Searching for multi-target antipsychotics: Discovery of orally active heterocyclic N-phenylpiperazine ligands of D2-like and 5-HT1A receptors

Article abstract of DOI:10.1016/j.bmc.2010.01.040

We described herein the design, synthesis, and pharmacological evaluation of N-phenylpiperazine heterocyclic derivatives as multi-target compounds potentially useful for the treatment of schizophrenia. The isosteric replacement of the heterocyclic ring at

Full text of DOI:10.1016/j.bmc.2010.01.040

Bioorganic & Medicinal Chemistry 18 (2010) 1925–1935
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Searching for multi-target antipsychotics: Discovery of orally active heterocyclic
N-phenylpiperazine ligands of D2-like and 5-HT1A receptors
Gilda Neves a,b,c, Ricardo Menegatti d,e, Camila B. Antonio a, Luiza R. Grazziottin a, Renan O. Vieira c,
Stela M. K. Rates a, François Noël c, Eliezer J. Barreiro e, Carlos A. M. Fraga e, ,1
*
a Laboratório de Psicofarmacologia Experimental, Faculdade de Farmácia, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil
b Programa de Pós-Graduação em Ciências Farmacêuticas, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil
c Laboratório de Farmacologia Bioquímica e Molecular, Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil
d Faculdade de Farmácia, Universidade Federal do Goiás, Goiânia, Brazil
e Laboratório de Avaliação e Síntese de Substâncias Bioativas (LASSBio), Faculdade de Farmácia, Universidade Federal do Rio de Janeiro, PO Box 68.023,
21941-902 Rio de Janeiro, RJ, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
We described herein the design, synthesis, and pharmacological evaluation of N-phenylpiperazine het-
erocyclic derivatives as multi-target compounds potentially useful for the treatment of schizophrenia.
The isosteric replacement of the heterocyclic ring at the biaryl motif generating pyrazole, 1,2,3-triazole,
and 2-methylimidazole[1,2-a]pyridine derivatives resulted in 21 analogues with different substitutions
at the para-biaryl and para-phenylpiperazine positions. Among the compounds prepared, 4 (LASSBio-
579) and 10 (LASSBio-664) exhibited an adequate binding profile and a potential for schizophrenia posi-
tive symptoms treatment without cataleptogenic effects. Structural features of this molecular scaffold are
discussed regarding binding affinity and selectivity for D2-like, 5-HT1A, and 5-HT2A receptors.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 16 October 2009
Revised 14 January 2010
Accepted 16 January 2010
Available online 25 January 2010
Keywords:
Atypical antipsychotic drugs
Schizophrenia
N-Phenylpiperazine derivatives
1-Arylpyrazole
1-Aryl-1,2,3-triazole
Imidazo[1,2-a]pyridine
1. Introduction
zapine (1) (Fig. 1), the first atypical antipsychotic.1,6 Initially it was
proposed that clozapine’s atypical profile, mainly the lack of extra-
The centenary Ehrlich’s magic bullet concept (‘one disease, one
target’) has been the basis of drug design in the last decades. How-
ever, there is an increasing change in this paradigm and the mod-
ulation of a multiplicity of targets has been considered as the best
option for treating a range of multifactorial diseases.1–3 For exam-
ple, most central nervous system disorders are highly complex in
its pathophysiology and the most effective drugs present a com-
plex pharmacology.4 In this way, schizophrenia can be considered
a challenging neuropsychiatric disease. It is characterized by the
development of three distinct kinds of symptoms: positive (hyper-
activity, delusions, hallucinations, disorganized speech), negative
(avolition, anhedonia, social isolation), and cognitive (attentional
impairment, memory deficits). The genesis of each symptom is
not fully understood, but several brain structures and neurotrans-
mitter systems are believed to be involved.5
pyramidal symptoms at therapeutic doses, was triggered by its
high affinity for D4 dopamine receptors.7,8 As a consequence, some
D4-selective compounds were developed but were not effective in
clinical trials.4,8 On the other hand, the role of 5-HT2A receptors in
ameliorating negative and cognitive symptoms of the disorder
emerged and selective 5-HT2A antagonists were designed.9 In clin-
ical trials, these compounds showed efficacy but no advance was
demonstrated in comparison with current available agents.4 Other
attempts to develop ‘magic bullets’ for treating schizophrenia also
failed and now most researches are back to the multi-target or
‘intramolecular polypharmacy’ approach.10 The importance of
multivalent ligands targeting G-protein-coupled receptors design
to deal with multifaceted neurodegenerative diseases, such as Par-
kinson’s disease, Alzheimer’s disease, and schizophrenia has been
pointed by many authors.11–13
Most of the multi-target drugs used clinically were not the re-
sult of a rational design but discovered by serendipity, such as clo-
Several targets are being explored for the development of new
antipsychotics and most of them are dopamine or serotonin recep-
tors.14,15 In this context, a significant interest has emerged from the
ability of aripiprazole (2) and bifeprunox (3) (Fig. 1) to activate the
5-HT1A receptor and to improve the cognitive and negative symp-
toms of schizophrenia and to reduce the extrapyramidal effects
* Corresponding author. Tel./fax: +55 21 2562 6503.
E-mail address: cmfraga@ccsdecania.ufrj.br (C.A.M. Fraga).
1
http://www.farmacia.ufrj.br/lassbio.
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.01.040

1926
G. Neves et al. / Bioorg. Med. Chem. 18 (2010) 1925–1935
Searching for new antipsychotic lead-compounds, our group de-
scribed the design and synthesis of three N-phenylpiperazine deriv-
atives—LASSBio-579 (4), LASSBio-580 (5) and LASSBio-581 (6)
(Fig. 1)—which differ by the isosteric replacement between pyra-
zole and 1,2,3-triazole heterocyclic rings.20 These compounds were
originally proposed to be selective ligands for the D2 or D4 recep-
tors. A preliminary pharmacological evaluation carried out by our
group demonstrated that 4 and 6 act as agonists at pre-synaptic
dopamine D2-like receptors while 5 acts as antagonist at the same
receptor.20 We also showed that compound 4 (30 mg/kg ip) inhibits
the induction of stereotyped behavior by amphetamine in rodents,
an effect predictive of efficacy for treating the positive symptoms of
schizophrenia.21 Subsequently, Löber et al.22 demonstrated that
compound 5 has a high affinity for the D4 receptor, presenting a
Ki value of 9.9 nM and a selectivity ratio of 86–150 when compared
to other D2-like receptors. However, in vivo studies demonstrated
that
4 and 6 modify behaviors and pharmacological effects
mediated by dopaminergic and serotonergic neurotransmission
in rodents, more specifically associated with D2-like, 5-HT1A, and
5-HT2A/C receptors23 pointing to an interesting pharmacological
profile characterized by the involvement of multiple receptors.
In this work, the molecular scaffold of compounds 4–6 was ex-
plored in the search of new antipsychotic lead-compounds with a
multi-receptor profile. The molecular diversity of the compounds
was achieved in three different subunits of the basic scaffold: (a)
isosteric replacement of the heterocyclic ring at the biaryl motif
generating pyrazole, 1,2,3-triazole and 2-methylimidazole[1,2-
a]pyridine analogues, (b) addition of different substituents at the
para-biaryl position (W), and (c) substitution at the para-phenyl-
piperazine position (Y) (Table 1). Substituents were selected
Figure 1. Molecular diversity of functionalized N-phenylpyrazole, N-phenyl-1,2,3-
triazole, and imidazo[1,2-a]pyridine N-phenylpiperazine derivatives (4–24), includ-
ing the structures of clozapine (1) and aripiprazole (2), two atypical antipsychotic
drugs, and bifeprunox (3), in phase III clinical trials.
characteristics of the typical antipsychotics.16,17 Studies with these
drugs demonstrated that compounds with combined 5-HT1A
agonism and D2 blockade might present an increased efficacy for
all kinds of symptoms as well as an improved side-effect
profile.16,18,19
Table 1
Structures of the functionalized N-phenylpiperazine derivatives 4–24 and apparent affinities for D2-like, 5-HT1A and 5-HT2A receptors
Compound
W
Y
Yielda (%)
Molecular formulab
Kic
(
lM)
Ratio
D2
5-HT1A
5-HT2A
D2/5-HT1A
D2/5-HT2A
5-HT1A/5-HT2A
1d
2d
4
—
—
Cl
H
Cl
H
H
H
F
F
Cl
Cl
H
H
F
—
—
H
H
H
H
F
Cl
H
—
—
—
—
0.12
0.001
0.11
0.73
0.95
0.11
0.51
3.74
0.07
0.48
>2.65
1.73
1.98
>7.94
0.74
>7.94
>2.65
1.74
>7.94
3.34
>7.94
>7.94
>7.94
0.38
0.0004
0.09
0.48
1.22
0.05
0.12
0.38
0.06
0.24
0.62
>4.44
1.50
4.25
0.89
3.13
>4.44
7.98
>13.31
2.78
0.014
0.007
2.32
5.66
10.91
1.75
1.04
0.92
0.92
0.53
>6.39
>6.39
3.32
2.06
5.62
0.31
2.50
1.21
1.54
0.78
2.31
4.42
9.89
1.07
2.05
8.57
0.14
0.05
0.13
0.09
0.06
0.48
4.04
0.07
0.91
27.14
0.057
0.04
0.08
0.11
0.03
0.12
0.41
0.07
0.44
77
72
77
90
83
79
79
63
86
85
78
78
82
72
76
38
88
76
—
C20H21ClN4
C19H21N5
C19H20ClN5
C20H22N4
C20H21FN4
C20H21ClN4
C20H21FN4
5e
6
7e
8e
9e
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24e
F
C
20H20F2N4
Cl
OCH3
F
Cl
H
C20H20Cl2N4
C21H23ClN4O
C19H20FN5
C19H20ClN5
C19H20FN5
1.32
0.82
0.60
0.13
0.45
2.07
0.16
0.67
F
F
C19H19F2N5
C19H19Cl2N5
4.67
3.22
Cl
Cl
Cl
NO2
NHAc
Cl
OH
OCH3
H
H
H
C
19H20ClN5O
>19.17
>19.17
>19.17
>19.17
11.57
4.92
0.22
1.20
C20H22ClN5O
C19H20N6O2
C21H24N6O
C19H22N4
>13.31
4.21
>13.31
48
45
0.36
Cl
C19H21ClN4
a
b
c
Isolated yield from the reductive amination step described in Scheme 1.
