Novel spirohydantoin derivative as a potent multireceptor-active antipsychotic and antidepressant agent

Article abstract of DOI:10.1016/j.bmc.2015.04.026

Abstract A series of novel spirohydantoin derivatives with arylpiperazinylbutyl moiety were synthesized and evaluated for serotonin 5-HT_1A, 5-HT_2A, 5-HT₇ and dopamine D₂ receptors. Based on these data, four comp

Full text of DOI:10.1016/j.bmc.2015.04.026

Accepted Manuscript
Novel Spirohydantoin Derivative as a Potent Multireceptor-Active Antipsy-
chotic and Antidepressant Agent
Anna Czopek, Marcin Kołaczkowski, Adam Bucki, Hanna Byrtus, Maciej
Pawłowski, Grzegorz Kazek, Andrzej J Bojarski, Agata Piaskowska, Justyna
Kalinowska-Tłuścik, Anna Partyka, Anna Wesołowska
PII:
S0968-0896(15)00324-7
DOI:
http://dx.doi.org/10.1016/j.bmc.2015.04.026
BMC 12238
Reference:
Bioorganic & Medicinal Chemistry
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8 January 2015
9 April 2015
11 April 2015
Please cite this article as: Czopek, A., Kołaczkowski, M., Bucki, A., Byrtus, H., Pawłowski, M., Kazek, G., Bojarski,
A.J., Piaskowska, A., Kalinowska-Tłuścik, J., Partyka, A., Wesołowska, A., Novel Spirohydantoin Derivative as a
Potent Multireceptor-Active Antipsychotic and Antidepressant Agent, Bioorganic & Medicinal Chemistry (2015),
doi: http://dx.doi.org/10.1016/j.bmc.2015.04.026
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Novel Spirohydantoin Derivative as a Potent Multireceptor-Active Antipsychotic and
Antidepressant Agent.
Anna Czopeka, Marcin Kołaczkowskia, Adam Buckia, Hanna Byrtusa, Maciej Pawłowskia,
Grzegorz Kazekb, Andrzej J Bojarskic, Agata Piaskowskad, Justyna Kalinowska-Tłuścikd,
Anna Partykae, Anna Wesołowskae
aDepartment of Pharmaceutical Chemistry, Jagiellonian University Medical College, 9
Medyczna Street, 30-688 Kraków, Poland
bDepartment of Pharmacodynamics, Jagiellonian University Medical College, 9 Medyczna
Street, 30-688 Kraków, Poland
cDepartment of Medicinal Chemistry, Institute of Pharmacology, Polish Academy of
Sciences, 12 Smętna Street, 31-343 Kraków, Poland
dFaculty of Chemistry, Jagiellonian University, 3 Ingardena Street, 30-060 Kraków, Poland
eDepartment of Clinical Pharmacy, Jagiellonian University Medical College, 9 Medyczna
Street, 30-688 Kraków, Poland
Corresponding author:
Tel. +48 12 620 54 50; Fax: +48 12 657 02 62; E–mail: anna.czopek@uj.edu.pl (Anna
Czopek) Department of Pharmaceutical Chemistry, Jagiellonian University Medical College,
9 Medyczna Street, 30-688 Kraków, Poland
1

Abstract
A series of novel spirohydantoin derivatives with arylpiperazinylbutyl moiety were
synthesized and evaluated for serotonin 5-HT1A, 5-HT2A, 5-HT7 and dopamine D2 receptors.
Based on these data, four compounds were selected for further binding affinity assays on
dopamine D1, D3, D4, and 5-HT2C, 5-HT6 as well as adrenergic α1 and α2C receptors, which are
involved in various CNS diseases such as schizophrenia, anxiety and/or depression. The
compound 14, 1-{4-[4-(2-metoxyphe-nyl)piperazin-1-yl]butyl}-3',4'-dihydro-2H,2'H,5H-
spiro[imidazolidine-4,1'-naphthalene]-2,5-dione, with the most promising functional profile,
mixed 5-HT2A/D2 antagonist and 5-HT1A partial agonist, was selected. In the mouse d-
amphetamine-induced locomotor hyperactivity model, compound 14 produced antipsychotic-
like activity, which is devoid of cataleptogenic effects and in the forced swim test in mice, it
showed a significant antidepressant-like effect unlike the reference drug aripiprazole.
Keywords: Antidepressant activity, Antipsychotic activity, Imidazolidine-2,4-dione
Spirohydantoines, D2/5-HT2A antagonist, 5-HT1A partial agonist
2

1. Introduction
Schizophrenia is considered one of the most serious mental illnesses, having a considerable
social and economic impact, and globally, it affects approximately 1% of the world
1
population . This psychotic illness is clinically described by the occurrence of positive
(hallucinations, disorganized thoughts, delusions, and irrational fears) and negative (social
withdrawal, diminished affect, poverty of speech, lack of energy, anhedonia) symptoms, as
well as cognitive impairments 2,3. The pathophysiology of schizophrenia is believed to be
associated with an imbalance of dopamine neurotransmission, manifested by mesolimbic
hyperactivity and mesocortical hypoactivity 1,4,5. Thus, introduction of drugs known as
classical antipsychotics, which antagonize dopamine D2 receptors, was the first advance in
schizophrenia treatment in the mid-twentieth century 1,6. These drugs are still used for
treatment of positive symptoms, but they have failed to manage the negative symptoms of
7
schizophrenia . The undesirable side effects of classical antipsychotics, such as tardive
dyskinesia and extrapyramidal symptoms, have led to development of atypical antipsychotic
agents, which are generally effective for both positive and negative symptoms of
schizophrenia 8.
A prototype of the entire class of atypical antipsychotic drugs was clozapine, which was
introduced in the 1990s as the next breakthrough in treatment of schizophrenia 9. The atypical
antipsychotic agents exhibit antagonism towards serotonin 5-HT2A and D2 receptors, and they
show less severe side effects than the classical ones. Furthermore, due to modulation of the
central serotonin neurotransmission, atypical antipsychotic drugs may also show anxiolytic
and/or antidepressant activity 8,10. Although clozapine and other atypical antipsychotic agents,
e.g. risperidone, olanzapine, and quetiapine have brought improvements in treatment of
negative symptoms of schizophrenia, they can still cause substantial weight gain, the
metabolic syndrome, blood dyscrasias, and some movement disorders 11,12. Moreover, they
have not fully improved the cognitive impairments and negative symptoms of schizophrenia
11. It seemed that a solution would be provided by introduction on the market of an atypical
drug, starting a new generation of antipsychotics – aripiprazole, which acts as a D2 and 5-
HT1A partial agonist and blocks 5-HT2A/5-HT7 receptors 13. Perhaps due to such unusual
mechanism of action among atypical antipsychotic agents, aripiprazole shows a favorable
safety and tolerability profile referring to the relatively low potential for causing
extrapyramidal symptoms, weight gain, QTc prolongation and sedation 14,15. Despite
improvement of the safety profile, aripiprazole may cause other potentially serious side
effects such as agitation, akathisia or insomnia 15. Therefore, development of more effective
3

antipsychotic compounds with less side effects remains a challenge and a goal for researchers
nowadays.
We previously reported on synthesis and biological evaluation of 5-(cyclo)alkyl-5-phenyl-
and 5-spiroimidazolidine-2,4-dione derivatives connected by three methylene chain with the
arylpiperazinylpropyl moiety. Among them, the most interesting compound I, (1-{3-[4-(2-
methoxyphenyl)piperazin-1-yl]propyl}-3’,4’-dihydro- 2’H-spiro[imidazolidine-4,1’-naphtha-
lene]-2,5-dione), behaved as a full, pre- and postsynaptic 5-HT1A receptor agonist and
displayed antidepressant- and anxiolytic-like activity in behavioral in vivo tests without any
unfavorable motor effects 16.
As a part of our ongoing studies on a group of 5-substituted imidazolidine-2,4-dione
derivatives of long-chain arylpiperazines (LCAPs), a new series of 4-arylpiperazinylalkyl
moiety with mono(bi)cyclic group spiro-connected to hydantoin ring was designed. The
structural modification in position 5 of the hydantoin core involved changing of the
cycloalkane ring size and position of the aromatic ring. To evaluate the influence of length of
spacer between hydantoin ring and arylpiperazine moiety on binding affinity, it was extended
from three to four methylene units, in comparison with the previously reported compounds
16,18. Herein we describe synthesis of new spirohydantoin derivatives and their biological
evaluation for 5-HT1A/2A/7 and D2 receptors. For the promising compounds, we present an
extended pharmacological in vitro profile, and for the most attractive one 14 − the in vivo
activity in common animal models of schizophrenia and affective disorders, i.e. depression
and anxiety. In these studies, aripiprazole was used as a reference drug. Additionally, based
on molecular modeling results, interactions between compound 14 and 5-HT1A and D2
receptors are discussed.
2. Chemistry and crystallographic study
New spirohydantoin derivatives 9 – 23 were prepared according to the synthetic route shown
in Scheme 1. Imidazolidine-2,4-diones (1-4) were prepared from the corresponding ketones
by means of the Bucherer-Berg reaction with modifications described by Goodson et al.
16,17,18, and after purification good yields were achieved (70-86%). Alkylation of the above at
N3 position with 1-bromo-4-chlorobutane resulted in obtaining compounds 5 – 8 in
satisfactory yields (65-85%). The following coupling with differently substituted
phenylpiperazines gave the final compounds 9-23 in moderate yields (53-84%). All the final
products were obtained as racemic mixtures and for further pharmacological studies they were
transformed into water-soluble hydrochloride salts.
4

