Novel 4-aryl-pyrido[1,2-c]pyrimidines with dual SSRI and 5-HT1A activity: Part 2

Article abstract of DOI:10.1016/j.ejmech.2009.07.007

Derivatives of 4-aryl-5,6,7,8-tetrahydro-pyrido[1,2-c]pyrimidine were synthesized. These compounds contain the 3-(4-piperidyl)-1H-indole residue or its 5-methoxy or 2-methyl derivative. In vitro binding tests were performed to determine the affinity of the compounds for the 5-HT_1A receptor and serotonin transporter (SERT) proteins in the rat brain cortex. In vivo studies, particularly the inducible hypothermia test and forced swimming test, were conducted to determine agonistic/antagonistic activity with pre- and postsynaptic 5-HT_1A receptors. Molecular modeling techniques were used to determine the binding modes of the selected compounds at the 5-HT_1A receptor and SERT. The SAR analysis showed that the presence of the 3-(4-piperidyl)-1H-indole group or its 5-methoxy derivative, as well as a para substitution with -OCH₃ or -F in the aryl ring of 4-aryl-5,6,7,8-tetrahydro-pyrido[1,2-c]pyrimidine, results in an increased affinity for both the 5-HT_1A receptors and SERT. In contrast, the presence of the 2-methyl-3-(4-piperidyl)-1H-indole group resulted in a considerable decrease in binding affinity.

Full text of DOI:10.1016/j.ejmech.2009.07.007

European Journal of Medicinal Chemistry 44 (2009) 4702–4715
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Novel 4-aryl-pyrido[1,2-c]pyrimidines with dual SSRI and 5-HT1A activity: Part 2q
,
a
a
a
a
Franciszek Herold a , qukasz Izbicki , Andrzej Chodkowski , Maciej Dawidowski , Marek Krol ,
*
´
Jerzy Kleps a, Jadwiga Tur1o a, Irena Wolska b, Gabriel Nowak c d, Katarzyna Stachowicz c,
,
d
d
e
f
g
_
_
´
Ma1gorzata Dyba1a , Agata Siwek , Mateusz Nowak , Elzbieta Pienia˛zek , Ma1gorzata Jaronczyk ,
Ingebrigt Sylte h, Aleksander P. Mazurek g
a Department of Drug Technology, Faculty of Pharmacy, Medical University of Warsaw, ul. Banacha 1, 02-097 Warszawa, Poland
b Department of Crystallography, Faculty of Chemistry, Adam Mickiewicz University, Grunwaldzka 6, 60-780 Poznan´, Poland
c Department of Neurobiology, Institute of Pharmacology, Polish Academy of Sciences, ul. Sme˛tna 12, 31-343 Krako´w, Poland
d Department of Cytobiology and Histochemistry, Collegium Medicum of Jagiellonian University, ul. Medyczna 9, 30-688 Krako´w, Poland
e
´
Department of Medicinal Chemistry, Institute of Pharmacology, Polish Academy of Sciences, ul. Sme˛tna 12, 31-343 Krakow, Poland
f
´
Department of Behavioral Neuroscience & Drug Development, Institute of Pharmacology, Polish Academy of Sciences, ul. Sme˛tna 12, 31-343 Krakow, Poland
g National Institute of Medicines, ul. Chełmska 30/34, 00-725 Warsaw, Poland
h Department of Pharmacology, Institute of Medical Biology, University of Tromsø, N-9037 Tromsø, Norway
a r t i c l e i n f o
a b s t r a c t
Article history:
Derivatives of 4-aryl-5,6,7,8-tetrahydro-pyrido[1,2-c]pyrimidine were synthesized. These compounds
contain the 3-(4-piperidyl)-1H-indole residue or its 5-methoxy or 2-methyl derivative. In vitro binding
tests were performed to determine the affinity of the compounds for the 5-HT1A receptor and serotonin
transporter (SERT) proteins in the rat brain cortex. In vivo studies, particularly the inducible hypothermia
test and forced swimming test, were conducted to determine agonistic/antagonistic activity with pre-
and postsynaptic 5-HT1A receptors. Molecular modeling techniques were used to determine the binding
modes of the selected compounds at the 5-HT1A receptor and SERT. The SAR analysis showed that the
presence of the 3-(4-piperidyl)-1H-indole group or its 5-methoxy derivative, as well as a para substi-
tution with –OCH3 or –F in the aryl ring of 4-aryl-5,6,7,8-tetrahydro-pyrido[1,2-c]pyrimidine, results in
an increased affinity for both the 5-HT1A receptors and SERT. In contrast, the presence of the 2-methyl-3-
(4-piperidyl)-1H-indole group resulted in a considerable decrease in binding affinity.
Received 27 January 2009
Accepted 9 July 2009
Available online 16 July 2009
Keywords:
Antidepressants
Pyrido[1,2-c]pyrimidines
Dual 5-HT1A/SERT activity
Ó 2009 Elsevier Masson SAS. All rights reserved.
1. Introduction
to the idea to develop newer, more efficacious, and safer antide-
pressants [1–3].
The introduction of selective serotonin reuptake inhibitors
(SSRIs) as second generation antidepressants was a breakthrough
in the treatment of depression. The SSRIs are characterized by
significantly reduced disadvantageous properties; firstly, these
drugs produce notably fewer adverse drug reactions (ADRs)
compared with older drugs such as tricyclic antidepressants (TCAs)
or nonselective monoaminooxidase inhibitors (MAOIs). However,
SSRIs also have several drawbacks, including lower clinical effec-
tiveness, some ADRs, and a relatively long onset of action, which led
The long latency of SSRIs is caused by their self-limiting action
on extracellular serotonin in the forebrain. The administration of an
SSRI leads to an increased serotonin concentration in the synaptic
clefts of the raphe nucleus, which in turn activates somatodendritic
serotoninergic autoreceptors (5-HT1A) [4,5]. The activation of
5-HT1A causes, due to negative feedback, the inhibition of serotonin
release from presynaptic terminals [6–8]. A renewed, permanent
increase in 5-HT concentrations in various brain synapses is ach-
ieved after a 2–4 week period, when the desensitization of 5-HT1A
autoreceptors occurs [9,10].
The latency can be shortened by the co-administration of an
SSRI with a 5-HT1A antagonist [11,12]. A pre-clinical in vivo
microdialysis study confirmed that the simultaneous administra-
tion of the 5-HT1A antagonist WAY 100635 with the SSRI fluoxetine
leads to an immediate increase in the serotonin concentration of
the rat brain cortex, whereas this effect was not observed after the
administration of fluoxetine alone [13]. Also, in clinical studies, the
Abbreviations: SERT, serotonin transporter; SAR, structure–activity relationship;
5-HT, serotonin; 8-OH-DPAT, 8-hydroxy-2-(di-n-propylamino)tetraline; Ki, Inhibitor
constant; TMS, tetramethylsilane.
Part of this work was presented at the XXth International Symposium on
Medicinal Chemistry, Vienna, Austria, August 31–September 4, 2008.
* Corresponding author.
q
E-mail address: franciszek.herold@wum.edu.pl (F. Herold).
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.07.007

F. Herold et al. / European Journal of Medicinal Chemistry 44 (2009) 4702–4715
4703
co-administration of a 5-HT1A antagonist (pindolol, WAY 100135, or
robalzotan) with an SSRI considerably shortened the latency period
to 3–7 days [14–16]. Hence, the idea of incorporating two parts into
one derivative molecule with a double mechanism of action may
lead to the development of a new generation of antidepressant
drugs [17–20].
Compounds 8c, 8d, 8e, 8f, 8h, and 8i were also investigated in vivo
using a hypothermic test in mice, whereas compounds 8c, 8f, 8h,
and 8i were investigated using a forced swimming test in mice
(behavioral study). Compounds 8d, 8e, 8h, 8l, 8n, 8q, and 8u were
docked to the 5-HT1A receptor model, whereas ligands 8d, 8e, 8h,
and 8l were also docked to the SERT model.
In our previous study, we described a series of new pyrido[1,2-
c]pyrimidine derivatives with a double mechanism of action: the
inhibition of 5-HT reuptake and agonistic activity towards both pre-
and postsynaptic 5-HT1A receptors [21]. These compounds can be
considered a good entry point for novel drug candidates. Presyn-
aptic agonistic effects can lead to faster desensitization of the
autoreceptors, which in turn could shorten the latency period. It
has also been demonstrated that postsynaptic agonistic activity
increases neurotransmission in the serotoninergic system [22].
The aim of our present study was to synthesize and assess the
biological properties of new tetrahydro-pyrido[1,2-c]pyrimidine
derivatives with dual activity. New ligands were obtained after the
modification of previously synthesized pyrido[1,2-c]pyrimidine
derivatives belonging to the long chain arylpiperazine (LCAP) group
with a high affinity for 5-HT1A receptors [23]. Modifications were
performed in the pharmacophore portion where the 3-(4-piper-
idyl)-1H-indole group or its 5-methoxy or 2-methyl derivative was
inserted. These groups reveal the molecule’s potential to inhibit the
serotonin transporter (SERT) protein, as well as bind 5-HT1A
receptors [24–26]. The pharmacophore portion of the molecule was
bound to the terminal tetrahydro-pyrido[1,2-c]pyrimidine moiety
by an n-butyl chain. Biological investigations of the newly synthe-
sized compounds (8c–8u) included determination of the binding
affinity for the 5-HT1A receptor and serotonin transporter SERT.
2. Chemistry
The respective nitriles (2a–2i) were synthesized by C-arylation
of the appropriate arylacetonitriles with 2-bromopyridine in an
aprotic polar solvent (Scheme 1) [27,28]. Next, the nitriles (2a–2i)
were hydrolyzed using a mixture of sulfuric acid and acetic acid to
produce amides (3a–3i) in good yield. Compounds 4a–4i were
formed by the intermolecular cyclization of 3a–3i with diethyl
carbonate. The nitrogen in the imide group of compounds 4a–4i
was alkylated with 1,4-dibromobutane to form bromobutyl deriv-
atives 5a–5i, which were subsequently hydrogenated to form
5,6,7,8-tetrahydro derivatives (6a–6i). The final compounds (8a–
8u) were obtained by the condensation of bromobutyl derivatives
6a–6i with the appropriate piperidyl-indole (7a–7c) synthesized
according to the method previously described [25].
3. Structural investigations
The new compounds (8a–8u) were characterized by physical
constants, elemental analysis, IR, and 1H, and 13C NMR spectroscopy
(see section 8.2). The NMR spectra mutually correlated and were in
agreement with the literature for similar systems [23,27] as well as
Scheme 1. aReagents: (i) 2-bromopyridine, KOH, DMSO,
60 atm., 50 ꢀC; (vi) 7a–7c, acetonitrile, K2CO3, KI,
. Numbering system of 8 for NMR analysis.
D; (ii) H2SO4, CH3COOH, D; (iii) diethyl carbonate, EtONa, EtOH, D; (iv) 1,4-dibromobutane, K2CO3, acetone, D; (v) H2,10% Pt/C,
D

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F. Herold et al. / European Journal of Medicinal Chemistry 44 (2009) 4702–4715
Fig. 1. A view of the 4-(4-chloro-phenyl)-2-{4-[4-(1H-indol-3-yl)-piperidin-1-yl]-butyl}-pyrido[1,2-c]pyrimidine-1,3-dione (9a) [21] molecule and its numbering scheme.
the theoretical spectra calculated according to the ACD/NMR
Predictor v. 8.09 program.
butyl chain at N16 occupies the equatorial position and adopts the
trans/gauche/trans conformation (see the torsion angles in Table 2).
The pyrido[1,2-c]pyrimidine fragment is essentially planar with
no atomic deviation greater than 0.044(1) Å (for C1 atom) from its
least-squares plane. The planar substituent at C4 creates an angle of
68.77(4)ꢀ with the best plane of the pyridopyrimidine system, and
the orientation of this substituent with respect to the mentioned
moiety can be described by the torsion angle C3–C4–C10–C20, which
is 68.1(2)ꢀ.
The XRD experiments carried out for compound 4-(4-chloro-
phenyl)-2-{4-[4-(1H-indol-3-yl)-piperidin-1-yl]-butyl}-pyrido[1,2-
c]pyrimidine-1,3-dione (9a) were previously described in part 1 of
this work [21]. The ORTEP view of the molecule, with drawings
created by the Mercury program [29], is shown in Fig. 1. Selected
bond lengths, bond angles, and torsion angles are listed in Table 2.
The piperidine ring adopts a chair conformation with N16 and
C19, 0.672(2) and 0.666(2) Å above and below the plane formed by
the piperidine ring carbons, respectively. The planarity of the indole
substituent at C19 with respect to the piperidine ring is indicated by
the value of the torsion angle C18–C19–C22–C23, 109.2(2)ꢀ. The
The crystal packing of the molecules is determined by different
N–H/O, C–H/Cl, and C–H/p intermolecular contacts (Fig. 2). The
geometric parameters of all these bonds are listed in Table 3.
4. Pharmacology
The in vitro affinity of target compounds 8c–8u for 5-HT1A
receptors and SERT in rat brain was assessed using radioligand
binding assays ([3H]8-OH-DPAT and [3H]citalopram, respectively).
Data were analyzed using iterative curve-fitting routines to obtain
IC50 values (GraphPAD/Prism, Version 3.0 – San Diego, CA, USA),
Table 1
Crystal data, data collection, and structure refinement for 4-(4-chloro-phenyl)-2-{4-[4-
(1H-indol-3-yl)-piperidin-1-yl]-butyl}-pyrido[1,2-c]pyrimidine-1,3-dione (9a) [21].
Compound
4-(4-Chloro-phenyl)-2-{4-[4-(1H-indol-3-yl)-
piperidin-1-yl]-butyl}-pyrido[1,2-c]
pyrimidine-1,3-dione
Empirical formula
Formula weight
T (K)
Wavelength (Å)
Crystal system, space group
C31H31N4O2Cl
527.05
293(2)
0.71073
Triclinic, P-1
Table 2
Selected bond lengths and angles and selected torsional angles for 4-(4-chloro-
phenyl)-2-{4-[4-(1H-indol-3-yl)-piperidin-1-yl]-butyl}-pyrido[1,2-c]pyrimidine-1,3-
dione (9a) [21].
Bond
C40–Cl
C4–C4A
C4–C10
C3–O11
C1–O10
C12–N2
C15–N16
C23–N24
C25–N24
Length (Å)
Unit cell dimensions
1.741(1)
1.374(2)
1.493(2)
1.231(2)
1.212(2)
1.484(2)
1.468(2)
1.360(2)
1.370(2)
a (Å)
b (Å)
c (Å)
10.4857(5)
10.9071(6)
12.4227(6)
77.821(5)
79.883(4)
76.435(5)
1338.1(1)
2, 1.308
a
b
g
(ꢀ)
(ꢀ)
(ꢀ)
Volume (Å3)
Z, Dx (Mg/m3)
m
(mmꢁ1
)
0.179
F(000)
range for data collection (ꢀ)
hkl Range
556
Bonds
Angle (deg)
Q
3.08–30.04
ꢁ13 < h < 14
ꢁ13 < k < 14
ꢁ16 < l < 17
C1–N2–C3
C3–C4–C10
C15–N16–C21
C15–N16–C17
C23–N24–C25
125.4(1)
118.0(1)
111.7(1)
109.8(1)
108.9(1)
Reflections
Collected
18056
6707 (0.024)
4480
6707/0/343
1.049
0.0461
Bonds
Torsional
angles (deg)
Unique (Rint
)
Observed (I > 2((I))
Data/restraints/parameters
Goodness-of-fit on F2
C20–C10–C4–C4A
ꢁ111.8(2)
ꢁ175.7(1)
71.5(2)
174.6(1)
109.2(2)
N2–C12–C13–C14
C12–C13–C14–C15
C13–C14–C15–N16
C18–C19–C22–C23
R(F) (I > 2
s
(I))
wR(F2) (all data)
0.1287
0.307/ꢁ0.259
Max/min. (e/Å3)