The analytical results for C, H, N were within 0.4% of calculated values.
For details about the protocol see Section 4.
d
e
Controls: clozapine (1) and aripiprazole (2).
Compounds previously described as high affinity D4 ligands.19,23

G. Neves et al. / Bioorg. Med. Chem. 18 (2010) 1925–1935
1927
according to their electronic, lipophilic and steric properties. This
2.2. Pharmacology
strategy lead to the synthesis and pharmacological evaluation of
16 compounds: seven pyrazolic derivatives 7–13 and nine 1,2,3-
triazole derivatives 14–22 (Fig. 1), including three compounds
(7–9) already described as selective D4 receptor ligands.22 Further-
more, two imidazole[1,2-a]pyridine derivatives 23 and 24 were de-
signed through fusion of phenyl and heteroarylazole rings (Fig. 1),
conferring a planar rigid structure similar to other previously
described dopamine ligands, such as indole,24 benzimidazole,25
pyrrolo[2,3-b]pyridine,24 and pyrazolo[1,5-a]pyridine26–29 deriva-
tives. All these compounds, including 24,26 were described as
selective D4 receptor ligands, including D4-selective imaging
agents.29
Affinity of the compounds for D2-like receptors was determined
via standard competition assays using rat striatum homogenate
with [3H]-YM-9151-2 (nemonapride) as the radioactive ligand.
The affinities for 5-HT1A and 5-HT2A receptors were determined
via competition assays using rat hippocampus homogenate with
[3H]-8-OH-DPAT and rat cortex homogenate with [3H]-ketanser-
ine, respectively. The results of these assays (Table 1) showed that
the N-phenylpiperazine derivatives 4–24 have a broad range of
affinities for these three receptors (Ki values between 0.07–
7.94 lM, 0.05–13.31 lM, and 0.53–19.71 lM for D2-like, 5-HT1A
and 5-HT2A receptors, respectively) and that substituents had ef-
fect on the binding profile.
Evaluating the isosteric replacement of the heterocyclic ring at
the biaryl motif, it is clear that pyrazole derivatives presented
higher affinities than their 1,2,3-triazole and 2-methylimidaz-
ole[1,2-a] pyridine analogues for both dopamine and serotonin
receptors (Table 1). The 1,2,3-triazole compounds presented Ki
values of 4–17-fold higher for D2, 9–15-fold higher for 5-HT1A
and 2–9-fold higher for 5-HT2A receptors than the corresponding
pyrazole analogues. Literature data demonstrated that substitu-
tion of the pyrazolic ring for a pyrrole in this same molecular
scaffold is also prejudicial to D2-like receptor binding.22,34 Besides
the previously described high affinity of 24 for D4 receptors
(Ki = 13 nM),26 the 2-methylimidazole[1,2-a]-pyridine ring addi-
tion strongly reduced the affinity for serotonin receptors and
abolished binding to D2-like receptors. Thus, derivatives 23 and
24 failed to comply with the multi-receptor profile intended in
this work. The observed differences between the affinity of 1-
phenylpyrazole and 1-phenyl-1,2,3-triazole derivatives described
herein for D2-like, 5-HT1A and 5-HT2A receptors could be due to
distinct conformational behavior of N-methylene-N-aryl-pipera-
zine side chain resultant from different stereoelectronic effects
of adjacent CH,CH versus CH,N groups, respectively, at pyrazole
and 1,2,3-triazole rings.
2. Results and discussion
2.1. Chemistry
The synthetic route planned to achieve the N-phenylpiperazine
derivatives 4–21, 23, and 24 explored the reductive amination of
the corresponding substituted 1-aryl-pyrazole-4-carbaldehydes
25a–c, 1-aryl-1,2,3-triazole-4-carbaldehydes 26a–d, and 2-meth-
ylimidazole[1,2-a]pyridine-3-carbaldehyde (27), which were ob-
tained from commercial sources or prepared as described
previously.20,30 The key step was performed through reduction of
the imine adducts formed from treatment of the heterocyclic alde-
hydes 25a–c, 26a–d, and 27 with functionalized N-phenylpipera-
zines 28a–e in dry methanol using sodium cyanoborohydride
and catalytic amounts of acetic acid,31 yielding the desired N-phe-
nylpiperazine derivatives 4–21 and 23–24 as shown in Table 1. On
the other hand, acetamide derivative 22 was prepared in 88% yield
(two steps) from reduction of the nitro compound 21,32 followed
by acetylation of the corresponding aniline intermediate 29
(Scheme 1).33
The structure of all 21 N-phenylpiperazine derivatives 4–24
were confirmed by the different analytical and spectroscopic tech-
niques described in Section 4.
The biarylmethylamine motif where the amine moiety is an
arylpiperazine group has been successfully explored for developing
dopamine receptor ligands as conformationally restricted ana-
logues of benzamide antipsychotics, such as sulpiride. Derivatives
presenting a biphenyl, 1-phenylpyrrole, 2-phenylpyrrole, 2-phen-
ylimidazole, 4-phenylthiazole, and 3-phenylpiridine systems were
successfully described as D4-selective ligands with different intrin-
sic activity.22,31,35–44 An important member of this series of com-
pounds is the 2-phenylimidazole derivative NGD-94-1 (30)
(Fig. 2), considered as a selective D4 full antagonist (Ki = 3.6 nM)
by some authors44 although others reported that it was able to
activate human recombinant dopamine D4.4 receptors expressed
in HEK293 cells.45 With regard to 1-phenylpyrazole moiety ex-
plored in this work, compound FAUC 2020 (31) (Fig. 2) has been re-
ported to be a selective D4 partial agonist (Ki = 0.59 nM).22
Some of the studies cited above carried on with biarylmethyl-
amine derivatives also describe compounds with considerable
affinity for the 5-HT1A receptors.22,35,39,42,44 Both NGD-94-1 (30)
and FAUC 2020 (31) bind with high affinity to this subtype of sero-
tonin receptor (Ki = 180 and 37 nM, respectively).22,44 Indeed, there
is a high molecular similarity between the compounds assayed in
present work, NGD-94-1 (30), FAUC 2020 (31), and bifeprunox
(3), a mixed D2 partial agonist (Ki = 2.2 nM) and 5-HT1A agonist
Scheme 1. Synthesis of functionalized N-phenylpiperazine derivatives 4–24
belonging to pyrazole, 1,2,3-triazole and imidazol [1,2-a] pyridine series.
Figure 2. Structure of D4-selective ligands NGD-94-1 (30) and FAUC 2020 (31).

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G. Neves et al. / Bioorg. Med. Chem. 18 (2010) 1925–1935
(Ki = 9.3 nM)42 currently in phase III clinical trials for schizophrenia
treatment.15 Differently from aripiprazole (2), bifeprunox (3) binds
to D4.4 receptor (Ki = 0.31 nM) acting as a partial agonist at this
dopamine receptor.46 Löber et al.22 reported an affinity 75–650-
fold higher for D4 receptor than for other D2-like receptors for
with the introduction of halogens at Y position (F—5 vs 14, 7 vs
8, 10 vs 11, 16 vs 17; Cl—4 vs 12, 5 vs 15, 6 vs 18, 7 vs 9; Table
1), an effect also reported by Lober et al.22 for binding to all D2-like
receptors (D2 short, D2 long, D3 and D4) and by Feenstra et al.42
with biphenyl derivatives. The presence of bulky groups at Y posi-
tion (Cl—4 vs 12, 5 vs 15, 6 vs 18, 7 vs 9; OCH3—4 vs 13, 6 vs 20,
Table 1) also contributed to decrease the binding to D2-like recep-
tors while a small electron-donor substituent (OH—6 vs 19, Table
1) seems to promote discreet effect. 5-HT2A receptor affinity is
slightly affected by substituents introduced at the Y position. How-
ever, an electronic effect can modulate the compounds selectivity
profile since electronegative substituents (F, Cl) improve (5 vs 14,
7 vs 8, 10 vs 11, 16 vs 17, 5 vs 15, 6 vs 18, 7 vs 9, Table 1) while
electron-donating substituents (OH, OCH3) impair (4 vs 13, 6 vs
19, 6 vs 20, Table 1) binding to the 5-HT2A receptor. As a result,
bulky electronegative substituents at the para-phenylpiperazine
position may lead to compounds with higher affinity for 5-HT2A
than for 5-HT1A or D2-like receptors, such as compound 15. This
observation is in accordance with the finding that the increase of
molecular volume is beneficial for 5-HT2A receptors recognition.47
Based on both the magnitude of binding affinity and the
selectivity profile of N-phenylpiperazine compounds so far stud-
ied (Table 1), five pyrazole derivatives were selected for in vivo
evaluation. Compounds 4, 7, and 10 were chosen taking into ac-
count their highest affinity for D2 and 5-HT1A receptors. They
have an equivalent selectivity profile, with similar affinities for
5-HT1A and D2-like and lower affinity for 5-HT2A receptors (5-
HT1A ffi D2-like > 5-HT2A). Compound 8 was selected due to its
approximately 10-fold higher affinity for 5-HT1A receptor than
the 1-phenyl-1,2,3-triazole derivative
5 (Ki D2-long = 1500 nM;
D3 = 1200 nM; D4 = 9.9 nM) and for the 1-phenylpyrazole
derivatives 7 (Ki D2-long = 140 nM; D3 = 140 nM; D4 = 1.0 nM), 8
(Ki D2-long = 450 nM; D3 = 540 nM; D4 = 2.9 nM) and 9 (Ki D2-long
=
2900 nM; D3 = 1900 nM; D4 = 2.9 nM). Based on this evidence, we
can propose that at least some of our compounds have a high affin-
ity for the D4 receptor. If this could be confirmed through a direct
binding assay for the D4 receptor, such compounds would combine
properties presented by clozapine (1, high affinity for D4 receptors)
and aripiprazole (2, high affinity for 5-HT1A receptors) and thus
provide a multi-receptor profile different from any antipsychotic
drug.
