New arylpiperazine derivatives as antagonists of the human cloned 5-HT4 receptor isoforms

Article abstract of DOI:10.1021/jm0009538

New derivatives of arylpiperazine 9 were designed from ML 10302, a potent 5-HT₄ receptor agonist in the gastrointestinal system. Compounds were synthesized by condensation of a number of available arylpiperazines or heteroarylpiperazines with 2

Full text of DOI:10.1021/jm0009538

J . Med. Chem. 2000, 43, 3761-3769
3761
New Ar ylp ip er a zin e Der iva tives a s An ta gon ists of th e Hu m a n Clon ed 5-HT4
Recep tor Isofor m s
Sophie Curtet,§ J ean-Louis Soulier,§ Ivan Zahradnik,† Mireille Giner,§ Isabelle Berque-Bestel,§ J eanne Mialet,‡
Frank Lezoualc’h,‡ Patrick Donzeau-Gouge,# Sames Sicsic,§ Rodolphe Fischmeister,‡ and Michel Langlois*,§
CNRS-BIOCIS (UPRES A 8076) and Laboratoire de Cardiologie Cellulaire et Mole´culaire, INSERM U-446,
Institut de Signalisation et Innovation The´rapeutique (IFR-ISIT), Faculte´ de Pharmacie, Universite´ de Paris-Sud,
92296 Chaˆtenay-Malabry, France
Received J une 19, 2000
New derivatives of arylpiperazine 9 were designed from ML 10302, a potent 5-HT4 receptor
agonist in the gastrointestinal system. Compounds were synthesized by condensation of a
number of available arylpiperazines or heteroarylpiperazines with 2-bromoethyl 4-amino-5-
chloro-2-methoxybenzoate. They were evaluated in binding assays on the recently cloned human
5-HT4(e) isoform stably expressed in C6 glial cells with [3H]GR 113808 as the radioligand. The
affinity values (Ki) depended upon the substituent on the aromatic ring. A chlorine atom
produced a marked drop in activity (Ki > 100 nM), while a m-methoxy group gave a compound
with nanomolar affinity (Ki ) 3 nM). The most potent compounds were the heterocyclic
derivatives with pyrimidine, pyrazine, pyridazine, or pyridine moieties (compounds 9r , 9t, 9u ,
9x, respectively). Ki values for 9a and 9r were determined for the 5-HT4(a), 5-HT4(b), 5-HT4(c)
,
and 5-HT4(d) receptor isoforms transiently expressed in COS cells. The results indicated that
the compounds were not selective. They produced an inhibition of the 5-HT-stimulated cyclic
AMP synthesis in the C6 glial cells stably expressing the 5-HT4(e) receptor and shifted the
5-HT concentration-effect curve on adenylyl cyclase activity with pKD values of 7.44 and 8.47,
respectively. In isolated human atrial myocytes, 9r antagonized the stimulatory effect of 5-HT
on the L-type calcium current (ICa) with a KD value of 0.7 nM.
In tr od u ction
The 5-HT4 receptor is a member of the seven trans-
membrane-spanning protein-coupled family of
and have been implicated in a number of pathological
disorders: memory deficits,12a irritable bowel syndrome,
gastroparesis, urinary incontinence,11 and cardiac atrial
arrhythmias.12b Recently, several groups reported the
cloning of the receptors from different species.1-3 They
are characterized by several splice variants which differ
in the length and sequence of their C termini. We
described2 the cloning and pharmacological character-
ization of four splice variants of the human 5-HT4
receptor: 5-HT4(a), 5-HT4(b), 5-HT4(c), and 5-HT4(d). More
recently another isoform (5-HT4(e)) has been character-
ized.13 Other 5-HT receptor subtypes also have splice
variants, in particular the 5-HT7 receptor which has four
isoforms14 produced by alternative splicing in the car-
boxyl terminus. The expression of 5-HT4 receptor iso-
forms depends on the tissue: three or four variants were
expressed in heart atrium, brain, and intestine, while
only one was found in kidney and bladder.2 On the other
hand, the 5-HT4(d) variant was only present in the
intestine.2
G
receptors.1-3 Initially, it was characterized by Dumuis4
in neuronal cells and shown to be linked positively to
adenylyl cyclase. This neuronal receptor was similar to
that discovered by Clarke5 in the gastrointestinal (GI)
system where it was shown to be responsible for
stimulating motility. Considerable interest has devel-
oped in this receptor because it provided a mechanism
of action for the gastric prokinetic drugs, a number of
which were found to be 5-HT4 receptor agonists.6 Many
of these compounds are members of the generic benza-
mide family, derived from metoclopramide.7 They are
amides or esters of 4-amino-5-chloro-2-methoxybenzoic
acid derivatives such as renzapride (2),8a SC-53116 (3),8b
ML 103028c (1), cisapride (4),8c prucalopride (5),8d and
mosapride (6) (Chart 1).8e The pharmacological charac-
terization of this receptor was facilitated by the syn-
thesis of potent and selective antagonists such as GR
113808 (7)9a and SB 207710 (8).9b
The availability of the cloned 5-HT4 receptors stimu-
lated more interest in the search for new ligands. In
particular the potential use of 5-HT4 receptor antago-
nists in the treatment of the human atrial arrhythmias
has become an emerging concept12b,15,16 since the 5-HT4
receptor stimulates two potentially arrhythmogenic ion
5-HT4 receptors are expressed in a wide variety of
tissues: brain, heart, bladder, intestine, and kidney10,11
* To whom correspondence should be addressed. Tel: 33 1 46 83 57
36. Fax: 33 1 46 83 57 40. E-mail: michel.langlois@cep.u-psud.fr.
§ CNRS-BIOCIS (UPRES A 8076).
channel currents in human atrial tissue: the L-type
‡ Laboratoire de Cardiologie Cellulaire et Mole´culaire, INSERM
U-446.
17
calcium current (ICa
)
and the pacemaker f channel
current (If).15 A very recent study has demonstrated the
potential therapeutic value of a selective 5-HT4 receptor
antagonist (RS-100302) as an antiarrhythmic drug.16b
Therefore, there is much interest in the clinical evalu-
† On leave from: Institute of Molecular Physiology and Genetics,
Slovak Academy of Sciences, Bratislava, Slovakia.
#
Permanent address: Institut Hospitalier J acques Cartier, Service
de Chirurgie Cardiaque, Avenue du Noyer Lambert, F-91349 Massy
Cedex, France.
10.1021/jm0009538 CCC: $19.00 © 2000 American Chemical Society
Published on Web 09/19/2000

3762 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 20
Curtet et al.
Ch a r t 1
Sch em e 1a
a
Reagents and conditions: (a) piperazine derivative, K2CO3, MeCN or DMF, 18-24 h, rt (method A); DIPEA, MeCN, reflux, 24 h
(method B); (b) R ) t-BOC, anisole, TFA, CH2Cl2, rt.
ation of compounds with cardiac 5-HT4 receptor antago-
nist properties.18 The design of molecules to be evalu-
ated on the available human cloned 5-HT4 receptors as
well as on native 5-HT4 receptors expressed in human
atrial myocytes constituted an original approach to
identify new leads capable of treating atrial arrhyth-
mias.
We report herein the preliminary results of this
approach where the piperazine derivatives 9 (Scheme
1) were synthesized and evaluated on the cloned and
native human 5-HT4 receptors.
synthesized by the process already reported20 and were
then condensed with various arylpiperazines which
were commercially available or were synthesized by
methods already described.21a-c Initially, the reaction
was carried out in DMF or acetonitrile in the presence
of K2CO3 at room temperature, and the compounds were
isolated after column chromatography as hydrochloride
salts (method A). Generally the yield of the pure
compound was low. A clear improvement of the yield of
the reaction was obtained with acetonitrile as solvent
at refluxing in the presence of diisopropylethylamine
(method B).
