Synthesis and pharmacological evaluation of new 5-(cyclo)alkyl-5-phenyl- and 5-spiroimidazolidine-2,4-dione derivatives. Novel 5-HT1A receptor agonist with potential antidepressant and anxiolytic activity

Article abstract of DOI:10.1016/j.ejmech.2009.11.053

The synthesis of 5-(cyclo)alkyl-5-phenyl- and 5-spiroimidazolidine-2,4-dione derivatives with an arylpiperazinylpropyl moiety (12-23) and their in vitro and in vivo pharmacological properties and molecular characteristics were described. The investigated compounds exhibited high affinity for 5-HT_1A (13-22) and 5-HT_2A (18, 20, 21, 23) receptors and diversified pharmacological profile. Compounds 17, 20 and 22 showed antagonistic, partial agonistic and agonistic activity, respectively, toward 5-HT_1A receptor and they were investigated as potential antidepressants and/or anxiolytics. The most interesting compound 22 (1-[3-(4-(2-methoxyphenyl)piperazin-1-yl)propyl]-3′,4′-dihydro-2′H-spiro[imidazolidine-4,1′-naphthalene]-2,5-dione), a pre- and postsynaptic 5-HT_1A receptor agonist produced an antidepressant-like effect, which was more pronounced than that of imipramine in the forced swim test in mice, without affecting locomotor activity. Moreover, compound 22 produced a weak anxiolytic-like effect in the four-plate test in mice. Molecular docking studies of compound 22 to the homology model of the 5-HT_1A receptor showed that a 3′,4′-dihydro-2′H-spiro[imidazolidine-4,1′-naphthalene]-2,5-dione moiety played an important role in stabilizing the ligand-receptor complex.

Full text of DOI:10.1016/j.ejmech.2009.11.053

European Journal of Medicinal Chemistry 45 (2010) 1295–1303
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and pharmacological evaluation of new 5-(cyclo)alkyl-5-phenyl- and
5-spiroimidazolidine-2,4-dione derivatives. Novel 5-HT1A receptor agonist with
potential antidepressant and anxiolytic activity
,
b
Anna Czopek a, Hanna Byrtus a, Marcin Ko1aczkowski a, Maciej Paw1owski a , Ma1gorzata Dyba1a ,
*
b
c
a,
c
c
´
´
Gabriel Nowak , Ewa Tatarczynska , Anna Weso1owska , Ewa Chojnacka-Wojcik
a Department of Pharmaceutical Chemistry, Jagiellonian University Medical College, 9 Medyczna Str, 30-688 Krako´w, Poland
b Department of Pharmacobiology, Jagiellonian University Medical College, 9 Medyczna Str, 30-688 Krako´w, Poland
c Department of New Drugs Research, Institute of Pharmacology, Polish Academy of Sciences, 12 Sme˛tna Str, 31-343 Krako´w, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis of 5-(cyclo)alkyl-5-phenyl- and 5-spiroimidazolidine-2,4-dione derivatives with an aryl-
piperazinylpropyl moiety (12–23) and their in vitro and in vivo pharmacological properties and
molecular characteristics were described. The investigated compounds exhibited high affinity for 5-HT1A
(13–22) and 5-HT2A (18, 20, 21, 23) receptors and diversified pharmacological profile. Compounds 17, 20
and 22 showed antagonistic, partial agonistic and agonistic activity, respectively, toward 5-HT1A receptor
and they were investigated as potential antidepressants and/or anxiolytics. The most interesting
compound 22 (1-[3-(4-(2-methoxyphenyl)piperazin-1-yl)propyl]-30,40-dihydro-20H-spiro[imidazolidine-
4,10-naphthalene]-2,5-dione), a pre- and postsynaptic 5-HT1A receptor agonist produced an antide-
pressant-like effect, which was more pronounced than that of imipramine in the forced swim test in
mice, without affecting locomotor activity. Moreover, compound 22 produced a weak anxiolytic-like
effect in the four-plate test in mice. Molecular docking studies of compound 22 to the homology model of
the 5-HT1A receptor showed that a 30,40-dihydro-20H-spiro[imidazolidine-4,10-naphthalene]-2,5-dione
moiety played an important role in stabilizing the ligand–receptor complex.
Received 24 May 2008
Received in revised form
18 November 2009
Accepted 27 November 2009
Available online 6 December 2009
Keywords:
Pre- and postsynaptic 5-HT1A receptor
agonists
Imidazolidine-2,4-diones
Anxiolytics
Antidepressants
Ó 2009 Elsevier Masson SAS. All rights reserved.
1. Introduction
305, II/III phase, MediciNova; AP-521, II phase, Asahi Kasei Corpo-
ration; Vilazodone, III phase, PGxHealth, LLC [8]; Gepirone ER, III
The role of serotonin (5-HT) in the central nervous system and
its importance for the pathogenesis and treatment of mood disor-
ders are unquestionable. 5-HT is believed to act via an interaction
with 14 identified 5-HT receptor subtypes, of which 5-HT1A one is
the most extensively studied because of its specific connotation of
affective disorders [1,2]. Despite a great number of currently mar-
keted antidepressants and anxiolytics, continual efforts are made to
develop new drugs with greater efficacy (especially in non-
responders), an earlier onset of therapeutic improvement and less
intensive side-effects [3]. Among the numerous potential drug
candidates currently explored, 5-HT1A receptor agonists are a group
of high interest, which is evidenced by not only the number of
scientific studies [4–6], but also the fact that some of them are
currently in different phases of clinical trials for the treatment of
depression and anxiety (OPC-14523, II phase, Pharmos [7]; MN-
phase, Fabre-Kramer [9]) as well as Alzheimer’s disease (Xalipro-
den, III phase, Sanofi-Aventis [10]).
In the course of our studies with active 5-HT1A and 5-HT2A
ligands, a series of
b
-tetralinohydantoin (30,40-dihydro-10H-spi-
ro[imidazolidine-4,20-naphthalene]-2,5-dione) derivatives were
synthesized. Among them, a few compounds were especially
interesting due to their 5-HT1A receptor agonistic activity and
potential anxiolytic/antidepressant properties [11,12]. As a contin-
uation of our research, in the present experiment we synthesized
a series with a novel terminal amide part containing 5-(cyclo)alkyl-
5-phenylimidazolidine-2,4-diones (5-cyclopropyl-5-phenyl (15–
17) and 5-methyl-5-phenylimidazolidine-2,4-diones (12–14)), or
their counterparts with 5-spiroimidazolidino-2,4-diones (20,30-
dihydrospiro[imidazolidine-4,10-indene]-2,5-dione (18–20) and
30,40-dihydro-20H-spiro[imidazolidine-4,10-naphthalene]-2,5-diones
(21–23)). We chose those terminal amide parts to examine the
influence of structural changes at the 5-position of the imidazoli-
dine ring on receptor affinity and pharmacological profile. The
planned compounds possess an asymmetric carbon atom at
* Corresponding author. Tel.: þ48 12 657 05 60; fax: þ48 12 657 02 62.
E-mail address: mfmpawlo@cyf-kr.edu.pl (M. Paw1owski).
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.11.053

1296
A. Czopek et al. / European Journal of Medicinal Chemistry 45 (2010) 1295–1303
terminal amide part which can exist in two stereoisomeric forms R
or S. The racemic compounds were chosen for the synthesis with
the aim to evaluate in vitro affinity at central serotonin and dopa-
mine receptors. To explore the effect of structural modifications at
terminal amide part, 5-HT1A, 5-HT2A and dopamine D2 receptor
affinities and in vivo intrinsic activities were determined for the
newly synthesized compounds. Three compounds (17, 20, 22) with
a diversified 5-HT1A functional profile were chosen and examined
using common behavioural tests for predicting antidepressant and/
or anxiolytic like activity in mice. Furthermore, to ascertain the
potential ligand binding mode of the studied compounds within
the 5-HT1A receptor, the most interesting one (22) was docked to
the homology model of that receptor, and molecular interactions,
potentially important to its pharmacological activity, were
described.
intermediates with the appropriate arylpiperazine (final bases
12–23). The structures of the compounds were established
using spectral 1H NMR spectra and an elemental analysis. The
detailed spectral data of each molecule are presented in
experimental section. For further pharmacological in vitro and
in vivo studies, bases 12–23 were transformed into water-well-
soluble hydrochlorides.
