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

Article abstract of DOI:10.1016/S0223-5234(99)80083-X

A number of new carboxamide derivatives were synthesized. The affinity of these compounds for the serotoninergic 5-HT₄ receptor was evaluated by use of radioligand-binding techniques. The agonistic activity was evaluated as the contractile effect of the ascending colon isolated from guinea-pigs. Among these compounds, 4-amino-5-chloro-2-methoxy-N-[l-[2- [(methylsulfonyl)amino]ethly]-4piperidinylmethyl]benzamide (24) showed a high affinity for the 5-HT₄ receptor (Ki = 9.6 nM). Compound 24 displayed a higher affinity for 5-HT₄ receptors than the other receptors, including, 5- HT₃ and dopamine D₂ receptors. In addition, compound 24 was confirmed to be a potent 5-HT₄ receptor agonist (ED⁵⁰ = 7.0 nM). An interaction model between compound 24 and 5-HT₄ receptor was proposed.

Full text of DOI:10.1016/S0223-5234(99)80083-X

Eur. J. Med. Chem. 34 (1999) 329−341
Elsevier, Paris
329
©
Original article
Synthesis and pharmacological evaluation of carboxamide derivatives
as selective serotoninergic 5-HT receptor agonists
4
Katsuhiko Itoh*, Koji Kanzaki, Tsuguo Ikebe, Takanobu Kuroita, Hideo Tomozane,
Shuji Sonda, Noriko Sato, Keiichiro Haga, Takeshi Kawakita
Research Laboratories, Yoshitomi Pharmaceutical Industries Ltd., 955 Koiwai, Yoshitomi-cho, Chikujo-gun, Fukuoka 871-8550, Japan
(Received 29 September 1998; revised 8 December 1998; accepted 9 December 1998)
Abstract – A number of new carboxamide derivatives were synthesized. The affinity of these compounds for the serotoninergic 5-HT4
receptor was evaluated by use of radioligand-binding techniques. The agonistic activity was evaluated as the contractile effect of the ascending
colon isolated from guinea-pigs. Among these compounds, 4-amino-5-chloro-2-methoxy-N-[1-[2-[(methylsulfonyl)amino]ethly]-4-
piperidinylmethyl]benzamide (24) showed a high affinity for the 5-HT receptor (Ki = 9.6 nM). Compound 24 displayed a higher affinity for
4
5
5
-HT receptors than the other receptors, including, 5-HT and dopamine D receptors. In addition, compound 24 was confirmed to be a potent
4
3
2
-HT receptor agonist (ED = 7.0 nM). An interaction model between compound 24 and 5-HT receptor was proposed. © Elsevier, Paris
4
50
4
5
-HT receptor / 5-HT receptor agonist / structure–activity relationship / carboxamide / receptor model construction
4
4
1
. Introduction
Cisapride and BIMU8 show obvious affinity for the
-HT receptor [9, 10]. While, ML 10302 is a selective
5
3
Serotonin (5-HT) is a neurotransmitter responsible for
5-HT receptor agonist, it shows limited pharmacological
4
a wide range of pharmacological reactions. Serotonergic
receptors are now classified into four broad subtypes such
as 5-HT , 5-HT , 5-HT , and 5-HT receptors, and clones
activities in vivo because of possible hydrolysis at its
benzoate moiety. Recently, some ketone derivatives were
reported to show a high affinity for the 5-HT receptor
1
2
3
4
4
for additional subtypes termed 5-HT , 5-HT , and 5-HT
and to be selective 5-HT partial agonists [11]. In addi-
5
6
7
4
receptors have been identified [1]. Among diverse 5-HT
tion, the original 5-TH receptor mapping by the active
4
receptors, we have investigated 5-HT function. Activa-
analogue approach by using several 5-HT4 receptor
antagonists and inactive ligands was reported [12].
4
tion of the 5-HT receptor mediates widespread effects in
4
the central and peripheral nervous systems [2]. Therefore,
One of our purposes is to supply a useful tool for
selective 5-HT receptor ligands would be useful for
4
elucidating the physiological function of the 5-HT re-
4
elucidating the physiological function of this receptor.
ceptor. In the course of our synthetic study on 5-HT3
receptor antagonists [13], it has been already clarified that
a benzoxazine-8-carboxamide derivative 1 (figure 2)
Agonists for the 5-HT receptor known to date are indole
4
derivatives such as 5-methoxytryptamine, benzamide de-
rivatives such as cisapride [3–5], benzimidazolone de-
rivatives such as BIMU8 [6], and benzoate derivatives
such as ML 10302 [7] (figure 1). However, nonselective
affinity or lability of these compounds prevents pure
pharmacological characterization of this receptor in vivo.
For example, 5-methoxytryptamine activates all 5-HT
receptor subtypes except the 5-HT3 receptor [8].
shows a weak affinity for the 5-HT receptor. This result
4
lead us to design selective 5-HT4 receptor agonists
having novel chemical structure and chemical stability.
Thus, compound 1, a leading compound for this study,
could further be optimized at four points: optimization of
(
1) the structure of the cycloamine moiety, (2) the
distance between the amide nitrogen in the carboxamide
moiety and the basic nitrogen in the cycloamine moiety,
(3) the variety and number of the substituents on the
*
Correspondence and reprints

3
30
BIMU 8
ML 10302
Figure 1. Chemical structures of 5-HT receptor agonists.
4
aromatic moiety, and (4) the structure of the side chain on
the cycloamine moiety. Herein, we describe the design,
synthesis, and pharmacological evaluation of carboxam-
ide derivatives as selective 5-HT4 receptor agonists.
(7–24), on which the synthetic data are listed in table I.
Among the acidic starting materials, 3,4-dihydro-1,4-
benzoxazine-8-carboxylic acid (2a) was prepared by our
method reported previously [14], and 2,3-dihydro-5-
chlorobenzofuran-7-carboxlic acid (2b) [15] and
5-chloro-2-methoxy-4-methylaminobenzoic acid (2h)
Interaction between putative 5-HT receptor models and
4
agonistic ligand is also discussed here.
[
16] were prepared by known procedures. As for the other
group of starting materials, pyrrolidine-3-methanol (3)
was condensed with phthalimide by the Mitsunobu reac-
tion [17] to afford a corresponding phthalimide deriva-
tive, which gave 1-benzyl-3-pyrrolidinylmethylamine
2. Chemistry
The general synthetic procedure used in this study is
illustrated in figure 3. By the mixed anhydrides method
method A) or the 1-ethyl-3-[3-(dimethylamino)propyl]
carbodiimide hydrochloride (WSC) method (method B),
carboxylic acids (2a–2h) were coupled with appropriate
amines (4a, 4b and 6a–6f) to afford target benzamides
(
4a) through a hydrazinolysis (figure 4). 1-Substituted-4-
(
piperidinylmethylamines (6a–6d) were prepared from
their corresponding isonipecotamides (5a–5d) by lithium
aluminium hydride reduction (figure 5). 2-(1-Benzyl-4-
piperidiyl)ethylamine (6f) and [1-(2-methansufonyl-
amino)ethyl]-4-piperidylmethylamine (6g) were prepared
using our procedures [18].
3. Pharmacological data and discussion
3.1. Structure–activity relationships
The affinity of compounds 7–24 for the 5-HT receptor
4
was determined as their ability to inhibit the binding of
3
[
H]GR113808 to the receptor. Their affinities for 5-HT3
and dopamine D receptors were similarly evaluated by
2
3
3
using [ H]granisetron and [ H]spiperone as radioligand,
respectively. Here, membrane preparations of the stria-
tum of guinea-pigs, the cerebral cortex of rats, and the
Figure 2. Chemical structure of compound 1.

