon
content in the top region and (2) to identify polar (less
lipophilic) substituents to help offset the contribution
of the top region side chains to the overall lipophilicity.
Substitution of chlorine at the 2-position of the bottom
aromatic (X ) Cl) with alternatives led to a decrease in
affinity regardless of the substituent. The electron-
withdrawing trifluoromethyl (23a, pKi ) 7.7) and fluoro
(23d, pKi ) 7.3) groups as well as electron-donating
groups such as methyl (23b, pKi ) 8.1) and methoxy
(23c, pKi ) 7.6) failed to increase the affinity of these
analogues for the receptor. In this region, it appears that
the shape of the substituent is more important than the
electronic nature for high-affinity antagonist binding.
This is also consistent with earlier observations that the
2-substituent steers the bottom aromatic ring out of
plane relative to the core heterocycle.23
In contrast to the 2-position, many of the similar
changes at the 4-position of the aromatic side chain
offered analogues equipotent to 12a independent of the
electronic nature of the substituents. The trifluoro-
methyl (23e, pKi ) 8.4), methyl (23f, pKi ) 8.2), methoxy
(23g, pKi ) 8.4), and fluoro (23h, pKi ) 7.9) substituted
analogues bound with comparable affinity as 12a.
Unfortunately the polar, electron-withdrawing carbo-
methoxy substituent reduced the binding affinity as
seen in 23i (pKi ) 6.7). In an attempt to elucidate a
synergistic relationship between the bottom region
aromatic substituents, the groups that yielded the

Design and Synthesis of CRF1 Antagonists
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 18 5785
Figure 3. Effects of 12a administered orally (3, 10, 30 mg/
kg) on CRF-induced ACTH release. Dose-dependent effects on
CRF-induced ACTH release 10 min following CRF administra-
tion: (†) p < 0.0001 vs vehicle; (/) p < 0.04 vs CRF; (//) p <
0.0004 vs CRF.
Figure 4. Effect of oral administration of 12a on increased
plasma ACTH levels following 45 min of restraint stress in
male CD-1 mice. Animals (n ) 10/group) were administered
either vehicle or 20 mg/kg 12a 1 h prior to 45 min of restraint
stress and assessed for plasma levels of ACTH. The increase
in ACTH due to restraint stress was completely attenuated
with prior oral administration of 12a: Veh, vehicle-treated;
NS, no Stress; S, stress.
the human or rat CRF2 receptor 12a was unable to
inhibit [125I]sauvagine binding, clearly establishing the
CRF subtype selectivity of this molecule. Recent studies
utilizing [3H]-12a have detailed the mechanism of the
inhibition of CRF-stimulated responses by non-peptide
molecules. The data demonstrated that non-peptide
antagonists and peptides bind to spatially distinct
binding sites with a weak interaction between their
binding and that the nature of this inhibition is con-
sistent with allosteric modulation of agonist activity.24
Compound 12a was then subjected to a pharmaco-
kinetic evaluation in rats prior to evaluating in vivo
functional antagonism. The estimated oral bioavailabil-
ity was 34% with a mean maximal plasma concentration
at 1 h of 560 ng/mL. Compound 12a has a volume of
distribution 17.8 L/kg, a plasma clearance of 17 mL
min-1 kg-1, and a half-life of 12 h. The compound also
penetrated the blood-brain barrier, resulting in a mean
maximal brain concentration of 700 ng/g, and in light
of these results compound 12a was further evaluated
in vivo.
The in vivo functional antagonism of compound 12a
was determined using both the CRF-induced ACTH
release in normal rats and the restraint stress model
of ACTH release in mice. Figure 3 shows that in rats
intravenous CRF administration produced a robust
increase in the plasma levels of ACTH (p < 0.0001). Oral
administration of compound 12a significantly attenu-
ated this effect in a dose-dependent manner with
statistically significant reductions observed at the 10
and 30 mg/kg doses (p < 0.04-0.004 vs CRF). The
magnitude of these attenuations ranged from 43% to
60%. We further characterized the in vivo functional
effects of compound 12a in a mouse restraint stress
model. While the end surrogate measure is the same
(plasma ACTH concentration), restraint stress is gener-
ally considered to be an emotional or psychological
stressor.25,26 For brief restraint-stress in mice, compound
12a (20 mg/kg, po) was able to completely attenuate the
stress-induced increase in plasma ACTH to basal levels,
which again demonstrated the functional activity of this
molecule as a CRF receptor antagonist (Figure 4).
In addition to the data described above, we have
recently examined the effects of CRF1 receptor antago-
nism in models of gastric and colonic motility. It is well-
documented that stress, mediated through the CRF
system, plays a major role in the physiology of the
gastrointestinal tract.27 Preclinical and clinical data
have demonstrated that a variety of acute stressors
inhibit gastric emptying and increase colonic motil-
ity.28,29 We have shown that CRF and related peptides,
injected directly into the central nervous system of mice
or rats, inhibit gastric emptying and stimulate distal
colonic motor function. Oral administration of the CRF1
receptor antagonist, NBI-35965 (compound 12a), re-
versed the icv CRF-induced colonic motility, demon-
strating that the effects of CRF on the lower gut are
mediated by the CRF1 receptor.30,31
Conclusions
The objective of this study was to see if the overall
lipophilicity of the pyrazolo[4,3-b]pyridine class of CRF1
antagonists could be decreased. Strategically, it was
anticipated that restricting the side chain conforma-
tional flexibility would ultimately lead to new series of
tricyclic antagonists that would possess intrinsically
higher affinity for the CRF1 receptor. The conclusion
from these studies is that the new tricyclic series of
antagonists are equipotent to their bicyclic counterparts
with an experimentally determined log D7.4 value of 4.4
due to the surprisingly basic nature of the core hetero-
cycle. Compound 12a (NBI 35965) proved to be one of
the highest affinity compounds for the receptor and was
studied further both in vitro and in vivo. These studies
confirmed that 12a is a functional CRF1 antagonist with
improved physicochemical properties and penetrates the
central nervous system. Additional studies will be
disclosed in due course.
Experimental Section
Synthetic Chemistry. General Methods. Nuclear mag-
netic resonance (1H, 13C NMR) spectra were obtained with a
Varian 300 MHz spectrometer (Mercury) or with a Bruker 500
MHz spectrometer at Numega Labs Inc., San Diego, CA. The
chemical shifts are reported in parts per million (δ) downfield

5786 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 18
Gross et al.
using TMS as the internal standard and CDCl3 as the solvent
except where indicated. Matrix-assisted laser desorption/
ionization (MALDI) FTMS experiments were performed on an
IonSpec FTMS mass spectrometer at The Scripps Research
Institute, San Diego, CA. Samples were irradiated with a
nitrogen laser operating at 337 nm, and the laser beam was
controlled by a variable attenuator and focused on the sample
target. Fast atom bombardment (FAB) analysis was carried
out on M-Scan’s VG Analytical ZAB 2-SE high-field mass
spectrometer at M-Scan Inc., West Chester, PA. A cesium ion
gun was used to generate ions for the acquired high-resolution
mass spectra. Optical activity was measured at 589 nm, 25
°C, using a Perkin-Elmer 341 polarimeter. LC-MS analyses
were performed on a Perkin-Elmer Sciex API-100 mass
spectrometer using the electron spray ionization technique or
on a SpectraSystem P4000 HPLC system coupled with a
Finnigan LCD/Deca mass spectrometer using the electrospray
ionization technique. Purification of final compounds was
conducted on a prep-LCMS Dionex system, using an Alpha
C18 30 mm × 75 mm reverse-phase column at a flow rate of
45 mL/min. The mobile phase was a gradient of 95/5 to 5/95
A/B: A) H2O-0.1%TFA, B ) CH3CN-0.1% TFA. All com-
pounds after purification were reanalyzed on a reverse-phase
HPLC-MS system (DIONEX with ESI+ ionization mode,
Agilent 1100 or Berger SFC with Agilent 1100). Chiral purity
was determined using chiral reverse phase HPLC utilizing a
Phenomenex Sumichiral R-VAL & S-NEA, 5 m, 4.6 mm × 150
mm with 87% buffer (10 mM NH4OAc, pH 4.0)/13% acetonitrile
at a flow rate of 1.0 mL/min (total run time of 25 min). The
log D7.4 and pKa determinations were performed at Robinson
Microlit Laboratories, Madison, NJ, using the GlpKa poten-
tiometric system. All commercially available reagents were
used without further purification.
and the pH of the aqueous phase was adjusted to >7.5 with
aqueous NaOH. Then the aqueous phase was extracted with
ethyl acetate (2 × 5 L), dried (MgSO4), and evaporated. The
crude material was crystallized from dichloromethane (4 L)
and hexane (3 L) to yield the desired product 8 as an off-white
solid (761 g, 91%): mp 106 °C; 1H NMR (CDCl3) δ 7.43 (d, J )
1.2 Hz, 1H), 7.28 (d, J ) 5.3 Hz, 1H), 7.20 (dd, J ) 5.0, 1.2 Hz,
1H), 7.13 (s, 1H); 13C NMR (CDCl3) δ 134.895, 133.799,
132.502, 129.808, 129.235, 127.434, 127.127; MS (Cl) m/z 228.0
(MH+). Anal. (C9H7Cl2N3) C, H, N.
3-(2,4-Dichlorophenyl)-5-methyl-7-hydroxypyrazolo-
[4,3-b]pyridine (9). Ethyl acetoacetate (739 g, 5.68 mol) and
p-toluenesulfonic acid (10.5 g, 0.055 mol) were added to a
solution of 8 (1.296 kg, 5.68 mol) in toluene (12.9 L). The
mixture was heated to reflux with a Dean-Stark trap and
after 4.5 h concentrated in vacuo. The resulting dark oil was
dissolved in diphenyl ether (4.0 L) and slowly added to a flask
with diphenyl ether (1 L) preheated to 250 °C. The addition
was performed over 2 h while maintaining internal reaction
temperature above 230 °C. The reaction mixture was cooled
to 70 °C, and heptane (6 L) was added. The resulting
precipitate was collected by filtration and the crude material
was triturated with THF (2.5 L) to afford 9 (1.28 kg, 77%) as
an off-white powder: mp 327 °C; 1H NMR (CD3OD) δ 7.67 (d,
J ) 1.2 Hz, 1H), 7.56 (d, J ) 5.0 Hz, 1H), 7.47 (dd, J ) 5.0,
1.2 Hz, 1H), 6.15 (s, 1H), 2.44 (s, 3H); 13C NMR (CD3OD) δ
170.91, 151.61, 137.06, 136.46, 134.58, 133.52, 131.00, 130.75,
129.59, 128.71, 119.96, 110.66, 19.57; MS (Cl) m/z 294.0 (MH+).
Anal. (C13H9Cl2N3O) C, H, N.
7-Chloro-3-(2,4-dichlorophenyl)-5-methylpyrazolo[4,3-
b]pyridine (10). Dimethylformamide (121 mL, 1.57 mol) was
added to a solution of 9 (1.85 kg, 6.29 mol) in anhydrous
acetonitrile (9 L), followed by the slow addition of phosphorus
oxychloride (2 kg, 13 mol), maintaining the internal temper-
ature below 50 °C. The mixture was heated at reflux for 3 h,
and then the mixture was cooled and concentrated in vacuo.
Ice (4.5 kg) and water (3 L) were added to the residue, and
the solution was carefully neutralized to approximately pH
7.0 with 10 N aqueous NaOH (4 L). The precipitate was
collected by filtration and washed with water. The product was
dried in vacuo to affording the desired product 10 (1.88 kg,
95%): mp 240 °C; 1H NMR (DMSO) δ 7.82-7.95 (m, 2H),
7.50-7.70 (m, 2H), 2.65 (s, 3H); MS (Cl) m/z 312.9 (MH+). Anal.
(C13H8Cl3N3) C, H, N.
2′-Phthalimide-2,4-dichloroacetophenone (6). To a so-
lution of potassium phthalimide (4.49 kg, 24.2 mol) in anhy-
drous DMF (8 L) was added 5 (3.56 kg, 15.9 mol) in anhydrous
DMF (6 L). The reaction mixture was stirred under room
temperature for 2 h, and then the reaction mixture was
partitioned between water (15 L) and ethyl acetate (20 L). The
aqueous phase was extracted three additional times with ethyl
acetate (15 L) each time. The organic extracts were combined
and volatiles were evaporated. The resulting orange solid was
slurried with 1 L of methyl tert-butyl ether (MTBE) and then
filtered and washed with MTBE to afford an off-white solid 6
1
(5.37 kg, 71%) mp 134 °C; H NMR (CDCl3) δ 7.85-7.86 (m,
2-(2,4-Dichlorophenyl)-4-methyl-7,8-dihydro-6H-1,3,6,8a-
tetraazaacenaphthylene (11a). Procedure A. A 1 L round-
bottom flask was charged with 10 (32.8 g, 105 mmol),
p-toluenesulfonic acid (40.0 g, 210 mmol), and ethanolamine
(38 g, 630 mmol). The mixture was heated at 140 °C for 3 h.
