Discovery of pyridone-containing imidazolines as potent and selective inhibitors of neuropeptide Y Y5 receptor

Article abstract of DOI:10.1016/j.bmc.2009.05.069

A series of 2-pyridone-containing imidazoline derivatives was synthesized and evaluated as neuropeptide Y Y5 receptor antagonists. Optimization of the 2-pyridone structure on the 2-position of the imidazoline ring led to identification of 1-(difluoromethy

Full text of DOI:10.1016/j.bmc.2009.05.069

Bioorganic & Medicinal Chemistry 17 (2009) 6106–6122
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Discovery of pyridone-containing imidazolines as potent and selective
inhibitors of neuropeptide Y Y5 receptor
*
Makoto Ando , Nagaaki Sato, Tsuyoshi Nagase, Keita Nagai, Shiho Ishikawa, Hirobumi Takahashi,
Norikazu Ohtake, Junko Ito, Mioko Hirayama, Yuko Mitobe, Hisashi Iwaasa, Akira Gomori,
Hiroko Matsushita, Kiyoshi Tadano, Naoko Fujino, Sachiko Tanaka, Tomoyuki Ohe, Akane Ishihara,
Akio Kanatani, Takehiro Fukami
Tsukuba Research Institute, Merck Research Laboratories, Banyu Pharmaceutical Company, Ltd, 3 Okubo, Tsukuba 300-2611, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 2-pyridone-containing imidazoline derivatives was synthesized and evaluated as neuro-
peptide Y Y5 receptor antagonists. Optimization of the 2-pyridone structure on the 2-position of
the imidazoline ring led to identification of 1-(difluoromethyl)-5-[(4S,5S)-4-(4-fluorophenyl)-4-(6-flu-
oropyridin-3-yl)-5-methyl-4,5-dihydro-1H-imidazol-2-yl]pyridin-2(1H)-one (7m). Compound 7m dis-
played statistically significant inhibition of food intake in an agonist-induced food intake model in
SD rats and no adverse cardiovascular effects in anesthetized dogs. In addition, markedly higher
brain penetrability and a lower plasma Occ90 value were observed in P-gp-deficient mdr1a (ꢀ/ꢀ)
mice compared to mdr1a (+/+) mice after oral administration of 7m.
Received 20 April 2009
Revised 27 May 2009
Accepted 28 May 2009
Available online 2 June 2009
Keywords:
Neuropeptide Y Y5 receptor
Anti-obesity
Ó 2009 Elsevier Ltd. All rights reserved.
Y5 antagonist
Imidazoline class
1. Introduction
exogenously administered Y5 agonists.12 In addition, chronic intra-
cerebroventricular administration of the Y5-specific agonist
Neuropeptide Y (NPY) is a highly conserved 36 amino acid pep-
tide neurotransmitter that was discovered in 1982 as a member of
the pancreatic polypeptide family, along with peptide YY, a pancre-
atic polypeptide.1 NPY is widely distributed in both the central and
the peripheral nervous systems and has various physiological func-
tions. The concentration of NPY and its mRNA level in the hypo-
thalamus are markedly increased during food deprivation and in
some genetic models of obesity in rodents.2–6 Chronic central infu-
sion of NPY in rodents results in a syndrome similar to that seen in
some genetic obesity models, which is characterized by hyperpha-
gia, insulin resistance, hyperinsulinemia, and reduced thermogenic
activity in brown adipose tissue.7 Furthermore, NPY-deficient ob/
ob mice are less obese and have reduced food intake compared
with ob/ob mice.8 Thus, NPY is thought to play a major role in
the physiological control of energy homeostasis and food intake.
Five distinct NPY receptor subtypes (Y1, Y2, Y4, Y5, and mouse
Y6) have been cloned to date,9 and pharmacological data suggest
that the NPY Y5 receptor (Y5) is involved in feeding regulation
and energy expenditure. Administration of Y5 antagonists sup-
presses Y5 agonist-induced food intake and diet-induced body
weight gain,10,11 and mice lacking Y5 show a reduced response to
D
-Trp34NPY produces obesity in rodents.13 These results suggest
that antagonism of Y5 may have considerable therapeutic benefits
in treating obesity. Thus, Y5 antagonists have been studied by
many pharmaceutical companies as potential anti-obesity drugs.14
Recently, it was reported that administration of a potent and
highly selective Y5 antagonist, MK-557, resulted in modest weight
loss in obese human subjects.15
We previously reported the identification of a potent imidazo-
line class of Y5 antagonists 1 (Fig. 1),16 which significantly reduced
body weight of established diet-induced obesity (DIO) mice both at
3 and 10 mg/kg oral doses. However, intravenous administration of
5 mg/kg of 1 produced significant cardiotonic effects and moderate
F
NH
N
F
N
N
CN
1
hY5 Ki: 1.3 nM
* Corresponding author. Tel.: +81 29 877 2000; fax: +81 29 877 2029.
E-mail address: andoumkt@gmail.com (M. Ando).
Figure 1. Structure and Y5 binding activity of compound 1.
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.05.069

M. Ando et al. / Bioorg. Med. Chem. 17 (2009) 6106–6122
6107
Table 1
that the pyridone structure might prevent cardiovascular toxicities
in this class. Therefore, we focused on optimization of a pyridone
on the 2-position of the imidazoline ring. Synthesis, structure–
activity relationships (SAR) of this series of Y5 antagonists, and
identification of a novel Y5 antagonist 7m with favorable cardio-
vascular safety profiles are described.
Effects of imidazolines on cardiovascular function in anesthetized ratsa
F
F
F
NH
NH
NH
N
N
N
O
S
O
F
N
F
N
F
N
N
CN
CN
NH2
N
2. Chemistry
1
2
3
The synthetic route for intermediates, which are not commer-
cially available, is described in Schemes 1–7. Diastereoselective
synthesis of the diamine intermediate 10 was performed using
(R)-tert-butyl sulfinamide, as illustrated in Scheme 1.17 Weinreb
amide 12 was converted to ketone 13, which was thermally con-
densed with (R)-tert-butylsulfinamide in the presence of titanium
tetraethoxide to afford ketimine 14. Ketimine 14 was reacted with
(6-fluoropyridin-3-yl)lithium in the presence of trimethylalumi-
num to give adduct 15 as a single diastereomer. Deprotection of
the Boc and tert-butylsulfinyl groups was achieved under acidic
conditions to furnish the key compound 10. The stereochemistry
of quaternary chiral center in 10 was determined through 1H
NMR comparison between diamine 10 and the authentic sample.16
Intermediates 11 and 18 were prepared from a 2,3-disubsti-
tuted pyridine (Scheme 2). Pyridines 16a and 16b were converted
to the corresponding cyanopyridines 17a and 17b by reaction con-
ditions a or b.18,19 The bromine atom of 17a was displaced with
benzyl alcohol to give benzyl ether 11. Compound 17b was con-
verted to the desired carboxylic acid 18 by hydrolysis with 10 N so-
dium hydroxide. Synthesis of compound 21 is shown in Scheme 3.
2-Cyano-5-fluoropyridine (19) was oxidized with urea hydrogen
peroxide addition compound (UHP) and trifluoroacetic anhydride
(TFAA) to give the corresponding N-oxide, which was treated with
acetic anhydride to afford acetoxy compound 20.18,20 The nitrile
and acetoxy groups of compound 20 were hydrolyzed under acidic
conditions to furnish pyridone carboxylic acid 21. Compounds
24a–c were prepared from pyridones 22a and 22b as shown in
Scheme 4. N-Alkylation of pyridones 22a and 22b followed by
hydrolysis of the ester afforded carboxylic acids 24a–c.21 The
known N-difluoromethyl pyridone 23d22 was converted to the cor-
responding carboxylic acid 24d in the same manner.
F
F
F
NH
NH
NH
N
N
N
N
NH
NH
F
N
F
N
F
O
O
N
4
5a
6
MAPb (%)
Plasma level (lM)
b
Compd
LV dp/dtmax (%)
1
18
15
20
30
5
21
ꢀ6
7
6.3
4.3
4.9
5.1
5.3
2.6
2
3c
4
19
5a
6
ꢀ15
ꢀ3
ꢀ19
a
The values represent the mean for n = 2ꢀ3 at 3 min after 5 mg/kg iv injection of
test compounds.
b
LV dp/dtmax and MAP values are shown as the percent change from baseline.
hY5 Ki = 1.7 0.2 nM.
c
QTc prolongation in anesthetized dogs, even though compound 1
displayed excellent selectivity with respect to a panel of 167
diverse, unrelated binding sites (IC50 > 1 lM for all the binding
sites tested). Therefore, we screened imidazolines with various
functional groups substituted on the 2-position of the imidazoline
ring using an anesthetized rat cardiovascular assay to identify imi-
dazoline analogs with a safe cardiovascular profile (Table 1). Intra-
venous administration of 1 (5 mg/kg) to rats resulted in an increase
in left ventricular cardiac contractility (LV dp/dtmax) and mean
arterial pressure (MAP) at a plasma concentration of 6.3 lM, sim-
ilar to those observed in anesthetized dogs.16 2-Cyanopyridine 2
and benzenesulfonamide 3 did not show a significant change in
MAP, while increases in LV dp/dtmax similar to 1 were observed.
Pyrazine 4 produced comparable hemodynamic effects at the same
plasma drug levels to 1. Pyridone 5a produced a nonsignificant
change in LV dp/dtmax and a slight decline in MAP at plasma levels
Preparation of substituted pyridones 27a, 27b, 30, 31, 34a, and
34b is described in Scheme 5. Bromination of 3-substituted pyri-
dones 25a and 25b afforded the 5-bromo compounds 26a and
26b. Compound 26b and commercially available reagent 26c were
reacted with n-BuLi followed by addition of CO2 gas to give carbox-
ylic acids 27a and 27b. 3-Trifluoromethylpyridone 26a was treated
of 5.3 lM. The related compound 6 with a bis(4-fluorophenyl)
group had a comparable cardiovascular profile to 5a, suggesting
F
F
O
a
b
NHBoc
N
NHBoc
O
N
NHBoc
S
O
O
12
13
14
F
F
c
d
NHBoc
NH2
NH
S
F
N
NH2
F
N
O
15
10
Scheme 1. Reagents and conditions: (a) 4-fluorophenylmagnesium bromide, THF, 0 °C to rt; (b) (R)-2-methylpropane-2-sulfinamide, Ti(OEt)4, toluene, 70 °C; (c) (i) 5-bromo-
2-fluoropyridine, n-BuLi, Et2O, ꢀ78 °C, (ii) AlMe3, 14, ꢀ78 °C; (d) (i) TFA, rt, (ii) 8 N HCl, 1,4-dioxane, 0 °C to rt.

6108
M. Ando et al. / Bioorg. Med. Chem. 17 (2009) 6106–6122
R3
R4
N
R3
R4
NC
N
OBn
NC
N
c
a or b
d
16a: R3 = Br, R4 = Me
16b: R3, R4 = Cl
17a: R3 = Br, R4 = Me
17b: R3, R4 = Cl
11
H
HO2C
N
O
Cl
18
Scheme 2. Reagents and conditions: (a) (i) mCPBA, CHCl3, rt, (ii) CH3OTf, CH2Cl2, 0 °C to rt, then NaCN, 0 °C to rt; (b) (i) UHP, TFAA, CH2Cl2, 0 °C to rt; (ii) (CH3)2SO4, 90 °C, then
NaCN, H2O, 0 °C; (c) NaH, BnOH, THF, reflux; (d) 10 N KOH, reflux.
H
2-fluoro-6-pyridone 41. Alkylation of 41 with benzyl bromide fol-
lowed by hydrolysis of the ester resulted in carboxylic acid 42.
The synthetic pathway for the desired imidazoline derivatives
reported here is shown in Schemes 8 and 9. The imidazoline rings
of 30, 5c–g, 7a–b, 7c0–d0, 7e–q, 8, and 90 were prepared by coupling
(1S,2S)-1-(4-fluorophenyl)-1-(6-fluoropyridin-3-yl)propane-1,2-
diamine (10) with aryl carboxylic acids, followed by thermal cycli-
zation of the resulting amides (Scheme 8).24 Removal of the Boc
group of 30 afforded the desired compound 3. The benzyl group
of 7c0 and 7d0 was removed under acidic conditions to yield 7c
and 7d. Demethylation of 90 was performed by treatment with
sodium iodide and trimethylsilyl chloride to produce the imidazo-
line analog 9. Imidazoline 5b was prepared by coupling of the dia-
mine 10 with an aryl imidate derived from 11,25 followed by
cleavage of the benzyl group of the resulting compound 5b0
(Scheme 9).
NC
N
NC
N
OAc
F
a
HO2C
N
O
F
b
F
19
20
21
Scheme 3. Reagents and conditions: (a) (i) UHP, TFAA, CH2Cl2, 0 °C to rt; (ii) Ac2O,
reflux; (b) 6 N HCl, reflux.
with phenylphosphonic dichloride followed by coupling with
p-methoxybenzyl alcohol to produce benzyl ether 28a. The known
compound 26d was alkylated with benzyl bromide in the presence
of silver carbonate to give benzyl ether 28b. Carbonylation of 28a
and 28b was achieved in the presence of a catalytic amount of pal-
ladium diacetate and 1,10-bis(diphenylphosphino)ferrocene to give
the corresponding n-propyl esters 29a and 29b. Removal of the
p-methoxybenzyl group of 29a followed by hydrolysis of the ester
afforded 2-pyridone carboxylic acid 30. Hydrolysis of 29b fur-
nished pyridine carboxylic acid 31. Alkylation of 3-methylpyridone
26c with ethyl bromide21 or sodium chlorodifluoroacetate fol-
lowed by carbonylation of the resulting N-substituted 3-methyl-
pyridones afforded the n-propyl esters 33a and 33b, which were
hydrolyzed under basic conditions, affording carboxylic acids 34a
and 34b.
3-Fluoropyridone 37 and N-alkoxypyridones 39a and 39b were
prepared according to Scheme 6. Pyridine methyl esters 35a and
35b were converted to the corresponding N-oxides 36a and 36b
using UHP and TFAA.18 Compound 36a was heated with acetic
anhydride followed by hydrolysis with 2 N sodium hydroxide to
give the desired carboxylic acid 37. Compound 36b was reacted
with TFAA to provide N-hydroxy-2-pyridone 38, which was alkyl-
ated with methyl iodide or ethyl bromide followed by hydrolysis
to produce carboxylic acids 39a and 39b.
3. Results and discussion
3.1. SAR of pyridone-containing imidazolines
The series of imidazoline compounds was tested in a [125I]PYY
binding assay using LMtkꢀ cell membranes expressing human re-
combinant Y5 receptors.26 Rat hepatic clearance was examined
by the in vitro serum incubation method to predict in vivo clear-
ance in the rat liver, as previously reported by our laboratory.27
The substitutions on the 4- and 5-positions of imidazoline,
(4S)-4-(4-fluorophenyl)-4-(6-fluoropyridin-3-yl), and (5S)-methyl
groups as in 5a were selected due to their favorable effects on Y5
potency, metabolic stability, and human Ether-à-go-go related
gene (hERG) affinity, as described in our previous report.16
We initially prepared pyridone regioisomers 7a, 8, and 9 (Table 2).
Compound 7a with 5-pyridin-2(1H)-one (5-pyridone) exhibited a
slightly reduced Y5 activity compared to the 6-pyridin-2(1H)-one
(6-pyridone) 5a. Replacement of the 6-pyridone with a 3-pyridin-
2(1H)-one (3-pyridone) or 4-pyridin-2(1H)-one (4-pyridone) as in 8
and 9 was deleterious to Y5 potency. We therefore focused on inves-
tigation of SAR for substitution effects on the 5- and 6-pyridone rings.
Note that metabolic stability of the 5-pyridone 7a was considerably
better than that of 6-pyridone 5a in rat hepatocytes.
The effects of substitutions on the 6-pyridone ring of 5a are
shown in Table 3. 3-Methyl substitution as in 5b resulted in a high-
er hepatic clearance than 5a, while retaining Y5 potency. Y5 affin-
ities for 4- and 5-methyl derivatives 5c and 5d were more than
fivefold less potent compared to 5a. Based on these observations,
additional 3-substituted pyridone derivatives were prepared and
evaluated. 3-Fluoro substitution led to a significant improvement
in hepatic clearance, although the derivative exhibited slightly
decreased Y5 activity. The 3-chloro compound 5f showed a similar
in vitro profile to 5e. The N-methyl derivative 5g showed a signif-
icant loss of Y5 potency.
The synthesis of 2-fluoropyridine carboxylic acid 42 is illus-
trated in Scheme 7. The methoxy group of compound 4023 was
removed using sodium iodide and trimethylsilyl chloride to afford
R6
H
N
O
N
O
6
a
R5
R5
5
22a: R5 = 6-CO2Me
22b: R5 = 5-CO2H
23a: R5 = 6-CO2Me
6 = Me
24a: R5 = 6-CO2H
R
R
6 = Me
23b: R5 = 5-CO2n-Pr 24b: R5 = 5-CO2H
R6 R6
-Pr -Pr
23c: R5 = 5-CO2i-Pr 24c: R5 = 5-CO2H
R6 R6
-Pr -Pr
23d: R5 = 5-CO2Me 24d: R5 = 5-CO2H
6 = CHF2 6 = CHF2
=
n
= n
=
i
= i
R
R
b
Scheme 4. Reagents and conditions: (a) R6X, CsF, DMF, rt; (b) 1 N NaOH, MeOH.