3. Pharmacology
3.1. In vitro tests
The affinities of all the synthesized compounds (12–23) for
central serotonin 5-HT1A and 5-HT2A and dopamine D2 receptors
were evaluated on the basis of their ability to displace [3H]-8-OH-
DPAT (8-hydroxy-2-(di-n-propylamino)tetralin), [3H]-ketanserin
and [3H]-spiperone, respectively. Radioligand binding studies were
2. Chemistry
conducted on rat brain using the cerebral cortex (5-HT1A, 5-HT2A
and the striatum (D2) [14–16].
)
The compounds were synthesized according to Scheme 1.
The
appropriate
5-(cyclo)alkyl-5-phenyl-
and
5-spi-
roimidazolidine-2,4-diones (4–7) were prepared from a ketone
by means of the Bucherer–Bergs reaction with modifications
described by Goodson et al. [11,13]. Compounds 12–23 were
obtained by a two-step procedure involving alkylation of imi-
dazolidino-2,4-diones at N3 position (intermediate compounds
8–11) with 1-bromo-3-chloropropane and condensation of
3.2. In vivo tests
Compounds with the highest affinities for 5-HT1A (13, 14, 15, 16,
17, 19, 20, 22) and 5-HT2A (18, 20, 21, 23) receptors were investi-
gated
in
in
vivo
models
to
establish
their
5-HT1A and/or 5-HT2A intrinsic activity.
O
O
a
b
A
A
NH
O
N
(CH2)3
Cl
ketone
N
H
N
H
O
4 - 7
8 - 11
c
A
Compd
4, 8
O
phenyl, methyl
A
N
(CH2)3
N
5, 9
phenyl, cyclopropyl
alpha-indanyl*
alpha-tetralinyl*
N
H
R
6, 10
7, 11
O
N
*spiro bond
12 - 23
Compd
A
R
H
Compd
18
A
R
H
12
13
14
15
16
phenyl, methyl
phenyl, methyl
phenyl, methyl
phenyl, cyclopropyl
phenyl, cyclopropyl
phenyl, cyclopropyl
α-indanyl*
α-indanyl*
α-indanyl*
α-tetralinyl*
α-tetralinyl*
α-tetralinyl*
19
2-OCH3
3-Cl
2-OCH3
3-Cl
20
21
H
H
22
2-OCH3
3-Cl
2-OCH3
3-Cl
23
17
*spiro bond
Reagents, reaction conditions:
(a) KCN, (NH4)2CO3, 50% ethyl alcohol;
(b) Br(CH2)3Cl, K2CO3, acetone, reflux;
(c) 1-phenylpiperazine derivative, anhydrous ethyl alcohol, reflux;
Scheme 1. The synthesis pathways of the investigated compounds.

A. Czopek et al. / European Journal of Medicinal Chemistry 45 (2010) 1295–1303
1297
It was already proved that the hypothermia induced by 8-
hydroxy-2-(di-n-propylamino)tetralin) (8-OH-DPAT, 5-HT1A
continue our investigations on
b
-tetralinohydantoins [11,12], we
a
designed, synthesized and tested in vitro and in vivo the activity of
new 5-substituted imidazolidine-2,4-dione derivatives with the
arylpiperazinepropyl moiety (12–23). The affinity of the synthe-
sized compounds for 5-HT1A receptors ranged from 11 to 457 nM.
The majority of compounds (except 23) with a 3-chloro or a 2-
methoxy group in the phenylpiperazine ring displayed very high
affinity for 5-HT1A receptors (Ki < 50 nM), the best ligands being 16
and 17 with contained 5-cyclopropyl-5-phenylimidazolidine-2,4-
dione (Ki ¼ 11 nM and 13 nM, respectively) (Table 1). In general,
introduction of a substituent into the phenylpiperazine moiety had
a positive impact on the binding at 5-HT1A receptor sites. In fact, the
unsubstituted analogues (12, 15, 18 and 21) showed lower 5-HT1A
receptor affinity. It is also noteworthy that high and significant
affinity for 5-HT2A receptors, which ranged from 25 to 53 nM, was
found for some of the 5-spiroimidazolidine-2,4-dione derivatives,
unsubstituted (18, 21) and 3-Cl substituted (20, 23) in phenyl-
piperazine fragment.
Moreover, few selected compounds (14, 18, 20, 21, 22, 23) were
evaluated in respect of their affinity for dopaminergic D2 receptors.
Compounds 21 and 22 demonstrated very low affinity for D2
receptors (Ki ¼ 675 ꢀ 82 nM and 965 ꢀ 6 nM, respectively),
whereas 14, 18, 20, and 23 were practically devoid of any affinity
(Ki > 1800 nM).
In the following study, selected compounds with the highest
affinity for 5-HT1A (Ki up to 70 nM) (13, 14, 15, 16, 17, 19, 20, 22) and/
or 5-HT2A (18, 20, 21, 23) receptors were further examined by
functional in vivo assays. The results of our in vivo experiments
clearly suggest that the investigated compounds show diversified
(full agonistic, partial agonistic or antagonistic) intrinsic activity,
agonistic or partial agonistic at 5-HT1A receptors and have 5-HT2A
antagonistic properties (Table 1). Compounds with a 2-methox-
yphenylpiperazine (13, 16, 19 and 22) induced effects characteristic
of presynaptic 5-HT1A receptor agonists (the hypothermia model in
receptor agonist) and abolished by (N-{2[4-(2-methoxyphenyl)-1-
piperazinyl]ethyl}-N-(2-pyridinyl)cyclohexanecarboxamide (WAY
100635, a selective 5-HT1A receptor antagonist) in mice was
mediated by activation of presynaptic 5-HT1A receptors [17,18].
Thus the ability of the tested compounds to produce that effect was
regarded as presynaptic 5-HT1A receptor agonistic activity. The
compounds that either did not change mouse body temperature or
abolished or attenuated the hypothermic effect of 8-OH-DPAT were
regarded as presynaptic 5-HT1A receptor antagonists. To determine
the postsynaptic 5-HT1A receptor agonistic effect of the tested
compounds, their ability to induce lower lip retraction (LLR) in rats
was tested. It had been commonly accepted that the 8-OH-DPAT-
induced LLR in rats depended on stimulation of postsynaptic 5-
HT1A receptors [19,20]. Moreover, it was shown that the later
symptom was sensitive to a 5-HT1A receptor antagonist [18]. The
ability of the tested compounds to inhibit the 8-OH-DPAT-induced
LLR was regarded as postsynaptic 5-HT1A receptor antagonistic
activity. In order to determine the central 5-HT2A receptor antag-
onistic properties of the tested compounds, their ability to inhibit
the head twitches induced by the 5-HT2A receptor agonist (ꢀ)-DOI
was studied in mice [21,22]. The results of in vitro and in vivo
studies on the newly synthesized compounds 12–23 are presented
in Table 1.
The potential antidepressant activity of compound 22 was
evaluated by the forced swim test in mice [23], and the potential
anxiolytic properties of compounds 17, 20, 22 were determined by
the four-plate test in mice [24]. To check possible occurrence of
drug-induced changes in the locomotor activity of mice, which may
have contributed to their behaviour in those two tests, the influ-
ence of compound 22 on locomotor activity was also examined.
4. Molecular modeling
mice), but only 22, containing a-tetralin in the terminal amide part
revealed features of an agonist of postsynaptic 5-HT1A receptors
(the LLR model in rats); on the other hand, the remaining analogues
were inactive (13, 16) or devoid of intrinsic activity at those sites
The potential ligand binding mode of the investigated
compounds within the 5-HT1A receptor was studied using in silico
modeling techniques, by means of the automated docking of the
most interesting compound 22 to the homology, a rhodopsin-based
model of the 5-HT1A receptor.