3
31
Figure 3. The general synthetic procedures used in this study. (a) method A: i-BuOCOCl, Et N, AcOEt or method B: WSC, HOBt,
3
DMF.
striatum of rats were used for 5-HT , 5-HT , and D
2
benzamide (clebopride), and 5-HT are listed in table II,
where clebopride and 5-HT are reference compounds. As
concerns the cycloamine part, we chose not the bicy-
cloamine group (tropane) but the monocycloamine group
(pyrrolidine and piperidine) to obtain the compounds
having a higher affinity for 5-HT receptor than for 5-HT
4
3
receptor binding assays, respectively. Either agonistic or
antagonistic activity of these compounds was evaluated
as the contractile ability of the ascending colon of
guinea-pigs.
Pharmacological data on compounds 1 and 7–24,
4
3
4-amino-N-(1-benzyl-4-piperidinyl)-5-chloro-4-methoxy-
Figure 4. Condensation of 3 by the Mitsunobu reaction. (a)
Figure 5. Lithium aluminium hydride reduction of 5a–5d. (a)
PPh , phthalimide, DEAD, THF; (b) NH NH H O, EtOH.
LiA1H , THF.
3
2
2
2
4

3
32
Table I. Physicochemical properties for compounds 7–24.
Compound Ar
n
0
X
R
Method
A
Mp (°C)
94–95
Formula
a
a
a
a
a
7
Bn
C H CIN O
3 2
21
24
f
8
9
0
1
1
2
Bn
Bn
Bn
Bn
A
A
A
A
232–233
186–187
97–98
C H CIN O C H O
22 26 3 2 4 4
4
g
C H CIN O C H O
22
26
3
2
2 4
4
1
0
C H CIN O
23 28 3
2
11
122–124
C H CIN O C H O 1/2EtOH
2
4
30
3
2
2
2
4
g
1
/2H O
2
a
a
a
a
g
g
1
1
1
1
2
3
4
5
1
1
1
2
Bn
Bn
Bn
Bn
B
B
B
B
176–178/DEC
161–163
C H CIN O C H O
22 25 2 2 2 2
4
C H CIN O C H O
2
0
24
3
2
2 2
4
155–156
C H CIN O
21 26 3
2
g
135–137
C H CIN O C H O
22 28 3 2 2 2
4
a
g
1
1
6
7
1
1
Bn
Bn
B
B
169–170
90–93
C H N O C H O
21 26 2 2 2 2 4
a
g
g
C H CIN O C H O 1/4H O
21
25
2
2
2
2
4
2
a
1
8
1
Bn
B
110–112/DEC
C H CIN O C H O 4/5H O
20 24 3 2 2 2 4 2

3
33
Table I. (Continued)
Compound Ar
n
1
X
R
Method
B
Mp (°C)
160–161
Formula
a
a
g
1
9
Bn
C H N O C H O
1 27 3 2 2 2 4
2
f
2
0
1
Bn
B
148–149
C H CIN O C H O
22 28 3 2 4 4 4
b
2
2
2
2
1
2
3
4
1
1
1
1
Et
B
B
B
B
115–116
C H N O
16 24 3 2
c
g
Bu
179–181
C H CIN O C H O
18
28
3
2
2
2
4
d
Hex
113–115
C H CIN O
20 32 3
2
e
g
MSAE
177–178/DEC
C H CIN O S2C H O
17 27 4 4 2 2 4
a
b
c
d
e
f
g
Bn, benzyl; Et, ethyl; Bu, butyl; Hex, hexyl; MSAE, (methylsulfonylamino)ethyl; fumalate; oxalate.
receptor [7]. As a result, monocycloamine derivatives (7
and 8) in benzoxazine series showed a low affinity for
Compound 10 was confirmed to be a 5-HT receptor
4
antagonist (pKB = 7.9). Compound 12, a benzofuran
analogue of benzoxazine derivative 10, was also con-
firmed to behave as a 5-HT receptor antagonist (pK =
5
-HT receptor. Furthermore, piperidine derivatives (10
3
and 14) showed a higher affinity for 5-HT receptor than
4
4
B
pyrrolidine derivatives (9 and 13).
8.2) although they showed a high affinity for the receptor.
However, compounds 13 and 14, both of which have a
classic 4-amino-5-chloro-2-methoxybenzamide skeleton,
Next, we investigated the distance requirements be-
tween the carbonyl group in the amide bond and the basic
nitrogen in the cyclic amine of pyrrolidine and piperidine
derivatives. Here, two benzoxazinecarboxamide deriva-
tives (7 and 8) and clebopride, where the hydrogen atom
on their carboxamide nitrogen is replaced by a 1-benzyl-
were confirmed to be a 5-HT receptor agonist. We next
4
focused on the effect of each substituent (4-amino,
5-chloro and 2-methoxy group) on the phenyl ring in
benzamide derivatives. A simple 2-methoxybenzamide
derivative 16 showed a low affinity to the receptor.
Although 5-chloro-2-methoxybenzamide derivative 17
3
-pyrrolidinyl or 1-benzyl-4-piperidinyl group, showed
affinities for 5-HT , 5-HT and D receptors. On the other
3
4
2
hand, compounds 9, 10 and 12–14, where the hydrogen
atom on their carboxamide nitrogen is replaced by a
exhibited a 5-HT receptor affinity similar to compound
4
14, the former never behaved as a agonist for the receptor.
On the other hand, such an affinity was practically
undetectable for the 4-amino-3-chlorobenzamide deriva-
tive 18, a 2-desmethoxy analogue of compound 14. The
moderate affinity of compound 16 and the low affinity of
compound 18 indicate that the 2-methoxy group is
1
-benzyl-3-pyrrolidinylmethyl or 1-benzyl-4-piperi-
dinylmethyl group, showed a higher affinity for the
-HT receptor than for the 5-HT receptor, and showed
5
4
3
a low affinity for the D receptor. Compounds 11 and 15,
2
where the hydrogen atom on their carboxamide nitrogen
is replaced by a 2-(1-benzyl-4-piperidinyl)ethyl group,
exhibited only low affinities for these kinds of receptors.
The results indicate that the optimum distance for selec-
essential for binding to the 5-HT receptor. Compound 17
4
showed a similar affinity to compound 14. This result
suggests that the 5-chloro group helped ligands to bind to
the receptor complementarily. The 4-Amino-2-
tive affinity for the 5-HT receptor was one carbon [9].
4