M. Ando et al. / Bioorg. Med. Chem. 17 (2009) 6106–6122
6109
H
N
OPro
N
OPro
N
O
g
f
Br
n-PrO2C
R7
R7
HO2C
CF3
28a: R7 = 3-CF3, Pro = PMB
28b: R7 = 4-Me, Pro = Bn
29a: R7 = 3-CF3, Pro = PMB
29b: R7 = 4-Me, , Pro = Bn
30
h
d or e
H
N
H
H
N
OBn
O
N
O
N
O
a or b
c
6
5
3
HO2C
R7
Br
HO2C
R7
R7
4
25a: R7 = 3-CF3
25b: R7 = 3-OMe
26a: R7 = 3-CF3
26b: R7 = 3-OMe
26c: R7 = 3-Me
26d: R7 = 4-Me
27a: R7 = 3-OMe
27b: R7 = 3-Me
31
i or j
R8
R8
R8
f
h
N
O
N
O
N
O
Br
32a: R8 = Et
Me
n-PrO2C
Me
HO2C
Me
33a: R8 = Et
34a: R8 = Et
34b: R8 = CHF2
32b: R8 = CHF2
33b: R8 = CHF2
Scheme 5. Reagents and conditions: (a) NaOAc, Br2, AcOH, 80 °C; (b) Br2, CH2Cl2, rt; (c) n-BuLi, THF, ꢀ78 °C, then CO2 gas; (d) (i) PhPOCl2, 140 °C, (ii) p-methoxybenzyl
alcohol, NaH, THF, 80 °C; (e) Ag2CO3, BnBr, benzene, rt; (f) Pd(OAc)2, dppf, Et3N, n-PrOH, DMF, CO gas, 100 °C; (g) (i) TFA, rt, (ii) 4 N NaOH, 1,4-dioxane, MeOH, rt; (h) 3 N
NaOH, MeOH, 0 °C–rt; (i) EtBr, CsF, DMF, rt; (j) CClF2CO2Na, NaH, LiBr, DMF, 145 °C.
Next, the effects of substitutions on the 5-pyridone ring of 7a
were investigated as shown in Table 4. 3-Methyl substitution as
in 7b led to slight improvement in Y5 potency, relative to the par-
H
ent compound 7a. However, 4- or 6-substitution, as in 7c and 7d,
resulted in significant loss of Y5 potency. Therefore, we focused
on the 3-substituted 5-pyridone derivatives. The 3-fluoro com-
pound 7e and the parent 7a were equipotent, and the 3-chloro,
3-trifluoromethyl, and 3-methoxy derivatives 7fꢀh exhibited
improved Y5 activities. The rat hepatic clearance of 7eꢀh was com-
parable to the unsubstituted pyridone 7a. Next, we investigated
the effects of N-substitution on the 5-pyridone of 7a. N-Methyla-
tion as in 7i led to a slight decrease in Y5 potency, whereas the
ethyl derivative 7j was 2.5-fold more potent than 7a. However,
the predicted metabolic turnover of 7j was much higher than that
of the parent 7a. The N-substituted pyridones 7k and 7l also
showed a comparable profile to 7j in terms of Y5 affinity and pre-
dicted rat hepatic clearance, suggesting that N-substituents of pyri-
dones of 7jꢀl were metabolically soft spots. Based on these results,
introduction of a difluoromethyl group on the nitrogen of the pyri-
done ring of 7a was attempted. The difluoromethyl compound 7m
showed improved Y5 inhibitory activity and an acceptable hepatic
clearance, compared to 7a. The N-methoxy analog 7n was found to
have a comparable profile to 7m. Extension of the alkyl group as in
7o decreased Y5 potency and increased hepatic clearance. The 1,3-
disubstituted 5-pyridones 7p and 7q exhibited reduced Y5 potency
compared to the 3-methyl compound 7b.
O
F
N
CO2H
37
b
O
N
2
3
6
N
4
a
R9
R9
5 CO2Me
CO2Me
35a: R9 = 3-F
36a: R9 = 3-F
36b: R9 = 2-Cl
35b: R9 = 2-Cl
c
OR10
N
OH
N
d
O
O
CO2Me
CO2H
39a: R10 = Me
39b: R10 = Et
38
Scheme 6. Reagents and conditions: (a) UHP, TFAA, CH2Cl2 or CH3CN, 0 °C to rt; (b)
(i) Ac2O, 140 °C, (ii) 2 N NaOH, rt; (c) TFAA, CH3CN, rt; (d) (i) R10X, K2CO3, DMF, rt,
(ii) 1 N NaOH, MeOH, rt.
Among the 5- and 6-pyridone derivatives described in Tables 3
and 4, the potent (hY5 Ki 6 4.0 nM) and metabolically stable
(predicted CLh 6 30 mL/min/kg) imidazolines 7f, 7g, 7m, and 7n
H
MeO
N
F
BnO
N
F
O
N
F
a
b
COOMe
COOH
COOMe
40
41
42
Scheme 7. Reagents and conditions: (a) NaI, TMSCl, CH3CN, rt; (b) (i) BnBr, CsF, DMF, rt; (ii) 1 N NaOH, MeOH, 0 °C to rt.

6110
M. Ando et al. / Bioorg. Med. Chem. 17 (2009) 6106–6122
Table 2
F
F
SAR of pyridone analoguesa
a or b
F
NH
NH2
N
F
NH2
F
N
R1
N
NH
Ar
N
3', 5c−g, 7a, 7b, 7c'−d', 7e−q, 8, 9'
10
F
N
hY5 bindingb Ki (nM)
CLH (mL/min/kg)
c
Compd
Ar
SO2NHBoc
SO2NH2
R1
R1
=
=
R1
R1
=
=
3'
3
H
N
O
5a
1.4 0.2
31
N
NH
c or d
7c', d'
7c, 7d
OBn
OMe
O
NH
R2
R2
7a
8
7.4 0.5
600
12
O
O
O
R1
=
R1
=
9'
9
d
N
NH
NH
Scheme 8. Reagents and conditions: (a) (i) R1CO2H, DMI, Et3N, CHCl3, 0 °C, (ii) neat,
150 °C; (b) (i) R1CO2H, WSCꢁHCl, pyridine, CHCl3, rt, (ii) Yb(OTf)3 or Sc(OTf)3,
toluene, 100ꢀ150 °C; (c) TFA, rt or 50 °C; (d) NaI, TMSCl, CH3CN, rt, then 90, rt.
O
d
9
42
4
NH
a
The values represent the mean SE for n = 3 or the mean for n = 2.
[
b
125I]PYY binding assay in LMtkꢀ cells expressing human recombinant Y5
were tested for P-glycoprotein (P-gp) susceptibility, which was as-
sessed using human MDR1- and mdr1a-transfected porcine renal
epithelial (LLC-PK1) cell monolayers and obtained transcellular
transport ratios.28 The transcellular transport ratios of the tested
compounds (B-to-A/A-to-B ratio) are summarized in Table 5. In
this P-gp transport assay, a compound with a B-to-A/A-to-B ratio
above 3 is considered to be a P-gp substrate. All the tested com-
pounds were mouse P-gp substrates (B-to-A/A-to-B ratio = 4ꢀ12),
and the N-unsubstituted 5-pyridones 7f and 7g were also found
to be human P-gp substrates. In contrast, the N-substituted com-
pounds 7m and 7n were found not to be human P-gp substrates.
Given that the N-unsubstituted 5-pyridone derivative 7a is a sub-
strate for human P-gp (B-to-A/A-to-B ratio = 8.5), the NH group on
the 5-pyridone ring may increase susceptibility to human P-gp.
The antagonistic activity of the negligible human P-gp sub-
strates 7m and 7n was determined by measuring the ability of
each compound to inhibit NPY-induced [Ca2+]i increase in LMtkꢀ
cells expressing the recombinant human Y5 receptor (Table 6).29
In this functional assay, both compounds showed potent antago-
nistic activities. Compounds 7m and 7n were also evaluated for
hERG K+ channel binding activity using the [35S]N-[(4R)-10-[(2R)-
6-cyano-1,2,3,4-tetrahydro-2-naphthalenyl]-3,4-dihydro-4-hydroxy-
spiro[2H-1-benzopyran-2,40-piperidin]-6-yl]methanesulfonamide
competitive binding assay to assess QTc prolongation liability
(Table 6).30 These compounds were found to have negligible hERG
inhibitory activities. Compound 7m was chosen for further studies
on the basis of its potent functional activity. This compound dis-
played excellent selectivity over other NPY receptors (hY1, hY2,
receptors.
c
CLH was determined by the serum incubation method using isolated rat
hepatocytes.
d
Not determined.
3.2. Pharmacokinetic, in vivo efficacy, and cardiovascular
safety studies with 7m
The pharmacokinetic parameters of 7m were evaluated in rats
and rhesus monkeys (Table 7).32 Compound 7m displayed excel-
lent profiles in both species. The brain penetrability of imidazoline
7m was assessed in Sprague-Dawley (SD) rats. Compound 7m had
a
brain-to-plasma ratio of 0.42 (brain = 0.91 nmol/g, plas-
ma = 2.20 M) 2 h after 10 mg/kg oral administration. This low ra-
tio is presumably due to the effect of P-gp efflux.
l
Having demonstrated the excellent potency, selectivity, and
suitable pharmacokinetic profiles of 7m, this compound was tested
in an agonist-induced acute food intake model with SD rats
(Fig. 2).33 The compound was orally administered 2 h before ani-
mals were treated with either the Y5 selective agonist D
-Trp34NPY
or artificial CSF, and cumulative food intake was measured for the
following 2 h. Compound 7m showed statistically significant and
dose-dependent inhibition of the Y5 selective agonist-induced food
intake in this feeding model.
The potential cardiovascular effects of 7m were evaluated in
anesthetized dogs (Table 8). QTc interval, mean arterial pressure
(MAP), left ventricular systolic pressure (LVSP), cardiac output
(CO), heart rate (HR), and cardiac contractility (LV dp/dtmax) were
assessed after cumulative intravenous infusion of 1, 2, and
and hY4 binding: >10
ing sites (IC50 > 1.0 M for all binding sites tested).
l
M)31 and over 168 diverse unrelated bind-
l
F
F
NC
N
OBn
a
b
NH
NH
N
N
F
F
N
N
N
NH
OBn
O
11
5b'
5b
Scheme 9. Reagents and conditions: (a) (i) NaOMe, MeOH, rt, then MsOH, (ii) 10, MeOH, rt; (b) TFA, rt or 50 °C.

M. Ando et al. / Bioorg. Med. Chem. 17 (2009) 6106–6122
6111
Table 3
Table 4
SAR of 6-pyridone derivativesa
SAR of 5-pyridone derivativesa
F
F
NH
Ar
NH
Ar
N
N
F
N
F
N
c
c
Compd
Ar
hY5 bindingb Ki (nM)
CLH (mL/min/kg)
31
Compd
Ar
hY5 bindingb Ki (nM)
CLH (mL/min/kg)
12
H
N
O
5a
1.4 0.2
7a
7.4 0.5
N
H
O
O
H
N
O
O
5b
5c
5d
5e
5f
2.6 0.3
8.6 0.7
55
7b
7cd
7d
7e
7f
4.3 0.8
630 83
>1000
24
N
H
H
N
d
e
N
H
O
H
N
O
O
d
28
5
e
F
N
O
H
H
N
6.9 0.9
8.6 1.8
350 29
19
F
F
11
2
9
N
O
H
N
H
O
19
Cl
O
Cl
O
3.4 0.5
3.8 1.0
5.0 0.7
19
13
N
H
N
d
5g
CF3
O
7g
7h
7i
a
The values represent the mean SE for n = 3 or the mean for n = 2.
N
H
b
[
125I]PYY binding assay in LMtkꢀ cells expressing human recombinant Y5
receptors.
c
O
O
CLH was determined by the serum incubation method using isolated rat
hepatocytes.
14
d
Not determined.
N
H
e
13
2
N
O
O
5 mg/kg/10 min of 7m, which produced peak plasma concentra-
tions of 7.7, 14.3, and 26.9 M at the end of the each dosing
period, respectively. In these cardiovascular studies, no significant
l
treatment-related adverse cardiovascular effects were observed.
7j
2.9 0.1
4.7 1.2
3.6 0.2
2.8 0.6
2.8 0.1
50
57
58
23
N
Et
3.3. Receptor occupancy of 7m in P-gp-deficient mdr1a (ꢀ/ꢀ)
and wild type mdr1a (+/+) CF-1 mice
7k
7l
N
O
O
O
O
Finally, brain NPY Y5 receptor occupancy was evaluated in ro-
dents using an ex vivo receptor occupancy method to estimate
plasma levels resulting in a high degree of receptor occupancy.
Our previous studies indicated that high and sustained Y5 receptor
occupancy is required for efficacy in both rodents with DIO and hu-
mans.11,15 Since 7m is a significant substrate for rodent P-gp as
shown in Table 5, brain penetration by 7m in rodents is limited
by P-gp mediated efflux, leading to limited receptor occupancy
and therefore limited efficacy in rodents. However, 7m is a weak
or negligible human P-gp substrate, and therefore, we speculated
that 7m shows higher brain penetrability and better receptor occu-
pancy in humans than in rodents. To demonstrate the potential of
7m, brain and plasma concentrations and receptor occupancy of
7m were studied in P-gp-deficient mdr1a (ꢀ/ꢀ) and wild type
mdr1a (+/+) CF-1 mice (Table 9). After oral administration of
10 mg/kg of 7m, the brain-to-plasma ratio was 2.05 in mdr1a
n-Pr
N
i-Pr
7m
7n
N
CHF2
25
N
O
(continued on next page)

