4-(1,3-dimethoxyprop-2-ylamino)-2,7-dimethyl-8-(2,4- dichlorophenyl)pyrazolo[1,5-α]-1,3,5-triazine: A potent, orally bioavailable CRF1 receptor antagonist

Article abstract of DOI:10.1021/jm9904351

Structure-activity studies in the pyrazolo[1,5-a]-1,3,5-triazine series led to the discovery that compound 11i (DMP696) is a potent hCRF₁ receptor antagonist (K(i) = 1.7 nM vs 7.5 nM for α-hel-CRF(9-41), hCRF₁ adenylate cyclase IC

Full text of DOI:10.1021/jm9904351

J . Med. Chem. 2000, 43, 449-456
449
4-(1,3-Dim eth oxyp r op -2-yla m in o)-2,7-d im eth yl-8-(2,4-d ich lor op h en yl)-
p yr a zolo[1,5-a ]-1,3,5-tr ia zin e: A P oten t, Or a lly Bioa va ila ble CRF 1 Recep tor
An ta gon ist
Liqi He, Paul J . Gilligan,* Robert Zaczek,† Lawrence W. Fitzgerald,† J ohn McElroy,† H-S. L. Shen,‡
J o Anne Saye,§ Ned H. Kalin,| Steven Shelton,| David Christ,‡ George Trainor, and Paul Hartig†
Chemical and Physical Sciences Department, DuPont Pharmaceuticals Company, Experimental Station, P.O. Box 80500,
Wilmington, Delaware 10880-0500
Received August 24, 1999
Structure-activity studies in the pyrazolo[1,5-a]-1,3,5-triazine series led to the discovery that
compound 11i (DMP696) is a potent hCRF1 receptor antagonist (Ki ) 1.7 nM vs 7.5 nM for
R-hel-CRF(9-41), hCRF1 adenylate cyclase IC50 ) 82 nM vs 286 nM for R-hel-CRF(9-41)).
Compound 11i has excellent oral pharmacokinetic profiles in rats and dogs (37% and 50% oral
bioavailabilities, respectively). This compound displays good activity in the rat situational
anxiety model (MED ) 3 mg/kg (po)), whereas a literature standard 1 (CP154526-1) was inactive
(MED > 30 mg/kg (po)). Analogue 11i reduced stereotypical mouth movements in rhesus
monkeys by 50% at 21 mg/kg (po) using the human intruder paradigm. Overall, the profile of
pyrazolotriazine 11i indicates that hCRF1 receptor antagonists may be anxiolytic agents, which
have reduced motor side effect profiles.
In tr od u ction
and non-human primates provide additional support for
the hypothesis that hypersecretion of CRF may be
Corticotropin-releasing factor (CRF), a 41-amino acid
peptide, is the primary physiological regulator of pro-
opiomelanocortin (POMC)-derived peptide secretion from
the anterior pituitary gland.1-3 This hormone also has
a broad extrahypothalamic distribution in the central
nervous system, apart from the pituitary gland, and
produces a broad spectrum of autonomic, electrophysi-
ological, and behavioral effects consistent with a neuro-
transmitter or neuromodulator role in the brain.4-6 CRF
also plays a significant role in the coordination of
immune system responses to physiological, psychologi-
cal, and immunological stressors.7,8
Clinical studies have provided evidence that CRF may
have a role in several psychiatric disorders and neuro-
logical diseases including depression, anxiety, and feed-
ing disorders.3 Hyposecretion of CRF has been postu-
lated in the etiology and pathophysiology of Alzheimer’s
disease and Parkinson’s disease.9 Postmortem studies
on the brains of Alzheimer’s patients uncovered a deficit
in CRF immunoreactivity in key cortical regions.10
Hypersecretion of CRF has been proposed to underlie
depression and anxiety-related disorders. In major
depression, the concentration of CRF is significantly
increased in the cerebrospinal fluid (CSF) of medication-
free individuals.11-14 Immunohistochemical studies have
shown that CRF-secreting cells are increased 4-fold in
the hypothalami of depressed patients.15 In addition,
there is a blunted effect of intravenous (iv) administra-
tion of CRF in depressed patients on adrenocorticotropin
(ACTH) plasma levels.16-18 Behavioral studies in rats
involved in the symptoms seen in human depression.19
Preclinical studies support a role for CRF in human
anxiety disorders. CRF produces “anxiogenic” effects in
animals after direct injection into the cerebral ventricles
(icv administration), and interactions between anxiolytic
drugs and CRF have been demonstrated in a variety of
behavioral models for anxiety.20,21 These “anxiogenic”
effects may be blocked with the peptidic CRF receptor
antagonist R-helical CRF(9-41) in a variety of behav-
ioral paradigms, demonstrating that the antagonist
produces “anxiolytic-like” effects that are qualitatively
similar to those of the benzodiazepines.22,23 Chlordiaz-
epoxide modulates the “anxiogenic” effects of CRF in
both the conflict test24,25 and the acoustic startle test26
in rats. Behavioral and physiological studies with trans-
genic mice which overexpress CRF and with mutant
mice which are null for CRF or one of its receptor
subtypes demonstrate a pivotal role for CRF in coordi-
nating neuronal, endocrine, and immune responses to
stress.3,27
The effects of CRF are mediated through seven-
transmembrane receptors, which are coupled to G-
proteins and are located in the central nervous system
(CNS) as well as the periphery.3,28 There are two
subtypes of CRF receptors: CRF1 and CRF2; three splice
variants have been found for the latter subtype: CRF2R
,
CRF2â, and CRF2γ. These receptor subtypes have been
cloned and expressed in cell systems.3 The distribution
of these subtypes in the CNS has been elucidated.3 The
function of these receptor subtypes in disease is cur-
rently the subject of intense research, and the evalua-
tion of subtype-selective compounds is necessary for
progress.
* Corresponding author. Tel: 302-695-1439. Fax: 302-695-4390.
E-mail: paul.j.gilligan@dupontpharma.com.
† Department of CNS Diseases Research.
‡ Drug Metabolism and Pharmacokinetic Section.
§ Department of Preclinical Pharmacology.
Nonpeptidic CRF1-selective receptor antagonists have
been reported in the literature,3 and the most exten-
| Department of Psychiatry, School of Medicine, University of
Wisconsin, Madison, WI 53719.
10.1021/jm9904351 CCC: $19.00 © 2000 American Chemical Society
Published on Web 01/26/2000

450 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 3
He et al.
Ch a r t 1
Sch em e 1a
sively studied compound is pyrrolopyrimidine
1
(CP154526-1).29-33 This high-affinity CRF1 antagonist
(human Ki ) 2.4 nM (IMR32 neuroblastoma cells))
blocks the behavioral effects of CRF in rats; it reverses
the r/hCRF-enhanced acoustic startle responses of rats
(dose of r/hCRF ) 1 mg in 2 mL (icv), complete block at
17.8 mg/kg (ip)). In comparison, the peptide antagonist
D-Phe-CRF(12-41) (3.3 mg (icv)) completely blocked this
effect when co-administered with the same dose of
r/hCRF. Compound 1 also antagonizes the fear-potenti-
ated acoustic startle response in rats (total block at 17.8
mg/kg (ip)). In the rat learned helplessness test, a
putative model for depression, acute administration of
1 (32 mg/kg (ip), 60-min pretest, single dose) reverses
the effects of exposure to inescapable foot shocks for 3
consecutive days. In the same paradigm, treatment with
the antidepressant imipramine (17.8 and 32 mg/kg (sc))
has no significant effect. The effects of 1 on behavior
suggest a potential anxiolytic or antidepressant indica-
tion for CRF1 antagonists.
a
(i) Na(0), EtOAc (65-77%); (ii) NH2NH2, HOAc, toluene (60-
70%); (iii) Me(CdNH)OEt (69-79%); (iv) CO(OEt)2, NaOEt, EtOH
(90-100%); (v) POCl3, Et2NPh, toluene (46-68%); (vi) HNR1R2,
THF (46-68%).