The biological activity of compound 9a (Table 1) was
compared to that of the corresponding amide 2-[N-(4-
phenyl)piperazino]ethyl 4-amino-5-chloro-2-methoxy ben-
zamide (11), which was synthesized by the condensation
of 2-amino-5-chloro-2-methoxybenzoic acid and N-phen-
ylpiperazineethylamine (see Experimental Section).
Ch em istr y
ML 10302 (1) has been characterized as a potent
5-HT4 receptor agonist in the GI system8c where it has
been shown to be more potent than cisapride while its
efficacy as an agonist on ICa in isolated human atrial
myocites was very weak.19 Moreover it antagonized the
stimulatory effect of 100 nM 5-HT on ICa with an IC50
value of 17.1 nM.19 Therefore, 1 seemed to be an
excellent lead for the search for new molecules with the
ability to block cardiac 5-HT4 receptors. As SARs
(structure-activity relationships) reported by us20 have
shown that various substituents could be introduced on
the 4 position of the piperidine ring of 1, the synthesis
of piperazine derivatives 9 in particular could constitute
a promising method for the design of new molecules.
They were prepared according to the synthetic pathway
reported in Scheme 1.
Biologica l Resu lts a n d Discu ssion
The compounds were evaluated routinely by binding
assays on the newly described human 5-HT4(e) isoform
stably expressed in C6 glial cells, using [3H]GR 113808
as the radioligand.2 These receptors have been charac-
terized in the human atrium,13 and consequently they
were relevant to select active compounds with thera-
peutic potential for the treatment of human atrial
arrhythmias. As shown in Table 1, substitution of the
piperidine ring of ML 10302 by an arylpiperazine moiety
gave compounds with good affinities for these receptors.
However, the potency was largely influenced by the
2-Bromoethyl and 3-bromopropyl 4-amino-5-chloro-
2-methoxybenzoate (10a and 10b, respectively) were

New Arylpiperazine Derivatives
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 20 3763
Ta ble 1. Binding Data for Compounds 9a -y on the Cloned
Human 5-HT4(e) Receptor Isoform
Ta ble 2. Binding Data for Compounds 9a and 9r on the
Cloned Human 5-HT4(a), 5-HT4(b), 5-HT4(c), and 5-HT4(d)
Receptors Expressed in COS-7 Cells
Ki (nM)a
compound
h5-HT4(a)
23
h5-HT4(b)
55
h5-HT4(c)
h5-HT4(d)
32
9a
12.5
[17-31 ]
3.5
[44-68]
8.1
[6.7-21]
2.47
[17-57]
7.03
9r
[2.8-4.5]
ML 10302 8.4
[6.55-9.9]
10.7
[1.8-3.3]
7.98
[3.2-15.4]
3.69
[6.9-9.9]
GR 113808 0.33
[7.8-13.6]
0.53
[5.2-10.7] [2.44-4.94]
0.41 0.078
[0.29-0.36] [0.43-0.63] [28-0.54] [0.054-0.1]
a
Membranes from transiently transfected COS-7 cells were
incubated with 50% of the KD value of [3H]GR 113808 for each
h5-HT4 receptor isoform (see ref 2). Nonspecific binding was
defined with 10 µM ML 10375 (see Experimental Section). Data
are the result of two or three experiments. Ki values are expressed
in nM and were calculated using the Cheng-Prussof equation from
the IC50 values with the PRISM program; 95% confidence limits
are in brackets.
receptors. Indeed, the corresponding amide 11 was
inactive (Ki > 1000 nM). On the other hand, substitution
of the piperazine by heteroaryl rings provided very
potent compounds; thus pyrimidine, pyrazine, py-
ridazine, and pyridine derivatives 9r , 9t, 9u , 9x, and
9y were compounds with nanomolar affinity.
An increase in the basicity of the second nitrogen
atom was not a favorable parameter as the N-Me and
NH derivatives 9n and 9o were inactive, while the
corresponding ethyl carbamate 9l had high affinity (Ki
) 23 nM). This point was confirmed with the N-benzyl
derivative 9q which was 1 order of magnitude less
potent than 9a .
Ki values for compounds 9a and 9r for the 5-HT4(a)
,
5-HT4(b), 5-HT4(c), and 5-HT4(d) receptor isoforms were
evaluated in binding assays on the different splice
variants of the human receptors transiently expressed
in COS-7 cells.2 The results are reported in Table 2. The
affinity values for the receptors were in the same range
as those obtained with the stable line where the 5-HT4(e)
isoform was expressed. Similar to the reference com-
pounds, 9a and 9r did not show any selectivity for the
different isoforms.2,13
a
Membranes from stably transfected C6 glial cells were incu-
bated with 0.2 nM [3H]GR 113808. Nonspecific binding was defined
with 10 µM ML 10375 (see Experimental Section). The data are
the result of two or three experiments. Ki values are expressed in
nM and were calculated using the Cheng-Prussof equation from
the IC50 values with the PRISM program; 95% confidence limits
are in brackets.
Previous results20 have shown that the pharmacologi-
cal profile of the molecule derived from ML 10302
depended upon the structure of the basic moiety;
therefore the pharmacological activities of compounds
9a and 9r were determined on the activity of adenylyl
cyclase, the effector of the 5-HT4 receptors. The produc-
tion of cAMP was measured in C6 glial cells stably
expressing the human 5-HT4(e) receptor, using a radio-
immunoassay technique as previously reported.2,19 In
contrast to 5-HT (10-6 M) which stimulated the syn-
thesis of cAMP, 9a and 9r did not possess intrinsic
activity at these receptors. On the contrary, both
compounds produced an antagonist effect on the 5-HT-
stimulated cAMP synthesis (Figure 1). 9r (1 µM) clearly
inhibited the effect of 5-HT (1 and 0.1 µM), while the
effect of 9a was less marked and only observed with 0.1
µM 5-HT. The competitive antagonist properties of both
compounds were demonstrated on the 5-HT concentra-
tion-effect curve which was shifted to the right by the
addition of 9a and 9r (70 and 5 nM, respectively). pKD
values of 7.54 and 8.47 were found respectively by
calculation of the Schild plots (Figure 2).
substituent on the aromatic ring and the position of the
substitution. A p-methoxy group and o- and p-fluorine
atom gave compounds 9e, 9i, and 9j equipotent to the
unsubstituted compound 9a (Ki ) 14.3 nM). A chlorine
atom on different positions (9b, 9c, 9d ), a m-trifluorom-
ethyl substituent (9h ), and an o-methoxy group (9g)
brought about a drop in activity which was particularly
marked with the chlorine atom while the m-methoxy
group provided a compound with nanomolar affinity (9f,
Ki ) 3 nM, respectively). The naphthalene derivative
9k was inactive, indicating the unfavorable influence
of steric hindrance in this part of the receptor site. The
affinity was not influenced by the lengthening of the
ethyl chain (9p , Ki ) 11.5 nM) which was equipotent to
that for 9a . As seen earlier with the esters derived from
piperidine,20,22 the presence of the ester function was
essential for recognition by the binding site of 5-HT4

3764 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 20
Curtet et al.
F igu r e 1. Evaluation of the antagonist effects of compounds
9a and 9r on cAMP synthesis in C6 glial cells stably trans-
fected with the 5-HT4(e) receptor isoform. The data are repre-
sented as % over basal activity. 5-HT was used alone at 1 or
0.1 µM (maximum effect at both doses). 9a and 9r were used
alone at 1 µM. Antagonist effects of 9a (1 µM) and 9r (1 µM)
were evaluated in the presence of 1 and 0.1 µM 5-HT.
