Synthesis and pharmacological properties of benzamide derivatives as selective serotonin 4 receptor agonists

Article abstract of DOI:10.1016/j.bmc.2004.02.036

A series of 4-amino-5-chloro-2-methoxy-N-(piperidin-4-ylmethyl)benzamides with a polar substituent group at the 1-position of the piperidine ring was synthesized and evaluated for its effect on gastrointestinal motility. The benzoyl, phenylsulfonyl, and benzylsulfonyl derivatives accelerated gastric emptying and increased the frequency of defecation. One of them, 4-amino-N-[1-[3-(benzylsulfonyl)propyl]piperidin-4-ylmethyl]-5-chloro-2- methoxybenzamide (13a, Y-36912), was a selective 5-HT₄ receptor agonist offering potential as a novel prokinetic with reduced side effects derived from 5-HT₃- and dopamine D₂ receptor-binding affinity. In the oral route of administration, this compound enhanced gastric emptying and defecation in mice, and has a possibility as a prokinetic agent, which is effective on both the upper and the lower gastrointestinal tract.

Full text of DOI:10.1016/j.bmc.2004.02.036

Bioorganic & Medicinal Chemistry 12 (2004) 2737–2747
Synthesis and pharmacological properties of benzamide derivatives
as selective serotonin 4 receptor agonists
Shuji Sonda,a,* Kenichi Katayama,b Toshio Kawahara,a Noriko Satoc and Kiyoshi Asanod
aPharmaceutical Development Laboratories, Technology and Production Division, Mitsubishi Pharma Corporation, 14, Sunayama,
Hasaki-machi, Kashima, Ibaraki 314-0255, Japan
bQuality Assurance Department, Pharmacovigilance and Quality Assurance Division, Mitsubishi Pharma Corporation,
1000, Kamoshida-cho, Aoba-ku, Yokohama 227-0033, Japan
cResearch Laboratory III, Research and Development Division, Mitsubishi Pharma Corporation, 1000, Kamoshida-cho, Aoba-ku,
Yokohama 227-0033, Japan
dResearch Coordination Department, Research and Development Division, Mitsubishi Pharma Corporation, 1000, Kamoshida-cho,
Aoba-ku, Yokohama 227-0033, Japan
Received 30 January 2004; revised 27 February 2004; accepted 27 February 2004
Available online 9 April 2004
Abstract—A series of 4-amino-5-chloro-2-methoxy-N-(piperidin-4-ylmethyl)benzamides with a polar substituent group at the 1-
position of the piperidine ring was synthesized and evaluated for its effect on gastrointestinal motility. The benzoyl, phenylsulfonyl,
and benzylsulfonyl derivatives accelerated gastric emptying and increased the frequency of defecation. One of them, 4-amino-N-[1-
[3-(benzylsulfonyl)propyl]piperidin-4-ylmethyl]-5-chloro-2-methoxybenzamide (13a, Y-36912), was a selective 5-HT4 receptor ago-
nist offering potential as a novel prokinetic with reduced side effects derived from 5-HT3- and dopamine D2 receptor-binding affinity.
In the oral route of administration, this compound enhanced gastric emptying and defecation in mice, and has a possibility as a
prokinetic agent, which is effective on both the upper and the lower gastrointestinal tract.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
have binding affinity for 5-HT4 receptors and the
pharmacological effect of these compounds is thought to
be based on 5-HT4 receptor agonism.
Serotonin (5-HT) is a neurotransmitter responsible for a
wide range of pharmacological reactions. Serotonergic
receptors are currently classified into four subtypes, 5-
HT1, 5-HT2, 5-HT3, and 5-HT4, and clones for the
subtypes, termed 5-HT5, 5-HT6, and 5-HT7 receptors,
have been identified.1 Activation of the 5-HT4 receptor
mediates diverse effects in the central and peripheral
nervous systems.2 At the periphery, the receptors play
an important role in the response functions of several
organs including the gastrointestinal tract. Stimulation
of the receptors present in the myenteric nerve causes the
release of neurotransmitters from the nerve endings and
finally bring about the contraction of the gastrointesti-
nal smooth muscle. Many gastrointestinal prokinetics
such as benzamides (e.g., metoclopramide,3 cisapride4)
However, these benzamides are also reported to have
binding affinity for dopamine D2-, 5-HT2-, and 5-HT3-
receptors.5 Dopamine D2 antagonism should cause
adverse reactions in the central nervous system such as
extrapyramidal syndrome, while 5-HT3 receptor anta-
gonism should reduce colonic transit in the lower gas-
trointestine.6;7
Accordingly, selective and potent 5-HT4 receptor
agonists would increase both upper and lower gastro-
intestinal motility and cause little adverse reaction, and
are therefore seen as promising new gastroprokinetic
candidates.
We have recently reported the pharmacological profile
of a series of orally active 4-amino-5-chloro-2-methoxy-
N-(piperidin-4-ylmethyl)benzamides with a benzoyl
or phenylsulfonyl moiety in the side chain part at the
Keyword: 5-HT4 agonist.
* Corresponding author. Tel.: +81-479-46-4618; fax: +81-479-46-4639;
e-mail: sonda.shuuji@ma.m-pharma.co.jp
0968-0896/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.02.036

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S. Sonda et al. / Bioorg. Med. Chem. 12 (2004) 2737–2747
1-position of the piperidine.8 These compounds were
5-HT4 receptor agonists with no other receptor binding
affinities.6;7 The present paper describes further studies
of benzoyl and phenylsulfonyl (or benzylsulfonyl)
derivatives.
using the MnO2 of the benzyl alcohol derivative 4, which
was prepared by coupling reaction of 3-chlorobenzal-
dehyde with Grignard reagent. The phenyl sulfonyl (6a–
d) and benzylsulfonyl (8a–b) derivatives were prepared
by alkylation of benzenethiol or benzyl mercaptan fol-
lowed by oxidation with H2O2/HCOOH. The key
intermediate 9 was prepared by a method reported
previously.8 The coupling reaction of compound 9 with
2a,c–e and 3a–o, with 6a–d, and with 8a–b in K2CO3/
DMF gave benzoyl (10a, 10c–e, 11a–o), phenylsulfonyl
(12a–d), and benzylsulfonyl (13a–b) derivatives, respec-
tively. (The preparation of 10e and 11d was described
previously.8) The compound 2b was protected with an
acetal group (14) and reacted with the benzamide 9. The
2. Chemistry
The general synthetic pathways used for preparation of
the benzamides are illustrated in Schemes 1 and 2. The
benzoyl derivatives 2a–e and 3a–e, 3g–o were prepared
by Friedel–Crafts reaction. The synthesis of the 3-chlo-
robenzoyl compound 3f was carried out by oxidation
Br(CH2)n
Cl
compd.
n
Br(CH2)n
2a
2b
2c
2d
2e
1
2
3
4
O
a
O
2a-e
5
Br(CH2)5
R3
R3
R2
R2
Cl
Br(CH2)5
O
R1
R1
a
O
3a-e, 3g-o
Cl
Cl
Cl
O
OH
(CH2)5Cl
c
b
CHO
( CH2)5Cl
4
3f
R2 R3
compd.
R1
Me
R2 R3
compd.
