5-HT3 antagonists derived from aminopyridazine-type muscarinic M1 agonists

Article abstract of DOI:10.1021/jm9705418

A conformational analysis, performed on muscarinic M₁ agonists, identified four structural features characteristic of the muscarinic M₁ pharmacophore: (i) a protonable basic or quaternary nitrogen acting as a cationic head; (ii) an electronegative dipole usually part of a planar mesomeric ester, amide, or amidine function which can be replaced by an ether (muscarine) or a dioxolane (AF 30); (iii) an intercharge distance of 5 ± 0.5 A? between the cationic head and the electronegative atom of the dipole; (iv) an elevation of 0.5 ± 0.03 A? of the cationic head over the plane containing the electronegative dipole. During a reinvestigation of the conformational behavior of published structures of 5-HT₃ antagonists, similar features were observed for the 5-HT₃ pharmacophore. However many 5-HT₃ antagonists possess additional aromatic planes not present in the muscarinic M₁ agonists. These observations brought us to predict the chemical modifications that would change muscarinic M₁ agonists into 5-HT₃ antagonists. Four of the predicted aminopyridazines were actually synthesized and submitted to testing. The observed IC₅₀ values for 5-HT₃ receptor binding ([³H] BRL 43694) ranged from 10 to 425 nM, whereas the affinities for the muscarinic receptor preparations ([²SH] pirenzepine) layed over 10 000 nM. In electrophysiological studies the two most active compounds 10 and 13 produced antagonist-like effects on the 5-HT receptor channel complexes responsible for the generation of the rapidly desensitizing ionic currents, and agonist- like effects on those responsible for the slowly desensitizing components.

Full text of DOI:10.1021/jm9705418

J . Med. Chem. 1998, 41, 311-317
311
5-HT3 An ta gon ists Der ived fr om Am in op yr id a zin e-typ e Mu sca r in ic M1 Agon ists
Yveline Rival, Re´my Hoffmann,† Bruno Didier, Volodymyr Rybaltchenko,‡,§ J ean-J acques Bourguignon, and
Camille G. Wermuth*
Laboratoire de Pharmacochimie Mole´culaire (UPR 421 du CNRS), Universite´ Louis Pasteur, 74 Route du Rhin,
67401 Illkirch Cedex, France, and Institute of Bioorganic Chemistry of National Ukrainian Academy of Sciences,
252660, 1, Murmanska, Kyı¨v-94, Ukraine
Received August 12, 1997X
A conformational analysis, performed on muscarinic M1 agonists, identified four structural
features characteristic of the muscarinic M1 pharmacophore: (i) a protonable basic or quaternary
nitrogen acting as a cationic head; (ii) an electronegative dipole usually part of a planar
mesomeric ester, amide, or amidine function which can be replaced by an ether (muscarine) or
a dioxolane (AF 30); (iii) an intercharge distance of 5 ( 0.5 Å between the cationic head and
the electronegative atom of the dipole; (iv) an elevation of 0.5 ( 0.03 Å of the cationic head
over the plane containing the electronegative dipole. During a reinvestigation of the
conformational behavior of published structures of 5-HT3 antagonists, similar features were
observed for the 5-HT3 pharmacophore. However many 5-HT3 antagonists possess additional
aromatic planes not present in the muscarinic M1 agonists. These observations brought us to
predict the chemical modifications that would change muscarinic M1 agonists into 5-HT3
antagonists. Four of the predicted aminopyridazines were actually synthesized and submitted
to testing. The observed IC50 values for 5-HT3 receptor binding ([3H] BRL 43694) ranged from
10 to 425 nM, whereas the affinities for the muscarinic receptor preparations ([3H] pirenzepine)
layed over 10 000 nM. In electrophysiological studies the two most active compounds 10 and13
produced antagonist-like effects on the 5-HT receptor channel complexes responsible for the
generation of the rapidly desensitizing ionic currents, and agonist-like effects on those
responsible for the slowly desensitizing components.
During the search for cholinergic agonists suitable for
a symptomatic treatment of Alzheimer’s disease, we
prepared some years ago a series of 3-aminopyridazines.
