The discovery of 4-(3-pentylamino)-2,7-dimethyl-8-(2-methyl-4- methoxyphenyl)-pyrazolo-[1,5-a]-pyrimidine: A corticotropin-releasing factor (hCRF1) antagonist

Article abstract of DOI:10.1016/S0968-0896(99)00271-0

Structure-activity relationship studies led to the discovery of 4-(3- pentylamino)-2,7-dimethyl-8-(2-methyl-4-methoxyphenyl)-pyrazolo-[1,5-a]- pyrimidine 11-31 (DMP904), whose pharmacological profile strongly supports the hypothesis that hCRF₁ antagonists may be potent anxiolytic drugs. Compound 11-31 (hCRF₁ K(i) = 1.0 ± 0.2 nM (n = 8)) was a potent antagonist of hCRF₁-coupled adenylate cyclase activity in HEK293 cells (IC₅₀ = 10.0 ± 0.01 nM versus 10 nM r/hCRF, n = 8); α-helical CRF(9-41) had weaker potency (IC₅₀ = 286 ± 63 nM, n = 3). Analogue 11-31 had good oral activity in the rat situational anxiety test; the minimum effective dose for 11-31 was 0.3 mg/kg (po). Maximal efficacy (approximately 57% reduction in latency time in the dark compartment) was observed at this dose. Chlordiazepoxide caused a 72% reduction in latency at 20 mg/kg (po). The literature compound 1 (CP154526-1, 30 mg/kg (po)) was inactive in this test. Compound 11-31 did not inhibit open-field locomotor activity at 10, 30, and 100 mg/kg (po) in rats. In beagle dogs, this compound (5 mg/kg, iv, po) afforded good plasma levels. The key iv pharmacokinetic parameters were t(1/2), CL and V(d,ss) values equal to 46.4 ± 7.6 h, 0.49 ± 0.08 L/kg/h and 23.0 ± 4.2 L/kg, respectively. After oral dosing, the mean C(max), T(max), t(1/2) and bioavailability values were equal to 1260 ± 290 nM, 0.75 ± 0.25 h, 45.1 ± 10.2 h and 33.1%, respectively. The overall rat behavioral profile of this compound suggests that it may be an anxiolytic drug with a low motor side effect liability. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0968-0896(99)00271-0

Bioorganic & Medicinal Chemistry 8 (2000) 181±189
The Discovery of 4-(3-Pentylamino)-2,7-dimethyl-8-(2-methyl-4-
methoxyphenyl)-pyrazolo-[1,5-a]-pyrimidine:
A Corticotropin-Releasing Factor (hCRF1) Antagonist
Paul J. Gilligan, a,* Caryn Baldauf, a,y Anthony Cocuzza, a Dennis Chidester, a
Robert Zaczek, b Lawrence W. Fitzgerald, b John McElroy, b Mark A. Smith, b
H.-S. L. Shen, c Jo Anne Saye, d David Christ, c George Trainor, a David W. Robertson a
and Paul Hartig b
aDuPont Pharmaceuticals Co., Chemical and Physical Sciences Department, Experimental Station, PO Box 80500, Wilmington,
DE 10880-0500, USA
bDuPont Pharmaceuticals Co., Department of CNS Diseases Research, Experimental Station, PO Box 80500, Wilmington,
DE 10880-0500, USA
cDuPont Pharmaceuticals Co., Drug Metabolism and Pharmacokinetic Department, Stine-Haskell Research Center, Newark,
DE 19714 USA
dDuPont Pharmaceuticals Co., Department of Preclinical Pharmacology, Experimental Station, PO Box 80500, Wilmington,
DE 10880-0500, USA
Received 3 August 1999; accepted 10 September 1999
AbstractÐStructure±activity relationship studies led to the discovery of 4-(3-pentylamino)-2,7-dimethyl-8-(2-methyl-4-methoxy-
phenyl)-pyrazolo-[1,5-a]-pyrimidine 11-31 (DMP904), whose pharmacological pro®le strongly supports the hypothesis that
hCRF1 antagonists may be potent anxiolytic drugs. Compound 11-31 (hCRF1 Ki=1.0‹0.2 nM (n=8)) was a potent antagonist of
hCRF1-coupled adenylate cyclase activity in HEK293 cells (IC50=10.0‹0.01 nM versus 10 nM r/hCRF, n=8); a-helical CRF(9-
41) had weaker potency (IC50=286‹63 nM, n=3). Analogue 11-31 had good oral activity in the rat situational anxiety test; the
minimum e€ective dose for 11-31 was 0.3 mg/kg (po). Maximal ecacy (approximately 57% reduction in latency time in the dark
compartment) was observed at this dose. Chlordiazepoxide caused a 72% reduction in latency at 20 mg/kg (po). The literature
compound 1 (CP154526-1, 30 mg/kg (po)) was inactive in this test. Compound 11-31 did not inhibit open-®eld locomotor activity at
10, 30, and 100 mg/kg (po) in rats. In beagle dogs, this compound (5 mg/kg, iv, po) a€orded good plasma levels. The key iv phar-
macokinetic parameters were t1/2, CL and Vd,ss values equal to 46.4‹7.6 h, 0.49‹0.08 L/kg/h and 23.0‹4.2 L/kg, respectively.
After oral dosing, the mean Cmax, Tmax, t1/2 and bioavailability values were equal to 1260‹290 nM, 0.75‹0.25 h, 45.1‹10.2 h and
33.1%, respectively. The overall rat behavioral pro®le of this compound suggests that it may be an anxiolytic drug with a low motor
side e€ect liability. # 2000 DuPont Pharmaceuticals Company. Published by Elsevier Science Ltd. All rights reserved.
