Synthesis of novel pyrido[1,2-c]pyrimidine derivatives with 6-fluoro-3-(4-piperidynyl)-1,2-benzisoxazole moiety as potential ssri and 5-ht1a receptor ligands

Article abstract of DOI:10.3390/ijms22052329

Two series of novel 4-aryl-2H-pyrido[1,2-c]pyrimidine (6a–i) and 4-aryl-5,6,7,8-tetrahydropyrido[1,2-c]pyrimidine (7a–i) derivatives were synthesized. The chemical structures of the new compounds were confirmed by¹H and¹³C NMR spectroscopy and ESI-HRMS spectrome-try. The affinities of all compounds for the 5-HT_1A receptor and serotonin transporter protein (SERT) were determined by in vitro radioligand binding assays. The test compounds demonstrated very high binding affinities for the 5-HT_1A receptor of all derivatives in the series (6a–i and 7a–i) and generally low binding affinities for the SERT protein, with the exception of compounds 6a and 7g. Extended affinity tests for the receptors D₂, 5-HT_2A, 5-HT₆ and 5-HT₇ were conducted with regard to selected compounds (6a, 7g, 6d and 7i). All four compounds demonstrated very high affinities for the D₂ and 5-HT_2A receptors. Compounds 6a and 7g also had high affinities for 5-HT₇, while 6d and 7i held moderate affinities for this receptor. Compounds 6a and 7g were also tested in vivo to identify their functional activity profiles with regard to the 5-HT_1A receptor, with 6a demonstrating the activity profile of a presynaptic agonist. Metabolic stability tests were also conducted for 6a and 6d.

Full text of DOI:10.3390/ijms22052329

International Journal of
Molecular Sciences
Article
Synthesis of Novel Pyrido[1,2-c]pyrimidine Derivatives with
6-Fluoro-3-(4-piperidynyl)-1,2-benzisoxazole Moiety as
Potential SSRI and 5-HT1A Receptor Ligands
*
, Jerzy Kleps 1, Szymon Ulenberg 2 , Mariusz Belka 2, Tomasz Ba˛czek 2,
1
1,
´
Marek Król , Grzegorz Slifirski
Agata Siwek 3, Katarzyna Stachowicz 4, Bernadeta Szewczyk 4, Gabriel Nowak 3,4, Beata Duszyn´ska 4
and Franciszek Herold 1
1
Department of Drug Technology and Pharmaceutical Biotechnology, Faculty of Pharmacy, Medical University
of Warsaw, 1, Banacha Street, 02-097 Warsaw, Poland; mkrol@wum.edu.pl (M.K.); jkleps@wum.edu.pl (J.K.);
fherold@wum.edu.pl (F.H.)
Department of Pharmaceutical Chemistry, Medical University of Gdan´sk, 107, J. Hallera Street,
2
80-416 Gdan´sk, Poland; szymon.ulenberg@gmail.com (S.U.); mariusz.belka@gumed.edu.pl (M.B.);
tomasz.baczek@gumed.edu.pl (T.B.)
Department of Pharmacobiology, Faculty of Pharmacy, Jagiellonian University Medical College, 9,
3
Medyczna Street, 30-688 Kraków, Poland; agat.siwek@uj.edu.pl (A.S.); nowak@if-pan.krakow.pl (G.N.)
Maj Institute of Pharmacology, Polish Academy of Sciences, 12, Sme˛tna Street, 31-343 Kraków, Poland;
4
stachow@if-pan.krakow.pl (K.S.); szewczyk@if-pan.krakow.pl (B.S.); duszyn@if-pan.krakow.pl (B.D.)
*
Correspondence: gslifirski@wum.edu.pl
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
´
Citation: Król, M.; Slifirski, G.; Kleps,
J.; Ulenberg, S.; Belka, M.; Ba˛czek, T.;
Siwek, A.; Stachowicz, K.; Szewczyk,
B.; Nowak, G.; et al. Synthesis of
Novel Pyrido[1,2-c]pyrimidine
Derivatives with 6-Fluoro-3-(4-
piperidynyl)-1,2-benzisoxazole
Moiety as Potential SSRI and 5-HT1A
Receptor Ligands. Int. J. Mol. Sci.
2021, 22, 2329. https://doi.org/
10.3390/ijms22052329
Abstract: Two series of novel 4-aryl-2H-pyrido[1,2-c]pyrimidine (6a–i) and 4-aryl-5,6,7,8-
tetrahydropyrido[1,2-c]pyrimidine (7a–i) derivatives were synthesized. The chemical structures
of the new compounds were confirmed by 1H and 13C NMR spectroscopy and ESI-HRMS spectrome-
try. The affinities of all compounds for the 5-HT1A receptor and serotonin transporter protein (SERT)
were determined by in vitro radioligand binding assays. The test compounds demonstrated very
high binding affinities for the 5-HT1A receptor of all derivatives in the series (6a–i and 7a–i) and
generally low binding affinities for the SERT protein, with the exception of compounds 6a and 7g
Extended affinity tests for the receptors D2, 5-HT2A, 5-HT6 and 5-HT7 were conducted with regard
to selected compounds (6a 7g 6d and 7i). All four compounds demonstrated very high affinities
.
,
,
for the D2 and 5-HT2A receptors. Compounds 6a and 7g also had high affinities for 5-HT7, while 6d
and 7i held moderate affinities for this receptor. Compounds 6a and 7g were also tested in vivo to
identify their functional activity profiles with regard to the 5-HT1A receptor, with 6a demonstrating
the activity profile of a presynaptic agonist. Metabolic stability tests were also conducted for 6a
and 6d.
Academic Editor: Geoffrey Brown
Received: 20 January 2021
Accepted: 22 February 2021
Published: 26 February 2021
Keywords: antidepressants; pyrido[1,2-c]pyrimidines; dual 5-HT1A/SERT activity; drug design
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
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iations.
1. Introduction
The serotonergic nervous system plays a substantial role in regulating mood, diurnal
rhythm, cognitive functions, memory, thermoregulation and anxiety, while also contribut-
ing to many other vital functions [1]. Numerous studies in recent years have confirmed
that disturbances in serotonergic neurotransmission are closely related to central nervous
system (CNS) disorders such as depression, anxiety, schizophrenia or obsessive compulsive
disorder (OCD) [2,3]. There is an increase in patients with these depressive disorders, which
represent the fourth most common class of medical conditions and affect approximately
20% of the population. Consequently, there is a growing interest in novel modulators of
Copyright:
© 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
the serotonergic system [4]. The World Health Organization predicts that by 2030, unipolar
depression will be the primary reason for inability to work worldwide, with depression
Int. J. Mol. Sci. 2021, 22, 2329. https://doi.org/10.3390/ijms22052329
https://www.mdpi.com/journal/ijms

Int. J. Mol. Sci. 2021, 22, 2329
2 of 21
and anxiety affecting approximately 100 million people in Europe alone [
5
]. At the same
time, drugs currently used to treat depression are far from satisfactory [2].
The introduction of selective serotonin reuptake inhibitors (SSRIs) such as fluoxetine
or citalopram in the 1980s marked a turning point in the pharmacotherapy of depression
(Figure 1) [
receptors, contributing to their limited adverse effects, better tolerability and higher thera-
peutic index than tricyclic antidepressants or monoamine oxidase inhibitors [ ]. Thera-
peutic efficacy of SSRIs has been documented for unipolar depression, anxiety disorder,
posttraumatic stress disorder, OCD and negative symptoms of schizophrenia [ ]. However,
SSRIs are not free from adverse effects, which include insomnia, nausea, sexual dysfunction
and a possible effect on myocardial ion channels [ 10]. One serious drawback of SSRIs is
6]. SSRIs demonstrate low affinities for adrenergic, histaminic and muscarinic
6–8
9
9
,
their long therapeutic latency (therapeutic effects become evident two to three weeks after
administration) and limited efficacy (visible effects are only seen in approximately 60% of
patients) [7]. Given the high probability of suicide in depressive patients, drugs without a
latency period are extremely important [1,4].
Figure 1. Chemical structures of fluoxetine and citalopram.
5-HT1A receptors play an important role in the self-regulation of the serotonergic sys-
tem [2]. They may function both as presynaptic (autoreceptors) or postsynaptic receptors.
The 5-HT1A presynaptic receptors are found in the neurons and dendrites of brainstem
raphe nuclei. Upon stimulation, they release endogenous serotonin into the synaptic cleft,
reducing transmission across serotonergic neurons. On the other hand, postsynaptic 5-
HT1A neurons are stimulated in somatodendric nerve endings in the cortico-limbic area
of the CNS, increasing transmission via serotonergic neurons [7,11,12]. In fact, a number
of 5-HT1A agonists are currently undergoing various phases of clinical studies or have
already been approved for marketing. Their pharmacological activity is not limited to the
treatment of depression, but may also be used in the treatment of anxiety (osemozotan,
phase II), schizophrenia (bifeprunox, phase III) or pain (befiradol, phase II) (Figure 2) [13].
Figure 2. Chemical structures of osemozotan, bifeprunox and befiradol.
The aforementioned SSRI latency period is associated with changes in adaptive pro-
cesses within the CNS that result in increased serotonergic neurotransmission via postsy-
naptic 5-HT1A receptors [8,14]. The therapeutic effects seen during SSRI administration
are the combined result of neurochemical changes in the brain, including desensitization
of 5-HT1A autoreceptors, downregulation of receptor responsivity to neurotransmitters,

Int. J. Mol. Sci. 2021, 22, 2329
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changes in signal transmission, neurotropism and hippocampal neurogenesis [15]. One key
consequence of desensitization of somatodendric 5-HT1A autoreceptors on brainstem raphe
nuclei is increasing serotonin levels in synaptic clefts [7,16].
In 1993, Artigas proposed that coadministration of a 5-HT1A receptor antagonist with
SSRIs should potentiate the antidepressant effect through accelerating the desensitization
of 5-HT1A autoreceptors, thus strengthening their function [17]. This hypothesis was con-
firmed upon coadministration of an SSRI with the partial 5-HT1A antagonist pindolol [
Unfortunately, 5-HT1A antagonists were nonselective and simultaneously blocked pre-
and postsynaptic receptors, an undesirable effect when treating depression [ ]. A more
7].
9
promising direction in the search for next-generation antidepressants looks at agonists
of both the 5-HT1A receptor and SSRI. Such compounds have a potential for accelerating
desensitization and downregulation of autoreceptors, while directly stimulating postsy-
naptic serotonergic neurons. As a result of this process, the concentration of endogenous
serotonin in the synaptic cleft increases [18]. Most importantly, the sensitivity of postsy-
naptic receptors does not decrease with prolonged administration [18
was positively verified by the introduction of vilazodone (Viibryd) to treat depression
Figure 3). Vilazodone was the first of several SSRI+ drugs whose mechanism of action
,19]. This approach
(
involves both agonism towards the 5-HT1A receptors and serotonin transporter protein
(SERT) inhibition [20–22]. In 2013, the Food and Drug Administration approved vortioxe-
tine as another SSRI+ agent with an extended receptor activity profile for pharmacotherapy
of depression (Figure 3). Vortioxetine acts as an SSRI, an agonist of the 5-HT1A receptor, a
partial agonist of the 5-HT1B and an antagonist of the 5-HT3 and 5-HT7 receptors [23].
Figure 3. Chemical structures of vilazodone and vortioxetine.
This work describes the synthesis and results of pharmacological testing of a series of
novel derivatives of 4-aryl-2H-pyrido[1,2-c]pyrimidine, characterized by double binding
for the 5-HT1A receptor and SERT protein. Selected compounds were tested further to
determine their activity towards other molecular targets, such as the 5-HT2A, 5-HT6, 5-HT7
and D2 receptors.
The research presented in this paper is a continuation of a long-term research project
conducted in our department, where ligands are tested for a double binding affinity for
both the SERT protein and 5-HT1A receptors [24
4-aryl-2H-pyrido[1,2-c]pyrimidine and 4-aryl-5,6,7,8-tetrahydropyrido[1,2-c]pyrimidine
were designed, based on lead compounds ( IV) that had been synthesized previously
–28]. Two series of novel derivatives of
I–
by the same research group and had demonstrated a high affinity for both the 5-HT1A
receptors and SERT protein (Figure 4) [26,27].
Modifications of lead compounds involved a change in the pharmacophore part via
the introduction of a 6-fluoro-3-(4-piperidinyl)-1,2-benzisoxazole residue. This addition
should increase affinity for the 5-HT1A receptors and SERT protein, and thus these new
compounds would potentially demonstrate dual binding affinity, appropriate functional
activity and affinity for other molecular targets (e.g., 5-HT2A, 5-HT6, 5-HT6, 5-HT7 and D2).
This study aimed to investigate the effect of (i.) introducing a 6-fluoro-3-(4-piperidinyl)-
1,2-benzisoxazole residue to the pharmacophore, (ii.) the degree of saturation of the
pyrido[1,2-c]pyrimidine residue in the terminal segment and (iii.) substituents in the 4-
aryl-pyrido[1,2-c]pyrimidine moiety on affinity for both the 5-HT1A receptor and SERT
protein and other receptors (5-HT2A, 5-HT6,5-HT7, D2) in extended receptor profile tests.
Further testing probed the effect of these modifications on the functional activity (agonism-
antagonism) and metabolic stability of the compounds of interest.

