Synthesis of novel pyrido[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.2019.01.031

The study enabled obtaining a number of new derivatives of 4-aryl-pyrido[1,2-c]pyrimidine 9.1–9.27 having conformationally restricted tryptamine moiety. In vitro studies (RBA) have shown that derivatives 9.1, 9.2, 9.4, 9.7, 9.9, 9.14 and 9.27 exhibit high affinity to molecular targets 5-HT_1A receptor and SERT protein. In general, compounds with an unsubstituted or a para-substituted benzene ring of the pyrido[1,2-c]pyrimidine residue in the terminal part were characterized by higher binding ability, which can be justified by the greater flexibility of the structure. For the selected compounds 9.1, 9.7, 9.9 and 9.27, further in vitro, in vivo and metabolic stability tests were performed. The in vitro studies in the extended receptor profile (D₂, 5-HT_2A, 5-HT₆ and 5-HT₇) indicated their selectivity toward the 5-HT_1A receptor and SERT protein. The in vivo studies (8-OH-DPAT-induced hypothermia in mice, FST) revealed that the compound 9.1 has the properties of presynaptic agonist of the 5-HT_1A receptor, and compound 9.7 demonstrated the properties of a presynaptic antagonist of the 5-HT_1A receptor. Metabolic stability studies, in turn, showed that compounds 9.1, 9.7 and 9.9, having an unsubstituted indole residue, were more resistant to biotransformation reactions of the first pass phase than was compound 9.27 containing a 5-methoxy-substituted indole residue. The obtained results allowed further optimization of the structure.

Full text of DOI:10.1016/j.ejmech.2019.01.031

Accepted Manuscript
Synthesis of novel pyrido[1,2-c]pyrimidine derivatives with rigidized tryptamine moiety
as potential SSRI and 5-HT receptor ligands
1A
Grzegorz Ślifirski, Marek Król, Jerzy Kleps, Szymon Ulenberg, 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)30041-8
DOI:
https://doi.org/10.1016/j.ejmech.2019.01.031
EJMECH 11038
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 21 September 2018
Revised Date: 11 January 2019
Accepted Date: 13 January 2019
Please cite this article as: G. Ślifirski, M. Król, J. Kleps, S. Ulenberg, M. Belka, T. Bączek, A. Siwek,
K. Stachowicz, B. Szewczyk, G. Nowak, A. Bojarski, A.E. Kozioł, J. Turło, F. Herold, Synthesis of
novel pyrido[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://doi.org/10.1016/
1A
j.ejmech.2019.01.031.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Synthesis of novel pyrido[1,2-c]pyrimidine derivatives with rigidized tryptamine moiety as
potential SSRI and 5-HT1A receptor ligands
a
a*
a
b
b
Grzegorz Ślifirski , Marek Król , Jerzy Kleps , Szymon Ulenberg , 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 Warsaw, 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. Tel.: +48 501499407 ; fax: +48 22 57 20 631.
E-mail address: marek.krol@wum.edu.pl
Keywords: Antidepressants, Pyrido[1,2-c]pyrimidines, Dual 5-HT1A/SERT activity
List of abbreviations: WHO, World Health Organization; 5-HT1AR, serotonin 5-HT1A receptors;
SERT, serotonin transporter; SAR, structure-activity relationship; Ki, inhibitory constant; NMR,
nuclear magnetic resonance; HRMS, high resolution mass spectroscopy; RBA, radioligand
binding assay; 8-OH-DPAT, 8-hydroxy-2-(di-n-propylamino)tetralin; TEA, triethylamine;
DMSO, dimethyl sulfoxide; XRD, X-ray diffraction; clogP, calculated logarithm of
octanol/water partition coefficient; RBA, radioligand binding assay; FST, forced swimming
test
Abstract
The study enabled obtaining a number of new derivatives of 4-aryl-pyrido[1,2-c]pyrimidine 9.1-
9.27 having conformationally restricted tryptamine moiety. In vitro studies (RBA) have shown that
derivatives 9.1, 9.2, 9.4, 9.7, 9.9, 9.14 and 9.27 exhibit high affinity to molecular targets 5-HT1A
receptor and SERT protein. In general, compounds with an unsubstituted or a para-substituted
benzene ring of the pyrido[1,2-c]pyrimidine residue in the terminal part were characterized by higher
binding ability, which can be justified by the greater flexibility of the structure. For the selected
compounds 9.1, 9.7, 9.9 and 9.27, further in vitro, in vivo and metabolic stability tests were
performed. The in vitro studies in the extended receptor profile (D2, 5-HT2A, 5-HT6 and 5-HT7)
indicated their selectivity toward the 5-HT1A receptor and SERT protein. The in vivo studies (8-OH-
DPAT-induced hypothermia in mice, FST) revealed that the compound 9.1 has the properties of
presynaptic agonist of the 5-HT1A receptor, and compound 9.7 demonstrated the properties of a
presynaptic antagonist of the 5-HT1A receptor. Metabolic stability studies, in turn, showed that
compounds 9.1, 9.7 and 9.9, having an unsubstituted indole residue, were more resistant to
1

ACCEPTED MANUSCRIPT
biotransformation reactions of the first pass phase than was compound 9.27 containing a 5-methoxy-
substituted indole residue. The obtained results allowed further optimization of the structure.
1. Introduction
Depression is a mental illness affecting over 350 million people around the world [1]. As
many as 80% of patients suffer recurrence after they have recovered [2]. Emotional, somatic and
functional disorders accompanying depression make this disease the primary cause of disability in
relation to the entire population. According to the WHO, by 2030 serious depressive illness will have
become the main cause of disability [3,4].
Fig. 1. SSRI drug structures.
A breakthrough in the treatment of depressive disorders was the introduction of SSRIs, which
are currently the first-line antidepressants (Fig. 1). Their mechanism is based on the serotoninergic
system and their molecular target is serotonin transporter protein (SERT). The effectiveness of these
therapeutics still leaves much to be desired, as up to 40% patients do not respond to treatment at all
[5]. Serotonin reuptake inhibitors are also seriously affected by the need to administer them for a
longer period of time (2–6 weeks) before a clear improvement of the patient’s mood can be
observed, which is due to latency.
Rodent models have shown that 5-HT1A receptor agonists (e.g. 8-OH-DPAT) may have
potential high antidepressant activity [6]. These effects are blocked by antagonists of 5-HT1A-R,
indicating that said activity is specific for transduction of the 5-HT1A receptor [7,8]. This is probably
connected to expression of 5-HT1A heteroreceptors in the limbic system [9]. The 5-HT1A
autoreceptors, on the other hand, work in opposition, causing prodepressive effects. Their
stimulation leads to hyperpolarization and reduced transmission of seam neurons, reducing the
release of serotonin in the projection regions [10]. Therefore, increased stimulation of 5-HT1A
autoreceptors during SSRI administration simultaneously inhibits activity of serotonin neurons [11].
Occurrence of the latency period is explained by the desensitization model of 5-HT1A autoreceptors
[12,13]. This indicates that long-term antidepressant therapy reduces negative feedback in the
serotoninergic system caused by the stimulation of 5-HT1A autoreceptors. This process occurs
through desensitization/negative regulation of 5-HT1A autoreceptors, which results in increased
serotonin transmission [13]. Accordingly, the use of the 5-HT1A autoreceptor activation mechanism
has been introduced as a strategy to supplement the inhibition of serotonin reuptake. The use of
agonists or partial agonists of the 5-HT1A receptor for this purpose appears to be a more favorable
choice, as the sensitivity of postsynaptic receptors is not reduced even after repeated exposure to
the 5-HT1A/SSRI agonist, as opposed to the response to the 5-HT1A/SSRI antagonist [11,14]. The
2

ACCEPTED MANUSCRIPT
legitimacy of this approach has been confirmed by the FDA registration of vilazodone and
vortioxetine (Fig. 2) [15–20].
Fig. 2. SSRI+ drug structures.
Our laboratory has been conducting research on double-binding ligands to the 5-HT1A
receptor and SERT protein among new pyrido[1,2-c]pyrimidine derivatives for a decade. Earlier works
described pyrido[1,2-c]pyrimidine derivatives with the fragment of 3-(piperidin-4-yl)-1H-indole in the
pharmacophore part; an array of high-binding compounds were obtained for both molecular targets,
as well as with an appropriate functional profile for the 5-HT1A receptor – pre- and postsynaptic
agonism [21–25].
Our current research has focused on pyrido [1,2-c] pyrimidine derivatives containing a 3-
(piperidin-3-yl)-1H-indole residue, where the aminoethyl chain on the indole is conformationally
restricted. The research objective was the synthesis of newly designed derivatives of the series 2H-
pyrido[1,2-c]pyrimidine based on previously obtained structures in which the pharmacophore part
had been redesigned. The introduced 3-(piperidin-3-yl)-1H-indole residue is more similar in its
structure to serotonin, and its conformationally constrained limitation (Fig. 3) aimed to increase the
activity of compounds by increasing the affinity for 5-HT1A and SERT, confirmed in numerous
references [26–31]. Moreover, residues with such a structure also have an effect on higher metabolic
stability [32].
Fig. 3. Comparison of the leading structure and structures examined in the project.
The SAR analysis investigated the effect of R3 (-H, -F, -OCH3) substituents present in the
pharmacophore part of the indole ring and in the terminal part of the benzene ring of the pyrido[1,2-
c]pyrimidine (-H, -Cl, -F, -CH3, -OCH3) residue on the affinity of the derivatives obtained for both
molecular targets (5-HT1A, SERT). For selected compounds, in vitro studies were also performed in the
extended receptor profile – D2, 5-HT2A, 5-HT6 and 5-HT7. Their functional profile to the 5-HT1A
3

ACCEPTED MANUSCRIPT
receptor (agonism/antagonism) and behavioral profile as well as their metabolic stability were also
tested.
2. Results and discussion
2.1. Chemistry
The designed compounds 9.1–9.27 were obtained by synthesis in accordance with Scheme 1.
Two synthon series were necessary for the syntheses thereof. The first series of syntons were N-
bromobutyl-4-aryl-pirydo[1,2-c]pyrimidine derivatives 5.1–5.9, followed by 3-(piperidin-3-yl)-1H-
indole derivatives 8.1–8.3 (Scheme 2).
Scheme 1. The synthesis pathway of compounds 9.1–9.27. 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) 8.1–8.3, acetonitrile, K2CO3, Δ.
Synthons 5.1–5.9 were obtained in accordance with pathways that we had developed earlier [21,23–
25]. Another group of synthons 8.1–8.3 was obtained in a two-step synthesis. 7.1–7.3 derivatives of
3-(1-phenyl-1,2,5,6-tetrahydropyridin-3-yl)-1H-indole were obtained based on the method of [33] in
condensation of indole derivatives 8.1–8.3 with 1-benzyl-3-piperidone hydrochloride. This formula
was modified by replacement of Na/MeOH with KOH and isopropanol, obtaining derivatives 7.1–7.3.
Hydrogenolysis with hydrogenation of compounds 7.1–7.3 was carried out with Pd/C 10% in
methanol at a pressure of 5 atm. The obtained bases 8.1–8.3 were converted into hydrochlorides, for
1
which melting points and composition were determined, and H, 13C NMR and XRD tests were
conducted. Physicochemical data for these compounds have not been reported in the literature so
far. There was only the application of base 8.1 indicated [34–36] (Scheme 2).
4

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Scheme 2. The synthesis pathway of substrates: 3-piperidyn-3-yl-1H-indole derivatives 8.1 (R3 = -H);
8.2 (R3 = -F); 8.3 (R3 = -OCH3). Reagents and conditions: (i) IPA, KOH, Δ, Ar; (ii.) 10% Pd/C, CH3OH, p, Δ;
(iii) CH3OH/HCl (g).
The structure and composition of all of the newly obtained derivatives 9.1–9.27 were proven by
1
HRMS and by H and 13C NMR spectroscopy. X-ray crystallography tests were performed for the
compounds
8.1–8.2.
Table 1
5-HT1A receptor and SERT binding affinities as well as clogP (“ACD/ChemSketch,” 2017) of 2H-
pyrido[1,2-c]pyrimidine derivatives.
Ki [nM]
Compound
9.1
R1
-H
-Cl
R2
-H
-H
-H
-H
-H
-Cl
-F
-CH3
-OCH3
-H
-H
-H
-H
-H
-Cl
-F
-CH3
-OCH3
-H
R3
-H
-H
-H
-H
-H
-H
-H
-H
-H
-F
-F
-F
-F
-F
-F
-F
-F
-F
5-HT1A
SERT
clog P
4.78
5.16
4.83
5.24
4.78
5.50
4.79
5.24
4.72
4.92
5.29
4.97
5.38
4.92
5.63
4.93
5.38
4.86
4.69
5.07
22.0 ± 2.7
50.0 ± 2.0
53.0 ± 5.0
102.0 ± 6.5
132.0 ± 10.7 411.0 ± 22.9
294.0 ± 24.5
46.0 ± 1.5
63.0 ± 1.5
42.0 ± 1.5
209.0 ± 10.5
378.0 ± 17.4 255.0 ± 17.9
218.0 ± 13.5 115.0 ± 22.0
376.0 ± 22.0 338.0 ± 45.0
80.0 ± 9.5
155.0 ± 8.5
147.0 ± 10.5
68.0 ± 7.5
103.0 ± 10.2
240.0 ± 6.5
76.0 ± 7.0
9.2
9.3
9.4
9.5
9.6
9.7
9.8
9.9
-F
-CH3
-OCH3
-H
-H
-H
-H
-H
-Cl
-F
-CH3
-OCH3
-H
-H
-H
-H
-H
-Cl
-F
82.0 ± 6.0
46.0 ± 2.8
207.0 ± 15.0
41.0 ± 1.8
8.0 ± 0.5
9.10
9.11
9.12
9.13
9.14
9.15
9.16
9.17
9.18
9.19
9.20
9.21
9.22
9.23
9.24
9.25
9.26
9.27
Imipramine
118.0 ± 9.4
86.0 ± 11.0
30.0 ± 1.4
136.0 ± 14.0 135.0 ± 14.5
140.0 ± 17.0 27.0 ± 1.5
-OCH3 261.0 ± 20.0 248.0 ± 20.8
-OCH3 176.0 ± 17.0 459.0 ± 32.5
-OCH3 130.0 ± 14.0 1670.0 ± 179.0 4.75
-OCH3 476.0 ± 4.0
-OCH3 69.0 ± 3.5
-OCH3 257.0 ± 14.0 490.0 ± 29.5
-OCH3 281.0 ± 16.5 192.0 ± 7.5
-H
-H
-H
-H
-Cl
-F
-CH3
-OCH3
-H
-H
-H
87.0 ± 6.5
386.0 ± 15.0
5.15
4.70
5.41
4.70
5.15
4.64
-CH3 -OCH3 333.0 ± 17.5 173.0 ± 16.8
-OCH3 -OCH3 62.0 ± 3.5
-H
86.0 ± 10.0
29.0 ± 2.0
5