Introduction
responses to stress. Rapid release of CRF mediates
many of the body's responses to stressors and intra-
cerebroventricular (icv) administration of CRF replicates
many of the behavioral and physiological e€ects of
stress.5±8 Intracerebroventricular administration of pep-
tidic CRF antagonists, such as a-helical CRF(9-41) and
astressin, block not only the e€ects of exogenously
administered CRF but also the e€ects of natural
stressors.3 Transgenic mice, which overexpress CRF,
demonstrate heightened stress-related behaviors and
have elevated function of the hypothalamus±adrenal±
pituitary (HPA) axis. Mice, which have null mutations
for CRF, exhibit a blunted endocrine response to stress.3
The above data, taken together with neuroanatomical
Corticotropin releasing factor (CRF) is a 41 amino acid
peptide, a pituitary secretagogue of adrenocorticotropin
hormone (ACTH) and a neurotransmitter.1±4 Since its
isolation and characterization, clinical and preclinical
studies have documented the pivotal role of CRF in the
regulation of endocrine, autonomic and behavioral
Keywords: corticotropin releasing factor; antagonists; pyrazolo[1,5-
a]pyrimidine.
*Corresponding author.
yCurrent address: Duke University School of Medicine, Durham, NC,
USA.
0968-0896/00/$ - see front matter # 2000 DuPont Pharmaceuticals Company.
Published by Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(99)00271-0

182
P. J. Gilligan et al. / Bioorg. Med. Chem. 8 (2000) 181±189
studies on the localization of CRF receptors and the
innervation of key brain centers by CRF-containing
®bers,9 support the proposal that CRF is the primary
component of the central response to stress.
Clinical studies have implicated CRF in the etiology of
depression and a€ective disorders.10±17 Maladaptive
response to stress and the cognate chronic elevation of
corticosteroid levels may be a common basis for the
production of major depression. This hypercortisolemia
is most likely the result of chronic hypersecretion of
hypothalamic CRF.18 CRF levels in the cerebrospinal
¯uid (CSF) are elevated in depressed patients as is the
number of CRF neurons in the hypothalami of such
individuals.14 Successful treatment of depressive symp-
toms is accompanied by a lowering of CSF CRF levels
and plasma cortisol levels to normal ranges. CSF CRF
levels are also elevated relative to controls during alco-
hol withdrawal, anorexia nervosa, obsessive±compulsive
disorder and post-traumatic stress syndrome.3
Our interest in pyrazolo-[1,5-a]-pyrimidines 11 as
potential CRF antagonists arose from an analysis of the
structure±activity relationships published for other
CRF antagonist series (Scheme 1).24±27 We have pre-
viously reported that anilino-pyrimidines/-triazines 2
and 3 as well as triazolo-pyrimidines 5 are potent
hCRF1 antagonists, some of which have excellent phar-
macokinetic pro®les in multiple species (Scheme 1).
Researchers at Sano® have identi®ed aryl thiazoles 4 to
be high anity CRF antagonists. Overlay of these
structures via molecular modeling techniques suggested
to us and others that alternate sca€olds, such as the
pyrazolo-[1,5-a]-pyrimidine core, are possible, providing
that the peripheral functional groups are held in
approximately the same spatial orientation to maximize
binding anity to the CRF1 receptor.28±30 We report
herein the structure±activity relationships leading to the
discovery of 4-(3-pentylamino)-2,7-dimethyl-8-(2-meth-
yl-4-methoxyphenyl)pyrazolo-[1,5-a]-pyrimidine 11-31
(DMP904), whose pharmacological pro®le strongly
supports the hypothesis that hCRF1 antagonists may be
potent anxiolytic drugs.31
The functions of CRF are mediated through two CRF
receptor subtypes and one binding protein.3,8,19 The
heptahelical G-protein-coupled receptor subtypes, CRF1
and CRF2, are distinguished by the relative anities
and ecacies of peptide agonists as well as anatomical
distribution. The latter type has three splice variants: a,
b, and g3,19 Signal transduction through each receptor
subtype involves stimulation of cyclic adenosine mono-
phosphate (c-AMP) production; agonists stimulate
c-AMP production for both subtypes. CRF1 sites are found
primarily in the rat pituitary, cerebral cortex, hippo-
campus and amygdala. In rats, the CRF2 receptor is
primarily a rat peripheral receptor with lower distribution
in discrete CNS regions: lateral septum, choroid plexus,
hypothalamus and sympathetic nuclei. The CRF binding
protein is widely distributed in the CNS, including hippo-
campus and amygdala and in human, but not rodent,
plasma. The central functions of the CRF receptor sub-
types and the central binding protein have been the
subject of intense investigation with peptidic antagonists
and antisense reagents.8 Modulation of CRF function
therefore o€ers the tantalizing prospect of developing new
therapeutic agents for anxiety, depression, Alzheimer's
disease and obesity but clinical studies with non-peptidic
selective agents are needed to truly test these hypotheses.3,8
Results and Discussion
Chemistry
Pyrazolo-[1,5-a]-pyrimidines 11 were synthesized
according to the procedures outlined in Scheme 2. Sub-
stituted phenylacetonitriles 6 were condensed with ethyl
acetate to generate keto-nitriles 7 in 45±80% yields.
Reaction of intermediates 7 with hydrazine-hydrate in
Pyrrolopyrimidine 1 (CP154526-1)20±23 is a non-peptidic
CRF1-selective antagonist, which has been studied at
length. This compound has high anity for hCRF1
receptors (human Ki=2.4 nM (IMR32 neuroblastoma
cells)) and blocks the behavioral e€ects of CRF in rats.
Compound 1 reduced the r/hCRF-enhanced acoustic
startle responses of rats. Compound 1 is also e€ective in
the fear-potentiated acoustic startle test in rats (max-
imal ecacy at 17.8 mg/kg (ip)). In the rat learned
helplessness model for depression, administration of 1
(32 mg/kg, ip, 60 min pretest, single dose) completely
counters the e€ects of inescapable foot shocks for three
consecutive days. In the same test, there was no sig-
ni®cant e€ect with the imipramine (17.8 and 32 mg/kg,
sc). These data suggest potential anxiolytic or anti-
depressant utilities for CRF1 antagonists.
Scheme 1.

P. J. Gilligan et al. / Bioorg. Med. Chem. 8 (2000) 181±189
183
analogues 11-2 and 11-1 had the best receptor binding
anity, but acylamino analogue 11-10 was a poorer
ligand.