The structure of the final compounds 9-26 was established on the basis of the results of
elemental (C, H, N) and spectral (1H NMR, 13C NMR, 19F NMR) analyses. The detailed
spectral data for each compound is listed in the experimental section.
Additionally, the molecular structure of selected compound, 14, has been crystallographically
confirmed. Hydrochloride salt of compound 14 crystallises as a racemic mixture, with centre
of chirality localised at the atom C10 of the molecule (Figure 1). The interesting feature of the
presented crystal structure is the mutual orientation of both rings of the arylpiperazine moiety,
which is highly conserved in the crystal structures of the 2-OCH3 substituted arylpiperazine
derivatives. This preferential orientation is related to the intramolecular hydrogen bond,
formed between equatorial hydrogen atom of piperazine and the oxygen atom of the 2-OCH3
group (C23-H23A...O31, Figure 1 and Supplementary materials). Single molecule of the
presented structure forms two strong hydrogen bonds N-H...Cl-, (from which one is charge-
assisted N19+-H19...Cl1-), creating a zig-zag chain C1 (10) 19 along [010] axis.
2
3. Pharmacology
3.1. In vitro evaluation
Radioligand binding assays were used for determining the affinity of all synthesized
compounds for native 5-HT1A/2A/7, and D2 receptors. This was accomplished by displacement
of [3H]-8-OH-DPAT from rat hippocampus for 5-HT1A receptors, [3H]-ketanserin from rat
cortex for 5-HT2A receptors, metylsergide from rat hypothalamus for 5-HT7 receptors, and
[3H]-spiperone from rat striatum for D2 receptors. Each compound was tested in triplicate at
7-8 concentrations (10-11-10-4) and inhibition constants (Ki, Table 1) were calculated.
Subsequently, the extended screening for receptor binding profile of compounds 10, 11, 14,
15 was determined at Cerep (Le Bois l’Eveque, 86600 Celle L’Evescault, France) according
to the previously published methods (Table 2) 20-22. Briefly, screening procedure was
accomplished by displacement of [3H]-8-OH-DPAT, [3H]-ketanserin, [3H]-mesulergine and
[3H]-pyrilamine from cloned human receptor stably expressed in HEK-293 cells for 5-HT1A,
5-HT2A, 5-HT2C and H1 receptors, respectively. Displacement of [3H]-SCH23 390, [3H]-
prazosine, [3H]-RX 821 002 and [3H]-4-DAMP evaluated in CHO cells which stably
expressed the human D1, α1, α2C and M3 receptors. In case of human 5-HT6/7 and D2/3/4
receptors stably expressed in CHO cells, [3H]-LSD and [3H]-metyl-spiperone were used,
respectively 20-22
.
3.2. Functional in vitro and in vivo evaluation
The in vitro functional activity of two selected compounds 10 and 14 on intracellular
cAMP/IP1/Ca2+ release levels, studied in CHO or HEK-293 cells which stably expressed the
5

human 5-HT1A/2A/2C/6/7, α1/2C, H1 and D1/2/3/4 receptors, was determined at Cerep (Le Bois
l’Eveque, 86600 Celle L’Evescault, France) according to the previously published methods
(Table 3) 20-23. Comparing affinity and in vitro obtained functional results, compound 14 has
been subjected to further functional in vivo studies on selected receptors, i.e. D2, 5-HT2A and
5-HT1A. To determinate D2 receptor antagonistic effect, its ability to abolish the apomorphine-
induced climbing in mice (Table 4) was tested. Antagonistic 5HT2A properties were assessed
in the ( )DOI-induced head twitch responses in mice (Table 4) 24. To examine pre- and
postsynaptic 5HT1A receptor activity, the ability to induce hypothermia in mice and lower lip
retraction (LLR) in rats, respectively, or to inhibit 8-OH-DPAT-induced effects was tested
(Table 4) 24.
3.3. Behavioral evaluation
In the successive phase of our investigations the derivative 14 was tested in vivo in behavioral
tests commonly used to predict antipsychotic-, antidepressant- and anxiolytic-like activity. To
evaluate potential antipsychotic activity in mice and cataleptogenic properties, the
hyperactivity induced by d-amphetamine and the bar test, respectively, were employed.
25
Antidepressant properties were established using the forced swim test (FST) in mice
.
Anxiolytic-like action was investigated in the four-plate test in mice 26. The influence of the
effective doses recorded in above-mentioned models were studied in spontaneous locomotor
activity in mice in order to exclude the possibility of competing behaviors such as general
locomotor activity and to determine specificity of the observed effects. Aripiprazole was used
as a reference antipsychotic drug in behavioral experiments.
4. Results and discussion
4.1. Structure-activity relationship studies and pharmacological in vitro evaluation
The series of spirohydantoin derivatives connected by a tetramethylene spacer with
arylpiperazine moiety was evaluated in radioligand binding assays to measure their affinity
for 5-HT1A, 5-HT2A, 5-HT7, and D2 receptors. The majority of compounds display high
affinity (Ki < 100 nM) for 5-HT1A and 5-HT2A receptors and diversified affinity for 5-HT7 and
D2 receptors, ranging from 27 nM to 1000 nM (Table 1). In general, in comparison with the
previously reported derivatives 16, elongation of the space length from three to four methylene
units between the spirohydantoin fragment and the arylpiperazine moiety significantly
increased the affinity for 5-HT1A, 5-HT7 and D2 receptors. At the same time, the affinity of
novel compounds for 5-HT2A sites was also slightly improved.
It seems that both the imide/amine fragments and the type of substituent in phenylpiperazine
system have a varying impact on the affinity of synthesized compounds for the receptors
6

examined. Analogues containing the indan or tetralin moiety connected to the hydantoin ring
by “spiro carbon” (9-16), were more active at 5-HT1A/2A/7 receptor sites than compounds 17-
23 with 2/4-phenyl-cyclohexane fragment. Interestingly, the combination of amide core with
2-phenyl-cyclohexane group and 2-OCH3- or 3-Cl-phenylpiperazine (22, 23) resulted in
stronger binding affinity for the 5-HT7 receptors and weaker for the 5-HT2A ones.
Introduction of 3-Cl into the phenylpiperazine moiety increased the affinity for both 5-HT1A
and 5-HT2A receptors (11, 15, 19, 23), while 2-OCH3 (10, 14, 18, 22) or 3-CF3 (12, 16, 20)
group favored only binding to 5-HT1A sites. Among the tested compounds, derivatives 11 and
15 showed the most balanced in vitro profile for serotonin 5-HT1A/2A/7 receptors. It was also
found that 2-methoxyphenylpiperazine moiety increased affinity for D2 receptors (10, 14, 18).
Among the structural counterparts, the unsubstituted phenylpiperazines were less active for
both 5-HT1A and 5-HT2A receptors, while the compounds containing 3-CF3-phenylpiperazine
moiety (12, 16, 20) showed lower affinity only for the 5-HT2A ones. The obtained affinity
results indicated that compounds 10, 14 and 18 were the most selective for 5-HT1A receptors,
whereas compounds 11 and 14 exhibited sustainable affinity for serotonin 5-HT1A/2A/7 and
dopamine D2 receptors.
Based on similarity in structure and in vitro results, four promising compounds (10, 11, 14,
15) were selected for the extended in vitro screening evaluation. The two compounds (10, 14)
possessed required high affinity for serotonin and dopamine receptors necessary for
antipsychotic action, and the other two (11, 15), their counterparts, showed promising affinity
for antidepressant activity. The above compounds were tested in an independent laboratory of
Cerep (France) (Table 2), and the obtained results confirm their strong binding affinities for
serotonin 5-HT1A/7 receptors, as well as significant affinity for dopamine D3/4, adrenergic α1/2C
and histaminic H1 receptors. Moreover, the tested compounds (10, 11, 14, 15) exhibited
moderate-to-high affinity for D2/1 and 5-HT2A receptors. Such multi-receptor profile may
indicate the need for further research in the direction of potential antipsychotic or
antidepressant effect. Moreover, the lowest percent of displacement for 5-HT2C receptors,
presented by compounds 10 and 14, may also be beneficial in this aspect due to a decreased
tendency for inducing weight gain 11. Thus both of the selected compounds 10 and 14 were
tested in vitro for intrinsic activity at a set of monoaminergic receptors 5-HT1A, 5-HT2A, 5-
HT7, D2-D4, α1, α2C and H1 (Table 3). The in vitro findings display a similar intrinsic activity
of both of the compounds studied, i.e. antagonist one at 5-HT1A, D2/4 and α1 receptors with
poorly marked agonist activity toward 5-HT1A and D2 sites. Moreover, despite significant
binding to 5-HT2A and 5-HT7 receptors, the antagonistic activity for these sites measured in
7

vitro was much weaker than it could be expected. A molecular characterization of new ligands
should be based on several observations made in different expression systems and using
different read-outs 27. In vitro model systems are very useful to differentiate between
functional properties of compounds, but some caution should be taken when extrapolating
results from recombinant systems to in vivo integrated ones. This fact is particularly
important for compounds with even low partial agonist activity, where detection could be
dependent on the method employed 28,29. Hence compound 14 has been tested in in vivo
functional models for assessing/confirming its inner activity toward 5-HT1A, 5-HT2A and D2
receptors.
4.2. Functional in vivo evaluation
Compound 14 (5-10 mg/kg) given alone evoked a decrease in body temperature in mice and
its effect was significantly attenuated by co-administration with the selective 5-HT1A receptor
antagonist, WAY-100635. However, it did not induce LLR per se but, at the same time,
significantly decreased the measured parameter produced by a 5-HT1A receptor agonist, 8-
OH-DPAT (Table 4). The above results allow us to classify compound 14 as a partial agonist
of 5-HT1A sites. Compound 14 did not reveal agonistic properties toward 5-HT2A and D2
receptors, lacking ability to induce head-twitch and climbing responses, respectively.
However, the tested agent (10-40 mg/kg) dose-dependently inhibited the ( )DOI-induced
head twitches in mice obtaining ID50=19.65 (18.6-20.7) mg/kg together with reduction of the
apomorphine-induced climbing behavior of mice after administration in doses of 1.25-60
mg/kg; ID50 dose for compound 14 was 37.93 (14.2-61.65) mg/kg (Table 4). Summarizing,
the results of our in vitro and in vivo functional study show that 14 behaves as a partial
agonist of the 5-HT1A receptor and displays D2/5-HT2A receptor antagonist activity.
4.3. Behavioral evaluation
There is a large body of evidence that agonist/antagonist of serotonin receptors produce
behavioral activity relevant to depression and anxiety 30,31. Moreover, several atypical
antipsychotic drugs with affinity for serotonin receptors (i.e. risperidone, olanzapine,
quetiapine, aripiprazole) have been used alone in clinical treatment of bipolar depression or in
combination with selective serotonin re-uptake inhibitors to augment their clinical efficacy
10,32,33
.
Considering the premises described above, as well as in vitro and in vivo data obtained for 14,
we undertook to examine its potential antipsychotic, antidepressant and anxiolytic activity in
behavioral models commonly used in mice, i.e. d-amphetamine-induced hyperactivity, the
forced swim test (FST) and the four-plate test, respectively. The tested compound, at doses of
8

