Tetrahydroisoquinolines as dopaminergic ligands: 1-Butyl-7-chloro-6-hydroxy-tetrahydroisoquinoline, a new compound with antidepressant-like activity in mice

Article abstract of DOI:10.1016/j.bmc.2009.05.079

Three series of 1-substituted-7-chloro-6-hydroxy-tetrahydroisoquinolines (1-butyl-, 1-phenyl- and 1-benzyl derivatives) were prepared to explore the influence of each of these groups at the 1-position on the affinity for dopamine receptors. All the compou

Full text of DOI:10.1016/j.bmc.2009.05.079

Bioorganic & Medicinal Chemistry 17 (2009) 4968–4980
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Bioorganic & Medicinal Chemistry
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Tetrahydroisoquinolines as dopaminergic ligands: 1-Butyl-7-chloro-6-hydroxy-
tetrahydroisoquinoline, a new compound with antidepressant-like activity
in mice
Inmaculada Berenguer a, Noureddine El Aouad a, Sebastián Andujar b,c, Vanessa Romero a, Fernando Suvire b,
Thomas Freret d, Almudena Bermejo a,e, María Dolores Ivorra a, Ricardo D. Enriz b,c, Michel Boulouard d,
Nuria Cabedo a,f, Diego Cortes a,
*
a Departamento de Farmacología, Facultad de Farmacia, Universidad de Valencia, 46100 Burjassot, Valencia, Spain
b Departamento de Química, Universidad Nacional de San Luis, Argentina
c IMIBIO-SL (CONICET), Chacabuco 915, 5700 San Luis, Argentina
d Groupe Mémoire et Plasticité Comportementale, UFR des Sciences Pharmaceutiques, Université de Caen Basse—Normandie, Caen, France
e Departamento de Citricultura, IVIA, crta Moncada-Náquera Km 4.5, 46113 Moncada, Valencia, Spain
f Centro de Ecología Química Agrícola-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:
Three series of 1-substituted-7-chloro-6-hydroxy-tetrahydroisoquinolines (1-butyl-, 1-phenyl- and 1-
benzyl derivatives) were prepared to explore the influence of each of these groups at the 1-position on
the affinity for dopamine receptors. All the compounds displayed affinity for D1-like and/or D2-like dopa-
mine receptors in striatal membranes, and were unable to inhibit [3H]-dopamine uptake in striatal syn-
aptosomes. Different structure requirements have been observed for adequate D1 or D2 affinities. This
paper details the synthesis, structural elucidation, dopaminergic binding assays, structure–activity rela-
tionships (SAR) of these three series of isoquinolines. Moreover, 1-butyl-7-chloro-6-hydroxy-tetrahydro-
isoquinoline (1e) with the highest affinity towards D2-like receptors (Ki value of 66 nM) and the highest
selectivity (49-fold D2 vs D1) by in vitro binding experiments was then evaluated in behavioral assays
(spontaneous activity and forced swimming test) in mice. Compound 1e increased locomotor activity
in a large dose range (0.04–25 mg/kg). Furthermore, this lead compound produced reduction in immobil-
ity time in the forced swimming test at a dose (0.01 mg/kg) that did not modify locomotor activity. The
haloperidol (0.03 mg/kg), a D2 receptor preferred antagonist, blocked the antidepressant-like effect of
compound 1e.
Received 26 January 2009
Revised 19 May 2009
Accepted 31 May 2009
Available online 6 June 2009
Keywords:
Tetrahydroisoquinolines
Dopamine receptors
Structure–activity relationships
Theoretical calculations
In vivo behavioral assays
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
antiparkinsonian drugs3 could be classified into several categories
in regard of their affinity and activity towards dopaminergic recep-
Dopamine-mediated neurotransmission plays an important role
in several psychiatric and neurological disorders affecting 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 schizo-
phrenia and Parkinson’s disease. 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 pharma-
cological families (D1 and D2-like) that are encoded by at least five
genes. The D2-like DR antagonists are used in the treatment of
schizophrenia (antipsychotics) and the agonists are utilized to
treat Parkinson’s disease.2 Dopaminergic agonist actually used as
tors4,5 but all of them exhibited D2-like agonist properties. Recent
studies have also evidenced the potential role of D2 agonists in the
treatment of depression. Besides, even thought the pathophysiol-
ogy of depression has been assigned to the noradrenalin and sero-
tonin system, several results also support
a role of the
dopaminergic system in this mood disorder.6 In particular, various
selective D2-type dopamine receptor agonists exert antidepres-
sant-like actions in diverse rodent models suggesting a specific role
of this subtype receptor in antidepressant-like activity.6–9
Substituted isoquinolines (IQ) represent a class of natural and
synthetic compounds that has been evaluated for their ability to
inhibit the dopamine transporter and to display affinity at D1 and
D2-like DR binding sites in rat brain tissue.10 Tetrahydroisoquino-
lines (THIQs), the most numerous naturally occurring alkaloids,
include 1-benzyl-THIQs and aporphines, both of which have struc-
tural similarities to dopamine and can interact with DR.11
* Corresponding author. Tel.: +34 963 54 49 75; fax: +34 963 54 49 43.
E-mail address: dcortes@uv.es (D. Cortes).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.05.079

I. Berenguer et al. / Bioorg. Med. Chem. 17 (2009) 4968–4980
4969
Previous results in our group suggested that some natural and
synthetic 1-benzyl-THIQs alkaloids were able to bind to DR.12–14
In this way, 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
series of 1-benzyl-THIQs, their (1S)-enantiomers were 5–15 times
more effective at the D1-like and D2-like DR than the (1R)-enanti-
omers.15 Moreover the different 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-meth-
ylated derivatives also showed affinity for the D1-like DR.16 We
also determined the role of certain structural requirements to
improve the affinity/selectivity for D1 and D2-like DR15–20 and we
have postulated that the presence of a hydroxyl (OH) and a halogen
group (Cl) in the THIQ A-ring could contribute to obtain molecules
which can bind selectively to one of the two groups of the afore-
mentioned receptors.18,19
The aim of the present work was to profound in the determina-
tion of the structural features that define the affinity and selectiv-
ity of these compounds for D1/D2 receptors, analyzing the influence
of the substitution at the 1-position over a 7-chloro-6-hydroxy-
THIQ core, in order to obtain more specific dopaminergic ligands.
We have prepared three series of 1-substituted: 1-butyl-, 1-phe-
nyl- and 1-benzyl-THIQs, which support constant structural factors
6-chloro and 7-oxygenated substitutions, as well as a basic second-
ary (NH) or tertiary (NCH3) amine. The structures of the resulting
twelve 1-substituted-THIQs were determined on the basis of their
NMR spectral data and mass spectrometry analysis. All the syn-
thesised compounds were tested for their ability to displace the
selective radioligands of D1 and D2-like DR from their specific bind-
ing sites in striatal membranes in order to establish their struc-
ture–activity relationships (SAR) as dopaminergic agents.
Molecular modeling of the possible stereo-electronic requirements
´
of dopamine D2 receptors ligand is also discussed with regard to
the different affinity.
Furthermore, and based on the implication of D2-like receptor li-
gands on spontaneous activity modulation,21,22 the effects of the
lead compound 1e (with the highest D2 receptor affinity and selec-
tivity) were investigated with a photoactimetry test in a large dose
range (0.01–25 mg/kg) in mice. Finally, in accordance with the
in vivo D2 receptor agonist activity observed and the therapeutic po-
tential of D2 receptor agonists in the treatment of depression6–9 the
antidepressant-like activity of this compound was evaluated in the
forced swimming test in mice.
2. Results and Discussion
In the present work we have studied the influence of the substi-
tution at the 1-position over a 7-chloro-6-hydroxy-THIQ core. By
preserving the chlorine and hydroxyl (or methoxyl) groups at the
C-6 and C-7 positions, respectively, of the THIQ A-ring with a sec-
ondary (NH) or a tertiary (NMe) amine, we decided to explore the
Cl
NH2
H3CO
2-(3-chloro-4-methoxy
phenyl)ethylamine
Cl
Cl
O
O
Cl
O
Cl
Cl
Cl
NH
NH
NH
H3CO
H3CO
O
H3CO
O
O
2
1
3
1-alkyl-THIQs
(a)
1-phenyl-THIQs
(a)
1-benzyl-THIQs
(a)
4
5
A
8
6
7
Cl
Cl
Cl
3
B
NR
NR
NR
H3CO
H3CO
H3CO
1
1a: R= H
3a: R= H
2a: R= H
2b: R= CH3
(b)
(b)
(b)
1b: R= CH3
3b: R= CH3
(c)
(d)
(d)
Cl
Cl
Cl
HO
NR
NR
NR
HO
HO
χ
θ1
φ1
φ2
θ2
φ3
2c: R= H
1c: R= H
3c: R= H
2d: R= CH3
1d: R= CH3
3d: R= CH3
Figure 1. Synthesis of 1-substituted 6-chloro-THIQ analogues. (a) POCl3–P2O5/toluene; NaBH4/MeOH; (b) HCHO/NaBH4; (c) BBr3, 18 h; (d) BBr3, 3 h.