F. Herold et al. / European Journal of Medicinal Chemistry 44 (2009) 4702–4715
4705
Fig. 2. The packing arrangement of 4-(4-chloro-phenyl)-2-{4-[4-(1H-indol-3-yl)-piperidin-1-yl]-butyl}-pyrido[1,2-c]pyrimidine-1,3-dione (9a) [21] with intermolecular interactions.
which in turn were used to calculate inhibition constant Ki
according to the Cheng–Prusoff formula (Table 4) [30].
Four ligands (8d, 8e, 8h, and 8l; Table 4) were also docked to the
SERT model using a docking procedure as implemented in the ICM
software [33]. The SERT model was based on the X-ray crystal
The final compounds were further evaluated in mice for their
agonist/antagonist properties towards pre- and postsynaptic
5-HT1A receptors using the inducible hypothermia and forced
swimming tests, respectively. In the inducible hypothermia test,
the effects of the administration of six compounds (8c, 8d, 8e, 8f,
8h, and 8i) on body temperature, as well as the effects of WAY
100635 (5-HT1A receptor antagonist) on the hypothermia induced
by the tested compounds were investigated. The results were
structure of the leucine transporter from Aquifex aeolicus (LeuTAa
)
[34,35]. This SERT model corresponds to the substrate-bound
conformation of the transporter model, closed on both sides of the
membrane. All compounds studied have a nitrogen atom attached
to the protonated butyl fragment. The functionally important
residue for ligand interaction with SERT, Asp981.45 in TMH1 [36],
was used as an anchoring point for docking fully flexible ligands to
the serotonin transporter.
expressed as changes in body temperature (Dt) compared with
basal body temperature as measured at the beginning of the
experiment. The forced swimming test was carried out according to
the method of Porsolt et al. for 8c, 8f, 8h, and 8i [31]. The effect of
these compounds on the spontaneous locomotor activity in mice
was also recorded. The obtained data were presented as the
mean ꢂ S.E.M. The significance of differences between groups was
evaluated by a one-way analysis of variance (ANOVA) followed by
intergroup comparisons using Dunnett’s or Newman–Keuls test;
p ꢃ 0.01 was accepted as significant.
6. Results and discussion
Several new derivatives of 4-aryl-tetrahydro-pyrido[1,2-
c]pyrimidine containing 3-(4-piperidyl)-1H-indole or its
5-methoxy or 2-methyl derivative in the pharmacophore portion of
the molecule were obtained (Fig. 1). In the 4-aryl-tetrahydro-pyr-
ido[1,2-c]pyrimidine system, an aryl substitute in compounds 8b
and 8o consisted of a non-substituted phenyl ring, whereas other
derivatives possessed additional –F (8f, 8i, 8n), –Cl (8m, 8t), –OCH3
(8p), or –CH3 (8j, 8k) groups at the ortho position of the phenyl ring,
as well as –F (8l, 8u), –Cl (8a, 8g, 8s), –OCH3 (8d, 8e, 8r), or –CH3 (8c,
8h, 8q) groups at the para position.
5. Molecular modeling
Ligand binding modes were studied by the fully flexible
molecular docking of seven ligands (8d, 8e, 8h, 8l, 8n, 8r, and 8v) to
the model of the transmembrane portion of the 5-HT1A receptor.
Additionally, three compounds described in Part 1 of this article
(8a, 10a, and 11a) were also docked [21]. One hundred 5-HT1A
receptor models were used that were previously ‘‘tuned’’ for aryl-
piperazine/arylpiperidine ligands [32]. Two pharmacophore
constraints were used to force the interactions crucial for the
recognition of arylpiperidine serotoninergic ligands: the ionic
interaction between Asp3.32 and the basic moiety of the ligand,
and the aromatic contact between the ligand and Phe6.52.
Table 4
Inhibition constant (Ki) values of the investigated compounds for 5-HT1A receptors
and SERT.
Derivative
R1
R2
R3
R4
Ki 5-HT1A (nM)
Ki SERT (nM)
8c
8d
8e
8f
8g
8h
8i
8j
8k
8l
8m
8n
8o
8p
8q
8r
8s
8t
H
H
H
F
H
H
F
CH3
CH3
H
Cl
F
H
OCH3
H
H
H
Cl
H
CH3
OCH3
OCH3
H
Cl
CH3
H
H
H
F
H
H
H
H
CH3
OCH3
Cl
H
F
OCH3
H
OCH3
OCH3
OCH3
H
H
H
OCH3
OCH3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
23.0 ꢂ 1.4
20.5 ꢂ 2.8
10.4 ꢂ 2.6
45.6 ꢂ 3.9
14.6 ꢂ 0.8
48.3 ꢂ 4.3
27.8 ꢂ 3.6
53.8 ꢂ 1.9
51.7 ꢂ 4.1
13.9 ꢂ 0.5
36.2 ꢂ 1.9
135.5 ꢂ 4.5
71.6 ꢂ 0.9
83.5 ꢂ 7.1
184.4 ꢂ 5.0
222.0 ꢂ 8.0
76.7 ꢂ 1.3
101.9 ꢂ 9.3
123.6 ꢂ 8.8
123.7 ꢂ 5.2
76.1 ꢂ 6.1
101.3 ꢂ 5.8
944 ꢂ 22
H
H
H
Table 3
Hydrogen-bonding geometry for 4-(4-chloro-phenyl)-2-{4-[4-(1H-indol-3-yl)-
piperidin-1-yl]-butyl}-pyrido[1,2-c]pyrimidine-1,3-dione (9a) [21].
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
2.2 ꢂ 0.2 (
1.4 ꢂ 0.1 (
1.6 ꢂ 0.1 (
1.5 ꢂ 0.1 (
835.6 ꢂ 64.4
1.5 ꢂ 0.3 (
2.1 ꢂ 0.1 (
3.2 ꢂ 0.2 (
mM)
mM)
mM)
mM)
273.6 ꢂ 4.5
719 ꢂ 48
D–H/A
D–H (Å)
H/A (Å)
D/A (Å)
<(D–H/A) (deg)
N24–H24A/O11i
C7–HA/Clii
0.86
0.93
0.93
2.16
2.95
2.64
2.845(2)
3.798(2)
3.535(2)
136
152
161
115.1 ꢂ 6.9
203 ꢂ 4
C60–H60A/Cgiii
mM)
mM)
mM)
460 ꢂ 25
480 ꢂ 43
Cg represents the centroid of the five-membered ring of the indole moiety.
Symmetry codes: (i) 1 ꢁ x, 1 ꢁ y, ꢁz; (ii) 1 ꢁ x, ꢁy, 2 ꢁ z; (iii) x, ꢁ1 þ y, 1 þ z.
8u
116.1 ꢂ 9.9

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F. Herold et al. / European Journal of Medicinal Chemistry 44 (2009) 4702–4715
Table 5
6.1. SAR analysis
The effect of the tested compounds on the body temperature of mice.
The binding values obtained for compounds 8c–8u for the
5-HT1A receptor and SERT provided the possibility of analyzing the
influence of substituents bound to the 3-(4-piperidyl)-1H-indole
moiety (–H or –OCH3 at 5 position, –CH3 or –H at 2 position), the
influence of various substitutes at the ortho/para position of the aryl
ring, and the impact of the degree of saturation of the tetrahydro-
pyrido[1,2-c]pyrimidine system (compare to Ref. [21]). The binding
tests for 8c–8u revealed a very high affinity of derivatives 8e
(Ki ¼ 10.4 nM), 8g (Ki ¼ 14.6 nM), and 8l (Ki ¼ 13.9 nM) for 5-HT1A
receptors. The remaining compounds possessed high to weak
Treatment
Dose (mg/kg)
D
t ꢂ SEM (ꢀC)
30 min
60 min
90 min
120 min
Vehicle
8c
–
5
10
20
0.0 ꢂ 0.0 ꢁ0.0 ꢂ 0.1 ꢁ0.1 ꢂ 0.0
0.0 ꢂ 0.1
ꢁ0.3 ꢂ 0.1 ꢁ0.2 ꢂ 0.1 ꢁ0.1 ꢂ 0.1 ꢁ0.1 ꢂ 0.0
ꢁ0.6 ꢂ 0.1b ꢁ0.4 ꢂ 0.1 ꢁ0.2 ꢂ 0.1 ꢁ0.1 ꢂ 0.1
ꢁ1.3 ꢂ 0.1c ꢁ1.3 ꢂ 0.2c ꢁ0.8 ꢂ 0.1c ꢁ0.7 ꢂ 0.1b
Vehicle
8d
–
5
10
20
ꢁ0.1 ꢂ 0.0 ꢁ0.0 ꢂ 0.1 ꢁ0.1 ꢂ 0.1 ꢁ0.1 ꢂ 0.1
ꢁ0.5 ꢂ 0.1a ꢁ0.2 ꢂ 0.1 ꢁ0.2 ꢂ 0.1 ꢁ0.1 ꢂ 0.1
ꢁ0.7 ꢂ 0.1b ꢁ0.5 ꢂ 0.1a ꢁ0.4 ꢂ 0.1 ꢁ0.3 ꢂ 0.1
ꢁ0.6 ꢂ 0.1b ꢁ0.7 ꢂ 0.1b ꢁ0.4 ꢂ 0.1 ꢁ0.3 ꢂ 0.1
Vehicle
8e
–
0.0 ꢂ 0.0 ꢁ0.0 ꢂ 0.0 ꢁ0.1 ꢂ 0.1
0.0 ꢂ 0.0
binding activity with Ki ranging from 20.5 nM to 3.2 mM
(compounds 8d, 8c, 8i, 8m, 8f, 8h, 8k, 8j, 8r, 8o, 8q, 8p, 8s, 8t, 8n,
and 8u according to increasing Ki values; see Table 4).
1.25
2.5
5
ꢁ0.3 ꢂ 0.1 ꢁ0.4 ꢂ 0.1 ꢁ0.4 ꢂ 0.1 ꢁ0.2 ꢂ 0.1
ꢁ0.8 ꢂ 0.1c ꢁ0.6 ꢂ 0.1b ꢁ0.6 ꢂ 0.1b ꢁ0.5 ꢂ 0.0a
ꢁ1.1 ꢂ 0.2c ꢁ0.5 ꢂ 0.1a ꢁ0.3 ꢂ 0.1 ꢁ0.1 ꢂ 0.0
Analyzing the effect of substituents at the indole moiety on
binding activity found that compounds bearing a non-substituted
3-(4-piperidyl)-1H-indole group or analogous 3-(4-piperidyl)-
5-methoxy-1H-indole derivatives possessed very high or high
affinity for 5-HT1A receptors. The presence of the 2-methyl-3-(4-
piperidyl)-1H-indole group resulted in a considerable loss in
affinity. An analysis of the effect of the substitution of the aryl ring
in tetrahydro-pyrido[1,2-c]pyrimidine found that, on the condition
that there is no –CH3 substituent at the 2 position of the 3-(4-
piperidyl)-1H-indole group, substitution with –OCH3 (8d, 8e), –F
(8l), or –Cl (8g) at the para position results in a higher affinity for
5–HT1A receptors than ligands with –F (8f, 8i), –CH3 (8j, 8k), or –Cl
(8m) at the ortho position and compounds with –CH3 (8c, 8h) at the
para position. In addition, we found that the degree of saturation of
the tetrahydro-pyrido[1,2-c]pyrimidine system has no major
influence on 5-HT1A receptor affinity compared to unsaturated
pyrido[1,2-c]pyrimidine derivatives previously described in Part 1
of this study [21]. The SERT binding studies demonstrated
a moderate to poor affinity for the newly synthesized compounds
with Ki values ranging from 71.6 to 943.7 nM (derivatives 8d, 8l, 8h,
8e, 8m, 8i, 8q, 8u, 8j, 8k, 8c, 8f, 8r, 8g, 8o, 8s, 8t, 8p, and 8n
according to increasing Ki values).
When the influence of the indole moiety substitution on SERT
binding was evaluated, we observed that the non-substituted 3-(4-
piperidyl)-1H-indole derivatives and 3-(4-piperidyl)-5-methoxy-
1H-indole analogues possessed relatively similar and substantial
affinities (Ki ¼ 71.6–222.0 nM), whereas the 2-methylindole deriva-
tives were mostly characterized by high Ki values. Analyzing the
influence of the aryl ring substitution on tetrahydro-pyrido[1,2-
c]pyrimidinerevealedthat substitutionwith –OCH3 (8d, 8e, 8r), –CH3
(8c, 8h, 8q), or –F (8l, 8u) at the para position results in relatively high
affinity. The remaining derivatives exhibited a lower affinity. Satu-
ration of the tetrahydro-pyrido[1,2-c]pyrimidine system decreased
SERT inhibition compared to the unsaturated pyrido[1,2-c]pyrimi-
dine derivatives previously described in Part 1 [21].
Vehicle
8f
–
5
10
20
ꢁ0.0 ꢂ 0.0 ꢁ0.0 ꢂ 0.1 ꢁ0.1 ꢂ 0.0 ꢁ0.0 ꢂ 0.1
ꢁ0.7 ꢂ 0.1b ꢁ0.6 ꢂ 0.1b ꢁ0.4 ꢂ 0.1 ꢁ0.3 ꢂ 0.1
ꢁ0.7 ꢂ 0.1b ꢁ0.8 ꢂ 0.1c ꢁ0.6 ꢂ 0.2b ꢁ0.4 ꢂ 0.1
ꢁ1.2 ꢂ 0.1c ꢁ1.0 ꢂ 0.1c ꢁ0.8 ꢂ 0.2c ꢁ0.7 ꢂ 0.2b
Vehicle
8h
–
5
10
ꢁ0.1 ꢂ 0.1 ꢁ0.1 ꢂ 0.0 ꢁ0.1 ꢂ 0.1 ꢁ0.1 ꢂ 0.1
ꢁ0.6 ꢂ 0.1b ꢁ0.6 ꢂ 0.1b ꢁ0.3 ꢂ 0.1 ꢁ0.2 ꢂ 0.1
ꢁ1.0 ꢂ 0.1c ꢁ0.5 ꢂ 0.1a ꢁ0.3 ꢂ 0.1 ꢁ0.2 ꢂ 0.1
Vehicle
8i
–
ꢁ0.0 ꢂ 0.0 ꢁ0.1 ꢂ 0.1 ꢁ0.1 ꢂ 0.1 ꢁ0.0 ꢂ 0.1
ꢁ0.3 ꢂ 0.1 ꢁ0.2 ꢂ 0.1 ꢁ0.2 ꢂ 0.1 ꢁ0.1 ꢂ 0.1
ꢁ1.2 ꢂ 0.2c ꢁ1.0 ꢂ 0.1c ꢁ0.8 ꢂ 0.1c ꢁ0.5 ꢂ 0.1a
5
10
Vehicle
8-OH-DPAT
WAY 100635 0.1
–
5
0.0 ꢂ 0.1
0.1 ꢂ 0.1
0.1 ꢂ 0.1
0.0 ꢂ 0.1
ꢁ1.5 ꢂ 0.1c ꢁ1.1 ꢂ 0.1c ꢁ0.7 ꢂ 0.1b ꢁ0.2 ꢂ 0.1
0.1 ꢂ 0.1
0.2 ꢂ 0.1
0.1 ꢂ 0.1
0.2 ꢂ 0.2
The tested compounds were administered by ip injection 30 min before the test. 8-
OH-DPAT and WAY 100635 were injected subcutaneously (sc). The absolute mean
body temperatures were 36 ꢂ 0.5 ꢀC; n ¼ 7–8 mice per group.
a
p < 0.05.
p < 0.01.
p < 0.001 vs. respective vehicle.
b
c
Table 6
The effect of WAY 100635 on the hypothermia induced in mice by the tested
compounds.
Compound and dose (mg/kg)
D
t ꢂ SEM (ꢀC)
30 min
60 min
Vehicle þ vehicle
ꢁ0.1
ꢁ1.3
ꢁ0.4
ꢂ0.1
ꢁ0.0
ꢁ0.8
ꢁ0.4
ꢂ0.0
ꢂ0.1a
ꢂ0.1
Vehicle þ 8c (20)
ꢂ0.2b
WAY 100635 (0.1) þ 8c (20)
ꢂ0.1d
Vehicle þ vehicle
ꢁ0.1
ꢁ1.1
ꢁ1.2
ꢂ0.1
ꢁ0.1
ꢁ0.6
ꢁ0.9
ꢂ0.1
Vehicle þ 8e (5)
ꢂ0.1b
ꢂ0.1a
WAY 100635 (0.1) þ 8e (5)
ꢂ0.2b
ꢂ0.2b
Vehicle þ vehicle
ꢁ0.0
ꢁ1.3
ꢁ0.5
ꢂ0.1
ꢁ0.1
ꢁ1.0
ꢁ0.4
ꢂ0.1
Vehicle þ 8f (20)
ꢂ0.2b
ꢂ0.1d
ꢂ0.2b
ꢂ0.1d
WAY 100635 (0.1) þ 8f (20)
Vehicle þ vehicle
ꢁ0.1
ꢁ1.0
ꢁ0.6
ꢂ0.0
ꢁ0.1
ꢁ0.5
ꢁ0.6
ꢂ0.1
ꢂ0.2
ꢂ0.1b
Vehicle þ 8h (10)
ꢂ0.1b
WAY 100635 (0.1) þ 8h (10)
ꢂ0.1b,c
6.2. Behavioral studies
Vehicle þ vehicle
ꢁ0.0
ꢁ1.2
ꢁ0.6
ꢂ0.1
ꢁ0.1
ꢁ1.0
ꢁ0.4
ꢂ0.1
ꢂ0.2b
ꢂ0.2b
Compounds with the most promising affinity for both 5–HT1A
receptor and SERT were selected for in vivo experiments (8c, 8d, 8e,
8f, 8h, and 8i) in models commonly used for evaluating 5-HT1A
receptor function. The 5-HT1A receptor agonist 8-OH-DPAT induces
hypothermia in mice, an effect mediated through the 5-HT1A
somatodendritic receptor [37,38]. This hypothermia is abolished by
the 5-HT1A receptor antagonist WAY 100635 [39]. Compounds 8c,
8e, 8f, 8h, and 8i induced hypothermia in mice similar to 8-OH-
DPAT, whereas 8d, analogous to WAY 100635, did not alter the body
temperature in mice (Table 5). The hypothermia induced by
compounds 8c (20 mg/kg), 8f (20 mg/kg), 8h (10 mg/kg), and 8i
(10 mg/kg) was attenuated by WAY 100635 (0.1 mg/kg) (Table 6).
Vehicle þ 8i (10)
WAY 100635 (0.1) þ 8i (10)
ꢂ0.1a,d
ꢂ0.1d
Vehicle þ vehicle
0.0
ꢁ1.5
ꢁ0.1
ꢂ0.1
ꢁ0.1
ꢁ1.1
ꢁ0.2
ꢂ0.1
Vehicle þ 8-OH-DPAT (5)
WAY 100635 (0.1) þ 8-OH-DPAT(5)
ꢂ0.1b
ꢂ0.1b
ꢂ0.1d
ꢂ0.1d
WAY 100635 was administered subcutaneously 15 min before the investigated
compounds, n ¼ 7–8 mice per group. The test was performed 30 min after the
injection of the tested compounds (ip). The absolute mean body temperatures were
36.0 ꢂ 0.4 ꢀC.
a
p < 0.01.
b
p < 0.001 vs. respective vehicle þ vehicle group.
c
p < 0.01.
d
p < 0.001 vs. respective vehicle þ compound group.

