Synthesis and preliminary pharmacological evaluation of imidazo[2,1-f]purine-2,4-dione derivatives

Article abstract of DOI:10.1016/j.ejmech.2009.07.014

A series of N-8-arylpiperazinylpropyl derivatives of 1,3-dimethyl-(1H,8H)-imidazo[2,1-f]purine-2,4-dione (2-10) and amide derivatives of 1,3-dimethyl-6,7-dihydroimidazo[2,1-f]purine-2,4-(1H,3H)-dione-7-carboxylic acid (11-13) were synthesized. Compounds (

Full text of DOI:10.1016/j.ejmech.2009.07.014

European Journal of Medicinal Chemistry 44 (2009) 4288–4296
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and preliminary pharmacological evaluation of imidazo[2,1-f]purine-
2,4-dione derivatives
a
a
a
,
b
b
*
´
Agnieszka Zagorska , S1awomir Jurczyk , Maciej Paw1owski , Ma1gorzata Dyba1a , Gabriel Nowak ,
c
c
c
´
´
Ewa Tatarczynska , Agnieszka Nikiforuk , Ewa Chojnacka-Wojcik
a Department of Medicinal Chemistry, Jagiellonian University Medical College, Medyczna 9, 30-688 Krako´w, Poland
b Department of Pharmacobiology, Jagiellonian University Medical College, Medyczna 9, 30-688 Krako´w, Poland
c Department of New Drugs Research, Institute of Pharmacology, Polish Academy of Sciences, Sme˛tna 12, 31-343 Krako´w, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of N-8-arylpiperazinylpropyl derivatives of 1,3-dimethyl-(1H,8H)-imidazo[2,1-f]purine-2,4-dione
(2–10) and amide derivatives of 1,3-dimethyl-6,7-dihydroimidazo[2,1-f]purine-2,4-(1H,3H)-dione-7-
carboxylic acid (11–13) were synthesized. Compounds (2–10) evaluated in vitro were potent 5-HT1A
receptor ligands. Preclinical studies indicated that 8-[3-(N4-phenyl)-piperazin-N1-yl-propyl]-1,3-dimethyl-
(1H,8H)-imidazo[2,1-f]purine-2,4-dione (2) exerts anxiolytic-like activity in the four-plate test in mice;
however its effect was weaker, than that produced by Diazepam. This compound and 8-[3-(N4-20-
metoxyphenyl)-piperazin-N1-yl-propyl]-1,3-dimethyl-(1H,8H)-imidazo[2,1-f]purine-2,4-dione (3) behaved
like antidepressants in the forced swimming test in mice; and their activity in that model was comparable
with the effect of Imipramine. The obtained results suggested that the long-chain arylpiperazines (LCAPs)
linked to tricyclic derivatives of the theophylline remain a worthy of future research for obtaining new
derivatives with potential anxiolytic/antidepressant activity.
Received 19 February 2008
Received in revised form
13 July 2009
Accepted 16 July 2009
Available online 21 July 2009
Keywords:
Theophylline
Imidazo[2,1-f]theophyllines
Arylalkylpiperazines
5-HT1A, 5-HT2A, D1, D2 receptor affinities
Anxiolytics/antidepressants
Ó 2009 Elsevier Masson SAS. All rights reserved.
1. Introduction
for 5-HT1A activity and selectivity for other receptors [3]. These
properties depended on the above-mentioned terminal fragments
A multitude of brain functions are influenced by serotonin
(5-HT) including sleep, cognition, sensory perception, appetite [1].
5-HT modulates the central nervous system through a number of
receptor subtypes (5-HTR) [2–5]. At present, seven 5-HTR classes,
including fourteen subtypes have been found, cloned and structure
described. Due to the receptor-binding studies and molecular
biology, close attention has been focused on site-selective agonists
and antagonists of 5-HTR, used as tool substances and as potential
new therapeutic drugs.
Long-chain arylpiperazines (LCAPs) have been extensively
studied especially as 5-HT1A and 5-HT2A receptor ligands due to
their potential antianxiety or antidepressant properties. Since the
launch of Buspirone, a large number of arylpiperazine derivatives
have been synthesized and some neuropsychiatric drugs e.g.
Gepirone [6], Ziprasidone [7], Aripiprazole [8], and compounds
with high therapeutical potential e.g. Adatanserin [9], Flesinoxan
[10] were developed. The SAR studies revealed the role of indi-
vidual fragments of this pharmacophore, essential for high affinity
(amide or imide) and also on the N4 aryl substituent as well as on
the length of the alkylen linker [11–15].
The incorporation of an appropriate long-chain substituent at
the basic nitrogen of the piperazine ring resulted a higher affinity
and selectivity [16]. From two to four methylene units can be found
in amide-arylpiperazine derivatives as optimal for 5-HT1A receptor
affinity. Furthermore, their selectivity between others’ receptors
depends on the length of the alkylen spacer and the nature of the
terminal fragment [17]. The chemical modification of Gepirone
showed, that the profile of 5-HT1A intrinsic activity was sensitive to
the mode of substitution in the aromatic part of the pharmaco-
phore [18,19]. It was found that ligands with the o-CH3 group in the
aryl moiety and cyclic imide system in the opposite terminal have
a tendency to block postsynaptic 5-HT1A receptors, whereas
unsubstituted, m-Cl or m-CF3 substituted derivatives show
agonistic or partial agonistic properties [20]. Furthermore, the
selectivity problem for the D2 and a1 receptors is not yet clearly
determined due to the high degree of homology between these
receptors [21].
Tricyclic theophylline derivatives, annelated six or seven-
membered heterocyclic ring at 7,8-position of theophylline generally
demonstrated a different profile of its central nervous system activity,
* Corresponding author. Tel.: þ48 12 620 54 51; fax: þ48 12 657 02 62.
E-mail address: mfmpawlo@cyf-kr.edu.pl (M. Paw1owski).
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.07.014

A. Zago´rska et al. / European Journal of Medicinal Chemistry 44 (2009) 4288–4296
4289
O
5-HT2A Ki ¼ 297 ꢀ 20 nM, D2 Ki ¼ 414 ꢀ120 nM) (Fig. 1) behaved
like an agonist of presynaptic and as a partial agonist of postsynaptic
5-HT1A receptors and resembled Ipsapirone in terms of functional
intrinsic activity [11]. The change of pyrimido moiety at 7,8-position
of the theophylline, into 1,3-diazepine ring in LCAPs derivatives,
resulted in a 12-fold decrease in 5-HT1A affinity [23]. It showed that
the third ring fused to theophylline plays an important role in
binding affinity and 5-HT1A/5-HT2A selectivity of these compounds.
The aim of the present work was the design, synthesis and bio-
logical evaluation of LCAPs with terminal part based on imidazo[2,1-
f]theophylline fragment (Fig. 2). We were particularly interested
whetherthepresenceofimidazoleringand/orexocyclicamidemoiety
in tricyclic theophylline derivatives can influence the profile of phar-
macological activity in comparison to described earlier compounds.
H3C
N
N
N
N
H3CO
O
N
N
N
CH3
5-HT1A Ki = 2,8 0,3 nM, 5-HT2A Ki = 486 31 nM [23]
Br
O
H
N
H3C
N
N
N
N
N
N
2. Chemistry
O
N
CH3
7-Acetic-8-bromotheophylline aldehyde (1a), 7-acetonyl-8-bro-
motheophylline (1b) and 7-phenacyl-8-bromotheophylline (1c)
were obtained according to the previously described procedures [24].
The final 8-substituted derivatives of 1,3-dimethyl-(1H,8H)-imi-
dazo[2,1-f]purine-2,4-dione (2–10) were obtained in the cyclo-
condensationreactionof1a,1b,1c, withdoubleamountofappropriate
arylpiperazinylpropylamine [25], in boiling 2-methoxyethanol
(Scheme 1).
