Synthesis, structure-activity relationships, and in vivo properties of 3,4-dihydro-1H-pyrido[2,3-b]pyrazin-2-ones as corticotropin-releasing factor-1 receptor antagonists

Article abstract of DOI:10.1021/jm049737f

Corticotropin releasing factor (CRF) is the primary regulator of the hypothalamus-pituitary-adrenal (HPA) axis, coordinating the endocrine, behavioral, and autonomic responses to stress. It has been postulated that small molecules that can antagonize the

Full text of DOI:10.1021/jm049737f

J. Med. Chem. 2004, 47, 5783-5790
5783
Synthesis, Structure-Activity Relationships, and in Vivo Properties of
3,4-Dihydro-1H-pyrido[2,3-b]pyrazin-2-ones as Corticotropin-Releasing Factor-1
Receptor Antagonists
Carolyn D. Dzierba,*, Amy G. Takvorian,† Maria Rafalski,† Padmaja Kasireddy-Polam,† Harvey Wong,‡
Thaddeus F. Molski,§ Ge Zhang,§ Yu-Wen Li,§ Snjezana Lelas,§ Yong Peng,§ John F. McElroy,§ Robert C. Zaczek,§
Rebecca A. Taub,§ Andrew P. Combs,† Paul J. Gilligan, and George L. Trainor
Discovery Chemistry, Metabolism and Pharmacokinetics Discovery, Neuroscience Biology, Bristol-Myers Squibb
Pharmaceutical Research Institute, 5 Research Parkway, Wallingford, Connecticut 06492
Received April 6, 2004
Corticotropin releasing factor (CRF) is the primary regulator of the hypothalamus-pituitary-
adrenal (HPA) axis, coordinating the endocrine, behavioral, and autonomic responses to stress.
It has been postulated that small molecules that can antagonize the binding of CRF1 to its
receptor may serve as a treatment for anxiety-related and/or affective disorders. Members within
a series of 3,4-dihydro-1H-pyrido[2,3-b]pyrazin-2-ones, exemplified by compound 2 (IC50 ) 0.70
nM), were found to be very potent antagonists of CRF1. Compound 8w showed high CRF1
receptor binding affinity and was examined further in vivo. The compound was efficacious in
a defensive withdrawal model of anxiety in rats and had a long half-life and reasonable oral
bioavailability in dog pharmacokinetic studies.
Introduction
Corticotropin releasing factor (CRF), a 41 amino acid
peptide first isolated in 1981,1 is the primary regulator
of the hypothalamus-pituitary-adrenal (HPA) axis, co-
ordinating the endocrine, behavioral and autonomic
responses to stress.2-5 There are two known receptor
subtypes of CRF, CRF1, and CRF2, the latter having
three splice variants, R, â, and γ.6 Of the two subtypes,
the role of CRF1 has been more extensively studied, and
numerous small molecule antagonists of CRF1 have
Figure 1.
been reported.3,6
There is both preclinical and clinical evidence to
suggest that CRF1 plays a role in anxiety-related
diseases.4,7-9 It has been shown that intracerebroven-
tricular (icv) injection of CRF in rats produces behav-
ioral and physiological changes that mimic the effects
of stress.9 Moreover, icv administration of antagonists
of CRF, such as R-helical ovine CRF9-41, blocks the
effects of exogenously administered CRF and the effects
of a variety of environmental sources of stress.7,8,10-12
It has also been postulated that hypersecretion of
CRF1 may be involved in affective disorders including
depression and anxiety.13-20 Patients suffering from
Figure 2.
depression have elevated levels of CRF in the cere-
bral spinal fluid (CSF). These levels are reduced
1) in depressed patients demonstrated reductions in
when the depression symptoms have been successfully
treated.17,21-23 Additionally, a small molecule CRF1
antagonist, CP-154,526 (Figure 1), has been shown to
block the behavioral effects of CRF1 in rat models of
anxiety.24-26 Moreover, an open-labeled clinical trial
studying the effects of CRF1 antagonist R121919 (Figure
depression and anxiety scores.27 This evidence strongly
suggests that small molecule antagonists of CRF1 would
serve as a treatment for anxiety-related disorders and/
or depression.
Design of a new series of small molecule CRF1
antagonists began with examination of the SAR of the
known monocyclic 2-anilinopyrimidine CRF1 antagonist
1 (Figure 2),28 which contains features found in several
known classes of mono- and bicyclic CRF antagonists.3,29
These features include (1) an sp2 nitrogen that may
accept a hydrogen bond from a residue on the receptor,
(2) an aryl ring that is minimally substituted in the
* Corresponding author: Tel: 203-677-7661. Fax: 203-677-7702,
E-mail carolyn.dzierba@bms.com.
Discovery Chemistry.
‡ Metabolism and Pharmacokinetics Discovery.
§ Neuroscience Biology.
† Present address: Discovery Chemistry, Incyte Corporation,
Wilmington, DE 19880.
10.1021/jm049737f CCC: $27.50 © 2004 American Chemical Society
Published on Web 09/29/2004

5784 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 23
Dzierba1 et al.
Table 1. SAR Studies of the 8-Amino Group of the
3,4-Dihydro-1H-pyrido[2,3-b]pyrazin-2-ones
Scheme 1. Synthesis of
3,4-Dihydro-1H-pyrido[2,3-b]pyrazin-2-onesa
mean rCRF1
IC50a (nM)
compd
R1
R2
R3
R4
8ab
8bb
8cb
8d
8eb
8fb
8gb
8h
8i
Bu
Et
Bu
Bu
Et
H
OMe
OMe
OMe
Cl
H
H
H
H
H
H
F
0.30 ( 0.05
>1000
H
>1000
Et
H
H
Et
Et
Et
Pr
Pr
0.49 ( 0.07
198 ( 23
68 ( 2
0.50 ( 0.07
1.5 ( 0.1
2.2 ( 0.1
1.2 ( 0.2
1.8 ( 0.3
3-pentyl
Cl
MeOCH2(Et)CH
Cl
Bu
OMe
OMe
CN
iPr
F
Bu
H
H
H
8j
Pr
CN
CN
8k
cyc-BuCH2-
a IC50 values (n ) 2, 3, or 4) ( SEM. b Trifluoroacetic acid salt.
a Reagents and conditions: (a) Cyclohexylamine, MeOH, room
temperature, 40 min, then POCl3, 20 °C, 80 h, 81%; (b) R1R2NH,
DIEA, CH3CN, reflux, 3-5 h; (c) POCl3, reflux, 3-5 h, 65-90%;
(d) R3-aniline (X ) CH) or R3-3-aminopyridine (X ) N), 130 °C,
6-12 h, 40-90% or R3-aniline (X ) CH), NaH, DMA, 0 °C, then
5, 100 °C, 10 h, 40-75%; (e) NaH, ethyl iodoacetate, DMF, 0 °C
f room temperature, 2 h, 70-95%; (f) Fe°, HCl, EtOH, 70 °C, 12-
24 h, 80-95%.
group with Fe° and acetic acid in refluxing ethanol also
affected cyclization to form the desired six-membered
ring of 8.
Biology. Our screening strategy began with evaluat-
ing the compounds in a receptor binding assay, by
measuring displacement of [125I]Tyr-o-CRF from rat
frontal cortex homogenate. Compounds with high bind-
ing affinities were evaluated in the defensive with-
drawal model of anxiety in rats. Compounds that
showed behavioral efficacy were examined in an ex vivo
binding assay to determine receptor occupancy and in
vivo plasma levels after oral dosing. Lead compounds
were then taken into pharmacokinetic (PK) studies to
evaluate their in vivo properties.
Structure-Activity Relationships. We set out to
examine the structure-activity relationships for this
series by evaluating the compounds in a CRF1 receptor
binding assay. We initially examined the effect of
varying the 8-amino group on binding potency (Table
1). It was found that a dialkylamino group attached to
the upper portion of the core led to better potency than
a monoalkylamino group. For example, N-ethylamino-
substituted (8b) or N-butylamino-substituted (8c) com-
pounds were both inactive (IC50 > 1 µM), while the
corresponding N-butyl-N-ethylamino compound (8a)
was highly potent (IC50 ) 0.30 nM). Branching some-
what improved the binding affinity of the monosubsti-
tuted amino analogues (compare 8e, IC50 ) 198 nM and
8f, IC50 ) 68 nM). Although these branched analogues
showed modest binding affinity, they were still 2 orders
of magnitude less potent than the corresponding disub-
stituted amino analogue 8d (IC50 ) 0.49 nM). Branching
on one of the amino substituents of a disubstituted
amino analogue did not have a detrimental effect on
binding affinity (compare 8g, IC50 ) 0.50 nM and 8h,
IC50 ) 1.5 nM). Other small aliphatic groups were
tolerated in this position as seen by comparing 8i (IC50
) 2.2 nM) with 8j and 8k, which bind to CRF1 with IC50
values of 1.3 and 1.8 nM, respectively.
ortho- and para-positions, and (3) a lipophilic group
attached to the core para to the sp2 nitrogen. SAR
studies of this series suggest that the ethyl group on
the lower anilinic nitrogen is necessary to achieve good
binding affinity to the CRF1 receptor. By tying the ethyl
group back into a ring, we hoped to reduce the flexibility
of both the ethyl and heteroaryl groups, thereby con-
straining these groups into an optimal conformation and
increase binding affinity. Thus, a series of 3,4-dihydro-
1H-pyrido[2,3-b]pyrazin-2-ones, exemplified by com-
pound 2 (Figure 2), was prepared. These compounds
were then examined for their ability to act as CRF1
receptor antagonists.