0.156-0.625 mg/kg, significantly decreased hyperactivity induced by d-amphetamine. By
comparison, aripiprazole was active in doses of 0.125-0.5 mg/kg, significantly and dose-
dependently attenuating the effect studied (Figure 2). These effects of both agents seem to be
specific, since minimum sedative doses for 14 and aripiprazole, investigated in the
spontaneous locomotor activity model in mice, were 2.5 mg/kg and 1 mg/kg, respectively
(Table 5). Our present results show that 14 (2.5 and 5 mg/kg) revealed antidepressant-like
properties, shortening significantly by 37% and 45%, respectively, the immobility time of
mice, while aripiprazole (0.01-0.1 mg/kg) was inactive in that test (Figure 3). Effective
antidepressant doses of the investigated agent did not stimulate the spontaneous locomotor
activity in mice during the 2-6-min session (i.e. at the time identical to the observation period
in the FST) (data not shown). The results obtained in the four-plate test indicate that neither
14 (0.312-5 mg/kg) nor aripiprazole (0.06-0.5 mg/kg) produced antianxiety-like effects in
mice (Table 6).
As enormous efforts have been made to develop novel antipsychotics that would not produce
extrapyramidal side effects (EPS), we also examined the cataleptogenic potential of 14 (3-30
mg/kg) in the bar test in mice, which is viewed as an important rodent model for predicting
EPS liability in humans 34. The minimum cataleptogenic dose of the investigated agent was 30
mg/kg; that is 50-200-fold higher than its effective antipsychotic doses and 30-fold higher
than minimal cataleptogenic dose of aripiprazole (1 mg/kg) (Table 7).
The results of pharmacological studies with 14 targeting D2 and 5-HT1A/5-HT2A receptors
show that the investigated agent, as well as aripiprazole, displayed antipsychotic-like activity
in d-amphetamine-induced locomotor hyperactivity test, which is believed to reflect the
potential efficacy of drugs in treating psychotic symptoms. Additionally, 14 produced
catalepsy in a much higher dose than aripiprazole. Furthermore, the compound examined, but
not aripiprazole, was effective in a mouse model of depression, i.e. FST. Similarly to our
study, the lack of aripiprazole’s antidepressant-like activity in FST was reported by Bourin et
36
al. 35. In contrast, Sarkisyan et al.
showed that aripiprazole was active in that test,
emphasizing its 5-HT7 receptor antagonistic properties as the potential mechanism of
antidepressant efficacy. These discrepancies may be caused by different strains of mice used
by both teams, i.e. Swiss albino at home and C57BL/6J by Sarkisyan et al. The results
obtained for derivative 14 are promising; however, they need to be corroborated by further
pharmacological studies in order to determine the detailed mechanism of its potential
antidepressant activity, as well as its activity in other species.
4.4. The molecular modeling study
9

The ligand binding mode of compound 14 in the most important biological targets was
determined using molecular docking studies. To this end, the previously described homology
models of 5-HT1A and D2 receptors were used 37,38,39,40. The compound was flexibly docked to
the binding sites of the model assemblies of both receptors, mimicking the conformational
flexibility of the proteins. The best scored complexes were analyzed to capture the proper
binding modes, characterized by favorable ligand-receptor interactions. Since the tested
compound was synthesized in a racemic form, both the enantiomers were considered in
computational studies. Due to better scores and a higher number of favorable interactions in
both receptors, the S enantiomer was regarded preferable and its binding mode was further
described.
The ligand binding mode in the both receptors proved to be consistent with the common one
for monoaminergic receptor ligands, which stands for reliability of the method 41,42. The main
anchoring interaction was a charge reinforced hydrogen bond between protonated nitrogen
atom of the ligand and carboxylic group of Asp3.32, together with the CH-π interactions of
arylpiperazine and aromatic aminoacid cluster, mainly Phe6.52 (Figure 4). The geometry of
14 molecule in both receptors was linear, extending from the deeper cavity formed by
transmembrane helices (TMHs) 3-6, to the second interaction area, located between TMHs 1,
2 and 7. The latter one is occupied by the spirohydantoin fragment, which finds both the
favorable hydrophobic, and polar contacts there. Besides the common interactions described
above, there are also additional contacts present, specific for each receptor type. In the case of
the 5-HT1A receptor, the o-methoxy group substituted at phenylpiperazine fragment interacts
with Lys191 from the second extracellular loop (ECL), carbonyl oxygen of hydantoin forms
an h-bond with the amide NH2 group of Asn7.39, while the aromatic ring of tetraline interacts
with the phenyl ring of Tyr2.64 (π-π stacking, Figure 4A). Indeed, in the D2 receptor, the
latter fragment forms a CH-π interaction with Tyr1.35, but the complex is lacking additional
favorable interactions of h-bond nature, which may contribute to a relatively lower affinity of
14 for this receptor type (Figure 4B).
5. Conclusions
Summing up, a number of spirohydantoin derivatives with differently substituted 4-
arylpiperazinylalkyl moiety have been developed. The applied modification, such as the
diversified bicyclic group spiro-connected to the hydantoin ring and the nature of substituent
in 4-arylpiperazine moiety, as well as elongation of the alkyl spacer between them, influenced
the affinity for 5-HT1A, 5-HT2A, 5-HT7, and D2 receptors. Among the derivatives developed,
the most promising compound 14 acts as a 5-HT1A receptor partial agonist, and a D2/5-HT2A
10

receptor antagonist. This multireceptor profile revealed the unique activity of compound 14 in
behavioural in vivo tests. It showed specific antipsychotic-like activity in an animal model of
schizophrenia without producing side effects, i.e. catalepsy and sedation in active doses.
Furthermore, compound 14, unlike the antipsychotic reference drug, behaved as a potential
antidepressant in a mouse model of depression. It should be underlined that in spirohydantoin
derivative, such combination of pharmacological activity - antipsychotic and antidepressant
effect - is unique and could be beneficial in treatment of schizophrenia.
6. Experimental Part
6.1. Chemistry
All chemicals and solvents were purchased from commercial suppliers (Aldrich and
Chempur) and were used without further purification. Melting points were determined in open
capillaries on an Electrothermal 9300 apparatus and were uncorrected.
Thin-layer chromatography (TLC) was performed on Merck silica gel 60 F254 aluminium
sheets (Merck; Darmstadt, Germany), using the following mixtures of solvents: (S1)
benzene/ethyl acetate/acetone (10:5:1), (S2) acetone/isopropanol/chloroform (20:10:1), (S3)
methanol/ammonium hydroxide (10:3drops), (S4) ethyl acetate/methanol (8:2), (S5)
chloroform/methanol (9:1).
Analytical HPLC were conducted on a Waters HPLC instrument with Waters 485 Tunable
Absorbance Detector UV, equipped with a Symetry column (C18, 3.5 µm, 4.6 x 30 mm)
using water/acetonitrile gradient with 0.1% TFA as mobile phase at a flow rate of 5 ml/min.
The purity of the investigated compounds (9-26) ranged from 96 to 99%. Additionally, the
liquid chromatography/mass spectrometry (LC/MS) analysis was performed on Waters
Acquity TQD system, with a Waters TQD quadrupole mass spectrometer with detection by
UV (DAD) using an Acquity UPLC BEH C18 column (1.7µm, 2.1mm x 100mm).
Water/acetonitrile gradient with 0.1% TFA was used as a mobile phase at a flow rate of 0.3
ml/min.
NMR spectra were recorded on Varian Mercury 300 MHz spectrometer (Varian Inc., Palo
Alto, CA, USA); chemical shifts are expressed in parts per million (ppm), using the solvent
(CDCl3 or DMSO-d6) signal as an internal standard. Signal multiplets are represented by the
following abbreviations: s (singlet), brs (broad singlet), d (doublet), t (triplet), m (multiplet).
Elemental analyses for C, H, N were carried on an Elementar Vario EL III apparatus (Hanau,
Germany). The results of elemental analyses were within 0.4% of the theoretical values.
6.1.1. General procedure for the synthesis of compounds 1-4
11

A solution of a suitable ketone (0.33 M) and ammonium carbonate (1.00 M) in 50% ethanol
was warmed to 50°C, at this point potassium cyanide (0.35 M), previously dissolved in 50 ml
of water was dropped in over a period of 15 min. The mixture was refluxed at
56-60°C for more than 20 h. After that time a water condenser was then replaced by an air
condenser and the temperature was raised to 80°C for 1h to remove the excess of ammonium
carbonate. Then the reaction solution was cooled to room temperature and acidified to pH of 6
with 16% sulphuric acid. The precipitated solids were filtered off, washed with water, and
recrystallized from a mixture of ethanol and water, giving an appropriate hydantoin.
Spectroscopic and analytical data matched those reported in the literature for 2’,3’-Dihydro-
2H,5H-spiro[imidazolidine-4,1’-indene]-2,5-dione (1) and 3’,4’-Dihydro-2H,2’H,5H-
spiro[imidazolidine-4,1’-naphthalene]-2,5-dione (2), see reference 16,17
.
6.1.1.2. 8-Phenyl-1,3-diazaspiro[4.5]decan-2,4-dione (3)
The free base was obtained in 70% yield as white powder; mp 287-288°C; TLC: Rf = 0.29
(S1); Anal. calcd for C14H16N2O2: C 68.83, H 6.60, N 11.47. found: C 68.90, H 6.90, N 11.57.
6.1.1.3. 6-Phenyl-1,3-diazaspiro[4.5]decan-2,4-dione (4)
The free base was obtained in 86% yield as white powder; mp 257-260°C; TLC: Rf = 0.26
(S1); Anal. calcd for C14H16N2O2: C 68.83, H 6.60, N 11.47; found: C 68.64, H 6.52, N 11.73.
6.1.2. General procedure for the synthesis of compounds 5-8
A suitable imidazolidine-2,4-dione (1-4) (20 mmol) and anhydrous K2CO3 (150 mmol) were
refluxed in acetone (120 ml) under intensive stirring for 30 min. Then a solution of 1-bromo-
3-chloropropane (55 mmol) in acetone (30 ml) was added in drops over the period of 30 min.
The reaction was refluxed for 7-20 h. The inorganic salts were filtered off, the solvent was
evaporated and the oily residue was purified by crystallization from 60% or 96% ethanol.
6.1.2.1.
1-(4-Chlorobutyl)-2',3'-dihydro-2H,5H-spiro[imidazolidine-4,1'-indene]-2,5-
dione (5)
Obtained from 1, in 85% yield, as white powder; mp 97 – 99°C; TLC: Rf = 0.60 (S1), 0.68
(S4); Anal. calcd for C15H17N2O2Cl: C 61.54, H 5.58, N 9.57, found: C 61.36, H 5.96, N 9.64.
6.1.2.2.
1-(4-Chlorobutylo)-3',4'-dihydro-2H,2'H,5H-spiro[imidazolidine-4,1'-
naphthalene]-2,5-dione (6)
Obtained from 2, in 78% yield, as white powder; mp 105 – 107°C; TLC: Rf = 0.77 (S1), 0.71
(S4); Anal. calcd for C16H19N2O2Cl: C 62.64, H 6.24, N 9.13, found: C 62.54, H 6.37, N 8.99.
6.1.2.3. 3-(4-Chlorobutylo)-8-phenylo-1,3-diazaspiro[4,5]dekan-2,4-dione (7)
Obtained from 3, in 65% yield, as white powder; mp 204 – 207°C; TLC: Rf = 0.64 (S1), 0.74
(S4); Anal. calcd for C18H23N2O2Cl: C 64.57, H 6.92, N 8.37, found: C 64.47, H 6.96, N 8.34.
12

