Arylcarbamate derivatives of 1-piperidineethanol as potent ligands for 5-HT4 receptors

Article abstract of DOI:10.1021/jm960853v

A series of carbamate derivatives (7) of 2-(1-piperidinyl)ethyl 4- amino-5-chloro-2-methoxybenzoates, which have been described as potent agonists and antagonists of 5-HT₄ receptors, were synthesized. They were evaluated using radioligand binding assays with [³H]GR 113808, a 5-HT₄ receptor selective ligand, in the rat striatum and the electrically stimulated myenteric plexus longitudinal muscle of the guinea pig. In contrast to the previously described ester derivatives, a drop in the affinity for 5-HT₄ receptors was observed and the compounds were inactive as agonists in the guinea pig ileum preparation. Unexpectedly, the ortho- substituted carbamates 8b,c (R' = H, RO = MeO or EtO, R'' = H) had nanomolar affinity for 5-HT₄ receptors (K(i) = 8.9 ± 0.5 and 2.6 ± 0.4 nM, respectively). As reported previously, the cis- or trans-3,5-dimethyl substitution of piperidine (8n,o) was particularly favorable (K(i) = 1.1 ± 0.6 nM for both isomers). 8c is an antagonist equipotent to the 5-HT₄ receptor antagonist SDZ 205-557 (1).

Full text of DOI:10.1021/jm960853v

J . Med. Chem. 1997, 40, 1755-1761
1755
Ar ylca r ba m a te Der iva tives of 1-P ip er id in eeth a n ol a s P oten t Liga n d s for 5-HT4
Recep tor s
J ean-Louis Soulier,† Donglai Yang,† Be´atrice Bre´mont,† Tiziano Croci,‡ Umberto Guzzi,‡ and Michel Langlois*,†
CNRS-BIOCIS, URA 1843, Faculte´ de Pharmacie, 5 rue J . B. Cle´ment, 92296 Chaˆtenay-Malabry, France, and Research Center
SANOFI-MIDY, 38, via G. B. Piranesi, 20137 Milan, Italy
Received December 17, 1996X
A series of carbamate derivatives (7) of 2-(1-piperidinyl)ethyl 4-amino-5-chloro-2-methoxyben-
zoates, which have been described as potent agonists and antagonists of 5-HT4 receptors, were
synthesized. They were evaluated using radioligand binding assays with [3H]GR 113808, a
5-HT4 receptor selective ligand, in the rat striatum and the electrically stimulated myenteric
plexus longitudinal muscle of the guinea pig. In contrast to the previously described ester
derivatives, a drop in the affinity for 5-HT4 receptors was observed and the compounds were
inactive as agonists in the guinea pig ileum preparation. Unexpectedly, the ortho-substituted
carbamates 8b,c (R′ ) H, RO ) MeO or EtO, R′′ ) H) had nanomolar affinity for 5-HT4 receptors
(Ki ) 8.9 ( 0.5 and 2.6 ( 0.4 nM, respectively). As reported previously, the cis- or trans-3,5-
dimethyl substitution of piperidine (8n ,o) was particularly favorable (Ki ) 1.1 ( 0.6 nM for
both isomers). 8c is an antagonist equipotent to the 5-HT4 receptor antagonist SDZ 205-557
(1).
Stimulation of 5-HT4 receptors is responsible for the
gastrokinetic effects of drugs such as the benzamides.1-3
In the guinea pig4b they are located on the ileal enteric
nerve where they mediate the release of acetylcholine,
thus bringing about the first phase of the muscle
contraction-concentration curve to 5-HT. The effect of
stimulation of 5-HT4 receptors seems to be species-
dependent as very different results have been obtained
in the rat where 5-HT4 receptor stimulation in the rat
ileum induced relaxation via an atropine-insensitive
mechanism, suggesting a localization1 on the smooth
muscle. On the other hand, in man 5-HT4 receptor-
mediated effects have not been observed in the ileum.
However 5-HT4 receptors are present in the colon1
where they appear to mediate a direct effect on the
smooth muscle, inducing muscle relaxation. Several
studies have shown the presence of 5-HT4 receptors in
the stomach of a variety of species, such as the guinea
pig, rat, dog, and man,1 where they mediate an increase
in gastric emptying. 5-HT4 receptors are also respon-
sible for the relaxation of the esophagus muscle in the
rat.4a
which appears to be an important structural require-
ment for 5-HT4 receptor agonist activity.
Recently, a new family of esters, derived from 2-(1-
piperidinyl) 4-amino-5-chloro-2-methoxybenzoate,11 were
described by us, as potent selective ligands for 5-HT4
receptors. Almost all of these derivatives were potent
agonists for this receptor, although it was shown that
the introduction of a 3,5-dimethylpiperidine moiety in
the basic chain brought about a dramatic change in the
pharmacological profile, as 2-(3,5-dimethylpiperidino)-
ethyl 4-amino-5-chloro-2-methoxybenzoate12 was dem-
onstrated to be a potent 5-HT4 receptor antagonist.
However, the presence of the ester function is an
important drawback for their administration in vivo due
to their putative short half-lives. To overcome this
problem, a series of carbamates (7) derived from the
esters previously described were synthesized, several
structural variations of the aromatic moiety were car-
ried out (compounds 8), and the role of the oxygen atom
in the basic chain was evaluated with the synthesis of
the amidic derivative 9.