Int. J. Mol. Sci. 2021, 22, 2329
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Figure 4. Comparison of the novel derivatives of 4-aryl-2H-pyrido[1,2-c]pyrimidine (6a–i) and 4-
aryl-5,6,7,8-tetrahydropyrido[1,2-c]pyrimidine (7a–i), designed in this paper with lead compounds
(I–IV).
2. Results and Discussion
2.1. Chemistry
The designed target compounds (6a–i) and (7a–i) were obtained in a multistage synthe-
sis process (Figure 5). The starting materials were phenylacetonitrile derivatives subjected
to C-arylation with 2-bromopyridine over KOH. This reaction yielded respective deriva-
tives of
α
-(2-pyridyl)-
α
-(aryl)-acetonitriles (1a
–
i), which were subsequently hydrolyzed
in an acidic environment to obtain respective
α
-(2-pyridyl)- -(aryl)-acetamides (2a ).
α
–i
These amides were then reacted with diethyl carbonate via a cyclocondensation process,
producing derivatives of 4-aryl-2H-pyrido[1,2-c]pyrimidine-1,3-dione (3a–i). All the com-
pounds (1a
3a ) were then N-alkylated with 1,4-dibromobuthane to produce N-bromobuthyl deriva-
tives of 4-aryl-2H-pyrido[1,2-c]pyrimidine-1,3-dione (4a ). Some of the 2-(4-bromobuthyl)-
4-aryl-pyrido[1,2-c]pyrimidine-1,3-dione (4a ) derivatives were subjected to catalytic
reduction over 10% Pd/C, yielding 2-(4-bromobuthyl)-4-aryl-5,6,7,8-tetrahydropyrido[1,2-
c]pyrimidine-1,3-dione (5a ) derivatives. The target compounds (6a–i) and (7a–i) were
obtained by reacting the bromobuthyl derivatives of (4a ) and (5a ) with 6-fluoro-3-(4-
–i), (2a–i), (3a–i) were obtained according to an original method. The imides
(
–i
–
i
–
i
–
i
–
i
–i
piperidinyl)-1,2-benzisoxazole. The chemical structures and purity of the newly synthe-
sized compounds (6a–i) and (7a–i) were confirmed by 1H and 13C NMR spectroscopy, as
well as LC/MS and HRMS spectrometry. The investigated compounds were subsequently
tested in vitro and in vivo as free bases.

Int. J. Mol. Sci. 2021, 22, 2329
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Figure 5. Schematic of the syntheses of compounds 6a–i and 7a–i. Reagents and conditions: (i) 2-bromopyridine, KOH,
DMSO, 50 ◦C; (ii) H2SO4, CH3COOH, 100 ◦C; (iii) (◦C2H5)2CO3, EtONa, EtOH reflux; (iv) 1,4-dibromobuthane, acetone,
K2CO3, reflux; (v) H2, 10% Pd/C, EtOH, 60 atm., 50 C; (vi) 6-fluoro-3-(4-piperidinyl)-1,2-benzisoxazole, K2CO3, CH3CN,
◦
45 C.
2.2. Biological Evaluation
2.2.1. Radioligand Binding Assay for 5-HT1A and SERT
The target compounds (6a–i) and (7a–i) were assessed for in vitro affinity for the
5-HT1A receptor and SERT protein by radioligand binding assays [26,29,30]. The results,
which were subsequently used for structure–activity relationship (SAR) analysis, can be
found in Table 1. Various substituents at the ortho or para position of the benzene ring—as
well as the degree of saturation of the pyrido[1,2-c]pyrimidine residue—were investigated
with regard to their effect on the binding affinity of the compounds (6a–i) and (7a–i).
The resulting data on 5-HT1AR binding affinity indicated very high affinity of the following
ligands: 6c (Ki = 7.0 nM), 6g (Ki = 10.0 nM), 6d (Ki = 11.0 nM), 6e (Ki = 15.0 nM), 6i
(Ki = 17.0 nM), 6h (Ki = 21.0 nM), 6a (Ki = 23.0 nM) and 6b (Ki = 30 nM), with 6f being the
only compound with a binding affinity at the level of Ki = 74 nM (Table 1). The compounds
(7a–i) (4-aryl-5,6,7,8-tetrahydropyrido[1,2-c]pyrimidine derivatives) demonstrated slightly
lower binding affinity for the 5-HT1AR compared to (6a–i Very high binding affinity
)
.
was noted for 7g (Ki = 5.0 nM), 7i (Ki = 9.5 nM), 7h (Ki = 25.0 nM), 7a (Ki = 27.0 nM), 7b
(Ki = 35.0 nM) and 7e (Ki = 36.0 nM); the other three derivatives demonstrated high binding
affinity, viz., 7f (Ki = 52.0 nM), 7c (Ki = 62.0 nM) and 7d (Ki = 71.0 nM). A comparison of

Int. J. Mol. Sci. 2021, 22, 2329
6 of 21
binding affinity data for the ligands (6a–i) and (7a–i) indicated that the degree of saturation
had little effect, with the 4-aryl-2H-pyrido[1,2-c]pyrimidine derivatives (6a–i) being slightly
superior to the 4-aryl-5,6,7,8-tetrahydropyrido[1,2-c]pyrimidine derivatives (7a–i).
Table 1. 5-HT1A receptor and SERT binding affinities, as well as cLogP [31] of 4-aryl-2H-pyrido[1,2-
c]pyrimidine derivatives 4-aryl-2H-pyrido[1,2-c]pyrimidine (6a–i) and 4-aryl-5,6,7,8-tetrahydro-
pyrido[1,2-c]pyrimidine derivatives (7a–i).
Ki [nM]
Compound
R
R1
5-HT1A
SERT
cLogP
6a
6b
6c
6d
6e
6f
6g
6h
6i
7a
7b
7c
7d
7e
7f
7g
7h
7i
–H
–CH3
–OCH3
–Cl
–F
–H
–H
–H
–H
–H
–CH3
–OCH3
–Cl
–F
–H
–H
–H
–H
–H
–H
–H
–H
–H
–CH3
–OCH3
–Cl
–F
–H
–H
–H
–H
–H
–CH3
–OCH3
–Cl
23.0 ± 1.0
30.0 ± 3.5
7.0 ± 1.0
32.0 ± 3.6
4.32
4.81
4.30
4.98
4.52
4.81
4.98
4.98
4.55
5.02
5.51
5.00
5.68
5.22
5.90
5.00
5.68
5.22
>5000
>5000
11.0 ± 1.4
15.0 ± 0.6
74.0 ± 4.0
10.0 ± 1.1
21.0 ± 1.5
17.0 ± 2.0
27.0 ± 2.0
35.0 ± 3.5
62.0 ± 6.0
71.0 ± 6.9
36.0 ± 4.1
52.0 ± 2.5
5.0 ± 0.5
373.0 ± 32.0
>1000
772.0 ± 36.0
730.0 ± 79.0
310.0 ± 1.0
342.0 ± 28.8
520.0 ± 58.8
>1000
878.0 ± 88.0
310.0 ± 34.5
>1000
1773.0
±
180.0
48.0 ± 2.4
290.0 ± 22.7
311.0 ± 35.0
25.0 ± 3.0
9.5 ± 1.1
–F
Reference
compound
Serotonin
Methiotepin
Imipramine
3.6 ± 0.4
4.8 ± 0.5
17.0 ± 1.3
An analysis of the effect of the substituents in the benzene ring of the 4-aryl-2H-
pyrido[1,2-c]pyrimidine residue on 5-HT1AR affinity of the ligands (6a–i) showed that
the presence of a substituent at the ortho position generally increased binding affinity,
compared to ligands with a substituent at the para position. The most marked effect on
affinity was exerted by substituents in the compounds (6a–i) in the following order: 6c
(o-OCH3) > 6g (p-OCH3) > 6d (o-Cl) > 6e (o-F) > 6i (p-F) > 6h (p-Cl) > 6a (-H) > 6b (o-CH3)
>
6f (p-CH3). Analysis of the effect of substituents on the binding affinity of the derivatives
of the 4-aryl-5,6,7,8-tetrahydro-pyrido[1,2-c]pyrimidine (7a–i) series revealed the most
marked effect on binding affinity of substituents at the para position in the following order:
7g (p-OCH3) > 7i (p-F) > 7h (p-Cl) > 7a (-H) > 7b (o-CH3) > 7e (o-F) > 7f (p-CH3) > 7c
(o-OCH3) > 7d (o-Cl).
Data on the affinity of the ligands (6a–i) and (7a–i) for SERT protein generally indicated
poor binding affinity of most of the compounds. Consequently, it is difficult to determine
the effect of the degree of saturation and substituents of the 4-aryl-pyrido[1,2-c]pyrimidine
residue in the test compounds on their binding affinity. High binding affinity was only
demonstrated for 6a (Ki = 32.0 nM) and 7g (Ki = 48.0 nM). The binding affinity values
for the other ligands in both series are low (Ki = from 310 to > 5000 nM for series
Ki = 290–1773 nM for series 7).
6 and
The compounds 6a, 6d, 7g and 7i were selected for in vitro studies. The compounds
6a and 7g showed very high affinity for the 5-HT1A receptor and SERT protein, while the
compounds 6d and 7i demonstrated very high affinity for the 5-HT1A receptor and poor
affinity for SERT. The compounds were tested for their multiple receptor binding affinity,

Int. J. Mol. Sci. 2021, 22, 2329
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with special regard to their affinity for the receptors 5-HT2A, 5-HT6, 5-HT7 and D2, whose
role in the pathomechanism of depression has been well documented [15,32] (Table 2). It has
been confirmed that disturbed dopaminergic neurotransmission in the mesolimbic and
nigrostriatal regions also contributes significantly to the development of depression [32].
Consequently, intensive research is under way on a new class of potential drugs which
exert their effects through interaction with D2/5-HT1A[33]; where the ligand would be
a partial agonist of the 5-HT1A receptor and would induce postsynaptic serotonergic
neurotransmission, which would increase dopamine levels in the mPFC (medial prefrontal
cortex) [34]. The role of 5-HT2A receptors in the treatment of schizophrenia has also been
well documented in numerous publications. Accordingly, multireceptor studies of the
selected ligands 6a, 6d, 7g and 7i—including this molecular target—appear well justified.
The role of the serotonergic receptors 5-HT6 and 5-HT7 in the pathomechanism of CNS
disorders, including depression, has been well documented and presented in numerous
papers [15], and so have the satisfactory results of treatment with multireceptor drugs,
such as Aripiprazole, Clozapine[35] or Vortioxetine[23]. This encouraged us to investigate
the extended affinity profile of selected compounds [36], targeting 5-HT2A, 5-HT6, 5-HT7
and D2 receptors.
Table 2. Binding affinity data on serotonin 5-HT1A, 5-HT2A, 5-HT6, 5-HT7 receptors, SERT protein, and dopamine D2 receptor of the
investigated 4-aryl-2H-pyrido[1,2-c]pyrimidine (6a, 6d) and 4-aryl-5,6,7,8-tetrahydro-pyrido[1,2-c]pyrimidine (7g, 7i) derivatives.
Ki [nM]
Compound
R1
R2
5-HT1A
SERT
5-HT2A
5-HT6
5-HT7
D2
6a
6d
7g
7i
–H
–Cl
–H
–H
–H
–H
–OCH3
–F
23.0 ± 1.0
11.0 ± 1.4
5.0 ± 0.5
9.5 ± 1.1
32.0 ± 3.6
17 ± 3
20 ± 4
16 ± 2
44 ± 6
376 ± 58
709 ± 135
400 ± 32
740 ± 183
62 ± 5
109 ± 14
94 ± 11
161 ± 27
7 ± 2
9 ± 2
373.0
±
32.0
48.0 ± 2.4
10 ± 1
17 ±2
311.0 35.0
±
Reference
Compound
Olanzapine [37]
Mianserin [37]
Clozapine [37]
Haloperidol [37]
Apomorphine [37]
Chlorpromazine
[37]
4.6 ± 0.9
2.8 ± 0.5
n.d.
n.d.
n.d.
7 ± 1
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
18 ± 2
n.d.
4.5 ± 0.7
n.d.
42 ± 6
n.d.
n.d.
n.d.
1.8 ± 0.3
n.d. = not determined.
Table 2 presents binding affinities of the compounds 6a and 7g, which exhibited very
high binding affinity for the receptors 5-HT1A, 5-HT2A, SERT and D2, and high binding
affinity for 5-HT7. The compounds 6d and 7i, in turn, demonstrated very high affinity
for the receptors 5-HT1A, 5-HT2A and D2 and moderate affinity for the receptor 5-HT7,
with low affinity for the receptors 5-HT6 and SERT. The in vitro data for both groups of
compounds can be seen as a good starting point for further research on multireceptor
ligands in the treatment of depressive disorder or schizophrenia. Additionally, cLogP
values for the compounds in the 4-aryl-2H-pyrido[1,2-c]pyrimidine (6a–i) series ranged
from 4.30 to 4.98, while cLogP values for the series of 4-aryl-5,6,7,8-tetrahydro-pyrido[1,2-
c]pyrimidine (7a–i) derivatives ranged from 5.00-5.90. These values are given in Table
1. A comparison of cLogP values for both ligand series shows that all cLogP values for
the (6a–i) series do not exceed 5.00—which, according to Lipin´ski [38,39] is a cut-off value
for transmembrane penetration required of candidate drugs. The derivatives 6a (R = -H,
R1 = -H) and 6c (R = -OCH3, R1 = H) had a cLogP value of 4.32 and 4.30, respectively,
which can be compared to the cLogP value of 4.26 [30] of Vortioxetine, a well-known
antidepressant of the SSRI/5-HT1A class. At the same time, ligands of the (7a–i) series

