Synthesis and biological evaluation of N-methyl-laudanosine iodide analogues as potential SK channel blockers

Article abstract of DOI:10.1016/j.bmc.2004.11.025

Neuronal action potentials are followed by an afterhyperpolarisation (AHP), which is mediated by small conductance Ca²⁺-activated K⁺ channels (SK channels or KCa2 channels). This AHP plays an important role in regulating neuronal act

Full text of DOI:10.1016/j.bmc.2004.11.025

Bioorganic & Medicinal Chemistry 13(2005) 1201–1209
Synthesis and biological evaluation of N-methyl-laudanosine
iodide analogues as potential SK channel blockers
a
b
b
a,c
A. Graulich,a, F. Mercier, J. Scuvee-Moreau, V. Seutin and J.-F. Liegeois
*
´
´
a
`
University of Liege, Natural and Synthetic Drugs Research Center, Laboratory of Medicinal Chemistry,
ˆ
`
avenue de l’Hopital, 1 (B36), B-4000 Liege 1, Belgium
University of Liege, Research Center for Cellular and Molecular Neurobiology, Laboratory of Pharmacology, avenue de l’Hopital,
b
`
ˆ
`
3 (B23), B-4000 Liege 1, Belgium
` ˆ
University of Liege, Laboratory of Physiology, avenue de l’Hopital, 3 (B23), B-4000 Liege 1, Belgium
c
`
Received 23June 2004; accepted 9 November 2004
Available online 8 December 2004
Abstract—Neuronal action potentials are followed by an afterhyperpolarisation (AHP), which is mediated by small conductance
Ca2+-activated K+ channels (SK channels or KCa2 channels). This AHP plays an important role in regulating neuronal activity
and agents modulating AHP amplitude could have a potential therapeutic interest. It was previously shown that N-methyl-bicucul-
line iodide blocks SK channels but its GABAA activity represents a serious drawback. In view of the structural analogy between
bicuculline and laudanosine 14, several N-quaternary analogues of the latter were developed. It was shown that N-methyl-laudano-
sine 15 (NML) and N-ethyl-laudanosine 16 induce a reversible and relatively specific blockade of the apamin sensitive AHP in dopa-
minergic neurones with mean IC50s of 15, and 47 lM, respectively. Laudanosine 14, N-butyl-17 and N-benzyl-18 derivatives were
less potent. In order to find pharmacophore elements, modifications were performed at different positions such as C-1, C-6 and C-7.
Intracellular recordings on rat midbrain dopaminergic neurones were made in order to evaluate the putative blockade of SK chan-
nels by these molecules. Simplified structures such as tetrahydroisoquinoline derivatives with H or Me at C-1 1–6 presented no sig-
nificant activity at 300 lM. The presence of a 1-(3,4-dimethoxybenzyl) moiety seems an important feature. Indeed, compound 8
showed a blockade of the AHP of only 33% at 300 lM while compound 13 blocked it by 67%, respectively, at the same concentra-
tion. Binding experiments were also performed. Binding affinities for SK channels are in good agreement with electrophysiological
data. These results indicate that the presence of a charged nitrogen group is an essential point for the affinity on SK channels.
Finally, because of the similar activity of both enantiomers of NML 19 and 20, the interaction site may present a symmetrical
configuration.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
members of the SK family have been cloned: SK1,
SK2 and SK3.3 In contrast to BK channels, SK channels
are voltage insensitive and are not blocked by low con-
centrations of tetraethylammonium5 or charybdotoxin,6
but are only activated by an increased intracellular cal-
cium concentration. The distribution of the SK channel
subtypes was investigated in the rat by using in situ
hybridization and immunohistochemistry and revealed
that SK1 and SK2 subtypes are mostly expressed in
the cortex and hippocampus7 while SK3channels
expression is higher in subcortical areas, especially in
the monoamine cell regions, for example, substantia
nigra, dorsal raphe and locus coeruleus. These charac-
teristics attract considerable attention to SK channels
as putative targets for indications in cognitive dysfunc-
tion,8–12 neuronal hyperexcitability,13 dopamine-related
disorders,14–16 and depression.11
Three families of calcium-activated potassium channels
have been identified, which can be separated on the basis
of their biophysical and pharmacological properties. So
the three families are called BK, IK and SK reflecting
their large, intermediate and low conductance,
respectively.1,2
Small conductance Ca2+-activated K+ (SK) channels
underlie the prolonged postspike afterhyperpolarisation
(AHP), which plays an important role in modulating the
firing rate and the firing pattern of neurones.3,4 Three
*
Corresponding author. Tel.: +32 (0) 43 66 43 77; fax: +32 (0) 43 66 43
62; e-mail: a.graulich@student.ulg.ac.be
0968-0896/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.11.025

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A. Graulich et al. / Bioorg. Med. Chem. 13 (2005) 1201–1209
NH
N
NH
N
I
I
UCL1684
O
O
O
O
I
N
N
Me
Me
Me
O
O
O
O
O
O
O
O
N-methyl-bicuculline
bicuculline
MeO
MeO
MeO
MeO
I
N
N
Me
Me
Me
OMe
OMe
OMe
OMe
(15)
(14)
Figure 1. Chemical structures of UCL 1684, bicuculline, N-methyl-bicuculline, laudanosine 14 and N-methyl-laudanosine iodide 15.
So far, the most potent SK channel blockers are venom
toxins such as apamin and leiurotoxin I. Apamin is an
octadecapeptide isolated from honey bee Apis mellifera
venom. This peptide presents two arginine residues in
contiguous positions and a rigid cyclic conformation
due to two disulfide bridges.17 Leiurotoxin I isolated
from scorpion Androctonus mauretanicus mauretanicus
potently blocks human SK2 and SK3but not SK1 chan-
nels.18 Dequalinium, a non-peptidic ligand, presents
some SK channel blocking properties and extensive
structure–activity relationship studies led to UCL 1684
(Fig. 1).19 On the other hand, N-methyl-bicuculline
(Fig. 1) was reported to potentiate burst firing in dopa-
minergic neurones by blocking the apamin-sensitive
Ca2+-activated K+ current.20 Because of the GABAA
antagonist activity of bicuculline quaternary salts, more
selective compounds were needed. Therefore we decided
to develop new SK blockers based on a SAR of the bicu-
culline structure. First studies started with laudanosine
14 (Fig. 1), an alkaloid having a structure quite related
to N-methyl-bicuculline. From the N-methyl quaternary
analogue, several structural modifications were made in
order to find some pharmacophore elements and the
corresponding molecules were tested for their effect on
the apamin-sensitive AHP of dopaminergic neurones.
