Synthesis and pharmacological evaluation of new N-phenylpiperazine derivatives designed as homologues of the antipsychotic lead compound LASSBio-579

Article abstract of DOI:10.1016/j.ejmech.2013.05.027

In an attempt to increase the affinity of our antipsychotic lead compound LASSBio-579 (1-((1-(4- chlorophenyl)-1H-pyrazol-4-yl)methyl)-4-phenylpiperazine; (2)) for the 5-HT2A receptor, we synthesized five new N-phenylpiperazine derivatives using a linear synthetic route and the homologation strategy. The binding profile of these compounds was evaluated for a series of dopaminergic, serotonergic and alpha-adrenergic receptors relevant for schizophrenia, using classical competition assays. Increasing the length of the spacer between the functional groups of (2) proved to be appropriated since the affinity of these compounds increased 3-10-fold for the 5-HT2A receptor, with no relevant change in the affinity for the D2-like and 5- HT1A receptors. A GTP-shift assay also indicated that the most promising derivative (1-(4-(1-(4-chlorophenyl)-1H- pyrazol-4-yl) butyl)-4-phenylpiperazine) (LASSBio-1635) (6) has the expected efficacy at the 5-HT2A receptors, acting as an antagonist. Intraperitoneal administration of (6) prevented apomorphine-induced climbing behavior and ketamine-induced hyperlocomotion in mice, in a dose dependent manner. Together, these results show that (6) could be considered as a new antipsychotic lead compound.

Full text of DOI:10.1016/j.ejmech.2013.05.027

European Journal of Medicinal Chemistry 66 (2013) 122e134
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and pharmacological evaluation of new N-phenylpiperazine
derivatives designed as homologues of the antipsychotic lead
compound LASSBio-579
,
b
,
,
Thais E.T. Pompeu a 1, Fernando R.S. Alves c 1, Carolina D.M. Figueiredo a,
Camila B. Antonio d, Vivian Herzfeldt d, Bruna C. Moura a, Stela M.K. Rates d,
**
Eliezer J. Barreiro e, Carlos A.M. Fraga f , François Noël
,
a,
*
a Laboratório de Farmacologia Bioquímica e Molecular, Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Avenida Carlos Chagas
Filho, 373, Sala J1-17, CEP 21941-912 Ilha do Fundão, Rio de Janeiro, Brazil
b Programa de Pós-Graduação em Farmacologia e Química Medicinal, Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Avenida
Carlos Chagas Filho, 373, Sala K2-27, CEP 21941-912, Ilha do Fundão, Rio de Janeiro, Brazil
c Programa de Pós-Graduação em Química, Instituto de Química, Universidade Federal do Rio de Janeiro, Avenida Athos da Silveira Ramos, 149 Bloco A e 6ꢀ
andar, CEP 21941-909, Ilha do Fundão, Rio de Janeiro, Brazil
d Programa de Pós-Graduação em Ciências Farmacêuticas, Universidade Federal do Rio Grande do Sul, Avenida Ipiranga 2752, Lab 505C, CEP 90610-000,
Porto Alegre, Brazil
e Laboratório de Avaliação e Síntese de Substâncias Bioativas (LASSBio), Faculdade de Farmácia, Universidade Federal do Rio de Janeiro, Avenida Carlos
Chagas Filho, 373, Bloco Bss 022, CEP 21944-971, Ilha do Fundão, Rio de Janeiro, Brazil
f Programa de Pesquisa em Desenvolvimento de Fármacos, Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Avenida Carlos Chagas
Filho, 373, Sala J1-01, CEP 21941-912, Ilha do Fundão, Rio de Janeiro, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
In an attempt to increase the affinity of our antipsychotic lead compound LASSBio-579 (1-((1-(4-
chlorophenyl)-1H-pyrazol-4-yl)methyl)-4-phenylpiperazine; (2)) for the 5-HT2A receptor, we synthe-
sized five new N-phenylpiperazine derivatives using a linear synthetic route and the homologation
strategy. The binding profile of these compounds was evaluated for a series of dopaminergic, seroto-
nergic and alpha-adrenergic receptors relevant for schizophrenia, using classical competition assays.
Increasing the length of the spacer between the functional groups of (2) proved to be appropriated since
the affinity of these compounds increased 3e10-fold for the 5-HT2A receptor, with no relevant change in
the affinity for the D2-like and 5-HT1A receptors. A GTP-shift assay also indicated that the most promising
derivative (1-(4-(1-(4-chlorophenyl)-1H-pyrazol-4-yl) butyl)-4-phenylpiperazine) (LASSBio-1635) (6)
has the expected efficacy at the 5-HT2A receptors, acting as an antagonist. Intraperitoneal administration
of (6) prevented apomorphine-induced climbing behavior and ketamine-induced hyperlocomotion in
mice, in a dose dependent manner. Together, these results show that (6) could be considered as a new
antipsychotic lead compound.
Received 16 January 2013
Received in revised form
6 May 2013
Accepted 22 May 2013
Available online 30 May 2013
Keywords:
Schizophrenia
Antipsychotics
N-Phenylpiperazine
LASSBio-579
Homologation
5-HT2A
Ó 2013 Elsevier Masson SAS. All rights reserved.
1. Introduction
anhedonia, avolition, alogia) and cognitive dysfunction (e.g. mem-
ory deficits, attention impairment) [2,3]. Since the discovery of
Schizophrenia is a severe neuropsychiatric disorder that affects
about 24 million people worldwide [1] and is characterized by
positive or psychotic symptoms (e.g. hallucinations, delusions and
disorganized thoughts), negative symptoms (e.g. social withdrawal,
chlorpromazine, in 1952, schizophrenia has been treated with
drugs now classified as typical or first-generation antipsychotics
(e.g. haloperidol, chlorpromazine) acting as postsynaptic dopamine
D2 receptor antagonists and effective against positive symptoms
[4]. However, these drugs have little effects on negative and
cognitive symptoms and elicit severe extrapyramidal side effects
due an excessive D2 receptor blockade in the striatum. A second
generation of antipsychotics (e.g. risperidone, olanzapine and
quetiapine) emerged after the introduction of clozapine (1) [4].
These drugs were named atypical based on their low propensity to
* Corresponding author. Tel.: þ55 21 2562 6732.
** Corresponding author. Tel.: þ55 21 25626447.
E-mail addresses: cmfraga@ccsdecania.ufrj.br (C.A.M. Fraga), fnoel@
pharma.ufrj.br, fgagnoel@gmail.com (F. Noël).
1
These two authors contributed equally to this work.
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.05.027

T.E.T. Pompeu et al. / European Journal of Medicinal Chemistry 66 (2013) 122e134
123
elicit extrapyramidal effects, claimed relative efficacy against
negative and cognitive symptoms and multi-target profile [5,6].
Initially, a key role has been attributed to the 5-HT2A receptors since
the potent blockade of these receptors coupled to weaker antago-
nism of D2 receptors has been found to be the feature shared by
these atypical antipsychotics [7]. More recently, the 5-HT1A re-
ceptors have attracted the attention since their activation should
also be a crucial element in the mechanism of action of these
atypical antipsychotics [7e9]. As a result, the present prevailing
idea is that improvement of positive symptoms is due to D2
antagonism whereas the lower liability to induce extrapyramidal
symptoms and the improvement of negative symptoms are a result
of an increase dopamine release in striatum and frontal cortex,
respectively, which can be achieved either by 5-HT2A blockade or 5-
HT1A activation [6e11]. However, there is a high level of treatment
discontinuation due to both low tolerability and insufficient effec-
tiveness, supporting the need for developing more effective and
safer antipsychotics [4,12,13]. In previous studies our group
described a series of N-phenylpiperazine derivatives in the search
for new atypical antipsychotic prototypes [14,15]. Based on these
results, (2) (Scheme 1), was selected for a more detailed charac-
terization of its pharmacological profile both in vitro and in vivo
[16,17]. Oral administration of (2) inhibited the apomorphine-
induced climbing, ketamine-induced hyperlocomotion and deficit
of prepulse inhibition of acoustic startle reflex induced by
apomorphine, (ꢁ)-DOI and ketamine, indicating that it acts more
like clozapine than haloperidol. The participation of a serotonergic
effect was compatible with preliminary results [18] and with its
detailed binding profile defining it as a dual D2-like/5-HT1A ligand
[17]. However, (2) has a relatively low affinity for the 5-HT2A re-
ceptor, classically considered as an important target for the atypical
antipsychotics (see above), and its pharmacodynamic profile could
be further optimized. For this purpose, homologation [19] could be
a suitable strategy since the presence of two planar aromatic or
heterocyclic ring systems separated by an aliphatic or alicyclic
chain containing basic protonable nitrogen has been reported to
favor binding to the 5-HT2A receptor [20,21]. In the present study,
we report the synthesis of five new derivatives obtained through
the extension of the methylene spacer between the 4-chloro-N-
phenylpyrazolyl and N-phenylpiperazine subunits of (2). The
binding profiles of these compounds to monoaminergic receptors
relevant to the treatment of schizophrenia as well as their effec-
tiveness in pharmacological models of schizophrenia symptoms in
mice were assessed. Present results indicate that a significant in-
crease in affinity for the 5-HT2A receptor has been achieved and one
of these compounds (6) is a better hit than (2) and is active in two
mice models of schizophrenia positive symptoms at doses without
effect on spontaneous locomotion.
2. Results and discussion
2.1. Chemistry
The novel series of pyrazole N-phenylpiperazine derivatives (3e
7) was prepared by classical synthetic methodologies, using 4-
formyl-N-(40-chlorophenyl)pyrazole (8) and N-(40-chlorophenyl)
pyrazole (14) as starting material (Schemes 2 and 3). The initial step
used for the construction of pyrazole N-phenylpiperazine oxana-
logue derivatives 3 (n ¼ 2) and 4 (n ¼ 3) consisted of the
Scheme 2. Synthesis of oxa-homologue pyrazole N-phenylpiperazine derivatives (3)
and (4). Reagents and Conditions: a) NaBH4, MeOH, 0 ꢀC, 15 min, 80%; b) 1) NaH, THF,
0 ꢀC, 1 h, 2) BrCH2CO2Et, 0 ꢀC, 4 h; 70%; c) 1) NaH, THF, 0 ꢀC, 1 h, 2) BrCH2CH]CH2, 0 ꢀC,
4h; 70%; d) LiAlH4, THF, 70 ꢀC, 4h, 70%; e) 1) BH3, THF, 0 ꢀC, 2h, 2) 30% aq. H2O2, 10% aq.
NaOH, reflux, 12h, 50%; f) TsCl, KOH, K2CO3, rt, 20 min [60% for (13a) and (13b)]; g) N-
phenylpiperazine, Li2CO3, CH3CN, 80 ꢀC, 20h [60% for (3) and (4)].
Scheme 1. Design Concept of novel oxa- and carba-homologue pyrazole N-phenyl-
piperazine derivatives (3e7).

