Closing in on the AMPA receptor: Synthesis and evaluation of 2-acetyl-1-(4′-chlorophenyl)-6-methoxy-7-[11C]methoxy-1 ,2,3,4-tetrahydroisoquinoline as a potential PET tracer

Article abstract of DOI:10.1016/j.bmc.2006.03.034

2-Acetyl-1-(4′-chlorophenyl)-6,7-dimethoxy-1,2,3,4-tetrahydroisoqu inoline, one of the most potent non-competitive AMPA antagonists described to date, has been labelled with carbon-11 and tritium and evaluated as a potential ligand for in vivo imaging of

Full text of DOI:10.1016/j.bmc.2006.03.034

Bioorganic & Medicinal Chemistry 14 (2006) 4712–4717
Closing in on the AMPA receptor: Synthesis and evaluation of
2-acetyl-1-(40-chlorophenyl)-6-methoxy-7-[11C]methoxy-1,2,3,4-
tetrahydroisoquinoline as a potential PET tracer
a,
b
b
b
c
˚
*
Erik Arstad, Rosaria Gitto, Alba Chimirri, Roberta Caruso, Andrew Constanti,
David Turton,a Sue P. Hume,a Rabia Ahmad,a Lyn S. Pilowskyd and Sajinder K. Luthraa
aHammersmith Imanet Ltd, Cyclotron Building, Du Cane Road, W12 ONN, London, UK
b
`
Dipartimento Farmaco-Chimico, Universita di Messina, Viale Annunziata, 98168 Messina, Italy
cDepartment of Pharmacology, The School of Pharmacy, 29/39 Brunswick Square, London WC1N 1AX, UK
dInstitute of Psychiatry, KCL De Crespigny Park, London SE5 8AF, and Institute of Nuclear Medicine,
UCL, Middlesex Hospital, W1N 8AA, UK
Received 12 October 2005; revised 27 February 2006; accepted 17 March 2006
Available online 18 April 2006
Abstract—2-Acetyl-1-(40-chlorophenyl)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline, one of the most potent non-competitive
AMPA antagonists described to date, has been labelled with carbon-11 and tritium and evaluated as a potential ligand for in vivo
imaging of AMPA receptors using PET. The carbon-11 labelled compound showed good initial brain uptake in rats, but with rapid
clearance and relatively homogenous distribution. In saturation binding studies, the tritiated racemic ligand was found to be highly
potent with a Kd of 14.8 1.8 nM. We conclude that the low receptor density labelled with this compound, its rapid clearance from
the CNS and low specific binding makes it unsuitable as an in vivo PET imaging agent for AMPA receptors.
Ó 2006 Elsevier Ltd. All rights reserved.
1. Introduction
nists as potential neuroprotective agents and to AMPA
receptor modulators (ampakines) as cognitive
enhancers.4
Glutamate is the principal excitatory neurotransmitter
in the central nervous system (CNS) and mediates its
actions via activation of three families of ionotropic
receptors (iGluRs): N-methyl-D-aspartate (NMDA),
AMPA receptors are composed of a four-subunit family
(GluR1–4) that are products of separate genes and are
believed to assemble as functional tetramers.10 Addi-
tional complexity is introduced by RNA editing and
alternative splicing, with all AMPA subunits being pres-
ent as two alternatively spliced forms known as flip and
flop. This heterogeneity of AMPA is important thera-
peutically because it conveys cation selectivity and rate
and extent of desensitization.2
a-amino-3-hydroxy-5-methyl-4-isoxazole
propionate
(AMPA) and kainate, as well as by G-protein-coupled
metabotropic receptors (mGluRs).1,2 Of these, AMPA
receptors principally mediate the majority of fast excit-
atory amino acid transmission in the CNS and play a
key role in memory, cognition and learning, as well as
synaptic plasticity and neurodevelopment.3 In the dis-
eased brain, the AMPA receptors are implicated in a
wide range of pathological processes, including epilepsy,
Parkinson’s disease, multiple sclerosis and schizophre-
nia.4–9 Consequently, AMPA receptors are attractive
for therapeutic intervention and particular attention
has been devoted to selective AMPA receptor antago-
Non-invasive in vivo imaging using selective AMPA
radioligands with positron emission tomography (PET)
offers a unique opportunity to study these receptors in
the living human brain and may therefore facilitate drug
development and provide new diagnostic tools.