R1
3h
3i
3j
3k
3l
3m
3n
3o
Me
H
H
Me
Et
OMe
OH
Cl
H
F
3a
H
H
H
H
H
H
H
H
Me
OMe
Cl
Me
3b
3c
3d
3e
3f
3g
OMe H
Cl
H
Cl
F
OMe
OMe
H
H
Cl
F
Cl
F
H
H
H
Cl
H
H
H
H
compd.
n
O
Cl
5a, 6a
5b, 6b
5c, 6c
5d, 6d
2
3
4
5
Br
(CH2)n
Cl
Cl
e
S
(CH2)n
(CH2)n
HS
S
O
d
5a-d
6a-d
compd.
n
Cl
(CH2)n Br
d
O
e
Cl
S
7a, 8a
7b, 8b
3
4
S
(CH2)n
Cl
HS
(CH2)n
O
7a-b
8a-b
Scheme 1. Reagents and conditions: (a) AlCl3, CH2Cl2, 25 ꢁC; (b) Mg, Br(CH2)5Cl, 50 ꢁC; (c) MnO2, 25 ꢁC, 5 days; (d) K2CO3/DMF, 50–60 ꢁC;
(e) 30% H2O2/HCOOH.

S. Sonda et al. / Bioorg. Med. Chem. 12 (2004) 2737–2747
2739
Cl
O
H
H2N
N
+
2a, 2c-e, 3a-o, 6a-d and 8a-b
2HCl
NH
O
9
Cl
R3
R2
O
H
N
n = 1-5
H2N
a
X
R1
N
(CH2)n
X = -CO-, -SO2-, -SO2CH2-
O
10a, 10c-e, 11a-o, 12a-d and 13a-b
Cl
Br(CH2)2
O
Br(CH2)2
O
H
c, d
b
O
N
H2N
N
(CH2)2
HCl
O
O
O
2b
14
10b
Scheme 2. Reagents and conditions: (a) K2CO3/DMF, 70–75 ꢁC; (b) ethyleneglycol, p-TsOH/benzene, reflux; (c) 9, K2CO3/DMF, 70–75ꢁC; (d) 1 N HCl.
derivative 10b was obtained by treatment with hydro-
chloric acid.
of the piperidine ring, possess dopamine D2 binding
affinity. Compound 10b (n ¼ 2) did not increase defe-
cation in mice at oral doses of 3 mg/kg. Compound 10e
(n ¼ 5) having the highest binding affinity (Ki ¼ 2:4 nM)
for the 5-HT4 receptor showed the strongest effect on
defecation (MED: 0.3 mg/kg). We therefore selected five
methylenes as the composition of the straight alkyl
chain at the 1-position of the piperidine ring.
3. Results and discussion
The synthesized compounds were evaluated for their 5-
HT4 receptor-binding affinity by use of [3H]GR-113808
binding assay in guinea-pig striatum membranes9 and
for in vitro 5-HT4 receptor-agonistic activity (EC50
value) by their ability to contract isolated guinea-pig
ascending colon.10 Evaluation for 5-HT3 receptor-
binding affinity was performed in rat cerebrocortical
membranes by [3H] Granisetron binding. The kinetic
activity on the upper gastrointestinal tract was evaluated
by determining the effect of the orally administered
compounds on gastric emptying rates of phenol red
semisolid meal through the stomach of rats or mice. The
effect on lower gastrointestinal motility was evaluated by
measuring the increase in defecation following oral
administration of the derivatives in mice.
For the further improvement of the compound, we
studied the influence of substitution in the benzoyl
group. The derivatives were measured for the rate of
increase in defecation induced in mice at oral
doses of 1 mg/kg as shown in Table 2 (percentage
increase in number, dry weight, and wet weight of
fecal deposits).
In this evaluation system, compound 10e significantly
increased defecation (respective increases of 49%, 90%,
76% in the three items measured). Introduction of
methyl (11a: 15%, 45%, 30%), ethyl (11b: 39%, 55%, 39%),
and fluoro (11g: 44%, 59%, 50%) groups at the 4-posi-
tion of the benzoyl group of 10e somewhat reduced the
effect on defecation.
As the first step, we optimized the length of the straight
alkyl chain at the 1-position of the piperidine ring of the
derivatives with an unsubstituted benzoyl group. Com-
pounds with a straight alkyl chain of from one to five
methylenes were prepared; pharmacological data are
listed in Table 1. Compounds 10a–e showed high bind-
ing affinities for the 5-HT4 receptor. Regarding effect on
gastric emptying, compound 10a (n ¼ 1) was weaker
than 10b–e (n ¼ 2–5). Compounds 10a (n ¼ 1) and 10c
(n ¼ 3) had undesirable binding affinity toward the
dopamine D2 receptor (Ki ¼ 15 and 21 nmol/L, respec-
tively).
Compounds substituted with chlorine at the 4-position
(11e: )5%, 3%, 6%) and the 3,4-position (11k: )7%, 4%,
6%) produced almost no increase in defecation. The
3-chloro (11f: 23%, 31%, 29%) and 2,4-dichloro (11l: 15%,
46%, 47%) derivatives did increase defecation, but the
effects were weaker than with compound 10e. These
results suggest that introduction of an electron-with-
drawing group into the benzoyl group does not con-
tribute to the effect on defecation.
This observation suggests that compounds 10a and 10c,
which have a short straight alkyl chain at the 1-position
A compound with a hydroxyl group at the 4-position of
the benzoyl (11d: 79%, 88%, 68%), like compound 10e,

2740
S. Sonda et al. / Bioorg. Med. Chem. 12 (2004) 2737–2747
Table 1. Pharmacological data of benzoyl derivatives 10a–e
Cl
O
O
H2N
NH
N
(CH2)
n
O
Compound
no
n
Binding affinitiesa
Potencyb
Gastric emptying Defecationd
5-HT4 Ki (nM)
5-HT3 Ki (nM)
D2 Ki (nM)
EC50 (nM)c
MED (mg/kg)
MED (mg/kg)
10a
10b
10c
10d
10e
1
2
3
4
5
3.0
6.1
2.5
4.0
2.4
270
15
>1000e
21
>1000e
>1000e
NT
35
10f
3f
NT
>3
NT
0.3
0.3
360
>1000e
>1000e
>1000e
10
NT
3g
3g
9.1
10
MED: minimum effective dose. NT: not tested.
a Each value is the mean from triplicate assay in single experiment.
b 5-HT4 receptor agonistic activities; contractile effects in guinea-pig ascending colon.
c EC50 values were determined by linear regression.
d Increase in defecation following oral administration of the derivatives in mice.
e IC50 value.
f Effect of the orally administered compound on gastric emptying rates of phenol red semisolid meal through the stomach of mice.
g Effect of the orally administered compound on gastric emptying rates of phenol red semisolid meal through the stomach of rats.
Table 2. Pharmacological data of benzoyl derivatives 10e, 11a–o
Cl
R3
R2
O
H
N
O
H2N
R1
N
O
Compound
no
R1
R2
R3
Binding affinitiesa
Rate of increased defecationb (%)
5-HT4 Ki (nM) 5-HT3 IC50 (nM)
Number
Dry weight
Wet weight
11a
11b
11c
11d
11e
11f
11g
11h
11i
Me
Et
H
H
H
H
H
H
H
H
H
Me
H
H
Cl
F
3.9
3.6
1.8
1.5
4.6
2.7
2.4
2.8
4.4
2.6
2.0
2.5
3.4
1.8
5.1
2.4
>1000
>1000
>1000
>1000
>1000
>1000
>1000
370c
15
39
26
79
)5
23
44
15
15
96
)7
15
37
24
31
49
45
55
71
88
3
30
39
48
68
6
H
OMe
OH
Cl
H
H
H
H
Cl
H
31
59
5
29
50
9
F
Me
Me
OMe
Cl
Me
H
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
15
106
4
20
87
6
11j
OMe
Cl
H
11k
11l
Cl
F
46
59
67
67
90
47
43
48
49
76
11m
11n
11o
10e
H
OMe
OMe
H
Cl
F
H
H
H
H
a Each value is the mean from triplicate assay in single experiment.
b Rate of increase in defecation induced in mice at oral doses of 1 mg/kg.
c Ki value.
increased the frequency of defecation, but the enhancing
effect on gastric emptying was weaker (MED: 10 mg/kg
po in rats) than with 10e (MED: 3 mg/kg po in rats). The
4-methoxy compound (11c: 26%, 71%, 48%, respec-
tively) showed moderate effect on defecation. Here, we
speculated that an electron-donating group on the benz-
oyl group might contribute to the effect on defecation.
