Design, Synthesis, and Biological Evaluation of Substituted 2-Cyclohexyl-4-phenyl-1H-imidazoles: Potent and Selective Neuropeptide Y Y5-Receptor Antagonists

Article abstract of DOI:10.1021/jm030490g

Antagonizing the robust stimulation of food intake by neuropeptide Y represents a new potential therapeutic approach for the treatment of obesity. Earlier pharmacological studies have pointed to the Y1 and Y5 receptors as the most likely mediators of the

Full text of DOI:10.1021/jm030490g

2318
J . Med. Chem. 2004, 47, 2318-2325
Design , Syn th esis, a n d Biologica l Eva lu a tion of Su bstitu ted
2-Cycloh exyl-4-p h en yl-1H-im id a zoles: P oten t a n d Selective Neu r op ep tid e Y
Y5-Recep tor An ta gon ists
Charles A. Blum,* Xiaozhang Zheng, and Ste´phane De Lombaert
Neurogen Corporation, 35 Northeast Industrial Road, Branford, Connecticut 06405
Received October 1, 2003
Antagonizing the robust stimulation of food intake by neuropeptide Y represents a new potential
therapeutic approach for the treatment of obesity. Earlier pharmacological studies have pointed
to the Y1 and Y5 receptors as the most likely mediators of the NPY orexigenic response. In
this paper, we describe a new series of small molecule Y5 antagonists derived from a 2,4-
diaryl-1H-imidazole lead. The main objectives of our structural optimization efforts were to
produce novel and potent Y5 antagonists with an improved oral pharmacokinetic profile and
less affinity for the hERG potassium channel compared to the lead 2,4-diarylimidazole
structures. These goals were accomplished by replacement of the 2-aryl ring with a cyclohexyl
ring and subsequent elaboration of the 4-position of the cyclohexyl ring with a variety of
hydrophilic functionalities. The resulting compound, N-(2-hydroxy-tert-butyl)(4-{4-[3-(trifluoro-
methyl)phenyl]imidazol-2-yl}cyclohexyl)carboxamide (20), displayed good potency at the Y5
receptor (Ki ) 3 nM), while interactions at the hERG channel were essentially eliminated (6%
inhibition at a concentration of 3 µM). Importantly, the pharmacokinetic properties of 20 (F )
36%) represented a marked improvement over that of the initial 2,4-diarylimidazole structures.
In tr od u ction
Although NPY interacts with at least six G-protein-
coupled receptors (Y1, Y2, Y3, Y4, Y5, y6), earlier
pharmacological studies have pointed to the Y1 and Y5
receptor subtypes as the most likely mediators of the
NPY orexigenic response. While the relative importance
of both receptor subtypes on feeding behavior remains
a matter of debate, we embarked several years ago in a
parallel research program aimed at producing small
molecule antagonists of the Y1 or Y5 receptors.
Neuropeptide Y (NPY), a 36-amino acid peptide, was
first isolated from porcine brain in the early 1980s. It
is widely distributed in the mammalian central and
peripheral nervous systems.1 More specifically, NPY is
highly expressed in the hypothalamus, an area of the
brain critical for the regulation of energy balance.2 The
release of NPY from these neuronal stores results in a
variety of physiological effects involved in the regu-
lation of blood pressure, anxiety, reproduction, thermo-
regulation, circadian rhythms, control of food intake,
and others.3 Of these, one of the most remarkable effects
of NPY, and the one which has stimulated the most
interest in the scientific community, is the strong
orexigenic effect that this peptide induces following
injections into the hypothalamus or cerebral ventricles
of animals.4 Indeed, chronic administration of NPY into
the brain induces hyperphagia and weight gain. In
addition, it has been shown that food deprivation in rats
results in a marked increase in the levels of both NPY
and its mRNA in the hypothalamus. Collectively these
and other data suggest an important role for NPY as a
regulator of physiological feeding behavior, particularly
during periods of starvation or food deprivation.
The initial evidence supporting the role of Y5 as an
important regulator of feeding behavior include the
positive correlation between the binding affinity of Y5
peptide agonists in vitro and their in vivo potency as
orexigenic agents in animal feeding models,6 the inhibi-
tion of NPY-stimulated food intake in rodents by oligo-
deoxynucleotides directed against the Y5 receptor,7-9
and more recently, the reduction of food intake observed
in ob/ob mice or Zucker obese rats following administra-
tion of small molecule Y5 antagonists.10-13
However, the importance of the Y5 receptor as a
regulator in more natural feeding states has also been
challenged recently. Indeed, despite reports of small
molecule Y5 antagonists inhibiting Y5 agonist-induced
feeding in rats, these same molecules have generally
failed to block feeding in more natural food intake
paradigms, or when feeding was elicited by exogenous
centrally administered NPY.14-18 Furthermore, in those
other instances where a block of natural feeding could
be demonstrated, the specificity of the compound toward
the Y5 receptor was insufficient or had not been
thoroughly evaluated.12,13,19 Finally, in feeding experi-
ments using Y5 knockout mice, NPY administration still
evoked a vigorous feeding response, suggesting that the
NPY feeding response is at least partially mediated
through a receptor other than Y5, possibly Y1.20
Considering that obesity is a major health problem
in advanced nations, and is known to increase the risk
of developing a variety of disorders including hyper-
tension, diabetes mellitus, and coronary heart failure
among others, new and more effective treatments are
sought.5 Antagonizing the stimulation of food intake by
neuropeptide Y could represent a potential therapeutic
approach for the treatment of obesity.
* To whom all correspondence should be addressed. Phone: (203)
488-8201. Fax: (203) 481-5290. E-mail: cblum@nrgn.com.
10.1021/jm030490g CCC: $27.50 © 2004 American Chemical Society
Published on Web 03/20/2004

Neuropeptide Y Y5-Receptor Antagonists
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 9 2319
Ta ble 1. In Vitro Data of Compounds 12-23
a
Compounds were assayed for their ability to compete with [125I]PYY at a recombinant human chimeric NPY5/NPY1 receptor expressed
b
in SF9 cells (ref 32). Data are expressed as the mean (n ) 2-3) ( SEM. Hepatic clearance predicted from human microsomal half-life
(n ) 1). hClhep ) 4 is the lower limit for this experiment and denotes a compound with no appreciable metabolism under the conditions
used (see Experimental Section). c Percentage of block at hERG K channel determined in an in vitro electrophysiological whole cell assay
using Cos7 cells (n g 3). Standard deviation. e Percent inhibition at 300 nM. f ND ) not determined. N-[4-[[1-[2-(6-Methyl-2-
d
g
pyridinyl)ethyl]-4-piperidinyl]carbonyl]phenyl]methanesulfonamide (see ref 31).
Ch a r t 1. NPY-5 Receptor Antagonists
In light of this conflicting evidence, the evaluation of
structurally diverse compound classes remains impor-
tant in order to better understand the role of the Y5
receptor in the control of appetite, and hence the
potential utility of these compounds for the treatment
of obesity.
