Preparation and affinity profile of novel nicotinic ligands

Article abstract of DOI:10.1016/j.bmcl.2007.12.075

Novel nicotinic ligands, characterized by the presence of an amino substituted cyclopropane ring connected to a pyridine nucleus, are described. Pharmacological investigation revealed that these compounds exhibit highest affinity for the rat α4β2 subtype of the nicotinic receptor with no affinity for the muscarinic receptor. No appreciable affinity for the muscular or for the ganglionic nicotinic receptor was observed at concentrations up to 10 μM. The increase in cortical ACh release as well as a positive effect on memory in a social recognition test in rat are exemplified.

Full text of DOI:10.1016/j.bmcl.2007.12.075

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 2188–2193
Preparation and affinity profile of novel nicotinic ligands
Yves Charton,a Claude Guillonneau,a Brian Lockhart,b
Pierre Lestageb and Solo Goldsteina,*
aSERVIER, Chemistry Research Division A, 11 rue des Moulineaux, 92150 Suresnes, France
bSERVIER, Division of Cerebral Pathology, 125 Chemin de Ronde, 78290 Croissy, France
Received 17 October 2007; revised 20 December 2007; accepted 21 December 2007
Available online 26 January 2008
Abstract—Novel nicotinic ligands, characterized by the presence of an amino substituted cyclopropane ring connected to a pyridine
nucleus, are described. Pharmacological investigation revealed that these compounds exhibit highest affinity for the rat a4b2 subtype
of the nicotinic receptor with no affinity for the muscarinic receptor. No appreciable affinity for the muscular or for the ganglionic
nicotinic receptor was observed at concentrations up to 10 lM. The increase in cortical ACh release as well as a positive effect on
memory in a social recognition test in rat are exemplified.
Ó 2008 Elsevier Ltd. All rights reserved.
Nicotine(S(ꢀ)nicotine) and nicotinic acetylcholine
receptors (nAChR) have been known and studied for a
long time, but it is only recently that renewed interest
and research has focalized on their potential role in dis-
ease and therapy.1
potent antagonist of receptors containing b2 and b4 sub-
units,3d possessing beneficial effects on neurodegeneres-
cence and cognitive dysfunction.3e
Our own research effort was aimed at developing novel
specific nicotinic ligands binding selectively to the a4b2
nAChR subtype.
Indeed, evidence coming from clinical investigations
demonstrated that S(ꢀ)nicotine is beneficial for age-re-
lated memory impairments and in particular attention
deficits.2 However, until recently clinical use of nicotinic
ligands has been thought to be severely limited by car-
diovascular and neuromuscular side effects resulting
from a non-selective activation of nAChR. Nevertheless,
recent studies indicate that it is possible to develop selec-
tive nicotinic agents with marked effects on cognition
but without some of the nicotine-related side effects.2,3a
Thus we have previously shown that ACh carbamate
analogs (I) with reduced conformational flexibility
induced by a cyclopropane ring are specific nicotinic
ligand versus muscarinic receptors, binding selectively
to the non-a7 subtype of nAChR. Investigation of
related compounds possessing a pyridine ring as a nico-
tinic element (II) further demonstrated the strength of
this approach for the design of new, powerful, and selec-
tive nicotinic ligands (Fig. 1).4,5
Thus ispronicline (TC-1734) showed good affinities for
the a4b2 nicotinic subtype and negligible affinities for
other subtypes. Oral administration increases cortical
ACh release (agonist effect) improving short and long
time memory. Furthermore, encouraging clinical results
have been reported recently.3b
In this paper, we present synthetic details for com-
pounds belonging to the general formula II as well as
binding studies permitting the determination of the
structural elements influencing potency and selectivity
with respect to nicotinic receptors.
Another example is the anabaseine analog GTS-21, a
weak partial agonist at the a7 subtype3c and a more
From the general formula II it is apparent that three
structural elements can be modulated, namely the linker,
the amino-head substitution, and the pyridine ring
substitution.
Keywords: Nicotinic; Subtypes; Ligands; Selectivity; a4b2; Substituted
cyclopropanes; Pyridine ring.
We therefore set up a synthetic program aimed at the
preparation of representative members of each class.
*
Corresponding author. Tel.: +33 1 55 72 25 99; fax: +33 1 55 72 24
30; e-mail: solo.goldstein@fr.netgrs.com
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.12.075

Y. Charton et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2188–2193
2189
H3C
O
CH3
CH3
N+
O
CH3
R1
R1
R3
R4
(linker)
N
(linker)
O
O
N
(CH3)2N
R2
R2
N
I
II
Figure 1. Nicotinic ligands with reduced conformational flexibility.