3
34
Table II. Pharmacological data of compound 1, compounds 7–24 and two reference compounds.
a
Binding affinities, Ki (nM)
Contractile effects in guinea pigs ascending colon
d
e
Compound
5-HT4
5-HT3
D2
EC 50 (nM)
Maximal response (%)
b
c
1
7
8
9
280
160
530
19
0.93
530
1.7
20
6.7
> 1000
66
8.1
> 1000
110
100
28
8.8
6.7
0.27
10
21
95
120
> 1000
340
160
290
> 1000
12
67
NT
–
–
–
c
NT
c
NT
b
b
b
> 1000
> 1000
> 1000
NT
> 10000
> 10000
NT
> 10000
47
10
b
1
1
–
12
13
14
15
16
17
18
19
20
21
22
23
24
b
b
b
> 1000
> 1000
> 1000
880
21
18
–
20
NT
b
b
c
470
b
b
b
b
b
b
b
b
b
c
> 1000
> 1000
> 1000
> 1000
> 1000
> 1000
> 1000
> 1000
> 1000
230
NT
–
c
NT
NT
NT
NT
NT
NT
NT
> 10000
c
c
NT
–
c
150
900
61
20
8.9
16
15
20
27
–
c
c
c
13
c
130
7.0
b
9.6
92
> 1000
c
clebopride
63
NT
5-HT
130
35
21
a
b
c
d
Each value is the mean from triplicate assays in a single experiment; IC value; not tested; EC values were determined by linear
50
50
e
regression; % of contraction to methacoline at 30 µM.
methoxybenzamide derivative 19, a 5-deschloro analogue
potent agonist at the 5-HT receptor than other com-
4
of compound 14, showed a potent 5-HT receptor ago-
pounds (14, 21–23). Therefore, compound 24 was as-
sayed for its binding ability for several kinds of receptors.
The results were as follows (IC50 value (nM), ligand):
4
nistic activity, but showed only a moderate affinity for the
receptor. These results indicate that the 4-amino group
3
3
played an important role in 5-HT receptor agonistic
α > 1 000, [ H]prazosin; 5-HT > 1 000, [ H]8-OH-
4
1 1A
3
activity. In addition, this consideration is supported by the
facts, which 2,3-dihydro-4-amino-5-chlorobenzofuran
DPAT; 5-HT > 1 000, [ H]ketanserin; MACh > 1 000,
[ H]QNB. Thus Compound 24 represented a selective
2
3
carboxamides showed 5-HT receptor agonistic activ-
5-HT receptor agonist.
4
4
ity [19], while 4-desamino-2,3-dihydro-5-chlorobenzo-
furan carboxamide 12 showed low 5-HT receptor ago-
3.2. Computational modelling study
4
nistic
activity.
Although
5-chloro-2-methoxy-4-
methylaminobenzamide derivative 20, an N-methyl
analogue of compound 14, also exhibited a moderate
3.2.1. 5-HT receptor model construction
4
A 5-HT receptor model was constructed in order to
4
affinity, it showed low 5-HT receptor agonistic activity.
obtain rational structure–activity relationships and to
clarify the interaction between ligands and the receptor.
4
Consequently, all the three substituents on the phenyl ring
are essential for both affinity and agonistic activity.
Finally, we examined the influence of the side chains of
N-substituted piperidinylmethyl group on the interaction
The 5-HT receptor is a member of the G-protein coupled
4
receptor (GPCR). Generally, GPCRs are assumed to
consist of seven transmembrane helices and eight intra
and extracellular loops and their models are constructed
from coordinates of bacteriorhodopsin [20–27]. The cred-
ibility of the model is evaluated by the consistency with
structure–activity relationships and mutational data. In
with the 5-HT receptor. Ethyl derivative 21 showed a
4
moderate affinity for the 5-HT receptor. Butyl derivative
4
2
2 was almost equal to compound 14 in the affinity, hexyl
derivative 23 was equal to compound 14 in the affinity,
but with reduced 5-HT receptor agonistic activity. When
the substituent was a polar methylsulfonylamino group,
agonistic activity increased. Compound 24 was a more
this study, affinity for the guinea-pig 5-HT receptor was
measured. Therefore the receptor model should be con-
structed from the sequence of guinea-pig. But the guinea-
4
4

3
35
pig sequence has not been reported yet. Although se-
quences of receptors are not identical among animals,
amino acids involved in ligand recognition are most
likely to be conserved. Therefore, in the agonist recogni-
tion site, the conserved amino acids between human and
assumed to interact with certain residues in the 5-HT4
receptor. From some mutational data, Asp308, Thr312,
Ser507, Ala510, and Phe619 are defined to be important
for agonist binding and receptor activation. Asp308 is
supposed to form a salt bridge with the ammonium
nitrogen of the ligand. Thr312 and Ser507 are supposed
to form a hydrogen bond. Ala510 is supposed to perform
a hydrophobic interaction with N-alkyl substituents in
some tryptamine derivatives. Phe619 is assumed to rec-
ognise an aromatic ring of the ligand. From these
structure–activity relationships and mutational data, an
rat 5-HT receptors should be conserved in guinea-pig
4
5
-HT receptors. The 5-HT receptor model was con-
4 4
structed with the residues in the human 5-HT receptor
4
and the conserved residues were used for speculation of
receptor-ligand interaction. The seven transmembrane
regions of the 5-HT receptor was determined from a
4
hydropathy plot [28]. To determine sequence alignment
interaction model between the 5-HT receptor and com-
4
of bacteriorhodopsin and the 5-HT receptor, over 70
4
pound 24 was proposed : (figure 6) the ammonium nitro-
gen in the piperidine ring forms a salt bridge with
Asp308. The 2-methoxy group forms an intramolecular
hydrogen bond with the NH group in the benzamide and
constrains the conformation of the benzamide group. The
GPCRs such as dopamine, adrenergic, muscarine recep-
tor, and rhodopsin were included in the study.
3
.2.2. Docking of compound 24 into the 5-HT receptor
4
model
5-chloro group performs a hydrophobic interaction with
Mutational studies for the 5-HT receptor have not
4
Ala510. The 4-amino group forms a hydrogen bond with
Ser507. The methylsulfonylamino group forms a hydro-
gen bond with Thr312. Furthermore, aromatic residues
such as Phe515, Phe612, Trp616, Phe619, and Phe620 are
presumed to locate near the ligand and they are supposed
to form an aromatic binding pocket or stabilize the
positive charge of the cationic amine. As for Trp409,
there may be a hydrogen bond to the CO group in the
benzamide.
been reported yet. Therefore, the structure–activity rela-
tionships for this series of compounds and mutational
data for the 5-HT1A and 5-HT2A receptors [29–33] were
used for the docking study of compound 24 into the
receptor model. From the structure–activity relationships,
the 2-methoxy, 5-chloro, and 4-amino groups on the
phenyl ring, and the methylsulfonylamino group in the
piperidinomethyl moiety were defined to be essential for
both affinity and agonistic activity. These groups are
Figure 6. Stereoview of the interaction of compound 24 with the 5-HT receptor.
4