Early work in our laboratories to identify lead Y5
antagonists through compound library screening had led
to the discovery of 2,4-diarylimidazoles, a structurally
simple yet potent series of Y5 receptor antagonists.18,21,22
The initial exploration of different substitution patterns
within this framework quickly revealed that electron-
withdrawing groups (e.g. CF3 or CN) at the 3-position
of the 4-aryl ring produced exceptionally potent com-
pounds, exemplified by 1 (Chart 1, Table 1).
poor oral bioavailability in rats. For example, an early
analogue of interest, 4-(4-chlorophenyl)-2-(4-fluorophen-
yl)imidazole, demonstrated an oral bioavailability in
rats of only 3%. In addition, many compounds from the
series significantly blocked the hERG potassium chan-
nel, thereby carrying a higher risk of adverse cardiac
events due to QT interval prolongation. Standard ap-
proaches to create a more viable series, for example
Although many of these compounds displayed high
in vitro potency, the series was generally plagued by

2320 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 9
Blum et al.
Sch em e 1. Synthesis of Compounds 12-23a
replacing the 2-aryl ring with a pyridine and incorpo-
rating a variety of polar ring substituents, were ulti-
mately successful, leading to some potent Y5 antago-
nists with excellent pharmacokinetic properties.18
A parallel approach, exploiting the 2,4-diarylimidazole
leads, was followed with the intent of improving on the
structural novelty as well as the druglike properties of
the series and reducing its interactions with the hERG
channel to an acceptable level. Because the 2-aryl side
of the molecule was much more amenable to the
addition of polar functionalites,18 this ring was the main
focus of subsequent efforts to achieve these goals.
Our point of divergence from the diarylimidazole
series came with the discovery that replacement of the
2-aryl ring with a saturated ring system produced
compounds with only a modest loss of activity at the
Y5 receptor (compare 1 and 2, Table 1). Placement of a
nitrogen atom into the cyclohexyl ring of 2 to generate
the corresponding piperidine analogues would offer a
handle from which to maximize potency and solubility.
However, with this approach, we were unable to gener-
ate analogues with the desired level of potency.23
Furthermore, a patent application disclosing similar
analogues appeared during the course of our work.24
Because the piperidines were no longer a viable option,
efforts were then focused on substituted cyclohexane
derivatives.
A common structural motif present in several series
of small molecule Y5 antagonists is the trans-(1,4-
disubstituted-cyclohexyl) ring.13 In most examples, one
or both of the 1,4-substituents contain a H-bond donor
two atoms away from the ring carbon, usually in the
form of an amine, a sulfonamide, or an amide function-
ality. In compound 2, we envisioned that this H-bond
donor would be represented by the imidazole N-H. The
presence of this N-H in the diarylimidazole series was
required for Y5 receptor affinity, as the replacement of
the imidazole with a oxazole moiety or methylation of
the N-H resulted in a complete loss of activity at Y5.23
For the introduction of a diversity of 4-functionalized
cyclohexyl substituents in 2, the common precursors,
10 and 11, were prepared (Scheme 1). A series of
compounds were synthesized from these intermediates
with the aim of achieveing the goals set forth above. In
this article we report the results of this work and the
optimization of a novel series of Y5-receptor antagonists
derived from the diarylimidazoles.
a
Reagents: (a) BOP, TEA, DMF, 80 °C; (b) 30 equiv of NH4OAc,
HOAc, reflux (16 h); (c) 9, 10% Pd/C, EtOH, 50 psi H2; (d) 8, LiOH-
THF-H2O, reflux; (e) 1.1 equiv of ArSO2Cl, 2 equiv of TEA,
CH2Cl2, room temp (compds 12-14); (f) BOP, TEA, DMF, 80 °C
(compds 16, 18-21) or EDCI/DMAP (1:1), CH2Cl2, room temp, 16
h (cmp 15).
tively methylating the sulfonamide nitrogen. Coupling
acid 11 with benzenesulfonamide, acetyl hydrazide, or
substituted amines afforded compounds 14, 15, and 16-
23, respectively.
Biologica l Resu lts a n d Discu ssion
Several other known Y5 receptor antagonists contain
1,4-disubstituted-cyclohexane spacers substituted with
benzenesulfonamide groups.12,13,26 Indeed, when 10 was
coupled with benzenesulfonyl chloride, a very potent Y5
antagonist (12) was obtained (Table 1). Compound 12
was about 30-fold more potent at Y5 than the lead
compound 2 and also showed excellent stability in in
vitro human microsomal preparations as indicated by
the low predicted plasma clearance value (4 mL/min/
kg) shown in Table 1. The methylated analogue 13
revealed that the sulfonamide N-H was not critical for
either Y5 receptor inhibition or hERG channel blockade.
Although compounds containing the benzenesulfon-
amide group dramatically increased receptor potency
(12 and 13), in vitro side-effect profiling showed that
they consistently inhibit hERG channel currents at very
low concentrations relative to lead compound 2 (Table
1). The blocking of this cardiac K+ channel (IKr) has
been linked to drug-induced long QT syndrome (LQT)27
which can lead to torsades de pointes, a life threatening
form of arrhythmia, and subsequent ventricular fibril-
lation. Compound 12, for example, inhibited the hERG
channel currents by 87% at a concentration of 300 nM.
A key finding resulted when close-in modifications of
12 demonstrated that the hERG activity could in fact
be substantially attenuated if the methylene linker
between the cyclohexyl ring and the sulfonamide nitro-
gen was converted to a carbonyl group to give an acyl-
Ch em istr y
Substituted R-aminoacetophenones (3) were pre-
pared by reaction of R-bromoacetophenones with hexa-
methylenetetramine, followed by acidic hydrolysis of the
resulting quaternary complex.25 Coupling of 3 with
trans-4-substituted cyclohexanecarboxylic acids 4 or 5
using the BOP reagent afforded keto-amides 6 and 7 in
good yields. Cyclization of 6 or 7 to the 2-cyclohexyl-5-
phenyl-1H-imidazoles 8 and 9 was accomplished upon
reflux with an excess of NH4OAc in acetic acid. Hy-
drolysis of 8 and the hydrogenolysis of 9 led to the acid
and amino derivatives 11 and 10, respectively. These
key intermediates were used to prepare the target
compounds 12-23 shown in Table 1. Specifically, com-
pounds 12 and 13 were prepared by coupling 10 (R2 )
3-CF3) with benzenesulfonyl chloride and then selec-

Neuropeptide Y Y5-Receptor Antagonists
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 9 2321
Ta ble 2. Single Dose Rat Pharmacokinetics of Compound 20
sulfonamide. The resulting compound (14, Table 1)
exhibited a markedly reduced interaction with the
hERG channel, but unfortunately a decreased affinity
for the Y5 receptor. Acyl-sulfonamides have been used
as bioisoteric replacements for carboxylic acids, and this
reduction in hERG interaction could be attributable to
the inherent acidity of 14.
after iv and po Administrationa
c
d
g
Cmax po,b
AUCpo
,
Fpo
,
CLe
Vss,f T(1/2)po
,
ng/mL
(ng × h)/mL
%
mL/min/kg L/kg
h
714
2380
36
50
6
13
SD ) (362)h
(1103)h
(17)h
(4.8)h
(0.5)h
(10)h
a
Rats were given either a single dose iv (3.0 mg/kg) (n ) 2) or
b
a single dose po (20.0 mg/kg) (n ) 3) of 20. Maximum plasma
In parallel with this work, an effort was being made
to replace the aryl-sulfonamide group with a more
hydrophilic ring system. A series of analogues was
synthesized in which five-membered ring heterocycles
were directly attached to the 4-position of the cyclohexyl
ring. Although these analogues were weak Y5 receptor
binders,23 the intermediate leading to an oxadiazole, i.e.,
the acyl hydrazide 15, exhibited a very promising in
vitro profile. Like the acyl-sulfonamide 14, compound
15 displayed low hERG affinity, but this time with a
much improved potency as a Y5 antagonist (Table 1).