Based on this speculation, we attempted to introduce a
methoxy group at the 3,4-positions of the benzoyl
group. As expected, the resulting compound (11j: 96%,
106%, 87%) powerfully enhanced defecation. Unfortu-
nately, the effect of compound 11j on gastric emptying in
mice (MED: 10 mg/kg) was weaker than that of 10e
(MED: 3 mg/kg).
The above results indicate the important influence on
gastric emptying and defecation of substituents in the
benzoyl group of benzoyl derivatives.

S. Sonda et al. / Bioorg. Med. Chem. 12 (2004) 2737–2747
2741
Table 3. Pharmacological data of phenylsulfonyl (12a–d) and benzylsulfonyl (13a–b) derivatives
Cl
O
H2N
NH
N
(CH2) n R1
O
Compound
no
R1
n
Binding affinitiesa
Potencyb
Gastric emptyingd Defecatione
5-HT4 Ki (nM) 5-HT3 Ki (nM) D2 IC50 (nM) EC50 (nM)c
MED (mg/kg)
MED (mg/kg)
O
S
12a
2
13
>1000f
>1000
NT
1
>10
O
O
S
O
12b
12c
3
4
1.7
240
>1000
>1000
9.5
12
>10
NT
O
S
O
3.0
1.4
>1000f
3
1
O
S
12d
5
>1000f
>1000
6.3
3
3
O
O
S
O
13a (Y-36912)
3
4
1.5
1.3
>1000f
>1000f
>1000
>1000
10.8
NT
3
0.3
O
S
O
13b
10
NT
MED: minimum effective dose. NT: not tested.
a Each value is the mean from triplicate assay in single experiment.
b 5-HT4 receptor agonistic activities; contractile effects in guinea-pig ascending colon.
c EC50 values were determined by linear regression.
d Effect of the orally administered compounds on gastric emptying rates of phenol red semisolid meal through the stomach of mice.
e Increase in defecation following oral administration of the derivatives in mice.
f IC50 value.
Table 3 shows the pharmaceutical properties of the
phenylsulfonyl (12a–d) and benzylsulfonyl (13a–b)
derivatives. Although compound 12a accelerated gastric
emptying in mice at a dose of 1 mg, it did not increase
defecation at a dose of 10 mg.
chain at the 1-position of the piperidine ring decreased
the effect on gastric emptying.
Next, we studied the pharmacokinetics of compound
13a (Y-36912). After oral administration in rats,
the unchanged compound concentrations reached the
Cmax of 1.6 lg/mL at 3.3 h and the t1=2 was 9.5 h.
The AUC was 12.3 lg/mL and bioavailability was
calculated to be 75.1% in oral administration (3 mg) in
dogs.11
Prolongation of the straight alkyl chain (12b–c)
enhanced 5-HT4 receptor binding affinities (Ki ¼ 1:7–
3.0 nM) relative to 12a (Ki ¼ 13 nM), but reduced the
effect on gastric emptying in mice. The benzylsulfonyl
derivative 13a showed a high affinity for the 5-HT4
receptor, with a Ki value of 1.5 nM, but little or no
affinity for other receptors. In the guinea-pig ascending
colon, 13a (Y-36912) induced contractions with an EC50
value of 10.8 nM and the concentration-effect curve was
shifted rightward by a 5-HT4 receptor antagonist
(GR113808). This compound accelerated gastric emp-
tying in mice at a dose of 3 mg/kg po and increased
defecation in mice at a dose of 0.3 mg/kg po.11 In con-
trast, prolongation (13b, n ¼ 4) of the straight alkyl
We therefore selected 4-amino-N-[1-[3-(benzylsulfo-
nyl)propyl]piperidin-4-ylmethyl]-5-chloro-2-methoxy-
benzamide 13a (Y-36912) as the candidate for additional
biological and pharmaceutical investigation.
This compound might be clinically effective in the
treatment of gastrointestinal motility disorders such as
constipation-predominant irritable bowel syndrome and
atonic constipation, and might improve postoperative

2742
S. Sonda et al. / Bioorg. Med. Chem. 12 (2004) 2737–2747
digestive function and the gastrointestinal symptoms
caused by chronic gastritis.
silica gel column chromatography (hexane/AcOEt: 9:1)
to give 3f (25%).
5.4. 2-(2-Bromoethyl)-2-phenyl-[1,3]dioxolane (14)
4. Conclusion
The compound 2b (10.0 g, 46.9 mmoL) was dissolved in
benzene (100 mL) and then ethylene glycol (2.90 g,
46.7 mmoL), p-TsOH were added. After refluxing for
70 h, the reaction mixture was washed with aqueous
K2CO3 solution. The solution was dried over MgSO4
and concentrated to afford crude product. Purification
by silica gel column chromatography (hexane/AcOEt:
10:1) provided the title compound 14 (50%).
A series of 4-amino-5-chloro-2-methoxy-N-(piperidin-
4-ylmethyl)benzamides with a benzoyl, phenylsulfonyl,
or benzylsulfonyl moiety in the side chain part at the 1-
position of the piperidine was synthesized and their
pharmacological properties evaluated. Structure–activ-
ity relationship studies gave useful information on the
structures required for effect on gastric emptying and
defecation. One of the series, the novel prokinetic agent
4-amino-N-[1-[3-(benzylsulfonyl)propyl]piperidin-4-yl-
methyl]-5-chloro-2-methoxybenzamide (13a, Y-36912),
was a selective 5-HT4 receptor agonist and potential
novel agent with reduced side effects due to 5-HT3, and
dopamine D2 receptor binding. Compound 13a
(Y-36912) could be developed as a novel proki-
netic, which can enhance the motor activity of both
the upper and lower gastrointestinal tract with few
side effects.
5.5. General procedure for preparation of intermediates
(6a–d, 8a–b)
A solution of benzenethiol (or benzyl mercaptan) and
bromochloroalkane in DMF was stirred at 50–60 ꢁC for
3–4 h in the presence of K2CO3. The reaction mixture
was poured into ice water and extracted with AcOEt.
The combined organic extracts were concentrated in
vaccuo and purified by silica gel column chromatogra-
phy (CHCl3/MeOH) to give thioether derivatives. 30%
H2O2 was added to this compound in HCOOH and the
reaction mixture was stirred at 25 ꢁC for 12 h. The
reaction mixture was poured into ice water and filtrated
to afford the title compound 6a–d, 8a–b.
5. Experimental section
5.1. General procedure for preparation of intermediates
(2a–e, 3a–e, 3g–o)
5.6. General procedure for preparation of benzamide
derivatives (10a–e, 11a–o, 12a–d, and 13a–b)
A cooled (5 ꢁC) solution of benzene and 6-chlorohexa-
noyl chloride in CH2Cl2 was treated with AlCl3 and
stirred at 25 ꢁC for 2–4 h. The reaction mixture was
poured into ice water and extracted with CH2Cl2. The
organic layer was concentrated in vacuo and purified by
silica gel column chromatography (CHCl3/MeOH) to
give pure product 2e.
