The search for TCP analogues binding to the low affinity PCP receptor sites in the rat cerebellum

Article abstract of DOI:10.1016/S0223-5234(99)80046-4

With the aim of obtaining selective ligands of the low affinity binding sites of [³H]-1-[1-(2-thienyl)cyclohexyl]piperidine ([³H]TCP) in the rat cerebellum, oxygen and sulfur atoms were introduced in the TCP structure and derivatives to obtain analogues with a lowered lipophilicity. These compounds, and others already obtained, were assayed comparatively to determine their affinities for three sites labeled with [³H]TCP: one in the forebrain, the originally described PCP receptor, and two in the rat cerebellum. Lowering the lipophilicity and modifying the hetero-aromatic moiety yielded some ligands with increased affinity for the low affinity sites in the rat cerebellum and decreased affinity for the high affinity sites in the forebrain. Particularly, two compounds displaying both a high affinity and a good selectivity might be valuable tools to elucidate the pharmacology of the low affinity PCP sites labeled with [³H]TCP in the rat cerebellum.

Full text of DOI:10.1016/S0223-5234(99)80046-4

Eur. J. Med. Chem. 34 (1999) 125−135
© Elsevier, Paris
125
Original article
The search for TCP analogues binding to the low affinity PCP receptor sites
in the rat cerebellum
Jacques Hamona, Florence Espazea, Jacques Vignonb, Jean-Marc Kamenkaa*
aCRBM, CNRS – UPR 1086, Ecole Nationale Supérieure de Chimie, 8, rue de l’Ecole Normale, 34296 Montpellier cedex 5, France
bINSERM U 336, Ecole Nationale Supérieure de Chimie, 8, rue de l’Ecole Normale, 34296 Montpellier cedex 5, France
(Received 18 December 1997; revised 25 September 1998; accepted 8 October 1998)
Abstract – With the aim of obtaining selective ligands of the low affinity binding sites of [3H]-1-[1-(2-thienyl)cyclohexyl]piperidine
([3H]TCP) in the rat cerebellum, oxygen and sulfur atoms were introduced in the TCP structure and derivatives to obtain analogues with a
lowered lipophilicity. These compounds, and others already obtained, were assayed comparatively to determine their affinities for three sites
labeled with [3H]TCP: one in the forebrain, the originally described PCP receptor, and two in the rat cerebellum. Lowering the lipophilicity
and modifying the hetero-aromatic moiety yielded some ligands with increased affinity for the low affinity sites in the rat cerebellum and
decreased affinity for the high affinity sites in the forebrain. Particularly, two compounds displaying both a high affinity and a good selectivity
might be valuable tools to elucidate the pharmacology of the low affinity PCP sites labeled with [3H]TCP in the rat cerebellum. © Elsevier,
Paris
TCP / TCP analogues / PCP receptor subsites / rat cerebellum
1. Introduction
potently to the low affinity sites might be crucial for their
pharmacological characterization. For clarity, the differ-
ent binding sites discussed here will be marked PCP1 (the
[3H]TCP high affinity binding sites in the forebrain, i.e.,
the PCP receptor within the NMDA receptor associated
ionic channel), PCP2 (the [3H]TCP high affinity binding
sites in the cerebellum), and PCP3 ([3H]TCP low affinity
binding sites in the cerebellum).
We have previously introduced oxygen and sulfur
atoms in the cyclohexyl and piperidinyl moieties of the
TCP structure to obtain analogues with a lowered lipo-
philicity. In rat forebrain membranes, the affinity of these
analogues for the PCP1 sites labeled with [3H]TCP was
decreased as a function of lipophilicity: the lower lipo-
philicity was, the lower affinity was [7]. However, their
affinity for PCP2 and PCP3 sites in rat cerebellum
membranes was not studied. The possibility that lowering
the lipophilicity decreased the affinity for PCP1 in the
forebrain while increasing the affinity for PCP3 in the
cerebellum was attractive since the lipophilic TCP and
MK-801 (log P = 4.56 ± 0.30 and 3.71 ± 0.39 respec-
tively) displayed very low affinities for PCP3 (table I).
Thus, we have decided to investigate this hypothesis by:
[3H]-5-methyl-10,11-dihydro-5H-dibenzo[a, d]cyclo-
hepten-5,10-imine ([3H]MK-801) and [3H]-1-[1-(2-
thienyl)cyclohexyl]piperidine ([3H]TCP) are the most
frequently used ligands for the labeling of the non-
competitive antagonists binding site within the N-methyl-
D-aspartate (NMDA) receptor associated Ca2+ channel
(the PCP receptor) [1, 2]. However, competition data
analysis using a two-site model revealed that both ligands
labeled at least two different binding sites in the rat brain:
(i) high affinity sites, well represented in the forebrain
and corresponding to the initially discovered PCP recep-
tor and (ii) lower affinity sites, more abundant in the
hindbrain and particularly in the cerebellum [3–8]. The
nature and the role of the low affinity binding sites are
poorly documented mostly because selective ligands are
not available. Particularly, their possible role in neuronal
protection is unknown whereas the high affinity sites is a
well-established target for neuroprotective agents [6].
Thus, the finding of ligands binding selectively and
*Correspondence and reprints

126
Table I. Inhibition of [3H]TCP binding in rat forebrain and cerebellum membranes by TCP and MK-801. The mean of at least three
independent determinations was analyzed according to a single-site model in the forebrain and cerebellum (IC50 a, nM, Hill’s number) or a
two-site model in the cerebellum. The two-site model was statistically more probable in the cerebellum: the proportion of PCP2 and PCP3 sites
(%) and affinities (IC50, nM) are given. SEM are in brackets.
Compound
Forebrain single-site model
Cerebellum single-site model
Cerebellum two-site model
IC50 (PCP2) % (PCP2)
IC50 (PCP1) nH
IC50
nH
IC50 (PCP3) % (PCP3)
b
b
TCP
MK-801
9.3
3.67 (0.65)
1.00
0.95 (0.04)
188 (63)
995 (458)
0.56 (0.05)
0.33 (0.04)
59 (17)
9.3 (4.6)
72.6 (3.6)
45.3 (4.9)
3716 (1195) 29.8 (4.5)
11125 (3179) 57.2 (5.8)
a
b
IC50: concentration of unlabeled drug that inhibited 50% of specific [3H]TCP binding on specified sites; from [20].
(i) preparing TCP analogues with varied lipophilicities by
means of O, S or hydroxyl substitution; (ii) using these
new TCP analogues and some of those previously ob-
tained for competition measurements in rat forebrain and
cerebellum membranes labeled with [3H]TCP; (iii) deter-
mining the more probable model of interaction (single- or
two-site) in the cerebellum and comparing the affinities of
compounds for PCP1, PCP2, and PCP3.
indeed, the hydrochlorides solutions are stabilized in
almost homogeneous conformations: the aromatic or
hetero-aromatic rings are essentially restrained to the
axial position. Consequently, the conformational trapping
induced by the protonation allows for structural compari-
sons (see below) between similar (axial aromatic rings)
conformations [16–19].
