Structure-activity relationship of dopaminergic halogenated 1-benzyl-tetrahydroisoquinoline derivatives

Article abstract of DOI:10.1016/j.ejmech.2009.06.033

Two series of halogenated 1-benzyl-7-chloro-6-hydroxy-tetrahydroisoquinolines were prepared to explore the influence of each series on the affinity for dopamine receptors. All the compounds displayed a high affinity for D₁-like and/or D₂

Full text of DOI:10.1016/j.ejmech.2009.06.033

European Journal of Medicinal Chemistry 44 (2009) 4616–4621
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Structure–activity relationship of dopaminergic halogenated
1-benzyl-tetrahydroisoquinoline derivatives
a
a
a
a
´
,
Noureddine El Aouad , Inmaculada Berenguer , Vanessa Romero , Paloma Marın ,
Angel Serrano , Sebastian Andujar c, Fernando Suvire b, Almudena Bermejo a
a
b,
,d
´
´
M. Dolores Ivorra a, Ricardo D. Enriz b c, Nuria Cabedo a e, Diego Cortes a
,
,
,
*
a
´
Departamento de Farmacologıa, Facultad de Farmacia, Universidad de Valencia, 46100 Burjassot, Valencia, Spain
b
´
Departamento de Quımica, Universidad Nacional de San Luis, Argentina
c IMIBIO-SL (CONICET), Chacabuco 915, 5700 San Luis, Argentina
d
´
Departamento de Citricultura, IVIA, crta Moncada-Naquera Km 4.5, 46113 Moncada, Valencia, Spain
e Centro de Ecolog´ıa Qu´ımica Agr´ıcola-Instituto Agroforestal del Mediterraneo, UPV, Campus de Vera, Edificio 6C, 46022 Valencia, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
Two series of halogenated 1-benzyl-7-chloro-6-hydroxy-tetrahydroisoquinolines were prepared to
explore the influence of each series on the affinity for dopamine receptors. All the compounds displayed
a high affinity for D1-like and/or D2-like dopamine receptors in striatal membranes, although they were
unable to inhibit [3H]-dopamine uptake in striatal synaptosomes. The halogen placed on the benzylic
ring in 1-benzyl-THIQs, compounds of the series 1, 20-bromobenzyl derivatives with Ki values into the
nanomolar range, and the series 2, 20,40-dichlorobenzyl-THIQ homologues, proves to be an important
factor to modulate affinity at dopamine receptor.
Received 3 April 2009
Received in revised form
25 June 2009
Accepted 29 June 2009
Available online 4 July 2009
Keywords:
Ó 2009 Elsevier Masson SAS. All rights reserved.
1-Halogenated benzyl-7-chloro-6-hydroxy-
tetrahydroisoquinolines
Binding
Dopamine receptors
Dopamine uptake
Structure–activity relationships
1. Introduction
the drugs (antagonists) used in the treatment of schizophrenia
(antipsychotics) and those (agonists) utilised to treat Parkinson’s
Dopamine-mediated neurotransmission plays an important role
in several psychiatric and neurological disorders which affect
several million people worldwide. Researchers have focused on
various approaches towards modulating dopaminergic activity via
the dopamine receptors (DR) as a potential means of treating
schizophrenia and Parkinson’s diseases. The consequences of an
activation or blockade of DR are wide-ranging, and a perturbation
of dopamine neurotransmission may result in profound neurolog-
ical, psychiatric, or physiological signs and symptoms. For these
reasons, much research has focused on the discovery of novel
dopaminergic ligands as potential drug candidates [1]. DR can be
classified into two pharmacological families (D1 and D2-like) that
are encoded by at least five genes. Which receptor(s) needs to be
activated to obtain therapeutic effects in Parkinson’s disease
remains controversial [2]. The D2-like DR shows high affinities for
disease [3].
Substituted isoquinolines (IQ) represent a class of natural and
synthetic compounds that have drawn considerable attention
because of their significant and powerful biological activities,
including the inhibition of cellular proliferation, and their anti-
tumour, antibiotic and anticonvulsant properties. A large number of
IQ alkaloids have been obtained from various plants species, and
have been evaluated for their ability to inhibit the dopamine
transporter and to display affinities at D1 and D2-like DR binding
sites in rat brain tissue [4]. The neurotransmitter dopamine (3,4-
dihydroxyphenethylamine) is involved in the regulation of several
functions, including locomotor activity, emotion, cognition and
neuroendocrine secretion. Tetrahydroisoquinolines (THIQs), the
most numerous naturally occurring alkaloids, include 1-benzyl-
THIQs and aporphines, both of which have structural similarities to
dopamine and can interact with DR [5].
Previous results of works done in our group suggest that some
natural and synthetic 1-benzyl-THIQs alkaloids are able to bind to
* Corresponding author. Tel.: þ34 963 54 49 75; fax: þ34 963 54 49 43.
E-mail address: dcortes@uv.es (D. Cortes).
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.06.033

N. El Aouad et al. / European Journal of Medicinal Chemistry 44 (2009) 4616–4621
4617
Cl
DR [6–8]. We described the enantioselective syntheses of pairs of
dopaminergic (1S)- and (1R)-benzyl-THIQs using (R)- and (S)-
phenylglycinol as the chiral source. We observed that in these 1-
benzyl-THIQs series, their (1S)-enantiomers were 5–15 times more
effective at the D1-like DR and D2-like DR than the (1R)-enantio-
mers [9]. Conversely, we described the preparation in a ‘one-pot’
sequence of 1-cyclohexylmethyl 7,8-dioxygenated-THIQs, which
was substituted and unsubstituted in the C ring by the application
of the photo-Fries transposition, followed by a tandem reduction–
cyclisation and further reduction. Indeed, we accomplished
a regioselective hydrogenation of the benzyl ring in the THIQ
system for the first time. All the synthesised 1-cyclohexylmethyl
THIQs were able to displace the D2-like DR radioligand from its
specific binding sites in rat striatal membranes, while the N-
methylated derivatives also showed affinity for the D1-like DR [10].
