Synthesis and structure-activity relationships of 8-(pyrid-3-yl)pyrazolo[1, 5-a]-1,3,5-triazines: Potent, orally bioavailable corticotropin releasing factor receptor-1 (CRF1) antagonists

Article abstract of DOI:10.1021/jm900025h

This report describes the syntheses and structure-activity relationships of 8-(substituted pyridyl)pyrazolo[1,5-a]-1,3,5-triazine corticotropin releasing factor receptor-1 (CRF1) receptor antagonists. These CRF₁ receptor antagonists may be potential anxiolytic or antidepressant drugs. This research resulted in the discovery of compound 13-15, which is a potent, selective CRF₁ antagonist (hCRF₁ IC₅₀ = 6.1 ± 0.6 nM) with weak affinity for the CRF-binding protein and biogenic amine receptors. This compound also has a good pharmacokinetic profile in dogs. Analogue 13-15 is orally effective in two rat models of anxiety: the defensive withdrawal (situational anxiety) model and the elevated plus maze test. Analogue 13-15 has been advanced to clinical trials.

Full text of DOI:10.1021/jm900025h

3084
J. Med. Chem. 2009, 52, 3084–3092
Synthesis and Structure-Activity Relationships of 8-(Pyrid-3-yl)pyrazolo[1,5-a]-1,3,5-triazines:
Potent, Orally Bioavailable Corticotropin Releasing Factor Receptor-1 (CRF1) Antagonists
Paul J. Gilligan,*,† Todd Clarke,† Liqi He,† Snjezana Lelas,† Yu-Wen Li,† Karen Heman,† Lawrence Fitzgerald,†,×
Keith Miller,† Ge Zhang,† Anne Marshall,† Carol Krause,† John F. McElroy,‡, Kathyrn Ward,‡ Kim Zeller,‡ Harvey Wong,‡
Steven Bai,‡ Joanne Saye,§,∞ Scott Grossman,§,∞ Robert Zaczek,§ Stephen P. Arneric,2 Paul Hartig,§ David Robertson,§,| 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-(substituted pyridyl)pyrazolo[1,5-
a]-1,3,5-triazine corticotropin releasing factor receptor-1 (CRF1) receptor antagonists. These CRF1 receptor
antagonists may be potential anxiolytic or antidepressant drugs. This research resulted in the discovery of compound
13-15, which is a potent, selective CRF1 antagonist (hCRF1 IC50 ) 6.1 ( 0.6 nM) with weak affinity for the
CRF-binding protein and biogenic amine receptors. This compound also has a good pharmacokinetic profile in
dogs. Analogue 13-15 is orally effective in two rat models of anxiety: the defensive withdrawal (situational
anxiety) model and the elevated plus maze test. Analogue 13-15 has been advanced to clinical trials.
Corticotropin-releasing factor (CRFa), also known as corticotro-
Scheme 1
pin-releasing hormone (CRH), is a 41-amino acid residue peptide
that has been implicated in the pathophysiology of anxiety and
depression.1-4 CRF functions as a pituitary ACTH secretagogue5,6
and a neurotransmitter.7-9 Preclinical studies have documented the
essential role of CRF in regulation of endocrine, autonomic, and
behavioral responses to stress.3 Peptidic CRF antagonists, such as
R-helical CRF9-41, not only block the effects of exogenous CRF
but also block the effects of various natural stressors. Preclinical
studies with selective, non-peptide CRF1 receptor antagonists, such
as 1 (CP 154526-1),10,11 2 (DMP696),12 and 3 (DMP 904),13
provide strong support for the hypothesis that this receptor subtype
may be involved in anxiety and depression. Some clinical studies
have implicated CRF in the pathophysiology of both depression
and anxiety. Maladaptation to chronic stress and related chronic
elevation of corticosteroids may be a major pathway leading to
some, but not all, forms of depression.14 Hypercortisolemia in these
forms of depression appears to be a direct result of chronic
hypersecretion of hypothalamic CRF.15 The small molecule CRF1
receptor antagonist 4 (R121919, Scheme 1) had some efficacy in
a small open label clinical study in anxiety and depression.16
However, another CRF1 antagonist 5 (CP316311) failed in a trial
for depression.17 Structurally diverse CRF1 antagonists may have
different clinical profiles; additional studies may be needed to define
the clinical utility of CRF antagonists.
This report describes the syntheses and structure-activity
relationships of 8-(pyrid-3-yl)pyrazolo[1,5-a]-1,3,5-triazine
CRF1 receptor antagonists, which may have utility as anxiolytic
and antidepressant drugs. Previous work on bicyclic CRF
antagonists led to the discovery of compounds such as 110,11
and 212 with high lipophilicity (HPLC log P > 5.0) or low water
solubility (<1 µg/mL, pH 7.0). We have independently reported
on several bicyclic CRF1 receptor antagonist chemotypes
including compound 6 (BMS-561388), which was advanced into
phase 1 clinical trials.18 We sought ways to improve water
solubility and to improve pharmacokinetic profiles by introduc-
tion of substituted pyridyl groups on a pyrazolotriazine core.
Our structure-activity studies led to improved physical proper-
ties and good efficacy profiles when a 2-methyl-6-methoxypyrid-
3-yl group was introduced at the 8-position of the pyrazolotri-
azine core. One of these compounds, 13-15, was advanced into
clinical trials.
* 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.
Present address: Pfizer Inc., Eastern Point Road, MS8220-4216, Groton,
×
CT 06430.
‡
Bristol-Myers Squibb Co., Wallingford, CT.
Present address: Jenrin Discovery, 3701 Market Street, Philadelphia,
PA 19104.
§
Bristol-Myers Squibb Co., Princeton, NJ.
Present address: Astra Zeneca, 1800 Concord Pike, Wilmington, DE
∞
19803.
2
Present address: Neuroscience Research, Eli Lilly and Company,
Indianapolis, IN 46285.
|
Results and Discussion
Deceased.
a Abbreviations: CRF, corticotropin-releasing factor; ACTH, adrenocor-
ticotropic hormone; c-AMP, cyclic adenosine monophosphate; CSF, cere-
brospinal fluid; HEK293, human embryonic kidney-293.
Chemistry. Pyridyl compounds were prepared according to
procedures that are outlined in Schemes 2 and 3.19,20 The pyridyl
10.1021/jm900025h CCC: $40.75
2009 American Chemical Society
Published on Web 04/10/2009

Pyridylpyrazolotriazines
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9 3085
Scheme 2
Scheme 3
starting materials were obtained from commercial sources or
by custom synthesis as described in the Experimental Section.
Cyanoketones 8 were prepared by acylation of pyridylacetoni-
triles 7 or by cleavage of isoxazoles 15. Aminopyrazoles 9 were
prepared by condensation of hydrazine with cyanoketones 8.
Treatment of these amines with ethyl acetamidate, followed by
cyclization with diethyl carbonate, generated pyrazolotriazinones
11. Conversion to the chloropyrazolotriazines by treatment with
phosphorus oxychloride, followed by displacement of the
chloride with various amines, gave the final products 13.
Pharmacology. Pyrazolo[1,5-a]-1,3,5-triazines 13 were stud-
ied in a series of in vitro and in vivo tests to identify preclinical
candidates. The compounds were first tested for their binding
affinity to rat cortical homogenate CRF receptors.21 Compounds
with high receptor binding affinity (IC50 e 20 nM) were then
evaluated in dog pharmacokinetic studies (1 mg/kg, po) using
a cassette dosing paradigm. Compounds that had AUC (po)
values greater than 1000 nM ·h and t1/2 (po) values greater than
10 h were advanced to secondary in vitro biochemical experi-
ments, rat behavioral pharmacology studies, and rat and
chimpanzee pharmacokinetic studies with individual compounds.
The leading compounds were tested for their affinity to CRF1
receptors endogenously expressed in human IMR32 neuroblas-
toma cells,10 since rat cortical membranes contain CRF1 and,
to a much lesser extent, CRF2 sites. Antagonist vs agonist
function was determined by measuring a compound’s effects
on CRF-stimulated ACTH production in rat pituitary cells.
