The role of aryl-topology in balancing between selective and dual 5-HT7R/5-HT1A actions of 3,5-substituted hydantoins

Article abstract of DOI:10.1039/c8md00168e

In order to search for active and selective serotonin 5-HT₇R antagonists among 3,5-disubstituted arylpiperazine-imidazolidine-2,4-diones, the role of the introduction/deletion and the mutual orientation of aromatic rings was analyzed. Chemical modifications of 2nd generation lead structure of 3-(3-(4-(diphenylmethyl)piperazin-1-yl)-2-hydroxypropyl)-5-(4-fluorophenyl)-5-methylimidazolidine-2,4-dione (2, KKB16) were performed. New derivatives (4-18) were designed and synthesized. X-ray crystallographic analysis of the representative compound 5-(4-fluorophenyl)-3-[2-hydroxy-3-(4-phenylpiperazin-1-yl)propyl]-5-methylimidazolidine-2,4-dione (3) was performed to support molecular modeling and SAR studies. The affinity for 5-HT₇R, D₂R and 5-HT_1AR in radioligand binding assays for the entire series and ADME-Tox parameters in vitro for selected compounds (7, 10, and 13) were evaluated. Molecular docking and pharmacophore model assessment were performed. According to the obtained results, 5-methyl-5-naphthylhydantoin derivatives were found to be the new highly active 5-HT₇R agents (K_i ≤ 5 nM) with significant selectivity over 5-HT_1AR and D₂R. On the contrary, the (1-naphthyl)piperazine moiety was gained with the potent dual 5-HT₇R/5-HT_1AR action (K_i: 11 nM/19 nM).

Full text of DOI:10.1039/c8md00168e

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Handzlik, Med. Chem. Commun., 2018, DOI: 10.1039/C8MD00168E.
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The role of aryl-topology for balancing between selective
and dual 5-HT7R/5-HT1A action of 3,5-substituted hydantoins
Katarzyna Kucwaj-Brysz1, Rafał Kurczab2, Ewa Żesławska3, Annamaria Lubelska1, Małgorzata Anna Marć1,
Gniewomir Latacz1, Grzegorz Satała2, Wojciech Nitek4, Katarzyna Kieć-Kononowicz1, Jadwiga Handzlik1,*
1
2
Department of Technology and Biotechnology of Drugs, Jagiellonian University Medical College, Medyczna 9, 30-688
Cracow, Poland
Department of Medicinal Chemistry Institute of Pharmacology, Polish Academy of Science, Smętna 12, 31-343, Cracow,
Poland
Department of Chemistry, Institute of Biology, Pedagogical University of Cracow, Podchorążych 2, 30-084 Cracow, Poland
Faculty of Chemistry, Jagiellonian University, Gronostajowa 2, 30-387, Cracow, Poland
3
4
* Correspondence: jhandzli@cm-uj.krakow.pl; Tel.: +012 620 55 80
Abstract:
In order to search for active and selective serotonin 5-HT7R antagonists among 3,5-disubstituted
arylpiperazine-imidazolidine-2,4-diones, the role of introduction/deletion and mutual orientation of
aromatic rings was analyzed. Chemical modifications of 2nd generation lead structure, 3-(3-(4-
(diphenylmethyl)piperazin-1-yl)-2-hydroxypropyl)-5-(4-fluorophenyl)-5-methylimidazolidine-2,4-
dione (2, KKB16) were performed. New derivatives (4-18) were designed and synthesized. The X-ray
crystallographic analysis for representative compound, 5-(4-fluorophenyl)-3-[2-hydroxy-3-(4-
phenylpiperazin-1-yl)propyl]-5-methylimidazolidine-2,4-dione (3), was performed to support
molecular modeling and SAR studies. The affinity for 5-HT7R, D2R and 5-HT1AR in the radioligand
binding assays for whole series and ADME-Tox parameters in vitro for selected compounds (7, 10,
13), were evaluated. The molecular docking and pharmacophore model assessment were performed.
As results, 5-methyl-5-naphtylhydantoin derivatives were found as new highly active 5-HT7R agents
(Ki ≤ 5 nM) with significant selectivity over 5-HT1AR and D2R. In contrary, the (1-naphthyl)piperazine
moiety gained with the potent dual 5-HT7R/5-HT1AR action (Ki: 11 nM/19 nM).
Introduction
Targeting receptor 5-HT7, the youngest member of serotoninergic system1–3 , seems to be a
promising approach in terms of treatment of CNS disorders such as depression4 and
schizophrenia-like cognitive impairments5. However considering design of 5-HT7R ligands,
the same dilemma still remains if selective6,7 or rather multitargeted therapy8,9 is better
solution for the CNS patients. The selective mechanism of action, targeting only into the
desired protein, gives hope for decrease probability of side-effects manifestation, caused by
interactions with off-targets. Going deeper according to functional selectivity approach, it is
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possible to activate not only chosen biological target, but specific signal transduction
pathway6. It is also worth mentioning that only one selective 5-HT7R ligand, compound JNJ-
18038683, has been studied in clinical trials, but unfortunately its antidepressant action has
not been proved due to the lack of sensitivity of performed studies7. Thus, searching for a
potent and selective 5-HT7R ligands seems to be significant direction in order to verify
potential advantages and disadvantages of selective therapy.
In contrary, polypharmacology is suggested to be an appropriate solution for an
achievement of high efficacy of complex therapy, e.g. of mood disorders and
schizophrenia8,9 or of both, cancer and CNS diseases10. Some trends in search for so called
« magic shotguns » against most common central nervous system disorders11 indicate an
8
9
importance of serotonin and dopamine targets, apart from 5-HT7, also 5-HT1A and D2
receptors. However, lines of evidence demonstrate various dual- and multitarget acting
compounds useful against CNS-disorders, which involve serotoninergic and other GPCRs, e.g.
muscarinic M4 receptors against schizophrenia12 or “non-monoaminergic” mechanisms, e.g.
neurokinin NK1 antagonists against depression13. To the date, 5-HT7R antagonists, acting in
multidirect way, are available on pharmaceutical market, e.g. vortioxetine, approved in 2013
by FDA for treatment of major depressive disorder14,15 or lurasidon, the second generation
antipsychotic drug16.
Our previous studies led to synthesis of 30-membered group of hydantoin-derived 5-HT7
ligands17–19. Nineteen of them are highly active (Ki < 100 nM) and selective over other GPCR’s
(5-HT1A, D2, 5-HT7, α1A-, α2B-, β2-adrenoceptors). Behavioral studies (Porsolt’s test),
performed for selected compounds, confirmed their antidepressant activitiy in mice. Worth
noting, that not the 1st generation lead (MF-8, 1, Fig. 1) with the strongest affinity towards 5-
18,20
HT7
but the lead derivative (KKB16, 2, Fig. 1) with the highest metabolic stability turned
up to cause the most significant antidepressant effect21. Moreover, the compound 2 showed
also the highest selectivity over 5-HT1AR (71-fold) within the whole series. According to SAR
analysis including 2 in comparison to majority of derivatives with (un)substituted phenyl
ring18,19, the observed selectivity seemed to be a consequence of presence of
diphenylmethyl group linked to piperazine.
2

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Fig. 1. Lead structures (MF-8, KKB-16) and areas of their modifications explored within this
work.
Hence, the aforementioned issues are worth to be further studied in order to verify (i) how
changes in number and (mutual) spatial orientation of aromatic rings influence activity and
selectivity for 5-HT7R among the hydantoin-derived ligands and (ii) the importance of
diphenylmethyl group for beneficial metabolic stability. For this purpose, the diphenylmethyl
derivative (2) was selected as the 2nd generation lead structure to be modified within
« antipodal » aromatic-containing areas (blue, Fig. 1). Both generations leads, 1 and 2, and
the previously investigated 5-phenyl derivative 318 (Table 1) were used as reference
compounds for these studies. Fifteen novel derivatives of lead 2 were designed and
synthesized (4-18, Table 1). The new compounds were tested in the radioligand binding
assay in order to assess affinity towards 5-HT7R, 5-HT1A and D2. X-ray crystallographic
analysis of 3 was performed. To elucidate differences in the 5-HT7R activity and selectivity,
molecular modeling, docking- and pharmacophore-based, studies were performed. Selected
compounds (7, 10 and 13) were examined on their metabolic stability and toxicity in vitro in
comparison to both leads (1 and 2).