F. Herold et al. / European Journal of Medicinal Chemistry 44 (2009) 4702–4715
4707
Table 7
(20 mg/kg) decreased the hypothermia induced by 8-OH-DPAT
(5 mg/kg) in mice (Table 7). Therefore, the decrease in body
temperature produced in mice by 8-OH-DPAT can be regarded as
a measure of its presynaptic 5-HT1A receptor antagonist activity.
WAY 100635 did not change the hypothermia induced by 8e (5 mg/
kg) in mice; therefore, it seems that the action of 8e on those
receptors is of no importance to its hypothermic effect.
Subsequently, to establish the agonist/antagonistic actions on
the postsynaptic 5-HT1A receptor, compounds 8c, 8f, 8h, and 8i
were investigated using the forced swimming test in mice, and
their effect on mouse locomotor activity was studied. Compound 8f
reduced the immobility time of the mice by 26% [F(2,27) ¼ 6.864,
p < 0.01] at a dose of 20 mg/kg but not 10 mg/kg. At the same time,
compounds 8h and 8i reduced the immobility time by 34% and 27%,
respectively [8h: F(2,27) ¼ 21.95, p < 0.001; 8i: F(2,27) ¼ 17.04,
p < 0.001] at a dose of 10 mg/kg but not 5 mg/kg. Compound 8c was
ineffective at doses of 10 mg/kg and 20 mg/kg. We found that
8-OH-DPAT reduced the immobility time of the mice by 45% and
57% (F(2,26) ¼ 53.92, p < 0.0001) at doses of 1 mg/kg and 2 mg/kg,
respectively (Fig. 3). All of the compounds tested (8c, 8f, 8h, and 8i)
reduced the spontaneous locomotor activity in mice during a 6-min
(i.e. for the time equivalent to the observation period in the forced
swimming test) or a 30-min observation period (Table 8).
The effect of compound 8d and WAY 100635 on 5 mg/kg 8-OH-DPAT-induced
hypothermia in mice.
Treatment and dose (mg/kg)
D
t ꢂ SEM (ꢀC)
15 min
30 min
45 min
60 min
Vehicle þ vehicle
ꢁ0.0 ꢂ 0.0
ꢁ0.1 ꢂ 0.1 ꢁ0.1 ꢂ 0.1 ꢁ0.1 ꢂ 0.1
Vehicle þ 8-OH-DPAT
8d (20) þ 8-OH-DPAT
ꢁ1.2 ꢂ 0.2b ꢁ1.6 ꢂ 0.1b ꢁ0.8 ꢂ 0.1a ꢁ0.2 ꢂ 0.1
0.6 ꢂ 0.1a,d ꢁ0.5 ꢂ 0.1a,c ꢁ0.3 ꢂ 0.1 ꢁ0.2 ꢂ 0.1
Vehicle þ vehicle
ꢁ0.1 ꢂ 0.0
ꢁ0.1 ꢂ 0.0 ꢁ0.1 ꢂ 0.0
0.0 ꢂ 0.1
Vehicle þ 8-OH-DPAT (5)
ꢁ1.2 ꢂ 0.1b ꢁ1.5 ꢂ 0.1b ꢁ0.7 ꢂ 0.1a ꢁ0.3 ꢂ 0.1
WAY100635(0.1) þ 8-OH-DPAT ꢁ0.1 ꢂ 0.1d ꢁ0.1 ꢂ 0.1d ꢁ0.1 ꢂ 0.1d ꢁ0.2 ꢂ 0.1d
Compound 8d was administered (ip) 45 min prior to 8-OH-DPAT, WAY 100635 (sc)
15 min prior to 8-OH-DPAT, n ¼ 8 mice per group. The absolute mean initial body
temperatures were 36.0 ꢂ 0.5 ꢀC.
a
p < 0.01.
b
p < 0.001 vs. respective vehicle þ vehicle group.
c
p < 0.01.
d
p < 0.001 vs. respective vehicle þ 8-OH-DPAT.
At the same time, the decrease in body temperature induced by
8-OH-DPAT (5 mg/kg) was completely blocked by WAY 100635
(0.1 mg/kg) (Table 6). Therefore, the decrease in body temperature
produced in mice by 8c, 8f, 8h, and 8i can be regarded as a measure
of its presynaptic 5-HT1A receptor agonist activity. Compound 8d
Fig. 3. Effects of compounds 8c (A), 8f (B), 8h (C), 8i (D), and 8-OH-DPAT (E) on Albino Swiss mice in the forced swimming test. Each bar represents the mean ꢂ SEM of 9–10 mice.
All compounds were injected 30 min before the test. *p < 0.01, **p < 0.001 vs. respective vehicle group (Dunnett’s test). X-Axis: dose [mg/kg]; Y-axis: immobility time [s].

4708
F. Herold et al. / European Journal of Medicinal Chemistry 44 (2009) 4702–4715
Table 8
pointing towards the interior of the binding pocket. This can be
seen in the top-scored pose of 8l (Fig. 4G) and in selected poses for
10a (Fig. 5). In such a conformation, the addition of the methyl
substituent will cause a steric hindrance with the surface of the
binding pocket.
Effect of compounds 8f, 8h, and 8i on locomotor activity in mice.
Treatment (mg/kg)
Locomotor activity
Number of crossings ꢂ S.E.M.
6 min
30 min
Another explanation for the influence of the methyl substitution
at the indole moiety could be a possible interaction with extracel-
lular loop 2 (EL2). Extracellular loops were not modeled due to the
unsatisfactory quality of loop modeling in Modeler [40]. The
superimposition of our receptor models on recent crystal structures
for the adrenergic receptors (data not shown) suggests that the area
where the methyl substituent is situated is not occupied by any
atoms of the EL2, suggesting that the steric hindrance of this loop is
not the reason for the loss of 5-HT1A affinity for ligands with
a methylated indole moiety.
Vehicle
8f (20)
8h (10)
8i (10)
414 ꢂ 34
1010 ꢂ 60
272 ꢂ 42a
520 ꢂ 80a
114 ꢂ 21a
221 ꢂ 42a
217 ꢂ 22a
333 ꢂ 60a
F(3,36) ¼ 16.57, p < 0.0001
F(3,36) ¼ 31.35, p < 0.0001
All compounds were injected (ip) 30 min before the test.
a
p < 0.01 vs. vehicle group, n ¼ 10.
6.3. Molecular modeling
The position of the 4-aryl-pyrido[1,2-c]pyrimidine or 4-aryl-
5,6,7,8-tetrahydro-pyrido[1,2-c]pyrimidine moiety was similar in
most of the top-scored poses for all ligands; it occupies the smaller
binding subpocket outlined by transmembrane helices (TMHs) 1–3
and 7. One of the carbonylic oxygen atoms of that moiety forms
bifurcated hydrogen bonds with Tyr7.43 and/or Asn7.39. The
phenyl ring substituted with a uracil moiety is, in most cases
(Fig. 4C–J), stacked between Phe3.28 and Tyr2.64 and forms
aromatic contacts with these residues.
The dockingstudiesindicated that favorableinteractionsbetween
ligands (8d, 8e, 8h, 8l) and the SERT model in closed conformation
occurred in the pore formed between TMHs 1, 3, 6, and 8 (Fig. 6). The
residues involved in ligand binding were (the generic number of the
The overall conformation of the alkyl spacer of the docked
ligands is extended and resembles the X-ray structure of 4-(4-
chloro-phenyl)-2-{4-[4-(1H-indol-3-yl)-piperidin-1-yl]-butyl}-
pyrido[1,2-c]pyrimidine-1,3-dione (9a; Fig. 1) described in Part 1
of this article [21]. The majority of the docking results do not
explicitly explain the influence of the methyl substituent at posi-
tion 2 of the indole ring that decreases the affinity to 5-HT1A to the
micromolar level. In approximately 80% of modeled complexes,
the methyl group was exposed towards the exterior of the binding
site, whereas the benzene portion of the indole ring penetrated
the binding pocket. In some of these models the position of the
indole moiety is additionally stabilized by H-bonding between the
indole NH group and Ser5.43 (Fig. 4C, H, I). However, in some
poses the alternative orientation of the indole moiety is visible in
the non-methylated ligands, with the NH group of the indole ring
residue [41] is shown as lower index): Tyr951.42, Asp981.45, Gly1001.48
Trp1031.50, and Arg1041.51 (TMH1); Ala1693.43, Ile1723.46, Tyr1753.49
Tyr1763.50, and Ile1793.53 (TMH3); Phe3356.53, Ser3366.54, Gly3386.56
,
,
,
Fig. 4. Ligand binding modes predicted by flexible docking. The transmembrane portion of the receptor is represented as ribbons. Side chains and the molecular surface are
visualized only for amino acids defined as being in the active site. Residues forming specific contacts with the ligand are shown as sticks. H-bonds are represented by yellow dotted
lines. (A) 10a [21], (B) 11a [21], (C) 8a [21], (D) 8d, (E) 8e, (F) 8h, (G) 8l, (H) 8n, (I) 8q.

F. Herold et al. / European Journal of Medicinal Chemistry 44 (2009) 4702–4715
4709
Fig. 5. Alternative binding modes present in approximately 20% of docking solutions, with a possible steric hindrance around position 2 in the indole ring.
Phe3416.59, and Val3436.62 (TMH6); Ser4388.60, Thr4398.61, and
Gly4428.64 (TMH8). All docked compounds are placed in the binding
site of SERT in a similar position. The ligand indole moiety was
located ina positioncorrespondingtotheSERTsubstrate bindingsite,
while the tetrahydro-pyrido[1,2-c]pyrimidine moiety was close to
the putative low affinity binding site [34,42]. For all docked ligands,
close interactions with Asp981.45 in TMH1 were observed. However,
the steric hindrance of Phe3556.53 in TMH6 with the ligands’ butyl
chains was observed. In addition, steric interactions between the
tetrahydro-pyrido[1,2-c]pyrimidine moiety and Arg1041.51 in TMH1
can affect the ligand binding affinity for SERT.
receptors, as well as a moderate to weak binding affinity for SERT.
The Ki values for both 5-HT1A receptor and SERT showed that
compounds 8e, 8d, 8i, and 8l had the highest affinity among new
derivatives. The remaining compounds either revealed a relatively
higher affinity for one of the proteins (8g and 8c for 5-HT1A
receptors; 8h and 8m for SERT) or bound both 5-HT1A receptor
and SERT with moderate or slight strength and were characterized
by Ki values ranging from intermediate to very high (majority of
compounds).
It was established that the presence of the 3-(4-piperidyl)-1H-
indole residue or its 5-methoxy derivative, as well as the fluorine
atom at the ortho/para position or –OCH3/–CH3 group at the para
position of the phenyl group in the aryl ring of the 4-aryl-5,6,7,8-
tetrahydro-pyrido[1,2-c]pyrimidine system, are at an advantage
with regard to binding affinity.
7. Conclusions
Several new 4-aryl-5,6,7,8-tetrahydro-pyrido[1,2-c]pyrimidine
derivatives possessing a 3-(4-piperidyl)-1H-indole group or its
5-methoxy or 2-methyl derivatives were synthesized. These
compounds are considered to have a double mechanism of action.
In vitro receptor binding studies of newly synthesized ligands
showed a very high to minimal binding affinity for 5-HT1A
We also found that the degree of saturation of the 5,6,7,8-tet-
rahydro-pyrido[1,2-c]pyrimidine system has no influence on the
binding activity for both 5-HT1A receptor and SERT compared to
unsaturated pyrido[1,2-c]pyrimidine derivatives described previ-
ously [21]. In contrast, the presence of the 2-methyl-3-(4-
Fig. 6. Ligand 8d binding to the SERT model.