Furthermore, the acyl hydrazide was a more compact,
hydrophilic functionality than the aryl-sulfonamides.
Interestingly, during the course of our work a patent
application appeared which disclosed a series of acyl-
aminocarboxylic hydrazides as neuropeptide Y receptor
ligands, wherein some of the examples utilized the
4-substituted-(acylhydrazido)cyclohexane ring system.28
We were concerned, however, by the known propensity
of acyl hydrazides to undergo hydrolysis in vivo with
subsequent release of an acetylhydrazine moiety. In the
liver, acetylhydrazine can be converted to a reactive
metabolite leading to liver necrosis.29 For this reason
we began to investigate direct replacements for the acyl
hydrazide group. Derivatives in which a hydrazide NH
was replaced with a methylene yielded keto-amides, for
example 16, which were both less active at Y5 and more
active at the hERG channel. However, reduction to the
corresponding alcohols produced compounds with a
more acceptable hERG profile (see 17 and 18, Table 1).
There was only a marginal chiral bias observed with
regard to Y5 affinity, with the R enantiomer (17)
exhibiting a 3-fold increase in potency relative to the S
isomer (18) (Table 1). Preparation of the achiral amido-
alcohol by removal of the terminal methyl group re-
sulted in a substantial increase in the hERG activity
(19, Table 1). Placement of the hydroxyl group â to the
amide nitrogen turned out to be optimal for Y5 receptor
affinity. Chain extension, for example, led to a much
less potent Y5 antagonist, compound 23. In addition,
methylation of the â-hydroxyl group to give 21, produced
a 9-fold loss of activity (Table 1). An attempt to replace
the hydroxyl group of 19 with NH2 in order to maximize
solubility resulted in a compound with significantly
reduced affinity for the Y5 receptor (compound 22).
When a gem-dimethyl group was incorporated at the
position R to the amide nitrogen in order to produce a
more rigid side chain, the resulting compound, 20,
exhibited an in vitro profile that was favorable both in
terms of Y5-receptor affinity (3 nM) and hERG activity
(6% inhibition @ 3 µM). In addition, compound 20 was
shown to be selective (<50% inhibition at 4 µM) in a
battery of more than 20 in-house in vitro screens
(including R1, D2, and GABA).
concentration after oral dosing. c Estimated area under the plasma-
d
concentration time curve after oral dosing. Oral bioavailability.
e Plasma clearance. f Volume of distribution at steady state. g Oral
h
half-life. Standard deviation.
compound with reasonable bioavailability (36%) and
moderately high clearance (50 mL/min/mg) which lim-
ited exposure in vivo. Nevertheless, based on human
liver microsomal data (Table 1), a lower clearance and
hence a longer oral half-life was predicted for humans.
The pharmacokinetic and in vitro pharmacological
properties of 20 are such that it can now be assessed in
pharmacological feeding models in rats. The results of
these experiments will help determine the potential
usefulness of these compounds as anorectic agents and
may also help to clarify the role of Y5 as a regulator of
feeding behavior in animals.
Con clu sion
We have succeeded in developing a low molecular
weight (MW ) 409.5) Y5 antagonist (20) with improved
hydrophilicity (MlogP ) 2.36) vs the lead compound 2
(MlogP ) 3.34). An increase in structural novelty was
achieved, and the hERG liability encountered with
many of the compounds from the series was ultimately
overcome by close-in structural modifications. Impor-
tantly, the pharmacokinetic properties of 20 represented
a great improvement over that of initial 1,4-diarylimid-
azole structures, although the in vivo clearance in rats
was substantially higher than predicted from in vitro
microsomal assays. These improvements were achieved
while maintaining low nanomolar potency at the Y5
receptor.
Exp er im en ta l Section
Ch em istr y. Melting points were determined using a MEL-
TEMP II melting point apparatus and are uncorrected. 1H
NMR spectra were recorded on a Varian Gemini operating at
300 MHz or a Varian Unity 400 operating at 400 MHz.
Atmospheric pressure chemical ionization (AP-CI) mass
spectra were determined on a Sciex 150. Electrospray mass
spectra were measured on a Waters single quad ZMD MKII.
Microanalyses were performed by Robertson Microlit Labora-
tories, Inc. (Madison, NJ ) for the elements indicated and are
within 0.4% of the theoretical values unless indicated. Chro-
matographic separations were performed on silica gel (0.040-
0.063 mm) columns by flash chromatography. Reactions and
product mixtures were routinely monitored by thin-layer
chromatography (TLC) on precoated silica gel plates (Analtech,
0.25 mm). Amino-ketones (3, R ) 3-CF3 and R ) 3-Br) were
prepared according to procedures analogous to those outlined
in the literature.25
2-P h en yl-4-[3-(tr iflu or om eth yl)p h en yl]im id a zole (1).
Compound 1 was synthesized according to general procedures
A and B using 3 (R2 ) 3-CF3) and benzoic acid as starting
materials. 1H NMR (300 MHz, DMSO-d6): δ 7.61-7.65 (m,
3H, aromatic); 7.74-7.63 (m, 2H, aromatic); 8.27-8.36 (m, 3H,
aromatic); 8.44 (s, 1H, NH). MS (CI): m/z (%) ) 289 (100) [M
+ 1]+. mp ) 234-236 °C; Anal. (C16H11N2F3‚HCl) C, H, N.
2-Cycloh exyl-4-[3-(tr iflu or om eth yl)p h en yl]im id a zole
(2). Compound 2 was synthesized according to general proce-
The pharmacokinetic properties of compound 20 were
examined in rats at doses of 3.0 mg/kg (iv) and 20.0 mg/
kg (po). The results shown in Table 2 suggest a

2322 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 9
dures A and B using 3 (R2 ) 3-CF3) and cyclohexanecarboxylic
Blum et al.
) 6 Hz, 2H), 4.91 (t, 1H, N-H), 5.10 (s, 2H), 7.22 (s, 1H), 7.32-
7.37 (m, 5H), 7.45 (d, J ) 4.9 Hz, 2H), 7.87 (m, 1H), 7.95 (s,
1H). MS 458 (M + H)+.