Diastereomers 16 and 17, bearing a methyl substitution
at the hetero-cyclohexyl ring (table IV), were prepared
using the azide synthesis shown in figure 2 [15, 20]. The
cis/trans configurations were attributed from the 13C-
NMR spectra of their HCl salts. The chemical shifts were
attributed by comparison with the spectra of GK-11 and
GK-12 hydrochlorides, two analogues whose diastereo-
meric configurations have been previously character-
ized [20, 21]. The cis (Me/Pip) configuration was attrib-
uted to compound 16 where the specific γ-interaction due
to the axial methyl substitution causes a clear upfield shift
of carbon 5 (table IV).
2. Chemistry
Two different synthetic strategies were used to obtain
TCP analogues according to the presence or absence of a
methyl substitution in the cyclohexyl or hetero-
cyclohexyl moiety (X, Y-substituted ring, see table II).
The unsubstituted compounds were easily obtained by
means of a Bruylants reaction [9]. It consists in the
replacement of a cyano group by an aryl- or hetero-aryl
group by the reaction of an α-aminonitrile with an aryl-
or hetero-arylmagnesium halide (figure 1). The suitable
α-aminonitrile resulted from a Strecker-like synthesis in
an organic or aqueous medium [10, 11]. When the hetero-
aryl moiety was a 2-furyl (8, 9), 4-methyl-2-thienyl (15)
or a 5-methyl-2-thienyl (14) group, the Grignard reagent
was best obtained by means of a magnesium/Lithium
exchange reaction [12, 13] between MgBr2 and the suit-
able 2-Li derivative. The pure (e.e. > 99%) enantiomers
of the 3-methyl-piperidine derivatives 10 and 11 were
obtained according to the same pathway (figure 1) but
starting from optically active 3-methyl-piperidines. The
optical resolution of the 3-methyl-piperidine racemate
was achieved by means of a crystallization procedure
with (+)- and (–)-mandelic acid in ethyl acetate resulting
in enantiomeric purities up to 98–99% [14, 15]. It should
be noticed that we have previously described 10-(–) and
10-(+) obtained by a different strategy [15] but with very
similar enantiomeric purities.
The synthesis of non-commercial ketonic or piperi-
dinic starting materials was required. Briefly, according
to reference [22], alkylation of ethyl 2-sulfanylacetate
with ethyl 4-chlorobutanoate gave a 76% yield of ethyl
4-[(2-ethoxy-2-oxoethyl)sulfanyl]butanoate which was
submitted to a Dieckmann cyclization to afford a 54%
The new compounds obtained according to figure 1
(6–9, 11–15) were all checked by 13C-NMR spectroscopy
of the hydrochloride salts (table III). In these series
Figure 1.

127
Table II. TCP derivatives and analogues unsubstituted at the cyclohexyl or heterocyclohexyl ring and their calculated log P.
a
Compound
X
Y
Z
K
R1
R2
R3
R4
log P
TCP
1
2
3
4
5
6
7
8
CH2
S
O
CH2
S
O
SO2
CH2
CH2
S
CH2
S
CH2
CH2
S
CH2
CH2
CH2
CH2
CH2
CH2
CH2
S
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
S
S
S
S
S
S
S
S
O
O
S
S
S
S
S
S
CH2
CH2
CH2
O
O
O
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
H
H
H
H
H
H
H
H
H
H
H
H
H
H
CH3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
CH3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OH
H
H
4.56 ± 0.30
3.66 ± 0.50
2.85 ± 0.39
3.02 ± 0.39
2.11 ± 0.56
1.31 ± 0.41
1.99 ± 0.40
3.52 ± 0.53
4.05 ± 0.29
3.14 ± 0.52
5.06 ± 0.30
4.15 ± 0.53
3.35 ± 0.37
3.21 ± 0.37
4.12 ± 0.53
4.12 ± 0.53
H
H
9
10
11
12
13
14
15
CH3
CH3
CH2OH
CH3
H
S
H
a
Calculated with the ACD/Log P program (ACD, Inc.) (95% confidence, octanol/water).
yield of ethyl 3-oxotetrahydro-2H-thiopyran-2-car-
boxylate. A decarboxylation in a 10% sulfuric acid
solution gave 54% of pure dihydro-2H-thiopyran-3(4H)-
one (figure 3). 3-Methyltetrahydro-4H-thiopyran-4-one
was obtained by reacting tetrahydro-4H-thiopyran-4-one
with methyl-iodide in THF in the presence of one
equivalent of LDA at the temperature of –80 °C.
3-Methyl-4-piperidinol was obtained essentially as its
a
b
Table III. 13C-NMR chemical shifts (hydrochloride in CDCl3, δ ppm from TMS).
c
Carbon
6
7
8
9
11
12
13
14
15
1
2
3
4
5
6
α
α’
â
â’
γ
R
CAr
–
22.7
–
23.4
22.5
29.8
67.6
29.8
22.5
47.0
47.0
22.3
22.3
21.7
–
–
–
22.2
23.0
33.4
64.2
33.0
23.0
49.7
46.8
36.6
25.3
24.0
69.7
22.5
30.8
32.8
69.0
32.8
30.8
50.6
45.3
35.4
32.8
70.0
15.1
–
–
48.0
30.5
66.6
30.5
48.0
47.3
47.3
22.8
22.8
21.5
–
24.5
30.2
66.8
30.2
24.5
46.7
46.7
22.0
22.0
21.3
–
24.9
33.4
68.9
33.2
24.9
52.3
46.2
28.2
30.5
22.1
18.9
24.7
32.8
68.8
32.8
24.7
46.5
46.5
22.3
22.3
21.7
14.7
25.2
33.6
69.4
33.6
25.2
47.2
47.2
22.8
22.8
21.1
15.6
33.4
68.0
32.4
26.8
47.0
47.0
25.5
25.5
23.9
–
132.9–128.4 134.6–128.6 146.6–110.7 145.2–110.7 134.5–127.9 135.8–128.0 135.1–127.5 142.9–125.8 138.9–123.9
a
b
c
For carbon atom numbering see table IV; italicized chemical shifts may be exchanged; DMSO-d6.

128
1
Table IV. Comparative 13C- (up) and H-NMR (down) chemical
shifts of GK11, GK12, and compounds 16, 17 (hydrochloride in
CDCl3, δ ppm from TMS).
Figure 3.