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Serotonin
3.0 ± 0.3
Table 2
Binding affinity data on serotonin 5-HT1A, 5-HT2A, 5-HT6, 5-HT7, and dopamine D2 receptors of the
investigated 2H-pyrido[1,2-c]pyrimidine derivatives.
Ki [nM]
Compound
9.1
R1
-H
-H
R2
-H
-F
R3
-H
-H
-H
5-HT2A 5-HT6 5-HT7
D2
435 1115 17670
697 1444 963
1709 1922 1129
433
435
21
9.7
9.9
-H -OCH3
9.27
-H -OCH3 -OCH3
314
4.6
2.8
n.d.
n.d.
n.d.
n.d.
690
7
958 11160
Olanzapine
Mianserine
Clozapine
Haloperidol
Apomorphine
Chloropromazine
n.d.
n.d.
18
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
4.5
42
n.d.
n.d.
n.d.
n.d.
n.d.
1.8
2.2 Biological evaluation
2.2.1 Radioligand binding assay for 5-HT1A receptor and SERT
Compounds 9.1–9.27 were tested for in vitro affinity for 5-HT1A receptor and SERT protein by
a radioligand binding assay (Table 1).
Binding affinity data for 5-HT1A and SERT for compounds 9.1–9.27 were used in SAR analysis.
The influence of substituents in the ortho (R1) and para (R2) positions in the terminal part of
the benzene ring of the pyrido [1,2-c] pyrimidine residue and the presence of R3 (-H, -F, -OCH3)
substituents in the indole residue of the pharmacophore part of the compounds were analyzed.
Analysis of the obtained results showed high affinity of compounds 9.1–9.4, 9.7–9.9, 9.14,
9.23 and 9.27 to the 5-HT1A receptor, with the Ki values of those compounds ranging from 22.0 to
102.0 nM. It can be clearly seen that compounds with an unsubstituted indole ring had higher affinity
to the 5-HT1A receptor than did their substituted analogs (R3 = -F, -OCH3). In turn, the analysis of the
effect of substituents on the terminal part present in the benzene ring showed that derivatives
containing a para-substituted ring had greater 5-HT1A binding ability than did ortho-substituted
derivatives. Compounds having a methoxy substituent in the terminal portion (9.5, 9.9, 9.14, 9.18,
9.23 and 9.27), regardless of its position in the benzene ring, generally had similar high to moderate
binding affinity values toward the 5-HT1A receptor.
While studying the effect of R1, R2 and R3 in both parts of the molecule for their affinity to the
SERT protein, it can be concluded that compound 9.10 showed the highest affinity (Ki = 8 nM) to
SERT, while the affinity of compounds 9.16 and 9.18 was high (Ki = 27.0–30.0 nM), and the affinity of
a series of compounds 9.1, 9.2, 9.4, 9.6, 9.7, 9.9, 9.12, 9.14–9.16, 9.18, 9.22 and 9.27 was high to
moderate. This molecular target shows that the highest binding ability was achieved by derivatives in
which R3 = -H or -F in the 3-(piperidin-3-yl)-1H-indole residue of the pharmacophore part. In turn,
methoxy-substituted analogs (R3 = -OCH3) showed significantly lower activity. As with the 5-HT1A
receptor, higher activity against this molecular target was demonstrated by compounds with a para-
6

ACCEPTED MANUSCRIPT
substituted benzene ring of the pyrido[1,2-c]pyrimidine residue in the terminal part, and this
tendency is even more visible in SERT. NMR studies show that the benzene ring in the ortho-
substituted compounds cannot rotate freely due to steric barriers (the NMR spectra show temporary
“magnetic stereoisomers” manifested by double instead of single signals of R1 hydrogens). This is
possible in para-substitute compounds, which may imply the hypothesis that a more flexible terminal
part can affect higher binding affinity to 5-HT1A and SERT.
The obtained clog P values for the compounds 9.1–9.27 are in the range from 4.64 to 5.63.
Value presented by the compounds 9.1, 9.3, 9.5, 9.7, 9.9, 9.10, 9.12, 9.14, 9.16, 9.18, 9.19, 9.21,
9.23, 9.25 and 9.27 were below 5, which meets the condition of proper penetration through the
membrane and complies with the Lipinski ‘rule of five’ [38]. In this respect, they are similar to
vortioxetine (clog P = 4.26), an SSRI+ antidepressant available on the market.
During the studies it was possible to obtain compounds 9.1, 9.2, 9.4, 9.7, 9.9, 9.14 and 9.27
with high binding affinity to both molecular targets (5-HT1A and SERT) and clog P values lower than 5
(9.1, 9.7, 9.9, 9.14 and 9.27).
Among the above-mentioned derivatives, compounds 9.1, 9.7, 9.9 and 9.27 were selected to
be subject to in vitro studies in the extended receptor profile to D2, 5-HT2A, 5-HT6 and 5-HT7 (Table 2).
The results of the tests showed that these compounds are selective ligands of molecular targets –
receptor 5-HT1A and SERT protein – due to the low affinity of the compounds to other receptors
tested in the profile.
2.2.2 In vivo studies
To determine the profile of functional activity of the selected ligands, behavioral tests were
performed.
Table 3
The effect of compounds: 9.1, 9.7, 9.9 and 9.27 on the body temperature in mice.
Treatment
Dose (mg/kg)
Δt ± SEM (0 C)
30 min
0.4ꢀ±ꢀ0.2
–0.1ꢀ±ꢀ0.2
–0.9ꢀ±ꢀ0.3c
P<0.0001
60 min
90 min
0.1ꢀ±ꢀ0.1
120 min
0.1ꢀ±ꢀ0.3
Vehicle
9.1
–
5
20
0.1ꢀ±ꢀ0.3
–0.4ꢀ±ꢀ0.1
–0.3ꢀ±ꢀ0.3
ns
–0.5ꢀ±ꢀ0.1b
0.1ꢀ±ꢀ0.2
–0.8ꢀ±ꢀ0.1c
0.2ꢀ±ꢀ0.1
Pꢀ=ꢀ0.0083
Pꢀ=ꢀ0.0003
Vehicle
9.7
–
5
20
0.4ꢀ±ꢀ0.2
0.4ꢀ±ꢀ0.1
–0.2ꢀ±ꢀ0.3
ns
0.1ꢀ±ꢀ0.3
0.4ꢀ±ꢀ0.2
0.5ꢀ±ꢀ0.2
ns
0.1ꢀ±ꢀ0.1
0.3ꢀ±ꢀ0.2
0.2ꢀ±ꢀ0.2
ns
0.1ꢀ±ꢀ0.3
0.2ꢀ±ꢀ0.1
0.1ꢀ±ꢀ0.2
ns
Vehicle
9.9
–
5
20
0.4ꢀ±ꢀ0.2
0.3ꢀ±ꢀ0.1
–1.6ꢀ±ꢀ0.3c
Pꢀ<ꢀ0.0001
0.1ꢀ±ꢀ0.3
0.4ꢀ±ꢀ0.1
0.3ꢀ±ꢀ0.2
Pꢀ=ꢀ0.0099
0.1ꢀ±ꢀ0.1
0.4ꢀ±ꢀ0.1
0.0ꢀ±ꢀ0.1
ns
0.1ꢀ±ꢀ0.3
0.5ꢀ±ꢀ0.1
0.3ꢀ±ꢀ0.2
ns
Vehicle
–
0.0ꢀ±ꢀ0.1
0.1ꢀ±ꢀ0.1
–0.1ꢀ±ꢀ0.1
–0.1ꢀ±ꢀ0.1
7