5-HT1A Ki = 11 0,1 nM, 5-HT2A Ki = 297 20 nM, D2 Ki = 414 120 nM
agonist of presynaptic and partial agonist of postsynaptic 5-HT1A receptors [11]
Fig. 1. Chemical structure and binding affinities of previously synthesized LCAPs with
amide part based on tricyclic theophylline derivatives.
in comparison to the reference compound (theophylline) [11,22,23].
The pharmacological evaluation of a series of tricyclic theophylline
derivatives with a pyrimido- ordiazepino-moietydemonstrated their
sedative, hypothermizing and neuroleptic-like effects on the CNS
[22]. Derivatives of pyrimido[2,1-f]theophylline with various LCAPs
moiety showed high or very high 5-HT1A receptor affinity and diver-
sified pharmacological profile. Some of the behavioral models
demonstrated that 1,3-dimethyl-9-{3-[4-(20-methoxyphenyl)-piper-
azin-1-yl]-propyl}-2,4-dioxo-1,3,6,7,8,9-hexahydropyrimido[2,1-f]
purine (Fig.1) was classified as presynaptic agonist and postsynaptic
partial agonist of 5-HT1A receptor while its unsaturated analog was
a pre- and postsynaptic agonist [23].
The amide derivatives of 1,3-dimethyl-6,7-dihydroimidazo
[2,1-f]purine-2,4-(1H,3H)-dione-7-carboxylic acid (11–13) were
obtained from 1d [26] with equimolar amount of appropriate
arylpiperazinylarylamine and HOBt in the presence of DCC in
dichloromethane (Scheme 1).
The structures of the compounds were confirmed by 1H NMR
and MS spectral as well as by elemental (C, H, N) (see Section 5)
analyses. For binding studies the synthesized free bases were
converted into hydrochloride salts.
The most potent for 5-HT receptors were the compounds with
aromatic six membered ring annelated at 7,8-position of the
theophylline [22] and the profile of their functional activity can be
3. Pharmacology
3.1. In vitro studies
attributed to
p-system present in an annelated third ring 1,3-
Dimethyl-7-bromo-8-[3-(4-phenylpiperazin-1-yl)-propylamino]-
The affinities of newly synthesized compounds for 5-HT1A
,
(1H,3H)-pyrimido[2,1-f]purine-2,4-dione (5-HT1A Ki ¼ 11 ꢀ0.1 nM,
5-HT2A and D2 receptors were determined by standard competitive
R1
O
R
N
H3C
O
N
N
N
N
N
R= -H, -CH3, -C6H5
N
R1
R1= -H, -2OCH3, -3Cl
CH3
2-10
N
O
N
O
N
N
H
H3C
N
N
N
NH
O
11-13
CH3
Fig. 2. General structure of the synthesized compounds.

4290
A. Zago´rska et al. / European Journal of Medicinal Chemistry 44 (2009) 4288–4296
R
O
O
O
R
H3C
O
R1
R1
N
N
H3C
O
N
N
N
(a)
N
Br
N
N
N
N
(CH2)3
N
NH2
+
N
N
CH3
CH3
1a-c
2-10
O
R1
COOH
O
N
N
O
N
H3C
O
N
N
R1
H
N
H3C
O
NH
(b)
N
N
N
N
(CH2)2
N
NH2
NH
+
N
N
CH3
1d
CH3
11-13
cmpd
R
2
3
-H
4
5
6
7
8
9
10
11
-
12
-
13
-H
-H
-H
-CH3
-H
-CH3
-CH3
-3-Cl
-C6H5
-H
-C6H5
-C6H5
-3-Cl
-
R1
-2-OMe
-3-Cl
-2-OMe
-2-OMe
-H
-2-OMe
-3-Cl
Scheme 1. Synthesis of imidazo[2,1-f]purine-2,4-dione derivatives. Reagents and conditions: (a) 2-methoxyethanol, reflux 6 h; (b) HOBt/DCC in DCM, stirring 12 h, room temp.
displacement assays. The results of in vitro binding studies of the
newly synthesized compounds (2–13) are shown in Table 1.
Compounds 2, 3 and 5–9 are potent 5-HT1A receptor ligands with
Ki within the range of 5.6–96.5 nM and demonstrate lack of affinity
for 5-HT2A subtype (except 4 Ki in micromolar value). Compounds
11–13 demonstrate lack of affinity for 5-HT1A subtype. Compounds
3, 6, 7 and 9 demonstrated low or very low affinity for D2 receptors
(from Ki ¼ 143 ꢀ 10.6 nM to839.0 ꢀ 61.0 nM)ordid not bindtothose
receptors (Ki in micromolar range 2, 4, 5, 8).
8-OH-DPAT-induced effects were abolished by 5-HT1A receptor
antagonists such as WAY 100635 [30,31]. Thus, the decrease in
body temperature in mice induced by the investigated compounds
and reduced by WAY 100635, was regarded as a measure of
presynaptic 5-HT1A receptor agonistic activity. Similarly, the ability
of the tested compounds to induce LLR in rats was regarded as
postsynaptic 5-HT1A receptor agonistic activity. The antagonistic 5-
HT1A receptor activity of the tested compounds was assessed on the
basis of the blockade of the above-mentioned 8-OH-DPAT-induced
in vivo effects. All the compounds tested in vivo administered alone
– like 8-OH-DPAT, a 5-HT1A receptor agonist – produced a dose-
dependent decrease in mouse body temperature, the maximal
effects were observed at 30 min after their administration.
The functional profile of the investigated compounds, suggests
that some of them may show anxiolytic- and/or antidepressant-
like activity. It is well established that 5-HT1A receptor ligands
possess such properties [32], but their underlying mechanism of
action is still unclear [33,34]. To date, there are a lot of evidences
indicating that partial agonists (e.g. Buspirone), as well as full
agonist (e. g. Flesinoxan) show intense activity in animal models of
anxiety and depression [32,35,36]. Considering the above
described premises we selected compounds 2 (a 5-HT1A receptor
partial agonist), 3 and 9 (full 5-HT1A receptor agonists) for further
in vivo preclinical studies.
3.2. In vivo studies
Selected compounds (2, 3, 6, 7 and 9) with the highest affinity
for 5-HT1A receptors (Ki down to 30 nM) were further tested in vivo.
To examine the functional activity of the compounds the
commonly used models were employed for measuring the
responses evoked by the activation of pre- and post-synaptic
5-HT1A
receptors.
8-Hydroxy-2-(di-n-propylamino)tetraline
(8-OH-DPAT) – a full 5-HT1A receptor agonist – evoked a hypo-
thermic effect in mice, a model representative of activity at
presynaptic 5-HT1A receptors [27] and lower lip retraction (LLR) in
rats, a model reflecting 5-HT1A postsynaptic activity [28,29]. Both
Table 1
Binding affinities of compounds 2–13.
3.2.1. Body temperature in mice
Ki nM (ꢀS.E.M.)
All the investigated compounds 2, 3, 6, 7 and 9 (5–20 mg/kg)
decreased rectal body temperature in mice in dose-dependent
manner. The maximum hypothermic effect was observed at 30 min
after their administration (Table 2). The hypothermia induced by 2,
3, 7, 9 or 8-OH-DPAT was reduced by 5-HT1A receptor antagonist
WAY 100635 (Table 3). At the same time, WAY 100635 did not
change the hypothermic effect induced by 6.
Cmpd 5-HT1A ([3H]-OH-DPAT) 5-HT2A ([3H]-Ketanserin) D2 ([3H]-Spiperone)
2
3
4
24.8 ꢀ 1.3
25.4 ꢀ 6.7
>1000
>1000
>1000
91.5 ꢀ 1.7
>1000
>1000
403 ꢀ 40
813.5 ꢀ 54.8
>1000
>1000
Nt
>1000
839 ꢀ 61
>1000
>1000
344.7 ꢀ 41
767.2 ꢀ 20.5
>1000
143.0 ꢀ 10.6
Nt
5
6
7
8
76.4 ꢀ 21.0
16.8 ꢀ 7.6
17.1 ꢀ 0.7
96.5 ꢀ 16
5.6 ꢀ 0.7
Nd
>1000
>1000
>1000
9
3.2.2. Lower lip retraction (LLR) in rats
10
11
12
13
Compounds 2, 3 and 9 (10–20 mg/kg) given alone induced weak
LLR in rats, while 6 and 7, in the same dose did not mimic the effect of
8-OH-DPAT in that test (Table 4). The LLR induced by 8-OH-DPAT was
partiallyreducedby2and7, theremainingcompounds(3, 6and9)did
Nt
Nt
Nt
Nt
Nt
Nd not detected; Nt not tested.

