Synthesis and pharmacological evaluation of new arylpiperazines. 3-{4-[4-(3-chlorophenyl)-1-piperazinyl]butyl}-quinazolidin-4-one - A dual serotonin 5-HT1A/5-HT2A receptor ligand with an anxiolytic-like activity

Article abstract of DOI:10.1016/S0968-0896(02)00349-8

On the basis of systematic studies on the structure-activity relationships in arylpiperazine group of serotonin ligands, 12 new derivatives containing quinazolidin-4(3H)-one (1-4), 2-phenyl-2,3-dihydrophthalazine-1,4-dione (5-8) or 1-phenyl-1,2-dihydropyridazine-3,6-dione (9-12) fragments were synthesized. The majority of the tested compounds (2, 4, 7, 8 and 10-12) showed a high affinity for 5-HT_1A receptors (K_i=11-54 nM) and two (1, 2) were found active at 5-HT_2A sites (16 and 68 nM, respectively). All the new 5-HT_1A ligands tested in vivo revealed an antagonistic activity at postsynaptic 5-HT_1A receptors, and three of them behaved as agonists at presynaptic ones. Additionally, both the meta-chlorophenylpiperazine derivatives containing quinazolidin-4-one fragment showed features of 5-HT_2A receptor antagonists. The dual 5-HT_1A/5-HT_2A receptor ligand (2) was further tested for its potential psychotropic activity. It showed a distinct anxiolytic-like activity in a conflict drinking test in rats and the observed effect was more potent in terms of the active dose, than that produced by diazepam (used as a reference drug).

Full text of DOI:10.1016/S0968-0896(02)00349-8

Bioorganic& Medicinal Chemistry 10 (2002) 3817–3827
Synthesis and Pharmacological Evaluation of New
Arylpiperazines. 3-{4-[4-(3-chlorophenyl)-1-piperazinyl]butyl}-
quinazolidin-4-one — A Dual Serotonin 5-HT1A/5-HT2A Receptor
Ligand with an Anxiolytic-Like Activity
Andrzej J. Bojarski,a,* Piotr Kowalski,b Teresa Kowalska,b Beata Duszynska,a
Sijka Charakchieva-Minol,a Ewa Tatarczynska,c Aleksandra K•odzinskac
and Ewa Chojnacka-Wojcikc
aDepartment of Medicinal Chemistry Institute of Pharmacology, Polish Academy of Sciences, 12 Smec˛tna Street,
´
31-343 Krakow, Poland
bInstitute of Organic Chemistry and Technology, Cracow University of Technology, 24 Warszawska Street,
´
31-155 Krakow, Poland
cDepartment of New Drug Research, Institute of Pharmacology, Polish Academy of Sciences, 12 Smec˛tna Street,
´
31-343 Krakow, Poland
Received 24 May 2002; accepted 2 August 2002
Abstract—On the basis of systematic studies on the structure–activity relationships in arylpiperazine group of serotonin ligands, 12
new derivatives containing quinazolidin-4(3H)-one (1–4), 2-phenyl-2,3-dihydrophthalazine-1,4-dione (5–8) or 1-phenyl-1,2-dihy-
dropyridazine-3,6-dione (9–12) fragments were synthesized. The majority of the tested compounds (2, 4, 7, 8 and 10–12) showed a
high affinity for 5-HT1A receptors (Ki=11–54 nM) and two (1, 2) were found active at 5-HT2A sites (16 and 68 nM, respectively).
All the new 5-HT1A ligands tested in vivo revealed an antagonisticactivity at postsynaptic5-HT
receptors, and three of them
1A
behaved as agonists at presynapticones. Additionally, both the meta-chlorophenylpiperazine derivatives containing quinazolidin-4-
one fragment showed features of 5-HT2A receptor antagonists. The dual 5-HT1A/5-HT2A receptor ligand (2) was further tested for
its potential psychotropic activity. It showed a distinct anxiolytic-like activity in a conflict drinking test in rats and the observed
effect was more potent in terms of the active dose, than that produced by diazepam (used as a reference drug).
# 2002 Published by Elsevier Science Ltd.
Introduction
with a desired receptor profile is extremely difficult.
Nevertheless, a potential multireceptor activity of aryl-
piperazines implicates their frequent use as a source of
new agents with different therapeuticproperties.
Arylpiperazine is a core fragment of many bioactive
compounds which exhibit a variety of pharmacological
effects. It has been shown that their action can be
mediated by different subpopulations of serotonin (5-
hydroxytryptamine, 5-HT), dopamine and adrenergic
receptors. Numerous studies have indicated that even
minor modifications in the chemical structure of arylpi-
perazine derivatives strongly affect the affinity and
selectivity for the above-mentioned targets (for a review
see Glennon et al.1). Therefore designing compounds
The most thoroughly studied group of arylpiperazine
derivatives, called long chain arylpiperazines (LCAPs),
can be found as serotonin receptor ligands, especially
5-HT1A and 5-HT2A ones. Their general chemical
structure consists of an alkyl chain (2–4 methylene
units) attached to the N4 atom of the piperazine moiety,
and a terminal amide or an imide fragment. The sig-
nificance of the respective parts of LCAPs for 5-HT1A
affinity, intrinsic activity and selectivity has been the
subject of many structure–activity relationship studies.2
Although the influence of the aryl group (typically a
*Corresponding author. Tel.: +48-12-637-40-22; fax: +48-12-637-45-
00; e-mail: bojarski@if-pan.krakow.pl
0968-0896/02/$ - see front matter # 2002 Published by Elsevier Science Ltd.
PII: S0968-0896(02)00349-8

3818
A. J. Bojarski et al. / Bioorg. Med. Chem. 10 (2002) 3817–3827
substituted phenyl or heteroaromaticmoiety), as well as
the length of the alkyl chain are rather well established,
the function of the terminal fragment is still unclear. A
great number of such fragments tested (even those
without the amide group) suggest that different forces
are engaged in stabilizing the ligand–receptor complex
in this region. If we assume that an arylpiperazine frag-
ment serves as an anchoring point, the highly flexible
alkyl chain allows a terminal fragment of LCAPs to
interact with different sites of a binding pocket.2
Subsequent alkylation with 1-bromo-3-chloropropane
or 1,4-dibromobutane in the presence of K2CO3 in ace-
tonitrile led to the formation of halogen intermediates
and the symmetrically disubstituted by-products.7 Tar-
get compounds were obtained upon condensation of
halogen derivatives with the respective 1-arylpiperazine.
1
The structure of free bases 1–12 was confirmed by H
NMR spectra, and by an elemental analysis after con-
version to hydrochloride salts. Physicochemical data of
1–12 are collected in Table 1.
During our systematic structure–affinity and structure–
intrinsicatcivity studies within the LCAP group of
5-HT1A ligands different termini were used.3ꢀ6 The main
structural features explored included changes in the
relative position of the amide group with regard to the
aromaticring, varied ring sizes (five- or six-membered)
and introduction of an additional carbonyl group and/
or the oxygen atom (Scheme 1). Although the studied
compounds exhibited diversified 5-HT1A affinities and
pharmacological profiles one generalization could be
observed. Ligands with a nitrogen atom attached
directly to the benzene ring (type A)—even those show-
ing a high 5-HT1A affinity—were not active in vivo.4 On
the contrary, compounds of type B were usually highly
potent in vivo 5-HT1A receptor ligands.6
Pharmacology
All the compounds were evaluated for their affinity at
5-HT1A and 5-HT2A receptors; additionally, the affinity
of derivative 2 for dopamine D2 sites was also deter-
mined. The most in vitro potent ligands were tested in
vivo to assign their agonistic/antagonistic properties
towards 5-HT1A and/or 5-HT2A receptors.