1
acid as starting materials. H NMR (300 MHz, DMSO-d6): δ
1.20-1.38 (m, 3H, CH2); 1.65-1.81 (m, 5H, CH2); 1.97-2.01
(m, 2H, CH2); 3.06-3.13 (m, 1H, CH); 7.69-7.75 (m, 2H,
aromatic); 8.23 (s, 1H, aromatic); 8.25 (s, 1H, aromatic); 8.32
(s, 1H, NH). MS (CI): m/z (%) ) 295 (100) [M + 1]+. mp )
245-247 °C; Anal. (C16H17N2F3‚HCl) C, H, N.
tr a n s-(4-{4-[3-(Tr iflu or om eth yl)p h en yl]im id a zol-2-yl}-
cycloh exyl)m et h yla m in e (10). An ethanolic solution of 9
(4.15 g, 0.0091 mol) and 10% Pd/carbon (400 mg) was shaken
overnight under 55 psi of hydrogen in a Paar apparatus. The
mixture was filtered through Celite and concentrated under
reduced pressure to give a foam. Trituration with EtOAc/
hexanes/MeOH produced the title compound as a filterable
solid. 1H NMR (300 MHz, DMSO-d6): δ 0.93-1.05 (m, 2H),
1.30-1.4 (m, 2H), 1.42-1.55 (m, 2H), 1.85 (d, J ) 12.8 Hz,
2H), 1.99 (d, J ) 11.6 Hz, 2H), 2.4-2.5 (m, 1H), 2.56-2.66
(m, 1H), 7.43-7.48 (m, 1H), 7.50-7.55 (m, 1H), 7.63 (s, 1H),
7.99 (d, J ) 7.9 Hz, 1H), 8.02 (s, 1H). MS 324 (M + H)+.
tr a n s-4-{4-[3-(Tr iflu or om eth yl)p h en yl]im id a zol-2-yl}-
cycloh exa n eca r boxylic a cid (11, R2 ) 3-CF 3). To a solution
of 8 (5.2 g, 13.4 mmol) in THF-H2O (1:1, 200 mL) was added
LiOH-H2O (3.4 g, 80.3 mmol). The resulting mixture was
stirred overnight at room temperature and then concentrated
under reduced pressure. The residue was diluted with H2O
and acidified to pH 5-6. The aqueous layer was extracted with
3 × 100 mL of EtOAc, and the EtOAc layer was washed with
brine. The EtOAc was dried (Na2SO4) and concentrated under
tr a n s-Cycloh exa n e-1,4-d ica r boxylic Acid Mon om eth yl
Ester (4). A solution of trans-cyclohexane-1,4-dicarboxylic acid
dimethyl ester (34.2 g, 0.171 mol) in dry methanol was brought
to reflux. A solution of KOH (11.2 g, 0.171 mol) in dry methanol
was added dropwise and the solution refluxed for an additional
5 h. The mixture was cooled and concentrated under reduced
pressure, and the resulting solid was taken up in water. The
mixture was washed with ether (3×) and brought to pH 6 with
6 N HCl. The precipitated white solid was collected via
filtration, washed with water (1×), and air-dried to afford 16.2
1
g (51%) of 4. H NMR (300 MHz, CDCl3): δ 1.4-1.5 (m, 4H);
2.08 (m, 4H); 2.2-2.37 (m, 2H, 2 methines); 3.67 (s, OCH3).
MS (CI, negative ion mode): m/z (%) ) 185 (100) [M - 1]-.
Gen er a l P r oced u r e A: Am id e F or m a tion Med ia ted by
BOP Reagen t. tr a n s-N-{2-Oxo-2-[3-(tr iflu or om eth yl)ph en -
yl]et h yl}(4-{[(p h en ylm et h oxy)ca r bon yla m in o]m et h yl}-
cycloh exyl)ca r boxa m id e (7). To a solution of 3 (R2 ) 3-CF3)
(3.4 g, 0.0144 mol) and trans-4-{[(phenylmethoxy)carbonyl-
amino]methyl}cyclohexanecarboxylic acid (5)30 (4.2 g, 0.0144
mol) in DMF was added benzotriazol-1-yloxytris(dimethyl-
amino)phosphonium hexafluorophosphate (BOP reagent, 6.4
g, 0.0144 mol) followed by triethylamine (4.0 mL, 0.0289 mol).
The reaction was stirred overnight and then poured into water
to produce a thick precipitate. The solid was collected on a
sintered glass funnel and washed consecutively with H2O, 3
N HCl (2×), H2O, NaHCO3 solution (2×), and H2O. The filter
cake was air-dried, added to a 500 mL round-bottom flask with
toluene, and concentrated under reduced pressure. Additional
toluene was added (200 mL), and the mixture was again
concentrated under reduced pressure to yield N-{2-oxo-2-
[3-(trifluoromethyl)phenyl]ethyl}(4-{[(phenylmethoxy)carbon-
ylamino]methyl}cyclohexyl)carboxamide as a solid. 1H NMR
(300 MHz, DMSO-d6): δ 0.8-0.93 (m, 2H); 1.2-1.37 (m, 3H);
1.71 (br s, 4H); 2.14 (t, 1H); 2.84 (br s, 2H); 4.57 (br s, 2H);
4.99 (s, 2H); 7.2-7.38 (m, 5H); 7.77(t, 1H); 8.01 (d, J ) 8.0
Hz, 1H); 8.14-8.26 (m, 3H). MS 477 (M + H)+.
tr a n s-Met h yl 4-{4-[3-(Tr iflu or om et h yl)p h en yl]im id -
a zol-2-yl}cycloh exa n eca r boxyla te (8, R2 ) 3-CF 3). Com-
pound 8 was synthesized according to general procedures A
and B using 3 (R2 ) 3-CF3) and 4 as starting materials. 1H
NMR (300 MHz, DMSO-d6): δ 1.38-1.56 (m, 2H, CH2); 1.63-
1.80 (m, 2H, CH2); 1.92-2.17 (m, 4H, CH2); 2.30-2.51 (m, 1H,
CH); 2.95-3.16 (m, 1H, CH); 3.60 (s, 3H, CH3); 7.65-7.85 (m,
3H, aromatic); 8.16-8.36 (m, 3H, aromatic). MS (CI): m/z (%)
) 354 (100) [M + 2]+.
tr a n s-Meth yl 4-[4-(3-Br om op h en yl)im id a zol-2-yl]cyclo-
h exa n eca r boxyla te (8, R2 ) 3-Br ). Compound 8 was syn-
thesized according to general procedures A and B using 3 (R2
) 3-Br) and 4 as starting materials. 1H NMR (300 MHz,
CD3OD): δ 1.41-1.81 (m, 4H, CH2); 2.00-2.15 (m, 4H, CH2);
2.48 (m, 1H, CH); 3.11 (m, 1H, CH); 3.68 (s, 3H, CH3); 7.41
(m, 1H, aromatic); 7.60 (d, J ) 4.1 Hz, 1H, aromatic); 7.780
(d, J ) 4.1 Hz, 1H, aromatic); 7.95 (s, 1H, aromatic); 7.99 (s,
1H, aromatic). MS (CI): m/z (%) ) 364 (100) [M + 1]+.