Carbon
GK11 (cis) GK12 (trans)
17 (trans)
16 (cis)
1
2
3
4
5
6
CH3
α
α’
â
â’
γ
2’
3’
4’
5’
17.8
30.2
35.4
72.9
26.5
22.6
15.8
48.9
46.7
22.3
22.1
22.5
137.1
127.6
127.2
130.1
22.5
30.0
36.3
74.5
30.7
22.0
17.1
48.2
47.9
21.72
21.69
21.9
136.4
126.8
126.4
130.6
–
29.8
34.7
73.3
29.6
22.7
16.8
48.6
47.2
21.9
21.6
21.2
135.8
127.3
127.0
130.3
–
3. Pharmacology
a
a
33.2
34.8
72.4
27.0
24.9
15.1
48.9
46.3
22.0
22.0
22.5
134.8
128.3
127.8
131.1
The binding assays in rat forebrain and cerebellum
membranes and the data analysis are described in experi-
mental protocols (Section 5.2). We have first checked that
a two-site model was more probable than a single-site
model to describe the competition of TCP and MK-801 in
the rat cerebellum membranes labeled with [3H]TCP
(table I). The results were consistent with those previ-
ously reported in membranes [3, 4] as well as in cultured
cerebellum cells [5]. The same treatment was applied to
21 compounds derived from the TCP structure; the results
are presented in table V.
a
a
δ-CH3
J (Hz)
1.6
6.9
1.1
6.6
1.2
6.2
1.8
6.7
4. Results and discussion
In forebrain homogenates, [3H]TCP inhibition curves
were better fitted to a single-site model (PCP1-sites)
(table V). This result was likely since Hill numbers were
most generally close to unity. However, in cerebellum
homogenates, Hill numbers were mostly lower than unity
and the inhibition curves were better fitted to a two-site
a
Italicized chemical shifts may be exchanged.
trans isomer by a 4-step synthesis starting from benza-
mide and ethyl acrylate (figure 4) [23]. The configuration
was attributed by comparison with the 13C-NMR spectra
of cis- and trans-2-methylcyclohexanol [24].
Figure 2.
Figure 4.

129
Table V. Inhibition of [3H]TCP binding in rat forebrain and cerebellum membranes. The mean of at least three independent determinations
was analyzed according to a single-site model in the forebrain and cerebellum (IC50 a, nM, Hill’s number) or a two-site model in the
cerebellum (SEM in brackets). When the two-site model was more probable, the proportion of PCP2 and PCP3 sites (%) and affinities (IC50
a, nM) are given. Very low affinities precluded the two-site computation (n.d.).
Compound
Forebrain single-site model
Cerebellum single-site model
Cerebellum two-site model
IC50 (PCP2) % (PCP2) IC50 (PCP3) % (PCP3)
IC50 (PCP1)
nH
IC50
nH
1
2
3
4
5
6
71.6 (10.5)
1223 (143)
294 (48)
8253 (320)
> 100 µM
1.05 (0.05)
0.98 (0.05)
0.77 (0.02)
0.78 (0.06)
_
178 (40)
0.68 (0.10)
0.58 (0.12)
0.69 (0.09)
0.72 (0.02)
586 (96)
5840 (1690) 80.3 (6.5)
183 (65)
n.d.
n.d.
75.2 (8.8)
8.5 (4.5)
35 (18)
2127 (1273) 33.0 (1.1)
n.d.
n.d.
n.d.
25.2 (7.2)
15.5 (5.7)
2396 (475)
1001 (408)
29700 (8022)
63800 (10400) 0.60 (0.08)
198500 (8500) 0.77 (0.22)
64.3 (3.4)
–
–
–
–
–
97700 (19066) 0.96 (0.18)
n.d.
7
8
9
73.4 (1.4)
47.8 (1.1)
133 (20)
5.5 (1.3)
5.2 (1.0)
158 (20)
132 (39)
24 (8.2)
529 (50)
28.4 (6.6)
2406 (218)
462 (88)
77.8 (9.3)
26.8 (4.5)
1.00 (0.05)
0.83 (0.06)
0.87 (0.06)
1.09 (0.07)
1.04 (0.06)
1.08 (0.09)
1.14 (0.11)
0.84 (0.03)
0.93 (0.05)
0.96 (0.09)
1.07 (0.04)
0.97 (0.07)
0.93 (0.06)
0.89 (0.01)
228 (43)
150 (78)
0.55 (0.03)
122 (38)
155 (10)
452 (53)
265 (80)
231 (71)
996 (314)
467 (158)
3362 (1013)
1170 (242)
762 (335)
206 (30)
0.65 (0.02)
0.70 (0.11)
0.55 (0.03)
0.80 (0.13)
0.62 (0.08)
0.90 (0.08)
0.69 (0.07)
0.59 (0.04)
0.85 (0.13)
0.61 (0.06)
1.02 (0.09)
0.63 (0.06)
0.62 (0.06)
0.71 (0.07)
0.60 (0.06)
659 (206)
187 (42)
77.8 (3.7)
66.3 (12.0) 5.6 (3.5)
13.8 (3.6)
8.0 (4.8)
23.4 (5.0)
30.7 (10.0)
32.3 (4.4)
1015 (262) 68.3 (2.8)
47.3 (5.2) 68.5 (9.6)
28.3 (17.1) 52.5 (10.5) 808 (47)
10-(±)
10-(+)
10-(–)
11-(±)
11-(+)
11-(–)
12
13
14
15
16
1450 (590) 33.7 (8.1)
51.0 (11.8)
b
b
–
–
–
265 (101)
69.1 (9.8)
2850 (433) 31.6 (11.1)
53.2 (15.8) 66.3 (1.1)
2076 (747) 34.6 (2.5)
b
b
–
–
–
–
2240 (330) 69.0 (4.0)
12.4 (5.7)
31.0 (5.0)
–
15.3 (6.9)
21.8 (3.8)
34.7 (9.5)
–
b
b
–
–
–
532 (20)
772 (202)
1355 (733) 62.8 (7.3)
n.d.
83 (3.5)
76.4 (2.6)
> 100 µM
5.4 (3.4)
17.6 (6.6)
n.d.
17
17233 (1417) 0.94 (0.09)
50467 (2663)
–
a
b
IC50: concentration of unlabeled drug that inhibited 50% of specific [3H]TCP binding on specified sites; a one-site model was more
probable.
model (PCP2- and PCP3-sites) which produced a signifi-
cant reduction in the sum of squares (P < 0.05, Student’s
t-test) and a Durbin–Watson coefficient between 1.5 and
2.5 (see Experimental protocols). The results presented in
table V confirmed that [3H]TCP labeled two sites in the
ligands for the PCP1-sites although their sulfur atoms (in
the six atoms ring) are located in two different positions.
Affinity for PCP2-sites could be determined for 16
compounds only (table V). Among them, 3, 11-(±), 11-
(+), 13, and 14 were unable to discriminate significantly
PCP1- and PCP2-sites since they displayed statistically
close affinities for both sites. At the contrary, the 12
remaining compounds had affinities significantly differ-
ent at PCP1- and PCP2-sites. These affinities were appar-
ently linearly correlated (P < 0.004, r = 0.76, see
figure 5). The lower affinity of these compounds for
PCP2-sites might be related to the distribution of NMDA
NR2 subunits. Indeed NR2A and NR2B subunits are highly
expressed in the forebrain while NR2C subunit is mainly
expressed in the cerebellum [25, 26]. NMDA receptors
involving NR2C subunits are less sensitive to MK-801
blockade than those comprising NR2A and/or NR2B
subunits [27]. These results are confirmed by competition
experiments with [3H]MK-801 [4, 5] and consistent with
MK-801 affinities in table I. Thus PCP1- and PCP2-sites
are likely to represent two different states of the NMDA
receptor discriminated by some of the new molecules.
cerebellum in
a 70:30 mean relative proportion
(PCP2/PCP3). Interestingly, most compounds were more
or less able to interact with these binding sites. However
compounds 4, 5, 6, and 17 displayed a very low affinity
whatever the binding sites and thus their affinity and
selectivity could not be evaluated. Compounds 10-(–),
11-(–), and 13 displayed Hill numbers close to unity and
their interaction was best described by a single-site
model.
