Synthesis and pharmacological evaluation of benzamide derivatives as selective 5-HT4 receptor agonists

Article abstract of DOI:10.1016/j.bmc.2005.02.016

It is thought that selective 5-HT₄ receptor agonists-such as 4-amino-5-chloro-2-methoxy-N-[1-(6-oxo-6-phenylhexyl)piperidin-4-ylmethyl] benzamide (2)-have the ability to enhance both upper and lower gastrointestinal motility without any significant adverse effects. Modification of 2 was performed. Variation of the piperidin-4-ylmethyl moiety of 2 led to a decrease in the binding affinity for the 5-HT₄ receptor. Following conversion of the carbonyl group on the benzoyl part to a hydroxyl or sulfoxide group, the binding affinity for the 5-HT₄ receptor was retained although the effect on defecation was reduced. Many of the 4-amino-5-chloro-2-methoxy-N- (piperidin-4-ylmethyl)benzamides that had a ether or sulfide moiety in the side-chain part at the 1-position of the piperidine exhibited high affinity for the 5-HT₄ receptor. Among these, phenylthio 41c and benzylthio derivative 44 were selective 5-HT₄ receptor agonists, and had a similar effect on defecation to compound 2.

Full text of DOI:10.1016/j.bmc.2005.02.016

Bioorganic & Medicinal Chemistry 13 (2005) 3295–3308
Synthesis and pharmacological evaluation of benzamide
derivatives as selective 5-HT4 receptor agonists
Shuji Sonda,a,* Toshio Kawahara,a Kenichi Katayama,b Noriko Satoc and Kiyoshi Asanoc
aPharmaceutical Development Laboratories, Technology and Production Division, Mitsubishi Pharma Corporation,
14 Sunayama, Hasaki-machi, Kashima-gun, 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 and Development Division, Mitsubishi Pharma Corporation, 1000, Kamoshida-cho, Aoba-ku,
Yokohama 227-0033, Japan
Received 6 October 2004; revised 8 February 2005; accepted 9 February 2005
Available online 19 March 2005
Abstract—It is thought that selective 5-HT4 receptor agonists—such as 4-amino-5-chloro-2-methoxy-N-[1-(6-oxo-6-phen-
ylhexyl)piperidin-4-ylmethyl]benzamide (2)—have the ability to enhance both upper and lower gastrointestinal motility without
any significant adverse effects.
Modification of 2 was performed. Variation of the piperidin-4-ylmethyl moiety of 2 led to a decrease in the binding affinity for the
5-HT4 receptor. Following conversion of the carbonyl group on the benzoyl part to a hydroxyl or sulfoxide group, the binding affin-
ity for the 5-HT4 receptor was retained although the effect on defecation was reduced. Many of the 4-amino-5-chloro-2-methoxy-N-
(piperidin-4-ylmethyl)benzamides that had a ether or sulfide moiety in the side-chain part at the 1-position of the piperidine exhib-
ited high affinity for the 5-HT4 receptor.
Among these, phenylthio 41c and benzylthio derivative 44 were selective 5-HT4 receptor agonists, and had a similar effect on
defecation to compound 2.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1.Introduction
D2 receptor produces unfavourable side effects, such as
extrapyramidal syndrome including parkinsonism, dys-
tonia, dyskinesia and akathisia in the central nervous
system. It is therefore thought that a selective 5-HT4
receptor agonist with no binding affinity for the 5-HT3
and dopamine D2 receptors should enhance both upper
and lower gastrointestinal motility without serious side
effects.
The 5-HT4 receptor is localized in the central nervous
system and peripheral tissues.1,2 At the periphery, stim-
ulation of the 5-HT4 receptor mediates gastric motility,
ileal motility and colonic transit. Recently, Grider et al.
indicated that 5-HT4 receptor agonists initiate the peri-
staltic reflex in human jejunum, rat and guinea-pig intes-
tine.3 Various compounds have been reported to act as
agonists or antagonists to 5-HT4 receptors.4–12 A num-
ber of benzamides known as 5-HT4 receptor agonists
(cisapride, BIMU-8, metoclopramide, etc.) have binding
affinity for other receptors, notably the 5-HT3 and dopa-
mine D2 receptors.13–16
In our previous reports, we have described the design and
characterization of a number of selective 5-HT4 receptor
agonists.17–21 The first step of our strategy was the opti-
mization of the aromatic ring and cyclic amine moiety of
the benzamide.18 Selectivity for the 5-HT4 receptor was
induced by using the piperidin-4-ylmethyl group as the
cyclic amine part [e.g., 4-amino-5-chloro-2-methoxy-N-
[1-[5-(1-methylindol-3-ylcarbonylamino)pentyl]piper-
idin-4-ylmethyl]benzamides (1) showed binding affinity
for 5-HT4 (Ki = 0.3 nM), 5-HT3 (IC50 > 1000 nM) and
dopamine D2 (IC50 > 1000 nM) Fig. 1]. It had been sup-
posed that compound 1 was a gastrointestinal motility
stimulant, which could enhance both upper and lower
It is known that 5-HT3 receptor antagonism reduces co-
lonic transit18 and that binding affinity for the dopamine
Keywords: 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 ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.02.016

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S. Sonda et al. / Bioorg. Med. Chem. 13 (2005) 3295–3308
in a good yield. The alcohol 4 was obtained by ring-
CH3
opening reaction of 3 with NH4OH followed by Boc-
protection of the amino group. Methylation of the 4-hy-
droxyl group on the piperidine ring of 4 provided com-
pound 5, from which the intermediate 6 was derived by
removing the benzyl group at the 1-position of the piper-
idine under reductive conditions. Similarly, compound 7
was synthesized by benzyl-deprotection of the interme-
diate 4. The piperidines 6 and 7 were treated with 6-bro-
mo-1-phenylhexan-1-one (8) in K2CO3/DMF followed
by Boc-deprotection with hydrochloric acid to afford
the corresponding amine derivatives 9 and 10, respec-
tively. Condensation of these with 4-amino-5-chloro-2-
methoxybenzoic acid using EDCÆHCl and HOBt affor-
ded the benzamides 11 and 12, respectively.
N
H
N
1 : R =
Cl
O
O
H2N
NH
N
R
O
CH3
2 : R =
O
Figure 1.
gastrointestinal motility with few side effects, but it dis-
played poor bioavailability in dogs (5.1%) because of
the low intestinal absorption rate.
The derivative 20, which had a methoxy group at the 3-
position of the piperidine part, was prepared from ethyl
1-benzyl-3-oxo-4-piperidinecarboxylate hydrochloride
(13) as the starting material according to the synthetic
pathway described in Scheme 2. The compound 13
was converted into the cis-diol 14 by reduction with
NaBH4. After removal of the benzyl group under reduc-
tive conditions, compound 14 was protected with the
tert-butoxycarbonyl group to give compound 15, from
which 16 was obtained by selective conversion through
mesylation of the hydroxylmethyl group at the 4-posi-
tion of the piperidine followed by azidation. Methyl-
ation of the hydroxyl group at the 3-position of 16 with
methyliodide provided derivative 17, hydrogenation of
which under atmospheric pressure in the presence of
Pd(OH)2 gave the amine 18 in a good yield.
In the next step, we replaced the 1-methylindol-3-ylcar-
bonylamino moiety of 1 with aralkylamino (or alkyl-
amino), benzoyl and phenylsulfonyl group to raise oral
bioavailability.20 Compounds with a benzoyl moiety
on the side-chain part at the 1-position of the piperidine
ring showed satisfactory intestinal absorption. Among
these, compound 2 showed 54% oral bioavailability in
dogs and displayed selective binding affinity for the 5-
HT4 receptor (Ki = 2.4nM).
We therefore carried out further optimization of com-
pound 2 to explore the structure–activity relationship.
In the present paper, we report the modification of the
piperidine and benzoyl moieties.
2.Chemistry
Compound 18 was condensed with 4-amino-5-chloro-2-
methoxybenzoic acid and treated with hydrochloric acid
to afford compound 19. A 6-oxo-6-phenylhexyl moiety
was introduced by alkylation of 19 with 8 to give the de-
sired benzamide 20.
The synthesis of derivatives 11 and 12 is outlined in
Scheme 1. The oxirane 3 was prepared from commer-
cially available 1-benzylpiperidin-4-one using Me3S(O)I
CH3
O
CH3
O
HO
O
d
N
c
a,b
N
N
NH
Boc N
Boc
N
H
Boc
N
H
H
4
3
5
6
d
HO
NH
e,f
4
Boc
N
H
Br
6,7
+
O
7
8
Cl
O
H2N
H2N
NH
N
g
N
O
R
O
O
CH3
O
R
O
.
2HCl
11:R = H
9:R = H
10:R = Me
12:R = Me
Scheme 1. Reagents and conditions: (a) NH4OH; (b) (BOC)2O; (c) MeI, 60% NaH; (d) 10% Pd–C, NH2NH2–H2O/EtOH; (e) K2CO3/DMF; (f) HCl/
2-propanol; (g) 4-amino-5-chloro-2-methoxybenzoic acid, EDC–HCl, HOBt, Et3N.

