Dopaminergic isoquinolines with hexahydrocyclopenta[ ij ]-isoquinolines as D2-like selective ligands

Article abstract of DOI:10.1016/j.ejmech.2016.06.009

Dopamine receptors (DR) ligands are potential drug candidates for treating neurological disorders including schizophrenia or Parkinson's disease. Three series of isoquinolines: (E)-1-styryl-1,2,3,4-tetrahydroisoquinolines (series 1), 7-phenyl-1,2,3,7,8,8a

Full text of DOI:10.1016/j.ejmech.2016.06.009

European Journal of Medicinal Chemistry 122 (2016) 27e42
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Dopaminergic isoquinolines with hexahydrocyclopenta[ij]-
isoquinolines as D2-like selective ligands
a
b
b
b
ꢀ
ꢀ
ꢀ
Javier Parraga , Sebastian A. Andujar , Sebastian Rojas , Lucas J. Gutierrez ,
Noureddine El Aouad c, M. Jesús Sanz d e, Ricardo D. Enriz b, Nuria Cabedo d
,
,
, *
,
**
Diego Cortes a
a Departamento de Farmacología, Laboratorio de Farmacoquímica, Facultad de Farmacia, Universidad de Valencia, 46100, Burjassot, Valencia, Spain
b Facultad de Química, Bioquímica y Farmacia, Universidad Nacional de San Luis-IMIBIO-SL, Chacabuco 915, 5700, San Luis, Argentina
c
ꢀ
Campus Universitaire Ait Melloul, Universite Ibn-Zohr, Agadir, Morocco
d Institute of Health Research-INCLIVA, University Clinic Hospital of Valencia, 46010, Valencia, Spain
e Departamento de Farmacología, Facultad de Medicina, Universidad de Valencia, 46010, Valencia, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
Dopamine receptors (DR) ligands are potential drug candidates for treating neurological disorders
including schizophrenia or Parkinson’s disease. Three series of isoquinolines: (E)-1-styryl-1,2,3,4-
tetrahydroisoquinolines (series 1), 7-phenyl-1,2,3,7,8,8a-hexahydrocyclopenta[ij]-IQs (HCPIQs) (series
2) and (E)-1-(prop-1-en-1-yl)-1,2,3,4- tetrahydroisoquinolines (series 3), were prepared to determine
their affinity for both D1 and D2-like DR. The effect of different substituents on the nitrogen atom (methyl
Received 14 March 2016
Received in revised form
7 June 2016
Accepted 8 June 2016
Available online 10 June 2016
or allyl), the dioxygenated function (methoxyl or catechol), the substituent at the b-position of the THIQ
skeleton, and the presence or absence of the cyclopentane motif, were studied. We observed that the
most active compounds in the three series (2c, 2e, 3a, 3c, 3e, 5c and 5e) possessed a high affinity for D2-
like DR and these remarkable features: a catechol group in the IQ-ring and the N-substitution (methyl or
allyl). The series showed the following trend to D2-RD affinity: HCPIQs > 1-styryl > 1-propenyl. There-
Keywords:
Tetrahydroisoquinolines
Hexahydrocyclopentaisoquinolines
Dopaminergic
fore, the substituent at the b-position of the THIQ and the cyclopentane ring also modulated this affinity.
Structure-activity relationships
Cytotoxicity
Molecular modeling
Among these dopaminergic isoquinolines, HCPIQs stood out for unexpected selectivity to D2-DR since the
Ki D1/D2 ratio reached values of 2465, 1010 and 382 for compounds 3a, 3c and 3e, respectively. None of
the most active THIQs in D2 DR displayed relevant cytotoxicity in human neutrophils and HUVEC. Finally,
and in agreement with the experimental data, molecular modeling studies on DRs of the most charac-
teristic ligands of the three series revealed stronger molecular interactions with D2 DR than with D1 DR,
which further supports to the encountered enhanced selectivity to D2 DR.
© 2016 Elsevier Masson SAS. All rights reserved.
1. Introduction
[5], NMDA [6] and dopamine receptors ligands [7e19].
Dopamine is a neurotransmitter that plays a key role in several
The tetrahydroisoquinoline (THIQ) structure has been identified
in a wide range of isoquinoline (IQ) alkaloids distributed in several
botanical families and marine animals [1,2]. This family of alkaloids
has been linked to important pharmacological activities, including
psychiatric and neurological disorders, and affects numerous peo-
ple worldwide. Modulation of the dopaminergic activity that acts at
dopamine receptors (DR) as potential targets for treating schizo-
phrenia or Parkinson’s disease is an area of enormous interest.
Therefore, the discovery of new dopaminergic ligands as potential
drug candidates for the treatment of these psychiatric and neuro-
logical disorders is widely required [20,21]. DRs can be classified
into two pharmacological families (D1 and D2-like) that include five
DR (D1-like: D1 and D5; D2-like: D2, D3 and D4). Therapeutically, the
D2-like DR antagonists have been seen to be effective to treat
schizophrenia (antipsychotics) and agonists in the treatment of
antitumor, antibiotic [3], b-adrenergic [4], a-glucosidase inhibition
* Corresponding author. Institute of Health Research-INCLIVA, University Clinic
Hospital of Valencia, 46010, Valencia, Spain
** Corresponding author. Departamento de Farmacología, Laboratorio de Farm-
acoquímica, Facultad de Farmacia, Universidad de Valencia, 46100, Burjassot,
Valencia, Spain.
Parkinson’s
disease
symptoms
[20,21].
Although
the
E-mail addresses: ncabedo@uv.es (N. Cabedo), dcortes@uv.es (D. Cortes).
http://dx.doi.org/10.1016/j.ejmech.2016.06.009
0223-5234/© 2016 Elsevier Masson SAS. All rights reserved.

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J. Parraga et al. / European Journal of Medicinal Chemistry 122 (2016) 27e42
28
pathophysiology of depression has been allocated to serotonin and
noradrenaline systems, nowadays the dopaminergic system also
seems to play an important role in this disorder [22,23]. Besides
dopamine re-uptake inhibitors, different selective D2-like DR ago-
nists have been found to display antidepressant-like behavioral
effects in several rodent models [24].
2. Results and discussion
2.1. Chemistry
The synthesis of THIQs (series 1 and 3) and HCPIQs (series 2)
was carried out as shown in the Schemes 1 and 2. THIQs (series 1)
and HCPIQs (series 2) (Scheme 1) were synthesized from 2-(3,4-
dimethoxyphenyl)ethylamine as starting material under
For several decades our research group has reported that THIQs,
which are closely related to the dopamine structure, have an af-
finity for DR [7e19]. In these studies, we determined the relevance
of different substituents in the IQ ring on DR affinity [11e16].
Presence of hydroxyl groups in the A-ring increases affinity to both
D1-like and D2-like DR families, whereas their blockade decreases
it. Affinity and selectivity to DR have been found to be modulated by
the presence of a secondary or tertiary amine in the THIQ structure
[9e16]. Molecular modeling studies have revealed the importance
of hydrophobic motifs since they seem to enhance their DR affinity
[17,18]. Recently, we introduced a new methodology to generate the
unusual 1,2,3,7,8,8a-hexahydrocyclopenta[ij]isoquinoline (HCPIQ)
skeleton [19] by Friedel-Crafts cyclization with Eaton’s reagent [25].
Tricyclic HCPIQ appears as an original scaffold that contains a THIQ
core connected to a cyclopentane motif and a phenyl substituent.
Preliminary results have indicated that this semi-rigid structure has
extraordinary affinity to DR [19], which encourages us to carry out
structure-activity relationship (SAR) and molecular modeling
studies. To this end, we prepared three series: (E)-1-styryl-1,2,3,4-
THIQs (series 1), 7-phenyl-1,2,3,7,8,8a-hexahydrocyclopenta[ij]-IQs
(series 2) and (E)-1-(prop-1-en-1-yl)-1,2,3,4-THIQs (series 3)
(Fig. 1) and we evaluated their potential dopaminergic activity. The
present study was designed to shed light onto the significant
groups that can affect the dopaminergic activity of the HCPIQ
skeleton. Based on previous SAR studies with dopaminergic IQ
[16,26,27], we evaluated the effect of different substituents on the
nitrogen atom (NH, N-methyl and N-allyl) and dioxygenated-IQ
Schotten-Baumann
conditions
to
generate
N-(3,4-
dimethoxyphenethyl)cinnamamide (1) [28,29]. Next, cinnama-
mide (1) was converted into the corresponding THIQ (2) by the
Bischler-Napieralski cyclodehydration reaction using POCl3 in dry
acetonitrile, followed by NaBH4 reduction [9]. Once obtained, the
(E)-6,7-dimethoxy-1-styryl-1,2,3,4-THIQ (2) was subjected to Frie-
deleCrafts cyclization conditions using Eaton’s Reagent [25]
(P2O5eCH3SO3H, 1:10, w/w) to generate the corresponding 5,6-
dimethoxy-7-phenyl-HCPIQ (3) (Scheme 1).
(E)-1-(Propenyl)-1,2,3,4-THIQs (series 3, Scheme 2) was syn-
thesized by a similar approach to that described above. The starting
material was also 2-(3,4-dimethoxyphenyl)ethylamine, but was
subjected to Schotten-Baumann conditions with crotonoyl chloride
to generate (E)-N-(3,4-dimethoxyphenethyl)but-2-enamide (4)
[15]. Amide 4 was treated with POCl3 followed by reduction with
NaBH4, to obtain (E)-6,7-dimethoxy-1-(prop-1-en-1-yl)-1,2,3,4-
THIQ (5) [9]. It seemed that the absence of the electron donating
group, such as the benzene ring in the series 3, prevented Friedel-
Crafts cyclization from take place. Therefore, the cyclopentane ring
did not form under these conditions.
After synthesizing THIQs 2 (series 1), 3 (series 2) and 5 (series 3),
methyl or allyl substituents were introduced into the nitrogen atom
to obtain tertiary amines. We obtained, on the one hand, the cor-
responding N-methyl THIQs 2b, 3b and 5b using formaldehyde and
formic acid, and subsequent NaBH4 reduction and, on the other
hand, the corresponding N-allyl-THIQs 2d, 3d and 5d using allyl
chloride under basic conditions with K2CO3.
Finally, all the THIQs (2, 2b, 2d, 3, 3b, 3d, 5, 5b and 5d) were O-
demethylated by the addition of four equivalents of BBr3 reagent for
2 h at room temperature [14] to obtain (E)-1-styryl-1,2,3,4-THIQs-
6,7-diol (2a, 2c, 2e for series 1) and 7-phenyl-HCPIQs-5,6-diol (3a,
3c, 3e for series 2) and (E)-1-(propenyl)-1,2,3,4-THIQs-6,7-diol (5a,
5c, 5e for series 3) with good yields.
functions (O-methyl and catechol), the substituent at the b-posi-
tion of the THIQ skeleton (methyl or phenyl) and the effect of the
presence or absence of the cyclopentane ring on both D1-like and
D2-like DR affinity. The toxicity of the most promising compounds
was also evaluated. The MTT ((3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide) assay and the cytofluorometric
analysis were performed to determine their impact on human cell
apoptosis and survival. Finally, in order to assess the different
molecular interactions that can stabilize or destabilize the distinct
ligand-receptor complexes in both receptor types, molecular
modeling studies that simulated the molecular interactions of the
most characteristic ligands of each series with both D2 and D1 DR
were carried out.
2.2. Binding affinities for dopamine receptors: structure-activity
relationship
The synthesized THIQs were assayed in vitro for their ability to
displace the selective radioligands of D1 and D2 DR from their
respective specific binding sites in striatal membranes. Dopamine
was used as the reference compound. All the synthetized
Fig. 1. 1-Styryl-THIQ (series 1), HCPIQ (series 2) and 1-propenyl-THIQ (series 3).