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I. Berenguer et al. / Bioorg. Med. Chem. 17 (2009) 4968–4980
impact of the inclusion at the 1-position of aliphatic and aromatic
groups, such as butyl-, phenyl- or benzyl- moieties, to determine
their influence on dopaminergic affinity. Thus, we prepared three
series of 1-substituted-THIQs: 1-butyl-THIQs (1a–d), 1-phenyl-
THIQs (2a–d) and 1-benzyl-THIQs (3a–d), which have enabled us
to draw conclusions about the influence of each of these groups
noted at the 1-position, a frequent occurrence in the structure of
natural and/or synthetic drugs.
Cl
NH
H3CO
O
1
1) Bischler-Napieralski
POCl3 / P2O5
2.1. Chemistry
2) NaBH4 / MeOH
The general synthetic plan for these compounds focused on the
preparation of the appropriate amides (1, 2, and 3) by standard
methods. Thus, the previously synthesised 2-(3-chloro-4-
methoxy)ethylamine was treated with three different acid
chlorides, these being alkanoyl (valeryl chloride), benzoyl and
phenylacetyl chloride, under Schotten–Baumann conditions
(Fig. 1), affording the three amide derivatives with good yields:
N-(3-chloro-4-methoxyphenethyl)pentanamide (1), N-(3-chloro-
4-methoxy-phenethyl)benzamide (2) and the N-(3-chloro-4-meth-
oxyphenethyl)-2-phenylacetamide (3). After a Bischler–Napieralski
cyclodehydration reaction, each N-phenylethylamide was converted
into the convenient 1-substituted-THIQ. At this stage, several
points should be emphasized: (i) A classical Bischler–Napieralski
cyclization failed in these compounds (refluxing with POCl3 in
CH2Cl2; which means having to use the most common Lewis acid
for this reaction) because of the halogen substitution over the THIQ
A-ring. A mixture of POCl3 and P2O5 was needed to obtain the
corresponding dihydroisoquinolines;23–25 (ii) An unusual Bisch-
ler–Napieralski cyclodehydration was performed given a high
concentration level of the P2O5 reagent.26 Under these conditions,
a mixture of THIQ isomers 6-OMe–7-Cl and 6-Cl–7-OMe was
obtained, after the Bischler–Napieralski reaction followed by
NaBH4 reduction (Figs. 1 and 2); (iii) The indispensable O-demeth-
ylation of all synthesised isoquinolines was performed with an
addition of 4 equiv of BBr3 reagent, and a longer reaction time
(18 h) was needed for the 1-butyl-THIQ series.
Cl
N
H3CO
6
6
Cl
H3CO
NH
NH
7
H3CO
7
Cl
1
1
1a
1a'
BBr3
BBr3
6
Cl
HO
Cl
NH
NH
HO
7
1
1
Concerning the synthesis, we observed the same fact during the
Bischler–Napieralski cyclization, as reported by Doi et al. in 1997,26
when we prepared the 1-butyl-THIQs starting from amide 1
(Fig. 2). It was necessary to add P2O5 (and POCl3, in a molar ratio
of 1:1) to the cyclodehydration reaction given the difficulty to cy-
clize the amide when there was a chlorine in the structure (origi-
nally at the C-6 position of the A-ring), and causes an aberrant
cyclization by means of the formation of a nitrilium intermediate
which gives two clearly identified positional isomers after the
reduction step: 6-chloro-7-hydroxy-1-butyl-THIQ (1c: expected
product), and 6-hydroxy,7-chloro-1-butyl-THIQ (1e: unexpected
cyclization product), in a 1:2 ratio. This finding was less significant
in series 2 and 3 where the mixture of isomers was 3:1 and 4:1,
respectively. Each isomer was unambiguously determined by
NOE DIFF experiments (Fig. 3). For compound 1c, irradiation of
H-8 (d 6.78, s) caused the enhancement of H-1 (d 3.88, dd) and
the H-10 (d 1.80–1.67, m) signals, while irradiation of the more
deshielded H-5 (d 7.03, s) only affected H-4 (d 2.73–2.65, m). How-
ever, NOE DIFF experiments for isomer 1e exhibited signal
enhancements of H-1 (d 3.85, dd) and H-10 (d 1.80–1.63, m) upon
irradiation of the more deshielded H-8 (d 7.07, s), and irradiation
of H-5 (d 6.68, s) which influenced H-4 (d 2.73–2.65, m).
1c
1e
Figure 2. Abnormal BN reaction: synthesis of 1-butyl-7-chloro-THIQ’s (1e).26
[3H]-dopamine uptake in rat striatal synaptosomes. Many of these
compounds were able to displace both [3H]-SCH 23390 and [3H]-
raclopride at nano- or micromolar (nM or lM) concentrations from
their specific binding sites in the rat striatum, but all the com-
pounds had a low or null effect on the [3H]-dopamine uptake.
The binding affinities for D1 and D2 DR are summarized in Table
1 and these results have illustrated some general trends of the
SAR: the effect of the hydroxyl or methoxyl group at the C-7 posi-
tion, the effect of the amine type (NH or NMe) and the effect of the
butyl, phenyl and benzyl group at the C-1 position.
2.2.1. 7-Hydroxyl group and amine type effects
In the 1-substituted THIQs synthesised, the presence of a hydro-
xyl (OH) group at C-7 position positively influences their ability to
displace the selective ligands of D1 and D2 DR from their specific
binding sites in the striatal membranes (Table 1). Generally, all
the tested 6-Cl and 7-OH-THIQs (1c,d; 2c,d and 3c,d) showed an
affinity for DR 5–15 times higher than their corresponding 6-Cl
and 7-OMe homologs. This result agrees with the established find-
ing that a hydrophilic area, usually provided by the phenolic
hydroxyl group in the THIQ A-ring, is required for a better binding
2.2. Binding affinities for dopamine receptors: SAR Studies
All the synthesised isoquinolines were assayed in vitro for their
ability to displace the selective radioligands of D1 and D2 DR from
their respective specific binding sites in rat striatal membranes.
They were also tested for their ability to inhibit an in vitro

I. Berenguer et al. / Bioorg. Med. Chem. 17 (2009) 4968–4980
4971
oxygen atom of the OH group of Ser 141 the proton–acceptor coun-
terpart. In contrast, in the case of compounds 1a, 2a and 3a the OH
group of Ser 141 is the proton–donor and the methoxyl group on
the ring-A is the acceptor.
δ
7.03 ( )
s
H5
δ
2.73-2.65 (
m
)
4
Cl
6
NH
7
HO
Figure 4a shows the ligand 3c interaction with the D2 DR opti-
mized using quantum mechanical calculations. The salt bridge
between the protonated amino group and the carboxyl group of
Asp 86 as well as the hydrogen bond between the 7-hydroxyl
group with Ser 141 might be appreciated in this figure. From Figure
4a it is clear that a strong salt bridge takes place for this compound
between the protonated amino groups and the carboxyl group of
Asp 86 (calculated distance of 3.47 Å). The hydrogen bond between
3c and Ser 141 is a bifurcated interaction in which the oxygen
atom of hydroxyl group and the oxygen of carbonyl group of Ser
141 are the proton–acceptors, giving interatomic distances of
2.28 Å and 2.40 Å, respectively. Figure 4b gives the ligand 3a inter-
action with the D2 DR. In this case the 7-methoxyl group acts as
proton–acceptor while the hydroxyl group of Ser 141 is the pro-
ton–donor displaying an interatomic distance of 2.32 Å.
1'
H1
δ
δ
3.88 (dd
)
δ
s
6.78 ( ) H8
1.80-1.67 ( )
m
H3C
1c
δ
s
6.68 ( )
H5
δ
2.73-2.65 (
m
)
4
HO
6
NH
7
Cl
1'
H
δ
1 δ 3.85 (dd
)
H8
δ
7.07 ( )
s
1.80-1.63 (
m
)
H3C
1e
Figure 3. NOE DIFF effects of compounds 1c and 1e.
Table 2 gives the energy binding calculated for the different
complexes using RHF/6–31G(d). All the compounds possessing 7-
methoxyl groups displayed higher binding energies with respect
to the 7- hydroxyl homologs (compare 1a with 1c, 2a with 2c
and 3a with 3c). Previously we reported that a 7-hydroxyl group
acting as proton–donor gives stronger hydrogen bond than those
derivatives possessing a 7-metoxyl group.19 The present results
are in agreement with those calculations previously reported for
isolated and solvated molecules, as well as, with the experimental
binding affinities reported here (Table 1).
In general, the comparative Ki values between pairs of NH and
NMe derivatives indicate that the attachment of a methyl group
at the nitrogen atom (tertiary amine) appears to be important for
improvement in the binding affinity to D1 DR. Thus in the 1-phe-
nyl-THIQ series (series 2), compounds with NMe amine showed
greater affinity for D1 receptors than their corresponding NH deriv-
atives. However in the 1-butyl- and 1-benzyl-THIQ series (series 1
and 3, respectively), secondary amine (NH) compounds showed a
greater affinity for D2-like DR than their corresponding homologs
with tertiary amine (NMe). Therefore, secondary amines 1c and
3c (Ki: 91 nM and 71 nM, respectively) displayed greater affinity
and selectivity for D2 receptors, whereas 2d tertiary amine (Ki:
47 nM) presented greater affinity and selectivity for D1 receptor
(Fig. 5). This finding might be explained if we take into account
the earliest conformational studies in which the N-methyl substi-
of the ligands to this type of receptors,16,20 which also occurs in the
molecule dopamine, a physiological mediator of the dopaminergic
via. A similar behavior was noted in compound 1e (1c isomer) with
inverted ring-A substitutions: the 7-Cl and 6-OH groups.
To better understand the above experimental results we per-
formed molecular modeling simulations combining both semiem-
pirical and ab initio quantum mechanical calculations for different
1-butyl-, 1-phenyl- and 1-benzyl-THIQs/D2 DR complexes. For
such calculations we use a reduced model from the complete D2
receptor model previously reported by Teeter et al.27 Consistent
with previous experimental28 and theoretical29 results, our simula-
tions indicate the importance of the negatively charged aspartate
86 for the binding of these ligands. A highly conserved aspartic acid
(Asp 86) in trans-membrane helix 3 (TM3) is important for the
binding of both agonists and antagonists to the D2 receptor,28,29
and its terminal carboxyl group may function as an anchoring
point for ligands with a protonated amino group.27
Our results indicate that in these complexes the strongest con-
tributor to the network of serines was Ser 141 which is consistent
with the experimental observation that a Ser 141 Ala mutated
receptor completely lost dopamine induced activation.28 It should
be noted that in compounds 1c, 1e, 2c and 3c the hydroxyl group
on the ring-A is acting as the proton–donor part; whereas the
Table 1
Dissociation constants (pKi) and selectivity of different compounds at the D1-like and D2-like dopaminergic receptors
Compounds
Series
Specific-D1 ligand [3H]-SCH 23390
Specific-D2 ligand [3H]-raclopride
Selectivity D1/D2
1a
1b
1c
1d
1-Butyl-THIQ
1-Butyl-THIQ
1-Butyl-THIQ
1-Butyl-THIQ
1-Butyl-THIQ
1-Phenyl-THIQ
1-Phenyl-THIQ
1-Phenyl-THIQ
1-Phenyl-THIQ
1-Benzyl-THIQ
1-Benzyl-THIQ
1-Benzyl-THIQ
1-Benzyl-THIQ
4.763 0.258
5.138 0.256
5.918 0.165i
5.944 0.09g
5.491 0.036
5.222 0.222
6.089 0.292d
6.607 0.205h
7.395 0.176d,h
5.628 0.397
5.785 0.359
6.487 0.075
6.953 0.149h
6.108 0.165c
5.424 0.026d
7.117 0.151c,h
6.403 0.204e,h
7.220 0.139c
5.212 0.124
5.670 0.406
5.950 0.198
6.298 0.187b
6.014 0.049
5.816 0.181
7.178 0.091a,h
6.683 0.139g
20
2.7
17
2.3
49
1.2
0.3
0.2
0.07
5
0.8
4.8
0.6
1e (1c isomer)
2a
2b
2c
2d
3a
3b
3c
3d
THIQ: tetrahydroisoquinoline.
The results are expressed as mean SEM from 3 to 6 experiments.
ANOVA, post Newmann Keuls multiple comparison test:
c
ap < 0.05, bp < 0.01, p < 0.001 versus D1-like dopaminergic receptor.
f
dp < 0.05, ep < 0.01, p < 0.001 versus corresponding –NH derivatives (compounds b vs a, and d vs c).
i
gp < 0.05, hp < 0.01, p < 0.001 versus corresponding –OCH3 derivatives (compounds c vs a, and d vs b).

