Design, synthesis and pharmacological profile of novel dopamine D2 receptor ligands

Article abstract of DOI:10.1016/S0968-0896(03)00487-5

The present study describes the synthesis and pharmacological profile of three novel heterocyclic compounds originally designed, on the basis of bioisosterism, as dopamine D2 receptor ligands: 1-[1-(4-chlorophenyl)-1H-pyrazol-4-ylmethyl]-4-phenyl-piperazi

Full text of DOI:10.1016/S0968-0896(03)00487-5

Bioorganic & Medicinal Chemistry 11 (2003) 4807–4813
Design, Synthesis and Pharmacological Profile of Novel Dopamine
D2 Receptor Ligands
Ricardo Menegatti,a,b Anna C. Cunha,c Vıtor F. Ferreira,c Edna F. R. Perreira,d
Ahmed El-Nabawi,d Amira T. Eldefrawi,d Edson X. Albuquerque,d,e Gilda Neves,f
Stela M. K. Rates,f Carlos A. M. Fragaa,b and Eliezer J. Barreiroa,b,
*
a
´
Laboratorio de Avaliac¸a˜o e Sıntese de Substancias Bioativas (LASSBio), Faculdade de Farmacia,
´
Universidade Federal do Rio de Janeiro, Rio de Janeiro, PO Box 68006, RJ 21944-970, Brazil
ˆ
´
b
´
Instituto de Quımica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil
c
´
Departamento de Quımica Orgaˆnica, Universidade Federal Fluminense, Rio de Janeiro, RJ, Brazil
dDepartment of Pharmacology and Experimental Therapeutic, University of Maryland, School of Medicine,
Baltimore, MD 21201, USA
e
´
Departamento de Farmacologia Basica e Clınica, Instituto de Ciencias Biomedicas,
´
Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil
ˆ
´
f
´
Faculdade de Farmacia, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil
Received 11 December 2002; accepted 27 June 2003
Abstract—The present study describes the synthesis and pharmacological profile ofthree novel heterocyclic compounds originally
designed, on the basis ofbioisosterism, as dopamine D2 receptor ligands: 1-[1-(4-chlorophenyl)-1H-pyrazol-4-ylmethyl]-4-phenyl-
piperazine (LASSBio-579), 1-phenyl-4-(1-phenyl-1H-[1,2,3]triazol-4-ylmethyl)-piperazine (LASSBio-580) and 1-[1-(4-chloro-
phenyl)-1H-[1,2,3]triazol-4-ylmethyl]-4-phenyl-piperazine (LASSBio-581). Binding studies performed on brain homogenate indi-
cated that all three compounds bind selectively to D2 receptors. In addition, electrophysiological studies carried out in cultured
hippocampal neurons suggested that LASSBio-579 and 581 act as D2 agonists, whereas LASSBio-580 acts as a D2 antagonist.
# 2003 Elsevier Ltd. All rights reserved.
Introduction
symptoms ofthe disease, including delusions and
hallucinations.3ꢀ5 The fact that all antipsychotic drugs
at clinically relevant concentrations block dopaminergic
receptors6 lends support to the concept that the dopa-
minergic system is involved in schizophrenia and in the
mechanism ofaction ofneuroleptics. To date, six types
ofdopaminergic receptors have been cloned from brain
tissue.7 These receptors are sub-divided into two famil-
ies, referred to as D1 and D2. The family of D1 recep-
tors includes the D1A and D1B subtypes (D1B is also
known as D5 in humans). The family of D2 receptors
includes the D2S, D2L, D3 and D4 subtypes.
Schizophrenia is a devastating mental disorder that
1
affects 1–2% ofthe population worldwide. The first
signs ofschizophrenia typically emerge in adolescence
or young adulthood,2 and include difficulties in orga-
nizing thoughts, which lead to hallucinations, delusions,
disordered thinking, and unusual speech or behavior.
Schizophrenia is the single most destructive disease to
young people.