336
4
. Conclusion
Hz), 3.55 (1H, d, J = 11 Hz), 3.70 (1H, d, J = 11 Hz), 4.35
2H, t, J = 5.6 Hz), 4.44–4.80 (1H, m), 6.62 (1H, d, J =
(
We describe the synthesis and biological evaluation
3.0 Hz), 7.20–7.30 (5H, m), 7.39 (1H, d, J = 3.0 Hz), 7.95
+
of a series of carboxamides as selective 5-HT receptor
agonists. Based on our results, we propose that 4-amino-
(1H, br-s); MS m/z: 385 (M ).
4
5
-chloro-2-methoxy-N-[(4-piperidinyl)methyl]benzamide
5
.1.1.2.
N-(1-Benzyl-4-piperidinyl)-6-chloro-3,4-dihy-
with a polar group at the 1-position on piperidine
moieties are necessary for the selective agonistic activity
for 5-HT receptors. Compound 24 represents a selective
dro-4-methyl-2H-1,4-benzoxazine-8-carboxamide fuma-
late 8
4
Similarly to 7, 8 was prepared starting from 2a (3.4 g,
5
-HT agonist and would be a useful tool for probing the
4
1
5 mmol), triethylamine (3.3 g, 33 mmol), isobutyl chlo-
roformate (2.3 g, 17 mmol), ethyl acetate (70 mL), and 6e
3.0 g, 15 mmol). The resulting oil was transformed into
fumalate and recrystallized from ethanol to give 8 (3.4 g,
5
-HT receptor function in vivo. Detailed analyses of
4
structure–activity relationships for the side chain moieties
will be reported in due course.
(
1
5
5%); H-NMR (DMSO-d ) δ: 1.54 (2H, dd, J = 12, 24
6
5
. Experimental protocols
Hz), 1.82 (2H, d, J = 12 Hz), 2.20–2.39 (2H, t, J = 12 Hz),
.80 (2H, d, J = 12 Hz), 2.87 (3H, s), 3.29 (2H, t, J = 4.0
2
5
.1. Chemistry
Hz), 3.50 (2H, s), 3.70–3.83 (1H, m), 4.29 (2H, t, J = 4.0
Hz), 6.60 (2H, s), 6.75 (1H, d, J = 3.0 Hz), 6.82 (1H, d,
J = 3.0 Hz), 7.22–7.38 (5H, m), 7.98 (1H, d, J = 7.2 Hz);
All melting points were measured in open capillaries
and uncorrected. Proton nuclear magnetic resonance
+
MS m/z: 399 (M ).
1
(
H-NMR) spectra were recorded on Jeol JNM-EX270
spectrometers and chemical shifts are expressed in ppm
with tetramethylsilane (TMS) as an internal standard.
Signal multiplicities are represented by s (singlet), d
5
3
.1.1.3. N-[(1-Benzyl-3-pyrrolidinyl)methyl]-6-chloro-
,4-dihydro-4-methyl-2H-1,4-benzoxazine-8-carboxamide
oxalate
9
(doublet), t (triplet), q (quartet), br-s (broad singlet) and
m (multiplet). Mass spectra (MS) were taken on Jeol
JMS-O1SG spectrometers. Elementary analysis was per-
formed for C, H and N, and were within 0.4% of the
calculated values. Silica-gel plates (Merck F254) and
silica gel 60 (Merck, 70–230 mesh) were used for
analytical and preparative column chromatography, re-
spectively.
Similarly to 7, 9 was prepared starting from 2a (1.8 g,
7.9 mmol), triethylamine (1.6 g, 16 mmol), isobutyl chlo-
roformate (1.1 g, 7.9 mmol), ethyl acetate (30 mL), and
4a (1.5 g, 7.9 mmol). The resulting oil was transformed
into oxalate and recrystallized from methanol to give 9
1
(2.4 g, 62%); H-NMR (DMSO-d ) δ: 1.60–2.22 (2H,
6
m), 2.64–2.75 (2H, m), 2.80–2.92 (2H, m), 2.90 (3H, s),
3
.17 (2H, d, J = 5.9 Hz), 3.55 (2H, s), 3.78–3.95 (1H, m),
.1.1. General procedure for the preparation of 7–11
4.21 (2H, t, J = 4.0 Hz), 4.29 (2H, t, J = 4.0 Hz), 6.74
(1H, d, J = 3.0 Hz), 6.78 (1H, d, J = 3.0 Hz), 7.25–7.60
+
(5H, m), 8.28 (1H, t, J = 6.0 Hz); MS m/z: 399 (M ).
N-(1-Benzyl-3-pyrrolidinyl)-6-chloro-3,4-
dihydro-4-methyl-2H-1,4-benzoxazine-8-carboxamide 7
Isobutyl chloroformate (1.2 g, 9.7 mmol) was added to
a mixture of 2a (2.0 g, 8.8 mmol), triethylamine (2.0 g,
5
3
1
.1.1.4.
N-[(1-Benzyl-4-piperidinyl)methyl]-6-chloro-
,4-dihydro-4-methyl-2H-1,4-benzoxazine-8-carboxamide
0
Similarly to 7, 10 was prepared starting from 2a (1.2 g,
1
9 mol), and ethyl acetate (40 mL) at –10 °C. The mix-
ture was stirred below –5 °C for 30 min and a solution of
b (1.5 g, 8.8 mmol) in ethyl acetate (10 mL) was added
4
5.3 mmol), triethylamine(0.80 g, 8.0 mmol), isobutyl
chloroformate (0.67 g, 5.3 mmol), ethyl acetate (25 mL),
and 6a (1.0 g, 5.3 mmol). The resulting solid was recrys-
with stirring at –10 °C. Stirring was continued at the same
temperature for 30 min. The resulting mixture was added
to water and extracted with ethyl acetate. The extract was
washed with brine, and dried over anhydrous magnesium
sulfate. After evaporation in vacuo, the residue was
tallized from ethyl acetate to give 10 (1.4 g, 64%);
1
H-NMR (CDCl ) δ: 1.23–1.32 (2H, m), 1.55 (1H, br-s),
3
1.73 (2H, d, J = 11 Hz), 2.00–2.42 (2H, m), 2.80 (2H, d,
J = 11 Hz), 2.90 (3H, s), 3.30 (2H, t, J = 5.6 Hz), 3.35
(2H, d, J = 11 Hz), 3.50 (2H, s), 4.35 (2H, t, J = 5.6 Hz),
6.64 (1H, d, J = 3.0 Hz), 7.20–7.30 (5H, m), 7.42 (1H, d,
chromatographed on silica gel (CHCl :MeOH = 10:1).
3
Recrystallized from ethyl acetate/diisopropylether to give
1
7
(0.50 g, 15%); H-NMR (CDCl ) δ: 1.70–1.82 (2H, m),
3
+
2.64 (2H, d, J = 11 Hz), 2.90 (3H, s), 3.35 (2H, t, J = 5.6
J = 3.0 Hz), 7.69 (1H, br-s); MS m/z: 413 (M ).