Dichlorophenylacetonitrile was acetylated in the pres-
ence of sodium metal and ethyl acetate to yield ketonitrile
6. Treatment of 6 with hydrazine monohydrate in
refluxing toluene afforded aminopyrazole 7. The ami-
dine acetate salt 8 was formed from condensation of
intermediate 7 with ethyl acetimidate in acetonitrile in
the presence of 1 equiv of glacial acetic acid. The
reaction of 8 with diethyl carbonate in the presence of
sodium ethoxide in refluxing ethanol formed pyrazolo-
triazinone 9. Chlorination with phosphorus oxychloride
in the presence of 2.0 equiv of N,N-diethylaniline at
reflux temperature generated the key intermediate 10.
The coupling of this water-sensitive intermediate with
various amines provided the final products.
Biology. The pharmacological evaluation of com-
pounds started with testing analogues in a cloned
human CRF1 (hCRF1) receptor binding assay to assess
receptor binding affinity, in which the displacements of
[
125I]0-Tyr-ovine CRF (oCRF) by our test compounds
were measured. Promising compounds in terms of
affinity were also evaluated in a rat ex vivo binding
assay to estimate plasma levels as a function of time.
This bioassay cannot distinguish parent compounds
from their active metabolites. Therefore, leading com-
pounds were studied in rat or dog pharmacokinetic
studies. Select analogues with good binding affinity (Ki
e 10 nM) were then evaluated in a CRF-stimulated
adenylate cyclase assay to measure antagonist potency.
Early SAR studies39,40 established that the 2,4-dichlo-
rophenyl group significantly enhanced hCRF1 binding
affinity, and subsequent analogue synthesis rapidly
focused on amino side chain substituents on the six-
membered portion of the bicycle (Table 1). Several high-
affinity CRF1 ligands were discovered. Substituted-
We focused on pyrazolo[1,5-a]-1,3,5-triazines 11 as
potential CRF receptor antagonists based on an analysis
of the structure-activity relationships (SARs) for anili-
nopyrimidines/triazines 2 and 3, triazolopyrimidines 4,
and arylthiazoles 5 (Chart 1).34-37 Overlay of these
structures suggested that alternate bicyclic nuclei, such
as the pyrazolo[1,5-a]-1,3,5-triazine core, might hold the
peripheral functional groups in the correct orientation
to promote binding affinity to the CRF1 receptor.38
Resu lts a n d Discu ssion
Ch em istr y. The chemical syntheses of pyrazolo[1,5-
a]-1,3,5-triazines 11 are illustrated in Scheme 1. 2,4-

Orally Bioavailable CRF1 Receptor Antagonist
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 3 451
Ta ble 1. hCRF1 Receptor Binding and Physical Dataa
mean hCRF1
example
R
Ki (nM)
formula
mp (°C)
11a
11b
11c
11d
11e
11f
11g
11h
11i
NEtBu
NPr2
NPr(CH2-c-Pr)
NH-3-pentyl
NEt2
NH-3-hexyl
NH-4-heptyl
NH-3-heptyl
NHCH(CH2OMe)2
N(CH2CH2OMe)2
NHCH(Et)CH2OMe
NHCH(CH2OEt)2
NMePh
0.4 ( 0.2 (5)
0.6 ( 0.1 (2)
0.7 ( 0.2 (5)
1.1 ( 0.4 (7)
1.2 ( 0.2 (4)
1.2 ( 0.5 (3)
1.6 ( 0.7 (6)
1.6 ( 0.4 (4)
1.7 ( 0.7 (16)
1.8 ( 0.5 (8)
1.9 ( 0.3 (4)
6.2 ( 0.3 (2)
44.3 ( 6.5 (2)
7.5 ( 1.0 (20)
1.6 ( 0.9 (11)
C
19H23Cl2N5
oil
oil
oil
C19H23Cl2N5
C20H23Cl2N5
C
18H21Cl2N5
139-140
133-134
130-132
114-116
90-92
120-121
oil
C17H19Cl2N5
C
C
C
19H23Cl2N5
20H25Cl2N5
20H25Cl2N5
C18H21Cl2N5O2
C19H23Cl2N5O2
C18H21Cl2N5O
C20H25Cl2N5O2
C
-
-
11j
11k
11l
179-181
oil
11m
20H17Cl2N5
60-62
-
-
R-hel-CRF(9-41)
1
-
-
a
Standard deviations are reported. Parenthetical values are the number of determinations.
Ta ble 2. Rat ex Vivo Binding Dataa
CRF-stimulated adenylate cyclase assay using rat corti-
cal membrane preparations against an identical agonist
concentration (IC50 ) 79 ( 27 nM, n ) 2). This
compound has no affinity for hCRF2R receptors ex-
pressed in HEK293 cells and no affinity for the human
CRF-binding protein. Compound 11i was also sub-
mitted to a broad battery of receptor binding assays in
the NovaScreen (Hanover, MD) and CEREP (Celle
L’Evescault, France) programs. No effects at 10 µM are
detected for monoamine (adrenergic (R1, R2, R3, â),
dopaminergic, or serotonergic receptors), histaminergic
H1 or H2, muscarinic, GABAa, GABAb, oxytocin, vaso-
pressin V1, glycine, CGRP, prostaglandin (LTB4, LTD4,
and thromboxane A2), or opioid receptors. Similarly, no
affinity is detected at 10 µM for ion channels (glutamate
(NMDA or Cl sites), calcium (types L or N), or calcium-
activated potassium sites), and no monoamine oxidase
A or B inhibition is detected at the same concentration.
Very weak binding is observed at adenosine A1 and A3
receptors (63% and 47% inhibition of CPA and NECA
binding, respectively, at 10 µM).