Most of the compounds have been obtained via a com-
mon alcohol intermediate, itself obtained from the com-
mercially available diester of cyclopropane 1,1
dicarboxylic acid (Scheme 1).
All synthesized compounds lack muscarinic activity;
a4b2 nAChR specificity versus a7 nAChR is observed
for all compounds with either type of linker.
The most powerful ligand in the series (Ki, 3 nM), is 1a
possessing an ether linker; carbon linkers (saturated or
unsaturated) containing compounds are less powerful
binders but are still selective for the a4b2 nAChR
subtype.
Thus seven different linkers and 14 N-monomethylated
analogs have been prepared from this key intermediate
(Scheme 2). An Eischweiler–Clark reaction allowed the
transformation of N-monomethylated compounds into
N-dimethylated derivatives (Scheme 3) while N-unsub-
stituted analogs were obtained via an N-Boc carbamate
already used for the synthesis of the common key alco-
hol (Scheme 4).
The amino-head substitution has been examined (Table 2)
on analogs of the most powerful ligand found, 1a.
Specific binding for a4b2 nAChR subtype versus a7
nAChR and muscarinic receptors is observed.
All compounds have been fully characterized by 1H, 13
C
NMR, and LCMS; final compounds possessed elemen-
tal analysis within accepted limits ( 0.4%) from theory.6
The order of binding is NHCH3 > NH2 > N(CH3)2,
compound 1a still remaining the best in the series.
Free bases were converted to their hydrochlorides or
fumarate salts and purified by crystallization.
Increasing the steric hindrance on the amino-head
decreases receptor binding (1a vs 15, 1b vs 16).
Binding experiments for a7, a4b2, and muscarinic recep-
tors were carried out in a final volume of 250 ll in 96-
well plates.7–9
Next parameter studied was the pyridine ring substitu-
tion on positions 5 and 6 (monomethylated amino-head,
Table 3).
The following tables illustrate the influence of each
parameter mentioned above (linker, amino-head substi-
tution, pyridine substitution) on the binding observed
for the a4b2, a7, and muscarinic receptors.
For the first time a muscarinic affinity was observed for
vinyl substituted pyridine 8, the affinity for the a4b2
nAChR subtype being roughly 2-fold higher (no affinity
for a7 subtype was observed).
Thus both heteroatoms containing and C-only linkers
have been investigated and compared (Table 1).
All other compounds are specific ligands for the a4b2
nAChR subtype. Substitution of the pyridine ring has
a beneficial effect on binding leading to 5a the most
powerful compound obtained.
a
b
H3COOC
H3COOC
COOCH3
NH-Boc
H3COOC
COOH
Although 5-bromo, 6-chloro di-substitution led to the
very powerful and specific ligand 6a, the order of
magnitude was comparable with that obtained for
mono-substituted, or non-substituted compounds 1a, 7
(slightly less than 5a).
d
c
Boc
Boc
HO
H3COOC
N
N
CH3
CH3
Subtle effects on a4b2 binding depending on the nature
of the substituents and their position was observed.
Thus 5a (Br in the 5 position) has the largest binding
while a nearly 40-fold drop in binding is observed with
Scheme 1. Synthesis of a key alcohol intermediate. Reagents and
condition: (a) NaOH 1 N, CH3OH (95%); (b) NEt3, DPPA, toluene,
tert-butanol (72%); (c) NaH, CH3I, DMF (82%); (d) LiBH4, THF
(82%).

