Synthesis and evaluation of 5,5-diphenylimidazolones as potent human neuropeptide Y5 receptor antagonists

Article abstract of DOI:10.1016/j.bmc.2006.04.042

A series of novel 5,5-diphenylimidazolones was synthesized and evaluated for activity against the human neuropeptide Y5 receptor. The 3-pyridyl analog 46 demonstrated an IC₅₀ of 8.3 nM with a favorable pharmacokinetic profile in rats, but was i

Full text of DOI:10.1016/j.bmc.2006.04.042

Bioorganic & Medicinal Chemistry 14 (2006) 5517–5526
Synthesis and evaluation of 5,5-diphenylimidazolones
as potent human neuropeptide Y5 receptor antagonists
Kevin W. Gillman,a,* Mendi A. Higgins,a Graham S. Poindexter,a Marc Browning,a
Wendy J. Clarke,a Sharon Flowers,a James E. Grace, Jr.,a John B.Hogan,a
Rachel T. McGovern,a Lawrence G. Iben,a Gail K. Mattson,a Astrid Ortiz,a
Stefanie Rassnick,a John W. Russella and Ildiko Antal-Zimanyib
aBristol-Myers Squibb Pharmaceutical Research Institute, Richard L. Gelb Center for Pharmaceutical Research and Development,
5 Research Parkway Wallingford, CT 06492, USA
bBristol-Myers Squibb Pharmaceutical Research Institute, PO Box 5400, Princeton, NJ 08543, USA
Received 28 December 2005; revised 19 April 2006; accepted 24 April 2006
Available online 11 May 2006
Abstract—A series of novel 5,5-diphenylimidazolones was synthesized and evaluated for activity against the human neuropeptide
Y5 receptor. The 3-pyridyl analog 46 demonstrated an IC50 of 8.3 nM with a favorable pharmacokinetic profile in rats, but was
ineffective in reducing food intake.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
2. Screening
Recently, there have been a number of publications dis-
cussing the potential of selective neuropeptide Y5
(NPY5) receptor antagonists as anorectic agents in a
variety of in vivo feeding models.1 The results of these
studies have only led to increased speculation as to the
function of the NPY5 receptor, given evidence both sup-
porting and negating the hypothesis that the NPY5
receptor is indeed involved in the regulation of food in-
take in rodents. Much of the data published on small
molecule, selective NPY5 antagonists to date has been
compiled using compounds that suffer from a variety
of pharmacokinetic issues such as poor brain penetra-
tion, or short in vivo half-lives which prevent definitive
interpretation of the results. In an effort to further eluci-
date the potential of NPY5 receptor antagonism and
how it relates to food intake, we set out to discover a
novel compound lacking in the aforementioned pharma-
cokinetic liabilities. Herein we disclose results on the
structure–activity and in vivo efficacy of a series of
5,5-diphenylimidazolone NPY5 selective antagonists.
High-throughput screening of the BMS compound col-
lection for binding against the human NPY5 receptor
identified a series of novel imidazolones as represented
by 1 possessing sub-micromolar affinity. These imidazol-
ones were attractive as a synthetic starting point based
on their chemical tractability as well as possessing low
affinity for the hNPY1 receptor. Particularly 5,5-diphe-
nylimidazolone 12 exhibited a >1000-fold selectivity
for the NPY5 receptor versus the NPY1 receptor
(IC50 = 6 nM NPY5 vs IC50 > 10 lM NPY1).
O
NH
N
1
Although compound 1 possessed potent and selective
binding affinity for the hNPY5 receptor, it suffered from
poor aqueous solubility (4 lg/mL, pH 2.0) as well as
negligible oral bioavailability, making it a poor candi-
date for either oral or parenteral formulations. In order
to overcome these obstacles, we embarked on a synthetic
campaign to enhance the aqueous solubility and oral
Keywords: Obesity; NPY5; Imidazolone; Orally bioavailable; Brain
penetrant.
*
Corresponding author. Tel.: +1 203 677 6785; fax: +1 203 677
7702; e-mail: gillmank@bms.com
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.04.042

5518
K. W. Gillman et al. / Bioorg. Med. Chem. 14 (2006) 5517–5526
Table 1. Human NPY5 binding affinity for select 2-substituted
imidazolone derivatives
bioavailability within the imidazolone chemotype, while
maintaining both potent binding affinity and selectivity
for the NPY5 receptor.
Compound
R1
a
hNPY5 IC50 (nM)
6
6 (1)
3. Results and discussion
MeO
Initial synthetic efforts around the central imidazolone
core focused on the removal of the lipophilic geminal
diphenyl substitution at the 5-position. Replacement of
the gem-diphenyl rings with 2-substituted pyridines
was accomplished via a pinacol-like rearrangement aris-
ing from the reaction of 2,20-pyridyl 2 and benzamidine/
HCl 3 (Scheme 1).3
7
8
434 57 (2)
170 6 (2)
0.66 0.04 (2)
304 40 (2)
108 16 (2)
2.9 0.2 (2)
0.59 0.04 (2)
262 2.7 (2)
>1000 (1)
OMe
CN
9
CN
Despite possessing improved aqueous solubility (10 mg/
mL in 0.1 N HCl) imidazolone 4 was completely devoid
10
11
12
13
14
15
16
17
18
19
of any significant NPY5 binding affinity (IC50
>
1000 nM). Additional analogs directed at removing either
one or both of the phenyl groups at the 5-position of the
imidazolone core also exhibited poor or greatly reduced
affinity at the hNPY5 receptor (data not shown).
F
CF3
Cl
Due to lack of tolerance for modification at the 5-posi-
tion, synthetic efforts quickly shifted to modifying sub-
stituents at the 2-position on the imidazolone core. We
developed an efficient two-step protocol starting from
aminocarboxamide 54 to construct a variety of diversely
substituted analogs 6–19 (Scheme 2). Treatment of
amine 5 with a polymer-supported carbodiimide re-
agent5 in the presence of excess carboxylic acid afforded
an intermediate a-amidoamide. These amides were iso-
lated by simple filtration to remove the polymer-sup-
ported reagent as well as any unreacted carboxylic
acid. Yields of the corresponding amides varied, but
usually were low (10–50%) due to the hindered nature
of the starting amine. The isolated a-amidoamides were
cyclized to the desired imidazolones in ethanol with
excess 1 N sodium hydroxide. All analogs were purified
by reverse-phase chromatography and evaluated for
their binding affinity against the human NPY5 receptor
(Table 1).
NH2
>1000 (1)
102 5.0 (2)
868 9.5 (2)
>1000 (1)
O
Cl
Cl
O
NH
NH3+Cl-
O
N
N
a
NH
a IC50 values are means SEM, number of results in parentheses.