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29
Scheme 1. Synthesis of 1-styryl-THIQs 2 and 2a-e (series 1), and HCPIQs 3 and 3a-e (series 2). Reagents and Conditions: (a) cinnamoyl chloride, CH2Cl2, 5% NaOH, rt, 3h; (b) POCl3,
CH3CN, N2, reflux, 5h; (c) NaBH4; MeOH, rt, 2h; (d) Eaton’s Reagent, reflux, 15h; (e) CH2Cl2, BBr3, rt, 2h; (f) CH3OH, CH2O, HCO2H, reflux, 1h; followed by NaBH4, reflux, 1h; (g) allyl
chloride, K2CO3, CH3CN, reflux, 10h.
Scheme 2. Synthesis of (E)-1-(propenyl)-1,2,3,4-THIQs 5 and 5a-e (series 3). Reagents and Conditions: (a) crotonoyl chloride, CH2Cl2, 5% NaOH, rt, 3h; (b) POCl3, CH3CN, N2, reflux,
5h; (c) NaBH4; MeOH, rt, 2h; (d) CH2Cl2, BBr3, rt, 2h; (e) CH3OH, CH2O, HCO2H, reflux, 1h; followed by NaBH4, reflux, 1h; (f) allyl chloride, K2CO3, CH3CN, reflux, 10 h.

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J. Parraga et al. / European Journal of Medicinal Chemistry 122 (2016) 27e42
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compounds were able to displace [3H]-SCH 23390 (a selective D1-
like DR radioligand) from its specific binding sites at micromolar
substitution in compounds 5,6-dihydroxy almost did not change
the D2 DR affinity (3a vs. 3e, Fig. 3) and preserved the high affinity
within the nanomolar range; c) in series 3, N-substitution in
compounds 6,7-dihydroxy increased the affinity to both DRs (5a vs.
5e, Fig. 3). Perhaps the reason was because, besides the salt bridge
between the protonated nitrogen atom of THIQ and Asp103, and
Asp114 for D1 and D2 DR, respectively, there were additional hy-
drophobic interactions between compounds/DRs (in general, more
with the D2) which contributed to the stabilization process
(described below in molecular modeling studies). This apparently
affected series 1 and 3 slightly more.
concentrations (
0.55 M for D1 DR, the compounds of series 1 (2,2a-e) displayed Ki
values between 3.18 and 9.57 M, and two compounds of series 3,
5c and 5e had Ki ¼ 1.28 and 1.71 M, respectively. Indeed both
mM). Although dopamine showed a Ki value of
m
m
m
series 1 and 3 possessed a more flexible structure than series 2 to
accommodate the binding pocket of D1 DR. However, several
compounds from series 1, 2 and 3 were able to displace [3H]-
raclopride (a selective D2-like DR radioligand) from its specific
binding sites at nanomolar (nM) concentrations, with 10e30 fold
more affinity for D2 DR than for dopamine (see Table 1 for the
binding affinities for D1 and D2 DR).
2.2.3. Effect of the substituent at the
THIQ (series 1 and 3)
b-position of 1-substituted
2.2.1. Effect of dioxygenated substituents in the THIQ and HCPIQ
nucleus
The comparison made between series 1 and 3 with a different
substituent at the -position of THIQs (methyl or phenyl) allowed
b
In general, all the tested O-methylated THIQs, including the
HCPIQs, showed a lower affinity to D1 and D2 DR than their cor-
responding homologs with a free catecholic group (see Table 1 and
Fig. 2), probably because of the possibility of forming hydrogen
bond interactions between catecholic hydroxyls and amino acid
residues (Ser193 and Ser197) at the D2 DR binding pocket
(described below in the molecular modeling studies).
us to demonstrate that the nature of the substituent affected ligand
DR binding. In fact, the aromatic substituents at this position
increased the affinity for both D1 and D2 DR. This effect was
observed in both compounds O-methylated and 6,7-dihydroxy
(Fig. 4). It could be related to the highest electronic density and
the steric hindrance of the aromatic group vs. methyl, which could
favor hydrophobic interactions with certain amino acids (depend-
ing on D1 or D2 DR) that promote stabilization to compound/DR
complexes (mentioned below in the molecular modeling studies).
2.2.2. Effect of the N-substitution
N-substitution was performed by introducing a methyl or allyl
group. The obtained results suggested that substitution for a methyl
or allyl group did not affect affinity to DR since no significant dif-
ferences were obtained. Therefore, it seemed that the affinity and
the selectivity for DR of the secondary or tertiary amines relied on
the presence or absence of catecholic groups in each THIQ struc-
ture. Despite these findings, a different affinity to D1 or D2 DR was
found in each synthesized series: a) in series 1, N-substitution
increased the affinity for D2 DR in both compounds 6,7-dimethoxy
(2 vs. 2d) and 6,7-dihydroxy (2a vs. 2e, Fig. 3); b) in series 2, N-
2.2.4. Effect of presence or absence of a cyclopentane on THIQ
Presence of a cyclopentane ring revealed a marked increase in
selectivity to D2 DR, with D1/D2 ratio values of 2465, 1010 and 382
for 3a, 3c and 3e (Fig. 5), respectively, which sharply contrasted
with the most active and selective compounds of series 1 and 3,
with Ki D1/D2 ratios of 56 for 2c, 147 for 2e, 18 for 5c and 95 for 5e
(Table 1). The best selectivity of series 2 was explained by the ri-
gidity of this skeleton.
Table 1
Values of affinity (Ki, pKi) and ratio Ki D1/D2 determined in binding experiments to D1 and D2 DR of series 1, 2 and 3.
THIQ
Specific ligand D1 [3H]-SCH 23390
Specific ligand D2 [3H]-raclopride
Ki D1/D2
Ki (
m
M)
pKi
Ki (
mM)
pKi
Dopamine
2
0.550 0.023
4.556 0.862
9.571 0.227
6.200 1.907
3.185 0.159
7.679 1.670
6.038 2.025
33.373 6.891
71.493 16.281
23.780 4.549
13.136 2.452
18.620 4.822
6.873 1.391
45.610 5.548
57.670 7.457
18.286 1.501
1.286 0.153
37.650 4.798
1.714 0.095
6.260 0.059
5.360 0.094
5.048 0.119
5.251 0.137
5.498 0.022
5.133 0.087
5.267 0.144
4.499 0.104d
4.174 0.117e
4.640 0.084h
4.895 0.076k,s
4.758 0.109
5.179 0.085s
4.347 0.050d
4.246 0.057e
4.740 0.034i,w
5.897 0.054l,x,y
4.432 0.059m
0.560 0.009
6.176 1.717
1.422 0.316
0.436 0.081
0.057 0.013
1.166 0.172
0.041 0.010
10.977 1.933
0.029 0.009
4.188 1.348
0.013 0.002
3.514 0.654
0.018 0.007
8.855 1.154
0.458 0.081
14.696 0.609
0.072 0.023
13.816 0.634
0.018 0.002
6.837 0.028
5.244 0.123
0.98
0.74
2a
2b
2c
2d
2e
3
3a
3b
3c
3d
3e
5
5a
5b
5c
5d
5e
5.869 0.099d
6.376 0.088d
7.264 0.094e,g
5.942 0.066d,h
7.408 0.097e,m
4.972 0.071
6.73b
14.22b
55.87c
6.58b
147.3c
3.04a
7.593 0.184e,q
5.422 0.137g,r
7.890 0.083j,t
5.472 0.092n,r
7.800 0.160p,u
5.059 0.054
2465.3c
5.67b
1010.5c
5.29b
381.8c
5.15b
6.352 0.077f,v
4.833 0.018g
7.181 0.129x,y
4.860 0.019m
7.749 0.069p,x,z,a’
125.9c
1.24
17.86c
2.72a
,x,a’
5.766 0.025ꢀ
95.2c
Data were displayed as mean SEM for 3 experiments.
Values of 3-3e from previous publication [19].
ANOVA, post Newmann-Keuls multiple comparison tests:
ap < 0.05 vs D1-like dopaminergic receptor. bp < 0.01 vs D1-like dopaminergic receptor. cp < 0.001 vs D1-like dopaminergic receptor. dp < 0.001 vs 2. ep < 0.001 vs 2a. fp < 0.01
vs 2a. gp < 0.001 vs 2b. hp < 0.01 vs 2b. ip < 0.05 vs 2b. jp < 0.001 vs 2c. kp < 0.01 vs 2c. lp < 0.05 vs 2c. mp < 0.001 vs 2d. np < 0.05 vs 2d. op < 0.01 vs 2e. pp < 0.05 vs 2e.
qp < 0.001 vs 3. rp < 0.05 vs 3. sp < 0.001 vs 3a. tp < 0.001 vs 3b. up < 0.001 vs 3d. vp < 0.001 vs 5. wp < 0.01 vs 5. xp < 0.001 vs 5a. yp < 0.001 vs 5b. zp < 0.001 vs 5c. a’p < 0.001 vs
5d.
Bold is to remark the good results

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Fig. 2. Displacement curves of [3H]-SCH 23390 (D1) and [3H] raclopride (D2) specific binding by compounds 3d vs 3e and 5d vs 5e. Data are presented as mean SEM of n ¼ 3
independent experiments.
Fig. 3. Displacement curves of [3H]-SCH 23390 (D1) and [3H] raclopride (D2) specific binding by compounds 2a vs 2e (series 1), 3a vs 3e (series 2) and 5a vs 5e (series 3). Data are
presented as mean SEM of n ¼ 3 independent experiments.