(19). Therefore it seems that the a-tetralin substituent in the amide
fragment may contribute the postsynaptic 5-HT1A agonism to those
compounds. The results of functional studies with derivatives with
the 3-chlorophenylpiperazine moiety (14, 17 and 20) indicate that
their presynaptic 5-HT1A receptor activity is diversified, since 14
was inactive,17 behaved like an antagonist and 20 like an agonist of
those sites. On the other hand, those compounds (14, 17 and 20)
showed antagonistic activity against postsynaptic 5-HT1A
5. Results and discussion
Since ligands at 5-HT1A/5-HT2A/D2 receptor sites seem to be of
therapeutic interest, extensive studies with chemically different
structures are under way in search of new potential drugs. To
Table 1
5-HT1A, 5-HT2A and D2 receptor affinities, and functional in vivo 5-HT1A/5-HT2A receptor activity of compounds 12–23.
Compd
Ki (nM)
5-HT1A
5-HT1A activity
Presynaptic
5-HT2A activity
5-HT2A
D2
Postsynaptic
12
13
14
15
16
17
18
19
20
21
22
23
457 ꢀ 142
47 ꢀ 1
69 ꢀ 6
730 ꢀ 68
312 ꢀ 21
100 ꢀ 12
710 ꢀ 88
78 ꢀ 1
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
Agonist
Non active
Non active
Agonist
Antagonist
nd
Agonist
Agonist
nd
Non active
Antagonist
Non active
Non active
Antagonist
nd
Antagonist
Antagonist
nd
29 ꢀ 5
3900 ꢀ 600
67 ꢀ 12
11 ꢀ 1
nd
nd
nd
13 ꢀ 1
98 ꢀ 16
49 ꢀ 2
30 ꢀ 2
15600 ꢀ 300
Antagonist
nd
Antagonist
Antagonist
nd
653 ꢀ 100
53 ꢀ 6
nd
38 ꢀ 1
6200 ꢀ 500
675 ꢀ 82
965 ꢀ 6
1800 ꢀ 300
88 ꢀ 21
23 ꢀ 5
25 ꢀ 5
284 ꢀ 9
35 ꢀ 6
Agonist
nd
Agonist
nd
350 ꢀ 123
Antagonist
nd: not determined; full agonist – compound administrated at doses up to 20 mg/kg behaved as agonist of pre- and postsynaptic 5-HT1A receptors; partial agonist – compound
administrated at doses up to 20 mg/kg behaved as both presynaptic agonist and postsynaptic antagonist of 5-HT1A receptors; non active – compound administrated at doses
up to 20 mg/kg behaved as neither agonist nor antagonist of pre- and/or postsynaptic 5-HT1A receptors.

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A. Czopek et al. / European Journal of Medicinal Chemistry 45 (2010) 1295–1303
Table 3
receptors. The above observations indicate that depending on the
combination of the structural features described in the present
paper yielded compounds with diversified 5-HT1A receptor prop-
erties, i.e. full agonistic (22, pre- and postsynaptic agonist), partial
agonistic (20, presynaptic agonist and postsynaptic antagonist), or
antagonistic (17, pre- and postsynaptic antagonist). It is noteworthy
that 20 is also an antagonist of 5-HT2A receptors.
The effects of 17, 20, 22 and diazepam in the four-plate test in mice.
Treatment
Dose (mg/kg)
Number of punished crossings mean ꢀ SEM
Vehicle
17
–
3.8 ꢀ 0.2
3.8 ꢀ 0.4
3.9 ꢀ 0.3
3.7 ꢀ 0.3
F(3,36) ¼ 0.0543
ns
10
20
30
It has been commonly accepted that 5-HT1A receptor agonists,
partial agonists and antagonists, as well as combined 5-HT1A/5-
HT2A receptor antagonists exhibit antidepressant- and/or anxio-
lytic-like effects [25–30]. Taking into account the intrinsic activity
of the investigated compounds, we selected compounds 22 (a full
5-HT1A receptor agonist), 20 (a partial 5-HT1A receptor agonist/5-
HT2A receptor antagonist) and 17 (a 5-HT1A receptor antagonist) for
further in vivo preclinical studies. The potential antidepressant (22)
and anxiolytic (17, 20, 22) activity of the selected compounds was
evaluated in the forced swim [23] and four-plate [24] tests in mice,
respectively. Our present results showed that compound 22 given
at doses of 10 and 20 mg/kg produced a distinct antidepressant-like
effect in the forced swim test in mice. Moreover, it shortened
mouse immobility time at a dose of 10 mg/kg which is lower than
that of the reference antidepressant imipramine (Table 2). Its anti-
immobility effect seems to be specific, since at doses effective in the
forced swim test (10 and 20 mg/kg) that compound did not change
the locomotor activity of mice during 6- or 30-min experimental
sessions (Table 4). Imipramine showed a weak sedative effect in
mice only when their locomotor activity was recorded for 30 min
(Table 4). Moreover, compound 22 exhibited anxiolytic-like activity
in the four-plate test in mice (Table 3); in terms of potency and
active doses, its effect was weaker than that of diazepam used as
a reference anxiolytic. Compounds 17 and 20 were ineffective in
that test (Table 3). Compound 22 displayed an anxiolytic-like effect
practically at one medium dose only, but produced antidepressant-
like activity in a dose-dependent manner. A limited number of data
concerning in vivo functional activity of compound 22 do not
permit us to find an explanation for the loss of anxiolytic-like
activity after administration of its higher doses. It is noteworthy
that some anxiolytics produce U-shaped dose–response effects in
animal models commonly used for the prediction of potential
anxiolytic activity [31].
Vehicle
20
–
4.2 ꢀ 0.4
4.0 ꢀ 0.3
4.2 ꢀ 0.2
4.2 ꢀ 0.2
F(3,36) ¼ 0.125
ns
10
20
30
Vehicle
22
–
5
10
20
3.8 ꢀ 0.2
3.9 ꢀ 0.4
5.9 ꢀ 0.5b
4.4 ꢀ 0.3
F(3,36) ¼ 6.065
p < 0.01
Vehiclec
Diazepam
–
3.5 ꢀ 0.4
1.25
2.5
5
5.5 ꢀ 0.5a
6.8 ꢀ 0.6b
6.7 ꢀ 0.6b
F(3,36) ¼ 9.514
p < 0.001
Compound 22 was administered 30 min, while 20, 17 – 60 min before the test.
n ¼ 10 mice per group.
ns: non-significant.
a
p < 0.05.
b
p < 0.01 vs. vehicle (Dunnett’s test).
Data taken from Ref. [34].
c
interaction anchoring the ligand in the receptor was a charge-rein-
forced hydrogen bond between the protonated nitrogen atom of the
piperazine ring and Asp3.32 (Ballesteros’s and Weinstein’s nomen-
clature). This interaction is widelyaccepted to be crucial in the group
of monoamine neurotransmitter receptors [32–35].
The phenyl ring substituted at the piperazine moiety was
localized in the close vicinity of the aromatic cluster of TMH 6; it
formed a CH-
substituent formed a hydrogen bond with Ser5.42. The putative
role of Phe6.52 and Ser5.42 in binding monoaminergic receptor
ligands was postulated in literature [33,34,36,37], however other
residues like: Phe6.51 and Phe3.28 or Ser/Thr5.43 were also
proposed as counterparts for aromatic and H-bond accepting
moieties of serotonin receptor ligands, respectively [38].
p
interaction with Phe6.52, while its 20-methoxy
Molecular docking studies with compound 22 in the homology
model of 5-HT1A receptor revealed that the investigated compound
interacted with the receptor at the binding site localized inside the
heptahelical bundle on the extracellular side of the receptor. The
ligand 22 was found to be placed along transmembrane helix (TMH)
3, its arylpiperazine moiety being located betweenTMHs 4–6 and the
30,40-dihydro-20H-spiro[imidazolidine-4,10-naphthalene]-2,5-dione
fragment localized in the vicinity of TMHs 7,1 and 2. (Fig.1) The main
Table 4
The effects of 22 and imipramine on the locomotor activity of mice.
