Synthesis and gastrointestinal prokinetic activity of novel benzamide derivatives with amphoteric side chains

Article abstract of DOI:10.1248/cpb.49.424

Novel benzamide derivatives (19-24, 32a-c, 43d-f), each possessing a cycloaminoalkanecarboxylic acid side chain, were synthesized and their gastrointestinal prokinetic and dopamine D₂ receptor antagonist activities were evaluated. 4-[(4-Amino-5

Full text of DOI:10.1248/cpb.49.424

424
Chem. Pharm. Bull. 49(4) 424—436 (2001)
Vol. 49, No. 4
Synthesis and Gastrointestinal Prokinetic Activity of Novel Benzamide
Derivatives with Amphoteric Side Chains
Jun SAKAGUCHI, ,a Nobuhiko IWASAKI,a Yuji IWANAGA,a Takaharu SAITO,a Eiji TAKAHARA,a
*
b
Hideo KATO,a and Miyoji HANAOKA
Research Division, Hokuriku Seiyaku Co., Ltd.,a Inokuchi 37–1–1, Katsuyama, Fukui 911–8555, Japan and Faculty of
Pharmaceutical Sciences, Kanazawa University,b Takara-machi 13–1, Kanzazawa, Ishikawa 920–0934, Japan.
Received October 19, 2000; accepted December 12, 2000
Novel benzamide derivatives (19—24, 32a—c, 43d—f), each possessing a cycloaminoalkanecarboxylic acid
side chain, were synthesized and their gastrointestinal prokinetic and dopamine D2 receptor antagonist activities
were evaluated. 4-[(4-Amino-5-chloro-2-methoxybenzoyl)amino]-1-piperidineacetic acid (19) exhibited the most
potent gastro- and colon-prokinetic activities, through intravenous administration to conscious dogs, and also
showed the reduced dopamine D2 receptor antagonistic activity. However, 19 showed only weak gastrointestinal
prokinetic activity after oral administration. Several ester prodrugs (44—62) of 19 were tested for pharmacologi-
cal activities as well as physicochemical and metabolic stability; the butyl ester (46) was consequently selected as
a promising gastrointestinal prokinetic agent with reduced side effects.
Key words gastroprokinetic activity; colon-prokinetic activity; 5-HT4 agonist; 4-[(4-amino-5-chloro-2-methoxybenzoyl)amino)]-1-
piperidineacetic acid
Metoclopramide (1a), possessing a dopamine D2 receptor atom of 1a, it had no significant gastrointestinal prokinetic
antagonist and weak serotonin 5-HT4 receptor agonist activi- activity. Cisapride (1b),4) which has a conformationally re-
ties,1a) is used clinically as a gastrointestinal prokinetic and stricted amino moiety in comparison with 1a, has been found
an antiemetic agent.1b) However, its clinical application is to show more potent gastrointestinal prokinetic activity than
limited due to its side effects, such as extrapyramidal syn- 1a. We therefore expected that some kind of conformational
drome, parkinsonism and elevated serum prolactin levels, restriction of 3 would lead to an increased gastrointestinal
caused by blockage of the dopamine D2 receptor.
prokinetic activity, and designed a series of [[(4-amino-5-
We previously reported that the introduction of a methyl- chloro-2-methoxybenzoyl)amino]cycloamino]alkanecar-
enephenoxy group between the benzamide moiety and the boxylic acid derivatives (4).
terminal aminoalkyl groups of 1a led to the discovery of ito-
In this paper, we describe the synthesis and gastrointesti-
pride (2), with an appropriate dopamine D2 receptor antago- nal prokinetic activity of a series of [[(4-amino-5-chloro-2-
nist activity and a distinctive acetylcholine esterase in- methoxybenzoyl)amino]cycloamino]alkanecarboxylic acids
hibitory activity.2) On the other hand, we suggested that am- (4). We also discuss conformational effects on gastrointesti-
photeric-ionization would be a useful approach to discover- nal prokinetic activity and the evaluation of ester prodrugs of
ing a new drug, and actually succeeded in finding selective 4-[(4-amino-5-chloro-2-methoxybenzoyl)amino]-1-
histamine H1 antagonists by applying this method to classical piperidineacetic acid (19), which exhibited the most excel-
antihistaminics.3) We initially applied the amphoteric-ioniza- lent gastrointestinal prokinetic activity.
tion to 1a in order to find a new type of gastrointestinal pro-
Chemistry New amphoteric compounds (19—24, 32a—
kinetic agent. Although we preliminarily synthesized an am- c, 38, 43d—f) and prodrugs (44—60) of compound 19 were
photeric-ionized compound (3) by the introduction of an synthesized as shown in Charts 2—6.
alkanecarboxylic acid moiety onto the terminal nitrogen
As shown in Chart 2, benzamides (19—24) with 1-
Chart 1
To whom correspondence should be addressed. e-mail: jun.sakaguchi@hokuriku-seiyaku.co.jp
© 2001 Pharmaceutical Society of Japan

April 2001
425
Chart 2
Chart 3
piperidinealkanecarboxylic acid side chains were prepared photeric benzamides (19—24).
by method A. Firstly, 4-amino-1-benzylpiperidine (5) was Benzamides (32a—c), possessing endo- or exo-9-azabicy-
treated with trifluoroacetic anhydride to give 6. Catalytic hy- clo[3.3.1]nonane-9-acetic acid, or an exo-8-azabicyclo-
drogenolysis of 6 with Pearlman’s catalyst, followed by con- [3.2.1]octane-8-acetic acid side chain, were prepared by
densation with trytyl chloride, afforded 8 via 7. Then deacy- method B as shown in Chart 3. 4-Acetylamino-2-methoxy-
lation of 8 with KOH gave 4-amino-1-tritylpiperidine (9). benzoic acid (25)6) was converted with ethyl chloroformate
Commercially available 4-amino-5-chloro-2-methoxybenzoic to the activated ester, which was condensed with endo- or
acid (10) was condensed with 9 to afford 11. Deprotection of exo-3-amino-9-benzyl-9-azabicyclo[3.3.1]nonane (26a, b)7)
11 with hydrochloric acid gave the secondary amine (12),5) or exo-3-amino-8-benzyl-8-azabicyclo[3.2.1]octane (26c)7) to
which was treated with ethyl or methyl w-bromoalkanecar- afford benzamides 27a—c. Catalytic hydrogenolysis of
boxylates, ethyl 2-bromopropionate or ethyl acrylate, to af- 27a—c with Pearlman’s catalyst gave secondary amines
ford various esters (13—18). The esters (13—18) were hy- 28a—c, and the subsequent treatment with ethyl bromoac-
drolyzed under basic conditions to provide the desired am- etate afforded compounds 29a—c. Chlorination of 29a—c

426
Vol. 49, No. 4
Chart 4
Chart 5
Chart 6
with sulfuryl chloride yielded 30a—c, which was deacety- yield 35. Compound 35 was treated with ethyl bromoacetate
lated with hydrochloric acid to give compounds 31a—c. to afford 36, which was subsequently deacetylated with hy-
Basic hydrolysis of 31a—c afforded the desired amphoteric drochloric acid to obtain ester (37). Compound 37 was hy-
benzamides (32a—c).
drolyzed with base to provide the desired amphoteric benza-
Benzamide (38), with an endo-8-azabicyclo[3.2.1]octane- mide (38).
8-acetic acid side chain, was synthesized by method C as
Heterocyclic carboxamides (43d—f), each possessing an
shown in Chart 4. Treatment of endo-4-acetylamino-5- amphoteric side chain, were prepared by method D as shown
chloro-2-methoxy-N-(8-methyl-8-azabicyclo[3.2.1]oct-3- in Chart 5. Acid chloride (39d—f)8,9) was condensed with
yl)benzamide (33)7) with methyl chloroformate gave ure- amine (26a) to afford amides (40d—f). Catalytic hydrogenol-
thane (34), which was exposed to trimethylsilyl iodide to ysis of 40d—f with Pearlman’s catalyst, followed by conden-

April 2001
427
sation with ethyl bromoacetate, afforded esters (42d—f) via N,N-dimethylformamide (DMF) in the presence of potassium
secondary amines (41d—f). The esters (42d—f) were subse- carbonate, to give the desired prodrugs (44—60).
quently hydrolyzed with base to give the desired amphoteric
carboxamides (43d—f).
Physicochemical data for compounds 13—24, 28a—c,
29a—c, 30a—c, 31a—c, 32a—c, 36—38, 42d—f, 43d—f
The ester prodrugs (44—60) of 19 were prepared by and 44—60 are given in the Experimental section or in Ta-
method E as shown in Chart 6. Compound 19 was esterified bles 1—4.
with alcohol using an acid catalyst, or with various halides in
Table 1. Physicochemical Data for Monocyclic Esters and Carboxylic Acids 15—18, 20—24
Analysis (%) Calcd (Found)
Compd.
R
Method
mp, °C (recryst. solvent)a)
Formula
C
H
N
15
16
17
18
20
21
22
23
24
(CH2)3CO2Et
(CH2)4CO2Et
(CH2)5CO2Me
CH(Me)CO2Et
(CH2)2CO2H
(CH2)3CO2H
(CH2)4CO2H
(CH2)5CO2H
CH(Me)CO2H
A
A
A
A
A
A
A
A
A
188.5—191.5
(E)
202—203.5
(E)
208.5—210.5
(M)
158—159
(E)
218—219.5
(W)
228.5—231.5
(W)
226—227.5
(W)
223—225
(W)
C19H28ClN3O4·HCl
52.54
(52.18
53.04
(52.99
51.01
(51.06
52.85
(52.67
48.99
(48.80
48.12
(47.97
49.32
(49.18
50.45
(50.33
50.79
(50.59
6.73
6.66
7.01
6.95
7.17
7.03
6.05
5.94
5.91
5.84
6.41
6.34
6.67
6.45
6.91
6.84
6.53
6.25
9.67
9.75)
9.28
9.21)
8.92
8.99)
8.40
8.39)
10.71
10.68)
9.90
10.12)
9.59
9.54)
9.29
9.25)
11.11
11.06)
C20H30ClN3O4·HCl·1·4H2O
C20H30ClN3O4·HCl·5·4H2O
b)
C18H26ClN3O4·C4H4O4
C16H22ClN3O4·HCl
C17H24ClN3O4·HCl·H2O
C18H26ClN3O4·HCl·H2O
C19H28ClN3O4·HCl·H2O
C16H22ClN3O4·5·4H2O
246—247
(W)
a) EϭEtOH, MϭMeOH, WϭWater. b) Fumaric acid.
Table 2. Physicochemical Data for Bicyclic Compounds 28—32
Analysis (%) Calcd (Found)
Compd.
n
R1 R2
R
endo/exo Method mp, °C (recryst. solvent)a)
Formula
C
H
N
28b
28c
29b
29c
30b
30c
31b
31c
32b
32c
3
2
3
2
3
2
3
2
3
2
Ac
Ac
Ac
Ac
H
H
H
H
H
exo
exo
exo
exo
exo
exo
exo
exo
exo
exo
B
B
B
B
B
B
B
B
B
B
194—196 (dec.)
(M)
223—225 (dec.)
(M)
oil
C18H25N3O3·C4H4O4b)·3/4H2O
57.32 6.67
(57.13 6.58
58.19 6.28
(58.05 6.45
417.2264d)
9.12
9.12)
9.69
9.71)
b)
H
C17H23N3O3·C4H4O4
CH2CO2Et
CH2CO2Et
C22H31N3O5
(417.2253)
oil
C21H29N3O5
403.2107d)
(403.2111)
Ac Cl CH2CO2Et
Ac Cl CH2CO2Et
125—128
(M)
174—175
(E)
161—164
(IP–DE)
188.5—190
(E–DE)
250—252 (dec.)
(M)
C22H30N3O5·3/4 C4H4O4b)·7/4H2O
52.63 6.45
(52.40 6.08
54.20 5.82
(54.03 5.85
56.73 7.02
(56.73 6.63
57.65 6.62 10.61
(57.60 6.57 10.49)
54.68 6.50 10.63
(54.68 6.25 10.94)
55.51 6.03 11.42
(55.20 6.05 11.20)
7.37
7.67)
7.58
7.56)
9.92
9.96)
c)
C21H28ClN3O5·C4H4O4
H
H
H
H
Cl CH2CO2Et
Cl CH2CO2Et
Cl CH2CO2H
Cl CH2CO2H
C20H28ClN3O4·3/4H2O
C19H26ClN3O4
C18H24ClN3O4·3/4H2O
C17H22ClN3O4
273—276 (dec.)
(M–DE)
a) DEϭEt2O, DIϭiso-Pr2O, EϭEtOH, IPϭiso-PrOH, MϭMeOH, WϭWater. b) Fumaric acid. c) Maleic acid. d) High resolution mass spectra.

