8-(4-Methoxyphenyl)pyrazolo[1,5-a]-1,3,5-triazines: Selective and centrally active corticotropin-releasing factor receptor-1 (CRF1) antagonists

Article abstract of DOI:10.1021/jm9000242

This report describes the syntheses and structure-activity relationships of 8-(4-methoxyphenyl)pyrazolo-[1,5-a]-1,3,5-triazine corticotropin releasing factor receptor-1 (CRF₁) receptor antagonists. CRF₁ receptor antagonists may be potential anxiolytic or antidepressant drugs. This research culminated in the discovery of analogue 12-3, which is a potent, selective CRF₁ antagonist (hCRF₁ IC₅₀ ) 4.7 ± 2.0 nM) with weak affinity for the CRF-binding protein and biogenic amine receptors. This compound also has a good pharmacokinetic profile in dogs. Analogue 12-3 is orally effective in two rat models of anxiety: the defensive withdrawal (situational anxiety) model and the elevated plus maze test. Analogue 12-3 has been advanced to clinical trials.

Full text of DOI:10.1021/jm9000242

J. Med. Chem. 2009, 52, 3073–3083
3073
8-(4-Methoxyphenyl)pyrazolo[1,5-a]-1,3,5-triazines: Selective and Centrally Active
Corticotropin-Releasing Factor Receptor-1 (CRF1) Antagonists
Paul J. Gilligan,*,‡ Liqi He,§ Todd Clarke,‡ Parcharee Tivitmahaisoon,‡ Snjezana Lelas,‡ Yu-Wen Li,‡ Karen Heman,‡
Lawrence Fitzgerald,# Keith Miller,† Ge Zhang,† Anne Marshall,‡ Carol Krause,‡ John McElroy,4 Kathyrn Ward,‡ Helen Shen,‡
Harvey Wong,‡ Scott Grossman,§ Gregory Nemeth,‡ Robert Zaczek,‡ Stephen P. Arneric,2 Paul Hartig,‡ David W. Robertson,3
and George Trainor§
Bristol-Myers Squibb Co., 311 Pennington Rocky Hill Road, Hopewell, New Jersey 08540, Bristol-Myers Squibb Co., 5 Research Parkway,
Wallingford, Connecticut 06492, and Bristol-Myers Squibb Co., Route 206 and ProVince Line Road, Princeton, New Jersey 08543
ReceiVed January 10, 2009
This report describes the syntheses and structure-activity relationships of 8-(4-methoxyphenyl)pyrazolo-
[1,5-a]-1,3,5-triazine corticotropin releasing factor receptor-1 (CRF1) receptor antagonists. CRF1 receptor
antagonists may be potential anxiolytic or antidepressant drugs. This research culminated in the discovery
of analogue 12-3, which is a potent, selective CRF1 antagonist (hCRF1 IC50 ) 4.7 ( 2.0 nM) with weak
affinity for the CRF-binding protein and biogenic amine receptors. This compound also has a good
pharmacokinetic profile in dogs. Analogue 12-3 is orally effective in two rat models of anxiety: the defensive
withdrawal (situational anxiety) model and the elevated plus maze test. Analogue 12-3 has been advanced
to clinical trials.
Corticotropin releasing factor (CRFa) receptor modulators
Scheme 1
may be useful in the treatment of stress-related disorders (e.g.,
anxiety or depression).1-5 Preclinical studies have documented
the essential role of CRF in the regulation of endocrine and
autonomic and behavioral responses to stress. Rapid, transient
release of CRF mediates several responses to natural stressors
in many species; intracerebroventricular (icv) administration of
CRF in rodents elicits behavioral and physiological effects
identical to those caused by natural stressors.6 Transgenic mice,
which overexpress CRF, demonstrate increased anxiety, whereas
mice in which the CRF gene is deleted exhibit a reduced
endocrine response to natural stressors.7,8 Peptidic CRF antago-
nists (e.g., R-helical CRF9-41, astressin) not only block the
effects of exogenous CRF but also block the effects of various
natural stressors.9,10 These data indicate that CRF receptors may
be important targets for the treatment of stress-related disorders.
CRF is a 41-amino acid peptide that was first characterized as
a pituitary adrenocorticotropic hormone (ACTH) secretagogue11
and then as a neurotransmitter.12-14 CRF function is mediated by
two G-protein-coupled receptor subtypes, CRF1 and CRF2 recep-
tors, which are positively coupled to cyclic adenosine monophos-
phate (c-AMP) production.15-22 Splice variants have been identified
for each subtype. The CRF1 receptor is the most abundant subtype
found in the rodent and primate pituitary and is involved in the
regulation of ACTH secretion from the pituitary.23-25 The relative
roles of the subtypes in neuropsychiatric and feeding disorders in
rodents have been reviewed at length.26 Great progress has been
made in defining the structure-activity relationships for peptidic
CRF1- and CRF2-selective agonists and antagonists (e.g., astressin
and anti-sauvagine1-30).9,10,27-29 However, the physical properties
of these agents limit their use in a clinical setting, which has
prompted a search for nonpeptidic antagonists. To date, only CRF1-
selective nonpeptidic antagonists have been reported. Preclinical
reports on CRF1-selective antagonists 1 (CP-154526),30,31 2 (DMP
696),32 and 3 (DMP 904)33 (Scheme 1) support the hypothesis that
CRF receptors may be good targets for treatment of stress-related
disorders. No data on CRF2-selective nonpeptidic agents have been
published.
* To whom correspondence should be addressed. Phone: 609-818-4670.
Fax: 609-818-3450. E-mail: paul.j.gilligan@bms.com.
†
Bristol-Myers Squibb Co., Hopewell, NJ.
Bristol-Myers Squibb Co., Wallingford, CT.
Bristol-Myers Squibb Co., Princeton, NJ.
Deceased.
‡
§
3
#
Present address: Pfizer Inc., Eastern Point Road, MS8220-4216, Groton,
CT 06430.
2
Present address: Neuroscience Research, Eli Lilly and Company,
Indianapolis, IN 46285.
4
Present address: Jenrin Discovery, 3701 Market Street, Philadelphia,
PA 19104.
Clinical studies have provided equivocal data on the role of
CRF in the pathophysiology of depression. A role for CRF in
major depression is inferred from the following observations:
a Abbreviations: CRF, corticotropin-releasing factor; ACTH, adrenocor-
ticotropic hormone; c-AMP, cyclic adenosine monophosphate; CSF, cere-
brospinal fluid; HEK293, human embryonic kidney-293.
10.1021/jm9000242 CCC: $40.75
2009 American Chemical Society
Published on Web 04/10/2009

3074 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9
Gilligan et al.
Scheme 2
Scheme 3
(a) maladaptation to chronic stress and related chronic elevation
of corticosteroids may be a major pathway leading to some,
but not all, forms of depression;34 (b) hypercortisolemia in these
forms of depression appears to be a direct result of chronic
hypersecretion of hypothalamic CRF;35 (c) the number of CRF-
expressing neurons in the hypothalamus of depressed patients
is increased relative to that in normal subjects;36 (d) concentra-
tions of CRF in cerebrospinal fluid (CSF) are increased in some
patients with major depression;37 and (e) successful treatment
of depression in these patients is associated with decreased CRF
levels in CSF.37-40 The small molecule CRF1 receptor antago-
nist 4 (R121919) had some efficacy in a small open label clinical
study in anxiety and depression.41 However, another CRF1
antagonist 5 (CP-316311) failed in a double-blind study
involving patients suffering from depression unlike the positive
control sertraline.42,43 Additional clinical studies with structurally
diverse CRF1 antagonists may better define the role of CRF in
human mental illness.
This report describes the syntheses and structure-activity
relationships of 8-(4-methoxyphenyl)pyrazolo[1,5-a]-1,3,5-tri-
azine CRF1 receptor antagonists, which may be potential
anxiolytic drugs. A preliminary report from this laboratory
outlined the structure-activity relationships for substitution on
the phenyl ring of 8-phenylpyrazolo[1,5-a]-1,3,5-triazines.44
Additional studies on 8-(4-methoxyphenyl) congeners are
reported below. This research has culminated in the discovery
of analogues 12-3, 12-16, 12-33, and 12-35, which are potent,
selective CRF1 antagonists with weak affinity for the CRF-
binding protein and biogenic amine receptors. These agents also
have good pharmacokinetic profiles in dogs. Analogue 12-3 is
effective in two rat models of anxiety: the defensive withdrawal
(situational anxiety) model and the elevated plus maze test.
These data strengthen the hypothesis that CRF1 antagonists may
be useful for the treatment of anxiety. Analogue 12-3 has been
advanced to clinical trials.
cases, nucleophilic displacement of benzyltrimethylammonium
halides was more efficient (Scheme 3). Alternatively, palladium-
mediated coupling of isoxazole 16 with phenylboronic acids
followed by basic hydrolysis could be used to generate
ꢀ-ketonitriles 6 (Scheme 4).45
Analogues with two different ortho substituents on the
8-phenyl group can exist as atropisomers. Compound 12-16
is one such example. Proton NMR spectroscopy reveals the
existence of atropisomers for 12-16 (which is a sulfuric acid
salt (1:1 molar ratio) crystallized from 2-propanol) due to
restricted rotation of the phenyl ring. Two signals are observed
in DMSO-d6 at 500 MHz for the methyl group at the 2-position
of the phenyl ring (δ 1.99, 1.98) and for the methoxy group at
the 6-position (δ 3.65, 3.64). Chiral HPLC (OD column, 1%
2-propanol-hexanes (v/v)) separates the free bases of the two
atropisomers. The melting points for the two atropisomers were
similar (60-61 °C for the first isomer; 59-60 °C for the second
isomer). Their optical rotations were significantly different ([R]2D5
-101° (0.256 g/dL, MeOH) for the first isomer; [R2D5] +42.2°
(0.252 g/dL, MeOH) for the second isomer). Variable temper-
ature NMR studies in DMSO-d6 on the first atropisomer with
the low retention time reveal that it is slowly converted to the
other atropisomer at 22 °C (t1/2 ) 2200 h) and rapidly converted
at 100 °C (t1/2 ) 0.5 h). This conversion raises questions about
long-term solid state stability of a potential dosage form for
this compound.
Results
Chemistry. Pyrazolo[1,5-a]-1,3,5-triazines 12 were synthe-
sized by the general method, which is illustrated in Scheme 2.
The phenylacetonitrile precursors were usually prepared by
nucleophilic displacement of benzyl halides with cyanide.
However, this method sometimes provided low yields, especially
when the phenyl ring has electron-donating substituents. In these
Pharmacology. Pyrazolo[1,5-a]-1,3,5-triazines 12 were pro-
gressed through a series of in vitro and in vivo tests to identify

Methoxyphenylpyrazolotriazines
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9 3075
Scheme 4
a preclinical candidate. The compounds were first tested for their
binding affinity to rat cortical homogenate CRF receptors.46
Compounds with high receptor binding affinity (IC50 e 10 nM)
were then evaluated in dog plasma ex vivo binding studies to
estimate plasma exposure after oral administration (5 mg/kg).