ACCEPTED MANUSCRIPT
9.27
5
15
17.5
20
–0.2ꢀ±ꢀ0.2
–0.5ꢀ±ꢀ0.3
–0.6ꢀ±ꢀ0.2 a
–2.4ꢀ±ꢀ0.3c
Pꢀ<ꢀ0.0001
0.2ꢀ±ꢀ0.3
0.2ꢀ±ꢀ0.3
–0.2ꢀ±ꢀ0.2
–1.0ꢀ±ꢀ0.4b
Pꢀ=ꢀ0.0176
0.2ꢀ±ꢀ0.2
0.3ꢀ±ꢀ0.4
–0.1ꢀ±ꢀ0.1
0.1ꢀ±ꢀ0.2
ns
–0.2ꢀ±ꢀ0.3
0.2ꢀ±ꢀ0.3
0.1ꢀ±ꢀ0.1
0.0ꢀ±ꢀ0.2
ns
Vehicle
WAY100635
8-OH-DPAT
–
0.1
5
0.2ꢀ±ꢀ0.1
0.1ꢀ±ꢀ0.2
0.1ꢀ±ꢀ0.2
0.1ꢀ±ꢀ0.2
0.1ꢀ±ꢀ0.2
–0.1ꢀ±ꢀ0.2
–0.1ꢀ±ꢀ0.1
ns
0.1ꢀ±ꢀ0.2
–0.2ꢀ±ꢀ0.1
0.3ꢀ±ꢀ0.3
ns
–1.7ꢀ±ꢀ0.2 c
Pꢀ<ꢀ0.0001
–1.1ꢀ±ꢀ0.2 c
Pꢀ<ꢀ0.005
The investigated compounds were administered 30 min before the test, apꢀ<ꢀ0.05 vs vehicle,
bpꢀ<ꢀ0.01 vs vehicle, cpꢀ<ꢀ0.001 vs vehicle, ns = non-significant.
It is known that 8-OH-DPAT as a 5-HT1A receptor agonist can induce hypothermia in mice,
through 5-HT1A somatodendritic receptors [39,40]. Moreover, this effect can be abolished by
WAY100635 [41], 5-HT1A receptor antagonist. Based on this knowledge, we tested compounds 9.1,
9.7, 9.9 and 9.27 in a commonly used in vivo panel of tests, to assess their functional 5-HT1A receptor
activity. Compounds 9.1, 9.9 and 9.27, as 8-OH-DPAT, induced hypothermia in mice (Table 3).
Hypothermia induced by compound 9.1 (20 mg/kg) was attenuated by WAY100635 (0.1 mg/kg)
(Table 4). Concluding, the decrease in mouse body temperature produced by compound 9.1 can be
accounted as a measure of its presynaptic 5-HT1A agonistic activity. Tested derivative 9.1 was
ineffective in the forced swimming test in mice, so we can conclude lack of postsynaptic 5-HT1A
receptor activity [39]. Compound 9.7 (20 mg/kg) decreased the hypothermia induced by 8-OH-DPAT
(5 mg/kg) in mice (Table 5) demonstrating its presynaptic 5-HT1A receptor antagonist activity.
Table 4
The effect of WAY100635 (0.1 mg/kg sc) on the hypothermia induced by compounds 9.1 and 9.9.
Treatment and dose (mg/kg)
Δt ± SEM (0C)
30 min
60 min
Vehicle
Vehicle + 9.1 (20)
WAY100635 + 9.1 (20)
0.3ꢀ±ꢀ0.1
–0.9ꢀ±ꢀ0.3 c
0.0ꢀ±ꢀ0.2 e
Pꢀ=ꢀ0.0002
0.3ꢀ±ꢀ0.1
–0.3ꢀ±ꢀ0.3
0.1ꢀ±ꢀ0.2
ns
Vehicle
Vehicle + 9.9 (20)
WAY100635 + 9.9
0.2ꢀ±ꢀ0.4
–1.6ꢀ±ꢀ0.3c
–1.4ꢀ±ꢀ0.2c
P<0.0001
0.1ꢀ±ꢀ0.2
0.3ꢀ±ꢀ0.2
–0.7ꢀ±ꢀ0.2 a
Pꢀ=ꢀ0.0216
Vehicle
WAY100635
0.1ꢀ±ꢀ0.1
0.3ꢀ±ꢀ0.3
ns
–0.0ꢀ±ꢀ0.1
0.2ꢀ±ꢀ0.3
ns
a
b
WAY100635 was administered 15 min before the tested compounds, pꢀ<ꢀ0.05 vs vehicle, pꢀ<ꢀ0.01
vs vehicle, pꢀ<ꢀ0.001 vs vehicle, pꢀ<ꢀ0.05 vs compound group, pꢀ<ꢀ0.01 vs compound group,
fpꢀ<ꢀ0.001 vs compound group, ns = non-significant.
c
d
e
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Table 5
The effect of compound 9.7 on the hypothermia induced by 8-OH-DPAT (5 mg/kg).
Treatment and dose (mg/kg)
Δt ± SEM (0C)
15 min
30 min
45 min
60 min
Vehicleꢀ+ vehicle
Vehicle +ꢀ8-OH-DPAT
9.7 (20) + 8-OH-DPAT
0.2ꢀ±ꢀ0.1
0.4ꢀ±ꢀ0.1
0.5ꢀ±ꢀ0.1
–2.6ꢀ±ꢀ0.2 c
–1.3ꢀ±ꢀ0.3 c, f
Pꢀ<ꢀ0.0001
0.3ꢀ±ꢀ0.2
–2.4ꢀ±ꢀ0.3 c
–1.3ꢀ±ꢀ0.3 c, e
Pꢀ<ꢀ0.0001
–1.9ꢀ±ꢀ0.2 c
–2.4ꢀ±ꢀ0.2 c
–1.2ꢀ±ꢀ0.2 c, e –1.2ꢀ±ꢀ0.3 c, e
Pꢀ<ꢀ0.0001
Pꢀ<ꢀ0.0001
a
Compound 9.7 was administered 45 min before to 8-OH-DPAT, nꢀ=ꢀ14–15, pꢀ<ꢀ0.05 vs vehicle,
bpꢀ<ꢀ0.01 vs vehicle, pꢀ<ꢀ0.001 vs vehicle, pꢀ<ꢀ0.05 vs compound group, pꢀ<ꢀ0.01 vs compound
c
d
e
group, fpꢀ<ꢀ0.001 vs compound group.
Fig. 4. Effect of compound 9.1 on forced swimming test in CD-1 mice
2.2.3 Metabolic stability evaluation
The results of compound incubations in the presence of pooled HLMs and NADPH are
presented in Table 6. Metabolic stability is presented in form of a biological half-life value, which
allows for easy comparison of compound structures and their susceptibility to phase 1
biotransformation reactions (the result of incubation on the presence of HLMs).
Table 6
Experimental t1/2 values along with corresponding SD and RSD%.
Average t1/2
[min] (nꢀ=ꢀ2)
6.12
Compound
SD [min]
RSD%
9.1
9.7
0.18
0.14
0.13
0.47
3.07
2.55
5.53
9.9
7.40
1.72
9.27
3.31
15.92
Table legend: SD – standard deviation, RSD% – relative
standard deviation, expressed as SD/average*100%.
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Results presented in Table 6 allow a quick assessment of metabolic stability. 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 9.1 (R1 = -H, R2 = -H, R3 = -H), 9.7 (R1 = -H, R2
= -F, R3 = -H) and 9.9 (R1 = -H, R2 = -OCH3, R3 = -H) proved to be more resistant to biotransformation
reactions. Compound 9.27 (R1 = -H, R2 = -OCH3, R3 = -OCH3) was most susceptible to phase 1
biotransformation reactions. Visual comparison of a derivative’s chemical structure and its biological
half-life allows for an assessment of basic structure–metabolic stability relationships. In the present
study, compounds with the highest resistance to phase 1 enzymes possessed a -OCH3 group in the R2
position of the terminal portion. Compounds 9.1 and 9.7 possessed a hydrogen and fluoride moiety
in that place, respectively. Addition of another -OCH3 moiety (in derivative 9.27) in position 5 of the
indole ring (R3) resulted in lowered metabolic stability (biological half-life value of 3.31 min).
Therefore, such direction of synthesis should rather be avoided to prevent development of further
metabolically unstable derivatives.
3. Materials and methods
3.1. General remarks
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. 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 >95% purity. The HPLC methods used a Phenomenex 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-pyrido[1,2- c]pyrimidine-1,3-diones (5.1–5.9)
The starting compounds 2.1–2.9, 3.1–3.9, 4.1–4.9 and 5.1–5.9 were obtained according to
procedures described in the literature[21–25].
3.2.2. General procedure for the synthesis of 3-piperidin-3-yl-1H-indole hydrochlorides (8.1–8.3)
A mixture of 0.01 mol of appropriate indole derivative (6.1–6.3), 0.03 mol of N-benzyl-3-
piperidone and 60 mL of 2 M KOH/isopropanole was stirred in 80 ⁰C under an argon atmosphere for
8 h. The mixture was further stirred for 8 h at room temperature and poured onto ice/water. The
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crude product was extracted with CHCl3 and the organic layers combined and dried with MgSO4. The
mixture was then filtered and the filtrate was evaporated to dryness. The crude residue was purified
by flash chromatography, using CH2Cl2/MeOH/TEA (9:1:0.2 v/v) and ethyl acetate/hexane (9:1 v/v)
mixtures. 0.01 mol of the obtained yellow solid (7.1–7.3)[33] was dissolved in 150 mL of methanol
and hydrogenated for 8 h at 30 ⁰C and 1 atm with 0.25 g of 10% Pd/C as catalyst. The catalyst was
then filtered off and the filtrate evaporated to one-third and then, after cooling, acidified with
HCl/MeOH. The evaporated-to-dryness crude product (8.1–8.3) was purified by crystallization from
methanol.
Fig. 5. Numbering system for NMR spectra interpretation of 3-piperidyn-3-yl-1H-indole derivatives 8.1
(R3 = -H); 8.2 (R3 = -F); 8.3 (R3 = -OCH3).
3.2.2.1. 3-(3-piperidyl)-1H-indole hydrochloride (8.1).
The title compound was isolated as white crystals. Yield: 68.0 %, m.p. 295.2-297.9 °C
1H NMR (500 MHz, D2O): δ 7.70 (C4”H, 1H, dd, 3J=8.0), 7.53 (C7”H,1H, m, 3J=7.0 , 4J=0.5), 7.27 (C6”H,
1H, m), 7.21 (C2”H, 1H, s), 7.19 (C5”H, 1H, m), 3.57 (CaH(E), 1H, m), 3.48 (CeH(E), 1H, m), 3.30
(CbH(A), 1H, tt, 3JA-A=12.0 , 3JA-E=4.0), 2.99 (CaH(A),CeH(A), 2H, m), 2.14 (CcH(E), 1H, m), 2.05
(CdH(E),1H, m), 1.89 (CdH(A), 1H, m), 1.76 (CcH(A), 1H, m).
13C NMR (125 MHz, D2O): δ 135.7 (C7”a, s), 124.9 (C3”a, s), 121.8 (C6”, s), 121.6 (C2”, s), 118.9 (C5”,
s), 118.0 (C4”, s), 114.4 (C3”, s), 111.6 (C7”, s), 48.3 (Ca,s), 43.7 (Ce, s), 30.6 (Cb,s), 28.3 (Cc, s), 21.9
(Cd, s).
ESI-HRMS m/z: Calcd for C13H17N2 [M+H]+ 201.13917. Found: 201.13874.
C13H17N2Cl: Mol. Wt.: 236.74 g mol-1; Anal. Calc.: Found% (Calc%): C, 65.87 (65.95); H, 7.24 (7.20); N,
11.83 (11.56).
3.2.2.2. 5-fluoro-3-(3-piperidyl)-1H-indole hydrochloride (8.2).
The title compound was isolated as white crystals. Yield: 70.2 %, m.p. 270.3-273.1 °C
1H NMR (500 MHz, D2O): δ 7.44 (C7”H, [1H], 4d, 3J=9.0, 4JH-F=5.0, 5J=0.5), 7.35 (C4”H, [1H], 4d, 3JH-
F=10.0, 4J=2.5, 5J=0.5), 7.24 (C2”H, [1H], s), 7.03 (C6”H, [1H], 8d, 3J=9.5, 3JH-F=8.0, 4J=2.5, pJ=0.5), 3.56
(CaH(E). [1H], m), 3.49 (CeH(E), [1H], m), 3.22 (CbH, [1H], tt, 3JA-A=12.0, 3JA-E=3.5), 3.01 (CeH(A), [1H],
td, 2J=3JA-A=13.0, 3JA-E=3.5), 2.98 (CaH(A), [1H], t, 2J=3JA-A=12.5), 2.01 – 2.14 (CcH(E),CdH(E), [2H], m),
1.83 – 1.95 (CdH(A), [1H], m), 1.72 (CcH(A), [1H], kd, 2J=3JA-A=12.0, 3JA-E=4.0).
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13C NMR (125 MHz, D2O): δ 156.9 (C5”, d, 1J=231.9), 132.2 (C3”, s), 125.1 (C3”a, d, 3J=9.9), 123.3 (C2”,
s), 114.6 (C7”a, d, 4J=4.8), 112.6 (C7”, d, 3J=9.9), 109.8 (C6”, d, 2J=26.4), 102.5 (C4”, d, 2J=23.8), 48.2
(Ca, s), 43.7 (Ce, s), 30.5 (Cb, s), 28.2 (Cc, s), 21.9 (Cd, s).
ESI-HRMS m/z: Calcd for C13H16FN2 [M+H]+ 219.12975. Found: 219.12935.
C13H16ClFN2: Mol. Wt.: 254.73 g mol-1; Anal. Calc.: Found% (Calc%): C, 61.26 (61.30); H, 6.30 (6.24); N,
11.10 (10.99).
3.2.2.3. 5-methoxy-3-(3-piperidyl)-1H-indole hydrochloride (8.3).
The title compound was isolated as white crystals. Yield: 68.0 %, m.p. 276.9-278.4 °C
1H NMR (500 MHz, D2O): δ 7.46 (C7”H, [1H], d, 3J=9.0), 7.27 (C2”H, [1H], s), 7.22 (C4”H, [1H], d,
4J=2.5), 6.96 (C6”H, [1H], dd, 3J=9.0, 4J=2.5), 3.90 (OCH3, [3H], s), 3.63 (CaH(E), [1H], m), 3.51 (CeH(E),
[1H], m), 3.32 (CbH, [1H], tt, 3JA-A=12.0, 3JA-E=4.0), 3.08 (CaH(A), [1H], t, 2J=3JA-A=12.5), 3.06 (CeH(A),
[1H], td, 2J=3JA-A=13.0, 3JA-E=3.0), 2.22 (CcH(E), [1H], m), 2.09 (CdH(E), [1H], m), 1.93 (CdH(A), [1H], m),
1.81 (CcH(A), [1H], kd, 2J=3JA-A=13.0, 3JA-E=3.5).
13C NMR (125 MHz, D2O): δ 153.0 (C5”, s), 131.6 (C7”a, s), 125.7 (C3”a, s), 123.0 (C2”, s), 114.8 (C3”,
s), 113.1 (C6”, s), 112.0 (C7”, s), 100.9 (C4”, s), 56.3 and 56.3 (OCH3, 2s*), 48.7 (Ca, s), 44.2 (Ce, s),
31.0 (Cb, s), 28.9 (Cc, s), 22.4 (Cd, s).
ESI-HRMS m/z: Calcd for C14H19N2O [M+H]+ 231.14974. Found: 231.14941.
C14H19ClN2O: Mol. Wt.: 266.77 g mol-1; Anal. Calc.: Found% (Calc%): C, 62.77 (63.03); H, 7.13 (7.18); N,
10.28 (10.50).
3.2.3. General procedure for the synthesis of 2-[4-[3-(1H-indol-3-yl)-1-piperidyl]butyl]-4-phenyl-
pyrido[1,2-c]pyrimidine-1,3-diones (9.1–9.27)
Compounds 5.1–5.9 (0.75 mmol), 8.1–8.3 (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 9.1–9.27.
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Fig. 6. Numbering system for NMR spectra interpretation of compounds 9.1–9.27
3.2.3.1. 2-[4-[3-(1H-indol-3-yl)-1-piperidyl]butyl]-4-phenyl-pyrido[1,2-c]pyrimidine-1,3-dione (9.1).
The title compound was isolated as a yellow powder. Yield: 66.0 %, m.p. 123.6-125.4 °C. HPLC tR =
5.52 min, 99.5% purity.
1H NMR (500 MHz, CDCl3): δ 8.31 (C8H, [1H], d, 3J=8.0), 8.26 (N1”H, [1H], bs), 7.65 (C4”H, [1H], d,
3J=7.5), 7.43 (C3’H,C5’H, [2H], t, 3J=7.0), 7.38 – 7.32 (C4’H,C7”H, [2H], m), 7.30 (C2’H,C6’H, [2H], d,
3J=7.0), 7.16 (C6”H, [1H], m), 7.08 (C5”H, [1H], m), 6.96 (C2”H, [1H], bs), 6.88 (C5H,C6H, [2H], m), 6.36
(C7H, [1H], m), 4.17 (C1xH2, [2H], t, 3J=7.0), 3.25 (CbH,CaH(E), [2H], bs), 3.05 (CeH(E), [1H], bs), 2.54
(C4xH2, [2H], bs), 2.16 (CaH(A),CeH(A), [2H], bs), 2.06 (CcH(E), [1H], pd), 2.00 – 1.60 (CdH2,C2xH2,C3xH2,
[6H], m), 1.51 (CcH(A), [1H], m).
13C NMR (125 MHz, CDCl3): δ 160.2 (C3, s), 148.9 (C1, s), 143.6 (C4a, s), 136.2 (C7”a, s), 132.8 (C6, s),
132.4 (C1’, s), 131.2 (C2’,C6’, s), 128.8 (C3’,C5’, s), 128.0 (C4’, s), 127.8 (C8, s), 126.6 (C3”a, s), 121.9
(C6”, s), 121.4 (C5, s), 120.4 (C2”, s), 119.2 (C5’, s), 119,1 (C4”, s), 111.2 (C7”, s), 110.7 (C7, s), 104.8
(C4, s), 60.2 (Ca, s), 58.4 (C4x, s), 53.9 (Ce, s), 42.1 (C1x, s), 33.5 (Cb, s), 30.9 (Cc, s), 25.4 (Cd, s), 25.0
(C2x, s), 24.0 (C3x, s).
ESI-HRMS m/z: Calcd for C31H33N4O2 [M+H]+ 493.26035. Found: 493.26028.
3.2.3.2. 4-(2-chlorophenyl)-2-[4-[3-(1H-indol-3-yl)-1-piperidyl]butyl]pyrido[1,2-c]pyrimidine-1,3-dione
(9.2).
The title compound was isolated as a yellow powder. Yield: 90.3 %, m.p. 175.2-177.7 °C. HPLC tR =
5.63 min, 99.9% purity.
1H NMR (500 MHz, CDCl3): δ 8.34 (C8H, [1H], 4t: (1): 8.347, 3J=7.5, 4J=5J=1.0, (2): 8.34, 3J=7.5,
4J=5J=1.0), 8.23 (N1”H, [1H], bs), 7.65 (C4”H, [1H], d, 3J=8.0), 7.49 (C3’H, [1H], m (14 lines)), 7.34 –
7.26 (C4’-6’H,C7”H, [4H], m), 7.15 (C6”H, [1H], m, 3J1=8.0, 3J2=7.0, 4J=1.0), 7.08 (C5”H, [1H], m), 6.96
(C2”H, [1H], bs), 6.93 (C6H, [1H}, m (11 lines)), 6.53 (C5H, [1H], dt, 3J=9.5, 4J=5J=1.0), 6.39 (C7H, [1H],
m(14 lines)), 4.18 (C1xH2, [2H], t, 3J=7.5), 3.20 (CaH(E),CbH, [2H], pd), 2.99 (CeH(E), [1H], bs), 2.47
(C4xH2, [2H], bs), 2.07 (CaH(A),CeH(A),CcH(E), [3H], m), 1.78 (CdH2,C2xH2, [4H], m), 1.66 (C3xH2, [2H],
m), 1.89 (CcH(A), [1H], m).
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13C NMR (125 MHz, CDCl3): δ 159.4 and 159.5 (C3, 2s), 148.9 (C1, s), 143.8 (C4a, s), 136.2 (C7”a, s),
135.2 and 135.7 (C2’, 2s), 133.4 (C6, 2s), 133.0 (C6’, s), 131.8 (C1’, s), 130.0 (C3’, s), 129.6 (C4’, s),
128.1 (C8, s), 127.3 (C5’, 2s), 126.7 (C3”a, s), 121.8 (C6”, s), 121.0 (C5, s), 120.3 (C2”, s), 119.1 (C5’, s),
119.1 (C4”, s), 111.2 (C7”, s), 110.7 (C7, s), 102.2 (C4, s), 60.7 (Ca, s), 58.5 (C4x, s), 54.0 (Ce, s), 42.3
(C1x, s), 33.7 (Cb, s), 31.2 (Cc, s), 25.5 (Cd, s), 25.0 (C2x,s), 24.5 (C3x, s).
ESI-HRMS m/z: Calcd for C31H32ClN4O2 [M+H]+ 527.22138. Found: 527.22162.
3.2.3.3. 4-(2-fluorophenyl)-2-[4-[3-(1H-indol-3-yl)-1-piperidyl]butyl]pyrido[1,2-c]pyrimidine-1,3-dione
(9.3).
The title compound was isolated as a yellow powder. Yield: 93.5 %, m.p. 137.8-139.7 °C. HPLC tR =
5.21 min, 99.9% purity.
1H NMR (500 MHz, CDCl3): δ 8.37 – 8.31 (C8H, [1H], m (12 lines)), 8.15 (N1”H, [1H], bs), 7.7 (C4”H,
[1H], d, 3J=8.0), 7.40 – 7.28 (C4’H,C6’H, [2H], m), 7.34 (C7”H, [1H], d, 3J=8.0), 7.24 – 7.19 (C5’H, [1H],
m), 7.19 – 7.11 (C3’H,C6”H, [2H], m), 7.09 (C5”H, [1H], m), 7.00 – 6.93 (C2”H,C6H, [2H], m), 6.72 (C5H,
[1H], m), 6.43 – 6.38 (C7H, [1H], m (14 lines)), 4.18 (C1xH2, [2H], t, 3J=7.5), 3.23 (CaH(E),CbH, [2H], bs),
3.02 (CeH(E), [1H], bs), 2.50 (C4xH2, [2H], bs), 2.20 – 2.00 (CaH(A),CeH(A),CcH(E), [3H], m), 1.80 – 1.60
(CdH2,C2xH2,C3xH2, [6H], m), 1.52 (CcH(A), [1H], m).
13C NMR (125 MHz, CDCl3): δ 160.9 (C2’, d, 1J=247.0), 159.6 (C3, s), 148.9 (C1, s), 144.0 (C4a, s), 136.2
(C7”a, s), 133.5 (C6’, d), 133.1 (C6, s), 130.1 (C4’, d, 3J=8.2), 128.2 (C8, s), 127.3 (C3”, s), 126.7 (C3”a,
s), 124.4 (C5’, d), 121.9 (C6”, s), 121.2 (C5, s), 120.3 (C1’, d, 2J=16.0), 120.3 (C2”, s), 119.2 (C4’,C5’, s),
116.1 (C3’, d, 2J=22.4), 111.2 (C7”, s), 110.8 (C7, s), 98.3 (C4, s), 61.0 (Ca, s), 58.5 (C4x, s), 54.0 (Ce, s),
42.3 (C1x, s), 33.0 (Cb, s), 31.0 (Cc, s), 25.5 (Cd, s), 25.0 (C2x, s), 24.0 (C3x, s).
ESI-HRMS m/z: Calcd for C31H32FN4O2 [M+H]+ 511.25092. Found: 511.25108.
3.2.3.4. 2-[4-[3-(1H-indol-3-yl)-1-piperidyl]butyl]-4-(o-tolyl)pyrido[1,2-c]pyrimidine-1,3-dione (9.4).
The title compound was isolated as a yellow powder. Yield: 93.7 %, m.p. 142.1-144.8 °C. HPLC tR =
6.08 min, 99.3% purity.
1H NMR (500 MHz, CDCl3): δ 8.43 (N1”H, [1H], bs), 8.31 (C8H, [1H], dt, 3J=7.5, 4J=5J=1.0), 7.64 (C4”H,
[1H], d, 3J=8.0), 7.31 (C7”H, [1H], d, 3J=8.0), 7.30 – 7.20 (C4’-6’H, [3H], m), 7.16 – 7.10 (C3’H,C6”H,
[2H], m), 7.06 (C5”H, [1H], m, 3J1=8.0, 3J2=7.5, 4J=1.0), 6,923 (C2”H, [1H], bs), 6.86 (C6H, [1H], 4d,
3J1=9.5, 3J2=6.0, 4J=1.0), 6.53 (C5H, [1H], dt, 3J=9.0, 4J=5J=1.0), 6.35 (C7H, [1H], m, 3J1=7.5, 3J2=9.0,
4J=1.0), 4.18 (C1xH2, [2H], t, 3J=7.5), 3.23 (CaH(E),CbH, [2H], m), 3.02 (CeH(E), [1H], pd), 2.51 (C4xH2,
[2H], bs), 2.13 and 2.10 ([6H], 2s), 2.07 – 2.00 (CaH(A),CeH(A),CcH(E), [3H], m), 1.85 – 1.73
(CdH2,C2xH2, [4H], m), 1.67 (C3xH2, [2H], m), 1.48 (CcH(A), [1H], kd, 2J=3JA-A=12.5, 3JA-E=3.5).
13C NMR (125 MHz, CDCl3): δ 159.7 (C3, s), 149.0 (C1, s), 143.5 (C4a, s), 138.4 and 138.5 (C2’, 2s),
136.2 (C7”a, s), 132.5 (C6, s), 131.5 and 131.6 (C3’, 2s), 130.5 (C6’, s), 128.3 (C4’, s), 128.0 (C8, s),
126.6 (C3”a, s), 126.3 (C5’, 2s), 121.8 (C6”, s), 121.3 (C5, s), 120.4 (C2”, s), 119.1 (C4”,C5”, s), 111.2
(C7”, s), 110.6 (C7, s), 104.0 (C4, s), 60.3 (Ca, s), 58.3 (C4x, s), 53.8 (Ce, s), 42.0 (C1x,s), 33.5 (Cb, s),
31.0 (Cc, s), 25.5 (Cd, s), 25.2 (C2x, s), 23.6 (C3x, s), 19.6 (CH3, 2s).
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ESI-HRMS m/z: Calcd for C32H35N4O2 [M+H]+ 507.27600. Found: 507.27636.
3.2.3.5. 2-[4-[3-(1H-indol-3-yl)-1-piperidyl]butyl]-4-(2-methoxyphenyl)pyrido[1,2-c]pyrimidine-1,3-
dione (9.5).
The title compound was isolated as a yellow powder. Yield: 66.6 %, m.p. 102.5-104.1 °C. HPLC tR =
5.28 min, 99.9% purity.
1H NMR (500 MHz, CDCl3): δ 8.38 (N1”H, [1H], bs), 8.29 (C8H, [1H], m(8 lines)), 7.64 (C4”H, [1H], d,
3J=8.0), 7.36 (C4’H, [1H], m), 7.31 (C7”H, [1H], d, 3J=8.0), 7.19 (C6”H, [1H], m , 3J1=7.5, 3J2=6.0, 4J=1.5),
7.14 (C6’H, [1H], m), 7.07 (C5”H, [1H], m), 7.02 (C5’H, [1H], m), 6.98 – 6.91 (C2”H,C3’H, [2H], m), 6.85
(C6H, [1H], m), 6.61 (C5H, [1H], m), 6.34 (C7H, [1H], m), 7.17 (C1xH2, [2H], m), 3.72 and 3.69 (OCH3,
[3H], 2s), 3.20 (CbH,CaH(E), [2H], m), 3.00 (CeH(E), [1H], bs), 2.18 – 2.00 (CaH(A),CeH(A),CcH(E), [3H],
m), 1.78 (CdH2,C2xH2, [4H], m), 1.67 (C3xH2, [2H], m), 1.48 (CcH(A), kd, 2J=3JA-A=12.5, 3JA-E=4.5).
13C NMR (125 MHz, CDCl3): δ 159.9 (C3, s), 157.9 (C2’, 2s), 149.1 (C1, s), 143.6 (C4a, s), 136.2 (C7”a, s),
133.0 (C6’, 2s), 132.0 (C6, s), 129.6 (C4’, s), 127.8 (C8, s), 126.6 (C3”a, s), 123.0 (C3”, s), 121.9 (C5, s),
121.7 (C6”, s), 121.4 (C1’, s), 120.9 (C5’, 2s), 120.4 (C2”, s), 119.1 (C5”, s), 119.1 (C4”, s), 111.4 and
111.3 (C3’, 2s), 111.2 (C7”, s), 110.5 (C7, s), 101.2 (C4, s), 60.4 (Ca, s), 58.5 (C4x, s), 55.6 (OCH3, 2s),
53.9 (Ce, s), 42.1 (C1x, s), 33.5 (Cb, s), 31.1 (Cc, s), 25.5 (Cd, s), 25.3 (C2x, s), 23.8 (C3x, s).
ESI-HRMS m/z: Calcd for C32H35N4O3 [M+H]+ 523.27091. Found: 523.27104.
3.2.3.6. 4-(4-chlorophenyl)-2-[4-[3-(1H-indol-3-yl)-1-piperidyl]butyl]pyrido[1,2-c]pyrimidine-1,3-dione
(9.6).
The title compound was isolated as a yellow powder. Yield: 87.8 %, m.p. 229.6-232.3 °C. HPLC tR =
6.87 min, 97.2% purity.
1H NMR (500 MHz, DMSO – d6): δ 10.82 (N1”H, [1H], bs), 8.29 (C8H, [1H], dt, 3J=7.5, 4J=5J=1.0), 7.53
(C4”H, [1H], d, 3J=7.5), 7.46 (C2’H,C6’H, [2H], dt, 3J=8.5, 4J=2.5), 7.33 (C7”H, [1H], d, 3J=8.0), 7.28
(C3’H,C5’H, [2H], dt, 3J=8.5, 4J=2.5), 7,139 (C6”H, [1H], 4d, 3J1=9.5, 3J2=6.0, 4J=1.0), 7.12 (C2”H, [1H], d,
3J=2.0), 7.05 (C5”H, [1H], m), 6.95 (C6H, [1H], m), 6.78 (C5H, [1H], dt, 3J=9.5, 4J=5J=1.0), 6.57 (C7H,
[1H], m, 3J1=7.5, 3J2=6.0, 4J=1.5), 4.00 (C1xH2, [2H], t, 3J=7.0), 3.34 (CaH(E),CbH, [2H], bs), 3.03 (CeH(E),
[1H], pt), 2.43 (C4xH2, [2H], bs), 2.07 (CaH(A),CeH(A), [2H], bs), 1.95 (CcH(E), [1H], pd), 1.72 (CdH(E),
[1H], bs), 1.65 (C2xH2, [2H], q, 3J=6.5), 1.53 (C3xH2,CcH(A),CdH(A), [4H], bs).
13C NMR (125 MHz, CDCl3): δ 160.0 (C3, s), 148.8 (C1, s), 143.7 (C4a, s), 136.2 (C7”a, s), 133.7 (C4’, s),
133.0 (C6, s), 132.7 (C3’,C5’, s), 131.3 (C1’, s), 129.0 (C2’,C6’, s), 128.1 (C8, s), 126.5 (C3”a, s), 122.0
(C6”, s), 121.0 (C5, s), 120.4 (C2”, s), 119.3 (C5”, s), 119.2 (C4”, s), 111.2 (C7”, s), 110.9 (C7, s), 103.4
(C4, s), 60.1 (Ca, s), 58.3 (C4x, s), 53.8 (Ce, s), 42.1 (C1x, s), 33.2 (Cb, s), 30.7 (Cc, s), 25.3 (Cd, s), 25.0
(C2x, s), 23.3 (C3x, s).
ESI-HRMS m/z: Calcd for C31H32ClN4O2 [M+H]+ 527.22138. Found: 527.22177.
3.2.3.7. 4-(4-fluorophenyl)-2-[4-[3-(1H-indol-3-yl)-1-piperidyl]butyl]pyrido[1,2-c]pyrimidine-1,3-dione
(9.7).
15