3
37
.1.1.5.
N-[2-(1-Benzyl-4-piperidinyl)ethyl]-6-chloro-
5.1.2.3. 4-Amino-N-[(1-benzyl-4-piperidinyl)methyl]-5-
chloro-2-methoxybenzamide 14
oxalate 11
Similarly to 13, 14 was prepared starting from 2c
(1.0 g, 5.0 mmol), 6a (1.0 g, 5.0 mmol), HOBt (0.86 g,
6.4 mmol), dimethylformamide (30 mL), and WSC
(1.1 g, 5.7 mmol). The resulting solid was recrystallized
Similarly to 7, 11 was prepared starting from 2a (1.0 g,
4
.6 mmol), triethylamine (1.0 g, 10 mmol), isobutyl chlo-
roformate (0.70 g, 5.1 mmol), ethyl acetate (50 mL), and
f (1.0 g, 4.6 mmol). The resulting oil was transformed
into oxalate and recrystallized from ethanol to give 11
1
6
from ethyl acetate to give 14 (1.4 g, 72%); H-NMR
(CDCl ) δ: 1.35–1.55 (5H, m), 2.00 (2H, d, J = 12 Hz),
3
1
(0.56 g, 24%); H-NMR (DMSO-d ) δ: 1.32–1.60 (4H,
2.90 (2H, d, J = 12 Hz), 3.30 (2H, t, J = 5.6 Hz), 3.50 (2H,
6
m), 1.84 (2H, d, J = 12 Hz), 2.80 (2H, d, J = 12 Hz), 2.84
2H, t, J = 11 Hz), 2.87 (3H, s), 3.29 (5H, m), 4.17 (2H,
s), 4.28 (2H, t, J = 4.6 Hz), 6.73 (1H, d, J = 2.7 Hz), 6.82
1H, d, J = 2.7 Hz), 7.41–7.49 (5H, m), 8.12 (1H, t, J =
s), 3.92 (3H, s), 4.38 (2H, br-s), 7.22–7.38 (5H, m), 7.70
+
(
(1H, br-s), 8.10 (1H, s); MS m/z: 387 (M ).
5
.1.2.4. 4-Amino-N-[2-(1-benzyl-4-piperidinyl)ethyl]-5-
(
5
+
chloro-2-methoxybenzamide oxalate 15
.2 Hz); MS m/z: 427 (M ).
Similarly to 13, 15 was prepared starting from 2c
(
0
(
.1.2. General procedure for the preparation of 12–24
5
.1.2.1. 4-Amino-N-[(1-benzyl-3-pyrrolidinyl)methyl]-5-
1
chloro-2-methoxybenzamide oxalate 13
A mixture of 2c (1.6 g, 7.9 mmol), 4a (1.5 g,
7
8
.9 mmol), 1-hydroxybenzotriazole (HOBt) (1.1 g,
.1 mmol), and dimethylformamide (50 mL) was stirred
under ice-cooling for 1 h and then 1-ethyl-3-[3-
(
dimethylamino)propyl] carbodiimide hydrochloride
WSC) (2.3 g, 8.4 mmol) was added at the same tempera-
5
.1.2.5. N-[(1-Benzyl-4-piperidinyl)methyl]-2-methoxy-
(
benzamide oxalate 16
ture. Stirring was continued overnight at room tempera-
ture. After evaporation, 5% aqueous sodium bicarbonate
was added to the residue and extracted with ethyl acetate.
The extraction was washed with brine, and was dried over
anhydrous magnesium sulfate. After evaporation in
vacuo, the residue was dissolved in ethanol, an alcoholic
solution of oxalic acid (2.5 equiv.) was added. The
Similarly to 13, 16 was prepared starting from 2d
(
5
(
0.74 g, 4.9 mmol), 6a (1.0 g, 4.9 mmol), HOBt (0.73 g,
.4 mmol), dimethylformamide (30 mL), and WSC
1.0 g, 5.4 mmol). The resulting oil was transformed into
oxalate and recrystallized from ethanol to give 16 (1.5 g,
1
7
1%); H-NMR (DMSO-d ) δ: 1.44 (2H, dd, J = 12, 24
6
Hz), 1.70–1.80 (1H, m), 1.82 (2H, d, J = 12 Hz), 2.78
precipitates were collected and recrystallized from etha-
nol to give 13 (1.9 g, 52%); H-NMR (DMSO-d ) δ:
(
(
2H, t, J = 12 Hz), 3.17–3.24 (4H, m), 3.85 (3H, s), 4.15
2H, s), 6.76 (1H, d, J = 8.0 Hz), 7.01 (1H, d, J = 7.2 Hz),
1
6
1
.60–2.22 (2H, m), 2.64–2.75 (2H, m), 2.80–2.92 (3H,
m), 3.17 (2H, d, J = 5.9 Hz), 3.55 (2H, s), 3.80 (3H, s),
.23 (2H, br-s), 6.48 (1H, s), 7.25–7.55 (5H, m), 7.62
7
.43–7.50 (6H, m), 7.54 (1H, dd, J = 2.0, 7.2 Hz), 8.22
+
(1H, t, J = 6.0 Hz); MS m/z: 338 (M ).
4
+
(1H, s), 8.02 (1H, t, J = 6.0 Hz); MS m/z: 373 (M ).
5.1.2.6. N-[(1-Benzyl-4-piperidinyl)methyl]-5-chloro-2-
methoxybenzamide oxalate 17
Similarly to 13, 17 was prepared starting from 2e
(0.82 g, 4.4 mmol), 6a (0.90 g, 4.4 mmol), HOBt (0.66 g,
4.8 mmol), dimethylformamide (30 mL), and WSC
(0.93 g, 4.8 mmol). The resulting oil was transformed
into oxalate and recrystallized from ethanol to give 17
5
3
.1.2.2.
N-[(1-Benzyl-4-piperidinyl)methyl]-5-chloro-
,4-dihydro-1,4-benzofuran-7-carboxamide oxalate 12
Similarly to 13, 12 was prepared starting from 2b
(
2
(
0.50 g, 2.5 mmol), 6a (0.49 g, 2.5mmol), HOBt (0.33 g,
.5 mmol), dimethylformamide (20 mL), and WSC
0.47g, 2.5 mmol). The resulting oil was transformed into
oxalate and recrystallized from isopropanol to give 12
1
(0.27 g, 16%); H-NMR (CDCl -CD OD) δ: 1.69 (2H,
3
3
dd, J = 12, 24 Hz), 1.93 (2H, d, J = 12 Hz), 2.00 (1H, m),
2.78 (2H, br-s), 3.32 (2H, d, J = 6.6 Hz), 3.48 (2H, d, J
= 12 Hz), 3.96(3H, s), 4.24 (2H, s), 6.97 (1H, d, J = 10
Hz), 7.41 (1H, dd, J = 2.7, 10 Hz), 7.50–7.62 (5H, m),
8.00 (1H, d, J = 2.7 Hz), 7.93 (1H, br-s); MS m/z: 372
1
(0.89 g, 77%), H-NMR (CDCl ) δ: 1.65–2.02 (6H, m),
3
2
.45–2.60 (2H, br-s), 3.16 (2H, t, J = 6.0 Hz), 3.30 (2H,
br-s), 3.58 (2H, br-s), 4.18 (2H, br-s), 4.72 (2H, t, J = 6.0
Hz), 7.30–7.42 (5H, m), 7.62 (1H, t, J = 6.0 Hz), 8.82
+
+
(1H, d, J = 2.0 Hz); MS m/z: 384 (M ).
(M ).