Results and Discussion
Chemistry. Synthesis of the desired pyridopyrazi-
nones started from commercially available 4-hydroxy-
6-methyl-3-nitro-2-pyridone, 3 (Scheme 1).30 The 4-
hydroxyl was selectively converted into a chloride
by formation of the cyclohexylamine salt followed by
treatment with POCl3 to afford chloropyridine 4. The
4-amino side chain was installed by displacement of
the chloride with a primary or secondary amine. The
resulting 2-pyridone was converted into the 2-chloro-
pyridine 5 by treatment with POCl3. The aryl ring was
installed by heating 5 with an aniline to give anilinopy-
ridine 6. For less reactive anilines, including those with
electron-withdrawing groups in the 4-position (para to
the nitrogen), it was necessary to initially deprotonate
the aniline with NaH in N,N-dimethylacetamide (DMA)
and then heat to 100 °C with 2-chloropyridine 5 to affect
conversion. Alkylation of the anilinic nitrogen with ethyl
iodoacetate gave nitro ester 7. Reduction of the nitro
To determine the effect of lipophilicity changes on
binding affinity, a series of analogues incorporating

CRF1 Receptor Antagonists
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 23 5785
Table 2. SAR Studies of the 8-Amino Group of the
3,4-Dihydro-1H-pyrido[2,3-b]pyrazin-2-ones
Table 3. SAR Studies of the 4-Aryl Group of the
3,4-Dihydro-1H-pyrido[2,3-b]pyrazin-2-ones
mean rCRF1
IC50a (nM)
mean rCRF1
compd
R1
R2
R3 R4
compd
R1
Br
X
R2
R3
R4
IC50a (nM)
8lb
Bu
Et
Pr
Cl i-Pr 0.91 ( 0.31
2
8s
CH
CH
CH
CH
CH
CH
CH
N
i-Pr
H
i-Pr
Cl
OMe
OMe
OMe
OMe
OMe
CN
H
H
H
H
H
F
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
0.70 ( 0.20
7.1 ( 0.4
49 ( 12
0.49 ( 0.07
0.30 ( 0.05
0.50 ( 0.07
0.92 ( 0.29
4.2 ( 0.8
0.82 ( 0.09
2.2 ( 0.1
10.0 ( 0.1
3.3 ( 0.3
8mb MeOCH2CH2
Cl i-Pr
2.6 ( 0.3
18 ( 3
4.1 ( 1.3
92 ( 21
Br
H
8nb MeOCH2CH2
MeOCH2CH2 Cl i-Pr
8tb
8d
8ab
8gb
8u
8vc
8w
8i
8o
8p
(rac)furanylCH2
NCCH2CH2CH2
Et
Et
Br i-Pr
Me OMe
Cl
Cl
8qb MeSO2CH2CH2CH2 Et
Br i-Pr 305 ( 92
Br i-Pr 126 ( 12
a IC50 values (n ) 2, 3 or 4) ( SEM. b Trifluoroacetic acid salt.
Cl
8r
morpholino
Me
Me
OMe
Cl
N
heteroatoms into the 8-amino group was examined
(Table 2). Addition of a methyl ether onto the amino
side chain (compound 8m) led to a small decrease in
binding affinity (IC50 ) 2.6 nM) versus the correspond-
ing N-butyl-N-ethylamino analogue 8l (IC50 ) 0.91 nM).
Addition of a second methyl ether (compound 8n) led
to an even greater decrease in affinity (IC50 ) 18 nM).
Constraining the ether substituent into a furanyl ring
was tolerated (8o, IC50 ) 4.1 nM). Incorporation of polar
groups such as cyano (8p) or methanesulfonyl (8q) led
to a significant loss in potency (IC50 ) 92 and 305 nM,
respectively). Constraining the heteroatoms into a ring
such as morpholine (8r, IC50 ) 126 nM) did not improve
the binding affinity. From examination of the SAR it
was determined that an 8-amino group disubstituted
with small lipophilic substituents is preferred to obtain
good CRF1 binding affinity.
Next we turned our attention to SAR studies around
the lower aryl ring, holding the 8-amino group constant
as N-butyl-N-ethylamino (Table 3). With many CRF
antagonists, for example in the pyrimidine series (com-
pound 1, Figure 2), it has been observed that both ortho-
and para-substitution is required for good binding
affinity. The group in the ortho-position helps to induce
a twist between the core ring system and lower aryl
group, while the substituent in the para-position helps
to increase binding affinity. This is exemplified by
comparison of compounds 2, 8s, and 8t. The 2-bromo-
4-isopropylphenyl analogue 2 has excellent potency,
with a binding affinity of 0.70 nM. Removal of the
4-isopropyl group (8s) led to a 10-fold loss in potency,
while removal of the 2-bromo substituent (8t) led to a
70-fold loss in potency. This suggests that both the
ortho- and para-substituents are required to obtain good
binding affinity.
CH
CH
CH
8xb
8yb
CN
Cl
Cl
CN
IC50 values (n ) 2, 3, or 4) ( SEM. b Trifluoroacetic acid salt.
a
c Bis-trifluoroacetic acid salt.
analogue 8v (IC50 ) 4.2 nM) was 5-fold less active than
the corresponding phenyl analogue 8u (IC50 ) 0.92 nM).
However, the more electron rich 2,4-dimethoxypyridyl
analogue 8w (IC50 ) 0.82 nM) was of similar potency
to analogue 8u.
Other polar substituents could be incorporated into
the phenyl group but were better tolerated in the para-
position than in the ortho-position. For example, 2-chloro-
4-cyanophenyl analogue 8i (IC50 ) 2.2 nM) was more
potent than the 4-chloro-2-cyanophenyl analogue 8x
(IC50 ) 10.0 nM). Addition of a second ortho-substituent
on the aryl ring was not detrimental to the overall
potency. For example, the 2,6-dichloro-4-cyanophenyl
analogue 8y (IC50 ) 3.3 nM) was essentially equipotent
to analogue 8i. Examination of the SAR for the lower
aryl group suggests that the optimal substitution pat-
tern is 2,4-disubstitution. Small groups are also toler-
ated in the 5- and 6-positions, while slightly larger
groups, such as isopropyl, are tolerated in the 4-position.
Polarity could be incorporated in this region either in
the para-position of the phenyl or as a pyridyl group.
In Vivo Studies. Compound 8w was selected for
further examination in a rat behavioral model based
on its high CRF1 binding affinity. Behavioral efficacy
and efficacious plasma concentrations were determined
using the defensive withdrawal model of anxiety in
rats.31-33 Briefly, rats were placed in a darkened
chamber located in an unfamiliar, brightly lit chamber
(a situation representing a heightened state of anxiety).
The amount of time required by each rat to emerge from
the chamber and explore the open field was measured.
A compound was considered efficacious if the latency
to emerge from the chamber was significantly reduced
relative to vehicle-treated animals.
Lipophilic groups such as isopropyl (compound 2) and
chloro (compound 8d) were preferred in the para-
position (IC50 ) 0.70 and 0.49 nM, respectively). How-
ever, mildly polar substituents, such as methoxy (com-
pound 8a), could also be incorporated into the para-
position without loss in activity (rCRF1 IC50 ) 0.30 nM).
Substitution at the 5-position was limited to small
groups, such as fluoro (8g, IC50 ) 0.50 nM).
Table 4 summarizes the results of the defensive
withdrawal model following oral dosing of compound 8w
at 1, 3, and 10 mg/kg. CRF1 receptor occupancy in the
parietal cortex was determined by ex vivo autoradiog-
raphy.33 The compound was effective at reducing exit
Introduction of a pyridyl group in the lower aryl
position was also examined. 2-Methyl-4-methoxypyridyl

5786 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 23
Dzierba1 et al.
