Novel serotonin 5-HT2A receptor antagonists derived from 4-phenylcyclohexane-5-spiro-and 5-methyl-5-phenyl-hydantoin, for use as potential antiplatelet agents

Article abstract of DOI:10.1007/s43440-021-00284-6

Background: Antiplatelet drugs have been used in the treatment of acute coronary syndromes and for the prevention of recurrent events. Unfortunately, many patients remain resistant to the available antiplatelet treatment. Therefore, there is a clinical need to synthesize novel antiplatelet agents, which would be associated with different pathways of platelet aggregation, to develop an alternative or additional treatment for resistant patients. Recent studies have revealed that 5-HT_2A receptor antagonists could constitute alternative antiplatelet therapy. Methods: Based on the structures of the conventional 5-HT_2A receptor ligands, two series of compounds with 4-phenylcyclohexane-5-spiro- or 5-methyl-5-phenyl-hydantoin core linked to various arylpiperazine moieties were synthesized and their affinity for 5-HT_2A receptor was assessed. Further, we evaluated their antagonistic potency at 5-HT_2A receptors using isolated rat aorta and cells expressing human 5-HT_2A receptors. Finally, we studied their anti-aggregation effect and compared it with ketanserin and sarpogrelate, the reference 5-HT_2A receptor antagonists. Moreover, the structure–activity relationships were studied following molecular docking to the 5-HT_2A receptor model. Results: Functional bioassays revealed some of the synthesized compounds to be moderate antagonists of 5-HT_2A receptors. Among them, 13, 8-phenyl-3-(3-(4-phenylpiperazin-1-yl)propyl)-1,3-diazaspiro[4.5]decane-2,4-dione, inhibited collagen stimulated aggregation (IC₅₀ = 27.3?μM) being more active than sarpogrelate (IC₅₀ = 66.8?μM) and comparable with ketanserin (IC₅₀ = 32.1?μM). Moreover, compounds 2–5, 9–11, 13, 14 inhibited 5-HT amplified, ADP- or collagen-induced aggregation. Conclusions: Our study confirmed that the 5-HT_2A antagonists effectively suppress platelet aggregation and remain an interesting option for the development of novel antiplatelet agents with an alternative mechanism of action.

Full text of DOI:10.1007/s43440-021-00284-6

Pharmacological Reports
https://doi.org/10.1007/s43440-021-00284-6
ARTICLE
Novel serotonin 5‑HT2A receptor antagonists derived
from 4‑phenylcyclohexane‑5‑spiro‑and 5‑methyl‑5‑phenyl‑hydantoin,
for use as potential antiplatelet agents
Anna Czopek1 · Monika Kubacka2 · Adam Bucki1 · Agata Siwek3 · Barbara Filipek2 · Maciej Pawłowski1 ·
Marcin Kołaczkowski1
Received: 31 December 2020 / Revised: 13 May 2021 / Accepted: 19 May 2021
© The Author(s) 2021
Abstract
Background Antiplatelet drugs have been used in the treatment of acute coronary syndromes and for the prevention of
recurrent events. Unfortunately, many patients remain resistant to the available antiplatelet treatment. Therefore, there is a
clinical need to synthesize novel antiplatelet agents, which would be associated with diferent pathways of platelet aggrega-
tion, to develop an alternative or additional treatment for resistant patients. Recent studies have revealed that 5-HT2A receptor
antagonists could constitute alternative antiplatelet therapy.
Methods Based on the structures of the conventional 5-HT2A receptor ligands, two series of compounds with 4-phenylcy-
clohexane-5-spiro- or 5-methyl-5-phenyl-hydantoin core linked to various arylpiperazine moieties were synthesized and their
afnity for 5-HT2A receptor was assessed. Further, we evaluated their antagonistic potency at 5-HT2A receptors using isolated
rat aorta and cells expressing human 5-HT2A receptors. Finally, we studied their anti-aggregation efect and compared it with
ketanserin and sarpogrelate, the reference 5-HT2A receptor antagonists. Moreover, the structure–activity relationships were
studied following molecular docking to the 5-HT2A receptor model.
Results Functional bioassays revealed some of the synthesized compounds to be moderate antagonists of 5-HT2A recep-
tors. Among them, 13, 8-phenyl-3-(3-(4-phenylpiperazin-1-yl)propyl)-1,3-diazaspiro[4.5]decane-2,4-dione, inhibited col-
lagen stimulated aggregation (IC50 =27.3 μM) being more active than sarpogrelate (IC50 =66.8 μM) and comparable with
ketanserin (IC50 =32.1 μM). Moreover, compounds 2–5, 9–11, 13, 14 inhibited 5-HT amplifed, ADP- or collagen-induced
aggregation.
Conclusions Our study confrmed that the 5-HT2A antagonists efectively suppress platelet aggregation and remain an inter-
esting option for the development of novel antiplatelet agents with an alternative mechanism of action.
Keywords Imidazolidinine-2,4-dione · 5-HT2A receptors · Hydantoin · Antiplatelet · Aggregation
Introduction
Antiplatelet drugs have long been used in the treatment of
acute coronary syndromes and for the prevention of recur-
rent events. Large clinical trials have confrmed that anti-
platelet agents such as clopidogrel and aspirin are capable of
reducing the risk of myocardial infarction, stroke, or death
[1, 2]. However, many patients treated with these agents still
experience recurrent atherothrombotic events [3]. Moreo-
ver, some are resistant to aspirin [cyclooxygenase-1 (COX-
1) inhibitor] or clopidogrel (P2Y12 receptor antagonist),
even when they are used in combination, which increases
their risk of further cardiovascular events [4–6]. Therefore,
there is still a clinical need to synthesize novel antiplatelet
* Anna Czopek
anna.czopek@uj.edu.pl
1
Department of Medicinal Chemistry, Faculty of Pharmacy,
Jagiellonian University Medical College, 9 Medyczna Street,
30-688 Kraków, Poland
2
Department of Pharmacodynamics, Jagiellonian University
Medical College, 9 Medyczna Street, 30-688 Kraków, Poland
3
Department of Pharmacobiology, Faculty of Pharmacy,
Jagiellonian University Medical College, Medyczna 9,
30-688 Krakow, Poland
Vol.:(0123456789)
1 3

