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

Article abstract of DOI:10.1016/j.ejmech.2008.09.021

A series of new derivatives of 4-aryl-pyrido[1,2-c]pyrimidine containing the 3-(4-piperidyl)-1H-indole residue or its 5-methoxy derivative were synthesized. They were characterized (i) in vitro by binding to 5-HT_1A receptors and 5-HT transporter proteins in rat brain cortex membranes and (ii) in vivo in the mouse by induced hypothermia and forced swimming models for antagonist/agonist activity against the 5-HT_1A autoreceptors and postsynaptic 5-HT_1A receptors, respectively. Structure activity relationship evaluation indicated that the presence of the 3-(4-piperidyl)-1H-indole residue and ortho- or para-substituents with -F or -CH₃ groups in the aryl ring as well as an unsubstituted aryl in the 4-aryl-pyrido[1,2-c]pyrimidine moiety promoted low K_i values for both receptors. In contrast, the presence of a 5-methoxy-3-(4-piperidyl)-1H-indole residue as well as -Cl or -OCH₃ substituents at the para position markedly reduced the receptor affinity.

Full text of DOI:10.1016/j.ejmech.2008.09.021

European Journal of Medicinal Chemistry 44 (2009) 1710–1717
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 1
,
a
a
a
a
Franciszek Herold a , Andrzej Chodkowski , qukasz Izbicki , Marek Krol , Jerzy Kleps ,
*
´
Jadwiga Tur1o a, Gabriel Nowak b c, Katarzyna Stachowicz b, Ma1gorzata Dyba1a c, Agata Siwek c
,
a Department of Drug Technology, Faculty of Pharmacy, Medical University of Warsaw, ul. Banacha 1, 02-097 Warszawa, Poland
b Institute of Pharmacology, Polish Academy of Sciences, ul. Sme˛tna 12, 31-343 Krako´w, Poland
c Department of Cytobiology and Histochemistry, Collegium Medicum of Jagiellonian University, ul. Medyczna 9, 30-688 Krako´w, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 27 April 2008
Received in revised form 10 September 2008
Accepted 15 September 2008
Available online 30 September 2008
A series of new derivatives of 4-aryl-pyrido[1,2-c]pyrimidine containing the 3-(4-piperidyl)-1H-indole
residue or its 5-methoxy derivative were synthesized. They were characterized (i) in vitro by binding to
5-HT1A receptors and 5-HT transporter proteins in rat brain cortex membranes and (ii) in vivo in the
mouse by induced hypothermia and forced swimming models for antagonist/agonist activity against the
5-HT1A autoreceptors and postsynaptic 5-HT1A receptors, respectively. Structure activity relationship
evaluation indicated that the presence of the 3-(4-piperidyl)-1H-indole residue and ortho- or para-
substituents with –F or –CH3 groups in the aryl ring as well as an unsubstituted aryl in the 4-aryl-pyr-
ido[1,2-c]pyrimidine moiety promoted low Ki values for both receptors. In contrast, the presence of a 5-
methoxy-3-(4-piperidyl)-1H-indole residue as well as –Cl or –OCH3 substituents at the para position
markedly reduced the receptor affinity.
Keywords:
Antidepressants
Depression
5-HT1A/5-HT-T dual activity
Pyrido[1,2-c]pyrimidines
Ó 2008 Elsevier Masson SAS. All rights reserved.
1. Introduction
recurrent inhibition (negative feedback). After desensitization of
autoreceptors (2–6 weeks after administration), release of 5-HT
Approximately 20% of the adult population suffers from
depression, a widespread affective psychiatric disease. According to
the monoamine theory, the pathomechanism of this disease is
connected with disturbances in serotoninergic transmission.
Correction of these disturbances has led to the design of new
compounds with antidepressant activity [1].
First generation drugs, tricyclic antidepressants (TCA) and
nonselective inhibitors of monoaminooxidase (MAO-I), exerted
several adverse drug reactions (ADR) because of low receptor
selectivity. Second generation antidepressants, including mainly
selective serotonin reuptake inhibitors (SSRI), produced markedly
less ADR than TCA or nonselective MAO-I. However, newer anti-
depressants exhibit relatively long onset of action (2–6 weeks) as
well as lower clinical effectiveness (on average only 70% of patients
respond positively to these drugs) [2,3].
from presynaptic terminals normalizes [4,5].
Artigas et al. found that latency can be shortened after coad-
ministration of SSRI with antagonist of 5-HT1A autoreceptor, e.g.
fluoxetine, paroxetine or fluvoxamine and pindolol, citalopram,
fluoxetine or fluvoxamine and WAY 100635 [6–8]. Results of these
investigations led to attempts to synthesize a molecule consisting
of two parts: one inhibiting 5-HT reuptake, and the second blocking
presynaptic 5-HT1A receptor. This innovation may result in
obtaining new antidepressant drugs with a double mechanism of
action, characterized by better clinical parameters than drugs
previously used.
Several compounds with double pharmacological properties
have been described [9–11]. Introduction of a moiety with 5-HT1A
antagonistic properties into the basic structure of the SSRI seemed
justified, although a component with unselective antagonistic
action on both pre- and postsynaptic receptors may abolish the
advantageous effects of presynaptic inhibition [12].
Long latency of antidepressive drugs is related to the desensi-
tization of somatodendritic serotoninergic autoreceptors. An initial
increase in serotonin concentration, after administration of the
drug, leads to attenuation of 5-HT neurotransmission due to
Another possibility is the synthesis of compounds with dual
activity consisting of a component with agonistic action toward the
5-HT1A receptor and a residue inhibiting the reuptake of 5-HT.
Presynaptic agonistic effects led to faster desensitization of the
autoreceptor which, in turn, shortened the latency period, whereas
postsynaptic agonistic activity may increase neurotransmission in
* Corresponding author.
E-mail address: franciszek.herold@wum.edu.pl (F. Herold).
0223-5234/$ – see front matter Ó 2008 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2008.09.021

F. Herold et al. / European Journal of Medicinal Chemistry 44 (2009) 1710–1717
1711
serotoninergic systems [13]. Several compounds possessing such
properties have been described (vilazodone, VN2222) (Fig. 1)
[12,14,15]. Promising results were obtained with vilazodone which
recently entered phase III of clinical trials.
USA), which in turn were used to calculate inhibition constant Ki
according to the Cheng–Prusoff formula (see Table 1) [22].
The final compounds were further evaluated in mice for their
agonist/antagonist properties toward presynaptic and postsynaptic
5-HT1A receptors, using the inducible hypothermia and forced
swimming tests, respectively.
In the inducible hypothermia test, the effects of administration
of 6 compounds (7a–12a) were recorded as well as the effect of
WAY 100635 (5-HT1A receptor antagonist) on the hypothermia
induced by the tested compounds. The results were expressed as
The aim of this study was to synthesize and evaluate the bio-
logical properties of new compounds with double activity: agonism
to 5-HT1A receptor and inhibition of 5-HT reuptake. New ligands
were obtained after modification of the design of the previously
acquired pyrido[1,2-c]pyrimidine derivatives belonging to the long
chain piperazine (LCP) group with high affinity to 5-HT1A receptors
[16]. Modifications were performed in the pharmacophore part of
the molecule by replacing the arylpiperazine residue with 3-(4-
piperidyl)-1H-indole group or its 5-methoxy derivative, resulting in
the formation of agents inhibiting the serotonin transporter protein
(5-HT-T) and possessing high affinity to 5-HT1A receptors [17–19].
Biological investigations of the newly synthesized compounds
(7a,b–12a,b) included determination of the binding affinity to 5-
HT1A receptor as well as to serotonin transporter (5-HT-T).
Compounds 7a–12a were also investigated in vivo using a hypo-
thermic test in mice, while compounds 8a, 10a, 11a and 12a were
investigated by means of a forced swimming test in mice.
a change in body temperature (Dt) with respect to 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. [23]. The effect of selected compounds on
spontaneous locomotor activity in mice was also recorded. The
obtained data were presented as the mean ꢀ SEM. Comparisons
between groups were carried out by a one-way analysis of variance
(ANOVA) followed by intergroup comparisons using the Dunnett’s
or Newman–Keuls test.
5. Results and discussion
Several new derivatives of 4-aryl-pyrido[1,2-c]pyrimidine con-
taining the 3-(4-piperidyl)-1H-indole residue or its 5-methoxy
counterpart were obtained. In the 4-aryl-pyrido[1,2-c]pyrimidine
system, aryl substituent in compounds 12a and 12b consisted of
a phenyl ring, whereas compounds 11a and 11b also possessed –F at
ortho position and the remaining compounds had –OCH3 (7a, 7b),
–CH3 (8a, 8b), –Cl (9a, 9b) or –F (10a, 10b) located at the para
position.
Binding values of compounds 7a,b–12a,b toward the 5-HT1A
receptor as well as to the 5-HT transporter were analyzed by
investigating the influence of substituents bound to the 3-(4-
piperidyl)-1H-indole moiety (–H or –OCH3 substituents at position
5) as well as substitutions in the aryl ring of the pyrido[1,2-
c]pyrimidine at the ortho and para positions (Table 1).