Ch em istr y
Considerable progress has been made in the localiza-
tion and study of the functions of 5-HT4 receptors with
the development of potent and selective antagonists
such as SDZ 205-557 (1),5 SB 204070A (2),6,7 SB207266
(3),8 and GR 113808 (4)9 and, in particular, by the use
of a number of them as radiolabeled ligands.7,9,10
The carbamates 7, structurally closely related to
benzoates derived from 4-amino-5-chloro-2-methoxy-
benzoic acid, were prepared according to the synthetic
pathway described in Scheme 1. 5-Chloro-o-anisidine
was acetylated and transformed into the corresponding
nitro amine derivative 10 according to the process
described by Hadley.13 It was treated with phosgene
in a toluene solution to give 5-chloro-2-methoxy-4-
nitrophenyl isocyanate (11) which reacted with the
different amino alcohols, easily synthesized by the
reaction of 2-bromoethanol and the secondary amines
in the presence of NaOH, to give compounds 12. The
nitro group of compounds 12 was reduced into the amino
derivatives 7a -g by the reaction of Na2S2O4 in a MeOH/
H2O14 mixture with a moderate yield. Compounds were
isolated as hydrochloride or oxalate salts.
Research into selective 5-HT4 receptor agonists as
gastrointestinal (GI) prokinetic drugs for the treatment
of motility disorders such as gastroesophageal reflux,
functional dyspepsia, and constipation has received
considerable interest recently. Many of the compounds
described as possessing these properties, such as
cisapride, renzapride, SC-53116 (5), BRL 24682 (6), and
zacopride, are members of the generic benzamide family
being amides of 4-amino-5-chloro-2-methoxybenzoic acid,1
† CNRS-BIOCIS.
Mono- or disubstituted carbamates 8 were prepared
from the isocyanates 13 which were either commercially
‡ SANOFI-MIDY.
X Abstract published in Advance ACS Abstracts, May 1, 1997.
S0022-2623(96)00853-9 CCC: $14.00 © 1997 American Chemical Society

1756 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 11
Notes
Ch a r t 1
Sch em e 1a
and the amide 9a which was isolated with a poor yield
and reduced by SnCl213 in an HCl mixture to the amino
derivative 9b. In contrast, 9c was prepared directly
from o-anisidine with an excellent yield.
Biologica l Meth od s
Assessment of the affinity of the compounds for 5-HT4
receptors was performed using radioligand binding
assays with [3H]GR 113808 and rat striatal mem-
branes.15 The existence of 5-HT4 receptors has been
clearly demonstrated in the central nervous system
(CNS), and these receptors have been shown to be
indentical to those in the periphery, in particular, those
in the intestine.16 Consequently, binding assays in the
brain constitute a valuable test to select compounds
which will be active in the GI tract.
The facilitatory role of 5-HT4 receptors in the peri-
staltic reflex was demonstrated by Craig and Clarke4
who found that both 5-HT3 and 5-HT4 receptor agonists
induced peristalsis in the guinea pig isolated ileum
maintained at a subthreshold intraluminal pressure.
The 5-HT4 receptor agonist activity of the tested com-
pounds was measured in the electrically stimulated
myenteric plexus and longitudinal muscle of the guinea
pig ileum, and it was assessed as the concentration
(EC50, nM) which gave a 50% increase in the response
a
(a) AcCl, CH2Cl2, rt; (b) HNO3, H2SO4, AcOH; (c) NaOH, EtOH/
H2O, reflux; (d) 20% toluene solution of COCl2, CH2Cl2, 0 °C; (e)
R2R1N(CH2)2OH, 0 °C, reflux; (f) Na2S2O4, MeOH/H2O.
available or prepared from the corresponding anilino
derivatives by reaction with phosgene. Their condensa-
tion (Scheme 2) with the corresponding amino alcohols
in THF under reflux gave compounds 8a -i,m -o. 8j-l
were obtained by the reduction of the corresponding
nitro derivatives 8p -r by Na2S2O4.14 The derivatives
of 3,5-dimethylpiperidine (compounds 7g, 8m -o) were
obtained as a 4:1 mixture of cis and trans compounds,
respectively, which were separated by column chroma-
tography and characterized by their 1H-NMR spectra.
Amide 9b was prepared from 5-chloro-2-methoxy-4-
nitroaniline (Scheme 3) which was condensed with
4-chlorobutyryl chloride to give the chloro amide 15. The
reaction of 15 (Ar ) 5-chloro-2-methoxy-4-nitrophenyl)
and piperidine gave a mixture of 1-(5-chloro-2-methoxy-
4-nitrophenyl)-2-pyrrolidone by a cyclization reaction
Sch em e 2a
a
(a) ROH, 0 °C, reflux; (b) Na2S2O4, MeOH/H2O, only with the compounds 8p -r .
Sch em e 3a
a
(a) ClCO-(CH2)3-Cl, benzene, rt; (b) piperidine, toluene, reflux for 4 h or piperidine, NaI, rt for 2 days; (c) 1-(3-(1-piperidinyl)pro-
panecarboxamido)-4-nitro-5-chloro-2-methoxybenzene, SnCl2, HCl.

Notes
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 11 1757
Ta ble 1. Pharmacological Activity of the Carbamates 7 at
5-HT4 Receptors
show that, in contrast to the esters, the carbamate
function is less favorable for recognition by the 5-HT4
receptor and the basic moiety does not influence the
pharmacological profile.
In the benzamide series derived from 4-amino-5-
chloro-2-methoxybenzoic acid, no clear structure-activ-
ity relationships have been described for the role of
aromatic substituents on pharmacological activity. Pre-
liminary studies on ML 10302 showed that such sub-
stituents were essential for maintaining affinity for
5-HT4 receptors, as a large drop in the affinity value
was observed with any mono- or disubstituted benzoate
esters of 2-piperidinoethanol.17 Different results were
obtained with the monosubstituted carbamates 8. The
data reported in Table 2 show the favorable influence
of monosubstitution by MeO or EtO in the ortho position
(compounds 8b,c,f,m ,n ), while substitution in the meta
or para position brought about a large decrease in the
biological activity (compounds 8d ,e). The presence of
the amino function was particularly harmful and could
explain the decrease in activity observed with the
carbamates 7 with regard to the corresponding esters.
