Synthesis and pharmacological evaluation of carboxamide derivatives as selective serotoninergic 5-HT4 receptor agonists (European Journal of Medicinal Chemistry (1999) 34 (329-987))

Eur. J. Med. Chem. 34 (1999) 977−989

977

Original article

Synthesis and pharmacological evaluation of carboxamide derivatives

as selective serotoninergic 5-HT₄receptor agonists

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-HT₄receptor (Ki = 9.6 nM). Compound 24 displayed a higher affinity for

5-HT₄receptors than the other receptors, including, 5-HT₃and dopamine D₂receptors. In addition, compound 24 was conﬁrmed to be a potent

scientiﬁques et médicales Elsevier SAS

5-HT₄receptor / 5-HT₄receptor agonist / structure–activity relationship / carboxamide / receptor model construction

1. Introduction

Cisapride and BIMU8 show obvious affinity for the

5-HT₃receptor [9, 10]. While, ML 10302 is a selective

5-HT₄receptor agonist, it shows limited pharmacological

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-HT₄receptor

and to be selective 5-HT₄partial agonists [11]. In addi-

tion, the original 5-TH₄receptor mapping by the active

analogue approach by using several 5-HT₄receptor

antagonists and inactive ligands was reported [12].

Serotonin (5-HT) is a neurotransmitter responsible for

a wide range of pharmacological reactions. Serotonergic

receptors are now classiﬁed into four broad subtypes such

as 5-HT₁, 5-HT₂, 5-HT₃, and 5-HT₄receptors, and clones

for additional subtypes termed 5-HT₅, 5-HT₆, and 5-HT₇

receptors have been identiﬁed [1]. Among diverse 5-HT

receptors, we have investigated 5-HT₄function. Activa-

tion of the 5-HT₄receptor mediates widespread effects in

the central and peripheral nervous systems [2]. Therefore,

selective 5-HT₄receptor ligands would be useful for

elucidating the physiological function of this receptor.

Agonists for the 5-HT₄receptor known to date are indole

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-HT₃receptor [8].

One of our purposes is to supply a useful tool for

elucidating the physiological function of the 5-HT₄re-

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)

shows a weak affinity for the 5-HT₄receptor. This result

lead us to design selective 5-HT₄receptor 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,

*Correspondence and reprints

978

BIMU 8

ML 10302

Figure 1. Chemical structures of 5-HT₄receptor agonists.

(3) the variety and number of the substituents on the

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-HT₄receptor agonists.

Interaction between putative 5-HT₄receptor models and

agonistic ligand is also discussed here.

amines (4a, 4b and 6a–6f) to afford target benzamides

(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)

[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

(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].

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

3. Pharmacological data and discussion

3.1. Structure–activity relationships

The affinity of compounds 7–24 for the 5-HT₄receptor

was determined as their ability to inhibit the binding of

[³H]GR113808 to the receptor. Their affinities for 5-HT₃

and dopamine D₂receptors were similarly evaluated by

using [³H]granisetron and [³H]spiperone as radioligand,

respectively. Here, membrane preparations of the stria-

Figure 2. Chemical structure of compound 1.

979

Figure 3. The general synthetic procedures used in this study. (a) method A: i-BuOCOCl, Et₃N, AcOEt or method B: WSC, HOBt,

DMF.

tum of guinea-pigs, the cerebral cortex of rats, and the

striatum of rats were used for 5-HT₄, 5-HT₃, and D₂

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.

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-HT₄receptor than for 5-HT₃

Pharmacological data on compounds 1 and 7–24,

4-amino-N-(1-benzyl-4-piperidinyl)-5-chloro-4-methoxy-

Figure 4. Condensation of 3 by the Mitsunobu reaction. (a)

PPh₃, phthalimide, DEAD, THF; (b) NH₂NH₂H₂O, EtOH.

Figure 5. Lithium aluminium hydride reduction of 5a–5d. (a)

LiA1H₄, THF.

980

Table I Physicochemical properties for compounds 7–24.

