Discovery of tetrasubstituted imidazolines as potent and selective neuropeptide Y Y5 receptor antagonists: Reduced human ether-a-go-go related gene potassium channel binding affinity and potent antiobesity effect

Article abstract of DOI:10.1021/jm900110t

A series of novel imidazoline derivatives was synthesized and evaluated as neuropeptide Y (NPY) Y5 receptor antagonists. Optimization of previously reported imidazoline leads, 1a and 1b, was attempted by introduction of substituents at the 5-position on the imidazoline ring and modification of the bis(4-fluorphenyl) moiety. A number of potent derivatives without human ether-a-go-go related gene potassium channel (hERG) activity were identified. Selected compounds, including 2a, were shown to have excellent brain and CSF permeability. Compound 2a displayed a suitable pharmacokinetic profile for chronic in vivo studies and potently inhibited D-Trp³⁴NPY-induced acute food intake in rats. Oral administration of 2a resulted in a potent reduction of body weight in a diet-induced obese mouse model.

Full text of DOI:10.1021/jm900110t

J. Med. Chem. 2009, 52, 3385–3396
3385
Discovery of Tetrasubstituted Imidazolines as Potent and Selective Neuropeptide Y Y5 Receptor
Antagonists: Reduced Human Ether-a-go-go Related Gene Potassium Channel Binding Affinity
and Potent Antiobesity Effect
Nagaaki Sato,* Makoto Ando, Shiho Ishikawa, Makoto Jitsuoka, Keita Nagai, Hirobumi Takahashi, Aya Sakuraba,
Hiroyasu Tsuge, Hidefumi Kitazawa, Hisashi Iwaasa, Satoshi Mashiko, Akira Gomori, Ryuichi Moriya, Naoko Fujino,
Tomoyuki Ohe, Akane Ishihara, Akio Kanatani, and Takehiro Fukami
Tsukuba Research Institute, Merck Research Laboratories, Banyu Pharmaceutical Company, Ltd., Okubo 3, Tsukuba 300-2611, Japan
ReceiVed January 28, 2009
A series of novel imidazoline derivatives was synthesized and evaluated as neuropeptide Y (NPY) Y5 receptor
antagonists. Optimization of previously reported imidazoline leads, 1a and 1b, was attempted by introduction
of substituents at the 5-position on the imidazoline ring and modification of the bis(4-fluorphenyl) moiety.
A number of potent derivatives without human ether-a-go-go related gene potassium channel (hERG) activity
were identified. Selected compounds, including 2a, were shown to have excellent brain and CSF permeability.
Compound 2a displayed a suitable pharmacokinetic profile for chronic in vivo studies and potently inhibited
D-Trp34NPY-induced acute food intake in rats. Oral administration of 2a resulted in a potent reduction of
body weight in a diet-induced obese mouse model.
Introduction
Neuropeptide Y (NPY),a identified in 1982, is a 36-amino
acid peptide neurotransmitter. NPY is a member of the
pancreatic polypeptide family, which includes peptide YY and
pancreatic polypeptide.1 NPY is widely distributed in both the
central and the peripheral nervous systems and has various
physiological functions. Concentrations of NPY and its mRNA
in the hypothalamus are affected by feeding status, including
food deprivation and refeeding.2,3 Chronic central infusion of
NPY in rodents results in a syndrome similar to that in some
genetic obesity models, characterized by hyperphagia, insulin
resistance, hyperinsulinemia, and reduced thermogenic activity
in brown adipose tissue.4 Furthermore, NPY-deficient ob/ob
mice are less obese and have reduced food intake compared
with ob/ob mice.5 Therefore, NPY is thought to have a major
role in the physiological control of energy homeostasis.
Five distinct NPY receptor subtypes (Y1, Y2, Y4, Y5, and
mouse y6) have been cloned to date,6 and pharmacological data
suggest that the NPY Y5 receptor is involved in regulation of
feeding and energy expenditure. Administration of Y5 antago-
Figure 1. Structures of Y5 antagonist 1a, 1b, 25, and 26.
nists suppressed Y5 agonist-induced food intake and diet-
induced body weight gain,7,8 and mice lacking the Y5 receptor
have considerable utility to examine the mechanism-based
showed a reduced response to exogenously administered Y5
receptor agonists.9 In addition, chronic intracerebroventricular
administration of a Y5-specific agonist, D-Trp34NPY, lead to
obesity.10 These results suggest that Y5 is a key regulator
involved in the development of obesity in rodents; hence, Y5
antagonists have been targeted by many pharmaceutical com-
panies as potential antiobesity drugs.11 There are conflicting
reports regarding the antiobesity effects of Y5 antagonists in
rodents.8,11c,d Therefore, Y5 specific antagonists, which are
effective in wild-type mice but ineffective in Y5 knockout mice,
efficacy of Y5 antagonists in vivo. Our laboratory has reported
two structurally diverse Y5 specific antagonists, 25 and 26.8
Chronic administration of compound 25 suppressed body weight
gain in diet-induced obese (DIO) rodents while having no effects
on lean and genetically obese rodents, highlighting the patho-
physiological roles of Y5 in the diet-induced obesity that may
represent the significant populations of obese human subjects
in the westernized countries.8a Recently, it was reported that
administration of a potent and highly selective NPY Y5 receptor
antagonist, MK-557, resulted in modest weight loss in obese
human subjects.12 Considering the well-tolerated Y5 antagonist
treatment in humans and possibility of the combination therapy
with Y1 antagonists, development of safe and mechanistically
validated Y5 antagonists is of considerable importance.11d
We previously reported the discovery of a potent imidazoline
class of Y5 antagonists, 1a and 1b (Figure 1). After oral
administration, compound 1b was shown to inhibit food intake
* To whom correspondence should be addressed. Phone: +81-29-877-
2004. Fax: +81-29-877-2029. E-mail: nagaaki_sato@merck.com.
a Abbreviations: aCSF, artificial cerebrospinal fluid; APCI, atmospheric
pressure chemical ionization; Boc, tert-butoxycarbonyl; CNS, central
nervous system; CSF, cerebrospinal fluid; DIO, diet-induced obesity; ESI,
electrospray ionization; hERG, human ether-a-go-go related gene potassium
channel; NOE, nuclear Overhauser effect; NPY, neuropeptide Y; SAR,
structuresactivity relationship; SD rats, SpraguesDawley rats.
10.1021/jm900110t CCC: $40.75
2009 American Chemical Society
Published on Web 05/06/2009

3386 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 10
Table 1. In Vitro Profiles of 5-Substituted Derivatives 1c-ha
Sato et al.
a The values represent the mean ( SEM or the mean (n ) 3-4). b
[
125I]PYY binding assay in LMtk- cells expressing human recombinant Y5 receptors;
c
see Experimental Section. Displacement binding assay of [35S]N-[(4R)-1′-[(2R)-6-cyano-1,2,3,4-tetrahydro-2-naphthyl]-3,4-dihydro-4-hydroxyspiro[2H-1-
benzopyran-2,4′-piperidin]-6-yl]methanesulfonamide in membranes derived from HEK293 cells stably transfected with the hERG gene expressing the IKr
channel protein. d Octanol-water distribution coefficient at pH 7.4; see ref 24 for experimental details. e CLH was determined by the serum incubation
method using isolated rat hepatocytes. f ND ) not determined. g Hydrochloride salt.
Scheme 1. Preparation of Aminoalcohol Intermediates 11a-fa
a Reagents and conditions: (a) 4-fluorophenylmagnesium bromide, THF, 0 °C to rt. (b) (i) 5-Bromo-2-fluoropyridine, n-BuLi, Et2O, -78 °C; (ii) 5, Et2O,
-78 °C to rt. (c) 4 N HClsEtOAc, rt. (d) 10% Pd/C, H2, MeOH, rt; (e) TFA, CH2Cl2, rt.
in rats induced by centrally administered Y5-preferring agonist,
D-Trp34NPY, where the minimum effective dose of 1b was 30
mg/kg.13 Further investigation of leads 1a and 1b was necessary
to address issues such as moderate in vivo potency and potent
human ether-a-go-go related gene potassium channel (hERG)
inhibitory activity14 in order to identify clinical candidates from
this class (Table 1). In this report, compounds 1a and 1b were
further optimized by modification of the 2, 4, and 5-positions
of the imidazoline substituents, resulting in the identification
of a potent and selective derivative, 2a.
Chemistry. The synthetic route for the derivatives reported
herein is illustrated in Schemes 1-4. The aminoalcohol
intermediates 11a-f having bis(4-fluorophenyl) or bis(6-fluoro-
3-pyridyl) groups were prepared from esters 5,15a 6, 7,15b 8,15c
and 915d (Scheme 1). Esters 5-8 were converted to 10a-d by
coupling with 4-fluorophenylmagnesium bromide, followed by