F igu r e 3. Representative experiment on the antagonism
between 5-HT and 9r (ML 10821) on the L-type Ca current
(ICa) in a human atrial myocyte. Each symbol corresponds to
a measure of ICa at 0 mV obtained every 8 s. The cell was first
superfused with control solution and then exposed to 10 nM
5-HT alone or 5-HT in the presence of increasing concentra-
tions of 9r (1 nM to 1 µM) during the periods indicated by the
solid lines. The individual current traces shown on the upper
part were obtained at the times indicated by the corresponding
letters in the bottom graph. The dotted line indicates the zero
current level.
F igu r e 2. Concentration-effect curves for 5-HT on adenylyl
cyclase activity in C6 glial cells stably transfected with the
5-HT4(e) receptor isoform: 9, 5-HT alone; 2, 5-HT + GR 113808
(2 nM); 0, 5-HT + 9a (70 nM); O, 5-HT + 9r (5 nM).
The effect of 9r (ML 10821) on the L-type calcium
current (ICa) in isolated human atrial myocytes was
examined using the whole-cell patch clamp technique.19
As previously reported,17,19 application of 5-HT to the
human atrial myocytes resulted in a large stimulation
of ICa. In the typical experiment of Figure 3, 10 nM 5-HT
increased basal ICa almost 4-fold. In contrast, 9r pro-
duced no effect on basal ICa. However, when it was
tested in the presence of 5-HT, a potent antagonist effect
was observed. The experiment of Figure 3 shows that
as low as 1 nM 9r produced an attenuation of the
stimulatory effect of 10 nM 5-HT. Increasing the con-
centration of 9r further reduced ICa until the effect of
5-HT was totally blunted at 100 nM. This effect was
reversible, since the amplitude of ICa increased again
upon washout of the drug (5-HT still present, Figure
3). Figure 4 summarizes the results of a number of
experiments in which the effects of increasing concen-
trations of 5-HT on ICa were successively examined on
the same cells in the absence (circles) or presence
(squares) of 10 nM 9r . The presence of 9r induced a
clear shift to the right of the concentration-effect curve
for the response of ICa to 5-HT. Maximal response of ICa
to 5-HT (Emax) and concentration of 5-HT necessary to
produce half-maximal response (EC50) were derived by
fitting the experimental points to the Hill equation. Hill
coefficients were close to unity in both conditions (0.71
( 0.15 and 0.89 ( 0.14, respectively). The presence of
9r increased the EC50 15-fold, from 4.5 to 66 nM.
Calculation of the Schild plot led to a KD value of 7 ×
10-10 for compound 9r in human atrial cells. This value
F igu r e 4. Concentration-effect curves for 5-HT on cumula-
tive ICa alone (b) or in the presence of 10 nM 9r (9) in human
atrial myocytes. The symbols indicate the mean values in
percentage stimulation of basal ICa (normalized to maximal
response), and the vertical line indicates the SEM of the
number of experiments indicated near the symbols. Data were
fitted with the Hill equation program Origin V.5. The concen-
tration-effect response of each cell was fitted with the Hill
equation and used to calculate the mean values and standard
errors. Maximal response of ICa to 5-HT (Emax) and concentra-
tion of 5-HT necessary to produce half-maximal response
(EC50) were derived from this analysis. EC50 values were 4.5
( 1.5 nM for 5-HT alone and 66 ( 13 nM for 5-HT in the
presence of 10 nM 9r .
is in the range of those found previously by Kaumann12b
for the antagonist reference compounds, and it con-
firmed the favorable role of the arylpiperazine and
particularly that of the heteroaryl substitution in the
design of new human 5-HT4 receptor antagonists. These
properties were in contrast to those observed with ML
10302 which was characterized as a potent agonist in
the GI system20 and a partial agonist in human atrium19
and the recombinant receptors.2

New Arylpiperazine Derivatives
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 20 3765
3-Br om op r op yl 4-Am in o-5-ch lor o-2-m eth oxyben zoa te
(10b). A mixture of 4-amino-5-chloro-2-methoxybenzoic acid
(2.02 g, 10 mmol) in 1,3-dibromopropane (10.2 mL) and dry
THF (20 mL) was heated to reflux. Then 1.5 mL of 1,8-
diazabicyclo[5.4.0]undec-7-ene (DBU) was added dropwise over
2 h, and the resulting mixture was maintained under reflux
overnight. The cooled mixture was then filtered, and the
filtrate was concentrated in vacuo. The residue was diluted
with CH2Cl2, washed with water, dried over MgSO4, and the
solvent evaporated. Purification by chromatography on silica
gel (CH2Cl2) and recrystallization from cyclohexane gave a
yellow solid (1.26 g, 39%): mp 104 °C; Rf 0.15 (CH2Cl2); 1H
NMR (CDCl3) δ 7.80 (s, 1H), 6.28 (s, 1H), 4.47 (br s, 2H), 4.36
(t, J ) 6 Hz, 2H), 3.84 (s, 3H), 3.54 (t, J ) 6 Hz, 2H), 2.27 (m,
2H); 13C NMR (CDCl3) δ 164.6, 160.3, 148.1, 133.3, 110.0,
109.4, 98.3, 62.1, 56.1, 31.9, 29.8. Anal. (C11H13BrNO3).
Con clu sion s
We have reported here a new class of antagonists of
the human 5-HT4 receptor isoforms, and on the basis
of the structural framework of ML 10302, we have
shown that the introduction of the heteroarylpiperazine
moiety gave compounds with high affinity for human
5-HT4 receptor isoforms. The antagonist properties of
9a and 9r were evaluated with an adenylyl cyclase
assay in C6 glial cells stably expressing the newly cloned
human 5-HT4(e) receptor. The antagonist profile of 9r
was confirmed electrophysiologically in human atrial
myocytes where it blocked the 5-HT-induced rise in ICa
with high affinity. Important SAR studies are in course
to evaluate the interest of these new compounds and
their therapeutic potential in preventing atrial arrhyth-
mia.
Gen er a l Meth od for th e P r ep a r a tion of Com p ou n d s
9a -n ,p -y. Meth od A. 2-[N-(4-Ch lor op h en yl)p ip er a zin o]-
et h yl 4-Am in o-5-ch lor o-2-m et h oxyb en zoa t e Dih yd r o-
ch lor id e Sa lt (9b). A mixture of 10a (1.23 g, 16 mmol),
4-chlorophenylpiperazine (1.29 g, 4.8 mmol) and K2CO3 (2.21
g, 16 mmol) in dry CH3CN or DMF (30 mL) was stirred for 18
h at room temperature. After concentration in vacuo, the
residue was dissolved in CH2Cl2 (100 mL), washed with water,
and dried over MgSO4. Chromatography on a column of silica
gel with Et2O/CH2Cl2/MeOH (40:50:10) afforded an oil which
was purified on neutral alumina with Et2O/CH2Cl2/MeOH (45:
50:5) to yield a pure white solid (1.19 g). This compound was
transformed into the hydrochloride salt with a 3 N HCl ethyl
acetate solution and recrystallized from MeOH/AcOEt/cyclo-
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
19F NMR spectra were recorded on a Bruker AC 200 spec-
trometer at 200 MHz. Chemical shifts are reported in parts
per million (δ) relative to tetramethylsilane as the internal
standard, and signals are quoted as s (singlet), d (doublet), br
s (broad singlet), or m (multiplet). Elemental analyses were
performed at the CNRS Analysis Services in Chaˆtenay-
Malabry (France) and were within 0.4% of the theoretical
values unless otherwise noted.