While considerable effort has been made to develop PET
tracers for the related NMDA receptor family,11 AMPA
imaging has remained largely unexplored. Compared
with NMDA, AMPA receptors are less well understood
Keywords: Glutamate; AMPA; PET; Isoquinolines; Carbon-11.
*
Corresponding author. Tel.: +44 0 208 383 3714; fax: +44 0 208 383
2029; e-mail: erik.arstad@csc.mrc.ac.uk
0968-0896/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.03.034

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E. Arstad et al. / Bioorg. Med. Chem. 14 (2006) 4712–4717
4713
synthesized in 4 steps from commercially available 3-
methoxytyramine (2) as outlined in Scheme 1. In short,
3-methoxytyramine (2) was reacted with 4-chlorobenzal-
dehyde (3) to give the corresponding imine 4, which
upon treatment with trifluoroacetic acid (TFA) under-
went intramolecular cyclization to afford the racemic
tetrahydroisoquinoline 5. Compound 5 was further
subjected to reaction with acetic anhydride to afford 2-
acetyl-1-(40-chlorophenyl)-7-acetoxy-6-methoxy-1,2,3,4-
tetrahydroisoquinoline (6). Derivative 6 was converted
into 2-acetyl-1-(40-chlorophenyl)-7-hydroxy-6-methoxy-
1,2,3,4-tetrahydroisoquinoline (7) under basic hydrolysis.
MeO
MeO
N
O
Me
Cl
1
Figure 1.
and few high affinity ligands are available. Non-compet-
itive antagonists are in general preferred as ligands for
PET imaging as they do not cause receptor internaliza-
tion and their binding is unaffected by endogenous
neurotransmitters. The recent discovery of the non-com-
petitive AMPA antagonist 2-acetyl-1-(40-chlorophenyl)-
6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline (1, Fig. 1)
as one of the most potent non-competitive AMPA
antagonists described to date12,13 indicated that this
compound might have the required brain uptake, affin-
ity and selectivity to allow in vivo imaging of AMPA
receptors. Herein we report the novel synthesis and
evaluation of N-acetyl-1-(40-chlorophenyl)-6-methoxy-
7-[11C]methoxy-1,2,3,4-tetrahydroisoquinoline ([11C]1)
as a potential PET ligand. In addition, the tritiated
derivative was prepared and used to determine affinity
for the AMPA receptor, specific binding and receptor
population in the rat brain. While this paper was under
review, the synthesis of the title compound and a related
fluorine-18 derivative was published elsewhere, albeit
without biological evaluation.14
2.2. Radiochemistry
Treatment of the phenolic precursor 7 with [11C]CH3I in
DMSO in the presence of sodium hydroxide followed by
HPLC purification provided the 11C labelled isoquino-
line in 17 5% (n = 8) decay corrected yield within
35 min after end-of-bombardment. The radiochemical
purity was 99% with a specific activity of 56 GBq/lmol.
The tritiated derivative 2-acetyl-1-(40-chlorophenyl)-6-
methoxy-7[3H]methoxy-1,2,3,4-tetrahydroisoquinoline
([3H]1) was obtained by replacing [11C]CH3I with
[3H]CH3I (custom synthesis by Tritium Custom Prepa-
ration Group, Amersham Biosciences).