Their binding affinities for SK channels were also
evaluated.
2. Chemistry
N-methylation of 1,2,3,4-tetrahydroisoquinoline deriva-
tives was performed by two methods depending on the
chemical series. In the first procedure (Scheme 1),
1,2,3,4-tetrahydroisoquinoline derivatives were con-
verted to the corresponding potassium amide by reaction
with potassium hydride and triethylamine in 1,2-dimeth-
oxyethane (DME) at 0 ꢁC and after addition of methyl
iodide the tertiary amines 1 and 3 were isolated. Finally
the N-methyl derivatives 2 and 4 were prepared by treat-
ment with methyl iodide.21 In the second case, 1-substi-
tuted tetrahydroisoquinoline analogues were methylated
by reductive alkylation using sodium cyanoborohydride
and paraformaldehyde to give the tertiary amines 5 and 7
(Scheme 2).22 The N-methyl derivatives 6 and 8 were pre-
pared by treatment with methyl iodide.21

A. Graulich et al. / Bioorg. Med. Chem. 13 (2005) 1201–1209
1203
R1
KH, DME, NEt3
R1
R1
R1
MeI, MeCN
reflux
I
N
N
NH
MeI
R1
Me
R1
Me
Me
0˚C to rt
2 R1 = H
4 R1 = OMe
1 R1 = H
3 R1 = OMe
R1 = H or OMe
Scheme 1. Preparation of 2,2-dimethyl-1,2,3,4-tetrahydroisoquinolinium analogues.
MeO
MeO
MeO
MeI, MeCN
reflux
Me
37 % HCOH, NaBH3CN
I
N
N
NH
MeO
Me
Me
MeCN
MeO
MeO
R2
R2
R2
6 R2 = Me
8 R2 = Bn
5 R2 = Me
7 R2 = Bn
R2 = Me or Bn
Scheme 2. Preparation of 1-substituted-2,2-dimethyl-1,2,3,4-tetrahydroisoquinolinium derivatives.
Compound 10 was synthesized by benzylation and
hydrolysis of the Reissert compound 9 obtained by reac-
tion of potassium cyanide and benzoyl chloride with iso-
quinoline in a mixture of methylene chloride and water
(Scheme 3).23 Compound 10 was further methylated by
methyl iodide in refluxing MeCN. The resulting isoquin-
olinium iodide 11 was then reduced by using sodium
borohydride in methanol to afford the methylated amine
12.
acetonitrile to give the (+)- and (ꢀ)-enantiomers 19
and 20.
3. Results
The biological data are summarized in Table 1. None of
the simplified structures such as tetrahydroisoquinoline
derivatives with a hydrogen in position 1 1–4 and com-
pound 5 showed any significant activity on the AHP at
concentrations up to 300 lM but compounds 6 and 12
presented a very slight blockade of 22.7% 4.6%
(n = 3) and 22.4% 1.8% (n = 3) of the AHP at
300 lM, respectively. In the 1-benzyl series, the tertiary
amine 7 induced a slight blockade of the AHP of 47%
while the quaternary derivatives compounds 8 and 13 in-
duced a blockade of the apamin-sensitive AHP of
33.3% 9.5% (n = 3) and 67.3% 9.3% (n = 3 ) at
300 lM, respectively (Fig. 2).
Quaternary ammonium iodides (2, 4, 6, 8, 13) were final-
ly obtained by treatment of the N-methyl-1,2,3,4-tetra-
hydroisoquinoline derivatives (1, 3, 5, 7, 12) with
methyl iodide in MeCN (Scheme 3).
N-alkyl or benzyl derivatives of laudanosine 15–18 were
synthesized from (+/ꢀ) laudanosine 14 in the presence
of the corresponding alkyl iodide or benzyl chloride.
Otherwise, from ( )-laudanosine 14 both enantiomers
were separated by chiral preparative HPLC and further
methylated by an excess of methyl iodide in refluxing
The enantiomers of N-methyl-laudanosine blocked the
AHP with an IC50 of 17.3 lM 2.3 lM (n = 3) and
1. NaH, 3,4-(MeO)2C6H3CH2Cl,
DMF, -10˚C
KCN, BzCl,
CH2Cl2, H2O
N
N
N
10
2. NaOH, H2O, EtOH
Bz
OMe
OMe
9
CN
MeI,
MeCN
reflux
NaBH4,
MeI, MeCN,
I
N
I
N
MeOH, rt
Me
reflux
OMe
N
Me
Me
Me
OMe
OMe
11
12
13
OMe
OMe
OMe
Scheme 3. Synthesis of 1-(3,4-dimethoxybenzyl)-2,2-dimethyl-1,2,3,4-tetrahydroisoquinolinium iodide 13.

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A. Graulich et al. / Bioorg. Med. Chem. 13 (2005) 1201–1209
Table 1. Effects of N-methyl laudanosine and analogues on the apamin-sensitive AHP of DA neurones and binding affinities for cortical apamin-
sensitive sites
Compounds
Blocking potential of the AHP in
dopaminergic neurones (IC50 SEM, lM)
In vitro binding on SK channels
(IC50 SD lM)
% of 125I-apamin displacedd
(1)
(2)
NAa
—
—
—
—
NAa
(3)
(4)
NAa
—
—
—
—
NAa
(5)
(6)
NAa
—
—
—
22.6 6.5%b
47.0% 6.2%b
33.3% 9.5%b
22.4% 1.8%b
67.3% 9.7%b
17%
21%
10%
10%
25%
10%
57%
—
(7)
(8)
—
—
(12)
(13)
—
—
40.2c
Laudanosine (14)
N-methyl-laudanosine (15)
N-ethyl-laudanosine (16)
N-butyl-laudanosine (17)
N-benzyl-laudanosine (18)
(+)-N-methyl-laudanosine (19)
(ꢀ)-N-methyl-laudanosine (20)
Apamin
152
15
8
c
)
1
8.7 2.3(3.9
47.8c
26.4 7.5
17.3 2.3
c
>250
56.4c
—
—
47
>300
6
7.6 0.9
7.6 1.2
20.4 5.7 pM
57%
54%
—
249 39
—
a NA means no activity at 300 lM.
b % of blockade of the AHP in dopaminergic neurones at 300 lM.
c
24
´
After Scuvee-Moreau et al. 2002.
d % of 125I-apamin displaced at 10 lM.
10 mV
50 ms
Apamin (300 nM)
Compound 13 (300 µM)
Compound 13 (100 µM)
Control
Figure 2. Effect of increasing concentrations of compound 13 on the AHP of midbrain dopaminergic neurons. While 100% blockade of the AHP is
obtained with 300 nM apamin, 67.3% blockade is obtained with 300 lM compound 13. Each trace is the mean of four sweeps. Action potentials are
truncated.
