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

Article abstract of DOI:10.1016/j.ejmech.2019.07.027

Extended studies in the 4-aryl-pyrido[1,2-c]pyrimidine group resulted in 27 new compounds (10.1-10.27), 5,6,7,8-tetrahydropyrido[1,2-c]pyrimidine derivatives. In vitro tests (RBA) were carried out for 10.1-10.27 compounds in order to determine their affinity to 5-HT_1A receptor and SERT protein. 10.1-10.3, 10.6, 10.7, 10.16 and 10.27 compounds had high binding ability to both molecular targets (5-HT_1A K_i = 8–87 nM; SERT K_i = 8–52 nM). For these compounds (10.1-10.3, 10.6, 10.7, 10.16, 10.27) further in vitro, in vivo and metabolic stability tests were performed. In vitro studies in the extended receptor profile (D₂, 5-HT_2A, 5-HT₆ and 5-HT₇) showed their selectivity towards 5-HT_1A receptor and SERT protein. In vivo tests revealed that compounds 10.7 and 10.16 had the properties of presynaptic antagonists of the 5-HT_1A receptor. The redesign of the 2H-pyrido[1,2-c]pyrimidine residue present in the terminal part towards 5,6,7,8-tetrahydropyrido[1,2-c]pyrimidine resulted in the improved metabolic stability and enhanced affinity to both molecular targets (5-HT_1A-R and SERT) compared to the precursors.

Full text of DOI:10.1016/j.ejmech.2019.07.027

Accepted Manuscript
Synthesis of new 5,6,7,8-tetrahydropyrido[1,2-c]pyrimidine derivatives with rigidized
tryptamine moiety as potential SSRI and 5-HT receptor ligands
1A
Grzegorz Ślifirski, Marek Król, Jerzy Kleps, Piotr Podsadni, Mariusz Belka, Tomasz
Bączek, Agata Siwek, Katarzyna Stachowicz, Bernadeta Szewczyk, Gabriel Nowak,
Andrzej Bojarski, Anna E. Kozioł, Jadwiga Turło, Franciszek Herold
PII:
S0223-5234(19)30649-X
DOI:
https://doi.org/10.1016/j.ejmech.2019.07.027
EJMECH 11524
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 27 May 2019
Revised Date: 8 July 2019
Accepted Date: 8 July 2019
Please cite this article as: G. Ślifirski, M. Król, J. Kleps, P. Podsadni, M. Belka, T. Bączek, A. Siwek,
K. Stachowicz, B. Szewczyk, G. Nowak, A. Bojarski, A.E. Kozioł, J. Turło, F. Herold, Synthesis of
new 5,6,7,8-tetrahydropyrido[1,2-c]pyrimidine derivatives with rigidized tryptamine moiety as potential
SSRI and 5-HT receptor ligands, European Journal of Medicinal Chemistry (2019), doi: https://
1A
doi.org/10.1016/j.ejmech.2019.07.027.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
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.
a
a*
a
a
b
Grzegorz Ślifirski , Marek Król , Jerzy Kleps , Piotr Podsadni , Mariusz Belka , Tomasz
b
c
c
c
c,d
Bączek , Agata Siwek , Katarzyna Stachowicz , Bernadeta Szewczyk , Gabriel Nowak
Andrzej Bojarski c, Anna E. Kozioł e, Jadwiga Turło a, Franciszek Herold a
,
a Department of Drug Technology and Pharmaceutical Biotechnology, Faculty of Pharmacy,
Medical University of Warsaw, 1, Banacha Street, 02-097 Warszawa, Poland
b
Department of Pharmaceutical Chemistry, Medical University of Gdańsk, 107, J. Hallera
Street, 80-416 Gdańsk, Poland
c Institute of Pharmacology, Polish Academy of Sciences, 12, Smętna Street, 31-343 Kraków,
Poland
d Chair of Pharmacobiology, Jagiellonian University Medical College, 9, Medyczna Street, 30-
688 Kraków, Poland
e Faculty of Chemistry, Maria Curie-Skłodowska University, 3, M. Curie-Skłodowskiej Sq., 20-
031 Lublin, Poland
*Corresponding author
E-mail address: marek.krol@wum.edu.pl
Keywords: Antidepressants, Pyrido[1,2-c]pyrimidines, Dual 5-HT1A/SERT activity
List of abbreviations: 5-HT1A-R, serotonin 5-HT1A receptor; 8-OH-DPAT, 8-hydroxy-2-(di-n-
propylamino)tetralin; DMSO, dimethyl sulfoxide; FST, forced swim test; Ki, inhibitory
constant; MAO-I, monoamine oxidase inhibitor; MDD, major depressive disorder; RBA,
radioligand binding assay; SAR, structure-activity relationship; SDARI, selective dopamine
reuptake inhibitor; SERT, serotonin transporter; SNRI, serotonin and norepinephrine reuptake
inhibitor; SSRI, selective serotonin reuptake inhibitor; TCA, tricyclic antidepressant; TEA,
triethylamine
Abstract
Extended studies in the 4-aryl-pyrido[1,2-c]pyrimidine group resulted in 27 new compounds
(10.1-10.27), 5,6,7,8-tetrahydropyrido[1,2-c]pyrimidine derivatives. In vitro tests (RBA) were carried
out for 10.1-10.27 compounds in order to determine their affinity to 5-HT1A receptor and SERT
protein. 10.1-10.3, 10.6, 10.7, 10.16 and 10.27 compounds had high binding ability to both molecular
targets (5-HT1A Ki = 8-87 nM; SERT Ki = 8-52 nM). For these compounds (10.1-10.3, 10.6, 10.7, 10.16,
10.27) further in vitro, in vivo and metabolic stability tests were performed. In vitro studies in the
extended receptor profile (D2, 5-HT2A, 5-HT6 and 5-HT7) showed their selectivity towards 5-HT1A
receptor and SERT protein. In vivo tests revealed that compounds 10.7 and 10.16 had the properties
of presynaptic antagonists of the 5-HT1A receptor. The redesign of the 2H-pyrido[1,2-c]pyrimidine
residue present in the terminal part towards 5,6,7,8-tetrahydropyrido[1,2-c]pyrimidine resulted in
the improved metabolic stability and enhanced affinity to both molecular targets (5-HT1A-R and SERT)
compared to the precursors.
1

ACCEPTED MANUSCRIPT
1. Introduction
There are currently several theories that attempt to explain the pathomechanism of Major
Depressive Disorder, a chronic, debilitating disease that impairs affective, cognitive and behavioral
processes, often resulting in suicide. The theory of monoamine deficiency in the central nervous
system seems to be currently the best justified one. Three classic groups of antidepressants used in
MDD pharmacotherapy, such as monoamine oxidase inhibitors (MAO-I), tricyclic antidepressants
(TCA) and selective serotonin reuptake inhibitors (SSRIs), increase the amount of serotonin in the
synaptic cleft [1]. However, although the maximum inhibition of serotonin reuptake in SSRI, the first-
line drug in MDD, occurs within hours of administration, it is necessary to wait for a therapeutic
response for several weeks due to latency. Latency results from long-lasting adaptation processes,
including primarily desensitization of 5-HT1A autoreceptors localized in the raphe nucleus, as well as
down-regulation of neurotransmitter receptors, changes in signal transmission, neurotropism,
mobilization and increase of neurogenesis in the hippocampus [2–4]. On the other hand, it has been
proved that sudden depletion of L-tryptophan, necessary for the synthesis of the amino acid
serotonin, may after a few hours induce a depressive episode in patients with remission following
SSRI [5,6]. These observations may suggest a model in which adaptive changes induced by long-term
treatment and maintenance of adequate serotonin availability are necessary for achieving lasting
antidepressant effect [7]. Due to numerous problems during SSRI treatment, such as delayed-onset
therapy, resistance to treatment or side effects (sexual dysfunctions, anxiety, gastro-intestinal
intolerances), there is a need to search for new generations of antidepressants that use, among
others, the mechanism of triple reuptake inhibition (SSRI/SNRI/SDARI), or combination of serotonin
reuptake inhibition with affinities to various 5-HT receptor subtypes [3,8,9]. A significant part of
recent studies shows that serotoninergic dysfunction, especially associated with the 5-HT1A
postsynaptic receptor, has an important role in MDD pathomechanism [10–18]. The combination of
SSRIs with both partial agonism and 5-HT1A receptor antagonism may result in an improvement in the
speed and effectiveness of antidepressant action in clinical studies [19–23]. Recently introduced into
pharmacotherapy, Vilazodone antidepressant confirms this approach, demonstrating high affinity
and selectivity to the 5-HT1A heteroreceptor and serotonin transporter, as well as fast efficacy with
limited side effects [24,25]. Also, vortioxetine, a new multi-target antidepressant that improves
cognitive parameters, is included in 5-HT1A receptor ligands and SSRIs [26,27]. The use of these
therapeutics in the current pharmacotherapy of depression justifies our team's choice of specific
molecular targets (5-HT1A and SERT) in the search for new drugs [17,18,24,27–29].
In previous works, we have described the pyrido[1,2-c]pyrimidine derivatives with the rest of
3-(piperidin-4-yl)-1H-indole in the pharmacophore part. A series of high-binding compounds were
obtained for both molecular targets (5-HT1A, SERT), also having an appropriate functional profile for
the 5-HT1A receptor - pre- and postsynaptic agonism [30–34]. Our recent research has focused on the
synthesis of 2H-pyrido[1,2-c]pyrimidine derivatives containing the fragment of 3-(piperidin-3-yl)-1H-
indole in the pharmacophore part, which is more similar in its structure to serotonin (Fig. 1)[35]. In
the inserted 3-(piperidin-3-yl)-1H-indole residue, the aminoethyl chain is conformationally
constrained, which may affect the affinity of the compounds to 5-HT1A receptor and SERT. In
addition, the conformational restriction may affect durability in the first pass phase, which is
confirmed by numerous references [36–41].
The aim of the work was the synthesis and biological evaluation of new 5,6,7,8-
tetrahydropyrido[1,2-c]pyrimidine derivatives, compounds based on the lead structure in which the
pharmacophore and terminal part had been redesigned (Fig. 1). The SAR analysis was conducted on
2

ACCEPTED MANUSCRIPT
R3 (-H, -F, -OCH3) substituents effect present on the indole ring in the pharmacophore part and on R1
and R2 (-Cl, -F, -CH3, -OCH3) benzene ring of the 5,6,7,8-tetrahydropyrido[1,2-c]pyrimidine terminal
part, as well as the effect of the hydrogenation degree of the pyrido[1,2-c]pyrimidine terminal part
on the affinity of the obtained derivatives to both molecular targets (5-HT1A, SERT). For selected
compounds, their functional profile to the 5-HT1A receptor (agonism/antagonism) and behavioural
profile as well as their metabolic stability were also tested.
3

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LEAD STRUCTURE
terminal
portion
pharmacophore
portion
R1, R2 = H, Cl, F, CH3, OCH3
R3 = H, F, OCH3
EXAMINED DERIVATIVES
R1, R2 = H, Cl, F, CH3, OCH3
R3 = H, F, OCH3
Fig. 1. Rational design of novel SSRI/5-HT1A dual activity compounds. Comparison of the leading
structure and structures examined in the project.
4

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2. Results and discussion
2.1. Chemistry
The final compounds 10.1-10.27 designed in the work were obtained through the multi-stage
synthesis shown in Scheme 1. The first series of synthons necessary for the synthesis were N-
bromobutyl-4-aryl-5,6,7,8-tetrahydropyrido[1,2-c]pyrimidine derivatives 6.1-6.9, and the next series
of synthons were 3-(piperidin-3-yl)-1H-indole derivatives 9.1-9.3.
6.1-6.9 synthons were obtained in accordance with the methods previously developed by us
[30–34], as synthons 9.1-9.3 [35]. The respective nitriles 1.1–1.9 were synthesized by C-arylation of
the appropriate arylacetonitriles with 2-bromopyridine in an aprotic polar solvent. Next, the nitriles
2.1–2.9 were hydrolyzed using a mixture of sulfuric acid and acetic acid. to produce amides 3.1–3.9.
Compounds 4.1–4.9 were formed by the intermolecular cyclization of 3.1–3.9 with diethyl carbonate.
The nitrogen in the imide group of compounds 4.1–4.9 was alkylated with 1,4-dibromobutane to give
bromobutyl derivatives 5.1–5.9, which were subsequently hydrogenated to form their 5,6,7,8-
tetrahydro analogues 6.1–6.9. 3-(piperidin-3-yl)-1H-indole derivatives 9.1-9.3 were obtained in a
two-step synthesis: 8.1–8.3 derivatives of 3-(1-phenyl-1,2,5,6-tetrahydropyridin-3-yl)-1H-indole were
synthesized based on the method of [35] in condensation of indole derivatives 7.1–7.3 with 1-benzyl-
3-piperidone hydrochloride. Hydrogenolysis with hydrogenation of compounds 8.1–8.3 was carried
out with Pd/C 10% in methanol at a pressure of 5 atm. The obtained bases were converted into
hydrochlorides 9.1–9.3. The final compounds 10.1–10.27 were synthesized by the condensation of
bromobutyl derivatives 6.1–6.9 with the appropriate 3-(piperidin-3-yl)-1H-indole derivatives 9.1-9.3.
5

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Scheme 1. The synthesis pathway of compounds 10.1-10.27. Reagents and conditions: (i.) 2-
bromopyridine, KOH, DMSO, Δ; (ii.) H2SO4, CH3COOH, Δ; (iii.) diethyl carbonate, EtONa, EtOH abs., Δ;
(iv.) 1,4-dibromobutane, K2CO3, acetone, Δ; (v.) H2, 10% Pd/C, CH3OH, p, Δ; (vi) 1-benzylpiperidin-3-
one, IPA, KOH, Δ, Ar; (vii.) 10% Pd/C, CH3OH, p, Δ; (viii) CH3OH/HCl (g); (ix.) acetonitrile, K2CO3, Δ.
Chemical structure and purity of all the newly synthesized derivatives 10.1-10.27 were
1
confirmed using H and 13C NMR spectroscopy, HRMS spectrometry as well as HPLC. Molecular
structure of the starting compound 9.3 has been determined by an X-ray structural analysis (Fig. 2).
6