338
5
.1.2.7. 4-Amino-N-[(1-benzyl-4-piperidinyl)methyl]-5-
Hz), 3.88 (3H, s), 4.38 (2H, br-s), 6.23 (1H, s), 7.68 (1H,
br-s), 8.03 (1H, s); MS m/z: 325 (M ).
+
chlorobenzamide oxalate 18
Similarly to 13, 18 was prepared starting from 2f
5
.1.2.11. 4-Amino-N-[(1-butyl-4-piperidinyl)methyl]-5-
(
4
(
0.76 g, 4.4 mmol), 6a (0.90 g, 4.4 mmol), HOBt (0.66 g,
.8 mmol), dimethylformamide (30 mL), and WSC
0.93 g, 4.8 mmol). The resulting oil was transformed
into oxalate and recrystallized from ethanol to give 18
chloro-2-methoxybenzamide oxalate 22
Similarly to 13, 22 was prepared starting from 2c
(
0.40 g, 2.0 mmol), 6c (0.31 g, 2.0 mmol), HOBt (0.29 g,
2.2 mmol), dimethylformamide (10 mL), and WSC
0.42 g, 2.2 mmol). The resulting oil was transformed
into oxalate and recrystallized from ethanol to give 22
1
(
0.41 g, 21%); H-NMR (CDCl -CD OD) δ: 1.74 (2H,
3 3
(
dd, J = 12, 24 Hz), 1.90 (2H, d, J = 12 Hz), 1.98 (1H, m),
.74 (2H, br-s), 3.24 (2H, d, J = 6.6 Hz), 3.44 (2H, d, J
12 Hz), 4.26 (2H, s), 6.76 (1H, d, J = 10 Hz), 7.32–7.48
5H, m), 7.54 (1H, dd, J = 2.7, 10 Hz), 7.79 (1H, d, J =
2
=
(
2
1
(
0.26 g, 29%); H-NMR (CD OD) δ: 1.23 (3H, t, J = 8.5
3
Hz), 1.35–1.42 (2H, m), 1.62 (2h, br-s), 1.70–1.85(2H,
m), 1.95 (1H, br-s), 1.97 (2H, d, J = 13Hz), 2.90 (2H,
br-s), 3.07 (2H, t, J = 8.5 Hz), 3.31 (2H, br-s), 3.35 (2H,
+
.7 Hz), 7.93 (1H, br-s); MS m/z: 357 (M ).
br-s), 3.91 (3H, s), 6.23 (1H, s), 7.78 (1H, s); MS m/z:
5
.1.2.8. 4-Amino-N-[(1-benzyl-4-piperidinyl)methyl]-2-
+
353 (M ).
methoxybenzamide oxalate 19
Similarly to 13, 19 was prepared starting from 2g
5
.1.2.12. 4-Amino-5-chloro-N-[(1-hexyl-4-piperidinyl)
(
5
(
0.82 g, 4.9 mmol), 6a (1.0 g, 4.9 mmol), HOBt (0.73 g,
.4 mmol), dimethylformamide (30 mL), and WSC
1.0 g, 5.4 mmol). The resulting oil was transformed into
oxalate and recrystallized from ethanol to give 19 (0.49 g,
methyl]-2-methoxybenzamide 23
Similarly to 13, 23 was prepared starting from 2c
(
1
1
1.7 g, 8.4 mmol), 6d (1.5 g, 8.4 mmol), HOBt (1.5 g,
1 mmol), dimethylformamide (30 mL), and WSC (2.1 g,
1 mmol). The resulting solid was recrystallized from
1
2
1
1
3
3%); H-NMR (DMSO-d ) δ: 1.43 (2H, t, J = 12 Hz),
6
.70 (1H, br-s), 1.77 (2H, d, J = 12 Hz), 2.78 (2H, t, J =
2 Hz), 3.18 (2H, t, J = 5.9 Hz), 3.25 (2H, d, J = 12 Hz),
.80 (3H, s), 4.16 (2H, br-s), 6.16 (1H, dd, J = 2.0, 8.0
ethyl acetate/diisopropylether to give 23 (2.1 g, 66%);
1
H-NMR (CDCl ) δ: 0.83 (3H, t, J = 12 Hz), 1.20–2.05
3
(
15H, m), 3.30 (2H, t, J = 12, 24 Hz), 3.92 (2H, d, J = 12
Hz), 3.33 (2H, t, J = 6.6 Hz), 3.90 (3H, s), 4.38 (2H, br-s),
.28 (1H, s), 7.70 (1H, br-s), 8.10 (1H, s); MS m/z: 381
Hz), 6.22 (1H, J = 2.0 Hz), 7.46 (5H, m), 7.59 (1H, d, J
+
=
8.0 Hz), 7.90 (1H, t, J = 6.0 Hz); MS m/z: 353 (M ).
6
+
(M ).
5
.1.2.9. N-[(1-Benzyl-4-piperidinyl)methyl]-5-chloro-2-
methoxy-4-methylaminobenzamide fumalate 20
5
.1.2.13.
4-Amino-5-chloro-N-[[2-[(methylsulfonyl)
Similarly to 13, 20 was prepared starting from 2h
amino]ethyl]-4-piperidinyl]-2-methoxybenzamide oxa-
late 24
(
6
(
1.2 g, 5.6 mmol), 6a (1.1 g, 5.6 mmol), HOBt (0.84 g,
.2 mmol), dimethylformamide (30 mL), and WSC
1.2 g, 6.2 mmol). The resulting oil was transformed into
fumalate and recrystallized from ethanol/acetone to give
Similarly to 13, 24 was prepared starting from 2c
(
1
1
2.0 g, 9.9 mmol), 6g (2.3 g, 9.9 mmol), HOBt (1.6 g,
2 mmol), dimethylformamide (50 mL), and WSC (2.3 g,
2 mmol). The resulting oil was transformed into oxalate
1
2
0 (0.31 g, 14%); H-NMR (DMSO-d6) δ: 1.27 (2H, dd,
J = 12, 21 Hz), 1.59 (1H, m), 1.65 (2H, d, J = 12 Hz),
.21 (2H, t, J = 11 Hz), 2.83 (3H, d, J = 4.7 Hz), 2.95 (2H,
d, J = 11 Hz), 3.18 (2H, t, J = 6.0 Hz), 3.69 (2H, s), 3.93
3H, s), 6.50 (1H, q, J = 4.7 Hz), 6.22 (1H, s), 6.59 (2H,
s), 7.31–7.70 (5H, m), 7.93 (1H, t, J = 6.0Hz); MS m/z:
and recrystallized from ethanol to give 24 (1.3 g, 26%);
2
1
H-NMR (DMSO-d ) δ: 1.20–2.00 (5H, m), 2.60–3.80
6
(
13H, m), 2.96 (3H, s), 3.79 (3H, s), 4.38 (2H, br-s), 6.42
(
(1H, s), 7.60 (1H, s), 7.94 (1H, t, J = 6.0 Hz); MS m/z:
+
418 (M ).
+
401 (M ).
5
.1.3. 1-Benzyl-3-pyrrolidinylmethylamine 4a
A mixture of 3 (5.0 g, 23 mmol), triphenylphosphine
5
.1.2.10.
4-Amino-5-chloro-N-[(1-ethyl-4-piperidinyl)
methyl]-2-methoxybenzamide 21
(6.6 g, 25 mmol), phthalimide (3.7 g, 25 mmol) and tet-
rahydrofuran (30 mL) was stirred under ice-cooling and
then diethyl azodicarboxylate (3.6 mL, 23 mmol) was
added at the same temperature. Stirring was continued
overnight at room temperature. After evaporation, the
residue was dissolved in ethyl alcohol and then hydrazine
monohydrate (3.3 mL, 68.4 mmol) was added. The mix-
ture was refluxed for 2 h. After cooling, the reaction
Similarly to 13, 21 was prepared starting from 2c
(
1
1
2.0 g, 9.9mmol), 6b (1.4 g, 9.9 mmol), HOBt (1.7 g,
3 mmol), dimethylformamide (30 mL), and WSC (2.3 g,
.2 mmol). The resulting solid was recrystallized from
1
ethyl acetate to give 21 (2.0 g, 62%); H-NMR (CDCl )
δ: 1.14 (3H, t, J = 12 Hz), 2.20–3.05 (7H, m), 2.20 (2H,
q, J = 12 Hz), 2.92 (2H, d, J = 12 Hz), 3.32 (2H, t, J = 6.6
3