Several models were proposed for measuring the
responses evoked by activation of pre- and postsynaptic
5-HT1A receptors in vivo.12,13 Administration of 8-
hydroxy-2-(di-n-propyloamino)tetraline (8-OH-DPAT)
—a full 5-HT1A agonist—evoked a hypothermiceffect
in mice (a model representative of activity at pre-
In order to further extend diversity of terminal amides B
we present the synthesis, 5-HT1A and 5-HT2A receptor
in vitro and in vivo studies of a new model arylpiper-
azines connected by three or four methylene group
spacer with quinazolidin-4-one (1–4), 2-phenyl-2,3-
dihydrophthalazine-1,4-dione (5–8) or 1-phenyl-1,2-
dihydropyridazine-3,6-dione (8–12) moieties. On the
basis of the obtained results, the most promising com-
pound 2 was then tested in several animal models of
anxiety and depression.
synaptic5-HT
receptors)14,15 and lower lip retraction
1A
(LLR) in rats,16 as well as a behavioral syndrome that is
a flat body posture (FBP) and forepaw treading (FT) in
reserpinized rats (models reflecting 5-HT1A postsynaptic
activity).17 The antagonistic5-HT
activity of the tes-
1A
ted compounds was assessed on the basis of the block-
ade of the above-mentioned 8-OH-DPAT-induced in
vivo effects. Their ability to mimic the 8-OH-DPAT-
induced action was regarded as a 5-HT1A agonistic
activity. Since hypothermia is a nonspecific in vivo
effect, we checked if it was abolished by the highly
selective 5-HT1A antagonist WAY 100635.18
Chemistry
The ability of the tested compounds to antagonize head
twitches in mice, observed after administration of (ꢁ)-
DOI [(ꢁ)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopro-
pane]—a 5-HT2A receptor agonist, was used to evaluate
5-HT2A receptor antagonistic properties.19
Final compounds 1–12 were obtained in a similar way
(Scheme 2) as their recently described phenylpiperazine
and pyrimidylpiperazine analogues.7 In short, the starting
quinazolidin-4(3H)-one, 2-phenyl-2,3-dihydrophtalazine-
1,4-dione and 1-phenyl-1,2-dihydropyridazine-3,6-dione
were prepared from antranilicaicd,
and maleicanhydride, 10,11 respectively, according to the
published procedures.
8
9
ftalicanhydride
The potential anxiolytic activity of compound 2 was
measured in two classic models in rats: the conflict
drinking (Vogel)20 and the elevated plus-maze21 tests.
The forced swimming test in mice was a behavioral
paradigm selected to determine potential antidepressant
properties.22 In order to measure the effect of com-
pound 2 administration on the exploratory activity in
rats, the open field test was also performed.23
Results and Discussion
The affinity of the tested compounds for 5-HT1A recep-
tors varied from 11 nM (12) to 415 nM (5), whereas for
5-HT2A receptors it ranged from 16 nM to 2100 for 1
and 11, respectively (Table 1). Generally, ortho-meth-
oxyphenylpiperazine derivatives were always more
Scheme 1.

A. J. Bojarski et al. / Bioorg. Med. Chem. 10 (2002) 3817–3827
3819
Scheme 2. Synthesis of the investigated compounds 1–12.
Table 1. Structure, physical data and 5-HT1A and 5-HT2A binding affinities of the compounds 1–12
Compound
R1
n
R
Yield (%)a
Mp (ꢂC)
Molecular formulab
KiꢁSEM (nM)
5-HT1A
5-HT2A
.
C21H23N4OCl HCl 0.5H2O
.
1
2
3
4
5
6
7
8
m-Cl
m-Cl
o-OCH3
o-OCH3
m-Cl
3
4
3
4
3
4
3
4
3
4
3
4
a
a
a
a
b
b
b
b
c79
c68
c74
c64
57
67
53
59
59
61
74
66
209–211
215–218
199–202
184–187
223–226
209–212
222–225
188–191
C
235ꢁ13
50ꢁ9
16ꢁ3
68ꢁ10
.
C22H25N4OCl HCl
.
C22H26N4O2 HCl H2O
.
100ꢁ3
36ꢁ1
460ꢁ4
.
C23H28N4O2 HCl
566ꢁ6
.
C27H27N4O2Cl HCl 0.5H2O
.
415ꢁ13
400ꢁ20
30ꢁ1
660ꢁ17
580ꢁ29
300ꢁ20
375ꢁ20
270ꢁ16
1830ꢁ26
2120ꢁ28
1450ꢁ24
.
C28H29N4O2Cl HCl
m-Cl
.
C28H30N4O3 HCl H2O
.
o-OCH3
o-OCH3
m-Cl
m-Cl
o-OCH3
o-OCH3
. .
C29H32N4O3 2 HCl
43ꢁ9
.
23H25N4O2Cl HCl 0.5H2O
.
9
207–210
206ꢁ9
50ꢁ8
. .
10
11
12
179–182
192–195
184–187
C
C
C
24H27N4O2Cl HCl H2O
.
.
24H28N4O3 HCl H2O
54ꢁ8
.
25H30N4O3 HCl 0.5H2O
.
11ꢁ2
aYield of a free base.
bAnal. C, H, N.
active at 5-HT1A receptors than the respective meta-
chloro analogues. Moreover, compounds with
5-HT2A ligands (Ki=1450–2130 nM), but at the same
time showing a high affinity for 5-HT1A receptors, hence
the highest 5-HT2A/1A selectivity (S2A/1A >35).
a
4-membered alkyl chain spacer (even numbers) were
more potent 5-HT1A ligands than were those containing
3 methylene groups. Thus the in vitro results were in
line with the general trends concerning affinities within
the arylpiperazine group of ligands. However, a closer
examination of the obtained 5-HT1A binding data
reveals that the influence of alkyl chain length, as well as
the arylpiperazine used depend on the terminal frag-
ment. In the case of series a and c, elongation of the
spacer caused app. 4-fold enhancement of 5-HT1A affi-
nity, but in series b it was without effect. On the other
hand, an increase in 5-HT1A affinity, connected with the
replacement of m-Cl by o-OCH3 in the phenyl ring, was
the most significant for series b (10-fold) and less
important for series c and a (4- and 2-fold, respectively).
The most in vitro active compounds (Ki<70 nM) were
further tested in several in vivo models to determine
their functional profile at 5-HT1A (2, 4, 7, 8, 10–12) and
5-HT2A (1, 2) receptors. All of the 5-HT1A ligands tested
given alone produced (like 8-OH-DPAT) a dose-depen-
dent decrease in body temperature in mice; the max-
imum hypothermiceffetc was observed 30 min after
injection (Table 2). The hypothermia evoked by 2 (1.25
mg/kg), 7 (5mg/kg), 10 (10 mg/kg), or the reference
8-OH-DPAT (5 mg/kg) was abolished by WAY100635
(0.1 mg/kg), hence those compounds were classified as
agonists of presynaptic5-HT
receptors. Since
1A
WAY100635 did not affect the decrease in body tem-
perature in mice induced by 4, 8 (10 mg/kg), 11 (5 mg/
kg) and 12 (2.5 mg/kg), it seems that 5-HT1A receptors
are not involved in these hypothermias (Table 3).
