New benzo[h][1,6]naphthyridine and azepino[3,2-c]quinoline derivatives as selective antagonists of 5-HT4 receptors: Binding profile and pharmacological characterization

Article abstract of DOI:10.1021/jm020954v

A series of benzo[h] [1,6] naphthyridine and azepino[3,2-c] quinoline derivatives were prepared and evaluated to determine the necessary requirements for high affinity on the 5-HT₄ receptors and high selectivity versus other receptors. The compounds were synthesized by substituting the chlorine atom of benzonaphthyridines and azepinoquinolines with various N-alkyl-4-piperidinylmethanolates. They were evaluated in binding assays with [³H]GR 113808 as the 5-HT₄ receptor radioligand. The affinity values (K_i or inhibition percentages) depended upon the substituent on the aromatic ring on one hand and the substituent on the lateral piperidine chain on the other hand. A chlorine atom produced a marked drop in activity while a N-propyl or N-butyl group gave compounds with nanomolar affinities (1 < K_i < 10 nM). Among the most potent ligands (3a, 4a, 5a), 4a was selected on the basis of its high affinity and selectivity for pharmacological screening and was evaluated in vivo in specific tests. This compound reveals itself as an antagonist/low partial agonist in the COS-7 cells stably expressing the 5-HT_4(a) receptor. Derivative 4a also showed in vivo potent analgesic activity in the writhing test at very low doses.

Full text of DOI:10.1021/jm020954v

138
J . Med. Chem. 2003, 46, 138-147
New Ben zo[h ][1,6]n a p h th yr id in e a n d Azep in o[3,2-c]qu in olin e Der iva tives a s
Selective An ta gon ists of 5-HT4 Recep tor s: Bin d in g P r ofile a n d P h a r m a cologica l
Ch a r a cter iza tion
Antoine Hinschberger,§,| Sabrina Butt,§,| Ve´ronique Lelong,§,† Michel Boulouard,§,| Aline Dumuis,‡
Franc¸ois Dauphin,|,† Ronan Bureau,§,| Bruno Pfeiffer, Pierre Renard, and Sylvain Rault*,§,|
ATBI, Universite´ de Caen, 5 rue Vaube´nard, 14032 Caen Cedex, France; Centre d’Etudes et de Recherche sur le Me´dicament de
Normandie, Universite´ de Caen, 5 rue Vaube´nard, 14032 Caen Cedex, France; UMR 6551 CNRS, Universite´ de Caen,
Centre Cyceron, Boulevard Henri Becquerel, 14074 Caen Cedex, France; UPR 9023, CCIPE, 141 rue de la Cardonille,
34094 Montpellier Cedex 5, France; and Les Laboratoires Servier 1, rue Carle Hebert 92415 Courbevoie Cedex
Received J une 7, 2002
A series of benzo[h][1,6]naphthyridine and azepino[3,2-c]quinoline derivatives were prepared
and evaluated to determine the necessary requirements for high affinity on the 5-HT4 receptors
and high selectivity versus other receptors. The compounds were synthesized by substituting
the chlorine atom of benzonaphthyridines and azepinoquinolines with various N-alkyl-4-
piperidinylmethanolates. They were evaluated in binding assays with [3H]GR 113808 as the
5-HT4 receptor radioligand. The affinity values (Ki or inhibition percentages) depended upon
the substituent on the aromatic ring on one hand and the substituent on the lateral piperidine
chain on the other hand. A chlorine atom produced a marked drop in activity while a N-propyl
or N-butyl group gave compounds with nanomolar affinities (1 < Ki < 10 nM). Among the
most potent ligands (3a , 4a , 5a ), 4a was selected on the basis of its high affinity and selectivity
for pharmacological screening and was evaluated in vivo in specific tests. This compound reveals
itself as an antagonist/low partial agonist in the COS-7 cells stably expressing the 5-HT4(a)
receptor. Derivative 4a also showed in vivo potent analgesic activity in the writhing test at
very low doses.
In tr od u ction
system were published5,7 and seemed to afford structure-
activity relationships that led to a common pharma-
cophore.18,19,20
The neurotransmitter serotonin (5-hydroxytryptamine,
5-HT) modulates the activity of the central nervous
system and peripheral tissues through its actions at
numerous receptor subtypes.1 During the past decades,
considerable attention has been centered around the
identification of agents which act selectively at each of
these receptor subtypes because of the wide range of
physiologic systems and pathologic conditions in which
5-HT is known to play a role. One of these receptor
subtypes, the 5-HT4 receptor,2 was first identified in
19883 and thereafter has been cloned.4 Various com-
pounds acting as antagonists on 5-HT4 receptors5-7 have
been described; a generic family of benzamides which
act as agonists has been discovered recently.8,9 Proposed
therapeutic applications for agents that bind to the
5-HT4 receptors include the treatment of memory
deficits,10,11 cardiac atrial arrhythmias,12,13 gastropare-
sis, urinary incontinence,14 irritable bowel syndrome,15
and pain.16,17
In this paper, we report the synthesis, the receptor
binding profile, and the in vitro and in vivo pharmaco-
logical evaluation of a series of tricyclic benzo[h][1,6]-
naphthyridine (BN), azepino[3,2-c]quinoline (AQ), and
tetracyclic dibenzo[b,h][1,6]naphthyridine (DBN) de-
rivatives, which are analogues of phenanthridines.
These derivatives have the tricyclic skeleton depicted
in Chart 2 and the 1-alkylpiperidinylmethyl moiety
encountered in carboxylate compounds (GR 113808, SB
204070, SB 207266).
In a manner similar to the one previously conducted
for the design of 5-HT3 receptor ligands,21,22 chemical
modifications via substitution of the BN’s, AQ’s, and
DBN’s skeletons were systematically carried out to
determine the necessary structural requirements for a
good affinity on 5-HT4 receptors and a high selectivity
toward other receptors.
The availability of the cloned 5-HT4 receptors stimu-
lated more interest in the search of new ligands.
Recently, new 5-HT4 antagonists (Chart 1) which pos-
sessed a side chain centered around a piperidine ring
5-HT4 Receptor antagonists already published (Chart
1) have relatively similar chemical structures. Indeed,
they all possess an ester or amide function attached to
an aromatic ring and an amine at a variable distance
from this ring.20 In our series, we explored the modifica-
tion of each of these elements: the aromatic acyl group
and the substituent on the basic nitrogen of the piperi-
dine ring system. Because of the expected lability of the
ester linkage in compounds such as SB 204070, we
focused our synthetic efforts on compounds in which the
acyl group was connected to the piperidine ring through
* Corresponding author. Universite´ de Caen, 5, rue Vaube´nard
14032 Caen Cedex, France. Te´l: 02 31 93 41 69. Fax.: 02 31 93 11 88.
E-mail: rault@pharmacie.unicaen.fr.
| ATBI, Universite´ de Caen.
§ Centre d’Etudes et de Recherche sur le Me´dicament de Normandie,
Universite´ de Caen.
† UMR 6551 CNRS, Universite´ de Caen.
‡ UPR 9023, CCIPE.
Les Laboratoires Servier 1.
10.1021/jm020954v CCC: $25.00 © 2003 American Chemical Society
Published on Web 12/07/2002

New Derivatives as Selective Antagonists of 5-HT4 Receptors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 1 139
Ch a r t 1
Ch a r t 2
Sch em e 1a
an iminoether linkage. Recent SAR studies that super-
impose iminoether derivatives with the pharmacophore
of 5-HT4 receptors showed that a cyclic iminoether group
could be considered as a bioisoster of the ester function
found in 5-HT4 antagonist structures.20
Indeed, influence of the substitutions on the phar-
macological nature of the interaction with the receptor
(partial agonist, antagonist) was also determined for the
most active compounds.
Ch em istr y
a
(i) DMF, reflux, 2 h; (ii) POCl3/pyridine, µw (700 W), 1 h 45;
(iii) N-alkyl-4-piperidinylmethanol, NaH, DMF, or toluene, 230-
240 °C, 4-6 bar, 1-2 h.
The general synthetic procedures used in this study
are issued of benzonaphthyridine and azepinoquinoline
chemistry that we reported previously23 and are il-
lustrated in Schemes 1-5. Thus, 5-substituted 1,2,3,4-
tetrahydrobenzo[h][1,6]naphthyridines 1a -8a (Scheme
1) were obtained in a three-step pathway starting with
condensation of isatoic anhydride and l-proline24-27 to
give the pyrrolo[2,1-c][1,4]benzodiazepine 9. Treatment
of 9 under drastic conditions with boiling phosphorus
oxychloride and a catalytic amount of pyridine gave the
rearranged 5-chloro-1,2,3,4-tetrahydrobenzo[h][1,6]-
naphthyridine 10. The displacement of the chlorine
atom was realized by treatment of 10 with an excess of
the appropriate alcoholate in anhydrous dimethylfor-
mamide (DMF) at 230-240 °C in a stainless autoclave
to give 1a -8a . The synthesis of corresponding alcoho-
lates was described in the literature or could be obtained
by well-known processes; thus, 1-alkyl-4-piperidinyl-
methanol was prepared from ethyl 4-piperidine car-
boxylate by N-alkylation with 1-halogenoalcane followed
by lithium aluminum hydride reduction of the interme-
diate.28,29
The (N-propargyl-4-piperidinyl) iminoether 7a was
also obtained by treatment of nonsubstituted (4-pip-
eridinyl) iminoether 1a with propargyl bromide in the
presence of K2CO3 at reflux of ethanol. No reaction on
the naphthyridic NH took place because of its poor
reactivity.23
Scheme 2 illustrates the synthesis of 9-chloro 5-sub-
stituted 1,2,3,4-tetrahydrobenzo[h][1,6]naphthyridines
4b, 5b which were obtained in a four-step pathway.
6-Chloroisatoic anhydride in the first step was prepared
by cyclization of 5-chloroanthranilic acid in the presence
of phosgene solution (20% in toluene) in dioxane30 at
room temperature. Then, the following steps of the
synthesis were realized in a similar manner as above.
6-Substituted 2,3,4,5-tetrahydro-1H-azepino[3,2-c]-
quinolines 3c-6c, 10-chloro 6-substituted tetrahydroaze-
pino[3,2-c]quinolines 4d , 5d and 9-chloro 6-substituted
tetrahydroazepino[3,2-c]quinoline 4e were obtained in
a very similar manner as their naphthyridine analogues

140 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 1
Hinschberger et al.