3
39
mixture was filtered by suction though celite and to the
filtrate was added diethylether. The precipitate was fil-
tered off and filtrate was extracted with 10% hydrochloric
acid. The aqueous layer was basified by 10% NaOH and
extracted with chloroform. The extraction was washed
with brine, and was dried over anhydrous magnesium
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 × 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. Non specific binding was
defined in the presence of unlabelled GR113808 to give a
final concentration of 1 µmol/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 = IC /(1 + L/Kd)), where L is the concen-
sulfate. After evaporation in vacuo to give 4a (2.8 g,
1
5
1
6%); H-NMR (CDCl ) δ: 1.10–1.29 (2H, m),
3
.38–1.52 (1H, m), 1.55 (2H, t, J = 7.2 Hz), 1.63 (2H, d,
J = 6.6 Hz), 1.92 (2H, dt, J = 1.0, 12 Hz), 2.50 (2H, br-s),
.72 (2H, t, J = 7.2 Hz), 2.82 (2H, d, J = 12 Hz), 3.61 (2H,
2
+
s), 7.12–7.23 (5H, m); MS m/z: 218 (M ).
50
tration of radioligand and Kd is the dissociation constant
of the radioligand.
5
5
.1.4. General procedure for the preparation of 6a–6d
.1.4.1. 4-Aminomethyl-1-benzylpipeidine 6a
A solution of 5a (5.7 g, 26 mmol) in tetrahydrofuran
5
.2.1.2. 5-HT receptor
3
3
[
H]Granisetron binding assays were performed ac-
(
50 mL) was added dropwise to a suspension of lithium
cording to the method of Nelson and Thomas [34]. 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 1 000 × 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
aluminium hydride (LiAlH ) (2.0 g, 52 mmol) in tetrahy-
4
drofuran (50 mL) at 0 °C, and the mixture was heated at
4
5 °C with stirring for 4 h. After cooling, water was
added dropwise to the mixture at a temperature below
°C to destroy the excess LiAlH . After filtration, the
0
4
filtrate was dried over anhydrous magnesium sulfate, and
after evaporation in vacuo, the residue was distilled under
reduced pressure to give 6a (5.0 g, 94%), b.p.
(
3
1
50 mmol/L, pH 7.4) and the suspension was incubated at
7 °C for 10 min and centrifuged at 40 000 × g for
5 min. The pellet was washed and centrifuged (40 000 ×
1
125 °C/1.0 mmHg; H-NMR (CDCl ) δ: 1.28 (2H, dd, J
3
g for 15 min). The final pellet was resuspended in 30
volumes of HEPES buffer and used as tissue homogenate.
=
4.0, 12 Hz), 1.30 (2H, br-s), 1.35–1.50 (1H, m), 1.72
(2H, d, J = 6.6 Hz), 1.92 (2H, dt, J = 2.0, 6.6 Hz), 2.64
3
The binding assay consisted of 50 µmol/L of [ H]Gran-
(2H, d, J = 6.6 Hz), 2.87 (2H, d, J = 6.6Hz), 3.00 (2H, t,
isetron, 50 µL of displacing drugs and 900 µL 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
+
J = 6.6 Hz), 7.18–7.35 (5H, m); MS m/z: 142 (M ).
The other piperidines (6b–6d) were prepared in a
similar manner.
5
5
5
.2. Pharmacology
(
50 mM, pH 7.4). ICS 205930 (100 mmol/L) was used
.2.1. Radioligand binding assays
for the determination of nonspecific binding.
.2.1.1. 5-HT receptor
4
Male Hartley guinea-pigs (Japan SLC, Ltd., Shizuoka,
5.2.1.3. D receptor
2
3
Japan) were sacrificed by cervical dislocation and the
striatum was separated from each brain. The striatum was
homogenized in 15 volumes 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 tubes contained 50 µL of
HEPES buffer or a solution of the test agents, 50 µL
[ H]Spiperone binding assays were performed accord-
ing to the method of Crees 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 recen-
trifuged (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
CaCl , 1 mmol/L MgCl , 1.1 mmol/L ascorbic acid and
3
solution of [ H]GR113808 (Amersham International,
UK) to give a final concentration of 0.1 nmol/L and
2
2
900 µL of tissue preparation. Each tube was incubated for
10 µmol/L pargyline, and incubated at 37 °C for 10 min.

