Interactions of N-{[2-(4-phenyl-piperazin-1-yl)-ethyl]-phenyl}-2-aryl-2-yl- acetamides and 1-{[2-(4-phenyl-piperazin-1-yl)-ethyl]-phenyl}-3-aryl-2-yl-ureas with dopamine D2 and 5-hydroxytryptamine 5HT1A receptors

Article abstract of DOI:10.1016/j.bmcl.2012.04.098

It is suggested that the ratio of dopamine D₂ to 5-hydroxytryptamine 5-HT_1A activity is an important parameter that determines the efficiency of antipsychotic drugs. Here we present the synthesis of N-{[2-(4-phenyl-piperazin-1-yl)-ethyl]-phenyl}-2-aryl-2-yl-acetamides and 1-{[2-(4-phenyl-piperazin-1-yl)-ethyl]-phenyl}-3-aryl-2-yl-ureas and their structure-activity relationship studies on dopamine D₂ and 5-hydrohytryptamine 5-HT_1A receptors. It was shown that ligand selectivity and affinity strongly depends on their topology and the presence of a pyridyl group in the head of molecules. Molecular modeling studies using homology modeling and docking simulation revealed a rational explanation for the ligand behavior. The observed binding modes and receptor-ligand interactions provided us with a clue for optimizing the optimal selectivity towards 5-HT _1A receptors.

Full text of DOI:10.1016/j.bmcl.2012.04.098

Bioorganic & Medicinal Chemistry Letters 22 (2012) 3967–3972
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Interactions of N-{[2-(4-phenyl-piperazin-1-yl)-ethyl]-phenyl}-2-aryl-2-yl-
acetamides and 1-{[2-(4-phenyl-piperazin-1-yl)-ethyl]-phenyl}-3-aryl-2-yl-
ureas with dopamine D2 and 5-hydroxytryptamine 5HT1A receptors
Vladimir Sukalovic a, Djurdjica Ignjatovic b, Gordana Tovilovic b, Deana Andric c, Kaveh Shakib d,
Sladjana Kostic-Rajacic a, Vukic Soskic e,
⇑
a ICTM—Department of Chemistry, University of Belgrade, Njegoseva 12, 11000 Belgrade, Serbia
b Department of Biochemistry, Institute for Biological Research, University of Belgrade, Serbia
c Faculty of Chemistry, University of Belgrade, Studentski trg 12–16, 11000 Belgrade, Serbia
d Barnet & Chase Farm Hospital, The Ridgeway, Enfield, Middlesex EN2 8JL, UK
e Proteosys AG, Carl-Zeiss-Str. 51, 55129 Mainz, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
It is suggested that the ratio of dopamine D2 to 5-hydroxytryptamine 5-HT1A activity is an important
parameter that determines the efficiency of antipsychotic drugs. Here we present the synthesis of
N-{[2-(4-phenyl-piperazin-1-yl)-ethyl]-phenyl}-2-aryl-2-yl-acetamides and 1-{[2-(4-phenyl-piperazin-
1-yl)-ethyl]-phenyl}-3-aryl-2-yl-ureas and their structure–activity relationship studies on dopamine D2
and 5-hydrohytryptamine 5-HT1A receptors. It was shown that ligand selectivity and affinity strongly
depends on their topology and the presence of a pyridyl group in the head of molecules. Molecular mod-
eling studies using homology modeling and docking simulation revealed a rational explanation for the
ligand behavior. The observed binding modes and receptor–ligand interactions provided us with a clue
for optimizing the optimal selectivity towards 5-HT1A receptors.
Received 29 March 2012
Revised 15 April 2012
Accepted 20 April 2012
Available online 30 April 2012
Keywords:
Dopamine
5-Hydroxytryptamine
Receptor
Arylpiperazine
Molecular docking
Ó 2012 Elsevier Ltd. All rights reserved.