2.3. Biology
High selectivity, good uptake and retention in target tis-
sue, and low unspecific binding are key requirements for
PET tracers. Our initial concern was therefore to con-
firm the selectivity of compound 1 for the AMPA recep-
tor system. A receptor binding assay covering 18
receptors and subtypes was therefore carried out (Nova-
Screen) and at a concentration of 10 lM no cross activ-
ity was observed. Next, [11C]1 was evaluated in adult
male Sprague–Dawley rats. The peripheral distribution
of the compound is shown in Figure 2. Of the tissues
sampled, skeletal muscle and skin had low initial
2. Results and discussion
2.1. Chemistry
Racemic
2-acetyl-1-(40-chlorophenyl)-6,7-dimethoxy-
1,2,3,4-tetrahydroisoquinoline (1) was prepared as
contents of less than
1
uptake unit15 (‘uptake
units’ = (cpm gꢀ1 wet weight tissue)/(injected cpm gꢀ1
previously described.12 The 7-desmethyl precursor was
MeO
MeO
HO
Cl
i
Cl
CHO
NH2
N
HO
2
3
4
ii
MeO
RO
MeO
MeO
HO
iii
iv
N
O
N
O
NH
O
O
Me
Me
Me
Cl
Cl
Cl
5
7: R = H
1: R = Me
Scheme 1. Reagents and conditions: (i) dry toluene, D, 3 h; (ii) TFA, D, 1.5 h; (iii) Ac2O, D, 1.5 h; (iv) K2CO3, MeOH, rt, 1 h.
6
[
11C]MeI

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E. Arstad et al. / Bioorg. Med. Chem. 14 (2006) 4712–4717
4714
liculi (2.52) and cingulate cortex (2.48), with uptake in
olfactory tubercles, thalamus and prefrontal cortex
closely following with about 2.40 uptake units. Hippo-
campus had the lowest uptake (1.84), comparable with
plasma concentration (1.40) at this time point. After
the initial uptake, no brain tissue showed any further
accumulation of radioactivity. Although there was
slightly slower loss in ‘pons with medulla’, the remaining
tissues showed a rapid clearance and relatively homoge-
neous distribution throughout the experiment. This is in
contrast to the known distribution of brain AMPA
receptors, with hippocampus, outer layers of cortex,
olfactory regions, lateral septum, basal ganglia and
amygdala enriched in GluR1, GluR2 and GluR3, while
reticular thalamic nuclei and cerebellum are enriched in
GluR4.3
Figure 2. The time course of radioactivity in representative peripheral
tissues after injection of [11C]1. Uptake is given in ‘uptake units’, see
text and reference.14
To assess the impact of metabolism on brain uptake and
distribution, the composition of radioactive species in
brain tissue and plasma was analysed. In plasma, 94%
was found to be intact tracer at 2 min, dropping to
81% at 5 min and 69% after 10 min. In brain, 91% of
the tracer was intact at 10 min, dropping to 86% after
30 min. A total of four metabolites were detected, all
more polar than the parent. This suggests that, at least
for the earlier time points, metabolism had a limited ef-
fect on the observed uptake and distribution of the com-
pound according to our recent studies.16 However, the
metabolic stability of compound 1 is likely be insuffi-
cient for PET studies in humans.
body weight)ꢀ1) and this concentration decreased to
ꢁ0.1 over 90 min. Similar profiles were observed in
stomach, thymus, intestine and liver although each
had higher initial contents of between 1 and 4 uptake
units. Kidney, spleen, heart and lung had an initial con-
tent of between 2 and 5 uptake units followed by a rapid
clearance. Fat was the only tissue that accumulated
radioactivity, from an initial low uptake of 0.30 uptake
units to 1.11 at 40 min after injection. During the course
of the experiment, there was only a small amount of
clearance via urine but high and rapid clearance via
the small intestine content. Initial clearance from body
fluids was rapid – dropping from 10 to 0.76 uptake units
from 0.2 to 2 min, thereafter clearance was slow reach-
ing 0.22 uptake units at the final sample time of 90 min.
To gain further understanding of the properties of
[11C]1, the tritiated racemic derivative [3H]1 was subject-
ed to a saturation binding study (MDS Pharma Servic-
es) using rat cortex membranes (Sprague–Dawley
derived rats, 6–8 weeks) (Fig. 4). In these experiments,
the Kd value for [3H]1 was found to be 14.8 1.8 nM,
specific to unspecific binding was 1:2 and Bmax was
148 33 fmol/mg protein.