One of the compounds prepared, the 3-((2-(diethyl-
amino)-2-methyl-1-propyl)amino)-5-propyl-6-phenyl-
pyridazine sesquifumarate (SR 46559A, 1) was selected
for further development.1,2 Presently it undergoes clini-
cal trials as muscarinic M1 agonist devoid of the cho-
linergic syndrome.3,4
Molecular modeling studies performed on 3-aminopy-
ridazine-derived muscarinic M1 agonists2,5 showed some
striking similarities between the muscarinic M1 agonist
and 5-HT3 antagonist pharmacophore models. This
analogy prompted us to use the 3-aminopyridazine core
also for the design of 5-HT3 receptor antagonists.
basic or quaternary nitrogen acting as a cationic head,
this cationic head is preferably surrounded by a lipo-
philic environment as found in amines such as piperi-
dine, diethylamine, N-ethylpyrrolidine, quinuclidine,
nortropane. (ii) An electronegative dipole usually part
of a planar mesomeric ester, amide or amidine function.
However, it can be replaced by an ether function
(muscarine) or a dioxolane (AF30). (iii) An intercharge
distance of 5 ( 0.5 Å between the cationic head and the
electronegative end of the dipole. (iv) An elevation of
0.5 ( 0.03 Å of the cationic head over the plane
containing the electronegative dipole.
Compound 1 and the related 3-aminopyridazines have
selective affinities for hippocampal M1 receptors.1-3 In
these compounds the protonated basic nitrogen can
mimic the quaternary ammonium of acetylcholine. In
assuming that the amidine function in the 3-amino-
pyridazine ring, unprotonated at pH 7.4,7 can function
as a bioisostere of the acetylcholine ester function in, it
becomes plausible that 1 can be recognized by the
muscarinic M1 receptors as an acetylcholine mimic
(Figure 2). Earlier studies from our laboratory have
already made a very successful use of the amidine
function in the 3-aminopyridazine ring as a bioisostere
of the amidinic function found in sulpiride-derived
dopamine antagonists.8
P h a r m a cop h or e of M1 Recep tor Agon ists
Starting from a set of 12 published muscarinic M1
agonists, the so-called “active analogue approach”6 led
us to a pharmacophore model for muscarinic M1 ago-
nists.2,5 The characteristics of this model can be sum-
marized as follows (Figure 1, left side): (i) A protonable
* Address correspondence to: Prof C. G. Wermuth, Laboratoire de
Pharmacochimie Mole´culaire, Faculte´ de Pharmacie, 74 Route du Rhin,
67401 Illkirch Cedex, France. Tel: +33 388 67 37 22. Fax: +33 388
67 47 94. E-mail: wermuth@pharma.u-strasbg.fr.
† Present address: Molecular Simulation, Parc Club Orsay Univer-
site´, 20, rue J ean Rostand, 91893 Orsay-Cedex, France.
‡ Present address: Institut de Physiologie (URA 1446 CNRS),
Universite´ Louis Pasteur, 21, rue Rene´ Descartes, 67084 Strasbourg,
France.
An ta gon ists of th e 5-HT3 Recep tor :
Con for m a tion a l Stu d y
In a previous paper, Hibert et al. characterized the
geometric elements accounting for 5-HT3 antagonism:9
§ Ukraine.
X Abstract published in Advance ACS Abstracts, December 15, 1997.
S0022-2623(97)00541-4 CCC: $15.00 © 1998 American Chemical Society
Published on Web 01/07/1998

312 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 3
Rival et al.
F igu r e 1. Pharmacophore model for muscarinic M1 agonists applied to acetylcholine (left) and for serotoninergic 5-HT3 antagonists
applied to quipazine (right). Plane P contains the x and z axes and the OdC-O or the NdC-N functions.
Ch a r t 1. Structure and Affinities of 5-HT3 Antagonistsa
F igu r e 2. Bioisosterism between the side chain of compound
1 and acetylcholine.