2190
Y. Charton et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2188–2193
e
O
CH3
CH3
CH3
O
d
c
f
NH-CH3
HOH2C
N
HOH2C
N
NH-CH3
H3COOC
N
Boc
Boc
Boc
11
N
N
12 trans
b
HO
O
j
g
h
i
CH3
N
Boc
CH3
O
CH3
N
NH-CH3
N
N
N
N
NH-CH3
Boc
Boc
10 cis
13 cis
k
a
NH-CH3
HO
CH3
N
9
N
Boc
m
l
q
r
n
O
Br
O
O
CH3
Br
CH3
NC
CH3
CH3
N
NH-CH3
N
N
N
HO
Boc
Boc
Boc
Boc
8
N
N
N
o
p
s
O
O
NH-CH3
NH-CH3
1a-6a
NH-CH3
R
7
14
N
N
R = H (1a), 6-CH3 (2a), 6-Cl (3a),
6-Br(4a), R= 5-Br (5a), RR' =
5-Br,6-Cl (6a)
Scheme 2. Synthesis of N-monomethylated compounds. Reagents: (a) ClCOCOCl, DMSO, CH2Cl2 (87%); (b) Ph3P@CHCOOEt, CH2Cl2 (trans/
cis > 90/10) (95%); (c) H2, Pd/C, EtOH (90%) then LiBH4/THF (97%); (d) 3-hydroxypyridine, Ph3P–PS resin, DIAD, THF (75%) then HCl/dioxan
and fumaric acid, EtOH (60%); (e) dibal/hexane, CH2Cl2 (65%); (f) 3-hydroxypyridine, Ph3P–PS resin, DIAD, THF (40%) then HCl/dioxan (50%);
(g) (PhO)2POCH2COOt-Bu, Triton B, THF (75%) then dibal/hexane (45%); (h) 3-hydroxypyridine, Ph3P–PS resin, DIAD, THF (45%) then HCl/
dioxan (40%); (i) 3-(Ph3P + CH2)-pyridine, Clꢀ, t-BuOK, DMSO (55%); (j) HCl/dioxan then fumaric acid, EtOH (50%); (k) H2, Pd/C, MeOH (90%)
then HCl/dioxan (95%); (l) CBr4, PPh3, ether (95%); (m) 3,5-diBr-pyridine, HNa, DMSO (81%); (n) Pd(Ph3P)4, H2C@CHSnBu3, toluene (80%) then
HCl/dioxan (65%); (o) Pd(PPh3)2Cl2, (CH3)3SiCCH, Et3N, CuI (78%) then TBAF, THF and HCl/dioxan (80%); (p) 5 or 6-substituted 3-
hydroxypyridine, Cs2CO3, butanone (70–90%) then HCl/EtOH (60–85%); (q) KCN, KI, DMSO (95%); (r) HCl/MeOH (73%) then Boc2O, DMAP,
CH2Cl2 (90%) and LiBH4/THF (84%); (s) 3-hydroxypyridine, PH3P, DIAD, THF (70%) then HCl/dioxan and fumaric acid, EtOH (80%).
surprisingly a 6-chloro substitution induced a better
binding.
O
O
CH3
NH-CH3
N
a,b
R
R
CH3
1a-6a
N
N
1b-6b
All these results are most probably due at least in part,
to subtle steric and stereoelectronic effects influencing
the interactions of the pyridine nitrogen and/or the pyr-
idine ring as a whole with the a4b2 receptor site.
R = H (1a,1b), 6-CH3 (2a,2b), 6-Cl (3a,3b), 6-Br (4a,4b),
R= 5-Br (5a,5b), RR' = 5-Br,6Cl (6a,6b)
Scheme 3. Synthesis of N-dimethylated compounds. Reagents: (a)
HCHO, HCOOH (75–95%); (b) HCl, EtOH (73–96%).
Table 4 below illustrates the results obtained when vary-
ing both the amino-head substitution (e.g., no substitu-
tion, methyl, dimethyl substitution) and the pyridine
ring substitution (e.g., no substitution, methyl, chloro,
bromo substitution).
O
a-d
NH2
R
H3COOC
NH-Boc
Regardless of the substitution pattern on the pyridine
ring, the binding ranking follows the order
NHCH3 > NH2 > N(CH3)2, most potent compounds in
the series being monomethylated 1a, 5a (5-bromo substi-
tution on the pyridine ring), and 6a (5-bromo, 6-chloro
substitution on the pyridine ring).
N
1c-4c
R = H (1c), 6-CH3 (2c), 6-Cl (3c), 6-Br(4c)
Scheme 4. Synthesis of N-unsubstituted compounds. Reagents: (a)
LiBH4, THF (95%); (b) CBr4, PH3P, ether (65%); (c) 6-substituted 3-
hydroxypyridine, HNa, DMSO (45–65%); (d) HCl/dioxan (75–85%).