The mixture was then cooled and diluted with aqueous
hydrobromic acid (48%, 500 mL) and reheated to 140 °C for
30 h. The mixture was then cooled, poured onto ice, neutralized
with 6 N NaOH, and then extracted with ethyl acetate. The
organic phase was dried over magnesium sulfate and concen-
trated in vacuo. The crude material was filtered through a plug
of silica gel, eluting with 1:4 EtOH/ethyl acetate and affording
11a (21.4 g, 64%) of as a light-brown foam. To 1.0 g of product
in 10 mL of dichloromethane at 0 °C was charged 1.7 mL (3.4
mmol, 1.1 equiv) of 2.0 M HCl in ether. The solvent was
removed in vacuo, affording a light-yellow foam. 1H NMR
(CDCl3) δ 7.86 (d, J ) 8.8 Hz, 1H), 7.51 (d, J ) 1.8 Hz, 1H),
7.32 (dd, J ) 1.8, 8.7 Hz), 6.26 (s, 1H), 4.93 (br s, 1H), 4.50 (t,
J ) 5.3 Hz, 2H), 3.86-3.82 (m, 2H), 2.56 (s, 3H); MS (Cl) m/z
319.0 (MH+); ESI-TOF-HRMS m/z calcd for C15H12Cl2N4 (MH+)
319.0512, found 319.0511. Anal. (C15H12Cl2N4‚HCl) C, H, N.
2H), 7.70-7.72 (m, 2H), 7.68 (d, J ) 5.0 Hz, 1H), 7.45 (d, J )
0.8 Hz, 1H), 7.33 (dd, J ) 5.0, 1.2 Hz, 1H), 5.03 (s, 2H); 13C
NMR (CDCl3) δ 192.5, 167.7, 139.1, 134.4, 134.0, 133.3, 132.2,
131.6, 130.9, 127.8, 123.7, 123.6, 47.1; MS (Cl) m/z 334.1
(MH+); GC tR ) 8.25 min; ESI-TOF-HRMS m/z calcd for C16H9-
Cl2NO3 (MH+) 334.0032, found 334.0031. Anal. (C16H19Cl2NO3‚
1/3C8H5NO2) C, H, N.
1-(2,4-Dichlorophenyl)-3-(dimethylamino)-2-phthal-
imidepropenone (7). A solution of 6 (5.0 kg, 15 mol) in
dimethylformamide dimethylacetal (11.0 kg, 92 mol) was
heated at 82 °C for 9 h. The reaction mixture was cooled to
ambient temperature, and the resulting solid was filtered and
washed with MTBE (2L) to affording 7 as a light-yellow,
1
granular solid (4.3 kg, 74%): mp 219 °C; H NMR (CDCl3) δ
7.92 (s, 2H), 7.76 (s, 2H), 7.45 (s, 1H), 7.38 (d, J ) 5.0 Hz,
1H), 7.25 (d, J ) 5.0 Hz, 1H), 7.03 (s, 1H), 2.97 (s, 6H); 13C
NMR (CDCl3) δ 1.86.3, 169.1, 154.4, 137.6, 135.5, 134.4, 132.5,
130.3, 130.0, 127.1, 123.9, 103.7; MS (Cl) m/z 389.0 (MH+).
Anal. (C19H14Cl2N2O3) C, H, N.
3-Amino-4-(2,4-dichlorophenyl)pyrazole (8). A suspen-
sion of 7 (1.42 kg, 3.65 mol) in ethanol (12 L) and water (1.2
L) was heated at 100 °C. Hydrazine monohydrochloride (500
g, 7.3 mol) was added, and the reaction mixture was refluxed
for 7 h. Then hydrazine monohydrate (355 mL, 7.3 mol) was
added dropwise over 10 min, and the mixture was allowed to
reflux for 1 h. The reaction mixture was cooled to room
temperature, water (2 L) was added, and the sample was
filtered through Celite and concentrated in vacuo. The residue
was partitioned between ethyl acetate (10 L) and water (5 L),
(S)-2-(2,4-Dichlorophenyl)-4,7-dimethyl-7,8-dihydro-
6H-1,3,6,8a-tetraazaacenaphthylene (11b). 11b was pre-
pared as a white powder in 13% yield from 10 and (S)-(+)-2-
amino-1-propanol according to procedure A. 1H NMR (CDCl3)
δ 7.88 (d, J ) 8.4 Hz, 1 H), 7.53 (d, J ) 1.8 Hz, 1 H), 7.35 (dd,
J ) 8.3, 2.3 Hz, 1 H), 6.28 (s, 1 H), 4.61-4.51 (m, 2 H), 4.10-
4.02 (m, 2 H), 2.58 (s, 3 H), 1.49 (d, J ) 6.3 Hz, 3 H); MS (CI)
m/z 332.9 (MH+). Anal. (C16H14Cl2N4‚0.5H2O) C, H, N.

Design and Synthesis of CRF1 Antagonists
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 18 5787
(S)-2-(2,4-Dichlorophenyl)-7-ethyl-4-methyl-7,8-dihydro-
6H-1,3,6,8a-tetraazaacenaphthylene (11c). 11c was pre-
pared as an off-white solid in a 72% yield from 10 according
to procedure A and (S)-(+)-2-amino-1-butanol. 1H NMR (CDCl3)
δ 7.89 (d, J ) 8.4 Hz, 1 H), 7.52 (d, J ) 2.1 Hz, 1 H), 7.34 (dd,
J ) 8.4 Hz, 1 H), 6.27 (s, 1 H), 4.63 (br s, 1 H), 4.60 (dd, J )
11.7, 3.3 Hz, 1 H), 4.10 (dd, J ) 11.9, 8.9 Hz, 1 H), 3.88-3.78
(m, 1 H), 2.57 (s, 3 H), 1.81 (dq, J ) 7.4, 7.4 Hz, 2 H), 1.14 (t,
J ) 7.5 Hz, 3 H); MS (CI) m/z 347.0 (MH+). Anal. (C17H16-
Cl2N4) C, H, N.
(R)-2-(2,4-Dichlorophenyl)-7-ethyl-4-methyl-7,8-dihydro-
6H-1,3,6,8a-tetraazaacenaphthylene (11d). 11d was pre-
pared as an off-white solid in a 50% yield from 10 according
to procedure A and (R)-(-)-2-amino-1-butanol. 1H NMR (CD3OD)
δ 7.62 (dd, J ) 1.0, 14.0 Hz, 1H), 7.63 (s, 1 H), 7.44 (dd, J )
14.0, 1.0 Hz, 1H), 6.34 (s, 1 H), 4.54 (dd, J ) 12.5, 4.0 Hz,
1H), 4.07 (dd, J ) 12.5, 9.0 Hz, 1H), 3.87-3.83 (m, 1H), 2.47
(s, 3H), 1.65-1.95 (m, 2H), 1.38 (t, J ) 7.0 Hz, 3 H); MS (Cl)
m/z 347.1 (MH+). ESI-TOF-HRMS m/z calcd for C17H16Cl2N4
(MH+) 347.0825, found 347.0821.
(S)-2-(2,4-Dichlorophenyl)-4-methyl-7-propyl-7,8-dihy-
dro-6H-1,3,6,8a-tetraazaacenaphthylene (11e). 11e was
prepared as a yellow powder in 18% yield from 10 and (S)-
(+)-2-amino-1-pentanol according to procedure A. 1H NMR
(CDCl3) δ 7.89 (d, J ) 8.4 Hz, 1 H), 7.53 (d, J ) 1.8 Hz, 1 H),
7.35 (dd, J ) 8.3 Hz, 1 H), 6.27 (s, 1 H), 4.60 (br s, 1 H), 4.59
(dd, J ) 12.0, 3.6 Hz, 1 H), 4.10 (dd, J ) 12.0, 9.0 Hz, 1 H),
3.96-3.87 (m, 1 H), 2.57 (s, 3 H), 1.78-1.69 (m, 2 H), 1.62-
1.52 (m, 2 H), 1.04 (t, J ) 7.4 Hz, 3 H); MS (CI) m/z 360.8
(MH+). Anal. (C18H18Cl2N4) C, H, N.
(S)-2-(2,4-Dichlorophenyl)-7-isopropyl-4-methyl-7,8-di-
hydro-6H-1,3,6,8a-tetraazaacenaphthylene (11f). 11f was
prepared as a yellow powder in 17% yield from 10 and (S)-
(+)-2-amino-3-methyl-1-butanol according to procedure A. 1H
NMR: (CDCl3) δ 7.90 (d, J ) 8.1 Hz, 1 H), 7.53 (d, J ) 1.8 Hz,
1 H), 7.35 (dd, J ) 8.3, 2.3 Hz, 1 H), 6.30 (s, 1 H), 4.60 (dd, J
) 12.3, 3.9 Hz, 1 H), 4.56 (br s, 1 H), 4.19 (dd, J ) 11.9, 9.2
Hz, 1 H), 3.76-3.69 (m, 1 H), 2.58 (s, 3 H), 2.09-2.00 (m, 1
H), 1.15 (d, J ) 6.6 Hz, 3 H), 1.14 (d, J ) 7.2 Hz, 3 H); MS
(CI) m/z 360.9 (MH+). Anal. (C18H18Cl2N4) C, H, N.
(S)-2-(2,4-Dichlorophenyl)-4-methyl-7-phenyl-7,8-dihy-
dro-6H-1,3,6,8a-tetraazaacenaphthylene (11g). 11g was
prepared as a yellow solid in a 42% yield from 10 according to
procedure A and (S)-(-)-2-amino-1-butanol. 1H NMR (CDCl3)
δ 7.84 (d, J ) 5.0 Hz, 1H), 7.50 (d, J ) 1.3 Hz, 1H), 7.44-7.40
(m, 1H), 7.31 (dd, J ) 1.3, 5.0 Hz, 1H), 6.33 (s, 1H), 5.05-4.96
(m, 1H), 4.66 (dd, J ) 2.4, 7.4 Hz, 1H), 4.28 (dd, J ) 5.8, 7.4
Hz, 1H), 2.58 (s, 3H); MS (Cl) m/z 395.1 (MH+); ESI-TOF-
HRMS m/z calcd for C21H16Cl2N4 (MH+) 395.0825, found
395.0825.
(S)-6-Cyclopropylmethyl-2-(2,4-dichlorophenyl)-7-
ethyl-4-methyl-7,8-dihydro-6H-1,3,6,8a-tetraazaacenaph-
thylene Mesylate (12a). Procedure B. A solution of 11c
(13.5 g, 38.9 mmol) in anhydrous DMF (60 mL) was cooled to
5 °C with an ice-water bath. NaH (95% suspension in oil, 1.1
g, 46.7 mmol) was added, and the mixture was stirred for 10
min. To the mixture was added bromomethylcyclopropane (6.3
g, 46.7 mmol), and the mixture was stirred at ambient
temperature for 1 h. Additional NaH (0.2 g, 7.8 mmol) and
bromomethylcyclopropane (1.0 g, 7.8 mmol) were added, and
the reaction mixture was stirred for an additional hour. The
reaction was quenched with saturated aqueous ammonium
chloride, and the mixture was concentrated in vacuo. The
residue was diluted with ethyl acetate, washed with brine,
dried (MgSO4), and concentrated in vacuo. The residue was
purified via flash chromatography on silica gel, eluting with
1:2 ethyl acetate/hexane to give 12a (13.1 g, 84%) as an oil.