1
reduced pressure to afford the title compound. H NMR (300
MHz, DMSO-d6): δ 1.38-1.6 (m, 4H, CH2); 196-2.10 (m, 4H,
CH2); 2.20-2.32 (m, 1H, CH); 2.60-2.74 (m, 1H, CH); 7.64-
7.60 (m, 2H, aromatic); 7.64 (s, 1H, aromatic); 7.96-8.08 (m,
2H, aromatic). MS (CI): m/z (%) ) 339 (100) [M + 1]+.
tr a n s-(P h en ylsu lfon yl)[(4-{4-[3-(tr iflu or om eth yl)p h en -
yl]im id a zol-2-yl}cycloh exyl)m eth yl]a m in e (12). A solution
of 10 (R2 ) 3-CF3) (351 mg, 1.09 mmol) and triethylamine (110
mg, 1.09 mmol) in CH2Cl2 was treated with benzenesulfonyl
chloride (192 mg, 1.09 mmol) via syringe in a dropwise fashion.
The resulting mixture was stirred at room temperature for 2
h, diluted with CH2Cl2, and washed in a separatory funnel
with aqueous NaHCO3 solution. The organic layer was dried
(Na2SO4) and concentrated under reduced pressure to give a
foam. Preparative plate chromatography (2 × 2 mm, 10%
MeOH/CH2Cl2 eluent) yielded the title compound as a clear
oil. 1H NMR (400 MHz, CDCl3): δ 0.95-1.08 (m, 2H, cyclohex.);
1.40-1.58 (m, 3H, cyclohex.); 1.85 (d, J ) 11.8 Hz, 2H); 2.09
(d, J ) 11.5 Hz, 2H); 2.67-2.78 (m, 1H, cyclohex.); 2.78 (t,
2H, CH2N); 7.21 (s, 1H, aromatic); 7.41 (m, 2H, aromatic); 7.50
(m, 2H, aromatic); 7.57 (m, 1H, aromatic); 7.8-7.9 (m, 4H,
aromatic). MS (ES): m/z ) 464 [M + 1]+. The oxalate salt
was prepared by adding a solution of oxalic acid (1 equiv) in
EtOAc/MeOH to a solution of the product in ether. Anal.
(C23H24N3O2F3S. C2H2O4) C, H, N.
tr a n s-Meth yl(p h en ylsu lfon yl)[(4-{4-[3-(tr iflu or om eth -
yl)p h en yl]im id a zol-2-yl}cycloh exyl)m eth yl]a m in e (13). A
solution of 12 (73 mg, 0.158 mmol) in anhydrous THF was
treated with sodium bis(trimethylsilyl)amide (1 N in THF, 158
µL) dropwise at room temperature. After 5 min, iodomethane
(22.3 mg, 0.158 mmol) was added and the reaction stirred
overnight. The reaction was partitioned between EtOAc and
NaHCO3 (aq) and the organic layer dried (Na2SO4) and
concentrated. Preparative plate chromatography (2 × 2 mm,
10% MeOH/CH2Cl2 eluent) yielded the title compound as a
clear oil in 40% yield. 1H NMR (400 MHz, CDCl3): δ 1.06-
1.08 (m, 2H, cyclohex); 1.54-1.72 (m, 3H, cyclohex); 1.97 (d, J
) 11.2 Hz, 2H); 2.19 (d, J ) 11.5 Hz, 2H); 2.74 (s, 3H, CH3);
2.80 (m, 1H, CH); 2.85 (d, J ) 7.51 Hz, 2H, CH2-N); 7.26 (s,
1H, aromatic); 7.45 (d, J ) 4.81 Hz, 2H); 7.52-7.62 (m, 3H,
aromatic); 7.79 (d, J ) 8.79 Hz, 2H); 7.89 (m, 1H); 7.96 (s, 1H).
MS (CI): m/z (%) ) 395 (54) [M + 1]+; 147 (100). The oxalate
salt was prepared by adding a solution of oxalic acid (1 equiv)
in EtOAc/MeOH to a solution of the product in ether. mp )
220-221 °C; Anal. (C24H26N3O2F3S. C2H2O4) C, H, N.
Gen er a l P r oced u r e B: Cycliza tion of Keto-a m id es
w ith Am m on iu m Aceta te. tr a n s-(P h en ylm eth oxy)-N-[(4-
{4-[3-(tr iflu or om eth yl)p h en yl]im id a zol-2-yl}cycloh exyl)-
m eth yl]ca r boxa m id e (9). To a solution of 7 (5.4 g, 0.0113
mol) in acetic acid was added ammonium acetate (26 g, 0.34
mol). The resulting homogeneous mixture was refluxed over-
night and then concentrated under reduced pressure. The
residue was partitioned between 10% NaOH and EtOAc (1:
1), and the EtOAc layer was washed with 10% NaOH (3×)
and brine. The EtOAc was dried (Na2SO4) and concentrated
under reduced pressure to afford the title compound. 1H NMR
(300 MHz, CDCl3): δ 1.02-1.18 (m, 2H), 1.46-1.62 (m, 3H),
1.86-1.96 (m, 2H), 2.12-2.21 (m, 2H), 2.74 (m, 1H), 3.10 (t, J
t r a n s-N -(P h e n ylsu lfon yl)(4-{4-[3-(t r iflu or om e t h yl)-
p h en yl]im id a zol-2-yl}cycloh exyl)ca r boxa m id e (14). To an
ice-water cooled mixture of 11 (100 mg, 0.30 mmol), benzene-
sulfonamide (47 mg, 0.30 mmol), and DMAP (36 mg, 0.30
mmol) in dichloromethane (10 mL) under nitrogen was added

Neuropeptide Y Y5-Receptor Antagonists
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 9 2323
EDCI (57 mg, 0.30 mmol). The mixture was stirred at room
temperature for overnight, concentrated, and chromato-
graphed on silica gel (19:1 EtOAc-MeOH eluent) to afford the
title compound. 1H NMR (400 MHz, CD3OD): δ 1.35-1.75 (m,
4H, cyclohex); 1.82-2.14 (m, 4H, cyclohex); 2.20-2.38 (m, 1H,
cyclohex); 2.65-2.80 (m, 1H, cyclohex); 7.30-7.55 (m, 5H,
aromatic); 7.75-7.90 (m, 3H, aromatic); 7.98 (s, 1H, aromatic).
MS (ES): m/z ) 478 (100) [M + 1]+. Anal. (C23H22N3O3F3S):
Calcd 57.85, 4.64, 8.80. Found 55.72, 4.40, 8.42
aromatic) 11.88 (s, 1H, -CONH). MS (CI): m/z (%) ) 396 (100)
[M + 1]+. Anal. (C20H24N3O2F3‚0.5H2O) C, H, N.
tr a n s-N-(2-Hydr oxyeth yl)(4-{4-[3-(tr iflu or om eth yl)ph en -
yl]im id a zol-2-yl}cycloh exyl)ca r boxa m id e (19). A mixture
of 11 (R2 ) 3-CF3) (50 mg, 0.148 mmol), benzotriazol-1-
yloxytris(dimethylamino)phosphonium hexafluorophosphate
(BOP reagent, 65 mg, 0.148 mmol), and ethanolamine (27 mg,
0.444 mmol) in DMF was stirred at room temperature for 16
h. The mixture was diluted with water and extracted with
EtOAc. The combined organics were washed with 10% NaOH
solution (3×) and brine (1×), dried (Na2SO4), and concentrated
under reduced pressure to give crude product. The residue was
triturated with ether to give the title compound as an off-white
solid. 1H NMR (300 MHz, CDCl3/CD3OD): δ 1.46-1.64 (m, 4H,
2 × CH2); 1.88-2.08 (m, 4H, 2 × CH2); 2.17 (m, 1H, CH); 2.71
(m, 1H, CH); 3.31 (dd, 2H, CH2); 3.62 (t, 2H, CH2); 7.17 (s,
2H, aromatic); 7.40 (d, J ) 4.7 Hz, 2H, aromatic); 7.77 (t, 1H,
-NH); 7.86 (s, 1H, aromatic). MS (CI): m/z (%) ) 382 (88) [M
+ 1]+; 293 (100). Anal. (C19H22N3O2F3) C, H, N.