In order to determine the effect of the substituents on binding,
the Ki values were carefully analyzed. Considering substitution at
para-biaryl position (W), neither Cl nor F altered significantly the
affinity of the compounds for the three target receptors (e.g., see
compounds 4 vs 7 vs 10, or compounds 5 vs 6 vs 16, Table 1),
whereas acetamide group decreased significantly the affinity for
D2 and 5-HT1A receptors (5 vs 22, Table 1), possibly due to a steric
hindrance at this position. No significant effect of the substitution
at this position could be noticed for 5-HT2A binding.
On the other hand, addition of different substituents at para-
phenylpiperazine position (Y) produced significant effects on the
selectivity profile. A volume restriction for 5-HT1A binding can also
be observed at this position with significant reduction of affinity
induced by addition of a methoxyl group (12 vs 4, 20 vs 6,
Table 1). A similar effect was reported with biphenyl derivatives,
where a fluorine atom at the para position of the biaryl subunit
slightly decreased the affinity for the 5-HT1A receptor.41 For bind-
ing to the D2-like receptors, a decrease in affinity was achieved
for the other ones,
a binding profile that could exhibit an
in vivo differential effect regarding cognitive symptoms and
extrapyramidal side effects. Finally, compound 11 was also se-
lected as a representative of compounds with similar affinities
for the three receptors, fitting with the multi-target approach
aimed in this work.
Figure 3. Effects of compounds 4, 7, 8, 10, and 11 (15 mg/kg po) in the apomorphine-induced climbing test. Clozapine (1) (15 mg/kg po) and haloperidol (0.5 mg/kg po) were
used as reference drugs. Data are expressed as mean + SEM (n = 08–13). ANOVA (F13,146 = 14.199, P <0.001): different from vehicle + vehicle group in post-hoc test *P <0.05, **P
<0.01, ***P <0.001; different from vehicle + apomorphine group in post-hoc test #P <0.05, ##P <0.01, ###P <0.001.

G. Neves et al. / Bioorg. Med. Chem. 18 (2010) 1925–1935
1929
The animal model chosen for the initial evaluation of selected
compounds was the apomorphine-induced climbing in mice. This
model is based on the induction of a hyperdopaminergic state by
apomorphine and has been classically linked to motor agitation,
one of the schizophrenia positive symptoms.47,48 N-Phenylpiper-
azine derivatives 4 and 10 (15 mg/kg, po) inhibited the apomor-
phine-induced climbing whereas none of the compounds had
significant effect on the climbing index (Fig. 3).
The presence of a halogen atom at the para-biaryl position
seems to be important in order to achieve an antidopaminergic
activity in vivo since compound 7, which has a binding affinity
and selectivity profile similar to 4 and 10, was not active in this
animal model. A similar result was reported by Wijngaaden
et al.36 with 2-phenylpyrrole derivatives where the fluorine addi-
tion at the para-biaryl position reduced fourfold the potency for
antagonizing the apomorphine-induced climbing. The lack of activ-
ity of compounds 8 and 11 at the tested dose is possibly due to
their low affinity for the D2-like receptor, approximately fivefold
lower than for compounds 4 and 10.
To further investigate their pharmacological profile, compounds
4 and 10 were also evaluated in the catalepsy test in mice, an ani-
mal model related to induction of extrapyramidal side effects, and
in the rota-rod test in order to evaluate eventual motor impair-
ments. Both derivatives did not induced catalepsy either at the
dose active in the apomorphine-induced climbing test or at twice
the dose (Table 2). Since 5-HT1A agonists can attenuate the cata-
lepsy induced by haloperidol and other antipsychotics,49–52 the
absence of catalepsy observed with 4 and 10 might be due to
5-HT1A receptor stimulation. On the other hand, the two com-
pounds produced impairment of the motor coordination, since
they reduced the longest permanence time of the animals in the
rota-rod apparatus (Table 2).
The potential of compound 4 (LASSBio-579) for treating the po-
sitive symptoms of schizophrenia had been previously supported
by its ability to inhibit amphetamine-induced stereotypy in rats.21
However, this substance has a limited oral bioavailability (0.6%) as
well as low brain penetration (ratio between brain and plasma
concentration = 6.3%).53 In this work, we showed that the fluorine
derivative compound 10 (LASSBio-664) also has a suitable pharma-
cological profile. Thus, further pharmacological evaluation of 10,
including functional in vitro assays, as well as determination of
its pharmacokinetic profile are necessary to characterize this struc-
turally-related prototype as an optimized analogue of N-phenyl-
piperazine derivative 4.
3. Conclusions
In this work we describe the design, synthesis, and pharmaco-
logical evaluation of N-phenylpiperazine heteroarylazole deriva-
tives as potential multi-target drugs and confirmed the potential
usefulness of this molecular scaffold for the development of new
second generation antipsychotic drugs.
Among the compounds prepared, 4 (LASSBio-579) and 10
(LASSBio-664) exhibited the highest affinity for binding to the
D2-like and 5-HT1A receptors. In mice, these derivatives demon-
strated a potential for treating positive symptoms of schizophrenia,
once they inhibited apomorphine-induced climbing behavior and
were devoid of cataleptogenic effects. Considering that compounds
4 and 10 produce some impairment of motor coordination and that
4 has a poor pharmacokinetic profile, further pharmacological
investigation of compound 10 is needed to verify its potential
advantages.
4. Experimental section
4.1. Chemistry
Melting points were determined with a Quimis Q340.M13
apparatus and are uncorrected. Proton magnetic resonance (1H
NMR), unless otherwise stated, was determined in deuterated
chloroform containing ca. 1% tetramethylsilane as internal stan-
dard in a Bruker DPX-200 spectrometer at 200 MHz. Splitting pat-
terns are as follows: s, singlet; d, doublet; dd, double doublet; m,
multiplet; br, broad. Carbon magnetic resonance (13C NMR) was
determined with Bruker DPX-200 spectrometer at 50 MHz, using
deuterated chloroform containing ca. 1% tetramethylsilane. Infra-
red (IR) spectra were obtained on a Nicolet-55a Magna spectropho-
tometer by using potassium bromide plates or liquid films. The
ultraviolet spectra were obtained on a Hitachi U-2000 Spectropho-
tometer by using methanol as solvent and as internal standard.
Table 2
Effect of N-phenylpiperazine derivatives 4 and 10 in the catalepsy and rota-rod tests (n = 10). Clozapine (1) and haloperidol were used as reference drugs
Treatment
Catalepsy (s)b
60 min
Rota-rod test
30 min
90 min
Permanence time (s)c
After
Number of fallsd
Before After
Before
Vehicle
4
1.8 1.1
5.6 4.8
2.7 2.9
23.6 27.5
2.4 2.6
32.2 45.4
233.3 21.3
220.8 27.8
241.4 18.0
149.3 33.2*
1.3 0.5
1.9 1.0
0.5 0.2
6.0 1.8
(15 mg/kg)
4
3.9 2.7
7.9 6.1
13.4 19.2
15.1 8.4
10.5 9.2
(30 mg/kg)
(15 mg/kg)
(30 mg/kg)
(15 mg/kg)
10
10
1
25.2 16.1
36.6 51.4
3.5 8.7
233.1 22.9
160.6 31.3*
2.4 0.9
8.2 2.6
13.1 10.3
23.4 36.5
54.4 68.2*
17.2 11.2
12.0 8.1
240.6 24.9
267.8 16.9
95.3 30.3***
73.5 20.6***
1.3 0.5
0.7 1.1
20.3 18.3***
15.3 3.8***
Haloa
(4 mg/kg)
89.7 79.1***
105.0 75.9***
a
Halo = haloperidol.
b
Data are expressed as mean SD. Two-way repeated measure ANOVA (treatment factor: F6,209 = 9.190, P <0.001; time factor: F2,209 = 7.052, P = 0.001; treatment  time
interaction: F12,209 = 2.238. P = 0.008): different from vehicle group at the same time of measure in post-hoc test *P <0.05, **P <0.01, ***P <0.001.
c
Data are expressed as mean SD. Two-way repeated measure ANOVA (treatment factor: F4,91 = 2.957, P = 0.031; time factor: F1,91 = 34.785, P <0.001; treatment  time
interaction: F4,91 = 4.981, P = 0.002): different from vehicle group at the same time of measure in post-hoc test *P <0.05, **P <0.01, ***P <0.001.
d
Data are expressed as mean SD. Two-way repeated measure ANOVA (treatment factor: F4,91 = 4.708, P = 0.003; time factor: F1,91 = 27.711, P <0.001; treatment  time
interaction: F4,91 = 5.091, P = 0.002): different from vehicle group at the same time of measure in post-hoc test *P <0.05, **P <0.01, ***P <0.001.

1930
G. Neves et al. / Bioorg. Med. Chem. 18 (2010) 1925–1935
Microanalysis data were obtained on a Thermofinnigan EA1112
analyzer, using a Metler MX5 electronic balance.