6112
M. Ando et al. / Bioorg. Med. Chem. 17 (2009) 6106–6122
Table 4 (continued)
4
3
2
1
0
hY5 bindingb Ki (nM)
CLH (mL/min/kg)
c
Compd
Ar
39%
68%
*
70%
*
7o
7.3 0.5
51
55
N
O
O
Et
7p
6.6 0.4
N
O
O
Et
7m(mg/kg, po)
D-Trp34NPY (µg/head)
0
0
0
1
1
1
3
1
10
1
e
7q
8.5
N
CHF2
Figure 2. Effect of 7m on food intake induced by the NPY selective agonist,
-Trp34NPY. Compound 7m was orally administered 2 h before 3rd ventricle
D
a
The values represent the mean SE for n = 3 or the mean for n = 2.
injection of
(n = 7ꢀ10). P < 0.05 (compared with only the
D lg/head). The values represent the mean SEM
-Trp34NPY (1
b
[
125I]PYY binding assay in LMtkꢀ cells expressing human recombinant Y5
*
D
-Trp34NPY treated group).
receptors.
c
CLH was determined by the serum incubation method using isolated rat
hepatocytes.
d
Trifluoroacetate.
Not determined.
Table 7
e
Pharmacokinetic parameters of 7ma
Species
IV CLP (mL/
min/kg)
Vdss (L/kg)
PO AUC0–1
M h)
Cmax
(l
M)
F (%)
(
l
Ratb
23
3.5
2.6
3.8
4.8
11
1.1
0.64
92
92
Table 5
Monkeyc
P-gp susceptibility of compounds 7f, 7g, 7m, and 7na
a
The values represent the mean for n = 3.
Oral dose = 3 mg/kg. Intravenous dose = 1 mg/kg.
Oral dose = 1 mg/kg. Intravenous dose = 0.3 mg/kg.
Compd
P-gp susceptibility transcellular transport ratio (B-to-A)/(A-to-B)
b
c
Human MDR1
Mouse mdr1a
7f
9.1
12
1.6
1.9
12
7g
7m
7n
5.2
7.0
4.0
4. Conclusion
a
The values represent the mean for n = 3. Transcellular transport ratios ((B-to-A)/
(A-to-B)) were obtained from MDR1- and mouse mdr1a-transfected LLC-PK1 cell
monolayers.
In summary, a series of 2-pyridone-containing imidazoline
derivatives was synthesized and evaluated as NPY Y5 antagonists.
Compound 7m was found to have potent Y5 antagonistic activity
and negligible susceptibility to human P-gp. In addition, imidazo-
line 7m showed statistically significant inhibition of food intake
in the agonist-induced food intake model and no adverse cardio-
vascular effects in anesthetized dogs. Because 7m is a significant
substrate for rodent P-gp, ex vivo receptor occupancy studies were
conducted in mdr1a (ꢀ/ꢀ) and mdr1a (+/+) mice to obtain plasma-
receptor occupancy relationships. Compound 7m displayed mark-
edly higher brain penetrability and a lower plasma Occ90 value in
mdr1a (ꢀ/ꢀ) than in mdr1a (+/+) mice, suggesting that 7m could
potentially show higher brain penetrability and receptor occu-
pancy in humans than in rodents. Based on the profiles described
in this report, compound 7m was selected as a clinical develop-
ment candidate for the treatment of obesity and CNS-related dys-
functions. Progress in the development of this compound will be
reported elsewhere.
(ꢀ/ꢀ) CF-1 mice, which is fourfold higher than that seen in mdr1a
(+/+) CF-1 mice. Encouraged by these results, ex vivo receptor
occupancy studies were carried out in mdr1a (ꢀ/ꢀ) and mdr1a
(+/+) CF-1 mice to obtain plasma level-receptor occupancy rela-
tionships ( Fig. 3A). As expected, a remarkable leftward shift of
the titration curve for mdr1a (ꢀ/ꢀ) mice was observed in the plas-
ma level-receptor occupancy relationship. The plasma level
required to achieve 90% receptor occupancy (Occ90) is 33 nM in
mdr1a (ꢀ/ꢀ) mice, whereas the Occ90 value (170 nM) is higher
in mdr1a (+/+) mice (Table 9). High and sustained receptor occu-
pancy was observed after oral administration of 1 and 3 mg/kg
7m in mdr1a (ꢀ/ꢀ) mice (Fig. 3B). These results suggest that 7m
may provide a high degree of brain receptor occupancy at a very
low plasma concentration in humans.
5. Experimental
5.1. Materials and methods
Table 6
Functional Y5 antagonistic activity and hERG affinity of 7m and 7na
Unless otherwise noted, all solvents, chemicals, and reagents
were obtained commercially and used without purification. Silica
gel column chromatography was carried out on a WakogelÒ C-
b
c
Compd
[Ca2+]i response IC50 (nM)
hERG IC50 (nM)
7m
7n
8.7 0.1
15.7 4.0
>10
>10
300 (Wako, mesh 45ꢀ75
lm) or with prepacked silica gel columns
(KP-Sil silica) from Biotage under the indicated conditions. Pre-
TM
a
The values represent the mean SE for n = 3.
Antagonistic activities (human recombinant Y5 receptor/Gqi5 in CHO cells) at
b
parative HPLC purification was carried out on a YMC-Pack Pro
100 nM NPY stimulation.
C18 (YMC, 50 mm ꢂ 30 mm id S-5
lm), eluting with a gradient of
Displacement binding assay of [35S]N-[(4R)-10-[(2R)-6-cyano-1,2,3,4-tetrahy-
c
0.1% CF3CO2H–CH3CN/0.1% aqueous CF3CO2H = 10/90 to 50/50 over
8 min at a flow rate of 40 mL/min. Preparative thin-layer chroma-
tography (TLC) was performed on a TLC Silica Gel 60 F (Merck
dro-2-naphthalenyl]-3,4-dihydro-4-hydroxyspiro[2H-1-benzopyran-2,40-piperi-
din]-6-yl]methanesulfonamide in membranes derived from HEK293 cells stably
transfected with the hERG gene expressing the IKr channel protein.

M. Ando et al. / Bioorg. Med. Chem. 17 (2009) 6106–6122
6113
Table 8
Cardiovascular effects of 7m in anesthetized dogsa
IV doseb (mg/kg)
Plasma concc
(l
M)
QTcd (%)
MAPd (%)
LVSPd (%)
COd (%)
HRd (%)
LV dp/dtmax (%)
d
1
2
5
7.7
14.3
26.9
+2
+2
+3
+4
+7
+5
+3
ꢀ2
ꢀ2
ꢀ3
ꢀ1
ꢀ2
ꢀ10
ꢀ16
ꢀ4
ꢀ5
ꢀ4
ꢀ2
a
The values represent the mean for n = 3.
Doses infused over 10 min were 1, 2, and 5 mg/kg, yielding rising cumulative doses of 1, 3, and 8 mg/kg.
Plasma concentrations were measured at the end of the dosing period.
b
c
d
The parameters were measured at 0, 10, and 20 min during the cardiovascular study. Each value is described as a maximum percentage change from baseline; see Section
5 for details.
KGaA). The 1H NMR spectra were obtained at 400 MHz on a MER-
CURY-400 (Varian), 400 MHz on a JMN-AL400 (JEOL), 200 MHz on
a Gemini 200 (Varian), or 300 MHz on a Gemini 300 (Varian) spec-
trometer, with chemical shift (d, 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 micro-
mass Q-Tof-2 instrument. High resolution mass spectra (HRMS)
were recorded with electron-spray ionization on a micromass
Q-Tof-2 instrument. The purity of target compounds was deter-
mined by HPLC with the two different eluting methods. Following
HPLC systems were used for purity assessment; Method A:
Hewlett–Packard agilent 1100 series with a SPELCO Ascentis
was stirred at 0 °C for 20 min. The resultant mixture was parti-
tioned 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 silica gel flash chroma-
tography with 33% ethyl acetate in hexanes to give N-[(1S,2S)-1-
amino-1-(4-fluorophenyl)-1-(6-fluoropyridin-3-yl)propan-2-yl]-3-
sulfamoylbenzamide as a colorless foam (893 mg). The neat amide
was heated to 150 °C for 3 days. The resulting reaction mixture was
purified by silica gel flash column chromatography with 50% ethyl
acetate in hexanes to give 30 (741 mg, 73% yield over 3 steps) as a
pale-yellow foam. 1H NMR (300 MHz, DMSO-d6): d 0.87 (3H, d,
J = 6.5 Hz), 1.24 (9H, s), 4.65 (1H, q, J = 6.5 Hz), 4.78 (1H, s), 5.27
(1H, s), 6.87 (1H, dd, J = 3.0, 8.5 Hz), 7.00 (2H, t, J = 8.7 Hz), 7.15–
7.30 (2H, m), 7.56 (1H, t, J = 7.5 Hz), 7.91 (1H, dt, J = 2.3, 8.2 Hz),
7.98 (1H, d, J = 7.7 Hz), 8.14 (1H, d, J = 7.7 Hz), 8.30 (1H, s), 8.39
(1H, s); MS (ESI): m/z 429 [MꢀBoc+H]+.
(4.6 mm ꢂ 150 mm id S-2.7
lm), eluting with a gradient of 0.1%
H3PO4/CH3CN = 95/5 to 10/90 over 7 min followed by 10/90 iso-
cratic over 1 min at a flow rate of 1.5 mL/min, 40 °C of column tem-
perature, detection with UV 210 nm; Method B: Hewlett–Packard
agilent 1100 series with a SPELCO Ascentis (4.6 mm ꢂ 150 mm id
S-2.7 lm), eluting with a gradient of 10 mM potassium phosphate
(pH 6.6)/CH3CN = 95/5 to 20/80 over 7 min followed by 20/80 iso-
cratic over 1 min at a flow rate of 1.5 mL/min, 40 °C of column tem-
perature, detection with UV 210 nm. Optical rotations of
compounds 7f, 7g, 7m, and 7n were recorded on a JASCO P-1020
polarimeter.
5.2.2. 3-[(4S,5S)-4-(4-Fluorophenyl)-4-(6-fluoropyridin-3-yl)-5-
methyl-4,5-dihydro-1H-imidazol-2-yl]benzenesulfonamide (3)
Compound 30 (198 mg, 0.409 mmol) was dissolved in TFA
(5.0 mL), and the mixture was stirred at room temperature for
20 h. After being concentrated, the residue was partitioned be-
tween ethyl acetate and saturated aqueous sodium hydrogen car-
bonate, 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 silica gel flash column chromatogra-
phy with 67% ethyl acetate in hexanes to give 3 (154 mg, 88% yield)
as a colorless foam. 1H NMR (300 MHz, CDCl3): d 0.84 (3H, d,
J = 6.6 Hz), 4.60–4.72 (1H, m), 5.60 (2H, brs), 6.82 (1H, dd, J = 3.0,
8.6 Hz), 6.98 (2H, t, J = 8.6 Hz), 7.15–7.30 (2H, m), 7.51 (1H, t,
J = 7.8 Hz), 7.77–7.87 (1H, m), 7.93 (1H, d, J = 7.7 Hz), 8.06 (1H, d,
J = 7.7 Hz), 8.25–8.38 (2H, m); HRMS (ES+) calcd for C21H19F2N4O2S
[M+H]+ m/z 429.1197, found m/z 429.1186; HPLC purity: (A) 99.2%
(tR = 3.9 min), (B) 97.3% (tR = 5.9 min).
5.2. Chemistry
5.2.1. tert-Butyl ({3-[(4S,5S)-4-(4-fluorophenyl)-4-(6-fluoropyri-
din-3-yl)-5-methyl-4,5-dihydro-1H-imidazol-2-yl]phenyl}sulfo-
nyl)carbamate (30)
To a stirred solution of 3-(chlorosulfonyl)benzoic acid (10.0 g,
45.3 mmol) in chloroform (100 mL) was added tert-butylamine
(16.3 mL, 320 mmol) at 0 °C, and the mixture was stirred at 0 °C
for 1 h. The reaction mixture was poured into 300 mL of 10% aque-
ous sodium hydrogen sulfate, and extracted with ethyl acetate. The
organic layer was washed with brine, dried over sodium sulfate,
concentrated, and vacuum-dried to give 3-[(tert-butoxycar-
bonyl)sulfamoyl]benzoic acid (12.0 g). To a stirred solution of the
crude benzoic acid (733 mg), (1S,2S)-1-(4-fluorophenyl)-1-(6-flu-
oropyridin-3-yl)propane-1,2-diamine (10) (500 mg, 1.90 mmol),
5.2.3. 2-(Benzyloxy)-6-[(4S,5S)-4-(4-fluorophenyl)-4-(6-fluoro-
pyridin-3-yl)-5-methyl-4,5-dihydro-1H-imidazol-2-yl]-3-meth-
ylpyridine (5b0)
and triethylamine (795 lL, 5.70 mmol) in chloroform (8.0 mL)
was added a 25 wt. % solution of 2-chloro-1,3-dimethylimidazolin-
ium chloride in dichloromethane (1.06 mL) at 0 °C, and the mixture
To a stirred solution of 6-(benzyloxy)-5-methylpyridine-2-car-
bonitrile (11) (52 mg, 0.23 mmol) in methanol (5.0 mL) was added
Table 9
Brain penetration and plasma Occ90 values of 7m in P-gp-deficient mdr1a (ꢀ/ꢀ) and wild type mdr1a (+/+) CF-1 micea
Plasmab
(lM)
Brainb (nmol/g)
Brain/plasmac
Plasma Occ90d (nM)
mdr1a (+/+)
mdr1a (ꢀ/ꢀ)
8.36
7.01
4.00
14.8
0.48
2.05
170
33
a
The values represent the mean for n = 3.
The brain and plasma concentrations were obtained 2 h after oral administration of 7m (10 mg/kg) in mice.
The ratios were obtained from the mean values.
b
c
d
Plasma Occ90 was determined by ex vivo receptor occupancy; see Section 5 for details.