The focus of our studies then shifted to the identi®ca-
tion of the optimal phenyl substitution pattern (Table
1). Replacement of a methyl group with a chlorine at
either the 2- or 4-positions had minimal e€ect on recep-
tor binding anity. Comparable anities were found
for the corresponding analogues in the 2,4-dimethyl-
phenyl, 4-chloro-2-methylphenyl, 2-chloro-4-methyl-
phenyl and 2,4-dichlorophenyl series (e.g. compare 11-2
(mean hCRF1 Ki=1.1 nM) with 11-11 (mean hCRF1
Ki=1.5 nM) or 11-17 (mean hCRF1 Ki=1.3 nM)). 2,4-
Disubstituted analogues generally had high receptor
binding anity than those compounds with 2,4,6-tri-
substitution.31 In the series where the amino side chain
is ®xed either as a diethylamino or a 3-pentylamino
group, various 2,4-disubstituted phenyl patterns
enhanced receptor binding anity in similar fashion
(e.g. analogues 11-1, 11-12, 11-18, 11-22, 11-29 and
11-31 had mean hCRF1 Ki values equal to 0.8, 1.5, 1.7,
1.3, 1.5, and 1.0 nM, respectively). Introduction of
alkoxy substituents at the 5-position reduced receptor
binding anity when the 2-substituent was bromine
(e.g. compare 11-36 (mean hCRF1 Ki=4.1 nM) with
11-39 (mean hCRF1 Ki=34.6 nM)). The e€ect was less
dramatic when chlorine was the 2-substituent (e.g.
compare 11-28 (mean hCRF1 Ki=1.2 nM) with 11-41
(mean hCRF1 Ki=2.6 nM)).
Scheme 2.
the presence of acetic acid and toluene produced ami-
nopyrazoles 8 in 40±85% yields. Subsequent condensa-
tion of pyrazoles 8 with ethyl acetoacetate in re¯uxing
acetic acid provided pyrazolopyrimidinones 9 in 80±
95% yields. Reaction of 9 with phosphorus oxychloride
in the presence of N,N0-diethylaniline a€orded chloro
intermediates 10, which were puri®ed by ¯ash chroma-
tography and stored at reduced temperature to mini-
mize hydrolysis back to the precursors 9. Reaction with
neat alkyl or dialkyl amines gave the desired target
compounds 11 in 30 to 80% overall yields.
Compounds were then evaluated in a rat ex vivo bind-
ing 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 post-dose. 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, which were tested, had superior exposure in
the rat relative to that of the reference compound 1
(CP154526-1), which had plasma exposures below the
limit of detection for this bioassay. While the exposures
of these compounds appeared to be high, the bioassay
can not discriminate parent compound from active
metabolite(s).
Pharmacology. A multivariate approach to screening
was utilized to eciently identify the optimal analogues.
A cloned human hCRF1 receptor binding assay, in
which inhibition of speci®c binding of 125I-tyr-o-CRF
by our test compounds was measured to determine their
receptor binding anity, began our testing sequence for
the pyrazolopyrimidines. High anity analogues were
next evaluated in a rat ex vivo binding assay to estimate
plasma levels as a function of time. However, this
bioassay was not able to discriminate parent com-
pounds from active metabolites. Therefore, rat or dog
pharmacokinetic studies were conducted on the leading
compounds. High anity ligands (Ki ꢀ10 nM) were also
evaluated in a hCRF1-mediated c-AMP production
assay to measure antagonist potency. Behavioral e€ects
of the test compounds were studied in rat models for
anxiety: the situational anxiety test and the elevated plus
maze model, while motor de®cits were monitored in the
rat locomotor activity or rotarod tests.
Pyrazolopyrimidine 11-31 (DMP904) (hCRF1 Ki=
1.0‹0.2 nM (n=8)) was advanced to secondary in vitro
studies based on its high anity for hCRF1 receptors
and its projected duration of exposure in the rat ex vivo
binding assay.31 This compound was a potent antago-
nist of hCRF1-coupled adenylate cyclase activity in
HEK293 cells (IC50=10.0‹0.01 nM versus 10 nM
r/hCRF, n=8); a-hCRF(9-41) had weaker potency
(IC50=286‹63 nM, n=3). The antagonist activity pro-
®le of 11-31 with hCRF1 receptors has been processed
through a Schild analysis; it has been shown to be a
non-competitive antagonist.31±33 This compound had