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Fig. 4. Displacement curves of [3H]-SCH 23390 (D1) and [3H] raclopride (D2) specific binding by compounds 2b vs 5b and 2d vs 5d. Data are presented as mean SEM of n ¼ 3
independent experiments.
Fig. 5. Displacement curves of [3H]-SCH 23390 (D1) and [3H] raclopride (D2) specific binding by compounds 2a vs 3a. Data are presented as mean SEM of n ¼ 3 independent
experiments.
2.3. Cytotoxicity assays
2.4. Molecular modeling
After determining the affinity to the DRs of the synthesized
THIQs, the cytotoxicity of the most active compounds in D2 DR (2c,
2e, 3a, 3c, 3e, 5c and 5e) was determined at 30 mM by the MTT assay
run on human neutrophils and primary cell cultures of human
umbilical vein endothelial cells (HUVEC). The MTT assay done with
HUVEC showed that the tested compounds did not displayed any
cytotoxicity, while in neutrophils only 3e had a significant effect
(Fig. 6). It is noteworthy that 3a, the most D2 selective compound,
did not display cytotoxicity in either neutrophils or HUVEC at
To better understand selectivity to D2-like DR, we performed a
molecular modeling study with the most characteristic ligands of
each series and compounds 2c, 2e, 3c, 3e, 5c and 5e (see Schemes 1
and 2) were selected. We herein simulated the molecular in-
teractions of these ligands with both receptors D1 and D2 to assess
the different molecular interactions that can stabilize or destabilize
the distinct ligand-receptor complexes. The molecular modeling
study was conducted in four different stages. In the first step, a
docking analysis was performed by the Autodock program [30]. The
compounds of series 1 and 3 only had one chiral center, whereas
those of series 2 presented two chiral carbons. Therefore, the
different enantiomers of each series were taken into account. Our
simulations also considered the possibility of the up or down po-
sition of the substituents in the N of amine group. In a second stage,
molecular dynamics (MD) simulations were performed using the
AMBER software package. Once again, all the different enantiomers
of each series were considered in these simulations. Our results
indicated that S enantiomer was the energetically preferred form
30
mM.
To confirm the cytotoxic effect of these THIQs, we investigated
the effect on neutrophil and HUVEC apoptosis, and the survival of
these most D2 DR active compounds (2c, 2e, 3a, 3c, 3e, 5c and 5e).
For this purpose, a cytofluorimetric method was employed. It
should be noted that most of the tested THIQs did not exhibit
cytotoxicity at the assayed concentration (30
trophils or HUVEC (Fig. 7).
mM) in either neu-

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33
Fig. 6. Effect of the synthesized THIQs on human neutrophil (A) and HUVEC (B) viability. Data are presented as mean SEM of n ¼ 3 independent experiments. *p < 0.05 relative to
the vehicle group.
for series 1 and 3, while R, S was the preferred form for series 2. It is
important to note that these theoretical results agree with those
previously reported for the compounds structurally related to se-
ries 1 and 3 [14,15,18], and with those for series 2 [19]. Based on the
data obtained with the MD simulations, the most representative
structures of each complex were selected by the cluster tool
implemented in the AMBER package [31]. Each structure was
optimized by employing hybrid method ONIOM [32]. Finally, a
QTAIM [33] study was carried out for the QM layers of the different
optimized complexes.
and Ser193 at D2 DR, which are significantly stronger than those
established with Asp103 and Ser198 at D1 DR. Once these com-
pounds were complexed with D2 DR, additional hydrophobic in-
teractions (Val115, Ile183, Ile184, Trp386, Phe389, Phe390, His393,
Thr412 and Tyr416) were detected, which favored stabilization to
the compound/receptor complexes. Although similar hydrophobic
interactions (Ile104, Asn185, Leu190, Phe288 and Trp321) were
established with D1 DR, they were much weaker than those for D2
DR. Similar results were detected for the complexes established
with compounds 2c, 3c and 5c (these results are shown in
Figs. 1Se3S as Supplementary Material).
In general, the compounds herein studied displayed their
pharmacophoric portions in a closely related spatial form to that
reported for dopamine [17,34] and other dopaminergic ligands [35].
Consistently with previous experimental [36] and theoretical [37]
data, the simulations indicated the relevance of negatively
charged aspartate 114 and 103 for D2 and D1 DR ligand binding,
respectively. In line with this, ligand binding to highly conserved
aspartic acid indicated that the terminal carboxyl group could
function as an anchoring point for the molecules that possess a
protonated amino group [36e39]. In fact, all the simulated com-
pounds were docked into the receptor with the protonated amino
group close to Asp114 for D2 DR, and to Asp103 for D1 DR. After
1.5 ns of MD simulations, ligands slightly and differently moved
from the initial position, but the strong interactions with both
aspartate residues were maintained for all the complexes. These
results further supported the role of aspartate residues as
anchoring points for ligands with a protonated amino group.
Since the different interactions that stabilize and destabilize the
formation of complexes with both receptors (D1 and D2) are rela-
tively weak, and MD simulations are not accurate enough to
distinguish the different affinities observed experimentally, addi-
tional studies were carried out. To do so, hybrid calculations (QM/
MM) and a subsequent QTAIM study were performed. Fig. 8 pro-
Next the molecular graphs obtained for the different compound/
receptor complexes were analyze by QTAIM techniques and the
intermolecular interactions were evaluated. Here the results ob-
tained for compound 2e are discussed since similar approaches can
be applied for the remainding compounds (see Figs. 4Se7S, Sup-
porting Information). Fig. 12 shows the main stabilizing in-
teractions such as Asp114 and Ser193 for D2 DR and Asp103 and
Ser198 for D1 DR, whereas Fig. 13 illustrates the most relevant hy-
drophobic interactions that contribute to the stabilization process.
Since the active site at D1 DR was greater than at D2 DR [40,41], it is
likely that compound interactions were favored to D2 DR vs. D1
(Fig. 12). Regarding the complexes shown in Fig. 13, more hydro-
phobic interactions were found with D2 DR than with D1 DR (nine
vs. five residues, respectively). Furthermore, the compound/D2 DR
interactions at the active site were stronger than those at D1 DR.
Similar data were obtained for compounds 3e and 5e (Figs. 5S and
7S, Supplementary Material). Taken together, the molecular
modeling study clearly indicated that the three series of com-
pounds herein evaluated established stronger molecular in-
teractions with D2 DR than with D1 which is consistent with the
presented experimental data and partly explained the selectivity
detected to the former DR.
vides the values obtained for
r for the six selected compounds,
which formed complexes with the two receptor types. Clearly all
the compounds that established complexes with D2 DR had higher
3. Conclusions
r
values than those observed with D1 DR. These theoretical results
In summary, we synthesized three series of IQs: (E)-1-styryl-
1,2,3,4-THIQs (series 1), 7-phenyl-1,2,3,7,8,8a-HCPIQs (series 2) and
(E)-1-(prop-1-en-1-yl)-1,2,3,4-THIQs (series 3) with different sub-
stituents on the nitrogen atom (methyl or allyl), the dioxygenated
fully coincided with the experimental data and could explain, at
least in part, the higher affinities displayed by these three series of
compounds for D2 DR. To better understand this differential
behavior at the molecular level, an analysis of the
residue was performed to discriminate their contribution to com-
plex interactions. Figs. 9e11 show the values of obtained for the
different molecular interactions of the complexes of compounds 2e,
3e and 5e with both receptors D1 and D2. These figures illustrate
clear molecular interactions between these compounds and Asp114
r value for each
function (methoxyl or catechol groups), the substituent at the b-
position of the THIQ skeleton and presence of the cyclopentane
motif. We evaluated the SAR for their potential dopaminergic af-
finity (D1 and D2-like DR). Presence of hydroxyls groups into the IQ-
ring resulted in increased affinity to DRs, while their blockade was
detrimental. Regarding N-substitution, the tertiary amines
r

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34
Fig. 7. Percentage of apoptotic (A) and survival neutrophils (B) and apoptotic (C) and survival HUVEC (D) after 24 h incubation with THIQs. Apoptotic cells were quantified as the
percentage of total population of annexin Vþ, PIꢁ cells, late apoptotic, and/or necrotic cells as annexin Vþ and PIþ, and viable nonapoptotic cells as annexin Vꢁ and PIꢁ.
Representative flow cytometry panels showing the effects of vehicle, 2c, 2e, 3a, 3c, 3e, 5c and 5e on neutrophil apoptosis/survival have been included. The columns are the
mean SEM of n ¼ 3 independent experiments. *p < 0.05 relative to the vehicle group.
improved affinity to DR compared to their corresponding second-
ary amines. Finally, of our series, the phenyl group at the position
of 1-substituted THIQs in series 1 increased the affinity to DRs
compared with the 1-alkyl-THIQs (series 3). It was noteworthy that
the presence of the cyclopentane motif (tricyclic HCPIQs, series 2)
generated the most active compounds, which displayed
b

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J. Parraga et al. / European Journal of Medicinal Chemistry 122 (2016) 27e42
35
molecular modeling study results were consistent with the exper-
imental data and provided further details about the molecular in-
teractions that stabilized the compound/DR complexes.
4. Experimental section
4.1. General instrumentation
EIMS and FAB mass were recorded on a VG Auto Spec Fisons
spectrometer instruments (Fisons, Manchester, United Kingdom).
1H NMR and 13C NMR spectra were recorded with CDCl3 or Meth-
anol-d4 as a solvent on a Bruker AC-300, AC-400 or AC-500. Mul-
tiplicities of 13C NMR resonances were assigned by DEPT
experiments. COSY, HSQC and HMBC correlations were recorded at
400 MHz and 500 MHz (Bruker AC-400ꢀ AC-500). The assignments
of all compounds were made by COSY, DEPT, HSQC and HMBC. All
the reactions were monitored by analytical TLC by silica gel 60 F254
P
Fig. 8. Sum of the values of charge density ( r(r)) at the bond critical points obtained
for the different compounds. The interactions are shown in orange for D1 DR and in
blue for D2 DR. (For interpretation of the references to colour in this figure legend, the
reader is referred to the web version of this article.)
(Merck 5554). Residues were purified by silica gel 60 (40e63 mm,
astonishing selectivity to D2-DR. None of the most active THIQs in
D2 DR (2c, 2e, 3a, 3c, 3e, 5c, and 5e) displayed any relevant cyto-
toxicity on both human neutrophils and HUVEC. Finally, the
Merck 9385) column chromatography. Quoted yields are of purified
material. The HCl salts of the synthesized compounds were pre-
pared from the corresponding base with 5% HCl in MeOH.
P
Fig. 9. Sum of the values of charge density (
interactions have been considered.
r(r)) at the bond critical points between compound 2e and the different residues of D1 DR (a) and D2 DR (b). Only the inter-molecular
P
Fig. 10. Sum of the values of charge density (
r(r)) at the bond critical points between compound 3e and the residues of D1 DR (a) and D2 DR (b).

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36
J. Parraga et al. / European Journal of Medicinal Chemistry 122 (2016) 27e42
P
Fig. 11. Sum of the values of charge density (
r(r)) at the bond critical points between compound 5e and the residues of D1 DR (a) and D2 DR (b).
Fig. 12. Molecular graph of the non-covalent interactions between compound 2e and main residues. a) Asp103, Ser198 and Ser202 of D1 DR. b) Asp114, Ser193 and Ser197 of D2 DR.
The elements of the topology of the electron density are shown: pink spheres represent the bond paths connecting the nuclei and the critical bond points are represented as red
spheres. The triangle in dot lines shows the interatomic distances obtained for the main residues. (For interpretation of the references to colour in this figure legend, the reader is
referred to the web version of this article.)
Fig. 13. Molecular graph of the molecular interactions between compound 2e and hydrophobic residues. a)Ile104, Asn185, Leu190, Phe288 and Trp321 of D1 DR. b) Val115, Ile183,
Ile184, Trp386, Phe389, Phe390, His393, Thr412 and Tyr416 of D2 DR.