Table 2
Treatment
Dose (mg/kg)
Locomotor activity: number of
crossings during:
The effects of 22 and imipramine in the forced swim test in mice.
Treatment
Dose (mg/kg)
Immobility time (s) Mean ꢀ SEM
6 min
30 min
Vehicle
22
–
5
10
20
181.4 ꢀ 4.9
Vehicle
22
–
10
20
107.1 ꢀ 8.5
110.0 ꢀ 7.0
111.6 ꢀ 9.8
F(2,27) ꢀ 0.072
ns
249.2 ꢀ 21.0
212.9 ꢀ 23.8
225.3 ꢀ 18.6
F(2,27) ꢀ 0.479
ns
161.2 ꢀ 14.0
121.4 ꢀ 12.1a
103.1 ꢀ 11.5a
F(3,32) ¼ 10.298
p < 0.0001
Vehicle
Imipramine
–
10
20
94.8 ꢀ 4.1
94.4 ꢀ 6.3
89.1 ꢀ 6.1
F(2,27) ¼ 0.321
ns
210.7 ꢀ 17.7
208.7 ꢀ 27.6
141.6 ꢀ 15.3a
F(2,27) ¼ 3.541
p < 0.05
Vehicle
Imipramine
–
10
20
167.1 ꢀ 6.7
149.1 ꢀ 10.7
107.8 ꢀ 12.4b
F(2,27) ¼ 8.760
p < 0.01
Compound 22 and imipramine were administered 30 min before the test. n ¼ 10
Compound 22 and imipramine were administered 30 min before the test. n ¼ 9–10
mice per group.
mice per group.
ns: non-significant.
a
a
p < 0.01 vs. vehicle (Dunnett’s test).
p < 0.05 vs. vehicle (Dunnett’s test).

A. Czopek et al. / European Journal of Medicinal Chemistry 45 (2010) 1295–1303
1299
functional profile is interesting, especially with respect to its
potential antidepressant and anxiolytic effects. In this connection,
new butyl derivatives of 5-spiroimidazolidine-2,4-dione are being
currently developed.
7. Experimental protocols
7.1. Chemistry
Melting points (m.p.) were determined in open capillaries on
the Electrothermal 9100 melting point apparatus (Jencons) and
were uncorrected. Elemental analysis was carried out using an
elemental Vario EI III Elementar analyzer (Hanau, Germany). An
ascending thin-layer chromatography method was performed on
Merck silica gel 60 F254 aluminium sheets (Merck; Darmstadt,
Germany) using the following solvents: benzene/ethyl acetate/
acetone (10:5:1), acetone/isopropanol/chloroform (20:10:1). Spots
Fig. 1. The ligand binding mode of compound 22 in the 5-HT1A receptor (a view from
the extracellular side); amino acids entering into specific interactions with ligands are
presented as ‘‘sticks’’. (A) ‘‘Cartoon’’ helices; (B) the surface of the binding site.
The substituted imidazolidine-2,4-dione moiety was found to
form a bifurcated hydrogen bond between the carbonyl oxygen
atom at 5-position of the imidazolidine ring, and Tyr7.43 and
Asn7.39. The spiro-substituted tetralin moiety had favorable van
der Waals’s contacts with the hydrophobic residues of TMHs 7,1
and 2, especially Phe3.28, yet, without entering into any specific
interaction.
The binding mode of compound 22 is in line with the binding
mode of long chain arylpiperazines proposed by us earlier [39,40],
and opposite to the binding mode of arylpiperazine derivatives
with hydantoin moiety proposed by Lopez-Rodriguez et al. [41–43],
considering orientation of the ligand in the binding site.
Since the investigated compounds possess an asymmetry center
at the 4-(spiro) position of the imidazolidine ring, they can interact
with the receptor in an R or S stereoisomer. To find out which
stereoisomer is most probably responsible for the activity of the
investigated compounds on the 5-HT1A receptor, compound 22 was
docked to the receptor model in both stereoisomeric forms. As
a result, a more favorable binding mode was found for the R
stereoisomer. As a consequence of above molecular modeling
results, we are going to synthesize the R stereoisomer of the most
promising compound 22 and further investigate its antidepressant
activity.
were detected by their absorption under UV light (
Analytical HPLC was conducted on a Waters HPLC instrument with
Waters 485 Tunable Absorbance Detector UV, equipped with
l
¼ 254 nm).
a Symetry column (C18, 3.5
m
m, 4.6 ꢁ 30 mm). A flow rate of 5 ml/
min and a 3 min gradient of water/0.1% TFA and acetonitrile/0.1%
TFA (from 0 to 100%) were used, detection at 214 nm. The purity of
the investigated compounds 12–23 ranged from 95 to 99 per cent.
1H NMR spectra were recorded with a Varian Mercury spec-
trometer (Varian Inc., Palo Alto, CA, USA) operating at 300 MHz in
a CDCl3 solution, with tetramethylsilane (TMS) as an internal
standard. Chemical shifts are shown as
d values (ppm), the J values
are expressed in Hertz (Hz). Signal multiplicities are represented as
s (singlet), d (doublet), t (triplet), q (quintet) or m (multiplet).
The substituted 1-phenylpiperazine derivatives, 1-bromo-3-
chloropropane,
a-indanone, a-tetralone, methyl(phenyl)ketone,
cyclopropyl(phenyl)ketone and other chemicals were commercially
available (Aldrich or Fluka).
7.1.1. General procedure for preparation of
imidazolidine-2,4-diones (4–7)
All imidazolidine-2,4-diones (4–7) were prepared from the
ketone by Bucherer–Bergs reaction with modifications described by
Goodson et al. [13].
6. Conclusions
A solution of ketone (330 mmol) and ammonium carbonate
(1 mol) in ethanol (330 ml) and water (220 ml) was warmed to
50 ꢂC, at which time potassium cyanide (350 mmol) dissolved in
50 ml of water was dropped in over a period of 15 min. The mixture
was heated under a reflux at 56–60 ꢂC for more than 20 h. The
reflux condenser was then replaced by an air condenser and the
temperature raised to 80 ꢂC for 1 h to remove the excess ammo-
nium carbonate. Then the reaction solution was cooled and acidi-
fied. The precipitated solid was filtered off, washed with water, and
recrystallized from a mixture of ethanol and water, giving 4–7
hydantoin.
A few new 5-(cyclo)alkyl-5-phenyl- and 5-spiroimidazolidine-
2,4-dione derivatives with the arylpiperazinylpropyl moiety were
designed and synthesized. The replacement of a flexible 5-alkyl-5-
phenyl by a rigid 5-spiroring in the imidazolidine-2,4-dione moiety
resulted in the diversification of intrinsic activity at the 5-HT1A
receptor binding site. The introduction of a (cyclo)alkyl-phenyl or
spiro substituent to the amide moiety and 2-methoxy or 3-chloro
group to the phenylpiperazine ring substantially changed the
pharmacological in vivo properties of the compounds tested i.e. 17
became a pre- and postsynaptic 5-HT1A antagonist and 22 was
a pre- and postsynaptic 5-HT1A agonist. Additionally, molecular
docking showed that the cyclic amide part (30,40-dihydro-20H-spi-
ro[imidazolidine-4,10-naphthalene]-2,5-dione moiety) played an
important role in stabilizing the 5-HT1A receptor–ligand complex.
Of the obtained compounds, 22 was the only one, that behaved like
a potent agonist of pre- and postsynaptic 5-HT1A receptors in the
behavioural tests used. Moreover, compound 22 showed antide-
pressant and anxiolytic like activity in animal models and seemed
to be devoid of any unfavourable motor effects. However, the
present data need to be corroborated by further pharmacological
studies in order to establish the position of compound 22 as a new
full 5-HT1A agonist and to determine the mechanism of its potential
antidepressant activity. The study shows that its in vitro affinity and
7.1.1.1. 5-Methyl-5-phenylimidazolidine-2,4-dione
(4). Following
the general procedure 4 was obtained from acetophenone (50ꢂ
EtOH). Yield: 51%; m.p. 180–182 ꢂC; 1H NMR (300 MHz, CDCl3):
d
10.52 (s, 1H), 8.15 (s, 1H), 7.55–7.30 (m, 5H), 1.85 (s, 3H). Anal.
calcd for C10H10N2O2: C, 63.15; H, 5.30; N, 14.73. Found: C, 63.52;
H, 5.45; N, 14.92.