N
+
N
O
N
Initial structure–activity studies around the 2-position
on the imidazolone core revealed a strong preference
for small, electron-withdrawing, meta-substituted phen-
yl rings. For example, both analogs 9 and 13 displayed
sub-nanomolar binding affinity to hNPY5, while 10
and 14 demonstrated considerably reduced affinity. Both
ortho and para substitution or combinations thereof
greatly diminished binding at the hNPY5 receptor.
Additionally, alkyl substitution either straight chain,
branched, or cyclic at the 2-position eliminated any sig-
nificant binding affinity for the hNPY5 receptor.
2
3
4
Scheme 1. 2-Phenyl-5,5-di-pyridin-2-yl-3,5-dihydro-imidazol-4-one synthe-
sis. Reagents and conditions: (a) EtOH, NaOH, reflux, 2 h (4, 90%).
O
O
O
a,b
NH
NH2
R1
+
HO
NH2
N
R1
5
6-19
We next turned our attention to aniline 14, which served
as a linchpin for further SAR studies. A variety of
mono-benzyl and alkyl substituted analogs generated
Scheme 2. Diphenylimidazolone synthesis. Reagents and conditions:
(a) P-EDC resin, CH2Cl2, rt, 24 h; (b) NaOH, EtOH, rt, 2 h.

K. W. Gillman et al. / Bioorg. Med. Chem. 14 (2006) 5517–5526
5519
Table 2. Human NPY5 binding affinity for select meta-substituted
aniline derivatives
from a reductive amination reaction with the corre-
sponding aldehyde (Scheme 3) were prepared. In our
hands we found that with aniline 14, the procedure de-
scribed by Abdel-Magid et al.6 gave the highest product
yields when using alkyl, electron-rich, or electron-defi-
cient substituted aryl aldehydes. From the substituted
aniline analogs synthesized and evaluated for binding
affinity, we found that in all cases, alkyl substitution
on the aniline nitrogen greatly decreased affinity for
the hNPY5 receptor. Interestingly, many of the benzyl
substituted analogs displayed moderate affinity, showing
a preference for extended substitution at the para posi-
tion on the nascent phenyl ring. Of all the aniline ana-
logs we synthesized where substitution on the nitrogen
varied, we did not see any increase in affinity for the
hNPY5 receptor over the starting unsubstituted aniline
14 (see Table 2).
a
hNPY5 IC50 (nM)
Compound
R2
Yield (%)
30
20
>1000 (1)
21
22
23
24
25
26
27
299 45 (2)
>1000 (1)
70
19
44
50
12
47
46
O
O
248 1.5 (2)
399 135 (2)
134 12 (2)
735 2.8 (2)
293 63 (2)
O
Structure–activity data generated from these ‘early’ ana-
logs indicated that there was tolerability for expansion
out from the 3-position of the phenyl ring, since sterical-
ly bulky aryl groups did not completely abolish binding
affinity for the hNPY5 receptor. With this in mind we
chose nitrile 9 as a versatile synthetic precursor for fur-
ther structural elaboration at the 3-phenyl position. Ni-
trile 9 was initially hydrolyzed by hydrogen peroxide/
sodium hydroxide7 affording primary carboxamide 28
(hNPY5, IC50 = 13 nM) (see Scheme 4). Reduction of
9 with Raney nickel in methanolic ammonia8 furnished
amine 29. Compound 29 provided us an additional
branching point in which to expand our structure–activ-
ity studies. Initial attempts to selectively mono-alkylate
amine 29 were unsuccessful using stoichiometric methyl
iodide and potassium carbonate in acetonitrile. Unfor-
tunately, we obtained the mono-methyl (32, Table 3)
and di-methyl derivatives (37, Table 4) in equal amounts
from the reaction mixture. The lack of product selectiv-
ity in the conversion of 29 to 32 precluded us from effi-
ciently synthesizing large numbers of analogs via this
procedure. To circumvent this problem, an alternative
synthetic strategy was developed to prepare both mono-
and di-alkylated 3-methylamino analogs independently.
This was accomplished through the use of meta-benzyl-
chloride intermediate 31. In the case where secondary
3-methylamino derivatives 32–36 were desired, we added
excess primary amine to the starting benzyl chloride 31
in the absence of base. Heating the mixture overnight
in acetonitrile at 60 ꢁC afforded the desired secondary
benzylamine as the major product. These conditions sig-
nificantly reduced the amount of dimerized product that
formed in the presence of the reacting amine with potas-
sium carbonate. Tertiary 3-methylamino analogs 37–43
O
F
a IC50 values are means SEM, number of results in parentheses.
were synthesized starting with an excess amount of the
desired secondary amine in the presence of potassium
carbonate. As before, heating the reaction mixture over-
night at 60 ꢁC in acetonitrile was necessary for the reac-
tion to proceed to completion (Scheme 4).
Structure–activity data generated from the secondary
3-methylamino series mirrored that found in the meta-
substituted reductive amination studies (see Table 3).
There was a clear preference for para-substituted aryl
rings over straight chain and branched alkyl substituents
for the hNPY5 receptor. In the case of the tertiary
3-methylamino series, cyclic substituted amines showed
enhanced binding affinity, producing several analogs
that exhibited binding affinities close to the lead com-
pound 1 (see Table 4). Although compounds 36 and
42 possessed potent binding affinity for the hNPY5
receptor, they offered no significant improvement in
physical properties when compared to imidazolone 1.
Parallel to SAR studies on maximizing the binding
affinity in our ‘meta’ 2-phenylimidazolone series, we
O
O
O
a
R2
NH
NH
H
N
N
H
N
NH2
R2
14
20-27
Scheme 3. 3-Substituted aniline synthesis. Reagents and conditions: (a) aldehyde, NaBH(OAc)3, C2H4Cl2, AcOH, rt, 96 h.

5520
K. W. Gillman et al. / Bioorg. Med. Chem. 14 (2006) 5517–5526
O
NH
N
CN
9
a
b
O
O
NH
NH
O
N
N
NH2
NH2
28
29
O
O
O
HO
c,d
NH
NH2
+
N
NH2
Cl
Cl
31
30
5
e
f
O
N
O
N
NH
NH
NHR3
R4
37-43
32-36
Scheme 4. Expanded 3-substituted analog synthesis. Reagents and conditions: (a) NaOH, 30% H2O2, EtOH, reflux, 3 h, 63%; (b) Raney Ni, H2,
NH4OH/MeOH, rt, 24 h, 64%; (c) EDC, HOBt, DMF, rt, 24 h, 30%; (d) NaOH, EtOH, rt, 1 h, 84%; (e) amine, CH3CN, 60 ꢁC, 24 h; (f) amine, excess
K2CO3, CH3CN, 60 ꢁC, 24 h.