Analogues, which displaced 125I-Tyr0-ovine-CRF ex vivo by
more than 30% at 10 h postdose, were submitted to discrete
dog pharmacokinetic studies. Select analogues with good
discrete dog pharmacokinetic profiles (oral bioavailability of
>10% at 5 mg/kg, po) were submitted to secondary pharma-
cological and behavioral tests. The leading compounds were
also tested for their affinity to CRF1 receptors endogenously
expressed in human IMR32 neuroblastoma cells,30 since rat
cortical membranes contain CRF1 and, to a much lesser extent,
CRF2 sites. Antagonist potency was determined by measuring
a compound’s effects on c-AMP production in rat cortical
homogenates. The potential anxiolytic activity of these agents
was assessed in the rat situational anxiety and elevated plus
maze models.47,48
Compounds 12-3, 12-16, 12-33, and 12-35 were profiled in
other receptor binding assays to determine their cross-reactivity
with other sites. All four compounds have high affinity for
human CRF1 receptors which are endogenously expressed in
human IMR32 cells. Analogues 12-3, 12-16, 12-33, and 12-35
have mean IC50 values ((standard deviation) equal to 4.7 (
2.0 nM (n ) 6), 3.4 ( 1.4 nM (n ) 6), 15.2 ( 1.6 nM (n ) 3),
and 1.2 ( 0.2 (n ) 5), respectively, while R-helical CRF9-41
has comparable affinity (IC50 ) 2.0 ( 0.5 nM, n ) 20). None
of the compounds binds human CRF2R receptors (IC50 > 1000
nM).26 These compounds have no significant affinity (IC50
>
1000 nM) for 30 other receptors or ion channels in a NovaScreen
battery of assays (e.g., adrenergic R1a, R1b, R2b, R2c; dopamine
D1, D2; serotonin, calcium type L or N, or vasopressin V1). None
of these compounds have affinity for the CRF binding protein
(IC50 > 1000 nM).49,50
The four leading compounds are silent and selective antago-
nists of rat CRF1 receptors. No agonist activity was detected in
a CRF-stimulated adenylate cyclase assay, which employed rat
brain homogenates. The compounds have no effect on isopro-
terenol-stimulated or basal adenylate cyclase activity. Com-
pounds 12-3, 12-16, 12-33, 12-35, and R-helical CRF9-41 have
rat IC50 values equal to 66 ( 10, 110 ( 8, 38 ( 3, 36 ( 5, and
264 ( 85 nM, respectively (n ) 3 for all compounds). These
analogues have no functional agonist or antagonist activity at
the cloned human CRF2R receptors expressed in human embry-
onic kidney-293 (HEK293) cells.50
Compounds that have superior magnitude and duration of
inhibition in the ex vivo binding assay (>40% inhibition at 10 h
postdose) were advanced to discrete dog pharmacokinetic
studies. Data for only the leading analogues are presented in
Table 3. Compound 12-35 had tolerability issues in the dog
and was deprioritized for further studies. The remaining
compounds were formulated as salts to facilitate oral dosing.
The 2-naphthalenesulfonic acid salt of analogue 12-3 was
initially used in animal studies. Later studies would demonstrate
that the benzenesulfonic acid salt of 12-3 had better pharma-
ceutic properties.51 Three compounds 12-3, 12-16, and 12-33
have moderate to low clearances in the dog when compared to
the hepatic blood flow (1.86 (L/h)/kg). The compounds also
have high volumes of distribution and long elimination half-
lives in the dog. Analogues 12-3 and 12-33 have oral bioavail-
ability in the dog (34% and 48%, respectively) which is superior
to that for compound 12-16 (4.7%). Analogue 12-16 has the
lowest maximal concentration (Cmax) after oral dosing (0.48 µM
normalized to 5 mg/kg). This compound was excluded from
further in vivo studies because of its inferior dog pharmacoki-
netic profile and the atropisomer issue (vide supra). Compound
12-3 was selected for additional studies over analogue 12-33
because of their divergent profiles in dog safety assessment
studies.
The rat receptor binding affinity data for compounds 12 are
summarized in Table 1. Compounds bearing a methoxy group
at the 4-position of the phenyl ring generally have high affinity
for rat CRF receptors. Hydrogen, methyl, chloro, and bromo
substituents are well tolerated at 2-position. Analogues 12-1,
12-3, 12-34, 12-35, 12-38, 12-40 and 12-42 have high affinity
for rat cortical CRF receptors. Methyl substitution at the
3-position reduces binding affinity in one case (compare 12-4
with 12-43). The introduction of a fluorine or methyl group at
the 5-position of the phenyl ring and the addition of methyl or
methoxy groups at the 6-position are also well tolerated (see
analogues 12-34 through 12-42). Diverse substituted alkyl amino
groups (secondary or tertiary) on the bicyclic core at the
6-position contribute to high receptor binding affinity in the
2-methyl-4-methoxyphenyl series and the 2-methyl-4,6-dimethox-
yphenyl series, which is consistent with SAR from other bicyclic
CRF antagonist series.1-3 Compounds in the 2-methyl-4,6-
dimethoxyphenyl series potentially could form atropisomers, but
we could only detect atropisomers in the case of analogue 12-
16. The atropisomers of 12-16 have similar affinity for rat
cortical homogenate CRF receptors (rat CRF IC50 ) 4.4 ( 0.6
nM (n ) 3) for the first isomer with low retention time and 5.2
( 1.0 nM (n ) 3) for the second isomer compared to 1.24 (
0.8 nM (n ) 8) for R-helical CRF9-41).
Compounds with high rat receptor binding affinity (IC50
<
10 nM) were next submitted to an ex vivo binding assay after
oral administration to dogs (5 mg/kg). Table 2 delineates the
data only for the leading compounds. Plasma samples were
drawn at 2 and 10 h postdose, diluted 10-fold with the receptor
binding assay buffer, and submitted to the standard binding
assay. Compounds that displaced 125I-Tyr0-ovine-CRF from rat
receptors were prioritized for discrete pharmacokinetic studies
based on the magnitude and duration of the binding inhibition.
While this assay cannot distinguish parent compound from active
metabolite, its high throughput made it a useful screening tool.
Several compounds significantly displace the radioligand at 10 h
postdose (i.e., >40% inhibition).
Compound 12-3 was evaluated in the rat elevated plus maze
test (Figure 1).52 Vehicle-treated animals spent less than 10%
of the test time in the open arms of the elevated-plus maze
(Figure 1, left panel). The mean percent time in open arms was
8.4 ( 2.3%. The positive control 2 (rat IC50 ) 4.2 ( 0.2 nM

3076 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9
Table 1. Rat Receptor Binding Affinity Dataa
Gilligan et al.
compd
R1
R2
R3
R4
R5
R6
rat IC50 + SD (nM)
n
12-1
12-2
12-3
12-4
12-5
12-6
12-7
12-8
N(CH2CH2OMe)2
NHCH(Et)2
N(CH2CH2OMe)2
NEt2
NPr2
NEtBu
NPr(CH2-c-C3H5)
NPr(CH2CH2CN)
N(c-C3H5)CH2CH2CN
pyrrolidinyl
NHCH(CH2OMe)2
NHCH(CH2OEt)2
NH-c-C4H7
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Me
Me
MeO
MeO
MeO
MeO
MeO
MeO
MeO
MeO
MeO
MeO
MeO
MeO
MeO
MeO
MeO
MeO
MeO
MeO
MeO
MeO
MeO
MeO
MeO
MeO
MeO
MeO
MeO
MeO
MeO
MeO
MeO
MeO
MeO
MeO
MeO
MeO
MeO
MeO
MeO
MeO
MeO
MeO
MeO
MeO
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
11.52 ( 2.46
3.80 ( 1.86
4.20 ( 0.40
6.08 ( 1.42
2.58 ( 0.90
2.30 ( 0.16
1.96 ( 1.02
2.16 ( 1.84
1.88 ( 0.76
80.66 ( 32.40
9.70 ( 9.68
19.34 ( 6.18
24.64 ( 5.94
5.00 ( 4.46
2.90 ( 0.84
5.70 ( 2.10
2.14 ( 0.58
17.42 ( 3.10
1.20 ( 0.14
3.72 ( 3.64
1.00 ( 0.34
1.48 ( 0.50
6.48 ( 2.26
4.92 ( 1.40
1.72 ( 0.72
1.78 ( 0.58
1.80 ( 0.48
7.96 ( 0.88
2.48 ( 0.34
3.26 ( 0.82
2.74 ( 0.94
4.22 ( 0.72
1.98 ( 0.14
1.90 ( 0.76
1.00 ( 0.22
1.64 ( 0.94
1.18 ( 0.38
8.00 ( 3.80
6.08 ( 1.48
4.06 ( 0.76
4.99 ( 1.44
13.68 ( 2.08
38.56 ( 8.22
11.32 ( 5.92
1.24 ( 0.8
3
3
9
3
3
3
3
3
4
4
4
4
4
4
3
3
3
4
4
3
3
5
3
3
3
4
5
3
3
3
3
3
3
3
5
4
3
3
3
3
3
3
3
3
8
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Cl
12-9
12-10
12-11
12-12
12-13
12-14
12-15
12-16
12-17
12-18
12-19
12-20
12-21
12-22
12-23
12-24
12-25
12-26
12-27
12-28
12-29
12-30
12-31
12-32
12-33
12-34
12-35
12-36
12-37
12-38
12-39
12-40
12-41
12-42
12-43
12-44
H
NHCH(Et)2
MeO
MeO
MeO
MeO
MeO
MeO
MeO
MeO
MeO
MeO
MeO
MeO
MeO
MeO
MeO
MeO
MeO
Me
Me
H
(S)-NHCH(Me)Et
(R)-NHCH(Me)Et
NHCH(Me)Pr
NH-c-C4H9
NEt2
NPr2
N(CH2CHdCH2)2
N(Pr)CH2-c-C3H5
N(Me)Pr
N(Me)CH2CHdCH2
NEtBu
N(Et)CH2CH2OMe
N(CH2-c-C3H5)CH2CH2OMe
N(CH2CH2OMe)2
N(Et)CH2CH2CN
2-Et-piperidinyl
N(CH2CH2OMe)2
NMeBu
N(CH2CH2OMe)2
NHCHEt2
NHCHEt2
Me
F
F
F
F
MeO
MeO
MeO
MeO
MeO
H
H
H
H
H
H
H
H
H
NEt2
Cl
Cl
Cl
Br
Br
Br
Me
Me
Me
N(CH2CH2OMe)2
N(CH2CH2OMe)2
NHCHEt2
NEt2
N(CH2CH2OMe)2
N(CH2CH2OMe)2
NEt2
H
H
H
NHCHEt2
H
R-helical CRF9-41
a SD ) standard deviation. n ) number of measurements.
(n ) 3) at a dose of 18 mg/kg significantly increased the percent
time spent in open arms (p ) 0.03). Pretreatment with 12-3
(30 mg/kg) significantly increased percent time in open arms
[F(5,79) ) 2.31, p ) 0.05, analysis of variance (ANOVA)
followed by individual mean comparisons using Fisher’s least
significant difference test]. The lowest effective dose of 12-3
was 30 mg/kg (p ) 0.02) (Figure 1, left panel).
Analogue 12-3 was also tested in the rat defensive withdrawal
(situational anxiety) model.52 Vehicle-treated animals showed
long latencies to exit the dark chamber and explore the open
field (Figure 1, right panel). The mean exit latency was 655 (
114 s (73% of the total test duration). The positive control 2 at
the dose of 10 mg/kg decreased exit latency by 52% relative to
vehicle control (p ) 0.04). Pretreatment with 12-3 decreases
exit latency (H(5) ) 11.9, p ) 0.04, the Kruskal-Wallis test,
followed by individual comparisons using the Mann-Whitney
U test). The lowest effective dose of 3.0 mg/kg decreased exit
latency by 62% relative to vehicle-treated animals (p ) 0.0009;
Figure 1, right panel). Higher doses of 12-3 (10 and 30 mg/kg)
decreased exit latency by 73% and 59%, respectively (p ) 0.01
and p ) 0.03, respectively).
The differences in the efficacies of compounds 12-3 and 2
between the two behavioral models may be explained by
differences in the type and severity of the stressor present in
the two tests. Similar observations have been reported for the
peptide antagonists R-hel-CRF9-41 and astressin.9,10 The same
strain of rats was employed in both tests.
The CRF receptor occupancy of compound 12-3 increases
with oral dose. In a separate experiment, rats were dosed at
0.3, 1, 3, 10, and 30 mg/kg (po) using the same vehicle and

Methoxyphenylpyrazolotriazines
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9 3077
Table 2. Dog ex Vivo Binding Data (5 mg/kg, po)a
% inhibition ( SD
and 121 ( 36 s for vehicle and chlordiazepoxide (30 mg/kg
free base weight, po), respectively (mean ( SEM, n ) 8).
Compound 12-3 is less likely than chlordiazepoxide to induce
motor side effects at 30 mg/kg (po).
compd
2 h postdose
10 h postdose
n
12-16
12-15
12-35
12-14
12-26
12-33
12-18
12-3
72 ( 6
72 ( 9
97 ( 3
80 ( 12
86 ( 5
84 ( 1
52 ( 8
75 ( 3
56 ( 23
81 ( 3
83 ( 6
74 ( 3
66 ( 7
56 ( 18
54 ( 1
44 ( 7
46 ( 9
37 ( 8
30 ( 17
25 ( 8
3
3
3
3
Analogue 12-3 was evaluated in a rat pharmacokinetic study.