The compound 9 was stirred with halide compounds
(2a–e, 3a–o, 6a–d, and 8a–b) at 70–75 ꢁC in K2CO3/
DMF for 5–12 h. The reaction mixture was poured into
ice water and extracted with AcOEt. The combined
extracts were washed with aqueous K2CO3 solution. The
organic layer was dried over MgSO4 and concentrated
in vacuo. The residue was purified by silica gel column
chromatography (CHCl3/MeOH) and recrystallized
with solvent, or added oxalate, hydrochloride to give
pure crystals 10a–e, 11a–o, 12a–d, and 13a–b.
5.2. 6-Chloro-1-(3-chlorophenyl)hexan-1-ol (4)
1-Bromo-5-chloropentane (7.00 g, 37.7 mmoL) in THF
(30 mL) was added dropwise to a suspension of Mg
turnings (920 mg, 37.7 mmoL) in THF (30 mL) at 40–
50 ꢁC under a nitrogen atmosphere. After 1 h 3-chloro-
benzaldehyde (5.31 g, 37.7 mmoL) in THF (30 mL) was
added dropwise and the mixture was stirred for 14 h at
25 ꢁC. The reaction mixture was poured into aqueous
NH4Cl solution and extracted with CHCl3. The organic
phase was washed with brine, dried (MgSO4), and
evaporated to crude oil, which was purified by silica gel
column chromatography (hexane/AcOEt: 90:10) to give
4 (32%).
5.7. 4-Amino-5-chloro-2-methoxy-N-[1-(2-oxo-2-phenyl-
ethyl)piperidin-4-ylmethyl]benzamide oxalate (10a)
The general procedure was followed for reaction time of
6 h with compound 2a as halide compound to give col-
orless oil (3.2 g, 57%). The pure oil was dissolved in
ethanol, then oxalic acid was added, and the suspension
was heated. After cooling, the resulting crystals were
filtered to obtain 10a; mp 183–185 ꢁC; 1H NMR
(DMSO-d6) d 1.40–1.62 (2H, m), 1.65–1.91 (3H, m),
2.73–3.00 (2H, m), 3.10–3.20 (2H, m), 3.35–3.50 (2H,
m), 3.83 (3H, s), 4.74 (2H, br s), 5.80–6.10 (2H, br s),
6.49 (1H, s), 7.53–7.80 (4H, m), 7.98–8.01 (3H, m);
anal. calcd for C22H26N3O3ClÆC2H2O4Æ1/2H2O: C,
55.98; H, 5.68; N, 8.16. Found: C, 55.96; H, 5.47; N,
8.07.
5.3. 6-Chloro-1-(3-chlorophenyl)hexan-1-one (3f)
The mixture of 4 (1.50 g, 6.10 mmoL) and MnO2 (7.99 g,
91.9 mmoL) in CHCl3 (30 mL) was stirred for 5 days at
25 ꢁC. The reaction mixture was filtered and purified by

S. Sonda et al. / Bioorg. Med. Chem. 12 (2004) 2737–2747
2743
5.8. 4-Amino-5-chloro-2-methoxy-N-[1-(3-oxo-3-phenyl-
propyl)piperidin-4-ylmethyl]benzamide hydrochloride
(10b)
1H NMR (CDCl3) d 1.31–1.47 (4H, m), 1.52–1.81 (6H,
m), 1.93–2.05 (2H, m) 2.28–2.38 (3H, m), 2.41 (3H, s),
2.80–3.02 (4H, m), 3.33 (2H, t, J ¼ 5:9 Hz), 3.90 (3H, s),
4.38 (2H, br s), 6.29 (1H, s), 7.25 (2H, d, J ¼ 7:9 Hz),
7.50–7.81 (1H, m), 7.84 (2H, d, J ¼ 7:9 Hz), 8.11(1H, s);
anal. calcd for C27H36N3O3ClÆ1/2H2O: C, 65.51; H, 7.53;
N, 8.49. Found: C, 65.55; H, 7.43; N, 8.44.
The general procedure was followed for reaction time of
3 h with compound 14 as halide compound to give col-
orless oil. The pure oil was treated with 1 mol/L
hydrochloric acid to obtain compound 10b (6.4 g, 80%);
1
mp 141–143 ꢁC; H NMR (DMSO-d6) d 1.40–1.70 (2H,
5.13. 4-Amino-5-chloro-N-[1-[6-(4-ethylphenyl)-6-oxo-
hexyl]piperidin-4-ylmethyl]-2-methoxybenzamide (11b)
m), 1.72–2.02 (3H, m), 2.85–3.06 (2H, m), 3.15–3.40
(4H, m), 3.51–3.61 (2H, m), 3.62–3.72 (2H, m), 3.83 (3H,
s), 5.93 (2H, br s), 6.49 (1H, s), 7.53–7.61 (2H, m), 7.65–
7.72 (2H, m), 7.98–8.03 (3H, m), 10.03–10.40 (1H, m);
anal. calcd for C23H28N3O3ClÆHClÆ2H2O: C, 54.98; H,
6.62; N, 8.36. Found: C, 55.09; H, 6.58; N, 8.40.
The general procedure was followed for reaction time of
5 h with compound 3b as halide compound to give solid.
The solid was recrystallized from EtOH/AcOEt to
obtain colorless prisms (340 mg, 21%); mp 140–145 ꢁC;
1H NMR (CDCl3) d 1.26 (3H, t, J ¼ 7:2 Hz), 1.34–1.90
(10H, m), 2.02–2.30 (2H, m), 2.42–2.60 (2H, m), 2.70
(2H, q, J ¼ 7:2 Hz), 2.95 (2H, t, J ¼ 7:2 Hz), 3.04–3.22
(2H, m), 3.29–3.41 (2H, m), 3.90 (3H, s), 4.42 (2H, br s),
6.31 (1H,s), 7.28 (2H, d, J ¼ 7:9 Hz), 7.70–7.82 (2H, m),
7.87 (2H, d, J ¼ 7:9 Hz), 8.09 (1H, s); anal. calcd for
C28H38N3O3ClÆH2O: C, 64.91; H, 7.78; N, 8.11. Found:
C, 64.73; H, 7.63; N, 8.24.
5.9. 4-Amino-5-chloro-2-methoxy-N-[1-(4-oxo-4-phenyl-
butyl)piperidin-4-ylmethyl]benzamide (10c)
The general procedure was followed for reaction time of
3 h with compound 2c as halide compound to give col-
orless oil. The pure oil was crystallized with ethanol to
obtain colorless crystals (1.8 g, 29%); mp 148–150 ꢁC; 1H
NMR (CDCl3) d: 1.19–1.39 (2H, m), 1.50–2.02 (7H, m),
2.40 (2H, t, J ¼ 7:3 Hz), 2.87–3.02 (4H, m), 3.30 (2H, t,
J ¼ 6:0 Hz), 3.88 (3H, s), 4.46 (2H, br s), 6.30 (1H, s),
7.42–7.60 (3H, m), 7.65–7.82 (1H, m), 7.95 (2H, dd,
J ¼ 7:3 Hz), 8.09 (1H, s); anal. calcd for
C24H30N3O3ClÆ1/10H2O: C, 64.67; H, 6.83; N, 9.43.
Found: C, 64.55; H, 6.79; N, 9.43.
5.14. 4-Amino-5-chloro-2-methoxy-N-[1-[6-(4-methoxy-
phenyl)-6-oxohexyl]piperidin-4-ylmethyl]benzamide (11c)
The general procedure was followed for reaction time of
3 h with compound 3c as halide compound to give solid.