A. Czopek et al.
agentsinvolving diferent pathways of platelet aggregation,
as an alternative or additional treatment for resistant patients.
Serotonin (5-HT) is a platelet activator, stored in plate-
let-dense granules [5]. Among the seven classes of 5-HT
receptors, only 5-HT2A has been found in platelets. Upon
platelet activation, 5-HT is released to plasma and, in an
autocrine manner, promotes further platelet activation,
acting at 5-HT2A receptors. Released serotonin is also a
potent vasoconstrictor of coronary arteries with a damaged
endothelium [5, 7, 8]. Furthermore, coronary artery disease
exerts a stimulating efect on platelets, triggering them to
release serotonin [9]. Several studies on animal models and
humans have shown that 5-HT2A receptor antagonists can
inhibit platelet aggregation [5, 8]. 5-HT may also be par-
tially responsible for the increased residual platelet reactiv-
ity observed in patients who are on clopidogrel treatment
after coronary stent placement, while 5-HT2A antagonists
reduce high on-treatment platelet reactivity. Therefore, in
addition to the established therapies, serotonin antagonism at
5-HT2A receptors might be a promising approach to improve
the treatment outcomes of patients with coronary artery dis-
ease [5, 10].
and exert an antagonistic efect. Among these structures, two
hydantoin derivatives with the highest afnity (Ki =50 and
69 nM, respectively) and selectivity for 5-HT2A receptors
were selected, namely, 4-phenylcyclohexane-5-spirohydan-
toin (compound A) and 5-methyl-5-phenylhydantoin (com-
pound B) linked with 4-phenypiperazin-1-yl-propyl moiety,
which displayed signifcant antagonistic activity for 5-HT2A
receptors (Fig. 1). The compounds A and B ft the pharmaco-
phore models of 5-HT2A receptor antagonists proposed over
the past two decades, which typically share two aryl rings
or hydrophobic fragments and basic amine moiety separated
from each other by a distance of 5.2–8.4 Å and 5.7–8.5 Å
[14] (Supplementary material Figure S1). These results were
a prerequisite for designing a new series of 5-methyl-5-phe-
nylhydantoin and 4-phenylcyclohexane-5-spirohydantoin
derivatives, which can serve as 5-HT2A serotonin receptor
ligands. The compounds have been designed as analogs of
the aforementioned hydantoin derivatives (compound A and
B) by combining structural fragments present in the con-
ventional 5-HT2A receptor antagonists, namely ketanserin
(5-HT2A: Ki = 2 nM), ritanserin (5-HT2A: Ki = 0.45 nM),
and fananserin (5-HT2A: Ki = 0.37 nM). The structural
modifcations carried out to develop novel 5-HT2A recep-
tor antagonists were (1) the extension of the alkyl spacer
linking hydantoin with arylpiperazine moiety and (2) the
Our previous studies [11–13] showed that a diferently
substituted hydantoin ring connected to arylpiperazine moi-
ety being the amine part, may interact with 5-HT2A receptors
Fig. 1 The general structures of designed compounds based on structure compounds 9 and 12 as well as conventional 5-HT2A antagonists: ketan-
serin, fananserin and ritanserin
1 3

Novel serotonin 5-HT2A receptor antagonists derived from 4-phenylcyclohexane-5-spiro-and…
introduction of a diferently substituted arylpiperazine frag-
ment in the amine part.
and D, scheme 1) [11, 12, 15] as well as (4-fuorophenyl)
(piperazin-1-yl)methanone [16] were described earlier and
the analytical results are consistent with those previously
published.
This manuscript describes the synthesis of the designed
compounds and their afnity for the 5-HT2A receptors. The
intrinsic activity of the most promising compounds was eval-
uated, and molecular modeling studies were undertaken to
identify the structural fragments responsible for their in vitro
afnity. Finally, the anti-aggregation efect was tested after
inducing the aggregation of whole rat blood with collagen
and with 5-HT and ADP or collagen at the sub-threshold
concentration.
General procedure for the synthesis of fnal compounds
(1–12)
The final compounds (1–12) were obtained within two
different synthetic pathways, described in procedure A
and B. Procedure A (1–6, 8, 9, 11): the fnal compounds
were synthesized in a CEM microwave reactor (Discover
model). The intermediate compound (0.3 mmol), trimeth-
ylamine (0.6 mmol) and appropriate piperazine derivatives
(0.36 mmol) in acetonitrile (4 mL) was added to the micro-
wave fask, and stirred for 65–100 min, at 100 °C, 200 W.
Upon cooling to room temperature, the reaction mixture was
concentrated in vacuum and purifed by column chromatog-
raphy. Procedure B (7, 10, 12): the reaction was carried out
in a round bottom fask on a magnetic stirrer. The intermedi-
ate compound (0.33 mmol), appropriate piperazine deriva-
tives (0.4 mmol), and potassium carbonate (0.66 mmol) in
acetonitrile (10 mL) were heated for 24–48 h at 80 °C, with
continuous stirring. After concentration in a vacuum, extrac-
tion was carried out with methylene chloride, the organic
layer was dried, concentrated, and purifed using column
chromatography.
Materials and methods
Chemistry
All chemicals and solvents were purchased from commer-
cial suppliers (Aldrich and Chempur) and were used without
further purifcation. Melting points were measured in open
capillaries on an Electrothermal 9300 apparatus. Thin-layer
chromatography (TLC) was run on Merck silica gel 60 F254
aluminium sheets (Merck; Germany), using the following
solvents (v:v): (S1) dichloromethane (9)/methanol (0.3), (S2)
ethyl acetate (9)/methanol (1), (S3) chloroform (7)/n-hex-
ane (2)/acetone (2). Analytical HPLC was conducted on a
Waters HPLC instrument with Waters 485 Tunable Absorb-
ance Detector UV, equipped with a Symmetry column (C18,
3.5 µm, 4.6×30 mm) using water/acetonitrile gradient with
0.1% TFA as mobile phase at a fow rate of 5 ml/min. Addi-
tionally, the liquid chromatography/mass spectrometry (LC/
MS) analysis was performed on the Waters Acquity TQD
system, with a Waters TQD quadrupole mass spectrometer
with detection by UV (DAD) using an Acquity UPLC BEH
C18 column (1.7 μm, 2.1×100 mm). Water/acetonitrile gra-
dient with 0.1% TFA was used as a mobile phase at a fow
rate of 0.3 ml/min. The UPLC/MS purity of the investigated
compounds (1–12) ranged to be over 95%. Elemental analy-
ses (C, H, and N) for fnal compounds were carried out by
a micro method using the elemental Vario EI III Elemental
analyzer (Hanau, Germany). NMR spectra were recorded
on Varian Mercury 300 MHz spectrometer (Varian Inc.,
Palo Alto, CA, USA) using the solvent (CDCl3) signal as
an internal standard; chemical shifts are expressed in parts
per million (ppm). Signal multiplets are represented by the
following abbreviations: s (singlet), brs (broad singlet), d
(doublet), t (triplet), m (multiplet). The Discover model,
CEM microwave reactor was used to carry out the pressur-
ized reaction.
The analytical results of 8-phenyl-3-(3-(4-phenylpipera-
zin-1-yl)propyl)-1,3-diazaspiro[4.5]decane-2,4-dione (13)
and 3-(3-(4-(3-chlorophenyl)piperazin-1-yl)propyl)-8-phe-
nyl-1,3-diazaspiro[4.5]decane-2,4-dione (14) were previ-
ously described [11]. Detailed descriptions of compounds
1, 6, 7, 8, and 12 are in Supplementary materials.
3‑(2‑(4‑Benzhydrylpiperazin‑1‑yl)ethyl)‑5‑methyl‑5‑phe‑
nylimidazolidine‑2,4‑dione (2) White powdery crystals.
Yield: 72%; mp 212–215 °C; TLC: Rf =0.31 (S1); HPLC:
tR =1.491; MS calcd for [M+H]+: C29H32N4O2 m/z: 468.25,
1
found: 469.27; H NMR (300 MHz, CDCl3-d) δ ppm 1.81
(s, 3 H, -CH3) 2.28 (br. s., 4 H, Pip) 2.47 (br. s., 4 H, Pip)
2.57 (t, J=6.45 Hz, 2 H, -CH2-Pip) 3.61 (t, J=6.45 Hz, 2 H,
Hyd-CH2-) 4.13 [s, 1 H, -CH-(Ph)2] 7.13—7.20 (m, 2 H, Ph,
Hyd) 7.22–7.30 (m, 5 H, Ph) 7.31–7.42 (m, 7 H, Ph) 7.47–
7.54 (m, 2 H, Ph). Anal. calcd for C29H32N4O2 (468.60): C:
74.33, H: 6.88, N: 11.96; Found C: 74.08, H: 7.02, N: 11.50.
3‑(3‑(4‑(2‑Fluorophenyl)piperazin‑1‑yl)propyl)‑5‑me‑
thyl‑5‑phenylimidazolidine‑2,4‑dione (3) White powdery
crystals. Yield: 83%; mp 126–129 °C; TLC: Rf =0.25 (S1);
HPLC: tR =1.217; MS calcd for [M+H]+: C23H27FN4O2
m/z: 410.21, found: 411.38; 1H NMR (300 MHz, CDCl3-d)
δ ppm 1.77–1.88 (m, 5 H, -CH3, -CH2-CH2-CH2-) 2.39 (t,
J=7.03 Hz, 2 H, -CH2-Pip) 2.56 (t, J=4.7 Hz, 4 H, Pip)
5-Methyl-5-phenylimidazolidine-2,4-dione (A,
scheme 1), 8-phenyl-1,3-diazaspiro[4.5]decane-2,4-dione
(C, scheme 1) and intermediate products (N3 halogealkyl
derivatives of substituted imidazolidyno-2,4-diones) (B
1 3