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T.E.T. Pompeu et al. / European Journal of Medicinal Chemistry 66 (2013) 122e134
The N-phenylpiperazine acetylene intermediates 16a and 16b
were prepared in one-pot regioselective Sonogashira coupling of
iodopyrazole derivative 15, propargyl bromide or 4-bromo-1-
butyne and N-phenylpiperazine, by using bis(triphenylphosphine)
palladium(II) dichloride as catalyst, in TEA containing copper iodide
[28]. These unsaturated compounds 16a and 16b, with C-3 and C-4
spacer length, were prepared in 75% and 80% yield, respectively. On
the other hand, acetylene derivative 17, with spacer C-5, was syn-
thesized in 78% yield by using the same method previously
described starting from commercially available 4-pentyn-1-ol
compound as acetylene source. By applying the sequence of tosy-
lation [26] and nucleophilic displacement with N-phenylpiperazine
[23], compound 17 was converted in the unsaturated N-phenyl-
piperazine derivative 16c, presenting C-5 spacer length, in 67%
yield (2 steps) (Scheme 3).
So, the target derivatives LASSBio-1528 (5), (6) and LASSBio-
1762 (7) were obtained in almost quantitative yields by catalytic
hydrogenation of the corresponding acetylene derivatives 16aec,
after treatment with molecular hydrogen and 10% Pd/C as catalyst
in protic media [29] (Scheme 3).
The structures of all five novel pyrazole N-phenylpiperazine
derivatives (3e7) were in agreement with analytical and spectral
data.
2.2. Pharmacology
2.2.1. Binding profile of the five new N-phenylpiperazine derivatives
The componund (2) and the N-phenylpiperazine derivatives bind
to dopaminergic, alpha-adrenergic and serotonergic receptors with
micromolar affinity (Table 1). These results are consistent with the
fact that the N-phenylpiperazine is a structurally privileged chemical
group which mimics biogenic amines. Indeed, the N-phenyl-
piperazine moiety has been shown to confer high affinity for 5-HT1A
receptors and alpha1-adrenoceptors [30,31]. The presence of aro-
maticring systems andbasicnitrogen makestheN-phenylpiperazine
scaffold the primary recognition element for the neurotransmitter
binding site of aminergic GPCRs [32]. Table 1 shows that the new N-
phenylpiperazine compounds have similar affinities as (2) for D2, D4,
5-HT1A, 5-HT2C and a2-adrenergic receptors, i.e. aminergic receptors
relevant for the treatment of schizophrenia.
Scheme 3. Synthesis of carba-homologue pyrazole N-phenylpiperazine derivatives (5),
(6) and (7). Reagents and Conditions: a) I2, 30% aq. H2O2, CH3CN, H2O, rt, 24h, 98%; b)
HC^CCH2Br or HC^C(CH2)2Br, PdCl2(PPh3)2, CuI, TEA, N-phenylpiperazine, rt, 20 h
[75% for (16a) and 80% for (16b)]; c) HC^C(CH2)3OH, PdCl2(PPh3)2, CuI, TEA, rt, 20h,
78%; d) TsCl, KOH, K2CO3, rt, 20 min, 80%; e) N-phenylpiperazine, Li2CO3, CH3CN, 80 ꢀC,
20 h, 84%; f) H2 (40 psi), Pd/C 10%, MeOH:EtOH (1:1), 3 h, rt, [99% for (5) and (6); 98%
for (7)].
These results indicate that the changes in the spacer length with
or without the introduction of a hydrogen bond acceptor (oxana-
logues) did not modify significantly the affinity for these receptors.
On the other hand, all the new compounds showed a three to twelve
fold increase in affinity for the 5-HT2A receptor when compared to
(2), so that we can conclude that the homologation strategy was
successful for both carbanalogues and oxanalogues compounds.
When comparing (2) with the carbanalogue compounds, we
observed that the insertion of two or three methylene groups (5)
and (6), respectively, between the 4-chlorophenylpyrazolyl and N-
phenylpiperazine subunits of (2) favored an appropriated confor-
mation for the interaction with the 5-HT2A receptors. A further in-
crease in the length of the spacer (4 methylenes, (7)) was without
effect. The 5-HT2A receptors are supposed to play a key role in the
mechanism of action of the atypical antipsychotics since blockade of
5-HT2A receptors of the nigrostriatal pathway increases dopamine
release, reducing the liability to induce extrapyramidal side effects,
whereas in the mesocortical pathway a similar mechanism would
chemoselective reduction of carbonyl derivative (8) with sodium
borohydride in methanol [22]. The obtained hydroxymethylene-
pyrazole derivative (9) was next O-alkylated with ethyl bromoa-
cetate or allyl bromide after treatment with sodium hydride in THF
[23], in order to introduce C-2 and C-3 in the spacer units leading to
spacer length of 4 and 5 atoms, respectively (Scheme 2). The
reduction of ethyl ester derivative 10 with lithium aluminum hy-
dride in tetrahydrofuran [24] furnished the primary alcohol 12a in
70% yield. On the other hand, the O-allyl derivative 11 was then
submitted to an oxidative hydroboration [23,25] to furnish the
corresponding primary alcohol 12b in 50% yield.
The alcohols 12a and 12b were triturated with tosyl chloride,
potassium hydroxide and potassium carbonate at room tempera-
ture [26] furnishing the corresponding tosylates 13a and 13b,
which were afterwards converted in target derivatives (3) and (4)
after nucleophilic displacement with N-phenylpiperazine in
acetonitrile [23] (Scheme 2).
improve the negative symptoms [7e11]. In the so-called “5-HT2A
/
D2” model, more than absolute affinities, it is a suitable relative
affinity for these two receptors that is needed, i.e. a relatively potent
blockade of 5-HT2A receptors coupled to weaker antagonism of
dopamine D2 receptors [7]. With this in mind, it is clear that
LASSBio-1635 has a better 5-HT2A/D2 relative affinity (0.4) than (2)
(unfavorable ratio of 0.056).
The pyrazole N-phenylpiperazine carbanalogue derivatives 5
(n ¼ 1), 6 (n ¼ 2) and 7 (n ¼ 3), with spacer length of 3, 4 and 5 atoms
respectively, were prepared from 4-iodo-N-(40-chlorophenyl)pyr-
azole (15), obtained by regioselective iodination of N-(40-chlor-
ophenyl)pyrazole (14), as previously reported by Kim et al. [27].

T.E.T. Pompeu et al. / European Journal of Medicinal Chemistry 66 (2013) 122e134
125
Table 1
Affinities of clozapine (1), LASSBio-579 (2) and the five new derivatives (3e7) for different receptors.
Compound
Ki (m
M)a
D2
D4
5-HT1A
5-HT2A
5-HT2C
a2
a1B
Muscarinic
0.022 [2]
Clozapine (1)
0.14 [3]
(0.11e0.17)
0.39 [4]
(0.26e0.57)
1.9 [4]
(1.4e2.7)
0.79 [3]
(0.57e1.1)
0.68 [3]
(0.53e0.88)
0.25 [3]
(0.19e0.32)
0.49 [3]
0.06 [2] (0.04e0.08)
0.26 [3] (0.22e0.30)
0.021 [2]
(0.016e0.027)
6.91 [3]
(4.4e10.7)
2.49 [3]
(1.67e3.71)
0.65 [3]
(0.49e0.84)
0.60 [3]
(0.43e0.83)
0.65 [5]
(0.57e0.74)
0.95 [6]
0.023 [2]
(0.019e0.029)
8.63 [4]
(6.61e11.27)
8.1 [2]
(5.7e11.3)
2.8 [2]
(1.7e4.7)
5.1 [2]
(3.4e7.6)
5.9 [3]
(4.2e8.1)
7.8 [2]
0.10 [3]
(0.08e0.13)
2.5 [3]
(2.0e3.1)
2.1 [2]
(1.4e3.1)
2.8 [2]
(2.1e3.7)
1.6 [2]
(1.0e2.5)
0.61 [4]
(0.48e0.77)
1.4 [3]
0.025 [3]
(0.018e0.034)
2.6 [3]
(1.9e3.5)
0.85 [2]
(0.67e1.1)
0.40 [2]
(0.22e0.73)
0.70 [2]
(0.40e1.2)
0.57 [4]
(0.40e0.81)
0.38 [3]
(0.008e0.064)
b
LASSBio-579 (2)
LASSBio-1526 (3)
LASSBio-1527 (4)
LASSBio-1528 (5)
LASSBio-1635 (6)
LASSBio-1762 (7)
0.18 [4] (0.12e0.26)
0.10 [3] (0.05e0.16)
1.5 [2] (1.0e2.2)
0.22 [4] (0.18e0.26)
0.11 [3] (0.07e0.17)
0.31 [3] (0.24e0.39)
0.18 [3] (0.12e0.26)
0.09 [3] (0.05e0.13)
0.16 [3] (0.11e0.22)
22% [2]
b
11% [2]
b
28% [2]
b
0.33 [3] (0.21e0.53)
0.80 [3] (0.61e1.04)
1.18 [3] (0.81e1.71)
39% [2]
b
2% [2]
b
26% [2]
(0.38e0.63)
(0.78e1.16)
(5.6e10.8)
(1.0e2.0)
(0.25e0.55)
a
Ki values calculated from the mean curves (obtained from two to six experiments [n] performed in triplicate) are expressed with their 95% confidence interval (in
parenthesis).
b
Ki value above 30 m mM).
M (% inhibition of [3H]-QNB binding at 30
Although the target of our homologation strategy was the 5-HT2A
purpose. The control curve of 5-HT has a shallow aspect charac-
terized by a Hill slope lower than 1 (nH ¼ 0.56). The experimental
curve was better fitted (p < 0.01, F-test) using a two independent
populations of binding sites characterized by a high affinity
receptors, an unexpected benefit occurred when increasing the
lengthofthespacer:thecarbanaloguecompounds(5),(6), (7)showed
a 1.6e4-fold higher affinity for a2 adrenoceptors when compared to
(2). In the case of (6), the affinity for the a2 receptors was similar tothe
affinity for the D2-like and 5-HT2A receptors. Such attribute could be
beneficial for an antipsychotic drug since the addition of a a2 antag-
onist to a D2 antagonist has been shown to improve the treatment of
schizophrenia by reducing negative symptoms, as indicated by a
meta-analysis [33]. These clinical data could be related to the obser-
vation that a2 antagonists increase the efflux of dopamine into the
pre-frontal cortex, in pre-clinical models [33].
Since there is a consensus that individualized treatment of
schizophrenia must be based not only on efficacy but also of min-
imal side-effects (and cost) of the different drugs [34], we also
evaluated the affinity of our new derivatives for muscarinic and a1B
receptors, that are associated with some side effects of the anti-
psychotic drugs (Table 1). Like (2), the new derivatives have no
affinity for the muscarinic receptors, unlike (1) which produces
severe anticholinergic adverse effects due to its high affinity for
these receptors [8,35]. On the other hand, our new compounds
have a 3e6-fold higher affinity for a1B adrenoceptors, when
compared to (2), indicating a higher risk of postural hypotension.
component (52%, IC50 ¼ 0.60
m
M) and a low affinity component
(IC50 ¼ 30.6 M). In the presence of 100
m
mM of Gpp(NH)p (a non-
hydrolysable analog of GTP), the competition curve of 5-HT was
shifted to the right indicating a loss of affinity for the 5-HT2A re-
ceptors. Note that the curve has now a normal aspect, with a Hill
slope not different from 1 and an IC50 of 13 mM. When an antagonist
is used as competitor, the addition of GTP has no effect, as observed
in Fig. 1B for ketanserin. The same pattern was observed with (6)
(Fig. 1C), since the competition curves in the absence and presence
of 1 mM GTP were superimposed, indicating that (6) is an antag-
onist of the 5-HT2A receptors. These results are in accordance with
the structural requirements reported for antagonists of the 5-HT2A
receptor, namely an aromatic ring, a basic nitrogen atom, an H-
bonding acceptor group and a hydrophobic region directly attached
to the basic center [20,21].
As the affinity of (6) for the a2 receptors was similar to its affinity
for the D2-like and 5-HT2A receptors, we also determined its intrinsic
activity at this receptor using the GTP-shift assay. Fig. 2 shows that
contrary to what occurred with the agonist oxymetazoline (Fig. 2A),
the competition curves in the absence and presence of 1 mM GTP
were superimposed for both antagonist (RX821002) and (6) (Fig. 2B
and C, respectively). Such results indicate that (6) is an antagonist of
the a2 receptors, an attribute that could be beneficial for an anti-
psychotic drug as mentioned in Section 2.2.1.
However, the affinity of (6) (Ki ¼ 0.57
mM) is still much lower than
M) for these receptors (Table 1), indicating a
(1) (Ki ¼ 0.025
m
theoretical higher safety margin for this adverse effect.
2.2.2. Functional binding assays for LASSBio-1635 (6)
Since efficacy is as important as affinity for the therapeutic effect
of a drug (see Section 2.2.1), we first decided to estimate the
intrinsic efficacy of our best derivative (6) for the 5-HT2A receptor,
target of present attempt to optimize our lead compound, using a
functional binding assay. The classical GTP-shift assay is based on
the ternary complex model for GPCRs [36e38] and has been vali-
dated for the 5-HT2A receptor [37], as well as for other receptors
[38]. This assay is based on the difference of affinity measured for
agonists (but not antagonists) in the absence and presence of a
large concentration of GTP (or a lower concentration of a non-
hydrolysable analog of GTP) that is capable to destabilize the
ternary complex ARG (high affinity state of the receptor) that is
formed by the agonist (A), the receptor (R) and the G-protein (G).
Fig. 1 shows the profiles of competition curves for [3H]-ketan-
serin binding to 5-HT2A receptors using either a full agonist (5-HT,
Fig. 1A) or an antagonist (ketanserin, Fig. 1B), for validation
Finally, we used the same experimental strategy to look at the 5-
HT1A receptors, for which a small increase of affinity has been
observed when comparing (6) to (2) (Table 1).
Fig. 3 shows that addition of GTP to the medium produced a
clear shift to the right of the competition curve of the agonist
(Fig. 3A) whereas it had no effect when the competitor was an
antagonist (Fig. 3B). With (6), we were not able to detect a signif-
icant shift of the competition curve, indicating that its intrinsic
activity would be very small, if any, at this receptor (Fig. 3C). As
previous in vivo data indicated that our lead compound (2) had
some agonistic activity at this receptor [18], we checked its
behavior in our in vitro GTP-shift assay. To our surprise, the pattern
of the competition curves in the presence and absence of GTP was
very similar to the one observed with (6), i.e. a very small shift of
the competition curve in the presence of GTP, if any. Although
difficult to explain (sensibility of the method for weak partial