Imidazolines as NPY Y5 Receptor Antagonists
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 10 3387
Scheme 2. Preparation of Compounds 1c-ia
a Reagents and conditions: (a) PPh3, Br2, Et3N, toluene, 0 °C. (b) NaN3, NH4Cl, DMF, H2O, 110 °C. (c) 10% Pd/C, H2, MeOH, rt. (d) Cat. Yb(OTf)3 or
Sc(OTf)3, ArCN, toluene, 100-110 °C. (e) Methyl arylcarboximidoate, MeOH, rt. (f) 6 N HCl, THF, rt.
Scheme 3. Preparation of Compounds 2a-oa
a Reagents and conditions: (a) (i) 5-Bromo-2-fluoropyridine, n-BuLi, Et2O, -78 °C; (ii) 15, Et2O, -78 °C to rt; (iii) DMSO, oxalyl chloride, CH2Cl2, -78
°C, then DIEA, -78 to 0 °C. (b) 4-Fluorophenylmagnesium bromide, THF, 0 °C. (c) TFA, CH2Cl2, rt. (d) PPh3, CCl3CCl3, Et3N, CH3CN, 50 °Csrt. (e)
NaN3, NH4Cl, DMF, H2O, 70 °C. (f) 10% Pd/C, H2, MeOH, rt. (g) Cat. Yb(OTf)3 or Sc(OTf)3, ArCN, toluene, 100-110 °C. (h) Methyl arylcarboximidoate,
MeOH, rt. (i) (i) ArCOOH, WSC ·HCl, pyridine, rt; (ii) Yb(OTf)3 or Sc(OTf)3, toluene, 130 °C. (j) TMSCN, Et3N, CH3CN, 80 °C. (k) (i) NH3, MeOH, -40
°C to rt; (ii) TFAA, Et3N, THF, -78 °C to rt. (l) NH3, MeOH, -40 °C to rt.
deprotection of the tert-butoxycarbonyl (Boc) group under acidic
conditions to afford the corresponding aminoalcohol 11a-d.
Ester 9 was converted to the corresponding N-protected ami-
noalcohol 10e in the same manner, which was hydrogenated to
give aminoalcohol 11e. Compound 11f was prepared from ethyl
ester 5. Ester 5 was coupled with (2-fluoropyridine-5-yl)lithium,
followed by deprotection of the Boc group to furnish the desired
intermediate 11f.
provided the corresponding aziridines 12a-f.16 The aziridine
ring was opened using sodium azide to give 13a-f,17 which
were reduced by hydrogenation to give diamines 14a-f.18
Formation of the imidazoline rings of 1c-f, 1g′, and 1h-i was
accomplished using reaction conditions d19 or e,20 as depicted
in Scheme 2. The methoxymethyl group of 1g′ was cleaved
under acidic conditions to give the desired imidazoline 1g.
Synthesis of compounds 2a-o is illustrated in Scheme 3.
Ketone 16 was prepared by the coupling of known aldehyde
15 with (2-fluoropyridin-5-yl)lithium, followed by oxidation of
the resultant alcohol. Coupling of 16 with 4-fluorophenylmag-
Synthesis of the imidazoline derivatives 1c-i from aminoal-
cohols 11a-f is shown in Scheme 2. Treatment of the
aminoalcohols 11a-f with triphenylphosphine and bromine

3388 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 10
Scheme 4. Preparation of Compounds 3 and 4a
Sato et al.
L-alanine. For compound 23, a characteristic NOE was observed
between the ortho-protons of the 6-fluoro-3-pyridyl group and
the methyl proton at the 5-position. For compound 24, a
characteristic NOE was observed between the ortho-protons of
the 4-fluorophenyl group and the methyl proton at the 5-position.
On the basis of these results, the absolute configurations of 23
and 24 were determined to be 5S,6R and 5S,6S, respectively.
Results and Discussion
The compounds were tested using the [125I]PYY binding assay
in membranes isolated from LMtk- cells transfected with cloned
human Y5 receptors. Selected compounds were evaluated for
hERG K+ channel binding activity using the [35S]N-[(4R)-1′-
[(2R)-6-cyano-1,2,3,4-tetrahydro-2-naphthyl]-3,4-dihydro-4-
hydroxyspiro[2H-1-benzopyran-2,4′-piperidin]-6-yl]methane-
sulfonamide competitive binding assay to assess QTc prolongation
liability.22 Rat hepatic clearance was assessed by the in vitro
serum incubation method previously reported by our labora-
tory.23 The 5-substituent of the imidazoline ring was investigated
initially (Table 1). Compound 1c, having a 5S-methyl group,
was twice as potent as the parent 1a and showed reduced hERG
activity. In contrast, the 5R isomer 1d showed a marked loss of
potency. Therefore, the 5S-methyl group of 1c was further
substituted as in the derivatives 1e, 1f, and 1g. The ethyl and
n-propyl derivatives (1e and 1f) were not as potent as 1c, and
their hERG activities were not improved. The hydroxymethyl
derivative 1g displayed ca. a 3-fold decrease in both hY5 and
hERG activities compared to 1c. A 5S-methyl group was
introduced into pyrazine lead 1b to afford 1h, resulting in loss
of the hERG activity while its hY5 activity was retained.
Unfortunately, compound 1h displayed relatively large predicted
rat hepatic clearance (predicted CLH ) 57 mL/min/kg). When
compounds 1c, 1g, and 1h were compared, hERG activity was
found to decrease as lipophilicity decreased. The literature also
suggests that a decrease in lipophilicity is effective for reducing
hERG activity.25 Hence, modification of the lipophilic 4,4-bis(4-
fluorophenyl) group was attempted (Table 2). Compounds 2a
and 2b, containing a 6-fluoropyridin-3-yl group,26 displayed
excellent hY5 binding and negligible hERG binding activities.
The log D7.4 values of 2a and 2b were found to be significantly
lower than that of 1c. It should be noted that the metabolic
stabilities of epimers 2a and 2b were strikingly different based
on their predicted rat hepatocyte clearance. Clearly, the nitrogen
atom of the fluoropyridine is recognized by metabolic enzymes.
The bis(fluoropyridine) derivative 1i showed a further decrease
in log D7.4 and negligible hERG activity. The methoxypyridine
derivative 3 exhibited potent hY5 activity; however, moderate
hERG activity and poor rat hepatic clearance were observed.
The pyridone derivative 4 was devoid of hY5 potency.
Modification of the 4- and 5-substituents of lead compounds
1a and 1b led to the identification of compound 2a, which has
both improved hY5 and hERG activities and good predicted
rat hepatic clearance. At this point, we decided to revisit the
structuresactivity relationship (SAR) for the 2-substituent, using
compound 2a as a template (Table 3). The 2-subsituents were
selected on the basis of our previous SAR study.13 The
cyanophenyl derivative 2c exhibited high affinity for hY5 but
moderate hERG affinity, probably due to its lipophilicity. In
contrast, the methanesulfonylphenyl derivative 2d had a slightly
reduced hY5 activity relative to 2a while its hERG activity was
negligible. When the cyanopyridine isomers 2e, 2f, and 2g were
compared, 2g was found to have a binding profile comparable
to the parent 2a, while 2e and 2f showed decreased activities.
In addition, 2g displayed good rat hepatic clearance. The
a Reagents and conditions: (a) 10% HClsMeOH, rt. (b) Pyridine-2,4-
dicarbonitrile, cat. Yb(OTf)3, toluene, 150 °C. (c) TMSCl, NaI, CH3CN,
rt.
Figure 2. Determination of the absolute configurations of compounds
23 and 24 by NOE.
nesium bromide was followed by removal of the Boc group to
give aminoalcohol 18 as a single isomer. Aminoalcohol 18 was
converted to diamines 21a and 21b using the same reaction
sequence as described for the preparation of 14a-f from 11a-f.
Aziridine formation of 18 was effected by treatment with a
preheated mixture of triphenylphosphine and hexachloroethane
to afford aziridines 19a and 19b in 39% and 22% yields,
respectively. Aziridines 19a and 19b were then reacted with
sodium azide followed by hydrogenation to give 21a and 21b,
which were converted to the imidazoline derivatives 2a-f, 2
g′-h′, 2i-j, 2k′, and 2l-o by reaction conditions g, h, or i
(Scheme 3). Compound 2g′ was converted to the pyridine
derivative 2g by treatment with trimethylsilyl cyanide. The ester
group of 2h′ was converted to the corresponding amide, which
was subsequently dehydrated by trifluoroacetic anhydride in the
presence of triethylamine to give cyanide 2h. Compound 2k′
was reacted with ammonia to give amide 2k. Compounds 3
and 4 were prepared from the diamine 21a as shown in Scheme
4. The 2-fluoro group of 21a was displaced by a methoxy group
by treatment with a methanolic solution of hydrogen chloride
to give 22, which was coupled with pyridine-2,4-dicarbonitrile
to give the imidazoline 3. Deprotection of the methoxy group
of 3 afforded 4.
Stereochemical Assignments of the Intermediates (18
and 21a). The stereochemistry of the 1-position of 18 and 21a
was supported by nuclear Overhauser effect (NOE) studies of
the corresponding morpholinone and piperazinone derivatives
23 and 24, as shown in Figure 2. Conversion to the cyclic
derivatives 23 and 24 was achieved without epimerization of
the chiral centers.21 The absolute configuration of the 5-methyl
group of 23 and 24 is S, which were derived from starting

