Novel multitarget 5-arylidenehydantoins with arylpiperazinealkyl fragment: Pharmacological evaluation and investigation of cytotoxicity and metabolic stability

Article abstract of DOI:10.1016/j.bmc.2019.07.046

On the basis of the structures of serotonin modulators or drugs (NAN-190, buspirone, aripiprazole) and phosphodiesterase 4 (PDE4) inhibitors (rolipram, RO-20-1724), a series of novel multitarget 5-arylidenehydantoin derivatives with arylpiperazine fragment was synthesized. Among these compounds, 5-(3,4-dimethoxybenzylidene-3-(4-(4-(2,3-dichlorophenyl)piperazine-1-yl)butyl)-imidazolidine-2,4-dione (13) and 5-(3-cyclopentyloxy-4-methoxybenzylidene-3-(4-(4-(2-methoxyphenyl)piperazine-1-yl)butyl)-imidazolidine-2,4-dione (18) were found to be the most promising showing very high affinity toward 5-HT_1A and 5-HT₇ receptors (K_i = 0.2–1.0 nM) but a negligible inhibitory effect on PDE4. The high affinity of the compounds for 5-HT_1A and 5-HT₇ receptors was further investigated by computer-aided studies. Moreover, compounds 13 and 18 showed no significant cytotoxicity in the MTT assay, but high clearance in the in vitro assay. In addition, these compounds behaved like 5-HT_1A and 5-HT₇ receptor antagonists and exhibited antidepressant-like activity, similar to the reference drug citalopram, in an animal model of depression.

Full text of DOI:10.1016/j.bmc.2019.07.046

Journal Pre-Proof
Novel Multitarget 5-Arylidenehydantoins with Arylpiperazinealkyl Fragment:
Pharmacological Evaluation and Investigation of Cytotoxicity and Metabolic
Stability
Anna Czopek, Adam Bucki, Marcin Kołaczkowski, Agnieszka Zagórska,
Marcin Drop, Maciej Pawłowski, Agata Siwek, Monika Głuch-Lutwin, Elżbieta
Pękala, Alicja Chrzanowska, Marta Struga, Anna Partyka, Anna Wesołowska
PII:
S0968-0896(19)30639-X
https://doi.org/10.1016/j.bmc.2019.07.046
BMC 15030
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
16 April 2019
17 July 2019
28 July 2019
Please cite this article as: Czopek, A., Bucki, A., Kołaczkowski, M., Zagórska, A., Drop, M., Pawłowski, M., Siwek,
A., Głuch-Lutwin, M., Pękala, E., Chrzanowska, A., Struga, M., Partyka, A., Wesołowska, A., Novel Multitarget
5
-Arylidenehydantoins with Arylpiperazinealkyl Fragment: Pharmacological Evaluation and Investigation of
Cytotoxicity and Metabolic Stability, Bioorganic & Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.bmc.
019.07.046
2
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JOURNAL PRE-PROOF
Novel
Multitarget
5-Arylidenehydantoins
with
Arylpiperazinealkyl
Fragment:
Pharmacological Evaluation and Investigation of Cytotoxicity and Metabolic Stability
a
a
a
a
a
Anna Czopek , Adam Bucki , Marcin Kołaczkowski , Agnieszka Zagórska , Marcin Drop ,
a
b
b
c
Maciej Pawłowski , Agata Siwek , Monika Głuch-Lutwin , Elżbieta Pękala , Alicja
d
d
e
e
Chrzanowska , Marta Struga , Anna Partyka , Anna Wesołowska
aDepartment of Pharmaceutical Chemistry, Jagiellonian University Medical College, 9
Medyczna Street, 30-688 Kraków, Poland
bDepartment of Pharmacobiology, Jagiellonian University Collegium Medicum, Krakow,
Poland
cDepartment of Pharmaceutical Biochemistry, Jagiellonian University Collegium Medicum,
Krakow, Poland
dChair and Department of Biochemistry, Medical University, 02-097 Warszawa, 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

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Abstract
On the basis of the structures of serotonin modulators or drugs (NAN-190, buspirone,
aripiprazole) and phosphodiesterase 4 (PDE4) inhibitors (rolipram, RO-20-1724), a series of
novel multitarget 5-arylidenehydantoin derivatives with arylpiperazine fragment was
synthesized. Among these compounds, 5-(3,4-dimethoxybenzylidene-3-(4-(4-(2,3-
dichlorophenyl)piperazine-1-yl)butyl)-imidazolidine-2,4-dione (13) and 5-(3-cyclopentyloxy-
4
-methoxybenzylidene-3-(4-(4-(2-methoxyphenyl)piperazine-1-yl)butyl)-imidazolidine-2,4-
dione (18) were found to be the most promising showing very high affinity toward 5-HT1A
and 5-HT receptors (K = 0.2–1.0 nM) but a negligible inhibitory effect on PDE4. The high
7
i
affinity of the compounds for 5-HT1A and 5-HT receptors was further investigated by
7
computer-aided studies. Moreover, compounds 13 and 18 showed no significant cytotoxicity
in the MTT assay, but high clearance in the in vitro assay. In addition, these compounds
behaved like 5-HT1A and 5-HT receptor antagonists and exhibited antidepressant-like
7
activity, similar to the reference drug citalopram, in an animal model of depression.
Key words: hydantoin, 5-HT /5-HT modulators, PDE4 inhibitors, cytotoxicity,
1
A
7
antidepressant activity

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1
. Introduction
Mental diseases, such as depression, schizophrenia, and anxiety disorders, are one of the
major global health problems. These are mostly characterized by a combination of abnormal
thoughts, behaviors, perceptions, emotions, and relationships with other people. According to
the latest estimates published by the WHO, depression is the most common form of mental
disorders and affected 322 million people in 2017. The number of affected patients increased
by more than 18% between 2005 and 2015 and is still expected to rise significantly, which
1
indicates that depression is one of the main causes of disability worldwide. To tackle the
global burden of depression, research investment needs to be substantially increased to
facilitate the development of new effective therapies. Although several pharmacological drug
classes, such as monoamine oxidase inhibitors, tricyclic antidepressants, selective serotonin
reuptake inhibitors (SSRIs), serotonin–norepinephrine reuptake inhibitors (SNRI), and
atypical antidepressants, have been developed, the treatment outcome of depression is still
suboptimal. The use of currently available antidepressants is limited because of their side
effects, slow response, and inadequate treatment efficacy.2
Because depression and other mental disorders are extremely complex and typically
associated with multiple drug targets, the use of multitarget-directed ligands (MTDLs) may be
a prospective solution. The MTDL strategy involves combining two or more pharmacophores
3
in one molecule, expressing a synergistic pharmacological effect in vivo. Currently, an
increasing number of multitarget drugs are being developed for the treatment of schizophrenia
4
and major depressive disorders. For example, ziprasidone, a typical antipsychotic used for
the treatment of schizophrenia and off-label for depression, is a full antagonist of D and 5-
2
5
HT receptors as well as a partial agonist of 5-HT , 5-HT , and 5-HT receptors. Another
2
A
1A
2C
1D
drug, vilazodone, approved for the treatment of major depressive disorders, exhibits SSRI-like
activity (serotonin transporter inhibition) and acts as a partial agonist of 5-HT1A receptor,
similar to the structurally related anxiolytic, buspirone.6
A growing body of evidence suggests that modulation of cyclic nucleotide (cAMP or cGMP)
signaling may play a role in the etiology of disorders of the central nervous system (CNS).7,8
Therefore, among the modern approaches, targeting cyclic nucleotide phosphodiesterase
(PDE), a key enzyme responsible for the degradation of cyclic nucleotides, can be considered
9
10
11
12
as an antipsychotic, antidepressant, anxiolytic, or a cognition-enhancing therapy. In the
context of antidepressant therapy, increasing the level of cAMP by inhibition of
phosphodiesterase 4 (PDE4) has gained significant interest. The PDE4 family comprises four
subtypes (PDE4A, PDE4B, PDE4C, and PDE4D) and is expressed almost exclusively in the

