Synthesis and binding affinity of new 1,4-disubstituted triazoles as potential dopamine D3 receptor ligands

Article abstract of DOI:10.1016/j.bmcl.2013.08.047

A series of new 1,4-disubstituted triazoles was prepared from appropriate arylacetylenes and aminoalkylazides using click chemistry methodology. These compounds were evaluated as potential ligands on several subtypes of dopamine receptors in in vitro competition assays, showing high affinity for dopamine D₃ receptors, lower affinity for D₂ and D₄, and no affinity for the D₁ receptors. Compound 18 displayed the highest affinity at the D₃ receptor with a K_i value of 2.7 nM, selectivity over D₂ (70-fold) and D₄ (200-fold), and behaviour as a competitive antagonist in the low nanomolar range.

Full text of DOI:10.1016/j.bmcl.2013.08.047

Bioorganic & Medicinal Chemistry Letters xxx (2013) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and binding affinity of new 1,4-disubstituted triazoles
as potential dopamine D3 receptor ligands
Ignacio Insua a, Mario Alvarado b, Christian F. Masaguer a, Alba Iglesias c, José Brea c, María I. Loza c,
Laura Carro a,
⇑
a Departamento de Química Orgánica, Facultad de Farmacia, Universidad de Santiago de Compostela, E-15782 Santiago de Compostela, Spain
b Grupo de Investigación en Compuestos Heterocíclicos, Semillero de Investigación en Química Médica, Facultad de Ciencias Básicas, Universidad del Atlántico,
A.A. 1890 Barranquilla, Colombia
c Departamento de Farmacología, Facultad de Farmacia, Universidad de Santiago de Compostela, E-15782 Santiago de Compostela, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of new 1,4-disubstituted triazoles was prepared from appropriate arylacetylenes and aminoalky-
lazides using click chemistry methodology. These compounds were evaluated as potential ligands on sev-
eral subtypes of dopamine receptors in in vitro competition assays, showing high affinity for dopamine D3
receptors, lower affinity for D2 and D4, and no affinity for the D1 receptors. Compound 18 displayed the
highest affinity at the D3 receptor with a Ki value of 2.7 nM, selectivity over D2 (70-fold) and D4 (200-
fold), and behaviour as a competitive antagonist in the low nanomolar range.
Received 31 May 2013
Revised 7 August 2013
Accepted 9 August 2013
Available online xxxx
Keywords:
1,2,3-Triazoles
Synthesis
Ó 2013 Elsevier Ltd. All rights reserved.
Click chemistry
Microwaves
Binding assays
Dopamine receptors
The neurotransmitter dopamine (DA) plays important roles in
behaviour and cognition, ranging from movement to emotion, sen-
sitization to addiction, and development to plasticity. All DA recep-
tors belong to the family of G protein coupled receptors (GPCR),
which are characterized by having seven hydrophobic transmem-
brane-spanning regions, as well as a functionally critical third
intracytoplasmic loop that interacts with G-proteins and other
effector molecules to mediate the physiological and neurochemical
effects of the receptors. Based on their pharmacological profiles,
including their effects on different signal transduction cascades,
these receptors are currently divided into two protein families:
the D1-like (D1 and D5) family or adenylyl-cyclase stimulators,
and the D2-like (D2, D3 and D4) family or adenylyl-cyclase
inhibitors.1
The D3 dopamine receptor was first identified and cloned by
Sokoloff et al.2 in 1990 and has been shown to be an interesting
target for different CNS diseases. Although the D2 and D3 receptor
subtypes are highly homologous, especially within the transmem-
brane segments, which serve as the binding site for dopamine, the
D3 receptor is generally less abundant than the D2 receptor, and the
difference is particularly striking in the caudate putamen, where
D2 receptors are displayed at high density, whereas D3 receptors
are poorly represented.3 Moreover, D3-binding sites and mRNA
encoding D3 receptors are concentrated in the limbic brain regions
known to be associated with cognitive and emotional functions.4
Due to this observation, the D3 receptor has been suggested to be
