Synthesis and anticonvulsant evaluation of N-substituted isoquinoline AMPA receptor antagonists

Article abstract of DOI:10.1016/j.bmc.2007.11.071

In our previous studies a ligand-based approach led to the identification of noncompetitive AMPA receptor antagonists containing isoquinoline scaffold. In an attempt to perform a systematic SAR study, we synthesized new N-substituted-isoquinolines bearing the most salient features described by our 3D pharmacophore model. All compounds were screened against audiogenic seizures and some derivatives showed anticonvulsant properties. Compound 24, the most active of the series, was also tested in vitro using the patch-clamp technique and proved to antagonize AMPA-mediated effects.

Full text of DOI:10.1016/j.bmc.2007.11.071

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 2379–2384
Synthesis and anticonvulsant evaluation
of N-substituted isoquinoline AMPA receptor antagonists
Rosaria Gitto,a Laura De Luca,a Benedetta Pagano,a Rita Citraro,b
Giovanbattista De Sarro,b Lara Costa,c Lucia Cirannac and Alba Chimirria,*
a
`
Dipartimento Farmaco-Chimico, Universita di Messina, Viale Annunziata, 98168 Messina, Italy
`
`
Dipartimento di Scienze Fisiologiche, Universita di Catania, Viale Andrea Doria 6, 95125 Catania, Italy
b
Dipartimento di Medicina Sperimentale e Clinica, Universita Magna Græcia, Via T. Campanella, 88100 Catanzaro, Italy
c
Received 17 July 2007; revised 21 November 2007; accepted 23 November 2007
Available online 20 February 2008
Abstract—In our previous studies a ligand-based approach led to the identification of noncompetitive AMPA receptor antagonists
containing isoquinoline scaffold. In an attempt to perform a systematic SAR study, we synthesized new N-substituted-isoquinolines
bearing the most salient features described by our 3D pharmacophore model. All compounds were screened against audiogenic sei-
zures and some derivatives showed anticonvulsant properties. Compound 24, the most active of the series, was also tested in vitro
using the patch–clamp technique and proved to antagonize AMPA-mediated effects.
Ó 2007 Elsevier Ltd. All rights reserved.
1. Introduction
In an attempt to describe the structural requirements for
noncompetitive antagonism, we have previously devel-
oped a 3D pharmacophore model which led to the iden-
tification of a new class of AMPAR ligands containing
the tetrahydroisoquinoline scaffold.13 Then we synthe-
sized a large series of tetrahydroisoquinolines and found
that some derivatives, such as the 2-acetyl-1-(40-chloro-
phenyl)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline
(3, Fig. 1) and 1-(40-bromophenyl)-6,7-dimethoxy-N-
(piperidin-1-yl-acetyl)-1,2,3,4-tetrahydroisoquinoline (4,
Fig. 1), demonstrated marked anticonvulsant effects in
various seizure models and noncompetitive AMPAR
antagonism.14–19
Glutamate (Glu), the major stimulatory neurotransmit-
ter in the central nervous system (CNS) of vertebrates,
interacts with ionotropic (iGluRs) and metabotropic
(mGluRs) receptors.1 Within the large family of iGluRs,
2-amino-3-hydroxy-5-methylisoxazole-4-propionic acid
receptors (AMPARs) constitute a subfamily character-
ized by specific pharmacological and functional proper-
ties.2,3 In particular, AMPARs play important roles in
neurotransmission in the CNS and in synaptic plasticity
that underlies learning processes and memory.4 How-
ever, the AMPARs over-activation determines neuronal
cell death related to various neurological diseases such
as stroke, Huntington’s chorea, epilepsy, etc. Therefore,
AMPAR antagonists have been considered useful as
therapeutic agents for these disorders.5–7 Moreover,
the unwanted side effects of competitive antagonists
steered the search for new agents such as 2,3-benzodiaz-
epine derivatives (e.g., GYKI 52466, 1, and CFM-2, 2)
that noncompetitively inhibit AMPARs.8–12
In our efforts to discover novel ligands with improved
pharmacological profile and to gain more insights into
the structure–activity relationships (SAR) for this class
of compounds, we report the synthesis of new N-substi-
tuted-isoquinolines (Fig. 2) that maintain the main
structural features suggested by our molecular model-
ling studies carried out by Catalyst software package
and shown in Figure 3 for compound 4.19 In particular,
we explored the effects of N-oxoacetamide chain con-
taining variable sizes of alkyl or cycloalkyl amines as
substituents in comparison with previously reported N-
acetamide-isoquinolines (e.g., 4). We evaluated their
anticonvulsant properties against audiogenic seizures
and studied noncompetitive AMPAR antagonist effects
Keywords: Isoquinolines; Microwave-assisted synthesis; Anticonvul-
sant agents; AMPA-antagonists; Molecular modelling.