6114
M. Ando et al. / Bioorg. Med. Chem. 17 (2009) 6106–6122
CD3OD): d 0.83 (3H, d, J = 6.6 Hz), 2.14 (3H, s), 4.79 (1H, q,
100
90
80
70
60
50
40
30
20
10
0
J = 6.6 Hz), 6.80–6.88 (1H, m), 6.98–7.15 (3H, m), 7.24–7.36 (2H,
m), 7.45–7.52 (1H, m), 7.95–8.06 (1H, m), 8.28–8.35 (1H, m);
HRMS (ES+) calcd for C21H19F2N4O [M+H]+ m/z 381.1527, found
m/z 381.1519; HPLC purity: (A) 98.4% (tR = 4.1 min), (B) 98.2%
(tR = 5.7 min).
mdr1a (-/-)
mdr1a (+/+)
5.2.5. 6-[(4S,5S)-4-(4-Fluorophenyl)-4-(6-fluoropyridin-3-yl)-5-
methyl-4,5-dihydro-1H-imidazol-2-yl]-4-methylpyridin-2(1H)-
one (5c)
To a stirred solution of 10 (150 mg, 0.57 mmol) and 4-methyl-6-
oxo-1,6-dihydropyridine-2-carboxylic acid (105 mg, 0.68 mmol) in
chloroform (5.0 mL) and pyridine (5.0 mL) was added 1-(3-diemthyla-
minopropyl)-3-ethylcarbodiimide hydrochloride (164 mg, 0.86 mmol)
at 0 °C, and the mixture was allowed to warm to room temperature and
stirred for 72 h. After being concentrated, the residue was partitioned
between ethyl acetate and saturated aqueous sodium hydrogen car-
bonate, andthelayerswere separated. The aqueouslayerwasextracted
with ethyl acetate, and the combined organic layers were washed with
brine, dried over sodium sulfate, and concentrated. The residue was
purified by silica gel flash column chromatography with 0–10% meth-
anol in chloroform to give N-[(1S,2S)-1-amino-1-(4-fluorophenyl)-1-
(6-fluoropyridin-3-yl)propan-2-yl]-4-methyl-6-oxo-1,6-dihydropyri-
dine-2-carboxamide (145 mg) as a pale-red amorphous. 1H NMR
(400 MHz, CDCl3): d 1.15 (3H, d, J = 6.3 Hz), 2.34 (3H, s), 5.30–5.45
(1H, m), 6.48–6.52 (1H, m), 6.76 (1H, dd, J = 3.2, 8.5 Hz), 6.98–7.08
(3H, m), 7.49–7.52 (2H, m), 7.80–7.90 (2H, m), 8.40 (1H, d,
J = 2.4 Hz); MS (ESI): m/z 399 [M+H]+. The amide was suspended in tol-
uene (5.0 mL) and treated with ytterbium tris(trifluoromethanesulfo-
nate) (46 mg, 0.073 mmol), and the mixture was heated to 150 °C for
6 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 chlo-
roform, and the combined organic layers were dried over sodium sul-
fate and concentrated. The residue was purified by preparative TLC
with 10% methanol in chloroform to give 5c (54 mg, 25% yield over 2
steps) as a colorless foam. 1H NMR (400 MHz, CD3OD): d 0.83 (3H, d,
J = 6.3 Hz), 2.28 (3H, d, J = 1.0 Hz), 4.82 (1H, q, J = 6.3 Hz), 6.49–6.51
(1H, m), 6.80–6.84 (1H, m), 7.01–7.09 (3H, m), 7.25–7.35 (2H, m),
7.99–8.04 (1H, m), 8.31 (1H, d, J = 2.4 Hz); HRMS (ES+) calcd for
C21H19F2N4O [M+H]+ m/z 381.1527, found m/z 381.1519; HPLC purity:
(A) 99.6% (tR = 4.3 min), (B) 99.2% (tR = 5.5 min).
-9
-8
-7
-6
-5
Log [Plasma] (M)
100
90
80
70
60
50
40
30
20
10
0
0.3 mg/kg (-/-)
1 mg/kg (-/-)
3 mg/kg (-/-)
0.3 mg/kg (+/+)
1 mg/kg (+/+)
3 mg/kg (+/+)
0
4
8
12
16
20
24
Time (h)
Figure 3. Brain Y5 receptor occupancy and drug exposure levels after oral
administration of 7m. (A): Relationship between plasma concentrations of 7m
and Y5 receptor occupancy in P-gp-deficient mdr1a (ꢀ/ꢀ) and wild type mdr1a (+/+)
CF-1 mice. Receptor occupancy and exposure were determined 1 or 24 h following
oral administration of vehicle or compound 7m (0.3, 1.0, and 3.0 mg/kg). See
Section
5 for details. (B) Receptor occupancy of 7m at 1 or 24 h after oral
administration (0.3, 1.0, and 3.0 mg/kg) in P-gp-deficient mdr1a (ꢀ/ꢀ) and wild
type mdr1a (+/+) CF-1 mice.
a 25 wt. % solution of sodium methoxide in methanol (9.0
0.038 mmol) at room temperature, and the mixture was heated
to 50 °C overnight. After methanesulfonic acid (17 L, 0.27 mmol)
lL,
l
was added, the mixture was treated with 10 (50 mg, 0.19 mmol),
and the resulting mixture was heated to 80 °C overnight. The reac-
tion mixture was partitioned between chloroform and saturated
aqueous sodium hydrogen carbonate, and the layers were sepa-
rated. The aqueous layer was extracted with chloroform, and the
combined organic layers were dried over sodium sulfate and con-
centrated. The residue was purified by silica gel flash column chro-
matography with 20% ethyl acetate in hexanes to give 5b0 (75 mg,
84% yield over 2 steps) as a colorless oil. 1H NMR (300 MHz,
CD3OD): d 0.83 (3H, d, J = 6.2 Hz), 2.24 (3H, s), 4.75 (1H, q,
J = 6.2 Hz), 5.48 (2H, s), 6.95–7.10 (3H, m), 7.20–7.38 (5H, m),
7.40–7.50 (2H, m), 7.55 (1H, d, J = 6.2 Hz), 7.66 (1H, d, J = 6.2 Hz),
8.02 (1H, dt, J = 2.5, 7.5 Hz), 8.32 (1H, d, J = 2.5 Hz); MS (ESI): m/z
471 [M+H]+.
5.2.6. 6-[(4S,5S)-4-(4-Fluorophenyl)-4-(6-fluoropyridin-3-yl)-5-
methyl-4,5-dihydro-1H-imidazol-2-yl]-5-methylpyridin-2(1H)-
one (5d)
Compound 5d was prepared from 10 and 3-methyl-6-oxo-1,6-
dihydropyridine-2-carboxylic acid using the procedure described
for 5c as a colorless solid (28% yield over 2 steps). 1H NMR
(400 MHz, CD3OD): d 0.86 (3H, d, J = 6.0 Hz), 2.16 (3H, s), 4.80–
4.90 (1H, m), 6.58 (1H, d, J = 9.2 Hz), 7.03 (1H, dd, J = 2.8 Hz,
8.8 Hz), 7.07 (2H, t, J = 8.8 Hz), 7.30–7.40 (2H, m), 7.48 (1H, d, J =
9.2 Hz), 7.88 (1H, s), 7.90–8.00 (1H, m), 8.31 (1H, d, J = 2.0 Hz);
HRMS (ES+) calcd for C21H19F2N4O [M+H]+ m/z 381.1527, found
m/z 381.1524; HPLC purity: (A) 99.7% (tR = 3.8 min), (B) 98.4%
(tR = 5.5 min).
5.2.4. 6-[(4S,5S)-4-(4-Fluorophenyl)-4-(6-fluoropyridin-3-yl)-5-
methyl-4,5-dihydro-1H-imidazol-2-yl]-3-methylpyridin-2(1H)-
one (5b)
Compound 5b0 (75 mg, 0.16 mmol) was dissolved in TFA
(10 mL), and the mixture was stirred at room temperature for
2 days. After being concentrated, the residue was partitioned be-
tween chloroform and saturated aqueous sodium hydrogen car-
bonate, 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 puri-
fied by preparative TLC with 10% methanol in chloroform to give
5b (37 mg, 61% yield) as a pale-brown solid. 1H NMR (300 MHz,
5.2.7. 3-Fluoro-6-[(4S,5S)-4-(4-fluorophenyl)-4-(6-fluoropyridin-
3-yl)-5-methyl-4,5-dihydro-1H-imidazol-2-yl]pyridin-2(1H)-one
(5e)
Compound 5e was prepared from 10 and 5-fluoro-6-oxo-1,6-
dihydropyridine-2-carboxylic acid (21) using the procedure de-
scribed for 5c as a colorless solid (6% yield over 2 steps). 1H NMR
(400 MHz, CD3OD): d 0.60–1.00 (3H, m), 4.90–5.10 (1H, m), 6.80–
7.40 (7H,m), 7.88 (1H, s), 8.00–8.40 (1H, m); HRMS (ES+) calcd

M. Ando et al. / Bioorg. Med. Chem. 17 (2009) 6106–6122
6115
for C20H16F3N4O [M+H]+ m/z 385.1276, found m/z 385.1270; HPLC
5.2.13. 5-[(4S,5S)-4-(4-Fluorophenyl)-4-(6-fluoropyridin-3-yl)-
purity: (A) 98.7% (tR = 4.0 min), (B) 98.0% (tR = 5.1 min).
5-methyl-4,5-dihydro-1H-imidazol-2-yl]-4-methylpyridin-
2(1H)-one trifluoroacetate (7c trifluoroacetate)
Compound 7c0 (49 mg, 0.10 mmol) was dissolved in TFA
(5.0 mL), and the mixture was stirred at room temperature over-
night. After being concentrated, the residue was purified by pre-
parative HPLC with 10–50% 0.1% CF3CO2H/CH3CN in 0.1%
aqueous CF3CO2H, and the combined fractions were concentrated
and vacuum-dried to give a trifluoroacetate salt of 7c (27 mg,
55% yield) as a pale-brown solid. 1H NMR (400 MHz, CD3OD): d
1.06 (3H, d, J = 6.4 Hz), 2.32 (3H, d, J = 1.2 Hz), 5.34 (1H, q,
J = 6.4 Hz), 6.48 (1H, s), 7.10–7.40 (5H, m), 8.00–8.10 (1H, m),
8.06 (1H, s), 8.35 (1H, d, J = 2.8 Hz); HRMS (ES+) calcd for
C21H19F2N4O [M+H]+ m/z 381.1527, found m/z 381.1526; HPLC pur-
ity: (A) 99.0% (tR = 3.7 min), (B) 98.9% (tR = 5.1 min).
5.2.8. 3-Chloro-6-[(4S,5S)-4-(4-fluorophenyl)-4-(6-fluoropyridin-
3-yl)-5-methyl-4,5-dihydro-1H-imidazol-2-yl]pyridin-2(1H)-one
(5f)
Compound 5f was prepared from 10 and 5-chloro-6-oxo-1,6-
dihydropyridine-2-carboxylic acid (18) using the procedure de-
scribed for 5c as a colorless solid (18% yield over 2 steps). 1H
NMR (300 MHz, CD3OD): d 0.89 (3H, d, J = 6.6 Hz), 4.93 (1H, q,
J = 6.6 Hz), 6.96 (1H, d, J = 7.6 Hz), 7.00–7.15 (3H, m), 7.20–7.35
(2H, m), 7.76 (1H, d, J = 7.6 Hz), 8.04 (1H, dt, J = 2.6, 8.5 Hz), 8.34
(1H, d, J = 2.6 Hz); HRMS (ES+) calcd for C20H16ClF2N4O [M+H]+ m/
z
401.0981, found m/z 401.0982; HPLC purity: (A) 99.2%
(tR = 4.1 min), (B) 98.8% (tR = 5.6 min).
5.2.14. 6-(Benzyloxy)-2-fluoro-3-[(4S,5S)-4-(4-fluorophenyl)-4-
(6-fluoropyridin-3-yl)-5-methyl-4,5-dihydro-1H-imidazol-2-
yl]pyridine (7d0)
5.2.9. 6-[(4S,5S)-4-(4-Fluorophenyl)-4-(6-fluoropyridin-3-yl)-5-
methyl-4,5-dihydro-1H-imidazol-2-yl]-1-methylpyridin-2(1H)-
one (5g)
Compound 7d0 was prepared from 10, 6-(benzyloxy)-2-fluoro-
pyridine-3-carboxylic acid (42), and scandium tris(trifluorometh
anesulfonate) in place of ytterbium tris(trifluoromethanesulfo-
nate) using the procedure described for 5c as a colorless oil (51%
yield over 2 steps). 1H NMR (400 MHz, CD3OD): d 0.83 (3H, d, J =
6.4 Hz), 4.76 (1H, q, J = 6.4 Hz), 5.37 (2H, s), 6.82 (1H, dd, J = 1.0,
8.3 Hz), 7.00–7.10 (3H, m), 7.25–7.40 (5H, m), 7.40–7.45 (2H, m),
7.95–8.05 (1H, m), 8.24 (1H, t, J = 9.0 Hz), 8.31 (1H, d, J = 2.9 Hz);
MS (ESI): m/z 475 [M+H]+.
Compound 5g was prepared from 10 and 1-methyl-6-oxo-1,6-
dihydropyridine-2-carboxylic acid (24a) using the procedure de-
scribed for 5c as a colorless solid (5% yield over 2 steps). 1H NMR
(300 MHz, CDCl3): d 0.87 (3H, d, J = 6.5 Hz), 2.68 (3H, s), 4.70 (1H,
q, J = 6.5 Hz), 5.19 (1H, s), 6.37 (1H, dd, J = 1.2 Hz, 6.7 Hz), 6.60–
6.65 (1H, m), 6.85–6.95 (1H,m), 7.00–7.10 (2H, m), 7.20–7.40
(3H, m), 7.75–7.85 (1H, m), 8.41 (1H, s); HRMS (ES+) calcd for
C21H19F2N4O [M+H]+ m/z 381.1527, found m/z 381.1529; HPLC pur-
ity: (A) 99.9% (tR = 3.6 min), (B) 99.8% (tR = 5.3 min).
5.2.15. 6-Fluoro-5-[(4S,5S)-4-(4-fluorophenyl)-4-(6-fluoropyridin-
3-yl)-5-methyl-4,5-dihydro-1H-imidazol-2-yl]pyridin-2(1H)-one
(7d)
5.2.10. 5-[(4S,5S)-4-(4-Fluorophenyl)-4-(6-fluoropyridin-3-yl)-
5-methyl-4,5-dihydro-1H-imidazol-2-yl]pyridin-2(1H)-one (7a)
Compound 7a was prepared from 10 and 6-oxo-1,6-dihydro-
pyridine-3-carboxylic acid using the procedure described for 5c
as a colorless solid (26% yield over 2 steps). 1H NMR (300 MHz,
CD3OD): d 0.83 (3H, d, J = 6.5 Hz), 4.74 (1H, q, J = 6.5 Hz), 6.56
(1H, d, J = 10.3 Hz), 7.00–7.10 (3H, m), 7.20–7.30 (2H, m), 7.89
(1H, s), 7.95–8.10 (2H, m), 8.27 (1H, d, J = 2.7 Hz); HRMS (ES+) calcd
for C20H17F2N4O [M+H]+ m/z 367.1370, found m/z 367.1360; HPLC
purity: (A) 99.9% (tR = 3.6 min), (B) 99.7% (tR = 5.0 min).
Compound 7d0 (84 mg, 0.18 mmol) was dissolved in TFA
(10 mL), and the mixture was heated to 50 °C for 3 days. After
being concentrated, the residue was purified by preparative HPLC
with 10–50% 0.1% CF3CO2H/CH3CN in 0.1% aqueous CF3CO2H, to
give 7d (2.3 mg, 3% yield) as
(400 MHz, CD3OD): 1.00 (3H, d, J = 6.4 Hz), 5.08 (1H, q,
a
pale-brown solid. 1H NMR
d
J = 6.4 Hz), 6.60–6.63 (1H, m), 7.05–7.15 (4H, m), 7.80–8.05 (3H,
m), 8.30–8.40 (1H, m); HRMS (ES+) calcd for C20H16F3N4O [M+H]+
m/z 385.1276, found m/z 385.1271; HPLC purity: (A) 97.0%
(tR = 4.0 min), (B) 96.6% (tR = 4.5 min).
5.2.11. 5-[(4S,5S)-4-(4-Fluorophenyl)-4-(6-fluoropyridin-3-yl)-
5-methyl-4,5-dihydro-1H-imidazol-2-yl]-3-methylpyridin-
2(1H)-one (7b)
5.2.16. 3-Fluoro-5-[(4S,5S)-4-(4-fluorophenyl)-4-(6-fluoropyridin-
3-yl)-5-methyl-4,5-dihydro-1H-imidazol-2-yl]pyridin-2(1H)-
one (7e)
Compound 7b was prepared from 10 and 5-methyl-6-oxo-1,6-
dihydropyridine-3-carboxylic acid (27b) using the procedure de-
scribed for 5c as a colorless solid (49% yield over 2 steps). 1H
NMR (400 MHz, CD3OD): d 0.83 (3H, d, J = 6.8 Hz), 2.14 (3H, s),
4.72 (1H, q, J = 6.8 Hz), 7.03 (1H, dd, J = 2.4, 8.4 Hz), 7.05–7.09
(2H, m), 7.23–7.27 (2H, m), 7.89 (1H, s), 7.90–7.91 (1H, m), 7.95–
8.00 (1H, m), 8.26 (1H, s); HRMS (ES+) calcd for C21H19F2N4O
[M+H]+ m/z 381.1527, found m/z 381.1528; HPLC purity: (A)
99.9% (tR = 3.7 min), (B) 99.7% (tR = 5.3 min).
Compound 7e was prepared from 10 and 5-fluoro-6-oxo-1,6-
dihydropyridine-3-carboxylic acid (37) using the procedure de-
scribed for 5c as a colorless amorphous (36% yield over 2 steps).
1H NMR (300 MHz, CD3OD): d 0.84 (3H, d, J = 6.3 Hz), 4.70–4.90
(1H, m), 7.00–7.15 (3H, m), 7.20–7.30 (2H, m), 7.82 (1H, dd,
J = 2.3, 11.2 Hz), 7.90–8.05 (2H, m), 8.28 (1H, d, J = 2.3 Hz); HRMS
(ES+) calcd for C20H16F3N4O [M+H]+ m/z 385.1276, found m/z
385.1278; HPLC purity: (A) 95.3% (tR = 3.7 min), (B) 95.3%
(tR = 5.1 min).
5.2.12. 2-(Benzyloxy)-5-[(4S,5S)-4-(4-fluorophenyl)-4-(6-fluoro-
pyridin-3-yl)-5-methyl-4,5-dihydro-1H-imidazol-2-yl]-4-meth-
ylpyridine (7c0)
5.2.17. 3-Chloro-5-[(4S,5S)-4-(4-fluorophenyl)-4-(6-fluoropyridin-
3-yl)-5-methyl-4,5-dihydro-1H-imidazol-2-yl]pyridin-2(1H)-
one (7f)
Compound 7f was prepared from 10 and 5-chloro-6-oxo-1,6-
dihydropyridine-3-carboxylic acid using the procedure described
for 5c as a colorless solid (29% yield over 2 steps). 1H NMR
Compound 7c0 was prepared from 10, 6-(benzyloxy)-4-methyl-
pyridine-3-carboxylic acid (31), and scandium tris(trifluorometh
anesulfonate) in place of ytterbium tris(trifluoromethanesulfo-
nate) using the procedure described for 5c as a pale-brown oil
(26% yield over 2 steps). 1H NMR (400 MHz, CD3OD): d 0.84 (3H,
d, J = 6.4 Hz), 2.35 (3H, s), 4.77 (1H, q, J = 6.4 Hz), 5.35 (2H, s),
6.73 (1H, s), 7.00–7.10 (3H, m), 7.20–7.40 (7H, m), 7.96 (1H, dt,
J = 2.4, 8.8 Hz), 8.17 (1H, s), 8.30 (1H, d, J = 3.2 Hz); MS (ESI): m/z
471 [M+H]+.
(300 MHz, CD3OD):
d 0.84 (3H, d, J = 6.6 Hz), 4.80 (1H, q,
J = 6.6 Hz), 7.00–7.15 (3H, m), 7.20–7.30 (2H, m), 7.89 (1H, s),
7.95–8.05 (1H, m), 8.04 (1H, d, J = 2.4 Hz), 8.21 (1H, d, J = 2.4 Hz),
8.25–8.30 (1H, m); HRMS (ES+) calcd for C20H16ClF2N4O [M+H]+