Sch em e 2a
Sch em e 4a
a
(i) COCl2 (20% in toluene)/dioxane, RT, 18 h; (ii) L-proline,
a (i) DMF, reflux, 2 h; (ii) POCl3/pyridine, µw (700 W), 1 h 45;
(iii) N-propyl-4-piperidinylmethanol, NaH, toluene, 240 °C, 6 bar,
1.5h.
DMF, reflux, 2 h; (iii) POCl3/pyridine, µw (700 W), 1 h 45; (iv)
N-alkyl-4-piperidinylmethanol, NaH, toluene, 240 °C, 6 bar, 1.5
h.
Sch em e 5a
Sch em e 3a
a
(i) DMF, reflux, 2 h; (ii) CrO3/H3PO4, acetone, 20 h; (iii) POCl3/
pyridine, µw (500 W), 50 min; (iv) N-alkyl-4-piperidinylmethanol,
NaH, toluene, reflux, 4 h.
above.39 Finally, the rearranged 5-chloro aromatized
product 23 was substituted selectively with alcoholates
to give the desired iminoethers 3g, 4g. We did not
observe reaction of substitution of the 3-chlorine atom.
However, the compound 23 exhibited a better reactivity
than the disymmetric rearranged product described
above since the reaction of substitution was realized at
reflux in toluene.
a
(i) COCl2 (20% in toluene)/dioxane, RT, 18 h; (ii) D,L-
pipecolinic acid, DMF, reflux, 2 h; (iii) POCl3/pyridines µw (700W),
1 h 45; (iv) N-alkyl-4-piperidinylmethanol, NaH, toluene, 240 °C,
6 bar, 1.5h.
(Scheme 3) starting from D,L-pipecolinic acid which was
first condensed with the appropriate isatoic anhydrides
to give the pyrido[2,1-c][1,4]benzodiazepines 13-15,31
which then rearranged into chlorotetrahydroazepino-
[3,2-c]quinolines 16-18.23,32 Finally, the nucleophilic
displacement of the chlorine atom gave 3c-6c, 4d , 5d ,
and 4e.
Scheme 4 illustrates the synthesis of 6-substituted
octahydrodibenzo[b,h][1,6]naphthyridine 4f realized in
a similar manner as above starting from condensation
of isatoic anhydride with trans-perhydroindole-2-car-
boxylic acid to give perhydroindolo[2,1-c][1,4]benzo-
diazepine 1933 that rearranged into 6-chloro-octahydro-
dibenzo[b,h][1,6]naphthyridine 20. Nucleophilic substi-
tution of the chlorine atom with N-propyl-4-piperidi-
nylmethanol gave 4f.
The synthesis of aromatized structures such as 5-sub-
stituted 3-chlorobenzo[h][1,6]naphthyridines 3g, 4g was
realized in a four-step pathway (Scheme 5) starting
with 2-hydroxy-hexahydro-5H-pyrrolo[2,1-c][1,4]benzo-
diazepine 21 prepared from condensation of isatoic
anhydride and trans-4-hydroxy-L-proline.34-36 Treat-
ment of alcohol 21 with chromic anhydride and phos-
phoric acid in acetone gave the ketone 22.34,37,38 The 3,5-
dichlorobenzo[h][1,6]naphthyridine 23 was obtained by
rearrangement of 22 in the same condition as described
Resu lts a n d Discu ssion
Twenty benzo[h][1,6]naphthyridines and azepino[3,2-
c]quinolines derivatives were designed, prepared, and
first evaluated in a prescreening procedure for their
affinity for 5-HT4 receptors (Tables 1-2). In addition,
selectivity toward other 5-HT receptor subtypes (5-HT1A
,
5-HT2A, 5-HT2B, 5-HT2C, 5-HT6, and 5-HT7) and nonse-
rotoninergic receptors as adrenergic, dopamine, hista-
mine, and vasopressin receptors was also evaluated in
one representative compound 4a (Table 3).
The discussion on the SARs are now axed around two
moieties: the lateral side chain and the tricyclic plat-
form.
Mod ifica tion of th e La ter a l Sid e Ch a in . These
studies identified a series of N-alkyl-4-piperidinyl-
methoxy derivatives whose affinities ranged between
10-8 and 10-9 M. We explored the substitution on the
piperidine moiety with linear alkyl saturated or non-
saturated groups. The selection of a flexible piperidine
system increased 5-HT4 receptor antagonist activity if
we compared with the rigid tropane skeleton encoun-

New Derivatives as Selective Antagonists of 5-HT4 Receptors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 1 141
Ta ble 1. Binding Properties of the
1,2,3,4-tetrahydrobenzo[h][1,6]naphthyridine Derivatives at
5-HT4 Receptorsa
Substitution with unsaturated groups (allyl and pro-
pargyl) led to a decrease in affinity at the 5-HT4
receptors. It seemed that the loss of affinity could be
linked to the unsaturated degree of the alkyl group (allyl
compound 8a , 100% inhibition at 10-6 M and 31%
inhibition at 10-8 M; propargyl compound 7a , 100%
inhibition at 10-6 M and 15% inhibition at 10-8 M).
Mod ifica tion of th e Ar om a tic P la tfor m . We also
investigated modifications around a quinoline cycle. We
thus studied the influence of fused saturated rings (C-6
cycle for the series a , b; C-7 cycle for the series c, d , e;
bicyclic C-6-C-6 for the series f and fused aromatic ring
for the series g). We also studied the influence of
substitution of the quinoline cycle with chlorine atoms.
In this respect, (a) substitution on the benzene part of
the tetrahydrobenzonaphthyridine skeleton with a 9-chlo-
ro (4b, 5b) and the tetrahydro-1H-azepinoquinoline
skeleton with a 9-chloro (4e) or 10-chloro (4d , 5d ) clearly
decreases the inhibition efficacy at 10-8 M; (b) replace-
ment of fused C-6 or C-7 saturated rings by an aromatic
ring in the benzonaphthyridine series (ethyl compound
3g, 81% inhibition at 10-6 M and 6% at 10-8 M; butyl
compound 4g, 100% inhibition at 10-6 M and 8%
inhibition at 10-8 M) also leads to weaker efficacies; and
(c) replacement of fused C-6 or C-7 saturated rings by
a bicyclic system in the octahydrodibenzonaphthyridine
derivative 4f results in a marked decrease in affinity
for the 5-HT4 receptors (29% inhibition at 10-6 M and
0% inhibition at 10-8 M).
% inhibition 5-HT4
compd
R
R1
10-6
M
10-8
M
Ki (nM)
1a
2a
3a
4a
5a
6a
7a
8a
4b
5b
H
CH3
H
H
H
H
H
H
H
H
Cl
Cl
96
24
52
55
70
77
51
15
31
0
100
100
100
100
100
100
100
100
100
79.7
13.1
2.89
10
CH2CH3
(CH2)2CH3
(CH2)3CH3
(CH2)4CH3
CH2-CtCH
CH2-CHdCH2
(CH2)2CH3
(CH2)3CH3
12.5
2
Percentage of inhibition of 0.6 nM [3H]GR 113808 binding (at
fixed concentrations of test compounds) and inhibition constants
(Ki, nM) are given.
a
Ta ble 2. Binding Properties of the
2,3,4,5-tetrahydro-1H-azepino[3,2-c]quinoline Derivatives at
5-HT4 Receptorsa
The above-mentioned results prove the important
contribution of the following pharmaphoric sites for
high-affinity 5-HT4 ligands: (1) a tricyclic heterocycle,
not halogenated on the benzene part and not entirely
aromatic; and (2) a voluminous substituent in the basic
nitrogen atom of the amino moiety of the lateral side
chain and the optimum distance from this nitrogen to
the aromatic ring that are important for high-affinity
for 5-HT4 receptors. SAR and QSAR studies recently
evaluated an ideal distance of around 8 Å between 1
and 2.18,19,20 Furthermore, the optimum distance from
the piperidinic nitrogen to the hydrogen-bond acceptor
represented by the “imine” group is 7.4 Å.20,40
% inhibition 5-HT4
compd
R
R1 R2
10-6
M
10-8
M
Ki (nM)
3c
4c
5c
6c
4d
5d
4e
CH2CH3
H
H
H
H
H
H
H
H
H
100
51
55
44
0
10.7
12.7
16.3
(CH2)2CH3
(CH2)3CH3
(CH2)4CH3
(CH2)2CH3
(CH2)3CH3
100
100
100
91
H
Cl
Cl
H
5
92
0
(CH2)2CH3 Cl
98
0
Percentage of inhibition of 0.6 nM [3H]GR 113808 binding (at
fixed concentrations of test compounds) and inhibition constants
(Ki, nM) are given.
a
In conclusion to this preliminary evaluation, the
chemical modifications that we carried out led us to the
discovery of new compounds having a very high affinity
at the 5-HT4 receptor (at least equivalent to the one of
SB 204070 or GR 113808) and a high selectivity toward
other 5-HT receptor subtypes. Indeed, among these
high-affinity compounds, the selectivity of 4a has been
checked versus seven serotonin receptors and reuptake
sites (Table 3): the IC50 values of this ligand are all
representative of low affinities, in the high micromolar
range except for the one on 5-HT2B receptors (IC50 ) 2
× 10-7 M). The selectivity of this compound was also
extended toward dopaminergic, adrenergic, histamin-
ergic, and vasopressin receptors (Table 3), where 4a
exhibits very limited affinity with IC50 values higher
than 10-6 M for all the receptors tested.
tered in Tropisetron that possess a 5-HT4 receptor
antagonist activity.5
With regard to the basic amino moiety of the molecule,
the presence of at least one methylene unit between the
acyl group or iminoether group and the 4-substituted
piperidine ring is necessary for selective binding at
5-HT4 sites over 5-HT3 receptors.7 We thus obtained
good results of affinities with methyl, ethyl, propyl, and
pentyl groups (2.89 < Ki < 16.3 nM) for series a (2a -
6a ) and c (3c-5c). Increasing the length of the linking
methylene chain to four and five carbons (5a , 6a , 5c,
6c) did not further increase the affinity at 5-HT4
receptors. For compound 6c, we observed a decrease in
affinity with a pentyl group (100% inhibition at 10-6
M
and 0% inhibition at 10-8 M). An increase in the
hydrophobic character around the distal nitrogen atom
is a favorable parameter since the NH derivative 1a
exhibited a lower affinity for the 5-HT4 receptors (96%
inhibition at 10-6 M and 24% inhibition at 10-8 M) than
N-alkyl compounds.
P h a r m a cology. Compound 4a , exhibiting the higher
affinity, has been selected for characterization of its
activity at the 5-HT4 receptor level. The agonist or
antagonist properties have been determined in a test
evaluating the production of cAMP measured in COS-7
cells expressing the mouse 5-HT4(a) receptor.41,42 In vivo

142 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 1
Ta ble 3. Binding Selectivity of 4a
Hinschberger et al.