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Table 1. Structures and radioligand binding results for compounds 1-18.
Ki [nM]
R1
R2
5-
HT1AR2
Cpd Group
D2R1
5-HT7R3
1*
2*
3*
4
A
4-Fluorophenyl
2-MeO-phenyl
715
261
121
3
5570
2733
79
A
A
A
A
A
A
B
B
A
A
A
A
A
A
A
A
C
4-Fluorophenyl
4-Fluorophenyl
4-Fluorophenyl
4-Fluorophenyl
4-Fluorophenyl
4-Fluorophenyl
4-Fluorophenyl
4-Fluorophenyl
1-Naphthyl
Diphenylmethyl
Phenyl
2906
223
Benzyl
20030 377500 2085
5
Benzoyl
10080
5233
295
6216
5577
19
3609
2172
11
6
(Naphthalene-1-yl)methyl
1-Naphthyl
7
8
Phenoxyl
4353
4116
256
5211
21470
326
165
172
5
9
4-Cl-Phenoxyl
2-MeO-Phenyl
2-CN-Phenyl
Diphenylmethyl
2-MeO-Phenyl
2-CN-Phenyl
2-MeO-Phenyl
2-CN-Phenyl
Diphenylmethyl
-
10
11
12
13
14
15
16
17
18
Refa-c
1-Naphthyl
416
1225
413200
128
19
1-Naphthyl
273
224
3
2-Naphthyl
153
2-Naphthyl
264
129
3
Methyl
2130
3429
1152
5848
9a
489
125
209
824
888
18c
Methyl
1155
15150
1551
20b
Methyl
4-Fluorophenyl
*Compounds from the previously published and pharmacologically described series.1-3Radioligands
used: [3H]-Raclopride (D2R), [3H]-8-OH-DPAT (5-HT1AR), [3H]-5-CT (5-HT7R). a-cReference ligands for
GPCRs investigated: aolanzapine, bbuspirone, cclozapine, nt - not tested.
Results and discussion
Synthesis
The designed new derivatives (4–18) were obtained based on previously optimized three-
step pathway19 starting from Bucherer-Bergs reaction, which led to hydantoin system
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formation, followed by N-alkylation with epichlorhydrin and solvent-free microwave-assisted
condensation with simultaneous epoxide opening at the end (Scheme 1). Compounds 15–17
were synthesized in two steps, starting from commercially available 5,5-dimethylhydantoin.
Pure products have been isolated during pH-dependent extraction. This method allowed to
eliminate time- and cost-consuming purufication by column chromatography used
previously18,19. All the compounds were obtained as racemic mixtures due to non-
stereoselectivity of Bucher-Bergs reaction and application of (+/-)-2-(chloromethyl)oxirane
as alkylating agent.
Scheme 1. Synthesis pathway for compounds 4–18: (i) KCN, (NH4)2CO3, 50% EtOH, 50oC, 24h;
(ii) 2-(chloromethyl)oxirane, NaOH, H2O, rt, 10h; (iii) R2-commercially available derivative of
piperazine/piperidine or isohexahydroquinoline,
irradiation 300-450W, 2 min.
, solvent-free reaction, microwave
X-ray crystallographic studies
The piperazine-hydantoin derivatives, obtained as modifications of lead 1, were hard to give
any crystal suitable for X-ray analysis, in particular, in the case of basic form that was also
rare to precipitate during synthesis work up. Before, 5-phenyl-3-(3-(4-(2-
ethoxyphenyl)piperazin-1-yl(-2-hydroxypropyl)-5-methylimidazolidine-2,4-dione
hydrochloride was only one that gave a sufficient crystal for X-ray analysis, but in the salt
form19. The present study has successfully provided the first representative crystal of basic
form (compound 3), appropriate for X-ray crystallographic analysis that allowed us to extend
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knowledge about 3D-properties of this chemical group, which also supported molecular
modeling.
The molecular geometry in the crystal structure of compound 3 with the atom
numbering scheme is shown in Fig. 2.
Fig. 2. The molecular structure of compound 3 showing the atom numbering scheme.
Displacement ellipsoids are drawn at the 30% probability level.
The flexible aliphatic linker, which binds hydantoin and piperazine rings, shows an extended
conformation, with the torsion angles being N3-C6-C7-C8 = 179.4(2)° and C6-C7-C8-N2 =
147.8(2)°. The bigger deviation of one value from 180° is due to the intramolecular contact
C9-H9A···O1, for which the following parameters are observed: H9A···O1 = 2.59 Å, C9···O1 =
3.128(3) Å and the angle C9-H9A···O1 = 114°. The mutual orientation of 4-fluorophenyl
substituent at C5 and hydantoin ring differs in comparison to geometry of the
aforementioned (2-ethoxyphenyl)piperazine compound with phenyl ring at C5 atom,
determined earlier 19. The angle between the planes of the aromatic ring and the hydantoin
ring is 62.2(1)°, while it is 86.73(5)° in the compared compound.
In the presented structure the piperazine ring adopts chair conformation, wherein the
substituents at N2 and N4 are equatorial. The torsion angles C14-C13-N4-C10 and C18-C13-
N4-C10 are 25.7(3)° and -157.3(3)°, respectively, which indicate that the phenyl ring at N4
atom is almost coplanar with piperazine moiety. The angle between the planes of aromatic
and piperazine (C9, C10, C11, C12) rings is 10.3(2)°. We observed a higher value of the
corresponding angle of 26.8(2)° for another hydantoin derivative with N-phenylpiperazine
moiety22.
The main intermolecular interactions are based on O-H···O and N-H···N hydrogen
bonds (Fig. 3). One oxygen atom (O2) of hydantoin is involved in hydrogen bond with
hydroxyl group, whereas the second oxygen atom (O4) is involved in C-H···O intermolecular
interactions. The nitrogen atom (N1) of hydantoin is engaged in hydrogen bond with
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nitrogen atom (N2) of piperazine ring. Furthermore, the fluorine atom makes C-H···F
contacts. The parameters of these interactions are listed in Table 2.
Fig. 3. Packing of molecules in the unit cell projected along [010] direction. Dashed lines
indicate hydrogen bonds.
Table 2. The parameters of intermolecular interactions in structure 3.
D-H···A
H···A (Å)
2.08(4)
1.90(4)
2.61
D···A (Å)
2.981(3)
2.756(3)
3.319(3)
3.464(3)
3.334(3)
3.498(4)
3.368(4)
D-H-A (°)
170(3)
167(4)
128.6
Symmetry code
-x+1, y-1/2, -z-1/2
-x+1, y-1/2, -z-1/2
-x+1, -y+1, -z
-x+1, -y+1, -z
-x+1, -y, -z
N1-H1N···N2
O1-H1···O2
C8-H8B···F1
C11-H11A···F1
C21-H21B···O4
C26-H26···O1
C27-H27···O4
2.77
127.6
2.36
169.9
2.81
130.3
-x+1, -y, -z
2.50
152.4
-x+1, -y, -z
Radioligand binding assays
The radioligand competition binding assays were applied to determine the affinity and
selectivity profiles of the newly synthesized compounds (4–18) for human serotonin 5-HT7bR,
5-HT1AR, and dopaminergic D2LR, stably expressed in HEK-293 cells (Table 1). Five compounds
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from the series (7, 10, 11, 13, 14) were highly active toward 5-HT7R (3 nM ≤ Ki ≤ 19 nM),
whereas other five (8, 9, 12, 15, 16) demonstrated rather moderate activity (125 nM ≤ Ki ≤
224 nM). Moreover, almost all the most active 5-HT7R agents (8–16), excluding compound 7,
showed selectivity over 5-HT1AR and D2R. The active compound 7 had also potent affinity
toward 5-HT1AR (Ki = 19 nM).