Compound 11i evinces anxiolytic activity in the rat
situational anxiety test.41,42 Rats placed in a small
darkened chamber located in an unfamiliar, brightly lit
open arena spend most of the time within the chamber,
an action consistent with a heightened state of anxiety.41
Systemic pretreatment with anxiolytic drugs such as the
benzodiazepines or intracerebroventricular injection of
the peptide CRF antagonist R-helical CRF(9-41) re-
duces the latency time to emerge from the chamber and
explore the open field. This model does not rely on
shocks or other aversive stimuli or conditioning para-
digms. Rats were dosed orally 1 h prior to testing with
chlordiazepoxide (20 mg/kg (po), n ) 16) and 11i (3, 5.6,
10, 18, and 45 mg/kg (po), n ) 8 each) in a methocel
suspension. The animals were placed in the chamber,
and the latency to exit was measured (Table 3). Total
test time was 900 s. The minimum effective dose for 11i
% binding inhibition
mean hCRF1
example
Ki (nM)
N
1 h
3 h
11a
11c
11d
11f
11g
11i
11j
1
0.4
0.7
1.1
1.2
1.6
1.7
1.8
1.6
5
5
8
4
5
7
8
10
73.2 ( 21.3
73.3 ( 17.1
65.9 ( 13.3
46.6 ( 14.8
63.9 ( 10.7
78.2 ( 13.6
85.4 ( 9.2
82.9 ( 14.9
95.4 ( 8.9
71.8 ( 6.7
67.0 ( 6.5
74.8 ( 11.1
81.3 ( 5.7
92.9 ( 6.8
<10
<10
a
Standard deviations are reported. N ) the number of rats
employed in the studies.
alkylamino and di(substituted-alkyl)amino groups greatly
enhanced binding affinity for the hCRF1 receptor, while
one example of arylamino substitution reduced affinity
(cf. 11m ).
Compounds were then evaluated in a rat ex vivo
binding assay to estimate plasma exposure after oral
administration. In this test, compounds were adminis-
tered orally at 10 mg/kg and plasma samples were
drawn at 1 and 3 h postdose. The plasma samples were
diluted 10-fold with assay medium and submitted to the
hCRF1 receptor binding assay. Table 2 delineates the
percent inhibition values as a function of time. All the
analogues had superior exposure in this rat test relative
to that of the reference compound 1 (CP154526-1), which
had plasma exposures below the limit of detection for
this bioassay.
Pyrazolotriazine 11i (DMP696) (hCRF1 Ki ) 1.7 ( 0.7
nM, n ) 16) was chosen for detailed in vitro studies
based on its high affinity for hCRF1 receptors and its
projected duration of exposure in the rat ex vivo binding
assay. This compound is a potent antagonist of hCRF1-
coupled adenylate cyclase activity in HEK293 cell
membranes (IC50 ) 82 ( 14 nM vs 10 nM for r/hCRF,
n ) 2) while R-hCRF(9-41) is less potent (IC50 ) 286
( 63 nM, n ) 3). This analogue is equally potent in a

452 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 3
Ta ble 3. Rat Situational Anxiety Test Dataa
He et al.
isotonic saline-0.25% methocel formulation of the test
compound. Total urine volume and osmolality as well
as glucose, urea, creatinine, sodium, potassium, and
chloride concentrations were within the ranges for
vehicle-treated animals. In contrast, the positive control
furosemide (30 mg/kg (po)) increased renal output
volume and decreased osmolality and glucose, urea,
creatinine, sodium, potassium, and chloride concentra-
tions. The data for furosemide are: renal ouput volume,
6.9 ( 0.4 mL/100 g body weight vs 2.0 ( 0.2 mL/100 g
body weight for vehicle (p < 0.05); osmolality, 211 ( 5
mosm/100 g body weight vs 320 ( 37 mosm/100 g body
weight for vehicle (p < 0.05); urea levels, 78 ( 6 mg/
dL/100 g body weight vs 290 ( 55 mg/dL/100 g body
weight for vehicle (p < 0.05); creatinine levels, 2.3 (
0.3 mg/dL/100 g body weight vs 9.5 ( 1.7 mg/dL/100 g
body weight for vehicle (p < 0.05); potassium levels, 26.0
( 1.2 mmol/dL/100 g body weight vs 53.3 ( 6.7 mmol/
dL/100 g body weight for vehicle (p < 0.05).
No statistically significant cardiovascular effects are
noted in pentobarbital-anesthetized dogs (n ) 4) after
intravenous administration of 11i. Escalating doses of
11i (0.3, 1.0, 3.0, and 5.0 mg/kg) in mixed solvent vehicle
(see Experimental Section) have been administered
intravenously at 30-min intervals. No effects are ob-
served on heart rate, mean arterial blood pressure,
contractility, and electrocardiographic parameters (e.g.
QT interval) relative to vehicle-treated animals.
dose
latency
compd
saline
(mg/kg (po))
to exit (s)
p vs saline
-
20.0
3.0
784 ( 55
216 ( 80
482 ( 100
426 ( 134
286 ( 96
408 ( 112
680 ( 100
-
chlordiazepoxide
<0.001
<0.05
<0.05
<0.001
<0.05
-
11i
11i
11i
11i
11i
5.6
10.0
18.0
45.0
a
Standard errors of the mean are reported. N ) 8. The total
test time was 900 s.
is 3 mg/kg (po), and the maximal efficacy is observed
after 10 mg/kg (po) (63% reduction in latency). The
literature compound 1 (CP154526-1; 30 mg/kg (po)) is
inactive in this test. Chlordiazepoxide causes a 72%
reduction in latency at 20 mg/kg (po). However, 11i does
not inhibit open-field locomotor activity at 10, 30, and
100 mg/kg (po), while chlordiazepoxide does at 30 mg/
kg (po) (50% reduction relative to controls). Compound
11i does not cause any significant neuromuscular effects
(e.g. ataxia, sedation, splayed limbs, catalepsy) up to
100 mg/kg (po) in fasted Sprague-Dawley rats over a
6-h observation period, except for loss of lift at 100 mg/
kg (po). These data suggest that analogue 11i, unlike
chlordiazepoxide, may be a potent anxiolytic drug, for
which there is a large separation between the dose
which causes sedation and that which is efficacious.
Compound 11i has been examined in a primate model
for anxiety, the human intruder test.43 Immature rhesus
monkeys display stereotypical behaviors (vocalizations
(coo or bark), freezing behavior, and exaggerated move-
ments of mouth muscles) when confronted with an
intruder or predator in a confined space. Eye contact
between a human technician and the caged monkey
immediately evokes these behaviors. Compound 11i
reduces stereotypical mouth movements (“lip smacking”)
by 50% at 21 mg/kg ((po) n ) 4), which is statistically
significant (p < 0.01) when compared to control animals.
Qualitative improvements in the other behavioral pa-
rameters are noted at this dose. Alprazolam is reported
to have efficacy at 1 mg/kg (im) in this model.46 Both
compounds were administered orally in 0.5% methocel
at 0.5 h prior to onset of the test.
Some peripheral effects of 11i have been examined
in the rat and dog since CRF receptors populate
peripheral as well as central tissues. CRF1 sites have
been found in rat testes, ovaries, and components of the
immune system, while CRF2 receptors have been re-
ported to be present in the rat GI tract, heart, and
skeletal muscle.3 Acute dosing of 11i (30 mg/kg (po)) has
no statistically significant effect on GI motility as
evaluated by the transit of an orally administered
charcoal suspension (10% w/v charcoal in 0.25% aqueous
methyl cellulose) when compared to vehicle control.
