Discovery and evaluation of pyrazolo[1,5-a]pyrimidines as neuropeptide Y1 receptor antagonists

Article abstract of DOI:10.1016/j.bmcl.2010.12.116

A novel series of pyrazolo[1,5-a]pyrimidine derivatives was synthesized and evaluated as NPY Y1R antagonists. High binding affinity and selectivity were achieved with C3 trisubstituted aryl groups and C7 substituted 2-(tetrahydro-2H-pyran-4-ylamino)ethyla

Full text of DOI:10.1016/j.bmcl.2010.12.116

Bioorganic & Medicinal Chemistry Letters 21 (2011) 2641–2645
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery and evaluation of pyrazolo[1,5-a]pyrimidines as neuropeptide
Y1 receptor antagonists
David A. Griffith a, Diane M. Hargrove b, Tristan S. Maurer c, Charles A. Blum d, Stéphane De Lombaert d,
John K. Inthavongsay a, Lee E. Klade a, Christine M. Mack d, Colin R. Rose a, Martin J. Sanders b,
Philip A. Carpino a,
⇑
a Department of Worldwide Medicinal Chemistry, Pfizer PharmaTherapeutics, Groton, CT 06340, USA
b Department of Cardiovascular, Metabolic, and Endocrine Diseases, Pfizer Global Research & Development, Groton, CT 06340, USA
c Department of Pharmacokinetics, Dynamics & Metabolism, Pfizer PharmaTherapeutics, Groton, CT 06340, USA
d Neurogen Corporation, Branford, CT 06405, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of pyrazolo[1,5-a]pyrimidine derivatives was synthesized and evaluated as NPY Y1R antag-
onists. High binding affinity and selectivity were achieved with C3 trisubstituted aryl groups and C7
substituted 2-(tetrahydro-2H-pyran-4-ylamino)ethylamine moieties. Efforts to find close analogs with
low plasma clearance in the rat and minimal p-glycoprotein efflux in the mouse were unsuccessful. Com-
pound 2f (CP-671906) inhibited NPY-induced increases in blood pressure and food intake after iv and icv
administration, respectively, in Sprague–Dawley (SD) rat models. Oral administration of compound 2f
resulted in a modest, but statistically significant, reduction in food intake in a Wistar rat model of feeding
behavior. Small inhibitions of food intake were also observed in an overnight fasting/refeeding model in
SD rats. These data suggest a potential role for Y1R in the regulation of food intake in rodents.
Ó 2011 Elsevier Ltd. All rights reserved.
Received 18 October 2010
Revised 18 December 2010
Accepted 21 December 2010
Available online 7 January 2011
Keywords:
Obesity
NPY Y1R
Pyrazolo[1,5-a]pyrimidine
Obesity is a body weight disorder characterized by excess adi-
pose tissue and a body mass index (BMI) greater than or equal to
30 kg/m2. Accumulation of fat mass is protective during periods
of starvation, but can otherwise lead to multiple co-morbidities
including hypertension, diabetes mellitus, and dyslipidemia. The
prevalence of obesity has steadily increased in the past decade to
the point where it is now considered to be the second leading
cause of preventable death after smoking.1 Weight loss through
diet, exercise, pharmacological treatment, or even bariatric surgery
has been shown to lead to significant improvements in the co-mor-
bidities associated with the disease. Diet and exercise are consid-
ered first line treatments for obesity while bariatric surgery is
only prescribed for the morbidly obese. Diet and exercise are rarely
successful over the long-term in maintaining weight loss, thus the
need for pharmacotherapy. Only a few drugs, including OrlistatÒ,
are currently approved to treat obesity, but these agents have lim-
itations which prevent their widespread use.2 A number of prom-
ising pharmacological agents have recently completed Phase III
clinical trials and are awaiting regulatory approval (QnexaÒ, Con-
traveÒ, and locaserin).3 A growing need exists for new and effective
weight loss drugs with improved toleration and excellent safety
profiles.