ACCEPTED MANUSCRIPT
The title compound was isolated as a yellow powder. Yield: 69.7 %, m.p. 209.6-212.2 °C. HPLC tR =
5.56 min, 99.7% purity.
1H NMR (500 MHz, CDCl3): δ 8.32 (N1”H, [1H], bs), 8.31 (C8H, [1H], dt, 3J=7.5), 7.65 (C7”H,[1H], d,
3J=8.0), 7.26 (C2’H,C6’H, [2H], pt), 7.16 (C6”H, [1H], m), 7.13 – 7.05 (C3’H,C5’H,C5”H, [3H], m), 6.96
(C2”H, [1H], d, 3J=1.5), 6.91 (C6H, [1H], 4d, 3J1=9.5, 3J2=6.5, 4J=1.0), 6.83 (C5H, [1H], dt, 3J=9.5), 6.37
(C7H, [1H], m, 3J1=7.5, 3J2=6.0, 4J=1.0), 4.16 (C1xH2, [2H], t, 3J=7.0), 3.26 (CaH(E),CbH, [2H], m), 3.07
(CeH(E), [1H], bs), 2.54 (C4xH2, [2H], bs), 2.16 (CaH(A),CeH(A), [2H], bs), 2.06 (CcH(E), [1H], pd), 2.00 –
1.60 (C2xH2,C3xH2,CdH2, [6H], m), 1.51 (CcH(A), [1H], kd, 2J=3JA-A=12.5, 3JA-E=3.5).
13C NMR (125 MHz, CDCl3): δ 162.3 (C4’, d, 1J=247.0), 160.2 (C3, s), 148.8 (C1, s), 143.7 (C4a, s), 136.2
(C7”a, s), 133.0 (C2’,C6’, d, 3J=8.0), 132.8 (C6, s), 128.6 (C1’, d, 4J=3.3), 128.1 (C8, s), 126.5 (C3”a, s),
121.9 (C6”, s), 121.1 (C5, s), 120.4 (C2”, s), 119.2 (C5”, s), 119.1 (C4”, s), 115.8 (C3’,C5’, d, 2J=21.5),
115.0 (C3”, s), 111.2 (C7”, s), 110.8 (C7, s), 103.6 (C4, s), 60.2 (Ca, s), 58.3 (C4x, s), 53.9 (Ce, s), 42.1
(C1x, s), 33.4 (Cb, s), 30.8 (Cc, s), 25.4 (Cd, s), 25.1 (C2x, s), 23.5 (C3x, s).
ESI-HRMS m/z: Calcd for C31H32FN4O2 [M+H]+ 511.25092. Found: 511.25112.
3.2.3.8. 2-[4-[3-(1H-indol-3-yl)-1-piperidyl]butyl]-4-(p-tolyl)pyrido[1,2-c]pyrimidine-1,3-dione (9.8).
The title compound was isolated as a yellow powder. Yield: 74.0 %, m.p. 217.8-219.9 °C. HPLC tR =
6.97 min, 96.6% purity.
1H NMR (500 MHz, CDCl3): δ 8.55 (N1”H, [1H],bs), 8.28 (C8H, [1H], d, 3J=7.5), 7.64 (C4”H, [1H], d,
3J=8.0), 7.33 (C7”H, [1H], d, 3J=8.0), 7.23 (C2’H,C6’H, [2H], pd), 7.17 (C3’H,C5’H, [2H], pd), 7.14 (C6”H,
[1H], m), 7.06 (C5”H, [1H], m), 6.92 (C2”H, [1H], bs), 6.88 (C5H,C6H, [2H], m), 6.34 (C7H, [1H], m,
3J1=7.5, 3J2=5.5, 4J=2.0), 4.16 (C1xH2, [2H], t, 3J=7.0), 3.35 (CbH, [1H], bs), 3.26 (CaH(E), [1H], pd), 3.12
(CeH(E), [1H], bs), 2.60 (C4xH2, [2H], bs), 2.38 (CH3, [3H], s), 2.19 (CaH(A),CeH(A), [2H], m), 2.04
(CcH(E), [1H], pd), 1.95 (CdH(E), [1H], bs), 1.83 – 1.65 (CdH(A),C2xH2,C3xH2, [5H],m), 1.48 (CcH(A),
[1H], kd, 2J=3JA-A=12.5, 3JA-E=3.5).
13C NMR (125 MHz, CDCl3): δ 160.3 (C3, s), 148.9 (C1, s), 143.5 (C4a, s), 137.5 (C4’, s), 136.2 (C7”a, s),
132.3 (C6, s), 131.0 (C3’,C5’, s), 129.7 (C1’, s), 129.5 (C2’,C6’, s), 127.9 (C8, s), 126.4 (C3”a, s), 121.9
C6”, s), 121.5 (C5, s), 120.5 (C2”, s), 119.2 (C5”, s), 119.1 (C4”, s), 118.0 (C3”, s), 111.3 (C7”, s), 110.7
(C7, s), 104.7 (C4, s), 59.8 (Ca, s), 58.0 (C4x, s), 53.5 (Ce, s), 41.8 (C1x, s), 32.9 (Cb, s), 30.5 (Cc, s), 25.2
{Cd, s), 24.7 (C2x, s), 23.1 (C3x, s), 21.3 (CH3, s).
ESI-HRMS m/z: Calcd for C32H35N4O2 [M+H]+ 507.27600. Found: 507.27579.
3.2.3.9. 2-[4-[3-(1H-indol-3-yl)-1-piperidyl]butyl]-4-(4-methoxyphenyl)pyrido[1,2-c]pyrimidine-1,3-
dione (9.9).
The title compound was isolated as a yellow powder. Yield: 92.6 %, m.p. 117.2-119.8 °C. HPLC tR =
5.70 min, 99.7% purity.
1H NMR (500 MHz, CDCl3): δ 8.29 (C8H, [1H], dt, 3J=7.5, 4J=5J=1.0), 8.23 (N1”H, [1H], bs), 7.65 (C4”H,
[1H], dd, 3J=8.0, 4J=0.5), 7.32 (C7”H, [1H], dt, 3J=8.0, 4J=5J=1.0), 7.22 (C2’H,C6’H, [2H], dt, 3J=9.0,
4J=3.0), 7.15 (C6”H, [1H], m, 3J1=8.0, 3J2=7.0, 4J=1.0), 7.08 (C5”H, [1H], m, 3J1=8.0, 3J2=7.0, 4J=1.0), 6.96
(C3’H,C5’H,C2”H, [3H], m), 6.89 (C5H, [1H], m, 3J=9.5, 4J=1.5, 5J=1.0), 6.86 (C6H, [1H], 4d, 3J1=9.5,
16