The drop in affinity of N(CH3)2 versus NHCH3
compounds can be anywhere between 10-fold and
300-fold (compare 5a, 5b); compared to NHCH3
compounds non-substituted amino-head derivates
4a (Br in the 6 position); the same drop in binding is
observed for the 6-methyl substituted pyridine 2a while

Y. Charton et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2188–2193
2191
Table 1. Linker dependence of binding
(linker)
NH-CH3
N
Compounda
Linker
Nicotinic affinity
Muscarinic affinity (Ki, nM)
a4b2 (Ki, nM)
a7 (Ki, nM)
1a
O–CH2
CH2–CH2
3
>10,000
1910
3460
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
85
9
10
251
1160
2460
1100
2280
109
39
CH@CH
11
12
O–CH2–CH2–CH2
O–CH2–CH@CHtrans
O–CH2–CH@CHcis
O–CH2–CH2
—
>10,000
>10,000
>10,000
>10,000
3509
13
14
ACh
Nicotine
—
4
1500
>10,000
a Compounds 1, 9, 12, and 13 were tested as HCl salts, compounds 10, 11, and 14 were tested as fumarates.
Table 2. Amino-head substitution dependence of binding
O
R1
N
R2
N
Compounda
N–R1R2
Nicotinic affinity
a4b2 (Ki, nM)
Muscarinic affinity
a7 (Ki, nM)
1a
1b
NHCH3
N(CH3)2
NH2
3
95
>10,000
>10,000
>10,000
>10,000
>10,000
3509
>10,000
>10,000
>10,000
>10,000
>10,000
85
1c
15
34
C2H5–NH
N-pyrrolidine
—
1010
1610
39
16
ACh
Nicotine
—
4
1500
>10,000
a Compounds 1, 9, 12, and 13 were tested as HCl salts, compounds 10, 11, and 14 were tested as fumarates. Synthesis of compounds 15 and 16 is
described in Ref. 5.
Table 3. Pyridine substitution dependence of binding
R3
R4
O
NH-CH3
N
Compounda
Pyridine substitution
Nicotinic affinity
Muscarinic affinity (Ki, nM)
a4b2 (Ki, nM)
a7 (Ki, nM)
1a
2a
—
3
40
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
3509
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
179
6-CH3
6-Cl
6-Br
3a
4a
13
38.8
1.07
5.37
6.56
70.1
39
5a
6a
5-Br
5-Br,6-Cl
5-Ethynyl
5-Vinyl
—
7
8
ACh
Nicotine
85
>10,000
—
4
1500
a All compounds were tested as HCl salts.

2192
Y. Charton et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2188–2193
Table 4. Binding dependence on cumulative pyridine and amino-head
substitution
3a amines or tertiary 1b–3b amines. Despite the lesser
steric hindrance this result might be explained by a lower
interaction energy with the receptor site due to less fit-
ting and/or loss of hydrophobic interaction coming from
the lacking methyl group.
R3
R4
O
R1
N
R2
N
All investigated compounds tested at concentrations up
to 10 lM did not demonstrate any appreciable affinity
neither for muscular ((a1)2bdc) (binding assays using
Compounda
Pyridine
substitution
NR1R2
Nicotinic
affinity a4b2
Ki (nM)
[
tissue) nor for the ganglionic nicotinic receptors (bind-
125I]a-bungarotoxin on Torpedo marmorata electroplax
1a
1b
1c
—
—
—
NHCH3
N(CH3)2
NH2
3
ing assays using [3H]-epibatidine on IMR-32 cells).
95
34
2a
2b
2c
6-CH3
6-CH3
6-CH3
NHCH3
N(CH3)2
NH2
40
300
1090
266
***
***
3a
3b
3c
6-Cl
6-Cl
6-Cl
NHCH3
N(CH3)2
NH2
13
250
200
150
100
50
***
***
***
*
304
98
**
4a
4b
6-Br
6-Br
NHCH3
N(CH3)2
38.8
394
**
5a
5b
5-Br
5-Br
NHCH3
N(CH3)2
1.07
273
6a
6b
5-Br,6-Cl
5-Br,6-Cl
NHCH3
N(CH3)2
5.37
110
a All compounds were tested as HCl salts.
controls (n=7)
compound 1a 0.3mk ip (n=6)
compound 1a 1mk ip (n=7)
compound 1a 3mk ip (n=8)
have somewhat lower but not negligible a4b2 binding
affinities.
i.p.
0
-90 -60 -30
0
30 60 90 120 150 180
As far as pyridine substitution is concerned it is appar-
ent from the data in Table 4 that Me or Br-substitution,
and to a lesser extent Cl-substitution, on the 6 position
of pyridine is detrimental for affinity. Br-substitution
on the 5-position is beneficial only for the NHCH3 but
not for the N(CH3)2 amino-head.
Time (min)
Figure 2. Effect of compound 1a on acetylcholine release in prefrontal
cortex of the freely moving rat. Values shown on percentage of control
value. *p 6 0.05, **p 6 0.01, ***p 6 0.001 versus control group.
