The low affinity PCP sites in the rat cerebellum not only bind TCP-like but also BTCP-like structures

Article abstract of DOI:10.1016/S0223-5234(00)00135-5

Congeners of the potent dopamine (DA) re-uptake inhibitor l-[1-(2- benzo[b]thiophenyl)cyclohexyl]piperidine (BTCP) are unexpectedly able to bind in the rat cerebellum, although this structure is devoid of dopaminergic nerve endings. In line with previous studies the hypothesis that they bind to low affinity PCP sites labelled with [³H]TCP in the rat cerebellum, even though they do not bind to the high affinity PCP sites in the forebrain, was considered. Analogues of l-[1-(2-thiophenyl)cyclohexyl]piperidine (TCP) and BTCP with a modified aromatic moiety and with O or S atoms substituted in the cyclohexyl ring were prepared and tested in competition experiments both in rat forebrain and cerebellum membranes labelled with [³H]TCP, and in rat striatum membranes labelled with [³H]BTCP. Results indicated that BTCP and congeners could bind to low affinity PCP sites labelled with [³H]TCP in the rat cerebellum with a decrease of the selectivity for the DA transporter. On the contrary, some TCP analogues displayed a very high selectivity for these low affinity sites: they might be important pharmacological tools to elucidate the nature and function at yet unknown of these sites. (C) 2000 Editions scientifiques et medicales Elsevier SAS.

Full text of DOI:10.1016/S0223-5234(00)00135-5

Eur. J. Med. Chem. 35 (2000) 323−331
© 2000 Éditions scientifiques et médicales Elsevier SAS. All rights reserved
S0223523400001355/FLA
323
Original article
The low affinity PCP sites in the rat cerebellum not only bind TCP-like but also
BTCP-like structures
Florence Espazea, Jacques Hamona, Hélène Hirbecb, Jacques Vignonb, Jean-Marc Kamenkaa*
a
´
´
CRBM, CNRS UPR 1086, Ecole nationale supérieure de chimie, 8, rue de l’Ecole-normale, 34296 Montpellier cedex 5, France
b
´
´
INSERM U 336, Ecole nationale supérieure de chimie, 8, rue de l’Ecole-normale, 34296 Montpellier cedex 5, France
Received 14 June 1999; revised 7 October 1999; accepted 18 October 1999
Abstract – Congeners of the potent dopamine (DA) re-uptake inhibitor 1-[1-(2-benzo[b]thiophenyl)cyclohexyl]piperidine (BTCP) are
unexpectedly able to bind in the rat cerebellum, although this structure is devoid of dopaminergic nerve endings. In line with previous studies
the hypothesis that they bind to low affinity PCP sites labelled with [3H]TCP in the rat cerebellum, even though they do not bind to the high
affinity PCP sites in the forebrain, was considered. Analogues of 1-[1-(2-thiophenyl)cyclohexyl]piperidine (TCP) and BTCP with a modified
aromatic moiety and with O or S atoms substituted in the cyclohexyl ring were prepared and tested in competition experiments both in rat
forebrain and cerebellum membranes labelled with [3H]TCP, and in rat striatum membranes labelled with [3H]BTCP. Results indicated that
BTCP and congeners could bind to low affinity PCP sites labelled with [3H]TCP in the rat cerebellum with a decrease of the selectivity for
the DA transporter. On the contrary, some TCP analogues displayed a very high selectivity for these low affinity sites; they might be important
pharmacological tools to elucidate the nature and function at yet unknown of these sites. © 2000 Éditions scientifiques et médicales Elsevier
SAS
TCP / TCP analogues / BTCP / BTCP analogues / PCP receptor subsites / DA transporter / rat cerebellum
1. Introduction
binding pattern obviously precluded the use of such
tracers for in vivo imaging and addressed the ques-
tion of binding sites for BTCP and congeners in the
cerebellum. We have recently shown that TCP ana-
logues with O or S atoms substituted in the cyclo-
hexyl ring and/or with a methyl-substituted hetero-
aromatic ring might yield selective ligands for low
affinity sites labelled with [3H]TCP in the rat cerebel-
lum [11]. Taken together these results opened the
hypothesis that BTCP and congeners could bind to
these low affinity sites in the rat cerebellum even
though they do not bind to the high affinity PCP sites
in the forebrain [1, 2]. Since the nature and the role of
the low affinity binding sites remains poorly docu-
mented we have decided to investigate this hypoth-
esis by: (i) preparing TCP analogues with enlarged
aromatic moieties to partly mimic bicyclic systems
like in BTCP and with O or S atoms substituted in the
cyclohexyl ring; (ii) preparing BTCP analogues with
O or S atoms substituted in the cyclohexyl ring;
(iii) using these new molecules, and some of those
1-[1-(2-Benzo[b]thiophenyl)cyclohexyl]piperidine
(BTCP) is a phencyclidine (1-(1-phenylcyclohexyl)
piperidine, PCP) analogue highly selective for the
dopamine (DA) neuronal transporter (DAT) when
compared to PCP, since it binds very poorly to the
PCP receptor within the NMDA-gated Ca++ chan-
nel [1, 2]. Accordingly, it inhibits DA re-uptake [1, 3,
4], increases striatal levels of extracellular DA in
rats [5] and evokes behaviour related to dopaminer-
gic stimulation [2, 6–9]. It was thus tempting to
hypothesize that positron-emitting radioligands de-
rived from BTCP might enable the selective deter-
mination of the degree of DA fibre loss in Parkin-
son’s disease. Unexpectedly the regional brain
distribution of radioligands in rats revealed a strik-
ingly similar accumulation of radioactivity in both
the striatum and the cerebellum [10], although the
latter is devoid of dopaminergic nerve endings. This
*Correspondence and reprints: kamenka@cit.enscm.fr

324
previously obtained, for competition experiments in
rat forebrain or cerebellum membranes labelled with
[3H]TCP, and in rat striatal membranes labelled with
[3H]BTCP. For clarity, since[3H]TCP labels three
sites in the rat CNS [11], the different binding sites
discussed here will be marked PCP1 (high affinity
binding sites in the forebrain, i.e. the original PCP
receptor within the NMDA receptor associated ionic
channel), PCP2 (high affinity binding sites in the
cerebellum, similar to PCP1 but of lower affin-
ity [11]), and finally PCP3 ([3H]TCP low affinity
binding sites in the cerebellum). Finally this work
was designed to determine how structural modifica-
tions of the aromatic moiety and/or hetero-
substitutions in the cyclohexyl ring affect the mo-
lecular targeting to the different binding sites labelled
by [3H]TCP in the rat forebrain or cerebellum, and to
the binding sites labelled by [3H]BTCP in the rat
striatum.