Imidazolines as NPY Y5 Receptor Antagonists
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 10 3389
Table 2. In Vitro Profiles of Imidazoline Derivatives 1i, 2a, 2b, 3, and 4a
a The values represent the mean ( SEM or the mean (n ) 3-4). b 125I]PYY binding assay in LMtk- cells expressing human recombinant Y5 receptors.
[
c Displacement binding assay of [35S]N-[(4R)-1′-[(2R)-6-cyano-1,2,3,4-tetrahydro-2-naphthyl]-3,4-dihydro-4-hydroxyspiro[2H-1-benzopyran-2,4′-
piperidin]-6-yl]methanesulfonamide in membranes derived from HEK293 cells stably transfected with the hERG gene expressing the IKr channel protein.
d Octanol-water distribution coefficient at pH 7.4; see ref 24 for experimental details. e CLH was determined by the serum incubation method using isolated
rat hepatocytes. f Hydrochloride salt. g ND ) not determined.
cyanopyrimidine derivative 2h showed decreased activity. The
4-substituted pyridin-2-yl derivatives 2i, 2j, and 2k were
prepared and evaluated. The methoxy and chloro derivatives
(2i and 2j) were relatively lipophilic (log D7.4 values >3.5) and
showed moderate hERG binding activities. The aminocarbonyl
derivative 2k showed decreased hY5 activity. The pyrazine and
pyridone derivatives (2l and 2m) displayed suitable hY5 and
hERG activities. Regarding five-membered heterocycles, the
thiadiazole derivative 2n exhibited a binding profile comparable
to its pyrazine bioisoster analogue 2l. The thiazole derivative
2o exhibited decreased activity. Thus, this 2-substituent SAR
study revealed that a variety of aryl groups are tolerated in terms
of hY5 activity, and that a log D7.4 value below 3.3 seems to
be necessary to eliminate hERG activity.
is 15.2).27 In addition to the brain concentration, free brain
concentration is a critical factor to take into consideration for
efficacy of central nervous system (CNS) targeting agents. The
free brain concentration is estimated from the CSF concentration
for moderate to high-permeability compounds.28 The CSF levels
of the 4-(6-fluoropyridin-3-yl)imidazoline derivatives 2a, 2c, 2e,
and 2g are remarkably high due to their high brain levels and
improved CSF-to-brain ratios. It should be noted that an
excellent correlation was observed between the log D7.4 values
and CSF-to-brain ratios (Figure 3). This correlation is generally
valid for a series of structurally close analogues that have similar
pKa values (data not shown), probably because basicity is an
important parameter that significantly influences brain tissue
binding.
Compounds 1g, 2a, 2c, 2e, 2g, and 2m were evaluated for
brain and CSF permeability in SpraguesDawley (SD) rats
(Table 4). The data for previously reported compounds 1a, 1b,
and 1j are included in Table 3 for comparison.13 With the
exception of 2m, all the newly reported test compounds showed
good brain permeability based on brain concentrations and brain-
to-plasma ratios. The relatively low brain permeability of 2m
is probably attributed to its mouse P-gp susceptibility (the mouse
P-gp transcellular transport ratio (B-to-A/A-to-B ratio) for 2m
Highly brain and CSF permeable 2a was chosen for further
studies based on its high Y5 affinity (Ki ) 1.3 nM), reduced
hERG binding (IC50 >10 µM), and good metabolic stability in
rat hepatocytes (CLH ) 18 mL/min/kg). Antagonistic activity
of 2a was assessed by measuring the antagonist inhibition of
NPY-induced [Ca2+
]
increase in LMtk- cells expressing
i
recombinant human Y5 receptor. In this functional assay, 2a
showed potent antagonistic activity, with an IC50 value of 2.0
( 0.3 nM. Compound 2a also displayed excellent selectivity

3390 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 10
Table 3. In Vitro Profiles of Imidazoline Derivatives 2c-oa
Sato et al.
both in rats and monkeys (Table 5). Note that the plasma
clearance value (CLP ) 20 mL/min/kg) is in good agreement
with the predicted in vitro rat hepatic clearance (CLH ) 18.0
mL/min/kg).
Compound 2a was tested in an agonist-induced acute food
intake model (Figure 4). The compound was orally administered
2 h prior to icv dosing of either theY5 selective agonist
D-Trp34NPY or artificial CSF, then cumulated food intake was
measured for 2 h. Compound 2a showed potent dose-dependent
inhibition of food intake, with 93% inhibition achieved at 1
mg/kg.
The excellent in vivo efficacy of 2a in the acute food intake
model prompted us to evaluate 2a for chronic efficacy. The
chronic antiobesity effect of 2a was evaluated in established
DIO mice fed a moderately high-fat diet (Figure 5). Compound
2a was orally administered once daily for 4 days, then twice
daily for an additional 15 days. Compound 2a significantly
reduced the body weight of the established DIO mice at doses
of 3 and 10 mg/kg (Figure 5a), with apparent maximum efficacy
at 3 mg/kg. Body weight reduction was not observed in DIO
Y5 receptor deficient mice following 3 mg/kg twice daily dosing
of 2a for 15 days, clearly demonstrating the Y5-specific efficacy
of compound 2a in DIO mice (Figure 5b). Plasma exposure on
the final day was monitored (Figure 6). The plasma exposure
was dose-proportional in the DIO wild-type mice (3 mg/kg vs
10 mg/kg), and the plasma levels after 3 mg/kg dosing were
comparable in the DIO wild-type and Y5 deficient mice.
Conclusion
Previously reported imidazoline leads 1a and 1b were
optimized to remove hERG activity and potentiate in vivo
efficacy. Introduction of substituents at the 5-position of the
imidazoline ring and modification of the lipophilic 4,4-bis(4-
fluorophenyl) group led to the identification of the potent and
selective Y5 receptor antagonist 2a, which showed negligible
hERG activity and good pharmacokinetic profile and brain
penetration in rats. Compound 2a potently suppressed Y5
selective agonist D-Trp34NPY-induced food intake in SD rats.
The Y5 specific chronic antiobesity effect of 2a was demon-
strated by body weight reduction in established DIO mice.
The potential cardiovascular effects of compound 2a were
evaluated in anesthetized and ventilated dogs.31 At 2 mg/kg iv
dosing (Cmax ) 12 µM), no adverse treatment-related cardio-
vascular effects were observed. However, at 5 mg/kg iv dosing
(Cmax ) 25 µM), 2a produced significant changes in mean
arterial pressure (+28%), left ventricular systolic pressure
(+29%), cardiac contractility (+50%), cardiac output (+46%),
femoral blood flow (-32%), and a moderate prolongation of
the QTc interval (+7%).
Further characterization of compound 2a is ongoing to assess
its potential as a clinical development candidate. Efforts to
mitigate the cardiovascular liability by further structural modi-
fication are also ongoing.
a The values represent the mean ( SEM or the mean (n ) 3-4).
b
[
125I]PYY binding assay in LMtk- cells expressing human recombinant
c
Y5 receptors. Displacement binding assay of [35S]N-[(4R)-1′-[(2R)-6-
cyano-1,2,3,4-tetrahydro-2-naphthyl]-3,4-dihydro-4-hydroxyspiro[2H-1-ben-
zopyran-2,4′-piperidin]-6-yl]methanesulfonamide in membranes derived
from HEK293 cells stably transfected with the hERG gene expressing the
IKr channel protein. d Octanol-water distribution coefficient at pH 7.4; see
ref 24 for experimental details. e CLH was determined by the serum
incubation method using isolated rat hepatocytes. f ND ) not determined.
g Hydrochloride salt.
Experimental Section
Chemistry. General Procedures. Unless otherwise noted, all
solvents, chemicals, and reagents were obtained commercially and
1
used without purification. The H NMR spectra were obtained at
400 MHz on a MERCURY-400 (Varian) or a JMN-AL400 (JEOL)
spectrometer, with chemical shift (δ, ppm) reported relative to TMS
as an internal standard. Mass spectra were recorded with electron-
spray ionization (ESI) or atmospheric pressure chemical ionization
(APCI) on a Waters micromass ZQ, micromass Quattro II or
micromass Q-Tof-2 instrument. Flash chromatography was carried
out with prepacked silica gel columns (KP-Sil silica) from Biotage.
with respect to other NPY receptor subtypes (hY1, hY2, and
hY4 binding: >10 µM)29 and to a panel of 167 diverse, unrelated
binding sites (IC50 >1 µM for all the binding sites tested).
Compound 2a was shown to have good pharmacokinetic profiles