(i) a basic amine, a carbonyl dipole (or an isosteric
equivalent), and an aromatic plane coplanar to the
carbonyl dipole; (ii) an intercharge distance of 5Å
between the cationic head and the negative end of the
carbonyl dipole; (iii) an elevation of 1.7 Å of the cationic
head over the aromatic plane. However, a major
drawback of this model is that, to locate the basic
nitrogen at 1.7 Å above the quinoline plane, an axial
attachment of the quinoline to the piperazine ring is
implied. An equatorial conformation would certainly be
more stable and actually is found in the crystal struc-
ture of other heterocyclic amidines related to qui-
pazine.10 For this latter reason we reinvestigated the
conformational behavior of a set of known 5-HT3 an-
tagonists (Chart 1), using systematic search. In the
present study, we focused only on low-energy conformers
(5 kcal/mol energy window above the computed global
minimum). The results of this conformational analysis
are shown in Table 1.
a
The biological data are taken from refs
9 and 11. (a)
In the conformational model of quipazine, there is a
conformer presenting the geometrical characteristics
found for muscarinic M1 agonists (d ) 4.8 Å and h )
0.48 Å) (Table 2). For each other compound, we looked
for the existence of a conformer presenting similar
geometrical constraints. These conformers were used
and mapped rigidly on the “proposed bioactive confor-
mation” found for quipazine (Figure 1, right side). Some
typical superpositions are shown in Figure 3B.
[3H]GR65630 in cerebral cortex; (b) potency on rabbit heart
serotonergic receptors; (c) [3H]ICS205930 in cerebral cortex.
“northwestern” part of the molecule whereas the sub-
stituted phenyl group of metoclopramide 7 (magenta)
is rather located in the “northeastern” part. These
additional space occupations constitute possible exten-
sions of the initial aromatic area found for quipazine.
It appears clearly that all of the compounds share a
common recognition part represented by the tertiary
amine, the dipole, and the aromatic system. However,
when compared to the aromatic system found in qui-
pazine, the aromatic rings of the other 5-HT3 antago-
nists occupy different positions in space. Thus, when
compared to quipazine (Figure 3A), the indolic benzo
group of indolyl carboxylate 4 (green) occupies to the
3-Am in op yr id a zin es a s 5-HT3 An ta gon ists
The comparison of the muscarinic M1 agonist phar-
macophore model with the one for 5-HT3 antagonists
guided us in the search for new potentially active 5-HT3
antagonists. Thus, taking into account the previously
defined geometrical elements, the 3-aminopyridazine 1
can be superimposed to quipazine in its serotononergic

5-HT
Antagonists
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 3 313
3
Ta ble 1. Conformational Analysis of Eight Representative
5-HT3 Antagonists
Sch em e 1. Synthesis of the
3-(4′-Methylpiperazin-1-yl)pyridazines
conformational analysis
selected active
conformations
no. of
elevation conform-
distance
compd rangea (Å) rangeb (Å)
d
h
∆E
ations
(Å)a (Å)b (kcal/mol)
2
3
4
5
6
7
8
9c
4.7-5.2 -0.9 to 0.9
5.3-5.8 0.0-2.5
184
701
580
554
625
3033
224
856
4.8 0.48
5.7 0.49
5.3 0.49
5.3 0.50
5.3 0.50
5.2 0.47
4.8 0.48
4.8 0.47
0.33
2.45
4.17
4.63
4.00
1.79
0.86
1.74
5.2-5.9 -2.2 to 3.2
5.1-5.8 -3.0 to 3.0
5.0-5.7 -2.9 to 2.9
2.9-5.3 -2.8 to 2.8
4.0-4.8 -2.0 to 2.0
2.9-5.3 -3.2 to 1.2
a
Distance between the cationic head and the negative end of
b
the dipole. Elevation of the cationic head over the plane contain-
ing the dipole. c In ondansetron 9, the cationic head is represented
by a delocalized imidazolic system, therefore the centroid of
NdC-N was used to map the cationic head.
“proposed bioactive conformation” as described above
(Figure 3B).
These findings suggest that minor structural changes
applied to aminopyridazines such as compound 1 should
turn them into 5-HT3 antagonists. Particularly, com-
pounds 10-13 (Figure 4) can be expected to be valuable
candidates for the switch from muscarinic M1 agonists
to 5-HT3 antagonists.
They all have a relatively rigid and tertiary N-
methylpiperazine side chain. According to the mapping
of structurally diverse 5-HT3 antagonists shown in
Figure 3A, the different substitution patterns of the
pyridazinic nucleus by an aromatic ring intend to
explore the size of the aromatic area (Figure 3C).