6116
M. Ando et al. / Bioorg. Med. Chem. 17 (2009) 6106–6122
m/z 401.0981, found m/z 401.0983; HPLC purity: (A) 98.7%
409.1839; HPLC purity: (A) 98.9% (tR = 4.1 min), (B) 98.3%
(tR = 6.0 min).
(tR = 3.9 min), (B) 98.8% (tR = 5.4 min); ½a D25
ꢀ239 ° (c 1.0, CH3OH).
ꢃ
5.2.18. 5-[(4S,5S)-4-(4-Fluorophenyl)-4-(6-fluoropyridin-3-yl)-
5-methyl-4,5-dihydro-1H-imidazol-2-yl]-3-(trifluoromethyl)-
pyridin-2(1H)-one (7g)
5.2.23. 5-[(4S,5S)-4-(4-Fluorophenyl)-4-(6-fluoropyridin-3-yl)-
5-methyl-4,5-dihydro-1H-imidazol-2-yl]-1-(propan-2-yl)pyridin-
2(1H)-one (7l)
Compound 7g was prepared from 10 and 6-oxo-5-(trifluoro-
methyl)-1,6-dihydropyridine-3-carboxylic acid (30) using the pro-
cedure described for 5c as a pale-yellow solid (25% yield over 2
steps). 1H NMR (300 MHz, CD3OD): d 0.85 (3H, d, J = 6.5 Hz), 4.73–
4.90 (1H, m), 7.02–7.14 (3H, m), 7.21–7.30 (2H, m), 7.99 (1H, ddd,
J = 2.7, 7.6, 8.6 Hz), 8.28 (1H, d, J = 2.5 Hz), 8.32 (1H, d, J = 2.6 Hz),
8.39 (1H, d, J = 1.9 Hz); HRMS (ES+) calcd for C21H16F5N4O [M+H]+
m/z 435.1244, found m/z 435.1250; HPLC purity: (A) 97.9%
Compound 7l was prepared from 10 and 6-oxo-1-(propan-2-
yl)-1,6-dihydropyridine-3-carboxylic acid (24c) using the proce-
dure described for 5c as a colorless solid (38% yield over 2 steps).
1H NMR (400 MHz, CD3OD): d 0.84 (3H, d, J = 6.4 Hz), 1.42 (3H, d,
J = 4.4 Hz), 1.44 (3H, d, J = 4.4 Hz), 4.75 (1H, q, J = 6.4 Hz), 5.18
(1H, qui, J = 4.4 Hz), 6.56 (1H, d, J = 9.2 Hz), 7.00–7.10 (3H, m),
7.20–7.30 (2H, m), 7.90–8.00 (2H, m), 8.27 (2H, d, J = 2.4 Hz);
HRMS (ES+) calcd for C23H23F2N4O [M+H]+ m/z 409.1840, found
m/z 409.1850; HPLC purity: (A) 99.4% (tR = 4.1 min), (B) 98.0%
(tR = 6.0 min).
(tR = 4.2 min), (B) 97.2% (tR = 5.9 min); ½a D25
ꢀ255 ° (c 0.45, CH3OH).
ꢃ
5.2.19. 5-[(4S,5S)-4-(4-Fluorophenyl)-4-(6-fluoropyridin-3-yl)-
5-methyl-4,5-dihydro-1H-imidazol-2-yl]-3-methoxypyridin-
2(1H)-one (7h)
5.2.24. 1-(Difluoromethyl)-5-[(4S,5S)-4-(4-fluorophenyl)-4-(6-
fluoropyridin-3-yl)-5-methyl-4,5-dihydro-1H-imidazol-2-
yl]pyridin-2(1H)-one (7m)
Compound 7h was prepared from 10 and 5-methoxy-6-oxo-
1,6-dihydropyridine-3-carboxylic acid (27a) using the procedure
described for 5c as a colorless solid (25% yield over 2 steps). 1H
NMR (400 MHz, CD3OD): d 0.85 (3H, d, J = 6.2 Hz), 3.88 (3H, s),
4.74 (1H, q, J = 6.2 Hz), 7.02–7.10 (3H, m), 7.24–7.28 (2H, m),
7.43 (1H, d, J = 2.0 Hz), 7.64 (1H, d, J = 2.0 Hz), 7.96–8.01 (1H, m),
8.27 (1H, d, J = 2.0 Hz); HRMS (ES+) calcd for C21H19F2N4O2
[M+H]+ m/z 397.1476, found m/z 397.1472; HPLC purity: (A)
95.4% (tR = 3.7 min), (B) 97.1% (tR = 5.1 min).
Compound 7m was prepared from 10 and 1-(difluoromethyl)-
6-oxo-1,6-dihydropyridine-3-carboxylic acid (24d) using the pro-
cedure described for 5c as a colorless amorphous (23% yield over
2 steps). 1H NMR (300 MHz, CD3OD): d 0.83 (1H, d, J = 6.5 Hz),
4.76 (1H, q, J = 6.5 Hz), 6.62 (1H, d, J = 9.9 Hz), 7.00–7.15 (3H, m),
7.20–7.30 (2H, m), 7.78 (1H, t, J = 59.9 Hz), 7.90–8.10 (2H, m),
8.25–8.35 (2H, m); HRMS (ES+) calcd for C21H17F4N4O [M+H]+ m/z
417.1338, found m/z 417.1336; HPLC purity: (A) 99.1%
(tR = 4.1 min), (B) 97.6% (tR = 6.4 min); ½a D25
ꢀ300 ° (c 0.63, CH3OH).
ꢃ
5.2.20. 5-[(4S,5S)-4-(4-Fluorophenyl)-4-(6-fluoropyridin-3-yl)-
5-methyl-4,5-dihydro-1H-imidazol-2-yl]-1-methylpyridin-
2(1H)-one (7i)
5.2.25. 5-[(4S,5S)-4-(4-Fluorophenyl)-4-(6-fluoropyridin-3-yl)-
5-methyl-4,5-dihydro-1H-imidazol-2-yl]-1-methoxypyridin-
2(1H)-one (7n)
Compound 7i was prepared from 10 and 1-methyl-6-oxo-1,6-
dihydropyridine-3-carboxylic acid using the procedure described
for 5c as a colorless amorphous (40% yield over 2 steps). 1H NMR
(300 MHz, CD3OD): d 0.83 (3H, d, J = 6.5 Hz), 3.60 (3H, s), 4.74
(1H, q, J = 6.5 Hz), 6.57 (1H, d, J = 9.6 Hz), 7.00–7.15 (3H, m),
7.20–7.30 (2H, m), 7.89 (1H, s), 7.90–8.05 (2H, m), 8.26 (2H, dd,
J = 2.4, 8.0 Hz); HRMS (ES+) calcd for C21H19F2N4O [M+H]+ m/z
381.1527, found m/z 381.1526; HPLC purity: (A) 98.0%
(tR = 3.7 min), (B) 97.8% (tR = 5.3 min).
Compound 7n was prepared from 10 and 1-methoxy-6-oxo-
1,6-dihydropyridine-3-carboxylic acid (39a) using the procedure
described for 5c as a colorless solid (9% yield over 2 steps). 1H
NMR (400 MHz, CD3OD): d 0.83 (3H, d, J = 6.4 Hz), 4.08 (3H, s),
4.75 (1H, q, J = 6.4 Hz), 6.69 (1H, d, J = 9.6 Hz), 7.02 (1H, dd,
J = 2.4, 8.4 Hz), 7.04–7.08 (2H, m), 7.24–7.27 (2H, m), 7.95–7.99
(2H, m), 8.26 (1H, d, J = 2.8 Hz), 8.47 (1H, d, J = 2.4 Hz); HRMS
(ES+) calcd for C21H19F2N4O2 [M+H]+ m/z 397.1476, found m/z
397.1469; HPLC purity: (A) 98.4% (tR = 3.7 min), (B) 96.1%
5.2.21. 1-Ethyl-5-[(4S,5S)-4-(4-fluorophenyl)-4-(6-fluoropyridin-
3-yl)-5-methyl-4,5-dihydro-1H-imidazol-2-yl]pyridin-2(1H)-
one (7j)
Compound 7j was prepared from 10 and 1-ethyl-6-oxo-1,6-
dihydropyridine-3-carboxylic acid using the procedure described
for 5c as a colorless solid (38% yield over 2 steps). 1H NMR
(tR = 5.5 min); ½a D25
ꢀ308 ° (c 0.75, CH3OH).
ꢃ
5.2.26. 1-Ethoxy-5-[(4S,5S)-4-(4-fluorophenyl)-4-(6-fluoropyridin-
3-yl)-5-methyl-4,5-dihydro-1H-imidazol-2-yl]pyridin-2(1H)-
one (7o)
Compound 7o was prepared from 10 and 1-ethoxy-6-oxo-1,6-
dihydropyridine-3-carboxylic acid (39b) using the procedure de-
scribed for 5c as a colorless solid (22% yield over 2 steps). 1H
(300 MHz, CD3OD):
d 0.83 (3H, d, J = 6.5 Hz), 1.37 (3H, t,
J = 7.2 Hz), 4.07 (2H, q, J = 7.2 Hz), 4.75 (1H, q, J = 6.5 Hz), 6.57
(1H, d, J = 9.6 Hz), 7.00–7.15 (3H, m), 7.20–7.30 (2H, m), 7.89
(1H, s), 7.90–8.00 (2H, m), 8.20–8.30 (2H, m); HRMS (ES+) calcd
for C22H21F2N4O [M+H]+ m/z 395.1683, found m/z 395.1675; HPLC
purity: (A) 99.5% (tR = 3.9 min), (B) 98.7% (tR = 5.7 min).
NMR (400 MHz, CD3OD):
d 0.80–0.90 (3H, m), 1.40 (3H, t,
J = 7.0 Hz), 4.33 (2H, d, J = 7.0 Hz), 4.80–4.90 (1H, m), 6.69 (1H, d,
J = 9.6 Hz), 7.00–7.10 (3H, m), 7.20–7.30 (2H, m), 7.94–8.02 (2H,
m), 8.25–8.30 (1H, m), 8.46 (1H, d, J = 2.4 Hz); HRMS (ES+) calcd
for C22H21F2N4O2 [M+H]+ m/z 411.1633, found m/z 411.1621; HPLC
purity: (A) 99.8% (tR = 3.9 min), (B) 99.6% (tR = 5.8 min).
5.2.22. 5-[(4S,5S)-4-(4-Fluorophenyl)-4-(6-fluoropyridin-3-yl)-
5-methyl-4,5-dihydro-1H-imidazol-2-yl]-1-propylpyridin-
2(1H)-one (7k)
5.2.27. 1-Ethyl-5-[(4S,5S)-4-(4-fluorophenyl)-4-(6-
Compound 7k was prepared from 10 and 6-oxo-1-propyl-1,6-
dihydropyridine-3-carboxylic acid (24b) using the procedure de-
scribed for 5c as a colorless solid (47% yield over 2 steps). 1H
NMR (400 MHz, CD3OD): d 0.84 (3H, d, J = 5.6 Hz), 0.97 (3H, t,
J = 7.6 Hz), 1.80 (2H, sextet, J = 7.6 Hz), 3.98 (2H, t, J = 7.6 Hz),
4.70–4.80 (1H, m), 6.56 (1H, d, J = 9.6 Hz), 7.00–7.10 (3H, m),
7.20–7.30 (2H, m), 7.90–8.00 (2H, m), 8.20–8.30 (2H, m); HRMS
(ES+) calcd for C23H23F2N4O [M+H]+ m/z 409.1840, found m/z
fluoropyridin-3-yl)-5-methyl-4,5-dihydro-1H-imidazol-2-yl]-3-
methylpyridin-2(1H)-one (7p)
Compound 7p was prepared from 10 and 1-ethyl-5-methyl-6-
oxo-1,6-dihydropyridine-3-carboxylic acid (34a) using the proce-
dure described for 5c as a colorless solid (23% yield over 2 steps).
1H NMR (400 MHz, CD3OD): d 0.84 (3H, d, J = 6.8 Hz), 1.37 (3H, t,
J = 7.2 Hz), 2.16 (3H, s), 4.08 (2H, q, J = 6.8 Hz), 4.74 (1H, q,
J = 6.8 Hz), 7.03 (1H, dd, J = 2.4, 8.0 Hz), 7.05–7.09 (2H, m),