Carbachol (0.3 mg/kg (ip)), the positive control for this
study, significantly increases GI motility (59.5 ( 4.9%
increase in the stomach to cecum distance traveled by
a charcoal meal (p < 0.01 relative to controls treated
with 0.25% methocel)), while atropine (3.0 mg/kg (po)),
the negative control, decreases GI motility (17.6 ( 3.1%
decrease (p < 0.01 relative to controls)) in the same
paradigm. Agent 11i (10 mg/kg (po)) has no statistically
significant effect on acute renal function up to 24 h
postdose in rats, which were volume-loaded with an
Intravenous infusion of 11i (1 and 5 mg/kg) does not
change pulmonary function in pentobarbital-anesthe-
tized dogs (n ) 8). No statistically significant changes
in respiration rate, intrapleural pressure, tidal volume,
minute volume, peak expiratory flow, arterial pO2,
arterial pCO2, or arterial pH are observed.
Compound 11i has a good rat pharmacokinetic profile
(Table 4). Administration of this compound to Sprague-
Dawley rats (5 mg/kg (iv, po), n ) 4) affords long
duration of plasma levels (mean t1/2 ) 12.3 h (iv), 16.5
h (po)). The volume of distribution at steady state (Vd,ss
)
is calculated to be 21.5 ( 3.0 L/kg, indicating that there
is very extensive distribution into tissues. Systemic
clearance (CL) is 2.0 ( 0.33 L/h/kg. The rat oral peak
plasma level is high (mean Cmax ) 317 nM at 0.44 h),
and the oral bioavailability is 37%. After continuous iv
infusion of 11i in solution (N,N-dimethylacetamide:
ethanol:PEG400: 1:1:8) in male CD rats (n ) 4) using
osmotic minipumps (0.47 mg/kg/h) for 69 h, the mean
plasma concentration is equal to 250 ( 53 nM at 69 h,
the total brain concentration is 579 ( 125 nM, and the
brain:plasma ratio is 2.3 ( 0.3. Thus, 11i is a highly
orally bioavailable and brain-penetrant compound in
rats.
In beagle dogs, 11i (1 mg/kg (iv, po), n ) 4) also
affords good plasma level profiles. Mean t1/2, CL, and
Vd,ss values are 34.7 ( 14.2 h, 0.95 ( 0.25 L/kg/h, and
27.1 ( 8.8 L/kg, respectively, after iv administration.
Oral dosing gives mean Cmax, mean t1/2, and bioavail-
ability parameters equal to 240 ( 78 nM, 33 ( 12 h,
and 50 ( 5%, respectively.
High plasma levels are also observed in rhesus
monkeys (n ) 6) when 11i is administered at 5 mg/kg
(po) (0.25% methocel). The maximal concentration is 365
+ 95 nM at 3 ( 0.6 h, the AUC value is 5576 ( 1974
nM‚h, and the half-life is 15 ( 2 h.

Orally Bioavailable CRF1 Receptor Antagonist
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 3 453
Ta ble 4. Single-Dose Pharmacokinetic Data for Compound 11ia
rat (5 mg/kg)
dog (1 mg/kg)
parameter
iv
po
iv
po
N
4
4
4
4
C5min (nM)
1/2 (h)
Cmax (nM)
Tmax (h)
AUCT (nM•h)
AUC (nM•h)
Vss (L/kg)
CL (L/h/kg)
% bioavail
2879 ( 100
12.3 ( 2.5
-
-
1371 ( 335
34.7 ( 14.2
-
-
t
16.5 ( 4.8
317 ( 42
0.44 (0.25-0.5)
1861 ( 354
2263 ( 473
-
33.4 ( 12.0
240 ( 78
0.88 (0.5-1.0)
469 ( 105
555 ( 158
-
-
-
5527 ( 604
6175 ( 939
21.5 ( 3.0
2.0 ( 0.33
-
2275 ( 299
2688 ( 556
27.1 ( 8.8
0.95 ( 0.25
-
-
36.7
-
49.7
a
The mean values are reported. N ) the number of animals used. Standard deviations are reported. The parenthetical values are the
ranges for the values.
formamide), HOAc (acetic acid), THF (tetrahydrofuran), and
TLC (thin-layer chromatography).
Con clu sion
Compound 11i (DMP696) is a high-affinity hCRF1
receptor antagonist, which is potent in rat and primate
models for anxiety. In the rat situational anxiety test,
this compound appears to be more potent after oral
administration than chlordiazepoxide and compound 1
(CP154526-1). In the human intruder test in rhesus
monkeys, analogue 11i inhibits a stress-induced behav-
ior at 21 mg/kg (po). On the basis of the currently
available data, the peripheral effects in rats and dogs
for this compound appear to be minimal after acute
dosing, even though high levels of exposure are achieved
in these two species. The overall profile of 11i supports
the proposal that hCRF1 receptor antagonists may be
effective anxiolytic drugs.
1-Cya n o-1-(2,4-d ich lor op h en yl)p r op a n -2-on e (6). To a
solution of 2,4-dichlorophenylacetonitrile (40.0 g, 0.22 mol) in
ethyl acetate (200 mL) were added sodium pellets (6.43 g, 0.28
mol) portionwise at room temperature. The reaction mixture
was heated to reflux for 1 h. Upon cooling to room temperature,
ethyl acetate (400 mL) was added to the reaction suspension
to facilitate stirring, and resultant mixture was stirred at room
temperature overnight. The collected white solid was washed
with copious amounts of ether and dissolved in water. The
water solution was acidified by adding acetic acid until pH )
5-6. The mixture was extracted three times with ethyl acetate
(200 mL); the combined organic layers were dried over
anhydrous magnesium sulfate, filtered, and concentrated to
dryness to provide a white solid (34.90 g, 70% yield): 1H NMR
(CDCl3) δ 2.38 (s, 3H), 5.15 (s, 1H), 7.36 (dd, 1H, J ) 8, 1 Hz),
7.25 (d, 1H, J ) 8 Hz), 7.50 (d, 1H, J ) 1 Hz); MS (CI) m/z
228 (M + H)+.
5-Am in o-4-(2,4-d ich lor op h en yl)-3-m eth ylp yr a zole (7).
A mixture of 1-cyano-1-(2,4-dichlorophenyl)propan-2-one (18.25
g, 80.3 mmol), hydrazine monohydrate (5.8 mL, 120.45 mmol),
acetic acid (14.9 mL, 200.75 mmol), and toluene (300 mL) was
heated at reflux temperature using a Dean-Stark trap for 5
h. The reaction mixture was cooled to room temperature and
concentrated in vacuo. The residue was dissolved in a 6 N HCl
solution (200 mL) and the resulting solution was extracted
with ether three times. The aqueous layer was basified by
concentrated ammonium hydroxide solution until pH ) 11.
The resulting mixture was extracted with EtOAc (300 mL)
three times. The combined organic layers were dried and
concentrated in vacuo to give a pale brown viscous oil (11.70
g, 61% yield): 1H NMR (CDCl3) δ 2.15 (s, 3H), 3.40-3.90 (brs,
1H), 6.20 (br s, 2H, concentration-dependent), 7.25 (d, 1H, J
) 7 Hz), 7.30 (dd, 1H, J ) 7, 1 Hz), 7.52 (d, 1H, J ) 1.0 Hz):
HRMS(CI) calcd 242.0252, found 242.0250 (M + H)+.
5-Ac e t a m id o -4-(2,4-d ic h lo r o p h e n y l)-3-m e t h y lp y r -
a zole, Acetic Acid Sa lt (8). Glacial acetic acid (4.0 mL, 70.5
mmol) was added into a stirred mixture of 5-amino-4-(2,4-
dichlorophenyl)-3-methylpyrazole (17.0 g, 70.5 mmol), ethyl
acetamidate (9.2 g, 105.8 mmol), and acetonitrile (400 mL).