% inhibition
receptor
5-HT1A
5-HT2A
5-HT2B
5-HT2C
5-HT4
5-HT6
5-HT7
recapture 5-HT
dopamine D1
dopamine D2
recapture DA
histamine H1
histamine H2
adrenergic â1
adrenergic â2
recapture NAD
vasopressin V1
vasopressin V2
radioligand
10-5
M
10-7
M
IC50 (M)
[3H]8-OH-DPAT
[3H]-ketanserin
[3H]5-HT
9.1 × 10-6
5 × 10-6
2 × 10-7
[3H]-mesulergine
[3H]-GR113808
3.4 × 10-6
5.6 × 10-10
[
[
125I]-LSD
125I]-LSD
12% at 10-6
36% at 10-6
79
M
M
0% at 10-8
20% at 10-8
0
M
M
[3H]-paroxetine
[3H]-SCH23390
[3H]-YM-0915-
[3H]-GBR12935
[3H]-mepyramine
[3H]-tiotidine
[3H]-CGP12177
[3H]-CGP12177
[3H]-nisoxetine
[3H]-AVP
>10-5
3.4 × 10-6
61
91
75
39
70
23
13
0
0
0
0
17
3.2 × 10-6
1.1 × 10-5
[3H]-AVP
F igu r e 1. Antagonist profile of 4a . Concentration-dependent
effect of 4a on adenylyl cyclase activity in COS-7 cells stably
transfected with the 5-HT4(a) receptor in the presence of 10-7
M of 5-HT.
F igu r e 2. Agonist profile of 4a . Concentration-dependent
effect of 4a on adenylyl cyclase activity in COS-7 cells stably
transfected with the 5-HT4(a) receptor in the absence of 5-HT.
studies have also been conducted to first determine
gross behavioral effects and acute toxicity.43 Second,
considering the demonstrated implication of 5-HT4
receptors in learning and memory processes,44-46 the
capacity of 4a to modulate such central action was
studied by testing these effects on spontaneous alterna-
tion47 (a working-memory model) in mice treated or not
by scopolamine. Finally, the nature of action profile of
4a was investigated by studying its analgesic potential
by simultaneously using the writhing test48 (peripheral
analgesic activity) and the hot plate method49(central
analgesic activity) in the mouse. Recent works have
described the potentiality of 5-HT4 receptor ligands to
decrease nociceptive responses in different models of
pain in the mouse.16,17
P h a r m a cologica l Resu lts. The nature of the inter-
action to the 5-HT4 receptors was studied in vitro on
COS-7 cells. In the presence of 5-HT (10-7 M), the
compound 4a decreased the production of cAMP into
COS-7 cells. The specific 5-HT4 antagonist effect of 4a
is ∼70% on 5-HT response (Ki ) 5 ( 1.5 nM) (Figure
1). In the absence of 5-HT, the compound 4a produced
a slight increase of cAMP which corresponds to ∼30%
of the 5-HT response that demonstrated an additional
partial agonist effect in this case (Figure 2).
Ta ble 4. Pharmacological and Toxicological Properties of 4a
doses
(mg/kg)
LD50
symptoms
symptoms
compd
(mg/kg) (subtoxic doses) (toxic doses)
hypoactivity
4a
12.5-25-50 37.5
relaxation
passivity
convulsions
hyperactivity
irritability
stereotypy
hypoactivity
passivity
methylphenidate 25
perphenazine
5
ptosis
The data concerning preliminary toxicological and
pharmacological screening of compound 4a (Table 4)
notably showed, at subtoxic doses (at <1/4 approxima-
tive LD50), that the major observation was hypoactivity.
Considering the LD50, subsequent studies were realized
at doses not higher than 1 mg/kg.
At the doses tested (see Experimental Section), com-
pound 4a had neither a per se effect on spontaneous
alternation performance in the mouse, nor an action on
the scopolamine-induced deficit in this model (data not
shown). In the writhing test (Table 5), 4a exhibited
antinociceptive activities at very weak doses and re-

New Derivatives as Selective Antagonists of 5-HT4 Receptors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 1 143
Ta ble 5. Antinociceptive Activity of Tested Compounds and
References in the Mouse Writhing Test after Intraperitoneal
Administration
washed three times with water, dried (MgSO4), decolorized
with vegetal charcoal, and filtered. The solvent was evaporated
under reduced pressure. The oil obtained was purified by
chromatography on a column of silicagel with CHCl3/MeOH
(90:10). The pure oil was dissolved in propan-2-ol; then 1.3
equivalent of fumaric acid was added, and the suspension was
refluxed for 10 min. After cooling, the precipitate was filtered
and dried first with anhydrous ethyl ether and then in a
laboratory oven (50 °C). This gave series a (2a -6a ): 11-23%
yield.
number of stretchesa
0.01
0.1
1
5
30
0
mg/kg mg/kg mg/kg mg/kg mg/kg
4a
24.6
19
16.5
18.2
13.8* 12.5**
11.5* 11.2*
12.8* 12.3*
GR 125487 20
GR 113808 21.8
a sp ir in e
p ir oxica m
25.8
24.6
8.7**
5-[(N -Me t h ylp ip e r id in -4-yl)m e t h oxy]-1,2,3,4-t e t r a -
h yd r ob en zo[h ][1,6]n a p h t h yr id in e Mon ofu m a r a t e (2a ).
Obtained as a white powder, 0.50 g, 19% yield. MP: 125°C
(i-PrOH). 1H NMR (DMSO-d6, 400 MHz): δ 1.53 (m, 2H, CH2),
1.84 (m, 5H, H2′, H3′, H6′), 2.47 (s, 3H, N-CH3), 2.50 (m, 2H,
2.4***
a
Number of stretches induced by a 0.6% acetic acid solution.
Statistical significance between control group and treated group
after a combined analysis of variance and a PLSD test.*p <
0.05;**p < 0.01;***p < 0.001.
H4′, H5′), 2.62 (m, 2H, CH2), 3.14 (d, 2H, H4′, H5′, J 4′,4′ ) J 5′,5′
)
11.10 Hz), 3.34 (m, 2H, CH2), 4.21 (d, 2H, O-CH2, J 1′,2′ ) 5.40
Hz), 6;53 (s, 2H, CHdCH, fumarate), 7.06 (s, 1H, NH), 7.22
(t, 1H, H9), 7.45 (t, 1H, H8), 7.51 (d, 1H, H7), 7.90 (d, 1H, H10).
IR: 3358 (m, NH), 1690 (s, CdO fumarate), 1636 (s, CdN)
cm-1. Anal. (C23H29N3O5) C, H, N.
vealed an activity profile close to the 5-HT4 receptor
antagonists GR 125487 and GR 113808. In the same
range of doses, no effect could be detected in the hot
plate test (data not shown). Taken together, these data
suggest a clear peripheral profile for 4a ; considering its
high activity in a visceral pain model, this compound
could constitute a lead in the search of new peripheral
analgesics with molecular targets different than clas-
sical nonsteroidal antiinflammatory drugs and thus,
potentially devoid of side gastrointestinal effects.
5-[(N -E t h y lp ip e r id in -4-y l)m e t h o x y ]-1,2,3,4-t e t r a -
h yd r ob en zo[h ][1,6]n a p h t h yr id in e Mon ofu m a r a t e (3a ).
Obtained as a white powder, 0.28 g, 14% yield. MP: 220 °C
1
(i-PrOH). H NMR (DMSO-d6, 400 MHz): δ 1.12 (t, 3H, CH3,
J ) 7.20 Hz), 1.56 (m, 2H, CH2), 1.86 (m, 5H, H2′, H3′, H6′),
2.47 (m, 2H, N-CH2), 2.62 (m, 2H, CH2), 2.74 (m, 2H, H4′, H5′),
3.23 (d, 2H, H4′, H5′, J 4′,4′ ) J 5′5′ ) 11.60 Hz), 3.34 (m, 2H, CH2),
4.21 (d, 2H, O-CH2, J 1′,2′ ) 5.90 Hz), 6;51 (s, 2H, CHdCH,
fumarate), 7.07 (s, 1H, NH), 7.22 (t, 1H, H9), 7.45 (t, 1H, H8),
7.51 (d, 1H, H7), 7.90 (d, 1H, H10). IR: 3378 (m, NH), 1690 (s,
CdO, fumarate), 1637 (s, CdN) cm-1. Anal. (C24H31N3O5) C,
H, N.
Con clu sion
All the compounds we have prepared within this new
family of tricyclic benzonaphthyridine and azepino-
quinoline derivatives lead us to clearly establish SARs
for selective and high-affinity 5-HT4 receptor ligands.
Experimental in vivo and in vitro data have revealed
the antagonist/partial agonist character of the best
5-HT4 derivatives. Among these, 4a proved to be of great
interest as a peripheral antinociceptive agent. Comple-
mentary studies are underway to evaluate the thera-
peutic potential of this compound. Moreover, these
results should be important for the research on the
pharmacophores of 5-HT4 receptors.
5-[(N -P r op ylp ip e r id in -4-yl)m e t h oxy]-1,2,3,4-t e t r a -
h yd r ob en zo[h ][1,6]n a p h t h yr id in e Mon ofu m a r a t e (4a ).
Obtained as a yellow powder, 1.10 g, 11% yield. MP: 200 °C
1
(i-PrOH). H NMR (DMSO-d6, 400 MHz): δ 0.86 (t, 3H, CH3,
J ) 7.30 Hz), 1.51 (m, 4H, 2CH2), 1.82 (m, 5H, H2′, H3′, H6′),
2.31 (m, 2H, N-CH2), 2.50 (m, 2H, H4′, H5′), 2.62 (m, 2H, CH2),
3.12 (d, 2H, H4′, H5′, J 4′,4′ ) J 5′,5′ ) 11.10 Hz), 3.34 (m, 2H, CH2),
4.19 (d, 2H, O-CH2, J 1′,2′ ) 5.70 Hz), 6;53 (s, 2H, CHdCH,
fumarate), 7.05 (s, 1H, NH), 7.22 (t, 1H, H9), 7.45 (t, 1H, H8),
7.51 (d, 1H, H7), 7.90 (d, 1H, H10). IR: 3379 (m, NH), 1705 (s,
CdO, fumarate), 1595 (s, CdN) cm-1. Anal. (C25H33N3O5) C,
H, N.