Dopamine (DA) and 5-hydrohytryptamine (5-HT) are two of the
major neurotransmitters in the central nervous system (CNS) and
play crucial roles in behavior and cognition. Receptors for DA and
5-HT are members of the superfamily of G-protein coupled recep-
tors (GPCRs) all shearing a rodopsinelike core structure. Based on
their pharmacological profiles and their effects on different signal
transduction cascades, these receptors are divided into different
subclasses. The focus of this study is the D2 DA and 5HT1A receptors
and their ligands, that are suggested to be a potential target in the
treatment of neurological disorders such as schizophrenia, depres-
sion, anxiety and drug abuse.1,2 It has been considered that D2 DA
receptors are the main mechanism responsible for the efficacy of
antipsychotics. That assumption was challenged by the discovery
of atypical antipsychotics with their activities at multiple recep-
tors. This notion was supported by clinical evidence that multi-tar-
get drugs are more effective than single target agents in the
treatment of CNS conditions.3,4 Among others, 5HT1A receptors
have been proposed as one of the targets for atypical antipsychotic
drugs. Some works suggest that the D2/5HT1A ratio is an important
parameter that determines the efficiency of antipsychotic drugs.5
Despite a rather high homology among D2 DA and 5HT1A receptors
there is a high diversity in ligands specificity towards these two
receptor subtypes. An understanding of molecular mechanisms of
ligand receptor interaction would contribute significantly to the
drug discovery process. Reliable molecular modeling of interac-
tions of ligands with D2 DA as well as 5HT1A receptor is facilitated
by the recent publishing of 3D structures of various eukaryotic
GPCRs.6–8 Starting from that point we published recently our re-
sults on molecular modeling of D2 DA9 and the 5HT1A receptor.10
In the model proposed by us D2 DA receptor can easily adopt radar
long linear ligands and form a stable complex through interactions
with two binding pockets, one located in the helical part and the
second in the extracellular loop (ecl2) of the receptor. Similarly,
for high affinity binding of large ligands two binding pockets are
required for the 5HT1A receptor. Both of these binding pockets
are located in the helical part of the 5HT1A receptor and require a
curved shaped ligand to access each of them. To confirm the qual-
ity of those two receptor models we designed two sets of ligands of
different molecular shape (Fig. 1) and performed the biological and
molecular docking test on the D2 DA and 5HT1A receptors. Newly
designed ligands consist of: a tail part (phenylpiperazine), a linker
⇑
Corresponding author. Tel.: +49 61315019247.
E-mail address: vukic.soskic@proteosys.com (V. Soskic).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.04.098

3968
V. Sukalovic et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3967–3972
The affinity of the new compounds for cloned human D2 DA and
5HT1A receptors was evaluated in in vitro binding assays using
[3H]spiperone for labeling D2 DA receptors and [3H]8-OH-DPAT
for labeling 5HT1A receptors, according to our previously described
procedures.12 In vitro receptor binding data are summarized in Ta-
ble 1. In this series of compounds, studies were carried out to
examine the effect of the phenyl ring substitution on the binding
affinity for the D2 DA and 5HT1A receptors. This molecular scaffold-
ing was identified on the basis of the unsubstituted compound 1-
phenethyl-4-phenyl-piperazine (13) having Ki values of 478 and
43.2 nM for the D2 DA and 5-HT1A receptor, respectively.13 The
compounds with arylureas and arylacetamides substituents at
the para-position of the phenyl ring (compounds 5a–8a and 5b–