Compared to mono-substitution on the pyridine ring,
dihalogen, 5-Br,6-Cl, substitution leads to the only com-
pounds (6a, 6b) having affinity of the same level as
unsubstituted pyridine compounds (1a, 1b).
Control
Compound 1a
10
0
(8)
(8)
(8)
(8)
In summary novel potent and selective a4b2 nicotinic
ligands have been obtained by combining structural
elements of ACh and of nicotine while restraining the
conformational mobility using a cyclopropane ring.
For the vast majority of the synthesized compounds
no muscarinic activity has been observed; most mole-
cules have exhibited high specificity toward the a4b2
subtype versus the a7 subtype. The a4b2 nAChR affinity
of these new ligands is modulated mainly by the nature
and length of the linker between the pyridine moiety and
the cyclopropane ring as well as by the degree of substi-
tution of the amine. The best a4b2 affinities are obtained
for the N-monomethylated compounds and an oxy-
methylene linker (ethers 1a–6a, 7). Good to excellent
affinities for the a4b2 nAChR subtypes are obtained
by the pyridine substitution on the 5 and/or 6 position.
The affinity observed for the primary amines 1c–3c is
intermediate between that observed for secondary 1a–
-10
-20
-30
-40
-50
-60
***
+++
+
***
+++
***
0
0.3
1
3
Dose (mg/kg i.p.)
Figure 3. Effect of compound 1a on the social Wistar rats. Values are
means sem with the number of animals/group in parentheses.
***p 6 0.001 versus control; +p 6 0.05 versus 1 mg/kg +++p 6 0.001
versus 3 mg/kg, ANOVA + Newman–Keuls.

Y. Charton et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2188–2193
2193
1H, NH+), 8.65 (m, 1H), 8.5 (m, 1H), 8.00 (m, 1H), 7.85
(m, 1H), 4.50 (s, 2H), 2.85 (s, 6H), 1.5–1.10 (m, 4H).
Other pharmaceutical properties are exemplified for
compound 1a. Thus 1a increased in a dose-dependant
manner (microdialysis, freely moving rats), the release
of cortical ACh (Fig. 2).10 This effect is indicative of
an agonist character. Moreover a positive effect on
memory was observed in the social recognition test in
Wistar rats (Fig. 3).11
1
Compound 1c: H NMR (DMSO d6-300 MHz) d 8.9 (sb,
3H, NH3þ), 8.65 (d, J = 2.0 Hz, 1H), 8.5 (d, J = 6.0 Hz,
1H), 8.10 (dd, J = 7.6, 2 Hz, 1H), 7.9 (m, 1H), 4.35 (s, 2H),
1.15–0.95 (m, 4H).
1
Compound 5a: H NMR (DMSO d6-300 MHz) d 9.7 (sb,
2H, NH2þ), 8.4 (m, 2H), 7.8 (m, 1H), 4.35 (s, 2H), 2.7 (s,
3H), 1.4–1.0 (m, 4H).
Compound 6a: 1H NMR (DMSO d6, 300 MHz) d 9.75 (sb,
2H, NH2þ), 8.25 (d, J = 2 Hz, 1H), 8.00 (d, J = 2 Hz, 1H),
4.35 (s, 2H), 2.65 (s, 3H), 1.3–0.95 (m, 4H).
Further pharmacological characterization of the above
compounds is ongoing and will be reported in due time.
7. For labeling of a7 nicotinic subtype rat brain cell mem-
branes (500 lg/ml) were incubated with [125I]a-bungaro-
toxine (2 nM) for 5 h at 37 °C. Non-specific binding was
determined by incubation of membrane preparations with
1 lM a-bungarotoxine Marks, M. J.; Stitzel, J. A.; Collins,
A. C. Pharmacol. Exp. Ther. 1985, 235, 619.
8. For labeling of a4b2 receptors, rat brain cell membranes
(250 lg/ml), were incubated with [3H]cytisine for 2 h at
room temperature. Non-specific binding was assessed by
the incubation of membrane preparations with 10 lM
S(ꢀ) nicotine Pabreza, L. A.; Dhawan, S.; Kellar, K. Mol.
Pharmacol. 1990, 39, 9.
Acknowledgments
We gratefully acknowledge the Cerebral Pathology
Division and the Chemistry Research Division A teams
as well as Dr. J.P. Bouchet and Mrs. C. Schaeffer for
analytical data and Mrs. D. Rostagni for the skillful typ-
ing of the manuscript.