1
hexane to give 0.110 g of a white solid (5%): mp 202 °C; H
NMR (CD3OD) (δ) 7.82 (s, 1H, ArH), 7.23 (d, 2H, J ) 8.69 Hz,
PhH), 7 (d, 2H, J ) 8.69 Hz, PhH), 6.44 (s, 1H, ArH), 4.6 (m,
2H, OCH2), 3.8 (s, 3H, OCH3), 3.82-3 (m, 8H, piperH), 3.64
(m, 2H, CH2N). Anal. (C20H23N3O3Cl2‚2HCl‚3.5H2O).
2-[N-(P h en yl)p ip er a zin o]et h yl 4-Am in o-5-ch lor o-2-
m eth oxyben zoa te (9a ). It was prepared according to the
process described for 9b and isolated as the hydrochloride salt
(11%) by crystallization from a MeOH/i-Pr2O mixture: mp 206
°C; 1H NMR (CD3OD) δ 7.83 (s, 1H), 7.27 (m, 2H), 7.02 (m,
1H), 6.89 (m, 2H), 6.48 (s, 1H), 4.62 (m, 2H), 3.82 (s, 3H), 3.8-
3.32 (m, 8H), 3.61 (m, 2H). Anal. (C20H24N3O3Cl‚HCl‚0.25H2O).
Ma ter ia ls. Acetonitrile, DMF, and the usual solvents were
purchased from SDS (Paris, France). Column chromatography
was performed on Merck silica gel 60 (70/230 mesh) and on
Aldrich neutral aluminum oxide (150 mesh).
2-Bromoethyl 4-amino-5-chloro-2-methoxybenzoate (10a )
was synthesized from 4-amino-5-chloro-2-methoxybenzoic acid
by condensation with 1,2-dibromoethane and 1,8-diazobicyclo-
[5.4.0]undec-7-ene (DBU) in dry THF.20 1-(1-Naphthyl)pipera-
zine hydrochloride was prepared according to a previously
reported procedure.23 N-Benzylpiperazine and N-(2-pyrimidyl)-
piperazine were commercially available. N-(2-Pyridyl)pipera-
zine, N-(4-pyridyl)piperazine, N-(4-pyrimidyl)piperazine, N-(2-
pyrazinyl)piperazine, N-(2-(6-chloro)pyridazinyl)piperazine, N-(3-
pyridazinyl)piperazine and N-(4-(2-chloro)pyrimidyl)piperazine
were prepared according to the methods already reported. 23a-c
4-P h en yl-1-[2-(2-p h th a lim id o)eth yl]p ip er a zin e. N-(2-
Bromoethyl)phthalimide (5.6 g, 22 mmol), potassium carbonate
(0.8 g, 6 mmol) and a catalytic quantity of KI were added
sequentially to a solution of 1-phenylpiperazine (3 mL, 20
mmol) in DMF (100 mL). The resulting mixture was main-
tained at 65 °C for 17 h. The reaction mixture was evaporated
in vacuo and partitioned between EtOAc and H2O. The
aqueous layer was extracted with EtOAc. The combined
organic layers were dried over Na2SO4, filtered, and evaporated
to give a yellow solid. Recrystallization from absolute EtOH
yielded 3.7 g (55%) of product as gold needles: mp 154 °C; 1H
NMR (CDCl3) δ 7.86-7.68 (m, 4H), 7.27-7.19 (m, 2H), 6.91-
6.78 (m, 3H), 3.86 (t, J ) 6.5 Hz), 3.16-3.11 (m, 4H), 2.73-
2.65 (m, 6H); 13C NMR (CDCl3) δ 168.3, 151.2, 133.8, 132.1,
128.9, 123.1, 119.5, 115.8, 55.6, 53, 49.1, 35.2.
1-(2-Am in oeth yl)-4-p h en ylp ip er a zin e. A solution of 85%
hydrazine hydrate (0.13 mL, 2.8 mmol) was added dropwise
to a stirred suspension of the previous compound (0.46 g, 1.4
mmol) in absolute EtOH (15 mL). After completing the
addition, the reaction mixture was heated for 1 h. The solution
was allowed to cool at room temperature and the solid material
was collected by filtration. The filtrate was evaporated under
reduced pressure to yield a yellow solid (0.3 g) used without
further purification: mp 203 °C; 1H NMR (CDCl3) δ 7.29-7.21
(m, 2H), 6.94-6.8 (m, 3H), 3.22-3.17 (m, 4H), 2.82 (t, J ) 6.1
Hz, 2H), 2.63-2.58 (m, 4H), 2.47 (t, J ) 6.1 Hz, 2H), 1.45 (br
s, 2H); 13C NMR (CDCl3) δ 151.4, 129.1, 119.6, 116, 61.2, 53.3,
49.2, 38.9.
2-[N-(3-Ch lor op h en yl)p ip er a zin o]eth yl
4-Am in o-5-
ch lor o-2-m eth oxyben zoa te (9c). It was prepared according
to the process described for 9b and isolated as the hydrochlo-
ride salt (19%) by crystallization from a MeOH/AcOEt/cyclo-
hexane mixture: mp 208 °C; 1H NMR (CD3OD) δ 7.82 (s, 1H),
7.23 (m, 1H), 7.03 (m, 1H), 6.9 (m, 2H), 6.46 (s, 1H), 4.6 (m,
2H), 3.80 (s, 3H), 3.8-3.13 (m, 10H). Anal. (C20H23N3O3Cl2‚
HCl‚0.25H2O).
2-[N-(2-Ch lor op h en yl)p ip er a zin o]eth yl
4-Am in o-5-
ch lor o-2-m eth oxyben zoa te (9d ). It was prepared according
to the process described for 9b and isolated as the hydrochlo-
ride salt (6%) by crystallization from a MeOH/AcOEt/cyclo-
hexane mixture: mp 234 °C; 1H NMR (CD3OD) δ 7.84 (s, 1H),
7.42 (m, 1H), 7.32 (m, 1H), 7.20 (m, 1H), 7.10 (m, 1H), 6.48 (s,
1H), 4.63 (m, 2H), 3.82 (s, 3H), 3.8 (m, 2H), 3.67 (m, 2H), 3.56
(m, 2H), 3.46 (m, 2H), 3.17 (m, 2H). Anal. (C20H23N3O3Cl2‚HCl‚
H2O).
2-[N-(4-Meth oxyp h en yl)p ip er a zin o]eth yl 4-Am in o-5-
ch lor o-2-m eth oxyben zoa te (9e). It was prepared according
to the process described for 9b and isolated as the hydrochlo-
ride salt (25%) by crystallization from a MeOH/AcOEt/cyclo-
hexane mixture: mp 198 °C; 1H NMR (CD3OD) δ 7.86 (s, 1H),
7.31 (d, 2H, J ) 7.18 Hz), 6.97 (d, 2H, J ) 7.18 Hz), 6.53 (s,
1H), 4.66 (m, 2H), 3.9-3.69 (m, 10H), 3.8 (s, 3H), 3.78 (s, 3H).
Anal. (C21H26N3O4Cl‚2HCl‚2H2O).
2-[N-(3-Meth oxyp h en yl)p ip er a zin o]eth yl 4-Am in o-5-
ch lor o-2-m eth oxyben zoa te (9f). It was prepared according
to the process described for 9b and isolated as the hydrochlo-
ride salt (13%) by crystallization from a MeOH/AcOEt/cyclo-
hexane mixture: mp 180 °C; 1H NMR (CD3OD) δ 7.84 (s, 1H,
ArH), 7.25-7.11 (m, 1H, PhH), 6.67-6.48 (m, 3H, PhH), 6.5
(s, 1H, ArH), 4.61 (m, 2H, OCH2), 3.82 (s, 3H, OCH3), 3.76 (s,

3766 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 20
Curtet et al.
3H, OCH3), 3.9-3.6 (m, 8H, piperH), 3.65 (m, 2H, CH2N). Anal.
(C21H26N3O4Cl‚2HCl‚H2O).