As previously shown, the affinity for PCP1-sites was
reduced with the decrease of lipophilicity [7] in homoge-
neous groups of structures. Indeed, TCP and derivatives
displayed greater affinities for PCP1 than their thio- or
oxa-analogues: TCP had a higher affinity for the PCP1-
sites than 1 or 2 (tables I and V), similarly 10 was a
higher affinity ligand than 11 whatever the chirality is
(table V). The same behavior was revealed when compar-
ing TCP and 8. Interestingly, 1 and 7 were equipotent

130
Figure 5. Linear relationships between affinities for PCP1- and
PCP2-sites (r = 0.76, p < 0.004) for 12 compounds discrimina-
ting significantly both sites.
Figure 6. Relationships between selectivity (PCP3/PCP1) and
log P (r = 0.84, p < 0.08) for molecules differing from the TCP
model only by substitution of one heteroatom in the cyclohexyl
ring and/or in the hetero-aromatic moiety.
The affinities for the PCP3-sites could not be correlated
with those for PCP1- or PCP2-sites and they appeared
very sensitive to structure. Indeed, molecules tested
appeared grossly separated into two groups: (i) molecules
differing from TCP only by substitution of one heteroa-
tom in the cyclohexyl ring and/or in the hetero-aromatic
moiety, (ii) molecules differing from TCP by introduction
of a methyl substitution in any of the constitutive rings or
by specific modifications of the piperidine ring. Com-
paratively to the TCP model structure, lowering the
lipophilicity (table II) clearly directed the first group of
molecules (1, 2, 7, 8, 9) toward the PCP3-sites since they
displayed higher affinities for these sites (5.6 to 35 nM)
than for PCP1- (47.8 to 1223 nM) or PCP2-sites (187 to
5840 nM) (table V).Moreover, plotting IC50 PCP3/IC50
PCP1 against log P confirmed this tendency: the lower
log P was, the higher the selectivity for PCP3 was
(figure 6), in line with our hypothesis. A similar tendency
was found with regard to PCP2 (not shown). Finally, this
group of compounds revealed interesting selectivities for
PCP3 when compared to PCP2 and PCP1. 2, the less
potent (35 nM) and the less lipophilic (log P = 2.85) in
this group, displayed a high selectivity for the PCP3-sites
when compared to the PCP1- and PCP2-sites (PCP3/PCP1
< 0.026; PCP3/PCP2 < 0.006).
PCP3-sites: 13, 10-(±), 10-(+), and their thio-analogues
11-(±), 11-(+). Interestingly, the affinities of 10-(–), 11-
(–), and 13 were better described by a single-site model in
the cerebellum. Compound 12 bearing an hydroxymethyl
substitution in the piperidine ring exhibited a high affinity
for both PCP3- and PCP1-sites with a high selectivity
with regard to PCP2-sites. The methyl substitution in the
thiopyranyl ring gave a high affinity cis-compound (16)
equipotent at the PCP1- and PCP3-sites and an inactive
trans-compound (17). The difference between cis and
trans diastereomers in the forebrain was higher than
previously observed in the cyclohexyl homologue se-
ries [15, 20] although affinities were lower. Finally, the
position of a methyl group in the hetero-aromatic moiety
was crucial. In the α position from the sulfur atom (14),
it lowered the affinity for PCP3 and at the contrary, in the
â position (15), it increased the affinity for these sites.
Moreover 15 displayed a good selectivity with regard to
both PCP1- and PCP2-sites (PCP3/PCP1 < 0.07;
PCP3/PCP2 < 0.007). This is a confirmation of the very
important role played by the aromatic or hetero-aromatic
ring in the arylcyclohexylamines selectivity of bind-
ing [28]. In this second group of molecule steric interac-
tions due to substitutions are likely to influence more
selctivity than lipophilicity.
In the second group, changing the piperidine for a
morpholine ring decreased considerably the affinities (3)
or precluded the two-site computation given very low
affinities (4, 5). Substitution of a methyl group in the
piperidine ring gave compounds with low affinities for
It is now well admitted that the NMDA receptor is an
hetero-oligomeric protein composed with 5 sub-units
differently expressed in forebrain, cerebellum, and spinal
cord [29–31]. The resulting diversity of NMDA receptors

131
in the CNS, and consequently the diversity of the PCP
receptors, is responsible for the heterogeneity of pharma-
cological responses [32]. The present study confirms the
binding sites heterogeneity since TCP analogues were
able to interact differently with subsites labeled with
[3H]TCP in the rat cerebellum. In this region interestingly
NMDA receptors display particular properties when com-
pared with NMDA receptors found in the forebrain [26].
Some of the structures we have prepared and tested might
be valuable tools to clarify the pharmacological role of
the low affinity sites in the rat cerebellum (PCP3). Indeed,
compounds 2 and 15 possess both a high affinity and a
good selectivity for these sites. Such molecules might
also be leads for new compounds able to interact with this
specific target.
After the solid was consumed, the mixture was cooled to
0 °C and ethyl 2-sulfanylacetate (22 mL, 200 mmol, 1
eq.) then ethyl 4-chlorobutanoate (30.1 g, 200 mmol, 1
eq.) was added slowly. The resulting mixture was stirred
for 20 h at room temperature. The NaCl precipitate was
filtered, the filtrate concentrated in vacuum, the oil
obtained was diluted in water (100 mL) and extracted
with ether (3 × 80 mL). The combined organic layers
were dried over MgSO4 and concentrated in vacuum. The
resulting yellow oil was distilled to yield 35.6 g (76%) of
a colorless oil.
5.1.1.2. Ethyl 3-oxotetrahydro-2H-thiopyran-2-carbo-
xylate
Ethyl 4-[(2-ethoxy-2-oxoethyl)sulfanyl]butanoate in
anhydrous ether (200 mL) was added dropwise in a
nitrogen atmosphere to a solution of sodium ethanolate
(20.7 g, 0.3 mol, 2 eq.) at 0 °C, stirred at 0 °C for 45 min
and at room temperature for 3 h. The mixture was
hydrolyzed with a water/acetic acid (80:20) solution, the
aqueous phase was separated and extracted with ether (3
× 50 mL), the combined organic layers were dried over
MgSO4 and concentrated in vacuum. The resulting yel-
low oil obtained was distilled under reduced pressure to
yield 15.3 g (54%) of a colorless liquid.
