Potent, orally active corticotropin-releasing factor receptor-1 antagonists containing a tricyclic pyrrolopyridine or pyrazolopyridine core

Article abstract of DOI:10.1021/jm050070m

Two new classes of tricyclic-based corticotropin-releasing factor (CRF ₁) receptor-1 antagonists were designed by constraining known 1H-pyrrolo[2,3-b]pyridine and 1H-pyrazolo[3,4-b]pyridine ligands. Pyrrole- and pyrazole-based molecules 19g and

Full text of DOI:10.1021/jm050070m

4
100
J. Med. Chem. 2005, 48, 4100-4110
Potent, Orally Active Corticotropin-Releasing Factor Receptor-1 Antagonists
Containing a Tricyclic Pyrrolopyridine or Pyrazolopyridine Core
Brian Dyck,*,# Dimitri E. Grigoriadis, Raymond S. Gross, Zhiqiang Guo, Mustapha Haddach,
‡
#
#
#
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#
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#
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Dragan Marinkovic, James R. McCarthy, Manisha Moorjani, Collin F. Regan, John Saunders,
#
#
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§
§
Michael K. Schwaebe, Tomas Szabo, John P. Williams, Xiaohu Zhang, Haig Bozigian, and Ta Kung Chen
Departments of Medicinal Chemistry, Pharmacology and Lead Discovery, and Preclinical Development, Neurocrine Biosciences,
12790 El Camino Real, San Diego, California 92130
Received January 25, 2005
1
Two new classes of tricyclic-based corticotropin-releasing factor (CRF ) receptor-1 antagonists
were designed by constraining known 1H-pyrrolo[2,3-b]pyridine and 1H-pyrazolo[3,4-b]pyridine
ligands. Pyrrole- and pyrazole-based molecules 19g and 22a, respectively, were discovered that
1 i
potently bind the recombinant CRF receptor (K ) 3.5, 2.9 nM) and inhibit adrenocorticotropic
hormone (ACTH) release from rat pituitary cell culture (IC50 ) 14, 6.8 nM). These compounds
show good oral bioavailabity (F ) 24%, 7.0%) and serum half-lives in rats (t1/2 ) 6.3, 12 h) and
penetrate the rat brain ([brain]/[plasma] ) 0.27, 0.52) but tend toward large volumes of
-
1
-1
-1
distribution (V
D
) 38, 44 L kg ) and rapid clearances (CL ) 70, 43 mL min kg ). When
given orally, both the pyrazole and the pyrrole leads dose-dependently inhibit stress-induced
ACTH release in vivo. ACTH reductions of 84-86% were observed for 30 mg kg doses.
-
1
Introduction
Corticotropin-releasing factor (CRF) is a 41-amino
acid peptide that has a multitude of effects in both the
central nervous system and periphery but has the
primary purpose of coordinating an organism’s response
to exogenous stressors.1-5 This action is mediated
through the binding of the CRF peptide on the pituitary,
which triggers secretion of adrenocorticotropic hormone
(
ACTH), the key hormone governing stress response.
terparts 44,5 (Figure 1). Both the above-mentioned
clinical compound (1) and the extensively researched
antalarmin (2)14 fall within this broad structural class.
A limited number of tricyclic derivatives have also been
The effect of CRF on the hypothalamic-pituitary-
adrenal (HPA) axis occurs via interaction with two
distinct G-protein-coupled receptors and, in particular,
via high-affinity binding to the first of these receptors
4
reported. We envisioned synthesizing compounds with
(CRF1), which is expressed in the pituitary. Consider-
a third ring installed (i.e., 5 and 6), anticipating that
the additional constraint would not only produce a
distinct SAR for this series relative to compounds of
classes 3 and 4 but may force conjugation of the 4-amino
nitrogen atom lone pair to the pyridine ring, potentially
affecting the hydrogen-bonding potential at this site.15
Although the precise nature of the interaction between
the pyridine or pyrimidine nitrogen atom and the
receptor is not known,16 its presence is essential for
high-affinity binding, and the basicity of this nitrogen
atom is important for the pharmacokinetics of the
molecule.17
able clinical evidence substantiates that hypersensitiv-
ity of the CRF signaling pathway results in anxiety and
stress-related disorders and that antagonism of the
CRF1 receptor represents a potential treatment for such
pathologies. Elevated levels of CRF have been found in
the cerebrospinal fluid (CSF) of patients suffering from
6
,7
8,9
depression and post-traumatic stress disorder, and
the efficacious treatment of clinically depressed patients
7
,10
led to a measurable decrease in these levels.
Post-
mortem analysis of suicide victims shows a reduced
presence of CRF1 receptors in the frontal cortex, pre-
sumed to be a consequence of down-regulation in
11
response to the elevated CRF levels. Finally, R121919
1), a non-peptide and selective CRF1 receptor antago-
Results and Discussion
(
Chemistry. We sought a general synthetic method
for the preparation of these compounds that would allow
late-stage differentiation to either the pyrazole 5 or the
pyrrole 6 and that would also permit rapid variation of
the aryl substituent to produce libraries of related
compounds. The facility with which nitrogen nucleo-
philes displace chloride from 2-chloropyridines, particu-
larly when the 3-position of the pyridine is substituted
with an electron-withdrawing group, suggested that
nist, proved to be effective in treating severely depressed
patients in an open labeled phase IIA clinical trial.1
2,13
Several companies have disclosed potent CRF1 recep-
tor antagonists based on monocyclic and bicyclic cores,
including substituted azines 3 and their bicyclic coun-
*
To whom correspondence should be addressed. Phone: 858-617-
7
778. Fax: 858-617-7619. E-mail: bdyck@neurocrine.com.
#
Department of Medicinal Chemistry.
‡
Department of Pharmacology and Lead Discovery.
§
18
Department of Preclinical Development.
ketone 7 would be a suitable key intermediate.
1
0.1021/jm050070m CCC: $30.25 © 2005 American Chemical Society
Published on Web 05/06/2005

CRF
1
Antagonists
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 12 4101
However, the modest yield for the cyclization step in
Scheme 1 prompted us to pursue an alternative syn-
thesis. Toward this end, bis(triflate) 13 was prepared
as a chemical equivalent of dichloride 8 by treating
hydroxypyridinone 1219 with trifluoroacetic anhydride
(
Scheme 2). When 13 was mixed with ethyl-N-(4-
methyl)-â-alanine under acid catalysis, the desired
-aminopyridine 14a was formed in a 3-fold excess over
4
the 2-aminopyridine isomer. Upon reaction with potas-
sium tert-butoxide, diester 14a underwent a Dieckmann
cyclization to give ketoester 15a. Heating this compound
with concentrated hydrochloric acid resulted in decar-
boxylation and exchange of the trifluoromethanesulfonyl
group for chlorine. The ketone 7a prepared by this route
was identical in every respect to that prepared in
Scheme 1.
1
Figure 1. Design of tricyclic CRF receptor antagonists.
Scheme 1a
The chloropyridine 16a, prepared either by addition
of the â-alanine derivatives to 8 or chlorinolysis of
sulfonate 14a, undergoes the cyclization/decarboxylation
procedure in substantially better yield. In general, the
intermediates 16 have the added advantage that they
are typically crystalline and preferentially crystallize
over their 4-chloropyridine isomers. As such, this ma-
nipulation is more suitable to large-scale synthesis and
purification of these intermediates. The bicyclic ketones
7
prepared from 16 are indistinguishable from those
prepared from sulfonates 14.
a
After generation of our prospective key intermediate
7, it remained to independently anneal both a pyrazole
and a pyrrole ring to give compounds 5 and 6, respec-
tively. In the pyrrole case, an additional carbon atom
was introduced via a Wittig reaction, which generated
the penultimate enol ether 18 (Scheme 3). Conversion
of 18 to the tricyclic compound 19 was effected in several
different ways depending on the choice of the R3
substituent. Typically the cyclization was conducted by
thermolysis in the presence of a suitable aniline or
amine. In some instances, the yield for this transforma-
tion was improved by using a microwave, and in the
case of particularly unreactive anilines, recourse was
made to palladium catalysis. Unfortunately, none of
these conditions were uniformly successful for all sub-
strates. However, despite the low yields for this process,
Reagents and conditions: (a) (i) LAH, THF, -78 °C; (ii) oxalyl
chloride, DMSO, TEA, DCM, -70 °C to room temp; (b) (i)
1
2
CH2dCHMgBr, THF, -78 °C; (ii) Jones’ reagent; (c) R R CHNH2,
ethanol, room temp.
The initial synthetic route to compound 7 is outlined
in Scheme 1. Ethyl 2,4-dichloro-6-methylnicotinate (8)
was converted to aldehyde 9 via lithium aluminum
hydride reduction and Swern oxidation of the resulting
alcohol. Addition of vinylmagnesium bromide to alde-
hyde 9, followed by oxidation of the resulting allylic
alcohol, afforded enone 10. This material readily cyclized
when treated with primary amines to generate a
mixture of regioisomers of the newly formed piperidine
ring. For example, when 4-heptylamine was used in this
reaction, the cyclized products were formed in 42%
combined yield and the 4-chloropyridine 11 was present
in slight excess.
3
the fact that the R group is introduced in the final step
Scheme 2a
a
Reagents and conditions: (a) trifluoroacetic anhydride, TEA, DCM, 10 °C; (b) ethyl-N-alkyl-â-alanine, TEA, ACN; (c) t-BuOK, THF;
(
d) 6 M HCl, reflux; (e) 4 M HCl in dioxane.

4
102 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 12
Dyck et al.
Scheme 3a
1
2
Table 1. Reference Compounds and SAR of R and R
Substitution for Tricyclic Compounds
a
Reagents and conditions: (a) Ph3PdCHOMe, THF, reflux; (b)
3
3
R NH2, PTSA, tetramethylene sulfone, 225 °C; (c) R NHNH2,
3
ethanol, reflux; (d) (i) R NHNH2, methanol, 60 °C; (ii) diphenyl
ether, 200 °C.
from readily available anilines and amines makes this
synthetic route amenable to parallel synthesis.
