Computer-aided insights into receptor-ligand interaction for novel 5-arylhydantoin derivatives as serotonin 5-HT7 receptor agents with antidepressant activity

Article abstract of DOI:10.1016/j.ejmech.2018.01.093

This paper presents a computer-aided insight into the receptor-ligand interaction for novel analogs of the lead structure 5-(4-fluorophenyl)-3-(2-hydroxy-3-(4-(2-methoxyphenyl)piperazin-1-yl)propyl)-5-methylimidazolidine-2,4-dione (1, MF-8), as part of the search for potent and selective serotonin 5-HT₇ receptor (5-HT₇R) agents. New hydantoin derivatives (4-19) were designed and synthesized. For 5-phenyl-3-(2-hydroxy-3-(4-(2-ethoxyphenyl)piperazin-1-yl)propyl)-5-methylimidazolidine-2,4-dione (4), its crystal structure was determined experimentally. Molecular modeling studies were performed, including both pharmacophore and structure-based approaches. New compounds were investigated in radioligand binding assays (RBA) for their affinity toward 5-HT₇R and selectivity over 5-HT_1AR, dopamine D₂R and α₁-, α₂-and β-adrenoceptors. Selected compounds (5-8) were assessed for their antidepressant and anxiolytic effects in vivo in mice. Most of the tested compounds displayed potent affinity and selectivity for 5-HT₇R in RBA, in particular seven compounds (4, 5, 7, 8 and 10-12, K_i ≤ 10 nM). Antidepressant-like activity in vivo for all tested compounds (5-8) was confirmed. SAR analysis based on both crystallography-supported molecular modeling and RBA results indicated that mono-phenyl substituents at both hydantoin and piperazine are more favorable for 5-HT₇R affinity than the di-phenyl ones.

Full text of DOI:10.1016/j.ejmech.2018.01.093

Accepted Manuscript
Computer-aided insights into receptor-ligand interaction for novel 5-arylhydantoin
derivatives as serotonin 5-HT receptor agents with antidepressant activity
7
Katarzyna Kucwaj-Brysz, Rafał Kurczab, Magdalena Jastrzębska-Więsek, Ewa
Żesławska, Grzegorz Satała, Wojciech Nitek, Anna Partyka, Agata Siwek, Agnieszka
Jankowska, Anna Wesołowska, Katarzyna Kieć-Kononowicz, Jadwiga Handzlik
PII:
S0223-5234(18)30115-6
10.1016/j.ejmech.2018.01.093
EJMECH 10170
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 16 December 2017
Revised Date: 25 January 2018
Accepted Date: 30 January 2018
Please cite this article as: K. Kucwaj-Brysz, Rafał. Kurczab, M. Jastrzębska-Więsek, E. Żesławska, G.
Satała, W. Nitek, A. Partyka, A. Siwek, A. Jankowska, A. Wesołowska, K. Kieć-Kononowicz, J. Handzlik,
Computer-aided insights into receptor-ligand interaction for novel 5-arylhydantoin derivatives as
serotonin 5-HT receptor agents with antidepressant activity, European Journal of Medicinal Chemistry
7
(2018), doi: 10.1016/j.ejmech.2018.01.093.
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ACCEPTED MANUSCRIPT
Graphical abstract

ACCEPTED MANUSCRIPT
Computer-aided insights into receptor-ligand interaction for novel 5-arylhydantoin
derivatives as serotonin 5-HT7 receptor agents with antidepressant activity
Katarzyna Kucwaj-Brysza Rafał Kurczabb, Magdalena Jastrzębska-Więsekc, Ewa Żesławskad,
,
Grzegorz Satałab, Wojciech Niteke, Anna Partykac, Agata Siwekf, Agnieszka Jankowskaa, Anna
Wesołowskac, Katarzyna Kieć-Kononowicza, Jadwiga Handzlika*
aDepartment of Technology and Biotechnology of Drugs, Jagiellonian University Medical
College, Medyczna 9, 30-688 Kraków, Poland
bDepartment of Medicinal Chemistry Institute of Pharmacology, Polish Academy of Sciences,
Smętna 12, 31-343 Kraków, Poland
cDepartment of Clinical Pharmacy, Jagiellonian University Medical College, Medyczna 9,
30-688 Cracow, Poland
dDepartment of Chemistry, Institute of Biology, Pedagogical University of Cracow,
Podchorążych 2, 30-084 Kraków, Poland
eFaculty of Chemistry, Jagiellonian University, Gronostajowa 2, 30-387 Kraków,
Poland
fDepartment of Pharmacobiology, Jagiellonian University Medical College, Medyczna 9,
30-688 Kraków, Poland
* Corresponding author: J. Handzlik
tel.: +48 012 620 55 80; fax: +48 012 620 55 96. E-mail: jhandzli@cm-uj.krakow.pl
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Abstract
This paper presents a computer-aided insight into the receptor-ligand interaction for novel
analogs of the lead structure 5-(4-fluorophenyl)-3-(2-hydroxy-3-(4-(2-
methoxyphenyl)piperazin-1-yl)propyl)-5-methylimidazolidine-2,4-dione (1, MF-8), as part of
the search for potent and selective serotonin 5-HT7 receptor (5-HT7R) agents. New hydantoin
derivatives (4-19) were designed and synthesized. For 5-phenyl-3-(2-hydroxy-3-(4-(2-
ethoxyphenyl)piperazin-1-yl)propyl)-5-methylimidazolidine-2,4-dione (4), its crystal
structure was determined experimentally. Molecular modeling studies were performed,
including both pharmacophore and structure-based approaches. New compounds were
investigated in radioligand binding assays (RBA) for their affinity toward 5-HT7R and
selectivity over 5-HT1AR, dopamine D2R and α1-, α2- and β-adrenoceptors. Selected
compounds (5-8) were assessed for their antidepressant and anxiolytic effects in vivo in mice.
Most of the tested compounds displayed potent affinity and selectivity for 5-HT7R in RBA, in
particular seven compounds (4, 5, 7, 8 and 10-12, Ki ≤ 10 nM). Antidepressant-like activity in
vivo for all tested compounds (5-8) was confirmed. SAR analysis based on both
crystallography-supported molecular modeling and RBA results indicated that mono-phenyl
substituents at both hydantoin and piperazine are more favorable for 5-HT7R affinity than the
di-phenyl ones.
Keywords:
serotonin receptors, 5-HT7R ligands, hydantoin, arylpiperazine, docking, antidepressant, CNS
drugability
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1. Introduction
The latest discovery from the serotoninergic system, the 5-HT7 receptor (5-HT7R) [1-3], is the
subject of many studies concerning CNS functioning. 5-HT7R plays a pivotal role in various
physiological processes, e.g. thermoregulation, circadian rhythm, learning and memory [4].
Moreover, it seems to contribute in pathological processes, including schizophrenia, migraine
or stress [5]. Preclinical studies involving commonly used behavioral tests on animals, the
forced swim test (FST) and the tail suspension test (TST), have shown that antagonism of the
5-HT7 receptor causes anti-depressant effects in rats and mice [6,7].
Serotonin receptor 5-HT7, as a member of the GPCR superfamily, features a structure
homologous to that of other receptors belonging to this group. Therefore, the design and
synthesis of agents, which are not only potent but also selective, is highly complicated. To
date, only one selective 5-HT7R ligand, compound JNJ-18038683, has been clinically
evaluated to confirm its efficacy in patients with major depressive disorder. However, the
obtained results are inconclusive because of the low sensitivity of the applied experiment [8].
Therefore, searching for novel selective 5-HT7R agents seems to be relevant in terms of future
effective treatment of depression.
Recently, our group discovered the first potent and selective 5-HT7R ligand among 5-
phenylhydantoin derivatives, the compound MF-8 (1, Fig. 1) [9]. Further studies led to the
synthesis, biological evaluation and structure-activity relationship (SAR) analysis of 14
derivatives of 1 [10], resulting in the selection of three substituents at the piperazine moiety
(Fig. 1) that provide the best potency and selectivity [10].
Fig. 1
This current study is the next step, in that it concerns an evaluation of the role of
substituents at position 5 of hydantoin in terms of the desired pharmacological action on 5-
HT7R (Fig. 1). Thus, sixteen new derivatives 4-19 (Table 1) were designed and synthesized.
For one representative compound (4), the crystal structure was determined experimentally to
support molecular modeling studies for the whole series, including both pharmacophore and
structure-based approaches, by using docking and molecular dynamics simulations for the
receptor-ligand complexes. All compounds (4-19) were investigated in radioligand binding
assays to estimate their 5-HT7R affinity and selectivity over 5-HT1AR and D2R as well as over
adrenoceptors α1-AR, α2-AR and β1-AR for representative members (1, 3-12). The most
active 5-HT7R agents (5-8) were also assessed for their antidepressant and anxiolytic-like
effects in vivo in mice. Finally, the computer-aided SAR analysis was discussed.