It has been hypothesized that decreased dopaminergic
tonus in prefrontal cortical areas accounts for the
negative symptoms ofschizophrenia, including apathy
and social withdraw, and that increased dopaminergic
activity in the striatum is associated with the positive
Positive symptoms ofschizophrenia respond well to
treatment with classical antipsychotic drugs such as
haloperidol, which act primarily as D2 receptor
antagonists.3 In contrast, the negative symptoms of
schizophrenia tend to worsen upon treatment with clas-
sical antipsychotics.8 Clozapine (1), an atypical anti-
psychotic agent that effectively controls the positive and
*Corresponding author. Tel.: +55-21-2562-6644; fax: +55-21-2562-
6445; e-mail: eliezer@ufrj.br
0968-0896/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0968-0896(03)00487-5

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R. Menegatti et al. / Bioorg. Med. Chem. 11 (2003) 4807–4813
some ofthe negative symptoms ofschizophrenia, in
addition to binding to D2 receptors, also binds to D4
receptors with an apparent affinity that is approximately
5-fold higher than that for D2 receptors (see Fig. 1).9,10
It was originally suggested that the therapeutic efficacy
ofclozapine was related to its ability to block D4
receptors.11 However, continued studies came to indi-
cate that the therapeutic efficacy ofthis atypical neuro-
leptic is likely due to its ability to induce high levels of
D2-receptor occupancy in the cerebral cortex.12 By and
large, atypical neuroleptics cause much less extra-
pyramidal side effects than classical neuroleptics.
Nevertheless, pharmacological intervention with cur-
rently available (classical and atypical) antipsychotics is
not effective against all symptoms ofthe disease. 6 There
is still need for development of more effective
antipsychotic drugs.
an aryl group on the position 1 to prevent the typical
prototropy ofthese unsubstituted azaheterocyclic sys-
tem.14 A para-chloro substituent was introduced in the
N-phenyl rings of 3 and 5 in order to increase the
structural similarity with 1. Finally, the d ring of 1 was
transposed to the distal nitrogen atom ofthe piperazine
c ring mimicking the structural subunit A present in 2
(see Fig. 1).
Chemistry
The synthetic route planned to achieve the new N-phe-
nylpiperazine derivatives 3–5 (Scheme 1) explored, in the
key step, the reductive amination ofthe 1-(4-chloro-
phenyl)pyrazole-4-carbaldehyde (8), 1-phenyl-1,2,3-tria-
zole-4-carbaldehyde (9) and 1-phenyl-1,2,3-triazole-4-
carbaldehyde (10).
In search for drugs that have better therapeutic effi-
cacy and less side effects than those that are currently
being used for treatment of schizophrenic patients,
new molecular templates that present functional
selectivity have been described. These include L-741
(2),13 a selective ligand for D2 receptors (Ki=2.4 nM)
(see Fig. 1).
Substituted 1,2,3-triazole-4-carbaldehyde derivatives 9
and 10 were prepared in 80 and 76% yield, respectively,
employing the reaction ofdiazomalonaldehydes with
appropriate aromatic amines in acetic acid, according to
the procedure described by Arnold and coworkers.15
The physical and spectroscopic properties ofheteroaro-
matic aldehydes 9 and 10 are in full agreement with
previously described by L’abbe and coworkers.16 On the
other hand, condensation of4-chlorophenylhydrazine
(6) with 1,1,3,3-tetramethoxipropane in the presence of
hydrochloric acid furnished 1-(4-chlorophenyl)-pyrazole
(7)17 which after regioselective formylation18 with
phosphorus oxychloride and DMF yielded 1-(4-chloro-
phenyl)-pyrazole-4-carbaldehyde (8).
In the scope ofa research program aimed at drug
development for treatment of neurological disorders, we
describe in the present study the synthesis and pharma-
cological evaluation ofnew N-phenylpiperazine deriva-
tives 3, 4 and 5, which were originally designed to be
selective D2 or D4 receptor ligands. These substances
resulted from hybridization of the lead compounds clo-
zapine (1) and L-741 (2). In designing compounds 3, 4
and 5, the central b ring ofclozapine was contracted
raising a pyrazole or 1,2,3-triazole ring, which received
Finally, sodium cyanoborohydride reduction19 ofthe
imine aducts formed from treatment of the heterocyclic
Figure 1. Structural design concept ofnew N-phenylpiperazine derivatives 3–5.