2. Chemistry
The newly synthesized compounds and some of those
previously obtained are presented in figure 1. They re-
sulted from various modifications on the TCP and BTCP
models. Oxygen and sulfur atoms were substituted at the
4 position of the cyclohexyl ring and the aromatic moiety
was chosen from (i) unsubstituted thiophenyl or furanyl
rings to yield TCP-like structures; (ii) substituted
thiophenyl or furanyl rings to partly mimic the steric bulk
existing in BTCP to yield pseudo-BTCP structures; (iii)
benzothiophenyl or benzofuranyl rings to yield BTCP-
like structures.
Each of these compounds was obtained by means of a
Bruylants reaction. Briefly, it consists of the replacement
of a cyano group by an aryl or hetero-aryl group during
the reaction between an α-aminonitrile and a Grignard
reagent [12]. The suitable α-aminonitrile resulted from a
Strecker-like synthesis in an organic medium as previ-
ously described [11, 13] (figure 2). Compounds 2 and 3
were previously prepared [11] from a Grignard reagent
easily obtained from 2-bromo-thiophene and Mg turn-
ings. In the present work, compounds 1 and 4–13 were
prepared from Grignard reagents best obtained by a
Mg/Li exchange reaction between MgBr2 and a suitable
hetero-aromatic Li derivative [14] (figure 2). The synthe-
sis of non-commercial hetero-aromatic starting materials
was required. 2,3-Dimethyl-furan was obtained in a
three-step preparation: (i) reduction with LiAlH4 of ethyl
2-methyl-3-furoate; (ii) reduction/chlorination with thio-
nyl chloride of 3-hydroxymethyl-2-methyl-furan in a pH
controlled medium; (iii) reduction with LiAlH4 in dig-
Figure 1. Structure of compounds.
lyme of the 3-chloromethyl-2-methyl-furan intermediate
(not isolated) [15–17]. 3-Ethyl-thiophene and 2,3-
dimethyl-thiophene were respectively obtained from the
commercially available 3-acetyl-thiophene and 3-methyl-
2-thiophene-carboxaldehyde by means of a Wolff-
Kishner reduction [18]. 3-Vinylthiophene resulted from
the reduction of 3-acetylthiophene followed by a dehy-
dration step [19]. The metallation reaction was done with
n-butyl-lithium in the presence of TMEDA. Under the
conditions used, the metallation occurred mainly at the α
position from the heteroatom (S or O). Consequently the
hetero-aromatic ring was linked by this same α position

325
Figure 2. Synthetic scheme (X and Y are sulfur or oxygen atoms, see figure 1).
to the cyclohexyl ring in the final compound. However, in
the case of 3-methyl-, 3-ethyl-, and 3-vinyl-thiophene the
metallation was conducted without TMEDA. This modi-
fication allowed us to obtain a pure isomer which was a
2,4-disubstituted thiophenyl compound (5, 6, 7 and 9).
This procedure was unsuccessful with 3-methyl-furan
(obtained from methyl 3-methyl-2-furoate [20]) which
gave, after completion of the reaction steps, and whatever
the conditions used, a non-separable isomeric mixture of
2,3- and 2,4-disubstituted furanyl compounds (not
shown). Structures of the new compounds were all
checked by 13C-NMR spectroscopy of the hydrochloride
salts since in these series protonation induces a confor-
mational trapping [11, 21, 22]. Purity was controlled by
GC/MS and elemental analysis.
Finally, affinities for the DA transporter were measured
by competition experiments in rat striatal membranes
labelled with [3H]BTCP (Experimental protocols, section
6.2.) and reported in table III.
4. Results and discussion
4.1. Inhibition of [3H]TCP binding
In forebrain homogenates (PCP1-sites), competition
curves yielded Hill numbers close or equal to unity with
the exception of 15 (table I) and therefore the single-site
model was the most probable. On the contrary, in
cerebellum homogenates, most Hill numbers were sig-
nificantly lower than unity and the inhibition curves were
best fitted to a two-site model (PCP2- and PCP3-sites)
which produced a significant reduction in the sum of
squares (P < 0.05, Student’s t-test) and a Durbin-Watson
coefficient between 1.5 and 2.5 (Experimental protocols,
section 6.2.1.4.). Most compounds, including BTCP and
BTCP-like compounds (13–16), were thus more or less
able to interact with PCP3-sites. However, the interaction
of compounds 8–10 and 12 in the cerebellum (Hill
numbers between 0.74 and 0.91) was better described by
a single-site model, thus suggesting similar affinities for
both sites. Interestingly compound 13, a moderately
effective ligand for PCP3-sites (Ki = 372 nM), was very
selective since it displayed a very low affinity (> 100 µM)
for PCP1- and PCP2-sites. In this respect compound 6 was
also found very selective for PCP3-sites, mostly because
it is a very high affinity ligand (Ki = 1.1 nM, see table I).