Only methoxy or ethoxy substitution in the ortho
position had a favorable effect, as NH2, Cl, CN,18 and
NO218 substitutions produced inactive compounds (Ki >
1000 nM) in the binding assays. On the other hand,
the length of the basic chain is an important structural
parameter in recognition by the binding site because the
carbamate 8s (Ki ) 81 nM), derived from 3-(1-piperidin-
yl)propanol, was 1 order of magnitude less potent than
the corresponding compound 8b (Ki ) 8.9 nM). As
observed previously, the introduction of 3,5-dimethyl-
piperidine was favorable, and compounds 8m -o had
nanomolar affinity, the effect of the cis or trans stere-
ochemistry not being very marked.
a
[3H]GR 113808 was used as the radioligand in the rat
striatum; Ki ( SEM values were determined from the Cheng-
Prussof equation. The agonist activity was assessed as the
b
concentration which gave a 50% increase in the response to
electrical stimulation (EC50) in the guinea pig ileum and is
expressed as a percent of the maximum 5-HT response; 95%
confidence limits are in brackets. c The antagonist activity (IC50
)
was calculated as the concentration which produced
reduction in the response to 5-HT in the guinea pig ileum; 95%
a 50%
d
confidence limits are in brackets. ne ) not capable of evaluation.
In contrast to the esters, no compound displayed
agonist activity in the functional assay for 5-HT4 recep-
tors. Thus, compounds 8b,c,f were antagonists in the
guinea pig ileum, and 8c was equipotent to 1. An
increase in the inhibitory effect was not observed with
the 3,5-dimethyl derivatives (compounds 8m -o) which
were equipotent to or less potent than compound 8c.
In a previous paper,12 we emphasized the important
role of the oxygen atom of the basic chain in the
recognition of the 5-HT4 receptor binding site by the
benzoate series. This finding was confirmed by the
results obtained with the amidic derivatives 9b,c which
were weak ligands for 5-HT4 receptors (Ki > 1000 nM).
e NT ) not tested. f I ) inactive up to 10-5 M.
to electrical stimulation with regard to its maximum
and related to the maximal effect of 5-HT (100%).
Antagonist activity (IC50, nM) was calculated as the
concentration which produced a 50% reduction in the
submaximal (80%) of 5-HT-induced contractions. The
activity of the compounds was compared to those of the
reference compounds: ML 10302 (2-(1-piperidinyl)ethyl
4-amino-5-chloro-2-methoxybenzoate),11 2-(cis-3,5-di-
methylpiperidino)ethyl 4-amino-5-chloro-2-methoxy-
benzoate,12 a recently described potent antagonist, and
compounds 1 and 4.
In summary, the present study has shown that the
carbamate derivatives of 1-piperidineethanol and 2-(3,5-
dimethylpiperidino)ethanol are potent ligands for 5-HT4
receptors. It emphasized the importance of the aromatic
substituents and, particularly, of o-methoxy or o-ethoxy
substituents for recognition by the 5-HT4 receptor. The
results demonstrated, unexpectedly, the unfavorable
role of the amino group in the 4-amino-5-chloro-2-
methoxybenzoic acid pharmacophore. Unlike the cor-
responding esters, no potent agonist was found with an
unsubstituted piperidine ring, suggesting the inability
of such molecules to occupy the putative agonist site
suggested previously.12 Several investigations are in
course to understand the cause of the weak functional
activity of these compounds on the GI receptors and
their potent affinity for central 5-HT4 receptors.
Resu lts a n d Discu ssion
The results in Table 1 show that the carbamates 7
are not as good ligands for 5-HT4 receptors as the
corresponding esters described previously;11b in particu-
lar, 7a (Ki ) 39 nM) was clearly less potent than ML
10302. However, as had already been noted,12 an
increase in the affinity for the receptor was observed
following methyl substitution, particularly with the
methylpiperidine derivatives 7e-g. The favorable role
of the 3,5-dimethyl substitution was again noted for 7g
which had nanomolar affinity. No compound displayed
significant agonist activity by increasing the response
to electrical stimulation in the guinea pig ileum. Only
compounds 7c,f,g produced a moderate inhibition of the
5-HT response in the guinea pig ileum. These data

1758 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 11
Notes
Ta ble 2. Pharmacological Activity of the Carbamates 8 at 5-HT4 Receptors
functional activity at 5-HT4 receptors
compd
Ar
R′
binding assays Ki, nM,a 5-HT4
EC50, nMb
IC50, nMc
8a
8b
8c
8d
8e
8f
8g
8h
8i
8j
8k
8l
8m
8n
8o
C6H5
H
H
H
H
H
H
H
H
H
H
H
H
31.7 ( 7.5
8.9 ( 0.5
2.6 ( 0.4
469 ( 44
>1000
>1000
∼1000
∼300
62 [40-95]
NT
NT
30 (60%)
NT
>1000
NT
NT
NT
2-OMeC6H4
2-OEtC6H4
3-OMeC6H4
4-OMeC6H4
2-OMe-5-ClC6H3
2-ClC6H4
∼100 (14%)
>1000
NTd
NT
4.2 ( 0.5
>1000
∼1000 (14%)
NT
3-ClC6H4
4-ClC6H4
60.8 ( 5.1
728 ( 63
>1000
>1000
>1000
2.6 ( 0.52
1.05 ( 0.68
1.16 ( 0.6
>1000
NT
NT
NT
NT
>1000
>1000
>1000
2-NH2C6H4
3-NH2C6H4
4-NH2C6H4
2-OMeC6H4
2-OEtC6H4
2-OEtC6H4
NT
Me, cis
Me, cis
Me, trans
160 (60%)
30 (70%)
100
a -d
See corresponding footnotes in Table 1.
residue was taken up with 10% aqueous NaHCO3 and ex-
tracted with CH2Cl2 (3 × 30 mL). The combined organic
extracts were dried over MgSO4 and concentrated in vacuo.