1H NMR (CDCl3) δ 7.56 (d, J ) 11.1 Hz, 1H), 7.55 (d, J ) 1.7
Hz, 1H), 7.31 (dd, J ) 11.1, 1.7 Hz, 1H), 6.28 (s, 1H), 4.70 (d,
J ) 12.9 Hz, 1H), 4.42 (dd, J ) 12.9, 4.7 Hz, 1H), 4.13 (p, J )
4.7 Hz, 1H), 3.75 (dd, J ) 14.6, 5.8 Hz, 1H), 3.25 (dd, J ) 14.6,
7.0 Hz, 1H), 2.85 (s, 3H), 2.40 (s, 3H), 1.65-1.95 (m, 2H), 1.15
(t, J ) 7.0 Hz, 1H), 1.09 (t, J ) 7.0 Hz, 3H), 0.65-0.80 (m,
2H), 05.0-0.35 (m, 2H); 13C NMR (CDCl3) δ 153.7, 142.5, 135.9,
134.4, 133.7, 132.2, 128.5, 127.0, 126.3, 124.6, 124.3, 97.2, 58.7,
52.2, 47.2, 38.1, 23.6, 19.7, 9.4, 8.5, 3.1, 2.9; MS (CI) m/z 401.1
(MH+); [R]250 -170.0° (c 1.0776 g/100 mL, methanol). Anal.
D
(C21H22Cl2N4‚CH4O3S) C, H, N. Chiral HPLC 99.88% S (AUC),
99.76% ee.
(R)-6-Cyclopropylmethyl-2-(2,4-dichlorophenyl)-7-
ethyl-4-methyl-7,8-dihydro-6H-1,3,6,8a-tetraazaacenaph-
thylene Mesylate (12b). 12b was prepared as a yellow oil in
40% yield from 11d according to procedures B and C to give
12b as a mesylate salt. The crude product was purified by flash
silica gel chromatography, eluting with 1:2 ethyl acetate/
hexane. 1H NMR (CDCl3) δ 7.56 (d, J ) 11.1 Hz, 1H), 7.55 (d,
J ) 1.7 Hz, 1H), 7.31 (dd, J ) 11.1, 1.7 Hz, 1H), 6.28 (s, 1H),
4.70 (d, J ) 12.9 Hz, 1H), 4.42 (dd, J ) 12.9, 4.7 Hz, 1H), 4.13
(p, J ) 4.7 Hz, 1H), 3.75 (dd, J ) 14.6, 5.8 Hz, 1H), 3.25 (dd,
J ) 14.6, 7.0 Hz, 1H), 2.85 (s, 3H), 2.40 (s, 3H), 1.65-1.95 (m,
2H), 1.15 (t, J ) 7.0 Hz, 1H), 1.09 (t, J ) 7.0 Hz, 3H), 0.65-
0.80 (m, 2 H), 05.0-0.35 (m, 2H); MS (CI) m/z 401.1 (MH+);
ESI-TOF-HRMS m/z calcd for C21H22Cl2N4 (MH+) 401.1289,
found 401.1289.
(6)-Cyclopropylmethyl-2-(2,4-dichlorophenyl)-4-meth-
yl-7,8-dihydro-6H-1,3,6,8a-tetraazaacenaphthylene (12c).
12c was prepared as a yellow oil in 40% yield from 11a
according to procedure B. The crude product was purified by
flash silica gel chromatography, eluting with 1:2 ethyl acetate/
1
hexane. H NMR (CDCl3) δ 7.89 (d, J ) 5.0 Hz, 1H), 7.52 (d,
J ) 1.2, 1H), 7.33 (dd, J ) 5.0, 1.2 Hz, 1H), 6.24 (s, 1H), 4.53
(t, J ) 3.3 Hz, 2H), 3.86 (t, J ) 3.3 Hz, 2H), 3.30 (d, J ) 4.0
Hz, 2H), 2.59 (s, 3H), 1.10 (p, J ) 4.1 Hz, 1H), 0.66-0.62 (m,
2H), 0.32-0.29 (m, 2H); MS (CI) m/z 373.1 (MH+). Anal.
(C19H18Cl2N4) C, H, N.
(S)-6-Cyclopropylmethyl-2-(2,4-dichlorophenyl)-4,7-di-
methyl-7,8-dihydro-6H-1,3,6,8a-tetraazaacenaphthyl-
ene (12d). 12d was prepared as a yellow powder in 47% yield
1
from 11b according to procedure B. H NMR (CDCl3) δ 7.89
(d, J ) 8.4 Hz, 1H), 7.53 (d, J ) 1.8 Hz, 1H), 7.36 (dd, J ) 8.4,
2.8 Hz, 1H), 6.21 (s, 1 H), 4.48 (dd, J ) 12.3, 2.7 Hz, 1H), 4.37
(dd, J ) 12.3, 2.7 Hz, 1H), 4.28-4.19 (m, 1H), 3.50 (dd, J )
14.6, 6.2 Hz, 1H), 3.10 (dd, J ) 14.4, 7.2 Hz, 1H), 2.61 (s, 3H),
1.37 (d, J ) 6.6 H, 3H), 1.18-1.04 (m, 1H), 0.74-0.60 (m, 2H),
0.42-0.26 (m, 2H); MS (CI) m/z 387.1 (MH+). Anal. (C20H20-
Cl2N4) C, H, N.
(S)-6-Cyclopropylmethyl-2-(2,4-dichlorophenyl)-4-
methyl-7-propyl-7,8-dihydro-6H-1,3,6,8a-tetraazaacenaph-
thylene (12e). 12e was prepared as a yellow oil in 55% yield
1
from 11e according to procedure B. H NMR (CDCl3) δ 7.90
(d, J ) 8.4 Hz, 1H), 7.54 (d, J ) 2.4 Hz, 1H), 7.36 (dd, J ) 8.4,
2.1 Hz, 1H), 6.19 (s, 1H), 4.56 (dd, J ) 12.3, 1.8 Hz, 1H), 4.36
(dd, J ) 12.3, 3.3 Hz, 1H), 4.04-3.97 (m, 1H), 3.58 (dd, J )
14.4, 5.7 Hz, 1H), 3.06 (dd, J ) 14.4, 6.9 Hz, 1H), 2.61 (s, 3H),
1.73-1.33 (m, 4H), 1.13-1.09 (m, 1H), 0.94 (t, J ) 7.2 Hz, 3H),
0.70-0.62 (m, 2H), 0.37-0.28 (m, 2H); 13C NMR (CDCl3) δ
157.9, 141.0, 139.6, 134.6, 134.2, 133.4, 130.4, 130.0, 127.4,
125.3, 98.2, 57.4, 52.1, 48.9, 32.9, 29.9, 25.4, 19.9, 14.1, 9.6,
4.9, 3.4; HRMS (FAB) m/z calcd for C22H24Cl2N4 (MH+)
415.1451, found 415.1447.
(S)-6-Cyclopropylmethyl-2-(2,4-dichlorophenyl)-7-iso-
propyl-4-methyl-7,8-dihydro-6H-1,3,6,8a-tetraazaacenaph-
thylene (12f). 12f was prepared as a yellow oil in 37% yield
from 11f according to procedure B. 1H NMR (CDCl3) δ 7.89
(d, J ) 8.9 Hz, 1H), 7.53 (d, J ) 2.1 Hz, 1H), 7.36 (dd, J ) 8.3,
2.3 Hz, 1H), 6.22 (s, 1H), 4.67 (dd, J ) 11.4, 1.8 Hz, 1H), 4.31
(dd, J ) 12.5, 4.7 Hz, 1H), 3.83-3.73 (m, 2H), 3.01 (dd, J )
14.6, 7.7 Hz, 1H), 2.61 (s, 3H), 2.22-2.10 (m, 1H), 1.18-1.07
(m, 1H), 1.02 (d, J ) 6.6 Hz, 3H), 0.85 (d, J ) 6.6 Hz, 3H),
0.68-0.57 (m, 2H), 0.37-0.25 (m, 2H); MS (CI) m/z 415.1
(MH+). Anal. (C22H24Cl2N4) C, H, N.
Procedure C. To a solution of 12a free-base (11.6 g, mmol)
in dichloromethane (50 mL) was added methanesulfonic acid
(1.74 mL, 26.8 mmol). The mixture was concentrated in vacuo,
and the resulting oil was triturated with 1:10 dichloromethane/
ether with stirring overnight. The solid was collected by
filtration and washed with ether to afford 12a methane-
sulfonate (13.7 g, 100%) as a light-yellow solid: mp 167 °C;

5788 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 18
Gross et al.
(S)-6-Methyl-2-(2,4-dichlorophenyl)-4-methyl-7-phen-
yl-7,8-dihydro-6H-1,3,6,8a-tetraazaacenaphthylene (12g).
12g was prepared as a yellow oil in 40% yield from 11a
according to procedure B. The crude product was purified by
flash silica gel chromatography, eluting with 1:1 ethyl acetate/
10.3, 0.8 Hz, 1H), 4.41 (dd, J ) 10.3, 3.5 Hz, 1H), 3.88 (m,
1H), 3.66 (dd, J ) 11.4, 5.2 Hz, 1H), 3.13 (dd, J ) 11.4, 6.8
Hz, 1H), 2.76 (s, 3H), 2.15 (m, 1H), 1.85 (m, 1H), 1.75 (m, 1H),
1.04-1.10 (m, 9H); 13C NMR: (CDCl3) δ 155.4.0, 143.4, 138.9,
136.7, 134.6, 133.1, 130.1, 127.7, 127.2, 126.9, 125.6, 97.9, 61.9,
56.9, 47.9, 31.2, 27.7, 24.5, 21.0, 20.3, 11.0; HRMS (FAB) m/z
calcd for C21H24Cl2N4 (MH+) 403.1451, found 403.1456.
(S)-6-Cyclobutylmethyl-2-(2,4-dichlorophenyl)-7-ethyl-
4-methyl-7,8-dihydro-6H-1,3,6,8a-tetraazaacenaphthyl-
ene Trifluoroacetate (12n). 12n was prepared from 11c
according to procedure B and then purified by preparative LC-
MS to obtain the desired product as a TFA salt (21 mg, 22%).
1H NMR (CDCl3) δ 7.54 (d, J ) 6.6 Hz, 1H), 7.54 (d, J ) 1.6
Hz, 1H), 7.37 (dd, J ) 6.6, 1.6 Hz, 1H), 6.25 (s, 1H), 4.66 (dd,
J ) 10.3, 1.0 Hz, 1H), 4.35 (dd, J ) 10.3, 3.3 Hz, 1H), 3.90 (m,
1H), 3.82 (dd, J ) 11.5, 5.4 Hz, 1H), 3.60 (m, 1H), 3.44 (dd, J
) 11.5, 6.4 Hz, 1H), 2.78 (m, 1H), 2.74 (s, 3H), 2.18 (m, 1H),
1.74-1.87 (m, 4H), 1.08 (t, J ) 6.0 Hz, 3H); 13C NMR (CDCl3)
δ 155.2, 143.5, 138.7, 136.7, 134.6, 133.1, 130.1, 127.7, 127.2,
126.7, 125.6, 97.8, 61.3, 54.2, 48.0, 33.9, 26.8, 26.5, 24.6, 21.0,
18.7, 10.9; HRMS (FAB) m/z calcd for C22H24Cl2N4 (MH+)
4415.1451, found 415.1445.
1
hexane. H NMR (CDCl3) δ 7.90 (d, J ) 5.0 Hz, 1H), 7.52 (d,
J ) 1.3 Hz, 1H), 7.36-7.34 (m, 1H), 7.22 (d, J ) 5.0 Hz, 1H),
6.38 (s, 1H), 5.17 (t, J ) 3.0, 1H), 4.69 (dd, J ) 2.8, 7.4 Hz,
1H), 4.50 (dd, J ) 3.3, 7.4 Hz, 1H), 3.65 (dd, J ) 3.0, 8.9 Hz,
1H), 2.78 (dd, J ) 4.8, 8.8 Hz, 1H), 2.65 (s, 3H), 1.06-1.02 (m,
1H), 0.56-0.54 (m, 1H), 0.50-0.47 (m, 1H), 0.15-0.12 (m, 1H),
0.08-0.05 (m, 1H); MS (CI) m/z 449 0.1 (MH+); ESI-TOF-
HRMS m/z calcd for C25H22Cl2N4 (MH+) 449.1294, found
449.1296.
(S)-6-Methyl-2-(2,4-dichlorophenyl)-7-ethyl-4-methyl-
7,8-dihydro-6H-1,3,6,8a-tetraazaacenaphthylene (12h).
Compound 12h was prepared as a white solid in 39% yield
1
from 11c according to procedure B. H NMR (CDCl3) δ 7.88
(d, J ) 8.1 Hz, 1H), 7.59 (d, J ) 2.0 Hz, 1H), 7.31 (dd, J ) 8.0
Hz, 1H), 6.20 (s, 1H), 4.61(dd, J ) 11.9, 3.0 Hz, 1H), 4.31 (dd,
J ) 12.5, 3.0 Hz, 1H), 4.00-3.82 (m, 1H), 3.44 (s, 3H), 2.59 (s,
3 H), 1.81 (dq, J ) 7.2, 7.3 Hz, 2H), 1.10 (t, J ) 7.6 Hz, 3H);
MS (CI) m/z 361.0 (MH+). Anal. (C18H18Cl2N4) C, H, N.