26.4 lM 7.5 lM (n = 3), respectively, for the (+) and
(ꢀ) isomers.
AHP in dopaminergic neurones with a poor selectivity.25
Then N-methyl-laudanosine 15 was synthesized and
tested for its blockade of apamin-sensitive AHP. This
compound 15 revealed an interesting binding profile
and a very quickly reversible effect on the apamin-sensi-
tive AHP.24 We therefore decided to synthesise and eval-
uate enantiomers of N-methyl-laudanosine and
compounds structurally close to N-methyl-laudanosine
template.
In binding experiments, the affinity of NML 15
(IC50 = 8.7 lM) and of apamin (IC50 = 20.4 pM) were
similar to those reported in a previous publication.24
Binding results did not show any significant difference
in affinity of both enantiomers 19 and 20
(IC50 = 7.6 lM) for apamin binding sites. Among re-
cently prepared derivatives only compounds 7 and 13
displaced more than 20% of 125I-apamin at 10 lM.
Apamin has two arginine residues and it was demon-
strated that these guanidinium functions are necessary
for the activity of this peptide.18,26,27 All other peptides
that block SK channels contain at least one charged res-
idue. This fact is also a common characteristic of non-
peptidic ligands such as dequalinium and its derivatives
such as UCL 1684 or with neuromuscular blockers
4. Discussion
Firstly it was demonstrated that N-methyl-bicuculline
was an interesting blocker of the apamin-sensitive

A. Graulich et al. / Bioorg. Med. Chem. 13 (2005) 1201–1209
1205
(atracurium, pancuronium and tubocurarine),28,29
which also block the AHP. From these studies, it was
also clearly shown that the quaternization of the amine
in this series seems a crucial point to produce an activity
on the AHP mediated by SK channels.
these results suggest the presence of one symmetry
element in the binding site.
5. Conclusion
We show here that a benzyl substituent at the C-1 posi-
tion (8, 13) is necessary for the activity of the com-
pounds while a smaller group (2,4,6) is unfavourable
to block the AHP of dopaminergic neurones. Moreover,
the 3,4-dimethoxybenzyl substituent (13) appears more
effective than an unsubstituted benzyl group (8) for the
blockade of the AHP. This observation suggests that a
bulky group or a H bond acceptor could be important
for interactions within the channel but more examples
are necessary to confirm this hypothesis.
Different compounds structurally related to laudanosine
were prepared and tested in order to find the structural
elements important for the activity on the apamin-sensi-
tive sites. These results show that both enantiomers of
NML have the same affinities for the apamin binding
sites and a quite similar effect on the AHP of dopami-
nergic neurons. Moreover quaternization of the nitrogen
in position 2 appears to be an important parameter for
affinities and activities on SK channels. Our data thus
suggest that the pharmacophore should present some
symmetrical environment. Finally, the 3,4-dimethoxy-
benzyl moiety seems to be crucial for affinities on apamin
binding sites in 1-substituted-1,2,3,4-tetrahydroisoquin-
oline series. Further chemical and biological studies
are in progress for examining other structural elements
of NML or related drugs.
By modulating the group on the nitrogen of laudanosine
it was previously shown24 that a methyl moiety (15) pre-
sents the best blocking potency. Ethyl (16), butyl (17)
and benzyl (18) groups induce a clear decrease of the
activity. Binding experiments on SK channels gave the
following IC50s: 3 .9lM for N-methyl-(15), 47.8 lM for
N-ethyl-(16), >250 lM for N-butyl-(17) and 56.4 lM
for N-benzyl-(18) analogues.24 The tertiary amine, lau-
danosine 14 was also tested and showed only a weak
affinity in binding experiments (IC50 of 40.2 lM). These
results are in agreement with the experiments performed
on rat brain slices and confirm the micromolar affinity
of N-methyl-laudanosine on SK channels. In addition
it was shown that N-methyl-laudanosine has no affinity
for voltage-sensitive potassium channels and a-bungaro-
toxin-insensitive nicotinic receptors, a weak affinity for
muscarinic receptors (IC50 = 114 lM) and a low affinity
6. Experimental sections
6.1. Chemistry
Melting points were determined on a Buchi-Tottoli cap-
¨
illary melting point apparatus in open capillary and are
uncorrected. NMR spectra were recorded on a Bruker
AM400 spectrometer at 400MHz with external standard.
IR spectra were obtained on a Perkin–Elmer FTIR-1750
spectrometer using KBr discs and only significant bands
from IR were reported. Elemental analyses were deter-
mined using a Carlo-Erba elemental analyser CHNS-O
model EA 1108 and were within 0.4% of theoretical val-
ues. Mass spectra were recorded on a QTOF II (micro-
mass, Manchester UK) spectrometer with electrospray
mode. Optical activity was determined on an optical
activity AA-10 polarimeter at room temperature. Sepa-
rations by flash chromatography were carried out using
Merck Kieselgel 60 (230–400 mesh). All starting materi-
als and reagents were obtained from Aldrich Chemical
Co. or Acros chemicals and were used without further
purification. Concentration and evaporation refer to re-
moval of volatile materials under reduced pressure
(10–15 mmHg at 30–50 ꢁC) on a Buchi Rotavapor.