The solid was recrystallized from AcOEt to obtain col-
1
orless crystals (1.38 g, 69%); mp 133–135 ꢁC; H NMR
(CDCl3) d 1.23–1.83 (11H, m), 1.89–2.08 (2H, m), 2.27–
2.47 (2H, m), 2.86–3.04 (4H, m), 3.32 (2H, t,
J ¼ 5:9 Hz), 3.87 (3H, s), 3.90 (3H, s), 4.38 (2H, br s),
6.29 (1H, s), 6.92 (2H, d, J ¼ 7:9 Hz), 7.68–7.88 (1H, m),
7.93 (2H, d, J ¼ 9:0 Hz), 8.10 (1H, s); anal. calcd for
C27H36N3O4ClÆ1/4H2O: C, 64.02; H, 7.26; N, 8.30.
Found: C, 63.95; H, 7.25; N, 8.32.
5.10. 4-Amino-5-chloro-2-methoxy-N-[1-(5-oxo-5-phenyl-
pentyl)piperidin-4-ylmethyl]benzamide (10d)
The general procedure was followed for reaction time of
3 h with compound 2d as halide compound to give col-
orless oil. The pure oil was crystallized with ethanol to
obtain colorless crystals (1.3 g, 64%); mp 100–102 ꢁC; 1H
NMR (CDCl3) d: 1.17 (2H, t, J ¼ 11 Hz), 1.44–1.59
(4H, m), 1.51–1.66 (5H, m), 1.81 (2H, t, J ¼ 11 Hz), 2.28
(2H, t, J ¼ 7:3 Hz), 2.83 (2H, d, J ¼ 11 Hz), 3.02 (2H, t,
J ¼ 7:2 Hz), 3.82 (3H, s), 5.90 (2H, s), 6.47 (1H, s), 7.49
(2H, dd, J ¼ 2:0, 7.7 Hz), 7.60 (1H, dd, J ¼ 2:0, 7.7 Hz),
7.66 (1H, s), 7.86 (1H, t, J ¼ 6:0 Hz), 7.96 (2H, dd,
J ¼ 1:6, 7.7 Hz); anal. calcd for C25H32N3O3ClÆH2O: C,
63.08; H, 7.20; N, 8.83. Found: C, 63.48; H, 7.10; N, 8.83.
5.15. 4-Amino-5-chloro-N-[1-[6-(4-hydroxylphenyl)-6-oxo-
hexyl]piperidin-4-ylmethyl]-2-methoxybenzamide (11d)
The general procedure was followed for reaction time of
6 h with compound 3d as halide compound to give solid.
The solid was recrystallized from EtOH/Et2O to obtain
colorless crystals (290 mg, 40%); mp 177–179 ꢁC; 1H
NMR (CDCl3/CD3OD) d 1.20–1.81 (12H, m), 1.91–2.09
(2H, m), 2.24–2.45 (2H, m), 2.82–3.02 (4H, m), 3.31–
3.38 (2H, m), 3.78 (3H, s), 4.77 (2H, br s), 6.37 (1H, s),
6.85 (2H, d, J ¼ 9:2 Hz), 7.85 (2H, d, J ¼ 9:2 Hz), 7.88–
7.99 (1H, m), 8.00 (1H, s); anal. calcd for
C26H34N3O4ClÆ1/2EtOH: C, 63.46; H, 7.30; N, 8.22.
Found: C, 63.24; H, 7.26; N, 8.33.
5.11. 4-Amino-5-chloro-2-methoxy-N-[1-(6-oxo-6-phen-
ylhexyl)piperidin-4-ylmethyl]benzamide (10e)
1
The preparation, H NMR and elemental analysis data
of 10e was described previously.8
5.12. 4-Amino-5-chloro-2-methoxy-N-[1-[6-(4-methylphen-
yl)-6-oxohexyl]piperidin-4-ylmethyl]benzamide (11a)
5.16. 4-Amino-5-chloro-N-[1-[6-(4-chlorophenyl)-6-oxo-
hexyl]piperidin-4-ylmethyl]-2-methoxybenzamide (11e)
The general procedure was followed for reaction time of
3 h with compound 3a as halide compound to give solid.
The solid was recrystallized from EtOH/AcOEt to
obtain colorless crystals (0.87 g, 45%); mp 130–131 ꢁC;
The general procedure was followed for reaction time of
8 h with compound 3e as halide compound to give solid.
After usual workup, title compound was obtained as

2744
S. Sonda et al. / Bioorg. Med. Chem. 12 (2004) 2737–2747
colorless powders (50%); mp 156–158 ꢁC; 1H NMR
(CDCl3) d 1.30–1.80 (11H, m), 1.90–2.09 (2H, m), 2.28–
2.48 (2H, m), 2.89–3.04 (4H, m), 3.32 (2H, t,
J ¼ 6:4 Hz), 3.90 (3H, s), 4.41 (2H, br s), 6.30 (1H, s),
7.43 (2H, d, J ¼ 8:6 Hz), 7.68–7.82 (1H, m), 7.98 (2H, d,
J ¼ 8:6 Hz), 8.00 (1H, s); anal. calcd for C26H33N3O3-
Cl2Æ1/2H2O: C, 60.58; H, 6.65; N, 8.15. Found: C, 60.82;
H, 6.61; N, 8.11.
orless solid. The solid was recrystallized from EtOH/
AcOEt to obtain colorless crystals (0.63 g, 38%); mp
106–110 ꢁC; H NMR (CDCl3) d 1.30–1.89 (10H, m),
2.01–2.21 (2H, m), 2.34 (3H, s), 2.47 (3H, s), 2.42–2.52
(2H, m), 2.87 (2H, t, J ¼ 7:3 Hz), 3.00–3.16 (2H, m),
3.33 (2H, t, J ¼ 6:3 Hz), 3.90 (3H, s), 4.43 (2H, br s),
6.31 (1H, s), 7.01–7.09 (2H, m), 7.56 (1H, d, J ¼ 7:9 Hz),
7.71–7.85 (1H, m), 8.09 (1H, s); anal. calcd for
C28H38N3O3ClÆ5/4H2O: C, 63.26; H, 7.87; N, 7.90.
Found: C, 63.19; H, 7.89; N, 7.91.
1
5.17. 4-Amino-5-chloro-N-[1-[6-(3-chlorophenyl)-6-oxo-
hexyl]piperidin-4-ylmethyl]-2-methoxybenzamide (11f)
5.21. 4-Amino-5-chloro-N-[1-[6-(3,4-dimethoxylphenyl)-
6-oxo-hexyl]piperidin-4-ylmethyl]-2-methoxybenzamide
(11j)
The general procedure was followed for reaction time of
8 h with compound 3f as halide compound to give solid.
The solid was recrystallized from EtOH to obtain col-
orless crystals (0.24 g, 31%); mp 128–132 ꢁC; H NMR
(CDCl3) d 1.31–1.92 (14H, m), 2.09–2.30 (2H, m), 2.48–
2.62 (2H, m), 2.95 (2H, t, J ¼ 7:0 Hz), 3.33 (2H, q,
J ¼ 6:0 Hz), 3.91 (3H, s), 4.39 (2H, br s), 6.30 (1H,s),
7.36–7.43 (1H, m), 7.49–7.55 (1H, m), 7.76–7.84 (1H,
m), 7.90–7.92 (1H, m), 8.09 (1H, s); anal. calcd for
C26H33N3O3Cl2Æ1.3H2O: C, 58.93; H, 6.77; N, 7.93.
Found: C, 58.92; H, 6.63; N, 8.05.