A. Zago´rska et al. / European Journal of Medicinal Chemistry 44 (2009) 4288–4296
4291
Table 2
Table 4
Effect of the investigated compounds and WAY 100635 on the body temperature in
mice.
Induction of lower lip retraction (LLR) by the investigated compounds and WAY
100635 (A) and their effect on the 8-OH-DPAT-induced LLR (B) in rats.
Treatment
Dose mg/kg
D
t ꢀ SEM (ꢁC)
Treatment
Dose mg/kg
Mean ꢀ SEM LLR score
30 min
60 min
90 min
120 min
A
B
Vehicle
2
–
ꢂ0.1 ꢀ 0.1
ꢂ0.6 ꢀ 0.2
0.0 ꢀ 0.1
0.1 ꢀ 0.1
ꢂ0.2 ꢀ 0.1
ꢂ0.4 ꢀ 0.2
ꢂ0.1 ꢀ 0.1
0.0 ꢀ 0.2
ꢂ0.2 ꢀ 0.2
0.1 ꢀ 0.1
Vehicle
2
–
0.0 ꢀ 0.0
0.2 ꢀ 0.1
0.8 ꢀ 0.2a
0.9 ꢀ 0.2a
1.5 ꢀ 0.2a
0.8 ꢀ 0.2a
0.8 ꢀ 0.2a
2.8 ꢀ 0.1
1.7 ꢀ 0.1a
1.8 ꢀ 0.3a
2.9 ꢀ 0.1
2.8 ꢀ 0.1
2.3 ꢀ 0.2
2.3 ꢀ 0.3
5
ꢂ0.4 ꢀ 0.1
10
20
10
20
10
20
10
10
20
ꢂ1.2 ꢀ 0.1b ꢂ0.4 ꢀ 0.2
3
ꢂ0.7 ꢀ 0.2b ꢂ0.6 ꢀ 0.2a ꢂ0.3 ꢀ 0.2
3
9
ꢂ1.8 ꢀ 0.2b ꢂ1.3 ꢀ 0.2b ꢂ1.0 ꢀ 0.1b ꢂ1.1 ꢀ 0.1b
Vehicle
6
–
ꢂ0.1 ꢀ 0.1
ꢂ0.4 ꢀ 0.1
0.0 ꢀ 0.1
ꢂ0.1 ꢀ 0.1
ꢂ0.3 ꢀ 0.1
ꢂ0.6 ꢀ 0.2
ꢂ0.5 ꢀ 0.2
0.0 ꢀ 0.0
ꢂ0.1 ꢀ 0.1
ꢂ0.2 ꢀ 0.1
ꢂ0.1 ꢀ 0.1
5
10
5
ꢂ0.4 ꢀ 0.2
ꢂ0.9 ꢀ 0.2b ꢂ0.5 ꢀ 0.2
Vehicle
6
–
0.0 ꢀ 0.0
0.1 ꢀ 0.1
0.1 ꢀ 0.1
0.0 ꢀ 0.0
0.0 ꢀ 0.0
0.1 ꢀ 0.1
2.8 ꢀ 0.2
2.7 ꢀ 0.2
2.7 ꢀ 0.2
1.6 ꢀ 0.1a
1.0 ꢀ 0.3a
0.3 ꢀ 0.2a
7
ꢂ0.5 ꢀ 0.2
ꢂ0.4 ꢀ 0.1
10
20
10
20
0.1
10
ꢂ1.0 ꢀ 0.1b ꢂ0.7 ꢀ 0.2a ꢂ0.8 ꢀ 0.2a ꢂ0.1 ꢀ 0.2
7
Vehicle
9
–
0.0 ꢀ 0.1
0.1 ꢀ 0.1
0.0 ꢀ 0.2
ꢂ0.1 ꢀ 0.1
0.1 ꢀ 0.1
ꢂ0.1 ꢀ 0.1
0.0 ꢀ 0.1
5
ꢂ0.7 ꢀ 0.3a
WAY 100635
10
0.1
ꢂ2.0 ꢀ 0.3b ꢂ0.6 ꢀ 0.1b ꢂ0.6 ꢀ 0.2a ꢂ0.5 ꢀ 0.2
WAY 100635
0.2 ꢀ 0.1
0.2 ꢀ 0.1
0.1 ꢀ 0.1
0.2 ꢀ 0.1
The investigated compounds (ip) and WAY 100635 (sc) were administrated 15 min
before the test (A), or 45 min before 8-OH-DPAT (1 mg/kg, sc) (B).
The investigated compounds (ip) and WAY 100635 (sc) were administered 30 min
before the test. The absolute mean initial body temperatures were within a range of
36.1 ꢀ 0.5 ꢁC.
a
p < 0.01 vs. vehicle (A) or vs. vehicle þ 8-OH-DPAT (B).
a
p < 0.05.
p < 0.01 vs. vehicle.
3.2.4. Forced swimming test in rats
b
Compounds 2 and 3 (10 and 20 mg/kg) reduced the immobility
time in mice. Activity of 2 and 3 in that model was comparable with
the effect of Imipramine (Table 7). Compound 9 (20–30 mg/kg) was
practically inactive in that test.
not change effects of 8-OH-DPAT in this test; while 5-HT1A receptor
antagonist WAY 100635 almost completely blocked that effect.
The results of the in vivo studies described above demonstrated
that the tested compounds present a diversified 5-HT1A receptor
functional activity (Table 5).
3.2.5. Locomotor activity in mice
Compound 2 at doses active in the four-plate and forced
swimming tests (20–30 mg/kg), potently reduced locomotor
activity of mice (during 6-min, as well as 30-min experimental
sessions). In this test compound 3 induced sedation only at the dose
of 30 mg/kg (Table 8).
3.2.3. Four-plate test in mice
Compound 2 (at doses 10–20 mg/kg) increase of spontaneous
punished crossings in the four-plate test in mice. However its effect
was weaker, in terms of its potency and active doses, than that
produced by Diazepam, used as reference drug (Table 6).
Compounds 3 and 9 were inactive in that test.
4. Result and discussion
The in vitro radioligand binding studies with arylpiperazines
containing a terminal imidazo[2,1-f]theophylline fragment and
trimethylene aliphatic spacer (2–9) showed that these compounds
were potent ligands at 5-HT1A recognition sites. The radioligand
binding data on the 5-HT1A receptor affinities showed large
differences and depend on both, 1-arylpiperazinylpropyl moiety
and the fragment of imidazo[2,1-f]theophylline. The chemical
structure of the terminal imidazo[2,1-f]theophylline moiety influ-
ences the binding parameters. The affinity of 2-methoxy- and
3-chloro-phenylpiperazinylpropyl derivatives for 5-HT1A receptor
was the lowest for unsubstituted at 7-position of 1,3-dimethyl-
(1H,8H)-imidazo[2,1-f]purine-2,4-dione derivatives (2–4) and one
inactive compound 4 belonged to this group (Ki ¼ 677.7 nM). The
most potent for 5-HT1A receptor was compound with phenyl
moiety at 7-position of the 1,3-dimethyl-(1H,8H)-imidazo[2,1-
f]purine-2,4-dione (9), and with substituted phenylpiperazine ring
(Ki ¼ 5.6 nM). Replacement of phenyl (9) by methyl moiety (6) in
7-position of the terminal tricyclic fragment afforded the 3-fold less
Table 3
Effect of WAY 100635 on the hypothermia induced by compounds 2, 3, 6, 7, 9 or
8-OH-DPAT in mice.