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CNS. The highest concentrations of PDE4B and PDE4D are found in specific brain regions
such as the nucleus accumbens shell and the frontal cortex, which suggests their role in
1
3
depression, schizophrenia, and cognitive process. To date, a plethora of different structurally
related molecules have been reported including the first prototypical PDE4-selective inhibitor,
rolipram, which has shown behavioral and antidepressant effect in rats and humans.14–16
Another molecule, RO-20-1724, has been shown to exhibit antipsychotic-like properties as
well as neuroprotective effect.17,18 Some newly developed molecules, for example, FCPR16,19
2
0
21
MK-0952, and HT-0712, with presumably higher potency and lower toxicity are currently
being researched.
The compounds discussed in the present study were designed as a series of MTDLs
combining the pharmacophore features of selective PDE4 inhibitors and serotonin (or 5-HT)
receptor modulators (figure 1). The first group was formed from chemical structures of
rolipram,22 RO-20-172417 and compound 6 (3-benzylideneindolin-2-one derivative)23 by
alternating their fragments and replacing pyrrolidin-2-one, imidazolidin-2-one, or 2,3-
dihydro-1H-indol-2-one system by imidazolidine-2,4-dione (hydantoin) as a bioisosteric
moiety. These modifications resulted in compounds that retain the geometry of their
prototypes, while hydantoin moiety enables additional functions which might lead to
improved physicochemical properties and facilitate additional interactions with residues at the
binding site. The main core was linked with 4-arylpiperazines by an aliphatic chain, which is
a characteristic of the pharmacophore of 5-HT modulators. The amine moieties were chosen
based on the structures of the drugs currently used for the treatment of depression,
2
4
schizophrenia, and anxiety, such as aripiprazole (2,3-dichlorophenylpiperazine moiety) and
buspirone (pyrimidin-2-yl-piperazine moiety),25 as well as a molecule widely used in
scientific studies, NAN-190 (2-methoxyphenylpiperazine moiety).26
O
O
O
NH
O
N
O
Rolipram
N
N
O
NAN-190
R2
O
X
N
O
X
O
NH
NH
Cl
Cl
O
N
O
O
N
Aripiprazole
O
H
n
N
N
R1
N
RO-20-1724
O
HN
O
O
O
N
O
O
N
N
O
N
N
N
H
Buspirone
compd 6

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Figure 1.
Taking into account the abovementioned facts, in the present work, a screening library of 5-
arylidenehydantoins with arylpiperazinealkyl moiety was synthesized and the ability of these
compounds to bind selected serotonin and dopamine receptors as well as inhibit PDE4
enzyme was evaluated. Furthermore, molecular modeling techniques were utilized to identify
structural fragments responsible for the in vitro affinity of the compounds. After selecting the
most active compounds, studies investigating cytotoxicity and metabolic stability were
undertaken. Moreover, the antidepressant activity of the selected compounds was determined
using in vivo pharmacological tests.
2
. Molecular modeling
The homology models of human 5-HT1A and 5-HT serotonin receptors were built on the
7
basis of β adrenergic receptor crystal structure (PDB ID: 2RH1). After optimization of each
2
receptor binding site, the selected 5-arylidenehydantoin derivatives were flexibly docked to it,
and best scored complexes were examined in order to describe possible binding mode which
may be helpful with understanding receptor-ligand interactions.
2
. Results and discussion
.1 Chemistry
2
New compounds with 5-arylidenehydantoin moieties (3, 4) were prepared according to the
synthetic route shown in Scheme 1 and Table 1. The benzaldehyde ethers (1, 2), were
2
7
synthesized in line with a known procedure. The 5-arylidenehydantoin (3, 4) were obtained
2
8
according to the Knoevenagel condensation, followed by the alkylation in position N3 of a
imidazolidine-2,4-dione ring. Then, the final compounds (9-23) were formed by coupling of
intermediate products (5-8) with differently substituted arylpiperazines. In line with literature
data, all target compounds occur in Z-configuration, due to benzylidene substituent attached
to hydantoin ring by C=C double bond.29,30
The standard synthetic procedures were adopted for optimization of the synthesis of new
series of compounds. The final compounds were obtained in multistep reactions with
satisfactory yields (42-90%), with high purity (above 95% in HPLC analysis). The structures
of all target compounds were assessed on the basis of chromatography (HPLC, LC/MS) and
1
spectral ( HNMR) and elemental analysis. For further pharmacological studies, selected
compounds 13 and 18 were transformed into water-soluble hydrochloride salts.

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R1
O
R1
O
HO
O
CHO
O
CHO
a
b
NH
HN
O
O
compd 1, 2
compd 3, 4
O
R = -CH ,
R = -CH ,
1
3
1
3
R2
X
c
X
N
R1
R1
O
O
N
O
Cl/Br
O
d
n
n
N
N
O
HN
O
HN
compd 9 - 20
compd 5 - 8
O
O
R = -CH ,
, n = 1, 2
R = -CH ,
, n = 1, 2
1
3
1
3
R = 2-OCH , 2,3-diCl, X = CH, N
Cl: compd 5, 7, and 8; Br; compd 6
2
3
Scheme 1. Reagents and conditions: a) methyl iodide or bromocyclopentane, K CO , DMF,
2
3
6
5°C, 16 h b) hydantoin, NaHCO , ETA, H O/EtOH, 120°C, 2 h c) appropriate
3 2
dihalogenoalkane, K CO , THF/ H O, 50°C, 12 h d) appropriate arylpiperazine, K CO , THF/
2
3
2
2
3
H O, 50°C, 24 h
2
2
.2 Structure–activity relationship studies
Our studies focused on developing a novel antidepressant by incorporating multifunctional
features to the structure of classical pharmacophore for monoaminergic receptor ligands
(arylpiperazine moiety). With the view of compounds exhibiting dual activity as a receptor
and an enzyme, we decided to use the imidazolidine-2,4-dione (hydantoin) scaffold as a
bioisostere for heterocycles found in PDE4 inhibitors such as rolipram and RO-20-1724. The
additional amide moiety in the imidazolidine-2,4-dione may enable a stronger interaction with
the molecular targets compared to rolipram, and especially, could stabilize the active ligand–
enzyme complex formed. Next, substituents which are present in the structures of the known
inhibitors of PDE were introduced at position-5 of the imidazolidine-2,4-dione system by an
additional unsaturated bond. This structural modification could provide a much more
favorable conformation to imidazolidine-2,4-dione, similar to that of rolipram. For studying
the structure–activity relationships, the influence of the substituents on arylpiperazine moiety,
length of the alkyl chain, and substituents in the position-5 of the imidazolidine-2,4-dione
system was taken into consideration.
The affinity of the newly synthesized 5-arylidenehydantoins for serotonin 5-HT1A and 5-HT7
receptors was estimated in binding assays. In addition, the binding affinity of all compounds