Table 3. Human NPY5 binding affinity for secondary 3-substituted
aryl- and akyl-methylamino derivatives
Table 4. Human NPY5 binding affinity for tertiary 3-substituted
methylamino derivatives
R3
a
hNPY5 IC50 (nM)
Compound
R4
a
hNPY5 IC50 (nM)
Compound
29
32
H
Me
48 3.6 (2)
>1000 (1)
37
>1000 (1)
N
N
33
34
35
36
>1000 (1)
38
39
40
41
42
43
395 70 (2)
326 112 (2)
35 2.6 (2)
15.4 6.3 (2)
8.6 1 (2)
Me
F
31 3.5 (2)
62 11 (2)
55 0.4 (2)
N
N
N
NO2
N
a IC50 values are means SEM, number of results in parentheses.
N
N
N
N
N
examined replacing the phenyl ring at the 2-position on
the imidazolone ring with a variety of aromatic hetero-
cycles. Through the incorporation of heteroatoms, spe-
cifically nitrogen on the aryl ring in the 2-position, it
was postulated that we could lower the clogP values
70 6.2 (2)
NO2
a IC50 values are means SEM, number of results in parentheses.

K. W. Gillman et al. / Bioorg. Med. Chem. 14 (2006) 5517–5526
5521
Table 5. Human NPY5 and NPY1 binding affinity for select
2-heterocyclic-5,5-diphenylimidazolone derivatives
in the imidazolone series, and thereby increase the aque-
ous solubility of these analogs. The synthesis of hetero-
cyclic analogs 44–50 was accomplished using either the
corresponding acid or acyl chloride based on commer-
cial availability. We found that on average the overall
yields from amine 5 to the desired imidazolone were bet-
ter using an acyl chloride to form the intermediate a-
amidoamide (Scheme 5). In the synthesis of imidazolone
47 (see Table 5) the starting pyrimidine-5-carboxylic
acid was not commercially available, and therefore
was prepared following the procedure described by
Kress.9 In the case of the heterocyclic imidazolone ser-
ies, the yield of the initial amide bond formation dictat-
ed the overall imidazolone yield, since the base-induced
cyclization step proceeded cleanly, and on average in
>80% yield for all analogs.
Compound Het
Yield (%) hNPY5
hNPY1
IC50 (nM)
c
IC50 (nM)
44
65a
45a
48a
8b
50 (1)
>1000 (1)
>1000 (1)
N
N
45
46
47
48
49
50
45 (1)
N
N
N
8.3 0.7 (6) >1000 (1)
76 28 (2)
>1000 (1)
N
20b
19b
5b
9.4 0.3 (2) >1000 (1)
Analogs synthesized in the 2-heterocyclic imidazolone
series were screened for binding affinity at both the
hNPY5 and hNPY1 receptor. Fortunately, a number
of analogs in the 2-heterocyclic imidazolone series dis-
played excellent affinity for the hNPY5 receptor while
maintaining exquisite selectivity over the hNPY1 recep-
tor (see Table 5). Pyridines 44 and 45, although modest-
ly active, were eclipsed by 46, their 3-substituted
homolog, further supporting the preference for meta
substitution in the 2-aryl imidazolone series. Interesting-
ly, pyrimidine 47 possessed significantly less binding
affinity for the hNPY5 receptor compared to pyridine
46. This suggests that there is an additional preference
for specific electrostatic interactions in the heteroaryl
ring binding pocket on the hNPY5 receptor. Similar to
pyrimidine 47, both thiophene 50 and furan analog 49
demonstrated only moderate affinity at the hNPY5
receptor.
N
d
109 0.8 (2)
O
d
171 19 (2)
S
a Synthesized starting with the corresponding acyl chloride.
b Synthesized starting with the corresponding carboxylic acid.
c IC50 values are means SEM, number of results in parentheses.
d hNPY1 binding data were not measured.
compound 1 at the hNPY5 receptor while devoid of
any hNPY1 binding affinity. Both equilibrium binding
by Scatchard analysis and functional activity based on
cAMP accumulation of 46 indicated that the compound
was a competitive antagonist against the hNPY5 recep-
tor with an app. Kb = 7.1 nM, which is in good agree-
ment with its binding affinity. Additionally, compound
46 displaced 125I-PYY in rat brain homogenates with a
measured IC50 = 6.5 3.5 nM.
Pyridine 46 was selected for further pharmacologic char-
acterization and found to be equipotent compared to
O
NH2
NH2
The measured aqueous solubility of pyridine 46 was
33 lg/mL (pH 2.0) an approximate 10-fold improvement
over starting imidazolone 1. Encouraged by this increase
in aqueous solubility, 46 was dosed in rats by both intra-
peritoneal (ip) and oral (po) administration. The abso-
lute brain levels as well as bioavailability were
measured. The pharmacokinetic data compiled on imi-
dazolone 46 demonstrated that this compound was far
superior to our lead imidazolone 1 in many respects.
The measured bioavailability of 46 dosed in rats by both
po and ip routes of administration was >90% (data not
shown). Imidazolone 46 demonstrated a relatively high
level of free drug concentration when measured in rat
serum (18.9%). The brain/plasma ratio at 120 min was
1.2 (see Table 6) with peak absolute brain levels of 46
achieving a concentration ꢀ500-fold over the experi-
mental Kb value of 7.1 nM out to 6 h. Oral dosing
[(25 mg/kg) 10:10:80 Cremophor ELTM/ethanol/water]
of 46 demonstrated that the compound was rapidly ab-
sorbed, achieving peak plasma levels of 10 lM at
70 min. The measured half-life (1.1 h) indicated the com-
O
O
b
5
Cl
Het
HO
Het
a
O
O
NH2
NH2
O
O
HN
HN
Het
Het
c
c
O
NH
N
Het
44-50
Scheme 5. 2-Heterocyclic-5,5-diphenylimidazolone analog synthesis.
Reagents and conditions: (a) Et3N, 4-DMAP, CH2Cl2, rt, 24 h; (b)
EDC, HOBt, CH2Cl2, rt, 24 h; (c) NaOH, EtOH, rt, 1 h.

5522
K. W. Gillman et al. / Bioorg. Med. Chem. 14 (2006) 5517–5526
Table 6. Absolute brain and plasma levels for 46 dosed ip (15 mg/kg)a in Sprague–Dawley rats
Time (min)
Plasma concnb (nM/L)
Whole brain concna (nM/L)
Free plasma concnc (nM/L)
Brain/plasma ratio
10
120
360
13,617
6585
2661
16,022
7396
4006
2574
1244
503
1.2
1.2
1.4
a Dosing vehicle, 2.0 mg/mL, 10:10:80 Cremophor ELTM/ethanol/water.
b Absolute concentration values are means of two measurements.
c Plasma-free fraction values calculated based on a serum-free fraction of 18.9%.
18.00
16.00
14.00
12.00
10.00
8.00
pound exhibited a modest rate of clearance (39 mL/min/
kg). When dosed either 25 mg/kg po or 15 mg/kg ip,
absolute brain levels were maintained at concentrations
>10-fold over the measured Kb as well as the protein
adjusted IC50 value (prot. adj. IC50 = 44 nM) for extend-
ed periods. Given the favorable pharmacokinetic data
for imidazolone 46, the compound was evaluated in
in vivo feeding studies in several accepted rodent models
of obesity (see Table 7).