ACCEPTED MANUSCRIPT
3J2=6.0, 4J=1.5), 6.33 (C7H, [1H], m, 3J1=7.5, 3J2=6.0, 4J=1.5), 4.17 (C1xH2, [2H], m), 3.83 (OCH3, [3H], s),
3,178 (CbH,CaH(E), [2H], m), 2.97 (CeH(E), [1H], pd), 2.44 (C4xH2, [2H], t, 3J=7.5), 2.13 – 1.98
(CaH(A),CeH(A),CcH(E), [3H], m), 1.82 – 1.73 (C2xH2,CdH2, [4H], m), 1.64 (C3xH2, [2H], q, 3J=7.5), 1.50
(CcH(A), [1H], m).
13C NMR (125 MHz, CDCl3): δ 160.4 (C3, s), 159.1 (C4’, s), 148.9 (C1, s), 143.5 (C4a, s), 136.2 (C7”a, s),
132.3 (C2’,C6’, s), 132.1 (C6, s), 127.9 (C8, s), 126.7 (C3”a, s), 124.9 (C1’, s), 121.8 (C6”, s), 121.6 (C5,
s), 120.3 (C2”, s), 119.6 (C3”, s), 119.1 (C5”, s), 119.1 (C4”, s), 114.3 (C3’,C5’, s), 111.1 (C7”, s), 110.5
(C7, s), 104.6 (C4, s), 60.8 (Ca, s), 58.7 (C4x, s), 55.3 (OCH3, s), 54.2 (Ce, s), 42.4 (C1x, s), 33.9 (Cb, s),
31.4 (Cc, s), 25.6 (Cd, s), 25.0 (C2x, s), 24.0 (C3x,s).
ESI-HRMS m/z: Calcd for C32H35N4O3 [M+H]+ 523.27092. Found: 523.27130.
3.2.3.10. 2-[4-[3-(5-fluoro-1H-indol-3-yl)-1-piperidyl]butyl]-4-phenyl-pyrido[1,2-c]pyrimidine-1,3-dione
(9.10).
The title compound was isolated as a yellow powder. Yield: 94.5 %, m.p. 103.8-105.4 °C. HPLC tR =
5.96 min, 99.9% purity.
1H NMR (500 MHz, CDCl3): δ 8.39 (N1”H, [1H], bs), 8.32 (C8H, [1H], d, 3J=7.5), 7.43 (C3’H,C5’H, [2H], t,
3J=7.5), 7.35 (C4’H, [1H], tt, 3J=7.5), 7.30 (C2’H,C6’H, [2H], dd, 3J=7.5), 7.26 (C4”H, [1H], dd, 3JH-F=10.0,
4J=2.5), 7.20 (C7”H, [1H], dd, 3J=9.0, 4JH-F=4.0), 7.00 (C2”H, [1H], bs), 6.88(C5H,C6H,C6”H, [3H], m),
6.36 (C7H, [1H], m), 4.18 (C1xH2, [2H], m), 3.13 (CbH,CaH(E), [2H], pd), 2.96 (CeH(E), [1H], pd), 2.46
(C4xH2, [2H], t, 3J=7.0), 2.11 (CaH(A),CeH(A), [2H], m), 2.02 (CcH(E), [1H], m), 1.78 (C2xH2,CdH2, [4H],
m), 1.65 (C3xH2, q, 3J=7.5), 1.47 (CcH(A), [1H], m).
13C NMR (125 MHz, CDCl3): δ 160.2 (C3, s), 157.5 (C5”, d, 1J=234.0), 148.9 (C1, s), 143.6 (C4a, s), 132.8
(C7”a, s), 132.7 (C1’, s), 132.4 (C6, s), 131.2 (C2’,C6’, s), 128.7 (C3’,C5’, s), 127.9 (C4’, s), 127.8 (C8, s),
127.0 (C3”a, d, 3J=9.4), 122.3 (C2”, s), 121.4 (C5, s), 119.4 (C3”, d), 111.7 (C7”, d, 3J=9.6), 110.7 (C7, s),
110.1 (C6”,d, 2J=26.3), 104.8 (C4, s), 103.9 (C4”, d, 2J=23.3), 60.4 (Ca, s), 58.5 (C4x, s), 54.0 (Ce, s), 42.3
(C1x, s), 33.6 (Cb, s), 31.1 (Cc, s), 25.5 (C2x,s), 25.5 (Cd,s), 24.0 (C3x, s).
ESI-HRMS m/z: Calcd for C31H32FN4O2 [M+H]+ 511.25093. Found: 511.25105.
3.2.3.11. 4-(2-chlorophenyl)-2-[4-[3-(5-fluoro-1H-indol-3-yl)-1-piperidyl]butyl]pyrido[1,2-c]pyrimidine-
1,3-dione (9.11).
The title compound was isolated as a yellow powder. Yield: 93.0 %, m.p. 114.6-116.9 °C. HPLC tR =
5.90 min, 99.9% purity.
1H NMR (500 MHz, CDCl3): δ 8.36 (N1”H, [1H], bs), 8.36 and 8.35 (C8H, [1H], m (10 lines)), 7.51 – 7.46
(C3’H, [1H], m (12 lines)), 7.35 – 7.26 (C4’-6’H,C4”H, [4H], m), 7.22 (C7”H, [1H], dd, 3J=8.5, 4JH-F=4.5),
7.01 (C2”H, [1H], pt), 6.95 (C6H, [1H], m (11 lines)), 6.89 (C6”H, [1H], td, 3J=9.0, 4J=2.5), 6.54 (C5H,
[1H], dt), 6.41 (C7H, [1H], 2m (14 lines), 3J1=8.0, 3J2=6.0, 4J=1.0), 4.19 (C1xH2, [2H], m), 3.14
(CbH,CaH(E), [2H], pd), 2.99 (CeH(E), [1H], bs), 2.48 (C4xH2, [2H], bs), 2.08 (CaH(A),CeH(A), [2H], m),
2.02 (CcH(E), [1H], pd), 1.78 (C2xH2,CdH2, [4H], m), 1.66 (C3xH2, [2H], q, 3J=7.0), 1.47 (CcH(A), [1H], m).
13C NMR (125 MHz, CDCl3): δ 159.5 (C3, 2s), 157.5 (C5’, d, 1J=234.1), 148.9 (C1, s), 143.9 (C4a, 2s),
135.7 (C2’, 2s),133.4 (C6’, 2s), 133.1 (C1’, s), 131.8 (C7”a, s), 130.0 (C3’, 2s), 129.6 (C4’, s), 128.1 (C8,
17