a potential target in the treatment of neurological disorders5 such
as schizophrenia,6 Parkinson’s disease,7 drug-induced dyskinesia,8
and drug abuse.9 Furthermore, some studies have shown that pro-
erectile effects of dopamine D2-like agonists are mediated by the
D3 receptor in rats and mice.10
An intensive effort has been directed toward the development
of selective ligands for dopamine D3 receptors in an attempt to
prove these hypotheses.11 Thus, a number of potent and selective
D3 ligands, including antagonists, partial agonists and full agonists
have been developed. Some of these well-known D3 ligands in-
clude pramipexole and ropinirole (Fig. 1), two highly potent D3 full
agonists but with only modest selectivity for the D3 receptor over
the D2 receptor.12 On the other hand, numerous antagonists and
partial agonists have been developed so far with a number of lead
compounds showing both great potency and selectivity for the D3
receptor (Fig. 1). Most of these compounds contain a piperazine
ring connected to a suitable benzamide-type moiety via variable
linker size.11,13 Among the compounds illustrated, it is worth high-
lighting the values displayed by the piperazines ABT 925 and RGH
⇑
Corresponding author. Tel.: +34 981563100; fax: +34 981594912.
E-mail address: laura.carro@usc.es (L. Carro).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.08.047
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2
I. Insua et al. / Bioorg. Med. Chem. Lett. xxx (2013) xxx–xxx
Figure 1. Structures of selective D3 receptor ligands.
237 exhibiting a good selectivity pattern of D3 over D2 with a ratio
of 200 and 1800, respectively. Nevertheless, despite the significant
number of D3 ligands reported, today there is still a need for more
selective molecular tools to facilitate a relevant differentiation
between D3 actions and those mediated by the D2 receptors, in
order to elucidate the function and potential therapeutic advanta-
ges of targeting D3 receptors, and for molecules enabling sufficient
D3 receptor occupancy to derive conclusions on a D3-mediated
therapeutic benefit.
In our laboratories, the synthesis and pharmacological evalua-
tion on D1, D2, D3 and D4 dopamine receptors of a series of cyclic
N-piperazinilalquil substituted benzolactams (A, Fig. 2), have
allowed us to determine some of the essential structural require-
ments for a high-affinity binding on the D3 receptor. The study of
the structure–activity relationships (SAR) in this series concluded
that both the length of the linker between the lactam and pipera-
zine ring, and the size of the lactam ring influence the affinity for
the D2 and D3 receptors.14 In a continuation of this work, we report
in this Letter the replacement of the lactam system for a 1,2,3-
triazole ring on the basis of previous studies15 which report that
carboxamide function could be successfully replaced by a five-
membered heterocycle in a series of dopamine D3 antagonists.
Therefore, new triazole analogues of our benzolactam derivatives
were obtained via click chemistry, using [3+2] azide–alkyne cyclo-
addition (Huisgen reaction) promoted by copper(I),16 and their
binding profile on dopamine receptors was determined.
In the search for a general method of synthesis of the new
disubtituted triazoles 15–20 (Fig. 2), two possible routes
(Scheme 1) were investigated starting from 4-clorobutilazide (1)
which was easily prepared by nucleophilic substitution of the bro-
mine atom in 1-bromo-4-chlorobutane by sodium azide in DMF in
92% yield. The general method was explored using phenylacety-
lene as alkyne and 1-(2-methoxyphenyl)piperazine as amine. Thus,
the reaction of 1 with 1-(2-methoxyphenyl)piperazine (route a)
under basic conditions afforded in 65% yield the 1-(4-azidobu-
tyl)-4-(2-methoxyphenyl)piperazine (2); subsequent Huisgen
1,3-dipolar cycloaddition17 with phenylacetylene 3 gave the corre-
sponding triazol 15 (see Scheme 1).
For this click reaction several conditions (Table 1) were evalu-
ated. The target compound 15 was obtained in a high yield of
89% (58% two-step yield) (entry 6).