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Table 1
2. Results and discussion
2.1. Synthesis
The new compounds 4-19 were synthesized via two- or three-step synthesis pathways
(Scheme 1) elaborated based on methods described previously [9, 10].
As the initial step for synthesis of compounds 4-17 (group A and B), 5-aryl-5-
methylhydantoins (20-26) were obtained within Bucherer-Bergs cyclic condensation of
appropriately substituted commercial acetophenones. In the case of derivatives 18 and 19
(group C), commercial 5,5-diphenylhydantoin (DPH) was used as a starting material. In the
next step, an introduction of a 2,3-epoxypropyl moiety into position 3 of hydantoin was
performed to obtain compounds 27-34. In contrast to the previous method [9, 10], Mitsunobu
reaction by using oxiran-2-ylmethanol was replaced by a more economical and
environmentally friendly N-alkylation method using 2-(chloromethyl)oxirane as an alkylation
agent. According to green chemistry rules, the reaction involved water in place of the
previously used dry THF, the purification process was much easier and yields were better
when compared to those of Mitsunobu ones. Similar to the synthesis of 1-3 [9,10], the
solvent-free N-alkylations of appropriate commercial arylpiperazines with intermediates (27-
34) were performed under microwave irradiation.
Scheme 1
The final products, obtained in amorphous basic forms, were converted into crystalline
hydrochloric salts (4-19) useful for biological studies. All the compounds were synthesized as
diastereoisomers (racemic mixtures in the cases of 18 and 19) due to the application of both a
non-stereospecific Bucherer-Bergs reaction and N-alkylation with a racemic mixture of 2-
(chloromethyl)oxirane.
2.2. X-ray crystallographic studies
The crystal structure of compound 4, the first and representative member of the newly
synthesized derivatives of lead 1, was determined by the X-ray diffraction method (Fig. 2,
Fig. 3 and Table 2). The unit cell consisted of four 4 molecules and two n-decane molecules,
which came from the solvent. The molecular geometry in the crystal structure of 4 with its
atom numbering is shown in Fig. 2.
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Fig. 2
The molecule possesses two chiral centers at C5 and C7. The crystal structure confirmed
(R,S) configuration at C5 and (S,R) at C7. The molecule of the investigated compound was
protonated at nitrogen atom N2 and created an extended conformation with the torsion angles
of the linker: N3-C6-C7-C8 = 179.4(1)°, C6-C7-C8-N2 = 171.3(1)°. The piperazine moiety
adopted chair conformation with equatorial substituents at N2 and N4 atoms. The phenyl ring
at N4 position was not coplanar with the piperazine moiety, and the torsion angles C14-C13-
N4-C10 and C18-C13-N4-C10 were -70.7(2)° and 111.2(1)°, respectively. This conformation
is less frequent in comparison to other crystal structures with a benzene ring at N4 in a
piperazine moiety deposited in the Cambridge Structural Database (CSD, ConQuest version
1.18) [11]. In most of the crystal structures with an arylpiperazine moiety, the phenyl ring is
more or less coplanar with the piperazine ring (torsion angles in the vicinity of 0° or 180°).
Moreover, a weak intramolecular hydrogen bond C-H···O was observed between the carbon
atom of the piperazine moiety (C10) as a donor, and the oxygen atom (O3) of the ethoxy
group as an acceptor (Table 2). The aromatic ring at C5 was almost perpendicular to the
hydantoin ring; the angle between the planes of these rings was 86.73(5)°. The angle between
the two aromatic rings was 43.80(5)°. The chlorine anion connected two molecules via
hydrogen bonds between the protonated nitrogen atom (N2) and the hydroxyl group of one
molecule and the nitrogen atom (N1) of the hydantoin ring of another molecule (Fig. 3).
Furthermore, the dimers were stabilized by both C-H···O and C-H···Cl interactions (Table 2).
The n-decane molecules were not engaged in any intermolecular contacts.
Fig. 3
Table 2
2.3. Molecular modeling studies
Docking studies were performed to gain insights into the mechanism of binding and to
estimate the binding mode description. The stability of the receptor-ligand complex was
investigated using molecular dynamics simulations. Next, it was determined how the newly
synthesized ligands fit into the known pharmacophore models of 5-HT7R antagonists. Finally,
CNS drugability for the series of arylpiperazine hydantoins (4-19) was examined.
2.3.1. Docking and molecular dynamics
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Molecular docking of the newly synthesized compounds (4-19) was performed with the
use of recently developed 5-HT7R homology models built on the dopaminergic D3 receptor
template (PDB ID: 3PBL) [12-17]. The binding mode of representative compound 4 is
presented in Fig. 4A.
Fig. 4
The main ligand-receptor interactions involved the following components: (i) a salt-bridge
with Asp3.32, (ii) CH–π/π–π interactions between the 2-ethoxyphenylpiperazine moiety and
the side chains of Phe6.51 and Phe6.52, (iii) π–π stacking interactions with Phe3.28 and (iv)
the hydrogen bond between the hydantoin fragment and Cys146 of ECL2. The analysis of 100
ns MD trajectory confirmed the key ligand-receptor interactions found in molecular docking;
however, it indicated that the hydantoin moiety is additionally stabilized by hydrogen bonds
with Arg7.36 and Arg6.58 (Fig. 4B, Fig. S1, Supplementary) and aromatic interaction with
Phe3.28 is less significant (see L-P interaction diagram, frequency less than 10%). Compound
4 stayed in the 5-HT7R binding cavity during the entire simulation (the ligand RMSD plot,
Fig. S2, Supplementary) and remained in very close conformation to those obtained from
docking and crystallographic studies (Fig. 5, see also Fig. S3, Supplementary).
Fig. 5
2.3.2. Pharmacophore-based simulation
Since 3-dimensional (3D) pharmacophore models for 5-HT7R antagonists have been
developed and successfully used in several studies [19-22], two of them (Fig. 6) were
reconstructed using the same datasets and parameters as had been reported in the original
papers [23, 24].
Fig. 6
The structural features of the compounds described here correlate with the predicted regions
in the aforementioned 5-HT7R antagonist pharmacophore models. A comparison of the fitting
of compound 11 to the model proposed by Lopez-Rodriguez with that of compound SB-
656104 (Fig. 6A) led to the conclusion that the hydantoin moiety, with a carbonyl group as
HBA, is a bioisostere of the sulfonamide group in the structure of SB-656104.
The mapping to the pharmacophore model of Bojarski’s research group (Fig. 6B) indicated
some differences between phenylpiperazine (4-15, 18, 19) and benzhydrylpiperazine
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derivatives (16 and 17) in terms of the position of the positive ionizable group relevant for the
5-HT7R affinity (PI). Thus, PI was placed on the piperazine nitrogen at the propyl chain for
the phenylpiperazine compounds (4-15, 18, 19), whereas the piperazine nitrogen at the
diphenylmethyl group fitted in with this pharmacophoric feature (PI) for the benzhydryl
compounds (16 and 17).
2.3.3. CNS-drugability prediction
The Multiparameter Optimization algorithm (MPO CNS), according to the procedure
developed by Wager et al., [25] was used to estimate the CNS drug-likeness properties of
newly synthesized series 4-19 (Table S1, Supplementary). The MPO CNS includes six
physicochemical properties (ClogP, ClogD, MW, TPSA, HBD, pKa) and its score ranges
from 0.00 to 6.00. For the synthesized library, the CNS MPO value changes from 4.19
(compound 19) to 4.84 (compound 5), which indicates their high CNS drugability.
2.4. Pharmacology
2.4.1. Radioligand binding assays
Radioligand binding assays were applied to determine the affinity and selectivity profiles of
the newly synthesized compounds for human serotonin 5-HT7bR, 5-HT1AR, and dopaminergic
D2LR, which were stably expressed in HEK293 cells. Additionally, ten representative
compounds (1, 3-12) were evaluated for their affinities for adrenergic receptors α1-, α2- and
β1-AR in the tissue rat cortex (Table 1).
Most of the tested compounds (4-15 and 17) demonstrated high affinities for 5-HT7R (Ki <
100 nM); very potent affinity was observed for 4, 5, 7, 8 and 10-12 (Ki ≤ 10 nM). Each of the
highly active compounds featured predominant affinity toward 5-HT7R in comparison to both
5-HT1AR and D2R. Compounds 16 and 17 showed the highest selectivity to 5-HT7R over 5-
HT1AR, and compounds 5 and 6 exerted 5-HT7R/5-HT1A selectivity at a similar level to that of
lead (1). The highest selectivity 5-HT7R/D2R rate, but lower than that of 1, was observed for
compounds 5, 6, 10 and 12. Results for selected compounds tested for their affinities for
adrenoceptors (1, 3-12) indicated a significantly weaker action on α1-AR than that on 5-HT7R
and a negligibly weak action on both α2-AR and β1-AR (Table 1).