5-[(N -B u t y lp ip e r id in -4-y l)m e t h o x y ]-1,2,3,4-t e t r a -
h yd r ob en zo[h ][1,6]n a p h t h yr id in e Mon ofu m a r a t e (5a ).
Obtained as a white powder, 0.30 g, 14% yield. MP: 210 °C
Exp er im en ta l Section
1
(i-PrOH). H NMR (DMSO-d6, 400 MHz): δ 0.88 (t, 3H, CH3,
Ch em istr y. Every compound was characterized by elemen-
tal analysis, IR spectra, and 1H NMR spectra; data are
reported only for the compounds tested in the pharmacological
study. IR spectra were recorded on a Genesis Series FTIR
infrared spectrometer using KBr pellets; the frequencies are
expressed in cm-1. The 1H NMR spectra were obtained on a
J EOL Lambda 400 spectrometer, with Me4Si as the internal
standard and DMSO-d6 as the solvent; the chemical shifts are
reported in ppm of Me4Si in δ units, and the coupling constants
J ) 7.30 Hz), 1.29 (sext, 2H, CH2, J 9′,8′ ) J 9′,10′ ) 7.30 Hz),
1.51 (m, 4H, 2CH2), 1.83 (m, 5H, H2′, H3′, H6′), 2.39 (m, 2H,
N-CH2), 2.61 (m, 4H, CH2, H4′, H5′), 3.18 (d, 2H, H4′, H5′, J 4′,4′
) J 5′,5′ ) 11.50 Hz), 3.34 (m, 2H, CH2), 4.20 (d, 2H, O-CH2,
J 1′,2′ ) 5.80 Hz), 6;53 (s, 2H, CHdCH, fumarate), 7.06 (s, 1H,
NH), 7.22 (t, 1H, H9), 7.45 (t, 1H, H8), 7.51 (d, 1H, H7), 7.90
(d, 1H, H10). IR: 3302 (m, NH), 1698 (s, CdO, fumarate), 1639
(s, CdN) cm-1. Anal. (C26H35N3O5) C, H, N.
1
are in hertz. The IR and H NMR spectra were consistent with
5-[(N -P e n t ylp ip e r id in -4-yl)m e t h oxy]-1,2,3,4-t e t r a -
h yd r ob en zo[h ][1,6]n a p h t h yr id in e Mon ofu m a r a t e (6a ).
Obtained as a white powder, 0.50 g, 23% yield. MP: 212 °C
assigned structures. Elementary analyses were within (0.4%
of the theoretical values except for compounds 2a , 7a , and 3c
for which the hydrogen analysis, respectively, exhibits (0.42,
(0.53, and (0.44% errors.
1
(i-PrOH). H NMR (DMSO-d6, 400 MHz): δ 0.87 (t, 3H, CH3,
J ) 6.90 Hz), 1.27 (m, 4H, 2CH2), 1.46 (m, 4H, 2CH2), 1.83
(m, 2H, H4′, H5′), 2.45 (t, 2H, N-CH2), 2.62 (m, 2H, CH2), 3.05
(d, 2H, H4′, H5′, J 4′,4′ ) J 5′,5′ ) 10.30 Hz), 3.34 (m, 2H, CH2),
4.19 (d, 2H, O-CH2, J 1′,2′ ) 5.50 Hz), 6;50 (s, 2H, CHdCH,
fumarate), 7.03 (s, 1H, NH), 7.21 (t, 1H, H9), 7.44 (t, 1H, H8),
7.51 (d, 1H, H7), 7.89 (d, 1H, H10). IR: 3300 (m, NH), 1694 (s,
CdO, fumarate), 1618 (s, CdN) cm-1. Anal. (C27H37N3O5) C,
H, N.
5-Su b st it u t ed 1,2,3,4-Tet r a h yd r ob en zo[h ][1,6]n a p h -
th yr id in es 2a -6a . Corresponding alcoholates were prepared
from alcohol (9 mmol) dissolved in anhydrous toluene or DMF
(25 mL) in the presence of 80% sodium hydride (31.6 mmol).
The suspension was stirred under argon and heated at 80-
100 °C for 45 min. 5-Chloro-1,2,3,4-tetrahydrobenzo[h][1,6]-
naphthyridine (10) (1.32 g, 6 mmol) diluted in toluene or DMF
(10 mL) was added, and the mixture was transferred into a
stainless autoclave under pressure (6 bar, 240 °C) for 1.5 h.
After cooling, the suspension was filtered and evaporated
under reduced pressure. The residue was taken-up with water
and extracted with ether (100 mL). The organic layer was
5-[(P ip er id in -4-yl)m et h oxy]-1,2,3,4-t et r a h yd r ob en zo-
[h ][1,6]n a p h th yr id in e (1a ). A suspension of 4-hydroxym-
ethylpiperidine (1.5 g, 12.8 mmol) and sodium hydride (1,17
g, 39 mmol) in anhydrous toluene (40 mL) was stirred under
argon and heated at 80-100 °C for 45 min. 5-Chloro-1,2,3,4-

144 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 1
Hinschberger et al.
tetrahydrobenzo[h][1,6]naphthyridine (10) (2 g, 9.1 mmol) was
added and the mixture was heated under reflux for 24 h. Then,
the mixture was transferred into a stainless autoclave under
pressure (4 bar, 230 °C) for 1.5 h. After cooling, the suspension
was filtered and the solvent evaporated to give a residual oil
that was crystallized by adding Et2O. This gave 1a as a yellow
powder: 0.72 g, 26% yield. MP: 140 °C (Et2O). 1H NMR
(DMSO-d6, 400 MHz): δ 1.15 (m, 2H, H3′, H6′), 1.67 (m, 2H,
H3′, H6′), 1.84 (m, 3H, H2′, H3′, H6′), 2.43 (m, 2H, H4′, H5′), 2.60
(m, 2H, CH2), 2.92 (d, 2H, H4′, H5′, J 4′,4′ ) J 5′,5′ ) 12.20 Hz),
3.33 (m, 2H, CH2), 4.12 (d, 2H, O-CH2, J 1′,2′ ) 6.10 Hz), 7.02
(s, 1H, NH), 7.20 (t, 1H, H9), 7.43 (t, 1H, H8), 7.50 (d, 1H, H7),
7.88 (d, 1H, H10). IR: 3274 (m, NH), 1620 (s, CdN) cm-1. Anal.
(C18H23N3O) C, H, N.
5-[(N -P r o p a r g y lp i p e r i d i n -4-y l)m e t h o x y ]-1,2,3,4-
tetr a h yd r oben zo[h ][1,6]n a p h th yr id in e (7a ). P r oced u r e
A. A suspension of N-propargyl-4-hydroxymethylpiperidine (1
g, 6.8 mmol) and sodium hydride (0.72 g, 24 mmol) in
anhydrous toluene (20 mL) was stirred under argon and
heated at 80-100 °C for 45 min. 5-Chloro-1,2,3,4-tetrahydro-
benzo[h][1,6]naphthyridine (10) (1 g, 4.5 mmol) was added and
the mixture was heated under reflux for 72 h. Then, the
mixture was transferred in a stainless autoclave under pres-
sure (4 bar, 228 °C) for 1 h 10 min. After cooling, the
suspension was filtered and evaporated to give a residue that
was purified by chromatography on a column of silica gel with
CHCl3/EtOAc/ Et3N (49.3:49.3:1.4). This gave 7a as a yellow
powder: 0.08 g, 5% yield.
9-Ch lor o-5-[(N-b u t ylp ip er id in -4-yl)m et h oxy]-1,2,3,4-
tetr a h yd r oben zo[h ][1, 6]n a p h th yr id in e Mon ofu m a r a te
(5b). Obtained as a yellow powder, 0.15 g, 8% yield. MP: 218
°C (i-PrOH). 1H NMR (DMSO-d6, 400 MHz): δ 0.88 (t, 3H,
CH3, J ) 7.20 Hz), 1.28 (m, 2H, CH2), 1.45 (m, 4H, 2CH2),
1.80 (m, 5H, H2′, H3′, H6′), 2.18 (m, 2H, N-CH2), 2.48 (m, 2H,
H4′, H5′), 2.61 (t, 2H, CH2), 3.05 (d, 2H, H4′, H5′, J 4′,4′ ) J 5′,5′
)
9.60 Hz), 3.33 (m, 2H, CH2), 4.18 (d, 2H, O-CH2, J 1′,2′ ) 5.50
Hz), 6;51 (s, 2H, CHdCH, fumarate), 7.15 (s, 1H, NH), 7.44
(d, 1H, H8), 7.52 (d, 1H, H7), 8.04 (s, 1H, H10). IR: 3279 (m,
NH), 1695 (s, CdO, fumarate), 1594 (s, CdN) cm-1. Anal.
(C26H34ClN3O5) C, H, Cl, N.
6-Su bstitu ted 2,3,4,5-Tetr ah ydr o-1H-azepin o[3,2-c]qu in -
olin es 3c-6c. The compounds c (3c-6c) were prepared from
16 by the same way described above for the derivatives a (2a -
6a ). This gave series c (3c-6c): 3-13% yield.
6-[(N-Eth ylp ip er id in -4-yl)m eth oxy]-2,3,4,5-tetr a h yd r o-
1H-a zep in o[3,2-c]qu in olin e Mon ofu m a r a te (3c). Obtained
as a yellow powder, 0.39 g, 13% yield. MP: 126 °C (i-PrOH).
1H NMR (DMSO-d6, 400 MHz): δ 1.10 (t, 3H, CH3, J ) 7.10
Hz), 1.51 (m, 2H, CH2), 1.83 (m, 5H, H2′, H3′, H6′, CH2), 2.35
(m, 2H, N-CH2), 2.66 (m, 2H, H4′, H5′), 2.88 (m, 2H, CH2), 3.16
(d, 2H, H4′, H5′, J 4′,4′ ) J 5′,5′ ) 11.10 Hz), 3.42 (m, 2H, CH2),
4.19 (d, 2H, O-CH2, J 1′,2′ ) 5.60 Hz), 6;50 (s, 2H, CHdCH,
fumarate), 6.61 (s, 1H, NH), 7.24 (t, 1H, H10), 7.46 (t, 1H, H9),
7.54 (d, 1H, H8), 8.02 (d, 1H, H11). IR: 3417 (m, NH), 1692 (s,
CdO, fumarate), 1591 (s, CdN) cm-1. Anal. (C25H33N3O5) C,
H, N.