126
T.E.T. Pompeu et al. / European Journal of Medicinal Chemistry 66 (2013) 122e134
(A)
(A)
100
75
50
25
0
100
Control
+Gpp(NH)p
Control
+ GTP
75
50
25
0
0.1
1
10
100
0.01
0.1
1
10
100
1000
5-HT (μM)
Oxymetazoline ( M)
(B)
100
75
50
25
0
(B)
100
Control
+ GTP
Control
+ GTP
75
50
25
0
0.1
1.0
10
100
Ketanserin (nM)
(C)
0.01
0.1
1
10
100
1000
100
75
50
25
0
RX821002 (nM)
Control
+ GTP
(C)
100
75
50
25
0
Control
+ GTP
0.01
0.1
1.0
10
LASSBio-1635 (μΜ)
Fig. 1. Effect of guanylyl nucleotide on 5-HT, ketanserin and LASSBio-1635 (6)
competition for [3H]-ketanserin binding to 5-HT2A receptors present in a rat cortex
membrane preparation. The assays were performed in the absence (control) or pres-
ence of 0.1 mM Gpp(NH)p (5-HT) or 1 mM GTP (ketanserin and LASSBio-1635). The
data were fitted assuming a single population of binding sites, except in the case of 5-
HT (control) where a better fit was obtained assuming two independent populations of
binding sites.
0.01
0.1
1
10
100
1000
LASSBio-1635 ( M)
Fig. 2. Effect of GTP on oximetazoline, RX821002 and LASSBio-1635 (6) competition
for [3H]-RX821002 binding to a2 adrenoceptors present in a rat cortex membrane
preparation. The assays were performed in the absence (control) or presence of 1 mM
GTP. The data were fitted assuming a single population of binding sites, except in the
case of oximetazoline (control) where a better fit was obtained assuming two inde-
pendent populations of binding sites.
agonists, role of metabolite(s) [16] for the in vivo action of (2)),
these results indicate that the in vitro profile of (6) is similar to the
one of its lead compound (2) at these 5-HT1A receptors.
With respect to the D2-like receptor, the antagonist behavior of
(6) has been verified in the in vivo assays described below.
the NMDA subtype of the glutamate receptor such as phencyclidine
(PCP), ketamine and dizocilpine (MK-801) [42]. In fact, PCP and
ketamine administration to healthy human subjects triggers a
behavioral state that mimics specific aspects of schizophrenia, such
as thought disorder and hallucinations [43].
Compound (6) prevented the apomorphine-induced climbing in
a dose dependent way (Fig. 4B). Mice treated with (6) (0.1 mg/kg)
plus apomorphine presented lower climbing index than mice
treated with apomorphine-vehicle, and higher than the vehicle
group; animals treated with intermediate doses of (6) (0.3 and
1.0 mg/kg) plus apomorphine presented a reduced climbing index
when compared to the apomorphine-vehicle group and did not
2.2.3. Effect of different doses of LASSBio-1635 on behaviors
correlated to schizophrenia positive symptoms
We also evaluated the effect of different doses of (6) on mice
behaviors correlated to positive symptoms of schizophrenia:
apomorphine-induced climbing and hyperlocomotion induced by
ketamine. Apomorphine-induced climbing is an animal model
classically linked to motor agitation, one of the schizophrenia
positive symptoms [39e41], being based on the induction of a
hyperdopaminergic state. Similarly to dopamine agonists, hyper-
activity can be induced in rodents by noncompetitive antagonists of

T.E.T. Pompeu et al. / European Journal of Medicinal Chemistry 66 (2013) 122e134
127
out any unspecific effect (Fig. 5A). At 10 mg/kg i.p. (6) significantly
reduced mice locomotor activity, expressed as number of crossings
whereas it was without effect at 3.0 mg/kg (p ¼ 0.199). Therefore,
only doses below 3 mg/kg i.p. were evaluated in the ketamine-
induced hyperlocomotion test (Fig. 5B). Compound (6) (1.0 mg/kg
and 3.0 mg/kg, i.p.) prevented the effect of ketamine in a dose
dependent manner. Considering that 1.0 mg/kg was also signifi-
cantly different from control group, lower doses were not tested.
With respect to reference drugs, only clozapine prevented ket-
amine’s motor effect, as expected [44].
(A)
100
75
50
25
0
Control
+ GTP
3. Conclusion
Five new N-phenylpiperazine compounds were obtained with a
linear synthetic route, using homologation strategy between the
functional groups of our lead compound (2). The homologation
strategy proved to be appropriated for increasing the interaction of
these compounds with the 5-HT2A receptors, with no significant
changes in the affinity for the D2-like, D4 and 5-HT1A receptors. In
this context, (6) was the most promising derivative being charac-
terized by a ten-fold higher affinity than its parent compound (2)
for the 5-HT2A receptor. Besides that, (6) has the expected efficacy
at the 5-HT2A receptors, being classified as an antagonist, stimu-
lating its characterization in animal models of schizophrenia.
LASSBio-1635 (6) has also a 4-fold higher affinity for a2 adreno-
ceptors when compared to (2) and the expected efficacy (antago-
nist) as identified by a binding functional assay. Compound (6) fully
prevented the apomorphine-induced climbing behavior in mice in
a dose dependent way and prevented the ketamine-induced
hyperlocomotion at doses with no effect on the mice locomotor
activity (1 and 3.0 mg/kg i.p.). Together, these results show that (6)
could be considered as a new antipsychotic lead compound.
1
10
100
1000
10000
5-HT (nM)
(B)
100
75
50
25
0
Control
+ GTP
0.01
0.1
1
10
100
pMPPF (nM)
(C)
100
4. Experimental protocols
Control
+ GTP
4.1. Substrate
75
50
25
0
Target pyrazole N-phenylpiperazine derivatives (3e7) were
synthesized at the Laboratório de Avaliação e Síntese de Sub-
stâncias Bioativas (LASSBio, Rio de Janeiro, Brazil).
4.2. Instrumentation, chemicals and solvents
Melting points were determined on a Quimis 340/23 apparatus
and are uncorrected. Infrared (IR) spectra were obtained on a IR
ABB FTLA2000-100 infrared spectrophotometer. Solid samples
were prepared in KBr pellets, whereas oily samples were analyzed
as film in NaCl plates. 1H and 13C NMR spectra were recorded on
Bruker AC-200 and Varian 400-MR instruments operated respec-
1
10
100
1000
10000
LASSBio-1635 (nM)
Fig. 3. Effect of GTP on 5-HT, pMPPF and LASSBio-1635 (6) competition for [3H]-pMPPF
binding to 5-HT1A receptors present in a rat hippocampal membrane preparation. The
assays were performed in the absence (control) or presence of 1 mM GTP. The data
were fitted assuming a single population of binding sites, except in the case of 5-HT
(GTP) where a better fit was obtained assuming two independent populations of
binding sites.
tively at 200 and 400 MHz. Chemical shifts (d) are expressed in
parts per million relative to internal tetramethylsilane; coupling
constants (J) are in hertz. Microanalyses were obtained with
Thermofinnigan EA1112 analyzer, using a Metler MX5 electronic
balance. Reactions were routinely monitored by thin-layer chro-
matography (TLC) in silica gel (F254 Merck plates) and the products
visualized with iodine or ultraviolet lamp (254 and 365 nm). For
normal pressure and flash column chromatography purifications,
Merck silica gel type 60 (size 70e230 and 230e400 mesh,
respectively) was used. Unless stated otherwise, starting materials
used were high-grade commercial products.
differ from vehicle group. The higher doses of (6) (3.0 and 10 mg/
kg) completely prevented the effect of apomorphine on climbing
behavior, as observed (Fig. 4A) with high doses of haloperidol
(0.5 mg/kg p.o.) and clozapine (15 mg/kg p.o.) used as reference
drugs. When comparing to the same doses of (2), in the same
experimental protocol, (6) had a better profile since its effect was
dose dependent, statistically significant and fully effective at the
higher doses, contrary to what was observed with (2) (Fig. 4C).
The effect of the higher doses of (6) (3.0 and 10 mg/kg i.p.) on
animals’ spontaneous locomotion was evaluated in order to rule
4.3. (1-(4-Chlorophenyl)-1H-pyrazol-4-yl)methanol (9)
Sodium borohydride (0.32 g; 8.49 mmol) was added stepwise to
a
stirred solution of 4-formylpyrazole derivative 8 (0.58 g;

128
T.E.T. Pompeu et al. / European Journal of Medicinal Chemistry 66 (2013) 122e134
Fig. 4. A) Effect of clozapine (1) (15 mg/kg p.o.) and haloperidol (0.5 mg/kg p.o.) on apomorphine-induced climbing behavior in mice. Clozapine and haloperidol were used as
reference drugs. Apomorphine (4 mg/kg) or vehicle was administered subcutaneously 30 min after drug pretreatment. Data are expressed as mean ꢁ S.E.M (n ¼ 8e9). ANOVA
(F3,33 ¼ 58.780, P < 0.001): different from vehicle (VEH) þ saline (SAL) group in post hoc test ***P < 0.001; different from VEH þ apomorphine group in post hoc test ###P < 0.001. B)
Effect of LASSBio-1635 (6) (0.1e10 mg/kg i.p.) on apomorphine-induced climbing behavior in mice. Data are expressed as mean ꢁ S.E.M (n ¼ 7e9). ANOVA (F6,55 ¼ 8.803, P < 0.001):
different from vehicle (VEH) þ saline (SAL) group in post hoc test *P < 0.05, ***P < 0.001; different from VEH þ apomorphine group in post hoc test #P < 0.05, ###P < 0.001;
different from LASSBio-1635 10 mg/kg i.p. group in post hoc test @P < 0.05, @@P < 0.01. C) Effect of LASSBio-579 (0.1e10 mg/kg i.p.) on apomorphine-induced climbing behavior in
mice. Data are expressed as mean ꢁ S.E.M (n ¼ 7e9). ANOVA (F8,53 ¼ 7.858, P < 0.001): different from vehicle (VEH) þ saline (SAL) group in post hoc test *P < 0.05, **P < 0.01,
***P < 0.001.
2.83 mmol) in methanol (40 mL) at 0 ꢀC. After complete addition,
the reaction mixture was allowed to warm to room temperature
and stirring continued for 15 min. The reaction was quenched by
adding a mixture of crushed ice and water, and the obtained pre-
cipitate was collected by filtration furnishing the desired com-
pound 9 in 80% yield, as a beige solid, m.p. ꢀC (Rf: 0.55 after elution
with 9:1 mixture of CH2Cl2:MeOH). IR (KBr, cmꢂ1) 3427, 3336, 3116,
2931, 1654, 1613, 1501, 1401, 1207, 1095, 954, 833; 1H NMR (CDCl3,
brown residue was purified by column chromatography on silica
gel eluted with a 9:1 mixture of CH2Cl2:MeOH to give the desired
ethyl ester 10 in 70% yield, as a yellow oil (Rf: 0.87 after elution with
9:1 mixture of CH2Cl2:MeOH). IR (NaCl, cmꢂ1) 2982, 1738, 1503,
1403, 1217, 1122, 1097, 1024, 951, 830; 1H NMR (DMSO-d6,
200 MHz):
d
1.19 (t, 3H, J ¼ 7.3 Hz, Ar-CH2OCH2CO2CH2CH3), 4.12 (q,
2H, J ¼ 7.3 Hz, Ar-CH2OCH2CO2CH2CH3), 4.14 (s, 2H, Ar-CH2OCH2
-
CO2CH2CH3), 4.51 (s, 2H, Ar-CH2OCH2CO2CH2CH3), 7.54 (d, 2H,
0
0
200 MHz):
d
4.68 (s, 2H, ArCH2OH), 7.44 (d, 2H, J ¼ 8.0 Hz, Ar-H3’
J ¼ 8.0 Hz, Ar-H3 and Ar-H5 ), 7.74 (s, 1H, pyrazole-H3), 7.87 (d, 2H,
J ¼ 8.0 Hz, Ar-H2 and Ar-H6 ), 8.52 (s, 1H, pyrazole-H5); 13C NMR
0
0
and Ar-H5’), 7.64 (d, 2H, J ¼ 8.0 Hz, Ar-H2’ and Ar-H6’), 7.71 (s, 1H,
pyrazole-H3), 7.90 (s, 1H, pyrazole-H5).
(DMSO-d6, 50 MHz):
d 14.0 (Ar-CH2OCH2CO2CH2CH3), 60.1 (Ar-
CH2OCH2CO2CH2CH3), 62.9 (Ar-CH2OCH2CO2CH2CH3), 66.5 (Ar-
CH2OCH2CO2CH2 CH3), 119.9 (Ar-C2’ and Ar-C6’), 120.0 (pyrazole-C4),
4.4. Ethyl 2-((1-(4-chlorophenyl)-1H-pyrazol-4-yl)methoxy)
acetate (10)
0
0
0
127.5 (pyrazole-C5), 129.4 (Ar-C3 and Ar-C5 ), 130.3 (Ar-C4 ), 138.4
0
(Ar-C1 ), 141.4 ( (pyrazole-C3), 170.0 (Ar-CH2OCH2 CO2CH2CH3).
To a suspension of sodium hydride (0.230 g, 9.58 mmol) in dry
THF (10 mL) was added a solution of alcohol 9 (0.400 g; 1.91 mmol)
in dry THF (10 mL) and the resulting mixture was stirred at 0 ꢀC for
1 h. Ethyl 2-bromoacetate (0.42 mL; 3.83 mmol) was added drop-
wise and the reaction mixture was maintained at 0 ꢀC for additional
4 h. Next, the reaction mixture was poured into crushed ice and
extracted with dichoromethane (2 ꢃ 30 mL). The combined organic
extracts were washed with brine and concentrated at reduced
pressure after drying with anhydrous sodium sulfate. The oily
4.5. 4-(Allyloxymethyl)-1-(4-chlorophenyl)-1H-pyrazole (11)
To a suspension of sodium hydride (0.173 g, 7.19 mmol) in dry
THF (10 mL) was added a solution of alcohol 9 (0.500 g; 2.39 mmol)
in dry THF (10 mL) and the resulting mixture was stirred at 0 ꢀC for
1 h. Then, allyl bromide (1.0 mL; 11.98 mmol) was added dropwise
and the reaction mixture was maintained at 0 ꢀC for additional 4 h.
Next, the reaction mixture was poured into crushed ice and
750
400
A)
B)
***
600
450
300
150
0
***
300
*
#
##
200
**
###
100
0
mg/kg
VEH
VEH
HAL
CZP
1.0
3.0
VEH
HAL
CZP L1635 3.0 L1635 10 mg/kg
LASSBio-1635
Sal
Ketamine
Fig. 5. Effect of different doses of LASSBio-1635 (6) i.p. on ketamine-induced hyperlocomotion in mice. Clozapine (1) (CZP e 1 mg/kg p.o.) and haloperidol (HAL e 0.01 mg/kg p.o.)
were used as reference drugs. Data are expressed as mean ꢁ S.E.M. (A) Spontaneous locomotor activity (n ¼ 8e9): ANOVA (F4,40 ¼ 4.149, P ¼ 0.007): different from vehicle in post
hoc test **P < 0.01. (B) Ketamine-induced hyperlocomotion (n ¼ 7e8). Ketamine (10 mg/kg s.c.) was administered 30 min after drug pretreatment. ANOVA (F5,45 ¼ 17.384, P < 0.001):
different from vehicle (VEH) þ saline (SAL) group in post hoc test *P < 0.05, ***P < 0.001; different from VEH þ ketamine group in post hoc test ##P < 0.01, ###P < 0.001.