Anxiolytic efficacy was assessed in the rat defensive withdrawal
and elevated plus maze models.22-24
dimethylaminopyrid-3-yl group at the 8-position of the pyra-
zolotriazine core affords analogues with moderate binding
affinity (compounds 13-1 to 13-7). The most potent analogues
in this subseries, 13-5, 13-6, and 13-7, have tertiary amine side
chains at the 4-position. Replacement of the 2-methyl group
with hydrogen on the pyrid-3-yl portion led to modest improve-
ments in receptor binding affinity in two cases (compare 13-9
with 13-3 as well as 13-8 with 13-4). Replacement of the
dimethylamino group with a methoxy substituent at the 6-posi-
tion of the pyrid-3-yl region gives small improvements in
receptor binding affinity in two cases (compare 13-11 with 13-
9, 13-12 with 13-8, and 13-13 with 13-10). However, introduc-
tion of the 2-methyl-6-methoxypyridyl group at the 8-position
of the pyrazolotriazine core affords analogues with superior
receptor binding affinity (compounds 13-16 to 13-30, Table 1).
Compounds with CRF binding IC50 values less 20 nM were
submitted to dog cassette dosing pharmacokinetic studies (1 mg/
kg, po). Data for only the leading compounds are presented in
Table 2, all of which have the 2-methyl-6-methoxypyrid-3-yl
substituent. Compounds 13-15, 13-16, 13-18, and 13-24 have
high AUC, Cmax, and t1/2 values. Analogue 13-15 has the best
AUC and Cmax values in this subset (AUC ) 2020.0 ( 558.6
nM·h and Cmax ) 301.3 ( 117.2 nM). The reference compound
6 has a comparable profile (AUC ) 1661.0 ( 210.6 nM·h and
Cmax ) 369.0 ( 92.6 nM).25
Compound 13-15 was profiled further in other receptor
binding assays to test for cross-reactivity with other sites. This
analogue potently displaces 125I-Tyr0-ovine-CRF (150 pM) from
human CRF1 receptors which are endogenously expressed in
human IMR32 cells10 (mean IC50 values ((standard deviation)
equal to 10.6 ( 4.5 nM (n ) 8) vs IC50 ) 2.0 ( 0.5 nM (n )
The rat CRF receptor binding data for compounds 13 are
summarized in Table 1. Introduction of a 2,4-dimethyl-6-

3086 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9
Table 1. Rat CRF Receptor Binding Affinity Dataa
Gilligan et al.
compd
R1
R2
R4
R5
R6
rat IC50 + SD (nM)
n
13-1
13-2
13-3
13-4
13-5
13-6
13-7
13-8
NHCHEt2
Me
Me
Me
Me
Me
Me
Me
H
H
H
H
H
NMe2
NMe2
NMe2
NMe2
NMe2
NMe2
NMe2
NMe2
NMe2
NMe2
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
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
H
H
H
H
H
H
H
H
H
25.6 ( 0.6
>10000
3
3
4
3
3
3
3
3
3
3
4
3
3
3
6
4
3
4
3
4
4
3
3
3
3
3
3
3
4
3
8
NHCH2CH2OMe
N(CH2CH2OMe)2
NEt2
NPr2
NEtBu
NPrBu
NEt2
N(CH2CH2OMe)2
NHCHEt2
N(CH2CH2OMe)2
NEt2
133.3 ( 18.8
58.0 ( 17.6
14.9 ( 4.0
32.8 ( 13.9
12.8 ( 1.6
13.8 ( 3.0
91.9 ( 4.5
24.8 ( 5.0
23.8 ( 15.4
14.5 ( 5.4
3.0 ( 2.0
3.1 ( 0.1
6.1 ( 0.6
7.0 ( 0.6
4.5 ( 1.6
3.5 ( 0.5
5.1 ( 0.9
12.0 ( 3.0
5.7 ( 1.4
8.5 ( 1.2
6.6 ( 1.3
3.1 ( 0.4
1.6 ( 1.5
81.6 ( 20.7
17.6 ( 4.3
8.1 ( 7.2
4.9 ( 1.4
2.4 ( 1.2
1.24 ( 0.8
13-9
13-10
13-11
13-12
13-13
13-14
13-15
13-16
13-17
13-18
13-19
13-20
13-21
13-22
13-23
13-24
13-25
13-26
13-27
13-28
13-29
13-30
NHCHEt2
NHCHEt2
H
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
(R)-NHCHMeEt
(S)-NHCHMeEt
(S)-NHCHMePr
(R,S)-NHCHMePr
NHCHMeBu
NEt2
NPr2
N(CH2CH2OMe)2
NEt(CH2CH2OMe)
N(CH2-c-C3H5)CH2CH2OMe
N(CH2-c-C3H5)Pr
NMeEt
NMePr
NMeBu
NEtBu
NPrBu
H
H
H
H
H
H
H
H
R-helical CRF9-41
a SD is the standard deviation, and n is the number of measurements.
Table 2. Dog Casette Dosing Pharmacokinetic Data (1 mg/kg, po)a
cells.26 The resultant Ki values were 2660 and 3520 nM. The
IC50 for the human neurokinin 2 receptor was determined to be
4.9 µM, based on two experiments.26
compd
mean AUC (nM ·h)
mean Cmax (nM)
mean t1/2 (h)
13-15
13-24
13-22
13-18
13-16
6
2020.0 ( 558.6
1726.9 ( 565.7
1465.1 ( 261.3
955.4 ( 347.6
916.3 ( 159.5
1661.0 ( 210.6
301.3 ( 117.2
313.7 ( 143.6
275.0 ( 106.4
155.7 ( 62.6
168.3 ( 46.9
369.0 ( 92.6
10.7 ( 1.8
11.4 ( 4.8
10.3 ( 2.5
11.8 ( 2.9
7.9 ( 0.8
Introduction of the 2-methyl-6-methoxypyrid-3-yl group at
the 8-position of the pyrazolotriazine core also afforded
improved solubility in the case of compound 13-15. The key
solubilities were 16 µg/mL in water (pH 7.4), 16.3 mg/mL in
0.01 N HCl (pH 2.5), 300 mg/mL in ethanol, and 460 mg/mL
in acetone. The solubilities for the reference compound 2 were
<1 µg/mL in water (pH 7.4) and 0.23 mg/mL in dilute HCl
(pH 2.3). Reference compound 6 had solubilities of <1 µg/mlL
in water (pH 7.4) and 2.5 mg/mL in dilute HCl (pH 2.3). HPLC
log P values are 4.32, 4.97, and 4.76 for compounds 13-15, 2,
and 6, respectively.
Analogue 13-15 is an antagonist of rat CRF1 receptors
expressed in rat brain homogenates. It inhibits CRF (0.3 nM)-
mediated adrenocorticotropic hormone (ACTH) release from
pituitary cell culture with an IC50 equal to 129 ( 17 nM (n )
3) and has no agonist properties. In the same experiments, the
r/hCRF concentration response was also evaluated and a
concentration at 50% maximum effect (EC50) equal to 0.3 (
0.1 nM (n ) 3) was observed. Analogue 13-15 does not exhibit
partial agonist or inverse agonist activity in the absence of CRF
stimulation. The reference compound 6 had an IC50 value equal
to 72.9 ( 10.4 nM (n ) 4), while r/hCRF had an EC50 value
equal to 0.3 ( 0.1 nM (n ) 4).
10.1 ( 1.7
a Vehicle ) Labrafil; n ) 3 dogs for each compound except reference
compound 6 where n ) 54 (18 separate studies, 3 dogs each). Standard
errors of the mean are reported.
20) for R-helical CRF9-41). Compound 13-15 does not displace
125I-Tyr0-sauvagine (150 pM) from CRF2ꢀ receptors expressed
in pig choroid plexus membranes (IC50 > 1000 nM).26 This
analogue does not bind to the human CRF binding protein (IC50
> 1000 nM).26 Analogue 13-15 was sent to NovaScreen
(Hanover, MD) for an extensive screening in a variety of assays
including receptor binding, ion channel effects, regulatory sites,
second messengers, uptake sites, and enzymes. This compound
was evaluated at 10-7 and 10-5 M to determine if it exhibited
significant activity, i.e., greater than 50% inhibition or enhance-
ment of binding, in 99 assays. Compound 13-15 at 10 µM
inhibited ligand binding in the adenosine A1 assay by 90% and
in the neurokinin 2 assay by 80%. This compound was then
tested twice in a dose response study for the adenosine A1
receptor using the cloned rat receptor transfected into CHO

Pyridylpyrazolotriazines
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9 3087
Figure 1. Rat behavioral data. Anxiolytic-like effects of 13-15 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 ) 12 animals per dose. Right panel ordinate displays the mean ((SEM) latency to exit the dark
chamber (in seconds) for n ) 8 animals per dose. Maximum latency is 900 s. Compounds 13-15 and 2 were administered in 0.25% Methocel, po,
60 min prior to testing: (*) p < 0.05; (**) p < 0.01.