For the purpose of comparing the binding affinities of 1-
substituted-THIQs for DR, and as a part of our research into the
synthesis of new dopaminergic THIQ derivatives, we prepared two
series of halogenated 1-benzyl-tetrahydroisoquinolines which
support constant structural factors 6-chloro and 7-oxygenated
substitutions, as well as a basic secondary (NH) or tertiary (NCH3)
amine.
NH
2
H CO
3
2-(3-chloro-4-methoxy
phenyl)ethylamine
Cl
Br
Cl
Cl
O
O
Cl
β
3
Cl
Cl
1
α
NH
NH
H CO
3
4
H CO
3
O
O
1'
Cl
Br
2'
Cl
1
2
2'-Br-1-benzyl-THIQ series
2',4'-diBr-1-benzyl-THIQ series
(a)
(a)
4
5
Cl
4a
6
7
Cl
3
A
B
NR
The structures of the resulting 1-benzyl-THIQs were determined
on the basis of their NMR spectral data and mass spectrometry
analysis. All the synthesised compounds were tested for their ability
to displace the selective radioligands of D1 and D2-like DR from their
specific binding sites in striatal membranes in order to establish their
structure–activity relationships (SARs) as dopaminergic agents.
NR
8a
6'
H CO
3
H CO
3
8
1
α
5'
4'
1a: R= H
1b: R= CH3
2a: R= H
2b: R= CH3
C
1'
2'
(b)
(b)
Br
Cl
Cl
3'
(c)
(c)
2. Results and discussion
Cl
Cl
NR
NR
In the present work, we have studied the effect of the attach-
ment of a chlorine or bromine atom to an aromatic C-ring in a series
of 1-benzyl-6-chloro-7-hydroxy-THIQ derivatives. In previous
works [10–13], we determined both the importance of the absolute
configuration of the carbon at position 1 and the role of certain
structural requirements for improving the affinity for the D1 and
D2-like DR families. Thus, it has been postulated that the substi-
tution into the A-ring for a hydroxyl (OH) and a halogen could lead
to molecules which selectively bind at least one of the two afore-
mentioned groups of receptors [12,13]. Previously, we have
observed that 1-substituted-THIQ derivatives with a benzyl moiety
at position 1 showed an affinity for D1 and D2 receptors [10,14].
Now, we decided to study the influence of the halogenation of this
benzyl group by maintaining the chlorine and OH groups at posi-
tions C-6 and C-7 in the THIQ A-ring, respectively.
In this way, we prepared two series of compounds (Scheme 1):
20-bromobenzyl-THIQs (series 1: 1a–1d) and 20,40-dichlorobenzyl-
THIQs (series 2: 2a–2d) with an identical substitution in the A-ring
(6-chloro, 7-hydroxy) and the nitrogen as a secondary or a tertiary
amine (NH and NMe). The structures of the resulting eight 1-
substituted-THIQs were determined on the basis of their NMR
spectral data and mass spectrometry analysis.
HO
HO
1c: R= H
1d: R= CH3
2c: R= H
2d: R= CH3
Br
Cl
Cl
Scheme 1. Synthesis of halogenated 1-benzyl-THIQ analogues (a) POCl3-P2O5/toluene;
NaBH4/MeOH; (b) HCHO/NaBH4; (c) BBr3 – 3 h.
(20-bromophenyl)acetamide (1) and N-(3-chloro-4-methoxy-phe-
nethyl)-2-(20,40-dichlorophenyl)acetamide (2). Afterwards, these
N-phenylethylamides (1 and 2) were converted into the corre-
sponding 1-substituted-THIQs using the Bischler–Napieralski
cyclodehydration reaction for which it was necessary to use
a mixture of POCl3 and P2O5 in dry toluene [15–17], followed by
NaBH4 reduction. Subsequently, these BTHIQ were treated with
formaldehyde and formic acid, followed by NaBH4 reduction, to
afford the N-methyl derivatives. Finally, the O-demethylation of all
the synthesised isoquinolines was performed by adding of 4
equivalents of BBr3 reagent for 3 h at room temperature to obtain
the resulting phenolic tertiary amines 1d and 2d.
2.1. Chemistry
2.2. Structure–activity relationship
The synthesis of 1-halogenated benzyl-7-chloro-6-hydroxy-
THIQ derivatives has been accomplished as an outline in Scheme 1,
starting from b-(3-chloro-4-methoxyphenyl) ethylamine, and the
2-bromobenzyl and 2,4-dichlorobenzyl acid chlorides for series 1
and 2, respectively. Firstly, that previously prepared by standard
methods 2-(3-chloro-4-methoxy)ethylamine (Scheme 1) was
treated with 2-bromobenzyl or 2,4-dichlorobenzyl acid chloride
under Schotten–Baumann conditions and afforded the two amide
derivatives with good yields: N-(3-chloro-4-methoxyphenethyl)-2-
All the synthesised compounds (see Scheme 1) were assayed in
vitro for their ability to displace the selective ligands of D1 and D2
DR from their respective specific binding sites in the striatal
membranes. All the compounds were unable to inhibit [3H]-
dopamine uptake in striatal synaptosomes. Many of these
compounds were able to displace both 3H-SCH 23390 and 3H-
raclopride from their specific binding sites in rat striatum at
micromolar (
mM) or nanomolar (nM) concentrations. The binding
affinities for D1 and D2 DR are summarised in Table 1, and these

4618
N. El Aouad et al. / European Journal of Medicinal Chemistry 44 (2009) 4616–4621
Table 1
isoquinolines [10,14]. The 1-substituted THIQs synthesised the
presence of a hydroxyl (OH) group at position C-7, and their
ability to displace the selective ligands of the D1 and D2
dopamine receptors from their specific binding sites in the
striatal membranes was positively influenced, while the
replacement with a methoxyl (OMe) group at position C-7 did
not favour dopaminergic activity.