ACCEPTED MANUSCRIPT
Fig. 2. Perspective view of compound 9.3 (two solid state conformers).
2.2 Biological evaluation
2.2.1 Radioligand binding assay for 5-HT1A receptor and SERT
In order to determine the in vitro binding of compounds 10.1-10.27 to the 5-HT1A receptor
and the SERT protein, studies using radioligand binding assay were carried out (Table 1).
Table 1
5-HT1A receptor and SERT binding affinities of 5,6,7,8-tetrahydro-pyrido[1,2-c]pyrimidine derivatives.
Ki [nM]
Compound
10.1
R1
-H
-Cl
R2
-H
-H
-H
-H
-H
-Cl
-F
-CH3
-OCH3
-H
-H
-H
-H
-H
-Cl
-F
R3
-H
-H
-H
-H
-H
-H
-H
-H
-H
-F
-F
-F
-F
-F
-F
-F
-F
-F
5-HT1A
27.0 ± 2.0 71.0 ± 6.3
8.0 ± 1.1 34.0 ± 3.7
15.0 ± 1.9 87.0 ± 5.4
62.0 ± 1.5 215.0 ± 18
25.0 ± 1.8 252.0 ± 35
30.0 ± 3.5 31.0 ± 2.0
SERT
10.2
10.3
10.4
10.5
10.6
10.7
10.8
10.9
10.10
10.11
10.12
10.13
10.14
10.15
10.16
10.17
10.18
10.19
10.20
-F
-CH3
-OCH3
-H
-H
-H
-H
-H
-Cl
-F
-CH3
-OCH3
-H
-H
-H
-H
-H
-Cl
13.0 ± 1.2
8.0 ± 0.9
50.0 ± 4.5 117.0 ± 10
52.0 ± 5.5 29.0 ± 1.0
105.0 ± 5.2 72.0 ± 8.0
47.0 ± 2.5 132.0 ± 15
28.0 ± 3.0 300.0 ± 28
142.0 ± 15 133.0 ± 8.3
40.0 ± 3.8 240.0 ± 18
259.0 ± 28 52.0 ± 2.0
31.0 ± 3.2 15.0 ± 1.0
233.0 ± 12 60.0 ± 6.5
42.0 ± 4.5 146.0 ± 7.5
-CH3
-OCH3
-H
-OCH3 102.0 ± 11 111.0 ± 6.0
-OCH3 50.0 ± 7.5 152.0 ± 16
-H
7

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10.21
10.22
10.23
10.24
10.25
-F
-CH3
-OCH3
-H
-H
-H
-H
-Cl
-F
-OCH3 236.0 ± 8.0 607.0 ± 34
-OCH3 66.0 ± 2.9 98.0 ± 4.0
-OCH3 239.0 ± 33
307 ± 36
-OCH3 23.0 ± 2.2 104.0 ± 7.0
-OCH3 76.0 ± 7.5 78.0 ± 5.1
-H
10.26
-H
-CH3 -OCH3 321.0 ± 10 602.0 ± 60
10.27
-H
-OCH3 -OCH3 52.0 ± 4.4 59.0 ± 3.2
Imipramine
Methiotepine
Serotonin
29.0 ± 2.0
4.8 ± 0.5
3.6 ± 0.4
In order to determine the in vitro binding of compounds 10.1-10.27 to the 5-HT1A receptor
and the SERT protein, studies using radioligand binding assay were carried out (Table 1).
The obtained results for compounds 10.1-10.27 affinity to the 5-HT1A receptor and SERT in
vitro allowed SAR analysis of the effect of ortho (R1) and para (R2) substituents on the benzene ring of
the 5,6,7,8-tetrahydropyrido[1,2-c]pyrimidine residue in the terminal part and analysis of the
presence of R3 (-H, -F, -OCH3) substituents in the indole residue of the pharmacophore part of the
compounds on their biological activity. The influence of the hydrogenation degree of the pyrido[1,2-
c]pyrimidine residue on 5-HT1A receptor and SERT binding in vitro with respect to the previously
described 2H-pyrido[1,2-c]pyrimidine derivatives was also analyzed [35].
After analyzing the obtained results, it was found that compound 10.7 (Ki = 8 nM) has the
highest affinity to the 5-HT1A receptor, high affinity was found for compounds 10.2, 10.6, 10.9, 10.16
with their Ki values ranging from 15.0 to 31.0 nM, and high to moderate affinity for compounds 10.1,
10.3, 10.10, 10.15, 10.17, 10.25 and 10.27 (Ki = 52.0-87.0 nM). A clear trend has been observed that
compounds with an unsubstituted indole ring have higher affinity to the 5-HT1A receptor than their
substituted analogues (R3 = -F, -OCH3), provided that the fluoro substituent confers a much higher
biological activity towards the 5-HT1A receptor than the methoxy substituent. On the other hand, the
analysis of the effect of substituents on the terminal part present in the benzene ring showed that
derivatives containing a para-substituted ring have much higher binding ability to 5-HT1A, than ortho-
substituted derivatives. Compounds having halogen substituents (R2 = -Cl, -F) in the para-position
were more biologically active than the unsubstituted analogues. Compounds containing the chloro
substituent in the terminal portion (10.2, 10.6, 10.11, 10.15, 10.20, 10.24), regardless of its position
on the benzene ring, generally have similar - high to moderate values of 5-HT1A receptor binding
ability. Compared to the 2H-pyrido[1,2-c]pyrimidine derivatives previously obtained [35], novel
hydrogenated 5,6,7,8-tetrahydropyrido[1,2-c]pyrimidine compounds have significantly higher affinity
to the 5-HT1A receptor.
While studying the effect of R1, R2 and R3 in both parts of the molecule on their affinity to the
SERT protein, it was found that compound 10.2 shows the highest affinity (Ki = 8 nM), series of
compounds 10.1, 10.3, 10.5-10.7, 10.11, 10.12, 10.14, 10.16, 10.18, 10.20 and 10.24 show high
affinity (Ki = 15.0-47.0 nM), and the compounds 10.4, 10.8, 10.9, 10.20, 10.22, 10.25, 10.27 show
high to moderate affinity (Ki = 50.0-76.0 nM) towards SERT. For this molecular target, it can be seen
that the highest affinities are achieved by unsubstituted (R3 = -H) derivatives in the 3-(piperidin-3-yl)-
1H-indole residue in the pharmacophore part. Presence of substituents R3 = -F in their analogues or -
OCH3 caused a three to four-fold decrease in activity. There is no significant difference in the degree
of affinity to the serotonin transporter protein between derivatives possessing ortho- and para-
substituted benzene rings at the 5,6,7,8-tetrahydropyrido[1,2-c]pyrimidine rest in the terminal part.
8

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In general, high activity is demonstrated by compounds having a substituent of R1 or R2 = -Cl, -F and -
OCH3 or an unsubstituted benzene ring. With regard to the 2H-pyrido[1,2-c]pyrimidine derivatives
previously obtained [35], their 5,6,7,8-tetrahydropyrido[1,2-c]pyrimidine analogues have on average
several times higher affinity to the SERT transporter.
NMR studies for compounds 10.1-10.27 show that the benzene ring in the ortho- substituted
compounds cannot rotate freely due to steric barriers, similar to the 2H-pyrido[1,2-c]pyrimidine
derivatives previously obtained [35] (the NMR spectra show temporary “magnetic stereoisomers”
manifested by double instead of single signals of R1 hydrogens). Such rotation is possible in para-
substituted compounds, more active than ortho- towards the 5-HT1A receptor and similarly active
towards the SERT. This seems to suggest the hypothesis that a more flexible terminal part can affect
the higher affinity to 5-HT1A and SERT [35]. This hypothesis would confirm the higher affinity of the
5,6,7,8-tetrahydropyrido[1,2-c]pyrimidine derivatives to both molecular targets than their 2H-
pyrido[1,2-c]pyrimidine precursors.
The study succeeded in obtaining the compounds 10.1-10.3, 10.6, 10.7, 10.16 and 10.27
showing high binding to both molecular targets (5-HT1A Ki = 8-87 nM; SERT Ki = 8-52 nM). The above-
mentioned derivatives were subjected to in vitro studies in the extended receptor profile to D2, 5-
HT2A, 5-HT6 and 5-HT7 (Table 2). The test results showed that these compounds are selective ligands
of the molecular targets - 5-HT1A receptor and SERT protein, due to the low affinity of the compounds
to other receptors examined in the profile.
Table 2
Binding affinity data on serotonin 5-HT2A, 5-HT6, 5-HT7, and dopamine D2 receptors of the investigated
5,6,7,8-tetrahydro-pyrido[1,2-c]pyrimidine derivatives.
Ki [nM]
Compound
10.1
R1
-H
-Cl
-F
-H
-H
-H
R2
-H
-H
-H
-Cl
-F
R3
-H
-H
-H
-H
-H
-F
5-HT2A
5-HT6
5-HT7
D2
725 ± 51
609 ± 34
325 ± 42
1146 ± 161 1092 ± 152 2349 ± 473
1030 ± 97 1361 ± 191 896 ± 138
1030 ± 78 2431 ± 337
10.2
10.3
10.6
10.7
10.16
10.27
> 10000
1488 ± 205 1320 ± 146 1384 ± 116 1651 ± 354
1277 ± 214 1651 ± 193 1712 ± 265 1977 ± 261
484 ± 72
-F
728 ± 89
452 ± 36
7 ± 1
n.d.
n.d.
n.d.
1112 ± 154 1047 ± 67
546 ± 85
n.d.
-H -OCH3 -OCH3 1191 ± 168
1802 ± 293
n.d.
Olanzapine
Mianserin
Clozapine
Haloperidol
Apomorphine
Chlorpromazine
4.6 ± 0.9
2.8 ± 0.5
n.d.
n.d.
n.d.
n.d.
18 ± 2
n.d.
n.d.
n.d.
n.d.
n.d.
4.5 ± 0.7
42 ± 6
1.8 ± 0.3
n.d.
n.d.
n.d.
n.d. – not determined
2.2.2 In vivo studies
To determine the profile of functional activity of selected ligands, behavioral tests for
selected compounds were performed.
Table 3
The effect of compounds: 10.1, 10.2, 10.3, 10.6, 10.7 and 10.16 on the body temperature in mice.
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Treatment
Dose (mg/kg)
Δt ± SEM (0 C)
30 min
60 min
90 min
120 min
Vehicle
10.1
-
5
20
0.2±0.1
0.2±0.2
0.0±0.1
0.3±0.1
-0.0±0.3
ns
-0.1±0.1
0.3±0.1
-0.2±0.2
ns
0.3±0.1
0.8±0.1a
-0.1±0.2
P=0.0028
-0.6±0.3b
P=0.0087
Vehicle
10.2
-
5
20
0.2±0.1
0.0±0.2
-0.8±0.2b
P=0.0021
0.0±0.1
0.3±0.3
0.2±0.2
ns
-0.1±0.1
0.9±0.3b
0.3±0.1
0.3±0.1
0.8±0.3
0.2±0.2
ns
P=0.0042
Vehicle
10.3
-
5
20
0.2±0.1
0.2±0.2
-0.8±0.3b
P=0.0024
0.0±0.1
0.3±0.1
0.0±0.3
P=0.0405
-0.1±0.1
0.7±0.2a
0.6±0.2a
P=0.0111
0.3±0.1
1.0±0.2a
0.7±0.2
P=0.0249
Vehicle
10.6
-
5
20
0.5±0.1
0.1±0.3
0.3±0.2
ns
0.1±0.2
0.3±0.2
0.7±0.1
ns
0.1±0.2
-0.1±0.3
0.5±0.2
ns
0.1±0.2
0.2±0.2
0.3±0.2
ns
Vehicle
10.7
-
5
20
0.5±0.1
0.3±0.1
0.1±0.2
ns
0.1±0.2
0.3±0.2
0.3±0.2
ns
0.1±0.2
0.3±0.2
-0.1±0.1
ns
0.1±0.2
0.5±0.2
0.1±0.2
ns
Vehicle
10.16
-
5
20
0.5±0.1
0.8±0.2
0.1±0.2
0.8±0.2a
0.1±0.2
0.1±0.2
0.6±0.1
0.1±0.2
ns
0.1±0.2
0.9±0.2b
0.0±0.3
-0.6±0.3c
P=0.0004
P=0.0179
P=0.0139
The investigated compounds were administered 30 min before the test, ap<0.05 vs vehicle, bp<0.01
vs vehicle, cp<0.001 vs vehicle.
To assess functional 5-HT1A receptor activity of tested compounds: 10.1, 10.2, 10.3, 10.6, 10.7
and 10.16 standard model of hypothermia in mice was used (Table 3). It is known that 8-OH-DPAT as
a 5-HT1A receptor agonist can induce hypothermia in mice, through 5-HT1A somatodendritic receptors
[42,43]. Moreover, this effect can be abolished by WAY100635 [44], 5-HT1A receptor antagonist.
Compounds: 10.7 and 10.16 decreased the hypothermia induced by 8-OH-DPAT (5 mg/kg) in mice
(Table 4) demonstrating its presynaptic 5-HT1A receptor antagonist activity. In addition, it was
observed that compound 10.16 in combination with 8-OH-DPAT caused a very strong forepaw
treading effect, lasting for about 30 minutes. The compound itself did not have such an effect.
Compound 10.7 caused a barely noticeable forepaw treading effect.
Table 4
The effect of compounds: 10.1, 10.3, 10.6, 10.7 and 10.16 on the hypothermia induced by 8-OH-DPAT
(5 mg/kg).
10

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Treatment and dose (mg/kg)
Δt ± SEM (0C)
15 min
30 min
45 min
60 min
Vehicle + vehicle
Vehicle + 8-OH-DPAT
10.1 (20) + 8-OH-DPAT
0.3±0.1
-0.8±0.4
-1.2±0.4a
P=0.0149
0.5±0.1
-0.8±0.4a
-1.1±0.3b
P=0.0031
0.7±0.3
-1.1±0.3b
-0.9±0.3b
P=0.0009
0.6±0.2
-0.6±0.5
0.7±0.4d
P=0.0335
Vehicle + vehicle
Vehicle + 8-OH-DPAT
10.3 (20) + 8-OH-DPAT
-0.2±0.1
-1.4±0.2b
-1.6±0.3c
P=0.0004
0.2±0.2
-2.1±0.2c
-1.8±0.3c
P<0.0001
-0.0±0.2
-2.7±0.2c
-1.9±0.3c
P<0.0001
0.2±0.2
-2.8±0.3c
-1.8±0.3c
P<0.0001
Vehicle + vehicle
Vehicle + 8-OH-DPAT
10.6 (20) + 8-OH-DPAT
0.6±0.1
-1.1±0.2c
-1.1±0.2c
P<0.0001
0.4±0.1
-1.3±0.2c
-1.1±0.3c
P<0.0001
0.5±0.2
-1.0±0.4b
-1.2±0.3b
P=0.0013
0.7±0.2
-1.0±0.3c
-1.2±0.3c
P=0.0002
Vehicle + vehicle
Vehicle + 8-OH-DPAT
10.7 (20) + 8-OH-DPAT
0.6±0.1
-1.1±0.2c
-0.2±0.2d
P<0.0001
0.4±0.1
-1.3±0.2c
-0.2±0.2
P<0.0001
0.5±0.2
-1.0±0.4b
-0.3±0.3
P=0.0048
0.7±0.2
-1.0±0.3c
-0.9±0.2b
P=0.0002
Vehicle + vehicle
Vehicle + 8-OH-DPAT
10.16 (20) + 8-OH-DPAT
0.6±0.1
-1.1±0.2c
-0.1±0.3
P=0.0001
0.4±0.1
-1.3±0.2c
0.2±0.4
0.5±0.2
-1.0±0.4b
-0.3±0.4
P=0.0131
0.7±0.2
-1.0±0.3b
-0.4±0.4a
P=0.0019
P=0.0004
Compounds: 10.1, 10.3, 10.6, 10.7 and 10.16 were administered 45 min before to 8-OH-DPAT,
ap<0.05 vs vehicle, bp<0.01 vs vehicle, cp<0.001 vs vehicle, dp<0.05 vs compound group, ep<0.01 vs
compound group, fp<0.001 vs compound group.
The effect of administration of 10.7 on the total duration of the immobility time in mice is
shown in Fig. 3. Compound 10.7 injected at the doses of 5 and 20 mg/kg had no effect on the
immobility time in the FST in mice [F(2,21)=0.5271, p=0.5979], so we can conclude lack of
postsynaptic 5-HT1A receptor activity [42].
11