428
Vol. 49, No. 4
Table 3. Physicochemical Data for Heterocyclic Compounds 42—43
Analysis (%) Calcd (Found)
Compd.
R
Method
D
mp, °C (recryst. solvent)a)
Oil
Formula
C21H27N3O3
C22H29N3O3
C
H
N
42e
Et
369.2052b)
(369.2057)
42f
43e
Et
H
D
D
155—156
(E)
68.90
(68.60
7.62
7.48
10.96
11.06)
235—240 (dec.)
(W)
C19H23N3O3·
HCl
60.39
(60.39
6.40
6.42
11.12
11.22)
43f
H
D
200—202
(W)
C20H25N3O3·
HCl·H2O
60.33
(60.27
7.36
7.39
9.59
9.71)
a) EϭEtOH, WϭWater. b) High resolution mass spectra.
Table 4. Physicochemical Data for Prodrugs 46—60
Analysis (%) Calcd (Found)
Compd.
R
Method
mp, °C (recryst. solvent)a)
Formula
C
H
N
46
47
48
49
50
51
52
53
54
55
56
57
n-Bu
F
F
F
F
F
F
F
F
F
F
F
F
209—211.5
(IP)
202.5—203
(IP)
133—137
(IP)
125—130
(IP)
207—208
(IP)
199.5—201
(IP)
147—150
(E)
174—175
(E)
174—175
(E)
196—201
(E)
190—191
(A)
124—129.5
C19H28ClN3O4
48.63
(48.37
49.65
(49.46
53.50
(53.26
47.37
(47.46
48.19
(48.14
48.36
(48.24
50.34
(50.40
56.99
(56.98
44.97
(44.88
45.84
(45.66
53.46
(53.59
50.37
(50.42
6.53
6.81
6.75
6.98
7.27
7.27
6.85
6.67
6.57
6.80
6.86
6.64
5.95
5.89
5.52
5.72
5.86
5.75
5.77
5.82
6.37
6.50
5.61
5.47
8.51
8.44)
8.27
8.22)
8.91
8.82)
9.21
9.14)
8.43
8.36)
8.06
8.02)
8.00
8.03)
7.67
7.67)
8.28
·CH4O3Sb)
n-Pentyl
C20H30ClN3O4
·CH4O3Sb)
n-Hex
C21H32ClN3O4·HCl
·1/2H2O
C18H26ClN3O4·HCl
·2H2O
iso-Pr
iso-Bu
C19H28ClN3O4
·CH4O3Sb)·1/4H2O
C20H30ClN3O4
·CH4O3Sb)·3/4H2O
C18H26ClN3O5
·C4H4O4c)·1/2H2O
C22H26ClN3O4
iso-Pentyl
CH2CH2OMe
Benzyl
c)
·C4H4O4
CH2COMe
CH2CO2Et
CH2CONMe2
C18H24ClN3O5
·CH4O3Sb)·3/4H2O
C19H26ClN3O6
·CH4O3Sb)
8.19)
8.02
7.88)
13.12
13.31)
7.66
C19H27ClN4O5
C19H26ClN3O6
·C4H4O4c)·1/4H2O
7.73)
(E)
58
59
60
F
F
F
160—164
(E)
C21H30ClN3O6·HCl
·5/4H2O
48.98
(48.74
6.56
6.25
8.16
8.20)
152—156.5
(E)
C21H30ClN3O7·2HCl
·1/4H2O
45.91
(45.81
5.96
5.83
7.65
7.70)
118.5—126
(M–IP)
C24H34ClN3O7·HCl
·H2O
50.89
(50.88
6.58
6.29
7.42
7.42)
a) AϭAcOEt, EϭEtOH, IPϭiso-PrOH, MϭMeOH. b) Methanesulfonic acid. c) Fumaric acid.

April 2001
429
Results and Discussion
ited excellent gastro- and colon-prokinetic activities in com-
The gastrointestinal prokinetic activities of all amphoteric parison with 1b. In addition, the introduction of a methyl
compounds (19—24, 32a—c, 38, 43d—f) in conscious dogs group (24) into the a-position of the carboxy group in 19 led
were determined using the method of Z. Itoh.10) The gastric to a slight reduction in gastro- and colon-prokinetic activi-
antrum motility of each compound was quantified by deter- ties. These results suggested that the distance between the
mining a motor index, which was equivalent to the integrated basic nitrogen atom and the carboxy group would be an im-
area between the contractile wave and the base line, and ex- portant factor for gastro- and colon-prokinetic activities.
pressed as a symbol based on a percentage against the basal
The dopamine D2 receptor affinity of the N-methyl analog
motor index. Colonic motility was expressed by the % ratio (61) was moderate (pIC50ϭ5.7), while none of the ampho-
of the number of dogs, in which the giant contraction on the teric compounds showed significant affinity. This result sug-
ascending colon was observed, versus the tested dogs. The gested that amphoteric-ionization caused a decrease in D2
dopamine D2 receptor binding affinity, which was responsible binding affinity.
for the side effect of benzamides, such as 1a and 1b, was es-
We selected the acetic acid moiety (nϭ1) as a substituent
timated and expressed by pIC50 (-logarithm of inhibitory con- on the basic nitrogen atom for further optimization, since 19
centration 50%) values. The pharmacological results are exhibited the most excellent gastro- and colon-prokinetic ac-
given in Tables 5—7.
tivities among monocyclic amphoteric compounds (19—24).
Firstly, we examined monocyclic amino compounds (Table
Secondly, we addressed our attention to the compounds
5). Amphoteric compounds (19—23) with a straight methyl- with a bicyclic amine moiety. As shown in Table 6, bicyclic
ene chain, except for 22, showed more potent gastro- and amphoteric compounds (32a—c, 38) showed similar or less
colon-prokinetic activities than N-methyl analog (61)11) or potent gastro- or colon-prokinetic activity in comparison
1a, and elongation of the methylene chain led to a decrease with the corresponding N-methyl analogs (62—65)7) in con-
in both prokinetic activities. Compound 19 especially exhib- trast to the case of monocyclic compounds. Especially, 32b
Table 5. Pharmacological Data for Monocyclic Benzamides
Gastric antruma) (mg/kg, i.v.) Ascending colonb) (mg/kg, i.v.)
Compd.
R
D2 binding pIC50
0.1
1.0
0.1
1.0
19
20
21
22
23
24
61
CH2CO2H
(CH2)2CO2H
(CH2)3CO2H
(CH2)4CO2H
(CH2)5CO2H
CH(Me)CO2H
Me
ϩϩ
ϩϩ
ϩ
NT
Ϫ
ϩ
Ϫ
Ϫ
ϩ
ϩϩ
ϩϩ
ϩϩ
Ϫ
ϩϩ
ϩ
ϩ
ϩ
ϩϩ
75
0
0
NT
0
0
0
0
0
100
25
100
0
50
75
0
Ͻ4
Ͻ4
Ͻ4
Ͻ4
4.4
Ͻ4
5.7
6.7
7.0
Metoclopramide (1a)
Cisapride (1b)
0
25
a) The symbols have the following meanings: ϩϩ, 150% or more; ϩ, 125% to 150%; Ϫ, less than 125%; NT, not tested. b) % ratio of number of dogs in which giant con-
traction was observed versus the tested dogs.
Table 6. Pharmacological Data for Bicyclic Benzamides
Gastric antruma)
(mg/kg, i.v.)
Ascending colonb)
(mg/kg, i.v.)
Compd.
n
R
endo/exo
D2 binding pIC50
0.1
1.0
0.1
1.0
32a
32b
32c
38
62
63
3
3
2
2
3
3
2
2
CH2CO2H
CH2CO2H
CH2CO2H
CH2CO2H
Me
Me
Me
Me
endo
exo
exo
endo
endo
exo
ϩ
NT
ϩ
Ϫ
ϩ
ϩ
ϩ
ϩϩ
ϩϩ
Ϫ
0
NT
33
0
0
0
75
0
67
100
50
0
5.0
Ͻ4
Ͻ4
Ͻ4
5.9
6.3
6.2
Ͻ4
ϩϩ
ϩϩ
ϩϩ
ϩϩ
ϩϩ
ϩϩ
64
65
exo
endo
67
0
67
33
a, b) See footnote a and b in Table 5.