Table 4. Mean Total and Free Plasma Exposures and CRF1
Receptor Occupancies Following Oral Doses of Compound 8w
in the Defensive Withdrawal Model in Rats
pressin V1 (49%, 53%, and 47% inhibition @ 10 µM,
respectively).
oral
mean total mean free
plasma plasma
CRF1
% decrease in
exit latency
Conclusion
dose
receptor
(mg/kg) concn (nM) concna (nM) occupancy (%) (p vs vehicle)
In conclusion, the structure-activity relationships for
this series of 3,4-dihydro-1H-pyrido[2,3-b]pyrazin-2-ones
was established by systematic variation of substituents
on the 8-amino side chain and the 4-aryl ring. A number
of highly potent analogues were identified, supporting
the hypothesis that reducing the conformational flex-
ibility of the system improves the receptor binding
affinity. Compound 8w was efficacious at 10 mg/kg in
a rat behavioral model for situational anxiety and was
found to occupy 59% of the CRF receptors at this dose.
This compound was further examined in dog pharma-
cokinetic studies and found to have a long half-life and
reasonable oral bioavailability in this species. Addition-
ally, 8w was shown to be a selective antagonist of CRF1
against a wide range of other receptors.
1
3.1 ( 1.0 0.05 ( 0.02
21.7 ( 24.6 0.36 ( 0.41
61.5 ( 21.8 1.02 ( 0.36
17 ( 13
23 ( 17
59 ( 19
0
3
14 (0.43)
81 (<0.0001)
10
a Based upon a rat unbound fraction in plasma of 1.66%
determined by equilibrium dialysis.
latency by 81% (relative to vehicle-treated control) at
10 mg/kg, (n ) 8 per dose). This dose provided a mean
CRF1 receptor occupancy of 59% (n ) 5) and a mean
free plasma concentration (plasma free fraction )
1.66%) similar to the in vitro CRF1 IC50 (i.e. 0.82 nM).
A similar correspondence between free plasma concen-
tration, occupancy, and behavioral efficacy has been
reported for the CRF1 antagonist DMP696.33
Compound 8w was examined in PK studies to further
characterize the in vivo properties of this CRF1 antago-
nist. On the basis of the in vitro metabolic properties of
this class of compounds in liver microsomes from
different species, dog was observed to be the species that
most closely correlated with human and was thus
chosen for the following study. Compound 8w was
administered to beagle dogs at an intravenous dose of
1.0 mg/kg (n ) 2) and at an oral dose of 5.0 mg/kg (n )
2). The following pharmacokinetic parameters were
derived noncompartmentally from the study: Cl ) 1.67
( 0.03 L/h/kg, Vss ) 40.6 ( 1.8 L/kg, t1/2 ) 27.6 ( 3.4 h,
F ) 24%. Upon the basis of the pharmacokinetic
parameters, compound 8w appeared to be a moderate/
high clearance compound with a large volume of distri-
bution, a long half-life, and reasonable oral bioavail-
ability in dogs. The long half-life of this compound is
likely due to its large volume of distribution. Although
the systemic clearance of 8w is not optimal, this
compound provides a reasonable starting point for
further optimization.
The antagonist properties of compound 8w were
assessed in a cell-based functional assay by measuring
the ability of the compound to inhibit CRF-stimulated
ACTH secretion in cultured rat pituitary cells.34 Com-
pounds 8w produced a concentration-dependent inhibi-
tion of CRF (0.3 nM)-induced ACTH release from rat
pituitary cells (IC50 of 38.7 ( nM, n ) 3 for CRF) and
completely suppressed CRF-stimulated ACTH secretion
at higher concentrations, indicating that this compound
behaves as an antagonist at pituitary CRF1 receptor.
To test for agonist activity, compound 8w was exposed
alone to pituitary cells. Basal secretion of ACTH from
cells was not altered, indicating a lack of agonist
properties. Compound 8w was also shown to be highly
selective for CRF1 over hCRF2R (hCRF2R IC50 > 10 µM)
and against a broad panel of receptors (CEREP, Celle
L’Evescault, France). At 10 µM no affinity was observed
for monoamine (adrenergic (R1, R2, â1), dopaminergic,
or serotonergic receptors), histaminergic H1, muscarinic,
GABA, oxytocin, CGRP, or opioid receptors. Addition-
ally, no affinity was detected at 10 µM for ion channels
(glutamate (NMDA), calcium type L, or calcium-
activated potassium sites). Only modest affinity was
observed for adenosine A3, canabanoid CB1 and vaso-
Experimental Section
Chemistry. All reactions were performed under nitrogen
with continuous magnetic stirring unless otherwise stated.
Anhydrous solvents from commercial sources were used for
all reactions. All other reagents and solvents were reagent
grade and used as received from commercial sources. Flash
chromatography was performed with E. Merck Kieselgel 60
silica gel (230-400 mesh). All 1H NMR and 13C NMR spectra
were obtained on a Varian Inova spectrometer (operating at
300 MHz) or a Bruker Avance (operating at 400 or 500 MHz),
and the signals are reported in ppm relative to TMS. All high-
resolution mass spectra (HRMS) were obtained on a VG 70-
VSE instrument with NH3 as the carrier gas for chemical
ionization. Compounds were purified using silica gel chroma-
tography (hexanes/ethyl acetate as the elutant) or by reverse
phase high-pressure liquid chromatography (HPLC) (0.1% TFA
in water-0.1% TFA in acetonitrile gradient-20-80% aceto-
nitrile over 30 min). Purity measurements were carried out
using reverse phase HPLC using two different eluting systems.
Method a: 0.1% TFA in water-0.1% TFA in acetonitrile
gradient (0-100% acetonitrile over 40 min) on an Agilent
HP1100 HPLC with a YMC C18 column; Method b: 0.2%
phosphoric acid and 10% methanol in water-0.2% phosphoric
acid and 10% water in methanol gradient (0-100% methanol
over 28 min) on a Shimadzu LC10 HPLC with a Zorbax SB-
C18 column.
4-Chloro-6-methyl-3-nitro-2-pyridone (4). To a 2-L round-
bottomed flask equipped with a magnetic stir bar was added
4-hydroxy-6-methyl-3-nitro-2-pyridone (163.3 g, 0.96 mol) in
MeOH (1.2 L). To the resulting slurry was added cyclohexyl-
amine (135.1 mL, 1.2 mol), and the mixture was stirred for 40
min. During this time, the mixture became homogeneous,
followed by precipitation of a yellow solid. The mixture was
then cooled in an ice bath and filtered. The mother liquor was
concentrated in vacuo (25-30 °C and 25 mmHg), affording a
yellow solid that was combined with the previous precipitate
and dried in a vacuum oven at room temperature. The solids
were added slowly in small portions to a 3-L round-bottomed
flask equipped with a mechanical stirrer, temperature probe,
N2 inlet, and POCl3 (1 L, 10.7 mol). The internal temperature
of the reaction was maintained <20 °C with external cooling.
The reaction was then stirred at room temperature for ∼80 h
and then poured slowly into a 5 gallon bucket containing 1 kg
of ice and 1 L of H2O. Additional ice was added to maintain
the temperature of the solution at room temperature. The
resultant slurry was filtered and the solids were dissolved in
EtOAc (1 L). The solution was dried over MgSO4, filtered, and
concentrated in vacuo (25-30 °C and 25 mmHg), affording
pyridone 4 (146.0 g, 81% yield) as a pale yellow solid. 1H NMR
(300 MHz, DMSO-d6) δ 2.25 (s, 3H), 6.40 (s, 1H), 12.95 (br s,
1H); 13C NMR (300 MHz, DMSO-d6) δ 18.7, 105.0, 137.8, 138.8,

CRF1 Receptor Antagonists
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 23 5787
150.3, 154.7; HRMS Calcd for (C6H5ClN2O3) [M + H]+ 189.0067;
vacuo. The crude yellow oil was purified by silica gel chroma-
tography (90% hexanes/10% ethyl acetate f 75% hexanes/25%
ethyl acetate) to afford 145 mg (81% yield) of 2 as a white solid.
1H NMR (300 MHz, DMSO-d6) δ 0.86 (t, J ) 7.3 Hz, 3H), 1.03
(t, J ) 7.3 Hz, 3H), 1.23 (d, J ) 7.3 Hz, 6H), 1.27 (sextet, J )
7.3 Hz, 2H), 1.38-1.47 (m, 2H), 2.18 (s, 3H), 2.94 (septet, J )
7.3 Hz, 1H), 3.12-3.36 (m, 4H), 3.96-4.14 (m, 1H), 4.26-4.42
(m, 1H), 6.56 (br s, 1H), 7.37 (br d, J ) 7.5 Hz, 1H), 7.42 (br
d, J ) 7.5 Hz, 1H), 7.59 (br s, 1H); 13C NMR (300 MHz, CD3-
OD) δ 13.0, 14.1, 19.0, 21.1, 24.0, 24.0, 30.7, 35.0, 47.1, 51.9,
55.1, 108.7, 111.5, 122.5, 129.4, 130.6, 133.6, 138.2, 143.8,
145.0, 151.7, 153.7, 164.2; HRMS Calcd for (C23H31BrN4O) [M
+ H]+ 459.1759; found 459.1766. HPLC: (a) >99%; (b) 97%.
found 189.0061.