The resultant mixture was stirred at room temperature for
16 h. Diethyl ether (150 mL) was added into the reaction
mixture. A white solid was collected, washed with diethyl
ether, and dried (16.50 g, 69%): 1H NMR (DMSO-d6) δ 1.82
(s, 3H), 2.05 (s, 3H), 3.20-3.45 (br s, 3H), 7.30 (d, 1H, J ) 7
Hz), 7.40 (dd, 1H, J ) 7, 1 Hz), 7.61 (d, 1H, J ) 1.0 Hz); MS(CI)
m/z 283 (M + H).
Exp er im en ta l Section
Ch em istr y. Analytical data were recorded for the com-
pounds described below using the following general procedures.
Proton NMR spectra were recorded on Varian VXR or Unity
300 FT-NMR instruments (300 MHz); chemical shifts were
recorded in ppm (δ) from an internal tetramethysilane stand-
ard in deuteriochloroform or deuteriodimethyl sulfoxide as
specified below. Coupling constants (J ) were recorded in hertz
(Hz). Mass spectra (MS) were recorded on a Finnegan MAT
8230 spectrometer or a Hewlett-Packard 5988A model spec-
trometer (both using chemical ionization (CI) with NH3 as the
carrier gas). Gas chromatography-mass spectrosopy (GC-MS)
was occasionally obtained using the former instrument. Chemi-
cal ionization high-resolution mass spectra (CI-HRMS) were
obtained on a VG 7-VSE instrument with NH3 as the carrier
gas. Combustion analyses were performed by Quantitative
Technologies Inc., Whitehouse, NJ . Melting points were meas-
ured on a Buchi model 510 melting point apparatus or a
Thomas-Hoover capillary apparatus and are uncorrected.
Boiling points are uncorrected.
Reagents were purchased from commercial sources and,
when necessary, purified prior to use according to the general
procedures outlined by Perrin and Armarego.44 Chromatog-
raphy 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.
All reactions were performed under a nitrogen atmosphere
using magnetic stirring. Reactions requiring anhydrous condi-
tions were performed in glassware, which had been flame-dried
or oven-dried with purging under a nitrogen atmosphere.
Reactions using aqueous media were run under the ambient
atmosphere. Anhydrous magnesium sulfate (MgSO4) was used
routinely to dry the combined organic layers from extractions.
Solvent was routinely removed in vacuo, using a rotary
evaporator, followed by evacuation with vacuum pump.
2,7-Dim et h yl-8-(2,4-d ich lor op h en yl)[1,5-a ]p yr a zolo-
1,3,5-tr ia zin -4(3H)-on e (9). Sodium pellets (11.0 g 0.048 mol)
were added in portions to stirred ethanol (400 mL) so that a
gentle reflux occurred. After all sodium reacted and the
reaction temperature subsided to the ambient value, 5-acet-
amido-4-(2,4-dichlorophenyl)-3-methylpyrazole acetic acid salt
(16.44 g, 0.048 mol) and diethyl carbonate (46.6 mL, 0.38 mol)
were added. The resultant reaction mixture was heated at
reflux temperature for 6 h. After cooling, the solvent was
Commonly used abbreviations are: EtOAc (ethyl acetate),
MeOH (methanol), EtOH (ethanol), DMF (N,N-dimethyl-

454 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 3
He et al.
removed in vacuo. The residue was dissolved in water and
acidified by slowly adding glacial acetic acid until pH ) 5-6.
The resulting mixture was extracted with EtOAc (300 mL)
three times, and the combined organic layers were dried over
magnesium sulfate, filtered, and concentrated to give a tan
solid (14.0 g, 95% yield): 1H NMR (CDCl3) δ 2.37 (s, 3H), 2.50
(s, 3H), 7.25 (d, 1H, J ) 7 Hz), 7.35 (dd, 1H, J ) 7, 1 Hz), 7.55
(d, 1H, J ) 1 Hz), 10.58 (s, 1H); HRMS(CI) calcd 308.0232,
found 309.0297 (M + H)+.
4-Ch lor o-2,7-d im et h yl-8-(2,4-d ich lor op h en yl)[1,5-a ]-
p yr a zolo-1,3,5-t r ia zin e (10). 2,7-Dimethyl-8-(2,4-dichloro-
phenyl)[1,5-a]pyrazolo-1,3,5-triazin-4(3H)-one (6.7 g, 21.7 mmol),
N,N-diethylaniline (7.5 mL, 47.2 mmol), and phosphorus
oxychloride (100 mL) was stirred at reflux temperature for 23
h. The excess phosphorus oxychloride was removed in vacuo.
The residue was poured onto ice and extracted quickly with
ethyl acetate (250 mL) three times. The combined organic
layers were washed with brine (100 mL) once and dried over
magnesium sulfate, filtered, and concentrated. The red brown
oil residue was quickly purified by flash chromatography using
EtOAc:hexanes (9:1) as eluent to afford a yellow oil (6.09 g,
86%): 1H NMR (CDCl3) δ 2.45 (s, 3H), 2.68 (s, 3H), 7.30 (d,
1H, J ) 7 Hz), 7.38 (dd, 1H, J ) 7, 1 Hz), 7.55 (d, 1H, J ) 1
Hz); MS(CI) m/z 327 (M + H)+.
4-(1,3-Dim eth oxy-2-p r op yla m in o)-2,7-d im eth yl-8-(2,4-
dich lor oph en yl)[1,5-a ]pyr azolo-1,3,5-tr iazin e (11i). A mix-
ture of 4-chloro-2,7-dimethyl-8-(2,4-dichlorophenyl)[1,5-a]-
pyrazolo-1,3,5-triazine (8.69 g, 26.5 mmol), 1,3-dimethoxypropyl-
2-aminopropane (5.4 g, 34.5 mmol), and THF (150 mL) was
stirred at room temperature for 45 min. The reaction mixture
was poured onto water and extracted with ethyl acetate (250
mL) for three times. The combined organic layer was dried
over magnesium sulfate, filtered, and concentrated. The
resulting residue was purified by column chromatography
using 20% hexane in ethyl acetate as eluent to give a pale
yellow solid. The solid was recrystallized from hexane to afford
white crystals (5.50 g, 51%): mp 120-121 °C; 1H NMR (CDCl3)
δ 2.35 (s, 3H), 2.50 (s, 3H), 3.43 (s, 6H), 3.55-3.70 (m, 4H),
4.58-4.70 (m, 1H), 6.75 (d, 1H, J ) 8 Hz), 7.28 (dd, 2H, J )
8, 1 Hz), 7.50 (s, 1H); HRMS(CI) calcd 410.1151, found
410.1147 (M + H)+. Anal. Calcd for C18H21Cl2N5O: C, 52.69;
H, 5.17; N, 16.62; Cl, 17.28. Found: C, 52.82; H, 5.06; N, 16.77;
Cl, 17.50.