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demonstrated cLogP values >5, which is not pharmacologically desirable, according to
Lipin´ski’s rule [38].
2.2.2. In Vivo Studies
To determine the profile of functional activity of the selected ligands, behavioral tests
were performed. It is known that 8-OH-DPAT, a 5-HT1A receptor agonist, can induce
hypothermia in mice, through 5-HT1A somatodendritic receptors [30,40]. Moreover, this
effect can be abolished by WAY-100635 [41], a 5-HT1A receptor antagonist. Based on this
knowledge, we tested compounds 6a (R = -H, R1 = -H) and 7g (R = -H, R1 = -OCH3) in a
commonly used in vivo panel of tests, to assess their functional 5-HT1A receptor activity.
Test compounds 6a and 7g, like 8-OH-DPAT, induced hypothermia in mice (Table 3).
Table 3. The effect of compounds 6a and 7g on body temperature in mice.
Dose
(mg/kg)
◦
Treatment
∆t ± SEM ( C)
30 min
60 min
90 min
120 min
Vehicle
6a
-
5
2.5
1.25
0.6
−0.2 ± 0.2
−2.0 ± 0.2 b
−1.6 ± 0.2 b
−1.7 ± 0.3 b
−1.0 ± 0.2 b
p < 0.0001
−0.0 ± 0.2
−2.1 ± 0.1 b
−2.2 ± 0.2 b
−1.5 ± 0.3 b
p < 0.0001
−0.0 ± 0.2
−3.2 ± 0.5 b
−2.5 ± 0.3 b
−1.7 ± 0.3 b
−1.2 ± 0.1 b
p < 0.0001
0.0 ± 0.1
−0.1 ± 0.1
−3.1 ± 0.5 b
−2.4 ± 0.3 b
−1.6 ± 0.2 b
−0.9 ± 0.1 a
p < 0.0001
−0.1 ± 0.1
−3.9 ± 0.5 b
−2.9 ± 0.3 b
−1.3 ± 0.4 b
p < 0.0001
0.1 ± 0.2
−0.0 ± 0.1
−2.8 ± 0.5 b
−2.0 ± 0.2 b
−1.6 ± 0.3 b
−0.8 ± 0.2 a
p < 0.0001
−0.1 ± 0.1
−4.2 ± 0.8 b
−3.0 ± 0.4 b
−1.0 ± 0.3
p < 0.0001
0.1 ± 0.2
Vehicle
7g
-
5
2.5
1.25
−3.4 ± 0.3 b
−2.8 ± 0.3 b
−1.7 ± 0.3 b
p < 0.0001
0.1 ± 0.2
Vehicle
WAY-100635
8-OH-DPAT
-
0.1
5
0.2 ± 0.1
0.1 ± 0.2
0.1 ± 0.2
−0.1 ± 0.2
−0.1 ± 0.1
ns
−0.2 ± 0.1
0.3 ± 0.3
−1.7 ± 0.2 b
−1.1 ± 0.2 b
p < 0.0001
p < 0.005
ns
The investigated compounds were administered 30 min before the test, a p < 0.05 vs vehicle, b p < 0.001 vs vehicle,
ns = non-significant.
WAY-100635 (0.1 mg/kg) diminished hypothermia induced by compound 6a (0.6
mg/kg) by 50% (Table 4).
Table 4. The effect of WAY-100635 (0.1 mg/kg sc) on the hypothermia induced by compounds 6a
and 7g.
◦
Treatment and Dose (mg/kg)
∆t ± SEM ( C)
30 min
60 min
Vehicle
Vehicle + 6a (0.6)
WAY-100635 + 6a
0.0 ± 0.1
−1.0 ± 0.2 c
−0.5 ± 0.2 b,d
p < 0.0001
−0.3 ± 0.1
−1.5 ± 0.3 c
−1.2 ± 0.2 b
p < 0.0001
0.1 ± 0.1
0.0 ± 0.0
−1.2 ± 0.1 c
−0.4 ± 0.2 a,e
p < 0.0001
−0.2 ± 0.1
−1.7 ± 0.3 c
−0.7 ± 0.3 d
p < 0.0005
−0.0 ± 0.1
0.2 ± 0.3
Vehicle
Vehicle + 7g (1.25)
WAY-100635 + 7g
Vehicle
WAY-100635
0.3 ± 0.3
ns
ns
WAY-100635 was administered 15 min before the tested compounds, a p < 0.05 vs vehicle, b p < 0.01 vs vehicle,
c
p < 0.001 vs vehicle, d p < 0.05 vs compound group, e p < 0.001 vs compound group, ns = non-significant.

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In conclusion, the decrease in mouse body temperature produced by compound 6a can
be accounted as a measure of its presynaptic 5-HT1A agonistic activity. Tested compound
6a was ineffective in the forced swimming test in mice (Figure 6), so we can conclude lack
of postsynaptic 5-HT1A receptor activity [40].
Figure 6. Effect of compound 6a on forced swimming test in CD-1 mice.
2.2.3. Metabolic Stability Evaluation
We decided to carry out preliminary tests of metabolic stability for selected com-
pounds, as metabolic stability is an important index of a compound’s pharmacokinetics.
Such studies are routinely performed at earlier stages of studies of potential new drugs.
They are extremely important, as many known valuable compounds possessing high de-
sirable pharmacological activity are disqualified at later clinical stages on account of an
undesirable pharmacokinetic profile with regard to metabolic stability. Thus, the prelimi-
nary studies in this regard performed by us appeared advisable.
A compound with poor metabolic stability will not reach appropriate therapeutic
levels for a given molecular target. High metabolic stability of a candidate drug or its
metabolite can, in turn, potentially cause higher toxicity or adverse effects. On the other
hand, the identification of inhibition or induction of cytochrome P-450 isoenzymes, which
mediate the metabolism of most drugs, allows for predicting potential drug-drug interac-
tions [42,43]. The results of a metabolic stability study in the presence of pooled human
liver microsomes (HLMs) and nicotinamide adenine dinucleotide phosphate (NADPH)
are shown in Table 5. Metabolic stability is presented in the form of biological half-life
value, which allows for easy comparison of compounds’ structure and their susceptibility
to phase 1 biotransformation reactions (the result of incubation on the presence of hu-
man liver microsomes). Results presented in Table 5 allow for the quick assessment of
metabolic stability.
Table 5. Experimental t1/2 values along with corresponding SD and RSD%.
Average t1/2 [min]
Compound
SD [min]
RSD%
(n = 2)
6a
6d
3.61
3.20
0.43
0.09
11.18
2.81
Table legend: SD—standard deviation, RSD%—relative standard deviation, expressed as SD/average × 100%.
Even though the biological half-life values for studied compounds were far from
high, it is worth noticing that biological half-life value depends on the compound’s initial
concentration in the incubation mix. As the initial studied compound concentration was
1 µM, such values were to be expected. Of the studied pyrido[1,2-c]pyrimidine derivatives,
compounds 6a (R = -H, R1 = -H) and 6d (R = -Cl, R1 = -H) were most susceptible to phase
1 biotransformation reactions. The metabolic stability investigations for the compounds
6a and 6d showed their sensitivity to the activity of human liver microsomes, which is
most likely associated with the presence of a 6-fluoro-3-(4-piperidinyl)-1,2-benzisoxazole
residue in the pharmacophore part. These results will prompt further studies in search of
compounds with greater metabolic stability in this group of derivatives. The justification

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for further research stems from the fact that the novel compounds revealed very high
affinity for a number of receptors and were also characterized by appropriate cLogP values.
3. Materials and Methods
3.1. General Remarks
Reagents and solvents were purchased from commercial suppliers: Sigma-Aldrich,
TCI, Alfa Aesar and Chempur. The purity of the obtained samples was routinely confirmed
by TLC using Merck plates (Kieselgel 60 F254). Melting points (m.p.) were determined
using the Electrothermal IA 9200 apparatus with open capillary tubes and were not cor-
1
rected. H and 13C NMR spectra were recorded on a Bruker AVANCE III HD (500 MHz)
instrument in CDCl3 (chemical shifts are reported in δ units), with the use of TMS as the
internal reference. The following abbreviations were used to describe peak patterns when
appropriate: s (singlet), 2s (double singlet), d (doublet), dd (double doublet), dt (double
triplet), t (triplet), td (triple doublet), 4d (quartet of doublets), m (multiplet), q (quartet), qu
(quintet). Coupling constants (J) are in hertz (Hz). Numbering system, which was used
in NMR spectra interpretation is shown in Figure 7. ESI-HRMS spectra were obtained on
a Thermo Q-Exactive instrument. Flash column chromatography was carried out using
Merck Silicagel (40–63 µ
m) Geduran® Si 60 and a mixture of toluene:ethylacetate:methanol
(10:4:3 v/v), methylene chloride:methanol:triethylamine (64:2:0.4 v/v), chloroform:methanol
(9:1 v/v) as an eluent. Thin layer chromatography was performed on Merck Silicagel (Kiesel-
gel 60 F254) plates, where the mobile phase was composed of toluene, dioxane, ethanol,
and 25% ammonia (9.0:5.0:1.0:0.3 v/v) or methylene chloride, methanol, triethylamine
(16.0:1.0:0.2 v/v). Plates were visualized by UV light (254 nm).
Figure 7. Numbering system for NMR spectra interpretation of compounds (6a–i) and (7a–i).
3.2. Synthesis of Compounds
3.2.1. Procedure for the Synthesis of
2-(4-Bromobutyl)-4-aryl-pyrido[1,2-c]pyrimidine-1,3-diones (4a–i) and
2-(4-bromobutyl)-4-aryl-5,6,7,8-tetrahydropyrido[1,2-c]pyrimidine-1,3-diones (5a–i)
Compounds (4a–i) and (5a–i) were obtained according to the previously described
procedures [26,37,44].
3.2.2. General Procedure for the Synthesis of Derivatives of
4-Aryl-2H-pyrido[1,2-c]pyrimidine-1,3-dione (6a–i) and
4-aryl-5,6,7,8-tetrahydropyrido[1,2-c]pyrimidine-1,3-dione (7a–i)
The appropriate bromobutyl derivatives (4a–i) or (5a–i) (0.75 mmol), 6-fluoro-3-(4-
piperidinyl)-1,2-benzisoxazole (0.75 mmol) and K2CO3 (2 mmol) were suspended in acetoni-
◦
trile (25 mL). The reaction mixture was carried out of at 45 C and stirred for 8–12 h. The re-
action time was determined using TLC. The mixture was filtered to remove inorganic salts,
and the solvent was removed from the filtrate under vacuum. The residue was purified by
column chromatography (flash or gravity technique) using toluene:ethylacetate:methanol
(10:4:3 v/v), methylene chloride:methanol:triethylamine (64:2:0.4 v/v), chloroform:methanol
(9:1 v/v) as an eluent. Appropriate fractions were identified by TLC and evaporated to give
compounds (6a–i) or (7a–i).