(S)-6-Benzyl-2-(2,4-dichlorophenyl)-7-ethyl-4-methyl-
7,8-dihydro-6H-1,3,6,8a-tetraazaacenaphthylene (12o).
12o was prepared as a white solid in 59% yield from 11c
(S)-6-Ethyl-2-(2,4-dichlorophenyl)-7-ethyl-4-methyl-
7,8-dihydro-6H-1,3,6,8a-tetraazaacenaphthylene (12i). 12i
was prepared as a white solid in 42% yield from 11c according
1
according to procedure B. H NMR (CDCl3) δ 7.89 (d, J ) 8.3
1
to procedure B. H NMR (CDCl3) δ 7.90 (d, J ) 8.3 Hz, 1H),
Hz, 1H), 7.54-7.49 (m, 4H), 7.31-7.29 (m, 3H), 6.20 (s, 1H),
4.62 (dd, J ) 11.9, 3.6 Hz, 1H), 4.37 (dd, J ) 12.1, 2.9 Hz,
1H), 3.99 (m, 1H), 3.58 (m, 2H), 2.64 (s, 3H), 1.74-1.53 (m,
2H), 1.01 (t, J ) 7.0 Hz, 3 H); MS (CI) m/z 437.1 (MH+). Anal.
(C24H22Cl2N4) C, H, N.
7.51 (d, J ) 2.1 Hz, 1H), 7.29 (dd, J ) 8.3 Hz, 1H), 6.15 (s,
1H), 4.51 (dd, J ) 11.8, 3.2 Hz, 1H), 4.38 (dd, J ) 11.8, 2.9
Hz, 1H), 3.98 (m, 1H), 3.58 (m, 2H), 2.65 (s, 3H), 1.73-1.32
(m, 5H), 0.91 (t, J ) 7.3 Hz, 3H); MS (CI) m/z 375.1 (MH+).
Anal. (C19H20Cl2N4) C, H, N.
(S)-2-(2,4-Dichlorophenyl)-7-ethyl-6-(2-methoxy-ethyl)-
4-methyl-7,8-dihydro-6H-1,3,6,8a-tetraazaacenaphthyl-
ene Trifluoroacetate (12p). 12p was prepared from 11c
according to procedure B and then purified by preparative LC-
MS to obtain the desired product as a TFA salt (25 mg, 40%).
1H NMR (CDCl3) δ 7.54 (d, J ) 6.6 Hz, 1H), 7.54 (d, J ) 1.6
Hz, 1H), 7.37 (dd, J ) 6.6, 1.6 Hz, 1H), 6.27 (s, 1H), 4.64 (dd,
J ) 10.3, 1.1 Hz, 1H), 4.39 (dd, J ) 10.3, 3.4 Hz, 1H), 4.10 (m,
1H), 3.94 (m, 1H), 3.64-3.68 (m, 3H), 3.38 (s, 3H), 2.73 (s, 3H),
2.17 (s, 3H), 1.87 (m, 1H), 1.77 (m, 1H), 1.08 (t, J ) 6.0 Hz,
3H); 13C NMR (CDCl3) δ 155.1, 143.4, 138.7, 136.7, 134.6,
133.1, 130.1, 127.7, 127.3, 126.7, 125.8, 97.8, 70.3, 62.0, 59.6,
49.2, 48.1, 24.7, 20.9, 10.8; HRMS (FAB) m/z calcd for
C20H22Cl2N4O (MH+) 405.1243, found 405.1238.
1-(2,4-Dichlorophenyl)-5-isobutyl-7-methyl-4,5-dihydro-
3H-1,2a,5,8-tetraazaacenaphthylene Trifluoroacetate
(12q). 12q was prepared from 11a according to procedure B
and then purified by preparative LC-MS to obtain the desired
product as a TFA salt (41 mg, 17%). 1H NMR (CD3OD) δ 7.71
(d, J ) 4.2 Hz, 1H), 7.63 (d, J ) 5.1 Hz, 1H), 7.52 (dd, J ) 4.2,
1.2 Hz, 1H), 6.77 (s, 1H), 4.63 (t, J ) 3.6 Hz, 2H), 4.15 (t, J )
3.6 Hz, 2H), 3.56 (d, J ) 4.5 Hz, 2H), 2.66 (s, 3H), 2.15-2.95
(m, 1H), 1.06 (d, J ) 3.9 Hz); ESI-HRMS m/z calcd for
C19H20Cl2N4 (MH+) 375.1138, found 375.1152; LC-MS m/z 375
(M + H+).
2-(2,4-Dichlorophenyl)-6-isopropyl-4-methyl-7,8-dihy-
dro-6H-1,3,6,8a-tetraazaacenaphthylene (12r). 12r was
prepared in 17% yield from 11a according to procedure B, and
the crude product was purified by flash silica gel chromatog-
raphy, eluting with 1:2 ethyl acetate/hexane. 1H NMR (CDCl3)
δ 7.87 (d, J ) 5.0 Hz, 1H), 7.51 (d, J ) 1.2 Hz, 1H), 7.33 (dd,
J ) 5.0, 1.2 Hz, 1H), 6.24 (s, 1H), 4.49 (t, J ) 3.2 Hz, 2H),
4.15 (p, J ) 4.0 Hz, 1H), 2.59 (s, 3H), 1.32 (d, J ) 4.0 Hz, 6H);
MS (CI) m/z 361.1 (MH+). Anal. (C18H18Cl2N4) C, H, N.
1-(2,4-Dichlorophenyl)-5-(1-ethylpropyl)-7-methyl-4,5-
dihydro-3H-1,2a,5,8-tetraazaacenaphthylene Trifluoro-
acetate (12s). 12s was prepared from 11a according to
procedure B and then purified by preparative LC-MS to
obtain the desired product as a TFA salt (39 mg, 16%). 1H
NMR (CD3OD) δ 7.72 (d, J ) 1.2 Hz, 1H), 7.64 (d, J ) 4.8 Hz,
1H), 7.52 (dd, J ) 1.2, 4.8 Hz), 6.89 (s, 1H), 4.62 (t, J ) 3.3
Hz, 2H), 4.152 (m, 1H), 4.03 (t, J ) 3.3 Hz, 2H), 2.67 (s, 3H),
(S)-6-Propyl-2-(2,4-dichlorophenyl)-7-ethyl-4-methyl-
7,8-dihydro-6H-1,3,6,8a-tetraazaacenaphthylene (12j). 12j
was prepared as a white solid in 48% yield from 11c according
1
to procedure B. H NMR (CDCl3) δ 7.89 (d, J ) 8.1 Hz, 1H),
7.51 (d, J ) 2.0 Hz, 1H), 7.31 (dd, J ) 8.3 Hz, 1H), 6.27 (s,
1H), 4.55 (dd, J ) 11.7, 3.3 Hz, 1H), 4.30 (dd, J ) 11.9, 3.2
Hz, 1H), 4.00 (m, 1H), 3.51 (m, 2H), 2.57 (s, 3H), 1.92 (dq, J )
7.1, 7.2 Hz, 2H), 1.73 (m, 2H), 1.14-1.01 (m, 6H); MS (CI) m/z
389.1 (MH+). Anal. (C20H22Cl2N4) C, H, N.
(S)-2-(2,4-Dichlorophenyl)-6-butyl-7-ethyl-4-methyl-
7,8-dihydro-6H-1,3,6,8a-tetraazaacenaphthylene TFA
(12k). 12k was prepared from 11c according to procedure B
and then purified by preparative TLC to obtain the desired
product as a pale-yellow oil (52 mg, 65%). 1H NMR (CDCl3) δ
7.91 (d, J ) 8.1 Hz, 1H), 7.53 (d, J ) 2.1 Hz, 1H), 7.35 (dd, J
) 8.1, 2.1 Hz, 1H), 6.14 (s, 1H), 4.54 (dd, J ) 12.5, 2.1 Hz,
1H), 4.33 (dd, J ) 12.5, 3.3 Hz, 1H), 3.70 (m, 1H), 3.62 (dt, J
) 14.6, 6.8 Hz, 1H), 3.21 (dt, J ) 14.6, 7.4 Hz, 1H), 2.59 (s,
3H), 1.58-1.80 (m, 4H), 1.42 (m, 2H), 1.03 (t, J ) 3.6 Hz, 3H),
0.99 (t, J ) 2.4 Hz, 3H); MS (CI) m/z 403.0 (MH+). Anal.
(C21H24Cl2N4) C, H, N.
(S)-2-(2,4-Dichlorophenyl)-7-ethyl-6-isopropyl-4-meth-
yl-7,8-dihydro-6H-1,3,6,8a-tetraazaacenaphthylene TFA
(12l). 12l was prepared from 11c according to procedure B
and then purified by preparative LC-MS to obtain the desired
1
product as a TFA salt (14 mg, 23%). H NMR (CDCl3) δ 7.55
(d, J ) 6.6 Hz, 1H), 7.54 (d, J ) 1.6 Hz, 1 H), 7.37 (dd, J )
6.6, 1.6 Hz, 1 H), 6.31 (s, 1 H), 4.74 (dd, J ) 10.3 Hz, 1 H),
4.27 (m, 1 H), 4.20 (dd, J ) 10.3, 3.1 Hz, 1H), 4.08 (m, 1H),
3.65 (m, 1H), 2.76 (s, 3H), 1.79 (pent, J ) 5.5 Hz, 1H), 1.53 (d,
J ) 5.5 Hz, 3H), 1.47 (d, J ) 5.5 Hz, 3H), 1.08 (t, J ) 6.0 Hz,
3H); 13C NMR (CDCl3) δ 155.2, 142.9, 138.7, 136.7, 134.6,
133.1, 130.1, 127.7, 127.2, 126.9, 125.6, 98.3, 55.8, 51.2, 47.9,
31.2, 26.4, 21.0, 20.7, 10.9; HRMS (FAB) m/z calcd for
C20H22Cl2N4 (MH+) 389.1294, found 389.1299.
(S)-2-(2,4-Dichlorophenyl)-7-ethyl-6-isobutyl-4-methyl-
7,8-dihydro-6H-1,3,6,8a-tetraazaacenaphthylene Trifluo-
roacetate (12m). 12m was prepared from 11c according to
procedure B and then purified by preparative LC-MS to
obtain the desired product as a TFA salt (12 mg, 19%). 1H
NMR: (CDCl3) δ 7.56 (d, J ) 6.7 Hz, H), 7.54 (d, J ) 1.6 Hz,
1H), 7.38 (dd, J ) 6.7, 1.6 Hz, 1H), 6.21 (s, 1H), 4.68 (dd, J )

Design and Synthesis of CRF1 Antagonists
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 18 5789
1.85-1.79 (m, 4H), 0.97 (t, 3H); ESI-HRMS m/z calcd for
C20H22Cl2N4 (MH+) 389.1294, found 389.1294; LC-MS m/z 389
(M + H+).
1H), 7.54 (d, J ) 8.1 Hz, 1H), 7.41 (m, 1H), 6.52 (s, 1H), 4.50-
4.66 (m, 2H), 4.14-4.23 (m, 1H), 3.86-4.06 (m, 2H), 3.67 (d,
J ) 6.0 Hz, 2H), 3.36 (s, 3H), 2.71 (s, 3H), 1.80-1.85 (m, 2H),
1.04 (t, 3H); MS (CI) m/z 405.0, 407.1 (MH+). Anal.
(C20H22Cl2N4O‚2TFA‚H2O) C, H, N.
2-(2,4-Dichlorophenyl)-4-methyl-6-(1-propylbutyl)-7,8-
dihydro-6H-1,3,6,8a-tetraazaacenaphthylene (12t). 12t
was prepared as a yellow oil in 58% yield from 11a according
to procedure B, and the crude product was purified by flash
silica gel chromatography, eluting with 1:2 ethyl acetate/
4-Nitro-1H-pyrazole (14). Pyrazole (425 g, 6.24 mol) was
added in portions to concentrated sulfuric acid (3 L) while
keeping the reaction temperature below 40 °C. To the solution
was then added 70% nitric acid (430 mL, 6.86 mol) dropwise
while maintaining the temperature below 55 °C. The mixture
was heated at 55 °C for 5 h, and then the solution was cooled
to 0 °C and 6 kg of ice was added. The mixture was neutralized
with 50% aqueous NaOH, and the resulting slurry was diluted
with 6 L of EtOAc with the precipitate being removed by
filtration. The filtrate was separated, and the organic phase
was washed with water (4 L) and brine (4 L), dried (Na2SO4),
and concentrated in vacuo. The crude product was crystallized
from ethanol to afford 14 (629 g, 89%) as a white solid. 1H
NMR (CDCl3) δ 7.85-7.86 (m, 2H), 7.70-7.72 (m, 2H), 7.68,
d, J ) 5.0 Hz, 1H), 7.45 (d, J ) 0.8 Hz, 1H), 7.33 (dd, J ) 5.0,
1.2 Hz, 1H), 5.03 (s, 2H); MS (Cl) m/z 114.0 (MH+). Anal.
(C3H3N3O2) C, H, N.