160.2, 148.1, 133.3, 109.9, 109.4, 98.2, 62, 56.6, 55, 53.2, 45.9.
Anal. (C15H22ClN3O3‚0.3H2O).
2-[N-(2-Meth oxyp h en yl)p ip er a zin o]eth yl 4-Am in o-5-
ch lor o-2-m eth oxyben zoa te (9g). It was prepared according
to the process described for 9b and isolated as the hydrochlo-
ride salt (19%) by crystallization from a MeOH/AcOEt/cyclo-
hexane mixture: mp 196 °C; 1H NMR (CD3OD) (δ) 7.81 (s,
1H), 7.75-6.82 (m,4H), 6.45 (s, 1H), 4.61 (m, 2H), 3.84 (s, 3H),
3.82 (s, 3H), 3.9-3 (m, 8H), 3.65 (m, 2H). Anal. (C21H26N3O4-
Cl‚2HCl‚2H2O).
2-[N-P ip er a zin o]et h yl 4-Am in o-5-ch lor o-2-m et h oxy-
ben zoa te (9o). A solution of 9m (1.3 g, 3.1 mmol) in CH2Cl2
(30 mL) was treated with anisole (0.1 mL) and trifluoroacetic
acid (2.43 mL) for 6 h. The solvent was removed in vacuo, and
the residue was taken up in CH2Cl2 and was made basic with
a saturated aqueous NaHCO3 solution and solid NaHCO3. The
organic phase was separated, washed with brine, dried with
MgSO4 and concentrated. Column chromatography on silica
gel with MeOH/CH2Cl2 (10:90) gave 0.9 g of 9o (96%) as a
white foam solid. This compound was transformed into its
hydrochloride salt with a 2 N HCl methanol solution and
precipitated with Et2O. Recrystallization from i-Pr2O/MeOH
afforded the dihydrochloride salt (14%): mp 244 °C; 1H NMR
(CD3OD) δ 7.81 (s, 1H), 6.47 (s, 1H), 4.57 (m, 2H), 3.88 (s, 3H),
3.57 (br m, 10H). Anal. (C14H20N3O3Cl‚2HCl‚H2O).
2-[N-(4-P h en yl)p ip er a zin o]p r op yl 4-Am in o-5-ch lor o-2-
m eth oxyben zoa te (9p ). A mixture of 10b (1.12 g, 3.4 mmol),
N-phenylpiperazine (0.6 mL, 3.7 mmol) and diisopropylethy-
lamine (1.2 mL, 6.8 mmol) in dry DMF (10 mL) was stirred
for 24 h at room temperature. The resulting mixture was
concentrated in vacuo. The residue was recristallized in AcOEt/
cyclohexane to give 0.7 g (50%) of a pale pink solid: mp 116
°C; 1H NMR (CDCl3) δ 7.80 (s, 1H), 7.29-7.21 (m, 2H), 6.94-
6.81 (m, 3H), 6.28 (s, 1H), 4.48 (s, 2H), 4.31 (t, J ) 6.4 Hz,
2H), 3.83 (s, 3H), 3.25 (m, 4H), 2.71-2.57 (m, 6H), 2.00 (m,
2H); 13C NMR (CDCl3) δ 164.7, 160.3, 151.4, 147.8, 133.3,
129.1, 119.7, 116.1, 109.9, 98.3, 62.9, 56.1, 55.3, 53.3, 49.2, 26.3.
Anal. (C21H26ClN3O3).
2-[N-(3-Tr iflu or op h en yl)p ip er a zin o]eth yl 4-Am in o-5-
ch lor o-2-m eth oxyben zoa te (9h ). It was prepared according
to the process described for 9b and isolated as the hydrochlo-
ride salt (10%) by crystallization from a MeOH/AcOEt/cyclo-
1
hexane mixture: mp 232-234 °C; H NMR (CD3OD) (δ) 7.84
(s, 1H), 7.46 (m, 1H), 7.28 (m, 2H), 7.19 (m, 1H), 6.48 (s, 1H),
4.62 (m, 2H), 3.82 (s, 3H), 3.8-3.4 (m, 10H); 19F NMR (CDCl3)
δ 62.937 (s, 3F). Anal. (C21H23N3O3F3‚HCl‚0.15H2O).
2-[N-(4-F lu or op h en yl)p ip er a zin o]eth yl
4-Am in o-5-
ch lor o-2-m eth oxyben zoa te (9i). It was prepared according
to the process described for 9b and isolated as hydrochloride
salt (17%) by crystallization from a MeOH/AcOEt/cyclohexane
mixture: mp 130 °C dec; 1H NMR (CD3OD) δ 7.86 (s, 1H),
7.16-6.94 (m, 4H), 6.54 (s, 1H), 4.64 (m, 2H), 3.83 (s, 3H),
3.92-3.5 (m, 8H), 3.65 (m, 2H); 19F NMR (CDCl3) δ 124.87 (s,
1F). Anal. (C20H23N3O3F‚2HCl‚0.75H2O).
2-[N-(2-F lu or op h en yl)p ip er a zin o]eth yl
4-Am in o-5-
ch lor o-2-m eth oxyben zoa te (9j). It was prepared according
to the process described for 9b and isolated as the hydrochlo-
ride salt (16%) by crystallization from a MeOH/AcOEt/cyclo-
hexane mixture: mp 235 °C; 1H NMR (CD3OD) δ 7.84 (s, 1H),
7.16-7 (m, 4H), 6.48 (s, 1H), 4.63 (m, 2H), 3.82 (s, 3H), 3.77
(m, 2H), 3.65 (m, 2H), 3.61(m, 2H), 3.46 (m, 2H), 3.16 (m, 2H);
19F NMR (CDCl3) δ 123.03 (s, 1F). Anal. (C20H23N3O3F‚HCl‚
0.25H2O).
2-[N-(2-P yr im id yl)p ip er a zin o]eth yl 4-Am in o-5-ch lor o-
2-m eth oxyben zoa te (9r ). It was prepared according to the
process described for 9b and isolated as the hydrochloride salt
(17%) by crystallization from a MeOH/AcOEt/cyclohexane
1
mixture: mp 244 °C; H NMR (CD3OD) δ 8.43 (m, 2H), 7.82
(s, 1H), 6.71 (m, 1H), 6.50 (s, 1H), 4.56 (m, 2H), 3.85 (s, 3H),
2-[N-(Na p h th yl)p ip er a zin o]eth yl 4-Am in o-5-ch lor o-2-
m eth oxyben zoa te (9k ). It was prepared according to the
process described for 9b and isolated as the hydrochloride salt
(21%) by crystallization from MeOH/AcOEt/Et2O: mp 174 °C
3.8-3.13 (m, 10H). Anal. (C18H22N5O3Cl‚4HCl‚0.25H2O).
Meth od B. 2-[N-(4-Ben zyl)p ip er a zin o]eth yl 4-Am in o-
5-ch lor o-2-m eth oxyben zoa te (9q). A mixture of 2-bromo-
ethyl 4-amino-5-chloro-2-methoxybenzoate (10a ) (0.62 g, 3.9
mmol), N-benzylpiperazine (0.38 mL, 2.2 mmol), and diiso-
propylethylamine (0.7 mL, 4 mmol) in dry CH3CN (10 mL) was
refluxed for 24 h under argon. The resulting mixture was
concentrated in vacuo. The residue was dissolved in methylene
chloride, washed successively with water and brine, and dried
over MgSO4. After evaporation of the solvent, the residue was
purified by chromatography (eluant CH2Cl2/i-PrOH 9:1) to give
a solid which was recrystallized in an AcOEt/cyclohexane
mixture to give 0.27 g (33%) of product as yellow needles: mp
117 °C; Rf 0.1 (CH2Cl2/i-PrOH 9:1); 1H NMR (CDCl3) δ 7.8 (s,
1H), 7.32-7.22 (m, 5H), 6.27 (s, 1H), 4.43 (s, 2H), 4.36 (t, J )
6 Hz, 2H), 3.82 (s, 3H), 3.51 (s, 2H), 2.75 (t, J ) 6 Hz, 2H),
2.61-2.50 (m, 8H); 13C NMR (CDCl3) δ 164.4, 160.2, 147.8,
138.12, 133.3, 129.2, 128.2, 126.9, 110, 98.3, 63, 62, 56.7, 56.1,
53.4, 53. Anal. (C21H26ClN3O3).