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Fig. 3. Effect of compound 10.7 on forced swimming test in CD-1 mice
2.2.3 Metabolic stability evaluation
The seven selected compounds were tested in in vitro metabolic stability assay. We aimed to
assess their susceptibility to undergo first-phase metabolism, thus human liver microsomes along
with NADPH were used as in vitro enzymatic model. Biotransformation reaction was followed by
liquid chromatography-mass spectrometry technique. Results derived from triplicate incubations,
expressed as in vitro metabolic half-life (t1/2), are gathered in Fig. 4. Our experimental results can be
also interpreted in the view of some in silico approaches. We decided to use Human Liver
Microsome-based model for CYP-mediated oxidations provided by Xenosite online tool to reveal
possible sites of first-phase metabolism [45].
Fig. 4. A: Summary of metabolic stability test. Bars shows average in vitro metabolic half-life (n=3),
whiskers represent standard deviation from independent enzymatic experiments.
B: Graphical output from Xenosite model for HLM. Intensity of green color indicates atoms more
vulnerable for metabolism.
All tested derivatives present moderate stability with the range 6.2-12.0 min. (Fig. 4. A) Previously,
we tested buspirone as a reference compound for arylpiperazine derivatives in the same metabolic
stability test with result around 6.6 min [46]. This study shows minor improvement in comparison
with reference compound, however our group have also proved that derivatives bearing 1,2,3,6-
tetrahydropyridine moiety presented much higher stability [33]. All of abovementioned observation
along with results achieved in silico using Xenosite model suggests that main part responsible for
metabolic liability is alkyl chain that joins piperazine (or its analogs: 1,2,3,6-tetrahydropyridine or
piperidine in the current study) with non-pharmacophore part of the molecule. Xenosite shows that
carbon atom next to piperidine nitrogen is the most liable part of the molecule (Fig. 4. B). Probably
this part of compound undergoes N-dealkylation which is common reaction catalyzed by CYP
isoforms [47] and was also reported for arylpiperazine derivatives [48]. Other constituents, i.e. –F, -
Cl and –OCH3, seem to have minor influence on metabolic stability. On the other hand, the redesign
of the structure of the 2H-pyrido[1,2-c]pyrimidine to 5,6,7,8-tetrahydropyrido[1,2-c]pyrimidine
performed in the work by means of hydrogenation made it possible to obtain twice as long-lasting
metabolically derived derivatives in relation to their precursors [35].
3. Materials and methods
3.1. General remarks
12

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Melting points were determined on an Electrothermal IA9200 apparatus with open capillary
tubes and are uncorrected. Elemental analyses were performed on a Elementar Vario EL III analyzer
1
and were within 0.4% of the theoretical values. H and 13C NMR spectra were obtained on Varian
Unity Plus 500 MHz instrument (chemical shifts are reported in δ units). Coupling constants (J) are in
hertz (Hz); the internal reference was TMS. The following abbreviations are used to describe peak
patterns when appropriate: s (singlet), bs (broad singlet), d (doublet), dd (double doublet), t (triplet),
td (triple doublet), ps (pseudotriplet), 4d (quartet of doublets), m (multiplet), E (equatorial), A (axial).
For the two-dimensional experiments, the pulse sequences, acquisition and processing parameters
were taken from the standard Varian software library. Numbering system, which was used in NMR
spectra interpretation is shown in Fig. 5. ESI-HRMS spectra were obtained on a Thermo Q-Exactive
instrument. Flash column chromatography was carried out on Merck silica gel 60 (230–400 mesh
ASTM) using the solvent methylene chloride/methanol/triethylamine (9:1:0.2, 8:2:0.1, v/v) and ethyl
acetate/hexane (9:1, v/v). Thin layer chromatography was run on Merck silica gel 60 F254 plates
using a mobile phase of methylene chloride/methanol/triethylamine (9:1:0.2, 8:2:0.1, v/v).
Compound purity was determined by high performance liquid chromatography (HPLC), and all final
test compounds were of >95% purity. The HPLC methods used a Phenomenex Gemini column C18 (3
μm, 150 mm x 2.00 mm); with a mobile phase of methanol/water/diisopropylamine (80:20:0.008,
v/v; detection at 280 nm; flow: 0.2 mL/min; temp 30 ⁰C).
3.2. Synthesis of compounds
3.2.1. Preparation of 2-(4-bromobutyl)-4-aryl-5,6,7,8-tetrahydro-pyrido[1,2-c]pyrimidine-1,3-diones
(6.1–6.9)
The starting compounds 2.1–2.9, 3.1–3.9, 4.1–4.9, 5.1–5.9 and 6.1–6.9 were obtained
according to procedures described in the literature [30,31,33–35,49].
3.2.2. Preparation of 3-piperidin-3-yl-1H-indole hydrochlorides (9.1–9.3)
The starting compounds 9.1-9.3 were obtained according to previously described procedures
[35]. Molecular structure of 9.3 was determined using a single crystal X-ray analysis.
Crystal data for 9.3: crystal system monoclinic, space group P 21/n, unit cell dimensions a = 17.786(3)
Å, b = 8.427(2) Å, c = 19.565(3) Å, β = 106.92(3)°, V = 2805.5(10) Å3, Z = 8, D(calc) = 1.263 g/cm3,
absorption coeff. 2.327 mm-1, F(000) = 1136. Reflections collected / independent 10248/ 5513; index
ranges -22 ≤ h ≤ 20, -9 ≤ k ≤ 10, -21 ≤ l ≤ 24. Goodness-of-fit on F2 = 1.091; final R indices [I>2s(I)] R1 =
0.0523, wR2 = 0.1383; R indices (all data) R1 = 0.0656, wR2 = 0.1675. ∆ρ max/min 0.42 / -0.38 e/ Å3.
The experimental details and final atomic parameters have been deposited with the Cambridge
Crystallographic Data Centre as supplementary material (CCDC ID: 1914576). Copies of the data can
be obtained free of charge on request via www.ccdc.cam.ac.uk/structures/.
3.2.3. General procedure for the synthesis of 2-[4-[3-(1H-indol-3-yl)-1-piperidyl]butyl]-4-phenyl-
5,6,7,8-tetrahydropyrido[1,2-c]pyrimidine-1,3-diones (10.1–10.27)
Compounds 6.1–6.9 (0.75 mmol), 9.1–9.3 (0.75 mmol), K2CO3 (1.65 mol) and 25 mL of
acetonitrile were stirred at 45 ⁰C for 4–5 h. Reaction time was monitored using TLC. After cooling, the
mixture was filtered, and the filtrate evaporated to dryness. The crude residue was purified by flash
chromatography, using CH2Cl2/MeOH/TEA (9:1:0.2, 8:2:0.1, v/v) mixture. Proper fractions were
identified by TLC and evaporated to dryness giving analytically pure compounds 10.1–10.27.
13

ACCEPTED MANUSCRIPT
R , R = -H, -Cl, -F, -CH , -OCH
4'
1
2
3
3
5'
6'
3'
2'
R = -H, -F, -OCH
3
3
1'
5
8
4
3
4a
6
7
e
d
2x
4x
2
c
1
1x
3x
a
b
4"
3"a
5"
6"
3"
2"
'
1"
7"a
7"
Fig. 5. Numbering system for NMR spectra interpretation of compounds 10.1–10.27
3.2.3.1. 2-[4-[3-(1H-indol-3-yl)-1-piperidyl]butyl]-4-phenyl-5,6,7,8-tetrahydropyrido[1,2-c]pyrimidine-
1,3-dione (10.1).
The title compound was isolated as a white powder. Yield: 88.1 %, m.p. 104.2-106.1 °C. HPLC tR = 5.16
min, 99.8% purity.
1H NMR (500 MHz, CDCl3): δ 8.31 (N1”H, [1H], bs), 7.65 (C4”H, [1H], d, 3J=7.5), 7.38 (C3’H,C5’H, [2H],
t, 3J=8.0), 7.35 – 7.30 (C4’H,C7”H, [2H], m), 7.20 – 7.13 (C2’H,C6’H,C6”H, [3H], m), 7.08 (C5”H, [1H],
td, 3J=8.0, 4J=0.5), 6.95 (C2”H, [1H], bs), 4.03 (C1xH2, [2H], t, 3J=7.0), 3.91 (C8H2, [2H], t, 3J=6.0), 3.23
(CaH(E),CbH, [2H], m), 3.03 (CeH(E), [1H], bs), 2.50 (C4xH2,C5H2, [4H], bs + t, 3J=7.0), 2.19 – 2.02
(CaH(A),CeH(A),CcH(E), [3H], m), 1.89 (C7H2, [2H], q, 3J=6.5), 1.78 (CdH2, [2H], m), 1.75 – 1.60
(C2xH2,C3xH2,C6H2, [6H], m), 1.50 (CcH(A), [1H], kd, 2J=3JA-A=12.5, 3JA-E=4.5).
13C NMR (125 MHz, CDCl3): δ 162.0 (C3, s), 151.7 (C1, s), 149.8 (C4a, s), 136.2 (C7”a, s), 133.4 (C1’, s),
130.7 (C2’,C6’, s), 128.5 (C3’,C5’, s), 127.7 (C4’, s), 126.6 (C3”a, s), 123.5 (C3”, s), 121.8 (C6”, s), 120.4
(C2”, s), 119.1 (C5”, s), 119.1 (C4”, s), 112.4 (C4, s), 111.2 (C7”, s), 60.3 (Ca, s), 58.4 (C4x, s), 53.8 (Ce,
s), 42.7 (C8, s), 41.2 (C1x, s), 33.4 (Cb, s), 30.9 (Cc, s), 26.7 (C5, s), 25.6 (C2x, s), 25.1 (C3x, s), 23.6 (Cd,
s), 21.7 (C7, s), 18.5 (C6, s).
ESI-HRMS m/z: Calcd for C31H37N4O2 [M+H]+ 497.29165. Found: 497.29139.
3.2.3.2. 4-(2-chlorophenyl)-2-[4-[3-(1H-indol-3-yl)-1-piperidyl]butyl]-5,6,7,8-tetrahydropyrido[1,2-
c]pyrimidine-1,3-dione (10.2).
The title compound was isolated as a white powder. Yield: 89.6 %, m.p. 125.3-127.5 °C. HPLC tR = 5.42
min, 99.9% purity.
1H NMR (500 MHz, CDCl3): δ 8.42 (N1”H, [1H], bs), 7.65 (C4”H, [1H], d, 3J=7.5), 7.46 – 7.40 (C3’H, [1H],
m), 7.33 (C7”H, [1H], dk, 3J=8.0, 4J=1.5, 5J=0.5), 7.31 – 7.24 (C5’H,C6’H, [2H], m), 7.20 – 7.13
(C4’H,C6”H, [2H], m), 7.07 (C5”H, [1H], m), 6.94 (C2”H, [1H], pd), 4.03 (C1xH2, [2H], t, 3J=7.5), 3.94
(C8H(2), [1H], tt), 3.87 (C8H(1), [1H], tt), 3.27 (CaH(E), CbH(E), [2H], m), 3.08 (CeH(E), [1H], bs), 2.55
(C4xH2, [2H], bs), 2.37 (C5H2, [2H], m), 2.15 (CaH(A),CeH(A), [2H], bs), 2.04 (CcH(E), [1H], pd), 1.89
(C7H2,CdH(E), [3H], m), 1.80 – 1.60 (CdH(A),C6H2,C2xH2,C3xH2, [7H], m), 1.49 (CcH(A), [1H], m).
14