1
hexane. H NMR (CDCl3) δ 7.60 (d, J ) 8.8 Hz, 1H), 7.46 (d,
J ) 2.3 Hz, 1H), 7.31 (dd, J ) 2.3, 8.8 Hz, 1H), 6.34 (s, 1H),
4.57 (t, J ) 5.3 Hz, 2H), 4.03 (p, J ) 7.0 Hz, 1H), 3.91 (t, J )
5.3 Hz, 1H), 2.90 (s, 3H), 1.67-1.76 (m, 4H), 1.32 (sextet, J )
7.0 Hz, 4H), 0.98 (t, J ) 7.0 Hz, 6H); MS (CI) m/z 417.1 (MH+).
Anal. (C22H26Cl2N4‚HCl) C, H, N.
5-(1-Butylpentyl)-1-(2,4-dichlorophenyl)-7-methyl-4,5-
dihydro-3H-1,2a,5,8-tetraazaacenaphthylene Trifluoro-
acetate (12u). 12u was prepared from 11a according to
procedure B and then purified by preparative LC-MS to
obtain the desired product as a TFA salt (21 mg, 15%). 1H
NMR (CD3OD) δ 7.72 (d, J ) 1.2 Hz, 1H), 7.64 (d, J ) 5.1 Hz,
1H), 7.52 (dd, J ) 1.2, 5.1 Hz), 6.89 (s, 1H), 4.60 (t, J ) 3.6
Hz, 2H), 4.28 (m, 1H), 4.03 (t, J ) 3.6 Hz, 2H), 2.67 (s, 3H),
1.80-1.75 (m, 4H), 1.41-1.31 (m, 8H), 0.91 (t, J ) 3.3 Hz, 6H);
ESI-HRMS m/z calcd for C24H30Cl2N4 (MH+) 445.1920, found
445.1915; LC-MS m/z 445 (M + H+).
5-Cyclohexyl-1-(2,4-dichlorophenyl)-7-methyl-4,5-di-
hydro-3H-1,2a,5,8-tetraazaacenaphthylene Trifluoro-
acetate (12v). A mixture of compound 10 (1.0 g, 3.2 mmol),
cyclohexylamine (2.9 g, 32 mmol), and p-toluenesulfonic acid
(973 mg, 5.1 mmol) was heated at 130 °C for 24 h. The reaction
mixture was cooled to room temperature, dissolved in EtOAc,
extracted with saturated NaHCO3, washed with brine, dried
(MgSO4), and concentrated in vacuo. The residue was purified
by silica gel chromatography using 70% EtOAc in hexanes to
yield 13a. Intermediate 13a (300 mg, 0.8 mmol) was treated
with dibromoethane (180 mg, 0.096 mmol) and K2CO3 (332 mg,
2.4 mmol) in 2-butanone (10 mL). The reaction mixture was
heated at 85 °C for 24 h, then concentrated in vacuo, dissolved
in EtOAc, extracted with water, dried (MgSO4), and concen-
trated again in vacuo. The product was purified by purified
by preparative LC-MS to obtain the desired product 12v as
a TFA salt (35 mg, 11%). 1H NMR (CD3OD) δ 7.72 (d, J ) 1.2
Hz, 1H), 7.62 (d, J ) 5.1 Hz, 1H), 7.52 (dd, J ) 1.2, 5.1 Hz),
6.84 (s, 1H), 4.10 (t, J ) 3.6 Hz, 2H), 4.10 (m, 3H), 2.69 (s,
3H), 1.97-1.94 (m, 4H), 1.81-1.78 (m, 3H), 1.31-1.29 (m, 1H);
ESI-HRMS m/z calcd for C21H22Cl2N4 (MH+) 401.1294, found
401.1294; LC-MS m/z 401.
[3-(2,4-Dichlorophenyl)-5-methyl-1H-pyrazolo[4,3-b]-
pyridin-7-yl]-(1-methoxymethylpropyl)amine (13b). A
mixture of compound 10 (0.50 g, 1.6 mmol), 2-amino-1-
methoxybutane (1.6 g, 16 mmol), and p-toluenesulfonic acid
monohydrate (0.5 g, 2.6 mmol) was heated at 130 °C in a sealed
tube overnight. The reaction mixture was diluted with ethyl
acetate, extracted with saturated aqueous NaHCO3, washed
with brine, dried (NaSO4), and concentrated in vacuo. The
residue was purified with flash chromatography on silica gel,
eluted with 50-100% ethyl acetate/hexane to yield 13b as a
light-tan solid (0.33 g, 53%). 1H NMR (CDCl3) δ 8.03 (br s,
1H), 7.43 (d, J ) 2.1 Hz, 1H), 7.23-7.26 (m, 2H), 6.18 (s, 1H),
3.60-3.70 (m, 1H), 3.45 (d, J ) 3.6 Hz, 2H), 3.30 (s, 3H), 2.56
(s, 3H), 1.66-1.74 (m, 1H), 1.53-1.61 (m, 1H), 0.95 (t, 3H);
MS (CI) m/z 378.9, 380.9 (MH+). Anal. (C18H20Cl2N4O‚0.25H2O)
C, H, N.
3-(1H-Pyrazol-4-ylamino)but-2-enoic Acid Ethyl Ester
(16). To a solution of 14 (50 g, 442 mmol) in ethanol (250 mL)
was added 10% Pd/C (2.5 g), and the mixture was hydro-
genated at 20-30 psi on a Parr shaking apparatus for 4 h.
The reaction mixture was filtered through Celite, washed with
ethanol, and concentrated in vacuo to yield 3-aminopyrazole
(15) as an oil: GC-MS m/z 84 (MH+). The crude product of
15 was dissolved in anhydrous toluene (450 mL). Then ethyl
acetoacetate (53.5 mL, 420 mol) and p-toluenesulfonic acid (250
mg, 1.3 mmol) were added, and the reaction mixture was
refluxed using a Dean-Stark trap for 2 h. The mixture was
then cooled to ambient temperature and concentrated in vacuo.
The residue was dissolved in ethyl acetate (250 mL) and
methanol (25 mL) and filtered through a silica gel plug using
ethyl acetate as the eluent. The fractions collected were
combined and concentrated to afford 16 (75.5 g, 88%) as an
1
off-white solid in two steps from 14. H NMR (CDCl3) δ 9.72
(s, 1H), 7.30 (s, 2H), 7.25 (s, 1H), 4.55 (s, 1H), 4.02 (q, J ) 5.4
Hz, 2H), 1.79 (s, 3H), 1.18 (t, J ) 5.4 Hz, 3H); MS (CI) m/z
196.0 (MH+). Anal. (C9H13N3O2) C, H, N.
5-Methyl-1H-pyrazolo[4,3-b]pyridine-7-ol (17). Enamine
16 (50 g, 256 mmol) was added portionwise to a flask with
diphenyl ether (100 mL) preheated at 250°C while maintaining
the solution temperature above 220 °C. The solution was
heated at 220 °C for an additional 10-15 min and then slowly
cooled to room temperature. The mixture was diluted with
MTBE (100 mL) and then stirred at room temperature for 1
h. The precipitate was collected by filtration and washed with
MTBE to afford the desired product 17 (35.7 g, 90%) as an
1
off-white solid. H NMR (CD3OD) δ 8.80 (s, 1H), 6.12 (s, 1H),
2.41 (s, 3H); MS (CI) m/z 150.0 (MH+). Anal. (C7H7N3O) C, H,
N.
7-Chloro-5-methyl-1H-pyrazolo[4,3-b]pyridine (18). To
a solution of 17 (131 g, 878 mmol) in anhydrous acetonitrile
(600 mL) was added phosphorus oxychloride (164 mL, 1.76
mol). After being heated at reflux for 3 h, the reaction mixture
was cooled to room temperature and then poured over 1.5 kg
of ice. The resulting mixture was neutralized to pH 6-7 with
approximately 50% NaOH (210 mL, 5.3 mol), resulting in a
precipitate. The solid was collected by filtration to yield 116 g
of the desired product after drying. The mother liquor was
extracted with ethyl acetate, affording an additional 24 g of
product. The combined product 18 (140 g, 95%) was a tan solid.
1H NMR (CDCl3) δ 8.20 (s, 1H), 7.20 (s, 1H), 2.71 (s, 3H); MS
(CI) m/z 168.0 (MH+). Anal. (C7H6ClN3) C, H, N.
3-Bromo-7-chloro-5-methyl-1H-pyrazolo[4,3-b]pyri-
dine (19). Bromine (15 mL, 296 mmol) in 50% aqueous
methanol (30 mL) was added dropwise at 0 °C to a solution of
18 (41 g, 247 mmol) in 50% aqueous methanol (500 mL). The
mixture was then stirred for 30 min, and the resulting
precipitate was collected by vacuum filtration. The resulting
filter cake was neutralized, first by resuspending in water and
2-(2,4-Dichlorophenyl)-6-(1-methoxymethylpropyl)-4-
methyl-7,8-dihydro-6H-1,3,6,8a-tetraazaacenaphthyl-
ene Trifluoroacetate (12w). A mixture of compound 13b
(0.20 g, 0.53 mmol), 1,2-dibromoethane (0.12 g, 0.63 mmol),
and potassium carbonate (0.22 g, 1.6 mmol) in 2-butanone (5
mL) was heated at 85 °C in a sealed tube for 24 h. Additional
1,2-dibromoethane (0.12 g, 0.63 mmol) and potassium carbon-
ate (0.22 g, 1.6 mmol) were added and heated overnight. The
reaction mixture was concentrated and purified by preparative
LC-MS to obtain the desired product 12w as a TFA salt (46
1
mg, 14%). H NMR (CDCl3 + CD3OD) δ 7.58 (d, J ) 2.1 Hz,

5790 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 18
Gross et al.
slowly adding saturated sodium bicarbonate while stirring.
The neutralized precipitate was filtered, washed with water,
and dried in vacuo at 45 °C, affording 19 (60 g, 98%) as a light-
yellow powder. 1H NMR (CDCl3) δ 10.82 (s, 1H), 7.32 (s, 1H),
2.73 (s, 3H); MS (CI) m/z 247.0, 249.0 (MH+). Anal.
(C7H5BrClN3) C, H, N.
(m, 2H), 0.48-0.34 (m, 2H); 13C NMR: (CDCl3) δ 161.4, 155.4,
143.2, 141.1, 139.3, 135.4, 132.3, 130.8, 127.3, 126.9, 126.4,
125.6, 97.9, 77.5, 77.2, 77.0, 60.1, 53.4, 48.0, 24.7, 20.8, 20.6,
10.8, 9.5, 4.9, 3.8; HRMS (FAB) m/z calcd for C22H25ClN4 (MH+)
381.1846, found 381.1836. Anal. (C22H25N4) C, H, N.
(S)-2-(4-Chloro-2-methoxyphenyl)-6-cyclopropylmethyl-
7-ethyl-4-methyl-7,8-dihydro-6H-1,3,6,8a-tetraazaacenaph-
thylene (23c). Procedure E. To a solution of 22 (66 mg, 0.18
mmol) in toluene (3.5 mL) and ethanol (0.5 mL) was added
2-methoxy-4-chlorophenylboronate ester (59 mg, 0.22 mmol),
aqueous sodium carbonate (3 M, 2 mL), and saturated aqueous
barium hydroxide (0.1 mL). The reaction mixture was degassed
with nitrogen for 10 min, and Pd(PPh3)4 (21 mg, 0.02 mmol)
was added. The mixture was heated under nitrogen at 100
°C, and once the starting materials were consumed, the
mixture was cooled, poured into water, and extracted with
ethyl acetate. The organic layer was washed with brine, dried,
and concentrated in vacuo.