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4-Phenyl-2-{4-[4-(6-fluoro-1,2-benzoxazol-3-yl)-1-piperidyl]butyl}-pyrido[1,2-
c]pyrimidine-1,3-dione 6a
The title compound was isolated as a yellow powder. Yield: 20.5%; m.p. 132–133 ◦C.
1H NMR (500 MHz, CDCl3): 8.33 (C8H, dt, 3J = 7.5, 4J = 5J = 1.5), 7.75 (C4”H, dd,
δ
3J = 8.5, 4JH-F = 5.0), 7.42–7.47 (C20H, C60H, m), 7.35 (C40H, tt, 3J = 7.5, 4J = 1.5), 7.30–7.33
3
4
5
3
(C30H, C50H, m), 7.22 (C7”H, 4d, JH-F = 8.5, J = 2.5, J = 0.5), 7.04 (C5”H, td, J = 9.0,
4J = 2.0), 6.89–6.91 (C5H, C6H, m), 6.38 (C7H, m, 3J1 = 8.0, 3J2 = 4.5, 4J = 3.5), 4.20 (C1xH2,
t, J = 7.5), 3.11 (CaH(E), CeH(E), CcH, m), 2.52 (C4xH2, t, J = 7.0), 2.02–2.30 (CaH(A),
CeH(A), CbH(A), CdH(A), CbH(E), CdH(E), m), 1,81 (C2xH2, q, 3J = 7.5), 1.68 (C3xH2, q,
3J = 7.5).
3
3
13C NMR (125 MHz, CDCl3):
δ
164.1 (C6”, d, 1J = 250.6*), 163.9 (C7”a, d, 3J = 13.6*),
160.9 (C3, s), 160.1 (C3”, s), 148.9 (C1, s), 143.5 (C4a, s), 132.8 (C10, s), 132,4 (C6, s), 131.2
(C20, C60, s), 128.8 (C30, C50, s), 127.9 (C40, s), 127.8 (C8, s), 122.8 (C4”, d, 3J = 10.2*), 121.4
(C5, s), 117.2 (C3”a, s), 112.3 (C5”, d, 2J = 25.3*), 110.7 (C7, s), 104.9 (C4, s), 97.3 (C7”, d,
2J = 26.7*), 58.3 (C4x, s), 53.4 i 53.4 (Ca i/lub Ce, 2s), 42.3 (C1x, s), 34.4 (Cc, s), 30.1 (Cb, Cd,
s), 25.4 (C2x, s), 24.1 (C3x, s).
ESI-HRMS m/z: Calcd for C30H30FN4O3 [M + H]+ 513.2296. Found: 513.2304
4-(2-Methylphenyl)-2-{4-[4-(6-fluoro-1,2-benzoxazol)-1-piperidyl]butyl}-pyrido[1,2-
c]pyrimidine-1,3-dione 6b
The title compound was isolated as a yellow powder. Yield: 59.7%; m.p. 120–122 ◦C.
1H NMR (500 MHz, CDCl3):
δ
8.33 (C8H, dt, 3J = 7.5, 4J = 5J = 1.0), 7.78 (C4”H, dd,
3J = 8.5, 4JH-F = 5.0), 7.21–7.33 (C40-60H, C7”H, [4H], m), 7.14 (C30H, dd, 3J = 8.0, 4J = 1.5),
7.05 (C5”H, td, 3J = 9.0, 4J = 2.0), 6.88 (C6H, 4d, 3J1 = 9.5, 3J2 = 6.0, 4J = 1.5), 6.56 (C5H, dt,
3J = 9.5, 4J = 5J = 1.0), 6.38 (C7H, m, 3J1 = 7.5, 3J2 = 6.0, 4J = 1.0), 4.20 (C1xH2, t, 3J = 7.5), 3.08
(CaH(E), CeH(E), CcH, m), 2.48 (C4xH2, t, 3J = 7.5), 2.15 (CH3, s), 2.0–2.2 (Ca(A), Ce(A),
CbH2, CdH2, m), 1.80 (C2xH2, q, 3J = 7.5), 1.65 (C3xH2, q, 3J = 7.5).
13C NMR (125 MHz, CDCl3): δ 164.0 (C6”, d, 1J = 250.6*), 163.8 (C7”a, d, 3J = 13.6*),
161.0 (C3”, s), 159.6 (C3, s), 149.1 (C1, s), 143.4 (C4a, s), 138.4 (C20, s), 132.4 (C10, s), 132.1
(C6, s), 131.5 (C60, s), 130.5 (C30, s), 128.4 (C40, s), 128.0 (C8, s), 126.4 (C50, s), 122.7 (C4”, d,
3J = 11.1*), 121.4 (C5, s), 117.2 (C3”a, s), 112.3 (C5”, d, 2J = 25.3*), 110.5 (C7, s), 104.1 (C4, s),
97.4 (C7”, d, 2J = 26.7*), 58.4 (C4x, s), 53.4 (Ca, Ce, s), 42.2 (C1x, s), 34.5 (Cc, s), 30.3 (Cb, Cd,
s), 25.5 (C2x, s), 24.2 (C3x, s), 19.6 (CH3, s).
ESI-HRMS m/z: Calcd for C31H32FN4O3 [M + H]+ 527.2453. Found: 527.2461
4-(2-Methoxphenyl)-2-{4-[4-(6-fluoro-1,2-benzoxazol)-1-piperidyl]butyl}-pyrido[1,2-
c]pyrimidine-1,3-dione 6c
The title compound was isolated as a yellow powder. Yield: 71.1%; m.p. 65–70 ◦C.
1H NMR (500 MHz, CDCl3):
δ
8.32 (C8H, dt, 3J = 7.5, 4J = 5J = 1.0), 7.73 (C4”H, dd,
4
3
3
4
3J = 8.5, JH-F = 5.0), 7.37 (C40H, 4d, J1 = 8.0, J2 = 7.5, J = 1.5), 7.22 (C60H, C7”H, m),
0
7.01–7.07 (C5 H, C5”H, m), 6.88 (C6H, 4d, 3J1 = 9.5, 3J2 = 6.5, 4J = 1.5), 6.63 (C5H, dt, 3J = 9.0,
4J = 5J = 1.5), 6.37 (C7H, m, 3J1 = 7.5, 3J2 = 6.5, 4J = 1.5), 4.19 (C1xH2, t, 3J = 7.5), 3.76 (OCH3,
s), 3.08 (CaH(E), CeH(E), CcH, m), 2.48 (C4xH2, t, 3J = 7.5), 2.0 -2.2 (Ca(A), Ce(A), CbH2,
CdH2, m), 1.80 (C2xH2, q, 3J = 7.0), 1.66 (C3xH2, q, 3J = 7.0).
13C NMR (125 MHz, CDCl3): δ 164.0 (C6”, d, 1J = 250.4*), 163.8 (C7”a, d, 3J = 13.6*),
161.1 (C3”, s), 159.9 (C3, s), 157.8 (C20, s), 149.1 (C1, s), 143.6 (C4a, 0s), 133.0 (C6’, s), 131.9
0
(C6, s), 129.6 (C4 , s), 127.8 (C8, s), 122.8 (C4”, d, 3J = 11.1*), 121.9 (C1 , s), 121.4 (C5, s), 120.9
(C50, s), 117.2 (C3”a, s), 112.3 (C5”, d, 2J = 25.1*), 111.4 (C30, s), 110.5 (C7, s), 101.2 (C4, s),
97.3 (C7”, d, 2J = 26.7*), 58.4 (C4x, s), 55.6 (OCH3, s), 53.5 (Ca, Ce, s), 42.3 (C1x, s), 34.5 (Cc,
s), 30.3 (Cb, Cd, s), 25.5 (C2x, s), 24.2 (C3x, s).
ESI-HRMS m/z: Calcd for C31H32FN4O4 [M + H]+ 543.2402. Found: 543.2410

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4-(2-Chlorophenyl)-2-{4-[4-(6-fluoro-1,2-benzoxazol)-1-piperidyl]butyl}-pyrido[1,2-
c]pyrimidine-1,3-dione 6d
The title compound was isolated as a yellow powder. Yield: 71.7%; m.p. 69–74 ◦C.
1H NMR (500 MHz, CDCl3): 8.37 (C8H, dt, 3J = 7.5, 4J = 5J = 1.0), 7.75 (C4”H, bs), 7.52
(C30H, m), 7.29–7.37 (C40H, C50H, C60H, m), 7.23 (C7”H, 4d, 3JH-F = 8.5, 4J = 2.5, 5J = 0.5),
δ
3
4
3
3
4
7.05 (C5”H, td, J = 8.5, J = 2.0), 6.97 (C6H, 4d, J1 = 9.5, J2 = 6.0, J = 1.0), 6.56 (C5H,
dt, 3J = 9.0, 4J = 5J = 1.0), 6.43 (C7H, m, 3J1 = 7.5, 3J2 = 6.5, 4J = 1.5), 4.21 (C1xH2, m), 3.10
(CaH(E), CeH(E), CcH, bs), 2.51 (C4xH2, bs), 2.0–2.3 (CaH(A), CeH(A), CbH2, CdH2), 1.81
(C2xH2, m), 1.67 (C3xH2, m).
13C NMR (125 MHz, CDCl3): δ 164.1 (C6”, d, 1J = 250.6*), 163.9 (C7”a, d, 3J = 13.6*),
161.0 (C3”, s), 159.5 (C3, s), 149.0 (C1, s), 143.8 (C4a, s), 135.7 (C20, s), 133.4 (C60, s), 133.1
(C10, s), 131.7 (C6, s), 130.0 (C30, s), 129.7 (C40, s), 128.1 (C8, s), 127.3 (C50, s), 122.8 (C4”, d,
3J = 10.7*), 121.1 (C5, s), 117.2 (C3”a, s), 122.3 (C5”, d, 2J = 25.1*), 110.8 (C7, s), 102.2 (C4, s),
97.4 (C7”, d, 2J = 26.7*), 58.3 (C4x, s), 53.4 (Ca, Ce, s), 42.2 (C1x, s), 34.4 (Cc, s), 30.2 (Cb, Cd,
s), 25.4 (C2x, s), 24.0 (C2x, s).
ESI-HRMS m/z: Calcd for C30H29ClFN4O3 [M + H]+ 547.1907. Found: 547.1915
4-(2-Fluorophenyl)-2-{4-[4-(6-fluoro-1,2-benzoxazol)-1-piperidyl]butyl}-pyrido[1,2-
c]pyrimidine-1,3-dione 6e
The title compound was isolated as a yellow powder. Yield: 63.2%; m.p. 58–62 ◦C.
1H NMR (500 MHz, CDCl3):
δ
8.37 (C8H, dt, 3J = 7.5, 4J = 5J = 1.0), 7.74 (C4”H, m),
7.35–7.43 (C40H, m), 7.33 (C50H, td, 3J = 7.5, 4J = 2.0), 7.23 (C60H, C7”H, m), 7.17 (C30H,
m, 3JH-F = 10.0, 3J = 8.5, 4J = 1.0), 7.04 (C5”H, td, 3J = 8.5, 4J = 2.5), 6.98 (C6H, 4d, 3J1 = 9.5,
3J2 = 6.5, 4J = 1.5), 6.74 (dt) i 6.74 (dt) (C5H, 3J = 9.0, 4J = 5J = 1.5**), 6.43 (C7H, m, 3J1 = 7.5,
3J2 = 6.5, 4J = 1.5), 4.20 (C1xH2, t, 3J = 7.5), 3.09 (CaH(E), CeH(E), CcH, pd), 2.49 (C4xH2,
bs), 2.00–2.30 (CaH(A), CeH(A), CbH2, CdH2, m), 1.81 (C2xH2, q, 3J = 7.5), 1.66 (C3xH2, q,
3J = 7.5).
13C NMR (125 MHz, CDCl3): δ 164.1 (C6”, d, 1J = 250.4*), 163.9 (C7”a, d, 3J = 13.5*),
1
161.1 (C3”, s), 160.9 (C20, d, J = 246.9*), 159.6 (C3, s), 148.9 (C1, s), 144.0 (C4a, s), 133.4
(C60, d, 3J = 3.0*), 133.1 (C6, s), 130.1 (C40, d, 3J = 8.2*), 128.2 (C8, s), 124.4 (C50, d, 4J = 3.5*),
0
0
122.8 (C4”, d, 3J = 11.1*), 121.2 (C5, s), 120.3 (C1 , d, 2J = 16.0*), 117.2 (C3”a, s), 116.1 (C3 , d,
2J = 22.3*), 112.3 (C5”, d, 2J = 25.1*), 110.8 (C7, s), 98.4 (C4, s), 97.4 (C7”, d, 2J = 26.8*), 58.4
(C4x, s), 53.5 (Ca, Ce, s), 42.4 (C1x, s), 34.5 (Cc, s), 30.3 (Cb, Cd, s), 25.5 (C2x, s), 24.2 (C3x, s).
ESI-HRMS m/z: Calcd for C30H29F2N4O3 [M + H]+ 531.2202. Found: 531.2212
4-(4-Methylphenyl)-2-{4-[4-(6-fluoro-1,2-benzoxazol)-1-piperidyl]butyl}-pyrido[1,2-
c]pyrimidine-1,3-dione 6f
The title compound was isolated as a yellow powder. Yield: 71.1%; m.p. 158–159 ◦C.
1H NMR (500 MHz, CDCl3):
δ
8.32 (C8H, dt, 3J = 7.5, 4J = 5J = 1.0), 7.78 (C4”H, bs),
7.18–7.26 (C20H, C30H, C50H, C60H, C7”H, m), 7.05 (C5”H, td, 3J = 9.0, 4J = 2.0), 6.86–6.93
(C5H, C6H, m), 6.37 (C7H, m, 3J1 = 7.5, 3J2 = 5.5, 4J = 1.5), 4.19 (C1xH2, t, 3J = 7.5), 3.14
(CaH(E), CeH(E), Cc, bs), 2.57 (C4xH2, bs), 2.39 (CH3, s), 2.03–2.35 (CaH(A), CeH(A), CbH2,
CdH2, m), 1.81 (C2xH2, q, 3J = 7.5), 1.70 (C3xH2, bs).
13C NMR (125 MHz, CDCl3): δ 164.1 (C6”, d, 1J = 250.7*), 163.9 (C7”a, d, 3J = 13.6*),
160.8 (C3”, s), 160.3 (C3, s), 149.0 (0C1, s), 143.5 (C4a, s), 137.6 (C6, s), 132.2 (C40, s), 131.0
0
0
0
0
(C2 , C6 , s), 129.7 (C1 , s), 129.5 (C3 , C5 , s), 127.9 (C8, s), 122.8 (C4”, d*), 121.6 (C5, s), 117.1
(C3”a, bs), 112.4 (C5”, d, 2J = 25.1*), 110.6 (C7H, s), 104.9 (C4, s), 97.4 (C7”, d, 2J = 26.8*),
58.2 (C4x, s), 53.2 (Ca, Ce, s), 42.1 (C1x, s), 34.2 (Cc, s), 30.0 (Cb, Cd, s), 25.4 (C2x, s), 23.7
(C3x, s), 21.3 (CH3, s).
ESI-HRMS m/z: Calcd for C31H32FN4O3 [M + H]+ 527.2453. Found: 527.2459