A. Czopek et al.
Scheme 1 The synthetic routes of novel 5-methyl-5-phenylhydantoin
(series 1) and 4-phenylcyclohexane-5-spirohydantoin (series 2) deriv-
atives. Reagents and conditions: A KCN, (NH2)2CO3, 50% EtOH,
70 °C, 3 h; B Br(CH2)nBr, K2CO3, DMF, 70 °C, 3 h; C arylpipera-
zine, TEA, MeCN, MW: 200 W, 100 °C, 65–100 min (compd 1–6, 8,
9, 11)/arylpiperazine, K2CO3, 80˚C, MeCN, 24–48 h (compd 7, 10,
12).
3.07 (t, J=4.7, 5.3 Hz, 4 H, Pip) 3.54 (t, J=7, 7.1 Hz, 2 H,
Hyd-CH2-) 6.86–7.08 (m, 5 H, Ph) 7.28–7.42 (m, 3 H, Hyd,
Ph) 7.47–7.54 (m, 2 H, Ph). Anal. calcd for C23H27FN4O2
(410.49): C: 67.30, H: 6.63, N: 13.65; Found C: 66.95, H:
6.72, N: 13.19.
3‑(3‑(4‑(4‑Fluorophenyl)piperazin‑1‑yl)propyl)‑5‑me‑
thyl‑5‑phenylimidazolidine‑2,4‑dione (5) White powdery
crystals. Yield: 71%; mp 141–143 °C; TLC: Rf =0.54 (S2);
HPLC: tR =1.235; MS calcd for [M+H]+: C23H27FN4O2
m/z: 410.21, found: 411.25; 1H NMR (300 MHz, CDCl3-d)
δ ppm 1.79–1.90 (m, 5 H, -CH3, CH2-CH2-CH2-) 2.41 (t,
J=7.33 Hz, 2 H, -CH2-Pip) 2.56 (t, J=4.1, 5.3 Hz, 4 H, Pip)
3.07 (t, J=4.7, 5.3 Hz, 4 H, Pip) 3.58 (t, J=7.03 Hz, 2 H,
Hyd-CH2-) 6.35 (s, 1 H, Hyd) 6.79–6.88 (m, 2 H, Ph) 6.90–
6.99 (m, 2 H, Ph) 7.29–7.42 (m, 3 H, Ph) 7.45–7.53 (m, 2
H, Ph). Anal. calcd for C23H27FN4O2 (410.49): C: 67.30, H:
6.63, N: 13.65; Found C: 66.82, H: 6.70, N: 13.36.
3‑(3‑(4‑(3‑Fluorophenyl)piperazin‑1‑yl)propyl)‑5‑me‑
thyl‑5‑phenylimidazolidine‑2,4‑dione (4) White powdery
crystals. Yield: 33%; mp 125–126 °C; TLC: Rf =0.26 (S1);
HPLC: tR =1.253; MS calcd for [M+H]+: C23H27FN4O2
m/z: 410.21, found: 411.18; 1H NMR (300 MHz, CDCl3-d)
δ ppm 1.77–1.89 (m, 5 H, -CH2-CH2-CH2-, -CH3) 2.38 (t,
J=7, 7.1 Hz, 2 H, -CH2-Pip) 2.47–2.55 (t, J=4.7, 5.3 Hz,
4 H, Pip) 3.08–3.16 (t, J=4.7, 5.3 Hz, 4 H, Pip) 3.58 (t,
J=7.33 Hz, 2 H, Hyd-CH2-) 6.46–6.67 (m, 4 H, Hyd, Ph)
7.11–7.22 (m, 1 H, Ph) 7.29–7.42 (m, 3 H, Ph) 7.47–7.53
(m, 2 H, Ph). Anal. calcd for C23H27FN4O2 x H2O (428.51):
C: 64.47, H: 6.82, N: 13.08; Found C: 64.66, H: 6.55, N:
12.63.
3‑(3‑(4‑(2‑Fluorophenyl)piperazin‑1‑yl)propyl)‑8‑phe‑
nyl‑1,3‑diazaspiro[4.5]decane‑2,4‑dione
(9) White
powdery crystals. Yield: 53%; mp 179–181 °C; TLC:
Rf =0.34 (S1); HPLC: tR =1.523; MS calcd for [M+H]+:
C27H33FN4O2 m/z: 464.26, found: 465.28; 1H NMR
(300 MHz, CDCl3-d) δ ppm 1.66–1.82 (m, 4 H, Cyclohex-
1 3