The 36 amino acid polypeptide neuropeptide Y (NPY), a mem-
ber of a family of pancreatic polypeptides, has been shown to play
a role in the regulation of food intake, energy homeostasis, vascular
tone, anxiety, and reproductive health.4,5 NPY is primarily synthe-
sized in the hypothalamus, a key area in the central nervous sys-
tem (CNS) involved in the control of energy homeostasis. NPY
stimulates food intake when injected centrally in satiated rats.6
Furthermore, chronic administration of NPY induces a sustained
increase in food intake and body weight.
The pharmacological actions of NPY are mediated through four
G-protein coupled receptors (Y1R, Y2R, Y4R, and Y5R).7 A fifth
cloned receptor called y6R is present in the mouse and rabbit,
but is non-functional in rats and humans.8 Y1R and Y5R are pri-
marily localized in the hypothalamus and vasculature, Y2R in the
gut and the hypothalamus, and Y4R in the nucleus of the solitary
tract and the hypothalamus.9 Studies with transgenic knockout
animals and subtype-selective peptidic analogs (i.e., antagonists
for Y1R and Y5R; agonists for Y2R and Y4R) have implicated the
four functional NPY receptors in the regulation of feeding behav-
ior.4 NPY receptors represent potential drug targets for the treat-
ment of obesity.
In the past few years, the discovery of non-peptidic, small mol-
ecule Y1R and Y5R antagonists has attracted considerable interest,
especially in the pharmaceutical industry. Two Y5R antagonists,
MK-0557 and S-2367, have been advanced into human clinical
⇑
Corresponding author. Tel.: +1 860 441 5694; fax: +1 860 441 8067.
E-mail address: philip.a.carpino@pfizer.com (P.A. Carpino).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.12.116

2642
D. A. Griffith et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2641–2645
trials, however MK-0557 was recently dropped because of a mod-
est effect in preventing weight re-gain following a very low calorie
diet.10,11 Several novel series of Y1R antagonists have been identi-
fied that reportedly modulate feeding behavior in rodent mod-
els.12,13 As part of an effort to validate Y1R as an anti-obesity
target, we sought Y1R antagonists with high functional activities
and CNS exposure to explore the role of the central Y1R on feeding
behavior in rodent models.
N-(2-Aminoethyl)-N-ethyl-9-mesityl-2-methyl-9H-pyrimido[4,
5-b]indol-4-amine 1 was identified as a Y1R antagonist during the
counterscreening of compounds prepared as corticotrophin-releas-
ing hormone receptor (CRHR) antagonists for another project
(Fig. 1).14 Compound 1 exhibited 29-fold selectivity for Y1R over
CRHR. Concerns about the high lipophilicity of 1 [calculated ElogD
(cElogD) = 3.9]15 led to the removal of the N-ethyl group and the
replacement of the tricyclic ring with bicyclic heterocyclic moieties
found in other known CRHR antagonists. These changes resulted in
the discovery of N-(2-aminoethyl)-3-mesityl-2,5-dimethylpyrazol-
o[1,5-a]pyrimidin-7-amine 2a which exhibited moderate Y1R
binding affinity, selectivity for Y1R over CRHR, and reduced lipo-
philicity compared to 1. Herein, we describe the structure–activity
relationships and biological characterizations of compounds 2a–m
and report on our efforts to ensure target engagement and confirm
specificity-of-action in behavioral studies measuring changes in
food intake.
N NH
O
a
c
b
CN
Ar
CN
Ar
NH2
Ar
3
4
5
H
N
R1 R2
NH2
R1 R2
OH
HN
HN
R
d,e
f
N
N
N
N
N
N
N
N
N
Ar
Ar
Ar
6
7
2a-f, 2i-m
Scheme 1. Reagents and conditions: (a) Na, EtOAc, 90 °C; (b) NH2NH2, HOAc,
toluene, 130 °C, Dean–Stark trap; (c) ethyl acetoacetate, HOAc, 130 °C; (d) POCl3,
130 °C; (e) H2NCH2C(R1R2)NH2, CH3CN, 80 °C; (f) R(@O), NaBH(OAc)3, HOAc, room
temperature.