*
Corresponding author. Tel.: +39 090 676 6412; fax: +39 090 355
613; e-mail: chimirri@pharma.unime.it
0968-0896/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.11.071

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R. Gitto et al. / Bioorg. Med. Chem. 16 (2008) 2379–2384
MeO
Me
O
NH
MeO
MeO
O
O
O
MeO
MeO
i
NH
N
N
MeO
OEt
N
N
O
ii
R1
5-8
9-12
R1
H2N
1, GYKI 52466
H2N
MeO
MeO
MeO
MeO
2, CFM-2
O
iii
O
N
N
R2
OH
MeO
MeO
MeO
O
O
N
Me
N
MeO
N
O
O
R1
R1
13-16
17-32
R1
3
Cl
17-20, R2 = NEt2
4
Br
5, 9, 13, 17, 21, 25, 29
H
21-24, R2 = 1-piperidinyl
25-28, R2 = 4-morpholinyl
29-32, R2 = 1-pyrrolidinyl
6, 10, 14, 18, 22, 26, 30 Br
7, 11, 15, 19, 23, 27, 31 Cl
Figure 1. Noncompetitive AMPAR antagonists.
8, 12, 16, 20, 24, 28, 32
F
MeO
MeO
O
Scheme 1. Reagents and conditions: (i) EtOCOCOCl, CH2Cl2, T.E.A,
rt, 90 min or MW, 280 W, rt, 5 min; (ii) NaOH, EtOH/H2O, rt, 2 h,
then HCl 6 N 2 h; (iii) N,N-diethylamine or cycloalkylamines, HBTU,
DMF, T.E.A, rt, 20 h or MW, 250 W, rt, 10 min.
R"
N
N
R'
O
R
a multistep approach starting from 1-aryl-6,7-dime-
thoxy-1,2,3,4-tetrahydroisoquinolines (5–8) that were
prepared16 according to previously reported procedure.
As drawn in Scheme 1 the reaction of ethyl oxalylchlo-
ride with 1-aryl-6,7-dimethoxy-1,2,3,4-tetrahydroiso-
quinolines (5–8) gave the ethyl 2-oxoacetates 9–12;
these intermediates were successively converted into
the corresponding 2-oxoacetic acid derivatives 13–16
which were coupled to appropriate amines using a stan-
dard amide coupling reaction to afford the correspond-
ing 2-oxoacetamide derivatives 17–32. We also carried
out a high-speed microwave-assisted synthesis of inter-
mediates 9–12 and desired compounds 17–32 thus opti-
mizing the chemical yield as well as greatly reducing the
reaction times more than 10-fold (see Scheme 1). The
structures of the compounds obtained were supported
by elemental analyses and spectroscopic measurements
(1H NMR).
Figure 2. General structure of the designed compounds.
The anticonvulsant effects of (1-aryl-6,7-dimethoxy-
1,2,3,4-tetrahydroisoquinolin-2-yl)-2-oxoacetamides 17–32
containing different substituents both on N-2-oxoaceta-
mide moiety and 1-aryl portion were evaluated against
audiogenic seizures in DBA/2 mice. This is considered
an excellent animal model for generalized epilepsy and
for screening new anticonvulsant drugs.20 The results
of pharmacological screening were compared with those
of other noncompetitive AMPA receptor antagonists
such as GYKI 52466 (1) as well as isoquinoline deriva-
tive 4. The data reported in Table 1 demonstrate that
these compounds generally protect DBA/2 mice in
audiogenic test after intraperitoneal (ip) administration
at higher doses than those of reference compounds 4
and GYKI 52466 (1). Compound 24 was the most active
derivative among this series of new N-substituted-iso-
Figure 3. Best HypoGen pharmacophore hypothesis aligned to com-
pound 4 (in the R configuration chosen by Catalyst). The pharmaco-
phore features are colour coded as follows: green, hydrogen bond
acceptors (HBA1 and HBA2); blue, hydrophobic aromatic region
(HYAr); cyan, hydrophobic groups (HY1 and HY2).