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4-(4-Methoxyphenyl)-2-{4-[4-(6-fluoro-1,2-benzoxazol)-1-piperidyl]butyl}-pyrido[1,2-
c]pyrimidine-1,3-dione 6g
The title compound was isolated as a yellow powder. Yield: 78.9%; m.p. 163–166 ◦C.
1H NMR (500 MHz, CDCl3):
δ
8.31 (C8H, dt, 3J = 7.5, 4J = 5J = 1.0), 7.73 (C4”H, dd,
3J = 8.5, 4JH-F = 5.0), 7.24 (C20H, C60H, dt, 3J = 8.5, 4J = 3.0), 7.21–7.24 (C7”H, m**), 7.04
(C5”H, td, 3J = 9.0, 4J = 2.0), 6.98 (C30H, C50H, dt, 3J = 8.5, 4J = 3.0), 6.86–6.94 (C5H, C6H,
m), 6.37 (C7H, m, 3J1 = 8.0, 3J2 = 6.0, 4J = 1.5), 4.19 (C1xH2, t, 3J = 7.5), 3.84 (OCH3, s), 3.09
(Ca(E), Ce(E), Cc, 3.09, m), 2.47 (C4xH2, t, 3J = 7.5), 2.00–2.20 (Ca(A), Ce(A), CbH2, CdH2,
m), 1.80 (C2xH2, q, 3J = 7.5), 1.65 (C3xH2, q, 3J = 7.7).
13C NMR (125 MHz, CDCl3): δ 164.1 (C6”, d, 1J = 250.4*), 163.8 (C7”a, d, 3J = 13.6*),
161.1 (C3”, s), 160.4 (C3, s), 159.1 (C40, s), 149.0 (C1, s), 143.5 (C4a, s), 132.3 (C20, C60, s),
132.1 (C6, s), 127.9 (C8, s), 124.8 (C10, s), 122.7 (C4”, d, 3J = 11.1*), 121.6 (C5, s), 117.2 (C3”a,
0
0
s), 114.3 (C3 , C5 , s), 112.3 (C5”, d, 2J = 25.3*), 110.6 (C7, s), 104.6 (C4, s), 97.4, d, 2J = 26.8*),
58.4 (C4x, s), 55.3 (OCH3, s), 53.5 (Ca, Ce, s), 42.4 (C1x, s), 34.5 (Cc, s), 30.4 (Cb, Cd, s), 25.5
(C2x, s), 24.3 (C3x, s).
ESI-HRMS m/z: Calcd for C31H32FN4O4 [M + H]+ 543.2402. Found: 543.2411
4-(4-Chlorophenyl)-2-{4-[4-(6-fluoro-1,2-benzoxazol)-1-piperidyl]butyl}-pyrido[1,2-
c]pyrimidine-1,3-dione 6h
The title compound was isolated as a yellow powder. Yield: 63.2%; m.p. 152–155 ◦C.
1H NMR (500 MHz, CDCl3):
δ
8.35 (C8H, dt, 3J = 7.5, 4J = 5J = 1.0), 7.76 (C4”H, bs),
7.25–7.29 (C30H, C50H, dt, 3J = 8.5, 4J = 2.5), 7.23 (C7”H, 4d, 3JH-F = 8.5, 4J = 2.0, 5J = 0.5),
7.40–7.44 (C20H, C60H, dt, 3J = 8.5, 4J = 2.5), 7.05 (C5”H, td, 3J = 8.5, 4J = 2.0), 6.95 (C6H,
4d, 3J1 = 9.5, 3J2 = 6.0, 4J = 1.0), 6.88 (C5H, m, 3J = 9.0, 4J = 5J = 1.5), 6.42 (C7H, m, 3J1 = 7.5,
3J2 = 6.0, 4J = 1.0), 4.19 (C1x H2, t, 3J = 7.5), 3.13 (CaH(E), CeH(E), CcH, bs), 2.55 (C4xH2,
bs), 2.0–2.4 (CaH(A), CeH(A), CbH2, CdH2, m), 1.80 (C2xH2, q, 3J = 7.0), 1.70 (C3xH2, bs).
13C NMR (125 MHz, CDCl3): δ 164.1 (C6”, d, 1J = 250.7*), 163.9 (C7”a, d, 3J = 13.7*),
160.8 (C3”, s), 160.0 (C3, s), 148.8 (C1, s), 143.7 (C4a, s), 133.7 (C40, s), 132.9 (C6, s), 132.6
(C30, C50, s), 131.2 (C10, s), 129.0 (C2’, C60, s), 128.1 (C8, s), 122.8 (C4”, d, 3J = 9.4*), 121.1
(C5, s), 117.1 (C3”a, s), 112.4 (C5”, d, 2J = 24.9*), 110.9 (C7, s), 103.5 (C4, s), 97.4 (C7”, d,
2J = 26.8*), 58.2 (C4x, s), 53.3 (Ca, Ce, s), 42.3 (C1x, s), 34.3 (Cc, s), 29.9 (Cb, Cd, s), 25.4 (C2x,
s), 23.8 (C3x, s).
ESI-HRMS m/z: Calcd for C30H29ClFN4O3 [M + H]+ 547.1907. Found: 547.1916
4-(4-Fluorophenyl)-2-{4-[4-(6-fluoro-1,2-benzoxazol)-1-piperidyl]butyl}-pyrido[1,2-
c]pyrimidine-1,3-dione 6i
The title compound was isolated as a yellow powder. Yield: 17.6%; m.p. 102–107 ◦C.
1H NMR (500 MHz, CDCl3):
δ
8.34 (C8H, dt, 3J = 7.0, 4J = 5J = 1.5), 7.75 (C4”H, bs),
7.29 (C20H, C60H, m), 7.23 (C7”H, 4d, 3JH-F = 8.5, 4J = 2.0, 5J = 0.5), 7.14 (C30H, C50H, tt,
3J = 8.5), 7.052 (C5”H, td, 3J = 8.5, 4J = 2.0), 6.94 (C6H, 4d, 3J1 = 9.0, 3J2 = 6.0, 4J = 1.5), 6.87
(C5H, dt, 3J = 9.0, 4J = 5J = 1.5), 6.41 (C7H, m, 3J1 = 7.5, 3J2 = 6.0, 4J = 1.5), 4.19 (C1xH2, t,
3J = 7.5), 3.12 (CaH(E), CeH(E)CcH, bs), 2.53 (C4xH2, bs), 2.0–2.3 (CaH(A), CeH(A), CbH2,
CdH2, m), 1.81 (C2xH2, q, 3J = 7.5), C3xH2, bs).
13C NMR (125 MHz, CDCl3): δ 164.1 (C6”, d, 1J = 250.8*), 163.9 (C7”a, d, 3J = 13.5*),
162.3 (C40H, d, 1J = 247.2*), ~161.0 (C3”, s), 160.2 (C3, s), 148.9 (C1, s), 143.7 (C4a, s), 133.0
3
4
(C20, C60, d, J = 8.2*), 132.8 (C6, s), 128.6 (C10, d, J = 3.5*), 128.1 (C8, s), 122.8 (C4”,
d, J = 10.8*), 121.2 (C5, s), 117.2 (C3”a, s), 115.8 (C30, C50, d, J = 21.5*), 112.4 (C5”, d,
2J = 24.9*), 110.8 (C7, s), 103.8 (C4, s), 97.4 (C7”, d, 2J = 26.8*), 58.4 (C4x, s), 53.4 (Ca, Ce, s),
42.3 (C1x, s), 34.4 (Cc, s), 30.1 (Cb, Cd, s), 25.4 (C2x, s), 24.1 (C3x, s).
3
2
ESI-HRMS m/z: Calcd for C30H29F2N4O3 [M + H]+ 531.2202. Found: 531.2211

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4-Phenyl-2-{4-[4-(6-fluoro-1,2-benzoxazol-3-yl)-1-piperidyl]butyl}-5,6,7,8-tetrahydro-
pyrido[1,2-c]pyrimidine-1,3-dione 7a
The title compound was isolated as a white powder. Yield: 81.6%; m.p. 110–113 ◦C.
3
1H NMR (500 MHz, CDCl3):
δ
7.78 (C4”H, bs), 7.40 (C30H, C50H, tt, J = 7.5), 7.33
(C40H, tt, 3J = 7.5), 7.23 (C7”H, dd, 3JH-F = 8.0, 4J = 2.0), 7.19 (C20H, C60H, dt, 3J = 7.5), 7.05
(C5”H, td, 3J = 9.0, 4J = 2.0), 4.05 (C1xH2, t, 3J = 7.5), 3.95 (C8H2, t, 3J = 6.5), 3.14 (CaH(E),
CeH(E), CcH, bs), 2.54 (C4xH2, C5H2, m), 2.05–2.35 (CaH(A), CeH(A), CdH2, m), 1.93
(C7H2, q, 3J = 6.5), 1.71 (C2xH2, C3xH2, C6H2, m).
13C NMR (125 MHz, CDCl3): δ 164.1 (C6”, d, 1J = 250.6*), 163.9 (C7”a, d, 3J = 13.4*),
162.0 (C3, s), 160.8 (C3”, s), 151.7 (C1, s), 149.8 (C4a, s), 133.3 (C10, s), 130.7 (C20, C60, s),
128.5 (C30, C50, s), 127.7 (C40, s), 122.9 (C4”, d*), 117.1 (C3”a, s), 112.4 (C5”, d, 2J = 25.2*),
112.4 (C4, s), 97.4 (C7”, d, 2J = 26.7*), 58.3 (C4x, s), 53.3 (Ca, Ce, s), 42.7 (C8, s), 41.3 (C1x, s),
34.1 (Cc, s), 29.8 (Cb, Cd, s), 26.7 (C5, s), 25.6 (C2x, s), 23.9 (C3x, s), 21.8 (C7, s), 18.6 (C6, s).
ESI-HRMS m/z: Calcd for C30H34FN4O3 [M + H]+ 517.2609. Found: 517.2617
4-(2-Methylphenyl)-2-{4-[4-(6-fluoro-1,2-benzoxazol)-1-piperidyl]butyl}-5,6,7,8-
tetrahydro-pyrido[1,2-c]pyrimidine-1,3-dione 7b
The title compound was isolated as an oil. Yield: 97.4%.
1H NMR (500 MHz, CDCl3):
δ
7.75 (C4”H, bs), 7.18–7.26 (C40-60H, C7”H, m), 7.05
(C30H, C5”H, m), 4.05 (C1xH2, t, J = 7.0), 3.93 (C8H2, m), 3.09 (CaH(E), CeH(E), CcH,
pd), 2.49 (C4xH2, bs), 2.45 (C5H(1), m), 2.27 (C5(2), m), 2.00–2.23 (CaH(A), CeH(A), CbH2,
CdH2, m), 2.14 (OCH3, s), 1.93 (C7H2, m), 1.60–1.78 (C2xH2, C3xH2, C6H2, m).
13C NMR (125 MHz, CDCl3): δ 164.1 (C6”, d, 1J = 250.4*), 163.9 (C7”a, d, 3J = 13.6*),
161.4 (C3, s), 161.0 (C3”, s), 151.9 (C1, s), 149.6 (C4a, s), 137.6 (C20, s), 132.9 (C10, s), 130.7
(C60, s), 130.3 (C30, s), 128.2 (C40, s), 126.2 (C50, s), 122.8 (C4”, d, 3J = 10.8*), 117.2 (C3”a, s),
112.4 (C5”, d, 2J = 25.3*), 111.8 (C4, s), 97.4 (C7”, d, 2J = 26.7*), 58.4 (C4x, s), 53.4 (Ca, Ce, s),
42.9 (C8, s), 41.3 (C1x, s), 34.5 (Cc, s), 30.2 (cb, Cd, s), 26.5 (C5, s), 25.7 (C2x, s), 24.1 (C3x, s),
21.9 (C7, s), 19.7 (CH3, s), 18.6 (C6, s).
3
ESI-HRMS m/z: Calcd for C31H36FN4O3 [M + H]+ 531.2766. Found: 531.2774
4-(2-Methoxphenyl)-2-{4-[4-(6-fluoro-1,2-benzoxazol)-1-piperidyl]butyl}-5,6,7,8-
tetrahydro-pyrido[1,2-c]pyrimidine-1,3-dione 7c
The title compound was isolated as an oil. Yield: 71.1%.
3
3
1H NMR (500 MHz, CDCl3):
δ
7.79 (C4”H, bs), 7.33 (C40H, 4d, J1 = 8.5, J2 = 7.5,
4J = 1.5), 7.23 (C7”H, 4d, 3JH-F = 8.5, 4J = 2.0, 5J = 0.5), 7.11 (C60H, dd, 3J = 7.5, 4J = 1.5),
7.06 (C5”H, td, 3J = 9.0, 4J = 2.0), 6.99 (C50H, td, 3J = 7.5, 4J = 1.0), 6.94 (C30H, dd, 3J = 8.5,
3
2
3
4J = 1.0), 4.03 (C1xH2, t, J = 7.0), 3.95 (C8H(1), dt, J = 13.5, J = 7.0), 3.90 (C8H(2), dt,
2J = 13.5, 3J = 7.0), 3.78 (OCH3, s), 3.14 (CaH(E), CeH(E), CcH, bs), 2.56 (C4xH2, bs), 2.43
(C5H2, m), 2.00 -2.35 (CaH(A), CeH(A), CbH2, CdH2, m), 1.92 (C7H2, q, 3J = 6.5), 1.61–1.80
(C2xH2, C3xH2, C6H2, m).
13C NMR (125 MHz, CDCl3): δ 164.1 (C6”, d, 1J = 250.8*), 163.9 (C7”a, d, 3J = 13.3*),
161.8 (C3, s), 160.8 (C3”, s), 157.3 (C20, s), 151.9 (C1, s), 150.2 (C4a, s), 132.3 (C60, s), 129.5
(C40, s), 122.9 (C4”, s), 122.0 (C10, s), 120.8 (C50, s), 117.1 (C3”a, s), 112.5 (C5”, d, 2J = 25.5*),
111.1 (C30, s), 108.5 (C4, s), 97.4 (C7”, d, 2J = 26.8*), 58.3 (C4x, s), 55.5 (OCH3, s), 53.3 (Ca,
Ce, s), 42.9 (C8, s), 41.1 (C1x, s), 34.2 (Cc, s), 29.9 (Cb, Cd, s), 26.3 (C5, s), 25.5 (C2x, s), ~23.8
(C3x, s), 21.8 (C7, s), 18.5 (C6, s).
ESI-HRMS m/z: Calcd for C31H36FN4O4 [M + H]+ 547.2715. Found: 547.2726
4-(2-Chlorophenyl)-2-{4-[4-(6-fluoro-1,2-benzoxazol)-1-piperidyl]butyl}-5,6,7,8-
tetrahydro-pyrido[1,2-c]pyrimidine-1,3-dione 7d
The title compound was isolated as an oil. Yield: 51.4%.