Table 3. Rat Spontaneous Locomotor Activity Dataa
Compound 13-15 was next evaluated in the rat elevated plus
maze test22-24 for possible anxiolytic efficacy. 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 7.3 ( 1.6 s. The positive control,
compound 2 at a dose of 18 mg/kg, significantly increased the
percent time spent in open arms (p ) 0.05). Pretreatment with
13-15 increased percent time in open arms [F(5,66) ) 2.35, p
) 0.05, analysis of variance (ANOVA) followed by individual
mean comparisons using Fisher’s least significant difference
test]. The lowest effective dose was 10 mg/kg (p ) 0.02; Figure
1, left panel). Higher doses of 13-15 (18 and 30 mg/kg) also
increased percent time in open arms of the elevated-plus maze
(p ) 0.03).23
compd
13-15
dose, po (mg/kg)
N
total distance ( SEM (cm)
0
8
8
8
8
8
8
8
8
8
8
8
5389 ( 950
4106 ( 407
5639 ( 942
3695 ( 244
4846 ( 627
4041 ( 419
5760 ( 963
4624 ( 738
3981 ( 259b
2162 ( 264c
1062 ( 161c
1.0
3.0
10
30
100
0
3.0
10
30
100
chlordiazepoxide
a The vehicle was 0.25% Methocel. Compounds were administered 1 h
pretest. SEM is the standard error of the mean, and N is the number of rats.
b Latency value differs significantly from that of the associated vehicle group
(zero dose) with p < 0.05. c Latency value differs significantly from that of
the associated vehicle group (zero dose) with p < 0.001.
The anxiolytic efficacy of analogue 13-15 was also assessed
in the rat defensive withdrawal model (Figure 1, right panel) 22-24
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 717 ( 94 s (80% of the total test
duration). The positive control 2 at a dose of 10 mg/kg decreased
exit latency by 72% relative to vehicle control (p ) 0.004).
Pretreatment with 13-15 decreased exit latency (H(5) ) 14.34,
p ) 0.02, 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 44%
relative to vehicle-treated animals (p ) 0.06; Figure 1, right
panel). A higher dose of 13-15 (10 mg/kg) decreased exit latency
by 65% (p ) 0.008).23
The CRF receptor occupancy of compound 13-15 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
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 protocol23 established receptor occupancy relative
to vehicle-control animals. The receptor occupancy ((standard
error of the mean) was calculated to be 37 ( 6%, 26 ( 15%,
59 ( 12%, 69 ( 9%, and 85 ( 10% 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 59 ( 12% receptor occupancy
in ex vivo studies.
Table 4. Rat Rotorod Performance Dataa
compd
13-15
dose, po (mg/kg)
N
time on rotorod ( SEM (s)
0
8
8
8
8
8
8
8
8
8
8
158 ( 30
133 ( 26
162 ( 14
158 ( 16
157 ( 27
121 ( 16
205 ( 23
227 ( 20
128 ( 35
114 ( 23b
1.0
3.0
10
30
100
0
3.0
10
30
chlordiazepoxide
a The vehicle was 0.25% Methocel. Compounds were administered 1 h
pretest. SEM is the standard error of the mean, and N is the number of rats.
b Latency value differs significantly from that of the associated vehicle group
(zero dose) with p < 0.05.
while chlordiazepoxide has pronounced effects at 30 and 100
mg/kg (po) (Table 3). In the rat rotorod test,22-24 compound
13-15 has no significant effect on rotorod performance up to
100 mg/kg (po) while chlordiazepoxide impaired performance
at 10 and 30 mg/kg (po) (Table 4). Thus, 13-15 has a side effect
profile in the locomotor and rotorod tests, which is superior to
chlordiazepoxide. Furthermore, the efficacy of 13-15 does not
appear to be influenced by motoric side effects.
Compound 13-15 was evaluated in rat pharmacokinetic
studies. Rats were dosed intravenously at 1 mg/kg and orally
at 5 mg/kg (n ) 3, Table 5). Reference compound 6 was also
administered separately. Compounds 13-15 and 6 have good
maximal oral exposure (Cmax ) 1260.8 ( 552.5 nM and 851 (
195 nM, respectively), good oral bioavailability (40% and 51%,
Compound 13-15 was evaluated for CNS side effects to
determine whether the apparent anxiolytic efficacy may be
confounded by other behavioral effects. In the rat spontaneous
locomotor activity test,22-24 compound 13-15 has modest but
not statistically significant effects at doses up to 100 mg/kg (po),

3088 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9
Gilligan et al.
Table 5. Rat Pharmacokinetic Parameters for Compounds 6 and 13-15
pressures, and the ECG was recorded. The following parameters
were determined: the first derivative of the left ventricular
pressure (to assess cardiac contractility); mean arterial blood
pressure; heart rate; and the PR, QRS, QT, and QTc intervals.
Each of four dogs received a 6 min continuous intravenous
infusion of 13-15 at escalating doses of 0.3, 1, 3, and 5 mg/kg,
with a 30 min interval between successive doses. A separate
group of four dogs received the vehicle on the same schedule.
Blood samples for determination of plasma concentration of 13-
15 were obtained after each infusion and at the end of the
experiment.
(1 mg/kg (iv), 5 mg/kg (po))a
parameter
6
13-15
CL ((mL/min)/kg)
Vss (L/kg)
t1/2 (h)
Cmax (po, nM)
tmax (po, h)
AUC (µM·h)
F (po, %)
20.0 ( 3.3
14.6 ( 3.9
9.7 ( 2.8
851 ( 195
0.5
17.9 ( 4.8
14.9 ( 1.0
13.5 ( 3.1
1260.8 ( 552.5
0.75
5330 ( 2070
51
5814.6 ( 1306.7
40
a n ) 3. Oral vehicle ) 0.5% methylcellulose. iv vehicle )10% DMAC,
70% propylene glycol, and deionized water.
Intravenous administration of 13-15 did not cause a significant
change in heart rate or PR, QRS, QT, or QTc interval in the
ECG. However, dogs administered 3 and 5 mg/kg doses showed
minimal and transient increases in mean arterial pressure of
approximately 10 mmHg, coincident with dose escalation. No
change in mean arterial pressure was detected in a similar time
frame in anesthetized, spontaneously breathing mongrel dogs
given 13-15 as a single intravenous dose of 5 mg/kg. Cardiac
contractility also increased (approximately 200 mmHg/s) co-
incident with dose escalation to the two highest doses. In
addition, contractility gradually increased in two of four dogs
during the last 2 h of the experiment, although the peak increase
did not correlate with the peak plasma concentration of 13-15.
Since this change was not observed consistently within the group
of dogs and was not accompanied by changes in other
cardiovascular parameters, it is probably unrelated to the
compound. Total pooled plasma concentrations at the end of
each infusion of 0.3, 1, 3, and 5 mg/kg were 149.9, 472.8, 1293,
and 2427 nM, respectively (0.1-1.7 times the mean single-
dose human Cmax at 100 mg). In conclusion, 13-15, when
administered intravenously in the anesthetized dog model,
produced a minimal and transient increase in mean arterial
pressure and cardiac contractility following dose escalation at
g3 mg/kg. There was no effect on heart rate or ECG parameters.