8b) were active for both D2 DA and 5-HT1A with Ki values within
the range of 3.6–4189 nM for the D2 DA receptor and 95.3–
2264 nM for 5HT1A receptors. Para-substituted, linear arylureas,
such as the 4-pyridyl (compound 8b), showed the best D2 DA activ-
ity in this series, with Ki values of 3.6 and 226.8 nM for D2 DA and
5-HT1A, respectively. Meanwhile, the phenyl derivatives, com-
pounds 5a and 5b, exhibited reduced affinities for both DA and
5HT receptors (482.5–4189 nM). Arylureas derivatives 5b–8b have
generally higher affinity (3.60–482.5 nM) towards D2 DA receptors
than arylacetamides 5a–8a (48.6–4189 nM). There is an opposite
but less clear trend in 5HT1A affinities as far as para-derivatives
are concerned. When the para-substitution was changed to meta-
substitution, the affinities were generally reduced for D2 DA recep-
tors and increased for 5-HT1A; 5HT1A Ki values of arylacetamides
9a–12a and arylureas 9b–12b drop in the low nM range (0.50–
7.53 nM), while D2 DA receptor affinity deceased several fold
(29.7–3575 nM). Phenylarylureas and phenylarylacetamides (com-
pounds 5a–5b and 9a–9b) have diminished affinities for both D2
DA receptors. In 5HT1A receptor assay para-derivatives (5a and
5b) are less active, while meta-substituted compounds (9a and
9b) are expressing 74 and sevenfold increase in affinity when com-
pared to 1-phenethyl-4-phenyl-piperazine. Pyridylarylureas and
pyridylarylacetamides (compound 6a–8a, 6b–8b, 10a–12a and
10b–12b) all have higher D2 DA and 5HT1A affinity than parent
Figure 1. Chemical structure of investigated N-{[2-(4-phenyl-piperazin-1-yl)-
ethyl]-phenyl}-2-aryl-2-yl-acetamides and 1-{[2-(4-phenyl-piperazin-1-yl)-ethyl]-
phenyl}-3-aryl-2-yl-ureas. Molecules are divided into three substructures: head
part (red), linker part (blue) and tail part (green) for clarity of discussion.
part (phenylcarbamate or phenylacetamid) and a head part (phe-
nyl or pyridyl group), Figure 1. Those structural motifs are chosen
because: mechanism of binding of phenylpiperazine to both types
of receptors is well studied,9,10 the linker part has optimal length
and rigidity properties and phenyl or pyridyl head can take part
in aryl–aryl but also polar type of interaction in the accessory
receptor binding pocket.
Synthetic route and chemical structures of the compounds
synthesized in the present study are shown in Scheme 1. Acylation
of N-phenylpiperazine using 4-nitrophenylacetic acid gave rise
to 2-(nitrophenyl)-1-(4-phenyl-piperazin-1-yl)-ethanones (2a,b).
Amides 2a,b were converted to 1-nitrophenethyl-4-phenyl-pipera-
zines 3a,b using diborane in tetrahydrofuran (THF). Reduction of
nitro compounds 3a,b by Ra-Ni/hydrazine provided 1-aminophen-
ethyl-4-phenyl-piperazines 4a,b. Target arylacetamides 5a–12a
were obtained by condensation of anilines 4a and 4b with corre-
sponding arylacetic acid in presence of propylphosphonic acid
anhydride (PPAA) in N,N-dimethylformamide (DMF). Arylureas
5b–12b were obtained by reacting anilines 4a,b with bis(trichloro-
methyl)-carbonate in pyridine, following by addition of aryl-
amine.11 All compounds were characterized by 1H and 13C NMR
spectroscopy and mass spectroscopy.
Scheme 1. Synthetic route and chemical structures of the N-{[2-(4-phenyl-piperazin-1-yl)-ethyl]-phenyl}-2-aryl-2-yl-acetamides and 1-{[2-(4-phenyl-piperazin-1-yl)-
ethyl]-phenyl}-3-aryl-2-yl-ureas. Reagents and conditions: (a) Phenylpiperazine, N,N0-dicyclohexylcarbodiimide, 4-dimethylaminopyridine, THF, rt, o/n; (b) B2H6, THF, 0 °C
for 6 h, rt for 1 h then reflux for 2 h; (c) Ra-Ni, N2H4ꢀH2O, EtOH, 1,2-dichloroethane, 30 °C, 45 °C, 1 h; (d) ArCH2CO2H, PPAA, DMF; (e) bis(trichloromethyl)-carbonate, dioxane,
pyridine, 0 °C for 2 min. rt for 30 min, following by addition of arylamine and triethylamine, 80 °C, o/n.