Treatment and dose (mg/kg)
D
t ꢀ SEM (ꢁC)
30 min
60 min
Vehicle þ vehicle
0.1 ꢀ 0.1
ꢂ1.2 ꢀ 0.2a
ꢂ0.3 ꢀ 0.1b
ꢂ0.1 ꢀ 0.1
ꢂ0.3 ꢀ 0.1
ꢂ0.1 ꢀ 0.1
Vehicle þ 2 (10)
WAY 100635 (0.1) þ 2 (10)
Vehicle þ vehicle
0.1 ꢀ 0.1
ꢂ1.8 ꢀ 0.2a
ꢂ0.9 ꢀ 0.2a,b
ꢂ0.1 ꢀ 0.1
ꢂ1.3 ꢀ 0.2a
ꢂ0.5 ꢀ 0.2b
Vehicle þ 3 (20)
WAY 100635 (0.1) þ 3 (20)
Vehicle þ vehicle
0.0 ꢀ 0.1
ꢂ0.9 ꢀ 0.1a
ꢂ1.0 ꢀ 0.2a
0.1 ꢀ 0.1
ꢂ0.6 ꢀ 0.1a
ꢂ0.9 ꢀ 0.1a
Vehicle þ 6 (10)
WAY 100635 (0.1) þ 6 (10)
Vehicle þ vehicle
0.0 ꢀ 0.1
ꢂ1.3 ꢀ 0.2a
ꢂ0.9 ꢀ 0.1a
ꢂ0.1 ꢀ 0.1
ꢂ1.2 ꢀ 0.2a
ꢂ0.5 ꢀ 0.1b
Vehicle þ 7 (10)
WAY 100635 (0.1) þ 7 (10)
Vehicle þ vehicle
0.1 ꢀ 0.1
ꢂ2.0 ꢀ 0.3a
ꢂ0.9 ꢀ 0.2a,b
0.1 ꢀ 0.1
ꢂ0.6 ꢀ 0.1a
0.1 ꢀ 0.1b
Table 5
Vehicle þ 9 (10)
Functional in vivo 5-HT1A-receptor activity of the investigated compounds.
WAY 100635 (0.1) þ 9 (10)
Cmpd
5-HT1A activity
Presynaptic
Vehicle þ 8-OH-DPAT (5)
ꢂ1.2 ꢀ 0.1a
ꢂ0.8 ꢀ 0.2a
Postsynaptic
WAY 100635 (0.1) þ 8-OH-DPAT (5)
ꢂ0.1 ꢀ 0.1b
0.1 ꢀ 0.1b
2
3
6
7
9
Agonist
Agonist
Non-active
Agonist
Agonist
Partial agonist
Agonist
Non-active
Antagonist
Agonist
WAY 100635 was administered (sc) 15 min before the investigated compounds (ip)
or 8-0H-DPAT (sc). The absolute mean initial body temperatures were within
a range of 36.4 ꢀ 0.5 ꢁC.
a
p < 0.01 vs. vehicle þ vehicle.
b
p < 0.01 vs. vehicle þ investigated compound.

4292
A. Zago´rska et al. / European Journal of Medicinal Chemistry 44 (2009) 4288–4296
Table 6
to those receptors (Ki in micromolar range). The present study
indicates that the five-membered heterocyclic ring at 7,8-position
of theophylline improved 5-HT1A receptor affinity especially in the
case of 2-methoxyphenylpiperazinylpropyl derivatives.
The results of the in vivo studies demonstrated that the hypo-
thermia induced by 2, 3, 7, 9 or 8-OH-DPAT was reduced by WAY
100635, a 5-HT1A receptor antagonist, hence those compounds may
be classified as presynaptic 5-HT1A receptor agonists. At the same
time, WAY 100635 did not change the hypothermic effect induced
by 6, so it seems that a contribution of presynaptic 5-HT1A receptor
to this effect should be excluded.
In behavioral experiment used to assess the function at post-
synaptic 5-HT1A receptors, compounds 2, 3 and 9 given alone
induced weak LLR in rats, while 6 and 7 did not mimic the effect of
8-OH-DPAT in that test. The LLR induced by 8-OH-DPAT was
partially reduced by 2 and 7, the remaining compounds (3, 6 and 9)
did not change effects of 8-OH-DPAT in this test; while 5-HT1A
receptor antagonist WAY 100635 almost completely blocked that
effect. The results obtained in the LLR model indicate that
compounds 3 and 9 might be regarded as potential agonists of
postsynaptic 5-HT1A receptors, whereas compound 2 behaves like
partial agonist and compound 7 like antagonist of these sites (6 was
inactive in that model).
The effect of compounds 2, 3, 9 and Diazepam in the four-plate test in mice.
Treatment
Dose mg/kg
Number of punished crossings mean ꢀ SEM
Vehicle
2
–
10
20
3.8 ꢀ 0.4
4.7 ꢀ 0.4
6.3 ꢀ 0.6b
F(2.27) ¼ 7.490
p < 0.01
Vehicle
3
–
20
30
3.0 ꢀ 0.3
2.5 ꢀ 0.3
2.5 ꢀ 0.4
F(2.27) ¼ 0.681
Ns
Vehicle
9
–
20
30
3.0 ꢀ 0.3
3.5 ꢀ 0.4
3.2 ꢀ 0.6
F(2.27) ¼ 0.328
Ns
Vehiclec
Diazepam
–
3.5 ꢀ 0.4
1.25
2.5
5
5.5 ꢀ 0.5a
6.8 ꢀ 0.6b
6.7 ꢀ 0.6b
F(3.36) ¼ 9.514
p < 0.001
The investigated compounds were administered 30 min, while Diazepam 60 min
The obtained results indicate that compound 2, but not 3 and 9,
exerts anxiolytic-like activity in the four-plate test in mice;
however its effect was weaker, than that produced by Diazepam,
used as reference anxiolytic drug. The results of our successive
experiments also show that compounds 2 and 3, but not 9, behaved
like antidepressants in the forced swimming test in mice; both
compounds – like a typical antidepressant Imipramine – shortened
the immobility time in mice. It is noteworthy that activity of 2 and 3
in that model was comparable with the effect of Imipramine. It has
also been demonstrated that 2, at doses active in the four-plate and
before the test. n ¼ 10 mice per group.
a
p < 0.05.
b
p < 0.01 vs. vehicle. Ns: not significant.
Data taken from ref [42].
c
of affinity for those sites (Ki ¼ 16.8 nM). The amide derivatives of
1,3-dimethyl-6,7-dihydroimidazo[2,1-f]purine-2,4-(1H,3H)-dione-
7-carboxylic acid (11–13) were generally not active at 5-HT1A
recognition sites.
The 5-HT2A receptor affinity of the target compounds was low
Ki > 1000 nM, whereas 4 displayed higher affinity for that receptor
subtype than for 5-HT1A (Ki ¼ 91.5 nM). Compounds 2–9 evaluated
for their D2 receptor affinity, were demonstrated low or very low
affinity (from Ki ¼ 143 ꢀ10.6 nM to 839.0 ꢀ 61 nM), or did not bind
Table 8
The effect of compounds 2, 3, Diazepam and Imipramine on the locomotor activity of
mice.
Treatment
Dose mg/kg
Locomotor activity: number of crossings
during:
Table 7
The effect of compounds 2, 3, 9 and Imipramine in the forced swimming test in mice.