430
Vol. 49, No. 4
Table 7. Pharmacological Data for Heterocyclic Carboxamides
Gastric antruma)
(mg/kg, i.v.)
Ascending colonb)
(mg/kg, i.v.)
Compd.
R
D2 binding pIC50
1.0
1.0
66
Me
Ϫ
Ϫ
0
0
Ͻ4
Ͻ4
43d
CH2COOH
67
Me
Ϫ
Ϫ
0
0
Ͻ4
Ͻ4
43e
CH2COOH
68
Me
Ϫ
Ϫ
0
0
4.2
Ͻ4
43f
CH2COOH
a, b) See footnote a and b in Table 5.
Table 8. Penetration of Compound 19 and Cisapride into Brain in Mice
Table 9. Effect of SB-207266 on Gastrointestinal Contraction Induced by
Intravenous Compound 19 Administration in Conscious Dogs
Plasma concentration Brain concentration
Compd.
Brain/Plasma
(mg/ml)
(mg/ml)
Dose
(mg/kg, i.v.)
Compd.
Gastric antruma) Ascending colonb)
19
Cisapride (1b)
23.25Ϯ1.32
2.24Ϯ0.46
0.42Ϯ0.059
5.73Ϯ1.77
0.018
2.40
19
19ϩSB-207266
1.0
1.0ϩ0.1
ϩϩ
Ϫ
100
0
a, b) See footnote a and b in Table 5.
showed no significant gastro- and colon-prokinetic activities.
Concerning D2 binding affinity, bicyclic amphoteric com-
pounds (32a—c, 38) showed less potent affinity than the cor-
responding bicyclic N-methyl derivatives (62—65), and am-
photeric-ionization of bicyclic compounds was found to
cause the decrease in dopamine D2 affinity. This observation
was consistent with the previous result obtained from mono-
cyclic compounds.
Also examined were the replacements of the benzene ring
of 32a, the most potent gastro- and colon-prokinetic active
compound among the bicyclic amphoteric compounds, by
some heterocyclic rings (Table 7). These replacements
caused the disappearance in both gastrointestinal prokinetic
activity and dopamine D2 affinity in not only N-methyl
analogs (66—68) but also the corresponding amphoteric
compounds (43d—f). Consequently, the benzene ring of 32a
seemed to be essential for the gastrointestinal prokinetic ac-
tivity.
On the basis of the above screening results, 19, with an
amphoteric monocyclic side chain, was selected as the candi-
date for a novel gastrointestinal prokinetic agent, exhibiting
both the gastro- and colon-prokinetic activities without D2
binding affinity. Furthermore, we examined its penetration
into the central nervous systems (CNS). As shown in Table 8,
19 showed a 100 times lower brain/plasma ratio than 1b and
thus amphoteric-ionization was also found to be effective in
lowering the penetration into the brain. Accordingly, 19 was
expected to show no significant CNS side effect.
influence of the selective 5-HT4 antagonist, SB-207266,12) on
the gastric and colonic contractions induced by 19 was evalu-
ated. As shown in Table 9, the selective 5-HT4 antagonist
blocked both gastric and colonic contractions. This result
suggested that the 5-HT4 receptor agonist activity would par-
ticipate in the mechanism for the gastrointestinal prokinetic
effect of 19.
The result shown in Table 9 allowed us to examine the re-
lationship between gastrointestinal effects and 5-HT4 agonist
activity by conformational analysis.
Serotonin 5-HT4 agonist activity of 19, 24, 32a—c and 38
was evaluated by their effects on carbachol-induced tone in
rat esophagus and expressed as the negative logarithm of the
molar concentration that exhibited 50% relaxation to the car-
bachol-induced contraction (pEC50), as shown in Table 10.
On the other hand, we implemented the conformational
analysis of 19, 24, 32a—c and 38 according to the method of
López-Rodríguez et al.13) The molecules were built de novo
in their protonated forms, which are believed to be bioactive
forms, using the CAChe system,14) and their geometry was
optimized by the MM2 force field of the CAChe system. For
the first purpose, we applied a systematic conformational
analysis around four rotatable bonds, as shown in Fig. 1, and
calculated the energy by 15° stepwise increments of the dihe-
dral angles. Then the lower energy conformers of each com-
pound were energy-minimized by the MM2 force field. We
tried to identify the lowest energy conformer of each com-
pound accordingly. Generally, the oxygen atom substituted at
5-HT4 Receptor Agonist Activity and Conformational
Analysis In order to clarify the mode of action for 19, the

April 2001
431
the ortho position of the benzamide is considered to form an and 5-HT4 receptor for enhancing 5-HT4 agonist activity (see
intramolecular hydrogen bond with the NH of the amide Fig. 3). The distances Ar–N of endo-bicyclic compounds
group. Concerning the dihedral angle (t), defined by [C1– (32a, 38) were also found to be shorter than that of mono-
C2–C3–O4] (see Fig. 1), we selected only the conformation cyclic compound (19), and it seemed to be one of the reasons
of each compound whose t was near 180°. López-Rodríguez they showed weak 5-HT4 agonist activity in comparison with
et al. studied a 5-HT4 pharmacophore model by their confor- 19. On the other hand, 5-HT4 agonist activities of all ampho-
mational analysis with various types of 5-HT4 ligands. They teric compounds almost correlated with their in vivo gas-
proposed four essential criteria for active conformation as trointestinal prokinetic activities. This result suggested that
shown in Fig. 2: 1) the basic nitrogen atom situated at an av- 5-HT4 agonist activities of amphoteric compounds could
erage distance of 8.0Ϯ0.1 Å from the centroid of the benzene contribute to their in vivo gastrointestinal prokinetic effects.
ring (Ar–N); 2) the oxygen atom of the amide’s carbonyl Compound 19, however, exhibited excellent in vivo gastroin-
group situated at an average distance of 3.6Ϯ0.1 Å from the testinal prokinetic activity after intravenous administration in
centroid of the benzene ring (Ar–O); 3) the oxygen atom of dogs in spite of weaker 5-HT4 agonist activity than 1b
the amide’s carbonyl group situated at an average distance of (pEC50ϭ7.26).15) Therefore, it was considered that other
5.4Ϯ0.1 Å from the basic nitrogen atom (O–N); and (4) the mechanisms contributed to the gastrointestinal activity of 19.
basic nitrogen atom deviated at a distance of 3.6—4.0 Å from
Examination of Prodrugs of Compound 19 As de-
the plane of the benzene ring (h). To identify the active con- scribed above, compound 19, with an amphoteric monocyclic
formation, we actually performed a conformational analysis side chain, was selected as a candidate for a novel gastroin-
and energy minimization of each compound around the dihe- testinal prokinetics. Unfortunately, 19 showed only weak gas-
dral angle (q), defined by [C3–N5–C6–C7] (see Fig. 1) of trointestinal activity by oral administration in further evalua-
each compound, by 1° stepwise increments of the dihedral tion. It seemed that poor oral absorption of 19 was attributed
angle, and then examined to see if the selected conformers of to its low lipophilicity. On the other hand, the ethyl ester
each compound were adapted to four structural criteria in the (13), its synthetic precursor, had potent gastrointestinal activ-
pharmacophore model, as described above.
ity after oral administration, but an undesired emetic side ef-
Except for the deviations (the criterion 4); h), the struc- fect was observed. We therefore set about examining the
tural parameters of 19, 24, 32b and 32c corresponded well other various ester prodrugs of 19.
with the above three criteria (1 to 3) of the 5-HT4 pharma-
We tested for the gastrointestinal prokinetic and emetic ac-
cophore model, but the 5-HT4 agonist activity of 24, 32b and tion as well as the physicochemical and metabolic stability of
32c was weaker than that of compound 19. The a-methyl the prodrugs. Their results were summarized in Table 11.
group of 24 and the alkylene-bridged chains of exo-bicyclic
Among the double ester type of prodrugs (57—60), 58
compounds (32b, 32c) were presumed to situate at the same showed the best pharmacological effect, but its physicochem-
side of their amide carbonyl groups and which might disturb
the tight interaction in the binding mode between the ligand
Fig. 2. 5-HT4 Pharmacophore Model
Fig. 1
Fig. 3. Presumed Active Conformation of Compounds 19, 24, 32a—c, 38 Calculated by the Molecular Modeling Program CAChe (Version 4.1.1)

432
Vol. 49, No. 4
Table 10. Energy and Structural Parameters of the Conformations and 5-HT4 Agonist Activity
Energy
(kcal/mol)
Ea)
(kcal/mol)
5-HT4 agonist activity
(pEC50)
Compd.
tb)
qb)
vb)
yb)
Ar–N (Å)c) Ar–O (Å)d) O–N (Å)e)
h (Å)f)
19
24
32a
32b
32c
38
Ϫ10.79
Ϫ7.56
7.87
0.29
0.03
0.06
0.04
0.01
0.16
Ϫ148.9
Ϫ146.8
Ϫ143.0 Ϫ127.7
Ϫ148.1
Ϫ146.5
Ϫ145.5 Ϫ122.8
89.7 167.3
108.8 164.9
65.3 Ϫ145.3
108.4 164.9
142.9
141.6
7.93
7.93
7.57
7.83
7.82
3.51
3.51
3.50
3.51
3.50
5.47
5.56
5.75
5.65
5.69
5.75
1.49
1.54
2.20
2.02
1.72
1.79
6.11
5.64
5.13
4.62
5.40
5.32
—
3.88
2.50
4.61
149.8
150.2
111.8 175.5
69.9 Ϫ149.3
7.48
8.0Ϯ0.1
3.50
3.6Ϯ0.1
Pharmacophore model
5.4Ϯ0.1 3.6—4.0
a) Increment of energy with respect to the lowest energy conformation. b) Torsion angle (°) defined in Fig. 1. c) Distance between the centroid of the benzene ring and
the basic nitrogen of the amine. d) Distance between the centroid of the benzene ring and the oxygen of the carboxyl group. e) Distance between the oxygen of the carboxyl
group and the basic nitrogen of the amine. f) Deviation of the basic nitrogen with respect to the benzene ring.
Table 11. Pharmacological Data for Prodrugs (13, 44—60)
Stability
Vomiting in ferrets
Gastric antruma)
(1 mg/kg, i.d.)
Ascending colonb)
(1 mg/kg, i.d.)
Compd.
R
(10 mg/kg, p.o.)
Physicochemicalc)
residual %
Metabolicd)
residual %
Response (%)
13
44
45
46
47
48
49
50
51
52
53
54
55
56
Et
Me
n-Pr
n-Bu
n-Pentyl
n-Hex
iso-Pr
iso-Bu
iso-Pentyl
CH2CH2OMe
Benzyl
CH2COMe
CH2CO2Et
CH2CONMe2
ϩϩ
ϩϩ
ϩϩ
ϩϩ
ϩϩ
ϩϩ
ϩϩ
ϩϩ
ϩϩ
ϩϩ
ϩϩ
ϩ
100
100
80
100
80
80
80
100
60
100
40
100
75
25
0
99
97
97
97
98
61
69
49
22
2
0
13
50
13
0
0
0
0
0
0
99
98
6
80
33
7
70
30
NDf)
25
97
NTe)
100
96
100
96
96
60
60
40
ϩϩ
ϩ
92
57
58
59
60
ϩϩ
ϩϩ
ϩϩ
ϩϩ
80
100
40
0
0
0
0
82
73
85
84
NDf)
NDf)
NDf)
7
60
a, b) See footnote a and b in Table 5. c) Physicochemical stability in buffer solution (pH 6.8). d) In vitro metabolic stability with human liver S9. e) Not tested.
f) Not detected.
ical stability was not so good. Although benzyl ester (53) and nal prokinetic activity without significant CNS or emetic side
alkyl esters, having functional groups (54—56), showed no effects.
emetic action, they had only moderate gastrointestinal proki-
netic action. Methoxyethyl ester (52) showed good pharma- Conclusion
cological actions but it was expected to be tolerant to hydrol-
1) The combination of amphoteric-ionization and cy-
ysis. Among straight or branched alkyl esters (44—51), n- clization of the terminal amine moiety of 1a caused an in-
butyl (46) and n-pentyl (47) esters were preferable in terms crease in gastro- and colon-prokinetic activities and a de-
of pharmacological and pharmacokinetic profiles. We se- crease in dopamine D2 receptor affinity. This combination
lected the n-butyl prodrug (46), which exhibited the most po- seemed to be an effective approach to a selective gastroin-
tent gastrointestinal prokinetic activity, as a developing can- testinal prokinetic activity devoid of other pharmacological
didate.
activity such as CNS side effects, and also to generating mar-
Compound 46 also did not show the emetic side effect velous colon-prokinetic activity.
after oral administration in dogs. After oral administration of
2) Amphoteric-ionization was also effective for reducing
46 (10 mg/kg) in rats, only 19 was detected in plasma. Ac- the penetration into the brain.
cordingly, 46 was expected to exhibit excellent gastrointesti- 3) The 5-HT4 receptor agonist activity of 19 was consid-