4-(N-Butyl-N-ethylamino)-2-chloro-6-methyl-3-nitro-
pyridine (5a). To a 2-L three-neck round-bottom flask
equipped with a mechanical stirrer, condenser, and N2 inlet
were added 4-chloro-6-methyl-3-nitro-2-pyridone 4 (29.7 g,
157.4 mmol), N-butyl-N-ethylamine (26.1 mL, 191.4 mmol),
CH3CN (800 mL), and DIEA (55 mL, 314.8 mmol). The mixture
was heated at reflux for 3 h. The mixture was cooled to room
temperature and concentrated in vacuo. The mixture was
treated with H2O and extracted with EtOAc (3×). The organic
fractions were combined, washed with saturated aqueous
NaCl, and concentrated to afford the aminopyridine as a yellow
powder (30.7 g, 77%). To a 1-L round-bottom flask equipped
with a magnetic stir bar and condenser was added POCl3 (174
mL, 1.8 mol). The aminopyridine (30.7 g, 121.9 mmol) was
added to the solution slowly at 0 °C (ice/H2O bath). The
mixture was heated at reflux for 3 h. The solution was cooled
and slowly added to ice/H2O (1.5 L). The mixture was neutral-
ized with 50% aq NaOH and extracted with EtOAc. The
combined organic layers were washed with brine and concen-
trated to afford aminopyridine 5a as yellow crystals (25.5 g,
72%): 1H NMR (500 MHz, CDCl3) δ 0.93 (t, J ) 7.3 Hz, 3H),
1.16 (t, J ) 7.0 Hz, 3H), 1.30 (sextet, J ) 7.3 Hz, 2H), 1.53
(pentet, J ) 7.3 Hz, 2H), 2.42 (s, 3H), 3.17 (t, J ) 7.3 Hz, 2H),
3.27 (q, J ) 7.0 Hz, 2H), 6.51 (s, 1H); HRMS Calcd for (C12H18-
ClN3O2) [M + H]+ 272.1166; found 272.1174.
N2-(2-Bromo-4-isopropylphenyl)-N4-butyl-N4-ethyl-6-
methyl-3-nitropyridine-2,4-diamine (6). 4-(N-n-Butyl-N-
ethylamino)-2-chloro-6-methyl-3-nitropyridine 5a (1.0 g, 3.7
mmol) and 2-bromo-4-isopropylaniline (1.6 g, 7.4 mmol) were
heated neat at 130 °C for 16 h. The viscous black oil was taken
up in EtOAc (50 mL) and washed with H2O (2×). The combined
aqueous layers were extracted with EtOAc (1×). The combined
organic layers were dried (MgSO4) and concentrated in vacuo.
The crude black oil was purified by silica gel chromatography
(98% hexanes/2% AcOH f 88% hexanes/10% ethyl acetate/
2% AcOH) to afford 1.2 g (68% yield) of 5a as an orange oil.
1H NMR (500 MHz, CDCl3) δ 0.91 (t, J ) 7.3 Hz, 3H), 1.19 (t,
J ) 7.0 Hz, 3H), 1.22 (d, J ) 6.7 Hz, 6H), 1.31 (m, 4H), 2.34
(s, 3H), 2.84 (septet, J ) 7.0 Hz, 1H), 3.19 (t, J ) 6.8 Hz, 2H),
3.26 (q, J ) 6.8 Hz, 2H), 6.23 (s, 1H), 7.13 (d, J ) 8.6 Hz, 1H),
7.40 (s, 1H), 8.31 (d, J ) 8.6 Hz, 1H), 9.52 (s, 1H); HRMS Calcd
for (C21H29BrN4O2) [M + H]+ 449.1552; found 449.1552.
{(2-Bromo-4-isopropylphenyl)-[4-(butylethylamino)-6-
methyl-3-nitropyridin-2-yl]amino}acetic Acid Ethyl Es-
ter (7). A solution of 5 (1.56 mmol, 701 mg) in DMF (3.1 mL)
was cooled to 0 °C. NaH (94 mg of a 60% dispersion in oil,
2.34 mmol) was added in portions and the red solution stirred
at 0 °C for 30 min. Ethyl iodoacetate (277 mL, 2.34 mmol) was
added dropwise over 5 min, and the resultant solution was
allowed to warm to room temperature and was stirred for an
additional 2 h. The reaction was quenched with H2O (10 mL)
and extracted with EtOAc (3×). The combined organic layers
were washed with H2O (5×), dried (MgSO4), and concentrated
in vacuo. The crude red-orange oil was purified by silica gel
chromatography (100% hexanes f 75% hexanes/25% ether)
to afford 677 mg (81% yield) of 7 as a red orange oil. 1H NMR
(400 MHz, CDCl3) δ 0.84 (t, J ) 7.3 Hz, 3H), 1.00 (t, J ) 7.1
Hz, 3H), 1.21 (d, J ) 6.9 Hz, 6H), 1.26 (t, J ) 7.1 Hz, 3H),
1.38 (sextet, J ) 7.1 Hz, 2H), 1.59 (pentet, J ) 7.3 Hz, 2H),
2.33 (s, 3H), 2.85 (septet, J ) 6.9 Hz, 1H), 2.94 (t, J ) 7.3 Hz,
2H), 3.02 (q, J ) 7.1 Hz, 2H), 4.16 (q, J ) 7.1 Hz, 2H), 4.38
(m, 2H), 6.34 (s, 1H), 7.04 (dd, J ) 8.3, 2.0 Hz, 1H), 7.27 (d, J
) 8.3 Hz, 1H), 7.43 (d, J ) 2.0 Hz, 1H); HRMS Calcd for
(C25H35BrN4O4) [M + H]+ 535.1920; found 535.1923.
Compounds 8a-c, 8g-h, and 8l-w were prepared in an
analogous fashion to compound 2.
8-(Butylethylamino)-4-(2-chloro-4-methoxyphenyl)-6-
methyl-3,4-dihydro-1H-pyrido[2,3-b]pyrazin-2-one, Tri-
1
fluoroacetic Acid Salt (8a). H NMR (300 MHz, CD3OD) δ
0.92 (t, J ) 7.3 Hz, 3H), 1.11 (t, J ) 7.3 Hz, 3H), 1.33 (sextet,
J ) 7.3 Hz, 2H), 1.51 (pentet, J ) 7.3 Hz, 2H), 2.24 (s, 3H),
3.22 (t, J ) 7.3 Hz, 2H), 3.27 (q, J ) 7.3 Hz, 2H), 3.85 (s, 3H),
4.78 (m, 2H), 6.55 (s, 1H), 6.99 (dd, J ) 2.9, 8.8 Hz, 2H), 7.12
(d, J ) 2.9 Hz, 1H), 7.38 (d, J ) 8.8 Hz, 1H); HRMS Calcd for
(C21H27ClN4O2) [M + H]+ 403.1901; found 403.1903; HPLC:
(a) 99%; (b) >99%.
4-(2-Chloro-4-methoxyphenyl)-8-(ethylamino)-6-meth-
yl-3,4-dihydropyrido[2,3-b]pyrazin-2(1H)-one, Trifluoro-
acetic Acid Salt (8b). 1H NMR (300 MHz, CD3OD) δ 1.31 (t,
J ) 7.3 Hz, 3H), 2.28 (s, 3H), 3.37 (q, J ) 7.3 Hz, 2H), 3.83 (s,
3H), 4.31 (m, 2H), 6.44 (s, 1H), 7.03 (dd, J ) 2.9, 8.8 Hz, 1H),
7.17 (d, J ) 2.9 Hz, 1H), 7.43 (d, J ) 8.8 Hz, 1H); HRMS Calcd
for (C17H19ClN4O2) [M + H]+ 347.1275; found 347.1276;
HPLC: (a) >98%; (b) >98%.