6.1.2.4. 3-(4-Chlorobutylo)-6-phenylo-1,3-diazaspiro[4,5]dekan-2,4-dion (8)
Obtained from 4, in 66% yield, as white powder; mp 195 – 197°C; TLC: Rf = 0.62 (S1), 0.80
(S4); Anal. calcd for C18H23N2O2Cl: C 64.57, H 6.92, N 8.37, found: C 64.43, H 7.18, N 8.25.
6.1.3. General procedure for the synthesis of final compounds 9-23
An intermediate 1-(3-chlorobutyl)-spirohydantoin (2.5 – 5 mmol) and the substituted
1-phenylpiperazine (5 – 10 mmol), potassium carbonate (7.5 – 15 mmol) and a bit of
potassium iodide in acetonitrile (9-20) or 2-methoxyethanol (21-23) were refluxed for 20 – 48
h separately. After cooling, the solvent was evaporated and the hydantoin derivatives were
separated by column chromatography using SiO2 and a mixture of EtOAc/MeOH = 9/1 or 8/2,
as eluting system. Then obtained products were recrystallized (96% ethanol) and converted
into the hydrochloride salts by passing of their solution in anhydrous ethanol with
hydrochloric gas.
6.1.3.1.
1-[4-(4-Phenylpiperazin-1-yl)butyl]-2',3'-dihydro-2H,5H-spiro[imidazolidine-
4,1'-indene]-2,5-dione (9)
White powdery crystals. Yield: 55%; mp 141 – 143°C; TLC: Rf = 0.64 (S2), 0.69 (S5); tR =
1
2.08; MS calcd for [M + H]+: C25H30N4O2 m/z: 418.53, found: 419.50; H NMR (CDCl3) δ
(ppm) 1.44 – 1.69 (m, 4H, CH2CH2CH2CH2), 2.13 – 2.23 (m, 1H, indane), 2.33 – 2.38 (t, J =
7 Hz, 2H, CH2N1pip(CH2)2), 2.50 – 2.54 (t, J = 5 Hz, 4H, N1pip(CH2)2), 2.55 – 2.63 (m, 1H,
indane), 2.93 – 3.02 (m, 1H, indane), 3.10 – 3.13 (m, 5H, indane, (CH2)2N4pip), 3.47 – 3.52
(t, J = 7 Hz, 2H, N3CH2), 6.75 – 6.87 (m, 4H, Ph, NH), 7.03 – 7.25 (m, 6H, Ph); Anal. calcd
for C25H30N4O2: C 71.74, H 7.22, N 13.39; found: C 71.73, H 7.15, N 13.28. Compd 9·HCl:
246 – 248°C; Anal. calcd for C25H30N4O2·H2O·HCl: C 63.48, H 7.03, N 11.84; found: C
63.68, H 7.04, N 11.89.
6.1.3.2.
1-{4-[4-(2-Methoxyphenyl)piperazin-1-yl]butyl}-2',3'-dihydro-2H,5H-
spiro[imidazolidine-4,1'-indene]-2,5-dione (10)
White powdery crystals. Yield: 64%; mp 132 – 134°C; TLC: Rf = 0.57 (S2); 0.86 (S5); tR =
1
2.07; H NMR (CDCl3) δ (ppm) 1.53 – 1.75 (m, 4H, CH2CH2CH2CH2), 2.18 – 2.28 (m, 1H,
indane), 2.41 – 2.46 (t, J = 7.45 Hz, 2H, CH2N1pip(CH2)2), 2.64 – 2.76 (m, 5H, indane,
N1pip(CH2)2), 2.99 – 3.09 (m, 5H, indane, (CH2)2N4pip), 3.20 – 3.47 (m, 1H, indane), 3.55 –
3.59 (t, J = 7.18 Hz, 2H, N3CH2), 3.85 (s, 3H, OCH3), 5.83 (s, 1H, NH), 6.84 – 7.02 (m, 4H,
Ph), 7.10 – 7.32 (m, 4H, Ph); 13C NMR (75 MHz, CDCl3) δ (ppm) 23.82, 26.16, 30.18, 36.98,
38.51, 50.51, 53.32, 55.31, 58.02, 71.02, 111.09, 118.20, 120.94, 122.61, 122.89, 125.46,
127.33, 129.54, 140.09, 141.25, 144.10, 152.21, 156.97, 175.58; Anal. calcdfor C26H32N4O3:
C 69.62, H 7.19, N 12.49; found: C 69.53, H 7.13, N 12.36. Compd 10·HCl: mp 235 – 236°C;
13

Anal. calcd for C26H32N4O3·2 HCl: C 59.88, H 6.57, N 10.74; found: C 59.85, H 6.92, N
10.70.
6.1.3.3.
1-{4-[4-(3-Chlorophenyl)piperazin-1-yl]butyl}-2',3'-dihydro-2H,5H-
spiro[imidazolidine-4,1'-indene]-2,5-dione (11)
White powdery crystals. Yield: 55%; mp 88 – 90°C; TLC: Rf = 0.70 (S2); 0.91 (S5); tR = 2.41;
1H NMR (CDCl3) δ (ppm) 1.52 – 1.75 (m, 4H, CH2CH2CH2CH2), 2.19 – 2.29 (m, 1H,
indane), 2.39 – 2.44 (t, J = 7.40 Hz, 2H, CH2N1pip(CH2)2), 2.54 – 2.57 (t, J = 5.00 Hz, 4H,
N1pip(CH2)2), 2.64 – 2.75 (m, 1H, indane), 3.00 – 3.09 (m, 1H, indane), 3.15 – 3.28 (m, 5H,
indane, (CH2)2N4pip), 3.55 – 3.60 (t, J = 7.00 Hz, 2H, N3CH2), 5.83 (s, 1H, NH), 6.75 – 6.86
(m, 3H, Ph), 7.12 – 7. 33 (m, 5H, Ph); Anal. calcd for C25H29N4O2Cl·H2O: C 63.75, H 6.63, N
11.90; found C 64.09, H 6.83, N 11.55. Compd 11·HCl: mp 191 – 193°C; Anal. calcd for
C29H25N4O2Cl·H2O·HCl: C 59.17, H 6.36, N 11.04; found: C 58.90, H 6.35, N 11.08.
6.1.3.4.
1-{4-[4-(3-Trifluoromethylphenyl)piperazin-1-yl]butyl}-2',3'-dihydro-2H,5H-
spiro[imidazolidine-4,1'-indene]-2,5-dione (12)
White powdery crystals. Yield: 60%; mp 103 – 105°C; TLC: Rf = 0.60 (S2); tR = 2.44; MS
calcd for [M + H]+: C26H29N4O2F3 m/z: 486.5, found: 487.5; 1H NMR (CDCl3) δ (ppm) 7,20 –
7,36 (m, 4H, Ph), 7,02 – 7,12 (m, 4H, Ph), 6,15 (s, 1H, NH), 3,54 – 3,58 (t, J = 7 Hz, 2H,
N3CH2), 3,20 – 3,23 (m, 5H, indane, (CH2)2N4pip), 2,99 – 3,10 (m, 1H, indane), 2,65 – 2,74
(m, 1H, indane), 2,57 – 2,60 (t, J = 5 Hz, 4H, N1pip(CH2)2), 2,41 – 2,46 (t, J = 7 Hz, 2H,
CH2N1pip(CH2)2), 2,18 – 2,36 (m, 1H, indane), 1,50 – 1,75 (m, 4H, CH2CH2CH2CH2); 19F
NMR (CDCl3) δ (ppm) -62.72 (s, 3F, CF3).
6.1.3.5.
1-[4-(4-Phenyopiperazin-1-yl)butyl]-3',4'-dihydro-2H,2'H,5H-
spiro[imidazolidine-4,1'-naphthalene]2,5-dione (13)
White powdery crystals. Yield: 59%; mp 147 – 149°C; TLC: Rf = 0.63 (S4); 0.32 (S5); tR =
1
2.15; MS calcd for [M + H]+: C26H32N4O2 m/z: 432.56, found: 433.6; H NMR (CDCl3) δ
(ppm) 1.54 – 1.64 (m, 2H, CH2CH2CH2CH2), 1.69 – 1.86 (m, 4H, tetralin, CH2CH2CH2CH2),
2.23 – 2.36 (m, 2H, tetralin), 2.43 – 2.48 (t, J = 7.20 Hz, 2H, CH2N1pip(CH2)2), 2.59 – 2.62
(t, J = 5.00 Hz, 4H, N1pip(CH2)2), 2.84 – 2.94 (m, 2H, tetralin), 3.18 – 3.22 (t, J = 5.00 Hz,
4H, (CH2)2N4pip), 3.58 – 3.63 (t, J = 7.20 Hz, 2H, N3CH2), 5.76 (s, 1H, NH), 6.82 – 6.94 (m,
3H, Ph), 7.03 – 7.29 (m, 6H, Ph); Anal. calcd for C26H32N4O2: C 72.19, H 7.46, N 12.95;
found: C 71.86, H 7.39, N 12.90. Compd 13·HCl: mp 252 – 253°C; Anal. calcd for
C26H32N4O2·HCl: C 66.58, H 7.09, N 11.95; found: C 66.63, H 7.05, N 12.01.
6.1.3.6.
1-{4-[4-(2-Metoxyphenyl)piperazin-1-yl]butyl}-3',4'-dihydro-2H,2'H,5H-
spiro[imidazoli-dine-4,1'-naphthalene]-2,5-dione (14)
14