13 show highly preferential affinities for 5-HT3 receptor
preparations. The factor which seems to be responsible
for the 5-HT3 preference, and which is common to the
four compounds, is the relatively rigid tertiary N-
methylpiperazinyl side chain. The lesser affinity of the
6-substituted pyridazines 11 and 12 suggests that a
phenyl ring attached to position 6 of the pyridazine ring
is rather detrimental. Concerning the agonist or an-
tagonist profile toward the 5-HT3 receptor preparations,
the Hill number, close to 1 for all four compounds,
suggests an antagonistic profile.
To confirm the antagonistic profile, and in order to
establish the possible physiological properties of these
molecules, a series of experiments on the dorsal root
ganglion (DRG) neurons was then undertaken on the
two most active compounds, 13 and 10. DRG neurons
express 5-HT3 type of receptors on their membranes
that are directly coupled with cationic channels.16 This
enables the ionic currents through the membrane,
evoked by the rapid application of 5-HT, to be registered.
Ch em istr y
The general procedure for the preparation of target
compounds is shown in Scheme 1. Reaction of 3(2H)-
pyridazinones 14, 15 and 1(2H)-phthalazinones 16, 17
with phosphorus oxychloride afforded the 3-chloro-
pyridazines 18, 19 and the 1-chlorophthalazines 20, 21
respectively.
These compounds were then treated with N-meth-
ylpiperazine to yield the expected 3-(4′-methylpiperazin-
1-yl)pyridazines or phthalazines 10-13 (Table 2). The
starting 3(2H)-pyridazinones 14 and 15 or 1(2H)-
phthalazinones 16 and 17 were prepared in condensing
the appropriate γ-oxo carboxylic acid with hydrazine
hydrate according to the literature procedures.12-15
The population of DRG neurons is known to be
heterogeneous, and not all neurons are 5-HT sensitive.16
Besides, it is presently considered that 5-HT3 receptors
may be coupled with different cationic channels at
different types of neurons.17 An example is the biphasic
(vs monophasic) rate of decay of currents resulting from
the desensitization of receptor channel complexes during
the sustained application of high concentrations of
5-HT.17-19 In our experiments, 18 out of 26 tested
neurons (69%) were 5-HT sensitive (5-HT: 50 µM).
Among them, seven neurons revealed slowly desensitiz-
ing currents, nine neurons a biphasic desensitization
profile in which the rapidly decaying component was
followed by a slow component, and two neurons pos-
Resu lts a n d Discu ssion
The results from the radioligand binding assays at
the 5-HT3, M1, and M2 receptors are given in Table 2.
For the four compounds 10-13, the observed IC50
5-HT3 receptor binding values ([3H]granisetron) range
from 10 to 425 nM, whereas the affinities for the
muscarinic receptor preparations([3H]pirenzepine) lay
over 10 000 nM. Thus, as predicted, compounds 10-
Ta ble 2. Binding Affinities for the 5-HT3, the M1, and the M2 Receptors of the 3-(4′-Methylpiperazin-1-yl)pyridazine and
1-(4′-Methylpiperazin-1-yl)phthalazine Hydrochlorides
a
d
compd no.
formula
5-HT3 IC50, nM (nH)b
M1c Ki, nM
M2 Ki, nM
10
11
12
13
C
C
C
C
15H18N4‚2HCl‚0.5H2O
15H18N4‚2HCl‚1.5H2O
19H20N4‚2HCl‚0.5H2O
13H16N4‚HCl
36 ( 12 (1.35)
425 ( 87 (0.97)
370 ( 73 (0.91)
10 ( 3 (1.29)
62000 ( 28000
15600 ( 970
13000 ( 240
10000 ( 3000
80000 ( 9000
71000 ( 7300
116000 ( 10780
38000 ( 3200
a
b
d
5-HT3 receptor ligand: [3H]granisetron. Hill number. c M1 receptor ligand: [3H]pirenzepine. M2 receptor ligand: [3H]NMS.

314 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 3
Rival et al.