no anity for hCRF2 receptors expressed in HEK293
cells and no anity for the CRF-binding protein.31,33
Analogue 11-31 was also submitted to several receptor
binding assays in the NovaScreen program (Hanover,
Structure±activity studies commenced in the 2,4-di-
methylphenyl series (Table 1). A variety of substituted
alkylamino or dialkylamino analogues were prepared
and evaluated in the hCRF1 receptor binding assay,
based on the published precedent in other bicyclic CRF
antagonist series. The diethylamino and 3-pentylamino

184
P. J. Gilligan et al. / Bioorg. Med. Chem. 8 (2000) 181±189
Table 1. hCRF1 Receptor binding and physical dataa±c
Example
R
Ar
Mean hCRF1 Ki (nM)
Formula
mp (ꢁC)
11-1
11-2
11-3
11-4
11-5
11-6
11-7
11-8
NH-3-pentyl
NEt2
2,4-Me2Ph
2,4-Me2Ph
2,4-Me2Ph
2,4-Me2Ph
2,4-Me2Ph
2,4Me2Ph
2,4-Me2Ph
2,4-Me2Ph
2,4-Me2Ph
2,4-Me2Ph
2-Cl-4-MePh
2-Cl-4-MePh
2-Cl-4-MePh
2-Cl-4-MePh
2-Cl-4-MePh
2-Cl-4-MePh
2-Me-4-ClPh
2-Me-4-ClPh
2-Me-4-ClPh
2-Me-4-ClPh
2,4-Cl2Ph
0.8‹0.2 (5)
1.1‹0.3 (4)
1.7‹0.5 (4)
1.8‹0.5 (3)
2.0‹0.7 (3)
3.4‹0.8 (3)
5.7‹2.9 (4)
6.4‹2.1 (4)
10.6‹4.2 (10)
28.9‹0.1 (3)
1.5‹1.1 (4)
1.5‹1.0 (4)
3.0‹1.1 (3)
3.2‹1.2 (3)
4.5‹2.2 (3)
4.6‹1.0 (4)
1.3‹0.4 (3)
1.7‹0.9 (5)
2.2‹0.9 (4)
2.3‹0.7 (3)
0.9‹0.1 (3)
1.3‹0.4 (3)
2.1‹0.5 (3)
2.3‹1.3 (4)
3.0‹1.3 (4)
3.1‹1.4 (3)
6.8‹0.4 (3)
1.2‹0.7 (4)
1.5‹0.6 (3)
0.8‹0.5 (4)
1.0‹0.2 (8)
1.8‹0.5 (3)
3.0‹0.9 (3)
3.5‹0.3 (4)
9.9‹0.9 (3)
4.1‹0.8 (3)
5.7‹2.0 (3)
4.9‹0.9 (3)
34.6‹5.4 (3)
41.0‹12.9 (3)
2.6‹0.4 (4)
4.5‹1.3 (8)
19.7‹1.0 (3)
7.5‹1.0 (20)
1.6‹0.9 (11)
C21H28N4
C20H26N4
C21H28N4O
C22H27N5
C23H29N5
Oil
92±94
Oil
NHCH(Et)CH2OMe
NPrCH2CH2CN
NBuCH2CH2CN
N(c-Pr)CH2CH2CN
N(CH2CH2OMe)2
NHCH(CH2OMe)2
NHEt
143
115±117
133 (dec)
87±89
103±105
Oil
C22H25N5
C22H30N4O2
C21H28N4O2
C18H22N4
C20H24N4O
C19H23ClN4
C20H25ClN4
11-9
11-10
11-11
11-12
11-13
11-14
11-15
11-16
11-17
11-18
11-19
11-20
11-21
11-22
11-23
11-24
11-25
11-26
11-27
11-28
11-29
11-30
11-31
11-32
11-33
11-34
11-35
11-36
11-37
11-38
11-39
11-40
11-41
11-42
11-43
a-hel-CRF(9-41)
1
NAcEt
NEt2
NH-3-pentyl
Oil
129±130
139±141
162
112±113
77±78
131±133
113±114
94±96
Oil
113±114
86±87
175±176
107
110±111
72±74
83±85
69
N(c-Pr)CH2CH2CN
NHCH(Et)CH2OMe
N(CH2CH2OMe)2
NHCH(CH2OMe)2
NEt2
C21H22ClN5
C20H25ClN4O
C21H27ClN4O2
C20H25ClN4O2
C19H23ClN4
C20H25ClN4
C21H27ClN4O2
C20H25ClN4O2
C20H24Cl2N4
C19H22Cl2N4
C19H22Cl2N4O2
C19H22Cl2N4O
C20H24Cl2N4O2
C20H21Cl2N5
C20H25ClN4O2
C19H23ClN4O
C20H25ClN4O
C20H26N4O
NH-3-pentyl
N(CH2CH2OMe)2
NHCH(CH2OMe)2
NEtBu
NH-3-pentyl
2,4-Cl2Ph
2,4-Cl2Ph
2,4-Cl2Ph
2,4-Cl2Ph
2,4-Cl2Ph
4-ClPh
NHCH(CH2OMe)2
NHCH(Et)CH2OMe
N(CH2CH2OMe)2
NPrCH2CH2CN
N(CH2CH2OMe)2
NEt2
2-Cl-4-MeOPh
2-Cl-4-MeOPh
2-Me-4-MeOPh
2-Me-4-MeOPh
2-Me-4-MeOPh
2-Me-4-MeOPh
2-Me-4-MeOPh
2-Me-4-MeOPh
2-Br-4-MeOPh
2-Br-4-MeOPh
2-Br-4,5-(MeO)2Ph
2-Br-4,5-(MeO)2Ph
2-Br-4,5-(MeO)2Ph
2-Br-4,5-(MeO)2ph
2-Br-4,5-(MeO)2Ph
2-Br-4,5-(MeO)2Ph
Ð
78±80
145±147
97±99
104±106
118±119
177
NH-3-pentyl
NEt2
NH-3-pentyl
C21H28N4O
C22H27N5O
C22H25N5O
NPrCH2CH2CN
N(c-Pr)CH2CH2CN
NHCH(Et)CH2OMe
NHCH(CH2OMe)2
NHCH(CH2OMe)2
N(CH2CH2OMe)2
NH-3-pentyl
NHCH(CH2OMe)2
N(CH2CH2OMe)2
NEt2
C21H28N4O2
C21H28N4O3
C20H25BrN4O3
C21H27BrN4O3
C21H27BrN4O2
C21H27BrN4O4
C22H29BrN4O4
C20H25ClN4O2
C21H27ClN4O2
C22H29ClN4O4
Ð
Oil
115±116
128±131
74±76
159±161
90±93
110
100±101
169±170
98±100
Ð
NH-3-pentyl
N(CH2CH2OMe)2
Ð
Ð
Ð
Ð
Ð
aStandard deviations are reported.
bParenthetical values are the number of determinations.
cc-Pr=cyclopropyl.
MD). No anity at 10 mM was detected for adenosine,
monoamine (adrenergic (a1, a2, a3, b), dopaminergic,
or serotonergic receptors), histaminergic H1 or H2,
muscarinic, GABAa, GABAb, oxytocin, vasopressin V1,
glycine, CGRP, prostaglandin (LTB4, LTD4 and
thromboxane A2) or opioid receptors. Similarly, the
compound had no anity at 10 mM for ion channels
(glutamate (NMDA or Cl sites), calcium (types L or N)
or calcium activated potassium sites) and no mono-
amine oxidase A or B inhibition was detected at the
same concentration. There was weak inhibition at
dopamine uptake sites (51% @ 10 mM).