Novel serotonin 5-HT2A receptor antagonists derived from 4-phenylcyclohexane-5-spiro-and…
ane) 1.75 (q, J=2.9, 11.7, 11.8 Hz, 4 H, Cyclohexane)
1.85–2.14 (m, 7 H, Cyclohexane, -CH2-CH2-CH2-, Ph-CH-)
2.47 (t, J=7.33 Hz, 2 H, -CH2-Pip) 2.61 (t, J=4.1 Hz, 4 H,
Pip) 3.08 (t, J=4.1, 4.6 Hz, 4 H, Pip) 3.65 (t, J=7.03 Hz,
2 H, Hyd-CH2-) 6.87–7.07 (m, 4 H, Ph) 7.19–7.25 (m, 1 H,
Ph) 7.30–7.36 (m, 4 H, Ph) 8.06 (s, 1 H, Hyd). Anal. calcd
for C23H25N4O3F (424.48): C: 69.80, H: 7.16, N: 12.06;
Found C: 69.58, H: 7.22, N: 12.18.
out in duplicates. Data were ftted to a one-site curve-ftting
equation with Prism 6 (GraphPad Software) and Ki values
were estimated from the Cheng−Prusof equation. The full
description of this assay is in Supplementary material.
Functional bioassays
In vitro functional bioassays at cells transfected
with human 5HT2A receptor (aequorin
3‑(3‑(4‑(3‑Fluorophenyl)piperazin‑1‑yl)propyl)‑8‑phe‑
nyl‑1,3‑diazaspiro[4.5]decane‑2,4‑dione (10) White pow-
dery crystals. Yield: 52%; mp 193–195 °C; TLC: Rf =0.37
(S1);HPLC:tR =1.546;MScalcdfor[M+H]+:C27H33FN4O2
m/z: 464.26, found: 465.27; 1H NMR (300 MHz, CDCl3-d)
δ ppm 1.74 (q, J=3.5, 11.7, 11.8 Hz, 4 H, Cyclohexane)
1.86–2.09 (m, 7 H, Cyclohexane, -CH2-CH2-CH2-, Ph-CH-)
2.44 (t, J=7.33 Hz, 2 H, -CH2-Pip) 2.55 (t, J=4.7, 5.3 Hz, 4
H, Pip) 3.16 (t, J=4.7, 5.3 Hz, 4 H, Pip) 3.65 (t, J=7.03 Hz,
2 H, Hyd-CH2-) 6.47–6.66 (m, 3 H, Ph) 7.12–7.25 (m, 2 H,
Ph) 7.30–7.35 (m, 4 H, Ph) 8.05 (s, 1 H, Hyd Anal. calcd for
C23H25N4O3F (424.48): C: 69.80, H: 7.16, N: 12.06; Found
C: 69.92, H: 7.34, N: 11.86.
and luminescence‑based intracellular calcium assay)
Tested and reference compounds were dissolved in DMSO
at a concentration of 10–2 M. Serial dilutions were prepared
in 96-well microplate in assay bufer and eight to ten con-
centrations were tested.
A cellular aequorin-based functional assay was performed
with recombinant Chinese hamster ovary cells express-
ing mitochondrially targeted aequorin, human G-protein-
coupled receptors, and the promiscuous G protein α16 for
5-HT2A. The full description of this assay is in Supplemen-
tary materials.
Functional bioassays at 5HT2A‑receptors in isolated rat
aorta
3‑(3‑(4‑(4‑Fluorophenyl)piperazin‑1‑yl)propyl)‑8‑phe‑
nyl‑1,3‑diazaspiro[4.5]decane‑2,4‑dione (11) White pow-
dery crystals. Yield: 60%; mp 216–217 °C; TLC: Rf =0.31
(S1);HPLC:tR =1.526;MScalcdfor[M+H]+:C27H33FN4O2
m/z: 464.26, found: 465.28; 1H NMR (300 MHz, CDCl3-d)
δ ppm 1.70–1.84 (q, J=9.4, 12.9, 14.6, 4 H, Cyclohexane)
1.85–2.13 (m, 7 H, Cyclohexane, -CH2-CH2-CH2-, Ph-CH-)
2.45 (t, J=7.33 Hz, 2 H, -CH2-Pip) 2.56 (t, J=4.7 Hz, 4 H,
Pip) 3.07 (t, J=4.7 Hz, 4 H, Pip) 3.65 (t, J=7.03 Hz, 2 H,
Hyd-CH2-) 6.78—6.87 (m, 2 H, Ph) 6.88–6.99 (m, 2 H, Ph)
7.18–7.25 (m, 1 H, Ph) 7.28–7.39 (m, 4 H, Ph) 8.38 (s, 1 H,
Hyd). Anal. calcd for C23H25N4O3F (424.48): C: 69.80, H:
7.16, N: 12.06; Found C: 69.87, H: 7.32, N: 11.81.
Isolated rat aorta was used to determine the antagonistic
activity of studied compounds for 5HT2A-receptors. The
male Wistar rats weighting 200–350 g were anesthetized
with thiopental sodium (75 mg/kg i.p.) and the aorta was
dissected and placed in a Krebs–Henseleit solution (NaCl
118 mM, KCl 4.7 mM, CaCl2 2.25 mM, MgSO4 1.64 mM,
KH2PO4 1.18 mM, NaHCO3 24.88 mM, glucose 10 mM,
C3H3O3Na 2.2 mM, EDTA 0.05 mM), denuded of the
endothelium, cleaned of surrounding fat tissues and cut into
4-mm-long rings. The aortic rings were incubated in 30 ml
chambers flled with a Krebs–Henseleit solution at 37 °C
and pH 7.4 with constant oxygenation (O2/CO2, 19:1). Two
stainless steel stirrups were inserted through the lumen of
each aortic segment: one stirrup was attached to the bot-
tom of the chamber and the other to an isometric FDT10-
A force–displacement transducer (BIOPAC Systems, Inc.,
COMMAT Ltd., Turkey). The aortic rings were stretched
and maintained at optimal tension of 2 g and allowed to
equilibrate for 2 h.
Radioligand binding assay
Preparation of solutions of test and reference compounds
Stock solutions of tested compounds (10 mM) were pre-
pared in DMSO. Serial dilutions of compounds were pre-
pared in 96-well microplate in assay bufers using an auto-
mated pipetting system epMotion 5070 (Eppendorf). Each
compound was tested in eight concentrations from 10–5 to
10–12 M (fnal concentration).
After the equilibration period, the aortic rings were con-
tracted to maximal tension with KCl (60 mM). The depo-
larizing solution KCl (60 mM) was obtained by equimolar
substitution of NaCl for KCl. Then the cumulative concen-
tration–response curves to 5-HT were determined in the
absence and presence of antagonist. Tissues were incubated
with antagonists for 30 min.
5‑HT2A receptor binding assay
Radioligand binding was performed using membranes
from CHO-K1 cells stably transfected with the human
5-HT2A receptor (PerkinElmer). All assays were carried
Concentration–response curves were analyzed using
GraphPad Prism 6.0 software (GraphPad Software Inc.,
1 3