for
a-bungarotoxin-sensitive
nicotinic
receptors
(IC50 = 3 67lM).24
The interest to find a selective blocker of the SK
channels with an IC50 in the micromolar range is to
have a highly reversible ligand that permits to explore
the pharmacology of these channels more easily in
comparison with apamin. Indeed, apamin is destroyed
by protease due to this peptidic nature30 and its interac-
tion with SK channels is very slowly reversible, making
´
it difficult to observe a wash-out of the drug (Scuvee-
Moreau, personal observation). Another advantage of
these ammonium iodides is their high solubility in water
and also the permanent charge on the nitrogen, both
parameters being interesting for iontophoresis
experiments.31
6.2. 2-Methyl-1,2,3,4-tetrahydroisoquinoline fumarate (1)
Moreover it was shown that, unlike N-methyl-bicucul-
line, N-methyl-laudanosine iodide has no antagonistic
effect on GABAA receptors.24
1,2,3,4-Tetrahydroisoquinoline (5 g; 37.5 mmol) was
dissolved in 1,2-dimethoxyethane (30 mL). Triethyl-
amine (5,2 mL; 375 mmol) and KH (7.5 g; 56.25 mmol)
were added under N2 at 0 ꢁC. After 30 min methyl
iodide (3.5 mL; 56.25 mmol) was added and the mixture
was stirred overnight at room temperature. The mixture
was extracted with CH2Cl2 (3 · 75 mL) after the addi-
tion of a saturated aqueous NH4Cl (30 mL) and a 10%
aqueous NaOH to basify. The organic solution was
dried on anhydrous MgSO4 and evaporated under
reduced pressure to give an oil, which was purified by
flash chromatography (eluent CH2Cl2/MeOH 97:3). The
N-methyl-laudanosine 15 being a racemic mixture, both
enantiomers 19 and 20 were isolated from laudanosine
14 by chiral preparative HPLC, and after methylation,
these isomers were tested in electrophysiological and in
binding experiments. No clear difference was found
between both enantiomers in binding studies and both
isomers appeared quite equivalent for blocking the
apamin-sensitive AHP in dopaminergic neurones. Thus

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A. Graulich et al. / Bioorg. Med. Chem. 13 (2005) 1201–1209
resulting oil was crystallized as fumarate salt from EtOH
(1.1 g; 11%). mp: 149–151 ꢁC. H NMR (DMSO-d6) d
7.16–7.14 (m, 4H, ArH); 6.57 (s, 2H, fumarate); 3.73
(s, 2H, phenyl–CH2–N); 2.89–2.84 (dd, 4H, CH2–CH2–
N); 2.51 (s, 3H, CH3). IR (KBr, cmꢀ1) 2670, 2597,
1705. Anal. C14H17NO4 (263.293) calcd: N, 5.32; C,
63.86; H, 6.51. Found: N, 5.41; C, 63.82; H, 6.79.
lowed by a 37% aqueous formaldehyde (1.84 mL;
24 mmol) and NaBH3CN (0.15 g; 2.4 mmol). After
30 min, acetic acid was added until neutral and the mix-
ture was stirred overnight. The solvent was removed un-
der reduced pressure and the crude residue was dissolved
in a 2N aqueous NaOH (15 mL). This suspension was
extracted with CHCl3 (3 · 15 mL). The organic layer
was washed with brine and dried with anhydrous
MgSO4. The solvent was evaporated under reduced
pressure to gave an oil. The compound was isolated as
a hydrochloride salt and recrystallized from MeCN/
Et2O (0.338 g; 71%). mp: 204–205 ꢁC. 1H NMR
(CDCl3) d 6.62 (s, 1H, ArH); 6.52 (s, 1H, ArH); 4.25
(d, 1H, phenyl–CH–N); 3.86 (s, 6H, 2 · OCH3); 3.36–
2.77 (m, 4H, phenyl–CH2–CH2–N); 1.96 (s, 3H,
1
6.3. 2,2-Dimethyl-1,2,3,4-tetrahydroisoquinolinium iodide
(2)
2-Methyl-1,2,3,4-tetrahydroisoquinoline fumarate (0.50
g; 1.89 mmol) was solubilized in water (20 mL) and then
basified with 10% aqueous NaOH. The base was ex-
tracted with CH2Cl2 (3 · 25 mL). The organic layer
was dried and evaporated under reduced pressure to
give an oil, which was dissolved in MeCN. Methyl
iodide (1.17 mL; 18.9 mmol) was added and the mixture
was refluxed overnight. Solvent was eliminated under
reduced pressure and the crude residue was crystallized
NCH3); 1.86–1.83(dd, H3 , C H3). IR (KBr, cmꢀ1
)
2458, 1522. Anal. C13H20NO2Cl (257.758) calcd: N,
5.43; C, 60.58; H, 7.82. Found: N, 6.08; C, 60.56; H,
8.03.
1
from MeOH/Et2O (0.43g; 86%). mp: 188–189 ꢁC. H
6.7. 6,7-Dimethoxy-1,2,2-trimethyl-1,2,3,4-tetrahydroiso-
quinolinium iodide (6)
NMR (DMSO-d6): d 7.36–7.17 (dd, 4H, ArH); 4.61 (s,
2H, phenyl–CH2–N); 3.70 (t, 2H, phenyl–CH2–CH2–
N); 3.33 (t, 2H, phenyl–CH2–CH2–N); 3.15 (s, 6H,
2 · NCH3). IR (KBr, cmꢀ1) 3001, 1478, 1460, 1436,
762. Anal. C11H16NI (289.156) calcd: N, 4.84; C,
45.69; H, 5.58, Found: N, 4.97; C, 45.47; H, 5.63.
A solution of 6,7-dimethoxy-1,2-dimethyl-1,2,3,4-tetra-
hydroisoquinoline (0.44 g; 2 mmol) in MeCN (20 mL)
was stirred with anhydrous K2CO3 (5.6 g; 40 mmol) dur-
ing 30 min. Methyl iodide (0.19 mL; 40 mmol) was
added. After 1 h the precipitate was filtered and the
resulting solution evaporated under reduced pressure.
Finally the residue was crystallized from EtOH
6.4. 6,7-Dimethoxy-2-methyl-1,2,3,4-tetrahydroisoquino-
line fumarate (3)
1
(0.224 g; 30%). mp: 203–204 ꢁC. H NMR (DMSO-d6)
This compound was obtained from 6,7-dimethoxy-
1,2,3,4-tetrahydroisoquinoline using the method de-
scribed for compound 1. The product was crystallized
from EtOH as fumarate salt (39%). mp: 175–177 ꢁC.
1H NMR (DMSO-d6) d 6.69 (s, 1H, ArH); 6.64 (s, 1H,
ArH); 6.52 (s, 2H, fumarate); 3.69 (s, 8H, 2 · OCH3 et
Ar–CH2–N); 2.87–2.8 (dd, 4H, CH2–CH2–N); 2.49 (s,
3H, NCH3). IR (KBr, cmꢀ1) 2598, 1702. Anal.
C16H21NO6 (323.341) calcd: N, 4.33; C, 59.43; H, 6.55.
Found: N, 4.65; C, 59.38; H, 6.74.
d 6.83(s, 1H, Ar H); 6.79 (s, 1H, ArH); 4.62 (s, 1H, phen-
yl–CH–N); 3.74 (s, 6H, 2 · OCH3); 3.58 (m, 2H, phen-
yl–CH2–CH2–N); 3.33 (d, 2H, phenyl–CH2–CH2–N);
3.11–3.05 (d, 6H, 2 · NCH3); 1.6 (d, 3H, CH3). IR
(KBr, cmꢀ1) 2990, 1520. Anal. C14H22NO2I (363.235)
calcd: N, 3.86; C, 46.29; H, 6.10. Found: N, 3.92; C,
45.90; H, 6.20.