It is noticeable that results presented in table I confirmed
that [3H]TCP labelled two sites in the cerebellum in about
a 70/30 mean relative proportion (PCP2/PCP3 = 71.2 ±
5.6/27.8 ± 5.4).
3. Pharmacology
The binding assays in rat forebrain and cerebellum
membranes and the data analysis are described in the
experimental protocols (section 6.2.). We have previously
shown that a two-site model was more probable than a
single-site model to describe the inhibitory effect of TCP
and MK-801 in the rat cerebellum membranes labelled
with [3H]TCP [11]. The IC50s obtained were consistent
with those previously reported [23–25]. Thus, the same
treatment was applied to BTCP and compounds belong-
ing to the TCP- and BTCP-like or pseudo-BTCP series
(figure 1). The results are summarized in table I. The
affinities attributed to the population of high affinity sites,
i.e. PCP2-sites, were checked by additional competition
experiments in rat cerebellum membranes labelled with
[3H]MK-801. Since MK-801 binds more than
1 000 times better to PCP2- than to PCP3-sites (see [11]:
IC50 = 9.3 nM and 11 125 nM respectively,) the inhibition
of [3H]MK-801 and [3H]TCP binding to PCP2-sites
should, respectively, appear in a similar range (table II).

326
Table I. Inhibition of [3H]TCP binding in rat forebrain and cerebellum membranes. The mean of at least three independent determinations
was analysed according to a single-site model in the forebrain and cerebellum (Kia, 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 (Kia, nM) are given.
Compound Forebrain single-site model
Cerebellum single-site model
Cerebellum two-site model
% (PCP2) Ki (PCP3)
Ki (PCP1)
nH
Kie
nH
Ki (PCP2)
56.5 (16)
% (PCP3)
TCP
1
7.7
1.00
188 (63)
0.56 (0.05)
72.6 (3.6) 3 700 (1 200) 29.8 (4.5)
51 000 (17 000) 70.6 (8.5) 19.5 (5.6)
80.3 (6.5) 35.0 (18)
75.2 (8.8) 8.5 (4.5)
14 400 (2 900) 77.0 (7.5) 266 (163)
11 600 (3 300) 65.3 (3.5) 183 (39)
5 100 (800)
1 000 (120)
59.3 (8.7)
0.85 (0.07)
0.98 (0.05)
1.05 (0.05)
10 300 (1 400) 0.63 (0.05)
29.3 (6.8)
15.5 (5.7)
25.2 (7.2)
23.0 (4.5)
33.5 (3.4)
19.4 (1.5)
2c
2 400 (480)
178 (40)
0.58 (0.12)
0.68 (0.10)
5 500 (1 600)
560 (92)
3c
4
15 000 (2 600) 0.89 (0.07)
10 100 (5 600) 0.63 (0.09)
5
6
7
8
1 700 (110)
880 (55)
49.6 (5.9)
315 (25)
0.95 (0.02)
0.88 (0.02)
1.01 (0.02)
0.98 (0.02)
0.85 (0.07)
3 600 (900)
1 800 (450)
270 (67)
265 (16.5)
4 700 (500)
0.48 (0.06)
0.58 (0.04)
0.45 (0.05)
0.89 (0.01)
0.79 (0.07)
2 500 (350)
62.2 (13.0)
76.8 (2.1) 1.1 (0.4)
56.4 (5.5) 12 000 (9 000) 43.1 (5.8)
b
b
–
–
–
–
–
–
–
–
–
–
b
b
b
9
1 700 (330)
–
b
10
11
12
13
14d
15d
16d
BTCP
12 000 (1 200) 1.01 (0.03)
15 000 (4 200) 0.74 (0.07)
–
13 600 (880)
1 200 (220)
> 100 µM
1.03 (0.01)
0.93 (0.03)
–
4 400 (760)
1 500 (560)
> 100 µM
0.63 (0.05)
0.91 (0.10)
–
6 400 (1 600)
80.5 (4.5) 15.0 (3.2)
22.4 (3.8)
–
b
b
–
–
–
> 100 µM
68.0 (3.9) 372 (167)
34.1 (5.0)
35.1 (5.2)
33.1 (6.3)
33.4 (8.4)
32.8 (7.1)
41 000 (3 000) 0.83 (0.11)
66 000 (6 000) 0.71 (0.05)
22 700 (4 900) 0.90 (0.10)
10 500 (1 200) 0.45 (0.04)
6 700 (990) 0.52 (0.07)
6 600 (2 900) 0.64 (0.16)
6 800 (2 800) 0.50 (0.07)
40 000 (23 000) 63.2 (7.0) 301 (112)
57 000 (22 000) 65.3 (3.8) 90 (47)
25 000 (7 900) 65.0 (8.0) 59.0 (29.3)
23 000 (2 800) 64.7 (9.5) 195 (85)
5 000
1.00
b
aInhibition by unlabelled drugs of specific [3H]TCP binding on specified sites; a single-site model was more probable, suggesting similar
c
d
e
affinities for both PCP2- and PCP3-sites; from [26]; from [11]; IC50 values computed according to a single-site model.
