Synthesis, binding properties and receptor docking of 4-halo-6-[2-(4-arylpiperazin-1-yl)ethyl]-1H-benzimidazoles, mixed ligands of D2 and 5-HT1A receptors

Article abstract of DOI:10.1016/j.ejmech.2007.09.027

In this publication we are describing synthesis, binding properties, and receptor docking of 4-halo-6-[2-(4-arylpiperazin-1-yl)ethyl]-1H-benzimidazoles, a new compounds with potential antipsychotics properties. Affinity towards the dopamine D₁-like and D₂-like, and serotonin 5-HT_1A receptors was evaluated using the radioligand binding assays. All compounds tested had affinity for the D₂-like and 5-HT_1A receptors, but were inactive towards the D₁-like receptor. Halogenated 6-[2-(4-arylpiperazin-1-yl)ethyl]-1H-benzimidazoles showed higher affinity compared to their nonhalogenated congeners. In silico docking analysis of selected ligands was performed in order to explain the results of binding assays. Our analysis suggests that stabilizing interactions between the halogen atom at the benzimidazole ring and the Ser-122 of the D₂-like and Trp-358 of the 5-HT_1A receptor. Energy contributions for these interactions were calculated using the ab initio method.

Full text of DOI:10.1016/j.ejmech.2007.09.027

Available online at www.sciencedirect.com
European Journal of Medicinal Chemistry 43 (2008) 1696e1705
http://www.elsevier.com/locate/ejmech
Original article
Synthesis, binding properties and receptor docking of
4-halo-6-[2-(4-arylpiperazin-1-yl)ethyl]-1H-benzimidazoles,
mixed ligands of D2 and 5-HT1A receptors
a
Deana Andric , Goran Roglic , Vladimir Sukalovic ,
a
b
ˇ
´
ˇ
´
Vukic Soskic , Sladjana Kostic-Rajacic
´
b
ˇ ´ c,
´
´
ꢀ
*
´
a Faculty of Chemistry, University of Belgrade, Studentski trg 16, 11000 Belgrade, Serbia
b Center for Chemistry, Institute for Chemistry, Technology and Metallurgy, Njegosˇeva 12, 11000 Belgrade, Serbia
c ProteoSys AG, Carl Zeiss Strasse 51, 55129 Mainz, Germany
Received 26 April 2007; received in revised form 14 September 2007; accepted 18 September 2007
Available online 7 October 2007
Abstract
In this publication we are describing synthesis, binding properties, and receptor docking of 4-halo-6-[2-(4-arylpiperazin-1-yl)ethyl]-1H-benz-
imidazoles, a new compounds with potential antipsychotics properties. Affinity towards the dopamine D1-like and D2-like, and serotonin 5-HT1A
receptors was evaluated using the radioligand binding assays. All compounds tested had affinity for the D2-like and 5-HT1A receptors, but were
inactive towards the D1-like receptor. Halogenated 6-[2-(4-arylpiperazin-1-yl)ethyl]-1H-benzimidazoles showed higher affinity compared to
their nonhalogenated congeners. In silico docking analysis of selected ligands was performed in order to explain the results of binding assays.
Our analysis suggests that stabilizing interactions between the halogen atom at the benzimidazole ring and the Ser-122 of the D2-like and Trp-
358 of the 5-HT1A receptor. Energy contributions for these interactions were calculated using the ab initio method.
Ó 2007 Elsevier Masson SAS. All rights reserved.
Keywords: Dopamine receptor; Serotonin receptor; Rational drug design
1. Introduction
serotonin system stabilizers’’ [5]. These derivatives possess
a combined antagonistic effect towards D2-like receptors and
serotonin 5-HT2 receptors (e.g., clozapine, risperidone, and
olanzapine), or act as partial agonists of D2-like receptors
and interacts with 5-HT1A receptors or other CNS targets (ari-
piprazole and bifeprunox) [2,3].
Another pharmacological approach, which has been gaining
ground steadily, involves combining D2 antagonism and 5-
HT1A agonism in the same molecule [6e8]. Therefore, para-
meters like D2/5-HT1A affinity ratio as well as 5-HT1A intrinsic
activities have been recognized as important in vitro indicators
for selection of lead candidates [8]. Our previous results have
shown that 5-[2-(4-arylpiperazin-1-yl)ethyl]-1H-benzimid-
azoles exert a mixed D2/5-HT1A affinity [9] and therefore can
be considered as candidates for this new class of antipsychotics.
We have also observed that halogenation of benzimidazole
The discovery of phenothiazine in 1952 marked the starting
point of the era of modern pharmacologic treatment of psy-
chosis. A series of drugs with similar propertiesemostly an-
tagonists of dopamine D2-like receptors, now referred to as
‘‘classical antipsychotics’’ewere subsequently developed [1].
The classical antipsychotics, however, are associated with se-
vere side effects and are inefficient against negative symptoms
of schizophrenia, such as social withdrawal [2e4]. The new
hope came with the antipsychotics of second generation,
also known as the ‘‘atypical antipsychotics’’ or ‘‘dopaminee
* Corresponding author. Tel.: þ49 6131 50 192 47; fax: þ49 6131 50 192
12.
ˇ
E-mail address: vukic.soskic@proteosys.com (V. Soskic).
ˇ
´
0223-5234/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2007.09.027

´
D. Andric et al. / European Journal of Medicinal Chemistry 43 (2008) 1696e1705
1697
moiety has a pronounced effect on the D2-like [10] and 5-HT1A
(data not yet published) receptor affinity.
diamines 10a,be14a,b with formic acid yielded 4-halo-6-[2-
(4-arylpiperazin-1-yl)ethyl]-1H-benzimidazoles 15a,be19a,b.
The effect of halogen on the stability of receptoreligand
complex can be explained by a charge transfer or hydrogen
bonding of the halogens (so-called ‘‘halogen bonding’’) and
the receptor molecules [11e13]. The aim of the present study
was to develop a molecular model that would explain the
affinity of 4-halo-6-[2-(4-arylpiperazin-1-yl)ethyl]-1H-benz-
imidazoles towards the D2-like and 5-HT1A receptors. There-
fore, newly synthesized 4-halo-6-[2-(4-arylpiperazin-1-yl)
ethyl]-1H-benzimidazoles were evaluated for their ability to
bind D1-like, D2-like and 5-HT1A receptors, and were further
subjected to docking analysis using the binding site models
of D2-like and 5-HT1A receptors and the ab initio
calculation for putative CeHalogen/N and CeHalogen/
HN interactions.
3. Pharmacology
The affinities (Ki values, Table 1) of the newly synthesized
4-halo-6-[2-(4-arylpiperazin-1-yl)ethyl]-1H-benzimidazoles
(15a,be19a,b) as well as for nonhalogenated analogues 5-[2-
(4-arylpiperazin-1-yl)ethyl]-1H-benzimidazoles (20e24) [9]
towards D1-like, D2-like and 5-HT1A receptors were evaluated
using in vitro binding assays.