2. Chemistry
The respective nitriles (2a–2f) were synthesized by the reaction
of C-arylation of appropriate arylacetonitriles (1a–1f) with 2-bro-
mopyridine in an aprotic polar solvent [20,21] (Fig. 2). Next, the
nitriles (2a–2f) were hydrolyzed using a mixture of sulfuric acid
and acetic acid to obtain the amides (3a–3f) in good yields.
Compounds 4a–4f were formed by the intermolecular cyclization
of 3a–3f with diethyl carbonate. The imide group of compounds
4a–4f was then N-alkylated by 1,4-dibromobutane to yield
bromobutyl derivatives 5a–5f. The final new target compounds
(7a,b–12a,b) were obtained by the condensation of bromobutyl
derivatives 5a–5f with the appropriate piperidyl-indole (6a, 6b)
synthesized according to the method previously described [19].
Compounds 11a, 8a, 10a, and 12a exhibited high affinity toward
the 5-HT1A receptors, with Ki values varying from 4.8 to 10.9 nM.
The remaining compounds possessed high to moderate binding
activity with Ki ranging from 16.7 to 78.1 nM (compounds 11b, 10b,
9a, 9b, 7a, 12b, 8b and 7b according to the increasing Ki values)
(Table 1).
An analysis of the effect of substituents at the indole moiety on
Ki values revealed that compounds with the 3-(4-piperidyl)-1H-
indole group had higher affinity for the 5-HT1A receptor than the
3. Structural investigation
All new compounds (7a,b–12a,b) were characterized by physical
constants, elemental analysis, IR, 1H, and 13C NMR spectroscopy
(see Section 7). The structures of these compounds were elucidated
from their analytical and spectroscopic data. The NMR spectra were
mutually correlated and were in agreement with the literature data
for similar systems [16,20], as well as with the theoretical spectra
calculated according to ACD/NMR Predictor v. 8.09 program.
analogous
5-methoxy-3-(4-piperidyl)-1H-indole
derivatives.
Substitution on the aryl ring in 4-aryl-pyrido[1,2-c]pyrimidine with
fluorine at both the para (10a, 10b) and ortho positions (11a, 11b) as
well as –CH3 at the para position (8a, 8b) or a lack of substitution
(12a, 12b) resulted in markedly higher binding values than with the
remaining ligands, i.e. derivatives with –Cl (9a, 9b) and –OCH3 (7a,
7b) at the para position.
Compounds 8a and 11a showed the best binding affinity for
both the serotonin protein transporter (Ki values less than 1.0 nM,
Table 1) and for the 5-HT1A receptor (Ki values w 5 nM). Compound
4. Pharmacology
Target compounds 7a,b–12a,b were assessed for in vitro affinity
for 5-HT1A and 5-HT-T receptors by radioligand binding assay, using
[3H]8-OH-DPAT and [3H]citalopram, respectively, in rat brain
tissues. Data were analyzed using iterative curve-fitting routines to
obtain IC50 values (GraphPAD/Prism, Version 3.0 – San Diego, CA,
Fig. 1. Compounds with double action (SSRI plus 5-HT1A agonist).

1712
F. Herold et al. / European Journal of Medicinal Chemistry 44 (2009) 1710–1717
Fig. 2. Synthetic scheme for novel compounds 7a–12a and 7b–12b. Reagents: (i) 2-bromopyridine, KOH, DMSO,
(iv) 1,4-dibromobutane, K2CO3, acetone, ; (v) 6a, 6b, acetonitrile, K2CO3, KI, . Numbering system of 7–12 for NMR analysis. R1, R2, R3: 7a H, OCH3, H; 8a H, CH3, H; 9a H, Cl, H; 10a
H, F, H; 11a F, H, H; 12a H, H, H; 7b H, OCH3, OCH3; 8b H, CH3, OCH3; 9b H, Cl, OCH3; 10b H, F, OCH3; 11b F, H, OCH3; 12b H, H, OCH3.
D; (ii) H2SO4, CH3COOH, D; (iii) diethyl carbonate, EtONa, EtOH, D;
D
D
10a also showed high levels of binding level whereas the binding
affinity of the remaining compounds to 5-HT transporter was
moderate to low. For the remaining compounds 12a, 10b, 11b, 12b,
7b, 7a, 9b and 9a, values of Ki lay between 54 and 280 nM (Table 1).
Evaluation of substitution in the indole moiety indicated that
some non-substituted 3-(4-piperidyl)-1H-indole derivatives
possessed significantly lower Ki values toward the serotonin
transporter protein than toward the 5-methoxy-3-(4-piperidyl)-
1H-indole analogues (compounds: 8a vs. 8b, 10a vs. 10b, 11a vs. 11b,
12a vs. 12b), while other compounds showed somewhat higher Ki
values (i.e. lower affinity) than the 5-methoxy analogues
(compounds 7a vs. 7b, 9a vs. 9b). A comparison of the substituents
on the aryl ring of the pyrido[1,2-c]pyrimidine moiety indicated
that compounds with the highest affinity for the 5-HT transporter
had a fluorine atom located at either the ortho (11a, 11b) or para
(10a, 10b) positions, –OCH3 or –CH3 substituent at para (7a, 7b, 8a)
position, or carried no substituents on the phenyl ring (12a, 12b).
Since radioligand binding technique does not distinguish
agonist/antagonist properties, we employed behavioral tests to
resolve this issue. Based on these results obtained in vitro,
compounds 7a–12a were selected for further in vivo experiments
because they had the most promising binding affinity for both the
5-HT1A receptor and 5-HT transporter.
completely blocked by WAY 100635 (0.1 mg/kg) (Table 3). There-
fore, the decrease in mouse body temperature, produced by 8a–
12a, can be regarded as a measure of their presynaptic 5-HT1A
receptor agonistic activity. However, since the hypothermia
induced by the tested compounds (especially by 9a, 10a and 12a)
was not completely antagonized by WAY 100635, receptors other
than presynaptic 5-HT1A receptors may participate in this effect.
Compound 7a (20 mg/kg) did not change the hypothermia induced
by 8-OH-DPAT (5 mg/kg) in mice; therefore, it seems that the
functional activity of 7a at those receptors was negligible in that
experimental paradigm.
Subsequently, to establish agonist/antagonistic action on the
postsynaptic 5-HT1A receptor, compounds 8a,10a,11a and 12a were
investigated in the forced swimming test in mice. Compounds 11a
and 12a at doses of 20 mg/kg (but not 10 mg/kg) reduced immo-
bility time by 40% and 29%, respectively, while compound 10a at
10 mg/kg reduced the immobility time by 34% in mice in the forced
swimming test (Fig. 3A–C). ANOVA revealed for the following
compounds: 12a: F(2,27) ¼ 14.57, p < 0.0001, 11a: F(2,27) ¼ 15.92,
p < 0.0001, 10a: F(2,26) ¼ 21.12, p < 0.0001. Compound 8a was
Table 1
5-HT1A and 5-HT-T binding affinities of compounds 7a–12a and 7b–12b.
Compounds 7a–12a were tested in in vivo models commonly
used for evaluating functional 5-HT1A receptor activity. Compound
8-OH-DPAT, a 5-HT1A receptor agonist, induces hypothermia in
mice; it is an effect mediated through 5-HT1A somatodendritic
receptor [24,25]. This hypothermia is abolished by 5-HT1A recep-
tor’s antagonist WAY 100635 [26]. Compounds 8a–12a, like 8-OH-
DPAT, induced hypothermia, whereas 7a, like WAY 100635, did not
change body temperature in mice (Table 2). The hypothermia
induced by compounds 8a (20 mg/kg), 9a (20 mg/kg), 10a (10 mg/
kg), 11a (20 mg/kg) and 12a (20 mg/kg) was attenuated by WAY
100635 (0.1 mg/kg) (Table 3). At the same time, the decrease in
body temperature induced by 8-OH-DPAT (5 mg/kg) was
Compound
R1
R2
R3
Ki for 5-HT1A (nM)
Ki for 5-HT-T (nM)
7a
8a
9a
10a
11a
12a
7b
8b
9b
H
H
H
H
F
H
H
H
H
H
F
OCH3
CH3
Cl
F
H
H
H
H
H
H
H
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
42.1 ꢀ 3.5
5.8 ꢀ 1.4
37.1 ꢀ 2.2
9.2 ꢀ 1.5
4.8 ꢀ 1.3
10.9 ꢀ 4.2
78 ꢀ 10
145.0 ꢀ 3.9
0.3 ꢀ 0.1
280.0 ꢀ 1.6
37.6 ꢀ 5.7
0.7 ꢀ 0.2
H
58.1 ꢀ 3.6
129.0 ꢀ 1.7
222.9 ꢀ 2.9
251 ꢀ 13
OCH3
CH3
Cl
F
H
59 ꢀ 20
41.1 ꢀ 1.6
34.6 ꢀ 2.1
16.7 ꢀ 4.9
52.3 ꢀ 2.9
10b
11b
12b
54.6 ꢀ 6.2
59.3 ꢀ 4.7
128.7 ꢀ 0.6
H
H

F. Herold et al. / European Journal of Medicinal Chemistry 44 (2009) 1710–1717
1713
Table 2
Table 3
The effect of the tested compounds on the body temperature in mice.
The effect of WAY 100635 on the hypothermia induced by the tested compounds in
mice.