Dissociation constants (Ki and pKi) and selectivity of different compounds at the
D1-like and D2-like dopaminergic receptors.
Compound Specific-D1 ligand [3H]-
SCH23390
Specific-D2 ligand [3H]-
raclopride
D1/D2
Ki (m
M)
pKi
Ki (
mM)
pKi
1a
1b
1c
1d
1.528 ꢀ 0.304 5.83 ꢀ 0.08 0.245 ꢀ 0.050 6.63 ꢀ 0.09c
1.319 ꢀ 0.145 5.89 ꢀ 0.05 1.336 ꢀ 0.296 5.90 ꢀ 0.09e
0.110 ꢀ 0.042 7.06 ꢀ 0.18f 0.046 ꢀ 0.012 7.39 ꢀ 0.14f
6.2
1.0
2.4
2. Both the 1 (20-bromobenzyl-THIQs) and 2 (20,40-dichlorobenzyl-
THIQs) compounds series showed a high affinity for dopami-
nergic receptors. Surprisingly however, the different type of
halogen at the benzyl level did not appear to be important for the
selective improvement of the affinities for the D1 or D2 receptors.
In addition, the secondaryamine (NH) showed an affinity similar
to that of its corresponding tertiary amine (NMe) homologues.
3. However, the absence of selectivity that these compounds dis-
played did not prevent them from behaving like molecules with
a high affinity for dopaminergic receptors, and actually showed
some Ki values in the nanomolar range (1d: 18 nM). We
observed that the compounds of series 1 (20-bromobenzyl
derivatives) showed an affinity that was 10 times higher than
those of their corresponding 20,40-dichlorobenzyl-THIQ homo-
logues (series 2) since the latter exhibited less potency to
displace the ligands of D1 and/or D2 DR from their specific
binding sites. It is likely that the larger size and atomic number
of the bromine atom could justify the better affinity for the D1
and D2 receptors (Table 1 and Fig. 1).
0.065 ꢀ 0.011 7.21 ꢀ 0.07f 0.018 ꢀ 0.004 7.76 ꢀ 0.08b,d,f 3.6
2a
2b
2c
2d
11.323 ꢀ 4.164 5.01 ꢀ 0.17 3.301 ꢀ 0.797 5.51 ꢀ 0.11a
15.113 ꢀ 2.860 4.84 ꢀ 0.08 6.132 ꢀ 1.381 5.23 ꢀ 0.10
0.183 ꢀ 0.014 6.74 ꢀ 0.03f 0.107 ꢀ 0.027 7.00 ꢀ 0.11f
0.241 ꢀ 0.054 6.71 ꢀ 0.14f 0.269 ꢀ 0.097 6.64 ꢀ 0.19f
3.4
2.5
1.7
0.9
The results are expressed as mean ꢀ SEM from 3 to 6 experiments.
ANOVA, post Newmann–Keuls Multiple comparison test:
a
p < 0.05.
p < 0.01.
b
c
p< 0.001 vs. D1-like dopaminergic receptor.
p < 0.05.
p < 0.001 vs. corresponding –NH derivatives (compounds b vs. a, and d vs. c).
p < 0.001 vs. corresponding –OCH3 derivatives (compounds c vs. a, and d vs. b).
d
e
f
results have illustrated some general trends of the structure–
activity relationship:
1. In general, all the tested 1-benzyl-6-chloro-7-methoxy-THIQ
(1a,b and 2a,b) showed a lower affinity for D1 and D2 DR than
their corresponding 7-hydroxy homologues (1c,d and 2c,d)
(about 70-fold). The higher affinity of compounds with free
phenol OH has been previously described in several
3. Conclusions
The halogen placed on the benzylic ring in 1-benzyl-THIQs
proves to be an important factor to modulate affinity at DR.
Compounds of the series 1, 20-bromobenzyl derivatives with Ki
values into the nanomolar range, and the series 2, 20,40-dichlor-
obenzyl-THIQ homologues, proves to be an important factor to
modulate affinity at dopamine receptor. The compounds of series 1
showed an affinity that was 10 times higher than those of their
corresponding series 2 homologues to displace the ligands of D1
and/or D2 DR from their specific binding sites.