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37
4.2. Chemistry
pressure. The aqueous phase was extracted with CH2Cl2 (3 ꢂ 15 ml),
and combined organic layers were dried over Na2SO4 and evapo-
rated to dryness. The crude product was purified by silica gel col-
umn chromatography (CH2Cl2/MeOH, 95:5) to furnish the
compound 2 (383 mg, 81%) as a yellow oil. 1H NMR (500 MHz,
4.2.1. General procedure for the synthesis of acetamides (1 and 4)
under Schotten-Baumann conditions
Formation of acetamides was carried out under Schotten-
Baumann conditions using 2-(3,4-dimethoxyphenyl)ethanamine
and the corresponding acid chloride.
CDCl3):
d
¼ 7.40 (d, J ¼ 7.6 Hz, 2H, CH-20 and CH-60), 7.29 (t,
J ¼ 7.6 Hz, 2H, CH-30 and CH-50), 7.24 (t, J ¼ 7.6 Hz, 1H, CH-40), 6.62
(s, 1H, CH-8), 6.60 (s, 1H, CH-5), 6.58 (d, J ¼ 14.8 Hz, 1H, CH-
b), 6.35
4.2.1.1. N-(3,4-dimethoxyphenethyl)cinnamamide (1). An amount of
458 mg of cinnamoyl chloride (2.76 mmol) dissolved in CH2Cl2
(1 mL) was added dropwise at 0 ꢀC to a solution of 2-(3,4-
dimethoxyphenyl)ethylamine (500 mg, 2.76 mmol) in CH2Cl2
(20 mL) and 5% aqueous NaOH (4.4 mL) with stirring at room
temperature for 3 h. Then, the mixture was stirred with 2.5%
aqueous HCl. The organic solution was washed with brine
(2 ꢂ 10 ml) and H2O (2 ꢂ 10 ml), dried over anhydrous Na2SO4 and
evaporated to dryness. The residue obtained was purified by silica
gel column chromatography (Hexane/CH2Cl2/EtOAc, 20:70:10) to
afford the acetamide 1 (781 mg, 91%) as a white oil. 1H NMR
(dd, J ¼ 14.8, 8.0 Hz, 1H, CH-
a
), 4.60 (d, J ¼ 8.0 Hz, 1H, CH-1), 3.84 (s,
), 3.07 (m, 1H,
b), 2.85 (m, 1H, CH-4a), 2.62 (m, 1H, CH-4b
); 13C NMR
3H, OCH3-7), 3.78 (s, 3H, OCH3-6), 3.28 (m, 1H, CH-3
a
CH-3
(125 MHz, CDCl3):
(CH- ), 131.9 (CH-a
), 128.5 (C-8a), 128.4 (CH-20 and CH-60), 127.6
d
¼ 147.7 (C-7), 147.1 (C-6), 136.8 (C-10), 132.3
b
(CH-30 and CH-50), 126.9 (C-4a), 126.5 (CH-40), 111.7 (CH-8), 110.4
(CH-5), 59.1 (CH-1), 55.9 (OCH3-6), 55.8 (OCH3-7), 41.5 (CH2-3),
29.0 (CH2-4); MS (FAB) m/z (%): 296 [MþH]þ, 279 (100), 200 (20),
151 (30); HRESIMS m/z 296.1644 [MþH]þ (296.1651, calc for
C19H22NO2).
(500 MHz, CDCl3):
d
¼ 7.62 (d, J ¼ 15.6 Hz, 1H, CH-
b
), 7.44 (m, 2H,
4.2.2.2. (E)-6,7-Dimethoxy-1-(prop-1-en-1-yl)-1,2,3,4-
tetrahydroisoquinoline (5).
CH-20 and CH-60), 7.31 (m, 3H, CH-30, CH-40 and CH-50), 6.79 (d,
J ¼ 7.9 Hz,1H, CH-8), 6.74 (d, J ¼ 7.9 Hz,1H, CH-9), 6.73 (s,1H, CH-5),
(E)-N-(3,4-dimethoxyphenethyl)but-2-enamide
(500
mg,
6.37 (d, J ¼ 15.6 Hz, 1H, CH-
a
), 6.02 (m, 1H, NH), 3.84 (s, 3H, OCH3-
2.00 mmol) was subjected to similar conditions to those described
above to obtain the THIQ 2. The residue was purified by silica gel
column chromatography (CH2Cl2/MeOH, 95:5) to afford the com-
pound 5 (410 mg, 88%) as a yellow oil. 1H NMR (500 MHz, CDCl3):
6), 3.83 (s, 3H, OCH3-7), 3.62 (q, J ¼ 7.0 Hz, 2H, CH2-2), 2.83 (t,
J ¼ 7.0 Hz, 2H, CH2-3); 13C NMR (125 MHz, CDCl3):
d
¼ 165.9 (CO),
148.9 (C-6), 147.6 (C-7), 140.8 (CH-b
), 134.7 (C-10), 131.3 (C-4), 129.5
(CH-40), 128.9 (CH-30 and CH-50), 127.6 (CH-20 and CH-60), 120.7
(CH-9), 120.6 (CH- ), 111.9 (CH-5), 111.3 (CH-8), 55.8 (OCH3-6), 55.7
d
¼ 6.56 (s, 1H, CH-5), 6.54 (s, 1H, CH-8), 5.73 (dd, J ¼ 15.2, 7.7 Hz,
1H, CH- ), 4.45 (d, J ¼ 7.9 Hz,1H,
), 5.62 (dd, J ¼ 15.2, 7.9 Hz,1H, CH-
CH-1), 3.83 (s, 3H, OCH3-7), 3.80 (s, 3H, OCH3-6), 3.25 (m, 1H, CH-
), 3.06 (m, 1H, CH-3 ), 2.84 (m, 1H, CH-4 ), 2.72 (m, 1H, CH-4 ),
1.74 (dd, J ¼ 7.7, 1.4 Hz, 3H, CH3-
); 13C NMR (125 MHz, CDCl3):
¼ 147.8 (C-7), 147.2 (C-6), 132.2 (CH- ), 129.7 (CH- ), 128.1 (C-8a),
126.3 (C-4a), 111.5 (CH-8), 110.5 (CH-5), 58.6 (CH-1), 55.9 (OCH3-6),
a
b
a
(OCH3-7), 40.9 (CH2-2), 35.1 (CH2-3); MS (FAB) m/z (%): 312
[MþH]þ, 295 (20), 165 (20), 131 (100).
3a
b
a
b
g
4.2.1.2. (E)-N-(3,4-dimethoxyphenethyl)but-2-enamide
(4).
d
a
b
2-(3,4-Dimethoxyphenyl)-ethylamine (500 mg, 2.76 mmol) and
crotonoyl chloride (0.26 mL, 2.76 mmol) were subjected to similar
conditions to those described above to obtain the compound 1. The
residue was purified by silica gel column chromatography (Hexane/
CH2Cl2/EtOAc, 20:70:10) to obtain the acetamide 4 (642 mg, 93%) as
55.8 (OCH3-7), 41.0 (CH2-3), 28.3 (CH2-4), 17.7 (CH3-g); MS (FAB) m/
z (%): 234 [MþH]þ, 217 (100), 189 (20); HRESIMS m/z 234.1486
[MþH]þ (234.1494, calc for C14H20NO2).
a white oil. 1H NMR (500 MHz, CDCl3):
d
¼ 6.82 (m, 1H, CH-
(m, 1H, CH-9), 6.71 (m, 1H, CH-8), 6.68 (m, 1H, CH-5), 5.70 (d,
J ¼ 15.1, 1H, CH- ), 5.52 (m, 1H, NH), 3.84 (s, 3H, OCH3-6), 3.83 (s,
3H, OCH3-7), 3.51 (q, J ¼ 7.0 Hz, 2H, CH2-2), 2.75 (t, J ¼ 7.0 Hz, 2H,
CH2-3), 1.80 (dd, J ¼ 6.1, 1.7 Hz, 3H, CH3-
); 13C NMR (125 MHz,
CDCl3): ), 131.4
¼ 165.9 (CO), 148.9 (C-6), 147.6 (C-7), 139.7 (CH-
(C-4), 125.0 (CH- ), 120.6 (CH-9), 111.9 (CH-5), 111.3 (CH-8), 55.8
b
), 6.77
4.2.3. General procedure for FriedeleCrafts cyclization
4.2.3.1. 5,6-Dimethoxy-7-phenyl-1,2,3,7,8,8a-hexahydrocyclopenta
[ij]isoquinoline (3). 5 mL of Eaton’s Reagent was used to dissolve an
amount of 500 mg of (E)-6,7-dimethoxy-1-styryl-1,2,3,4-
tetrahydroisoquinoline (2) (1.69 mmol) at room temperature and
the reaction mixture was stirred at 45 ꢀC overnight. Then, 5%
aqueous NaOH was added and the mixture was then extracted with
CH2Cl2 (3 ꢂ 15 mL). Combined CH2Cl2 layers were dried over
Na2SO4 and the solvent was evaporated in vacuo obtaining reddish
oil. The residue obtained was purified by silica gel column chro-
a
g
d
b
a
(OCH3-6), 55.7 (OCH3-7), 40.6 (CH2-2), 35.4 (CH2-3), 17.6 (CH3-g);
MS (FAB) m/z (%): 272 [MþNa]þ, 254 (50), 135 (20).
4.2.2. General procedure for the synthesis of 1,2,3,4-
tetrahydroisoquinoleines (2 and 5) by Bischler-Napieralski
cyclization
matography
hexahydrocyclopenta-IQ 3 (339 mg, 68%) as a brown oil. 1H NMR
and HRESIMS [19]; 13C NMR (125 MHz, CDCl3):
(CH2Cl2/MeOH,
95:5)
to
afford
the
d
¼ 152.7 (C-6a),
4.2.2.1. (E)-6,7-Dimethoxy-1-styryl-1,2,3,4-tetrahydroisoquinoline
(2). The corresponding acetamide 1 (500 mg, 1.61 mmol) was
added in dry acetonitrile (40 mL) to a 100 mL three-neck round-
bottom flask at 0 ꢀC under N2, and treated with POCl3 (1.15 mL,
11.2 mmol). The mixture was stirred and refluxed under N2 for 5 h
and then cooled to room temperature. Acetonitrile was evaporated
under reduced pressure and the reaction mixture was slowly
poured into a mixture of crushed ice. The solid residue was
neutralized with 10% aqueous NaOH to obtain a suspension
(pH z 8e9) which was then extracted with CH2Cl2 (3 ꢂ 15 mL).
Combined CH2Cl2 layers were dried over Na2SO4 and the solvent
was evaporated in vacuo obtaining reddish oil. The residue was
dissolved in MeOH (10 mL), and then it was treated with NaBH4
(189 mg, 5 mmol). The reaction mixture was stirred for 2 h. Next,
water (15 mL) was added and methanol evaporated under reduced
143.9 (C-6), 143.8 (C-5), 135.6 (C-10), 134.7 (C-3a1), 128.3 (CH-30 and
CH-50), 127.3 (CH-20 and CH-60), 126.8 (C-3a), 126.0 (CH-40), 111.0
(CH-4), 60.3 (OCH3-5), 57.5 (CH-8a), 56.1 (OCH3-6), 46.3 (CH-7),
44.6 (CH2-2), 44.4 (CH2-8), 25.8 (CH2-3); MS (FAB) m/z (%): 296
[MþH]þ, 279 (100), 267 (20), 192 (30), 151 (30).
4.2.4. General procedure for N-methylation
4.2.4.1. (E)-6,7-Dimethoxy-2-methyl-1-styryl-1,2,3,4-
tetrahydroisoquinoline (2b). To a stirred solution of 2 (100 mg,
0.34 mmol) in MeOH (15 mL), 37% formaldehyde (4.7 mL) and one
drop of formic acid were added. The mixture was refluxed for 1 h,
cooled to room temperature, treated with NaBH4 (120 mg,
3.4 mmol), and refluxed an additional hour. The reaction mixture
was cooled to room temperature and the solvent was removed
under reduced pressure. Then, water (3 mL) was added, and the