For the pyrazole compound class, direct cyclization
of 7 with substituted hydrazines in refluxing ethanol
resulted in almost exclusive formation of the 1-aryl
isomers 20. Under these conditions, the reaction ap-
pears to proceed by initial displacement of chloride by
the more nucleophilic primary nitrogen of the hydrazine,
followed by cyclization and dehydration. To drive the
reaction to the desired 2-aryl isomer 22, ketone 7 and
the hydrazine were allowed to react at low temperature
until formation of the corresponding hydrazone 21 was
complete. Thermolysis of this intermediate then af-
forded 22, which was accompanied by smaller amounts
of 20. The use of these modified conditions allowed the
preparation of a variety of tricyclic-based pyrazolopy-
ridines 22 for evaluation in the CRF1 receptor binding
assay.
In Vitro Results. When comparing the biological
activity of these new tricyclic compounds to previously
reported monocyclic and bicyclic analogues, we antici-
pated that the alkyl bridge forming the new fused ring
would compensate for one of the alkyl chains on the sp3
nitrogen atom of compounds such as 3 and 4. To
evaluate this hypothesis, a number of representatives
containing alternative substitutions in this position
were synthesized, and the binding constants of some key
examples are presented in Table 1. The simple N-methyl
analogue 19a is inactive, but the introduction of an
N-butyl group (19b) or a 3-pentyl group (19c) results
in binding constants of 49 and 62 nM, respectively.
When the size of this substituent is increased to a
a
b
Averaged from a minimum of two replicates. R121919.
c
d
Antalarmin. Not active (<30% inhibition at 10 µM).
more hydrophilic groups into this portion of the molecule
also met with limited success.
On the other hand, when the group R is considered,
3
the SAR of these new compounds demonstrates a
similarity to that of the bicyclic counterparts (Table 2).20
In particular, 2,4-disubstituted aryl groups provide the
most potent analogues, particularly when the substit-
uents are small (19g-l). The electronic nature of these
groups does not appear to have a significant impact on
the binding potency. For example, a cyano, a methoxy,
or a methanesulfonyl group are all well-tolerated (19i-
k). In fact, even a dimethoxypyridine group can be used
to replace this substituted phenyl, as evidenced by 19l.
This provides an opportunity to balance the hydropho-
bicity inherent to the R-branched alkyl group on the
piperidine ring. As one would expect from the previously
reported SAR of the bicycle-based compounds, deletion
of the 4-substituent (19m) or the 2-substituent (19n)
reduces inhibition constants by more than 5-fold, and
3
-substituted examples (19o) are typically 50-fold less
potent. Moreover, attempting to replace the aryl group
with an alternative lipophilic counterpart drastically
alters the affinity of these compounds as exemplified
by 19p, and even introducing a methylene spacer
between the aryl ring and the tricyclic core reduces the
potency by several orders of magnitude (19q).
Binding data for a representative pyrazole-based
compound 22a and its regioisomer 20a are presented
in Table 3. The SAR of the pyrazole-based tricycles
parallels that of their pyrrole counterparts and does not
need to be discussed in detail. Hence, the analogue
containing a 2,4-dichlorophenyl group and a 4-heptyl
group as the pyrazole and piperidine substituents,
respectively, has a Ki of 2.9 nM (22a). The regioisomer
20a, formed during preliminary investigations into the
4
1
-heptyl group, as in 19d, binding potency improves to
3 nM. Regrettably, the relatively lower potency of 19b
indicates that in the fused tricyclic series, the new alkyl
bridge is incapable of compensating for one of the
substituents on the exocyclic nitrogen atom of bicyclic
ligands such as 4, and branched aliphatic groups on the
piperidine ring nitrogen atom are essential for high-
affinity binding. In an attempt to reduce the significant
impact of a heptyl group on the log D of these molecules,
oxygen atoms were incorporated into one (19e) or both
(
19f) arms of the branched group. This leads to signifi-
cant loss of binding potency. Other attempts to introduce

CRF
1
Antagonists
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 12 4103
3
Table 2. SAR of R Substitution for Tricyclic Compounds
4). Pyrrole 19g is simultaneously one of the most
lipophilic compounds from the series and the most
potent, with a Ki of 3.5 nM and an IC50 of 14 nM in a
functional assay of ACTH release in rat pituitary cell
culture. When administered orally to conscious rats, the
compound showed good bioavailability (F ) 24%) and
nearly an ideal serum half-life (t1/2 ) 6.3 h). The
-
1
-1
molecule was rapidly cleared (CL ) 70 mL min kg
)
and demonstrated a very high volume of distribution
-
1
(
VD ) 38 L kg ), assuming linear pharmacokinetics at
these doses. In an attempt to gauge how the volume of
distribution and clearance of these compounds relate to
their substituents, less lipophilic analogues were simi-
larly evaluated. Surprisingly, introduction of more polar
3
groups into the R site failed to lower the volume of
distribution, and the clearance of these molecules was
either increased or unchanged (19j-l). For 19j and 19l,
the increased clearance may be a result of the metaboli-
cally labile methoxy groups. Indeed, when the methoxy
substituent was replaced with the trifluoromethoxy
-
1
group as in 19r, the clearance dropped to 28 mL min
-
1
kg , which also resulted in the highest oral bioavail-
ability of the group (76%). In terms of the R and R
1
2
sites of substitution, the introduction of less lipophilic
substituents had a somewhat unpredictable effect on the
pharmacokinetics of the molecules (19r and 19s). In
some cases, these substitutions lead to lower volumes
of distribution (19s) or clearances (19r) but never both.
Again, the presence of a labile methoxy group, this time
in the R1 substituent, may be the reason for the
extensive clearance of 19s. In addition, compounds 19r
and 19s were evaluated in a cassette paradigm in lower
doses and in the presence of other compounds. This may
have influenced the determined pharmacokinetic prop-
erties, and these data are not necessarily directly
comparable to the data from noncassette studies.
Pyrazole 22a possesses a more moderate clearance,
a longer half-life, but a lower bioavailability than pyrrole
1
9g. This may indicate that the pyrazole class is not as
easily absorbed as the corresponding pyrrole-based
compounds. As well, most of the tricyclic compounds
tested are able to penetrate the brain. Pyrrole 19g and
pyrazole 22a show brain-to-plasma ratios of 0.27 and
0
.52, respectively, when administered intravenously.
a
A: site of attachment to tricyclic core. b Averaged from a
minimum of two replicates.
In Vivo Efficacy. Compound 19g demonstrates high
potency in binding and ACTH release and good oral
bioavailability and serum half-life and penetrates the
blood-brain barrier. It is rapidly cleared but still shows
the best overall profile of the various pyrrole compounds.
Pyrazole 22a has lower oral bioavailability than 19g,
but is modestly more potent and has better clearance
and brain penetration values. The complementary na-
ture of these compounds prompted us to evaluate them
in vivo. A number of different animal efficacy models
have been reported in the literature, although the
performance of CRF1 antagonists in these models is
somewhat indiscriminate.21 In our hands, the most
predictive models are those involving the antagonist-
mediated suppression of elevated serum ACTH levels
induced by an exogenous stressor. As such, we chose to
evaluate the tricyclic compounds in a restraint-induced
anxiety model.
Table 3. CRF1 Binding Data for 20a and 22a
a
b
A: site of attachment to tricyclic core. Averaged from a
c
minimum of two replicates. Not active (<30% inhibition at 10
µM).
synthesis of 22a, is inactive in the CRF1 receptor
binding assay.
Pharmacokinetics. On the basis of their in vitro
profiles, several of the tricyclic CRF1 receptor ligands
were evaluated in rat pharmacokinetic studies (Table
The effects of oral dosing of 19g and 22a in the
restraint-induced ACTH release mouse model are pre-

4
104 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 12
Dyck et al.
Table 4. In Vitro and in Vivo Properties of Selected Tricyclic CRF1 Receptor Antagonists
a
b
c
A: site of attachment to tricyclic core. d Averaged from a minimum of two replicates unless otherwise noted. Measuring ACTH
e
release in isolated rat pituitary cell culture. By administration of 10 mg/kg po and 10 mg/kg iv. By coadministration of 2 mg/kg po and
mg/kg iv each of five compounds. f Single determination. g By administration of 10 mg/kg po and 5 mg/kg iv.
1
Figure 2. Effect of compounds 19g (a) and 22a (b) on stress-induced ACTH release. Data are presented as the mean ( SEM:
(
†) p < 0.0001 vs vehicle with no stress induction; (/) p < 0.001 vs vehicle with stress induction; (//) p < 0.0001 vs vehicle with
stress induction.
sented in Figure 2. In both experiments, a robust
increase in ACTH (p < 0.0001) is observed when mice
are subjected to a 45 min period of restraint. Both
compounds attenuate this ACTH elevation in a dose-
dependent manner from oral doses of 3-30 mg/kg, and
the effect is statistically significant at 10 mg/kg. At 30
mg/kg, the pyrrole 19g and the pyrazole 22a showed
clearly demonstrated that they are efficacious attenu-
ators of stress-induced ACTH release.
Conclusion
In summary, we have presented the design, synthesis,
pharmacology, pharmacokinetics, and in vivo efficacy
of two novel classes of fused tricyclic CRF1 receptor
ligands. Both compound classes have generated potent,
8
4% and 86% reduction in ACTH levels, respectively,
functional CRF receptor antagonists, which are orally
1
with p-values below 0.0001. These compounds have
absorbed, penetrate the brain, and normalize elevated

CRF
1
Antagonists
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 12 4105
ACTH levels brought on by exogenous sources of stress.
The introduction of a third ring and a branched lipo-
philic substituent gives these molecules an unusual
pharmacokinetic profile in rodents. It is unclear at this
stage whether these data will be predictive of the
pharmacokinetics in humans, but the synthesis of
compounds designed to optimize these properties is
currently underway.
δ 10.45 (s, 1 H), 7.26 (s, 1 H), 2.58 (s, 3 H); LC-MS (APCI)
+
m/z 189.9 (MH ). Anal. (C
7
H
5
Cl
2
NO) C, H, N.