2.4.2. Behavioral tests in vivo
For representative compounds (5-8), screening in vivo behavioral tests were performed with
Albino Swiss mice. To assess antidepressant- and anxiolytic-like activity, the forced swim test
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(FST, Fig. 7; Table S2, Supplementary) [26] and the four-plate test (FPT, Table S3,
Supplementary) [27, 28], respectively, were conducted. Moreover, the spontaneous locomotor
activity assay was performed to exclude hyperactivity of mice (Table S4, Supplementary).
Results indicated that the whole group (5-8) significantly decreased immobility time of mice
at the highest doses (10 and/or 20 mg/kg), while the lower doses had no pronounced effect in
this test (Fig. 7). The observed decrease in immobility time after i.p. injection of 20 mg/kg of
compounds 5 (F(3,31)=39.754; p<0.0001), 7 (F(3,31)=19.394; p<0.0001), and 8
(F(3,31)=15.535; p<0.001) was similar to the same dose of the reference compound
imipramine (F(3,36)=16.757; p<0.001). The most potent antidepressant-like activity was
observed for compound 6 (at the dose of 20 mg/kg) which significantly decreased the
immobility time of mice by about 80% vs the respective vehicle group (F(3,28)=32.753;
p<0.0001). Moreover, compound 7 showed antidepressant-like activity at the lowest dose of
all investigated compounds (10 mg/kg) and decreased the immobility time by almost 50% vs
the respective vehicle group, but this activity was not as high as for 20 mg/kg of 6 (Fig. 7).
Fig. 7
In the FPT, compounds 5-8 were investigated in comparison to diazepam as a reference
anxiolytic drug. The number of punished crossings observed for animals after i.p.
administration of 5-8 was lower than that for diazepam. In the case of compounds 5, 6 and 8,
there is a tendency to increase the measure parameter but the results did not reach a
significant level (Table S3, Supplementary). Thus, the hydantoin 5-HT7R agents (5-8) did not
demonstrate anxiolytic-like properties.
The compounds (5-8) did not change the spontaneous locomotor activity in mice (Table S4,
Supplementary data). Thus, the observed anti-immobility effect in the FST (Fig. 7) seems to
be specific, which is in accordance to the results of previously tested compound 3 [29].
2.5. Structure-activity relationship (SAR) analysis
The performed research provided data for a comprehensive SAR-analysis. The radioligand
binding results distinctly showed a highly potent action on 5-HT7R, comparable to or higher
than that of the reference clozapine, for most tested (2-cyano)- and (2-
alkoxyphenyl)piperazine derivatives (4-13). Our chemical modifications were focused on the
substitution at position 5 of hydantoin. A difference of the affinity, related to properties of that
substitution, is especially visible for changes of the p-substituent position at the 5-phenyl ring
(1 and 14, 15) as well as for an addition of one more aromatic moiety (18, 19). In contrast, an
exchange of fluorine at para position of the 5-phenylhydantoin phenyl ring with a bigger
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sized halogen atom (chlorine, bromine) or with the methoxyl- or methyl- group did not affect
the activity significantly. However, the p-methoxyl substituent seems to be the most profitable
one (11 and 12; Table 1).
Molecular modeling indicated a different orientation of the 5-phenylhydantoin ring in the
binding site depending on the type of substituents in para position (Fig. 8A). Both the methyl-
and methoxyl- derivatives formed π-π interaction with Phe3.28, whereas halogenated
derivatives (F-, Cl- and Br-) pointed toward a channel formed by ECL2 and Arg7.36 and
Glu7.35. In contrast, the affinity for 5-HT7R was distinctly decreased due to the shift of
fluorine from para to ortho (15) or meta (18) position, thus indicating position para as
optimal. The docking study suggested that the decrease in the considered affinity may be a
result of interplay between steric and polar interactions formed by C-F bonds, depending on
their position (Fig. 8B). An approximate 10-fold decrease in the affinity was observed for
analogs representing modification A (9) and C (16). A comparison of its binding modes
showed that the benzhydrylpiperazine derivative (16) was placed in a less favorable
orientation to interact with Asp3.32 (Fig. 8C). Replacement of the 5-methylhydantoin methyl
group with a phenyl one within the structure of compounds 5 and 6, respectively, caused a
great reduction of the affinity for 5-HT7R (18 and 19), and this was especially pronounced
compared with the affinities of the two (2-cyanophenyl)piperazine compounds 6 and 19
(Table 1).
Fig. 8
As far as selectivity is concerned, this chemical group had a very weak tendency to act on α2-
AR and β1-adrenoceptors. Thus, both the α2-AR/5-HT7R and β1-AR/5-HT7R selectivity values
were large for all tested compounds, without any regular SAR. The best results toward 5-
HT7R in respect to all D2R, 5-HT1AR and α1-AR were demonstrated by compounds with the
unsubstituted phenyl ring of the 5-methyl-5-phenylhydantoin and the 2-MeO or 2-CN
substituent at phenylpiperazine (5 or 6). In general, a tendency was observed for the (2-
cyanophenyl)piperazine derivatives to higher D2R/5-HT7R selectivity, to as much as 140- to
190-fold (6, 10 and 12). The 4-methoxyphenyl derivative (12) was also the most selective 5-
HT7R agent over α1-adrenoceptors. In contrast, the highest selectivity toward 5-HT7R in
respect to 5-HT1AR was observed for the benzhydrylpiperazine derivatives 16 and 17. In
particular, the 5-(4-methoxyphenyl)hydantoin moiety together with the benzhydrylpiperazine
one (17) conditioned the most potent 5-HT1AR/ 5-HT7R selectivity (>200-fold, Table 1). A
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similar tendency for the benzhydryl compound (3) was found in our previous studies [10, 29].
The significant selectivity together with high metabolic stability seems to be the main reason
for the impressive in vivo action recently described for compound 3 [29]. This is also a good
prognosis for its 4-methoxy analog (17), which marks this new compound as a good candidate
for further extended studies, including in vivo assays on animals and pharmaceutical profile
evaluation in vitro.
Analysis of CNS MPO results (Table S1, Supplementary) led to the conclusion that the
described new hydantoin compounds are good CNS-agent candidates (CNS MPO scores >
4.00). The highest scores were observed for derivatives with (2-methoxyphenyl)piperazine.
Moreover, the type of substituents at the phenyl ring in position 5 of hydantoin also
influenced CNS drugability, revealing the unsubstituted phenyl ring (compound 5 – score
4.81) to be the most profitable one.
On the other hand, our current in vivo studies, performed for two pairs of 2-methoxy- (5, 7)
and (2-cyanophenyl)piperazine (6, 8) derivatives with 4-bromo or unsubstituted 5-
phenylhydantoin phenyl ring, respectively, did not show any regular SAR. Introduction of a
halogen atom in para position of 5-phenylhydantoin phenyl group did not change the
antidepressant-like activity in the FST; neither did this cause anxiolytic-like properties or
changes to the spontaneous locomotor activity.
3. Conclusions
The performed comprehensive studies, including chemical synthesis, crystallographic analysis
and molecular modeling together with the pharmacological evaluation, enabled a new series
of potent 5-HT7R agents to be found with significant selectivity over five other competitive
GPCR targets in vitro and confirmed their pharmacological properties in vivo.
Results of molecular modeling studies, supported by the crystallographic analysis and data
from the radioligand binding assays, provided a deeper insight into the ligand-receptor
interaction for the novel derivatives (4-19) of lead (1, MF-8). Molecular dynamic simulation
confirmed the stability of the protein-ligand complex, whereas pharmacophore mapping
resulted in the conclusion that the hydantoin moiety might be a bioisostere of the sulfonamide
group. Special attention was paid to the role of substituents at hydantoin position 5. Thus on
the one hand, favorable properties for mono-phenyl substituents of the 5-methyl-5-
phenylhydantoin moiety, in comparison to those of di-aromatic ones of 5,5-
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diphenylhydantoin, were indicated. However on the other hand, para position for substituents
within the mono-aromatic moiety (hydantoin position 5) as predominant in respect to both
ortho and meta ones was found and explained in detail by molecular modeling simulations.
Furthermore, the computer-aided insights into the ligand-receptor interaction provided some
hypotheses that could be helpful to understand (i) the role of substituent types at position para
of 5-phenylhydantoin and (ii) the decrease in the 5-HT7R affinity caused by the di-aromatic
benzhydryl substituent at piperazine moiety. Most of the 5-phenyl-3-(3-(4-phenylpiperazin-1-
yl)-2-hydroxypropyl)-5-methylhydantoin derivatives (4-12) displayed an excellent and
selective action on 5-HT7R (Ki ≤ 20 nM). In addition, an antidepressant-like activity in vivo
was confirmed for those tested in behavioral studies (5-8). A beneficial CNS-drugability for
the whole series (4-19) was also predicted by the MPO method in silico.
Considering the promising results of the primary pharmacological screening, the most active
members of the series should proceed to further stages of drug research and be included in
development processes in the search for new antidepressant drugs acting via 5-HT7R.