95:5). IR (KBr) cmÀ1: 3094–3067 (k C–H), 1599–1501 (k C@C and
C@N), 1097 (k C–Cl); 1H NMR (200 MHz, CDCl3) d: 2.72–2.77 (4H,
m, Ar-CH2N(CH2–CH2)2NPh), 3.20–3.25 (4H, m, Ar-CH2N(CH2–
CH2)2NPh), 3.83 (2H, s, Ar-CH2N(CH2–CH2)2NPh), 6.82–6.95 (3H,
m, H-200, H-400 and H-600), 7.22–7.30 (2H, m, H-300 and H-500), 7.50
(2H, d, J = 8.7 Hz, H-30 and H-50), 7.71 (2H, d, J = 8.7 Hz, H-20 and
H-60), 7.95 (1H, s, H-5); 13C NMR (50 MHz, CDCl3) d: 49.2 (Ar-
CH2N(CH2–CH2)2NPh), 53.2 (Ar-CH2N(CH2–CH2)2NPh), 53.4 (Ar-
CH2N(CH2–CH2)2NPh), 116.3 (C-200 and C-600), 120.0 (C-400), 121.0
(C-5), 121.8 (C-20 and C-60), 129.3 (C-30 and C-50), 130.1 (C-300 and
C-500), 134.6 (C-40), 135.7 (C-10), 145.4 (C-4), 151.3 (C-100); UV
(MeOH) kmax: 252 nm. Anal. Calcd for C19H20ClN5: C, 64.49; H,
5.70; N, 19.79. Found: C, 64.61; H, 5.59; N, 19.75.
The progress of all reactions was monitored by TLC performed on
2.0 cm  6.0 cm aluminum sheets precoated with Silica Gel 60 (F-
254, Merck) to a thickness of 0.25 mm. The developed chromato-
grams were viewed under ultraviolet light at 254 and 365 nm. For
column chromatography Merck Aluminum Oxide 60 (70–230
mesh) was used. The usual workup means that the organic extracts
prior to concentration under reduced pressure had been treated
with a saturated aqueous sodium chloride solution, referred to as
brine, dried over anhydrous sodium sulfate and filtered.
4.2. General procedure for the preparation of N-phenylpiperazine
derivatives 4–21 and 23–24
4.2.4. 1-Phenyl-4-[(1-phenyl-1H-pyrazol-4-yl)methyl]piperazine
(7)
A solution of the corresponding heterocyclic aldehyde 25a–c,
26a–d, or 27 (0.7 mmol) and N-phenylpiperazine derivative 28a–
e (0.7 mmol) in dry methanol (2.5 mL) was adjusted to pH 6.0 by
dropwise addition of concentrated acetic acid. Then, sodium cya-
noborohydride (0.25 g, 4 mmol) was added and the resultant mix-
ture stirred at 60 °C for 2–4 h. After removal of the solvent under
reduced pressure, the residue was partitioned between dichloro-
methane and 10% aqueous potassium phosphate. The organic layer
was separated and submitted to usual workup to yield a crude pre-
cipitate, which was purified by recrystallization in ethanol/water.
Reductive amination between 25a and 28a afforded derivative 7
in 90% yield, as a beige solid, mp 106 °C (literature 109 °C22),
Rf = 0.34 (CH2Cl2/MeOH 95:5). IR (KBr) cmÀ1: 3096–3038 (
1600–1499 (m C@C and C@N), 808 (m C–H), 758 (m
m
C–H),
C–H); 1H NMR
(200 MHz, CDCl3) d: 2.64–2.66 (4H, m, Ar-CH2N(CH2–CH2)2NPh),
3.20–3.23 (4H, m, Ar-CH2N(CH2–CH2)2NPh), 3.56 (2H, s, Ar-
CH2N(CH2–CH2)2NPh), 6.88 (3H, m, H-200, H-400 and H-600), 7.26
(3H, m, H-30, H-40 and H-50), 7.44 (2H, m, H-300 and H-500), 7.67
(1H, s, H-3), 7.68 (2H, m, H-20 and H-60), 7.90 (1H, s, H-5); 13C
NMR (50 MHz, CDCl3) d: 49.3 (Ar-CH2N(CH2–CH2)2NPh), 52.8 (Ar-
CH2N(CH2–CH2)2NPh), 53.1 (Ar-CH2N(CH2–CH2)2NPh), 116.2 (C-200
and C-600), 119.1 (C-20 and C-60), 119.9 (C-400), 126.5 (C-40), 126.7
(C-5), 129.3 (C-300 and C-500), 129.6 (C-30 and C-50), 140.3 (C-10),
142.0 (C-3), 151.5 (C-100); UV (MeOH) kmax: 252.0 nm. Anal. Calcd
for C20H22N4: C, 75.44; H, 6.96; N, 17.60. Found: C, 75.29; H,
6.93; N, 17.63.
4.2.1. 1-[(1-(4-Chlorophenyl)-1H-pyrazol-4-yl)methyl]-4-phe
nylpiperazine (4)
Reductive amination between 25c and 28a afforded derivative 4
in 77% yield, as a white solid, mp 122 °C, Rf = 0.38 (CH2Cl2/MeOH
95:5). IR (KBr) cmÀ1: 3106–3022 (
m
C–H), 1598–1497 (
m C@C and
C@N), 1093 (
m
C–Cl); 1H NMR (200 MHz, CDCl3) d: 2.67–2.71 (4H,
m, Ar-CH2N(CH2–CH2)2NPh), 3.21–3.26 (4H, m, Ar-CH2N(CH2–
CH2)2NPh), 3.59 (2H, s, Ar-CH2N(CH2–CH2)2NPh), 6.83–6.95 (3H,
m, H-200, H-400 and H-600), 7.23–7.30 (2H, m, H-300 and H-500), 7.42
(2H, d, J = 8.9 Hz, H-30 and H-50), 7.64 (2H, d, J = 8.9 Hz, H-20 and
H-60), 7.68 (1H, s, H-3), 7.90 (1H, s, H-5); 13C NMR (50 MHz, CDCl3)
d: 49.2 (Ar-CH2N(CH2–CH2)2NPh), 52.7 (Ar-CH2N(CH2–CH2)2NPh),
53.1 (Ar-CH2N(CH2–CH2)2NPh), 116.3 (C-200 and C-600), 119.9 (C-
4), 120.2 (C-20 and C-60), 126.6 (C-5), 129.3 (C-30 and C-50), 129.7
(C-300 and C-500), 132.0 (C-40), 138.8 (C-10), 142.3 (C-3), 151.4 (C-
100); UV (MeOH) kmax: 255.0 nm. Anal. Calcd for C20H21ClN4: C,
68.08; H, 6.00; N, 15.88. Found: C, 67.89; H, 5.98; N, 15.93.
4.2.5. 1-(4-Fluorophenyl)-4-[(1-phenyl-1H-pyrazol-4-yl)
methyl]piperazine (8)
Reductive amination between 25a and 28b afforded derivative
8 in 83% yield, as a beige solid, mp 111–113 °C, Rf = 0.34 (CH2Cl2/
MeOH 95:5). IR (KBr) cmÀ1: 3097–3050 (
m
C–H), 1597–1499 (
m
C@C and C@N), 1250–1241 (
m
C–F), 814 (
m
C–H), 755 (
m
C–H); 1H
NMR (200 MHz, CDCl3) d: 2.64–2.66 (4H, m, Ar-CH2N(CH2–
CH2)2NPh), 3.12–3.14 (4H, m, Ar-CH2N(CH2–CH2)2NPh), 3.55 (2H,
s, Ar-CH2N(CH2–CH2)2NPh), 6.90 (4H, m, H-200, H-300, H-500 and H-
600), 7.28 (1H, m, H-40), 7.44 (2H, m, H-3 and H-5), 7.69 (2H, m,
H-20 and H-60), 7.70 (1H, s, H-3), 7.89 (1H, s, H-5); 13C NMR
(50 MHz, CDCl3) d: 50.3 (Ar-CH2N(CH2–CH2)2NPh), 52.7 (Ar-
CH2N(CH2–CH2)2NPh), 53.0 (Ar-CH2N(CH2–CH2)2NPh), 115.6 (2C,
d, J = 21.9 Hz, C-300 and C-500), 118.0 (2C, d, J = 7.5 Hz, C-200 and C-
600), 119.0 (C-20 and C-60), 126.5 (C-40), 126.6 (C-5), 129.6 (C-30
and C-50), 140.3 (C-10), 142.0 (C-3), 148.1 (1C, d, J = 2.2 Hz, C-100),
157.6 (1C, d, J = 237.3 Hz, C-400); UV (MeOH) kmax: 249.0 nm. Anal.
Calcd for C20H21FN4: C, 71.41; H, 6.29; N, 16.65. Found: C, 71.52; H,
6.32; N, 16.72.
4.2.2. 1-Phenyl-4-[(1-phenyl-1H-1,2,3-triazol-4-yl)methyl]
piperazine (5)
Reductive amination between 26a and 28a afforded derivative 5
in 72% yield, as a white solid, mp 150 °C, Rf = 0.48 (CH2Cl2/MeOH
95:5). IR (KBr) cmÀ1: 3083–3035 (
m C–H), 1601–1501 (m C@C and
C@N); 1H NMR (200 MHz, CDCl3) d: 2.73–2.78 (4H, m, Ar-
CH2N(CH2–CH2)2NPh), 3.21–3.26 (4H, m, Ar-CH2N(CH2–CH2)2NPh),
3.84 (2H, s, Ar-CH2N(CH2–CH2)2NPh), 6.82–6.95 (3H, m, H-200, H-400
and H-600), 7.22–7.30 (2H, m, H-300 and H-500), 7.44–7.54 (3H, m, H-
30, H-40 and H-50), 7.73–7.77 (2H, m, H-20 and H-60), 7.98 (1H, s, H-
5); 13C NMR (50 MHz, CDCl3) d: 49.2 (Ar-CH2N(CH2–CH2)2NPh),
53.2 (Ar-CH2N(CH2–CH2)2NPh), 53.4 (Ar-CH2N(CH2–CH2)2NPh),
116.3 (C-200 and C-600), 120.0 (C-400), 120.6 (C-20 and C-60), 121.1
(C-5), 128.9 (C-40), 129.3 (C-30 and C-50), 130.0 (C-300 and C-500),
137.2 (C-10), 145.1 (C-4), 151.4 (C-100); UV (MeOH) kmax: 246 nm.
Anal. Calcd for C19H21N5: C, 71.45; H, 6.63; N, 21.93. Found: C,
71.56; H, 6.54; N, 21.77.