The time course of radioactivity in several brain regions
after injection of [11C]1 was also examined (Fig. 3). The
highest initial uptake (1 min p.i.) was found in inferior
colliculi with 3.12 uptake units followed by superior col-
750
Total bound
Non-specifically bound
Specifically bound
500
250
0
0
10
20
30
40
50
60
Ligand concentration (nM)
Figure 4. Characterization of [3H]1 binding to rat cortex membranes.
These are representative experiments repeated 4 times. [3H]1 (0.61–
52 nM) was incubated for 90 min at 4 °C before filtration. Total
binding (j), non-specific binding (m) measured with [3H]1 (5 nM) in
the presence of 100 lM 1 and specific binding (d) are expressed in
fmol/mg protein. Ki was 14.8 1.8 nM, specific binding was 50% and
Bmax was 148 33 fmol/mg protein. Data are means SD.
Figure 3. The time course of radioactivity in representative brain
regions after injection of [11C]1. Uptake is given in ‘uptake units’, see
text and reference.14

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E. Arstad et al. / Bioorg. Med. Chem. 14 (2006) 4712–4717
4715
Although the receptor density measured with [3H]1 is
comparable to that found for the high affinity site of
the ligand AMPA itself (Bmax 200 fmol/mg),17 it is sig-
nificantly lower than for other selective high affinity
AMPA antagonists such as Ro 48-8587 (Bmax
1000 fmol/mg)18 and CP-526,427 (Bmax 7000 fmol/
mg).19 Neuroimaging using PET requires a certain pro-
portion of the radioligand to be receptor bound in order
to give an accumulation or increased retention of the li-
gand in target tissue. As the concentration of the radio-
ligand itself is minute, the binding potential (BP) is
dictated by the affinity of the ligand for the receptor
and the receptor density according to the formula
BP = Bmax/Kd.20 In order to obtain a sufficient signal
in vivo, the binding potential should exceed 0.5.21
Although results obtained in vitro only serve as an indi-
cation of what might be found in vivo, the substantial
difference between the measured Bmax and Kd for [3H]1
clearly suggests insufficient binding potential and may
explain the rapid clearance of the radioligand [11C]1
from the CNS, observed in our experiments. Hence,
the binding pattern of [11C]1 in the CNS is likely to
represent a combination of regional blood flow and
unspecific binding.
pid clearance of the ligand from the CNS. In conclusion,
the low receptor density labelled with this compound, its
rapid clearance from the CNS and low specific binding
make it unsuitable as an in vivo imaging agent for
AMPA receptors using PET.
4. Experimental
4.1. General
4.1.1. Chemistry. 4-Hydroxy-3-methoxyphenylethyl-
amine was obtained from Fluorochem limited, UK. All
other reagents and solvents were obtained from Aldrich
Chemical Co. or Fisher Scientific and used without fur-
ther purification. Melting points were determined on a
Stuart SMP10 apparatus and are uncorrected. Elemental
analyses (C, H, N) were carried out on a Carlo Erba
Model 1106 Elemental Analyzer and the results are with-
in 0.4% of the theoretical values. Merck silica gel 60
F254 plates were used for analytical TLC; column chro-
matography was performed on Merck silica gel 60 (70–
1
230 mesh). H NMR spectra were measured in CDCl3
with a Varian Gemini 300 spectrometer; chemical shifts
are expressed in d (ppm) relative to TMS as internal stan-
dard and coupling constants (J) in Hz. All exchangeable
protons were confirmed by addition of D2O.
This study raises several issues for future development
of PET imaging of AMPA receptors. First, it is clear
that the measured receptor density varies substantially
between AMPA specific antagonists, despite the current
notion that only AMPA modulators are subtype selec-
tive.4 The assessment of future PET candidates should
therefore be based on a combination of their affinity
and the density of the receptor population they bind
to. Second, as different AMPA antagonists label differ-
ent populations of AMPA receptors, a detailed pharma-
cological profile will be required for any future imaging
agent for AMPA receptors. Finally, the low density of
AMPA receptors that in general are found with current-
ly available high affinity AMPA ligands suggests that a
ligand with sub-nanomolar affinity might be required
for successful in vivo imaging of AMPA receptors.