A. Czopek et al.
San Diego, CA, USA). Contractile responses to 5-HT (in
the presence or absence of tested compounds) are expressed
as a percentage of the maximal KCl efect reached in the
concentration–response curves obtained before incubation
with the tested compounds (Emax = 100%). Data are the
mean SEM of three separate experiments. The afnity
was estimated with the equation pKB = log(concentration
ratio–1)-log(molar antagonist concentration), where the con-
centration ratio is the ratio of equi-efective agonist concen-
trations in the absence and in the presence of the antagonist.
procedure for obtaining ligand-optimized models of high
predictive value was characterized in detail previously [17,
18]. The 5-HT2A receptor model was based on the 5-HT2B
receptor crystal structure 4IB4 [19]. Glide XP fexible dock-
ing was carried out using the OPLS3 force feld and default
parameters. H-bond constraint, as well as centroid of a
grid box for docking to the receptor models were located
on Asp863.32. The selection of the poses was based on the
prevalence of well-scored (glide gscore) complexes and
visual evaluation of binding interactions. Ligand structures
were optimized using LigPrep. The presented computational
tools were implemented in Small-Molecule Drug Discovery
Suite (Schrodinger, Inc.), which was licensed for Jagiellon-
ian University Medical College.
In vitro whole blood aggregation tests
In vitro aggregation assays were performed using freshly
drawn whole rat blood with a Multiplate platelet function
analyzer (Roche Diagnostic), the fve-channel aggregometer
based on measurements of electric impedance. Blood was
collected from carotid arteries with a hirudin blood tube
(Roche Diagnostic). 300 μl of hirudin anticoagulated blood
was mixed with 300 μl prewarmed isotonic saline solution
containing studied compound or vehicle (deionized water
or DMSO 0.1%) and preincubated for 3 min at 37 °C with
continuous stirring. Aggregation was induced by adding
collagen (Hyphen-Biomed, France) (fnal concentration
1.6 µg/ml), 5-HT (30 µM) and sub-threshold concentra-
tion of collagen (0.8 µg/ml), 5-HT (6 µM) and sub-thresh-
old concentration of ADP (ADP test, Roche Diagnostic),
(1.6 µM). The aggregation process was recorded for 6 min.
Ketanserin (ketanserin ( +) tartrate salt, Sigma-Aldrich,
Germany), sarpogrelate (sarpogrelate hydrochloride, Sigma-
Aldrich, Germany), and aspirin (Tocris, UK) were used as
reference compounds. The Multiplate software analyzed the
area under the curve (AUC) of the clotting process of each
measurement and calculated the mean values. Each concen-
tration of studied compounds was tested at least three times.
The exact sample size (n value) is presented on Figs. 2–4.
Concentration-inhibition curves were constructed and ana-
lyzed by non-linear curve ftting using GraphPad Prism 6.0
(GraphPad Software Inc., San Diego, CA, USA).
In silico toxicity prediction
In silico toxicity was assessed with a novel approach
(pkCSM) which uses graph-based signatures to develop
predictive models of ADMET properties or toxicity of intro-
duced structures [20].
Results and discussion
Chemistry
The fnal compounds (series 1 and 2) were synthesized as
outlined in Scheme 1. The substituted imidazolidine-2,4-di-
one derivatives (A and C) used as starting compounds were
prepared from 1-phenylethanone or 4-phenylcyclohexanone,
respectively, by the Bucherer–Bergs reaction followed by
N3-alkylation with dihalogenoalkanes in the hydantoin ring.
The resulting intermediates (B and D) were coupled with
appropriate arylpiperazines in the presence of potassium
carbonate or triethylamine to obtain the fnal compounds.
The crude fnal products were purifed using column chro-
matography. The structure and purity of the synthesized
compounds were confrmed by spectral and chromatographic
analyses, and the reaction progress was monitored by thin-
layer chromatography. The detailed physicochemical and
spectral data are summarized in the Experimental Part.
Statistical analysis
Data were presented as mean standard error the mean
(SEM). Statistical comparisons were made by the one-way
analysis of variance (one-way ANOVA) and the signifcance
of the diferences between the control group and treated
groups was determined by the Dunnet post hoc test. p<0.05
was considered signifcant.
In vitro 5‑HT2A receptor activity of compound 1–14
The novel series of compounds targeting serotonin 5-HT2A
receptors were designed by coupling the amide part of
4-phenylcyclohexane-5-spiro- or 5-methyl-5-phenylimida-
zolidine-2,4-dione with various arylpiperazine moieties,
using linkers of diferent lengths. The heterocyclic imide
ring, or substituted imidazolidine-2,4-dione in this case,
seemed to be a good starting point for designing 5-HT2A
receptor ligands because it is also present in the structures
Molecular modeling studies
Molecular docking was performed to the previously
developed homology models of the 5-HT2A receptor. The
1 3

Novel serotonin 5-HT2A receptor antagonists derived from 4-phenylcyclohexane-5-spiro-and…
of the conventional 5-HT2A receptor antagonists such as ket-
anserin and fananserin (Fig. 1). In the amine part, the pip-
erazine analogs (4-fuorophenyl)(piperidin-4-yl)methanone
and 4-(bis(4-fuorophenyl)methylene)-piperidine, found in
ketanserin and ritanserin, were introduced. Furthermore, a
key structural fragment of fananserin, 4-fuorophenyl-1-pip-
erazine, was chosen as the amine part, as well as its analogs
with a fuorine atom at positions 2 and 3.
5-methyl-5-phenylimidazolidine-2,4-dione moiety, showed
a moderate affinity for the tested serotonin receptors
(Ki = 450 nM, Table 1). However, the replacement of the
aforementioned piperazines by fuorophenylpiperazine led
to an increase in their afnity. Interestingly, compounds 5
and 11 containing a 4-fuorophenyl-1-piperazine fragment,
which is present in fananserin, as well as compounds 13
and 14 having 1-phenylpiperazine and 3-chlorophenyl-
1-piperazine moiety, respectively, displayed the highest
afnity for the 5-HT2A receptors in the prepared series
(Ki = 21–50 nM). Already published compounds 13 and
14 showed selectivity for serotonin 5-HT1A and dopamine
D2 receptors [11] (for details see Supplementary materi-
als Table S1). Moreover, the position of the fuorine atom
in the 1-phenylpiperazine fragment seemed to determine
the afnity of compounds for 5-HT2A receptors as shift-
ing the fuorine atom of this fragment from position 4 to
position 3 or 2 caused a signifcant (6- to 10-fold) decrease
in afnity (Table 1). The infuence of the linker length
on the compounds’ afnity for 5-HT2A receptors was also
investigated. It was observed that compounds with a linker
having two methylene groups showed no afnity for the
receptors (except compound 2), but the elongation of the
linker to three methylene groups resulted in an increased
binding afnity.
In the synthesized series, ten compounds (2–6, 9–14)
displayed low-to-high binding affinity for the 5-HT2A
receptors (Ki = 21–2584 nM, Table 1). Compounds con-
taining phenyl(piperazin-1-yl)methanone (1, 6, 7, 12) or
1-(diphenylmethyl)piperazine (2) fragment (except com-
pound 2) showed low or no afnity, while compound 2,
which is a 1-(diphenylmethyl)piperazine derivative with
Table 1 The radioligand binding data of 4-phenylcyclohexane-
5-spirohydantoin and 5-methyl-5-phenylhydantoin derivatives and
reference drug (mianserin) for serotonin 5-HT2A receptors
Compd
n
R
Ki [nM] SEM
1
0
–CO4FPh nd
For further characterization of the compounds, Ki
(5-HT2A)<1000 nM was set as selection criteria. Based on
this, compounds 2–5, 9–11 were chosen for further determi-
nation of functional activity for the 5-HT2A receptors. The
binding afnity of compounds 13 and 14 has been studied
and published in our previous work [11].
2
3
4
5
6
7
8
0
1
1
1
1
0
0
–CH(Ph)2 450.0 43.9
–2FPh
–3FPh
–4FPh
516.0 52.0
340.0 57.0
48.0 4.9
–CO4FPh nd
–CO4FPh nd
–CH(Ph)2 nd
Biofunctional assays
The antagonistic activity of compounds 2–5, 9–11 for
5-HT2A receptors was analyzed by evaluating the inhibition
of serotonin-induced contraction in rat aorta. The tested
compounds shifted the 5-HT concentration–response curve
to the right without lowering the maximal response, which
indicated that they are competitive antagonists of the rat
5-HT2A receptors (Figure S2). The afnities of these com-
pounds were expressed as pKB values, estimated from the
5-HT concentration–response curves and a single concentra-
tion of the compounds. The pKB values ranged from 6.161
(compound 2) to 7.430(compound 11) (Table 2) and were
in line with the radioligand binding data. Additionally,
compounds 2–5, 9, and 11 were studied in cells transfected
with human 5-HT2A receptors. The results of this bioassay
revealed that compounds 3 and 11 are moderate antagonists
of the human 5-HT2A receptors (pKB ≈ 6.758, Table 2). The
antagonistic activity of compounds 13 and 14 has been stud-
ied and published in our previous work [11].
9
1
1
1
1
1
1
–2FPh
–3FPh
–4FPh
220.0 19.6
297.0 19.9
21.0 1.7
10
11
12
–CO4FPh 2584.0 302.0
–Ph
–3ClPh
13 (Compd A)a,b
20.0 4.8a
24.0 2.5a
1.2 0.2
14a
Mianserin
The data for 13, 14, were already published in [11] and re-printed in
the current manuscript for comparison, with permission from Acta
Poloniae Pharmaceutica—Drug Research
nd no afnity detected
aThe data for 13, 14, were already published in [11] and re-printed
in the current manuscript for comparison, with permission from Acta
Poloniae Pharmaceutica—Drug Research
bCompd A Fig. 1
1 3