T.E.T. Pompeu et al. / European Journal of Medicinal Chemistry 66 (2013) 122e134
129
0
0
extracted with dichoromethane (2 ꢃ 30 mL). The combined organic
extracts were washed with brine and concentrated at reduced
pressure after drying with anhydrous sodium sulfate. The oily
brown residue was purified by column chromatography on silica gel
eluted with a 9:1 mixture of CH2Cl2:MeOH to give the desired allyl
ether 11 in 70% yield, as a yellow oil (Rf: 0.85 after elution with 9:1
mixture of CH2Cl2:MeOH). IR (NaCl, cmꢂ1) 2869, 1502, 1403, 1337,
CH2OCH2CH2CH2OH), 7.40 (d, 2H, J ¼ 7.9 Hz, Ar-H3 and Ar-H5 ), 7.61
0
0
(d, 2H, J ¼ 7.9 Hz, Ar-H2 and Ar-H6 ), 7.68 (s, 1H, pyrazole-H3), 7.87
(s, 1H, pyrazole-H5); 13C NMR (CDCl3, 50 MHz):
d
32.3 (Ar-
CH2OCH2CH2CH2OH), 61.7 (Ar-CH2OCH2CH2CH2OH), 64.0 (Ar-
CH2OCH2CH2CH2OH), 69.2 (Ar-CH2OCH2CH2CH2OH), 120.4 (Ar-C2’
and Ar-C6’), 120.9 (pyrazole-C4), 126.3 (pyrazole-C5), 127.7 (Ar-C3
0
0
0
0
and Ar-C5 ), 132.1 (Ar-C4 ), 138.7 (Ar-C1 ), 141.3 (pyrazole-C3).
1214, 1077, 1013, 991, 953, 837; 1H NMR (CDCl3, 400 MHz):
d 4.05 (d,
2H, J ¼ 8.0 Hz, Ar-CH2OCH2CH]CH2), 4.49 (s, 2H, Ar-CH2OCH2CH]
CH2), 5.23 (d, 1H, J ¼ 10.2 Hz, Ar-CH2OCH2CH]CH2), 5.32 (d, 2H,
J ¼ 17.2 Hz, Ar-CH2OCH2CH]CH2), 5.94 (m, 1H, J ¼ 17.2, 10.2 and
4.8. Generalprocedureforthepreparationof tosylates(13a),(13b)and
(18)
0
8.0 Hz, Ar-CH2OCH2CH]CH2), 7.41 (d, 2H, J ¼ 8.0 Hz, Ar-H3 and Ar-
A mortar was charged with a finely powder potassium hydrox-
ide (0.304 g; 5.44 mmol) and corresponding alcohol (1.08 mmol),
grinded with a pestle for 5 min, then dry potassium carbonate
(0.500 g; 3.61 mmol) was added, and mixed thoroughly. Tosyl
chloride (0.311 g; 1.63 mmol) was added in one portion and grinded
vigorously for 20 min. After completion of the reaction, water was
added and the reaction mixture was extracted with dichloro-
methane (3 ꢃ 30 mL). The combined organic extracts were washed
with 10% aqueous hydrochloric acid solution, and brine. After dry-
ing under sodium sulfate and evaporation of the solvent under
reduced pressure, the residue was purified by silica gel column
chromatography after elution with dichloromethane to furnish the
corresponding tosylates 13a, 13b and 18, as described next.
0
0
0
H5 ), 7.61 (d, 2H, J ¼ 8.0 Hz, Ar-H2 and Ar-H6 ), 7.70 (s, 1H, pyrazole-
H3), 7.89 (s, 1H, pyrazole-H5); 13C NMR (CDCl3, 100 MHz):
d 62.9 (Ar-
CH2OCH2CH]CH2), 71.3 (Ar-CH2OCH2CH]CH2), 117.7 (Ar-
0
0
CH2OCH2CH]CH2), 120.4 (Ar-C2 and Ar-C6 ), 121.1 (pyrazole-C4),
0
0
0
126.4 (pyrazole-C5), 129.7 (Ar-C3 and Ar-C5 ), 132.1 (Ar-C4 ), 134.7
0
(Ar-CH2OCH2CH]CH2), 138.8 (Ar-C1 ), 141.4 (pyrazole-C3).
4.6. 2-((1-(4-Chlorophenyl)-1H-pyrazol-4-yl)methoxy)ethanol
(12a)
To
8.04 mmol) in anhydrous THF (15 mL) was added during 15 min a
solution of -oxoester derivative 10 (0.500 g; 2.01 mmol) in anhy-
a suspension of lithium aluminum hydride (0.305 g,
b
drous THF (15 mL). The reaction mixture was stirred at 70 ꢀC during
additional 4 h, when the excess of reductive agent was quenched
with methanol (1 mL) and 10% aqueous NaOH solution (1 mL) until
formation of aluminum hydroxide, which was neutralized with 10%
aqueous hydrochloric acid solution (ca. 2 mL). The obtained mixture
was extracted with ethyl acetate (3 ꢃ 20 mL) and the combined
organic extracts were washed with brine and concentrated at
reduced pressure after drying with anhydrous sodium sulfate. The
oily brown residue was purified by column chromatography on
silica gel eluted with a 99:1 mixture of CH2Cl2:MeOH to give the
desired alcohol 12 in 70% yield, as a yellow oil (Rf: 0.22 after elution
with 7:3 mixture of n-hexane:AcOEt). IR (NaCl, cmꢂ1) 3388, 2928,
1588, 1501, 1401, 1207, 1095, 1035, 984, 954, 833; 1H NMR (CDCl3,
4.8.1. 2-((1-(4-Chlorophenyl)-1H-pyrazol-4-yl)methoxy)ethyl 4-tol-
uenesulfonate (13a)
Yellow oil; 60% yield (Rf ¼ 0.82 after elution with 98:2 mixture
of CH2Cl2:MeOH). IR (NaCl, cmꢂ1) 2948, 2920, 2870, 1596, 1503,
1403, 1348, 1193, 1170, 1093, 1037, 954, 834; 1H NMR (CDCl3,
200 MHz):
d
2.34 (s, 3H, O2SPh-CH3), 3.63 (t, 2H, J ¼ 6.0 Hz, Ar-
CH2OCH2CH2OTs), 4.14 (t, 2H, J ¼ 6.0 Hz, Ar-CH2OCH2CH2OH), 4.39
00
(s, 2H, Ar-CH2OCH2CH2OTs), 7.25 (d, 2H, J ¼ 8.0 Hz, OTs-H3 and
00
00
00
OTs-H5 ), 7.35 (d, 2H, J ¼ 8.0 Hz, OTs-H2 and OTs-H6 ), 7.51e7.56
0
0
(m, 3H, Ar-H3 , Ar-H5 and pyrazole-H3), 7.73 (d, 2H, J ¼ 8.0 Hz, Ar-
H2 and Ar-H6 ), 7.79 (s, 1H, pyrazole-H5); 13C NMR (CDCl3, 50 MHz):
21.8 (O2SPh-CH3), 64.2 (Ar-CH2OCH2CH2OTs), 67.5 (Ar-
CH2OCH2CH2CH2OTs), 69.4 (Ar-CH2OCH2CH2CH2OTs), 119.2 (pyr-
0
0
d
0
0
200 MHz):
d
3.69 (t, 2H, J ¼ 5.8 Hz, Ar-CH2OCH2CH2OH), 3.79 (t, 2H,
azole-C4), 120.3 (Ar-C2 and Ar-C6 ), 126.7 (pyrazole-C5), 128.1 (OTs-
00
00
00
00
J ¼ 5.8 Hz, Ar-CH2OCH2CH2OH), 4.50 (s, 2H, Ar-CH2OCH2CH2OH),
C2 and OTs-C6 ), 129.6 (OTs-C3 and OTs-C4 ), 130.0 (Ar-C3’ and Ar-
0
0
0
00
0
0
7.44 (d, 2H, J ¼ 8.0 Hz, Ar-H3 and Ar-H5 ), 7.65 (d, 2H, J ¼ 8.0 Hz, Ar-
C5 ), 132.2 (OTs-C1 ), 133.1 (Ar-C4 ), 138.7 (Ar-C1 ), 141.2 (pyrazole-
0
0
00
H2 and Ar-H6 ), 7.70 (s, 1H, pyrazole-H3), 7.89 (s, 1H, pyrazole-H5).
C3), 145.1 (OTs-C4 ).
4.7. 3-((1-(4-Chlorophenyl)-1H-pyrazol-4-yl)methoxy)propan-1-ol
(12b)
4.8.2. 3-((1-(4-Chlorophenyl)-1H-pyrazol-4-yl)methoxy)propyl 4-tol-
uenesulfonate (13b)
Yellow oil; 60% yield (Rf ¼ 0.70 after elution with 98:2 mixture
of CH2Cl2:MeOH). IR (NaCl, cmꢂ1) IR (KBr, cmꢂ1) 2960, 2926, 2899,
2869, 1598, 1501, 1400, 1356, 1189, 1177, 1095, 952, 832; 1H NMR
To a solution of the corresponding allyl ether 11 (0.660 g;
2.65 mmol) in dry THF (15 mL) was added a 1 M borane-THF so-
lution in THF (3.70 mL, 3.70 mmol). The mixture was vigorously
stirred at 0 ꢀC, under nitrogen atmosphere and protected from light.
After 4 h, the excess of reductive agent was quenched with meth-
anol (1 mL) and 10% NaOH solution (1.3 mL) and 30% aqueous H2O2
(1.3 mL). The suspension was stirred at room temperature for 12 h.
The mixture was extracted with dichloromethane (3 ꢃ 30 mL) and
the combined organic extracts were washed with 10% aqueous
hydrochloric acid solution, and brine. After drying under sodium
sulfate and evaporation of the solvent under reduced pressure, the
residue was purified by silica gel column chromatography after
elution with n-hexane:AcOEt (1:1) to give the corresponding pri-
mary alcohol 12b in 50% yield, as a yellow oil (Rf: 0.61 after elution
with 9:1 mixture of CH2Cl2:MeOH). IR (NaCl, cmꢂ1) 3343, 3241,
2932, 2879, 1504, 1404, 1350, 1222, 1096, 1066, 1010, 953, 822; 1H
(CDCl3, 400 MHz):
d 1.92 (m, 2H, Ar-CH2OCH2CH2CH2OTs), 2.41 (s,
3H, O2SPh-CH3), 3.46 (t, 2H, J ¼ 8.0 Hz, Ar-CH2OCH2CH2CH2OTs),
4.08 (t, 2H, J ¼ 8.0 Hz, Ar-CH2OCH2CH2CH2OTs), 4.33 (s, 2H, Ar-
00
00
CH2OCH2CH2CH2OTs), 7.25 (d, 2H, J ¼ 8.0 Hz, OTs-H3 and OTs-H5 ),
00
00
7.33 (d, 2H, J ¼ 8.0 Hz, OTs-H2 and OTs-H6 ), 7.51e7.58 (m, 3H, Ar-
0
0
H3’, Ar-H5 and pyrazole-H3), 7.70 (d, 2H, J ¼ 8.0 Hz, Ar-H2 and Ar-
0
H6 ), 7.79 (s, 1H, pyrazole-H5).
4.8.3. 5-(1-(4-Chlorophenyl)-1H-pyrazol-4-yl)pent-4-ynyl 4-toluen-
esulfonate (18)
Brown solid; 80% yield (Rf ¼ 0.67 after elution with 98:2 mixture
of CH2Cl2:MeOH). IR (NaCl, cmꢂ1) 2931, 2919, 1598, 1501, 1351, 1188,
1170,1095,1004, 957, 927, 832; 1H NMR (CDCl3, 400 MHz):
d 1.85 (q,
2H, J ¼ 5.7 Hz, Ar-C^CCH2CH2CH2OTs), 2.33 (s, 3H, O2SPh-CH3),
NMR (CDCl3, 200 MHz):
d 1.86 (m, 2H, Ar-CH2OCH2CH2CH2OH),
3.63 (t, 2H, J ¼ 5.7 Hz, Ar-C^CCH2CH2CH2OTs), 4.12 (t, 2H,
00
3.67 (t, 2H, J ¼ 5.7 Hz, Ar-CH2OCH2CH2CH2OH), 3.77 (t, 2H,
J ¼ 5.7 Hz, Ar-C^CCH2CH2CH2OTs), 7.26 (d, 2H, J ¼ 8.0 Hz, OTs-H3
00
0
0
J ¼ 5.7 Hz, Ar-CH2OCH2CH2CH2OH), 4.48 (s, 2H, 3.70 (t, 2H, Ar-
and OTs-H5 ), 7.35 (d, 2H, J ¼ 8.0 Hz, Ar-H3 and Ar-H3 ), 7.52 (d, 2H,