Treatment
Dose (mg/kg)
D
t ꢀ SEM (ꢁC)
Compound and dose (mg/kg)
D
t ꢀ SEM (ꢁC)
30 min
60 min
90 min
120 min
30 min
60 min
Vehicle
7a
Ki
5
10
20
0.0 ꢀ 0.1
ꢂ0.3 ꢀ 0.1
ꢂ0.3 ꢀ 0.1
ꢂ0.3 ꢀ 0.1
0.0 ꢀ 0.1
ꢂ0.1 ꢀ 0.1
0.0 ꢀ 0.1
ꢂ0.6 ꢀ 0.1a ꢂ0.6 ꢀ 0.1a ꢂ0.4 ꢀ 0.1
Vehicle þ vehicle
0.0 ꢀ 0.1
ꢂ1.0 ꢀ 0.1b
ꢂ0.3 ꢀ 0.1d
0.0 ꢀ 0.1
ꢂ0.8 ꢀ 0.1b
ꢂ0.2 ꢀ 0.1d
ꢂ0.3 ꢀ 0.1
ꢂ0.2 ꢀ 0.1
ꢂ0.1 ꢀ 0.0 ꢂ0.2 ꢀ 0.0
Vehicle þ 8a (20)
ꢂ0.2 ꢀ 0.1
ꢂ0.1 ꢀ 0.1
WAY 100635 (0.1) þ 8a (20)
Vehicle
8a
–
0.0 ꢀ 0.1
0.0 ꢀ 0.1
ꢂ0.1 ꢀ 0.1
ꢂ0.1 ꢀ 0.1
ꢂ0.1 ꢀ 0.1
ꢂ0.1 ꢀ 0.1
ꢂ0.1 ꢀ 0.0
ꢂ0.1 ꢀ 0.1
Vehicle þ vehicle
ꢂ0.1 ꢀ 0.1
ꢂ1.2 ꢀ 0.1b
ꢂ0.4 ꢀ 0.1d
0.0 ꢀ 0.1
ꢂ1.0 ꢀ 0.1b
ꢂ0.2 ꢀ 0.1d
5
ꢂ0.4 ꢀ 0.1
ꢂ0.2 ꢀ 0.1
Vehicle þ 9a (20)
10
20
ꢂ0.5 ꢀ 0.1a ꢂ0.3 ꢀ 0.1
WAY 100635 (0.1) þ 9a (20)
ꢂ1.0 ꢀ 0.1c ꢂ0.8 ꢀ 0.1c ꢂ0.7 ꢀ 0.1b ꢂ0.8 ꢀ 0.1c
Vehicle þ vehicle
0.0 ꢀ 0.1
ꢂ1.0 ꢀ 0.1b
ꢂ0.8 ꢀ 0.1b,c
ꢂ0.1 ꢀ 0.0
ꢂ1.0 ꢀ 0.1b
ꢂ0.8 ꢀ 0.1b,c
Vehicle
9a
–
ꢂ0.1 ꢀ 0.1
0.0 ꢀ 0.1
ꢂ0.2 ꢀ 0.1
ꢂ0.4 ꢀ 0.1
ꢂ0.1 ꢀ 0.1
ꢂ0.1 ꢀ 0.0
ꢂ0.2 ꢀ 0.1
ꢂ0.1 ꢀ 0.0
Vehicle þ 10a (10)
5
ꢂ0.5 ꢀ 0.1a ꢂ0.4 ꢀ 0.1
WAY 100635 (0.1) þ 10a (10)
10
20
ꢂ0.4 ꢀ 0.1
ꢂ0.1 ꢀ 0.1
ꢂ1.2 ꢀ 0.1c ꢂ1.0 ꢀ 0.1c ꢂ1.1 ꢀ 0.1c ꢂ0.7 ꢀ 0.1b
Vehicle þ vehicle
ꢂ0.1 ꢀ 0.1
ꢂ1.1 ꢀ 0.1b
ꢂ0.3 ꢀ 0.1d
ꢂ0.1 ꢀ 0.1
ꢂ0.8 ꢀ 0.2a
ꢂ0.1 ꢀ 0.1c
Vehicle þ 11a (20)
Vehicle
10a
–
0.0 ꢀ 0.1
ꢂ0.1 ꢀ 0.0
ꢂ0.3 ꢀ 0.1
0.0 ꢀ 0.1
0.0 ꢀ 0.1
WAY 100635 (0.1) þ 11a (20)
5
ꢂ0.4 ꢀ 0.1
ꢂ0.4 ꢀ 0.1
ꢂ0.3 ꢀ 0.1
10
20
ꢂ1.2 ꢀ 0.2c ꢂ1.2 ꢀ 0.2c ꢂ1.0 ꢀ 0.2c ꢂ0.8 ꢀ 0.2c
ꢂ1.2 ꢀ 0.2c ꢂ1.0 ꢀ 0.1c ꢂ0.8 ꢀ 0.1c ꢂ0.6 ꢀ 0.1b
Vehicle þ vehicle
ꢂ0.1 ꢀ 0.1
ꢂ0.1 ꢀ 0.1
ꢂ1.3 ꢀ 0.2b
ꢂ0.3 ꢀ 0.1d
Vehicle þ 12a (20)
ꢂ1.2 ꢀ 0.2b
ꢂ0.5 ꢀ 0.1a,d
WAY 100635 (0.1) þ 12a (20)
Vehicle
11a
–
5
10
20
ꢂ0.1 ꢀ 0.1
ꢂ0.1 ꢀ 0.1
ꢂ0.1 ꢀ 0.1
ꢂ0.1 ꢀ 0.1
ꢂ0.3 ꢀ 0.1
ꢂ0.1 ꢀ 0.1
ꢂ0.1 ꢀ 0.0
ꢂ0.1 ꢀ 0.0
ꢂ0.5 ꢀ 0.1a ꢂ0.3 ꢀ 0.1
ꢂ0.8 ꢀ 0.1c ꢂ0.2 ꢀ 0.2
Vehicle þ vehicle
0.0 ꢀ 0.1
ꢂ1.5 ꢀ 0.1b
ꢂ0.1 ꢀ 0.1d
ꢂ0.1 ꢀ 0.1
ꢂ1.1 ꢀ 0.1b
ꢂ0.2 ꢀ 0.1d
Vehicle þ 8-OH-DPAT (5)
WAY 100635 (0.1) þ 8-OH-DPAT(5)
ꢂ1.1 ꢀ 0.1c ꢂ0.8 ꢀ 0.2b ꢂ0.7 ꢀ 0.1b ꢂ0.7 ꢀ 0.1b
WAY 100635 was administered (sc) 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 within a range
of 36.0 ꢀ 0.4 ꢁC.
Vehicle
12a
–
5
10
20
ꢂ0.1 ꢀ 0.1
ꢂ0.4 ꢀ 0.1
ꢂ0.1 ꢀ 0.1
ꢂ0.1 ꢀ 0.1
0.0 ꢀ 0.1
ꢂ0.1 ꢀ 0.0
ꢂ0.1 ꢀ 0.0
ꢂ0.5 ꢀ 0.2a ꢂ0.4 ꢀ 0.1
ꢂ0.8 ꢀ 0.1c ꢂ0.4 ꢀ 0.1
ꢂ0.3 ꢀ 0.1
ꢂ1.2 ꢀ 0.2c ꢂ1.3 ꢀ 0.2c ꢂ1.1 ꢀ 0.3c ꢂ1.9 ꢀ 0.8c
a
p < 0.01 vs. respective vehicle þ vehicle group.
b
p < 0.001 vs. respective vehicle þ vehicle group.
Vehicle
8-OH-DPAT
WAY 100635
–
0.0 ꢀ 0.1
0.1 ꢀ 0.1
0.1 ꢀ 0.1
0.0 ꢀ 0.1
c
p < 0.01 vs. respective vehicle þ compound group.
5
0.1
ꢂ1.5 ꢀ 0.1c ꢂ1.1 ꢀ 0.1c ꢂ0.7 ꢀ 0.1b ꢂ0.2 ꢀ 0.1
d
p < 0.001 vs. respective vehicle þ compound group.
0.1 ꢀ 0.1
0.2 ꢀ 0.1
0.1 ꢀ 0.1
0.2 ꢀ 0.2
The tested compounds were administered (ip) 30 min before the test. The absolute
mean body temperatures were within a range of 36 ꢀ 0.5 ꢁC; n ¼ 7–8 mice per
group.
The presence of the 3-(4-piperidyl)-1H-indole residue, as well
as a fluorine atom or methyl group at ortho/para positions of the
phenyl group (or unsubstituted phenyl) in the aryl ring of 4-aryl-
pyrido[1,2-c]pyrimidine system is of special advantage with regard
to binding affinity.
a
p < 0.05 vs. respective vehicle.
p < 0.01 vs. respective vehicle.
b
c
p < 0.001 vs. respective vehicle.
In the induced hypothermia model in mice compounds 8a–12a
showed agonistic properties to presynaptic 5-HT1A receptor.
Compound 10a decreased body temperature when administered at
10 mg/kg dose, whereas for the remaining compounds (8a, 9a, 11a
and 12a) doses of 20 mg/kg were needed.
Agonistic effects on postsynaptic 5-HT1A receptor of compounds
10a, 11a and 12a were also demonstrated in the forced swimming
test in mice. Compound 10a has an effect at 10 mg/kg dose, while
compounds 11a and 12a required higher doses of 20 mg/kg to be
effective.
ineffective at doses of 10 mg/kg and 20 mg/kg in that test (data not
shown). Compound 8-OH-DPAT, at doses of 1 mg/kg and 2 mg/kg,
reduced the immobility time by 45% and 57%, respectively (Fig. 3D;
ANOVA: F(2,26) ¼ 53.92, p < 0.0001).