Alternatively (route b), cycloaddition reaction of phenylacety-
lene with the chlorobutylazide 1 afforded 4-phenyl-1-(4-chlorobu-
tyl)-1H-1,2,3-triazole (9). In this case, the reaction took place in the
presence of Cu2O and benzoic acid in water;18 after stirring for 30
min at room temperature, triazole 9 was obtained in 96% yield. The
chlorine atom in 9 was replaced by 1-(2-methoxyphenyl)pipera-
zine leading to the desired compound 15 in 94% yield. The utiliza-
tion of microwave irradiation at 130 °C allowed to reduce the
reaction time from 16 h (conventional thermal heating conditions)
to 30 min. Having successfully established a suitable catalyst and
the optimal conditions to synthesize the target compound 15
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I. Insua et al. / Bioorg. Med. Chem. Lett. xxx (2013) xxx–xxx
3
Figure 2. Structures of the D3 dopamine benzolactam ligands and proposed 1,2,3-triazole analogues.
Scheme 1. Reagents and conditions: (i) NaN3, DMF, rt, 72 h, 92%; (ii) K2CO3, KI, methylisobutylketone, reflux, 16 h, 65%; (iii) CuSO4Á5H2O, Cu(0), t-BuOH:H2O (1:1),
microwave, 125 °C, 10 min, 89%; (iv) Cu2O, benzoic acid, H2O, rt, 30 min, 96%; (v) K2CO3, KI, DMF, microwave (100 W), 130 °C, 30 min, 94%.
Table 1
Optimization assays of the click reaction leading to triazole 15
3
N
N
N
OCH3
N
N
N
OCH3
N3
N
15
2
Entry
Heating conditions
Catalyst
Solvent
Temp (°C)
Time
Yield (%)
1
2
3
4
5
6
Thermal
Thermal
MW
MW
MW
CuSO4Á5H2O, sodium ascorbate
CuSO4Á5H2O, sodium ascorbate
CuSO4Á5H2O, sodium ascorbate
CuSO4Á5H2O, sodium ascorbate
CuSO4Á5H2O, sodium ascorbate
CuSO4Á5H2O, Cu(0)
t-BuOH/H2O (1:1)
t-BuOH/H2O (1:1)
DMF
DMF
t-BuOH/H2O (1:1)
t-BuOH/H2O (1:1)
rt
15 h
2 h
30 min
15 min
10 min
10 min
73
87
58
73
88
89
120
110
150
125
125
MW
(90% yield in two steps), we tackled the issues of scope and group
tolerance, being this second approach the selected procedure for
the synthesis of the other 1,4-disubstituted triazoles.19
3-ethynylpyridine (7) under these conditions did not allow isolat-
ing the expected triazole 13, so the amount of Cu2O was in-
creased from 1% to 10%. Under these conditions triazoles 12–14
were obtained from alkynes 6–8 in yields ranging from 17% to
75%. Finally, subsequent displacement of the chlorine atom by
1-(2-methoxyphenyl)piperazine in basic medium under micro-
wave irradiation furnished the final products 16–20 in 42–81%
yield (Table 2).20
In vitro dopamine receptor binding studies: The affinity of the new
compounds for cloned human D1, D2 and D3 receptors was evalu-
ated in in vitro binding assays using [3H]SCH23390 for labelling
D1 receptors and [3H]spiperone for labelling D2, D3 and D4 recep-
To apply the route b to the new series of alkynes (4–8) the sol-
vent in the click reaction had to be modified: while phenylacety-
lene is a liquid under normal conditions, which enables the
distribution of reagents in the flask, the rest of acetylenes studied
are solid and precipitate in water. Changing the media to dichlo-
romethane facilitated the contact between the reagents. Conse-
quently, using the same catalytic system as in the preparation