S. Sonda et al. / Bioorg. Med. Chem. 13 (2005) 3295–3308
3297
O
HO
.
HO
HCl
HO
O
O
b,c
d,e
a
HO
N
N
N
Boc
14
13
15
CH3
O
CH3
O
HO
N3
N3
g
H2N
f
N
Boc
N
Boc
N
Boc
16
17
18
CH3
O
CH3
O
Cl
Cl
h,i
O
O
H
N
H
j
H2N
H2N
N
NH
N
O
O
O
.
2HCl
HC3
H3C
19
20
Scheme 2. Reagents and conditions: (a) NaBH4; (b)10% Pd–C, NH2NH2–H2O; (c) (BOC)2O; (d) MsCl, Et3N; (e) NaN3, NH4Cl/DMF; (f) MeI, 60%
NaH/DMF; (g) H2, Pd(OH)2; (h) 4-amino-5-chloro-2-methoxybenzoic acid, EDC–HCl, HOBt, Et3N; (i) HCl/2-propanol; (j) 8, K2CO3/DMF.
The derivative 27, which possessed an unsaturated
piperidine, was synthesized from ethyl 4-oxopiperidine-
1-carboxylate (21) as shown in Scheme 3. The derivative
22 was prepared by nitro-aldol reaction with nitrometh-
ane from 21. The dehydroxylation of 22 in the presence
of P2O5 provided the unsaturated piperidine 23, the ni-
tro group of which was reduced with Fe and NH4Cl
to give the amine derivative 24. Condensation reaction
of 4-amino-5-chloro-2-methoxybenzoic acid with 24
afforded compound 25, which was treated with KOH/
2-propanol to provide 26. Coupling reaction of the
benzamide 26 with compound 8 in the presence of
K2CO3 gave the desired compound 27.
from the intermediate 29 by deprotection of the Boc
group with hydrochloric acid, was attached to 4-amino-
5-chloro-2-methoxybenzoic acid under standard conden-
sation conditions using EDCÆHCl–HOBt to provide 31.
Scheme 5 shows the preparation of the alcohol 32 and
the oxime 33. The reduction of the carbonyl group of
compound 2 with NaBH4 afforded the racemic second-
ary alcohol derivative 32. Condensation of the carbonyl
group of 2 with hydroxylamine hydrochloride using pyr-
idine afforded the oxime derivative 33.
The preparation of the compounds in Table 3 proceeded
as shown in Scheme 6. The ethers 34a–e and sulfides
35a–d were synthesized by alkylation of phenol or benz-
enethiol with the corresponding bromochloroalkanes
(n = 2–6). The sulfide derivative 35c was oxidized with
1 equiv of m-CPBA to give the sulfoxide 36. The
The piperidin-3-ylmethyl compound 31 was synthesized
as shown in Scheme 4. Alkylation of the piperidine 28
with the bromide 8 under a typical condition (K2CO3/
DMF) gave compound 29. Compound 30, produced
O2N
b
O2N
a
N
O
COOEt
N
COOEt
N
COOEt
Cl
HO
21
23
22
O
H2N
d
c
H2N
NH
N COOEt
N
R
O
CH3
24
25 : R = COOEt
26 : R = H
e
Cl
O
f
H2N
NH
N
O
CH3
O
27
Scheme 3. Reagents and conditions: (a) CH3NO2, Na/EtOH; (b) P2O5, benzene reflux; (c) Fe, NH4Cl, H2O/toluene reflux; (d) 4-amino-5-chloro-2-
methoxybenzoic acid, EDC–HCl, Et3N, HOBt; (e) KOH, 2-propanol reflux; (f) 8, K2CO3/DMF.

3298
S. Sonda et al. / Bioorg. Med. Chem. 13 (2005) 3295–3308
Cl
H
O
R N
H
N
Boc
NH
H2N
c
a
N
NH
N
O
CH3
O
O
29 : R = Boc
30 : R = H
b
28
31
Scheme 4. Reagents: (a) 8, K2CO3/DMF; (b) HCl (aq); (c) 4-amino-5-chloro-2-methoxybenzoic acid, EDC–HCl, HOBt, Et3N.
Cl
HO
O
a or b
H2N
NH
32: R =
33: R =
2
N
OH
R
O
CH3
N
Scheme 5. Reagents and conditions: 32: (a) NaBH4; 33: (b) NH2OH–HCl, pyridine/MeOH reflux.
compounds 37a–c and 37e–f were synthesized from the
corresponding benzyl alcohols (37d: from cyclo-
hexylmethanol). Alkylation of benzylmercaptane with
the corresponding bromochloroalkane in 60% NaH/
THF afforded 38. The key intermediate 39 was prepared
using a method reported previously.20 Coupling reaction
of compound 39 with 34a–e, 35a–d, 36, 37a–f and 38 in
K2CO3–DMF gave phenoxy (40a–e), phenylthio (41a–
d), phenylsulfinyl (42), benzyloxy (43a–c,e,f), cyclo-
hexylmethyl (43d) and benzylthio (44) derivatives,
respectively.
3.Results and discussion
The synthesized compounds were evaluated for their 5-
HT4 receptors-binding affinity by use of [3H]GR113808.
Their affinity for the 5-HT3 and dopamine D2 receptors
Br
X
(CH )
c
Cl
2 n
(CH )
S
Cl
Cl
35c
2 n
X
a
O
34a-e : X=O
35a-d : X=S
36
Br
Br
Ar
(CH )
Cl
(CH )
2 5
Cl
2 n
O
Ar
(CH )
Cl
2 5
Y
HO
Y
b
a or b
(CH )
2 n
Cl
37d-f
37a-c : Y=O
38
: Y=S
Cl
O
H2N
NH
34a-e, 35a-d, 36, 37a-f and 38
+
NH
2HCl
O
39
Cl
O
NH
d
H2N
(CH )
2 n
N
Ar
n = 2-6
Z
O
Ar =
40a-e : Z = O
41a-d : Z = S
42 : Z = SO
Ar =
43d : Z = OCH2-
Cl
Ar =
Ar =
Ar =
Ar =
43e : Z = OCH2-
43f : Z = OCH2-
44 : Z = SCH2-
43a-c : Z = OCH2- Ar =
Ar =
Scheme 6. Reagents and conditions: (a) K2CO3/DMF; (b) 60% NaH; (c) m-CPBA; (d) K2CO3/DMF.

S. Sonda et al. / Bioorg. Med. Chem. 13 (2005) 3295–3308
3299
was similarly evaluated using [3H]Granisetron and
[3H]Spiperone, respectively, as radioligands. Membrane
preparations of guinea-pig striatum, rat cerebral cortex
and rat striatum were used for the 5-HT4, 5-HT3 and
dopamine D2 receptor-binding assays, respectively.
the piperidine ring could decrease selectivity for the 5-
HT4 receptor.
The piperidin-3-ylmethyl derivative 31 exhibited an 11-
fold decrease in binding affinity compared to the piper-
idin-4-ylmethyl derivative 2. These finding suggests that
use of an unsubstituted piperidin-4-ylmethyl part as the
cyclic amine on the benzamide is optimal for 5-HT4
receptor-binding affinity.
Initially, we proceeded with the modification of the
piperidine moiety (or R1) of the parent compound 2
to investigate the influence of a steric hindrance (or elec-
tric atmosphere) by substituents such as OH, OCH3,
introduction of double bond or transformation to the
piperidin-3-ylmethyl group.
The structure–activity relationships revealed by trans-
formation to bioisosters of the carbonyl group (R2 part)
on the benzamides are summarized in Table 2. The
derivatives were measured for the rate of increase in def-
ecation induced in mice at oral doses of 1 mg/kg (per-
centage increase in number, dry weight and wet weight
of faecal deposits). Binding affinity for the 5-HT4 recep-
tor was retained after reduction of the carbonyl group of
2 (racemic 32, Ki = 3.6 nM). Similarly, the oxime 33 and
the racemic sulfoxide derivative 42 were nearly equipo-
tent (Ki = 2.5 and 1.9, respectively) with the derivative
2. However, the effect on defecation of 32 and 42 was
considerably less than that of compound 2. Compound
43b, which possessed benzyloxy in the side-chain part
of the 1-position of the piperidine ring, had moderate
effect on defecation.
The results of modification are shown in Table 1. Com-
pound 2 showed high binding affinity and high selectiv-
ity for the 5-HT4 receptor compared to the 5-HT3 and
dopamine D2 receptors. Introduction of a hydroxyl
group at the 4-position (11) of the piperidine ring re-
sulted in a 3.7-fold decrease in the binding affinity for
the 5-HT4 receptor. The decrease in binding affinity
was particularly drastic when the methoxy group occu-
pied the 4-position of the piperidine ring (12).
A methoxy substituent (cis form) at the 3-position (20)
caused an 18-fold decrease in binding affinity and a de-
cline in the selectivity of the binding affinity for the 5-
HT4 receptor. Incorporation of a double bond at the
3,4-position of the piperidine ring (27) resulted in a loss
of binding affinity for the 5-HT4 receptor (Ki = 15 nM).
These results suggest that the introduction of a steric
hindrance (or electric atmosphere) at the 3-position of
On the basis of the above results, the development of the
structure–activity relationship was focused on the ether
derivatives. As shown in Table 3, the synthesized phen-
oxy (or phenylthio) derivatives and benzyloxy (or benz-
ylthio) derivatives possessed strong affinity for the 5-
HT4 receptor but showed little binding affinity for the
5-HT3 receptor.
Table 1. Pharmacological data of benzoyl derivatives
Cl
O
H
A series of phenoxy derivatives, 40a–e, with 2–6 methyl-
enes as a spacer between the piperidine and phenoxy
moieties, possessed moderate binding affinity (Ki = 15,
4.5, 9.4, 6.2 and 4.1 nM) for the 5-HT4 receptor. Com-
pounds containing ethylene (40a) and propylene spacers
(40b) displayed binding affinity for the dopamine D2
receptor (43 and 89 nM, respectively), insertion of 4–6
methylenes as a spacer (40c–e) was effective in decreas-
ing this affinity (IC50 > 1000 nM). These results suggest
that in the hydrophobic region of the dopamine D2
receptor there is not enough space available for a phen-
oxy group linked with 4–6 methylenes. The replacement
of the phenyl group with a benzyl group increased the
binding affinity for the 5-HT4 receptor (40c vs 43a,
40d vs 43b and 40e vs 43c).
H2N
N
R1
O
N
O
Compd
R1
Binding affinitya
5-HT4
Ki (nM)
5-HT3
IC50 (nM)
D2
IC50 (nM)
2
2.4>1000
>1000
N
HO
11
12
20
8.8
>1000
>1000
180b
>1000
>1000
>1000
N
N
O
>100
Similarly, the effect on defecation (percentage increase in
dry weight and wet weight of faecal deposits) of benzyl-
oxy derivatives was greater than that of phenoxy com-
pounds. The effect of 43c linked with six methylenes
was particularly strong (respective increases of 93%,
110% and 87% in the three items measured).
O
44
N
N
27
31
15
28
240b
NTc
>1000
>1000
The saturation of the benzyl group of 43b to the cyclo-
hexylmethyl group resulted in a small decrease in bind-
ing affinity for the 5-HT4 receptor (43d, Ki = 10 nM).
This observation suggests that there may be a favour-
able interaction between the p-electron density of the
N
a Each value is the mean from triplicate assay in a single experiment.
b Ki value.
c Not tested.