8-(Butylamino)-4-(2-chloro-4-methoxyphenyl)-6-meth-
yl-3,4-dihydropyrido[2,3-b]pyrazin-2(1H)-one, Trifluoro-
acetic Acid Salt (8c). 1H NMR (300 MHz, CD3OD) δ 0.97 (t,
J ) 7.3 Hz, 3H), 1.45 (sextet, J ) 7.3 Hz, 2H), 1.64 (pentet, J
) 7.3 Hz, 2H), 2.28 (s, 3H), 3.34 (t, J ) 7.3 Hz, 2H), 3.83 (s,
3H), 4.84 (m, 2H), 6.44 (s, 1H), 7.03 (dd, J ) 2.9, 8.8 Hz, 1H),
7.17 (d, J ) 2.9 Hz, 1H), 7.42 (d, J ) 8.8 Hz, 1H); HRMS Calcd
for (C19H23ClN4O2) [M + H]+ 375.1588; found 375.1579;
HPLC: (a) 95%; (b) 98%.
N4-Butyl-N2-(2,4-dichlorophenyl)-N4ethyl-6-methyl-3-
nitropyridine-2,4-diamine (6d). To 2,4-dichloroaniline (1.2
g, 7.4 mmol) in N,N-dimethylacetamide (35 mL) cooled to 0
°C was added NaH (588 mg of 60% NaH in oil, 14.7 mmol) in
small portions. The solution was stirred at 0 °C for 15 min,
and then 2-chloro-4-(N-ethyl-N-butylamino)-5-methyl-3-nitro-
pyridine 5a (1.0 g, 3.68 mmol) in N,N-dimethylacetamide (2
mL) was added slowly. The mixture was allowed to warm to
ambient temperature then heated at 100 °C for 3 h. The
reaction was carefully quenched with H2O and extracted with
EtOAc (3×). The combined organic layers were washed with
H2O (4×), dried (MgSO4), and concentrated in vacuo. The crude
brown oil was purified by silica gel chromatography (98%
hexanes/2% AcOH f 88% hexanes/10% ethyl acetate/2%
AcOH) to afford 949 mg (65% yield) of 6d as an orange oil. 1H
NMR (400 MHz, CDCl3) δ 0.91 (t, J ) 7.3 Hz, 3H), 1.20 (t, J
) 7.1 Hz, 3H), 1.31 (sextet, J ) 7.3 Hz, 2H), 1.58 (pentet, J )
7.3 Hz, 2H), 2.36 (s, 3H), 3.20 (dd, J ) 7.3, 7.3 Hz, 2H), 3.27
(q, J ) 7.1 Hz, 2H), 6.28 (s, 1H), 7.20 (dd, J ) 8.9, 2.5 Hz,
1H), 7.38 (d, J ) 2.5 Hz, 1H), 8.55 (d, J ) 8.9 Hz, 1H), 9.68
(br s, 1H). HRMS Calcd for (C18H22Cl2N4O2) [M + H]+
397.1198; found 397.1196.
Compound 6d was converted into compound 8d in the same
fashion as compound 5a was converted into compound 2.
8-(Butylethylamino)-4-(2,4-dichlorophenyl)-6-methyl-
3,4-dihydro-1H-pyrido[2,3-b]pyrazin-2-one (8d). 1H NMR
(300 MHz, CD3OD) δ 0.93 (t, J ) 7.3 Hz, 3H), 1.19 (t, J ) 7.3
Hz, 3H), 1.32 (sextet, J ) 7.3 Hz, 2H), 1.58 (pentet, J ) 7.3
Hz, 2H), 2.33 (s, 3H), 3.47 (t, J ) 7.3 Hz, 2H), 3.53 (q, J ) 7.3
Hz, 2H), 4.37 (s, 2H), 6.70 (s, 1H), 7.53 (m, 2H), 7.71 (dd, J )
2.6, 1.1 Hz, 1H); 13C NMR (300 MHz, CD3OD) δ 13.0, 14.1,
18.9, 21.1, 30.8, 47.2, 51.9, 54.8, 109.0, 111.8, 130.7, 131.4,
4-(2-Bromo-4-isopropylphenyl)-8-(butylethylamino)-6-
methyl-3,4-dihydro-1H-pyrido[2,3-b]pyrazin-2-one (2). A
solution of ester 7 (200 mg, 0.37 mmol) and iron powder (104
mg, 1.87 mmol) in EtOH (4 mL) was heated at reflux for 12 h
using a heating mantle and boiling chips. The mixture was
allowed to cool to room temperature and filtered through Celite
eluting with EtOAc. The organics were washed with 1 N NaOH
(2×) then brine (1×), dried (MgSO4), and concentrated in

5788 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 23
Dzierba1 et al.
132.2, 133.7, 136.4, 138.2, 143.4, 145.3, 152.1, 164.3; HRMS
Calcd for (C20H24Cl2N4O) [M + H]+ 407.1405; found 407.1409;
HPLC: (a) >99%; (b) >99%.
Compounds 8e-f, 8i-k, and 8x-y were prepared in an
analogous fashion to compound 8d.
8-(Butylethylamino)-4-(2-chloro-4-isopropylphenyl)-6-
methyl-3,4-dihydro-1H-pyrido[2,3-b]pyrazin-2-one, Tri-
1
fluoroacetic Acid Salt (8l). H NMR (300 MHz, CD3OD) δ
0.93 (t, J ) 7.3 Hz, 3H), 1.19 (t, J ) 7.3 Hz, 3H), 1.28 (d, J )
7.3 Hz, 6H), 1.32 (sextet, J ) 7.3 Hz, 2H), 1.58 (pentet, J )
7.3 Hz, 2H), 2.33 (s, 3H), 2.99 (septet, J ) 7.3 Hz, 1H), 3.41-
3.59 (m, 4H), 4.34 (m, 2H), 6.68 (br s, 1H), 7.38 (dd, J ) 8.4,
1.8 Hz, 1H), 7.42 (d, J ) 8.4 Hz, 1H), 7.59 (d, J ) 1.8 Hz,1H);
13C NMR (300 MHz, CD3OD) δ 13.0, 14.2, 19.4, 21.2, 24.1 (2C),
30.8, 35.2, 47.3, 52.0, 55.1, 108.9, 111.8, 128.6, 130.2, 130.4,
132.7, 137.0, 144.2, 145.7, 151.5, 153.2, 164.5; HRMS Calcd
for (C23H31ClN4O) [M + H]+ 415.2265; found 415.2254;
HPLC: (a) >99%; (b) >99%.
4-(2,4-Dichlorophenyl)-6-methyl-8-(pentan-3-ylamino)-
3,4-dihydropyrido[2,3-b]pyrazin-2(1H)-one, Trifluoro-
1
acetic Acid Salt (8e). H NMR (300 MHz, DMSO-d6) δ 0.84
(t, J ) 7.3 Hz, 6H), 1.33-1.59 (m, 4H), 1.97 (s, 3H), 3.21-3.32
(m, 1H), 4.11 (m, 2H), 5.46 (d, J ) 8.0 Hz, 1H), 6.03 (s, 1H),
7.36-7.43 (m, 2H), 7.58 (s, 1H), 9.96 (br s, 1H); HRMS Calcd
for (C19H22Cl2N4O) [M + H]+ 393.1249; found 393.1264;
HPLC: (a) >99%; (b) 96%.
4-(2-Chloro-4-isopropylphenyl)-8-[(2-methoxyethyl)-
propylamino]-6-methyl-3,4-dihydro-1H-pyrido[2,3-b]-
pyrazin-2-one, Trifluoroacetic Acid Salt (8m). 1H NMR
(300 MHz, CD3OD) δ 0.96 (t, J ) 7.3 Hz, 3H), 1.27 (d, J ) 7.3
Hz, 6H), 1.59 (sextet, J ) 7.3 Hz, 2H), 2.28 (s, 3H), 2.97 (septet,
J ) 7.3 Hz, 1H), 3.24-3.34 (m, 3H), 3.48-3.56 (m, 4H), 3.73
(t, J ) 7.3 Hz, 2H), 4.31 (m, 2H), 6.61 (br s, 1H), 7.34 (dd, J )
1.8, 8.8 Hz, 1H), 7.41 (d, J ) 8.8 Hz, 1H), 7.46 (d, J ) 1.8 Hz,
1H); 13C NMR (300 MHz, CD3OD) δ 11.7, 21.7, 23.6, 24.2, 53.0,
30.7, 54.2, 55.8, 59.2, 71.2, 108.3, 113.9, 127.3, 129.5, 130.2,
133.4, 139.8, 146.8, 147.6, 150.6, 152.2, 166.1; HRMS Calcd
for (C23H31ClN4O2) [M + H]+ 431.2214; found 431.2221;
HPLC: (a) 97%; (b) 97%.