White powdery crystals. Yield: 53%; mp 108 – 109°C; TLC: Rf = 0.56 (S3); 0.41 (S5); tR =
1
2.17; MS calcd for [M + H]+: C27H34N4O3 m/z: 462.27, found: 463.22; H NMR (CDCl3) δ
(ppm) 1.53 – 1.67 (m, 2H, CH2CH2CH2CH2), 1.72 – 1.88 (m, 4H, tetralin, CH2CH2CH2CH2),
2.22 – 2.30 (m, 2H, tetralin), 2.43 – 2.47 (t, J = 7.05 Hz, 2H, CH2N1pip(CH2)2), 2.62 (br s,
4H, N1pip(CH2)2), 2.83 – 2.95 (m, 2H, tetralin), 3.07 (br s, 4H, (CH2)2N4pip), 3.54 – 3.62 (t,
J = 7.18 Hz, 2H, N3CH2), 3.85 (s, 3H, OCH3), 5.74 (s, 1H, NH), 6.80 – 6.96 (m, 5H, Ph),
7.05 – 7.27 (m, 3H, Ph); 13C NMR (75 MHz, CDCl3) δ (ppm) 19.03, 23.65, 26.16, 28.81,
34.12, 38.48, 50.33, 53.25, 55.31, 57.96, 62.65, 111.09, 118.23, 120.95, 122.96, 126.42,
126.84, 128.58, 129.75, 133.13, 138.11, 141.13, 152.19, 156.91, 176.30; C27H34N4O3·H2O: C
67.48, H 7.55, N 11.66; found: C 67.71, H 7.77, N 11.53. Compd 14·HCl: mp 241 – 244°C.
Anal. calcd for C27H34N4O3·2 HCl C 60.56, H – 6.78, N – 10.46; found: C 60.26, H 7.03, N
10.27.
6.1.3.7.
1-{4-[4-(3-Chlorophenyl)piperazin-1-yl]butyl}-3',4'-dihydro-2H,2'H,5H-
spiro[imidazolidine-4,1'-naphthalene]-2,5-dione (15)
White powdery crystals. Yield: 68%; mp 88 – 91°C; TLC: Rf = 0.65 (S2); tR = 2.39; MS calcd
1
for [M + H]+: C26H31N4O2Cl m/z: 466.21, found: 467.21; H NMR (CDCl3) δ (ppm) 1.54 –
1.66 (m, 2H, CH2CH2CH2CH2), 1.68 – 1.84 (m, 2H, tetralin), 1.92 – 2.04 (m, 2H,
CH2CH2CH2CH2), 2.24 – 2.32 (m, 2H, tetralin), 2.46 (br. s., 2H, CH2N1pip(CH2)2), 2.60
(br.s., 4H, N1pip(CH2)2), 2.82 – 2.87 (m, 2H, tetralin), 3.21 (br. s., 4H, (CH2)2N4pip), 3.55 –
3.61 (t, J = 7.05 Hz, 2H, N3CH2), 5.78 (s, 1H, NH), 6.72 – 6.90 (m, 3H, Ph), 7.02 – 7.26 (m,
5H, Ph). Anal. calcd for C26H31N4O2Cl: C 66.87, H 6.69, N 12.00; found: C 66.48, H 7.00, N
– 11.92. Compd 15·HCl: mp 207 – 209°C; Anal. calcd for C26H31N4O2Cl·H2O·HCl: C 59.88,
H 6.57, N 10.74; found: C 59.49, H 6.62, N 10.53.
6.1.3.8. 1-{4-[4-(3-Trifluoromethylphenyl)piperazin-1-yl]butyl}-3',4'-dihydro-2H,2'H,5H-
spiro[imidazolidine-4,1'-naphthalene]-2,5-dione (16)
White powdery crystals. Yield: 63%; mp 180 – 183°C; TLC: Rf = 0.68 (S2); 0.64 (S4); 0.60
(S3); tR = 2.52; MS calcd for [M + H]+: C27H31N4O2F3 m/z: 500.56, found: 501.50; 1H NMR
(CDCl3) δ (ppm) 1.59 – 1.66 (m, 2H, CH2CH2CH2CH2), 1.70 – 1.87 (m, 4H, tetralin,
CH2CH2CH2CH2), 2.23 – 2.33 (m, 2H, tetralin), 2.48 – 2.52 (t, J = 7.20 Hz, 2H,
CH2N1pip(CH2)2), 2.62 – 2.65 (t, J = 5.00 Hz, 4H, N1pip(CH2)2), 2.84 – 2.91 (m, 2H,
tetralin), 3.24 – 3.27 (t, J = 5.00 Hz, 4H, (CH2)2N4pip), 3.59 – 3.64 (t, J = 7.10 Hz, 2H,
N3CH2), 5.88 (s, 1H, NH), 7.01 – 7.36 (m, 8H, Ph); 19F NMR (CDCl3) δ (ppm) -61.63 (s, 3F,
CF3).
6.1.3.9. 3-[4-(4-Phenylpiperazin-1-yl)butyl]-8-phenyl-1,3-diazaspiro[4,5]dekan-2,4-dione
15

(17)
White powdery crystals. Yield: 84%; mp 238 – 240°C; TLC: Rf = 0.66 (S2); 0.68 (S5); tR =
1
2.45; MS calcd for [M + H]+: C28H36N4O2 m/z: 460.61, found: 461.60; H NMR (CDCl3) δ
(ppm) 1.53 – 1.80 (m, 8H, cyclohexane, CH2CH2CH2CH2), 1.97 – 2.12 (m, 4H, cyclohexane),
2.38 – 2.43 (t, J = 7.20 Hz, 2H, CH2N1pip(CH2)2), 2.52 – 2.55 (t, J = 5.00 Hz, 4H,
N1pip(CH2)2), 2.61 – 2.69 (m, 1H, cyclohexane), 3.14 – 3.17 (t, J = 5.00 Hz, 4H,
(CH2)2N4pip), 3.58 –3.62 (t, J = 7.00 Hz, 2H, N3CH2), 6.82 – 6.92 (m, 3H, Ph), 7.23 – 7.34
(m, 7H, Ph), 7.80 (s, 1H, NH); Anal. calcd for C28H36N4O2·H2O: C 70.26, H 8.00, N 11.71;
found: C 70.30, H 8.06, N – 11.70. Compd 17·HCl: mp 293 – 294°C; Anal. calcd for
C28H36N4O2·H2O·HCl: C 65.29, H 7.63, N 10.88; found: C 65.60, H 7.58, N 10.9.
6.1.3.10.
3-{4-[4-(2-Metoxyphenyl)piperazin-1-yl]butyl}-8-phenyl-1,3-
diazaspiro[4,5]dekan-2,4-dione (18)
1
White powdery crystals. Yield: 72%; mp 209 – 211°C; TLC: Rf = 0.69 (S2); tR = 2.47; H
NMR (CDCl3) δ (ppm) 1.51 – 1.80 (m, 8H, cyclohexane, CH2CH2CH2CH2), 1.97 – 2.12 (m,
4H, cyclohexane), 2.39 – 2.44 (t, J = 7.10 Hz, 2H, CH2N1pip(CH2)2), 2.58 – 2.69 (m, 5H,
cyclohexane, N1pip(CH2)2), 3.05 (br s, 4H, (CH2)2N4pip), 3.58 – 3.62 (t, J = 7.00 Hz, 2H,
N3CH2), 3.85 (s, 3H, OCH3), 6.84 – 7.02 (m, 3H, Ph), 7.20 – 7.33 (m, 6H, Ph), 7.84 (s, 1H,
NH); Anal. calcd for C29H38N4O3: C 70.99, H 7.81, N 11.42; found: C 70.73, H 7.59, N –
11.21. Compd 18·HCl: mp 273 – 274°C; Anal. calcd for C29H38N4O3·H2O·HCl: C 63.90, H
7.58, N 10.28; found: C 63.82, H 7.61, N – 10.17.
6.1.3.11.
3-{4-[4-(3-Chlorophenyl)piperazin-1-yl]butyl}-8-phenyl-1,3-
diazaspiro[4,5]dekan-2,4-dione (19)
White powdery crystals. Yield: 63%; mp 227 – 228°C; TLC: Rf = 0.73 (S4); 0.42 (S5); tR =
2.65; MS calcd for [M + H]+: C28H35N4O2Cl m/z: 494.24, found: 495.19; 1H NMR (CDCl3) δ
(ppm) 1.63 –1.81 (m, 8H, cyclohexane, CH2CH2CH2CH2), 1.97 – 2.12 (m, 4H, cyclohexane),
2.43 (br. s., 2H, CH2N1pip(CH2)2), 2.50 – 2.71 (m, 5H, cyclohexane, N1pip(CH2)2), 3.26 (br
s, 4H, (CH2)2N4pip), 3.57 – 3.62 (t, J = 6,80 Hz, 2H, N3CH2), 6.73 – 6.86 (m, 3H, Ph), 7.13 –
7.33 (m, 6H, Ph), 7.66 (s, 1H, NH); Anal. calcd for C28H35N4O2Cl: C 67.93, H 7.13, N 11.32;
found: C 67.69, H 7.29, N – 11.24. Compd 19·HCl: mp 283 – 285°C; Anal. calcd for
C28H35N4O2Cl·HCl: C 63.27, H 6.83, N – 10.54; found: C 62.97, H 6.84, N – 10.53.
6.1.3.12.
3-{4-[4-(3-Trifluoromethylphenyl)piperazin-1-yl]butyl}-8-phenyl-1,3-
diazaspiro[4,5]dekan-2,4-dione (20)
White powdery crystals. Yield: 81%; mp 217 – 219°C; TLC: Rf = 0.65 (S2); 0.74 (S5); tR =
2.72; MS calcd for [M + H]+: C29H35N4O2F3 m/z: 528.61, found: 529.50; 1H NMR (CDCl3) δ
16