V. Sukalovic et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3967–3972
3969
phenethyl-4-phenyl-piperazine. With regard to the selectivity be-
tween D2 DA and 5-HT1A, the compound arylacetamides (5a–8a
and 5b–8b) showed better affinity for D2 DA over 5-HT1A receptors.
Compound 8b had the highest selectivity value (63) in this series
(226.8 vs 3.60 nM). However, opposite results were observed with
arylureas 9a–12a and 9b–12b, the most selective was compound
9a having 6163 times higher 5HT1A receptor affinity (3575 vs
0.58 nM).
To find the structural basis for further optimization of selectiv-
ity, we set out to use molecular modeling methods. For the sake of
clarity we divided the ligand structure into three distinctive parts:
the tail part formed by aryl-piperazine structure, the linker part
connecting the aryl-piperazine structure and the head part that
is shown by the arylurea and arylacetamide structural motive
(Fig. 1). In this series of compounds the tail part was kept constant
while the linker and head parts were altered to investigate the
structure to activity relationship.
Receptor–ligand interactions were investigated by structure-
based molecular modeling methods. Because crystal structures
for our target proteins are not published so far, homology modeling
is used for building the three-dimensional protein structures. The
human D3 DA receptor was used as a template crystal structure
(PDB code: 3PBL) for modeling the D2 DA receptor as described
in our previous publication.9 The Serotonin 5HT1A model was
adopted from our previous publication.10
Ligand 3D structures were generated using the Discovery Studio
program.14 Assuming physiological conditions, the basic aliphatic
nitrogen atom of the piperazine was protonated. The geometry
was optimized using the CHARMM force field applying the conju-
gate gradient method until the energy difference between succes-
sive cycles was below 0.0042 kJ/mol.15
Docking of the selected ligands as presented in Table 1 was
done by simulated annealing using the LIBDOCK module from
Discovery Studio. All ligands were docked as protonated, using
the CHARMM force field. The initial position of the ligand in the
binding site, was arbitrary, while protonated nitrogen on the ligand
part was kept in close proximity to Asp 114 of the D2 receptor and
Asp 116 of the 5HT1A receptor. After initial ligand placement, no
further ligand constraints were applied and the 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 to remove steric interaction with a gradient limit of
0.0042 kJ/mol or 4000 optimization steps. The obtained docked
structures were examined, and 10 of those with the lowest total
energy were further filtered to obtain docking structures with
the best ligand fit. We selected structure based on the following
criteria: lowest total energy of the complex, shortest salt bridge
formed between Asp 114 of the D2 DA receptor (Asp 116 of the
5HT1A receptor) and protonated piperazine nitrogen, chair confor-
mation of arylpiperazine ring, and aryl part of the molecule posi-
tioned in the rear hydrophobic pocket of the receptors; Phe 386,
Trp 390 and Tyr 420 for D2 DA, and Phe 112 and Tyr 390 for
5HT1A receptors.16,10 After those initial criteria were satisfied, a
second step was performed to examine other interactions that
can be formed between receptor and ligand (hydrogen bonds,
aromatic–aromatic interactions, etc.). In that way, the best possible
docking structures were selected. Structures were visualised using
DS Visualise v2.5.114 and the obtained images were rendered using
PovRay Raytracer v3.6.17
Table 1
Chemical structure and binding affinity of the examined arylpiperazine ligands for the
D2 DA and 5-HT1A receptors
No.