Compounds 10a and 12a at a dose of 20 mg/kg did not change
the spontaneous locomotor activity during a 6-min (i.e. the time
equivalent to the observation period in the forced swimming test)
or a 30-min observation period in mice (Table 4). In contrast,
compound 11a reduced the spontaneous locomotor activity of mice
during both the 6-min and 30-min observation period in mice
(Table 4).
Compounds 8a, 10a and 11a are currently being tested for multi-
receptor binding profile (D1, D2, a1 and b1 receptors) as well as for
chronic toxicity. These results will be required for further neuro-
biological evaluation.
6. Conclusions
Several new derivatives of 4-aryl-pyrido[1,2-c]pyrimidine pos-
sessing either 3-(4-piperidyl)-1H-indole group or its 5-methoxy
derivative were synthesized (7a,b–12a,b). These compounds are
considered to possess double biological activity. In in vitro experi-
ments newly synthesized compounds possessed high to moderate
binding activity to the 5-HT1A receptor as well as to the 5-HT
transporter.
Based on Ki values in the low nM range, we determined that
compounds 8a and 11a possessed the highest binding affinity for
both the 5-HT1A receptor and the 5-HT transporter. The remaining
compounds also bind to both the 5-HT1A receptor and 5-HT
transporter with moderate to relatively low affinity.
7. Experimental protocols
7.1. Chemistry
7.1.1. General remarks
Melting points were determined on an Electrothermal 9100
apparatus with open capillary tubes and are uncorrected. Ele-
mentar analyses were performed on a Perkin–Elmer 2400 analyzer
and were within ꢀ0.4% of the theoretical values. Infrared spectra
were recorded on Shimadzu FTIR-8300 spectrometer. 1H and 13C
NMR spectra were obtained on Bruker AVANCE DMX 400WB
instrument in CDCl3 (chemical shifts are reported in
d units).

1714
F. Herold et al. / European Journal of Medicinal Chemistry 44 (2009) 1710–1717
Fig. 3. Effects of compounds 12a (A), 11a (B), 10a (C) and 8-OH-DPAT (D) on immobility time in the forced swimming test in Albino Swiss mice. Each bar represents the mean ꢀ SEM
of 9–10 mice. All compounds were injected 30 min before the test. **p < 0.001 vs. respective vehicle group (Dunnett’s test).
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), d (doublet), t (triplet), q
(quartet), qt (quintet), m (multiplet), p (pseudo-), b (broad-). For
the two-dimensional experiments, the pulse sequences, acquisi-
tion, 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 the solvent methylene chlo-
ride/methanol (99:1, 97:3, 95:5, v/v). Thin layer chromatography
was run on Merck Silica gel 60 F254 plates using mobile phase 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).
water. The crude product was extracted with CHCl3. Organic layers
were combined and dried with MgSO4. The mixture was then
filtered and the filtrate was evaporated to dryness. The crude
residue was purified with diethylether. The obtained yellow solid
was dissolved in 300 ml of methanol, hydrogenated for 8 h in 45 ꢁC
and 1 atm on 10% Pd/C as catalyst. The catalyst was then filtered off
and the filtrate was evaporated to dryness. The crude product was
purified by crystallization from ethyl acetate.
7.1.4. General procedure for the synthesis of 2-{4-[4-(1H-indol-3-
yl)-piperidin-1-yl]-butyl}-4-aryl-pyrido[1,2-c]pyrimidine-1,3-
diones (7a,b–12a,b)
Compounds 5a–5f (0.0026 mol), 6a, 6b (0.0026 mol), K2CO3
(0.005 mol), 70 ml of 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) mixture. Proper
fractions were identified by TLC and evaporated to dryness giving
analytically pure compounds 7a,b–12a,b.
7.1.2. Preparation of 2-(4-bromobutyl)-4-aryl-pyrido[1,2-
c]pyrimidine-1,3-diones (5a–5f)
The starting compounds 2a–2f, 3a–3f, 4a–4f and 5a–5f were
obtained according to procedures described in Refs. [20,21].
7.1.3. General procedure for the synthesis of 3-piperidin-4-yl-1H-
indoles (6a, 6b)
A mixture of 0.1 mol of appropriate indole, 0.2 mol of N-benzyl-
4-piperidone and 150 ml of 2 N KOH/MeOH was refluxed while
stirring under an atmosphere of nitrogen for 8 h. The mixture was
further stirred for 8 h at room temperature and poured onto ice/
7.1.4.1. 2-{4-[4-(1H-Indol-3-yl)-piperidin-1-yl]-butyl}-4-(4-methox-
yphenyl)-pyrido[1,2-c]pyrimidine-1,3-dione (7a). Yield: 90%; yellow
crystals, m.p. 235–236 ꢁC; 1H NMR (400 MHz)
d: 1.67 (m, C-3 x H2),
1.79 (m, 4H, C-2 x H2, Cb,dHax), 2.07 (m, 4H, Ca,eHax, Cb,dHeq), 2.44
(t, 3J ¼ 7.6 Hz, C-4 x H2), 2.82 (pt, CcH), 3.06 (pd, 2H, Ca,eeq), 3.84 (s,
OCH3), 4.19 (t, 3J ¼ 7.2 Hz, C-1 x H2), 6.36 (t, 3J ¼ 7.2 Hz, C-7H), 6.90
(m, C-5H, C-6H), 6.96 (bs, C-200H), 6.98 (d, C-30H, C-50H), 7.09 (t,
3J ¼ 7.6 Hz, C-500H), 7.17 (t, 3J ¼ 7.6 Hz, C-600H), 7.24 (d, 3J ¼ 8.4 Hz, C-
20H, C-60H), 7.34 (d, 3J ¼ 8.0 Hz, C-700H), 7.65 (d, 3J ¼ 7.6 Hz, C-400H),
8.0 (bs, NH), 8.34 (d, 3J ¼ 7.6 Hz, C-8H).
Table 4
Effect of compounds 10a, 11a and 12a on the locomotor activity in mice.
Treatment (mg/kg)
Locomotor activity
Number of crossings ꢀ SEM during
13C NMR (100 MHz)
d: 24.8 (C-3x), 25.9 (C-2x), 33.3 (Cb, Cd),
6 min
30 min
33.7 (Cc), 42.7 (C1x), 54.7 (Ca, Ce), 55.6 (OCH3), 59.0 (C-4x), 104.8
(C-4), 110.8 (C-7), 111.4 (C-700), 114.5 (C-30, C-50), 119.2 (C-500), 119.3
(C-400), 119.7 (C-300), 119.9 (C-200), 121.8 (C-5), 122.0 (C-600), 125.1 (C-
10),126.9 (C-300a),128.1 (C-8),132.3 (C-6),132.6 (C-20, C-60),136.5 (C-
700a), 143.7 (C-4a), 149.2 (C-1), 159.3 (C-40), 160.6 (C-3); IR v: 1635
(C]O), 1705 (C]O). Anal. Calcd for C32H34N4O3: C, 73.5; H, 6.6; N,
10.7. Found: C, 72.9; H, 6.3; N, 10.2.
Vehicle
10a (10)
11a (20)
12a (20)
343 ꢀ 41
889 ꢀ 111
376 ꢀ 46
965 ꢀ 125
179 ꢀ 50
399 ꢀ 131
409 ꢀ 23**
926 ꢀ 70**
F(3,36) ¼ 6.184; P < 0.001
F(3,36) ¼ 5.656; P < 0.001
All compounds were injected ip 30 min before the test.
** p < 0.01 vs. vehicle group (n ¼ 10).

F. Herold et al. / European Journal of Medicinal Chemistry 44 (2009) 1710–1717
1715
7.1.4.2. 2-{4-[4-(5-Methoxy-1H-indol-3-yl)-piperidin-1-yl]-butyl}-
4-(4-methoxyphenyl)-pyrido[1,2-c]pyrimidine-1,3-dione (7b). Yield:
13C NMR (100 MHz)
d
: 24.8 (C-3x), 25.9 (C-2x), 33.3 (Cb, Cd),
33.8 (Cc), 42.8 (C-1x), 54.7 (Ca, Ce), 59.0 (C-4x), 103.8 (C-4), 111.0 (C-
7), 111.4 (C-700), 119.2 (C-500), 119.9 (C-200), 121.3 (C-5), 121.8 (C-300),
122.0 (C-600), 126.9 (C-300a), 128.4 (C-8), 129.2 (C-20, C-60), 131.6 (C-
10), 132.9 (C-30, C-50), 133.1 (C-6), 133.9 (C-40), 136.6 (C-700a), 143.8
(C-4a), 149.1 (C-1), 160.2 (C-3); IR v: 1635 (C]O), 1713 (C]O). Anal.
Calcd for C31H31N4O2Cl: C, 70.6; H, 5.9; N, 10.6. Found: C, 70.0; H,
6.2; N, 10.6.