Compound Ar

n

0

X

R

Method

A

Mp (°C)

94–95

Formula

7

Bn^a

C₂₁H₂₄CIN₃O₂

f

8

0

1

2

Bn^a

A

232–233

186–187

97–98

C₂₂H₂₆CIN₃O₂C₄H₄O₄

C₂₂H₂₆CIN₃O₂C₂H₄O₄

C₂₃H₂₈CIN₃O₂

g

9

10

11

122–124

C₂₄H₃₀CIN₃O₂C₂H₂O₄1/2EtOH

1/2H₂O^g

Bn^a

B

176–178/DEC

161–163

C₂₂H₂₅CIN₂O₂C₂H₂O₄

g

12

13

14

15

1

2

g

C₂₀H₂₄CIN₃O₂C₂H₂O₄

155–156

C₂₁H₂₆CIN₃O₂

g

135–137

C₂₂H₂₈CIN₃O₂C₂H₂O₄

g

16

17

1

Bn^a

B

169–170

90–93

C₂₁H₂₆N₂O₂C₂H₂O₄

C₂₁H₂₅CIN₂O₂C₂H₂O₄1/4H₂O^g

C₂₀H₂₄CIN₃O₂C₂H₂O₄4/5H₂O^g

18

1

Bn^a

B

110–112/DEC

981

Table I (Continued)

Compound Ar

n

1

X

R

Method

B

Mp (°C)

160–161

Formula

g

19

Bn^a

C₂₁H₂₇N₃O₂C₂H₂O₄

f

20

1

Bn^a

B

148–149

C₂₂H₂₈CIN₃O₂C₄H₄O₄

21

22

23

24

1

Et^b

B

115–116

C₁₆H₂₄N₃O₂

Bu^c

179–181

C₁₈H₂₈CIN₃O₂C₂H₂O₄

C₂₀H₃₂CIN₃O₂

g

Hex^d

MSAE^e

113–115

g

177–178/DEC

C₁₇H₂₇CIN₄O₄S2C₂H₂O₄

b

c

d

e

f

g

^aBn, 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

5-HT₃receptor. Furthermore, piperidine derivatives (10

and 14) showed a higher affinity for 5-HT₄receptor than

pyrrolidine derivatives (9 and 13).

Compound 10 was conﬁrmed to be a 5-HT₄receptor

antagonist (pK_B= 7.9). Compound 12, a benzofuran

analogue of benzoxazine derivative 10, was also con-

ﬁrmed to behave as a 5-HT₄receptor antagonist (pK_B=

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,

were conﬁrmed to be a 5-HT₄receptor agonist. We next

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

exhibited a 5-HT₄receptor affinity similar to compound

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

essential for binding to the 5-HT₄receptor. Compound 17

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-

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-

3-pyrrolidinyl or 1-benzyl-4-piperidinyl group, showed

affinities for 5-HT₃, 5-HT₄and D₂receptors. On the other

hand, compounds 9, 10 and 12–14, where the hydrogen

atom on their carboxamide nitrogen is replaced by a

1-benzyl-3-pyrrolidinylmethyl or 1-benzyl-4-piperi-

dinylmethyl group, showed a higher affinity for the

5-HT₄receptor than for the 5-HT₃receptor, and showed

a low affinity for the D₂receptor. Compounds 11 and 15,

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-

tive affinity for the 5-HT₄receptor was one carbon [9].

982

Table II Pharmacological data of compound 1, compounds 7–24 and two reference compounds.