4. Experimental
4.1. Synthesis
1H NMR and 13C NMR spectra were recorded on a Varian Mercury VX 300 MHz PFG
instrument (Varian Inc., Palo Alto, CA, USA) in DMSO-d6 at ambient temperature using the
solvent signal as an internal standard. Data are reports using the following abbreviations: s,
singlet; d, doublet; t, triplet; m, multiplet; Ph, phenyl; Pp, piperazine, Ar, aromatic Thin-layer
.
chromatography was performed on pre-coated Merck silica gel 60 F254 aluminum sheets, the
solvent systems used were methylene chloride/methanol 95:5. The mass for compounds 4-34
were recorded on a Waters ACQUITYTM UPLC (Waters, Milford, MA, USA) coupled to a
Waters TQD mass spectrometer (electrospray ionization mode ESI-tandem quadrupole).
Retention times (tR) are given in minutes. The UPLC/MS purity of all final compounds was
determined (%) Syntheses under microwave irradiation were performed in a Samsung
.
MW71B household microwave oven. The procedure for preparation of hydantoins 20-26 has
already been published [9]. Physicochemical data for compounds 20-25 are available in the
literature [30-33].
4.1.1. General procedure for synthesis of (oxiran-2-ylmethyl)imidazolidine-2,4-diones (27-34)
An appropriated hydantoin (5.0 mmol) was suspended in water (~10 mL) followed by
addition of sodium hydroxide (5.0 mmol) in one portion. The solution was stirred at room
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temperature until dissolution of the reagents. Then, 2-(chloromethyl)oxirane (5.0 mmol) was
added and the reaction mixture was stirred for 24 hours at room temperature. Progress of the
reaction was monitored by thin layer chromatography (TLC) in methylene chloride/methanol
9:1. If precipitate occurred, the reaction mixture was filtered giving the desired product
(Method A). Otherwise, the product was extracted from water phase with DCM. The resulting
organic phase was washed with brine, filtered, dried over Na2SO4 and concentrated under a
vacuum to provide a crude product. The desired product was purified with column
chromatography using DCM/MeOH (98:2→95:5) as the eluent (Method B).
4.1.1.1. 5-Phenyl-5-methyl-3-(oxiran-2-ylmethyl)imidazolidine-2,4-dione (27)
Method B. Colorless oil. Yield 54%. LC/MS±: purity 93.27%, tR=4.03, (ESI) m/z [M+H]
247.09. 1H NMR δ (ppm) 8.97 (s, 1H, N1H), 7.48-7.27 (m, 5H, Ar), 3.55-3.53 (d, J = 4.5 Hz,
2H, CH2), 3.11-3.05 (m, 1H, CH), 2.72-2.64 (m, 1H, CH2a), 2.41-2.37 (m, 1H, CH2b), 1.66 (s,
3H, CH3).
4.1.1.2. 5-(4-Bromophenyl)-5-methyl-3-(oxiran-2-ylmethyl)imidazolidine-2,4-dione (28)
Method A. White solid. Yield 81%. LC/MS±: purity 90.09%, tR=5.05, (ESI) m/z [M+H]
326.97. 1H NMR δ (ppm): 9.00 (s, 1H, N1H), 7.61-7.58
(d, J = 7.5 Hz, 2H, Ar), 7.44-7.41 (d,
J = 8.0 Hz, 2H, Ar), 3.58 – 3.48 (d, J = 4.6 Hz, 2H, CH2), 3.10-3.05 (m, 1H, CH), 2.69-2.65
(m, 1H, CH2a), 2.46-2.39 (m, 1H, CH2b), 1.63 (s, 3H, CH3).
4.1.1.3. 5-(4-Chlorophenyl)-5-methyl-3-(oxiran-2-ylmethyl)imidazolidine-2,4-dione (29)
Method A. White solid. Yield 75%. LC/MS±: purity 89.21%, tR=4.90, (ESI) m/z [M+H]
281.05. 1H NMR (ppm) 8.98 (s, 1H, N1H), 7.37-7.34 (d, J = 8.9 Hz, 2H, Ar), 6.94-6.92 (d, J =
7.8 Hz, 2H, Ar) 3.58-3.51(d, J = 4.5 Hz, 2H, CH2), 3.09-3.06 (m, 1H, CH), 2.68-2.65 (m, 1H,
CH2a), 2.44-2.38 m, 1H, CH2b), 1.64 (s, 3H, CH3).
4.1.1.4. 5-(4-Methoxyphenyl)-5-methyl-3-(oxiran-2-ylmethyl)imidazolidine-2,4-dione (30)
Method B. White solid. Yield 57%. LC/MS±: purity 89.32%, tR=4.07, (ESI) m/z [M+H]
1
277.13. H NMR δ (ppm) 8.94 (s, 1H, N1H), 7.37-7.33 (m, 4H, Ar), 3.72 (s, 3H, OCH3) 3.56-
3.52 (d, J = 4.5 Hz, 2H, CH2), 3.07-3.02 (m, 1H, CH), 2.69-2.64 (m, 1H, CH2a), 2.43-2.37 (m,
1H, CH2b), 1.63 (s, 3H, CH3).
4.1.1.5. 5-(4-Methylphenyl)-5-methyl-3-(oxiran-2-ylmethyl)imidazolidine-2,4-dione (31)
Method B. White solid. Yield 59%. LC/MS±: purity 91.27%, tR=4.61, (ESI) m/z [M+H]
1
261.11. H NMR δ (ppm) 8.92 (s, 1H, N1H), 7.34-7.32 (d, J = 7.6 Hz, 2H, Ar), 7.20-7.17 (d, J
= 8.4 Hz, 2H, Ar), 3.54-3.52 (d, J = 4.5 Hz, 2H, CH2), 3.10-3.06 (m, 1H, CH), 2.69-2.65 (m,
1H, CH2a), 2.44-2.37 (m, 1H, CH2b), 2.27 (s, 3H, CH3), 1.66 (s, 3H, CH3).
4.1.1.6. 5-(2-Fluorophenyl)-5-methyl-3-(oxiran-2-ylmethyl)imidazolidine-2,4-dione (32)
12

ACCEPTED MANUSCRIPT
Method B. White solid. Yield 77%. LC/MS±: purity 97.82%, tR=4.30, (ESI) m/z [M+H]
265.17. 1H NMR δ (ppm) 8.92 (s, 1H, N1H), 7.32-7.17 (m, 4H, Ar), 3.56-3.53 (d, J = 4.2 Hz,
2H, CH2), 3.11-3.06 (m, 1H, CH), 2.69-2.65 (m, 1H, CH2a), 2.45-2.38 (m, 1H, CH2b), 1.66 (s,
3H, CH3).
4.1.1.7. 5-(3-Fluorophenyl)-5-methyl-3-(oxiran-2-ylmethyl)imidazolidine-2,4-dione (33)
Method B. White solid. Yield 64%. LC/MS±: purity 93.46%, tR=4.64, (ESI) m/z [M+H]
1
265.17. H NMR δ (ppm): 9.03 (s, 1H, N1H ), 7.47-7.40 (m, 1H, Ph), 7.36-7.28 (m, 2H, Ar),
7.20-7.13 (m, 1H, Ar), 7.03-6.85 (m, 4H, Ar), 3.54-3.52 (d, J = 4.5 Hz, 2H, CH2), 3.10-3.06
(m, 1H, CH), 2.69-2.65 (m, 1H, CH2a), 2.43-2.36 (m, 1H, CH2b), 2.27 (s, 3H, CH3), 1.65 (s,
3H, CH3).
4.1.1.8. 5-Phenyl-5-phenyl-3-(oxiran-2-ylmethyl)imidazolidine-2,4-dione (34)
Method B. White solid. LC/MS ±: purity 92.60%, tR=5.52, (ESI) m/z [M+H] 309.22.
1H NMR δ (ppm): 9.68 (s, 1H, N1H), 7.44-7.28 (m, 10H, Ar), 3.65-3.61 (m, 2H, CH2), 3.15-
3.11 (m, 1H, CH), 2.71-2.68 (m, 1H, CH2a), 2.45-2.42 (m, 1H, CH2b).
4.1.2. General procedure for synthesis of final compounds (4-19)
5-(4-Fluorophenyl)-5-methyl-3-(oxiran-2-ylmethyl)imidazolidine-2,4-dione 17 (3.5 mmol,
1.0 eq) with appropriate piperazine derivative (3.0 mmol, 0.9 eq) in 50 mL flat-bottom flask
were irradiated in household microwave: 450MW (2-3min). Progress of reaction was
controlled by TLC (DCM/MeOH 95:5). Pure products were obtained using column
chromatography with DCM/MeOH (99:1→95:5). All the final products were transformed into
hydrochloride salts by dissolving in 3-4 mL of 1.25 M HCl solution in ethanol. After 30
minutes of stirring at room temperature, resulted precipitates were filtered off, washed with
diethyl ether and dried.