4.2. Inhibition of [3H]MK-801 binding
Table II. Inhibition by unlabelled drugs of specific [3H]MK-801
(Ki nM, Hill’s number) and [3H]TCP (Ki nM from table I) binding
As expected, competition experiments in rat cerebel-
lum membranes labelled with [3H]MK-801 revealed that
ligand interactions were better described by a single-site
model (PCP2-sites) (table II). Interestingly the Ki com-
puted from competition experiments in cerebellum mem-
branes labelled either by [3H]MK-801 or [3H]TCP
(PCP2-sites) were in the same range or even similar in
several instances (table II). Taken together these results
confirmed that: (i) MK-801 binds essentially one family
of sites in the cerebellum; (ii) these sites are very likely
similar to the 70% population, marked PCP2-sites, bound
by TCP itself.
to PCP2 in rat cerebellum membranes (SEM in brackets).
Compound PCP2 ([3H]MK801)
PCP2 ([3H]TCP)
Ki
Ki
nH
TCP
MK-801
1
2
25.7 (4.8)
5.6 (0.5)
1.00 (0.10)
0.97 (0.05)
0.96 (0.01)
1.00 (0.02)
59 (17)
9.3 (4.6)
53 000 (18 000)
5 800 (1 700)
586 (96)
7 300 (930)
2 900 (600)
b
b
3
–
–
4
5
6
7
8
9
9 600 (600)
2 400 (170)
1 170 (100)
59.1 (3.2)
300 (3.6)
2 600 (260)
0.98 (0.01)
0.98 (0.02)
0.98 (0.01)
0.99 (0.01)
1.01 (0.02)
1.00 (0.01)
15 000 (3 000)
12 100 (3 400)
2 600 (370)
65 (14)
b
–
4.3. Inhibition of [3H]BTCP binding
b
–
b
10
11
12
13
14
15
16
BTCP
28 000 (1 100) 0.98 (0.01)
–
Inhibition of [3H]BTCP binding to rat striatal mem-
branes (table III) revealed important structural features:
(i) an oxygen atom introduced in the six-membered ring
in place of a sulfur atom or a methylene group generally
decreased affinities for the DAT (1, 2, 4–6, 9–11, 13 and
14) whatever the aromatic moiety was; (ii) as expected,
BTCP-like compounds (13–16) interacted potently with
the DAT, 15 being at least as efficient as BTCP; (iii) when
comparing compounds differing only by substitutions on
the thiophenyl or furanyl moiety the pseudo-BTCP com-
9 800 (600)
1 100 (130)
> 100 µM
1.00 (0.01)
0.99 (0.01)
–
6 700 (1 700)
b
–
> 100 µM
40 000 (8 000) 0.97 (0.02)
42 000 (24 000)
59 000 (23 000)
26 600 (8 200)
24 000 (2 900)
> 100 µM
–
–
b
b
–
10 400 (1 400) 1.04 (0.02)
aA single-site model was more probable for 8, 9, 10, and 12 with
respective Hill’s numbers: 0.89 (0.01), 0.79 (0.07), 0.74 (0.07), and
b
0.91 (0.10) (table I); not tested.

327
Table III. Inhibition of [3H]BTCP binding to rat striatal membranes (IC50 nM, Hill’s number) by the unlabelled drugs (SEM in brackets) and
selectivity for DAT and PCP3-sites with respect to other sites.
Compound
DA transporter
nH
DA selectivity
PCP2
PCP3 selectivity
a
b
c
e
f
Ki
PCP3
0.34
PCP1
DAd
PCP2
PCP1
TCP
1
2
3
4
5
6
7
8
1 250
0.82
22.1
1.13
1.31
2.5
162
11.3
7.2
2.96
65.5
480
58 000 (10 000) 0.93 (0.07)
3 000
206
0.0003
0.006
0.006
0.029
0.19
0.0004
0.006
0.015
0.018
0.016
0.0004
192.9
0.004
0.035
0.14
0.018
0.11
0.001
241.9
–
7 200 (240)
1 400 (120)
9 100 (540)
940 (57)
1 800 (270)
403 (48.1)
295 (15)
0.89 (0.03)
1.00 (0.06)
0.89 (0.04)
0.91 (0.05)
0.93 (0.05)
1.06 (0.08)
1.01 (0.05)
0.95 (0.04)
1.07 (0.06)
0.91 (0.01)
0.94 (0.04)
0.90 (0.02)
0.93 (0.02)
0.97 (0.05)
1.0 (0.10)
165
23.6
0.61
0.55
2.04
8.12
0.93
0.39
0.13
0.02
34.2
5.14
1 636
0.03
0.63
0.08
0.72
6.50
0.0006
29.8
g
g
g
g
g
–
–
–
–
–
g
g
g
g
g
9
670 (75)
–
–
–
–
–
g
g
g
g
g
10
11
12
13
14
15
16
BTCP
1 500 (170)
295 (5.0)
31.2 (1.3)
210 (15.4)
98.2 (18)
5.9 (0.6)
–
–
–
–
19.7
0.05
0.05
0.002
–
0.0009
–
g
g
g
g
g
–
–
0.026
–
0.59
0.33
0.07
0.23
0.04
< 0.002
0.0025
0.0001
0.0005
0.0003
< 0.002
0.0024
0.0001
0.0006
0.002
1.77
2.89
15.3
4.3
< 0.004
0.007
0.0016
0.002
< 0.004
0.006
0.001
0.003
0.04
13.7 (3.7)
7.9 (0.2)
0.98 (0.01)
24.7
0.008
aKi(DA)/Ki(PCP3); bKi(DA)/Ki(PCP2); cKi(DA)/Ki(PCP1); dKi(PCP3)/Ki(DA); eKi(PCP3)/Ki(PCP2); fKi(PCP3)/Ki(PCP1); gnot determined, a
single-site model was more probable (see table I).
pounds 10–12 were the most potent (10 > 4 > 1; 11 > 5
> 2; 12 > 7 and 8 > 3)
5. Conclusion
Two lines of evidence can be drawn from the present
work which confirms hypothesis and previous observa-
tions:
4.4. Binding selectivities
— BTCP and BTCP-like structures are able to bind in
the rat cerebellum to sites marked here PCP3-sites.