A. Czopek et al.
Table 2 In vitro functional antagonist data of tested 4-phenyl-
cyclohexane-5-spirohydantoin and 5-methyl-5- phenylhydantoin
derivatives and reference drugs (ketanserin, sapogrelate) for 5-HT2A
receptors [a] in rat aorta and [b] in cells expressing human 5-HT2A
receptors
exert antiplatelet activity due to 5-HT2A antagonism as well
as aspirin (COX-1 inhibitor) were used as reference com-
pounds for this analysis.
When studying aggregation, various agonists can be
used individually to identify a pathway that may be poten-
tially afected by a test compound. Collagen is one of the
most important, natural platelet agonist. The fnal efect of
collagen stimulation is increased intracellular Ca2+ con-
centration, which leads to morphological changes, ADP
and 5-HT secretion, and the synthesis of thromboxane A2
(TXA2) [21]. Therefore using collagen as an aggregation
agonist, provides a general means of assessing platelet func-
tion and anti-aggregation efect of test compounds in vitro
[21]. Figure 2 shows the efect of compound 13, ketanserin,
sarpogrelate, and aspirin on aggregation induced by colla-
gen. In our study, only aspirin and the most potent 5-HT2A
receptor antagonists were able to inhibit collagen-induced
aggregation. These compounds significantly decreased
platelet aggregation (F12,38 = 9.873, p < 0.0001). Among
the obtained compounds, only compound 13, which was
identifed as the most potent 5-HT2A antagonist, was able
to inhibit collagen-stimulated aggregation of whole rat
blood (IC50 = 27.3 3.8 μM). Its anti-aggregation efect
was greater than that of sarpogrelate (IC50 =66.8 12.9 μM)
and was comparable to ketanserin (IC50 = 32.1 3.0 μM).
For comparison, the IC50 value for aspirin was equal to
14.5 1.1 μM (Table 3, Fig. 2).
Compd
pKB SEM
a
b
2
6.161 0.06
6.666 0.12
6.357 0.18
6.957 0.23
7.018 0.08
6.300 0.36
7.430 0.32
7.665 0.03a
7.110 0.05a
9.439 0.09b
8.220 0.08b
5.706 0.21
6.758 0.15
5.035 0.23
5.032 0.30
5.702 0.16
nt
3
4
5
9
10
11
6.757 0.06
nt
13 (Compd A)
14
nt
Ketanserin
Sarpogrelate
7.839 0.03b
7.296 0.04b
nt not tested
a
b
a,bThe data for 13, 14, as well as for ketanserin and sarpogrelate
were already published in [7, 11], respectively, and re-printed in the
current manuscript for comparison, with permission from European
Journal of Pharmacology and Acta Poloniae Pharmaceutica—Drug
Research
In vitro whole blood aggregation tests
Further, the antiplatelet activity was studied using 5-HT
as a co-agonist of aggregation induced by collagen or ADP
at the sub-threshold concentration. Physiologically, many
factors that induce platelet aggregation act synergisti-
cally. 5-HT itself is only a weak platelet activator, but it
can enhance the aggregation process induced by collagen
or ADP, thus activating platelet 5-HT2A receptors [5, 22,
23]. Collagen used at a concentration of 0.8 μg/ml did not
induce aggregation of whole rat blood, while 5-HT used
Compounds with confirmed 5-HT2A antagonistic activ-
ity (2–5, 9–11, 13, 14) were subjected to further studies.
First, to evaluate the antiplatelet efect, freshly isolated rat
whole blood was incubated with the selected compounds
(3–200 μM) or vehicle (DMSO), and the aggregation
responses were recorded. Platelet aggregation was triggered
by collagen or using collagen or ADP at the subthreshold
concentration and 5-HT. Ketanserin and sarpogrelate, which
Fig. 2 Efects of compound 13,
sarpogrelate, ketanserin and
aspirin on in vitro whole rat
blood aggregation induced by
collagen (1.6 μg/ml) Results
are expressed as mean SEM,
***p<0.001, ****p<0.0001
versus control group (0.1%
DMSO in saline), one-way
ANOVA, post hoc Dunnet test.
AUC area under curve
1 3

Novel serotonin 5-HT2A receptor antagonists derived from 4-phenylcyclohexane-5-spiro-and…
Table 3 Potencies of studied and reference compounds in inhibiting
whole rat blood aggregation in vitro induced by [a] collagen (1.6 μg/
ml), [b] 5-HT (30 μM) and collagen (0.8 μg/ml), [c] 5-HT (6 μM) and
ADP (1.6 μM)
of 1.3 0.01 μM, which confrmed its high antagonistic
activity for 5-HT2A receptors found in functional studies.
The tested compounds also inhibited 5-HT-amplifed
and ADP-induced platelet aggregation. At a concentra-
tion of 1.6 μM, ADP used alone could cause only partial
and transient aggregation of rat blood in vitro, while 5-HT
used alone did not induce aggregation at any concentration
tested. However, combining 5-HT at a concentration of
6 μM with ADP at the sub-threshold concentration resulted
in a maximal aggregation response. The serotonin-mediated
amplifcation of ADP-stimulated aggregation was attenu-
ated when rat blood was preincubated with compounds 3,
4, 5, 10, 13, and 14 (F24,54 = 20.60, p < 0.0001), with the
IC50 values ranging from 9.9 2.5 μM (compound 13) to
140.5 44.6 μM (compound 11). For comparison, the IC50
value of ketanserin was similar to that of compound 13 and
was equal to 9.7 2.1 μM [7]. As observed for induction
with collagen alone, compound 13 was more potent than
sarpogrelate, which IC50 value, found in our previous work
was equal to 22.7 2.1 [7]. On the other hand, even at a
concentration of 200 μM, compound 2 did not exhibit any
signifcant inhibitory efect on ADP- and 5-HT-induced
blood aggregation, which proved its lowest intrinsic activ-
ity toward 5-HT2A receptors found in the biofunctional
study. Besides compound 13, compound 3 was observed to
be the most potent in the entire series of newly synthesized
compounds, with an IC50 value of 17.4 10.9 μM against
collagen- and 5-HT-induced blood aggregation and a value
of 37.9 6.5 μM against ADP- and 5-HT induced blood
aggregation. Thus, the efect of compound 3 on ADP- and
5-HT-induced aggregation was comparable to that of sar-
pogrelate. The results of this analysis are presented in Fig. 4
and Table 3.
Compound
a (collagen) b (5-HT+collagen) c (5-HT+ADP)
IC50 [μM]
IC50 [μM]
IC50 [μM]
2
3
4
5
9
10
11
na
na
na
na
na
na
na
139.8 18.9
17.4 10.9
153.1 26.4
127.2 32.7
138.6 16.1
94.3 53.5
91.3 7.1
na
37.9 6.5
83.1 8.1
123.7 36.2
na
118.6 32.1
140.5 44.6
9.9 2.5
93.8 9.4
22.7 2.1a
9.7 2.1a
nt
13 (Compd A) 27.3 3.8
14
Sarpogrelate
Ketanserin
Aspirin
53.3 4.6
75.8 16.6
na
66.8 12.9 nt
32.1 3.0
14.5 1.1
1.3 0.0
nt
na not active, nt not tested
aThe data for ketanserin and sarpogrelate were already published in
[7] and re-printed in the current manuscript for comparison, with per-
mission from European Journal of Pharmacology
alone could not cause aggregation at any concentration
tested. However, the combination of 5-HT at a concentra-
tion of 30 μM with collagen at the subliminal concentra-
tion resulted in the maximal aggregation response (Fig. 3).
This aggregation efect was attenuated by the studied com-
pounds (2–5, 9–11, 13, 14) and ketanserin [(F31,84 =7.808,
p < 0.0001)], (Fig. 3). The obtained IC50 values ranged
from 17.4 10.9 μM (compound 3) to 153.1 26.4 μM
(compound 4), (Table 3). The reference compound ket-
anserin inhibited blood aggregation with an IC50 value
It was found that the most potent 5-HT2A recep-
tor antagonists—compound 13, ketanserin, and
Fig. 3 Efects of compounds
2, 3, 4, 5, 9, 10, 11, 13, 14 and
ketanserin on in vitro whole rat
blood aggregation induced by
simultaneous addition of 5-HT
(30 μM) and collagen (0.8 μg/
ml). Results are expressed
as mean SEM, *p<0.05,
**p<0.01, ****p<0.0001 ver-
sus control group (0.1% DMSO
in saline), one-way ANOVA,
post hoc Dunnet test. AUC area
under curve
1 3