4.1.2.1. 5-phenyl-3-(3-(4-(2-ethoxyphenyl)piperazin-1-yl(-2-hydroxypropyl)-5-
methylimidazolidine-2,4-dione hydrochloride (4)
1
White solid. Yield 66%. LC/MS±: purity 99.26%, tR=4.51, (ESI) m/z [M+H] 453.40. H
NMR δ (ppm): 9.91 (s, 1H, NH+), 8.96 (s, 1H, N1H), 7.51-7.47 (dd, J = 8.2, 1.2 Hz, 2H, Ar),
7.43-7.27 (m, 3H, Ar), 7.00-6.82 (m, 4H, Ar), 5.92 (d, J = 5.6 Hz, 1H, CH-OH), 4.23 (m, 1H,
CH-OH), 4.01 (q, J = 6.9 Hz, 2H, OCH2CH3), 3.61 – 3.34 (m, 6H, Pp-3,5-H, Pp-CH2), 3.27-
13
2.89 (m, 6H, N3-CH2, Pp-2,6-H), 1.71 (s, 3H, CH3), 1.34 (t, J = 6.9 Hz, OCH2CH3, 3H). C
NMR δ (ppm): 175.91, 167.70, 155.94, 151.42, 140.07, 139.98, 128.82, 128.35, 126.04,
123.68, 121.27, 118.63, 113.42, 63.77, 63.32, 55.37, 52.98, 47.03, 42.47, 25.24, 15.21.
13

ACCEPTED MANUSCRIPT
4.1.2.2.
5-phenyl-3-(3-(4-(2-methoxyphenyl)piperazin-1-yl(-2-hydroxypropyl)-5-
methylimidazolidine-2,4-dione hydrochloride (5)
White solid. Yield 74%. LC/MS±: purity 100% tR=4.06, (ESI) m/z [M+H] 439.38. 1H NMR δ
(ppm): 10.14 (s, 1H, NH+), 8.97 (s, 1H, N1H), 7.51-7.47 (dd, J = 8.3, 1.3 Hz, 2H, Ar), 7.41-
7.29 (m, 3H, Ar), 7.07-6.82 (m, 4H, Ar), 4.25 (m, 1H, CHOH), 3.77 (s, 3H, OCH3), 3.61-3.31
(m, 6H, Pp-3,5-H, Pp-CH2), 3.26-2.94 (m, 6H, N3-CH2, Pp-2,6-H), 1.71 (s, 3H, CH3). 13C
NMR δ (ppm): 175.94, 155.93, 152.24, 140.06, 139.55, 128.92, 128.35, 126.04, 121.28,
118.75, 116.55, 112.43, 63.32, 55.84, 52.88, 51.59, 47.23, 42.47, 25.24.
4.1.2.3.
5-phenyl-3-(3-(4-(2-cyanophenyl)piperazin-1-yl(-2-hydroxypropyl)-5-
methylimidazolidine-2,4-dione hydrochloride (6)
White solid. Yield 62%. LC/MS±: purity 100% tR=4.04, (ESI) m/z [M+H] 434.39. 1H NMR δ
(ppm): 10.22 (s, 1H, NH+), 8.96 (s, 1H, N1H), 7.75 (dd, J = 7.7, 1.5 Hz, 1H, Ar), 7.62 (m,
1H), 7.52-7.46 (m, 2H, Ar), 7.43-7.28 (m, 3H, Ar), 7.25-7.12 (m, 2H, Ar), 5.94 (s, 1H,
CHOH), 4.24 (m, 1H, CHOH), 3.75-3.38 (m, 6H, Pp-3,5-H; Pp-CH2), 3.26-2.89 (m, 6H, N3-
CH2, Pp-2,6-H), 1.71 (s, 3H, CH3). 13C NMR δ (ppm): 175.99, 175.95, 156.03, 155.94,
154.17, 140.09, 134.96, 134.73, 128.93, 128.36, 126.04, 123.46, 119.83, 118.35, 105.57,
63.33, 48.32, 25.25.
4.1.2.4.
5-(4-Bromophenyl)-3-(3-(4-(2-methoxyphenyl)piperazin-1-yl(-2-hydroxypropyl)-5-
methylimidazolidine-2,4-dione hydrochloride (7)
White solid. Yield 59%. LC/MS±: purity 100% tR=4.73, (ESI) m/z [M+H] 519.12. 1H NMR δ
(ppm): 10.20 (s, 1H, NH+ ), 9.01 (s, 1H, N1H ), 7.61-7.57 (d, J = 7.59 Hz, 2H, Ar), 7.46-7.43
(d, J = 7.45 Hz, 2H, Ar), 7.09-6.80 (m, 4H, Ar), 4.24 (s, 1H, CHOH), 3.77 (s, 3H, OCH3),
3.64-3.32 (m, 6H, Pp-3,5-H; Pp-CH2 ), 3.30-2.93 (m, 6H, N3-CH2, Pp-2,6-H), 1.69 (s, 3H,
CH3). 13C NMR δ (ppm): 175.60, 155.93, 155.85, 152.25, 139.57, 131.82, 128.42, 123.72,
121.77, 121.28, 118.74, 112.43, 63.03, 55.84, 52.92, 46.90, 42.66, 25.27.
4.1.2.5.
5-(4-Bromophenyl)-3-(3-(4-(2-cyanophenyl)piperazin-1-yl(-2-hydroxypropyl)-5-
methylimidazolidine-2,4-dione hydrochloride (8)
White solid. Yield 47%. LC/MS±: purity 98.81% tR=4.67, (ESI) m/z [M+H] 514.30. 1H NMR
δ (ppm): 10.50 (s, 1H, NH+), 9.00 (s, 1H, N1H), 7.77-7.62 (m, 2H, Ar), 7.76-7.62 (m, 2H, Ar),
7.62-7.42 (m, 4H, Ar), 7.26-7.11 (m, 2H, Ar), 5.93 (s, 1H, CHOH), 4.26 (s, 1H, CHOH),
3.75-3.34 (m, 6H, Pp-3,5-H; Pp-CH2), 3.30-3.05 (m, 6H, N3-CH2, Pp-2,6-H), 1.69 (s, 3H,
CH3). 13C NMR δ (ppm): 175.56, 155.94, 155.86, 154.18, 139.57, 139.52, 134.96, 134.72,
131.82, 128.42, 123.19, 121.79, 119.83, 118.35, 105.56, 63.04, 59.33, 52.57, 48.03, 47.86,
42.44, 25.31.
14

ACCEPTED MANUSCRIPT
4.1.2.6. 5-(4-Chlorophenyl)-3-(3-(4-(2-methoxyphenyl)piperazin-1-yl(-2-hydroxypropyl)-5-
methylimidazolidine-2,4-dione hydrochloride (9)
White solid.
Yield 52%. LC/MS±: purity 98.31% tR=4.63, (ESI) m/z [M+H] 473.12. 1H NMR
δ (ppm): 10.47 (s, 1H, NH+), 9.02 (s, 1H, N1H ), 7.53-7.45 (m, 4H, Ar), 7.02-6.85 (m, 4H,
Ar), 4.26 (s, 1H, CHOH), 3.77 (s, 3H, OCH3), 3.66-3.32 (m, 6H, Pp-3,5-H; Pp-CH2), 3.30 -
13
2.96 (m, 6H, 6H, N3-CH2, Pp-2,6-H), 1.70 (s, 3H, CH3). C NMR δ (ppm): 175.58, 156.29,
156.24, 152.36, 141.64, 139.30, 133.12, 128.88, 128.85, 127.94, 122.74, 121.24, 118.27,
112.29, 64.84, 63.05, 62.72, 62.69, 55.71, 54.00, 50.39, 43.63, 25.57.
4.1.2.7.
5-(4-Chlorophenyl)-3-(3-(4-(2-cyanophenyl)piperazin-1-yl(-2-hydroxypropyl)-5-
methylimidazolidine-2,4-dione hydrochloride (10)
White solid. Yield 58%. LC/MS±: purity 95.39% tR=4.52, (ESI) m/z [M+H] 468.36. 1H NMR
δ (ppm): 10.54 (s, 1H, NH+), 9.00 (s, 1H, N1H), 7.74 (d, J = 7.6 Hz, 1H, Ar), 7.68-7.57 (m,
1H, Ar), (7.53-7.40, m, 4H, Ar), 7.27-7.09 (m, 2H, Ar), 5.93 (s, 1H, CH-OH), 4.27 (m, 1H,
CH-OH), 3.76-3.36 (m, 6H, Pp-3,5-H; Pp-CH2), 3.27-2.96 (s, 6H, N3-CH2, Pp-2,6-H), 1.70 (s,
3H, CH3). 13C NMR δ (ppm): 175.62, 155.93, 155.86, 154.18, 139.13, 134.95, 134.72,
133.19, 128.88, 128.08, 123.45, 119.82, 118.34, 105.56, 62.98, 52.73, 48.38, 25.37.
4.1.2.8.