Although the nature and function of these low affinity
TCP binding sites in the cerebellum remain unknown
they can probably play a role in the pharmacology of
BTCP-like molecules in addition to their well known
properties of DA re-uptake inhibition.
— importantly, a TCP-like and a pseudo-BTCP com-
pound built from a tetrahydropyranyl six-membered ring
displayed a high affinity and selectivity for low affinity
PCP binding sites in the rat cerebellum. Such specific
ligands might be valuable tools to elucidate the nature
and function of these sites.
Finally, the binding selectivity of the molecules for: (i)
the DAT with regard to the three PCP sites and (ii) the
selectivity for the PCP3-sites with regard to the DAT,
PCP1- and PCP2-sites were examined (table III). Among
the molecules tested, BTCP and 15 were the most
selective ligands for the DAT. However, this selectivity
was moderate because they bind only 26 and 15 times
better to the DAT than to the PCP3-sites, respectively.
Interestingly, a progressive increase in the selectivity for
the DAT is observed when passing from TCP-like to
pseudo-BTCP molecules and finally to BTCP-like mol-
ecules. Suitable pharmacomodulations might thus yield,
in the future, molecules devoid of affinity in the cerebel-
lum. On the contrary, interesting selectivities for the
PCP3-sites were encountered in TCP-like compounds
especially with molecules 1 and 6. It is noticeable that
both are built from a tetrahydropyranyl moiety which
hypothetically might contribute, partly at least, to the
observed selectivities.
6. Experimental protocols
6.1. Chemistry
Melting points (uncorrected) were determined with a
digital melting point apparatus (Electrothermal, IA9300).

328
Table IV. 13C-NMR spectra of hydroclorides in CDCl3 at 50.323 MHz (δa ppm from TMS).
Carbon
1
4
5
6
7
8
9
10
11
12
13
2,6
3,5
4
α
â
64.3
30.8
65.6
47.3
22.7
22.3
146.3
114.1
111.4
144.6
–
64.3
30.6
65.6
46.9
22.5
22.2
143.7
115.1
107.4
154.6
–
64.2
33.3
67.1
46.7
22.7
22.4
134.4
132.4
138.9
124.0
15.2
64.1
33.2
67.0
46.6
23.4
22.6
134.2
131.2
145.7
122.6
22.3
14.2
–
25.3
33.5
69.3
47.1
22.9
22.3
134.5
131.4
145.8
122.5
22.3
14.2
–
25.2
33.4
69.4
47.0
23.3
22.8
131.7
124.4
130.4
151.0
–
64.1
33.2
67.0
46.8
22.6
22.3
135.3
128.1
141.3
124.7
130.0
115.5
–
64.3
30.6
65.7
46.9
22.6
22.3
142.4
117.2
115.8
150.1
9.7
64.2
33.1
67.1
46.5
22.6
22.2
134.3
133.0
136.7
128.8
13.6
25.2
33.3
69.2
46.9
22.8
22.3
134.3
133.1
136.6
129.2
13.5
64.3
30.8
66.0
46.7
22.6
22.2
148.1
111.3
126.9
154.8
–
γ
2′
3′
4′
5′
R1
R2
–
13.6
–
22.3
15.3
11.5
13.1
13.0
–
aItalicized chemical shifts may be exchanged.
Yields were not optimized. Elemental analysis was per-
formed at the CNRS Microanalytical Section in Mont-
pellier on the hydrochloride salts and were within ± 0.4%
of theoretical values. 13C-NMR spectra were obtained on
a Bruker AC200 spectrometer at 50.32 MHz in 5 mm
sample tubes in the FT mode. For some 13C-signal
assignments, a spin-echo sequence (Jmod) was used.
Chemical shifts (δ) are reported in ppm downfield from
TMS and collected in table IV. GC/MS analyses were
performed on a Hewlett-Packard 5890 instrument
equipped with a 9825B computer through a 25 m OV-1
capillary column. For NMR measurements and binding
experiments, compounds were used as their hydrochlo-
ride salts precipitated by adding a dry HCl ethereal
solution to a solution of base in ether. After filtration, the
solid collected was dried under vacuum.
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
then extracted with ether and the organic layer was
washed with water until neutrality, dried over MgSO4,
filtered, and concentrated under vacuum to yield 5.3 g
(98%) of 4-piperidin-1-yl-tetrahydro-thiopyran-4-carbo-
nitrile as an orange oil pure enough for use in the next
step.
4-Piperidin-1-yl-tetrahydro-pyran-4-carbonitrile
was similarly obtained from tetrahydro-pyran-4-one
(80.6%, F = 47 °C).