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for 5-HT , 5-HT , and D receptors was evaluated in screening experiments (Table 2). All
2
A
6
2
the tested compounds exhibited moderate-to-high affinity for either 5-HT1A or 5-HT7
receptor. Compounds 13 and 18 were found to be the most potent in binding to both of the
serotonin receptors, with their K values in the range of 0.2–1 nM (Table 2). Other compounds
i
exhibited a 7- to 933-fold decreased affinity for either 5-HT or 5-HT receptor (18 vs 12 and
1
A
7
1
8 vs 14). Among the tested compounds, only 16 and 17 selectively bound to either 5-HT or
7
5
-HT1A receptor. A general trend observed for the affinity toward 5-HT1A receptor was as
follows: compounds with 2-methoxy moiety (9, 12, 15, 18) showed the highest affinity for 5-
HT1A receptor, followed by the 2-pyrimidine derivatives (11, 14, 17, 20) which exhibited
median affinity, whereas compounds containing 2,3-dichloro moiety (except 13) showed the
lowest affinity for these receptors. A contrasting trend was observed for the affinity toward 5-
HT receptor consistent with the literature data:30,31 the highest affinity was shown by
7
compounds with 2-methoxy and 2,3-dichloro moieties (9, 10, 12, 13, 15, 18, 19).
To determine the influence of the linker type on receptor affinity and/or selectivity,
compounds with a three- and four-methylene linker between the arylpiperazine moiety and
the imidazoline-2,4-dione ring were synthesized. In general, compounds with a four-carbon
linker (12–14, 18–20) showed higher affinity than their counterparts with a three-carbon
linker (9–11, 15–17), with the most potent compounds 13 and 18 belonging to this group.
In addition, screening studies investigating the binding affinity of compounds to 5-HT , 5-
2
A
HT , and D receptors showed that except 10 and 13, none of the tested compounds exhibited
6
2
significant affinity (over 50%) for these receptors at a concentration of 1.E-07 M. Only
compounds 10 and 13 showed a moderate affinity for 5-HT2A receptors (61% and 82%,
respectively).
Structural analysis and docking results of different classes of PDE4 inhibitors have revealed
3
2
the common features of inhibitors binding to the enzyme. The proposed model of Liao et al.
showed that cyclopentoxy and methoxy substituents connected via an aromatic ring to
scaffold could form integral H-bonds in the lipophilic pockets of PDE4. Moreover, many
potent PDE4 inhibitors have bulk aromatic substituents linked to the scaffold that may form
integral H-bonds with residues such as His234, His238, His274, and Asp257 depositing in the
large M-pocket of the enzyme. Although the newly synthesized compounds 9–20 meet the
structural requirements referred above, screening data revealed that these compounds
exhibited only a weak or negligible inhibitory effect on PDE4B (1–14%) in comparison with
the reference drug rolipram (85%, table 2). The highest but still weak inhibitory effect was
shown by compounds 14 and 18 (14%). Results showed that even though the additional

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unsaturated bond could provide a much more favorable conformation to 5-
arylideneimidazolidine-2,4-dione, it did not positively influence PDE4 inhibition.
As a continuation of the above studies, compounds with the highest affinity for 5-HT and 5-
1
A
HT receptors (13 and 18) were chosen for extended functional evaluation. It was found that
7
both compounds behaved like potent 5-HT1A receptor antagonists (K = 1.3 and 1.7 nM,
b
respectively) and moderate 5-HT receptor antagonists (K = 54.3 and 73.0 nM, respectively)
7
b
(Figure 2, Supplementary materials, Table 2).
Antagonist response
Antagonist response
8
0
1
50
00
SB 269970
NAN-190
13
13
60
18
1
18
4
0
0
0
50
2
0
-
-
12
-10
-8
-6
-4
12
-10
-8
-6
-4
Log [Antagonist] (M)
Log [Antagonist] (M)
Figure 2.
All the above observations indicated that for designing compounds that can act as both
receptor ligands and enzyme inhibitors, not only the combination of structural features but
also an appropriate substitution in position-5 of imidazolidine-2,4-dione scaffold, which
determines the overall effect, is important.
2
.3 Cytotoxicity
The early implementation of in vitro cytotoxicity testing has become an integral aspect of
drug discovery of new chemical entities (NCE) and NCE have been under investigation of
their safety to the host cell or the cytotoxic effect in a cancer cell. The in vitro cytotoxicity
testing has greatly streamlined drug discovery process and is now considered to be a nearly
compulsory activity starting at target validation.
Cytotoxic activity of tested compounds (table 3) was estimated by determining the half-
maximal inhibitory concentration IC . Additionally, the cytotoxic potential of cancer cells for
5
0
tested compounds was expressed as a selectivity factor (selectivity index, SI) and calculated
by the formula: SI = IC of normal cells / IC of cancer cells. Doxorubicin (anticancer drug)
5
0
50
was used as a reference compound.

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In the MTT assay, both examined compounds (13, 18) exhibited significantly less cytotoxic
activity to cancer and normal cells than selected positive reference compound – doxorubicin
(
the IC values for both compounds were > 100 µM, table 3). Compound 13 was lower toxic
50
against normal cells (SI > 1.0), than 18 and doxorubicin (SI < 1). This study showed that
compounds 13 and 18 can be used during long-term exposure without any harmful effect on
normal cells (IC > 100 µM).
5
0
2
.4 In vivo pharmacology
The most potent compounds (13, 18) were evaluated for their antidepressant activity in mice
using the forced swim test (FST). To determine the specificity of the antidepressant-like effect
and to exclude the possibility of competing behaviors, the influence of the effective doses
estimated using the FST on spontaneous locomotor activity was studied. Citalopram was used
as a reference antidepressant drug in these behavioral experiments.
A number of preclinical and clinical studies have shown that mainly serotonin receptor
3
3
ligands produce antidepressant-like effects. Moreover, such behavioral effects have been
associated with the antagonistic activity of 5-HT1A and 5-HT receptors and are similar to
7
3
4
those of SSRIs (e.g., citalopram). On the basis of these data and functional in vitro profile as
well as the results of the cytotoxic study, the most potent compounds 13 and 18 were selected
for in vivo behavioral studies for investigating their antidepressant effect. In the FST,
compound 13 administered at a dose of 0.625 mg/kg shortened the immobility time of mice
by 27%, while compound 18 was active at doses of 0.625, 1.25, and 2.5 mg/kg decreasing the
immobility time by approximately 30%. The antidepressant effects of compounds 13 and 18
were similar to that of the reference drug citalopram administered at a dose of 1.25 mg/kg
(figure 3). Moreover, these effects seemed to be specific because compounds 13 and 18 and
citalopram, used in active doses, did not influence the locomotor activity of mice measured
during the time equal to the observation period in the FST (data not shown).

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Figure 3.
2
.5 Metabolism in vitro study in mouse liver microsomes
Taking into account the fact, that for many drugs the primary site of metabolism is liver, in
the next phase of the investigation, the preliminary metabolic stability study of selected
compound 18 was performed using mouse liver microsomes. In accordance with previously
3
6
described procedure tested compound, potassium phosphate buffer, NADPH-regenerating
system and levallorphan as an internal standard were incubated with microsems, and further
examined by means of liquid chromatography mass spectrometry. Then, analyzing the
depletion of compound 18 allowed for calculation of the in vitro half-time (t ) and intrinsic
1
/2
clearance (Cl ) values.
int
One of the active molecules, compound 18, as an example, was selected for further metabolic
stability studies with mouse liver microsomes in order to find out whether parent compound
or its metabolites determine antidepressant-like activity. For this purpose, sample of
compounds 18 with mouse liver microseomes were incubated and examined at a three time
points (5, 15 and 30 min) using liquid chromatography mass spectrometry. Analysis of full
chromatogram after 30 min of incubation revealed the presence of seven metabolites (M1-M7,
+
+
figure 4) with the following molecular ions: M1 [M+H] = 389.11 m/z, M2 [M+H] = 565.25
+
+
+
m/z, M3 [M+H] = 193.06 m/z, M4 [M+H] = 565.32 m/z, M5 [M+H] = 443.28 m/z, M6
+
+
[
M+H] = 565.38 m/z, M7 [M+H] = 481.17 m/z. Based on liquid chromatography mass
spectrometry results, N-dealkylation and hydroxylation of compound 18 are considered as
main metabolic pathways. Furthermore, to assess hepatic first pass effect, the intrinsic
clearance (Cl ) value of compound 18 was estimated (table 4).
int