Binding affinities, Ki (nM)^a

Contractile effects in guinea pigs ascending colon

Compound

5-HT₄

5-HT₃

D₂

EC (nM)^d

Maximal response (%)^e

50

1

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

280

160

530

19

0.93

530

1.7

0.27

10

21

95

> 1000^b

12

NT^c

> 10000

NT

–

67

> 1000^b

NT

120

> 1000^b

340

–

> 10000

47

20

6.7

160

290

470

> 1000^b

880

21

18

–

20

> 1000^b

66

NT^c

> 10000

NT^c

150

> 1000^b

230

–

8.1

NT^c

> 1000^b

110

100

28

NT^c

–

NT^c

20

8.9

16

15

20

27

–

NT^c

900

61

13

130

NT^c

22

23

24

8.8

6.7

9.6

92

NT^c

> 1000^b

63

7.0

clebopride

5-HT

NT^c

35

130

21

b

c

d

^aEach value is the mean from triplicate assays in a single experiment; IC₅₀value; not tested; EC₅₀values were determined by linear

e

regression; % of contraction to methacoline at 30 µM.

methoxybenzamide derivative 19, a 5-deschloro analogue

of compound 14, showed a potent 5-HT₄receptor ago-

nistic activity, but showed only a moderate affinity for the

receptor. These results indicate that the 4-amino group

played an important role in 5-HT₄receptor agonistic

activity. In addition, this consideration is supported by the

facts, which 2,3-dihydro-4-amino-5-chlorobenzofuran

carboxamides showed 5-HT₄receptor agonistic activ-

ity [19], while 4-desamino-2,3-dihydro-5-chlorobenzo-

furan carboxamide 12 showed low 5-HT₄receptor ago-

potent agonist at the 5-HT₄receptor than other com-

pounds (14, 21–23). Therefore, compound 24 was as-

sayed for its binding ability for several kinds of receptors.

The results were as follows (IC₅₀value (nM), ligand):

α₁> 1 000, [³H]prazosin; 5-HT_1A> 1 000, [³H]8-OH-

DPAT; 5-HT₂> 1 000, [³H]ketanserin; MACh > 1 000,

[³H]QNB. Thus Compound 24 represented a selective

5-HT₄receptor agonist.

3.2. Computational modelling study

nistic

activity.

Although

5-chloro-2-methoxy-4-

3.2.1. 5-HT₄receptor model construction

methylaminobenzamide derivative 20, an N-methyl

analogue of compound 14, also exhibited a moderate

affinity, it showed low 5-HT₄receptor agonistic activity.

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

with the 5-HT₄receptor. Ethyl derivative 21 showed a

moderate affinity for the 5-HT₄receptor. Butyl derivative

22 was almost equal to compound 14 in the affinity, hexyl

derivative 23 was equal to compound 14 in the affinity,

but with reduced 5-HT₄receptor agonistic activity. When

the substituent was a polar methylsulfonylamino group,

agonistic activity increased. Compound 24 was a more

A 5-HT₄receptor model was constructed in order to

obtain rational structure–activity relationships and to

clarify the interaction between ligands and the receptor.

The 5-HT₄receptor is a member of the G-protein coupled

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

this study, affinity for the guinea-pig 5-HT₄receptor was

measured. Therefore the receptor model should be con-

structed from the sequence of guinea-pig. But the guinea-

983

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

rat 5-HT₄receptors should be conserved in guinea-pig

5-HT₄receptors. The 5-HT₄receptor model was con-

structed with the residues in the human 5-HT₄receptor

and the conserved residues were used for speculation of

receptor-ligand interaction. The seven transmembrane

regions of the 5-HT₄receptor was determined from a

hydropathy plot [28]. To determine sequence alignment

of bacteriorhodopsin and the 5-HT₄receptor, over 70

GPCRs such as dopamine, adrenergic, muscarine recep-

tor, and rhodopsin were included in the study.

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

interaction model between the 5-HT₄receptor and com-

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

5-chloro group performs a hydrophobic interaction with

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.

3.2.2. Docking of compound 24 into the 5-HT₄receptor

model

Mutational studies for the 5-HT₄receptor have not

been reported yet. Therefore, the structure–activity rela-

tionships for this series of compounds and mutational

data for the 5-HT_1Aand 5-HT_2Areceptors [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-HT₄receptor.

984

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 =

3.0 Hz), 7.20–7.30 (5H, m), 7.39 (1H, d, J = 3.0 Hz), 7.95

(1H, br-s); MS m/z: 385 (M⁺).