5. Experimental protocols
5.1. Chemistry
Melting points (uncorrected) were determined with a
Büchi–Tottoli apparatus. Yields were not optimized. El-
emental analysis was performed at the CNRS Microana-
lytical Section in Montpellier on the hydrochloride salts
1
and were within ±0.4% of theoretical values. H- and
13C-NMR spectra were obtained on a Brucker AC 200
spectrometer at 200.13 and 50.32 MHz respectively in
5-mm sample tubes in the FT mode. For some 13C-signal
assignments, a spin-echo sequence (Jmod) was used.
Chemical shifts are reported in (δ) ppm downfield from
TMS. Enantiomeric purities were determined on a Shi-
madzu HPLC equipment (LC-1O AD pump, SPD-6A UV
spectrometer), computer-controlled by the Class LC-10
program. Analysis were made on a Chiralcel-OD column
(10 mm, 4.6 × 250 mm) (Daicel Chemical Industries) in
heptane (0.6 mL /min) at 36 °C. UV-detection was made
at 240 nm. Typical injection volumes were 5 mL of a 30
µM solution of base compound in heptane. Optical
rotations were obtained in methanol with a Perkin-Elmer
241 polarimeter in a 1-dm microcell at 20 °C. For NMR
and in vitro experiments, compounds were used as their
hydrochloride salts; salts were precipitated by adding a
dry HCl ethereal solution in ether to a solution of base in
ether. After filtration, the solids collected were dried in
vacuum.
5.1.1.3. Dihydro-2H-thiopyran-3(4H)-one
Ethyl
3-oxotetrahydro-2H-thiopyran-2-carboxylate
(15.2 g, 80.9 mmol) was refluxed in a 15% sulfuric acid
solution for 18 h then cooled to room temperature. A 10%
NaOH solution in water was then added dropwise to
reach pH 6. The mixture was extracted with ether (3 ×
50 mL), the organic phases washed with water, dried over
MgSO4, and concentrated in vacuum. The resulting oil
was distilled under reduced pressure to yield 5.1 g (54%)
of a colorless oil.
5.1.2. Synthesis of 1,1-dioxo-tetrahydro-1λ6-thiopy-
ran-4-one
A solution of hydrogen peroxide (11.4 mL, 0.1 mol, 2
eq.) was added dropwise to a mixture of tetrahydro-4H-
thiopyran-4-one(5.6 g, 48 mmol, 1 eq.) and acetic acid
(25 mL) keeping the temperature below 30 °C. The
mixture was stirred for 4 h, the acetic acid distilled under
reduced pressure, and the crystallized yellow residue was
filtered and washed with ether to yield 4.7 g (67%) of
white crystals.
5.1.1. Synthesis of dihydro-2H-thiopyran-3(4H)-one
5.1.1.1. Ethyl 4-[(2-ethoxy-2-oxoethyl)sulfanyl]buta-
noate
Sodium (4.6 g, 200 mmol, 1 eq.) was added cautiously
by portion in a nitrogen atmosphere to ethanol (100 mL).

132
5.1.3. Synthesis of 3-methyl-4-piperidinol
5.1.3.1. Ethyl 1-benzoyl-4-oxo-3-piperidinecarboxy-
few drops of a 5% HCl aqueous solution, and concen-
trated in vacuum. The resulting yellow solid was dis-
solved in hot ethanol and precipitated at room tempera-
ture by the addition of drops of ether to yield a white solid
(1.9 g, 87%).
late
Sodium hydride (4 g, 0.1 mol, 1 eq.) was added in a
nitrogen atmosphere to a solution of benzamide (12.1 g,
0.1 mol, 1 eq.) in toluene (200 mL), the mixture was
refluxed for 1 h, cooled to 0 °C and ethyl acrylate
(32.6 mL, 0.3mol, 3 eq.) was then rapidly added. The
solution was stirred at 60 °C for 24 h, cooled to 0 °C,
diluted with ice-cold water (100 mL) and stirred for 0.5 h.
The aqueous phase was separated, washed with ether
(50 mL), acidified until pH 3 and extracted with CH2Cl2
(3 × 50 mL). The combined organic layers were dried
over Na2SO4, filtered and concentrated in vacuum. The
yellow oil obtained was purified by column chromato-
graphy (SDS Chromagel 70–100 µ) in ether to yield 9.4 g
(34%) of a red oil.
5.1.4. Synthesis of α-aminonitriles
α-aminonitriles were obtained by means of two differ-
ent synthetic methods. Since the same method was
applied to various compounds we describe only one
example in each case.
5.1.4.1. Method A
Acetone cyanohydrine (2.2 g, 25.8 mmol, 1 eq.) was
added dropwise to a stirred mixture of tetrahydro-4H-
thiopyran-4-one (3 g, 25.8 mmol, 1 eq.), anhydrous
MgSO4 (9.3 g, 77.4 mmol, 3 eq.), dimethylacetamide
(2.25 g, 25.8 mmol, 1 eq.) and piperidine (4.4 g,
51.6 mmol, 2 eq.). The pasty mixture was heated at 45 °C
for 48 h, cooled to room temperature, poured onto ice and
stirred for 30 min. The aqueous mixture obtained was
extracted with ether and the organic layer was washed
with water until neutrality, dried over Na2SO4, filtered,
and concentrated in vacuum to yield 5.3 g (98%) of an
orange oil of 4-piperidinotetrahydro-2H-thiopyran-4-
carbonitrile.
5.1.3.2. Ethyl 1-benzoyl-3-methyl-4-oxo-3-piperidine-
carboxylate
A
mixture of ethyl 1-benzoyl-4-oxo-3-piperi-
dinecarboxylate (9.3 g, 33.8 mmol, 1 eq.) and sodium
hydride (1.35 g, 34 mmol, 1 eq.) in dimethoxyethane
(50 mL) was refluxed for 2 h then cooled to 0 °C. ICH3
(5.2 mL, 84 mmol, 2.5 eq.) was added and the mixture
heated at 60 °C for 40 h. The mixture, after concentration
in vacuum, was diluted with water (100 mL) and ex-
tracted with CH2Cl2 (3 × 50 mL). The combined organic
layers were washed successively with a 5% NaOH water
solution (50 mL), a 5% HCl water solution (50 mL), with
water (50 mL), then dried over Na2SO4, and concentrated
in vacuum. The resulting brown oil (9.4 g) was purified
by column chromatography (SDS Chromagel 70–200 µ)
in ether to yield 7.7 g (79%) of a slightly yellow oil.
The following α-aminonitriles were similarly synthe-
sized:
4-piperidinotetrahydro-2H-pyran-4-carbonitrile
(79%, F = 46–47 °C) from piperidine and tetrahydro-4H-
pyran-4-one; 4-(3-methylpiperidino)tetrahydro-2H-thio-
pyran-4-carbonitrile (> 98%, oil) from 3-methyl-
piperidine and tetrahydro-4H-thiopyran-4-one; R-4-(3-
methylpiperidino)tetrahydro-2H-thiopyran-4-carbonitrile
(> 98%, oil) and S-4-(3-methylpiperidino)tetrahydro-2H-
thiopyran-4-carbonitrile (> 98%, oil) from R-(–)-3-
methylpiperidine and S-(+)-3-methylpiperidine [7, 8, 21],
and tetrahydro-4H-thiopyran-4-one; 4-(3-methylpipe-
5.1.3.3. 3-Methyl-piperidin-4-one hydrochloride
A solution of ethyl 1-benzoyl-3-methyl-4-oxo-3-
piperidinecarboxylate (7.7 g, 26.6 mmol, 1 eq.) in a 6 N
HCl aqueous solution was refluxed for 72 h, then the
benzoic acid precipitate was filtered, the filtrate washed
with ether (3 × 50 mL) and concentrated in vacuum. The
brown solid obtained was crystallized in ethanol to get
white crystals (2.85 g, 72%).
ridino)tetrahydro-2H-thiopyran-4-carbonitrile
(81%,
solid) from 3-hydroxymethylpiperidine and cyclohex-
anone.