6.8. 1-Benzyl-6,7-dimethoxy-2-methyl-1,2,3,4-tetrahydro-
isoquinoline hydrochloride (7)
6.5. 6,7-Dimethoxy-2,2-dimethyl-1,2,3,4-tetrahydroiso-
quinolinium iodide (4)
Compound 7 was obtained from 1-benzyl-6,7-dime-
thoxy-1,2,3,4-tetrahydroisoquinoline using the method
described for compound 5. Finally the residue was cryst-
allized from EtOH/Et2O (53%). mp: 184–186 ꢁC. 1H
NMR (CDCl3) d 6.98 (s, 5H, ArH); 6.52 (s, 2H, ArH);
5.43(s, 1H, benzyl–C H–N); 3.74 (s, 6H, 2 · OCH3);
3.29 (s, 2H, phenyl–CH2–CH2–N); 3.24 (s, 2H, C6H5–
CH2–); 2.98–2.88 (d, 2H, phenyl–CH2–CH2–N); 2.79
(d, 3H, NCH3). IR (KBr, cmꢀ1) 2434, 1520, 1266. Anal.
C19H24NO2Cl (333.859) calcd: N, 4.19; C, 68.35; H,
7.25. Found: N, 4.60; C, 68.07; H, 7.88.
Compound 4 was prepared from 6,7-dimethoxy-2-
methyl-1,2,3,4-tetrahydroisoquinoline fumarate using
the method described for compound 1 but in the pres-
ence of a large excess of methyl iodide (10equiv). The
product was crystallized from MeOH/Et2O (25%). mp:
234–236 ꢁC. 1H NMR (DMSO-d6) d 6.88 (s, 1H,
ArH); 6.75 (s, 1H, ArH); 4.48 (s, 2H, phenyl–CH2–N);
3.73 (d, 6H, 2 · OCH3); 3.6 (t, 2H, phenyl–CH2–CH2–
N); 3.1 (s, 6H, 2 · NCH3); 3.0 (s, 2H, phenyl–CH2–
CH2–N). IR (KBr, cmꢀ1) 2968, 1519, 1116. Anal.
C13H20NO2I (349.208) calcd: N, 4.01; C, 44.71; H,
5.77. Found: N, 4.05; C, 44.87; H, 5.93.
6.9. 1-Benzyl-6,7-dimethoxy-2,2-dimethyl-1,2,3,4-tetra-
hydroisoquinolinium iodide (8)
Compound 8 was prepared from compound 7 using the
method described for compound 6. Finally the residue
was crystallized from EtOH/Et2O (18%). mp: 146–
147 ꢁC. 1H NMR (DMSO-d6) d 7.66–7.64 (d, 2H,
ArH); 7.40–7.26 (m, 5H, ArH); 6.9 (s, 1H, benzyl–
CH–N); 3.8 (s, 2H, C6H5–CH2–); 3.42 (s, 2H, phenyl–
6.6. 6,7-Dimethoxy-1,2-dimethyl-1,2,3,4-tetrahydroiso-
quinoline hydrochloride (5)
6,7-Dimethoxy-1-methyl-1,2,3,4-tetrahydroisoquinoline
(0.41 g; 2 mmol) was dissolved in MeCN (20 mL) fol-

A. Graulich et al. / Bioorg. Med. Chem. 13 (2005) 1201–1209
1207
CH2–CH2–N); 3.35 (s, 6H, 2 · OCH3); 3.19 (s, 6H,
N(CH3)2); 3.11 (s, 2H, phenyl–CH2–CH2–N). IR
(KBr, cmꢀ1) 2985, 1517. Anal. C20H26NO2I (439.339)
calcd: N, 3.19; C, 54.68; H, 5.97. Found: N, 3.25; C,
54.80; H, 5.94.
6.14. 1-(3,4-Dimethoxybenzyl)-2,2-dimethyl-1,2,3,4-tetra-
hydroisoquinolinium iodide (13)
Compound 13 was prepared using the experimental pro-
cedure described for compound 2 and was recrystallized
from EtOH/Et2O (55%). mp: 182 ꢁC. 1H NMR (CDCl3)
d 7.26–7.19 (m, 2H, ArH); 7.02–6.97 (m, 1H, ArH); 6.71
(d, 1H, ArH); 6.55 (d, 2H, ArH); 6.47 (d, 1H, ArH); 5.3
(m, 1H, benzyl–CH–N); 4.08 (m, 1H, phenyl–CH2–
CH2–N); 3.88 (s, 3H, OCH3); 3.85 (s, 3H, OCH3); 3.74
(s, 4H, NCH3 et phenyl–CH2–CH2–N); 3.66 (d, 1H,
phenyl–CH2–CH2–N); 3.49 (s, 3H, NCH3); 3.33–3.25
(m, 2H, C6H3–CH2–); 2.97 (dd, 1H, phenyl–CH2–
CH2–N). IR (KBr, cmꢀ1) 2933, 1517, 1025. Anal.
C20H26NO2I (439.333) calcd: N, 3.19; C, 54.68; H, 5.97.
Found: N, 3.35; C, 54.70; H, 6.33.
6.10. 2-Benzoyl-1-cyano-1,2-dihydroisoquinoline (9)
This product was obtained by using a method previously
described.23 The compound was crystallized from EtOH
(47.04 g; 72%). mp: 124–125 ꢁC. 1H NMR (CDCl3)
d 7.0–7.7 (m, 9H, ArH); 6.60 (s, 1H, H3); 6.55 (s, 1H,
H1); 6.05 (d, 1H, H4). IR (KBr, cmꢀ1) 2239, 1660,
1632. Anal. C17H12N2O (260.297) calcd: N, 10.76; C,
78.44; H, 4.65. Found: N, 10.86; C, 78.21; H, 4.94.
6.11. 1-(3,4-Dimethoxybenzyl)-isoquinoline (10)
6.15. N-Methyl-laudanosine iodide (15)
Compound 10 was prepared as previously described.23
The crude residue was recrystallized in petroleum ether
100–140 ꢁC. (84%). mp: 70–72 ꢁC. H NMR (CDCl3) d
8.47 (d, 1H, ArH); 8.16 (d, 1H, ArH); 7.79 (d, 1H,
ArH); 7.61 (m, 1H, ArH); 7.52 (m, 2H, ArH); 6.72–
6.82 (m, 3H, benzyl CH); 4.59 (s, 2H, benzyl CH2);
3.78 (d, 6H, 2 · OCH3) IR (KBr, cmꢀ1) 1517, 1234,
1139, 1023. Anal. C18H17NO2 (279.339) calcd: N,
5.01; C, 77.40; H, 6.13. Found: N, 5.22; C, 77.45; H,
6.52.