4710
F. Herold et al. / European Journal of Medicinal Chemistry 44 (2009) 4702–4715
piperidyl)-1H-indole group results in a drastic affinity decrease
with regard to 5-HT1A receptors and a significant reduction in the
SERT-inhibiting action of relevant compounds.
www.ccdc.cam.ac.uk/data_request/cif, by emailing data_request@
ccdc.cam.ac.uk, or by contacting The Cambridge Crystallographic
Data Centre, 12, Union Road, Cambridge CB2 1EZ, UK; fax: þ44 1223
336033.
In an induced hypothermia test in mice, compounds 8c, 8f, 8h,
and 8i exhibited agonistic properties towards presynaptic 5-HT1A
receptors when administered at doses of 20 mg/kg, 20 mg/kg,
10 mg/kg, and 10 mg/kg, respectively. In contrast, compound 8d
exhibited antagonistic properties when administered at a dose of
20 mg/kg. The agonistic effects of compounds 8f, 8h, and 8i on
postsynaptic 5-HT1A receptors were also demonstrated in forced
swimming tests in mice. Compounds 8h and 8i have an effect at
a 10 mg/kg dose, whereas compound 8f has an effect a dose of
20 mg/kg.
8.1.2. Preparation of 2-(4-bromobutyl)-4-aryl-pyrido[1,2-c]
pyrimidine-1,3-diones (5a–f)
The starting compounds 2a–i, 3a–i, 4a–i, and 5a–i were
obtained according to previously described procedures [27,28].
8.1.3. Preparation of 2-(4-bromobutyl)-4-aryl-5,6,7,8-tetrahydro-
pyrido[1,2-c]pyrimidine-1,3-diones (6a–i)
The derivatives 5a–5i (0.01 mol) were hydrogenated in 100 ml
acetic acid with 0.1 g 10% Pt/C, and the catalytic reduction was
carried out under 60 atmospheres of hydrogen pressure at 50 ꢀC for
10 h. The catalyst was removed by filtration and the solvent by
evaporation in vacuo. Compounds 6a–6i were purified by column
chromatography (chloroform:methanol, 97:3 v/v).
8. Experimental protocols
8.1. Chemistry
8.1.1. General remarks
Catalytic hydrogenation reactions were performed using a Roth
Model IV apparatus. Melting points were determined on an Elec-
trothermal 9100 apparatus with open capillary tubes and are
uncorrected. Elemental analyses were performed on a Perkin–Elmer
2400 analyzer and were within ꢂ0.4% of the theoretical values.
Infrared spectra (KBr) were recorded on a Shimadzu FTIR-8300
spectrometer. The 1H and 13C NMR spectrawere obtained on a Bruker
AVANCE DMX 400WB instrument in CDCl3 with TMS as an internal
8.1.4. General procedure for the synthesis of 3-piperidin-4-yl-1H-
indoles (7a, 7b)
The starting compounds 7a and 7b were obtained according to
previously described procedures [25].
8.1.5. General procedure for the synthesis of 2-methyl-3-piperidin-
4-yl-1H-indole (6c)
The starting compound 7c was obtained according to previously
described procedures [47].
reference. Chemical shifts are expressed in
d units and coupling
constants (J) are expressed in hertz (Hz). The following abbreviations
are used to describe peak patterns when appropriate: s (singlet),
d (doublet), t (triplet), q (quartet), qt (quintet), m (multiplet),
p (pseudo-), and b (broad-). For the two-dimensional experiments,
the pulse sequences, acquisition, and processing parameters were
taken from the standard Bruker software library.
Flash column chromatography was carried out on Merck Silica
gel 60 (230–400 mesh ASTM) using methylene chloride/methanol
(99:1, 97:3, 95:5 v/v) as the eluent. Thin layer chromatography
was run on Merck Silica gel 60 F254 plates with a mobile phase
consisting of dioxane, toluene, ethanol, and 25% NH4OH
(6.0:3.2:0.5:0.2, v/v). Compounds were visualized by UV light
(254 nm).
Crystals of 4-(4-chloro-phenyl)-2-{4-[4-(1H-indol-3-yl)-piper-
idin-1-yl]-butyl}-pyrido[1,2-c]pyrimidine-1,3-dione (9a) [21] suit-
able for X-ray analysis were grown by the slow evaporation of an
EtOH solution. All details of the measurements, crystal data, and
structure refinement are given in Table 1. The data were collected on
an Oxford Diffraction KM4CCD diffractometer [43] at 293 K using
graphite-monochromated MoKa radiation. The unit cell parameters
were determined by the least-squares treatment of setting angles at
the highest-intensity reflections chosen from the whole experiment.
Intensity data were corrected for the Lorentz and polarization
effects [44]. The structure was solved by direct methods using the
SHELXS97 program [45] and refined by the full-matrix least-squares
method with the SHELXL97 program [46]. The function
8.1.6. General procedure for the synthesis of 2-{4-[4-(1H-indol-3-
yl)-piperidin-1-yl]-butyl}-4-aryl-5,6,7,8-tetrahydro-pyrido[1,2-c]
pyrimidine-1,3-diones (8a–8v)
Compounds 6a–6i (0.0026 mol) and 7a–7c (0.0026 mol) and
K2CO3 (0.005 mol), 70 ml acetonitrile, and a catalytic amount of KI
were stirred and refluxed for 4–5 h. Reaction time was monitored
using TLC. After cooling, the mixture was filtered, and the filtrate
was evaporated to dryness. The crude residue was purified by flash
chromatography using CH2Cl2/MeOH (96:4 v/v) as an eluent. Proper
fractions were identified by TLC and evaporated to dryness giving
analytically pure compounds 8a–8v.
8.2. 1H NMR, 13C NMR, IR and elementary analysis data
8.2.1. 2-(4-Bromobutyl)-4-(4-chloro-phenyl)-5,6,7,8-tetrahydro-
pyrido[1,2-c]pyrimidine-1,3-dione (6a)
The title compound was isolated as white powder (64%); mp
91–92 ꢀC. 1H NMR (400 MHz, CDCl3)
d
: mp: 91–92 ꢀC, 1.71 (q,
3J ¼ 6.8, C7H2), 1.83 (q, C2 ꢄ H2), 1.93 (m, C6H2, C3 ꢄ H2), 2.52 (t,
3J ¼ 6.6, C5H2), 3.44 (t, 3J ¼ 6.6, C4 ꢄ H2), 3.94 (t, 3J ¼ 6.4, C1 ꢄ H2),
3
4.03 (t, 3J ¼ 7.0, C8H2), 7.14 (d, J ¼ 8.4, C20H, C60H), 7.37 (d, C30H,
C50H). 13C NMR (400 MHz, CDCl3)
27.0 (C5), 30.4 (C3ꢄ), 33.4 (C4ꢄ), 41.0 (C8), 42.9 (C1ꢄ), 111.5 (C4),
128.9 (C30, C50), 131.9 (C10), 132.4 (C20, C60), 134.0 (C40), 150.3 (C4a),
151.8 (C1), 162.0 (C3).
d
: 18.7 (C7), 21.9 (C6), 26.6 (C2ꢄ),
2
2 2
S
w(jFoj ꢁ jFcj ) was minimized with wꢁ1 ¼ [
s
2(Fo)2 þ (0.0691P)2],
where P ¼ (Fo2 þ 2Fc2)/3.
8.2.2. 2-(4-Bromobutyl)-4-(4-fluoro-phenyl)-5,6,7,8-tetrahydro-
pyrido[1,2-c]pyrimidine-1,3-dione (6b)
All non-hydrogen atoms were refined with anisotropic thermal
parameters. The coordinates of the hydrogen atoms were calcu-
lated in idealized positions and refined as a riding model with their
thermal parameters calculated as 1.2 times Ueq of the respective
carrier carbon atom.
The title compound was isolated as white powder (85%); mp
64.0–66.0 ꢀC. 1H NMR (400 MHz, CDCl3)
d
: 1.71 (q, 3J ¼ 6.4 Hz, 2H,
C–6H2), 1.84 (q, 2H, C2 ꢄ H2), 1.93 (m, 4H, C–7H2, C–3 ꢄ H2), 2.52 (t,
3J ¼ 6.6 Hz, 2H, C–5H2), 3.44 (t, 3J ¼ 6.6 Hz, 2H, C–4 ꢄ H2), 3.94 (t,
3J ¼ 6.4 Hz, 2H, C–8H2), 4.03 (t, 3J ¼ 6.8 Hz, 2H, C–1 ꢄ H2), 7.09 (m,
2H, C–30H, C–50H), 7.17 (m, 2H, C–20H, C–60H). 13C NMR (400 MHz,
The deposition number CCDC 686643 for 4-(4-chloro-phenyl)-2-
{4-[4-(1H-indol-3-yl)-piperidin-1-yl]-butyl}-pyrido[1,2-c]pyrimidine-
1,3-dione (9a) [21] contains the supplementary crystallographic
data for this paper. These data can be obtained free of charge via
CDCl3)
d
: 18.7 (C-6), 21.9 (C-7), 26.6 (C-2ꢄ), 26.9 (C-5), 30.4
(C-3ꢄ), 33.4 (C-4ꢄ), 40.9 (C-1ꢄ), 42.9 (C-8), 111.5 (C-4), 115.7