5-(4-Chlorophenyl)-3-(3-(4-(diphenylmethyl)piperazin-1-yl)-2-hydroxypropyl)-5-
methylimidazolidine-2,4-dione hydrochloride (11)
White solid. Yield 38%. LC/MS±: purity 96.29% tR=5.62, (ESI) m/z [M+H] 533.38. 1H NMR
δ (ppm): 9.96 (s, 1H, NH+), 8.96 (s, 1H, N1H), 7.55-7.39 (m, 8H, Ar), 7.32-7.27 (t, J = 7.29
Hz, 4H, Ar), 7.21-7.16 (m, 2H, Ar), 5.87 (s, 1H, CH-OH), 4.41 (s, 1H, CH(Ph)2), 4.17 (m,
1H, CH-OH), 3.48-3.34 (m, 4H, Pp-3,5-H)), 3.17-2.92 (m, 4H, Pp-2,6-H), 2.84-2.70 (m, 2H,
Pp-CH2,), 2.43-2.29 (m, 2H, N3-CH2), 1.68 (s, 3H, CH3).13C NMR δ (ppm): 175.78, 163.39,
160.95, 156.28, 156.23, 143.33, 136.56, 128.92, 128.19, 128.11, 128.00, 127.24, 115.77,
115.54, 75.59, 64.99, 64.83, 62.98, 62.68, 53.87, 51.87, 43.68, 25.88, 25.82.
4.1.2.9. 5-(4-methoxyphenyl)-3-(3-(4-(2-methoxyphenyl)piperazin-1-yl(-2-hydroxypropyl)-5-
methylimidazolidine-2,4-dione hydrochloride (12)
White solid. Yield 53%. LC/MS±: purity 97.85% tR=4.11, (ESI) m/z [M+H] 469.14. 1H NMR
δ (ppm): 10.76 (s, 1H, NH+), 8.94 (s, 1H, N1H), 7.38 (d, J = 8.8 Hz, 2H, Ar), 7.08-6.82 (m,
6H, Ar), 4.36-4.21 (m, 1H, CH-OH), 3.74 (s, 3H, OCH3), 3.71 (s, 3H, CH3), 3.67-3.35 (m,
6H, Pp-3,5-H; Pp-CH2), 3.17 (m, 6H, N3-CH2, Pp-2,6-H), 1.67 (s, 3H, CH3). 13C NMR δ
(ppm): 176.26, 159.33, 156.00, 155.92, 152.25, 139.41, 132.02, 127.33, 121.28, 118.80,
114.25, 112.46, 62.92, 55.84, 55.62, 25.18.
15

ACCEPTED MANUSCRIPT
4.1.2.10. 5-(4-Methoxyphenyl)-3-(3-(4-(2-cyanophenyl)piperazin-1-yl(-2-hydroxypropyl)-5-
methylimidazolidine-2,4-dione hydrochloride (13)
White solid. Yield 51%. LC/MS±: purity 96.85% tR=4.08, (ESI) m/z [M+H] 464.37. 1H NMR
δ (ppm): 10.39 (s, 1H, NH+), 8.90 (s, 1H, N1H), 7.76 -7.73 (dd, J = 7.7, 1.5 Hz, 1H, Ar), 7.68-
7.58 (m, 1H, Ar), 7.44-7.34 (m, 2H, Ar), 7.26-7.11 (m, 2H, Ar), 6.93 (d, J = 8.9 Hz, 2H, Ph),
5.92 (s, 1H, CHOH), 4.26 (s, 1H, CHOH), 3.72 (s, 3H, OCH3), 3.70-3.35 (m, 6H, Pp-3,5-H;
Pp-CH2), 3.31-3.03 (m, 6H, N3-CH2, Pp-2,6-H), 1.63 (s, 3H, CH3). 13C NMR δ (ppm):
176.21, 159.34, 156.01, 154.18, 134.95, 134.72, 132.05, 127.31, 123.45, 119.82, 118.35,
114.25, 105.56, 62.92, 55.62, 47.99, 25.21.
4.1.2.11. 5-(4-Methoxyphenyl)-3-(3-(4-(diphenylmethyl)piperazin-1-yl)-2-hydroxypropyl)-5-
methylimidazolidine-2,4-dione hydrochloride (14)
White solid. Yield 47%. LC/MS±: purity 96.80% tR=5.39, (ESI) m/z [M+H] 529.36. 1H NMR
δ (ppm): 8.84 (s, 1H, s, 1H, N1H), 8.02-7.50 (m, 4H, Ar), 7.38-7.35 (m, 8H, Ar), 6.93-6.90 (d,
J = 8.8 Hz, 2H), 4.20 (m, 1H, CH-OH), 4.02-4.00 (m, 1H, CH(Ph)2), 3.69 (s, 3H, CH3), 3.61 -
13
2.84 (m, 12H, Pp-3,5-H, Pp-2,6-H, Pp-CH2, N3-CH2), 1.66 (s, 3H, CH3). C NMR δ (ppm):
176.26, 159.33, 156.00, 155.92, 152.25, 152.24, 139.41, 132.02, 127.33, 124.16, 121.28,
118.80, 114.25, 112.46, 62.92, 55.84, 55.62, 47.13, 25.18.
4.1.2.12. 5-(4-Methylphenyl)-3-(3-(4-(2-methoxyphenyl)piperazin-1-yl(-2-hydroxypropyl)-5-
methylimidazolidine-2,4-dione hydrochloride (15)
White solid. Yield 41%. LC/MS±: purity 95.44% tR=4.38, (ESI) m/z [M+H] 453.54. 1H NMR
δ (ppm): 10.26 (s, 1H, NH+), 8.91 (s, 1H, N1H ), 7.27 (dd, J = 53.2, 8.3 Hz, 4H, Ar), 7.04-6.81
(m, 4H, Ar), 4.26 (s, 1H, CHOH), 3.65-3.32 (m, 6H, Pp-3,5-H; Pp-CH2), 3.10 (m, 6H, N3-
CH2, Pp-2,6-H), 2.27 (s, 3H, Ph-CH3), 1.66 (s, 3H, CH3). 13C NMR δ (ppm): 176.12, 156.01,
155.93, 152.24, 139.23, 137.62, 137.14, 129.44, 125.94, 124.22, 121.28, 118.86, 112.47,
63.14, 55.85, 25.11, 21.00.
4.1.2.13. 5-(2-fluorophenyl)-3-{3-[4-(2-methoxyphenyl)piperazin-1-yl]-2-hydroxypropyl}-5-
methylimidazolidine-2,4-dione hydrochloride (16)
White solid. Yield 59%, LC/MS±: purity 97.34% tR=3.99, (ESI) m/z [M+H] 457.17. 1H NMR
δ (ppm): 10.46 (s, 1H, NH+), 8.75 (d, J = 3.8 Hz, 1H, N1H ), 7.58-7.53 (d, J = 16.2 Hz, 1H,
Ar), 7.47-7.40 (m, 1H, Ar), 7.22 (m, 2H, Ar), 6.95 (m, 4H, Ar), 4.28 (s, 1H, CHOH), 3.77 (s,
3H, OCH3), 3.62-3.39 (m, 6H, Pp-3,5-H; Pp-CH2), 3.30-2.95 (m, 6H, N3-CH2, Pp-2,6-H),
1.78 (m, 3H, CH3). 13C NMR δ (ppm): 176.15, 159.07, 158.84, 156.00, 152.26, 139.42,
131.35, 129.38, 124.83, 123.73, 121.29, 118.81, 112.48, 63.37, 60.78, 60.68, 59.23, 55.86,
47.30, 23.29.
16

ACCEPTED MANUSCRIPT
4.1.2.14. 5-(3-fluorophenyl)-3-{3-[4-(2-methoxyphenyl)piperazin-1-yl]-2-hydroxypropyl}-5-
methylimidazolidine-2,4-dione hydrochloride (17)
White solid. Yield 64%. LC/MS±: purity 99.02% tR=4.24, (ESI) m/z [M+H] 457.17. 1H NMR
δ (ppm): 10.37 (s, 1H, NH+), 9.03 (s, 1H, N1H ), 7.47-7.40 (m, 1H, Ar), 7.36-7.28 (m, 2H,
Ar), 7.20-7.13 (m, 1H, Ar), 7.03-6.85 (m, 4H, Ar), 4.24 (s, 1H, CHOH), 3.77 (s, 3H, OCH3),
3.63 – 3.29 (m, 6H, Pp-3,5-H; Pp-CH2), 3.21-2.99 (m, 6H, N3-CH2, Pp-2,6-H), 1.71 (m, 3H,
CH3). 13C NMR δ (ppm): 174.98, 163.81, 160.46, 156.11, 152.15, 143.04, 138.75, 131.07,
124.46, 121.93, 119.05, 115.28, 114.97, 113.38, 113.07, 111.98, 63.18, 55.74, 47.45, 42.54,
25.23.
4.1.2.15.