through an electrophysiological study to establish the
mechanism of action.
2. Results and discussion
A series of N-substituted-2-oxoacetamide-1,2,3,4-tetra-
hydroisoquinolines (17–32) were synthesized employing

R. Gitto et al. / Bioorg. Med. Chem. 16 (2008) 2379–2384
2381
Table 1. Anticonvulsant activity of compounds 1, 4, and 17–32 against
audiogenic seizures in DBA/2 mice
influence the anticonvulsant effects and in particular
the 1-piperidinyl moiety generally characterized the
most active compounds (e.g., 24). Significant activity
was also shown by some N,N-diethylamine and 1-pyrro-
lidinyl derivatives, whereas the presence of the 4-mor-
pholinyl moiety was detrimental for anticonvulsant
efficacy. The trends were not clear when we considered
the effects of the different substitution pattern of 40-posi-
tion of phenyl ring linked to the C-1 of isoquinoline
skeleton.
a
ED50 (lmol/kg)
LogPb
Clonus
Tonus
1c
4c
35.8 (24.4–52.4)
12.7 (6.09–26.3)
65.1 (50.8–83.4)
>100
25.3 (16.0–40.0)
8.17 (4.03–16.6)
53.3 (40.4–70.3)
>100
0.55
4.93
2.54
3.31
3.13
2.59
2.25
3.02
2.84
2.30
0.70
1.47
1.29
0.75
1.68
2.45
2.28
1.73
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
75.9 (60.8–94.7)
>100
67.7 (52.4–87.5)
>100
98.1 (78.4–123)
60.5 (26.2–101)
88.4 (63.2–124)
44.6 (29.0–68.7)
>100
55.3 (38.1–80.3)
41.6 (29.7–58.1)
65.3 (47.2–90.4)
38.7 (22.8–65.7)
72.1 (55.9–93.0)
>100
In spite of the fact that these new derivatives fitted well
the main structural features suggested by our 3D phar-
macophore hypothesis (two hydrogen bond acceptors,
hydrophobic aromatic region and hydrophobic groups
shown in Fig. 3), their less activity with respect to the
N-acetamide-isoquinolines (e.g., 4) could be due to the
presence of an additional carbonyl moiety which nega-
tively influences their pharmacokinetic properties.
>100
91.0 (68.4–121)
>100
>100
46.9 (33.3–66.2)
90.2 (56.8–144)
54.6 (41.5–71.9)
45.4 (29.7–69.4)
37.2 (24.0–57.7)
73.0 (45.7–117)
89.2 (64.1–124)
58.1 (37.3–90.5)
>100
With the aim to define the mechanism of action of com-
pound 24 electrophysiological study has also been per-
formed.22 The application of AMPA (5 lM, 5 s)
induced in CA1 pyramidal neurons an inward current,
the amplitude of which ranged between 30 9 and
209 13 pA in different neurons (mean SEM of 3–6
consecutive responses to AMPA). When compound 24
(50 lM, 5 min) was simultaneously applied through
the bath perfusion system, AMPA-mediated current
was reversibly reduced (Fig. 4A). The amplitude of
AMPA-mediated current (at least 3 values from consec-
utive responses) was compared with amplitude values (at
a All data were calculated according to the method of Litchfield and
Wilcoxon. At least 32 animals were used to calculate each ED50 and
95% confidence limits are given in parentheses.
b LogP data are predicted from a commercially available program
(ACD/Lab).
c Ref. 19.