1
-(2,4-Dichloro-6-methylpyridin-3-yl)prop-2-en-1-ol. Vi-
nylmagnesium bromide in THF (1.0 M, 6.7 mL) was added to
a stirred solution of aldehyde 9 (1.15 g, 6.05 mmol) in THF
20 mL) at -78 °C. The mixture was stirred at this temper-
(
ature for 30 min and warmed to room temperature, and the
reaction was quenched with aqueous sodium bicarbonate (40
mL). The mixture was extracted twice with ethyl acetate, and
the combined extracts were dried (MgSO
4
) and concentrated
under vacuum to afford 1.37 g of the crude allylic alcohol as a
Experimental Section
1
yellow oil: H NMR (300 MHz, CDCl
(ddd, J ) 17.1, 10.5, 5.3 Hz, 1 H), 5.94-5.88 (m, 1 H), 5.34-
.27 (m, 2 H), 3.01 (d, J ) 9.9 Hz, 1 H), 2.51 (s, 3 H); LC-MS
3
) δ 7.16 (s, 1 H), 6.18
Chemistry. General Methods. 1H and 13C NMR spectra
5
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 using TMS as the internal standard and
+
(
APCI) m/z 218.1 (MH ). Anal. (C
9 9 2
H Cl NO) C, H, N.
1-(2,4-Dichloro-6-methylpyridin-3-yl)propenone (10).
The allylic alcohol from the previous step and N-methylmor-
pholine N-oxide (NMO, 1.06 g, 9.05 mmol) were dissolved in
dichloromethane (27 mL) and treated with 4 Å molecular
sieves (1.3 g). The mixture was stirred for 20 min, and
tetrapropylammonium perruthenate (TPAP, 65 mg) was added.
The mixture was stirred for 1 h. Some starting material
persisted, so additional NMO (1.06 g) and TPAP (65 mg) were
added and stirring was continued for 1 h. The mixture was
filtered (Celite) and concentrated under vacuum, and the
residue was purified on a silica gel column (elution with 10%
3
CDCl 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 operated 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. LC-MS analyses were per-
formed on a Perkin-Elmer Sciex API-100 mass spectrometer
using the electron spray ionization technique or on a Spectra-
System 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-LC-MS Dionex system, using an Alpha C18 30 mm ×
ethyl acetate in hexanes) to afford 0.50 g (38%) of 10 as a pale-
1
yellow oil: H NMR (300 MHz, CDCl
3
) δ 7.21 (s, 1 H), 6.60
(dd, J ) 17.7, 10.5 Hz, 1 H), 6.22 (d, J ) 10.5 Hz, 1 H), 6.02
(d, 17.4 Hz, 1 H), 2.57 (s, 3 H); LC-MS (APCI) m/z 215.9
+
(MH ). Anal. (C
9
H
7
Cl
2
NO) C, H, N.
5-Chloro-7-methyl-1-(1-propylbutyl)-2,3-dihydro-1H-
[1,6]naphthyridin-4-one (7d) and 5-Chloro-7-methyl-1-(1-
propylbutyl)-2,3-dihydro-1H-[1,8]naphthyridin-4-one(11d).
Enone 10 (920 mg, 4.26 mmol) was dissolved in ethanol (20
mL) and treated with 4-heptylamine (0.64 mL, 4.3 mmol). The
mixture was heated at 60 °C for 16 h and concentrated under
vacuum. The residue was taken up in ethyl acetate (50 mL),
washed with aqueous sodium bicarbonate (20 mL), dried
7
5 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 ) H O-
.1%TFA, B ) CH CN-0.1% TFA. All compounds after
2
0
3
purification were reanalyzed on a reverse-phase HPLC-MS
system (DIONEX with ESI+ ionization mode) and shown to
be at least 85% pure on the basis of both UV wavelengths (220
and 254 nM) and total ion current (TIC) integration from the
mass spectrum. All commercially available reagents were used
without further purification. Microwave reactions were per-
formed using a Personal Chemistry Emrys optimizer. Hydro-
chloride salts of final compounds used for pharmacokinetic
measurements were prepared by treatment of the free bases
in DCM with excess HCl in ether, concentration of the mixture,
and recrystallization as necessary. Methanesulfonic acid salts
were prepared by addition of a single equivalent of the sulfonic
acid from a stock solution, concentration, and recrystallization
as necessary.
4
(MgSO ), and again concentrated. The residue was purified
on a silica gel column (elution with 5% ethyl acetate in hexanes
for 11d and 25% ethyl acetate in hexanes for 7d) to afford 314
mg (25%) of 11d as a yellow oil followed by 214 mg (17%) of
7d as a white solid.
1
3
Compound 7d: H NMR (300 MHz, CDCl ) δ 6.54 (s, 1 H),
3.99 (pent, J ) 6.5 Hz, 1 H), 3.40 (dd, J ) 7.1, 7.1 Hz, 2 H),
2.64 (dd, J ) 7.1, 7.1 Hz, 2 H), 2.39 (s, 3 H), 1.64-1.486 (m, 4
H), 1.38-1.24 (m, 4 H), 0.93 (t, J ) 7.1 Hz, 6 H); LC-MS
+
16 23 2
(APCI) m/z 295.1 (MH ). Anal. (C H ClN O) C, H, N. The
singlet at 6.54 (pyridyl-H) and the multiplet at 3.99 (CHPr2)
showed an NOE effect that was absent in 11d, confirming the
identity of this compound as 7d.
2,4-Dichloro-6-methylpyridine-3-carbaldehyde (9). Eth-
1
yl 2,4-dichloro-6-methylnicotinate (8.04 g, 34.3 mmol) was
dissolved in THF (40 mL) and added to a stirred suspension
of LAH (6.52 g, 0.170 mmol) in THF (80 mL) at -78 °C. The
mixture was stirred for 6 h at this temperature and 1 h at
Compound 11d: H NMR (300 MHz, CDCl ) δ 6.42 (s, 1
3
H), 5.38-5.22 (m, 1 H), 3.36-3.31 (m, 2 H), 2.63-2.59 (m, 2
H), 2.31 (s, 3 H), 1.58-1.26 (m, 4 H), 1.33-1.21 (m, 4 H), 0.90
+
(t, J ) 7.4 Hz, 6 H); LC-MS (APCI) m/z 295.1 (MH ). Anal.
-
30 °C and treated cautiously with water (5.5 mL), 15%
(C H ClN O‚0.2H O) C, H, N.
1
6
23
2
2
aqueous sodium hydroxide (5.5 mL), and water (17 mL) with
vigorous stirring. The mixture was warmed to room temper-
ature and filtered. The white precipitate was washed liberally
with ethyl acetate. The combined organic portions were dried
Ethyl 6-Methyl-2,4-bis(trifluoromethanesulfonyloxy)-
nicotinate (13). In a 5 L round-bottom three-neck flask
equipped with a mechanical stirrer and nitrogen bubbler was
charged 1 L of anhydrous dichloromethane. To the solution
was charged 320 g (1.62 mol) of 12a and 490 mL (3.5 mol, 2.2
equiv) of triethylamine. The mixture was cooled below 5 °C
with an ice bath, and 950 g (3.37 mol, 2.08 equiv) of trifluo-
romethanesulfonic anhydride in 500 mL of dichloromethane
was added via a 2 L addition funnel over 2 h. The mixture
was allowed to warm to ambient temperature and was stirred
for an additional 2 h. The reaction was quenched with 500
mL of water, and the organic phase was separated. The organic
phase was washed twice with 200 mL of 1 N HCl and 100 mL
of brine, dried over magnesium sulfate, and filtered over a pad
of silica gel (80 mm × 120 mm), eluting with ethyl ether. The
(
4
MgSO ) and concentrated under vacuum to afford 6.40 g of
the crude alcohol as a colorless oil that solidified on standing.
DMSO (14.2 mL, 200 mmol) was added to a stirred solution
of oxalyl chloride (8.7 mL, 99 mmol) in dichloromethane (100
mL) at -70 °C. After 15 min, the above alcohol (6.40 g, 33.3
mmol) in dichloromethane (25 mL) was added, followed by
triethylamine (56 mL), and the mixture was allowed to warm
to room temperature and stirred for 1 h. The mixture was
washed with aqueous sodium bicarbonate (75 mL), dried
(
4
MgSO ), and concentrated under vacuum. The residue was
purified by column chromatography (elution with 10% ethyl
acetate in hexanes) to afford 5.00 g (78%) of 9 as a pale-yellow
oil which solidified on standing: H NMR (300 MHz, CDCl
solvent was removed in vacuo to afford 790 g (100%) of 13 as
1
1
3
)
a brown oil: H NMR (300 MHz, CDCl
3
) δ 7.22 (s, 1H), 4.45

4
106 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 12
Dyck et al.
(
q, J ) 6.9 Hz, 2H), 2.65 (s, 3H), 1.42 (t, J ) 6.9 Hz, 3H); GC-
extracts were dried (MgSO
4
) and concentrated, and the residue
MS (EI) m/z 461.
was purified by flash chromatography (elution with 50% ethyl
Ethyl 3-Methylaminoproprionate.22 The compound was
acetate in hexanes) to afford 178 mg (35%) of 7a as a white
1
powder: H NMR (300 MHz, CDCl
3
) δ 6.34 (s, 1 H), 3.60 (t, J
prepared according to the general procedure of Rosenberg and
2
3
) 7.2 Hz, 2 H), 3.07 (s, 3 H), 2.75 (t, J ) 7.2 Hz, 2 H), 2.42 (s,
Rapoport. Ethyl acrylate (10 mL, 92 mmol) was dissolved in
ethanol (50 mL) and treated with N-benzylmethylamine (11
mL, 99 mmol). The mixture was stirred for 18 h, after which
+
3
2
H); LC-MS (APCI) m/z 210.9 (MH ). Anal. (C10H11ClN O)
C, H, N. Using the same procedure with 16a as starting
+
material afforded 86% of the title compound.
LC-MS indicated complete product formation (MH , 222.0).
Palladium on carbon (10%, 5.2 g) was added, followed by
ammonium formate (8.2 g, 124 mmol), and the mixture was
placed in a preheated oil bath and heated to reflux for 1 h.
The mixture was cooled to room temperature, diluted with
ethyl acetate, filtered (Celite), and concentrated under vacuum
5-Chloro-4-[1-methoxymeth-(E)-ylidene]-1,7-dimethyl-
1,2,3,4-tetrahydro[1,6]naphthyridine (18a). Method D.