The single dose intravenous (iv) pharmacokinetics of compound
12-3 in male (n ) 3) and female (n ) 3) Sprague-Dawley CD
rats was investigated following administration of a 1 mg/kg iv
bolus dose. The single dose oral (po) pharmacokinetics in male
rats (n ) 3) was investigated at a dose level of 5 mg/kg.
Compound 12-3 was formulated in 20% ethanol in physiological
saline (apparent pH 3.4) for iv administration and in 0.5%
aqueous methylcellulose containing 1% Tween-80 for oral
administration. Following the iv bolus administration of 12-3,
plasma concentrations exhibited a biexponential decline with
time. The apparent volume of distribution at steady-state (Vss)
was high with a mean value of 14.6 ( 3.9 and 12.2 ( 2.3 L/kg
for the male and female rats, respectively, suggesting extensive
extravascular tissue distribution (standard deviations are re-
ported). Analogue 12-3 was cleared from the systemic circula-
tion in rats with mean values of systemic clearance (CL) of 1.2
( 0.2 (L/h)/kg in males and 0.87 ( 0.28 (L/h)/kg in females
(hepatic blood flow ) 3.3 (L/h)/kg). The mean elimination half-
life was 9.7 ( 2.8 h in males and 11.9 ( 2.9 h in females.
Overall, the iv pharmacokinetics were similar between the male
and female rats. Following oral administration of 5 mg/kg in
male rats, 12-3 appeared to be rapidly absorbed as indicated by
a mean Tmax of 0.5 h. The mean Cmax (observed peak plasma
concentrations) of 12-3 after the oral dose was 0.851 µM in
male rats. The apparent bioavailability (F) in the male rats was
51% relative to the 1 mg/kg iv dose.
3
3b
3
3b
3
12-19
12-34
3
a The doses are expressed as free base weights. The vehicle is Labrafil
unless otherwise noted. SD ) standard deviation. n ) number of
measurements. b Vehicle ) Tween-80/0.5% aqueous methylcellulose, 1:99
(v/v).
Table 3. Dog Discrete Pharmacokinetic Data (5 mg/kg, po)a
12-3b
12-16c
12-33d
dose (iv, mg/kg)
n
5
4
1
3
5
4
CL ((L/h)/kg)
Vss (L/kg)
t1/2 (h)
1.20 ( 0.25
18.45 ( 3.95
27.3 ( 6.8
0.60 ( 0.10
14.80 ( 0.10
33.3 ( 8.6
0.66 ( 0.11
23.71 ( 7.69
40.2 ( 9.60
dose (po, mg/kg)
n
5
4
20
3
5
5
Cmax (µM)
2.38 ( 0.39
0.5 ( 0.2
34.0
1.94 ( 0.30
4.0 ( 1.0
4.7
1.22 ( 0.29
0.8 ( 0.2
48.0
tmax (h)
F, po (%)
a The vehicle for iv studies was N,N-dimethylformamide/ethanol/
propylene glycol/water, 10:10:55:25. The vehicle for oral studies was 0.5%
Methocel-Tween-80 (99:1). Free base weights are reported for the doses.
n ) number of measurements. b The 2-naphthalenesulfonic acid salt was
used. c The sulfuric acid salt was used. d The benzenesulfonic acid salt was
used.
Compound 12-3 (BMS 561388)51,53 was advanced to clinical
studies in humans on the basis of its favorable preclinical profile.
It is a potent and selective antagonist of CRF1 receptors. It is
rapidly absorbed in both rats and dogs after oral administration.
Furthermore, this analogue did not cause toxicity in dogs unlike
some closely related analogues. The details of the safety
assessment studies and clinical trials will be presented in
separate publications.
protocol as those used in the defensive withdrawal test. Rats
were sacrificed and their brains were removed, sectioned, and
frozen at -80 °C. Subsequent ex vivo binding studies using
the published protocol52 established receptor occupancy relative
to vehicle-control animals. The receptor occupancy ((standard
error of the mean) was calculated to be 12 ( 13%, 17 ( 8%,
48 ( 12%, 60 ( 8%, and 82 ( 12% for the 0.3, 1, 3, 10, and
30 mg/kg (po). Thus, the minimal effective dose of compound
12-3 (3 mg/kg, po) corresponds to 48 ( 12% receptor occupancy
in ex vivo studies.
Compound 12-3 does not cause adverse motor effects at doses
that are efficacious in the anxiety models. The compound has
no effect in a rat open-field locomotor activity test47,48 or in a
rat rotorod performance test47,48 up to 30 mg/kg (po, free base
weight). The compound was administered by oral gavage in
0.5% methocel-Tween-80 (99:1), and behavior was monitored
at 1 h postdose as in the behavioral studies. There was no
difference between the vehicle control and compound 12-3 in
these two tests, while chlordiazepoxide blocked motor function
in these two tests at 30 mg/kg (po) when dosed in the same
vehicle. In the rat locomotor activity test, the total distances
traveled were 5286 ( 751 and 4986 ( 931 cm for vehicle and
compound 12-3 (30 mg/kg free base weight, po), respectively
(mean ( SEM, n ) 8). In a separate experiment in this test,
the total distances traveled were 5785 ( 941 and 2249 ( 286
cm for vehicle and chlordiazepoxide (30 mg/kg free base weight,
po), respectively (mean ( SEM, n ) 8). In the rat rotorod test,
the times on the rotorod were 153 ( 29 and 158 ( 93 s for
vehicle and compound 12-3 (30 mg/kg free base weight, po),
respectively (mean ( SEM, n ) 8). In a separate experiment
in this second model, the times on the rotorod were 198 ( 43
Experimental Section
Analytical data were recorded for the compounds described
below using the following general procedures. Proton NMR spectra
were recorded on a Varian VXR or Unity 300 FT-NMR instruments
(300 MHz); chemical shifts were recorded in ppm (δ) from an
internal tetramethysilane standard in deuterochloroform or deu-
terodimethyl sulfoxide as specified below. Mass spectra (MS) or
high resolution mass spectra (HRMS) were recorded on a Finnegan
MAT 8230 spectrometer or a Hewlett-Packard 5988A model
spectrometer (using chemi-ionization (CI) with NH3 as the carrier
gas, electrospray (ESI), atmospheric pressure chemi-ionization
(APCI) or gas chromatography (GC)). Melting points were recorded
on a MelTemp 3.0 heating block apparatus and are uncorrected.
Boiling points are uncorrected. All pH determinations during
workup were made with indicator paper.
Reagents were purchased from commercial sources and, where
necessary, purified prior to use according to previously described
general procedures.54 Chromatography was performed on silica gel
using the solvent systems indicated below. For mixed solvent
systems, the volume ratios are given. Otherwise, parts and percent-
ages are by weight.
The purity of final compounds was assessed by two analytical HPLC
methods or combustion analysis and was found to be greater than or
equal to 95% for all cases. Analytical HPLC analyses were performed
on a Rainin HPLC machine (dual SD-200 pumps) using a C18 column
(Dynamax 60A, 83-201C, 250 mm × 4.6 mm, 100 Å pore size, flow
rate ) 1 mL/min, solvent A ) 0.1% TFA-H2 O, solvent B ) 0.1%

3078 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9
Gilligan et al.
Figure 1. Anxiolytic-like effects of 12-3 in the elevated-plus maze (left) and defensive withdrawal tests (right) in rats. Abscissae indicate the dose
(mg/kg). cpd2@18 is compound 2 at 18 mg/kg, and cpd2@10 is compound 2 at 10 mg/kg. Left panel ordinate displays the mean ((SEM) percent
time in open arms for n ) 13-15 animals per dose. Right panel ordinate displays the mean ((SEM) latency to exit the dark chamber (in seconds)
for n ) 12 animals per dose. Maximum latency is 900 s. Analogue 12-3 and compound 2 were administered in 0.25% Methocel, po, 60 min prior
to testing: (*) p < 0.05; (**) p < 0.01.
TFA-CH3CN, gradient of 15-95% B over 15 min) (method A).
Analytical HPLC analyses were also performed on a Shimadzu HPLC
machine (model LC-10AT) using a C18 column (Zorbax SB, 700 mm
× 4.6 mm, 100 Å pore size, flow rate ) 2.5 mL/min, solvent A )
0.1% TFA-H2 O, solvent B ) 0.1% TFA-MeOH, gradient of
5-95% B over 15 min) (method B). Combustion analyses were
performed by Quantitative Technologies, Whitehouse, NJ.
Commonly used abbreviations are AIBN (azobis(isobutyro)ni-
trile), DMF (N,N-dimethylformamide), EtOH (ethanol), MeOH
(methanol), EtOAc (ethyl acetate), HOAc (acetic acid), DME (1,2-
diethoxyethane), and THF (tetrahydrofuran).
Step C: 5-Amino-4-(2-methyl-4-methoxyphenyl)-3-meth-
ylpyrazole. A mixture of 1-cyano-1-(2-methyl-4-methoxyphenyl)-
propan-2-one (4.5 g, 22.2 mmol), hydrazine hydrate (2.25 mL, 44.4
mmol), glacial acetic acid (4.3 mL, 75 mmol), and toluene (57 mL)
was stirred in an apparatus fitted with a Dean-Stark trap at reflux
temperature for 18 h under a nitrogen atmosphere. The reaction
mixture was cooled to ambient temperature, and solvent was
removed in vacuo. The residue was dissolved in 4 N HCl, and the
resulting solution was extracted with ether three times. A concen-
trated sodium hydroxide solution was added to the aqueous layer
until pH 11 (test paper) was attained. The resulting semisolution
was extracted three times with ethyl acetate (100 mL). The
combined organic layers were dried over MgSO4 and filtered.
Solvent was removed in vacuo to give a yellow solid (4.0 g, 83%
4-(Bis(2-methoxyethyl)amino)-2,7-dimethyl-8-(2-methyl-4-
methoxyphenyl)[1,5-a]pyrazolo-1,3,5-triazine, Benzenesulfonic
Acid Salt (12-3). Step A: 4-Methoxy-2-methylbenzyl Cyanide.
A mixture of 3,4-dimethylanisole (24.5 g, 0.18 mol), N-bromosuc-
cinimide (32 g (0.18 mol), and AIBN (1 g, 0.006 mol) in carbon
tetrachloride (350 mL) was stirred at reflux temperature for 2 h.
The reaction mixture was cooled to ambient temperature and
filtered. Solvent was removed in vacuo to provide a yellow oil
(30 g), which was used immediately without further purification.
1H NMR (CDCl3, 300 MHz): δ 7.21 (d, 1H, J ) 8), 6.70 (m, 2H),
4.5 (s, 2H), 3.80 (s, 3H), 2.4 (s, 3H).
1
yield). H NMR (CDCl3, 300 MHz): δ 7.10 (d, 1H, J ) 8), 6.85
(d, 1H, J ) 1), 6.80 (dd, 1H, J ) 8,1), 3.85 (s, 3H), 2.20 (s, 3H),
2.18 (m, 2H), 2.05 (s, 3H). APCI-MS (NH3): 218 (M+ + H).
Step D: 5-Acetamidino-4-(2,5-dimethyl-4-methoxyphenyl)-3-
methylpyrazole, Acetic Acid Salt. A mixture of glacial acetic acid
(1.04 mL, 18.4 mmol), 5-amino-4-(2-methyl-4-methoxyphenyl)-3-
methylpyrazole (4.0 g, 18.4 mol), ethyl acetamidate (2.37 g, 27.2
mol), and acetonitrile (80 mL) was stirred at room temperature for
20 h. Solvent was removed in vacuo to about one-third of the
original volume. The resulting suspension was filtered, and the col-
lected solid was washed with copious amounts of ether. The white
The yellow oil was dissolved in DMF (75 mL). The solution
was added dropwise to a mechanically stirred solution of sodium
cyanide (12.3 g, 0.35 mol) in ethanol (500 mL) and water (250
mL) at 90 °C. The mixture was stirred at this temperature for 18 h.