8-[Bis-(2-methoxyethyl)amino]-4-(2-chloro-4-isopropyl-
phenyl)-6-methyl-3,4-dihydro-1H-pyrido[2,3-b]pyrazin-2-
one, Trifluoroacetic Acid Salt (8n). 1H NMR (300 MHz,
CD3OD) δ 1.28 (d, J ) 7.3 Hz, 6H), 2.31 (br s, 3H), 2.98 (septet,
J ) 7.3 Hz, 1H), 3.43 (s, 6H), 3.64-3.76 (m, 8H), 4.32 (m, 2H),
6.77 (br s, 1H), 7.38 (dd, J ) 1.8, 8.4 Hz, 1H), 7.43 (d, J ) 8.4
Hz, 1H), 7.49 (d, J ) 1.8 Hz, 1H); 13C NMR (300 MHz, CD3-
OD) δ 22.2, 22.8, 33.5, 51.8, 52.7, 57.7, 69.8 106.9, 112.3, 125.9,
128.0, 128.7, 131.9, 138.3, 145.2, 146.2, 149.1, 151.0, 164.6;
HRMS Calcd for (C23H31ClN4O3) [M + H]+ 447.2163; found
447.2152; HPLC: (a) >98%; (b) >99%.
4-(2-Bromo-4-isopropylphenyl)-8-[ethyl(tetrahydrofu-
ran-2-ylmethyl)amino]-6-methyl-3,4-dihydro-1H-pyrido-
[2,3-b]pyrazin-2-one (8o). 1H NMR (500 MHz, CDCl3) δ 1.10
(t, J ) 7.1 Hz, 3H), 1.26 (d, J ) 6.9 Hz, 6H), 1.47 (m, 1H),
1.97 (m, 3H), 2.19 (s, 3H), 2.92 (m, 3H), 3.09 (sextet, J ) 6.9
Hz, 1H), 3.24 (sextet, J ) 6.9 Hz, 1H), 3.88 (m, 1H), 4.21 (m,
2H), 4.20 (m, 2H), 6.29 (s, 1H), 7.20 (dd, J ) 1.7, 8.1 Hz, 1H),
7.22 (d, J ) 8.1 Hz, 1H), 7.48 (d, J ) 1.7 Hz, 1H), 9.83 (s, 1H);
13C NMR (400 MHz, CDCl3) δ ppm 13.0, 23.8, 23.8, 24.2, 25.9,
29.4, 33.6, 44.7, 53.4, 68.1, 76.3, 77.2, 106.3, 112.3, 122.9, 126.6,
129.6, 131.6, 140.0, 144.4, 145.7, 149.0, 150.6, 163.9; HRMS
Calcd for (C24H31BrN4O2) [M + H]+ 487.1709; found 487.1703;
HPLC: (a) >98%; (b) >98%.
4-(2,4-Dichlorophenyl)-8-(1-methoxybutan-2-ylamino)-
6-methyl-3,4-dihydropyrido[2,3-b]pyrazin-2(1H)-one, Tri-
1
fluoroacetic Acid Salt (8f). H NMR (300 MHz, CD3OD) δ
1.01 (t, J ) 7.3 Hz, 3H), 1.56-1.72 (m, 2H), 1.72-1.86 (m, 2H),
2.32 (s, 3H), 3.36 (s, 3H), 3.43-3.58 (m, 2H), 3.86-3.92 (m,
1H), 4.40 (m, 2H), 6.12 (s, 1H), 7.53 (m, 2H), 7.73 (s, 1H); 13C
NMR (300 MHz, CD3OD) δ 10.6, 18.8, 25.5, 54.1, 56.2, 59.4,
75.2, 102.4, 107.1, 130.7, 131.2, 132.3, 133.8, 136.2, 138.0,
139.9, 145.4, 147.8, 165.1; HRMS Calcd for (C19H22Cl2N4O2)
[M + H]+ 409.1198; found 409.1190; HPLC: (a) 95%; (b) 95%.
8-(Butylethylamino)-4-(2-chloro-5-fluoro-4-methoxy-
phenyl)-6-methyl-3,4-dihydro-1H-pyrido[2,3-b]pyrazin-2-
one, Trifluoroacetic Acid Salt (8g). 1H NMR (300 MHz,
CD3OD) δ 0.91 (t, J ) 7.3 Hz, 3H), 1.17 (t, J ) 7.3 Hz, 3H),
1.31 (sextet, J ) 7.3 Hz, 2H), 1.55 (pentet, J ) 7.3 Hz, 2H),
2.32 (s, 3H), 3.47 (m, 4H), 3.92 (s, 3H), 4.30 (m, 2H), 6.66 (s,
1H), 7.35 (d, JH-F ) 8.4 Hz, 1H), 7.41 (d, JH-F ) 11.4 Hz, 1H);
13C NMR (500 MHz, CD3OD) δ 13.0, 14.2, 19.2, 21.2, 30.9, 47.2,
52.0, 54.8, 57.5, 108.9, 111.5, 116.7, 117.9 (d, JC-F ) 21.2 Hz),
128.8, 131.0, 144.0, 145.3, 150.7 (d, JC-F ) 12.2 Hz), 151.6,
153.1 (d, JC-F ) 249.2 Hz), 164.1; HRMS Calcd for (C21H26-
ClFN4O2) [M + H]+ 421.1807; found 421.1805; HPLC: (a)
>99%; (b) 95%.
4-(2-chloro-5-fluoro-4-methoxyphenyl)-8-(ethyl(isopro-
pyl)amino)-6-methyl-3,4-dihydropyrido[2,3-b]pyrazin-
1
2(1H)-one (8h). H NMR (300 MHz, CD3OD) δ 0.92 (t, J )
7.3 Hz, 3H), 1.11 (d, J ) 7.3 Hz, 6H), 2.16 (s, 3H), 3.05 (q, J )
7.3 Hz, 2H), 3.31 (septet, J ) 7.3 Hz, 1H), 3.88 (s, 3H), 4.23
(m, 2H), 6.51 (s, 1H), 7.22 (s, 1H), 7.24 (s, 1H); HRMS Calcd
for (C20H24ClFN4O2) [M + H]+ 407.1650; found 407.1673;
HPLC: (a) 95%; (b) 97%.
4-[8-(Butylethylamino)-6-methyl-2-oxo-2,3-dihydro-1H-
1
pyrido[2,3-b]pyrazin-4-yl]-3-chloro-benzonitrile (8i). H
NMR (300 MHz, CDCl3) δ 0.88 (t, J ) 7.3 Hz, 3H), 1.01 (t, J
) 7.3 Hz, 3H), 1.27 (sextet, J ) 7.3 Hz, 2H), 1.40 (pentet, J )
7.3 Hz, 2H), 2.21 (s, 3H), 2.90 (t, J ) 7.3 Hz, 2H), 2.99 (q, J )
7.3 Hz, 2H), 4.35 (m, 2H), 6.44 (s, 1H), 7.37 (d, J ) 8.0 Hz,
1H), 7.59 (dd, J ) 8.0, 1.9 Hz, 1H), 7.73 (d, J ) 1.9 Hz, 1H),
7.84 (br s, 1H); 13C NMR (300 MHz, CDCl3) δ 12.2, 13.9, 20.4,
24.1, 29.3, 47.3, 51.9, 52.8, 100.7, 110.1, 114.5, 117.5, 128.1,
131.3, 132.6, 134.2, 143.9, 145.2, 145.8, 151.5, 163.2; HRMS
Calcd for (C21H24ClN5O) [M + H]+ 398.1748; found 398.1761;
HPLC: (a) >99%; (b) >99%.
4-{Ethyl-[4-(4-methoxy-2-methylphenyl)-6-methyl-2-
oxo-1,2,3,4-tetrahydro-pyrido[2,3-b]pyrazin-8-yl]amino}-
1
butyronitrile (8p). H NMR (300 MHz, CD3OD) δ 1.04 (t, J
) 7.3 Hz, 3H), 1.78 (pentet, J ) 7.3 Hz, 2H), 2.06 (s, 3H), 2.16
(s, 3H), 2.49 (t, J ) 7.3 Hz, 3H), 3.05-3.22 (m, 4H), 3.78 (s,
3H), 4.00-4.40 (m, 2H), 6.81 (s, 1H), 6.80 (d, J ) 8.1 Hz, 1H),
6.82 (s, 1H), 7.14 (d, J ) 8.1 Hz, 1H); 13C NMR (300 MHz,
CDCl3) δ 12.3, 15.5, 19.0, 23.7, 24.7, 49.1, 50.4, 54.3, 55.7,
107.5, 112.4, 113.8, 116.6, 119.6, 127.8, 135.3, 143.4, 146.5,
152.1, 158.4, 164.0; HRMS Calcd for (C22H27N5O2) [M + H]+
394.2243; found 394.2238; HPLC: (a) >98%; (b) >98%.