April 2001
433
recrystallized from MeOH to afford the hydrochloride (20.8 g, 72%) of 7 as
a colorless crystal, mp 236—240 °C. Anal. Calcd For C7H11F3N2O·HCl: C,
36.14; H, 5.20; N, 12.04. Found: C, 36.01; H, 5.12; N, 12.25. MS m/z: 196
(Mϩ). IR n (liq) cmϪ1: 1720. 1H-NMR (DMSO-d6): 1.75—1.95 (2H, m),
2.05—2.20 (2H, m), 3.05—3.20 (2H, m), 3.40—3.55 (2H, m), 3.95—4.15
ered to participate partially in the gastrointestinal prokinetic
effect.
4) According to our conformational analyses, the a-
methyl group in 24 and the alkylene-bridged chain in exo-bi-
cyclic compounds (32b, 32c) were found to be located at the (1H, m).
N-(1-Trityl-4-piperidyl)trifluoroacetamide (8)
A
mixture of
7
same side of their amide carbonyl groups. These conforma-
tions might disturb the interaction in the binding mode be-
tween the ligand and 5-HT4 receptor, leading to their lower
gastrointestinal activity.
5) The n-butyl ester (46) of 19 was found to improve
oral availability compared to 19, without an emetic side ef-
fect.
Further studies of the candidate, the ester 46 (Code No:
AU-224), as a new gastrointestinal prokinetic agent for pre-
clinical evaluation, is now in progress, and the mechanism
for the active metabolite 19 is also being examined in detail.
(186.4 g, 950 mmol), trityl chloride (264.8 g, 950 mmol) and Et3N (145.7 ml,
1.05 mol) in CH2Cl2 was stirred at room temperature for 3 h. Water
(1000 ml) was poured into the reaction mixture and the mixture was ex-
tracted with CH2Cl2. The extracts were evaporated and the resulting residue
was washed with diisopropyl ether (iso-Pr2O) to afford 303.5 g (73%) of 8 as
a colorless crystal, which was used for the next step without further purifica-
tion. MS m/z: 438 (Mϩ). IR n (KBr) cmϪ1: 1716, 1696. H-NMR (CDCl3):
1
1.35—1.60 (2H, m), 1.70—1.80 (2H, m), 1.90—2.05 (2H, m), 2.95—3.20
(2H, m), 3.60—3.70 (1H, m), 6.05—6.15 (1H, m), 7.15—7.50 (15H, m).
4-Amino-1-tritylpiperidine (9) A mixture of 8 (206.4 g, 471 mmol),
KOH (62.13 g, 941 mmol) in H2O (1 l) and EtOH (1 l) was refluxed for 2.5 h.
After cooling, the resulting precipitate was collected by filtration and
washed successively with water and n-hexane, to afford 151.1 g (94%) of 9
as a colorless crystal, which was used for the next step without further pu-
rification. MS m/z: 342 (Mϩ). IR n (KBr) cmϪ1: 1596. H-NMR (CDCl3):
1.30—1.95 (6H, m), 2.40—2.60 (1H, m), 2.85—3.20 (2H, m), 7.10—7.55
(15H, m).
1
Experimental
Chemistry All melting points were measured on a Yanagimoto melting
point apparatus and are uncorrected. Spectral data were obtained using the
following apparatus: 1H-NMR spectra with JEOL FX-90Q (90 MHz), JEOL
EX-270 (270 MHz) and JEOL A-500 (500 MHz) spectrometers; mass spec-
tra (MS) with JEOL JMS-DX 300 mass spectrometer; IR spectra with Hi-
tachi 270-30 spectrometer. Chemical shifts are expressed as d (ppm) values
with tetramethylsilane (TMS) as an internal standard. Column chromatogra-
phy was carried out with Kieselgel 60 (Merck) or Aluminium oxide 90
(Merck). Elemental analyses were performed using Yanagimoto MT-3 and
MT-5 elemental analysis apparatus, and analytical results were within
Ϯ0.4% of theoretical values. TLC was conducted on a 0.25 mm pre-coated
silica gel plate (60F254, Merck), and spots were detected by inspection under
short (254 nm) wavelength UV light, or by the colors developed with iodine.
Organic extracts were dried over anhydrous Na2SO4. The solvent was evapo-
rated under reduced pressure.
4-Amino-5-chloro-2-methoxy-N-(1-trityl-4-piperidyl)benzamide (11)
A mixture of 10 (16.4 g, 81.3 mmol), Et3N (13.6 ml, 97.7 mmol) and
ClCO2Et (8.55 ml, 89.9 mmol) in anhydrous tetrahydrofuran (THF, 330 ml)
was stirred under ice cooling. After 2 h, 9 (30.6 g, 89.3 mmol) was added
dropwise to the mixture and stirred at room temperature for 20 h. The insol-
uble substance was filtered off and the filtrate was evaporated. The resulting
residue was dissolved in CH2Cl2. The CH2Cl2 solution was washed with
water, dried and evaporated. The residue was washed with n-hexane to af-
ford 39.4 g (92%) of 11 as a colorless crystal, which was used for the next
step without further purification. MS m/z: 525, 527 (3 : 1, Mϩ). IR n (KBr)
cmϪ1: 1646, 1624. 1H-NMR (CDCl3): 1.55—1.80 (2H, m), 1.90—2.20 (2H,
m), 2.70—3.40 (2H, m), 3.55—4.10 (3H, m), 4.33 (2H, br s), 6.25 (1H, s),
7.05—7.55 (15H, m), 7.67 (1H, br s), 8.06 (1H, s).
4-Amino-5-chloro-2-methoxy-N-(4-piperidyl)benzamide (12) A mix-
ture of 11 (39.4 g, 74.9 mmol) and concentrated HCl (10 ml) in acetone
(600 ml) was refluxed for 40 min. After cooling, the resulting precipitate was
collected by filtration and washed with acetone to afford the hydrochloride
(25.8 g, 78%) of 125) as a colorless crystal, which was recrystallized from
EtOH to afford colorless crystals, mp 214—217 °C. Anal. Calcd for
C13H18ClN3O2·HCl·1/2H2O: C, 47.43; H, 6.12; N, 12.76. Found: C, 47.64;
H, 6.39; N, 12.97. MS m/z: 283, 285 (3 : 1, Mϩ). IR n (KBr) cmϪ1: 2948,
2812, 1640. 1H-NMR (DMSO-d6): 1.60—1.85 (2H, m), 1.90—2.15 (2H, m),
2.85—3.10 (2H, m), 3.10—3.35 (2H, m), 3.83 (3H, s), 3.85—4.10 (1H, m),
6.35 (1H, s), 7.62 (1H, s), 7.65—7.80 (1H, m).
The following known intermediates and reference compounds were pre-
pared essentially according to the literature: 4-amino-5-chloro-N-[2-(diethyl-
amino)ethyl]-2-methoxybenzamide (metoclopramide 1a),6) cis-4-amino-5-
chloro-N-[1-[3-(4-fluorophenoxy)propyl]-3-methoxy-4-piperidyl]-2-
methoxybenzamide (cisapride 1b),4) 4-acetylamino-2-methoxybenzoic acid
(25),6) endo- or exo-3-amino-9-benzyl-9-azabicyclo[3.3.1]nonane (26a, b),7)
exo-3-amino-8-benzyl-8-azabicyclo[3.2.1]octane (26c),7) 1-methyl-1H-inda-
zole-3-carbonyl chloride (39d),8 1H-indole-3-carbonyl chloride (39e),9) 1-
methyl-1H-indole-3-carbonyl chloride (39f),9) 4-amino-5-chloro-N-(1-
methyl-4-piperidyl)-2-methoxybenzamide (61),11) endo-4-amino-5-chloro-2-
methoxy-N-(9-methyl-9-azabicyclo[3.3.1]nonan-3-yl)benzamide (62),7) exo-
4-amino-5-chloro-2-methoxy-N-(9-methyl-9-azabicyclo[3.3.1]nonan-3-
yl)benzamide (63),7) exo-4-amino-5-chloro-2-methoxy-N-(8-methyl-8-azabi-
cyclo[3.2.1]oct-3-yl)benzamide (64),7) endo-4-amino-5-chloro-2-methoxy-
N-(8-methyl-8-azabicyclo[3.2.1]oct-3-yl)benzamide (BRL24682; 65),7)
endo-1-methyl-N-(9-methyl-9-azabicyclo[3.3.1]non-3-yl)-1H-indazole-3-
carboxamide (66),8) endo-N-(9-methyl-9-azabicyclo[3.3.1]non-3-yl)indole-
3-carboxamide (67),7) endo-1-methyl-N-(9-methyl-9-azabicyclo[3.3.1]non-
3-yl)indole-3-carboxamide (68).7)
Method A. N-(1-Benzyl-4-piperidyl)trifluoroacetamide (6) A solu-
tion of 4-amino-1-benzylpiperidine 5 (25.0 g, 131 mmol) and trifluoroacetic
anhydride (20.4 ml, 147 mmol) in CH2Cl2 (100 ml) was stirred at room tem-
perature for 30 min. The reaction mixture was concentrated and saturated
aqueous NaHCO3 was added to the residue. The mixture was extracted with
CH2Cl2. The extract was dried and evaporated. The resulting residue was
converted to the hydrochloride in the usual way to give the hydrochloride
(41.6 g, 98%) of 6 as a colorless crystal, which was recrystallized from
AcOEt to give colorless needles, mp 205—207 °C. Anal. Calcd For
C14H17F3N2O·HCl: C, 52.10; H, 5.62; N, 8.68. Found: C, 51.89; H, 5.53; N,
8.67. MS m/z: 286 (Mϩ). IR n (liq) cmϪ1: 1714. 1H-NMR (DMSO-d6):
1.25—2.65 (10H, m), 3.35 (1H, br s), 3.55—3.95 (2H, m), 4.13 (3H, s),
4.25—4.80 (1H, m), 7.15—7.80 (3H, m), 8.05—8.35 (1H, m), 8.40—9.40
(1H, m).
Ethyl 4-[(4-Amino-5-chloro-2-methoxybenzoyl)amino]-1-piperidineac-
etate (13) A mixture of the hydrochloride of 12 (3.20 g, 9.91 mmol), ethyl
bromoacetate (1.22 ml, 11.0 mmol) and K2CO3 (3.04 g, 22.0 mmol) in DMF
(32 ml) was stirred at 60 °C for 2.5 h. After evaporation of the solvent, water
was added to the residue and extracted with CH2Cl2. The organic layer was
washed with water, dried and evaporated to afford yellow oily residue. The
residue was converted to the hydrochloride in the usual manner to afford the
hydrochloride (3.31 g, 73%) of 13 as a pale yellow crystal, which was re-
crystallized from EtOH to afford colorless flakes, mp 196.5—198.5 °C.
Anal. Calcd for C17H24ClN3O4·HCl: C, 50.25; H, 6.20; N, 10.34. Found: C,
49.98; H, 6.23; N, 10.36. MS m/z: 369, 371 (3 : 1, Mϩ). IR n (KBr) cmϪ1
1758, 1650. H-NMR (DMSO-d6): 1.26 (3H, t, Jϭ7.0 Hz), 1.75—2.20 (4H,
m), 3.05—3.70 (4H, m), 3.75—4.20 (1H, m), 3.84 (3H, s), 4.17 (2H, s), 4.24
(2H, q, Jϭ7.0 Hz), 6.54 (1H, s), 7.63 (1H, s), 7.74 (1H, br s).
:
1
Compounds 15—18 were prepared in a manner similar to that described
above and their physicochemical data were summarized in Table 1.
Ethyl 4-[(4-Amino-5-chloro-2-methoxybenzoyl)amino]-1-piperidine-
propionate (14) A mixture of 12 (2.79 g, 9.80 mmol) and ethyl acrylate
(1.35 ml, 12.5 mmol) in EtOH (28 ml) was refluxed for 4 h. The reaction
mixture was concentrated and the residue was washed with iso-Pr2O to af-
ford pale yellow crystals (3.34 g, 87%), which were recrystallized from a
mixture of acetone and Et2O to give colorless needles, mp 116—117.5 °C.
Anal. Calcd for C18H26ClN3O4: C, 56.32; H, 6.83; N, 10.95. Found: C,
N-(4-Piperidyl)trifluoroacetamide (7) A mixture of the hydrochloride
of 6 (40.0 g, 124 mmol), 10% Pd–C (5.0 g) and H2O (50 ml) in MeOH
(200 ml) was stirred at 35 °C and 2 atm of H2 for 3 h. The catalyst was re-
moved by filtration and the filtrate was evaporated. The resulting residue was
56.28; H, 6.78; N, 10.87. MS m/z: 383, 385 (3 : 1, Mϩ). IR n (KBr) cmϪ1
:
1
1730, 1628. H-NMR (DMSO-d6): 1.18 (3H, t, Jϭ7.0 Hz), 1.40—1.60 (2H,
m), 1.70—1.90 (2H, m), 2.05—2.15 (2H, m), 2.35—2.80 (6H, m), 3.65—