R
Ki D2 (nM)
Ki 5HT1A(nM)
N
N
O
R
C
N
H
H2
5a
6a
4189
70.7
902.6
95.3
N
N
7a
8a
69.6
48.6
159.8
122.5
N
N
N
O
R
N
H
N
H
5b
6b
482.5
14.2
2264
N
N
N
125.8
N
7b
8b
8.2
3.6
397.9
226.8
N
H
H
C2
N
N
N
R
O
9a
3575
0.58
4.73
10a
11a
12a
277.8
N
82.4
1.39
2.26
N
201.6
H
H
N
N
N
N
R
O
9b
3250
364
5.78
7.53
10b
11b
12b
N
29.7
48.7
0.95
0.50
N
N
N
R
Docking analysis results on D2 DA receptor ( Fig. 2) show
common interactions, shared by all investigated ligands. Those
interactions are: salt bridge formed between Asp 114 and proton-
ated piperazine nitrogen, hydrophobic interactions between Phe
386, Trp 390, Tyr 420 and aryl-piperazine, Ser 194 or Ser 167
hydrogen bond bridges with ligand carbamide as for amide
function, and aromatic type interactions of Phe 189 and His 397
13
478.0
43.2
Structure of N-{[2-(4-phenyl-piperazin-1-yl)-ethyl]-phenyl}-2-aryl-2-yl-aceta-
mides and 1-{[2-(4-phenyl-piperazin-1-yl)-ethyl]-phenyl}-3-aryl-2-yl-ureas tested
for the binding to the D2 DA and 5-HT1A serotonin receptors is shown. Ki values are
the mean of three independent experiments done in triplicate performed at eight to
8 competing ligand concentrations.

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V. Sukalovic et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3967–3972
Figure 2. Results from docking compound 8a and 8b in binding pocket of the homology model of D2 DA receptor. (A) Docking of ligand 8a in homology model of D2 DA
receptor active site. (B) Docking of ligand 8a in homology model of D2 DA receptor active site. Only key amino acid residues are shown.
and polar type of interaction of Asn 175 with head of ligands.
Amino acid residues Asp 114, Phe 386, Trp 390 and Tyr 420 are
all located in the transmembrane domain of while Ser 194, Ser
167, Phe 189 and His 397 are part of the extracellular loops of
the D2 DA receptor.
In the 5HT1A receptor (Fig. 3) ligand–receptor interactions are:
salt bridge between Asp 116 and protonated piperazine nitrogen,
hydrophobic interactions between ligand aryl-piperazine and
receptor hydrophobic pocket formed by Phe 112 and Tyr 390,
hydrogen bonds between ligand and Ser 199 and Thr 200 and aro-
matic type of interaction between Phe 204 and Phe 362 and the
head of ligands. All listed amino acid residues are located in the
transmembrane domain of receptor molecules and are described
in our earlier publications.13
Figure 4. Overlay of compounds 8b (green) and 12b (red). Overlaying the tail part
of the compounds 8b and 12b that occupies the same space in the receptor
hydrophobic pocket, shows the difference in overall shape of para- (5a–8a and 5b–
Ligand affinity towards the D2 or 5HT1A receptor is in the first
place influenced by the shape and length of the ligand (Fig. 4).
para-Substituted ligands 5a–8b have an elongated, linear shape
(Fig. 4), that protrudes into the extracellular part of the D2 DA
receptor wherein additional stabilizing interactions are taking
place. These stabilizing effects are absent in 5HT1A receptor
binding because the binding pocket of the 5HT1A receptor is deeper
than the binding pocket of the D2 DA receptors and therefore
ligands do not protrude into the extracellular part. Stabilizing
effect of the D2 DA receptor ecl2 domain is also absent in
meta-substituted ligands 9a–12b since they are bent in shape
( Fig. 4), and therefore not long enough to occupy extracellular
8b) and meta-substituted (9a–12a and 9b–12b) ligands. Ligand overlay was
obtained using targeted overlay method for flexible compounds in DISCOVERY STUDIO
2.5 software.
receptor domains. Due to the existence of the lateral accessory
binding pocket formed by Ser 199, Thr 200, Phe 204 and Phe 362
that can adopt head of only meta-substituted ligands, that shape
is preferred by the 5HT1A receptor (Fig. 3). A similar structure is
not present in D2 DA receptors leading to lower D2 DA activity of
compounds with meta-substitution compared to para-counterparts
Figure 3. Results from docking compound 12a and 12b in binding pocket of the homology model of 5HT1A receptor. (A) Docking of ligand 12a in homology model of 5HT1A
receptor active site. (B) Docking of ligand 12b in homology model of 5HT1A receptor active site. Only key amino acid residues are shown.