ACCEPTED MANUSCRIPT
13C NMR (125 MHz, CDCl3): δ 161.3 (2xC3, 2s), 151.7 (2xC1, 2s), 150.6 (2xC4a, 2s), 136.2 (2xC7’a, 2s),
135.1 and 135.0 (2xC2’, 2s), 132.5 (2xC1’, 2s), 132.4 (C6’, s), 129.7 (2xC3’, 2s), 129.5 (2xC4’, 2s), 127.2
and 127.1 (2xC5’, 2s), 126.5 (2xC3”a, 2s), 121.8 (C6”, s), 120.4 (C2”, s), 119.2 (C5”, s), 119.1 (C4”, s),
111.3 (C7”, s), 110.0 (2xC4, 2s), 60.0 (Ca, s), 58.2 (C4x, s), 53.7 (Ce, s), 43.0 (2xC8, 2s), 41.0 (C1x, s),
33.2 (Cb, s), 30.7 (Cc, s), 26.4 (C5, s), 25.5 (Cd, s), 24.9 (C2x, s), 23.2 (C3x, s), 21.8 (C7, s), 18.4 (C6, s).
ESI-HRMS m/z: Calcd for C31H36ClN4O2 [M+H]+ 531.25268. Found: 531.25272.
3.2.3.3. 4-(2-fluorophenyl)-2-[4-[3-(1H-indol-3-yl)-1-piperidyl]butyl]-5,6,7,8-tetrahydropyrido[1,2-
c]pyrimidine-1,3-dione (10.3).
The title compound was isolated as a white powder. Yield: 68.4 %, m.p. 120.8-122.0 °C. HPLC tR = 4.94
min, 99.7% purity.
1H NMR (500 MHz, CDCl3): δ 8.46 (N1”H, [1H], bs), 7.66 (C4”H, [1H], d, 3J=8.0), 7.37 – 7.29 (C4’H,C7”H,
[2H], m), 7.23 – 7.14 (C5’H,C6’H,C6”H, [3H], m), 7.14 – 7.04 (C3’H,C5”H, [2H], m), 6.95 (C2”H, [1H], d,
3J=4.0), 4.02 (C1xH2, [2H], t, 3J=6.5), 3.91 (C8H2, [2H], m(13 linii) *), 3.45 – 3.21 (CaH(E),CbH(E),
[2H],m), 3.13 (CeH(E), [1H], bs), 2.60 (C4xH2, [2H], bs), 2.47 (C5H2, [2H], m *), 2.30 – 2.01
(CaH(A),CeH(A),CcH(E), [3H], m), 1.92 (C7H2, [2H], m), 1.85 – 1.60 (CdH2,C6H2C2xH2,C3xH2, [8H], m),
1.50 (CcH(A), [1H], m).
13C NMR (125 MHz, CDCl3): δ 161.5 and 161.4 (2xC3, 2s), 160.4 (C2’, d, 1J=246.0), 151.6 (2xC1, 2s),
151.1 (2xC4a, 2s), 136.2 (C7”a, s), 132.9 (2xC6’,2d, 3J=5.0), 130.0 (C4’, d, 3J=8.3), 126.4 (C3”a, s),
124.2 (2xC5’, 2d, 4J=3.6), 123.6 (C3”, s), 121.9 (C6”, s), 120.8 (C1’, d, 2J=16.2), 120.8 (C2”, s), 119.2
(C5”, s), 119.1 (C4”, s), 115.8 and 115.7 (2xC3’, 2d, 2J=22.3), 111.3 (C7”, s), 106.0 (2xC4, 2s), 59.7 (Ca,
s), 58.0 (C4x, s), 53.5 (Ce, s), 42.9 (2xC8, 2s), 41.0 (C1x, s), 33.0 (Cb, s), 30.5 (Cc, s), 26.5 (C5, s), 25.4
(Cd, s), 24.6 (C2x, s), 22.9 (C3x, s), 21.7 (C7, s), 18.3 (C6, s).
ESI-HRMS m/z: Calcd for C31H36FN4O2 [M+H]+ 515.28223. Found: 515.28214.
3.2.3.4. 2-[4-[3-(1H-indol-3-yl)-1-piperidyl]butyl]-4-(o-tolyl)-5,6,7,8-tetrahydropyrido[1,2-c]pyrimidine-
1,3-dione (10.4).
The title compound was isolated as a white powder. Yield: 68.7 %, m.p. 114.1-115.9 °C. HPLC tR = 5.49
min, 98.3% purity.
1H NMR (500 MHz, CDCl3): δ 8.33 (N1”H, [1H], bs), 7.65 (C4”H, [1H], d, 3J=7.5), 7.32 (C7”H, [1H], dt,
3J=8.0, 4J=5J=1.0), 7.27 – 7.17 (C4”- 6”H, [3H], m), 7.15 (C6”H, [1H], m), 7.07 (C3’H, [1H], m), 7.02
(C5”H, [1H], m), 6.94 (C2”H, [1H], bs), 4.03 (C1xH2, [2H], t, 3J=7.0), 3.90 (C8H2, [2H], m(13 lines)), 3.23
(CaH(E),CbH(E). [2H], m), 3.03 (CeH(E), [1H], bs), 2.50 (C4xH2, [2H], bs), 2.42 (C5H(2), [1H], m, 2J=17.5),
2.24 (C5H(1), [1H], m, 2J=17.5), 2.12 and 2.08 (2xCH3, [2x3H], 2s *), 2.15 – 2.00
(CaH(A),CeH(A),CcH(E), [3H], m), 1.89 (C7H2, [2H], m), 1.81 – 1.63 (CdH2,C6H2,C2xH2,C3xH2, [8H], m),
1.49 (Cc(A), [1H], m).
13C NMR (125 MHz, CDCl3): δ 161.5 (C3, 2s), 151.8 (C1, 2s), 149.6 (C4a, s), 137.7 and 137.6 (C2’, 2s),
136,219 (C7”a, s), 132.9 (C1’, s), 130.7 (C6’, 2s), 130.2 (C3’, s), 128.2 (C4’, s), 126.6 (C3”a, s), 126.2
(C5’, 2s), 122.9 (C3”, s), 121.8 (C6”, s), 120.4 (C2”, s), 119.1 (C4”,C5”, s), 111.7 (C4, s), 111.2 (C7”, s),
15