The residue was chromatographed on a column of silica gel
with a CH2Cl2/MeOH mixture (9:1) to give a crude product
which was reduced to the amino derivative according to the
following procedure. It was dissolved in a mixture of MeOH
(30 mL), H2O (25 mL), and 1.92 g of Na2S2O4, and the solution
was stirred for 4 h at room temperature (7d -f, 8j-l) or at 60
°C (7a ,b,g). The solvent was removed by distillation in vacuo,
and the residue was taken up with brine and extracted with
CH2Cl2 (4 × 25 mL). The aqueous layer was made alkaline
with a 1 N NaOH aqueous solution and extracted again with
CH2Cl2 (4 × 25 mL). The combined organic layers were dried
over Na2SO4 and concentrated to give the amino derivative
which was transformed into the hydrochloride salt with a 2 N
HCl methanol solution or the oxalate salt by treatment with
oxalic acid in acetone. The salt was recrystallized according
to the usual procedure.
Gen er a l Meth od for th e P r ep a r a tion of Ca r ba m a tes
8b-e,g-i,n -s. These compounds were prepared from the
corresponding anilines according to the previous procedure
except for the nitro reduction step and were transformed into
the hydrochloride or oxalic salt according to classical methods.
Gen er a l Meth od for th e P r ep a r a tion of Ca r ba m a tes
8a ,f,m . To a stirred solution of commercial isocyanate (10
mmol) in anhydrous CH2Cl2 was added dropwise the corre-
sponding amino alcohol (20 mmol). The reaction mixture was
refluxed for 1.5 h. After evaporation of the solvent in vacuo,
the residue was chromatographed on a column of silica gel with
a CH2Cl2/MeOH mixture (9:1), and the compound was trans-
formed into the hydrochloride salt with a 2 N HCl methanol
solution or the oxalate salt by treatment with oxalic acid in
acetone. The salt was recrystallized according to the usual
procedure.
Exp er im en ta l Section
Ch em istr y. Melting points were determined on a Mettler
FP 61 melting point apparatus and are uncorrected. 1H- and
13C-NMR spectra were recorded on a Bruker AC 200 spec-
trometer at 200 and 50 MHz, respectively. Chemical shifts
are reported in parts per million (δ) relative to tetramethyl-
silane as the internal standard, and signals are quoted as s
(singlet), ds (dedoublet singlet), d (doublet), dd (dedoublet
doublet), t (triplet), dt (dedoublet triplet), q (quartet), br s
(broad singlet), or m (multiplet). Elemental analyses were
performed at the CNRS’s analysis services in Chaˆtenay-
Malabry (France) and were within 0.4% of the theoretical
values unless otherwise noted.
Ma ter ia ls. Tetrahydrofuran (THF) was distilled from
sodium/benzophenone. Acetonitrile, dimethylformamide, tolu-
ene, and the usual solvents were purchased from SDS (Paris,
France). Column chromatography was performed on Merck
silica gel 60 (70/230 mesh). Thin-layer chromatography was
done on silica gel 60F-254 (0.26 mm thickness) plates.
4-Amino-5-chloro-2-methoxybenzoic acid, 2-bromoethanol,
the mixture of cis- and trans-3,5-dimethylpiperidine, hexa-
methylenimine, 4-methylpiperidine, 3-methylpiperidine, 2-me-
thylpiperidine, pyrrolidine, 1-piperidineethanol, 1-pyrrolidine-
ethanol, and N,N-diethylaminoethanol were purchased from
Aldrich (France). 2-(Hexamethyleneimino)ethanol, 2-(cis,trans-
3,5-dimethylpiperidino)ethanol, 2-(4-methylpiperidino)ethanol,
2-(3-methylpiperidino)ethanol, and 2-(2-methylpiperidino)et-
hanol were prepared from 2-bromoethanol according to the
process already reported.19 N-Acetyl-5-chloro-4-nitro-2-anisi-
dine was synthesized from 5-chloro-2-anisidine according to
the process reported by Hadley.13
5-Ch lor o-4-n itr o-2-a n isid in e. N-Acetyl-5-chloro-4-nitro-
2-anisidine was refluxed with stirring for 2 h in a solution of
10% NaOH in an EtOH/H2O mixture (7:3) to give, after
evaporation and extraction with CH2Cl2, 5-chloro-4-nitro-2-
anisidine which was recrystallized in an acetone/AcOEt mix-
ture (68%) as yellow spangles: 1H NMR (CDCl3) δ 7.57 (s, 1H),
6.69 (s, 1H), 4.53 (m, 2H), 3.91 (s, 3H).
Gen er a l Meth od for th e P r ep a r a tion of Ca r ba m a tes
7a -g a n d 8j-l. 5-Chloro-4-amino-2-anisidine or another
appropriate aniline (5 mmol) was added to a solution of
phosgene (2.74 mL, 20% in toluene) in anhydrous CH2Cl2 (20
mL) at 0 °C under argon. The solution was stirred for 15 min,
and NEt3 (1.6 mL, 11.5 mmol) was added. After 15 min, a
solution of the amino alcohol (1.3 mL, 10 mmol) in anhydrous
CH2Cl2 (5 mL) was added dropwise at 0 °C. The reaction
mixture was stirred for 10 min and refluxed overnight. The
solvent was evaporated under reduced pressure, and the
2-P ip er id in oet h yl
N-(4-a m in o-5-ch lor o-2-m et h oxy-
ph en yl)car bam ate (7a): hydrochloride salt (11%) from i-Pr2O/
MeOH; mp 196-198 °C dec; 1H NMR (CD3OD) δ 8.12 (s, 1H),
7.03 (s, 1H), 4.53 (m, 2H), 3.92 (s, 3H), 3.64 (m, 2H), 3.47 (m,
2H), 3.03 (m, 2H), 2.1-1.4 (m, 6H). Anal. (C15H22ClN3O3‚
HCl‚3H2O).