4972
I. Berenguer et al. / Bioorg. Med. Chem. 17 (2009) 4968–4980
Figure 4. Interactions of compound 3c (a) and 3a (b) with the binding pocket D2 DR. Spatial view of two interactions: salt bridge (Asp 86 with protonated amino group) to the
right and hydrogen bond between hydroxyl groups with Ser 141 to the left.
Table 2
It is interesting to note that the only structural difference be-
tween compounds 1c, 2c and 3c are the substituents at C-1.
Whereas compound 2c has a relatively rigidly held phenyl ring,
the corresponding butyl and benzyl substituents on compounds
1c and 3c, respectively, are free to rotate allowing to better
accommodate these hydrophobic moieties to interact with the
cluster of aromatic and non polar residues located in the cleft.
These results might be better appreciated from the different con-
formational behavior obtained for the torsional angles of their
respective hydrophobic moieties from the calculations. Both
1-butyl and 1-benzyl groups displayed a high molecular flexibility
from the calculations. In contrast, the conformational behavior
obtained for the phenyl ring of compound 2c displayed a very re-
stricted molecular flexibility, keeping a spatial ordering almost
perpendicular with respect to the rest of the molecule from our
calculations (Fig. 6c). The different affinities obtained for com-
pounds 2c and 3c suggest that the orientation of the substituent
at C-1 may be the more important factor in the different effects
on receptor affinity for the two ligands. This argument also ap-
plies to 1c and 1e, where the orientation of the butyl substituents
is more favorable for hydrophobic interactions. Thus, the different
affinities and selectivities obtained for these compounds might be
explained, at least in part, by the different spatial orientations
adopted by the varied hydrophobic portions located at C-1 which
give different molecular interactions with the D2 receptor. Figure
6a gives again the ligand 3c interactions with the binding pocket.
However, in this case a different spatial view is shown in order to
better appreciate the hydrophobic interactions of this compound.
From this figure we can observe that the benzyl group of 3c
adopts an adequate conformation to interact with Phe 186, Phe
82 and His 189. The butyl group of 1c displays a spatial ordering
very similar to that of the benzyl group of 3c, giving also closely
related hydrophobic interactions with the same hydrophobic res-
idues (Fig. 6b). In contrast, the phenyl group of 2c displayed a
different spatial ordering giving an adequate distance to interact
only with Phe 186 (Fig. 6c). Interestingly, the bonding energies
obtained for these complexes are: 3c/D2 DR stronger than 1c/D2
DR and this complex stronger than 2c/D2 DR (Table 2), which is
in agreement with the experimental results. The butyl portion
of compound 1e interacts with three aromatic residues: Trp
182, Phe 82 and Phe 186 (Fig. 6d).
Relative binding energies obtained for the different complexes from RHF/6-31G(d)
calculations
Compounds
Relative binding energy (BE) (kcal/mol)
BEQM
BEQM
D
(RHF/6-31G(d))
(RHF/6-31G(d))
1a
1c
1e
2a
2c
3a
3c
ꢀ98.91
ꢀ111.92
ꢀ114.03
ꢀ75.46
ꢀ90.48
17.45
4.43
2.32
40.89
25.87
16.41
0.00
ꢀ99.94
ꢀ116.35
tuent takes a more stable equatorial orientation in the N-methyl-
THIQs; therefore, the lone pair on the nitrogen is found at the axial
position, thus favouring the interaction with D1 receptor binding
sites, whereas the nitrogen lone pair in the secondary amine pre-
cursors (NH) may be in equilibrium with axial and equatorial
orientations.19,25,30
2.2.2. 1-Butyl, 1-phenyl and 1-benzyl group effects
Our results could indicate that the substituent at the C-1 posi-
tion is an important factor to modulate the selectivity at dopamine
receptors. A greater affinity towards D2 receptors is evident in the
1-butyl-THIQ and 1-benzyl-THIQ series (series 1 and 3, respec-
tively). However, 1-phenyl-THIQ derivatives (series 2) show selec-
tivity for D1 over the D2 receptors (Fig. 5 and Table 1). Moreover, it
is important to note that the compounds of series 1 (1-butyl-THI-
Qs) show a lower D1 affinity, and as a result, a more important D2
selectivity than the compounds of the series 3 (1-benzyl-THIQs).
The greater planar structure in series 2, as well as the electronic
influence on the tertiary amine nitrogen, could justify its higher
selectivity for D1 receptors.
Aromatic side chains are bulky, have low barriers for rotation,
and are ideal to adjust to the changing conformation of the hydro-
phobic moiety of the ligand. In the dopamine D2 receptor, the bind-
ing site proved to be lined with aromatic side chains and such
residues can adjust to the different shapes and flexibility of the
agonists in the binding site. Thus, Phe 82, Val 83 and Val
87(TM3); Phe 145 (TM5); and Trp 182, Phe 185, Phe 186 and His
189 (TM6) form a mostly hydrophobic pocket for ligands.