Imidazolines as NPY Y5 Receptor Antagonists
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 10 3391
Table 4. Brain and CSF Permeability of Selected Compoundsa
brain levelb
(nmol/g)
CSF levelb
(µM)
brain-to-plasma
ratio
CSF-to-brain
ratio
compd
plasmab (µM)
log D7.4
1ac
1bc
1g
1.3
1.2
1.1
1.4
2.6
2.6
4.4
3.7
3.7
6.4
5.9
2.3
8.1
7.0
6.1
5.3
3.2
0.36
0.025
0.064
0.057
0.027
0.21
0.13
0.46
0.22
0.050
4.9
4.9
2.1
5.8
2.7
2.3
1.2
0.9
0.1
0.004
0.011
0.025
0.003
0.029
0.022
0.087
0.069
0.139
4.0
3.5
3.4
4.3
3.2
3.6
2.9
3.2
2.4
1jc
2a
2c
2e
2g
2m
a The plasma, brain, and CSF concentrations were determined 2 h after oral administration of 10 mg/kg of test compounds. b The values represent the
mean (n ) 2-3). c The synthesis and brain penetration data of, 1a, 1b, and 3-[4,4-bis(4-fluorophenyl)-4,5-dihydro-1H-imidazol-2-yl]benzonitrile (1j) were
reported previously. See ref 13 for details.
the target compounds was determined by HPLC with the two
different eluting methods as follows. Analytical HPLC was
performed on a SPELCO Ascentis Express (150 mm × 4.6 mm
id), eluting with a gradient of (A) 0.1% H3PO4/CH3CN ) 95/5 to
10/90 over 7 min followed by 10/90 isocratic over 1 min and (B)
10 mM potassium phosphate buffer (pH 6.6)/CH3CN ) 95/5 to
20/80 over 7 min followed by 20/80 isocratic over 1 min (detection
at 210 nm). All tested compounds had a purity of >95%. High
resolution mass spectra were recorded with electron-spray ionization
(ESI) on a micromass Q-Tof-2 instrument.
2-[(5S)-4,4-Bis(4-fluorophenyl)-5-methyl-4,5-dihydro-1H-imida-
zol-2-yl]-4-pyridinecarbonitrile (1c). To a solution of 14a (200 mg,
0.76 mmol) and 2,4-pyridinedicarbonitrile (108 mg, 0.84 mmol)
in toluene (5.0 mL) was added ytterbium tris(trifluoromethane-
sulfonate) (47 mg, 0.076 mmol), and the mixture was heated to
100 °C for 15 h in a sealed tube. The resultant mixture was
partitioned between ethyl acetate and saturated aqueous sodium
hydrogen carbonate, and the layers were separated. The aqueous
layer was extracted with ethyl acetate, and the combined organic
layers were washed with brine, dried over sodium sulfate, and
concentrated. The residue was purified by preparative HPLC with
Figure 3. Relationship between log D7.4 values and CSF-to-brain ratios
of compounds in Table 4.
10-50% 0.1% CF3CO2H-CH3CN in 0.1% aq CF3CO2H to give 1c
Table 5. Phamacokinetic Parameters of 2aa
(49.6 mg, 17% yield) as a colorless solid; HPLC purity: 98.8%.
1H NMR (400 MHz, CD3OD): δ 0.89 (3H, d, J ) 6.4 Hz),
4.83-4.90 (1H, m), 7.00-7.10 (4H, m), 7.20-7.25 (2H, m),
7.50-7.55 (2H, m), 7.82 (1H, dd, J ) 1.2, 4.8 Hz), 8.37-8.43
(1H, m), 8.87-8.90 (1H, m). HRMS (ES+) calcd for C22H17N4F2
[M + H]+ m/z 375.1421, found m/z 375.1418. [R]25D: -341° (c
1.00, CH3OH).
iv CLP
Vdss
PO AUC0-∞
Cmax
(µM)
species
(mL/min/kg)
(L/kg)
(µM·h)
F (%)
ratb
20
8.0
6.2
4.4
1.8
4.6
0.50
0.59
77
76
monkeyc
a The values represent the mean (n ) 3). b 1 mg/kg po and 1 mg/kg iv
in n ) 3 animals/dose. c 1 mg/kg po and 0.3 mg/kg iv in n ) 3 animals/
dose.
2-[(5R)-4,4-Bis(4-fluorophenyl)-5-methyl-4,5-dihydro-1H-imida-
zol-2-yl]-4-pyridinecarbonitrile (1d). Compound 1d was prepared
from 14b using the procedure described for 1c as a pale-pink solid
(22% yield); HPLC purity: 98.5%. 1H NMR (400 MHz, CD3OD):
δ 0.88 (3H, d, J ) 6.8 Hz), 4.80-4.90 (1H, m), 6.98-7.10 (4H,
m), 7.18-7.26 (2H, m), 7.48-7.54 (2H, m),7.78-7.83 (1H, m),
8.40 (1H, s), 8.86 (1H, d, J ) 4.8 Hz); HRMS (ES+) calcd for
C22H17N4F2 [M + H]+ m/z 375.1421, found m/z 375.1413. [R]25
+317° (c 0.60, CH3OH).
:
D
2-[(5S)-5-Ethyl-4,4-bis(4-fluorophenyl)-4,5-dihydro-1H-imidazol-
2-yl]-4-pyridinecarbonitrile (1e). Compound 1e was prepared from
14c using the procedure described for 1c as a pale-yellow solid
(28% yield); HPLC purity: 97.7%. 1H NMR (400 MHz, CD3OD):
δ 0.90-1.10 (4H, m), 1.16-1.28 (1H, m), 4.50-4.65 (1H, m),
6.97-7.10 (4H, m), 7.15-7.23 (2H, m), 7.47-7.55 (2H, m), 7.83
(1H, dd, J ) 1.2, 4.8 Hz), 8.42 (1H, s), 8.85-8.90 (1H, m). HRMS
(ES+) calcd for C23H19N4F2 [M + H]+ m/z 389.1578, found m/z
389.1571. [R]25D: -255° (c 1.00, CH3OH).
2-[(5S)-4,4-Bis(4-fluorophenyl)-5-propyl-4,5-dihydro-1H-imida-
zol-2-yl]-4-pyridinecarbonitrile (1f). Compound 1f was prepared
from 14d using the procedure described for 1c as a colorless
amorphous (39% yield); HPLC purity: 98.2%. 1H NMR (400 MHz,
CD3OD): δ 0.83 (3H, t, J ) 7.2 Hz), 0.95-1.20 (2H, m), 1.30-1.60
(2H, m), 4.60-4.70 (1H, m), 6.98-7.12 (4H, m), 7.14-7.22 (2H,
m), 7.47-7.56 (2H, m), 7.83 (1H, dd, J ) 1.6, 4.8 Hz), 8.42 (1H,
Figure 4. Effect of 2a on food intake induced by D-Trp34NPY.
Compound 2a was orally administered 2 h before third ventricle
injection of D-Trp34NPY (1 µg/head). The values represent the mean
( SEM (n ) 5-9). ** P < 0.01 (compared with only D-Trp34NPY
treated group).
Preparative thin-layer chromatography (TLC) was performed on a
TLC Silica Gel 60 F (Merck KGaA). Preparative HPLC purification
was carried out on a YMC-Pack Pro C18 (YMC, 50 mm × 30
mm id), eluting with a gradient of CH3CN/0.1% aq CF3CO2H )
10/90 to 50/50 over 8 min at a flow rate of 40 mL/min. Purity of

3392 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 10
Sato et al.