2-P yr r olid in oet h yl N-(4-a m in o-5-ch lor o-2-m et h oxy-
p h en yl)ca r ba m a te (7b): hydrochloride salt (5%) from i-Pr2O/
1
MeOH; mp 180 °C; H NMR (CD3OD) δ 7.62 (s, 1H), 6.56 (s,
1H), 4.46 (m, 2H), 3.84 (s, 3H), 3.56 (m, 2H), 3.47 (m, 4H),
2.35-2 (m, 4H). Anal. (C14H20ClN3O3‚HCl).
2-(Hexa m eth ylen eim in o)eth yl N-(4-a m in o-5-ch lor o-2-
m eth oxyp h en yl)ca r ba m a te (7c): oxalate salt (10%) from
1
i-Pr2O/MeOH; mp 174-176 °C; H NMR (CD3OD) δ 7.57 (s,

Notes
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 11 1759
1H), 6.51 (s, 1H), 4.46 (m, 2H), 3.92 (s, 3H), 3.79 (m, 2H), 3.49
(m, 2H), 3.34 (m, 4H), 1.92 (m, 4H), 1.73 (m, 4H). Anal.
(C16H24ClN3O3‚C2H2O4).
2-(Diet h yla m in o)et h yl N-(4-a m in o-5-ch lor o-2-m et h -
oxyp h en yl)ca r ba m a te (7d ): oxalate salt (28%) from i-Pr2O/
MeOH; mp 146-148 °C; 1H NMR (CD3OD) δ 7.60 (s, 1H), 6.57
(s, 1H), 4.51 (m, 2H), 3.85 (s, 3H), 3.54 (m, 2H), 3.37 (m, 4H),
1.4 (m, 6H). Anal. (C14H22ClN3O3‚C2H2O4).
(m, 1H), 4.46 (m, 2H), 3.6-3 (m, 6H), 2.1-1.5 (m, 6H). Anal.
(C14H21N3O2‚HCl).
2-P ip er id in oet h yl
N-(3-a m in op h en yl)ca r b a m a t e
(8k ): hydrochloride salt (88%) from i-Pr2O/MeOH; mp 230 °C;
1H NMR (DMSO) δ 10.8 (m, 1H), 10.16 (s, 1H), 7.59 (s, 1H),
7.42 (m, 1H), 7.35 (m, 1H), 7 (m, 1H), 4.5 (m, 2H), 3.46 (m,
2H), 3.35 (m, 2H), 2.97 (m, 2H), 2-1.5 (m, 5H), 1.3 (m, 1H).
Anal. (C14H21N3O2‚2HCl).
2-P ip er id in oeth yl N-(4-a m in op h en yl)ca r ba m a te (8l):
hydrochloride salt (34%) from i-Pr2O/MeOH; mp >230 °C; 1H
NMR (DMSO) δ 10.55 (s, 1H), 10.06 (s, 1H), 7.55 (d, 2H), 7.31
(d, 2H), 4.48 (m, 2H), 3.46 (m, 2H), 3.34 (m, 2H), 2.95 (m, 2H),
2-1.6 (m, 5H), 1.77 (m, 1H). Anal. (C14H21N3O2‚2HCl).
2-(3-Met h ylp ip er id in o)et h yl N-(4-a m in o-5-ch lor o-2-
m eth oxyp h en yl)ca r ba m a te (7e): oxalate salt (9%) from
i-Pr2O/MeOH; mp 166-168 °C; H NMR (CD3OD) δ 7.56 (s,
1H), 6.51 (s, 1H), 4.47 (m, 2H), 3.79 (s, 3H), 3.7-3.45 (m, 2H),
3.42 (m, 2H), 3-2.8 (m, 1H), 2.5-2.72 (m, 1H), 2.1-1.7 (m,
4H), 1.35-1.05 (m, 1H), 0.99 (d, 3H). Anal. (C16H24ClN3O3‚
C2H2O4).
2-(4-Met h ylp ip er id in o)et h yl N-(4-a m in o-5-ch lor o-2-
m eth oxyp h en yl)ca r ba m a te (7f): oxalate salt (30%) from
i-Pr2O/MeOH; mp 160 °C; 1H NMR (CD3OD) δ 7.58 (s, 1H),
6.52 (s, 1H), 4.46 (m, 2H), 3.8 (s, 3H), 3.75-3.52 (m, 2H), 3.52-
3.35 (m, 2H), 3.15-2.9 (m, 2H), 2.05-1.8 (m, 2H), 1.8-1.35
(m, 3H), 1.02 (d, 3H). Anal. (C16H24ClN3O3‚C2H2O4‚1.5H2O).
2-(cis-3,5-Dim eth ylp ip er id in o)eth yl N-(4-a m in o-5-ch lo-
r o-2-m eth oxyp h en yl)ca r ba m a te (7g): oxalate salt (24%)
from i-Pr2O/MeOH; mp 136-138 °C; 1H NMR (CD3OD) δ 7.57
(s, 1H), 6.51 (s, 1H), 4.48 (m, 2H), 3.79 (s, 3H), 3.65-3.4 (m,
4H), 2.56 (m, 2H), 2.1-1.8 (m, 3H), 1.05-0.8 (m, 7H). Anal.