The mixture was diluted with water (1000 mL) and extracted three
times with EtOAc (200 mL). The combined organic layers were
dried over MgSO4 and filtered. Solvent was removed in vacuo to
give a light-brown oil. Column chromatography (EtOAc/hexane,
1:9) provided the desired product as a yellow oil (6.5 g, 22% overall
1
solid was dried in vacuo (4.2 g, 76% yield). H NMR (DMSO-
d6,300 MHz): δ 6.95 (d, 1H, J ) 8), 6.8 (d, 1H, J ) 1), 6.75 (dd,
1H, J ) 8, 1), 3.75 (s, 3H), 2.10 (s, 3H), 2.0 (s, 3H), 1.9 (s, 3H).
APCI-MS (NH3): 259 (M+ + H, free base).
Step E: 2,7-Dimethyl-8-(2-methyl-4-methoxyphenyl)[1,5-a]-
pyrazolo[1,3,5]triazin-4(3H)-one. Sodium pellets (4.0 g, 0.175
mol) were added portionwise to ethanol (100 mL) with vigorous
stirring. After all the sodium reacted, the reaction mixture was
cooled to room temperature. 5-Acetamidino-4-(2-methyl-4-meth-
oxyphenyl)-3-methylpyrazole, acetic acid salt (1.65 g, 5.48 mmol),
and diethyl carbonate (8.6 mL, 430 mmol) were added sequentially.
The resulting reaction mixture was heated to reflux temperature
and stirred for 69 h. The mixture was cooled to room temperature.
Solvent was removed in vacuo. The residue was dissolved in water,
and a concentrated HCl solution was added slowly until pH 7 (test
paper) was attained. The aqueous mix was extracted with ethyl
acetate (100 mL) three times; the combined organic layers were
dried over MgSO4 and filtered. Solvent was removed in vacuo to
give a white solid (1.33 g, 85% yield). 1H NMR (CDCl3, 300 MHz):
δ 7.10 (d, 1H, J ) 8), 6.83 (m, 2H), 3.85 (s, 3H), 2.50 (s, 3H),
2.30 (s, 3H), 2.15 (s, 3H). CI-MS (NH3): 285 (M+ + H).
1
yield). H NMR (CDCl3, 300 MHz): δ 7.25 (d, 1H, J ) 8), 6.75
(m, 2H), 3.8 (s, 3H), 3.6 (s, 2H), 2.30 (s, 3H). APCI-MS: 162
(M + H).
Step B: 1-Cyano-1-(2-methyl-4-methoxyphenyl)propan-2-one.
The above nitrile (6.5 g, 40.4 mmol) was dissolved in EtOAc
(200 mL). Sodium pellets (1.2 g, 52.2 mmol) were added
portionwise over 15 min. The reaction mixture was heated to
reflux temperature and stirred for 3 h under a nitrogen
atmosphere. The reaction mixture was cooled to room temper-
ature, and the creme colored suspension was filtered. The
precipitate was dissolved in a minimum amount of water. A
concentrated HCl solution was added dropwise with stirring until
pH 5 (test paper) was attained. The mixture was extracted three
times with EtOAc (100 mL). The combined organic layers were
dried over MgSO4 and filtered. Solvent was removed in vacuo
1
to provide a yellow oil (4.5 g, 55% yield). H NMR (CDCl3,
Step F: 4-Chloro-2,7-dimethyl-8-(2-methyl-4-methoxyphe-
nyl)[1,5-a]pyrazolotriazine. A mixture of 2,7-dimethyl-8-(2-meth-
yl-4-methoxyphenyl)[1,5-a]pyrazolo-1,3,5-triazin-4-one (8.2 g, 28.9
mmol), N,N-diethylaniline (18.5 mL, 116 mmol), phosphorus
300 MHz): δ 7.30 (dd, 1H, J ) 8, 1), 6.80 (m, 2H), 4.75
(s, 1H), 3.8 (s, 3H), 2.3 (s, 3H), 2.2 (s, 3H). APCI-MS: 203
(M + H).

Methoxyphenylpyrazolotriazines
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9 3079
oxychloride (10.8 mL, 116 mmol), and toluene (200 mL) was stirred
at reflux temperature for 42 h. The reaction mixture was concen-
trated in vacuo to give an oil. Flash column chromatography (ethyl
acetate.hexanes, 1:9) gave a yellow oil after removal of solvent in
vacuo (5.1 g, 58% yield). 1H NMR (CDCl3, 300 MHz): δ 7.10 (d,
1H, J ) 7), 6.90 (d, 1H, J ) 1), 6.85 (dd, 1H, J ) 7,1), 3.85 (s,
3H), 2.65 (s, 3H), 2.45 (s, 3H), 2.15 (s, 3H). CI-MS: 303, 305 (M+
+ H).
The following compounds were prepared by methods analogous
to those for compound 12-3, by converting the appropriate
phenylacetonitrile precursor to the chloropyrazolotriazine intermedi-
ate, followed by nucleophilic displacement with an amine.
12-1: oil. NMR (CDCl3, 300 MHz): δ7.58 (d, 2H, J ) 9), 6.99
(d, 2H, J ) 9), 4.32 (s, 4H), 3.84 (s, 3H), 3.76 (t, 4H, J ) 6), 3.37
(s, 6H), 2.47 (s, 3H), 2.43 (s, 3H). CI-MS calcd for C20H28N5O3:
386. Found: 386 (M + H). Anal.: HPLC.
Step G: 4-(Bis(2-methoxyethyl)amino)-2,7-dimethyl-8-(2,5-
dimethyl-4-methoxyphenyl)[1,5-a]pyrazolo-1,3,5-triazine (the
Free Base of 12-3). Sodium hydride (60% in oil, 1.32 g, 33 mmol)
was washed with hexanes, and the solvent was decanted. The solid
was immediately taken up in THF (50 mL). Bis(2-methoxyethyl)-
amine (4.55 g, 33 mmol) was added dropwise with vigorous stirring.
Gas evolution ensued. The reaction mixture was stirred for 30 min.
A mixture of 4-chloro-2,7-dimethyl-8-(2,5-dimethyl-4-methoxyphe-
nyl)[1,5-a]pyrazolo-1,3,5-triazine (5.1 g, 17 mmol), and tetrahy-
drofuran (50 mL) was added dropwise, and stirring was continued
at ambient temperature for 18 h. The reaction mixture was poured
carefully onto water (250 mL) and extracted three times with ethyl
acetate. The combined organic layers were dried over MgSO4 and
filtered. Solvent was removed in vacuo to give an oil. The product
became a white solid after prolonged standing (4.2 g, 32% yield):
12-2: oil. NMR (CDCl3, 300 MHz): δ 7.57 (d, 2H, J ) 9), 7.00
(d, 2H, J ) 9), 6.16 (d, 1H, J ) 9), 4.20 (m, 1H), 3.84 (s, 3H),
2.53 (s, 3H), 2.51 (s, 3H), 1.76 (m, 2H), 1.62 (m, 2H), 0.99 (t, 6H,
J ) 7). CI-MS calcd for C19H26N5O: 340. Found: 340 (M + H).
Anal.: HPLC.
12-4: oil. NMR (CDCl3, 300 MHz): δ 7.10 (d, 1H, J ) 8), 6.85
(d, 1H, J ) 1), 6.75 (dd, 1H, J ) 8,1), 4.20-4.00 (br m, 4H), 3.85
(s, 3H, 2.40 (s, 3H), 2.30 (s, 3H), 2.20 (s, 3H), 1.35 (t, 6H, J ) 7).
CI-HRMS calcd for C19H26N5O: 340.2137. Found: 340.2122
(M + H). Anal.: HPLC.
12-5: oil. NMR (CDCl3, 300 MHz): δ 7.03 (d, 1H, J ) 8), 6.86
(d, 1H, J ) 1), 6.76 (dd, 1H, J ) 8, 1), 4.94 (m, 4H), 3.75 (s, 3H),
2.31 (s, 3H), 2.18 (s, 3H), 2.11 (s, 3H), 1.70 (m, 4H), 0.91 (t, 6H,
J ) 7). CI-HRMS calcd for C21H30N5O: 368.2450. Found: 368.2454
(M + H). Anal.: HPLC.
12-6: oil. NMR (CDCl3, 300 MHz): δ 7.12 (d, 1H, J ) 8), 6.85
(d, 1H, J ) 1), 6.78 (dd, 1H, J ) 8, 1), 4.24 (m, 4H), 3.75 (s, 3H),
2.39 (s, 3H), 2.25 (s, 3H), 2.18 (s, 3H), 1.75 (m, 2H), 1.4 (m, 5H),
1.00 (t, 3H, J ) 7). CI-HRMS calcd for C21H30N5O: 368.2451.
Found: 368.2473 (M + H). Anal.: HPLC.
12-7: oil. NMR (CDCl3, 300 MHz): δ 7.11 (d, 1H, J ) 9), 6.84
(d, 1H, J ) 3), 6.78 (dd, 1H, J ) 9, 3), 4.10 (m, 2H), 3.93
(m, 2H), 3.82 (s, 3H), 2.39 (s, 3H), 2.25 (s, 3H), 2.18 (s, 3H), 1.80
(m, 2H), 1.25 (m, 1H), 0.97 (t, 3H, J ) 7), 0.56 (m, 2H), 0.37 (m,
2H). CI-HRMS calcd for C22H30N5O: 379.5. Found: 380.2 (M +
H). Anal.: HPLC.
12-8: oil. NMR (CDCl3, 300 MHz): δ 7.10 (d, 1H, J ) 8), 6.85
(d, 1H, J ) 1), 6.80 (dd, 1H, J ) 8,1), 4.15 (m, 4H), 3.85 (s, 3H),
3.00 (t, 2H, J ) 7), 2.45 (s, 3H), 2.30 (s, 3H), 2.20 (s, 3H), 1.85
(m, 2H), 1.00 (t, 3H, J ) 7). CI-HRMS calcd for C21H27N6O:
379.2246. Found: 379.2248 (M + H). Anal.: HPLC.
12-9: solid; mp 129 °C. NMR (CDCl3, 300 MHz): δ 7.10 (d,
1H, J ) 8), 6.90 (d, 1H, J ) 1), 6.80 (dd, 1H, J ) 8,1), 4.45 (m,
2H), 3.85 (s, 3H), 3.35 (m, 1H), 2.85 (t, 2H, J ) 7), 2.50 (s, 3H),
2.30 (s, 3H), 2.20 (s, 3H), 1.05 (m, 2H), 0.70 (m, 2H). CI-HRMS
calcd for C21H25N6O: 377.2090. Found: 377.2092 (M + H). Anal.:
HPLC.
12-10: oil. NMR (CDCl3, 300 MHz): δ 7.11 (d, 1H, J ) 9),
6.84 (d, 1H, J ) 3), 6.78 (dd, 1H, J ) 9,3), 4.10 (m, 2H), 3.82 (s,
3H), 2.40 (s, 3H), 2.25 (s, 3H), 2.18 (s, 3H), 2.03 (m, 4H), 1.20
(m, 2H). CI-MS calcd for C18H24N5O: 338. Found: 338 (M + H).
Anal.: HPLC.
12-11: solid, mp 45-46 °C. NMR (CDCl3, 300 MHz): δ 7.10
(d, 1H, J ) 8), 6.90 (d, 1H, J ) 1), 6.80 (dd, 1H, J ) 8,1), 6.70
(br d, 1H, J ) 8), 4.65 (m, 1H), 3.85 (s, 3H), 3.63 (m, 4H), 3.45
(s, 6H), 2.50 (s, 3H), 2.30 (s, 3H), 2.15 (s, 3H). CI-HRMS calcd
for C20H28N5O3: 386.2192. Found: 386.2181 (M + H). Anal.:
HPLC.
12-12: oil. NMR (CDCl3, 300 MHz): δ 7.10 (d, 1H, J ) 8),
6.85 (d, 1H, J ) 1), 6.75 (dd, 1H, J ) 8,1), 4.60 (m, 1H), 3.85 (s,
3H), 3.72 (m, 4H), 3.60 (q, 4H, J ) 7), 2.50 (s, 3H), 2.30 (s, 3H),
2.20 (s, 3H), 1.25 (t, 6H, J ) 7). CI-HRMS calcd for C22H32N5O3:
413.2427. Found: 413.2416 (M + H). Anal.: HPLC.
12-13: solid; mp 94.5-96 °C. NMR (CDCl3, 300 MHz): δ 7.11
(d, 1H, J ) 8), 6.85 (d, 1H, J ) 3), 6.78 (dd, 1H, J ) 8,3), 6.53
(d, 1H, J ) 8), 4.77 (m, 1H), 3.82 (s, 3H), 2.51 (m, 2H), 2.48 (s,
3H), 2.30 (s, 3H), 2.17 (s, 3H), 2.10 (m, 2H), 1.95 (m, 2H). CI-
MS calcd for C19H24N5O: 366 (M + H)+. Anal.: HPLC.