58.8%; yellow crystals, m.p. 162–164 ꢁC; 1H NMR (400 MHz)
d: 1.64
(q, C-3 x H2), 2.03 (m, C-2 x H2, Cb,dHax), 2.03 (pd, Cb,dHeq), 2.44 (t,
3J ¼ 7.6 Hz, C-4 x H2), 2.77 (tt, CcH), 3.83 (s, OCH3), 3.86 (s, OC-
10H3), 4.19 (t, 3J ¼ 7.4 Hz, C-1 x H2), 6.35 (t, 3J ¼ 7.2 Hz, C-7H), 6.83
(dd, 3J ¼ 8.8 Hz, 4J ¼ 2.0 Hz, C-600H), 6.90 (m, C-5H, C-6H), 6.92 (s, C-
4
200H), 7.05 (d, J ¼ 1.6 Hz, C-400H), 7.05 (d, C-30H, C-50H), 7.21 (d,
3J ¼ 8.4 Hz, C-700H), 7.26 (d, 3J ¼ 8.4 Hz, C-20H, C-60H), 8.04 (bs, NH),
8.31 (d, 3J ¼ 7.2 Hz, C-8H).
7.1.4.6. 4-(4-Chloro-phenyl)-2-{4-[4-(5-methoxy-1H-indol-3-yl)-
piperidin-1-yl]-butyl}-pyrido[1,2-c]pyrimidine-1,3-dione (9b). Yield:
13C NMR (100 MHz)
d: 24.8 (C-3x), 25.9 (C-2x), 33.2 (Cb, Cd), 33.7
(Cc), 42.7 (C-1x), 54.7 (Ca, Ce), 55.6 (OCH3), 56.3 (OC-10H3), 59.1 (C-
4x), 101.3 (C-400), 104.8 (C-4), 110.8 (C-7), 112.1 (C-700), 112.2 (C-600),
114.5 (C-30, C-50), 121.8 (C-5), 125.1 (C-10), 127.3 (C-300a), 128.1 (C-8),
131.8 (C-700a),132.6 (C-20, C-60),143.7 (C-4a),149.2 (C-500),153.9 (C-1),
159.4 (C-40), 160.6 (C-3); IR v: 1647 (C]O), 1720 (C]O). Anal. Calcd
for C33H36N4O4: C, 71.7; H, 6.6; N, 10.1. Found: C, 71.1; H, 6.4; N, 10.0.
53.8%; yellow crystals, m.p. 110–112 ꢁC; 1H NMR (400 MHz)
d: 1.64
(q, 3J ¼ 7.2 Hz, C-3 x H2), 1.77 (m, 4H, C-2 x H2, Cb,dHax), 2.03 (pd,
Cb,dHeq), 2.11 (pt, Ca,eHax), 2.44 (t, 3J ¼ 7.8 Hz, C-4 x H2), 2.77 (tt, 3JA–
3
¼ 12.0 Hz, JA–E ¼ 3.6 Hz, CcH), 3.04 (pd, Ca,eeq), 3.86 (s, OCH3),
A
4.19 (t, 3J ¼ 7.4 Hz, C-1 x H2), 6.40 (td, 3J ¼ 6.8 Hz, 4J ¼ 1.0 Hz, C-7H),
6.84 (dd, 3J ¼ 8.8 Hz, 4J ¼ 2.4 Hz, C-600H), 6.88 (d, 3J ¼ 9.6 Hz, C-5H),
6.94 (s, C-200H), 6.94 (td, 3J ¼ 7.8 Hz, 4J ¼ 0.8 Hz, C-6H), 7.06 (d,
3
7.1.4.3. 2-{4-[4-(1H-Indol-3-yl)-piperidin-1-yl]-butyl}-4-p-tolyl-
pyrido[1,2-c]pyrimidine-1,3-dione (8a). Yield: 50%; yellow crystals,
4J ¼ 2.4 Hz, C-400H), 7.23 (d, J ¼ 8.4 Hz, C-700H), 7.27 (d, C-30H, C-
3
50H), 7.42 (d, J ¼ 8.4 Hz, C-20H, C-60H), 7.94 (bs, NH), 8.35 (d,
m.p. 178–179 ꢁC; 1H NMR (400 MHz)
d
: 1.64 (q, 3J ¼ 7.6 Hz, C3 x H2),
3J ¼ 7.6 Hz, C-8H).
1.78 (m, 4H, C-2 x H2, Cb,dHax), 2.07 (m, 4H, Ca,eHax, Cb,dHeq), 2.38
(s, CH3), 2.43 (t, 3J ¼ 7.6 Hz, C-4 x H2), 2.82 (pt, CcH), 4.20 (t,
3J ¼ 7.4 Hz, C-1 x H2), 6.35 (td, 3J ¼ 6.0 Hz, 4J ¼ 0.8 Hz, C-7H), 6.88
(m, C-5H, C-6H), 6.93 (bs, C-200H), 7.08 (t, 3J ¼ 7.4 Hz, C-500H), 7.16 (t,
3J ¼ 7.6 Hz, C-600H), 7.20 (d, C-30H, C-50H), 7.25 (d, 3J ¼ 7.2 Hz, C-20H,
C-60H), 7.33 (d, 3J ¼ 8.0 Hz, C-700H), 7.63 (d, 3J ¼ 7.6 Hz, C-400H), 8.09
(bs, NH), 8.31 (d, 3J ¼ 7.2 Hz, C-8H).
13C NMR (100 MHz)
d: 24.5 (C-3x), 25.6 (C-2x), 32.9 (Cb, Cd),
33.5 (Cc), 42.5 (C-1x), 54.5 (Ca, Ce), 56.0 (OCH3), 58.8 (C-4x), 101.0
(C-400), 103.5 (C-4), 110.8 (C-7), 111.8 (C-700), 112.0 (C-600), 121.0 (C-5),
121.3 (C-300), 125.5 (C-300a), 128.1 (C-8), 129.0 (C-20, C-60), 131.2 (C-
10), 131.5 (C-700a), 132.7 (C-30, C-50), 132.9 (C-6), 133.7 (C-40), 143.6
(C-4a), 148.8 (C-500), 153.7 (C-1), 160.0 (C-3); IR v: 1624 (C]O), 1709
(C]O). Anal. Calcd for C32H33N4O3Cl: C, 69.0; H, 6.0; N, 10.1. Found:
C, 68.6; H, 6.1; N, 9.5.
13C NMR (100 MHz)
d: 21.5 (CH3), 24.8 (C-3x), 25.9 (C-2x), 33.3
(Cb, Cd), 33.8 (Cc), 42.7 (C-1x), 54.7 (Ca, Ce), 59.1 (C-4x), 105.2 (C-4),
110.8 (C-7), 111.4 (C-700), 119.2 (C-500), 119.3 (C-400), 119.7 (C-300), 119.9
(C-200), 121.8 (C-5), 122.0 (C-600), 126.9 (C-300a), 128.1 (C-8), 129.7 (C-
20, C-60), 129.9 (C-10), 131.3 (C-30, C-50), 132.3 (C-6), 136.6 (C-700a),
137.8 (C-40), 143.6 (C-4a), 149.2 (C-1), 160.5 (C-3); IR v: 1635 (C]O),
1709 (C]O). Anal. Calcd for C32H34N4O2: C, 75.8; H, 6.7; N, 11.0.
Found: C, 75.3; H, 6.7; N, 11.1.
7.1.4.7. 4-(4-Fluoro-phenyl)-2-{4-[4-(1H-indol-3-yl)-piperidin-1-yl]-
butyl}-pyrido[1,2-c]pyrimidine-1,3-dione (10a). Yield: 56.5%; yellow
crystals, m.p. 182–183 ꢁC; 1H NMR (400 MHz)
d: 1.64 (q, C-3 x H2),
1.79 (m, 4H, C-2 x H2, Cb,dHax), 2.02 (pd, Cb,dHeq), 2.10 (pt, Ca,eHax),
2.44 (t, 3J ¼ 7.6 Hz, C-4 x H2), 2.83 (tt, CcH), 3.05 (pd, Ca,eHeq), 4.19 (t,
3J ¼ 7.4 Hz, C-1 x H2), 6.39 (t, 3J ¼ 6.8 Hz, 4J ¼ 0.8 Hz, C-7H), 6.86 (d,
3
3J ¼ 9.2 Hz, C-5H), 6.92 (t, J ¼ 6.0 Hz, C-6H), 6.95 (s, C-200H), 7.05–
7.1.4.4. 2-{4-[4-(5-Methoxy-1H-indol-3-yl)-piperidin-1-yl]-butyl}-4-
p-tolyl-pyrido[1,2-c]pyrimidine-1,3-dione (8b). Yield: 78%; yellow
7.23 (m, C-500H, C-600H), 7.27–7.33 (m, C-20H, C-60H), 7.34 (d,
3J ¼ 8.4 Hz, C-700H), 7.64 (d, 3J ¼ 7.6 Hz, C-400H), 8.02 (bs, NH), 8.34 (d,
3J ¼ 7.6 Hz, C-8H).