quinolines (17–32) in which the different anticonvulsant
potencies were not apparently related to their relative
lipophilicity (LogP, Table 1).21 These results suggested
that the nature of (cyclo)alkylamino substituents might
A
AMPA
5 μM
24 (50 μM)
30 pA
10 s
control
B
C
175
120
100
80
60
40
20
0
control
24
control
24
**
150
125
**
100
*
75
**
**
50
**
***
25
0
1
2
3
4
5
6
1
2
3
4
5
6
neurons
neurons
Figure 4. Effect of compound 24 on AMPA-mediated current. (A) Application of AMPA (5 lM, 2 s) induced in CA1 pyramidal neurons an inward
current, the amplitude of which was reduced by application of the antagonist 24 (50 lM, 5 min). (B) The amount of reduction of AMPA-mediated
current amplitude by 24 was statistically significant in 5 out of 6 neurons. (C) In 2 of the responsive neurons, AMPA-mediated current was also
reduced in duration in the presence of compound 24 (***P < 0.0001; **P < 0.001; *P < 0.01; unpaired t test, two-tailed).

2382
R. Gitto et al. / Bioorg. Med. Chem. 16 (2008) 2379–2384
least 3) during antagonist application: the amount of de-
crease was statistically significant in 5 out of 6 neurons
tested (Student’s t test, Fig. 4B). In the presence of 24,
the amplitude of AMPA-mediated current was reduced
on average by 60 8% (mean SEM, n = 5, range 41–
89, median 60.0); Wilcoxon matched pairs test, per-
formed on data from all the tested neurons, confirmed
a significant reduction of AMPA-mediated current.
The duration of AMPA-mediated current was reduced
during 24 application in 3 of the neurons studied (num-
bered as 2, 4 and 6 in Fig. 4C). In the remaining neu-
rons, the duration of AMPA response was either not
modified (neuron No. 1 and 5, Fig. 4C) or slightly in-
creased (neuron No. 3, Fig. 4C). Recovery of AMPA-
mediated current amplitude and duration was observed
between 2 and 5 min after washout of 24. In line with
in vivo, study GYKI 52466 (1) demonstrated better ef-
fects than compound 24 in the same assay (see Supple-
mentary material).
dried over Na2SO4, and the solvent was removed until
dryness under reduced pressure to give ethyl (1-aryl-
6,7-dimethoxy-1,2,3,4-dihydroisoquinolin-2-yl)-oxoace-
tate intermediates (9–12). The crude product from the
previous step was dissolved in 50% EtOH/water
(5 mL) and stirred at 25 °C for 2 h. Then the mixture
was made acidic by the addition of HCl (6.0 N); the
resultant yellow solid was removed by filtration, washed
with water to afford 1-aryl-6,7-dimethoxy-1,2,3,4-tetra-
hydroisoquinolin-2-yl-oxoacetic acids (13–16). To a
solution in DMF (5 mL) of crude material obtained in
previous step (1 mmol, 13–16) and HBTU (1 mmol),
the suitable alkyl- or cycloalkyl amine (1 mmol) was
added; the reaction mixture was placed in a cylindrical
quartz tube (B 2 cm), then stirred and irradiated in a
microwave oven at 250 W for 10 min at 25 °C; the
cooled organic layer was extracted with EtOAc, dried
over Na2SO4 and the solvent was removed until dryness
under reduced pressure; the resultant solid was crystal-
lized from 50% diethyl ether/cyclohexane to give
ethyl(1-aryl-6,7-dimethoxy-1,2,3,4-tetrahydroisoquino-
lin-2-yl)-2-oxoacetamide derivatives 17–32. 1H NMR
spectral data confirmed the structure of intermediates
9–16 (see Supplementary material).
3. Conclusion
In conclusion, a series of new N-substituted isoquinoline
17–32 AMPA receptor antagonists was prepared and
tested as anticonvulsant agents. In spite of maintenance
of structural requirements for recognition of noncom-
petitive binding site of AMPAR, the results obtained
suggest that the planned modification of the linkage be-
tween tetrahydroisoquinoline system and alkylamino
moiety negatively affects the anticonvulsant efficacy. In
fact only compound 24 proved to prevent audiogenic
seizures in DBA/2 mice with a potency comparable to
that of GYKI 52466 (1) and reduce AMPA-mediated
current in electrophysiological assay.