Potassium tert-butoxide (319 mg, 2.86 mmol) and (methoxy-
methyl)triphenylphosphonium chloride (982 mg, 2.86 mmol)
were combined under nitrogen. THF (10 mL) was added, and
the mixture was stirred for 10 min to generate a deep-red
solution. Ketone 7a (302 mg, 1.43 mmol) was added, and the
mixture was heated to reflux for 1 h. After cooling, the mixture
was poured into water and extracted with ethyl acetate. The
to afford the title compound as a colorless oil, which was used
1
without further purification: H NMR (300 MHz, CDCl
3
) δ 4.14
(
q, J ) 7.1 Hz, 2 H), 2.84 (t, J ) 6.5 Hz, 2 H), 2.50 (t, J ) 6.5
Hz, 2 H), 2.43 (s, 3 H), 1.26 (t, J ) 7.2 Hz, 3 H); GC-MS (EI)
+
m/z 131 (M ).
combined extracts were dried (MgSO
the residue was purified by flash chromatography (elution with
0% ethyl acetate in hexanes) to afford 191 mg (56%) of 18a
4
) and concentrated, and
Ethyl 2-[(2-Ethoxycarbonylethyl)methylamino]-6-meth-
yl-4-trifluoromethanesulfonyloxynicotinate and Ethyl
5
1
as a white solid: H NMR (300 MHz, CDCl
.18 (s, 1 H), 3.72 (s, 3 H), 2.34 (t, J ) 6.2 Hz, 2 H), 2.93 (s, 3
H), 2.56 (t, J ) 6.3 Hz, 2 H), 2.35 (s, 3 H); LC-MS (APCI) m/z
3
) δ 6.94 (s, 1 H),
4
-[(2-Ethoxycarbonylethyl)methylamino]-6-methyl-2-tri-
6
fluoromethanesulfonyloxynicotinate (14a). Method A.
Compound 13 (8.79 g, 19.1 mmol) was dissolved in acetonitrile
+
2
38.9 (MH ). Anal. (C12
2
H15ClN O) C, H, N.
(
9 mL) and cooled in an ice bath. Triethylamine (3.2 mL, 23
mmol) was added, followed by ethyl 3-methylaminopropionate
3.00 g, 22.9 mmol) in acetonitrile (5 mL). The mixture was
1-(4-Chloro-2-cyanophenyl)-5,7-dimethyl-1,3,4,5-tet-
(
rahydro-1,5,8-triazaacenaphthylene (19a). Method E.
Enol ether 18a (123 mg, 0.52 mmol), 2-amino-4-chloroben-
zonitrile (102 mg, 0.67 mmol), and 4-toluenesulfonic acid
monohydrate (10 mg, 0.053 mmol) were dissolved in tetra-
methylenesulfone (2 mL) and heated to 225 °C for 1 h in a
microwave reactor. After cooling, the mixture was poured into
1 N sodium hydroxide solution and extracted with ethyl
allowed to warm to room temperature over 1 h and was stirred
at this temperature for 18 h. It was diluted with ethyl acetate
(
100 mL), washed with water (75 mL) and aqueous sodium
chloride (75 mL), dried (MgSO ), and concentrated under
vacuum. The residue was purified by flash chromatography
4
(
(
elution with 20% ethyl acetate in hexanes) to afford 1.33 g
16%) of ethyl 2-[(2-ethoxycarbonylethyl)methylamino]-6-
4
acetate. The combined extracts were dried (MgSO ) and
methyl-4-trifluoromethanesulfonyloxynicotinate as a white
concentrated, and the residue was purified by flash chroma-
tography followed by preparative TLC (elution with 30% ethyl
1
powder: H NMR (300 MHz, CDCl
)
2
(
(
second more polar fraction afforded 4.01 g (47%) of 14a as a
white powder: H NMR (300 MHz, CDCl
3
) δ 6.39 (s, 1 H), 4.36 (q, J
7.1 Hz, 2 H), 4.12 (q, J ) 7.2 Hz, 2 H), 3.86 (t, J ) 7.2 Hz,
H), 2.95 (s, 3 H), 2.67 (t, J ) 7.1 Hz, 2 H), 2.40 (s, 3 H), 1.37
acetate in hexanes) to afford 24 mg (14%) of 19a as a pale-
1
yellow solid: H NMR (300 MHz, CDCl
3
) δ 7.93 (d, J ) 9.0
t, J ) 7.2 Hz, 2 H), 2 H), 1.23 (t, J ) 7.2 Hz, 2 H); LC-MS
APCI) m/z 442.9 (MH ). Anal. (C16
Hz, 1 H), 7.72 (d, J ) 2.4 Hz, 1 H), 7.65 (dd, J ) 8.7, 2.4 Hz,
1 H), 6.94 (s, 1 H), 6.16 (s, 1 H), 3.40 (t, J ) 6.1 Hz, 2 H), 3.09
+
21 3 2 7
H F N O S) C, H, N. A
(t, J ) 6.1 Hz, 2 H), 3.00 (s, 3 H), 2.52 (s, 3 H); LC-MS (APCI)
1
+
) δ 6.49 (s, 1 H), 4.37
m/z 322.9 (MH ). Anal. (C18
H
15ClN
4
) C, H, N.
3
24
(
q, J ) 7.1 Hz, 2 H), 4.14 (q, J ) 7.1 Hz, 2 H), 3.61 (t, J ) 7.42
Ethyl 3-Butylaminoproprionate. Method F. The com-
H), 2.96 (s, 3 H), 2.63 (t, J ) 7.4 Hz, 2 H), 2.39 (s, 3 H), 1.39
pound was prepared according to the general procedure of
+
22 1
(
t, J ) 7.1 Hz, 2 H); LC-MS (APCI) m/z 442.9 (MH ). Anal.
S) C, H, N. Compound 14a showed an NOE
) and the singlet at
.49 (pyridyl-H). An analogous signal was absent in the other
isomer isolated from this reaction.
3
Holley and Holley: H NMR (300 MHz, CDCl ) δ 4.10 (dd, J
(
16
C H
21
F
3 2
N O
7
) 7, 14.1 Hz, 2 H), 2.83 (t, J ) 6.4 Hz, 2 H), 2.57 (t, J ) 6.8
Hz, 2 H), 3.47 (t, J ) 6.4 Hz, 2 H), 1.58 (broad s, 1 H), 1.48-
1.15 (m, 4 H), 1.22 (t, J ) 7 Hz, 3 H), 0.87 (t, J ) 7.2 Hz, 3 H);
signal between the singlet at 2.96 (N-CH
3
6
+
LC-MS (APCI) m/z 173.9 (MH ).
Ethyl 2-Chloro-4-[(2-ethoxycarbonylethyl)methylami-
Ethyl 4-[Butyl-(2-ethoxycarbonylethyl)amino]-6-meth-
no]-6-methylnicotinate (16a). Using Method A and com-
yl-2-trifluoromethanesulfonyloxynicotinate (14b). 14b
1
pound 8 as starting material afforded 23% of 16a as a colorless
was prepared using method A: H NMR (300 MHz, CDCl
3
) δ
1
oil: H NMR (300 MHz, CDCl
3
) δ 6.42 (s, 1 H), 4.37 (q, J )
6.46 (s, 1 H), 4.35 (q, J ) 7.2 Hz, 2 H), 4.13 (q, J ) 7.2 Hz, 2
H), 3.57 (q, J ) 7.5 Hz, 2 H), 3.23 (q, J ) 7.5 Hz, 2 H), 2.38 (s,
3 H), 1.63-1.50 (m, 2 H), 1.38 (t, J ) 7.2 Hz, 3 H), 1.37-1.18
7
.1 Hz, 2 H), 4.13 (q, J ) 7.2 Hz, 2 H), 3.60 (t, J ) 7.5 Hz, 2
H), 2.95 (s, 3 H), 2.60 (t, J ) 7.5 Hz, 2 H), 2.41 (s, 3 H), 1.39
t, J ) 7.4 Hz, 2 H), 1.25 (t, J ) 7.2 Hz, 2 H); LC-MS (APCI)
(
(m, 4 H), 1.24 (t, J ) 7.2 Hz, 3 H), 0.93 (t, J ) 7.4 Hz, 3 H);
+
+
m/z 328.9 (MH ). Anal. (C15
H
21ClN
2
O
4
) C, H, N.
LC-MS (APCI) m/z 405.0 (MH ). Anal. (C19
H
27
F
3
N
2
O
7
S‚
0
2
.4H O) C, H, N.
Ethyl 2-Chloro-4-[(2-ethoxycarbonylethyl)methylami-
no]-6-methylnicotinate (16a). Method B. Diester 14a (2.11
g, 4.77 mmol) was dissolved in 4 M hydrogen chloride in
dioxane and stirred at room temperature for 16 h. The mixture
was basified to pH 10 with 1 N aqueous sodium hydroxide and
extracted three times with DCM. The combined extracts were
1-Butyl-5-chloro-7-methyl-2,3-dihydro-1H-[1,6]naph-
1
thyridin-4-one (7b). 7b was prepared using method C:
H
NMR (300 MHz, CDCl ) δ 6.33 (s, 1 H), 3.61-3.52 (m, 1 H),
3
3.45 (pent, J ) 3 Hz, 1 H), 3.37 (t, J ) 7.5 Hz, 2 H), 2.70 (dd,
J ) 3.6, 2.4 Hz, 2 H), 2.39 (s, 3 H), 1.90-1.63 (m, 2 H), 1.43
(hex, J ) 7.4 Hz, 2 H), 0.99 (t, J ) 7.4 Hz, 3 H); LC-MS (APCI)
4
dried (MgSO ) and concentrated, and the residue was purified
+
by flash chromatography (elution with 50% ethyl acetate in
hexanes) to afford 174 mg (11%) of 16a, which was identical
to material prepared using method A.
m/z 252.8 (MH ).