(C17H26ClN3O3‚C2H2O4.H2O).
1
2-(cis-3,5-Dim et h ylp ip er id in o)et h yl
N-(2-m et h oxy-
p h en yl)ca r ba m a te (8m ): oxalate salt (22%) from i-Pr2O/
EtOH; mp 188-190 °C; 1H NMR (CD3OD) δ 7.83 (m, 1H), 7.1-
6.8 (m, 3H), 4.51 (m, 2H), 3.86 (s, 3H), 3.56 (m, 2H), 3.45 (m,
2H), 2.54 (t, 2H), 2.15-1.75 (m, 3H), 0.98 (d, 6H), 0.86 (m,
1H). Anal. (C17H26N2O3‚C2H2O4).
2-(cis-3,5-Dim eth ylp ip er id in o)eth yl N-(2-eth oxyp h en -
yl)ca r ba m a te (8n ): oxalate salt (30%) from i-Pr2O/MeOH;
1
mp 164 °C; H NMR (CD3OD) δ 7.82 (m, 1H), 7.09-6.84 (m,
3H), 4.51 (m, 2H), 4.11 (q, 2H), 3.53 (m, 2H), 3.46 (m, 2H),
2.55 (t, 2H), 2.1-1.75 (m, 3H), 1.42 (t, 3H), 0.99 (d, 6H), 0.86
(m, 1H). Anal. (C18H28N2O3‚C2H2O4).
2-(tr a n s-3,5-Dim et h ylp ip er id in o)et h yl N-(2-et h oxy-
p h en yl)ca r ba m a te (8o): oxalate salt (6%) from i-Pr2O/
MeOH; mp 174-176 °C; 1H NMR (CD3OD) δ 7.81 (m, 1H),
7.1-6.8 (m, 3H), 4.69-4.4 (m, 2H), 4.11 (q, 2H), 3.5-3.4 (m,
2H), 3.4-2.9 (m, 2H), 2.35-2.1 (m, 2H), 1.53 (m, 2H), 1.42 (t,
3H), 1.08 (d, 6H). Anal. (C18H28N2O3‚C2O4H2).
2-P ip er id in oet h yl N-(2-n it r op h en yl)ca r b a m a t e (8p ):
hydrochloride salt (83%) from i-Pr2O/EtOH; mp 164 °C; 1H
NMR (CD3OD) δ 8.2 (m, 1H), 8.12 (m, 1H), 7.68 (m, 1H), 7.27
(m, 1H), 4.58 (m, 2H), 3.51 (m, 2H), 3.8-2.8 (m, 4H), 2.2-1.4
(m, 6H). Anal. (C14H19N3O4‚HCl).
2-P ip er id in oet h yl N-(3-n it r op h en yl)ca r b a m a t e (8q ):
hydrochloride salt (61%) from i-Pr2O/MeOH; mp >210 °C; 1H
NMR (CD3OD) δ 8.49 (m, 1H), 7.87 (m, 1H), 7.75 (m, 1H), 7.51
(m, 1H), 4.56 (m, 2H), 3.49 (m, 2H), 3.9-2.9 (m, 4H), 2.2-1.4
(m, 6H). Anal. (C14H19N3O4‚HCl).
2-P ip er id in oet h yl N-(4-n it r op h en yl)ca r b a m a t e (8r ):
base (71%) from petroleum ether; mp 104 °C; 1H NMR (CDCl3)
δ 8.15 (d, 2H), 7.5 (d, 2H), 7.45 (s, 1H), 4.43 (t, 2H), 2.65 (t,
2H), 2.45 (m, 4H), 1.7-1.3 (m, 6H). Anal. (C14H19N3O4).
3-P ip er id in op r op yl N-(2-m et h oxyp h en yl)ca r b a m a t e
(8s): oxalate salt (34%) from i-Pr2O/MeOH; mp 170 °C; 1H
NMR (CD3OD) δ 7.82 (d, 1H), 6.81-7.1 (m, 3H), 4.22 (m, 2H),
3.86 (s, 3H), 3.8-3.35 (m, 2H), 3.27-3.15 (m, 2H), 3.15-2.6
(m, 2H), 2.3-2.23 (m, 2H), 1.6-1.4 (m, 6H). Anal. (C16H24N2O3‚
C2H2O4).
1-(3-(1-P ip er id in yl)p r op a n eca r boxa m id o)-4-a m in o-5-
ch lor o-2-m eth oxyben zen e (9b). 4-Chlorobutyryl chloride
(0.7 g, 5 mmol) in 10 mL of benzene was added to 1 g (5 mmol)
of 5-chloro-4-nitro-2-anisidine in 20 mL of benzene at room
temperature, and the solution was stirred for 30 min. Then
50 mL of a saturated aqueous solution of Na2CO3 was
introduced, and the mixture was stirred for 15 min. The
organic solution was separated, washed with a 1 N HCl
aqueous solution and water, and dried over MgSO4. The
solvent was evaporated to give 1 g of chloroamide 15 (65%)
which was added to a mixture of 4 mL of piperidine and
toluene and refluxed for 4 h. The precipitate of piperidine
hydrochloride was filtered, and the organic solution was
evaporated. The crude product was dissolved in AcOEt, and
the addition of 3 N HCl gave 9a hydrochloride (0.5 g) which
was isolated by filtration as an orange powder. The evapora-
tion of the filtrate provided 0.6 g of 1-(5-chloro-2-methoxy-4-
nitrophenyl)-2-pyrrolidone as the product of the cyclization
reaction; 0.5 g of 9a hydrochloride was dissolved in a mixture
of HOAc and concentrated HCl and stirred in the presence of
2-P ip er id in oeth yl N-p h en ylca r ba m a te (8a ): hydrochlo-
1
ride salt (28%) from i-Pr2O/MeOH; mp 216-218 °C; H NMR
(CD3OD) δ 8.6 (s, 1H), 7.48 (m, 2H), 7.28 (m, 2H), 7.04 (m,
1H), 4.58 (m, 2H), 3.27 (m, 2H), 4-1.3 (m, 10H). Anal.