A mixture of ( )-laudanosine 14 (0.1 g; 0.28 mmol) and
an excess of methyl iodide (0,17 mL; 28 mmol) in aceto-
nitrile (10 mL) was refluxed overnight. Excess of reagent
and solvent were removed under reduced pressure. The
crude quaternary salt was isolated in an acetone/diethyl-
ether mixture. Finally, the product was recrystallized
1
1
from EtOH (0.115 g; yield: 82%). mp: 209–210 ꢁC. H
NMR (CDCl3) d 6.89 (s, 1H, ArH); 6.69–6.62 (d, 3H,
ArH); 6.46 (s, 1H, ArH); 5.2 (d, 1H, –CH–N); 3.9–3.8
(m, 14H, 4 · OCH3 and benzyl CH2); 3.52 (s, 2H, phen-
yl–CH2–CH2–N); 3.38 (s, 8H, 2 · NCH3 and phenyl–
CH2–CH2–N). IR (KBr, cmꢀ1) 2920, 1515. Anal.
C22H30NO4I (499.385) calcd: N, 2.80; C, 52.91; H,
6.06. Found: N, 3.03; C, 52.95; H, 6.14.
6.12. 1-(3,4-Dimethoxybenzyl)-2-methyl-isoquinolinium
iodide (11)
This product was obtained from compound 10 using
the method described for compound 2 but the crude
residue was used in the next step without further
purification.
6.16. N-Ethyl-laudanosine iodide (16)
Compound 16 was prepared using the experimental pro-
cedure described for compound 15 with an excess of
ethyl iodide in place of methyl iodide and was recrystal-
lized from EtCOMe (0.112 g; yield: 78%). mp: 157–
6.13. 1-(3,4-Dimethoxybenzyl)-2-methyl-1,2,3,4-tetrahy-
droisoquinoline hydrochloride (12)
1
NaBH4 (5 equiv) was added to a solution of crude 1-
(3,4-dimethoxybenzyl)-2-methyl-isoquinolinium iodide
in ice-cold MeOH (20 mL). After 30 min the solution
was evaporated under reduced pressure and the residue
was dissolved in 1 N aqueous HCl (100 mL). The acidic
solution was washed with Et2O (2 · 30 mL) and then
basified by addition of a 10% aqueous NaOH. The
resulting suspension was extracted with CH2Cl2
(3 · 50 mL) and the organic layer was dried with anhy-
drous MgSO4 before the evaporation of solvent under
reduced pressure. The crude oil was isolated as an
hydrochloride salt and was recrystallized in EtOH/
Et2O (24%). mp: 226–227 ꢁC. 1H NMR (CDCl3) d
7.32–7.27 (t, 1H, ArH); 7.22–7.20 (d, 1H, ArH); 7.13–
7.08 (t, 1H, ArH); 6.75–6.73(d, 1H, Ar H); 6.64–6.60
(d, 2H, ArH); 6.51–6.48 (d, 1H, ArH); 4.29–4.13(d,
1H, benzyl–CH–N); 3.97–3.92 (d, 1H, C6H3–CH2–);
3.85 (s, 3H, OCH3); 3.74 (s, 3H, OCH3); 3.64 (m, 1H,
C6H3–CH2–); 3.34–3.30 (m, 1H, phenyl–CH2–CH2–N);
3.11–3.04 (m, 3H, phenyl–CH2–CH2–N); 2.87 (s, 3H,
NCH3). IR (KBr, cmꢀ1) 2934, 2467, 1510. Anal.
C19H24NO2Cl (333.856) calcd: N, 4.20; C, 68.36; H,
7.25. Found: N, 4.42; C, 68.22; H, 7.96.
158 ꢁC. H NMR (CDCl3) d 6.87 (s, 1H, ArH); 6.66–
6.60 (m, 2H, ArH); 6.45 (d, 1H, ArH); 5.70 (s, 1H,
ArH); 5.01 (dd, 1H, –CH–N); 3.86–3.63 (m, 18H,
4 · OCH3, phenyl–CH2–CH2–N and benzyl CH2); 3.36
(s, 3H, NCH3); 3.10 (q, 2H, CH2CH3); 1.41 (t, 3H,
CH2CH3). IR (KBr, cmꢀ1
)
2931, 1517. Anal.
C23H32NO4I (513.412) calcd: N, 2.73; C, 53.81; H,
6.28. Found: N, 3.09; C, 53.95; H, 6.14.
6.17. N-Butyl-laudanosine iodide (17)
Compound 17 was prepared using the experimental pro-
cedure described for compound 15 with an excess of
freshly distilled butyl iodide in place of methyl iodide
and was recrystallized from Me2CO/Et2O (0.08 g;
53%). mp: 100–102 ꢁC. 1H NMR (CDCl3) d 6.89 (s,
1H, ArH); 6.70–6.57 (m, 2H, ArH); 6.45–6.42 (m, 1H,
ArH); 5.69 (s, 1H, ArH); 5.03(dd, 1H, –C H–N); 3.82–
3.77 (m, 18H, 4 · OCH3, phenyl–CH2–CH2–N and ben-
zyl CH2); 3.39 (s, 3H, NCH3); 3.10 (t, 2H, CH2CH2-
CH2CH3); 1.78 (quintuplet, 2H, CH2CH2CH2CH3);
1.29 (sextuplet, 2H, CH2CH2CH2CH3); 0.89 (t, 3H,
CH2CH2CH2CH3). m/z 414 (M+). IR (KBr, cmꢀ1
)

1208
A. Graulich et al. / Bioorg. Med. Chem. 13 (2005) 1201–1209
2959, 1517, 1262. Anal. C25H36NO4IÆ3/4H2O (554.979)
calcd: N, 2.52; C, 54.05; H, 6.76. Found: N, 2.78; C,
54.28; H, 7.16.
25,000 · g for 10 min. The resulting pellet was dispersed
in 5 mL 0.32 M sucrose to be aliquoted. Protein concen-
tration was determined by the method of Hartree with
bovine serum albumin as a standard.32
6.18. N-Benzyl-laudanosine chloride (18)
7.1.2. Binding experiments. The buffer consisted of a
10 mM Tris–HCl (pH 7.5) solution containing 5.4 mM
KCl and 0.1% bovine serum albumin. The radioligand
was 125I-apamin (Perkin–Elmer, Specific activity
81.4 TBq mmolꢀ1). Glass fibre filters (Whatman GF/C)
used in these experiments were coated for 1 h in 0.5%
polyethylenimine and then washed with 2.5 mL of the
ice-cold buffer just before use. Binding experiments were
always terminated as follows. Aliquots were filtered un-
der reduced pressure through Whatman filters. Filters
were rapidly washed twice with 2.5 mL of buffer. The
radioactivity remaining on the filter was evaluated with
a Packard Tri-Carb 1600TR liquid scintillation analyser
with an efficacy of 69%. 125I-apamin binding to the fil-
ters was also estimated in the absence of synaptosomes.