6 min
30 min
Treatment
Dose mg/kg
Immobility time (s), mean ꢀ SEM
Vehicle
2
–
20
30
360.9 ꢀ 19.0
146.4 ꢀ 17.9b
139.2 ꢀ 21.9b
F(2.24) ¼ 41.03
p < 0.001
973.1 ꢀ 70.5
255.6 ꢀ 25.2b
221.0 ꢀ 31.8b
F(2.24) ¼ 81.956
p < 0.001
Vehicle
2
–
159.9 ꢀ 11.4
147.5 ꢀ 22.4
87.9 ꢀ 17.3a
88.9 ꢀ 15.3a
F(3.28) ¼ 4.966
p < 0.01
10
20
30
Vehicle
3
–
20
30
324.3 ꢀ 19.7
353.4 ꢀ 15.5
198.0 ꢀ 20.1b
F(2.21) ¼ 19.850
p < 0.001
931.1 ꢀ 101.5
925.8 ꢀ 114.2
567.6 ꢀ 50.8a
F(2.21) ¼ 5.018
p < 0.05
Vehicle
3
–
166.3 ꢀ 6.5
134.2 ꢀ 13.0
104.8 ꢀ 18.4a
101.3 ꢀ 5.2a
F(3.28) ¼ 6.363
p < 0.01
10
20
30
Vehicle
Diazepam
917.9 ꢀ 59.1
686.3 ꢀ 72.1
666.8 ꢀ 87.3
589.5 ꢀ 76.7a
F(3.36) ¼ 3.477
p < 0.05
1.25
2.5
5
NT
Vehicle
9
–
20
30
166.6 ꢀ 11.6
169.3 ꢀ 8.2
172.0 ꢀ 9.3
F(2.21) ¼ 0.075
Ns
Vehicle
Imipramine
340.6 ꢀ 34.1
338.0 ꢀ 36.4
314.9 ꢀ 24.1
300.9 ꢀ 34.5
F(3.36) ¼ 0.3398
ns
892.7 ꢀ 76.6
876.8 ꢀ 67.0
625.3 ꢀ 53.6a
602.3 ꢀ 71.6a
F(3.36) ¼ 5.361
p < 0.01
10
20
30
Vehicle
Imipramine
–
167.1 ꢀ 6.7
149.1 ꢀ 10.7
107.8 ꢀ 12.4a
76.4 ꢀ 7.1a
10
20
30
F(3.36) ¼ 18.200
p < 0.001
The investigated compounds and Imipramine were administered 30 min, while
Diazepam 60 min before the test. n ¼ 8–10 mice per group. NT: not tested. ns: not
significant.
The investigated compounds and Imipramine were administered 30 min before the
a
test. n ¼ 8–10 mice per group.
p < 0.05.
p < 0.01 vs. vehicle.
a
b
p < 0.01 vs. vehicle.

A. Zago´rska et al. / European Journal of Medicinal Chemistry 44 (2009) 4288–4296
4293
forced swimming tests, potently reduced locomotor activity of mice
during a 6-min, as well as 30-min experimental sessions, while 3
induced sedation only at the higher dose used. In comparison,
Diazepam produces sedative effect in mice at dose 4-fold higher
than that evoking a minimal, but statistically significant, anxiolytic-
like effects, whereas Imipramine at doses active in the forced
swimming test induces a weak sedation in mice only when loco-
motor activity was recorded for 30 min. The sedative effects exerted
by compounds 2 and 3 excluded these ligands from being regarded
as potential drugs.
represented by the following abbreviations: s (singlet), d (doublet),
dd (double doublet), m (multiplet), t (triplet), br s (broad singlet).
The mass spectra (MS) were recorded on MDX SCIEX API 2000
(Concord, ON, Canada).
6.1.2. General procedure for the synthesis of 8-N4-arylpiperazin-
N1-yl-propyl derivatives of 1,3-dimethyl-(1H,8H)-imidazo[2,1-f]
purine-2,4-dione (2–10)
A mixture of compound 1a–c [24] (0.005 mol) and 0.01 mol of
suitable arylpiperazinylpropylamine in 20 ml of 2-methoxyethanol
was refluxed for 6 h. The products were isolated from the mixture
according to the method: A or B. Method A: A reaction mixture was
refrigerated (ꢂ15 ꢁC) for 12 h. The precipitated product was filtered
off, washed with a small amount of cold anhydrous ethanol and
recrystallized from ethanol. Method B: From the reaction mixture
the solvent was distilled under reduced pressure and to the oily
residue 50 ml of acetone was added. The precipitated product was
filtered off; the filtrate was evaporated and the oily residue was
dissolved in anhydrous ethanol (20 ml). After cooling the separated
product was recrystallized from ethanol.
5. Conclusion
The present study indicates that the reduction of the ring size
from six to five member at 7,8-position of the theophylline had
limited influence on receptors affinity and the binding profile, in
comparison to the pyrimido[2,1-f]purine analogs. The impact of
a
double bond of the five-membered ring of imidazo[2,1-
f]theophylline moiety was evaluated. We established in turn that
the double bond in imidazo[2,1-f]theophylline had the most
favourable influence on the affinity of derivatives for the 5-HT1A
receptor. The arylpiperazinylalkyl amides of imidazo[2,1-
f]theophylline-7-carboxylic acid were inactive for 5-HT1A receptors.
Compounds showed high invitroactivityat 5-HT1A receptorsand
its intrinsic in vivo activity at this receptor subtype was diversified.
8-[3-(N4-Phenyl)-piperazin-N1-yl-propyl]-1,3-dimethyl-(1H,8H)-
imidazo[2,1-f]purine-2,4-dione (2) exerts anxiolytic-like activity
in the four-plate test in mice; however its effects were weaker,
in terms of its potency and active doses, than that produced by
Diazepam, used as reference anxiolytic drug. This compound
and 8-[3-(N4-20-methoxyphenyl)-piperazin-N1-yl-propyl]-1,3-
dimethyl-(1H,8H)-imidazo[2,1-f]purine-2,4-dione (3) behaved
like antidepressants in the forced swimming test in mice and
their activity in that model was comparable with the effect of
Imipramine. It has also been demonstrated the sedative effects
exerted by those compounds at doses active in the four-plate
and forced swimming tests. The obtained results suggested that
the LCAPs linked to tricyclic derivatives of the theophylline
remain a worthy of future research for obtaining new deriva-
tives with potential anxiolytic/antidepressant activity.
6.1.2.1. 8-[3-(N4-phenyl)-piperazin-N1-yl-propyl]-1,3-dimethyl-
(1H,8H)-imidazo[2,1-f]purine-2,4-dione (2). Method A, yield 60%,
M.p. 189–190 ꢁC, 1H NMR (CDCl3)
d: 2.13–2.20 (m, 2H, CH2CH2CH2),
2.45–2.47 (t, J ¼ 13.2 Hz, 2H, CH2CH2CH2), 2.49–2.64 (m, 4H,
(CH2)2N1pip), 3.23–3.26 (m, 4H, N4pip(CH2)2), 3.47 (s, 3H, N3CH3),
3.64 (s, 3H, N1CH3), 4.19–4.24 (t, J ¼ 13.7 Hz, 2H, N8CH2), 6.86–6.98
(m, 4H, Haromat.), 7.27–7.33(m, 2H, Haromat. þ C7H), 7.44 (d, J ¼ 2.4 Hz,
1H, C6H). MS (M þ Hþ): 423.5. Anal. calcd. for C22H27N7O2: C, 62.69;
H, 6.45; N, 23.26%. Found: C, 62.33; H, 6.58; N, 23.02%.
6.1.2.2. 8-[3-(N4-20-methoxyphenyl)-piperazin-N1-yl-propyl]-1,3-
dimethyl-(1H,8H)-imidazo[2,1-f]purine-2,4-dione (3). Method A,
yield 73%, M.p. 230–231 ꢁC, 1H NMR (CDCl3)
d: 2.12–2.17 (m, 2H,
CH2CH2CH2), 2.45–2.48(t, J ¼ 13.7 Hz,2H, CH2CH2CH2),2.50–2.68(m,
4H, (CH2)2N1pip), 3.13–3.20 (m, 4H, N4pip(CH2)2), 3.47 (s, 3H, N3CH3),
3.64 (s, 3H, N1CH3), 3.89 (s, 3H, OCH3), 4.19–4.23 (t, J ¼ 13.7 Hz, 2H,
N8CH2), 6.87–7.07(m, 5H,Haromat. þ C7H), 7.43–7.44 (d, J ¼ 2.4 Hz,1H,
C6H). MS (M þ Hþ): 452.5. Anal. calcd. for C23H29N7O3: C, 61.18; H,
6.47; N, 21.71%. Found: C, 60.88; H, 6.51; N, 21.72%.