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J. Parraga et al. / European Journal of Medicinal Chemistry 122 (2016) 27e42
38
aqueous mixture was extracted with CH2Cl2 (3 ꢂ 15 mL). Combined
organic layers were dried over Na2SO4 and concentrated under
reduced pressure to give the crude residue which was further pu-
rified by silica gel column chromatography (CH2Cl2/MeOH, 98:2) to
afford the compound 2b (83 mg, 79%). 1H NMR (500 MHz, CDCl3):
the solvent removed under reduced pressure. Then, water (3 mL)
was added and the aqueous mixture was extracted with CH2Cl2
(3 ꢂ 15 mL). Combined organic layers were dried over Na2SO4 and
concentrated under reduced pressure to give the crude residue
which was further purified by silica gel column chromatography
(CH2Cl2/MeOH 99:1) to afford the THIQ 2d (97 mg, 87%) as a white
d
¼ 7.42 (d, J ¼ 7.5 Hz, 2H, CH-20 and CH-60), 7.31 (t, J ¼ 7.5 Hz, 2H,
CH-30 and CH-50), 7.25 (m, 1H, CH-40), 6.62 (s, 1H, CH-8), 6.61 (s, 1H,
oil. 1H NMR (500 MHz, CDCl3):
d
¼ 7.33 (d, J ¼ 7.2 Hz, 2H, CH-20 and
CH-5), 6.59 (d, J ¼ 15.8 Hz, 1H, CH-
CH- ), 3.85 (s, 3H, OCH3-7), 3.81 (m, 1H, CH-1), 3.74 (s, 3H, OCH3-6),
3.09 (m, 1H, CH-3 ), 3.05 (m, 1H, CH-4 ), 2.74 (m, 1H, CH-4 ), 2.57
(m, 1H, CH-3
b
), 6.14 (dd, J ¼ 15.8, 8.9 Hz, 1H,
CH-60), 7.23 (t, J ¼ 7.2 Hz, 2H, CH-30 and CH-50), 7.15 (m, 1H, CH-40),
a
6.54 (s, 1H, CH-8), 6.52 (s, 1H, CH-5), 6.48 (d, J ¼ 15.8 Hz, 1H, CH-
b),
a
a
b
6.14 (dd, J ¼ 15.8, 8.6 Hz, 1H, CH-
a
), 5.86 (m, 1H, CH-200), 5.14 (d,
b
), 2.46 (s, 3H, NCH3); 13C NMR (125 MHz, CDCl3):
J ¼ 17.2 Hz, 1H, CH-300
a
), 5.10 (d, J ¼ 10.2 Hz, 1H, CH-300
b), 4.09 (d,
d
¼ 147.5 (C-7), 147.0 (C-6), 136.6 (C-10), 133.1 (CH-
b
), 131.3 (CH-
a
),
J ¼ 8.6 Hz, 1H, CH-1), 3.81 (s, 3H, OCH3-7), 3.67 (s, 3H, OCH3-6), 3.41
128.3 (CH-30 and CH-50), 128.1 (C-8a), 127.4 (CH-40), 126.4 (C-4a),
126.3 (CH-20 and CH-60), 111.1 (CH-8), 110.7 (CH-5), 68.4 (CH-1),
55.7 (OCH3-6), 55.0 (OCH3-7), 51.2 (CH2-3), 44.0 (NCH3), 28.7 (CH2-
4); MS (FAB) m/z (%): 310 [MþH]þ, 279 (90), 206 (50), 201 (50), 151
(100); HRESIMS m/z 310.1793 [MþH]þ (310.1807, calc for
(dd, J ¼ 13.9, 5.4 Hz, 1H, CH-100
a
), 3.05 (m, 1H, CH-3
), 2.72 (m, 1H, CH-
¼ 147.7 (C-
), 131.1 (CH- ),
a), 3.00 (dd,
J ¼ 13.9, 7.5 Hz, 1H, CH-100
b), 2.80 (m, 1H, CH-4a
4b), 2.54 (m, 1H, CH-3b d
); 13C NMR (125 MHz, CDCl3):
7), 147.1 (C-6), 136.8 (C-10), 135.6 (CH-200), 133.1 (CH-
b
a
128.5 (CH-20 and CH-60), 128.3 (C-8a), 127.5 (CH-40), 126.7 (C-4a),
126.4 (CH-30 and CH-50), 117.7 (CH2-300), 111.3 (CH-8), 111.1 (CH-5),
65.4 (CH-1), 57.7 (CH2-100), 55.9 (OCH3-6), 55.8 (OCH3-7), 46.3 (CH2-
3), 28.4 (CH2-4); MS (FAB) m/z (%): 336 [MþH]þ, 281 (20), 189 (15),
151 (100); HRESIMS m/z 336.1953 [MþH]þ (336.1964, calc for
C
20H24NO2).
4.2.4.2. 5,6-dimethoxy-1-methyl-7-phenyl-1,2,3,7,8,8a-hexahy-
drocyclopenta-[ij]isoquinoline (3b).
5,6-Dimethoxy-7-phenyl-1,2,3,7,8,8a-hexahydrocyclopenta-[ij]-
isoquinoline (3) (100 mg, 0.34 mmol) was subjected to similar
conditions to those described above to obtain the compound 2b.
The residue was purified by silica gel column chromatography
(CH2Cl2/MeOH, 95:5) to afford the THIQ 3b (86 mg, 82%) as a white
C22H26NO2).
4.2.5.2. 1-Allyl-5,6-dimethoxy-7-phenyl-1,2,3,7,8,8a-hexahy-
drocyclopenta[ij]-isoquinoline (3d).
5,6-Dimethoxy-7-phenyl-1,2,3,7,8,8a-hexahydrocyclopenta-[ij]-
isoquinoline (3) (100 mg, 0.34 mmol) was subjected to similar
conditions to those described above to obtain the THIQ 2d. The
residue was purified by silica gel column chromatography (CH2Cl2/
MeOH, 99:1) to afford compound 3d (101 mg, 89%) as a brown oil
oil [19]. 1H NMR (500 MHz, CDCl3):
d
¼ 7.18 (m, 2H, CH-30 and CH-
50), 7.09 (m, 2H, CH-20 and CH-60), 7.08 (m, 1H, CH-40), 6.53 (s, 1H,
CH-4), 4.51 (d, J ¼ 8.4 Hz,1H, CH-7), 3.74 (s, 3H, OCH3-5), 3.48 (s, 3H,
OCH3-6), 3.33 (dd, J ¼ 9.8, 6.6 Hz, 1H, CH-8a), 3.05 (dd, J ¼ 12.0,
6.6 Hz, 1H, CH-2
(m, 1H, CH-2
(125 MHz, CDCl3):
a
), 2.89 (m, 1H, CH-3
a
), 2.72 (m, 1H, CH-3
b
), 2.42
[19]. 1H NMR (500 MHz, CDCl3):
d
¼ 7.18 (m, 2H, CH-30 and CH-50),
b
), 2.31 (m, 1H, CH2-8), 2.26 (s, 1H, NCH3); 13C NMR
7.10 (m, 2H, CH-20 and CH-60), 7.08 (m,1H, CH-40), 6.53 (s, 1H, CH-4),
d
¼ 152.7 (C-6a), 144.1 (C-6), 144.0 (C-5), 135.1
5.82 (m, 1H, CH-200), 5.11 (d, J ¼ 17.2 Hz, 1H, CH-300
a), 5.07 (d,
(C-10), 128.4 (CH-30 and CH-50), 128.1 (C-3a1), 127.5 (CH-20 and CH-
60), 127.4 (C-3a), 126.1 (CH-40), 110.6 (CH-4), 65.7 (CH-8a), 60.3
(OCH3-6), 56.1 (OCH3-5), 54.8 (CH2-2), 46.4 (CH-7), 43.6 (CH2-8),
43.2 (NCH3), 27.2 (CH2-3); MS (FAB) m/z (%): 310 [MþH]þ, 291 (45),
267 (100), 206 (45); HRESIMS m/z 310.1797 [MþH]þ (310.1807, calc
for C20H24NO2).
J ¼ 10.1 Hz, 1H, CH-300
), 4.51 (d, J ¼ 8.5 Hz, 1H, CH-7), 3.74 (s, 3H,
b
OCH3-5), 3.50 (dd, J ¼ 10.2, 6.3 Hz, 1H, CH-8a), 3.47 (s, 3H, OCH3-6),
3.36 (dd, J ¼ 13.6, 3.1 Hz, 1H, CH-100
a
), 3.18 (dd, J ¼ 11.8, 6.5 Hz, 1H,
), 2.65 (dd, J ¼ 13.6,
b), 2.36 (m, 1H, CH-2b
), 2.26 (m, 2H, CH2-8); 13C
CH-2a), 2.82 (m, 1H, CH-3a), 2.74 (m, 1H, CH-3b
8.2 Hz, 1H, CH-100
NMR (125 MHz, CDCl3):
d
¼ 152.7 (C-6a), 144.2 (C-6), 144.0 (C-5),
135.4 (C-10), 135.3 (CH-200), 128.4 (CH-30 and CH-50), 128.1 (C-3a1),
127.5 (CH-20 and CH-60), 127.1 (C-3a), 126.0 (CH-40), 117.8 (CH2-300),
110.7 (CH-4), 64.4 (CH-8a), 60.3 (OCH3-6), 58.5 (CH2-100), 56.1
(OCH3-5), 51.0 (CH2-2), 46.4 (CH-7), 43.8 (CH2-8), 27.5 (CH2-3); MS
(FAB) m/z (%): 336 [MþH]þ, 279 (100), 265 (10); HRESIMS m/z
336.1956 [MþH]þ (336.1964, calc for C22H26NO2).
4.2.4.3. (E)-6,7-Dimethoxy-2-methyl-1-(prop-1-en-1-yl)-1,2,3,4-
tetrahydroisoquinoline (5b).
(E)-6,7-Dimethoxy-1-(prop-1-en-1-yl)-1,2,3,4-
tetrahydroisoquinoline (5) (100 mg, 0.43 mmol) was subjected to
similar conditions to those described above to obtain the THIQ 2b
and 3b. The residue was purified by silica gel column chromatog-
raphy (CH2Cl2/MeOH, 95:5) to afford the compound 5b (91 mg,
4.2.5.3. (E)-6,7-Dimethoxy-2-methyl-1-(prop-1-en-1-yl)-1,2,3,4-
tetrahydroisoquinoline (5d).
(E)-6,7-Dimethoxy-1-(prop-1-en-1-yl)-1,2,3,4-
tetrahydroisoquinoline (5) (100 mg, 0.43 mmol) was subjected to
similar conditions to those described above to obtain the THIQ 2d
and 3d. The residue was purified by silica gel column chromatog-
raphy (CH2Cl2/MeOH, 99:1) to afford the compound 5d (100 mg,
86%) as a white oil. 1H NMR (500 MHz, CDCl3):
d
¼ 6.56 (s, 1H, CH-
5), 6.55 (s, 1H, CH-8), 5.68 (dt, J ¼ 15.1, 6.4 Hz, 1H, CH- ), 5.35 (dd,
J ¼ 15.1, 8.8 Hz,1H, CH- ), 3.82 (s, 3H, OCH3-7), 3.79 (s, 3H, OCH3-6),
3.59 (d, J ¼ 8.8 Hz, 1H, CH-1), 2.98 (m, 1H, CH-3 ), 2.91 (m, 1H, CH-
), 2.66 (m, 1H, CH-4 ), 2.48 (m, 1H, CH-3 ), 2.38 (s, 3H, NCH3),
1.76 (dd, J ¼ 6.4, 1.6 Hz, 3H, CH3-
); 13C NMR 7(125 MHz, CDCl3):
¼ 147.5 (C-7), 146.9 (C-6), 132.7 (CH- ), 129.2 (CH- ), 128.8 (C-8a),
126.3 (C-4a), 111.1 (CH-8), 110.8 (CH-5), 68.3 (CH-1), 55.8 (OCH3-6),
b
a
a
4
a
b
b
g
d
a
b
86%) as a yellow oil. 1H NMR (500 MHz, CDCl3):
d
¼ 6.56 (s, 1H, CH-
5), 6.54 (s, 1H, CH-8), 5.88 (m, 1H, CH-200), 5.64 (dt, J ¼ 15.3, 6.3 Hz,
55.7 (OCH3-7), 51.2 (CH2-3), 43.9 (NCH3), 28.7 (CH2-4), 17.6 (CH3-
g);
1H, CH-
b
), 5.44 (dd, J ¼ 15.3, 8.6 Hz, 1H, CH- ), 5.20 (d, J ¼ 17.1 Hz,
a
MS (FAB) m/z (%): 270 [MþNa]þ, 213 (10); HRESIMS m/z 248.1647
1H, CH-300
a
), 5.14 (dd, J ¼ 17.1, 10.3 Hz, 1H, CH-300
b), 3.92 (d,
[MþH]þ (248.1651, calc for C15H22NO2).
J ¼ 8.6 Hz,1H, CH-1), 3.82 (s, 3H, OCH3-7), 3.79 (s, 3H, OCH3-6), 3.42
(m, 1H, CH-100
CH-100
), 2.78 (m, 1H, CH-4
a
), 3.08 (m, 1H, CH-3
a
), 3.03 (dd, J ¼ 13.8, 7.4 Hz, 1H,
), 2.53 (m, 1H, CH-
); 13C NMR (125 MHz, CDCl3):
4.2.5. General procedure for N-allylation
b
a), 2.70 (m, 1H, CH-4b
4 . 2 . 5 .1. ( E ) - 2 - A l l yl - 6 , 7 - d i m e t h o x y - 1 - s t y r yl - 1, 2 , 3 , 4 -
tetrahydroisoquinoline (2d). To a stirred solution of 2 (100 mg,
0.34 mmol) in CH3CN (10 mL), K2CO3 (300 mg, 2.17 mmol) and allyl
chloride (0.1 mL, 1.23 mmol) were added. The mixture was refluxed
for 10 h. The reaction mixture was cooled to room temperature and
3
b
), 1.75 (dd, J ¼ 6.3, 1.4 Hz, 3H, CH3-
g
d
¼ 147.5 (C-7), 146.9 (C-6), 135.7 (CH-200), 132.3 (CH-
a), 129.1 (CH-
b
), 128.9 (C-8a), 126.6 (C-4a), 117.5 (CH2-300), 111.2 (CH-8), 111.1 (CH-
5), 65.2 (CH-1), 57.5 (CH2-100), 56.0 (OCH3-6), 55.8 (OCH3-7), 46.1
(CH2-3), 28.1 (CH2-4), 17.7 (CH3-
g
); MS (FAB) m/z (%): 274 [MþH]þ,