125
100
[3H]-SCH 23390
binding
75
50
25
0
[3H]-raclopride
binding
4. Experimental section
4.1. General instrumentation
Melting points were taken on a Cambridge microscope instru-
ment coupled with a Reichert-Jung. EI and FAB mass spectra were
recorded on a VGAuto Spec Fisons spectrometer instruments (Fisons,
Manchester, United Kingdom). 1H NMR and 13C NMR spectra were
recorded with CDCl3 as a solvent on a Bruker AC-300, AC-400 or AC-
500. Multiplicities of 13C NMR resonances were assigned by DEPT
experiments. NOE DIFF irradiations, COSY, HMQC, HSQC and HMBC
correlations were recorded at 400 MHz and 500 MHz (Bruker AC-
400 or AC-500). All the reactions were monitored by analytical TLC
with silica gel 60 F254 (Merck 5554). Residues were purified by silica
-10.0
-7.5
-5.0
compound 1c log [M]
-2.5
125
100
75
50
25
0
[3H]-SCH 23390
binding
gel 60 (40–63 mm, Merck 9385) column chromatography. Solvents
and reagents used and purchased from commercial sources. The cited
yields were of purified material. The HCl salts of the synthesised
compounds were prepared from the corresponding base with 5% HCl
in MeOH.
[3H]-raclopride
binding
4.2. General procedure for the synthesis of amides 1 and 2
-10.0
-7.5
compound 1d log [M]
-5.0
-2.5
2-(3-Chloro-4-methoxyphenyl) ethylamine was obtained by
standard methods [8]. Amides 1 and 2 were prepared under
Schotten–Baumann conditions by condensing 2-(3-chloro-4-
methoxyphenyl)ethylamine with the appropriate benzoyl chloride.
Fig. 1. Displacement of specific binding of [3H]-SCH23390 (D1-like DR specific ligand)
and [3H]-raclopride (D2-like DR specific ligand) by compounds 1c and 1d. The results
are expressed as mean ꢀ SEM from 3–6 experiments.

N. El Aouad et al. / European Journal of Medicinal Chemistry 44 (2009) 4616–4621
4619
4.2.1. 2-(20-Bromophenyl)-N-(3-chloro-4-
methoxyphenylethyl)acetamide (1)
34%) as a yellow oil. 1H NMR (300 MHz, CDCl3)
d
7.60 (dd, 1H, J ¼ 8.0,
0.9 Hz, H-30), 7.30–7.16 (m, 3H, H-60, H-50, H-40), 7.11 (s,1H, H-5), 6.73
A mixture of 2-(2-bromophenyl)acetic acid (1 g, 4.65 mmol) and
SOCl2 (0.4 mL, 5.58 mmol) in anhydrous CH2Cl2 was refluxed for 3 h.
Then, the solvent was removed to obtain the 2-(2-bromophenyl)a-
cetyl chloride as a pale yellow oil, which was used in the next step
without further purification. Then, an amount of 2-bromophenyla-
cetyl chloride (0.6 mL, 4.03 mmol) was added dropwise at 0 ꢁC to
(s,1H, H-8), 4.28 (dd,1H, J ¼ 9.7, 4.0 Hz, H-1), 3.81 (s, 3H, OCH3), 3.34
(dd,1H, J ¼ 13.6, 4.0 Hz, H-
aa), 3.25–3.19 (m,1H, H-3a), 3.04–2.76 (m,
2H, H-a
b, H-3b), 2.74–2.71 (m, 2H, H-4); 13C NMR (75 MHz, CDCl3)
d
152.7 (C-7), 138.4 (C-10), 137.9 (C-8a), 133.5 (CH-30), 132.4 (CH-60),
130.8 (CH-5), 128.8 (C-40), 128.4 (C-4a), 127.9 (C-50), 124.8 (C-20),
120.2 (C-6),110.7 (CH-8), 56.5 (OCH3), 55.4 (CH-1), 43.5 (CH2-3), 40.1
a solution of the
b-(3-chloro-4-methoxyphenyl)ethylamine (500 mg,
(CH2-
a
), 29.2 (CH2-4); FAB-MS m/z 366 [M þ H]þ,196; HRFABMS m/z
2.69 mmol) in CH2Cl2 (20 mL) and 5% aqueous NaOH (4.5 mL), and
was stirred at room temperature for 3 h. Next, 2.5% aqueous HCl was
added, and the organic solution was washed with brine (2 ꢂ10 mL)
and H2O (2 ꢂ10 mL), dried over Na2SO4 and evaporated to dryness.
The residue was purified via silica gel column chromatography
(hexane/EtOAc, 50:50) to afford 753 mg of the amide 1 (1.97 mmol,
366.0238 [M þ H]þ (C17H18BrClNO, calc 366.0260).
4.3.2. 1-(20,40-Dichlorobenzyl)-6-chloro-7-methoxy-1,2,3,4-
tetrahydroisoquinoline (2a)
The title compound was prepared according to the procedure for
1a using the corresponding amide 2 (500 mg, 1.34 mmol), P2O5
(3.8 g, 13.4 mmol) and POCl3 (1.3 mL, 13.4 mmol). The residue
obtained as a reddish oil was treated with NaBH4 (76 mg, 2 mmol).