12-14: solid; mp 145-146 °C. NMR (CDCl3, 300 MHz): δ 6.47
(d,1H, J ) 3), 6.39 (d, 1H, J ) 3), 6.16 (d, 1H, J ) 10), 4.18 (m,
1H), 3.83 (s, 3H), 3.72 (s, 3H), 2.45 (s, 3H), 2.23 (s, 3H), 2.12 (s,
3H), 1.63 (m, 4H), 1.00 (t, 6H, J ) 8). CI-HRMS calcd for
1
mp 57.3 °C (DSC); HPLC log P ) 4.76. H NMR (CDCl3, 300
MHz): δ 7.10 (d, 1H, J ) 8), 6.85 (d, 1H, J ) 3), 6.78 (dd, 1H, J
) 8,3), 4.45-4.25 (br s, 4H), 3.82 (s, 3H), 3.76 (t, 4H, J ) 5),
3.38 (s, 6H), 2.39 (s, 3H), 2.24 (s, 3H), 2.17 (s, 3H). 13C NMR
(CDCl3, 75.44 MHz): δ 162.2, 159.0, 153.2, 149.4, 149.0, 139.5,
132.2, 123.5, 115.7, 111.2, 106.8, 71.9, 58.9 (2C), 55.2 (2C), 50.8
(2C), 25.7, 20.6, 13.3. IR (neat, KBr, cm-1): 2926 (m), 2893 (m),
2830 (m), 1616 (sh), 1604 (s), 1563 (s), 1534 (s), 1465 (s), 1442
(s), 1404 (m), 1373 (m), 1356 (m), 1310 (m), 1292 (m), 1278 (m),
1241 (m), 1204 (m), 1186 (m), 1162 (m), 1116 (m), 1047 (m),
1013 (m), 1002 (m). CI-HRMS (NH3) calcd for C21H29N5O3:
400.2349. Found: 400.2336 (M+ + H). Anal. (C21H29N5O3) C,
H, N.
Step H: 4-(Bis(2-methoxyethyl)amino)-2,7-dimethyl-8-(2-
methyl-4-methoxyphenyl)[1,5-a]pyrazolo-1,3,5-triazine, Benze-
nesulfonic Acid Salt (12-3). A mixture of 4-(bis(2-methoxyethyl)-
amino)-2,7-dimethyl-8-(2-methyl-4-methoxyphenyl)[1,5-a]pyrazolo-
1,3,5-triazine (1.5 g, 3.7 mmol), benzenesulfonic acid (0.5 g, 3.7
mmol), ether (50 mL), and ethanol (10 mL) was stirred at ambient
temperature for 1 h. Solvent was removed in vacuo; the residue
was triturated with copious amounts of ether. Filtration and drying
in vacuo afforded a white crystalline solid (1.2 g, 82% yield) which
1
is a hemihydrate: mp 97.5 °C (DSC). H NMR (DMSO-d6, 300
MHz): δ 7.60 (m, 2H), 7.31 (m, 3H), 7.10 (d, 1H, J ) 9), 6.92 (d,
1H, J ) 3), 6.84 (dd, 1H, J ) 9,3), 4.30 (br s, 4H), 3.79 (s, 3H),
3.69 (t, 4H, J ) 5), 3.28 (s, 6H), 2.35 (s, 3H), 2.19 (s, 3H), 2.09
(s, 3H). Anal. (C21H29N5O3 ·C10H8O3S·0.5H2O) C, H, N, S.
4-(Bis(2-methoxyethyl)amino)-2,7-dimethyl-8-(2-methyl-4-
methoxyphenyl)[1,5-a]pyrazolo-1,3,5-triazine, 2-Naphthalene-
sulfonic Acid Salt (2-Naphthalenesulfonic Acid Salt Analogue
of 12-3). A mixture of 4-(bis(2-methoxyethyl)amino)-2,7-dimethyl-
8-(2-methyl-4-methoxyphenyl)[1,5-a]-pyrazolo-1,3,5-triazine (132
mg, 0.23mmol), 2-naphthalenesulfonic acid (36.1 mg, 0.23 mmol),
and ether (5 mL) was stirred at ambient temperature for 1 h. Solvent
was removed in vacuo; the residue was triturated with copious
amounts of ether. Filtration and drying in vacuo afforded a white
crystalline solid (100 mg, 77% yield): mp 129.5 °C (differential
scan calorimetry). 1H NMR (DMSO-d6, 300 MHz): δ 8.14 (s, 1H),
8.00-7.84 (m, 4H), 7.70 (d, 1H, J ) 8), 7.55-7.50 (m, 2H), 7.10
(d, 1H, J ) 8), 6.93 (d, 1H, J ) 3), 6.84 (dd, 1H, J ) 8,3),
4.45-4.25 (br s, 4H), 3.79 (s, 3H), 3.71 (t, 4H, J ) 5), 3.28 (s,
6H), 2.36 (s, 3H), 2.19 (s, 3H), 2.09 (s, 3H). IR (neat, KBr, cm-1):
3440 (br, m), 3056 (m), 2926 (m), 2836 (m), 2552 (m), 1660 (s),
1606 (s), 1568 (s), 1504 (m), 1454 (s), 1374 (m), 1358 (m), 1344
(m), 1316 (s), 1244 (s), 1202 (m), 1166 (s), 1118 (s), 1134 (s),
1118 (s), 1090 (s), 1016 (s). CI-HRMS (NH3) calcd for C21H29N5O3:
400.2349. Found: 400.2336 (M+ + H). Anal. (C21H29N5O3 ·
C10H8O3S) C, H, N, S.

3080 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9
Gilligan et al.
C21H30N5O2: 368.2450. Found: 368.2454 (M + H). Anal.
(C21H29N5O2) C, H, N.
12-27: oil. NMR (CDCl3, 300 MHz): δ 6.47 (d,1H, J ) 2), 6.39
(d, 1H, J ) 2), 4.34 (m, 2H), 4.04 (m, 2H), 3.86 (s, 3H), 3.79 (t,
2H, J ) 6), 3.71 (s, 3H), 3.39 (s, 3H), 2.38 (s, 3H), 2.17 (s, 3H),
0.86 (m, 1H), 0.54 (m, 2H), 0.40 (m, 2H). CI-HRMS calcd for
C23H32N5O3: 426.2488. Found: 426.2488 (M + H). Anal.: HPLC.
12-28: oil. NMR (CDCl3, 300 MHz): δ 6.47 (d,1H, J ) 2), 6.39
(d, 1H, J ) 2), 4.30 (m, 4H), 3.83 (s, 3H), 3.77 (t, 4H, J ) 6),
3.71 (s, 3H), 3.39 (s, 6H), 2.37 (s, 3H), 2.16 (s, 3H), 2.09 (s, 3H).
CI-HRMS calcd for C22H32N5O4: 430.2454. Found: 430.2468(M
+ H). Anal.: HPLC.
12-29: oil. NMR (CDCl3, 300 MHz): δ 6.47 (d,1H, J ) 2), 6.39
(d, 1H, J ) 2), 4.15 (m, 4H), 3.83 (s, 3H), 3.71 (s, 3H), 3.01 (t,
2H, J ) 7), 2.39 (s, 3H), 2.18 (s, 3H), 2.09 (s, 3H), 1.38 (t, 3H, J
) 7). CI-HRMS calcd for C21H27N6O2: 395.4883. Found: 395.2192
(M + H). Anal.: HPLC.
12-30: solid. NMR (CDCl3, 300 MHz): δ 6.47 (d,1H, J ) 2),
6.39 (d, 1H, J ) 2), 3.83 (s, 3H), 3.20 (s, 3H), 3.20 (m, 1H), 2.39
(s, 3H), 2.19 (s, 3H), 2.10 (s, 3H), 1.80 (m, 8H), 0.94 (dt, 3H, J )
7, 3). CI-HRMS calcd for C23H32N5O2: 410.5438. Found: 410.2558
(M + H). Anal.: HPLC.
12-31: solid; mp 117.7-118 °C. NMR (CDCl3, 300 MHz): δ
6.67 (s, 2H), 4.30 (m, 4H), 3.80 (s, 3H), 3.78 (t, 4H, J ) 8), 3.40
(s, 6H), 2.37 (s, 3H), 2.12 (s, 3H), 2.02 (s, 6H). CI-HRMS calcd
for C22H32N5O3: 414.2505. Found: 414.2492 (M + H). Anal.:
HPLC.
12-32: oil. NMR (CDCl3, 300 MHz): δ 6.47 (d, 1H, J ) 2),
6.38 (d, 1H, J ) 2), 4.10 (m, 2H), 3.82 (s, 3H), 3.71 (s, 3H), 3.56
(s, 3H), 2.39 (s, 3H), 2.18 (s, 3H), 2.09 (s, 3H), 1.75 (m, 2H), 1.40
(m, 2H), 0.98 (t, 3H, J ) 7). CI-HRMS calcd for C21H30N5O2:
384.2419. Found: 384.2413 (M + H). Anal.: HPLC.
12-33, free base: solid; mp 72 °C. NMR (CDCl3, 300 MHz): δ
6.93 (s, 1H), 6.77 (s, 1H), 4.20-4.45 (m, 4H), 3.84 (s, 3H), 3.76
(t, J ) 8, 4H), 3.40 (s, 6H), 2.40 (s, 3H), 2.25 (s, 3H), 2.19 (s,
3H), 2.15 (s, 3H). CI-HRMS calcd for C22H32N5O3: 414.2505.
Found: 414.2493 (M + H). Anal.: HPLC.
12-33: white crystalline solid, mp 168.6 °C. NMR (DMSO-d6,
300 MHz): δ 7.59 (m, 2H), 7.31 (m, 3H), 6.90 (d, 2H, J ) 8), 3.82
(s, 3H), 3.74 (t, 4H, J ) 6), 3.28 (s, 6H), 2.32 (s, 3H), 2.18 (s,
3H), 2.13 (s, 3H), 2.07 (s, 3H). CI-HRMS (NH3) calcd for
C22H31N5O3: 414.2505. Found: 414.2509 (M + H)+. Anal.
(C22H31N5O3 ·C6H5SO3H) C, H, N.
12-34: oil. NMR (CDCl3, 300 MHz): δ 6.94 (d, 1H, J ) 12),
6.89 (d, 1H, J ) 9), 6.18 (d, 1H, J ) 9), 4.20 (m, 2H), 3.91 (s,
3H), 2.47 (s, 3H), 2.30 (s, 3H), 2.17 (s, 3H), 1.65 (m, 4H), 1.00 (t,
3H, J ) 7). CI-HRMS calcd for C20H27FN5O: 372.2199. Found:
372.2203 (M + H). Anal.: HPLC.
12-35: solid. NMR (CDCl3, 300 MHz): δ 7.10 (d, 1H, J ) 11),
7.08 (d, 1H, J ) 8), 6.18 (d, 1H, J ) 10), 4.20 (m, 2H), 3.92 (s,
3H), 2.48 (s, 3H), 2.34 (s, 3H), 1.65 (m, 4H), 1.00 (t, 6H, J ) 7).
CI-HRMS calcd for C19H24ClFN5O: 392.1653 Found: 392.1649 (M
+ H). Anal. (C19H23ClFN5O) C, H, N.
12-36: solid, mp 96-98 °C. NMR (CDCl3, 300 MHz): δ 7.09
(d, 1H, J ) 12), 7.08 (d, 1H, J ) 8), 4.07 (m, 4H), 3.91 (s, 3H),
2.41 (s, 3H), 2.30 (s, 3H), 1.35 (t, 6H, J ) 7). CI-HRMS calcd for
C18H22ClFN5O: 378.1497. Found: 378.1500 (M + H). Anal.: HPLC.