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Figure 4.
The obtained result of compound 18 indicates its high susceptibility to metabolism by mouse
liver microsomes. The intrinsic clearance value of the tested compound was slightly higher
than that of imipramine, reference antidepressant drug (135.5 vs 125.5 µl/mg/min). The
metabolic stability depends on the type of species (e.g. mouse vs human) and shows
significance differences presumably due to varied specific activity of cytochrome P450
enzymes.36 According to aforementioned data, it is highly likely that metabolites of
compound 18 affect its in vivo antidepressant-like activity.
2
.6 Molecular modeling
Rational design of the presented 5-arylidenehydantoin derivatives resulted in the series of
compounds showing pronounced affinity for 5-HT and 5-HT serotonin receptors. Analysis
1
A
7
of in vitro binding data has shown superior activity of two compounds – 13 and 18. Therefore,
modelling of their molecular interactions with the target proteins was performed in order to
assess their binding modes and thus explain receptor affinity. To this end, structure-based
method based on ligand docking was applied. The conformational flexibility of the 5-HT1A
and 5-HT receptor homology models was imitated by sets of several models obtained by
7
ligand-steered optimization. Flexible docking, probing conformational states of the ligands
resulted in complexes, which were characterized by favourable ligand-receptor interactions.
The proposed binding mode of the ligands in the both receptor sites proved to be consistent
with the common one for monoaminergic receptor ligands and results published
previously.37,38
Docking studies revealed that the molecules of compounds 13 and 18 bound to the 5-HT1A
and 5-HT receptor’s sites by two common interactions: i) a charge-reinforced hydrogen bond
7
between protonated nitrogen atom of the ligand and carboxyl group of Asp3.32, and ii) CH-π
stacking of an arylpiperazine fragment and aromatic rings of aminoacid residues from
transmembrane helix (TMH) 6, mainly Phe6.52 (figure 5). The studies exposed several

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additional interactions characteristic for each receptor type, which distinctly stabilized the
particular complexes and therefore were responsible for developing pronounced affinity of the
compounds in question. In the case of the 5-HT1A receptor, the substituted phenylpiperazine
moiety formed interactions with Lys191 of dual nature – hydrogen bond, where chlorine atom
acts as electronegative acceptor (specific hydrogen bond formed with halogen atom of
compound 13 – figure 5A), and aromatic cation-π (compound 13 and 18, figure 1A and 1B,
respectively). On the other side of the binding site, the compounds form analogous
interactions – H-bond between carbonyl group in position-2 of the hydantoin moiety and
Asn7.39, as well as aromatic π-π between benzylidene moiety and Tyr2.64 (figure 5A, 5B). In
the case of 5-HT receptor, the specific interactions concern the part of receptor active site
7
formed between TMHs 2,3 and 7. In particular, Arg7.36 donates hydrogen bond to the 2-
carbonyl group of the hydantoin fragment, and additionally binds the molecule (benzylidene
fragment) via cation-π aromatic interaction (figure 5C and 5D, showing compound 13 and 18,
respectively).
Figure 5.
3
. Conclusion
In the current study, a series of 5-arylidenehydantoins with arylpiperazinealkyl moiety was
synthesized and evaluated as serotonin receptor ligands and potential inhibitors of PDE4B
enzyme. The binding studies revealed that all compounds exhibited satisfactory affinities for

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either 5-HT1A or 5-HT receptor, but did not significantly inhibit PDE4B. However, the
7
studies allowed identifying two compounds (13, 18) with very high affinity for both 5-HT1A
and 5-HT receptors (compound 13: 0.2 and 0.8 nM, respectively; compound 18: 1.0 and 1.0
7
nM, respectively), which were classified as the antagonists of these receptors. In addition, the
interactions of compounds 13 and 18 with 5-HT1A and 5-HT receptors were investigated
7
using a molecular modeling approach which explained their affinity for the targets of interest.
After testing their cytotoxicity using the MTT test, compounds 13 and 18 were further
evaluated by preliminary pharmacological in vivo studies using a mouse model of depression
which showed that they exhibited specific antidepressant-like activity, similar to the reference
drug citalopram. Although both compounds showed significant antidepressant-like activity,
they need to be further optimized to improve their activity and metabolic stability.
Acknowledgments
This study was financially supported by National Science Center grant (No. DEC-
2
012/07/B/NZ7/01173). Part of this work was presented at 2nd School of Medicinal
Chemistry, 17-19 June 2015, Wroclaw, Poland. This work was also supported by the Medical
University of Warsaw and carried out with the use of CePT infrastructure financed by the
European Union – the European Regional Development Fund within the Operational
Programme Innovative Economy for 2007-2013.
Conflict of interest
Authors declare no conflict of interest.
4
. Experimental Part
.1 Chemistry
4
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. Thin-layer chromatography (TLC) was
performed on Merck silica gel 60 F254 aluminium sheets (Merck; Darmstadt, Germany), using
the following mixtures of solvents: (S ) petroleum ether / ethyl acetate (5:5), (S ) methylene
1
2
chloride / methanol (9:0.5), (S ) methylene chloride / methanol (9:0.7). Analytical HPLC
3
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 (up to 100%) with 0.1% TFA as mobile phase at a flow rate of 5 ml/min. The purity
of the investigated compounds (9-20) ranged from 95 to 99%. Additionally, the liquid
chromatography/mass spectrometry (LC/MS) analysis was performed on Waters Acquity

JOURNAL PRE-PROOF
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 (from 95% to 0% of water for 10 min, then 100% water for 2 min)
with 0.1% TFA was used as a mobile phase at a flow rate of 0.3 ml/min. The retention time
(tR) was measured in minutes. NMR spectra were recorded on either Varian Mercury 300
MHz or JOEL 500 Hz spectrometer; chemical shifts are expressed in parts per million (ppm),
using the solvent (CDCl or DMSO-d ) signal as an internal standard. Signal multiplets are
3
6
represented by the following abbreviations: s (singlet), brs (broad singlet), d (doublet), t
triplet), quin (quintet), 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.
The analytical data of starting compounds: 3,4-dimethoxybenzaldehyde (1),39 3-
2
7
(
cyclopentyloxy)-4-methoxybenzaldehyde (2), 5-(3,4-dimethoxybenzylidene)imidazolidine-
2
9
40
2
,4-dione (3) 5-(3-cyclopentyloxy-4-methoxybenzylidene)imidazolidine-2,4-dione (4) and
intermediate product: 3-(3-chloropropyl)-5-(3,4-dimethoxybenzylidene)-imidazolidine-2,4-
4
1
dione (5) were previously published (Supplementary materials, chemistry).
.1.1 General procedure for alkylation of 5-arylidenehydantoin (6-8)
The intermediate products (6-8) were obtained according to the methods described before
4
3
2
with slightly modifications. A mixture of the appropriate 5-arylidenehydantoin (3 or 4; 2
mmol) and potassium carbonate (0.304 g, 2.2 mmol) in solution of tetrahydrofuran/water (4:1)
was heated to 50°C. After 30 minutes of heating, an appropriate dihalogenoalkane (2.2 mmol)
was dropwise added to reaction mixture, which was kept in 50°C for 12 hours. Subsequently
the reaction mixture was extracted with methylene chloride and concentrated under vacuum.
The resulting residue was purified by column chromatography on silica gel, using methylene
chloride / methanol (9:0.5) as eluent.
4
.1.1.1
(Z)-3-(4-Bromobutyl)-5-(3,4-dimethoxybenzylidene)-imidazolidine-2,4-
dione (6)
Creamy powdery crystals. Yield: 54%; mp 167.4-168.2°C; TLC: R = 0.54 (S ); HPLC: t =
f
2
R
+
1
1
.686; MS calcd for [M + H] : C H BrN O m/z: 383.24, found: 385.06; H NMR (300
1
6
19
2
4
MHz, CDCl -d) δ ppm 1.77 - 1.91 (m, 4 H), 3.53 - 3.60 (m, 2 H), 3.63 - 3.71 (m, 2 H), 3.94
3
(
s, 3 H), 3.95 (s, 3 H), 6.72 (s, 1 H), 6.90 - 6.97 (m, 2 H), 7.03 - 7.12 (m, 1 H), 8.48 (s, 1 H).
.1.1.2 (Z)-3-(3-Chloropropyl)-5-(3-cyclopentyloxy-4-methoxybenzylidene)-
imidazolidine-2,4-dione (7)
4