Compound 11-31 had good oral activity in the rat
situational anxiety test and the rat elevated plus maze
model.31,34,35 The minimum e€ective dose for 11-31 was
0.3 mg/kg (po) in the rat situational anxiety test.34
Maximal ecacy (approximately 57% reduction in
latency time in the dark compartment) was observed at
this dose and after 10 and 30mg/kg po. Chlordiazepoxide

P. J. Gilligan et al. / Bioorg. Med. Chem. 8 (2000) 181±189
185
Table 2. Rat ex vivo bnding dataa,b
Compound 11-31 had a promising dog pharmacoki-
netic pro®le (Table 3). In beagle dogs, this compound
(5 mg/kg, iv, po) a€orded good plasma levels. Intra-
venous administration using a mixed solvent vehicle
(N,N-dimethylacetamide:ethanol:propylene glycol:water:
1:2:6:1) generated mean t1/2, CL and Vd,ss values equal
to 46.4‹7.6 h, 0.49‹0.08 L/kg/h and 23.0‹4.2 L/kg,
respectively, after iv administration. After oral dosing,
the mean Cmax, Tmax, t1/2 and bioavailability values
were equal to 1260‹290 nM, 0.75‹0.25 h, 45.1‹10.2 h
and 33.1%, respectively.37
Example
Mean hCRF1 Ki (nM)
Nc
1 h
3 h
11-2
11-3
11-5
11-7
11-18
11-19
11-25
11-28
11-30
11-31
11-42
1
1.1
1.7
2.0
5.4
1.7
2.2
3.0
1.2
0.8
1.0
4.5
1.6
4
5
5
4
9
5
5
5
4
57.0‹9.2
42.3‹23.0
29.1‹3.4
16.3‹10.9
88.9‹7.4
48.3‹10.5
57.9‹11.4
86.7‹7.8
64.2‹11.5
82.2‹7.9
57.9‹12.6
<10
32.2‹6.1
27.3‹4.0
18.7‹11.5
23.0‹5.0
89.9‹13.5
49.3‹14.7
54.5‹12.3
91.3‹3.1
52.8‹17.9
87.4‹4.6
62.5‹15.7
<10
11
5
10
aStandard deviations are reported.
Conclusion
bSee Table 1 for the statistics associated with the mean Ki values.
cN=the number of rats employed in the studies.
Analogue 11-31 (DMP904) is a potent hCRF1 antago-
nist, which has good ecacy in two rat models for
anxiety. In the rat situational anxiety test, this com-
pound appears to be a more potent anxiolytic agent
after oral administration than chlordiazepoxide and
compound 1 (CP154526-1). Based on the currently
available data, the peripheral e€ects in rats and dogs for
this compound appear to be minimal. Compound 11-31
has an excellent oral pharmacokinetic pro®le in dogs.
The overall pro®le of this compound suggests that it
may be an anxiolytic drug with a low motor side e€ect
liability.
caused a 72% reduction in latency at 20 mg/kg (po). The
literature compound 1 (CP154526-1, 30 mg/kg (po)) was
inactive in this test. Compound 11-31 was also e€ective
in the rat elevated plus maze model, the ED50 was 5 mg/
kg (po).31,35 However, 11-31 did not inhibit open-®eld
locomotor activity at 10, 30 and 100 mg/kg (po), while
chlordiazepoxide did at 30 mg/kg (po, 50% reduction
relative to controls). This compound did not cause any
signi®cant motor de®cits at 30 mg/kg (po) in the rat
rotarod model.34 These results indicate that 11-31 may
be a potent anxiolytic drug, for which there is a good
separation between the doses which cause motor side
e€ects and those which lead to behavioral ecacy.
Experimental
Chemistry
Some peripheral e€ects of 11-31 were examined in the
rat since CRF receptors populate peripheral as well as
central tissues. CRF sites are present in rat testes, ovar-
ies and components of the immune system, the GI tract,
heart and skeletal muscle tissues.3 Single dosing of 11-31
(30 mg/kg, po) had no statistically signi®cant e€ect on
gastrointestinal (GI) motility; carbachol (0.3 mg/kg, ip)
signi®cantly increased GI motility, while atropine
(3.0 mg/kg, po) decreased GI motility in the same
experimental protocol. Compound 11-31 (30 mg/kg, po)
had no statistically signi®cant e€ect on acute renal
function up to 24 h post-dose in rats. In contrast, the
positive control furosemide (30 mg/kg, po) elevated
renal output and reduced osmolality and glucose, urea,
creatinine, sodium, potassium and chloride levels.36
Analytical data were recorded for the compounds
described below using the following general procedures
(Table 4). Proton NMR spectra were recorded on
Varian VXR or Unity 300 FT-NMR instruments
(300 MHz); chemical shifts were recorded in ppm (d)
from an internal tetramethysilane standard in deutero-
chloroform or deuterodimethylsulfoxide as speci®ed
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 spectrometer (both using chemi-ionization (CI)
with NH3 as the carrier gas). Gas chromatography±mass
spectroscopy was run occasionally using the former
instrument. Chemi-ionization high resolution mass
Compound 11-31 did not cause statistically signi®cant
cardiovascular or pulmonary e€ects in anesthetized
dogs (n=4) after intravenous administration of 11-31.
Escalating doses of 11-31 (0.3, 1.0, 3.0 and 5.0 mg/kg)
in a mixed solvent vehicle (N,N-dimethylacetamide:
polyethylene glycol:water: 1:7:2) were administered at
30 min intervals. Peak plasma concentrations were
1.55‹0.24, 2.60‹0.16, 10.85‹2.82 and 13.82‹4.39nM
for the 0.3, 1.0, 3.0, and 5.0 mg/kg doses, respectively.
Mean arterial blood pressure, contractility, heart rate
and the lead II ECG wave form, including QT interval
were within the ranges for control values. Respiration
rate, tidal volume, minute volume, peak expiratory ¯ow
as well as arterial pO2, pCO2 and pH were unchanged
for the duration of the experiment.36
Table 3. Single dose dog pharmacokinetic data for compound 11-31
(5 mg/kg)a
Parameter
iv
po
Nb
t1/2 (h)
Cmax (nM)
Tmax
AUCT (nM h)
AUC (nM h)
Vd,ss (L/kg)
CL (L/h/kg)
% Bioavailability
4
46.4‹7.6
Ð
4
45.1‹10.2
1260‹290
0.75 (0.5±1.0)c
8120‹270
9780‹310
Ð
Ð
24,420‹3260
29,520‹4790
23.0‹4.2
0.49‹0.08
Ð
Ð
33.1
aThe mean values are reported. Standard deviations are reported.
bN is the number of animals used in the study.
cThe parenthetical value is the range for the value.