4. Molecular modeling and ab initio calculations
All ligands were docked into the binding-pocket model of
the D2-like receptor defined by Teeter et al. [14], according
to the procedure previously described [15]. Docking to the
5-HT1A receptor was performed as previously described
[16]. Ab initio MP2 calculations of relative stabilizing energy
contribution of the CeHalogen/HN and CeHalogen/HO
were performed using Gaussian 03W suit. In these calcula-
tions MP2/6-31þG* basic set was used, including counter-
poise correction to BSSE. Calculations were performed on
the couples: ligandeSer-122 (D2-like receptor, Table 2) and li-
gandeTrp-358 (5-HT1A receptor, Table 2) that were incised
from docked ligandereceptor complexes, taking care that
the original geometry is preserved.
2. Chemistry
Synthetic route and chemical structures of the compounds
synthesized in the present study are shown in Scheme 1. Key
intermediary compounds, 2-halo-4-(2-chloroethyl)-6-nitroani-
lines (4a,b), were obtained as previously described [10].
Alkylation of arylpiperazines using those compounds gave
rise to 2-halo-6-nitro-4-[2-(4-arylpiperazin-1-yl)ethyl]anilines
5a,be9a,b. Reduction of nitroanilines 5a,be9a,b by Ra-Ni/
hydrazine provided 2-amino-3-halo-5-[2-(4-arylpiperazin-1-yl)-
ethyl]phenylamines 10a,be14a,b. Finally, a reaction of
Scheme 1. Synthetic pathways for 4-halo-6-[2-(4-arylpiperazin-1-yl)ethyl]-1H-benzimidazoles; (a) SnCl2, EtOH, Ac2O; (b) SO2Cl2, CHCl3; (c) Br2, AcOH;
(d) Ac2O, HNO3/H2SO4; (e) 4 N HCl; (f) DMF, KI, K2CO3, arylpiperazine; (g) Ra-Ni, hydrazine; (h) HCO2H. Hal is Cl for all compounds labeled with No.a
and Br for the one labeled with No.b.

´
D. Andric et al. / European Journal of Medicinal Chemistry 43 (2008) 1696e1705
1698
Table 1
Structure and binding properties of the ligands described in this study
No
Ar
Ki (nM)
D1
D2
5-HT1A
15a
16a
17a
18a
19a
Phe
2-MeOPhe
2-ClPhe
>1000
>1000
>1000
>1000
>1000
84.7 ꢀ 11
5.49 ꢀ 0.8
19.1 ꢀ 2.4
200 ꢀ 19
550 ꢀ 25
89.6 ꢀ 18
5.2 ꢀ 0.4
45.6 ꢀ 3.2
20.7 ꢀ 2.1
529.6 ꢀ 29
3-CF3Phe
Pyrimidin-2-yl
15b
16b
17b
18b
19b
Phe
2-MeOPhe
2-ClPhe
>1000
>1000
>1000
>1000
>1000
61.9 ꢀ 3.3
0.56 ꢀ 0.09
5.77 ꢀ 1.2
14.6 ꢀ 3.2
26.7 ꢀ 22
24.1 ꢀ 3.3
1.4 ꢀ 0.2
0.14 ꢀ 0.06
10.3 ꢀ 0.5
129 ꢀ 15
3-CF3Phe
Pyrimidin-2-yl
20
21
22
23
24
Phe
2-MeOPhe
2-ClPhe
>1000
nd
nd
138 ꢀ 23
13.1 ꢀ 1.0
59.7 ꢀ 3.2
273 ꢀ 38
>1000
197 ꢀ 52
20.3 ꢀ 3.0
243 ꢀ 54
33.0 ꢀ 4.0
>1000
3-CF3Phe
Pyrimidin-2-yl
>1000
nd
Values are the means ꢀ S.E.M. of 3e4 independent experiments done in triplicate, performed at five ([3H]SCH 2339) and eight competing ligand concentrations
([3H]spiperone and [3H]8-OH-DPAT).
5. Results and discussion
some 5-HT1A receptor selectivity ( p < 0.05; Table 1). The most
potent 5-HT1A receptor ligand was 4-bromo-6-{2-[4-(2-chloro-
phenyl)piperazin-1-yl]ethyl}-1H-benzimidazole (17b), where
halogenation increased the affinity towards the D2-like and 5-
HT1A receptor by 12.0 and 1735 times, respectively. The
most potent ligand for the D2-like receptor was 4-bromo-
6-{2-[4-(2-methoxyphenyl)piperazin-1-yl]ethyl}-1H-benzimid-
azole (16b), where halogenation increased the affinity towards
the D2-like and 5-HT1A receptor by 40.0 and 12.9 times,
respectively.
5.1. Pharmacology
Herein tested compounds were poor competitors of specific
radioligands for the D1-like receptor, while showing affinity to-
wards D2-like and 5-HT1A receptor. Halogenation increased the
affinity of new ligands towards both types of receptors in the
following order: Br > Cl > H. Besides, N-trifluoromethylpiper-
azine 18a and 4-bromoimidazoles (15b, 17b and 19b) showed

´
D. Andric et al. / European Journal of Medicinal Chemistry 43 (2008) 1696e1705
1699
Table 2
Relative stabilizing energy calculation for 4-halo-benzimidazoleeSer-141 (D2-like receptor) and 4-halo-benzimidazoleeTrp-358 (5-HT1A receptor) interactions
LigandeD2 -like receptor interaction
Ligande5-HT1A receptor interaction
Molecular model
Calculated stabilizing energy (kcal molꢂ1
)
X ¼ H; E ¼ 0.0
X ¼ H; E ¼ 0.0
X ¼ Cl; E ¼ ꢂ0.99
X ¼ Br; E ¼ ꢂ1.13
A: Sketch of the benzimidazole part of ligands (X: H, Cl or Br) and Ser-122 (D2-like receptor), B: sketch of the benzimidazole part of ligand (X: H, Cl or Br) and
X ¼ Cl; E ¼ ꢂ0.46
X ¼ Br; E ¼ ꢂ0.63
Trp-358 (5-HT1A receptor) that were incised from docked ligandereceptor complexes.
This increase in affinity suggests an attractive interaction
between the halogen atom and some molecular moiety in the
binding pocket of both D2-like and 5-HT1A receptors. Charge
transfer and halogen bonding interactions have been already
described as important in some chemical and biological sys-
tems [11e13]. To test the involvement of those interactions
in our system, we performed in silico docking of halogenated
ligands to the models of D2-like and 5-HT1A receptors.