of 9, triazoles 10 and 11 were obtained in 94% and 53% yield,
respectively, (Table 1, entries 2 and 3). However, the reaction of
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I. Insua et al. / Bioorg. Med. Chem. Lett. xxx (2013) xxx–xxx
Table 2
Reaction conditions for the preparation of 1,2,3-triazoles 9–14 and 15–20
N
R
N
HN
OCH3
3-8
Cl
N
OCH3
Cl
N
N
9-14
N
N
R
R
N3
Conditions "a"
Conditions "b"
N
N
1
15-20
Entry
R
Conditions ‘a’
Compound (Yield)
9 (96%)
Conditions ‘b’
Compound (Yield)
15 (94%)
1
2
3
4
Benzoic acid, 1% Cu2O, H2O, rt, 30 min
Benzoic acid, 1% Cu2O, CH2Cl2, rt, 85 min
Benzoic acid, 1% Cu2O, CH2Cl2, rt, 72 h
Benzoic acid, 10% Cu2O, CH2Cl2, rt, 40 h
K2CO3, KI, DMF, MW (100 W), 130 °C, 30 min
K2CO3, KI, DMF, MW (100 W), 130 °C, 30 min
K2CO3, KI, DMF, MW (100 W), 130 °C, 30 min
K2CO3, KI, DMF, MW (100 W), 130 °C, 45 min
10 (94%)
16 (81%)
Br
11 (53%)
17 (80%)
H3CO
H2N
12 (37%)
18 (55%)
5
6
Benzoic acid, 10% Cu2O, CH2Cl2, rt, 48 h
Benzoic acid, 10% Cu2O, CH2Cl2, rt, 60 h
13 (75%)
14 (17%)
K2CO3, KI, DMF, MW (100 W), 130 °C, 55 min
K2CO3, KI, DMF, MW (100 W), 130 °C, 60 min
19 (42%)
20 (73%)
N
O
N
tors, according to our previously described procedures.21 Ki values
were calculated according to the Cheng–Prusoff equation.22 For the
compounds that showed little affinity, a percentage of inhibition at
150
100
50
the highest concentration tested (1
lM) is reported. The in vitro
receptor binding data are summarized in Table 3.
The new synthesized triazoles showed no significant affinity at
the D1 receptor, while on D2–D4 subtypes the affinities were in the
sub-micromolar order. In general, the order of affinity for the six
new prepared compounds was D3 > D2 > D4 > D1.
0
-11 -10 -9
-8
-7
-6
-5
-4
The six new compounds showed good affinity and selectivity
for the D3 receptor, but it is worth highlighting the profile of
log [dopamine] (M)
-50
compound 18, with
a strong affinity for the D3 receptor
Figure 3. Concentration–response curves of dopamine in the absence (j) and in
the presence (N) of 5 nM triazole 18 by measuring GTP S specific binding to
dopamine D3 receptors. Points represent the mean standard deviation (vertical
bars) of duplicate measurements of a representative experiment.
(Ki = 2.7 nM) and a selectivity for this receptor subtype of approx-
imately 70-times on the D2, and almost 200-times on the D4
c
receptors, showing little affinity (>1
lM) for D1 receptors. In this
compound, the amine group critically influences the affinity for
the D3 dopamine receptors, while it has little influence on the
other dopamine receptor subtypes. This behaviour could be
attributed to the establishment of an additional H bonding be-
tween the NH2 and an acceptor group in the D3 receptor binding
site. On the other hand, it seems to be some relationship between
the electron density of the aromatic ring attached to the triazole
and the D3 receptor affinity such that the higher the electron
density (compounds 18 and 17) the higher the affinity, which
could be related to the existence of a pi-stacking interaction with
an aromatic residue in the binding site.