4.1.2. 2-(6,7-Dimethoxy-1-phenyl-3,4-dihydroisoquinolin-
2(1H)-yl)-N,N-diethyl-2-oxoacetamide (17). Mp 136–
1
138 °C. Yield 80%. H NMR: d 1.10 (t, 3H, J = 7.14,
CH3), 1.22 (t, 3H, J = 7.14, CH3), 2.75–3.69 (m, 8H,
CH2), 3.80 (s, 3H, CH3O-6), 3.94 (s, 3H, CH3O-7),
6.54 (s, 1H, H-1), 6.70 (s, 1H, H-5), 6.82 (s, 1H, H-8),
7.31–7.34 (m, 5H, Ar). Anal. (C23H28N2O4) C, H, N.
4.1.3. 2-[1-(4-Bromophenyl)-6,7-dimethoxy-3,4-dihydro-
isoquinolin-2(1H)-yl]-N,N-diethyl-2-oxoacetamide (18).
Mp 82–84 °C. Yield 83%. 1H NMR: d 1.09 (t, 3H,
J = 7.14, CH3), 1.18 (t, 3H, J = 7.14, CH3), 2.70–3.61
(m, 8H, CH2), 3.76 (s, 3H, CH3O-6), 3.90 (s, 3H,
CH3O-7), 6.45 (s, 1H, H-1), 6.66 (s, 1H, H-5), 6.72 (s,
1H, H-8), 7.17 (d, 2H, J = 7.7, H20–H60), 7.43 (d, 2H,
J = 7.7, H30–H50). Anal. (C23H27BrN2O4) C, H, N.
4. Experimental
4.1. Chemistry
Microwave-assisted reactions were carried out in a CEM
focused Microwave Synthesis System. 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 within 0.4% of the theoretical values.
Merck silica gel 60 F254 plates were used for analytical
TLC. 1H NMR spectra were measured in CDCl3 with a
Varian Gemini 300 spectrometer; chemical shifts are ex-
pressed in d (ppm) relative to TMS as internal standard
and coupling constants (J) in Hz.
4.1.4. 2-[1-(4-Chlorophenyl)-6,7-dimethoxy-3,4-dihydro-
isoquinolin-2(1H)-yl]-N,N-diethyl-2-oxoacetamide (19).
Mp 135–137 °C. Yield 82%. H NMR: d 1.13 (t, 3H,
J = 7.14, CH3), 1.23 (t, 3H, J = 7.14, CH3), 2.74–3.66
(m, 8H, CH2), 3.80 (s, 3H, CH3O-6), 3.94 (s, 3H,
CH3O-7), 6.49 (s, 1H, H-1), 6.70 (s, 1H, H-5), 6.79 (s,
1H, H-8), 7.27 (d, 2H, J = 8.2, H20–H60), 7.32 (d, 2H,
J = 8.2, H30–H50). Anal. (C23H27ClN2O4) C, H, N.
1
4.1.5. N,N-Diethyl-2-[1-(4-fluorophenyl)-6,7-dimethoxy-
3,4-dihydroisoquinolin-2(1H)-yl]-2-oxoacetamide (20).
1
4.1.1. General procedure for the preparation of 2-(6,
7-dimethoxy-1-aryl-1,2,3,4-tetrahydroisoquinolin-2-yl)-2-
oxoacetamide derivatives 17–32. A mixture of suitable
isoquinoline derivatives (5–8)16 (1 mmol) and ethyloxa-
lyl chloride (14 mmol) in CH2Cl2 (10 mL) was placed
in a cylindrical quartz tube (B 2 cm), then stirred and
irradiated in a microwave oven at 280 W for 5 min at
25 °C. After cooling the reaction was quenched by add-
ing a saturated sodium bicarbonate solution, and the
mixture extracted with EtOAc. The organic layer was
Mp 141–143 °C. Yield 81%. H NMR: d 1.08 (t, 3H,
J = 7.14, CH3), 1.18 (t, 3H, J = 7.14, CH3), 2.70–3.59
(m, 8H, CH2), 3.75 (s, 3H, CH3O-6), 3.90 (s, 3H, CH3O-
7), 6.45 (s, 1H, H-1), 6.66 (s, 1H, H-5), 6.72 (s, 1H, H-8),
7.15–7.44 (m, 4H, Ar). Anal. (C23H27FN2O4) C, H, N.