5.3. Docking to the 5-HT1A receptor
Docking of 4-halo-6-{2-[4-arylpiperazin-1-yl]ethyl}-1H-
benzimidazoles (15a,be19a,b) and their nonhalogenated con-
geners (20e24) to the 5-HT1A receptor binding site revealed:
(i) a salt bridge between the protonated N1 of the piperazine
ring and negatively charged Asp-116, (ii) hydrogen bonds be-
tween the substituents on the benzimidazole part and Ser-199
and Trp-358, and (iii) edge-to-face interactions of the aromatic
ring or arylpiperazine part with Phe-361 and Tyr-390. Dis-
tances between the receptoreligand atom pairs are shown in
Fig. 2. Docking analysis showed that this increase in affinity
is likely due to a charge transfer interaction between the hal-
ogen atoms of the ligands and partially positive charges of hy-
drogen atoms linked to the nitrogen of Trp-358 (Fig. 2). The
distances between CeCl (ligand 16a) and CeBr (ligand
5.2. Docking to the D2-like receptor
Docking of 4-halo-6-{2-[4-arylpiperazin-1-yl]ethyl}-1H-
benzimidazoles (15a,be19a,b) and their nonhalogenated
congeners (20e24) to the to the D2-like receptor binding
site revealed that (i) close interaction of the protonated N1
of the piperazine ring with Asp-86, (ii) hydrogen bonds be-
tween the benzimidazole nitrogens and Ser-141 and Ser-122,
and (iii) edge-to-face interactions of the aromatic ring or the
arylpiperazine part of and the rings of Phe-178, Tyr-216 and
Trp-182 represent main stabilizing forces. Distances between
receptoreligand atom pairs are shown in Fig. 1.
˚
16b) and HN of Trp-358 (ligand 16a) areꢁ 3.61 A and
˚
3.53 A, respectively, with the angle of about 90 falling well
in the range of this type of interactions [11e13]. The small an-
gle measured between CeHalogen/Trp-358 eliminates the
possibility of halogen binding, which is considered to be
a near-linear interaction (nucleophileeCehalogen angle of
160ꢁe180ꢁ) [13].
Therefore, a disproportionally high affinity of ligand 17b is
difficult to explain solely on the basis of the formation of one
new charge transfer interaction. Addressing this issue in fine
detail will be possible with the publication of a high-resolution
model of the receptoreligand complex.
In our previous publication [15] we reported an interaction
of benzimidazole catechol bioisostere with both Ser-122 and
Ser-144 of the D2-like receptor. Docking of halogenated com-
pounds indicates close interaction only of Ser-122, which can
be explained by halogen-directed reorientation of the benz-
imidazole part of the ligand towards the formation of hydrogen
bond with Ser-122 (distance Ser-122(OH)/Nbzi, 2.63 A (16a)
5.4. Ab initio calculations of CeHalogen/N and
CeHalogen/HeN interactions
˚
and Ser-122(OH)/Nbzi, 2.67 A (16b); Fig. 1) and with the
˚
charge transfer interaction [11,12] of Ser-122 and the halogen
˚
(distance Ser-122(OH)/Cl, 2.95 A (16a) and Ser-122(OH)/Br,
˚
2.87 A (16b); Fig. 1).
In support our hypothesis that charge transfer interactions
play a significant stabilizing role in the formation of

´
D. Andric et al. / European Journal of Medicinal Chemistry 43 (2008) 1696e1705
1700
Fig. 1. View of the interaction between D2-like receptor binding site and 16b. Docking of ligand 16b to D2-like receptor is presented. Images are showing only key
amino acid residues of the receptor binding pocket. Figures a and b shows docking of 16b viewed from different angles.
complexes between our ligands and D2-like and 5-HT1A recep-
tors, ab initio MP2 calculations were performed. Relative sta-
bilizing energy contribution of the Cehalogen/HN(Trp-358)
and Cehalogen/HO(Ser-122) interactions are presented in
Table 2. The calculations show that there is a gain in energy
of the system, due to the charge transfer interaction between
halogen atoms of the ligand and the counterpart polar group
of the receptor, wherein Br has higher effect than Cl.
receptors, than their nonhalogenated analogues. Docking anal-
ysis using the at D2-like receptor model revealed that introduc-
tion of halogen atom in the benzimidazole part of the ligand
promotes formation of a hydrogen bond and charge transfer in-
teraction of between the halogen and the Ser-122. Similarly,
charge transfer interaction was observed between halogenated
ligands and the Trp-358 residue of 5-HT1A receptor. Results
of the docking analysis were supported by ab initio calcula-
tions of putative charge transfer interactions. Bromo deriva-
tives showed a higher affinity than their chloro-counterparts,
but no satisfactory explanation could be found at this point.
Based on the high affinity and favorable D2/5-HT1A affinity ra-
tio, compound 17b is a good candidate for further pharmaco-
logical studies.
6. Conclusion
4-Halo-6-[2-(4-arylpiperazin-1-yl)ethyl]-1H-benzimid-
azoles have higher affinity towards both D2-like and 5-HT1A

´
D. Andric et al. / European Journal of Medicinal Chemistry 43 (2008) 1696e1705
1701
Fig. 2. View of the interaction between 5-HT1A receptor binding site and ligand 16b. Docking of ligand 16b to 5-HT1A receptor is presented. Images are showing
only key amino acid residues of the receptor binding pocket. Figures a and b shows docking of 16b viewed from different angles.
7. Experimental protocols
run on a Perkin Elmer 457 Grating FT Infrared Spectrophotom-
eter (Perkin Elmer, Beaconsfield, UK). The mass spectra were
determined by field desorption mass spectroscopy on Finnigan
Mat 8230 mass spectrometer (Finnigan, Bremen, Germany).
For analytical thin-layer chromatography Merck (Darmstadt,
Germany) F-256 plastic-backed thin-layer silica gel plates
were used. Chromatographic purifications were performed on
Merck-60 silica gel columns, 230e400 mesh ASTM, under
medium pressure (dry column flash chromatography). All re-
agents and solvents used in this work were obtained from Al-
drich and were used without further purification. Solutions
7.1. General
A Boetius PHMK apparatus (VEB Analytic, Dresden, Ger-
many) was used to determine melting points, presented here
as uncorrected. 1H NMR (at 200 MHz) and 13C NMR (at
50 MHz) spectra were recorded on a Gemini 2000 spectrometer
(Varian, Palo Alto, CA, USA) with CDCl3 as a solvent, unless
otherwise stated, are reported in parts per million using tetra-
methylsilane as the internal standard. The IR spectra were

´
D. Andric et al. / European Journal of Medicinal Chemistry 43 (2008) 1696e1705
1702
were routinely dried over anhydrous Na2SO4 prior to
evaporation.
docking procedure based on Monte Carlo methodology was
carried out. Up to 100 structures were produced in every run
and each finally optimized in order remove steric interaction
with gradient limit of 0.0042 kJ molꢂ1 or 4000 optimization
steps.