1-Butyl-5-chloro-4-[1-methoxymeth-(E)-ylidene]-7-meth-
yl-1,2,3,4-tetrahydro[1,6]naphthyridine (18b). 18b was
1
5
-Chloro-1,7-dimethyl-2,3-dihydro-1H-[1,6]naphthyri-
din-4-one (7a). Method C. Potassium tert-butoxide (0.293 g,
.61 mmol) was added to an ice-cooled, stirred solution of 14a
1.07 mg, 2.41 mmol) in THF (5 mL). The mixture was warmed
prepared using method D: H NMR (300 MHz, CDCl
3
) δ 6.91
(s, 1 H), 6.18 (s, 1 H), 3.71 (s, 3 H), 3.34 (t, J ) 6 Hz, 2 H),
3.24 (t, J ) 7.5 Hz, 2 H), 2.52 (t, J ) 6 Hz, 2 H), 2.34 (s, 3 H),
1.66-1.50 (m, 2 H), 1.45-1.27 (m, 2 H), 0.96 (t, J ) 7.4 Hz, 3
2
(
+
to room temperature and stirred for 3 h. Hydrochloric acid (6
N, 3 mL) was added, and the mixture was heated to reflux for
8 h. It was cooled, basified with 1 N aqueous sodium
hydroxide, and extracted three times with DCM. The combined
H); LC-MS (APCI) m/z 281.0 (MH ).
5-Butyl-1-(4-chloro-2-cyanophenyl)-7-methyl-1,3,4,5-
1
tetrahydro-1,5,8-triazaacenaphthylene (19b). 19b was
1
prepared using method E: H NMR (300 MHz, CDCl
3
) δ 7.92

CRF
1
Antagonists
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 12 4107
(
d, J ) 9 Hz, 1 H), 7.71 (d, J ) 2.4 Hz, 1 H), 7.64 (dd, J ) 9,
J ) 4.2 Hz, 3 H), 1.26-1.20 (m, 4 H), 0.87 (t, J ) 4.2 Hz, 6 H);
+
2.4 Hz, 1 H), 6.91 (s, 1 H), 6.13 (s, 1 H), 3.46 (t, J ) 3.4 Hz, 2
LC-MS (APCI) m/z 413.0 (MH ). Anal. (C21
H
33ClN
2
O
4
) C, H,
H), 3.35 (t, J ) 7.5 Hz, 2 H), 3.05 (t, J ) 5.7 Hz, 2 H), 2.51 (s,
N.
3
3
H), 1.72-1.60 (m, 2 H), 1.43 (pent, J ) 4.6 Hz, 2 H), 0.98 (s,
5
-Chloro-1-(1-propylbutyl)-4-[1-methoxymeth-(E)-yli-
+
4
H); LC-MS (APCI) m/z 365.0 (MH ). Anal. (C21
O) C: calcd, 64.87; found, 65.23. H: calcd, 6.13; found, 6.64.
N: calcd, 14.41; found, 13.64.
4
H21ClN ‚ /
dene]-7-methyl-1,2,3,4-tetrahydro[1,6]naphthyridine (18d).
H
2
18d was prepared using method D: H NMR (300 MHz,
1
3
CDCl ) δ 6.83 (s, 1 H), 6.31 (s, 1 H), 3.85-3.80 (m, 1 H), 3.65
3
Ethyl 3-(1-Ethyl-propylamino)proprionate. The com-
(s, 3 H), 3.07 (t, J ) 3.5 Hz, 2 H), 2.41 (t, J ) 3.7 Hz, 2 H),
2.27 (s, 3 H), 1.50-1.41 (m, 2 H), 1.41-1.38 (m, 2 H), 1.23-
1
pound was prepared using method F: H NMR (300 MHz,
CDCl
2
H), 1.26 (t, J ) 6.9 Hz, 3 H), 0.94-0.85 (m, 6 H); GC-MS (EI)
m/z 187 (M ).
) δ 4.15 (q, J ) 6.9 Hz, 2 H), 2.85 (t, J ) 6.3 Hz, 2 H),
1.17 (m, 4 H), 0.83 (t, J ) 4.4 Hz, 6 H); LC-MS (APCI) m/z
3
+
.60-2.50 (m, 1 H), 2.49 (t, J ) 6.3 Hz, 2 H), 1.50-1.30 (m, 4
323.0 (MH ). Anal. (C18
H
27ClN
2
O) C, H, N.
1-(4-Chloro-2-cyanophenyl)-5-(1-propylbutyl)-7-meth-
yl-1,3,4,5-tetrahydro-1,5,8-triazaacenaphthylene Hydro-
+
1
Ethyl 4-[(2-Ethoxycarbonylethyl)(1-ethylpropyl)amino]-
chloride (19d). 19d was prepared using method E: H NMR
6
-methyl-2-trifluoromethanesulfonyloxynicotinate (14c).
(300 MHz, CDCl
3
) δ 7.82-7.81 (m, 1 H), 7.79 (s, 1 H), 7.72-
7
3
.71 (m, 1 H), 6.72 (s, 1 H), 6.20 (s, 1 H), 4.00-3.90 (m, 1 H),
.50 (t, J ) 5.0 Hz, 2 H), 3.05 (t, J ) 4.8 Hz, 2 H), 2.81 (s, 3
1
4c was prepared using method A and used without further
purification in the preparation of 16c.
H), 1.72-1.55 (m, 4 H), 1.36-1.27 (m, 4 H), 0.94 (t, 5.7 Hz, 6
Ethyl 2-Chloro-4-[(2-ethoxycarbonylethyl)(1-ethylpro-
+
H); LC-MS (APCI) m/z 407.1 (MH ). Anal. (C24
O) C, H, N.
Ethyl 3-(1-Methoxymethylpropylamino)proprionate.
4
H27ClN ‚HCl‚
pyl)amino]-6-methylnicotinate (16c). 16c was prepared
1
H
2
using method B: H NMR (300 MHz, CDCl
3
) δ 6.51 (s, 1 H),
4
.33(q, J ) 4.4 Hz, 2 H), 4.12 (q, J ) 4.4 Hz, 2 H), 3.44-3.30
1
The compound was prepared using method F: H NMR (300
MHz, CDCl ) δ 4.13 (q, J ) 6.9 Hz, 2 H), 3.39-3.23 (m, 2 H),
(
m, 3 H), 2.50-2.47 (m, 2 H), 2.03 (s, 3 H), 1.59-1.51 (m, 4
H), 1.36 (t, J ) 4.2 Hz, 3 H), 1.24 (t, J ) 3.6 Hz, 3 H), 0.84 (t,
3
+
3.33 (s, 3 H), 2.91-2.85, (m, 2 H), 2.67-2.58, (m, 1 H), 2.49 (t,
J ) 6.3 Hz, 2 H), 1.47-1.41 (m, 2 H), 1.26, (t, J ) 7.2 Hz, 3
H), 0.89 (t, J ) 7.5 Hz, 3 H).
J ) 4.4 Hz, 6 H); LC-MS (APCI) m/z 385.1 (MH ). Anal.
(C H29ClN O ‚0.5C H O ) C, H, N.
19 2 4 4 8 2
5
-Chloro-1-(1-ethylpropyl)-7-methyl-2,3-dihydro-1H-
Ethyl 4-[(2-Ethoxycarbonylethyl)(1-methoxymethyl-
propyl)amino]-6-methyl-2-trifluoromethanesulfonyloxy-
[
1,6]naphthyridin-4-one (7c). 7c was prepared using method
1
C: H NMR (300 MHz, CDCl
H), 3.37 (t, J ) 4.1 Hz, 2 H), 2.61 (t, J ) 4.1 Hz, 2 H), 2.36
s, 3 H), 1.66-1.54 (m, 4 H), 0.89 (t, J ) 4.4 Hz, 6 H); LC-MS
3
) δ 6.54 (s, 1 H), 3.80-3.70 (m,
1
nicotinate (14e). 14e was prepared using method A: H NMR
1
(
3
300 MHz, CDCl ) δ 6.69 (s, 1 H), 4.35 (q, J ) 6.9 Hz, 2 H),
(
(
+
4.13 (q, J ) 7.2 Hz, 2 H), 3.69-3.59 (m, 1 H), 3.55-3.42 (m, 4
H), 3.30 (s, 3 H), 2.62-2.37 (m, 2 H), 2.40 (s, 3 H), 1.65-1.60
APCI) m/z 267.0 (MH ). Anal. (C14
-Chloro-1-(1-ethylpropyl)-4-[1-methoxymeth-(E)-yli-
dene]-7-methyl-1,2,3,4-tetrahydro[1,6]naphthyridine (18c).
8c was prepared using method D: 1H NMR (300 MHz, CDCl
δ 6.88 (s, 1 H), 6.36 (s, 1 H), 3.70 (s, 3 H), 3.70-3.60 (m, 1 H),
.12 (t, J ) 3.6 Hz, 2 H), 2.47 (t, J ) 3.6 Hz, 2 H), 2.32 (s, 3
2
H19ClN O) C, H, N.
5
(
m, 2 H), 1.38 (t, J ) 6.9 Hz, 3 H), 1.25 (t, J ) 7.1 Hz, 3 H),
+
0
.87 (t, J ) 7.2 Hz, 3 H); LC-MS (APCI) m/z 515.1 (MH ).
Anal. (C20 S) C, H, N.
-Chloro-1-(1-methoxymethylpropyl)-7-methyl-2,3-di-
hydro-1H-[1,6]naphthyridin-4-one (7e). 7e was prepared
1
3
)
29 3 2 8
H F N O
5
3
H), 1.55 (p, J ) 4.4 Hz, 4 H), 0.84 (t, J ) 4.4 Hz, 6 H); LC-
1
+
using method C: H NMR (300 MHz, CDCl
3
) δ 6.54 (s, 1 H),
.08-4.03 (m, 1 H), 3.54-3.45 (m, 4 H), 3.33 (s, 3 H), 2.68-
.62 (m, 2 H), 2.39 (s, 3 H), 1.74-1.61 (m, 2 H), 0.98 (t, J )
2 7 16
MS (APCI) m/z 385.1 (MH ). Anal. (C16H23ClN O‚0.25C H )
C, H, N.
4
2
7
1
-(4-Chloro-2-cyanophenyl)-5-(1-ethylpropyl)-7-meth-
yl-1,3,4,5-tetrahydro-1,5,8-triazaacenaphthylene (19c).
9c was prepared using method E: 1H NMR (300 MHz, CDCl
+
.2 Hz, 3 H); LC-MS (APCI) m/z 283.1 (MH ). Anal. (C14
) C, H, N.