6.1.2. Synthesis of 3-hydroxymethyl-2-methyl-furan
Ethyl
2-methyl-furan-3-carboxylate
(24.5 g,
159 mmol, 1 eq.) was added to an ethereal solution (1 M)
of lithium aluminium hydride (4.8 g, 127 mmol, 0.8 eq.)
and the mixture was refluxed for 2 h. The vessel was
cooled in an ice bath and 5 mL of ice-cold water, then
5 mL of 15% NaOH, and 14.5 mL of water were succes-
sively added with caution. The white precipitate of LiOH
was filtered and rinsed with ether. The organic layer was
dried over MgSO4 and concentrated under vacuum to
yield an oily compound distilled under reduced pressure
to obtain 15.5 g (87%) of a colourless oil. GC/MS Rt =
Synthesis of compounds 2 [26, 27], 3, 14, 15 and
16 [11, 26] have been previously described. A typical
synthesis of new compounds is detailed in section 6.1.7.,
however their purification and properties are collected in
table V.
6.1.1. Synthesis of α-aminonitriles
Acetone cyanohydrine (2.2 g, 25.8 mmol, 1 eq.) was
added dropwise to a stirred mixture of tetrahydro-
thiopyran-4-one (3 g, 25.8 mmol, 1 eq.), anhydrous

329
Table V. Purification and properties of new compounds.
Purification method Solvent (v/v)
Fbase (°C)
FHCl (°C)
GC/MS (min, m/e)
Yield (%)
1
4
5
6
7
8
9
column chromatography (SiO2) petroleum ether/ether (60/40) –
column chromatography (SiO2) petroleum ether/ether (75/25) 76.6–77.0
column chromatography (SiO2) petroleum ether/ether (90/10) –
column chromatography (SiO2) petroleum ether/ether (80/20) oil
column chromatography (SiO2) petroleum ether/ether (95/5) oil
180.7–180.9
230
14.11, 235
14.49, 249
20.91, 285
17.84, 279
19.40, 295
21.63, 295
19.02, 277
15.67, 263
19.33, 279
21.81, 295
19.01, 285
42
34.8
22.7
34.6
56.1
64.4
37.5
63.9
49.1
67.8
38.3
209.4–209.8
165.2–165.5
154.4–154.6
164.8–165.1
177.8–178.0
160
170
175
209.6–210.2
crystallization
petroleum ether
69.5–69.7
column chromatography (SiO2) petroleum ether/ether (70/30) oil
10 column chromatography (SiO2) petroleum ether/ether (70/30) 106.2–106.6
11 column chromatography (SiO2) petroleum ether/ether (70/30) 97.7–98.0
12 crystallization
ether
118.4–118.7
13 column chromatography (Al2O3) petroleum ether/ether (90/10) 111.2–111.5
5.06 min, m/e = 112; 1H-NMR 7.22 (d, 1H 5), 6.32 (d, 1H
4), 4.34 (s, 2H), 3.20 (s, 1H, OH), 2.22 (s, 3H); 13C-NMR
148.9 (C2), 140.1 (C5), 118.6 (C3), 110.8 (C4), 55.9
(CH2OH), 11.2 (CH3).
trated under vacuum to yield 3.1 g (86%) of a pale yellow
1
oil. H-NMR 7.35 (s, 1H 5), 7.00 (s, 1H), 6.97 (s, 1H),
2.70 (q, 2H), 1.29 (t, 3H); 13C-NMR 144.5 (C3), 127.8
(C4), 125.1 (C5), 119.1 (C2), 23.3 (CH2), 14.6 (CH3).
6.1.3. Synthesis of 2,3-dimethyl-furan
6.1.5. Synthesis of 3-vinyl-thiophene
Pyridine (11.3 mL, 140 mmol, 1 eq.), thionyl chloride
(17.2 g, 140 mmol, 1 eq.), 3-hydroxymethyl-2-methyl-
furan (15.5 g, 138 mmol, 1 eq.) in 26 mL of petroleum
ether (30–40 °C), and 17 mL of anhydrous ether were
successively added to a stirred mixture of 175 mL of
petroleum ether (30–40 °C) and Na2CO3 (16.1 g,
152 mmol, 1.1 eq.) at –20 °C. The mixture obtained was
stirred at 0 °C for 45 min, then at room temperature for
30 min and filtered. The filtrate, kept at 0 °C, was
concentrated under vacuum to yield a brown oil. Lithium
aluminium hydride (3.9 g, 105 mmol, 0.75 eq.) was
added to a solution of the crude oil dissolved in diglyme
(87 mL) at 0 °C. The mixture was stirred for 16 h at room
temperature, then cooled at 0 °C, treated with 17.5 mL of
water and distilled. The organic layer was washed with
water, then with a copper (II) sulfate aqueous solution,
and three times with water. The organic layer was then
stirred for 10 min with coal, copper and potassium
carbonate, filtered, and distilled to yield 8.2 g (61.9%) of
A solution of 3-acetyl-thiophene (12 g, 95 mmol, 1 eq.)
in 20 mL of ether was added dropwise to a solution of
LiAlH4 (5.2 g, 137 mmol, 1.45 eq.) in ether (40 mL). The
mixture was stirred at room temperature for 4 h, then
cooled to 0 °C, and 5 mL of water, 5 mL of NaOH 15%,
and 15 mL of water were successively added with cau-
tion. The white precipitate of LiOH was filtered and
washed with ether. The combined organic phases were
dried over MgSO4, concentrated in vacuum to yield
11.0 g (90.5%) of 1-thiophene-3-yl-ethanol as a yellow
oil. A mixture of 1-thiophene-3-yl-ethanol (10 g,
78 mmol) and hydroquinone (5 mg) was distilled at
190 °C and the distillate decanted. The organic layer,
dried over MgSO4, was concentrated in vacuum to yield
2.8 g (33%) of a colourless oil. GC/MS Rt = 6.03 min,
m/e = 128.