1
dec; H NMR (CD3OD) δ 8.22 (m, 1H), 7.84 (m, 2H), 7.63 (m,
1H), 7.49 (m, 2H), 7.41 (m, 1H), 7.21 (m, 1H), 6.75 (s, 1H),
4.65 (m, 2H), 3.82 (s, 3H), 3.94-3.27 (m, 10H). Anal.
(C24H26N3O3Cl‚HCl‚0.5H2O).
2-[N-(E t h oxyca r b on yl)p ip er a zin o]et h yl 4-Am in o-5-
ch lor o-2-m eth oxyben zoa te (9l). The compound was syn-
thesized according to the process used for 9b. It was purified
by chromatography (CH2Cl2/Et2O 1:1) to give a colorless oil
which was recrystallized in an acetone/pentane mixture to give
a white solid (yield 28%): mp 92 °C; Rf 0.2 (CH2Cl2/i-PrOH
95:5); 1H NMR (CDCl3) δ 7.78 (s, 1H), 6.27 (s, 1H), 4.52 (s,
2H), 4.34 (t, J ) 6 Hz, 2H), 4.11 (q, J ) 7 Hz, 2H); 3.80 (s,
3H), 3.46 (t, J ) 5 Hz, 4H), 2.73 (t, J ) 6 Hz, 2H), 2.50 (t, J )
5 Hz, 4H), 1.24 (t, J ) 7 Hz, 3H); 13C NMR (CDCl3) δ 164.5,
160.3, 155.5, 148, 133.3, 109.9, 109.5, 98.2, 62, 61.3, 56.8, 56,
53.1, 43.7,14.7. Anal. (C17H24ClN3O5).
2-[N-(4-P yr im id yl)p ip er a zin o]eth yl 4-Am in o-5-ch lor o-
2-m eth oxyben zoa te (9s). It was synthesized according to the
previous process: the reaction between 2-bromoethyl 4-amino-
5-chloro-2-methoxybenzoate (10a ) (0.53 g, 1.71 mmol), N-(4-
pyrimidyl)piperazine (306 mg, 1.9 mmol), and diisopropyleth-
ylamine (0.6 mL, 3.42 mmol) in dry CH3CN (10 mL) gave, after
purification by chromatography (eluant CH2Cl2/i-PrOH 9:1)
200 mg (33%) of product as a white solid: mp 218-220 °C; Rf
0.63 (eluant CH2Cl2/MeOH 9:1); 1H NMR (CDCl3) δ 8.58 (s,
1H), 8.18 (d, J ) 5.3 Hz, 1H), 7.80 (s, 1H), 6.48 (d, J ) 6 Hz,
1H), 6.28 (s, 1H), 4.48 (s, 2H), 4.39 (t, J ) 6 Hz, 2H), 3.83 (s,
3H), 3.68-3.63 (m, 4H), 2.79 (t, J ) 6 Hz, 2H), 2.66-2.61 (m,
4H); 13C NMR (CDCl3) δ 164.4, 161.6, 161.3, 160.3, 155.5,
147.8, 133.3, 110, 101.9, 98.3, 61.8, 56.7, 56.7, 52.9, 43.7. Anal.
(C19H23ClN4O3‚2HCl‚1H2O).
2-[N-(ter t-Bu tyloxycar bon yl)piper azin o]eth yl 4-Am in o-
5-ch lor o-2-m eth oxyben zoa te (9m ). It was synthesized ac-
cording to the previous process (yield 58%): mp 102 °C; 1H
NMR (CDCl3) δ 7.80 (s, 1H), 6.28 (s, 1H), 4.48 (br.s, 2H), 4.36
(m, 2H), 3.83 (s, 3H), 3.43 (m, 4H), 2.74 (m, 2H), 2.47 (m, 4H),
1.45 (s, 9H). Anal. (C19H28N3O5Cl‚0.25H2O).
2-[N-(4-Meth yl)p ip er a zin o]eth yl 4-Am in o-5-ch lor o-2-
m eth oxyben zoa te (9n ). A mixture of 10a (1.2 g, 3.9 mmol),
N-methylpiperazine (0.5 mL, 4.3 mmol) and diisopropylethy-
lamine (1.4 mL, 7.8 mmol) in dry DMF (10 mL) was stirred
for 24h at room temperature. The resulting mixture was
concentrated in vacuo. The residue was dissolved in methylene
chloride, washed successively with water and brine, and dried
over MgSO4. After evaporation of the solvent, the residue was
purified by chromatography (eluant CH2Cl2/MeOH 9:1) to give
0.6 g (46%) of a white powder: mp 118 °C; Rf 0.10 (CH2Cl2/
MeOH 9:1); 1H NMR (CDCl3) δ 7.78 (s, 1H), 6.25 (s, 1H), 4.50
(s, 2H), 4.34 (t, J ) 6 Hz, 2H), 3.79 (s, 3H), 2.72 (t, J ) 6 Hz,
2H), 2.70-2.40 (m, 8H), 2.27 (s, 3H); 13C NMR (CDCl3) δ 164.5,
2-[N-(2-P yr a zin yl)p ip er a zin o]eth yl 4-Am in o-5-ch lor o-
2-m eth oxyben zoa te (9t). It was synthesized according to the
previous process: the reaction between 2-bromoethyl 4-amino-
5-chloro-2-methoxybenzoate (10a ) (0.63 g, 2 mmol), N-(2-

New Arylpiperazine Derivatives
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 20 3767
pyrazinyl)piperazine (373 mg, 2.3 mmol), and diisopropyleth-
ylamine (0.71 mL, 4.08 mmol) in dry CH3CN (10 mL) gave,
after purification by chromatography (eluant CH2Cl2/i-PrOH
9:1) 318 mg (40%) of product as a pale yellow solid: mp 163
previous process: the reaction between 2-bromoethyl 4-amino-
5-chloro-2-methoxybenzoate (10a ) (0.64 g, 2 mmol), N-(4-
pyridyl)piperazine (375 mg, 2.3 mmol), and diisopropylethy-
lamine (0.72 mL, 4.1 mmol) in dry CH3CN (10 mL) gave, after
purification by chromatography (eluant CH2Cl2/MeOH 9:1) 200
mg (24%) of product as a white solid: mp °C; Rf 0.07 (CH2-
Cl2/MeOH 9:1); 1H NMR (CDCl3) δ 8.24 (d, J ) 4 Hz, 1H), 7.82
(s, 1H), 6.65 (d, J ) 5.5 Hz, 1H), 6.29 (s, 1H), 4.54 (s, 2H),
4.39 (t, J ) 6 Hz, 2H), 3.82 (s, 3H), 3.38-3.33 (m, 4H), 2.78 (t,
J ) 6 Hz, 2H), 2.70-2.65 (m, 4H); 13C NMR (CDCl3) δ 163.9,
160.3, 155.1, 149, 147.9, 133.2, 110.1, 108.3, 98.3, 61.7, 56.6,
56, 52.7, 46. Anal. (C19H23ClN4O3‚0.4H2O).