Regarding 5-HT2A receptors, only two relatively potent
compounds were found (1 and 2, Ki=16 and 68 nM,
respectively), either containing
a
meta-chloro-
phenylpiperazine and a quinazolidin-4-one (R=a) frag-
ments. Other compounds displayed a low affinity for 5-
HT2A receptors. Derivatives 10–12 were the least potent
All the investigated compounds given alone in doses up
to 10 or 20 mg/kg, evoked neither LLR in rats nor flat
body posture (FBP) or forepaw treading (FT) in reser-

3820
A. J. Bojarski et al. / Bioorg. Med. Chem. 10 (2002) 3817–3827
Table 2. The effect of the investigated compounds on the body temperature in mice
Treatment
Dose (mg/kg)
ÁtꢁSEM (ꢂC)
30 min
60 min
90 min
120 min
Vehicle
2
—
1.25
2.5
0.0ꢁ0.1
ꢀ1.3ꢁ0.3b
ꢀ2.3ꢁ0.1b
0.1ꢁ0.1
ꢀ1.4ꢁ0.1b
0.0ꢁ0.1
ꢀ0.1ꢁ0.1
ꢀ1.0ꢁ0.1b
0.1ꢁ0.1
0.1ꢁ0.1
ꢀ0.5ꢁ0.2
ꢀ0.5ꢁ0.1
Vehicle
4
—
5
10
0.0ꢁ0.1
ꢀ0.9ꢁ0.2a
ꢀ2.0ꢁ0.2b
0.1ꢁ0.1
ꢀ1.4ꢁ0.2b
0.0ꢁ0.1
0.1ꢁ0.1
0.6ꢁ0.1a
ꢀ0.1ꢁ0.1
0.0ꢁ0.2
0.3ꢁ0.2
ꢀ0.7ꢁ0.2
Vehicle
7
—
5
10
ꢀ0.2ꢁ0.1
ꢀ0.9ꢁ0.1b
ꢀ1.3ꢁ0.3b
ꢀ0.1ꢁ0.1
ꢀ0.6ꢁ0.1b
ꢀ1.1ꢁ0.3b
ꢀ0.1ꢁ0.1
ꢀ0.7ꢁ0.1b
ꢀ0.8ꢁ0.2b
0.1ꢁ0.1
ꢀ0.4ꢁ0.1b
ꢀ0.2ꢁ0.2
Vehicle
8
—
5
10
ꢀ0.2ꢁ0.1
ꢀ0.6ꢁ0.3
ꢀ1.5bꢁ0.2
ꢀ0.1ꢁ0.1
ꢀ0.8ꢁ0.1b
ꢀ1.3bꢁ0.2
ꢀ0.1ꢁ0.1
ꢀ0.2ꢁ0.1
ꢀ0.9bꢁ0.2
0.1ꢁ0.1
ꢀ0.1ꢁ0.1
ꢀ0.4ꢁ0.1
Vehicle
10
—
5
10
ꢀ0.1ꢁ0.2
ꢀ1.1ꢁ0.2b
ꢀ1.5ꢁ0.2b
ꢀ0.2ꢁ0.1
ꢀ0.7ꢁ0.2a
ꢀ0.9ꢁ0.2
0.01ꢁ0.1
ꢀ0.6ꢁ0.2a
ꢀ0.9ꢁ0.2b
ꢀ0.1ꢁ0.1
ꢀ0.5ꢁ0.2
ꢀ0.7ꢁ0.2
Vehicle
11
—
5
10
ꢀ0.1ꢁ0.2
ꢀ1.5ꢁ0.2b
ꢀ2.5ꢁ0.3b
ꢀ0.2ꢁ0.1
ꢀ0.7ꢁ0.2a
ꢀ1.7ꢁ0.3b
0.01ꢁ0.1
ꢀ0.4ꢁ0.2
ꢀ0.9ꢁ0.2b
ꢀ0.1ꢁ0.1
ꢀ0.1ꢁ0.2
ꢀ0.4ꢁ0.2
Vehicle
12
—
2.5
5
ꢀ0.1ꢁ0.1
ꢀ1.8ꢁ0.2b
ꢀ2.3ꢁ0.2b
ꢀ0.1ꢁ0.1
ꢀ1.4ꢁ0.1b
ꢀ2.4ꢁ0.2b
ꢀ0.1ꢁ0.2
ꢀ0.8ꢁ0.1b
ꢀ1.5ꢁ0.2b
ꢀ0.1ꢁ0.1
ꢀ0.6ꢁ0.1
ꢀ1.1ꢁ0.2b
WAY 100635
0.1
0.2ꢁ0.1
0.2ꢁ0.1
0.1ꢁ0.1
0.2ꢁ0.1
The investigated compounds (ip) and WAY 100635 (sc) were administered 30 min before the test. The absolute mean body temperatures were within
a range of 36.8ꢁ0.3 ꢂC.
ap<0.05 versus vehicle.
bp<0.01 versus vehicle.
pinized rats (data not shown); therefore it seems that
they have no agonisticactivity at postsynaptic5-HT
receptors.
Compounds 1 and 2 behaved like 5-HT2A receptor
antagonists, since either of them—like the reference
compound ketanserin- dose-dependently antagonized
head twitches in mice, observed after (ꢁ)-DOI (2.5 mg/kg)
administration. ED50 values were 6.2 (4.4–8.7) mg/kg,
9.2 (6.1–13.8) mg/kg and 0.14 (0.07–0.20) mg/kg for 1, 2
and ketanserin, respectively.
1A
The LLR induced by 8-OH-DPAT (1 mg/kg, the max-
imum possible score being 93%) was dose-dependently
antagonized by compounds 2, 4 (1.25–10 mg/kg), 7, 10
and 11 (10–20 mg/kg) and 12 (5–10 mg/kg), whereas the
effect of 8 (10–20 mg/kg) was weak irrespective of the
dose used (Table 4). Both the symptoms of the 8-OH-
DPAT-induced behavioral syndrome in reserpinized
rats (5 mg/kg, the maximum behavioral scores for FBP
and FT being 12.0–14.3 and 12.7–14.4, respectively)
were attenuated by 4 (5–20 mg/kg), 11 (10–20 mg/kg)
and 12 (5–10 mg/kg). Compounds 8 (10–20 mg/kg), 2
(10 mg/kg) and 7 (20 mg/kg) reduced the FT, but failed
to inhibit the FBP, whereas 10 (10–20 mg/kg) did not
affect any component of the 5-HT syndrome (Table 4).
It is noteworthy that only derivative 4 in the higher dose
used (20 mg/kg) produced an almost complete blockade
of the FBP and FT.
On the basis of the results of a functional study, and
considering the premises described below, compound 2
—an agonist of presynapticand an antagonist of post-
synaptic5-HT
receptors with a 5-HT2A receptor
1A
antagonistic activity—was selected for further in vivo
preclinical studies as a potential psychotropic agent.