12-37: solid. NMR (CDCl3, 300 MHz): δ 7.10 (d, 1H, J ) 4),
7.08 (d, 1H, J ) 8), 4.07 (m, 4H), 3.91 (s, 3H), 2.41 (s, 3H), 2.30
(s, 3H), 1.35 (t, 6H, J ) 7). CI-HRMS calcd for C20H26ClFN5O3:
438.1708. Found: 438.1701 (M + H). Anal.: HPLC.
12-38: solid, NMR (CDCl3, 300 MHz): δ 6.97 (s, 1H), 6.84 (s,
1H), 4.00 (m, 4H), 3.90 (s, 3H), 3.86 (s, 3H), 2.40 (s, 3H), 2.30 (s,
3H), 1.75 (m, 4H), 1.40 (m, 4H), 0.99 (t, 6H, J ) 7). CI-HRMS
calcd for C23H33ClN5O2: 446.2323. Found: 446.2326 (M + H).
Anal.: HPLC.
12-39: solid, mp 50-52 °C. NMR (CDCl3, 300 MHz): δ 7.14
(s, 1H), 6.82 (s, 1H), 6.18 (d, 1H, J ) 7), 4.20 (m, 1H), 3.91 (s,
3H), 3.85 (s, 3H), 2.49 (s, 3H), 2.35 (s, 3H), 1.75 (m, 4H), 1.00 (t,
6H, J ) 7). CI-HRMS calcd for C20H25BrN5O2: 448.1348. Found:
448.1355 (M + H). Anal.: HPLC.
12-40: solid, mp 52-58 °C. NMR (CDCl3, 300 MHz): δ 7.14
(s, 1H), 6.82 (s, 1H), 3.91 (s, 3H), 3.85 (s, 3H), 2.96 (q, 4H, J )
12-15: solid. NMR (CDCl3, 300 MHz): δ 6.47 (d,1H, J ) 2),
6.39 (d, 1H, J ) 2), 6.21 (d, 1H, J ) 9), 4.30 (m, 1H), 3.83 (s,
3H), 3.71 (d, 3H, J ) 2), 2.46 (s, 3H), 2.22 (s, 3H), 2.11 (d, 3H,
J ) 3), 1.69 (m, 2H), 1.33 (t, 3H, J ) 7), 1.01 (q, 3H, J ) 7).
CI-HRMS calcd for C20H28N5O2: 370.2243. Found: 370.2244 (M
+ H). Anal.: HPLC.
12-16, free base: solid; mp 111-112 °C. NMR (CDCl3, 300
MHz): δ 6.47 (d,1H, J ) 2), 6.39 (d, 1H, J ) 2), 6.22 (d, 1H, J )
9), 4.29 (m, 1H), 3.83 (s, 3H), 3.71 (s, 3H), 2.46 (s, 3H), 2.27 (s,
3H), 2.11 (d, 3H, J ) 3), 1.68 (m, 2H), 1.33 (t, 3H, J ) 7), 1.01
(q, 3H, J ) 7). CI-HRMS calcd for C20H28N5O2: 370.2243. Found:
370.2242 (M + H). Anal.: HPLC.
12-16: solid; mp 202-203 °C. NMR (DMSO-d6, 300 MHz): δ
6.57 (d, 1H, J ) 2), 6.53 (d, 1H, J ) 2), 4.33 (m, 1H), 3.82 (s,
3H), 3.68 (s, 3H), 2.44 (s, 3H), 2.16 (s, 3H), 2.03. 2.01 (2s, 3H),
1.73 (m, 2H), 1.3 (d, 3H, J ) 3), 0.90 (t, 3H, J ) 7). Anal.
(C20H25N5O2 ·H2SO4) C, H, N.
12-17: oil. NMR (CDCl3, 300 MHz): δ 6.47 (d,1H, J ) 2), 6.39
(d, 1H, J ) 2), 6.20 (d, 1H, J ) 9), 4.39 (m, 1H), 3.83 (s, 3H),
3.71 (d, 3H, J)2), 2.46 (s, 3H), 2.22 (s, 3H), 2.11 (d, 3H, J ) 2),
1.62 (m, 2H), 1.44 (m, 2H), 1.33 (t, 3H, J ) 7), 0.95 (m, 3H).
CI-HRMS calcd for C21H30N5O2: 384.5056. Found: 384.2396 (M
+ H). Anal.: HPLC.
12-18: solid. NMR (CDCl3, 300 MHz): δ 6.53 (d,1H, J ) 8),
6.47 (d, 1H, J ) 2), 6.39 (d, 1H, J ) 2), 4.76 (m, 1H), 3.83 (s,
3H), 3.70 (s, 3H), 2.49 (m, 2H), 2.46 (s, 3H), 2.22 (s, 3H), 2.14
(m, 2H), 2.09 (s, 3H), 1.81 (m, 2H). CI-HRMS calcd for
C20H26N5O2: 368.4625. Found: 368.2100 (M + H). Anal.: HPLC.
12-19: solid; mp 114 °C. NMR (CDCl3, 300 MHz): δ 6.47 (d,
1H, J ) 2), 6.38 (d, 1H, J ) 2), 4.05 (m, 4H), 3.80 (s, 3H), 3.70
(s, 3H), 2.38 (s, 3H), 2.18 (s, 3H), 2.10 (s, 3H), 1.34 (t, 6H, J )
7). CI-HRMS calcd for C20H28N5O2: 370.4785. Found: 370.2200
(M + H). Anal. (C20H28N5O2) C, H, N.
12-20: oil. NMR (CDCl3, 300 MHz): δ 6.46 (d, 1H, J ) 2),
6.38 (d, 1H, J ) 2), 3.95 (m, 4H), 3.82 (s, 3H), 3.71 (s, 3H), 2.37
(s, 3H), 2.17 (s, 3H), 2.10 (s, 3H), 1.77 (m, 4H), 0.98 (t, 6H, J )
7). CI-HRMS calcd for C22H32N5O2: 398.2553. Found: 398.5326
(M + H). Anal.: HPLC.
12-21: oil. NMR (CDCl3, 300 MHz): δ 6.47 (d, 1H, J ) 2),
6.39 (d, 1H, J ) 2), 6.01 (m, 2H), 5.29 (m, 4H), 4.66 (m, 4H),
3.83 (s, 3H), 3.71 (s, 3H), 2.39 (s, 3H), 2.18 (s, 3H), 2.09 (s, 3H).
CI-HRMS calcd for C22H28N5O2: 394. Found: 394 (M + H). Anal.:
HPLC.
12-22: oil. NMR (CDCl3, 300 MHz): δ 6.47 (d, 1H, J ) 2),
6.38 (d, 1H, J ) 2), 4.03 (m, 4H), 3.83 (s, 3H), 3.71 (s, 3H), 2.38
(s, 3H), 2.18 (s, 3H), 2.10 (s, 3H), 1.26 (m, 1H), 0.97 (t, 3H, J )
7), 0.55 (m, 2H), 0.38 (m, 2H). CI-MS calcd for C22H28N5O2: 410.
Found: 410 (M + H). Anal.: HPLC.
12-23: oil. NMR (CDCl3, 300 MHz): δ 6.47 (d,1H, J ) 3), 6.38
(d, 1H, J ) 3), 4.06 (m, 2H), 3.82 (s, 3H), 3.70 (s, 3H), 3.56 (s,
3H), 2.39 (s, 3H), 2.18 (s, 3H), 2.09 (s, 3H), 1.78 (m, 2H), 0.97 (t,
3H, J ) 7). CI-HRMS calcd for C20H28N5O2: 370.2246. Found:
370.4785 (M + H). Anal.: HPLC.
12-24: oil. NMR (CDCl3, 300 MHz): δ 6.47 (d,1H, J ) 3), 6.39
(d, 1H, J ) 3), 5.98 (m, 1H), 5.28 (m, 2H), 4.76 (m, 2H), 3.83 (s,
3H), 3.70 (s, 3H), 3.52 (s, 3H), 2.40 (s, 3H), 2.12 (s, 3H), 2.09 (s,
3H). CI-HRMS calcd for C20H26N5O2: 368.2090. Found: 368.4625
(M + H). Anal.: HPLC.
12-25: oil. NMR (CDCl3, 300 MHz): δ 6.47 (d,1H, J ) 2), 6.38
(d, 1H, J ) 2), 4.04 (m, 4H), 3.83 (s, 3H), 3.71 (s, 3H), 2.37 (s,
3H), 2.17 (s, 3H), 2.10 (s, 3H), 1.76 (m, 2H), 1.42 (m, 2H), 1.33
(t, 3H, J ) 7), 1.01 (t, 3H, J ) 7). CI-HRMS calcd for C22H32N5O2:
398.5326. Found: 398.2559 (M + H). Anal.: HPLC.
12-26: oil. NMR (CDCl3, 300 MHz): δ 6.47 (d,1H, J ) 2), 6.39
(d, 1H, J ) 2), 4.10 (m, 4H), 3.83 (s, 3H), 3.78 (t, 2H, J ) 6),
3.71 (s, 3H), 3.40 (s, 3H), 2.38 (s, 3H), 2.17 (s, 3H), 2.10 (s, 3H),
1.33 (t, 3H, J ) 7). CI-HRMS calcd for C21H30N5O3: 400.2343.
Found: 400.2353 (M + H). Anal.: HPLC.

Methoxyphenylpyrazolotriazines
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9 3081
7), 2.49 (s, 3H), 2.35 (s, 3H), 1.00 (t, 6H, J ) 7). CI-HRMS calcd
for C19H24BrN5O2: 433.1113. Found: 433.1109 (M, 79Br). Anal.:
HPLC.
were administered orally by gavage (po) 1 h before behavioral
testing in a volume of 2 mL/kg body weight. Doses of all drugs
were calculated and are expressed in terms of the free base weight.
The elevated-plus maze, constructed of black, opaque Plexiglas,
consisted of four arms at right angles to each other and was elevated
30 cm off the floor. Two of the arms were enclosed and two arms
had no walls (open arms). The illumination was 60 lx in both closed
and open arms. Rats were placed individually in the center of the
maze facing one of the open arms. Behavior was assessed for 5
min by a trained observer (unaware of treatment assignment) via a
video monitor. The time in open arms was recorded (in seconds),
and data are expressed as percent time in open arms. Entry into an
open or a closed arm was defined as placement of all four paws in
the open or closed arm. The maze was cleaned with 1.0% glacial
acetic acid between animals to prevent olfactory cues from
influencing the behavior of subsequently tested animals. The
positive control for both studies was compound 2 at a dose of 18
mg/kg, po, administered 1 h before testing. Analogue 12-3 (1.0,
3.0, 10, or 30 mg/kg) was administered acutely po 1 h prior to
behavioral testing.
The defensive withdrawal procedure was used with minor
modifications.47 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 50 lx reading at the entr-
ance to the cylinder. Rats were habituated to handling by gently
stroking their dorsal surface for approximately 1 min the day 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. The positive control for both studies was compound 2 at
10 mg/kg, po, administered 1 h before testing. Analogue 12-3 (1.0,
3.0, 10, or 30 mg/kg) was administered acutely po 1 h prior to
behavioral testing.
Results for percent time in open arms and exit latency are
expressed as the mean ( SEM. The percent time in open arms
data were subjected to analysis of variance (ANOVA) followed
by individual mean comparisons using Fisher’s least significant
difference test (Kirk, 1968) where appropriate. The significance
level was set at p < 0.05. The exit latency data were subjected to
the Kruskal-Wallis test, followed by individual comparisons using
the Mann-Whitney U test. The significance level was set at p <
0.05.
Rat Pharmacokinetic Studies. The intravenous (iv) bolus dose
consisted of compound 12-3 at 0.5 mg/mL (free base equivalent)
in 20% ethanol in physiological saline. This was administered to
six rats (three male, three female). The oral dose consisted of 12-3
at 1 mg/mL in 0.5% aqueous methylcellulose containing 1% Tween-
80. This was administered the three male rats. Blood was collected
from the jugular vein into EDTA containing tubes. Samples were
collected at predose, 6, 15, and 30 min and 1, 2, 4, 6, 8, 12, 24,
and 32 h postdose. The dosage of 12-3 is expressed as free base
equivalents. Individual rat plasma samples were analyzed by
analysts in the Drug Metabolism and Pharmacokinetics (DM&PK)
Section of DuPont Pharmaceuticals Company, Newark, DE. The
free base of 12-3 was quantified using a validated LC/MS method.
The assay method involved robotic extraction using a Chemelute
solid phase extraction cartridge followed by LC/MS detection.