F. Herold et al. / European Journal of Medicinal Chemistry 44 (2009) 4702–4715
4711
2
4
(d*, J ¼ 21.4 Hz, C-30, C-50), 129.3 (d*, J ¼ 3.5 Hz, C-10), 132.7
C3 ꢄ H2), 1.91 (m, C7H2, CbHax, CdHax), 2.04 (pd, CbHeq, CdHeq), 2.15
(pt, CaHax, CeHax), 2.48 (t, C4 ꢄ H2), 2.54 (t, 3J ¼ 6.6, C5H2), 2.83 (pt,
CcH), 3.09 (pd, CaHeq, CeHeq), 3.81 (s, OCH3), 3.93 (t, 3J ¼ 6.2, C8H2),
4.05 (t, 3J ¼ 6.8, C1 ꢄ H2), 6.93 (d, C30H, C50H), 6.94 (s, C200H), 7.06 (t,
(d*, 3J ¼ 8.1 Hz, C-20, C0-60), 150.2 (C4a), 151.8 (C-1), 162.1 (C-3), 162.4
(d*, 1J ¼ 246.9 Hz, C-4 ). IR (KBr)
n: 1635 (C]O), 1695 (C]O). Anal.
Calc. for C18H20FN2O2Br: C, 54.7; H 5.1; N 7.1. Found: C, 54.5; H,
5.1; N, 7.1.
3
3
3J ¼ 8.0, C600H), 7.09 (t, J ¼ 8.0, C500H), 7.12 (d, J ¼ 8.4, C20H, C60H),
3
3
7.34 (d, J ¼ 8.0, C700H), 7.63 (d, J ¼ 8.0, C400H), 8.19 (bs, NH). 13C
8.2.3. 4-(4-Chloro-phenyl)-2-{4-[4-(1H-indol-3-yl)-piperidin-1-
yl]-butyl}-5,6,7,8-tetrahydro-pyrido[1,2-c]
pyrimidine-1,3-dione (8a)
NMR (400 MHz, CDCl3)
d
: 18.8 (C6), 22.0 (C7), 24.4 (C2ꢄ), 25.9
(C3ꢄ), 26.9 (C5), 32.9 (Cb, Cd), 33.5 (Cc), 41.6 (C1ꢄ), 42.8 (C8), 54.5
(Ca, Ce), 58.8 (C4ꢄ), 111.4 (C700), 112.2 (C4), 114.2 (C30, C50), 119.2
(C500), 119.2 (C400), 120.0 (C200), 122.0 (C600), 123.5 (C300), 125.7 (C10),
126.8 (C300a), 132.0 (C20, C60), 136.5 (C700a), 149.9 (C4a), 151.9 (C1),
159.2 (C40), 162.4 (C3). IR v: 1636 (C]O), 1693 (C]O), Anal. Calcd.
for C32H38N4O3: C, 73.0; H, 7.3; N,10.7. Found: C, 72.7; H, 7.3; N,10.7.
The title compound was isolated as white powder (67.5%); mp
131–132 ꢀC. 1H NMR (400 MHz, CDCl3)
d: 1.71 (m, CbHax, CdHax),
1.93 (m, C7H2, C2 ꢄ H2), 2.06 (pd, CbHeq, CdHeq), 2.22 (pt, CaHax
,
,
CeHax), 2.51 (m, C5H2, C4 ꢄ H2), 2.86 (m, CcH), 3.13 (pd, CaHeq
CeHeq), 3.94 (t, 3J ¼ 6.4, C8H2), 4.04 (t, 3J ¼ 6.8, C1 ꢄ H2), 6.96 (bs,
3
3
C200H), 7.10 (t, J ¼ 7.2, C500H), 7.14 (d, J ¼ 8.4, C20H, C60H), 7.17 (t,
8.2.7. 2-{4-[4-(5-Methoxy-1H-indol-3-yl)-piperidin-1-yl]-butyl}-
4-(4-methoxy-phenyl)-5,6,7,8-tetrahydro-pyrido[1,2-c]pyrimidine-
1,3-dione (8e)
3J ¼ 7.2, C600H), 7.37 (d, C30H, C50H), 7.63 (d, 3J ¼ 7.6, C400H), 8.15 (bs,
NH). 13C NMR (400 MHz, CDCl3)
d
: 18.7 (C6), 21.9 (C7), 24.1 (C2ꢄ),
25.8 (C3ꢄ), 27.0 (C5), 32.6 (Cb, Cd), 33.4 (Cc), 41.6 (C1ꢄ), 42.9 (C8),
54.4 (Ca, Ce), 58.7 (C4ꢄ), 111.4 (C4, C700), 119.2 (C500), 119.3 (C400),
120.0 (C200), 121.1 (C300), 126.8 (C300a), 129.0 (C30, C50), 132.0 (C10),
132.4 (C20, C60), 133.9 (C40), 136.6 (C700a), 150.2 (C4a), 151.8 (C1),
162.1 (C3). IR v: 1635 (C]O), 1697 (C]O), Anal. Calcd. for
C31H35ClN4O2: C, 70.1; H, 6.6; N, 10.6. Found: C, 69.8; H, 6.7; N, 10.6.
The title compound was isolated as white powder (81.1%); mp
179–181 ꢀC. 1H NMR (400 MHz, CDCl3)
d
: 1.68 (m, C6H2, C2 ꢄ H2,
C3 ꢄ H2), 1.81 (pt, CbHax, CdHax), 1.90 (q, 3J ¼ 6.8, C7H2), 2.02 (pd,
CbHeq, CdHeq), 2.12 (pt, CaHax, CeHax), 2.45 (t, 3J ¼ 7.6, C4 ꢄ H2), 2.53
(t, 3J ¼ 6.8, C5H2), 2.76 (tt, 3Jax–ax ¼ 12.0, 3Jax–eq ¼ 3.6, CcH), 3.06 (pd,
CaHeq, CeHeq), 3.80 (s, OCH3), 3.85 (s, OC11H3), 3.92 (t, 3J ¼ 6.4,
C8H2), 4.05 (t, 3J ¼ 7.4, C1 ꢄ H2), 6.82 (dd, 3J ¼ 8.8, J ¼ 2.0, C600H),
4
8.2.4. 2-{4-[4-(1H-Indol-3-yl)-piperidin-1-yl]-butyl}-4-phenyl-
5,6,7,8-tetrahydro-pyrido[1,2-c]pyrimidine-1,3-dione (8b)
The title compound was isolated as white powder (70%); mp
6.90 (d, 3J ¼ 2.0, C200H), 6.92 (d, C30H, C50H), 7.05 (d, 4J ¼ 2.0, C400H),
7.11 (d, 3J ¼ 8.8, C20H, C60H), 7.20 (d, 3J ¼ 8.8, C700H), 8.3 (bs, NH). 13C
NMR (400 MHz, CDCl3)
d
: 18.8 (C6), 22.0 (C7), 24.6 (C2ꢄ), 26.0
189–190 ꢀC. 1H NMR (400 MHz, CDCl3)
d
: 1.70 (m, CbHax, CdHax),
(C3ꢄ), 27.0 (C5), 33.0 (Cb, Cd), 33.6 (Cc), 41.7 (C1ꢄ), 42.8 (C8), 54.6
(Ca, Ce), 55.5(C10), 56.3 (C11), 59.0 (C4ꢄ), 101.2 (C400), 112.1 (C600,
C700), 112.2 (C4), 114.2 (C30, C50), 121.0 (C200), 121.1 (C300), 125.7
(C300a), 127.2 (C10), 131.8 (C700a), 132.1 (C20, C60), 149.9 (C4a), 151.9
(C1), 153.8 (C500), 159.2 (C40), 162.5 (C3). IR v: 1636 (C]O), 1690
(C]O), Anal. Calcd. for C33H40N4O4: C, 71.2; H, 7.2; N, 10.1. Found: C,
70.8; H, 7.2; N, 10.1.
1.83 (q, C2 ꢄ H2), 1.91 (q, C7H2), 2.03 (pd, CbHeq, CdHeq), 2.11 (pt,
CaHax, CeHax), 2.44 (t, 3J ¼ 7.4, C4 ꢄ H2), 2.52 (t, 3J ¼ 6.4, C5H2), 2.82
(m, CcH), 3.05 (pd, CaHeq, CeHeq), 3.93 (t, 3J ¼ 6.2, C8H2), 4.05 (t,
3J ¼ 7.2, C1 ꢄ H2), 6.91 (bs, C200H), 7.07 (t, J ¼ 7.6, C500H), 7.15 (t,
3
3J ¼ 7.6, C600H), 7.20 (d, J ¼ 7.2, C20H, C60H), 7.33 (pd, C40H, C700H),
3
7.39 (t, 3J ¼ 7.0, C30H, C50H), 7.63 (d, 3J ¼ 7.6, C400H), 8.28 (bs, NH). 13C
NMR (400 MHz, CDCl3)
d
: 18.8 (C6), 22.0 (C7), 24.7 (C2ꢄ), 26.0
(C3ꢄ), 26.9 (C5), 33.2 (Cb, Cd), 33.7 (Cc), 41.8 (C1ꢄ), 42.9 (C8), 54.7
(Ca, Ce), 59.1 (C4ꢄ), 111.5 (C700), 112.7 (C4), 119.1 (C500), 119.3 (C400),
120.0 (C200), 121.5 (C300), 121.9 (C600), 126.9 (C300a), 127.9 (C40), 128.7
(C30, C50), 131.0 (C20, C60),133.6 (C10),136.6 (C700a), 149.9 (C4a),152.0
(C1), 162.2 (C3). IR v: 1663 (C]O), 1690 (C]O), Anal. Calcd. for
C31H36N4O2: C, 75.0; H, 7.3; N, 11.3. Found: C, 74.6; H, 7.2; N, 11.1.
8.2.8. 4-(2-Fluoro-phenyl)-2-{4-[4-(5-methoxy-1H-indol-3-yl)-
piperidin-1-yl]-butyl}-5,6,7,8-tetrahydro-pyrido[1,2-c]pyrimidine-
1,3-dione (8f)
The title compound was isolated as white powder (71%); mp 115–
118 ꢀC.1H NMR (400 MHz, CDCl3)
d
: 1.74 (m, C6H2, C2 ꢄ H2, C3 ꢄ H2),
1.94 (q, C7H2), 1.94 (bs, CbHeq, CdHeq), 2.37 (bs, CaHax, CeHax), 2.48
(pt, C5H2), 2.67 (pt, C4 ꢄ H2), 2.82 (m, CcH), 3.22 (pd, CaHeq, CeHeq),
3.87 (s, OCH3), 3.94 (m, C8H2), 4.00 (pt, C1 ꢄ H2), 6.83 (dd, 3J ¼ 8.8,
8.2.5. 2-{4-[4-(5-Methoxy-1H-indol-3-yl)-piperidin-1-yl]-butyl}-
4-p-tolyl-5,6,7,8-tetrahydro-pyrido[1,2-c]pyrimidine-1,3-dione (8c)
The title compound was isolated as white powder (74%); mp
3
4
4J ¼ 4.8, C600H), 6.93 (d, J ¼ 4.0, C200H), 7.05 (d, J ¼ 4.8, C400H), 7.13
(pt, C30H), 7.20 (m, C50H, C60H), 7.24 (m, C40H), 7.24 (d, 3J ¼ 8.8, C700H),
205–207 ꢀC. 1H NMR (400 MHz, CDCl3)
d
: 1.68 (m, C6H2, C2 ꢄ H2,
8.24 (bs, NH). 13C NMR (400 MHz, CDCl3)
d: 18.6 (C6), 21.9 (C7), 23.2
C3 ꢄ H2), 1.87 (pt, CbHax, CdHax), 1.91 (q, C7H2), 2.02 (pd, CbHeq
,
(C3ꢄ), 25.5 (C2ꢄ), 26.7 (C5), 31.7 (bs, Cb, Cd), 32.9 (Cc), 41.1 (C1ꢄ),
CdHeq), 2.15 (pt, CaHax, CeHax), 2.36 (s, CH3), 2.47 (t, 3J ¼ 6.8,
43.2 (C8), 54.0, 54.2 (Ca, Ce), 56.4 (OCH3), 58.2 (C4ꢄ), 101.1 (C400),
C4 ꢄ H2), 2.53 (t, 3J ¼ 6.6, C5H2), 2.77 (m, CcH), 3.08 (pd, CaHeq
,
106.2 (C4), 112.2 (C700), 112.2 (C600), 116.0 (d*, J ¼ 22.3, C30), 119.9
2
CeHeq), 3.86 (s, OCH3), 3.93 (t, 3J ¼ 6.4, C8H2), 4.05 (t, 3J ¼ 6.8,
(C200), 121.0 (d*, J ¼ 16.0, C10), 121.1 (C300), 124.5 (d*, J ¼ 3.4, C50),
126.9 (C300a), 130.3 (d*, 3J ¼ 8.2, C40), 131.7 (C700a), 133.1 (d*, 3J ¼ 2.7,
C60),151.4 (C4a),151.8 (C1),154.0 (C500),160.6 (d*,1J ¼ 16.0, C10),161.8
(C3). IR v: 1651 (C]O), 1701 (C]O), Anal. Calcd. for C31H35FN4O2: C,
70.6; H, 6.9; N, 10.3. Found: C, 70.2; H, 6.9; N, 10.1.
2
4
C1 ꢄ H2), 6.83 (dd, 3J ¼ 8.4, J ¼ 1.2, C600H), 6.90 (s, C200H), 7.07 (m,
4
C30H, C50H, C400H), 7.21 (m, C20H, C60H, C700H), 8.20 (bs, NH). 13C
NMR (400 MHz, CDCl3) d: 18.8 (C6), 21.4 (CH3), 21.9 (C7), 24.3
(C2ꢄ), 25.9 (C3ꢄ), 26.9 (C5), 32.8 (Cb, Cd), 33.5 (Cc), 41.6 (C1ꢄ), 42.8
(C8), 54.5 (Ca, Ce), 56.3 (OCH3), 58.8 (C4ꢄ), 101.2 (C400), 112.1 (C600,
C700), 112.5 (C4), 120.9 (C200, C300), 127.1 (C300a), 129.4 (C20, C60), 130.5
(C10), 130.7 (C30, C50), 137.6 (C40), 149.8 (C4a), 151.9 (C1), 153.9 (C500),
162.3 (C3). IR v: 16433 (C]O), 1693 (C]O), Anal. Calcd. for
C33H40N4O3: C, 73.3; H, 7.5; N, 10.4. Found: C, 73.0; H, 7.3; N, 10.4.
8.2.9. 4-(4-Chloro-phenyl)-2-{4-[4-(5-methoxy-1H-indol-3-yl)-
piperidin-1-yl]-butyl}-5,6,7,8-tetrahydro-pyrido[1,2-c]pyrimidine-
1,3-dione (8g)
The title compound was isolated as white powder (79%); mp
199.0–200.3 ꢀC. 1H NMR (400 MHz, CDCl3)
d: 1.71 (m, C6H2,
8.2.6. 2-{4-[4-(1H-Indol-3-yl)-piperidin-1-yl]-butyl}-4-(4-
methoxy-phenyl)-5,6,7,8-tetrahydro-pyrido[1,2-c]pyrimidine-1,3-
dione (8d)
C2 ꢄ H2, C3 ꢄ H2), 1.93 (q, 3J ¼ 6.6, C7H2), 2.04 (pd, CbHeq, CdHeq),
2.28 (bps, CaHax, CeHax), 2.52 (t, 3J ¼ 6.6, C5H2), 2.59 (bps, C4 ꢄ H2),
2.82 (bps, CcH), 3.19 (pd, CaHeq, CeHeq), 3.87 (s, OCH3), 3.94 (t,
3J ¼ 6.4, C8H2), 4.05 (t, 3J ¼ 6.4, C1 ꢄ H2), 6.85 (dd, 3J ¼ 8.6, 4J ¼ 2.2,
C600H), 6.95 (bs, C200H), 7.06 (d, C400H), 7.15 (d, 3J ¼ 8.4, C20H, C60H),
The title compound was isolated as white powder (79.2%); mp
218–219 ꢀC. 1H NMR (400 MHz, CDCl3)
d: 1.69 (m, C6H2, C2 ꢄ H2,