7.2. Molecular modeling
7.2.1. D2-like receptor binding site
Obtained docked structures were examined, and those with
lowest total energy were further filtered to obtain docking
structures with best ligand fit. We selected structures based
on following criteria: lowest total energy of the complex, short-
est salt bridge formed between Asp-86 of D2-like or Asp-116 of
5-HT1A receptor and proton on nitrogen, chair conformation of
arylpiperazine ring and aryl part of the molecule positioned in
rear hydrophobic pocket of the ligand. After an initial criterion
was satisfied, second step was examination of different interac-
tions that can be formed between receptor and ligand (hydro-
gen bonds, aromaticearomatic interactions, etc.). In that way,
the best possible docking structures were selected. Structures
were rendered using povray raytracer v3.6 [25].
All ligands were docked into the binding pocket of the
D2-like receptor, according to the procedure we described ear-
lier [15]. Shortly, the model of the D2 DAR transmembrane
helices was constructed directly from the bacteriorhodopsin
coordinates derived from two-dimensional electron diffraction
experiments, but the orientations of all TM domains were sub-
sequently adjusted in order to mimic the topology of the TM
domains of rhodopsin [14,17]. This model was tested for its
ability to accommodate rigid agonist and semi-rigid antago-
nist molecules which were docked into the putative binding
pocket with stabilizing interactions. The model is consistent
with structureeactivity relationships of agonists and antago-
nists that interact with the receptor [14] and with site-directed
mutagenesis data [18e20].
7.3. Ab initio calculations
7.2.2. 5-HT1A receptor binding site
Gaussian 03 suite [26] was used to carry on calculation of en-
ergy contribution of the chosen LigandeHalogen/AA dimer.
Mutual orientation interacting groups were taken from docking
analysis results. The stabilization energies of the paired struc-
tures were calculated as a difference between dimmer and sepa-
rate molecular entities using a MP2HF method and 6-31þG*
basis set. To compensate for basis set superposition error, coun-
terpoise method suggested by Boys and Bernardi [27] was used.
The model of the human 5-HT1A was built as described pre-
viously [16] using crystal structures of bovine rhodopsin (PDB
codes 1F88, 1HZX, and 1L9H) was a template. Comparative
modeling by means of the MODELER program [21], which
is part of the Insight II package from Accelrys, has been
used. Our model includes seven transmembrane helices end
second extracellular loop e2-loop that can be in direct contact
with the ligand [22]. The binding site of the ligand in the 5-
HT1A receptor was determined starting from the fact that for
the ligand activity formation of the salt bridge between proton-
ated piperazine nitrogen and Asp-116 (Asp 3.32) is necessary
[23], active site search procedure from binding site analysis
module (Insight II) [24] was used to select all amino acid res-
idues forming the cavity near Asp-116. The binding site de-
fined in the previous step was further refined by manually
excluding all amino acid residues that cannot come in direct
contact with the inside of the cavity.
7.4. Chemistry
7.4.1. General procedure for the synthesis of 2-halo-6-nitro-
4-[2-(4-arylpiperazin-1-yl)ethyl]anilines 5a,be9a,b
To 10.0 mmol solution of arylpiperazine in 50.0 ml DMF,
12.0 mmol of 2-halo-4-(2-chloroethyl)-6-nitroaniline (4a,b),
6.0 g K2CO3 and 0.1 g of KI were added. The mixture was
stirred at 80 ꢁC for 12 h. After cooling, the precipitate was re-
moved and the filtrate was evaporated in vacuo. The residue
was dissolved in CH2Cl2 and obtained products purified by
MPLC using CH2Cl2 as the eluent.
7.2.3. Docking
All ligands used in docking analysis were modeled using
Accelrys Insight II program build module. Initial geometry
was optimized until energy minima were reached using inbuilt
geometry optimization routine. Geometry obtained in this
manner was starting point for docking analysis.
Docking of the selected ligands presented in Table 1 was
done by simulated annealing using the Affinity module from
Insight on SGI Octane2 workstation [24]. All ligands were
docked as a protonated, using the CFF91 force field. Amino
acid residues charges were adjusted where needed. Protein
binding site was determined by combining results from exper-
imental data and Insight II bind site analysis module. Initial
position of the ligand in the bind site, was arbitrary, while pro-
tonated nitrogen on ligand part was kept in close proximity of
Asp-86 of D2-like or Asp-116 of 5-HT1A receptor. After initial
ligand placement no further constrains were applied and
7.4.1.1. 2-Chloro-6-nitro-4-[2-(4-phenylpiperazin-1-yl)ethyl]-
aniline (5a). Yield: 69%; m.p. 104 ꢁC; 1H NMR: d 2.57e2.80
(m, 8H), 3.20e3.25 (m, 4H), 6.45 (s, 2H, NH2), 6.87e6.97 (m,
3H, ArH), 7.22e7.32 (m, 2H, ArH), 7.45 (d, 1H, J ¼ 2.2 Hz,
ArH), 7.97 (d, 1H, J ¼ 2 Hz, ArH).
7.4.1.2. 2-Bromo-6-nitro-4-[2-(4-phenylpiperazin-1-yl)ethyl]-
aniline (5b). Yield: 72%; m.p. 110 ꢁC; 1H NMR: d 2.57e2.80
(m, 8H), 3.20e3.25 (m, 4H), 6.51 (s, 2H, NH2), 6.83e6.96 (m,
3H, ArH), 7.24e7.32 (m, 2H, ArH), 7.62 (d, 1H, J ¼ 2 Hz,
ArH), 8.01 (d, 1H, J ¼ 2 Hz, ArH).
7.4.1.3. 2-Chloro-6-nitro-4-{2-[4-(2-methoxyphenyl)piperazin-
1-yl]ethyl}aniline (6a). Yield: 76%; m.p. 97 ꢁC; 1H NMR:
d 2.59e2.81 (m, 8H), 3.10e3.21 (m, 4H), 3.87 (s, 3H, OCH3),

´
D. Andric et al. / European Journal of Medicinal Chemistry 43 (2008) 1696e1705
1703
6.45 (s, 2H, NH2), 6.85e7.02 (m, 4H, ArH), 7.45 (d, 1H,
J ¼ 2 Hz, ArH), 7.97 (d, 1H, J ¼ 2 Hz, ArH).
filtered through celite. The filtrate was evaporated in vacuo
and crude products were used for further syntheses.
7.4.3. Synthesis of 4-chloro-6-[2-(4-arylpiperazin-1-yl)-
ethyl]-1H-benzimidazoles (15ae19a) and 4-bromo-6-[2-
(4-arylpiperazin-1-yl)ethyl]-1H-benzimidazoles (15be19b)
Diamine of 2 mmol (10a,be14a,b) and 0.44 ml (7.3 mmol)
of 98% formic acid were heated in an oil bath at 100 ꢁC for
2 h. After cooling to ambient temperature, 15 ml of 10%
NaHCO3 was added and the product extracted with CH2Cl2.
The solvent was removed in vacuo and the residue chromato-
graphed on silica gel.