434
Vol. 49, No. 4
3.85 (1H, m), 3.84 (3H, s), 4.06 (2H, q, Jϭ7.0 Hz), 5.70—5.85 (2H, br s),
6.49 (1H, s), 7.60—7.75 (1H, m), 7.66 (1H, s).
2 Hz), 7.65—7.80 (1H, m), 7.83 (1H, d, Jϭ2 Hz), 8.04 (1H, d, Jϭ8.5 Hz),
8.77 (1H, s).
4-[(4-Amino-5-chloro-2-methoxybenzoyl)amino]-1-piperidineacetic
Compounds 29b, c were prepared in a manner similar to that described
Acid (19) A mixture of the hydrochloride of 13 (2.23 g, 5.49 mmol) and above and their physicochemical data were summarized in Table 2.
2 N aqueous NaOH solution (9.86 ml) in MeOH (22 ml) was refluxed for 1 h. Ethyl endo-3-[(4-Acetylamino-5-chloro-2-methoxybenzoyl)amino]-9-
The reaction mixture was concentrated and the resulting residue was dis- azabicyclo[3.3.1]nonnane-9-acetate (30a) The mixture of 29a (13.0 g,
solved in a small amount of water. The solution was adjusted to pH 2 with 31.1 mmol) and SO2Cl2 (3.42 ml, 34.3 mmol) in CH2Cl2 (140 ml) was stirred
10% HCl. The resulting precipitate was collected by filtration and washed at room temperature for 1.5 h. The reaction mixture was basified with aque-
with water to afford the hydrochloride (1.47 g, 78%) of 19 as a pale brown ous NaHCO3. The organic layer was dried and evaporated. The resulting
crystal, which was recrystallized from water to afford pale yellow pillars, mp residue was purified by column chromatography [SiO2, CH2Cl2–MeOH
248—251 °C (dec.). Anal. Calcd for C15H20ClN3O4·HCl·1/4H2O: C, 47.07;
H, 5.66; N 10.98. Found: C, 47.34; H, 5.58; N, 11.08. MS m/z: 341, 343
(3 : 1, Mϩ). IR n (KBr) cmϪ1: 1734, 1628. 1H-NMR (DMSO-d6): 1.75—2.20
(4H, m), 3.05—3.35 (2H, m), 3.35—3.60 (2H, m), 3.85 (3H, s), 3.85—4.10
(1H, m), 3.98 (2H, s), 6.55 (1H, s), 7.63 (1H, s), 7.65—7.80 (1H, m).
(50 : 1)] to afford 11.4 g (81%) of 30a as a pale yellow amorphous solid.
High resolution MS m/z: Calcd for C22H30ClN3O5: 451.1874, 453.1844.
Found: 451.1859, 453.1853. MS m/z: 451, 453 (Mϩ, 3 : 1). IR n (KBr) cmϪ1
:
1750, 1646. 1H-NMR (CDCl3): 1.05—2.20 (8H, m), 1.28 (3H, t, Jϭ7.5 Hz),
2.28 (3H, s), 2.40—2.65 (2H, m), 3.10—3.35 (2H, m), 3.49 (2H, s), 3.98
Compounds 20—24 were prepared in a manner similar to that described (3H, s), 4.17 (2H, q, Jϭ7.5 Hz), 7.55—7.70 (1H, m), 7.80 (1H, s), 8.22 (1H,
above and their physicochemical data were summarized in Table 1.
s), 8.32 (1H, s).
Method B. endo-4-Acetylamino-N-(9-benzyl-9-azabicyclo[3.3.1]non-
Compounds 30b, c were prepared in a manner similar to that described
above and physicochemical data were summarized in Table 2.
Ethyl endo-3-[(4-Amino-5-chloro-2-methoxybenzoyl)amino]-9-azabi-
3-yl)-2-methoxybenzamide (27a) A mixture of 4-acetylamino-2-methoxy
-
benzoic acid 25 (11.0 g, 52.6 mmol), ClCO2Et (5.28 ml, 55.2 mmol) and
Et3N (8.43 ml, 60.5 mmol) in THF (210 ml) was stirred for 1.5 h at room cyclo[3.3.1]nonane-9-acetate (31a) A mixture of 30a (11.0 g, 24.4 mmol)
emperature. Under ice-cooling, endo-3-amino-9-benzyl-9-azabicyclo[3.3.1]- and 20% ethanolic hydrochloride (66 ml) in EtOH (22 ml) was refluxed for
t
nonane 26a (12.7 g, 55.2 mmol) was added dropwise to the mixture and the 1 h. The reaction mixture was evaporated and the resulting residue was dis-
resulting mixture was stirred at room temperature for 2.5 d. The reaction solved in water. The solution was adjusted to pH 10 with K2CO3. The result-
mixture was concentrated and water was added to the residue. The solution ing precipitate was collected by filtration and washed successively with
was adjusted to pH 9 with 10% aqueous K2CO3. The resulting precipitate water and iso-Pr2O to give 8.88 g (89%) of 31a as a pale brown crystal,
was collected by filtration, washed successively with water and ethanol and which was recrystallized from EtOH to afford colorless needles, mp 163.5—
dried to afford 15.7 g (71%) of 27a as a colorless crystal, which was used in 164.5 °C. Anal. Calcd for C20H28ClN3O4: C, 58.60; H, 6.88; N, 10.25.
the next step without further purification. MS m/z: 421 (Mϩ). IR n (KBr) Found: C, 58.39; H, 6.84; N, 10.26. MS m/z: 409, 411 (Mϩ, 3 : 1). IR n
1
cmϪ1: 1690, 1624. 1H-NMR (DMSO-d6): 0.90—1.15 (2H, m), 1.25—1.60 (KBr) cmϪ1: 1744, 1646. H-NMR (CDCl3): 1.00—1.65 (5H, m), 1.27 (3H,
(3H, m), 1.80—2.40 (5H, m), 2.06 (3H, s), 2.95—3.15 (2H, m), 3.81 (2H, t, Jϭ7.5 Hz), 1.75—2.15 (3H, m), 2.35—2.65 (2H, m), 3.05—3.30 (2H, m),
s), 3.86 (3H, s), 4.25—4.50 (1H, m), 7.10—7.45 (5H, m), 7.18 (1H, dd,
Jϭ8.5, 2 Hz), 7.49 (1H, d, Jϭ2 Hz), 7.60—7.80 (1H, m), 7.72 (1H, d,
Jϭ8.5 Hz), 10.04 (1H, s).
3.47 (2H, s), 3.89 (3H, s), 4.16 (2H, q, Jϭ7.5 Hz), 4.30—4.55 (1H, m), 6.30
(1H, s), 7.40—7.60 (1H, m), 8.10 (1H, s).
Compounds 31b, c were prepared in a manner similar to that described
Compounds 27b, c were prepared in a manner similar to that described above and physicochemical data were summarized in Table 2.
above. 27b: MS m/z: 421 (Mϩ). IR n (KBr) cmϪ1: 1692, 1666. 1H-NMR
(DMSO-d6): 1.35—1.60 (2H, m), 1.55—2.15 (8H, m), 2.07 (3H, s), 2.75—
endo-3-[(4-Amino-5-chloro-2-methoxybenzoyl)amino]-9-azabicy-
clo[3.3.1]nonane-9-acetic Acid (32a) mixture of 31a (8.00 g,
A
2.95 (2H, m), 3.83 (2H, s), 3.87 (3H, s), 4.55—4.85 (1H, m), 7.10—7.45 19.5 mmol) and 2 N aqueous NaOH solution (19.5 ml, 39.0 mmol) in MeOH
(5H, m), 7.18 (1H, dd, Jϭ8.5, 1.5 Hz), 7.50 (1H, d, Jϭ2 Hz), 7.60—7.75 (80 ml) was refluxed for 1 h. The reaction mixture was concentrated and the
(1H, m), 7.70 (1H, d, Jϭ8.5 Hz), 10.04 (1H, s). 27c: MS m/z: 407 (Mϩ). IR
resulting residue was dissolved in a small amount of water. The solution was
adjusted to pH 1 with 10% HCl. The resulting precipitate was collected by
n (KBr) cmϪ1: 1694, 1630. 1H-NMR (DMSO-d6): 1.50—1.85 (6H, m),
1.90—2.15 (2H, m), 2.06 (3H, s), 3.05—3.35 (2H, m), 3.55 (2H, s), 3.85 filtration, washed with water and iso-Pr2O, and recrystallized from a mixture
(3H, s), 4.05—4.30 (1H, m), 7.10—7.45 (5H, m), 7.17 (1H, dd, Jϭ8.5, of MeOH and iso-Pr2O to afford the hydrochloride (5.75 g, 68%) of 32a as a
1.5 Hz), 7.48 (1H, d, Jϭ1.5 Hz), 7.60—7.80 (1H, m), 7.70 (1H, d, colorless crystalline powder, mp 189—194 °C (dec.). Anal. Calcd for
Jϭ8.5 Hz), 10.04 (1H,s).
C18H24ClN3O4·HCl·H2O: C, 49.55; H, 6.23; N, 9.63. Found: C, 49.38; H,
1
endo-4-Acetylamino-N-(9-azabicyclo[3.3.1]non-3-yl)-2-methoxybenza- 5.99; N, 9.49. IR n (KBr) cmϪ1: 1732, 1626. H-NMR (DMSO-d6): 1.40—
mide (28a) A mixture of 27a (15.5 g, 36.8 mmol), acetic acid (20 ml) and 1.65 (3H, m), 1.70—1.90 (2H, m), 1.95—2.30 (3H, m), 2.35—2.60 (2H, m),
Pearlman’s catalyst (2.0 g) in MeOH (180 ml) was stirred at room tempera- 3.75—3.90 (2H, m), 3.84 (3H, s), 4.17 (2H, s), 4.35—4.55 (1H, m), 6.53
ture and an atmospheric pressure of H2 for 1 h. The catalyst was removed by (1H, s), 7.65 (1H, s), 7.55—7.85 (1H, m).
filtration and the filtrate was evaporated. The resulting residue was dissolved
in water and the solution was adjusted to pH 9 with 10% aqueous K2CO3. above and their physicochemical data were summarized in Table 2.
The resulting precipitate was collected by filtration, washed with water and Method C. Methyl endo-3-[(4-Acetylamino-5-chloro-2-methoxyben-
Compounds 32b, c were prepared in a manner similar to that described
dried to give 11.7 g (95%) of 28a as a pale yellow crystal, which was recrys- zoyl)amino]-8-azabicyclo[3.2.1]octane-8-carboxylate (34) A mixture of
tallized from 1,2-dichloroethane to afford colorless prisms, mp 196— 337) (40.0 g, 0.109 mmol), K2CO3 (16.0 g, 0.116 mol) and ClCO2Me
200 °C. Anal. Calcd for C18H25N3O3·1/4H2O: C, 64.36; H, 7.65; N, 12.51. (80.0 ml, 1.04 mol) in chloroform (400 ml) was refluxed for 19 h. The result-
Found: C, 64.32; H, 7.53; N, 12.47. MS m/z: 331 (Mϩ). IR n (KBr) cmϪ1
:
ing precipitate was filtered off and washed with CH2Cl2. The filtrate and
washing CH2Cl2 were combined and evaporated, and the residue was washed
1692, 1634. 1H-NMR (DMSO-d6): 1.10—1.70 (7H, m), 1.80—2.30 (3H, m),
2.06 (3H, s), 3.00—3.40 (2H, m), 3.86 (3H, s), 3.95—4.20 (1H, m), 7.17 successively with AcOEt and MeOH to give 17.5 g (39%) of 34 as a color-
(1H, dd, Jϭ8.5, 1.5 Hz), 7.48 (1H, d, Jϭ1.5 Hz), 7.55—7.75 (1H, m), 7.72 less crystal, which was recrystallized from a mixture of MeOH and CH2Cl2
(1H, d, Jϭ8.5 Hz), 10.04 (1H, br s).
to afford colorless crystalline powder, mp 250—252 °C. Anal. Calcd for
Compounds 28b, c were prepared in a manner similar to that described C19H24ClN3O5·1/2H2O: C, 54.48; H, 6.02; N, 10.03. Found: C, 54.17; H,
above and their physicochemical data were summarized in Table 2.
Ethyl endo-3-[(4-Acetylamino-2-methoxybenzoyl)amino]-9-azabicyclo-
5.77; N, 9.79. MS m/z: 409, 411 (Mϩ, 3 : 1). IR n (liq) cmϪ1: 1704, 1682,
1
1652. H-NMR (DMSO-d6): 1.70—1.85 (2H, m), 1.90—2.15 (6H, m), 2.15
[3.3.1]nonane-9-acetate (29a) A mixture of 28a (11.6 g, 34.5 mmol), (3H, s), 3.61 (3H, s), 3.90 (3H, s), 4.05—4.25 (3H, m), 7.80 (1H, s), 7.81
ethyl bromoacetate (4.60 ml, 41.4 mmol) and K2CO3 (5.72 g, 41.4 mmol) in (1H, s), 8.25—8.40 (1H, m), 9.45 (1H, br s).
DMF (75 ml) was stirred at 60 °C for 2 h. The reaction mixture was poured
into water. The resulting precipitate was collected by filtration and washed methoxybenzamide (35)
endo-4-Acetylamino-N-(8-azabicyclo[3.2.1]oct-3-yl)-5-chloro-2-
mixture of 34 (16.3 g, 39.8 mmol) and
A
with water to afford 13.4 g (91%) of 29a as a pale yellow crystal, which was trimethylsilyl iodide (22.0 ml, 155 mmol) in CH2Cl2 (400 ml) was stirred at
recrystallized from benzene to give colorless prisms, mp 85—88 °C. Anal. room temperature for 6 h. Sodium hydrogensulfite solution was added to the
Calcd for C22H31N3O5·1/2H2O: C, 61.95; H, 7.56; N, 9.85. Found: C, 61.85; reaction mixture. The mixture was stirred at room temperature for 15 min
H, 7.55; N, 9.86. MS m/z: 417 (Mϩ). IR n (liq) cmϪ1: 1732, 1694, 1636. 1H- and adjusted to pH 2 with 10% HCl. The aqueous layer was basified with
NMR (CDCl3): 1.00—1.65 (4H, m), 1.26 (3H, t, Jϭ7 Hz), 1.75—2.15 (2H, K2CO3 and extracted with CH2Cl2. The extract was washed with water, dried
m), 2.22 (3H, s), 2.40—2.65 (2H, m), 3.05—3.30 (2H, m), 3.47 (2H, s), 3.92
and evaporated to give a yellow brown oil, which was purified by column
(3H, s), 4.15 (2H, q, Jϭ7 Hz), 4.35—4.60 (1H, m), 6.83 (1H, dd, Jϭ8.5, chromatography [alumina, CH2Cl2–MeOH (50 : 1)] to afford 8.41 g (60%) of