This binding was also subtracted from the total binding.
Curve fitting was carried out using GraphPad Prismꢂ.
Compound 18 was prepared using the experimental pro-
cedure described for compound 15 with an excess of
benzyl chloride in place of methyl iodide and was recrys-
tallized from CH2Cl2/Et2O (0.22 g; yield: 80%). mp:
178–180 ꢁC. 1H NMR (CDCl3) d 7.50–7.36 (m, 5H,
N–CH2–C6H5); 6.68–6.64 (m, 2H, ArH); 6.54 (s, 1H,
ArH); 6.37 (d, 1H, ArH); 5.70 (s, 1H, ArH); 5.08 (dd,
1H, benzyl–CH–N); 4.93(d, 1H, N–C H2–C6H5); 4.79
(d,1H, N–CH2–C6H5) 3.85–3.79 (m, 12H, 4 · OCH3);
3.42 (s, 3H, NCH3) 3.32–2.86 (m, 6H, phenyl–CH2–
CH2–N and benzyl CH2). m/z 448 (M+). IR (KBr,
cmꢀ1) 2938, 1518, 1264. Anal. C28H34NO4ClÆ1/2H2O
(493.042) calcd: N, 2.82; C, 68.15; H, 7.30. Found: N,
3.03; C, 68.21; H, 7.16.
6.19. Racemic resolution of laudanosine
The enantiomers were separated from ( )-laudanosine
14 (600 mg) by chiral preparative HPLC using the fol-
lowing conditions: column CHIRACELꢂ OD
250 · 50 mm, 20 lm; eluent heptane/isopropanol/dieth-
ylamine 80/20/0.05; flow rate 120 mL/min; UV detection
at 300 nm and room temperature. (+)-Laudanosine
(300 mg) was firstly eluted at 7.7 min and (ꢀ)-laudano-
sine (300 mg) was eluted at 16.5 min.
Saturation binding experiments: Synaptosomes (0.2 mg
of protein/mL) were incubated with increasing concen-
trations of 125I-apamin (25 lL) with 975 lL of incuba-
tion buffer for 1 h at 0 ꢁC. Samples were then filtered
on Whatman GF/C filter and the radioactivity was
measured as described above. Nonspecific binding
was determined in parallel experiments in the presence
of an excess of unlabeled apamin (0.1 lM) and sub-
tracted from the total binding to obtain the specific
binding.
6.20. (+)-N-Methyl-laudanosine iodide (19)
A mixture of (+)-laudanosine (0.25 g; 0.70 mmol) with
an excess of methyl iodide (0,17 mL; 28 mmol) in aceto-
nitrile (10 mL) was refluxed overnight. Excess of reagent
and solvent was removed under reduced pressure. The
Competition experiments between 125I-apamin and drugs:
Synaptosomes (0.2 mg of protein/mL) were incubated
for 1 h at 0 ꢁC with 10 pM of 125I-apamin (25 lL)
and nine concentrations of drugs (10ꢀ4 to 10ꢀ7 M).
Nonspecific binding was determined in the presence of
an excess of unlabeled apamin (0.1 lM). Samples were
then filtered on Whatman filter and the radioactivity
was measured as described above.
crude quaternary salt was recrystallized from MeCN/
20
D
Et2O (0.31 g; yield: 89%). mp: 216–218 ꢁC. ½aꢁ : +95.0
Anal. C22H30NO4I (499.385) calcd: N, 2.80; C, 52.91;
H, 6.06. Found: N, 2.97; C, 52.55; H, 6.15.
6.21. (ꢀ)-N-Methyl-laudanosine iodide (20)
7.2. Electrophysiological experiments
The (ꢀ)stereoisomer was prepared using the experimen-
The procedure is largely described in a previous paper.24
Briefly, male Wistar rats (150–200 g) were anaesthetised
with chloral hydrate (400 mg/kg IP) and decapitated.
The brain was excised quickly and placed in cold
(ꢂ4 ꢁC) artificial cerebro-spinal fluid (ACSF) at the fol-
lowing composition (in mM): NaCl 126, KCl 2.5, NaH2-
PO4 1.2, MgCl2 1.2, CaCl2 2.4, glucose 11, NaHCO3 18,
saturated with 95% O2 and 5% CO2 (pH 7.4). A block of
tissue containing the midbrain was cut in horizontal
slices (thickness 350 lm) in a Vibratome (Lancer). The
slice containing the region of interest was placed on a
nylon mesh in a recording chamber (volume 500 ll).
The tissue was completely immersed in a continuously
flowing (ꢂ2 mL/min) ACSF, heated at 35 ꢁC. Most
recordings were made from dopaminergic neurones lo-
cated in the substantia nigra pars compacta. Identifica-
tion of dopaminergic cells was performed as described
previously.33 Intracellular recordings were performed
tal procedure described for compound 18. The crude
quaternary salt was recrystallized from MeCN/Et2O
20
D
yield: 80%. mp: 216–218 ꢁC. ½aꢁ : ꢀ86.7 Anal.
C22H30NO4I (499.385) calcd: N, 2.80; C, 52.91; H,
6.06. Found: N, 3.12; C, 52.58; H, 6.10.
7. Biological evaluation
7.1. Radioligand binding studies and data analysis
7.1.1. Synaptosome preparation. Rats (male Wistar,
250 g) were killed by decapitation and the brains were
quickly removed and kept on ice during dissection.
Crude cortex was dispersed in 0.32 M sucrose by using
a Potterꢂ homogeniser. After a first centrifugation at
1500 · g for 10 min, the supernatant was centrifuged at

A. Graulich et al. / Bioorg. Med. Chem. 13 (2005) 1201–1209
1209
4. Stocker, M.; Krause, M.; Pedarzani, P. Proc. Natl. Acad.
Sci. U.S.A. 1999, 96, 4662.
5. Bretschneider, F.; Wrisch, A.; Lehmann-Horn, F.; Griss-
mer, S. Biophys. J. 1999, 76, 2351.
6. Sah, P.; Faber, L. Prog. Neurobiol. 2002, 66, 345.
7. Stocker, M.; Pedarzani, P. Mol. Cell. Neurosci. 2000, 15,
476.