ADME-Tox studies in vitro
The preliminary in vitro studies on ADME-Tox parameters were performed for selected, most
active 5-HT7R ligands (7, 10 and 13) in comparison to the results for both leads (1 and 2) and
the metabolically stable drug, aripiprazole. For this purpose, mouse liver microsomes
(MLMs) and human embryonic kidney (HEK-293) or hepatoma (HepG2) cell lines were used
according to the described previously protocols20,21
.
Metabolic stability
The incubation of the 5-HT7R ligands (2, 7, 10 and 13) in the presence of MLMs
followed by the UPLC-MS analyses, including ion fragmentation and supported by in silico
simulation with MetaSite software, allowed to determine the metabolic stability, metabolic
pathways and the most probable structures of the 5-HT7R ligands’ metabolites (details in
Supplementary materials: Table S3, Fig. S1-S12).
Regarding the metabolic stability, the new compounds (7, 10 and 13) were less stable
than arirpiprazole (Table S3, Fig.S1-S9 v. Fig.S10, Supplementary materials). It was seen that
~65-70% of compounds 7 and 13 were metabolized into four or six metabolites, respectively,
whereas ~40% of compound 10 into four metabolites. However, the metabolic stability of 10
was the closest to the most stable lead 2 (~20% remaining, Table 3) that metabolized into
one metabolite (Table S3, Fig.S11 and S12, Supplementary).
Furthermore, the metabolic stability of the lead 2 and compounds 7, 10 and 13 were
determined in pharmacokinetic in vitro studies, performed by using MLMs in order to
calculate the half-life period (t1/2) and hepatic metabolic clearance CLint and compared to the
obtained previously data for the reference aripiprazole and the 1st generation lead 120 (Table
3).
A comparison of the pharmacokinetic values (t1/2, CLint) for both leads (120 and 2) has
shown their similar metabolic stability in MLMs. In contrast, the new synthesized derivatives
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(7, 10 and 13) were less stable than both leads (1 and 2) and the reference aripiprazole but
they demonstrated rather satisfying values of both t1/2 and CLint. The CLint value of the most
stable compound 10 was only ~1.7 fold higher than CLint of the leads (1 and 2) and ~2.3 fold
higher than that of metabolically stable aripiprazole, whereas the CLint of the most labile
compound 7 was ~3.5 fold higher than that of both leads and almost 5 fold higher in
comparison to the value of aripiprazole (Table 3).
Table 3. Pharmacokinetic properties of the 5-HT7R ligands and the reference drug
aripiprazole estimated in vitro by MLMs
Aripiprazole
1*
2
7
10
13
t1/2
217.0*
157.5*
157.5
45.0
94.0
62.5
(min)
mouse CLint
(ml/min/kg)
% of
remaining
substrate
after 120
min of
12.5*
ND**
17.2*
ND**
17.2
60.3
~65
29.0
~40
43.4
~70
~20
incubation
* values in MLMs estimated previously20 **ND = no determined
;
Toxicity
Compounds 7, 10 and 13 were tested for their safety in HEK-293 and HepG2 cell lines,
at four concentration intervals (0.1–100 µM) for 72 h. The antiproliferative drug doxorubicin
(DX) was used as a positive control at 1 µM. Additionally, the mitochondrial toxin carbonyl
cyanide 3-chlorophenylhydrazone (CCCP), was used at 10 µM for assays with HepG2 (Fig. 4).
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Fig. 4. The effect of 7 , 10 , 13 and doxorubicin (DX) on HEK-293 viability after 72h of incubation (A) .
The effect of 7 , 10 , 13, doxorubicin (DX) and carbonyl cyanide 3-chlorophenylhydrazone (CCCP) on
HepG2 viability after 72h of incubation (B) The statistical significance was evaluated by a one-way
ANOVA, followed by Bonferroni’s Comparison Test (∗∗∗p < 0.001 compared with negative control).
All examined compounds significantly influenced both cell lines viability, but only at
the highest used dose (100 μM). However, the comparison of the obtained results to the
effect of DX at the 1 µM or CCCP at 10 μM indicated rather weak cytotoxic and hepatotoxic
effects of the examined 5-HT7R ligands (Fig. 4). Regarding described previously effects of
leads 1 and 2 on HEK-293 cells viability20,21, compounds 7, 10 and 13 were in the range of
toxicity of the lead 2.
Molecular modeling and SAR analysis
To support the discussion about aromatic ring localization and its influence on affinity
towards 5-HT7R, appropriate interfeature distances have been measured and compared with
the known pharmacophore model requirements23. Moreover, all the compounds (1–18)
were docked to homology models of 5-HT7R in order to define key protein–ligand
interactions. Additionally, due to the occurred 5-HT7/5-HT1A dual action for compound 7, the
docking studies to 5-HT1AR homology models have been applied, as well.
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Previously, we confirmed19 that analogs of lead structure MF-8 (1) fit very well to the
pharmacophore model of 5-HT7R antagonists reported by Lopez-Rodriguez23. For this model,
the optimal distance between basic center (N2) and hydrophobic (aromatic) area (ring
centroid) is 5.4–6.4 Å, thus the arylpiperazine moiety met perfectly this requirement (Fig.
5A). The radioligand binding results showed significant relationship between N2–ring
centroid distance and 5-HT7R affinities of compounds 2–8 and 18. Both, an increase and a
decrease of this distance, beyond the optimum range, resulted in a significant reduction of
5-HT7R affinity, e.g. compound 4 (Fig. 5B) and 6 (Fig. 5C), with N2-ring centroid distance > 6.4
Å, showed Ki = 2085 nM and 2172 nM, respectively, while compound 18 (Fig. 5D) with N2-
ring centroid distance < 5.4 Å was also less active (Ki = 888 nM). Intriguing results were
observed for the diphenylmethyl-containing lead 2 (Ki = 79 nM, Fig. 5E) if comparing to the
inactive benzyl compound (4, Fig.5B). The presence of an additional aromatic ring changed
geometry in the way that one of the phenyl group was closer to the basic center (6.30 Å),
resulting with a significant 5-HT7R activity improvement. Moreover, not only the presence of
additional phenyl ring was profitable, but also the distance between this two phenyl rings
(e.g. compound 2 vs 6). Fused rings, as is observed in naphthalene moiety, linked to
piperazine via methylene group (6, Fig. 5C), brought strong decrease of the 5-HT7R affinity,
whereas an introduction of diphenylmethyl group (2, 12, and 17) improved the 5-HT7R
affinity.
On the other hand, the naphthyl group linked directly to piperazine (7, Fig. 5F) provided very
high affinity (Ki = 11 nM) and additionally, this structure turned out to be the first dual 5-HT-
7/5-HT1A ligand coming from the herein- and previously described series18,19
.
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Fig. 5. Comparison of distances between basic center (N2) and aromatic centroids (presented as
green lines and expressed in [Å]) for compounds 2–4, 6,7 and 18.
An analysis of the binding modes for compounds 4, 6, 7 and 18 (Fig. 6A) showed the
influence of above-discussed distances on the receptor–ligand interactions. Although all
compounds (4, 6, 7 and 18) interact with aromatic cluster formed by Phe6.51 and Phe6.52,
the significant decrease of activity for compounds 4, 6, and 18 seems the be a consequence
of loss of the salt bridge with Asp3.32, the key interaction linked with high 5-HT7R affinity
that is maintained only for the most active one (7).