3300
S. Sonda et al. / Bioorg. Med. Chem. 13 (2005) 3295–3308
Table 2. Pharmacological data of benzamide derivatives
Cl
O
H
N
H2N
N
R2
O
Compd
R2
Binding affinitya
5-HT3 IC50 (nM)
Rate of increase in defecationb (%)
5-HT4 Ki (nM)
D2 IC50 (nM)
NTc
Number
Dry weight
Wet weight
OH
32
33
3.6
>1000
>1000
À3
15
13
HO
NTc
33
N
2.5
1.9
>1000
>1000
O
S
42
>1000
40
59
25
68
43b
2
3.6
270d
>1000
81
O
O
2.4>1000
>10900
90
4
76
a Each value is the mean from triplicate assay in a single experiment.
b Rate of increase in defecation induced in mice at oral dose of 1 mg/kg.
c Not tested.
d Ki value.
benzyl group of 43b and the corresponding hydrophobic
binding region of the 5-HT4 receptor.
decrease in the binding affinity for the 5-HT4 receptor.
Incorporation of a 3-methoxy group or double bond
in the piperidine moiety also decreased the binding affin-
ity. The conversion of the carbonyl group of the benzoyl
part to a hydroxyl or sulfoxide (32 and 42) group main-
tained most of the binding affinity for the 5-HT4 recep-
tor, but the effect on defecation was reduced compared
to compound 2. Many of the ether and sulfide deriva-
tives exhibited high affinity for the 5-HT4 receptor.
Among these, phenylthio 41c and benzylthio derivative
44 were selective 5-HT4 receptor agonists, and had a
similar effect on defecation to compound 2. Accord-
ingly, these derivatives would be a useful tool for search
of the 5-HT4 receptor functions.
With introduction of a chlorine atom (43e), the binding
affinity for the 5-HT4 receptor was nearly retained than
in the counterpart 43b. Meanwhile, 43e provided a po-
tent effect on defecation (increases of 93% in number,
93% in dry weight and 77% in wet weight of faecal
deposits). The naphthalen-2-ylmethoxy derivative 43f
also maintained binding affinity for the 5-HT4 receptor
(Ki = 4.7 nM) and displayed a moderate effect on defeca-
tion. This result demonstrates that the corresponding
hydrophobic binding region of the 5-HT4 receptor has
enough space to fit a naphthalen-2-ylmethoxy moiety
and this compound can produce agonistic activity.
The phenylthio derivatives 41a–d possessed higher bind-
ing affinity (Ki = 2.6, 3.4, 4.1 and 2.3 nM) for the 5-HT4
receptor than the corresponding phenoxy derivatives
40b–e. The compound containing propylene spacer
(41a) displayed a similar binding affinity for the dopa-
mine D2 receptor (Ki = 23 nM) to compounds 40a,b.
The replacement of the phenylthio group (41c) with a
benzylthio group (44) increased the binding affinity
(Ki = 1.7 nM). Compound 44 displayed moderate effect
on defecation (increases of 80% in number, 89% in dry
weight and 71% in wet weight of faecal deposits).
5.Experimental
5.1. Chemistry
Melting points were determined in open capillaries and
are uncorrected. Proton nuclear magnetic resonance
(1H NMR) spectra were recorded at 270 MHz on JEOL
JNM-EX270 spectrometer. Coupling constants are re-
ported in hertz (Hz) and chemical shifts are expressed
in ppm downfield from tetramethylsilane as an internal
standard. Mass spectra (MS) were obtained by a JMS-
O1SG spectrometer. Elementary analysis was performed
for C, H and N by our laboratory.
4.Conclusion
5.1.1. tert-Butyl (1-benzyl-4-hydroxypiperidin-4-yl)meth-
ylcarbamate (4). To a solution of 6-benzyl-1-oxa-6-aza-
spiro[2.5]octane (3) (39.1 g, 0.192 mmol) in ethanol
(170 mL) was added 28% NH4OH (117 mL). The reac-
We performed modification of the parent compound 2.
The introduction of a hydroxyl or methoxy group at
the 4-position of the piperidin-4-ylmethyl moiety led to

S. Sonda et al. / Bioorg. Med. Chem. 13 (2005) 3295–3308
3301
Table 3. Pharmacological data of benzamide derivatives
Cl
O
H
N
H2N
R3
(CH2)
n
N
O
Compd
R3
n
Binding affinitya
5-HT3 IC50 (nM)
Rate of increase in defecationb (%)
5-HT4 Ki (nM)
D2 IC50 (nM)
43
Number
Dry weight
NTd
Wet weight
40a
40b
40c
40d
40e
2
15
>1000
140
O
O
O
O
O
3
4.5
89
NTd
49.4
5
>1000
>1000
76
31
73
24
6.2
310c
>1000
>1000
73
66
14
6
4.1
>1000
18
24
43a
43b
43c
43d
43e
42.2
>1000
>1000
6
4
64
56
O
5
6
5
5
3.6
2.3
270c
>1000
59
81
110
NTd
93
68
87
O
O
O
O
>1000
>1000
>1000
NTd
93
10
>1000
NTd
Cl
5.2
93
77
43f
41a
41b
5
4.7
2.6
>1000
200c
>1000
23
37
32
65
49
43
34
O
3
S
43.4
>1000
>1000
64
86
91
S
41c
41d
5
6
5
4.1
2.3
1.7
>1000
>1000
340c
>1000
>1000
61
32
80
90
49
89
69
34
71
S
S
44
2
>1000
904
S
2.4>1000
>10090
76
a Each value is the mean from triplicate assay in a single experiment.
b Rate of increase in defecation induced in mice at oral dose of 1 mg/kg.
c Ki value.
d Not tested.
tion mixture was stirred at room temperature for 9 h
and evaporated to give crude aminoalcohol derivative.
This material was dissolved in CH2Cl2 (250 mL) and
di-tert-butyl dicarbonate (43.5 g, 0.199 mmol) was
added at 0 ꢁC. The mixture was stirred at room temper-
ature for 3 h, washed with aqueous K2CO3 and dried
over MgSO4. The solvent was removed, and the residue
was purified by column chromatography (CHCl3–
MeOH) to afford the title compound 4 as a pale yellow
1
solid (36.5 g, 59% from 3). H NMR (CDCl3): d 1.43

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S. Sonda et al. / Bioorg. Med. Chem. 13 (2005) 3295–3308
(9H, s), 1.54–1.68 (4H, m), 2.28–2.45 (3H, m), 2.55–2.70
(2H, m), 3.05–3.11 (2H, m), 3.52 (2H, s), 4.83–5.00 (1H,
m), 7.18–7.36 (5H, m); MS (EI) m/z 320 (M+).
7.70 (1H, m), 7.90–7.99 (2H, m), 8.12 (3H, br s), 10.60
(1H, br).
5.1.6. 6-(4-Aminomethyl-4-methoxypiperidin-1-yl)-1-phen-
ylhexan-1-one dihydrochloride (10). A mixture of 6
(2.30 g, 9.41 mmol), 8 (2.50 g, 9.80 mmol) and K2CO3
(2.60 g, 18.8 mmol) in DMF (40 mL) was stirred at
60 ꢁC for 2 h. The mixture was cooled to room temper-
ature and diluted with water. The product was extracted
with CHCl3. The combined extracts were washed with
brine, dried over MgSO4 and the solvent was evapo-
rated. The residue was purified by column chromatogra-
phy (CHCl3–MeOH) to afford tert-butyl [4-methoxy-
1-(6-oxo-6-phenylhexyl)piperidin-4-ylmethyl]carbamate
(3.4g, 85%) as an oil.
5.1.2. tert-Butyl (1-benzyl-4-methoxypiperidin-4-yl)meth-
ylcarbamate (5). To a suspension of 60% NaH (1.87 g,
77.9 mmol) in DMF (50 mL) was added a solution of
4 (15.0 g, 46.8 mmol) in DMF (100 mL) at 0 ꢁC, and
resulting solution was stirred at room temperature for
1 h. The solution was cooled to 0 ꢁC, and MeI (6.64g,
46.8 mmol) was added slowly. The reaction mixture
was stirred at room temperature for 5 h and diluted with
water. The product was extracted with ethyl acetate. The
combined extracts were washed with brine and dried
over MgSO4. The solvent was evaporated, and the
residue was purified by column chromatography (ethyl
acetate–methanol) to afford the title compound 5 as a
pale yellow solid (6.0 g, 38%). 1H NMR (CDCl3): d
1.43 (9H, s), 1.49–1.85 (4H, m), 2.20–2.40 (2H, m),
2.45–2.68 (2H, m), 3.16 (3H, s), 3.17–3.22 (2H, m),
3.52 (2H, s), 4.59–4.74 (1H, m), 7.19–7.39 (5H, m);
To the solution of this compound (3.35 g, 8.0 mmol) in
2-propanol (15 mL) was added 15% HCl/2-propanol
(15 mL). This mixture was stirred at 60 ꢁC for 1 h and
evaporated to give the title compound 10 as a colourless
1
solid (2.25 g, 72%); mp 185–188 ꢁC. H NMR (DMSO-
+
MS (EI) m/z 334(M ).
d6): d 1.22–2.16 (10H, m), 2.78–3.13 (10H, m), 3.14(3H,
s), 7.48–7.58 (2H, m), 7.59–7.70 (1H, m), 7.95–8.02 (2H,
m), 8.05–8.19 (2H, m), 8.75–9.04(m, 1H), 10.71–11.05
(1H, m).
5.1.3. tert-Butyl (4-methoxypiperidin-4-yl)methylcarba-
mate (6). A mixture of 5 (6.0 g, 17.9 mmol), 10% Pd–C
(2.0 g) and hydrazine hydrate (0.87 mL) in ethanol
(70 mL) was refluxed for 3 h. The mixture was cooled
to room temperature and the resulting precipitate was
filtered. The filtrate was evaporated to give 6 (3.8 g,
87%) as a colourless oil. 1H NMR (CDCl3): d 1.44
(9H, s), 1.46–1.84 (4H, m), 2.85–3.01 (4H, m), 3.18
(3H, s), 3.19–3.28 (1H, m), 3.29–3.56 (2H, m), 4.61–
4.80 (1H, m); MS (EI) m/z 244 (M+).
5.1.7. 4-Amino-5-chloro-N-[4-hydroxy-1-(6-oxo-6-phen-
ylhexyl)piperidin-4-ylmethyl]-2-methoxybenzamide (11).
To a solution of 9 (2.90 g, 7.69 mmol), 4-amino-5-
chloro-2-methoxybenzoic acid (1.55 g, 7.67 mmol),
Et3N (3.2 mL, 23.0 mmol) and HOBt (1.09 g,
7.12 mmol) in DMF (50 mL) was added EDC hydro-
chloride (1.55 g, 8.09 mmol) at 0 ꢁC and stirred at room
temperature for 6 h. The mixture was diluted with water
and extracted with CHCl3. The combined extracts were
washed with brine and dried over MgSO4. The solvent
was evaporated, and purified by column chromatogra-
phy (CHCl3–MeOH). Crystallization from ethyl acetate
gave the title compound 11 (2.38 g, 63%) as a colourless
5.1.4. tert-Butyl (4-hydroxypiperidin-4-yl)methylcarba-
mate (7). The same procedure, as described for synthesis
of 6, was followed using 4 (4.0 g, 12.5 mmol), 10% Pd–C
(1.0 g) and hydrazine hydrate (0.60 mL) in ethanol
(40 mL). The filtrate was concentrated to give 7 (2.8 g,
97%) as a colourless oil. 1H NMR (CDCl3): d 1.44
(9H, s), 1.50–1.62 (4H, m), 2.55–2.75 (2H, m), 2.80–
3.02 (4H, m), 3.09–3.19 (2H, m), 5.05–5.19 (1H, m);
MS (EI) m/z 242 (M+).
1
solid; mp 102–103 ꢁC. H NMR (CDCl3): d 1.29–1.84
(10H, m), 2.26–2.49 (4H, m), 2.54–2.69 (2H, t,
J = 7.6 Hz), 2.97 (2H, t, J = 7.6 Hz), 3.36 (1H, br s),
3.46 (2H, d, J = 6.0 Hz), 3.89 (3H, s), 4.47 (2H, br s),
6.30 (1H, s), 7.40–7.50 (2H, m), 7.51–7.60 (1H, m),
7.90–7.98 (2H, m), 7.99–8.06 (1H, m), 8.08 (1H, s);
MS (EI) m/z 487 (M+). Anal. Calcd for
C26H34ClN3O4Æ1/4H2O: C, 63.40; H, 7.06; N, 8.53.
Found: C, 63.32; H, 7.10; N, 8.20.
5.1.5. 6-(4-Aminomethyl-4-hydroxypiperidin-1-yl)-1-phen-
ylhexan-1-one dihydrochloride (9). A mixture of 7
(2.50 g, 10.2 mmol), 6-bromo-1-phenylhexan-1-one (8)
(2.90 g, 11.4mmol) and K 2CO3 (3.00 g, 21.7 mmol) in
DMF (40 mL) was stirred at 60 ꢁC for 2 h. The mixture
was cooled to room temperature and diluted with water.
The product was extracted with CHCl3. The combined
extracts were washed with brine and dried over MgSO4.
The solvent was removed, the residue was purified by
column chromatography (CHCl3–MeOH) to afford
5.1.8. 4-Amino-5-chloro-2-methoxy-N-[4-methoxy-1-(6-
oxo-6-phenylhexyl)piperidin-4-ylmethyl]benzamide (12).
To a solution of 10 (2.25 g, 5.75 mmol), 4-amino-5-
chloro-2-methoxybenzoic acid (1.16 g, 5.75 mmol),
Et3N (2.40 mL, 17.2 mmol) and HOBt (0.815 g,
5.32 mmol) in DMF (50 mL) was added EDC hydro-
chloride (1.16 g, 6.05 mmol) at 0 ꢁC. The reaction mix-
ture was stirred at room temperature for 4h and
poured into ice/water. The product was extracted with
CHCl3. The combined extracts were washed with brine,
dried over MgSO4 and the solvent was evaporated to
obtain a crude oil. This oil was purified by column chro-
matography (CHCl3–MeOH). Crystallization from
ethyl acetate/n-hexane gave the title compound 12
tert-butyl
[4-hydroxy-1-(6-oxo-6-phenyl)piperidine-4-
ylmethyl]carbamate as an oil. To the solution of this
material in 2-propanol (30 mL) was added 15% HCl/
2-propanol (10 mL) and stirred at 60 ꢁC for 3 h. After
cooling to room temperature, the resulting crystals were
filtrated to give the title compound 9 (2.92 g, 76%) as a
colourless solid. 1H NMR (DMSO-d6): d 1.20–1.47 (2H,
m), 1.55–2.05 (8H, m), 2.68–2.87 (2H, m), 2.94–3.18
(8H, m), 5.40–5.74 (1H, m), 7.45–7.57 (2H, m), 7.59–