Table 6. Pharmacokinetic Parameters for Compound 13-15 in
Chimpanzee (1 mg/kg, po)a
parameter
iv chimpanzee oral chimpanzee oral chimpanzee
dose (mg/kg)
AUCinf (µM·h)
Cmax (µM)
CL ((L/h)/kg)
Vss (L/kg)
0.5
11.9
1.0
11.7
0.84
1.0
16.1
1.3
0.12
4.2
30
t1/2 (h)
F (po, %)
26
49
26
68
a Oral vehicle ) 50-50 (v/v) mixture of 0.5% methylcellulose and Tang
orange juice containing 1% Tween-80. iv vehicle ) 10% DMAC, 70%
propylene glycol, and deionized water over 10 min (0.2 mL/kg).
Table 7. Pharmacokinetic Parameters for Compound 6 in Chimpanzee
(0.5 mg/kg, iv, or 1 mg/kg, po)a
parameter
iv
po
N
2
3
dose (mg/kg)
AUCinf (µM·h)
Cmax (µM)
CL ((L/h)/kg)
Vss (L/kg)
0.5
2.04
1.0
0.212 ( 0.089
0.027 ( 0.006
0.61
9.52
20.2
t1/2 (h)
F (po, %)
16.5 ( 3.4
5.2 ( 2.2
a Oral vehicle ) 50-50 (v/v) mixture of 0.5% methylcellulose and Tang
orange juice containing 1% Tween-80. iv vehicle ) 10% DMAC, 70%
propylene glycol, and deionized water over 10 min (0.2 mL/kg).
The effects of 13-15 were examined on respiratory function
in spontaneously breathing, anesthetized mongrel dogs (n ) 4).
The procedures followed those previously described for refer-
ence compound 2.12 Infusion of 13-15 at 5.0 mg/kg to
barbiturate-anesthetized dogs over 6 min resulted in a transient
(less than 10 min) increase in respiration rate (37.5 ( 21.7%)
and in minute volume (28.0 ( 9.5%) when compared to dogs
receiving a vehicle infusion. These effects disappeared by 15
min postdose. No other respiratory parameters were affected.
Neither mean arterial pressure nor heart rate was affected by
compound 13-15 in this study. Total pooled plasma concentra-
tions were 1790, 834.8, 412.1, and 229.0 nM at 6, 15, 30, and
60 min, respectively. The reference compound 2 was previously
reported to have no effects in this model, but a different vehicle
was employed because of solubility differences.12 Thus, com-
pound 13-15 (5.0 mg/kg, iv) has a minimal effect acutely on
respiratory function with no effect upon mean arterial pressure
and heart rate.
respectively), and acceptable elimination half-lives (13.5 ( 3.1 h
and 9.7 ( 2.8 h, respectively).
Analogue 13-15 also has an excellent oral pharmacokinetic
profile in chimpanzees (Table 6). The compound was dosed in
two animals at 1 mg/kg (po), using a Tang orange drink-Meth-
ocel vehicle. In addition, the compound was administered
intravenously to one chimpanzee at a dose of 0.5 mg/kg in 10%
dimethylacetamide, 70% propylene glycol, and deionized water
over 10 min (0.2 mL/kg). The key parameters are as follows:
CL ) 0.12 (L/kg)/h, Cmax(po) ) 840 and 1300 nM, elimination
t1/2 ) 26 h, and percent oral bioavailability F ) 49% and 68%
(animals 1 and 2, respectively). In contrast, reference compound
6, which was administered at the same doses, had higher
clearance (0.61 (L/kg)/h), lower Cmax(po) (270 ( 6 nM), shorter
t1/2 (16.5 ( 3.4 h), and lower oral bioavailability (5.2 ( 2.2%)
(Tables 6 and 7).
The effects of 13-15 on renal function in rats (n ) 9) were
also examined according to previously published procedures.12
Administration of analogue 13-15 (30 mg/kg, po) resulted in
an increase in urine volume (3.0 ( 0.1 mL/100 g body weight
vs 1.7 ( 0.2 mL/100 g body weight for vehicle). The compound
also caused decreases in the concentration of glucose (4.4 (
0.4 mg/dL per 100 g body weight vs 9.9 ( 1.9 mg/dL per 100 g
body weight for vehicle), urea (166 ( 18 mg/dL per 100 g body
weight vs 321 ( 62 mg/dL per 100 g body weight for vehicle),
creatinine (8.0 ( 0.9 mg/dL per 100 g body weight vs 16.2 (
3.3 mg/dL per 100 g body weight for vehicle), sodium (58.4 (
Corticotropin-releasing factor has peripheral as well as central
pharmacological effects. It is therefore important to assess the
peripheral effects of potential CRF1 antagonist candidates.
Analogue 13-15 was tested for effects on cardiovascular,
pulmonary, renal, and gastrointestinal functions.
Compound 13-15 was examined in dogs for effects on
cardiovascular function. The procedures followed those de-
scribed for reference compound 2.12 In an anesthetized dog
study, mongrel dogs were maintained on sodium pentobarbital
anesthesia and mechanically ventilated. Transducers were
implanted for measurement of left ventricular and arterial blood

Pyridylpyrazolotriazines
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9 3089
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-H2O,
solvent B ) 0.1% TFA-CH3CN, gradient 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-H2O, solvent B ) 0.1%
TFA-MeOH, gradient 5-95% B over 15 min) (method B).
Combustion analyses were performed by Quantitative Technologies,
Whitehouse, NJ.
3.4 mg/dL per 100 g body weight vs 49.9 ( 3.1 mg/dL per
100 g body weight for vehicle), and potassium (32.1 ( 2.8 mg/
dL per 100 g body weight vs 60.3 ( 11.1 mg/dL per 100 g
body weight for vehicle) in the urine (p < 0.5 for all cases).
Plasma concentrations were determined in a different cohort of
rats at 1 and 5 h postdosing. The mean plasma concentrations
were 3994 and 4952 nM at 1 and 5 h after oral administration
of 30 mg/kg analogue 13-15 dosed as described above.
Treatment with the control compound furosemide affected renal
function in a predictable fashion. Furosemide (30 mg/kg) in this
model significantly increased urine volume and decreased
osmolality, glucose, urea, protein, and potassium (see Supporting
Information). The reference compound 2 was previously
reported to have no effects in this model.12 Therefore, analogue
13-15 has an effect on acute renal function at 30 mg/kg (po);
furosemide affects renal function in a predictable fashion.
Compound 13-15 did not significantly affect gastrointestinal
motility when compared to vehicle controls. Fasted male
Sprague-Dawley (Cesarean-derived, 171-217 g) rats (n ) 10)
were used to determine the effects of 13-15 and standard controls
(carbachol and atropine) on gastrointestinal (GI) motility as
evaluated by gross observation and transit of an orally admin-
istered charcoal suspension (10% w/v charcoal in 0.25%
methylcellulose).10 Carbachol (0.3 mg/kg, ip) significantly
increased GI motility from 38.4 ( 3.8% (vehicle group, mean
( SEM) to 55.8 ( 3.6% (carbachol), while atropine (3.0 mg/
kg, po) significantly decreased GI motility in this animal model
from 42.1 ( 1.8% (vehicle) to 27.5 ( 2.5% (atropine). The
reference compound 2 was previously reported to have no effects
in this model, similar to compound 13-15.12
Commonly used abbreviations are DMF (N,N-dimethylforma-
mide), EtOH (ethanol), MeOH (methanol), EtOAc (ethyl acetate),
HOAc (acetic acid), DME (1,2-diethoxyethane), and THF (tetrahy-
drofuran).
4-((R)-2-Butylamino)-2,7-dimethyl-8-(2-methyl-6-methoxypy-
rid-3-yl)[1,5-a]pyrazolo-1,3,5-triazine (13-15). Step A: 2-Meth-
oxy-6-methylpyridine. Sodium (31.0 g, 1.35 mol) was added
portionwise to methanol (500 mL) over 30 min with stirring in a
flask equipped with a reflux condenser. After the addition was
complete, the reaction mixture was allowed to cool to ambient
temperature. 2-Fluoro-6-methylpyridine (50 g, 450 mmol) was
added portionwise with stirring. The reaction mixture was then
heated to reflux temperature and stirred for 48 h. The mixture was
then cooled to ambient temperature and solvent was removed in
vacuo to provide 2-methoxy-6-methylpyridine, a yellow oil. The
residue was taken up in water (500 mL), and three extractions with
ether (200 mL) were performed. The combined organic layers were
dried over MgSO4 and filtered, and solvent was removed in vacuo
1
from the filtrate to give a yellow liquid. H NMR (CDCl3, 300
MHz): δ 7.44 (dd, 1H, J ) 8, 7), 6.71 (d, 1H, J ) 7), 6.53 (d, 1H,
J ) 8), 3.91 (s, 3H), 2.45 (s, 3H).