JOURNAL PRE-PROOF
Creamy powdery crystals. Yield: 57%; mp 168.0-169.2°C; TLC: R = 0.62 (S ); HPLC: t =
f
2
R
+
1
1
.736; MS calcd for [M + H] : C H ClN O m/z: 378.85, found: 379.35; H NMR (300
1
9
23
2
4
MHz, CDCl -d) δ ppm 1.57 - 1.68 (m, 2 H) 1.79 - 1.98 (m, 6 H) 2.17 (quin, J=6.67 Hz, 2 H)
3
3
.59 (t, J=6.41 Hz, 2 H) 3.80 (t, J=6.92 Hz, 2 H) 3.88 (s, 3 H) 4.77 - 4.84 (m, 1 H) 6.69 (s, 1
H) 6.87 - 6.95 (m, 2 H) 7.01 - 7.06 (m, 1 H) 8.03 (s, 1 H).
.1.1.3 (Z)-3-(4-Chlorobutyl)-5-(3-cyclopentyloxy-4-methoxybenzylidene)-
4
imidazolidine-2,4-dione (8)
Creamy powdery crystals. Yield: 60%; mp 177.5-178.7°C; TLC: R = 0.63 (S ); HPLC: t =
f
2
R
+
1
1
.820; MS calcd for [M + H] : C H ClN O m/z: 392.88, found: 393.38; H NMR (300
2
0
25
2
4
MHz, CDCl -d) δ ppm 1.59 - 1.65 (m, 2 H) 1.78 - 1.97 (m, 10 H) 3.54 - 3.60 (t, J=6.05, 2 H)
3
3
.63 - 3.70 (t, J=6.65, 2 H) 3.88 (s, 3 H) 4.78 - 4.84 (m, 1 H) 6.68 (s, 1 H) 6.88 - 6.94 (m, 2
H) 6.99 - 7.05 (m, 1 H) 8.06 (s, 1 H).
.1.2 General procedure for obtaining the final compounds (9-20)
A mixture of the appropriate intermediate (5-8; 0.3 mmol), potassium carbonate (0.054 g,
.39 mmol) and arylpiperazine (0.39 mmol) in a solution of tetrahydrofuran/water (4:1) was
4
0
heated to 50°C. After 24 hours of heating, the reaction mixture was extracted with methylene
chloride and concentrated under vacuum. The resulting residue was purified by column
chromatography on silica gel, using methylene chloride / methanol (9:0.7) as eluent.
4
.1.2.1 (Z)-5-(3,4-dimethoxybenzylidene-3-(3-(4-(2-methoxyphenyl)piperazine-1-
yl)propyl)-imidazolidine-2,4-dione (9)41
White powdery crystals. Yield: 52%; mp 135.8-136.6°C; TLC: R = 0.33 (S ); HPLC: t =
f
3
R
+
1
1
.032; MS calcd for [M + H] : C H N O m/z: 480.56, found: 481.17; H NMR (300 MHz,
26 32 4 5
CDCl -d) δ ppm 1.86 - 1.98 (m, 2 H) 2.52 (t, J=6.92 Hz, 2 H) 2.66 (br. s., 4 H) 3.05 (br. s., 4
3
H) 3.70 (t, J=6.80 Hz, 2 H) 3.84 (s, 3 H) 3.90 (s, 6 H) 6.68 (s, 1 H) 6.80 - 6.86 (m, 3 H) 6.91
1
3
(
d, J=3.33 Hz, 2 H) 6.93 - 6.99 (m, 1 H) 7.04 (dd, J=8.21, 1.80 Hz, 1 H) 8.52 (br. s., 1 H); C
NMR (126 MHz, CDCl -d)  ppm 24.83, 37.51, 50.50, 53.41, 55.41, 56.04, 56.07, 56.15,
3
1
1
11.20, 111.61, 112.09, 112.31, 112.34, 118.28, 121.01, 122.49, 123.04, 125.00, 125.99,
49.49, 150.07, 152.31, 155.85, 164.30.
4
.1.2.2 (Z)-5-(3,4-dimethoxybenzylidene-3-(3-(4-(2,3-dichlorophenyl)piperazine-1-
yl)propyl)-imidazolidine-2,4-dione (10)
White powdery crystals. Yield: 42%; mp 187.8-188.2°C; TLC: R = 0.39 (S ); HPLC: t =
f
3
R
+
1
1
.221; MS calcd for [M + H] : C H Cl N O m/z: 519.42, found: 519.12; H NMR (300
25 28 2 4 4
MHz, CDCl -d) δ ppm 1.84 - 1.96 (m, 2 H) 2.52 (t, J=6.80 Hz, 2 H) 2.61 (br. s., 4 H) 3.01 (br.
3
s., 4 H) 3.72 (t, J=6.80 Hz, 2 H) 3.91 (d, J=1.54 Hz, 6 H) 6.69 (s, 1 H) 6.82 (dd, J=7.95, 1.80

JOURNAL PRE-PROOF
1
3
Hz, 1 H) 6.88 - 6.94 (m, 2 H) 7.02 - 7.09 (m, 2 H) 7.10 - 7.15 (m, 1 H) 8.45 (br. s., 1 H); C
NMR (126 MHz, CDCl -d)  ppm 24.77, 37.45, 51.12, 53.25, 55.92, 56.09, 56.19, 111.62,
3
1
1
12.07, 112.44, 118.67, 122.59, 124.73, 124.90, 125.96, 127.47, 127.59, 134.07, 149.53,
50.14, 151.16, 155.90, 164.26.
4
.1.2.3
yl)propyl)-imidazolidine-2,4-dione (11)
Yellow powdery crystals. Yield: 73%; mp 168.8-169.2°C; TLC: R = 0.27 (S ); HPLC: t =
(Z)-5-(3,4-dimethoxybenzylidene-3-(3-(4-(pirimidin-2-yl)piperazine-1-
f
3
R
+
1
0
.916; MS calcd for [M + H] : C H N O m/z: 452.5, found: 453.19; H NMR (300 MHz,
23 28 6 4
CDCl -d) δ ppm 1.90 (quin, J=6.73 Hz, 2 H) 2.26 - 2.53 (m, 6 H) 3.63 - 3.81 (m, 6 H) 3.90 (d,
3
J=4.87 Hz, 6 H) 6.40 - 6.48 (m, 1 H) 6.68 (s, 1 H) 6.74 - 6.94 (m, 2 H) 6.95 - 7.08 (m, 1 H)
1
3
8
4
1
.23 - 8.30 (m, 2 H) 8.54 (br. s., 1 H); C NMR (126 MHz, CDCl -d)  ppm 24.93, 37.44,
3
3.51, 53.06, 55.91, 56.07, 56.15, 111.61, 112.03, 112.37, 122.44, 124.92, 125.95, 149.14,
49.51, 150.10, 157.77, 161.62, 164.21.
4
.1.2.4 (Z)-5-(3,4-dimethoxybenzylidene-3-(4-(4-(2-methoxyphenyl)piperazine-1-
yl)butyl)-imidazolidine-2,4-dione (12)
Creamy powdery crystals. Yield: 90%; mp 495.19 80.7-81.7°C; TLC: R = 0.35 (S ); HPLC:
f
3
+
1
t = 1.060; MS calcd for [M + H] : C H N O m/z: 494.58, found: 495.19; H NMR (300
R
27 34
4
5
MHz, CDCl -d) δ ppm 1.55 - 1.66 (m, 2 H) 1.67 - 1.79 (m, 2 H) 2.44 - 2.53 (m, 2 H) 2.69 (br.
3
s., 4 H) 3.10 (br. s., 4 H) 3.65 (t, J=6.80 Hz, 2 H) 3.85 (s, 3 H) 3.91 (s, 3 H) 3.93 (s, 3 H) 6.68
(s, 1 H) 6.83 - 6.87 (m, 1 H) 6.88 - 6.94 (m, 4 H) 6.96 - 7.01 (m, 1 H) 7.02 - 7.07 (m, 1 H)
1
3
8
5
1
.38 (br. s., 1 H); C NMR (126 MHz, CDCl -d)  ppm 23.73, 26.23, 38.61, 50.31, 53.36,
3
5.43, 56.07, 56.20, 57.96, 111.23, 111.61, 111.63, 112.14, 112.55, 112.56, 118.36, 121.08,
22.62, 123.13, 124.81, 125.96, 149.50, 150.12, 152.31, 164.22.
4
.1.2.5 (Z)-5-(3,4-dimethoxybenzylidene-3-(4-(4-(2,3-dichlorophenyl)piperazine-1-
yl)butyl)-imidazolidine-2,4-dione (13)
Creamy powdery crystals. Yield: 51%; mp 167.2-169.0°C; TLC: R = 0.34 (S ); HPLC: t =
f
3
R
+
1
1
.240; MS calcd for [M + H] : C H Cl N O m/z: 533.45, found: 533.06; H NMR (300
2
6
30
2
4
4
MHz, CDCl -d) δ ppm 1.57 - 1.68 (m, 2 H) 1.69 - 1.80 (m, 2 H) 2.52 (br. s., 2 H) 2.70 (br. s.,
3
4
6
H) 3.10 (br. s., 4 H) 3.66 (t, J=6.92 Hz, 2 H) 3.92 (d, J=3.08 Hz, 6 H) 6.69 (s, 1 H) 6.89 -
1
3
.97 (m, 3 H) 6.99 - 7.06 (m, 1 H) 7.09 - 7.19 (m, 2 H) 8.09 (br. s., 1 H); C NMR (75 MHz,
CDCl -d) δ ppm 23.86, 26.13, 38.56, 51.07, 53.18, 55.99, 56.11, 57.80, 111.51, 112.01,
3
1
12.54, 118.60, 122.63, 124.60, 124.63, 125.84, 127.42, 127.45, 133.99, 149.42, 150.07,