S. Sonda et al. / Bioorg. Med. Chem. 13 (2005) 3295–3308
3303
(1.43 g, 50%) as a colourless solid; mp 91–93 ꢁC. 1H
NMR (CDCl3): d 1.30–1.91 (11H, m), 2.23–2.43 (4H,
m), 2.47–2.65 (2H, m), 2.97 (2H, t, J = 7.3 Hz), 3.22
(3H, s), 3.51 (2H, d, J = 5.3 Hz), 3.89 (3H, s), 4.40
(1H, br s), 6.29 (1H, br s), 7.40–7.61 (3H, m), 7.85–
8.01 (3H, m), 8.10 (1H, s); MS (EI) m/z 501 (M+). Anal.
Calcd for C27H36ClN3O4Æ0.15H2O: C, 64.25; H, 7.25; N,
8.32. Found: C, 64.26; H, 7.23; N, 8.36.
to room temperature and diluted with water. The prod-
uct was extracted with CHCl3. The combined extracts
were washed with brine, dried over MgSO4 and the sol-
vent was removed in vacuo. The resulting residue was
purified by column chromatography (CHCl3–MeOH)
to give 16 (3.4g, 92%) as a colourless oil. 1H NMR
(CDCl3): d 1.46 (9H, s), 1.50–1.85 (3H, m), 2.65–2.88
(2H, m), 2.99–3.16 (1H, m), 3.19–3.30 (1H, m), 3.40–
3.50 (1H, m), 4.05–4.64 (3H, m).
5.2. General procedure for the preparation of compounds
20, 27
5.2.4.
(3R*,4S*)-4-(Azidomethyl)-1-(tert-butoxycar-
bonyl)-3-methoxypiperidine (17). To a suspension of
60% NaH (580 mg, 24.1 mol) in DMF (10 mL) was
added 16 (3.4g, 13.3 mmol) in DMF (10 mL) and the
mixture was stirred at room temperature for 1 h. To this
solution was added MeI (3.72 mL, 59.8 mmol) and mix-
ture was stirred at room temperature for 17 h. The mix-
ture was diluted with water and extracted with CHCl3.
The combined organic extracts were washed with brine,
dried over MgSO4 and the solvent was evaporated. The
residue was purified by column chromatography eluted
with CHCl3 to afford the title compound 17 (1.50 g,
42%) as a colourless oil. 1H NMR (CDCl3): d 1.47
(9H, s), 1.51–1.62 (2H, m), 1.68–1.85 (1H, m), 2.51–
2.83 (2H, m), 3.08–3.23 (1H, m), 3.28–3.39 (1H, m),
3.40–3.55 (1H, m), 3.36 (3H, s), 3.89–4.59 (2H, m).
A suspension of benzamide (2.70 mmol), 8 (758 mg,
2.97 mmol) and K2CO3 (0.41 g, 2.97 mmol) in DMF
(20 mL) stirred at 60–70 ꢁC for 2–8 h. The resulting
solution was cooled, then treated with aqueous K2CO3
and extracted with CHCl3. The combined extracts were
evaporated and residue was purified by column chroma-
tography. Crystallization from ethyl acetate or ethanol
gave the title compound as a colourless solid.
5.2.1. (3R*,4S*)-1-Benzyl-4-(hydroxymethyl)piperidin-3-
ol (14). To a solution of 13 (21.0 g, 80.4mmol) in EtOH
(200 mL) was added NaBH4 (9.1 g, 0.24mol) at 0 ꢁC
and stirred at room temperature for 9 h. The mixture
was diluted with water and extracted with CHCl3. The
combined extracts were washed with brine, dried over
MgSO4 and the solvent was evaporated. The resulting
residue was purified by silica gel column chromatogra-
phy (CHCl3–MeOH) to afford the title compound 14
(10.6 g, 59%) as a colourless oil. (The configuration of
compound 14 was assessed by NOESY–NMR
experiment.)
5.2.5.
bonyl)-3-methoxypiperidine (18).
(3R*,4S*)-4-(Aminomethyl)-1-(tert-butoxycar-
mixture of 17
A
(1.50 g, 5.55 mmol) and 10% Pd(OH)2 (0.6 g) in MeOH
(50 mL) was stirred at room temperature for 2 h. The
mixture was filtered and filter pad was washed with
MeOH. The filtrate and washings were concentrated to
give 18 (1.20 g, 88%) as a colourless oil. 1H NMR
(DMSO-d6): d 1.39 (9H, s), 1.50–2.15 (3H, m), 2.55–
2.97 (4H, m), 2.98–3.10 (1H, m), 3.30 (3H, s), 3.45–
4.65 (4H, m).
5.2.2.
methyl)piperidin-3-ol (15). A mixture of 14 (7.1 g,
32.1 mmol), hydrazine monohydrate (3.12 mL,
(3R*,4S*)-1-(tert-Butoxycarbonyl)-4-(hydroxy-
64.3 mmol) and 10% Pd–C (6.0 g) in ethanol (120 mL)
was refluxed for 5 h. The reaction mixture was cooled
to room temperature and resulting solid was filtered.
The filtrate was concentrated to give 4-(hydroxy-
methyl)piperidin-3-ol as a colourless oil. To a solution
of this material in DMF (100 mL) was added di-tert-bu-
tyl dicarbonate (8.4g, 38.5 mmol), and the solution was
stirred at room temperature for 22 h. The solvent was
evaporated and the residue was purified by column chro-
matography (CHCl3–MeOH) to afford the title com-
5.2.6.
4-Amino-5-chloro-2-methoxy-N-[(3R*,4S*)-3-
methoxypiperidin-4-ylmethyl]benzamide dihydrochloride
(19). To a mixture of 4-amino-5-chloro-2-methoxyben-
zoic acid (0.99 g, 4.9 mmol), 18 (1.2 g, 4.9 mmol) and
HOBt (0.70 g, 5.2 mmol) in DMF (30 mL) was added
EDC hydrochloride (0.99 g, 5.2 mmol) at 0 ꢁC and the
mixture was stirred at room temperature for 4h. The
solution was diluted with water and extracted with
CHCl3. The combined extracts were washed with brine,
dried over MgSO4 and the solvent was evaporated in va-
cuo. The residue was purified by column chromatogra-
phy (CHCl3–MeOH) to afford pure oil. To the solution
of this compound in 2-propanol was added 15% HCl/2-
propanol. This mixture was stirred at 60 ꢁC for 1 h and
evaporated to give the title compound 19 (0.88 g, 45%
1
pound 15 (6.50 g, 88%) as a colourless oil. H NMR
(DMSO-d6): d 1.55 (9H, s), 1.57–1.85 (2H, m), 2.48–
2.95 (4H, m), 3.62–3.79 (3H, m), 4.01–4.27 (2H, m).
5.2.3.
(3R*,4S*)-4-(Azidomethyl)-1-(tert-butoxycar-
bonyl)piperidin-3-ol (16). To a solution of 15 (6.5 g,
28 mmol), Et3N (5.57 mL) in CH2Cl2 (100 mL) was
added methanesulfonyl chloride (3.54g, 30.9 mmol)
and the mixture was stirred at room temperature for
6 h. The mixture was diluted with water and extracted
with CHCl3. The combined extracts were washed with
brine, dried over MgSO4 and evaporated to obtain me-
syl compound as a crude oil. A mixture of mesyl com-
pound (4.44 g, 14.4 mmol), NaN3 (1.40 g, 21.5 mmol)
and NH4Cl (1.15 g, 21.5 mmol) in DMF (60 mL) was
stirred at 60 ꢁC for 6 h. The reaction mixture was cooled
1
from 18) as a colourless solid. H NMR (DMSO-d6): d
1.45–1.71 (2H, m), 1.86–2.05 (1H, m), 2.70–2.95 (2H,
m), 3.00–3.58 (6H, m), 3.35 (3H, m), 3.84(3H, s), 5.92
(2H, br s), 6.54(1H, s), 7.67 (1H, s), 7.90–8.05 (1H, m),
8.14–8.50 (1H, m), 9.48–9.75 (1H, m).
5.2.7. 4-Amino-5-chloro-2-methoxy-N-[3-methoxy-1-(6-
oxo-6-phenylhexyl)piperidin-4-yl-methyl]benzamide fum-
alate (20). Prepared from 19 and 8 according to the gen-
eral procedure. The resulting oil was transformed into