7.1.1.2. 5-Cyclopropyl-5-phenylimidazolidine-2,4-dione (5). Following
the general procedure 5 was obtained from cyclopropyl(phenyl)
methanone (50ꢂ EtOH). Yield: 66%; m.p. 184–185 ꢂC; 1H NMR
(300 MHz, CDCl3):
d 10.78 (s, 1H), 8.30 (s, 1H), 7.60–7.50 (m, 2H),
7.45–7.30 (m, 3H), 1.75–1.52 (m, 1H), 0.60–0.24 (m, 4H). Anal. calcd

1300
A. Czopek et al. / European Journal of Medicinal Chemistry 45 (2010) 1295–1303
for C12H12N2O2: C, 66.65; H, 5.59; N, 12.96. Found: C, 65.12; H, 4.92;
N, 12.97.
7.1.3.1. 5-Methyl-5-phenyl-3-[3-(4-phenylpiperazin-1-yl)propyl]-
imidazolidine-2,4-dione (12). Following the general procedure 12
was obtained from 8 (EtOH). Yield: 53%; m.p. 140–144 ꢂC; 1H NMR
7.1.1.3. 20,30-Dihydrospiro[imidazolidine-4,10-indene]-2,5-dione
(300 MHz, CDCl3): d 7.54–7.25 (m, 7H), 6.95–6.85 (m, 3H), 6.22 (s,
(6). This compound was previously described [13].
1H), 3.65–3.60 (t, J ¼ 7 Hz, 2H) 3.20–3.17 (t, J ¼ 5 Hz, 4H), 2.60–2.57
(t, J ¼ 5 Hz, 4H), 2.46–2.41 (t, J ¼ 7 Hz, 2H), 1.93–1.83 (m, 5H). Anal.
calcd for C23H28N4O2: C, 70.38; H, 7.19; N, 14.27. Found: C, 70.25; H,
7.00; N, 14.36.
7.1.1.4. 30,40-Dihydro-20H-spiro[imidazolidine-4,10-naphthalene]-2,5-
dione (7). This compound was previously described [13].
7.1.2. General procedure for synthesis of 3-(3-chloropropyl)imidazol-
idine-2,4-dione derivatives (8–11)
7.1.3.2. 5-Methyl-5-phenyl-3-{3-[4-(2-methoxyphenyl)piperazin-1-
yl]propyl}-imidazolidine-2,4-dione (13). Following the general
procedure 13 was obtained from 8 (EtOH). Yield: 46%; m.p. 156–
A suspension of appropriate imidazolidine-2,4-dione (50 mmol)
and anhydrous K2CO3 (150 mmol) was refluxed in acetone (160 ml)
under intensive stirring for 30 min. Afterwards a solution of 1-
bromo-3-chloropropane (55 mmol) in acetone (50 ml) was instilled
during the period of 20 min. The reaction was carried on for 8–14 h.
The hot reaction mixture was then filtered off, the solvent was
evaporated and the oily residue was purified by crystallization from
a 40ꢂ or 50ꢂ ethanol.
159 ꢂC; 1H NMR (300 MHz, CDCl3):
d 7.51–7.33 (m, 5H), 7.03–6.81
(m, 4H), 5.56 (s, 1H), 3.88–3.82 (s, 3H), 3.64–3.55 (t, J ¼ 7 Hz, 2H)
3.10–3.01 (m, 4H), 2.62–2.54 (m, 4H), 2.43–2.35 (t, J ¼ 7 Hz, 2H),
1.89–1.80 (m, 5H). Anal. calcd for C24H30N4O3: C, 68.22; H, 7.16; N,
13.26. Found: C, 67.70; H, 7.24; N, 13.15.
7.1.3.3. 5-Methyl-5-pheny-3-{3-[4-(3-chlorophenyl)piperazin-1-yl]-
propyl}-imidazolidine-2,4-dione (14). Following the general proce-
dure 14 was obtained from 8 (EtOH). Yield: 45%; m.p. 142–145 ꢂC;
7.1.2.1. 3-(3-Chloropropyl)-5-methyl-5-phenylimidazolidine-2,4-
dione (8). Following the general procedure 8 was obtained from 4
(40ꢂ EtOH). Yield: 98%; m.p.107–109 ꢂC; 1H NMR (300 MHz, CDCl3):
1H NMR (300 MHz, CDCl3):
d 7.52–7.27 (m, 5H), 7.03–6.82 (m, 4H),
5.86 (s, 1H), 3.68–3.61 (t, J ¼ 6.8 Hz, 2H) 3.22–3.17 (m, 4H), 2.66–
2.55 (m, 4H), 2.52–2.44 (t, J ¼ 6.8 Hz, 2H), 1.89–1.81 (m, 5H). Anal.
calcd for C23H27N4O2Cl: C, 64.70; H, 6.37; N, 13.12. Found: C, 64.46;
H, 6.14; N, 13.09.
d
7.53–7.33 (m, 5H), 6.58 (s, 1H), 3.70–3.63 (t, J ¼ 6.8 Hz, 2H), 3.53–
3.47 (t, J ¼ 6.5 Hz, 2H), 2.16–2.03 (q, J ¼ 6.6 Hz, 2H), 1.83 (m, 3H).
Anal. calcd for C13H15N2O2Cl: C, 58.54; H, 5.67; N, 10.50. Found:
C, 58.27; H, 5.60; N, 10.46.
7.1.3.4. 5-Cyclopropyl-5-phenyl-3-[3-(4-phenylpiperazin-1-yl)pro-
pyl]imidazolidine-2,4-dione (15). Following the general procedure
15 was obtained from 9 (EtOH). Yield: 41%; m.p. 131–134 ꢂC; 1H
7.1.2.2. 3-(3-Chloropropyl)-5-cyclopropyl-5-phenylimidazolidine-
2,4-dione (9). Following the general procedure 9 was obtained
from 5 (40ꢂ EtOH). Yield: 86%; m.p. 96–101 ꢂC; 1H NMR (300 MHz,
NMR (300 MHz, CDCl3): d 7.61–7.25 (m, 7H), 6.96–6.85 (m, 3H), 5.92
CDCl3):
d
7.58–7.53 (m, 2H), 7.45–7.26 (m, 3H), 5.78 (s, 1H), 3.72–
(s, 1H), 3.66–3.61 (t, J ¼ 7 Hz, 2H), 3.22–3.19 (m, 4H), 2.63–2.59 (m,
4H), 2.48–2.43 (t, J ¼ 7 Hz, 2H), 1.92–1.87 (q, J ¼ 7 Hz, 2H), 1.73–1.71
(m, 1H), 0.74–0.35 (m, 4H). Anal. calcd for C25H30N4O2: C, 71.74; H,
7.22; N, 13.39. Found: C, 71.53; H, 7.53; N, 13.41.
3.64 (t, J ¼ 7 Hz, 2H), 3.55–3.49 (t, J ¼ 7 Hz, 2H), 2.18–2.05 (q,
J ¼ 7 Hz, 2H), 1.74–1.59 (m, 1H), 0.75–0.52 (m, 3H), 0.37–0.28 (m,
1H). Anal. calcd for C15H17N2O2Cl: C, 61.54; H, 5.85; N, 9.57. Found:
C, 61.27; H, 5.70; N, 9.37.
7.1.3.5. 5-Cyclopropyl-5-phenyl-3-{3-[4-(2-methoxyphenyl)piper-
7.1.2.3. 1-(3-Chloropropyl)-20,30-dihydrospiro[imidazolidine-4,10-
indene]-2,5-dione (10). Following the general procedure 10 was
obtained from 6 (40ꢂ EtOH). Yield: 70%: m.p. 101–102 ꢂC; 1H NMR
azin-1-y]propyl}–imidazolidine-2,4-dione
general procedure 16 was obtained from 9 (EtOH). Yield: 49%; m.p.