2-[N-(4-P h en yl)p ip er a zin o]p r op yl 4-Am in o-5-ch lor o-2-
m eth oxyben za m id e (11). A mixture of 4-amino-5-chloro-2-
methoxybenzoic acid (0.7 g, 3.5 mmol), triethylamine (0.54 mL,
3.9 mmol) and DMF (30 mL) was cooled to 4 °C. Ethyl
chloroformate (0.33 mL, 3.5 mmol) was added and the mixture
stirred for 30 min at 4 °C. 1-(2-Aminoethyl)-4-phenylpiperazine
(0.8 g, 3.9 mmol) was then added at room temperature and
the mixture stirred overnight. The reaction mixture was
washed with a 10% Na2CO3 solution and the aqueous layer
was extracted several times with CH2Cl2. The combined
organic layers were dried over MgSO4 and concentrated in
vacuo. Recrystallization from absolute EtOH yielded 0.52 g
(38%) of product as yellow needles: mp 185 °C; 1H NMR
(CDCl3) δ 8.22 (br s, 1H), 8.12 (s, 1H), 7.31-7.23 (m, 2H), 6.96-
6.86 (m, 3H), 6.27 (s, 1H), 4.35 (s, 2H), 3.87 (s, 3H), 3.58 (m,
2H); 3.40-3.20 (m, 4H), 2.70-2.60 (m, 6H); 13C NMR (CDCl3)
δ 176.8, 164.3, 151.1, 146.5, 132.9, 129.07, 119.7, 115.9, 111.5,
110.7, 97.8, 56.5, 56.1, 52.8, 49.3, 36.4. Anal. (C20H25ClN4O2).
1
°C; Rf 0.15 (CH2Cl2/i-PrOH 9:1); H NMR (CDCl3) δ 8.09 (s,
1H), 8.02-8.01 (m, 1H), 7.70 (d, J ) 2.5 Hz, 1H), 7.77 (s, 1H),
6.24 (s, 1H), 4.56 (s, 2H), 4.39 (t, J ) 6 Hz, 2H), 3.77 (s, 3H),
3.60-3.54 (m, 4H), 2.76 (t, J ) 6 Hz, 2H), 2.70-2.60 (m, 4H);
13C NMR (CDCl3) δ 164.4, 160.2, 154.9, 147.9, 141.6, 133.2,
132.8, 130.9, 109.9, 109.5, 98.2, 61.8, 56.7, 56, 52.9, 44.5. Anal.
(C18H23ClN5O3‚0.2H2O).
2-[N-(3-P yr idazin yl)piper azin o]eth yl 4-Am in o-5-ch lor o-
2-m eth oxyben zoa te (9u ). It was synthesized according to the
previous process: the reaction between 2-bromoethyl 4-amino-
5-chloro-2-methoxybenzoate (10a ) (0.62 g, 2 mmol), N-(3-
pyridazinyl)piperazine (0.36 g, 1.8 mmol), and diisopropyleth-
ylamine (0.7 mL, 4 mmol) in dry CH3CN (10 mL) gave, after
purification by chromatography (eluant CH2Cl2/i-PrOH 9:1
then CH2Cl2/MeOH 9:1) and formation of the chlorhydrate 176
mg (20%) of product as a yellow solid: mp 210 °C dec; Rf 0.22
(CH2Cl2/i-PrOH 9:1); 1H NMR (CDCl3) δ 8.49 (dd, J ) 3.3 Hz,
J ) 1.2 Hz, 1H), 7.75 (s, 1H), 7.14 (dd, J ) 9.3 Hz, J ) 4.4 Hz,
1H), 6.84 (dd, J ) 9.4 Hz, J ) 1.2 Hz, 1H), 6.27 (s, 1H), 4.71
(s, 2H), 4.35 (t, J ) 5.7 Hz, 2H), 3.74 (s, 3H), 3.65-3.55 (m,
4H), 2.74 (t, J ) 5.7 Hz, 2H), 2.66-2.60 (m, 4H). Anal. (C18H22
ClN5O3‚3HCl).
-
2-[N-(4-(2-Ch lor o)pyr im idyl)piper azin o]eth yl 4-Am in o-
5-ch lor o-2-m eth oxyben zoa te (9v). It was synthesized ac-
cording to the previous process: the reaction between 2-bro-
moethyl 4-amino-5-chloro-2-methoxybenzoate (10a ) (0.62 g, 2
mmol), N-(4-(2-chloro)pyrimidyl)piperazine (0.36 g, 1.8 mmol),
and diisopropylethylamine (0.7 mL, 4 mmol) in dry CH3CN
(13 mL) gave, after purification by chromatography (eluant
CH2Cl2/i-PrOH 9:1), 155 mg (20%) of product as a white solid:
mp 97 °C; Rf 0.59 (CH2Cl2/i-PrOH 9:1); 1H NMR (CDCl3) δ 8.02
(d, J ) 6 Hz, 1H, CH(6′)), 7.80 (s, 1H, ArH), 6.37 (d, J ) 6 Hz,
1H), 6.28 (s, 1H), 4.46 (s, 2H), 4.39 (t, J ) 5.8 Hz, 2H), 3.84 (s,
3H), 3.68-3.64 (m, 4H), 2.79 (t, J ) 6 Hz, 2H), 2.66-2.60 (m,
4H); 13C NMR (CDCl3) δ 164.4, 162.6, 160.8, 160.3, 157.2,
147.8, 133.3, 109.09, 109.6, 101.1, 98.3, 61.7, 56.6, 56.1, 52.7,
42.8. Anal. (C18H21Cl2N5O3‚0.9H2O).
Mem br a n e P r ep a r a tion a n d Ra d ioliga n d Bin d in g As-
sa ys. Briefly, cells grown to confluence were washed twice with
phosphate-buffered-saline (PBS) and centrifuged at 300g for
5 min. The pellet was used immediately or stored at -80 °C.
The pellet was resuspended in 10 volumes of ice-cold HEPES
buffer (50 mM, pH 7.4) and centrifuged at 40000g for 20 min
at 4 °C. The resulting pellet was resuspended in 15 volumes
of HEPES buffer (50 mM, pH 7.4). The protein concentration
were determined by the method of Lowry24 using bovine serum
albumin as the standard.
Radioligand binding studies were performed in 500 µL of
buffer (50 mM HEPES, pH 7.4), 20 µL of competing agent (7
concentrations), 20 µL of [3H]GR 113808 at a concentration of
0.2 nM for the 5-HT4(e) receptor isoform or at a concentration
of half the value of the KD values for the other isoforms
expressed in the COS cell lines and 50 µL of membrane
preparation (100-200 µg of protein). Nonspecific binding was
defined with 10 µM ML 10375 2-(cis-3,5-dimethylpiperidino)-
ethyl 4-amino-5-chloro-2-methoxybenzoate.19 Tubes were in-
cubated at 25 °C for 30 min, and the reaction was terminated
by filtration through Whatman GF/B filter paper using the
Brandel 48R cell harvester. Filters were presoaked in a 0.1%
solution of polyethylenimine. Filters were subsequently washed
with ice-cold buffer (50 mM Tris-HCl, pH 7.4) and placed
overnight in 4 mL of ready-safe scintillation cocktail. Radio-
activity was measured using a Beckman model LS 6500C
liquid scintillation counter. Binding data (Ki) were analyzed
by computer-assisted nonlinear regression analysis (Prism,
Graphpad Software, San Diego, CA). The data are the results
of two or three determinations.