130
T.E.T. Pompeu et al. / European Journal of Medicinal Chemistry 66 (2013) 122e134
0
0
J ¼ 8.0 Hz, Ar-H2 and Ar-H6 ), 7.57 (s, 1H, pyrazole-H3), 7.74 (d, 2H,
J ¼ 8.0 Hz, Ar-C^CCH2CH2CH2N-piperazine-Ph), 2.57 and 3.15 (2t,
8H, J ¼ 4.0 Hz, Ar-C^CCH2CH2CH2N-piperazine-Ph), 6.77e6.87 (m,
3H, Ar-C^CCH2CH2CH2N-piperazine-Ph), 7.22 (t, 2H, J ¼ 8.0 Hz, Ar-
00
00
J ¼ 8.0 Hz, OTs-H2 and OTs-H6 ), 7.82 (s, 1H, pyrazole-H5).
0
4.9. General procedure for preparation of functionalized N-phenylpi-
perazine derivatives (3), (4) and (19)
C^CCH2CH2CH2N-piperazine-Ph), 7.35 (d, 2H, J ¼ 8.0 Hz, Ar-H3 , Ar-
0
0
0
H5 ), 7.52 (d, 2H, J ¼ 8.0 Hz, Ar-H2 and Ar-H6 ), 7.66 (s, 1H, pyrazole-
H3), 7.86 (s, 1H, pyrazole-H5). Anal. Calcd. for C24H25ClN4O: C, 71.19;
H, 6.22; N, 13.84. Found: C, 71.08; H, 6.25; N, 13.88.
To a suspension of corresponding tosylate derivative 13a, 13b or
18 (0.238 mmol) and lithium carbonate (0.015 g, 0.238 mmol) in
acetonitrile (10 mL), the adequate N-phenylpiperazine derivative
(0.26 mmol) was added. The mixture was kept at reflux for 20 h
with vigorous stirring under nitrogen atmosphere. Then the solu-
tion was concentrated in a rotatory evaporator, dissolved in
dichloromethane and mixed with silica gel. The material was
separated by chromatography on silica gel eluted with a mixture
dichloromethane:MeOH (95:5), to give the desired N-phenyl-
piperazine derivatives, as described next.
4.10. 1-(4-Chlorophenyl)-4-iodo-1H-pyrazole (15)
To a solution of 4-chlorophenyl-1H-pyrazole (14) (1.00 g;
5.598 mmol) and iodine (1.875 g, 6.158 mmol) in a mixture of
acetonitrile (10 mL) and water (20.0 mL) was added 30% aq. H2O2
(1.0 mL). The reaction mixture was stirred for 24 h at room tem-
perature. After this time, a solution of 5% aq. NaHSO3 was added
until the disappearance of orange color. Then, the reaction mixture
was extracted with dichloromethane (3 ꢃ 50 mL). The combined
organic extracts were washed with water, dried under sodium
sulfate and evaporated under reduced pressure to furnish the
desired iodopyrazole derivative 15 in 98% yield, as a pale-yellow
4.9.1. 1-(2-((1-(4-Chlorophenyl)-1H-pyrazol-4-yl)methoxy)ethyl)-4-
phenylpiperazine, LASSBio-1526 (3)
Beige solid; 60% yield, mp 90e92 ꢀC. 1H NMR (CDCl3, 200 MHz):
d
2.60e2.65 (m, 6H, Ar-CH2OCH2CH2N-piperazine-Ph), 3.16 (t, 4H,
solid, mp 66e68 ꢀC. 1H NMR (CDCl3, 200 MHz):
d
7.32 (d, 2H,
0
0
0
0
J ¼ 6.0 Hz, Ar-CH2OCH2CH2N-piperazine-Ph), 3.60 (t, 2H, J ¼ 6.0 Hz,
Ar-CH2OCH2CH2N-piperazine-Ph), 4.46 (s, 2H, Ar-CH2OCH2CH2N-
piperazine-Ph), 6.75e6.87 (m, 3H, Ar-CH2OCH2CH2N-piperazine-
Ph), 7.15e7.24 (m, 2H, Ar-CH2OCH2CH2N-piperazine-Ph), 7.36 (d,
J ¼ 8.9 Hz, Ar-H3 , Ar-H5 ), 7.49 (d, 2H, J ¼ 8.9 Hz, Ar-H2 and Ar-H6 ),
7.63 (s, 1H, pyrazole-H3), 7.85 (s, 1H, pyrazole-H5); 13C NMR (CDCl3,
0 0
d 59.5 (pyrazole-C4), 120.4 (Ar-C2 and Ar-C6 ), 129.8
(pyrazole-C5), 131.4 (Ar-C3 and Ar-C5 ), 132.8 (Ar-C4 ), 138.2 (Ar-C1 ),
146.4 (pyrazole-C3).
50 MHz):
0
0
0
0
0
0
0
2H, J ¼ 8.0 Hz, Ar-H3 , Ar-H5 ), 7.57 (d, 2H, J ¼ 8.0 Hz, Ar-H2 and Ar-
H6 ), 7.64 (s, 1H, pyrazole-H3), 7.83 (s, 1H, pyrazole-H5); 13C NMR
0
(CDCl3, 50 MHz):
d
49.1 and 53.7 (Ar-CH2OCH2CH2N-piperazine-Ph),
4.11. General procedure for preparation of functionalized alkynyl-N-
58.9 (Ar-CH2OCH2CH2N-piperazine-Ph), 63.9 (Ar-CH2OCH2CH2N-
piperazine-Ph), 67.6 (Ar-CH2OCH2CH2N-piperazine-Ph), 116.2 and
phenylpiperazine derivatives (16a) and (16b)
0
119.8 (Ar-CH2OCH2CH2N-piperazine-Ph), 120.3 (Ar-C2’ and Ar-C6 ),
121.0 (pyrazole-C4), 126.3 (pyrazole-C5), 129.2 (Ar-CH2OCH2CH2N-
Under argon atmosphere, propargyl bromide or 4-bromobut-1-
yne (0.196 mmol) was slowly added, at 0 ꢀC, to a solution containing
1-(4-chlorophenyl)-4-iodo-1H-pyrazole (15) (0.164 mmol),
PdCl2(PPh3)2 (5 mol %, 0.0082 mmol), CuI (10 mol %, 0.0164 mmol),
and N-phenylpiperazine (2 mL). The reaction mixture was stirred at
room temperature for 20 h until TLC analysis indicated complete
consumption of starting materials then concentrated under
reduced pressure. The reaction mixture was filtered under diato-
maceous earth and washed with dichloromethane. The residue
obtained after concentration of organic extract under reduced
pressure was purified by chromatography on silica gel eluted with
dichloromethane to afford the desired alkynyl-N-phenylpiperazine,
as described next.
0
0
0
piperazine-Ph), 129.6 (Ar-C3 and Ar-C5 ), 132.1 (Ar-C4 ), 138.7 (Ar-
0
C1 ), 141.4 (pyrazole-C3), 151.4 (Ar-CH2OCH2CH2N-piperazine-Ph).
Anal. Calcd. for C22H25ClN4O: C, 66.57; H, 6.35; N, 14.12. Found: C,
66.61; H, 6.37; N, 14.09.
4.9.2. 1-(3-((1-(4-Chlorophenyl)-1H-pyrazol-4-yl)methoxy)propyl)-
4-phenylpiperazine, LASSBio-1527 (4)
Light yellow solid; 60% yield, mp 80e82 ꢀC. 1H NMR (CDCl3,
200 MHz):
d 1.97 (m, 2H, Ar-CH2OCH2CH2CH2N-piperazine-Ph),
2.51 (t, 2H, J ¼ 6.0 Hz, Ar-CH2OCH2CH2CH2N-piperazine-Ph), 2.64
and 3.27 (2t, 8H, J ¼ 6.0 Hz, Ar-CH2OCH2CH2CH2N-piperazine-Ph),
3.59 (t, 2H, J ¼ 6.0 Hz, Ar-CH2OCH2CH2CH2N-piperazine-Ph), 4.50
(s, 2H, Ar-CH2OCH2CH2CH2N-piperazine-Ph), 6.96e6.84 (m, 3H, Ar-
CH2OCH2CH2CH2N-piperazine-Ph), 7.28 (t, 2H, J ¼ 8.0 Hz, Ar-
4.11.1. 1-(3-(1-(4-Chlorophenyl)-1H-pyrazol-4-yl)prop-2-ynyl)-4-
phenylpiperazine (16a)
0
CH2OCH2CH2CH2N-piperazine-Ph), 7.42 (d, 2H, J ¼ 10.0 Hz, Ar-H3 ,
Yellow solid, 75% yield. 1H NMR (CDCl3, 400 MHz):
d 2.78 and
0
0
0
Ar-H5 ), 7.63 (d, 2H, J ¼ 10.0 Hz, Ar-H2 and Ar-H6 ), 7.72 (s, 1H,
3.22 (2t, 8H, J ¼ 4.0 Hz, Ar-C^CCH2N-piperazine-Ph), 3.54 (s, 2H,
Ar-C^CCH2N-piperazine-Ph), 6.80 (t, 1H, J ¼ 8.0 Hz, Ar-C^CCH2N-
piperazine-Ph), 6.87 (d, 2H, J ¼ 8.0 Hz, Ar-C^CCH2N-piperazine-
Ph), 7.19 (d, 2H, J ¼ 8.0 Hz, Ar-C^CCH2N-piperazine-Ph), 7.34 (d,
2H, J ¼ 8.0 Hz, Ar-H3’, Ar-H5’), 7.54 (d, 2H, J ¼ 8.0 Hz, Ar-H2’ and Ar-
H6’), 7.69 (s, 1H, pyrazole-H3), 7.91 (s, 1H, pyrazole-H5). Anal. Calcd.
for C22H21ClN4:C, 70.11; H, 5.62; N,14.87. Found: C, 70.19; H, 5.58; N,
14.91.
pyrazole-H3), 7.90 (s, 1H, pyrazole-H5); 13C NMR (CDCl3, 50 MHz):
d
27.2 (Ar-CH2OCH2CH2CH2N-piperazine-Ph), 49.2 and 53.3 (Ar-
CH2OCH2CH2CH2N-piperazine-Ph), 55.5 (Ar-CH2OCH2CH2CH2N-
piperazine-Ph), 63.7 (Ar-CH2OCH2CH2CH2N-piperazine-Ph), 68.6
(Ar-CH2OCH2CH2CH2N-piperazine-Ph), 116.2 and 119.8 (Ar-
0
0
CH2OCH2CH2CH2N-piperazine-Ph), 120.3 (Ar-C2 and Ar-C6 ), 121.0
(pyrazole-C4), 126.2 (pyrazole-C5), 129.2 (Ar-CH2OCH2CH2CH2N-
0
0
0
piperazine-Ph), 129.6 (Ar-C3 and Ar-C5 ), 132.1 (Ar-C4 ), 138.7 (Ar-
0
C1 ), 141.4 (pyrazole-C3), 151.4 (Ar-CH2OCH2CH2N-piperazine-Ph).
4.11.2. 1-(4-(1-(4-Chlorophenyl)-1H-pyrazol-4-yl)but-3-ynyl)-4-
Anal. Calcd. for C23H27ClN4O:C, 67.22; H, 6.62; N, 13.63. Found: C,
66.99; H, 6.67; N, 13.59.
phenylpiperazine (16b)
Yellow solid, 80% yield. 1H NMR (CDCl3, 400 MHz):
d 2.55e2.70
(m, 8H, Ar-C^CCH2CH2N-piperazine-Ph), 3.20 (t, 4H, J ¼ 6.9 Hz, Ar-
4.9.3. 1-(5-(1-(4-Chlorophenyl)-1H-pyrazol-4-yl)pent-4-ynyl)-4-
C^CCH2CH2N-piperazine-Ph), 6.78 (t, 1H,
C^CCH2CH2N-piperazine-Ph), 6.82 (d, 2H,
C^CCH2CH2N-piperazine-Ph), 7.20 (d, 2H, J
J
¼
¼
¼
8.0 Hz, Ar-
8.0 Hz, Ar-
8.0 Hz, Ar-
0
phenylpiperazine (19)
J
Light yellow solid; 84% yield. 1H NMR (CDCl3, 400 MHz):
d
1.80
(q, 2H, J ¼ 8.0 Hz, Ar-C^CCH2CH2CH2N-piperazine-Ph), 2.41 (t, 2H,
C^CCH2CH2N-piperazine-Ph), 7.30 (d, 2H, J ¼ 8.0 Hz, Ar-H3 , Ar-
0
0
0
J ¼ 8.0 Hz, Ar-C^CCH2CH2CH2N-piperazine-Ph), 2.47 (t, 2H,
H5 ), 7.49 (d, 2H, J ¼ 8.0 Hz, Ar-H2 and Ar-H6 ), 7.70 (s, 1H, pyrazole-