JOURNAL PRE-PROOF
1
5
51.13, 155.84, 164.10. Anal. calcd for C H Cl N O : C 58.54, H 5.67, N 10.50; found: C
2
6
30
2
4
4
8.23, H 5.74, N – 10.23.
.1.2.6 (Z)-5-(3,4-dimethoxybenzylidene-3-(4-(4-(pirimidin-2-yl)piperazine-1-
yl)butyl)-imidazolidine-2,4-dione (14)
Creamy powdery crystals. Yield: 43%; mp 175.8-176.2°C; TLC: R = 0.28 (S ); HPLC: t =
4
f
3
R
+
1
0
.948; MS calcd for [M + H] : C H N O m/z: 466.53, found: 467.21; H NMR (300 MHz,
24 30 6 4
CDCl -d) δ ppm 1.50 - 1.65 (m, 2 H) 1.66 - 1.80 (m, 2 H) 2.35 - 2.58 (m, 6 H) 3.65 (t, J=6.92
3
Hz, 2 H) 3.83 (br. s., 4 H) 3.91 (s, 3 H) 3.93 (s, 3 H) 6.47 (t, J=4.74 Hz, 1 H) 6.68 (s, 1 H)
6
.87 - 6.95 (m, 2 H) 7.04 (dd, J=8.21, 1.80 Hz, 1 H) 8.29 (d, J=4.62 Hz, 2 H) 8.42 (br. s., 1
1
3
H); C NMR (126 MHz, CDCl -d)  ppm 26.13, 29.40, 38.46, 43.24, 52.97, 56.10, 56.19,
3
5
1
7.92, 111.66, 112.03, 112.46, 122.35, 124.85, 125.95, 149.55, 150.14, 155.40, 157.83,
61.58, 164.09.
4
.1.2.7
methoxyphenyl)piperazine-1-yl)propyl)-imidazolidine-2,4-dione (15)
White powdery crystals. Yield: 68%; mp 76.8-77.9°C; TLC: R = 0.30 (S ); HPLC: t = 1.262;
(Z)-5-(3-cyclopentyloxy-4-methoxybenzylidene-3-(3-(4-(2-
f
3
R
+
1
MS calcd for [M + H] : C H N O m/z: 534.65, found: 535.21; H NMR (300 MHz, CDCl -
3
0
38
4
5
3
d) δ ppm 1.54 - 1.67 (m, 2 H) 1.77 - 1.97 (m, 8 H) 2.50 (t, J=6.80 Hz, 2 H) 2.62 (br. s., 4 H)
.04 (br. s., 4 H) 3.71 (t, J=6.80 Hz, 2 H) 3.85 (d, J=8.46 Hz, 6 H) 4.75 - 4.82 (m, 1 H) 6.67
s, 1 H) 6.81 - 6.86 (m, 3 H) 6.89 (d, J=3.08 Hz, 2 H) 6.93 - 6.99 (m, 1 H) 7.02 (d, J=8.21 Hz,
3
(
1
3
1
5
1
H) 8.21 (br. s., 1 H); C NMR (126 MHz, CDCl -d)  ppm 24.17, 24.94, 32.90, 37.56,
3
0.64, 53.46, 55.41, 56.09, 56.15, 80.78, 111.19, 112.26, 115.85, 118.27, 121.01, 121.90,
22.96, 124.97, 125.89, 141.34, 141.35, 148.15, 151.11, 152.33, 155.43, 164.26.
4
.1.2.8
dichlorophenyl)piperazine-1-yl)propyl)-imidazolidine-2,4-dione (16)
White powdery crystals. Yield: 51%; mp 189.9-190.4°C; TLC: R = 0.33 (S ); HPLC: t =
(Z)-5-(3-cyclopentyloxy-4-methoxybenzylidene-3-(3-(4-(2,3-
f
3
R
+
1
1
.422; MS calcd for [M + H] : C H Cl N O m/z: 573.51, found: 573.09; H NMR (300
29 34 2 4 4
MHz, CDCl -d) δ ppm 1.55 - 1.66 (m, 2 H) 1.81 - 1.96 (m, 8 H) 2.51 (t, J=6.80 Hz, 2 H) 2.60
3
(br. s., 4 H) 3.00 (br. s., 4 H) 3.72 (t, J=6.80 Hz, 2 H) 3.87 (s, 3 H) 4.78 (td, J=5.77, 3.08 Hz,
1
7
3
1
H) 6.67 (s, 1 H) 6.81 (dd, J=7.95, 1.54 Hz, 1 H) 6.86 - 6.92 (m, 2 H) 7.01 - 7.07 (m, 2 H)
1
3
.09 - 7.14 (m, 1 H) 8.20 (br. s., 1 H); C NMR (126 MHz, CDCl -d)  ppm 24.15, 24.78,
3
2.89, 37.48, 51.20, 53.27, 55.95, 56.16, 80.81, 112.27, 112.32, 115.92, 118.67, 121.96,
21.98, 124.67, 124.95, 125.87, 127.46, 127.59, 134.05, 148.16, 151.16, 151.23, 164.30.