M. Ando et al. / Bioorg. Med. Chem. 17 (2009) 6106–6122
6117
7.24–7.28 (2H, m), 7.84 (1H, dd, J = 1.2, 2.4 Hz), 7.95–8.01 (1H, m),
8.12 (1H, dd, J = 0.8, 2.4 Hz), 8.27 (1H, d, J = 2.8 Hz); HRMS (ES+)
calcd for C23H23F2N4O [M+H]+ m/z 409.1840, found m/z 409.1835;
HPLC purity: (A) 98.3% (tR = 4.1 min), (B) 97.2% (tR = 6.1 min).
solved in dioxane (13 mL), and the mixture was cooled to 0 °C.
8 N HCl (13 mL) was added slowly to the mixture at 0 °C, and the
resulting mixture was allowed to warm up to room temperature
and stirred for 2 h. After being cooled to 0 °C, the mixture was basi-
fied with 8 N NaOH (20 mL). The resultant mixture was saturated
with solid NaCl and extracted twice with ethyl acetate. The com-
bined organic layers were washed with brine, dried over sodium
sulfate, concentrated, and vacuum-dried to give 10 (3.02 g, 100%)
as a yellow oil. 1H NMR (400 MHz, CDCl3): d 1.00 (3H, d,
J = 6.2 Hz), 4.04 (1H, q, J = 6.2 Hz), 6.85 (1H, dd, J = 3.4, 8.8 Hz),
7.00–7.05 (2H, m), 7.30–7.40 (2H, m), 7.90–8.00 (1H, m), 8.39
(1H, d, J = 2.0 Hz); MS (ESI): m/z 264 [M+H]+.
5.2.28. 1-(Difluoromethyl)-5-[(4S,5S)-4-(4-fluorophenyl)-4-(6-
fluoropyridin-3-yl)-5-methyl-4,5-dihydro-1H-imidazol-2-yl]-3-
methylpyridin-2(1H)-one (7q)
Compound 7q was prepared from 10 and 1-(difluoromethyl)-5-
methyl-6-oxo-1,6-dihydropyridine-3-carboxylic acid (34b) using
the procedure described for 5c as a yellow solid (17% yield over
2 steps). 1H NMR (400 MHz, CD3OD): d 0.81–0.88 (3H, m), 2.16
(3H, s), 4.72–4.78 (1H, brs), 7.10–7.09 (3H, m), 7.23–7.28 (2H,
m), 7.80 (1H, t, J = 60 Hz), 7.92–8.20 (2H, m), 8.16–8.19 (1H, br
s), 8.25–8.30 (1H, brs); HRMS (ES+) calcd for C22H19F4N4O [M+H]+
m/z 431.1495, found m/z 431.1485; HPLC purity: (A) 99.6%
(tR = 4.3 min), (B) 99.1% (tR = 6.9 min).
5.2.33. 6-(Benzyloxy)-5-methylpyridine-2-carbonitrile (11)
To a stirred solution of 17a (1.31 g, 6.7 mmol) and benzyl alco-
hol (763 lL, 7.4 mmol) in THF (50 mL) was added sodium hydride
(60% dispersion in oil, 320 mg, 8.0 mmol), and the mixture was
heated to reflux for 1 h. After being cooled to room temperature,
the mixture was partitioned between ethyl acetate and saturated
aqueous sodium hydrogen carbonate, and the layers were sepa-
rated. 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 sil-
ica gel flash column chromatography with 5% ethyl acetate in hex-
anes to give 11 (1.05 g, 70% yield) as a pale-pink oil. 1H NMR
(400 MHz, CDCl3): d 2.28 (3H, s), 5.40 (2H, s) 7.20–7.30 (1H, m),
7.30–7.40 (3H, m), 7.40–7.50 (3H, m); MS (ESI): m/z 225 [M+H]+.
5.2.29. 3-[(4S,5S)-4-(4-Fluorophenyl)-4-(6-fluoropyridin-3-yl)-
5-methyl-4,5-dihydro-1H-imidazol-2-yl]pyridin-2(1H)-one (8)
Compound 8 was prepared from 10 and 2-oxo-1,2-dihydropyr-
idine-3-carboxylic acid using the procedure described for 5c as a
pale-yellow solid (11% yield over 2 steps). 1H NMR (300 MHz,
CD3OD): d 1.03 (3H, d, J = 6.6 Hz), 5.24 (1H, q, J = 6.6 Hz), 6.65
(1H, dd, J = 6.4, 7.4 Hz), 7.10–7.40 (5H, m), 7.89 (1H, s), 7.96 (1H,
dd, J = 2.1, 6.3 Hz), 8.05–8.15 (1H, m), 8.38 (1H, d, J = 2.8 Hz), 8.45
(1H, dd, J = 2.1, 7.4 Hz); HRMS (ES+) calcd for C20H17F2N4O
[M+H]+ m/z 367.1370, found m/z 367.1361; HPLC purity: (A)
95.1% (tR = 3.8 min), (B) 95.8% (tR = 5.1 min).
5.2.34. tert-Butyl [(2S)-1-(4-fluorophenyl)-1-oxopropan-2-
yl]carbamate (13)
5.2.30. 4-[(4S,5S)-4-(4-Fluorophenyl)-4-(6-fluoropyridin-3-yl)-
5-methyl-4,5-dihydro-1H-imidazol-2-yl]-2-methoxypyridine
(90)
To a stirred solution of N0-(tert-butoxycarbonyl)-N-methoxy-N-
methyl-L-alaninamide (12) (20.0 g, 86.1 mmol) in THF (300 mL)
was added a 2 M THF solution of 4-fluorophenylmagnesium bro-
mide (100 mL, 200 mmol) at 0 °C, and the mixture was allowed
to warm up to room temperature and stirred overnight. After being
cooled to 0 °C, saturated aqueous ammonium chloride was added
to the mixture. The resulting mixture was extracted twice with
ethyl acetate, and the combined organic layers were washed with
brine, dried over sodium sulfate, concentrated, and vacuum-dried
to give 13 (23.1 g, 100% yield) as a pale-brown solid. 1H NMR
(400 MHz, CDCl3): d 0.95 (3H, t, J = 7.6 Hz), 1.30 (9H, s), 5.24 (1H,
qui, J = 7.6 Hz), 5.20–5.40 (1H, m), 7.16 (2H, t, J = 8.0 Hz), 7.95–
8.05 (2H, m); MS (ESI): m/z 268 [M+H]+.
Compound 90 was prepared from 10 and 2-methoxypyridine-4-
carboxylic acid using the procedure described for 5c as a colorless
amorphous (46% yield over 2 steps). 1H NMR (300 MHz, CD3OD): d
0.80–0.90 (3H, m), 3.92 (3H, s), 4.70–4.90 (1H, m), 7.00–7.40 (7H,
m), 7.95–8.05 (1H, m), 8.20–8.40 (2H, m); MS (ESI): m/z 381 [M+H]+.
5.2.31. 4-[(4S,5S)-4-(4-Fluorophenyl)-4-(6-fluoropyridin-3-yl)-
5-methyl-4,5-dihydro-1H-imidazol-2-yl]pyridin-2(1H)-one (9)
To a stirred solution of sodium iodide (352 mg, 2.35 mmol) in
acetonitrile (10 mL) was added trimethylsilyl chloride (298 lL,
2.35 mmol) at room temperature, and the mixture was stirred at
room temperature for 15 min. To the resulting mixture was added
a solution of 90 (180 mg, 0.47 mmol) in acetonitrile (10 mL) at room
temperature, and the mixture was stirred at room temperature for
3 days. After being concentrated, the residue was partitioned be-
tween chloroform and saturated aqueous sodium hydrogen car-
bonate, 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 puri-
fied by preparative TLC with 10% methanol in chloroform to give 9
(153 mg, 89% yield) as a colorless solid. 1H NMR (300 MHz,
CD3OD): d 0.83 (3H, d, J = 6.3 Hz), 4.70–4.90 (1H, m), 6.83 (1H,
dd, J = 1.3, 6.9 Hz), 6.92 (1H, s), 7.00–7.10 (3H, m), 7.20–7.30 (2H,
m), 7.49 (1H, d, J = 6.7 Hz), 7.84 (1H, s), 7.90–8.10 (1H, m), 8.28
(1H, s); HRMS (ES+) calcd for C20H17F2N4O [M+H]+ m/z 367.1370,
found m/z 367.1373; HPLC purity: (A) 99.8% (tR = 3.6 min), (B)
99.5% (tR = 5.1 min).
5.2.35. tert-Butyl [(1E,2S)-1-{[(R)-tert-butylsulfinyl]imino}-1-
(4-fluorophenyl)propan-2-yl]carbamate (14)
To a stirred solution of 13 (64.8 g, 243 mmol) and (R)-2-methyl-
propane-2-sulfinamide (35.4 g, 292 mmol) in toluene (130 mL) was
added titanium tetraethanolate (127 mL, 607 mL), and the mixture
was heated to 70 °C for 17 h under a nitrogen atmosphere. After
being cooled to room temperature, brine (240 mL) and ethyl acetate
(480 mL) was added to the mixture, and the resulting mixture was
stirred at room temperature for 20 min. The resultant suspension
was filtered though Celite pad, and the filtrate was washed with
water and brine, dried over sodium sulfate, and concentrated. The
residue was purified by silica gel flash column chromatography
with 10–15% ethyl acetate in hexanes to give 14 (54.3 g, 60% yield)
as a pale-yellow solid. 1H NMR (300 MHz, CDCl3): d 1.32 (9H, s), 1.44
(9H, s), 1.51 (3H, d, J = 7.2 Hz), 5.00–5.40 (1H, br s), 7.11 (2H, t,
J = 8.0 Hz), 7.20–8.00 (2H, m); MS (ESI): m/z 371 [M+H]+.
5.2.32. (1S,2S)-1-(4-Fluorophenyl)-1-(6-fluoropyridin-3-yl)-
propane-1,2-diamine (10)
5.2.36. tert-Butyl [(1S,2S)-1-{[(R)-tert-butylsulfinyl]amino}-1-(4-
fluorophenyl)-1-(6-fluoropyridin-3-yl)propan-2-yl]carbamate (15)
Compound 15 (5.37 g, 11.5 mmol) was dissolved in TFA (11 mL)
at 0 °C, and the mixture was allowed to warm to room temperature
and stirred for 1 h. After being concentrated, the residue was dis-
To a stirred solution of 5-bromo-2-fluoropyridine (7.87 g,
44.7 mmol) in diethyl ether (200 mL) was added slowly a 2.66 M
solution of n-BuLi in hexanes (18.5 mL, 49.2 mmol) at ꢀ78 °C,

6118
M. Ando et al. / Bioorg. Med. Chem. 17 (2009) 6106–6122
and the mixture was stirred at ꢀ78 °C for 30 min. A solution of 14
(5.52 g, 14.9 mmol) and trimethylaluminum (1 M hexane solution,
19.4 mL, 19.4 mmol) in diethyl ether (100 mL), which was stirred
at 0 °C for 15 min in advance, was added to the resultant orange
suspension at ꢀ78 °C. The resulting mixture was stirred at
ꢀ78 °C for 30 min before adding saturated aqueous sodium sulfate
and solid magnesium sulfate. After being warmed to room temper-
ature, the suspension was filtered through Celite pad, and the fil-
trate was concentrated. The residue was triturated with
diisopropyl ether, and the resulting precipitate was collected by fil-
tration. The resultant solid was vacuum-dried to give 15 (5.37 g,
77% yield) as a pale-brown solid. 1H NMR (300 MHz, CDCl3): d
0.99 (3H, d, J = 6.7 Hz), 1.44 (9H, s), 1.48 (9H, s), 4.37 (1H, s), 5.03
(1H, m), 6.81 (1H, brs), 6.87 (1H, dd, J = 3.2, 8.8 Hz), 7.03 (2H, m),
7.29 (2H, m), 7.97 (1H, m), 8.22 (1H, d, J = 2.6 Hz); MS (ESI): m/z
468 [M+H]+.
washed with water and vacuum-dried. The solid was triturated
with 5% methanol in diisopropyl ether, and the precipitate was
collected and dried in vacuo to give 17b (4.32 g, 37% yield over
2 steps) as a colorless solid. 1H NMR (300 MHz, CDCl3): d 7.61
(1H, d, J = 10.6 Hz), 7.94 (1H, d, J = 10.6 Hz); MS (ESI): m/z 173
[M+H]+.
5.2.39. 5-Chloro-6-oxo-1,6-dihydropyridine-2-carboxylic acid
(18)
Compound 17b (500 mg, 2.48 mmol) in 10 N KOH (10 mL)
was heated to reflux for 2 h. The mixture was cooled to 0 °C
and acidified to pH 2ꢀ3 with concentrated HCl. The resulting
precipitate was collected, washed with water, and vacuum-dried.
The resultant crude was dissolved in water (30 mL) and few
drops of 28% aqueous ammonia solution, and the mixture was
acidified to pH 2ꢀ3 with concentrated HCl. The precipitate was
collected, washed with water, and dried in vacuo to give 18
(290 mg, 52% yield) as pale-yellow solid. 1H NMR (300 MHz,
DMSO-d6): d 6.60–6.70 (1H, m),7.70–7.80 (1H, m); MS (ESI):
m/z 172 [MꢀH]ꢀ.
5.2.37. 6-Bromo-5-methylpyridine-2-carbonitrile (17a)
To
a stirred solution of 2-bromo-3-methylpyridine (16a)
(2.94 g, 17.1 mmol) in chloroform (30 mL) was added mCPBA
(4.43 g, 25.7 mmol) at room temperature, and the mixture was
stirred at room temperature overnight. After being cooled to 0 °C,
saturated aqueous sodium hydrogen sulfite was added to the mix-
ture. The resulting mixture was extracted twice with chloroform,
and the combined organic layers were washed with saturated
aqueous sodium hydrogen carbonate, dried over sodium sulfate,
concentrated, and vacuum-dried to give 2-bromo-3-methylpyri-
dine 1-oxide (2.43 g). To a stirred solution of the crude in dichloro-
methane (30 mL) was added methyl trifluoromethanesulfonate
(1.61 mL, 14.1 mmol) at 0 °C, and the mixture was allowed to
warm up to room temperature and stirred for 2 h. After being con-
centrated, the residue was dried in vacuo. The resulting crude was
dissolved in DMF (30 mL) and treated with NaCN (946 mg,
19.3 mmol) at 0 °C, and the mixture was allowed to warm to room
temperature and stirred for 19 h. The mixture was partitioned be-
tween ethyl acetate and saturated aqueous sodium hydrogen car-
bonate, 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 silica gel flash column chromatogra-
phy with 10% ethyl acetate in hexanes to give 17a (1.31 g, 52%
yield over 3 steps) as a pale-yellow oil. 1H NMR (400 MHz, CDCl3):
d 2.49 (3H, s), 7.56 (1H, d, J = 8.0 Hz), 7.63 (1H, d, J = 8.0 Hz); MS
(ESI): m/z 197 [M+H]+.
5.2.40. 6-Cyano-3-fluoropyridin-2-yl acetate (20)
To a stirred solution of 5-fluoropyridine-2-carbonitrile (19)
(421 mg, 3.45 mmol) and urea hydrogen peroxide addition com-
pound (649 mg, 6.90 mmol) in dichloromethane (40 mL) was
added dropwise TFAA (1.02 mL, 7.25 mmol) at 0 °C, and the mix-
ture was allowed to warm to room temperature and stirred for
3 h. Saturated aqueous sodium hydrogen sulfite was added to the
mixture, and the layers were separated. The aqueous layer was ex-
tracted twice with chloroform, and the combined organic layers
were dried over sodium sulfate, concentrated, and vacuum-dried
to give 5-fluoropyridine-2-carbonitrile 1-oxide (272 mg) as a yel-
low solid. The crude was treated with acetic anhydride (10 mL),
and the mixture was heated to reflux for 16 h. After being concen-
trated, the residual oil was purified by silica gel flash column chro-
matography with 10% ethyl acetate in hexanes to give 20 (290 mg,
81% yield over 2 steps) as a colorless solid. 1H NMR (400 MHz,
CDCl3): d 2.41 (3H, s), 7.42–7.56 (2H, m); MS (ESI): m/z 139
[MꢀAc+H]+.
5.2.41. 5-Fluoro-6-oxo-1,6-dihydropyridine-2-carboxylic acid
(21)
Compound 20 (227 mg, 1.26 mmol) was treated with 6 N HCl
(20 mL), and the mixture was heated to reflux for 16 h. After being
cooled to room temperature, the resulting precipitate was col-
lected, washed with water, and vacuum-dried to give 21
(151 mg, 76% yield) as a colorless solid. 1H NMR (400 MHz,
DMSO-d6): d 6.81 (1H, dd, J = 3.2, 9.2 Hz), 7.66 (1H, t, J = 9.2 Hz);
MS (ESI): m/z 158 [M+H]+.
5.2.38. 5,6-Dichloropyridine-2-carbonitrile (17b)
To a stirred solution of 2,3-dichloropyridine (16b) (10.0 g,
67.6 mmol) and urea hydrogen peroxide addition compound
(13.3 g, 142 mmol) in dichloromethane (100 mL) was added drop-
wise TFAA (19.1 mL, 135 mmol) at 0 °C, and the mixture was al-
lowed to warm to room temperature and stirred for 3 h. After
being cooled to 0 °C, saturated aqueous sodium hydrogen sulfite
was added to the mixture. The resulting mixture was poured into
0.5 N HCl, and the layers were separated. The aqueous layer was
extracted twice with chloroform, and the combined organic layers
were stirred with saturated sodium hydrogen carbonate for
5 min. The layers were separated, and the organic layer was dried
over sodium sulfate, concentrated, and vacuum-dried to give 2,3-
dichloropyridine 1-oxide (11.0 g) as a colorless solid. The crude
5.2.42. Methyl 1-methyl-6-oxo-1,6-dihydropyridine-2-
carboxylate (23a)
To a stirred solution of methyl 6-oxo-1,6-dihydropyridine-2-
carboxylate (22a) (200 mg, 1.30 mmol) and methyl iodide
(243 lL 3.90 mmol) in DMF (5.0 mL) was added CsF (592 mg,
3.90 mmol) at room temperature, and the mixture was stirred at
room temperature for 2 days. The mixture was partitioned be-
tween ethyl acetate and saturated aqueous sodium hydrogen car-
bonate, and the layers were separated. The organic layer was
washed with water, dried over sodium sulfate, and concentrated.
The residue was purified by silica gel flash column chromatogra-
phy with 5% methyl alcohol in chloroform to give 23a (103 mg,
48% yield) as a colorless solid. 1H NMR (400 MHz, CDCl3): d 3.69
(3H, s), 3.93 (3H, s), 6.70–6.80 (2H, m), 7.33 (1H, dd, J = 6.8,
8.8 Hz); MS (ESI): m/z 168 [M+H]+.
pyridine 1-oxide was added to
a stirred dimethyl sulfate
(6.67 mL, 70.4 mmol) in small portions, and the mixture was
heated to 90 °C for 30 min. After being cooled to room tempera-
ture, the mixture was diluted with water (50 mL). To the stirred
aqueous solution was added NaCN (3.94 g, 80.5 mmol) at 0 °C,
and the mixture was stirred at 0 °C for 1 h under a nitrogen atmo-
sphere. The resulting precipitate was collected by filtration and