The crude product was purified by silica gel column chromatog-
raphy CH2Cl2/MeOH/NH3 (100:2:0.5) to furnish 193 mg of 2a
73.2%). 1H NMR (300 MHz, CDCl3)
d
7.58 (dd, J ¼ 7.80,1.1 Hz,1H, H-30),
7.27–7.16 (m, 3H, H-60, H-50, H-40), 7.08 (d, J ¼ 2.2 Hz, 1H, H-2), 6.93
(dd, J ¼ 8.4, 2.2 Hz, 1H, H-6), 6.78 (d, J ¼ 8.4 Hz, 1H, H-5), 3.94 (s, 3H,
OCH3-4), 3.72 (s, 2H, CH2CO), 3.37 (td, J ¼ 6.9, 6.0 Hz, 2H, H-
a
), 2.83 (t,
169.6 (CO), 153.6 (C-
4), 134.5 (C-10), 133.8, 133.1, 131.5, 130.4, 129.2, 128.1, 127.6, 124.9 (C-
20), 122.3 (C-3), 112.3 (CH-5), 56.2 (OCH3), 43.9 (CH2-
), 40.7 (CH2),
34.9 (CH2-
); FAB-MS m/z 383 [M]þ, 381, 368, 168; HREIMS m/z
J ¼ 6.9 Hz, 2H, H-
b
); 13C NMR (75 MHz, CDCl3)
d
(0.51 mmol, 38%) as a yellow oil.1H NMR (400 MHz, CDCl3)
d 7.43 (d,
1H, J ¼ 1.7 Hz, H-30), 7.21–7.20 (m, 2H, H-50, H-60), 7.12 (s, 1H, H-5),
6.66 (s,1H, H-8), 4.24 (dd,1H, J ¼ 9.8, 4.1 Hz, H-1), 3.83 (s, 3H, OCH3),
a
b
3.29 (dd, 1H, J ¼ 13.7, 4.1 Hz, H-
aa), 3.25–3.19 (m, 1H, H-3a), 3.00–
381.0139 (C17H17BrClNO2, calc 381.0131).
2.94 (m, 2H, H-3b, H-
NMR (100 MHz, CDCl3)
a C
b), 2.74–2.71 (m, 2H, H-4), 1.7 (s, 1H, NH); 13
d
152.9 (C-7), 137.8 (C-10), 135.4 (C-8a),134.9
4.2.2. 2-(20,40-Dichlorophenyl)-N-(3-chloro-4-
methoxyphenylethyl)acetamide (2)
(C-20), 133.1 (C-60), 132.6 (CH-40), 130.6 (CH-5), 129.5 (CH-30), 128.2
(C-50), 127.1 (CH-4a), 120.6 (C-6), 110.2 (CH-8), 56.1 (OCH3), 54.9
The title compound was prepared according to the procedure for 1,
using 2-(2,4-dichlorophenyl)acetyl chloride (0.6 mL, 4.03 mmol) and
-(3-chloro-4-methoxyphenyl) ethylamine (500 mg, 2.69 mmol). The
(CH-1), 40.1 (CH2-3), 39.6 (CH2-a), 28.6 (CH2-4); FAB-MS m/z 356
[M þ H]þ, 196; HRFABMS m/z 356.0321 [M þ H]þ (C17H17Cl3NO, calc
b
356.0375).
residue was purified via silica gel column chromatography (hexane/
EtOAc, 50:50) to afford 682 mg of the amide 2 (1.83 mmol, 68%). 1H
4.4. General procedure for N-methylation
NMR (300 MHz, CDCl3)
d
7.41 (d, J ¼ 1.1 Hz, 1H, H-30), 7.22 (m, 2H, H-50,
H-60), 7.10 (d, J ¼ 2.2 Hz,1H, H-2), 6.92 (dd, J ¼ 8.4, 2.2 Hz,1H, H-6), 6.79
4.4.1. 1-(20-Bromobenzyl)-6-chloro-7-methoxy-N-methyl-1,2,3,4-
tetrahydroisoquinoline (1b)
(d, J ¼ 8.4 Hz, 1H, H-5), 3.88 (s, 3H, OCH3-4), 3.60 (s, 2H, CH2CO), 3.43
(td, J ¼ 6.7, 6.0 Hz, 2H, H-
a
), 2.68 (t, J ¼ 6.7 Hz, 2H, H-
b
); 13C NMR
To a stirred solution of 1a (500 mg, 1.37 mmol) in MeOH
(20 mL), 37% formaldehyde (15 mL) and one drop of formic acid
were added. The mixture was refluxed for 1 h, cooled to room
temperature, treated with NaBH4 (520 mg, 13.7 mmol), and
refluxed for an additional 1 h. The reaction mixture was left to
warm up to room temperature, and the solvent was removed under
reduced pressure. Water (3 mL) was added to the residue and the
aqueous mixture was extracted with CH2Cl2 (3 ꢂ15 mL). The
combined organic extracts were dried over Na2SO4 and concen-
trated under reduced pressure to give the crude product which was
further purified by silica gel column chromatography (CH2Cl2/
MeOH/NH4OH, 100:1:0.3) to afford 437 mg of 1b (1.23 mmol, 90%).
(75 MHz, CDCl3)
d
169.3 (CO), 154.0 (C-4), 135.3 (C-20), 134.4 (C-10),
132.8 (C-60), 132.0 (CH-40), 131.8 (C-1), 130.8 (CH-2), 130.0 (CH-30),
128.3 (CH-6), 128.1 (CH-50), 122.7 (C-3), 112.5 (CH-5), 56.6 (OCH3), 41.2
(CH2), 41.0 (CH2-a), 34.7 (CH2-
b
); FAB-MS m/z 372 [M þ H]þ, 168;
HREIMS m/z 372.0312 (C17H17Cl3NO2, calc 372.0325).
4.3. General procedure for Bischler–Napieralski cyclisation
4.3.1. 1-(20-Bromobenzyl)-6-chloro-7-methoxy-1,2,3,4-
tetrahydroisoquinoline (1a)
To a 250 mL three-neck round-bottom flask under N2, the cor-
responding amide 1 (500 mg, 1.31 mmol) was added in dry toluene
(20 mL), and was treated with P2O5 (3.7 g, 13.1 mmol), which was
added in portions and followed by the dropwise addition of POCl3
(1.2 mL, 13.1 mmol). The mixture was stirred and refluxed under N2
for 6–8 h, and then cooled to room temperature. Toluene was
concentrated under reduced pressure and the reaction mixture was
slowly poured into a mixture of crushed ice. The solid residue was
triturated with 10% aqueous NaOH to afford a suspension (pH z 8–
9), then extracted with CH2Cl2 (3 ꢂ15 mL). The combined CH2Cl2
extracts were dried over Na2SO4 and the solvent evaporated in vacuo
to afford reddish oil. The residue was dissolved in MeOH (20 mL), and
was then cooled to ꢃ78 ꢁC and treated with NaBH4 (76 mg, 2 mmol).