H
H
N
N
HN
HN
O
O
N
N
N
N
a, b
N
N
Cl
Cl
Cl
Cl
O 4'
R3O
The pyrazolo[1,5-a]pyrimidine derivatives 2a–m were synthe-
sized from key intermediate 5 which was prepared in two steps
from benzonitrile derivative 3 (Scheme 1). Cyclocondensation of 5
with ethyl acetoacetate afforded compound 6 which was treated
first with POCl3 and then with an appropriately substituted diamine
derivative to give a family of analogs encompassed by 2a or 7. When
substituted ethylenediamine derivatives were used, the predomi-
nant product came from the addition of the less sterically hindered
amino group. Reductive amination of 7 with appropriately substi-
tuted aldehyde or ketone derivatives provided target compounds
2a–f and 2i–m. Methyl ether 2f was transformed into ethyl and
n-propyl ethers 2g and 2h in two steps that involved demethylation
of the aryl ether group using aqueous HBr, followed by alkylation
with an appropriately substituted alkyl halide (Scheme 2).16
The pyrazolo[1,5-a]pyrimidine derivatives were evaluated in
125I-PYY and 125I-sauvagine binding displacement assays using
Sf9 membranes over-expressing recombinant human or rat Y1
receptors (hY1R or rY1R) or membranes from IMR-32 cells endog-
enously expressing CRHR.14,17 In vitro functional activity was as-
sessed in human neuroblastoma SK-N-MC cells by measuring the
ability of test compounds to antagonize NPY-induced mobilization
of intracellular Ca2+ concentrations using a Fluorometric Imaging
Plate Reader (FLIPR).18 In vivo functional activity was evaluated
in Sprague–Dawley (SD) rats by measuring the ability of test com-
pounds to antagonize NPY-induced increases in blood pressure
(BP) (NPY pressor model).5,19
2f
2g, 2h
Scheme 2. Reagents and conditions: (a) aq HBr, 100 °C; (b) R3I, aq NaOH, isopropyl
alcohol, reflux.
and the c-pentylamino analog 2c were significantly more potent
than 2a in the Y1R binding assay and exhibited high selectivity
for Y1R over CRHR (data not shown). The mesityl group in 2c could
be replaced by a 2,6-di-Cl-phenyl moiety without resulting in a
loss of binding affinity. Compounds 2b–d exhibited high lipophilic-
ities, outside the preferred range for drugs that act at pharmacolog-
ical targets located in the CNS.20 Replacement of the c-pentyl group
in 2d with a tetrahydropyran-4-yl (4-THP) group decreased lipo-
philicity and led to a surprising increase in binding affinity. Further
fine-tuning of the lipophilicity of 2e by incorporation of a methoxy
substituent at the C40 position in the 2,6-di-Cl-phenyl ring pro-
vided 2f (CP-671906). Substitution of the methoxy group in 2f with
larger alkoxyl groups led to a slight decrease in binding affinity (2g,
2h).
Compound 2f was selected for further characterization based on
its affinity for hY1R. This compound exhibited low nanomolar affin-
ity for rY1R (Ki = 3.5 nM) in a binding displacement assay and
showed selectivity versus the human Y2 and Y5 receptors (IC50’s
>10,000 nM). It did not display any significant activity across a pa-
nel of 42 receptors, ion channels, transporters, and enzymes (<50%
A series of N2-alkylated derivatives of 2a was initially investi-
gated (Table 1). The 4-methoxyphenethylamino derivative 2b
inhibition at 1 l l
M)21 or against CRHR (<50% inhibition at 10 M).22
Compound 2f demonstrated potent Y1R functional antagonism in
SK-N-MC cells (Table 2). In an anesthetized rat model, 2f was shown
to block the NPY-induced increase in blood pressure. Compound 2f
was the first compound in the pyrazolo[1,5-a]pyrimidine series to
demonstrate functional activity in an animal model. This assay
was introduced into the screening paradigm in order to confirm tar-
get engagement, albeit in the periphery and not in the CNS.