It is well established that 5-HT1A ligands possess anxio-
24,25
lyticand antidepressive properties,
but their under-
lying mechanism of action is still unclear.26 To date, there
are a lot of evidences indicating that partial agonists
(such as the well-known drug buspirone), as well as full
agonists (flesinoxan) show intense activity in a number
of models of anxiety and depression.27ꢀ29 Moreover, it
has been demonstrated that 5-HT1A antagonists (WAY
100635, WAY100135 and MM77) produce anxiolytic-
like effects in some animal studies,30,31 however a num-
ber of data to the contrary have also been reported.32,33
In those models, all the tested ligands—like the refer-
ence compound WAY 100635—showed features char-
acteristic of postsynaptic 5-HT1A receptor antagonists.
Moreover, it should be stressed that only quinazolinone
derivatives 2 and 4 with a tetramethylene spacer
demonstrated a high potency, since they inhibited the
effects of 8-OH-DPAT already in a dose of 1.25 mg/kg,
however still higher than in the case of WAY 100635.
Furthermore, not only 5-HT1A, but also 5-HT2A recep-
tors have been proposed to be involved in the regulation
of anxiety and depression.34 Studies with nonselective

A. J. Bojarski et al. / Bioorg. Med. Chem. 10 (2002) 3817–3827
3821
Table 3. The effect of WAY 100635 on the hypothermia-induced by
investigated compounds in mice
In the conflict drinking test in rats, compound 2 (0.31–
2.5 mg/kg) dose-dependently increased the number of
punished licks (Table 5), but used in a higher dose (5
mg/kg) it induced sedation and other behavioral dis-
turbances (e.g., abduction, weak tremor), so that dose
was not tested. It seems that the observed effect was
specifically anxiolytic, since when compound 2 was
given in doses evoking an anticonflict activity, it affected
neither the shock threshold nor the non-punished water
consumption. It should be noted here that the antic-
onflict effect of 2 was even more potent in terms of
active dose than that produced by diazepam, used as a
reference drug (Table 5), and comparable to that of the
partial 5-HT1A receptor agonists buspirone and
MM199,40 or of mixed 5-HT1A/5-HT2A ligands, for
example, adatanserin.41
Treatment dose (mg/kg)
ÁtꢁSEM (ꢂC)
30 min
60 min
Vehicle+vehicle
Vehicle+2 (1.25)
WAY 100635 (0.1)+2 (1.25)
0.1ꢁ0.1
ꢀ1.3ꢁ0.3b
ꢀ0.5ꢁ0.2c
ꢀ0.1ꢁ0.1
ꢀ0.5ꢁ0.2
ꢀ0.2ꢁ0.1
Vehicle+vehicle
Vehicle+4 (10)
WAY 100635 (0.1)+4 (10)
ꢀ0.1ꢁ0.1
ꢀ3.2ꢁ0.2b,c
ꢀ0.1ꢁ0.1
ꢀ1.5ꢁ0.2b
ꢀ2.5ꢁ0.3b
ꢀ2.1ꢁ0.2b
Vehicle+vehicle
Vehicle+7 (5)
WAY 100635 (0.1)+7 (10)
ꢀ0.2ꢁ0.1
ꢀ0.9ꢁ0.2a
ꢀ0.2ꢁ0.1c
0.2ꢁ0.1
ꢀ0.7ꢁ0.1a
ꢀ0.1ꢁ0.1c
Vehicle+vehicle
Vehicle+8 (10)
WAY 100635 (0.1)+8 (10)
0.0ꢁ0.1
ꢀ1.7ꢁ0.2b
ꢀ1.4ꢁ0.1b
ꢀ0.1ꢁ0.1
ꢀ1.0ꢁ0.1b
ꢀ0.5ꢁ0.1
On the other hand, compound 2 (0.31–1.25 mg/kg) was
practically inactive in the plus-maze test in rats, in
which diazepam (2.5–5 mg/kg) showed a marked
anxiolytic-like activity. However there also exist con-
tradictory data about the effects of 5-HT1A partial ago-
Vehicle+vehicle
Vehicle+10 (10)
WAY 100635 (0.1)+10 (10)
ꢀ0.2ꢁ0.1
ꢀ1.7ꢁ0.2b
ꢀ0.3ꢁ0.1c
ꢀ0.1ꢁ0.1
ꢀ2.0ꢁ0.1b
ꢀ1.4ꢁ0.2b
ꢀ0.1ꢁ0.1
ꢀ1.8ꢁ0.2b
ꢀ1.5ꢁ0.2b
ꢀ0.2ꢁ0.1
ꢀ0.8ꢁ0.2b
ꢀ0.1ꢁ0.1c
ꢀ0.1ꢁ0.1
ꢀ0.8ꢁ0.2b
ꢀ0.4ꢁ0.1
0.1ꢁ0.1
ꢀ1.4ꢁ0.1b
ꢀ1.0ꢁ0.1b
Vehicle+vehicle
Vehicle+11 (5)
WAY 100635 (0.1)+11 (5)
nists (e.g., buspirone) in this test, since anxiolytic-like,42
lack of effect43 or even anxiogeniceffetcs
described.
were
44ꢀ46
Vehicle+vehicle
Vehicle+12 (2.5)
WAY 100635 (0.1)+12 (2.5)
Vehicle
8-OH-DPAT (5)
WAY100635 (0.1)+8-OH-DPAT (5)
0.1ꢁ0.1
ꢀ1.0ꢁ0.1b
ꢀ0.1ꢁ0.1c
0.1ꢁ0.1
ꢀ0.7ꢁ0.2
0.1ꢁ0.1c
The results of our experiments also showed that 2 (2.5–
10 mg/kg) did not change immobility time in the Porsolt
test in mice, while the typical antidepressant imipramine
(30 mg/kg, a 70% decrease in immobility time,
P<0.01), used as a reference drug, showed distinct
activity in that model. However, such a result could be
expected, since the 5-HT1A receptor partial agonists
buspirone and ipsapirone after systemicadministration
did not reduce immobility, either; 40,47ꢀ49 they exhibited
antidepressant-like activity in animals pretreated with
an inhibitor of drug metabolism.47
WAY 100635 was administered (sc) 15 min before investigated com-
pounds. The absolute mean body temperatures were within a range of
36.7ꢁ0.4 ꢂC.
ap<0.05 versus vehicle+vehicle.
bp<0.01 versus vehicle+vehicle.
cp<0.01 versus respective vehicle+compound.
serotonin drugs (e.g., ritanserin,35 adantanserin,36 nefa-
zodon37) have shown that their therapeuticeffects can
also be related with the antagonisticactivities at 5-HT
Compound 2 administered in a dose of 1.25 mg/kg did
not change exploratory locomotor activity in the open
field test in rats, but in doses of 2.5 and 5 mg/kg it
potently and dose-dependently attenuated general
exploratory behavior (time spent walking, ambulation
and rearing+peeping; Table 6). Although the latter
dose produced almost complete reduction of rats activ-
ity, it should be noted that it was 16-fold higher than
that evoking a minimal, but statistically significant,
anticonflict effect. In comparison, diazepam adminis-
tered in a dose of 2.5 mg/kg practically did not affect the
exploratory activity of rats, but its dose of 5 mg/kg sig-
nificantly reduced that exploration in that test (Table 6).
2A
receptors. Regarding compounds with such a ‘mixed
activity’, much attention has been given to drugs com-
bining 5-HT1A agonistic/antagonistic and 5-HT2A/2C
antagonisticproperties, as it is believed that they may
increase the therapeutic response of psychotropic drugs.