P r oced u r e B. 5-[(Piperidin-4-yl)methoxy]-1,2,3,4-tetra-
hydrobenzo[h][1,6]naphthyridine (1a ) (0.40 g, 1.30 mmol) was
dissolved in ethanol (6 mL). Potassium carbonate (0.28 g, 1.90
mmol) and then propargyl bromide (0.17 mL, 1.40 mmol) were
added. The mixture was refluxed for 24 h, then filtered. The
solvent was evaporated to give a residue that was extracted
by Et2O. We obtained 7a as a yellow powder washed with
petroleum ether: 0.33 g, 73% yield. MP: 50 °C (Et2O). 1H NMR
(DMSO-d6, 400 MHz): δ 1.33 (m, 2H, H3′, H6′), 1.69-1.84 (m,
5H, H2′, H3′, H6′, CH2), 2.10 (t, 2H, H4′, H5′), 2.60 (m, 2H, CH2),
2.80 (d, 2H, H4′, H5′), 3.12 (s, 1H, H9′), 3.23 (s, 2H, N-CH2),
3,34 (m, 2H, CH2), 4.15 (d, 2H, O-CH2, J 1′,2′ ) 5.80 Hz), 7.03
(s, 1H, NH), 7.20 (t, 1H, H9), 7.43 (t, 1H H8), 7.50 (d, 1H, H7),
7.88 (d, 1H, H10). IR: 3287 (m, NH), 1618 (s, CdN) cm-1. Anal.
(C21H25N3O) C, H, N.
6-[(N-P r opylpiper idin -4-yl)m eth oxy]-2,3,4,5-tetr ah ydr o-
1H-a zep in o[3,2-c]qu in olin e Mon ofu m a r a te (4c). Obtained
as a beige powder, 0.17 g, 4% yield. MP: 194 °C (i-PrOH). H
1
NMR (DMSO-d6, 400 MHz): δ 0.93 (t, 3H, CH3, J ) 7.40 Hz),
1.58 (m, 4H, 2CH2), 1.89 (m, 5H, H2′, H3′, H6′ CH2), 2.44 (m,
2H, N-CH2), 2.63 (m, 2H, H4′, H5′), 2.95 (m, 2H, CH2), 3.23
(d, 2H, H4′, H5′, J 4′,4′ ) J 5′,5′ ) 9.15 Hz), 3.48 (m, 2H, CH2),
4.26 (d, 2H, O-CH2, J 1′,2′ ) 3.80 Hz), 6;60 (s, 2H, CHdCH,
fumarate), 6.67 (s, 1H, NH), 7.31 (t, 1H, H10), 7.53 (t, 1H, H9),
7.61 (d, 1H, H8), 8.09 (d, 1H, H11). IR: 3394 (m, NH), 1704 (s,
CdO, fumarate), 1593 (s, CdN) cm-1. Anal. (C26H35N3O5) C,
H, N.
6-[(N-Bu tylp ip er id in -4-yl)m eth oxy]-2,3,4,5-tetr a h yd r o-
1H-a zep in o[3,2-c]qu in olin e Mon ofu m a r a te (5c). Obtained
as a beige powder, 0.23 g, 5.5% yield. MP: 202 °C (i-PrOH).
1H NMR (DMSO-d6, 400 MHz): δ 0.88 (t, 3H, CH3, J ) 7.30
Hz), 1.29 (sext, 2H, H9′ CH2, J 9′,8′ ) J 9′,10′ ) 6.80 Hz), 1.51 (m,
4H, 2CH2), 1.83 (m, 5H, H2′, H3′, H6′ CH2), 2.41 (t, 2H, N-CH2),
2.61 (m, 2H, H4′, H5′), 2.88 (m, 2H, CH2), 3.17 (d, 2H, H4′, H5′,
J 4′,4′ ) J 5′,5′ ) 11.30 Hz), 3.42 (m, 2H, CH2), 4.19 (d, 2H, O-CH2,
J 1′,2′ ) 5.70 Hz), 6;53 (s, 2H, CHdCH, fumarate), 6.61 (s, 1H,
NH), 7.25 (t, 1H, H10), 7.46 (t, 1H, H9), 7.54 (d, 1H, H8), 8.02
(d, 1H, H11). IR: 3340 (m, NH), 1696 (s, CdO, fumarate), 1641
(s, CdN) cm-1. Anal. (C27H37N3O5) C, H, N.
5-[(N -Ally lp i p e r i d i n -4-y l)m e t h o x y ]-1,2,3,4-t e t r a -
h yd r oben zo[h ][1,6]n a p h th yr id in e (8a ). This compound
was synthesized with the procedure A used for 7a . 8a was
purified by chromatography on a column of silicagel with
CHCl3/MeOH/ EtOAc (85.7:9.3:5) to give a brown powder: 0.66
1
g, 21% yield. MP < 50 °C. H NMR (DMSO-d6, 400 MHz): δ
1.17 (m, 2H, H3′, H6′), 1.73-1.92 (m, 7H, H2′, H3′, H4′, H5′, H6′,
CH2), 2.61 (t, 2H, CH2), 2.87 (d, 2H, H4′, H5′), 2.93 (d, 2H,
N-CH2, J 7′,8′ ) 5.80 Hz) 3.35 (m, 2H, CH2), 4.16 (d, 2H,
O-CH2, J 1′,2′ ) 5.50 Hz), 5.10 (d, 1H, H9′, J cis ) 10.10 Hz),
5.16 (d, 1H, H9′, J trans ) 17.30 Hz), 5.82 (m, 1H, H8′), 7.04 (s,
1H, NH), 7.21 (t, 1H, H9), 7.44 (t, 1H H8), 7.51 (d, 1H, H7),
7.90 (d, 1H, H10). IR: 3339 (m, NH), 1618 (s, CdN) cm-1. Anal.
(C21H27N3O) C, H, N.
6-[(N-P en tylpiper idin -4-yl)m eth oxy]-2,3,4,5-tetr ah ydr o-
1H-a zep in o[3,2-c]qu in olin e Mon ofu m a r a te (6c). Obtained
1
as a beige powder, 0.06 g, 3% yield. MP: 174 °C (i-PrOH). H
NMR (DMSO-d6, 400 MHz): δ 0.87 (t, 3H, CH3, J ) 6.0 Hz),
1.27 (m, 4H, 2CH2), 1.50 (m, 4H, 2CH2), 1.83 (m, 5H, H2′, H3′,
H6′ CH2), 2.33 (m, 2H, N-CH2), 2.56 (m, 2H, H4′, H5), 2.88 (m,
2H, CH2), 3.14 (d, 2H, H4′, H5′, J 4′,4′ ) J 5′,5′ ) 11.10 Hz), 3.42
(m, 2H, CH2), 4.19 (d, 2H, O-CH2, J 1′,2′ ) 4.50 Hz), 6;53 (s,
2H, CHdCH, fumarate), 6.60 (s, 1H, NH), 7.24 (t, 1H, H10),
7.46 (t, 1H, H9), 7.54 (d, 1H, H8), 8.02 (d, 1H, H11). IR: 3406
(m, NH), 1701 (s, CdO, fumarate), 1639 (s, CdN) cm-1. Anal.
(C28H39N3O5) C, H, N.
6-Su bstitu ted 10-Ch lor o-2,3,4,5-tetr ah ydr o-1H-azepin o-
[3,2-c]qu in olin es 4d , 5d . The compounds d (4d -5d ) were
prepared from 17 by the same way described above for the
derivatives a (2a -6a ). This gave series d (4d -5d ): 5% yield.
10-Ch lor o-6-[(N-P r op ylp ip er id in -4-yl)m eth oxy]-2,3,4,5-
t et r a h yd r o-1H -a zep in o[3,2-c]q u in olin e Mon ofu m a r a t e
(4d ). Obtained as a white powder, 0.08 g, 5% yield. MP: 190
°C (i-PrOH). 1H NMR (DMSO-d6, 400 MHz): δ 0.87 (t, 3H,
CH3, J ) 7.30 Hz), 1.55 (m, 4H, 2CH2), 1.83 (m, 5H, CH2, H2′,
H3′, H6′), 2.40 (t, 2H, N-CH2), 2.59 (m, 2H, H4′, H5′), 2.88 (t,
5-Su bstitu ted 9-Ch lor o-1,2,3,4-tetr a h yd r oben zo[h ][1,6]-
n a p h th yr id in es 4b,5b. The compounds b (4b, 5b) were
prepared from 12 by the same way described above for the
derivatives a (2a -6a ). This gave series b (4b, 5b): 8-12%
yield.
9-Ch lor o-5-[(N-p r op ylp ip er id in -4-yl)m eth oxy]-1,2,3,4-
tetr a h yd r oben zo[h ][ 1,6]n a p h th yr id in e Mon ofu m a r a te
(4b). Obtained as a white powder, 0.23 g, 12% yield. MP: 230
°C (i-PrOH). 1H NMR (DMSO-d6, 400 MHz): δ 0.86 (t, 3H,
CH3, J ) 7.20 Hz), 1.49 (m, 4H, 2CH2), 1.80 (m, 5H, H2′, H3′,
H6′), 2.18 (m, 2H, N-CH2), 2.43 (m, 2H, H4′, H5′), 2.61 (t, 2H,
CH2), 3.05 (d, 2H, H4′, H5′, J 4′,4′ ) J 5′,5′ ) 10.70 Hz), 3.33 (m,
2H, CH2), 4.18 (d, 2H, O-CH2, J 1′,2′ ) 5.50 Hz), 6;51 (s, 2H,
CHdCH, fumarate), 7.14 (s, 1H, NH), 7.44 (d, 1H, H8), 7.52
(d, 1H, H7), 8.03 (s, 1H, H10). IR: 3271 (m, NH), 1695 (s, Cd
O, fumarate), 1594 (s, CdN) cm-1. Anal. (C25H32ClN3O5) C, H,
Cl, N.

New Derivatives as Selective Antagonists of 5-HT4 Receptors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 1 145
2H, CH2), 3.18 (d, 2H, H4′, H5′, J 4′,4′ ) J 5′,5′ ) 11,70 Hz), 3.42
(m, 2H, CH2), 4.19 (d, 2H, O-CH2, J 1′,2′ ) 5.80 Hz), 6;53 (s,
2H, CHdCH, fumarate), 6.68 (s, 1H, NH), 7.46 (d, 1H, H9),
7.54 (d, 1H, H8), 8.17 (s, 1H, H11). IR: 3361 (m, NH), 1702 (s,
CdO, fumarate), 1638 (s, CdN) cm-1. Anal. (C26H34ClN3O5)
C, H, Cl, N.