F igu r e 3. Molecular modeling. All molecules were superimposed (FIT program of the SYBYL software) using the N-methyl
nitrogen atom of the piperazine ring and the two oxygen atoms of the ester function (or the corresponding atoms of the ester
bioisoteres) as fitting points. The molecular volumes are displayed as Conolly surfaces (MOLCAD). (A) Overlay of compounds 4,
7, 8, 9, and quipazine (white). The aromatic extensions are coded as follows: 4 (green), 7 (magenta), 8 (yellow), 9 (red). (B) Overlay
of quipazine (white), a 5-HT3 antagonist, and the 3-aminopyridazine 1 (white and red), a muscarinic agonist (stereoview). (C)
Overlay of the four 3-aminopyridazines 10-13 and quipazine (white). The aromatic extensions are coded as follows: 10 (red), 11
(green), 12 (yellow), and 13 (magenta).

5-HT
Antagonists
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 3 315
3
molar potency. In electrophysiological studies, the two
most active compounds, 13 and 10, produced antagonist-
like effects on the 5-HT receptor channel complexes
responsible for the generation of the rapidly desensitiz-
ing ionic currents, and agonist-like effects on those
responsible for the slowly desensitizing components. The
present findings highlight, once again, the utility of the
3-aminopyridazines in the design of ligands of the
biogenic amine receptors.
Exp er im en ta l Section
Ch em istr y. Melting points were determined with a Mettler
FP62 apparatus and are uncorrected. All 1H NMR spectra
were recorded on a Bruker AC 200 instrument (200 MHz), and
chemical shifts are reported in parts per million (δ) relative
to Me4Si for CDCl3 and Me2SO-d6 solutions (DMSO-d6).
Signals are quoted as s (singlet), d (doublet), t (triplet), q
(quartet), or m (multiplet). Flash chromatography was carried
out on silica gel (70-230 mesh ASTM). Elemental analyses
are indicated only by the symbols of the elements; analytical
results were within (0.4% of the theoretical values.
I. P yr id a zin on es a n d P h th a la zin on es. The starting
pyridazinones are already known and were prepared by
literature procedures.
F igu r e 4. 3-Aminopyridazines predicted to be active as 5-HT3
antagonists.
sessed only the rapidly desensitizing component of the
current. At all 5-HT sensitive neurons, both the ph-
thalazine 13 and the 5-phenyl analogue 10 produced
clear pharmacological effects. In 10 µM concentration,
both compounds mimicked the slowly desensitizing
component of the current at the neurons at which this
component could be evoked by 5-HT. In these cases
coapplication of 5-HT (50 µM) during a sustained
application of the phthalazine 13 and the 5-phenyl
analogue 10 produced only negligible effects. At the
neurons at which 5-HT evoked a biphasic character of
receptor desensitization, both substances evoked only
the slowly desensitizing component of current and
blocked the rapidly desensitizing component of the
current when 5-HT was coapplied. At the neurons at
which an application of 5-HT (50 µM) evoked only the
rapidly desensitized component of current, both of the
ligands we tested (10 or 50 µM) did not produce
appreciable currents and blocked them when simulta-
neously coapplied with 5-HT (50 mM). This kind of
response was registered only at the small DRG neurons
which are supposed to be C-type nociceptive neurons.
Summarizing the electrophysiological observations, it
can be concluded that the phthalazine 13 and the
5-phenyl analogue 10 are effective modulators of the
5-HT3 receptor functioning. They produce antagonist-
like effects on the 5-HT receptor channel complexes
responsible for the generation of the rapidly desensitiz-
ing ionic currents and agonist-like effects on those
responsible for the slowly desensitizing components. A
similar duality of pharmacological properties has re-
cently been reported even for quipazine, which is
classically referred to as a 5-HT3 receptor blocker, and
which is able to activate receptors in NG108-15 cells.19
The physiological role of the rapidly and slowly desen-
sitizing components of the neuronal responses produced
by application of 5-HT is presently not clear. Of
particular importance is the ability of phthalazine 13
and 5-phenyl analogue 10 to produce opposite effects
on the two components of 5-HT-evoked currents. This
property may be used for the discrimination between
different types of 5-HT3 receptor channel membrane
complexes, as well as for a more detailed classification
of DRG neurons that express 5-HT3 receptor complexes
on their membranes.
5-P h en yl-3(2H)-p yr id a zin on e (14): yield 78% (EtOH);
1
mp 193 °C (lit.12 mp 194 °C); H NMR (CDCl3) δ 8.13 (1H, s),
7.58-7.54 (5H, m), 7.11 (1H, s).