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1H NMR (500 MHz, CDCl3):
δ
4
7.71 (C4”H, pt), 7.46 (C30H, m), 7.28–7.33 (C50H, C60H,
m), 7.23 (C7”H, 4d, JH-F = 8.5, J = 2.0, J = 0.5), 7.18–7.21 (C40H, m), 7.04 (C5”H, td,
3J = 9.0, 4J = 2.0), 4.05 (C1xH2, t, 3J = 7.0), 3.98 (C8H(1), dt, 2J = 13.5, 3J = 6.5), 3.90 (C8H(2),
dt, 2J = 13.5, 3J = 6.5), 3.06 (CaH(E), CeH(E), CcH, pd), 2.44 (C4xH2, pt), 2.41 (C5H2, m),
2.00–2.20 (CaH(A), CeH(A), CbH2, CdH2, m), 1.93 (C7H2, m), 1.67–1.79 (C2xH2, C6H2 m),
1.61 (C3xH2, q, 3J = 7.0).
3
5
13C NMR (125 MHz, CDCl3): δ 164.1 (C6”, d, 1J = 250.4*), 163.9 (C7”a, d, 3J = 13.6*),
161.2 (C3, C3”, pd), 151.8 (C1, s), 150.4 (C4a, s), 135.1 (C20, s), 132.6 (C60, s), 132.5 (C10, s),
129.7 (C30, s), 129.5 (C40, s), 127.1 (C50, s), 122.7 (C4”, d, 3J = 10.9*), 117.3 (C3”a, s), 112.3
(C5”, d, 2J = 25.3*), 110.1 (C4, s), 97.4 (C7”, d, 2J = 26.7*), 58.5 (C4x, s), 53.5 (Ca, Ce, s), 43.0
(C8, s), 41.5 (C1x, s), 34.7 (Cc, s), 30.5 (Cb, Cd, s), 26.4 (C5, s), 25.7 (C2x, s), 24.4 (C3x, s), 21.8
(C7, s), 18.5 (C6, s).
ESI-HRMS m/z: Calcd for C30H33ClFN4O3 [M + H]+ 551.2220. Found: 551.2230
4-(2-Fluorophenyl)-2-{4-[4-(6-fluoro-1,2-benzoxazol)-1-piperidyl]butyl}-5,6,7,8-
tetrahydro-pyrido[1,2-c]pyrimidine-1,3-dione 7e
The title compound was isolated as an oil. Yield: 76.3%.
1H NMR (500 MHz, CDCl3):
δ
7.72 (C4”H, dd, 3J = 9.0, 4JH-F = 5.5), 7.31–7.37 (C40H,
m), 7.20–7.25 (C60H, C7”H, m), 7.19 (C50H, td, 3J = 7.5, 4J = 1.0), 7.12 (C30H, m, 3JH-F = 9.5,
3J = 8.0, 4J = 1.0), 7.04 (C5”H, td, 3J = 9.0, 4J = 2.0), 4.04 (C1xH2, t, 3J = 7.5), 3.97 (C8H(1), dt,
2J = 14.0, 3J = 7.0 **), 3.92 (C8H(2), dt, 2J = 14.0, 3J = 7.0 **), 3.07 (CaH(E), CeH(E), CcH, pd),
2.47–2.57 (C5H2, m**), 2.45 (C4xH2 t, 3J = 7.0), 2.00–2.19 (CaH(A), CeH(A), CbH2, CdH2,
m), 1.94 (C7H2, q, 3J = 7.0), 1.65–1.81 (C2xH2, C6H2, m), 1.61 (C3xH2, q, 3J = 7.5).
13C NMR (125 MHz, CDCl3): δ0 164.1 (C6”, d, 1J = 250.4*), 163.9 (C7”a, d, 3J = 13.4*),
0
161.4 (C3, s), 161.1 (C3”, s), 160.4 (C2 , d, 1J = 245.8*), 151.7 (C1, s), 150.9 (C4a, s), 132.9 (C6 ,
d, 3J = 3.1*), 130.0 (C40, d, 3J = 8.3*), 124.2 (C50, d, 4J = 3.6*), 122.7 (C4”, d, 3J = 11.2*), 120.8
(C10, d, 2J = 16.2*), 117.3 (C3”a, s), 115.8 (C30, d, 2J = 22.4*), 112.3 (C5”, d, 2J = 25.1*), 106.1
(C4, s), 97.4 (C7”, d, 2J = 26.7*), 58.5 (C4x, s), 53.5 (Ca, Ce, s), 42.8 (C8, s), 34.6 (Cc, s), 30.5
(Cb, Cd, s), 26.5 (C5, s), 25.7 (C2x, s), 24.4 (C3x, s), 21.8 (C7, s), 18.4 (C6, s).
ESI-HRMS m/z: Calcd for C30H33F2N4O3 [M + H]+ 535.2515. Found: 535.2522
4-(4-Methylphenyl)-2-{4-[4-(6-fluoro-1,2-benzoxazol)-1-piperidyl]butyl}-5,6,7,8-
tetrahydro-pyrido[1,2-c]pyrimidine-1,3-dione 7f
The title compound was isolated as a white powder. Yield: 55.3%; m.p. 107–110 ◦C.
1H0NMR0(500 MHz, CDCl3):
δ
7.71 (C4”H, pt), 7.23 (C7”H, dd, 3JH-F = 8.5, 4J = 2.0),
0
0
7.20 (C2 H, C6 H, d, 3J = 8.0), 7.08 (C3 H, C5 H, d, 3J = 8.0), 7.04 (C5”H, td, 3J = 9.0, 4J = 2.0),
4.04 (C1xH2, t, 3J = 7.0), 3.94 (C8H2, t, 3J = 6.0), 3.06 (CaH(E), CeH(E), CcH, pd), 2.54 (C5H2,
t, 3J = 6.5), 2.440 (C4xH2, pt), 2.36 (CH3, s), 2.00–2.17 (CaH(A), CeH(A), CbH2, CdH2, m),
1.92 (C7H2,q, 3J = 6.5), 1.65–1.77 (C2xH2, C6H2, m), 1.605 (C3xH2, q, 3J = 6.5).
13C NMR (125 MHz, CDCl3): δ 164.1 (C6”, d, 1J = 250.4*), 163.9 (C7”a, d, 3J = 13.4*),
162.1 (C3, s), 161.1 (C3”, s), 151.7 (C1, s), 149.5 (C4a, s), 137.4 (C40, s), 130.6 (C20, C60, s),
0
0
0
130.3 (C1 , s), 129.2 (C3 , C5 , s), 122.7 (C4”, d, 3J = 10.9*), 117.3 (C3”a, s), 112.4 (C4, s), 112.3
(C5”, d, 2J = 25.1*), 97.4 (C7”, d, 2J = 26.7*), 58.6 (C4x, s), 53.6 (Ca, Ce, s), 42.6 (C8, s), 41.5
(C1x, s), 34.7 (Cc, s), 30.5 (Cb, Cd, s), 26.7 (C5, s), 25.7 (C2x, s), 24.4 (C3x, s), 21.8 (C7, s), 21.3
(CH3, s), 18.6 (C6, s).
ESI-HRMS m/z: Calcd for C31H36FN4O3 [M + H]+ 531.2766. Found: 531.2775
4-(4-Methoxyphenyl)-2-{4-[4-(6-fluoro-1,2-benzoxazol)-1-piperidyl]butyl}-5,6,7,8-
tetrahydro-pyrido[1,2-c]pyrimidine-1,3-dione 7g
The title compound was isolated as a white powder. Yield: 78.9%; m.p. 108–109 ◦C.
1H NMR (500 MHz, CDCl3):
δ
7.75 (C4”H, bs), 7.23 (C7”H, dd, 3JH-F = 8.5, 4J = 2.0),
7.12 (C20H, C60H, dt, 3J = 8.5, 4J = 3.0), 7.05 (C5”H, td, 3J = 9.0, 4J = 2.0), 6.93 (C30H, C50H,

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dt, 3J = 9.0, 4J = 2.5), 4.04 (C1xH2, t, 3J = 7.5), 3.94 (C8H2, t, 3J = 6.5), 3.82 (OCH3, s), 3.10
(CaH(E), CeH(E), CcH, bs), 2.55 (C5H2, t, 3J = 7.0), 2.49 (C4xH2, bs), 2.00–2.25 (CaH(A),
CeH(A), CbH2, CdH2, m), 1.93 (C7H2, q, 3J = 7.0), 1.60–1.80 (C2xH2, C3xH2, C6H2, m).
13C NMR (125 MHz, CDCl3): δ 164.1 (C6”, d, 1J = 250.5*), 163.9 (C7”a, d, 3J = 13.6*),
162.2 (C3, s), 161.0 (C3”, s), 151.7 (C1, s), 149.7 (C4a, s), 131.8 (C20, C60, s), 125.5 (C10, s),
122.8 (C4”, d, 3J = 11.2*), 117.2 (C3”a, s), 114.0 (C30, C50, s), 112.4 (C5”, d, 2J = 25.1*), 112.0
(C4, s), 97.4 (C7”, d, 2J = 26.7*), 58.5 (C4x, s), 55.3 (OCH3, s), 53.4 (Ca, Ce, s), 42.7 (C8, s),
41.4 (C1x, s), 34.5 (Cc, s), 30.2 (Cb, Cd, s), 26.8 (C5, s), 25.7 (C2x, s), 24.2 (C3x, s), 21.8 (C7, s),
18.6 (C6, s).
ESI-HRMS m/z: Calcd for C31H36FN4O4 [M + H]+ 547.2715. Found: 547.2724
4-(4-Chlorophenyl)-2-{4-[4-(6-fluoro-1,2-benzoxazol)-1-piperidyl]butyl}-5,6,7,8-
tetrahydro-pyrido[1,2-c]pyrimidine-1,3-dione 7h
The title compound was isolated as a white powder. Yield: 89.2%; m.p. 116–120 ◦C.
1H NMR (500 MHz, CDCl3):
δ
7.77 (C4”H, bs), 7.37 (C20H, C60H, dt, 3J = 9.0, 4J = 2.5),
7.23 (C7”H, 4d, 3JH-F = 8.5, 4J = 2.0, 5J = 0.5), 7.15 (C30H, C50H, dt, 3J = 8.5, 4J = 2.5), 7.06
(C5”H, td, 3J = 9.0, 4J = 2.0), 4.04 (C1xH2, t, 3J = 7.5), 3.94 (C8H2, t, 3J = 6.5), 3.14 (CaH(E),
CeH(E), CcH, bs), 2.52 (C4xH2, C5H2, m), 2.02–2.40 (CaH(A), CeH(A), CbH2, CdH2, m),
1.94 (C7H2, q, 3J = 6.5), 1.72 (C2xH2, C3xH2, C6H2, m).
13C NMR (125 MHz, CDCl3): δ 164.1 (C6”, d, 1J = 250.8*), 163.9 (C7”a, d, 3J = 13.6*),
161.8 (C3, s), 160.8 (C3”, s), 151.6 (C1, s), 150.0 (C4a, s), 133.715 (C40, s), 132.176 (C30,C50, s),
0
0
0
131.776 (C1 , s), 128.731 (C2 , C6 , s), 122.8 (C4”, d, 3J = 10.3*), 117.1 (C3”a, s), 112.5 (C5”, d,
2J = 25.7*), 111.2 (C4, s), 97.4 (C7”, d, 2J = 26.8*), 58.3 (C4x, s), 53.2 (Ca, Ce, s), 42.7 (C8, s),
41.3 (C1x, s), 34.2 (Cc, s), 29.8 (Cb, Cd, s), 26.8 (C5, s), 25.5 (C2x, s), 23.8 (C3x, s), 21.7 (C7, s),
18.5 (C6, s).
ESI-HRMS m/z: Calcd for C30H33ClFN4O3 [M + H]+ 551.2220. Found: 551.2227
4-(4-Fluorophenyl)-2-{4-[4-(6-fluoro-1,2-benzoxazol)-1-piperidyl]butyl}-5,6,7,8-
tetrahydro-pyrido[1,2-c]pyrimidine-1,3-dione 7i
The title compound was isolated as a white powder. Yield: 71.1%; m.p. 57–60 ◦C.
1H NMR (500 MHz, CDCl3):
δ
7.71 (C4”H, pt), 7.23 (C7”H, 4d, 3JH-F = 8.5, 4J = 2.0,
5J = 0.5), 7.17 (C20H, C60H, kt, 3J = 9.0, 4JH-F = 5.5, 4J = 2.0), 7.09 (C30H, C50H, tt, 3J = 8.5,
4J = 2.0), 7.04 (C5”H, td, 3J = 8.5, 4J = 2.0), 4.04 (C1xH2, t, 3J = 7.5), 3.94 (C8H2, t, 3J = 6.5),
3.07 (CaH(E), CeH(E), CcH, pd), 2.52 (C5H2, t, 3J = 6.5), 2.44 (C4xH2, bs), 2.20–2.22 (CaH(A),
CeH(A), CbH2, CdH2, pd), 1.93 (C7H2, q, 3J = 7.0), 1.72 (C2xH2, C6H2, m), 1.61 (C3xH2, q,
3J = 7.0).
13C NMR (125 MHz, CDCl3): δ 164.1 (C6”, d, 1J = 250.6*), 163.9 (C7”a, d, 3J = 13.6*),
0
0
162.3 (C4 , d, 1J = 246.9*), 162.0 (C3, s), 161.1 (C3”, s), 151.6 (C1, s), 149.9 (C4a, s), 132.5 (C2 ,
C6 , d, 3J = 8.0*), 129.2 (C1 , d, 4J = 3.5*), 122.7 (C4”, d, 3J = 11.3*), 117.3 (C3”a, s), 115.5 (C3 ,
C50, d, 2J = 21.5*), 112.3 (C5”, d, 2J = 25.5*), 111.4 (C4, s), 97.4 (C7”, d, 2J = 26.7*), 58.5 (C4x,
s), 53.6 (Ca, Ce, s), 42.7 (C8, s), 41.6 (C1x, s), 34.6 (Cc, s), 30.5 (Cb, Cd, s), 26.8 (C5, s), 25.7
(C2x, s), 24.4 (C3x, s), 21.7 (C7, s), 18.6 (C6, s).
0
0
0
ESI-HRMS m/z: Calcd for C30H33F2N4O3 [M + H]+ 535.2515. Found: 535.2523
3.3. Biological Tests
3.3.1. In Vitro Tests
5-HT1A Binding Assay
Radioligand binding was performed using membranes from CHO-K1 cells stably
transfected with the human 5-HT1A receptor (PerkinElmer, Fremont, CA, USA). All assays
were carried out in duplicate. Then, 50
50
L [3H]-8-OH-DPAT (final concentration 1 nM) and 150
g protein per well) prepared in assay buffer (50 mM Tris, pH 7.4, 10 mM MgSO4, 0.5
mM EDTA, 0.1% ascorbic acid) were transferred to a polypropylene 96-well microplate
µL working solution of the tested compounds,
µ
µL diluted membranes (20
µ