The reaction mixture was stirred for 2 h. Water (15 mL) was added
and volatiles were evaporated under reduced pressure. The aqueous
phase was extracted with CH2Cl2 (3 ꢂ 15 mL), and the combined
organic layers were dried over Na2SO4 and evaporated to dryness.
The crude product was purified by silica gel column chromatography
CH2Cl2/MeOH/NH3 (100:2:0.5) to furnish 164 mg of 1a (0.50 mmol,
1H NMR (300 MHz, CDCl3)
d
7.56 (dd, 1H, J ¼ 7.5, 1.6 Hz, H-30), 7.16
(dd, 1H, J ¼ 7.5, 1.6 Hz, H-50), 7.09 (s, 1H, H-5), 7.07 (dd, 1H,
J ¼ 7.5 Hz, 1.6 Hz, H-60), 6.97 (dd, 1H, J ¼ 7.5 Hz, 1.6 Hz, H-40), 5.91 (s,
1H, H-8), 3.92 (m, 1H, H-1), 3.49 (s, 3H, OCH3), 3.35–3.30 (m, 2H, H-
3a, H-
a
a), 2.93–2.80 (m, 3H, H-4a, H-3b, H-
a
b), 2.64–2.59 (m,1H, H-
4b), 2.54 (s, 3H, NCH3); 13C NMR (75 MHz, CDCl3)
d
151.8 (C-7),138.8
(C-10), 136.2 (C-8a), 132.7 (CH-30 and CH-60), 130.0 (CH-5), 127.9
(CH-40), 127.1 (CH-50), 125.1 (C-20), 120.1 (C-6), 111.8 (CH-8), 62.3
(CH-1), 55.6 (OCH3), 45.5 (CH2-3), 42.4 (NCH3), 40.5 (CH2-a), 24.4
(CH2-4); FAB-MS m/z 380 [M þ H]þ, 210; HRFABMS m/z 380.0318
[M þ H]þ (C18H20BrClNO, calc 380.0417).
4.4.2. 1-(20,40-Dichlorobenzyl)-6-chloro-7-methoxy-N-methyl-
1,2,3,4-tetrahydroisoquinoline (2b)
The title compound was prepared according to the procedure for
1b using the corresponding secondary amine 2a (500 mg,
1.41 mmol), 37% formaldehyde (15 mL) and NaBH4 (535 mg,
14.1 mmol). The crude product was purified by silica gel column

4620
N. El Aouad et al. / European Journal of Medicinal Chemistry 44 (2009) 4616–4621
chromatography (CH2Cl2/MeOH/NH4OH, 100:1:0.3) to afford
(m, 1H, H-1), 3.35–3.25 (m, 2H, H-3), 3.09–2.89 (m, 2H, H-
a
), 2.79–
469 mg of 2b (1.27 mmol, 90%). 1H NMR (400 MHz, CDCl3)
d
7.38 (d,
2.75 (m, 2H, H-4); 13C NMR (100 MHz, CDCl3)
d 150.4 (C-7),138.1 (C-
10), 136.0 (C-8a), 134.0 (C-20), 132.4 (C-60), 132.0 (C-40), 130.5 (CH-
4a), 130.2 (CH-5), 130.1 (CH-30), 126.8 (CH-50), 118.7 (C-6), 114. 1
1H, J ¼ 2.1 Hz, H-30), 7.13 (dd, 1H, J ¼ 8.2 Hz, 2.1 Hz, H-50), 6.96 (d,
1H, J ¼ 8.2 Hz, H-60), 7.09 (s, 1H, H-5), 6.04 (s, 1H, H-8), 3.91–3.82
(m, 1H, H-1), 3.59 (s, 3H, OCH3), 3.27–3.20 (m, 2H, H-3a, H-
a
a),
(CH-8), 61.6 (CH-1), 42.7 (CH2-3), 37.1 (CH2-a), 26.8 (CH2-4); FAB-
2.91–2.77 (m, 3H, H-3b, H-
a
b, H-4a), 2.61–2.56 (m, 1H, H-4b), 2.49
152.2 (C-7), 136.2 (C-10),
MS m/z 343 [M þ 1]þ, 183, 148.
(s, 3H, NCH3); 13C NMR (100 MHz, CDCl3)
d
135.9 (C-8a), 135.0 (C-20), 133.2 (CH-60), 132.6 (C-40), 130.1 (CH-5),
129.1 (CH-30), 127.1 (C-4a), 126.7 (CH-50), 120.4 (C-6), 111.6 (CH-8),
4.5.4. 1-(20,40-Dichlorobenzyl)-6-chloro-7-hydroxy-N-methyl-
1,2,3,4-tetrahydroisoquinoline (2d)
62.5 (CH-1), 55.7 (OCH3), 45.7 (CH2-3), 42.4 (NCH3), 37.7 (CH2-
a
),
The title compound was prepared according to the procedure for
1c using the corresponding isoquinoline 2b (260 mg, 0.70 mmol)
and BBr3 (0.28 mL, 2.8 mmol). The residue was purified by silica gel
column chromatography (CH2Cl2/MeOH/NH4OH, 100:3:0.5) to
afford 198 mg of 2d (0.56 mmol, 80%). 1H NMR (400 MHz, CDCl3)
24.2 (CH2-4); FAB-MS m/z 370 [M þ H]þ, 210; HRFABMS m/z
370.0520 [M þ H]þ (C18H19Cl3NO, calc 370.0532).