April 2001
435
35 as a pale yellow viscous oil. MS m/z: 351, 353 (Mϩ, 3 : 1). IR n (KBr)
cmϪ1: 1694, 1644. 1H-NMR (CDCl3): 1.65—2.30 (9H, m), 2.28 (3H, s),
3.60—3.75 (2H, m), 4.02 (3H, s), 4.30—4.45 (1H, m), 7.82 (1H, br s), 8.21
(1H, s), 8.34 (1H, s), 8.40—8.50 (1H, m).
by filtration and the filtrate was evaporated. The resulting residue was dis-
solved in 10% HCl and the insoluble substance was removed by filtration.
The filtrate was basified with K2CO3 and the precipitate was collected by fil-
tration, washed with water and dried to give 9.90 g (100%) of 41d as a color-
less amorphous solid, which was converted to the hydrochloride in the usual
manner. The hydrochloride was recrystallized from EtOH to afford colorless
needles, mp Ͼ300 °C. Anal. Calcd for C17H22N4O·HCl: C, 60.98; H, 6.92;
N, 16.73. Found: C, 60.77; H, 7.01; N, 16.55. MS m/z: 298 (Mϩ). IR n
(KBr) cmϪ1: 1640. 1H-NMR (DMSO-d6): 1.25—2.65 (10H, m), 3.35 (1H,
br s), 3.55—3.95 (2H, m), 4.13 (3H, s), 4.25—4.80 (1H, m), 7.15—7.80
(3H, m), 8.05—8.35 (1H, m), 8.40—9.40 (1H, m).
Ethyl endo-3-[(4-Acetylamino-5-chloro-2-methoxybenzoyl)amino]-8-
azabicyclo[3.2.1]octane-8-acetate (36) A mixture of 35 (8.00 g, 22.7
mmol), ethyl bromoacetate (4.18 g, 25.0 mmol) and K2CO3 (3.46 g, 25.0
mmol) in DMF (50 ml) was stirred at 60 °C for 2 h. After the addition of
water, the reaction mixture was extracted with AcOEt. The extract was
washed with water, dried and evaporated. The resulting residue was washed
with iso-Pr2O to give 5.20 g (52%) of 36 as a colorless crystal, which was re-
crystallized from EtOH to afford colorless prisms, mp 144—145 °C. Anal.
Calcd for C21H28ClN3O5: C, 57.60; H, 6.44; N, 9.60. Found: C, 57.45; H,
6.32; N, 9.49. MS m/z: 437, 439 (Mϩ, 3 : 1). IR n (KBr) cmϪ1: 1738, 1704,
1640. 1H-NMR (CDCl3): 1.29 (3H, t, Jϭ7 Hz), 1.65—2.45 (9H, m), 2.28
(3H, s), 3.26 (2H, s), 3.30—3.50 (2H, m), 4.02 (3H, s), 4.21 (2H, q,
Jϭ7 Hz), 4.25—4.40 (1H, m), 7.80 (1H, br s), 8.21 (1H, s), 8.34 (1H, s),
8.35—8.50 (1H, m).
Ethyl endo-3-[(4-Amino-5-chloro-2-methoxybenzoyl)amino]-8-azabi-
cyclo[3.2.1]octane-8-acetate (37) A mixture of 36 (4.60 g, 10.5 mmol)
and 10% ethanolic hydrochloride (30 ml) in EtOH (10 ml) was refluxed for
1 h. The reaction mixture was evaporated and water was added to the result-
ing residue. The aqueous layer was washed with AcOEt and then adjusted to
pH 9 with 10% aqueous K2CO3. The resulting crystals were collected by fil-
tration and washed with water and iso-Pr2O to give 4.09 g (98%) of 37 as a
pale yellow crystal, which was recrystallized from EtOH to afford colorless
crystals, mp 187—188 °C. Anal. Calcd for C19H26ClN3O4: C, 57.65; H, 6.62;
N, 10.61. Found: C, 57.57; H, 6.52; N, 10.42. MS m/z: 395, 397 (Mϩ, 3 : 1).
IR n (KBr) cmϪ1: 1748, 1640. 1H-NMR (CDCl3): 1.28 (3H, t, Jϭ7 Hz),
1.60—2.50 (8H, m), 3.26 (2H, s), 3.25—3.50 (2H, m), 3.93 (3H, s), 4.20
(2H, q, Jϭ7 Hz), 4.20—4.40 (1H, m), 4.41 (2H, br s), 6.31 (1H, s), 8.10
(1H, s), 8.20—8.35 (1H, m).
endo-3-[(4-Amino-5-chloro-2-methoxybenzoyl)amino]-8-azabicy-
clo[3.2.1]octane-8-acetic Acid (38) A mixture of 37 (3.60 g, 9.09 mmol)
and 2 N aqueous NaOH solution (9.1 ml) in MeOH (36 ml) was refluxed for
1 h. The reaction mixture was concentrated and the resulting residue was
dissolved in a small amount of water. The solution was adjusted to pH 1
with 10% HCl. The resulting precipitate was collected by filtration and
washed with water to give crude crystals, which were recrystallized from
water to afford the hydrochloride (3.19 g, 81%) of 38 as a pale yellow crys-
tal, mp 254—256 °C (dec.). Anal. Calcd for C17H22ClN3O4·HCl·3/2H2O: C,
47.34; H, 6.08; N, 9.74. Found: C, 47.36; H, 5.96; N, 9.79. MS m/z: 367,
369 (Mϩ, 3 : 1). IR n (KBr) cmϪ1: 1736, 1628. 1H-NMR (DMSO-d6): 1.95—
2.60 (8H, m), 3.85 (3H, s), 3.95—4.15 (1H, m), 4.01 (2H, s), 5.90 (2H, br),
6.56 (1H, s), 7.65 (1H, s), 8.05—8.20 (1H, m).
Method D. endo-N-(9-Benzyl-9-azabicyclo[3.3.1]non-3-yl)-1-methyl-
1H-indazole-3-carboxamide (40d) A mixture of endo-3-amino-9-benzyl-
9-azabicyclo[3.3.1]nonane (13.8 g, 59.9 mmol), Et3N (6.60 g, 65.2 mmol)
and 1-methyl-1H-indazole-3-carbonyl chloride 39d (10.2 g, 52.2 mmol) in
CH2Cl2 (150 ml) was stirred at room temperature for 2 h. The reaction mix-
ture was washed with water, dried and evaporated. The resulting residue was
purified by column chromatography (SiO2, CHCl3) and crystallized from
iso-Pr2O to give 14.75 g (73%) of 40d as a colorless crystal, which was re-
crystallized from MeOH to afford colorless needles, mp 161—162 °C. Anal.
Calcd for C24H28N4O: C, 74.20; H, 7.26; N, 14.42. Found: C, 74.15; H, 7.32;
N, 14.41. MS m/z: 388 (Mϩ). IR n (KBr) cmϪ1: 1672. 1H-NMR (CDCl3):
0.75—2.25 (8H, m), 2.25—2.75 (2H, m), 2.90—3.35 (2H, m), 3.87 (2H, s),
4.09 (3H, s), 4.50—4.95 (1H, m), 6.70—6.95 (1H, m), 7.05—7.65 (8H, m),
8.35—8.55 (1H, m).
Compounds 41e, f were prepared in a manner similar to that described
above. 41e: mp 232—236 °C (dec., aqueous EtOH). Anal. Calcd for
C17H21N3O·H2O: C, 67.75; H, 7.69; N, 13.94. Found: C, 67.85; H, 7.76; N,
1
13.86. MS m/z: 283 (Mϩ). IR n (KBr) cmϪ1: 1606. H-NMR (DMSO-d6):