5-phenyl-3-(3-(4-(2-methoxyphenyl)piperazin-1-yl(-2-hydroxypropyl)-5-
phenylimidazolidine-2,4-dione hydrochloride (18)
White solid. Yield 67%. LC/MS±: purity 95.36% tR=4.98, (ESI) m/z [M+H] 501.40. 1H NMR
δ (ppm): 10.87 (s, 1H, NH+), 9.71 (s, 1H, N1H), 7.45-7.23 (m, 10H, Ar), 7.08-6.86 (m, 4H,
Ar), 4.37 (m, 1H, CHOH), 3.77 (s, 3H, OCH3), 3.66-3.35 (m, 6H, Pp-3,5-H; Pp-CH2), 3.33 -
3.06 (m, 6H, N3-CH2, Pp-2,6-H). 13C NMR δ (ppm): 173.86, 155.69, 152.24, 140.18, 140.01,
139.00, 128.93, 128.55, 127.34, 127.28, 124.46, 121.29, 118.96, 112.52, 69.72, 63.05, 55.88,
47.18, 42.59.
4.1.2.16.
5-phenyl-3-(3-(4-(2-cyanophenyl)piperazin-1-yl(-2-hydroxypropyl)-5-
phenylimidazolidine-2,4-dione hydrochloride (19)
White solid. Yield 71%. LC/MS±: purity 97.16% tR=4.96, (ESI) m/z [M+H] 496.35. 1H NMR
δ (ppm): 10.40 (s, 1H, NH+), 9.67 (s, 1H, N1H), 7.76 -7.73 (dd, J = 7.74 Hz, 1.5 Hz, 1H, Ar),
7.66-7.60 (m, 1H, Ar), 7.42-7.31 (m, 10H, 2Ph), 7.24-7.14 (m, 2H, Ar), 5.92 (s, 1H, CHOH),
4.33 (m, 1H, CHOH), 3.68-3.36 (m, 6H, Pp-3,5-H; Pp-CH2), 3.25-3.14 (m, 6H, N3-CH2, Pp-
2,6-H)13C NMR δ (ppm): 173.86, 155.71, 154.17, 140.20, 140.02, 134.96, 134.73, 128.94,
128.56, 127.34, 127.28, 123.46, 119.82, 118.35, 105.57, 69.72, 63.06, 59.58, 48.40, 42.62.
4.2. X-ray analysis
Single crystals suitable for X-ray analysis were obtained from a mixture of 2-propanol and n-
decane using the method of slow evaporation of the solvent at room temperature. Intensity
data for single crystal were collected on a Bruker-Nonius Kappa CCD four circle
diffractometer equipped with a Mo (0.71069 Å) Kα radiation source. The phase problem was
solved by direct methods with SHELXS and the final refinements were performed by the
SHELXL program [34]. All non-hydrogen atoms were refined anisotropically using weighted
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full-matrix least-squares on F2, while the hydrogen atoms attached to carbon atoms were
refined isotropically in calculated positions using the riding model with Uiso(H) fixed at 1.2
Ueq of C and 1.5 Ueq for methyl groups. Hydrogen atoms attached to oxygen and nitrogen
atoms were located in a difference Fourier map and refined without any restraints. For
molecular graphics, ORTEP [35] and MERCURY [36] programs were used.
+
Crystallographic data: C25H32N4O4 Cl‾·0.5C10H22, Mr = 560.14, monoclinic, space group
P21/c, a = 20.8070(2) Å, b = 14.6810(3) Å, c = 10.0540(3) Å, β = 90.557(1)°, V = 3071.0(1)
Å3, Z = 4, T = 100(2)K, 22726 reflections collected, 7006 unique reflections (Rint = 0.0403),
R1 = 0.0366, wR2 = 0.0807 [I > 2σ(I)], R1 = 0.0567, wR2 = 0.0887 [all data].
CCDC 1582676 contains the supplementary crystallographic data. These data can be obtained
free of charge from The Cambridge Crystallographic Data Center via
www.ccdc.cam.ac.uk/data_request/cif.
4.3. Radioligand binding assays
4.3.1. Affinities for human 5-HT1A, 5-HT7b and D2L receptors
HEK293 cells with stable expression of human 5-HT1A, 5-HT7b and D2L receptors
(prepared with the use of Lipofectamine 2000) were maintained at 37°C in a humidified
atmosphere with 5% CO2 and grown in Dulbecco’s Modifier Eagle Medium containing 10%
dialyzed fetal bovine serum and 500 µg/ml G418 sulfate. For membrane preparation, cells
were subcultured in 150 cm2 flasks, grown to 90% confluence, washed twice with phosphate
buffered saline (PBS) prewarmed to 37°C and pelleted by centrifugation (200 g) in PBS
containing 0.1 mM EDTA and 1 mM dithiothreitol. Prior to membrane preparation, pellets
were stored at −80°C.
Cell pellets were thawed and homogenized in 10 volumes of assay buffer using an Ultra
Turrax tissue homogenizer and centrifuged twice at 35,000 × g for 15 min at 4°C, with
incubation for 15 min at 37°C in-between. The composition of the assay buffers was as
follows: for 5-HT1AR: 50 mM Tris HCl, 0.1 mM EDTA, 4 mM MgCl2, 10 µM pargyline and
0.1% ascorbate; for 5- HT7bR: 50 mM Tris HCl, 4 mM MgCl2, 10 µM pargyline and 0.1%
ascorbate; for dopamine D2LR: 50 mM Tris HCl, 1 mM EDTA, 4 mM MgCl2, 120 mM NaCl,
5 mM KCl, 1.5 mM CaCl2 and 0.1% ascorbate. All assays were incubated in a total volume of
200 µL in 96-well microtiter plates for 1 h at 37°C, except those for 5-HT1AR which were
incubated at room temperature. The process of equilibration was terminated by rapid filtration
through Unifilter plates with a FilterMate Unifilter 96 Harvester (PerkinElmer). The
radioactivity bound to the filters was quantified on a Microbeta TopCount instrument
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(PerkinElmer, USA). For competitive inhibition studies, the assay samples contained the
following as radioligands (PerkinElmer, USA): 2.5 nM [3H]-8-OH-DPAT (135.2 Ci/ mmol)
for 5-HT1AR; 0.8 nM [3H]-5-CT (39.2 Ci/mmol) for 5-HT7R or 2.5 nM [3H]-raclopride (76.0
Ci/mmol) for D2LR. Non-specific binding was defined with 10 µM of 5-HT in 5-HT1AR and
5-HT7R binding experiments, whereas 10 µM of haloperidol was used in D2L assays. Each
compound was tested in triplicate at 7 concentrations (10-10–10-4 M). The inhibition constants
(Ki) were calculated from the Cheng-Prusoff equation [37]. For all binding assays, results
were expressed as means of at least two separate experiments (SD ≤ 19%).
4.3.2. Affinities for adrenergic receptors
The radioligand binding was performed using rat cortex tissue. Rats’ brains were
homogenized in 20 volumes of ice-cold 50 mM Tris-HCl buffer (pH 7.6), and centrifuged at
20.000 × g for 20 min (0–4°C). The cell pellet was re-suspended in Tris-HCl buffer and
centrifuged again.
For competitive inhibition studies, the assay samples contained as radioligands (PerkinElmer,
USA), respectively: 0.2 nM [3H]-prazosin for the α1 receptor; 2.0 nM [3H]-clonidine for the α2
receptor and 0.8 nM [3H]-CGP-12177 for the β1 receptor. Non-specific binding was defined
with 10 µM of phentolamine in the α1 receptor; 10 µM of clonidine in the α2 receptor and 1
µM of propranolol in the β1 receptor. All assays were incubated in a total volume of 250 µL in
96-well microplates for 30 min at 30°C, except for the β1 receptor which was incubated for 1
h at 37°C.
The process of equilibration was terminated by rapid filtration through GF/B filter mate using
96-well FilterMate harvester (PerkinElmer, USA). The filter mates were dried at 37°C in a
forced air fan incubator and then solid scintillator MeltiLex was melted on the filter mates at
90°C for 4 min. The radioactivity on the filter was measured in a MicroBeta TriLux 1450
scintillation counter (PerkinElmer, USA). Data were fitted to a one-site curve-fitting equation
with Prism 6 (GraphPad Software) and Ki values were estimated from the Cheng−Prusoff
equation. Each compound was tested in duplicate at 6 concentrations (10-10–10-5 M).
4.4. Behavioral tests
The experiments were performed on male Swiss Albino mice (22–26 g,). All animals
were kept in an environmentally controlled room (ambient temperature 21 ± 2°C; relative
humidity 50–60%; 12:12 light–dark cycle, lights on at 8:00) and filtered water was freely
available. All the experimental procedures were approved by the 1st Local Ethics
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Commission at the Jagiellonian University in Krakow. All the experiments were conducted in
the light phase between 09.00 and 14.00 h. Each experimental group consisted of 6–10
animals/dose, and the animals were used only once in each test.