Figure 5. Effect of compound 2a on body weight of established DIO mice (a) and established DIO Y5 receptor deficient mice (b) fed a moderately
high-fat diet. The values represent the mean ( SEM (n ) 7-10). * P < 0.05, ** P < 0.01 (compared with vehicle).
2-[(5S)-4,4-Bis(4-fluorophenyl)-5-methyl-4,5-dihydro-1H-imida-
zol-2-yl]pyrazine Hydrochloride (1h Hydrochloride). To a stirred
solution of 14a (50.0 mg, 0.191 mmol) in methanol (1.0 mL) was
added methyl pyrazine-2-carboximidoate methanesulfonate30 (44.0
mg, 0.191 mmol) at room temperature, and the mixture was stirred
at room temperature for 2 days under a nitrogen atmosphere. The
reaction mixture was partitioned between ethyl acetate and saturated
aqueous sodium hydrogen carbonate, and the layers were separated.
The aqueous layer was extracted with ethyl acetate, and the
combined organic layers were washed with brine, dried over sodium
sulfate, and concentrated. The residue was purified by flush
chromatography with 33% ethyl acetate in hexanes to give 1h. A
hydrochloride salt of 1h was prepared by treatment with 4 N
hydrogen chloride in dioxane as a colorless foam (49.0 mg, 73%
yield); HPLC purity: 99.4%. 1H NMR (300 MHz, CDCl3):
tautomers δ 0.90 (2.1H, d, J ) 6.4 Hz), 0.99 (0.9H, d, J ) 6.9
Hz), 4.70 (0.7H, q, J ) 6.4 Hz), 5.03 (0.3H, q, J ) 6.9 Hz),
5.97-6.00 (0.7H, m), 6.43-6.45 (0.3H, m), 6.95-7.08 (4H, m),
7.25-7.37 (2.8H, m), 7.51-7.56 (1.2H, m), 8.53-8.55 (1H, m),
8.66 (1H, d, J ) 2.5 Hz), 9.42 (0.3 H, d, J ) 1.7 Hz), 9.54 (0.7H,
d, J ) 1.5 Hz). HRMS (ES+) calcd for C20H17N4F2 [M + H]+ m/z
351.1421, found m/z 351.1412. [R]25D: -289° (c 0.21, CH3OH).
2-[(5S)-4,4-Bis(6-fluoro-3-pyridyl)-5-methyl-4,5-dihydro-1H-imi-
dazol-2-yl]-4-pyridinecarbonitrile (1i). Compound 1i was prepared
from 14f using the procedure described for 1c as a pale-yellow
solid (46% yield); HPLC purity: 99.6%. 1H NMR (300 MHz,
CDCl3) tautomers δ 0.93 (2.7H, d, J ) 6.4 Hz), 1.01 (0.3H, d, J )
6.8 Hz), 4.77 (0.9H, d, J ) 6.4 Hz), 5.00-5.10 (0.1H, m), 6.25
(0.9H, brs), 6.60 (0.1H, brs), 6.89-6.92 (2H, m), 7.62 (1H, dd, J
) 1.3, 5.0 Hz), 7.69-7.76 (1H, m), 7.89-7.95 (1H, m), 8.14-8.15
(1H, m), 8.40-8.43 (1H, m), 8.53 (1H, s), 8.77 (1H, d, J ) 5.0
Hz). HRMS (ES+) calcd for C20H15N6F2 [M + H]+ m/z 377.1326,
found m/z 377.1327. [R]25D: -333° (c 0.50, CH3OH).
2-[(4S,5S)-4-(4-Fluorophenyl)-4-(6-fluoro-3-pyridyl)-5-methyl-
4,5-dihydro-1H-imidazol-2-yl]-4-pyridinecarbonitrile (2a). Com-
pound 2a was prepared from 21a using the procedure described
for 1c as a colorless amorphous (69% yield); HPLC purity: 98.1%.
1H NMR (300 MHz, CDCl3): tautomers δ 0.87 (2.4H, d, J ) 6.7
Hz), 0.98 (0.6H, d, J ) 7.1 Hz), 4.73 (0.8H, q, J ) 6.7 Hz), 4.98
(0.2H, d, J ) 7.1 Hz), 6.15 (0.8H, s), 6.52 (0.2H, s), 6.89 (0.8H,
dd, J ) 2.8, 8.5 Hz), 6.85-6.95 (0.2H, m), 6.95-7.10 (2H, m),
7.15-7.30 (2H, m), 7.61 (1H, d, J ) 4.4 Hz), 7.72-7.82 (0.2H,
m), 7.90 (0.8H, dt, J ) 2.8, 8.5 Hz), 8.20-8.30 (0.2H, m), 8.39
(0.8H, d, J ) 3.5 Hz), 8.42-8.47 (0.2H, m), 8.54 (0.8H, d, J )
1.6 Hz), 8.70-8.80 (1H, m). HRMS (ES+) calcd for C21H16N5F2
[M + H]+ m/z 376.1374, found m/z 376.1369. [R]25D: -417° (c
1.00, CHCl3).
Figure 6. Plasma drug levels after the final oral dosing of compound
2a in chronic in vivo efficacy studies in MHF diet-fed C57BL/6N mice
and MHF diet-fed Y5 receptor deficient mice. See Experimental Section
for details.
s), 8.84-8.90 (1H, m). HRMS (ES+) calcd for C24H21N4F2 [M +
H]+ m/z 403.1734, found m/z 403.1729. [R]25D: -262° (c 0.56,
CH3OH).
2-[(5R)-4,4-Bis(4-fluorophenyl)-5-(hydroxymethyl)-4,5-dihydro-
1H-imidazol-2-yl]-4-pyridinecarbonitrile (1g). To a solution of 14e
(1.50 g, 4.65 mmol) and 2,4-pyridinedicarbonitrile (601 mg, 4.65
mmol) in toluene (30 mL) was added ytterbium tris(trifluo-
romethanesulfonate) (289 mg, 0.465 mmol), and the mixture was
heated to reflux for 2 days in a sealed tube. The resultant mixture
was partitioned between ethyl acetate and saturated aqueous sodium
hydrogen carbonate, and the layers were separated. The aqueous
layer was extracted with ethyl acetate, and the combined organic
layers were washed with brine, dried over sodium sulfate, concen-
trated, and vacuum-dried. The crude material was dissolved in THF
(20 mL) and treated with 6 N hydrochloric acid (10 mL), and the
mixture was stirred for 1 day. After being neutralized with solid
sodium hydrogen carbonate, the resulting mixture was partitioned
between ethyl acetate and saturated aqueous sodium hydrogen
carbonate. The layers were separated, and the aqueous layer was
extracted with ethyl acetate. The combined organic layers were
washed with brine, dried over sodium sulfate, and concentrated.
The residue was purified by flash chromatography with 50% ethyl
acetate in hexanes to give 1g (385 mg, 21% yield over 2 steps) as
1
a pale-yellow foam; HPLC purity: 96.7%. H NMR (300 MHz,
CDCl3): tautomers δ 3.08-3.20 (0.6H, m), 3.30 (1.4H, dd, J )
4.5, 10.8 Hz), 4.69 (0.7H, dd, J ) 4.5, 8.9 Hz), 4.68 (0.3H, dd, J
) 4.3, 8.2 Hz), 6.59 (0.7H, s), 6.66 (0.3H, s), 6.93-7.09 (4H, m),
7.18-7.30 (2H, m), 7.30-7.40 (0.6H, m), 7.45-7.55 (1.4H, m),
7.55-7.63 (1H, m), 8.45 (0.3H, s), 8.57 (0.7H, s), 8.73-8.80 (1H,
m). HRMS (ES+) calcd for C22H17N4OF2 [M + H]+ m/z 391.1370,
found m/z 391.1367. [R]25D: -313° (c 1.00, CHCl3).