Veh
BMS 60
BMS 100
6.00
4.00
2.00
0.00
We initially evaluated the effect of 46 on reversing
NPY-induced feeding in Sprague–Dawley rats via
Treatment
two routes of administration. Food intake was in-
creased by administration of NPY1ꢁ36 (1 nmol) into
the cerebral ventricle of rats.10 Compound 46 was
either administered into the paraventricular nucleus
of the hypothalamus (30 nmol, 46), or orally (60 mg/
kg 46). In both cases there was no statistically signif-
icant reduction in food intake between the 46 treated
and vehicle control animals as compared to the NPY
treated groups.
Figure 1. Total nocturnal food intake following oral dosing (60 and
100 mg/kg) of compound 46 in rats (n = 12 animals/group). Vehicle:
20% DMSO/40% PEG400/40% TrappsolTM
.
groups was observed. The results of the study, depicted
below (Fig. 2) are representative of all doses evaluated
(0.001–15.0 mg/kg ip) in this model.
15
We additionally looked at the effect of 46 when dosed at
either 60 or 100 mg/kg po on spontaneous overnight
food intake in a 24 h light/dark free-feeding model with
Sprague–Dawley rats (see Fig. 1). As in the NPY-
induced feeding study, there was no significant reduction
in total food intake in the treated animals when com-
pared to the vehicle dosed control group in either the
60 or 100 mg/kg dosed animals.
Vehicle
5mg/kg
15mg/kg
10
5
Last, we looked extensively at the effect of 46 using a
broad range of ip doses (0.001–15 mg/kg) in a time
dependent, diet-induced model of obesity in Sprague–
Dawley rats. This study has the potential advantage of
depicting any time dependent differences of food intake
that arise during a normal 12 h light/dark feeding
regimen.
0
0
2
3
5
6
8
9
11 12 14 15 17 18 20 21 23 24
Time (h)
Figure 2. Cumulative food consumption in diet-induced obese rats
following compound 46 (5 and 15 mg/kg, ip) injections before onset of
dark cycle (n = 11–12 animals/group). Vehicle: 20% NMP/30%
PEG400/50% (5% Tween 80TM) in water.
Similar to previous feeding studies, no statistical differ-
ence between the compound 46 and vehicle dosed
Table 7. In Vivo feeding studies with imidazolone 46
Animal model
Route of administration
Dose
Significant effect
NPY-induced food intake in SDa rats
NPY-induced food intake in SDa rats
Spontaneous overnight food intake in SDa rats
Diet-induced obese model in SDa rats
NPY (1 nmol) icv, 46 po
NPY (1 nmol) icv, 46 PVNb
60 mg/kg
30 nmol
None
None
None
None
46 po
46 ip
60 and 100 mg/kg
5 and 15 mg/kg
a Sprague–Dawley.
b Paraventricular nucleus.

K. W. Gillman et al. / Bioorg. Med. Chem. 14 (2006) 5517–5526
5523
4. Conclusion
5.1.2. General procedure for the preparation of imidazol-
ones (7–19). a-Amino-a,a-diphenylacetamide 5 (0.050 g,
0.22 mmol) was added to a solution of the correspond-
ing carboxylic acid (0.44 mmol), 0.690 g of P-EDC5 res-
in (1.4 meq/g, 0.88 mmol) in 5 mL of dry CH2Cl2. The
reaction mixture was shaken for 36 h at rt, then the
crude reaction mixture was filtered and the filter cake
was washed with excess CH2Cl2. The resulting filtrate
was evaporated in vacuo to yield a crude solid. This sol-
id was dissolved in 3 mL EtOH and 0.5 mL of 1 N
NaOH (aq). The resulting solution was stirred for 16 h
then neutralized with 1 N HCl (aq). The solvent was
evaporated in vacuo and the crude solid was purified
by reverse-phase chromatography YMC Inc.,
Imidazolone 46 is a potent and selective hNPY5 recep-
tor antagonist with excellent pharmacokinetic proper-
ties. The compound exhibits high oral bioavailability
(>90%) and brain penetration, achieving sufficient brain
concentrations compared to the protein adjusted
IC50 = 44 nM of the compound (IC50/plasma-free frac-
tion) after both ip and po administration. In spite of
its pharmacokinetic profile, 46 demonstrated no effect
on appetite control in a variety of animal models,
including the diet-induced animal model that is believed
to mimic human obesity. Multiple pharmacological,
molecular, and physiological studies1 suggest the
involvement of the NPY5 receptor in the reduction of
food intake and body weight. Imidazolone 46 possesses
many favorable pharmacokinetic properties including a
high plasma-free fraction, good brain penetration, as
well as in vivo half-life. Dosed both orally and ip, 46
achieved free drug concentrations in plasma along with
sufficient whole brain concentrations to cover the pro-
tein adjusted IC50 for the NPY5 receptor. Despite this,
in our hands imidazolone 46 was ineffective in reducing
appetite, food intake or body weight in a variety of
accepted feeding models. Based on these data we suggest
that the NPY5 receptor in and of itself is not solely
responsible for the modulation of food intake in rats.
Further studies may be necessary to conclude unequivo-
cally the role of selective NPY5 antagonists and their
ability to modulate food intake.
˚
20 · 100 mm, 5 lm particle size, 120 A pore size C18 sta-
tionary phase ODS-A fast elution: 50–100% B A = (10%
MeOH/90% H2O–0.1% TFA), B = (90% MeOH/10%
H2O–0.1% TFA) providing the desired pure imidazol-
ones 7–19 in yields ranging from 2% to 42% (hNPY5
binding data-summarized in Table 1).
5.1.3. 2-(3-Aminophenyl)-5,5-diphenyl-3,5-dihydro-imi-
dazol-4-one (14). To a 250-mL flask were added a-ami-
no-a,a-diphenylacetamide 5 (1.0 g, 4.42 mmol) and
3-nitrobenzoic acid (1.11 g, 6.64 mmol). The solids were
dissolved in 40 mL of dry CH2Cl2 and the reaction mix-
ture was stirred until homogeneous. After cooling to
0 ꢁC, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide
hydrochloride (EDC) (1.74 g, 7.07 mmol) was added in
one portion and the reaction was allowed to warm slow-
ly to rt overnight. The organic layer was washed with
0.5 N HCl, dried (Na2SO4), and evaporated in vacuo
to yield a crude solid. Treatment of the solid with
4.0 mL of 1 N NaOH in 20 mL EtOH for 16 h followed
by neutralization with 1 N HCl and evaporation of the
solvent produced a crude yellow oil. The oil was
chromatographed with silica gel (3:1 hexane/acetone)
5. Experimental
5.1. Chemistry
Melting points were determined using a Thomas–Hoo-
ver capillary melting apparatus and are uncorrected.