I. Berenguer et al. / Bioorg. Med. Chem. 17 (2009) 4968–4980
4973
[3H]-SCH 23390
binding
[3H]-SCH 23390
100
75
50
25
0
binding
100
75
50
25
0
[3H]-raclopride
binding
[3H]-raclopride
binding
-9
-8
-7
-6
-5
-4
-9
-8
-7
-6
-5
-4
log [compound 1c] (M)
log [compound 1e] (M)
100
[3H]-SCH 23390
binding
100
[3H]-raclopride
binding
75
50
25
0
75
50
25
0
[3H]-SCH 23390
binding
[3H]-raclopride
binding
-9
-8
-7
-6
-5
-4
-9
-8
-7
-6
-5
-4
log [compound 2d] (M)
log [compound 3c] (M)
Figure 5. Displacement of specific binding of [3H]-SCH 23390 (D1-like DR specific ligand) and [3H]-raclopride (D2-like DR specific ligand) by compounds 1c, 1e, 2d and 3c. The
results are expressed as mean SEM from 3 to 6 experiments.
2.2.3. Behavioral assays
nyl-THIQ derivatives (series 2) show a selectivity for D1 over D2
receptors. The different activities and selectivity obtained for these
compounds might be explained, at least in part, by the different
spatial orientations adopted by the varied hydrophobic portions
located at C-1 which lead to different molecular interactions with
the D2 receptors (quantum mechanics calculations). Nonetheless in
the 1-butyl- and 1-benzyl-THIQ (series 1 and 3), the greater flexi-
bility of these substituents, along with their slight electronic effect
towards the A-ring aromatic, confer more affinity for D2.
1-Butyl-7-chloro-6-hydroxy-tetrahydroisoquinoline (1e), with
the highest affinity towards the D2 receptor (Ki value of 66 nM)
and the highest selectivity (49-fold), was then evaluated by behav-
ioral assays (spontaneous activity and forced swimming test) in
mice. Compound 1e increased the spontaneous activity as from
0.2 mg/kg and demonstrated an antidepressant-like activity at a
low dose (0.01 mg/kg), which is likely because of an activation of
the D2-type receptor. Further experiments will be necessary to
evaluate the real therapeutic potential of this compound as a
new antidepressant drug.
The major compound 1e presents a similar dopaminergic profile
to its isomer 1c, and shows a similar affinity and selectivity to-
wards D2-like receptors (Table 1 and Fig. 4). For this reason, we
have undertaken a large-scale synthesis of 1e to perform in vivo
tests (Fig. 7).
Acute administration of compound 1e induced a dose-depen-
dant hyperlocomotor activity (two-way ANOVA, p < 0.05, Table
3), that was observed as from 0.2 mg/Kg and which reached its
maximum at 5 mg/Kg. Furthermore at the dose of 0.2 mg/Kg, this
effect appeared as from the first 6 min of the test (post-hoc PLSD
of Fischer, p < 0.05). In order to investigate the involvement of D2
receptors in this hyperlocomotor activity, we examined the effect
of a concomitant administration of compound 1e with haloperidol
at a non-sedative dose (Fig. 7). Haloperidol (0.03 mg/kg) totally re-
versed the hyperactivity induced by compound 1e at 0.2 mg/Kg.
Given that 1) compound 1e displayed in vitro selectivity D2 versus
D1 (49-fold), 2) haloperidol showed in vitro selectivity D2 versus D1
(at least, 20-fold), and 3) low doses of the two compounds were
used, these facts suggest an agonist D2-receptor activity in vivo
for compound 1e.
4. Experimental
Forced swimming test: compound 1e at 0.0 1 mg/Kg (the high-
est dose that did not increase spontaneous activity in the first
6 min) and imipramine at 16 mg/Kg (used as pharmacological ref-
erence), induced a significant reduction of the immobility time in
the forced swimming test (Fig. 8). This antidepressant-like effect
of 1e was reversed by haloperidol (0.03 mg/Kg) (ANOVA followed
by post-hoc PLSD of Fischer’s test, p < 0.05, Fig. 9).
4.1. General instrumentation
Melting points were taken on a Cambridge microscope instru-
ments coupled with a Reichert-Jung. EI and FAB mass spectra were
recorded on a VG Auto Spec Fisons spectrometer instruments
(Fisons, Manchester, United Kingdom). 1H NMR and 13C NMR
spectra were recorded with CDCl3 as solvent on a Bruker AC-300,
AC-400 or AC-500. Multiplicities of 13C NMR resonances were as-
signed 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 reactions were monitored
by analytical TLC with Silica gel 60 F254 (Merck 5554). The residues
3. Conclusions
The replacement of THIQs at the C-1 position is an important
factor to modulate the selectivity at DR. Series 1 and 3 show a
greater affinity towards D2 receptors when a butyl or benzyl
moiety, respectively, is located in that position; however, 1-phe-
were purified through Silica gel 60 (40–63 lm, Merck 9385)

4974
I. Berenguer et al. / Bioorg. Med. Chem. 17 (2009) 4968–4980
Figure 6. Interactions of compound 3c (a), 1c (b), 2c (c) and 1e (d) with the binding pocket D2 DR. Different spatial views to show the hydrophobic interactions; the aromatic
rings of Phe 82, His 189, Phe 186 and Trp 182 are denote in thick green lines in this figure.
Table 3
Number of light beam crossing
Dose effect of compound 1e on spontaneous locomotor activity assessed by the
number of light beam crossing in a photoelectronic actimeter during a 30 min test
session
300
*
#
250
0–6 min
0–12 min
0–18 min
0–24 min
0–30 min
Vehicle
66 29
72 38
102 39
105 58
142 39
186 58*
200 43*
276 82*
260 73*
131 55
133 81
170 47
219 69*
257 48*
345 80*
319 106*
153 63
147 85
185 42
255 88*
307 74*
411 114*
367 139*
161 70
153 88
200 46
200
150
100
50
0.01 mg/Kg
0.04 mg/Kg
0.2 mg/Kg
1 mg/Kg
5 mg/Kg
25 mg/Kg
102 24*
115 36*
122 29*
153 57*
170 39*
277 115*
340 87*
464 145*
426 164*
*
ANOVA, PLSD of Fisher: p < 0.05 versus vehicle group, for each time section.
0
haloperidol
-
-
+
-
+
+
-
(0.03 mg/Kg)
4.1.1. 2-(3-Chloro-4-methoxy-phenyl)ethylamine
A
mixture of 3-chloro-4-methoxy-benzaldehyde (1.0 g,
compound1e
+
(0.2 mg/Kg)
5.87 mmol), nitromethane (1 mL, 18.41 mmol) and NH4OAc
(1.2 g, 15.57 mmol) in AcOH (15 mL) was refluxed for 4 h. After
cooling, the mixture was diluted with H2O (10 mL) and extracted
with CH2Cl2 (3 ꢁ 10 mL). The organic solution was washed with
brine (2 ꢁ 10 mL) and H2O (2 ꢁ 10 mL), dried over anhydrous
Na2SO4, and evaporated to dryness to obtain 3-chloro-4-methoxy-
b-nitrostyrene from EtOH as yellow needles (1.1 g, 88%) which
was used in the following step; mp: 143–145 °C; 1H NMR*
Figure 7. Effect of haloperidol (0.03 mg/kg; sc) on hyperlocomotor activity induced
by compound 1e (0.2 mg/kg; ip).*p < 0.05 versus control group, #p < 0.05 versus
‘haloperidol + 1e’ group (PLSD of Fischer). The results are expressed as mean SD
(n = 10 animals for each group).
column chromatography. Solvents and reagents were used as pur-
chased from commercial sources. Quoted yields are of purified
material. The HCl salts of the synthesised compounds were
prepared from the corresponding base with 5% HCl in MeOH.
(400 MHz, CDCl3):
d 7.91 (d, J = 13.74 Hz, 1H, H-), 7.59 (d,
J = 2.16 Hz, 1H, H-2), 7.52 (d, J = 13.74 Hz, 1H, H-), 7.44 (dd,