Imidazolines as NPY Y5 Receptor Antagonists
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 10 3393
2-[(4R,5S)-4-(4-Fluorophenyl)-4-(6-fluoro-3-pyridyl)-5-methyl-
4,5-dihydro-1H-imidazol-2-yl]-4-pyridinecarbonitrile (2b). Com-
pound 2b was prepared from 21b using the procedure described
for 1c as a yellow solid (55% yield); HPLC purity: 95.5%. 1H NMR
(300 MHz, CDCl3): tautomers δ 0.96 (2.4H, d, J ) 6.5 Hz), 1.01
(0.6H, d, J ) 6.9 Hz), 4.76 (0.8H, q, J ) 6.5 Hz), 5.07 (0.2H, q,
J ) 6.9 Hz), 6.14 (0.8H, brs), 6.54 (0.2H, brs), 6.84-6.91 (1H,
m), 7.00-7.10 (2H, m), 7.30-7.37 (0.4H, m), 7.48-7.56 (1.6H,
m), 7.60-7.70 (2H, m), 8.08 (0.8H, d, J ) 2.6 Hz), 8.15 (0.2H, d,
J ) 2.6 Hz), 8.45 (0.2H, s), 8.57 (0.8H, s), 8.74-8.78 (1H, m).
HRMS (ES+) calcd for C21H16N5F2 [M + H]+ m/z 376.1374, found
m/z 376.1378. [R]25D: -291° (c 0.80, CH3OH).
Hz), 7.85-8.00 (2H, m), 8.39 (1H, d, J ) 1.6 Hz), 8.51 (1H, d, J
) 7.3 Hz). HRMS (ES+) calcd for C21H16N5F2 [M + H]+ m/z
376.1374, found m/z 376.1380. [R]25D: -335° (c 0.20, CH3OH).
4-[(4S,5S)-4-(4-Fluorophenyl)-4-(6-fluoro-3-pyridyl)-5-methyl-
4,5-dihydro-1H-imidazol-2-yl]pyridine 1-oxide (2g′). To a stirred
solution of 4-pyridinecarbonitrile 1-oxide (100 mg, 0.83 mmol) in
methanol (4.0 mL) was added sodium methoxide (13.5 mg, 0.25
mmol) at room temperature, and the mixture was stirred at room
temperature for 16 h before being neutralized with methanesulfonic
acid (70 µL, 1.1 mmol). To the mixture was added 21a (158 mg,
0.60 mmol) in methanol (2.0 mL) at room temperature, and the
mixture was stirred at room temperature for an additional 16 h and
concentrated. The residue was partitioned between chloroform and
saturated aqueous sodium hydrogen carbonate, and the layers were
separated. The aqueous layer was extracted with chloroform, and
the combined organic layers were dried over sodium sulfate, filtered,
and concentrated. The residue was purified by flush chromatography
with 10% methanol in chloroform to give 2g′ (216 mg, 77% yield)
as a colorless oil. 1H NMR (300 MHz, CDCl3): δ 0.89 (3H, d, J )
7.5 Hz), 4.69 (1H, q, J ) 7.5 Hz), 5.10-5.25 (1H, m), 6.87 (1H,
dd, J ) 3.0, 7.5 Hz), 7.15-7.30 (2H, m), 7.75 (2H, d, J ) 6.8
Hz), 7.80-7.92 (1H, m), 8.20 (2H, J ) 6.8 Hz), 8.37 (1H, s).MS
(ESI): m/z 367 [M + H]+.
4-[(4S,5S)-4-(4-Fluorophenyl)-4-(6-fluoro-3-pyridyl)-5-methyl-
4,5-dihydro-1H-imidazol-2-yl]-2-pyridinecarbonitrile (2g). To a
stirred solution of 2g′ (172 mg, 0.47 mmol) and triethylamine (332
µL, 2.4 mmol) in acetonitrile (5.0 mL) was added trimethylsilan-
ecarbonitrile (301 µL, 2.4 mmol) at room temperature, and the
mixture was heated to 90 °C for 17 h in a sealed tube. The reaction
mixture was concentrated, and the resulting residue was purified
by flash chromatography with 50% ethyl acetate in hexanes to give
2g (108 mg, 61% yield) as a colorless foam; HPLC purity: 99.1%.
1H NMR (300 MHz, CDCl3): tautomers δ 0.89 (2.7H, d, J ) 6.5
Hz), 0.96 (0.3H, d, J ) 6.7 Hz), 4.74 (0.9H, q, J ) 6.5 Hz),
4.90-5.00 (0.1H, m), 5.12 (0.9H, s), 5.40 (0.1H, s), 6.88 (1H, dd,
J ) 3.0, 8.6 Hz), 7.00-7.10 (2H, m), 7.18-7.30 (2H, m),
7.80-7.95 (2H, m), 8.12 (0.1H, s), 8.18 (0.9H, d, J ) 1.0 Hz),
8.20-8.25 (0.1H, s), 8.38 (0.9H, d, J ) 2.1 Hz), 8.82 (1H, d, J )
5.1 Hz). HRMS (ES+) calcd for C21H16N5F2 [M + H]+ m/z
376.1374, found m/z 376.1369. [R]25D: -322° (c 0.68, CH3OH).
Ethyl 2-[(4S,5S)-4-(4-Fluorophenyl)-4-(6-fluoro-3-pyridyl)-5-
methyl-4,5-dihydro-1H-imidazol-2-yl]-4-pyrimidinecarboxylate (2h′).
Compound 2h′ was prepared from 21a, ethyl 2-cyanopyrimidine-
4-carboxylate,30 and scandium tris(trifluoromethanesulfonate) in
place of ytterbium tris(trifluoromethanesulfonate) using the proce-
dure described for 1c as a brown amorphous (85% yield). 1H NMR
(300 MHz, CDCl3): δ 0.90 (3H, d, J ) 6.3 Hz), 1.46 (3H, t, J )
7.2 Hz), 4.51 (2H, q, J ) 7.2 Hz), 4.80 (1H, q, J ) 6.3 Hz),
6.80-6.90 (1H, m), 6.95-7.10 (3H, m), 7.20-7.40 (2H, m),
7.90-8.00 (1H, m), 8.03 (1H, d, J ) 4.9 Hz), 8.43 (1H, brs), 9.14
(1H, d, J ) 4.9 Hz). MS (ESI): m/z 424 [M + H]+.
3-[(4S,5S)-4-(4-Fluorophenyl)-4-(6-fluoro-3-pyridyl)-5-methyl-
4,5-dihydro-1H-imidazol-2-yl]benzonitrile (2c). To a stirred solution
of the 21a (100 mg, 0.380 mmol) and 3-cyanobenzoic acid (84.0
mg, 0.571 mmol) in chloroform (2.0 mL) and pyridine (2.0 mL)
was added 1-(3-diemthylaminopropyl)-3-ethylcarbodiimide hydro-
chloride (109 mg, 0.570 mmol) at 0 °C, and the mixture was
allowed to warm to room temperature and stirred for 18 h. After
being concentrated, the residue was partitioned between ethyl
acetate and saturated aqueous sodium hydrogen carbonate, and the
layers were separated. The aqueous layer was extracted with ethyl
acetate, and the combined organic layers were washed with brine,
dried over sodium sulfate, concentrated, and vacuum-dried. The
crude material was dissolved in toluene (2.0 mL) and treated with
ytterbium tris(trifluoromethanesulfonate) (24.0 mg, 0.0380 mmol),
and the mixture was heated to 130 °C for 15 h in a sealed tube.
The reaction mixture was partitioned between chloroform and
saturated aqueous sodium hydrogen carbonate, and the layers were
separated. The aqueous layer was extracted with chloroform, and
the combined organic layers were dried over sodium sulfate and
concentrated. The residue was purified by flash chromatography
with 50% ethyl acetate to give 2c (140 mg, 98% yield over 2 steps)
1
as a colorless foam; HPLC purity: 95.3%. H NMR (300 MHz,
CDCl3): δ 0.89 (3H, d, J ) 6.5 Hz), 4.66-4.74 (1H, m), 5.10 (1H,
brs), 6.85-6.90 (1H, m), 7.02 (2H, t, J ) 8.4 Hz), 7.20-7.30 (2H,
m), 7.57 (1H, t, J ) 7.9 Hz), 7.74-7.79 (1H, m), 7.85-7.95 (1H,
m), 8.07-8.12 (1H, m), 8.19 (1H, s), 8.37-8.42 (1H, m). HRMS
(ES+) calcd for C22H17N4F2 [M + H]+ m/z 375.1421, found m/z
375.1419. [R]25D: -296° (c 1.00, CHCl3).
2-Fluoro-5-{(4S,5S)-4-(4-fluorophenyl)-5-methyl-2-[3-(methylsul-
fonyl)phenyl]-4,5-dihydro-1H-imidazol-4-yl}pyridine (2d). Com-
pound 2d was prepared from 21a and 3-(methylsulfonyl)benzoic
acid using the procedure described for 2c as a pale-yellow
amorphous (24% yield over 2 steps); HPLC purity: 97.5%. 1H NMR
(300 MHz, CDCl3): δ 0.90 (3H, d, J ) 6.3 Hz), 3.10 (3H, s),
4.70-4.80 (1H, m), 5.25 (1H, brs), 6.87 (1H, dd, J ) 2.8, 8.3 Hz),
7.02 (2H, t, J ) 8.6 Hz), 7.20-7.30 (2H, m), 7.66 (1H, t, J ) 7.8
Hz), 8.89 (1H, dt, J ) 2.8, 8.3 Hz), 8.07 (1H, dd, J ) 1.2, 7.8 Hz),
8.23 (1H, d, J ) 7.8 Hz), 8.37 (2H, s). HRMS (ES+) calcd for
C22H20N3O2F2S [M + H]+ m/z 428.1244, found m/z 428.1238.
[R]25D: -263° (c 0.90, CHCl3).
5-[(4S,5S)-4-(4-Fluorophenyl)-4-(6-fluoro-3-pyridyl)-5-methyl-
4,5-dihydro-1H-imidazol-2-yl]-3-pyridinecarbonitrile (2e). Com-
pound 2e was prepared from 21a and 5-cyanonicotinic acid30 using
the procedure described for 2c as a colorless foam (17% yield over
2 steps); HPLC purity: 98.6%. 1H NMR (300 MHz, CDCl3) δ 0.90
(3H, d, J ) 6.3 Hz), 4.75 (1H, q, J ) 6.3 Hz), 5.38 (1H, s), 6.89
(1H, dd, J ) 2.9, 8.6 Hz), 7.03 (2H, t, J ) 8.6 Hz), 7.20-7.30
(2H, m), 7.85-7.95 (1H, m), 8.35-8.45 (1H, m), 8.54 (1H, s),
8.96 (1H, d, J ) 2.0 Hz), 9.23 (1H, d, J ) 2.0 Hz). HRMS (ES+)
calcd for C21H16N5F2 [M + H]+ m/z 376.1374, found m/z 376.1370.
[R]25D: -245° (c 0.56, CH3OH).
2-[(4S,5S)-4-(4-Fluorophenyl)-4-(6-fluoro-3-pyridyl)-5-methyl-
4,5-dihydro-1H-imidazol-2-yl]-4-pyrimidinecarbonitrile (2h). Com-
pound 2h′ (405 mg, 0.96 mmol) was dissolved in methanol (10
mL) and cooled to -78 °C. Ammonia gas was bubbled into the
solution for 20 min, and the mixture was allowed to warm to room
temperature and stirred for 22 h. The resulting mixture was
concentrated, and the residue was dissolved in ethyl acetate and
filtered. The filtrate was concentrated, and the residue was purified
by flash chromatography with 50% ethyl acetate in chloroform and
then 10% methanol in chloroform to give 2-[(4S,5S)-4-(4-fluo-
rophenyl)-4-(6-fluoro-3-pyridyl)-5-methyl-4,5-dihydro-1H-imidazol-
2-yl]-4-pyrimidinecarboxamide (351 mg) as a colorless amorphous.
1H NMR (300 MHz, CDCl3): δ 0.90 (3H, d, J ) 6.3 Hz), 4.70-4.85
(1H, m), 5.80-5.90 (1H, m), 6.24 (1H, brs), 6.80-6.95 (1H, m),
6.98-7.10 (3H, m), 7.20-7.35 (2H, m), 7.90-8.00 (1H, m), 8.21
(1H, d, J ) 4.9 Hz), 8.42 (1H, brs), 9.08 (1H, d, J ) 4.9 Hz). MS
(ESI): m/z 395 [M + H]+. To a stirred solution of the amide (337
mg, 0.85 mmol) and triethylamine (1.20 mL, 8.62 mmol) in THF
(1.7 mL) was added trifluoroacetic anhydride (360 µL, 2.55 mmol)
at -78 °C, and the mixture was allowed to warm to room
6-[(4S,5S)-4-(4-Fluorophenyl)-4-(6-fluoro-3-pyridyl)-5-methyl-
4,5-dihydro-1H-imidazol-2-yl]-2-pyridinecarbonitrile (2f). Com-
pound 2f was prepared from 21a and pyridine-2,6-dicarbonitrile
using the procedure described for 1c as a colorless oil (13% yield);
1
HPLC purity: 97.3%. H NMR (300 MHz, CDCl3): tautomers δ
0.88 (2.4H, d, J ) 6.5 Hz), 0.95-1.00 (0.6H, m), 4.72 (0.8H, q, J
) 6.5 Hz), 4.95-5.05 (0.2H, m), 6.17 (1H, s), 6.82-6.92 (1H,
m), 6.98-7.10 (2H, m), 7.20-7.30 (2H, m), 7.79 (1H, d, J ) 6.6