6. Experimental protocols
6.1.2.3. 8-[3-(N4-30-chlorophenyl)-piperazin-N1-yl-propyl]-1,3-
dimethyl-(1H,8H)-imidazo[2,1-f]purine-2,4-dione (4). Method A,
6.1. Chemistry
yield 69%, M.p. 244–245 ꢁC, 1H NMR (CDCl3)
d: 1.95–2.03 (m, 2H,
CH2CH2CH2), 2.50–2.53 (t, J ¼ 13.3 Hz, 2H, CH2CH2CH2), 2.55–2.70
(m, 4H, (CH2)2N1pip), 3.13–3.20 (m, 4H, N4pip(CH2)2), 3.47 (s, 3H,
N3CH3), 3.64 (s, 3H, N1CH3), 4.15–4.21 (t, J ¼ 13.7 Hz, 2H, N8CH2),
6.83–7.07 (m, 5H, Haromat. þ C7H), 7.44 (d, J ¼ 2.3 Hz, 1H, C6H). MS
(M þ Hþ): 456.9. Anal. calcd. for C22H26N7O2Cl: C,57.95; H,5.74;
N,21.50%. Found: C, 57.88; H, 5.95; N, 21.72%.
6.1.1. General remarks
All the chemicals and solvents were purchased from Merck
(Darmstad, Germany) or Fluka (Buchs, Switzerland) and were used
without further purification. Melting points (M.p.) were measured
in open capillaries on an Electrothermal 9100 melting point
apparatus without corrections. The purity of the compounds was
confirmed by the thin-layer chromatography (TLC) performed on
Kieselgel 60 F254 plates (Merck) and the solvents: benzene/acetone
(7:3) and chloroform/methanol/99.5% acetic acid (60:20:10) were
used. Flash column chromatography was carried out on SilicaGel
100-Fluka using the solvent acetic acid/ethanol (1:2). All the
compounds used (bases 2–13 and hydrochlorides 2a–13a) were
subjected to a quantitative elemental (C, H, N) analysis by a micro-
method using the elemental Vario EI III Elementar analyzer (Hanau,
Germany). The results of elemental analyses were within ꢀ0.4% of
the theoretical values. 1H NMR spectra were obtained in a Varian
Mercury spectrometer (Varian Inc., Palo Alto, CA, USA) operating at
6.1.2.4. 7-Methyl-8-[3-(N4-phenyl)-piperazin-N1-yl-propyl]-1,3-
dimethyl-(1H,8H)-imidazo[2,1-f]purine-2,4-dione (5). Method
B, yield 87%, M.p. 197–198 ꢁC, 1H NMR (CDCl3)
d: 2.12–2.16
(m, 2H, CH2CH2CH2), 2.38 (s, 3H, 7CH3) 2.43–2.48 (t, J ¼ 13.3 Hz,
2H, CH2CH2CH2), 2.61–2.64 (m, 4H, (CH2)2N1pip), 3.21–3.25
(m, 4H, N4pip(CH2)2), 3.46 (s, 3H, N3CH3), 3.63 (s, 3H, N1CH3),
4.12–4.16 (t, J ¼ 13.3 Hz, 2H, N8CH2), 6.87–6.97 (m, 3H, Haromat.),
7.18 (s, 1H, C6H), 7.26–7.30 (m, 2H, Haromat.). MS (M þ Hþ):
437.9. Anal. calcd. for C23H29N7O2: C, 63.38; H, 6.71; N, 22.51%.
Found C, 63.58; H, 6.94; N, 22.59%.
300 MHz. Chemical shifts are expressed in parts per million (
d)
6.1.2.5. 7-Methyl-8-[3-(N4-20-methoxyphenyl)-piperazin-N1-yl-propyl]
downfield of tetramethylsilane (TMS) used as an internal standard.
The J values are expressed in Hertz (Hz). Signal multiplicities are
-1,3-dimethyl-(1H,8H)-imidazo[2,1-f]purine-2,4-dione (6). Method
B, yield 85%, M.p. 195–196 ꢁC, 1H NMR (CDCl3)
d: 2.11–2.16

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A. Zago´rska et al. / European Journal of Medicinal Chemistry 44 (2009) 4288–4296
(m, 2H, CH2CH2CH2), 2.39 (s, 3H, 7CH3) 2.39–2.47 (t, J ¼ 13.3 Hz, 2H,
CH2CH2CH2), 2.49–2.67 (m, 4H, (CH2)2N1pip), 3.05–3.1 (m, 4H,
N4pip(CH2)2), 3.46 (s, 3H, N3CH3), 3.63 (s, 3H, N1CH3), 3.89
(s, 3H, OCH3), 4.12–4.16 (t, J ¼ 13.7 Hz, 2H, N8CH2), 6.88–7.07
(m, 4H, Haromat.), 7.18 (s, 1H, C6H). MS (M þ Hþ): 466.5. Anal. calcd.
for C24H31N7O3: C, 61.9; H, 6.71; N, 21.06%. Found: C, 61.72; H, 6.56;
N, 20.82%.
with an excess of triethylamine and free bases were recrystallized
from acetone.
6.1.3.1. N-2-[(N4-phenyl)-piperazin-N1-yl]-ethylamideof1,3-dimethyl-
6,7-dihydroimidazo[2,1-f]purine-2,4-(1H,3H)-dione-7-carboxylic acid
(11). Yield 51%, M.p. 215–216 ꢁC, 1H NMR (CDCl3)
d: 2.50–2.53
(t, J ¼ 13.3 Hz, 2H, CH2CH2), 2.64–2.7 (m, 4H, (CH2)2N1pip), 3.06–3.12
(m, 4H, N4pip(CH2)2), 3.20–3.30 (m, 6H, N3CH3 þ CH2CH2 þ C7Himi-
dazole), 3.55 (s, 3H, N1CH3), 4.21 (dd, J ¼ 9.9 Hz, J ¼ 5.9 Hz, 1H,
C6Himidazole), 4.44–4.5 (m, 1H, NHimidazole), 4.86 (dd, J ¼ 9.9 Hz,
J ¼ 5.9 Hz, 1H, C6Himidazole), 6.67–6.80 (m, 3H, Haromat.), 7.06–7.10 (m,
2H, Haromat.), 8.22 (br s, 1H, NH). MS (M þ Hþ): 453.52. Anal. calcd. for
C22H28N8O3: C, 58.39; H, 6.23; N, 24.76%. Found: C, 58.33; H, 6.30; N,
24.62%.
6.1.2.6. 7-Methyl-8-[3-(N4-30-chlorophenyl)-piperazin-N1-yl-propyl]
-1,3-dimethyl-(1H,8H)-imidazo[2,1-f]purine-2,4-dione (7). Method
B, yield 80%, M.p. 194–195 ꢁC, 1H NMR (CDCl3)
d: 2.10–2.16 (m, 2H,
CH2CH2CH2), 2.39 (s, 3H, 7CH3) 2.45–2.52 (m, 2H, CH2CH2CH2),
2.58–2.70 (m, 4H, (CH2)2N1pip), 3.20–3.30 (m, 4H, N4pip(CH2)2),
3.47 (s, 3H, N3CH3), 3.62 (s, 3H, N1CH3), 4.19–4.25 (t, J ¼ 12.7 Hz,
2H, N8CH2), 6.87–6.97 (m, 3H, Haromat.), 7.18 (s, 1H, C6H), 7.28–7.30
(m, 1H, Haromat.). MS (M þ Hþ): 470.9. Anal. calcd. for
C23H28N7O2Cl: C, 58.78; H, 6.00; N, 20.86%. Found: C, 58.72;
H, 6.26; N, 20.74%.