6-P h en yl-3(2H)-p yr id a zin on e (15): yield 81% (EtOH); mp
201.5 °C (lit.14 mp 202 °C).
4-P h en yl-1(2H)-p h th a la zin on e (16): yield 90% (i-PrOH);
mp 241.5 °C (lit.13 mp 236-237 °C); 1H NMR (CDCl3) δ 8.58-
8.52 (1H, m), 7.88-7.76 (4H, m), 7.63-7.52 (5H, m).
1(2H)-P h th a la zin on e (17): yield 68% (EtOH); mp 185 °C
(lit.15 mp 183-184 °C); 1H NMR (CDCl3) δ 8.47-8.42 (1H, m),
8.18 (1H, s), 7.91-7.72 (3H, m), 1.64 (1H, s).
II. 3-Ch lor op yr id a zin es a n d 1-Ch lor op h th a la zin es.
The appropriate substituted 3(2H)-pyridazinone or 1(2H)-
phthalazinone was treated with an excess of phosphorus
oxychloride (10 mL for 1 g of compound). The mixture was
heated at 85 ( 3 °C for 4 h, then carefully poured onto ice
(200 g for 1 g of compound), and finally rendered alkaline with
20% NaOH. The crude 3-chloropyridazine or 1-chlorophthala-
zine was collected by filtration, washed with water, and dried
under vacuum. It was purified by crystallization in ethanol
or 2-propanol or by chromatography on silica gel using a
mixture of hexane-ethyl acetate (1:1).
3-Ch lor o-5-p h en ylp yr id a zin e (18): 61%, yellow crystal-
line powder (EtOH); mp 181 °C; 1H NMR (CDCl3) δ 9.385 (1H,
J ) 2 Hz, d), 7.71-7.64 (3H, m), 7.59-7.55 (3H, m).
3-Ch lor o-6-p h en ylp yr id a zin e (19): 82%, white crystals
1
(EtOH); mp 159 °C (lit.13 mp 158 -160 °C); H NMR (CDCl3)
δ 8.08-8.03 (2H, m), 7.84 (1H, J ) 10 Hz, d), 7.73-7.52 (4H,
m).
1-Ch lor o-4-p h en ylp h th a la zin e (20): 79%, white crystals
(EtOH); mp 158 °C (lit.14 mp 160-161 °C); 1H NMR (CDCl3) δ
8.41 (1H, J ) 8 Hz, d), 8.12-7.71 (5H, m), 7.60-7.55 (3H, m).
1-Ch lor op h th a la zin e (21): 28%, yellowish crystals (i-
PrOH); mp 117 °C (lit.20 mp 119-120 °C); 1H NMR (CDCl3) δ
8.35-8.29 (1H, m), 8.06-7.99 (4H, m).
IV. Su bstitu ted 3-(4-Meth ylp ip er a zin -1-yl)p yr id a zin e
a n d 1-(4-Meth ylp ip er a zin -1-yl)p h th a la zin e Sa lts. The
N-methylpiperazinyl derivatives 10, 11, 12, and 13 were
prepared from the corresponding 3-chloropyridazines 18, 19
and 1-chlorophthalazines 20, 21.
Gen er a l P r oced u r e. To a solution of the appropriate
3-chloropyridazine (5 × 10-3 mol) in 1-butanol (30 mL) was
added 8 × 10-3 mol of N-methylpiperazine and 5 × 10-3 mol
of ammonium chloride. The reaction mixture was refluxed for
48 h. The solvent was removed under reduced pressure, and
the residue was diluted in 100 mL of water. After alkaliniza-
tion with solid K2CO3, the mixture was extracted with ethyl
acetate (EtOAc) The combined organic layers were treated with
a 10% solution of citric acid in water in order to recover the
Taken together our results confirm a certain degree
of relationship between the 5-HT3 and the M1 pharma-
cophores. They demonstrate the easy possibility, in the
3-aminopyridazine series, to switch from muscarinic M1
agonists to serotoninergic 5-HT3 antagonists of nano-

316 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 3
Rival et al.
obtained base. The separated aqueous phase was then ren-
dered alkaline with K2CO3 and extracted with EtOAc. After
drying over Na2SO4 and evaporation of the solvent, the
obtained crude free base was purified by chromatography on
silica gel using a mixture of ethyl acetate-triethylamine, 98:2
(v/v).