NH2
NH2
N
N
HN
N
N
N
N
N
3
Pharmacokinetic evaluation of 2f in the SD rat revealed moder-
ate plasma clearance (Table 2), a short half-life (t1/2 = 1.2 h), and
poor oral bioavailability (F = 13%).23 A close analog with reduced
plasma clearance was sought for in vivo studies. Studies with 2f
in rat microsomal preparations showed that a principle route of
metabolism was O-demethylation of the alkoxy group on the C3
aryl ring. Plasma clearances of the ethoxy analog 2g and the
1 hY1R Ki = 154 nM
2a, hY1R Ki = 2300 nM
CRH Ki = 4474 nM
CRH Ki >10000 nM
Figure 1. Y1R screening hit and selective bicyclic derivative.

D. A. Griffith et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2641–2645
2643
Table 1
hY1R binding affinities and calculated lipophilicities
H
N
HN
N
R
R1 R2
N
N
Ar
R1, R2
Compd
Ar
R
cE log D15
Kia,b (nM)
2a
2b
2c
2d
2,4,6-Me-Ph
2,4,6-Me-Ph
2,4,6-Me-Ph
2,6-Cl-Ph
H, H
H, H
H, H
H, H
H, H
H, H
H, H
H, H
H, Mec
H
2.4
4.1
3.3
3.4
2.8
2.6
2.8
3.2
3.4
2.9
4.2
3.2
3.4
2300
29
21
38
7
1
3
8
5
141
5
22
383
CH2CH2-4-MeO-Ph
c-Pentyl
c-Pentyl
4-THPc
4-THP
4-THP
2e
2,6-Cl-Ph
2f (CP-671906)
2,6-Cl-4-MeO-Ph
2,6-Cl-4-EtO-Ph
2,6-Cl-4-n-PrO-Ph
2,6-Cl-4-MeO-Ph
2,6-Cl-4-MeO-Ph
2,6-Cl-4-MeO-Ph
2,6-Cl-4-MeO-Ph
2,6-Cl-4-MeO-Ph
2g
2h
2i
2j
2k
2l
4-THP
4-THP
4-THP
Me, Me
H, H
H, H
2-F-cyclohex-1-yld
CH2CH2F
CH2CHF2
2m
H, H
a
b
c
Human Y1R radioligand binding assay.17
Data represent single determinations obtained in triplicate.
Mixture of enantiomers.
d
Mixture of cis/trans isomers.
Table 2
Y1R functional activities and plasma clearance and protein binding values
was actively transported from the CNS by PGP at the level of the
blood–brain barrier.
Several strategies were investigated for decreasing PGP efflux of
2f in the mouse and improving CNS penetration: (1) introduction
of steric hindrance around the tetrahydro-2H-pyran-4-amino
group in 2f; and (2) reduction of the pKa of this amino group.27
The basic amino group was hypothesized to play a role in the rec-
ognition of the compounds by PGP efflux transporters as com-
Compd FLIPR IC50 Rat Clp
Rat fu,p Rat Clp,u
NPY pressor
(nM)a
(mL/min/kg)
(mL/min/kg) response
(%inh at 3 mg/kg iv)19
2f
2g
2h
7
15
207
45
28
25
0.059
0.029
0.011
763
996
2273
92
47
0
FLIPR Ca2+ mobilization assay in SK-N-MC cells; IC50 curves were constructed
pounds from other series that lacked such a group were
a
using the logistic equation from eight or nine concentrations of test compound with
generally found to be weak PGP efflux substrates (data not shown).
The methyl derivative 2i showed similar PGP efflux to 2f but the
gem-dimethyl analog 2j exhibited reduced efflux (Table 3). Unfor-
tunately, 2j was significantly less potent in the binding assay com-
pared to 2f. The pKa of the amino group in 2f was modulated by
replacing the 4-THP group with fluorinated cycloalkyl and alkyl
groups (Table 3).27 The 2-F-cyclohex-1-yl derivative 2k was shown
to be a PGP efflux substrate while the 2-F-ethylamino analog 2l
and the 2,2-F-ethylamino compound 2m exhibited reduced affinity
for the PGP transporter. Unfortunately, 2l and 2m also displayed
weaker binding affinities for Y1R (Table 1).