R. Menegatti et al. / Bioorg. Med. Chem. 11 (2003) 4807–4813
4809
Figure 2. Displacement by LASSBio-579, -580 and -581 ofbinding of
quinuclidinyl benzilate, epibatidine, SCH-23390 and YM-09151-2 to
muscarinic, nicotinic, D1 and D2 receptors, respectively, in rat brain
membranes.
affinities ofLASSBio-579 and -581 were the same ( Fig.
3). In contrast, the D2 receptor binding affinity of
LASSBio-580 was approximately 5-fold lower than that
ofLASSBio-579 and -581 ( Fig. 3).
An electrophysiological assay was used to determine
whether the novel compounds act as D2 receptor ago-
nists or antagonists. Numerous studies have demon-
strated that D2 receptor agonists interacting with
presynaptically located D2 receptors can reduce gluta-
mate release in the ventral tegmental area, the striatum
and the hippocampus.25,26 Thus, to determine whether
LASSBio-579, -580 and -581 can alter glutamate release,
spontaneous postsynaptic currents (PSCs) were recor-
ded by means ofthe patch-clamp technique rfom cul-
tured hippocampal neurons before, during and after
their exposure to physiological solution containing one
ofthe test compounds. All PSCs were recorded rfom
neurons continuously perfused with external solution
containing the GABAA receptor antagonist picrotoxin
(100 mM). Under this experimental condition, as shown
in previous studies from our laboratory, the PSCs are
Scheme 1. Reagents and conditions: (i) 1,1,3,3-tetramethoxipropane
(1 equiv), concd HCl, reflux, 1 h, 86%; (ii) phosphorus oxychloride
(4 equiv), DMF (4 equiv), 70 ꢁC, 12 h, 78%; (iii) N-phenylpiperazine
(1 equiv), acetic acid, methanol, sodium cyanoborohydride (5.7 equiv),
4 h, 3 (79%), 4 (72%) and 5 (78%).
aldehydes 8–10 with N-phenylpiperazine in dry metha-
nol containing catalytic amount ofacetic acid give tar-
get-compounds 3, 4, and 5, herein referred to as
LASSBio-579, -580 and -581, respectively.
Pharmacology
Receptor binding selectivity ofLASSBio-579, -580 and
-581 was assayed according to a protocol recently
developed to allow for rapid screening of binding of
different compounds to various neurotransmitter recep-
tors in a single membrane preparation from rat brain.
The following radioactive ligands were used: [3H]qui-
nuclidinyl benzilate (0.4 nM, selective for muscarinic
receptors20) [3H]epibatidine (10 nM, selective for a4b2
nicotinic receptors21); [3H]SCH-23390 (3 nM, selective
for D1 receptors22); and [3H]YM-09151-2 (2.5 nM,
selective for D2 receptors23). The concentrations ofthe
radioactive ligands were selected such that their specific
24
binding corresponded to 82–96% ofthe total binding.
At 10 mM, LASSBio-579, -580 and -581 were unable to
displace binding ofquinuclidinyl benzilate to muscarinic
receptors, epibatidine to nicotinic receptors and SCH-
233390 to D1 receptors (Fig. 2). However, at the same
concentration, all three compounds effectively displaced
the binding ofYM-0915-2 to D2 receptors ( Fig. 2).
Analysis ofthe concentration-response relationship of r
displacement of[ 3H]YM-09151-2 binding to the rat
brain membranes revealed that the D2 receptor binding
Figure 3. Concentration–response relationship for binding of LASS-
Bio-579, 580- and -581 to D2 receptors in rat brain membranes.