4712
F. Herold et al. / European Journal of Medicinal Chemistry 44 (2009) 4702–4715
7.25 (d, C700H), 7.37 (d, C30H, C50H), 8.03 (bs, NH). 13C NMR
(400 MHz, CDCl3)
2.77 (pt, CcH), 3.06 (pd, CaHeq, CeHeq), 3.85 (s, OCH3), 3.92 (m,
C8H2), 4.06 (t, C1 ꢄ H2), 6.82 (d, 3J ¼ 8.8, C600H), 6.88 (bs, C200H), 7.04
(bps, C30H, C400H), 7.21 (d, 3J ¼ 8.4, C700H), 7.25 (m, C40H, C60H), 8.40
d: 18.7 (C6), 21.9 (C7), 23.8 (C2ꢄ), 25.7 (C3ꢄ), 27.0
(C5), 32.3 (Cb, Cd), 33.3 (Cc), 41.5 (C1ꢄ), 43.0 (C8), 54.4 (Ca, Ce), 56.3
(OCH3), 58.6 (C4ꢄ), 101.2 (C400), 111.5 (C4), 112.1 (C700), 112.3 (C600),
121.0 (C200, C300), 127.1 (C300a), 129.0 (C30, C50), 132.0 (C10), 132.4 (C20,
C60), 133.4 (C40), 150.3 (C4a), 151.8 (C1), 154.0 (C500), 162.1 (C3). IR
v: 1636 (C]O), 1697 (C]O), Anal. Calcd. for C32H37ClN4O3: C, 68.5;
H, 6.7; N, 10.0. Found: C, 68.4; H, 6.6; N, 10.0.
(bs, NH). 13C NMR (400 MHz, CDCl3)
d: 18.7 (C6), 19.8 (CH3), 22.0
(C7), 23.9 (C2ꢄ), 25.8 (C3ꢄ), 26.7 (C5), 32.4 (Cb, Cd), 33.2 (Cc), 41.3
(C1ꢄ), 43.1 (C8), 54.3 (Ca, Ce), 56.2 (OCH3), 58.6 (C4ꢄ), 101.0 (C400),
111.8 (C4), 112.1 (C700), 112.2 (C600), 120.4 (C300), 121.0 (C200), 126.4
(C50), 127.0 (C300a), 128.4 (C40), 130.4 (C30), 130.9 (C60), 131.7 (C700a),
133.1 (C10), 137.8 (C20), 152.0 (C4a), 153.8 (C1), 161.7 (C3). IR v: 1635
(C]O), 1693 (C]O), Anal. Calcd. for C33H40N4O3: C, 73.3; H, 7.5; N,
10.4. Found: C, 73.0; H, 7.4; N, 10.3.
8.2.10. 2-{4-[4-(1H-Indol-3-yl)-piperidin-1-yl]-butyl}-4-p-tolyl-
5,6,7,8-tetrahydro-pyrido[1,2-c]pyrimidine-1,3-dione (8h)
The title compound was isolated as white powder (80%); mp
167–168 ꢀC. 1H NMR (400 MHz, CDCl3)
d
: 1.70 (q, 3J ¼ 6.4, C6H2),
8.2.14. 4-(4-Fluoro-phenyl)-2-{4-[4-(5-methoxy-1H-indol-3-yl)-
piperidin-1-yl]-butyl}-5,6,7,8-tetrahydro-pyrido[1,2-c]pyrimidine-
1,3-dione (8l)
1.75 (pd, 6H, CbHax, CdHax), 1.92 (q, 3J ¼ 6.4, C7H2), 2.05 (bps, CbHeq
,
CdHeq), 2.32 (bpt, CaHax, CeHax), 2.36 (s, CH3), 2.54 (t, 3J ¼ 6.4, C5H2),
2.64 (bps, C4 ꢄ H2), 2.87 (m, CcH), 3.20 (bpd, CaHeq, CeHeq), 3.94 (t,
The title compound was isolated as white powder (54%); mp
3
3J ¼ 6.4, C8H2), 4.05 (t, J ¼ 6.4, C1 ꢄ H2), 6.96 (bs, C200H), 7.08 (d,
162–163 ꢀC. 1H NMR (400 MHz, CDCl3)
d
: 1.63 (m, C2 ꢄ H2, C3 ꢄ H2),
3
C30H, C50H), 7.09 (t, C500H), 7.17 (t, J ¼ 7.6, C600H), 7.21 (d, 3J ¼ 8.0,
1.70 (q, C6H2), 1.80 (pt, CbHax, CdHax), 1.91 (q, 3J ¼ 6.4, C7H2), 2.02
(pd, CbHeq, CdHeq), 2.14 (pt, CaHax, CeHax), 2.46 (t, 3J ¼ 7.2, C4 ꢄ H2),
2.51 (t, 3J ¼ 6.8, C5H2), 2.78 (tt, 3Jax–ax ¼ 11.6, 3Jax–eq ¼ 3.6, CcH), 3.08
(pd, CaHeq, CeHeq), 3.86 (s, OCH3), 3.93 (t, 3J ¼ 6.4), 4.04 (t, 3J ¼ 7.2,
C20H, C60H), 7.36 (d, 3J ¼ 8.0, C700H), 7.61 (d, 3J ¼ 7.6, C400H), 8.27 (bs,
NH). 13C NMR (400 MHz, CDCl3)
d: 18.8 (C6), 21.5 (CH3), 22.0 (C7),
26.2 (C3ꢄ), 26.6 (C2ꢄ), 27.0 (C5), 31.8 (Cc), 32.1 (Cb, Cd), 41.2 (C1ꢄ),
42.9 (C8), 55.0 (Ca, Ce), 58.0 (C4ꢄ), 111.5 (C700), 112.5 (C4), 119.1
(C500), 119.3 (C400), 120.2 (C200), 121.8 (C300), 122.1 (C600), 126.7 (C300a),
129.5 (C20, C60), 130.5 (C10), 130.7 (C30, C50), 136.5 (C700a), 137.7 (C40),
148.0 (C4a), 152.0 (C1), 162.4 (C3). IR v: 1639 (C]O), 1701 (C]O),
Anal. Calcd. for C32H38N4O2: C, 75.3; H, 7.5; N, 11.0. Found: C, 74.8;
H, 7.4; N, 10.6.
4
4
C1 ꢄ H2), 6.84 (dd, 3J ¼ 8.6, J ¼ 2.2, C600H), 7.05 (d, J ¼ 2.0, C400H),
7.16 (m, C20H, C60H), 7.22 (d, C700H), 8.15 (bs, NH). 13C NMR
(400 MHz, CDCl3)
d
: 18.7 (C6), 21.9 (C7), 24.5 (C2ꢄ), 25.9 (C3ꢄ), 26.9
(C5), 32.9 (Cb, Cd), 33.6 (Cc), 41.7 (C1ꢄ), 42.9 (C8), 54.6 (Ca, Ce), 56.3
(OCH3), 58.9 (C4ꢄ), 101.2 (C400), 111.6 (C4), 112.1 (C600, C700), 115.7 (d*,
2J ¼ 21.5, C30, C50), 120.8 (C200), 121.1 (C300), 127.1 (C300a), 129.4 (d*,
4J ¼ 3.4, C10), 131.7 (C700a), 132.7 (d*, J ¼ 7.9, C20, C60), 150.1 (C500),
3
1
8.2.11. 4-(2-Fluoro-phenyl)-2-{4-[4-(1H-indol-3-yl)-piperidin-1-
yl]-butyl}-5,6,7,8-tetrahydro-pyrido[1,2-c]pyrimidine-1,3-dione (8i)
The title compound was isolated as white powder (73%); mp
151.8 (C4a), 153.9 (C1), 162.2 (C3), 162.4 (d*, J ¼ 246.9, C40). IR v:
1645 (C]O), 1718 (C]O), Anal. Calcd. for C32H37FN4O3: C, 70.6; H,
6.9; N, 10.1. Found: C, 69.7; H, 6.8; N, 10.1.
106–108 ꢀC. 1H NMR (400 MHz, CDCl3)
d
: 1.72 (m, C6H2, C3 ꢄ H2),
1.92 (q, C7H2), 2.01 (m, 6H, CbHeq, CdHeq C2 ꢄ H2), 2.48 (m, C5H2),
2.65 (t, 3J ¼ 7.4, C4 ꢄ H2), 2.84 (tt, CcH), 3.18 (m, CaHax, CeHax), 3.21
(pd, CaHeq, CeHeq), 3.93 (m, C8H2), 4.04 (t, 3J ¼ 7.2, C1 ꢄ H2), 6.90
(bs, C200H), 7.12 (pt, C30H), 7.15 (C600H), 7.20 (m, C50H, C60H), 7.22 (bs,
8.2.15. 4-(2-Chloro-phenyl)-2-{4-[4-(1H-indol-3-yl)-piperidin-1-
yl]-butyl}-5,6,7,8-tetrahydro-pyrido[1,2-c]pyrimidine-
1,3-dione (8m)
The title compound was isolated as white powder (60%); mp
NH), 7.34 (m, C40H, C700H), 7.59 (d, J ¼ 8.0, C400H). 13C NMR
92.4–93.1 ꢀC. 1H NMR (400 MHz, CDCl3)
d
: 1.72 (m, C6H2, C2 ꢄ H2,
3
(400 MHz, CDCl3)
d
: 18.5 (C6), 21.8 (C7), 22.9 (C2ꢄ), 25.5 (C3ꢄ), 26.6
C3 ꢄ H2), 1.91 (m, C7H2, CbHax, CdHax), 2.04 (pd, CbHeq, CdHeq), 2.19
(C5), 31.6 (Cb, Cd), 32.8 (Cc), 41.2 (0C0 1ꢄ), 43.1 (C8), 53.6, 53.7 (Ca, Ce),
(pt, CaHax, CeHax), 2.40 (dt, C5H2), 2.52 (t, 3J ¼ 6.8, C4 ꢄ H2), 2.83 (t,
2
3
57.8 (C4ꢄ), 106.020 (C4), 111.5 (C7 ), 115.9 (d*, J ¼ 22.2, C30), 119.0
3Jax–ax ¼ 11.6, Jax–eq ¼ 3.6, CcH), 3.1 (pd, CaHeq, CeHeq), 3.93 (m,
2
(C400), 120.0 (C2 ), 120.3(C300), 120.9 (d*, J ¼ 16.2, C10), 124.3 (d*,
2J ¼ 13.8, 3J ¼ 6.8, C8H2), 4.06 (t, 3J ¼ 7.0, C1 ꢄ H2), 6.94 (d, 3J ¼ 1.6),
7.08 (t, 3J ¼ 7.2, C500H), 7.16 (t, 3J ¼ 7.4, C600H), 7.21 (m, C60H), 7.30 9
4J ¼ 3.4, C50), 126.6 (C300a), 130.2 (d*, 3J ¼ 8.1, C40), 133.0 (d*, 3J ¼ 2.7,
C60), 136.6 (C700a), 151.4 (C4a), 151.8 (C1), 160.6 (d*, 1J ¼ 247.9, C20),
161.7 (C3). IR v: 1651 (C]O), 1701 (C]O), Anal. Calcd. for
C31H35FN4O2: C, 72.4; H, 6.9; N, 10.9. Found: C, 71.9; H, 6.7; N, 11.0.
3
(m, C40H, C50H), 7.34 (d, J ¼ 8.4, C700H), 7.45 (m, C30H), 7.62 (d,
3J ¼ 8.0, C400H), 8.29 (bs, NH). 13C NMR (400 MHz, CDCl3)
d: 18,4
(C6), 21.8 (C7), 23.0 (C2ꢄ), 25.6 (C3ꢄ), 26.4 (C5), 33.1 (Cb, Cd), 33.2
(Cc), 40.0 (C1ꢄ), 55.0 (Ca, Ce), 58.3 (C4ꢄ), 110.0 (C700), 111.2 (C4),
118.9 (C400, C500), 119.8 (C200), 121.7 (C600), 121.8 (C300), 127.0 (C300a),
127.1 (C50), 129.5 (C40), 129.7 (C40), 132.4 (C10), 132.5 (C60), 135.0
(C20), 136.3 (C700a), 151.7 (C1), 161.2 (C3). IR v: 1632 (C]O), 1704
(C]O), Anal. Calcd. for C31H35ClN4O2: C, 70.1; H, 6.6; N, 10.6. Found:
C, 69.4; H, 6.9; N, 10.5.
8.2.12. 2-{4-[4-(1H-Indol-3-yl)-piperidin-1-yl]-butyl}-4-o-tolyl-
5,6,7,8-tetrahydro-pyrido[1,2-c]pyrimidine-1,3-dione (8j)
The title compound was isolated as white powder (5.2%); mp
128.0–130.2 ꢀC. 1H NMR (400 MHz, CDCl3)
d: 1.74 (m, C2 ꢄ H2,
C3 ꢄ H2), 1.94 (q, C7H2), 2.03 (m, CbH2, CdH2), 2.14 (s, CH3), 2.25 (pk
CaHax, CeHax), 2.28 (m, C5(00)H), 2.42 (C5(0)H), 2.84 (m, CcH), 3.17
(pd, CaHeq, CeHeq), 3.94 (m, C8H2), 4.06 (t, 3J ¼ 6.0, C1 ꢄ H2), 6.92 (s,
C200H), 7.05 (d, 3J ¼ 7.2, C30H), 7.09 (t, 3J ¼ 7.6, C500H), 7.16 (t, 3J ¼ 7.6,
8.2.16. 4-(2-Fluoro-phenyl)-2-{4-[4-(2-methyl-1H-indol-3-yl)-
piperidin-1-yl]-butyl}-5,6,7,8-tetrahydro-pyrido[1,2-c]pyrimidine-
1,3-dione (8n)
3
C600H), 7.18–7.30 (m, C40H–C60H), 7.35 (d, J ¼ 8.0, C700H), 7.60 (d,
3J ¼ 7.6, C400H), 8.31 (d, NH). IR v: 1635 (C]O), 1697 (C]O), Anal.
Calcd. for C32H38N4O2: C, 75.3; H, 7.5; N, 11.0. Found: C, 74.9; H, 7.5;
N, 11.1.
The title compound was isolated as white powder (48%); mp
192.2–194.8 ꢀC. 1H NMR (400 MHz, CDCl3)
d: 1.74 (m, 8H, CbHax,
CdHax), 1.94 (q, 3J ¼ 6.3, C7H2), 2.24 (bps, CbHeq, CdHeq), 2.33 (bps,
CaHax, CeHax), 2.40 (m þ s, C4 ꢄ H2, CH3), 2.49 (td, 3J ¼ 5.4, 4J ¼ 2.1),
2.77 (bps, CcH), 3.19 (bps, CaHeq, CeHeq), 3.93 (m, C8H2), 4.05 (t,
3J ¼ 6.3, C1 ꢄ H2), 7.03 (t, C500H), 7.10 (t, C500H), 7.20 (m, C30H), 7.23
8.2.13. 2-{4-[4-(5-Methoxy-1H-indol-3-yl)-piperidin-1-yl]-butyl}-
4-o-tolyl-5,6,7,8-tetrahydro-pyrido[1,2-c]pyrimidine-1,3-dione (8k)
The title compound was isolated as white powder (55%); mp
4
(dd, 3J ¼ 6.0, J ¼ 1.2, C700H), 7.35 (m, C40H, C50H, C60H), 7.73 (d,
101.5–103.2 ꢀC. 1H NMR (400 MHz, CDCl3)
d
: 1.71 (m, C2 ꢄ H2,
3J ¼ 7.5, C400H), 7.94 (bs, NH). 13C NMR (400 MHz, CDCl3)
d: 12.6
C3 ꢄ H2), 1.90 (m, CdHax), 2.01 (pd, CbHeq, CdHeq), 2.13 (s, CH3), 2.20
(CH3), 18.6 (C6), 21.9 (C7), 25.8 (C2ꢄ), 26.7 (C3ꢄ, C5), 31.5 (Cb, Cd),
(pt, CaHax, CeHax), 2.27 (m, C500H), 2.43 (m, C50H), 2.53 (t, C4 ꢄ H2),
34.2 (Cc), 43.0 (C1ꢄ), 43.1 (C8), 54.8 (Ca, Ce), 58.8 (C4ꢄ), 106.3 (C4),