ACCEPTED MANUSCRIPT
s), 127.3 (C5’, 2s), 127.0 (C3”a, d, 3J=9.2), 122.3 (C2’, s), 121.0 (C5, s), 119.3 (C3”, s), 111.8 (C7”, d,
3J=9.7), 110.8 (C7, s), 110.1 (C6”, d, 2J=26.3), 104.0 (C4”, d, 2J=23.1), 102.2 and 102.1 (C4, 2s), 60.3
(Ca, s), 58.4 (C4x, s), 53.9 (Ce, s), 42.2 (C1x, s), 33.6 (Cb, s), 31.1 (Cc, s), 25.5 (Cd, s), 25.2 (C2x, s), 23.7
(C3x, s).
ESI-HRMS m/z: Calcd for C31H31ClFN4O2 [M+H]+ 545.21196. Found: 545.21194.
3.2.3.12. 4-(2-fluorophenyl)-2-[4-[3-(5-fluoro-1H-indol-3-yl)-1-piperidyl]butyl]pyrido[1,2-c]pyrimidine-
1,3-dione (9.12).
The title compound was isolated as a yellow powder. Yield: 92.8 %, m.p. 109.3-111.1 °C. HPLC tR =
5.42 min, 99.9% purity.
1H NMR (500 MHz, CDCl3): δ 8.54 (N1”H, [1H],bs), 8.35 (C8H, [1H], m (10 lines)), 7.40 – 7.28
(C4’H,C6’H, [2H], m), 7.26 (C4”H, [1H], dd, 3JH-F-9.5, 4J=2.5), 7.24 – 7.17 (C5’H,C7”H, [2H], m), 7.17 –
7.10 (C3’H, [1H], m), 6.99 (C2”H, [1H], bs), 6.96 (C6H, [1H], m), 6.87 (C6”H, [1H], td, 3J=9.0, 4J=2.4),
6.72 (C5H, [1H], d, 3J=9.5), 6.41 (C7H, [1H], m(13 lines)), 4.17 (C1xH2, [2H], m), 3.14 (CbH,CaH(E), [2H],
pd), 2.98 (CeH(E), [1H], pd), 2.48 (C4xH2, [2H], pt), 2.17 – 1.96 (CaH(A),CeH(A),CcH(E), [3H], m), 1.78
(CdH2,C2xH2, [4H], m), 1.66 (C3xH2, [2H], q, 3J=7.5), 1.45 (CcH(A), [1H], m).
13C NMR (125 MHz, CDCl3): δ 160.8 (C2’, d, 1J=248.0), 159.6 (C3, 2s), 157.4 (C5’, d, 1J=234.0), 148.8
(C1, s), 144.0 (C4a, s), 133.4 (C6’, 2d, 3J=2.5 i 3J=2.8), 133.2 (C6, s), 132.7 (C7”, s), 130.0 (C4’, d, 3J=8.3),
128.1 (C8, s), 126.9 (C3”a, d, 3J=9.6), 124.4 (C5’, d, 4J=2.4), 122.3 (C2”, s), 121.1 (C5, s), 120.3 (C1’, d,
2J=16.1), 119.1 (C3”, s), 116.0 (C3’, 2d, 2J=22,1 i 2J=22.3), 111.8 (C7”, d, 3J=9.6), 110.8 (C7, s), 110.0
(C6”, d, 2J=26.4), 103.9 (C4”, d, 2J=23.3), 98.2 (C4, 2s), 60.3 (Ca, s), 58.4 (C4x, s), 53.8 (Ce, s), 42.3 (C1x,
s), 33.5 (Cb, s), 31.0 (Cc, s), 25.4 (Cd, s), 25.2 (C2x, s), 23.8 (C3x, s).
ESI-HRMS m/z: Calcd for C31H31F2N4O2 [M+H]+ 529.24151. Found: 529.24169.
3.2.3.13. 2-[4-[3-(5-fluoro-1H-indol-3-yl)-1-piperidyl]butyl]-4-(o-tolyl)pyrido[1,2-c]pyrimidine-1,3-
dione (9.13).
The title compound was isolated as a yellow powder. Yield: 97.6 %, m.p. 127.5-130.2 °C. HPLC tR =
6.35 min, 98.7% purity.
1H NMR (500 MHz, CDCl3): δ 8.57 (N1”H, [1H], bs), 8.35 – 8.30 (C8H, [1H], m), 7.32 – 7.20 (C4’-
6’H,C4”H, [4H], m), 7.18 (C7”H, [1H], dd, 3J=9.0, 4JH-F=4.5), 7.12 (C3’H, [1H], m), 6.95 (C2”H, [1H], bs),
6.90 – 6.82 (C6H,C6”H , [2H], m), 6.53 (C5H, [1H], d, 3J=9.5), 6.36 (C7H, [1H], m, 3J1=7.5, 3J2=6.0,
4J=1.0), 4.19 (C1xH2, [2H], m), 3.11 (CbH,CaH(E), [2H], m), 2.96 (CeH(E), [1H], pd), 2.46 (C4xH2, [2H], t,
3J=6.0), 2.13 and 2.10 (CH3, [3H], 2s), 2.02 (CaH(A),CeH(A),CcH(E), [3H], m), 1.77 (C2xH2,CdH2, [4H],
m), 1.65 (C3xH2, [2H], m), 1.45 (CcH(A), [1H], m).
13C NMR (125 MHz, CDCl3): δ 159.7 (C3, s), 157.4 (C5”, d, 1J=234.0), 149.0 (C1, s), 143.5 (C4a, s), 138.5
(C2’, 2s), 132.7 (C1’, s), 132.5 (C6, s), 132.1 (C7”a, s), 131.6 (C3’ 2s), 130.5 (C6’, s), 128.3 (C4’, s), 128.0
(C8, s), 126.9 (C3”a, d, 3J=9.6), 126.3 (C5’, 2s), 122.4 (C2”, s), 121.3 (C5, s), 119.1 (C3”, s), 111.8 (C7”,
d, 3J=9.6), 110.6 (C7, s), 109.9 (C6”, d, 2J=26.3), 104.0 (C4, s), 103.8 (C4”, d, 2J=23.3), 60.4 (Ca, s), 58.5
(C4x, s), 53.9 (Ce, s), 42.1 (C1x, s), 33.6 (Cb, s), 31.1 (Cc, s), 25.5 (Cd, s), 25.3 (C2x, s), 23.9 (C3x, s), 19.6
(CH3, 2s).
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ESI-HRMS m/z: Calcd for C32H34FN4O2 [M+H]+ 525.26658. Found: 525.26687.
3.2.3.14. 2-[4-[3-(5-fluoro-1H-indol-3-yl)-1-piperidyl]butyl]-4-(2-methoxyphenyl)pyrido[1,2-
c]pyrimidine-1,3-dione (9.14).
The title compound was isolated as a yellow powder. Yield: 92.0 %, m.p. 110.0-112.3 °C. HPLC tR =
5.48 min, 99.9% purity.
1H NMR (500 MHz, CDCl3): δ 8.31 (C8H, [1H], m (8 lines)), 8.27 (N1”H, [1H], bs), 7.36 (C4’H, [1H], m (8
lines)), 7.27 (C4”H, [1H], m), 7.22 (C7”H, [1H], dd, 3J=9.0, 4JH-F=4.5), 7.21 (C6’H, [1H], m), 7.03
(C3’H,C2”H, [2H], m), 6.96 (C5’H, [1H], m,(6 lines)), 6.92 – 6.82 (C6H,C6”H, [2H], m), 6.61 (C5H, [1H],
dt, 3J=9.5, 4J=5J=1.0), 6.35 (C7H, [1H], m (10 lines)), 4.17 (C1xH2, [2H], m (11 lines)), 3.73 and 3.70
(OCH3, [3H], 2s), 3.15 (CbH,CaH(E), [2H], bs), 2.99 (CeH(E), [1H], bs), 2.50 (C4xH2, [2H], bs), 2.13
(CaH(A),CeH(A), [2H], pd), 2.02 (CcH(E), [1H], pd), 1.78 (C2xH2,CdH2, [4H], m), 1.67 (C3xH2, [2H], m),
1.48 (CcH(A), [1H], m).
13C NMR (125 MHz, CDCl3): δ 159.5 (C3, s), 157.4 (C2’, 2s), 157.04 (C5”, d, 1J=231.1), 148.6 (C1, s),
143.2 (C4a, s), 132.5 (C6, 2s), 132.2 (C7”a, s), 131.5 (C6’, s), 129.2 (C4’, s), 127.4 (C8, s), 126.5 (C3”a,
s), 121.9 (C2’, s), 121.4 (C5, s), 121.0 (C1’, s), 120.5 (C5’, 2s), 118.0 (C3”, s), 111.3 (C7”, d, 3J=9.7),
110.9 (C3’, 2s), 110.0 (C7, s), 109.7 (C6”, d, 2J=26.4), 103.5 (C4”, d, 2J=23.3), 100.7 (C4, s), 60.0 (Ca, s),
58.0 (C4x, s), 55.1 (OCH3, 2s), 53.4 (Ce, s), 41.5 (C1x, s), 33.0 (Cb, s), 30.5 (Cc, s), 25.0 (Cd, s), 25.0 (C2x,
s), 24.5 (C3x, s).
ESI-HRMS m/z: Calcd for C32H34FN4O3 [M+H]+ 541.26149. Found: 541.26142.
3.2.3.15. 4-(4-chlorophenyl)-2-[4-[3-(5-fluoro-1H-indol-3-yl)-1-piperidyl]butyl]pyrido[1,2-c]pyrimidine-
1,3-dione (9.15).
The title compound was isolated as a yellow powder. Yield: 59.0 %, m.p. 233.3-234.9 °C. HPLC tR =
7.18 min, 98.5% purity.
1H NMR (500 MHz, DMSO d6): δ 10.96 (N1”H, [1H], bs), 8.29 (C8H, [1H], dt, 3J=7.0, 4J=5J=1.5), 7.46
(C2’H,C6’H, [2H], dt, 3J=8.5, 4J=2.5), 7.32 (C7”H, [1H], dd, 3J=8.5, 4JH-F=4.5), 7.28 (C4”H, [1H], dd), 7.27
{C3’H,C5’H, [2H], dt, 3J=8.0, 4J=2.5), 7.21 (C2”H, [1H], d, 3J=2.5), 7.13 (C6H, [1H], 4d, 3J1=9.5, 3J2=6.5,
4J=1.0), 6.89 (C6”H, [1H], td, 3J=9.0, 4J=2.5), 6.77 (C5H, [1H], dt, 3J=9.5, 4J=5J=1.0), 6.57 (C7H, [1H], m,
3J1=7.5, 3J2=6.5, 4J=1.0), 4,000 (C1xH2, [2H], t, 3J=7.5), 3.15 – 2.90 (CbH,CaH(E),CeH(E), [3H], m), 2.49
(C4xH2, [2H], bs), 2.16 (CaH(A),CeH(A), [2H], bs), 1.93 (CcH(E), [1H], pd), 1.80 – 1.60 (C2xH2,CdH2, [4H],
m), 1.55 (C3xH2, [2H], bs), 1.49 (CcH(A), [1H], m).
13C NMR (125 MHz, DMSO d6): δ 159.1 (C3, s), 156.5 (C5”, d, 1J=230.8), 148.4 (C1, s), 143.3 (C4a, s),
134.1 (C7”a, s), 133.2 (C3’,C6’, s), 132.8 (C6, s), 132.3 (C4’, s), 131.9 (C1’, s), 128.4 (C2’,C6’, s), 128.1
(C8, s), 126.4 (C3”a, d, 3J=9.6), 123.4 (C2”, s), 120.3 (C5, s), 118.0 (C3”, s), 112.4 (C7”, d, 3J=9.8), 111.3
(C7, s), 109.0 (C6”, d, 2J=26.2), 103.1 (C4”, d, 2J=23.0), 101.9 (C4, s), 60.0 (Ca, s), 57.4 (C4x, s), 53.1 (Ce,
s), 41.5 (C1x, s), 33.0 (Cb, s), 30.7 (Cc, s), 25.0 (C2x, s), 24.8 (Cd, s), 24.0 (C3x, s).
ESI-HRMS m/z: Calcd for C31H31ClFN4O2 [M+H]+ 545.21196. Found: 545.21225.
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3.2.3.16. 4-(4-fluorophenyl)-2-[4-[3-(5-fluoro-1H-indol-3-yl)-1-piperidyl]butyl]pyrido[1,2-c]pyrimidine-
1,3-dione (9.16).
The title compound was isolated as a yellow powder. Yield: 72.1 %, m.p. 102.3-104.7 °C. HPLC tR =
5.79 min, 99.9% purity.
1H NMR (500 MHz, DMSO d6): δ 10.92 (N1”H, [1H], bs), 8.28 (C8H, [1H], dt, 3J=7.5, 4J=5J=1.0), 7.31
(C7”H, [1H], dd, 3J=9.0, 4JH-F=5.0), 7.28 (C2’H,C6’H,C4”H, [3H], m), 7.23 (C3’H,C5’H, [2H], tt, 3J=9.0,
4J=2.5), 7.20 (C2”H, [1H], 3J=2.5), 7.12 (C6H, [1H], 4d, 3J1=8.5, 3J2=6.0, 4J=1.0), 6.88 (C6”H, [1H], td,
3J=9.5, 4J=2.5), 6.74 (C5H, [1H], dt, 3J=9.5, 4J=5J=1.0), 6.55 (C7H, [1H], m, 3J1=7.5, 3J2=6.0, 4J=1.0), 4.00
(C1xH2, [2H], t, 3J=7.5), 2.96 (CbH,CaH(E),CeH(E), [3H], m), 2.36 (C4xH2, [2H], bs), 2.01 (CaH(A),CeH(A),
[2H], bs), 1.92 (CcH(E), [1H], pd), 1.65 (C2xH2,CdH2, [4H], m), 1.51 (C3xH2, [2H], m), 1.43 (CcH(A), [1H],
m).
13C NMR (125 MHz, DMSO d6): δ 161.4 (C4’, d, 1J=244.0), 159.3 (C3, s), 156.5 (C5”, d, 1J=230.9), 148.4
(C1, s), 143.3 (C4a, s), 133.8 (C7”a, s), 133.3 (C2’,C6’, d, 3J=8.0), 132.8 (C6, s), 129.6 (C1’, d, 4J=3.0),
128.0 (C8, s), 126.5 (C3”a, d, 3J=9.7), 123.3 (C2”, s), 120.3 (C5, s), 118.0 (C3”, s), 115.3 (C3’,C5’, d,
2J=21.2), 112.3 (C7”, d, 3J=9.7), 111.1 (C7, s), 108.9 (C6”, d, 2J=26.0), 103.1 (C4”, d, 2J=23.0), 102.2 (C4,
s), 60.2 (Ca, s), 57.8 (C4x, s), 53.5 (Ce, s), 41.6 (C1x, s), 33.3 (Cb, s), 31.1 (Cc, s), 25.2 (C2x, s), 25.0 (Cd,
s), 23.8 (C3x, s).
ESI-HRMS m/z: Calcd for C31H31F2N4O2 [M+H]+ 529.24151. Found: 529.24167.
3.2.3.17. 2-[4-[3-(5-fluoro-1H-indol-3-yl)-1-piperidyl]butyl]-4-(p-tolyl)pyrido[1,2-c]pyrimidine-1,3-
dione (9.17).
The title compound was isolated as a yellow powder. Yield: 68.6 %, m.p. 129.1-132.7 °C. HPLC tR =
7.27 min, 99.3% purity.
1H NMR (500 MHz, DMSO d6): δ 10.93 (N1”H, [1H], bs), 8.26 (C8H, [1H], dt, 3J=7.5, 4J=5J=1.0), 7.32
(C7”H, [1H], dd, 3J=8.5, 4JH-F=5.0), 7.27 (C4”H, [1H], dd), 7.21 (C2’H,C6’H,C2”H, [3H], m), 7.13
(C3’H,C5’H, [2H], dt, 3J=8.0, 4J=1.5), 7.08 (C6H, [1H], 4d, 3J1=9.5, 3J2=6.5, 4J=1.0), 6.89 (C6”H, [1H], td,
3J=9.5, 4J=2.5), 6.75 (C5H, [1H], dt, 3J=9.5, 4J=5J=1.0), 6.52 (C7H, [1H], m, 3J1=7.5, 3J2=6.5, 4J=1.5), 3.99
(C1xH2, [2H], t, 3J=7.5), 2.98 (CbH,CaH(E),CeH(E), [3H], m), 2.36 (C4xH2, [2H], bs), 2.34 (CH3, [3H], s),
2.03 (CaH(A),CeH(A), [2H], bs), 1.92 (CcH(E), [1H], pd), 1.64 (C2xH2,CdH2, [4H], m), 1.52 (C3xH2, [2H],
bs), 1.45 (CcH(A), [1H], bs).
13C NMR (125 MHz, DMSO d6): δ 159.3 (Cs, s), 156.5 (C5”, d, 1J=230.9), 148.4 (C1, s), 143.1 (C4a, s),
136.5 (C4’, s), 133.4 (C7”a, s), 132.8 (C6, s), 131.1 (C3’,C5’, s), 130.3 (C1’, s), 129.0 (C2’,C6’, s), 127.8
(C8, s), 126.5 (C3”a, d, 3J=9.3), 123.3 (C2”, s), 120.6 (C5, s), 117.0 (C3”, s), 112.3 (C7’, d, 3J=9.9), 111.0
(C7, s), 108.9 (C6”, d, 2J=26.2), 107.2 (C4, s), 103.1 (C4”, d, 2J=23.0), 60.0 (Ca, s), 57.7 (C4x, s), 53.4 (Ce,
s), 41.5 (C1x, s), 33.2 (Cb, s), 31.1 (Cc, s), 25.0 (C2x, s), 24.9 (Cd, s), 24.0 (C3x, s), 20.8 (CH3, s).
ESI-HRMS m/z: Calcd for C32H34FN4O2 [M+H]+ 525.26658. Found: 525.26668.
3.2.3.18. 2-[4-[3-(5-fluoro-1H-indol-3-yl)-1-piperidyl]butyl]-4-(4-methoxyphenyl)pyrido[1,2-
c]pyrimidine-1,3-dione (9.18).
20