7.4.1.4. 2-Bromo-6-nitro-4-{2-[4-(2-methoxyphenyl)piperazin-
1-yl]ethyl}aniline (6b). Yield: 67%; m.p. 95 ꢁC; 1H NMR:
d 2.58e2.79 (m, 8H), 3.12 (s, 4H), 3.87 (s, 3H, OCH3), 6.51
(s, 2H, NH2), 6.85e7.02 (m, 4H, ArH), 7.61 (d, 1H,
J ¼ 2 Hz, ArH), 8.00 (d, 1H, J ¼ 2 Hz, ArH).
7.4.1.5. 2-Chloro-6-nitro-4-{2-[4-(2-chlorophenyl)piperazin-1-
yl]ethyl}aniline (7a). Yield: 79%; m.p. 98 ꢁC; 1H NMR:
d 2.60e2.81 (m, 8H), 3.09e3.13 (m, 4H), 6.46 (s, 2H,
NH2), 6.93e7.09 (m, 2H, ArH), 7.19e7.27 (m, 1H, ArH),
7.36 (dd, 1H, J ¼ 4 Hz, J ¼ 2 Hz, ArH), 7.46 (d, 1H,
J ¼ 2 Hz, ArH), 7.98 (d, 1H, J ¼ 2 Hz, ArH).
7.4.3.1. 4-Chloro-6-[2-(4-phenylpiperazin-1-yl)ethyl]-1H-ben-
zimidazole (15a). Yield: 80%; m.p. 186 ꢁC; IR (cmꢂ1): 634,
693, 1139, 1239, 1410, 1494, 1593, 2826; 1H NMR
(DMSO-d6): d 2.55e2.65 (m, 6H), 2.89 (t, 2H, J ¼ 8.2 Hz),
3.13 (s, 4H), 6.77 (t, 1H, J ¼ 7.2 Hz, ArH), 6.93 (d, 2H,
J ¼ 7.6 Hz, ArH), 7.17e7.25 (m, 3H, ArH), 7.38 (s, 1H,
ArH), 8.25 (s, 1H, CH), 12.67 (s, 1H, NH); 13C NMR
(DMSO-d6): d 32.60 (CH2), 48.29 (2CH2), 52.81 (2CH2),
60.07 (CH2), 110.82 (CH), 113.24 (CeCl), 115.53 (2CH),
118.99 (CH), 122.58 (CH), 128.60 (C), 129.14 (2CH),
133.85 (C), 135.98 (C), 142.87 (CH), 151.29 (CeN); MS:
m/e (100) 340.1 (Mþ).
7.4.1.6. 2-Bromo-6-nitro-4-{2-[4-(2-chlorophenyl)piperazin-1-
yl]ethyl}aniline (7b). Yield: 76%; m.p. 128 ꢁC; 1H NMR:
d 2.60e2.80 (m, 8H), 3.08e3.13 (m, 4H), 6.52 (s, 2H,
NH2), 6.94e7.09 (m, 2H, ArH), 7.19e7.23 (m, 1H, ArH),
7.36 (dd, 1H, J ¼ 6.2 Hz, J ¼ 1.8 Hz, ArH), 7.62 (d, 1H,
J ¼ 2 Hz, ArH), 8.02 (d, 1H, J ¼ 2 Hz, ArH).
7.4.1.7. 2-Chloro-6-nitro-4-(2-{4-[3-(trifluoromethyl)phenyl]-
1
piperazin-1-yl}ethyl)aniline (8a). Yield: 59%; oil; H NMR:
d 2.58e2.78 (m, 8H), 3.23e3.28 (m, 4H), 6.44 (s, 2H,
NH2), 7.08e7.21 (m, 3H, ArH), 7.42 (t, 1H, J ¼ 8 Hz, ArH),
7.59 (d, 1H, J ¼ 2 Hz, ArH), 7.97 (d, 1H, J ¼ 2 Hz, ArH).
7.4.3.2. 4-Bromo-6-[2-(4-phenylpiperazin-1-yl)ethyl]-1H-benz-
imidazole (15b). Yield: 85%; m.p. 188 ꢁC; IR (cmꢂ1): 762,
1
1235, 1499, 1599, 2815, 3384; H NMR: d 2.63e2.74 (m,
6H), 2.92e2.99 (m, 2H), 3.13e3.27 (m, 4H), 6.83e6.96 (m,
3H, ArH), 7.13e7.45 (m, 4H, ArH), 8.07 (s, 1H, CH); 13C
NMR (DMSO-d6): d 32.56 (CH2), 48.35 (2CH2), 52.85
(2CH2), 60.17 (CH2), 110.86 (CeBr), 111.35 (CH), 115.50
(2CH), 118.94 (CH), 125.46 (CH), 128.63 (C), 129.13
(2CH), 133.49 (C), 136.69 (C), 142.67 (CH), 151.26 (CeN);
MS: m/e (100) 385.9 (Mþ).
7.4.1.8. 2-Bromo-6-nitro-4-(2-{4-[3-(trifluoromethyl)phenyl]-
1
piperazin-1-yl}ethyl)aniline (8b). Yield: 64%; oil; H NMR:
d 2.58e2.71 (m, 8H), 3.24e3.29 (m, 4H), 6.52 (s, 2H,
NH2), 7.05e7.39 (m, 4H, ArH), 7.62 (d, 1H, J ¼ 2 Hz,
ArH), 8.04 (d, 1H, J ¼ 2 Hz, ArH).
7.4.1.9. 2-Chloro-6-nitro-4-{2-[4-(2-pyrimidinyl)piperazin-1-
yl]ethyl}aniline (9a). Yield: 69%; m.p. 115 ꢁC; 1H NMR:
d 2.54e2.65 (m, 6H), 2.72e2.80 (m, 2H), 3.83e3.88 (m, 4H),
6.45e6.52 (m, 3H, NH2, ArH), 7.45 (d, 1H, J ¼ 2 Hz, ArH),
7.97 (d, 1H, J ¼ 2 Hz, ArH), 8.28 (d, 2H, J ¼ 4.6 Hz, ArH).
7.4.3.3. 4-Chloro-6-{2-[4-(2-methoxyphenyl)piperazin-1-yl]-
ethyl}-1H-benzimidazole (16a). Yield: 78%; m.p. 84 ꢁC; IR
1
(cmꢂ1): 750, 1027, 1241, 1500, 2814; H NMR: d 2.77e2.85
(m, 6H), 2.98e3.10 (m, 2H), 3.21 (s, 4H), 3.91 (s, 3H,
OCH3), 6.89e7.11 (m, 4H, ArH), 7.18 (s, 1H, ArH), 7.30 (s,
1H, ArH), 8.10 (s, 1H, CH); 13C NMR: d 32.10 (CH2), 50.29
(CH3), 53.31 (2CH2), 55.33 (2CH2), 60.52 (CH2), 111.19
(CH), 112.12 (CH), 113.22 (CeCl), 118.29 (CH), 121.04
(CH), 123.23 (CH), 123.48 (CH), 128.62 (C), 133.96 (C),
135.92 (C), 140.23 (CeO), 141.25 (CH), 152.23 (CeN); MS:
m/e (100) 370.1 (Mþ).