A. Czopek et al.
Fig. 4 Efects of compounds 3,
4, 5, 10, 11, 13, 14 on in vitro
whole rat blood aggregation
induced by simultaneous addi-
tion of 5-HT (6 μM) and ADP
(1.6 μM). Results are expressed
as mean SEM, *p<0.05,
**p<0.01, ***p<0.001,
****p<0.0001 versus control
group (0.1% DMSO in saline),
one-way ANOVA, post hoc
Dunnet test. AUC area under
curve
sarpogrelate—inhibited collagen-induced platelet aggrega-
tion, whereas other 5-HT2A antagonists—compounds 2–5,
9–11, and 14—inhibited 5-HT-potentiated platelet aggre-
gation. Among these, compound 3 was the most active and
inhibited collagen- or ADP-induced and 5-HT-amplifed
aggregation with the lowest IC50 values.
In silico toxicity prediction
In preliminary studies, the balance between activity, potency,
pharmacokinetics, and toxicity is crucial for efective drug
candidates. Therefore, computational approaches to optimize
pharmacokinetics and toxicity properties have been devel-
oped that allow the initial estimation of these parameters for
the newly synthesized structures. One of these predictive
models was used to assess the in silico toxicity of the most
active compounds 2–5, 9–11, 13, and 14 (for detailed infor-
mation please see Table S2 in the Supplementary materials)
with a hydantoin core.
Molecular modeling studies
The lead compounds 3 and 13, although having difer-
ent arylpiperazine substitutions, both showed a signifcant
afnity for 5-HT2A receptors in the radioligand binding
assays. Nevertheless, compared to compound 3, compound
13 showed over 25-fold higher afnity for the target pro-
tein. To analyze this diference at the molecular level,
docking to the homology model was performed. In the
case of both compounds, the arylpiperazine moiety bound
in the orthosteric binding site, as expected for the mono-
aminergic ligands. This moiety was anchored through the
salt bridge (charge-assisted hydrogen bond) between the
basic nitrogen atom and Asp863.32, as well as through
CH–π stacking between the aromatic ring and Phe2286.52
(Fig. 5A). The hydantoin-containing fragments of the
compounds were found to be located in the accessory
cavity of the receptor. In the case of compound 13 (but
not compound 3), the spiro-cyclohexane structure in this
region provided a favorable conformation, allowing the
formation of π–π interactions between the phenyl ring and
Tyr70. Moreover, the hydantoin itself formed an H-bond
with Ser157 and overlapped with 1,2,3,4-tetrahydroquina-
zoline-2,4-dione moiety of the reference 5-HT2A receptor
ligand ketanserin (Fig. 5B). The above relationships may
help in the design of further 5-HT2A receptor ligands of
the proposed chemotype.
Based on the prediction results, the tested compounds
showed no toxicity in the AMES test, which measures the
potential mutagenic activity. Moreover, one of the hERG
predictive models indicated no cardiotoxicity of the tested
compounds, while the other suggested a possible interaction
with the potassium channel. Besides, this model predicted
the possibility of hepatotoxicity of some compounds, but
these results require further evaluation in in vitro tests.
Conclusion
A series of compounds with 5-methyl-5-phenylhydantoin
and 4-phenylcyclohexane-5-spirohydantoin core con-
nected to arylpiperazine moiety via a methylene linker
were designed and synthesized (1–14) as potential 5-HT2A
receptor antagonists. The analysis of binding afnity and
functional bioassays showed that a majority of these com-
pounds exhibited moderate afnity and antagonistic activ-
ity for rat and human serotonin 5-HT2A receptors, which
further led to the evaluation of their anti-aggregation efect.
In the entire series, compound 13 was found to be the most
potent, as it inhibited collagen-stimulated aggregation of
1 3

Novel serotonin 5-HT2A receptor antagonists derived from 4-phenylcyclohexane-5-spiro-and…
Fig. 5 The predicted binding
mode of compound 13 (green)
displayed together with (A)
compound 3 (orange) and (B)
reference compound ketanserin
(pink) in the 5-HT2A recep-
tor homology model based on
4IB4. The arylpiperazine moie-
ties interact with Asp863.32 (salt
bridge/charge-reinforced hydro-
gen bond) and Phe2286.52 (CH-π
stacking) while hydantoin-con-
taining fragment of compound
13 interacts exclusively with
Tyr70 (π-π stacking) and Ser157
(H-bond) from extracellular
loops 1 and 2, respectively.
Amino acid residues engaged in
ligand binding (within 4 Å from
the ligand atoms) are repre-
sented as sticks
Supplementary Information The online version contains supplemen-
whole rat blood. The anti-aggregation efect of compound
13 was comparable to that of ketanserin and greater than
sarpogrelate. The tested compounds were also analyzed
for their inhibitory efect on 5-HT-amplifed, and ADP- or
collagen-induced platelet aggregation. The 5-HT-dependent
aggregation studies showed compounds 3 and 13 as the most
active. From the results of this study, it can be concluded
that 5-HT2A antagonists may effectively inhibit platelet
aggregation and are an interesting target for the development
of novel antiplatelet agents with an alternative mechanism
of action.
tary material available at https://doi.org/10.1007/s43440-021-00284-6.
Author contributions Substantial contributions to the conception or
design of the work ACz MK AB; or the acquisition and funding ACz
MK MP BF, analysis, or interpretation of data for the work ACz MK
AB AS MKoł; drafting the work or revising it critically for important
intellectual content ACz MK AB; fnal approval of the version to be
published; and ACz MK, agreement to be accountable for all aspects of
the work in ensuring that questions related to the accuracy or integrity
of any part of the work are appropriately investigated and resolved
ACz MK AS AB.
1 3