tr a n s-N-(2-Hyd r oxy-ter t-bu tyl)(4-{4-[3-(tr iflu or om eth -
yl)ph en yl]im idazol-2-yl}cycloh exyl)car boxam ide (20). This
compound was synthesized from 2-amino-2-methylpropan-1-
ol and 11 (R2 ) 3-CF3) using a method analogous to that used
for 19. 1H NMR (300 MHz, CDCl3): δ 1.28 (s, 6H, (CH3)2); 1.62
(m, 4H, 2 × CH2); 1.94 (m, 2H, cyclohex); 2.10 (m, 3H,
cyclohex); 2.78 (m, 1H, CH); 3.57 (s, 2H, CH2O); 6.02 (s, 1H,
-OH); 7.23 (s, 1H, aromatic); 7.42 (m, 2H, aromatic); 7.81 (t,
1H, aromatic); 7.91 (s, 1H, aromatic). MS (CI): m/z (%) ) 410
(100) [M + 1]+. The oxalate salt was prepared by adding a
solution of oxalic acid (1 equiv) in EtOAc/MeOH to a solution
of the product in ether. mp ) 181-183 °C; Anal. (C21H26N3O2F3‚
C2H2O4) C, H, N.
tr a n s-4-[4-(3-Tr iflu or om et h ylp h en yl)-1H -im id a zol-2-
yl]cycloh exa n eca r boxylic Acid (2-Meth oxyeth yl)a m id e
(21). This compound was synthesized from 2-methoxyethyl-
amine and 11 (R2 ) 3-CF3) using a method analogous to that
used for 19. 1H NMR (300 MHz, CD3OD): δ 1.57-1.75 (m,
4H, CH2); 1.90-1.97 (m, 2H, CH2); 2.05-2.15 (m, 2H, CH2);
2.30 (m, 1H, CH); 2.79 (m, 1H, CH); 3.25-3.40 (m, 2H, CH2);
3.38 (s, 3H, OCH3); 3.45-3.50 (m, 2H, CH2); 7.41 (s, 1H,
aromatic); 7.47-7.58 (m, 2H, aromatic), 7.91 (d, J ) 6.8 Hz,
1H, aromatic); 8.00 (s, 1H, 11.90 aromatic). MS (CI): m/z (%)
) 396 (100) [M + 1]+. The oxalate salt was prepared by adding
a solution of oxalic acid (1 equiv) in EtOAc/MeOH to a solution
of the product in ether. mp ) 176-178 °C; Anal. (C20H24N3O2F3‚
C2H2O4‚0.5H2O) C, H, N.
tr a n s-4-[4-(3-Tr iflu or om et h ylp h en yl)-1H -im id a zol-2-
yl]cycloh exan ecar boxylic Acid (2-Am in oeth yl)am ide (22).
This compound was synthesized from ethylenediamine and 11
(R2 ) 3-CF3) using a method analogous to that used for 19.
1H NMR (300 MHz, CD3OD): δ 1.60-1.76 (m, 4H, CH2); 1.96-
2.08 (m, 4H, CH2); 2.30 (m, 1H, CH); 2.64 (m, 1H, CH); 2.78-
2.85 (m, 2H, CH2); 3.25-3.35 (m, 2H, CH2); 7.41 (s, 1H,
aromatic); 7.50-7.58 (m, 2H, aromatic), 7.90 (d, J ) 7.4 Hz,
1H, aromatic); 8.00 (s, 1H, 11.90 aromatic). MS (CI): m/z (%)
) 389 (100) [M-1]+. The oxalate salt was prepared by adding
a solution of oxalic acid (1 equiv) in EtOAc/MeOH to a solution
of the product in ether. Anal. (C19H23N4OF3‚2C2H2O4) C, H,
N.
tr a n s-N-[(4-{4-[3-(Tr iflu or om eth yl)p h en yl]im id a zol-2-
yl}cycloh exyl)ca r bon yla m in o]a ceta m id e (15). A mixture
of 11 (127 mg, 0.376 mmol), acetic hydrazide (28 mg, 0.376
mmol), benzotriazol-1-yloxytris(dimethylamino)phosphonium
hexafluorophosphate (BOP reagent, 166 mg, 0.376 mmol), and
triethylamine (38 mg, 0.376 mmol) in DMF (4 mL) was heated
at 60 °C for 1.5 h with stirring. The mixture was poured into
water and extracted with EtOAc (3×). The combined organic
layers were washed with 10% NaOH solution (2×) and brine
(1×) and dried (Na2SO4). After concentrating under reduced
pressure, the crude product was chromatographed using
preparative TLC plates (2 × 2 mm, 10% MeOH/CH2Cl2 eluent)
1
to afford the title compound. H NMR (300 MHz, CD3OD): δ
1.72 (m, 4H, cyclohex.); 1.98-2.15 (m, 4H, cyclohex.); 2.0 (s,
3H, CH3); 2.37 (m, 1H, CH); 2.81 (m, 1H, CH); 7.42 (s, 1H,
aromatic); 7.52 (m, 2H, aromatic); 7.92 (d, 1H, aromatic); 8.03
(s, 1H, aromatic). MS (CI): m/z (%) ) 395 (54) [M + 1]+; 147
(100). mp ) 230-232 °C; Anal. (C19H21N4O2F3): Calcd 57.86,
5.37, 14.21. Found 57.00, 5.06, 13.87.
tr a n s-{4-[4-(3-Br om op h en yl)im id a zol-2-yl]cycloh exyl}-
N-(2-oxop r op yl)ca r boxa m id e (16). To a cooled solution
(-78 °C to -60 °C) of oxalyl chloride (2.0 M, 61.5 µL, 0.12
mmol) in CH2Cl2 (5 mL) was added DMSO (17.5 µL, 0.25
mmoL) dropwise. The mixture was stirred 2 min at the same
temperature. N-(2-Hydroxypropyl)(4-[4-(3-bromophenyl)imid-
azol-2-yl}cyclohexyl]carboxamide (prepared from 1-amino-
propan-2-ol and 11 [R2 ) Br] using procedures analogous to
those used for 17 below) (50 mg, 0.12 mmol) in 2 mL of CH2Cl2
was added. The mixture was stirred for 15 min at the same
temperature, and then TEA (85 µL, 0.60 mmol) was added.