4.5. General procedure for O-demethylation
d
7.43 (d, 1H, J ¼ 1.7 Hz, H-30), 7.31–7.20 (m, 2H, H-50, H-60), 7.18 (s,
4.5.1. 1-(20-Bromobenzyl)-6-chloro-7-hydroxy-1,2,3,4-
tetrahydroisoquinoline (1c)
1H, H-5), 6.92 (s, 1H, H-8), 4.27 (dd, 1H, J ¼ 9.8, 4.0 Hz, H-1), 3.20–
2.81 (m, 4H, H-3, H-
a), 2.77–2.73 (m, 2H, H-4), 2.46 (s, 3H, NCH3);
A
solution of the appropriate isoquinoline 1a (260 mg,
13C NMR (100 MHz, CDCl3) 150.8 (C-7), 139.9 (C-10), 135.3 (C-8a),
d
0.71 mmol) in dry CH2Cl2 (10 mL) was cooled to ꢃ78 ꢁC. To this
stirring solution, BBr3 (0.28 mL, 2.8 mmol) was added dropwise.
After 15 min, the reaction mixture was left to warm up to room
temperature and stirred for 3 h. The reaction was terminated by the
addition of MeOH (5 mL), dropwise and the mixture was stirred for
another 30 min. The solvent was concentrated to dryness. The
residue was dissolved in EtOAc (2 mL) and made alkaline with 37%
aqueous NH4OH to pH z 11, and was subsequently neutralised with
1 M HCl to pH z 7–8. The aqueous layer was then extracted with the
EtOAc (3 ꢂ 10 mL). The combined EtOAc extracts were dried over
Na2SO4, and evaporated under reduced pressure. The residue was
purified by silica gel column chromatography (CH2Cl2/MeOH/
NH4OH, 100:6:0.5) to afford 185 mg of 1c (0.53 mmol, 75%). 1H NMR
134.1 (C-20), 132.9 (C-60), 132.8 (C-40), 131.5 (CH-4a), 130.3 (CH-30),
130.1 (CH-5), 126.8 (CH-50), 118.3 (C-6), 115.8 (CH-8), 64.1 (CH-1),
48.3 (CH2-3), 43.6 (NCH3), 36.5 (CH2-
a), 26.5 (CH2-4); FAB-MS m/z
357 [M þ 1]þ, 196.
4.6. Pharmacological in vitro assays
4.6.1. Animals
Female Wistar rats (200–220 g), bred in a standard experi-
mental animal room of the Faculty of Pharmacy, were used for [3H]-
dopamine uptake assays and radioligand binding experiments. Rats
were housed under a 12-h light/dark cycle at 22 ꢁC and 60%
humidity. All the protocols complied with European Community
guidelines for the use of experimental animals and were approved
by the University of Valencia’s Ethics Committee.
(300 MHz, CDCl3)
d
7.56 (dd,1H, J ¼ 7.6,1.2 Hz, H-30), 7.27 (s,1H, H-5),
7.22–7.02 (m, 3H, H-60, H-50, H-40), 6.91 (s, 1H, H-8), 4.29 (dd, 1H,
J ¼ 9.7, 4.0 Hz, H-1), 3.25–3.04 (m, 2H, H-3a, H-
H-3b, H-
b), 2.79–2.75 (m, 2H, H-4); 13C NMR (75 MHz, CDCl3)
150.4 (C-7), 139.3 (C-10), 136.2 (C-8a), 132.4 (CH-30), 131.1 (CH-60),
130.7 (C-4a), 130.4 (CH-5), 128.1 (CH-40), 127.6 (CH-50), 124.4 (C-20),
aa), 3.02–2.76 (m, 2H,
a
d
4.6.2. [3H]-dopamine uptake assay
[3H]-dopamine uptake was studied using a preparation of rat
striatal synaptosomes. All the experimental procedures for synap-
tosomes preparation were carried out at 0–4 ꢁC. The rat striatum
was dissected, homogenised in 10 volumes (w/v) of 0.32 M sucrose
with an ultraturrax T25 (Janke & Kinkel) (4 s, maximal scale) and
centrifuged at 1000g for 10 min. The supernatant was stored
and the pellet was resuspended in 10 volumes of 0.32 M sucrose,
and was centrifuged again at 1000g for 10 min. The two superna-
tants were combined and the mixture was centrifuged at 16,000g
for 30 min. The resultant pellet was suspended in 10 volumes of
ice-cold Krebs medium (pH 7.6) containing (mM): 118 mM NaCl;
4.75 mM KCl; 1.2 mM KH2PO4; 1.8 mM CaCl2; 1.2 mM MgCl2;
25 mM NaHCO3; and 11 mM glucose. Aliquots were pre-incubated
117.2 (C-6), 114.1 (CH-8), 57.6 (CH-1), 42.1 (CH2-3), 39.4 (CH2-a), 27.9
(CH2-4); FAB-MS m/z 353 [M þ H]þ, 183.