3-Chloro-4-(8-(dipropylamino)-6-methyl-2-oxo-2,3-di-
hydropyrido[2,3-b]pyrazin-4(1H)-yl)benzonitrile (8j). 1H
NMR (300 MHz, CDCl3) δ 0.87 (t, J ) 7.3 Hz, 6H), 1.45 (sextet,
J ) 7.3 Hz, 4H), 2.21 (s, 3H), 2.89 (t, J ) 7.3 Hz, 4H), 4.35 (m,
2H), 6.44 (s, 1H), 7.37 (d, J ) 8.5 Hz, 1H), 7.59 (dd, J ) 8.5,
1.8 Hz, 1H), 7.73 (d, J ) 1.8 Hz, 1H), 7.81 (br s, 1H); HRMS
Calcd for (C21H24ClN5O) [M + H]+ 398.1748; found 398.1753;
HPLC: (a) >99%; (b) >99%.
3-Chloro-4-(8-((cyclobutylmethyl)(propyl)amino)-6-
methyl-2-oxo-2,3-dihydropyrido[2,3-b]pyrazin-4(1H)-yl)-
benzonitrile (8k). 1H NMR (300 MHz, CDCl3) δ 0.85 (t, J )
7.3 Hz, 3H), 1.43 (sextet, J ) 7.3 Hz, 2H), 1.53-1.68 (m, 2H),
1.71-1.85 (m, 2H), 1.85-2.03 (m, 2H), 2.21 (s, 3H), 2.41 (t, J
) 7.3 Hz, 1H), 2.86 (t, J ) 7.3 Hz, 2H), 2.95 (d, J ) 7.3 Hz,
2H), 4.35 (m, 2H), 6.44 (s, 1H), 7.37 (d, J ) 8.0 Hz, 1H), 7.59
(dd, J ) 8.0, 1.8 Hz, 1H), 7.72 (d, J ) 1.8 Hz, 1H), 7.83 (br s,
1H); HRMS Calcd for (C23H26ClN5O) [M + H]+ 424.1904; found
424.1909; HPLC: (a) >99%; (b) >99%.
4-(2-Bromo-4-isopropylphenyl)-8-[ethyl-(3-methane-
sulfonylpropyl)amino]-6-methyl-3,4-dihydro-1H-pyrido-
[2,3-b]pyrazin-2-one, Trifluoroacetic Acid Salt (8q). 1H
NMR (400 MHz, CDCl3) δ ppm 1.14 (t, J ) 7.1 Hz, 3H), 1.25
(d, J ) 6.9 Hz, 6H), 2.10 (m, 2H), 2.35 (s, 3H), 2.92 (s, 3H),
2.92 (septet, J ) 6.9 Hz, 1H), 3.11 (dd, J ) 7.1, 7.1 Hz, 2H),
3.33 (m, 2H), 3.42 (m, 2H), 4.40 (m, 2H), 6.40 (s, 1H), 7.29
(dd, J ) 8.2, 1.7 Hz, 1H), 7.41 (d, J ) 8.2 Hz, 1H), 7.52 (d, J
) 1.7 Hz, 1H), 8.08 (s, 1H); HRMS Calcd for (C23H31BrN4O3S)
[M + H]+ 523.1378; found 523.1372; HPLC: (a) >98%; (b) 95%.
4-(2-Bromo-4-isopropylphenyl)-6-methyl-8-morpholin-
4-yl-3,4-dihydro-1H-pyrido[2,3-b]pyrazin-2-one (8r). 1H
NMR (300 MHz, DMSO-d6) δ 1.27 (d, J ) 7.3 Hz, 6H), 2.33 (s,

CRF1 Receptor Antagonists
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 23 5789
3H), 2.97 (septet, J ) 7.3 Hz, 2H), 3.26-3.50 (m, 4H), 3.78-
3.96 (m, 4H), 4.20-4.48 (m, 2H), 6.72 (s, 1H), 7.40-7.47 (m,
2H), 7.65 (d, J ) 1.4 Hz, 1H); HRMS Calcd for (C21H25BrN4O2)
[M + H]+ 445.1239; found 445.1242; HPLC: (a) >99; (b) 95%.
4-(2-Bromophenyl)-8-(butylethylamino)-6-methyl-3,4-
dihydro-1H-pyrido[2,3-b]pyrazin-2-one (8s). 1H NMR (400
MHz, CDCl3) δ 0.94 (t, J ) 7.3 Hz, 3H), 1.18 (t, J ) 7.1 Hz,
3H), 1.35 (sextet, J ) 7.3 Hz, 2H), 1.53 (pentet, J ) 7.3 Hz,
2H), 2.36 (s, 3H), 3.18 (dd, J ) 7.3, 7.3 Hz, 2H), 3.27 (q, J )
7.1 Hz, 2H), 4.41 (m, 2H), 6.36 (s, 1H), 7.29 (m, 1H), 7.46 (m,
2H), 7.63 (br s, 1H) 7.68 (d, J ) 8.1 Hz, 1H); HRMS Calcd for
(C20H25BrN4O) [M + H]+ 417.1290; found 417.1287; HPLC: (a)
97%; (b) >98%.
Hz, 2H), 1.37 (pentet, J ) 7.3 Hz, 2H), 2.16 (s, 3H), 2.91 (t, J
) 7.3 Hz, 2H), 2.98 (q, J ) 7.3 Hz, 2H), 4.31 (m, 2H), 6.37 (s,
1H), 7.69 (s, 2H); HRMS Calcd for (C21H23Cl2N5O) [M + H]+
432.1358; found 432.1353; HPLC: (a) 98%; (b) 97%.
Biology. Rat CRH Receptor Binding Assay. Frozen rat
frontal cortex (source of CRF1 receptor) or frozen porcine
choroid plexus (source of CRF2 receptor) were thawed rapidly
in assay buffer containing 50 mM Hepes (pH 7.0 at 23 °C), 10
mM MgCl2, 2 mM EGTA, 1 µg/mL aprotinin, 1 µg/mL leupep-
tin, 1 µg/mL pepstatin A, 0.005% Triton X-100, 10 U/mL
bacitracin, and 0.1% ovalbumin and homogenized. The sus-
pension was centrifuged at 32000g for 30 min. The resulting
supernatant was discarded and the pellet resuspended by
homogenization in assay buffer and centrifuged again. The
supernatant was discarded and the pellet resuspended by
homogenization in assay buffer and frozen at -70 °C. On the
day of the experiment aliquots of the homogenate were thawed
quickly and homogenate (25 µg/well rat frontal cortex or 10
µg/well porcine choroid plexus) added to ligand (150 pM 125I-
ovine-CRF for CRF1 binding or 100 pM 125I-sauvagine for CRF2
binding) and drugs in a total volume of 100 µL assay buffer.
The assay mixture was incubated for 2 h at 21 °C. Bound and
free radioligand were then separated by rapid filtration, using
glass fiber filters (Whatman GF/B, pretreated with 0.3% PEI)
on a Brandel Cell Harvester. Filters were then washed
multiple times with ice cold wash buffer (PBS w/o Ca2+ and
Mg2+, 0.01% Triton X-100 (pH 7.0 at 23 °C)). Nonspecific
binding was defined using 1 µM DMP696 in the CRF1 binding
assay and 1 µM R-helical CRF (9-41) in the CRF2 binding
assay. Filters were then counted in a Wallac Wizard gamma
counter.
Behavioral Studies. Subjects. Male Sprague-Dawley
rats weighing 180-300 g were purchased from Charles River
Laboratories (Wilmington, MA.). The rats were housed indi-
vidually in suspended wire cages in a colony room maintained
at constant temperature (21 ( 2 °C) and humidity (50 ( 10%).
The room was illuminated 12 h per day (lights on at 0600 h).
The rats had ad libitum access to food and water throughout
the study. Behavioral studies were conducted between 0600
and 1300 h. Animals were maintained in accordance with the
guidelines of the Committee on Animals of the Bristol-Myers
Squibb Company, the “Guide for Care and Use of Laboratory
Animals” (Institute of Animal Laboratory Resources, 1996),
and the guidelines published in the National Institutes of
Health Guide for the Care and Use of Laboratory Animals.
Research protocols were approved by the Bristol-Myers Squibb
Company Institutional Animal Care and Use Committee.