Biology. The cloned human receptor binding assay was
performed as described previously.34 The rat and human CRF-
coupled adenylate cyclase assays have been reported else-
where.45
The rat ex vivo assay is a variation of the cloned human
receptor binding assay. Test compounds (10 mg/kg) were orally
administered in a 0.5% methocel suspension. Plasma samples
were drawn from Sprague-Dawley rats at 1 and 3 h postdose.
Plasma samples were then diluted 10-fold with assay medium
and aliquots of these diluted samples were analyzed in the
standard binding assay.
Pharmacokinetic parameters were determined in rats after
intravenous or oral doses of 5 mg/kg. The iv vehicle (2.5 mg/
mL) was a mixture of N,N-dimethylacetamide, ethanol, pro-
pylene glycol 400, and water (1:1:6:2, v/v/v/v). The oral vehicle
was a 0.25% methocel suspension. At 15 min, 30 min, 1, 2, 4,
6, 8, 10, 12, 16, 24, and 32 h after dosing, blood samples were
collected from the tail vein into heparinized tubes containing
EDTA. Steady-state brain-to-plasma ratios in rats were de-
termined at the end of a 69-h iv infusion. Blood and brain
tissue were collected at the end of the infusion. Plasma was
separated by centrifugation and stored frozen until analysis.
Male beagle dogs (n ) 4) were given 1 mg/kg of compound
iv in a cosolvent vehicle (N,N-dimethylacetamide:ethanol:
polyethylene glycol 400:water: 1:1:6:2, 2.5 mg/mL) or 1 mg/
kg po in 0.25% methylcellulose suspension (2.5 mg/mL). Blood
samples were collected from jugular veins at predose, 5, 15,
and 30 min, and 1, 2, 4, 8, 10, 12, 16, 24, 32, 48, 56, and 72 h
after dosing.
samples were drawn via an in-dwelling cannula in an arm at
0, 0.25, 0.5, 1, 2, 4, 8, 24, 36, and 48 h.
Plasma samples and brain tissues were analyzed after
extraction of test compounds by simple liquid-liquid extrac-
tion. LC/MS/MS analysis was performed on a Sciex (Thornhill,
Ontario) model APIIII triple quadrapole mass spectrometer
interfaced with a turbo ion spray ionization source. The liquid
chromatography consisted of a Perkin-Elmer series 200 solvent
delivery system (Norwalk, CT), a Perkin-Elmer ISS 200
autoinjector, and a Waters Symmetry octyl minibore column
(2.1 × 50 mm).
Cardiovascular pharmacodynamic testing was performed in
anesthetized dogs. Mongrel dogs (n ) 8) of both sexes (weights
) 7.0-12.3 kg; HRP Inc., Cumberland VA) were anesthetized
with sodium pentobarbital (30 mg/kg (iv)) and mechanically
ventilated (15 breaths/min) using a Harvard respirator (Har-
vard Apparatus, South Natick, MA). The cephalic veins were
cannulated for compound administration and infusion of
sodium pentobarbital at 6 mg/kg/h. A micromanometer tip
catheter (Millar Instruments, Houston, TX) was placed in the
left ventricle to measure left ventricular pressure. The first
derivative of the left ventriuclar pressure signal, LV + dP/dt,
was obtained by the Modular Instruments software. Arterial
blood pressure was monitored from a cannulated brachial
artery with a Statham pressure transducer (Spectamed-Viggio,
Oxnard, CA). Blood pressure, left ventricular pressure, left
ventricular dP/dt, and a lead II ECG were recorded on a
Modular Instruments polygraph. Compound 11i was admin-
istered iv at 0.3, 1.0, 3.0, and 5.0 mg/kg at intervals of 30 min.
The vehicle was composed of N,N-dimethylacetamide, pro-
pylene glycol, ethanol, and water (1:6:1:2) and the infusion rate
was 0.033 mL/kg/min over 6 min for the 0.3 and 1.0 mg/kg
doses, 0.06 mL/kg/min over 10 min for the 3 mg/kg dose, and
0.0625 mL/kg/min over 16 min for the 5.0 mg/kg dose.
Respiratory function was monitored also in anesthetized
dogs. Mongrel dogs were treated with sodium pentobarbital
as described above but were allowed to breathe spontaneously.
Test compound was administered as described above for the
cardiovascular test. The femoral artery and vein were cannu-
lated to collect blood samples for determination of blood gases,
pH, and compound plasma levels. Arterial blood pressure was
measured as described previously. A balloon was positioned
in the lower third of the esophagus to record pleural pressure.
The endotracheal tube was attached to a Hans Rudolph
pneumotachograph (3500 series; Kansas City, MO) connected
to a Validyne differential pressure transducer (DP45-14) for
the measurement of air flow. The esophageal balloon was
similarly attached to one side of a second differential pressure
transducer (Validyne, DP45-24), the other side of which was
connected to a sidearm inserted in the tracheal cannula
adjacent to the pneumotachograph to measure intrapleural
pressure. The vehicle was composed of N,N-dimethylacet-
amide, polyethylene glycol 400 (PEG400), ethanol, and water
(1:6:1:2) and the infusion rate was 0.033 mL/kg/min over 6
min for 0.3 and 1.0 mg/kg doses. Arterial blood samples were
taken at -10, 0, 6, 15, 30, 60, and 90 min to measure arterial
pH, pO2, and pCO2 using a Ciba-Corning 278 pH/blood gas
analyzer (Ciba-Corning Diagnostics Corp., Medfield, MA).
Renal function was assessed in conscious male Sprague-
Dawley rats (caesarian-derived, 199-224 g). The animals were
volume-loaded with isotonic saline containing compound 11i
(10 mg/kg (po)) or furosemide (30 mg/kg (po)) in methylcellu-
lose at a total volume of 25 mL/kg. The initial urine samples
were collected during the first 5-h period postdose, then
between 5 and 24 h. Total volume and osmolality (Fiske 2400
osmometer, Fiske Associates, Norwood, MA) were measured
and glucose, urea, creatinine, sodium, potassium, and chloride
concentrations were determined (dimension clinical analyzer,
DuPont, Wilmington, DE).
Gastrointestinal motility was evaluated by gross observation
and transit of an orally administered charcoal suspension (10%
w/v charcoal in 0.25% methylcellulose) in fasted male
Sprague-Dawley rats (178-222 g, caesarian-derived). Com-
pound 11i (30 mg/kg (po)), atropine (3.0 mg/kg (po)), and
Rhesus monkeys were dosed at 5 mg/kg ((po) n ) 6, 3 males,
3 females, 0.25% methocel) via a naso-gastric tube. Blood

Orally Bioavailable CRF1 Receptor Antagonist
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 3 455
(13) France, R. D.; Urban, B.; Krishnan, R. R.; Bissette, G.; Banki,
C. M.; Nemeroff, C. B.; Spielman, F. J . CSF corticotropin-
releasing factor-like immunoreactivity in chronic pain patients
with and without major depression. Biol. Psychiatry 1988, 23,
86-88.
(14) Arato, M.; Banki, C. M.; Bissette, G.; Nemeroff, C. B. Elevated
CSF CRF in suicide victims. Biol. Psychiatry 1989, 25, 355-
359.