6.1.3.2. N-2-[N4-(2-methoxy)-phenyl-piperazin-N1-yl]-ethyl amide
of 1,3-dimethyl-6,7-dihydroimidazo[2,1-f]purine-2,4-(1H,3H)-dione-
7-carboxylic acid (12). Yield 51%, M.p. 217–218 ꢁC, 1H NMR (CDCl3)
d:
2.50–2.53 (t, J ¼ 13.3 Hz, 2H, CH2CH2), 2.64–2.7 (m, 4H,
6.1.2.7. 7-Phenyl-8-[3-(N4-phenyl)-piperazin-N1-yl-propyl]-1,3-
(CH2)2N1pip), 3.06–3.12 (m, 4H, N4pip(CH2)2), 3.20–3.30 (m, 6H,
N3CH3 þ CH2CH2 þ C7Himidazole), 3.55 (s, 3H, N1CH3), 3.89 (s, 3H,
OCH3), 4.21 (dd, J ¼ 9.9 Hz, J ¼ 5.9 Hz, 1H, C6Himidazole), 4.44–4.5 (m,
1H, NHimidazole), 4.86 (dd, J ¼ 9.9 Hz, J ¼ 5.9 Hz, 1H, C6Himidazole),
6.67–6.80 (m, 3H, Haromat.), 7.06–7.10 (m, 2H, Haromat.), 8.22 (br s,1H,
NH). MS (M þ Hþ): 483.5. Anal. calcd. for C23H30N8O4: C, 57.25; H,
6.27; N, 23.22%. Found: C, 57.03; H, 5.98; N, 22.91%.
dimethyl-(1H,8H)-imidazo[2,1-f]purine-2,4-dione (8). Method B,
yield 49%, M.p. 161–162 ꢁC, 1H NMR (CDCl3)
d: 1.99–2.04 (m, 2H,
CH2CH2CH2), 2.36–2.45 (m, 6H, CH2CH2CH2 þ (CH2)2N1pip), 3.05–
3.10 (m, 4H, N4pip(CH2)2), 3.48 (s, 3H, N3CH3), 3.67 (s, 3H, N1CH3),
4.24–4.29 (t, J ¼ 14.5 Hz, 2H, N8CH2), 6.87–6.94 (m, 3H, Haromat.),
7.26–7.29 (m, 2H, Haromat.), 7.46 (s, 1H, C6H), 7.49–7.55 (m, 5H,
Haromat.). MS (M þ Hþ): 499.6. Anal. calcd. for C28H31N7O2: C, 67.59;
H, 6.28; N, 19.70%. Found: C, 67.83; H, 6.18; N, 19.79%.
6.1.3.3. N-2-[N4-(3-chloro)-phenyl-piperazin-N1-yl]-ethyl amide of
1,3-dimethyl-6,7-dihydroimidazo[2,1-f]purine-2,4-(1H,3H)-dione-
7-carboxylic acid (13). Yield 58%, M.p. 203–205 ꢁC, 1H NMR (DMSO)
6.1.2.8. 7-Phenyl-8-[3-(N4-20-methoxyphenyl)-piperazin-N1-yl-propyl]
-1,3-dimethyl-(1H,8H)-imidazo[2,1-f]purine-2,4-dione (9). Method
d
:
2.7 (s, 3H, N1CH3), 3.06–3.12 (m, 9H, (CH2)2N1pip
þ
B, yield 54%, M.p. 171–172 ꢁC, 1H NMR (CDCl3)
d
: 2.00–2.04 (m, 2H,
N3CH3 þ CH2CH2), 3.31–3.42 (m, 7H, N4pip(CH2)2 þ CH2CH2 þ
C7Himidazole), 4.21 (dd, J ¼ 9.9 Hz, J ¼ 5.9 Hz, 1H, C6Himidazole), 4.22–
4.32 (m, 1H, NHimidazole), 4.86 (dd, J ¼ 9.9 Hz, J ¼ 5.9 Hz, 1H,
C6Himidazole), 6.67–6.94 (m, 2H, Haromat.), 7.16–7.20 (m, 2H, Haromat.),
8.24 (br s, 1H, NH). MS (M þ Hþ): 487.5. Anal. calcd. for
C22H27N8O3Cl: C, 54.26; H, 5.59; N, 23.01%. Found: C, 54.16; H, 5.22;
N, 22.86%.
CH2CH2CH2), 2.36–2.45 (m, 6H, CH2CH2CH2 þ (CH2)2N1pip), 3.05–
3.1 (m, 4H, N4pip(CH2)2), 3.48 (s, 3H, N3CH3), 3.67 (s, 3H, N1CH3),
3.89 (s, 3H, OCH3), 4.24–4.29 (t, J ¼ 13.8 Hz, 2H, N8CH2), 6.87–6.94
(m, 3H, Haromat.), 7.26–7.29 (m, 1H, Haromat.), 7.46 (s, 1H, C6H), 7.49–
7.55 (m, 5H, Haromat.). MS (M þ Hþ): 528.6. Anal. calcd. for
C29H33N7O3: C, 67.07; H, 6.30; N,18.58%. Found: C, 66.94; H, 6.56; N,
18.72%.
6.1.4. Preparation of hydrochlorides (2a–13a)
The bases (2–13) were dissolved in an excess (2 ml per
0.001 mol) of conc. HCl. Then anhydrous ethanol was added until
salt precipitation was observed. The product was recrystallized
from anhydrous ethanol.
6.1.2.9. 7-Phenyl-8-[3-(N4-30-chlorophenyl)-piperazin-N1-yl-propyl]
-1,3-dimethyl-(1H,8H)-imidazo[2,1-f]purine-2,4-dione (10). Method
B, yield 51%, M.p. 228–229 ꢁC, 1H NMR (CDCl3)
d: 1.99–2.04 (m, 2H,
CH2CH2CH2), 2.36–2.45 (m, 6H, CH2CH2CH2 þ (CH2)2N1pip), 3.05–3.1
(m, 4H, N4pip(CH2)2), 3.48 (s, 3H, N3CH3), 3.67 (s, 3H, N1CH3), 4.24–
4.29 (t, J ¼ 13.3 Hz, 2H, N8CH2), 6.87–6.94 (m, 3H, Haromat.), 7.26
(s, 1H, Haromat.), 7.46 (s, 1H, C6H), 7.49–7.55 (m, 5H, Haromat.). MS
(M þ Hþ): 533.0. Anal. calcd. for C28H30N7O2Cl: C, 63.79; H, 5.91; N,
17.95%. Found: C, 63.94; H, 6.06; N, 18.02%.
6.2. In vitro experiments
Radioligand binding experiments were conducted in the rat
brain tissue (cerebral cortex tissue for 5-HT1A, 5-HT2A and striatum
tissue for D2) according to the published procedures [37,38,39]. The
following radioligands were used: [3H]-8-Hydroxy-2-(di-n-propy-
lamino)-tetralin ([3H]-8-OH-DPAT, 106 Ci/mmol, NEN Chemicals),
[3H]-Ketanserin (60 Ci/mmol, NEN Chemicals) and [3H]-Spiperone
(15 Ci/mmol, NEN). Radioactivity was measured in a WALLAC 1409
DSA – liquid scintillation counter. Ki values were determined on the
basis of at least three competition binding experiments in which 10
drug concentrations, run in triplicate, were used. The Cheng and
Prusoff equation was used for Ki calculations. Radioligand binding
data were analyzed using interactive curve fitting routines
(GraphPAD/Prism, Version 3.0, San Diego, CA, USA).
6.1.3. General procedure for the synthesis of 11–13
1-Hydroxy-1H-benzotriazole (HOBt) (0.8 g, 5.8 mmol) was
added to the mixture of acid 1d [26] (1.0 g, 3.8 mmol) and 1.0 g
arylpiperazinylalkylamine in 20 ml of dichloromethane. A mixture
was stirred for 30 min in 0 ꢁC then N,N0-dicyclohexylcarbodiimide
(DCC) (1.2 g, 5.8 mmol) was added and the mixture was stirred at
room temperature for 12 h. The solvent was distilled, the solid
residue was washed with 10% NaOH (30 ml) and filtered off. The
precipitate was treated with 20 ml of acetic acid and the mixture
was stirred at room temperature for 15 min, diluted with water
(20 ml), the precipitate was filtered off. The filtrate was evaporated
to the oily residue which was purified by flash column chroma-
tography on silica gel, using acetic acid/ethanol (1:2 v/v) as eluent.