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J. Parraga et al. / European Journal of Medicinal Chemistry 122 (2016) 27e42
39
219 (100), 192 (20), 177 (20); HRESIMS m/z 274.1806 [MþH]þ
(100); HRESIMS m/z 308.1637 [MþH]þ (308.1651, calc for
(274.1807, calc for C17H24NO2).
C20H22NO2).
4.2.6. General procedure for O-Demethylation
4.2.6.1. (E)-1-Styryl-1,2,3,4-tetrahydroisoquinoline-6,7-diol
4.2.6.4. 7-Phenyl-1,2,3,7,8,8a-hexahydrocyclopenta[ij]isoquinoline-
5,6-diol (3a).
(2a).
A solution of the appropriate THIQ 2 (100 mg, 0.34 mmol) in dry
CH2Cl2 (10 mL) was cooled to ꢁ78 ꢀC. To this solution, BBr3 (0.16 mL,
1.19 mmol) was added dropwise. After 15 min at ꢁ78 ꢀC, the re-
action mixture was cooled to room temperature and stirred for 2 h.
The reaction was finished by the addition of MeOH (1.5 mL) drop-
wise and the mixture was stirred for another 30 min. The solvent
was concentrated to dryness. The residue was purified by silica gel
column chromatography (CH2Cl2/MeOH, 90:10) to afford the
5,6-Dimethoxy-7-phenyl-1,2,3,7,8,8a-hexahydrocyclopenta[ij]iso-
quinoline (3) (100 mg, 0.34 mmol) was subjected to similar con-
ditions to those described above to obtain the THIQ 2a, 2c and 2e.
The residue was purified by silica gel column chromatography
(CH2Cl2/MeOH, 90:10) to afford the compound 3a (77 mg, 88%) as a
brown oil. 1H NMR and HRESIMS [19]; 13C NMR (125 MHz, CD3OD):
d
¼ 147.7 (C-6a), 143.5 (C-6), 141.8 (C-5), 129.5 (C-10), 129.2 (CH-30
and CH50), 128.9 (C-3a1), 128.2 (CH-20 and CH-60), 127.2 (CH-40),
121.6 (C-3a), 114.2 (CH-4), 58.1 (CH-8a), 46.4 (CH-7), 44.4 (CH2-2),
42.5 (CH2-8), 23.6 (CH2-3); MS (FAB) m/z (%): 268 [MþH]þ, 251
(100), 239 (80), 190 (30), 154 (50).
compound 2a (83 mg, 92%). 1H NMR (500 MHz, CD3OD):
d
¼ 7.40 (d,
J ¼ 7.7 Hz, 2H, CH-20 and CH-60), 7.28 (t, J ¼ 7.7 Hz, 2H, CH-30 and
CH-50), 7.18 (t, J ¼ 7.7 Hz, 1H, CH-40), 6.55 (d, J ¼ 15.8 Hz, 1H, CH-
b),
6.52 (s, 1H, CH-8), 6.51 (s, 1H, CH-5), 6.30 (dd, J ¼ 15.8, 7.9 Hz, 1H,
CH-
1H, CH-3
(125 MHz, CD3OD):
(CH- ), 132.4 (CH-b
), 129.4 (CH-30 and CH-50), 128.5 (C-8a), 128.2
a
), 4.47 (d, J ¼ 7.9 Hz, 1H, CH-1), 3.17 (m, 1H, CH-3
a
), 2.93 (m,
4.2.6.5. 1-Methyl-7-phenyl-1,2,3,7,8,8a-hexahydrocyclopenta[ij]iso-
quinoline-5,6-diol (3c).
b
), 2.69 (m, 1H, CH-4a), 2.54 (m, 1H, CH-4b
); 13C NMR
d
¼ 144.7 (C-7), 144.0 (C-6), 138.1 (C-10), 133.1
5,6-Dimethoxy-1-methyl-7-phenyl-1,2,3,7,8,8a-hexahy-
drocyclopenta[ij]isoquinoline (3d) (100 mg, 0.32 mmol) was sub-
jected to similar conditions to those described above to obtain THIQ
2a, 2c, 2e and 3a. The residue was purified by silica gel column
chromatography (CH2Cl2/MeOH, 90:10) to afford compound 3c
(84 mg, 94%) as a green oil [19]. 1H NMR (500 MHz, CD3OD):
a
(CH-40), 127.2 (CH-20 and CH-60), 126.8 (C-4a), 116.2 (CH-8), 114.8
(CH-5), 59.8 (CH-1), 42.2 (CH2-3), 29.2 (CH2-4); MS (FAB) m/z (%):
268 [MþH]þ, 251 (100), 173 (20), 123 (30); HRESIMS m/z 268.1335
[MþH]þ (268.1338, calc for C17H18NO2).
d
¼ 7.26 (t, J ¼ 7.5 Hz, 2H, CH-30 and CH-50), 7.16 (m, 2H, CH-20 and
4.2.6.2. (E)-2-Methyl-1-styryl-1,2,3,4-tetrahydroisoquinoline-6,7-
diol (2c).
CH-60), 7.14 (m, 1H, CH-40), 6.48 (s, 1H, CH-4), 4.42 (d, J ¼ 8.4 Hz, 1H,
CH-7), 3.38 (m, 1H, CH-8a), 3.07 (m, 1H, CH-2a), 2.72 (m, 1H, CH-
(E)-6,7-Dimethoxy-2-methyl-1-styryl-1,2,3,4-
3a), 2.63 (m, 1H, CH-3b), 2.49 (m, 1H, CH-2b), 2.36 (m, 1H, CH-8a),
tetrahydroisoquinoline (2b) (100 mg, 0.32 mmol) was subjected to
similar conditions to those described above to obtain the THIQ 2a.
The residue was purified by silica gel column chromatography
(CH2Cl2/MeOH, 90:10) to afford the compound 2c (82 mg, 91%) as a
2.20 (s,1H, NCH3), 2.14 (m,1H, CH-8b
); 13C NMR (125 MHz, CD3OD):
d
¼ 145.4 (C-6), 143.7 (C-5), 140.1 (C-6a), 128.5 (C-10), 128.1 (CH-30
and CH-50), 128.0 (C-3a1), 127.2 (CH-20 and CH-60), 125.8 (CH-40),
121.1 (C-3a), 112.9 (CH-4), 65.6 (CH-8a), 54.4 (CH2-2), 45.1 (CH-7),
42.7 (CH2-8), 39.9 (NCH3), 26.0 (CH2-3); MS (FAB) m/z (%): 282
[MþH]þ, 265 (50), 253 (65), 239 (100), 175 (20); HRESIMS m/z
282.1495 [MþH]þ (282.1494, calc for C18H20NO2).
brown oil. 1H NMR (500 MHz, CD3OD):
d
¼ 7.47 (d, J ¼ 7.5 Hz, 2H,
CH-20 and CH-60), 7.31 (t, J ¼ 7.5 Hz, 2H, CH-30 and CH-50), 7.24 (m,
1H, CH-40), 6.73 (d, J ¼ 15.8 Hz, 1H, CH-
b), 6.61 (s, 1H, CH-8), 6.56 (s,
1H, CH-5), 6.27 (dd, J ¼ 15.8, 8.9 Hz,1H, CH-
a), 4.18 (d, J ¼ 8.9 Hz,1H,
CH-1), 3.23 (m, 1H, CH-3
a
), 3.01 (m, 1H, CH-4
a
), 2.81 (m, 1H, CH-
4.2.6.6. 1-Allyl-7-phenyl-1,2,3,7,8,8a-hexahydrocyclopenta[ij]iso-
quinoline-5,6-diol (3e).
3
b), 2.70 (m, 1H, CH-4b
), 2.55 (s, 3H, NCH3); 13C NMR (125 MHz,
CD3OD):
d
¼ 145.2 (C-7),144.3 (C-6),137.3 (C-10),135.5 (CH-
b
),129.4
1-Allyl-5,6-dimethoxy-7-phenyl-1,2,3,7,8,8a-hexahydrocyclopenta
[ij]isoquinoline (3d) (100 mg, 0.29 mmol) was subjected to similar
conditions to those described above to obtain THIQ 2a, 2c, 2e, 3a
and 3c. The residue was purified by silica gel column chromatog-
raphy (CH2Cl2/MeOH, 90:10) to afford the compound 3e (82 mg,
90%) as a brown oil. 1H NMR and HRESIMS [19]; 13C NMR (125 MHz,
(CH-30 and CH-50), 129.0 (CH- ), 128.6 (CH-40), 127.4 (CH-20 and CH-
a
60), 126.1 (C-8a), 125.0 (C-4a), 115.7 (CH-8), 115.1 (CH-5), 68.5 (CH-
1), 51.4 (CH2-3), 43.1 (NCH3), 27.7 (CH2-4); MS (FAB) m/z (%): 282
[MþH]þ, 251 (100), 178 (70), 173 (100), 123 (90); HRESIMS m/z
282.1487 [MþH]þ (282.1494, calc for C18H20NO2).
CD3OD):
d
¼ 145.7 (C-6), 144.9 (C-5), 140.8 (C-6a), 136.7 (CH-200),
4.2.6.3. (E)-2-Allyl-1-styryl-1,2,3,4-tetrahydroisoquinoline-6,7-diol
(2e).
135.4 (C-10), 129.2 (C-3a1), 128.9 (CH-30 and CH-50), 128.4 (CH-20
and CH-60), 126.7 (CH-40), 123.5 (C-3a), 117.6 (CH2-300),113.6 (CH-4),
65.5 (CH-8a), 59.2 (CH2-100), 52.2 (CH2-2), 46.6 (CH-7), 44.7 (CH2-8),
27.8 (CH2-3); MS (FAB) m/z (%): 308 [MþH]þ, 251 (100), 175 (10).
(E)-2-Allyl-6,7-dimethoxy-1-styryl-1,2,3,4-tetrahydroisoquinoline
(2d) (100 mg, 0.29 mmol) was subjected to similar conditions to
those described above to obtain the THIQ 2a and 2c. The residue
was purified by silica gel column chromatography (CH2Cl2/MeOH,
90:10) to afford the compound 2e (85 mg, 93%) as a brown oil. 1H
4.2.6.7. (E)-1-(prop-1-en-1-yl)-1,2,3,4-tetrahydroisoquinoline-6,7-
diol (5a).
(E)-6,7-Dimethoxy-1-(prop-1-en-1-yl)-1,2,3,4-
tetrahydroisoquinoline (5) (100 mg, 0.43 mmol) was subjected to
similar conditions to those described above to obtain THIQ 2a, 2c,
2e, 3a, 3c and 3e. The residue was purified by silica gel column
chromatography (CH2Cl2/MeOH, 90:10) to afford compound 5a
NMR (500 MHz, CD3OD):
d
¼ 7.24 (d, J ¼ 7.2 Hz, 2H, CH-20 and CH-
60), 7.19 (t, J ¼ 7.2 Hz 2H, CH-30 and CH-50), 7.13 (m, 1H, CH-40), 6.39
(d, J ¼ 15.8 Hz, 1H, CH-
b), 6.37 (s, 1H, CH-8), 6.22 (s, 1H, CH-5), 6.06
(dd, J ¼ 15.8, 8.9 Hz, 1H, CH-
a
), 5.86 (m, 1H, CH-200), 5.12 (m, 2H,
CH2-300), 3.99 (d, J ¼ 8.9 Hz, 1H, CH-1), 3.39 (dd, J ¼ 13.6, 5.6 Hz, 1H,
CH-100
a), 3.02 (m,1H, CH-3
a
), 2.99 (m,1H, CH-100
b
), 2.59 (m,1H, CH-
(78 mg, 89%) as a brown oil. 1H NMR (500 MHz, CD3OD):
d
¼ 6.61 (s,
1H, CH-5), 6.54 (s, 1H, CH-8), 6.03 (dq, J ¼ 13.2, 6.6 Hz, 1H, CH- ),
5.60 (ddd, J ¼ 15.2, 8.4, 1.6 Hz, 1H, CH- ), 4.35 (m, 1H, CH-1), 3.48
(m, 1H, CH-3 ), 3.33 (m, 1H, CH-3 ), 2.99 (m, 1H, H-4 ), 2.89 (dd,
J ¼ 17.0, 5.3 Hz, 1H, H-4 ), 1.83 (dd, J ¼ 6.5, 1.3 Hz, 3H, CH3-
NMR (125 MHz, CD3OD):
¼ 146.8 (C-7), 145.7 (C-6), 137.0 (CH-
127.9 (CH-
4
a
), 2.48 (m, 1H, CH-4
b
), 2.47 (m, 1H, CH-3
b
); 13C NMR (125 MHz,
b
CD3OD):
d
¼ 143.5 (C-7), 143.0 (C-6), 136.3 (C-10), 134.5 (CH-
b
),
a
133.4 (CH-200), 128.6 (CH-30 and CH-50), 128.5 (CH-
a
), 127.8 (CH-40),
a
b
a
126.9 (C-8a), 126.5 (CH-20 and CH-60), 125.5 (C-4a), 119.7 (CH2-300),
115.0 (CH-8), 114.5 (CH-5), 65.6 (CH-1), 57.9 (CH2-100), 46.4 (CH2-3),
27.4 (CH2-4); MS (FAB) m/z (%): 308 [MþH]þ, 253 (50), 161 (30), 123
b
g
); 13
C
),
d
b
a
), 123.8 (C-8a), 123.3 (C-4a), 116.1 (CH-8), 114.8 (CH-5),