Such was the case with adatanserin, flibanserin38 and a
number of other 5-HT1A and 5-HT2A receptor ligands
which showed robust activity in preclinical models of
anxiety and/or depression and are now awaiting clinical
validation.28 In addition, as follows from a number of
preclinical and clinical investigations, ligands combining
D2/5-HT2A antagonism with 5-HT1A receptor activity for
example, ziprasidone39 may have potential antipsychotic
properties.
Conclusions
Thus, as a first step, the radioligand binding profile of
compound 2 was completed by the determination of its
affinity for dopamine D2 receptors. Since the affinity
found (Ki=430ꢁ10 nM) was low, further in vivo
examination in that direction was unfounded.
Here we reported the preparation, structure–activity
studies and discussion of pharmacological results of a
series of 12 new model arylpiperazines containing qui-
nazolidin-4-one, 2-phenyl-2,3-dihydrophthalazine-1,4-
dione or 1-phenyl-1,2-dihydropyridazine-3,6-dione moi-
eties. The differences in 5-HT1A binding constants were
explained on the basis of the known effects of the length
of an alkyl linker and the substituents used (m-Cl or
The successive phase of our studies with compound 2
focused on its evaluation as a potential anxiolytic and/
or antidepressant agent.

3822
A. J. Bojarski et al. / Bioorg. Med. Chem. 10 (2002) 3817–3827
Table 4. The effect of the tested compounds on the 8-OH-DPAT-induced lower lip retraction (LLR) in rats (A) and on the 8-OH-DPAT-induced
behavioral syndrome in reserpinized rats (B)
Compound
Dose mg/kg
Behavioral score, meanꢁSEM
A: LLR
B: Flat body posture
Forepaw treading
Vehicle
2
—
1.25
2.5
5
2.8ꢁ0.1
1.6ꢁ0.2b
1.3ꢁ0.3b
1.3ꢁ0.2b
0.8ꢁ0.2b
14.3ꢁ0.3
NT
NT
12.7ꢁ0.3
NT
NT
13.8ꢁ0.8
11.8ꢁ0.7
10
13.7ꢁ0.9
8.7ꢁ0.6b
Vehicle
4
—
1.25
2.5
5
10
20
2.8ꢁ0.1
1.7ꢁ0.1b
1.2ꢁ0.1b
1.0ꢁ0.2b
0.5ꢁ0.2b
NT
14.3ꢁ0.3
NT
12.7ꢁ0.3
NT
NT
NT
8.2ꢁ1.1b
3.5ꢁ1.2b
0.8ꢁ0.7b
8.7ꢁ0.7b
8.3ꢁ0.7b
2.0ꢁ0.7b
Vehicle
7
—
10
20
2.8ꢁ0.1
2.0ꢁ0.2b
1.5ꢁ0.4b
14.3ꢁ0.3
14.2ꢁ0.5
14.2ꢁ0.4
13.2ꢁ0.7
10.8ꢁ0.8
10.3ꢁ0.8a
Vehicle
8
—
10
20
2.8ꢁ0.1
1.9ꢁ0.2b
2.1ꢁ0.2a
13.8ꢁ0.8
13.7ꢁ0.5
13.3ꢁ0.7
13.2ꢁ0.7
8.5ꢁ0.7b
9.3ꢁ0.7b
Vehicle
10
—
10
20
2.8ꢁ0.1
1.2ꢁ0.2b
0.8ꢁ0.3b
12.0ꢁ1.0
10.2ꢁ0.6
11.7ꢁ0.8
13.2ꢁ0.7
10.3ꢁ0.4
9.5ꢁ0.9
Vehicle
11
—
10
20
2.8ꢁ0.1
1.4ꢁ0.2b
1.5ꢁ0.3b
12.8ꢁ1.1
9.0ꢁ0.8a
9.2ꢁ1.1a
14.4ꢁ0.2
8.7ꢁ1.6
5.0ꢁ1.5b
Vehicle
12
—
5
10
2.8ꢁ0.2
2.1ꢁ0.1a
0.4ꢁ0.1b
0.3ꢁ0.2b
12.0ꢁ1.0
9.0ꢁ1.1
6.0ꢁ0.4b
0.7ꢁ0.4b
13.2ꢁ0.7
6.7ꢁ1.3b
2.0ꢁ0.4b
1.3ꢁ0.8b
WAY 100635
0.1
(A) The tested compounds (ip) and WAY 100635 (sc) were administered 45 min, before 8-OH-DPAT (1 mg/kg, sc); (B) reserpine (1 mg/kg, sc), the
tested compounds (ip) and WAY 100635 (sc) were administered 18 h, 60 min and 30 min, respectively, before 8-OH-DPAT (5 mg/kg, sc); n=6 rats
per group. NT — not tested.
ap<0.05 versus vehicle.
bp<0.01 versus vehicle.
Table 5. The effect of compound 2 and diazepam in the conflict drinking test (A) and plus-maze test (B) in rats
Treatment
Dose mg/kg
A
B
Number of shocks accepted/5 min
% Of time in open arms
% Of open arms entries
Vehicle
2
—
0.08
0.16
0.31
7.6ꢁ0.9
14.7ꢁ3.1
18.3ꢁ4.7
33.6ꢁ6.3b
10.1ꢁ2.3
NT
NT
16.6ꢁ6.8
41.1ꢁ5.6
NT
NT
28.5ꢁ8.0
Vehicle
—
0.625
1.25
2.5
9.6ꢁ1.8
33.5ꢁ6.5b
34.4ꢁ3.8b
39.3ꢁ6.4b
33.5ꢁ15.8
25.4ꢁ15.6
NT
37.3ꢁ13.8
30.6ꢁ15.6
NT
Vehicle
Diazepam
—
2.5
5.0
9.3ꢁ2.1
29.7ꢁ3.5b
38.3ꢁ6.9b
10.9ꢁ1.1
38.5ꢁ3.1
73.8ꢁ4.2a
76.2ꢁ8.8b
47.2ꢁ5.3a
70.4ꢁ10.9b
Compound 2 and diazepam were administered 30 and 60 min, respectively before the tests.
ap<0.05 versus vehicle.
bp<0.01 versus vehicle.
o-OCH3) on a phenylpiperazine fragment. All the com-
pounds tested in functional in vivo models (2, 4, 7, 8,
10–12) behaved like antagonists of postsynaptic5-HT 1A
receptors and moreover 2, 7 and 10 may be regarded as
agonists of presynapticsites. Since the quinazolidin-4-
one derivative 2 also showed features of a 5-HT2A
receptor antagonist, it was further examined in in vivo
tests as a potential psychotropic agent. This dual
5-HT1A/5-HT2A ligand displayed distinct anxiolytic-like
activity in the Vogel test, but was inactive in the plus-
maze model, nor did it exert antidepressant-like prop-
erties in the forced swimming test in mice. Although

A. J. Bojarski et al. / Bioorg. Med. Chem. 10 (2002) 3817–3827
3823
Table 6. The effect of compound 2 and diazepam on the exploratory activity in the open field test in rats
Treatment
Dose mg/kg
Exploratory activity
Ambulation
Time of walking (s)
Peeping+rearing
Vehicle
2
—
1.25
2.5
37.7ꢁ4.7
39.2ꢁ7.2
23.0ꢁ7.1
2.3ꢁ1.0b
14.7ꢁ3.1
13.7ꢁ3.1
6.0ꢁ1.7a
0.5ꢁ0.2b
13.3ꢁ1.9
13.2ꢁ3.3
4.3ꢁ1.9a
0.3ꢁ0.2b
5.0
Vehicle
diazepam
—
2.5
5.0
37.0ꢁ4.0
27.0ꢁ4.1
17.8ꢁ4.6b
17.0ꢁ2.3
12.0ꢁ1.9
5.0ꢁ1.3b
11.2ꢁ2.4
7.8ꢁ2.1
4.4ꢁ2.0a
Compound 2 and diazepam were administered 30 and 60 min, respectively before the test.
ap<0.05 versus vehicle.
bp<0.01 versus vehicle.