HPLC was carried out using a Beckman 110B HPLC
pump connected to a SA 6506 UV detector (Severn Ana-
lytical) set at 254 nm, a GM tube and a Servogor 124
twin pen chart recorder (Spectronic). Analytical HPLC
for quality control was carried out on an Agilent 1100
series HPLC system equipped with a Bioscan GM tube
detector.
4.1.2. Radiochemistry. [11C]Methyl iodide was prepared
using the standard ‘wet’ method as described by Turton
et al.22 The radio-synthesis was carried out using a
remotely controlled apparatus housed within a lead
shielded enclosure. [3H]1 was synthesized by Amersham
Biosciences (The Maynard Centre, UK) by reaction of
precursor 7 with [3H]CH3I. The radiochemical purity
was 99% and the specific activity was 3.1 GBq/lmol.
3. Conclusion
2-Acetyl-1-(40-chlorophenyl)-6,7-dimethoxy-1,2,3,4-tet-
rahydroisoquinoline (1), one of the most potent non-
competitive AMPA antagonists described to date, has
been labelled with carbon-11 and tritium and evaluated
as a potential ligand for in vivo imaging of AMPA
receptors using PET. Labelling with carbon-11 was
achieved in 17% decay corrected yield (35 min after
end-of-bombardment) by [11C]methylation of the desm-
ethyl precursor. In biodistribution studies in rats, the la-
belled compound showed good brain uptake and rapid
clearance from the periphery; however, the distribution
in the CNS was uniform and no retention was observed
in regions known to be rich in AMPA receptors. In sat-
uration binding studies, the tritiated racemic ligand was
found to be highly potent with a Kd of 14.8 1.8 nM,
but the AMPA receptor population labelled with this
compound was only 148 33 fmol/mg protein. This sug-
gests a low binding potential, which may explain the ra-
4.1.3. Biology
4.1.3.1. Biodistribution. Male Sprague–Dawley rats
(n = 10; body weight range, 256–290 g SE = 275 4 g)
were given tail vein and artery catheters under isoflurane
and N2O/O2 anaesthesia. The animals were allowed to
recover whilst lightly restrained in Bollman cages. Each
rat received ꢁ12 MBq of [11C]1 in a volume of 0.20 ml
associated with a mass of co-injected compound 1 of
0.77 0.03 nmol kgꢀ1
.
Radiochemical purity was
ꢁ99% and, at the time of injection, specific activity
was ꢁ56 MBq nmolꢀ1. At graded times 5–6 blood sam-
ples per animal with an arterial catheter (n = 4) were
withdrawn at designated times, composite blood and
plasma curves for radioactivity as a function of time were
obtained by pooling data from animals. At times ranging
between 1 and 90 min after radioligand injection, rats
were killed and the following brain tissues were sampled;
olfactory bulbs, olfactory tubercles, hypothalamus,

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E. Arstad et al. / Bioorg. Med. Chem. 14 (2006) 4712–4717
4716
thalamus, prefrontal cortex, septal nuclei, cingulate cor-
tex, striata, somatosensory cortex, amygdala with piri-
form cortex, inferior colliculi, superior colliculi,
hippocampus, visual with temporal cortex, pons with
medulla and cerebellum. In addition, the following
peripheral tissues were sampled; skeletal muscle, skin, ur-
ine, fat, testis, small intestine, small intestine content,
large intestine, large intestine content, spleen, liver,
kidney, stomach, thymus, lung and heart ventricle.
Radioactivity content was measured using a Wallac gam-
ma-counter, with automatic correction for radioactive
decay, and results normalized for the amount injected
relative to body weight, giving15
acid. The incubations were carried out for 90 min at
4 °C. The results provided are the average of four exper-
iments, each carried out in doublets with 6 concentra-
tions of compound 1. The data were analysed using
GraphPad Prism (GraphPad Prism version 4.03, San
Diego, CA, USA). All animal procedures were carried
out in full compliance with institutional guidelines relat-
ing to the conduct of animal experiments.