8. Messier, C.; Mourre, C.; Bontempi, B.; Sif, J.; Lazdunski,
M.; Destrade, C. Brain Res. 1991, 551, 322.
9. Deschaux, O.; Bizot, J. C.; Goyffon, M. Neurosci. Lett.
1997, 222, 159.
using glass microelectrodes filled with KCl 2 M (resis-
tance 70–150 MX). All recordings were made in the
bridge balance mode, using a npi SEC1L amplifier
(Tamm, Germany). The accuracy of the bridge was
checked throughout the experiment. Membrane poten-
tials and injected currents were recorded on a Gould
TA240 chart recorder and on a Fluke Combiscope oscil-
loscope. The Flukeview software was used for off-line
analysis in most cases. Drug effects on the prominent
apamin-sensitive AHP in dopaminergic neurones were
quantified as the percent reduction of the surface area
of the AHP (in mV s), which was blocked by a maxi-
mally active concentration of apamin (300 nM).33 Aver-
ages of four sweeps were considered in all cases. The
spontaneous firing of the neurones was usually reduced
by constant current injection (ꢀ20 to ꢀ100 pA) in order
to increase the amplitude of the AHP. Because the
amplitude of the AHP is very sensitive to the firing rate,
care was taken to compare all AHPs of one cell at the
same firing rate. All drugs were applied by superfusion;
complete exchange of the bath solution occurred within
2–3min. Curve fitting was carried out using GraphPad
10. Ikonen, S.; Riekkinen, P., Jr. Eur. J. Pharmacol. 1999, 382,
151.
11. Van der Staay, F. J.; Fanelli, R. J.; Blokland, A.; Schmidt,
B. H. Neurosci. Biobehav. Rev. 1999, 23, 1087.
12. Fournier, C.; Kourrich, S.; Soumireu-Mourat, B.;
Mourre, C. Behav. Brain Res. 2001, 121, 81.
13. Pedarzani, P.; Mosbacher, J.; Rivard, A.; Cingolani, L. A.;
Oliver, D.; Stocker, M.; Adelman, J. P.; Fakler, B. J. Biol.
Chem. 2001, 276, 9762.
14. Steketee, J. D.; Kalavas, P. W. J. Pharmacol. Exp. Ther.
1990, 254, 711.
15. Shepard, P. D.; Bunney, B. S. Exp. Brain Res. 1991, 86,
141.
16. Seutin, V.; Johnson, S. V.; North, R. A. Brain Res. 1993,
630, 341.
17. Vincent, J. P.; Schweitz, H.; Lazdunski, M. Biochemistry
1975, 14, 2521.
18. Zerrouk, H.; Lavaba-Djerbari, F.; Fremont, V.; Meki, A.;
Darbon, H.; Mansuelle, P.; Oughuideni, R.; Van Riet-
schoten, J.; Rochat, H.; Martin-Eauclaire, M. F. Int. J.
Pept. Protein Res. 1996, 48, 514.
Prismꢂ and the standard equation: E = Emax
/
[1 + (IC50/x)h], where x is the concentration of the drug
and h the Hill coefficient. Numerical values are ex-
pressed as means SEM. Apamin (Sigma) and all other
drugs were dissolved in water.
19. Chen, J. Q.; Galanakis, D.; Ganellin, C. R.; Dunn, P. M.;
Jenkinson, D. H. J. Med. Chem. 2000, 43, 3478.
20. Johnson, S. W.; Seutin, V. Neurosci. Lett. 1997, 231, 13 .
21. Mohri, K.; Suzuki, K.; Usui, M.; Isobe, K.; Tsuda, Y.
Chem. Pharm. Bull. 1995, 43, 159.
22. Cheng, Y.-X.; Dukat, M.; Dowd, M.; Fiedler, W.; Martin,
B.; Damaj, M. I.; Glennon, R. A. Eur. J. Med. Chem.
1999, 34, 177.
Acknowledgements
The technical assistance of Y. Abrassart, S. Counerotte,
L. Massotte, and J.-C. Van Heugen is gratefully
acknowledged. A.G. is a Research Fellow of the ꢀFonds
`
pour la formation a la Recherche Industrielle et Agricole
(F.R.I.A.)ꢁ, F.M. was supported by a grant from the
´
`
ꢀPatrimoine de lꢁUniversite de Liegeꢁ and J.-F.L. is a Se-
nior Research Associate of the ꢀFonds National de la
Recherche Scientifique de Belgique (F.N.R.S.)ꢁ. This
work was financially supported in part by F.N.R.S.
grants no. 3.4525.98 and 9.4560.03 and by grants from
23. Uff, B. C.; Kershaw, J. R.; Neumeyer, J. L. Org. Syn.
1977, 56, 19.
´
´
24. Scuvee-Moreau, J.; Liegeois, J.-F.; Massotte, L.; Seutin,
V. J. Pharmacol. Exp. Ther. 2002, 302, 1176.
25. Seutin, V.; Johnson, S. W. Trends Pharmacol. Sci. 1999,
20, 268.
´
the ꢀFonds Speciauxꢁ 2002 and 2003of the University
`
of Liege.
26. Granier, C.; Pedroso Muller, E.; Van Rietschoten, J. Eur.
J. Biochem. 1978, 82, 293.
27. Habermann, E. Pharmacol. Ther. 1984, 25, 255.
28. Nohmi, M.; Kuba, K. Brain Res. 1984, 301, 146.
29. Cook, N. S.; Haylett, D. G. J. Physiol. 1985, 358, 373.
30. Hugues, M.; Duval, D.; Kitabgi, P.; Lazdunski, M.;
Vincent, J.-P. J. Biol. Chem. 1982, 257, 2762.
References and notes
1. Sah, P. Trends Neurosci. 1996, 19, 150.
2. Vergara, C.; Latorre, R.; Marrion, N. V.; Adelman, J. P.
Curr. Opin. Neurobiol. 1998, 8, 321.
3. Kolher, M.; Hirschberg, B.; Bond, C. T.; Kinzie, J. M.;
Marrion, N. V.; Maylie, J.; Adelman, J. P. Science 1996,
273, 1709.
´
´
31. Seutin, V.; Massotte, L.; Liegeois, J.-F.; Scuvee-Moreau,
J. Soc. Neurosci. Abstr. 2002, 28, 340.5.
32. Hartree, E. F. Anal. Biochem. 1972, 48, 422.
´
33. Seutin, V.; Scuvee-Moreau, J.; Dresse, A. Neuropharma-
cology 1997, 36, 1653.