The crude product was purified by flash chromatography
(silica, 5% MeOH/DCM with 1% TEA) to obtain 23c as a free
base (26 mg, 37%). 1H NMR (CDCl3) δ 7.98 (d, J ) 8.3 Hz,
1H), 7.06 (dd, J ) 8.3, 1.8 Hz, 1H), 7.02 (d, J ) 1.8 Hz, 1H),
6.17 (s, 1H), 4.58 (dd, J ) 12.3, 1.6 Hz, 1H), 4.33 (dd, J )12.3,
4 Hz, 1H), 3.92 (s, 3H), 3.89-3.93 (m, 1H), 3.60 (dd, J ) 14.4,
5.9 Hz, 1H), 3.07 (dd, J ) 14.4, 7.3 Hz, 1H), 2.63 (s, 3H), 1.76
(m, 1H), 1.65 (m, 1H), 1.10 (m, 1H), 1.02 (t, J ) 7.4 Hz, 3H),
0.59-0.72 (m, 2H), 0.27-0.38 (m, 2H); MALDI-HRMS m/z
calcd for C22H25ClN4O (MH+) 397.1790, found 397.1784.
(S)-2-(4-Chloro-2-fluorophenyl)-6-cyclopropylmethyl-
7-ethyl-4-methyl-7,8-dihydro-6H-1,3,6,8a-tetraazaacenaph-
thylene (23d). 23d was prepared according to procedure E
with 2-fluoro-4-chlorophenylboronate ester (56 mg, 0.22 mmol).
The final product was purified by flash chromatography (silica,
5% MeOH/DCM with 1% TEA) to obtain 23d as a free base
(34 mg, 49%). 1H NMR (CDCl3) δ 8.43 (t, J ) 8.1 Hz, 1H), 7.28
(dd, J ) 8.4, 2 Hz, 1H), 7.24 (dd, J ) 10.5, 2 Hz, 1H), 6.20 (s,
1H), 4.60 (dd, J ) 12.4, 1.6 Hz, 1H), 5.35 (dd, J ) 12.3, 3.9
Hz, 1H), 3.90-3.96 (m, 1H), 3.59 (dd, J ) 14.4, 5.9 Hz, 1H),
3.07 (dd, J ) 14.4, 7.3 Hz, 1H), 2.65 (s, 3H), 1.71-1.82 (m,
1H), 1.53-1.67 (m, 1H), 1.05-1.16 (m, 1H), 1.01 (t, J ) 7.5
Hz, 3H), 0.60-0.72 (m, 2H), 0.26-0.38 (m, 2H); MALDI-HRMS
m/z calcd for C21H22ClFN4 (MH+) 385.1590, found 385.1592.
(S)-2-Bromo-7-ethyl-4-methyl-7,8-dihydro-6H-1,3,6,8a-
tetraazaacenaphthylene (21). A mixture of 19 (20.0 g, 81.1
mmol), p-toluenesulfonic acid (17.0 g, 89.2 mmol), and (S)-(+)-
2-amino-1-butanol (21.7 g, 243 mmol) was heated at 150 °C
for 16 h. Aqueous hydrobromic acid (48%, 125 mL) was added
to the mixture and then heated at 120 °C for 3 days. The
mixture was cooled to room temperature and poured onto
crushed ice. The resulting mixture was neutralized with 4 N
KOH and extracted with EtOAc. The combined organic layers
were washed with brine, dried (MgSO4), and concentrated in
vacuo. The crude product was purified by flash silica gel
chromatography (3:2 ethyl acetate/hexanes) to afford 21 (9.8
g, 43% yield) over two steps. 1H NMR (CD3OD) δ 6.25 (s, 1 H),
4.62 (s, 1 H), 4.50 (dd, J ) 2.0, 4.0 Hz, 1 H), 4.05 (dd, J ) 4.0,
12.5 Hz, 1 H), 3.85-3.75 (m, 1 H), 2.47 (s, 3 H), 1.75 (q, J )
7.0 Hz, 2 H), 1.15 (t, J ) 7.0 Hz, 3 H); MS (CI) m/z 282.0,
284.0 (MH+); ESI-TOF-HRMS m/z calcd for C11H13BrN4 (MH+)
281.0396, found 281.0397.
(S)-6-Cyclopropylmethyl-2-bromo-7-ethyl-4-methyl-7,8-
dihydro-6H-1,3,6,8a-tetraazaacenaphthylene (22). A solu-
tion of 21 (2.81 g, 10 mmol) in 50 mL of anhydrous DMF was
added to NaH (60% in mineral oil, 600 mg, 15 mmol) and
stirred at room temperature for 15 min. Bromomethylcyclo-
propane (2.99 g, 22 mmol) was then added slowly, and the
mixture was stirred at room temperature for 1 h. The reaction
was quenched with water, and the sample layer was extracted
with ethyl acetate, washed with brine, dried (Na2SO4), and
concentrated in vacuo. Flash chromatography (9:1 DCM/
methanol) provided 22 (2.13 g, 64%). 1H NMR (CDCl3) δ 6.17
(s, 1H), 4.48 (dd, J ) 8, 1.8 Hz, 1H), 4.22 (dd, J ) 12.3, 3.9
Hz, 1H), 3.90-3.84 (m, 1H), 3.54 (dd, J ) 14.5, 6 Hz, 1H), 3.03
(dd, J ) 14.5, 7.5 Hz, 1H), 2.60 (s, 3H), 1.80-1.68 (m, 1H),
1.64-1.51 (m, 1H), 1.12-1.02 (m, 1H), 0.99 (t, J ) 7.5 Hz, 3H),
0.71-0.54 (m, 2H), 0.36-0.24 (m, 2H); MS (CI) m/z 334.9,
337.0 (MH+); HRMS m/z calcd for C15H19BrN4 (MH+) 335.0866,
found 335.0877.
(S)-2-(4-Chloro-2-trifluoromethylphenyl)-6-cyclo-
propylmethyl-7-ethyl-4-methyl-7,8-dihydro-6H-1,3,6,8a-
tetraazaacenaphthylene (23a). Procedure D. To a solution
of compound 22 (60 mg, 0.18 mmol) in dioxane/water (2 mL/1
mL) was added sodium carbonate (106 mg, 1 mmol) and
4-chloro-2-trifluoromethyphenylboronic acid (58 mg, 0.26 mmol).
The reaction mixture was degassed with nitrogen for 10 min,
tetrakis(triphenylphosine)palladium(0) (21 mg, 0.02 mmol)
was added, and the reaction mixture was heated at 100 °C
overnight. The reaction mixture was partitioned between brine
and EtOAc. The organic layer was dried (sodium sulfate),
evaporated, and purified by flash chromatography (silica, 5%
MeOH/DCM with 1% TEA) to give compound 23a (32 mg,
(S)-2-(2-Chloro-4-trifluoromethylphenyl)-6-cyclo-
propylmethyl-7-ethyl-4-methyl-7,8-dihydro-6H-1,3,6,8a-
tetraazaacenaphthylene Trifluoroacetate (23e). 23e was
prepared according to procedure D with 4-chloro-2-(trifluoro-
methyl)phenylboronic acid. The final product was purified by
preparative LC-MS to give compound 23e as a TFA salt (53
mg, 37%). 1H NMR (CDCl3) δ 7.77 (d, J ) 9.0 Hz, 1H), 7.65 (d,
J ) 9.0 Hz, 1H), 6.30 (s, 1H), 4.71 (d, J ) 12.9, 1H), 4.45 (dd,
J ) 12.9, 3.9 Hz, 1H), 4.15-4.11 (m, 1H), 3.74 (dd, J ) 14.7,
6 Hz, 1H), 3.67-3.60 (m, 1H), 3.26 (dd, J ) 14.7, 7.8 Hz, 1H),
2.78 (s, 3H), 1.94-1.84 (m, 1H), 1.81-1.68 (m, 1H), 1.41 (dd,
J ) 13.5, 6.3 Hz, 1H), 1.09 (t, J ) 7.4 Hz, 3H), 0.82-0.71 (m,
2H), 0.48-0.36 (m, 2H); 13C NMR (CDCl3) δ 155.7, 143.2, 138.6,
134.7, 133.0, 132.4, 127.3, 125.7, 124.0, 97.9, 77.5, 77.2, 77.0,
60.2, 53.4, 48.2, 24.6, 21.0, 10.9, 9.5, 4.9, 3.8; HRMS (FAB)
m/z calcd for C22H22ClF3N4 (MH+) 435.1563, found 435.1564.
1
49%). H NMR (CDCl3) δ 7.78 (d, J ) 8.7 Hz, 1H), 7.70-7.64
(m, 1H), 7.56-7.45 (m, 1H), 6.26 (s, 1H), 4.69 (d, J ) 12.9 Hz,
1H), 4.43 (dd, J ) 12.9, 2.1 Hz, 1H), 4.24 (br s, 1H), 3.75 (dd,
J ) 14.7, 6 Hz, 1H), 3.26 (dd, J ) 14.7, 7.8 Hz, 1H), 2.82 (s,
3H), 1.86-1.76 (m, 1H), 1.73-1.62 (m, 1H), 1.26 (s, 3H), 1.08-
1.01 (m, 1H), 0.88-0.77 (m, 2H), 0.43-0.36 (m, 2H); HRMS
(FAB) m/z calcd for C22H22ClF3N4 (MH+) 435.1563, found
435.1557.
(S)-2-(2-Chloro-4-methylphenyl)-6-cyclopropylmethyl-
7-ethyl-4-methyl-7,8-dihydro-6H-1,3,6,8a-tetraazaacenaph-
thylene Trifluoroacetate (23f). 23f was prepared according
to procedure D with 4-chloro-2-(trifluoromethyl)phenylboronic
acid. The final product was purified by preparative LC-MS
1
(S)-2-(4-Chloro-2-methylphenyl)-6-cyclopropylmethyl-
7-ethyl-4-methyl-7,8-dihydro-6H-1,3,6,8a-tetraazaacenaph-
thylene (23b). 23b was prepared according to procedure D
with 4-chloro-2-(trifluoromethyl)phenylboronic acid. The final
product was purified by preparative LC-MS to give compound
to give compound 23f as a TFA salt (49 mg, 43%). H NMR
(CDCl3) δ 7.48 (d, J ) 7.8 Hz, 1H), 7.34 (s, 1H), 7.19 (d, J )
8.4 Hz, 1H), 6.29 (s, 1H), 4.69 (d, J ) 12.9, 1H), 4.41 (dd, J )
12.9, 3.9 Hz, 1H), 4.15-4.10 (m, 1H), 3.74 (dd, J ) 14.7, 6.3
Hz, 1H), 3.65 (s, 3H), 3.26 (dd, J ) 14.7, 7.8 Hz, 1H), 2.74 (s,
3H), 2.39 (s, 1H), 1.94-1.82 (m, 1H), 1.80-1.70 (m, 1H), 1.07
1
23b (112 mg, 49%). H NMR (CDCl3) δ 7.51 (d, J ) 7.5 Hz,
1H), 7.28 (d, J ) 8.7 Hz, 2H), 6.29 (s, 1H), 4.67 (d, J ) 12.9,
1H), 4.40 (dd, J ) 12.3, 4.2 Hz, 1H), 4.15-4.12 (m, 1H), 3.74
(dd, J ) 14.7, 6.3 Hz, 1H), 3.26 (dd, J ) 14.7, 6.9 Hz, 1H),
2.75 (s, 3H), 2.37 (s, 3H), 1.94-1.82 (m, 1H), 1.79-1.69 (m,
1H), 1.18-1.15 (m, 1H), 1.07 (t, J ) 7.4 Hz, 3H), 0.83-0.70
(t, J ) 7.4 Hz, 3H), 0.82-0.69 (m, 2H), 0.47-0.33 (m, 2H); 13
C
NMR (CDCl3) δ 155.1, 143.2, 141.8, 140.1, 133.5, 132.0, 130.7,
128.1, 127.1, 125.6, 125.5, 97.8, 77.5, 77.2, 77.0, 60.2, 53.4, 48.0,
24.6, 21.3, 20.9, 10.9, 9.5, 4.9, 3.8; HRMS (FAB) m/z calcd for
C22H25ClN4 (MH+) 381.1846, found 381.1832.

Design and Synthesis of CRF1 Antagonists
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 18 5791
(S)-2-(2-Chloro-4-methoxyphenyl)-6-cyclopropylmethyl-
7-ethyl-4-methyl-7,8-dihydro-6H-1,3,6,8a-tetraazaacenaph-
thylene Trifluoroacetate (23g). 23g was prepared according
to procedure D with 4-chloro-2-trifluoromethyphenylboronic
acid. The final product was purified by preparative LC-MS.