4.2.6. 1-(4-Chlorophenyl)-4-[(1-phenyl-1H-pyrazol-4-yl)
methyl]piperazine (9)
Reductive amination between 25a and 28c afforded derivative 9
in 79% yield, as a white solid, mp 125 °C, Rf = 0.36 (CH2Cl2/MeOH
95:5). IR (KBr) cmÀ1: 3099–3044 (
m
C–H), 1641–1499 (
m C@C and
C@N), 1117 (
m
C–Cl), 808 (
m
C–H), 751
(m
C–H); 1H NMR
(200 MHz, CDCl3) d: 2.62–2.65 (4H, m, Ar-CH2N(CH2–CH2)2NPh),
3.16–3.18 (4H, m, Ar-CH2N(CH2–CH2)2NPh), 3.55 (2H, s, Ar-
CH2N(CH2–CH2)2NPh), 6.83 (2H, d, J = 9.0 Hz, H-200 and H-600), 7.19
(2H, d, J = 9.0 Hz, H-300 and H-500), 7.28 (1H, m, H-40), 7.44 (2H, m,
H-30 and H-50), 7.68 (2H, s, H-20 and H-60), 7.70 (1H, s, H-3), 7.89
(1H, s, H-5); 13C NMR (50 MHz, CDCl3) d: 49.3 (Ar-CH2N(CH2–
CH2)2NPh), 52.7 (Ar-CH2N(CH2–CH2)2NPh), 52.9 (Ar-CH2N(CH2–
4.2.3. 1-[(1-(4-Chlorophenyl)-1H-1,2,3-triazol-4-yl)methyl]-4-
phenylpiperazine (6)
Reductive amination between 26c and 28a afforded derivative 6
in 77% yield, as a white solid, mp 151 °C, Rf = 0.48 (CH2Cl2/MeOH

G. Neves et al. / Bioorg. Med. Chem. 18 (2010) 1925–1935
1931
CH2)2NPh), 117.4 (C-200 and C-600), 119.0 (C-20 and C-60), 124.7 (C-
400), 126.5 (C-40), 126.6 (C-5), 129.1 (C-300 and C-500), 129.6 (C-30
and C-50), 140.3 (C-10), 142.0 (C-3), 150.1 (C-100); UV (MeOH) kmax
255.5 nm. Anal. Calcd for C20H21ClN4: C, 68.08; H, 6.00; N, 15.88.
Found: C, 67.85; H, 6.09; N, 15.75.
4.2.10. 1-{[1-(4-Chlorophenyl)-1H-pyrazol-4-yl]methyl}-4-(4-
methoxyphenyl)piperazine (13)
:
Reductive amination between 25c and 28e afforded derivative
13 in 85% yield, as a white solid, mp 145 °C, Rf = 0.32 (CH2Cl2/
MeOH 95:5). IR (KBr) cmÀ1: 3107–3078 (
m
C–H), 1595–1498 (
m
C@C and C@N), 1245 (
m
C–O–C), 1093 (
m
C–Cl), 1036 ( C–O–C);
m
1H NMR (200 MHz, CDCl3) d: 2.72 (4H, s, Ar-CH2N(CH2–CH2)2NPh),
3.15 (4H, s, Ar-CH2N(CH2–CH2)2NPh), 3.61 (2H, s, Ar-CH2N(CH2–
CH2)2NPh), 3.76 (3H, s, OCH3), 6.82 (2H, d, J = 9.3 Hz, H-200 and H-
600), 6.90 (2H, d, J = 9.3 Hz, H-300, and H-500), 7.41 (2H, d, J = 8.8 Hz,
H-30 and H-50), 7.63 (2H, d, J = 8.8 Hz, H-20 and H-60), 7.67 (1H, s,
H-3), 7.95 (1H, s, H-5); 13C NMR (50 MHz, CDCl3) d: 50.7 (Ar-
CH2N(CH2–CH2)2NPh), 52.6 (Ar-CH2N(CH2–CH2)2NPh), 53.1 (Ar-
CH2N(CH2–CH2)2NPh), 55.7 (OCH3), 114.6 (C-200 and C-600), 118.4
(C-300 and C-500), 120.1 (C-20 and C-60), 126.7 (C-5), 129.6 (C-30
and C-50), 131.9 (C-40), 138.8 (C-10), 142.3 (C-3), 145.7 (C-100),
4.2.7. 1-[(1-(4-Fluorophenyl)-1H-pyrazol-4-yl)methyl)-4-
phenylpiperazine (10)
Reductive amination between 25b and 28a afforded derivative
10 in 79% yield, as a white solid, mp 106 °C, Rf = 0.31 (CH2Cl2/
MeOH 95:5). IR (KBr) cmÀ1: 3096–3081 (
m
C–H), 1599–1513 (
m
C@C and C@N), 1236 (
m
C–F), 837 (
m
C–H), 757 (
m
C–H); 1H NMR
(200 MHz, CDCl3) d: 2.64–2.66 (4H, m, Ar-CH2N(CH2–CH2)2NPh),
3.21–3.23 (4H, m, Ar-CH2N(CH2–CH2)2NPh), 3.55 (2H, s, Ar-
CH2N(CH2–CH2)2NPh), 6.88 (3H, m, H-200, H-400 and H-600), 7.14
(2H, m, H-300 and H-500), 7.26 (2H, m, H-30 and H-50), 7.62 (2H, m,
H-20 and H-60), 7.66 (1H, s, H-3), 7.83 (1H, s, H-5); 13C NMR
(50 MHz, CDCl3) d: 49.3 (Ar-CH2N(CH2–CH2)2NPh), 52.7 (Ar-
CH2N(CH2–CH2)2NPh), 53.1 (Ar-CH2N(CH2–CH2)2NPh), 116.3 (C-200
and C-600), 116.4 (2C, d, J = 22.8 Hz, C-30 and C-50), 119.9 (C-400),
120.8 (2C, d, J = 8.2 Hz, C-20 and C-60), 126.8 (C-5), 129.3 (C-300
and C-500), 136.6 (C-10), 142.0 (C-3), 151.4 (C-100), 161.2 (1C, d,
J = 244.2 Hz, C-40); UV (MeOH) kmax: 250.0 nm. Anal. Calcd for
C20H20FN4: C, 71.41; H, 6.29; N, 16.65. Found: C, 71.48; H, 6.25;
N, 16.62.
154.0 (C-400); UV (MeOH) kmax
: 255.0 nm. Anal. Calcd for
C21H23ClN4O: C, 65.87; H, 6.05; N, 14.63. Found: C, 65.95; H,
6.02; N, 14.71.
4.2.11. 1-(4-Fluorophenyl)-4-[(1-phenyl-1H-1,2,3-triazol-4-
yl)methyl]piperazine (14)
Reductive amination between 26a and 28b afforded derivative
14 in 78% yield, as a white solid, mp 150 °C, Rf = 0.39 (CH2Cl2/
MeOH 95:5). IR (KBr) cmÀ1: 3082–3050 (
m
C–H), 1595–1500 (
m
C@C and C@N), 1241 (
m
C–F), 813 (
m
C–H), 757 (
m
C–H); 1H NMR
(200 MHz, CDCl3) d: 2.73–2.76 (4H, m, Ar-CH2N(CH2–CH2)2NPh),
3.13–3.16 (4H, m, Ar-CH2N(CH2–CH2)2NPh), 3.83 (2H, s, Ar-
CH2N(CH2–CH2)2NPh), 6.92 (4H, m, H-200, H-300, H-500 and H-600),
7.48 (2H, m, H-30, H-40 and H-50), 7.75 (2H, m, H-20 and H-60),
7.98 (1H, s, H-5); 13C NMR (50 MHz, CDCl3) d: 50.3 (Ar-
CH2N(CH2–CH2)2NPh), 53.2 (Ar-CH2N(CH2–CH2)2NPh), 53.5 (Ar-
CH2N(CH2–CH2)2NPh), 115.9 (2C, d, J = 22.0 Hz, C-300 and C-500),
118.0 (2C, d, J = 7.6 Hz, C-200 and C-600), 120.6 (C-20 and C-60),
121.0 (C-5), 129.9 (C-30 and C-50), 145.3 (C-4), 148.1 (C-100); UV
(MeOH) kmax: 242.5 nm. Anal. Calcd for C19H20FN5: C, 67.64; H,
5.97; N, 20.76. Found: C, 67.49; H, 6.01; N, 20.82.
4.2.8. 1-(4-Fluorophenyl)-4-{[1-(4-fluorophenyl)-1H-pyrazol-4-
yl]methyl}piperazine (11)
Reductive amination between 25b and 28b afforded derivative
11 in 63% yield, as a white solid, mp 110 °C, Rf = 0.31 (CH2Cl2/
MeOH 95:5). IR (KBr) cmÀ1: 3096–3083 (
m
C–H), 1566–1516 (
m
C@C and C@N), 1247 (
m
C–F); 1H NMR (200 MHz, CDCl3) d: 2.66
(4H, s, Ar-CH2N(CH2–CH2)2NPh), 3.13 (4H, s, Ar-CH2N(CH2–
CH2)2NPh), 3.55 (2H, s, Ar-CH2N(CH2–CH2)2NPh), 6.94 (4H, m, H-
200, H-300, H-500 and H-600), 7.13 (2H, m, H-30 and H-50), 7.63 (2H,
m, H-20 and H-60), 7.66 (1H, s, H-3), 7.83 (1H, s, H-5); 13C NMR
(50 MHz, CDCl3) d: 50.3 (Ar-CH2N(CH2–CH2)2NPh), 52.6 (Ar-
CH2N(CH2–CH2)2NPh), 53.0 (Ar-CH2N(CH2–CH2)2NPh), 115.6 (2C,
d, J = 21.9 Hz, C-300 and C-500), 116.4 (2C, d, J = 22.8 Hz, C-30 and C-
50), 118.0 (2C, d, J = 7.6 Hz, C-200 and C-600), 120.8 (2C, d, J = 8.2 Hz,
C-20 and C-60), 126.8 (C-5), 136.6 (1C, d, J = 2.8 Hz, C-10), 142.0 (C-
3), 148.1 (1C, d, J = 2.2 Hz, C-100), 156.8 (1C, d, J = 189.6 Hz, C-400),
161.7 (1C, d, J = 196.6 Hz, C-40); UV (MeOH) kmax: 248.0 nm. Anal.