Recently, we also indicated that introduction of additional phenyl ring into position 5 of
hydantoin caused significant decrease of 5-HT7R affinity19. Additionally, an exchange of
phenyl into methyl group (resulting with 5,5-dimethylhydantoin moiety) led to decrease of
5-HT7R activity (Ki = 125 nM for 15 vs Ki = 3 nM for the 1st generation lead 1), but not so
strong as in the case of presence of two phenyl rings (5,5-diphenylhydantoin, 189-fold
decrease in respect to 1)19. Interestingly, an exchange of the phenyl ring at position 5 of
hydantoin into 1- or 2-naphthyl was profitable and did not disrupt the high 5-HT7R affinity,
making compounds 10, 11, 13 and 14 the most active ones in the whole series (4–18). For
the naphthyl derivatives with the (2-methoxyphenyl)piperazine fragment (10 and 13), the 5-
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HT7R affinity was almost identical as that for the 1st generation lead 1. No visible difference
in 5-HT7R affinity between compounds 10 and 13 seems to be a result of very well fitting of
both aromatic moieties (1-naphthyl and 2-naphthyl) into the hydrophobic pocket formed by
ECL2 and TM2, 3, and 7 (Fig. 6B). In comparison, an absence of the aromatic group in the
case of compound 15 (with methyl group) influenced on lack of this additional stabilization,
which resulted in decrease of activity. For the compounds with the (2-
cyanophenyl)piperazine moiety (11 and 14), a little difference was visible, indicating that 2-
naphtyl (14) was more preferable in respect to 1-naphthyl (11) group. The molecular docking
of this pair of compounds (11 and 14), resulted in coherent binding mode to 5-HT7R.
However, there was a slight difference in hydantoin orientation, which might cause a weaker
stabilization of this moiety by Arg7.36 (reported previously by our group19) for compound
11, resulting in ~4-fold lower 5-HT7R affinity than that for compound 14 (Fig. 6C).
Fig. 6. (A) Influence of basic center-aromatic centroid distance on binding mode for
compounds 4 (brown), 6 (pink), 7 (red) and 18 (olive); (B) The comparison of binding mode
of lead structure, 1 (yellow) and its analogs with 2-naphthyl hydantoin moiety - 10 (cyan)
and dimethylhydantoin - 15 (violet); (C) The comparison of binding mode of 1-naphthyl and
2-naphthyl hydantoin derivative with (2-cyanophenyl)piperazine moiety – 11 (orange) and
14 (green).
Worth mentioning that an introduction of the 1- and 2-naphthyl group, instead of the 4-
fluorophenyl ring, allowed for keeping the 5-HT7R selectivity over the 5-HT1AR and D2R,
however slightly less than that in the case of both, the 1st generation lead 1 (the highest 5-
HT7/D2 selectivity ratio) and the 2nd generation lead 2 (the highest 5-HT7/5-HT1A selectivity
ratio).
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As the naphthylpiperazine derivative (7), the molecular docking to the 5-HT1AR and 5-HT7R
homology models confirmed that structure 7 fit very well into the both binding pockets, and
formed the following interactions: (i) a salt bridge with Asp3.32 and (ii) CH-π/π-π interaction
between naphthyl group linked with piperazine and Phe6.52 side chain (Fig. 7). In
comparison to the binding mode of dual ligands to 5-HT1AR from different chemical class (2-
benzoxazolones and 2-benzothiazolones) published previously24, the compound 7 interacted
also with Tyr7.43, while the hydrogen bond formation with Trp7.40 and Pro144 from EL2
was not observed. Regarding docking to the 5-HT7R, the hydantoin moiety was hydrogen
bonded by Arg7.43, similarly as the terminal heterocycle moiety of above-mentioned 2-
benzoxazolones and 2-benzothiazolones derivatives.
Fig. 7. Binding mode of compound 7 in 5-HT7R (A) and 5-HT1AR (B) homology models.
The size, type and spatial orientation of the aromatic rings influenced not only activity and
selectivity but also metabolic stability properties of the investigated series (1-18). The lead 2,
with two unfused phenyl rings linked via methylene group to the piperazine and with mono-
aromatic substituents at position 5 of hydantoin, has shown the high metabolic stability,
predominantly in human21, but also in mouse liver microsomes. In contrary, the presence of
fused naphthyl rings either on the piperazine (7) or on hydantoin (10, 13) sides resulted with
some decrease of metabolic stabilities, especially distinct in the case of the β-naphthyl
derivative (13). In contrary, an introduction of the naphthyl moiety, regardless of the
14

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topology, did not significantly affect the safety, which was in the range of the lead 2 for all
new derivatives (7, 10 and 13).
Conclusions
Based on the 2nd generation lead (KKB16, 2) and SAR analysis for the previous series, we
have enriched the library of hydantoin-derived 5-HT7R ligands by introducing 15 new
members with diverse receptor affinities, especially potent in the case of 5-
naphthylhydantoin derivatives. The performed X-ray analysis, molecular modeling and,
consequently, comprehensive SAR-studies enabled to validate the role of changes in the
number and (mutual) spatial orientation of aromatic rings for the 5-HT7R activity and
selectivity. We indicated that the 1- or 2-naphthyl moieties in position 5 of hydantoin were
crucial feature for the potent action on 5-HT7R with maintenance of distinct selectivity
towards 5-HT1AR and D2R. In turn, the introduction of 1-naphthyl group on the piperazine
side effected with significant 5-HT7R and 5-HT1AR dual action. The ADMET studies in vitro,
performed for selected compounds (7, 10, 13) have confirmed their low cytotoxicity. The
precise pharmacokinetic studies in vitro revealed in a general lower metabolic stability of
naphthyl derivatives (7, 10, 13) in comparison to the leads (1 and 2) and aripiprazole, but
more satisfying from “druglikeness” point of view for 1-naphthyl derivatives, especially, in
the case of the naphthylpiperazine derivative (7). Bearing in mind these results, it would be
worth to further investigate if both, the dual receptor action and lower (comparing with the
leads) metabolic stability in vitro, are reflected in the pharmacological action in vivo, with
special respect to antidepressant properties.
Material and methods
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; bs, broad singlet; d, doublet; t, triplet; m, multiplet; Ph, phenyl; Pp, piperazine, Ar,
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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 4-24 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 5-methyl-5-naphthylhydantoins (20, 21) and 5-(4-fluorophenyl)-5-methylhydantoin (19)
and their spectral data has already been published17,25,26. Procedure for N-alkylation has
been described before19,27. Physicochemical data for the oxirane 22 is available in the
literature27 and for oxiranes 23, 24 can be found in Supplementary.
General procedure for synthesis of final compounds (4-18)
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). The progress of reaction was
controlled by TLC (DCM/MeOH 95:5). The crude product was dissolved in methylene
chloride. The resulted organic phase was washed with 2% HCl water solution, followed by
water, then dried over Na2SO4, filtered through cotton and concentrated under vacuum to
provide the desired product. Majority of 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
2-propanol and dried.
3-[3-(4-benzylpiperazin-1-yl)-2-hydroxypropyl]-5-(4-fluorophenyl)-5-methylimidazolidine-
2,4-dione hydrochloride (4)
White solid. Yield 32%. LC/MS±: purity 98.05% tR=3.69, (ESI) m/z [M+H] 441.29. 10.44 (s, 1H,
NH+), 9.01 (s, 1H, N1H), 7.56-7.52 (m, 2H, Ar), 7.51-7.43 (m, 5H, Ar), 7.31-7.19 (m, 2H, Ar),
5.94 (s, 1H, CHOH), 4.23 (s, 1H, CHOH), 3.65-3.28 (m, 10H, N3-CH2, Ph-CH2, Pp-2,6-H), 3.21-
3.04 (s, 4H, Pp-CH2, Pp-3,5-H), 1.72 (s, 3H, CH3). 13C NMR δ (ppm): 13C NMR δ (ppm): 175.89,
169.58, 163.42, 160.99, 155.95, 155.86, 136.35, 136.32, 136.28, 135.19, 130.47, 128.98,
128.34, 128.27, 127.58, 115.81, 115.60, 62.95, 62.46, 59.35, 42.47, 25.95.