crystals, m.p. 110–112 ꢁC; 1H NMR (400 MHz)
d: 1.67 (q, C3 x H2),
1.79 (m, C-2 x H2, Cb,dHax), 2.03 (pd, Cb,dHeq), 2.15 (pt, Ca,eHax),
2.39 (s, CH3), 2.46 (t, 3J ¼ 7.8 Hz, C-4 x H2), 2.78 (pt, CcH), 3.06 (pd,
Ca,eeq), 3.87 (s, OCH3), 4.19 (t, 3J ¼ 7.2 Hz, C-1 x H2), 6.39 (m, C-7H),
6.83 (dd, 3J ¼ 8.8 Hz, 4J ¼ 2.4 Hz, C-600H), 6.91 (bps, C-50H, C-6H), 7.07
13C NMR (100 MHz)
d: 24.8 (C-3x), 25.9 (C-2x), 33.3 (Cb, Cd),
33.7 (Cc), 42.8 (C-1x), 54.7 (Ca, Ce), 59.0 (C-4x), 104.0 (C-4), 110.9
2
(C-7), 111.3 (C-700), 116.0 (d^, J ¼ 21.4 Hz, C-30, C-50), 119.2 (C-500),
119.3 (C-400), 119.7 (C-300), 119.8 (C-200), 121.4 (C-5), 122.1 (C-600),
4
3
4
(t, J ¼ 2.0 Hz, C-400H), 7.20 (d, J ¼ 8.0 Hz, C-30H, C-5 H), 7.22–7.31
126.9 (C-300a), 128.3 (C-8), 128.9 (d^, J ¼ 3.1 Hz, C-10), 132.9 (C-6),
3
3
(m, C-20H, C-60H, C-700H), 7.33 (d, J ¼ 8.0 Hz, C-700H), 8.32 (d,
133.2 (d^, J ¼ 8.0 Hz, C-20, C-60), 136.5 (C-700a), 143.9 (C-4a), 149.1
3J ¼ 7.2 Hz, C-8H), 8.40 (bs, NH).
(C-1), 160.4 (C-3), 162.5 (d^, 1J ¼ 246.9 Hz, C-40); IR v: 1643 (C]O),
1720 (C]O). Anal. Calcd for C31H31N4O2F: C, 72.8; H, 6.1; N, 11.0.
Found: C, 72.3; H, 6.1; N, 10.8.
13C NMR (100 MHz)
d: 21.4 (CH3), 24.3 (C-3x), 25.8 (C-2x), 32.8
(Cb, Cd), 33.6 (Cc), 42.6 (C-1x), 54.5 (Ca, Ce), 56.2 (OCH3), 58.9 (C-
4x),101.2 (C-400), 105.1 (C-4), 110.9 (C-7), 112.0 (C-600, C-700), 120.8 (C-
200), 120.9 (C-300), 121.8 (C-5), 127.0 (C-300a), 128.0 (C-8), 129.6 (C-20,
C-600), 129.7 (C-10), 131.2 (C-700a), 132.5 (C-6), 137.8 (C-40), 143.8 (C-
4a), 149.1 (C-500), 153.7 (C-1), 160.6 (C-3); IR v: 1620 (C]O), 1709
(C]O). Anal. Calcd for C33H36N4O3: C, 73.9; H, 6.8; N,10.4. Found: C,
73.3; H, 6.8; N, 10.3.
7.1.4.8. 4-(4-Fluoro-phenyl)-2-{4-[4-(5-methoxy-1H-indol-3-yl)-
piperidin-1-yl]-butyl}-pyrido[1,2-c]pyrimidine-1,3-dione (10b).
Yield: 78.7%; yellow crystals, m.p. 151–152 ꢁC; 1H NMR
(400 MHz)
d: 1.82 (ps, 4H, C-3 x H2, Cb,Hax, CdHax), 2.08 (ps,
C2 x H2, CbHeq, CdHeq), 2.41 (bps, C-4 x H2), 2.72 (ps, CaHax
,
CeHax), 2.86 (ps, CcH), 3.26 (ps, Ca,Heq, CeHeq), 3.88 (s, OCH3),
4.20 (t, C-1 x H2), 6.41 (t, 3J ¼ 6.4 Hz, C-7H), 6.86 (m, C-5H, C-
600H), 6.94 (m, C-6H), 6.97 (s, C-200H), 7.06 (d, 4J ¼ 1.6 Hz, C-400H),
7,14 (t, C-30H, C-50H), 7.27 (m, C-20H, C-60H, C-700H), 8.08 (bs,
NH), 8.35 (t, 3J ¼ 7.6 Hz, C-8H).
7.1.4.5. 4-(4-Chloro-phenyl)-2-{4-[4-(1H-indol-3-yl)-piperidin-1-yl]-
butyl}-pyrido[1,2-c]pyrimidine-1,3-dione (9a). Yield: 75.5%; yellow
crystals, m.p. 173–174 ꢁC; 1H NMR (400 MHz)
d: 1.69 (q, C-3 x H2),
1.78 (m, 4H, C-2 x H2, Cb,dHax), 2.01 (pd, Cb,dHeq), 2.43 (t,
3J ¼ 7.6 Hz, C-4 x H2), 2.82 (tt, CcH), 4.19 (t, 3J ¼ 7.4 Hz, C-1 x H2),
6.39 (td, 3J ¼ 6.8 Hz, 4J ¼ 0.8 Hz, C-7H), 6.87 (d, 3J ¼ 9.2 Hz, C-5H),
13C NMR (100 MHz)
d: 25.4 (C-3x), 25.5 (C-2x), 36.0 (Cb, Cd),
54.2 (Ca, Ce), 56.4 (OCH3), 101.14 (C-400), 103.9 (C-4), 111.1 (C-7),
112.2 (C-700), 112.4 (C-600), 116.1 (d^, 2J ¼ 21.4 Hz, C-30, C-50), 119.4 (C-
300), 121.1 (C-200), 121.4 (C-5), 127.0 (C-300a), 128.3 (C-8), 128.9 (d^,
4J ¼ 3.0 Hz, C-10), 132.1 (C-6), 133.2 (d^, 3J ¼ 8.2 Hz, C-20, C-60), 131.7
(C-700a), 144.1 (C-4a), 154.1 (C-1), 160.5 (C-3); IR v: 1635 (C]O),
3
6.93 (t, C-6H), 6.93 (s, C-200H), 7.08 (t, J ¼ 7.2 Hz, C-500H), 7.16 (t,
3J ¼ 7.2 Hz, C-600H), 7.26 (d, C-30H, C-50H), 7.33 (d, 3J ¼ 8.0 Hz, C-
700H), 7.41 (d, 3J ¼ 8.4 Hz, C-20H, C-60H), 7.64 (d, 3J ¼ 8.0 Hz, C-400H),
8.11 (bs, NH), 8.34 (d, 3J ¼ 7.2 Hz, C-8H).

1716
F. Herold et al. / European Journal of Medicinal Chemistry 44 (2009) 1710–1717
1697 (C]O). Anal. Calcd for C32H33N4O3F: C, 71.1; H, 6.2; N, 10.4.
Found: C, 67.3; H, 6.1; N, 9.8.
yellow crystals, m.p. 158–160 ꢁC; 1H NMR (400 MHz)
d: 1.87 (q,
3J ¼ 6.8 Hz, C-3 x H2), 1.98 (ps, C-2 x H2,), 2.10 (bps, CbHax, CdHax),
2.47 (bps, CbHeq, CdHeq), 2.94 (bps, CcH), 3.05 (bps, CaHax, CeHax),
3.45 (bps, CaHeq, CeHeq), 3.89 (s, OCH3), 4.21 (t, 3J ¼ 6.8 Hz, C-1 x H2),
6.43 (td, 3J ¼ 6.8 Hz, 4J ¼ 1.2 Hz, C-7H), 6.85 (dd, 3J ¼ 8.8 Hz,
4J ¼ 2.0 Hz, C-600H), 6.89 (ps, C-200H), 6.94 (m, C-5H, C-6H), 7.07 (ps,
7.1.4.9. 4-(2-Fluoro-phenyl)-2-{4-[4-(1H-indol-3-yl)-piperidin-1-yl]-
butyl}-pyrido[1,2-c]pyrimidine-1,3-dione (11a). Yield: 37.6%; yellow
crystals, m.p. 191–192.6 ꢁC; 1H NMR (400 MHz)
d: 1.65 (q, C-3 x H2),
3
3
1.79 (m, 4H, C-2 x H2, Cb,dHax), 2.01 (pd, Cb,dHeq), 2.10 (pt, Ca,eHax),
2.44 (t, 3J ¼ 7.2 Hz, C-4 x H2), 2.82 (tt, CcH), 3.05 (pd, Ca,eHeq), 4.20
(t, 3J ¼ 7.2 Hz, C-1 x H2), 6.41 (t, 3J ¼ 6.8 Hz, C-7H), 6.73 (d,
3J ¼ 9.2 Hz, C-5H), 6.96 (t, 3J ¼ 8.8 Hz, C-6H), 6.94 (s, C-200H), 7.08 (t,
3J ¼ 7.2 Hz, C-500H), 7.16 (m, C-30H, C-60H), 7.26 (t, 3J ¼ 7.2 Hz, C-50H),
7.30–7.42 (m, C-40H, C-60H, C-700H), 7.64 (d, 3J ¼ 8.0 Hz, C-400H), 8.07
(bs, NH), 8.36 (d, 3J ¼ 7.2 Hz, C-8H).
C-400H), 7.26 (d, J ¼ 8.8 Hz, C-700H), 7.31 (d, J ¼ 7.6 Hz, C-20H, C-
60H), 7.37 (t, C-40H), 7.45 (t, 3J ¼ 7.2 Hz, C-30H, C-50H), 8.26 (bs, NH),
8.35 (d, 3J ¼ 7.6 Hz, C-8H); IR v: 1620 (C]O), 1709 (C]O). Anal.