I. Berenguer et al. / Bioorg. Med. Chem. 17 (2009) 4968–4980
Immobility time (s)
4975
120
100
80
60
40
20
0
*
* *
0.001mg/kg
0.0033mg/kg
0.01mg/kg
compound 1e
-
-
-
Imipramine
16 mg/kg
-
-
-
Figure 8. Effect of compound 1e (0.001–0.0033–0.01 mg/kg; ip) and imipramine (16 mg/kg) on immobility time in the forced swimming test. * and ** illustrated statistical
difference as compared to the control group (respectively, p < 0.05 and p < 0.001; PLSD of Fischer). The results are expressed as mean SD (n = 10 animals for each group).
2.8 ppm (m, 2H, H-); 13C NMR* (100 MHz, CDCl3): d 153.8 (C-4),
133.3 (C-1), 130.8 (CH-2), 128.4 (CH-6), 122.6 (C-3), 112.5 (CH-
Immobility time (s)
120
5), 56.5 (OCH3), 43.8 (CH2-a), 39.1 ppm (CH2-b); *The assignments
were made by COSY and DEPT; MS (EI) m/z (%): 185 (45) [M]+.
100
80
60
40
20
0
*
#
4.2. General procedure for synthesis of amides 1–3
Amides 1–3 were prepared under Schotten–Baumann condi-
tions by condensation of the 2-(3-chloro-4-methoxyphenyl) ethyl-
amine with the appropriate acid chloride: valeryl chloride (series
1), phenylacetyl chloride (series 2) or benzoyl chloride (series 3).
4.2.1. N-(3-Chloro-4-methoxyphenylethyl)butylacetamide (1)
An amount of valeryl chloride (0.44 mL, 3.75 mmol) was added
dropwise at 0 °C to a solution of the b-(3-chloro-4-methoxy-
phenyl)ethylamine (580 mg, 3.13 mmol) in CH2Cl2 (20 mL) and
5% aqueous NaOH (4.5 mL), stirring at room temperature for 3 h.
After, 2.5% aqueous HCl was added, and the organic solution was
washed with brine (2 ꢁ 10 mL) and H2O (2 ꢁ 10 mL), dried over
Na2SO4 and evaporated to dryness. The residue was purified
through silica gel column chromatography (hexane/EtOAc, 60:40)
to afford the amide 1 as a yellow powder (730 mg, 2.71 mmol,
86.7%); 1H NMR* (400 MHz, CDCl3): d 7.19 (d, J = 2.07 Hz, 1H,
H-2), 7.04 (dd, J = 8.28, 2.07 Hz, 1H, H-6), 6.88 (d, J = 8.28 Hz, 1H,
H-5), 3.88 (s, 3H, OCH3-4), 3.47 (m, 2H, H-), 2.73 (t, J = 6.97 Hz,
2H, H-), 2.13 (t, J = 7.62 Hz, 2H, CH2-10), 1.59 (m, 2H, CH2-20),
1.30 (m, 2H, CH2-30), 0.91 ppm (t, J = 7.26 Hz, 2H, CH3); 13C NMR*
(100 MHz, CDCl3): d 173.5 (CO), 154.0 (C-4), 132.1 (C-1), 130.4
(CH-2), 128.3 (CH-6), 122.7 (C-3), 112.5 (CH-5), 56.5 (OCH3), 40.9
haloperidol
(0.03 mg/Kg)
-
-
+
-
-
+
+
compound 1e
(0.01 mg/Kg)
+
Figure 9. Effect of haloperidol (0.03 mg/kg; sc) on decrease of immobility time
induced by compound 1e (0.01 mg/kg ip) in the forced swimming test. *p < 0.05 as
compared to the control group. #p < 0.05 versus ‘haloperidol + 1e’ group (PLSD of
Fischer). The results are expressed as mean SD (n = 10 animals for each group).
J = 8.58, 2.16 Hz, 1H, H-6), 6.97 (d, J = 8.58 Hz, 1H, H-5), 3.90 ppm
(s, 3H, OCH3-4); 13C NMR* (100 MHz, CDCl3) d 158.4 (C-4), 138.4
(CH-b), 136.4 (CH-a), 130.9 (CH-2), 130.1 (CH-6), 124.2 (C-1),
123.7 (C-3), 112.7 (CH-5), 56.8 ppm (OCH3); *The assignments
were made by COSY and DEPT; MS (EI) m/z (%): 213 (55) [M]+,
(CH2-a
), 36.8 (CH2-10), 34.9 (CH2-b), 28.1 (CH2-20), 22.7 (CH2-30),
185 (100).
A solution of 3-chloro-4-methoxy-b-nitrostyrene
14.3 ppm (CH3-40); *The assignments were made by COSY and
(1.0 g, 4.7 mmol) in anhydrous THF (14 mL) was added dropwise
to a well-stirred suspension of LiAlH4 (0.7 g, 18.5 mmol) in anhy-
drous Et2O (20 mL) under nitrogen atmosphere, and after refluxed
for 2 h. After cooling, the excess of reagent was destroyed by drop-
wise addition of H2O and 15% aqueous NaOH. After partial evapo-
ration of the filtered, the aqueous solution was extracted with
CH2Cl2 (3 ꢁ 10 mL) and the organic layers were treated with 5%
aqueous HCl. The resulting aqueous acid layer was made basic
(5% aqueous NH4OH, pH ꢂ 9) and extracted with CH2Cl2. The
organic solution was washed with brine (2 ꢁ 10 mL) and H2O
(2 ꢁ 10 mL), dried over Na2SO4, and the solvent was removed in
vacuo to give b-(3-chloro-4-methoxyphenyl)ethylamine as a yellow
powder (630 mg, 72%); The compound was used in further reaction
DEPT; MS (FAB) m/z (%): 270 [M+H]+, 268 (35), 256 (21).
4.2.2. N-(3-Chloro-4-methoxyphenylethyl)benzylacetamide (2)
The title compound was prepared according to the procedure
for 1, using benzoyl chloride (0.43 mL, 3.75 mmol). The residue
was purified through silica gel column chromatography (hexane/
EtOAc, 50:50) to give the amide 2 as white powder (488 mg,
1.68 mmol, 54%); 1H NMR* (300 MHz, CDCl3): d 8.13 (dd, J = 8.46,
1.14, 2H, H-20, H-60), 7.43 (m, 3H, H-30, H-40, H-50), 7.25(d,
J = 2.20 Hz, 1H, H-2), 7.08 (dd, J = 8.42, 2.20 Hz, 1H, H-6), 6.86 (d,
J = 8.42 Hz, 1H, H-5), 3.94 (s, 3H, OCH3-4), 3.65 (m, 2H, H-),
2.89 ppm (t, J = 6.96 Hz, 2H, H-); 13C NMR* (75 MHz, CDCl3): d
168.0 (CO), 154.1 (C-4), 134.9 (C-10), 132.6 (C-1), 132.0, 129.0,
127.8 (5CH–Ar), 128.8 (CH-2), 127.2 (CH-6), 122.3 (C-3), 112.6
without purification; 1H NMR* (400 MHz, CDCl3):
J = 2.24 Hz, 1H, H-2), 7.2 (dd, J = 8.46, 2.24 Hz, 1H, H-6), 6.8 (d,
J = 8.46 Hz, 1H, H-5), 3.9 (s, 3H, OCH3-4), 3.1 (m, 2H, H- ),
d 7.3 (d,
(CH-5), 56.6 (OCH3), 41.5 (CH2-a), 34.9 ppm (CH2-b); *The assign-
a