Elemental analyses were performed at Robertson
Microlabs, Madison, NJ, USA, and are within 0.4% of
affording
imidazol-4-one (0.744 g, 69%) as a light yellow solid:
2-(3-nitrophenyl)-5,5-diphenyl-3,5-dihydro-
1
LRMS m/z (ESI) 358.3 (M+H)+; H NMR (DMSO-
1
theoretical C, H, and N values. H NMR spectra were
recorded in CDCl3 (unless otherwise noted) with the sol-
vent resonance of CDCl3 (7.27 ppm) as the internal stan-
d6, 300 MHz) d = 12.17 (br s, 1H), 8.93 (s, 1H), 8.52
(d, 1H, J = 6.0 Hz), 8.47 (d, 1H, J = 6.0 Hz), 7.88 (t,
1H, J = 6.0 Hz), 7.48 (m, 4H), 7.35 (m, 6H); Anal. Calcd
for C21H15N3O3: C, 70.58; H, 4.23; N, 11.76. Found: C,
70.29; H, 4.39; N, 11.49.
dard on
a
Bruker AC 300 MHz spectrometer.
Electrospray mass spectra (MS) were recorded on a
Finnigan SSQ-7000 mass spectrometer. Reagents and
solvents were used as obtained from commercial suppli-
ers without further purification. Column chromatogra-
phy was performed using silica gel and the flash
technique. Representative procedures and physical
properties for selected compounds are described.
To a 100-mL flask were added 2-(3-nitrophenyl)-5,5-di-
phenyl-3,5-dihydro-imidazol-4-one
(0.614 g,
1.72
mmol) and 0.092 g of platinum(IV) oxide in 20 mL of
a 8:1 EtOH/THF solution. The reaction vessel was
charged with 5 psi hydrogen and stirred overnight.
Upon completion the reaction was filtered through Cel-
ite and the solvent was evaporated in vacuo. The crude
solid was chromatographed with silica gel (4:1 hexane/
acetone) affording (0.480 g, 85%) of the desired amine
14 as a white solid: LRMS m/z (ESI) 328.3 (M+H)+;
1H NMR (CDCl3, 300 MHz) d = 7.60 (d, 4H,
J = 6.0 Hz), 7.41 (s, 1H), 7.35 (m, 6H), 7.19 (m, 1H),
6.85 (m, 1H), 6.41 (d, 1H, J = 6.0 Hz).
5.1.1. 2,5,5-Triphenyl-3,5-dihydro-imidazol-4-one (1).
Compound 1 was prepared by standard procedure as
referenced in the literature.2 All spectroscopic data were
consistent with that as indicated by structure 1: white
solid (mp 238–239 ꢁC); 1H NMR (CDCl3, 300 MHz)
d = 7.98 (d, 2H, J = 6.0 Hz), 7.51 (d, 4H, J = 6.0 Hz),
7.41 (m, 3H), 7.26 (m, 6H); 13C NMR (CDCl3,
75 MHz) d = 188.36, 140.34, 132.54, 129.21, 128.75,
128.31, 128.13, 127.61, 127.56; LRMS m/z (ESI) 311
(MꢁH)ꢁ; Anal. Calcd for C21H16N2O: C, 80.75; H,
5.16; N, 8.97. Found: C, 80.47; H, 5.06; N, 8.94.
5.1.4. General procedure for the preparation of imidazol-
ones (20–27). Aniline 14 (0.025 g, 0.076 mmol) was
added to a solution of the corresponding aldehyde

5524
K. W. Gillman et al. / Bioorg. Med. Chem. 14 (2006) 5517–5526
(0.11 mmol), acetic acid (0.10 mL, 0.11 mmol), and sodi-
um triacetoxyborohydride (0.097 g, 0.46 mmol) in 5 mL
of dry CH2Cl2. The reaction mixture was stirred for 96 h
at rt, then the crude reaction mixture was loaded onto
an SCX cartridge pretreated with CH2Cl2. The cartridge
was washed with 5 mL CH2Cl2 followed by 5 mL
CH3OH. The product was eluted with 2% NH4OH in
CH3OH. The filtrate was evaporated in vacuo and the
crude solid was purified by reverse phase chromatogra-
in vacuo to yield a crude solid. The solid was purified
by chromatography with silica gel (4:1 hexane/acetone)
affording (2.5 g, 30%) of the desired amide intermedi-
ate as a white solid. Treatment of the purified amide
with 10.0 mL of 1 N NaOH in 50 mL EtOH for 1 h
followed by neutralization with 1 N HCl and evapora-
tion of the solvent produced a crude solid which was
chromatographed with silica gel (4:1 hexane/acetone)
affording (2.0 g, 84%) of the desired imidazolone 31
as a white solid: LRMS m/z (ESI) 361.2 (M+H)+;
1H NMR (CDCl3, 300 MHz) d = 8.10 (s, 1H), 7.91
(d, 1H, J = 6.0 Hz), 7.65–7.51 (m, 6H), 7.40–7.30 (m,
6H), 4.65 (s, 2H).
˚
phy YMC Inc., 20 · 100 mm, 5 lm particle size, 120 A
pore size C18 stationary phase ODS-A fast elution: 50–
100%
B A = (10% MeOH/90% H2O–0.1% TFA)
B = (90% MeOH/10% H2O–0.1% TFA) providing the
desired pure imidazolones 20–27 in yields ranging from
12% to 70% (hNPY5 binding data are summarized in
Table 2).
5.1.8. 2-[3-((Methylamino)methyl)-phenyl]-5,5-diphenyl-
3,5-dihydro-imidazol-4-one (32). To a glass bomb were
added benzylchloride 31 (0.107 g, 0.297 mmol) and ex-
cess anhydrous methylamine (4.0 mL of a 2.0 M solu-
tion in THF). The reaction vessel was sealed and
stirred at rt overnight. The solvent was evaporated
in vacuo producing an off-white solid. The crude solid
was purified by reverse-phase chromatography YMC
5.1.5. 3-(5-Oxo-4,4-diphenyl-4,5-dihydro-1H-imidazol-2-
yl)-benzamide (28). To a 50-mL flask were added imi-
dazolone 9 (0.30 g, 0.889 mmol) and 6 mL of 95%
EtOH. The solution was stirred until homogeneous
and then 1 mL of 6 N NaOH and 1 mL of 30% hydro-
gen peroxide were added and the solution was heated
to reflux for 3 h. The reaction was cooled to rt and neu-
tralized with concd HCl. The solvent was evaporated in
vacuo, and the crude residue was chromatographed with
silica gel (4:1 hexane/acetone) producing (0.20 g, 63%) of
the desired amide 28 as a white solid: LRMS m/z (ESI)
356.2 (M+H)+; 1H NMR (DMSO-d6, 300 MHz)
d = 8.74 (s, 1H), 8.32 (d, 1H, J = 6.0 Hz), 8.10 (d, 1H,
J = 6.0 Hz), 7.66 (t, 1H, J = 6.0 Hz), 7.48 (d, 4H,
J = 6.0 Hz), 7.33 (m, 6H).