(ppm) 1.59 – 1.80 (m, 8H, cyclohexane, CH2CH2CH2CH2) 1.96 – 2.12 (m, 4H, cyclohexane),
2.36 – 2.40 (m, 2H, CH2N1pip(CH2)2), 2.44 – 2.70 (m, 5H, cyclohexane, N1pip(CH2)2), 3.23
(br s, 4H, (CH2)2N4pip), 3.58 –3.63 (t, J = 6.80 Hz, 2H, N3CH2), 7.01 – 7.08 (m, 3H, Ph),
7.35 – 7.20 (m, 6H, Ph), 7.63 (s, 1H, NH); 19F NMR (CDCl3) δ (ppm) -62,76 (s, 3F, CF3).
6.1.3.13.
3-[4-(4-Phenylpiperazin-1-yl)butyl]-6-phenyl-1,3-diazaspiro[4,5]dekan-2,4-
dione (21)
White powdery crystals. Yield: 60%; mp 185 – 187°C; TLC: Rf = 0.65 (S2); 0.57 (S4); tR =
1
2.29; MS calcd for [M + H]+: C28H36N4O2 m/z: 460.61, found: 461.60; H NMR (CDCl3) δ
(ppm) 1.05 – 1.20 (m, 4H, CH2CH2CH2CH2), 1.41 – 1.56 (m, 3H, cyclohexane), 1.66 – 1.91
(m, 5H, cyclohexane), 2.19 – 2.24 (m, 2H, CH2N1pip(CH2)2), 2.56 (br. s., 4H, N1pip(CH2)2),
3.06 – 3.09 (m, 1H, cyclohexane), 3.15 – 3.32 (m, 6H, (CH2)2N4pip, N3CH2), 6.81 – 6.95 (m,
3H, Ph), 7.16 – 7.28 (m, 8H, Ph, NH); Anal. calcd for C28H36N4O2: C 73.01, H 7.88, N 12.16;
found: C 73.10, H 7.87, N – 11.92. Compd 21·HCl: mp 208 – 210°C; Anal. calcd for
C28H36N4O2·2 HCl: C 63.03, H 7.18, N 10.50; found: C 63.18, H 7.53, N 10.53.
6.1.3.14.
3-{4-[4-(2-Metoxyphenyl)piperazin-1-yl]butyl}-6-phenyl-1,3-
diazaspiro[4,5]dekan-2,4-dione (22)
White powdery crystals. Yield: 57%; mp 173 – 174°C; TLC: Rf = 0.59 (S2); 0.57 (S5); tR =
1
2.27; H NMR (CDCl3) δ (ppm) 1.07 – 1.22 (m, 4H, CH2CH2CH2CH2), 1.43 – 1.60 (m, 3H,
cyclohexane), 1.75 – 1.94 (m, 5H, cyclohexane), 2.21 – 2.26 (t, J = 7.20 Hz, 2H,
CH2N1pip(CH2)2), 2.57 (br s, 4H, N1pip(CH2)2), 3.07 (br s, 4H, (CH2)2N4pip), 3.13 – 3.20
(m, 1H, cyclohexane), 3.27 – 3.31 (t, J = 6.90 Hz, 2H, N3CH2), 3.85 (s, 3H, OCH3), 6.83 –
7.01 (m, 3H, Ph), 7.16 – 7.25 (m, 7H, Ph, NH); Anal. calcd for C29H38N4O3: C 70.99, H 7.81,
N 11.42; found: C 70.63, H 8.05, N – 11.26. Compd 22·HCl: mp 218 – 220°C; Anal. calcd for
C29H38N4O3·2 HCl: C 61.81, H 7.15, N 9.94; found: C 61.72, H 6.92, N – 9.73.
6.1.3.15.
3-{4-[4-(3-Chlorophenyl)piperazin-1-yl]butyl}-6-phenyl-1,3-
diazaspiro[4,5]dekan-2,4-dione (23)
White powdery crystals. Yield:65%; mp 108 – 109°C; TLC: Rf = 0.61 (S4); 0.76 (S5); tR =
1
2.49; H NMR (CDCl3) δ (ppm) 1.03 – 1.18 (m, 4H, CH2CH2CH2CH2), 1.42 – 1.58 (m, 3H,
cyclohexane), 1.72 – 1.95 (m, 5H, cyclohexane), 2.18 – 2.23 (t, J = 6.90 Hz, 2H,
CH2N1pip(CH2)2), 2.48 – 2.51 (t, J = 4.90 Hz, 4H, N1pip(CH2)2), 3.07 – 3.09 (m, 1H,
cyclohexane), 3.14 – 3.17 (t, J = 4.90 Hz, 4H, (CH2)2N4pip), 3.22 – 3.30 (t, J = 6.80 Hz, 2H,
N3CH2), 6.74 – 6.85 (m, 3H, Ph), 7.12 – 7.24 (m, 7H, Ph, NH); Anal. calcd for
C28H35N4O2Cl: C 67.93, H 7.13, N 11.32; found: C 67.87, H 7.23, N 11.22. Compd 23·HCl:
mp 215 – 218°C; Anal. calcd for C28H35N4O2Cl·2 HCl: C 59.21, H 6.57, N 9.86; found: C
17

59.41, H 6.65, N 9.82.
6.2. X-ray structure determination and refinement
Hydrochloride salt of compound 14 crystallises as a racemic mixture, with centre of chirality
localised at the atom C10 of the molecule. A good quality single crystal of compound 14 was
obtained from ethanol by slow evaporation of the solvent under ambient conditions. A few
drops of water were added in order to increase solubility. X-ray diffraction data were
collected at 120(2) K using a SuperNova diffractometer with CuKα radiation (λ=1.54184 Å)
and processed with CrysAlisPro software 43. The phase problem was solved by direct methods
with SHELXS-97 44. Parameters of the obtained model were refined by full-matrix least-
squares on F2 using SHELXL-97 44. All non-hydrogen atoms were refined anisotropically.
Hydrogen atoms attached to nitrogen atoms N11 and N19 were found on the difference
Fourier map and refined with isotropic displacement parameter Uiso[H] = 1.2 Ueq[N]. The
distance restraints on bond length were applied here.
Positions of hydrogen atoms attached to carbon atoms were calculated with C-H = 0.93 Å for
aromatic, C-H = 0.97 Å for methylene, C-H = 0.96 Å for methyl groups and were refined
using the riding model with the isotropic displacement parameter Uiso[H] = 1.2 Ueq[C] or
Uiso[H] = 1.5 Ueq[C] (methyl groups only).
45
All crystallographic data are presented in suppl. materials. WinGX software was used to
prepare materials for publication. The figure showing asymmetric unit was obtained with
ORTEP-3 for Windows 45.
6.3. Pharmacology.
6.3.1. Serotonin 5-HT1A, 5-HT2A, and 5-HT7 receptors binding assays. Radioligand
binding studies with native 5-HT1A, 5-HT2A, and 5-HT7 receptors were conducted according
to the methods previously described by us 46. Briefly: 5-HT1A assays used rat hippocampal
membranes, [3H]-8-OH-DPAT (170 Ci/mmol, NEN Chemicals) and 5-HT for non-specific
binding; 5-HT2A assays used rat cortical membranes, [3H]-ketanserin (88.0 Ci/mmol, NEN
Chemicals) and methysergide for nonspecific binding; 5-HT7 receptor assay was performed
using rat hypothalamic membranes, [3H]-5-CT (34.5 Ci/mmol; NEN) and 5-HT for non-
specific binding. Each compound was tested in triplicate at 7–8 concentrations (10–11–10–4
M). The radioactivity was measured by liquid scintillation counting (Beckman LS 6500
apparatus) in 4 mL scintillation fluid (Akwascynt, BioCare). Binding isotherms were analyzed
by nonlinear regression (Prism, GraphPad Software Inc., San Diego, USA), using the Cheng-
Prusoff equation to calculate Ki values. Results were expressed as means of at least two
separate experiments.
18

6.3.2. Other in vitro studies. The extended receptor binding and functional profile with
respect to 5-HT1A/2A/2C/6/7, α1/2C, H1 and D1/2/3/4 receptors was determined at Cerep (Le Bois
l’Eveque, 86600 Celle L’Evescault, France). The data is expressed as mean % inhibition of
control specific binding at the test concentration 1.0E-06 in duplicates. Methodological details
of these studies are available on the company’s web site (www.cerep.fr).
6.3.3. In vivo studies. The experiments were performed on male Swiss albino mice (22–26 g)
purchased from a licensed breeder Staniszewska (Ilkowice, Poland) or male CD-1 mice
(accredited animal facility Jagiellonian University Medical College, Kraków, Poland) and
mice were kept in groups of ten to Makrolon type 3 cages (dimensions 26.5 × 15 × 42 cm).
The animals were kept in an environmentally controlled rooms (ambient temperature 22 2°C;
relative humidity 50–60%; 12:12 light:dark cycle, lights on at 8:00). They were allowed to
acclimatize with the environment for one week before commencement of the experiments.
Standard laboratory food (Ssniff M-Z) and filtered water were freely available. All the
experimental procedures were approved by the I Local Ethics Commission at the Jagiellonian
University in Krakow.
All the experiments were conducted in the light phase between 09.00 and 14.00 hours.
Depending on the type of the test, each experimental group consisted of: 7-9 animals/dose in
the body temperature and forced swim tests, 5-6 animals/dose in the lower lip retraction, head
twitch response and apomorphine-induced climbing tests and 8-10 animals/dose in the
spontaneous locomotor activity, amphetamine-induced hyperlocomotor activity, catalepsy and
four-plate tests .The animals were used only once. The experiments were performed by an
observer unaware of the treatment administered.
6.3.3.1. Body temperature in CD-1 mice. Effects of the tested compounds given alone on
the rectal body temperature in mice (measured with an Ellab thermometer) were recorded 30,
60, 90, and 120 min after their administration. In a separate experiment, the effect of WAY
100635 (0.3 mg/kg) on the hypothermia induced by compounds 14 or 8-OH-DPAT was
tested. WAY 100635 was administered 15 min before the compounds or 8-OH-DPAT and
rectal body temperature was recorded 30 and 60 min after injection of the tested compounds.
The results were expressed as a change in body temperature (∆t) with respect to the basal
body temperature, as measured at the beginning of the experiment.
6.3.3.2. Lower lip retraction (LLR) in Wistar rats. LLR was assessed according to the
method described by Berendsen et al. 47. The rats were individually placed in cages (30 cm ×
25 cm × 25 cm) and they were scored three times (at 15, 30 and 45 min) after the
administration of the tested compounds or 8-OH-DPAT as follows: 0 = lower incisors not
19