Calcd for C32H34N4O3: C, 73.5; H, 6.6; N, 10.7. Found: C, 73.2; H, 6.4;
N, 10.6.
7.2. Pharmacology
13C NMR (100 MHz)
d: 24.7 (C-3x), 25.8 (C-2x), 33.3 (Cb, Cd), 33.7
(Cc), 42.8 (C-1x), 54.7 (Ca, Ce), 59.0 (C-4x), 98.6 (C-4), 111.0 (C-7),
7.2.1. In vitro experiments
2
111.3 (C-700), 116.3 (d^, J ¼ 22.2 Hz, C-30), 119.2 (C-500), 119.3 (C-400),
7.2.1.1. 5-HT1A binding assay. [3H]-8-hydroxy-2-(di-n-propyla-
mino)-tetralin ([3H]-8-OH-DPAT, spec. act. 106 Ci/mmol, NEN
Chemicals) was used for labeling 5-HT1A receptors. The membrane
preparation and the assay procedure were carried out according to
the published procedure [27] with slight modifications. Briefly, the
hippocampus tissue was homogenized in 20 vol. of 50 mM Tris–HCl
buffer (pH 7.7 at 25 ꢁC) using Ultra-TurraxÒ T 25, and was then
centrifuged at 32,000 x g for 10 min. The supernatant fraction was
discarded, and the pellet was resuspended in the same volume of
Tris–HCl buffer and was then centrifuged. Before the third centri-
fugation, the samples were incubated at 37 ꢁC for 10 min. The final
2
119.9 (C-200), 120.5 (d^, J ¼ 16.0 Hz, C-10), 121.4 (C-5), 121.8 (C-300),
4
122.0 (C-600), 124.6 (d^, J ¼ 3.5 Hz, C-50), 126.9 (C-300a), 128.4 (C-8),
3
130.3 (d^, J ¼ 9.3 Hz, C-40), 133.3 (C-6), 133.7 (d^, 3J ¼ 3.0 Hz, C-60),
136.5 (C-700a), 144.2 (C-4a), 149.1 (C-1), 159.8 (C-3), 161.0 (d^,
1J ¼ 249.5 Hz, C-20); IR v: 1647 (C]O), 1713 (C]O). Anal. Calcd for
C31H31N4O2F: C, 72.9; H, 6.3; N, 11.0. Found: C, 73.0; H, 6.2; N, 10.7.
7.1.4.10. 4-(2-Fluoro-phenyl)-2-{4-[4-(5-methoxy-1H-indol-3-yl)-
piperidin-1-yl]-butyl}-pyrido[1,2-c]pyrimidine-1,3-dione (11b).
Yield: 65%; yellow crystals, m.p. 88–92 ꢁC; 1H NMR (400 MHz)
d:
1.73 (q, C-3 x H2), 1.79 (q, C-2 x H2), 1.94 (pt, CbHax,CdHax), 2.04
(pd, CbHeq, CdHeq), 2.27 (pt, CaHax, CeHax), 2.59 (t, 3J ¼ 7.2 Hz, C-
4 x H2), 2.81 (m, CcH), 3.16 (pd, CaHeq, CeHeq), 3.87 (s, OCH3), 4.20
(t, 3J ¼ 7.0 Hz, C-1 x H2), 6.44 (t, 3J ¼ 6.8 Hz, C-7H), 6.74 (d,
pellet was resuspended in Tris–HCl buffer containing 10
pargyline, 4 mM CaCl2 and 0.1% ascorbic acid. One milliliter of the
tissue suspension (5 mg of wet weight), 100 l of 10 M serotonin
(for unspecific binding), 100 l of
l of [3H]-8-OH-DPAT and 100
analyzed compound were incubated at 37 ꢁC for 15 min. The
incubation was followed by a rapid vacuum filtration through
Whatman GF/B glass filters, and was then washed three times with
5 ml of a 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 10ꢂ10
to 10ꢂ4 M.
mM
m
m
m
m
4
3J ¼ 9.6 Hz, C-5H), 6.83 (dd, 3J ¼ 8.8 Hz, J ¼ 2.0 Hz, C-600H), 6.94
(s, C-200H), 6.99 (dd, C-6H), 7.05 (d, 4J ¼ 1.6 Hz, C-400H), 7.16 (m, C-
30H), 7.26 (m, C-50H, C-70H), 7.33 (t, C-40H), 7.37 (m, C-60H), 8.18
(bs, NH), 8.37 (d, 3J ¼ 7.6 Hz, C-8H).
13C NMR (100 MHz)
d: 24.0 (C-3x), 25.5 (C-2x), 33.3 (Cb, Cd),
32.0 (Cb, Cd), 32.5 (Cc), 42.2 (C-1x), 54.2 (Ca, Ce), 56.3 (C-4x, OCH3),
99.8 (C-4), 101.1 (C-400), 111.2 (C-7), 112.2 (C-600, C-700), 116.3 (d^,
2J ¼ 22.3 Hz, C-30), 119.6 (C-200), 120.4 (C-300), 121.0 (d^, 2J ¼ 16.0 Hz,
4
C-10), 121.4 (C-5), 124.7 (d^, J ¼ 3.5 Hz, C-50), 127.0 (C-300a), 128.4
7.2.1.2. 5-HT-T binding assay. The assay was performed according
to the method of Owens et al. [28] with slight modifications. Rat
cerebral cortex was homogenized in 30 vol. of ice-cold 50 mM Tris–
HCl containing 150 mM NaCl and 5 mM KCl, pH 7.7 at 25 ꢁC and
centrifuged at 20,000 x g for 20 min. The supernatant was decanted
and the pellet was resuspended in 30 vol. of buffer and centrifuged
again. The resulting pellet was resuspended in the same quantity of
the buffer and centrifuged for the third time in the same conditions.
[3H]-citalopram (spec. act. 50 Ci/mmol, NEN Chemicals) was used
for labeling 5-HT-transporter. Aliquots of the tissue suspension
3
(C-8), 130.4 (d^, J ¼ 7.5 Hz, C-40), 131.7 (C-700a), 133.5 (C-6), 133.6
(d^, 3J ¼ 3.0 Hz, C-60), 144.4 (C-4a), 149.1 (C-1), 158.6 (d^,
1J ¼ 249.0 Hz, C-20), 159.9 (C-3); IR v: 1643 (C]O), 1713 (C]O).
Anal. Calcd for C32H33N4O3F: C, 71.1; H, 6.1; N, 10.4. Found: C, 70.8;
H, 6.0; N, 10.1.
7.1.4.11. 2-{4-[4-(1H-Indol-3-yl)-piperidin-1-yl]-butyl}-4-phenyl-
pyrido[1,2-c]pyrimidine-1,3-dione (12a). Yield: 70.3%; yellow crys-
tals, m.p. 219–220 ꢁC; 1H NMR (400 MHz)
d: 1.67 (q, C-3 x H2), 1.80
(m, 4H, C-2 x H2, Cb,dHax), 2.03 (pd, 2H, Cb,dHeq), 2.15 (pt, 2H,
Ca,eHax), 2.49 (t, 3J ¼ 7.6 Hz, C-4 x H2), 2.83 (tt, 3J ¼ 11.6 Hz,
4J ¼ 7.6 Hz), 3.08 (pd, 2H, Ca,eHeq), 4.20 (t, 3J ¼ 7.2 Hz, C-1 x H2), 6.37
(q, 3J ¼ 7.6 Hz, 4J ¼ 3.6 Hz, C-7H), 6.89 (pd, C-5H, C-6H), 6.92 (bs, C-
200H), 7.08 (t, 3J ¼ 7.4 Hz, C-500H), 7.16 (t, 3J ¼ 7.4 Hz, C-600H), 7.32 (m,
4H, C-60H, C-700H), 7.62 (d, 3J ¼ 7.6 Hz, C-40H), 8.18 (bs, NH), 8.33 (d,
3J ¼ 7.6 Hz, C-8H).
(240
ml), 30
ml of 1
mM imipramine (displacer), 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 analyzed compounds ranged
from 10ꢂ10 to 10ꢂ4 M. Incubations were terminated by vacuum
filtration over Whatman GF/B filters and washed two times with
100 ml of ice-cold buffer. Radioactivity was measured in a WALLAC
1409 DSA liquid scintillation counter. All assays were done in
duplicates. Radioligand binding data were analyzed using iterative
curve-fitting routines (GraphPAD/Prism, Version 3.0 – San Diego,
CA, USA). Ki values were calculated from the Cheng and Prusoff
equation [21].
13C NMR (100 MHz)
d: 24.4 (C-3x), 25.7 (C-2x), 32.9 (Cb, Cd),
33.5 (Cc), 42.6 (C1x), 54.5 (Ca, Ce), 58.8 (C-4x), 105.1 (C-4), 110.9 (C-
7), 111.4 (C-700), 119.2 (C-400, C-500), 120.0 (C-200), 121.3 (C-300), 121.6
(C-5), 122.0 (C-600), 126.8 (C-300a), 128.0 (C-40), 128.1 (C-8), 129.0 (C-
30, C-50), 131.4 (C-20, C-60), 132.6 (C-6), 133.0 (C-10), 136.5 (C-700a),
143.7 (C-4a),149.1 (C-1), 160.4 (C-3); IR v: 1639 (C]O),1709 (C]O).