4.1.6. 6,7-Dimethoxy-2-[oxo(piperidin-1-yl)acetyl]-1-phe-
nyl-1,2,3,4-tetrahydroisoquinoline (21). Mp 115–117 °C.
1
Yield 46%. H NMR: d 1.21–1.71 (m, 6H, CH2), 2.72–
3.64 (m, 8H, CH2), 3.75 (s, 3H, CH3O-6), 3.89 (s, 3H,

R. Gitto et al. / Bioorg. Med. Chem. 16 (2008) 2379–2384
2383
CH3O-7), 6.49 (s, 1H, H-1), 6.66 (s, 1H, H-5), 6.76 (s,
1H, H-8), 7.28–7.30 (m, 5H, Ar). Anal. (C24H28N2O4)
C, H, N.
CH2), 2.72–3.64 (m, 8H, CH2), 3.75 (s, 3H, CH3O-6),
3.90 (s, 3H, CH3O-7), 6.49 (s, 1H, H-1), 6.66 (s, 1H,
H-5), 6.77 (s, 1H, H-8), 7.21–7.30 (m, 5H, Ar). Anal.
(C23H26N2O4) C, H, N.
4.1.7. 1-(4-Bromophenyl)-6,7-dimethoxy-2-[oxo(piperi-
din-1-yl)acetyl]-1,2,3,4-tetrahydroisoquinoline (22). Mp
94–96 °C. Yield 48%. H NMR: d 1.18–1.60 (m, 6H,
CH2), 2.69–3.65 (m, 8H, CH2), 3.75 (s, 3H, CH3O-6),
3.89 (s, 3H, CH3O-7), 6.44 (s, 1H, H-1), 6.65 (s, 1H,
H-5), 6.70 (s, 1H, H-8), 7.16 (d, 2H, J = 8.5, H20–H60),
7.43 (d, 2H, J = 8.5, H30–H50). Anal. (C24H27BrN2O4)
C, H, N.
4.1.15. 1-(4-Bromophenyl)-6,7-dimethoxy-2-[oxo(pyrroli-
din-1-yl)acetyl]-1,2,3,4-tetrahydroisoquinoline (30). Mp
97–99 °C. Yield 46%. H NMR: d 1.42–1.91 (m, 4H,
CH2), 2.69–3.66 (m, 8H, CH2), 3.75 (s, 3H, CH3O-6),
3.89 (s, 3H, CH3O-7), 6.45 (s, 1H, H-1), 6.66 (s, 1H,
H-5), 6.72 (s, 1H, H-8), 7.17 (d, 2H, J = 8.0, H20–H60),
7.43 (d, 2H, J = 8.0, H30–H50). Anal. (C23H25BrN2O4)
C, H, N.
1
1
4.1.8. 1-(4-Chlorophenyl)-6,7-dimethoxy-2-[oxo(piperi-
din-1-yl)acetyl]-1,2,3,4-tetrahydroisoquinoline (23). Mp
88–90 °C. Yield 47%. H NMR: d 1.25–1.64 (m, 6H,
CH2), 2.70–3.65 (m, 8H, CH2), 3.75 (s, 3H, CH3O-6),
3.90 (s, 3H, CH3O-7), 6.44 (s, 1H, H-1), 6.65 (s, 1H,
H-5), 6.72 (s, 1H, H-8), 7.22 (d, 2H, J = 8.2, H20–H60),
7.29 (d, 2H, J = 8.2, H30–H50). Anal. (C24H27ClN2O4)
C, H, N.