186
P. J. Gilligan et al. / Bioorg. Med. Chem. 8 (2000) 181±189
Table 4. Analytical data
Example MS (M+H)+
Quantitative analysis
theory (found)
mp (ꢁC)
Example MS (M+H)+
Quantitative analysis
theory (found)
mp (ꢁC)
175±176
no.
calcd (found)
no.
calcd (found)
11-1
337.2388
(337.2392)
323.2233
(323.2236)
353.2333
(353.2341)
362.2337
(362.2345)
376.2486
(376.2501)
360.2182
(360.2188)
383.2427
(383.2447)
369.2291
(369.2291)
295.1919
(295.1923)
337.2017
(337.2028)
343.1685
(343.1690)
357.1846
Oil
92±94
Oil
11-22
C 60.48 H 5.89 N 14.85
(C 60.62 H 5.88 N 14.82)
11-2
C 74.50 H 8.137 N 17.38
(C 74.43 H 7.90 N 17.16)
11-23
11-24
(409,411)
107
110±111
C 58.02 H 5.65 N 14.24
(C 58.11 H 5.52 N 14.26)
11-3
11-25
11-26
11-27
11-28
11-29
11-30
11-31
11-32
11-33
11-34
11-35
11-36
11-37
11-38
11-39
11-40
11-41
11-42
11-43
423.1355
(423.1337)
72±74
83±85
69
11-4
C 73.10 H 7.538 N 19.37
(C 73.18 H 7.59 N 18.81)
C 73.57 H 7.78 N 18.65
(C 73.55 H 7.79 N 18.64)
C 73.51 H 7.01 N 19.48
(C 73.57 H 7.15 N 19.12)
C 69.08 H 7.915 N 14.65
(C 68.85 H 7.83 N 14.54)
C 68.45 H 7.669 N 15.21
(C 68.35 H 7.49 N 14.91)
143
C 59.71 H 5.26 N 14.85
(C 59.94 H 5.09 N 17.23)
11-5
115±117
133 (dec)
87±89
103±105
Oil
389.1720
(389.1744)
358.1547
(358.1560)
372.1701
(372.1717)
339.2182
(339.2185)
353.2334
(353.2341)
378.2274
(378.2294)
376.2125
(376.2137)
369.2278
(369.2291)
385.2243
(385.2240)
448.1092
11-6
C 63.59 H 6.46 N 15.61
(C 63.22 H 6.24 N 15.27)
C 64.42 H 6.77 N 15.02
(C 64.08 H 6.63 N 14.96)
C 70.98 H 7.74 N 16.55
(C 71.15 H 7.46 N 16.56)
C 71.56 H 8.017 N 15.90
(C 71.48 H 7.99 N 15.88)
C 70.00 H 7.219 N 18.55
(C 70.05 H 7.22 N 18.36)
C 70.38 H 6.71 N 18.65
(C 70.35 H 6.82 N 18.83)
78±80
145±147
97±99
108
11-7
11-8
11-9
11-10
11-11
11-12
11-13
11-14
11-15
11-16
11-17
11-18
11-19
11-20
11-21
Oil
118±119
177
C 66.59 H 6.76 N 16.34
(C 66.69 H 6.82 N 16.20)
C 67.31 H 7.06 N 15.70 Cl 9.93
129±130
139±141
162
(357.1846) (C 67.32 H 6.95 N 15.50 Cl 9.93)
380.1634 C 66.39 H 5.847 N 18.44 Cl 9.33
(380.1642) (C 66.29 H 5.51 N 18.36 Cl 9.31)
373.1800
(373.1795)
403.1899
(403.1901)
389.1742
Oil
C 65.60 H 7.34 N 1457
(C 65.42 H 7.24 N 14.37)
C 53.46 H 5.62 N 12.47 Br 17.78
115±116
128±131
74±76
159±161
90±93
110
C 64.42 H 6.767 N 15.02
(C 64.59 H 6.51 N 14.81)
112±113
77±78
131±133
113±114
94±96
Oil
(448.1110) (C 53.91 H 5.81 N 12.12 Br 18.00)
462.1238
(462.1267)
447.1396
C 54.43 H 5.87 N 12.09
(C 54.52 H 5.86 N 11.91)
C 56.38 H 6.08 N 12.52 Br 17.86
C 61.77 H 6.49 N 14.41 Cl 9.13
(389.1744) (C 61.90 H 6.66 N 13.62 Cl 9.25)
342.1597
(342.1611)
357.1848
(357.1846)
403.1887
(403.1901)
C 66.56 H 6.76 N 16.34
(C 66.70 H 6.73 N 16.13)
C 67.31 H 7.06 N 15.70
(C 67.12 H 6.86 N 15.53)
(447.1396) (C 56.42 H 5.77 N 12.46 Br 18.15)
479.1289
(479.1294)
493.1441
C 53.55 H 5.92 N 11.36 Br 16.20
(493.1450) (C 53.64 H 5.92 N 11.29 Br 16.33)
388.1649
(388.1666)
403.1834
(403.1902)
449.1938
(449.1956)
C 61.77 H 6.49 N 14.41
(C 62.13 H 6.42 N 14.11)
C 62.30 H 6.75 N 13.91
(C 62.30 H 6.66 N 13.61)
100±101
169±170
98±100
389.1733 C 61.77 H 6.489 N 14.41 Cl 9.126
(389.1744) (C 62.06 H 6.37 N 14.25 Cl 9.12)
C 61.38 H 6.18 N 14.32
113±114
86±87
(C 61.54 H 6.12 N 14.37)
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 measured on
a Buchi Model 510 melting point apparatus or a Thomas
Hoover capillary apparatus and are uncorrected.
Reagents were purchased from commercial sources and,
when necessary, puri®ed prior to use according to the
general procedures outlined by D. Perrin and W. L. F.
Armarego.38 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. All
reactions were performed under a nitrogen atmosphere
using magnetic stirring. Reactions requiring anhydrous
conditions were performed in glassware, which had been
¯ame-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.