˚
Inc., 20 · 100 mm, 5 lm particle size, 120 A pore size
C18 stationary phase ODS-A fast elution: 50–100% B
A = (10% MeOH/90% H2O–0.1% TFA) B = (90%
MeOH/10% H2O–0.1% TFA) producing (0.070 g,
67%) of the desired amine 32 as a white solid: LRMS
m/z (ESI) 356.2 (M+H)+; 1H NMR (MeOH-d4,
300 MHz) d = 8.26 (s, 1H), 8.10 (d, 1H, J = 6.0 Hz),
7.79 (d, 1H, J = 6.0 Hz), 7.71 (t, 1H, J = 6.0 Hz),
7.49 (d, 4H, J = 6.0 Hz), 7.32 (m, 6H), 4.31 (s, 2H),
2.77 (s, 3H).
5.1.6. 2-[3-(Aminomethyl)-phenyl]-5,5-diphenyl-3,5-dihy-
dro-imidazol-4-one (29). To a Parr hydrogenator were
added 9 (0.100 g, 0.297 mmol) and 0.010 g of freshly
washed Raney nickel in 6 mL of a 5:1 MeOH/NH4OH
(concd) solution. The reaction vessel was charged with
50 psi hydrogen and shaken overnight. The reaction
was filtered through Celite and the solvent was evapo-
rated in vacuo. The crude solid was chromatographed
with silica gel (2:1 hexane/acetone) affording (0.065 g,
64%) of the desired amine 29 as an off-white solid:
LRMS m/z (ESI) 342.3 (M+H)+; 1H NMR (CDCl3,
300 MHz) d = 8.02 (s, 1H), 7.90 (m, 2H), 7.59–7.56 (m,
4H), 7.47–7.40 (m, 2H), 7.35–7.24 (m, 5H), 3.93 (s,
2H), 2.18 (s, 2H).
5.1.9. General procedure for the synthesis of secondary
amino analogs 33–36. Benzylchloride 31 (0.015 g,
0.042 mmol) was added to a solution of the correspond-
ing primary amine (0.083 mmol), in 2 mL of dry
CH3CN. The reaction vessel was sealed and gently heat-
ed to 60 ꢁC with stirring for 16 h. The reaction mixture
was reduced in vacuo and the crude oil was purified
by reverse-phase chromatography (YMC Inc.,
˚
20 · 100 mm, 5 lm particle size, 120 A pore size C18 sta-
tionary phase ODS-A fast elution: 50–100% (10%
MeOH/90% H2O–0.1% TFA): (90% MeOH/10% H2O–
0.1% TFA)) providing the desired pure secondary-amin-
oimidazolones 33–36 in yields ranging from 6% to 13%
(hNPY5 binding data are summarized in Table 3).
5.1.7. 2-[3-(Chloromethyl)-phenyl]-5,5-diphenyl-3,5-dihy-
dro-imidazol-4-one (31). To a 500 mL flask were added
a-amino-a,a-diphenylacetamide 5 (5.0 g, 22.1 mmol)
5.1.10. General procedure for the synthesis of tertiary-
amino analogs 37–43. Benzylchloride 31 (0.025 g,
0.069 mmol) was added to a solution of the correspond-
ing primary amine (0.138 mmol) and K2CO3 (0.038 g,
0.277 mmol), in 2 mL of dry CH3CN. The reaction ves-
sel was sealed and gently heated to 60 ꢁC with stirring
for 16 h. The reaction mixture was reduced in vacuo
and the crude oil was purified by reverse-phase chroma-
tography YMC Inc., 20 · 100 mm, 5 lm particle size,
and
3-(chloromethyl)benzoic
acid
30
(4.15 g,
24.3 mmol). The solids were dissolved in 100 mL of
dry DMF and the reaction mixture was stirred until
homogeneous and then cooled to 0 ꢁC. 1-(3-Dimeth-
ylaminopropyl)-3-ethylcarbodiimide
hydrochloride
(EDC) (4.65, 24.3 mmol) and 1-hydroxybenzotriazole
hydrate (HOBt) (3.28 g, 24.3 mmol) were added in
one portion and the reaction was allowed to warm
slowly to rt and stirred overnight. After concentrating
in vacuo, the crude residue was dissolved in 250 mL
CH2Cl2 and washed with 0.5 N HCl. The organic
layer was separated, dried (Na2SO4), and evaporated
˚
120 A pore size C18 stationary phase ODS-A fast elu-
tion: 50–100% B A = (10% MeOH/90% H2O–0.1%
TFA) B = (90% MeOH/10% H2O–0.1% TFA) providing
the desired pure tertiary-aminoimidazolones 37–43 in
yields ranging from 6% to 100% (hNPY5 binding data
are summarized in Table 4).

K. W. Gillman et al. / Bioorg. Med. Chem. 14 (2006) 5517–5526
5525
5.1.11. General procedure for the synthesis of heterocyclic
imidazolones 44 and 45 (procedure A). a-Amino-a,a-
diphenylacetamide 5 (0.30 g, 1.24 mmol) was added to
a solution of triethylamine (0.503 g, 4.98 mmol), 4-di-
methylaminopyridine (4-DMAP) (0.015 g, 0.124 mmol),
and 15 mL dry CH2Cl2. The solution was cooled to 0 ꢁC
and then the corresponding acid chloride (2.48 mmol)
was added in one portion. The reaction mixture was stir-
red at 0 ꢁC for 1 h then warmed to rt and stirred for an
additional 16 h. The reaction was then quenched with
5% NaHCO3 (aq) and the aqueous layer was extracted
with CH2Cl2. The organic fractions were combined,
dried with anhydrous Na2SO4, and evaporated in vacuo
to yield a red oil. This oil was chromatographed with sil-
ica gel (4:1 hexane/acetone) affording the desired inter-
mediate amide. The resulting amide was dissolved in
20 mL EtOH and 1 N NaOH (aq) (3.0 mL, 3.0 mmol)
was added. The reaction mixture was stirred for 3 h at
rt, then the reaction was neutralized with 1 N HCl
(aq). The reaction solvent was evaporated in vacuo
and the crude solid was dissolved in CH2Cl2 and washed
with H2O. The organic phase was dried with anhydrous
Na2SO4 and the solvent was evaporated in vacuo. The
resulting solid was chromatographed with silica gel
(6:1 hexane/acetone) affording the desired imidazolones
44 and 45 in yields ranging from 46% to 65% (hNPY5
binding data are summarized in Table 5).