(15) 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. Neuroendo-
crinology 1994, 60, 436-444.
carbachol (0.3 mg/kg (ip)) were administered in the standard
vehicles. The charcoal suspension (5 mL/kg) was administered
orally 60 min after the dose of SK696 and 30 min after the
doses of carbachol or atropine. Five minutes after the charcoal
meal, the rats were euthanized with CO2 and the gastrointes-
tinal tract (stomach to sigmoid colon) was removed. The
distance from the pyloric sphincter to the charcoal at its most
distal location in the small intestine was measured and
compared to the pyloric sphincter to cecum measurement. The
results are expressed as a percentage relative to methocel
controls.
(16) Gold, P. W.; Chrousos, G.; Kellner, C.; Post, R.; Roy, A.;
Augerinos, P.; Schultz, H.; Oldfield, E.; Loriaux, D. L. Psychiatric
implications of basic and clinical studies with corticotropin
releasing factor. Am. J . Psychiatry 1984, 141, 619-627.
(17) Holsboer, F.; Muller, O. A.; Doerr, H. G. ACTH and multisteroid
responses to corticotropin releasing factor in depressive illness:
relationship to multisteroid responses after ACTH stimulation
and dexamethasone suppression. Psychoneuroendocrinology 1984,
9, 147-160.
(18) 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. N.
Engl. J . Med. 1986, 314, 1329-1335.
Ack n ow led gm en t. The authors gratefully acknowl-
edge the expert technical contributions of T. Boop, F.
Brown, C. Chi, Benjamin Chien, Nancy Contel, G.
Demond, T. Donovan, C.-M. Lai, L. Liang, Anne Mar-
shall, Carol Krause, Susan Keim, J ohn Patterson, N.
Raghavan, Cindy Rominger, David Rominger, Wanda
West, and Han G. Zeng.
Su p p or tin g In for m a tion Ava ila ble: Combustion analy-
ses, melting points, and spectral data (NMR or HRMS). This
material is available free of charge via the Internet at
http://pubs.acs.org.
(19) Sapolsky, R. M. Hypercortisolism among socially subordinate
wild baboons originates at the CNS level. Arch. Gen. Psychiatry
1989, 46, 1047-1051.
(20) Britton, D. R.; Koob, G. F.; Rivier, J .; Vale, W. Intracerebroven-
tricular corticotropin releasing factor enhances behavioral effects
of novelty. Life Sci. 1982, 31, 363-376.
(21) Berridge, C. W.; Dunn, A. J . Corticotropin-releasing factor elicits
naloxone-sensitive stress-like alterations in exploratory behavior
in mice. Regul. Pept. 1986, 16, 83-93.
(22) Berridge, C. W.; Dunn, A. J . A corticotropin-releasing factor
antagonist reverses the stress-induced changes in exploratory
behavior in mice. Horm. Behav. 1987, 21, 393-401.
(23) Berridge, C. W.; Dunn, A. J . Physiological and behavioral
responses to corticotropin-releasing factor administration: is
CRF a mediator of anxiety or distress responses. Brain Res. Rev.
1990, 15, 71-100.
(24) Britton, K. T.; Morgan, J .; Rivier, J .; Vale, W.; Koob, G. F.
Chlordiazepoxide attenuates response suppression induced by
corticotropin-releasing factor in the conflict test. Psychophar-
macology 1985, 86, 170-174.
(25) Britton, K. T.; Lee, G.; Koob, G. F. Corticotropin-releasing factor
and amphetamine exaggerate partial agonist properties of the
benzodiazepine antagonist Ro 15-1788 in the conflict test.
Psychopharmacology 1988, 94, 306-311.
(26) Swerdlow, N. R.; Geyer, M. A.; Vale, W. W.; Koob, G. F.
Corticotropin releasing factor potentiates acoustic startle in
rats: blockade by chlordiazepoxide. Psychopharmacology 1986,
88, 147-152.
Refer en ces
(1) Rivier, J .; Spiess, J .; Vale, W. Characterization of rat hypothala-
mus corticotropin releasing factor. Proc. Natl. Acad. Sci. U.S.A.
1983, 80, 4851-4855.
(2) Vale, W.; Spiess, J .; Rivier, C.; Rivier, J . Characterization of a
41-residue ovine hypothalamic peptide that stimulates secretion
of corticotropin and beta-endorphin. Science 1981, 213, 1394-
1397.
(3) Gilligan, P. J .; Hartig, P. R.; Robertson, D. W.; Zaczek, R.
Corticotropin Releasing Hormone (CRH) Receptors and the
Discovery of Selective Non-Peptide CRH1 Antagonists. In Annual
Reports in Medicinal Chemistry; Bristol, J . A., Ed.; Academic
Press: San Diego, CA, 1997; Vol. 32, pp 41-50.
(4) Sawchenko, P. E.; Swanson, L. W. Organization of CRF immuno-
reactive cells and fibers in the rat brain: Immunohistochemical
studies. In Corticotropin-releasing factor: basic and clinical
studies of a neuropeptide; Desouza, E. B., Nemeroff, C. B., Eds.;
CRC Press: Boca Raton, FL, 1990; pp 29-51.
(5) Koob, G. F.; Heinrichs, S. C.; Pich, E. M.; Menzaghi, F.; Baldwin,
H. Miczek, K.; Britton, K. T. The role of corticotropin releasing
factor in behavioural responses to stress. In Corticotropin-
releasing factor, Ciba Foundation Symposium 172; Chadwick,
D. J ., Marsh, J ., Ackrill, K., Eds.; J . Wiley and Sons: Chicester,
U.K., 1993; pp 277-291.
(27) Timpl, P.; Spanagel, R.; Sillaber, I.; Kresse, A.; Reul, J . M. H.
M.; Stalla, G. K.; Blanquet, V.; Steckler, T.; Holsboer, F.; Wurst,
W. Impaired stress response and reduced anxiety in mice lacking
a functional corticotropin-releasing hormone receptor 1. Nature
Genetics 1998, 19, 162-166.
(28) 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.
(29) Chen, Y. L.; Mansbach. R. S.; Winter, S. M.; Brooks, E.; Collins,
J .; Corman, M. L.; Dunaiskis, A. R.; Faraci, W. S.; Gallaschun,
R. J .; Schmidt, A.; Schulz, D. W. Synthesis and Oral Efficacy of
a 4-(Butylethylamino)pyrrolo[2,3-d]pyrimidine: A Centrally Ac-
tive Corticotropin Releasing Factor1 Receptor Antagonist. J .
Med. Chem. 1997, 40, 1749-1754.
(6) De Souza, E. B.; Insel, T. R.; Perrin, M. H.; Rivier, J . Vale, W.
W.; Kuhar, M. J . Corticotropin-releasing factor receptors are
widely distributed within the rat central nervous system: an
autoradiographic study. J . Neurosci. 1985, 5, 3189-3203.
(7) Webster, E. L.; Torpy, D. J .; Elenkov, I. J .; Chrousos, G. P.
Corticotropin-releasing hormone and inflammation. Ann. N. Y.
Acad. Sci. 1998, 840, 21-32.
(8) Chrousos, G. P. The hypothalamic-pituitary-adrenal axis and
immune-mediated inflammation. N. Engl. J . Med. 1995, 332,
1351-1362.