Based on the binding results, the next goal of our study was to
evaluate the functional activity at the D3 receptor of the most
promising compound of the collection. Consequently, triazole 18
was tested in [35S]GTP S functional assay to characterize its ability
c
to stimulate D3 receptor in comparison to the endogenous ligand
DA according to standard protocols.23
As it is illustrated in the Figure 3, compound 18 right-shifted the
dopamine concentration–response curve, behaving as an antago-
nist of dopamine D3 receptors, showing a KB value of 1.4 0.26
Table 3
Human D1–D4 receptor binding affinities of new compounds (pKi or percent displacement at 1 l
M)a
Compound
Affinity
Selectivity
D1 (% at 1
lM)
D2 (Ki nM)
D3 (Ki nM)
D4 (Ki nM)
D2/D3
D4/D3
15 (1T1401)
16 (2T1401)
17 (3T1401)
18 (4T1401)
19 (5T1401)
20 (6T1401)
39.41
44.26
44.26
38.43
40.85
50.01
228.3
226.6
211.7
198.8
181.5
136.6
81.2
110.7
40.1
2.7
132.5
134.6
639.3
386.7
394.8
532.5
1898.2
426.4
2.8
2.0
5.3
73.6
1.4
1.0
7.9
3.5
9.8
197.2
14.3
3.2
a
All values are means of two or three separate competition experiments.
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I. Insua et al. / Bioorg. Med. Chem. Lett. xxx (2013) xxx–xxx
5
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nM, this value is in agreement with the affinity of the compound
measured in binding experiments.
In conclusion, a new series of 1,4-disubstituted-1,2,3-triazoles
was synthesized by a short, efficient, microwave-assisted process,
and evaluated as dopamine D3 receptor ligands. The binding affin-
ities of the synthesized compounds on the dopamine receptor sub-
types unveiled that triazole 18 (4T1401), bearing a 4-NH2-phenyl
group, has a high D3 receptor affinity together with an excellent
selectivity on other dopamine receptors. Furthermore, functional
assay showed its behaviour as a competitive antagonist at D3
receptor in the nanomolar range.
According to our results, the presence of an electron-rich substi-
tuent in the phenyl ring could favor both potency and selectivity
on D3 receptor. These data enhance our understanding of the D3
pharmacophore and are expected to lead to novel compounds with
higher affinity and selectivity on this receptor taking triazole 18 as
a lead molecule. Further optimization of this series is in progress
and is directed to the synthesis of new counterparts and will be re-
ported in due course.
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Acknowledgments
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3782.
This work was supported in part by grants from the CICYT
(Spain, SAF2009-13609-C04) and by the Xunta de Galicia (Spain,
INCITE09 203184 PR). Ph.D. fellowships (FPU) have been granted
to L.C. and to A.I. by the Ministerio de Educación y Ciencia (Spain)
and the Ministerio de Ciencia e Innovación (Spain) respectively.
19. Additionally, at this point, we explored
a one pot, microwave-assisted
synthesis of triazole 9 (Scheme 2) by using CuSO4Á5H2O/Cu(0) as catalyst
system, in tert-butanol/water or DMF as solvent, and varying other parameters
such as temperature, time and power of irradiation. This method would allow
to avoid the purification of the intermediate chlorobutylazide 1, saving
resources and shortening the length of the route. Thus, the best result was
obtained at 125 °C (constant power of irradiation of 100 W) in DMF for 15 min,
References and notes
which afforded compound
9 in 46% yield. To our disappointment, the
application of this method to other alkynes (4-aminophenylacetylene or
pyridin-3-ylacetylene) gave the corresponding triazoles in poor yields, and
therefore, this method for the preparation of the target triazoles was
discarded.
1. Missale, C.; Nash, S. R.; Robinson, S. W.; Jaber, M.; Caron, M. G. Physiol. Rev.
1998, 78, 189.
2. Sokoloff, P.; Giros, B.; Martres, M. P.; Bouthenet, M. L.; Schwartz, J. C. Nature
1990, 347, 146.