ACCEPTED MANUSCRIPT
60.3 (Ca, s), 58.4 (C4x, s), 53.8 (Ce, s), 42.9 (C8, s), 41.1 (C1x, s), 33.4 (Cb, s), 30.9 (Cc, s), 26.5 (C5, s),
25.6 (Cd, s), 25.2 (C2x, s), 23.6 (C3x, s), 21.8 (C7, s), 19.7 and 19.6 (2xCH3, 2s), 18.6 (C6, s).
ESI-HRMS m/z: Calcd for C32H39N4O2 [M+H]+ 511.30730. Found: 511.30742.
3.2.3.5. 2-[4-[3-(1H-indol-3-yl)-1-piperidyl]butyl]-4-(2-methoxyphenyl)-5,6,7,8-tetrahydropyrido[1,2-
c]pyrimidine-1,3-dione (10.5).
The title compound was isolated as a white powder. Yield: 70.4 %, m.p. 110.3-112.2°C. HPLC tR = 4.89
min, 99.7% purity.
1H NMR (500 MHz, CDCl3): δ 8.36 (N1”H, [1H], bs), 7.65 (C4”H, [1H], dt, 3J=7.5, 4J=2.0, 5J=0.5), 7.35 –
7.29 (C4’H,C7”H, [2H], m), 7.15 (C6”H, [1H], m), 7.11 – 7.05 (C5”H,C6’H, [2H], m), 7.01 – 6.88
(C2”H,C3’H,C5’H, [3H], m), 4.02 (C1xH2, [2H], m), 3.89 (C8H2, [2H], m(18 lines)), 3.74 and 3.70
(2xOCH3, [2x3H], 2s), 3.23 (CaH(E),CbH(E), [2H], m), 3.04 (CeH(E), [1H], bs), 2.51 (C4xH2, [2H], bs), 2.41
(C5H2, [2H], t, 3J=7.0), 2.20 – 2.00 (CaH(A),CeH(A),CcH(E), [3H], m), 1.89 (C7H2, [2H], m), 1.82 – 1.60
(CdH2,C6H2,C2xH2,C3xH2, [8H], m), 1.49 (CcH(A), [1H],m).
13C NMR (125 MHz, CDCl3): δ 161.8 (C3, s), 157.3 (C2’, s), 151.8 (C1, s), 150.2 (C4a, s), 136.2 (C7”a, s),
132.3 (2xC6’, 2s), 129.5 (C4’, s), 126.5 (C3”a, s), 123.0 (C3”, s), 122.1 (C1’, s), 121.8 (C6”, s), 120.7 (C5’,
s), 120.5 (C2”, s), 119.1 (C4”,C5’, s), 111.2 (C7”, s), 111.1 (C3’, s), 108.5 (C4, s), 60.3 (Ca, s), 58.4 (C4x,
s), 55.5 and 55.4 (2xOCH3, 2s), 53.8 (Ce, s), 42.9 (C8, s), 41.1 (C1x, s), 33.3 (Cb, s), 30.9 (Cc, s), 26.3 (C5,
s), 25.6 (Cd, s), 25.1 (C2x, s), 23.5 (C3x, s), 21.8 (C7, s), 18.5 (C6, s).
ESI-HRMS m/z: Calcd for C32H39N4O3 [M+H]+ 527.30222. Found: 527.30210.
3.2.3.6. 4-(4-chlorophenyl)-2-[4-[3-(1H-indol-3-yl)-1-piperidyl]butyl]-5,6,7,8-tetrahydropyrido[1,2-
c]pyrimidine-1,3-dione (10.6).
The title compound was isolated as a white powder. Yield: 87.8 %, m.p. 232.6-234.5 °C. HPLC tR = 6.13
min, 97.9% purity.
1H NMR (500 MHz, CDCl3): δ 8.18 (N1”H, [1H], bs), 7.66 (C4”H, [1H], d, 3J=8.0), 7.38 – 7.30
(C2’H,C6’H,C7”H, [3H], m), 7.17 (C6”H, [1H], m, 3J1=8.0, 3J2=7.0, 4J=1.0), 7.14 – 7.07 (C3’H,C5’H,C5”H,
[3H], m), 6.97 (C2”H, [1H], d, 3J=2.0), 4.02 (C1xH2, [2H], t, 3J=7.0), 3.91 (C8H, [1H], t, 3J=6.5), 3.27
(CaH(E),CbH(E), [2H], m), 3.08 (CeH(E), [1H], bs), 2.53 (C4xH2, [2H], bs), 2.49 (C5H2, [2H], t, 3J=6.5),
2.25 – 2.01 (CaH(A),CeH(A),CcH(E), [3H], m), 1.90 (C7H2, [2H], q, 3J=6.5), 1.82 (CdH2, [2H], m), 1.75 –
1.60 (C6H2,C2xH2,C3xH2, [6H], m), 1.53 (CcH(A), [1H], m).
13C NMR (125 MHz, CDCl3): δ 160.9 (C3, s), 150.9 (C1,C4a, s), 136.2 (C7”a, s), 132.8 (C3’,C5’, s), 132.7
(C4’, s), 132.0 (C1’, s), 128.1 (C2’,C6’, s), 126.3 (C3”a, s), 121.1 (C2”, s), 120.1 (C6”, s), 118.5 (C5”, s),
118.2 (C4”, s), 111.4 (C7”, s), 109.5 (C4, s), 60.0 (Ca, s), 58.0 (C4x, s), 53.0 (Ce, s), 42.3 (C8, s), 40.7
(C1x, s), 33.5 (Cb, s), 31.2 (Cc, s), 26.2 (Cd, s), 26.2 (C5, s), 25.2 (C2x, s), 26.0 (C3x, s), 21.0 (C7, s), 17.8
(C6, s).
ESI-HRMS m/z: Calcd for C31H36ClN4O2 [M+H]+ 531.25268. Found: 531.25287.
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3.2.3.7. 4-(4-fluorophenyl)-2-[4-[3-(1H-indol-3-yl)-1-piperidyl]butyl]-5,6,7,8-tetrahydropyrido[1,2-
c]pyrimidine-1,3-dione (10.7).
The title compound was isolated as a white powder. Yield: 92.8 %, m.p. 106.8-108.4 °C. HPLC tR = 5.05
min, 99.9% purity.
1H NMR (500 MHz, CDCl3): δ 8.29 (N1”H, [1H], bs), 7.65 (C4”H, [1H], d, 3J=8.0), 7.33 (C7”H. [1H], dt,
3J=8.0, 4J=5J=1.0), 7.18 – 7.12 (C2’H,C6’H,C6”H, [3H], m), 7.10 – 7.03 (C3’H,C5’H,C5”H, [3H], m), 6.96
(C2”H, [1H], d, 3J=2.0), 4.02 (C1xH2, [2H], t, 3J=7.5), 3.91 (C8H2, [2H], t, 3J=6.5), 3.23 (CaH(E),CbH(E),
[2H], m), 3.02 (CeH(E), [1H], pd), 2.48 (C4xH2,C5H2,[4H], bs+t, 3J=6.5), 2.15 – 2.02
(CaH(A),CeH(A),CcH(E), [3H], m), 1.90 (C7H2, [2H], q, 3J=6.5), 1.80 (CdH2, [2H], m), 1.75 – 1.60
(C2xH2,C3xH2,C6H2, [6H], m), 1.50 (CcH(A), [1H], m).
13C NMR (125 MHz, CDCl3): δ 162.2 (C4’,d, 1J=246.9 *), 162.0 (C3, s), 151.6 (C1, s), 149.9 (C4a, s),
136.2 (C7”a, s), 132.5 (C2’,C6’, d, 3J=8.0), 129.2 (C1’, d, 4J=3.5), 126.7 (C3”a, s), 121.9 (C6”, s), 120.3
(C2”, s), 119.5 (C3”, s), 119.2 (C4”,C5”, s), 115.4 (C3’,C5’, d, 2J=21.5), 111.4 (C4, s), 111.2 (C7”, s), 60.6
(Ca, s), 58.6 (C4x, s), 54.0 (Ce, s), 42.7 (C8, s), 41.4 (C1x, s), 33.7 (Cb, s), 31.2 (Cc, s), 26.7 (C5, s), 25.7
(Cd, s), 25.4 (C2x, s), 24.0 (C3x, s), 21.7 (C7, s), 18.5 (C6, s).
ESI-HRMS m/z: Calcd for C31H36FN4O2 [M+H]+ 515.28223. Found: 515.28199.
3.2.3.8. 2-[4-[3-(1H-indol-3-yl)-1-piperidyl]butyl]-4-(p-tolyl)-5,6,7,8-tetrahydropyrido[1,2-c]pyrimidine-
1,3-dione (10.8).
The title compound was isolated as a white powder. Yield: 94.5 %, m.p. 227.2-229.0 °C. HPLC tR = 6.08
min, 95.7% purity.
1H NMR (500 MHz, CDCl3): δ 8.26 (N1”H, [1H], bs), 7.66 (C4”H, [1H], d, 3J=8.0), 7.33 (C7”H, [1H], dt,
3J=8.0, 4J=5J=0.5), 7.18 (C2’H,C6’H, [2H], d, 3J=8.0), 7.16 (C6”H, [1H], m, 3J1=8.0, 3J2=7.0, 4J=1.0), 7.08
(C5”H, [1H], m, 3J1=7.5, 3J2=7.0, 4J=1.0), 7.06 (C3’H,C5’H, [2H], d, 3J=8.0), 6.96 (C2”H, [1H], bs), 4.02
(C1xH2, [2H], t, 3J=6.5), 3.90 (C8H2, [2H], t, 3J=6.5), 3.24 (CaH(E),CbH(E), [2H], bs), 3.05 (CeH(E), [1H],
bs), 2.52 (C5H2,C4xH2, [4H], t+bs), 2.36 (CH3, [3H], s), 2.24 – 2.00 (Ca(A),Ce(A),Cc(E), [3H], m), 1.89
(C7H2, [2H], q, 3J=7.0), 1.79 (CdH2, [2H], m), 1.67 (C6H2, C2xH2,C3xH2, [6H], m), 1.51 (CcH(A), [1H], m).
13C NMR (125 MHz, CDCl3): δ 162.1 (C3,s), 151.7 (C1,s), 149.7 (C4a, s), 137.4 (C4’, s), 136.2 (C7”a, s),
130.5 (C3’,C5’, s), 130.3 (C1’, s), 129.2 (C2’,C6’, s), 126.5 (C3”a, s), 123.4 (C3’, s), 121.9 (C6”, s), 120.5
(C2”, s), 119.2 (C5’, s), 119.2 (C4”, s), 112.3 (C4, s), 111.2 (C7”, s), 60.1 (Ca, s), 58.5 (C4x, s), 53.8 (Ce,
s), 42.7 (C8, s), 41.1 (C1x, s), 33.3 (Cb, s), 30.8 (Cc, s), 26.7 (C5, s), 25.5 (Cd, s), 24.9 (C2x, s), 23.4 (C3x,
s), 21.7 (C7, s), 21.3 (CH3, s), 18.6 (C6, s).
ESI-HRMS m/z: Calcd for C32H39N4O2 [M+H]+ 511.30730. Found: 511.30735.
3.2.3.9. 2-[4-[3-(1H-indol-3-yl)-1-piperidyl]butyl]-4-(4-methoxyphenyl)-5,6,7,8-tetrahydropyrido[1,2-
c]pyrimidine-1,3-dione (10.9).
The title compound was isolated as a white powder. Yield: 89.9 %, m.p. 113.2-114.8 °C. HPLC tR = 5.07
min, 99.8% purity.
1H NMR (500 MHz, CDCl3): δ 8.26 (N1”H, [1H], bs), 7.65 (C4”H, [1H], dd, 3J=7.5, 4J=0.5), 7.32 (C7”H,
[1H], dt, 3J=8.0, 4J=5J=1.0), 7.15 (C6”H, [1H], m, 3J1=8.0, 3J2=7.0, 4J=1.0), 7.10 (C2’H,C6’H, [2H], dt,
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ACCEPTED MANUSCRIPT
3J=8.5, 4J=2.0), 7.08 (C5”H, [1H], m, 3J1=8.0, 3J2=7.0, 4J=1.0), 6.95 (C2”H, [1H], d, 3J=2.0), 6.91
(C3’H,C5’H, [2H], dt, 3J=8.0, 4J=2.5), 4.02 (C1xH2, [2H], t, 3J=7.5), 3.91 (C8H2, [2H], t, 3J=6.5), 3.81
(OCH3, [3H], s), 3.21 (CaH(E),CbH(E), [2H], pd), 3.00 (CeH(E), [1H], bs), 2.52 (C5H2, [2H], t, 3J=6.5), 2.46
(C4xH2, [2H], pt), 2.16 – 2.10 (CaH(A),CeH(A),CcH(E), [3H], m), 1.89 (C7H2, [2H], q, 3J=7.0), 1.79 (CdH2,
[2H], m), 1.75 – 1.58 (C2xH2,C3xH2,C6H2, [6H], m), 1.50 (CcH(A), [1H], m).
13C NMR (125 MHz, CDCl3): δ 162.2 (C3, s), 159.0 (C4’, s), 151.7 (C1, s), 149.6 (C4a, s), 136.2 (C7”a, s),
131.8 (C3’,C5’, s), 126.7 (C3”a, s), 125.5 (C1’, s), 121.8 (C6”, s), 120.3 (C2”, s), 119.5 (C3”, s), 119.2
(C4”, s), 119.1 (C5”, s), 113.9 (C2’,C6’, s), 112.0 (C4, s), 111.1 (C7”, s), 60.6 (Ca, s), 58.7 (C4x, s), 55.3
(OCH3, s), 54.1 (Ce, s), 42.6 (C8, s), 41.4 (C1x, s), 33.7 (Cb, s), 31.2 (Cc, s), 26.7 (C5, s), 25.7 (Cd, s), 25.5
(C2x, s), 24.1 (C3x, s), 21.7 (C7, s), 18.6 (C6, s).
ESI-HRMS m/z: Calcd for C32H39N4O3 [M+H]+ 527.30222. Found: 527.30208.
3.2.3.10. 2-[4-[3-(5-fluoro-1H-indol-3-yl)-1-piperidyl]butyl]-4-phenyl-5,6,7,8-tetrahydropyrido[1,2-
c]pyrimidine-1,3-dione (10.10).
The title compound was isolated as a white powder. Yield: 93.0 %, m.p. 119.9-121.4 °C. HPLC tR = 5.30
min, 99.9% purity.
1H NMR (500 MHz, CDCl3): δ 8.42 (N1”H, [1H], bs), 7.38 (C3’H,C5’H, [2H], t, 3J=7.5), 7.32 (C4’H, [1H],
tt, 3J=7.5, 4J=1.5), 7.26 (C4”H, [1H], dd, 3JH-F=10.0, 4J=2.5), 7.21 (C7”H, [1H], dd, 3J=8.5, 4JH-F=4.5), 7.18
(C2’H,C6’H, [2H], dt, 3J=7.0, 4J=2.0), 6.99 (C2”H, [1H], bs), 6.89 (C6”H, [1H], td, 3J=9.5, 4J=2.5), 4.03
(C1xH2, [2H], m), 3.92 (C8H2, [2H], t, 3J=6.5), 3.14 (CaH(E),CbH(E), [2H], pd), 2.98 (CcH(E), [1H], bs),
2.51 (C5H2, [2H], t, 3J=6.5), 2.48 (C4xH2, [2H], t, 3J=7.0), 2.17 – 1.98 (CaH(A),CeH(A),CcH(E), [3H], m),
1.91 (C7H2, [2H], q, 3J=7.0), 1.84 – 1.59 (C2xH2,C3xH2,C6H2,CdH2, [8H], m), 1.46 (CcH(A), [1H], kd, 2J=3JA-
A=12.5, 3JA-E=3.5).
13C NMR (125 MHz, CDCl3): δ 162.15 (C3, s), 157.7 (C5”, d, 1J=234.0), 151.8 (C1, s), 149.9 (C4a, s),
133.5 (C7”a, s), 132.8 (C1’, s), 130.9 (C2’,C6’, s), 128.6 (C3’,C5’, s), 127.8 (C4’, s), 127.0 (C3”a, d), 122.5
(C2”, s), 112.5 (C4, s), 111.9 (C7”, d, 3J=9.7), 110.3 (C6”, d, 2J=26.4), 104.1 (C4”, d, 2J=23.3), 60.4 (Ca,
s), 58.7 (C4x, s), 54.1 (Ce, s), 42.8 (C8, s), 41.5 (C1x, s), 33.7 (Cb, s), 31.2 (Cc, s), 26.9 (C5, s), 25.8 (Cd, s),
25.4 (C2x, s), 24.1 (C3x, s), 21.9 (C7, s), 18.7 (C6, s).
ESI-HRMS m/z: Calcd for C31H32FN4O2 [M+H]+ 515.28223. Found: 515.28206.
3.2.3.11. 4-(2-chlorophenyl)-2-[4-[3-(5-fluoro-1H-indol-3-yl)-1-piperidyl]butyl]-5,6,7,8-
tetrahydropyrido[1,2-c]pyrimidine-1,3-dione (10.11).
The title compound was isolated as a white powder. Yield: 87.9 %, m.p. 120.6-122.3 °C. HPLC tR = 5.55
min, 99.9% purity.
1H NMR (500 MHz, CDCl3): δ 8.37 (N1”H, [1H], bs), 7.46 – 7.41 (C3’H, [1H], m), 7.31 – 7.25
(C5’H,C6’H,C4”H, [3H], m), 7.22 (C7”H, [1H], dd, 3J=9.0, 4JH-F=4.5), 7.18 (C4’H, [1H], m), 7.03 (C2”H,
[1H], pt), 6.89 (C6”H, [1H], td, 3J=9.0, 4J=2.0), 4.03 (C1xH2, [2H], m), 3.99 – 3.84 (C8H2, [2H], m), 3.13
(CaH(E),CbH(E), [2H], pd), 2.96 (CeH(E), [1H], bs), 2.45 (C4xH2, [2H], pt), 2.38 (C5H2, [2H], m), 2.14 –
1.98 (CaH(A),CeH(A),CcH(E), [3H], m), 1.91 (C7H2, [2H], m), 1.83 – 1.65 (CdH2,C2xH2,C6H2, [6H], m),
1.62 (C3xH2, [2H], q, 3J=7.5), 1.46 (CcH(A), [1H], m).
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ACCEPTED MANUSCRIPT
13C NMR (125 MHz, CDCl3): δ 161.4 (C3, s), 157.7 (C5”, d, 1J=234.1), 151.9 (C1, s), 150.6 (C4a, s), 135.2
(C2’, 2s), 132.8 (C7”a, s), 132.7 (C1’, 2s), 132.6 (C6’, s), 129.8 (C3’, s), 129.6 (C4’, s), 127.3 (C5’, 2s),
127.2 (C3”a, d), 122.4 (C2”, s), 120.0 (C3”, bs), 111.8 (C7”, d, 3J=9.7), 110.3 (C6”, d, 2J=23.5), 110.2
(C4, 2s), 104.2 (C4”, d, 2J=23.3), 60.7 (Ca, s), 58.8 (C4x, s), 54.1 (Ce, s), 43.1 (C8, s), 41.6 (C1x, s), 33.9
(Cb, s), 31.4 (Cc, s), 26.5 (C5, s), 25.8 (Cd, s), 25.6 (C2x, s), 24.3 (C3x, s), 22.0 (C7, s), 18.6 (C6, s).
ESI-HRMS m/z: Calcd for C31H35ClFN4O2 [M+H]+ 549.24325. Found: 549.24333.
3.2.3.12. 4-(2-fluorophenyl)-2-[4-[3-(5-fluoro-1H-indol-3-yl)-1-piperidyl]butyl]-5,6,7,8-
tetrahydropyrido[1,2-c]pyrimidine-1,3-dione (10.12).
The title compound was isolated as a white powder. Yield: 87.6 %, m.p. 122.3-124.2 °C. HPLC tR = 5.07
min, 99.9% purity.
1H NMR (500 MHz, CDCl3): δ 8.40 (N1”H, [1H], bs), 7.36-7.30 (C4’H, [1H], m), 7.29 (C4”H, [1H], dd, 3JH-
F=11.0, 4J=2.5), 7.23 (C7”H, [1H], dd, 3J=8.5, 4JH-F=4.0), 7.22 – 7.13 (C6’H,C5’H, [2H], m), 7.09 (C3’H,
[1H], m), 7.00 (C2”H, [1H], m), 6.89 (C6”H, [1H], td, 3J=9.0, 4J=2.5), 4.02 (C1xH2, [2H], t, 3J=7.0), 3.91
(C8H2, [2H], m), 3.15 (CaH(E),CbH(E), [2H], pd), 3.00 (CeH(E), [1H], pd), 2.48 (C4xH2,C5H2, [4H], m),
2.18 – 1.98 (CaH(A),CeH(A),CcH(E), [3H], m), 1.91 (C7H2, [2H], m), 1.88 – 1.60 (CdH2,C2xH2,C3xH2,C6H2,
[8H], m), 1.47 (CcH(A), [1H], m).
13C NMR (125 MHz, CDCl3): 161.4 (C3, s), 160.4 (2 x C2’, 2d, 2J=249.9), 157.5 (C5”, d, 1J=234.1), 151.6
(C1, s), 151.0 (C4a, 2s), 132.9 (C6’, C7”a, d, 3J=2.5), 130.0 (C4’, d, 3J=8.3), 126.9 (C3”a, 2s), 124.2
(2xC5’, 2d, 4J=3.4), 122.3 (C2”, s), 120.8 (C1’, d, 2J=16.2), 119.5 (C3”, bs), 115.7 (2xC3’, 2d, 2J=22.4),
111.8 (C7”, d, 3J=9.4), 110.1 (C6”, d, 2J=26.3), 106.4 (C4, d, 3J=2.5), 103.9 (C4”, d, 3J=23.4), 60.2 (Ca, s),
58.4 (C4x, s), 54.0 (Ce, s), 42.9 (C8, s), 41.4 (C1x, s), 33.5 (Cb, s), 31.0 (Cc, s), 26.5 (C5, 2s), 25.6 (Cd, s),
25.1 (C2x, s), 23.7 (C3x, s), 21.7 (C7, s), 18.3 (C6, s).
ESI-HRMS m/z: Calcd for C31H35F2N4O2 [M+H]+ 533.27281. Found: 533.27267.
3.2.3.13. 2-[4-[3-(5-fluoro-1H-indol-3-yl)-1-piperidyl]butyl]-4-(o-tolyl)-5,6,7,8-tetrahydropyrido[1,2-
c]pyrimidine-1,3-dione (10.13).
The title compound was isolated as a white powder. Yield: 88.5 %, m.p. 117.5-119.3 °C. HPLC tR = 5.68
min, 99.5% purity.
1H NMR (500 MHz, CDCl3): δ 8.34 (N1”H, [1H], bs), 7.29 – 7.16 (C4’H,C5’H,C6’H,C4”H,C7”H, [5H], m),
7.01 (C3’H,C2”H, [2H], m), 6.89 (C6”H, [1H], td, 3J=9.0, 4J=2.5), 4.03 (C1xH2, [2H], m), 3.91 (C8H2, [2H],
m), 3.12 (CaH(E),CbH(E), [2H], pd), 2.96 (CeH(E), [1H], bs), 2.50 – 2.38 (C4xH2,C5H(2), [3H], m), 2.24
(C5H(1), [1H], m), 2.12 and 2.09 (2 x CH3, [3H], 2s), 2.15 – 1.98 (CaH(A),CeH(A),CcH(E), [3H], m), 1.90
(C7H2, [2H], m), 1.82 – 1.56 (C6H2,CdH2,C2xH2,C3xH2, [8H], m), 1.47 (CcH(A), [1H], m).
13C NMR (125 MHz, CDCl3): δ 161.5 (C3, s), 157.5 (C5”, d, 1J=234.1), 151.9 (C1, 2s), 149.6 (C4a, s),
137.7 (C2’, 2s), 133.0 (C1’, s), 132.7 (C7”a, s), 130.7 (C6’, 2s), 130.3 (C3’, s), 128.2 (C4’, s), 127.0 (C3”a,
d, 3J=9.2), 126.2 (C5’, 2s), 122.4 (C2”, s), 119.5 (C3”, bs), 111.7 (C4, C7”, d, 3J=8.8), 119.1 (C6”, d,
2J=26.3), 104.0 (2 x C4”, 2d, 2J=24.5), 60.4 (Ca, s), 58.6 (C4x, s), 54.0 (Ce, s), 42.9 (C8, s), 41.3 (C1x, s),
33.7 (Cb, s), 31.2 (Cc, s), 26.5 (C5, s), 25.8 (Cd, s), 25.4 (C2x, s), 24.1 (C3x, s), 21.9 (C7, s), 19.7 and 19.6
(2 x CH3, 2s), 18.6 (C6, s).
ESI-HRMS m/z: Calcd for C32H38FN4O2 [M+H]+ 529.29788. Found: 525.29768.
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3.2.3.14. 2-[4-[3-(5-fluoro-1H-indol-3-yl)-1-piperidyl]butyl]-4-(2-methoxyphenyl)-5,6,7,8-
tetrahydropyrido[1,2-c]pyrimidine-1,3-dione (10.14).
The title compound was isolated as a white powder. Yield: 83.2 %, m.p. 137.7-139.4 °C. HPLC tR = 5.08
min, 99.9% purity.
1H NMR (500 MHz, CDCl3): δ 8.52 (N1”H, [1H], bs), 7.32 (C4’H, [1H], 4d, 3J1=8.0, 3J2=7.5, 4J=2.0), 7.26
(C4”H, [1H], m), 7.21 (C7”H, [1H], dd, 3J=8.5, 4JH-F=4.5), 7.09 (C6’H, [1H], m, 4J=2.0), 7.00 – 6.94
(C3’H,C2”H, [2H], m), 6.93 – 6.85 (C5’H,C6”H, [2H], m), 4.02 (C1xH2, [2H], m), 3.90 (C8H2, [2H], m),
3.73 and 3.70 (OCH3, [3H], 2s), 3.15 (CaH(E),CbH(E), [2H], pd), 3.00 (CeH(E), [1H], bs), 2.50
(C4xH2,[2H], bs), 2.41 (C5H2, [2H], t, 3J=7.0), 2.20 – 1.96 (CaH(A),CeH(A),CcH(E), [3H], m), 1.90 (C7H2,
[2H], q, 3J=6.0), 1.80 – 1.60 (CdH2,C2xH2,C3xH2,C6H2, [8H], m), 1.46 (CcH(A), [1H], m).
13C NMR (125 MHz, CDCl3): δ 161.8 (C3, s), 157.5 (C5”, d, 1J=234.1), 157.4 (C2’, 2s), 151.8 (C1, s), 150.3
(C4a, s), 132.7 (C7”a, s), 132.3 (C6’, 2s), 129.5 (C4’, s), 126.8 (C3”a, d, 3J=9.0), 122.4 (C2”, s), 122.1
(C1’, 2s), 120.7 (C5’, 2s), 118.8 (C3”, bs), 111.8 (C7”, d, 3J=9.6), 111.1 (C3’, 2s), 110.0 (C6”, d, 2J=26.4),
108.5 (C4, 2s), 103.9 (C4”, d, 2J=23.6), 60.1 (Ca, s), 58.4 (C4x, s), 55.4 (OCH3, 2s), 53.8 (Ce, s), 42.9 (C8,
s), 41.2 (C1x, s), 33.4 (Cb, s), 30.9 (Cc, s), 26.3 (C5, s), 25.6 (Cd, s), 25.0 (C2x, s), 23.6 (C3x, s), 21.8 (C7,
s), 18.5 (C6, s).
ESI-HRMS m/z: Calcd for C32H38FN4O3 [M+H]+ 545.29279. Found: 545.29258.
3.2.3.15. 4-(4-chlorophenyl)-2-[4-[3-(5-fluoro-1H-indol-3-yl)-1-piperidyl]butyl]-5,6,7,8-
tetrahydropyrido[1,2-c]pyrimidine-1,3-dione (10.15).
The title compound was isolated as a white powder. Yield: 67.5 %, m.p. 237.6-239.5 °C. HPLC tR = 6.37
min, 98.5% purity.
1H NMR (500 MHz, CDCl3): δ 8.18 (N1”H, [1H], bs), 7.34 (C2’H,C6’H, [2H], d, 3J=8.0), 7.28 (C4”H, [1H],
dd, 3JH-F=9.5, 4J=2.0), 7.25 (C7”H, [1H], dd, 3J=9.0, 4JH-F=4.5), 7.12 (C3’H,C5’H, [2H], d, 3J=8.0), 7.03
(C2”H, [1H], bs), 6.92 (C6”H, [1H], td, 3J=9.0, 4J=2.5), 4.02 (C1xH2, [2H], t, 3J=7.0), 3.92 (C8H2, [2H], t,
3J=6.5), 3.18 (CaH(E),CbH(E), [2H], bs), 3.03 (CeH(E), [1H], bs), 2.50 (C4xH2,C5H2, [4H], bs+t), 2.20 –
2.00 (CaH(A),CeH(A),CcH(E), [3H], m), 1.91 (C7H2, [2H], q, 3J=7.0), 1.85 – 1.76 (CdH2, [2H], m), 1.75 –
1.60 (C2xH2,C3xH2,C6H2, [6H], m), 1.50 (CcH(A), [1H], m).
13C NMR (125 MHz, CDCl3): δ 161.8 (Cs, s), 157.6 (C5”, d, 1J=234.2), 151.6 (C1, s), 149.9 (C4a, s), 133.7
(C4’, s), 132.7 (C7”, s), 132.2 (C3’,C5’, s), 131.8 (C1’, s), 128.7 (C2’,C6’, s), 127.0 (C3”a, s), 122.2 (C2”,
s), 120.0 (C3”, s), 111.7 (C7”, d, 3J=9.7), 111.3 (C4, s), 110.3 (C6”, d, 2J=26.7), 104.1 (C4”, d, 2J=23.4),
60.5 (Ca, s), 58.7 (C4x, s), 54.1 (Ce, s), 42.7 (C8, s), 41.5 (C1x, s), 34.0 (Cb, s), 31.0 (Cc, s), 26.7 (C5, s),
25.7 (Cd, s), 25.5 (C2x, s), 24.5 (C3x, s), 21.7 (C7, s), 18.5 (C6, s).
ESI-HRMS m/z: Calcd for C31H35ClFN4O2 [M+H]+ 549.24325. Found: 549.24337.
3.2.3.16. 4-(4-fluorophenyl)-2-[4-[3-(5-fluoro-1H-indol-3-yl)-1-piperidyl]butyl]-5,6,7,8-
tetrahydropyrido[1,2-c]pyrimidine-1,3-dione (10.16).
The title compound was isolated as a white powder. Yield: 83.1 %, m.p. 216.5-218.3 °C. HPLC tR = 5.29
min, 99.9% purity.
20