2-[N-(3-(6-Ch lor o)p yr id a zin yl)p ip er a zin o]eth yl
4-
Am in o-5-ch lor o-2-m eth oxyben zoa te (9w ). It was synthe-
sized according to the previous process: the reaction between
2-bromoethyl 4-amino-5-chloro-2-methoxybenzoate (10a ) (0.62
g, 2 mmol), N-(3-(6-chloro)pyridazinyl)piperazine (0.36 g, 1.8
mmol), and diisopropylethylamine (0.7 mL, 4 mmol) in dry
CH3CN (10 mL) gave, after purification by chromatography
(eluant CH2Cl2/i-PrOH 95:5 then CH2Cl2/i-PrOH 9:1), 0.5 g
(65%) of product as a pale yellow solid: mp 179 °C; Rf 0.54
(CH2Cl2/i-PrOH 9:1); 1H NMR (CDCl3) δ 7.81 (s, 1H), 7.19 (d,
J ) 9.5 Hz, 1H), 6.87 (d, J ) 9.5 Hz, 1H), 6.28 (s, 1H), 4.46 (s,
2H), 4.41 (t, J ) 6 Hz, 2H), 3.84 (s, 3H), 3.67-3.62 (m, 4H),
2.80 (t, J ) 6 Hz, 2H), 2.71-2.67 (m, 4H); 13C NMR (CDCl3) δ
164.6, 160.4, 159.7, 148, 146.8, 133.3, 128.8, 115.3, 109.9,
109.3, 98.2, 61.8, 56.7, 56, 52.8, 45. Anal. (C18H21Cl2N5O3).
2-[N-(2-P yr id yl)p ip er a zin o]eth yl 4-Am in o-5-ch lor o-2-
m eth oxyben zoa te (9x). It was synthesized according to the
previous process: the reaction between 2-bromoethyl 4-amino-
5-chloro-2-methoxybenzoate (10a ) (0.62 g, 2 mmol), N-(2-
pyridyl)piperazine (0.33 mL, 2.2 mmol), and diisopropylethy-
lamine (0.7 mL, 4 mmol) in dry CH3CN (10 mL) gave, after
purification by chromatography (eluant CH2Cl2/i-PrOH 95:5
then CH2Cl2/i-PrOH 9:1) and recrystallization in an AcOEt/
cyclohexane mixture, 0.33 g (42%) of product as a yellow
powder: mp 143 °C; Rf 0.3 (CH2Cl2/i-PrOH 9:1); 1H NMR
(CDCl3) δ 8.18 (dd, J o ) 4.8 Hz, J m ) 1.6 Hz, 1H), 7.82 (s, 1H),
7.46 (m, 1H), 6.66-6.58 (m, 2H), 6.27 (s, 1H), 4.47 (s, 2H), 4.41
(t, J ) 6 Hz, 2H), 3.83 (s, 3H), 3.58-3.53 (m, 4H), 2.79 (t, J )
6 Hz, 2H), 2.70-2.66 (m, 4H); 13C NMR (CDCl3) δ 165.1, 160.9,
160.1, 148.6, 148.4, 138, 133.9, 110.6, 110.4, 107.7, 98.9, 62.6,
57.4, 56.7, 53.8, 45.8. Anal. (C19H23ClN4O3‚0.2H2O).
Mea su r em en t of cAMP . For the measurement of intrac-
ellular cAMP accumulation, C6 glial cells stably transfected
with human 5-HT4(e) receptors were grown to confluency and
incubated with serum-free medium for 4 h before the beginning
of the assay. Then the cells were preincubated for 15 min with
serum-free medium supplemented with 5 mM theophylline and
10 µM pargyline. 5-HT (1 µM) and/or compounds 9a and 9r
were added and incubated for an additional 15 min at 37 °C
in 5% CO2. The reaction was stopped by aspiration of the
medium and addition of 500 µL of ice-cold ethanol. After 1 h
at room temperature, the ethanol fraction was collected and
lyophilized. The pellet was reconstituted and cAMP was
quantified using a radioimmunoassay. The 5-HT concentra-
2-[N-(4-P yr id yl)p ip er a zin o]eth yl 4-Am in o-5-ch lor o-2-
m eth oxyben zoa te (9y). It was synthesized according to the

3768 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 20
Curtet et al.
tion-effect curve was calculated using seven concentrations
(10-8-10-5 M) alone or in the presence of 9a (70 nM) or 9r (5
nM).
mean ( SEM. In each experimental condition, the effects of
5-HT on ICa are expressed in percent (%) change with respect
to the values of the current under basal conditions, i.e., in the
absence of any drug.
Exp er im en ts on Hu m a n Atr ia l Myocytes. Su r ger y: All
protocols for obtaining human cardiac tissue were approved
by the ethics committee of our institution (GREBB, Hoˆpital
de Biceˆtre, Universite´ de Paris-Sud). Specimens of right atrial
appendages were obtained from patients undergoing heart
surgery for congenital defects, coronary artery diseases or
valve replacement. Most patients received a pharmacological
pretreatment (Ca channel blockers, digitalis, â-adrenergic
antagonists, diuretics, ACE inhibitors, NO donors, and/or
antiarrhythmic drugs). In addition, all patients received
sedatives, anasthesia, and antibiotics. Dissociation of the cells
was realized immediately after surgery.
Ack n ow led gm en t. We thank Florence Lefebvre for
helpful technical assistance in isolating human atrial
myocytes. Sophie Curtet and Ivan Zahradnik were
supported by grants from the French Ministe`re de
l’Education Nationale, de l’Enseignement Supe´rieur et
de la Recherche. Ivan Zahradnik is grateful for a
INSERM grant (poste jaune).
Refer en ces
Hu m a n a tr ia l cell d issocia tion : Myocytes were isolated
as desribed previously25 with some modifications. Briefly,
quickly after excision, the tissue was cut up and washed in a
calcium-free Tyrode solution supplemented with 30 mM 2,3-
butanedionemonoxine which was carefully removed after
cutting. Small (∼1 mm3) pieces of atria were then incubated
in the same Tyrode solution containing 40 IU/mL collegenase,
15 IU/mL protease and 5 mg/mL BSA. After 30 min, this
solution was removed and replaced by fresh enzymatic solution
containing only collagenase (200 UI/mL) for 10-20 min until
a satisfactory cell yield was obtained. All steps were carried
out at 37 °C, with continuous stirring at 200 rpm and gassing
with 95% O2 and 5% CO2. The cell suspension was filtered and
centrifuged (1 min at 600-700 rpm) and the pellet resus-
pended in DMEM supplemented with 10% fœtal calf serum,
nonessential amino acids, 1 nM insulin and antibiotics (peni-
cillin, 100 IU/mL, and streptomycin, 01 µg/mL). For patch-
clamp experiments 20-100 mL of this cell suspension was put
in a Petri dish containing control external solution.
Electr op h ysiologica l exp er im en ts: The whole-cell con-
figuration of the patch-clamp technique26 was used to record
the high-threshold calcium current (ICa) on Ca2+-tolerant
human atrial myocytes. In the routine protocols the cells were
depolarized every 8 s from a holding potential of -50 to 0 mV
for 400 ms. This holding potential was chosen to completely
inactivate the fast Na current. K currents were blocked by
replacing all K+ ions with intracellular Cs+ and extracellular
Cs+. Voltage-clamp protocol was generated by a challenger/
09-VM programmable function generator (Kinetic Software,
Atlanta, GA). The cells were voltage-clamped using a patch-
clamp amplifier (model RK-400, Biologic, Claix, France).
Currents were sampled at a frequency of 10 kHz using a 12-
bit analogue-to-digital converter (DT2827, Data Translation,
Malboro, MA) connected to a PC compatible computer (3865/
33 System-pro, Compaq, Houston, TX). All experiments were
done at room temperature (19-25 °C).
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107.1 NaCl, 10 Hepes, 40 CsCl, 4 NaHCO3, 0.8 NaH2PO4, 1.8
CaCl2, 1.8 MgCl2, 5 D-glucose, 5 sodium pyruvate, pH 7.4
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