-Chloro-1-(1-methoxymethylpropyl)-4-[1-meth-
oxymeth-(E)-ylidene]-7-methyl-1,2,3,4-tetrahydro[1,6]-
19
H -
2 2
ClN O
1
3
)
5
δ 7.95 (d, J ) 8.7 Hz, 1 H), 7.71 (d, J ) 2.4 Hz, 1 H), 7.64 (dd,
J ) 8.7, 2.4 Hz, 1 H), 6.92 (s, 1 H), 6.18 (s, 1 H), 3.63 (pentet,
J ) 7.5 Hz, 1 H), 3.33 (t, J ) 6.2 Hz, 2 H), 3.01 (t, J ) 5.7 Hz,
1
naphthyridine (18e). 18e was prepared using method D:
NMR (300 MHz, CDCl
H
3
) δ 6.90 (s, 1 H), 6.37 (s, 1 H), 3.96-
2
H), 2.50 (s, 3 H), 1.69-1.59 (m, 4 H), 0.93 (t, J ) 7.4 Hz, 6
3
3
1
.88 (m, 1 H), 3.71 (s, 3 H), 3.64-3.37 (m, 2 H), 3.32 (s, 3 H),
.25-3.20 (m, 2 H), 2.49 (t, J ) 6.6 Hz, 2 H), 2.33 (s, 3 H),
+
H); LC-MS (APCI) m/z 379.0 (MH ). Anal. (C18
.5H O‚0.5C O) C, H, N.
Ethyl 3-(1-Propylbutylamino)proprionate. The com-
4
H15ClN ‚
0
2
3 6
H
.65-1.58 (m, 2 H), 0.90 (t, J ) 7.2 Hz, 3 H); LC-MS (APCI)
+
m/z 311.1 (MH ). Anal. (C16
H
23ClN
2
O
2
) C, H, N.
1
pound was prepared using method F: H NMR (300 MHz,
CDCl ) δ 4.13 (q, J ) 7.5 Hz, 2 H), 2.84 (t, J ) 6.6 Hz, 2 H),
1
-(4-Chloro-2-cyanophenyl)-5-(1-methoxymethylpropyl)-
3
7
-methyl-1,3,4,5-tetrahydro-1,5,8-triazaacenaphthyl-
2
.48 (t, J ) 6.6 Hz, 2 H), 2.50-2.42 (m, 1 H), 1.38-1.30 (m, 8
1
ene (19e). 19e was prepared using method E: H NMR (300
MHz, CDCl ) δ 7.93 (d, J ) 9.0 Hz, 1 H), 7.72 (d, J ) 2.4 Hz,
H), 7.64 (dd, J ) 8.7, 2.4 Hz, 1 H), 6.91 (s, 1 H), 6.21 (s, 1
H), 1.26 (t, J ) 7.5 Hz, 3 H), 0.94-0.86 (m, 6 H); GC-MS (EI)
3
+
m/z 215 (M ).
1
Ethyl 4-[(2-Ethoxycarbonylethyl)(1-propylbutyl)amino]-
H), 3.93-3.88 (m, 1 H), 3.61-3.35 (m, 4 H), 3.35 (s, 3 H), 3.02-
6
-methyl-2-trifluoromethanesulfonyloxynicotinate (14d).
2
7
.98 (m, 2 H), 2.50 (s, 3 H), 1.75-1.68 (m, 2 H), 0.95 (t, J )
1
4d was prepared using method A: 1H NMR (300 MHz, CDCl
3
)
+
.5 Hz, 3 H); LC-MS (APCI) m/z 395.0 (MH ). Anal. (C22
23
H -
δ 6.53 (s, 1 H), 4.31 (q, J ) 4.3 Hz, 2 H), 4.12 (q, J ) 4.3 Hz,
4 3 6
ClN O‚0.5C H O) C, H, N.
2
H), 3.50-3.44 (m, 3 H), 2.49-2.45 (m, 2 H), 2.37 (s, 3 H),
.54-1.46 (m, 4 H), 1.35 (t, J ) 4.3 Hz, 3 H), 1.24 (t, J ) 4.3
Ethyl 3-(2-Methoxy-1-methoxymethylethylamino)pro-
1
1
prionate. The compound was prepared using method F:
H
Hz, 3 H), 1.24-1.18 (m, 4 H), 0.86 (t, J ) 4.3 Hz, 6 H); GC-
NMR (300 MHz, CDCl ) δ 4.16 (q, J ) 7.1 Hz, 2 H), 3.71-3.55
3
+
MS (EI) m/z 526 (M ). Anal. (C22
-Chloro-1-(1-propylbutyl)-7-methyl-2,3-dihydro-1H-
1,6]naphthyridin-4-one (7d). 7d was prepared using method
33 3 2 7
H F N O S) C, H, N.
(
m, 5 H), 3.37 (s, 6 H), 3.22 (t, J ) 6.2 Hz, 2 H), 2.67 (t, J )
5
6.2 Hz, 2 H), 1.27 (t, J ) 7.1 Hz, 3 H); LC-MS (APCI) m/z
+
[
220.0 (MH ).
C. This material was identical to the material prepared
according to Scheme 1.
Ethyl 4-[(2-Ethoxycarbonylethyl)(2-methoxy-1-meth-
oxymethylethyl)amino]-6-methyl-2-trifluoromethane-
Ethyl 2-Chloro-4-[(2-ethoxycarbonylethyl)(1-propyl-
sulfonyloxynicotinate (14f). 14f was prepared using method
1
butyl)amino]-6-methylnicotinate (16d). 16d was prepared
A: H NMR (300 MHz, CDCl
3
) δ 6.70 (s, 1 H), 4.36 (q, J ) 7.1
1
using method B: H NMR (300 MHz, CDCl
3
) δ 6.49 (s, 1 H),
Hz, 2 H), 4.13 (q, J ) 7.1 Hz, 2 H), 3.87-3.80 (m, 1H), 3.63-
3.48 (m, 6 H), 3.28 (s, 6 H), 2.48 (t, J ) 7.7 Hz, 2 H), 2.40 (s,
3 H), 1.37 (t, J ) 7.1 Hz, 3 H), 1.24 (t, J ) 7.1 Hz, 3 H); LC-
4
.32 (q, J ) 4.3 Hz, 2 H), 4.12 (q, J ) 4.3 Hz, 2 H), 3.54 (p, J
)
4.2 Hz, 1 H), 3.44-3.40 (m, 2 H), 2.48-2.45 (m, 2 H), 2.40
+
(
s, 3 H), 1.52-1.45 (m, 4 H), 1.36 (t, J ) 4.2 Hz, 3 H), 1.25 (t,
MS (APCI) m/z 531.0 (MH ).

4
108 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 12
Dyck et al.
5
-Chloro-1-(2-methoxy-1-methoxymethylethyl)-7-meth-
J ) 4.8 Hz, 3 H), 2.81 (s, 3 H), 1.67-1.59 (m, 4 H), 1.34-1.30
yl-2,3-dihydro-1H-[1,6]naphthyridin-4-one (7f). 7f was
(m, 4 H), 0.93 (t, J ) 6.0 Hz, 6 H); LC-MS (APCI) m/z 409.2
1
+
prepared using method C: H NMR (300 MHz, CDCl
3
) δ 6.51
(MH ). Anal. (C24
H
32
4
N O
2
‚2HCl‚0.5H
2
O) C, H, N.
(
6
s, 1 H), 4.27 (p, J ) 6.1 Hz, 1 H), 3.66-3.56 (m, 6 H), 3.35 (s,
1-(2-Methoxyphenyl)-7-methyl-5-(1-propylbutyl)-1,3,4,5-
H), 2.65 (t, J ) 7.0 Hz, 2 H), 2.38 (s, 3 H); LC-MS (APCI)
tetrahydro-1,5,8-triazaacenaphthylene Hydrotrifluoro-
+
1
m/z 298.9 (MH ). Anal. (C14
H
19ClN
2
O
3
) C, H, N.
-Chloro-1-(2-methoxy-1-methoxymethylethyl)-4-[1-
methoxymeth-(E)-ylidene]-7-methyl-1,2,3,4-tetrahydro-
1,6]naphthyridine (18f). 18f was prepared using method
acetate (19m). 19m was prepared using method E: H NMR
5
3
(300 MHz, CDCl ) δ 7.60 (m, 1 H), 7.30 (m, 1 H), 7.04-7.09
(m, 2 H), 6.75 (s, 1 H), 6.11 (s, 1 H), 3.83 (s, 3 H), 3.35 (dd, J
) 5.4, 6.0 Hz, 2 H), 3.01 (dd, J ) 5.4, 6.0 Hz, 2 H), 2.51 (s, 3
[
1
D: H NMR (300 MHz, CDCl
3
) δ 6.90 (s, 1 H), 6.35 (s, 1 H),
.16 (p, J ) 6.1 Hz, 1 H), 3.71 (s, 3 H), 3.62-3.51 (m, 4 H),
.36-3.32 (m, 8 H), 2.48 (t, J ) 6.3 Hz, 2 H), 2.33 (s, 3 H);
H), 1.25-1.80 (m, 8 H), 0.92 (t, J ) 7.5 Hz, 6 H); LC-MS
+
4
3
(APCI) m/z 378.0 (MH ). Anal. (C24
H
31
N
3
O‚0.13C
2
HF
3 2
O ‚
2
0.13H O) C, H, N.
+
LC-MS (APCI) m/z 326.8 (MH ).