The desired product (23g) was obtained as a TFA salt (28 mg,
(S)-2-(2,4-Bis-trifluoromethyl-phenyl)-6-cyclopropyl-
methyl-7-ethyl-4-methyl-7,8-dihydro-6H-1,3,6,8a-tetra-
azaacenaphthylene (23l). 23l was prepared according to
procedure D with 2,4-bistrifluoromethylphenylboronic acid (57
mg, 0.22 mmol). The final product was purified by flash
chromatography (silica, 5% MeOH/DCM with 1% TEA) to
obtain 23l as a free base (24 mg, 23%). 1H NMR (CDCl3) δ
8.15 (d, J ) 8.1 Hz, 1H), 8.06 (s, 1H), 7.91 (d, J ) 7.9 Hz, 1H),
6.22 (s, 1H), 4.58 (dd, J ) 12.4, 1.6 Hz, 1H), 4.38 (dd, J )
12.2, 4.1 Hz, 1H), 3.92-3.98 (m, 1H), 3.60 (dd, J ) 14.5, 5.9
Hz, 1H), 3.08 (dd, J ) 14.4, 7.3 Hz, 1H), 2.60 (s, 3H), 1.73-
1.83 (m, 1H), 1.54-1.68 (m, 1H), 1.07-1.17 (m, 1H), 1.02 (t, J
) 3.9 Hz, 3H), 0.58-0.73 (m, 2H), 0.28-0.40 (m, 2H); MALDI-
HRMS m/z calcd for C23H22F6N4 (MH+) 469.1821, found
469.1828.
1
30%). H NMR (CDCl3 + CD3OD) δ 7.51 (d, J ) 8.7 Hz, 1H),
7.10 (d, J ) 2.4 Hz, 1H), 6.96 (m, 1H), 6.41 (s, 1H), 4.71 (d, J
) 12.9 Hz, 1H), 4.43 (m, 1H), 4.16-4.22 (m, 1H), 3.87 (s, 3H),
3.79 (m, 1H), 3.31 (m, 1H), 2.71 (s, 3H), 1.87-1.94 (m, 1H),
1.71-1.81 (m, 1H), 1.14-1.20 (m, 1H), 1.09 (t, 3H), 0.73-0.78
(m, 2H), 0.37-0.48 (m, 2H); 13C NMR (CDCl3) δ 161.43, 154.87,
143.35, 139.66, 134.56, 132.99, 126.81, 125.56, 120.73, 115.76,
113.31, 97.70, 60.20, 55.81, 53.37, 47.91, 24.54, 20.83, 10.85,
9.44, 4.83, 3.75; MALDI-HRMS m/z calcd for C22H25ClN4O
(MH+) 397.1790, found 397.1781; LC-MS m/z 397.1 (MH+).
(S)-6-Cyclopropylmethyl-7-ethyl-2-(4-methoxy-2-tri-
fluoromethylphenyl)-4-methyl-7,8-dihydro-6H-1,3,6,8a-
tetraazaacenaphthylene (23m). 23m was prepared accord-
ing to procedure D with 4-methoxy-2-trifluoromethylphenyl-
boronic acid. The final product was purified by flash chroma-
tography (silica, 5% MeOH/DCM with 1% TEA) to obtain 23m
(S)-2-(2-Chloro-4-fluorophenyl)-6-cyclopropylmethyl-
7-ethyl-4-methyl-7,8-dihydro-6H-1,3,6,8a-tetraazaacenaph-
thylene (23h). 23h was prepared according to procedure E
with 2-chloro-4-fluorophenylboronate ester (56 mg, 0.22 mmol).
The final product was purified by flash chromatography (silica,
5% MeOH/DCM with 1% TEA) to obtain 23h as a free base
1
as a free base (29 mg, 37%). H NMR (CDCl3) δ 7.78 (d, J )
1
8.3 Hz, 1H), 7.32 (d, J ) 2.6 Hz, 1H), 7.18 (dd, J ) 2.6, 8.3 Hz,
1H), 6.19 (s, 1H), 4.57 (dd, J ) 1.8, 12.3 Hz, 1H), 4.35 (dd, J
) 3.9, 12.3 Hz, 1H), 3.92-4.00 (m, 1H), 3.89 (s, 3H), 3.61 (dd,
J ) 6.1, 14.5 Hz, 1H), 3.06-3.14 (m, 1H), 2.64 (s, 3H), 1.75-
1.81 (m, 1H), 1.59-1.69 (m, 1H), 1.10-1.15 (m, 1H), 1.02 (t, J
) 7.5 Hz, 3H), 0.64-0.73 (m, 2H), 0.32-0.38 (m, 2H); MS (CI)
m/z 431.2 (MH+). Anal. (C23H25F3N4O) C, H, N.
(18.7 mg, 27%). H NMR (CDCl3) δ 7.80-7-92 (m, 1H), 7.27
(dd, J ) 8.5, 2.6 Hz, 1H), 7.10 (dt, J ) 2.5, 8.3 Hz, 1H), 6.19
(s, 1H), 4.59 (dd, J ) 12.3, 1.4 Hz, 1H), 4.36 (dd, J ) 12.3, 4
Hz, 1H), 3.91-3.98 (m, 1H), 3.61 (dd, J ) 14.4, 5.8 Hz, 1H),
3.08 (dd, J ) 14.4, 7.3 Hz, 1H), 2.63 (s, 3H), 1.72-1.82 (m,
1H), 1.56-1.70 (m, 1H), 1.07-1.18 (m, 1H), 1.03 (t, J ) 7.4
Hz, 3H), 0.57-0.73 (m, 2H), 0.26-0.39 (m, 2H); MALDI-HRMS
m/z calcd for C21H22ClFN4 (MH+) 385.1590, found 385.1594.
(S)-7-Ethyl-6-isobutyl-2-(4-isopropyl-2-trifluorometh-
ylphenyl)-4-methyl-7,8-dihydro-6H-1,3,6,8a-tetraaza-
acenaphthylene Hydrochloride (23n). A mixture of 2-chloro-
5-bromobenzotrifluoride (24) (4.5 g, 17.5 mmol), tributyl(1-
ethoxyvinyl)tin (7 g, 19.4 mmol), and PdCl2(PPh3)2 (0.61 g,
0.875 mmol) in toluene (30 mL) was refluxed for 2 h. The
mixture was then cooled and filtered through a pad of silica
gel with EtOAc. The organic layer was washed with 1 N HCl
and saturated aqueous potassium fluoride solution, dried,
filtered, and concentrated in vacuo. The residue was stirred
for 3 h in a mixture of THF and 1 N HCl and then partitioned
between EtOAc and water. The organic layer was separated,
dried, filtered, and evaporated. Silica gel chromatography of
the residue (10% EtOAc/hexanes) afforded compound 25 (5.16
g, 100%).
A mixture of the 25 (5.15 g, 17.5 mmol), bis(pinacolato)-
diboron (5.3 g, 21 mmol), PdCl2(dppf) (1.3 g, 1.75 mmol), and
KOAc (2.0 g, 21 mmol) in toluene (60 mL) was refluxed for 48
h with an additional 0.65 g of PdCl2(dppf) being added after
24 h. The mixture was partitioned between EtOAc and water,
and the organic layer was separated, dried (MgSO4), filtered,
and concentrated in vacuo. Silica gel chromatography of the
residue (15-20% EtOAc/hexanes) afforded the boronate ester
26 (4.74 g, 62%), which was shown to be 70% pure by GC-
MS, with the impurity being bis(pinacolato)diborane. 1H NMR
(CDCl3) δ 8.22 (s, 1H), 8.06-8.09 (m, 1H), 7.82 (d, J ) 8 Hz,
1H), 2.65 (s, 3H), 1.27 (s, 12H).
To a solution of the 2-trifluoromethyl-4-acetylphenylboronic
acid pinacol ester 26 (5.52 g, 12.5 mmol) and 22 (3.0 g, 8.96
mmol) in toluene (35 mL) and ethanol (10 mL) was added 2 M
aqueous sodium carbonate solution (12.5 mL), saturated
aqueous barium hydroxide solution (8 mL), and Pd(PPh3)4 (1.0
g, 0.9 mmol). The mixture was heated under nitrogen at 100
°C, and after disappearance of the starting material the
mixture was cooled, poured into water, and extracted with
ethyl acetate. The organic layer was washed with brine, dried,
and evaporated. Silica gel chromatography of the residue (40%
EtOAc/hexanes) afforded 27 (3.78 g, 95%). MS (CI) m/z 443.2
(MH+).
A solution of methylmagnesium bromide (1.4 M in toluene/
THF, 75:25, 12.2 mL, 17.1 mmol) was added dropwise to a
solution of 27 (3.78 g, 8.55 mmol) in THF (60 mL) at -78 °C.
The mixture was then warmed to room temperature and
allowed to stir for 2 h. An additional 3 mL (4.2 mmol) of
methylmagnesium bromide was added, and the mixture was
(S)-2-(2-Chloro-4-methoxycarbonylphenyl)-6-cyclo-
propylmethyl-7-ethyl-4-methyl-7,8-dihydro-6H-1,3,6,8a-
tetraazaacenaphthylene (23i). 23i was prepared according
to procedure D with 2-chloro-4-carbomethoxyphenylboronic
acid. The final product was purified by flash chromatography
(silica, 5% MeOH/DCM with 1% TEA) to obtain 23i as a free
base (29 mg, 37%). 1H NMR (CDCl3) δ 8.19 (d, J ) 1.2 Hz,
1H), 8.11 (d, J ) 8.1 Hz, 1H), 8.03 (m, 1H), 6.20 (s, 1H), 4.61
(d, J ) 12.3 Hz, 1H), 4.38 (m, 1H), 3.95 (s, 3H), 3.90-3.95 (m,
1H), 3.59 (m, 1H), 3.06 (m, 1H), 2.60 (s, 3H), 1.71-1.82 (m,
1H), 1.57-1.67 (m, 1H), 1.09-1.14 (m, 1H), 1.02 (t, 3H), 0.61-
0.70 (m, 2H), 0.30-0.37 (m, 2H); 13C NMR (CDCl3) δ 166.24,
158.49, 141.30, 139.04, 137.62, 136.14, 133.37, 132.56, 131.84,
130.62, 127.93, 125.22, 98.31, 58.74, 52.55, 51.94, 48.49, 26.06,
23.61, 11.11, 9.53, 4.78, 3.32; MALDI-HRMS m/z calcd for
C23H25ClN4O2 (MH+) 425.1739, found 425.1730.
(S)-6-Cyclopropylmethyl-2-(2,4-dimethylphenyl)-7-eth-
yl-4-methyl-7,8-dihydro-6H-1,3,6,8a-tetraazaacenaphth-
ylene (23j). 23j was prepared according to procedure D with
2,4-dimethylphenylboronic acid. The final product was purified
by flash chromatography (silica, 5% MeOH/DCM with 1%
1
TEA) to give compound 23j (32 mg, 49%). H NMR (CDCl3) δ
7.71 (d, J ) 7.5 Hz, 1H), 7.08-7.12 (m, 2H), 6.16 (s, 1H), 4.54
(dd, J ) 1.8, 12.3 Hz, 1H), 4.31 (dd, J ) 3.9, 12.3 Hz, 1H),
3.88-3.93 (m, 1H), 3.59 (dd, J ) 5.7, 14.5 Hz, 1H), 3.05 (dd, J
) 7.2, 14.0 Hz, 1H), 2.59 (s, 3H), 2.49 (s, 3H), 2.25 (s, 3H),
1.71-1.82 (m, 1H), 1.60-1.69 (m, 1H), 1.09-1.14 (m, 1H), 1.02
(t, J ) 7.5 Hz, 3H), 0.61-0.69 (m, 2H), 0.30-0.37 (m, 2H);
MS (CI) m/z 361.2 (MH+). Anal. (C23H28N4) C, H, N.