Anal. Calcd for C31H32N4O2: C, 75.6; H, 6.5; N,11.4. Found: C, 75.6; H,
6.6; N, 11.2.
IC50
Ki ¼
LO
1 þ K
D
7.1.4.12. 2-{4-[4-(5-Methoxy-1H-indol-3-yl)-piperidin-1-yl]-butyl}-
LO – labeled ligand concentration
4-phenyl-pyrido[1,2-c]pyrimidine-1,3-dione
(12b). Yield:
55%;
KD – dissociation constant of labeled ligand

F. Herold et al. / European Journal of Medicinal Chemistry 44 (2009) 1710–1717
1717
7.2.2. In vivo experiments
7.2.2.1. Induced hypothermia in mice
when it remained floating on water, making only small movements
to keep its head above it. The total duration of immobility was
measured by an experimenter running the final 4 min of 6-min test
session, after a 2-min habituation period.
7.2.2.1.1. Animals and housing. The experiments were per-
formed on male Albino Swiss mice (24–28 g; purchased from
licenced dealer Staniszewska, Ilkowice, Poland). The animals were
kept at room temperature (20 ꢀ 1 ꢁC) on a natural day-light cycle
(January–February) and housed under standard laboratory condi-
tions. They had free access to food and tap water before the
experiment. Each experimental group consisted of 7–8 animals/
dose, and all the animals were used only once. 8-Hydroxy-2-(di-n-
propylamino)-tetralin hydrobromide (8-OH-DPAT, Research
Biochemical Inc.) and N-{2-[4[(2[methoxyphenyl)-1-piperazinyl]-
ethyl}-N-(2-pyridinyl)-cyclohexane-carboxamide trihydrochloride
(WAY 100635, synthesized by Dr. J. Boksa, Institute of Pharma-
7.2.2.2.4. Locomotor activity in mice. The spontaneous loco-
motor activity of mice was recorded in photoresistor actiometers
(24 cm in diameter, illuminated by two light beams), which were
connected to a counter for the recording of light-beam interrup-
tions. The mice were placed individually in the actiometers, and the
number of light-beam crossings was counted twice: during the first
6-min, i.e. at the time equal to the observation period in the forced
swimming test, and during 30-min experimental session.
7.2.2.2.5. Statistical analysis. The obtained data were presented
as the mean ꢀ SEM. Comparisons between groups were carried out
by a one-way analysis of variance (ANOVA) followed by intergroup
comparisons using the Dunnett0s test. A p value < 0.05 was
considered significant.
´
cology, Polish Academy of Sciences, Krakow, Poland) were used as
aqueous solutions. Compounds 7a–12a were suspended in a 1%
aqueous solution of Tween 80. Compounds 8-OH-DPAT and WAY
100635 were injected subcutaneously (sc), 7a–12a were given
interperitoneally (ip) in a volume of 10 mg/kg (mice). The obtained
data were analyzed by Dunnett’s test (when only one drug was
given) or by the Newman–Keuls test (when two drugs were
administered).
7.2.2.1.2. Body temperature in mice. The effects of the tested
compounds given alone on the rectal body temperature in mice
(measured with Ellab thermometer) were recorded 30, 60, 90 and
120 min after their administration. In an independent experiment,
the effect of WAY 100635 (0.1 mg/kg) on the hypothermia induced
by compounds 8a–12a or 8-OH-DPAT was tested. WAY 100635 was
administered 15 min before 8a–12a or 8-OH-DPAT and the rectal
body temperature was recorded 30 and 60 min after injection of
the tested compounds. In another experiment, the effect of 7a
(which did not change body temperature in mice) on the 8-OH-
DPAT (5 mg/kg)-induced hypothermia was tested. Compound 7a
was administered 45 min before 8-OH-DPAT, and the rectal body
temperature was measured 15, 30, 45 and 60 min after injection of
8-OH-DPAT. The results were expressed as a change in body
Acknowledgements
This research was supported in part by a grant from the Medical
University of Warsaw (FW-26/W/2007) and the Polish Pharma-
ceutical Society.
References
[1] H.U. Wittchen, in: F.A. Helmchen, H. Henn, H. Lauter, N. Sartorious (Eds.),
Contemporary Psychiatry, Springer, Berlin/London, 2000, pp. 232–234.
[2] J. Feighner, J. Clin. Psychiatry Suppl. 4 (60) (1999) 4–11.
[3] K. Briner, R. Dodel, Curr. Pharm. Des. 4 (1998) 291–302.
[4] P. Blier, C. De Montigny, Y. Chaput, J. Clin. Psychol. 51 (1990) 14–20.
[5] F. Artigas, Trends Pharmacol. Sci. 14 (1993) 262–263.
[6] F. Artigas, P. Celada, M. Laruelle, A. Adell, Trends Pharmacol. Sci. 22 (2001)
224–228.
[7] F. Artigas, V. Perez, E. Alvarez, Arch. Gen. Psychiatry 51 (1994) 248–251.
[8] F. Artigas, L. Romero, C. De Montigny, P. Blier, Trends Neurosci. 19 (1996)
378–383.
[9] 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.
[10] 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.
temperature (
Dt) with respect to basal body temperature, as
measured at the beginning of the experiment.
7.2.2.2. Forced swimming test in mice
[11] D. Spinks, G. Spinks, Curr. Med. Chem. 9 (2002) 799–810.
[12] L. Romero, P. Celada, R. Martin-Ruiz, L. Diaz-Mataix, M. Mourelle, J. Delgadillo,
I. Hervas, F. Artigas, Neuropsychopharmacology 28 (2003) 445–456.
[13] J.F. Heiser, C.S. Wilcox, CNS Drugs 10 (5) (1998) 343–353.
[14] T. Heinrich, H. Bo¨ttcher, R. Gericke, G.D. Bartoszyk, S. Anzali, C.A. Seyfried,
H.E. Greiner, C. van Amsterdam, J. Med. Chem. 47 (19) (2004) 4684–4692.
[15] P. Grof, R. Joffe, S. Kennedy, E. Persad, J. Syrotiuk, D. Bradford, Int. Clin. Psy-
chopharmacol. 8 (1993) 167–172.
7.2.2.2.1. Animals and housing. The experiments were per-
formed on male Albino Swiss mice (24–28 g; purchased from
licenced dealer Staniszewska, Ilkowice, Poland). The animals were
kept in groups of 20 in a cage (60 x 38 x 20 cm) at a temperature of
20 ꢀ 1 ꢁC, and had free access to food (standard laboratory pallets)
and tap water before experiment. All investigations were con-
ducted in the light phase, on a natural light/dark cycle (January–
February), between 9 AM and 2 PM. Each experimental group
consisted of 9–10 animals per drug dose. Animals were tested in
a counterbalanced order and were used only once in each test. The
experiments were performed by an observer unaware of the
treatment. All the experimental procedures were approved by the
Local Bioethics Commission at the Institute of Pharmacology, Polish
´
[16] F. Herold, M. Krol, J. Kleps, G. Nowak, Eur. J. Med. Chem. 41 (2006) 125–134.
[17] 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.
[18] 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.K. Torgersen, B.K. Koe, J. Med. Chem. 33
(1990) 2087–2093.
[19] J. Guillaume, C. Dumont, J. Laurent, L. Nedelec, Eur. J. Med. Chem. Chim. Ther.
22 (1987) 33–43.
[20] F. Herold, I. Wolska, E. Helbin, M. Kro´l, J. Kleps, J. Heterocycl. Chem. 26 (1999)
389–396.
[21] F. Herold, J. Kleps, R. Anulewicz-Ostrowska, B. Szcze˛sna, J. Heterocycl. Chem.
39 (2002) 773–782.
[22] Y.C. Cheng, W.H. Prusoff, Biochem. Pharmacol. 22 (1973) 3099–3108.
[23] R.D. Porsolt, A. Bertin, M. Jalfre, Arch. Int. Pharmacodyn. Ther. 229 (1977)
327–336.
[24] G.M. Goodwin, R.J. De Souza, A.R. Green, Neuropharmacology 24 (1985) 1187–
1194.
[25] 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.
´
Academy of Sciences in Krakow.
7.2.2.2.2. Drug treatment. The following drugs were used 8-OH-
DPAT (Research Biochemical Inc.), compounds 8a, 10a, 11a and 12a.
All compounds were suspended in a 1% aqueous solution of Tween
80. Compound 8-OH-DPAT was injected subcutaneously (sc), 8a,
10a, 11a, and 12a were given interperitoneally (ip), 30 min before
the test in a volume of 10 ml/kg (mice).
7.2.2.2.3. Forced swimming test in mice. The experiment was
carried out according to the method of Porsolt et al. [23]. Briefly, the
mice were individually placed in a glass cylinder (25 cm high, 10 cm
in diameter) containing 6 cm of water maintained at 23–25 ꢁC, and
were left therein for 6 min. A mouse was regarded as immobile
[26] 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.
[27] D.N. Middlemiss, J.R. Fozard, Eur. J. Pharmacol. 90 (1983) 150–153.
[28] M.J. Owens, W.N. Morgan, S.J. Plott, C.B. Nemeroff, J. Pharmacol. Exp. Ther. 283
(1997) 1305–1322.