A. Czopek et al.
10. Lee DH, Chun EJ, Hur JH, Min SH, Lee JE, Oh TJ, et al. Efect of
sarpogrelate a selective 5-HT2A receptor antagonist on character-
istics of coronary artery disease in patients with type 2 diabetes.
Atherosclerosis. 2017;257:47–54. https://doi.org/10.1016/j.ather
osclerosis.2016.12.011.
Funding This work was supported by Statutory Funds of the Faculty
of Pharmacy, grants number: K/ZDS/006208, K/ZDS/006235, N42/
DBS/000020, N42/DBS/000139, Jagiellonian University Medical Col-
lege, Krakow, Poland.
11. Czopek A, Zagorska A, Kolaczkowski M, Bucki A, Gryzlo B,
Rychtyk J, et al. New spirohydanoin derivatives—synthesis phar-
macological evaluation and molecular modeling study. Acta Pol
Pharm. 2016;73:1545–54.
Declarations
Conflict of interest The authors declare no confict of interest relevant
to the ideas or the contents of this manuscript.
12. Czopek A, Byrtus H, Kołaczkowski M, Pawłowski M, Dybała
M, Nowak G, et al. Synthesis and pharmacological evaluation of
new 5-(cyclo)alkyl-5-phenyl- and 5-spiroimidazolidine-2 4-dione
derivatives Novel 5-HT1A receptor agonist with potential antide-
pressant and anxiolytic activity. Eur J Med Chem. 2010;45:1295–
303. https://doi.org/10.1016/j.ejmech.2009.11.053.
Open Access This article is licensed under a Creative Commons Attri-
bution 4.0 International License, which permits use, sharing, adapta-
tion, distribution and reproduction in any medium or format, as long
as you give appropriate credit to the original author(s) and the source,
provide a link to the Creative Commons licence, and indicate if changes
were made. The images or other third party material in this article are
included in the article’s Creative Commons licence, unless indicated
otherwise in a credit line to the material. If material is not included in
the article’s Creative Commons licence and your intended use is not
permitted by statutory regulation or exceeds the permitted use, you will
need to obtain permission directly from the copyright holder. To view a
copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
13. Czopek A, Kołaczkowski M, Bucki A, Byrtus H, Pawłowski M,
Kazek G, Bojarski AJ, et al. Novel spirohydantoin derivative as
a potent multireceptor-active antipsychotic and antidepressant
agent. Bioorganic Med Chem. 2015;23:3436–47. https://doi.org/
10.1016/j.bmc.2015.04.026.
14. Shah UH, Gaitonde SA, Moreno JL, Glennon RA, Dukat M,
González-Maeso J. Revised pharmacophore model for 5-HT2A
receptor antagonists derived from the atypical antipsychotic agent
risperidone. ACS Chem Neurosci. 2019;10:2318–31. https://doi.
org/10.1021/acschemneuro.8b00637.
15. Bagley JR, Thomas SA, Rudo FG, Spencer HK, Doorley BM,
Ossipov MH, et al. New l-(heterocyclylalkyl)-4-(propionanilido)-
4-piperidinyl methyl ester and methylene methyl ether analgesics. J
Med Chem. 1991;34:827–41. https://doi.org/10.1021/jm00106a051.
16. Kurys BE, Fink DM, Freed BS, Merriman GH (1998)
N-(pyridinylamino)isoindolines and related compounds. World
patent, WO 98/29407, 9 July.
References
1. Mo F, Li J, Yan Y, Wu W, Lai S. Efect and safety of antithrom-
botic therapies for secondary prevention after acute coronary
syndrome: a network meta-analysis. Drug Des Devel Ther.
2018;12:3583–94. https://doi.org/10.2147/DDDT.S166544.
2. Wiviott SD, Steg PG. Clinical evidence for oral antiplatelet ther-
apy in acute coronary syndromes. Lancet. 2015;386:292–302.
https://doi.org/10.1016/S0140-6736(15)60213-6.
17. Truchon JF, Bayly CI. Evaluating virtual screening methods: good
and bad metrics for the “early recognition” problem. J Chem Inf
Model. 2007;47:488–508. https://doi.org/10.1021/ci600426e.
18. Bucki A, Marcinkowska M, Śniecikowska J, Zagórska A, Jam-
rozik M, Pawłowski M, et al. Multifunctional 6-fluoro-3-[3-
(pyrrolidin-1-yl)propyl]-1 2-benzoxazoles targeting behavioral
and psychological symptoms of dementia (BPSD). Eur J Med
Chem. 2020. https://doi.org/10.1016/j.ejmech.2020.112149.
19. Wacker D, Wang C, Katritch V, Han GW, Huang XP, Vardy E,
et al. Structural features for functional selectivity at serotonin
receptors. Science (80-). 2013;340:615–9. https://doi.org/10.1126/
science.1232808.
3. Luna M, Holper EM. Use of novel antiplatelet agents in acute
coronary syndromes. Curr Atheroscler Rep. 2015;17:12. https://
doi.org/10.1007/s11883-014-0483-4.
4. Capranzano P, Capodanno D. Dual antiplatelet therapy in patients
with diabetes mellitus: special considerations. Expert Rev Cardio-
vasc Ther. 2013;11:307–17. https://doi.org/10.1586/erc.13.3.
5. Duerschmied D, Ahrens I, Mauler M, Brandt C, Weidner S, Bode
C, Moser M. Serotonin antagonism improves platelet inhibition
in clopidogrel low-responders after coronary stent placement: an
in vitro pilot study. PLoS ONE. 2012;7:e32656. https://doi.org/
10.1371/journal.pone.0032656.
20. Stegner D, Nieswandt B. Platelet receptor signaling in thrombus
formation. J Mol Med. 2011;89:109–21. https://doi.org/10.1007/
s00109-010-0691-5.
6. Floyd CN, Ferro A. Antiplatelet drug resistance: molecular
insights and clinical implications. Prostaglandins Other Lipid
Mediat. 2015;120:21–7. https://doi.org/10.1016/j.prostaglandins.
2015.03.011.
21. Belcher P, Drake-Holland A, Noble M. The antiplatelet drug target
in atherosclerotic diseases. Cardiovasc Hematol Disord Targets.
2012;6:43–55. https://doi.org/10.2174/187152906776092631.
22. Nagatomo T, Rashid M, Abul Muntasir H, Komiyama T. Func-
tions of 5-HT 2A receptor and its antagonists in the cardiovascu-
lar system. Pharmacol Ther. 2004;104:59–81. https://doi.org/10.
1016/j.pharmthera.2004.08.005.
7. Kubacka M, Kazek G, Kotańska M, Filipek B, Waszkielewicz AM,
Mogilski S. Anti-aggregation efect of aroxyalkyl derivatives of
2-methoxyphenylpiperazine is due to their 5-HT 2A and α 2-adreno-
ceptor antagonistic properties A comparison with ketanserin sar-
pogrelate prazosin yohimbine and ARC239. Eur J Pharmacol.
2018;818:263–70. https://doi.org/10.1016/j.ejphar.2017.10.053.
8. Saini HK, Takeda N, Goyal RK, Kumamoto H, Arneja AS, Dhalla
NS. Therapeutic potentials of sarpogrelate in cardiovascular dis-
ease. Cardiovasc Drug Rev. 2006;22:27–54. https://doi.org/10.
1111/j.1527-3466.2004.tb00130.x.
23. Adams JW, Ramirez J, Shi Y, Thomsen W, Frazer J, Morgan M,
et al. APD791 3-methoxy-N-(3-(1-methyl-1H-pyrazol-5-yl)-
4-(2-morpholinoethoxy) phenyl)benzamide a novel 5-hydroxy-
tryptamine 2A receptor antagonist: pharmacological profle phar-
macokinetics platelet activity and vascular biology. J Pharmacol
Exp Ther. 2009;331:96–103. https://doi.org/10.1124/jpet.109.
153189.
9. Mauler M, Herr N, Schoenichen C, Witsch T, Marchini T, Härdt-
ner C, et al. Platelet serotonin aggravates myocardial ischemia/
reperfusion injury via neutrophil degranulation. Circulation.
2019;139:918–31. https://doi.org/10.1161/CIRCULATIONAHA.
118.033942.
Publisher’s Note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional afliations.
1 3