Conclusion
Alternatively, a mixture of 2-hydroxy-6-methylpyridine (6.85 g,
62.8 mmol), silver carbonate (22.5 g, 81.6 mmol), iodomethane
(39.1 mL, 628 mmol), and chloroform (200 mL) was stirred at
ambient temperature for 40 h in the dark. The reaction mixture
was filtered through Celite. The collected solid was washed with
ether. The combined filtrates were concentrated in vacuo to give
2-methoxy-6-methylpyridine, a liquid (6.25 g), which gave NMR
data identical to the product from the above procedure.
Step B: 3-Bromo-6-methoxy-2-methylpyridine. A mixture of
2-methoxy-6-methylpyridine (17.0 g, 138 mmol) and a solution of
disodium hydrogen phosphate (0.15 M in water, 250 mL) was
stirred at room temperature. Bromine (7.1 mL, 138 mmol) was
added dropwise over 15 min via an addition funnel. The reaction
mixture was then stirred at room temperature for 4 h. The clear
colorless solution was diluted with water (500 mL) and extracted
with dichloromethane (200 mL) three times. The combined organic
layers were dried over MgSO4 and filtered, and solvent was removed
in vacuo from the filtrate to give a yellow liquid. Flash chroma-
tography on silica gel (EtOAc/hexane, 1:20) and removal of solvent
from the desired combined fractions afforded 6-methoxy-3-bromo-
2-methylpyridine, a clear colorless liquid (15.4 g). 1H NMR (CDCl3,
300 MHz): δ 7.60(d, 1H, J ) 8), 6.46 (d, 1H, J ) 8), 3.89 (s, 3H),
2.54 (s, 3H).
Step C: 6-Methoxy-2-methylpyridine-3-boronic Acid. A solu-
tion of 6-methoxy-3-bromo-2-methylpyridine (59.8 g, 296 mmol)
in dry THF (429 mL) was cooled with stirring to about -78 °C
under a nitrogen atmosphere. A solution of n-butyllithium (2.5 M,
130.4 mL, 326 mmol) in hexane was added dropwise over 30 min.
The reaction mixture was stirred for 3 h at about -78 °C. A solution
of triisopropyl borate (102.7 mL, 445 mmol) in dry THF (100 mL)
was added dropwise over 30 min. The reaction mixture was warmed
to ambient temperature with stirring over 16 h. Acetic acid (37.35
g, 622 mmol) and then water (110 mL) were added to the reaction
mixture with stirring. After 2 h, the layers were separated and the
organic layer was concentrated in vacuo. The residue was taken
up in 2-propanol (750 mL), and solvent was removed on a rotary
Compound 13-15 is a potent, orally bioavailable antagonist
of CRF1 receptors. The compound has anxiolytic efficacy in
the rat elevated plus maze and defensive withdrawal models
with little effect on locomotor or rotorod activity. It has
promising oral pharmacokinetic profiles in rats, dogs, and
chimpanzees. The compound has small or no effects on dog
cardiovascular and respiratory functions and no effect on rat
gastrointestinal function. This compound has a mild acute effect
on renal function at 30 mg/kg (po). Compound 13-15 (BMS-
562086)28 has been advanced into clinical trials, the results of
which will be reported separately.
Experimental Section
Chemistry. 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 instrument (300 MHz); chemical shifts were recorded in ppm
(δ) from an internal tetramethysilane standard in deuterochloroform
or deuterodimethyl 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-ioniza-
tion (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 the general procedures
outlined by D. Perrin and WLF Armarego.29 Chromatography was
performed on silica gel using the solvent systems indicated below.
For mixed solvent systems, the volume ratios are given. Otherwise,
parts and percentages are by weight.

3090 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9
Gilligan et al.
evaporator (bath temperature of ∼50 °C). The residue was triturated
with ether. The product, 6-methoxy-2-methylpyridine-3-boronic
acid, was collected by filtration and dried in vacuo (48.4 g): mp
and stirred for 18 h. The mixture was cooled to room temperature,
and solvent was removed in vacuo. The residue was dissolved in
water, and a 1 N HCl solution was added slowly until pH ∼ 6.
The aqueous layer was extracted with EtOAc three times; the
combined organic layers were dried over MgSO4 and filtered.
Solvent was removed in vacuo to give a solid. Trituration with
ether, filtration, and drying in vacuo afforded a white solid (3.9 g).
1H NMR (CD3OD, 300 MHz): δ 7.49 (d, 2H, J ) 8), 6.69 (d, 2H,
J ) 8), 3.93 (s, 3H), 2.35 (s, 3H), 2.28 (s, 3H), 2.24 (s, 3H). APCI+-
MS: 286 (M+ + H). Anal. (C14H15N5O2 ·H2O) C, H, N.
Step I: 4-Choro-2,7-dimethyl-8-(2-methyl-6-methoxypyrid-
3-yl)[1,5-a]pyrazolo-1,3,5-triazine. A mixture of 2,7-dimethyl-8-
(2-methyl-6-methoxypyrid-3-yl)[1,5-a]pyrazolo-1,3,5-triazin-4-
one (example 1, 3.9 g, 13.7 mmol), diisopropylethylamine (9.5 mL,
54.7 mmol), phosphorus oxychloride (5.1 mL, 54.7 mmol), and
toluene (75 mL) was stirred at reflux temperature for 4 h. The
volatiles were removed in vacuo. The residue was loaded on a pad
of silica gel on Celite and eluted with a 1:1 mixture of EtOAc and
hexane. Solvent was removed in vacuo from the filtrate to give
4-chloro-2,7-dimethyl-8-(2-methyl-6-methoxypyrid-3-yl)[1,5-a]-
pyrazolotriazine, an oil, which was used without further purification.
1
>200 °C. H NMR (CD3OD, 300 MHz): δ 7.83 (d, 1H, J ) 8),
6.56 (d, 1H, J ) 8), 3.85 (s, 3H), 2.44 (s, 3H). GC-MS: 168 (M+
+ H).
Step D: 2-Methyl-3-(5-methylisoazolyl)-6-methoxypyridine.
A mixture of 4-iodo-5-methylisoxazole (18.2 g, 87 mmol), 6-meth-
oxy-2-methylpyridine-3-boronic acid (14.6 g, 87 mmol), sodium
bicarbonate (22.0 g, 262 mmol), water (150 mL), and DME (150
mL) was degassed three times with stirring by the application of a
vacuum and then introduction of a nitrogen atmosphere. [1,1-
Bis(diphenylphosphino)ferrocene]dichloropalladium(II) (2.14 g, 2.6
mmol) was added in one portion. The reaction mixture was degassed
as before. The reaction mixture was then stirred at 80 °C for 4 h,
and then it was cooled to ambient temperature. Three extractions
with EtOAc, drying the combined organic layers over MgSO4,
filtration, and removal of solvent in vacuo afforded an oil. Flash
chromatography (EtOAc/hexane, 1:9) and removal of solvent in
vacuo from the desired fractions gave the product 2-methyl-3-(5-
methylisoxazol-4-yl)-6-methoxypyridine (7.15 g). 1H NMR (CDCl3,
300 MHz): δ 8.16 (s, 1H), 7.33 (d, 1H, J ) 8), 6.63 (d, 1H, J )
8), 3.95 (s, 3H), 2.35 (s, 6H). APCI+-MS: 205 (M+ + H).
Step E: 1-Cyano-1-(2-methyl-6-methoxypyrid-3-yl)propanone,
Sodium Salt. A mixture of sodium methoxide (25% w/w, 13 mL,
70 mmol), 2-methyl-3-(5-methylisoxazol-4-yl)-6-methoxypyridine
(7.15 g, 35 mmol), and methanol (50 mL) was stirred at room
temperature for 16 h. Solvent was removed in vacuo to give a
yellow oil. Trituration with ether, filtration, and drying in vacuo
afforded the crude product as a white solid (9.3 g).