V. Sukalovic et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3967–3972
3971
or the parent compound (13). Except for playing a crucial role in
defining the overall shape of the molecules, the linker structure
itself determines compounds’ biological activity through effective
hydrogen bonding with Ser 194 and Ser 167 of the D2 DA receptor.
In this prospect higher affinity of arylureas (5b–8b) compared to
arylacetamides (5a–8a) in the D2 DA receptor assay (Table 1) can
be explained by three hydrogen bonds in arylureas compared to
only two in arylacetamides (Fig. 2).
Results presented in this publication are consistent with our
10
previously presented models of D2 DA9 and 5HT1A receptors.
We can understand rather well the influence of the linker structure
to the biological activity of ligands but the huge impact of a minor
modification in the head part remains to be explained in future
studies.
Acknowledgments
The head part of the ligands plays an influential role in ligand
binding to both D2 DA and 5HT1A receptors. The head part of
para-substituted ligands 5a–8a and 5b–8b can occupy the hydro-
phobic pocket formed by flexible ecl2 of the D2 DA receptor but
not the lateral hydrophobic pocket of the 5HT1A receptors, giving
those compounds a more dopaminergic character. On the other
hand aromatic heads of meta-substituted compounds fit well into
the lateral hydrophobic pocket of 5HT1A receptors but do not
interact with the extracellular domain of D2 DA receptors. As a
onsequence this group of ligands has a more pronounced seroto-
nergic activity.
This research was part of project 172032 funded by the Ministry
for Science and Technology, Republic of Serbia. There are no com-
peting interests.
Supplementary data
Supplementary data associated with this article can be found, in
the
online
version,
at
http://dx.doi.org/10.1016/
j.bmcl.2012.04.098. These data include MOL files and InChiKeys
of the most important compounds described in this article.
Higher affinity of arylureas (5b–8b) compared to arylacetmides
(5a–8a) can be accounted for by stronger hydrogen bonds between
urea linker and the receptor molecules as their more rigid structure
can contribute to better positioning of the aromatic ring of the
head of the molecules into the receptor binding pocket.
References and notes
1. Missale, C.; Nash, S. R.; Robinson, S. W.; Jaber, M.; Caron, M. G. Physiol. Rev.
1998, 78, 189.
2. Hayes, D. J.; Greenshaw, A. J. Neurosci. Biobehav. Rev. 2011, 35, 1419.
3. Farah, A. J. Clin. Psychiatry 2005, 7, 268.
The introduction of nitrogen atoms in the aromatic ring into the
head of ligands, dramatically increases their affinity towards D2 DA
receptors. For example, 4-pyridyl derivatives 8a and 8b are 86 and
134 times more active than corresponding phenyl derivatives 5a
and 5b. Most probably this is due to favorable aromatic type inter-
actions of ligand pyridyl residue with counterpart amino acid
residues (e.g., Phe 189 and His 397, Fig. 2) in ecl2 part of D2 DA
receptors. Compounds 8a and 8b were also more active in D2 DA
receptor binding assay than 2-pyridyl (6a and 6b) and 3-pyridyl
(7a and 7b) derivatives most likely due to their interaction with
Asn 175 (Fig. 2). On the other hand there is no clearly obvious cor-
relation between replacing the phenyl ring with a pyridyl one on
5HT1A receptor affinity (Table 1). It is postulated that for high
5HT1A receptor affinity, aromatic interactions between the head
of the ligands and the hydrophobic pocket of receptor formed by
Phe 204 and Phe 362, are necessary (Fig. 3, Ref. 13). The introduc-
tion of nitrogen into the position 3 and 4 of the phenyl ring of
arylacetamide 9a and arylcarbamates 9b has the opposite effect
on their 5HT1A affinity (Table 1). Those results show the complex
nature of these interactions, wherein at least two factors play a
role; for example rigidity of the linker part of the molecule as well
as the electrostatic surface potential in the head part of the mole-
cule that can both favor or disfavor ligand–receptor interactions.