M. Ando et al. / Bioorg. Med. Chem. 17 (2009) 6106–6122
6119
5.2.43. Propyl 6-oxo-1-propyl-1,6-dihydropyridine-3-
carboxylate (23b)
in acetic acid (30 mL) was added bromine (900
l
L, 18.0 mmol) at
room temperature, and the mixture was heated to 80 °C for 14 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 dried
over magnesium sulfate and concentrated. The residue was tritu-
rated with hexanes, collected by filtration, and vacuum-dried to
To a stirred solution of 6-oxo-1,6-dihydropyridine-3-carboxylic
acid (22b) (2.00 g, 14.4 mmol) and n-propyl bromide (5.23 mL,
57.6 mmol) in DMF (50 mL) was added CsF (8.75 g, 57.6 mmol) at
room temperature, and the mixture was stirred at room tempera-
ture for 17 h. The mixture was partitioned between ethyl acetate
and water, and the layers were separated. The organic layer was
washed with brine, dried over sodium sulfate, and concentrated.
The residue was purified by silica gel flash column chromatogra-
phy with 10% ethyl acetate in hexanes to give 23b (240 mg, 7%
yield) as a colorless oil. 1H NMR (300 MHz, CDCl3): d 0.90–1.10
(6H, m), 1.70–1.90 (4H, m), 3.95 (2H, t, J = 6.7 Hz), 4.23 (2H, t,
J = 6.7 Hz), 6.55 (1H, d, J = 9.2 Hz), 7.81 (1H, dd, J = 2.8, 9.2 Hz),
8.15 (1H, d, J = 2.8 Hz); MS (ESI): m/z 224 [M+H]+.
give 26a (3.52 g, 91% yield) as
a
colorless solid. 1H NMR
(300 MHz, CDCl3): d 7.73 (1H, d, J = 2.6 Hz), 7.90 (1H, dd, J = 0.7,
2.6 Hz); MS (ESI): m/z 242 [M+H]+.
5.2.50. 5-Bromo-3-methoxypyridin-2(1H)-one (26b)
To a stirred solution of 3-methoxypyridin-2(1H)-one (25b)
(1.00 g, 8.00 mmol) in dichloromethane (10 mL) was added drop-
wise bromine (453 lL, 8.79 mmol) at room temperature, and the
5.2.44. Propan-2-yl 6-oxo-1-(propan-2-yl)-1,6-dihydropyridine-
3-carboxylate (23c)
mixture was stirred at room temperature for 3.5 h. After being
cooled to 0 °C, saturated aqueous sodium hydrogen carbonate
was added to the mixture. The resulting mixture was extracted
with ethyl acetate, and the organic layer was washed with brine,
dried over sodium sulfate, and concentrated. The residue was puri-
fied by silica gel flash column chromatography with 33% ethyl ace-
tate in hexanes to give 26b (600 mg, 37% yield) as a pale-brown
solid. 1H NMR (400 MHz, CDCl3): d 3.86 (3H, s), 6.79 (1H, d,
J = 2.0 Hz), 7.11 (1H, d, J = 2.0 Hz); MS (ESI): m/z 204 [M+H]+.
Compound 23c was prepared from 6-oxo-1,6-dihydropyridine-
3-carboxylic acid (22b) and i-propyl bromide using the procedure
described for 23b as a colorless oil (0.7% yield). 1H NMR (300 MHz,
CDCl3): d1.35 (6H, d, J = 4.6 Hz), 1.42 (6H, d, J = 4.4 Hz), 5.10–5.30
(2H, m), 6.52 (1H, d, J = 9.2 Hz), 7.80 (1H, dd, J = 1.6, 9.2 Hz), 8.21
(1H, d, J = 1.6 Hz); MS (ESI): m/z 224 [M+H]+.
5.2.45. 1-Methyl-6-oxo-1,6-dihydropyridine-2-carboxylic acid
(24a)
5.2.51. 5-Methoxy-6-oxo-1,6-dihydropyridine-3-carboxylic acid
(27a)
To a stirred solution of 23a (98 mg, 0.59 mmol) in methyl alcohol
(3.0 mL) was added 1 N NaOH (1.18 mL, 1.18 mmol) at 0 °C, and the
mixture was allowed to warm to room temperature and stirred for
30 min. The mixture was partitioned between ethyl acetate and
0.5 N HCl, and the layers were separated. The aqueous layer was ex-
tracted with ethyl acetate, and the combined organic layers were
washed with brine, dried over sodium sulfate, concentrated, and vac-
uum-dried to give 24a (18 mg, 20% yield) as a colorless solid. 1H NMR
(300 MHz, DMSO-d6): d 2.49 (3H, s), 6.55–6.60 (1H, m), 6.65–6.75
(1H, m), 7.43 (1H, dd, J = 6.8, 9.4 Hz); MS (ESI): m/z 154 [M+H]+.
To a stirred solution of 26b (422 mg, 2.07 mmol) in THF (15 mL)
was added a 1.59 M solution of n-BuLi in hexanes (2.74 mL,
4.35 mmol) at ꢀ78 °C, and the mixture was stirred at ꢀ78 °C for
30 min. After CO2 gas was bubbled into the reaction mixture at
ꢀ78 °C for 1 h, the resulting mixture was stirred at ꢀ78 °C for 1 h
and warmed up to room temperature. After being concentrated,
the residue was dissolved in water and acidified with 4 N HCl.
The mixture was extracted five times with ethyl acetate, and the
combined organic layers were washed with brine, dried over so-
dium sulfate, concentrated, and vacuum-dried to give 27a
(131 mg, 37% yield) as a brown solid. 1H NMR (400 MHz, CD3OD):
d 3.85 (3H, s), 7.28 (1H, d, J = 2.0 Hz), 7.76 (1H, d, J = 2.0 Hz); MS
(ESI): m/z 170 [M+H]+.
5.2.46. 6-Oxo-1-propyl-1,6-dihydropyridine-3-carboxylic acid
(24b)
Compound 24b was prepared from 23b using the procedure de-
scribed for 24a as a colorless solid (62% yield). 1H NMR (300 MHz,
DMSO-d6): d 1.43 (3H, t, J = 7.5 Hz), 1.82 (2H, sextet, J = 7.5 Hz),
3.95 (2H, t, J = 7.5 Hz), 6.19 (1H, d, J = 9.5 Hz), 7.87 (1H, dd,
J = 1.6, 9.6 Hz), 8.21 (1H, d, J = 1.6 Hz), 12.40 (1H, s); MS (ESI): m/
z 182 [M+H]+.
5.2.52. 5-Methyl-6-oxo-1,6-dihydropyridine-3-carboxylic acid
(27b)
Compound 27b was prepared from 26c using the procedure de-
scribed for 27a as a pale-pink solid (61% yield). 1H NMR (200 MHz,
CD3OD): d 2.11 (3H, s), 7.80–7.90 (1H, m), 7.95–8.05 (1H, m); MS
(ESI): m/z 152 [MꢀH]ꢀ.
5.2.47. 6-Oxo-1-(propan-2-yl)-1,6-dihydropyridine-3-carboxylic
acid (24c)
Compound 24c was prepared from 23c using the procedure de-
scribed for 24a as a colorless solid (81% yield). 1H NMR (300 MHz,
DMSO-d6): d 1.66 (6H, d, J = 4.4 Hz), 4.49 (1H, septet, J = 4.4 Hz),
6.19 (1H, d, J = 9.5 Hz), 7.87 (1H, dd, J = 1.6, 9.5 Hz), 8.14 (1H, d,
J = 1.6 Hz), 12.43 (1H, brs); MS (ESI): m/z 182 [M+H]+.
5.2.53. 5-Bromo-2-[(4-methoxybenzyl)oxy]-3-(trifluoromethyl)-
pyridine (28a)
Compound 26a (1.98 g, 8.17 mmol) in phenylphosphonic
dichloride (4.0 mL) was heated to 140 °C for 4 h. After being cooled
to room temperature, the mixture was poured into ice water, and
extracted three times with chloroform. The combined organic lay-
ers were washed with saturated aqueous sodium hydrogen car-
bonate and brine, dried over sodium sulfate, concentrated, and
vacuum-dried to give 5-bromo-2-chloro-3-(trifluoromethyl)pyri-
dine (1.96 g) as a brown oil. To a stirred solution of the crude
5.2.48. 1-(Difluoromethyl)-6-oxo-1,6-dihydropyridine-3-
carboxylic acid (24d)
Compound 24d was prepared from 23d using the procedure de-
scribed for 24a as a colorless solid (73% yield). 1H NMR (400 MHz,
DMSO-d6): d 6.56 (1H, d, J = 9.6 Hz), 7.82 (1H, t, J = 58.8 Hz), 7.86
(1H, dd, J = 1.6, 9.6 Hz), 8.24 (1H, d, J = 1.6 Hz); MS (ESI): m/z 190
[M+H]+.
and 4-methoxybenzyl alcohol (940 lL, 7.54 mmol) in THF
(15 mL) was added sodium hydride (60% dispersion in oil,
350 mg, 8.75 mmol) at room temperature, and the mixture was
heated to 80 °C for 1 h. After being cooled to room temperature,
the mixture was quenched with water. The mixture was extracted
three times with ethyl acetate, and the combined organic layers
were washed with brine, dried over magnesium sulfate, and
5.2.49. 5-Bromo-3-(trifluoromethyl)pyridin-2(1H)-one (26a)
To a stirred solution of 3-(trifluoromethyl)pyridin-2(1H)-one
(25a) (2.61 g, 16.0 mmol) and sodium acetate (1.48 g, 18.0 mmol)

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M. Ando et al. / Bioorg. Med. Chem. 17 (2009) 6106–6122
concentrated. The residue was purified by silica gel flash column
chromatography with 4% ethyl acetate in hexanes to give 28a
(2.18 g, 80% yield over 2 steps) as a colorless solid. 1H NMR
(300 MHz, CDCl3): d 3.81 (3H, s), 5.42 (2H, s), 6.86–6.94 (2H, m),
7.34–7.40 (2H, m), 7.95 (1H, d, J = 2.4 Hz), 8.35 (1H, d, J = 2.4 Hz);
MS (ESI): not detected.
m), 12.80–12.90 (1H, m), 13.10–13.25 (1H, m); MS (ESI): m/z 206
[MꢀH]ꢀ.
5.2.58. 6-(Benzyloxy)-4-methylpyridine-3-carboxylic acid (31)
Compound 31 was prepared from 29b using the procedure de-
scribed for 24a as a pale-brown solid. (81% yield). 1H NMR
(400 MHz, DMSO-d6): d 2.50 (3H, s), 5.39 (2H, s), 6.82 (1H, s),
7.31–7.44 (5H, m), 8.64 (1H, s), 12.87 (1H, s); MS (ESI): m/z 244
[M+H]+.
5.2.54. 2-(Benzyloxy)-5-bromo-4-methylpyridine (28b)
To a stirred solution of 26d (1.00 g, 5.30 mmol) and benzyl bro-
mide (1.90 mL, 15.9 mmol) in benzene (20 mL) was added silver
carbonate (1.50 g, 5.30 mmol) at room temperature, and the mix-
ture was stirred at room temperature for 4 days under light shield-
ing conditions. After being filtered through a pad of Celite, the
filtrate was concentrated. The residue was purified by silica gel
flash column chromatography with 5% ethyl acetate in hexanes
to give 28b (1.40 g, 93% yield) as a colorless oil. 1H NMR
(400 MHz, CDCl3): d 2.34 (3H, s), 5.33 (2H, s), 6.71 (1H, s), 7.30–
7.45 (5H, m), 8.18 (1H, s); MS (ESI): m/z 278 [M+H]+.
5.2.59. 5-Bromo-1-ethyl-3-methylpyridin-2(1H)-one (32a)
Compound 32a was prepared from 26c and ethyl bromide using
the procedure described for 23a as a colorless solid (69% yield). 1H
NMR (400 MHz, CDCl3): d 1.38 (3H, t, J = 7.8 Hz), 2.15 (3H, s), 3.96
(2H, q, J = 7.8 Hz), 7.20–7.25 (1H, m), 7.25–7.30 (1H, m); MS (ESI):
m/z 216 [M+H]+.
5.2.60. Propyl 1-ethyl-5-methyl-6-oxo-1,6-dihydropyridine-3-
carboxylate (33a)
5.2.55. Propyl 6-[(4-methoxybenzyl)oxy]-5-(trifluoromethyl)-
pyridine-3-carboxylate (29a)
Compound 33a was prepared from 32a using the procedure de-
scribed for 29a as a brown oil (90% yield). 1H NMR (400 MHz,
CDCl3): d 1.01 (3H, t, J = 7.6 Hz), 1.39 (3H, t, J = 6.8 Hz), 1.75 (2H,
sextet, J = 6.8 Hz), 2.16 (3H, s), 4.03 (2H, q, J = 7.6 Hz), 4.21 (2H, t,
J = 6.8 Hz), 7.69 (1H, dd, J = 1.2, 2.4 Hz), 8.04 (1H, d, J = 2.4 Hz);
MS (ESI): m/z 224 [M+H]+.
To a stirred solution of 28a (1.01 g, 2.78 mmol), 1,10-bis(diphen-
ylphosphino)ferrocene (308 mg, 0.555 mmol), and triethylamine
(0.77 mL, 5.53 mmol) in DMF (3.7 mL) and n-propyl alcohol
(7.4 mL) was added palladium diacetate (62.9 mg, 0.280 mmol),
and the mixture was heated to 100 °C under an atmospheric pres-
sure of carbon monoxide for 13 h. After being cooled to room tem-
perature, the black solution was filtered through a pad of SiO2 and
washed with ethyl acetate. The filtrate was washed with water and
brine, dried over magnesium sulfate, and concentrated. The residue
was purified by silica gel flash column chromatography with 6%
ethyl acetate in hexanes to give 29a (919 mg, 89% yield) as a
pale-yellow oil. 1H NMR1H NMR (300 MHz, CDCl3): d 1.02 (3H, t,
J = 6.7 Hz), 1.78 (2H, sextet, J = 6.7 Hz), 3.81 (3H, s), 4.30 (2H, t,
J = 6.7 Hz), 5.53 (2H, s), 6.90 (2H, d, J = 8.5 Hz), 7.39 (2H, d,
J = 8.5 Hz), 8.44 (1H, d, J = 2.0 Hz), 8.96 (1H, d, J = 2.0 Hz); MS
(ESI): m/z 370 [M+H]+.
5.2.61. Propyl 1-(difluoromethyl)-5-methyl-6-oxo-1,6-
dihydropyridine-3-carboxylate (33b)
To a stirred solution of 26c (2.30 g, 12.2 mmol) and lithium bro-
mide (2.12 g, 24.4 mmol) in DMF (10 mL) was sodium hydride (60%
dispersion in oil, 540 mg, 13.5 mmol) at 0 °C, and the mixture was
allowed to warm to room temperature and stirred for 15 min. To
the resultant mixture was added sodium chloro(difluoro)acetate
(3.72 g, 24.2 mmol), and the mixture was heated to 140 °C for
30 min. After being concentrated, the residue was partitioned be-
tween ethyl acetate and saturated aqueous ammonium chloride,
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 resi-
due was purified by silica gel flash column chromatography with
0–10% ethyl acetate in hexanes to give 32b (870 mg) as a pale-yel-
low oil. Compound 33b was prepared from the crude 32b using the
procedure described for 29a as a brown oil (18% yield over 2 steps).
1H NMR (400 MHz, CDCl3): d 1.03 (3H, t, J = 7.2 Hz), 1.77 (2H, sex-
tet, J = 7.2 Hz), 2.17 (3H, s), 4.25 (2H, t, J = 7.2 Hz), 7.70 (1H, t,
J = 63.2 Hz), 7.70–7.75 (1H, m), 8.15–8.20 (1H, m); MS (ESI): m/z
246 [M+H]+.
5.2.56. Propyl 6-(benzyloxy)-4-methylpyridine-3-carboxylate
(29b)
Compound 29b was prepared from 28b using the procedure de-
scribed for 29a as a pale-yellow solid (89% yield). 1H NMR
(300 MHz, CDCl3): d 1.03 (3H, t, J = 7.3 Hz), 1.74–1.81 (2H, m),
2.57 (3H, s), 4.25 (2H, t, J = 6.6 Hz), 5.42 (2H, s), 6.65 (1H, s),
7.26–7.40 (3H, m), 7.41–7.43 (2H, m), 8.79 (1H, s); MS (ESI): m/z
286 [M+H]+.
5.2.57. 6-Oxo-5-(trifluoromethyl)-1,6-dihydropyridine-3-
carboxylic acid (30)
5.2.62. 1-Ethyl-5-methyl-6-oxo-1,6-dihydropyridine-3-
carboxylic acid (34a)
Compound 29a (492 mg, 1.33 mmol) was dissolved in TFA
(1.3 mL), and the mixture was stirred at room temperature for
1 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 dried
over magnesium sulfate and concentrated to give propyl 6-oxo-
5-(trifluoromethyl)-1,6-dihydropyridine-3-carboxylate as a yellow
amorphous. The crude product was dissolved in methyl alcohol
(5.0 mL) and treated with 4 N NaOH (0.67 mL), and the mixture
was stirred at room temperature for 4 days. After being concen-
trated, the residue was diluted with ethyl acetate and water. The
layers were separated, and the organic layer was washed with
water. The combined aqueous layers were acidified with concen-
trated HCl, and the resulting precipitate was collected and dried
in vacuo to give 30 (115 mg, 42% yield over 2 steps) as a colorless
solid. 1H NMR (300 MHz, DMSO-d6): d 8.15 (1H, s), 8.20–8.25 (1H,
Compound 34a was prepared from 33a using the procedure de-
scribed for 24a as a colorless solid (97% yield). 1H NMR (400 MHz,
CD3OD): d 1.34 (3H, t, J = 7.2 Hz), 2.12 (3H, s), 4.07 (2H, q,
J = 7.2 Hz), 7.79 (1H, d, J = 2.8 Hz), 8.29 (1H, d, J = 2.8 Hz); MS
(ESI): m/z 182 [M+H]+.
5.2.63. 1-(Difluoromethyl)-5-methyl-6-oxo-1,6-dihydropyridine-
3-carboxylic acid (34b)
Compound 34b was prepared from 33b using the procedure de-
scribed for 24a as a colorless solid (72% yield). 1H NMR (400 MHz,
CD3OD): d 2.14 (3H, s), 7.77 (1H, t, J = 60.0 Hz), 7.83–7.84 (1H, m),
8.25 (1H, d, J = 2.0 Hz); MS (ESI): m/z 204 [M+H]+.
5.2.64. Methyl 5-fluoropyridine-3-carboxylate 1-oxide (36a)
To a stirred solution of methyl 5-fluoropyridine-3-carboxylate
(35a) (880 mg, 5.7 mmol) and urea hydrogen peroxide addition