All drugs were suspended in 1% Tween 80 immediately before administration in a
volume of 10 ml/kg. All the compounds were administered intraperitoneally (i.p.).
Imipramine was given 30 min before the test, while the remaining compounds were injected
60 min before. Control animals received a vehicle (1% Tween-80) according to the same
schedule.
4.4.1 Forced swim test
The antidepressant-like activity of the selected 5-HT7R ligands was evaluated in the forced
swim test (FST) in mice and their activity was compared with imipramine. The experiment
was performed according to the method of Porsolt et al. (1977) [26]. Mice (Swiss Albino)
were individually placed in a glass cylinder (25 cm high; 10 cm in diameter) containing 10 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 the water. The total duration of immobility was recorded during the last 4 min of a
6-min test session.
4.4.2 Four-plate test in mice
The anxiolytic-like activity of the selected compounds was evaluated in a four-plate test in
mice and their activity was compared with diazepam. The experiment was performed
according to the method of Boissier et al. [27, 28]. Single mice were placed gently onto the
plate, and each animal was allowed to explore for 15 s. Afterwards, each time a mouse passed
from one plate to another, the experimenter electrified the whole floor for 0.5 s (current 0.8
mA), which evoked a visible flight reaction for the animal. If the animal continued running, it
received no new shock for the following 3 s. The number of punished crossings was counted
for 60 s.
4.4.3 Spontaneous locomotor activity
The locomotor activity was recorded with an Opto M3 multi-channel activity monitor
(MultiDevice Software v.1.3, Columbus Instruments). The investigated compounds or vehicle
were administered intraperitoneally (i.p.) 60 min before the test. The mice were individually
placed in plastic cages (22 x 12 x 13 cm) for a 30 min habituation period, and then the
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crossings of each channel (ambulation) were measured for a 3–6 min test session for Albino
Swiss mice. The cages were cleaned with 70% ethanol after each mouse.
4.4.4 Statistical analysis
The data are presented as means ± SEM. The obtained data were analyzed by one-way
analysis of variance (ANOVA) followed by Bonferroni’s post-hoc test. P<0.05 was
considered statistically significant.
4.5. In silico studies
4.5.1. Molecular docking
Homology models of 5-HT6 receptor were built on GPCR crystal structures, i.e. 5-HT1B (PDB
ID: 4IAR, 4IAQ), 5-HT2B (PDB ID: 4IB4), A2A (PDB ID: 3QAK, 2YDV, 4UHR), β2 (PDB
ID: 3P0G, 4LDE, 3PDS), M2 (PDB ID: 4MQS), CXC4 (PDB ID: 3OE0), D3 receptor (PDB
ID:3PBL) and H1 receptor (PDB ID: 3RZE). For each template, 20 models were generated
using Modeller v9.9 software [38], and validated by docking of an active/inactive set
containing 5-HT6R ligands sourced from the ChEMBL database [39]. The quality of each
model was determined by the Area Under ROC curve (AUROC) calculated based on the
Glide Score values of docked compounds (the undocked actives and inactives were
considered false negatives and true negatives, respectively). Three models with the highest
AUROC value per template were selected for final validation. The final validation was
performed by docking of the synthetized library of compounds. Only those models were kept,
for which binding modes were observed that were coherent for the whole set of compounds
and that explained the main structure-activity relationships.
3-dimensional structures of the ligands were prepared using LigPrep v3.6 [40], and the
appropriate ionization states at pH=7.4±1.0 were assigned using Epik v3.4 [41]. Compounds
with unknown absolute configuration were docked in R and S configurations. One low energy
ring conformation per ligand was generated. The Protein Preparation Wizard was used to
assign the bond orders, appropriate amino acid ionization states and to check for steric
clashes. The receptor grid was generated (OPLS3 force field [42]) by centering the grid box
with a size of 12 Å on Asp3.32 side chain. Automated flexible docking was performed using
Glide v6.9 at SP level [43].
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4.5.2. Pharmacophore mapping
The two published 5-HT7R pharmacophore models were reconstructed using the 3D QSAR
Pharmacophore Generation protocol implemented in Discovery Studio 3.5. The same ligand
structures and parameters as described in the original papers were used. Next, the synthetized
compound library was screened by the obtained pharmacophore models using the
Pharmacophore Mapping protocol from Discovery Studio. During the screening, the
conformation space of each compound was generated using the BEST algorithm within a
relative energy threshold of 20 kcal/mole above the global energy minimum and with the
maximum number of generated conformations per ligand set to 255, and no omitting features
choice turned on.
4.5.3. Molecular Dynamic simulations
A 100 ns Molecular Dynamics (MD) simulation was performed using Schrödinger Desmond
software. The ligand-protein complex was immersed into a POPC (300K) membrane bilayer,
where the position was calculated using the PPM web server (accessed 05-10-2017) [44]. The
system was solvated by water molecules described by the TIP4P potential and the OPLS3
force field parameters were used for all atoms. 0.15 M NaCl was added to mimic the ionic
strength inside the cell. The output trajectory was hierarchically clustered into 10 clusters
according to the ligand and this was used to calculate the simulation interaction diagram using
trajectory analysis tools available in the Maestro Schrödinger Suit.
4.5.4. CNS Multiparameter optimization
The Central Nervous System Multiparameter Optimization (CNS MPO) score was
calculated based on the six molecular descriptors, i.e. logP, logD, molecular weight (MW),
topological polar surface area (TPSA), the count of hydrogen bond donor (HBD) and the
strongest basic pKa using Calculator Plugins [45] implemented in ChemAxon. For logP, logD,
MW, HBD, and pKa a monotonic decreasing function was employed, whereas a hump
function for TPSA was used to transform the values of the descriptors into a dimensionless
scale. The most desirable and the least desirable ranges for each descriptor were adopted from
the original paper [25]. Transformed values (desirability score) for the descriptors were
determined and summed for each compound to obtain the final CNS MPO, which can range
from 0 (the worst CNS drug) to six 6 (the best CNS drug).
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Acknowledgements
The authors would like to thank Prof. Andrzej J. Bojarski for his helpful suggestions
concerning this study. Agnieszka Jankowska, currently a PhD-student at the Department of
Medicinal Chemistry, Faculty of Pharmacy, JU MC, performed a part of the synthesis of
intermediates within the Student Medicinal Chemistry Scientific Group at the Department of
Technology and Biotechnology of Drugs, JU MC (Studenckie Koło Chemii Medycznej,
UJCM). This study was supported by National Science Center (Poland) Grant No. UMO-
2014/15/N/NZ7/03072 and statutory projects K/ZDS/006134 and K/ZDS/005593.
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ACCEPTED MANUSCRIPT
List of Figures, Tables and Schemes
1. Table 1 Structures and receptor binding properties for compounds 1-19.
2. Table 2 Parameters of intra- and inter-molecular interactions for compound 4.
3. Fig. 1. General structure of the investigated hydantoin compounds and beneficial
substituents at piperazine found during previous modifications of lead MF-8 (1) [10].
The area of present modifications marked in gray.
4. Fig. 2. Atom numbering scheme for 4. Displacement ellipsoids are drawn at the 50%
probability level.
5. Fig. 3. Intermolecular interactions between two molecules of compound 4. Dashed
lines indicate hydrogen bonds.
6. Fig. 4. Binding mode of compound 4 in the 5-HT7R homology model (A). The
hydrogen bonds are marked with a yellow dotted line. The 2D ligand-protein
interaction diagram obtained by averaging all frames from molecular dynamics
simulation. Only interactions which occurred in at least 50% (the frequency of the
particular interaction occurrence was marked by magenta font) of the MD frames are
shown. The detailed ligand-protein interaction diagram is available in the
Supplementary materials (Fig. S1).
7. Fig. 5. Alignment of compound 4 conformations obtained in molecular docking
(green), through crystallization (cyan) and in molecular dynamics (magenta). The
molecular dynamic trajectory was hierarchically clustered, fixing the number of
resultant clusters to 10, and the centroid of the most populated cluster was selected as
a representative conformation of compound 4.
8. Fig. 6. Reconstructed 3-dimensional pharmacophore model for 5-HT7R antagonists
including four key pharmacophore features: a positive ionizable group (PI, red), an
aromatic ring (AR, orange), a hydrogen bond acceptor group (HBA, green) and
hydrophobic groups (HYD, blue); (A) compound 11 (green) and known strong
selective 5-HT7R antagonist SB-656104 (gray) [18] mapped onto the 5-HT7R
antagonist pharmacophore model of Lopez-Rodriguez [23]; (B) compounds 11 and 17
(in yellow and gray, respectively) mapped onto the 5-HT7R antagonist pharmacophore
model of Bojarski’s research group [24].
9. Fig. 7. Effect of investigated compounds and imipramine in the forced swim test
(FST) in Albino Swiss mice. The compounds were injected i.p. 60 min, while
imipramine 30 min before the test. Values represent means ± SEM during the last 4-
min test session compared to the respective vehicle group (one-way ANOVA is
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