In conclusion, the molecular shape of the herein described
arylureas and arylacetmides together with the correct orientation
of a number of functional groups determines affinity towards D2
DA and 5HT1A receptors. High affinity ligand must form numerous
interactions with different parts of the receptor. In case of the D2
receptor, the ligand must occupy two hydrophobic pockets, one lo-
cated deep inside the binding site, and the second formed by an
ecl2 loop. Only by forming interactions with both parts of the
receptor, together with salt bridge with Asp 144 and hydrogen
bonds with Ser 167, 194 or 197, can high affinity be achieved.
A ligand bound to the 5HT1A receptor with high affinity also has
to fit into two hydrophobic pockets, both placed within the mem-
brane part of the receptor. One of these sites is located near Asp
116, while the second is adjacent to Ser 199 and Thr 200. Salt
bridge between ligand and Asp 116, hydrogen bond with Ser 199
or Thr 200 together with aromatic interactions with hydrophobic
pockets plays a key role. Extracellular loops in the 5HT1A receptor
do not play a significant role in ligand binding.
4. Nasrallah, H. A. Mol. Psychiatry 2008, 13, 27.
5. Newman-Tancredi, A.; Assié, B.; Leduc, N.; Ormière, A. M.; Danty, N.; Cosi, C.
Int. J. Neuropsychopharmacol. 2005, 8, 341.
6. Rasmussen, S. G.; Choi, H. J.; Fung, J. J.; Pardon, E.; Casarosa, P.; Chae, P. S.;
Devree, B. T.; Rosenbaum, D. M.; Thian, F. S.; Kobilka, T. S.; Schnapp, A.;
Konetzki, I.; Sunahara, R. K.; Gellman, S. H.; Pautsch, A.; Steyaert, J.; Weis, W. I.;
Kobilka, B. K. Nature 2011, 469, 175.
7. Chien, E. Y.; Liu, W.; Zhao, Q.; Katritch, V.; Han, G. W.; Hanson, M. A.; Shi, L.;
Newman, A. H.; Javitch, J. A.; Cherezov, V.; Stevens, R. C. Science 2010, 1091, 330.
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V.; Abagyan, R.; Cherezov, V.; Liu, W.; Han, G. W.; Kobayashi, T.; Stevens, R. C.;
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´
9. Sukalovic, V.; Soskic, V.; Andric, D.; Roglic, G.; Kostic-Rajacic, S. J. Serb. Chem.
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11. General procedure for the synthesis of N-{[2-(4-phenyl-piperazin-1-yl)-ethyl]-
phenyl}-2-aryl-2-yl-acetamides (5a–5b and 9a–12a). Arylacetic acids
(2.2 mmol), amines 4a or 4b (560 mg, 2.0 mmol), 1.0 ml triethyl amine, and
1.8 ml 50% PPAA, ware stirred in 7 ml DMF at room temperature for 16 h,
whereupon diluted with 200 ml ethyl acetate and extracted 2 times with 50 ml
8% NaHCO3 and 50 ml H2O, each. Organic phase was dried over MgSO4, filtered
and concentrated in vacuo. Obtained products ware purified by silica gel
column chromatography using
a
gradient of methanol (0–5%) in
dichloromethane. Obtained amides (5a–5b and 9a–12a) were crystallized
from ethyl acetate as free bases.
General procedure for the synthesis of 1-{[2-(4-phenyl-piperazin-1-yl)-ethyl]-
phenyl}-3-aryl-2-yl-ureas (5b–8b and 9b–12b). Bis(trichloromethyl)-
carbonate (200 mg, 0.7 mmol) was added upon stirring into a solution of
amine 4a or 4b (560 mg, 2.0 mmol) in 2.0 ml dry pyridine and 8.0 ml dry
dioxane. Reaction mixture was stirred for 2 min. at ice bath, 30 min. at room
temperature, following by addition of 200 mg of arylamine and 1 ml
triethylamine. Reaction mixture was transferred to an oil bath at 80 °C and
left at that temperature for another 12 h, whereupon diluted with 200 ml ethyl
acetate and extracted 2 times with 50 ml 8% NaHCO3 and 50 ml H2O, each.