1
The general procedure was followed for reaction time of
8 h with compound 3j as halide compound to give col-
orless solid. The solid was recrystallized from EtOH/
AcOEt to obtain colorless crystals (0.50 g, 29%); mp
1
102–105 ꢁC; H NMR (CDCl3) d 1.24–2.27 (13H, m),
2.28–2.45 (2H, m), 2.88–3.05 (4H, m), 3.32 (2H, t,
J ¼ 6:3 Hz), 3.89 (3H, s), 3.93 (3H, s), 3.94 (3H, s), 4.40
(2H, br s), 6.30 (1H, s), 6.88 (1H, d, J ¼ 8:6 Hz), 7.52
(1H, d, J ¼ 2:0 Hz), 7.57 (1H, dd, J ¼ 2:0 Hz, 8.6 Hz),
7.68–7.85 (1H, m), 8.10 (1H, s); anal. calcd for
C28H38N3O5ClÆ3/2H2O: C, 60.15; H, 7.39; N, 7.52.
Found: C, 60.06; H, 7.60; N, 7.45.
5.18. 4-Amino-5-chloro-N-[1-[6-(4-fluorophenyl)-6-oxo-
hexyl]piperidin-4-ylmethyl]-2-methoxybenzamide (11g)
The general procedure was followed for reaction time of
3 h with compound 3g as halide compound to give solid.
The solid was recrystallized from EtOH/AcOEt to ob-
tain colorless crystals (1.0 g, 50%); mp 137–139 ꢁC, H
5.22. 4-Amino-5-chloro-N-[1-[6-(3,4-dichlorophenyl)-6-
oxo-hexyl]piperidin-4-ylmethyl]-2-methoxybenzamide
hydrochloride (11k)
1
NMR (CDCl3) d 1.25–1.82 (11H, m), 1.88–2.08 (2H, m),
2.25–2.45 (2H, m), 2.86–3.03 (4H, m), 3.33 (2H, t,
J ¼ 6:3 Hz), 3.90 (3H, s), 4.39 (2H, br s), 6.30 (1H, s),
7.04–7.20 (2H, m), 7.69–7.83 (1H, m), 7.92–8.03 (2H,
m), 8.10 (1H, s); anal. calcd for C26H33N3O3FClÆ1/
2H2O: C, 62.58; H, 6.87; N, 8.42. Found C, 62.77; H,
6.78; N, 8.44.
The general procedure was followed for reaction time of
3 h with compound 3k as halide compound to give col-
orless oil (110 mg, 15%). The pure oil was converted into
hydrochloride salt 10% HCl in EtOH to obtain colorless
1
crystals; mp 203–205 ꢁC; H NMR (DMSO-d6) d 1.23–
1.90 (11H, m), 2.70–3.55 (10H, m), 3.83 (3H, s), 5.93
(2H, br s), 6.48 (1H, s), 7.66 (1H, s), 7.81 (1H, d,
J ¼ 8:5 Hz), 7.92 (1H, dd, J ¼ 2:0 Hz, 8.5 Hz), 7.95–8.05
(1H, m), 8.15 (1H, d, J ¼ 2:0 Hz); anal. calcd for
C26H32N3O3Cl3ÆHClÆ1/4H2O: C, 53.67; H, 5.80; N, 7.22.
Found: C, 53.69; H, 6.05; N, 7.10.
5.19. 4-Amino-5-chloro-N-[1-[6-(3,4-dimethylphenyl)-6-
oxo-hexyl]piperidin-4-ylmethyl]-2-methoxybenzamide (11h)
The general procedure was followed for reaction time of
3 h with compound 3h as halide compound to give solid.
The solid was recrystallized from EtOH/AcOEt to
obtain colorless crystals (0.82 g, 30%); mp 115–117 ꢁC.
5.23. 4-Amino-5-chloro-N-[1-[6-(2,4-dichlorophenyl)-6-
oxo-hexyl]piperidin-4-ylmethyl]-2-methoxybenzamide
hydrochloride (11l)
1H NMR (CDCl3) d 1.21–1.47 (4H, m), 1.48–1.81 (9H,
m), 1.82–2.01 (2H, m), 2.30 (6H, s), 2.31 (6H, s), 2.82–
3.02 (4H, m), 3.32 (2H, q, J ¼ 6:0 Hz), 3.90 (3H, s), 4.36
(2H, br s), 6.29 (1H, s), 7.20 (1H, d, J ¼ 7:9 Hz), 7.62–
7.81 (3H, m), 8.11 (1H, s); anal. calcd for
C28H38N3O3Cl2: C, 67.25; H, 7.66; N, 8.40. Found: C,
67.23; H, 7.73; N, 8.52.
The general procedure was followed for reaction time of
3 h min with compound 3l as halide compound to give
colorless oil. The pure oil was converted into hydro-
chloride salt with 10% HCl in EtOH to obtain colorless
crystals (0.11 g, 33%); mp 150–155 ꢁC; 1H NMR
(DMSO-d6) d 2.65–3.09 (17H, m), 3.10–3.22 (2H, m),
3.30–3.52 (2H, m), 3.83 (3H, s), 5.75–6.08 (2H, br s),
6.49 (1H, s), 7.55 (1H, dd, J ¼ 1:3 Hz, 9.9 Hz), 7.68 (1H,
d, J ¼ 9:9 Hz), 7.73 (1H, d, J ¼ 1:3 Hz), 7.92–8.07
(1H, m), 9.50–13.00 (1H, m); anal. calcd for
C26H32N3O3Cl3ÆHClÆH2O: C, 52.45; H, 5.80; N, 7.22.
Found: C, 52.61; H, 6.06; N, 7.31.
5.20. 4-Amino-5-chloro-N-[1-[6-(2,4-dimethylphenyl)-6-
oxo-hexyl]piperidin-4-ylmethyl]-2-methoxybenzamide (11i)
The general procedure was followed for reaction time of
7 h with compound 3i as halide compound to give col-

S. Sonda et al. / Bioorg. Med. Chem. 12 (2004) 2737–2747
2745
5.24. 4-Amino-5-chloro-N-[1-[6-(2,4-difluorophenyl)-6-
oxo-hexyl]piperidin-4-ylmethyl]-2-methoxybenzamide
(11m)
SClÆHClÆ3/2H2O: C, 49.90; H, 6.09; N, 7.94. Found: C,
49.63; H, 6.04; N, 7.78.
The general procedure was followed for reaction time
5 h with compound 3m as halide compound to give
solid. The solid was recrystallized from EtOH/AcOEt to
obtain colorless crystals (0.25 g, 18%); mp 115–117 ꢁC;
1H NMR (CDCl3) d 1.21–2.00 (13H, m), 2.21–2.40 (2H,
m), 2.82–3.00 (4H, m), 3.32 (2H, t, J ¼ 6:3 Hz), 3.90
(3H, s), 4.37 (2H, br s), 6.29 (1H, s), 6.80–6.99 (2H, m),
7.68–7.80 (1H, m), 7.86–7.92 (1H, m), 8.10 (1H, s); anal.
calcd for C26H32N3O3ClF2: C, 61.47; H, 6.35; N,8.27.
Found: C, 61.15; H, 6.36; N, 8.22.
5.28. 4-Amino-5-chloro-2-methoxy-N-[1-(3-phenylsulfon-
ylpropyl)piperidin-4-ylmethyl]benzamide methanesulfo-
nate (12b)
The general procedure was followed for reaction time of
4 h with compound 6b as halide compound to give col-
orless oil (18 g, 71%). The pure oil was transformed into
methanesulfonate and recrystallized from 2-propanol-
diisopropylether to obtain title compound; mp 190–
192 ꢁC; 1H NMR (DMSO-d6) d 1.25–1.50 (2H, m),
1.70–1.89 (3H, m), 1.90–2.11 (2H, m), 2.33 (3H, s), 2.73–
3.01 (2H, m), 3.03–3.60 (5H, m), 3.82 (3H, s), 6.48 (1H,
s), 7.56–8.04 (4H, m), 7.84 (4H, m), 7.88–8.05 (3H, m),
5.25. 4-Amino-5-chloro-N-[1-[6-(3-chloro-4-methoxyphen-
yl)-6-oxo-hexyl]piperidin-4-ylmethyl]-2-methoxybenz-
amide (11n)
8.85–9.20
(1H,
m);
anal.
calcd
for
C23H30N3O4SClÆCH3SO3HÆ1/4H2O: C, 49.65; H, 5.99;
N, 7.24. Found: C, 49.61; H, 5.99; N, 7.22.