0.90—2.35 (10H, m), 2.95—3.55 (2H, m), 3.80—4.50 (1H, m), 6.90—7.55
(4H, m), 7.99 (1H, s), 8.00—8.25 (1H, m), 11.39 (1H, br s). 41f: mp 218—
219 °C (EtOH). Anal. Calcd for C18H23N3O: C, 72.70; H, 7.80; N, 14.13.
Found: C, 72.54; H, 7.84; N, 13.96. MS m/z: 297 (Mϩ). IR n (KBr) cmϪ1
:
1614. 1H-NMR (CDCl3): 1.00—2.10 (8H, m), 1.90 (1H, s), 2.10—2.60 (2H,
m), 3.20—3.55 (2H, m), 3.80 (1H, s), 4.00—4.60 (1H, m), 5.60—5.95 (1H,
m), 7.10—7.50 (3H, m), 7.67 (1H, s), 7.80—8.05 (1H, m).
Ethyl endo-3-[[(1-Methyl-1H-indazol-3-yl)carbonyl]amino]-9-azabicy-
clo[3.3.1]nonane-9-acetate (42d) A mixture of 41d (2.50 g, 8.38 mmol),
ethyl bromoacetate (1.54 g, 9.22 mmol) and K2CO3 (1.16 g, 8.39 mmol) in
DMF (20 ml) was stirred at 70 °C for 4.5 h. After the addition of water, the
reaction mixture was extracted with Et2O. The extract was washed with
water, dried and evaporated. The residue was purified by column chromatog-
raphy [SiO2, CH2Cl2] to afford 2.69 g (84%) of 42d as a pale yellow viscous
oil. High resolution MS m/z: Calcd for C21H28N4O3: 384.2161. Found:
384.2173. MS m/z: 384 (Mϩ). IR n (KBr) cmϪ1: 1748, 1662. 1H-NMR
(CDCl3): 0.90—2.25 (8H, m), 1.28 (3H, t, Jϭ7.5 Hz), 2.25—2.80 (2H, m),
3.05—3.45 (2H, m), 3.49 (2H, s), 4.08 (3H, s), 4.17 (2H, q, Jϭ7.5 Hz),
4.20—4.80 (1H, m), 6.65—6.95 (1H, m), 7.10—7.60 (3H, m), 8.20—8.50
(1H, m).
Compounds 42e, f were prepared in a manner similar to that described
above and their physicochemical data were summarized in Table 3.
endo-3-[[(1-Methyl-1H-indazol-3-yl)carbonyl]amino]-9-azabicyclo-
[3.3.1]nonane-9-acetic Acid (43d) A mixture of 42d (2.00 g, 5.20 mmol)
and 2 N aqueous NaOH solution (5.2 ml) in MeOH (20 ml) was refluxed for
1 h. The reaction mixture was evaporated and the resulting residue was dis-
solved in a small amount of water. The solution was adjusted to pH 2 with
10% aqueous HCl. The resulting precipitate was collected by filtration to af-
ford 1.50 g (67%) of 43d as a colorless amorphass solid. Anal. Calcd for
C19H24N4O3·HCl·2H2O: C, 53.21; H, 6.81; N, 13.06. Found: C, 53.34; H,
1
6.66; N, 13.04. MS m/z: 357 (Mϩϩ1). IR n (KBr) cmϪ1: 1744, 1652. H-
NMR (DMSO-d6): 1.25—2.70 (10H, m), 3.70—4.05 (2H, m), 4.14 (3H, s),
4.18 (2H, s), 4.35—5.05 (1H, m), 7.10—7.85 (3H, m), 8.00—8.50 (2H, m).
Compounds 43e, f were prepared in a manner similar to that described
above and their physicochemical data was summarized in Table 3.
Method E. Methyl 4-[(4-Amino-5-chloro-2-methoxybenzoyl)amino]-
1-piperidineacetate (44) To a suspension of 19 (5.00 g, 14.6 mmol) in
MeOH (75 ml) was added concentrated sulfuric acid (1.80 g). The mixture
was refluxed for 16 h. After cooling, the solvent was evaporated to give the
residue, which was basified (pHϭ9) with aqueous K2CO3 and extracted with
CH2Cl2. The extract was washed with saturated aqueous NaCl, dried and
evaporated to afford 3.82 g (74%) of 44 as pale brown residue, which was re-
crystallized from MeOH to afford 2.95 g of pale brown prisms, mp 196—
197 °C. Anal. Calcd for C16H22ClN3O4: C, 54.01; H, 6.23; N,11.81. Found:
C, 53.80; H, 6.14; N, 11.83. MS m/z: 355, 357 (3 : 1, Mϩ). IR n (KBr) cmϪ1
:
Compounds 40e, f were prepared in a manner similar to that described
above. 40e: mp 212—214 °C (CH2Cl2–MeOH). Anal. Calcd for C24H27N3O:
C, 77.18; H, 7.29; N, 11.25. Found: C, 77.08; H, 7.34; N, 11.24. MS m/z:
373 (Mϩ). IR n (KBr) cmϪ1: 1620. 1H-NMR (DMSO-d6): 0.70—2.75 (10H,
m), 2.85—3.30 (2H, m), 3.83 (2H, s), 4.05—4.85 (1H, m), 6.90—7.60 (9H,
m), 7.99 (1H, d, Jϭ3 Hz), 8.10—8.30 (1H, m), 11.38 (1H, br s). 40f: mp
210—211 °C (EtOH). Anal. Calcd for C25H29N3O: C, 77.49; H, 7.54; N,
10.84. Found: C, 77.45; H, 7.57; N, 11.10. MS m/z: 387 (Mϩ). IR n (KBr)
cmϪ1: 1614. 1H-NMR (CDCl3): 0.80—2.20 (8H, m), 2.30—2.80 (2H, m),
3.00—3.30 (2H, m), 3.76 (3H, s), 3.86 (2H, s), 4.25—5.10 (1H, m), 5.70—
5.80 (1H, m), 7.10—7.50 (8H, m), 7.63 (1H, s), 7.80—8.15 (1H, m).
endo-N-(9-Azabicyclo[3.3.1]non-3-yl)-1-methyl-1H-indazole-3-carbox-
amide (41d) A mixture of 40d (12.9 g, 3.32 mmol), Pearlman’s catalyst
(2.0 g) and acetic acid (25 ml) in MeOH (225 ml) was stirred at room tem-
perature and atmospheric pressure of H2 for 3 h. The catalyst was removed
3400, 3320, 3216, 1756. 1H-NMR (DMSO-d6): 1.45—1.55 (2H, m), 1.75—
1.85 (2H, m), 2.30—2.35 (2H, m), 2.70—2.80 (2H, m), 3.23 (2H, s), 3.62
(3H, s), 3.70—3.85 (1H, m), 3.85 (3H, s), 5.80 (2H, br s), 6.50 (1H, s), 7.67
(1H, s), 7.68 (1H, d, Jϭ7.5 Hz).
n-Propyl 4-[(4-Amino-5-chloro-2-methoxybenzoyl)amino]-1-piperidineac-
etate (45) A solution of n-propyl bromide (1.78 g, 14.5 mmol) in DMF
(15 ml) was added to a suspension of 19 (4.50 g, 13.2 mmol) and K2CO3
(2.00 g, 14.5 mol) in DMF (135 ml) at 60 °C over 1 h. The mixture was
stirred at 60 °C for 2 h. After cooling, the reaction mixture was added to
water and extracted with toluene. The extract was washed with saturated
aqueous NaCl, dried and evaporated to afford colorless residue. The residue
was washed with n-heptane to afford 3.94 g (78%) of the crude 45, which
was converted to the mesylate in the usual manner. The mesylate of 45 was
recrystallized from iso-PrOH to give a colorless crystal, mp 209—210 °C.