4.1.3.4. Receptor screen. Adenosine, A2; Adrenergic;
Alpha 1, non-selective; Adrenergic, Beta, non-selective;
Dopamine, D1; Dopamine, D2s; GABA A; GABA B;
Muscarinic, Non-selective, Central; Nicotinic; Opioid,
Non-selective; Serotonine, 5HT1, Non-selective; Gluta-
mate, AMPA site; Glutamate, Chloride Dependent
site; Glutamate, Kainate Site; Glutamate, MK-801 site;
Glutamate, NMDA Agonist site; Glutamate, PCP site;
Glutamate, NMDA, Glycine site.
‘uptake units’ ¼ ðcpm/g wet weight tissueÞ
=ðinjected cpm/g body weightÞ.
All biological work was carried out by licensed investi-
gators in accordance with the UK Home office’s ‘‘Guid-
ance on the operation of Animals (Scientific procedures)
Act 1986’’ (HMSO, Feb 1990).
4.1.4. Synthesis of 1-(40-chlorophenyl)-7-hydroxy-6-meth-
oxy-1,2,3,4-tetrahydroisoquinoline (5). A mixture of 2-(30-
methoxy-40-hydroxyphenyl)ethylamine
(2)
(1.8 g,
4.1.3.2. Radiolabelled metabolite analysis. Blood sam-
ples were taken at 2, 5, 10 and 30 min after i.v. injection
of [11C]1). An aliquot of blood was taken for measure-
ment of radioactivity, the remaining sample was dis-
pensed into a heparinized tube and centrifuged (2000g
for 2 min). Plasma samples (450 lL) were deproteinated
with ice-cold acetonitrile (10 mL) and centrifuged
(2000g for 2 min). The resulting pellets and duplicate ali-
quots (100 lL) of acetonitrile supernatant were taken
for measurement of radioactivity. The remaining aceto-
nitrile supernatant containing the extracted [11C]1 was
rotary evaporated, the residue dissolved in the HPLC
mobile phase (2.5 mL, acetonitrile/0.1 M ammonium
formate 6:4 volume/volume) and filtered. Duplicate ali-
quots were taken for counting. Brain samples (cerebel-
lum) were homogenized with ice-cold acetonitrile
(14 mL) and the resulting mixture was centrifuged
(2000g for 2 min). Duplicate aliquots (100 lL) were
taken for counting. The acetonitrile supernatant was
concentrated in vacuo, the residue dissolved in HPLC
eluent (2.5 mL) and filtered.
10 mmol) and 4-chlorobenzaldehyde (3) (1.68 g,
12 mmol) in anhydrous toluene (50 mL) was refluxed
for 3 h, then cooled and evaporated in vacuo. The oil res-
idue was treated with diethyl ether to give a solid crude
product, which was crystallized from EtOH to afford
the desired imine 4. Trifluoroacetic acid (10 mL) was add-
ed to a solution of 4-chlorobenziliden[2-(30-methoxy-40-
hydroxyphenyl)ethyl]amine (4) (0.924 g, 3.2 mmol), and
the mixture was refluxed for 90 min. By adding water
the reaction was quenched, and the mixture was basified
(pH 8–9) with NaOH to give the isoquinoline derivative 5
as a solid. The crude product was collected by filtration
and purified by crystallization with MeOH. Mp 90–
94 °C. Yield 80%. 1H NMR: d: 2.74–3.24 (m, 5H, CH2–
CH2 + NH), 3.87 (s, 3H, MeO-6), 4.97 (s, 1H, H-1),
6.26 (s, 1H, H-5), 6.60 (s, 1H, H-8), 7.19 (d, 2H, J = 8.2,
H20–H60), 7.29 (d, 2H, J = 8.2, H30–H50).