6;55 (s, 2H, CHdCH, fumarate), 7.62 (t, 1H, H9), 7.81 (m, 2H,
H7, H8), 8.66 (s, 1H, H4), 8.82 (d, 1H, H10), 9.21 (s, 1H, H2). IR:
3539 (m, NH+), 1683 (s, CdO, fumarate), 1603 (s, CdN) cm-1
.
Anal. (C25H28ClN3O5) C, H, Cl, N.
P h a r m a cologica l Meth od s: Mem br a n e P r ep a r a tion
a n d Ra d ioliga n d Bin d in g Assa ys. Briefly, male guinea pigs
(220-224 g, Iffa Credo, France) were subjected to euthanasia
and decapitated. Brains were rapidly removed at 4 °C and
striatal regions carefully dissected and pooled. The tissues
were then suspended in 10 volumes of HEPES buffer (50 mM,
pH 7.4) at 4 °C. After homogenization at 4 °C (Ultra-Turrax,
maximal speed, 15 s) and ultracentrifugation (23000 x g, 60
min, 4 °C), the pellet was resuspended in HEPES buffer (50
mM, pH 7.4) at 4 °C to obtain a tissue concentration of about
15 mg protein/ml. The protein concentration was determined
by the method of Lowry50 using bovine serum albumin as the
standard.
For radioligand binding studies, membrane preparations
were incubated in duplicate (HEPES buffer: 50 mM, pH 7.4)
at 37 °C for 30 min with 0.6 nM [3H]-GR 113808 (Amersham,
France) and fixed concentrations of compounds under study.51
Incubation was terminated by rapid filtration through 0.5%
polyethylenimine-presoaked Whatman GF/B filters using a
Brandel cell harvester. Filters were subsequently washed three
times with 4 mL of HEPES buffer (50 mM, pH 7.4) at 4 °C.
Nonspecific binding of [3H]-GR 113808 was defined in the
presence of 10 µM 5-HT. Results were expressed as the
percentage of inhibition of the [3H]-GR 113808 binding (at 10-6
and 10-8 M of compounds under study, concentrations chosen
to first screen for intermediate and high affinity compounds)
and as inhibition constants (Ki) for drugs that exhibit inhibi-
tion higher than 40% at 10-8M.
Cell Cu ltu r e a n d Tr a n sfection . cDNA, subcloned into
pRK5, was introduced into COS-7 cells by electroporation.52
Briefly, cells were trypsinized, centrifuged, and resuspended
in electroporated buffer (50 mM K2HPO4, 20 mM CH3CO2K,
20 mM KOH, 26.7 mM MgSO4, pH 7.4) with 25-2000 ng of
receptor cDNA. The total amount of DNA was kept constant
at 15 µg/transfection with wild-type pRK5 vector. After 15 min
at room temperature (RT), 300 µl of cell suspension (107 cells)
was transferred to a 0.4-cm electroporation cuvette (Bio-Rad,
Heidemannstrabe, Munchem) and pulsed with a Gene pulser
apparatus (setting 1000 µf, 280 V). Cells were diluted in
Dulbecco’s modified Eagle’s medium (DMEM; 106 cells/ml)
containing 10% dialyzed fetal bovine serum (dFBS) and plated
on 15-cm Falcon Petri dishes or into 12-well clusters at the
desired density.
cAMP F or m a tion . Intracellular cAMP levels were deter-
mined by measuring the conversion of the [3H]adenine nucle-
otide precursor [3H]ATP to [3H]cAMP, as described previously.3
On the sixth day of culture and before each experiment,
neurons were incubated at 37 °C for 2 h with culture medium
containing 2 µCi/ml [3H]adenine (24 Ci/mmol) (Amersham,
UK). After 2 h, the cultures were washed and incubated with
0.75 mM IBMX, 0.1 µM FK, and test agents (agonists or
antagonists prepared in culture medium), in a volume of 1 mL,
for 5 min at 37 °C. The reaction was stopped by aspiration of
the medium and addition of 1 mL of ice-cold 5% trichloracetic
acid. Cells were loosened with the aid of a rubber scraper and
100 µl of 5 mM ATP/5 mM cAMP were added to the mixture.
Cellular protein was centrifuged at 500 x g and the superna-
tant was eluted through sequential chromatography on Dowex
and alumina columns, which separated [3H]ATP from [3H]-
cAMP. We have previously shown that, in neuronal cultures,
0.1 µM FK does not modify basal cAMP concentrations but
increases neurotransmitter efficacy in cAMP production; po-
tency remains unaffected.53
10-Ch lor o-6-[(N-bu tylp ip er id in -4-yl)m eth oxy]-2,3,4,5-
t et r a h yd r o-1H -a zep in o[3,2-c]q u in olin e Mon ofu m a r a t e
(5d ). Obtained as a white powder, 0.07 g, 5% yield. MP: 186
°C (i-PrOH). 1H NMR (DMSO-d6, 400 MHz): δ 0.89 (t, 3H,
CH3, J ) 7.30 Hz), 1.29 (m, 2H, CH2), 1.50 (m, 4H, 2CH2),
1.83 (m, 5H, H2′, H3′, H6′ CH2), 2.34 (t, 2H, N-CH2), 2.57 (m,
2H, H4′, H5′), 2.88 (m, 2H, CH2), 3.14 (d, 2H, H4′, H5′, J 4′,4′
)
J 5′,5′ ) 10.30 Hz), 3.42 (m, 2H, CH2), 4.18 (d, 2H, O-CH2, J 1′,2′
) 5.20 Hz), 6;54 (s, 2H, CHdCH, fumarate), 6.67 (s, 1H, NH),
7.47 (d, 1H, H9), 7.54 (d, 1H, H8), 8.17 (d, 1H, H11). IR: 3346
(m, NH), 1710 (s, CdO, fumarate), 1594 (s, CdN) cm-1. Anal.
(C27H36ClN3O5) C, H, Cl, N.
9-Ch lor o-6-[(N-P r op ylp ip er id in -4-yl)m eth oxy]-2,3,4,5-
t et r a h yd r o-1H -a zep in o[3,2-c]q u in olin e Mon ofu m a r a t e
(4e). This compound was prepared from 18 by the same way
described above for the derivatives a (2a -6a ). This gave 4e
as a yellow powder, 0.2 g, 26% yield. MP: 248 °C (i-PrOH).
1H NMR (DMSO-d6, 400 MHz): δ 0.87 (t, 3H, CH3, J ) 7.30
Hz), 1.55 (m, 4H, 2CH2), 1.82 (m, 5H, H2′, H3′, H6′ CH2), 2.41
(t, 2H, N-CH2), 2.57 (m, 2H, H4′, H5′), 2.87 (t, 2H, CH2), 3.18
(d, 2H, H4′, H5′, J 4′,4′ ) J 5′,5′ ) 10,70 Hz), 3.43 (m, 2H, CH2),
4.19 (d, 2H, O-CH2, J 1′,2′ ) 5.80 Hz), 6;53 (s, 2H, CHdCH,
fumarate), 6.74 (s, 1H, NH), 7.28 (d, 1H, H10), 7.54 (d, 1H, H8),
8.06 (s, 1H, H11). IR: 3328 (m, NH), 1699 (s, CdO, fumarate),
1641 (s, CdN) cm-1. Anal. (C26H34ClN3O5) C, H, Cl, N.
6-[(N-P r opylpiper idin -4-yl)m eth oxy]-7,7a,8,9,10,11,11a,-
12-oct a h yd r od ib en zo[b,h ][1,6]n a p h t h yr id in e Mon ofu -
m a r a te (4f). The synthesis of this compound followed the
same pathway described above for the iminoethers a (2a -6a ).
This gave 4f as a white powder, 0.20 g, 17% yield. MP: 184
°C (i-PrOH). 1H NMR (DMSO-d6, 400 MHz): δ 0.87 (t, 3H,
CH3, J ) 7.20 Hz), 1.38-1.57 (m, 13H, H piperidine and
cyclohexane, CH2), 1.83-2.00 (m, 3H, H piperidine, H7,7a), 2.50
(m, 2H, H4′, H5′), 2.65 (m, 2H, N-CH2), 3.24 (d, 2H, H4′, H5′,
J 4′,4′ ) J 5′,5′ ) 11.10 Hz), 3.51 (m, 1H, H11a), 4.21 (d, 2H,
O-CH2), 6;54 (s, 2H, CHdCH, fumarate), 6.76 (s, 1H, NH),
7.22 (t, 1H, H2), 7.45 (d, 1H, H4), 7.51 (t, 1H, H3), 8.06 (d, 1H,
H1). IR: 3355 (m, NH), 1705 (s, CdO, fumarate), 1641 (s, Cd
N) cm-1. Anal. (C29H39N3O5) C, H, N.
5-Su bstitu ted 3-Ch lor oben zo[h ][1,6]n aph th yr idin es 3g,
4g. Corresponding alcoholates was prepared from alcohol (1.3
mmol) dissolved in anhydrous toluene (5 mL) in the presence
of 80% sodium hydride (4.7 mmol). The suspension was stirred
under argon and heated at 80-100 °C for 45 min. 3,5-
Dichlorobenzo[h][1,6]naphthyridine (23) (0.21 g, 0.8 mmol) was
added, and the mixture was heated under reflux for 4 h. After
cooling, the solvent was evaporated to give a residue that was
extracted with Et2O. After the usual treatments, the obtained
solid was salified by fumaric acid in propan-2-ol. This gave
series g (3g, 4 g): 51-70% yield.
3-Ch lor o-5-[(N-Eth ylp ip er id in -4-yl)m eth oxy]ben zo[h ]-
[1,6]n a p h th yr id in e Mon ofu m a r a te (3g). Obtained as a
white powder, 0.28 g, 70% yield. MP: 196 °C (i-PrOH). 1H
NMR (DMSO-d6, 400 MHz): δ 1.13 (t, 3H, CH3, J ) 7.30 Hz),
1.62 (m, 2H, H piperidine), 1.99 (m, 3H, H piperidine), 2.43
(m, 2H, N-CH2), 2.74 (m, 2H, H4′, H5′), 3.25 (d, 2H, H4′, H5′,
J 4′,4′ ) J 5′,5′ ) 11.60 Hz), 4.46 (d, 2H, O-CH2, J 1′,2′ ) 5.90 Hz),
6;53 (s, 2H, CHdCH, fumarate), 7.61 (t, 1H, H9), 7.79 (t, 1H,
H8), 7.84 (d, 1H, H7), 8.66 (s, 1H, H4), 8.82 (d, 1H, H10), 9.21
(s, 1H, H2). IR: 1687 (s, CdO, fumarate), 1602 (s, CdN) cm-1
.
Anal. (C24H26ClN3O5) C, H, Cl, N.