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3-[3-(4-benzoylpiperazin-1-yl)-2-hydroxypropyl]-5-(4-fluorophenyl)-5-methylimidazolidine-
2,4-dione hydrochloride (5)
1
White solid. Yield 29%. LC/MS±: purity 96.84% tR=3.59, (ESI) m/z [M+H] 455.31. H NMR δ
(ppm): 10.54 (s, 1H, NH+), 9.02 (s, 1H, N1H), 7.56-7.53 (m, 2H, Ar), 7.37-7.12 (m, 7H, Ar), 5.98
(s, 1H, CHOH), 4.53 (m, 1H, CHOH), 4.35 (s, 2H, N3-CH2), 3.80-3.32 (m, 6H, Pp-3,5-H, Pp-CH2),
3.30-2.92 (m, 4H, Pp-2,6-H), 1.72 (s, 3H, CH3). 13C NMR δ (ppm): 175.91, 175.87, 163.42,
160.99, 155.97, 155.89, 136.35, 128.94, 128.35, 128.27, 128.03, 127.03, 115.81, 115.59,
65.37, 63.42, 62.98, 62.95, 58.84, 58.73, 25.51, 15.63.
5-(4-fluorophenyl)-3-(2-hydroxy-3-{4-[(naphthalen-1-yl)methyl]piperazin-1-yl}propyl)-5-
methylimidazolidine-2,4-dione (6)
1
White solid. Yield 43%. LC/MS±: purity 95.24% tR=4.77, (ESI) m/z [M+H] 491.34. H NMR δ
(ppm): 9.02 (s, 1H, N1H), 8.43 (s, 1H, Ar), 8.11-7.99 (m, 2H, Ar), 7.95 (s, 1H, Ar), 7.73-7.48 (m,
5H, Ar), 7.22 (t, J = 8.9, 2H, Ar), 4.88 (s, 2H, CH2-Naphthyl), 4.26 (s, 1H, CHOH), 4.00-3.01 (m,
12H, Pp-2,3,5,6-H, Pp-CH2, N3-CH2), 1.69 (s, 3H, CH3). 13C NMR δ (ppm): 175.90, 175.85,
163.40, 160.97, 155.97, 155.88, 136.38, 136.35, 136.33, 136.30, 133.90, 132.55, 130.80,
129.18, 128.36, 128.34, 128.28, 128.26, 127.47, 126.73, 125.82, 124.57, 115.80, 115.59,
62.96, 62.93, 42.43, 25.44.
5-(4-fluorophenyl)-3-{2-hydroxy-3-[4-(naphthalen-1-yl)piperazin-1-yl]propyl}-5-
methylimidazolidine-2,4-dione hydrochloride (7)
1
White solid. Yield 36%. LC/MS±: purity 97.74% tR=4.77, (ESI) m/z [M+H] 477.31. H NMR δ
(ppm): 10.82 (bs, 1H, NH+), 9.07 (s, 1H, N1H), 8.14-8.12 (d, J = 8.7 Hz, 1H, Ar), 7.96-7.87 (m,
1H, Ar), 7.67-7.65 (d, J = 8.2 Hz, 1H, Ar), 7.63-7.49 (m, 4H, Ar), 7.46-7.43 (t, J = 7.8 Hz, 1H, Ar),
7.24 (t, J = 7.6 Hz, 2H, Ar), 7.17-7.16 (d, J = 7.2 Hz, 1H, Ar), 6.00 (s, 1H, CHOH), 4.38 (s, 1H,
CHOH), 3.82-3.13 (m, 12H, Pp-2,3,5,6-H, Pp-CH2, N3-CH2), 1.75 (s, 3H, CH3). 13C NMR δ (ppm):
175.95, 175.90, 163.44, 161.01, 155.99, 155.91, 148.16, 136.40, 136.37, 136.33, 134.75,
128.86, 128.37, 128.31, 126.55, 126.40, 126.23, 124.46, 123.62, 115.83, 115.61, 115.52,
63.28, 62.99, 53.26, 42.62, 25.53.
5-(4-fluorophenyl)-3-[2-hydroxy-3-(4-phenoxypiperidin-1-yl)propyl]-5-
methylimidazolidine-2,4-dione hydrochloride (8)
1
LC/MS±: purity 100.00% tR=4.55, (ESI) m/z [M+H] 442.28. H NMR δ (ppm): δ 10.56 (s, 1H,
NH+), 9.05 (s, 1H, N1H), 7.62-7.50 (m, 2H, Ar), 7.30 (dd, J = 14.3, 7.4 Hz, 8H), 7.27 – 7.19 (m,
2H, Ar), 7.07-6.90 (m, 3H, Ar), 5.92 (s, 1H, CHOH), 4.74 (m, 1H, CHaOH), 4.55 (m, CHbOH, 1H),
4.29 (s, 1H, CH-O), 3.55-3.25 (m, 4H, N3-CH2, Pp-CH2), 3.25-2.88 (m, 4H, Pp-2,6-H), 2.29-2.09
(m, 2H, Pp-3-H), 2.01-1.98 (m, 2H, Pp-5-H), 1.72 (s, 3H, CH3). 13C NMR δ (ppm): 175.86,
163.42, 160.99, 157.10, 156.79, 155.96, 136.36, 130.09, 128.35, 128.26, 121.68, 121.47,
116.73, 116.13, 115.81, 115.59, 70.53, 66.87, 63.43, 63.18, 63.09, 62.95, 59.54, 59.21, 51.61,
50.86, 48.75, 47.75, 47.63, 42.52, 26.56, 25.48.
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3-{3-[4-(4-chlorophenoxy)piperidin-1-yl]-2-hydroxypropyl}-5-(4-fluorophenyl)-5-
methylimidazolidine-2,4-dione hydrochloride (9)
1
White solid. Yield 58%. LC/MS±: purity 100.00% tR=5.10, (ESI) m/z [M+H] 476.25 H NMR δ
(ppm): 10.55 (bs, 1H, N+H), 9.04 (s, 1H, N1H), 7.60-7.48 (m, 2H, Ar), 7.40-7.28 (m, 2H, Ar),
7.26-7.21 (m, 2H, Ar), 7.11-6.97 (m, 2H, Ar), 5.92 (s, 1H, CHOH), 4.74 (m, 1H, CHaOH), 4.55
(m, CHbOH, 1H), 4.28 (s, 1H, CH-O), 3.55-3.25 (m, 4H, N3-CH2, Pp-CH2), 3.27-2.97 (m, 4H, Pp-
2,6-H), 2.26-2.09 (m, 2H, Pp-3-H), 2.01-1.98 (m, 2H, Pp-5-H), 1.72 (s, 3H, CH3). 13C NMR δ
(ppm): 175.86, 163.42, 160.99, 155.99, 155.96, 155.88, 155.67, 136.35, 129.86, 128.34,
128.26, 125.36, 125.13, 118.54, 117.90, 115.81, 115.59, 71.04, 67.44, 63.41, 63.17, 62.95,
59.49, 59.15, 51.57, 50.79, 48.67, 47.62, 42.52, 28.28, 28.20, 26.44, 25.95, 25.48.
3-{2-hydroxy-3-[4-(2-methoxyphenyl)piperazin-1-yl]propyl}-5-methyl-5-(naphthalen-1-
yl)imidazolidine-2,4-dione (10)
1
White solid. Yield 48%. LC/MS±: purity 99.04% tR=4.69, (ESI) m/z [M+H] 489.57. H NMR δ
(ppm): 8.79 (s, 1H, N1H), 7.98-7.94 (m, 2H, Ar), 7.92-7.79 (m, 1H, Ar), 7.75-7.72 (d, J = 8.1 Hz,
1H, Ar), 7.60-7.44 (m, 3H, Ar), 6.98-6.78 (m, 4H, Ar), 4.92 (t, J = 5.2 Hz, 1H, CHOH), 4.16-3.99
(m, 1H, CHOH), 3.74 (s, 3H, OCH3), 3.67-3.47 (m, 2H, N3-CH2), 2.94 (m, Pp-2,6-H, 4H), 2.57-
2.49 (m, 4H, Pp-3,5-H), 2.40-2.39 (d, J = 5.9 Hz, 2H, Pp-CH2), 1.92 (s, 3H, CH3). 13C NMR δ
(ppm): 177.07, 156.58, 156.54, 152.39, 141.68, 134.46, 133.98, 133.93, 131.02, 130.93,
130.22, 129.63, 127.07, 126.10, 125.37, 124.50, 122.77, 121.26, 118.32, 112.28, 65.41,
65.21, 63.46, 63.39, 55.72, 54.07, 50.48, 26.72.