4976
I. Berenguer et al. / Bioorg. Med. Chem. 17 (2009) 4968–4980
ments were made by COSY and DEPT; MS (EI) m/z (%): 289 [M]+,
275 (15), 168 (60).
*The assignments were made by COSY 45, DEPT, HSQC and HMBC;
MS (FAB) m/z (%): 254 (100) [M+H]+, 196 (73); HRMS-FAB m/z
[M+H]+ calcd for C14H21NOCl: 254.1311, found 254.1313.
4.2.3. N-(3-Chloro-4-methoxyphenylethyl)phenylacetamide (3)
The title compound was prepared according to the procedure
for 1 and 2, using the phenylacetyl chloride (0.5 mL, 3.75 mmol).
The residue was purified through silica gel column chromatogra-
phy (hexane/EtOAc, 60:40) to afford 3 as white powder (655 mg,
2.16 mmol, 69%); 1H NMR* (400 MHz, CDCl3): d 7.35–7.14 (m,
5H, H–Ar), 7.05 (d, J = 2.7 Hz, 1H, H-2), 6.89 (dd, J = 8.5, 2.7 Hz,
1H, H-6), 6.78 (d, J = 8.5 Hz, 1H, H-5), 3.54 (s, 2H, CH2CO), 3.90
(s, 3H, OCH3-4), 3.4 (m, 2H, H-), 2.64 ppm (t, J = 6.79 Hz, 2H, H-);
13C NMR* (100 MHz, CDCl3): d 171.3 (CO), 154.0 (C-4), 135.0
(C-10), 132.2 (C-1), 127.7, 129.4, 129.5, 129.8, 130.8 (5CH–Ar),
128.2 (CH-2), 127.3 (CH-6), 122.7 (C-3), 112.5 (CH-5), 56.5
4.3.2. 1-Phenyl-6-chloro-7-methoxy-1,2,3,4-
tetrahydroisoquinoline (2a)
The title compound was prepared according to the same proce-
dure for 1a using the corresponding amide 2 (500 mg, 1.73 mmol),
P2O5 (4.9 g, 17.3 mmol) and POCl3 (1.6 mL, 17.3 mmol). The residue
was purified through silica gel column chromatography (CH2Cl2/
MeOH, 90:10) to obtain 2a as a yellow oil (110 mg, 0.403 mmol,
23.3%); 1H NMR* (400 MHz, CDCl3): d 7.34–7.23 (m, 5H, H–Ar),
7.15 (s, 1H, H-5), 6.30 (s, 1H, H-8), 5.05 (s, 1H, H-1), 3.63 (s, 3H,
OCH3), 3.23–3.18 (m, 1H, H-4a), 3.05–2.87 (m, 2H, H-3), 2.76–
2.71 ppm (m, 1H, H-4b); 13C NMR* (100 MHz, CDCl3): d 152.7 (C-
7), 144.1 (C-10), 137.5 (C-8a), 135.1 (C-4a), 130.2 (CH-5), 128.9,
128.4, 127.5 (5CH–Ar), 120.4 (C-6), 111.6 (CH-8), 61.6 (CH-1),
56.0 (OCH3), 41.6 (CH2-3), 28.6 ppm (CH2-4); *The assignments
were made by COSY 45, DEPT, HSQC and HMBC; MS (FAB) m/z
(%): 274 (100) [M+H]+, 196 (13); HRMS-FAB m/z [M]+ calcd for
C16H16NOCl: 273.098, found 273.104.
(OCH3), 44.2 (CH2-a), 41.0 (CH2), 34.7 ppm (CH2-b); *The assign-
ments were made by COSY and DEPT; MS (EI) m/z (%): 303 (100)
[M]+, 289 (10).
4.3. Synthesis of 1-butyl, 1-phenyl and 1-benzyl-THIQs series
(compounds 1a–3d)
4.3.3. 1-Benzyl-6-chloro-7-methoxy-1,2,3,4-
tetrahydroisoquinoline (3a)
THIQs series were prepared in two steps by standard methods
starting the corresponding amides 1–3.
The title compound was prepared according to the same proce-
dure for 1a using the corresponding amide 3 (500 mg, 1.65 mmol),
P2O5 (4.7 g, 16.5 mmol) and POCl3 (1.5 mL, 16.5 mmol). The residue
was purified through silica gel column chromatography (CH2Cl2/
MeOH, 90:10) to obtain 3a as a yellow oil (138 mg, 0.48 mmol,
29%); 1H NMR* (400 MHz, CDCl3): d 7.35–7.24 (m, 5H, H–Ar),
7.11 (s, 1H, H-5), 6.63 (s, 1H, H-8), 4.18 (dd, J = 4.81, 9.22 Hz, 1H,
4.3.1. General procedure for Bischler–Napieralski cyclization. 1-
Butyl-6-chloro-7-methoxy-1,2,3,4-tetrahydroisoquinoline (1a)
To a 250 mL three-neck round-bottomed flask under N2, the
corresponding amide 1 (500 mg, 1.86 mmol) was added in dry
toluene (20 mL) and treated with P2O5 (5.2 g, 18.6 mmol) which
was added in portions followed by the dropwise addition of POCl3
(1.7 mL, 18.6 mmol). The mixture was stirred and refluxed under
N2 for 6–8 h and then, cooled to room temperature. The toluene
was concentrated under reduced pressure and the reaction mix-
ture was slowly poured into a mixture of crushed ice. The solid res-
idue was triturated with 10% aqueous NaOH to afford a suspension
(pH ꢂ 8–9) and extracted with CH2Cl2 (3 ꢁ 15 mL). The combined
CH2Cl2 extracts were dried over NaSO4 and the solvent evaporated
in vacuo to afford a reddish oil. The residue was dissolved in MeOH
(20 mL), and then, cooled to ꢀ78 °C and treated with NaBH4
(76 mg, 2 mmol). The reaction mixture was stirred for 2 h. Water
(15 mL) was added and the volatiles were evaporated under re-
duced pressure. The aqueous phase was extracted with CH2Cl2
(3 ꢁ 15 mL), and the combined organic layers were dried over
Na2SO4 and evaporated to dryness. The crude was purified by silica
gel column chromatography (CH2Cl2/MeOH, 90:10) to furnish 1a
and 1a0 (223 mg, 0.88 mmol, 47.3%); 1a: 1H NMR* (400 MHz,
CDCl3): d 7.06 (s, 1H, H-5), 6.65 (s, 1H, H-8), 3.90 (m, 1H, H-1),
3.84 (s, 3H, OCH3), 3.25–3.17 (m, 1H, H-3a), 2.97–2.88 (m, 1H, H-
3b), 2.72–2.59 (m, 2H, H-4), 1.80–1.68 (m, 1H, H-10), 1.47–1.28
(m, 4H, H-20, H-30), 0.92 ppm (t, J = 6.90 Hz, 3H, H-40); 13C NMR*
(100 MHz, CDCl3): d 152.8 (C-7), 139.3 (C-8a), 130.3 (CH-5),
128.3 (C-4a), 119.9 (C-6), 109.9 (CH-8), 56.1 (OCH3), 55.6 (CH-1),
40.7 (CH2-3), 36.1 (CH2-10), 28.8 (CH2-4), 28.2 (CH2-20), 22.8
(CH2-30), 14.0 ppm (CH3-40); *The assignments were made by COSY
45, DEPT, HSQC and HMBC; MS (FAB) m/z (%): 254 (100) [M+H]+,
196 (73); HRMS-FAB m/z [M+H]+ calcd for C14H21NOCl: 254.1311,
found 254.1317; 1a0: 1H NMR* (400 MHz, CDCl3): d 7.07 (s, 1H,
H-8), 6.57 (s, 1H, H-5), 3.87 (m, 1H, H-1), 3.80 (s, 3H, OCH3),
3.18–3.15 (m, 1H, H-3a), 2.93–2.88 (m, 1H, H-3b), 2.78–2.66 (m,
2H, H-4), 1.76–1.60 (m, 1H, H-10), 1.37–1.31 (m, 4H, H-20, H-30),
0.89 ppm (t, J = 6.90 Hz, 3H, H-40); 13C NMR* (100 MHz, CDCl3): d
152.7 (C-6), 134.7 (C-8a), 132.8 (C-4a), 127.5 (CH-8), 119.5 (C-7),
112.3 (CH-5), 55.9 (CH-1), 54.9 (OCH3), 40.8 (CH2-3), 35.9 (CH2-
10), 29.8 (CH2-4), 28.1 (CH2-20), 22.7 (CH2-30), 14.0 ppm (CH3-40);
H-1), 3.80 (s, 3H, OCH3), 3.24–3.16 (m, 2H, H-
aa, H-3a), 2.98–
2.81 (m, 2H, H-
a
b, H-3b), 2.76–2.69 ppm (m, 2H, H-4); 13C NMR*
(100 MHz, CDCl3): d 152.6 (C-7), 138.7 (C-10), 138.0 (C-8a), 129.3
(CH-5), 129.0, 128.6, 126, 5 (5CH–Ar), 128.3 (C-4a), 120.2 (C-6),
110.1 (CH-8), 57.0 (CH-1), 56.1 (OCH3), 42.7 (CH2-3), 40.3 (CH2-
a
), 28.8 ppm (CH2-4); *The assignments were made by COSY 45,
DEPT, HSQC and HMBC; MS (FAB) m/z (%): 288 (84) [M+H]+, 196
(100); HRMS-FAB m/z [M+H]+ calcd for C17H19NOCl: 288.1155,
found 288.1148.
4.3.4. General procedure for N-methylation. 1-Butyl-6-chloro-
7-methoxy-N-methyl-1,2,3,4-tetrahydroisoquinoline (1b)
To a stirred solution of 1a (500 mg, 1.98 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 tem-
perature, treated with NaBH4 (750 mg, 19.8 mmol), and refluxed
an additional 1 h. The reaction mixture was warmed up to ambient
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 ꢁ 15 mL). The combined organic
extracts were dried over Na2SO4 and concentrated under reduced
pressure to give the crude product which was further purified by
silica gel column chromatography (CH2Cl2/MeOH/NH4OH, 98:2:
0.2) to afford 1b (480 mg, 1.8 mmol, 91%); 1H NMR* (300 MHz,
CDCl3): d 7.08 (s, 1H, H-5), 6.61 (s, 1H, H-8), 3.86 (s, 3H, OCH3),
3.35 (t, 1H, J = 5.46 Hz, H-1), 3.12–3.06 (m, 1H, H-3a), 2.77–2.73
(m, 2H, H-4), 2.72–2.64 (m, 1H, H-3b), 2.42 (s, 3H, NCH3), 1.75–
1.68 (m, 2H, H-10), 1.34–1.19 (m, 4H, H-20, H-30), 0.88 ppm (t,
J = 6.97 Hz, 3H, H-40); 13C NMR* (75 MHz, CDCl3): d 152.6 (C-7),
134.3 (C-8a), 131.6 (C-4a), 128.6 (CH-5), 119.6 (C-6), 111.9 (CH-
8), 62.8 (CH-1), 56.2 (OCH3), 48.0 (CH2-3), 42.6 (NCH3), 34.4
(CH2-10), 27.56 (CH2-20), 26.06 (CH2-4), 22.94 (CH2-30), 14.05 ppm
(CH3-40); *The assignments were made by COSY 45, DEPT, HSQC
and HMBC; MS (EI) m/z (%): 268 (100) [M+H]+, 210 (68); HRMS-
FAB m/z [M+H]+ calcd for C15H23NOCl: 268.1468, found 268.1475.