Since an analog of 2f with reduced plasma clearance in the rat,
reduced affinity for PGP efflux transporters in mouse models, and
comparable pharmacological activity could not be readily identi-
fied, compound 2f was selected for in vivo studies exploring the
role of Y1R in the regulation of food intake.28 To demonstrate
four replicates per concentration.18
n-propoxy derivative 2h, both of which showed good Y1R binding
affinities and moderate half-lives in rat microsomal preparations
(data not shown), were reduced, although the unbound clearance
of 2h was greater than that of 2g or 2f. Compounds 2g and 2h were
2- and 30-fold less potent in the FLIPR assay compared to 2f. Com-
pound 2g was unable to completely block the NPY pressor re-
sponse in anesthetized rats at the single dose tested, while 2h
was devoid of activity in this model, most likely as a result of weak
functional potency and high unbound clearance (Table 2). Neither
2g nor 2h proved to be suitable tool compounds for assessing the
role of Y1R. Pharmacokinetic properties of other close analogs of
2f, including those lacking 4-methoxy groups on the C3 aryl ring,
were not investigated due to either weak Y1R binding affinities
or high microsomal clearance values (data not shown).
Table 3
Calculated pKa values and ratios of B/P ratios in Mdr1a/1b KO and
As the Y1R mediating the orexigenic activity of NPY is located
within the hypothalamus, brain penetration of 2f was measured.
Brain-to-plasma (B/P) ratios were shown to be poor in SD rats
and FVB mice (0.13 and 0.1, respectively).24,25 The low B/P values
suggested the compound might be a substrate for efflux transport-
ers such as p-glycoprotein (PGP), an ATP-dependent drug transport
protein widely distributed in the body including the brain capillary
endothelial cells.26 Efflux was confirmed in studies measuring
brain and plasma concentrations in multidrug resistant Mdr1a/1b
knockout (KO) mice and wild-type FVB mice following a subcuta-
neous (sc) dose (Note–the mdr gene encodes for PGP). The B/P ratio
was ninefold higher in Mdr1a/1b knockout mice compared to the
B/P ratio in FVB mice. The large ratio of B/P ratios showed that 2f
wild-type FVB mice
a
Compd
Calculated pKa
[B/P] in Mdr1a/1b knockout
mice/[B/P] in FVB miceb
2f
2i
2j
2k
2l
8.9
9.0
9.0
8.0
7.7
6.3
9
10
4
8
4
2m
2
a
Calculated using the software program ACD labs pKa
predictor.
b
Brain and plasma levels were measured at 0.5 h following a
5 mg/kg sc dose.

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D. A. Griffith et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2641–2645
target engagement in the CNS where Y1R is hypothesized to regu-
late feeding behavior, 2f was co-injected with NPY into the hypo-
thalamus of SD rats via an intracerebroventricular (icv)
cannula.29 Compound 2f significantly suppressed NPY-induced
feeding, confirming the ability of the drug to modulate the target
in SD rats, albeit at very high concentrations as administered via
the icv route (Fig. 2). The effects of oral administration of 2f
(40 mg/kg) were tested in lean rats subjected to two different feed-
ing conditions. Compound 2f induced a small, transitory decrease
in food intake in fasting/refeeding SD rats (12% decrease versus
vehicle treated controls at the 2 h timepoint following return of
food or the 4 h timepoint post-dose; no change at the 4 h timepoint
following return of food).30 No significant changes were observed
in locomotor activity, a signal of potential off-target activity. Com-
pound 2f also induced a modest, but statistically significant inhibi-
tion, of nocturnal feeding behavior in Wistar rats, again without
affecting locomotor activity (Fig. 3).31
To confirm that 2f was exposed at the target site in SD rats fol-
lowing a 40 mg/kg oral dose, concentrations of the drug in both
cerebral spinal fluid (CSF) and brain interstitial fluid (ISF) were
measured, although in a separate group of animals from the feed-
ing studies.32 Concentrations of 2f were 26 nM at the 2 h time-
point, significantly higher than the Ki value measured in the
radioligand binding assay, but notably lower than that expected
from unbound plasma concentrations (95 nM), consistent with
the impairment observed in the MDR1a/1b knockout and FVB
mouse exposure studies. ISF concentrations of 2f (collected at the
2 h timepoint from paraventricular nucleus using microdialysis
probes) were found to be similar to the concentrations of 2f in
the CSF ([ISF] = 17 nM). Brain and CSF exposure of 2f in the Wistar
rat model of nocturnal feeding behavior was not measured.