3-{2-hydroxy-3-[4-(2-cyanophenyl)piperazin-1-yl]propyl}-5-methyl-5-(naphthalen-1-
yl)imidazolidine-2,4-dione hydrochloride (11)
1
White solid. Yield 44%. LC/MS±: purity 97.53% tR=4.65, (ESI) m/z [M+H] 484.52. H NMR δ
(ppm) 10.93 (s, 1H, NH+), 8.95 (s, 1H, N1H), 8.95 (s, 1H, Ar), 7.99-7.95 (m, 2H, Ar), 7.88-7.70
(m, 3H, Ar), 7.68-7.45 (m, 4H, Ar), 7.27-7.07 (m, 2H, Ar), 6.05 (s, 1H, CHOH), 4.47 (s, 1H,
CHOH), 3.82-3.49 (m, 6H, Pp-3,5-H; Pp-CH2), 3.49-3.11 (m, 6H, N3-CH2, Pp-2,6-H), 1.98 (s, 3H,
CH3). 13C NMR δ (ppm): 177.10, 177.06, 156.16, 154.20, 134.99, 134.74, 134.47, 133.77,
130.95, 130.90, 130.33, 129.73, 129.66, 127.25, 127.17, 126.94, 126.85, 126.14, 125.43,
124.55, 124.36, 123.48, 119.83, 118.37, 105.58, 63.69, 48.40, 26.62
3-{3-[4-(diphenylmethyl)piperazin-1-yl]-2-hydroxypropyl}-5-methyl-5-(naphthalen-1-
yl)imidazolidine-2,4-dione hydrochloride (12)
1
White solid. Yield 33%. LC/MS±: purity 98.69% tR=5.86, (ESI) m/z [M+H] 549.60 H NMR δ
(ppm): 10.57 (bs, 1H, NH+), 8.94 (s, 1H, N1H), 7.97 (m, 2H, Ar), 7.87-7.70 (m, 2H, Ar), 7.62-
7.47 (m, 3H, Ar), 7.44-7.42 (d, J = 7.5 Hz, 4H, Ar), 7.33-7.23 (t, J = 7.4 Hz, 4H, Ar), 7.22-7.17
(m, 2H, Ar), 6.00 (s, 1H, CHOH), 4.43 (m, 2H, CHOH, Ph-CH-Ph ), 3.63-3.40 (m, 4H, Pp-3,5-H),
3.20-3.13 (m, 4H, Pp-2,6-H ), 2.79-2.77 (m, 2H, Pp-CH2), 1.96 (s, 3H, CH3). 13C NMR δ (ppm):
177.07, 177.03, 156.15, 142.41, 134.47, 133.75, 130.95, 130.89, 130.33, 129.72, 129.65,
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129.16, 129.02, 127.63, 127.18, 126.90, 126.12, 125.42, 124.46, 124.32, 74.17, 63.68, 52.63,
48.26, 42.33, 26.27.
3-{2-hydroxy-3-[4-(2-methoxyphenyl)piperazin-1-yl]propyl}-5-methyl-5-(naphthalen-2-
yl)imidazolidine-2,4-dione hydrochloride (13)
1
White solid 42%. LC/MS±: purity 97.39% tR=4.84, (ESI) m/z [M+H] 489.57. H NMR δ (ppm):
10.63 (bs, 1H, NH+ ), 9.11 (s, 1H, s, 1H, N1H), 8.03 (s, 1H, Ar), 7.99-7.88 (m, 3H, Ar), 7.66-7.62
(m, 1H, Ar), 7.53-7.50 (m, 2H, Ar), 6.99-6.93 (m, 2H, Ar), 6.88-6.87 (m, 2H, Ar), 5.97 (s, 1H,
CHOH), 4.33 (s, 1H, CHOH), 3.76 (s, 3H, OCH3), 3.61 (s, 1H, CHOH), 3.55-3.39 (m, 6H, , Pp-3,5-
H; Pp-CH2 ), 3.21 (s, 2H, N3-CH2), 3.14-2.91 (m, 4H, Pp-2,6-H), 1.83 (s, 3H, CH3). 13C NMR δ
(ppm): 175.97, 175.97, 156.08, 156.01, 156.01, 152.20, 139.84, 137.54, 137.49, 132.95,
132.76, 128.58, 127.84, 126.94, 124.83, 124.33, 123.82, 121.25, 118.58, 112.30, 63.51,
63.09, 55.78, 52.95, 47.17, 42.52, 24.98.
3-{2-hydroxy-3-[4-(2-cyanophenyl)piperazin-1-yl]propyl}-5-methyl-5-(naphthalen-2-
yl)imidazolidine-2,4-dione hydrochloride (14)
White solid. Yield 38%. LC/MS±: purity 99.09% tR=4.77, (ESI) m/z [M+H] 484.52.
1H NMR δ (ppm): 10.82 (bs, 1H, NH+), 9.10 (s, 1H, N1H), 8.01 (s, 1H, Ar), 8.00-7.83 (m, 3H, Ar),
7.75-7.72 (t, J = 11.5 Hz, 1H), 7.65-7.60 (m, 2H, Ar), 7.53-7.50 (m, 2H, Ar), 7.22-7.12 (m, 2H,
Ar), 5.98 (s, 1H, CHOH), 4.34 (s, 1H, CHOH), 3.51 (m, 6H, Pp-3,5-H; Pp-CH2), 3.23 (m, 6H, N3-
CH2; Pp-2,6-H), 1.81 (s, 3H, CH3). 13C NMR δ (ppm): 176.02, 156.09, 156.03, 154.18, 137.53,
137.48, 134.97, 134.73, 132.95, 132.76, 131.25, 128.67, 128.58, 127.84, 126.95, 124.87,
124.34, 123.45, 119.81, 118.36, 105.54, 63.51, 63.16, 59.05, 52.71, 48.37, 42.63, 24.97.
3-{2-hydroxy-3-[4-(2-methoxyphenyl)piperazin-1-yl]propyl}-5,5-dimethylimidazolidine-2,4-
dione hydrochloride (15)
1
White solid. Yield 39%. LC/MS±: purity 98.25% tR=3.04, (ESI) m/z [M+H] 377.28. H NMR δ
(ppm): 10.59 (s, 1H, NH+), 8.37 (s, 1H, N1H), 7.08-6.88 (m, 4H, Ar), 4.28-4.27 (m, 1H, CHOH ),
3.79 (s, 3H, OCH3), 3.66-3.63 (d, J = 11.6 Hz, 1H, CHOH), 3.52-3.40 (m, 4H, Pp-3,5-H), 3.42-
3.05 (m, 8H, Pp-CH2, N3-CH2, Pp-2,6-H), 1.30 (s, 6H, diCH3).13C NMR δ (ppm): 178.03, 155.70,
152.29, 139.66, 124.04, 121.30, 118.75, 112.45, 63.18, 59.30, 58.31, 55.85, 52.92, 51.64,
47.27, 47.15, 42.31, 25.04.