3304
S. Sonda et al. / Bioorg. Med. Chem. 13 (2005) 3295–3308
fumalate and recrystallized from ethanol/EtOAc to give
the title compound 20 (84%) as a colourless solid; mp
99–101 ꢁC. H NMR (DMSO-d6): d 1.21–1.72 (9H, m),
4.15 (2H, q, J = 7.2 Hz), 4.31–4.49 (2H, br s), 5.49–
5.62 (1H, m), 6.31 (1H, s), 7.66–7.81 (1H, m), 8.12
(1H, s); MS (EI) m/z 367 (M+).
1
2.28–2.45 (2H, m), 2.51–2.65 (2H, m), 2.92–3.10 (3H,
m), 3.17–3.35 (6H, m), 3.39–3.48 (1H, m), 3.83 (3H, s),
5.92 (2H, br s), 6.48 (1H, s), 7.46–7.58 (2H, m), 7.59–
7.71 (2H, m), 7.85–8.04(3H, m). Anal. Calcd for
C27H36ClN3O4ÆC4H4O4Æ3/2H2O: C, 57.71; H, 6.72; N,
6.51. Found: C, 57.78; H, 6.54; N, 6.41.
5.2.12. 4-Amino-5-chloro-2-methoxy-N-[(1,2,3,6-tetrahy-
dropyridin)-4-ylmethyl]benzamide (26). A mixture of 25
(1.1 g, 3.0 mmol), 85% KOH (0.84g, 13 mmol) in 2-pro-
panol (11 mL) was refluxed for 2.5 h. The mixture was
cooled to room temperature and diluted with water.
The product was extracted with CHCl3. The combined
extracts were washed with brine, dried over MgSO4
and the solvent was evaporated. The residue was puri-
fied by column chromatography (CHCl3–MeOH) to af-
ford 26 (0.40 g, 31%) as a yellow oil. 1H NMR (CDCl3):
d 1.60–1.85 (1H, m), 2.00–2.10 (2H, m), 2.97 (2H, t,
J = 6.0 Hz), 3.20–3.39 (2H, m), 3.90 (3H, s), 3.94–4.02
(2H, m), 4.39 (2H, br s), 5.57–5.68 (1H, m), 6.30 (1H,
s), 7.64–7.80 (1H, m), 8.12 (1H, s); MS (EI) m/z 295
(M+).
5.2.8. 1-Ethoxycarbonyl-4-hydroxy-(4-nitromethyl)piper-
idine (22). To a solution of Na (4.6 g, 0.20 mol) in etha-
nol (80 mL), was added 1-ethoxycarbonylpiperidine-4-
one (21) (34.2 g, 0.20 mol), nitromethane (16 g,
0.26 mol) and stirred at 50 ꢁC for 1 h. The mixture was
diluted with water and extracted with ethyl acetate
The combined extracts were washed with brine, dried
over MgSO4 and evaporated to obtain compound 22
as a crude oil. 1H NMR (CDCl3): d 1.26 (3H, t,
J = 7.3 Hz), 1.51–1.78 (4H, m), 2.95–3.32 (3H, m),
3.87–4.05 (2H, m), 4.13 (2H, q, J = 7.3 Hz), 4.43 (2H,
s); MS (EI) m/z 232 (M+).
5.2.13. 4-Amino-5-chloro-2-methoxy-N-[1-(6-oxo-6-phen-
ylhexyl)-(1,2,3,6-tetrahydropyridin)-4-yl-methyl]benzam-
ide (27). Compound 27 was prepared in 60% yield
according to the general procedure from 26 and 8: col-
5.2.9. 1-Ethoxycarbonyl-4-nitromethyl-1,2,3,6-tetrahy-
dropyridine (23). A mixture of 22 (10.0 g, 43.1 mmol)
and P2O5 (30.6 g, 108 mmol) in benzene (130 mL) was
refluxed for 2.5 h. The mixture was cooled to room tem-
perature and was filtered. The filtrate was washed with
brine, dried over MgSO4 and the solvent was evaporated
1
ourless solid; mp 112–114 ꢁC (ethyl acetate). H NMR
(CDCl3): d 1.34–1.49 (2H, m), 1.52–1.68 (2H, m),
1.70–1.85 (2H, m), 2.12–2.25 (2H, m), 2.38–2.50 (2H,
m), 2.55–2.69 (2H, m), 2.92–3.04(4H, m), 3.88 (3H, s),
3.95–4.05 (2H, m), 4.42 (2H, br s), 5.53–5.63 (1H, m),
6.30 (1H, s), 7.39–7.41 (2H, m), 7.51–7.61 (1H, m),
7.65–7.79 (1H, m), 7.89–8.01 (2H, m), 8.11 (1H, s);
MS (EI) m/z 469 (M+). Anal. Calcd for
C26H32ClN3O3Æ1/2H2O: C, 65.19; H, 6.94; N, 8.77.
Found C, 65.33; H, 6.85; N, 8.87.
1
to obtain 23 (4.27 g, 46%) as a colourless oil. H NMR
(CDCl3): d 1.27 (3H, t, J = 7.3 Hz), 2.15–2.30 (2H, m),
3.51–3.68 (2H, m), 3.99–4.10 (2H, m), 4.16 (2H, q,
J = 7.3 Hz), 4.89 (2H, br s), 5.85–6.00 (1H, m); MS
+
(EI) m/z 214(M ), 168 ([M-NO2]+).
5.2.10. 4-Aminomethyl-1-ethoxycarbonyl-1,2,3,6-tetrahy-
dropyridine (24). A mixture of 23 (2.0 g, 9.3 mmol), Fe
(2.5 g), NH4Cl (2.5 g, 47 mmol) and water (20 mL) in
toluene (100 mL) was refluxed for 6 h. The mixture
was cooled to room temperature and filtered. The fil-
trate was washed with aqueous NaOH, dried over
MgSO4 and the solvent was evaporated to obtain 24
(1.53 g, 89%) as a colourless oil. 1H NMR (CDCl3):
d 1.26 (3H, t, J = 7.0 Hz), 1.91–2.21 (2H, m), 3.14–3.26
(2H, m), 3.40–3.65 (2H, m), 3.81–4.00 (2H, m), 4.13
(2H, q, J = 7.0 Hz), 5.35–5.69 (1H, m), 7.00–7.32 (2H,
5.2.14. tert-Butyl [1-(6-oxo-6-phenylhexyl)piperidin-3-
ylmethyl]carbamate (29). A mixture of 28 (5.66 g,
26.4mmol), 6-bromo-1-phenylhexan-1-one ( 8) (6.10 g,
29.1 mmol) and K2CO3 (8.03 g, 58.1 mmol) in DMF
(40 mL) was stirred at 70 ꢁC for 10 h. The mixture was
cooled to room temperature and diluted with water.
The product was extracted with ethyl acetate. The com-
bined extracts were washed with brine and dried over
MgSO4. The solvent was removed and the residue was
purified by column chromatography (CHCl3–MeOH)
to afford title compound 29 (8.30 g, 81%) as a colourless
+
1
m); MS (EI) m/z 184(M ), 167 ([MÀNH3]+).
oil. H NMR (CDCl3): d 1.25–1.96 (11H, m), 1.44 (9H,
s), 2.21–2.41 (2H, m), 2.75–2.90 (2H, m), 2.94–3.10 (4H,
m), 4.54–4.74 (2H, m), 7.41–7.51 (2H, m), 7.52–7.62
(2H, m), 7.92–8.04(2H, m).
5.2.11.
4-Amino-5-chloro-2-methoxy-N-[1-ethoxycar-
bonyl-(1,2,3,6-tetrahydropyridin)-4-ylmethyl]benzamide
(25). To a solution of 24 (1.53 g, 8.3 mmol), 4-amino-5-
chloro-2-methoxybenzoic acid (1.67 g, 8.3 mmol) and
HOBt (1.18 g, 8.7 mmol) in DMF (50 mL) was added
EDC hydrochloride (1.67 g, 8.71 mmol) and the mixture
was stirred at room temperature for 14h. The mixture
was diluted with water and extracted with CHCl3. The
combined extracts were washed with aqueous K2CO3,
dried over MgSO4 and the solvent was evaporated to
obtain. The residue was purified by column chromato-
graphy (CHCl3–MeOH) to afford 25 (2.18 g, 71%) as a
colourless solid. 1H NMR (CDCl3): d 1.26 (3H, t,
J = 7.2 Hz), 1.55–1.68 (2H, m), 2.00–2.19 (2H, m),
3.47–3.61 (2H, m), 3.91 (3H, s), 3.85–4.05 (2H, m),
5.2.15. 6-(3-Aminomethylpiperidin-1-yl)-1-phenylhexan-
1-one 2hydrochloride (30). To a solution of 29 (8.29 g,
21.4mmol) in water (50 mL) was added hydrochloric
acid (50 mL) and mixture was stirred at room tempera-
ture for 4h. The mixture was evaporated to obtain
crude oil. Crystallization from ethanol gave the title
compound 30 (4.29 g, 62%) as a colourless solid. 1H
NMR (CDCl3/CD3OD): d 1.25–1.55 (3H, m), 1.70–
2.19 (8H, m), 2.41–2.68 (1H, m), 2.78–3.18 (9H, m),
2.41–2.68 (1H, m), 2.78–3.18 (1H, m), 3.45–3.60 (1H,
m), 3.65–3.79 (1H, m), 7.39–7.52 (2H, m), 7.57–7.65
(1H, m), 7.91–8.04(2H, m).