4.5.2. 1-(20-Bromobenzyl)-6-chloro-7-hydroxy-N-methyl-1,2,3,4-
tetrahydroisoquinoline (1d)
The title compound was prepared according to the procedure for
1c using the corresponding compound 1b (260 mg, 0.73 mmol) and
BBr3 (0.29 mL, 2.9 mmol). The residue was purified by silica gel
column chromatography (CH2Cl2/MeOH/NH4OH, 100:3:0.5) to
afford 190 mg of 1d (0.56 mmol, 77%). 1H NMR (300 MHz, CDCl3)
d
7.60 (dd, 1H, J ¼ 8.0, 0.8 Hz, H-30), 7.30–7.16 (m, 2H, H-60, H-50, H-
40), 7.27 (s, 1H, H-5), 6.92 (s, 1H, H-8), 4.30–4.02 (m, 1H, H-1), 3.35–
3.30 (m, 2H, H-3a, H-
2.64–2.59 (m, 1H, H-4b), 2.53 (s, 3H, NCH3); 13C NMR (75 MHz,
CDCl3)
150.3 (C-7), 139.6 (C-10), 135.3 (C-8a), 132.6 (C-30), 131.4 (C-
4a), 131.1(C-60), 130.1 (C-5), 128.2 (C-40), 127.6 (C-50), 124.4 (C-20),
a
a), 2.93–2.80 (m, 3H, H-4a, H-3b, H-
a
b),
for 10 min at 37 ꢁC in Krebs buffer containing 10
mM pargyline (to
block the metabolism of dopamine by monoamine oxidase), [3H]-
dopamine (47 Ci/mmol, Amersham) was added to a final concen-
tration of 0.5 nM, and incubation continued for another 10 min.
d
119.9 (C-6), 115.8 (C-8), 64.8 (C-1), 47.5 (CH2-3), 39.1 (CH2-
a
), 26.5
Compounds were screened at 100 mM. Incubation was terminated
(CH2-4); FAB-MS m/z 367 [M þ 1]þ, 183.
by dilution into ice-cold Krebs medium, and the samples were
filtered rapidly through fibreglass filters (Schleicher & Schuell
Grade 30) using a Brandel cell harvester (model M-24, Biochemical
Research and Development Laboratories, Inc.). Filters were washed
twice with 3 mL cold Krebs medium and dried. Non-specific [3H]-
4.5.3. 1-(20,40-Dichlorobenzyl)-6-chloro-7-hydroxy-1,2,3,4-
tetrahydroisoquinoline (2c)
The title compound was prepared according to the procedure for
1c using the corresponding isoquinoline 2a (260 mg, 0.69 mmol)
and BBr3 (0.27 mL, 2.7 mmol). The residue was purified by silica gel
column chromatography (CH2Cl2/MeOH/NH4OH, 100:6:0.5) to
afford 182 mg of 2c (0.5 mmol, 73%). 1H NMR (400 MHz, CDCl3)
dopamine uptake was determined in the presence of 10 mM
nomifensine (dopamine uptake inhibitor). Filters were placed into
a scintillation mixture (Optiphase ‘‘Hisafe’’ 2, Perkin Elmer), and
radioactivity was determined by scintillation spectrometry [6,9].
Protein concentrations were determined using the Bradford protein
assay (BioRad).
d
7.51 (d, 1H, J ¼ 2.1 Hz, H-30), 7.31 (dd, 1H, J ¼ 8.2, 2.1 Hz, H-50), 7.25
(d, 1H, J ¼ 8.2 Hz, H-60), 7.18 (s, 1H, H-5), 6.45 (s, 1H, H-8), 4.01–3.96

N. El Aouad et al. / European Journal of Medicinal Chemistry 44 (2009) 4616–4621
4621
4.6.3. Radioligand binding assays
Acknowledgments
[3H]-SCH23390 and [3H]-raclopride binding experiments were
performed on rat striatal membranes. The rat striatum was
homogenised in 10 volumes (w/v) of Tris–HCl buffer (50 mM, pH
7.4 at 22 ꢁC) with an ultraturrax T25 (Janke & Kinkel) (4 s, maximal
scale). The homogenate was centrifuged twice at 49,000g for
15 min at 4 ꢁC with a resuspension cycle in the same volume of
Tris–HCl buffer between each centrifugation. The final pellet was
resuspended in Tris-ions buffer containing 120 mM NaCl; 2 mM
CaCl2, 5 mM KCl; 1 mM MgCl2; and 0.1% ascorbic acid (pH 7.4). For
This research was supported by the Spanish ‘‘Ministerio de
´
Educacion
y Ciencia’’ grant SAF 2007-63142. I. Berenguer
acknowledges the fellowship of Generalitat Valenciana. S. Andujar
acknowledges the fellowship of CONICET-Argentina.
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determined in the presence of 50 mM apomorphine (Sigma). In both
cases, incubations were stopped by the addition of 3 mL ice-cold
Tris-ions buffer followed by a rapid filtration through fibreglass
filters (Schleicher & Schuell Grade 30) using a Brandel cell harvester
(model M-24, Biochemical Research and Development Laborato-
ries, Inc.). Filters were washed twice with 3 mL cold Tris-ions buffer.
After the filters had been dried, radioactivity was counted in 4 mL
scintillation liquid (Optiphase ‘‘Hisafe’’ 2, Perkin Elmer). All the
compounds were used as hydrochloride salts. Data were analysed
by Prim (Graph Pad Software; San Diego, California, U.S.A), and Ki
values were determined using the KD value for [3H]-SCH23390 of
0.36 nM and for [3H]-raclopride of 1.25 nM. Values are expressed as
the mean ꢀ SEM of three to six independent determinations per-
formed in duplicate.