3-{2-hydroxy-3-[4-(2-cyanophenyl)piperazin-1-yl]propyl}-5,5-dimethylimidazolidine-2,4-
dione hydrochloride (16)
1
White solid. Yield 51%. LC/MS±: purity 100.00% tR=3.02, (ESI) m/z [M+H] 372.30. H NMR δ
(ppm): 10.93 (s, 1H, NH+), 8.38 (s, 1H, N1H), 7.76 (dd, J = 7.7, 1.5 Hz, 1H, Ar), 7.68-7.58 (m,
1H, Ar), 7.24 (d, J = 8.3 Hz, 1H, Ar), 7.18 (t, J = 7.5 Hz, 1H, Ar), 5.97 (s, 1H, CHOH), 4.30 (d, J =
7.0 Hz, 1H, CHOH), 3.75-3.59 (m, 4H, Pp-3,5-H), 3.45-3.10 (m, 8H, Pp-CH2, N3-CH2, Pp-2,6-H),
1.32 (s, 6H, 2xCH3). 13C NMR δ (ppm): 178.02, 155.68, 154.21, 134.98, 134.74, 123.45,
119.83, 118.36, 105.57, 63.26, 59.32, 58.31, 52.68, 51.58, 48.42, 42.32, 25.04.
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3-{3-[4-(diphenylmethyl)piperazin-1-yl]-2-hydroxypropyl}-5,5-dimethylimidazolidine-2,4-
dione hydrochloride (17)
1
White solid. Yield 54%. LC/MS±: purity 100.00% tR=4.64, (ESI) m/z [M+H] 437.30. H NMR δ
(ppm): 8.33 (s, 2H, N1H), 7.40-7.32 (m, 10H), 4.21-4.18 (m, 2H, CHOH, Ph-CH-Ph ), 3.49-2.96
(m, 12H, Pp-CH2, N3-CH2, Pp-2,3,5,6-H ), 1.31 (s, 6H, 2xCH3).13C NMR δ (ppm): 178.01, 155.69,
129.69, 128.66, 65.37, 63.47, 58.31, 42.23, 25.05, 25.03, 15.64.
5-(4-fluorophenyl)-3-[2-hydroxy-3-(1,2,3,4-tetrahydroisoquinolin-2-yl)propyl]-5-
methylimidazolidine-2,4-dione (18)
1
White solid. Yield 42%. LC/MS±: purity 96.84% tR=3.88, (ESI) m/z [M+H] 398.22 . H NMR δ
(ppm): 9.03 (bs, 1H, N1H), 7.67 (s, 2H, Ar), 7.55-7.52 (m, 2H, Ar), 7.48-7.41 (m, 2H, Ar), 7.23
(td, J = 8.9, 1.0 Hz, 2H, Ar), 4.41-4.39 (m, 2H, N3-CH2), 4.26 (s, 1H, CHOH), 3.78-3.07 (m, 10H,
Pp-CH2, Pp-2,3,5,6-H), 1.70 (s, 3H, CH3). 13C NMR δ (ppm): 175.90, 175.85, 163.40, 160.97,
155.97, 155.88, 136.38, 136.35, 136.33, 136.30, 131.98, 130.04, 129.26, 128.36, 128.27,
115.81, 115.59, 63.39, 62.96, 62.93, 25.57, 25.44.
Crystallographic studies
The crystals of compound 3 suitable for X-ray structure analysis were obtained from a
mixture of propan-2-ol and water solution by slow evaporation of the solvent at room
temperature. The intensity data for single crystal were collected using the Oxford Diffraction
SuperNova four circle diffractometer, equipped with the Mo (0.71069 Å) Kα radiation source
and graphite monochromator. The phase problem was solved by direct methods using SIR-
201428 and all non-hydrogen atoms were refined anisotropically using weighted full-matrix
least-squares on F2. Refinement and further calculations were carried out using SHELXL29.
The hydrogen atoms bonded to carbons were included in the structure at idealized positions
and were refined using a riding model with Uiso(H) fixed at 1.2 Ueq of C with the exception of
hydrogen atoms in methyl group for which Uiso(H) fixed at 1.5 Ueq. Hydrogen atoms attached
to nitrogen and oxygen atoms were found from the difference Fourier map and refined
without any restraints. For molecular graphics ORTEP30 and MERCURY31 programs were
used.
C23H27FN4O3, Mr = 426.48, crystal size = 0.39 x 0.13 x 0.02 mm3, monoclinic, space
group P21/c, a = 17.4230(7) Å, b = 6.0728(2) Å, c = 20. 2071(7) Å, V = 2077.9(1) Å3, Z = 4, T =
130(2)K, 26816 reflections collected, 4984 unique reflections (Rint = 0.0787), R1 = 0.0738,
wR2 = 0.1793 [I > 2σ(I)].
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CCDC 1831620 contains the supplementary crystallographic data. These data can be
obtained free of charge from The Cambridge Crystallographic Data Centre via
www.ccdc.cam.ac.uk/data_request/cif.
Molecular modeling studies
Pharmacophore modeling
The 3-dimensional pharmacophore model was reconstructed by using the 3D QSAR
Pharmacophore Generation protocol implemented in Discovery Studio 3.5. The structures of
training compounds, as well as parameters were fetched from23. The conformation space of
both training and synthetized compounds was generated using the BEST algorithm within a
relative energy threshold of 20 kcal/mol above the global energy minimum and with a
maximum number of generated conformations per ligand set to 255. The minimum distance
between features was fixed at 2.5 Å, and the top 10 pharmacophore hypotheses were
returned by the generation process and further used in the evaluation with reference
pharmacophore model23 with respect to the inter-feature distances and angles.
Molecular docking
The 3-dimensional structures of the ligands were prepared using LigPrep v3.632, and the
appropriate ionization states at pH = 7.4 ± 1.0 were assigned using Epik v3.433. 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 field34) 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 level35.
Radioligand binding assays
Affinities for human 5-HT1A, 5-HT7b and D2L receptors
HEK-293 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
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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 (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 equation36. For all binding assays, results were expressed as means
of at least two separate experiments (SD ≤ 19%).
ADME-Tox properties studies
The reference compound aripiprazole was synthesized and provided by Adamed Ltd.
(Pieńków, Poland). The reference cytostatic drug doxorubicin (DX) and mitochondrial toxin
carbonyl cyanide 3-chlorophenylhydrazone (CCCP )were provided by Sigma-Aldrich (St. Louis,
MO, USA).
Mouse liver microsomes (MLMs): Microsomes from Liver, Pooled; biological source: from
mouse mus musculus (CD-1), male; gene information: Mouse MGST-1 (56615) were
purchased form Sigma-Aldrich (St. Louis, MO, USA).
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The NADPH Regeneration System was purchased from Promega (Madison, WI, USA). All
experiments were performed as described before20,21
.
Human embryonic kidney HEK-293 cell line ATCC CRL−1573 was kindly donated by Prof. Dr.
Christa Müller (Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn).
HepG2 (ATCC HB-8065) cell line was kindly donated by the Department of Pharmacological
Screening, Jagiellonian University Medical College. The cell cultures’ growth conditions were
applied as described before20,21. The CellTiter 96® AQueous Non-Radioactive Cell
Proliferation Assay (MTS) used for the cells’ viability determination was purchased from
Promega (Madison, WI, USA). The assays were performed as described previously20,21. The
absorbance of the samples was measured using a microplate reader EnSpire (PerkinElmer,
Waltham, MA USA) at 490 nm. GraphPad Prism™ software (version 5.01, San Diego, CA, USA)
was used to calculate statistical significance.
Acknowledgements:
The authors would like to thank Prof. Dr. Christa Müller as well as to the research team of
Department of Pharmacological Screening, Jagiellonian University Medical College for kind
donating cell lines for the ADMET studies. The authors would like to thank Prof. Andrzej J.
Bojarski for opportunity to carry out RBA studies in his Department and for all his helpful
suggestions concerning this study. Described herein research was supported by National
Science Center (Poland) Grant No. UMO-2014/15/N/NZ7/03072.
Conflict of Interest
The authors declare no competing interest.
References
1
2
3
4
5
M. Ruat, E. Traiffort, R. Leurs, J. Tardivel-Lacombe, J. Diaz, J. M. Arrang and J. C.
Schwartz, Proc Natl Acad Sci U S A, 1993, 90, 8547–8551.
S. B. Lovenberg TW1, Baron BM, de Lecea L, Miller JD, Prosser RA, Rea MA, Foye PE,
Racke M, Slone AL, Neuron, 1993, 11, 449–458.