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using 96-wells pipetting station Rainin Liquidator (MettlerToledo, Columbus, OH, USA).
Serotonin (10
µ
M) was used to define nonspecific binding. The microplate was covered
◦
with a sealing tape, mixed and incubated for 60 min at 27 C. The reaction was terminated
by rapid filtration through GF/B filter mate, presoaked with 0.5% polyethyleneimine for 30
◦
min. Ten rapid washes with 200
µ
L 50 mM Tris buffer (4 C, pH 7.4) were performed using
an automated harvester system—Harvester-96 MACH III FM (Tomtec, Hamden, CT, USA).
◦
The filter mates were dried at 37 C in a forced-air fan incubator, and then solid scintillator
◦
Meltilex was melted on filter mates at 90 C for 6 min. The radioactivity on the filter
was measured in a MicroBeta TriLux 1450 scintillation counter (PerkinElmer, Waltham,
MA, USA). Data were fitted to a one-site curve-fitting equation with Prism 6 (GraphPad
Software, San Diego, CA, USA) and Ki values were estimated from the Cheng–Prusoff
equation.
SERT Binding Assay
Radioligand binding was performed using rat cortex tissue. All assays were carried
out in duplicate. First, 50
µL working solution of the tested compounds, 50
µ
L [3H]-
citalopram (final concentration 1.0 nM) and 150
µL tissue suspension, prepared in assay
buffer (50 mM Tris. pH 7.7; 150 mM NaCl; 5 mM KCl), were transferred to a polypropylene
96-well microplate using 96-wells pipetting station Rainin Liquidator (MettlerToledo).
Imipramine (10 µM) was used to define nonspecific binding. The microplate was covered
with sealing tape, mixed and incubated for 60 min at 24 ◦C. The reaction was terminated
by rapid filtration through GF/B filter mate presoaked with 0.3% polyethyleneimine for
30 min. Ten rapid washes with 200 µ
L 50 mM Tris buffer (4 ◦C, pH 7.7) were performed
using an automated harvester system—Harvester-96 MACH III FM (Tomtec). The filter
mates were dried at 37 ◦C in a forced-air fan incubator and then solid scintillator MeltiLex
◦
was melted on filter mates at 90 C for 6 min. The radioactivity on the filter was measured
in MicroBeta TriLux 1450 scintillation counter (PerkinElmer, USA). Data were fitted to
a one-site curve-fitting equation with Prism 6 (GraphPad Software) and Ki values were
estimated from the Cheng–Prusoff equation.
5-HT2A, 5-HT6, 5-HT7 and D2 Binding Assay
In vitro radioligand binding assays for 5-HT2A, 5-HT6, 5-HT7 and D2 receptors were
carried out using methods published by Zajdel et al. [45]. For the assays, HEK293 cell
cultures stably expressing the investigated human receptors were used. Cell pellets were
thawed and homogenized in 20 vol of assay buffer us◦ing an Ultra Turrax tissue homoge-
nizer, then centrifuged twice at 35000 g for 20 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-HT2A receptors—
50 mM Tris-HCl, 0.1 mM EDTA, 4 mM MgCl2 and 0.1% ascorbate; for 5-HT6 receptors—50
mM Tris-HCl, 0.5 mM EDTA, 4 mM MgCl2; for 5-HT7 receptors—50 mM Tris-HCl, 4 mM
MgCl2, 10 µM pargyline and 0.1% ascorbate; for D2 receptors—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 total volume of 200 mL in 96-well microtitre plates for 1 h at
37 ◦C, except for 5-HT2A receptors which were incubated at room temperature for 1.5 h.
The process of equilibration was terminated by rapid filtration through Unifilter plates
with a 96-well cell harvester, and radioactivity retained on the filters was quantified on a
Microbeta plate reader (PerkinElmer, USA). For displacement studies, the assay samples
contained as radioligands (PerkinElmer): 2 nM [3H]-ketanserin (spec. act. 53.4 Ci/mmol)
for 5-HT2A receptors; 2 nM [3H]-LSD (spec. act. 83.6 Ci/mmol) for 5-HT6 receptors; 0.6 nM
[3H]-5-CT (spec. act. 39.2 Ci/mmol) for 5-HT7 receptors and [3H]-raclopride (spec. act. 76.0
Ci/mmol) for D2 receptors. Nonspecific binding was defined with 10 µM chlorpromazine,
10 µM methiotepine, or 1 µM (+) butaclamol used in 5-HT2A, 5-HT6 and D2 receptors as-
says, respectively. Each compound was tested in triplicate at 7–8 concentrations (10-11-10-4
M). The inhibition constants (Ki) were calculated from the Cheng–Prusoff equation [46].
Results were expressed as means of at least two separate experiments.

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3.3.2. In Vivo Tests
All studies were performed according to the guidelines of the European Community
Council (Directive 86/609/EEC) and were approved by the Ethical Committee of the
Institute of Pharmacology (88/2016, 05/31/2016). The experiments were performed on
male CD-1 mice (23–40 g). The animals were kept at room temperature (21
±
2 ◦C) on a
natural day-night cycle (March–October) and housed under standard laboratory conditions.
They had free access to food and tap water before the experiment. Each experimental group
consisted of 6–8 animals/dose. All the animals were used only once. 8-Hydroxy-2-(di-n-
propylamino)tetralin hydro-bromide (8-OH-DPAT, Research Biochemical Inc.) was used as
aqueous solution. Compounds 6a and 7g were suspended in a 10% aqueous solution of
dimethyl sulphoxide (DMSO). Vehicle group was administered as 10% aqueous solution
of dimethyl sulphoxide (DMSO). 8-OH-DPAT was injected subcutaneously (sc); 6a and
7g were given intraperitoneally (ip) in a volume of 10 mL/kg/mice. The obtained data
were analyzed by Dunnett’s test (one drug administration) or by the Newman–Keuls
test (two drugs administrations). Forced swim test: the obtained data was evaluated by
one-way analysis of variance (ANOVA) followed by the Dunnett’s multiple comparisons
test: p < 0.05 was considered significant.
Body Temperature in Mice
The effects of the tested compounds 6a and 7g given alone, on the rectal body temper-
ature in mice (measured with an Ellab thermometer) were recorded 30, 60, 90 and 120 min
after their administration. In a separate experiment the effect of WAY-100635 (0.1 mg/kg
s.c.) on the hypothermia induced by tested compounds was measured. WAY-100635 was
administered 15 min before the tested compounds and the rectal body temperature was
recorded 30 min and 60 min after injection. The absolute mean body temperatures were
◦
within a range 36.7
ture (
±
0.5 C. The results were expressed as a change in body tempera-
∆
t) with respect to the basal body temperature, as measured at the beginning of the
experiment.
Forced Swim Test in Mice
The forced swim test (FST) was carried out according to the method of Porsolt at
al. [47]. Mice were placed individually into glass cylinders (height 25 cm, diameter 10 cm)
◦
containing 20 cm of water and maintained at 23
±
1 C. The animals were left in the
cylinder for 6 min. After the first 2 min adaptation period, the total duration of immobility
was measured during the last 4 min test. The mouse was judged to be immobile when it
remained floating passively, performing slow motions to keep its head above the water.
Tested compounds were administered 30 min before test.
Metabolic Stability
Stock solutions of studied compounds were prepared at concentration of 100
1:1 acetonitrile/water mixture. Incubation mixes consisted 1 M of a studied compound,
100 M of NADPH in phosphate buffer and 1 mg/mL of pooled HLMs (Sigma-Aldrich, St.
µM in
µ
µ
Louis, MO, USA) in potassium phosphate buffer (0.1 M, pH 7.4). Incubation was carried
out in 96-well plates at 37 ◦C. Incubation mixtures (excluding compound solution) were
subjected to 5 min preincubations, and started by addition of 10
solution. After 0, 5, 10, 15, and 30 min, 25 L samples of incubation reaction were added to
the equal volume of ice-cold acetonitrile containing 1 M of IS (buspirone hydrochloride).
µL of compound stock
µ
µ
Control incubations were performed without NADPH to assess possible chemical insta-
bility. All samples were immediately centrifuged (10 min, 10,000 rpm) and the resulting
supernatant was directly subjected to LC-MS analysis.
LC-MS analysis was performed on an Agilent 1260 system coupled to SingleQuad
6120 mass spectrometer (Agilent Technologies, Santa Clara, CA, USA). A Poroshell C18
EC120 column (3.0
×
100 mm2, 2.7
µm, Agilent Technologies, Santa Clara, CA, USA) was
used in reversed-phase mode with gradient elution starting with 90% of phase A (0.1%

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formic acid in deionised water) and 10% of phase B (0.1% formic acid in acetonitrile).
The gradient elution program was: 0.00–10.00 min, 10–95% B; 10.01 min–10.02 min, 95–
◦
10% B; 10.02–15.00 min, 10% B. Total analysis time was 15 min at 40 C, flow rate was
0.4 mL/min and the injection volume was 5 µL. The mass spectrometer was equipped
with an electrospray ionization source and was in positive ionization mode. The mass
analyzer was set individually to each derivative to detect pseudomolecular ions [M +
H]+. Mass spectrometry detector (MSD) parameters of the ESI source were as follows:
nebulizer pressure—35 psig (N2); drying gas 12 L/min (N2); drying gas temperature—300
◦C; capillary voltage—3.0 kV; fragmenter voltage—70 V.
4. Conclusions
The use of antidepressants or neuroleptic drugs acting through an extended receptor
profile is now becoming a widely used therapeutic approach. The paper describes the
synthesis and biological studies in vitro and in vivo (binding affinity for receptor, functional
profile and metabolic stability) of new 4-aryl-2H-pyrido[1,2-c]pyrimidine derivatives (6a–i),
as well as 4-aryl-5,6,7,8-tetrahydro-pyrido[1,2-c]pyrimidines (7a–i), having a 6-fluoro-3-(4-
piperidinyl)-1,2-benzisoxazole residue in the pharmacophore ligand part. Receptor studies
of derivatives 6a (5-HT1A Ki = 23.0 nM; SERT Ki = 32.0 nM) and 7g (5-HT1A Ki = 5.0 nM;
SERT Ki = 48.0 nM) showed their desirable very high binding to both molecular targets.
Analyses of the obtained results of affinities of the derivatives (6a–i) and (7a–i) for the
5-HT1A receptor, showed that the derivatives of both series have very high bindings (6a–i
5-HT1A Ki = 7.0–30.0 nM, and for 7a–i Ki = 5.0–52.0 nM), where the series (6a–i) showed
higher (more active derivatives) activity against compounds of the series (7a–i).
Thus, when examining the effect of the degree of hydrogenation of the terminal part
of ligands on the affinity for the 5-HT1A receptor for both series (6a–i) and (7a–i), it can be
concluded that it was low. Derivatives of both series (6a–i) and (7a–i) generally showed
low affinity for SERT protein with the exception of 6a (SERT Ki = 32.0 nM) and 7g (SERT
Ki = 48.0 nM). Analysis of the effect of substituents (R, R1) bound to the benzene ring of
4-aryl-2H-pyrido[1,2-c]pyrimidine residue, (6a–i) derivatives on affinity for the 5-HT1A
receptor, showed a significant effect in the ortho position. However, in the case of 4-aryl-
5,6,7,8-tetrahydro-pyrido[1,2-c]pyrimidine derivatives, compounds (7a–i), an increase in
affinity for the 5-HT1A receptor was observed for derivatives having substituents in the
para position.
Functional profile study for compound 6a in induced hypothermia tests showed that
it is a presynaptic agonist of the 5-HT1A receptor. In turn, studies of the 6a compound
in the FST showed its inactivity, which indicated the lack of postsynaptic activity to the
5-HT1A receptor.
Metabolic stability studies were performed for the 6a and 6d derivatives, which
showed their sensitivity to the action of human liver microsomes. Low stability may be
due to the introduction of the 6-fluoro-3-(4-piperidinyl)-1,2-benzisoxazole residue into the
pharmacophore part.
For the selected compounds 6a
In vitro affinity studies in an extended receptor profile (D2, 5-HT2A, 5-HT6 and 5-HT7)
indicated that derivatives 6a and 7g have very high binding affinity to the 5-HT1A, 5-HT2A
SERT, D2 receptors, high binding affinity to the 5-HT 7 receptor and low binding affinity to
, 6d, 7g and 7i, further in vitro studies were performed.
,
5-HT6. For compounds 6d and 7i, ligands showed very high affinity for 5-HT1A, 5-HT2A
D2 receptors, medium affinity for 5-HT7 and low affinity for 5-HT6 and SERT. By analyzing
the results, it can be concluded that they are a good starting point for further research on
ligands with a multireceptor profile.
,
The obtained study results encourage further optimization of the obtained ligand
structures in the search for new pyrido[1,2-c]pyrimidine derivatives with potential antide-
pressant activity from the SSRI+ group.