[8-(4-Methoxy-2,3-dimethylphenyl)-2,7-dimethylpyrazolo[1,5-a]-
[1,3,5]triazinyl]bis(2-methoxyethyl)amine, a structural analogue,
was used as the internal standard. The assay range was 5 to 5000
nM using 0.25 mL of rat plasma. The pharmacokinetic parameters
were calculated using a noncompartmental model on the WATSON
12-41: oil. NMR (CDCl3, 300 MHz): δ 7.13 (s, 1H), 6.80 (s,
1H), 4.35 (m, 4H), 3.90 (s, 3H), 3.85 (s, 3H), 3.76 (t, 4H, J ) 7),
3.39 (s, 6H), 2.41 (s, 3H), 2.29 (s, 3H). CI-HRMS calcd for
C21H29BrN5O4: 494.1403. Found: 494.1397 (M + H). Anal.: HPLC.
12-42: oil. NMR (CDCl3, 300 MHz): δ 6.81 (s, 1H), 6.70
(s, 1H), 4.35 (m, 4H), 3.90 (s, 3H), 3.83 (s, 3H), 3.76 (t, 4H, J )
7), 3.39 (s, 6H), 2.40 (s, 3H), 2.26(s, 3H), 2.13 (s, 3H). CI-HRMS
calcd for C22H32N5O4: 430.2454. Found: 430.2453 (M + H). Anal.:
HPLC.
12-43: oil. NMR (CDCl3, 300 MHz): δ 7.04 (d, J ) 8, 1H),
6.79 (d, J ) 8, 1H), 4.11 (m, 4H), 3.83 (s, 3H), 2.39 (s, 3H), 2.22
(s, 3H), 2.20 (s, 3H), 2.07 (s, 3H), 1.35 (t, J ) 8, 6H). CI-HRMS
calcd for C21H28N5O: 354.2216. Found: 354.2211 (M + H). Anal.:
HPLC.
12-44: solid; mp 123-124 °C. NMR (CDCl3, 300 MHz): δ 7.05
(d, J ) 8, 1H), 6.79 (d, J ) 8, 1H), 6.15 (d, J ) 8, 1H), 4.19
(m, 1H), 3.85 (s, 3H), 2.46 (s, 3H), 2.30 (s, 3H), 2.22 (s, 3H), 2.10
(s, 3H), 1.70 (m, 4H). CI-HRMS calcd for C21H30N5O: 368.2372.
Found: 368.2372(M + H). Anal. (C21H29N5O) C, H, N.
Biological Testing. Some experimental details for the CRF
receptor binding assays (rat cortical membranes and human IMR32
cell membranes), the in vitro adenylate cyclase assay, the dog ex
vivo binding assay, the defensive withdrawal test, the elevated plus
maze test and receptor occupancy studies, have been reported
previously.46-48,52 Additional details are provided below.
Receptor binding affinity to rat cortical receptors was assayed
according to the published methods.46 Curves of the inhibition of
125I-Tyr0-ovine-CRF binding to cell membranes at various dilutions
of test drug were analyzed by the iterative curve fitting program
LIGAND55 which provides IC50 values for inhibition which are
then used to assess biological activity.
Inhibition of CRF-stimulated adenylate cyclase activity was
performed as described by Battaglia and co-workers.56 Assays are
carried out at 37 °C for 10 min in 200 mL of buffer containing
100 mM Tris-HCl (pH 7.4 at 37 °C), 10 mM MgCl2, 0.4 mM
EGTA, 0.1% BSA, 1 mM isobutylmethylxanthine (IBMX), 250
units/mL phosphocreatine kinase, 5 mM creatine phosphate, 100
mM guanosine 5′-triphosphate, 100 nM ovine-CRF, antagonist
peptides (concentration range from 10-9 to 10-6 M), and 0.8 mg
of original wet weight tissue (approximately 40-60 mg of protein).
Reactions are initiated by the addition of 1 mM ATP/[32P]ATP
(approximately 2-4 mCi/tube) and terminated by the addition of
100 mL of 50 mM Tris-HCL, 45 mM ATP, and 2% sodium dodecyl
sulfate. In order to monitor the recovery of c-AMP, 1 µL of [3H]c-
AMP (approximately 40 000 dpm) is added to each tube prior to
separation. The separation of [32P]c-AMP from [32P]ATP is
performed by sequential elution over Dowex and alumina columns.
Behavioral Pharmacology. Male Sprague-Dawley rats weigh-
ing 180-300 g were purchased from Charles River Laboratories
(Wilmington, MA). The rats were housed individually in suspended
wire cages (for defensive withdrawal studies) or three to a plastic
cage (for elevated-plus maze studies) in colony rooms maintained
at constant temperature (21 ( 2 °C) and humidity (50 ( 10%).
The rooms were illuminated 12 h per day (lights on at 0600 h).
The rats had ad libitum access to food and water throughout the
studies. 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.
Test compounds were prepared as suspensions in an aqueous
vehicle of 0.25% Methocel (methyl cellulose, type A15c, Dow
Chemicals). Suspensions were bead-milled overnight to ensure even
suspension using three layers of 4 mm glass beads. Compounds

3082 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9
Gilligan et al.
corticotropin and beta-endorphin. Science 1981, 213, 1394–1397.
(12) Valentino, R. J.; Foote, S. L. Brain Noradrenergic Neurons, Corti-
cotropin Releasing Factor and Stress. In Neural and Endocrine
Peptides and Receptors; Moody, T., Ed.; Plenum Press: New York,
1986; pp 101-120.
program, version 5.4 v2, 1998, by Pharmaceutical Software
Systems, Inc., Wayne, PA. Systemic clearance (CL), volume of
distribution at steady state (Vss), apparent half-life (t1/2), and area
under the plasma concentration time curve from time 0 to infinity
(AUC0-∞) were determined. Harmonic mean and pseudostandard
deviation were calculated for the half-lives using Excel software
(MS Office 97).
(13) Davies, M. The role of the amygdala in fear and anxiety. Annu. ReV.
Neurosci. 1993, 15, 353–375.
(14) Lehnert, H.; Schulz, C.; Dieterich, K. Physiological and neurochemical
aspects of corticotropin-releasing factor actions in the brain: the role
of the locus coeruleus. Neurochem. Res. 1998, 23, 1039–1052.
(15) Chalmers, D. T.; Lovenberg, T. W.; De Souza, E. B. Localization of
novel corticotropin-releasing factor receptor (CRF2) mRNA expression
to specific subcortical nuclei in rat brain; comparison with CRF1
receptor mRNA expression. J. Neurosci. 1995, 15, 6340–6350.
(16) Chen, R.; Lewis, K. A.; Perrin, M. H.; Vale, W. W. Expression cloning
of a human corticotropin-releasing factor receptor. Proc. Natl. Acad.
Sci. U.S.A. 1993, 90, 8967–8971.
(17) Kishimoto, T.; Pearse, R. V.; Lin, C. R.; Rosenfeld, M. G. A sauvagine/
corticotropin releasing factor receptor expressed in heart and skeletal
muscle. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 1108–1112.
(18) Kostich, W. A.; Chen, A.; Sperle, K.; Largent, B. L. Molecular
identification and analysis of a novel human corticotropin-releasing
factor (CRF) receptor: the CRF2γ receptor. Mol. Endocrinol. 1998,
12, 1077–1085.
(19) Liaw, C. W.; Lovenberg, T. W.; Barry, G.; Oltersdorf, T.; Grigoriadis,
D. E.; De Souza, E. B. Cloning and characterization of the human
corticotropin releasing factor-2 receptor complementary deoxyribo-
nucleic acid. Endocrinology 1996, 137, 72–77.
(20) Lovenberg, T. W.; Chalmers, D. T.; Liu, C.; De Souza, E. B. CRF2R
and CRF2ꢀ receptor mRNAs are differentially distributed between the
rat central nervous system and peripheral tissues. Endocrinology 1995,
136, 4139–4142.
Dog Pharmacokinetic Studies. The test compounds were
administered to male beagle dogs (8.0-11.0 kg) at 1 mg/kg
intravenously or 5 mg/kg orally (n ) 4/group), and blood was
collected up to 72 h postdose. The iv solution (25 mg/mL) was
formulated in a mixture of N,N-dimethylacetamide/propylene glycol/
0.12 N sodium hydroxide (10/65/25, v/v/v) with a final pH of 3.0.
The oral suspension (5 mg/mL) was prepared in 0.5% methylcel-
lulose containing 1% Tween-80. All plasma concentrations are
expressed as the free base concentrations. Dog plasma samples were
collected at 0.25, 0.5, 1, 2, 4, 6, 8, 12, and 24 h postdose and
analyzed by LC/MS/MS. Sodium hydroxide (0.5 mL, 0.1 N) and
0.1 mL of the internal standard, 2 (500 nM), were added to the
plasma (250 µL) and buffer (500 µL) samples. The test compound
was extracted with 7 mL of hexane/ether (80/20, v/v). After shaking
for at least 10 min at low speed and centrifuging for 5 min at ∼3000
rpm, the organic layer was transferred to a 15 mL conical tube and
evaporated under nitrogen. The residue was reconstituted with 200
µL of mobile phase (acetonitrile/10 mM ammonium acetate/acetic
acid, 70/30/0.1, v/v/v), and test compound concentrations were
determined by LC/MS/MS using a Waters Symmetry C8 column
(2.1 mm × 150 mm) and a Sciex API 300 mass spectrometer. The
quantitation limit was 1 ng/mL (2.42 nM). The calculation of
pharmacokinetic parameters followed the general methods described
for the rat pharmacokinetic studies described above.
(21) Lovenberg, T. W.; Liaw, C. W.; Grigoriadis, D. E.; Clevenger, W.;
Chalmers, D. T.; De Souza, E. B.; Oltersdorf, T. Cloning and
characterization of a functionally distinct corticotropin releasing factor
receptor subtype from rat brain. Proc. Natl. Acad. Sci. U.S.A. 1995,
92, 836–840.
(22) Perrin, M.; Donaldson, C.; Chen, R.; Blount, A.; Bergren, T.;
Bilezikjian, L.; Sawchenko, P.; Vale, W. W. Identification of a second
corticotropin-releasing factor receptor gene and characterization of a
c-DNA expressed in heart. Proc. Natl. Acad. Sci. U.S.A. 1995, 92,
2969–2973.
(23) Chalmers, D. T.; Lovenberg, T. W.; Grigoriadis, D. E.; Behan, D. P.;
DeSouza, E. B. Corticotropin-releasing factor receptors: from molec-
ular biology to drug design. Trends Pharmacol. Sci. 1996, 17, 166–
172.
Note Added after ASAP Publication. An author name was
omitted in the version of this paper released to the web on April
10, 2009. The revised version posted on April 15, 2009.
Supporting Information Available: HPLC purity data of final
products. This material is available free of charge via the Internet
at http://pubs.acs.org.
(24) Millan, M. A.; Jacobowitz, D. M.; Hauger, R. L.; Catt, K. J.; Aguilera,
G. Distribution of corticotropin-releasing factor receptors in the primate
brain. Proc. Natl. Acad. Sci. U.S.A. 1986, 83, 1921–1925.
(25) Grigoriadis, D. E.; Dent, G. W.; Turner, J. G.; Uno, H.; Shelton, S. E.;
De Souza, E. B.; Kalin, N. H. Corticotropin-releasing factor (CRF)
receptors in infant rhesus monkey brain and pituitary gland: biochemi-
cal characterization and autoradiographic localization. DeV. Neurosci.
1995, 17, 357–367.
(26) Grigoriadis, D. E.; Lovenberg, T. W.; Chalmers, D. T.; Liaw, C.; De
Souza, E. B. Characterization of corticotropin-releasing factor receptor
subtypes. Ann. N.Y. Acad. Sci. 1996, 780, 60–80.
(27) Miranda, A.; Lahrichi, S. L.; Gulyas, J.; Koerber, S. C.; Craig, A. G.;
Corrigan, A.; Rivier, C.; Vale, W.; Rivier, J. Constrained corticotropin-
releasing factor antagonists with i-(i+3) Glu-Lys bridges. J. Med.
Chem. 1997, 40, 3651–3658.
References
(1) Dzierba, C. D.; Hartz, R. A.; Bronson, J. J. Recent advances in
corticotropin-releasing factor antagonists. Annu. Rep. Med. Chem.