The obtained products, in the form of acetic acid salt, were treated
6.2.1. 5-HT1A receptor-binding experiments
The membrane preparation and the assay procedure were
carried out according to the published procedure [37] with slight
modifications. [3H]-8-OH-DPAT was used for labeling 5-HT1A

A. Zago´rska et al. / European Journal of Medicinal Chemistry 44 (2009) 4288–4296
4295
receptors. Briefly, the cerebral cortex tissue was homogenized in
20 vol. of 50 mM tris–HCl buffer (pH 7.7 at 25 ꢁC) using ULTRA
TURAX homogeniser, and was then centrifuged at 32000 ꢃ g for
10 min. The supernatant fraction was discarded, and pellet was
resuspended in the same volume of tris–HCl buffer and was then
centrifuged. Before the third centrifugation, the samples were
incubated at 37 ꢁC for 10 min. The final pellet was resuspended in
100635, synthesized by Dr. J. Boksa, Institute of Pharmacology,
´
Polish Academy of Sciences, Krakow, Poland) and Imipramine
(hydrochloride, Polfa-Starogard, Poland) were dissolved in distilled
´
water. Diazepam (Polfa-Poznan, Poland) and the investigated
compounds (2, 3, 6, 7 and 9) were suspended in 1% aqueous solu-
tion of Tween 80. 8-OH-DPAT and WAY 100635 were injected
subcutaneously (sc), Diazepam, Imipramine and the tested
compounds were given intraperitoneally (ip) in a volume of 2 ml/kg
(rats) or 10 ml/kg (mice). Each experimental group consisted of six
to ten animals, and all the animals were used only once.
tris–HCl buffer containing 10
ascorbic acid. One milliliter of the tissue suspension (9 mg of wet
weight), 100 l of 10 M serotonin (for unspecific binding), 100 l of
[3H]-8-OH-DPAT and 100
l of analyzed compound were incubated
mM Pargyline, 4 mM CaCl2 and 0.1%
m
m
m
m
at 37 ꢁC for 15 min. The incubation was followed by a rapid vacuum
filtration through Whatman GF/B glass filters, and was when
washed 3 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 concen-
tration was 1 nM, and the concentrations of the analyzed
compounds ranged from 10ꢂ10 to 10ꢂ4 M.
6.3.1. Body temperature in mice
Effects of the tested compounds given alone on the rectal body
temperature in mice (measured with an Ellab thermometer) were
recorded 30, 60, 90, and 120 min after their administration. In
separate experiment the effect of WAY 100635 (0.1 mg/kg) on the
hypothermia induced by compounds 2, 3, 6, 7 and 9 or 8-OH-DPAT
was tested. WAY 100635 was administered 15 min before the
compounds or 8-OH-DPAT and rectal body temperature was
recorded 30 and 60 min after injection of the tested compounds.
6.2.2. 5-HT2 receptor-binding experiments
The assay was performed according to the method of Laysen
et al. [38] with slight modifications. [3H]-ketanserin was used for
labeling 5-HT2 receptors. The cerebral cortex tissue was homoge-
nized in 20 vol. of 50 mM tris–HCl buffer (pH 7.7 at 25 ꢁC) and
centrifuged at 32000 ꢃ g for 20 min. The resulting pellet was
resuspended in the same quantity of the buffer, preincubated at
37 ꢁC for 10 min and centrifuged for 20 min. The final pellet was
resuspended in 50 vol. of the same buffer. One milliliter of the
The results were expressed as a change in body temperature (
Dt)
with respect to the basal body temperature, as measured at the
beginning of the experiment.
6.3.2. Lower lip retraction (LLR) in rats
LLR was assessed according to the method described by
Berendsen et al. [28]. The rats were individually placed in cages
(30 cm ꢃ 25 cm ꢃ 25 cm) and they were scored three times (at 15,
30 and 45 min) after the administration of the tested compounds or
8-OH-DPAT as follows: 0 ¼ lower incisors not visible, 0.5 ¼ partly
visible, 1 ¼ completely visible. The total maximum scores amoun-
ted to 3 for each rat. In a separate experiment, the effect of the
tested compounds or WAY 100635 on the LLR induced by 8-OH-
DPAT (1 mg/kg) was tested. The compounds 2, 3, 6, 7, 9 and WAY
100635 were administered 45 min and 15 min respectively before
8-OH-DPAT and the animals were scored 15, 30 and 45 min after 8-
OH-DPAT administration.
tissue suspension, 100
m
l of 1
mM Mianserin (displacer), 100 ml of
[3H]-Ketanserin and 100
ml of the analyzed compound were incu-
bated at 37 ꢁC for 20 min, followed by a rapid vacuum filtration
through Whatman GF/B glass filters, and were then washed three
times with 5 ml of a cold tris–HCl buffer. The final [3H]-Ketanserin
concentration was 0.6 nM and the concentrations of analyzed
compounds ranged from 10ꢂ10 to 10ꢂ4 M.
6.2.3. D-2 receptor-binding experiments
The assay was performed according to the method described
before [39]. Rat striatum tissue was thawed in 50 vol. of ice-cold
50 mM potassium phosphate buffer, pH 7.4, homogenized and
centrifuged at 20000 ꢃ g for 20 min. The resulting pellet was
resuspended in the same quantity of the buffer, and centrifuged
again for 20 min. Assay tubes contained membrane suspension
(3 mg of wet weight), [3H]-Spiperone (15 Ci/mmol, NEN) in
6.3.3. Four-plate test in mice
The box was made of an opaque plastic and was rectangular
(25 cm ꢃ 18 cm ꢃ 16 cm) in shape. The floor was covered with four
rectangular metal plates (11 cm ꢃ 8 cm), separated by a 4 mm gap.
The plates were connected to a source of continuous current which
enabled a 120 V difference of potential between two adjacent
plates for 0.5 s when the experimenter pressed the switch. Indi-
vidual mice were placed gently onto the plate, and were allowed to
explore for 15 s. Afterwards, each time a mouse passed from one
plate to another, the experimenter electrified the whole floor which
evoked a visible flight reaction of the animal. If the animal
continued running, it received no new shocks for the following 3 s.
The episodes of punished crossing were counted for 60 s [40].
concentration of 1 nM, (þ)Butaclamol (10
mM) or analyzed
compound in a final volume of 0.5 ml. Samples were incubated for
30 min in 37 ꢁC. The incubation was terminated by rapid filtration
over glass fiber filters (Whatman GF/B) using Brandel manifold. The
filters were then washed 2 times with 5 ml ice-cold buffer. The
filters obtained from the above-described procedures were placed
in scintillation vials with liquid scintillation cocktail.
6.3. In vivo experiments
6.3.4. Forced swimming test in mice
The experiments were performed on male Wistar rats (290–
310 g) or male Albino Swiss mice (24–28 g). Before the experiment
animals were kept at a room temperature of 20 ꢀ 1 ꢁC, and had free
access to food (standard laboratory pellets) and tap water. All the
investigations were conducted in the light phase, on a natural day–
night cycle (from February to May), between 9 a.m. and 2 p.m. All
the experimental procedures were approved by the local Bioethics
Commission at the Institute of Pharmacology, Polish Academy of
The experiment was carried out according to the method of
Porsolt et al. [41]. Briefly, 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 there for 6 min. A mouse was
regarded as immobile when it remained floating on the water,
making only small movements to keep its head above it. The total
duration of immobility was recorded during the last 4 min of a
6-min test session.
´
Sciences in Krakow. 8-Hydroxy-2-(di-n-propylamino)tetralin
(hydrobromide, 8-OH-DPAT, Tocris, Cookson Ltd., UK) was dis-
solved in saline, N-{2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl}-
N-(2-pyridinyl)cyclohexanecarboxamide (trihydrochloride, WAY
6.3.5. Locomotor activity in mice
The spontaneous locomotor activity of mice was recorded by
Opto-M3 multi-channel activity monitor (MultiDevice Software v.

4296
A. Zago´rska et al. / European Journal of Medicinal Chemistry 44 (2009) 4288–4296
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´
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This work was supported by Grant KBN No. 2P05F04226 from
the State Committee for Scientific Research, Poland.
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