CO3 (aq) and the aqueous layer was extracted with
CH2Cl2. The organic fractions were combined, dried
with anhydrous Na2SO4, and evaporated in vacuo to
yield a red oil. This oil was chromatographed with sil-
ica gel (4:1 hexane/acetone) affording the desired inter-
mediate amide as a white solid (1.2 g, 58%). The
resulting amide (1.2 g, 3.63 mmol) was dissolved in
30 mL EtOH and 1 N NaOH (aq) (4.0 mL, 4.0 mmol)
was added. The reaction mixture was stirred for 2 h
at rt and then neutralized with 1 N HCl (aq). The reac-
tion solvent was evaporated in vacuo and the crude
solid was dissolved in CH2Cl2 and subsequently
washed with H2O. The organic phase was dried with
anhydrous Na2SO4 and the solvent was evaporated in
vacuo. The resulting solid was chromatographed with
silica gel (6:1 hexane/acetone) producing pure imidazo-
lone 46 as a white solid (0.940 g, 83%): mp 205–206 ꢁC;
1H NMR (CDCl3, 300 MHz) d = 9.40 (br s, 1H), 8.83
(d, 1H, J = 3 Hz), 8.59 (d, 1H, J = 9.0 Hz), 7.56 (d,
4H, J = 6.0 Hz), 7.32 (m, 8H); 13C NMR (CDCl3,
75 MHz) d = 193.19, 186.16, 152.74, 148.31, 139.91,
135.04, 128.67, 128.09, 127.33, 124.77, 123.97; LRMS
m/z (ESI) 314 (M+H)+; Anal. Calcd for C20H15N3O:
C, 76.66; H, 4.82; N, 13.41. Found: C, 76.51; H,
4.83; N, 13.36.
6. Biological methods
6.1. Membrane harvest
5.1.12. General procedure for the synthesis of heterocy-
clic imidazolones 48–50 (procedure B). To a 50 mL flask
were added a-amino-a,a-diphenylacetamide 5 (0.33 g,
1.46 mmol) and the corresponding carboxylic acid
(1.61 mmol). The solids were dissolved in 10 mL dry
CH2Cl2 and the reaction mixture was stirred until
homogeneous, then cooled to 0 ꢁC. 1-(3-Dimethylami-
nopropyl)-3-ethylcarbodiimide hydrochloride (0.31 g,
1.61 mmol) and 1-hydroxybenzotriazole hydrate
(0.22 g, 1.61 mmol) were then added in one portion.
The reaction was allowed to warm slowly to rt and
stirred overnight. The reaction mixture was poured
into a separatory funnel, diluted with 100 mL CH2Cl2,
and washed with 0.5 N HCl. The organic layer was
separated, dried (Na2SO4), and evaporated in vacuo
to yield a crude solid which was chromatographed with
silica gel (4:1 hexane/acetone) affording the desired
amide intermediate. Treatment of the purified amide
with 2.0 mL of 1 N NaOH in 10 mL EtOH for 4 h
was followed by neutralization with 1 N HCl. Evapora-
tion of the solvent produced a crude solid which was
chromatographed with silica gel (4:1 hexane/acetone)
affording the desired imidazolones 48–50 in yields rang-
ing from 5% to 20% (hNPY5 binding data are summa-
rized in Table 5).
Membranes were harvested from either Hi5 or CHO
cells transfected with the human NPY5 receptor. Hi5
cells were pelleted and washed with phosphate-buffered
saline (138 mM NaCl, 8.1 mM Na2HPO4, and 1.2 mM
KH2PO4, pH 7.4). Adherent cells were washed twice
with ice-cold (4 ꢁC) phosphate-buffered saline. Cells
were scraped in ice-cold hypotonic buffer (20 mM Tris
and 5 mM EDTA, pH 7.7), lysed in a Dounce homog-
enizer, and membranes were pelleted by centrifugation
(18,000 rpm, SS-34 rotor, 15 min, 4 ꢁC). Both pellets
were resuspended, homogenized in ice-cold hypotonic
buffer, and again centrifuged. The final membrane pel-
let was resuspended through a 27 G needle into a
small volume of ice-cold buffer (10 mM NaCl,
20 mM Hepes, 0.22 mM KH2PO4, 1.26 mM CaCl2,
and 0.81 mM MgSO4, pH 7.4), approximately 100 lL
per T175 flask (5–8 mg/mL protein concentration).
Protein concentration was measured by the Bradford
method (Bradford, 1976) using Bio-Rad reagent with
a BSA standard curve. Membranes were held on ice
for up to 2 h or flash-frozen in liquid nitrogen and
stored at ꢁ80 ꢁC.
5.1.13. 2-(3-Pyridinyl)-3,5-dihydro-5,5-diphenyl-4H-imi-
5
6.2. Radioligand binding
dazol-4-one (46). a-Amino-a,a-diphenylacetamide
(1.40 g, 6.19 mmol) was added to a solution of triethyl-
amine (2.50 g, 24.8 mmol) and 30 mL dry CH2Cl2. The
solution was cooled to 0 ꢁC and then nicotinoyl chlo-
ride hydrochloride (1.43 g, 8.05 mmol) was added in
one portion. The reaction mixture was stirred at 0 ꢁC
for 1 h then warmed to rt and stirred for a total of
16 h. The reaction was then quenched with 5% NaH-
Membrane solutions were diluted in binding buffer con-
sisting of 50 mM Tris–HCl (pH 7.4), 10 mM NaCl,
5 mM MgCl2, and 2.5 mM CaCl2, supplemented with
Aprotinin (10lg/lL), Leupeptin (10lg/lL), and 0.1%
BSA. Compounds were diluted in 100% DMSO to a
10 mM stock concentration, then assayed at 1–
10,000 nM. The assay was run in 96-deep well plates

5526
K. W. Gillman et al. / Bioorg. Med. Chem. 14 (2006) 5517–5526
containing 25 lL NSB, Total, Reference, or compound,
and 25 lL of 0.5 nM [125I]PYY (0.05 nM final). Finally,
200 lL Hi 5 membrane preparation (5 lg total protein)
was added to each well for a final DMSO concentration
of 1%. The assay plates were incubated at 22 ꢁC for
90 min. Incubation was terminated by filtration over
Whatman GF/C filters (pre-soaked in 1% polyethylene-
imine for at least 1 h), followed by four washes with
ice-cold 50 mM Tris–HCl at pH 7.4 on the Brandel 96
Harvester. The filters were counted on the WALLAC
Trilux 1450 microbeta counter. Non-specific binding
was defined in the presence of 1 lM NPY. Binding data
were analyzed by non-linear regression using the
KaleidaGraph or the Ligand program.
Acknowledgment
The authors thank Dr. Richard Dalterio for assistance
in obtaining key analytical data.
References and notes
1. For comprehensive reviews on current data in the NPY5
field, see: (a) Guba, W.; Neidhart, W.; Nettekoven, M.