Compound 2 revealed anxiolytic-like properties already
at low doses (from 0.31 mg/kg), the sedative effect
exerted by its dose of 5 mg/kg, excluded that ligand from
being regarded as a potential drug. New derivatives of
quinazolidin-4-one are currently being developed.
3-{3-[4-(3-Chlorophenyl)-1-piperazinyl]propyl}-quinazoli-
din-4-one (1). Base 1 was obtained in 57% yield, mp 59–
61 ꢂC (acetone–H2O 5:1); 1H NMR: d 1.94–2.18 (m, 2H,
CH2CH2CH2), 2.36–2.63 (m, 6H, CH2N(CH2)2 and
CH2N(CH2)2), 3.09–3.23 (m, 4H, (CH2)2NAr), 4.11 (t,
2H, CH2NC¼O, J=6.6 Hz), 8.13 (s, 1H, CH=N),
6.75–8.37 (m, 8HArom); MS: m/z (I%); M 382 (21), 209
(16), 187 (100); hydrochloride mp 209–211 ꢂC (2-propa-
Experimental
Chemistry
.
.
nol). Anal. calcd for C21H23N4OCl HCl 0.5H2O
(428.36): C, 58.88; H, 5.88; N, 13.08. Found: C, 59.01;
H, 5.94; N, 12.89.
Elemental analyses were performed on a Perkin–Elmer
2400 analyzer located in Regional Laboratory of
Jagiellonian University. 1H NMR spectra were recorded
in deuterochloroform with a Tesla 487C (80 MHz)
spectrometer and tetramethylsilane (TMS) as an inter-
nal standard; chemical shifts are reported in ppm (d);
coupling constants were taken from the expanded spec-
trum. Melting points were determined in a Boetius
apparatus and are uncorrected.
3-{4-[4-(3-Chlorophenyl)-1-piperazinyl]butyl}-quinazoli-
din-4-one (2). Base 2 was obtained in 67% yield, mp 87–
89 ꢂC (acetone); 1H NMR: d 1.58–2.04 (m, 4H,
CH2CH2CH2CH2), 2.34–2.63 (m, 6H, CH2N(CH2)2 and
CH2N(CH2)2), 3.11–3.25 (m, 4H, (CH2)2NAr), 4.05 (t,
2H, CH2NC¼O, J=6.6 Hz), 8.04 (s, 1H, CH=N),
6.72–8.37 (m, 8HArom); MS: m/z (I%); M 396 (33), 209
(100); hydrochloride mp 215–218 ꢂC (2-propanol–acetone
.
1:1). Anal. calcd for C22H25N4OCl HCl (433.38): C, 60.97;
H, 6.05; N, 12.93. Found: C, 60.72; H, 5.89; N, 13.01.
For biological experiments, free bases 1–12 were con-
verted into hydrochloride salts, and their molecular
formulas and molecular weights were established on the
basis of an elemental analysis.
3-{3-[4-(2-Methoxyphenyl)-1-piperazinyl]propyl}-quina-
zolidin-4-one (3). Base 3 was obtained as an oil in 53%
yield; 1H NMR: d 1.94–2.19 (m, 2H, CH2CH2CH2),
2.35–2.62 (m, 6H, CH2N(CH2)2 and CH2N(CH2)2),
3.04–3.24 (m, 4H, (CH2)2NAr), 3.86 (s, 3H, CH3O),
4.12 (t, 2H, CH2NC¼O, J=6.6 Hz), 8.17 (s, 1H,
CH=N), 6.78–8.37 (m, 8HArom); MS: m/z (I%); M 378
(100), 205 (31), 187 (94); hydrochloride mp 199–202 ꢂC
General procedure for the preparation of compounds 1–12
The mixture of the corresponding chloro derivative
(0.01 mol), arylpiperazine (0.01 mol), powdered K2CO3
(4.14 g, 0.03 mol) and catalytic amount of KI in 30 mL
of acetonitrile was stirred for 48 h at 50–60 ꢂC (Scheme
2). Then the inorganicprecipitate was filtered off, the
filtrate was evaporated, and the residue was purified
using silica gel chromatography with chloroform/
methanol=9:1 (3, 10, 12) or crystallized from the
appropriate solvent.
(acetone–ethanol
.
10:1).
Anal.
calcd
for
.
C22H26N4O2 HCl H2O (432.95): C, 61.03; H, 6.75; N,
12.94. Found: C, 60.89; H, 6.82; N, 12.92.
3-{4-[4-(2-methoxyphenyl)-1-piperazinyl]butyl}-quinazoli-
din-4-one (4). Base 4 was obtained in 59% yield, mp 59–
62 ꢂC (acetone); 1H NMR: d 1.62–2.03 (m, 4H,
CH2CH2CH2CH2), 2.37–2.70 (m, 6H, CH2N(CH2)2 and
CH2N(CH2)2), 3.03–3.18 (m, 4H, (CH2)2NAr), 3.86 (s,
3H, CH3O), 4.05 (t, 2H, CH2NC¼O, J=6.6 Hz), 8.05 (s,
1H, CH=N), 6.88–8.37 (m, 8HArom); MS: m/z (I%); M
392 (92), 205 (100); hydrochloride mp 184–187 ꢂC (2-
General procedure for the preparation of hydrochlorides
Free bases 1–12 were converted into hydrochlorides by
dissolving the corresponding base in acetone (10 mL/g)
and treating with ethanol saturated with HCl. The pre-
cipitate was filtered off and washed with acetone. Some
of the hydrochlorides were additionally purified by
crystallization.
.
propanol–acetone 1:1). Anal. calcd for C23H28N4O2 HCl
(428.96): C, 64.40; H, 6.81; N, 13.06. Found: C, 64.13;
H, 6.68; N, 13.29.

3824
A. J. Bojarski et al. / Bioorg. Med. Chem. 10 (2002) 3817–3827
3-{3-[4-(3-Chlorophenyl)-1-piperazinyl]propyl}-2-phenyl-
2,3-dihydrophtalazine-1,4-dione (5). Base 5 was obtained
in 59% yield, mp 52–54 ꢂC (methanol); 1H NMR: d
2.00–2.28 (m, 2H, CH2CH2CH2), 2.50–2.71 (m, 6H,
CH2N(CH2)2 and CH2N(CH2)2), 3.12–3.31 (m, 4H,
(CH2)2NAr), 4.44 (t, 2H, CH2NC¼O, J=6.6 Hz),
6.78–8.55 (m, 13HArom); MS: m/z (I%); M 474 (7), 279
(12), 209 (100); hydrochloride mp 223–226ꢂC (ac etone–
CH2N(CH2)2 and CH2N(CH2)2), 3.12–3.27 (m, 4H,
(CH2)2NAr), 4.20 (t, 2H, CH2NC¼O, J=6.6 Hz),
6.84–7.73 (m, 11HArom); MS: m/z (I%); M 438 (13), 209
(100); hydrochloride mp 179–182 ꢂC (acetone–ethanol
4:1). Anal. calcd for C24H27N4O2Cl HCl H2O (493.43):
C, 58.42; H, 6.13; N, 11.35. Found: C, 58.31; H, 6.21; N,
11.09.