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J. Parraga et al. / European Journal of Medicinal Chemistry 122 (2016) 27e42
40
59.1 (CH-1), 41.3 (CH2-3), 25.8 (CH2-4), 18.0 (CH3-
g
); MS (FAB) m/z
immediately diluted with Tris buffer. The homogenate was centri-
fuged either twice ([3H] SCH 23390 binding experiments) or four
times ([3H] raclopride binding experiments) at 20000 g for
10 min at 4 ꢀC with resuspension in the same volume of Tris buffer
between centrifugations. For [3H] SCH 23390 binding experiments,
the final pellet was resuspended in Tris buffer containing 5 mM
MgSO4, 0.5 mM EDTA and 0.02% ascorbic acid (Tris-Mg buffer), and
the suspension was briefly sonicated and diluted to a protein
(%): 206 [MþH]þ, 191 (35), 189 (100), 161 (55); HRESIMS m/z
206.1177 [MþH]þ (206.1181, calc for C12H16NO2).
4 . 2 . 6 . 8 . ( E ) - 2 - M e t h y l - 1 - ( p r o p - 1 - e n - 1 - y l ) - 1, 2 , 3 , 4 -
tetrahydroisoquinoline-6,7-diol (5c).
(E)-6,7-Dimethoxy-2-methyl-1-(prop-1-en-1-yl)-1,2,3,4-
tetrahydroisoquinoline (5b) (100 mg, 0.40 mmol) was subjected to
similar conditions to those described above to obtain THIQ 2a, 2c,
2e, 3a, 3c, 3e and 5a. The residue was purified by silica gel column
chromatography (CH2Cl2/MeOH, 90:10) to afford the compound 5c
concentration of 1 mg/mL. An aliquot of 100
membrane suspension (100 g of striatal protein) was incubated for
1 h at 25 ꢀC with 100 L Tris buffer containing [3H] SCH 23390
(0.25 nM final concentration) and 800 L of Tris-Mg buffer con-
taining the required drugs. Non-specific binding was determined in
mL of freshly prepared
m
m
(81 mg, 92%) as a brown oil. 1H NMR (500 MHz, CD3OD):
d
¼ 6.58 (s,
1H, CH-5), 6.51 (s, 1H, CH-8), 5.98 (dq, J ¼ 13.4, 6.2 Hz, 1H, CH- ),
5.47 (dd, J ¼ 13.4, 8.8, Hz, 1H, CH- ), 4.39 (d, J ¼ 8.8 Hz, 1H, CH-1),
3.43 (m, 1H, CH-3 ), 3.00
), 3.10 (ddd, J ¼ 14.2, 9.6, 5.2 Hz, 1H, CH-3
(dd, J ¼ 9.6, 5.8 Hz, 1H, CH-4 ), 2.85 (dt, J ¼ 17.0, 5.2 Hz, 1H, CH-4 ),
2.72 (s, 3H, NCH3), 1.84 (dd, J ¼ 6.2, 1.6 Hz, 3H, CH3-
); 13C NMR
(125 MHz, CD3OD): ), 128.2
¼ 146.5 (C-7), 145.5 (C-6), 136.6 (CH-
(CH- ), 124.6 (C-8a), 123.7 (C-4a), 115.9 (CH-8), 115.2 (CH-5), 68.7
m
b
a
the presence of 30 mM SK&F 38393 and represented around 2e3%
a
b
of total binding. For [3H] raclopride binding experiments, the final
pellet was resuspended in Tris buffer containing 120 mM NaCl,
5 mM KCl, 1 mM CaCl2, 1 mM MgCl2 and 0.1% ascorbic acid (Tris-
ions buffer), and the suspension was treated as described above. A
a
b
g
d
b
a
200
striatal protein) was incubated for 1 h at 25 ꢀC with 200
buffer containing [3H] raclopride (0.5 nM final concentration) and
400 L of Tris-ions buffer containing the drug being investigated.
Non-specific binding was determined in the presence of 50
mL aliquot of freshly prepared membrane suspension (200
mg of
(CH-1), 51.4 (CH2-3), 41.9 (NCH3), 26.7 (CH2-4), 18.0 (CH3-
g); MS
mL of Tris
(FAB) m/z (%): 220 [MþH]þ, 189 (100), 178 (20), 151 (45), 149 (20);
HRESIMS m/z 220.1332 [MþH]þ (220.1338, calc for C13H18NO2).
m
m
M
4 . 2 . 6 . 9 . ( E ) - 2 - A l l y l - 1 - ( p r o p - 1 - e n - 1 - y l ) - 1, 2 , 3 , 4 -
tetrahydroisoquinoline-6,7-diol (5e).
(E)-2-Allyl-6,7-dimethoxy-1-(prop-1-en-1-yl)-1,2,3,4-
tetrahydroisoquinoline (5d) (100 mg, 0.37 mmol) was subjected to
similar conditions to those described above to obtain THIQ 2a, 2c,
2e, 3a, 3c, 3e, 5a and 5c. The residue was purified by silica gel
column chromatography (CH2Cl2/MeOH, 90:10) to afford the
compound 5e (84 mg, 94%) as a brown oil. 1H NMR (500 MHz,
apomorphine and represented around 5e7% of total binding. In
both cases, incubations were stopped by addition of 3 mL of ice-
cold buffer (Tris-Mg buffer or Tris-ions buffer, as appropriate) fol-
lowed by rapid filtration through Whatman GF/B filters using a
Brandel harvester (model M ꢁ 24, Biochemical Research and
Development Laboratories, Inc.). Tubes were rinsed with 3 mL ice-
cold buffer, and filters were washed with 3 ꢂ 3 mL ice-cold buffer.
After the filters had been dried, radioactivity was counted in 4 mL
scintillation liquid (Optiphase ‘Hisafe’ 2, Perkin Elmer). Filter blanks
corresponded to approximately 0.5% of total binding and were not
modified by drugs.
CD3OD):
200), 5.76 (m, 1H, CH-
5.29 (m, 1H, CH-300
), 4.10 (d, J ¼ 8.9 Hz, 1H, CH-1), 3.56 (m, 1H, CH-
100), 3.19 (m, 1H, CH-3 ), 3.14 (m, 1H, CH-100
), 2.73 (m, 1H, CH-4 ),
2.69 (m,1H, CH-4 ), 2.67 (m, 1H, CH-3
CH3-
); 13C NMR (125 MHz, CD3OD):
133.6 (CH-200), 133.2 (CH-
), 131.1 (CH-
d
¼ 6.52 (s, 1H, CH-5), 6.49 (s, 1H, CH-8), 5.93 (m, 1H, CH-
b
), 5.44 (m, 1H, CH- ),
a
), 5.39 (m, 1H, CH-300
a
b
a
b
a
b
b
d
), 1.79 (dd, J ¼ 6.5,1.6 Hz, 3H,
4.3.3. Cytotoxicity studies
The cytotoxity of 2c, 2e, 3a, 3c, 3e, 5c and 5e was determined at
30 mM on neutrophils and HUVEC by the use of two different ap-
proaches: the MTT colorimetric assay and flow cytometry analysis
of cell apoptosis and survival [43].
g
¼ 145.9 (C-7), 145.0 (C-6),
b
a
), 127.2 (C-8a), 125.4 (C-4a),
121.2 (CH2-300), 115.7 (CH-8), 115.5 (CH-5), 66.8 (CH-1), 58.2 (CH2-
100), 47.7 (CH2-3), 27.8 (CH2-4), 17.9 (CH3-
g
); MS (FAB) m/z (%): 246
[MþH]þ, 191 (100), 161 (10), 149 (25); HRESIMS m/z 246.1498
[MþH]þ (246.1494, calc for C15H20NO2).
4.3.4. MTT assay
The viability of neutrophils and HUVEC was determined using
the previously described MTT (3[4,5-dimethylthiazol-2-yl]-2,5-
diphenyltetrazolium bromide) colorimetric assay [44]. Neutro-
phils were obtained from buffy coats of healthy donors by Ficoll-
Hypaque density gradient centrifugation as described [45]. Neu-
trophils and HUVEC suspension in supplemented RPMI medium
4.3. Biological assays
All research with human samples in this study, were complied
with the principles of the Declaration of Helsinki and was approved
by the institutional ethics committee of the University Clinic Hos-
pital of Valencia (Valencia, Spain). Written, informed consent was
obtained from volunteers.
(105 cells/100
mL) was added to each well of a 96-well microtiter
plate. Cells were incubated in the absence or presence of the
compounds at 37 ꢀC for 24 h. MTT was freshly prepared at 2 mg/mL
4.3.1. Cell culture
in PBS. 100
bated at 37 ꢀC for another 3 h. The supernatants were discarded and
200 L of DMSO was added to each well to dissolve formazan. The
optical densities at dual wavelengths (560 and 630 nm) were
determined in a spectrophotometer (Infinite M200, Tecan, Man-
nedorf, Switzerland).
mL of MTT solution was added to each well and incu-
Human umbilical venous endothelial cells (HUVEC) were iso-
lated by collagenase treatment [42] and maintained in human
specific endothelial basal medium (EBM-2) supplemented with
endothelial growth media (EGM-2) and 10% FBS. Cells up to passage
one were grown to confluence on 24-well culture plates. Prior to
every experiment, cells were incubated 16 h in medium containing
1% FBS and returned at the beginning of experimental protocols to
the 10% FBS medium.
m
4.3.4.1. Cytofluorometric analysis of apoptosis and survival.
Freshly isolated neutrophils and HUVEC were resuspended in
supplemented RPMI medium at 2 ꢂ 106 cells/mL. 25
mL and
4.3.2. Binding assays
cultured in a 96-well plate containing 200 mL of supplemented
Binding experiments were performed on striatal membranes.
Each striatum was homogenized in 2 mL ice-cold Tris-HCl buffer
(50 mM, pH ¼ 7.4 at 22 ꢀC) with a Polytron (4s, maximal scale) and
RPMI medium for 24 h in the absence or presence of the com-
pounds as described previously [46]. Assessment of apoptosis was
performed by flow cytometry using annexin V-FITC and propidium