M. Ando et al. / Bioorg. Med. Chem. 17 (2009) 6106–6122
6121
compound (1.1 g, 12 mmol) in dichloromethane (14 mL) was
added dropwise TFAA (1.6 mL, 11 mmol) at 0 °C, and the mixture
was allowed to warm to room temperature and stirred for 17 h.
After being cooled to 0 °C, saturated aqueous sodium hydrogen sul-
fite was added to the mixture. The layers were separated, and the
aqueous layer was extracted three times with chloroform. The
combined organic layers were washed with saturated aqueous so-
dium hydrogen carbonate, dried over sodium sulfate, concentrated,
and vacuum-dried to give 36a (740 mg, 76% yield) as a colorless so-
lid. 1H NMR (400 MHz, CDCl3): d 3.98 (3H, s), 7.60–7.65 (1H, m),
8.20–8.30 (1H, m), 8.60 (1H, t, J = 2.0 Hz); MS (ESI): m/z 172
[M+H]+.
was stirred at room temperature overnight. After being concen-
trated, the residue was partitioned between ethyl acetate and
water, and the layers were separated. The aqueous layer was ex-
tracted with ethyl acetate, and the combined organic layers were
washed with brine, dried over sodium sulfate, and concentrated.
The residue was purified by silica gel flash column chromatogra-
phy with 0–75% ethyl acetate in hexanes to give methyl 1-meth-
oxy-6-oxo-1,6-dihydropyridine-3-carboxylate (418 mg) as a pale-
yellow solid. The resultant ester was dissolved in methanol
(5 mL) and treated with 1 N NaOH (4.48 mL, 4.48 mmol), and the
mixture was stirred at room temperature for 6 h. After being con-
centrated, 8 N HCl was added to the residue. The resulting precip-
itate was collected by filtration, washed with water, and dried in
vacuo to give 39a (254 mg, 62% yield over 2 steps) as a colorless so-
lid. 1H NMR (400 MHz, CD3OD): d 4.07 (3H, s), 6.63 (1H, d,
J = 9.6 Hz), 7.94 (1H, dd, J = 2.4, 9.6 Hz), 8.58 (1H, d, J = 2.4 Hz);
MS (ESI): m/z 170 [M+H]+.
5.2.65. Methyl 6-chloropyridine-3-carboxylate 1-oxide (36b)
To a stirred solution of methyl 6-chloropyridine-3-carboxylate
(35b) (9.80 mg, 49.1 mmol) and urea hydrogen peroxide addition
compound (9.70 g, 103 mmol) in acetonitrile (100 mL) was added
dropwise TFAA (13.9 mL, 98.2 mmol) at 0 °C, and the mixture
was allowed to warm to room temperature and stirred for 3 h.
After being concentrated, the residue was partitioned between
ethyl acetate and saturated aqueous sodium hydrogen sulfite,
and the layers were separated. The aqueous layer was extracted
with ethyl acetate, and the combined organic layers were dried
over sodium sulfate and concentrated. The residue was purified
by silica gel flash column chromatography with 0–80% ethyl ace-
tate in hexanes to give 36b (9.45 g, 89% yield) as a colorless solid.
1H NMR (400 MHz, CD3OD): d 3.96 (3H, s), 7.86 (1H, d, J = 8.6 Hz),
7.95 (1H, dd, J = 1.8, 8.6 Hz), 8.88 (1H, d, J = 1.8 Hz); MS (ESI): m/z
188 [M+H]+.
5.2.69. 1-Ethoxy-6-oxo-1,6-dihydropyridine-3-carboxylic acid
(39b)
Compound 39b was prepared from 38 and ethyl bromide using
the procedure described for 39a as a colorless solid (30% yield over
2 steps). 1H NMR (400 MHz, CD3OD): d 1.38 (3H, t, J = 7.2 Hz), 4.31
(2H, t, J = 7.2 Hz), 6.63 (1H, d, J = 9.6 Hz), 7.93 (1H, dd, J = 2.2,
9.6 Hz), 8.55 (1H, d, J = 2.2 Hz); MS (ESI): m/z 184 [M+H]+.
5.2.70. Methyl 2-fluoro-6-oxo-1,6-dihydropyridine-3-
carboxylate (41)
To a stirred solution of sodium iodide (8.50 g, 56.5 mmol) in
acetonitrile (60 mL) was added trimethylsilyl chloride (7.17 mL,
56.5 mmol) at 0 °C, and the mixture was allowed to warm to room
temperature and stirred for 15 min. To the resulting mixture was
added 40 (2.09 g, 11.3 mmol) at room temperature, and the mix-
ture was stirred at room temperature for 17 h. After being concen-
trated, water and saturated aqueous sodium hydrogen sulfite were
added to the residue. The resulting precipitate was collected by fil-
tration and vacuum-dried to give 41 (1.33 g, 69% yield) as a pale-
yellow solid. 1H NMR (400 MHz, CDCl3): d 3.93 (3H, s), 6.71 (1H,
dd, J = 1.0, 8.4 Hz), 8.34 (1H, dd, J = 8.4, 9.5 Hz), 10.84 (1H, brs);
MS (ESI): m/z 172 [M+H]+.
5.2.66. 5-Fluoro-6-oxo-1,6-dihydropyridine-3-carboxylic acid
(37)
Compound 36a (334 mg, 1.95 mmol) in acetic anhydride
(15 mL) was heated to 140 °C for 5 h. The mixture was concen-
trated, and the residual oil was heated to 50 °C for 15 min and
concentrated. The residual brown solid was suspended in
dichloromethane and filtered. The resulting solid was dried in va-
cuo to give methyl 5-fluoro-6-oxo-1,6-dihydropyridine-3-carbox-
ylate (360 mg) as a brown solid. The crude ester was dissolved in
2 N NaOH (2.5 mL) and stirred at room temperature for 17 h. Con-
centrated HCl was added to adjust pH to ca. 3, and the resulting
precipitate was collected, washed with water and diethyl ether,
and dried in vacuo to give 37 (270 mg, 88% over 2 steps) as a
pale-brown solid. 1H NMR (300 MHz, DMSO-d6): d 7.62 (1H, dd,
J = 2.3, 11.2 Hz), 7.86 (1H, s), 12.60 (1H, brs), 12.99 (1H, brs); MS
(ESI): m/z 158 [M+H]+.
5.2.71. 6-(Benzyloxy)-2-fluoropyridine-3-carboxylic acid (42)
Methyl 6-(benzyloxy)-2-fluoropyridine-3-carboxylate was pre-
pared from 41 and benzyl bromide using the procedure described
for 23a as a colorless oil. Subsequently, compound 42 was pre-
pared from the resultant benzyl ether using the procedure de-
scribed for 24a as a colorless solid (55% yield over 2 steps). 1H
NMR (400 MHz, DMSO-d6): d 5.36 (2H, s), 6.90 (1H, dd, J = 1.2,
8.5 Hz), 7.34–7.41 (3H, m), 7.43–7.50 (2H, m), 8.28 (1H, dd,
J = 8.5, 10.0 Hz), 13.23 (1H, s); MS (ESI): m/z 248 [M+H]+.
5.2.67. Methyl 1-hydroxy-6-oxo-1,6-dihydropyridine-3-
carboxylate (38)
To a stirred solution of 36b (2.60 g, 12.1 mmol) in acetonitrile
(10 mL) was added TFAA (20 mL) at room temperature, and the
mixture was stirred at room temperature for 1 h. After being con-
centrated, solid sodium hydrogen carbonate and methyl alcohol
was added to the residue, and the resulting suspension was fil-
tered. The filtrate was concentrated, and the residue was purified
by silica gel flash column chromatography with 0–5% methyl alco-
hol in chloroform to give 38 (1.34 g, 65% yield) as a pale-yellow so-
lid. 1H NMR (400 MHz, CD3OD): d 3.83 (3H, s), 6.52 (1H, d,
J = 9.2 Hz), 7.80 (1H, dd, J = 2.4, 9.2 Hz), 8.51 (1H, d, J = 2.4 Hz);
MS (ESI): m/z 170 [M+H]+.
5.3. Biology
5.3.1. Anesthetized rats cardiovascular assay
Male Sprague-Dawley rats were anesthetized with the long-act-
ing barbiturate, inactin (100 mg/kg). Catheters were inserted into
the femoral artery and vein for measurement of arterial pressure
and compound administration, respectively. Additional catheter
was placed in the left ventricle through the carotid artery for mea-
surement of left ventricular pressure (LVP). The index of cardiac
contractility, left ventricular dp/dtmax (LV dp/dtmax) is derived from
LVP. Rats were randomly chosen for each treatment group. Follow-
ing a control period, vehicle (60% propylene glycol/distilled water;
1 mL/kg) was administered. Forty-five minutes later, compound
was administered. A small volume blood sample was collected at
3 and 30 min after administration for drug level determination.
5.2.68. 1-Methoxy-6-oxo-1,6-dihydropyridine-3-carboxylic acid
(39a)
To a stirred solution of 38 (420 mg, 2.48 mmol) and iodometh-
ane (465
lL, 7.44 mmol) in DMF (10 mL) was added potassium car-
bonate (1.03 g, 7.44 mmol) at room temperature, and the mixture

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M. Ando et al. / Bioorg. Med. Chem. 17 (2009) 6106–6122
5.3.2. Anesthetized dog cardiovascular assay
(0.3, 1 and 3 mg /kg) was orally administered to three animals
per strain, and blood samples were collected at 1 and 24 h. Plasma
levels of the Y5 antagonist after oral dosing were measured by
HPLC.
Male beagle dogs (n = 2–3/group) were anesthetized by 5% iso-
flurane vapor mixed with oxygen (2 L/min) and N2O (3 L/min).
Anesthesia was maintained by 1.5–2.0% isoflurane vapor through
an endotracheal tube with an artificial ventilator (SAFER100, Aika,
Tokyo). Ventilatory volume and/or rate were adjusted to maintain
the level of PCO2 within the normal range. Briefly, polyethylene
catheters were inserted into the left femoral artery and vein for
measurement of aortic blood pressure and infusion of compound
7m, respectively. A 7-F Swan-Gantz catheter was advanced into
the pulmonary artery through the left jugular vein for measure-
ment of cardiac output via thermodilution utilizing a cardiac out-
put computer (MTC-6210, Nihon Kohden, Tokyo, Japan). Central
venous pressure and pulmonary arterial pressure were measured
through the distal and proximal ports of the catheter, respectively.
A microtip catheter (Miller, Model MPC-500, 5F) was advanced into
the left ventricle via the left carotid artery for measurement of left
ventricular pressure. A standard limb lead II electrocardiogram
(ECG) was recorded via subcutaneous limb leads. The QT and PR
intervals were determined using commercial software (SBP-4/8,
Softron, Tokyo, Japan). The rate-corrected QT interval (QTc) was
calculated using a Bazzett’s formula: QTc = QT/(RR1/2). Cardiac con-
tractility (LV dp/dtmax) was obtained by differentiating the LVP sig-
nal with an electronic differentiator (EQ-601G, Nihon Kohden,
Tokyo, Japan). Following the completion of the surgical protocol,
animals were allowed to stabilize for at least 1 h and baseline data
were collected. Compounds were infused over 10 min at 1, 2 and
5 mg/kg, yielding rising cumulative doses of 1, 3 and 8 mg/kg,
respectively. The parameters were measured at 0, 10 and 20 min
during the cardiovascular study. A blood sample was collected
immediately after the end of dosing.
Acknowledgement
We thank Mr. Hirokazu Ohsawa for the measurement of HRMS.
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Male mdr1a (+/+) and mdr1a (-/-) CF-1 mice (Charles River,
Tokyo, Japan) were administered vehicle or the Y5 antagonist (0.3, 1
and 3 mg /kg) by oral gavage. One and 24 h after drug administra-
tion animals were killed by collecting whole blood from the heart
under isoflurane anesthesia. Whole brains rapidly removed and
were frozen in dry ice-cold isopentane and stored at ꢀ80 °C until
use. Serial coronal sections, 20 lm in thickness, of the anterior stri-
atal (0.2ꢄ1.0 mm anterior to Bregma) regions were cut by cryostat
microtome and stored at ꢀ80 °C. Because we have confirmed that
equivalent Y5 receptor occupancy was observed in both striatal
and posterior hypothalamic regions after oral administration of
the Y5 antagonist in the previous studies (data not shown), striatal
sections were used for the Y5 receptor occupancy assay. Striatal
sections were incubated with 100 pM of concentration of another
Y5 antagonist with 35S label (compound A; Banyu Pharmaceutical)
in 50 mM Tris HCl buffer for 30 min at room temperature. Com-
pound A is a spiroindoline class Y5 antagonist, binds to Y5 receptor
with high affinity (IC50 for human Y5 = 0.99 nM), and is highly
selective for Y5 because the binding was displaceable by Y5-selec-
tive compounds and was completely abolished in the Y5 receptor
KO mouse brain (data not shown). Nonspecific binding was evalu-
ated by using an adjacent brain slice by the addition of a 10,000-
fold excess of nonlabeled Y5 antagonist. Following incubation,
the striatal sections were washed, air-dried and exposed to BAS
5000 imaging plates (Fuji, Kanagawa, Japan), and autoradiographic
images were quantified. Ex vivo receptor labeling by 35S compound
A in drug-treated animals was calculated and expressed as follows:
RO (%) = 100 ꢂ [1 ꢀ (receptor labeling of drug-treated animals/
receptor labeling of vehicle-treated animals)]. Compound 7m
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26. For experimental details for the [125I]PYY binding assay, see Ref. 16.
27. Shibata, Y.; Takahashi, H.; Ishii, Y. Drug Metab. Dispos. 2000, 28, 1518.
28. For experimental details describing the determination of the transcellular
transport ratio (B-to-A/A-to-B ratio), see: Ohe, T.; Sato, M.; Tanaka, S.; Fujino,
N.; Hata, M.; Shibata, Y.; Kanatani, A.; Fukami, T.; Yamazaki, M.; Chiba, M.;
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29. For experimental details for the functional assay, see Ref. 16.
30. Butcher, J. W.; Claremon, D. A.; Connolly, T. M.; Dean, D. C.; Karczewski, J.;
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31. Binding affinities for human Y1, Y2, and Y4 receptors were determined as
described in: Kanatani, A.; Ishihara, A.; Iwassa, H.; Nakamura, K.; Okamoto, O.;
Hidaka, M.; Ito, J.; Fukuroda, T.; MacNeil, D. J.; Van der Ploeg, L. H. T.; Fukami,
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32. For the experimental details of the pharmacokinetic studies, see Ref. 16.
33. For the experimental details of the agonist-induced food intake study, see
Ref. 16.