7.4.1.10. 2-Bromo-6-nitro-4-{2-[4-(2-pyrimidinyl)piperazin-1-
yl]ethyl}aniline (9b). Yield: 63%; m.p. 118 ꢁC; 1H NMR:
d 2.54e2.65 (m, 6H), 2.71e2.80 (m, 2H), 3.82e3.88 (m,
4H), 6.47e6.52 (m, 3H, NH2, ArH), 7.62 (d, 1H, J ¼ 2 Hz,
ArH), 8.01 (d, 1H, J ¼ 2 Hz, ArH), 8.31 (d, 2H, J ¼ 4.6 Hz,
ArH).
7.4.3.4. 4-Bromo-6-{2-[4-(2-methoxyphenyl)piperazin-1-yl]-
ethyl}-1H-benzimidazole (16b). Yield: 97%; m.p. 49 ꢁC; IR
(cmꢂ1): 754, 1244, 1455, 1500, 1593, 3392; 1H NMR
(DMSO-d6): d 2.86 (s, 6H), 2.95e32.99 (m, 2H), 3.07 (s,
4H), 3.78 (s, 3H, OCH3), 6.87e7.00 (m, 4H, ArH), 7.36 (s,
1H, ArH), 7.47 (s, 1H, ArH), 8.27 (s, 1H, CH); 13C NMR
(DMSO-d6): d 30.43 (CH2), 47.98 (CH3), 50.35 (CH2),
52.24 (CH2), 55.24 (CH2), 57.79 (CH2), 60.03 (CH2), 110.03
7.4.2. General procedure for synthesis of 2-amino-3-halo-5-
[2-(4-arylpiperazin-1-yl)ethyl]phenylamines 10a,be14a,b
Raney-Ni (0.06e0.08 g) was added in small portions to
a stirring solution of 2 mmol of nitro compound (5a,be9a,b)
in 5 ml EtOH, 10 ml 1,2-dichloro-ethane and 0.9 ml hydrazine
hydrate at 30 ꢁC. After the addition of Ra-Ni was completed,
the mixture was heated in a water bath (50 ꢁC, 60 min) and

´
D. Andric et al. / European Journal of Medicinal Chemistry 43 (2008) 1696e1705
1704
(CeBr), 111.18 (CH), 113.14 (CH), 118.33 (CH), 120.93
(CH), 123.90 (CH), 125.67 (CH), 128.27 (C), 132.86 (C),
136.16 (C), 139.21 (CeO), 142.35 (CH), 151.91 (CeN);
MS: m/e (100) 413.9 (Mꢂ2), (90.78) 415.9 (Mþ).
3.87 (t, 4H, J ¼ 5.4 Hz), 6.50 (t, 1H, J ¼ 5 Hz, ArH), 7.19 (s,
1H, ArH), 7.27 (s, 1H, ArH), 8.07 (s, 1H, CH), 8.33 (d, 2H,
J ¼ 5 Hz, ArH), 10.31 (br s, 1H, NH); 13C NMR: d 32.58
(CH2), 43.52 (2CH2), 52.68 (2CH2), 60.20 (CH2), 110.31
(CH), 110.75 (CH), 113.20 (CeCl), 122.40 (CH), 122.71 (C),
125.45 (CH), 134.89 (CH), 136.24 (C), 142.76 (CH), 158.16
(CeN), 161.49 (CeN); MS: m/e (100) 342.1 (Mþ).
7.4.3.5. 4-Chloro-6-{2-[4-(2-chlorophenyl)piperazin-1-yl]eth-
yl}-1H-benzimidazole (17a). Yield: 71%; m.p. 122 ꢁC; IR
1
(cmꢂ1): 692, 1039, 1283, 1446, 1479, 2816, 2931; H NMR:
d 2.70e2.77 (m, 6H), 2.91e2.99 (m, 2H), 3.09e3.13 (m,
4H), 6.92e7.05 (m, 2H, ArH), 7.17e7.27 (m, 2H, ArH),
7.33e7.38 (m, 2H, ArH), 8.10 (s, 1H, CH); 13C NMR:
d 33.30 (CH2), 50.98 (2CH2), 53.26 (2CH2), 60.56 (CH2),
110.62 (CH), 113.22 (CeCl), 120.30 (CH), 123.36 (CH),
123.74 (C), 127.58 (2CH), 128.67 (C), 130.60 (CH), 130.95
(C), 136.14 (C), 141.58 (CH), 149.02 (CeN); MS: m/e (100)
374.0 (Mþ).
7.4.3.10. 4-Bromo-6-{2-[4-(2-pyrimidinyl)piperazin-1-yl]ethyl}-
1H-benzimidazole (19b). Yield: 74%; m.p. 166 ꢁC; IR
1
(cmꢂ1): 954, 982, 1258, 1357, 1482, 1548, 1587, 2811; H
NMR (DMSO-d6): d 2.53e2.65 (m, 2H), 2.88 (t, 4H,
J ¼ 8.2 Hz), 3.41e3.51 (m, 2H), 3.71e3.76 (m, 4H), 6.62 (t,
1H, J ¼ 4.6 Hz, ArH), 7.33 (s, 1H, ArH), 7.45 (s, 1H, ArH),
8.27 (s, 1H, CH), 8.36 (d, 2H, J ¼ 4.8 Hz, ArH), 12.72 (s,
1H, NH); 13C NMR (DMSO-d6): d 32.47 (CH2), 43.51
(2CH2), 52.65 (2CH2), 60.22 (CH2), 110.27 (CeBr), 110.95
(CH), 111.12 (CH), 123.07 (C), 123.85 (CH), 125.48 (CH),
135.02 (CH), 136.58 (C), 142.67 (CH), 158.12 (CeN),
161.47 (CeN); MS: m/e (100) 386.8 (Mþ).
7.4.3.6. 4-Bromo-6-{2-[4-(2-chlorophenyl)piperazin-1-yl]ethyl}-
1H-benzimidazole (17b). Yield: 93%, m.p. 92 ꢁC; IR (cmꢂ1):
626, 758, 1231, 1283, 1447, 1480, 1581, 2948, 3100, 3410;
1H NMR (DMSO-d6): d 2.81 (s, 6H), 2.92e2.99 (m, 2H),
3.06 (m, 4H), 7.01e7.44 (m, 6H, ArH), 8.26 (s, 1H, CH),
12.77 (s, 1H, NH); 13C NMR (DMSO-d6): d 31.76 (CH2),
50.28 (2CH2), 52.58 (2CH2), 59.33 (CH2), 110.12 (CeBr),
111.22 (CH), 121.05 (CH), 124.23 (C), 125.47 (CH), 127.92
(CH), 128.30 (CH), 130.35 (C), 130.56 (CH), 134.03 (C),
137.04 (C), 141.52 (CH), 149.28 (CeN); MS: m/e (100)
419.7 (Mþ).