ꢀ
J. Parraga et al. / European Journal of Medicinal Chemistry 122 (2016) 27e42
41
iodide (PI). The protocol indicated by the manufacturer (FITC
remained fixed during optimization.
Annexin V Apoptosis Detection Kit I; BD Biosciences) was used as
outlined previously [47]. Cells (1 ꢂ 104) were analyzed in a BD
FACSVerse Flow Cytometer (BD Biosciences, San Jose, CA) and
differentiated as early or viable apoptotic (annexin Vþ and PIꢁ), late
apoptotic and/or necrotic (annexin Vþ and PIþ), and viable non-
apoptotic (annexin Vꢁ and PIꢁ) cells.
4.4.5. Atoms in molecules theory
After the QM/MM calculation, the optimized geometry for each
complex was used as input for quantum theory atoms in molecule
(QTAIM) analysis [33], which was performed with the help of
Multiwfn software [56], using the wave functions generated at the
M062X-D/6-31G(d) level. This type of calculations have been used
in recent studies since it ensures a reasonable compromise be-
tween the wave function quality required to obtain reliable values
4.4. Molecular modeling
4.4.1. Set up of dopaminergic receptors D1 and D2
of the derivatives of r(r) and the computer power available, which
3D models of the human D1 and D2 DRs were used for the
molecular modeling study. Both models are based on the homology
is due to the extension of the system in study [57,58].
model from the crystallized D3 DR, b2 adrenoceptor and A2
4.4.6. Additional materials
a
adenosine receptor as templates [48]. These models were suc-
cessfully used previously to perform molecular modeling studies of
different dopamine receptor ligands [14,15,17,18,32,33].
FITC-Annexin and propidium iodide staining solution were from
BD Bioscience (San Jose, CA). Unless stated, all other reagents were
from Sigma-Aldrich.
4.4.2. Molecular docking
Acknowledgements
AutoDock 4 [30] was used to dock each compound to the re-
ceptor active site using a Lamarckian genetic algorithm with
pseudo-Solis and Wets local search [49]. The following parameters
were used: the initial population of trial ligands was constituted by
250 individuals; the maximum number of generations was set to
2.7 ꢂ 104. The maximum number of energy evaluations was
10.0 ꢂ 106. For each docking job, 100 conformations were gener-
ated. All other run parameters were maintained at their default
setting. The resulting docked conformations were clustered into
families by considering the backbone rmsd. The lowest docking-
energy conformation was considered the most favorable orienta-
tion [50].
This study was supported by grants SAF2014-57845-R from the
Spanish Ministry of Economy and Competiveness, the European
Regional Development Fund (FEDER) and research grants from
Generalitat Valenciana (PROMETEO II/2013/014). This study was
also supported by the Spanish Ministry of Economy and Competi-
tiveness through a Miguel Servet Program (CP15/00150) ISCIII
(Carlos III Institute of Health) co-funded by the European Regional
Development Fund (FEDER)/European Social Fund (ESF). Grants
from the Universidad Nacional de San Luis (UNSL-Argentina)
partially supported this work. R.D.E. and SAA are members of the
ꢀ
Consejo Nacional de Investigaciones Científicas y Tecnicas (CONI-
CET-Argentina) staff and L.J.G acknowledges to CONICET-Argentina
by a postdoctoral fellowship. The authors would like to thanks MSc.
Daniel O. Zamo for technical assistance (CONICET-Argentina).
4.4.3. MD simulations
The complex geometries from docking were soaked in boxes of
explicit water using the TIP3P model [51] and subjected to MD
simulation. All MD simulations were performed with the Amber
software package [31] using periodic boundary conditions and
cubic simulation cells. The particle mesh Ewald method (PME) [52]
was applied using a grid spacing of 1.2 Å, a spline interpolation
order of 4 and a real space direct sum cutoff of 10 Å. The SHAKE
algorithm was applied allowing for an integration time step of 2 fs.
MD simulations were carried out at 310 K temperature. Three MD
simulations of 3 ns were conducted for each system under different
starting velocity distribution functions; thus, in total 9 ns were
simulated for each complex. The NPT ensemble was employed us-
ing Berendsen coupling to a baro/thermostat (target pressure
1 atm, relaxation time 0.1 ps). Post MD analysis was carried out
with program PTRAJ.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2016.06.009.
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