1-(4-Methylphenyl)-7-methyl-5-(1-propylbutyl)-1,3,4,5-
1
-(4-Chloro-2-cyanophenyl)-5-(2-methoxy-1-methoxy-
tetrahydro-1,5,8-triazaacenaphthylene (19n). 19n was
1
methylethyl)-7-methyl-1,3,4,5-tetrahydro-1,5,8-triaza-
prepared using method E: H NMR (300 MHz, CDCl
3
) δ 7.57
acenaphthylene (19f). 19f was prepared using method E:
(d, J ) 8.1 Hz, 2 H), 7.19 (d, J ) 8.1 Hz, 2 H), 6.73 (s, 1 H),
6.05 (s, 1 H), 3.78 (m, 1 H), 3.27 (dd, J ) 6.0, 6.0 Hz, 2 H),
2.93 (dd, J ) 6.0, 6.0 Hz, 2 H), 2.48 (s, 3 H), 2.30 (s, 3 H),
1
3
H NMR (300 MHz, CDCl ) δ 7.88 (d, J ) 8.7 Hz, 1 H), 7.73
(
1
d, J ) 2.4 Hz, 1 H), 7.68-7.64 (m, 1 H), 6.90 (s, 1 H), 6.22 (s,
H), 4.22-4.17 (m, 1 H), 3.72-3.63 (m, 4 H), 3.57 (t, J ) 5.8
1.18-1.64 (m, 8 H), 0.844 (t, J ) 7.2 Hz, 6 H); LC-MS (APCI)
+
Hz, 2 H), 3.37 (s, 6 H), 3.02 (t, J ) 5.8 Hz, 2 H), 2.52 (s, 3 H);
m/z 362.1 (MH ). Anal. (C24
H
31
N
3
‚0.25H
2
O) C, H, N.
+
LC-MS (APCI) m/z 411.1 (MH ). Anal. (C22
H
23ClN
4
2
O ‚H
2
O)
1-(3-Methoxyphenyl)-7-methyl-5-(1-propylbutyl)-1,3,4,5-
C, H, N.
1
tetrahydro-1,5,8-triazaacenaphthylene (19o). 19o was
1
-(2-Chloro-4-methylphenyl)-7-methyl-5-(1-propylbu-
prepared using method E: H NMR (300 MHz, CDCl
3
) δ 7.56-
tyl)-1,3,4,5-tetrahydro-1,5,8-triazaacenaphthylene Hy-
drochloride (19g). 19g was prepared using method E:
NMR (300 MHz, CDCl
7.54 (m, 1 H), 7.36-7.32 (m, 2 H), 6.85 (s, 1 H), 6.77-6.74 (m,
1 H), 6.13 (s, 1 H), 3.85 (s, 3 H), 3.84-3.82 (m, 1 H), 3.32 (t, J
) 6.0 Hz, 2 H), 2.99 (t, J ) 5.1 Hz, 2 H), 2.53 (s, 3 H), 1.63-
1
H
3
) δ 7.46-7.40 (m, 2 H), 7.30-7.26 (m,
1
3
1
H), 6.60 (s, 1 H), 6.14 (s, 1 H), 4.00-3.88 (m, 1 H), 3.51-
.47 (m, 2 H), 3.07-3.03 (m, 2 H), 2.81 (s, 3 H), 2.44 (s, 3 H),
1.30 (m, 8 H), 0.91 (t, J ) 7.2 Hz, 6 H); LC-MS (APCI) m/z
+
378.0 (MH ). Anal. (C24
31 3 2
H N O‚0.2H O) C, H, N.
.75-1.54 (m, 4 H), 1.39-1.27 (m, 4 H), 0.93 (t, J ) 7.2 Hz, 6
1-(3-Isopropoxypropyl)-7-methyl-5-(1-propylbutyl)-
+
H); LC-MS (APCI) m/z 396.2 (MH ). Anal. (C24
3
H30ClN ‚2HCl‚
1,3,4,5-tetrahydro-1,5,8-triazaacenaphthylene (19p). 19p
1
1
.5H
-(2,4-Dichlorophenyl)-7-methyl-5-(1-propylbutyl)-
,3,4,5-tetrahydro-1,5,8-triazaacenaphthylene Hydro-
2
O) C, H, N.
was prepared using method E: H NMR (300 MHz, CDCl ) δ
3
1
6.48 (s, 1 H), 6.02 (s, 1 H), 4.23 (t, J ) 7.2 Hz, 2 H), 3.85-3.75
(m, 1 H), 3.56-3.52 (m, 1 H), 3.42 (t, J ) 6.3 Hz, 2 H), 3.27 (t,
J ) 6.0 Hz, 2 H), 2.92 (t, J ) 5.4 Hz, 2 H), 2.52 (s, 3 H), 2.15-
2.04 (m, 2 H), 1.60-1.23 (m, 8 H), 1.16 (d, J ) 6.3 Hz, 6 H),
1
1
chloride (19h). 19h was prepared using method E: H NMR
(
3
300 MHz, CDCl ) δ 7.56 (d, J ) 8.7 Hz, 1 H), 7.53 (d, J ) 2.1
+
Hz, 1 H), 7.33 (dd, J ) 9, 2.4 Hz, 1 H), 6.67 (s, 1 H), 6.12 (s, 1
H), 3.90-3.78 (m, 1 H), 3.33 (t, J ) 6.3 Hz, 2 H), 2.99 (t, J )
0.89 (t, J ) 7.2 Hz, 6 H); LC-MS (APCI) m/z 372.1 (MH ).
Anal. (C23
37 3 2
H N O‚0.2H O) C, H, N.
5
.7 Hz, 2 H), 2.47 (s, 3 H), 1.76-1.42 (m, 8 H), 0.93 (t, J ) 7.5
1-(4-Chloro-2-methylbenzyl)-7-methyl-5-(1-propylbutyl)-
+
Hz, 6 H); LC-MS (APCI) m/z 416.0 (MH ). Anal. (C23
2 3
H27Cl N ‚
1,3,4,5-tetrahydro-1,5,8-triazaacenaphthylene (19q). 19q
1
0
.5H
-(2-Chloro-4-cyanophenyl)-7-methyl-5-(1-propylbutyl)-
,3,4,5-tetrahydro-1,5,8-triazaacenaphthylene Hydro-
2
O) C, H, N.
was prepared using method E: H NMR (300 MHz, CDCl ) δ
3
1
7.17 (m,1 H), 7.09 (dd, J ) 8.1, 2.1 Hz, 1 H), 6.89 (d, J ) 8.1
Hz, 1 H), 6.27 (s, 1 H), 6.08 (s, 1 H), 5.32 (br s, 2 H), 3.82 (m,
1 H), 3.30 (dd, J ) 6.0, 6.0 Hz, 2 H), 2.91 (dd, J ) 6.0, 6.0 Hz,
1
1
chloride (19i). 19i was prepared using method E: H NMR
(
300 MHz, CDCl
.0, 1.4 Hz, 1 H), 7.67 (d, J ) 6.4 Hz, 1 H), 6.61 (s, 1 H), 6.19
s, 1 H), 3.95-3.93 (m, 1 H), 3.35 (t, J ) 5.0 Hz, 2 H), 3.05 (t,
3
) δ 7.84 (d, J ) 1.4 Hz, 1 H), 7.74 (dd, J )
2 H), 2.55 (s, 3 H), 2.30 (s, 3 H), 1.26-1.80 (m, 8 H), 0.91 (t, J
+
6
) 7.5 Hz, 6 H); LC-MS (APCI) m/z 410.1 (MH ). Anal. (C25
32
H -
(
3 2
ClN ‚0.33H O) C, H, N.
J ) 5.0 Hz, 2 H), 2.75 (s, 3 H), 1.69-1.56 (m, 4 H), 1.34-1.27
5-(1-Ethylpropyl)-7-methyl-1-(2-methyl-4-trifluo-
(
(
m, 4 H), 0.92 (t, J ) 5.9 Hz, 6 H); LC-MS (APCI) m/z 407.1
romethoxyphenyl)-1,3,4,5-tetrahydro-1,5,8-triazaacenaph-
+
MH ). Anal. (C24
1
H
27ClN
4
‚HCl‚1.67H
2
O) C, H, N.
thylene Hydrochloride (19r). 19r was prepared using
1
-(4-Methoxy-2-methylphenyl)-7-methyl-5-(1-propyl-
method E: H NMR (300 MHz, CDCl
3
) δ 7.35 (d, J ) 8.5 Hz,
butyl)-1,3,4,5-tetrahydro-1,5,8-triazaacenaphthylene Hy-
1 H), 7.22 (s, 1 H), 7.17 (d, J ) 8.3 Hz, 1 H), 6.54 (s, 1 H), 6.18
(s, 1 H), 3.78-3.74 (m, 1 H), 3.49 (t, J ) 5.8 Hz, 2 H), 3.05 (t,
J ) 5.8 Hz, 2 H), 2.76 (s, 3 H), 2.17 (s, 3 H), 1.76-1.64 (m, 4
dromethanesulfonate (19j). 19j was prepared using method
1
E: H NMR (300 MHz, CDCl
3
) δ 7.25 (d, J ) 7.5 Hz, 1 H),
+
6
1
2
1
.92-6.86 (m, 2 H), 6.54 (s, 1 H), 6.16 (s, 1 H), 4.00-3.88 (m,
H), 0.92 (t, J ) 7.2 Hz, 6 H); LC-MS (APCI) m/z 418.1 (MH ).
H), 3.87 (s, 3 H), 3.50-3.45 (m, 2 H), 3.06-3.03 (m, 2 H),
.74 (s, 3 H), 2.47 (s, 3 H), 2.10 (s, 3 H), 1.74-1.53 (m, 4 H),
26 3 3 2
Anal. (C23H F N O‚0.25HCl‚2H O) C, H, N.
1-(4-Chloro-2,6-dimethylphenyl)-5-(1-methoxymethyl-
.40-1.26 (m, 4 H), 0.93 (t, J ) 6.0 Hz, 6 H); LC-MS (APCI)
propyl)-7-methyl-1,3,4,5-tetrahydro-1,5,8-triazaacenaph-
+
m/z 392.2 (MH ). Anal. (C25
1
H
33
4
N O‚CH
4
SO
3
) C, H, N.
thylene Hydrochloride (19s). 19s was prepared using
1
-(2-Chloro-4-methanesulfonylphenyl)-7-methyl-5-(1-
method E: H NMR (300 MHz, CDCl
3
) δ 7.18 (s, 2 H), 6.42 (s,
propylbutyl)-1,3,4,5-tetrahydro-1,5,8-triazaacenaphthyl-
1 H), 6.21 (s, 1 H), 4.08-4.00 (m, 1 H), 3.61-3.53 (m, 4 H),
3.35 (s, 3 H), 3.08-3.04 (m, 2 H), 2.75 (s, 3 H), 2.02 (d, J ) 3.9
ene Hydrochloride (19k). 19k was prepared using method
1
E: H NMR (300 MHz, CDCl
3
) δ 8.14 (d, J ) 1.9 Hz, 1 H),
Hz, 6 H), 1.77-1.71 (m, 2 H), 0.99 (t, J ) 7.2 Hz, 3 H); LC-
+
8
(
2
1
.00 (dd, J ) 8.5, 1.5 Hz, 1 H), 7.69 (d, J ) 8.5 Hz, 1 H), 6.69
MS (APCI) m/z 398.1 (MH ). Anal. (C23
3 2
H28ClN O‚HCl‚1.2H O)
s, 1 H), 6.17 (s, 1 H), 3.94-3.92 (m, 1 H), 3.50 (t, J ) 6.1 Hz,
C, H, N.