(S)-6-Cyclopropylmethyl-7-ethyl-2-(4-methoxy-2-
methylphenyl)-4-methyl-7,8-dihydro-6H-1,3,6,8a-
tetraazaacenaphthylene (23k). 23k was prepared accord-
ing to procedure D with 2-methyl-4-methoxyphenylboronic acid
(36.5 mg, 0.22 mmol). The final product was purified by flash
chromatography (silica, 5% MeOH/DCM with 1% TEA) to
1
obtain 23k as a free base (35 mg, 52%). H NMR (CDCl3) δ
7.76 (d, J ) 9.2 Hz, 1H), 6.82-6.87 (m, 2H), 6.17 (s, 1H), 4.54
(dd, J ) 12.2, 1.7 Hz, 1H), 4.30 (dd, J ) 12.3, 3.8 Hz, 1H),
3.88-3.95 (m, 1H), 3.83 (s, 3H), 3.59 (dd, J ) 14.4, 5.9 Hz,
1H), 3.07 (dd, J ) 14.5, 7.3 Hz, 1H), 2.62 (s, 3H), 2.51 (s, 3H),
1.73-1.83 (m, 1H), 1.58-1.70 (m, 1H), 1.06-1.17 (m, 1H), 1.02
(t, J ) 7.4 Hz, 3H), 0.58-0.72 (m, 2H), 0.27-0.39 (m, 2H);
MALDI-HRMS m/z calcd for C23H28N4O (MH+) 377.2336, found
377.2334.

5792 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 18
Gross et al.
heated to 60 °C for 30 min to consume all of the starting
material. After cooling, the mixture was poured onto water,
extracted with ethyl acetate, dried (MgSO4), and conctrated
in vacuo. This material was dissolved in toluene (50 mL) and
TFA (1.25 mL). Then MgSO4 (1.1 g. 9.1 mmol) and p-
toluenesulfonic acid (50 mg, 0.26 mmol) were added, and the
mixture was heated for 16 h. The mixture was filtered, and
the solvent was evaporated. Silica gel chromatography of the
residue (50% EtOAc/hexanes + 2% Et3N) afforded 28 (1.68 g,
added in a volume of 25 µL of cAMP assay buffer at various
concentrations for inhibition, and the cells were incubated for
30 min at 37 °C in 5% CO2. Total cAMP produced was
measured by chemiluminescent immunoassay (Tropix, Bed-
ford, MA) and measured on an Analyst (LJL Biosystems Inc.,
CA). All IC50 values were calculated using the nonlinear curve-
fitting algorithm software Prism as above.
CRF-Stimulated ACTH Release from Cultured Rat
Anterior Pituitary Cells. For the inhibition of ACTH release
from primary rat pituitary cell cultures, five whole pituitaries
are collected from 7-week-old female Sprague Dawley rats.
Pituitaries are washed six times with HEPES buffer (2.5 g/L
BSA, 10 mg/L deoxyribonuclease I, 8.0 g/L NaCl, 0.37 g/L KCl,
100 mg/L sodium phosphate dibasic, 6.0 g/L HEPES, 2.0 g/L
glucose) and minced. The tissue is then digested with 10 mL
of collagenase for 1.5 h at 37 °C with trituration every 30 min.
The digest is then transferred to a 50 mL conical centrifuge
tube and centrifuged at 1000 rpm for 4 min. The supernatant
is discarded, and the pellet is resuspended in 10 mL of
neuraminidase solution and incubated for 9 min at 37 °C. The
suspension is centrifuged at 1000 rpm for 5 min, and the pellet
was washed once with 10 mL of BBM-T medium (11.49 g/L
Custom Media Mixture, Irvine Scientific, CA; 1.83 g of NaCO3/
L; 2.4 g of HEPES/L; 2.0 g/L BSA; 10.0 mg/L transferrin;
50 000 IU/L penicillin and streptomycin; 1 µg/L insulin; 0.1
µg/L EGF; 0.4 µg/L T3; 0.7 µg/L PTH; 10 µg/L glucagon). The
resulting pellet is finally resuspended in 3% FCS/BBM-T
medium and cultured in 96-well tissue culture plates for 2-3
days at a density of 40 000 cells/well in a final volume of 200
µL of medium.
1
45%) as an oil. H NMR (CDCl3) δ 7.87 (dd, J ) 4.5, 3 Hz, 2
H), 7.71 (dd, J ) 8.1, 1.8 Hz), 6.19 (s, 1H), 5.47 (s, 1H), 5.18-
5.19 (m, 1 H), 4.55 (dd, J ) 12.3, 2.4 Hz, 1H), 2.35 (dd, J )
12.3, 4.2 Hz, 1H), 3.88-3.95 (m, 1 H), 3.59 (dd, J ) 14.1, 6
Hz, 1 H), 3.05 (dd, J ) 14.1, 7.5 Hz, 1 H), 2.20 (s, 3H), 2.58 (s,
3H), 1.70-1.80 (m, 1 H), 1.56-1.67 (m, 1 H), 1.07-1.14 (m, 1
H), 1.01 (t, J ) 7.5 Hz, 3 H), 0.86-0.90 (m, 2H), 0.58-0.73
(m, 2H), 0.28-0.38 (m, 2H); MS (CI) m/z 441.2 (MH+).
A solution of 28 (1.65 g, 3.75 mmol) and PtO (0.10 g) in
ethanol (30 mL) was shaken under an atmosphere of hydrogen
at 40 psi for 1 h. The mixture was filtered through a plug of
silica gel and Celite, and the filtrate was evaporated to
dryness. The residue was dissolved in diethyl ether (50 mL)
and treated with a 1.0 M solution of HCl in ether (3.56 mL,
3.56 mmol). After trituration, the organic solvent was decanted
off and the remaining solid was washed three times with ether
and dried under vacuum to afford the hydrochloride salt of
the compound as a pale-yellow powder (23n, 1.25 g, 70%).
HPLC showed the compound to be 96.3% pure. 1H NMR
(CDCl3) δ 7.76 (d, J ) 8.1 Hz, 1 H), 7.63 (s, 1H), 7.48 (d, J )
8.4 Hz, 1 H), 6.18 (s, 1H), 4.53 (dd, J ) 12.3, 1.8 Hz, 1H), 4.34
(dd, J ) 12.3, 4.2 Hz, 1H), 3.88-3.96 (m, 1 H), 3.58 (dd, J )
14.1, 6 Hz, 1H), 2.96-3.08 (m, 2H), 2.57 (s, 3H), 1.70-1.81
(m, 1H), 1.57-1.67 (m, 1H), 1.30 (d, J ) 7.2 Hz, 6H), 1.07-
1.17 (m, 1 H), 1.01 (t, J ) 7.5 Hz, 3 H), 0.58-0.73 (m, 2H),
0.26-0.39 (m, 2H); MS (CI) m/z 443.2 (MH+). Anal. (C25H29N4F3‚
HCl) C, H, N.
For the assay of antagonists, cells are washed once with
BBM-T and test samples are added in various concentrations
(1 µM to 1 pM) with 0.5 nM r/hCRF in 200 mL BBM-T and
incubated for 4 h at 37 °C. The medium is then aspirated and
assessed for ACTH release using a standard RIA kit (MP
Biomedicals, NY). Again, all data were analyzed using the
nonlinear curve-fitting algorithm software Prism as above.
Biology. In Vitro Binding and Functional Studies.
Radioligand binding assays and functional inhibition of CRF-
induced cAMP production were performed essentially as
previously described for the cloned CRF1 receptor in L-CRF1
cell membranes.22 Equilibrium binding of unlabeled ligands
was measured in duplicate by inhibition of radioligand binding
([125I]sauvagine) to LtK- cells expressing the human CRF1
receptor. Assay buffer (30 µL of DPBS, 1.5 mM KH2PO4, 8.1
mM Na2HPO4, 2.7 mM KCl, 138 mM NaCl) supplemented with
10 mM MgCl2, 2 mM ethylene glycol bis[â-aminoethyl]-
N,N,N′,N′-tetraacetic acid (pH 7.4), 20 µL of unlabeled ligand,
50 µL of radioligand, and 100 µL of L-CRF1 cell membranes
were sequentially added to low protein-binding 96-well plates
(Corning no. 3605). The final concentration of radioligand was
approximately 90 pM for [125I]sauvagine with a total of 5 µg
of membrane. Unlabeled compounds were serially diluted for
final concentrations of 10 pM to 1 µM. Following a 2 h
incubation at room temperature, bound and free radioligands
were separated by rapid vacuum filtration. In all assays the
total radioligand bound to the filter (total binding) was less
than 20% of the total amount of radioligand added. Nonspecific
binding was determined in the presence of an excess of the
unlabeled analogue of the radioligand. Bound and nonspecific
radioactivity was monitored using a Packard Cobra II γ
counter (78% efficiency) and analyzed using the nonlinear
curve-fitting algorithm software Prism (GraphPad Inc., CA).
CRF-Stimulated cAMP Production in Cells Express-
ing Human CRF1 Receptors. For the inhibition of CRF-
stimulated cAMP formation, the assays were again performed
as described.22 One day prior to assay, L-hCRF1 cells were
transferred to 96-well tissue culture plates (100 000 cells/well
in 200 µL of medium). On the day of assay, the medium was
removed and the cells were washed with 200 µL of DPBS.
Following aspiration of DPBS, 75 µL of cAMP assay buffer was
added to each well (DMEM without phenol red, supplemented
with 2 mM glutamine, 1 mM sodium pyruvate, 10 mM HEPES,
50 IU/mL penicillin, 50 µg/mL streptomycin, and 1 mM IBMX).
Corticotropin-releasing factor and non-peptides were then
In Vivo CRF-Induced ACTH Release in Rats. Three
days prior to testing with compound, rats were anesthetized
(n ) 6 per group) with isoflurane and implanted with a femoral
vein catheter (IITC no. 26A; PE 10 silastic) in the right groin
area. The catheter was secured in place with 4-0 suture. A
gastric catheter was placed in the stomach and sutured with
a purse string suture (4-0 suture) to secure the cannula in
place. The cannulae were fed subcutaneously to the dorsal
section of the rat (behind the ears), where they exited and were
sutured in place. All external incisions were closed using
standard wound clips. On the day of testing, fed rats were
weighed and then connected to PE50 tubing via manosil
tubing. They were then placed in opaque collection containers,
with the PE50 tubing drawn through the top of the container,
and habituated to the containers for 1 h. Following a baseline
blood draw, Compound 12a (3, 10, or 30 mg/kg) or vehicle (2
mL/kg) was infused via the intragastric tube. Sixty minutes
later, CRF (0.3 nmol/kg) or vehicle (0.5 mL/kg) was injected
iv. The CRF vehicle was a 0.1% BSA and 10 mM acetic acid
solution. Blood was drawn at 2, 10, and 30 min following the
CRF injection, collected in EDTA-coated tubes, and centrifuged
at 2500 rpm (4 °C) for 20 min. Plasma was frozen (-80 °C)
until the time of assay. The ACTH levels were determined in
these samples using a standard ACTH RIA kit (MP Biomedi-
cals, NY), with sample ACTH values calculated from a log-
transformed standard curve. ACTH values over time were
analyzed using repeated measures and mixed-design ANOVA.
Peak (10 min time point) ACTH values were analyzed using
one-way ANOVA, with Fischer’s PLSD as the post hoc method
of testing dose group differences.
Restraint Stress-Induced ACTH Release in Mice. Male
CD-1 mice (24-26 g, 10/group) were weighed and handled once
a day prior to the restraint stress. On the day of testing,
animals were dosed by oral gavage with 12a (20 mg/kg) or
vehicle (10 mL/kg 5% mannitol-d (w/v) in water) 60 min prior
to the initiation of the stressor. Restraint stress was evoked
by allowing the mice to enter a 50 mL plastic conical tube (tip

Design and Synthesis of CRF1 Antagonists
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 18 5793
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removed to allow free flow of air) and held in place for 45 min.
The animals cannot alter their position once inside the tube.
Following the 45 min of restraint stress, the animals are
removed and immediately placed in an isoflurane chamber for
a maximum of 2 min and blood is collected via cardiac puncture
into 0.5 M EDTA-coated tubes. The blood is kept on ice, and
plasma is separated by centrifugation (6000g at 4 °C for 15
min). Mice in the nonstress control group remained in their
home cages for the duration of the experiment following oral
gavage with the vehicle. Plasma ACTH concentration was
measured using a standard ACTH RIA kit (MP Biomedicals,
NY). The ACTH values over time were analyzed using
repeated measures and mixed-design ANOVA with peak
ACTH values analyzed using a one-way ANOVA with post hoc
Fischer’s test for group differences.
Rat Pharmacokinetics. Compound 12a was dosed as a
mesylate salt in aqueous solution. The pharmacokinetics
profile was determined in male Sprague Dawley rats (N )
3/time points at a dose of 10 mg/kg). The dosing solution was
prepared in purified water and filtered through a 0.2 µm nylon
filter prior to administration (2 mL/kg) via the tail vein (iv) or
oral gavage (po).
Supporting Information Available: Tables of HPLC
purity and elemental analyses. This material is available free
of charge via the Internet at http://pubs.acs.org.
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