S. Sonda et al. / Bioorg. Med. Chem. 13 (2005) 3295–3308
3305
5.2.16. 4-Amino-5-chloro-2-methoxy-N-[1-(6-oxo-6-phen-
ylhexyl)piperidin-3-ylmethyl]benzamide (31). To a solu-
tion of 30 (4.25 g, 11.8 mmol), 4-amino-5-chloro-2-
methoxybenzoic acid (2.62 g, 13.0 mmol), Et3N (5.26 g,
52.0 mmol) and HOBt (1.76 g, 13.0 mmol) in DMF
(100 mL) was added EDC hydrochloride (2.49 g,
13.0 mmol) and stirred at room temperature for 22 h.
The mixture was diluted with water and extracted with
CHCl3. The combined extracts were washed with aque-
ous K2CO3, dried over MgSO4 and the solvent was
evaporated. Crystallization from ethyl acetate/n-hexane
gave the title compound 31 (3.46 g, 62%) as a colourless
solid; mp 73–76 ꢁC. 1H NMR (CDCl3): d 0.91–1.97
(13H, m), 2.25–2.41 (2H, m), 2.75–3.02 (4H, m), 3.22–
3.44 (2H, m), 3.88 (3H, s), 4.42 (2H, br s), 6.29 (1H,
s), 7.39–7.50 (2H, m), 7.51–7.59 (1H, m), 7.65–7.75
(1H, m), 7.90–7.99 (2H, m), 8.10 (1H, s). Anal. Calcd
for C26H34ClN3O3Æ1/2H2O: C, 64.92; H, 7.12; N, 8.74.
Found: C, 64.96; H, 7.38; N, 8.74.
solution was cooled, then treated with aqueous K2CO3
and extracted with CHCl3. The combined extracts were
evaporated and residue was purified by column chroma-
tography. Crystallization from ethyl acetate or ethanol
gave the title compound as a colourless solid.
5.3.1. 4-Amino-5-chloro-2-methoxy-N-[1-[5-(phenylsulfin-
yl)pentyl]piperidin-4-ylmethyl]benzamide (42). Com-
pound 42 was prepared according to the general
procedure from 39 and 36; amorphous. 1H NMR
(CDCl3): d 1.20–2.02 (13H, m), 2.24–2.39 (2H, m),
2.78 (2H, t, J = 7.9 Hz), 2.85–2.98 (2H, m), 3.32 (2H,
t, J = 6.3 Hz), 3.90 (3H, s), 4.38 (2H, br s), 6.29 (1H,
s), 7.45–7.58 (3H, m), 7.59–7.65 (2H, m), 7.69–8.22
(1H, m), 8.10 (1H, m); MS (FAB) m/z 492 (M+H+).
Anal. Calcd for C25H34ClN3O3SÆ1.1H2O: C, 58.66; H,
7.13; N, 8.22. Found: C, 58.37; H, 6.88; N, 8.07.
5.3.2.
4-Amino-5-chloro-2-methoxy-N-[1-(4-phenoxy-
ethyl)piperidin-4-ylmethyl]benzamide (40a). Compound
40a was prepared according to the general procedure
from 39 and 34a: colourless solid; mp 71–73 ꢁC (ethyl
acetate). 1H NMR (CDCl3): d 1.26–1.45 (2H, m),
1.51–1.97 (3H, m), 2.01–2.21 (2H, m), 2.74–2.85 (2H,
m), 2.94–3.05 (2H, m), 3.33 (2H, t, J = 6.3 Hz), 3.89
(3H, s), 4.10 (2H, t, J = 6.3 Hz), 4.40 (2H, br s), 6.29
(1H, s), 6.87–6.97 (3H, m), 7.23–7.31 (2H, m), 7.66–
7.84(1H, m), 8.11 (1H, s). Anal. Calcd for
C22H28ClN3O3Æ1/5H2O: C, 62.68; H, 6.79; N, 9.97.
Found: C, 62.59; H, 6.76; N, 9.98.
5.2.17. 4-Amino-5-chloro-N-[1-(6-hydroxy-6-phenylhex-
yl)piperidin-4-ylmethyl]-2-methoxybenzamide (32). To a
solution of 2 (1.0 g, 2.1 mmol) in ethanol (20 mL) was
added NaBH4 (0.16 g, 4.2 mmol) and mixture was stir-
red at room temperature for 1 h. The mixture was di-
luted with water and extracted with CHCl3. The
combined extracts were washed with brine, dried over
MgSO4 and the solvent was evaporated to obtain crude
oil. Crystallization from ethyl acetate gave the title com-
pound 32 (0.95 g, 95%) as a colourless solid; mp 157–
1
158 ꢁC. H NMR (DMSO-d6): d 1.20–1.95 (15H, m),
2.12–2.40 (3H, m), 2.81–2.99 (2H, m), 3.30 (2H, t,
J = 6.3 Hz), 3.88 (3H, s), 4.40 (2H, br s), 4.60–4.72
(1H, m), 6.28 (1H, s), 7.19–7.40 (5H, m), 7.66–7.82
(1H, m), 8.10 (1H, m). Anal. Calcd for
C26H36ClN3O3Æ1/3H2O: C, 65.13; H, 7.69; N, 8.76.
Found: C, 65.01; H, 7.60; N, 8.73.
5.3.3. 4-Amino-5-chloro-2-methoxy-N-[1-(4-phenoxyprop-
yl)piperidin-4-ylmethyl]benzamide (40b). Compound
40b was prepared in 46% yield according to the general
procedure from 39 and 34b: colourless solid; mp 132–
1
134 ꢁC (ethyl acetate). H NMR (CDCl3): d 1.26–1.46
(2H, m), 1.54–1.80 (3H, m), 1.90–2.08 (4H, m), 2.48–
2.59 (2H, m), 2.90–3.04(2H, m), 3.33 (2H, t,
J = 6.3 Hz), 3.89 (3H, s), 4.00 (2H, t, J = 6.3 Hz), 4.40
(2H, br s), 6.29 (1H, s), 6.85–6.96 (3H, m), 7.21–7.30
(2H, m), 7.70–7.84(1H, m), 8.10 (1H, s); MS (EI) m/z
431 (M+). Anal. Calcd for C23H30ClN3O3Æ2/5H2O: C,
62.90; H, 6.89; N, 9.57. Found: C, 62.98; H, 7.01; N,
9.53.
5.2.18. 4-Amino-5-chloro-N-[1-[6-(hydroxyimino)-6-phen-
ylhexyl]piperidin-4-ylmethyl]-2-methoxybenzamide (33).
To a solution of 2 (1.0 g, 2.1 mmol) and pyridine
(2 mL) in MeOH (10 mL) was added hydroxylamine
hydrochloride (0.15 g, 2.1 mmol), and refluxed for 6 h.
The mixture was cooled to room temperature, diluted
with aqueous K2CO3 and extracted with MeOH–
CHCl3. The combined extracts were washed with brine,
dried over MgSO4 and evaporated to obtain crude oil.
Crystallization from ethanol gave the title compound
33 (0.80 g, 76%) as a colourless solid; mp 150–151 ꢁC.
1H NMR (CDCl3): d 1.28–1.78 (10H, m), 1.80–2.03
(3H, m), 2.25–2.39 (2H, m), 2.70–2.83 (2H, m), 2.89–
3.05 (2H, m), 3.32 (2H, t, J = 5.9 Hz), 3.85 (3H, s),
4.40 (2H, br s), 6.26 (1H, s), 7.30–7.41 (3H, m), 7.55–
7.65 (2H, m), 7.71–7.84(1H, m), 8.10 (1H, s), 10.08
(1H, br s). Anal. Calcd for C26H35ClN4O3: C, 64.12;
H, 7.24; N, 11.50. Found: C, 63.90; H, 7.24; N, 11.44.
5.3.4. 4-Amino-5-chloro-2-methoxy-N-[1-(4-phenoxybut-
yl)piperidin-4-ylmethyl]benzamide (40c). Compound 40c
was prepared according to the general procedure from
39 and 34c: colourless solid; mp 71–73 ꢁC (ethyl ace-
tate). 1H NMR (DMSO-d6): d 1.22–2.02 (11H, m),
2.30–2.45 (2H, m), 2.85–3.03 (2H, m), 3.32 (2H, t,
J = 6.3 Hz), 3.88 (3H, s), 3.97 (2H, t, J = 6.0 Hz), 4.41
(2H, br s), 6.28 (1H, s), 6.83–6.99 (3H, m), 7.20–7.36
(2H, m), 7.66–7.82 (1H, m), 8.10 (1H, s). Anal. Calcd
for C24H32ClN3O3Æ1/4H2O: C, 63.99; H, 7.27; N, 9.33.
Found: C, 63.98; H, 7.31; N, 9.41.
5.3. General procedure for the preparation of compounds
40a–e, 41a–d, 42, 43a–f, 44
5.3.5. 4-Amino-5-chloro-2-methoxy-N-[1-(5-phenoxypen-
tyl)piperidin-4-ylmethyl]benzamide (40d). Compound 40d
was prepared according to the general procedure from
39 and 34d: colourless solid; mp 129–132 ꢁC (ethyl ace-
A suspension of 39 (1.00 g, 2.70 mmol), aralkyl chloride
(2.97 mmol) and K2CO3 (0.41 g, 2.97 mmol) in DMF
(20 mL) was stirred at 60–70 ꢁC for 2–8 h. The resulting
1
tate). H NMR (CDCl3): d 1.34–1.86 (11H, m), 1.94–
2.06 (2H, m), 2.36–2.45 (2H, m), 2.96–3.04 (2H, m),