148–150 ꢂC; 1H NMR (300 MHz, CDCl3):
7.60–7.32 (m, 5H), 7.02–
(16). Following
the
d
(300 MHz, CDCl3):
d
7.33–7.15 (m, 4H), 5.98 (s, 1H), 3.67–3.61 (t,
6.82 (m, 4H), 6.01 (s, 1H), 3.87–3.83 (s, 3H), 3.62–3.56 (t, J ¼ 7 Hz,
2H) 3.11–3.02 (m, 4H), 2.66–2.58 (m, 4H), 2.47–2.39 (m, 2H), 1.91–
1.80 (t, J ¼ 7 Hz, 2H), 1.75–1.64 (m, 1H), 0.76–0.29 (m, 4H). Anal.
calcd for C26H32N4O3: C, 69.62; H, 7.19; N, 12.49. Found: C, 69.98; H,
7.34; N, 12.67.
J ¼ 6.8 Hz, 2H), 3.55–3.50 (t, J ¼ 6.6 Hz, 2H), 3.29–3.18 (m, 1H), 3.08–
2.98 (m, 1H) 2.72–2.64 (m, 1H), 2.28–2.22 (m, 1H), 2.18–2.06 (q,
J ¼ 6.8 Hz, 2H). Anal. calcd for C14H15N2O2Cl: C, 60.33; H, 5.42; N,
10.05. Found: C, 60.34; H, 5.64; N, 10.03.
7.1.2.4. 1-(3-Chloropropyl)-30,40-dihydro-20H-spiro[imidazolidine-4,10-
naphthalene]-2,5-dione (11). Following the general procedure 11
was obtained from 7 (50ꢂ EtOH). Yield: 81%; m.p. 150–152 ꢂC; 1H
7.1.3.6. 5-Cyclopropyl-5-phenyl-3-{3-[4-(3-chlorophenyl)piperazin-
1-yl]propyl}-imidazolidine-2,4-dione (17). Following the general
procedure 17 was obtained from 9 (EtOH). Yield: 31%; m.p. 122–
NMR (300 MHz, CDCl3):
d
7.31–7.05 (m, 4H), 5.92 (s, 1H), 3.65–3.59
125 ꢂC; 1H NMR (300 MHz, CDCl3):
d 7.61–7.32 (m, 5H), 7.20–7.11
(t, J ¼ 7 Hz, 2H), 3.24–3.18 (q, J ¼ 7.2 Hz, 2H), 2.66–2.54 (t, J ¼ 7 Hz,
2H), 2.40–2.29 (m, 2H), 2.15–1.96 (m, 4H). Anal. calcd for
C15H17N2O2Cl: C, 61.54; H, 5.85; N, 9.57. Found: C, 61.62; H, 5.66; N,
9.30.
(m, 1H), 6.87–6.72 (m, 3H), 6.05 (s, 1H), 3.64–3.57 (m, 2H) 3.29–3.17
(m, 4H), 2.74–2.46 (m, 6H), 1.99–1.88 (m, 2H), 1.73–1.65 (m, 1H),
0.75–0.31 (m, 4H). Anal. calcd for C25H29N4O2Cl: C, 66.29; H, 6.45;
N, 12.37. Found: C, 66.13; H, 6.19; N, 12.28.
7.1.3. General procedure for synthesis of compounds 12–23
A mixture of appropriate derivatives of 3-(3-chloropropyl)-
hydantoin (5 mmol), the substituted 1-phenylpiperazine (1 mmol)
in anhydrous ethanol (30 ml) was refluxed for 28–30 h. After
cooling, the solvent was evaporated and the residue was treated
with water (50 ml); and the solution was extracted with CHCl3
(3 ꢁ 15 ml). The combined organic phases were dried over anhy-
drous Na2SO4. Then the solution was filtered off, the solvent was
evaporated and the crude product was purified by crystallization
from anhydrous ethanol.
7.1.3.7. 1-[3-(4-Phenylpiperazin-1-yl)propyl]-20,30-dihydrospiro[imi-
dazolidine-4,10-indene]-2,5-dione (18). Following the general
procedure 18 was obtained from 10 (EtOH). Yield: 59%; m.p. 134–
135 ꢂC; 1H NMR (300 MHz, CDCl3):
d 7.35–7.10 (m, 6H), 6.92–6.83
(m,,3H), 6.25 (s, 1H), 3.64–3.59 (t, J ¼ 7.2 Hz, 2H), 3.30–3.22 (m,
1H), 3.18–3.15 (t, J ¼ 5 Hz, 4H), 3.08–2.99 (m, 1H), 2.75–2.67 (m,
1H), 2.59–2.57 (t, J ¼ 5 Hz, 4H), 2.46–2.41 (t, J ¼ 7.2 Hz, 2H), 2.28–
2.18 (m, 1H), 1.93–1.83 (q, J ¼ 7.15 Hz, 2H). Anal. calcd for
C24H28N4O2: C, 71.26; H, 6.98; N, 13.85. Found: C, 70.94; H, 6.86;
N, 13.87.

A. Czopek et al. / European Journal of Medicinal Chemistry 45 (2010) 1295–1303
1301
7.1.3.8. 1-{3-[4-(2-Methoxyphenyl)piperazin-1-yl]propyl}-20,30-dihy-
drospiro[imidazolidine-4,10-indene]-2,5-dione (19). Following the
general procedure 19 was obtained from 10 (EtOH). Yield: 72%; m.p.
were homogenized in 20 volumes of 50 mM Tris–HCl buffer
(pH 7.7 at 25 ꢂC) using Ultra-TurraxÒ T 25, and were then centri-
fuged at 30,000g for 10 min. The supernatant fraction was dis-
carded, and the pellet was resuspended in the same volume of the
Tris–HCl buffer, afterwards it was centrifuged as above. Prior to
the third centrifugation, the samples were incubated at 37 ꢂC for
10 min. The final pellet was resuspended in the Tris–HCl buffer
containing 10 mM pargyline, 4 mM CaCl2 and a 0.1% ascorbic acid.
The final incubation mixture consisted of 1 mL of the tissue
160–161 ꢂC; 1H NMR (300 MHz, CDCl3):
d 7.32–7.06 (m, 5H), 7.01–
6.80 (m, 3H), 5.91 (s, 1H), 3.85 (s, 3H), 3.65–3.60 (t, J ¼ 7 Hz, 2H),
3.28–3.20 (m, 1H), 3.06 (br.s, 4H), 3.02–2.99 (m, 1H), 2.76–2.67 (m,
1H), 2.63 (br.s, 4H), 2.48–2.43 (t, J ¼ 7 Hz, 2H), 2.29–2.22 (m, 1H),
1.94–1.84 (q, J ¼ 7 Hz, 2H). Anal. calcd for C25H30N4O3: C, 69.10; H,
6.96; N, 12.89. Found: C, 69.20; H, 6.89; N, 12.84.
suspension (9 mg of wet weight), 100
m
l of 10
m
M serotonin (for
l of the
7.1.3.9. 1-{3-[4-(3-Chlorophenyl)piperazin-1-yl]propyl}-20,30-dihydr-
ospiro[imidazolidine-4,10-indene]-2,5-dione (20). Following the
general procedure 20 was obtained from 10 (EtOH). Yield: 53%; m.p.
unspecific binding), 100 m
m
l of [3H]-8-OH-DPAT and 100
analyzed compounds. The sample was incubated at 37 ꢂC for
15 min. The incubation was followed by rapid vacuum filtration
through Whatman GF/B glass filters. The filters were then washed
3 times with 5 ml of an ice-cold buffer (50 mM Tris–HCl, pH 7.7)
using a Brandel cell harvester. The final [3H]-8-OH-DPAT concen-
tration was 1 nM, and the concentrations of the analyzed
compounds ranged from 10ꢃ10 to 10ꢃ4 M. Then the filters were
placed in scintillation vials with a scintillation cocktail. Radioac-
tivity was measured in a WALLAC 1409 DSA – liquid scintillation
counter. All the assays were carried out in duplicate. Radioligand
binding data were analyzed using iterative curve fitting routines
(GraphPAD/Prism, version 3.0 – San Diego, CA, USA).