Calcd for C20H20F2N4: C, 67.78; H, 5.69; N, 15.81. Found: C, 67.55;
H, 5.72; N, 15.72.
4.2.12. 1-(4-Chlorophenyl)-4-[(1-phenyl-1H-1,2,3-triazol-4-
yl)methyl]piperazine (15)
Reductive amination between 26a and 28c afforded derivative
15 in 78% yield, as a white solid, mp 150 °C, Rf = 0.35 (CH2Cl2/
MeOH 95:5). IR (KBr) cmÀ1: 3083 (
m
C–H), 1598–1498 (
m C@C
and C@N), 1044 (
m
C–Cl), 811 (
m
C–H), 759 (
m
C–H); 1H NMR
(200 MHz, CDCl3) d: 2.65–2.67 (4H, m, Ar-CH2N(CH2–CH2)2NPh),
3.09–3.12 (4H, m, Ar-CH2N(CH2–CH2)2NPh), 3.75 (2H, s, Ar-
CH2N(CH2–CH2)2NPh), 6.74 (2H, d, J = 8.9 Hz, H-200 and H-600), 7.11
(2H, d, J = 8.9 Hz, H-300 and H-500), 7.39 (3H, m, H-30, H-40 and H-
50), 7.66 (2H, m, H-20 and H-60), 7.90 (1H, s, H-5); 13C NMR
(50 MHz, CDCl3) d: 49.2 (Ar-CH2N(CH2–CH2)2NPh), 53.0 (Ar-
CH2N(CH2–CH2)2NPh), 53.4 (Ar-CH2N(CH2–CH2)2NPh), 117.4 (C-200
and C-600), 120.6 (C-20 and C-60), 121.1 (C-5), 124.7 (C-400), 128.9
(C-40), 129.1 (C-300 and C-500), 129.9 (C-30 and C-50), 137.2 (C-10),
145.0 (C-4), 150.0 (C-100); UV (MeOH) kmax: 252.0 nm. Anal. Calcd
for C19H20ClN5: C, 64.49; H, 5.70; N, 19.79. Found: C, 64.61; H,
5.72; N, 19.77.
4.2.9. 1-(4-Chlorophenyl)-4-{[1-(4-chlorophenyl)-1H-pyrazol-
4-yl]methyl}piperazine (12)
Reductive amination between 25c and 28c afforded derivative
12 in 86% yield, as a white solid, mp 130–132 °C, Rf = 0.37
(CH2Cl2/MeOH 95:5). IR (KBr) cmÀ1: 3106–3078 (
1499 (m C@C and C@N), 1096 (m
m
C–H), 1596–
C–Cl); 1H NMR (200 MHz, CDCl3)
d: 2.63–2.65 (4H, m, Ar-CH2N(CH2–CH2)2NPh), 3.16–3.19 (4H, m,
Ar-CH2N(CH2–CH2)2NPh), 3.55 (2H, s, Ar-CH2N(CH2–CH2)2NPh),
6.83 (2H, d, J = 8.9 Hz, H-200 and H-600), 7.20 (2H, d, J = 8.9 Hz, H-
300 and H-500), 7.41 (2H, d, J = 8.9 Hz, H-30 and H-50), 7.63 (2H, d,
J = 8.9 Hz, H-20 and H-60), 7.68 (1H, s, H-3), 7.86 (1H, s, H-5); 13C
NMR (50 MHz, CDCl3) d: 49.4 (Ar-CH2N(CH2–CH2)2NPh), 52.7 (Ar-
CH2N(CH2–CH2)2NPh), 53.0 (Ar-CH2N(CH2–CH2)2NPh), 117.5 (C-200
and C-600), 120.2 (C-20 and C-60), 124.8 (C-400), 126.6 (C-5), 129.2
(C-300 and C-500), 129.7 (C-30 and C-50), 132.0 (C-40), 138.9 (C-10),
142.3 (C-3), 150.1 (C-100); UV (MeOH) kmax: 258.0 nm. Anal. Calcd
for C20H20Cl2N4: C, 62.02; H, 5.20; N, 14.47. Found: C, 62.14; H,
5.18; N, 14.52.
4.2.13. 1-{[1-(4-Fluorophenyl)-1H-1,2,3-triazol-4-yl]methyl}-4-
phenylpiperazine (16)
Reductive amination between 26b and 28a afforded derivative
16 in 82% yield, as a white solid, mp 143 °C, Rf = 0.48 (CH2Cl2/
MeOH 95:5). IR (KBr) cmÀ1: 3096–3011 (
m
C–H), 1600–1503 (
m
C@C and C@N), 1257 (
m
C–F), 841 (
m
C–H), 757 (
m
C–H); 1H NMR
(200 MHz, CDCl3) d: 2.73–2.76 (4H, m, Ar-CH2N(CH2–CH2)2NPh),
3.22–3.24 (4H, m, Ar-CH2N(CH2–CH2)2NPh), 3.83 (2H, s, Ar-
CH2N(CH2–CH2)2NPh), 6.88 (3H, m, H-200, H-400 and H-600), 7.24

1932
G. Neves et al. / Bioorg. Med. Chem. 18 (2010) 1925–1935
(2H, m, H-300 and H-500), 7.24 (2H, m, H-30 and H-50), 7.73 (2H, dd,
J = 4.6 Hz and 8.7 Hz, H-20 and H-60), 7.93 (1H, s, H-5); 13C NMR
(50 MHz, CDCl3) d: 49.0 (Ar-CH2N(CH2–CH2)2NPh), 53.1 (Ar-
CH2N(CH2–CH2)2NPh), 53.3 (Ar-CH2N(CH2–CH2)2NPh), 116.5 (C-200
and C-600), 116.9 (2C, d, J = 23.0 Hz, C-30 and C-50), 120.3 (C-400),
121.8 (C-5), 122.7 (2C, d, J = 8.6 Hz, C-20 and C-60), 129.4 (C-300
and C-500), 133.5 (C-10), 144.4 (C-4), 162.6 (1C, d, J = 247.6 Hz, C-
40); UV (MeOH) kmax: 245.0 nm. Anal. Calcd for C19H20FN5: C,
67.64; H, 5.97; N, 20.76. Found: C, 67.55; H, 5.94; N, 20.71.
4.2.17. 1-(4-Methoxyphenyl)-4-{[1-(4-chlorophenyl)-1H-1,2,3-
triazol-4-yl]methyl}piperazine (20)
Reductive amination between 26c and 28e afforded derivative
20 in 88% yield, as a white solid, mp 153 °C, Rf = 0.48 (CH2Cl2/
MeOH 95:5). IR (KBr) cmÀ1: 3092–3068 (
m
C–H), 1514–1501 (
C–O–C), 1097 ( C–Cl), 1036–1022
m
C–O–C); 1H NMR (200 MHz, CDCl3) d: 2.76 (4H, s, Ar-
m
C@C and C@N), 1252–1228 (
m
m
(
CH2N(CH2–CH2)2NPh), 3.12 (4H, s, Ar-CH2N(CH2–CH2)2NPh), 3.76
(2H, s, Ar-CH2N(CH2–CH2)2NPh), 3.82 (3H, s, OCH3), 6.88 (4H, m,
H-200, H-300, H-500 and H-600), 7.49 (2H, d, J = 8.0 Hz, H-30 and H-50),
7.70 (2H, d, J = 8.0 Hz, H-20 and H-60), 7.95 (1H, s, H-5); 13C NMR
(50 MHz, CDCl3) d: 50.7 (Ar-CH2N(CH2–CH2)2NPh), 53.3 (Ar-
CH2N(CH2–CH2)2NPh), 53.4 (Ar-CH2N(CH2–CH2)2NPh), 55.7
(OCH3), 114.6 (C-200 and C-600), 118.3 (C-300 and C-500), 120.8 (C-5),
121.7 (C-20 and C-60), 130.0 (C-30 and C-50), 134.5 (C-40), 135.7 (C-
10), 145.6 (C-4), 154.0 (C-400); UV (MeOH) kmax: 244.5 nm. Anal.
Calcd for C20H22ClN5O: C, 62.58; H, 5.78; N, 18.24. Found: C,
62.71; H, 5.83; N, 18.19.
4.2.14. 1-(4-Fluorophenyl)-4-{[1-(4-fluorophenyl)-1H-1,2,3-
triazol-4-yl]methyl}piperazine (17)
Reductive amination between 26b and 28b afforded derivative
17 in 72% yield, as a white solid, mp 148 °C, Rf = 0.39 (CH2Cl2/
MeOH 95:5). IR (KBr) cmÀ1: 3082–3059 (
m
C–H), 1604–1518 (
m
C@C and C@N), 1247 (
m
C–F); 1H NMR (200 MHz, CDCl3) d: 2.73–
2.75 (4H, m, Ar-CH2N(CH2–CH2)2NPh), 3.13–3.15 (4H, m, Ar-
CH2N(CH2–CH2)2NPh), 3.82 (2H, s, Ar-CH2N(CH2–CH2)2NPh), 6.89
(4H, m, H-200, H-300, H-500 and H-600), 7.20 (2H, d, J = 8.6 Hz, H-30
and H-50), 7.72 (2H, dd, J = 4.6 Hz and 8.6 Hz, H-20 and H-60), 7.92
(1H, s, H-5); 13C NMR (50 MHz, CDCl3) d: 50.3 (Ar-CH2N(CH2–
CH2)2NPh), 53.2 (Ar-CH2N(CH2–CH2)2NPh), 53.4 (Ar-CH2N(CH2–
CH2)2NPh), 115.7 (2C, d, J = 22.0 Hz, C-300 and C-500), 116.9 (2C, d,
J = 23.1 Hz, C-30 and C-50), 118.0 (2C, d, J = 7.6 Hz, C-200 and C-600),
121.2 (C-5), 122.6 (2C, d, J = 8.6 Hz, C-20 and C-60), 133.6 (C-10),
145.5 (C-4), 148.1 (C-100), 157.4 (1C, d, J = 237.4 Hz, C-400), 162.6
(1C, d, J = 247.5 Hz, C-40); UV (MeOH) kmax: 241.5 nm. Anal. Calcd
for C19H19F2N5: C, 64.21; H, 5.39; N, 19.71. Found: C, 64.08; H,
5.44; N, 19.80.