3306
S. Sonda et al. / Bioorg. Med. Chem. 13 (2005) 3295–3308
3.33 (2H, t, J = 6.6 Hz), 3.89 (3H, s), 3.94(2H, t,
J = 6.6 Hz), 4.40 (2H, s), 6.29 (1H, s), 6.85–6.96 (3H,
m), 7.22–7.30 (2H, m), 7.71–7.79 (1H, m), 8.10 (1H, s);
MS (EI) m/z 459 (M+). Anal. Calcd for
C25H34ClN3O3Æ1/2H2O: C, 64.02; H, 7.52; N, 8.96.
Found: C, 63.83; H, 7.50; N, 8.99.
(DMSO-d6): d 0.78–2.00 (21H, m), 2.66–3.08 (4H, m),
3.11–3.22 (4H, m), 3.33 (2H, t, J = 6.0 Hz), 3.39–3.50
(2H, m), 3.83 (3H, s), 4.20–4.60 (2H, m), 6.52 (1H, s),
7.66 (1H, s), 7.91–8.09 (1H, m), 10.40–10.72 (1H, m).
Anal. Calcd for C26H42ClN3O3ÆHClÆ3/2H2O: C, 57.45;
H, 8.53; N, 7.73. Found: C, 57.46; H, 8.50; N, 7.80;
MS (EI) m/z 479 (M+).
5.3.6. 4-Amino-5-chloro-2-methoxy-N-[1-(6-phenoxyhex-
yl)piperidin-4-ylmethyl]benzamide (40e). Compound 40e
was prepared according to the general procedure from
39 and 34e: colourless solid; mp 113–115 ꢁC (ethyl ace-
5.3.11.
4-Amino-5-chloro-N-[1-[5-(4-chlorobenzyloxy)-
pentyl]piperidin-4-ylmethyl]-2-methoxybenzamide (43e).
Compound 43e was prepared according to the general
procedure from 39 and 37e: colourless solid; mp 131–
132 ꢁC (ethyl acetate). H NMR (DMSO-d6): d 1.01–
1.64(11H, m), 1.68–1.89 (2H, m), 2.11–2.29 (2H, m),
2.71–2.89 (2H, m), 3.06–3.19 (2H, m), 3.41 (2H, t,
J = 6.6 Hz), 3.82 (3H, s), 4.43 (2H, br s), 5.90 (2H, br
s), 6.50 (1H, s), 7.32 (2H, d, J = 8.5 Hz), 7.41 (2H, d,
J = 8.5 Hz), 7.66 (1H, s), 7.82–7.95 (1H, m). Anal. Calcd
for C26H35Cl2N3O3: C, 61.41; H, 6.94; N, 8.26. Found:
C, 61.20; H, 7.07; N, 8.27.
1
tate). H NMR (CDCl3): d 1.29–1.86 (13H, m), 1.95–
1
2.16 (2H, m), 2.45–2.51 (2H, m), 2.97–3.13 (2H, m),
3.33 (2H, t, J = 6.0 Hz), 3.89 (3H, s), 3.94(2H, t,
J = 6.6 Hz), 4.39 (2H, br s), 6.30 (1H, s), 6.81–6.98
(3H, m), 7.19–7.33 (2H, m), 7.69–7.82 (1H, m), 8.10
(1H, s). Anal. Calcd for C26H36ClN3O3ÆH2O: C, 63.47;
H, 7.78; N, 8.54. Found: C, 63.67; H, 7.47; N, 8.54.
5.3.7.
4-Amino-N-[1-(4-benzyloxybutyl)piperidin-4-yl-
methyl]-5-chloro-2-methoxybenzamide oxalate (43a). Pre-
pared from 39 and 37a according to the general
procedure. The resulting oil was transformed into oxa-
late and recrystallized from ethanol to give 43a: colour-
less solid; mp 175–176 ꢁC. 1H NMR (DMSO-d6): d
1.32–1.91 (9H, m), 2.69–2.89 (2H, m), 2.91–3.05 (2H,
m), 3.12–3.27 (2H, m), 3.31–3.50 (4H, m), 3.82 (3H, s),
4.45 (2H, s), 5.81–6.06 (2H, br), 6.49 (1H, s), 7.22–7.42
(5H, m), 7.66 (1H, s), 7.94–8.05 (1H, s). Anal. Calcd
for C25H34ClN3O3ÆC2H2O4Æ1/5H2O: C, 58.58; H, 6.63;
N, 7.59. Found: C, 58.58; H, 6.57; N, 7.61.
5.3.12. 4-Amino-5-chloro-2-methoxy-N-[1-[5-(naphtha-
len-2-ylmethoxy)pentyl]piperidin-4-ylmethyl]-benzamide
hydrochloride (43f). Prepared from 39 and 37f according
to the general procedure. The resulting oil was treated
with hydrochloric acid and recrystallized from ethanol
1
to give 43f: colourless solid; mp 161–165 ꢁC. H NMR
(DMSO-d6): d 1.25–2.03 (11H, m), 2.65–3.62 (8H, m),
2.11–2.29 (2H, m), 3.83 (3H, s), 4.62 (2H, s), 5.65–6.25
(2H, br s), 6.50 (1H, s), 7.38–7.55 (3H, m), 7.67 (1H,
s), 7.83 (1H, s), 7.90–7.95 (3H, m), 7.96–8.04(1H, s),
10.12–10.46 (1H, m). Anal. Calcd for C30H38ClN3O3ÆH-
ClÆH2O: C, 62.28; H, 7.14; N, 7.26. Found: C, 62.23; H,
7.27; N, 7.22.
5.3.8. 4-Amino-N-[1-(5-benzyloxypentyl)piperidin-4-yl-
methyl]-5-chloro-2-methoxybenzamide oxalate (43b). Pre-
pared from 39 and 37b according to the general
procedure. The resulting oil was transformed into oxa-
late and recrystallized from ethanol to give 43b: colour-
less solid; mp 185–188 ꢁC. 1H NMR (DMSO-d6): d
1.24–1.93 (10H, m), 2.70–3.03 (4H, m), 3.11–3.25 (2H,
m), 3.30–3.53 (5H, m), 3.83 (3H, s), 4.44 (2H, s), 5.72–
6.11 (2H, m), 6.49 (1H, s), 7.23–7.44 (5H, m), 7.67
(1H, s), 7.94–8.09 (1H, m). Anal. Calcd for
C26H36ClN3O3ÆC2H2O4Æ1/2H2O: C, 58.68; H, 6.86; N,
7.33. Found: C, 58.86; H, 6.78; N, 7.45.
5.3.13.
4-Amino-5-chloro-2-methoxy-N-[1-[3-(phenyl-
thio)propyl]piperidin-4-ylmethyl]benzamide (41a). Com-
pound 41a was prepared in 47% yield according to the
general procedure from 39 and 35a: colourless solid;
1
mp 146–148 ꢁC (ethanol). H NMR (CDCl3): d 1.19–
1.40 (2H, m), 1.48–2.00 (7H, m), 2.34–2.48 (2H, m),
2.78–2.99 (4H, m), 3.31 (2H, t, J = 6.3 Hz), 3.88 (3H,
s), 4.41 (2H, br s), 6.29 (1H, s), 7.09–7.39 (5H, m),
7.65–7.81 (1H, m), 8.10 (1H, s); MS (EI) m/z 447 (M+).
Anal. Calcd for C23H30ClN3O2SÆ1/10H2O: C, 61.41; H,
6.67; N, 9.34. Found: C, 61.31; H, 6.78; N, 9.42.
5.3.9.
4-Amino-N-[1-(6-benzyloxyhexyl)piperidin-4-yl-
methyl]-5-chloro-2-methoxybenzamide (43c). Compound
43c was prepared according to the general procedure
from 39 and 37c: colourless solid; mp 91–95 ꢁC (ethyl
acetate). 1H NMR (CDCl3): d 1.21–2.03 (15H, m),
2.21–2.36 (2H, m), 2.85–3.00 (2H, m), 3.32 (2H, t,
J = 6.3 Hz), 3.46 (2H, t, J = 6.3 Hz), 3.88 (3H, s), 4.40
(2H, s), 4.49 (2H, s), 6.28 (1H, s), 7.21–7.42 (5H, m),
7.68–7.83 (1H, s), 8.10 (1H, s); MS (EI) m/z 487 (M+).
Anal. Calcd for C27H38ClN3O3: C, 66.44; H, 7.85; N,
8.61. Found: C, 66.29; H, 7.96; N, 8.58.
5.3.14.
4-Amino-5-chloro-2-methoxy-N-[1-[4-(phenyl-
thio)butyl]piperidin-4-ylmethyl]benzamide hydrochloride
(41b). Prepared from 39 and 35b according to the general
procedure. The resulting oil was treated with hydrochlo-
ric acid and recrystallized from ethanol to give 41b as a
1
colourless solid; mp 102–105 ꢁC. H NMR (DMSO-d6):
d 1.36–1.92 (11H, m), 2.63–3.57 (8H, m), 3.83 (3H, s),
5.60–6.17 (2H, m), 6.48 (1H, s), 7.11–7.27 (1H, m),
7.28–7.41 (4H, m), 7.66 (1H, s), 7.90–8.07 (1H, m),
9.65–10.00 (1H, m); MS (EI) m/z 461 (M+). Anal. Calcd
for C24H32ClN3O2SÆHClÆ3/2H2O: C, 54.85; H, 6.90; N,
8.00. Found: C, 54.71; H, 6.66; N, 8.06.
5.3.10. 4-Amino-5-chloro-N-[1-[5-(cyclohexylmethoxy)-
pentyl]piperidin-4-ylmethyl]-2-methoxybenzamide hydro-
chloride (43d). Prepared from 39 and 37d according to
the general procedure. The resulting oil was treated with
hydrochloric acid and recrystallized from ethanol to give
43d as a colourless solid; mp 103–105 ꢁC. 1H NMR
5.3.15.
4-Amino-5-chloro-2-methoxy-N-[1-[5-(phenyl-
thio)pentyl]piperidin-4-ylmethyl]benzamide (41c). Com-
pound 41c was prepared according to the general