The mixture was stirred another 5 min, warmed to room
temperature, and diluted with water. The organic layer was
separated, dried over Na2SO4, concentrated under vacuum,
and purified by flash chromatography (90:10:1 CH2Cl2/MeOH/
NH4OH) to afford the title compound. 1H NMR (300 MHz,
DMSO-d6): δ 1.40-1.55 (m, 4H, CH2); 1.78-1.86 (m, 2H, CH2);
2.04 (s, 3H, CH3); 2.22 (m, 1H, CH); 2.61 (m, 1H, CH); 3.21-
3.39 (m, 2H, CH2); 3.85 (d, J ) 5.7 Hz, 2H, -COCH2); 7.25-
7.38 (m, 2H, aromatic,); 7.55 (s, 1H, aromatic); 7.77 (d, J )
4.5 Hz, aromatic); 7.89 (s, 1H, NH), 8.09 (m, 1H, aromatic)
1190 (br, 1H, CONH). MS (ES): m/z ) 406 (100) [M + 1]+.
Anal. (C19H22N3O3Br) C, H, N.
t r a n s-N -((2R )-2-H yd r oxyp r op yl)(4-{4-[3-(t r iflu or o-
m et h yl)p h en yl]im id a zol-2-yl}cycloh exyl)ca r b oxa m id e
(17). Compound 17 was synthesized from 11 (R2 ) 3-CF3) and
1
1-aminopropan-2-ol using general procedure A. H NMR (300
MHz, DMSO-d6): δ 0.98 (d, J ) 6.3 Hz, 3H, CH3); 1.39-1.60
(m, 4H, CH2); 1.76-1.85 (m, 2H, CH2); 1.86-2.05 (m, 2H, CH2);
2.12 (m, 1H, CH); 2.65 (m, 1H, CH); 2.93-3.05 (m, 2H, CH2);
3.65 (m, 1H, CH); 4.65 (d, J ) 5.1 Hz, 1H, -OH); 7.40-7.56
(m, 2H, aromatic); 7.67 (s, 1H, NH); 7.70-7.81 (m, 1H,
aromatic), 7.95-8.09 (m, 2H, aromatic) 11.90 (s, 1H, CONH).
MS (CI): m/z (%) ) 396 (100) [M + 1]+. The oxalate salt was
prepared by adding a solution of oxalic acid (1 equiv) in EtOAc/
MeOH to a solution of the product in ether. mp ) 164-167
°C; Anal. (C20H24N3O2F3. C2H2O4) C, H, N.
tr a n s-4-[4-(3-Tr iflu or om et h ylp h en yl)-1H -im id a zol-2-
yl]cycloh exa n eca r boxylic Acid (3-Hyd r oxyp r op yl)a m id e
(23). 1H NMR (300 MHz, DMSO-d6): δ 1.42-1.5 (m, 6H, CH2);
1.75-1.82 (m, 2H, CH2); 1.90-2.04 (m, 2H, CH2); 2.12 (m, 1H,
CH); 2.63 (m, 1H, CH); 2.98-3.09 (m, 2H, CH2); 3.35-3.42 (m,
2H, CH2); 4.42 (t, 1H, OH); 7.40-7.56 (m, 2H, aromatic); 7.68
(s, 1H, NH); 7.75 (m, 1H, aromatic), 7.96-8.05 (m, 2H,
aromatic) 11.93 (s, 1H, CONH). MS (CI): m/z (%) ) 396 (100)
[M + 1]+. Anal. (C120H24N3O2F3) C, H, N,: calcd, 60.75, 6.12,
10.63; found, 58.97, 6.36, 9.81.
Biology. Ra d ioliga n d Bin d in g Assa y. Sf9 cell pellets (ref
32) are resuspended in homogenization buffer (10 mM HEPES,
250 mM sucrose, 0.5 µg/mL leupeptin, 2 µg/mL Aprotinin, 200
µM PMSF, and 2.5 mM EDTA, pH 7.4) and homogenized using
a Polytron homogenizer (setting 5 for 30 s). The homogenate
t r a n s-N -((2S)-2-H yd r oxyp r op yl)(4-{4-[3-(t r iflu or o-
methyl)phenyl]imidazol-2-yl}cyclohexyl)carboxamide (18).
Compound 18 was synthesized by the general procedure A.
1H NMR (300 MHz, DMSO-d6): δ 0.99 (d, J ) 6.0 Hz, 3H,
CH3); 1.38-1.62 (m, 4H, CH2); 1.78-1.90 (m, 2H, CH2); 1.95-
2.08 (m, 2H, CH2); 2.20 (m, 1H, CH); 2.64 (m, 1H, CH); 2.90-
3.08 (m, 2H, CH2); 3.60 (m, 1H, CH); 4.65 (d, J ) 4.8 Hz, 1H,
-OH); 7.46-7.79 (m, 4H, aromatic, NH); 7.90-8.17 (m, 2H,

2324 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 9
Blum et al.
is centrifuged (536g/10 min/4 °C) to pellet the nuclei. The
supernatant containing isolated membranes is decanted to a
clean centrifuge tube, centrifuged (48 000g/ 30 min, 4 °C) and
resuspended in 30 mL of homogenization buffer. This cen-
trifugation and resuspension step is repeated twice. The final
pellet is resuspended in ice cold Dulbecco’s PBS containing 5
mM EDTA and stored in frozen aliquots until needed at -80
°C. The protein concentration of the resulting membrane
preparation (P2 preparation) is measured using the Bradford
protein assay (Bio-Rad Laboratories, Hercules, CA). By this
measure, a 1-L culture of cells typically yields 50-75 mg of
total membrane protein.
H2O. The solution was sonicated for 15 min prior to dosing.
Samples of iv and po dosing solutions were collected im-
mediately after preparation and immediately before dosing and
stored in polypropylene microcentrifuge tubes at 4 °C. The iv
dosing solutions were administered via catheter (2 mL/kg)
followed by a 1 mL saline wash. The oral dosing solutions were
administered via oral gavage. Sample collection: blood samples
(0.4 mL/animal) were collected at predose, 5, 10, 20, and 40
min, 1, 1.5, 2, 4, 6, 8, 10, and 24 h after iv dosing and at
predose, 15, and 30 min, 1, 1.5, 2, 3, 4, 6, 8, 10, and 24 h after
oral dosing. Blood samples from each animal were placed into
tubes containing 20 units of sodium heparin. Each animal was
injected intravenously with 0.4 mL of 0.9% saline after blood
sample collection. Plasma samples were obtained by centrifu-
gation of 0.4 mL of whole blood at 12000 rpm for 10 min and
stored at -20 °C in polypropylene microcentrifuge tubes until
needed. Sample preparation: Methanol/water 50/50 (20 µL)
was added to a microcentrifuge tube containing 200 µL of
plasma sample, followed by 200 µL of acetonitrile. The tubes
were capped, vortexed, and centrifuged at 10 000 rpm for 20
min. The supernatant was transferred to injection vials for
LC analysis.
Purified P2 membranes are thawed, centrifuged, and washed
with PBS and resuspended by homogenization in binding
buffer (50 mM Tris-HCl, 5 mM KCl, 120 mM NaCl, 2 mM
CaCl2, 1 mM MgCl2, 0.1% BSA, pH 7.4).