Beh a vior a l Stu d ies. An im a ls a n d Dr u g Ad m in istr a -
tion . In all studies, male OF1 mice (20-24 g, Iffa Credo,
France) were used. All compounds tested were dissolved in
saline solution and administered intraperitoneally (10 mL /kg).
CNS-Activity a n d Acu te Toxicity Test. Behavioral and
neurological changes induced by graded doses (12.5, 25, 50 mg/
kg) of the tested derivatives were evaluated in mice, in groups
3-Ch lor o-5-[(N-P r op ylp ip er id in -4-yl)m et h oxy]b en zo-
[h ][1,6]n a p h th yr id in e Mon ofu m a r a te (4g). Obtained as a
white powder, 1 g, 51% yield. MP: 190 °C (i-PrOH). H NMR
(DMSO-d6, 400 MHz): δ 0.88 (t, 3H, CH3, J ) 5.80 Hz), 1.59
(m, 4H, CH2, H piperidine), 1.98 (m, 3H, H piperidine), 2.50
(m, 2H, N-CH2), 2.62 (m, 2H, H4′, H5′), 3.22 (d, 2H, H4′, H5′,
J 4′,4′ ) J 5′,5′ ) 9.80 Hz), 4.46 (d, 2H, O-CH2, J 1′,2′ ) 4.20 Hz),
1

146 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 1
Hinschberger et al.
of four, by a standardized observation technique43 at different
times (30 min, 3 and 24 h) after intraperitoneal administration.
Major changes of behavioral data (for example, hypo- or
hyperactivity, ataxia, tremors, convulsion, etc.) were noted in
comparison to the control group. The approximate LD50 of the
compounds were also calculated through the quantification of
deaths after 24 h.
tors. Trends Pharmacol. Sci. 1995, 16, 391-398. (c) Eglen, R.
M.; Hegde, S. S. 5-Hydroxytryptamine (5-HT)4 receptors: physi-
ology, pharmacology and therapeutic potential. Exp. Opin.
Invest. Drugs 1996, 5 (4), 373-388.
(3) Dumuis, A.; Bouhelal, R.; Sebben, M.; Cory, R.; Bockaert, J . A.
Non classical 5-hydroxytryptamine receptor positively coupled
with adenylate cyclase in the central nervous system. Mol.
Pharmacol. 1988, 34, 880-887.
(4) (a) Gerald, C.; Adham, N.; Kao, H.-T.; Olsen, M. A.; Laz, T. M.;
Schechter, L. E.; Bard, J . A.; Vaysse, P. J . J .; Hartig, P. R.;
Branchek, T. A.; Weinshank, R. L. The 5-HT4 receptor: molec-
ular cloning and pharmacological characterization of two splice
variants. EMBO J . 1995, 14, 2806-2815. (b) Ullmer, C.; Sch-
muck, K.; Kalkman, H. O.; Lubbert, H. Expression of serotonin
receptor mRNAs in blood vessels. FEBS Lett. 1995, 370, 215-
221.
(5) Schaus, J . M., Thompson, D. C.; Bloomquist W. E.; Susemichel,
A. D.; Calligaro, D. O.; Cohen, M. L. Synthesis and structure-
activity relationships of potent and orally active 5-HT4 receptor
antagonists: indazole and benzimidazolone derivatives. J . Med.
Chem. 1998, 41, 1943-1955.
(6) Curtet, S.; Soulier J . L.; Zahradnick, I.; Giner, M.; Berque-Bestel,
I.; Mialet, J .; Lezoualc’h, F.; Donzeau-Gouge, P.; Sicsic, S.;
Fischmeister, R.; Langlois, M. New arylpiperazine derivatives
as antagonists of the human cloned 5-HT4 receptor isoforms. J .
Med. Chem. 2000, 43, 3761-3769.
(7) Lopez-Rodriguez, M. L.; Benhamu, B.; Viso, A.; Morcillo, M. J .;
Murcia, M.; Orensanz, L.; Alfaro, M. J .; Martin, M. I. Benzimi-
dazole derivatives. Part 1: Synthesis and structure-activity
relationships of new benzimidazole-4-carboxamides and car-
boxylates as potent and selective 5-HT4 receptor antagonists.
Bioorg. Med. Chem. 1999, 7, 2271-2281.
(8) Hiroshi, H.; Hiroshi, Y.; Yoshihito, T.; Hirotaka, T.; Yukiko, M.;
Naoyuki, Y.; Shiro, K. Novel N-[1-(1-substituted 4-piperidinyl-
methyl)-4-piperidinyl]benzamides as potent colonic prokinetic
agents. Bioorg. Med. Chem. Lett. 2002, 12 (6), 967-970.
(9) Katsuhiko, I.; Hideo, T.; Hidetoshi, H.; Shuji, S.; Kiyoshi, A.;
Masatake, F.; Noriko, S.; Keiichiro, H.; Takeshi, K. Synthesis
and pharmacological properties of novel benzamide derivatives
acting as ligands to the 5-hydroxytryptamine 4 (5-HT4) receptor.
Eur. J . Med. Chem. 1999, 34 (12), 1101-1108.
(10) Marchetti-Gauthier, E.; Roman, F. S.; Dumuis, A.; Bockaert, J .;
Soumireu-Mourat, B. BIMU1 increase associative memory in
rats by activating 5-HT4 receptors. Neuropharmacology 1997,
36, 697-706.
(11) Siniscalchi, A.; Badini, I.; Beani, L.; Bianchi, C. 5-HT4 receptor
modulation of acetylcholine outflow in guinea pig brain slices.
NeuroReport 1999, 10 (3), 547-51.
(12) Kaumann, A. J . Do human atrial 5-HT4 receptors mediate
arrhytmias? Trends Pharmacol. Sci. 1994, 15, 451-455.
(13) Ohuoha, D. C.; Schindler, C. W.; Rothman, R. B.; Serotonin-4
receptor antagonists reverse cocaine-induced cardiac arrhyth-
mia. Life Sci. 1998, 63, 19, 1673-1678.
(14) Hedge, S.; Eglen, R. Peripheral 5-HT4 receptors. FASEB J . 1996,
10, 1398-1407.
(15) Scott, L. J .; Perry, C. M. Tegaserod. Drugs 1999, 58 (3), 491-
496; discussion 497-498.
(16) Espejo, E. F.; Gil, E. Antagonism of peripherical 5-HT4 receptors
reduces visceral and cutaneous pain in mice, and induces visceral
analgesia after simultaneous inactivation of 5-HT3 receptors.
Brain Res. 1998, 788, 20-24.
(17) Ghelardini, C.; Galeotti, N.; Casamenti, F.; Malmmberg-Aiello,
P.; Pepeu, G.; Gualtieri, F.; Bartolini, A. Central cholinergic
antinociception induced by 5-HT4 agonists: BIMU1 and BIMU8.
Life Sci. 1996, 58, 2297-2309.
(18) Lopez-Rodriguez, M. L.; Morcillo, M. J .; Benhamu, B.; Rosado
M. L. Comparative receptor mapping of serotoninergic 5-HT3 and
5-HT4 binding sites. J . Comput.-Aided Mol. Des. 1997, 11, 589-
599.
(19) Gaster, L. M.; King, F. D. Serotonin 5-HT3 and 5-HT4 receptor
antagonists. Med. Res. Rev. 1997, 17, 163-214.
Sp a tia l Wor k in g Mem or y. Promnesiant activity of tested
compounds was evaluated by reversal of scopolamine-induced
deficit on spontaneous alternation behavior in the Y maze
test.47 The black wooden maze consisted of three equally
spaced arms (22-cm long, 6.5-cm wide with walls 10-cm high).
The mouse was placed at the end of one of the arms and
allowed to move freely through the maze during a 5 min
session while the sequence of arm entries was recorded by an
observer. An arm entry was scored when all four feet crossed
into the arm. An alternation was defined as entries into all
three arms on a consecutive occasion. The number of possible
alternation is thus the total number of arm entries minus two;
the percentage of alternation was calculated as (actual alter-
nation/possible alternation) × 100. The percentage of alterna-
tion of scopolamine-treated mice (1 mg/kg) was significantly
reduced in comparison with control mice (52% vs 66%,
respectively, p ) 0.0049 (PLSD of Fisher)). Compound 4a was
tested at 0.25, 0.5, and 1 mg/kg. The administration was
realized 30 min before testing. For each dose, four groups were
constituted: control (saline + saline), scopolamine (saline +
scopolamine), tested compound (compound + saline), and
association (compound + scopolamine). In this condition,
arecoline (1 mg/kg), used as pharmacological reference, sig-
nificantly reversed the scopolamine-induced deficit (48% for
scopolamine group vs 64% for arecoline + scopolamine group,
p < 0.0001 (PLSD of Fisher).
Wr ith in g Test. The test employed was essentially that
described by Hendershot and Forsaith;54 however, acetic acid48
rather than phenylquinone was used to elicit stretching.
Groups of eight mice (20-24 g) were injected ip with 10 mL/
kg of 0.6% aqueous acetic acid. The mice were placed in an
observation beaker, and the number of stretches per animal
was counted during a 10-min period starting 10 min after
acetic acid treatment. A stretch was defined as a sequence of
arching of the back, pelvic rotation, and hind limb extension.
Tested and reference compounds were administered 15 min
before acetic acid solution.
Hot P la te Test. The method employed for measuring
central analgesic effect was first described by Woolfe and
McDonald.49 Briefly, every mouse was individually placed on
a plate heated to 55 °C and the time until forepaws licking
occurs was recorded by a stop-watch. We measured the
reaction times of groups of 10 mice twice before injections (mice
must react between 4 and 12 s). The compounds were tested
at 0.01, 0.1, and 1 mg/kg ip and reaction times were deter-
mined at 15 and 30 min after injection. If an animal did not
respond by 30 s (cutoff time), it was removed from the plate
to avoid tissue damage. Morphine used as reference at 8 mg/
kg induced abolition of avoidance behavior (mean reaction
times, 30 s and 26 s at 15 and 30 min, respectively; p < 0.0001
vs control at the two times, PLSD of Fisher).
Sta tistica l An a lyses. All quantitative data were expressed
as mean (SEM and were analyzed using analysis of variance
(ANOVA) followed, in case of significant effects, post-hoc
multiple comparison tests (PLSD of Fisher). p-Values less than
0.05 were considered to be significant.
(20) Bureau, R.; Daveu, C.; Lemaˆıtre, S.; Dauphin, F.; Landelle, H.;
Lancelot, J . C.; Rault, S. Molecular Design based on 3D-
Pharmacophore. Application to 5-HT4 receptors. J . Chem. Inf.