3. Levant, B. Pharmacol. Rev. 1997, 49, 231.
20. Spectral data for compound 15: mp 89–90 °C. 1H NMR (300 MHz, CDCl3) d 7.81
(d, J = 8.1 Hz, 2H); 7.76 (s, 1H); 7.75–7.36 (m, 2H); 7.32–7.26 (m, 1H), 6.99–
6.81 (m, 4H); 4.38 (t, J = 7.0 Hz, 2H); 3.81 (s, 3H); 3.06 (br s, 4H); 2.59 (br s,
4H); 2.42 (t, J = 7.0 Hz, 2H); 1.97 (t, J = 7.3 Hz, 2H); 1.56 (t, J = 7.3 Hz, 2H). 13C
NMR (75 MHz, CDCl3) d 152.2, 141.2, 130.6, 128.8, 128.1, 125.7, 122.9, 120.9,
119.5, 118.2, 111.1, 109.9, 55.3, 53.4, 50.6, 50.3, 29.7, 28.3, 23.7. IR: 1499.4,
1448.3, 1238.1, 1153.2, 1025.9, 748.2. MS: 392.2 (M+1), 393.2, 391.2, 247,
204. Anal. (C23H29N5OÁ2H2O) C, H, N. Compound 16: mp 102–104 °C. 1H NMR
(300 MHz, CDCl3) d 7.75 (s, 1H); 7.69 (d, J = 8.7 Hz, 2H); 7.53 (d, J = 8.7 Hz,
2H); 6.95–6.85 (m, 4H); 4.42 (t, J = 7.1 Hz, 2H); 3.84 (s, 3H); 3.06 (br s, 4H);
2.61 (br s, 4H); 2.43 (t, J = 7.2 Hz, 2H); 2.05–1.95 (m, 2H); 1.62–1.55 (m, 2H).
13C NMR (75 MHz, CDCl3) d 152.2, 146.7, 141.2, 132.0, 129.7, 127.2, 123.0,
122.0, 121.0, 119.6, 118.2, 111.2, 57.7, 55.3, 53.4, 50.6, 50.3, 28.3, 23.7. IR:
1498.4, 1462.7, 1237.1, 1222.6, 822.5, 745.4. MS: 470.2 (M+1), 472.2, 471.2,
469.2, 247.3, 205.2, 164.1, 150.1. Anal. (C23H28BrN5O) C, H, N. Compound 17:
mp 108–110 °C. 1H NMR (300 MHz, CDCl3) d 7.76 (d, J = 8.9 Hz, 2H); 7.68 (s,
1H); 6.99–6.84 (m, 6H); 4.43 (t, J = 7.1 Hz, 2H); 3.85 (s, 3H, –OCH3); 3.84 (s,
3H, –OCH3); 3.08 (br s, 4H); 2.62 (br s, 4H); 2.45 (t, J = 7.2 Hz, 2H); 2.06–1.96
(m, 2H); 1.62–1.57 (m, 2H). 13C NMR (75 MHz, CDCl3) d 159.5, 152.2, 147.6,
141.3, 127.0, 123.4, 122.9, 121.0, 118.7, 118.1, 114.2, 111.2, 57.7, 55.3, 53.4,
50.6, 50.2, 28.3, 23.8 ppm. IR: 1498.4, 1238.1, 1179.3, 1027.9, 830.2, 747.3.
MS: 422.1 (M+1), 421.1, 246.9, 204.8, 163.7, 134.7, 83.9 (37). Anal.
(C24H31N5O2Á0.2CH3OH) C, H, N. Compound 18: mp 60–61 °C. 1H NMR
(300 MHz, CDCl3) d 7.63–7.59 (m, 3H); 7.02–6.84 (m, 4H); 6.74–6.70 (m,
2H); 4.40 (t, J = 7.1 Hz, 2H); 3.85 (s, 3H); 3.70 (br s, 2H, exchange with D2O);
3.07 (br s, 4H); 2.62 (br s, 4H); 2.44 (t, J = 7.4 Hz, 2H); 2.04–1.94 (m, 2H);
1.63–1.53 (m, 2H). 13C NMR (75 MHz, CDCl3) d 152.2, 148.1, 146.5, 141.2,
126.9, 122.9, 121.1, 121.0, 118.2, 115.2, 113.7, 111.2, 57.7, 55.3, 53.4, 50.5,
50.1, 28.3, 23.7. IR: 1616.1, 1498.4, 1239.0, 1022.1, 750.2. MS: 407.1 (M+1);
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Please cite this article in press as: Insua, I.; et al. Bioorg. Med. Chem. Lett. (2013), http://dx.doi.org/10.1016/j.bmcl.2013.08.047

6
I. Insua et al. / Bioorg. Med. Chem. Lett. xxx (2013) xxx–xxx
256.9, 246.9, 204.8, 163.7. Anal. (C23H30N6OÁ0.1H2O) C, H, N. Compound 19:
mp 90–91 °C. 1H NMR (300 MHz, CDCl3)
9.01–9.00 (m, 1H); 8.58 (d,
111.2, 57.7, 55.3, 53.4, 50.6, 50.4, 28.3, 23.8. IR: 1498.4, 1238.1, 1098.3,
d
1030.8, 940.1, 825.4, 737.6, 726.1. MS: 459.1 (M+1), 460.1, 458.1, 246.9,
204.8, 163.7, 149.7. Anal. (C26H30N6O2Á0.85H2OÁ0.3CH3OH) C, H, N.