Step J: 4-((R)-2-Butylamino)-2,7-dimethyl-8-(2-methyl-6-
methoxypyrid-3-yl)[1,5-a]pyrazolo-1,3,5-triazine (13-15). A mix-
ture of 4-chloro-2,7-dimethyl-8-(2-methyl-6-methoxypyrid-3-yl)[1,5-
a]pyrazolotriazine,(R)-2-butylamine(2.0mL,20.5mmol),diisopropyl-
ethylamine (9.5 mL, 54.7 mmol), and dry THF (25 mL) was stirred
at ambient temperature for 18 h. Solvent was removed in vacuo.
Column chromatography of the residue (first using EtOAc/hexane,
1:2, then using EtOAc/hexane, 1:4) afforded the product. Removal
of solvent in vacuo gave the title product, a white solid (2.3 g):
mp 118.3 °C. 1H NMR (CDCl3, 300 MHz): δ 7.41 (d, 1H, J ) 8),
6.63 (d, 1H, J ) 8), 6.25 (br d, 1H, J ) 9), 4.35-4.30 (m, 1H),
3.95 (s, 3H), 2.49 (s, 3H), 2.35 (s, 3H), 2.30 (s, 3H), 1.76-1.66
(m, 2H), 1.34 (d, 3H, J ) 7), 1.02 (t, 3H, J ) 7). 13C NMR (CDCl3,
100.52 MHz): δ 163.8, 163.0, 155.7, 153.7, 147.8, 146.6, 141.6,
118.5, 107.4, 106.6, 53.3, 48.2, 29.7, 26.1, 22.9, 20.4, 13.1, 10.3.
IR (neat, KBr, cm-1): 3380 (m), 3371 (m), 2968 (m), 2928 (m),
2872 (w), 1621 (s), 1588 (s), 1544 (s), 1489 (s), 1460 (s), 1425
(s), 1413 (s), 1364 (s), 1346 (m), 1304 (s), 1275 (s), 1247 (s), 1198
(m), 1152 (m), 1134 (m), 1112 (m), 1034 (s), 1003 (m). ESI(+)-
Step F: 5-Amino-4-(2-methyl-6-methoxypyrid-3-yl)-3-meth-
ylpyrazole. A mixture of 1-cyano-1-(2-methyl-6-methoxypyrid-3-
yl)propan-2-one, sodium salt (9.3 g), hydrazine hydrate (6 mL,
123.3 mmol), and glacial acetic acid (150 mL) was stirred at room
temperature for 4 h. The reaction mixture was concentrated in
vacuo. The residue was dissolved in 1 N HCl, and the resulting
solution was extracted with EtOAc two times. A 1 N NaOH solution
was added to the aqueous layer until pH 12. The resulting
semisolution was extracted three times with ethyl acetate. The
combined organic layers were dried over MgSO4 and filtered.
1
HRMS calcd for C18H24N6O: 341.2089. Found: 341.2093 (M+
H). Anal. (C18H24N6O) C, H, N.
+
Solvent was removed in vacuo to give a viscous oil (5.8 g). H
NMR (CDCl3, 300 MHz): δ 7.37 (d, 2H, J ) 8), 6.62 (d, 2H, J )
8), 3.95 (s, 3H), 2.36 (s, 3H), 2.08 (s, 3H). APCI+-MS: 219 (M+
+ H), 260 (M+ + CH3CN).
The following analogues were prepared according to the methods
described above from the appropriate pyridine precursors and
amines.
13-1: oil. NMR (CDCl3, 300 MHz): δ 6.31 (s, 1H), 6.17 (d, 1H,
J ) 10), 4.20 (m, 1H), 3.09 (s, 6H), 2.46 (s, 3H), 2.20 (s, 3H),
2.17 (s, 3H), 1.99 (s, 3H), 1.76 (m, 2H), 1.64 (m, 2H), 1.02 (t, 3H,
J ) 7). CI-HRMS calcd for C21H32N7: 382.2719. Found: 382.2712
(M + H). Anal.: HPLC.
13-2: oil. NMR (CDCl3, 300 MHz): δ 6.69 (m, 1H), 6.31 (s,
1H), 3.86 (q, 2H, J ) 5), 3.65 (t, 2H, J ) 5), 3.44 (s, 3H), 3.08 (s,
6H), 2.46 (s, 3H), 2.20 (s, 3H), 2.15 (s, 3H), 1.97 (s, 3H). CI-
HRMS calcd for C19H28N7O: 370.2355. Found: 370.2378 (M +
H). Anal.: HPLC.
13-3: oil. NMR (CDCl3, 300 MHz): δ 6.31 (s, 1H), 4.33 (s, 4H,
J ) 5), 3.77 (t, 4H, J ) 6), 3.39 (s, 6H), 3.08 (s, 6H), 2.37 (s, 3H),
2.15 (s, 3H), 2.14 (s, 3H), 1.97 (s, 3H). CI-HRMS calcd for
C22H34N7O2: 428.2774. Found: 428.2775 (M + H). Anal.: HPLC.
13-4: oil. NMR (CDCl3, 300 MHz): δ 6.31 (s, 1H), 4.07 (m,
4H), 3.08 (s, 6H), 2.38 (s, 3H), 2.16 (s, 3H), 2.15 (s, 3H), 1.98 (s,
3H), 1.36 (t, 6H, J ) 7). CI-HRMS calcd for C20H30N7: 368.2563.
Found: 368.2565 (M + H). Anal.: HPLC.
Step G: 5-Acetamidino-4-(2-methyl-6-methoxypyrid-3-yl)-3-
methylpyrazole, Acetic Acid Salt. Ethyl acetamidate hydrochloride
(6.46 g, 52.2 mmol) was added quickly to a rapidly stirred mixture
of potassium carbonate (6.95 g, 50.0 mol), dichloromethane (60
mL), and water (150 mL). The layers were separated, and the
aqueous layer was extracted with dichloromethane (2 × 60 mL).
The combined organic layers were dried over MgSO4 and filtered.
Solvent was removed by simple distillation, and a clear pale-yellow
liquid was used without further purification.
Glacial acetic acid (1.0 mL, 17.4 mmol) was added to a stirred
mixture of 5-amino-4-(2-methyl-6-methoxypyrid-3-yl)-3-meth-
ylpyrazole (3.8 g, 17.4 mmol), ethyl acetamidate free base, and
dichloromethane (100 mL). The resulting reaction mixture was
stirred at room temperature for 16 h; at the end of that time, it was
concentrated in vacuo. The residue was triturated with ether, and
the product was filtered and washed with copious amounts of ether.
1
The white solid was dried in vacuo (5.4 g). H NMR (CD3OD,
300 MHz): δ 7.43 (d, 2H, J ) 8), 6.69 (d, 2H, J ) 8), 4.9 (br s,
2H), 3.93 (s, 3H), 2.31 (s, 3H), 2.24 (s, 3H), 2.13 (s, 3H), 1.88 (s,
3H). APCI+-MS: 260 (M+ + H).
13-5: oil. NMR (CDCl3, 300 MHz): δ 6.31 (s, 1H), 4.18 (m,
4H), 3.08 (s, 6H), 2.37 (s, 3H), 2.16 (s, 3H), 2.15 (s, 3H), 1.98 (s,
3H), 1.79 (m, 4H), 0.98 (t, 3H, J ) 7). CI-HRMS calcd for
C22H34N7: 396.2875. Found: 396.2878 (M + H). Anal.: HPLC.
13-6: oil. NMR (CDCl3, 300 MHz): δ 6.31 (s, 1H), 4.08 (m,
4H), 3.08 (s, 6H), 2.38 (s, 3H), 2.16 (s, 3H), 2.15 (s, 3H), 1.98 (s,
3H), 1.76 (m, 2H), 1.42 (m, 2H), 1.33 (t, 3H, J ) 7), 1.00 (t, 3H,
Step H: 2,7-Dimethyl-8-(2-methyl-6-methoxypyrid-3-yl)[1,5-
a]pyrazolo[1,3,5]triazin-4(3H)-one. Sodium pellets (3.9 g, 169
mmol) were added portionwise to ethanol (200 mL) with vigorous
stirring. After all the sodium reacted, 5-acetamidino-4-(2-methyl-
6-methoxypyrid-3-yl)-3-methylpyrazole, acetic acid salt (5.4 g, 16.9
mmol) and diethyl carbonate (16.4 mL, 135.3 mmol) were added.