S. Sonda et al. / Bioorg. Med. Chem. 13 (2005) 3295–3308
3307
procedure from 39 and 35c: colourless solid; mp 143–
144 ꢁC (ethyl acetate). H NMR (CDCl3): d 1.21–2.00
(1 + L/Kd)), where L is the concentration of radioligand
and Kd is the dissociation constant of the radioligand.
1
(13H, m), 2.21–2.48 (2H, m), 2.82–3.00 (4H, m), 3.32
(2H, t, J = 6.3 Hz), 3.90 (3H, s), 4.36 (2H, br s), 6.29
(1H, s), 7.11–7.20 (1H, m), 7.22–7.38 (4H, m), 7.68–
7.80 (1H, m), 8.11 (1H, s); MS (EI) m/z (M+). Anal.
Calcd for C25H34ClN3O2SÆ1/4H2O: C, 62.48; H, 7.18;
N, 8.74. Found: C, 62.57; H, 7.16; N, 8.68.
5.5. 5-HT3 receptor-binding assay
[3H]Granisetron binding assay was performed according
to the method of Nelson and Thomas.22 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 1000g for 10 min. The supernatant
was centrifuged at 40,000g 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 40,000g for 15 min. The pellet
was washed and centrifuged (40,000g 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]Granisetron, 50 lL of
displacing drugs and 900 lL of tissue homogenate. Fol-
lowing 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% polyethyleneimine. Filters were washed immedi-
ately with 3 · 3 mL of ice-cold Tris–HCl buffer (50 mM,
pH 7.4). ICS 205930 (100 lm mol/L) was used for the
determination of non-specific binding.
5.3.16.
4-Amino-5-chloro-2-methoxy-N-[1-[6-(phenyl-
thio)hexyl]piperidin-4-ylmethyl]benzamide (41d). Com-
pound 41d was prepared according to the general
procedure from 39 and 35d: colourless solid; mp 68–
70 ꢁC (ethyl acetate). 1H NMR (DMSO-d6): d 1.21–
2.05 (15H, m), 2.21–2.36 (2H, m), 2.82–2.98 (4H, m),
3.32 (2H, t, J = 6.4 Hz), 3.89 (3H, s), 4.41 (2H, br s),
6.29 (1H, s), 7.10–7.19 (2H, m), 7.21–7.38 (3H, m),
7.68–7.80 (1H, m), 8.10 (1H, s); MS (EI) m/z (M+).
Anal. Calcd for C26H36ClN3O2SÆ3/4H2O: C, 62.01; H,
7.51; N, 8.34. Found: C, 62.12; H, 7.49; N, 8.44.
5.3.17. 4-Amino-N-[1-[5-(benzylthio)pentyl]piperidin-4-
ylmethyl]-5-chloro-2-methoxybenzamide oxalate (44).
Prepared from 39 and 38 according to the general proce-
dure. The resulting oil was transformed into oxalate and
recrystallized from ethanol to give 44 as a colourless so-
1
lid; mp 169–172 ꢁC. H NMR (DMSO-d6): d 1.21–1.92
(11H, m), 2.48–2.52 (2H, m), 2.71–3.01 (4H, m), 3.10–
3.26 (2H, m), 3.32–3.49 (2H, m), 3.71 (2H, s), 3.83
(3H, s), 5.74–6.04 (2H, m), 6.49 (1H, s), 7.18–7.49
(5H, m), 7.66 (1H, s), 7.98–8.05 (1H, m); MS (EI) m/z
(M+). Anal. Calcd for C26H36ClN3O2SÆC2H2O4: C,
57.61; H, 6.58; N, 7.19. Found: C, 57.60; H, 6.50; N,
7.29.
5.6. 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 (500g, 10 min,
0 ꢁC). The supernatant was centrifuged at 50,000g for
15 min. The pellet was suspended in 100 volumes of
ice-cold Tris–HCl buffer (50 mmol/L, pH 7.7) and recen-
trifuged (500g, 10 min, 0 ꢁC). The final pellet was resus-
pended in 150 volumes (50 mmol/L, pH 7.7) containing
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
5.4. 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 35,000g 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
HEPES buffer or a solution of the test agents, 50 lL
solution 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 filtration through a Whatmann GF/B filter
(presoaked in 0.01% v/v polyethyleneimine) followed
by washing with 1 · 4mL of ice-cold HEPES buffer.
Then the filter was placed in 3 mL of scintillator and
the radioactivity was determined by scintillation count-
ing in a Beckman model LS3801 scintillation counter.
Non-specific binding was defined in the presence of
unlabelled GR113808 to give a final concentration of
1 lmol/L. The IC50 value was determined by non-linear
regression of the displacement curve, and the Ki value
was calculated according to the formula (Ki = IC50/
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, fol-
lowed by 50 lL of [3H]Spiperone (40 Ci/mmol) at a final
concentration 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). Sul-
piride (100 lmol/L) was used for the determination of
non-specific binding. The radioactivity trapped on the
filters was measured by liquid scintillation spectrometry.
5.7. 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
30 min. The number, wet weight and dry weight of feces
excreted for 2 h from immediately after the administra-
tion were measured. Results are expressed as means
SEM and were compared by Dunnett method.

3308
S. Sonda et al. / Bioorg. Med. Chem. 13 (2005) 3295–3308
9. Berque-Bestel, I.; Soulier, J. L.; Giner, M.; Rivail, L.;
Acknowledgements
Langlois, M.; Sicsic, S. J. Med. Chem. 2003, 46, 2606–2620.
10. Suzuki, M.; Ohuchi, Y.; Asanuma, H.; Kaneko, T.;
Yokomori, S.; Ito, C.; Isobe, Y.; Muramatsu, M. Chem.
Pharm. Bull. 2000, 48, 2003–2008.
11. Suzuki, M.; Ohuchi, Y.; Asanuma, H.; Kaneko, T.;
Yokomori, S.; Ito, C.; Isobe, Y.; Muramatsu, M. Chem.
Pharm. Bull. 2001, 49, 29–39.
We thank Ms. F. Matsugaki, Mrs. M. Miyoshi and Mrs.
Y. Hattori for some of the biological results. We also
thank Dr. K. Adachi, Mr. K. Haga and Mr. K. Ito
for helpful discussion.
12. Tapia, I.; Alonso-Cires, L.; Lopez-Tudanca, P. L.; Mos-
quera, R.; Labeaga, L.; Innerarity, A.; Orjales, A. J. Med.
Chem. 1999, 42, 2870–2880.
References and notes
13. Harrington, R. A.; Hamilton, C. W.; Brogden, R. N.;
Linkewich, J. A.; Romankiewicz, J. A.; Heel, R. C. Drugs
1983, 25, 451.
14. Schuurkes, J. A.; Van Nueten, J. M.; Van Daele, P. G. H.;
Reyntjens, A. J.; Janssen, P. A. J. J. Pharmacol. Exp. Ther.
1985, 234, 775.
1. Alexander, S.; Peters, J.; Mead, A.; Lewis, S. TiPS
Receptor Ion Channel Nomencl. Suppl. 1998, 19, 4 6–
48.
2. Eglen, R. M.; Wong, E. F.; Dumuis, A.; Bockaert, J.
Trends Pharmacol. Sci. 1995, 16, 391.
3. Grider, J. R.; Foxx-Orenstein, A. E.; Jin, J. G. Gastroen-
terology 1998, 115, 370–380.
4. Harada, H.; Yamazaki, H.; Toyotomi, Y.; Tateishi, H.;
Mine, Y.; Yoshida, N.; Kato, S. Bioorg. Med. Chem. Lett.
2002, 12(6), 967–970.
15. Taniyama, K.; Nakayama, S.; Takeda, K.; Matsuyama,
S.; Shinakawa, J.; Sano, I.; Tanaka, C. J. Parmacol. Exp.
Ther. 1991, 258, 1098.
16. Katayama, K.; Morio, Y.; Haga, K.; Fukuda, T. Nippon
Yakurigaku Zasshi Folia 1995, 105, 461.
5. Lopez-Rodriguez, M. L.; Murcia, M.; Benhamu, B.; Viso,
A.; Campillo, M.; Pardo, L. J. Med. Chem. 2002, 45(22),
4806–4815.
17. Itoh, K.; Kanzaki, K.; Ikebe, T.; Kuroita, T.; Tomozane,
H.; Sonda, S.; Sato, N.; Haga, K.; Kawakita, T. Eur. J.
Med. Chem. 1999, 34, 977.
6. Schaus, J. M.; Thompson, D. C.; Bloomquist, W. E.;
Susemichel, A. D.; Calligaro, D. O.; Cohen, M. L. J. Med.
Chem. 1998, 41, 1943–1955.
18. Itoh, K.; Tomozane, H.; Hakira, H.; Sonda, S.; Asano,
K.; Fujimura, M.; Sato, N.; Haga, K.; Kawakita, T. Eur.
J. Med. Chem. 1999, 34, 1101.
7. Curtet, S.; Soulier, J. L.; Zahradnick, I.; Giner, M.;
Berque-Bestel, I.; Mialet, J.; Lezoualcꢀh, F.; Donzeau-
Gouge, P.; Sicsic, S.; Fischmeister, R.; Langlois, M. J.
Med. Chem. 2000, 43, 3761–3769.
8. Lopez-Rodriguez, M. L.; Benhamu, B.; Viso, A.;
Morcillo, M. J.; Murcia, M.; Orensanz, L.; Alfaro,
M. J.; Martin, M. I. Bioorg. Med. Chem. 1999, 7, 2271–
2281.
19. Itoh, K.; Sato, N.; Murozono, T. Drugs Future 1999, 24,
155.
20. Sonda, S.; Kawahara, T.; Murozono, T.; Sato, N.; Asano,
K.; Haga, K. Bioorg. Med. Chem. 2003, 11, 4225.
21. Sonda, S.; Katayama, K.; Kawahara, T.; Sato, N.; Asano,
K. Bioorg. Med. Chem. 2004, 12, 2737.
22. Nelson, D. R.; Thomas, D. R. Biochem. Pharmacol. 1989,
38, 1693.