T.E.T. Pompeu et al. / European Journal of Medicinal Chemistry 66 (2013) 122e134
131
0
0
0
H3), 7.85 (s, 1H, pyrazole-H5). Anal. Calcd. for C23H23ClN4: C, 70.67;
H, 5.93; N, 14.33. Found: C, 70.77; H, 5.89; N, 14.27.
H5 ), 7.47 (d, 2H, J ¼ 8.0 Hz, Ar-H2 and Ar-H6 ), 7.72 (s, 1H, pyrazole-
H3), 7.90 (s, 1H, pyrazole-H5). 13C NMR (CDCl3, 50 MHz):
d 24.1 (Ar-
CH2CH2CH2CH2N-piperazine-Ph), 26.2 (Ar-CH2CH2CH2CH2N-piper-
azine-Ph), 28.8 (Ar-CH2CH2CH2CH2N-piperazine-Ph), 22.2 (Ar-
CH2CH2CH2N-piperazine-Ph), 27.9 (Ar-CH2CH2CH2N-piperazine-
Ph), 49.0 and 53.7 (Ar-CH2CH2CH2CH2N-piperazine-Ph), 58.5 (Ar-
4.12. 5-(1-(4-Chlorophenyl)-1H-pyrazol-4-yl)pent-4-yn-1-ol (17)
Under argon atmosphere, 4-pentyn-1-ol (0.049 g; 0.590 mmol)
was slowly added, at
0
ꢀC, to
a
solution containing 1-(4-
CH2CH2CH2CH2N-piperazine-Ph),
116.3
and
(Ar-CH2CH2CH2CH2N-
0
0
chlorophenyl)-4-iodo-1H-pyrazole (15) (0.15 g; 0.492 mmol),
PdCl2(PPh3)2 (0.035 g; 10 mol %), CuI (0.009 g; 10 mol %), 1,4-
dioxane (10 mL) and triethylamine (1 mL). The reaction mixture
was stirred at room temperature for 12 h until TLC analysis indi-
cated complete consumption of starting materials then concen-
trated under reduced pressure. The reaction mixture was filtered
under diatomaceous earth and washed with dichloromethane. The
residue obtained after concentration of organic extract under
reduced pressure was purified by chromatography on silica gel
eluted with dichloromethane to afford the desired alkynyl-alcohol
17 in 80% yield, as a yellow solid. IR (NaCl, cmꢂ1) 3200, 2926, 1500,
piperazine-Ph), 118.9
(Ar-C2
Ar-C6 ), 120.1
(Ar-
CH2CH2CH2CH2N-piperazine-Ph), 123.6 (pyrazole-C4), 124.9 (pyr-
0
azole-C5), 126.2 (Ar-C4 ), 129.3 (Ar-CH2CH2CH2CH2N-piperazine-Ph),
0
0
0
129.5 (Ar-C3 and Ar-C5 ),140.1 (Ar-C1 ),141.4 (pyrazole-C3),151.2 (Ar-
CH2CH2CH2CH2N-piperazine-Ph). Anal. Calcd. for C23H27ClN4: C,
69.95; H, 6.89; N, 14.19. Found: C, 70.09; H, 6.91; N, 14.13.
4.13.3. 1-(4-(1-(4-Chlorophenyl)-1H-pyrazol-4-yl)butyl)-4-phenylpi-
perazine, LASSBio-1762 (7)
Beige solid; 98% yield, mp 58e60 ꢀC. 1H NMR (CDCl3, 400 MHz):
d
1.45 (q, 4H, J ¼ 5.0 Hz, Ar-CH2CH2CH2CH2CH2N-piperazine-Ph),
1401, 1230, 1094, 1032, 953, 829; 1H NMR (CDCl3, 400 MHz):
d
1.86
1.62 (q, 4H, J ¼ 7.0 Hz, Ar-CH2CH2CH2CH2CH2N-piperazine-Ph), 1.68
(q, 4H, J ¼ 7.0 Hz, Ar-CH2CH2CH2CH2CH2N-piperazine-Ph), 2.45 (t,
2H, J ¼ 7.0 Hz, Ar-CH2CH2CH2CH2CH2N-piperazine-Ph), 2.60 (t, 2H,
J ¼ 7.0 Hz, Ar-CH2CH2CH2CH2CH2N-piperazine-Ph), 2.65 and 3.27
(2t, 8H, J ¼ 5.0 Hz, Ar-CH2CH2CH2CH2CH2N-piperazine-Ph), 6.85 (t,
1H, J ¼ 6.8 Hz, Ar-CH2CH2CH2CH2CH2N-piperazine-Ph), 6.93 (d, 2H,
J ¼ 6.8 Hz, Ar-CH2CH2CH2CH2CH2N-piperazine-Ph), 7.25e7.35 (m,
(q, 2H, J ¼ 7.6 Hz, Ar-C^CCH2CH2CH2OH), 2.54 (t, 2H, J ¼ 7.6 Hz, Ar-
C^CCH2CH2CH2OH), 3.82 (t, 2H, J ¼ 7.6 Hz, Ar-C^CCH2CH2CH2OH),
0
0
0
7.42 (d, 2H, J ¼ 8.0 Hz, Ar-H3 , Ar-H5 ), 7.59 (d, 2H, J ¼ 8.0 Hz, Ar-H2
0
and Ar-H6 ), 7.71 (s, 1H, pyrazole-H3), 7.92 (s, 1H, pyrazole-H5). Anal.
Calcd. for C14H13ClN2O: C, 64.49; H, 5.03; N, 6.14. Found: C, 64.27; H,
5.05; N, 6.13.
0
2H, Ar-CH2CH2CH2N-piperazine-Ph), 7.36 (d, 2H, J ¼ 8.0 Hz, Ar-H3 ,
0
0
0
4.13. General procedure for preparation of saturated N-phenylpipera-
zine derivatives (5), (6) and (7)
Ar-H5 ), 7.53 (d, 2H, J ¼ 8.0 Hz, Ar-H2 and Ar-H6 ), 7.66 (s, 1H, pyr-
azole-H3), 7.86 (s, 1H, pyrazole-H5). Anal. Calcd. for C24H29ClN4: C,
70.48; H, 7.15; N, 13.70. Found: C, 70.31; H, 7.17; N, 13.76.
To a solution of the corresponding alkynyl-N-phenylpiperazine
derivative 16a, 16b or 19 (0.494 mmol) in an 1:1 mixture of MeOH:
EtOH (40 mL) catalytic amount of 10% Pd-C (0.055 g) was added.
The reaction mixture was maintained at room temperature and
kept under an atmosphere of hydrogen under pressure (40 psi) for
3 h. The reaction mixture was filtered under diatomaceous earth
and washed with dichloromethane. After concentration of organic
extract under reduced pressure the respective saturated N-phe-
nylpiperazine derivative was obtained, as described next.
4.14. In vitro pharmacological evaluation
4.14.1. Radioligands and drugs
[3H]-YM-09151-2 (82.7 Ci/mmol), [3H]-8-OH-DPAT (154.2 Ci/
mmol), [3H]-ketanserin (67 Ci/mmol) [3H]-RX821002 (60 Ci/mmol),
[3H]-QNB (48 Ci/mmol), [3H]-prazosin (85 Ci/mmol) and [3H]-
mesulergine (84.3 Ci/mmol) were purchased from New England
Nuclear Life Science Products, PerkinElmer, USA. Ketanserin
tartrate, pargilyne hydrochloride, prazosin hydrochloride, 5-HT
creatinine sulphate, (ꢁ)-sulpiride, (ꢂ)-epinephrine, clozapine,
atropine sulfate and guanosine 50-triphosphate sodium salt hydrate
were purchased from Sigma, São Paulo, Brazil. Stock solutions were
prepared in DMSO (test substances, clozapine and (ꢁ)-sulpiride),
ethanol (prazosin) or purified water (other compounds). At the
final concentration used, DMSO and ethanol (below 0.5%) had no
effect in our assays.
4.13.1. 1-(3-(1-(4-Chlorophenyl)-1H-pyrazol-4-yl)propyl)-4-phenyl-
piperazine, LASSBio-1528 (5)
Light yellow solid; 99% yield, mp 45e47 ꢀC. 1H NMR (CDCl3,
200 MHz):
d
1.87 (t, 2H, J ¼ 8.0 Hz, Ar-CH2CH2CH2N-piperazine-Ph),
2.35e2.59 (m, 8H, Ar-CH2CH2CH2N-piperazine-Ph), 3.15 (t, 4H,
J ¼ 5.0 Hz, Ar-CH2CH2CH2N-piperazine-Ph), 6.75e6.88 (m, 3H, Ar-
CH2CH2CH2N-piperazine-Ph), 7.18e7.22 (m, 2H, Ar-CH2CH2CH2N-
0
0
piperazine-Ph), 7.34 (d, 2H, J ¼ 8.0 Hz, Ar-H3 , Ar-H5 ), 7.49 (d, 2H,
0
0
J ¼ 8.0 Hz, Ar-H2 and Ar-H6 ), 7.55 (s, 1H, pyrazole-H3), 7.62 (s, 1H,
4.14.2. Tissue preparation
pyrazole-H5). 13C NMR (CDCl3, 50 MHz):
d
22.2 (Ar-CH2CH2CH2N-
Adult male Wistar rats (250e300 g) were anaesthetized with
ether and killed by decapitation. The brains were immediately
removed on ice and hippocampus, striatum and cortex were
dissected, weighted and stored in liquid nitrogen until use. The liver
was immediately removed on ice, weighted and stored in liquid
nitrogen until use. This procedure was approved by the Institu-
tional Ethical Committee for Animal Care from the Federal Uni-
versity of Rio de Janeiro and satisfied the EU Directive 2010/63/
EUfor animal experiments.
piperazine-Ph), 27.9 (Ar-CH2CH2CH2N-piperazine-Ph), 49.2 and
53.4 (Ar-CH2CH2CH2N-piperazine-Ph), 57.9 (Ar-CH2CH2CH2N-
piperazine-Ph), 116.3 and 119.8 (Ar-CH2CH2CH2N-piperazine-Ph),
0
0
120.0 (Ar-C2 and Ar-C6 ), 123.9 (pyrazole-C4), 124.9 (pyrazole-C5),
0
0
129.3 (Ar-CH2CH2CH2N-piperazine-Ph), 129.6 (Ar-C3 and Ar-C5 ),
0
0
131.6 (Ar-C4 ), 138.9 (Ar-C1 ), 141.4 (pyrazole-C3), 151.4 (Ar-
CH2CH2CH2N-piperazine-Ph). Anal. Calcd. for C22H25ClN4: C, 69.37;
H, 6.62; N, 14.71. Found: C, 69.55; H, 6.59; N, 14.67.
Striatum were homogenized in a Potter apparatus with a motor-
driven Teflon pestle at 4 ꢀC in 20 volumes per gram of tissue of ice-
cold TriseHCl 50 mM buffer (pH 7.4) containing MgCl2 8 mM and
EDTA 5 mM. The resulting suspension was ultracentrifuged at
48,000 gav at 4 ꢀC for 20 min. The pellet was resuspended in 20
volumes of buffer and incubated at 37 ꢀC during 10 min for
endogenous neurotransmitters removal. This suspension was
cooled on ice and ultracentrifuged twice at 48,000 gav for 20 min at
4.13.2. 1-(4-(1-(4-Chlorophenyl)-1H-pyrazol-4-yl)butyl)-4-phenyl-
piperazine, LASSBio-1635 (6)
Beige solid; 99% yield, mp 78e80 ꢀC. 1H NMR (CDCl3, 200 MHz):
d
1.51e1.65 (m, 4H, Ar-CH2CH2CH2CH2N-piperazine-Ph), 3.28 (t, 4H,
J ¼ 5.0 Hz, Ar-CH2CH2CH2CH2N-piperazine-Ph), 6.84e6.93 (m, 3H,
Ar-CH2CH2CH2N-Piperazine-Ph), 7.32 (t, 2H,
CH2CH2CH2CH2N-piperazine-Ph), 7.42 (d, 2H, J ¼ 8.0 Hz, Ar-H3 , Ar-
J
¼
8.0 Hz, Ar-
0