(C14H21N2O2‚HCl).
2-P ip er id in oet h yl N-(2-m et h oxyp h en yl)ca r b a m a t e
(8b): oxalate salt (38%) from i-Pr2O/MeOH; mp 180-182 °C;
1H NMR (CD3OD) δ 7.83 (m, 1H), 7.2-6.85 (m, 3H), 4.5 (m,
2H), 3.87 (s, 3H), 3.6-3 (m, 6H), 2-1.55 (m, 6H). Anal.
(C15H22N2O3‚C2H2O4).
2-P ip er id in oet h yl
N-(2-et h oxyp h en yl)ca r b a m a t e
(8c): oxalate salt (46%) from i-Pr2O/MeOH; mp 295 °C; 1H
NMR (CD3OD) δ 7.83 (m, 1H), 7.1-6.8 (m, 3H), 4.49 (m, 2H),
4.1 (q, 2H), 3.44 (m, 2H), 3.45-3.1 (m, 4H), 2-1.5 (m, 6H),
1.42 (t, 3H). Anal. (C16H24N2O3‚C2H2O4).
2-P ip er id in oet h yl N-(3-m et h oxyp h en yl)ca r b a m a t e
(8d ): hydrochloride salt (43%) from i-Pr2O/MeOH; mp 182 °C;
1H NMR (CD3OD) δ 7.19 (m, 1H), 7.13 (s, 1H), 6.97 (m, 1H),
6.61 (m, 1H), 4.5 (m, 2H), 3.76 (s, 3H), 3.46 (m, 2H), 3.9-2.8
(m, 4H), 2.1-1.55 (m, 6H). Anal. (C15H22N2O3‚HCl).
2-P ip er id in oet h yl N-(4-m et h oxyp h en yl)ca r b a m a t e
(8e): oxalate salt (56%) from i-Pr2O/MeOH; mp 160 °C; 1H
NMR (CD3OD) δ 7.33 (d, 2H), 6.84 (d, 2H), 4.45 (m, 2H), 3.75
(s, 3H), 3.41 (m, 2H), 3.7-2.9 (m, 4H), 2-1.45 (m, 6H). Anal.
(C15H22N2O3‚C2O4H2).
2-P ip er id in oeth yl N-(5-ch lor o-2-m eth oxyp h en yl)ca r -
ba m a te (8f): oxalate salt (47%) from i-Pr2O/MeOH; mp 226-
1
228 °C; H NMR (CD3OD) δ 7.96 (d, 1H), 7.05 (dd, 1H), 6.96
(d, 1H), 4.51 (m, 2H), 3.87 (s, 3H), 3.44 (m, 2H), 3.5-3.15 (m,
4H), 1.96-1.6 (m, 6H). Anal. (C15H21ClN2O3‚C2O4H2).
2-P ip er id in oet h yl
N-(2-ch lor op h en yl)ca r b a m a t e
(8g): hydrochloride salt (85%) from i-Pr2O/EtOH; mp >220
°C dec; 1H NMR (CD3OD) δ 7.85 (m, 1H), 7.4 (m, 1H), 7.3 (m,
1H), 7.1 (m, 1H), 4.5 (m, 2H), 3.3 (m, 2H), 3.15 (m, 4H), 2.3
(m, 4H), 1.65 (m, 2H). Anal. (C14H19ClN2O2‚HCl).
2-P ip er id in oet h yl
N-(3-ch lor op h en yl)ca r b a m a t e
(8h ): hydrochloride salt (84%) from i-Pr2O/MeOH; mp 222 °C;
1H NMR (CD3OD) δ 7.6 (m, 1H), 7.31 (m, 1H), 7.25 (t, 1H), 7
(m, 1H), 4.5 (m, 2H), 3.45 (m, 2H), 3.9-2.7 (m, 4H), 2.1-1.4
(m, 6H). Anal. (C14H19ClN2O2‚HCl).
2-P ip er id in oeth yl N-(4-ch lor op h en yl)ca r ba m a te (8i):
hydrochloride salt (72%) from i-Pr2O/EtOH; mp 214 °C; 1H
NMR (CD3OD) δ 7.45 (d, 2H), 7.25 (d, 2H), 4.5 (m, 2H), 3.48
(m, 2H), 3.9-2.9 (m, 4H), 2.1-1.4 (m, 6H). Anal. (C14H19
ClN2O2‚HCl).
-
SnCl2
overnight. Then, it was poured onto ice, basified, and
extracted by EtOAc. The organic solution was dried over
MgSO4 and evaporated. The crude material was transformed
into the hydrochloride salt and recrystallized from a i-Pr2O/
MeOH mixture to give 150 mg of 9b (33%): mp 190-200 °C;
2-P ip er id in oeth yl N-(2-a m in op h en yl)ca r ba m a te (8j):
hydrochloride salt (77%) from i-Pr2O/MeOH; mp 172 °C; 1H
NMR (CD3OD) δ 7.15 (m, 1H), 7 (m, 1H), 6.82 (m, 1H), 6.69

1760 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 11
Notes
1H NMR (CD3OD) δ 8.4 (s, 1H), 7.1 (s, 1H), 3.55 (m, 2H), 3.45
(s, 3H), 3.15 (m, 2H), 2.95 (m, 2H), 2.65 (m, 2H), 2.2-1.6 (m,
7H), 1.55 (m, 1H). Anal. (C16H24ClN3O2‚2HCl‚2H2O).