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The title compound was isolated as a yellow powder. Yield: 88.3 %, m.p. 118.7-122.1 °C. HPLC tR =
5.90 min, 99.9% purity.
1H NMR (500 MHz, CDCl3): δ 8.48 (N1”H, [1H], bs), 8.30 (C8H, [1H], dt, 3J=7.5, 4J=5J=1.0), 7.26 (C4”H,
[1H], dd, 3JH-F=10.0, 4J=2.5), 7.23 (C7”H, [1H], dd, 3J=8.5, 4JH-F=4.5), 7.21 (C2’H,C6’H, [2H], d, 3J=8.5),
6.99 (C2”H, [1H], bs), 6.95 (C3’H,C5’H, [2H], d, 3J=8.5), 6.91 – 6.84 (C5H,C6H,C6”H, [3H], m), 6.35
(C7H, [1H], m, 3J1=7.5, 3J2=5.5, 4J=2.5), 4.17 (C1xH2, [2H], m), 3.83 (OCH3, [3H], s), 3.18 CaH(E),CbH,
[2H], pd), 3.03 (CeH(E), [1H],bs), 2.53 (C4xH2, [2H], bs), 2.16 (CaH(A),CeH(A), [2H], m), 2.02 (CcH(E),
[1H], pd), 1.84 (CdH(E),[1H], bs), 1.78 (CdH(A),C2xH2, [3H],m), 1.69 (C3xH2, [2H], m), 1.47 (CcH(A),
[1H], kd, 3JA-A=12.5, 3JA-E=4.0).
13C NMR (125 MHz, CDCl3): δ 160.5 (C3,s), 159.1 (C4’, s), 157.5 (C5”, d, 1J=234.2), 148.9 (C1, s), 143.6
(C4a, s), 132.7 (C7”a, s), 132.3 (C2’,C6’, s), 132.2 (C6, s), 127.9 (C8, s), 126.8 (C3”a, d, 3J=9.9), 124.8
(C1’, s), 122.4 (C2”, s), 121.5 (C5, s), 117.0 (C3”, d), 114.3 (C3’,C5’ s), 111.8 (C7”, d, 3J=9.7), 110.7 (C7,
s), 110.1 (C6”, d, 2J=26.5), 104.4 (C4, s), 103.9 (C4”, d, 2J=23.4), 60.0 (Ca, s), 58.3 (C4x, s), 55.3 (OCH3,
s), 53.8 (Ce, s), 42.1 (C1x, s), 33.4 (Cb, s), 30.9 (Cc, s), 25.4 (Cd, s), 25.0 (C2x, s), 24.0 (C3x, s).
ESI-HRMS m/z: Calcd for C32H34FN4O3 [M+H]+ 541.26149. Found: 541.26178.
3.2.3.19. 2-[4-[3-(5-methoxy-1H-indol-3-yl)-1-piperidyl]butyl]-4-phenyl-pyrido[1,2-c]pyrimidine-1,3-
dione (9.19).
The title compound was isolated as a yellow powder. Yield: 85.0 %, m.p. 94.9-96.1 °C. HPLC tR = 5.29
min, 99.9% purity.
1H NMR (500 MHz, CDCl3): δ 8.30 (N1”H, [1H], bs), 8.30 (C8H, [1H], dt, 3J=7.5, 4J=5J=1.0), 7.43
(C3’H,C5’H, [2H], t, 3J=7.0), 7.35 (C4’H, [1H], tt, 3J=7.0, 4J=1.5), 7.29 (C2’H,C6’H, [2H], m, 3J=7.0), 7.21
(C7”H, [1H], d, 3J=8.5), 7.10 (C4”H, [1H], d, 4J=2.5), 6.92 (C2”H, [1H], d, 3J=2.0), 6.88 (C5H,C6H, [2H],
m), 6.82 (C6”H, [1H], dd, 3J=9.0, 4J=2.5), 6.36 (C7H, [1H], m, 3J1=7.5, 3J2=5.0, 4J=2.5), 4.17 (C1xH2, [2H],
m), 3.83 (OCH3, [3H], s), 3.26 (CbH,CaH(E), [2H], pd), 3.10 (CeH(E), [1H], bs), 2.57 (C4xH2, [2H], bs),
2.13 (CaH(A),CeH(A), [2H], m), 2.05 (CcH(E), [1H], pd), 1.85 – 1.55 (CdH2,C2xH2,C3xH2, [6H], m), 1.47
(CcH(A), [1H], kd, 3JA-A=13.0, 3JA-E=4.0).
13C NMR (125 MHz, CDCl3): δ 160.2 (C3, s), 153.8 (C5”, s), 148.9 (C1, s), 143.6 (C4a, s), 132.8 (C6, s),
132.5 (C1’, s), 131.3 (C7”a, s), 131.2 (C2’,C6’, s), 128.8 (C3’,C5’, s), 127.9 (C4’, s), 127.8 (C8, s), 126.9
(C3”a, s), 121.4 (C5, s), 121.1 (C2”, s), 119.0 (C3”, s), 112.3 (C6”, s), 112.0 (C7”, s), 110.8 (C7, s), 104.8
(C4, s), 100.7 (C4”, s), 60.2 (Ca, s), 58.2 (C4x, s), 56.0 (OCH3, s), 53.6 (Ce, s), 42.0 (C1x, s), 33.1 (Cb, s),
30.6 (Cc, s), 25.3 (Cd, s), 24.9 (C2x, s), 23.3 (C2x, s).
ESI-HRMS m/z: Calcd for C32H35N4O3 [M+H]+ 523.27092. Found: 523.27118.
3.2.3.20. 4-(2-chlorophenyl)-2-[4-[3-(5-methoxy-1H-indol-3-yl)-1-piperidyl]butyl]pyrido[1,2-
c]pyrimidine-1,3-dione (9.20).
The title compound was isolated as a yellow powder. Yield: 76.1 %, m.p. 117.3-120.2 °C. HPLC tR =
5.23 min, 99.9% purity.
1H NMR (500 MHz, CDCl3): δ 8.35 and 8.34 (C8H, [1H], m (10 lines)), 8.14 (N1”H< [1H], bs), 7.49 (C3’H,
[1H], m), 7.35 – 7.26 (C4’-6’H, [3H], m), 7.21 (C7”H, [1H], d, 3J=8.5), 7.09 (C2”H, [1H], d, 3J=2.5), 6.94
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ACCEPTED MANUSCRIPT
(C6H,C4”H, [2H], m), 6.82 (C6”H, [1H], dd, 3J=8.5, 4J=2.5), 6.53 (C5H, [1H], dt, 3J=9.5, 4J=5J=1.0), 6.40
(C7H, [1H], m (13 lines)), 4.18 (C1xH2, [2H] m), 3.84 and 3.83 (OCH3, [3H], 2s), 3.19 (CbH,CaH(E), [2H],
pd), 3.00 (CeH(E), [1H], bs), 2.48 (C4xH2, [2H], bs), 2,05 (CaH(A),CeH(A),CcH(E), [3H], pd), 1.79
(CdH2,C2xH2, [4H], m), 1.66 (C3xH2, [2H], m), 1.47 (CcH(A), [1H], m).
13C NMR (125 MHz, CDCl3): δ 159.5 (C3, 2s), 153.8 (C5”, s), 148.9 (C1, s), 143.8 (C4a, s), 135.7 (C2’, 2s),
133.4 (C6, s), 133.1 (C6’, s), 131.7 (C1’, s), 131.3 (C7”a, s), 130.0 (C3’, s), 129.6 (C4’, s), 128.1 (C8, s),
127.3 (C5’, 2s), 127.0 (C3”a, s), 121.0 (C5,C2”, s), 119.0 (C3”, s), 112.2 (C6”, 2s), 111.9 (C7”, s), 110.8
(C7, s), 102.2 (C4, s), 100.7 (C4”, 2s), 60.8 (Ca, s), 58.5 (C4x, s), 55.9 (OCH3, s), 53.8 (Ce, s), 42.2 (C1x, s),
33.6 (Cb, s), 31.1 (Cc, s), 25.5 (Cd, s), 25.0 (C2x, s), 23.9 (C3x, s).
ESI-HRMS m/z: Calcd for C32H34ClN4O3 [M+H]+ 557.23194. Found: 557.23212.
3.2.3.21. 4-(2-fluorophenyl)-2-[4-[3-(5-methoxy-1H-indol-3-yl)-1-piperidyl]butyl]pyrido[1,2-
c]pyrimidine-1,3-dione (9.21).
The title compound was isolated as a yellow powder. Yield: 77.9 %, m.p. 126.5-128.7 °C. HPLC tR =
4.85 min, 99.9% purity.
1H NMR (500 MHz, CDCl3): δ 8.35 and 8.34 (C8H, [1H], m(12 lines)), 8.33 (N1”H, [1H], bs), 7.37 (C4’H,
[1H], m(22 lines)), 7.30 (C6’H, [1H], m(10 lines)), 7.23 (C7”H, [1H], d, 3J=9.0), 7.19 (C5’H, [1H], m(8
lines)), 7.16 – 7.10 (C3’H,C4”H, [2H], m), 6.97 (C6H, [1H], m(14 lines)), 6.93 (C2”H, [1H], m), 6.82
(C6”H, [1H], dd, 3J=8.5, 4J=2.0), 6.72 (C5H, [1H], m), 6.42 (C7H, [1H], m(12 lines)), 4.17 (C1xH2, [2H],
pt), 3.84 (OCH3, [3H], s), 3.30 (CbH,CaH(E), [2H], pd), 3.15 (CeH(E), [1H], bs), 2.62 (C4xH2, [2H], bs),
2.18 (CaH(A),CeH(A), [2H], bs), 2.06 (CcH(E), [1H], pd), 1.96 (CdH(E), [1H], bs), 1.85 – 1.65
(C2xH2,C3xH2,CdH(A), [5H], m), 1.48 (CcH(A), [1H], m).
13C NMR (125 MHz, CDCl3): δ 160.8 (C2’, d, 1J=246,9), 159.7 and 159.6 (C3, 2s), 153.9 (C5”, s), 148.8
(C1, s), 144.1 (C4a, 2s), 133.4 (C6’, 2d, 3J=2.6, 3J=2.8), 133.3 and 133.2 (C6, 2s), 131.0 (C4’, d, 3J=8.2),
128.2 (C8, 2s), 126.8 (C3”a, s), 124.5 and 124.4 (C5’, 2d, 4J=3.1, 4J=3.4), 121.1 (C5,C2”, 2s), 120.3 (C1’,
d, 2J=16.0), 116.0 (C3’, 2d, 2J=22.3, 2J=22.1), 112.3 (C6”, 2s), 112.0 (C7”, s), 110.9 (C7, 2s), 100.7 and
100.6 (C4”, 2s), 98.2 (C4, 2s), 60.0 (Ca, s), 58.0 (C4x, s), 56.0 (OCH3, s), 53.5 (Ce, s), 41.9 (C1x, s), 32.9
(Cb, s), 25.2 (Cc, s), 24.7 (Cd, s), 25.0 (C2x, s), 22.8 (C3x, s).
ESI-HRMS m/z: Calcd for C32H34FN4O3 [M+H]+ 541.26149. Found: 541.26166.
3.2.3.22. 2-[4-[3-(5-methoxy-1H-indol-3-yl)-1-piperidyl]butyl]-4-(o-tolyl)pyrido[1,2-c]pyrimidine-1,3-
dione (9.22).
The title compound was isolated as a yellow powder. Yield: 73.0 %, m.p. 104.5-105.6 °C. HPLC tR =
5.59 min, 99.6% purity.
1H NMR (500 MHz, CDCl3): δ 8.31 (C8H, [1H], dt, 3J=7.5, 4J=5J=1.0), 8.12 (N1”H, [1H], bs), 7.30 – 7.22
(C4’-6’H, [3H], m(20 lines)), 7.21 (C7”H, [1H], dd, 3J=9.0, 5J=0.5), 7.12 (C3’H, [1H], m(9 lines)), 7.09
(C4”H, [1H], d, 4J=2.5), 6.94 (C2”H, [1H], bs), 6.87 (C6H, [1H], 4d, 3J1=9.0, 3J2=6.5, 4J=1.5), 6.82 (C6”H,
[1H], dd, 3J=8.5, 4J=2.5), 6.54 (C5H, [1H], dt, 3J=9.5, 4J=5J=1.0), 6.36 (C7H, [1H], m, 3J1=7.5, 3J2=6.5,
4J=1.5), 4.18 (C1xH2, [2H], m), 3.84 and 3.83 (OCH3, [3H], 2s), 3.20 (CbH,CaH(E), [2H] pd), 2.49 (C4xH2,
[2H], bs), 2.13 and 2.11 (CH3, [3H], 2s), 2.14 – 2.00 (CaH(A),CeH(A),CcH(E), [3H], m), 1.79 (C2xH2,CdH2,
[4H], m), 1.67 (C3xH2, ]2H], m), 1.49 (CcH(A), [1H], m).
22

ACCEPTED MANUSCRIPT
13C NMR (125 MHz, CDCl3): δ 159.7 (C3, s), 153.8 (C5”, s), 149.1 C1, s), 143.5 (C4a, s), 138.5 (C2’, 2s),
132.5 (C1’, s), 132.1 (C6, s), 131.6 (C3’, 2s), 131.3 (C7”a, s), 130.5 (C6’, s), 128.3 (C4’, s), 128.0 (C8, s),
127.0 (C3”a, s), 126.4 (C5’, s), 121.3 (C5, s), 121.1 (C3”, s), 112.2 (C6”, s), 111.9 (C7”, s), 110.5 (C7, s),
104.0 (C4, s), 100.7 (C4”, 2s), 60.7 (Ca, s), 58.5 (C4x, s), 56.0 (OCH3, 2s), 53.9 (Ce, s), 42.1 (C1x, s), 33.5
(Cb, s), 31.0 (Cc, s), 25.5 (Cd, s), 25.0 (C2x, s), 23.8 (C3x, s), 19.6 (CH3, 2s).
ESI-HRMS m/z: Calcd for C33H37N4O3 [M+H]+ 537.28657. Found: 537.28658.
3.2.3.23. 2-[4-[3-(5-methoxy-1H-indol-3-yl)-1-piperidyl]butyl]-4-(2-methoxyphenyl)pyrido[1,2-
c]pyrimidine-1,3-dione (9.23).
The title compound was isolated as a yellow powder. Yield: 62.5 %, m.p. 106.6-108.3 °C. HPLC tR =
4.90 min, 99.9% purity.
1H NMR (500 MHz, CDCl3): δ 8.42 (N1”H, [1H], bs), 8.29 (C8H, [1H], m (14 lines)), 7.36 (C4’H, [1H], m
(12 lines)), 7.20 (C7”H, [1H], dd, 3J=8.5, 5J=0.5), 7.18 (C6’H, [1H], m (6 lines)), 7.10 (C4”H, [1H], pt),
7.02 (C5’H, [1H], m (8 lines)), 6.96 (C3’H, [1H], m (8 lines)), 6.89 (C2”H, [1H], bs), 6.87 (C6H, [1H], m
(12 lines)), 6.81 (C6”H, [1H], dd, 3J=8.5, 4J=2.0), 6.61 (C5H, [1H], dt), 6.35 (C7H, [1H], m (10 lines)),
4.16 (C1xH2, [2H], m), 3.84 (C5”-OCH3, [3H], s), 3.72 and 3.67 (C2’-OCH3, [3H], 2s), 3.27 (CbH,CaH(E),
[2H], m), 3.12 (CeH(E), [1H], bs), 2.60 (C4xH2, [2H], bs), 2.15 (CeH(A), [1H], bs), 2.04 (CcH(E), [1H], pd),
1.85 – 1.65 (CdH2,C2xH2,C3xH2, [6H], m), 1.45 (CcH(A), [1H], kd, 3JA-A=12.5, 3JA-E=3.5).
13C NMR (125 MHz, CDCl3): δ 159.9 (C3, s), 157.9 (C2’, 2s), 153.8 (C5”, s), 149.1 (C1, s), 143.6 (C4a, s),
133.0 (C6’, 2s), 132.0 (C6, s), 131.3 (C7”a, s), 129.7 (C4’, s), 127.8 (C8, s), 127.0 (C3”a, s), 121.9 (C5, s),
121.4 (C1’, s), 121.1 (C2”, s), 121.0 and 120.9 (C5’, 2s), 119.0 (C3”, s), 112.3 (C6”, s), 111.9 (C7’, s),
111.4 (C3’, 2s), 110.5 (C7, s), 101.2 (C4, s), 100.7 (C4”, 2s), 60.7 (Ca, s), 58.5 (C4x, s), 56.0 (5”-OCH3, s),
55.6 (C2’-OCH3, 2s), 53.8 (Ce, s), 42.1 (C1x, s), 33.5 (Cb, s), 31.0 (Cc, s), 25.5 (Cd, s), 25.0 (C2x, s), 23.7
(C3x, s).
ESI-HRMS m/z: Calcd for C33H37N4O4 [M+H]+ 553.28148. Found: 553.28179.
3.2.3.24. 4-(4-chlorophenyl)-2-[4-[3-(5-methoxy-1H-indol-3-yl)-1-piperidyl]butyl]pyrido[1,2-
c]pyrimidine-1,3-dione (9.24).
The title compound was isolated as a yellow powder. Yield: 65.1 %, m.p. 111.8-114.4 °C. HPLC tR =
6.24 min, 98.4% purity.
1H NMR (500 MHz, CDCl3): δ 8.32 (C8H, [1H], dt, 3J=7.5, 4J=5J=1.0), 8.13 (N1”H, [1H], bs), 7.9
(C2’H,C6’H, [2H], dt, 3J=8.5), 7.25 – 7.21 (C3’H,C5’H,C7”H, [3H], m), 7.10 (C4”H, [1H], d, 4J=2.0), 6.95 –
6.91 (C6H,C2”H, [2H], m), 6.87 – 6.81 (C5H,C6”H, [2H], m), 6.387 (C7H, [1H], m, 3J1=7.5, 3J2=6.0,
4J=1.0), 4.16 (C1xH2, [2H], t, 3J=7.5), 3.84 (OCH3, [3H], s), 3.24 (CbH,CaH(E), [2H], bs), 3.07 (CeH(E),
[1H], bs), 2.53 (C4xH2, [2H], bs), 2.08 (CaH(A),CeH(A),CcH(E), [3H], m), 1.85 – 1.73 (CdH2,C2xH2, [4H],
m), 1.69 (C3xH2, [2H], bs), 1.49 (CcH(A), [1H], kd, 3JA-A=12.5, 3JA-E=4.0).
13C NMR (125 MHz, CDCl3): δ 160.0 (C3, s), 153.8 (C5”, s), 148.8 (C1, s), 143.7 (C4a, s), 133.7 (C4’, s),
133.0 (C6, s), 132.7 (C3’,C5’, s), 131.3 (C1’, s), 131.3 (C7”a, s), 129.0 (C2’,C6’, s), 128.1 (C8, s), 127.0
(C3”a, s), 121,036 (C2”, s), 121.0 (C5, s), 118.5 (C3”, s), 112.2 (C6”, s), 111.9 (C7”, s), 110.9 (C7, s),
103.4 (C4, s), 100.7 (C4”, s), 60.5 (Ca, s), 58.4 (C4x, s), 56.0 (OCH3, s), 53.8 (Ce, s), 42.2 (C1x, s), 33.4
(Cb, s), 30.8 (Cc, s), 25.4 (Cd, s), 25.2 (C2x, s), 23.6 (C3x, s).
23