ACCEPTED MANUSCRIPT
1H NMR (500 MHz, CDCl3): δ 8.37 (N1”H, [1H], bs), 7.27 (C4”H, [1H], dd, 3JH-F=10.0, 4J=2.5), 7.22 (C7”H,
[1H], 4d, 3J=8.5, 4JH-F=4.5, 5J=0.5), 7.15 (C2’H,C6’H, [2H], dd, 3J=8.5, 4JH-F=5.5), 7.06 (C3’H,C5’H, tt,
3J=9.0, 4J=2.5), 7.01 (C2”H, [1H], d, 3J=2.0), 6.89 (C6”H, [1H], td, 3J=9.0, 4J=2.5), 4.02 (C1xH2, [2H], td,
3J=7.0), 3.92 (C8H2, [2H], t, 3J=6.5), 3.13 (CaH(E),CbH(E), [2H], m), 2.97 (CeH(E), [1H], pd), 2.49 (C5H2,
[2H], t, 3J=7.0), 2.44 (C4xH2, [2H], t, 3J=7.5), 2.13 – 1.99 (CaH(A),CeH(A),CcH(E), [3H], m), 1.78 (CdH2,
[2H], m), 1.69 (C2xH2,C6H2, [4H], m), 1.61 (C3xH2, [2H], q, 3J=7.5), 1.47 (CcH(A), [1H], m).
13C NMR (125 MHz, CDCl3): δ 162.2 (C4’, d, 1J=247.0), 162.0 (C3, s), 157.5 (C5”, d, 1J=234.2), 151.6 (C1,
s), 150.0 (C4a, s), 132.7 (C7”a, s), 132.5 (C2’,C6’, d, 3J=8.2), 129.2 (C1’, d, 4J=3.5), 127.0 (C3”a, d,
3J=9.6), 122.3 (C2”, s), 119.6 (C3”, s), 115.5 (C3’,C5’, d, 2J=21.5), 111.7 (C7”, d, 3J=9.7), 111.4 (C4, s),
110.2 (C6”, d, 2J=26.4), 104.0 (C4”, d, 2J=23.4), 60.5 (Ca, s), 58.6 (C4x, s), 54.0 (Ce, s), 42.7 (C8, s), 41.5
(C1x, s), 33.7 (Cb, s), 31.2 (Cc, s), 26.8 (C5, s), 25.7 (Cd, s), 25.4 (C2x, s), 24.1 (C3x, s), 21.7 (C7, s), 18.6
(C6, s).
ESI-HRMS m/z: Calcd for C31H35F2N4O2 [M+H]+ 533.27281. Found: 533.27251.
3.2.3.17. 2-[4-[3-(5-fluoro-1H-indol-3-yl)-1-piperidyl]butyl]-4-(p-tolyl)-5,6,7,8-tetrahydropyrido[1,2-
c]pyrimidine-1,3-dione (10.17).
The title compound was isolated as a white powder. Yield: 77.4 %, m.p. 227.2-229.7 °C. HPLC tR = 6.36
min, 99.2% purity.
1H NMR (500 MHz, CDCl3): δ 8.30 (N1”H, [1H], bs), 7.27 (C4”H, [1H], dd, 3JH-F=9.5, 4J=2.5), 7.20 (C7”H,
[1H], dd, 3J=9.0, 4JH-F=4.5), 7.19 (C2’H,C6’H, [2H], d, 3J=7.0), 7.07 (C3’H,C5’H, [2H], d, 3J=8.0), 7.01
(C2”H, [1H], bs), 6.89 (C6”H, [1H], td, 3J=9.0, 4J=2.5), 4.03 (C1xH2, [2H], m), 3.92 (C8H2, [2H], t, 3J=6.5),
3.12 (CaH(E),CbH(E), [2H], pd), 2.95 (CeH(E), [1H], pd), 2.52 (C5H2, [2H], t, 3J=6.5), 2.43 (C4xH2, [2H], t,
3J=7.0), 2.36 (CH3, [3H], s), 2.14 – 1.80 (CaH(A),CeH(A),CcH(E), [3H], m), 1.90 (C7H2, [2H], q, 3J=6.5),
1.76 (CdH2, [2H], m), 1.68 (C2xH2,C6H2, [4H], m), 1.61 (C3xH2, [2H], q, 3J=7.5), 1.47 (CcH(A), [1H], m).
13C NMR (125 MHz, CDCl3): δ 162.1 (C3, s), 157.5 (C5”, d, 1J=234.1), 151.7 (C1, s), 149.6 (C4a, s), 137.4
(C4’, s), 132.7 (C7”a, s), 130.6 (C2’,C6’, s), 130.3 (C1’, s), 129.2 (C3’,C5’, s), 127.1 (C3”a, d, 3J=9.4),
122.4 (C2”, s), 120.0 (C3”, d), 112.3 (C4, s), 111.7 (C7”, d, 3J=9.6), 115.0 (C6”, d, 2J=26.4), 104.0 (C4”, d,
2J=23.4), 60.5 (Ca, s), 58.7 (C4x, s), 54.1 (Ce, s), 42.6 (C8, s), 41.5 (C1x, s), 33.8 (Cb, s), 31.3 (Cc, s), 26.7
(C5, s), 25.7 (Cd, s), 25.4 (C2x, s), 24.2 (C3x, s), 21.8 (C7, s), 21.3 (CH3, s), 18.6 (C6, s).
ESI-HRMS m/z: Calcd for C32H38FN4O2 [M+H]+ 529.29788. Found: 529.29773.
3.2.3.18. 2-[4-[3-(5-fluoro-1H-indol-3-yl)-1-piperidyl]butyl]-4-(4-methoxyphenyl)-5,6,7,8-
tetrahydropyrido[1,2-c]pyrimidine-1,3-dione (10.18).
The title compound was isolated as a white powder. Yield: 85.7 %, m.p. 124.8-125.9 °C. HPLC tR = 5.28
min, 99.9% purity.
1H NMR (500 MHz, CDCl3): δ 8.43 (N1”H, [1H], bs), 7.27 (C4”H, [1H], dd, 3JH-F=10.0, 4J=2.5), 7.22 (C7”H,
[1H], 4d, 3J=8.5, 4JH-F=4.5), 7.10 (C2’H,C6’H, [2H], dt, 3J=8.5, 4J=3.0), 6.99 (C2”H, [1H], bs), 6.91
(C3’H,C5’H, [2H], dt, 3J=9.0, 4J=3.0), 6.89 (C6”H, [1H], td, 3J=9.0, 4J=2.5), 4.02 (C1xH2, [2H], m), 3.91
(C8H2, [2H], t, 3J=6.5), 3.81 (OCH3, [3H], s), 3.15 (CaH(E),CbH(E), [2H], pd), 2.99 (CeH(E), [1H], bs), 2.52
(C5H2, [2H], t, 3J=7.0), 2.48 (C4xH2, [2H], pt), 2.18 – 1.98 (CaH(A),CeH(A),CcH(E), [3H], m), 1.90 (C7H2,
21

ACCEPTED MANUSCRIPT
[2H], q, 3J=6.5), 1.78 (CdH2, [2H], m), 1.75 – 1.60 (C2xH2,C3xH2,C6H2, [6H], m), 1.46 (CcH(A), [1H], kd,
2J=3JA-A=11.5, 3JA-E=3.5).
13C NMR (125 MHz, CDCl3): δ 162.3 (C3, s), 159.0 (C4’, s), 157.5 (C5” d, 1J=234.1), 151.7 (C1, s), 149.8
(C4a, s), 132.7 (C7’a, s), 131.8 (C3’,C5’, s), 126.9 (C3”a, d, 3J=9.4), 125.5 (C1’, s), 122.4 (C2’, s), 119.2
(C3”, s), 114.0 (C2’,C6’, s), 111.9 (C4, s), 111.8 (C7”, d, 3J=9.7), 110.1 (C6”, d, 2J=26.4), 103.9 (C4”, d,
2J=23.4), 60.2 (Ca, s), 58.5 (C4x, s), 55.3 (OCH3, s), 53.9 (Ce, s), 42.7 (C8, s), 41.3 (C1x, s), 33.5 (Cb, s),
31.0 (Cc, s), 26.7 (C5, s), 25.6 (Cd, s), 25.1 (C2x, s), 23.8 (C3x, s), 21.7 (C7, s), 18.6 (C6, s).
ESI-HRMS m/z: Calcd for C32H38FN4O3 [M+H]+ 545.29279. Found: 545.29256.
3.2.3.19. 2-[4-[3-(5-methoxy-1H-indol-3-yl)-1-piperidyl]butyl]-4-phenyl-5,6,7,8-tetrahydropyrido[1,2-
c]pyrimidine-1,3-dione (10.19).
The title compound was isolated as a white powder. Yield: 68.4 %, m.p. 126.8-128.2 °C. HPLC tR = 4.81
min, 99.9% purity.
1H NMR (500 MHz, CDCl3): δ 8.11 (N1”H, [1H], bs), 7.38 (C3’H,C5’H, [2H], t, 3J=7.5), 7.32 (C4’H, [1H],
tt, 3J=7.5, 4J=1.5), 7.22 (C7”H, [1H], dd, 3J=8.5, 5J=0.5), 7.18 (C2’H,C6’H, [2H], dt, 3J=7.0, 4J=1.5), 7.1
(C4”H, [1H], d, 4J=2.5), 6.9 (C2”H, [1H], d, 3J=1.5), 6.83 (C6”H, [1H], dd, 3J=9.0, 4J=2.5), 4.02 (C1xH2,
[2H], m), 3.91 (C8H2, [2H], t, 3J=6.5), 3.84 (OCH3, [3H], s), 3.26 (CaH(E),CbH(E), [2H], bs), 3.08 (CeH(E),
[1H], bs), 2.51 (C4xH2,C5H2, [4H], m), 2.18 – 2.02 (CaH(A),CeH(A), CcH(E), [3H], m), 1.90 (C7H2, [2H], q,
3J=7.0), 1.84 – 1.77 (CdH2, [2H], m), 1.68 (C6H2,C2xH2,C3xH2, [6H], m), 1.49 (CcH(A), [1H], kd, 3JA-A=12.0,
3JA-E=3.5).
13C NMR (125 MHz, CDCl3): δ 162.0 (C3, s), 153.8 (C5”, s), 151.7 (C1, s), 149.7 (C4a, s), 133.4 (C7”a, s),
131.3 (C1’, s), 130.8 (C2’,C6’, s), 128.5 (C3’,C5’, s), 127.5 (C4’, s), 127.1 (C3”a, s), 121.1 (C2”, s), 119.0
(C3”, s), 112.4 (C4, s), 112.2 (C6”, s), 111.9 (C7”, s), 100.7 (C4”, s), 60.9 (Ca, s), 56.7 (C4x, s), 55.9
(OCH3, s), 54.0 (Ce, s), 42.6 (C8, s), 41.5 (C1x, s), 33.7 (Cb, s), 31.1 (Cc, s), 26.7 (C5, s), 25.7 (Cd, s), 25.5
(C2x, s), 24.0 (C3x, s), 21.7 (C7, s), 18.6 (C6, s).
ESI-HRMS m/z: Calcd for C32H39N4O3 [M+H]+ 527.30222. Found: 527.30227.
3.2.3.20. 4-(2-chlorophenyl)-2-[4-[3-(5-methoxy-1H-indol-3-yl)-1-piperidyl]butyl]-5,6,7,8-
tetrahydropyrido[1,2-c]pyrimidine-1,3-dione (10.20).
The title compound was isolated as a white powder. Yield: 49.2 %, m.p. 109.1-111.0 °C. HPLC tR = 5.01
min, 99.9% purity.
1H NMR (500 MHz, CDCl3): δ 8.09 (N1”H, [1H], bs), 7.46 – 7.42 (C3’H, [1H], m), 7.31 – 7.26 (C5’H, C6’H,
[2H], m), 7.21 (C7”H, [1H], d, 3J=8.5), 7.20 – 7.16 (C4’H, [1H], m), 7.10 (C4”H, [1H], d, 4J=2.5), 6.95
(C2”H, [1H], pt), 6.83 (C6”H, [1H], m, 3J=8.5, 4J=2.0, 5J=0.5), 4.03 (C1xH2, [2H], m), 3.98 – 3.86 (C8H2,
[2H], m), 3.84 (C5”-OCH3, [3H], 2s), 3,20 (CaH(E),CbH(E), [2H], pd), 3.02 (CeH(E), [1H], bs), 2.47 (C4xH2,
[2H], bs), 2.39 (C5H2, [2H], m), 2.05 (CaH(A),CeH(A),CcH(E), [3H], m), 1.90 (C7H2, [2H], m), 1.80 (CdH2,
[2H], bs), 1.75 – 1.60 (C2xH2,C3xH2,C6H2, [6H], m), 1.48 (CcH(A), [1H], m).
13C NMR (125 MHz, CDCl3): δ 161.2 (C3, s), 153.8 (C5”, s), 153.8 (C5”, s), 151.8 (C1, s), 150.4 (C4a, s),
135.1 (C2’, s), 132.6 (C6’, s), 132.5 (C7”a, s), 131.3 (C1’, s), 129.7 (C3’, s), 129.5 (C4’, s), 127.1 (C5’, s),
127.0 (C3”a, s), 119.5 (C3”, s), 112.2 (C6”, s), 111.8 (C7”, s), 110.1 (C4, s), 100.7 (C4”, 2s), 61.0 (Ca, s),
22