7.5. Membrane preparation, binding assays and
data analysis
Specific binding affinities (Ki values, Table 1) were deter-
mined as described previously [28,29], by measuring the extent
of displacement of the specific tritiated ligands [3H]SCH 23390
(spec. act. 82.1 Ci mmolꢂ1) for D1-like, [3H]spiperone (spec.
act. 80.5 Ci mmolꢂ1) for D2-like and [3H]8-OH-DPAT (spec.
act. 223 Ci mmolꢂ1) for 5-HT1A like receptors (all products
of Amersham Buchles GmbH, Germany) to the fresh mem-
brane preparations of the bovine caudate nuclei or hippocampi.
The competitive radioassays were performed in sample tripli-
cates. Retained radioactivity was measured by introducing
dry filters into 5 ml toluene-based scintillation liquid and
counting in a 1219 Rackbeta Wallac scintillation counter.
7.4.3.7. 4-Chloro-6-(2-{4-[3-(trifluoromethyl)phenyl]pipera-
zin-1-yl}ethyl)-1H-benzimidazole (18a). Yield: 94%; m.p.
92 ꢁC; IR (cmꢂ1): 694, 949, 1355, 1450, 1583, 1612, 2820;
1H NMR: d 2.63e2.75 (m, 6H), 2.91e2.98 (m, 2H), 3.24e
3.29 (m, 4H), 7.04e7.10 (m, 3H, ArH), 7.20 (s, 1H, ArH),
7.30e7.41 (m, 2H, ArH), 8.12 (s, 1H, CH); 13C NMR:
d 32.53 (CH2), 47.69 (2CH2), 52.56 (2CH2), 59.93 (CH2),
110.95 (CH), 111.02 (CH), 113.20 (CeCl), 114.79 (CH),
118.90 (CH), 121.96 (CH), 122.42 (C), 127.39 (CH), 129.77
(C), 130.14 (C), 130.39 (C), 136.59 (C), 142.78 (CH),
151.42 (CeN); MS: m/e (100) 408.0 (Mþ).
7.5.1. [3H]SCH 23390 receptor binding assay
Each tube contained 1.0 mM EDTA, 4 mM MgCl2, 1.5 mM
CaCl2, 5 mM KCl, 120 mM NaCl, 25 mM TriseHCl, pH 7.4,
caudate nuclei synaptosomal membranes (prot. conc 0.7 mg
mlꢂ1), 1.0 nM [3H]SCH 23390 and various concentrations
(10ꢂ5e10ꢂ8 M) of the tested compounds in a final volume
of 0.5 ml. The tubes were incubated (20 min, 37 ꢁC) and the
reaction terminated by vacuum filtration through Whatman
GF/B filters. The filters were washed three times with 5 ml
of ice-cold 25 mM TriseHCl buffer, pH 7.4, and bound radio-
activity measured by liquid scintillation spectrometry. Specific
binding to D1-like receptors was defined as the difference be-
tween the binding in the absence and in the presence of cold
1 mM SCH 23390. The Kd value determined for SCH 23390
was 1.1 nM.
7.4.3.8. 4-Bromo-6-(2-{4-[3-(trifluoromethyl)phenyl]pipera-
zin-1-yl}ethyl)-1H-benzimidazole (18b). Yield: 94%; m.p.
74 ꢁC; IR (cmꢂ1): 695, 950, 1290, 1316, 1353, 1450, 1497,
1
1575, 2886, 2961; H NMR (DMSO-d6): d 2.53e2.65 (m,
6H), 2.89 (t, 2H, J ¼ 8.2 Hz), 3.20e3.25 (m, 4H), 7.05e7.45
(m, 6H, ArH), 8.25 (s, 1H, CH), 12.75 (br s, 1H, NH); 13C
NMR (DMSO-d6): d 32.58 (CH2), 47.78 (2CH2), 52.63
(2CH2), 60.13 (CH2), 110.93 (CeBr), 111.06 (CH), 111.38
(CH), 114.70 (CH), 118.87 (CH), 122.00 (CH), 125.53 (C),
127.41 (CH), 129.79 (C), 130.15 (C), 130.41 (C), 136.37
(C), 142.72 (CH), 151.48 (CeN); MS: m/e (100) 453.9 (Mþ).
7.4.3.9. 4-Chloro-6-{2-[4-(2-pyrimidinyl)piperazin-1-yl]ethyl}-
1H-benzimidazole (19a). Yield: 82%; m.p. 157 ꢁC; IR (cmꢂ1):
956, 982, 1358, 1395, 1512, 1549, 1587, 2936; 1H NMR:
d 2.58e2.63 (m, 4H), 2.68e2.73 (m, 2H), 2.92e2.99 (m, 2H),
7.5.2. [3H]Spiperone receptor binding assay
[3H]Spiperone binding was assayed in 1.0 mM EDTA, 4 mM
MgCl2, 1.5 mM CaCl2, 5 mM KCl, 120 mM NaCl, 25 mM
TriseHCl solution, pH 7.4, caudate nuclei synaptosomal

´
D. Andric et al. / European Journal of Medicinal Chemistry 43 (2008) 1696e1705
1705
membranes (prot. conc 0.7 mg mlꢂ1), at 37 ꢁC for 20 min in
a total volume of incubation mixture of 1.0 ml. Binding of the
radioligand to 5-HT2 receptors was prevented by 50 mM ketan-
serin. The Ki values of the tested compounds were determined
by competition binding at 0.2 nM of the radioligand and
8e10 different concentrations of each compound (10ꢂ5e
10ꢂ10 M). Nonspecific binding was measured in the presence
of 1.0 mM (þ)-butaclamol. The reaction was terminated by
rapid filtration through Whatman GF/C filters, which were fur-
ther washed three times with 5.0 ml of ice-cold incubation
buffer, and retained radioactivity was measured by liquid scin-
tillation spectrometry. The Kd value determined for spiperone
was 0.9 nM.
[7] M.B. Assie, V. Ravailhe, V. Faucillon, A. Newman-Tancredi, J. Pharma-
col. Exp. Ther. 315 (2005) 265e272.
[8] S. Cuisiat, N. Bourdiol, V. Lacharme, A. Newman-Tancredi, F. Colpaert,
B. Vacher, J. Med. Chem. 50 (2007) 865e876.
ˇ
ˇ
´
´
[9] V. Soskic, J. Joksimovic, Curr. Med. Chem. 5 (1998) 493e512.
ˇ
ˇ
´
´
´
´
´
ˇ
´
ꢀ
[10] V. Sukalovic, D. Andric, G. Roglic, S. Kostic-Rajacic, V. Soskic, Arch.
Pham. Pharm. Med. Chem. 337 (2004) 376e382.
[11] P. Metrangolo, H. Neukirch, T. Pilati, G. Resnati, Acc. Chem. Res. 38
(2005) 386e395.
[12] P. Auffinger, F.A. Hays, E. Westhof, P.S. Ho, Proc. Natl. Acad. Sci.