138–139 ꢂC; 1H NMR (300 MHz, CDCl3):
d 7.39–7.14 (m, 5H), 6.90–
6.78 (m, 3H), 5.72 (s, 1H), 3.69–3.64 (t, J ¼ 7.2 Hz, 2H), 3.38–3.35 (t,
J ¼ 5 Hz, 4H), 3.31–3.20 (m, 1H), 3.13–3.04 (m, 1H) 2.80–2.71 (m,
1H), 2.63–2.59 (t, J ¼ 5 Hz, 4H), 2.51–2.46 (t, J ¼ 7.2 Hz, 2H), 2.33–
2.23 (m, 1H), 1.97–1.87 (q, J ¼ 7.10 Hz, 2H). Anal. calcd for
C24H27N4O2Cl: C, 65.67; H, 6.20; N, 12.76. Found: C, 65.66; H, 6.22;
N, 12.78.
7.1.3.10. 1-[3-(4-Phenylpiperazin-1-yl)propyl]-30,40-dihydro-20H-spi-
ro[imidazolidine-4,10-naphthalene]-2,5-dione (21). Following the
general procedure 21 was obtained from 11 (EtOH). Yield: 56%; m.p.
142–143 ꢂC; 1H NMR (300 MHz, CDCl3):
d
7.28–7.04 (m, 6H), 6.94–
7.2.2. 5-HT2A receptor binding experiments
6.82 (m, 3H), 5.68 (s, 1H), 3.68–3.63 (t, J ¼ 7.2 Hz, 2H), 3.21–3.18 (t,
J ¼ 5 Hz, 4H), 2.90–2.84 (m, 2H), 2.62–2.59 (t, J ¼ 5 Hz, 4H), 2.49–
2.44 (t, J ¼ 7.2 Hz, 2H), 2.39–2.24 (m, 2H), 2.02–1.77 (m, 4H). Anal.
calcd for C25H30N4O2: C, 71.75; H, 7.22; N, 13.39. Found: C, 71.68; H,
7.22; N, 13.35.
[3H]-Ketanserin (spec. act. 60 Ci/mM, NEN Chemicals) was used
for labeling 5-HT2A receptors. The assay was performed according
to the method described previously by Leysen et al. [15] Rat
cerebral cortex tissues were homogenized in 20 volumes of
50 mM Tris–HCl buffer (pH 7.7 at 25 ꢂC) and centrifuged at
30,000g for 20 min. The resulting pellet was resuspended in the
same quantity of the buffer, preincubated at 37 ꢂC for 10 min and
centrifuged for 20 min as above. The final pellet was resuspended
in 50 volumes of the same buffer. The final incubation mixture
7.1.3.11. 1-{3-[4-(2-Methoxyphenyl)piperazin-1-yl]propyl}-30,40-dih-
ydro-20H-spiro[imidazolidine-4,10-naphthalene]-2,5-dione (22). Follo-
wing the general procedure 22 was obtained from 11 (EtOH). Yield:
53%; m.p. 205–209 ꢂC; 1H NMR (300 MHz, CDCl3):
d
7.26–7.12 (m,
consisted of 1 mL of the tissue suspension, 100 M mian-
m
l of 1
m
3H), 7.05–6.83 (m, 5H), 6.08 (s, 1H), 3.85 (s, 3H), 3.61–3.57 (t,
J ¼ 7.2 Hz, 2H), 3.08 (br.s, 4H), 2.92–2.79 (m, 2H), 2.65 (br.s, 4H),
2.48–2.43 (t, J ¼ 7.2 Hz, 2H), 2.32–2.21 (m, 2H), 2.01–1.83 (m, 4H).
Anal. calcd for C26H32N4O3: C, 69.62; H, 7.19; N, 12.49. Found: C,
69.28; H, 7.15; N, 12.27.
serin (displacer), 100 ml of the
m
l of [3H]-ketanserin and 100
analyzed compounds. The sample was incubated at 37 ꢂC for
20 min, followed by a rapid vacuum filtration through Whatman
GF/B glass filters, and was then washed three times with 5 ml of an
ice-cold Tris–HCl buffer. The final [3H]-ketanserin concentration
was 0.6 nM, and the concentrations of the analyzed compounds
changed from 10ꢃ10 to 10ꢃ4 M. Then the filters were placed in
a scintillation vials with scintillation cocktail. Radioactivity was
measured in a WALLAC 1409 DSA – liquid scintillation counter. All
the assays were carried out in duplicate. Radioligand binding data
were analyzed using iterative curve fitting routines (GraphPAD/
Prism, version 3.0 – San Diego, CA, USA).
7.1.3.12. 1-{3-[4-(3-Chlorophenyl)piperazin-1-yl]propyl}-30,40-dihydro-
20H-spiro[imidazolidine-4,10-naphthalene]-2,5-dione (23). Following
the general procedure 23 was obtained from 11 (EtOH). Yield: 52%;
m.p. 158–159 ꢂC; 1H NMR (300 MHz, CDCl3):
d 7.26–7.03 (m, 5H),
6.91–6.76 (m, 3H) 5.72 (br.s, 1H), 3.63–3.58 (t, J ¼ 7.2 Hz, 2H),
3.20–3.16 (t, J ¼ 4.9 Hz, 4H), 2.90–2.84 (m, 2H), 2.58–2.55
(t, J ¼ 4.9 Hz, 4H), 2.45–2.41 (t, J ¼ 7.2 Hz, 2H), 2.35–2.23 (m, 2H),
2.02–1.75 (m, 4H). Anal. calcd for C25H29N4O2Cl: C, 66.29; H, 6.45; N,
12.37. Found: C, 66.16; H, 6.40; N, 12.36.
7.2.3. D2 receptor binding experiments
The assay was conducted according to the method described by
Ossowska et al. [16]. Rat striatum tissues were thawed in 50
volumes of an ice-cold 50 mM potassium phosphate buffer (pH 7.4)
homogenized and centrifuged at 20,000g for 20 min. The resulting
pellet was resuspended in the same quantity of the buffer, and was
centrifuged again for 20 min as described above.
7.1.4. General procedure for the preparation of hydrochlorides
Free bases were dissolved in an excess of concentrated HCl upon
heating and were cooled down. Afterwards anhydrous ethanol was
added until the salt began to separate. Then hydrochloride salts
were recrystallized from anhydrous ethanol.
Assay tubes contained a membrane suspension (3 mg of wet
weight), [3H]-spiperone (15 Ci/mM, NEN) at a concentration of 1 nM,
7.2. In vitro experiments
(þ)butaclamol (10
m
M) or the analyzed compound (from 10ꢃ10 to
10ꢃ4 M) at a final volume of 0.5 ml. The sample was incubated at
37 ꢂC for 30 min. The incubation was terminated by rapid filtration
over glass filters (Whatman GF/B) using Brandel’s manifold. The
filters were then washed twice with 5 ml of an ice-cold buffer and
were placed in scintillation vials with a liquid scintillation cocktail.
Radioactivity was measured in a WALLAC 1409 DSA – liquid scin-
tillation counter. All the assays were carried out in duplicate.
7.2.1. 5-HT1A receptor binding experiments
[3H]-8-Hydroxy-2-(di-n-propylamino)-tetralin ([3H]-8-OH-DPAT,
spec. act. 106 Ci/mM, NEN Chemicals) was used for labeling 5-HT1A
receptors. The membrane preparation and the assay procedure
were carried out according to the previously published procedure
[14] with slight modifications. Briefly, rat cerebral cortex tissues

1302
A. Czopek et al. / European Journal of Medicinal Chemistry 45 (2010) 1295–1303
Radioligand binding data were analyzed using iterative curve fitting
routines (GraphPAD/Prism, version 3.0 – San Diego, CA, USA).
treatment. Head twitches in mice were induced by (ꢀ)-DOI
(2.5 mg/kg). Immediately after the treatment, the head twitches
were counted throughout 20 min [21]. The tested compounds (18,
20, 21, 23) were administered 60 min before (ꢀ)-DOI.