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R. Menegatti et al. / Bioorg. Med. Chem. 11 (2003) 4807–4813
Figure 4. Effects ofLASSBio-579, -580 and -581 on frequency and amplitude ofspontaneous EPSCs recorded from cultured hippocampal neurons.
referred to as EPSCs) were reduced during exposure of
the neurons to LASSBio-579 and -581 (Fig. 4). At
10 mM, each compound reduced by 65 and 68%,
respectively, the area-under-the-curve ofhistograms of
number ofEPSCs versus amplitude ( Fig. 4). In contrast,
EPSC frequency and amplitudes were increased by
LASSBio-580. At 10 mM, LASSBio-580 increased by
54% the area-under-the-curve ofhistograms ofnumber
ofEPSCs versus amplitude ( Fig. 4). On average, it took
40 s ofr the onset ofthe effects ofLASSBio-579, -580
and -581 on glutamatergic transmission. In addition, the
effects were fully reversible after 1 min of perfusion of
the neurons with drug-free physiological solution. In
agreement with the concept that the effects were medi-
ated by the interaction ofthe test compounds with pre-
synaptically located dopaminergic receptors, were the
findings that LASSBio-579, -580 and -581 were unable
to affect the frequency or amplitude of spontaneous
EPSCs recorded from neurons continuously perfused
with physiological solution containing the dopamine
receptor antagonist spiperone (10 mM, data not shown).
These results taken together indicate that LASSBio-579
and 581- act as D2 receptor agonists, whereas LASS-
Bio-580 acts as a D2 receptor antagonist.
Figure 5. Minimum energy conformers of the derivatives LASSBio-
580 and LASSBio-581. Molecular electrostatic maps ofLASSBio-580
(A) and LASSBio-581 (B). LUMO distribution maps ofLASSBio-580
(C) and LASSBio-581 (D-).
Molecular Modeling
the result ofactivat ion ofpostsynaptic AMPA recep-
tors by glutamate released from glutamatergic neurons
synapsing onto the neurons under study. The frequency
and the amplitude ofthese excitatory PSCs (herein
To elucidate the possible structural reasons ofthe dopa-
mine receptor ligand profile ofthese new azaheterocyclic
derivatives, we submitted representative compounds

R. Menegatti et al. / Bioorg. Med. Chem. 11 (2003) 4807–4813
4811
LASSBio-580 and LASSBio-581 to theoretical study
using semiempirical AM1 method27 at the SCF-MO
level, with full geometry optimization implemented on a
Pentium III 900 MHz computer.
tetramthylsilane as an internal standard with Bruker
AC 200, Bruker DRX 300 spectrometers at 200 MHz,
respectively. Splitting patterns are as follows: s, singlet;
d, doublet; dd, double doublet; m, multiplet. Carbon
magnetic resonance (13C NMR) was determined with
the same spectrometer described above at 50 MHz,
using deuterated chloroform containing ca. 1% tetra-
methylsilane. Infrared (IR) spectra were obtained with a
Nicolet-55a Magna spectrophotometer by using potas-
sium bromide plates. The ultraviolet spectra were
obtained with a Hitachi U-2000 Spectrophotometer by
using methanol with solvent and an internal standard.
Microanalysis data was obtained with Perkin–Elmer
240 analyzer, using Perkin-Elmer AD-4 balance.
From this studies we were able to observe that the
minimal energy conformer of LASSBio-580 and LASS-
Bio-581 presented a very closed extended shape, with
piperazine group adopting a chair orientation (Fig. 5).
Additionally, the measurement ofthe torsional angles
(y1) between 1,2,3-triazole ring and its phenyl attached
group ofLASSBio-580 and LASSBio-581, that is ꢀ25.4
and ꢀ24.1ꢁ respectively, indicated that no significative
effect ofthe chloro substituent on the conformation was
evidenced. On the other hand, chloro substituent of
LASSBio-581 contributes to the increase ofits molar
volume in ca. 23 A3 and to the significant decrease ofthe
overall dipole moment.
The progress ofall reactions was monitored by TLC
performed on 2.0 cm ꢂ 6.0 cm aluminum sheets pre-
coated with silica gel 60 (HF-254, Merck) to a thickness
of0.25 mm. The developed chromatograms were viewed
under ultraviolet light at 254 nm. For column chroma-
tography Merck alumine was used. The usual workup
means that the organic extracts prior to concentration,
under reduced pressure, were treated with a saturated
aqueous sodium chloride solution, referred to as brine,
dried over anhydrous sodium sulfate and filtered.