5.1.4.2. Method B
5% HCl (a few drops) was added to a stirred mixture of
3-methyl-4-piperidinol (0.8 g, 7 mmol, 2 eq.) in 10 mL of
water and cyclohexanone (2.7 g, 23.6 mmol, 1 eq.) to
reach pH 3. KCN (0.47 g, 7.3 mmol, 1.05 eq.) was added
(pH reached 11). The mixture was stirred at room
temperature for 24 h then extracted with CH2Cl2, dried
over Na2SO4, filtered, and concentrated in vacuum to
yield 1.43 g (93%) of 1-(4-hydroxy-3-methylpiperi-
dino)cyclohexanecarbonitrile as a colorless oil.
5.1.3.4. 3-Methyl-4-piperidinol
A solution of 5% NaOH in water (7.6 mL) was added
dropwise to a solution of 3-methyl-piperidin-4-one hy-
drochloride (2.85 g, 19 mmol, 1 eq.) in methanol (30 mL)
and the mixture was stirred for 0.5 h at room temperature.
A solution of NaBH4 (2.46 g, 6.5 mmol, 1.4 eq.) in
methanol (30 mL) was added, the mixture was stirred for
4 h at room temperature, cooled to 0 °C, made acidic with

133
Table VI. Purification and properties of new compounds.
Purification
Solvent (v/v)
Fbase (°C)
FHCl (°C)
Yield (%)
6
7
8
9
Crystallization
AcOEt
167
58–60
Oil
79–81
Oil
Oil
Oil
Oil
120–122
93–95
89–91
179–183
184–186
166–167
171–173
168–169
170–172
169–171
176–178
170–172
160–162
144–146
48
83
76
71
57
59
64
56
71
67
55
c
Column chromatograpy (Al2O3)
Column chromatograpy (Al2O3)
Column chromatograpy (Al2O3)
Column chromatograpy (Al2O3)
Column chromatograpy (Al2O3)
Column chromatograpy (Al2O3)
Column chromatograpy (Al2O3)
Column chromatograpy (Al2O3)
Column chromatograpy (Al2O3)
Column chromatograpy (Al2O3)
Petroleum ether/ether (40:60)
CH2Cl2
Petroleum ether/ether (98:2)
Petroleum ether/ether (95:5)
Petroleum ether/ether (95:5)
Petroleum ether/ether (95:5)
Petroleum ether/ether (50:50)
Petroleum ether/ether (20:80)
Petroleum ether/ether (98:2)
Petroleum ether/ether (90:10)
11-(±)
11-(+)
11-(–)
12
13
14
a
b
15
a
20
b
HPLC (chiral phase): Rt = 29.1 min, e.e. > 99%; [αD
]
= +12° (c 1, CH3OH); HPLC (chiral phase): Rt = 32.3 min, e.e. > 99%;
base
20
c
[αD
]
= –11° (c 1, CH3OH); aluminium oxide 90, 2–3 Merck.
base
1,1-Dioxo-4-piperidinohexahydro-1λ6-thiopyran-4-
carbonitrile (64%, F = 148 °C) was similarly prepared
from piperidine and 1,1-dioxo-tetrahydro-1λ6-thiopyran-
4-one.
5.1.5.2. Method 2: TCP analogues with a furanyl (8, 9)
or a substituted thiophenyl (14, 15) hetero-aromatic ring
1-[4-(2-furyl)tetrahydro-2H-thiopyran-4-yl]piperidine
9: Firstly, a MgBr2 solution was prepared from 1,2-
dibromo-ethane (6.76 g, 36 mmol, 4 eq.) and magnesium
turnings (0.88 g, 36 mmol, 4 eq.) in ether (80 mL) in a
nitrogen atmosphere. Secondly, a solution of 2-furyl-
Lithium was prepared in a nitrogen atmosphere by the
dropwise addition of a n-butyl-lithium solution 1.6 M in
hexane (28 mL, 45 mmol, 5 eq.) to a mixture of furane
(3.1 g, 45 mmol, 5 eq.) and TMEDA (5.2 g, 45 mmol, 5
eq.) in anhydrous ether (100 mL) at –20 °C (without
TMEDA in the synthesis of 15). The mixture was then
refluxed for 2 h, cooled to room temperature, and added
dropwise to the MgBr2 solution in ether. A solution of
4-piperidinotetrahydro-2H-thiopyran-4-carbonitrile (1.9 g,
9 mmol, 1 eq.) in ether was added dropwise at room
temperature, the mixture refluxed for 16 h, cooled to
room temperature, and treated as described above. The
crude product was purified as described in table VI to
yield 1.6 g (71%) of 9 as a white solid.
5.1.5. Synthesis of TCP analogues from α-amino-
nitriles
Compounds 1 [7], 2 [7, 33], 3–5 [7] as well as com-
pounds 10-(±), 10-(–), 10-(+) [15] have been previously
described. TCP analogues were obtained by means of two
different synthetic methods. Since the same method was
applied to various compounds we describe only one
example to illustrate each strategy. Compounds purifica-
tions and properties are given in table VI.
5.1.5.1. Method 1: TCP analogues with a thiophenyl
hetero-aromatic ring (6, 7, 11–13)
1-[3-(2-thienyl)tetrahydro-2H-thiopyran-3-yl]piperidi-
ne 7: An ethereal solution of 3-piperidinotetrahydro-2H-
thiopyran-3-carbonitrile (1 g, 4.76 mmol, 1 eq.) was
added dropwise at room temperature to a well stirred
solution containing a Grignard reagent prepared from
2-bromo-thiophene (2.3 g, 14.3 mmol, 3 eq.) and Mg
turnings (0.35 g, 14.3 mmol, 3 eq.). The mixture was
refluxed for 20 h, cooled to room temperature and treated
as follows: the mixture was poured carefully on an
ice-cold saturated solution of NH4Cl, stirred for 30 min,
extracted with ether, the combined ether layers were
extracted 3 times with 10% HCl, 20% NH4OH was added
to the aqueous phase until neutrality. The aqueous phase
was extracted with ether, the organic phase washed with
water, dried over Na2SO4, filtered, and concentrated in
vacuum. The crude product obtained was purified as
described in table VI to yield 1.05 g (83%) of 7 as a
white solid.