We describe the synthesis and biological evaluation

of a series of carboxamides as selective 5-HT₄receptor

agonists. Based on our results, we propose that 4-amino-

5-chloro-2-methoxy-N-[(4-piperidinyl)methyl]benzamide

with a polar group at the 1-position on piperidine

moieties are necessary for the selective agonistic activity

for 5-HT₄receptors. Compound 24 represents a selective

5-HT₄agonist and would be a useful tool for probing the

5-HT₄receptor function in vivo. Detailed analyses of

structure–activity relationships for the side chain moieties

will be reported in due course.

5.1.1.2.

N-(1-Benzyl-4-piperidinyl)-6-chloro-3,4-dihy-

dro-4-methyl-2H-1,4-benzoxazine-8-carboxamide fuma-

late 8

Similarly to 7, 8 was prepared starting from 2a (3.4 g,

15 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,

1

55%); H-NMR (DMSO-d₆) δ: 1.54 (2H, dd, J = 12, 24

5. Experimental protocols

Hz), 1.82 (2H, d, J = 12 Hz), 2.20–2.39 (2H, t, J = 12 Hz),

2.80 (2H, d, J = 12 Hz), 2.87 (3H, s), 3.29 (2H, t, J = 4.0

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);

MS m/z: 399 (M⁺).

5.1. Chemistry

All melting points were measured in open capillaries

and uncorrected. Proton nuclear magnetic resonance

(¹H-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

(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.

5.1.1.3. N-[(1-Benzyl-3-pyrrolidinyl)methyl]-6-chloro-

3,4-dihydro-4-methyl-2H-1,4-benzoxazine-8-carboxamide

oxalate

9

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-d₆) δ: 1.60–2.22 (2H,

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),

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⁺).

5.1.1. General procedure for the preparation of 7–11

(method A)

5.1.1.1.

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,

19 mol), and ethyl acetate (40 mL) at –10 °C. The mix-

ture was stirred below –5 °C for 30 min and a solution of

4b (1.5 g, 8.8 mmol) in ethyl acetate (10 mL) was added

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

chromatographed on silica gel (CHCl₃:MeOH = 10:1).

Recrystallized from ethyl acetate/diisopropylether to give

7 (0.50 g, 15%); ¹H-NMR (CDCl₃) δ: 1.70–1.82 (2H, m),

2.64 (2H, d, J = 11 Hz), 2.90 (3H, s), 3.35 (2H, t, J = 5.6

5.1.1.4.

N-[(1-Benzyl-4-piperidinyl)methyl]-6-chloro-

3,4-dihydro-4-methyl-2H-1,4-benzoxazine-8-carboxamide

10

Similarly to 7, 10 was prepared starting from 2a (1.2 g,

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-

tallized from ethyl acetate to give 10 (1.4 g, 64%);

¹H-NMR (CDCl₃) δ: 1.23–1.32 (2H, m), 1.55 (1H, br-s),

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,

J = 3.0 Hz), 7.69 (1H, br-s); MS m/z: 413 (M⁺).

985

5.1.1.5.

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

3,4-dihydro-4-methyl-2H-1,4-benzoxazine-8-carboxamide

oxalate 11

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

6f (1.0 g, 4.6 mmol). The resulting oil was transformed

into oxalate and recrystallized from ethanol to give 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

1

from ethyl acetate to give 14 (1.4 g, 72%); H-NMR

(CDCl₃) δ: 1.35–1.55 (5H, m), 2.00 (2H, d, J = 12 Hz),

2.90 (2H, d, J = 12 Hz), 3.30 (2H, t, J = 5.6 Hz), 3.50 (2H,

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⁺).