(9) DeSouza, E. B.; Grigoriadis, D. E. Corticotropin-Releasing
Factor. Physiology, Pharmacology and Role in Central Nervous
System and Immune Disorders. In Psychopharmacology; The
Fourth Generation of Progress; Bloom, F. E., Kupfer, D. J ., Eds.;
Raven Press: New York, NY, 1995; pp 505-517.
(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) Griebel, G.; Perrault, G.; Sanger, D. J . Characterization of the
behavioral profile of the non-peptide CRF antagonist CP154,526
in anxiety models in rodents: Comparison with diazepam and
buspirone. Psychopharmacology 1998, 138, 55-66.
(33) Guanowsky, V.; Chen, Y. L.; Seymour, P. A. Anxiolytic effect of
the CRF antagonist, CP154526, in a light/dark anxiety test in
mice. Soc. Neurosci. Abstr. 1997, 23, 203.7.
(10) DeSouza, E. B.; Bisette, G.; Whitehouse, P. J .; Powers, R. E.;
Price, D. L.; Vale, W. W.; Nemeroff, C. B. Abnormalities in
corticotropin releasing factor (CRF) in neurodegenerative dis-
eases. In Corticotropin-releasing factor: basic and clinical studies
of a neuropeptide; Desouza, E. B., Nemeroff, C. B., Eds.; CRC
Press: Boca Raton, FL, 1990; pp 335-350.
(11) Owens, M. J .; Nemeroff, C. B. The role of corticotropin-releasing
factor in the pathophysiology of affective and anxiety disorders:
laboratory and clinical studies. In Corticotropin-releasing factor,
Ciba Foundation Symposium 172; Chadwick, D. J ., Marsh, J .,
Ackrill, K., Eds.; J . Wiley and Sons: Chicester, U.K., 1993; pp
296-316.
(12) Banki, C. M.; Bisette, G.; Arato, M.; O’Connor, L.; Nemeroff, C.
B. CSF Corticotropin-releasing Factor-like Immunoreactivity in
Depression and Schizophrenia. Am. J . Psychiatry 1987, 144,
873-877.

456 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 3
He et al.
(34) Arvanitis, A. G.; Gilligan, P. J .; Chorvat, R. J .; Cheeseman, R.
S.; Christos, T. E.; Bakthavatchalam, R.; Beck, J . P.; Cocuzza,
A. J .; Hobbs, F. W.; Wilde, R. G.; Arnold, C.; Chidester, D.; Curry,
M. He, L.; Hollis, A.; Klaczkiewicz, J .; Krenitsky, P. J .; Rescinito,
J . P.; Scholfield, E.; Culp, S.; De Souza, E. B.; Fitzgerald, L.;
Grigoriadis, D.; Tam, S. W.; Wong, Y. N.; Huang, S.-M.; Shen,
H. Non-Peptide Corticotropin-Releasing Hormone (CRH) An-
tagonists: The Syntheses and Structure-Activity Relationships
of 2-AnilinoPyrimidines and -Triazines. J . Med. Chem. 1999, 42,
808-818.
(35) Hodge, C. N.; Aldrich, P. E.; Wasserman, Z. R.; Fernandez, C.
H.; Arvanitis, A.; Chorvat, R. J .; Cheeseman, R. S.; Christos, T.
E.; Scholfield, E.; Krenitsky, P.; Gilligan, P. J .; Ciganek, E.;
Strucely, P. CRH Antagonists. II. New Framework Design and
Synthesis Guided by Anilinopyrimidine Conformational Studies.
J . Med. Chem. 1999, 42, 819-832.
(36) Chorvat, R. J .; Bakthavatchalam, R.; Beck, J . P.; Gilligan, P.
J .; Wilde, R. G.; Cocuzza, A.; Hobbs, F. W.; Cheeseman, R. S.;
Curry, M.; Rescinito, J . P.; Krenitsky, P.; Chidester, D.; Yarem,
J .; Klackewicz, J . D.; Hodge, C. N.; Aldrich, P. E.; Wasserman,
Z. R.; Fernandez, C. H.; Zaczek, R.; Fitzgerald, L.; Huang, S.-
M.; Shen, H. L.; Wong, Y. N.; Chien, B. M.; Quon, C. Y.;
Arvanitis, A. Synthesis, Corticotropin Releasing Factor Receptor
Binding Affinity and Pharmacokinetic Properties of Triazolo-,
Imidazo- and Pyrrolo-Pyrimidines and Pyridines. J . Med. Chem.
1999, 42, 833-848.
(39) He, L.; Gilligan, P. J .; Fitzgerald, L.; Zaczek, R.; Shen, H.;
Trainor, G. L.; Hartig, P. R. Pyrazolo-[1,5-a]-s-triazines as novel
hCRF1 receptor antagonists: Design, synthesis and structure
activity relationships. American Chemical Society National
Meeting, Anaheim, CA, Mar 21-25, 1999; Abstr. MEDI 107.
(40) He, L.; Gilligan, P. J .; Zaczek, R.; Fitzgerald, L.; Kalin, N.;
McElroy, J .; Saye, J .; Shen, H.; Shelton, S.; Smith, M.; Trainor,
G.; Hartig, P. DMP696: A potent orally bioavailable pyrazolo-
[1,5-a]-s-triazine corticotropin releasing factor (CRF) antagonist.
American Chemical Society National Meeting, Anaheim, CA,
Mar 21-25, 1999; Abstr. MEDI 4.
(41) Takahashi, L. K.; Kalin, N. H.; VandenBurgt, J . A.; Sherman,
J . E. Corticotropin-releasing factor modulates defensive-with-
drawal and exploratory behavior in rats. Behav. Neurosci. 1989,
103, 648-654.
(42) Zaczek, R.; Fitzgerald, L. W.; McElroy, J . F.; Smith, M. A.;
Gilligan, P. J .; He, L.; Olson, R. E.; Robertson, D. W.; Kalin, N.
H.; Shelton, S. E.; Hartig, P. R. Novel Small molecule CRF1
antagonists: Identification and pharmacological characteriza-
tion. American Neuroendocrine Society, Neuroendocrine Work-
shop on Stress, New Orleans, LA, J une 21-23, 1998; Abstr. 6.
(43) Kalin, N. H.; Shelton, S. E.; Turner, J . G. Effects of alprazolam
on fear-related behavioral, hormonal, and catecholamine re-
sponses in infant rhesus monkeys. Life Sci. 1991, 49, 2031-
2044.
(44) Perrin, D.; Armarego, W. L. F. Purification of Laboratory
Chemicals, 3rd ed.; Pergamon Press: New York, NY, 1988.
(45) Battaglia, G.; Webster, E. L.; De Souza, E. B. Characterization
of Corticotropin-Releasing Factor Receptor-mediated Adenylate
Cyclase Activity in the Rat Central Nervous System. Synapse
1987, 1, 572-581.
(37) Courtmanche, G.; Gautier, C.; Gully, D.; Roger, P.; Valette, G.;
Wermuth, C.-G. Branched alkylamino derivatives of thiazole,
processes for preparing them and pharmaceutical compositions
containing them. U.S. Patent 5464847, 1995.
(38) Structures were minimized using the PCSpartan Pro software
package (v. 1.0.1; Wavefunction Inc., Irvine, CA) and super-
imposed such that the pendant phenyl rings were coincident.
J M9904351