5.1.6. Preparation of compounds 16 and 17
5.1.6.1. 3-Methyltetrahydro-4H-thiopyran-4-one
n-Butyl-lithium (37.5 mL, 60 mmol, 1 eq.) was added
dropwise in a nitrogen atmosphere to a stirred solution of
diisopropylamine (8.4 mL, 60 mmol, 1 eq.) in THF
(74 mL) and the mixture was stirred 0.5 h at room
temperature, then cooled to –80 °C. After slow addition
of tetrahydro-4H-thiopyran-4-one (6.96 g, 60 mmol, 1
eq.) and stirring for 0.5 h, ICH3 (5.6 mL, 90 mmol, 1.5
eq.) was added, the mixture allowed to warm up to room
temperature while stirred for 5 h. The mixture was diluted
with a NaCl saturated 5% solution of sodium bicarbonate

134
in water and the organic phase separated, dried over
Na2SO4 and concentrated in vacuum. The orange oil
obtained was purified by chromatography (SDS Chroma-
gel 70–200 µ) in petroleum ether/ethyl acetate (90:10) to
yield 3.2 g (41%) of a colorless oil.
necessary to destroy lithium aluminium hydride was
added slowly, the precipitate filtered, washed with
CH2Cl2 (300 mL), and the filtrate concentrated in
vacuum. The brown oil obtained was diluted in ether,
extracted with a 10% HCl solution (3 × 100 mL) and 20%
NH4OH added to the aqueous phase until neutrality. The
aqueous phase was extracted with ether (3 × 100 mL) and
the combined organic layers washed with water, dried
over MgSO4, filtered, and concentrated in vacuum. The
crude diastereomeric oily mixture was purified by chro-
matography (SDS Chromagel 70–200 µ) in petroleum
ether/ether (50:50) to yield 3.7 g (75%) of a cis/trans
primary amines mixture as a colorless oil.
5.1.6.2. Cis- and trans-3-methyl-4-(2-thienyl)tetra-
hydro-2H-thiopyran-4-ol
2-Thienyl-magnesium bromide was prepared by add-
ing dropwise in a nitrogen atmosphere a solution of
2-bromo-thiophene (6.1 g, 37.2 mmol, 1.1 eq.) in ether
(100 mL) to Mg turnings (0.9 g, 37.2 mmol, 1.1 eq.).
After the mixture was gently refluxed for 3 h, a solution
of
3-methyltetrahydro-4H-thiopyran-4-one
(4.2 g,
32 mmol, 1 eq.) dissolved in ether (50 mL) was added at
room temperature. The mixture was refluxed for 16 h,
cooled to room temperature, poured onto a saturated
NH4Cl solution in water, stirred 0.5 h. The aqueous phase
was separated, extracted with ether (3 × 50 mL) and the
combined organic layers extracted with 15% HCl (3 ×
50 mL). 25% NH4OH was added to the aqueous phase
until neutrality and the solution extracted with ether. The
final organic phase was washed with water, dried over
Na2SO4, filtered, and concentrated in vacuum. The green
oil obtained was purified by chromatography (SDS Chro-
magel 70–200 µ) in petroleum ether/ether (90:10) to yield
6.8 g (98%) of a slightly blue oil containing the diaste-
reomeric alcohols.
5.1.6.5. Cis- and trans-1-[3-methyl-4-(2-thienyl)tetra-
hydro-2H-thiopyran-4-yl]piperidine 16, 17
Potassium carbonate (5.8 g, 42 mmol, 2 eq.) and 1,5-
dibromopentane (3.6 mL, 26.2 mmol, 1.25 eq.) was
added in a nitrogen atmosphere to cis- and trans3-methyl-
4-(2-thienyl)tetrahydro-2H-thiopyran-4-amine
(4.5 g,
21 mmol, 1 eq.) dissolved in HMPT (50 mL) and the
resulting mixture stirred at 60 °C for 48 h then cooled to
room temperature. The mixture was poured onto water
(200 mL) and extracted with ether (3 × 100 mL). The
combined organic layers were extracted 3 times with 10%
HCl and 20% NH4OH was added to the aqueous phase
until neutrality. The aqueous phase was extracted with
ether and the final organic phase was washed with water,
dried over MgSO4, filtered, and concentrated in vacuum.
The resulting yellow oil was chromatographied (SDS
Chromagel 70–200 µ) in petroleum ether/ether (90:10) to
yield the pure diastereomers as white solids: 17 (2 g,
34%) and 16 (0.7 g, 12%).
5.1.6.3. Cis- and trans-3-methyl-4-(2-thienyl)tetra-
hydro-2H-thiopyran-4-azide
Sodium azide (4 g, 61.7 mmol, 2 eq.) was cautiously
added to a solution of trichloro-acetic acid (15.1 g,
92.4 mmol, 3 eq.) in CHCl3 (100 mL), the mixture was
cooled to 10 °C, and a solution of diastereomeric alcohols
3-methyl-4-(2-thienyl)tetrahydro-2H-thiopyran-4-ol(6.6g,
30.8 mmol, 1 eq.) in CHCl3 (50 mL) was added. The
mixture was stirred for 72 h at 10–12 °C, then a 10%
NH4OH solution was added until neutrality and the
aqueous phase extracted with CH2Cl2 (3 × 100 mL).
The pooledorganic phases were washed with water,
dried over Na2SO4, filtered, and concentrated in
vacuum. The oil obtained (6.8 g, 92%) was used in the
next step without further purification.
5.2. Biochemistry
5.2.1. Binding assays
[3H]TCP (Amersham, 48 Ci/mmol) binding to the PCP
receptor subtypes was measured as previously de-
scribed [3]. Briefly, the rat (wistar) brain (minus the
cerebellum) or the cerebellum was homogenized with an
Ultraturax (Ika Werke, maximum setting) in a 50 mM
Tris/HCl, pH 7.7 buffer for 20 s at 4 °C. The homogenate
was then centrifuged at 49000 g for 20 min. The pellet
was resuspended in the same buffer and the homogeni-
zation–centrifugation steps performed a second time. The
final pellet was resuspended in 10 volumes of a 50 mM
Tris/Hepes, pH 7.7 buffer and used without further
purification.
5.1.6.4. Cis- and trans-3-methyl-4-(2-thienyl)tetra-
hydro-2H-thiopyran-4-amine
A solution of cis- and trans-3-methyl-4-(2-thienyl)
tetrahydro-2H-thiopyran-4-azide (5.5 g, 23 mmol, 1 eq.)
in THF (30 mL) was added dropwise at 0 °C to a solution
of lithium aluminium hydride (0.87 g, 23 mmol, 4 eq.) in
THF in a nitrogen atmosphere stirred for 24 h at room
temperature. The strict minimum amount of NH4OH
The forebrain or cerebellum homogenate (0.5–0.8 mg
protein/mL) was incubated with [3H]TCP (1 nM or 2.5
nM respectively) in a 5 mM Tris/Hepes, pH 7.7 buffer
(0.5 mL or 1 mL respectively) in the absence (total

135
[4] Ebert B., Wong E.H.F., Krogsgaard-Larsen P., Eur. J. Pharmacol.