1

(0.56 g, 24%); H-NMR (DMSO-d₆) δ: 1.32–1.60 (4H,

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 =

5.2 Hz); MS m/z: 427 (M⁺).

5.1.2.4. 4-Amino-N-[2-(1-benzyl-4-piperidinyl)ethyl]-5-

chloro-2-methoxybenzamide oxalate 15

Similarly to 13, 15 was prepared starting from 2c

(0.93 g, 4.6 mmol), 6f (1.0 g, 4.6 mmol), HOBt (0.65 g,

0.48 mmol), dimethylformamide (30 mL), and WSC

(0.92 g, 4.8 mmol). The resulting oil was transformed

into oxalate and recrystallized from ethanol to give 15

5.1.2. General procedure for the preparation of 12–24

(method B)

5.1.2.1. 4-Amino-N-[(1-benzyl-3-pyrrolidinyl)methyl]-5-

chloro-2-methoxybenzamide oxalate 13

1

(0.56 g, 24%); H-NMR (DMSO-d₆) δ: 1.35–1.55 (5H,

m), 1.85 (2H, d, J = 12 Hz), 2.75 (2H, t, J = 6.8 Hz),

3.19–3.30 (4H, m), 3.88 (3H, s), 4.14 (2H, s), 5.91 (2H,

br-s), 6.47 (1H, s), 7.41–7.47 (5H, m), 7.66 (1H, s), 7.89

(1H, t, J = 5.9Hz); MS m/z: 401 (M⁺).

A mixture of 2c (1.6 g, 7.9 mmol), 4a (1.5 g,

7.9 mmol), 1-hydroxybenzotriazole (HOBt) (1.1 g,

8.1 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-

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

precipitates were collected and recrystallized from etha-

5.1.2.5. N-[(1-Benzyl-4-piperidinyl)methyl]-2-methoxy-

benzamide oxalate 16

Similarly to 13, 16 was prepared starting from 2d

(0.74 g, 4.9 mmol), 6a (1.0 g, 4.9 mmol), HOBt (0.73 g,

5.4 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

71%); H-NMR (DMSO-d₆) δ: 1.44 (2H, dd, J = 12, 24

Hz), 1.70–1.80 (1H, m), 1.82 (2H, d, J = 12 Hz), 2.78

(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),

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⁺).

1

nol to give 13 (1.9 g, 52%); H-NMR (DMSO-d₆) δ:

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),

4.23 (2H, br-s), 6.48 (1H, s), 7.25–7.55 (5H, m), 7.62

(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

5.1.2.2.

N-[(1-Benzyl-4-piperidinyl)methyl]-5-chloro-

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

3,4-dihydro-1,4-benzofuran-7-carboxamide oxalate 12

Similarly to 13, 12 was prepared starting from 2b

(0.50 g, 2.5 mmol), 6a (0.49 g, 2.5mmol), HOBt (0.33 g,

2.5 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 (CDCl₃-CD₃OD) δ: 1.69 (2H,

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

(M⁺).

1

(0.89 g, 77%), H-NMR (CDCl₃) δ: 1.65–2.02 (6H, m),

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⁺).

986

5.1.2.7. 4-Amino-N-[(1-benzyl-4-piperidinyl)methyl]-5-

chlorobenzamide oxalate 18

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⁺).

Similarly to 13, 18 was prepared starting from 2f

(0.76 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 18

5.1.2.11. 4-Amino-N-[(1-butyl-4-piperidinyl)methyl]-5-

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

(0.26 g, 29%); ¹H-NMR (CD₃OD) δ: 1.23 (3H, t, J = 8.5

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,

br-s), 3.91 (3H, s), 6.23 (1H, s), 7.78 (1H, s); MS m/z:

353 (M⁺).