In conclusion, pyrazolo[1,5-a]pyrimidines have been identified
as a new chemotype with Y1R activity. One compound, 2f, inhib-
ited NPY-induced increases in blood pressure and food intake after
iv and icv administration, respectively, confirming functional activ-
ity in an animal model. Analogs of 2f with reduced plasma clear-
ances in the rat or reduced PGP efflux liabilities in a mouse
model were also discovered, but these derivatives were not suit-
able tool compounds. In studies of feeding behavior with 2f, mod-
est inhibitions of food intake were observed in several lean rodent
models. Exposure of 2f at the target site and direct modulation of
the target were both demonstrated. Overall, the results suggest
that Y1R antagonists may have modest effects on feeding behavior
in rat models. Longer term studies will be required to determine if
the small anorectic effects observed with an acute dose can be sus-
tained during repeat dosing and translate into longer term reduc-
tions in body weight.
Acknowledgments
We thank David Hepworth for helpful discussions and Hans
Rollema for the microdialysis data.
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2
1
0
veh/NPY
CP-671906/NPY
treatment group
Figure 2. Icv inhibition by 2f (CP-671906, 120 lg) of NPY(5 lg)-induced increase in
daytime feeding in Sprague–Dawley rats [⁄p <0.05 for vehicle/NPY versus 2f/NPY
(paired two-tailed t-test)].
24
vehicle
13. (a) Lee, J.; Song, K.-S.; Kang, J.; Lee, S. H.; Lee, J. Curr. Top. Med. Chem. 2009, 9,
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14. Compound 1 was synthesized by the methods described in WO199951600 and
the binding affinity against the CRH receptor was measured according to the
assays described therein.
CP-671906
20
*
*
16
12
8
15. (a) ElogD was calculated from
a model built according to the methods
described in Ref. 15b, based on Cubist [Rulequest Research, www.rulequest.
com]. The training set for the model was 33,000 ElogD values measured using
methodology described in Ref. 15c.; (b) Gao, H.; Yao, L.; Mathieu, H. W.; Zhang,
Y.; Maurer, T. S.; Troutman, M. D.; Scott, D. O.; Ruggeri, R. B.; Lin, J. Drug Metab.
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Med. Chem. 2001, 44, 2490.
p = 0.06
n.s.
n.s.
*
4
16. All compounds described in this manuscript gave satisfactory spectral data (1H
NMR, MS).
dark phase
*
0
17. For details of this assay, see WO 2001023387.
18. Antagonism of NPY-induced mobilization of intracellular Ca2+ concentrations
0
2
4
6
8
10
12
14
by 2f was determined in Fluo-3 loaded SK-N-MC cells using
a 96-well
time (hr)
Fluorometric Imaging Plate Reader (FLIPR, Molecular Devices). SK-N-MC cells
were maintained in culture under standard conditions and plated at a density
of 25,000 cells per well 4 days prior to experimentation. On the day of the
experiment, cells were washed with assay buffer (5 mM KCl, 115 mM NaCl,
0.96 mM NaH2PO4, 1 mM MgSO4, 2 mM CaCl2, 1 mM carbachol, 5 mM
dosing
Figure 3. Time course of the effects of 2f (CP-671906, 40 mg/kg po) on nocturnal
feeding in Wistar rats in a continuous food intake monitoring system [⁄p <0.05 for
2f versus time-matched vehicle point (two-tailed, unpaired t-test)].