4.2.18. 1-{[1-(4-Nitrophenyl)-1H-1,2,3-triazol-4-yl]methyl}-4-
phenylpiperazine (21)
Reductive amination between 26d and 28a afforded derivative
21 in 76% yield, as a yellow solid, mp 153 °C, Rf = 0.38 (CH2Cl2/
MeOH 95:5). IR (KBr) cmÀ1: 3085 (
m
C–H), 1598–1500 (
m C@C
and C@N), 1345 (
m
C–NO2), 855 (
m
C–H), 761 (
m
C–H); 1H NMR
(200 MHz, CDCl3) d: 2.75–2.77 (4H, m, Ar-CH2N(CH2–CH2)2NPh),
3.22–3.24 (4H, m, Ar-CH2N(CH2–CH2)2NPh), 3.86 (2H, s, Ar-
CH2N(CH2–CH2)2NPh), 6.88 (3H, m, H-200, H-400 and H-600), 7.26
(2H, m, H-300 and H-500), 7.99 (2H, d, J = 9.0 Hz, H-20 and H-60),
8.12 (1H, s, H-5), 8.41 (2H, d, J = 9.0 Hz, H-30 and H-50); 13C NMR
(50 MHz, CDCl3) d: 49.22 (Ar-CH2N(CH2–CH2)2NPh), 53.2 (Ar-
CH2N(CH2–CH2)2NPh), 53.3 (Ar-CH2N(CH2–CH2)2NPh), 116.3 (C-200
and C-600), 120.0 (C-400), 120.5 (C-20 and C-60), 120.9 (C-5), 125.7
(C-30 and C-50), 129.3 (C-300 and C-500), 141.4 (C-40), 146.3 (C-4),
147.3 (C-10), 151.3 (C-100); UV (MeOH) kmax: 248.5 and 283.0 nm.
Anal. Calcd for C19H20N6O2: C, 62.62; H, 5.53; N, 23.06. Found: C,
62.73; H, 5.48; N, 23.12.
4.2.15. 1-(4-Chlorophenyl)-4-{[1-(4-chlorophenyl)-1H-1,2,3-
triazol-4-yl]methyl}piperazine (18)
Reductive amination between 26c and 28c afforded derivative
18 in 76% yield, as a white solid, mp 143 °C, Rf = 0.37 (CH2Cl2/
MeOH 95:5). IR (KBr) cmÀ1: 3101–3085 (
m
C–H), 1595–1496 (
m
C@C and C@N), 1096 (
m
C–Cl); 1H NMR (200 MHz, CDCl3) d:
2.72–2.75 (4H, m, Ar-CH2N(CH2–CH2)2NPh), 3.17–3.20 (4H, m,
Ar-CH2N(CH2–CH2)2NPh), 3.82 (2H, s, Ar-CH2N(CH2–CH2)2NPh),
6.83 (2H, d, J = 8.9 Hz, H-200 and H-600), 7.19 (2H, d, J = 8.9 Hz, H-
300 and H-500), 7.50 (2H, d, J = 8.8 Hz, H-30 and H-50), 7.70 (2H, d,
J = 8.8 Hz, H-20 and H-60), 7.96 (1H, s, H-5); 13C NMR (50 MHz,
CDCl3) d: 49.2 (Ar-CH2N(CH2–CH2)2NPh), 53.0 (Ar-CH2N(CH2–
CH2)2NPh), 53.4 (Ar-CH2N(CH2–CH2)2NPh), 124.7 (C-400), 129.1 (C-
300 and C-500), 130.1 (C-30 and C-50), 134.6 (C-40), 135.8 (C-10),
145.5 (C-4), 150.0 (C-100); UV (MeOH) kmax: 253.5 nm. Anal. Calcd
for C19H19Cl2N5: C, 58.77; H, 4.93; N, 18.04. Found: C, 58.55; H,
4.99; N, 18.06.
4.2.19. 2-Methyl-3-(4-phenylpiperazinylmethyl)imidazo[1,2-
a]-pyridine (23)
Reductive amination between 27 and 28a afforded derivative
23 in 48% yield, as a yellow solid, mp 103–105 °C, Rf = 0.33
(CH2Cl2/MeOH 95:5). IR (KBr) cmÀ1: 3090–3062 (
1504 (m C@C and C@N), 754 (m
m
C–H), 1634–
C–H); 1H NMR (200 MHz, CDCl3)
d: 2.46 (3H, s, CH3), 2.59–2.61 (4H, m, Ar-CH2N(CH2–CH2)2NPh),
3.15–3.17 (4H, m, Ar-CH2N(CH2–CH2)2NPh), 3.81 (2H, s, Ar-
CH2N(CH2–CH2)2NPh), 6.84 (3H, m, H-20, H-40 and H-60), 6.84 (1H,
m, H-6), 7.20 (2H, m, H-30 and H-50), 7.20 (1H, m, H-7), 7.52 (1H,
d, J = 8.9 Hz, H-8), 8.26 (1H, d, J = 6.6 Hz, H-5); 13C NMR (50 MHz,
CDCl3) d: 13.6 (CH3), 49.3 (Ar-CH2N(CH2–CH2)2NPh), 51.6 (Ar-
CH2N(CH2–CH2)2NPh), 53.0 (Ar-CH2N(CH2–CH2)2NPh), 111.6 (C-
6), 116.0 (C-2), 116.2 (C-20 and C-60), 116.6 (C-8), 119.9 (C-40),
124.1 (C-7), 124.9 (C-5), 129.3 (C-30 and C-50), 142.4 (C-3), 144.9
(C-9), 151.4 (C-10); UV (MeOH) kmax: 228.0 and 246.5 nm. Anal.
Calcd for C19H22N4: C, 74.48; H, 7.24; N, 18.29. Found: C, 74.37;
H, 7.29; N, 18.22.
4.2.16. 1-(4-Hydroxyphenyl)-4-{[1-(4-chlorophenyl)-1H-1,2,3-
triazol-4-yl]methyl}piperazine (19)
Reductive amination between 26c and 28d afforded derivative
19 in 38% yield, as a white solid, mp 228–230 °C, Rf = 0.16
(CH2Cl2/MeOH 95:5). IR (KBr) cmÀ1: 3530 (
m
O-H), 1510–1498 (
C–O–C), 1110 ( C–Cl), 1048–1029
m
C–O–C); 1H NMR (200 MHz, CDCl3) d: 2.18 (1H, br, OH), 2.98
m
C@C and C@N), 1248–1216 (
m
m
(
(4H, s, Ar-CH2N(CH2–CH2)2NPh), 3.10 (4H, s, Ar-CH2N(CH2–
CH2)2NPh), 3.83 (2H, s, Ar-CH2N(CH2–CH2)2NPh), 6.80 (4H, m, H-
200, H-300, H-500 and H-600), 7.50 (2H, d, J = 8.8 Hz, H-30 and H-50),
7.69 (2H, d, J = 8.8 Hz, H-20 and H-60), 7.94 (1H, s, H-5); 13C NMR
(50 MHz, CDCl3) d: 49.9 (Ar-CH2N(CH2–CH2)2NPh), 52.2 (Ar-
CH2N(CH2–CH2)2NPh), 53.8 (Ar-CH2N(CH2–CH2)2NPh), 115.0 (C-200
and C-600), 118.8 (C-300 and C-500), 121.3 (C-5), 121.7 (C-20 and C-
60), 133.9 (C-40), 135.9 (C-10), 144.8 (C-4), 155.1 (C-400); UV (MeOH)
kmax: 239.5 nm. Anal. Calcd for C19H20ClN5O: C, 61.70; H, 5.45; N,
18.94. Found: C, 61.73; H, 5.47; N, 19.01.
4.2.20. 2-Methyl-3-[4-(4-chlorophenyl) piperazinylmethyl]imidazo-
[1,2-a]-pyridine (24)
Reductive amination between 27 and 28b afforded derivative
24 in 45% yield, as a yellow solid, mp 122–123 °C, Rf = 0.34
(CH2Cl2/MeOH 95:5); 1H NMR (200 MHz, CDCl3 )
2.46 (3H, s,
d:
CH3), 2.57–2.60 (4H, m, Ar-CH2N(CH2–CH2)2NPh), 3.10–3.13 (4H,
m, Ar-CH2N(CH2–CH2)2NPh), 3.80 (2H, s, Ar-CH2N(CH2–CH2)2NPh),
6.78 (1H, m, H-6), 6.81 (2H, d, J = 8.8 Hz, H-20 and H-60), 7.16 (1H,
m, H-7), 7.18 (2H, d, J = 8.8 Hz, H-30 and H-50), 7.52 (1H, d,

G. Neves et al. / Bioorg. Med. Chem. 18 (2010) 1925–1935
1933
J = 9.1 Hz, H-8), 8.24 (1H, d, J = 6.8 Hz, H-5); 13C NMR (50 MHz,
CDCl3) d: 13.6 (CH3), 49.3 (Ar-CH2N(CH2–CH2)2NPh), 51.5 (Ar-
CH2N(CH2–CH2)2NPh), 52.8 (Ar-CH2N(CH2–CH2)2NPh), 111.6 (C-
6), 115.7 (C-2), 116.7 (C-8), 117.4 (C-20 and C-60), 124.2 (C-7),
124.7 (C-40), 124.8 (C-5), 129.1 (C-30 and C-50), 142.9 (C-3), 144.9
(C-9), 150.0 (C-10). Anal. Calcd for C19H21ClN4: C, 66.95; H, 6.21;
N, 16.44. Found: C, 67.07; H, 6.26; N, 16.50.
assay) and cortex (for 5-HT2A) were dissected and stored in liquid
nitrogen until use. The structures were homogenized in ice-cold
Tris–HCl 50 mM buffer (pH 7.4) containing different salts accord-
ing to specific procedures for the three receptors. The resulting sus-
pension was ultracentrifuged (48,000gav at 4 °C), the pellet was
resuspended and incubated at 37 °C for 10 min for removal of
endogenous neurotransmitters. This suspension was cooled on
ice and ultracentrifuged (48,000gav at 4 °C) twice. The final pellet
was resuspended in buffer yielding a proportion of 1.5 mL/g tissue
and stored in liquid nitrogen until use.