3
40
A portion of this membrane suspension (900 µmol/L) was
placed in a tube, and 50 µmol/L of either test compound
5.3. Construction of receptor model
or vehicle solution was added, followed by 50 µL of
5.3.1. Sequence
3
[
H]Spiperone (40 Ci/mmol) at a final concentration of
.2 nmol/L. The tubes were incubated at 37 °C for 20 min
The sequence in this study, except for the 5-HT4
receptor, was from the Swiss-Prot Protein Sequence Data
Bank [35]. The coordinates of Bacteriorhodopsin (entry
1BRD) [36] were from the Protein Data Bank [37] at
Brookhaven National Laboratory. Modelling was
achieved with the molecular package SYBYL 6.2 [38].
The interactive modelling and display were performed on
a Silicon Graphics IRIS INDIGO/Elan 4 000 computer.
Five main steps were used: stretch of α-helical structure
where putative transmembrane region is longer than
bacteriorhodopsin, amino acid substitution, local geom-
etry optimization, docking of ligand, and side-chain
rotation to minimize overlaps between helices. Some
manual adjustments were made to remove bad steric
interactions in geometry optimization. In Energy minimi-
zation procedure, the backbone was aggregated until the
0
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). Sulpiride (100 µmol/L) was
used for the determination of nonspecific binding. The
radioactivity trapped on the filters was measured by
liquid scintillation spectrometry.
5
5
.2.2. Contractile effects
.2.2.1. 5-HT receptor agonism
4
Male Hartley guinea-pigs (Japan SLC, Ltd., Shizuoka,
Japan) were killed by cervical dislocation and the ascend-
ing 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 di-
vided into four segments of about 2.5 cm. Four muscle
strip preparations were individually mounted vertically
for isotonic measurement into a tissue bath containing
2
RMS gradient was less than 0.05 (kcal/mol Å ). A
dielectric constant of compound 24 was calculated where
a cut off distance of 8 Å was used and no solvent
molecules were included in the calculation.
1
0 mL Tyrode solution. Only 5-HT was tested in the
Tyrode solution containing methysergide (1 µmol/L) and
granisetron (1 µmol/L) to inhibit responses mediated by
Acknowledgements
5
-HT and 5-HT -like and 5-HT receptors, respectively.
2 1 3
We thank Mrs. F. Matsugaki for some of the biological
results. We also thank Dr. M. Terasawa and Dr. K. Adachi
for helpful discussion.
This solution was kept at 37 °C and gassed with 95% O2,
% CO . The strips were subjected to a preload of 1 g and
5
2
allowed to stabilize for 20 min. After stabilization, the
response of the longitudinal muscle to 30 µmol/L metha-
choline was measured. Agonist concentration-effect
curves were constructed using sequential dosing, leaving
References
15 min between doses. A 15 min dosing cycle was
[1] Alexander S.H., Peters J.A., TPiS Receptor and Ion Chanenel
required to prevent desensitization. The agonist was left
in contact with a preparation until the response had
reached a maximum, the preparation was washed. Forty
minutes was left between the determination of
concentration-effect curves. GR113808 (10 nmol/L) were
incubated for 10 min before repeating agonist
concentration-effect curves. After each determination of
concentration-effect curve, 30 µmol/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 µmol/L methacholine. The
EC50 value, the concentration causing 50% of the maxi-
mal response, was determined by linear regression analy-
sis.
Nomenclature Supplement, Elsevier, 1998, pp. 46–48.
[2] Eglen R.M., Wong E.F., Dumuis A., Bockaert J., Trends Pharmacol.
Sci. 16 (1995) 391–398.
[
[
[
3] Craig D.A., Clarke D.E., J. Pharmacol. Exp. Ther. 252 (1990)
378–1386.
4] Elswood C.J., Bunce K.T., Humphrey P.P.A., Eur. J. Pharmacol. 196
1991) 149–155.
1
(
5] Briejer M.R., Akkerman L.S., Meulemans A.L., Lefebvre R.A.,
Schuurkes L.J., Naunyn-Schmiederg’s Arch. Pharmacol. 347 (1993)
464–470.
[
[
[
[
6] Rizzi C.A., Coccini T., Onori L., Manzo L., Tonini M., J. Pharmacol.
Exp. Ther. 261 (1992) 412–419.
7] Langlois M., Zhang L., Yang D., Brémont B., Shen S., Manara L.,
Croci T., Bioorg. Med. Chem. Lett. 4 (1994) 1433–1436.
8] Dumuis A., Bouhelal R., Sebben M., Cory R., Bockaert J., Mol.
Pharmacol. 34 (1988) 880–887.
9] Flynn D.L., Zabroski D.L., Becker D.P., Nosal R., Villamil C.L.,
Gullikson G.W., Moummi C., Yang D., J. Med. Chem. 35 (1992)
5
.2.2.2. 5-HT receptor antagonism
4
1487–1491.
5
-HT receptor antagonism was expressed in the form
B
4
[10] Turconi M., Schiantarelli P., Borsini F., Rizzi C.A., Ladinsky H.,
Donetti A., Drugs Future 16 (1991) 1011–1026.
of pK value on the contractile response to 5-MeOT.

3
41
[
11] Clark R.D., Jahangir A., Langston J.A., Weinhardt K.K., Miller
[25] Glennon R.A., Dukat M., Westkaemper R.B., Ismaiel A.M., Izzarelli
D.G., Parker E.A., Mol. Pharmacol. 49 (1996) 198–206.
A.B., Leung E., Eglen R.M., Bioorg. Med. Chem. Lett. 4 (1994)
2
477–2480.
[26] Teeter M.M., Froimowitz M.B., Durand C.J., J. Med. Chem. 37
[
[
[
[
[
12] López-Rodríguez M.L., Morcillo M.J., Benhaú B., Rosado M.L., J.
(1996) 2874–2888.
Comput.-Aided Mol. Des. 11 (1997) 589–599.
[27] Kotoyianni M., Deweese C., Penzotti J.E., Lybrand T.P., J. Med.
Chem. 39 (1996) 4406–4420.
13] Kuroita T., Sakamori M., Kawakita T., Chem. Pharm. Bull. 44
(1996) 756–764.
[28] Kyte J., Doolittle R.F., J. Mol. Biol. 157 (1982) 105–132.
14] Kuroita T., Ikebe T., Murakami S., Takehara S., Kawakita T., Bioorg.
Med. Chem. Lett. 5 (1995) 1245–1250.
[29] Begonia Y.H., Karschin A., Branchek T., Davidson N., Lester H.A.,
FEBS. Lett. 312 (1992) 259–262.
15] Tahara T., Hayano K., Murakami S., Fukuda T., Setoguti M., Ikeda
K., Marubayasi N., Chem. Pharm. Bull. 38 (1990) 1609–1615.
[30] Johnson M.P., Loncharich R.J., Baez M., Nelson D.L., Mol. Phar-
macol. 45 (1993) 277–286.
16] Iwanami S., Takasima M., Hirata Y., Hasegawa O., Usuda S., J.
Med. Chem. 24 (1981) 1224–1230.
[31] Almaula N., Ebersole B.J., Zhang D., Weinstein H., Sealfon S.C., J.
Biol. Chem. 271 (1996) 14672–14675.
[
[
[
[
[
[
32] Almaula N., Ebersole B.J., Ballesteros J.A., Weinstein H., Sealfon
[
17] Mitsunobu O., Synthesis (1981) 1–28.
S.C., Mol. Pharmacol. 50 (1996) 34–42.
[18] Kawakita T., Kuroita T., Murozono T., Hakira H., WO 95/26953
Chem. Abstr. 124 (1996) 14913.
33] Choudhary M.S., Craigo S., Roth B.L., Mol. Pharmacol. 43 (1993)
7
55–761.
34] Nelson D.R., Thomas D.R., Biochem. Pharmacol. 38 (1989)
693–1695.
35] Bairoch A., Boeckmann B., Nucleic Acids Res. 20 (1992)
019–2022.
[
[
[
[
[
[
19] Fancelli D., Caccia C., Fornaretto M.G., McArthur R., Severino D.,
Vaghi F., Varasi M., Bioorg. Med. Chem. Lett. 6 (1996) 236–266.
1
20] Hibert M.F., Trumpp-Kallmeyer S., Bruinvels A., Hoflack J., Mol.
Pharmacol. 40 (1991) 8–15.
2
21] Ijzerman A.P., Van Gallen P.M., Jacobson K.A., Drug Design Dev. 9
36] Henderson R., Baldwin J.M., Ceska T.A., Zemlin F., Beckmann
E.D., J. Mol. Biol. 213 (1990) 899–929.
(1992) 49–67.
22] Trumpp-Kallmeyer S., Hoflack J., Bruinvels A., Hibert M., J. Med.
Chem. 35 (1992) 3448–3462.
37] Bernstein F.C., Koetzle T.F., Williams G.B., Meyer E.F. Jr., Brice
M.D., Rodgers J.R., Kennard O., Shimanouchi T., Tasumi M., J.
Mol. Biol. 112 (1977) 535–542.
23] Yamamoto Y., Kamiya K., Terao S., J. Med. Chem. 36 (1993)
8
20–825.
[38] Tripos E & S, 6548 Clayton Road, St. Louis, MO 62117. SYBYL
24] Nordvall G., Hacksell U., J. Med. Chem. 36 (1993) 976–976.
molecular modelling software.