visible, 0.5 = partly visible, 1 = completely visible. The total maximum scores amounted to 3
for each rat. In a separate experiment, the effect of the tested compounds or WAY 100635 on
the LLR induced by 8-OH-DPAT (1 mg/kg) was tested. The compounds 14 and WAY
100635 were administered 45 min and 15 min, respectively before 8-OH-DPAT and the
animals were scored 15, 30 and 45 min after 8-OH-DPAT administration.
6.3.3.3. Head twitch response in Swiss albino mice. In order to habituate mice to the
experimental environment, each animal was randomly transferred to a 12 (diameter × 20 cm
(height) glass cage, lined with sawdust 20 min before the treatment. Head twitches in mice
were induced by ( )DOI (2.5 mg/kg). Immediately after treatment, the number of head
twitches was counted during 20 min. ID50 (the dose inhibiting the head twitches in mice by
50%) was calculated using Graph Pad Prism 5 Software.
6.3.3.4. Apomorphine-induced climbing behavior in Swiss albino mice. For observation,
mice were placed in separate cages with walls made of metal bars. Apomorphine (3 mg/kg)
was injected 10 min after the drugs. Twenty minutes after injection of apomorphine, time of
climbing was determined for 2 min. Climbing time was defined as the period during which
the animal held the 2, 3 or 4 paws on the wall. ID50 (the dose inhibiting climbing behavior in
mice by 50%) was calculated using Graph Pad Prism 5 Software.
6.3.3.5. Locomotor activity in mice. The locomotor activity was recorded with an Opto M3
multi-channel activity monitor (MultiDevice Software v.1.3, Columbus Instruments). The
Swiss albino mice were individually placed in plastic cages (22 × 12 × 13 cm), and then the
crossings of each channel (ambulation) were counted from 2 to 6 min, i.e. the time equal to
the observation period in the forced swim test and during 30-min experimental sessions. The
CD-1 mice were individually placed in plastic cages (22 × 12 × 13 cm) for 30 min habituation
period, and then ambulation were counted during 1 h with data recording every 5 min. The
cages were cleaned up with 70% ethanol after each mouse.
6.3.3.6. d-Amphetamine-induced hyperlocomotor activity in CD-1 mice. The locomotor
activity was recorded according to the method described above.
6.3.3.7. Catalepsy. The investigated compound was administered to 10 mice per treatment
group. Animal’s forelimbs were draped over a thin, cylindrical horizontal rod elevated 4 cm
above the tabletop at 30, 60 and 120 min after administration of a test compound. The length
of time the animal touched the bar with both front paws was measured up to a pre-set cut-off
time of 60 s. A maximum of three trials was used for each animal.
48
In the bar test, a scoring system used by Ögren et al. was employed. Results of each trial
were scored as follows: 0 for holding the position for <15 s, 1 for holding for 15-29.9 s, 2 for
20

holding for 30-59.9 s, and a maximum score of 3 for staying on the bar for >60 s. The
minimum cataleptogenic dose was defined as the lowest dose inducing a catalepsy mean score
of >1 at 30, 60 or 120 min post-treatment.
6.3.3.8. Forced swim test in Swiss albino mice. The experiment was carried out according to
25
the method of Porsolt et al. . Briefly, mice were individually placed in a glass cylinder (25
cm high; 10 cm in diameter) containing 6 cm of water maintained at 23–25°C, and were left
there for 6 min. A mouse was regarded as immobile when it remained floating on the water,
making only small movements to keep its head above it. The total duration of immobility was
recorded during the last 4 min of a 6-min test session
6.3.3.8. Four-plate test in Swiss albino mice.
The four-plate apparatus (BIOSEB) consists of a cage (25 x 18 x 16 cm) floored by four
identical rectangular metal plates (8 x 11 cm) separated from one another by a gap of 4 mm.
The top of the cage is covered by a transparent Perspex lid that prevents escape behavior. The
plates are connected to a device that can generate electric shocks. Following a 15-s
habituation period, the animal’s motivation to explore a novel environment is suppressed by
an electric foot shock (0.8 mA, 0.5 s) every time it moves from one plate to another during a
1-min test session. This action is referred to as a ‘punished crossing’, and is followed by a 3 s
shock interval, during which the animal can move across plates without receiving a shock.
Drugs. The following drugs were used: d-amphetamine (sulfate, Sigma-Aldrich), aripiprazole
(hydrochloride, Adamed Pharmaceuticals), apomorphine (hydrochloride, Tocris) and tested
compound 14. Apomorphine and d-amphetamine were dissolved in distilled water; remaining
compounds were suspended in a 1% aqueous solution of Tween 80 immediately before
administration. 14 was administered intraperitoneally (i.p.) 60 min, before the test,
apomorphine was injected i.p. 20 min before testing, and d-amphetamine was injected
subcutaneously (s.c.) 30 min before the test. All compounds were injected at a volume of 10
ml/kg. Control animals received a vehicle injection according to the same schedule.
Statistics. All the data are presented as the mean SEM. The statistical significance of the
results was evaluated by a one-way ANOVA, followed by Bonferroni’s Comparison Test.
6.4. Molecular modeling
The homology models of human D2 dopamine and 5-HT1A serotonin receptors used herein
were generated and described in previously published papers 37,38. The D2 receptor models
were built using dopamine D3 receptor crystal structure as a template (Protein Data Bank
(PDB) database ID: 3PBL) 49. The 5-HT1A receptor models were built on the basis of β2
adrenergic receptor crystal structure (PDB ID: 2RH1) 50. Sequence alignments between D2
21

and 5-HT1A receptors (UniProt database accession numbers P14416 and P08908 respectively)
51
and their templates were performed by hhsearch tool via GeneSilico Metaserver 52. The
crude receptor models were obtained using SwissModel 53, and were validated by processing
in Protein Preparation Wizard 54. For each receptor type, a set of bioactive compounds was
selected for ligand-steered binding site optimization, which was performed using induced fit
docking (IFD) workflow 55. That procedure resulted in a variety of conformational models
that served as molecular targets in docking studies.
Ligand structures were optimized using LigPrep tool. Glide docking procedure was carried
out using default parameters, setting docking precision XP (extra precision) and flexible
docking option retaining original conformations of amide bonds. H-bond constraints, as well
as centroid of a grid box (22 x 22 x 22 Å) for docking studies were located on Asp3.32.
Glide, induced fit docking, LigPrep and Protein Preparation Wizard were implemented in
Schrödinger Suite 2011, which was licensed for Jagiellonian University Collegium Medicum.
Acknowledgments
This study was financially supported by National Science Center grants (No. UMO-
2011/01/B/NZ4/00695, UMO-2012/07/B/NZ7/01173) and the research was carried out with
the equipment purchased thanks to the financial support of the European Regional
Development Fund in the framework of the Polish Innovation Economy Operational Program
(contract no. POIG.02.01.00-12-023/08).
22

References
1. Butini, S.; Gemma, S.; Campiani, G.; Franceschini, S.; Trotta, F.; Borriello, M.; Ceres,
N.; Ros, S.; Coccone, SS.; Bernetti, M.; De Angelis, M.; Brindisi, M.; Nacci, V.;
Fiorini, I.; Novellino, E.; Cagnotto, A.; Mennini, T.; Sandager-Nielsen, K.; Andreasen,
J. T.; Scheel-Kruger, J.; Mikkelsen, J. D.; Fattorusso, C. J. Med. Chem. 2009, 52(1),
151–69.
2. Freedman, R. N. Engl. J. Med. 2003, 349, 1738–1749.
3. Laviolette, S. R. Schizophr. Bull. 2007, 33(4), 971–81.
4. Abi-Dargham, A. Int. J. Neuropsychopharmacol. 2004, 7(Suppl 1), 1–5.
5. Haifeng, Sun.; Liyuan, Zhu.; Huicui, Yang.; Wangke, Qian.; Lin, Guo.; Shengbin,
Zhou.; Bo, Gao.; Zeng, Li.; Yu, Zhou.; Hualiang, Jiang.; Kaixian, Chen.; Xuechu,
Zhen.; Hong, Liu. Bioorg. Med. Chem. 2012, 21(4), 856–68.
6. Garzya, V.; Forbes, I. T.; Gribble, A. D.; Hadley, M. S.; Lightfoot, A. P.; Payne, A.
H.; Smith, A. B.; Douglas, S. E.; Cooper, D. G.; Stansfield, I. G.; Meeson, M.; Dodds,
E. E.; Jones, D. N.; Wood, M.; Reavill, C.; Scorer, C. A.; Worby, A.; Riley, G.;
Eddershaw, P.; Ioannou, C.; Donati, D.; Hagan, J. J.; Ratti, E. A. Bioorg. Med. Chem.
Lett. 2007, 17(2), 400–5.
7. Taylor, D. M. Schizophrenia in Focus; Pharmaceutical Press: London, 2006; Chapters
1–5, pp 1–45.
8. Tomić, M.; Ignjatović, D.; Tovilović, G.; Andrić, D.; Roglić, G.; Kostić-Rajacić, S.
Bioorg. Med. Chem. Lett. 2007, 17(21), 5749–53.
9. Ablordeppey, S. Y.; Altundas, R.; Bricker, B.; Zhu, X. Y.; Kumar, E. V.; Jackson, T.;
Khan, A.; Roth, B. L. Bioorg. Med. Chem. 2008, 16(15), 7291–301.
10. Leucht, S.; Corves, C.; Arbter, D.; Engel, R. R.; Li, C.; Davis, J. M.. Lancet 2009,
373, 31–41.
11. Carro, L.; Raviña, E.; Domínguez, E.; Brea, J.; Loza, M. I.; Masaguer, C. F. Bioorg.
Med. Chem. Lett. 2009, 19(21), 6059–62.
12. Wirshing, D. A.; Wirshing, W. C.; Kysar L.; Berisford, M. A.; Goldstein, D.; Pashdag,
J.; Mintz, J.; Marder S. R. J. Clin. Psychiatry 1999, 60(6), 358–63.
13. Vangveravong, S.; Zhang, Z.; Taylor, M.; Bearden, M.; Xu, J.; Cui, J.; Wang, W.;
Luedtke, R. R.; Mach, R. H. Bioorg. Med. Chem. 2011, 19(11), 3502–11.
14. Zajdel, P.; Partyka, A.; Marciniec, K.; Bojarski, A. J.; Pawlowski, M.; Wesolowska,
A. Future Med. Chem. 2014, 6(1), 57–75.
23