Organic phase was dried over MgSO4, filtered and concentrated in vacuo.
Obtained products ware purified by silica gel column chromatography using a
gradient of methanol (0–10%) in dichloromethane. Carbamates (5b–8b and
9b–12b) were crystallized from ethyl acetate as free bases.
12. Biological assays: Radioligands [3H]spiperone and [3H]8OH-DPAT were
purchased from Amersham Biosciences (Buckinghamshire, UK). CHO-hD2S
cells stably expressing native human D2 DA receptor and CHO-K1 clonal cell
line that stably expresses 5-HT1A receptors were obtained from Professor
Phillip G. Strange (University of Reading, UK) and Dr. Kelly Berg (University of
Texas Health Science Center, San Antonio, Texas), respectively. CHO cells were
grown in DMEM containing 5% fetal calf serum and 400 lg/ml active geneticin
(to maintain selection pressure). The cell lines were maintained at 37 °C in a
humidified atmosphere with 5% CO2. Isolation of membranes with D2 DA and
5-HT1A receptors and competition binding assays at the D2 DA and serotonin
5-HT1A receptor were performed using [3H]-spiperone (0.4 nM) and [3H]8OH-
DPAT (0.8 nM), respectively by the protocol provided in.18,19 Briefly, receptor
membranes (50 lg) were incubated at 25 °C for 60 min in a final volume of

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1.0 ml reaction mixture containing radioligands and various concentrations of
13. Soskic, V.; Joksimovic, J. Curr. Med. Chem. 1998, 5, 493.
14. Accelrys Software Inc.; Discovery Studio Modeling Environment, Release 2.5,
San Diego: Accelrys Software Inc.; 2009.
15. Brooks, B. R.; Bruccoleri, R. E.; Olafson, B. D.; States, D. J.; Swaminathan, S.;
Karplus, M. J. Comput. Chem. 1983, 4, 187.
the new compounds in 20 mM HEPES (pH 7.4) buffer containing 1 mM EGTA,
1 mM EDTA, 10 mM MgCl2 and 100 mM NaCl. The samples were incubated at
25° C for 60 min and incubation was followed by rapid filtration through
Whatman GF/B filters under vacuum. The filters were washed twice with
3.5 ml of ice-cold phosphate buffer saline and counted using PPO/POPOP/
toluene based cocktail at liquid scintillation counter (LKB 1219 RACKBETA
16. Sukalovic, V.; Andric, D.; Roglic, G.; Kostic-Rajaci, c S.; Schrattenholz, A.; Soskic,
V. Eur. J. Med. Chem. 2005, 40, 481.
LSC). Nonspecific binding was determined in the presence of
3
l
M
(+)
17. Pov-Ray; The Persistence of Vision Ray-Tracer, Version 3.6 2003-2011, http://
www.povray.org/ (Accessed Jun 12, 2011).
butaclamol or 10 M buspirone for D2 DA and 5HT1A receptors, respectively.
l
The analysis of the data was performed using GraphPadPrism 4.0 software for
MS Windows (GRAPHPAD PRISM Program, San Diego, USA). Competition binding
studies were carried out with 8 varied concentrations of the test compounds
run in triplicate tubes, and isotherms from three assays were calculated by
computerized non-linear regression analysis to yield Ki values.
18. Roberts, D. J.; Lin, H.; Strange, P. G. Mol. Pharmacol. 2004, 66, 1573.
19. Evans, K. L.; Cropper, J. D.; Berg, K. A.; Clarke, W. P. J. Pharmacol. Exp. Ther. 2001,
1025, 297.