U.S.A. 101 (2004) 16789e16794.
[13] P. Politzer, P. Lane, M.C. Concha, Y. Ma, J.S. Murray, J. Mol. Model. 13
(2007) 305e311.
[14] M.M. Teeter, M. Froimowitz, J.B. Stec, C.J. DuRand, J. Med. Chem. 37
(1994) 2874e2888.
ˇ
´
´
´
´
´
ꢀ ´
[15] V. Sukalovic, M. Zlatovic, D. Andric, G. Roglic, S. Kostic-Rajacic,
ˇ
V. Soskic, Arch. Pham. Pharm. Med. Chem. 337 (2004) 502e512.
ˇ
´
7.5.3. 8-OH-[3H]DPAT receptor binding assay
ˇ
[16] M.V. Zlatovic, V.V. Sukalovic, C. Schneider, G.M. Roglic, Bioorg. Med.
´
´
´
Chem. 14 (2006) 2994e3001.
[17] H. Lan, C.J. Durand, M.M. Teeter, K.A. Neve, Mol. Pharmacol. 69
(2006) 185e194.
Each tube contained 1.0 mM EDTA, 4 mM MgCl2, 1.5 mM
CaCl2, 5 mM KCl, 120 mM NaCl, 25 mM TriseHCl, pH 7.4,
hippocampi synaptosomal membrane (prot. conc 0.7 mg mlꢂ1),
0.6 nM 8-OH-[3H]DPAT and various concentrations (10ꢂ5e
10ꢂ11 M) of the tested compounds in a final volume of
0.5 ml. The tubes were incubated (20 min, 37 ꢁC) and the reac-
tion terminated by vacuum filtration through Whatman GF/B
filters. The filters were washed three times with 5 ml of ice-
cold 25 mM TriseHCl buffer, pH 7.4, and bound radioactivity
measured by liquid scintillation spectrometry. 5-HT1A specific
binding was defined as the difference between the binding in
the absence and in the presence of 10 mM 5-hydroxytryptamine.
The Kd value determined for 8-OH-DPAT was 1.6 nM.
[18] R.E. Wilcox, W.H. Huang, M.Y. Brusniak, D.M. Wilcox, R.S. Pearlman,
M.M. Teeter, C.J. DuRand, B.L. Wiens, K.A. Neve, J. Med. Chem. 43
(2000) 3005e3019.
[19] K.A. Neve, M.G. Cumbay, K.R. Thompson, R. Yang, D.C. Buck, V.J. Watts,
C.J. DuRand, M.M. Teeter, Mol. Pharmacol. 60 (2001) 373e381.
[20] J.A. Ballesteros, L. Shi, J.A. Javitch, Mol. Pharmacol 60 (2001) 1e19.
[21] A. Sali, T.L. Blundell, J. Mol. Biol. 234 (1993) 779e815.
[22] L. Shi, J.A. Javitch, Proc. Natl. Acad. Sci. U.S.A. 101 (2004) 440e445.
[23] K. Bondensgaard, M. Ankersen, H. Thøgersen, B.S. Hansen, B.S. Wulff,
R.P. Bywater, J. Med. Chem. 47 (2004) 888e899.
[24] SGI Octane2 is trademark of Silicon Graphics, Inc, CA, USA (<http://
www.sgi.com>).
[25] PovRay, Persistence of Vision Raytrace is trademark of PovRay team,
(<http://www.povray.org>).
The inhibition curve and statistical (Student’s t-test) analy-
sis was performed by GraphPad Prism program packet [30].
[26] M.J. Frisch, G.W. Trucks, H.B. Schlegel, G.E. Scuseria, M.A. Robb,
J.R. Cheeseman, J.A. Montgomery Jr., T. Vreven, K.N. Kudin,
J.C. Burant, J.M. Millam, S.S. Iyengar, J. Tomasi, V. Barone,
B. Mennucci, M. Cossi, G. Scalmani, N. Rega, G.A. Petersson,
H. Nakatsuji, M. Hada, M. Ehara, K. Toyota, R. Fukuda, J. Hasegawa,
M. Ishida, T. Nakajima, Y. Honda, O. Kitao, H. Nakai, M. Klene, X. Li,
J.E. Knox, H.P. Hratchian, J.B. Cross, V. Bakken, C. Adamo,
J. Jaramillo, R. Gomperts, R.E. Stratmann, O. Yazyev, A.J. Austin,
R. Cammi, C. Pomelli, J.W. Ochterski, P.Y. Ayala, K. Morokuma,
G.A. Voth, P. Salvador, J.J. Dannenberg, V.G. Zakrzewski, S. Dapprich,
A.D. Daniels, M.C. Strain, O. Farkas, D.K. Malick, A.D. Rabuck,
K. Raghavachari, J.B. Foresman, J.V. Ortiz, Q. Cui, A.G. Baboul,
S. Clifford, J. Cioslowski, B.B. Stefanov, G. Liu, A. Liashenko,
P. Piskorz, I. Komaromi, R.L. Martin, D.J. Fox, T. Keith, M.A. Al-Laham,
C.Y. Peng, A. Nanayakkara, M. Challacombe, P.M.W. Gill, B. Johnson,
W. Chen, M.W. Wong, C. Gonzalez, J.A. Pople, Gaussian 03, Revision
C.02, Gaussian Inc., Wallingford CT, 2004.
Acknowledgements
This work was supported by the Ministry of Science, Tech-
nology and Development of Serbia, grant # 142009. The
authors wish to thank Dr. V. Pejovic for fruitful discussions
and his help in preparation of this manuscript.
References
[1] A. Zhang, J.L. Neumeyer, R.J. Baldessarini, Chem. Rev. 107 (2007)
274e302.
[2] M.S. Stahl, J. Clin. Psychiatry 5 (2003) 9e13.
[3] A. Farah, J. Clin. Psychiatry 7 (2005) 268e274.
[4] R.R. Conley, D.L. Kelly, Curr. Drug Targets CNS Neurol. Disord. 1
(2002) 123e128.
[27] S.F. Boys, F. Bernardi, Mol. Phys. 19 (1970) 553e566.
ˇ
[28] Z. Tuce, V. Soskic, J. Joksimovic, Drug Des. Discov. 11 (1994) 251e258.
ˇ
´
´
ˇ
[29] S. Dukic, S. Kostic-Rajacic, D. Dragovic, V. Soskic, J. Joksimovic, J.
´
´
ꢀ ´
Pharm. Pharmacol. 49 (1997) 1036e1041.
´
ˇ
´
´
[5] C.A. Tamminga, A. Carlsson, Curr. Drug Targets CNS Neurol. Disord. 1
(2002) 141e147.
[30] GraphPad Prism is a biostatistical and curve fitting software packet by
GraphPad Software (<http://www.graphpad.com>).
[6] S.L.A. Bruins, M.S. Kleven, A. Newman-Tancredi, Neuropharmacology
49 (2005) 996e1006.