132
T.E.T. Pompeu et al. / European Journal of Medicinal Chemistry 66 (2013) 122e134
4
ꢀC. The final pellet was resuspended in buffer yielding a propor-
Non-specific binding was estimated in the presence of 100
HT. For binding to a2 adrenoreceptors, cortical membranes from
rat brain (150
g protein) and 1 nM [3H]-RX821002 were incubated
at 30 ꢀC for 45 min under yellow light in a solution containing Trise
HCl 50 mM (pH 7.4), in a final volume of 500 L. Non-specific
binding was estimated in the presence of 100
M (ꢂ)-epineph-
mM 5-
tion of 1.5 mL/g tissue and stored in liquid nitrogen until use.
Hippocampus and cortex were homogenized in a Potter apparatus
with a motor-driven Teflon pestle at 4 ꢀC in 20 volumes (hippo-
campus) or 10 volumes (cortex) of ice-cold TriseHCl 50 mM (pH
7.4) buffer per gram of tissue. The resulting suspension was
centrifuged twice at 900 gmax at 4 ꢀC for 10 min. The resulting su-
pernatants were combined and ultracentrifuged at 48,000 gav for
10 min. The pellet was resuspended in buffer and incubated at 37 ꢀC
during 10 min for endogenous neurotransmitters removal. This
suspension was cooled on ice and ultracentrifuged twice at
48,000 gav for 10 min at 4 ꢀC. The final pellet was resuspended in
buffer yielding a proportion of 1.5 mL/g tissue and stored in liquid
nitrogen until use.
The livers were thawed in a buffer (sucrose 0.25 M, EGTA 1 mM,
TriseHCl 5 mM, pH 7.2) and minced in ice-cold buffer EDTA 2 mM,
NaCl 100 mM, TriseHCl 50 mM (pH 7.2) discarding the fibrous part.
The organs were homogenized in an Ultra-Turrax apparatus, 3
times for 15 s. The resulting suspension was filtrated and centri-
fuged at 5000 gav at 4 ꢀC for 20 min. The resulting supernatants
were ultracentrifuged at 100,000 gav at 4 ꢀC for 60 min. The pellet
was resuspended in ice-cold buffer (EDTA 1 mM, TriseHCl 50 mM,
pH 7.2) and ultracentrifuged as described above. The final pellet
was resuspended in ice-cold buffer (sucrose 0.25 M, TriseHCl
5 mM, pH 7.4). The protein concentration was determined by the
method of Lowry and co-workers [45] using bovine serum albumin
as standard.
m
m
m
rine. In the GTP shift assay, paired competition curves for the
binding of 1 nM [3H]-RX821002 were performed in the presence
and absence of 1 mM GTP.
For a1B receptors, rat liver membranes (150
mg protein) and
0.1 nM [3H]-prazosin were incubated at 30 ꢀC for 45 min in a so-
lution containing EDTA 1 mM and TriseHCl 50 mM (pH 7.4), in a
final volume of 500
presence of 1 M prazosin. For the muscarinic receptors, cortical
membranes from rat brain (150
g protein) and 0.1 nM [3H]-QNB
were incubated at room temperature for 60 min in a solution
containing TriseHCl 50 mM (pH 7.4), in a final volume of 500 L.
Non-specific binding was estimated in the presence of 1 M atro-
pine sulfate. In all assays, different concentrations of the test
compounds were used and a 30 M value was assumed as the cut
mL. Non-specific binding was estimated in the
m
m
m
m
m
off for evaluation. After incubation, samples were rapidly diluted
with 3 ꢃ 4 mL TriseHCl 5 mM and immediately filtered under
vacuum on glass fibre filters (GMF 3, Filtrak, Germany) previously
soaked in 0.5% polyethyleneimine (only for the binding to 5-HT1A
5-HT2A, 5-HT2C, and a2 receptors). Filters were then dried and
immersed in scintillation mixture (POPOP (1,4-bis-[2-(5-
,
a
phenyloxazolyl)]-benzene) 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. Competition curves with clozapine and LASSBio-579 were
also performed for receptor profile comparisons.
4.14.3. Binding assays
For the binding to D2-like receptors, striatal membranes from
rat brain (50
m
g protein) and [3H]-YM-09151-2 (0.1 nM) were
incubated at 37 ꢀC for 60 min under yellow light in a solution
containing 120 mM NaCl, 5 mM KCl, 5 mM MgCl2, 1.5 mM CaCl2,
1 mM EDTA and TriseHCl 50 mM (pH 7.4) in a final volume of
500
30
branes from rat brain (40
were incubated at 37 ꢀC for 15 min under yellow light in a solution
containing 1 mM CaCl2, 1 mM MnCl2, 10 M pargyline and TriseHCl
50 mM (pH 7.4), in a final volume of 500 L. Non-specific binding
was estimated in the presence of 10 M 5-HT. In the GTP shift assay,
m
L. Non-specific binding was estimated in the presence of
M (ꢁ)-sulpiride. For 5-HT1A receptors, hippocampal mem-
g protein) and 1 nM [3H]-8-OH-DPAT
4.15. In vivo pharmacological evaluation
m
m
4.15.1. Animals
Adult male CF1 mice (25e35 g) from Fundação Estadual de
Produção e Pesquisa em Saúde (FEPPS-RS) breeding colony were
used. Animals (05 per cage) were housed in plastic cages
(17 cm ꢃ 28 cm ꢃ 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 lightedark cycle (lights off at 7:00
pm) and were adapted to local conditions for at least 72 h before
the experiments. All experimental protocols were approved by
CEUA e UFRGS (Protocol 22650/2012) and performed according to
guidelines of The National Research Ethical Committee (published
by National Heath Council-MS, 1998) and Brazilian law [46], which
are in compliance with the International Guiding Principles for
Biomedical Research Involving Animals [47].
m
m
m
paired competition curves for the binding of 0.5 nM [3H]-pMPPF
were performed in the presence and absence of 1 mM GTP. The
incubation was performed at 37 ꢀC for 45 min under yellow light in
TriseHCl 50 mM (pH 7.4). Non-specific binding was estimated in
the presence of 10
branes from rat brain (150
were incubated at 37 ꢀC for 15 min in a solution containing 100 nM
m
M 5-HT. For 5-HT2A receptors, cortical mem-
m
g protein) and [3H]-ketanserin (1 nM)
prazosin and TriseHCl 50 mM (pH 7.4), in a final volume of 500
Non-specific binding was estimated in the presence of 1
mL.
mM
ketanserin. In the GTP shift assay (Fig. 1), paired competition curves
were performed in the presence and absence of a guanylyl nucle-
otide (0.1 mM Gpp(NH)p or 1 mM GTP had the same effects). For
the binding to D4 receptors, membrane preparations of cells
4.15.2. Drugs and treatments
Apomorphine hydrochloride hemihydrate (Sigma, São Paulo,
Brazil), ketamine hydrochloride (Cristália, São Paulo, Brazil), clo-
zapine (Novartis, São Paulo, Brazil) and haloperidol (Galena, São
Paulo, SP, Brazil) were used. LASSBio1635 and haloperidol were
suspended in saline with addition of 1% (v/v) polissorbate 80. Ke-
tamine was directly dissolved in saline. Apomorphine was dis-
solved in saline with addition of 0.1% ascorbic acid and clozapine in
saline with addition of 0.1% acetic acid 0.1 M. Vehicle groups
received 1% (v/v) polissorbate 80 in saline. The drugs were
administered by intraperitoneal and oral routes (10 ml/kg body
weight) or subcutaneously (5 ml/kg body weight). All animals were
fasted for 2 h before oral drug administration. All doses are
expressed as free base.
transfected with human receptors (ChemiscreenÔ, Millipore) (5 mg
protein) and [3H]-YM-09151-2 (0.1 nM) were incubated at room
temperature for 120 min under yellow light in a solution containing
120 mM NaCl, 5 mM KCl, 5 mM MgCl2, 1.5 mM CaCl2, 1 mM EDTA
and TriseHCl 50 mM (pH 7.4) in a final volume of 500
specific binding was estimated in the presence of 10
pine. For 5-HT2C receptors, membrane preparations of cells trans-
mL. Non-
m
M cloza-
fected with human receptors (ChemiscreenÔ, Millipore) (5
mg
protein) and 1 nM [3H]-mesulergine were incubated at 37 ꢀC for
60 min under yellow light in a solution containing 50 mM HEPES
(pH 7.4), 5 mM MgCl2 and 1 mM CaCl2 in a final volume of 500 mL.