Comput. Sci. 2002, 42, 962-967.
(21) Rault, S.; Lancelot, J . C.; Prunier, H.; Robba, M.; Renard, P.;
Delagrange, P.; Pfeiffer, B.; Caignard, D. H.; Guardiola-Lemaˆı-
tre, B.; Hamon, M. Novel selective and Partial Agonists of 5-HT3
Receptors. Part 1. Synthesis and Biological Evaluation of Pip-
erazinopyrrolothienopyrazines. J . Med. Chem. 1996, 39, 2068-
2080.
(22) Prunier, H.; Rault, S.; Lancelot, J . C.; Robba, M.; Renard, P.;
Delagrange, P.; Pfeiffer, B.; Caignard, D. H.; Misslin, R.;
Guardiola-Lemaˆıtre, B.; Hamon, M. Novel selective and Partial
Agonists of 5-HT3 Receptors. Part 2. Synthesis and Biological
Evaluation of Piperazinopyridopyrrolopyrazines, Piperazinopy-
rroloquinoxalines, and piperazinopyridopyrroloquinoxalines. J .
Med. Chem. 1997, 40, 1808-1819.
Refer en ces
(1) Hoyer, D.; Clarke, D. E.; Fozard, J . R.; Hartig, P. R.; Martin, G.
R.; Mylecharane, E. J .; Saxena, P. R.; Humphrey, P. P. A.
International Union of Pharmacology Classification of Receptors
for 5-Hydroxytryptamine (Serotonin). Pharmacol. Rev. 1994, 46
(2), 157-203.
(2) For reviews, see: (a) Ford, A. P. D. W.; Clarke, D. E. The 5-HT4
Receptor. Med. Res. Rev. 1993, 13, 633-662. (b) Eglen, R. M.;
Wong, E. H. F.; Dumuis, A.; Bockaert, J . Central 5-HT4 recep-

New Derivatives as Selective Antagonists of 5-HT4 Receptors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 1 147
(23) Hinschberger, A.; Gillard, A. C.; Bureau, I.; Rault, S. Reinves-
tigation of rearrangement of benzodiazepinediones into quinoleine
under microwave heating or conventional heating conditions.
Tetrahedron 2000, 56, 1361-1367.
(24) Wright, W. B., J r.; Brabander, H. J .; Greenblatt, E. N.; Day, I.
P.; Hardy, R. A., J r. Derivatives of 1,2,3,11a-tetrahydro-5H-
pyrrolo[2,1-c][1,4]benzodiazepine-5,11(10H)-dione as anxiolytic
agents. J . Med. Chem. 1978, 21, 1087-1089.
(25) Reed, J . N.; Snieckus, V. Sequential directed ortho metalation
reactions. A synthesis of anthramycin. Tetrahedron Lett. 1984,
25, 5505-5508.
(26) Schultz, A. G.; McCloskey, P. J .; Sundararaman, P.; Springer,
J . P. Enantioselection via Birch reduction-alkylation of a chiral
anthranilic acid derivative. Synthesis of enantiomerically pure
aminocyclohexanes. Tetrahedron Lett. 1985, 26, 1619-1622.
(27) J ones, G. B.; Daney, C. L.; J enkins, T. C.; Kamal, A.; Kneale, G.
G.; Neidle, S.; Webster, G. D.; Thurston, D. E. The non-covalent
interaction of pyrrolo[2,1-c][1,4]benzodiazepine-5,11-diones with
DNA. Anti-Cancer Drug Des. 1990, 5, 249-264.
(28) Crider, A. M.; Lamey, R.; Floss, H. G.; Cassady, J . M.; Bradner,
W. J . Synthesis of nitrosourea derivatives of pyridine and
piperidine as potential anticancer agents. J . Med. Chem. 1980,
23, 848-851.
(29) Gaster, L. M.; J ennings, A. J .; J oiner, G. F.; King, F. D.;
Mulholland, K. R.; Rahman, S. K.; Starr, S.; Wyman, P. A.;
Wardle, K. A.; Ellis, E. S.; Sanger, G. J . (1-Butyl-4-piperidinyl)-
methyl 8-amino-7-chloro-1,4-benzodioxane-5-carboxylate hydro-
chloride: a highly potent and selective 5-HT4 receptor antagonist
derived from metoclopramide. J . Med. Chem. 1993, 36, 4121-
4123.
(30) Baronnet, R.; Callendret, R.; Blanchard, L.; Foussard-blanpin,
O.; Bretaudeau, J . Synthe`se et pharmacodynamie de dialky-
lamino-3-1H, 3H quinazolinediones-2,4 et de de´rive´s. Eur. J .
Med. Chem. Chim. Ther. 1983, 18 (3), 241-247.
(31) Mori, M.; Kimura, M.; Uozumi, Y.; Born, Y. A one step synthesis
of 1,4-benzodiazepines: synthetic studies on neothramycin.
Tetrahedron Lett. 1985, 26, 5947.
(32) Bureau, I.; Chardon, J .; Leclaire, A.; Hinschberger, A.; Rault,
S. Structure of 6-chloro-5H-tetrahydroazepino[3,2-c]quinoleine.
Acta Crystallogr. 1999, C55, part 4, IUC 9900022.
(33) Wright, W. B. Pyrrolobenzodiazepines. U.S. Patent N° 3,947,408,
1976.
(34) Kamal, A.; Thurston, D. E. Reaction of pyrrolo[2,1-c][1,4]-
benzodiazepine-2,5,11-triones with phosphorus oxychloride. Tet-
rahedron Lett. 1989, 30, 6221-6222.
(35) Thurston, D. E.; Bose, D. S. Synthesis of DNA-interactive
pyrrolo[2,1-c][1,4]benzodiazepines. Chem. Rev. 1994, 94, 433-
467.
(39) Rault, S.; Hinschberger, A.; Dauphin, F.; Boulouard, M.; Dumuis,
A. Benzo[h][1,6]naphthyridine and azepino[3,2c]quinoline imino-
ethers, preparation method, and therapeutic use thereof as
5-HT4 receptor antagonists. PCT Int. Appl. WO 0179205 A1
20011025, 2001.
(40) Yang, D.; Soulier, J . L.; Sicsic, S.; Mathe´-Allainmat, M.; Bre´mont,
B.; Croci, T.; Cardamone, R.; Aureggi, G.; Langlois, M. Arylcar-
bamate derivatives of 1-piperidineethanol as potent ligands for
5-HT4 receptors. J . Med. Chem. 1997, 40, 608-621.
(41) Blondel, O.; Gastineau, M.; Langlois, M.; Fischmeister, R. The
5-HT4 receptor antagonist ML10375 inhibits the constitutive
activity of human 5-HT4(c) receptor. Br. J . Pharmacol. 1998, 125,
595-597.
(42) Blondel, O.; Vandecasteele, G.; Gastineau, M.; Leclerc, S.;
Dahmoune, Y.; Langlois, M.; Fischmeister, R. Molecular and
functional characterization of a 5-HT4 receptor cloned from
human atrium. FEBS Lett. 1997, 412, 465-474.
(43) Morpugo, C. A new design for the screening of CNS-active drugs
in mice. Arzneim.-Forsch. (Drug Res.) 1971, 11, 1727-1734.
(44) Galeotti, N.; Ghelardini, C.; Bartolini, A. Role of 5-HT4 receptors
in mouse passive avoidance test. J . Pharmacol. Exp. Ther. 1998,
286, 1115-1121.
(45) Marchetti, E.; Dumuis, A.; Bockaert, J .; Soumireu-Mourat, B.;
Roman, F. S. Differential modulation of the 5-HT4 receptor
agonists and antagonists on rats learning and memory in rats.
Neuropharmacology 2000, 39, 2017-2027.
(46) Lelong, V.; Dauphin, F.; Boulouard, M. RS 67333 and D-
Cycloserine accelerate learning acquisition in the rat. Neurop-
harmacology 2001, 41, 517-522.
(47) Hooper, N.; Fraser, C.; Stone, T. Effect of purines analogues on
spontaneous alternation in mice. Psychopharmacology 1996, 123,
250-257.
(48) Koster, R.; Anderson, M.; De Beer, E. J . Acetic acid for analgesic
screening. Fed. Proc. 1959, 18, 412.
(49) Woolfe, G.; McDonald, A. D. The evaluation of the analgesic
action of petidine hydrochloride (demerol). J . Pharmacol. Exp.
Ther. 1944, 80, 300-307.
(50) Lowry, O. H.; Rosebrought, N. J .; Farr, A. L.; Randall, R. J .
Protein measurement with the folin reagent. J . Biol. Chem.
1951, 193, 265-275.
(51) Grossman, C. J .; Kilpatrick, G. J .; Bunce, K. T. Development of
a radioligand binding assay for 5-HT4 receptors in guinea-pig
and rat brain. Br. J . Pharmacol. 1993, 109, 618-624.
(52) Claeysen, S.; Sebben, M.; J ournot, L.; Bockaert, J .; Dumuis, A.
Cloning, expression and pharmacology of the mouse 5-HT4L
receptor. FEBS Lett. 1996, 398, 19-25.
(53) Weiss, S.; Sebben, M.; Garcia-Sainz, J . A.; Bockaert, J . D2-
dopamine receptor-mediated inhibition of cyclic AMP formation
in striatal neurons in primary culture. Mol. Pharmacol. 1985,
27, 595-599.
(36) Thurston, D. E.; Bose, D. S.; Howard, P. W.; J enkins, T. C.; Leoni,
A.; Baraldi, P. G.; Guiotto, A.; Cacciari, B.; Kelland, L. R.;
Foloppe, M. P.; Rault, S. Effect of A-ring modifications on the
DNA-binding behavior and cytotoxicity of pyrrolo[2,1-c][1,4]-
benzodiazepines. J . Med. Chem. 1999, 42, 1951-1964.
(37) Bowden, K.; Heilbron, I. M.; J ones, E. R. H.; Weedon, B. C. L.
Researches on acetylenic compounds. Part I. The preparation
of acetylenic ketones by oxidation of acetylenic carbinols and
glycols. J . Chem. Soc. 1946, 39-45.
(54) Hendershot, L. C.; Forsaith, S. Antagonism of the frequency of
phenylquinone-induced writhing in the mouse by weak analge-
sics and non analgesics. J . Pharmacol. Exp. Ther. 1959, 125,
237-240.
(38) Rault, S.; Prunier, H. In some cases, sulfuric acid can be
fruitfully replaced by phosphoric acid in the J ones oxidation
procedure. Communication personnelle.
J M020954V