Commonly used abbreviations are: EtOAc (ethyl ace-
tate), MeOH (methanol), EtOH (ethanol), DMF (N,N-
dimethylformamide), HOAc (acetic acid), THF (tetra-
hydrofuran), and TLC (thin-layer chromatography).
2-Methyl-4-methoxyphenylacetonitrile. A mixture of
1-bromomethyl-2-methyl-4-methoxybenzene39 (35 g,
163 mmol) and sodium cyanide (39.9 g, 813 mmol) in
DMF (800 mL) was stirred at re¯ux temperature for
23 h. Solvent was removed in vacuo and the residue was
treated with a 1 N NaOH solution (500 mL). The aqu-
eous mix was extracted three times with CH2Cl2. The
combined organic layers were washed twice with brine,
dried and ®ltered. Solvent was removed in vacuo. The
residue was puri®ed by column chromatography
(EtOAc:hexanes: 1:9) to give a solid, which was used
1
without further puri®cation: H NMR (CDCl3) d 7.25
(br d, 1H, J=8, C6-H), 6.80±6.70 (m, 2H, C3-H and C5-
H), 3.80 (s, 3H, OCH3), 3.60 (s, 2H, CH2CN); MS (GC)
m/z 162 (M+H)+.
1-Cyano-1-(2-methyl-4-methoxyphenyl)propane-2-one.
To a solution of 2-methyl-4-methoxyphenylacetonitrile

P. J. Gilligan et al. / Bioorg. Med. Chem. 8 (2000) 181±189
187
(7.0 g, 43.4 mmol) in ethyl acetate (150 mL) were added
sodium pellets (1.2 g, 52.1 mmol) portionwise at room
temperature. The reaction mixture was heated to re¯ux
for 16 h. Upon cooling to room temperature, the result-
ing suspension was ®ltered and washed with copious
amounts of ether. The collected white solid was dis-
solved in water. The water solution was acidi®ed by
adding acetic acid until pH 5±6. The mixture was
extracted with ethyl acetate; the combined organic lay-
ers were dried, ®ltered and concentrated to dryness to
provide a white solid (5.3 g, 60% yield): 1H NMR
(CDCl3) d 7.30 (dd, 1H, J=8,1, C6-H), 6.85±6.75 (m,
2H, C3-H and C5-H), 4.75 (s, 1H, CHCN), 3.80 (s, 3H,
OCH3), 2.35, 2.30, 2.25, 2.20 (4s, 6H, CH3 and
COCH3); MS (CI) m/z 204 (M+H)+.
phenyl C6-H), 6.88 (d, 1H, J=3, phenyl C3-H), 6.81 (dd,
1H, J=8,3, phenyl C5-H), 6.78 (s, 1H, C6-H), 3.84 (s,
3H, OCH3), 2.54 (s, 3H, CH3), 2.42 (s, 3H, CH3), 2.17
(s, 3H, CH3); CI-MS: 302 (M+H)+.
7-(Pentyl-3-amino)-2,5-dimethyl-3-(2-methyl-4-methoxy-
phenyl)pyrazolo[1,5-a]pyrimidine (11-31). A solution of
3-pentylamine (12.7 g, 146 mmol) and 7-chloro-5-methyl-
3-(2-methyl-4-methoxyphenyl)pyrazolo[1,5-a]pyrimidine
(8.8 g, 29.2 mmol) was stirred at re¯ux temperature for
16 h; then it was cooled to ambient temperature. The
reaction mixture was then poured onto water (100 mL)
and mixed. Three extractions with EtOAc, washing the
combined organic layers with brine, drying, ®ltration
and removal of solvent in vacuo produced a yellow
solid. Recrystallization from hexane a€orded a white
solid (8.8 g, 45% yield): mp 108 ꢁC; H NMR (CDCl3,
1
5-Amino-4-(2-methyl-4-methoxyphenyl)-3-methylpyrazole.
A mixture of 1-cyano-1-(2-methyl-4-methoxyphenyl)-
propane-2-one (5.3 g, 26.1 mmol), hydrazine mono-
hydrate (2.6 mL, 52.2 mmol), acetic acid (5.1 mL,
88.7 mmol) and toluene (125 mL) was heated at re¯ux
using a Dean±Stark trap for 5 h. The reaction mixture
was cooled to room temperature and concentrated. The
residue was dissolved in 6 N HCl and the resulting
solution was extracted with ether:hexanes (1:1) three
times (25 mL). The aqueous layer was basi®ed by addi-
tion of concentrated ammonium hydroxide solution
until pH 11. The resulting mixture was extracted with
ethyl acetate three times. The combined organic layers
were dried and concentrated in vacuo to give a pale
300 Hz) d 6.86 (d, 1H, J=3 Hz, phenyl C3-H), 6.77±6.81
(dd, 1H, J=9, 3 Hz, phenyl C5-H), 5.99 (bd, 1H,
J=9 Hz, phenyl C6-H), 5.77 (s, 1H, C6-H), 3.83 (s, 3H,
OCH3), 3.40±3.48 (m, 1H, NHCH), 2.44 (s, 3H, CH3),
2.32 (s, 3H, CH3), 2.21 (s, 3H, CH3), 1.58±1.82 (m, 4H,
CH2CH3), 1.02 (t, 6H, J=7, CH2CH3). Anal. calcd for
C21H28N4O: C 71.56, H, 8.02, N 15.90, Found: C,
71.48, H, 7.99, N 15.88.
Biology
The cloned human receptor binding assay was per-
formed as described previously.24 The rat and human
CRF-coupled adenylate cyclase assays have been
reported elsewhere.24,40
1
brown viscous oil (3.6 g, 66% yield). H NMR (CDCl3)
d 7.08 (d, 1H, J=8, C6-H), 6.75±6.85 (m, 2H C3-H
and C5-H), 3.83 (s, 3H), 2.19 (s, 3H); MS(CI) 218
(M+H)+.
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 suspen-
sion. Plasma samples were drawn from Sprague±
Dawley rats at 1 and 3 h post-dose. Plasma samples
were then diluted 10-fold with assay medium and aliquots
of these diluted samples were analyzed in the standard
binding assay.