J. A. Bard, J. Zgombick, N. Adham, P. Vaysse, T. A. Branchek and R. L. Weinshank, J.
Biol. Chem., 1993, 268, 23422–23426.
A. Wesołowska, A. Nikiforuk, K. Stachowicz and E. Tatarczyńska, Neuropharmacology,
2006, 51, 578–586.
A. Nikiforuk, T. Kos, K. Fijał, M. Hołuj, D. Rafa and P. Popik, PLoS One, 2013, 8, e66695.
23

MedChemComm
Page 24 of 26
View Article Online
DOI: 10.1039/C8MD00168E
6
7
J. Śniecikowska, M. Głuch-lutwin, A. Bucki, P. Mierzejewski and M. Kołaczkowski,
2017, 26, 165–178.
P. Bonaventure, C. Dugovic, M. Kramer, P. De Boer, J. Singh, S. Wilson, K. Bertelsen, J.
Di, J. Shelton, L. Aluisio, L. Dvorak, I. Fraser, B. Lord, D. Nepomuceno, A. Ahnaou, W.
Drinkenburg, W. Chai, C. Dvorak, S. Sands, N. Carruthers and T. W. Lovenberg, J.
Pharmacol. Exp. Ther., 2012, 342, 429–440.
8
9
A. Zagórska, A. Bucki, M. Kołaczkowski, A. Siwek, M. Głuch-Lutwin, G. Starowicz, G.
Kazek, A. Partyka, A. Wesołowska, K. Słoczyńska, E. Pękala and M. Pawłowski, J.
Enzyme Inhib. Med. Chem., 2016, 31, 10–24.
A. Zagórska, M. Kołaczkowski, A. Bucki, A. Siwek, G. Kazek, G. Satała, A. J. Bojarski, A.
Partyka, A. Wesołowska and M. Pawłowski, Eur. J. Med. Chem., 2015, 97, 142–154.
A. Anighoro, J. Bajorath and G. Rastelli, J. Med. Chem., 2014, 57, 7874–7887.
B. L. Roth, D. J. Sheffler and W. K. Kroeze, Nat. Rev. Drug Discov., 2004, 3, 353–359.
C. J. Suckling, J. A. Murphy, A. I. Khalaf, S. ze Zhou, D. E. Lizos, A. N. van Nhien, H.
Yasumatsu, A. McVie, L. C. Young, C. McCraw, P. G. Waterman, B. J. Morris, J. A. Pratt
and A. L. Harvey, Bioorganic Med. Chem. Lett., 2007, 17, 2649–2655.
M. J. Millan, Neurotherapeutics, 2009, 6, 53–77.
10
11
12
13
14
B. Bang-Andersen, T. Ruhland, M. Jorgensen, G. Smith, K. Frederiksen and K. G.
Jensen, J. Med. Chem., 2011, 54, 3206–21.
15
16
17
A. D’Agostino, C. D. English and J. A. Rey, P T, 2015, 40, 36–40.
A. Loebel and L. Citrome, BJPsych Bull., 2015, 39, 237–241.
J. Handzlik, A. J. Bojarski, G. Satała, M. Kubacka, B. Sadek, A. Ashoor, A. Siwek, M.
Wiȩcek, K. Kucwaj, B. Filipek and K. Kieć-Kononowicz, Eur. J. Med. Chem., 2014, 78,
324–339.
18
19
20
21
K. Kucwaj-Brysz, D. Warszycki, S. Podlewska, J. Witek, K. Witek, A. G. Izquierdo, G.
Satała, M. I. Loza, A. Lubelska, G. Latacz, A. J. Bojarski, M. Castro, K. Kieć-Kononowicz
and J. Handzlik, Eur. J. Med. Chem., , DOI:10.1016/j.ejmech.2016.02.024.
K. Kucwaj-Brysz, R. Kurczab, M. Jastrzębska-Więsek, E. Żesławska, G. Satała, W. Nitek,
A. Partyka, A. Siwek, A. Jankowska, A. Wesołowska, K. Kieć-Kononowicz and J.
Handzlik, Eur. J. Med. Chem., , DOI:10.1016/j.ejmech.2018.01.093.
G. Latacz, A. Lubelska, M. Jastrzębska-Więsek, A. Partyka, K. Kucwaj-Brysz, A.
Wesołowska, K. Kieć-Kononowicz and J. Handzlik, Bioorg. Med. Chem. Lett., 2018, 28,
878–883.
G. Latacz, A. Lubelska, M. Jastrzębska-Więsek, A. Partyka, A. Sobiło, A. Olejarz, K.
Kucwaj-Brysz, G. Satała, A. J. Bojarski, A. Wesołowska, K. Kieć-Kononowicz and J.
Handzlik, Chem. Biol. Drug Des., , DOI:10.1111/cbdd.13106.
22
23
E. Żesławska, A. Kincses, G. Spengler, W. Nitek, K. Wyrzuc, K. Kieć-Kononowicz and J.
Handzlik, Bioorg. Med. Chem., 2016, 24, 2815–2822.
M. L. López-Rodríguez, E. Porras, M. J. Morcillo, B. Benhamú, L. J. Soto, J. L. Lavandera,
J. A. Ramos, M. Olivella, M. Campillo and L. Pardo, J. Med. Chem., 2003, 46, 5638–
5650.
24
L. Salerno, V. Pittalà, M. N. Modica, M. A. Siracusa, S. Intagliata, A. Cagnotto, M.
Salmona, R. Kurczab, A. J. Bojarski and G. Romeo, Eur. J. Med. Chem., 2014, 85, 716–
726.
25
26
R. G. Murray, D. M. Whitehead, F. Le Strat and S. J. Conway, Org. Biomol. Chem.,
2008, 6, 988.
J. Lee, J. Kim, J. S. Koh, H.-H. Chung and K.-H. Kim, Bioorg. Med. Chem. Lett., 2006, 16,
24

Page 25 of 26
MedChemComm
View Article Online
DOI: 10.1039/C8MD00168E
1954–1956.
27
28
R. Li, P. Hu, X. Ren, S. D. Worley and T. S. Huang, Carbohydr. Polym., 2013, 92, 534–
539.
M. C. Burla, R. Caliandro, B. Carrozzini, G. L. Cascarano, C. Cuocci, C. Giacovazzo, M.
Mallamo, A. Mazzone, G. Polidori and IUCr, J. Appl. Crystallogr., 2015, 48, 306–309.
G. M. Sheldrick, Acta Crystallogr. Sect. C Struct. Chem., 2015, 71, 3–8.
L. J. Farrugia and IUCr, J. Appl. Crystallogr., 2012, 45, 849–854.
C. F. Macrae, P. R. Edgington, P. McCabe, E. Pidcock, G. P. Shields, R. Taylor, M.
Towler, J. van de Streek and IUCr, J. Appl. Crystallogr., 2006, 39, 453–457.
Schrödinger Release 2017-3: LigPrep, Schrödinger, LLC, New York, NY, 2017, (n.d.).
Schrödinger Release 2017-3: Epik, Schrödinger, LLC, New York, NY, 2017, (n.d.).
E. Harder, W. Damm, J. Maple, C. Wu, M. Reboul, J. Y. Xiang, L. Wang, D. Lupyan, M. K.
Dahlgren, J. L. Knight, J. W. Kaus, D. S. Cerutti, G. Krilov, W. L. Jorgensen, R. Abel and
R. A. Friesner, J. Chem. Theory Comput., 2016, 12, 281–296.
29
30
31
32
33
34
35
36
Schrödinger Release 2017-3: Glide, Schrödinger, LLC, New York, NY, 2017., (n.d.)
Y. Cheng and W. H. Prusoff, Biochem. Pharmacol., 1973, 22, 3099–108.
25

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Table of contents information
Influence of presence and spatial orientation of aromatic ring(s) on 5-HT7/5-HT1A selectivity and
activity were investigated within novel hydantoin-derived series.