The resulting reaction mixture was heated to reflux temperature

Pyridylpyrazolotriazines
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9 3091
J ) 7). CI-HRMS calcd for C22H34N7: 396.2875. Found: 396.2881
(M + H). Anal.: HPLC.
6), 3.39 (s, 6H), 2.40 (s, 3H), 2.33 (s, 3H), 2.23 (s, 3H). CI-HRMS
calcd for C20H29N6O3: 401.2301. Found: 401.2301 (M + H). Anal.:
HPLC.
13-23: oil. NMR (CDCl3, 300 MHz): δ 7.40 (d, 1H, J ) 8),
6.63 (d, 1H, J ) 8), 4.19 (m, 4H), 3.95 (s, 3H), 3.78 (t, 4H, J )
6), 3.40 (s, 3H), 2.40 (s, 3H), 2.33 (s, 3H), 2.24 (s, 3H), 1.34 (t,
3H, J ) 7). CI-HRMS calcd for C19H27N6O2: 371.2195. Found:
371.2204 (M + H). Anal.: HPLC.
13-24: oil. NMR (CDCl3, 300 MHz): δ 7.40 (d, 1H, J ) 9),
6.62 (d, 1H, J ) 8), 4.35 (m, 2H), 4.06 (m, 2H), 3.95 (s, 3H), 3.78
(t, 2H, J ) 6), 3.39 (s, 3H), 2.41 (s, 3H), 2.33 (s, 3H), 2.25 (s,
3H), 1.28 (m, 1H), 0.55 (m, 2H), 0.41 (m, 2H). CI-HRMS calcd
for C21H29N6O2: 397.2352. Found: 397.2361 (M + H). Anal.:
HPLC.
13-25: oil. NMR (CDCl3, 300 MHz): δ 7.41 (d, 1H, J ) 8),
6.62 (d, 1H, J ) 8), 4.09 (m, 4H), 3.95 (s, 3H), 2.39 (s, 3H), 2.38
(s, 3H), 2.25 (s, 3H), 1.80 (m, 2H), 1.25 (m, 1H), 0.98 (t, 3H, J )
7), 0.55 (m, 2H), 0.40 (m, 2H). CI-HRMS calcd for C21H29N6O:
381.2403. Found: 381.2396 (M + H). Anal.: HPLC.
13-26: oil. NMR (CDCl3, 300 MHz): δ 7.40 (d, 1H, J ) 8),
6.62 (d, 1H, J ) 8), 4.16 (m, 2H), 3.95 (s, 3H), 3.56 (s, 3H), 2.41
(s, 3H), 2.33 (s, 3H), 2.26 (s, 3H), 1.34 (t, 3H, J ) 7). CI-HRMS
calcd for C17H23N6O: 327.1933. Found: 327.1928 (M + H). Anal.:
HPLC.
13-27: oil. NMR (CDCl3, 300 MHz): δ 7.40 (d, 1H, J ) 9),
6.62 (d, 1H, J ) 9), 4.08 (m, 2H), 3.95 (s, 3H), 3.55 (s, 3H), 2.41
(s, 3H), 2.33 (s, 3H), 2.25 (s, 3H), 1.80 (m, 2H), 0.98 (t, 3H, J )
7). CI-HRMS calcd for C18H25N6O: 341.2890. Found: 341.2083
(M + H). Anal.: HPLC.
13-28: oil. NMR (CDCl3, 300 MHz): δ 7.40 (d, 1H, J ) 8),
6.63 (d, 1H, J ) 8), 4.12 (m, 2H), 3.95 (s, 3H), 3.55 (s, 3H), 2.41
(s, 3H), 2.33 (s, 3H), 2.25 (s, 3H), 1.75 (m, 2H), 1.41 (m, 2H),
0.99 (t, 3H, J ) 7). CI-HRMS calcd for C19H27N6O: 355.2246.
Found: 355.2240 (M + H). Anal.: HPLC.
13-29: oil. NMR (CDCl3, 300 MHz): δ 7.40 (d, 1H, J ) 8),
6.62 (d, 1H, J ) 8), 4.04 (s, 4H), 3.95 (s, 3H), 2.40 (s, 3H), 2.33
(s, 3H), 2.24 (s, 3H), 1.76 (m, 2H), 1.43 (m, 2H), 1.33 (t, 3H, J )
7), 1.00 (t, 3H, J ) 7). CI-HRMS calcd for C20H29N6O: 369.2403.
Found: 369.2399 (M + H). Anal.: HPLC.
13-30: oil. NMR (CDCl3, 300 MHz): δ 7.41 (d, 1H, J ) 8),
6.62 (d, 1H, J ) 8), 3.97 (s, 4H), 3.95 (s, 3H), 2.39 (s, 3H), 2.33
(s, 3H), 2.24 (s, 3H), 1.76 (m, 4H), 1.42 (m, 2H), 0.92 (m, 6H).
CI-HRMS calcd for C21H31N6O: 383.2533. Found: 383.2546 (M
+ H). Anal.: HPLC.
Biological Evaluation. The CRF receptor binding assay, the
CRF-stimulated adenylate cyclase assay, the rat defensive with-
drawal, the rat elevated plus maze tests, the rat receptor occupancy
studies, and the discrete pharmacokinetic studies have been
described previously.18,21-24,27 The dog cardiovascular, dog pul-
monary, rat renal, and rat gastrointestinal studies were performed
by previously described procedures.12
Dog n-in-1 Pharmacokinetic Studies. The dog n-in-1 pharma-
cokinetic studies were analyzed with an LC/MS/MS method at
Primedica Corporation (Worcester, MA). The test compounds and
the reference compound (0.1 mg/kg each (free base weight)) were
administered as a homogeneous mixture in Labrafil (test samples
were vortexed for 15 min) via oral gavage to each of three beagle
dogs. Blood samples were collected into EDTA tubes predose and
at 0.5, 1, 2, 4, 6, 9, 12, and 24 h postdose. Samples were extracted
using a protein precipitation technique. A sample aliquot was
transferred to a plastic test tube, and 25 µL of internal standard
was added. Next, 1 mL of acetonitrile was added to the samples
and they were vortexed briefly and centrifuged for 5 min. The
organic layer was transferred to a clean plastic tube and evaporated
to dryness at approximately 65 °C under nitrogen. The dried samples
were reconstituted in formic acid/acetonitrile/water (0.1:10:90) and
vortexed for approximately 1 min, and the resulting solutions were
transferred to autosampler vials.
13-7: solid. NMR (CDCl3, 300 MHz): δ 6.31 (s, 1H), 3.98 (s,
4H), 3.08 (s, 6H), 2.37 (s, 3H), 2.16 (s, 3H), 2.15 (s, 3H), 1.98 (s,
3H), 1.76 (m, 4H), 1.42 (m, 2H), 0.99 (m, 6H). CI-HRMS calcd
for C23H36N7: 410.3032. Found: 410.3034 (M + H). Anal.: HPLC.
13-8: solid; mp 117 °C. NMR (CDCl3, 300 MHz): δ 7.98 (s,
1H), 6.47 (s, 1H), 4.08 (m, 4H), 3.10 (s, 6H), 2.39 (s, 3H), 2.28 (s,
3H), 2.15 (s, 3H), 1.35 (t, 6H, J ) 8). CI-HRMS calcd for C19H28N7:
354.2406. Found: 354.2388 (M + H). Anal. Calcd for C19H27N7:
C, 64.56; H, 7.70; N, 27.74. Found: C, 64.89; H, 7.57; N, 27.63.
13-9: solid; mp 103-104 °C. NMR (CDCl3, 300 MHz): δ 7.97
(s, 1H), 6.47 (s, 1H), 4.30 (m, 4H), 3.76 (d, 4H, J ) 8), 3.39 (s,
6H), 3.10 (s, 6H), 2.37 (s, 3H), 2.27 (s, 3H), 2.15 (s, 3H). CI-
HRMS calcd for C21H32N7O2: 414.2617. Found: 414.2600 (M +
H). Anal.: HPLC.