T.E.T. Pompeu et al. / European Journal of Medicinal Chemistry 66 (2013) 122e134
133
4.15.3. Apomorphine-induced climbing
fellowships. Barreiro, E.J.; Fraga, C.A.M.; Noël, F. and Rates, S.M.K.
are senior CNPq fellows (Brazil).
This test was conducted as described by Neves et al. [15]. Briefly,
mice were treated with test compounds or vehicle (first treatment)
and immediately put in cages (29 cm ꢃ 23 cm ꢃ 19 cm) with the
floor, walls and top consisting of parallel metal bars (2 mm diam-
eter). Animals were allowed to freely explore the cages for 30 min.
After that they were treated with apomorphine 4 mg/kg or vehicle
s.c. (second treatment). The climbing behavior score was evaluated
as: normal behavior (0 point), increased activity and sniffing (1
point), occasional clinging to sides of cage with forepaws (2 points),
intermittent clinging to sides or top of cage with all four paws (3
points) and uninterrupted climbing with all four paws (4 points).
Climbing behavior was scored at 5, 10, 15, 20, 25 and 30 min after
second treatment administration. The period of observation in each
interval was 1 min. The climbing index was calculated as the sum of
all scores obtained by the same animal at each time interval.
References
[1] WHO (World Health Organization). http://www.who.int/mental_health/
management/schizophrenia/en/ (accessed 13.10.12).
[2] A.H.C. Wong, H.H.M. Van Tol, Schizophrenia: from phenomenology to
neurobiology, Neurosci. Biobehav. Rev. 27 (2003) 269e306.
[3] D.T.Y. Tsoi, M.D. Hunter, P.W.R. Woodruff, History, aetiology, and symptom-
atology of schizophrenia, Psychiatry 7 (2008) 404e409.
[4] R. Tandon, H.A. Nasrallah, M.S. Keshavan, Schizophrenia, “just the fact” 5.
Treatment and prevention. Past, present, and future, Schizophr. Res. 122
(2010) 1e23.
[5] B.L. Roth, D.J. Sheffler, W.K. Kroeze, Magic shotguns versus magic bullets:
selectively non-selective drugs for mood disorders and schizophrenia, Nat.
Rev. Drug Discov. 3 (2004) 353e359.
[6] G. Gründer, H. Hippius, A. Carlsson, The ‘atypicality’ of antipsychotics:a concept
re-examined and re-defined, Nat. Rev. Drug Discov. 8 (2009) 197e202.
[7] H.Y. Meltzer, Serotonergic mechanisms as targets for existing and novel an-
tipsychotics, Handb. Exp. Pharmacol. 212 (2012) 87e124.
[8] D.H. Kim, M.J. Maneen, S.M. Stahl, Building a better antipsychotic: receptor
targets for the treatment of multiple symptom dimensions of schizophrenia,
Neurotherapeutics 6 (2009) 78e85.
[9] A. Newman-Tancredi, The importance of 5-HT1A receptor agonism in anti-
psychotic drug action: rationale and perspectives, Curr. Opin. Invest. Drugs 11
(2010) 802e812.
4.15.4. Spontaneous locomotor activity
Locomotor activity was monitored in an area made of acrylic
(transparent walls and black floor, 30 cm ꢃ 30 cm ꢃ 45 cm) with
the floor divided into 24 squares of equal area. Mice were treated
with test compounds or vehicle and immediately positioned at the
apparatus center. Mice were allowed to freely explore during
30 min and then observed during 20 min. The total number of
squares crossings during the last 20 min was recorded by a trained
observer unaware of the treatments. All procedures were done in a
room dimly lit.
[10] S. Kapur, G. Remington, Serotonin-dopamine interaction and its relevance to
schizophrenia, Am. J. Psychiatry 153 (1996) 466e476.
[11] N.M. Richtand, J.A. Welge, A.D. Logue, P.E. Keck Jr., S.M. Strakowski,
R.K. Mcnamara, Dopamine and serotonin receptor binding and antipsychotic
efficacy, Neuropsychopharmacology 32 (2007) 1715e1726.
[12] J.A. Lieberman, T.S. Stroup, J.P. McEvoy, M.S. Swartz, R.A. Rosenheck,
D.O. Perkins, R.S.E. Keefe, S.M. Davis, C.E. Davis, B.D. Lebowitz, J. Severe,
J.K. Hsiao, For the Clinical Antipsychotic Trials of Intervention Effectiveness
(CATIE) Investigators, Effectiveness of antipsychotic drugs in patients with
chronic schizophrenia, N. Engl. J. Med. 353 (2005) 1209e1223.
[13] R. Tandon, S.D. Targum, H.A. Nasrallah, R. Ross, Strategies for maximizing
clinical effectiveness in the treatment of schizophrenia, J. Psychiatr. Pract. 12
(2006) 348e363.
[14] R. Menegatti, A.C. Cunha, V.F. Ferreira, E.F.R. Pereira, A. El-Nabawi,
A.T. Eldefrawi, E.X. Albuquerque, G. Neves, S.M.K. Rates, C.A.M. Fraga,
E.J. Barreiro, Design, synthesis and pharmacological profile of novel dopamine
D2 receptor ligands, Bioorg. Med. Chem. 11 (2003) 4807e4813.
[15] G. Neves, R. Menegatti, C.B. Antonio, L.R. Grazziottin, R.O. Vieira, S.M.K. Rates,
F. Noël, E.J. Barreiro, C.A.M. Fraga, Searching for multi-target antipsychotics:
discovery of orally active heterocyclic N-Phenylpiperazine ligands of D2-like
and 5-HT1A receptors, Bioorg. Med. Chem. 18 (2010) 1925e1935.
[16] T.F. Gomes, T.E.T. Pompeu, D.A. Rodrigues, F. Noël, R. Menegatti,
C.A. Andrade, J.R. Sabino, E.S. Gil, T. Dalla Costa, A.H. Betti, C.B. Antonio,
S.M.K. Rates, C.A.M. Fraga, E.J. Barreiro, V. De Oliveira, Biotransformation of
LASSBio-579 and pharmacological evaluation of p-hydroxylated metabolite a
N-phenylpiperazine antipsychotic lead compound, Eur. J. Med. Chem. 62
(2013) 214e221.
[17] G. Neves, C.B. Antonio, A.H. Betti, M.A. Pranke, C.A.M. Fraga, E.J. Barreiro,
F. Noël, S.M.K. Rates, New insights into pharmacological profile of LASSBio-
579, a multi-target N-phenylpiperazine derivative active on animal models
of schizophrenia, Behav. Brain Res. 237 (2013) 86e95.
[18] G. Neves, M. Kliemann, A.H. Betti, D.J. Conrado, L. Tasso, C.A.M. Fraga,
E.J. Barreiro, T. Dalla Costa, S.M.K. Rates, Serotonergic neurotransmission
mediates hypothermia induced by the N-phenylpiperazine antipsychotic
prototypes LASSBio-579 and LASSBio-581, Pharmacol. Biochem. Behav. 89
(2008) 23e30.
4.15.5. Ketamine-induced hyperlocomotion
The same apparatus and conditions for the measure of sponta-
neous locomotor activity were used. Mice were treated with test
compounds or vehicle, positioned at the apparatus center and
allowed to free exploration. Thirty minutes later they were treated
with ketamine 10 mg/kg s.c. and observed during 20 min. The total
number of squares crossings was recorded by a trained observer
unaware of the treatments. Doses of clozapine (1 mg/kg p.o.) and
haloperidol (0.01 mg/kg p.o.) were chosen on the basis of absence
of effect on animals’ spontaneous locomotion [17].
4.16. Statistical analysis
The median inhibitory concentrations (IC50) were estimated
from the competition curves, using a computerized non-linear
regression analysis of the untransformed data (Prism 4.0, Graph-
Pad Software Inc.), assuming one or two populations of binding
sites (“one, or two, site competition” equation). The Ki values were
calculated using the ChengePrusoff equation: Ki
¼
IC50/
(1 þ [radioligand]/Kd). The Kd values were obtained in saturation
experiments.
Apomorphine-induced climbing, locomotor activity and
ketamine-induced hyperlocomotion tests results were analyzed by
one-way ANOVA followed by Student-NewmaneKeuls post hoc
test. The analyses were performed using Sigma Stat 2.03 software
(Jandel Scientific Corporation). Differences were considered sta-
tistically significant at P < 0.05.
[19] C.G. Wermuth, The Practice of Medicinal Chemistry, third ed., Academic Press,
UK, 2008.
[20] M.M. El-Kerdawya, E.R. El-Bendary, A.A.-M. Abdel-Aziz, D.R. El-wasseef,
N.I. Abd El-Aziz, Synthesis and pharmacological evaluation of novel fused
thiophene derivatives as 5-HT2A receptor antagonists: molecular modeling
study, Eur. J. Med. Chem. 45 (2010) 1805e1820.
[21] F.M. Awadallah, Synthesis, pharmacophore modeling, and biological evalua-
tion of novel 5H-Thiazolo[3,2-a]pyrimidin-5-one derivatives as 5-HT2A re-
ceptor antagonists, Sci. Pharm. 76 (2008) 415e438.
[22] S.N. Aslam, P.C. Stevenson, S.J. Phythian, N.C. Veitch, D.R. Hall, Synthesis of
cicerfuran, an antifungal benzofuran, and some related analogues, Tetrahe-
dron 62 (2006) 4214e4226.
Conflict of interest
The authors declare to have no conflict of interest.
[23] L.A.S. Romeiro, M.S. Ferreira, L.L. Silva, H.C. Castro, A.L.P. Miranda, C.L.M. Silva,
F. Noël, J.B. Nascimento, C.V. Araújo, E. Tibiriçá, E.J. Barreiro, C.A.M. Fraga,
Discovery of LASSBio-772, a 1,3-benzodioxole N-phenylpiperazine derivative
with potent alpha 1A/D-Adrenergic receptor blocking properties, Eur. J. Med.
Chem. 46 (2011) 3000e3012.
Acknowledgments
[24] C.D. Duarte, H. Verli, J.X. Araújo-Junior, I.A. Medeiros, E.J. Barreiro,
C.A.M. Fraga, New optimized piperamide analogues with potent in vivo hy-
potensive properties, Eur. J. Pharm. Sci. 23 (2004) 363e369.
The authors thank CAPES (BR), CNPq (BR), FAPERJ (BR) and INCT-
INOFAR (BR, #573.564/2008-6) for financial support and

134
T.E.T. Pompeu et al. / European Journal of Medicinal Chemistry 66 (2013) 122e134
[25] H. Brown, B. Rao, A new technique for the conversion of olefins into orga-
noboranes and related alcohols, J. Am. Chem. Soc. 78 (1956) 5694e5695.
[26] F. Kazemi, A.R. Massah, M. Javaherian, Chemoselective and scalable prepara-
tion of alkyl tosylates under solvent-free conditions, Tetrahedron 63 (2007)
5083e5087.
[27] M.M. Kim, R.T. Ruck, D. Zhao, M.A. Ruffman, Green iodination of pyrazoles
with iodine/hydrogen peroxide in water, Tetrahedron Lett. 49 (2008) 4026e
4028.
[28] N. Olivi, P. Spruyt, J.-F. Peyrat, M. Alami, J.-D. Brion, Tandem amine
propargylation-Sonogashira reactions: new three-component coupling lead-
ing to functionalized substituted propargylic amines, Tetrahedron Lett. 45
(2004) 2607e2610.
[29] D. Teschner, E. Vass, M. Hävecker, S. Zafeiratos, P. Schnörch, H. Sauer, A. Knop-
Gericker, R. Schlögl, M. Chaman, A. Wootsch, A.S. Canning, J.J. Gamman,
S.D. Jackson, J. McGregor, L.F. Gladden, Alkyne hydrogenation over Pd cata-
lysts: a new paradigm, J. Catal. 242 (2006) 26e37.
[36] A. De Lean, J.M. Stadel, R.J. Lefkowitz, A ternary complex model explains the
agonist-specific binding properties of the adenylate cyclase-coupled
adrenergic receptor, J. Biol. Chem. 255 (1980) 7108e7117.
b-
[37] T. Branchek, N. Adham, M. Macchi, H.-T. Kao, P.R. Hartig, [3H]-DOB(4-Bromo-
2, 5-Dimethoxyphenylisopropylamine) and [3H]ketanserin label two affinity
states of the cloned human 5-Hydroxytryptamine2 receptor, Mol. Pharmacol.
38 (1990) 604e609.
[38] T.P. Kenakin, A pharmacology primer: theory, applications, and methods,
in: Pharmacological Assay Formats: Binding, third ed., London, UK. 2009,
pp. 61e79.
[39] B. Costall, R.J. Naylor, V. Nohria, Climbing behaviour induced by apomorphine
in mice: a potential model for the detection of neuroleptic activity, Eur. J.
Pharmacol. 50 (1978) 39e50.
[40] M.A. Geyer, B. Ellenbroek, Animal behavior models and the mechanisms un-
derlying antipsychotic atypicality, Prog. Neuropsychopharmacol. Biol. Psy-
chiatry 27 (2003) 1071e1079.
[30] J.L. Mokrosz, M.J. Mokrosz, S. Charakchieva-Minol, M.H. Paluchowska,
A.J. Bojarski, B. Duszynska, Structureeactivity relationship studies of CNS
agents, XIX: quantitative analysis of the alkyl chain effects on the 5-HT1A and
5-HT2 receptor affinities of 4-alkyl-1arylpiperazines and their analogs, Arch.
Pharm. 328 (1995) 143e148.
[41] J.M. Weiss, C.D. Kilts, Animal models of depression and schizophrenia, in:
A.F. Schatzberg, C.B. Nemeroff (Eds.), Textbook of Psychopharmacology, sec-
ond ed., American Psychiatric Press, Washington, 1998.
[42] R.D. Porsolt, P.C. Moser, V. Castagné, Behavioral indices in antipsychotic drug
discovery, J. Pharmacol. Exp. Ther. 333 (2010) 632e638.
[31] M.J. Mokrosz, M.H. Paluchowska, S. Charakchieva-Minol, A. Bien, Structuree
activity relationship studies of central nervous system agents, Part 32: Effect
[43] J.T. Coyle, G. Tsai, D. Goff, Converging evidence of NMDA receptor hypo-
function in the pathophysiology of schizophrenia, Ann. N. Y. Acad. Sci. 1003
(2003) 318e327.
[44] A. Satow, G. Suzuki, S. Maehara, H. Hikichi, T. Murai, T. Murai, H. Kawagoe-Takaki,
M. Hata, S. Ito, S. Ozaki, H. Kawamoto, H. Ohta, Unique antipsychotic activities of
of structural modifications on
(1997) 177e180.
a1-adrenoceptor affinity, Arch. Pharm. 330
[32] K. Ehrlich, A. Gotz, S. Bollinger, N. Tschammer, L. Bettinetti, S. Harterich,
H. Hubner, H. Lanig, P. Gmeiner, Dopamine D2, D3, and D4 selective phe-
nylpiperazines as molecular probes to explore the origins of subtype specific
receptor binding, J. Med. Chem. 52 (2009) 4923e4935.
the selective metabotropic glutamate receptor
1 allosteric antagonist 2-
cyclopropyl-5-[1-(2-fluoro-3-pyridinyl)-5-methyl-1H-1,2,3-triazol-4yl]-2,3-
dihydro-1H-isoindol-1-one, J. Pharmacol. Exp. Ther. 330 (2009) 179e190.
[45] O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall, Protein measurement with
the folin phenol reagent, J. Biol. Chem. 193 (1951) 265e275.
[33] E.M. Hecht, D.C. Landy, Alpha-2 receptor antagonist add-on therapy in the
treatment of schizophrenia;
a meta-analysis, Schizophr. Res. 134 (2012)
202e206.
[46] Brasil, Congresso Nacional Lei n. 11.794. Regulamenta o inciso VII do x 1o do
art. 225 da Constituição Federal, estabelecendo procedimentos para o uso
científico de animais; revoga a Lei no 6.638, de 8 de maio de 1979; e dá outras
providências. D.O.U., 2008.
[34] S. Leucht, C. Corves, D. Arbter, R.R. Engel, C. Li, J.M. Davis, Second-generation
versus first-generation antipsychotic drugs for schizophrenia: a meta-anal-
ysis, Lancet 373 (2009) 31e41.
[35] R.J. Baldessarini, R. Frankenburg, Clozapine e a novel antipsychotic agent,
N. Eng. J. Med. 324 (1991) 746e754.
[47] CIOMS, International Guiding Principles for Biomedical Research Involving
Animals, Council for International Organizations of Medical Sciences, 1985.