I. Berenguer et al. / Bioorg. Med. Chem. 17 (2009) 4968–4980
4977
4.3.5. 1-Phenyl-6-chloro-7-methoxy-N-methyl-1,2,3,4-
tetrahydroisoquinoline (2b)
by COSY 45, DEPT and NOE; MS (EI) m/z (%): 238 (5) [MꢀH]+,
182 (100); HRMS-EI m/z [MꢀH]+ calcd for C13H17NOCl: 238.0999,
found: 238.0972.
The title compound was prepared according to the same proce-
dure for 1b using the compound 2a (500 mg, 1.83 mmol), 37%
formaldehyde (15 mL), one drop of formic acid and NaBH4
(700 mg, 18.3 mmol). The residue was purified by silica gel column
chromatography (cyclohexane/OEtAc/Et2N, 92:6:2) to obtain 2b
(460 mg, 1.60 mmol, 87.4%); 1H NMR* (400 MHz, CDCl3): d 7.3–
7.23 (m, 5H, H–Ar), 7.13 (s, 1H, H-5), 6.16 (s, 1H, H-8), 4.19 (s,
1H, H-1), 3.57 (s, 3H, OCH3), 3.24–3.13 (m, 2H, H-4), 2.80–2.74
(m, 2H, H-3), 2.23 ppm (s, 3H, NCH3); 13C NMR* (100 MHz, CDCl3):
d 152.9 (C-7), 143.2 (C-10), 134.0 (C-8a), 131.8 (C-4a), 129.9 (CH-5),
129.5, 128.4, 127.5 (5CH–Ar), 119.8 (C-6), 111.4 (CH-8), 71.1 (CH-
1), 56.1 (OCH3), 51.9 (CH2-3), 44.2 (NCH3), 28.3 ppm (CH2-4); *The
assignments were made by COSY 45, DEPT, HSQC and HMBC; MS
(FAB) m/z (%): 288 (100) [M+H]+, 210 (48); HRMS-FAB m/z
[M+H]+ calcd for C18H20NOCl: 288.0013, found 288.1064.
1H NMR* (400 MHz, CD3OD) for 1e: d 7.07 (s, 1H, H-8), 6.68 (s,
1H, H-5), 3.85 (dd, J = 8.70, 3.60 Hz, 1H, H-1), 3.23–3.17 (m, 1H, H-
3a), 2.98–2.90 (m, 1H, H-3b), 2.73–2.65 (m, 2H, H-4), 1.80–1.63 (m,
2H, H-10), 1.40–1.33 (m, 4H, H-20 and H-30), 0.93 ppm (t, J = 6.97 Hz,
3H, H-40); 13C NMR* (100 MHz, CD3OD): d 152.60 (C-6), 135.62 (C-
4a), 131.71 (C-8a), 128.32 (CH-8), 119.60 (C-7), 117.56 (CH-5),
56.12 (CH-1), 41.65 (CH2-3), 36.72 (CH2-10), 29.47 (CH2-4), 29.04
(CH2-20), 23.83 (CH2-30), 14.38 ppm (CH3-40); *The assignments
were made by COSY 45, DEPT, HSQC, HMBC and NOE; MS (EI) m/
z (%): 238 (3) [MꢀH]+, 182 (100); HRMS-EI m/z [MꢀH]+ calcd for
C13H17NOCl: 238.0999, found: 238.0986.
4.3.8. 1-Butyl-6-chloro-7-hydroxy-N-methyl-1,2,3,4-
tetrahydroisoquinoline (1d)
This compound was prepared as the same procedure for the
synthesis of 1c using the compound 1b (260 mg, 0.97 mmol) and
BBr3 (0.39 mL, 3.9 mmol). The residue was purified by silica gel col-
umn chromatography (CH2Cl2/MeOH/NH4OH, 100/6/0.5) to give 1d
(194 mg, 0.77 mmol, 79%); 1H NMR* (300 MHz, CDCl3): d 7.03 (s,
1H, H-5), 6.72 (s, 1H, H-8), 3.35 (t, 1H, J = 5.46 Hz, H-1), 3.12–
3.06 (m, 1H, H-3a), 2.77–2.63 (m, 3H, H-4, H-3b), 2.42 (s, 3H,
NCH3), 1.74–1.67 (m, 2H, H-10), 1.33–1.26 (m, 4H, H-20, H-30),
0.88 ppm (t, J = 7.15 Hz, 3H, H-40); 13C NMR* (75 MHz, CDCl3): d
149.0 (C-7), 135.1 (C-8a), 131.8 (C-4a), 127.3 (CH-5), 117.2 (C-6),
115.7 (CH-8), 62.9 (CH-1), 47.8 (CH2-3), 42.5 (NCH3), 34.5 (CH2-
10), 27.7 (CH2-20), 25.6 (CH2-4), 22.9 (CH2-30), 14.1 ppm (CH3-40);
MS (FAB) m/z (%): 254 (19) [M+H]+; MS (EI) m/z (%): 196 (100);
HRMS-FAB m/z [M+H]+ calcd for C14H21NOCl: 254.1311, found:
254.1312.
4.3.6. 1-Benzyl-6-chloro-7-methoxy-N-methyl-1,2,3,4-
tetrahydroisoquinoline (3b)
The title compound was prepared according to the same pro-
cedure for 1b using the compound 3a (500 mg, 1.74 mmol), 37%
formaldehyde (15 mL), one drop of formic acid and NaBH4
(660 mg, 17.4 mmol). The residue was purified through silica
gel column chromatography (cyclohexane/OEtAc/Et2N, 98:1:1)
to obtain 3b (451 mg, 1.5 mmol, 86.2%); 1H NMR* (400 MHz,
CDCl3): d 7.29–7.09 (m, 5H, H–Ar), 7.07 (s, 1H, H-5), 5.92 (s,
1H, H-8), 3.77–3.74 (m, 1H, H-1), 3.47 (s, 3H, OCH3), 3.25–3.15
(m, 2H, H-
2.63–2.57 (m, 1H, H-4b), 2.54 ppm (s, 3H, NCH3); 13C NMR*
(100 MHz, CDCl3):
151.9 (C-7), 139.6 (C-10), 136.7 (C-8a),
aa, H-3a), 2.88–2.71 (m, 3H, H-ab, H-3b, H-4a),
d
129.9, 129.9, 128.2 (5CH–Ar), 126.8 (C-4a), 126.1 (CH-5), 120.0
(C-6), 111.7 (CH-8), 65.1 (CH-1), 55.6 (OCH3), 46.3 (CH2-3),
42.5 (NCH3), 40.7 (CH2-a), 24.9 ppm (CH2-4); *The assignments
4.3.9. 1-Phenyl-6-chloro-7-hydroxy-1,2,3,4-
tetrahydroisoquinoline (2c)
were made by COSY 45, DEPT, HSQC and HMBC; MS (EI) m/z
(%): 301 (5) [M]+, 210 (100); MS (FAB) m/z (%): 302 (100)
[M+H]+, 210 (43); HRMS-FAB m/z [M+H]+ calcd for C18H20NOCl:
301.8105, found: 301.7903.
This compound was prepared as the above procedure for the
synthesis of 1c using the compound 2a (260 mg, 0.95 mmol) and
BBr3 (0.38 mL, 3.8 mmol). The residue was purified through silica
gel column chromatography (CH2Cl2/MeOH/NH4OH, 100:7:0.5) to
give 2c (170 mg, 0.66 mmol, 69%); 1H NMR* (300 MHz, CD3OD): d
7.28–7.15 (m, 5H, H–Ar), 7.01 (s, 1H, H-5), 6.17 (s, 1H, H-8), 4.88
(s, 1H, H-1), 3.15–3.06 (m, 1H, H-3a), 2.94–2.79 (m, 2H, H-3b, H-
4a), 2.70–2.60 ppm (m, 1H, H-4b); 13C NMR* (75 MHz, CD3OD): d
150.0 (C-7), 142.6 (C-10), 136.5 (C-8a), 128.6 (CH-5), 127.4, 127.3,
126.5 (5CH–Ar), 126.2 (C-4a), 114.6 (CH-8), 60.4 (CH-1), 40.6
(CH2-3), 26.5 ppm (CH2-4); HRMS-EI m/z [MꢀH]+ calcd for
C15H14NOCl: 259.0764, found: 259.0728.
4.3.7. General procedure for O-demethylation. 1-Butyl-6-
chloro-7-hydroxy-1,2,3,4-tetrahydroisoquinoline (1c) and 1-
butyl-6-hydroxy-7-chloro-1,2,3,4-tetrahydro-isoquinoline (1e)
A solution of the appropriate isoquinoline 1a and 1a0 (260 mg,
1.02 mmol) in dry CH2Cl2 (10 mL) was cooled to ꢀ78 °C. To this
stirring solution, BBr3 (0.4 mL, 4.08 mmol) was added dropwise.
After 15 min, the reaction mixture was warmed up to ambient
temperature and stirred for 18 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 ꢂ 11, and subsequently neutralized
with 1 M HCl to pH ꢂ 7–8. The aqueous layer was then extracted
with the EtOAc (3 ꢁ 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, 100:8:0.5) to afford a mixture of the two
isomers 1c and 1e (220 mg, 0.92 mmol, 90%) in a 1:2 ratio; 1c
(73 mg, 0.3 mmol, 30%) and 1e (147 mg, 0.6 mmol, 60%); 1H
NMR* (300 MHz, CDCl3) for 1c: d 7.03 (s, 1H, H-5), 6.78 (s, 1H, H-
8), 3.88 (dd, J = 8.85, 3.75 Hz, 1H, H-1), 3.23–3.17 (m, 1H, H-3a),
2.98–2.90 (m, 1H, H-3b), 2.73–2.65 (m, 2H, H-4), 1.80–1.67 (m,
2H, H-10), 1.40–1.33 (m, 4H, H-20, H-30), 0.93 ppm (t, J = 6.97 Hz,
3H, H-40); 13C NMR* (100 MHz, CDCl3): d 149.2 (C-7), 140.1 (C-
8a), 129.0 (CH-5), 128.3 (C-4a), 117.5 (C-6), 113.6 (CH-8), 55.5
(C-1), 41.5 (CH2-3), 35.9 (CH2-10), 28.9 (CH2-4), 28.1 (CH2-20),
22.8 (CH2-30), 14.0 ppm (CH3-40); *The assignments were made
4.3.10. 1-Phenyl-6-chloro-7-hydroxy-N-methyl-1,2,3,4-
tetrahydroisoquinoline (2d)
This compound was prepared in a similar manner as de-
scribed for the synthesis of 1c using the compound 2b
(260 mg, 0.9 mmol) and BBr3 (0.36 mL, 3.6 mmol). The residue
was purified through silica gel flash column (CH2Cl2/MeOH/
NH4OH, 100:2:0.5) to give 2d (130 mg, 0.47 mmol, 52%); 1H
NMR* (500 MHz, CDCl3): d 7.30–7.20 (m, 5H, H–Ar), 7.06 (s,
1H, H-5), 6.23 (s, 1H, H-8), 4.12 (s, 1H, H-1), 3.16–3.09 (m, 1H,
H-4a), 3.07–3.04 (m, 1H, H-3a), 2.72–2.70 (m, 1H, H-4b), 2.55
(td, 1H, J = 11.13, 3.62 Hz; H-3b) 2.16 ppm (s, 3H, NCH3); 13C
NMR* (75 MHz, CDCl3): d 149.07 (C-7), 143.06 (C-10), 138.94
(C-8a), 129.48, 128.40, 127.52 (5CH–Ar), 128.25 (CH-5), 127.60
(C-4a), 118.02 (C-6), 115.94 (CH-8), 71.06 (CH-1), 52.16 (CH2-
3), 44.20 (NCH3), 28.42 ppm (CH2-4); *The assignments were
made by COSY 45 and HSQC; HRMS-EI m/z [MꢀH]+ calcd for
C16H16NOCl: 273.0920, found: 272.0923.