F. Herold et al. / European Journal of Medicinal Chemistry 44 (2009) 4702–4715
4713
2
110.4 (C70000), 116.0 (d*, J ¼ 22.1, C30), 119.1 (C300), 119.4 (C400, C500),
(CH3), 18.6 (C6), 21.8 (C7), 24.3 (C2ꢄ), 25.6 (C3ꢄ), 26.7 (C5), 32.2
(Cb, Cd), 34.8 (Cc), 41.6 (C1ꢄ), 42.6 (C8), 55.0 (Ca, Ce), 55.3 (OCH3),
59.0 (C4ꢄ), 110.3 (C4), 112.0 (C700), 114.0 (C30, C50), 115.3 (C300), 118.6
(C500), 119.3 (C400), 120.4 (C600), 125.5 (C10), 127.6 (C300a), 130.0 (C700a),
131.9 (C20, C60), 149.7 (C4a), 151.7 (C1), 159.0 (C40), 162.2 (C3). IR v:
1636 (C]O), 1690 (C]O), Anal. Calcd. for C33H40N4O3: C, 73.3; H,
7.5; N, 10.4. Found: C, 72.7; H, 7.4; N, 10.3.
2
4
120.9 (C6 ), 121.0 (d*, J ¼ 17.7, C10), 124.4 (d*, J ¼ 3.3, C50), 127.6
(C300a), 130.2 (d*, 3J ¼ 8.3, C40), 130.3 (C700a), 133.9 (d*, 3J ¼ 3.3, C60),
1
151.4 (C4a), 151.8 (C1), 160.6 (d*, J ¼ 245.5, C20), 161.7 (C3). IR v:
1634 (C]O), 1715 (C]O), Anal. Calcd. for C32H37FN4O2: C, 72.7; H,
7.1; N, 10.6. Found: C, 72.2; H, 7.0; N, 10.3.
8.2.17. 2-{4-[4-(2-Methyl-1H-indol-3-yl)-piperidin-1-yl]-butyl}-4-
phenyl-5,6,7,8-tetrahydro-pyrido[1,2-c]pyrimidine-1,3-dione (8o)
The title compound was isolated as white powder (68%); mp
8.2.21. 4-(4-Chloro-phenyl)-2-{4-[4-(2-methyl-1H-indol-3-yl)-
piperidin-1-yl]-butyl}-5,6,7,8-tetrahydro-pyrido[1,2-c]pyrimidine-
1,3-dione (8s)
196.0–197.8 ꢀC. 1H NMR (400 MHz, CDCl3)
d: 1.64 (pd, CbHax,
CdHax), 1.72 (m, C2 ꢄ H2, C6H2, C3 ꢄ H2), 1.93 (q, C7H2), 2.04 (pt,
The title compound was isolated as white powder (43%); mp
CbHeq, CdHeq), 2.23 (pk, CaHax, CeHax), 2.40 (s, CH3), 2.42 (t,
173.5–174.6 ꢀC. 1H NMR (500 MHz, CDCl3)
d
: 1.62 (m, C3 ꢄ H2),
C4 ꢄ H2), 2.54 (t, 3J ¼ 6.6, C5H2), 2.73 (m, CcH), 3.08 (pd, CaHeq
,
1.68–1.77 (m, 6H, CbHax, CdHax), 1.92 (q, 3J ¼ 6.6, C7H2), 2.03 (m,
CbHeq, CdHeq), 2.22 (m, CaHax, CeHax), 2.38 (s, CH3), 2.42 (t, 3J ¼ 7.8,
CeHeq), 3.95 (t, 3J ¼ 6.6, C8H2), 4.05 (t, 3J ¼ 7.0, C1 ꢄ H2), 7.02 (t,
3
3
3
3J ¼ 7.4, C500H), 7.07 (t, J ¼ 7.2, C600H), 7.21 (d, J ¼ 7.6, C20H, C60H),
C4 ꢄ H2), 2.51 (t, 3J ¼ 6.6, C5H2), 2.72 (tt, Jax–ax ¼ 12.4, 3Jax–eq ¼ 3.7,
3
3
7.24 (d, C700H), 7.33 (t, J ¼ 7.2, C40H), 7.40 (t, J ¼ 7.2, C30H, C50H),
CcH), 3.07 (pd, CaHeq, CeHeq), 3.94 (t, C8H2), 4.05 (t, 3J ¼ 7.1,
7.71 (s, NH), 7.74 (d, C400H). 13C NMR (400 MHz, CDCl3)
d: 12.8 (CH3),
C1 ꢄ H2), 7.00 (m, C500H), 7.06 (td, 3J ¼ 7.1, J ¼ 1.0, C600H), 7.15 (dt,
4
3
4
18.9 (C6), 21.9 (C7), 25.7 (C2ꢄ), 26.9 (C3ꢄ, C5), 33.0 (Cb, Cd), 34.7
(Cc), 41.8 (C1ꢄ), 42.6 (C8), 54.3 (Ca, Ce), 57.1 (C4ꢄ),110.5 (C700),111.2
(C4), 119.3 (C500), 119.4 (C400), 120.1 (C200), 121.7 (C300), 121.9 (C600),
126.5 (C300a), 127.9 (C40), 128.7 (C30, C50), 131.1 (C20, C60), 133.9 (C10),
136.6 (C700a), 149.6 (C4a), 152.4 (C1), 162.9 (C3). IR v: 1648 (C]O),
1695 (C]O), Anal. Calcd. for C32H38N4O2: C, 75.3; H, 7.5; N, 11.0.
Found: C, 74.8; H, 7.2; N, 10.7.
C30H, C50H), 7.23 (d, J ¼ 8.1, C700H), 7.37 (dt, 3J ¼ 8.5, J ¼ 2.4, C20H,
C60H), 7.70 (d, J ¼ 7.8, C400H), 7.77 (bs, NH). 13C NMR (500 MHz,
3
CDCl3)
d
: 12.4 (CH3),18.6 (C6), 21.7 (C7), 24.6 (C2ꢄ), 25.9 (C3ꢄ), 26.7
(C5), 32.2 (Cb, Cd), 34.8 (Cc), 41.7 (C1ꢄ), 42.7 (C8), 55.0 (Ca, Ce), 59.0
(C4ꢄ), 110.2 (C4), 111.3 (C700), 115.5 (C300), 118.8 (C500), 119.4 (C400),
120.6 (C600), 127.6 (C300a), 128.7 (C30, C50), 129.85 (C700a), 131.9 (C10),
132.2 (C20, C60), 133.7 (C40), 149.9 (C4a), 151.6 (C1), 161.8 (C3). IR v:
1643 (C]O), 1703 (C]O), Anal. Calcd. for C32H37ClN4O2: C, 72.7; H,
7.0; N, 10.6. Found: C, 72.0; H, 6.8; N, 10.7.
8.2.18. 4-(2-Methoxy-phenyl)-2-{4-[4-(2-methyl-1H-indol-3-yl)-
piperidin-1-yl]-butyl}-5,6,7,8-tetrahydro-pyrido[1,2-c]pyrimidine-
1,3-dione (8p)
8.2.22. 4-(2-Chloro-phenyl)-2-{4-[4-(2-methyl-1H-indol-3-yl)-
piperidin-1-yl]-butyl}-5,6,7,8-tetrahydro-pyrido[1,2-c]pyrimidine-
1,3-dione (8t)
The title compound was isolated as white powder (41%); mp
189.7–191.3 ꢀC. 1H NMR (400 MHz, CDCl3)
d: 1.76 (m, 8H, CbHax,
CdHax), 1.92 (q, 3J ¼ 6.4, C7H2), 2.00 (bps, CbHeq, CdHeq), 2.25 (pk,
CaHax, CeHax), 2.39 (m þ s, C4 ꢄ H2, CH3), 2.43 (t, 3J ¼ 6.8, C5H2),
2.77 (bps, CcH), 3.20 (bps, CaHeq, CeHeq), 3.76 (s, OCH3), 3.91 (t,
The title compound was isolated as white powder (67%); mp
170.0–173.3 ꢀC. 1H NMR (400 MHz, CDCl3)
d: 1.71 (m, 8H, CbHax,
CdHax), 1.95 (q, 3J ¼ 6.3, C7H2), 2.06 (bps, CbHeq, CdHeq), 2.25 (pk,
CaHax, CeHax), 2.41 (m þ s, C4 ꢄ H2, CH3), 2.43 (td, 3J ¼ 6.6, 4J ¼ 1.8,
C5H2), 2.75 (tt, CcH), 3.00 (pd, CaHeq, CeHeq), 3.96 (m, C8H2), 4.08
3
C8H2), 4.05 (t, 3J ¼ 6.0, C1 ꢄ H2), 6.93 (d, J ¼ 8.4, C30H), 6.99 (t,
4
3J ¼ 7.6, C50H), 7.05 (td, 3J ¼ 7.2, J ¼ 0.9, C500H), 7.10 (td, 3J ¼ 7.6,
4J ¼ 1.6, C600H), 7.23 (dd, 3J ¼ 7.6, 4J ¼ 0.8, C60H), 7.32 (m, C40H, C70H),
7.74 (d, 3J ¼ 6.8, C400H), 7.99 (bs, NH). IR v: 1631 (C]O), 1693 (C]O),
Anal. Calcd. for C33H40N4O3: C, 73.3; H, 7.5; N, 10.4. Found: C, 73.0;
H, 7.2; N, 10.2.
(t, 3J ¼ 7.2, C1 ꢄ H2), 7.03 (t, J ¼ 6.9, C500H), 7.09 (t, J ¼ 6.3, C600H),
3
3
7.22 (d, C60H), 7.24 (d, C700H), 7.32 (m, C40H, C50H),7.48 (dd, C30H),
7.73 (d, 3J ¼ 7.5, C400H), 7.80 (bs, NH). 13C NMR (400 MHz, CDCl3)
d:
12.6 (CH3), 18.7 (C6), 22.0 (C7), 24.6 (C2ꢄ), 26.0 (C3ꢄ), 26.6 (C5),
32.3 (Cb, Cd), 35.0 (Cc), 41.7 (C1ꢄ), 43.2 (C8), 55.1 (Ca, Ce), 59.1
(C4ꢄ), 110.3 (C4), 110.4 (C700), 115.7 (C300), 118.9 (C500), 119.6 (C400),
120.8 (C600), 127.3 (C50), 127.8 (C300a), 129.7 (C40), 129.9 (C30), 130.1
(C700a), 132.7 (C60), 132.8 (C10), 135.3 (C20), 135.4 (C200), 150.6 (C4a),
152.0 (C1), 161.4 (C3). IR v: 1639 (C]O), 1695 (C]O), Anal. Calcd.
for C32H37ClN4O2: C, 70.5; H, 6.8; N, 10.3. Found: C, 71.0; H, 6.5;
N, 10.0.
8.2.19. 2-{4-[4-(2-Methyl-1H-indol-3-yl)-piperidin-1-yl]-butyl}-4-
p-tolyl-5,6,7,8-tetrahydro-pyrido[1,2-c]pyrimidine-1,3-dione (8q)
The title compound was isolated as white powder (41%); mp
163.5–166.9 ꢀC. 1H NMR (400 MHz, CDCl3)
d: 1.61 (ps, CbHax,
CdHax),1.72 (m, C2 ꢄ H2, C6H2, C3 ꢄ H2),1.93 (q, 3J ¼ 6.4, C7H2), 2.03
(pt, CbHeq, CdHeq), 2.22 (pk, CaHax, CeHax), 2.40 (t þ s, C4 ꢄ H2, CH3),
2.55 (t, 3J ¼ 6.6, C5H2), 2.73 (m, CcH), 3.08 (pd, CaHeq, CeHeq), 3.82 (s,
OCH3), 3.94 (t, 3J ¼ 6.4, C8H2), 4.05 (t, 3J ¼ 7.2, C1 ꢄ H2), 6.94 (d,
8.2.23. 4-(4-Fluoro-phenyl)-2-{4-[4-(2-methyl-1H-indol-3-yl)-
piperidin-1-yl]-butyl}-5,6,7,8-tetrahydro-pyrido[1,2-c]pyrimidine-
1,3-dione (8u)
3
3
3J ¼ 8.8, C30H, C50H), 7.02 (t, J ¼ 7.2, C500H), 7.07 (t, J ¼ 6.8, C600H),
7.13 (d, C20H, C60H), 7.25 (d, C700H), 7.72 (m, C400H, NH). IR v: 1642
(C]O), 1697 (C]O), Anal. Calcd. for C33H40N4O2: C, 75.5; H, 7.7; N,
10.7. Found: C, 75.3; H, 7.6; N, 10.8.
The title compound was isolated as white powder (53%); mp
199.3–200.0 ꢀC. 1H NMR (400 MHz, CDCl3)
d: 1.71 (m, 8H, CbHax,
CdHax), 1.94 (q, 3J ¼ 6.3, C7H2), 2.04 (pt, CbHeq, CdHeq), 2.23 (pk,
CaHax, CeHax), 2.39 (s, CH3), 2.43 (t, C4 ꢄ H2), 2.52 (t, 3J ¼ 6.9, C5H2),
2.73 (tt, CcH), 3.09 (pd, CaHeq, CeHeq), 3.95 (t, 3J ¼ 6.3, C8H2), 4.06 (t,
3J ¼ 6.9, C1 ꢄ H2), 7.02 (t, 3J ¼ 7.2, C500H), 7.08 (t, C600H), 7.09 (d, C30H,
8.2.20. 4-(4-Methoxy-phenyl)-2-{4-[4-(2-methyl-1H-indol-3-yl)-
piperidin-1-yl]-butyl}-5,6,7,8-tetrahydro-pyrido[1,2-c]pyrimidine-
1,3-dione (8r)
3
The title compound was isolated as white powder (72%); mp
C50H), 7.18 (d, C20H, C60H), 7.24 (d, J ¼ 8.1, C700H), 7.74 (d, 3J ¼ 7.5,
180–182.3 ꢀC. 1H NMR (500 MHz, CDCl3)
d
: 1.58–1.78 (m, 8H, CbHax
,
C400H), 7.79 (bs, NH), 13C NMR (400 MHz, CDCl3)
d: 12.6 (CH3), 18.8
CdHax), 1.89 (q, 3J ¼ 6.6, C7H2), 2.02 (m, CaHeq, CeHeq), 2.21 (m,
CaHax, CeHax), 2.34 (s, CH3), 2.41 (t, 3J ¼ 7.8, C4 ꢄ H2), 2.52 (t, 3J ¼ 6.8,
C5H2), 2.71 (tt, 3Jax–ax ¼ 12.4, 3Jax–eq ¼ 3.7, CcH), 3.79 (s, OCH3), 3.92
(C6), 21.9 (C7), 24.8 (C2ꢄ), 26.0 (C3ꢄ), 27.0 (C5), 32.3 (Cb, Cd), 35.0
(Cc), 41.9 (C1ꢄ),040 2.9 (C8), 55.2 (Ca, Ce), 59.1 (C4ꢄ), 110.4 (C4), 111.6
2
(C70000), 115.7 (C3 ), 115.7 (d0*0, J ¼ 21.6, C30, C50), 118.9 (C500), 119.6
3
(t, 3J ¼ 6.3, C8H2), 4.06 (t, J ¼ 7.8, C4 ꢄ H2), 6.91 (dt, C30H, C50H),
(C4 ), 120.8 (C600), 127.8 (0C3 a), 129.4 (d*, 4J ¼ 3.9, C10), 130.1 (C700a),
132.7 (d*, 3J ¼ 8.3, C20, C6 ), 135.4 (C200), 150.1 (C4a), 151.9 (C1), 162.2
(C3) 162.4 (d*, 1J ¼ 246.6, C40). IR v: 1635 (C]O), 1692 (C]O), Anal.
3
6.98 (m, C500H), 7.03 (m, C600H), 7.08 (d, J ¼ 8.1, C700H), 7.11 (dt,
3J ¼ 8.8, 4J ¼ 1.9), 8.04 (bs, NH). 13C NMR (500 MHz, CDCl3)
d: 12.2