436
Vol. 49, No. 4
Anal. Calcd for C18H26ClN3O4·CH4O3S: C, 47.54; H, 6.30; N, 8.75. Found: HPLC.
C, 47.47; H, 6.41; N, 8.69. MS m/z: 383, 385 (3 : 1, Mϩ). IR n (KBr) cmϪ1
:
Metabolic Stability with Human Liver S9 The tested compounds were
1
3408, 3324, 3216, 1754. H-NMR (DMSO-d6): 0.93 (3H, t, Jϭ7 Hz), 1.66 added to human liver S9 (HBI, 0.1 mg protein/ml), pre-incubated at 37 °C,
(2H, sextet, Jϭ7 Hz), 1.70—2.00 (2H, m), 2.00—2.10 (2H, m), 2.33 (3H, s),
and the mixture was incubated at 37 °C for 30 min. The initial concentration
3.10—3.40 (2H, m), 3.40—3.70 (2H, m), 3.84 (3H, s), 3.90—4.10 (1H, m),
4.17 (2H, br s), 4.22 (2H, br s), 5.82 (2H, br s), 6.52 (1H, s), 7.63 (1H, s), determined by HPLC.
of prodrug was 1 mmol/l. The concentration of prodrug that remained was
7.74 (1H, br s), 9.92 (1H, br s).
Brain Distribution The tested compounds were administrated intra-
Compounds 46—60 were prepared in a manner similar to that described venously to CD-1(ICR) mice (CharlesRiver Japan, male, 10 mg/kg, nϭ4).
above and their physicochemical data were summarized in Table 4.
Pharmacology
Blood and brain were taken 2 min after administration. The concentrations
of plasma and brain were determined by HPLC or liquid chromatography/
Gastrointestinal Contractile Activity in Dogs The gastrointestinal mass spectrometory/mass spectrometory (LC/MS/MS). Brain distribution
motility was measured, according to the method of Z. Itoh et al., in dogs of was estimated from the concentration ratio of each compound in brain to
both sexes, weighing 7.9 to 12.0 kg. Under general anesthesia, a strain-gauge plasma.
force transducer was chronically sutured on the ascending colon 10 cm distal
to the cecum in a direction to measure circular muscle contractions. A
Acknowledgments We are grateful to Dr. N. Kado, Research Division,
sailastic cannula for intravenous (i.v.) or intra-duodenal (i.d.) administration Hokuriku Seiyaku Co., Ltd., for his encouragement and valuable comments
of the tested compound was placed into the superior vena cava or duodenum. through out this work. Thanks are also due to the staff of our analytical sec-
The administration of the tested compounds to the dogs was performed more
than 2 weeks after the surgery. The tested compounds were solved in saline,
tion for elemental analyses and spectral measurements.
including 1% lactic acid, and were intravenously or intra-duodenally admin- References
istered to the animals more than 2 h after feeding. Gastric motility was ex-
pressed as a symbol based on a motor index, representing the area sur-
rounded by the construction wave and the base line during a 20 min period.
Colonic motility was expressed as a ratio of the number of dogs, showing
giant contractions in the ascending colon during 40 min after the administra-
tion, versus the number of tested dogs.
1) a) Dumuis A., Sebben M., Bockaert J., Naunyn-Schmideberg’s Arch.
Pharmacol., 340, 403—410 (1989); b) Harrington R. A., Hamilton C.
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2) Sakaguchi J., Nishino H., Ogawa N., Iwanaga Y., Yasuda S., Kato H.,
Ito Y., Chem. Pharm. Bull., 40, 202—211 (1992).
Dopamine D2 Binding Assay The test compounds at the concentration
of 1—100 mM were tested in binding assays using rat brain synaptic mem-
branes for competition with [3H]Spiperon in the rat striatum at their respec-
tive binding sites. Each assay was started by an addition of tissue preparation
and terminated by rapid filtration through a Whatman GF/B glass fiber filter
under the reduced pressure. The filters were washed three times with 5 ml of
ice-cold buffer and transferred to scintillation vials that contained 7 ml of
scintillator, and the radioactivity in the filter was counted with a scintillation
counter. IC50 values (the concentration causing 50 inhibition of H-labeled
ligand specific binding) of the test compounds were calculated with the
equation of Cheng and Prusoff.
5-HT4 Agonist Activity The esophagus was removed from a male Wis-
ter rat, and two preparations, obtained from each animal, was used as a lon-
gitudinal preparation. The tissues were mounted in organ baths containing
10 ml of Krebs–Henseleit solution. The bathing medium was maintained at
37 °C, pH 7.4, and was equilibrated with a gas mixture consisting of 95% O2
3) Iwasaki N., Sakaguchi J., Ohashi T., Takahara E., Ogawa N., Yasuda
S., Koshinaka E., Kato H., Ito Y., Sawanishi H., Chem. Pharm. Bull.,
42, 2276—2284 (1994); Iwasaki N., Sakaguchi J., Ohashi T., Ya-
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Sawanishi H., ibid., 42, 2285—2290 (1994).
4) Van Daele G. H. P., De Bruyn M. F. L., Sommen F. M., Janssen M.,
Van Nueten J. M., Schuurkes J. A. J., Niemegeers C. J. E., Leysen J. E.,
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3
5) Spickett R. G., Moragues J., Prieto J., Japan. Patent 50—129573
(1975).
6) Luwig Heumann & Co GmbH, Japan. Patent 50—32144 (1975).
7) Hadley M. S., King F. D., Japan. Patent 55—92384 (1980).
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W. D., Sanger G. J., J. Med. Chem., 33, 1924—1929 (1990).
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Patent 59—67284 (1984).
and 5% CO2. An initial tension of 1.0 g was applied and the responses were 10) Itoh Z., Jap. J. Smooth Muscle Res., 13, 33—43 (1976).
recorded isometrically with a force-displacement transducer. All prepara- 11) Prieto J., Moragues J., Spickett R. G., Vega A., Colombo M., Salazar
tions were equilibrated for at least 60 min before the start of the experi-
ments. Once the carbachol (10Ϫ6 M)-induced contractions reached a steady
state, drugs were added cumulatively, and relaxation was calculated as a per-
centage of the initial carbachol-induced force.
W., Roberts D. J., J. Pharm. Pharmac., 29, 147—152 (1977).
12) Gaster L. M., Joiner G. F., King F. D., Wyman P. A., Sutton J. M.,
Bingham S., Ellis E. S., Sanger G. J., Wardle K. A., J. Med. Chem., 38,
4760—4763 (1995).
Emetic Action in Ferrets Male ferrets, weighing 0.9 to 1.3 kg, and 13) López-Rodríguez M. L., Morcillo M. J., Benhamú B., Rosado M. L., J.
fasted for 16 h, were used. The tested compounds were dissolved with 10% Comput.-Aided Mol. Des., 11, 589—599 (1997).
aqueous DMSO solution and were administered orally (p.o.). Then, the inci- 14) Fujitsu Limited, 1–9–3 Nakase, Mihama-ku, Chiba 261–8588, Japan,
dence of vomiting during the 2 h after the administration was observed. version 4.1.1
Physicochemical Stability The tested compounds were dissolved in the 15) Flynn D. L., Zabrowski D. L., Becker D. P., Nosal R., Villamil C. I.,
pH6.8 buffer solution (Japanese Pharmacopoeia 2nd fluid), and the solution
was heated at 37 °C for 3 h. The initial concentration of prodrug was
100 mg/ml. The concentration of prodrug that remained was determined by
Gullikson G. W., Moummi C., Yang D. C., J. Med. Chem., 35, 1486—
1489 (1992).