6.1.6. Synthesis of 2,3-dimethyl-thiophene
3-Methyl-thiophene-2-carboxaldehyde
(25 g,
1
198 mmol, 1 eq.) and hydrazine hydrate 88% (21.7 mL,
500 mmol, 2.5 eq.) were added to a solution of KOH
(28.05 g, 0.50 mol, 2.5 eq.) in 150 mL of ethylene glycol
at 60 °C. The mixture was heated at 160 °C for 1 h, then
distilled at 180 °C; the distillate was decanted, the aque-
ous phase was extracted with ether and the combined
organic layers washed with water until neutrality, then
dried over MgSO4, concentrated under vacuum to yield a
yellow oil. A distillation yielded 19.6 g (88%) of a
2,3-dimethylfurane as a colourless oil. H-NMR 7.23 (d,
1H 5), 6.18 (d, 1H 4), 2.22 (s, 3H 2-Me), 1.97 (s, 3H
3-Me).
6.1.4. Synthesis of 3-ethyl-thiophene
3-Acetylthiophene (4.03 g, 32 mmol, 1 eq.) and hydra-
zine hydrate 88% (3.44 mL, 71 mmol, 2.5 eq.) was added
to a solution of KOH (3.65 g, 65 mmol, 2 eq.) in ethylene
glycol (24.3 mL) at 60 °C, heated for 1 h at 160–180 °C,
and distilled at the same temperature. The distillate was
decanted, the organic layer separated, and the aqueous
layer extracted with ether. The combined organic layers
were washed with water, dried over MgSO4, and concen-
1
colourless oil. H-NMR 7.05 (d, 1H 5), 6.86 (d, 1H 4),
2.43 (s, 2-CH3), 2.23 (d, 3-CH3); 13C-NMR 132.1 (C3),
131.7 (C2), 129.1 (C4), 119.7 (C5), 12.7 (3-CH3), 12.1
(2-CH3).

330
6.1.7. Typical synthesis of TCP and BTCP analogues:
1-[4-(2-furyl)-tetrahydro-pyran-4-yl]piperidine 1
filtration over GF/B (Whatman) glass fibre pre-soaked in
0.05% polyethyleneimine (Aldrich) with an MR24 Bran-
del cell harvester. The filters were rinsed three times with
5 mL of 50 mM NaCl, Tris HCl 10 mM, pH 7.7 buffer
and the radioactivity retained was counted in 3.5 mLACS
(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.
Firstly, a MgBr2 solution was prepared from 1,2-
dibromo-ethane (9.0 g, 48 mmol, 4 eq.) in ether (100 mL)
and magnesium turnings (1.2 g, 48 mmol, 4 eq.) 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 (40 mL, 60 mmol, 5 eq.) to a mixture of furane
(4.4 mL, 60 mmol, 5 eq.) and TMEDA (9.0 mL,
60 mmol, 5 eq.) in anhydrous ether (130 mL) at –20 °C
(without TMEDA in the synthesis of 5, 6, 7 and 9). The
mixture was then stirred for 2 h at room temperature and
added dropwise to the MgBr2 solution in ether. The new
mixture was stirred at room temperature for 0.5 h, then a
solution of 4-piperidin-1-yl-tetrahydro-pyran-4-carbo-
nitrile (2.3 g, 12 mmol, 1 eq.) in ether was added drop-
wise at room temperature. The mixture, refluxed for 16 h,
cooled to room temperature, was treated as follows: the
mixture was poured carefully onto an ice-cold saturated
solution of NH4Cl, stirred for 30 min, extracted with
ether, the combined organic layers were extracted 3 times
with 10% HCl and 20% NH4OH was added to the acidic
aqueous phase until neutrality. The aqueous phase was
extracted with ether, the organic phase washed with
water, dried over Na2SO4, filtered and concentrated under
vacuum. The crude product obtained was purified as
described in table V to yield 1.18 g (42%) of 1 as a white
solid.
6.2.1.2. [3H]MK-801
[3H]MK-801 (New England Nuclear, 20.3 Ci/mmol)
binding to the PCP1-, PCP2- and PCP3-sites was mea-
sured as described above for [3H]TCP (see 6.2.1.1.) with
incubation times fixed at 3 h instead of 30 min.
6.2.1.3. [3H]BTCP
[3H]BTCP binding to the DAT was measured accord-
ing to a previously described method [4]. Rat striata
stored at –80 °C were homogenized with an ultraturax in
a 320 mM sucrose, 10 mM Tris/HCl (pH 7.4) buffer and
centrifuged at 1 000 g for 10 min. The supernatant was
then centrifuged at 49 000 g for 20 min. The resulting
pellet (crude membrane preparation) was resuspended in
the same buffer (1 mL per striatum). The homogenate
(0.05–0.1 mg protein/mL) was incubated with [3H]BTCP
(0.2–0.5 nM) (C.E.A, 55 Ci/mmol) in the absence or in
the presence of the competing drug in a 50 mM Na2HPO4
(pH 7.4) buffer in a volume of 2 mL for 90 min at 4 °C.
The incubation was terminated by filtration over GF/B
glass fibre filters (Whatman) pre-soaked in 0.5% polyeth-
yleneimine (Aldrich) with an MR24 Brandel cell har-
vester (Beckman). The filters were rinsed three times with
5 mL of a 50 mM NaCl, Tris/HCl 10 mM (pH 7.7) buffer
and the radioactivity retained counted in 3.5 mL ACS
(Amersham) with an Excel 1410 (LKB) liquid scintilla-
tion spectrophotometer. The non-specific binding was
determined in parallel incubations in the presence of
100 µM unlabelled BTCP.