JOURNAL PRE-PROOF
4
.1.2.9
(Z)-5-(3-cyclopentyloxy-4-methoxybenzylidene-3-(3-(4-(pirimidin-2-
yl)piperazine-1-yl)propyl)-imidazolidine-2,4-dione (17)
Creamy powdery crystals. Yield: 62%; mp 118.8-119.6°C; TLC: R = 0.25 (S ); HPLC: t =
f
3
R
+
1
1
.163; MS calcd for [M + H] : C H N O m/z: 506.6, found: 507.22; H NMR (300 MHz,
27 34 6 4
CDCl -d) δ ppm 1.55 - 1.64 (m, 2 H) 1.81 - 1.96 (m, 8 H) 2.46 (t, J=4.87 Hz, 6 H) 3.71 (t,
3
J=6.80 Hz, 2 H) 3.74 - 3.80 (m, 4 H) 3.85 (s, 3 H) 4.75 - 4.83 (m, 1 H) 6.44 (t, J=4.74 Hz, 1
H) 6.66 (s, 1 H) 6.84 - 6.90 (m, 2 H) 7.01 (dd, J=8.34, 1.92 Hz, 1 H) 8.26 (d, J=4.87 Hz, 2 H)
1
3
8
5
1
.33 (br. s., 1 H); C NMR (126 MHz, CDCl -d)  ppm 24.14, 24.93, 32.87, 37.40, 43.53,
3
3.06, 56.07, 56.13, 80.78, 109.92, 112.23, 112.49, 115.97, 122.07, 124.85, 125.82, 148.09,
51.15, 155.64, 157.76, 161.59, 164.31.
4
.1.2.10
methoxyphenyl)piperazine-1-yl)butyl)-imidazolidine-2,4-dione (18)
Creamy powdery crystals. Yield: 67%; mp 77.9-79.1°C; TLC: R = 0.44 (S ); HPLC: t =
(Z)-5-(3-cyclopentyloxy-4-methoxybenzylidene-3-(4-(4-(2-
f
3
R
+
1
1
.289; MS calcd for [M + H] : C H N O m/z: 548.67, found: 549.3; H NMR (300 MHz,
31 40 4 5
CDCl -d) δ ppm 1.55 - 1.67 (m, 4 H) 1.68 - 1.78 (m, 2 H) 1.79 - 1.96 (m, 6 H) 2.48 (t, J=6.54
3
Hz, 2 H) 2.69 (br. s., 4 H) 3.10 (br. s., 4 H) 3.66 (t, J=6.92 Hz, 2 H) 3.87 (s, 3 H) 3.85 (s, 3 H)
4
.77 - 4.85 (m, 1 H) 6.67 (s, 1 H) 6.82 - 6.93 (m, 5 H) 6.95 - 7.03 (m, 2 H) 7.95 (br. s., 1 H);
1
3
C NMR (75 MHz, CDCl -d) δ ppm 24.06, 24.10, 26.23, 32.80, 38.66, 50.61, 53.41, 55.31,
3
5
1
6
6.05, 58.06, 80.72, 111.14, 112.16, 112.38, 115.93, 118.19, 120.95, 121.91, 122.83, 124.72,
25.79, 141.36, 148.03, 151.08, 152.25, 155.43, 164.10. Anal. calcd for C H N O x H 0: C
3
1
40
4
5
2
5.70, H 7.47, N 9.89; found: C 66.00, H 7.32, N – 9.71.
.1.2.11 (Z)-5-(3-cyclopentyloxy-4-methoxybenzylidene-3-(4-(4-(2,3-
dichlorophenyl)piperazine-1-yl)butyl)-imidazolidine-2,4-dione (19)
4
Creamy powdery crystals. Yield: 51%; mp 156.7-157.3°C; TLC: R = 0.44 (S ); HPLC: t =
f
3
R
+
1
1
.432; MS calcd for [M + H] : C H Cl N O m/z: 587.54, found: 587.19; H NMR (300
3
0
36
2
4
4
MHz, CDCl -d) δ ppm 1.53 - 1.64 (m, 4 H) 1.67 - 1.77 (m, 2 H) 1.80 - 1.94 (m, 6 H) 2.38 -
3
2
4
1
2
1
1
.45 (m, 2 H) 2.46 - 2.52 (m, 4 H) 3.65 (t, J=6.92 Hz, 2 H) 3.78 - 3.83 (m, 4 H) 3.87 (s, 3 H)
.76 - 4.84 (m, 1 H) 6.46 (t, J=4.74 Hz, 1 H) 6.66 (s, 1 H) 6.86 - 6.91 (m, 2 H) 6.97 - 7.02 (m,
1
3
H) 7.96 (br. s., 1 H) 8.28 (d, J=4.62 Hz, 2 H); C NMR (126 MHz, CDCl -d)  ppm 24.16,
3
6.21, 32.90, 38.58, 51.08, 53.26, 56.16, 57.88, 80.83, 112.26, 112.52, 115.94, 118.73,
22.00, 124.75, 124.79, 125.83, 127.55, 127.58, 134.10, 148.15, 151.16, 151.20, 155.33,
64.16.
4
.1.2.12
(Z)-5-(3-cyclopentyloxy-4-methoxybenzylidene-3-(4-(4-(pirimidin-2-
yl)piperazine-1-yl)butyl)-imidazolidine-2,4-dione (20)

JOURNAL PRE-PROOF
Creamy powdery crystals. Yield: 68%; mp 144.4-146.8°C; TLC: R = 0.37 (S ); HPLC: t =
f
3
R
+
1
1
.185; MS calcd for [M + H] : C H N O m/z: 520.62, found: 521.18; H NMR (300 MHz,
28 36 6 4
CDCl -d) δ ppm 1.52 - 1.65 (m, 4 H) 1.68 - 1.77 (m, 2 H) 1.79 - 1.94 (m, 6 H) 2.37 - 2.52 (m,
3
6
H) 3.65 (t, J=7.05 Hz, 2 H) 3.77 - 3.83 (m, 4 H) 3.87 (s, 3 H) 4.77 - 4.84 (m, 1 H) 6.46 (t,
J=4.87 Hz, 1 H) 6.65 - 6.73 (m, 1 H) 6.85 - 6.91 (m, 2 H) 6.94 - 7.03 (m, 1 H) 7.96 (br. s., 1
1
3
H) 8.28 (d, J=4.62 Hz, 2 H); C NMR (126 MHz, CDCl -d)  ppm 24.15, 26.20, 32.89,
3
3
1
8.56, 43.47, 53.04, 56.14, 58.01, 80.81, 109.99, 112.23, 112.55, 115.97, 122.04, 124.78,
25.82, 148.11, 151.18, 155.44, 157.79, 161.64, 164.19.
4
4
.2 Pharmacology
.2.1 In vitro assays
Radioligand binding studies were employed to determine the affinity of the newly synthesized
compounds (9-20) for serotonin 5-HT , 5-HT , 5-HT , 5-HT and dopamine D receptors
1
A
7
2A
6
2
(table 2). This was accomplished by displacement of respective radioligands (Supplementary
materials, table 2) from cloned human receptors, according to a previously described
4
2
methods. For serotonin 5–HT and 5–HT receptors, the inhibition constant values were
1
A
7
-6
-11
determined at 11 concentrations (10 M – 10 M) whereas for 5–HT , 5–HT and D
2
2
A
6
-6
receptors the percent inhibition of specific binding was estimated in two concentration (10 M
-7
and 10 M). Briefly: binding experiments were conducted in 96-well microplates in a total
volume of 250 μl of appropriate buffers. Reaction mix included 50 μl solution of test
compound, 50 μl of radioligand and 150 μl of diluted membranes. Specific assay conditions
for each receptors are shown in supplementary materials. Recombinant human proteins were
used for serotonin and dopamine receptors. The radioactivity was measured in MicroBeta2
scintillation counter (PerkinElmer, USA). Each compound was tested in assay at eleven (10-
6
-11
-6
-7
M – 10 M for 5–HT and 5–HT ) and two (10 M and 10 M, for 5–HT , 5–HT and D )
1
A
7
2A
6
2
final concentrations. Results were expressed as constant affinity or percent inhibition of
specific binding.
4
.2.2 Functional assays for 5-HT receptor
1A
Tested and reference compounds were dissolved in dimethyl sulfoxide (DMSO) at
a concentration of 1 mM. Serial dilutions were prepared in 96-well microplate in assay buffer
and 8 to 10 concentrations were tested.
A cellular aequorin-based functional assay was performed with recombinant CHO-K1 cells
expressing mitochondrially targeted aequorin, human GPCR and the promiscuous G protein
4
2
α16 for 5-HT . Assay was executed according to previously described protocol. After
1
A
thawing, cells were transferred to assay buffer (DMEM/HAM’s F12 with 0.1% protease free