208 (1991) 49–52.
binding) or in the presence of the competing drug for
30 min at 25 °C. The incubation was terminated by
filtration over GF/B (Whatman) glass fibre presoaked in
0.05% polyethyleneimine (Aldrich) with an MR24 Bran-
del cell harvester. The filters were rinsed three times with
5 mL 50 mM NaCl, Tris HCl 10 mM, pH 7.7 buffer and
the radioactivity retained was counted in 3.5 mL ACS
(Amersham) with an Excel 1410 (LKB) liquid scintilla-
tion spectrophotometer. The non-specific binding was
determined in parallel experiments in the presence of 100
µM unlabelled TCP.
[5] Berman F.W., Murray T.F., J. Biochem. Toxicol. 11 (1996)
217–226.
[6] Choi D.W., Cerebrovasc. Brain Metab. Rev. 2 (1990) 105–147.
[7] Hamon J., Vignon J., Kamenka J.M., Eur. J. Med. Chem. 31 (1996)
489–495.
[8] Vignon J., Chaudieu I., Allaoua H., Journod L., Javoy-Agid F.,
Agid Y., Chicheportiche R., Life Sci. 45 (1989) 2547–2555.
[9] Bruylants P., Bruylants P., Bull. Soc. Chim. Belg. 33 (1924)
467–478; Bull. Soc. Chim. Belg. 35 (1926) 139–154.
[10] Geneste P., Kamenka J.M., Dessapt P., Bull. Soc. Chim. Fr. 3& 4
II (1980) 186–191.
[11] Ilagouma A.T., Dornand J., Liu C.F., Zénone F., Mani J.C.,
Kamenka J.M., Eur. J. Med. Chem. 25 (1990) 609–615.
5.2.2. Data analysis
In each experiment, values are the mean of three
independent determinations. Each experiment was per-
formed 3–5 times. The data from competition experi-
ments were first analyzed by Hill’s representation accord-
ing to a single-site model, then by a non linear regression
method (Marquardt–Levenberg algorithm) according to a
two-site model using the Sigmaplott 4 software (Jandel).
The two-site interaction was represented by:
[12] Minato A., Tarrao K., Hayashi T., Susuki K., Kumada M.,
Tetrahedron Lett. 52 (1981) 5319–5322.
[13] Coderc E., Martin-Fardon R., Vignon J., Kamenka J.M., Eur. J.
Med. Chem. 28 (1993) 893–898.
[14] Marwaha J., Palmer M., Hoffer B., Freedman R., Rice K.C., Paul
S., Skolnick P., Arch. Pharmacol. 315 (1981) 203–209.
[15] Coderc E., Cerruti P., Vignon J., Rouayrenc J.F., Kamenka J.M.,
Eur. J. Med. Chem. 30 (1995) 463–470.
[16] Geneste P., Kamenka J.M., Org. Magn. Res. 7 (1975) 579–584.
[LB] = (B1 + B2) – {([I] × B1/(IC501 + [I]))
[17] Kamenka J.M., Geneste P., In: Domino E.F. (Ed.), PCP (Phencyc-
lidine): Historical and Current Perspectives, NPP Books, Ann Arbor, 1981,
pp. 47–82.
+ ([I] × B2/(IC502 + [I]))}
where [LB] was the percentage of radioligand concen-
tration specifically bound, [I] the competitor concentra-
tion, B1 and B2 the percentage of each binding site, IC501
and IC502 the concentrations of unlabeled competitor that
inhibited 50% of specific [3H]TCP binding on specified
sites. Two constraints were fixed: (i) IC501, IC502, B1, B2
> 0; (ii) 95% < (B1 + B2) < 105% because of the
uncertaincy on the total binding (10%). Experimental
results were submitted to an ANOVA test followed by a
Durbin–Watson test. The two-site model was preferred
when it produced a significant reduction in the sum of
squares (P < 0.05, Student’s t-test) and when the Durbin-
–Watson coefficient was closer to 2 than in the single-site
model and comprised between 1.5 and 2.5.
[18] Manoharan M., Eliel E.L., Carrol F.I., Tetrahedron Lett. 24 (1983)
1855–1858.
[19] Manoharan M., Eliel E.L., Kanan W., Tetrahedron Lett. 25 (1984)
3267–3268.
[20] Michaud M., Warren H., Drian M.J., Rambaud J., Cerruti P.,
Nicolas J.P., Vignon J., Privat A., Kamenka J.M., Eur. J. Med. Chem. 29
(1994) 869–876.
[21] Geneste P., Kamenka J.M., Ung S.N., Herrmann P., Goudal R.,
Trouiller G., Eur. J. Med. Chem. 14 (1979) 301–308.
[22] Young T.E., Heitz L.J., J. Org. Chem. 38 (1973) 1562–1566.
[23] Baty J.D., Jones G., Moore C., J. Chem. Soc. (C) (1967)
2645–2647.
[24] Pouchert C.J., Behnke J. (Eds.), The Aldrich library of 13C and 1H
FT NMR Spectra, Vol. 1, Aldrich Chemical Company, 1993, pp. 235–236.
[25] Conley E.C., In: Conley E.C. (Ed.), The Ion Channel Facts Book
I, Academic Press, New York, 1996, pp. 140–233.
[26] Sekiguchi M., Okamoto K., Sakai Y., J. Neurosci. 10 (1990)
2148–2155.
Acknowledgements
[27] Yamakura T., Mori Y., Masaki H., Shimoji K., Mishima M.,
Neuroreport 4 (1993) 687–690.
This work was supported by D.R.E.T. (grant 94/141).
Thanks are due to M. Michaud for her excellent technical
assistance.
[28] Chaudieu I., Vignon J., Chicheportiche M., Kamenka J.M., Trouil-
ler G., Chicheportiche R., Pharmacol. Biochem. Behav. 32 (1989) 699–705.
[29] Moriyoshi K., Masu M., Ishii T., Shigemoto R., Mizuno N.,
Nakanishi S.,, Nature 354 (1991) 31–37.
[30] Durand G.M., Bennett M.V.L., Zukin R.S., Proc. Natl. Acad. Sci.
USA 90 (1993) 6731–6735.
References
[31] Widdowson P.S., Trainor A., Lock E.A., J. Neurochem. 64 (1995)
651–661.
[1] Vignon J., Chicheportiche R., Chicheportiche M., Kamenka J.M.,
Geneste P., Lazdunski M., Brain Res. 280 (1983) 194–197.
[32] Lynch D.R., Lawrence J.J., Lenz S., Anegawa N.J., Dichter M.,
Pritchett D.B., J. Neurochem. 64 (1995) 1462–1467.
[2] Wong E.H.F., Kemp J.A., Priestley T., Knight A.R., Woodruff
G.N., Iversen L.L., Proc. Natl. Acad. Sci. USA 83 (1986) 7104–7108.
[33] Eiden F., Schmidt M., Buchborn H., Arch. Pharmacol. 320 (1987)
348–361.
[3] Vignon J., Privat A., Chaudieu I., Thierry A., Kamenka J.M.,
Chicheportiche R., Brain Res. 378 (1986) 133–141.