The comparative analysis ofthe electrostatic potential
surface maps of the minimal energy conformers of
LASSBio-580 (Fig. 5, A) and LASSBio-581 (Fig. 5, B),
obtained after single-point ab initio calculation using
3-21G* basis set28 with SPARTAN 1.0.5 program,29 did
not allow to evidence significative differences in the
overall charge distribution. Despite this, plot ofthe
LUMO delineate areas ofLASSBio-580 ( Fig. 5, C) and
LASSBio-581 (Fig. 5, D), indicated that chloro atom
increase significantly the charge density on its phenyl-
attached ring and additionally in the C-5 of1,2,3-tria-
zole ring, which could be evidenced by the attenuation
of‘blue’ color in the assigned regions.
1-(4-Chlorophenyl)-1H-pyrazole (7).
A solution of
1,1,3,3-tetramethoxypropane (2.54 g, 15.5 mmol) and 4-
chlorophenylhydrazine (6) (2.21 g, 15.5 mmol) in 95%
ethanol (15.5 mL) containing hydrochloric acid (0.8 mL)
was refluxed until TLC analysis indicated the total con-
sumption ofthe starting materials (ca. 1 h). Then, atfer
cooling at room temperature the reaction mixture was
neutralized with 10% aq NaHCO3 and extracted with
CH2Cl2 (3 ꢂ 20 mL). After usual workup of the organic
extracts, the precipitate was crystallized from hexanes to
give 2.37 g (86%) ofthe compound 7, as yellow crystals,
mp 57–58 ꢁC (lit.,16 54 ꢁC), Rf=0.62 (CH2Cl2/MeOH
95:5): IR (KBr) cmꢀ1: 3101–3069 (n C–H), 1523–1498 (n
This distinct electronic distribution profile associated
with steric effect promoted by introduction of para-
chloro substituent in the compound LASSBio-581 sug-
gested to us that intrinsic activity could be modulated
by its different molecular recognition by dopamine D2
receptors, probably due to modifications in charge-
transfer type and/or hydrophobic interations with com-
plementary aminoacid residues.
1
C¼C and C¼N), 1095 (n C–Cl); H NMR (200 MHz,
CDCl3): d 6.49 (1H, s, H-4), 7.43 (2H, d, J=8.9 Hz, H-
30 and H-50), 7.66 (2H, d, J=8.9 Hz, H-20 and H-60),
7.74 (1H, s, H-3), 7.90–7.91 (1H, m, H-5); 13C NMR
(50 MHz, CDCl3): d 108.1 (C-4), 120.5 (C-20 and C-60),
126.8 (C-5), 129.7 (C-30 and 50), 132.0 (C-40), 138.9 (C-
10), 141.5 (C-3).
As concluding remarks, our results suggest that this new
series ofheterocyclic N-phenylpiperazine derivatives 3–5
designed by applying molecular hybridization and sim-
plification approach on clozapine (1) are selective
dopamine D2 receptor ligands, which presents its
intrinsic agonist/antagonist activity modulated by
introduction ofa chloro atom on the heterocyclic-
attached phenyl ring. Additionally, the dopamine D2
receptor agonist LASSBio-581 was selected for further
in vivo pharmacological investigations30 to well char-
acterize its potential use for treatment of schizophrenia.
1-(4-Chlorophenyl)-1H-pyrazole-4-carbaldehyde
(8).