ACCEPTED MANUSCRIPT
ESI-HRMS m/z: Calcd for C32H34ClN4O3 [M+H]+ 557.23194. Found: 557.23219.
3.2.3.25. 4-(4-fluorophenyl)-2-[4-[3-(5-methoxy-1H-indol-3-yl)-1-piperidyl]butyl]pyrido[1,2-
c]pyrimidine-1,3-dione (9.25).
The title compound was isolated as a yellow powder. Yield: 63.1 %, m.p. 105.5-107.7 °C. HPLC tR =
5.17 min, 99.9% purity.
1H NMR (500 MHz, CDCl3): δ 8.31 (C8H, [1H], dt, 3J=7.0, 4J=5J=1.5), 8.15 (N1”H, [1H], bs), 7.26
(C2’H,C6’H, [2H], m), 7.23 (C7”H, [1H], dd, 3J=8.5, 5J=0.5), 7.14 – 7.08 (C3’H,C5’H,C4”H, [3H], m), 6.94
(C2”H, [1H], d, 3J=2.0), 6.92 (C6H, [1H], 4d, 3J1=9.5, 3J2=6.0,4J=1.0), 6.85 – 6.81 (C5H,C6”H, [2H], m),
6.38 (C7H, [1H], m, 3J1=7.5, 3J2=6.5, 4J=1.5), 4.16 (C1xH2, [2H], t, 3J=7.0), 3.84 (OCH3, [3H], s), 3.26
(CbH,CaH(E), [2H], pd), 3.09 (CeH(E), [1H], bs), 2.55 (C4xH2, [2H], bs), 2.12 (CaH(A),CeH(A), [2H], bs),
2.07 (CcH(E), [1H], pd), 1.95 – 1.60 (CdH2,C2xH2,C3xH2, [6H], m), 1.50 (CcH(A), [1H], kd, 3JA-A=12.5, 3JA-
E=4.0).
13C NMR (125 MHz, CDCl3): δ 162.3 (C4’, d, 1J=247.0), 160.2 (C3, s), 153.8 (C5”, s), 148.8 (C1, s), 143.7
(C4a, s), 133.0 (C2’,C6’, d, 3J=8.2), 132.8 (C6, s), 131.3 (C7”a, s), 128.6 (C1’, d, 4J=3.4), 128.0 (C8, s),
126.9 (C3”a, s), 121.1 (C5, s), 121.1 (C2”, s), 118.4 (C3”, s), 115.8 (C3’,C5’, d, 2J=21.5), 112.3 (C6”, s),
111.9 (C7”, s), 110.8 (C7, s), 103.6 (C4, s), 100.7 (C4”, s), 60.3 (Ca, s), 58.3 (C4x, s), 56.0 (OCH3, s), 53.8
(Ce, s), 42.1 (C1x, s), 33.3 (Cb, s), 30.7 (Cc, s), 25.4 (Cd, s), 25.1 (C2x, s), 23.5 (C3x, s).
ESI-HRMS m/z: Calcd for C32H34FN4O3 [M+H]+ 541.26149. Found: 541.26171.
3.2.3.26. 2-[4-[3-(5-methoxy-1H-indol-3-yl)-1-piperidyl]butyl]-4-(p-tolyl)pyrido[1,2-c]pyrimidine-1,3-
dione (9.26).
The title compound was isolated as a yellow powder. Yield: 67.8 %, m.p. 112.1-113.9 °C. HPLC tR =
6.40 min, 97.3% purity.
1H NMR (500 MHz, CDCl3): δ 8.29 (C8H, [1H], dt, 3J=7.5, 4J=5J=1.0), 8.28 (N1”H, [1H], bs), 7.23
(C2’H,C6’H, [2H], d, 3J=8.5), 7.21 (C7”H, [1H], d, 3J=9.0), 7.18 (C3’H,C5’H, [2H], d, 3J=8.5), 7.10 (C4”H,
[1H], d, 4J=2.0), 6.92 (C2”H, [1H], d, 3J=2.0), 6.88 (C5H,C6H, [2H], m), 6.82 (C6”H. [1H], dd, 3J=8.5,
4J=2.0), 6.35 (C7H, [1H], m, 3J1=7.5, 3J2=6.0, 4J=2.5), 4.16 (C1xH2, [2H], m), 3.84 (OCH3, [3H], s), 3.25
(CbH,CaH(E), [2H], pd), 3.09 (CeH(E), [1H], bs), 2.56 (C4xH2, [2H], bs), 2.38 (CH3, [3H], s), 2.12
(CaH(A),CeH(A), [2H], m), 2.05 (CcH(E), [1H], pd), 1.85 – 1.65 (CdH2,C2xH2,C3xH2, [6H], m), 1.47
(CcH(A), [1H], kd, 3JA-A=12.5, 3JA-E=4.0).
13C NMR (125 MHz, CDCl3): δ 160.29 (C3, s), 153.8 (C5”, s), 148.9 (C1, s), 143.5 (C4a, s), 137.5 (C4’, s),
132.2 (C6, s), 131.3 (C7”a, s), 131.0 (C3’,C5’, s), 129.7 (C1’, s), 129.5 (C2’,C6’, s), 127.9 (C8, s), 126.9
(C3”a, s), 121.5 (C5, s), 121.1 (C2”, s), 118.5 (C3”, s), 112.2 (C6”, s), 112.0 (C7”, s), 110.7 (C7, s), 104.8
(C4, s), 100.7 (C4”, s), 60.3 (Ca, s), 58.2 (C4x, s), 56.0 (OCH3, s), 53.6 (Ce, s), 42.0 (C1x, s), 33.2 (Cb, s),
30.6 (Cc, s), 25.3 (Cd, s), 25.0 (C2x, s), 23.4 (C3x, s), 21.3 (CH3, s).
ESI-HRMS m/z: Calcd for C33H37N4O3 [M+H]+ 537.28657. Found: 537.28677.
3.2.3.27. 2-[4-[3-(5-methoxy-1H-indol-3-yl)-1-piperidyl]butyl]-4-(4-methoxyphenyl)pyrido[1,2-
c]pyrimidine-1,3-dione (9.27).
24

ACCEPTED MANUSCRIPT
The title compound was isolated as a yellow powder. Yield: 59.8 %, m.p. 90.4-91.6 °C. HPLC tR = 5.33
min, 99.5% purity.
1H NMR (500 MHz, CDCl3): δ 8.29 (N1”H, [1H], bs), 8.29 (C8H, [1H], dt, 3J=7.5, 4J=5J=1.0), 7.21
(C2’H,C6’H,C7”H, [3H], m), 7.11 (C4”H, [1H], d, 4J=2.5), 6.96 (C3’H,C5’H, [2H], d, 3J=8.5), 6.91 (C2”H,
[1H], d, 3J=1.5), 6.89 – 6.86 (C5H,C6H, [2H], m), 6.822 (C6”H, [1H], dd, 3J=8.5, 4J=2.5), 6.35 (C7H, [1H],
m, 3J1=7.5, 3J2=4.5, 4J=3.0), 4.16 (C1xH2, [2H], t, 3J=7.0), 3.84 (C5”-OCH3, [3H], s), 3.83 (C4’-OCH3, [3H],
s), 3.29 (CbH,CaH(E), [2H], m), 3.13 (CeH(E), [1H], bs), 2.61 (C4xH2, [2H], bs), 2.18 (CaH(A),CeH(A),
[2H], bs), 2.06 (CcH(E), [1H], pd), 1.85 – 1.70 (CdH2,C2xH2,C3xH2, [6H], m), 1.48 (CcH(A), [1H], kd, 3JA-
A=12.5, 3JA-E=3.5).
13C NMR (125 MHz, CDCl3): δ 160.4 (C3, s), 159.13 (C4’, s), 153.8 (C5”, s), 148.9 (C1, s), 143.5 (C4a, s),
132.3 (C2’,C6’, s), 132.2 (C6, s), 131.3 (C7”a, s), 127.9 (C8, s), 127.0 (C3”a, s), 124.9 (C1’, s), 121.6 (C5,
s), 121.1 (C2”, s), 119.0 (C3”, s), 114.3 (C3’,C5’, s), 112.3 (C6”, s), 111.9 (C7”, s), 110.6 (C7, s), 104.5
(C4, s), 100.7 (C4”, s), 60.6 (Ca, s), 58.5 (C4x, s), 56.0 (C5’-OCH3, s), 55.3 (4’-OCH3, s), 53.9 (Ce, s), 42.2
(C1x, s), 33.6 (Cb, s), 31.0 (Cc, s), 25.5 (Cd, s), 25.0 (C2x, s), 23.9 (C3x, s).
ESI-HRMS m/z: Calcd for C33H37N4O4 [M+H]+ 553.28148. Found: 553.28186.
2.3. X-ray crystallography
The diffraction data for 8.1 and 8.2 were collected at room temperature with a KM4 (Cu Kα, λ
= 1.54173 Å) diffractometer. The data were corrected for Lorentz and polarization effects and a
multi-scan absorption correction applied. The structure was solved using direct methods
implemented in SHELXS-97, and refined by the full matrix least-squares on F2 with the SHELXL-97
program [42]. 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 oxygen-bonded H atoms were found in the difference-Fourier map and included at
fixed positions with isotropic displacement parameters. The data were included in supplementary
information.
2.4. Biological tests
2.4.1. In vitro tests
2.4.1.1. 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
25

ACCEPTED MANUSCRIPT
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 equaꢁon [43]:
ꢂꢃꢄꢅ
ꢀ =
ꢁ
ꢆꢇ
1 +
ꢀꢈ
LO – labeled ligand concentration
KD – dissociation constant of labeled ligand
Kd value was determined in our experimental conditions.
2.4.1.2. 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 equaꢁon [43]. Kd value was
determined in our experimental conditions.
2.4.1.3. 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.[44]. 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 mM 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
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(PerkinElmer, USA). For displacement studies, the assay samples contained as radioligands
(PerkinElmer): 2 nM [3 H]-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 mM of 5-HT in 5-HT7 receptors binding experiments, whereas 10 mM
chlorpromazine, 10 mM methiothepine, or 1 mM (+)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–Prushoff equation [43]. Results
were expressed as means of at least two separate experiments.
2.4.2. 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. The experiments were performed on male CD-1 mice (23–40 g). The animals were
kept at room temperature (21 ± 2 ⁰C) 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 (except 14–15 animals/dose in
the hypothermia induced by 8-OH-DPAT and 9.7). 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 solution. Compounds 9.1, 9.7, 9.9 and 9.27 were suspended in a 10% aqueous solution of
dimethyl sulfoxide (DMSO). Vehicle group was administered as 10% aqueous solution of dimethyl
sulfoxide (DMSO). 8-OH-DPAT was injected subcutaneously (sc); 9.1, 9.7, 9.9 and 9.27 were given
intraperitoneally (ip) in a volume of 10 mL/kg/mouse. The obtained data were analyzed by Dunnett’s
test (one drug administration) or by the Newman-Keuls test (two drugs administration).
2.4.2.1. Body temperature in mice
The effects of the tested compounds 9.1, 9.7, 9.9 and 9.27 given alone on the rectal body
temperature 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 WAY100635 (0.1 mg/kg sc) on the
hypothermia induced by the tested compounds and/or 8-OH-DPAT was measured. WAY100635 was
administered 15 min before the tested compounds and the rectal body temperature was recorded
30 min and 60 min after injection. In an independent experiment, the effect of compound 9.7 (with
no effect on body temperature) on the hypothermia induced by 8-OH-DPAT (5 mg/kg sc) was tested.
Compound 9.7 was 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
36.7 ± 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.
2.4.2.2. Forced swimming test in mice
The experiments were performed according to the method of [45]. Mice were placed
individually into glass cylinders (height 25 cm, diameter 10 cm) containing 20 cm of water and
maintained at 23 ⁰C. 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 a 4-min test. The mouse was judged to
be immobile when it remained floating passively, performing slow motion to keep head above the
water. Tested compounds were administered 30 min before the test.
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2.4.3. Metabolic stability
Stock solutions of studied compounds were prepared at a concentration of 100 µM in 1:1
acetonitrile/water. Incubation mixes consisted of 1 µM of studied compound, 100 µM of NADPH in
phosphate buffer and 1 mg/mL of pooled human liver microsomes (HLMs) (Sigma-Aldrich, St. Louis,
MO, USA) in potassium phosphate buffer (0.1 M, pH 7.4). Incubations were carried out in 96-well
plates at 37 °C. Incubation mixtures (excluding compound solution) were subjected to 5 min pre-
incubation and started by addition of 10 µL of compound stock solution. After 0, 5, 10, 15, and 30
min 25 μl samples of incubation reaction were added to the equal volume of ice-cold acetonitrile
containing 1 μM of IS (buspirone hydrochloride). Control incubations were performed without
NADPH to assess possible chemical instability. 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 x 100
mm, 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% formic acid in deionized 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;
10.01–10.02 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 in positive ionization mode. The mass analyzer was set individually
to each derivative to detect pseudomolecular ions [M+H+]. 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 study enabled us to obtain a number of new derivatives of 4-aryl-pyrido[1,2-c]pyrimidine
9.1–9.27 having a conformationally restricted residue of 3-(3-piperidinyl)-1H-indole (9.1–9.9), 5-
fluoro-3-(3-piperidinyl)-1H-indole (9.10–9.18) or 5-methoxy-3-(3-piperidinyl)-1H-indole (9.19–9.27).
In vitro studies have shown that derivatives 9.1, 9.2, 9.4, 9.7, 9.9, 9.14 and 9.27 have high affinity to
both molecular targets 5-HT1A receptor and SERT protein (5-HT1A Ki = 41–118 nM; SERT Ki = 22–
102 nM). The remaining compounds showed moderate to weak affinity to 5-HT1A and SERT. In
general, compounds with an unsubstituted or a para-substituted benzene ring of the pyrido[1,2-
c]pyrimidine residue in the terminal part were characterized by higher binding affinity, which can be
justified by the greater flexibility of the structure. For the selected compounds 9.1, 9.7, 9.9 and 9.27,
further in vitro, in vivo and metabolic stability tests were performed. The in vitro studies in the
extended receptor profile (D2, 5-HT2A, 5-HT6 and 5-HT7) indicated their selectivity toward the 5-HT1A
receptor and SERT protein. The in vivo studies of these compounds (8-OH-DPAT-induced
hypothermia in mice, FST) revealed that compound 9.1 had the properties of presynaptic agonist of
the 5-HT1A receptor, but did not show any postsynaptic activity [39]. In turn, compound 9.7
demonstrated the properties of a presynaptic antagonist of the 5-HT1A receptor. Metabolic stability
studies showed that compounds 9.1, 9.7 and 9.9, having an unsubstituted indole residue (R3 = -H),
were more resistant to biotransformation reactions of the first pass phase than compound 9.27,
which contains a 5-methoxy substituted indole residue (R3 = -OCH3). The presented results allow
further optimization of the structure in order to obtain a novel potential antidepressant agent within
the group of pyrido[1,2-c]pyrimidine derivatives.
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