The general procedure was followed for reaction time of
3 h with compound 3n as halide compound to give col-
orless solid. The solid was recrystallized from EtOH/
AcOEt to obtain colorless crystals (0.45 g, 33%); mp
104–106 ꢁC; 1H NMR (CDCl3) d 1.30–1.50 (2H, m),
1.51–1.83 (10H, m), 1.90–2.07 (2H, m), 2.31–2.43 (2H,
m), 2.90 (2H, t, J ¼ 7:3 Hz), 2.95–3.05 (2H, m), 3.32
(2H, t, J ¼ 6:3 Hz), 3.90 (3H, s), 3.97 (3H, s), 4.40 (2H,
br s), 6.29 (1H, s), 6.95 (1H, d, J ¼ 8:6 Hz), 7.85 (1H, dd,
J ¼ 2:0 Hz, 8.6 Hz), 7.98 (1H, d, J ¼ 2:0 Hz), 8.09 (1H,
s); anal. calcd for C27H35N3O4Cl2ÆH2O: C, 58.48; H,
6.73; N, 7.58. Found: C, 58.62; H, 6.66; N, 7.81.
5.29. 4-Amino-5-chloro-2-methoxy-N-[1-(4-phenyl-
sulfonylbutyl)piperidin-4-ylmethyl]benzamide hydrochlo-
ride (12c)
The general procedure was followed procedure of
compound 6c as halide compound to give solid (9.2 g,
46%). The solid was converted to the hydrochloride salt
with 1 N HCl in EtOH to obtain colorless crystals; mp
158–159 ꢁC; 1H-NMR (DMSO-d6) d: 1.52–1.63 (4H, m),
1.77–1.95 (5H, m), 2.77 (2H, dd, J ¼ 10 Hz, 22 Hz),
2.90–3.02 (2H, m), 3.10–3.20 (3H, m), 3.83 (3H, s), 5.95
(2H, s), 6.51 (1H, s), 7.66 (2H, dd, J ¼ 2:0 Hz, 6.6 Hz),
7.70 (1H, dd, J ¼ 2:0 Hz, 6.6 Hz), 7.89 (2H, dd, J ¼ 2:0,
6.6 Hz), 7.94 (1H, t, J ¼ 5:3 Hz), 10.42 (1H, br s); anal.
calcd for C24H32ClN3O4SÆHClÆH2O: C, 52.55; H, 6.43;
N, 7.66. Found: C, 52.30; H, 6.45; N, 7.58.
5.26. 4-Amino-5-chloro-N-[1-[6-(3-fluoro-4-methoxyphen-
yl)-6-oxo-hexyl]piperidin-4-ylmethyl]-2-methoxybenz-
amide (11o)
The general procedure was followed for reaction time of
5 h with compound 3o as halide compound to give col-
orless solid. The solid was recrystallized from EtOH/
AcOEt to obtain colorless crystals (0.90 g, 64%); mp
5.30. 4-Amino-5-chloro-2-methoxy-N-[1-(5-phenylsulfon-
ylpentyl)piperidin-4-ylmethyl]benzamide hydrochloride
(12d)
1
112–113 ꢁC; H NMR (CDCl3) d 1.20–1.92 (10H, m),
1.93–2.04 (2H, m), 2.24–2.45 (2H, m), 2.80–3.02 (4H,
m), 3.32 (2H, t, J ¼ 6:0 Hz), 3.81 (3H, s), 3.95 (3H, s),
4.43 (2H, br s), 6.30 (1H, s), 6.99 (1H, d, J ¼ 8:3 Hz),
7.64–7.89 (4H, m), 8.10 (1H, s); anal. calcd for
C27H35N3O4ClFÆ1/2H2O: C, 61.30; H, 6.86; N, 7.94.
Found: C, 61.46; H, 6.93; N, 7.85.
1
The preparation, H NMR and elemental analysis data
of 12d was described previously.8
5.31. 4-Amino-N-[1-[3-(benzylsulfonyl)propyl]piperidin-4-
ylmethyl]-5-chloro-2-methoxybenzamide (13a, Y-36912)
5.27. 4-Amino-5-chloro-2-methoxy-N-[1-(2-phenylsulfonyl-
ethyl)piperidin-4-ylmethyl]benzamide hydrochloride (12a)
The general procedure was followed for reaction time of
7.5 h with compound 8a as halide compound to give
solid. The resulting solid was recrystallized from ethanol
to obtain colorless powders (45 g, 63%); mp 170–171 ꢁC;
1H NMR (CDCl3) d: 1.17–1.32 (2H, m), 1.50–1.63 (1H,
m), 1.69 (2H, d, J ¼ 13 Hz), 1.86–2.00 (4H, m), 2.37
(2H, t, J ¼ 6:9 Hz), 2.82 (2H, d, J ¼ 12 Hz), 2.88–2.94
(2H, m), 3.31 (2H, t, J ¼ 6:3 Hz), 3.88 (3H, s), 4.22 (2H,
s), 4.42 (2H, s), 6.30 (1H, s), 7.37–7.43(5H, m), 7.73
(1H, t, J ¼ 5:6 Hz), 8.10 (1H, s). Anal. Calcd for
C24H32ClN3O4S: C, 58.34; H, 6.53; N, 8.51. Found: C,
58.15; H, 6.56; N, 8.49.
The general procedure was followed for reaction time of
5 h with compound 6a as halide compound to give col-
orless oil (2.74 g, 31%). The pure oil was converted to
the hydrochloride salt with 1 N HCl in EtOH to obtain
colorless crystals; mp 116–117 ꢁC; 1H NMR (DMSO-d6)
d 1.35–2.05 (5H, m), 2.70–3.02 (2H, m), 3.03–3.60 (5H,
m), 3.80 (3H, s), 3.88–4.15 (2H, m), 5.70–6.15 (2H, m),
6.50 (1H, s), 7.55–7.89 (4H, m), 7.90–8.09 (3H, m),
10.50–11.70 (1H, m); anal. calcd for C22H28N3O4-

2746
S. Sonda et al. / Bioorg. Med. Chem. 12 (2004) 2737–2747
5.32. 4-Amino-N-[1-[4-(benzylsulfonyl)butyl]piperidin-4-
ylmethyl]-5-chloro-2-methoxybenzamide hydrochloride
(13b)
tron, 50 lL of displacing drugs and 900 lL of tissue
homogenate. Following a 30 min incubation at 25 ꢁC,
the assay mixture was rapidly filtered under reduced
pressure through Whatman GF/B glass filters which had
been presoaked in 0.1% polyethyleneimie. Filters were
washed immediately with 3 ꢀ 3 mL of ice-cold Tris–HCl
buffer (50 mM, pH 7.4). ICS 205930 (100 lmmol/L) was
used for the determination of nonspecific binding.