6.2. Pharmacology
6.2.1. Binding assays
6.2.1.1. [3H]TCP
[3H]TCP (Amersham, 48 Ci/mmol) binding to the
PCP1-, PCP2- and PCP3-sites was measured as previously
described [23]. Briefly, the rat (Wistar) brain (minus the
cerebellum) or the cerebellum, stored at –80 °C, 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 49 000 g
for 20 min. The pellet was resuspended in the same buffer
and the homogenization–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.
6.2.1.4. 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 analysed by the Hill’s representation and
then by a non-linear regression method (Marquardt-
Levenberg algorithm) according to a single or two-site
model using the Sigmaplott 4 software (Jandel). The
two-site interaction was represented by: [LB] = (B1 + B2)
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
binding) or in the presence of the competing drug for
30 min at 25 °C. The incubation was terminated by
– {([I] × B1/(IC50 + [I])) + ([I] × B2/(IC50 + [I]))}, where
1
[LB] was the % of radioligand specificall2y bound, [I] the
competitor concentration, B1 and B2 the % of each
binding site, IC50 and IC50 the concentrations of unla-
1
2

331
belled competitor that inhibited 50% of specific [3H]TCP
binding on specified sites. Two constraints were fixed: (i)
IC50 , IC50 , B1, B2 > 0, (ii) 95% < (B1 + B2) < 105%
beca1use of2 the uncertainty on the total binding (10%).
Experimental results were submitted to an ANOVA fol-
lowed 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 was between 1.5 and 2.5. Finally Ki
values were computed from the Cheng-Prusov equation:
Ki = IC50/(1 + L/KD).
[7] Duterte-Boucher
D.,
Kamenka
J.M.,
Costentin
J.,
Psychopharmacology 101 (1990) 344–353.
[8] French E.D., Lopez M., Peper S., Kamenka J.M., Roberts D.C.S.,
Behav. Pharmacol. 6 (1995) 223–228.
[9] Prinssen E.P.M., Kleven M.S., Vignon J., Kamenka J.M., Koek W.,
J. Pharmacol. Exp. Ther. 276 (1996) 904–911.
[10] Loustau-Then I., Ponchant M., Fuseau C., Kamenka J.M., Vignon J.,
Crouzel C., Nucl. Med. Biol. 24 (1997) 513–518.
[11] Hamon J., Espaze F., Vignon J., Kamenka J.M., Eur. J. Med. Chem.
34 (1999) 125–135.
[12] Bruylants P., Bull. Soc. Chim. Belg. 33 (1924) 467–478 and 35
(1926) 139–154.
[13] Geneste P., Kamenka J.M., Dessapt P., Bull. Soc. Chim. Fr. 3 & 4 II
(1980) 186–191.
[14] Minato A., Tarrao K., Hayashi T., Susuki K., Kumada M.,
Tetrahedron Lett. 52 (1981) 5319–5322.
[15] Winberg H.E., Fawcett F.S., Mochel W.E., Theobald C.W., J. Am.
Chem. Soc. 82 (1960) 1428–1435.
Acknowledgements
[16] Rice K.C., Dyer J.R., J. Heterocycl. Chem. 12 (1975) 1325–1326.
[17] Mann J., Holland H., Tetrahedron 43 (1987) 2533–2542.
The authors wish to thank D.R.E.T. for the financial
support given to F. Espaze. Thanks are due to M.
Michaud for her excellent technical assistance.
[18] Mohamadi F., Spees M., Grindey J., J. Med. Chem. 35 (1992)
3012–3016.
[19] Yoshironi T., Milton L., Raymond N., J. Heterocycl. Chem. 18
(1981) 967–972.
References
[20] Rabjohn N. (Ed.), Organic Synthesis, Coll. Vol. IV, Wiley, London,
1963, p. 628.
[1] Vignon J., Pinet V., Cerruti C., Kamenka J.M., Chicheportiche R.,
Eur. J. Pharmacol. 148 (1988) 427–436.
[21] Geneste P., Kamenka J.M., Org. Magn. Res. 7 (1975) 579–584.
[22] Manoharan M., Eliel E.L., Kanan W., Tetrahedron Lett. 25 (1984)
3267–3268.
[2] Ilagouma A.T., Maurice T., Duterte-Boucher D., Coderc E., Vignon
J., Costentin J., Kamenka J.M., Eur. J. Med. Chem. 28 (1993)
377–385.
[23] Vignon J., Privat A., Chaudieu I., Thierry A., Kamenka J.M.,
Chicheportiche R., Brain Res. 378 (1986) 133–141.
[3] Cerruti C., Drian M.J., Kamenka J.M., Privat A., Brain Res. 55
(1991) 51–57.
[24] Ebert B., Wong E.H.F., Krogsgaard-Larsen P., Eur. J. Pharmacol.
208 (1991) 49–52.
[4] Maurice T., Vignon J., Kamenka J.M., Chicheportiche R., J. Neu-
rochem. 56 (1991) 553–559.
[25] Berman F.W., Murray T.F., J. Biochem. Toxicol. 11 (1996) 217–226.
[5] Martin-Fardon R., Arnaud M., Rousseau E., Kamenka J.M., Privat
A., Vignon J., Neurosci. Lett. 211 (1996) 179–182.
[26] Hamon J., Vignon J., Kamenka J.M., Eur. J. Med. Chem. 31 (1996)
489–495.
[6] Koek W., Colpaert F.C., Woods J.H., Kamenka J.M., J. Pharmacol.
Exp. Ther. 250 (1989) 1019–1027.
[27] Eiden F., Schmidt M., Buchborn H., Arch. Pharmacol. 320 (1987)
348–361.