.
.
.
.
ethanol 10:1). Anal. calcd for C27H27N4O2Cl HCl 0.5H2O
(520.46): C, 62.31; H, 5.62; N, 10.76. Found: C, 62.41; H,
5.58; N, 10.58.
2-{3-[4-(2-Methoxyphenyl)-1-piperazinyl]propyl}-1-phenyl-
1,2-dihydropyridazine-3,6-dione (11). Base 11 was
obtained in 74% yield, mp 57–60 ꢂC (acetone); 1H
NMR: d 1.91–2.12 (m, 2H, CH2CH2CH2), 2.50–2.75
(m, 6H, CH2N(CH2)2 and CH2N(CH2)2), 3.03–3.19 (m,
4H, (CH2)2NAr), 3.86 (s, 3H, CH3O), 4.25 (t, 2H,
CH2NC¼O, J=6.6 Hz), 6.91–7.73 (m, 11HArom); MS:
m/z (I%); M 420 (63), 229 (ꢀ), 205 (100); hydrochloride
mp 192–195 ꢂC (acetone–ethanol 10:1). Anal. calcd for
3-{4-[4-(3-Chlorophenyl)-1-piperazinyl]butyl}-2-phenyl-
2,3-dihydrophtalazine-1,4-dione (6). Base 6 was obtained
in 61% yield, mp 78–80 ꢂC (acetone); 1H NMR: d 1.75–
2.00 (m, 4H, CH2CH2CH2CH2), 2.41–2.67 (m, 6H,
CH2N(CH2)2 and CH2N(CH2)2), 3.14–3.26 (m, 4H,
(CH2)2NAr), 4.40 (t, 2H, CH2NC¼O, J=6.6 Hz),
6.79–8.47 (m, 13HArom); MS: m/z (I%); M 488 (6), 209
(100); hydrochloride mp 209–212 ꢂC (1-butanol). Anal.
.
.
C24H28N4O3 HCl H2O (474.99): C, 60.69; H, 6.58; N,
11.80. Found: C, 60.60; H, 6.59; N, 11.83.
.
calcd for C28H29N4O2Cl HCl (528.48): C, 64.00; H,
5.75; N, 10.66. Found: C, 63.81; H, 5.82; N, 10.48.
2-{4-[4-(2-Methoxyphenyl)-1-piperazinyl]butyl}-1-phenyl-
1,2-dihydropyridazine-3,6-dione (12). Base 12 was
1
obtained as an oil in 64% yield; H NMR: d 1.66–1.94
3-{3-[4-(2-Methoxyphenyl)-1-piperazinyl]propyl}-2-
phenyl-2,3-dihydrophtalazine-1,4-dione (7). Base 7 was
obtained in 74% yield, mp 116–118 ꢂC (acetone); 1H
NMR: d 2.00–2.26 (m, 2H, CH2CH2CH2), 2.57–2.76
(m, 6H, CH2N(CH2)2 and CH2N(CH2)2), 3.10–3.26 (m,
4H, (CH2)2NAr), 3.87 (s, 3H, CH3O), 4.44 (t, 2H,
CH2NC¼O, J=6.6 Hz), 6.94–8.55 (m, 13HArom); MS:
m/z (I%); M 470 (4), 279 (5), 205 (100); hydrochloride
mp 222–225 ꢂC (acetone–ethanol 10:1). Anal. calcd for
(m, 4H, CH2CH2CH2CH2), 2.38–2.75 (m, 6H,
CH2N(CH2)2 and CH2N(CH2)2), 3.04–3.21 (m, 4H,
(CH2)2NAr), 3.85 (s, 3H, CH3O), 4.20 (t, 2H,
CH2NC¼O, J=6.6 Hz), 6.91–7.74 (m, 11HArom); MS:
m/z (I%); M 434 (12), 205 (100); hydrochloride mp 184–
187 ꢂC (2-propanol–acetone 1:1). Anal. calcd for
.
.
C25H30N4O3 HCl 0.5H2O (480.00): C, 62.56; H, 6.72; N,
11.67. Found: C, 62.79; H, 6.56; N, 11.66.
.
.
C28H30N4O3 HCl H2O (525.05): C, 64.05; H, 6.34; N,
10.67. Found: C, 64.30; H, 6.22; N, 10.71.
In vitro studies — receptor binding
5-HT1A and 5-HT2A receptor binding assays. Radi-
oligand binding experiments were conducted in rat hip-
pocampus for 5-HT1A receptors, and in the cortex for
5-HT2A receptors according to the published proce-
dures.50 The radioligands used were [3H]-8-OH-DPAT
(190 Ci/mmol, Amersham) and [3H]-ketanserin (60 Ci/
mmol, NEN Chemicals) for 5-HT1A and 5-HT2A recep-
tors, respectively.
3-{4-[4-(2-Methoxyphenyl)-1-piperazinyl]butyl}-2-phenyl-
2,3-dihydrophtalazine-1,4-dione (8). Base 8 was obtained
in 66% yield, mp 91–93 ꢂC (acetone); 1H NMR: d 1.72–
1.99 (m, 4H, CH2CH2CH2CH2), 2.41–2.72 (m, 6H,
CH2N(CH2)2 and CH2N(CH2)2), 3.12–3.22 (m, 4H,
(CH2)2NAr), 3.86 (s, 3H, CH3O), 4.40 (t, 2H,
CH2NC¼O, J=6.6 Hz), 6.90–8.44 (m, 13HArom); MS:
m/z (I%); M 484 (11), 205 (100); hydrochloride mp 188–
191 ꢂC (2-propanol–acetone 1:1). Anal. calcd for
D2 dopaminergic receptor binding assay. Competition
binding studies were performed in rat striatal mem-
branes prepared according to the previously published
procedure.51 An assay was carried out in a 96-well filter
plate (containing glass fiber type C, Millipore), pre-
soaked with 100 mL of ice-cold 50 mM potassium
phosphate buffer (pH 7.4) and filtered using a Millipore
Vacuum Manifold prior to sample addition. 150 mL ali-
quots of striatal membrane preparations, 50 mL of the
radioligand ([3H]-spiperone, 15.70 Ci/mmol, NEN Che-
micals), and either 50 mL of the buffer (for total binding
assay) or 50 mL of (ꢁ)-butaclamol (5 mM) to determine
the unspecific binding, or 50 mL of the compounds to be
tested, were added to each well. Additionally, to prevent
[3H]-spiperone binding to 5-HT2A receptors, ketanserin
(50 nM) was included in the assay buffer. After incu-
bation at 37 ꢂC for 30 min, binding reaction was termi-
nated by vacuum filtration and washed 3 times with 200
mL of buffer. Radioactivity was determined by liquid
scintillation counting in a Beckman LS 6500 apparatus.
.
C29H32N4O3 2HCl (557.52): C, 62.48; H, 6.15; N, 10.05.
Found: C, 62.76; H, 6.23; N, 10.03.