The general procedure was followed for reaction time of
6 h with compound 8b as halide compound to give col-
orless oil. The pure oil was converted into hydrochloride
salt with 10% HCl/EtOH to obtain colorless crystals
1
(0.96 g, 33%); mp 114–116 ꢁC; H NMR (DMSO-d6) d
1.45–2.02 (9H, m), 2.70–3.48 (12H, m), 3.83 (3H, s), 4.49
(2H, br s), 6.51 (1H, s), 7.30–7.51 (5H, m), 7.66 (3H, m),
7.91–8.09 (3H, m), 10.30–10.60 (1H, m); anal. calcd for
C25H34N3O4SClÆHClÆ5/4H2O: C, 52.95; H, 6.67; N, 7.41.
Found C, 52.93; H, 6.72; N, 7.30.
5.35. Dopamine D2 receptor binding assay
[3H]Spiperone binding assay was performed according
to the method of Creese et al. Male Wistar rat (Japan
SLC, Ltd, Shizuoka, Japan) striatal membrane was
homogenized in 100 volumes of ice-cold Tris–HCl buffer
(50 mmol/L, pH 7.7) and centrifuged (500·g, 10 min,
0 ꢁC). The supernatant was centrifuged at 50,000·g
for 15 min. The pellet was suspended in 100 volumes of
ice-cold Tris–HCl buffer (50 mmol/L, pH 7.7) and re-
centrifuged (500·g, 10 min, 0 ꢁC). The final pellet was
resuspended in 150 volumes (50 mmol/L, pH 7.7) con-
taining 120 mmol/L NaCl, 5 mmol/L KCl, 2 mmol/L
CaCl2, 1 mmol/L MgCl2, 1.1 mmol/L ascorbic acid, and
10 lmol/L pargyline, and incubated at 37 ꢁC for 10 min.
A portion of this membrane suspension (900 lmol/L)
was placed in a tube, and 50 lmol/L of either test
compound or vehicle solution was added, followed by
50 lL of [3H]Spiperone (40 Ci/mmol) at a final concen-
tration of 0.2 nmol/L. The tubes were incubated at 37 ꢁC
for 20 min and filtered through Whatman GF/B
glass filters, which were then washed three times with
3 mL of Tris–HCl buffer (50 mmol/L, pH 7.7). Sulpi-
ride (100 lmol/L) was used for the determination of
nonspecific binding. The radioactivity trapped on
the filters was measured by liquid scintillation spec-
trometry.
5.33. 5-HT4 receptor binding assay
Male Hartley guinea pigs (Japan SLC, Ltd, Shizuoka,
Japan) were sacrificed by cervical dislocation and the
striatum was separated from each brain. The striatum
was homogenized in 15 volume of 50 mmol/L ice-cold
HEPES buffer (pH 7.4) with Polytron PT-10 and then
centrifuged at 35,000·g for 20 min. The resulting pellet
was resuspended in the HEPES buffer and finally diluted
to the appropriate concentration for assay (6 mg wet
weight per assay tube). This suspension was used as the
tissue preparation. Assay tube contained 50 lL of HE-
PES buffer or a solution of the test agents, 50 lL solu-
tion of [3H]GR113808 (Amersham International, UK)
to give a final concentration of 0.1 nmol/L and 900 lL of
tissue preparation. Each tube was incubated for 30 min
at 37 ꢁC and the reaction was terminated by rapid fil-
tration through a Whatmann GF/B filter (presoaked in
0.01% v/v polyethyleneimine) followed by washing with
1 ꢀ 4 mL of ice-cold HEPES buffer. Then the filter was
placed in 3 mL of scintillator and the radioactivity was
determined by scintillation counting in a Beckman
model LS3801 scintillation counter. Nonspecific binding
was defined in the presence of unlabelled GR113808 to
give a final concentration of 1 lmol/L. The IC50 value
was determined by nonlinear regression of the dis-
placement curve, and the Ki value was calculated
according to the formula (Ki ¼IC50/(1 þ L/Kd)), where L
is the concentration of radioligand and Kd is the disso-
ciation constant of the radioligand.
5.36. 5-HT4 receptor agonistic activities in vitro contrac-
tion of isolated guinea-pig ascending colon
Male Hartley guinea pigs (Japan SLC, Ltd, Shizuoka,
Japan) were killed by cervical dislocation and the
ascending colon (a 10 cm segment starting 1 cm from the
caecum) was removed. The longitudinal muscle layer
was separated from the underlying circular muscle and
divided into four segments of about 2.5 cm. Four muscle
strip preparations were individually mounted vertically
for isotonic measurement into a tissue bath containing
10 mL Tyrode solution. Only 5-HT was tested in the
Tyrode solution with containing methysergide (1 lmol/
L) and granisetron (1 lmol/L) to inhibit responses
mediated by 5-HT2 and 5-HT1-like and 5-HT3 receptors,
respectively. This solution was kept at 37 ꢁC and gassed
with 95% O2, 5% CO2. The strips were subjected to a
preload of 1 g and allowed to stabilize for 20 min. After
stabilization, the response of the longitudinal muscle to
30 lmol/L methacholine was measured. Agonist con-
centration-effect curves were constructed using sequen-
tial dosing, leaving 15 min between doses. A 15 min
dosing cycle was required to prevent desensitization.
The agonist was left in contact with a preparation until
5.34. 5-HT3 receptor binding assay
[3H]Granisetron binding assay was performed according
to the method of Nelson and Thomas.12 Male Wistar rat
(Japan SLC, Ltd, Shizuoka, Japan) cerebral cortex was
homogenized in 20 volumes of 0.32 mol/L sucrose and
the centrifuged at 1000·g for 10 min. The supernatant
was centrifuged at 40,000·g for 15 min. The pellet was
suspended in 20 volumes of HEPES buffer (50 mmol/L,
pH 7.4) and suspension was incubated at 37 ꢁC for
10 min, was centrifuged at 40,000·g for 15 min. The
pellet was washed and centrifuged (40,000·g for 15 min).
The final pellet was resuspended in 30 volumes of
HEPES buffer and used as tissue homogenate. The
binding assay consisted of 50 lmmol/L of [3H]Granise-

S. Sonda et al. / Bioorg. Med. Chem. 12 (2004) 2737–2747
2747
the response had reached a maximum, the preparation
was washed. Forty minutes was left between the deter-
mination of concentration-effect curves. GR113808
(10 nmol/L) were incubated for 10 min before repeating
agonist concentration-effect curves. After each determi-
nation of concentration-effect curve, 30 lmol/L of
methacholine was added to the tissue bath again. All
responses were expressed as a percentage of the mean of
the two contractions induced by 30 lmol/L methacho-
line. The EC50 value, the concentration causing 50% of
the maximal response, was determined by linear
regression analysis.
30 min. The number, wet weight, and dry weight of
feces excreted for 2 h from immediately after the
administration were measured. Results are expressed
as means ꢁ SEM and were compared by Dunnett
method.
Acknowledgements
We thank Mrs. F. Matsugaki, Mrs. M. Miyoshi, Mrs.
Y. Hattori and T. Murozono for some of the biological
results. We also thank Mr. K. Haga and Mr. K. Itoh for
helpful discussion.
5.37. Effect of compounds on gastric emptying of liquid
meal in mice
Male Sea:ddY mice were deprived food for about 18 h
before use and were orally administered test com-
pounds. Half an hour later, mice were given 0.1 mL of
test meal containing 0.05% phenol red in 1.5%
hydroxypropyl methylcellulose solution. Animals were
sacrificed 15 min after administration of test meal and
the stomach was removed. Phenol red remaining in
stomach was measured by colorimetric assay. Results
are expressed as means ꢁ SEM and were compared by
Dunnett method.
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5.38. Effect of compounds on gastric emptying of solid
meal in rats
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5.39. Effect on defecation in mice
Male Crj:CD-1(ICR) mice were orally or subcutane-
ously administered test compounds after being adapted
to experimental surroundings in partition box for