ACCEPTED MANUSCRIPT
58.8 (C4x, s), 55.9 (C5”-OCH3, s), 54.0 (Ce, s), 43.0 (C8, s), 41.5 (C1x, s), 33.8 (Cb, s), 31.2 (Cc, s), 26.4
(C5, s), 25.8 (Cd, s), 25.7 (C2x, s), 24.3 (C3x, s), 21.8 (C7, s), 18.4 (C6, s).
ESI-HRMS m/z: Calcd for C32H38ClN4O3 [M+H]+ 561.26324. Found: 561.26320.
3.2.3.21. 4-(2-fluorophenyl)-2-[4-[3-(5-methoxy-1H-indol-3-yl)-1-piperidyl]butyl]-5,6,7,8-
tetrahydropyrido[1,2-c]pyrimidine-1,3-dione (10.21).
The title compound was isolated as a white powder. Yield: 49.4 %, m.p. 113.7-115.4 °C. HPLC tR = 4.64
min, 99.8% purity.
1H NMR (500 MHz, CDCl3): δ 8.00 (N1”H, [1H], bs), 7.36 – 7.30 (C4’H, [1H],m), 7.22 (C7”H, [1H], d,
3J=9.0), 7.22 – 7.14 (C5’H,C6’H, [2H], m), 7.13 – 7.07 (C3’H,C4”H, [2H], m), 6.97 (C2”H, [1H], bs), 6.83
(C6”H, [1H], dd, 3J=9.0, 4J=2.5), 4.01 (C1xH2, [2H], m), 3.90 (C8H2, [2H], m), 3.84 (OCH3, [3H], s), 3.19
(CaH(E),CbH(E), [2H], m), 2.97 (CeH(E), [1H], bs), 7.55 – 7.35 (C5H2,C4xH2, [4H], m), 2.10 – 1.95
(CaH(A),CeH(A),CcH(E), [3H], m), 1.92 (C7H2, [2H], m), 1.82 – 1.57 (CdH2C6H2,C2xH2,C3xH2, [8H], m),
1,478 (CcH(A), [1H], m).
13C NMR (125 MHz, CDCl3): δ 161.4 (C3, s), 160.4 (C2’, d, 1J=249.9), 153.8 (C5”, s), 151.7 (C1, s), 150.9
(C4a, s), 132.9 (C6’, d, 3J=2.9), 131.3 (C7”a, s), 129.9 (C4’, d, 3J=9.3), 127.1 (C3”a, s), 124.2 (C5’, d,
4J=3.5), 121.0 (C2”, s), 120.8 (C1’, d, 2J=16.2), 119.5 (C3”, s), 115.8 (C3’, d, 2J=22.3), 112.2 (C6”, s),
111.8 (C7”, s), 106.1 (C4, s), 100.7 (C4”, 2s), 61.0 Ca, s), 58.8 (C4x, s), 55.9 (OCH3, s), 54.0 (Ce, s), 42.8
(C8, s), 41.6 (C1x, s), 33.4 (Cb, s), 31.2 (Cc, s), 26.5 and 26.4 (C5, 2s), 25.7 (Cd, s), 25.7 (C2x, s), 24.3
(C3x, s), 21.7 (C7, s), 18.4 (C6, s).
ESI-HRMS m/z: Calcd for C32H38FN4O3 [M+H]+ 545.29279. Found: 545.29245.
3.2.3.22. 2-[4-[3-(5-methoxy-1H-indol-3-yl)-1-piperidyl]butyl]-4-(o-tolyl)-5,6,7,8-tetrahydropyrido[1,2-
c]pyrimidine-1,3-dione (10.22).
The title compound was isolated as a white powder. Yield: 54.7 %, m.p. 116.8-118.6 °C. HPLC tR = 5.11
min, 99.5% purity.
1H NMR (500 MHz, CDCl3): δ 8.12 (N1”H, [1H], s), 7.21 (C7”H, [1H], d, 3J=9.0), 7.25 – 7.16 (C4’-6’H,
[3H], m(18 lines)), 7.09 (C4”H, [1H], d, 4J=2.0), 7.02 (C3’H, [1H], m(6 lines)), 6.94 (C2”H, [1H], m), 6.82
(C6”H, [1H], dd, 3J=9.0, 4J=2.5), 4.03 (C1xH2, [2H], t, 3J=7.0), 3.90 (C8H2, [2H], m), 3.84 and 3.83 (OCH3,
[3H], 2s), 3.19 (CaH,CbH(E), [2H] pd), 3.00 (CeH(E), [1H] bs), 2.46 (C4xH2, [2H], bs), 2.42 and 2.24
(C5H2, [2H], m(14 lines)), 2.10 (CH3, [3H], 2s), 2.10 – 1.98 (CaH(A),CeH(A),CcH(E), [3H], m), 1.90 (C7H2,
[2H], m), 1.90 (CdH2, [2H], m), 1.75 – 1.60 (C2xH2,C3xH2,C6H2, [6H], m), 1.47 (CcH(A), [1H], m).
13C NMR (125 MHz, CDCl3): δ 161.4 (C3, s), 153.8 (C5”, s), 151.9 C1, 2s), 149.5 (C4a, s), 137.7 (C2’, 2s),
133.0 (C1’, s), 131.3 (C7”a, s), 130.7 (C6’, 2s), 130.3 (C3’, s), 128.2 (C4’, s), 127.1 (C3”a, s), 126.2 (C5’,
s), 121.1 (C2”, s), 119.5 (C3”, s), 112.2 (C6”, s), 111.8 (C7”, s), 111.7 (C4, s), 100.7 (C4”, 2s), 60.9 (Ca,
s), 58.7 (C4x, s), 56.0 (OCH3, 2s), 53.9 (Ce, s), 42.9 (C8, s), 41.3 (C1x, s), 33.7 (Cb, s), 31.1 (Cc, s), 26.5
(C5, s), 25.8 (Cd, C2x, s), 24.2 (C3x, s), 21.9 (C7, s), 19.7 (CH3, 2s), 18.6 (C6, s).
ESI-HRMS m/z: Calcd for C33H41N4O3 [M+H]+ 541.31787. Found: 541.31780.
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3.2.3.23. 2-[4-[3-(5-methoxy-1H-indol-3-yl)-1-piperidyl]butyl]-4-(2-methoxyphenyl)-5,6,7,8-
tetrahydropyrido[1,2-c]pyrimidine-1,3-dione (10.23).
The title compound was isolated as a white powder. Yield: 55.8 %, m.p. 110.9-112.3 °C. HPLC tR = 4.62
min, 99.9% purity.
1H NMR (500 MHz, CDCl3): δ 8.13 (N1”H, [1H], bs), 7.32 (C4’H, [1H], m, 3J1=7.5, 3J2=8.0, 4J=2.0), 7.20
(C7”H, [1H], d, 3J=8.5), 7.09 (C6’H,C4”H, [2H], m), 6.99 (C5’H, [1H], m), 6.95 – 6.89 (C3’H,C2”H, [2H],
m), 6.82 (C6”H, [1H], dd, 3J=8.5, 4J=2.0), 4.02 (C1xH2, [2H], m), 3.89 (C8H2, [2H], m), 3.84 (C5”-OCH3,
[3H], s), 3.74 and 3.72 (C2’-OCH3, 2x[3H], 2s), 3.19 (CaH(E),CbH(E), [2H], bs), 3.02 (CeH(E), [1H], bs),
2.47 (C4xH2, [2H], bs), 2.45 (C5H2, [2H], t, 3J=6.5), 2.05 (CaH(A),CeH(A),CcH(E), [3H], pd), 1.89 (C7H2,
[2H], q, 3J=7.0), 1.80 (CdH2, [2H], bs), 1.75 – 1.60 (C2xH2,C3xH2,C6H2, [6H], m), 1.47 (CcH(A), [1H], m).
13C NMR (125 MHz, CDCl3): δ 161.7 (C3, s), 157.4 (C2’, s), 153.8 (C5”, s), 151.9 (C1, s), 150.1 (C4a, s),
132.3 (C6, 2s), 131.3 (C7”a, s), 129.5 (C4’, s), 127.1 (C3”a, s), 122.1 (C1’, s), 121.1 (C2”, s), 120.7 (C5’,
s), 119.0 (C3”, s), 112.2 (C6”, s), 111.8 (C7”, s), 111.1 (C3’, 2s), 108.6 (C4, s), 100.7 (C4”, 2s), 60.9 (Ca,
s), 55.9 (C5”-OCH3, s), 55.5 (C2’-OCH3, 2s), 53.9 (Ce, s), 42.8 (C8, s), 41.4 (C1x, s), 33.8 (Cb, s), 31.2 (Cc,
s), 26.3 (C5, s), 25.8 (Cd, s), 25.7 (C2x, s), 24.0 (C3x, s), 21.8 (C7, s), 18.5 (C6, s).
ESI-HRMS m/z: Calcd for C33H41N4O4 [M+H]+ 557.31278. Found: 557.31244.
3.2.3.24. 4-(4-chlorophenyl)-2-[4-[3-(5-methoxy-1H-indol-3-yl)-1-piperidyl]butyl]-5,6,7,8-
tetrahydropyrido[1,2-c]pyrimidine-1,3-dione (10.24).
The title compound was isolated as a white powder. Yield: 65.1 %, m.p. 125.2-127.4 °C. HPLC tR = 5.73
min, 99.4% purity.
1H NMR (500 MHz, CDCl3): δ 8.04 (N1”H, [1H], bs), 7.35 (C2’H,C6’H, [2H], dt, 3J=8.5), 7.22 (C7”H, [1H],
dd, 3J=9.0, 5J=0.5), 7.12 (C3’H,C5’H, [2H], dt, 3J=8.5), 7.09 (C4”H, [1H], d, 4J=2.5), 6.95 (C2’H, [1H], d,
3J=2.0), 6.83 (C6”H, [1H], 4d, 3J=9.0, 4J=2.5, 5J=0.5), 4.02 (C1xH2, [2H], m), 3.91 (C8H2, [2H], t, 3J=6.0),
3.84 (OCH3, [3H], s), 3.19 (CaH(E),CbH(E), [2H], pd), 3.01 (CeH(E), [1H], bs), 2.49 (C5H2, [2H], t, 3J=6.5),
2.45 (C4xH2, [2H], bs), 2.05 (CaH(A),CeH(A),CcH(E), [3H], pd), 1.90 (C7H2, [2H], q, 3J=6.5), 1.80 (CdH2,
[2H], bs), 1.75 – 1.65 (C2xH2,C6H2, [4H], m), 1.63 (C3xH2, [2H], bs), 1.48 (CcH(A), [1H], m).
13C NMR (125 MHz, CDCl3): δ 161.8 (C3, s), 153.8 (C5”, s), 151.6 (C1, s), 149.9 (C4a, s), 133.7 (C4’, s),
132.2 (C3’,C5’, s), 131.8 (C1’, s), 131.3 (C7”a, s), 128.7 (C2’,C6’, s), 127.1 (C3”a, s), 121.0 (C2”, s),
119.4 (C3”, s), 112.2 (C6”, s), 111.9 (C7”, s), 111.2 (C4, s), 100.8 (C4”, s), 60.9 (Ca, s), 58.7 (C4x, s), 56.0
(OCH3, s), 54.0 (Ce, s), 42.7 (C8, s), 41.5 (C1x, s), 33.8 (Cb, s), 31.1 (Cc, s), 26.7 (C5, s), 25.7 (Cd, s), 25.5
(C2x, s), 24.2 (C3x, s), 21.7 (C7, s), 18.5 (C6, s).
ESI-HRMS m/z: Calcd for C32H38ClN4O3 [M+H]+ 561.26324. Found: 561.26306.
3.2.3.25. 4-(4-fluorophenyl)-2-[4-[3-(5-methoxy-1H-indol-3-yl)-1-piperidyl]butyl]-5,6,7,8-
tetrahydropyrido[1,2-c]pyrimidine-1,3-dione (10.25).
The title compound was isolated as a white powder. Yield: 53.6 %, m.p. 124.9-127.4 °C. HPLC tR = 4.79
min, 99.9% purity.
1H NMR (500 MHz, CDCl3): δ 8.00 (N1”H, [1H], bs), 7.22 (C7”H, [1H], d, 3J=8.5), 7.18 – 7.12 (C2’H,C6’H,
[2H], m), 7.10 – 7.04 (C3’H,C5’H,C4”H, [3H], m), 6.96 (C2”H, [1H], d, 3J=1.5), 6.83 (C6”H, [1H], dd,
24