1

(0.41 g, 21%); H-NMR (CDCl₃-CD₃OD) δ: 1.74 (2H,

dd, J = 12, 24 Hz), 1.90 (2H, d, J = 12 Hz), 1.98 (1H, m),

2.74 (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 =

2.7 Hz), 7.93 (1H, br-s); MS m/z: 357 (M⁺).

5.1.2.8. 4-Amino-N-[(1-benzyl-4-piperidinyl)methyl]-2-

methoxybenzamide oxalate 19

Similarly to 13, 19 was prepared starting from 2g

(0.82 g, 4.9 mmol), 6a (1.0 g, 4.9 mmol), HOBt (0.73 g,

5.4 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,

5.1.2.12. 4-Amino-5-chloro-N-[(1-hexyl-4-piperidinyl)

methyl]-2-methoxybenzamide 23

Similarly to 13, 23 was prepared starting from 2c

(1.7 g, 8.4 mmol), 6d (1.5 g, 8.4 mmol), HOBt (1.5 g,

11 mmol), dimethylformamide (30 mL), and WSC (2.1 g,

11 mmol). The resulting solid was recrystallized from

ethyl acetate/diisopropylether to give 23 (2.1 g, 66%);

¹H-NMR (CDCl₃) δ: 0.83 (3H, t, J = 12 Hz), 1.20–2.05

(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),

6.28 (1H, s), 7.70 (1H, br-s), 8.10 (1H, s); MS m/z: 381

(M⁺).

1

23%); H-NMR (DMSO-d₆) δ: 1.43 (2H, t, J = 12 Hz),

1.70 (1H, br-s), 1.77 (2H, d, J = 12 Hz), 2.78 (2H, t, J =

12 Hz), 3.18 (2H, t, J = 5.9 Hz), 3.25 (2H, d, J = 12 Hz),

3.80 (3H, s), 4.16 (2H, br-s), 6.16 (1H, dd, J = 2.0, 8.0

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⁺).

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

(1.2 g, 5.6 mmol), 6a (1.1 g, 5.6 mmol), HOBt (0.84 g,

6.2 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

20 (0.31 g, 14%); ¹H-NMR (DMSO-d6) δ: 1.27 (2H, dd,

J = 12, 21 Hz), 1.59 (1H, m), 1.65 (2H, d, J = 12 Hz),

2.21 (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:

401 (M⁺).

amino]ethyl]-4-piperidinyl]-2-methoxybenzamide oxa-

late 24

Similarly to 13, 24 was prepared starting from 2c

(2.0 g, 9.9 mmol), 6g (2.3 g, 9.9 mmol), HOBt (1.6 g,

12 mmol), dimethylformamide (50 mL), and WSC (2.3 g,

12 mmol). The resulting oil was transformed into oxalate

and recrystallized from ethanol to give 24 (1.3 g, 26%);

¹H-NMR (DMSO-d₆) δ: 1.20–2.00 (5H, m), 2.60–3.80

(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⁺).

5.1.3. 1-Benzyl-3-pyrrolidinylmethylamine 4a

5.1.2.10.

methyl]-2-methoxybenzamide 21

Similarly to 13, 21 was prepared starting from 2c

(2.0 g, 9.9mmol), 6b (1.4 g, 9.9 mmol), HOBt (1.7 g,

13 mmol), dimethylformamide (30 mL), and WSC (2.3 g,

1.2 mmol). The resulting solid was recrystallized from

ethyl acetate to give 21 (2.0 g, 62%); H-NMR (CDCl₃)

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

4-Amino-5-chloro-N-[(1-ethyl-4-piperidinyl)

A mixture of 3 (5.0 g, 23 mmol), triphenylphosphine

(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

1

987

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

sulfate. After evaporation in vacuo to give 4a (2.8 g,

56%); ¹H-NMR (CDCl₃) δ: 1.10–1.29 (2H, m),

1.38–1.52 (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),

2.72 (2H, t, J = 7.2 Hz), 2.82 (2H, d, J = 12 Hz), 3.61 (2H,

s), 7.12–7.23 (5H, m); MS m/z: 218 (M⁺).

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 IC₅₀value was

determined by non-linear regression of the displacement

curve, and the Ki value was calculated according to the

formula (Ki = IC₅₀/(1 + L/Kd)), where L is the concen-

tration of radioligand and Kd is the dissociation constant

of the radioligand.