H), 3.20 (s, 3 H), 3.06 (t, J ) 5.7 Hz, 2 H), 2.69 (s, 3 H),
1-(2,4-Dichlorophenyl)-4-methyl-6-(1-propylbutyl)-
1,6,7,8-tetrahydro-1,2,3,6-tetraazaacenaphthylene (20a).
Ketone 7d (308 mg, 1.04 mmol) and 2,4-dichlorophenylhydra-
zine hydrochloride (231 mg, 1.08 mmol) were dissolved in
ethanol (5 mL) and heated to reflux for 18 h. The mixture was
cooled, poured into aqueous sodium bicarbonate (20 mL), and
extracted three times with ethyl acetate (10 mL). The com-
bined extracts were dried (MgSO ) and concentrated, and the
4
residue was purified by flash chromatography (elution with
1% methanol and 0.5% aqueous ammonia in DCM) to afford
.69-1.57 (m, 4 H), 1.34-1.27 (m, 4 H), 0.92 (t, J ) 7.2 Hz, 6
+
H); LC-MS (APCI) m/z 460.1 (MH ). Anal. (C24
H
30ClN
3
O
2
S‚
HCl‚1.5H
-(2,6-Dimethoxypyridin-3-yl)-7-methyl-5-(1-propylbu-
tyl)-1,3,4,5-tetrahydro-1,5,8-triazaacenaphthylene Hy-
2
O) C, H, N.
1
1
drochloride (19l). 19l was prepared using method E:
NMR (300 MHz, CDCl ) δ 7.60 (d, J ) 6.4 Hz, 1 H), 6.56 (s, 1
H), 6.45 (d, J ) 6.2 Hz, 1 H), 6.13 (s, 1 H), 4.00 (s, 3 H), 3.98
s, 3 H), 3.98-3,88 (m, 1 H), 3.47 (t, J ) 5.0 Hz, 2 H), 3.03 (t,
H
3
(

CRF
1
Antagonists
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 12 4109
1
95 mg (45%) of 20a as a yellow oil: 1H NMR (300 MHz,
tube and centrifuged at 1000 rpm for 4 min. The supernatant
is discarded, and the pellet was 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
CDCl ) δ 7.57 (d, J ) 2.1 Hz, 1 H), 7.52 (d, J ) 8.4 Hz, 1 H),
.36 (dd, J ) 8.4, 2.4 Hz, 1 H), 6.08 (s, 1 H), 3.87-3.78 (m, 1
3
7
H), 3.51 (dd, J ) 6.3, 6.3 Hz, 2 H), 3.15 (dd, J ) 6.5, 6.5 Hz,
2
0
H), 2.51 (s, 3 H), 1.72-1.50 (m, 4 H), 1.42-1.25 (m, 4 H),
+
.93 (t, 7.2 Hz, 6 H); LC-MS (APCI) m/z 417.0 (MH ). Anal.
custom media mixture, Irvine Scientific, CA; 1.83 g of Na
CO /L; 2.4 g of HEPES/L; 2.0 g/L BSA; 10.0 mg/L transferrin;
2
-
(
C
22
H
26Cl
2
N
4
‚0.25CH
2
Cl
2
) C, H, N. A second fraction afforded
3
8
2 mg (19%) of 22a, which was identical to the material
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.
For the assay of antagonists, cells are washed once with
BBM-T, test samples are added in various concentrations (1
µM to 1 pM) with 0.5 nM r/hCRF in 200 µL of 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.
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 compound or vehicle
prepared below. The two regioisomers were identified by the
characteristic downfield shift of the pyridine hydrogen atoms
in the H NMR spectra of 2-substituted pyrazolo[3,4-b]-
pyridines relative to their 1-substituted isomers.
1
2
5
2
-(2,4-Dichlorophenyl)-4-methyl-6-(1-propylbutyl)-
2
,6,7,8-tetrahydro-1,2,3,6-tetraazaacenaphthylene (22a).
Pyridine 7d (503 mg, 1.63 mmol) and 2,4-dichlorophenylhy-
drazine hydrochloride (390 mg, 1.83 mmol) were dissolved in
methanol (5 mL) and heated at 60 °C for 18 h. The mixture
was concentrated under vacuum, the residue was diluted with
diphenyl ether (2 mL), and the mixture was placed in a
preheated oil bath at 200 °C. After 2 h at this temperature,
the mixture was cooled, diluted with DCM, and purified by
flash chromatography (elution with 3% methanol and 0.5%
aqueous ammonia in DCM) to afford 439 mg (65%) of 22a as
1
a tan solid: H NMR (300 MHz, CDCl
3
) δ 7.56 (d, J ) 9.0 Hz,
H), 7.55 (d, J ) 1.8 Hz, 1 H), 7.40 (dd, J ) 8.4, 2.1 Hz, 1 H),
.92 (s, 1 H), 3.92-3.82 (m, 1 H), 3.57 (dd, J ) 6.3, 6.3 Hz, 2
(
10 mL/kg 5% D-mannitol (w/v) in water) 60 min prior to the
1
5
initiation of the stressor. Restraint stress was evoked by
allowing the mice to enter a 50 mL plastic conical tube (tip
removed to allow free flow of air) and to be 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 was analyzed using a one-way ANOVA with post
hoc Fischer’s test for group differences.
H), 3.09 (dd, J ) 6.3, 6.3 Hz, 2 H), 2.66 (s, 3 H), 1.72-1.52 (m,
H), 1.40-1.25 (m, 4 H), 0.93 (t, 7.2 Hz, 6 H); LC-MS (APCI)
4
+
2 4 2
m/z 417.0 (MH ). Anal. (C22H26Cl N ‚0.33H O) C, H, N.
This material was converted to the methanesulfonic acid
1
salt as described in General Methods: H NMR (300 MHz,
CDCl
8
)
3
Hz, 6 H); LC-MS (APCI) m/z 417.1 (MH ). Anal. (C22
Cl
3
) δ 7.61 (s, 1 H), 7.58 (d, J ) 8.3 Hz, 1 H), 7.48 (dd, J )
.6, 1.5 Hz, 1 H), 6.20 (s, 1 H), 4.00-3.85 (m, 1 H), 3.69 (t, J
6.7 Hz, 2 H), 3.22 (t, J ) 6.7 Hz, 2 H), 2.79 (s, 3 H), 2.46 (s,
H), 1.72-1.64 (m, 4 H), 1.36-1.29 (m, 4 H), 0.95 (t, J ) 7.3
+
26
H -
2
N
4
‚CH
4 3
SO ) C, H, N.
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 CRF receptor in heterolo-
1
gously expressed cell membranes. Equilibrium binding of
unlabeled ligands was measured in duplicate by inhibition of
2
6
Rat Pharmacokinetics. The pharmacokinetics and blood-
brain barrier (BBB) penetrations were determined in male
Sprague Dawley rats following an intravenous (iv, N ) 3/time
point) and oral (po, N ) 3/time point) dose. The dosing solution
was prepared in purified water and filtered through a 0.2 µm
Nylon filter before administration (2 mL/kg) via tail vain (iv)
or a gavage (po). Terminal blood and brain tissue samples were
taken at predetermined time for composite sampling. All
plasma and tissue samples were flash-frozen in liquid nitrogen
within 10 min of sampling and stored in -70 °C or below until
analysis. The bioanalytical method applied for the measure-
ment of test articles in plasma along with added internal
standard consisted of precipitation with 200 µL of acetonitrile
from 50 µL of plasma, centrifugation, recovery of the super-
natant, drying down in a vacuum, and then reconstitution in
acetonitrile-water solutions before introduction into an LC-
MS/MS system for analysis. The lower limit of quantification
1
25
-
radioligand binding ([ I]sauvagine) to LtK cells expressing
the human CRF receptor. Thirty microliters of assay buffer
DPBS, 1.5 mM KH PO , 8.1 mM Na HPO , 2.7 mM KCl, 138
mM NaCl) supplemented with 10 mM MgCl , 2 mM ethylene
1
(
2
4
2
4
2
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-CRF receptor cell membranes were sequentially added to
1
low-protein-binding 96-well plates (Corning no. 3605). The
final concentration of radioligand was approximately 90 pM
125
for [ I]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 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 moni-
tored using a Packard Cobra II γ counter (78% efficiency) and
analyzed using the nonlinear curve-fitting algorithm software
Prism (GraphPad Inc., CA).
(
LLOQ) for the analytical methods was 5 ng/mL of test article
in plasma. The bioanalytical method applied for the measure-
ment of test articles in brain tissue along with added internal
standard consisted of homogenization of half of the brain tissue
(
longitude cut) in 2 mL of acetonitrile/water (50:50), centrifu-
gation, and recovery of the supernatant before introduction
into an LC-MS/MS system for analysis. The lower limit of
quantification for the analytical methods was 5 ng/g of test
article in brain tissue. All pharmacokinetic parameters were
calculated from a noncompartmental model in WinNonlin
program. Brain-to-plasma ratio was obtained by comparing
brain AUC to plasma AUC.
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,
1
00 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
Supporting Information Available: Table of elemental
analysis data for reported compounds. This material is avail-
able free of charge via the Internet at http://pubs.acs.org.

4
110 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 12
Note Added after ASAP Publication. This manu-
Dyck et al.
(14) 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 (CRH)
receptor antagonist: suppression of pituitary ACTH release and
peripheral inflammation. Endrocrinology 1996, 137, 5747-
script was released ASAP on May 6, 2005, with an
incomplete author byline. The correct version was
posted on May 13, 2005.
5
750.
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