D
-glucose) and incubated in dye loading solution (assay buffer with 220 nM

D. A. Griffith et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2641–2645
2645
Fluo-3 AM, 1 mM probenecid and an additional 10 mM HEPES) for 1 h at 37 °C
25. Kalvass, J. C.; Maurer, T. S. Biopharm. Drug Dispos. 2002, 23, 327.
26. Schinkel, A. H. Adv. Drug Delivery Rev. 1999, 36.
27. Morgenthaler, M.; Schweizer, E.; Hoffmann-Roder, A.; Benini, F.; Martin, R. E.;
Jaeschke, G.; Wagner, B.; Fischer, H.; Bendels, S.; Zimmerli, D.; Schneider, J.;
Diederich, F.; Kansy, M.; Muller, K. ChemMedChem 2007, 2, 1100.
28. All animal protocols were reviewed and approved by the Pfizer or Neurogen
Institutional Animal Care and Use Committees.
29. SD rats with icv cannula were fed powdered rat chow ad libitum. Between 10–
in a 5% CO2 atmosphere. After a 1 h incubation period, plates were washed and
re-suspended in assay buffer containing test compound at the desired
concentration. Fluorescence responses were elicited by an EC50 concentration
of NPY (range 0.5–1.5 nM), which was determined just prior to each
experiment.
19. SD rats, fasted overnight, were anesthetized with inactin (100 mg/kg ip),
surgically prepared with tracheal (PE205) and femoral arterial and venous
cannula (PE50) and placed on a water-jacketed heating pad. The arterial
pressure and heart rate signals were fed into a PONEMAH Physiology Platform
(Gould Instrument Systems, Valley View OH) and data were recorded every 5
11 am during the light cycle, 2f (120
were co-infused with NPY (5 g in 4
volume 5 L over 1 min). After dosing, food intake over the next 4 h was
l
g) or vehicle (1
lL of propylene glycol)
l
lL sterile water) via the icv cannula (total
l
seconds during the experiment. NPY (5
l
g
in 0.25 mL of saline), was
measured. Rats received two treatments in a cross-over experiment separated
by 2 days.
administered to the rats at 15 min intervals as an iv bolus. Following the
third bolus injection of NPY, the test compound (3 mg/kg in a saline vehicle,
1 mL/kg) was dosed iv and three subsequent NPY injections were
administered.
30. SD rats were individually housed in suspended wire cages with powdered
chow and water provided ad libitum. Before the experiment, rats were fasted
overnight. On the day of the experiment, rats were dosed with 2f (40 mg/kg,
po) or vehicle (0.5% methyl cellulose in water, dosing volume 1.5 mL). Food, in
pre-weighed jars, was restored to the cages 2 or 4 h post dose. Food intake was
recorded 2 and 4 h after restoring food.
31. Wistar rats that had been acclimated to a continuous food intake monitoring
system (Columbus Instruments, Columbus OH) were dosed with 2f (40 mg/kg,
po) or vehicle, 30 min prior to the dark cycle. The system recorded food intake
every 10 min for 18 h post-dose.
20. Wager, T. T.; Chandrasekaran, R. Y.; Hou, X.; Troutman, M. D.; Verhoest, P. R.;
Villalobos, A.; Will, Y. ACS Chem. Neurosci. 2010, 1.
21. Selectivity assays (42 receptors, ion channels and enzymes) were performed by
MDS Pharma (Taipei, Taiwan).
22. CRH receptor binding activity was measured at Cerep (France); www.cerep.fr.
23. For the determination of pharmacokinetic parameters, jugular vein cannulated
SD rats were dosed with 2f by oral gavage (3 mg/kg in 0.5% methyl cellulose at
2 mL/kg, n = 2) or via the jugular vein (1.0 mg/kg in 50% PEG vehicle at 1 ml/kg,
n = 2). Prior to dosing, rats were fasted overnight with water available ad
libitum.
32. CSF samples were obtained 2 h post dose (n = 2). Rats were anesthetized with
metofane. CSF was drawn directly from the cisterna magna through a 26-gauge
needle.
24. B/P ratios were calculated from unbound plasma - to - brain fraction ratios;
equilibrium dialysis was used to measure unbound fraction of compound in
brain homogenates and whole plasma from rats and mice.