4.2.21. 4-[4-(4-Phenylpiperazinilmetil)-1H-1,2,3-triazol-4-
yl]aniline (29)
Derivative 21 (0.17 g, 0.47 mmol) was mixed with 95% ethanol
(5.0 mL) and a catalytic amount of Pd/C10% under heating and re-
flux. When the mixture boiled, 80% aq hydrazine hydrate (0.38 mL,
11.5 mmol) was added dropwise. Four hours later, the reaction
mixture was filtered in hot using CeliteÒ and washed with CH2Cl2.
10% aq NaHCO3 was added to the filtrate and afterwards it was ex-
tracted with CH2Cl2. The organic layer was separated and submit-
ted to the usual workup. Compound 29 was obtained in 96% yield
as a beige solid, mp 157 °C, Rf = 0.33 (CH2Cl2/MeOH 95:5). IR (KBr)
4.3.1.2. D2 -like receptor. Different concentrations of test com-
pounds were incubated at 37 °C for 60 min with striatal mem-
branes (50 lg
protein) and 0.1 nM [3H]-YM-09151-2 ([3H]-
nemonapride, 82.5 Ci/mmol, New England Nuclear) in a buffer
solution of Tris–HCl 50 mM (pH 7.4) containing 120 mM NaCl,
5 mM KCl, 5 mM MgCl2, 1.5 mM CaCl2 and 1 mM EDTA, in a final
volume of 500
lL. Nonspecific binding was estimated in the pres-
ence of 30
l
M (À)sulpiride.
cmÀ1: 3477–3417 (
and C@N), 757 (
m
N–H), 3213–3148 (
m C–H), 1602–1521 (m C@C
m
C–H); 1H NMR (200 MHz, CDCl3) d: 2.72–2.74
4.3.1.3. 5-HT1A receptor. Different concentrations of test com-
pounds were incubated at 37 °C for 15 min with hippocampal
(4H, m, Ar-CH2N(CH2–CH2)2NPh), 3.21–3.23 (4H, m, Ar-
CH2N(CH2–CH2)2NPh), 3.81 (2H, s, Ar-CH2N(CH2–CH2)2NPh), 3.89
(2H, s, NH2), 6.77 (3H, m, H-200, H-400 and H-600), 6.93 (2H, d,
J = 8.4 Hz, H-30 and H-50), 7.26 (2H, m, H-300 and H-500), 7.47 (2H,
d, J = 8.4 Hz, H-20 and H-60), 7.83 (1H, s, H-5); 13C NMR (50 MHz,
CDCl3) d: 49.3 (Ar-CH2N(CH2–CH2)2NPh), 53.2 (Ar-CH2N(CH2–
CH2)2NPh), 53.6 (Ar-CH2N(CH2–CH2)2NPh), 115.4 (C-30 and C-50),
116.2 (C-200 and C-600), 119.9 (C-400), 121.1 (C-5), 122.4 (C-20 and
C-60), 128.9 (C-10), 129.3 (C-300 and C-500), 144.8 (C-4), 147.2 (C-
40), 151.4 (C-100). UV (MeOH) kmax: 251.6 and 277.8 nm.
membranes (50
mmol, New England Nuclear) in a buffer solution of Tris–HCl
50 mM (pH 7.4) containing 1 mM CaCl2, 1 mM MnCl2, 10 M par-
L. Nonspecific binding was esti-
M serotonin. The Kd and Bmax
l
g protein) and 1 nM [3H]-8-OH-DPAT (170.2 Ci/
l
gyline, in a final volume of 500
mated in the presence of 10
l
l
values obtained in a saturation experiment were 0.80 0.22 nM
and 166 16 fmol/mg protein, respectively.
4.3.1.4. 5-HT2A receptor. Different concentrations of test com-
pounds were incubated at 37 °C for 15 min with cortex membranes
4.2.22. 4-[4-(4-Phenylpiperazinilmetil)-1H-1,2,3-triazol-4-
yl]acetamide (22)
(150
land Nuclear) in a buffer solution of Tris–HCl 50 mM (pH 7.4) con-
taining 100 nM prazosin, in a final volume of 500 L. Nonspecific
binding was estimated in the presence of 1 M unlabeled ketan-
l
g protein) and 1 nM [3H]-ketanserin (67 Ci/mmol, New Eng-
Compound 29 (0.10 g, 0.30 mmol) was mixed with acetic anhy-
dride (1.5 mL, 13.6 mmol) and the reaction mixture heated to 55 °C
for 45 min. After neutralization with 10% aq solution of NaHCO3
(80 mL), the mixture was extracted with CH2Cl2. The organic layer
was separated and submitted to the usual workup. Compound 22
was obtained in 92% yield as a beige solid, mp 252 °C, Rf = 0.18
l
l
serin. The Kd and Bmax values obtained in a saturation experiment
were 1.77 0.67 nM and 348 51 fmol/mg protein, respectively.
After incubation, samples were rapidly diluted with 3 Â 4 mL of
cold 5 mM Tris–HCl buffer and immediately filtered under vacuum
on glass fiber filters (GMF 3, Filtrak, Germany) previously soaked in
0.5% polyethyleneimine. Filters were then dried and immersed in a
scintillation mixture (POPOP (1,4-bis-[2-(5-phenyloxazolyl)]-ben-
zene) 0.1 g/L and POP (2,5-diphenyloxazole) 4.0 g/L in toluene).
The radioactivity retained in the filters was counted with a Packard
Tri-Carb 1600 TR liquid scintillation analyzer. The assays were con-
(CH2Cl2/MeOH 95:5). IR (KBr) cmÀ1: 3444–3394 (
3123 ( C–H), 1665 ( C@O), 825 ( C–H), 753 (
m
N–H), 3311–
m
m
m
m
C–H); 1H NMR
(200 MHz, CDCl3) d: 2.22 (3H, s, NHCOCH3), 2.72–2.74 (4H, m,
Ar-CH2N(CH2-CH2)2NPh), 3.20–3.22 (4H, m, Ar-CH2N(CH2–
CH2)2NPh), 3.81 (2H, s, Ar-CH2N(CH2–CH2)2NPh), 6.88 (3H, m, H-
200, H-400 and H-600), 7.26 (2H, m, H-300 and H-500), 7.65 (2H, d,
J = 9.1 Hz, H-20 and H-60), 7.72 (2H, d, J = 9.1 Hz, H-30 and H-50),
7.94 (1H, s, H-5), 8.04 (1H, s, NHCOCH3); 13C NMR (50 MHz, CDCl3)
d: 24.7 (NHCOCH3), 49.2 (Ar-CH2N(CH2–CH2)2NPh), 53.2 (Ar-
CH2N(CH2–CH2)2NPh), 53.5 (Ar-CH2N(CH2–CH2)2NPh), 120.8 (C-30
and C-50), 121.1 (C-5), 121.3 (C-20 and C-60), 129.3 (C-300 and C-
500), 133.1 (C-10), 138.8 (C-40), 145.3 (C-4), 151.4 (C-100), 169.0
ducted to a maximum concentration of 30
to the solubility of the compounds.
lM or 10 lM according
4.3.1.5. Statistical analysis. The median inhibitory concentra-
tions (IC50) for binding assays were estimated using a computer-
ized nonlinear regression analysis of the untransformed data
(Prism 4.0, GraphPad Software Inc.), assuming a single population
of binding sites. The Ki values were calculated using the Cheng
and Prusoff equation: Ki = IC50/(1 + [radioligand]/Kd). The Kd values
used were obtained from saturation experiments performed in our
tissue preparations for [3H]-8-OH-DPAT and [3H]-ketanserin (see
details above) or from the literature54 for [3H]-YM-09151-2
(Kd = 0.036 nM).
(NHCOCH3). UV (MeOH) kmax
:
257.0 nm. Anal. Calcd for
C21H24N6O: C, 67.00; H, 6.43; N, 22.32. Found: C, 67.19; H, 6.38;
N, 22.41.
4.3. Pharmacology
4.3.1. In vitro binding assays
This procedure was approved by the Institutional Ethics Com-
mittee for Animal Care from the Federal University of Rio de
Janeiro.
4.4. Behavioral experiments
4.4.1. Animals
4.3.1.1. Tissue preparation. Adult male Wistar rats (200–300 g)
were killed by decapitation. The brains were immediately removed
on ice and hippocampus (for 5-HT1A assay), striatum (for D2-like
Adult male CF1 mice (25–35 g) from Fundação Estadual de
Produção e Pesquisa em Saúde (FEPPS-RS) breeding colony were
used. Animals were housed in groups of eight individuals in plastic

1934
G. Neves et al. / Bioorg. Med. Chem. 18 (2010) 1925–1935
cages (17 Â 28 Â 13 cm) with free access to food (NuvitalÒ) and
water. Mice were kept at constant room temperature (22 2 °C)
and humidity (60%), under a 12 h light–dark cycle (lights off at
7:00 pm) and were adapted to local conditions for at least 72 h be-
fore the experiments. All experimental protocols were approved by
CONEP—Brazil (National Commission of Research Ethics, Protocol
2006541) and performed according to guidelines of the National
Research Ethics Committee (published by National Heath Council,
MS, 1998) and of the Brazilian law,55 which are in compliance with
the International Guiding Principles for Biomedical Research
Involving Animals.56
authors are also grateful for the fellowships granted by CAPES,
CNPq, FAPERJ and FAPERGS.
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