References and notes
9. For labeling of muscarinic receptors (M2/M4) rat brain cell
membranes (250 lg/ml) were incubated with [3H]oxotre-
morine (2 nM) for 2 h at room temperature non-specific
binding being determined by incubation of membrane
preparations with 1 lM atropine Lockhart, B.; Closier,
M.; Howard, K.; Stewart, C.; Lestage, P. Naunyn Schmi-
edeberg Arch. Pharmacol. 2001, 363, 429.
1. (a) Jensen, A. A.; Frølund, B.; Liljefors, T.; Krogsgaard-
Larsen, P. J. Med. Chem. 2005, 48, 4705; (b) Romanelli,
N.; Gratteri, P.; Guandalini, L.; Martini, E.; Bonaccini, C.
ChemMedChem 2007, 2, 746.
2. (a) Dani, J. A. Biol. Psychiatry 2001, 49, 166; (b) Rezvani,
A. H.; Levin, E. D. Biol. Psychiatry 2001, 49, 258.
3. (a) Levin, E. D.; Bradley, A.; Addy, N.; Sigurani, N.
Neuroscience 2002, 109, 757; (b) Gatto, G. J.; Bohme, G.
A.; Caldwell, W. S.; Letchworth, S. R.; Traina, V. M.;
Obinu, M. C.; Laville, M.; Reibaud, M.; Pradier, L.;
Dunbar, G.; Bencherif, M. CNS Drug Rev. 2004, 10, 147;
(c) de Fiebre, C. M.; Meyer, E. M.; Henry, J. C.;
Muraskin, S. I.; Kem, W. R.; Papke, R. L. Mol.
Pharmacol. 1995, 47, 164; (d) Astles, P. C.; Baker, S. R.;
Boot, J. R.; Broad, L. M.; Dell, C. P.; Keenan, M. Curr.
Drug Targets CNS Neurol. Disord. 2002, 1, 337; (e)
Kitagawa, H.; Takenouchi, T.; Azuma, R.; Wesnes, K. A.;
Kramer, W. G.; Clody, D. E.; Burnett, A. L. Neuropsy-
chopharmacology 2003, 28, 542.
10. ACh levels: A guide-cannula was stereotaxically implanted
in the prefrontal cortex (AP: +3.3, L: ꢀ0.6, V: ꢀ0.5
relative to bregma). After 5–7 days of recovery, the
microdialysis probe (CMA11, 4 mm length, ; 0.23 mm)
was lowered in the guide-cannula. The dialysis probe was
continuously perfused at a flow rate of 1 ll/min with an
artificial cerebrospinal fluid (composition in mM: NaCl
147, KCl 2.7, MgCl2 1, CaCl2 1.2, adjusted at pH 7.4 with
2 mM phosphate buffer). Two hours after probe implan-
tation (stabilization period) in the rat prefrontal cortex,
dialysates were collected every 30 min and analyzed by
HPLC coupled to electrochemical detection. The first four
dialysates were considered as baseline. Drug administra-
tion began at the end of the baseline. Data are expressed in
percentage of the mean value of the baseline samples.
11. Social recognition: Adult male Wistar rats placed in their
standard home cages were exposed twice to a juvenile rat
(25–30 days old) in two 5 min sessions separated by a
retention interval of 120 min. Manual chronometric eval-
uation of exploration times (s) of the juvenile rats was
performed through a video-camera system. Decrease of
the exploration time toward the juvenile rate on the 2nd
session indicated that the adult rat recognized the juvenile
(increase of memory retention).
4. Goldstein, S.; Guillonneau, C.; Charton, Y.; Lockhart, B.;
Lestage, P. Eur. Patent 117081 A1, 2002; Chem. Abstr.
2002, 136, 86 625.
5. Lockhart, B.; Bonhomme, N.; Lebrun, C.; Roger, A.;
Guillonneau, C.; Charton, Y.; Goldstein, S.; Lestage, P.
Pharmacologist 2002, 44, A97.
6. Selected data: Compound 1a: 1H NMR (DMSO d6-
300 MHz) d 9.85 (sb, 2H, NH2þ), 8.65 (d, J = 2.0 Hz, 1H),
8.5 (d, J = 6.0 Hz, 1H), 8.05 (d, J = 7.6 Hz, 1H), 7.9 (m,
1H), 4.45 (s, 2H), 2.65 (s, 3H), 1.35–1.00 (m, 4H).
Compound 1b: 1H NMR (DMSO d6-300 MHz) d 11.4 (sb,