13-10: solid; mp 153-154 °C. NMR (CDCl3, 300 MHz): δ 7.99
(s, 1H), 7.26 (s, 1H), 6.16 (d, 1H, J ) 8), 4.20 (m, 1H), 3.11 (s,
6H), 2.46 (s, 3H), 2.33 (s, 3H), 2.17 (s, 3H), 1.69 (m, 4H). CI-
HRMS calcd for C20H30N7: 367.2484. Found: 367.2477 (M + H).
Anal.: HPLC.
13-11: oil. NMR (CDCl3, 300 MHz): δ 7.96 (s, 1H), 6.69 (s,
1H), 4.32 (m, 4H), 3.94 (s, 3H), 3.76 (t, 2H, J ) 8), 3.39 (s, 6H),
2.39 (s, 3H), 2.26 (s, 3H), 2.17 (s, 3H). CI-HRMS calcd for
C20H28N6O3: 401.2301. Found: 401.2302 (M + H). Anal.: HPLC.
13-12: oil. NMR (CDCl3, 300 MHz): δ 7.97 (s, 1H), 6.69 (s,
1H), 4.07 (m, 2H), 3.94 (s, 3H), 2.40 (s, 3H), 2.28 (s, 3H), 2.17 (s,
3H), 1.36 (t, 6H, J ) 8). CI-HRMS calcd for C18H24N6O: 341.2090.
Found: 341.2091 (M + H). Anal.: HPLC.
13-13: oil. NMR (CDCl3, 300 MHz): δ 7.98 (s, 1H), 6.70 (s,
1H), 6.19 (d, 1H, J ) 8), 4.22 (m, 1H), 3.95 (s, 3H), 2.47 (s, 3H),
2.32 (s, 3H), 2.19 (s, 3H), 1.73 (m, 4H), 1.00 (t, 6H, J ) 8). CI-
HRMS calcd for C19H27N6O: 355.2246. Found: 355.2248 (M +
H). Anal.: HPLC.
13-14: oil. NMR (CDCl3, 300 MHz): δ 7.41 (d, 1H, J ) 9),
6.63 (d, 1H, J ) 9), 6.19 (d, 1H, J ) 10), 4.22 (m, 1H), 3.95 (s,
3H), 2.48 (s, 3H), 2.35 (s, 3H), 2.29 (s, 3H), 1.73 (m, 4H), 1.00 (t,
6H, J ) 8). CI-HRMS calcd for C19H27N6O: 355.2246. Found:
355.2238 (M + H). Anal.: HPLC.
13-16: oil. NMR (CDCl3, 300 MHz): δ 7.41 (d, 1H, J ) 8),
6.63 (d, 1H, J ) 8), 6.22 (br d, 1H, J ) 9), 4.33 (m, 1H), 3.95 (s,
3H), 2.48 (s, 3H), 2.34 (s, 3H), 2.29 (s, 3H), 1.71 (m, 2H), 1.34 (d,
3H, J ) 7), 1.03 (t, 3H, J ) 7). CI-HRMS calcd for C18H24N6O:
341.2090. Found: 341.2088 (M + H). Anal.: HPLC.
13-17: oil. NMR (CDCl3, 300 MHz): δ 7.40 (d, 1H, J ) 8),
6.63 (d, 1H, J ) 8), 6.20 (br d, 1H, J ) 9), 4.40 (m, 1H), 3.95 (s,
3H), 2.48 (s, 3H), 2.34 (s, 3H), 2.29 (s, 3H), 1.65 (m, 2H), 1.48
(m, 2H), 1.34 (d, 3H, J ) 7), 0.97 (t, 3H, J ) 7). CI-HRMS calcd
for C19H27N6O: 355.2246. Found: 355.2244 (M + H). Anal.: HPLC.
13-18: oil. NMR (CDCl3, 300 MHz): δ 7.40 (d, 1H, J ) 8),
6.63 (d, 1H, J ) 8), 6.20 (br d, 1H, J ) 9), 4.40 (m, 1H), 3.95 (s,
3H), 2.48 (s, 3H), 2.34 (s, 3H), 2.29 (s, 3H), 1.65 (m, 2H), 1.48
(m, 2H), 1.34 (d, 3H, J ) 7), 0.97 (t, 3H, J ) 7). CI-HRMS calcd
for C19H27N6O: 355.2246. Found: 355.2250 (M + H). Anal.: HPLC.
13-19: oil. NMR (CDCl3, 300 MHz): δ 7.40 (d, 1H, J ) 8),
6.63 (d, 1H, J ) 8), 6.22 (d, 1H, J ) 8), 4.37 (m, 1H), 3.95 (s,
3H), 2.48 (s, 3H), 2.34 (s, 3H), 2.29 (s, 3H), 1.65 (m, 2H), 1.38
(m, 4H), 1.34 (d, 3H, J ) 7), 0.92 (t, 3H, J ) 7). CI-HRMS calcd
for C20H29N6O: 369.2403. Found: 369.2427 (M + H). Anal.: HPLC.
13-20: oil. NMR (CDCl3, 300 MHz): δ 7.40 (d, 1H, J ) 8),
6.62 (d, 1H, J ) 8), 4.07 (m, 4H), 3.95 (s, 3H), 2.40 (s, 3H), 2.33
(s, 3H), 2.25 (s, 3H), 1.35 (t, 6H, J ) 7). CI-HRMS calcd for
C18H25N6O: 341.2090. Found: 341.2086 (M + H). Anal.: HPLC.
13-21: oil. NMR (CDCl3, 300 MHz): δ 7.40 (d, 1H, J ) 8),
6.62 (d, 1H, J ) 8), 4.00 (m, 4H), 3.95 (s, 3H), 2.39 (s, 3H), 2.33
(s, 3H), 2.24 (s, 3H), 1.79 (m, 4H), 0.98 (t, 3H, J ) 7). CI-HRMS
calcd for C20H29N6O: 369.2403. Found: 369.2397 (M + H). Anal.:
HPLC.
Gradient reversed-phase LC/MS/MS conditions were employed
to quantitate these analytes. A gradient consisting of 0.1% formic
acid in water and acetonitrile was used to elute the analytes from
13-22: oil. NMR (CDCl3, 300 MHz): δ 7.39 (d, 1H, J ) 8),
6.62 (d, 1H, J ) 8), 4.33 (m, 4H), 3.95 (s, 3H), 3.76 (t, 4H, J )

3092 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9
Gilligan et al.
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
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a Phenomonex Luna C8 column (3 µm, 20 mm × 2 mm). Analytes
were quantitated with a SCIEX API 3000 LC/MS/MS using
TurboIonSpray under positive ion, MRM conditions.
Chimpanzee Pharmacokinetic Studies. The chimpanzee phar-
macokinetic studies were analyzed with an LC/MS/MS method at
Primedica Corporation (Worcester, MA). Samples were prepared
and analyzed as for the dog n-in-1 pharmacokinetic studies The
following mass transitions were used: compound 13-15, 341.2 f
285.3; internal standard (8-(5-chloro-2,4-dimethoxyphenyl)-2,7-
pyrazolo[1,5-a][1,3,5]triazinyl]bis(2-methoxyethyl)amine), 450.3 f
319.1.
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zees at 1 mg/kg, and blood samples were collected into EDTA tubes
predose and at 2, 4, 6, 9, 12, 24, 36, 48, 72, and 96 h postdose.
The oral suspension (1 mg/mL) was prepared in a 50-50 (v/v)
mixture of 0.5% methylcellulose and Tang orange juice containing
1% Tween-80. No adverse effects, including emesis, were observed
in the chimpanzees. In addition, the compound was administered
intravenously to one chimpanzee at a dose of 0.5 mg/kg in 10%
DMAC, 70% propylene glycol, and deionized water over 10 min
(0.2 mL/kg). Blood sampling was similar to that described after
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Note Added after ASAP Publication. An author name was
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10, 2009. The revised version posted on April 15, 2009.
Supporting Information Available: HPLC purity data of final
products and the dog cardiovascular function, dog pulmonary
function, rat renal function, and rat gastrointestinal motility assess-
ments. This material is available free of charge via the Internet at
http://pubs.acs.org.
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