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BSA) and centrifuged. The cell pellet was resuspended in assay buffer and coelenterazine h
was added at final concentrations of 5 μM. The cells suspension was incubated at 16 °C,
protected from light with constant agitation for 16 h and then diluted with assay buffer to the
concentration of 5000 cells/ml. After 1 h of incubation, 50 μl of the cells suspension was
dispensed using automatic injectors built into the radiometric and luminescence plate counter
MicroBeta2 LumiJET (PerkinElmer, USA) into white opaque 96-well microplates preloaded
with test compounds. Immediate light emission generated following calcium mobilization was
recorded for 30 s. In antagonist mode, after 25 min of incubation the reference agonist was
added to the above assay mix and light emission was recorded again. Final concentration of
the reference agonist was equal to EC80 (100 nM serotonin).
4
.2.3 Functional assays for 5-HT receptor
7
Tested and reference compounds were dissolved in dimethyl sulfoxide (DMSO) to the
concentration of 1 mM. Serial dilutions were prepared in 96-well microplate in assay buffers
and 8 concentrations were tested.
For the 5-HT adenylyl cyclase activity were monitored using cryopreserved CHO-K1 cells
7
with expression of the human serotonin 5-HT receptor. Functional assay based on cells with
7
expression of the human 5-HT receptor was performed. CHO - K1 cells were transfected
7
with a beta lactamase (bla) reporter gene under control of the cyclic AMP response
element (CRE) (Life Technologies). Thawed cells were resuspended in stimulation buffer
(HBSS, 5 mM HEPES, 0.5 IBMX, and 0.1% BSA at pH 7.4) at 2x105 cells/ml for 5-HT7
receptor. The same volume (10 μl) of cell suspension was added to tested compounds for 5-
HT receptor. Samples were loaded onto a white opaque half area 96-well microplate. The
7
antagonist response experiment was performed with 10 nM serotonin as the reference agonist
for 5-HT receptor. The agonist and antagonist were added simultaneously. Cell stimulation
7
was performed for 60 min at room temperature. After incubation, cAMP measurements were
performed with homogeneous TR-FRET immunoassay using the LANCE Ultra cAMP kit
(PerkinElmer, USA). 10 μl of EucAMP Tracer Working Solution and 10 μl of ULight-anti-
cAMP Tracer Working Solution were added, mixed, and incubated for 1 h. The TR-FRET
signal was read on POLAR STAR BMG microplate reader. IC 50 and EC 50 were determined
by nonlinear regression analysis using GraphPad Prism 6.0 software.
4
.2.4 Protocols for measuring PDE 4B1 inhibition in vitro
The phosphodiesterase (PDE) inhibitory activity was determined (table 2) using recombinant
human PDE 4B1 (5ng/well) expressed in Baculovirus infected Sf9 cells (BPS Biosciences),
and combined with substrate: 10 µM cAMP (Sigma). Using a PDELight® kit (Lonza), the

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amount of AMP, produced in the reaction of the cAMP hydrolysis, was quantified using
PDELight AMP Detection Reagent which converts the AMP directly to ATP. The assay used
luciferase, which catalyzed the formation of light from the newly formed ATP and luciferin.
Briefly: Tested and reference compounds were dissolved in dimethyl sulfoxide (DMSO) at a
concentration of 1 mM and further diluted in assay buffer (10 mM Tris-HCl, 10 mM
magnesium chloride and 0,05% Tween-20; pH 7,4). The final concentration of compounds
-5
was 10 M. All reactions were carried out at 37 °C in white, half-area 96-well plate (Perkin
Elmer). The inhibition of PDE enzyme was measured using PDElight HTS cAMP
phosphodiesterase assay kit (Lonza) according to manufacturer’s recommendations. 5 ng of
PDE4B1enzyme (Sigma-Aldrich) in appropriate buffer was incubated with reference and
tested compound for 20 minutes. After incubation the cAMP substrate (final concentration 10
µM for PDE4B1) was added and incubated for 1 hour. Then PDELight AMP Detection
Reagent was added and incubated 10 minutes. Luminescence was measured in a multifunction
plate reader (POLARstar Omega, BMG Labtech, Germany).
4
.3 Cell culture condition for MTT assay
The human primary colon cancer (SW480), metastatic prostate cancer (PC3) and immortal
keratinocyte (HaCaT) cell lines were obtained from the American Type Culture Collection
(ATCC Rockville MD, USA). SW480 cells were cultured in MEM, PC3 cells in RPMI 1640
and HaCaT cells in DMEM High Glucose medium. All media were supplemented with 10%
FBS, penicillin (100 U/mL), streptomycin (100 μg/mL), and HEPES (20 mM) (Thermo Fisher
Sci Waltham, MA, USA). The cells were cultured in 50 mL tissue flasks in 37°C/5% CO2
humidified incubator.
After reaching 80-90% confluence cells were passaged using trypsin (0.25%)–EDTA (0.02%)
and seeded in 96-well plates at density of 1 × 104 cells per well for MTT assay. The cells
were treated with serial concentrations (10, 20, 40, 60, 80, 100, 140 µM) of tested compounds
followed by 24 h pre-incubation. Untreated cancer and normal cells were used as a control.
4
.3.1 MTT assay
MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Sigma-Aldrich, St
4
3
Louis MO, USA) assay was performed according to Denizot and Lang. The assay is based
on the metabolic reduction of soluble MTT by mitochondrial dehydrogenase activity from
viable cells, into an insoluble purple formazan product.
After 72 h incubation in the presence of tested compounds the cells were given free-serum
medium containing MTT (0.5 mg/mL) and incubated for 4 h at 37°C in a CO humidified
2
incubator. Subsequently, medium was removed and obtained formazan crystals were

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dissolved in DMSO and isopropanol (1:1). Optical density of the solution of each well was
measured at 570 nm using UVM 340 reader (ASYS Hitech GmbH, Austria) at a wavelength
of 570 nm. The IC50 value was estimated using CompuSyn version 1.0 and expressed in µM.
4
.4 In vivo studies
The experiments were performed on male Swiss albino mice (22–26 g) purchased from a
licensed breeder Staniszewska (Ilkowice, Poland). 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%;
1
2:12 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. Each
experimental group consisted of 7–10 animals/dose and the animals were used only once. The
experiments were performed by an observer unaware of the treatment administered.
4
.4.1 Forced swim test in Swiss albino mice.
The experiment was carried out according to the method of Porsolt et al.44 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.
4
.4.2 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.
.4.3 Drugs.
(
(
4
The following drugs were used: citalopram (hydrochloride, Adamed Pharmaceuticals) and
tested compounds 13 and 18. Citalopram was dissolved in distilled water; remaining
compounds were suspended in a 1% aqueous solution of Tween 80 immediately before
administration. Compounds 13 and 18 were administered intraperitoneally (ip) 60 min and

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citalopram ip 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.
4
.4.4 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.
4
.5 Metabolic stability screen in mouse liver microsomes (MLMs)
Mouse liver microsomes (MLMs) and all reagents such as NADPH-regenerating system
components and levallorphan were supplied by Sigma Aldrich.
Stock solution was performed in destiled water. Microsomes were carefully thawed on ice
before the experiment. Test compound (final concentration of 20µM) and 0.4 mg/mL of
appropriate microsomal proteins (MLMs) in potassium phosphate buffer (pH 7.4,100 mM)
were preincubated at 37 oC for 15 min. The incubation reaction was initiated with the
addition of NADPH-regenerating system (NADP, glucose-6-phosphate, glucose-6-phosphate
dehydrogenase, 100 mM potassium phosphate buffer) and mixtures were incubated at 37°C
for various time periods (up to 30 min). Next, an internal standard (levallorphan, 20µM) was
added, and the reactions were terminated with perchloric acid. Negative control incubations
containing no NADPH-regenerating system were conducted.45,46 The samples were
centrifuged and the supernatants were subjected to LC-MS/MS analysis (UPLC/MS, Waters
Corporation, Milford, MA, USA). The in vitro half-time (t ) and intrinsic clearance (Cl ) of
1
/2
int
test compound in liver microsomes were determined according to literature procedures.36,47
.6 Molecular modeling
The homology models of human 5-HT1A and 5-HT serotonin receptors used herein were
4
7
4
8
generated based on ligand-steered optimization method and were presented in previously
published papers.49–51
The homology models were built on the basis of β adrenergic receptor crystal structure (PDB
2
ID: 2RH1).52 Sequence alignments between target receptors (UniProt database accession
numbers P08908 and P34969 respectively) and the template were performed by hhsearch tool
5
3
54
via GeneSilico Metaserver. The crude receptor models were obtained using SwissModel,
5
5
and were validated by processing in Protein Preparation Wizard. For each receptor type, a
set of bioactive compounds was selected for ligand-steered binding site optimization, which
5
6
was performed using induced fit docking (IFD) workflow. 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

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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
Small Molecule Drug Discovery Suite (Schrödinger Ltd.), which was licensed for Jagiellonian
University Medical College.

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