F or m a tion of th e Hyd r och lor id e Sa lts. To the free base
(0.01 mol) dissolved in 10 mL of i-PrOH was added 2 mL (0.024
mol) of 37% HCl. The hydrochloride salt usually crystallizes
on standing, eventually after addition of anhydrous ether. The
collected solids were recrystallized in absolute EtOH or
i-PrOH.
3-(4-Meth ylp ip er a zin -1-yl)-5-p h en ylp yr id a zin e Dih y-
d r och lor id e (10). The free base was purified by chromatog-
raphy on silica gel and elution with a 8:2:2 (v/v) mixture of
ethyl acetate-methanol and triethylamine: yield 22% of
yellow needles; 1H NMR (CDCl3) δ 8.875 (1H, s), 7.65-7.49
(5H, m), 7.02 (1H, s), 3.875 (4H, J ) 6 Hz, t), 2.73 (4H, J ) 6
Hz, t), 2.48 (3H, s). Dihydrochloride (EtOH): 70%; mp 285
°C.
3-(4-Meth ylp ip er a zin -1-yl)-6-p h en ylp yr id a zin e Dih y-
d r och lor id e (11). The free base (89%) was recrystallized in
i-PrOH: 1H NMR (CDCl3) δ 8.02-7.39 (6H, m), 6.98 (1H, J )
10 Hz, d), 3.74 (4H, J ) 6 Hz, t), 2.37 (3H, s). Dihydrochloride
(EtOH): 40%; mp 250 °C.
by means of a 2 h soaking in a 0.3% polyethylenimine solution.
The filters were then washed two times with room-tempera-
ture HEPES buffer (7.5 mL), and the radioactivity on the
filters was counted in a liquid scintillation counter (Tri-Carb
2200 CA; Packard Co., Ltd.). The specific binding was
determined by subtracting the nonspecific binding (in the
presence of 100 nM zacopride) from the total binding persisting
on the filters measured by liquid scintillation counting.
Mu sca r in ic Recep tor Bin d in g Assa ys. The cortical M1
receptor was identified with the binding of [3H]pirenzepine in
homogenates. Adult rat hippocampus was dissected on an ice-
cold block and homogenized (1:200, w/v) with a polytron in 50
mM sodium-potassium phosphate buffer (pH ) 7.4), EDTA
(1 mM), and 0.1 mM PMSF (phenylmethanesulfonyl fluoride).
Competition between [3H]pirenzepine (0.7-1 nM) and the
unlabeled test compounds was measured in an assay volume
of 1 mL of this buffer with 0.5 mg of tissue; after 2 h 30 min
at 25°C, the bound ligand was separated from free ligand with
filtration over 0.3% polyethylenimine-treated Whatman GF/C
filters. Atropine (10 mM) was used to determine the level of
nonspecific binding.
The brainstem M2 receptor was studied with [3H]NMS
binding. Adult rat cardiac cells were dissected and homog-
enized (1:200, ww/v) with
a polytron in 50 mM sodium
phosphate buffer, pH 7.4. The competition essay was con-
ducted with 0.2 nM [3H]NMS, 0.5 mg of tissue, and various
concentrations of unlabeled ligand in an essay volume of 1 mL
at 37 °C for 1 h. The bound [3H]NMS was separated from free
by filtration over Whatman GF/C filters. Atropine (10 mM)
was used to determine the level of nonspecific binding.
1-(4-Meth ylp ip er a zin -1-yl)-4-p h en ylp h th a la zin e Dih y-
d r och lor id e (12). The free base (61%) was purified by
chromatography on silica gel and elution with a 8:2:2 (v/v)
mixture of ethyl acetate-methanol and triethylamine: 1H
NMR (CDCl3) δ 8.07 (2H, J ) 7 Hz, d), 7.87-7.72 (3H, m),
7.60-7.52 (3H, m), 3.65 (4H, J ) 5 Hz, t), 2.75 (4H, J ) 5 Hz,
t), 2.44 (3H, s). Dihydrochloride (EtOH): 90%; mp 255 °C.