Phosphorus oxychloride (1.04 mL, 1.72 g, 11.22 mmol)
was added dropwise to dry N,N-dimethylformamide
(0.86 mL, 0.82 g, 11.22 mmol) at ꢀ10 ꢁC. Then, 1-(4-
chlorophenyl)-1H-pyrazole (7) (0.5 g, 2.8 mmol) was
added to the Vilsmeier–Haack complex and the reaction
mixture heated at 70 ꢁC for 12 h. After neutralization
with 10% aq NaHCO3, the precipitate was filtered out
and recrystallised from hexanes furnishing derivative 8
(0.42 g, 78%) as a white crystals, mp 113–114 ꢁC,
Rf=0.38 (CH2Cl2/MeOH 95:5): IR (KBr) cmꢀ1: 3123–
3067 (n C–H), 1681 (n C¼O), 1630–1501 (n C¼C and
Experimental
Chemistry
1
All melting points were determined with a Thomas-
Hoover apparatus and are uncorrected. Proton mag-
netic resonance (1H NMR), unless otherwise stated, was
determined in deuterated chloroform containing ca. 1%
C¼N), 1099 (n C–Cl); H NMR (200 MHz, CDCl3) d
7.47–7.62 (2H, m, H-30 and 50), 7.51–7.79 (3H, m, H-3,
H-20 and H-60), 8.56 (1H, s, H-5), 10.22 (1H, s, CHO);
13C NMR (50 MHz, CDCl3): d 120.8. (C-40), 121.0 (C-20

4812
R. Menegatti et al. / Bioorg. Med. Chem. 11 (2003) 4807–4813
and C-60), 123.3 (C-4), 130.0 (C-5), 130.1 (C-30 and C-
1599–1501 (n C¼C and C¼N), 1097 (n C–Cl); 1H NMR
50), 136.4 (C-10), 148.3 (C-3), 185.2 ðCHOÞ.
(200 MHz, CDCl3):
d
2.72–2.77 (4H, m, Ar-
CH2N(CH)2–CH2)2NPh), 3.20–3.25 (4H, m, Ar-
CH2N(CH)2–CH2)2NPh), 3.83 (2H, s, Ar-CH2N(CH)2–
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,
General procedure for the preparation of compounds 3–5
A solution ofthe corresponding heterocyclic aldehyde
derivative 8–10 (0.7 mmol) and N-phenylpiperazine
(0.11 g, 0.7 mmol) in dry methanol (2.5 mL) was adjus-
ted to pH 6.0 by dropwise addition ofconcentrated
acetic acid. Then, sodium cyanoborohydride (0.25 g,
4 mmol) was added and the resultant mixture stirred at
60 ꢁC for 2–4 h. After removal of the solvent under
reduced pressure, the residue was partitioned between
dichloromethane and 10% aqueous potassium phos-
phate. The organic layer was separated and submitted
to usual workup to furnish a crude precipitate, which
was purified by recrystallization in ethanol/water.
À
Á
CDCl3): d 49.2 Ar-CH2NðCH2ꢀCH2Þ2NPh , 53.2
À
Á
Ar-CH2NðCH2ꢀCH2Þ NPh , 53.4 Ar-CH2N(CH)2–
CH2)2NPh, 116.3 (C-2002 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) lmax: 252 (log e=4.34). Anal.
calcd for C19H20ClN5: C, 64.49%; H, 5.70%; N,
19.79%. Found: C, 64.61%; H, 5.59%; N, 19.75%.
Pharmacology
1-[1-(4-Chlorophenyl)-1H-pyrazol-4-ylmethyl]-4-phenyl-
piperazine (3). Derivative 3 was obtained in 77% yield,
as a white solid, mp 122 ꢁC, Rf=0.38 (CH2Cl2/MeOH
95:5): IR (KBr) cmꢀ1: 3106–3022 (n C–H), 1598–1497 (n
Rat brain membrane preparation. For each experiment,
one male Sprague–Dawley rat (200–300 g) was anesthe-
tized with CO2 and decapitated by guillotine. The whole
brain was rapidly removed onto ice. Then, using a glass
Teflon Potter-Elvehjem homogenizer (10 strokes at
600 rpm), the brain was homogenized in 20 volumes of
ice-cold Tris–Krebs Ringer containing 118 mM NaCl,
4.8 mM KCl, 2.5 mM CaCl2, 1.2 mM MgSO4, 1 mM
EDTA (pH, 7.4). The homogenate was centrifuged at
40,000 g for 20 min at 4 ꢁC. The resultant pellet (P1) was
resuspended in ice-cold 20 mM Tris–HCl pH 7.4 by
homogenization with a Brinkmann PT-10 Polytron at a
setting of5 s, and subsequently centriufged at 40,000 g
for 20 min at 4 ꢁC. This washing step was repeated four
times with ice-cold 20 mM Tris–HCl buffer pH 7.5. The
final pellet was resuspended in the homogenization buf-
fer and kept on ice until assayed. Protein content was
determined using BCA reagents (Pierce Chemical,
Rockford, IL, USA) with bovine serum albumin as a
standard.