21. Brea, J.; Castro, M.; Loza, M. I.; Masaguer, C. F.; Raviña, E.; Dezi, C.; Pastor, M.;
Sanz, F.; Cabrero-Castel, A.; Galán-Rodríguez, B.; Fernández-Espejo, E.;
Maldonado, R.; Robledo, P. Neuropharmacology 2006, 51, 251.
J = 4.8 Hz, 2H); 8.24–8.20 (m, 1H), 7.86 (d, J = 1.0 Hz, 1H); 7.40-7.35 (m, 1H);
7.03–6.84 (m, 4H); 4.48 (t, J = 7.1 Hz, 2H); 3.85 (d, 3H, J = 1.0 Hz); 3.09 (br s,
4H); 2.63 (br s, 4H); 2.47 (t, J = 7.4 Hz, 2H); 2.09–2.00 (m, 2H); 1.66–1.56 (m,
2H). 13C NMR (75 MHz, CDCl3) d 152.2, 149.2, 147.0, 144.7, 141.2, 133.0,
126.8, 123.8, 122.9, 121.0, 119.8, 118.1, 111.1, 57.7, 55.3, 53.4, 50.6, 50.4,
28.3, 23.8. IR: 1499.4, 1237.1, 1030.8, 808.0, 738.6, 703.9. MS: 393.1 (M+1),
394.1, 392.1, 246.9, 204.8. Anal. (C22H28N6OÁ0.4H2O) C, H, N. Compound 20:
mp 157–158 °C. 1H NMR (300 MHz, CDCl3) d 7.93–7.89 (m, 3H); 7.82 (s, 1H);
7.73–7.70 (m, 2H); 7.39 (s, 1H); 7.02–6.84 (m, 4H); 4.46 (t, J = 7.1 Hz, 2H);
3.85 (s, 3H); 3.08 (br s, 4H); 2.63 (br s, 4H); 2.46 (t, J = 7.6 Hz, 2H); 2.06–2.00
(m, 2H); 1.63–1.58 (m, 2H). 13C NMR (75 MHz, CDCl3) d 152.2, 151.2, 150.5,
147.0, 141.2, 130.9, 127.3, 126.1, 124.8, 122.9, 121.7, 121.0, 119.7, 118.1,
22. Cheng, Y.; Prusoff, W. H. Biochem. Pharmacol. 1973, 22, 3099.
23. Membranes expressing human D3 receptors (Perkin Elmer) were pre-
incubated (10 lg/well) in assay buffer (20 mM HEPES, 3 lM GDP, 3 mM
MgCl2, 100 mM NaCl; pH 7.4) with compounds 18 for 15 min at 22 °C in
a 96-well polypropilene plate. Then 1 nM [35S]GTP S (Perkin Elmer) was
c
added to the well and the mixture was incubated for 40 min at 22 °C.
The reaction was terminated by filtration on
plate.
a multiscreen FB 96-well
Please cite this article in press as: Insua, I.; et al. Bioorg. Med. Chem. Lett. (2013), http://dx.doi.org/10.1016/j.bmcl.2013.08.047