1-(4-Meth ylp ip er a zin -1-yl)p h th a la zin e Hyd r och lor id e
(13). The free base (46%) was purified by chromatography
on silica gel and elution with a 8:2:2 (v/v) mixture of ethyl
acetate-methanol and triethylamine: 1H NMR (CDCl3) δ
9.145 (1H, J ) 2 Hz, d), 8.06-8.00 (1H, m), 7.90-7.76 (3H,
m), 3.54 (4H, J ) 5 Hz, t), 2.685 (4H, J ) 5 Hz, t), 2.44 (3H,
s). Hydrochloride (i-PrOH): 80%; needles; mp 150 °C.
Com p u ter Gr a p h ics Stu d y. The study has been per-
formed using the SYBYL 5.4 package21 running on a Vax
Station 2000/E&S PS390 configuration. All the molecules
were built by assembling standard fragments, and the result-
ing geometries were optimized by molecular mechanics (Tripos
Force Field). Systematic conformational analysis was per-
formed by rotating each rotatable bond with increments
ranging from 1 to 15° depending upon the flexibility of the
molecules (SEARCH option of SYBYL). Detailed descriptions
of the pharmacophore construction are given in the previous
papers.2,5,9
Biology. 5-HT3 Recep tor Bin d in g Test. The radioligand
binding studies were performed according to a slightly modi-
fied version of the technique described by Nelson and Tho-
mas.22 Cerebral cortical tissue was obtained from male
Sprague-Dawley rats (Charles River, France) and homog-
enized (Polytron, 30 s at speed 7) in 10 volumes of cold 50 mM
HEPES buffer (pH ) 7.5). The homogenate was centrifugated
(10 min at 48000g, at 4 °C). The obtained pellet was washed
three times by resuspending it in 10 volumes of HEPES buffer
(Vortex) and centrifugating again.The final pellet was sus-
pended in 20 mL of the previous HEPES buffer so as to obtain
about 2.5 mg of protein/mL and stored on ice until required.
The binding experiments were performed using [3H]gran-
isetron ([3H]BRL 43694),23 a high-affinity ligand for the 5-HT3
receptors. In a series of 16 × 100 mm polypropylene tubes
aliquots of 0.4 mL of the membrane suspension were incubated
during 45 min at 25 °C, in the presence of 0.3 nM of [3H]BRL
43694 (NEN, specific activity 61 Ci/mmol) and increasing
concentrations of the test compounds, the final volume being
adjusted to 2 mL with the HEPES buffer. For the measure-
ment of the nonspecific binding, the incubations were per-
formed in the presence of 100 nM of zacopride. The incubated
suspensions were then individually filtered on Whatman GF/B
filters (diameter: 2.5 cm) which were previously impregnated
E lect r op h ysiologica l In vest iga t ion . The experiments
were carried out on the freshly isolated, dissociated, and kept
in culture (2-10 h) DRG neurons from rats (10-14 days old).
The procedures of isolation of neurons24 and electrophysiologi-
cal recording25 (patch-clamp, whole-cell configuration) were
standard. Recordings of the ionic currents of neurons were
performed in the extracellular low-calcium solution, containing
(mM) the following: NaCl (140), KCl (3), CaCl2 (0.1), MgCl2
(3), HEPES (8), glucose (10). Neurons were perfused with the
intracellular solution, containing (mM) the following: KCI
(140), CaCl2 (1), EGTA (10), HEPES (10), MgATP (1). The
membrane potential of voltage-clamped neurons was held at
-80 mV.
The modified method for rapid application of substances on
the neuron26 was used, being necessary because of the ex-
tremely rapid time rate of activation and subsequent desen-
sitization of receptors (200 ms range). Ionic currents activated
by 5-HT and tested substances reached maximal amplitude
within 35-50 ms for the rapidly desensitizing and 200-300
ms for the slowly desensitizing currents. Blocking effects of
phthalazine 17 and 5-phenyl analogue 14 on the rapidly
decaying 5-HT-activated currents were completely reversible
after washout of the drugs.
Ack n ow led gm en t. This study was supported by the
postdoctoral fellowship for V. Rybaltchenko from the
Fondation pour la Recherche Me´dicale (France). We
thank Dr. Philippe Soubrie´ (Sanofi Recherche, Mont-
pellier) for the 5-HT3 receptor binding assays and Dr.
J . P. Gies (Louis Pasteur University, Strasbourg) for the
muscarinic receptor binding assays.
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