5.1.4. General procedure for the preparation of 6a–6d

5.1.4.1. 4-Aminomethyl-1-benzylpipeidine 6a

5.2.1.2. 5-HT₃receptor

A solution of 5a (5.7 g, 26 mmol) in tetrahydrofuran

(50 mL) was added dropwise to a suspension of lithium

aluminium hydride (LiAlH₄) (2.0 g, 52 mmol) in tetrahy-

drofuran (50 mL) at 0 °C, and the mixture was heated at

45 °C with stirring for 4 h. After cooling, water was

added dropwise to the mixture at a temperature below

0 °C to destroy the excess LiAlH₄. After ﬁltration, the

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. 125 °C/

1.0 mmHg; ¹H-NMR (CDCl₃) δ: 1.28 (2H, dd, J = 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 (2H, d, J =

6.6 Hz), 2.87 (2H, d, J = 6.6Hz), 3.00 (2H, t, J = 6.6 Hz),

7.18–7.35 (5H, m); MS m/z: 142 (M⁺).

[³H]Granisetron binding assays were performed ac-

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

(50 mmol/L, pH 7.4) and the suspension was incubated at

37 °C for 10 min and centrifuged at 40 000 × g for

15 min. The pellet was washed and centrifuged (40 000 ×

g for 15 min). The ﬁnal pellet was resuspended in 30

volumes of HEPES buffer and used as tissue homogenate.

The binding assay consisted of 50 µmol/L of [³H]Gran-

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

(50 mM, pH 7.4). ICS 205930 (100 mmol/L) was used

for the determination of nonspeciﬁc binding.

The other piperidines (6b–6d) were prepared in a

similar manner.

5.2. Pharmacology

5.2.1. Radioligand binding assays

5.2.1.1. 5-HT₄receptor

5.2.1.3. D₂receptor

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 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

solution of [³H]GR113808 (Amersham International,

UK) to give a ﬁnal concentration of 0.1 nmol/L and

900 µL of tissue preparation. Each tube was incubated for

[³H]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

CaCl₂, 1 mmol/L MgCl₂, 1.1 mmol/L ascorbic acid and

10 µmol/L pargyline, and incubated at 37 °C for 10 min.

988

A portion of this membrane suspension (900 µmol/L) was

placed in a tube, and 50 µmol/L of either test compound

or vehicle solution was added, followed by 50 µL 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). Sulpiride (100 µmol/L) was

used for the determination of nonspeciﬁc binding. The

radioactivity trapped on the ﬁlters was measured by

liquid scintillation spectrometry.

5.3. Construction of receptor model

5.3.1. Sequence

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

RMS gradient was less than 0.05 (kcal/mol Å²). 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.

5.2.2. Contractile effects

5.2.2.1. 5-HT₄receptor agonism

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

10 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

5-HT₂and 5-HT₁-like and 5-HT₃receptors, respectively.

This solution was kept at 37 °C and gassed with 95% O₂,

5% CO₂. The strips were subjected to a preload of 1 g and

allowed to stabilize for 20 min. After stabilization, the

response of the longitudinal muscle to 30 µmol/L metha-

choline was measured. Agonist concentration-effect

curves were constructed using sequential dosing, leaving

15 min between doses. A 15 min dosing cycle was

required to prevent desensitization. The agonist was left

in contact with a preparation until 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

EC₅₀value, the concentration causing 50% of the maxi-

mal response, was determined by linear regression analy-

sis.

Acknowledgements

We thank Mrs. F. Matsugaki for some of the biological

results. We also thank Dr. M. Terasawa and Dr. K. Adachi

for helpful discussion.

References

[1] Alexander S.H., Peters J.A., TPiS Receptor and Ion Chanenel

Nomenclature Supplement, Elsevier, 1998, pp. 46–48.

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Article Doi

Synthesis and pharmacological evaluation of carboxamide derivatives as selective serotoninergic 5-HT4 receptor agonists (European Journal of Medicinal Chemistry (1999) 34 (329-987))

DOI: 10.1016/S0223-5234(99)00158-0

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