incubation with 10-7 M phenoxybenzamine, the voltage was
reduced to obtain contractions 50% of those elicited by supra-
maximal stimulation. In control experiments, at least three
fully reproducible cumulative concentration-effect curves to
5-HT (3 × 10-10-10-7 M, contact time of about 60 s) were
obtained. After two fully reproducible cumulative concentra-
tion-effect curves to 5-HT, a cumulative concentration-effect
curve to the 5-HT4 receptor agonist under test was carried out
(contact time of 60-120 s). The agonist activity (EC50, nM)
was assessed as the concentration which gave a 50% increase
in the response to the electrical stimulation with regard to the
maximum, and the results are expressed as a percentage of
the maximal effect of 5-HT (100%) found in the second
reference curve. In antagonist studies, the compound under
test was added 30 min before the last cumulative concentra-
tion-effect curve to 5-HT (3 × 10-10-10-5 M). Several
concentrations (n g 3) of antagonist were tested, and the IC50
value was calculated as the concentration producing a 50%
reduction of the submaximal (80%) 5-HT contraction. The
submaximal (80%) 5-HT concentration (3 × 10-8 M) was
calculated on the second reference cumulative concentration-
response curve.
1-(3-(1-P ip er id in yl)p r op a n eca r boxa m id o)-2-m eth oxy-
ben zen e (9c). A 3.4 mL aliquot (30 mmol) of 4-chlorobutyryl
chloride in 50 mL of benzene was added to 4.9 g (40 mmol) of
2-anisidine in 100 mL of benzene at room temperature, and
the solution was stirred for 30 min. Then 50 mL of a saturated
Na2CO3 aqueous solution was added, and the mixture was
stirred for 15 min. The organic solution was separated,
washed with a 1 N HCl aqueous solution and water, and dried
over MgSO4; 6.8 g of crude product was obtained after the
evaporation of the solvent. It was added to 10 mL of piperidine
and stirred at room temperature in the presence of 0.1 g of
NaI for 2 days. The piperidine was evaporated, and the
residue was taken up in ether, a 10% Na2CO3 aqueous solution,
and water, and dried over MgSO4. After evaporation of the
solvent, the crude compound was transformed into the hydro-
chloride salt with 3 N HCl/AcOEt solution and recrystallized
1
from an AcOEt/EtOH mixture (7 g, 74.6%): mp >220 °C; H
NMR (CD3OD) δ 7.9 (m, 1H), 7.15 (m, 1H), 7 (m, 1H), 6.9 (m,
1H), 3.85 (s, 3H), 3.1 (m, 4H), 3 (m, 2H), 2.6 (m, 2H), 2.05 (m,
2H), 1.8 (m, 4H), 1.62 (m, 2H). Anal. (C16H24N2O2‚HCl).
P h a r m a cologica l Meth od s. 1. 5-HT4 Recep tor Bin d -
in g Assa ys. Male Sprague-Dawley rats from J anvier Labo-
ratories (France) were used. Animals were housed at 22 ( 1
°C, with 55% humidity, on a 12 h light-dark cycle with free
access to food and water for 4 days before the experiments.
Membranes were prepared from rat striatum and olfactory
tubercules which were pooled separately and stored at -80
°C. Tissues were thawed at 0 °C, homogenized in 15 volumes
of ice-cold 50 mM Hepes (pH 7.4) using a Polytron homog-
enizer, and then centrifuged once at 40000g at 4 °C for 15 min.
The resulting pellet was resuspended in 5 volumes of Hepes
buffer to a final concentration of 4-5 mg of protein/mL.
Membrane aliquots of 2.6 mL were kept frozen at -80 °C for
subsequent use.
Refer en ces
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previously described12 with modifications. The binding of [3H]-
GR 113808 (85 Ci/mmol; Amersham) was measured using
membranes (50 µL aliquots, equivalent to 0.1-0.2 mg of
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soaked in 0.1% poly(ethylenimine) using a Brandel Cell
harvester. The filters were then washed twice with 5 mL of
50 mM Tris-HCl (pH 8.4) at room temperature and dried. The
filters were placed in polyethylene vials to which was added 4
mL of a scintillation cocktail (Beckman, Ready-Safe), and after
equilibration, the radioactivity was determined using liquid
scintillation spectrometry. The data were analyzed by a
computer-assisted curve-fitting program in Lotus 1.2.3. to
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the Cheng-Prussof equation.
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125I]-SB 207710 of a small 5-HT4 receptor population in piglet
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K. A. N-[(1-Butyl-4-piperidinyl)methyl]-3,4-dihydro-2H-[1,3]ox-
azino[3,2-a]indole-10-carboxamide hydrochloride: The first po-
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Bingham, S.; Ellis, E. S.; Gaster, L. M.; Rushan, B.; Smith, M.
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Lon gitu d in a l Mu scle P r ep a r a tion fr om Gu in ea P ig Il-
eu m . Tissues were obtained from male Crl (HA) SPF guinea
pigs (400-600 g) housed as described previously. According
to Craig and Clarke,4a segments (2 cm long, 10 cm from the
ileo-cecal junction) of myenteric plexus and longitudinal muscle
from the ileum were carefully dissected and mounted in a 20
mL organ bath containing a warm (37 °C), aerated (95% O2,
5% CO2) Krebs solution. The tissue was stretched with a 0.5
g weight, and contractions elicited by transmural electrical
stimulation (0.2 Hz, pulse duration of 1.5 ms) were recorded
by means of an isotonic transducer connected to a polygraph
(Gemini, Basile, Italy). The preparations were subjected to a
supramaximal voltage stimulation. After stabilization and

Notes
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 11 1761
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R. W.; Cruz Sa´nchez, F.; Tolosa, E.; Palacios, J . M. Identification
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J M960853V