2008, 43, 3–23.
(2) Tellew, J. E.; Luo, Z. Small molecule antagonists of the corticotropin-
releasing factor (CRF) receptor: recent medicinal chemistry develop-
ments. Curr. Top. Med. Chem. 2008, 8, 506–520.
(3) Gilligan, P. J.; Robertson, D. W.; Zaczek, R. Corticotropin releasing
factor (CRF) receptor modulators: progress and opportunities for new
therapeutic agents. J. Med. Chem. 2000, 43, 1641–1660.
(4) McCarthy, J. R.; Heinrichs, S. C.; Grigoriadis, D. E. Recent advances
with the CRF1 receptor: Design of small molecule inhibitors, receptor
subtypes and clinical indications. Curr. Pharm. Des. 1999, 5, 289–
315.
(28) Rivier, J.; Lahrichi, S. L.; Gulyas, J.; Erchegyi, J.; Koerber, S. C.;
Craig, A. G.; Corrigan, A.; Rivier, C.; Vale, W. W. Minimal size-
constrained corticotropin-releasing factor agonists with i-(i+3) Glu-
Lys and Lys-Gln bridges. J. Med. Chem. 1998, 41, 2614–2620.
(29) Ruhmann, A.; Bonk, I.; Lin, C. R.; Rosenfeld, M. G.; Spiess, J.
Structural requirements for peptidic antagonists of the corticotrpoin-
releasing factor receptor (CRFR): development of CRFR2ꢀ-selective
antisauvagine-30. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 15264–
15269.
(5) Gilligan, P. J. Corticotropin-releasing factor receptor antagonists.
Expert Opin. Ther. Pat. 2006, 16, 913–924.
(6) Dunn, A. J.; Berridge, C. W. Physiological and behavioral responses
to corticotropin-releasing factor administration: Is CRF a mediator of
anxiety or stress responses? Brain Res. ReV. 1990, 15, 71–100.
(7) Stenzel-Poore, M. P.; Heinrichs, S. C.; Rivest, R.; Koob, G. F.; Vale,
W. W. Overproduction of corticotropin-releasing factor in transgenic
mice: a genetic model of anxiogenic behavior. J. Neurosci. 1994, 14,
2579–2584.
(30) Schulz, D. W.; Mansbach, R. S.; Sprouse, J.; Braselton, J. P.; Collins,
J.; Corman, M.; Dunaiskis, A.; Faraci, S.; Schmidt, A. W.; Seeger,
T.; Seymour, P.; Tingley, F. D.; Winston, E. N.; Chen, Y. L.; Heym,
J. CP-154,526: a potent and selective nonpeptide antagonist of
corticotropin releasing factor receptors. Proc. Natl. Acad. Sci. U.S.A.
1996, 93, 10477–10482.
(31) Mansbach, R. S.; Brooks, E. N.; Chen, Y. L. Antidepressant-like effects
of CP154526: a selective CRF1 receptor antagonist. Eur. J. Pharmacol.
1997, 323, 21–26.
(32) He, L.; Gilligan, P. J.; Zaczek, R.; Fitzgerald, L. W.; McElroy, J. F.;
Shen, H.-S. L.; Saye, J. A.; Kalin, N. H.; Shelton, S.; Christ, D.;
Trainor, G.; Hartig, P. 4-(1,3-Dimethoxypropyl-2-amino)-2,7-dimethyl-
8-(2,4-dichlorophenyl)pyrazolo-[1,5-a]-1,3,5-triazine: a potent, orally
(8) Majzoub, J. A.; Muglia, L. J.; Martinez, C.; Jacobson, L. Molecular
and transgenic studies of the corticotropin-releasing hormone gene.
Ann. N.Y. Acad. Sci. 1995, 771, 293–300.
(9) Gulyas, J.; Rivier, C.; Perrin, M.; Koerber, S. C.; Sutton, S.; Corrigan,
A.; Lahrichi, S. L.; Craig, A. J.; Vale, W.; Rivier, J. Potent, structurally
constrained agonists and competitive antagonists of corticotropin-
releasing factor. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 10575–10579.
(10) Perrin, M. H.; Sutton, S. W.; Cervini, L. A.; Rivier, J.; Vale, W. W.
Comparison of an agonist, urocortin, and an antagonist, astressin, as
radioligands for characterization of corticotropin-releasing factor
receptors. J. Pharmacol. Exp. Ther. 1999, 288, 729–734.
(11) Vale, W.; Spiess, J.; Rivier, C.; Rivier, J. Characterization of a 41-
residue ovine hypothalamic peptide that stimulates secretion of

Methoxyphenylpyrazolotriazines
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9 3083
bioavailable CRF1 antagonist. J. Med. Chem. 2000, 430, 449–456.
(33) Gilligan, P. J.; Baldauf, C.; Cocuzza, A.; Chidester, D.; Zaczek, R.;
Fitzgerald, L. W.; McElroy, J. F.; Smith, M. A.; Shen, H.-S.L.; Saye,
J. A.; Christ, D.; Trainor, G.; Robertson, D. W.; Hartig, P. The
discovery of 4-(3-pentylamino)-2,7-dimethyl-8-(2-methyl-4-methox-
yphenyl)pyrazolo-[1,5-a]-pyrimidine: a corticotropin-releasing factor
antagonist (hCRF1) antagonist. Bioorg. Med. Chem. 2000, 8, 181–
189.
(34) Gold, P. W.; Loriaux, D. L.; Roy, A.; Kling, M. A.; Calkabrese, J. R.;
Kellner, C. H.; Nieman, L. K.; Post, R. M.; Pickar, D.; Gallucci, W.;
Augerinos, P.; Paul, S.; Oldfiled, E. H.; Cutler, G. B.; Chrousos, G. P.
Responses to corticotropin-releasing hormone in the hypercortisolism
of depression and Cushing’s disease. New Engl. J. Med. 1986, 314,
1329–1335.
Winston, E.; Schulz, D. W. Synthesis and SAR of 2-aryloxy-4-
alkoxypyridines as potent orally active corticotropin-releasing factor
1 receptor antagonists. J. Med. Chem. 2008, 51, 1377–1384.
(44) He, L.; Gilligan, P. J.; Fitzgerald, L.; Zaczek, R.; Shen, H.; Trainor,
G. L.; Hartig, P. R. Pyrazolo-[1,5-a]-s-triazine as Novel hCRF1
Receptor Antagonists: Design, Synthesis and Structure Activity
Relationships. Presented at the American Chemical Society National
Meeting, Anaheim, CA, March 21-25, 1999; MEDI 107.
(45) Zhou, J.; Oh, L. M.; Ma, P. Convergent Synthesis of R-Aryl-ꢀ-
ketonitriles from Aryl Boronic Acids and Isoxazoles. US 6562965,
2003.
(46) DeSouza, E. B. Corticotropin-releasing factor receptors in the rat
central nervous system: characterization and regional distribution.
J. Neurosci. 1987, 7, 88–100.
(47) McElroy, J. F.; Ward, K.; Zeller, K. L.; Jones, K. W.; Gilligan, P. J.;
He, L.; Lelas, S. The CRF1 receptor antagonist DMP696 produces
anxiolytic effects and inhibits the stress-induced hypothalamic-
pituitary-adrenal axis activation without sedation or ataxia in rats.
Psychopharmacology 2002, 165, 86–92.
(48) Li, Y.-W.; Fitzgerald, L.; Wong, H.; Lelas, S.; Zhang, G.; Lindner,
M. D.; Wallace, T.; McElroy, J.; Lodge, N. J.; Gilligan, P. J.; Zaczek,
R. The pharmacology of DMP696 and DMP904, non-peptidergic CRF1
receptor antagonists. CNS Drug ReV. 2005, 11, 21–52.
(35) Carroll, B. J.; Curtis, G. C.; Davies, B. M.; Mendels, J.; Sugarman,
A. A. Urinary free cortisol secretion in depression. Psychol. Med. 1976,
6, 43–50.
(36) Raadsheer, F. C.; Hoogendijk, W. J. G.; Stam, F. C.; Tilders, F. J. H.;
Swaab, D. F. Increased numbers of corticotropin-releasing hormone
expressing neurons in the hypothalamic paraventricular nucleus of
depressed patients. Neuroendocrinology. 1994, 60, 436–444.
(37) Nemeroff, C. B.; Bisette, G.; Akil, H.; Fink, M. Neuropeptide
concentrations in the cerebrospinal fluid of depressed patients treated
with electroconvulsive therapy: corticotropin-releasing factor, ꢀ-en-
dorphin,and somatostatin. Br. J. Psychiatry 1991, 158, 59–63.
(38) Banki, C. M.; Karmacsi, L.; Bissette, G.; Nemeroff, C. B. CSF
corticotropin releasing hormone and somatostatin in major depression:
response to antidepressant treatment and relapse. Eur. Neuropsychop-
harmacol. 1992, 2, 107–113.
(39) DeBellis, M. D.; Gold, P. W.; Geracioti, T. D.; Listwak, S. J.; Kling,
M. A. Association of fluoxetine treatment with reductions in CSF
concentrations of corticotropin-releasing hormone and arginine vaso-
pressin in patients with major depression. Am. J. Psychiatry. 1993,
150, 656–657.
(40) Kling, M. A.; Geracioti, T. D.; Licinio, D.; Michelson, E. H.; Oldfield,
E. H.; Gold, P. W. Effects of electroconvulsive therapy on the CRH-
ACTH-cortisol sytem in melancholic depression: preliminary findings.
Psychopharmacol. Bull. 1994, 30, 489–494.
(41) Zobel, A. W.; Nickel, T.; Kunzel, H. E.; Ackl, N.; Sonntag, A.; Ising,
M.; Holsboer, F. Effects of the high affinity corticotropin releasing
hormone receptor 1 antagonist R121919 in major depression: the first
20 patients treated. J. Psychiatr. Res. 2000, 34, 171–181.
(42) Binneman, B.; Feltner, D.; Kolluri, S.; Shi, Y.; Qiu, R.; Stiger, T. A
6-week randomized, placebo-controlled trial of CP-316,311 (a selective
CRH1 antagonist) in the treatment of major depression. Am. J.
Psychiatry 2008, 165, 617–620.
(49) Sutton, S. W.; Behan, D. P.; Lahrichi, S. L.; Kaiser, R.; Corrigan, A.;
Lowry, P.; Potter, E.; Perrin, M. H.; Rivier, J.; Vale, W. Ligand
requirements of the human corticotropin-releasing factor-binding
protein. Endocrinology 1995, 136, 1097–1102.
(50) Fitzgerald, L. Unpublished results.
(51) Gilligan, P. J.; Rabel, S. R.; Meenan, P. A. Salt and Crystalline Form
Thereof of a Corticotropin Releasing Factor Receptor Antagonist. U.S.
Patent Application US2005113375, 2005.
(52) Li, Y.-W.; Hill, G.; Wong, H.; Kelly, N.; Ward, K.; Pierdomenico,
M.; Ren, S.; Gilligan, P.; Grossman, S.; Trainor, G.; Taub, R.;
McElroy, J.; Zazcek, R. Receptor occupancy of nonpeptide corticotro-
pin-releasing factor 1 antagonist DMP696: correlation with drug
exposure and anxiolytic efficacy. J. Pharmacol. Exp. Ther. 2003, 305,
86–96.
(53) He, L.; Gilligan, P. J.; Arvanitis, A. G.; Chorvat, R. J. Azolo Triazines
and Pyrimidines. US 6124289, 2000.
(54) Perrin, D.; Armarego, W. L. F. Purification of Laboratory Chemicals,
3rd ed.; Pergamon Press: New York, 1988.
(55) Munson, P. J.; Rodbard, D. LIGAND: a versatile computerized
approach for characterization of ligand binding systems. Anal. Bio-
chem. 1980, 107, 220–239.
(56) Battaglia, G.; Webster, E. L.; De Souza, E. B. Characterization of
corticotropin-releasing factor (CRF) receptor-mediated stimulation of
adenylate cyclase activity in rat brain. Synapse 1987, 1, 572–581.
(43) Chen, Y. L.; Braselton, J.; Forman, J.; Gallaschun, R. J.; Mansbach,
R.; Schmidt, A. W.; Seeger, T. F.; Sprouse, J. S.; Tingley, F. D.;
JM9000242