4.1.5. Synthesis of 7-acetoxy-2-acetyl-1-(40-chlorophe-
nyl)-6-methoxy-1,2,3,4-tetrahydroisoquinoline (6).
A
solution of 1-(40-chlorophenyl)-7-hydroxy-6-methoxy-
1,2,3,4-tetrahydroisoquinoline (5) (0.375 g, 1.3 mmol)
in Ac2O (10 mL) was refluxed for 90 min and then
cooled, the reaction was quenched by adding water
and the organic layer was extracted with CHCl3. The
organic layer was dried over Na2SO4, and the solvent
was removed until dryness under reduced pressure.
The oil residue was washed with Et2O, and the crude
was crystallized with EtOH to afford compound 6. Mp
82–84 °C. Yield 64%. 1H NMR: d: 2.16 (s, 3H,
MeCON), 2.27 (s, 3H, MeCOO), 2.71–3.71 (m, 4H,
CH2–CH2), 3.84 (s, 3H, MeO-6), 6.72 (s, 1H, H-5),
6.77 (s, 1H, H-8), 6.81 (s, 1H, H-1), 7.18 (d, 2H,
J = 8.5, H20–H60), 7.24 (d, 2H, J = 8.5, H30–H50).
The processed samples were injected into an HPLC col-
umn (‘l’ Bondapak C18). The column was eluted at a
flow rate of 3 mL/min. The HPLC eluent was monitored
sequentially for radioactivity and UV absorbance at
254 nm. Both detectors were linked to a computer-based
integrator that recorded the chromatogram and allowed
the correction of the data for physical decay, back-
ground radioactivity. The methodology has been report-
ed elsewhere.16,23
4.1.3.3. In vitro studies. The radioligand binding stud-
ies were carried out by NovaScreen, USA. Determina-
tion of AMPA affinity, Bmax and specific binding for
compound [3H]1 was carried out by MDS Pharma Ser-
vices, Taiwan, using literature procedures17,24 with the
following modifications: rat cortex membranes from
Sprague–Dawley derived rats (6–8 weeks) were used in
the experiments. Non-specific binding was measured
with 100 lM compound 1 in place of 1 mM glutamic
4.1.6. Synthesis of 2-acetyl-1-(40-chlorophenyl)-7-hy-
droxy-6-methoxy-1,2,3,4-tetrahydroisoquinoline (7).
mixture of 7-acetoxy-2-acetyl-1-(40-chlorophenyl)-6-
methoxy-1,2,3,4-tetrahydroisoquinoline (6) (0.1 g,
A
0.268 mmol) and K2CO3 (0.081 g, 0.587 mmol), in
MeOH (10 mL), was stirred at room temperature for

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E. Arstad et al. / Bioorg. Med. Chem. 14 (2006) 4712–4717
4717
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1 h. The reaction mixture was filtered and then evapo-
rated in vacuo. The residue was made acidic (pH 2–3)
with HCl to give the desired compound 7 as a white sol-
id, which was collected by filtration. Mp 64–66 °C. Yield
1
60%. H NMR: d: 2.16 (s, 3H, MeCON), 2.70–3.74 (m,
4H, CH2–CH2), 3.90 (s, 3H, MeO-6), 5.56 (s, 1H, OH),
6.59 (s, 1H, H-5), 6.65 (s, 1H, H-8), 6.76 (s, 1H, H-1),
7.17 (d, 2H, J = 8.5, H20–H60), 7.23 (d, 2H, J = 8.5,
H30–H50).
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4.1.7. 2-Acetyl-1-(40-chlorophenyl)-6-methoxy-7-[11C]meth-
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nolic precursor 7 (2 mg) were added NaOH (4 mg) and
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Pak column (C18 classic), the column was washed with
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compound in 17 5% (n = 8) decay corrected yield. In
this system, the retention time of the product was
12.0 min and that of the precursor 7 was 8.3 min. The
radioactive product co-eluted with an authentic sample
of compound 1.
Acknowledgment
Financial support for this research by MIUR
(PRIN2004, Roma, Italy) is gratefully acknowledged.
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