7.3. In vivo experiments
The experiments were performed on male Wistar rats (290–
310 g) or male Albino Swiss mice (24–28 g). The animals were kept
at a room temperature of 20 ꢀ 1 ꢂC and had free access to food
(standard laboratory pellets) and tap water before the experiment.
All the investigations were conducted in the light phase on a natural
day–night cycle (from May to September), between 9 a.m. and
7.3.4. Four-plate test in mice
The box was made of opaque plastic and was rectangular
(25 cm ꢁ 18 cm ꢁ 16 cm) in shape. Its floor was covered with four
rectangular metal plates (11 cm ꢁ 8 cm), separated by a 4mm gap.
The plates were connected to the source of a continuous current,
which enabled a 120 V potential difference between two adjacent
plates for 0.5 s when the experimenter pressed the switch. Indi-
vidual mice were gently placed on the plate and were allowed to
explore for 15 s. Afterwards, each time a mouse passed from one
plate to another, the experimenter electrified the whole floor,
which evoked a visible flight reaction of the animal. If the animal
continued running, it received no new shocks during the following
3 s. The episodes of punished crossing were counted for 60 s [24].
2
p.m. All the experimental procedures were approved by
the Local Bioethics Commission for Animal Experiments at the
´
Institute of Pharmacology, Polish Academy of Sciences in Krakow. 8-
Hydroxy-2-(di-n-propylamino)tetralin (hydrobromide; 8-OH-
DPAT, Tocris, Cookson Ltd., UK) and (ꢀ)-2,5-dimethoxy-4-iodoam-
phetamine (hydrochloride; (ꢀ)-DOI, Sigma–Aldrich, Inc., USA) were
dissolved
in
saline;
N-{2-[4-(2-methoxyphenyl)-1-piper-
azinyl]ethyl}-N-(2-pyridinyl)cyclohexanecarboxamide (trihydro-
chloride, WAY 100635, synthesized by Dr. J. Boksa, Institute of
7.3.5. Forced swim test in mice
´
Pharmacology, Polish Academy of Sciences, Krakow, Poland) and
The experiment was carried out according to the method of
Porsolt et al. [23] Briefly, the mice were individually placed in
a glass cylinder (25 cm high; 10 cm in diameter) containing 6 cm of
water kept at 23–25 ꢂC, and were left therein for 6 min. A mouse
was regarded as immobile when it remained floating in the water,
making only small movements to keep its head above the surface.
The total duration of immobility was recorded during the last 4 min
of a 6-min test session.
imipramine (hydrochloride; Polfa-Starogard, Poland) were dis-
solved in distilled water. The investigated compounds were sus-
pended in a 1% aqueous solution of Tween 80. 8-OH-DPAT and WAY
100635 were injected subcutaneously (sc), (ꢀ)-DOI, imipramine and
the tested compounds were given intraperitoneally (ip) at a volume
of 2 ml/kg (rats) or 10 ml/kg (mice). Each experimental group con-
sisted of 6–10 animal per dose, and each animal was used only once.
7.3.1. Body temperature in mice
7.3.6. Locomotor activity in mice
The effects of the tested compounds given alone on rectal body
temperature (measured with an Ellab thermometer) were recorded
at 30, 60, 90, and 120 min after their administration to mice. In
a separate experiment, the effect of WAY 100635 (0.1 mg/kg) on the
hypothermia induced by compounds 13, 14, 16, 19, 22 and 8-OH-
DPAT was tested. WAY 100635 was administered 15 min before the
former compounds and 8-OH-DPAT, and rectal body temperature
was recorded at 30 and 60 min after injection of the tested
compounds. In another experiment, the effect of 18, 20 and 23
(which did not change mouse body temperature) on the 8-OH-
DPAT (5 mg/kg)-induced hypothermia was assessed. The tested
compounds were administered 45 min before 8-OH-DPAT, and
rectal body temperature was measured 15, 30, 45, and 60 min after
8-OH-DPAT injection. The results were expressed as a change in
The locomotor activity of mice was recorded in photoresistor
actometers (24 cm in diameter), illuminated by two light beams
which were connected to a counter for the recording of light beam
interruptions. The mice were individually placed in the actometers,
and the number of crossings of the light beams was assessed twice:
during the first 6 min, and during 30-min experimental sessions.
7.4. Statistics
The obtained data were analyzed by a one-way analysis of
variance, followed by Dunnett’s test (when only one drug was
given), or by the Newman–Keuls test (when two drugs were
administered). ID50 values were calculated by the method of
Litchfield and Wilcoxon.
body temperature (
Dt) with respect to basal body temperature, as
measured at the beginning of the experiment.
7.5. Molecular modeling
7.3.2. Lower lip retraction (LLR) in rats
Docking studies of compound 22 were carried out using
a homology, rhodopsin-based model of rat 5-HT1A serotonin
receptor described previously [40]. To explore the conformational
space of the binding site, a ligand was docked to the population of
100 models which differed significantly in their side-chain
conformations, while the polypeptide backbone differed only
insignificantly from the original template.
LLR was assessed according to the method described by
Berendsen et al. [19] The rats were individually placed in cages
(30 cm ꢁ 25 cm ꢁ 25 cm) and were scored three times after
administration (at 15, 30 and 45 min) of the tested compounds (13,
14, 15, 16, 17, 19, 20, 22) and 8-OH-DPAT as follows: 0 ¼ lower
incisors not visible; 0.5 ¼ partly visible; 1 ¼ completely visible. The
total maximum score was 3 for each rat. In a separate experiment,
the effect of the tested compounds and WAY 100635 on the LLR
induced by 8-OH-DPAT (1 mg/kg) was tested. The compounds and
WAY 100635 were administered at 45 min and 15 min before 8-OH-
DPAT, respectively, and the animals were scored at 15, 30 and
45 min after 8-OH-DPAT administration.
A molecular model of compound 22 was built with CORINA
(www2.chemie.uni-erlangen.de/software/corina), followed by
geometry optimization using
a PM5 quantum semiempirical
method with the COnductorlike Screening MOdel (COSMO)
approach, to simulate a water environment (MOPAC 2002, imple-
mented in the CAChe Worksystem Pro 6.1; www.cachesoftware.
com). The þ1 formal charge was located on a protonated nitrogen
atom of the piperazine moiety.
7.3.3. Head twitch response in mice
To habituate mice to the experimental environment, each
animal was randomly transferred to a 12 cm (diameter) ꢁ 20 cm
(height) glass cage lined with sawdust, at 30 min before the
Docking was carried out using FlexX (www.biosolveit.de), with
default parameters. FlexX rapidly and exhaustively samples the
conformational space of a ligand by means of an incremental

A. Czopek et al. / European Journal of Medicinal Chemistry 45 (2010) 1295–1303
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1303
construction algorithm that builds the ligand in the site [44]. An
interaction constraint (the FlexX-Pharm module of FlexX) was
applied on the hydrogen bond between Asp3.32 and the proton-
ated nitrogens of the ligands during docking, since that interaction
was considered crucial for all the monoamine neurotransmitter
receptors [35]. The obtained ligand–receptor complexes were
scored using five scoring functions: F_score, D_score, G_score,
Chem_score, and PMF_score, with a subsequent consensus scoring
as implemented in the CScore module of SYBYL 7.1. Only complexes
with the highest ‘‘5’’ CScore value were considered, and the ranking
of compounds was based on the PMF_score, since that scoring
function was reported to provide the best enrichment factors in
virtual screening experiments [45].
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The authors wish to thank the Polish Ministry of Science and
Higher Education for financial support (grant No. 2P05F04329).
Part of this work was presented at the Joint Meeting on Medicinal
Chemistry in Vienna 2005, and at the MKNOL in 2006.
Anna Czopek is a scholarship holder of the project which is co-
financed from the European Social Fund and national budget in the
frame of The Integrated Regional Operational Programme.
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