3394 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 10
Sato et al.
temperature and stirred for 19 h. The reaction mixture was
partitioned between ethyl acetate and water, and the layers were
separated. The aqueous layer was extracted with ethyl acetate, and
the combined organic layers were washed with brine, dried over
magnesium sulfate, and concentrated. The residue was purified by
flash chromatography with 50% ethyl acetate in chloroform to give
2h (253 mg, 62% yield over 2 steps) as a brown amorphous; HPLC
(1H, m), 8.80 (1H, d, J ) 2.6 Hz), 9.51 (1H, s). HRMS (ES+)
calcd for C19H16N5F2 [M + H]+ m/z 352.1374, found m/z 352.1371.
[R]25D: -335° (c 0.53, CH3OH).
6-[(4S,5S)-4-(4-Fluorophenyl)-4-(6-fluoro-3-pyridyl)-5-methyl-
4,5-dihydro-1H-imidazol-2-yl]-2(1H)-pyridinone (2m). Compound
2m was prepared from 21a and 6-oxo-1,6-dihydropyridine-2-
carboxylic acid using the procedure described for 2c as a colorless
amorphous (58% yield over 2 steps); HPLC purity: 97.6%. 1H NMR
(300 MHz, CDCl3): δ 0.87 (3H, d, J ) 6.5 Hz), 4.70-4.80 (1H,
m), 5.65-5.85 (1H, m), 6.65-6.80 (2H, m), 6.86-6.92 (1H, m),
6.98-7.06 (2H, m), 7.15-7.24 (2H, m), 7.45-7.52 (1H, m),
7.80-7.88 (1H, m), 8.33 (1H, s). HRMS (ES+) calcd for
1
purity: 97.1%. H NMR (300 MHz, CD3OD): δ 0.86 (3H, d, J )
6.6 Hz), 4.80-4.93 (1H, m), 6.95-7.13 (3H, m), 7.26-7.35 (2H,
m), 8.00-8.10 (2H, m), 8.35-8.40 (1H, m), 9.18 (1H, d, J ) 4.9
Hz). HRMS (ES+) calcd for C20H15N6F2 [M + H]+ m/z 377.1326,
found m/z 377.1326. [R]25D: -351° (c 1.00, CH3OH).
2-[(4S,5S)-4-(4-Fluorophenyl)-4-(6-fluoro-3-pyridyl)-5-methyl-
4,5-dihydro-1H-imidazol-2-yl]-4-(methyloxy)pyridine (2i). Com-
pound 2i was prepared from 21a and 4-(methyloxy)-2-pyridine-
carbonitrile30 using the procedure described for 1c as a colorless
foam (62% yield); HPLC purity: 95.5%. 1H NMR (300 MHz,
CDCl3): tautomers δ 0.87 (3H, d, J ) 6.3 Hz), 3.92 (3H, s),
4.60-4.75 (0.8H, m), 4.90-5.00 (0.2H, m), 5.55-5.70 (0.2H, m),
6.22 (0.8H, brs), 6.83-7.08 (4H, m), 7.20-7.30 (2H, m), 7.75-7.85
(1H, m), 7.90-8.00 (1H, m), 8.35-8.45 (2H, m). HRMS (ES+)
calcd for C21H19N4OF2 [M + H]+ m/z 381.1527, found m/z
381.1523. [R]25D: -334° (c 0.52, CH3OH).
C20H17N4OF2 [M + H]+ m/z 367.1370, found m/z 367.1374. [R]25
-259° (c 0.70, CH3OH).
:
D
2-Fluoro-5-[(4S,5S)-4-(4-fluorophenyl)-5-methyl-2-(1,2,5-thiadia-
zol-3-yl)-4,5-dihydro-1H-imidazol-4-yl]pyridine (2n). Compound 2n
was prepared from 21a and 1,2,5-thiadiazole-3-carboxylic acid using
the procedure described for 2c as a colorless oil (54% yield over 2
steps); HPLC purity: 99.5%. 1H NMR (300 MHz, CDCl3):
tautomers δ 0.88 (2.4H, d, J ) 6.5 Hz), 0.98 (0.6H, d, J ) 6.9
Hz), 4.70 (0.8H, q, J ) 6.5 Hz), 4.90-5.00 (0.2H, m), 5.72 (0.8H,
s), 6.08 (0.2H, s), 6.85-6.95 (1H, m), 7.00-7.10 (2H, m),
7.20-7.30 (2H, m), 7.73-7.82 (0.2H, m), 7.90 (0.8H, dt, J ) 2.6,
7.6 Hz), 8.25-8.30 (0.2H, m), 8.39 (0.8H, d, J ) 1.7 Hz), 9.14
(0.2H, s), 9.20 (0.8H, s). HRMS (ES+) calcd for C17H14N5F2S [M
+ H]+ m/z 358.0938, found m/z 358.0937. [R]25D: -369° (c 1.00,
CH3OH).
2-Fluoro-5-[(4S,5S)-4-(4-fluorophenyl)-5-methyl-2-(1,3-thiazol-
2-yl)-4,5-dihydro-1H-imidazol-4-yl]pyridine (2o). Compound 2o was
prepared from 21a and 1,3-thiazole-2-carboxylic acid using the
procedure described for 2c as a pale-yellow amorphous (9% yield over
2 steps); HPLC purity: 97.7%. 1H NMR (300 MHz, CDCl3): tautomers
δ 0.88 (2.4H, d, J ) 6.6 Hz), 0.97 (0.6H, d, J ) 6.7 Hz), 4.69 (0.8H,
q, J ) 6.6 Hz), 4.88-4.98 (0.2H, m), 5.98 (0.8H, brs), 6.40 (0.2H,
brs), 6.85-6.95 (1H, m), 7.01 (2H, t, J ) 8.7 Hz), 7.20-7.30 (2H,
m), 7.51 (1H, d, J ) 3.1 Hz), 7.70-8.00 (2H, m), 8.27 (0.2H, brs),
8.40 (0.8H, brs). HRMS (ES+) calcd for C18H15N4F2S [M + H]+ m/z
357.0985, found m/z 357.0984. [R]25D: -339° (c 0.57, CH3OH).
2-{(4S,5S)-4-(4-Fluorophenyl)-5-methyl-4-[6-(methyloxy)-3-py-
ridyl]-4,5-dihydro-1H-imidazol-2-yl}-4-pyridinecarbonitrile Hy-
drochloride (3 Hydrochloride). Compound 3 was prepared from 22
using the procedure described for 1c. A hydrochloride salt of 3 was
prepared by treatment with 4 N hydrogen chloride in dioxane as a
pale-yellow solid (76% yield over 2 steps); HPLC purity: 97.3%. 1H
NMR (300 MHz, CD3OD): δ 0.84 (3H, d, J ) 6.3 Hz), 3.87 (3H, s),
4.79-4.88 (1H, m), 6.75 (1H, d, J ) 8.9 Hz), 7.00-7.10 (2H, m),
7.22-7.32 (2H, m), 7.74 (1H, dd, J ) 2.6, 8.9 Hz), 7.80 (1H, dd, J )
1.4, 5.0 Hz), 8.25 (1H, d, J ) 2.6 Hz), 8.40 (1H, s), 8.85 (1H, d, J )
5.0 Hz). HRMS (ES+) calcd for C22H19N5OF [M + H]+ m/z 388.1574,
found m/z 388.1566. [R]25D: -255° (c 0.97, CH3OH).
2-[(4S,5S)-4-(4-Fluorophenyl)-5-methyl-4-(6-oxo-1,6-dihydro-3-
pyridyl)-4,5-dihydro-1H-imidazol-2-yl]-4-pyridinecarbonitrile (4).
To a stirred solution of sodium iodide (90 mg, 0.60 mmol) in
acetonitrile (5.0 mL) was added chloro(trimethyl)silane (76 µL, 0.60
mmol) at 0 °C, and the mixture was stirred at room temperature for
15 min. To the resultant mixture was added a free base of 3 (45 mg,
0.12 mmol) in acetonitrile (2.0 mL), and the mixture was allowed to
warm up to room temperature and stirred for 2 days. The resulting
mixture was concentrated, and the residue was poured into a mixture
(1:1) of saturated aqueous sodium thiosulfate and saturated aqueous
sodium hydrogen carbonate, which was extracted with chloroform
twice. The combined organic layers were dried over sodium sulfate
and concentrated. The residue was purified by preparative TLC with
10% methanol in chloroform to give 4 (14.6 mg, 33% yield) as a pale-
yellow solid; HPLC purity: 98.1%. 1H NMR (400 MHz, CD3OD): δ
0.80 (3H, d, J ) 6.8 Hz), 4.60-4.70 (1H, m), 6.50 (1H, d, J ) 9.2
Hz), 7.10 (2H, t, J ) 8.8 Hz), 7.35 (2H, dd, J ) 5.4, 8.8 Hz), 7.52
(1H, d, J ) 2.6 Hz), 7.62 (1H, dd, J ) 2.6, 9.2 Hz), 7.84 (1H, dd, J
) 1.5, 5.2 Hz), 8.41 (1H, s), 8.87 (1H, d, J ) 1.5, 5.2 Hz). HRMS
(ES+) calcd for C21H17N5OF [M + H]+ m/z 374.1417, found m/z
374.1412. [R]25D: -333° (c 0.41, CH3OH).
4-Chloro-2-[(4S,5S)-4-(4-fluorophenyl)-4-(6-fluoro-3-pyridyl)-5-
methyl-4,5-dihydro-1H-imidazol-2-yl]pyridine Hydrochloride (2j
Hydrochloride). Compound 2j was prepared from 21a and 4-chloro-
2-pyridinecarboxylic acid using the procedure described for 2c. A
hydrochloride salt of 2j was prepared by treatment with 4 N
hydrogen chloride in dioxane as a colorless foam (39% yield over
1
2 steps); HPLC purity: 97.9%. H NMR (300 MHz, CD3OD): δ
1.09 (3H, d, J ) 6.7 Hz), 5.42 (1H, q, J ) 6.7 Hz), 7.18-7.35
(5H, m), 7.85-7.90 (1H, m), 8.12 (1H, ddd, J ) 2.9, 8.8, 8.8 Hz),
8.31 (1H, dd, J ) 0.7, 2.0 Hz), 8.40-8.45 (1H, m), 8.81 (1H, d, J
) 5.3 Hz). HRMS (ES+) calcd for C20H16N4F2Cl [M + H]+ m/z
385.1032, found m/z 385.1029. [R]25D: -161° (c 1.01, CH3OH).
Methyl 2-[(4S,5S)-4-(4-fluorophenyl)-4-(6-fluoro-3-pyridyl)-5-
methyl-4,5-dihydro-1H-imidazol-2-yl]-4-pyridinecarboxylate (2k′).
Compound 2k′ was prepared from 21a and 4-methoxypyridine-2-
carbonitrile31 using the procedure described for 1c as a yellow oil
(87% yield). 1H NMR (300 MHz, CDCl3): tautomers δ 0.88 (2.4H,
d, J ) 6.3 Hz), 0.90-1.05 (0.6H, m), 3.99 (3H, s), 4.70 (0.8H, q,
J ) 6.3 Hz), 4.92-5.05 (0.2H, m), 6.19 (0.8H, brs), 6.60 (0.2H,
brs), 6.82-6.95 (1H, m), 6.95-7.10 (2H, m), 7.20-7.32 (2H, m),
7.90-8.00 (2H, m), 8.38 (0.2H, s), 8.41 (0.8H, s), 8.73 (1H, d, J
) 5.0 Hz), 8.79 (0.8H, s), 8.81 (0.2H, s). MS (ESI): m/z 409 [M +
H]+.
2-[(4S,5S)-4-(4-Fluorophenyl)-4-(6-fluoro-3-pyridyl)-5-methyl-
4,5-dihydro-1H-imidazol-2-yl]-4-pyridinecarboxamide (2k). Com-
pound 2k′ (339 mg, 0.83 mmol) was dissolved in methanol (5.0
mL) and cooled to -40 °C. Ammonia gas was bubbled into the
solution for 15 min, and the mixture was allowed to warm to room
temperature and stirred for 3 days. The resulting mixture was
concentrated, and the residue was dissolved in ethyl acetate and
filtered. The filtrate was concentrated, and the residue was purified
by flash chromatography with 20% methanol in chloroform to give
2k (265 mg, 81% yield) as a colorless solid; HPLC purity: 99.3%.
1H NMR (400 MHz, CDCl3): tautomers δ 0.88 (2.6H, d, J ) 6.4
Hz), 0.95-1.05 (0.4H, m), 4.65-4.75 (0.8H, m), 4.90-5.05 (0.2H,
m), 5.99 (1H, brs), 6.27 (0.8H, s), 6.50-6.80 (1.2H, m), 6.88 (1H,
brd, J ) 7.6 Hz), 7.02 (2H, t, J ) 8.8 Hz), 7.20-7.30 (2H, m),
7.85-8.00 (2H, m), 8.30-8.40 (1H, m), 8.40-8.60 (1H, m), 8.73
(1H, d, J ) 4.8 Hz). HRMS (ES+) calcd for C21H18N5OF2 [M +
H]+ m/z 394.1479, found m/z 394.1476. [R]25D: -287° (c 0.32,
CH3OH).
2-[(4S,5S)-4-(4-Fluorophenyl)-4-(6-fluoro-3-pyridyl)-5-methyl-
4,5-dihydro-1H-imidazol-2-yl]pyrazine (2l). Compound 2l was
prepared from 21a using the procedure described for 1h as a pale-
yellow oil (94% yield); HPLC purity: 97.9%. 1H NMR (300 MHz,
CDCl3): δ 0.88 (3H, d, J ) 6.0 Hz), 4.60-4.80 (1H, m), 6.11 (1H,
s), 6.88 (1H, dd, J ) 2.6, 8.6 Hz), 7.02 (2H, t, J ) 8.6 Hz),
7.20-7.35 (2H, m), 7.90-8.00 (1H, m), 8.41 (1H, s), 8.50-8.60