15. Leucht, S.; Cipriani, A.; Spineli, L.; Mavridis, D.; Orey, D.; Richter, F.; Samara, M.;
Barbui, C.; Engel, R. R.; Geddes, J. R.; Kissling, W.; Stapf, M. P.; Lässig, B.; Salanti,
G.; Davis, J. M. Lancet 2013, 382, 951–62.
16. Czopek, A.; Byrtus, H.; Kołaczkowski, M.; Pawłowski, M.; Dybała, M.; Nowak, G.;
Tatarczyńska, E.; Wesołowska, A.; Chojnacka-Wójcik, E. Eur. J. Med. Chem. 2010,
45(4), 1295–303.
17. Goodson, L. H.; Honigberg, J. L.; Lehman, J. L.; Burton, W. H. J. Org. Chem. 1960,
25, 1920–1924.
18. Byrtus, H.; Pawłowski, M.; Czopek, A.; Bojarski, A. J.; Duszyńska, B.; Nowak, G.;
Kłodzińska, A.; Tatarczyńska, E.; Wesołowska, A.; Chojnacka-Wójcik, E. Eur. J.
Med. Chem. 2005, 40, 820-829.
19. Etter, M. C.; MacDonald, J.C.; Bernstein, J. Acta Cryst. 1990, B46, 256–262.
20. MacKenzie, R. G.; VanLeeuwen, D.; Pugsley, T. A.; Shih, Y. H.; Demattos, S.; Tang,
L.; Todd, R. D.; O’Malley, K. L. Eur. J. Pharmacol. 1994, 266, 79–85.
21. Van Tol, H. H.; Wu, C. M.; Guan, H. C.; Ohara, K.; Bunzow, J. R.; Civelli, O.;
Kennedy, J.; Seeman, P.; Niznik, H. B.; Jovanovic, V. Nature 1992, 358, 149–52.
22. Kołaczkowski, M.; Marcinkowska, M.; Bucki, A.; Pawłowski, M.; Mitka, K.;
Jaśkowska, J.; Kowalski, P.; Kazek, G.; Siwek, A.; Wasik, A.; Wesołowska, A.;
Mierzejewski, P.; Bienkowski, P. J. Med. Chem. 2014, 57(11), 4543–57.
23. Adham, N.; Zgombick, J. M.; Bard, J.; Branchek, T. A. J. Pharmacol. Exp. Ther.
1998, 287, 508–14.
24. Darmani, N. A.; Martin, B. R.; Pandey, U.; Glennon, R. A. Pharmacol. Biochem.
Behav. 1990, 36, 901-906.
25. Porsolt, R. D.; Bertin, A.; Jalfre, M. Arch. Int. Pharmacodyn. Ther. 1977, 229(2),
327–36.
26. Boissier, J.R.; Simon, P.; Aron, C. Eur. J. Pharmacol. 1968, 4, 145–51.
27. Romero, G.; Sánchez, E.; Pujol, M.; Pérez, P.; Codony, X.; Holenz, J.; Buschmann,
H.; Pauwels, P. J. Br. J. Pharmacol. 2006, 148(8), 1133–43.
28. Jastrzębska-Więsek, M.; Siwek, A.; Kazek, G.; Nawieśniak, B.; Partyka, A.;
Marcinkowska, M.; Kołaczkowski, M.; Wesołowska, A. Pharmacol. Rep. 2013, 65(4),
998–1005.
29. Dupuis, D. S.; Mannoury la Cour, C.; Chaput, C.; Verriele, L.; Lavielle, G.; Millan,
M. J. Eur. J. Pharmacol. 2008, 588, 170–77.
30. Akimowa, E.; Lanzenberger, R.; Kasper, S. Biol. Psychiat. 2009, 7, 627–35.
24

31. Carr, G. V.; Lucki, I. Psychopharmacol. 2011, 213, 265–87.
32. Goodwin, F. K.; Whitham, E. A.; Ghaemi, S. N. CNS Drugs 2011, 25(10), 819–27.
33. Nelson, J. C.; Papakostas, G. I. Am. J. Psych. 2009, 166, 980–91.
34. Hoffman, D. C.; Donovan, H. Psychopharmacol .1995, 120, 128–133.
35. Bourin, M.; Chenu, F.; Prica, C.; Hascoët, M. Psychopharmacol. 2009, 206, 97–107.
36. Sarkisyan, G.; Roberts, A. J.; Hedlund, P. B. Behav. Brain. Res. 2010, 209, 99–108.
37. Kołaczkowski, M.; Bucki, A.; Feder, M.; Pawłowski, M. J. Chem. Inf. Model. 2013,
53, 638–648.
38. Kowalski, P.; Jaśkowska, J.; Bojarski, A. J.; Duszyńska, B.; Bucki, A.; Kołaczkowski,
M. J. Heterocycl. Chem. 2011, 48, 192–198.
39. Kołaczkowski, M.; Marcinkowska, M.; Bucki, A.; Śniecikowska, J.; Pawłowski, M.;
Kazek, G.; Siwek, A.; Jastrzębska-Więsek, M.; Partyka, A.; Wasik, A.; Wesołowska,
A.; Mierzejewski, P.; Bienkowski, P. Eur. J. Med. Chem. 2015, 92, 221-235.
40. Czopek, A.; Kołaczkowski, M.; Bucki, A.; Byrtus, H.; Pawłowski, M.; Siwek, A.;
Bojarski, A. J.; Bednarski, M.; Wróbel, D.; Wesołowska, A. Arch Pharm (Weinheim).
2013, 346(2), 98-109.
41. Strader, C. D.; Sigal, I. S.; Candelore, M. R.; Rands, E.; Hill, W. S.; Dixon, R. A. J.
Biol. Chem. 1988, 263, 10267–10271.
42. Braden, M. R.; Parrish, J. C.; Naylor, J. C.; Nichols, D. E. Mol. Pharmacol. 2006, 70,
1956 –1964.
43. CrysAlisPro. Agilent Technologies UK Ltd, 2011, Yarnton, England
44. Sheldrick, G. M. Acta Cryst. 2008, A64, 112–22.
45. Farrugia, L. J. J. Appl. Cryst. 2012, 45, 849–854.
46. Bojarski, A. J.; Paluchowska, M. H.; Duszyńska, B.; Kłodzińska, A.; Tatarczyńska,
E.; Chojnacka-Wójcik, E. Bioorg. Med. Chem. 2005, 13, 2293–2303.
47. Berendsen, H. H.; Jenck, F.; Broekkamp, C. L. Pharmacol. Biochem. Behav. 1989, 33,
821–27.
48. Ogren, S. O.; Hall, H.; Köhler, C.; Magnusson, O.; Sjöstrand S. E.
Psychopharmacology 1986, 90, 287–94.
49. Chien, E. Y. T.; 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, 330, 1091–
95.
25

50. Cherezov, V.; Rosenbaum, D. M.; Hanson, M. A.; Rasmussen, S. G. F.; Thian, F. S.;
Kobilka, T. S.; Choi, H.-J.; Kuhn, P.; Weis, W. I.; Kobilka, B. K.; Stevens, R. C.
Science 2007, 318, 1258–65.
51. Jain, E.; Bairoch, A.; Duvaud, S.; Phan, I.; Redaschi, N.; Suzek, B. E.; Martin, M. J.;
McGarvey, P.; Gasteiger, E. BMC Bioinformatics 2009, 10, 136–42.
52. Kurowski, M. A.; Bujnicki, J. M. Nucleic Acids Res. 2003, 31, 3305–07.
53. Schwede, T.; Kopp, J.; Guex, N.; Peitsch, M. C. Nucleic Acids Res. 2003, 31, 3381–
3385.
54. Sastry, G. M.; Adzhigirey, M.; Day, T.; Annabhimoju, R.; Sherman, W. J. Comput.
Aided Mol. Des. 2013, 27, 221–234.
55. Sherman, W.; Day, T.; Jacobson, M. P.; Friesner, R. A.; Farid, R. J. Med. Chem. 2006,
49, 534–553.
26

Table 1. Binding of the spirohydantoin derivatives for serotonin (5-HT) and dopamine D2
receptors
Table 2. Extended receptor binding profile of the selected 10, 11, 14, 15 spirohydantoin
derivatives.
Table 3. Extended functional profile of compounds 10 and 14.
Table 4. Functional in vivo profile of the compound 14
Table 5. Effects of compound 14 and aripiprazole on the spontaneous locomotor activity in
CD-1 mice
Table 6. Effects of compound 14 and aripiprazole on the number of punished crossings in the
four-plate test in Swiss albino mice.
Table 7. Cataleptogenic effect of compound 14 and aripiprazole in CD-1 mice.
27

Figure 1. Molecular geometry in the crystal structure of compound 14 showing the atom
labelling scheme. Dashed lines represent a charge-assisted hydrogen bond N+-H...Cl- and the
weak intramolecular hydrogen bond C-H...O. Displacement ellipsoids of non-hydrogen atoms
are drawn at the 30 % probability level. Hydrogen atoms are presented as small spheres with
an arbitrary radius.
Figure 2. Effects of compound 14 and aripiprazole on the hyperlocomotor activity induced by
d-amphetamine in CD-1 mice.
Figure 3. Effects of compound 14 and aripiprazole on the immobility time in the forced swim
test in Swiss albino mice.
Figure 4. Binding modes of 14 in the binding sites of 5-HT1A (A) and D2 (B) receptors.
Amino acid residues engaged in ligand binding (within 4Å from the ligand atoms) are shown
as thick sticks. Dotted yellow lines represent H-bonds with polar residues. For the sake of
clarity a part of ECL2 was hidden. TMH – transmembrane helix; ECL – extracellular loop.
Scheme 1. Synthetic pathways of compounds 9-23.
28

Table 1. Binding of the spirohydantoin derivatives for serotonin (5-HT) and dopamine D2
receptors.
Ki SD [nM]a
5-HT1A
75
Compd
D2
5-HT2A
111
5-HT7
93
9
NT
5
12
6
12
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
2.6
12
6
0.2
1
98
21
71
27
104
539
412
743
59
7.5
46
12
49
2
3
2
1
87
6
67
9
13
2.2
15
5.3
35
3
2
77
8
116
47
16
8
0.2
2
59
4
27
2
37
4
1100
608
100
17
0.7
3
157
48
17
3
117
515
130
768
379
1504
63
13
65
23
112
47
193
7
NT
0.4
2
75
6
79
6
>10000
>10000
30
22
306
42
113
38
4
2
122
414
498
171
78
32
63
9
23
3
NT
NT
NT
15
43
5
NT – not tested, a Data expressed as the mean SD of at least two independent experiment in duplicate.
29