4.1.16. 1-(4-Chlorophenyl)-6,7-dimethoxy-2-[oxo(pyrroli-
din-1-yl)acetyl]-1,2,3,4-tetrahydroisoquinoline (31). Mp
91–93 °C. Yield 45%. H NMR: d 1.43–1.93 (m, 4H,
CH2), 2.70–3.65 (m, 8H, CH2), 3.75 (s, 3H, CH3O-6),
3.89 (s, 3H, CH3O-7), 6.45 (s, 1H, H-1), 6.67 (s, 1H, H-
5), 6.73 (s, 1H, H-8), 7.21 (d, 2H, J = 8.2, H20–H60), 7.30
(d, 2H, J = 8.2, H30–H50). Anal. (C23H25ClN2O4) C, H, N.
1
1
4.1.9. 1-(4-Fluorophenyl)-6,7-dimethoxy-2-[oxo(piperidin-
1-yl)acetyl]-1,2,3,4-tetrahydroisoquinoline (24). Mp 140–
142 °C. Yield 46%. 1H NMR: d 1.25–1.62 (m, 6H,
CH2), 2.70–3.60 (m, 8H, CH2), 3.75 (s, 3H, CH3O-6),
3.89 (s, 3H, CH3O-7), 6.45 (s, 1H, H-1), 6.65 (s, 1H,
H-5), 6.73 (s, 1H, H-8), 6.96–7.01 (m, 4H, Ar). Anal.
(C24H27FN2O4) C, H, N.
4.1.17. 1-(4-Fluorophenyl)-6,7-dimethoxy-2-[oxo(pyrroli-
din-1-yl)acetyl]-1,2,3,4-tetrahydroisoquinoline (32). Mp
76–78 °C. Yield 43%. H NMR: d 1.42–1.91 (m, 4H,
CH2), 2.76–3.65 (m, 8H, CH2), 3.75 (s, 3H, CH3O-6),
3.89 (s, 3H, CH3O-7), 6.46 (s, 1H, H-1), 6.66 (s, 1H,
H-5), 6.74 (s, 1H, H-8), 6.99–7.28 (m, 4H, Ar). Anal.
(C23H25FN2O4) C, H, N.
1
4.1.10. 6,7-Dimethoxy-2-[morpholin-4-yl(oxo)acetyl]-1-
phenyl-1,2,3,4-tetrahydroisoquinoline (25). Mp 153–
155 °C. Yield 51%. 1H NMR: d 2.73–3.71 (m, 12H,
CH2), 3.75 (s, 3H, CH3O-6), 3.90 (s, 3H, CH3O-7),
6.48 (s, 1H, H-1), 6.67 (s, 1H, H-5), 6.76 (s, 1H, H-8),
7.25–7.31 (m, 5H, Ar). Anal. (C23H26N2O5) C, H, N.
4.2. Pharmacology
4.2.1. Testing of anticonvulsant activity. All experiments
were performed with DBA/2 mice which are genetically
susceptible to sound-induced seizures.20 DBA/2 mice (8–
12 g; 22 to 25 days old) were purchased from Harlan
Italy (Corezzano, Italy). Groups of 10 mice of either
sex were exposed to auditory stimulation 30 min follow-
ing administration of vehicle or each dose of drugs stud-
ied. The compounds were given intraperitoneally (ip)
(0.1 mL/10 g of body weight of the mouse) as a freshly
prepared solution in 50% dimethylsulfoxide (DMSO)
and 50% sterile saline (0.9% NaCl). Individual mice were
placed under a hemispheric perspex dome (diameter
58 cm), and 60 s was allowed for habituation and assess-
ment of locomotor activity. Auditory stimulation (12–
16 kHz, 109 dB) was applied for 60 s or until tonic
extension occurred, and induced a sequential seizure re-
sponse in control DBA/2 mice, consisting of an early
wild running phase, followed by generalized myoclonus
and tonic flexion and extension sometimes followed by
respiratory arrest. The control and drug-treated mice
were scored for latency to and incidence of the different
phases of the seizures. The experimental protocol and all
the procedures involving animals and their care were
conducted in conformity with the Institutional Guide-
lines and the European Council Directive of laws and
policies.