2,5-Dimethyl-3-(2-methyl-4-methoxyphenyl)-pyrazolo[1,5-
ꢀ]pyrimidin-7-one. 5-Amino-4-(2-methyl-4-methoxy-
phenyl)-3-methylpyrazole (5.3 g, 24.4 mmol) was dis-
solved in glacial acetic acid (30 mL) with stirring. Ethyl
acetoacetate (3.4 mL, 26.8 mmol) was then added drop-
wise to the resulting solution. The reaction mixture was
then heated to re¯ux temperature and stirred for 16 h,
then cooled to room temperature. Ether (100 mL) was
added and the resulting precipitate was collected by ®l-
tration. Drying in vacꢁuo a€orded a white solid (4.7 g,
The rat situational anxiety, elevated plus maze and
rotarod tests were performed as described previously.41±43
Pharmacokinetic parameters were determined in dogs
after intravenous (iv) or oral (po) doses of 5 mg/kg each.
Dogs (n=4) were given the compound intravenously in
a cosolvent vehicle (N,N-dimethylacetamide:ethanol:
propylene glycol:water: 1:2:6:1), or orally in 0.25%
methylcellulose suspension (with 0.1% Tween 80 in
some cases). Blood samples were collected from jugular
veins at pre-dose, 5, 15, 30 min and 1, 2, 4, 8, 10, 12, 16,
24, 32, 48, 56, and 72 h after dosing.
1
68% yield): mp >200 C; H NMR (CDCl3, 300 Hz) d
9.69 (s, 1H, NH), 7.08 (d, 1H, J=8, phenyl C6-H), 6.72±
6.67 (m, 2H, phenyl C3-H and C5-H), 5.49 (s, 1H, C6-
H), 3.77 (s, 3H, OCH3), 2.37 (s, 3H, CH3), 2.14 (s, 3H,
CH3), 2.10 (s, 3H, CH3); CI-MS: 284 (M+H)+.
7-Chloro-2,5-dimethyl-3-(2-methyl-4-methoxyphenyl)-
pyrazolo[1,5-ꢀ]pyrimidine. A mixture of 2,5-dimethyl-3-
(2-methyl-4-methoxyphenyl)-pyrazolo[1,5-a]pyrimidin-7-
one (3.0 g, 10.6 mmol), phosphorus oxychloride (8.2 g,
5.0 mL, 52.9 mmol), N,N-diethylaniline (1.9 g, 2.0 mL,
12.7 mmol) and toluene (20 mL) was stirred at re¯ux
temperature for 3 h, then it was cooled to ambient tem-
perature. The volatiles were removed in vacuo. Flash
chromatography (EtOAc:hexane: 1:2) on the residue
gave 7-chloro-5-methyl-3-(2-methyl-4-methoxyphenyl)-
pyrazolo[1,5-a]pyrimidine (2.87 g, 90% yield) as a yel-
Plasma samples were analyzed after extraction of test
compounds by simple liquid±liquid extraction. LC/MS/
MS analysis was performed on a Sciex (Thornhill,
Ontario) Model APIIII triple quadrapole mass spectrom-
eter 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 Sym-
metry octyl mini-bore column (2.1Â50 mm).
1
low oil: H NMR (CDCl3, 300 Hz) d 7.17 (d, 1H, J=8,

188
P. J. Gilligan et al. / Bioorg. Med. Chem. 8 (2000) 181±189
Cardiovascular pharmacodynamic testing was per-
formed in anesthetized dogs. Mongrel dogs (n=8) of
both sexes (weights=7.0 to 12.3 kg, HRP Inc., Cum-
berland, VA) were anesthetized with sodium pento-
barbital (30 mg/kg, iv) and mechanically ventilated
(15 breaths/min) using a Harvard respirator (Harvard
Apparatus, South Natick, MA). The cephalic veins were
cannulated for compound administration and infusion
of sodium pentobarbital at 6 mg/kg/h. A micro-
manometer tip catheter (Millar Instruments, Houston,
TX) was placed in the left ventricle to measure left ven-
tricular pressure. The ®rst derivative of the left ven-
tricular pressure signal, LV+dP/dt, was obtained by
the modular Instruments software. Arterial blood pres-
sure 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 11-31
was administered 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, propylene 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 for 16 min for the 5.0 mg/kg dose.
Osmometer, Fiske Associates, Norwood, MA) were
measured and glucose, urea, creatinine, sodium, potas-
sium and chloride concentrations were determined
(Dimension Clinical analyzer, DuPont, Wilmington, DE).
Gastrointestinal motility was evaluated by gross obser-
vation and transit of an orally administered charcoal
suspension (10% w/v charcoal in 0.25% methylcellu-
lose) in fasted male Sprague±Dawley rats (178±222 g,
Caesarian-derived). Compound 11-31 (30 mg/kg, po),
atropine (3.0 mg/kg, po) and carbachol (0.3 mg/kg, ip)
were administered in the standard vehicles. The char-
coal suspension (5 mL/kg) was administered orally
60 min after the dose of 11-31 and 30 min after the
doses of carbachol or atropine. Five minutes after the
charcoal meal, the rats were euthanized with CO2 and
the gastrointestinal 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 were
expressed as a percentage relative to methocel controls.
Acknowledgements
The authors gratefully acknowledge the expert technical
contributions of T. Boop, F. Brown, C. Chi, Nancy
Contel, G. Demond, T. Donovan, C.-M. Lai, L. Liang,
Anne Marshall, Carol Krause, Susan Keim, John Pat-
terson, N. Raghavan, Cindy Rominger, David Romin-
ger, Wanda West and Han Zeng.
Respiratory function was monitored in anesthetized
dogs. Mongrel dogs were treated with sodium pento-
barbital 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 cannulated 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 posi-
tioned 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 di€erential
pressure transducer (DP45-14) for the measurement of
air ¯ow. The esophageal balloon was similarly attached
to one side of a second di€erential pressure transducer
(Validyne, DP45-24), the other side of which was con-
nected to a side arm inserted in the tracheal cannula
adjacent to the pneumotachograph to measure intra-
pleural pressure. The vehicle was composed of N,N-
dimethylacetamide, 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 dose.
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., Med®eld, MA).
References
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