4978
I. Berenguer et al. / Bioorg. Med. Chem. 17 (2009) 4968–4980
4.3.11. 1-Benzyl-6-chloro-7-hydroxy-1,2,3,4-
Brandel cell harvester (model M-24, Biochemical Research and
tetrahydroisoquinoline (3c)
Development Laboratories, Inc.). Filters were washed twice with
This compound was prepared in a similar manner as described
for the synthesis of 1c using the compound 3a (260 mg, 0.9 mmol)
and BBr3 (0.36 mL, 3.6 mmol). The residue obtained was purified
through silica gel column (CH2Cl2/MeOH/NH4OH, 100:6:0.5) to
give 3c (183 mg, 0.77 mmol, 74%); 1H NMR* (300 MHz, CDCl3): d
7.34–7.22 (m, 5H, H–Ar), 7.06 (s, 1H, H-5), 6.85 (s, 1H, H-8), 4.11
3 mL cold Krebs medium and dried. Non-specific [3H]-dopamine
uptake was determined in the presence of 10 lM nomifensine
(dopamine uptake inhibitor). Filters were placed into scintillation
mixture (Optiphase ‘Hisafe’ 2, Perkin Elmer) and radioactivity
was determined by scintillation spectrometry12,15 Protein concen-
trations were determined using the Bradford protein assay
(Biorad).
(dd, J = 10.17, 3.75 Hz, 1H, H-1), 3.22–3.14 (m, 2H, H-aa, H-3a),
2.92–2.81 (m, 2H, H-a
b, H-3b), 2.75–2.65 ppm (m, 2H, H-4); 13C
NMR* (75 MHz, CDCl3): d 149.7 (C-7), 138.2 (C-10), 138.2 (C-8a),
4.4.3. Radioligand binding assays
129.4 (CH-5), 129.3, 128.8, 126.7 (5CH–Ar), 127.8 (C-4a), 118.5
(C-6), 114.1 (CH-8), 56.7 (CH-1), 42.0 (CH2-3), 40.2 (CH2-a),
28.5 ppm (CH2-4); *The assignments were corroborated by NOE
DIFF; HRMS-EI m/z [MꢀH]+ calcd for C16H15NOCl: 272.0842, found:
272.0804.
[3H]-SCH23390 and [3H]-raclopride binding experiments were
performed on rat striatal membranes. The rat striatum was homog-
enized 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 resuspension in the same volume of TRIS–
HCl buffer between each centrifugation. The final pellet was resus-
pended 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 D1-like
4.3.12. 1-Benzyl-6-chloro-7-hydroxy-N-methyl-1,2,3,4-
tetrahydroisoquinoline (3d)
This compound was prepared in a similar manner as described
for the synthesis of 1c using the compound 3b (260 mg,
0.86 mmol) and BBr3 (0.34 mL, 3.4 mmol). The residue was purified
through silica gel column chromatography (CH2Cl2/MeOH/NH4OH,
100:3:0.5) to give 3d (150 mg, 0.52 mmol, 61%); 1H NMR*
(300 MHz, CDCl3): d 7.28–7.05 (m, 5H, H–Ar), 7.01 (s, 1H, H-5),
receptor binding assays, membranes (100 lg/mL) were incubated
with [3H]-SCH 23390 (0.25 nM; 66 Ci/mmol, Amersham, GE
Healthcare, UK) and various concentrations of competition com-
pound (10ꢀ10M–10ꢀ4M) for 1 h at 23 °C. Non-specific binding
was determined in the presence of 30
l
M SK&F38393. For D2-like
6.28 (s, 1H, H-8), 3.75 (m, 1H, H-1), 3.27–3.09 (m, 2H, H-
a
a,
receptor binding assays, membranes (200 lg/mL) were incubated
H-3a), 2.87–2.62 (m, 2H, H-
ab, H-4a, H-3b), 2.58–2.52 (m, 1H,
with [3H]-raclopride (0.5 nM; 62.2 Ci/mmol, Perkin Elmer) and
various concentrations of competition compound (10ꢀ10M–
10ꢀ4M) for 1 h at 23 °C. Non-specific binding was determined in
H-4b), 2.48 ppm (s, 3H, NCH3); 13C NMR* (75 MHz, CDCl3): d
148.79 (C-7), 139.44 (C-10), 137.95 (C-8a), 129.56, 128.14, 126.10
(5CH–Ar), 128.69 (CH-5), 127.31 (C-4a), 117.86 (C-6), 115.29
the presence of 50 lM apomorphine (Sigma). In both cases, incuba-
(CH-8), 64.67 (CH-1), 46.52 (CH2-3), 42.51 (NCH3), 41.01 (CH2-
a
),
tions were stopped by the addition of 3 mL ice-cold TRIS-ions
buffer followed by rapid filtration through fiberglass 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 buf-
fer. 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 ana-
lyzed by Prim (Graph Pad Software; San Diego, California, USA)
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 at the mean SEM of three to six independent deter-
minations performed in duplicate.
24.67 ppm (CH2-4); HRMS-FAB m/z [M+H]+ calcd for C17H19NOCl:
288.1155, found: 288.1151.
4.4. Pharmacological in vitro assays
4.4.1. Animals
Female Wistar rats (200–220 g) bred in a standard experimental
animal room of the Faculty of Pharmacy were used for [3H]-dopa-
mine uptake assays and radioligand binding experiments. The rats
were housed under a 12-h light/dark cycle at 22 °C and 60% humid-
ity. All protocols complied with European Community guidelines
for the use of experimental animals and were approved by the
Ethics Committee of the University of Valencia.
4.5. Behavioral studies
4.4.2. [3H]-Dopamine uptake assay
[3H]-Dopamine uptake was studied using a preparation of rat
striatal synaptosomes. All experimental procedures for the synap-
tosomes preparation were carried out at 0–4 °C. The rat striatum
was dissected, homogenized 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
recentrifuged at 1000g for 10 min. The two supernatants were
combined and the mixture centrifuged at 16,000g for 30 min. The
resultant pellet was suspended in 10 volumes of ice-cold Krebs
medium (pH 7.6) contained (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 preincubated during 10 min at
4.5.1. Animals
Naïve male NMRI mice (Centre d’Elevage René Janvier) weigh-
ing 25–30 g were used for all experiments. Mice were housed by
groups of 10 animals in standard polycarbonate cages and main-
tained in a regulated environment (22 1 °C) under 12–12 h
light/dark cycle (light on between 20:00 and 8:00) with food and
water freely available in the home cage. Behavioral tests were con-
ducted during the dark phase of the cycle, between 10 h and 16 h.
Each animal was used only once. All experiments complied with
the European Community guidelines and the French law on animal
experimentation (personal authorization n° 14–17 and 14–26 for
MB and TF, respectively).
37 °C in Krebs buffer containing 10
l
M pargyline (to block metab-
4.5.2. Drug administration
olism of dopamine by monoamine oxidase), [3H]-dopamine (47 Ci/
mmol, Amersham) was added to a final 0.5 nM concentration and
the incubation was continued for another 10 min. Compounds
Animals were randomly divided into groups (n = 10/group). All
injections (10 mL/Kg) were realized 30 min before behavioral tests.
In dose–response study, mice were injected intra-peritaneously, by
either the 1e compound (0.001–25 mg/kg), or vehicle (saline
water, 0.9% NaCl). In order to investigate the implication of
D2-receptors in hyperlocomotor and anti-depressant like activities,
were screened at 100 lM. Incubation was terminated by dilution
into ice-cold Krebs medium and the samples were filtered rapidly
through fiberglass filters (Schleicher & Schuell Grade 30) using a

I. Berenguer et al. / Bioorg. Med. Chem. 17 (2009) 4968–4980
4979
BEQM ¼ EL=D2DR ꢀ ðED2DR þ ELÞ
where BEQM is the binding energy, EL/D2DR the complex energy,
ED2DR the energy of the reduced receptor model (binding pocket)
and EL is the energy of the ligand.
a D2-receptor antagonist (haloperidol) was used. In both cases, hal-
operidol was injected sub-cutaneously, just before ip administra-
tion (1e compound or saline water), at the dose of 0.03 mg/Kg on
basis preliminary study (data not shown).
All the simulations and optimizations reported here have been
carried out on the R isomer of each compound which displayed
the better stereo-electronic complementarities with the D2 DR.
All the calculations reported here were carried out using the
GAUSSIAN 03 program.35
4.5.3. Spontaneous locomotor activity
In order to assess the spontaneous horizontal activity, mice
were tested using a photoelectronic actimeter (APELABÒ), initially
designed by Boissier and Simon (1965).31 The apparatus consisted
of a perspex enclosure (25.5 ꢁ 20.5 ꢁ 9 cm3). Two infrared perpen-
dicular light beams emerging from two consecutive walls, crossed
in the center of the box and were connected to two photoelectric
cells. Locomotor activity was recorded as the number of times
the mouse crossed each beam, and consecutively interrupting the
light beam. Each mouse was tested for a total period of 30 min
and the numbers of light beams interruptions were noted all
6 min. In this condition, chlorpromazine (4 mg/Kg, ip) and amphet-
amine (10 mg/Kg, ip), used as pharmacological references (depres-
sive and stimulative, respectively), induce, respectively a decrease
(490%, PLSD of Fisher, p < 0.001) and an increase (260%, PLSD of
Fisher, p < 0.001) of spontaneous activity measured during 30 min.
Spatial views shown in Figures 4 and 6 were constructed using
the UCSF CHIMERA program36 as graphic interface.
Acknowledgments
This research was supported by the Spanish ‘Ministerio de Edu-
cación y Ciencia’ Grant SAF 2007-63142. I. Berenguer acknowl-
edges the fellowship of Generalitat Valenciana. S. Andujar
acknowledges the fellowship of CONICET-Argentina.
References and notes
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4.5.4. Forced swimming test
The FST32,33 was carried out in mice which were individually
forced to swim during 6 min in an open cylindrical container
(diameter 12 cm, height 20 cm) with a water depth of 13 cm at
23–24 °C. The duration of immobility, after a delay of 2 min, was
measured during the last 4 min. Each mouse was judged to be
immobile when it ceased struggling and remained floating motion-
less in the water, making only those movements necessary to keep
its head above water. Animals were not pre-tested. In this condi-
tion, imipramine (16 mg/kg), used as pharmacological reference,
significantly decreased the immobility time (32% versus control
group, p < 0.001—PLSD of Fisher).
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angles of backbones as well as the bond angles and bond lengths
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