1
C¼C and C¼N), 1093 (n C–Cl); H NMR (200 MHz,
CDCl3) d 2.67–2.71 (4H, m, Ar-CH2NðCH2ꢀCH2Þ2NPh),
3.21–3.26 (4H, m, Ar-CH2NðCH2ꢀCH2Þ2NPh), 3.59
(2H, s, Ar-CH2NðCH2ꢀCH2Þ2NPh), 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(CH)2–CH2)2NPh),
52.7
(Ar)-CH2N(CH)2–
À
Á
CH2)2NPh), 53.1 Ar-CH2NðCH2ꢀCH2Þ2NPh , 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) lmax: 255 (log e=4.47). Anal. calcd for
C20H21ClN4: C, 68.08%; H, 6.00%; N, 15.88%. Found:
C, 66.99%; H, 6.15%; N, 15.80%.
1-Phenyl-4-(1-phenyl-1H-[1,2,3]triazol-4-ylmethyl)-piper-
azine (4). Derivative 4 was obtained in 72% yield, as a
white solid, mp 150 ꢁC, Rf=0.48 (CH2Cl2/MeOH 95:5):
IR (KBr) cmꢀ1: 3083–3035 (n C–H), 1601–1501 (n C¼C
Binding of radioactive ligands to neurotransmitter re-
ceptors. All radioactive ligands were purchased from
New England Nuclear. Nonspecific binding was deter-
mined as the amount ofradioactive ligand bound in the
presence ofa high concentration ofa displacer that
would inhibit all nonspecific binding. Binding ofselec-
tive radiolabeled ligands to their receptors was mea-
sured by a filtration assay. Rat brain membranes were
incubated for 60 min with the radiolabeled ligand in a
total volume of250 mL buffer in the absence or pres-
ence ofthe specific displacer (i.e., binds to the same
receptor and saturates all sites). The most selective
radioactive ligand and displacer were used under opti-
mum incubation conditions.24 All incubations were ter-
minated by vacuum filtration over Whatman GF/B
glass-fiber filters (presoaked in 0.05% polyethylenimine
for at least 30 min to eliminate nonspecific filter bind-
ing), followed by three 4-mL washes with ice-cold 0.9%
NaCl solution. Radioactivity was counted by liquid
scintillation spectroscopy.
1
and C¼N); H NMR (200 MHz, CDCl3): d 2.73–2.78
(4H, m, Ar-CH2NðCH2ꢀCH2Þ2NPh), 3.21–3.26 (4H, m,
Ar-CH2NðCH2ꢀCH2Þ NPh),
3.84
(2H,
s,
Ar-CH2NðCH2ꢀCH2Þ22NPh), 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(CH)2–CH2)2NPh), 53.2 (Ar)-
CH2N(CH)2–CH2)2NPh), 53.4 (Ar)-CH2N(CH)2–
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) lmax: 246 (log e=4.29).
Anal. calcd for C19H21N5: C, 71.45%; H, 6.63%; N,
21.93%. Found: C, 71.56%; H, 6.54%; N, 21.77%.
1-[1-(4-Chlorophenyl)-1H-[1,2,3]triazol-4-ylmethyl]-4-
phenyl-piperazine (5). Derivative 5 was obtained in 77%
yield, as a white solid, mp 151 ꢁC, Rf=0.48 (CH2Cl2/
MeOH 95:5): IR (KBr) cmꢀ1: 3094–3067 (n C–H),
Cultured hippocampal neurons. Primary cultures ofneu-
rons dissociated from the hippocampi of 16–19-day-old

R. Menegatti et al. / Bioorg. Med. Chem. 11 (2003) 4807–4813
4813
rat fetuses (Sprague–Dawley) were prepared according
to the procedure described in Pereira et al.31 Experi-
ments were performed on hippocampal neurons cul-
tured for 15–20 days.