Bioorg. Med. Chem. Lett. 2005, 15, 1599; (b) Elliot, R. L.;
Oliver, R. M.; Hammond, M.; Patterson, T. A.; She, L.;
Hargrove, D. M.; Martin, K. A.; Maurer, T. S.; Kalvass,
J. C.; Morgan, B. P.; DaSilva-Jardine, P. A.; Stevenson,
R. W.; Mack, C. M.; Casella, J. V. J. Med. Chem. 2003,
46, 670; (c) Elliot, R. L.; Oliver, R. M.; LaFlamme, J. A.;
Gillaspy, M. L.; Hammond, M.; Hank, R. F.; Maurer, T.
S.; Baker, D. L.; DaSilva-Jardine, P. A.; Stevenson, R. W.;
Mack, C. M.; Casella, J. V. Bioorg. Med. Chem. Lett.
2003, 13, 3593; (d) Hammond, M. Idrugs 2001, 4, 920; (e)
Dax, S. L. Drugs Future 2002, 27, 273; (f) Chamorro, S.;
Della-Zuana, O.; Fauchere, J.-L.; Feletou, M.; Galizzi, J.-
P.; Levens, N. Int. J. Obes 2002, 26, 281; (g) Antal-
Zimanyi, I.; Poindexter, G. S. Drug Dev. Res. 2000, 51, 94;
(h) Parker, E.; Van Heek, M.; Stamford, A. Eur. J.
Pharmacol. 2002, 440, 173; (i) Weinland, H. A.; Hamilton,
B. S.; Krist, B.; Doods, H. N. Exp. Opin. Invest. Drugs
2000, 9, 1327; (j) Duhault, J.; Boulanger, B.; Chamorro,
C.; Boutin, J. A.; Zuana, O. D.; Fauchere, J.-L.; Feletou,
M.; Germain, M.; Husson, B.; Vega, A. M.; Renard, P.;
Tisserand, F. Can. J. Pharmacol. 2000, 78, 173; (k)
Turnbull, A. V.; Ellershaw, L.; Masters, D. J.; Birtles,
S.; Boyer, S.; Carroll, D.; Clarkson, P.; Loxham, S. J. G.;
McAulay, P.; Teague, J. L.; Foote, K. M.; Pease, E.;
Block, M. H. Diabetes 2002, 51, 2441; (l) Block, M. H.;
Boyer, S.; Brailsford, W.; Brittain, D. R.; Carroll, D.;
Chapman, S.; Clarke, D. S.; Donald, C. S.; Foote, K. M.;
Godfrey, L.; Ladner, A.; Marsham, P. R.; Masters, D. J.;
Mee, C. D.; O’Donovan, M. R.; Pease, J. E.; Pickup, A.
G.; Rayner, J. W.; Roberts, A.; Schofiled, P.; Suleman, A.;
Turnbull, A. W. J. Med. Chem. 2002, 45, 3509; (m) Zuana,
D. O.; Sadlo, M.; Germain, M.; Feletou, M.; Chamorro,
S.; Tisserand, F.; de Montrion, C.; Boivin, J. F.; Duhault,
J.; Boutin, J. A.; Levens, N. Int. J. Obes. Relat. Metab.
Disord. 2001, 25, 84; (n) Kask, A.; Vasar, E.; Heidmets,
L.-T.; Allikmets, L.; Wikberg, J. E. S. Eur. J. Pharmacol.
2001, 414, 215; (o) Kanatani, A.; Ishihara, A.; Iwaasa, H.;
Nakamura, K.; Okamoto, O.; Hidaka, M.; Ito, J.;
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Ihara, M. Biochem. Biophys. Res. Commun. 2000, 272, 169.
2. Biltz, H. Liebigs Ann. 1909, 368, 225.
6.3. cAMP assay
CHO cells expressing the human NPY5 receptor were
seeded on 48-well cell culture plates (Polyfiltronics cata-
logue #PF-150-SCC9; ordered from VWR) at a density
of 5· 104 to 1· 105 cells/mL, and they were incubated at
37 ꢁC, 5% CO2overnight. The medium was removed and
displaced with fresh serum-free medium containing
20 mM Hepes, 100 lM IBMX, 10 lM forskolin, and
various concentrations of NPY with or without various
concentrations (100–1500 nM) of compound 46. The
cells were incubated for 10 min at 37 ꢁC. The drug solu-
tion was aspirated from cells and the reaction was termi-
nated by addition of 700 lL of 0.1 N HCl. Cells were
incubated for at least 1 h at room temperature before
starting radioimmunoassay to measure cyclic-AMP.
Each data point was performed in duplicate. cAMP lev-
els were determined using an RIA assay kit (BIOTRAK
cAMP [125I] Assay System; Amersham RPA 509 kit)
according to the manufacturer’s directions. cAMP levels
were expressed in femtomoles based on a standard
curve.
6.4. Feeding studies
All test subjects were male Sprague–Dawley rats
(Harlan, USA). Rats were individually housed with
free access to food and water in a temperature
and humidity controlled environment (12 h light/
12 h dark). Rats in nocturnal food intake and diet-
induced obesity studies were dosed 45–60 min prior
to the onset of darkness. NPY-Induced Feeding
studies were performed during the daylight portion
of the cycle. For the diet-induced obesity studies rats
were fed a high fat diet (45% calories from fat) for
16 weeks. A control group was fed a low fat diet
(10% calories from fat). After 16 weeks, the mean
bodyweights were calculated. Animals in the high
fat group with bodyweights exceeding means + 1
standard deviation of the control group were consid-
ered obesity prone and used in the diet-induced
obesity studies. All protocols and procedures were
approved by the Bristol-Myers Squibb Animal Care
and Use Committee and conducted in a facility ap-
proved by the American Association for the Accred-
itation of Laboratory Animal Care (AAALAC).
3. Rio, P. G.; Ranjon, A. Bull. Soc. Chim. Fr. 1958, 543.
4. Edward, J. T.; Lantos, I. Can. J. Chem. 1967, 45, 1925.
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48, 7685.
6. Abdel-Magid, A. F.; Maryanoff, C. A.; Carson, K. G.
Tetrahedron Lett. 1990, 39, 5595.
7. Hajek, M.; Silhavy, P.; Malek, J. Collect. Czech. Chem.
Commun. 1974, 39, 2667.
8. Bergeron, R. J.; Garlich, J. R. Synthesis 1984, 9, 782.
9. Kress, T. J. Heterocycles 1994, 38, 1375.
10. Criscione, L.; Rigollier, P.; Batzl-Hartman, C.; Rueger,
H.; Stricker-Krongrad, A.; Wyss, P.; Brunner, L.; White-
bread, S.; Yamaguchi, Y.; Gerald, C.; Heurich, R.;
Walker, M.; Chiesi, M.; Schilling, W.; Hofbauer, K.;
Levens, H. J. Clin. Invest. 1998, 102, 2136.