Imidazolines as NPY Y5 Receptor Antagonists
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 10 3395
(5S,6R)-6-(4-Fluorophenyl)-6-(6-fluoro-3-pyridyl)-5-methyl-3-
morpholinone (23). To a stirred solution of 18 (30 mg, 0.11 mmol)
and triethylamine (40 µL, 0.28 mmol) in chloroform (3.0 mL) was
added chloroacetyl chloride (11 µL, 0.14 mmol) at 0 °C, and the
mixture was allowed to warm to room temperature and stirred for 2 h.
The mixture was poured into saturated aqueous sodium hydrogen
carbonate and extracted with chloroform twice. The combined organic
layers were washed with brine, dried over magnesium sulfate,
concentrated, and vacuum-dried. The crude material was dissolved in
dimethylformamide (3.0 mL) and treated with sodium hydride (60%
dispersion in oil, 4.0 mg, 0.28 mmol) at 0 °C, and the mixture was
allowed to warm to room temperature and stirred for 1 h. The mixture
was poured into water and extracted with diethyl ether. The organic
layer was washed with brine, dried over magnesium sulfate, and
concentrated. The residue was purified by preparative TLC with 50%
ethyl acetate in hexanes to give 23 (14 mg, 40% yield) as a colorless
amorphous. 1H NMR (300 MHz, CDCl3) δ 1.10 (3H, d, J ) 6.5 Hz),
3.87 (1H, d, J ) 17.2 Hz), 4.33 (1H, d, J ) 17.2 Hz), 4.32-4.42 (1H,
m), 6.87 (1H, dd, J ) 3.0, 8.6 Hz), 7.00-7.10 (2H, m), 7.35-7.45
(2H, m), 7.60-7.70 (1H, m), 8.13 (1H, s). MS (ESI) m/z 305 [M +
H]+.
with 2.0 mL of water by ultrasonification, and an aliquot of the
homogenate was deproteinized with 3-fold amount of ethanol contain-
ing an internal standard. The cerebrospinal fluid was mixed with 3-fold
amount of ethanol containing an internal standard. These samples were
allowed to stand at -20 °C for 20 min and then centrifuged (4 °C,
12000g, 10 min). The supernatant was analyzed by LC/MS/MS, and
the concentration of the test compound in the plasma, brain, and
cerebrospinal fluid were measured by the method using a relative
calibration curve.
Pharmacokinetics. Pharmacokinetic characterizations were con-
ducted in male SD rats and male rhesus monkeys following single
oral and single intravenous administration. Single doses of 2a were
administered either intravenously in a vehicle of PEG400/EtOH/H2O
) 50/10/40 (rat) or 60% propylene glycol (pH 3) (monkey) or orally
by gavage in a vehicle of 0.5% methylcellurose aqueous suspension.
Doses of 1 (iv) and 1 (po) mg/kg for rats and 0.3 (iv) and 1 (po) mg/
kg for monkeys were used. Blood samples for the determination of
drug plasma concentrations were obtained at multiple time points up
to 8 h after administration. Blood samples were centrifuged to separate
the plasma, and the plasma samples were deproteinized with ethanol
containing an internal standard. Compound 2a and the internal standard
were detected by LC-MS/MS in a positive ionization mode using the
electrospray ionization probe, and the precursor to product ion
combinations were monitored in multiple reaction monitoring mode.
(5S,6S)-6-(4-Fluorophenyl)-6-(6-fluoro-3-pyridyl)-5-methyl-2-
piperazinone (24). To a stirred solution of 21a (30 mg, 0.11 mmol)
and triethylamine (40 µL, 0.29 mmol) in chloroform (3.0 mL) was
added chloroacetyl chloride (11 µL, 0.14 mmol) at 0 °C, and the
mixture was allowed to warm to room temperature and stirred for 1 h.
The reaction mixture was poured into saturated aqueous sodium
hydrogen carbonate, and the layers were separated. The aqueous layer
was extracted with chloroform, and the combined layers were washed
with brine, dried over magnesium sulfate, and concentrated. The residue
was dissolved in methanol (3.0 mL) and treated with triethylamine
(32 µL, 0.23 mmol), and the mixture was heated to 85 °C for 3 days
in a sealed tube. After being cooled to room temperature, the reaction
mixture was concentrated. The residue was purified by flash chroma-
tography with 10% methanol in chloroform to give 24 (13 mg, 61%
In Vivo Efficacy Study: Acute Feeding in SD Rats.
Sprague-Dawley (SD) rats (10-15 weeks old) were anesthetized
with sodium pentobarbital (50 mg/kg, ip), and a sterile 26-gauge
guide cannula (Plastics One Inc., Roanoke, VA) was stereotaxically
implanted into the third ventricle. The stereotaxic coordinates used
were 2.2 mm posterior to the bregma and 8.0 mm from the surface
of the skull, using a flat skull position. Cannula was attached to
the skull with dental cement, and an antibiotic (Cefamedin R, 50
mg/kg) was subcutaneously injected. The experiments were con-
ducted during light cycle at least 1 wk after the surgery. Test
compounds were suspended in 0.5% methylcellulose in water, and
D-Trp34NPY was dissolved in artificial cerebrospinal fluid (aCSF).
Test compounds or the vehicle were administered orally to SD rats.
Two hours after the oral dosing, D-Trp34NPY (1 µg/0.4 µL) or the
vehicle was injected into the third ventricle. Food intake was
monitored for 2 h after the D-Trp34NPY injection. Data are expressed
as means ( SEM. Significant differences in D-Trp34NPY-induced
feeding were analyzed by Dunnett’s test.
In Vivo Efficacy Study: Chronic Dosing in DIO Mice. Male
C57BL/6N and Y5R KO mice (18-20 weeks old) were fed moderately
high-fat diet (MHF diet; Oriental Bioservice Kanto Inc.) ad libitum
for about 6 months before the experiment was initiated. The MHF
diet provides 52.4% energy as carbohydrate, 15.0% as protein, and
32.6% as fat (4.4 kcal/g). The MHF diet-fed C57BL/6N mice were
divided into three groups matched for body weight, and each group
was orally administered either vehicle (0.5% methylcellulose in water)
or a test compound at doses of 3 and 10 mg/kg, qid for 5 days and bid
for another 15 days. The MHF diet-fed Y5R KO mice were treated
with the test compound at 3 mg/kg bid for 15 days. Administration
was performed about 1 h before the start of the dark period after
measurement of body weight and about 2 h after the start of the light
period. At the end of the experiment, plasma drug levels were measured
at 1, 2, 4, and 15 h after the final dosing. Data are expressed as means
( SEM (body weight change) or means (plasma levels). Body weight
changes were analyzed by repeated-measures one-way ANOVA
followed by Bonferroni test.
1
yield) as a pale-brown amorphous. H NMR (300 MHz, CDCl3): δ
1.04 (3H, d, J ) 6.6 Hz), 3.08 (1H, d, J ) 18.3 Hz), 3.56 (1H, d, J )
18.3 Hz), 4.40-4.50 (1H, m), 6.92 (1H, dd, J ) 3.0, 8.6 Hz), 7.01
(2H, t, J ) 8.9 Hz), 7.15-7.30 (2H, m), 7.80-7.90 (1H, m), 8.37
(1H, d, J ) 2.6 Hz). MS (ESI): m/z 304 [M + H]+.
NPY Y5 Receptor Binding Assay. The assay was performed
according to previously described procedures with membranes prepared
as follows;29 LMtk- cells were washed with 10 mM MOPS buffer
(pH 7.4) containing 154 mM NaCl, 10 mM KCl, 0.8 mM CaCl2, and
20% sucrose, homogenized and centrifuged at 1000 g for 15 min. The
supernatant was centrifuged at 90000g for 50 min. The pellets were
resuspended in 5 mM Hepes/Tris buffer (pH 7.4) and centrifuge again.
The membrane fraction was resuspended by a homogenizer in the same
buffer and used for the assay.
[Ca2+]i Response Assay. CHO-K1 cells stably expressing hu-
manY5 and chimeric G protein (Gqi5) were seeded into 96-well plates
and incubated with a cytoplasmic calcium indicator, Fluo-4AM. After
the cells were washed four times, the intracellular Ca2+ mobilization
evoked by 100 nM of human NPY was monitored as a change in cell
fluorescence intensity by FLIPR (Molecular Devices). Varying con-
centration of Y5 antagonists were added to the plate 5 min prior to
the addition of human NPY. The antagonistic activities were calculated
as IC50 values.
Plasma, Brain, and Cerebrospinal Fluid Concentrations in SD
Rats. Test compound was suspended in 0.5% methylcellulose and
orally administered to male Sprague-Dawley rats (7-10 weeks old,
200-400 g) at 10 mg/kg. At designated time after administration, blood
was collected with a heparinized syringe from the abdominal aorta
under isoflurane anesthesia. Then, the head skin was cut open, and a
dental 30 G needle was inserted between the cervical vertebrae, and it
was further inserted into the cavum subarachnoideale. After 50-100
µL cerebrospinal fluid had been collected by a 1 mL syringe through
a tube connected to the needle, the brain was extracted. The blood
sample was centrifuged (4 °C, 6000 rpm, 10 min) to separate plasma,
and the plasma sample was deproteinized with 3-fold amount of ethanol
containing an internal standard. The brain sample was homogenized
Acknowledgment. We thank Hirokazu Ohsawa and Atsushi
Hirano for the measurement of HRMS and log D7.4 values.
Supporting Information Available: Procedures for the synthesis
of intermediates, biological methods, HPLC retention times, purity,
and HPLC traces for compounds 1g, 2a, 2c, 2e, 2g, and 2m. This
material is available free of charge via the Internet at http://
pubs.acs.org.

3396 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 10
Sato et al.
Inhibitors of Hepatitis C Virus Full-Length NS3 (Protease-Helicase/
NTPase): Model Compounds towards Small Molecule Inhibitors.
Bioorg. Med. Chem. 2003, 11, 2955–2963. (c) Compound 8 was
synthesized following the synthetic procedure described for 7 (see
Supporting Information for details). (d) Hwang, D. R.; Helquist, P.;
Shekhani, M. S. Total Synthesis of (+)-Sparsomycin. Approaches
using Cysteine and Serine Inversion. J. Org. Chem. 1985, 50, 1264–
1271.
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