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´
Author Contributions: Conceptualization, M.K.; synthesis of compounds M.K., G.S. and F.H.;
´
methodology, investigation, and data analysis, M.K., G.S., J.K., S.U., M.B., T.B., A.S., K.S., B.S.,
G.N., B.D. and F.H.; writing—original draft preparation, M.K.; writing—review and editing, F.H. and
´
G.S. All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by the Polish National Science Center, grant OPUS 6, No.
UMO-2013/11/B/NZ7/01638.
Institutional Review Board Statement: In vivo studies were performed according to the guidelines
of the European Community Council (Directive 86/609/EEC) and were approved by the Ethical
Committee of Maj Institute of Pharmacology (88/2016, 05/31/2016).
Informed Consent Statement: Not applicable.
Data Availability Statement: Data is contained within the article.
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
Akimova, E.; Lanzenberger, R.; Kasper, S. The Serotonin-1A Receptor in Anxiety Disorders. Biol. Psychiatry 2009, 66, 627–635.
[CrossRef] [PubMed]
2.
3.
4.
Artigas, F. Serotonin receptors involved in antidepressant effects. Pharmacol. Ther. 2013, 137, 119–131. [CrossRef] [PubMed]
Breier, A. Serotonin, schizophrenia and antipsychotic drug action. Schizophr. Res. 1995, 14, 187–202. [CrossRef]
Pacher, P.; Kohegyi, E.; Kecskemeti, V.; Furst, S. Current Trends in the Development of New Antidepressants. Curr. Med. Chem.
2001, 8, 89–100. [CrossRef]
5.
6.
Doucet, M.V.; Harkin, A.; Dev, K.K. The PSD-95/nNOS complex: New drugs for depression? Pharmacol. Ther. 2012, 133, 218–229.
[CrossRef] [PubMed]
Romero, L.; Celada, P.; Martin-Ruiz, R.; Diaz-Mataix, L.; Mourelle, M.; Delgadillo, J.; Hervas, I.; Artigas, F. Modulation of seroton-
ergic function in rat brain by VN2222, a serotonin reuptake inhibitor and 5-HT1A receptor agonist. Neuropsychopharmacology 2003
28, 445–456. [CrossRef]
,
7.
Artigas, F.; Adell, A.; Celada, P. Pindolol Augmentation of Antidepressant Response. Curr. Drug Targets 2006, 7, 139–147.
[CrossRef] [PubMed]
8.
9.
Blier, P. The pharmacology of putative early-onset antidepressant strategies. Eur. Neuropsychopharmacol. 2003, 13, 57–66. [CrossRef]
Spinks, D.; Spinks, G. Serotonin Reuptake Inhibition: An Update on Current Research Strategies. Curr. Med. Chem. 2002, 9,
799–810. [CrossRef]
10. Pacher, P.; Kecskemeti, V.; Pal Pacher, V.K. Trends in the development of new antidepressants. Is there a light at the end of the
tunnel? Curr. Med. Chem. 2004, 11, 925–943. [CrossRef]
11. Nichols, D.E.; Nichols, C.D. Serotonin Receptors. Chem. Rev. 2008, 108, 1614–1641. [CrossRef]
12. Ohno, Y. Therapeutic Role of 5-HT1A Receptors in The Treatment of Schizophrenia and Parkinson’s Disease. CNS Neurosci. Ther.
2011, 17, 58–65. [CrossRef]
13. Newman-Tancredi, A. Biased agonism at serotonin 5-HT 1A receptors: Preferential postsynaptic activity for improved therapy of
CNS disorders. Neuropsychiatry 2011, 1, 149–164. [CrossRef]
14. Adell, A.; Castro, E.; Celada, P.; Bortolozzi, A.; Pazos, A.; Artigas, F. Strategies for producing faster acting antidepressants.
Drug Discov. Today 2005, 10, 578–585. [CrossRef]
15. Carr, G.V.; Lucki, I. The role of serotonin receptor subtypes in treating depression: A review of animal studies. Psychopharmacology
2011, 213, 265–287. [CrossRef]
16. Lacivita, E.; Leopoldo, M.; Berardi, F.; Perrone, R. 5-HT1A receptor, an old target for new therapeutic agents. Curr. Top. Med.
Chem. 2008, 8, 1024–1034. [CrossRef] [PubMed]
17. Artigas, F.; Bramwell, S.R.; Grahame-Smith, D.G.; Probst, A.; Palacios, J.M. 5-HT and antidepressants: New views from
microdialysis studies. Trends Pharmacol. Sci. 1993, 14, 262. [CrossRef]
18. Blier, P.; Ward, N.M. Is there a role for 5-HT1A agonists in the treatment of depression? Biol. Psychiatry 2003, 53,
193–203. [CrossRef]
19. Blier, P.; De Montigny, C. Modification of 5-HT neuron properties by sustained administration of the 5-HT1A agonist gepirone:
Electrophysiological studies in the rat brain. Synapse 1987, 1, 470–480. [CrossRef] [PubMed]
20. Rickels, K.; Athanasiou, M.; Reed, C. Vilazodone, a novel, dual-acting antidepressant: Current status, future promise and potential
for individualized treatment of depression. Per. Med. 2009, 6, 217–224. [CrossRef] [PubMed]
21. Howland, R.H. Vilazodone: Another novel atypical antidepressant drug. J. Psychosoc. Nurs. Ment. Health Serv. 2011, 49, 19–22.
[CrossRef] [PubMed]
22. Frampton, J.E. Vilazodone: In major depressive disorder. CNS Drugs 2011, 25, 615–627. [CrossRef] [PubMed]

Int. J. Mol. Sci. 2021, 22, 2329
21 of 21
23. Bang-Andersen, B.; Ruhland, T.; Jørgensen, M.; Smith, G.; Frederiksen, K.; Jensen, K.G.; Zhong, H.; Nielsen, S.M.; Hogg, S.; Mørk,
A.; et al. Discovery of 1-[2-(2,4-Dimethylphenylsulfanyl)phenyl]piperazine (Lu AA21004): A Novel Multimodal Compound for
the Treatment of Major Depressive Disorder. J. Med. Chem. 2011, 54, 3206–3221. [CrossRef] [PubMed]
24. Herold, F.; Chodkowski, A.; Izbicki, Ł.; Król, M.; Kleps, J.; Turło, J.; Nowak, G.; Stachowicz, K.; Dybała, M.; Siwek, A. Novel
4-aryl-pyrido[1,2-c]pyrimidines with dual SSRI and 5-HT1A activity, Part 1. Eur. J. Med. Chem. 2009, 44, 1710–1717. [CrossRef]
25. Herold, F.; Izbicki, Ł.; Chodkowski, A.; Dawidowski, M.; Król, M.; Kleps, J.; Turło, J.; Wolska, I.; Nowak, G.; Stachowicz, K.
Novel 4-aryl-pyrido[1,2-c]pyrimidines with dual SSRI and 5-HT1A activity: Part 2. Eur. J. Med. Chem. 2009, 44, 4702–4715.
[CrossRef] [PubMed]
26. Herold, F.; Chodkowski, A.; Izbicki, Ł.; Turło, J.; Dawidowski, M.; Kleps, J.; Nowak, G.; Stachowicz, K.; Dybała, M.; Siwek,
A.; et al. Novel 4-aryl-pyrido[1,2-c]pyrimidines with dual SSRI and 5-HT1A activity. Part 3. Eur. J. Med. Chem. 2011, 46,
142–149. [CrossRef]
27. Chodkowski, A.; Wróbel, M.Z.; Turło, J.; Kleps, J.; Siwek, A.; Nowak, G.; Belka, M.; Ba˛czek, T.; Mazurek, A.P.; Herold, F. Novel
4-aryl-pyrido[1,2-c]pyrimidines with dual SSRI and 5-HT1A activity. Part 4. Eur. J. Med. Chem. 2015, 90, 21–32. [CrossRef]
28. Gomolka, A.; Ciesielska, A.; Wróbel, M.Z.; Chodkowski, A.; Kleps, J.; Dawidowski, M.; Siwek, A.; Wolak, M.; Stachowicz, K.;
Slawinska, A.; et al. Novel 4-aryl-pyrido[1,2-c]pyrimidines with dual SSRI and 5-HT1A activity. Part 5. Eur. J. Med. Chem. 2015
98, 221–236. [CrossRef]
,
29. Wróbel, M.Z.; Chodkowski, A.; Herold, F.; Gomółka, A.; Kleps, J.; Mazurek, A.P.; Plucin´ski, F.; Mazurek, A.; Nowak, G.; Siwek,
A.; et al. Synthesis and biological evaluation of novel pyrrolidine-2,5-dione derivatives as potential antidepressant agents. Part 1.
Eur. J. Med. Chem. 2013, 63, 484–500. [CrossRef]
30. Martin, K.F.; Heal, D.J. 8-OH-DPAT-Induced Hypothermia in Rodents. A Specific Model of 5-HTlA Autoreceptor Function.
In Serotonin: Molecular Biology, Receptors and Functional Effects; Birkhäuser Basel: Basel, Switzerland, 1991; pp. 483–490. ISBN
9783034872614.
31. ACD/ChemSketch 2017; Version 2017.1.2; Advanced Chemistry Development, Inc.: Toronto, ON, Canada, 2017.
32. Di Giovanni, G.; Esposito, E.; Di Matteo, V. Role of Serotonin in Central Dopamine Dysfunction. CNS Neurosci. Ther. 2010, 16,
179–194. [CrossRef]
33. Etievant, A.; Bétry, C.; Haddjeri, N. Partial Dopamine D2/Serotonin 5-HT1A Receptor Agonists as New Therapeutic Agents.
Open Neuropsychopharmacol. J. 2010, 3, 1–12. [CrossRef]
34. Sakaue, M.; Somboonthum, P.; Nishihara, B.; Koyama, Y.; Hashimoto, H.; Baba, A.; Matsuda, T. Postsynaptic 5-hydroxytryptamine
1A receptor activation increases in vivo dopamine release in rat prefrontal cortex. Br. J. Pharmacol. 2000, 129, 1028–1034. [CrossRef]
35. Fountoulakis, K.N.; Kelsoe, J.R.; Akiskal, H. Receptor targets for antidepressant therapy in bipolar disorder: An overview. J.
Affect. Disord. 2012, 138, 222–238. [CrossRef] [PubMed]
36. Millan, M.J. Multi-target strategies for the improved treatment of depressive states: Conceptual foundations and neuronal
substrates, drug discovery and therapeutic application. Pharmacol. Ther. 2006, 110, 135–370. [CrossRef]
´
37. Slifirski, G.; Król, M.; Kleps, J.; Podsadni, P.; Belka, M.; Ba˛czek, T.; Siwek, A.; Stachowicz, K.; Szewczyk, B.; Nowak, G.; et al.
Synthesis of new 5,6,7,8-tetrahydropyrido[1,2-c]pyrimidine derivatives with rigidized tryptamine moiety as potential SSRI and
5-HT1A receptor ligands. Eur. J. Med. Chem. 2019, 180, 383–397. [CrossRef] [PubMed]
38. Lipinski, C.A.; Lombardo, F.; Dominy, B.W.; Feeney, P.J. Experimental and computational approaches to estimate solubility and
permeability in drug discovery and development settings. Adv. Drug Deliv. Rev. 1997, 23, 3–25. [CrossRef]
39. Chang, Y.; Pi, W.; Ang, W.; Liu, Y.; Li, C.; Zheng, J.; Xiong, L.; Yang, T.; Luo, Y. Synthesis and evaluation of amide side-chain
modified Agomelatine analogues as potential antidepressant-like agents. Bioorg. Med. Chem. Lett. 2014, 24, 1672–1676. [CrossRef]
40. Goodwin, G.M.; De Souza, R.J.; Green, A.R. The pharmacology of the hypothermic response in mice to 8-hydroxy-2-(DI-n-
propylamino)tetralin (8-OH-DPAT). Neuropharmacology 1985, 24, 1187–1194. [CrossRef]
41. Forster, E.A.; Cliffe, I.A.; Bill, D.J.; Dover, G.M.; Jones, D.; Reilly, Y.; Fletcher, A. A pharmacological profile of the selective silent
5-HT1A receptor antagonist, WAY-100635. Eur. J. Pharmacol. 1995, 281, 81–88. [CrossRef]
42. Yap, C.; Xue, Y.; Chen, Y. Application of Support Vector Machines to In Silico Prediction of Cytochrome P450 Enzyme Substrates
and Inhibitors. Curr. Top. Med. Chem. 2006, 6, 1593–1607. [CrossRef] [PubMed]
43. Ulenberg, S.; Belka, M.; Georgiev, P.; Król, M.; Herold, F.; Ba˛czek, T. In Vitro approach for identification of a leading cytochrome
P450 isoenzyme responsible for biotransformation of novel arylpiperazine drug candidates and their inhibition potency towards
CYP3A4. Acta Pol. Pharm. Drug Res. 2020, 77, 69–76. [CrossRef]
´
44. Slifirski, G.; Król, M.; Kleps, J.; Ulenberg, S.; Belka, M.; Ba˛czek, T.; Siwek, A.; Stachowicz, K.; Szewczyk, B.; Nowak, G.; et al.
Synthesis of novel pyrido[1,2-c]pyrimidine derivatives with rigidized tryptamine moiety as potential SSRI and 5-HT1A receptor
ligands. Eur. J. Med. Chem. 2019, 166, 144–158. [CrossRef] [PubMed]
45. Zajdel, P.; Kos, T.; Marciniec, K.; Satała, G.; Canale, V.; Kamin´ski, K.; Hołuj, M.; Lenda, T.; Koralewski, R.; Bednarski, M.; et al.
Novel multi-target azinesulfonamides of cyclic amine derivatives as potential antipsychotics with pro-social and pro-cognitive
effects. Eur. J. Med. Chem. 2018, 145, 790–804. [CrossRef]
46. Yung-Chi, C.; Prusoff, W.H. Relationship between the inhibition constant (KI) and the concentration of inhibitor which causes 50
per cent inhibition (I50) of an enzymatic reaction. Biochem. Pharmacol. 1973, 22, 3099–3108. [CrossRef]
47. Porsolt, R.D.; Bertin, A.; Jalfre, M. Behavioral despair in mice: A primary screening test for antidepressants. Arch. Int.
Pharmacodyn. Ther. 1977, 229, 327–336.