Defensive Withdrawal. The defensive withdrawal proce-
dure was used as previously described.32 Briefly, the testing
apparatus consisted of an opaque plexiglass open field (106
cm length × 92 cm width × 50 cm height), containing a
cylindrical galvanized chamber (14 cm length, 10 cm diameter)
that was positioned lengthwise against one wall, with the open
end 40 cm from the corner. The open field was illuminated by
a 60-W incandescent bulb and illumination was titrated by a
powerstat transformer to a 23-lux reading at the entrance to
the cylinder. Rats were habituated to handling by gently
stroking their dorsal surface for approximately one minute
daily for 5-6 consecutive days before testing. To initiate
testing, each rat was placed within the cylinder that was then
secured to the floor. Behavior was assessed for 15 min by a
trained observer (unaware of treatment assignment) via a
video monitor in an adjacent room. The latency to exit the
chamber, defined by the placement of all four paws into the
open field was recorded (in seconds). The plexiglass chamber
and the cylinder were cleaned with 1.0% glacial acetic acid
between animals to prevent olfactory cues from influencing
the behavior of subsequently tested animals. All compounds
were prepared in 0.25% methocel suspension and beadmilled
overnight. They were administered po, 1 h before testing in a
volume of 2 mL/kg body weight. Data were analyzed using an
analysis of variance, followed by individual mean comparisons
using Fisher’s Least Significant Difference Test. The signifi-
cance level was set at p < 0.05.
8-(Butylethylamino)-4-(4-isopropylphenyl)-6-methyl-
3,4-dihydro-1H-pyrido[2,3-b]pyrazin-2-one, Trifluoroace-
tic Acid Salt (8t). 1H NMR (300 MHz, DMSO-d6) δ 0.84 (t, J
) 7.3 Hz, 3H), 1.04 (t, J ) 7.3 Hz, 3H), 1.18 (d, J ) 7.3 Hz,
6H), 1.23 (sextet, J ) 7.3 Hz, 2H), 1.43 (pentet, J ) 7.3 Hz,
2H), 2.25 (s, 3H), 2.89 (septet, J ) 7.3 Hz, 1H), 3.25-3.45 (m,
4H), 4.28 (m, 2H), 6.67 (br s, 1H), 7.22 (d, J ) 8.8 Hz, 2H),
7.27 (d, J ) 8.8 Hz, 2H). HRMS Calcd for (C22H32N4O) [M +
H]+ 381.2654; found 381.2650; HPLC: (a) 97%; (b) >95%.
8-(Butylethylamino)-4-(4-methoxy-2-methylphenyl)-6-
methyl-3,4-dihydro-1H-pyrido[2,3-b]pyrazin-2-one (8u).
1H NMR (300 MHz, CDCl3) δ 0.88 (t, J ) 7.3 Hz, 3H), 1.00 (t,
J ) 7.3 Hz, 3H), 1.27 (sextet, J ) 7.3 Hz, 2H), 1.40 (pentet, J
) 7.3 Hz, 2H), 2.09 (s, 3H), 2.18 (s, 3H), 2.89 (t, J ) 7.3 Hz,
2H), 2.96 (q, J ) 7.3 Hz, 2H), 3.80 (s, 3H), 4.15-4.42 (m, 2H),
6.60 (s, 1H), 6.76 (dd, J ) 7.7, 2.9 Hz, 1H), 6.79 (dd, J ) 2.9,
1.1 Hz, 1H), 7.13 (dd, J ) 7.7, 1.1 Hz, 1H), 7.91 (br s, 1H); 13
C
NMR (300 MHz, CDCl3) δ 12.4, 13.9, 18.6, 20.4, 24.3, 29.5,
47.7, 52.3, 54.0, 55.3, 107.6, 112.0, 113.5, 116.2, 127.3, 135.1,
137.5, 144.7, 145.9, 151.3, 157.9, 163.6; HRMS Calcd for
(C22H30N4O2) [M + H]+ 383.2447; found 383.2451; HPLC: (a)
97%; (b) >95%.
8-(Butylethylamino)-4-(6-methoxy-2-methylpyridin-
3-yl)-6-methyl-3,4-dihydro-1H-pyrido[2,3-b]pyrazin-2-
one, bis-Trifluoroacetic Acid Salt (8v). 1H NMR (300 MHz,
CD3OD) δ 0.91 (t, J ) 7.3 Hz, 3H), 1.16 (t, J ) 7.3 Hz, 3H),
1.31 (sextet, J ) 7.3 Hz, 2H), 1.55 (pentet, J ) 7.3 Hz, 2H),
2.30 (s, 3H), 2.31 (s, 3H), 3.43 (t, J ) 7.3 Hz, 2H), 3.47 (q, J )
7.3 Hz, 2H), 3.92 (s, 3H), 4.29 (m, 2H), 6.63 (s 1H), 6.76 (d, J
) 8.8 Hz, 1H), 7.63 (d, J ) 8.8 Hz, 1H); HRMS Calcd for
(C21H29N5O2) [M + H]+ 384.2400; found 384.2389; HPLC: (a)
97%; (b) >98%.
8-(Butylethylamino)-4-(2,6-dimethoxypyridin-3-yl)-6-
methyl-3,4-dihydro-1H-pyrido[2,3-b]pyrazin-2-one (8w).
1H NMR (300 MHz, CD3OD) δ 0.93 (t, J ) 7.3 Hz, 3H), 1.17
(t, J ) 7.3 Hz, 3H), 1.32 (sextet, J ) 7.3 Hz, 2H), 1.55 (pentet,
J ) 7.3 Hz, 2H), 2.32 (s, 3H), 3.42 (t, J ) 7.3 Hz, 2H), 3.48 (q,
J ) 7.3 Hz, 2H), 3.97 (s, 3H), 3.98 (s, 3H), 4.27 (m, 2H), 6.46
(d, J ) 8.4 Hz, 1H), 6.64 (s 1H), 7.63 (d, J ) 8.4 Hz, 1H); 13C
NMR (300 MHz, CD3OD) δ 13.0, 14.1, 18.8, 21.1, 30.8, 47.0,
48.2, 51.8, 54.5, 54.7, 104.1, 108.5, 111.2, 115.9, 140.9, 144.1,
144.4, 151.3, 159.8, 164.4, 164.6; HRMS Calcd for (C21H29N5O3)
[M + H]+ 400.2349; found 400.2364; HPLC: (a) >98%; (b)
>95%.
2-[8-(Butylethylamino)-6-methyl-2-oxo-2,3-dihydro-1H-
pyrido[2,3-b]pyrazin-4-yl]-5-chloro-benzonitrile, Trifluo-
roacetic Acid Salt (8x). 1H NMR (300 MHz, CD3OD) δ 0.87
(t, J ) 7.3 Hz, 3H), 1.02 (t, J ) 7.3 Hz, 3H), 1.28 (sextet, J )
7.3 Hz, 2H), 1.42 (pentet, J ) 7.3 Hz, 2H), 2.20 (s, 3H), 3.06
(t, J ) 7.3 Hz, 2H), 3.12 (q, J ) 7.3 Hz, 2H), 4.34 (m, 2H),
6.57 (s, 1H), 7.37 (d, J ) 8.8 Hz, 1H), 7.67 (dd, J ) 8.8, 2.6
Hz, 1H), 7.72 (d, J ) 2.6 Hz, 1H), 7.84 (br s, 1H); 13C NMR
(300 MHz, DMSO-d6) δ 12.5, 13.8, 20.3, 20.6, 29.5, 46.2, 50.8,
53.6, 109.5, 111.1, 113.1, 115.1, 127.8, 133.3, 133.8, 135.0,
142.1, 142.8, 147.9, 149.7, 162.4; HRMS Calcd for (C21H24-
ClN5O2) [M + H]+ 398.1748; found 398.1749; HPLC: (a) >98%;
(b) >98%.
4-[8-(Butylethylamino)-6-methyl-2-oxo-2,3-dihydro-1H-
pyrido[2,3-b]pyrazin-4-yl]-3,5-dichloro-benzonitrile, Tri-
fluoroacetic Acid Salt (8y). 1H NMR (300, CDCl3) δ 0.88 (t,
J ) 7.3 Hz, 3H), 1.01 (t, J ) 7.3 Hz, 3H), 1.29 (sextet, J ) 7.3

5790 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 23
Dzierba1 et al.
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Pharmacokinetic Studies. Pharmacokinetic parameters
were estimated in beagle dogs following intravenous (1 mg/
kg; n ) 2) and oral (5 mg/kg; n ) 2) doses. Intravenous doses
were prepared in a vehicle consisting of propylene glycol:water:
N,N-dimethylacetamide, 80:10:10 (v/v). The oral vehicle con-
sisted of Labrafil:ethanol:Tween 80, 85:10:5 (v/v). Blood samples
were collected via jugular venipuncture at 0, 0.1, 0.25, 0.5, 1,
2, 4, 6, 8, and 24 h post-dose for the intravenous experiment,
and at 0, 0.5, 1, 2, 4, 6, 8, and 24 h post-dose for the oral
experiment. Plasma was separated by centrifugation and
stored frozen at -20 °C until analysis. Concentrations of
compound were determined by LC/MS/MS.
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JM049737F