4714
F. Herold et al. / European Journal of Medicinal Chemistry 44 (2009) 4702–4715
Calcd. for C32H37FN4O2: C, 72.7; H, 7.0; N, 10.6. Found: C, 72.6; H,
6.8; N, 10.3.
light/dark cycle. Animals had free access to food and water before
the experiment. Each experimental group consisted of 7–8 animals/
dose, and all the animals were used only once.
8.3. Pharmacology
8.3.2.1.2. Drug
treatment. 8-Hydroxy-2-(di-n-propylamino)-
tetraline hydrobromide (8-OH-DPAT, Research Biochemical Inc.)
and N-{2-[4-(2-methoxy-phenyl)-1-piperazinyl]ethyl}-N-(2-pyr-
idinyl)cyclohexanecarboxamide trihydrochloride (WAY 100635,
synthesized by Dr. J. Boksa, Institute of Pharmacology, Polish
Academy of Sciences, Cracow, Poland) were used as aqueous solu-
tions and injected subcutaneously (sc). Compounds 8c, 8d, 8e, 8f,
8h, and 8i were suspended in a 1% aqueous solution of Tween 80
and given intraperitoneally (ip) at a dose of 10 mg/kg. The data
were analyzed by Dunnett’s test when only one drug was given or
by the Newman–Keuls test when two drugs were administered.
8.3.2.1.3. Effect of investigated compounds on body temperature
in mice. The effects of the tested compounds on the rectal body
temperature in mice when given alone were measured with an
Ellab thermometer 30, 60, 90, and 120 min after their administra-
tion. In an independent experiment, the effect of WAY 100635
(0.1 mg/kg) on the hypothermia induced by compounds 8c, 8e, 8f,
8h, and 8i or 8-OH-DPAT was tested. WAY 100635 was adminis-
tered 15 min before 8c, 8e, 8f, 8h, 8i, or 8-OH-DPAT and the rectal
body temperature recorded 30 min and 60 min after the injection
of the investigated compounds. In another experiment, the effect of
8d, which did not change the body temperature in mice, on the
hypothermia induced by 8-OH-DPAT (5 mg/kg) was tested.
Compound 8d was administered 45 min before 8-OH-DPAT, and the
rectal body temperature was measured 15, 30, 45, and 60 min after
the injection of 8-OH-DPAT. The results were expressed as a change
8.3.1. In vitro experiments
8.3.1.1. 5-HT1A binding assay. [3H]8-Hydroxy-2-(di-n-propylami-
no)tetraline ([3H]-8-OHꢁDPAT, spec. act. 106 Ci/mmol, NEN
Chemicals) was used for labeling 5-HT1A receptors. The membrane
preparation and assay procedure were carried out according to
a previously described procedure [48] with slight modifications. In
brief, the hippocampus tissue was homogenized in 20 volumes of
50 mM Tris–HCl buffer (pH 7.7 at 25 ꢀC) using Ultra-TurraxÒ T 25
and then centrifuged at 32 000g for 10 min. The supernatant frac-
tion was discarded, the pellet resuspended in the same volume of
Tris–HCl buffer, and the solution centrifuged again. Before the third
centrifugation, the samples were incubated at 37 ꢀC for 10 min. The
final pellet was resuspended in Tris–HCl buffer containing 10
pargyline, 4 mM CaCl2, and 0.1% ascorbic acid. Samples containing
1 ml of the tissue suspension (5 mg wet weight), 100 l of 10
serotonin for non-specific binding, 100
l of [3H]8-OH-DPAT, and
100
l of analyzed compound were incubated at 37 ꢀC for 15 min.
mM
m
mM
m
m
The incubation was followed by rapid vacuum filtration through
Whatman GF/B glass filters, and the remainder was washed three
times with 5 ml cold buffer (50 mM Tris–HCl, pH 7.7) using
a Brandel cell harvester. The final [3H]8-OH-DPAT concentration
was 1 nM, and the concentrations of the analyzed compounds
ranged from 10ꢁ10 to 10ꢁ4 M.
8.3.1.2. SERT binding assay. The assay was performed according to
the method of Owens et al. [49] with slight modifications. Rat
cerebral cortex was homogenized in 30 volumes of ice-cold 50 mM
Tris–HCl buffer containing 150 mM NaCl and 5 mM KCl (pH 7.7,
25 ꢀC) and centrifuged at 20 000g for 20 min. The supernatant was
decanted and the pellet resuspended in 30 volumes of buffer and
centrifuged again. The resulting pellet was resuspended in the
same volume of buffer and centrifuged a third time in the same way
as above. [3H]-citalopram (spec. act. 50 Ci/mmol, NEN Chemicals)
was used for labeling the 5-HT-transporter. Samples consisting of
in body temperature (
Dt) compared to basal body temperature
measured at the beginning of the experiment.
8.3.2.2. Forced swimming test in mice
8.3.2.2.1. Animals and housing. The experiments were per-
formed in male Albino Swiss mice (24–28 g; purchased from
licensed dealer Staniszewska, Ilkowice, Poland). The animals were
kept in groups of twenty per cage (dimensions: 60 ꢄ 38 ꢄ 20 cm) at
a temperature of 20 ꢂ 1 ꢀC and had free access to food (standard
laboratory pellets) and water before the experiments. All investi-
gations were conducted in the light phase of a natural light/dark
cycle (January to February) between 9 AM and 2 PM. Each experi-
mental group consisted of 9–10 animals per drug dose. Animals
were tested in a counter-balanced order and were used in only one
experiment. The experiments were conducted by an investigator
blinded to treatment. All of the experimental procedures were
approved by the Local Bioethics Commission at the Institute of
Pharmacology, Polish Academy of Sciences, Cracow.
8.3.2.2.2. Drug treatment. The following drugs were used:
8-OH-DPAT (Research Biochemical Inc.) and compounds 8c, 8f, 8h,
and 8i. All compounds were suspended in a 1% aqueous solution of
Tween 80. 8-OH-DPAT was injected subcutaneously (sc), and 8c, 8f,
8h, and 8i were given intraperitoneally (ip) 30 min before the test
at a dose of 10 ml/kg.
8.3.2.2.3. Effect of the investigated compounds on forced swim-
ming test results. The experiment was carried out according to the
method of Porsolt et al. [31]. In brief, the mice were placed indi-
vidually in a glass cylinder (25 cm high, 10 cm in diameter, filled
with water to a height of 6 cm) maintained at 23–25 ꢀC and were
left inside for 6 min. A mouse was regarded as immobile when it
remained floating on the water, making only small movements to
keep its head above. The total immobility time was measured by an
experimenter during the final 4 min of a 6-min test session after
a 2-min habituation period.
the tissue suspension (240
m
l), 30
m
l of 1
m
M imipramine (dis-
placer), 30
m
l of 1 nM [3H]-citalopram, and 100
m
l of the analyzed
compound were incubated at 22 ꢀC for 1 h. The concentrations of
the analyzed compounds ranged from 10ꢁ10 to 10ꢁ4 M. Incubations
were terminated by vacuum filtration through Whatman GF/B
filters and washed two times with 100 ml ice-cold buffer. Radioac-
tivity was measured in a WALLAC 1409 DSA liquid scintillation
counter. All assays were performed in duplicates. Radioligand
binding data were analyzed using iterative curve-fitting routines
(GraphPAD/Prism, Version 3.0 – San Diego, CA, USA). The Ki values
were calculated from the Cheng and Prusoff equation [30].
IC50
Ki ¼
LO
1 þ
KD
LO ¼ labeled ligand concentration.
KD ¼ dissociation constant of labeled ligand.
8.3.2. In vivo experiments
8.3.2.1. Induced hypothermia in mice
8.3.2.1.1. Animals and housing. The experiments were per-
formed in male Albino Swiss mice (24–28 g; purchased from
licensed dealer Staniszewska, Ilkowice, Poland). The animals were
housed under standard laboratory conditions in a room maintained
at a temperature of 20 ꢂ 1 ꢀC with a natural (January to February)
8.3.2.2.4. Locomotor activity in mice. The spontaneous loco-
motor activity of mice was recorded for compounds 8c, 8f, 8h, and

F. Herold et al. / European Journal of Medicinal Chemistry 44 (2009) 4702–4715
4715
[11] M.B. Tome, M.T. Isaac, R. Harte, C. Holland, Int. Clin. Psychopharmacol. 12
(1997) 81–89.
[12] R. Zanardi, F. Artigas, L. Franchini, J. Clin. Psychopharmacol. 17 (1997) 446–450.
[13] L.A. Dawson, D.L. Nguyen, D.L. Smith, L.E. Schechter, J. Psychopharmacol. 16
(2002) 145–152.
[14] F. Artigas, L. Romero, C. De Montigny, P. Blier, Trends Neurosci. 19 (1996)
378–383.
[15] F. Artigas, V. Perez, E. Alvarez, Arch. Gen. Psychiatry 51 (1994) 248–251.
[16] F. Artigas, P. Celada, M. Laruelle, A. Adell, Trends Pharmacol. Sci. 22 (2001)
224–228.
[17] D. Zhou, B.L. Harrison, U. Shah, T.H. Andree, G.A. Hornby, R. Scerni,
L.E. Schechter, D.L. Smith, K.M. Sullivan, R.E. Mewshaw, Bioorg. Med. Chem.
Lett. 16 (2006) 1338–1341.
[18] V.P. Rocco, P.G. Spinazze, T.J. Kohn, N.A. Honigschmidt, D.L. Nelson,
D.B. Wainscott, L.J. Ahmad, J. Shaw, P.G. Threlkeld, D.T. Wong, K. Takeuchi,
Bioorg. Med. Chem. Lett. 14 (2004) 2653–2656.
8i in photoresistor actometers (24 cm in diameter, illuminated by
two light beams) connected to a counter for the recording of light
beam interruptions. The mice were placed individually in the
actometers, and the number of light beam crossings was counted
twice: first during the first 6-min period, i.e. the time equal to the
observation period in the forced swimming test, and during
a 30-min experimental session.
8.3.2.2.5. Statistical analysis. The obtained data were presented
as the mean ꢂ S.E.M. The statistical significance of differences
between groups was evaluated by one-way analysis of variance
(ANOVA) followed by comparisons using Dunnett’s test. A p value
<0.05 was accepted as significant.
8.3.2.2.6. Molecular docking. The population of one hundred
5-HT1AR models described previously was reused in the present
work [32]. The models were constructed on the basis of a slightly
modified bovine rhodopsin template. All of the docking experiments
were conducted using the FlexX software with the FlexX-Pharm
extension. Pharmacophore constraints were applied: H-bond
[19] D. Spinks, G. Spinks, Curr. Med. Chem. 9 (2002) 799–810.
[20] T. Heinrich, H. Bo¨ttcher, R. Gericke, G.D. Bartoszyk, S. Anzali, C.A. Seyfried,
H.E. Greiner, J. Med. Chem. 47 (2004) 4684–4692.
[21] F. Herold, A. Chodkowski, q. Izbicki, M. Kro´l, J. Kleps, I. Wolska, G. Nowak, K.
Stachowicz, M. Dyba1a, A. Siwek, Eur. J. Med. Chem. 44 (2009) 1710–1717.
[22] J.F. Heiser, C.S. Wilcox, CNS Drugs 10 (1998) 343–353.
[23] F. Herold, M. Kro´l, J. Kleps, G. Nowak, Eur. J. Med. Chem. 41 (2006) 125–134.
[24] L. Matzen, C. van Amsterdam, W. Rautenberg, H.E. Greiner, J. Harting,
C.A. Seyfried, H. Bo¨ttcher, J. Med. Chem. 43 (2000) 1149–1157.
[25] J. Guillaume, C. Dumont, J. Laurent, L. Nedelec, Eur. J. Med. Chem. Chim. Ther.
22 (1987) 33–43.
acceptor at the carboxylic oxygen of Asp3.32, CH-p edge-to-face
interaction with Phe6.52, and H-bond donor at the hydroxylic group
of Ser5.43. The results were rescored with four additional scoring
functions and subjected to a consensus scoring procedure as
implemented in SYBYL. The highest PMF-scored solutions out of
those having a consensus score of 5 were considered representative.
The PMF score was used for the final scoring of the docking solutions
because it was proven to give the best enrichment factors in virtual
screening experiments [50].
The ligands were docked to the SERT model using a flexible
Monte Carlo docking procedure as implemented in the ICM
program [35]. The functionally important residue for ligand
anchoring was Asp981.45 in TMH1. The ligand molecules were fully
flexible and the protein was represented by grid interaction
potentials. The geometrically different and low-energy conforma-
tions of the ligand in the complex ligand-serotonin transporter
accumulated in the conformational stack. For each ligand, the best
pose was selected using a visual inspection of the interactions with
amino acid residues inside the binding site and the scoring func-
tions implemented in the Xscore program [51].
[26] J.E. Macor, C.A. Burkhart, J.H. Heym, J.L. Ives, L.A. Lebel, M.E. Newman,
J.A. Nielsen, K. Ryan, D.W. Schulz, L. Torgersen, K. Koe, J. Med. Chem. 33 (1990)
2087–2093.
´
[27] F. Herold, I. Wolska, E. Helbin, M. Krol, J. Kleps, J. Heterocycl. Chem. 26 (1999)
389–396.
[28] F. Herold, J. Kleps, R. Anulewicz-Ostrowska, B. Szcze˛sna, J. Heterocycl. Chem.
39 (2002) 773–782.
[29] C.F. Macrae, P.R. Edgington, P. McCabe, E. Pidcock, G.P. Shields, R. Taylor,
M. Towler, J. van de Streek, J. Appl. Crystallogr. 39 (2006) 453–457.
[30] Y.C. Cheng, W.H. Prusoff, Biochem. Pharmacol. 22 (1973) 3099–3108.
[31] R.D. Porsolt, A. Berlin, M. Jalfre, Arch. Int. Pharmacodyn. Ther. 229 (1977)
327–336.
[32] M. Nowak, M. Ko1aczkowski, M. Paw1owski, A.J. Bojarski, J. Med. Chem. 49
(2006) 205–214.
[33] R. Abagyan, M. Totrov, D.N. Kuznetsov, J. Comput. Chem. 15 (1994) 488–506.
[34] A. Yamashita, S.K. Singh, T. Kawate, Y. Jin, E. Gouaux, Nature 437 (2005)
215–223.
´
´
[35] M. Jaronczyk, Z. Chilmonczyk, A.P. Mazurek, G. Nowak, A.W. Ravna,
K. Kristiansen, I. Sylte, Bioorg. Med. Chem. 16 (2008) 9283–9294.
[36] E.L. Barker, K.R. Moore, F. Rakhshan, R.D. Blakely, J. Neurosci. 19 (1999)
4705–4717.
[37] G.M. Goodwin, R.J. De Souza, A.R. Green, Neuropsychopharmacology 24 (1985)
1187–1194.
[38] K.F. Martin, D.L. Heal, in: J.R. Fozar, P.R. Saxena (Eds.), Serotonin: Molecular
Biology, Receptors and Functional Effects, Birkhauser Verlag, Basel, 1991, pp.
483–490.
Acknowledgements
[39] E.A. Forster, I.A. Cliffe, D.J. Bill, G.M. Dover, D. Jones, Y. Reilly, A. Flether, Eur. J.
Pharmacol. 281 (1995) 81–88.
[40] Y. Zhang, J. Skolnick, Proc. Natl. Acad. Sci. U.S.A. 101 (2004) 7594–7599.
[41] E. Beuming, L. Shi, A.J. Javitch, H. Weinstein, Mol. Pharmacol. 70 (2006)
1630–1642.
This research was financially supported by KBN Grant No. N
N405 064934, Statutory Activity the Institute of Pharmacology PAS
and Collegium Medicum Jagiellonian University.
[42] H.A. Neubauer, C.G. Hansen, O. Wiborg, Mol. Pharmacol. 69 (2006) 1242–1250.
[43] Oxford Diffraction Poland, CrysAlisCCD, CCD data collection GUI, version 1.171.
23, 2003.
References
[44] Oxford Diffraction Poland, CrysAlisRED, CCD data reduction GUI, version
1.171.27, 2005.
[45] G.M. Sheldrick, Acta Crystallogr. A46 (1990) 467–473.
[46] G.M. Sheldrick, SHELXL97: Program for the Refinement of Crystal Structures,
University of Go¨ttingen, Germany, 1997.
[47] K. Freter, V. Fuchs, E. Barsumian, J.T. Oliver, Arzneim.-Forsch./Drug Res. 35
(1985) 272–276.
[48] D.N. Middlemiss, J.R. Fozard, Eur. J. Pharmacol. 90 (1983) 150–153.
[49] M.J. Owens, W.N. Morgan, S.J. Plott, C.B. Nemeroff, J. Pharmacol. Exp. Ther. 283
(1997) 1305–1322.
[1] J.P. Feighner, J. Clin. Psychiatry 60 (Suppl. 4) (1999) 4–11.
[2] K. Briner, R.C. Dodel, Curr. Pharm. Des. 4 (1998) 291–302.
[3] L.A. Labbate, J.B. Grimes, G.W. Arana, Biol. Psychiatry 43 (1998) 904–907.
[4] R. Invernizzi, S. Belli, R. Samanin, Brain Res. 584 (1992) 322–324.
[5] I. Hervas, F. Artigas, Eur. J. Pharmacol. 358 (1998) 9–18.
[6] P. Blier, C. De Montigny, Y. Chaput, J. Clin. Psychiatry 51 (1990) 14–20.
[7] F. Artigas, Trends Pharm. 14 (1993) 262.
[8] S.E. Gartside, V. Umbers, M. Hajos, T. Sharp, Br. J. Pharmacol. 115 (1995)
1064–1070.
[50] A. Evers, T. Klabunde, J. Med. Chem. 48 (2005) 1088–1097.
[51] R. Wang, L. Lai, S. Wang, J. Comput.-Aided Mol. Des. 16 (2002) 11 [X-Score].
[9] N. Bel, F. Artigas, Synapse 15 (1993) 243–245.
[10] D.S. Kreiss, I. Lucki, J. Pharmacol. Exp. Ther. 269 (1994) 1268–1279.