4.1.11. 1-(4-Bromophenyl)-6,7-dimethoxy-2-[morpholin-
4-yl(oxo)acetyl]-1,2,3,4-tetrahydroisoquinoline (26). Mp
161–163 °C. Yield 53%. 1H NMR: d 2.72–3.72 (m,
12H, CH2), 3.75 (s, 3H, CH3O-6), 3.90 (s, 3H, CH3O-
7), 6.43 (s, 1H, H-1), 6.66 (s, 1H, H-5), 6.69 (s, 1H, H-
8), 7.16 (d, 2H, J = 8.2, H20–H60), 7.43 (d, 2H, J = 8.5,
H30–H50). Anal. (C23H25BrN2O5) C, H, N.
4.1.12. 1-(4-Chlorophenyl)-6,7-dimethoxy-2-[morpholin-
4-yl(oxo)acetyl]-1,2,3,4-tetrahydroisoquinoline (27). Mp
1
99–100 °C. Yield 52%. H NMR: d 2.76–3.72 (m, 12H,
CH2), 3.75 (s, 3H, CH3O-6), 3.89 (s, 3H, CH3O-7),
6.43 (s, 1H, H-1), 6.65 (s, 1H, H-5), 6.81 (s, 1H, H-8),
7.25 (d, 2H, J = 8.5, H20–H60), 7.29 (d, 2H, J = 8.5,
H30–H50). Anal. (C23H25ClN2O5) C, H, N.
4.1.13. 1-(4-Fluorophenyl)-6,7-dimethoxy-2-[morpholin-4-
yl(oxo)acetyl]-1,2,3,4-tetrahydroisoquinoline (28). Mp
191–193 °C. Yield 48%. 1H NMR: d 2.73–3.72 (m, 12H,
CH2), 3.75 (s, 3H, CH3O-6), 3.90 (s, 3H, CH3O-7), 6.45
(s, 1H, H-1), 6.66 (s, 1H, H-5), 6.69 (s, 1H, H-8), 6.73–
7.27 (m, 4H, Ar). Anal. (C23H25FN2O5) C, H, N.
4.2.2. Statistical analysis. Statistical comparisons be-
tween groups of control and drug-treated animals were
made using Fisher’s exact probability test (incidence of
the seizure phases). The ED50 values of each phase of
4.1.14. 6,7-Dimethoxy-2-[oxo(pyrrolidin-1-yl)acetyl]-1-
phenyl-1,2,3,4-tetrahydroisoquinoline (29). Mp 114–
116 °C. Yield 42%. 1H NMR: d 1.43–1.92 (m, 4H,

2384
R. Gitto et al. / Bioorg. Med. Chem. 16 (2008) 2379–2384
audiogenic seizures were determined for each dose of
compound administered, and dose–response curves were
fitted using a computer program by Litchfield and Wil-
coxon’s method.23
Acknowledgments
Financial support for this research by Fondo di Ateneo
`
per la Ricerca (PRA 2004—Universita di Messina) and
Fondo per gli Investimenti della Ricerca di Base (FIRB
2003) is gratefully acknowledged.
4.2.3. Electrophysiology. Young Wistar rats (age 9–17
days) were decapitated under ether anaesthesia; brains
were rapidly removed, placed in oxygenated ice-cold
artificial cerebrospinal fluid (ACSF; composition in
mM: NaCl 124; KCl 3.0; NaH2PO4 1.2; MgSO4 1.2;
CaCl2 2.0; NaHCO3 26; D-glucose 10, pH 7.3) and cut
into 300 lm slices with a vibratome. One slice contain-
ing the hippocampus was then transferred to the record-
ing chamber of a patch–clamp set-up, continually
perfused with oxygenated ACSF and viewed under an
infrared differential interference contrast microscope
(Leica DMLFS). CA1 pyramidal neurons were visually
identified by their location and by their typical shape
and dimension. Membrane current was recorded from
single CA1 pyramidal neurons with the patch–clamp
technique in the whole-cell configuration, using an L/
M-EPC7 amplifier (List Electronic, Germany). The
recording electrode was a glass micropipette having a fi-
nal tip resistance between 1.5 and 3 MX, filled with
intracellular solution (composition in mM: K-gluconate
170; HEPES 10; NaCl 10; MgCl2 2; EGTA 0.2; Mg-
ATP 3.5; Na-GTP 1; pH 7.3).
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/
j.bmc.2007.11.071.
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