2-{3-[4-(3-Chlorophenyl)-1-piperazinyl]propyl}-1-phenyl-
1,2-dihydropyridazine-3,6-dione (9). Base 9 was obtained
in 79% yield, mp 92–94 ꢂC (acetone–methanol 1:1); H
1
NMR: d 1.87–2.09 (m, 2H, CH2CH2CH2), 2.47–2.69
(m, 6H, CH2N(CH2)2 and CH2N(CH2)2), 3.12–3.28 (m,
4H, (CH2)2NAr), 4.25 (t, 2H, CH2NC¼O, J=6.6 Hz),
6.81–7.73 (m, 11HArom); MS: m/z (I%); M 424 (11), 229
(5), 209 (100); hydrochloride mp 207–210 ꢂC (ac etone–
.
.
ethanol 10:1). Anal. calcd for C23H25N4O2Cl HCl 0.5H2O
(470.40): C, 58.73; H, 5.79; N, 11.91. Found: C, 58.95; H,
5.89; N, 11.69.
2-{4-[4-(3-Chlorophenyl)-1-piperazinyl]butyl}-1-phenyl-
1,2-dihydropyridazine-3,6-dione (10). Base 10 was
1
obtained as an oil in 68% yield; H NMR: d 1.62–1.86
(m, 4H, CH2CH2CH2CH2), 2.34–2.66 (m, 6H,

A. J. Bojarski et al. / Bioorg. Med. Chem. 10 (2002) 3817–3827
3825
Ki values were determined from at least three competi-
tion binding experiments in which 10 drug concentra-
tions, run in triplicate, were used. The Cheng and
Prusoff equation was used for Ki calculations.52
8-OH-DPAT. Observation sessions, lasting 45 s each,
began 3 min after drug administration and were repe-
ated every 3 min. Reciprocal forepaw treading and flat
body posture were scored using a ranked intensity scale,
where 0=absent, 1=equivocal, 2=present, and
3=intense. The maximum score, summed up over five
observation periods, amounted to 15 for each symptom/
animal.17 The effect of the tested compounds and WAY
100635 on the behavioral syndrome induced by 8-OH-
DPAT (5 mg/kg) was assessed in a separate experiment.
The investigated compounds were administered 60 min
before 8-OH-DPAT, and the animals were scored at 3,
6, 9, 12 and 15 min after 8-OH-DPAT administration.
Reserpine (1 mg/kg) was given 18 h before the test.
In vivo studies
The experiments were carried out on male Wistar rats
(250–300 g) or male Albino-Swiss mice (25–30 g). The
animals were kept at a room temperature of 20ꢁ1 ꢂC
and were housed under standard laboratory conditions
with free access to food and tap water before the
experiment. All experiments were conducted in the light
phase, on a natural light–dark cycle (from May to
December), between 9 am and 2 pm. All the experi-
mental procedures were approved by the Animal Care
and Use Committee at the Institute of Pharmacology,
Polish Academy of Sciences in Krakow. (ꢁ)-DOI
(Research Biochemical, Inc.), 8-OH-DPAT (Research
Biochemical, Inc), reserpine (Ciba, ampules) and WAY
100635 (synthesized by Dr. J. Boksa, Institute of Phar-
macology, PAS, Krakow) were dissolved in saline. Dia-
zepam (Polfa, SA) and the investigated salts of the
tested compounds were used in the form of freshly pre-
pared suspensions in a 1% Tween 80. 8-OH-DPAT,
reserpine and WAY 100635 were injected sub-
cutaneously (sc), diazepam and the tested compounds
intraperitoneally (ip) in a volume of 2 mL/kg (rats) or
10 mL/kg (mice). Each group consisted of 6–9 animals.
The obtained data were analyzed by Dunnett’s test.
Head twitches in mice
In order to habituate the mice to the experimental
environment, each animal was randomly transferred to
a 12 cm (diameter)ꢃ20 cm (height) glass cage, lined with
sawdust, 30 min prior to treatment. Head twitches in
mice were induced by (ꢁ)-DOI (2.5 mg/kg). Immedi-
ately after the treatment, the number of head twitches
was counted for 30 min.19 The tested compounds or
saline were administered 60 min before (ꢁ)-DOI.
Conflict drinking test (Vogel test)
A modification of the method of Vogel et al.20 was used.
On the first day of the experiment, the rats were adapted
to the test chamber for 10 min. It was a Plexiglass box
(27ꢃ27ꢃ50 cm), equipped with a grid floor of stainless
steel bars and with a drinking bottle containing tap
water. After the initial adaptation period, the animals
were deprived of water for 24 h and were then placed in
the test chamber for a further 10-min adaptation period
during which they had free access to the drinking bottle.
Afterwards, they were allowed a 30 min free-drinking
session in their home cage. After another 24-h period of
water deprivation, the rats (those that drank water the
day before) were placed again in the test chamber and
allowed to drink for 30 s. Immediately afterwards, their
drinking attempts were punished with an electric shock
(0.5 mA). Impulses were released every 2 s (timed from
the moment when a preceding shock was delivered) in
1-s periods, between the grid floor and the spout of the
drinking bottle. The number of shocks accepted
throughout a 5-min experimental session was counted
by an experimenter who observed a behavioral reaction
(e.g., body jerks) of rats to an electric shock. The tested
compounds were administered 60 min before the test.
The effect on the body temperature in mice
Rectal body temperature (measured with an Ellab ther-
mometer) was recorded in mice 30, 60, 90 and 120 min
after injection of the tested compounds. In a separate
experiment, the effect of WAY 100635 (0.1 mg/kg) on
the hypothermia induced by 2, 4, 7, 8 and 10–12 or
8-OH-DPAT was investigated. Rectal body temperature
was measured 30 and 60 min after injection of the drugs
tested. WAY 100635 was given 15 min before the tested
compounds.
Lower lip retraction in rats
LLR was assessed according to the method described by
Berendsen et al.16 The rats were placed individually in
cages (30ꢃ25ꢃ25 cm) and were scored three times (at
15, 30 and 45 min after the tested compounds or 8-OH-
DPAT) as follows: 0=lower incisors invisible,
0.5=partly visible, 1=clearly visible. The summed up
maximum scores amounted to 3 for each rat. The effect
of the tested compounds and WAY 100635 on the LLR
induced by 8-OH-DPAT (1 mg/kg) was assessed in a
separate experiment. The investigated compounds were
administered 45 min before 8-OH-DPAT, and the ani-
mals were scored at 15, 30 and 45 min after 8-OH-
DPAT administration.
Shock threshold and free-drinking tests
To control the possibility of drug-induced changes in
the perception of a stimulus or in the thirst drive, which
might have contributed to the activity in the conflict
drinking test, stimulus threshold measurements and a
free-drinking experiment were also carried out. In both
those cases, the rats were treated before the experiment
in the same manner as described in the conflict drinking
test, including two 24-h water deprivation periods sepa-
rated by 30 min of water availability. In the shock
Behavioral syndrome in reserpinized rats
The rats were placed individually in cages (30ꢃ25ꢃ25
cm) 5 min before injection of the tested compounds or

3826
A. J. Bojarski et al. / Bioorg. Med. Chem. 10 (2002) 3817–3827
threshold test, the rats were placed individually in the
box and electric shocks were delivered through the grid
floor. The shock threshold was determined stepwise at
15 s shock-free intervals by increasing manually the
current (0.1, 0.2, 0.3, 0.4 and 0.5 mA); the shock lasted
for 1 s and was delivered through the grid-floor until a
rat showed an avoidance reaction (jump, or jerk) to the
electric stimulus.
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Acknowledgements
This study was partially supported by the Polish State
Committee for Scientific Research (KBN), Grant No.
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