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3J=9.0, 4J=2.5), 4.02 (C1xH2, [2H], m), 3.91 (C8H2, [2H], t, 3J=6.5), 3.84 (OCH3, [3H], s), 3.17
(CaH(E),CbH(E), [2H], m), 2.98 (CeH(E), [1H], bs), 2.49 (C5H2, [2H], t, 3J=7.0), 2.41 (C4xH2, [2H], bs),
2.10 – 1.94 (CaH(A),CeH(A),CcH(E), [3H], m), 1.91 (C7H2, [2H], q, 3J=6.5), 1.79 (CdH2, [2H], bs), 1.75 –
1.65 (C2xH2,C6H2, [4H], m), 1.61 (C3xH2, [2H], bs), 1.48 (CcH(A), [1H], m).
13C NMR (125 MHz, CDCl3): δ 162.2 (C4’H, d, 1J=246.8), 162.0 (C3, s), 153.8 (C5”, s), 151.6 (C1, s),
149.9 (C4a, s), 132.5 (C2’,C6’, d, 3J=8.2), 131.3 (C7”a, s), 129.2 (C1’, d, 4J=3.4), 127.1 (C3”a, s), 121.0
(C2”, s), 119.4 (C3”, s), 115.5 (C3’,C5’, d, 2J=21.5), 112.2 (C6”, s), 111.8 (C7”, s), 111.4 (C4, s), 100.8
(C4”, s), 60.9 (Ca, s), 58.8 (C4x, s), 55.9 (OCH3, s), 54.0 (Ce, s), 42.7 (C8, s), 41.6 (C1x, s), 33.8 (Cb, s),
31.2 (Cc, s), 26.7 (C5, s), 25.7 (Cd, s), 25.7 (C2x, s), 24.2 (C3x, s), 21.7 (C7, s), 18.6 (C6, s).
ESI-HRMS m/z: Calcd for C32H38FN4O3 [M+H]+ 545.29279. Found: 545.29269.
3.2.3.26. 2-[4-[3-(5-methoxy-1H-indol-3-yl)-1-piperidyl]butyl]-4-(p-tolyl)-5,6,7,8-tetrahydropyrido[1,2-
c]pyrimidine-1,3-dione (10.26).
The title compound was isolated as a white powder. Yield: 51.8 %, m.p. 113.9-115.6 °C. HPLC tR = 5.72
min, 99.5% purity.
1H NMR (500 MHz, CDCl3): δ 8.01 (N1”H, [1H], bs), 7.18 (C2’H,C6’H,C7”H, [3H], m), 7.09 (C4”H, [1H],
d, 4J=2.5), 7.07 (C3’H,C5’H, [2H], dt, 3J=8.0), 6.96 (C2”H, [1H], d, 3J=2.0), 6.83 (C6”H, [1H], 4d, 3J=8.5,
4J=2.5, 5J=0.5), 4.02 (C1xH2, [2H], m), 3.91 (C8H2, [2H], t, 3J=6.5), 3.84 (OCH3, [3H], s), 3.16
(CaH(E),CbH(E), [2H], m), 2.97 (CeH(E), [1H], pd), 2.52 (C5H2, [2H], t, 3J=7.0), 2.41 (C4xH2, [2H], bs),
2.360 (CH3, [3H], s), 2.10 – 1.92 (CaH(A),CeH(A),CcH(E), [3H], m), 1.89 (C7H2, [2H], q, 3J=7.0), 1.78
(CdH2, [2H], m), 1.74 – 1.64 (C6H2,C2xH2, [4H], m), 1.60 (C3xH2, [2H], m), 1.40 (CcH(A), [1H], kd, 3JA-
A=12.5, 3JA-E=5.0).
13C NMR (125 MHz, CDCl3): δ 162.1 (C3, s), 153.8 (C5”, s), 151.7 (C1, s), 149.5 (C4a, s), 137.4 (C4’, s),
131.3 (C7”a, s), 130.6 (C2’,C6’, s), 130.3 (C1’, s), 127.1 (C3”a, s), 121.1 (C2”, s), 119.5 (C3”, s), 112.3
(C4, s), 112.2 (C6”, s), 111.8 (C7”, s), 100.7 (C4”, s), 61.0 (Ca, s), 58.8 (C4x, s), 55.9 (OCH3, s), 54.0 (Ce,
s), 42.6 (C8, s), 41.5 (C1x, s), 33.8 (Cb, s), 31.3 (Cc, s), 26.7 (C5, s), 25.8 (Cd, s), 25.5 (C2x, s), 24.3 (C3x,
s), 21.8 (C7, s), 21.3 (CH3, s), 18.6 (C6, s).
ESI-HRMS m/z: Calcd for C33H41N4O3 [M+H]+ 541.31787. Found: 541.31768.
3.2.3.27. 2-[4-[3-(5-methoxy-1H-indol-3-yl)-1-piperidyl]butyl]-4-(4-methoxyphenyl)-5,6,7,8-
tetrahydropyrido[1,2-c]pyrimidine-1,3-dione (10.27).
The title compound was isolated as a white powder. Yield: 57.0 %, m.p. 121.0-122.7 °C. HPLC tR = 4.84
min, 99.7% purity.
1H NMR (500 MHz, CDCl3): δ 8.09 (N1”H, [1H], bs), 7.21 (C7”H, [1H], dd, 3J=8.5, 5J=0.5), 7.10
(C2’H,C6’H,C4”H, [3H], m), 6.94 (C2”H, [1H], bs), 6.91 (C3’H,C5’H, [2H], dt, 3J=9.0, 4J=2.0), 6.82 (C6”H,
[1H], m, 3J=9.0, 4J=2.5, 5J=0.5), 4,021 (C1xH2, [2H], m), 3.91 (C8H2, [2H], t, 3J=7.5), 3.84 (C5”-OCH3,
[3H], s), 3.81 (C4’-OCH3, [3H], s), 3.19 (CaH(E),CbH(E), [2H], bs), 3.01 (CeH(E), [1H], bs), 2.52 (C5H2,
[2H], t, 3J=7.0), 2.45 (C4xH2, [2H], bs), 2.05 (CaH(A),CeH(A),CcH(E), [3H], m), 1.90 (C7H2, [2H], q,
3J=7.0), 1.80 (CdH2, [2H], bs), 1.75 – 1.58 (C2xH2,C3xH2,C6H2, [6H], m), 1.48 (CcH(A), [1H], m).
13C NMR (125 MHz, CDCl3): δ 162.2 (C3, s), 159.0 (C4’, s), 153.8 (C5”, s), 151.7 (C1, s), 149.7 (C4a, s),
131.9 (C3’,C5’, s), 131.3 (C7”a, s), 127.1 (C3”a, s), 125.5 (C1’, s), 121.1 (C2”, s), 119.3 (C3”, s), 114.0
25

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(C2’,C6’, s), 112.2 (C4, s), 112.0 (C6”, s), 111.9 (C7”, s), 100.7 (C4”, s), 61.0 (Ca, s), 58.7 (C4x, s), 56.0
(C5”-OCH3, s), 55.3 (C4’-OCH3, s), 53.9 (Ce, s), 42.6 (C8, s), 41.4 (C1x, s), 33.7 (Cb, s), 31.2 (Cc, s), 26.7
(C5, s), 25.7 (Cd, s), 25.6 (C2x, s), 24.0 (C3x, s), 21.8 (C7, s), 18.6 (C6, s).
ESI-HRMS m/z: Calcd for C33H41N4O4 [M+H]+ 557.31278. Found: 557.31245.
3.3. X-ray crystallography
The diffraction data for 9.3 were collected at room temperature with a SuperNova diffractometer
and Cu Kα radiation (λ = 1.54184 Å). The data were corrected for Lorentz and polarization effects,
and a multi-scan absorption correction was applied. The structure was solved using direct methods
implemented in the SHELXS, and refined by the full matrix least-squares on F2 with the SHELXL-
2018/3 program [50]. All non-H atoms were refined with anisotropic displacement parameters. The
H-atoms attached to carbon were positioned geometrically and refined using the riding model with
Uiso(H) = 1.2 Ueq(C). The nitrogen bonded H-atoms were found in the difference-Fourier map and
their positions and isotropic displacement parameters were refined.
3.4. Biological tests
3.4.1. In vitro tests
3.4.1.1. 5-HT1A binding assay
Radioligand binding was performed using membranes from CHO-K1 cells stably transfected
with the human 5-HT1A receptor (Perkin Elmer). All assays were carried out in duplicate. Working
solution (50 µL) of the tested compounds, 50 µL [3H]-8-OH-DPAT (spec. act. 139.7 Ci/mmol, final
concentration 1 nM) and 150 µL diluted membranes (10 µg 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 using a Rainin Liquidator 96-well pipetting station (Mettler
Toledo). Serotonin (10 μM) 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/C filter mate presoaked with 0.3% polyethyleneimine for 30 min. Ten rapid
washes with 200 µL 50 mM Tris buffer (4 °C, pH 7.4) were performed using a Harvester-96 MACH III
FM automated harvester system (Tomtec). The filter mates were dried at 37 °C in a forced-air fan
incubator and then MeltiLex solid scintillator was melted onto the filter mates at 90 °C for 4 min.
Radioactivity was counted in a MicroBeta2 scintillation counter (PerkinElmer). Data were fitted to a
one-site curve-fitting equation with Prism 6 (GraphPad Software) and Ki values were estimated from
the Cheng−Prusoff equaꢀon [51]:
ꢂꢃꢄꢅ
ꢀ =
ꢁ
ꢆꢇ
1 +
ꢀꢈ
LO – labeled ligand concentration
KD – dissociation constant of labeled ligand
Kd value was determined in our experimental conditions.
26

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3.4.1.2. SERT binding assay
Radioligand binding was performed using rat cortex tissue. All assays were carried out in
duplicate. Working solution (50 µL) of the tested compounds, 50 µL [3H]-citalopram (spec. act. 84.5
Ci/mmol, 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 a
Rainin Liquidator 96-well pipetting station (Mettler Toledo). 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 a GF/B filter mate presoaked with
0.3% polyethyleneimine for 30 min. Ten rapid washes with 200 µL of 50 mM Tris buffer (4 °C, pH 7.7)
were performed using a Harvester-96 MACH III FM automated harvester system (Tomtec). The filter
mates were dried at 37 °C in a forced-air fan incubator and then solid scintillator MeltiLex was
melted onto the filter mates at 90 °C for 5 min. Radioactivity was counted in MicroBeta2 scintillation
counter (PerkinElmer). Data were fitted to a one-site curve-fitting equation with Prism 6 (GraphPad
Software) and Ki values were estimated from the Cheng−Prusoff equaꢀon [51]. Kd value was
determined in our experimental conditions.
3.4.1.3. 5-HT2A, 5-HT6, 5-HT7 and D2 binding assays
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. [52]. For the assays, HEK293 cell cultures stably expressing
the investigated human receptors were used. Cell pellets were thawed and homogenized in 20
volumes of assay buffer using an Ultra Turrax tissue homogenizer and 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 and 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 a 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 of 5-HT in 5-HT7 receptors binding experiments, whereas 10 µM chlorpromazine,
10 µM methiothepine, or 1 µM (+)butaclamol were used in 5-HT2A, 5-HT6 and D2 receptors assays,
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 [51]. Results were
expressed as means of at least two separate experiments.
3.4.2. In vivo experiments
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. The experiments were performed on male CD-1 mice (28–40 g) on a natural day-night
cycle (October - November) and housed under standard laboratory conditions. They had free access
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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
hydrobromide (8-OH-DPAT, Research Biochemical Inc.) was used as aqueous solutions. Compounds:
10.1-10.3, 10.6, 10.7 and 10.16 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), 10.1-10.3, 10.6, 10.7 and 10.16 were given
intraperitoneally (ip) in a volume of 10 mL/kg/mice. Induced hypothermia test: the obtained data
were analyzed by Dunnett’s test (one drug administration) or by the Newman-Keuls test (two drugs
administration). Forced swim test: the obtained data was evaluated by the one-way analysis of
variance (ANOVA) followed by the Dunnett's multiple comparisons test: p<0.05 was considered
significant.
3.4.2.1. Body temperature in mice
The rectal body temperature in mice (measured with an Ellab thermometer) were recorded
30, 60, 90 and 120 min after administration of compounds: 10.1-10.3, 10.6, 10.7 and 10.16.
Separately, the effect of compounds 10.1, 10.3, 10.6, 10.7 and 10.16 which had no effect on baseline
body temperature on the hypothermia induced by 8-OH-DPAT (5 mg/kg s.c.) was tested. Compounds:
10.1, 10.3, 10.6, 10.7 and 10.16 were administered 45 min before 8-OH-DPAT, and the rectal body
temperature was recorded 15, 30, 45 and 60 min later. The absolute mean body temperatures were
within a range 36.9 ± 0.5 ⁰C. The results were expressed as a change in body temperature (Δt) with
respect to the basal body temperature, as measured at the beginning of the experiment.
3.4.2.2. Forced swim test in mice
Forced swim test (FST) was carried out according to the method of Porsolt et al. [53]. Mice
were placed individually into glass cylinders (height 25 cm, diameter 10 cm) containing 20 cm of
water, maintained at 23 ± 1 ⁰C. The animals were left in the cylinder for 6 min. The total duration of
immobility was measured during the last 4 min. The animals were considered to be immobile
when they floated passively in the water. Tested compounds were administered 30 min before the
test.
3.4.3. Metabolic stability
Stock solutions of studied compounds were prepared at concentration of 10 mM in DMSO.
Working solutions were prepared by dilution of stock with reaction buffer or acetonitrile, final
concentration of organic solvent did not exceed 0.1%. Incubation mixture contained 1 μM of a
studied derivative, 1 mM of NADPH (Sigma-Aldrich) and 0.5 mg/mL of human liver microsomes (HLM,
Sigma-Aldrich) in potassium phosphate buffer (0.1 M, pH 7.4). Incubation was carried out in shaking
thermostat at 37 ⁰C and started by addition of compound of interest. 20 μL samples were taken after
5, 15, 30, 45 and 60 min. Enzymatic reaction was terminated by the addition of the equal volume of
ice-cold acetonitrile containing 10 μM of buspirone as internal standard. Control incubations were
performed without NADPH to assess chemical instability. After collection, samples were immediately
centrifuged (10 min, 10,000 rpm) and resulted supernatant was directly analyzed or kept in −80 ⁰C
until LC-MS analysis. Natural logarithm of a compound over IS peak area ratio was plotted vs.
28