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were recorded according to the whole-cell mode of
the patch-clamp technique32 using an LM-EPC-7
amplifier (List Electronics, Darmstadt, FRG). Signals
were filtered at 1–2 kHz, and either stored in a VCR
for later analysis or directly sampled by a Pentium-
III computer using the PCLAMP6 program (Axon
Instruments, Foster City, CA, USA). The composi-
tion ofthe external solution used to bathe the neu-
11. Liegeois, J.; Eyrolles, L.; Bruhwyler, J.; Delarge, J. Curr.
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rons was (in mM): NaCl 165, KCl 5, CaCl2 2H2O 1, 4-
(2-hydroxyethyl)-1-piperazineethanesulfonic acid
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(HEPES) 5, dextrose 10 (pH was adjusted to 7.3 with
NaOH; 340 mOsM). The internal solution used to fill the
patch pipettes had the following composition (in mM):
CsCl 80, CsF 80, ethyleneglycoltetraacetic acid (EGTA)
10, CsOH 22.5 and HEPES 10 (pH was adjusted to 7.3
with CsOH; 340 mOsM). Results were not used when
the access resistance changed significantly during the
experiment.
Data analysis. Peak amplitude and frequency of PSCs
were analyzed using the Continuous Data Recording
program.33
19. Thurkauf, A.; Yuan, J.; Chen, N.; Wasley, J. W. F.;
Meade, R.; Woodruff, K. H.; Huston, K.; Ross, P. C. J. Med.
Chem. 1995, 38, 4950.
Computational chemistry
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The geometry optimization ofLASSBio-580 and
LASSBio-581 was performed using the AM1Hamilton-
ian27 within SPARTAN 1.0.5 program29 on a Pentium
III 900 MHz. In order to better evaluate the electronic
properties ofthe AM1 minimal energy conformations of
LASSBio-580 and LASSBio-581, they were submitted
to a single point ab-initio calculation with a 3-21G*
basis set28 with the same program described above.
Molecular electrostatic potential maps (MEPs) and
LUMO density isosurfaces were generated and plotted
employing default parameters in SPARTAN.
26. Koga, E.; Momiyama, T. J. Physiol. (Lond.) 2000, 523, 163.
27. Dewar, M. J. S.; Zoebisch, E. G.; Healy, E. F.; Stewart,
J. J. P. J. Am. Chem. Soc. 1985, 107, 3902.
Acknowledgements
28. Hessian matrix analyses were analyses were employed to
unequivocally characterize them as true minima potential
energy surface.
We gratefully acknowledge financial support and
fellowships from the PRONEX (Grant #0888, Bra-
zil), CNPq (Grants 301.327/1996-3 and 50.0033/1995-
0, Brazil), CAPES (PROCAD #0082/01-5, Brazil),
FAPERJ (Brazil) and FUJB (Brazil). We also thank
the NPPN Analytical Center ofUniversidade Fed-
eral do Rio de Janeiro (Brazil) for spectroscopic
facilities.
29. Wavefunction Inc.: Irvine, CA, 2000.
30. Neves, G.; Fenner, R.; Heckler, A. P.; Viana, A. F.;
Tasso, L.; Menegatti, R.; Fraga, C. A. M.; Barreiro, E. J.;
Dalla-Costa, T.; Rates, S. M. K. Braz. J. Med. Biol. Res. 2003,
36, 625.
31. Pereira, E. F. R.; Reinhardt-Maelicke, S.; Schrattenholz,
A.; Maelicke, A.; Albuquerque, E. X. J. Pharmacol. Exp.
Ther. 1993, 265, 1474.
32. Hamill, O. P.; Marty, A.; Neher, E.; Sakmann, B.; Sig-
worth, F. J. Pflugers Arch. 1981, 391, 85.
References and Notes
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