For competition analysis membranes (5-50 µg) are added
to polypropylene tubes containing 0.050 nM [125I]PYY (porcine)
and 2 µL of test compound in DMSO (1- 4 µM final concen-
trated). Nonspecific binding is determined in the presence of
1 µM NPY (human, American Peptide Co., Sunnyvale, CA) and
accounts for less that 10% of total binding. Following a 2-h
incubation at room temperature, the reaction is terminated
by rapid vacuum filtration. Samples are filtered over presoaked
(in 1.0% polyethyeneimine for 2 h prior to use) GF/C Whatman
filters and rinsed two times with 5 mL of cold binding buffer
without BSA. Remaining bound radioactivity is quantified by
gamma counting. Ki and Hill coefficient (nH) are determined
by fitting the Hill equation to the measured values with the
aid of Sigmaplot software (SPSS Inc., Chicago).
Ack n ow led gm en t. The technical support of Chu
Ngo, Charles Cheng, J ennifer Kwash, J ohn Dessaint,
Weifeng Yu (electrophysiology) and Melissa Hill (in vitro
pharmacology) is gratefully acknowledged. The authors
also thank J ean Simmermacher, Christine Orozco,
Fengang Zhu, Phil J oyce, Yi Luo, and Du-Shieng Chien
for DMPK support.
For saturation binding analysis, membranes (5-50 µg) are
added to polypropylene tubes containing 0.010-0.500 nM
Refer en ces
[
125I]PYY (porcine, New England Nuclear Corp., Boston, MA).
(1) Stanley, B. G. Neuropeptide Y in Multiple Hypothalamic Sites
Controls Eating Behavior, Endocrine, and Autonomic Systems
for Body Energy Balance. The Biology of Neuropeptide Y and
Related Peptides; Humana: Totowa, NJ , 1993; pp 457-509.
(2) Widdowson, P. S.; Williams, G. Regulation of appetite. The
Diabetes Annual, 12th ed.; Elsevier Science: New York, 1999.
(3) The Biology of Neuropeptide Y and Related Peptides; Humana
Press: Totowa, NJ , 1993.
(4) Stanley, B. G.; Magdalin, W.; Seirafi, A.; Nguyen, M. M.;
Leibowitz, S. F. Evidence for Neuropeptide Y Mediation of
Eating Produced by Food Deprivation and for a Variant of the
Y1 Receptor Mediating This Peptide’s Effect. Peptides 1992, 13,
581-587.
(5) Kordik, C. P.; Reitz, A. B. Pharmacological treatment of obesity:
therapeutic strategies. J . Med. Chem. 1999, 42, 181-201.
(6) Gerald, C.; Walker, M. W.; Criscione, L.; Gustafson, E. L.; Batzl-
Hartmann, C. et al. A receptor subtype involved in neuropeptide-
Y-induced food intake. Nature 1996, 382, 168-171.
(7) Schaffhauser, A. O.; Stricker-Krongrad, A.; Brunner, L.; Cumin,
F.; Gerald, C. et al. Inhibition of food intake by neuropeptide Y
Y5 receptor antisense oligodeoxynucleotides. Diabetes 1997, 46,
1792-1798.
(8) Tang-Christensen, M.; Kristensen, P.; Stidsen, C. E.; Brand, C.
L.; Larsen, P. J . Central administration of Y5 receptor antisense
decreases spontaneous food intake and attenuates feeding in
response to exogenous neuropeptide Y. J . Endocrinol. 1998, 159,
307-312.
(9) Flynn, M. C.; Turrin, N. P.; Plata-Salaman, C. R.; Ffrench-
Mullen, J . M. Feeding response to neuropeptide Y-related
compounds in rats treated with Y5 receptor antisense or sense
phosphothio-oligodeoxynucleotide. Physiol. Behav. 1999, 66,
881-884.
(10) Daniels, A. J .; Grizzle, M. K.; Wiard, R. P.; Matthews, J . E.;
Heyer, D. Food intake inhibition and reduction in body weight
gain in lean and obese rodents treated with GW438014A, a
potent and selective NPY-Y5 receptor antagonist. Regul. Pept.
2002, 106, 47-54.
(11) Criscione, L.; Rigollier, P.; Batzl-Hartmann, C.; Rueger, H.;
Stricker-Krongrad, A. et al. Food intake in free-feeding and
energy-deprived lean rats is mediated by the neuropeptide Y5
receptor. J . Clin. Invest. 1998, 102, 2136-2145.
(12) Kordik, C. P.; Luo, C.; Zanoni, B. C.; Dax, S. L.; McNally, J . J .
et al. Aminopyrazoles with high affinity for the human neu-
ropeptide Y5 receptor. Bioorg. Med. Chem. Lett. 2001, 11, 2283-
2286.
Electr op h ysiology Recor d in gs. Standard whole-cell re-
cording31 was carried out on the COS cells transiently trans-
fected with HERG K+ channels. Microelectrodes were pulled
from 1.5 mm glass (World Precision Instruments, TW100-4)
on a horizontal puller (Sutter Instrument Company, Model
P-87) and had resistances between 2 and 3 MΩ when filled
with internal solution. The internal solution contains: 100 mM
KF, 40 mM KCl, 5 mM NaCl, 10 mM EGTA, and 10 mM
HEPES, adjusted to pH 7.4 with KOH. The cells were perfused
with external solution containing 140 mM NaCl, 2 mM CaCl2,
5 mM KCl, 2 mM MgCl2, 10 mM glucose, and 10 mM HEPES,
adjusted to pH 7.4 with NaOH. Currents were filtered at 5
kHz on an Axopatch-1D amplifier (Axon Instruments) and
recorded onto a PC with sample rate of 1 kHz using pClamp
8 (Axon Instruments). Data were analyzed using Clampfit
(Axon Instruments) and Origin softwares (Microcal). Stock
solutions for compounds were made in DMSO at a concentra-
tion of 10 mM. Final test concentration was achieved by
diluting the stock solution directly into the external solution.
Meta bolic Sta bility. Compounds were incubated with
pooled human liver microsomes. The rates of oxidative me-
tabolism were measured under the following conditions: com-
pound, 1 µM final concentration; final microsomal protein
concentration approximately 1 mg/mL; NADPH, 0.5 mM; pH
7.4 sodium phosphate buffer. Incubations were performed at
39 °C and were initiated by the addition of NADPH. Samples
(50 µL) were taken from each incubation well at about 0.25,
1, 5, 10, and 30 min and added to 75 µL of ice-cold acetonitrile.
The aliquot mixtures were mixed and then centrifuged at 3000
rpm for 15 min. The supernatant was analyzed for parent
compound using LC/MS, and the percent metabolized was
calculated based on disappearance of parent compound.
P h a r m a cok in etics. The pharmacokinetics of 20 was in-
vestigated in Sprague-Dawley rats following a single intra-
venous dose of 3 mg/kg (n ) 2 rats) and a single oral dose of
20 mg/kg (n ) 3 rats). Preparation of iv dosing solution: 7.5
mg of 20 was weighed and transferred to a 5 mL volumetric
flask. Vehicle (50% PEG/H2O) was added and brought up to
volume. Preparation of po dosing solution: 20 mg of 20 was
weighed out and transferred to a 10 mL volumetric flask, 20%
vitamin E added, and the solution brought up to volume with
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