New 8-aminoalkyl derivatives of purine-2,6-dione with arylalkyl, allyl or propynyl substituents in position 7, their 5-HT1A, 5-HT2A, and 5-HT7 receptor affinity and pharmacological evaluation

Article abstract of DOI:10.1016/S1734-1140(13)70960-5

Background: Our previous studies in a group of arylpiperazine derivatives of 1,3-dimethyl-3,7-dihydro-purine-2,6-diones, aimed at chemical diversification of the purine-2,6-dione by introduction of hydrophobic substituent in a 7- or 8- position or elongation of the linker length between arylpiperazine and purine core, allowed a selection of potent 5-HT_1A, 5-HT_2A and 5-HT₇ receptor ligands displaying anxiolytic and antidepressant properties. Continuing our research in this field, in the present studies we designed a new series of 8-aminoalkylamino (15-35) and 8-arylpiperazinylpropoxy (36-42) derivatives of 7-substituted 1,3-dimethyl-3,7-dihydropurine-2,6-dione as potential 5-HT_1A, 5-HT_2A and 5-HT₇ receptor ligands with potential psychotropic activity. Methods: Radioligand binding assays were employed for determining the affinity and the selectivity profile of the synthesized compounds for native 5-HT_1A, 5-HT_2A, and cloned 5-HT₆ and 5-HT₇ receptors. The functional activity of the selected compounds at 5-HT_1A and 5-HT_2A receptors was tested in the commonly used in vivo models. Antidepressant and anxiolytic properties were evaluated in the forced swim (FST) and the four-plate test (FPT) in mice, respectively. Results: Among the evaluated series, selected 7-benzyl-8-((4-(4-(3-chlorophenyl)piperazin-1-yl)butyl)amino)-1, 3-dimethyl-1H-purine-2,6(3H,7H)-dione (21), a mixed 5-HT_1A/5-HT _2A/5-HT₇ receptor ligand, produced an antidepressant-like effect in FST, and exerted anxiolytic-like activity in FPT. Another pharmacologically evaluated compound 42 (a mixed 5-HT_1A/5-HT ₇ ligand) slightly, but non-significantly attenuated the immobility time of mice in FST and was devoid of activity in FPT. Conclusions: Study revealed advantage of mixed 5-HT_1A/5-HT_2A/5-HT₇ receptor ligands over 5-HT_1A/5-HT₇ agents to display antidepressant-and anxiolytic-like activity. Modification of arylalkyl/allyl substituent in position 7 of purine-2,6-dione opens possibility for designing new 5-HT ligands with preserved p electron system and lower molecular weight. Copyright

Full text of DOI:10.1016/S1734-1140(13)70960-5

New 8-aminoalkyl derivatives of purine-2,6-dione
with arylalkyl, allyl or propynyl substituents in
position 7, their 5-HT1A, 5-HT2A, and 5-HT7
receptor affinity and pharmacological evaluation
1
1
2
Gra¿yna Ch³oñ-Rzepa , Pawe³ ¯mudzki , Grzegorz Sata³a ,
2
3
3
Beata Duszyñska , Anna Partyka , Dagmara Wróbel ,
3
3
2
Magdalena Jastrzêbska-Wiêsek , Anna Weso³owska , Andrzej J. Bojarski ,
1
1
Maciej Paw³owski , Pawe³ Zajdel
Department of Medicinal Chemistry, Department of Clinical Pharmacy, Jagiellonian University Medical College,
Medyczna 9, PL 30-688 Kraków, Poland
Department of Medicinal Chemistry, Institute of Pharmacology, Polish Academy of Sciences, Smêtna 12,
PL 31-343 Kraków, Poland
Correspondence: Pawe³ Zajdel: e-mail: pawel.zajdel@uj.edu.pl
Abstract:
Background: Our previous studies in a group of arylpiperazine derivatives of 1,3-dimethyl-3,7-dihydro-purine-2,6-diones, aimed
at chemical diversification of the purine-2,6-dione by introduction of hydrophobic substituent in a 7- or 8- position or elongation of
the linker length between arylpiperazine and purine core, allowed a selection of potent 5-HT1A, 5-HT2A and 5-HT7 receptor ligands
displaying anxiolytic and antidepressant properties. Continuing our research in this field, in the present studies we designed a new
series of 8-aminoalkylamino (15–35) and 8-arylpiperazinylpropoxy (36–42) derivatives of 7-substituted 1,3-dimethyl-3,7-dihydro-
purine-2,6-dione as potential 5-HT1A, 5-HT2A and 5-HT7 receptor ligands with potential psychotropic activity.
Methods: Radioligand binding assays were employed for determining the affinity and the selectivity profile of the synthesized com-
pounds for native 5-HT1A, 5-HT2A, and cloned 5-HT6 and 5-HT7 receptors. The functional activity of the selected compounds at
5-HT1A and 5-HT2A receptors was tested in the commonly used in vivo models. Antidepressant and anxiolytic properties were evalu-
ated in the forced swim (FST) and the four-plate test (FPT) in mice, respectively.
Results: Among the evaluated series, selected 7-benzyl-8-((4-(4-(3-chlorophenyl)piperazin-1-yl)butyl)amino)-1,3-dimethyl-
1H-purine-2,6(3H,7H)-dione (21), a mixed 5-HT1A/5-HT2A/5-HT7 receptor ligand, produced an antidepressant-like effect in FST,
and exerted anxiolytic-like activity in FPT. Another pharmacologically evaluated compound 42 (a mixed 5-HT1A/5-HT7 ligand)
slightly, but non-significantly attenuated the immobility time of mice in FST and was devoid of activity in FPT.
Conclusions: Study revealed advantage of mixed 5-HT1A/5-HT2A/5-HT7 receptor ligands over 5-HT1A/5-HT7 agents to display an-
tidepressant- and anxiolytic-like activity. Modification of arylalkyl/allyl substituent in position 7 of purine-2,6-dione opens possibil-
ity for designing new 5-HT ligands with preserved p electron system and lower molecular weight.
Key words:
purine-2,6-diones, 5-HT , 5-HT , 5-HT receptor ligands, depression, forced swim test, anxiety, four-plate test
15

R
O
N
N
m
O
O
N
N
N
O
N
N
N
n
N
N
N
N
N
N
O
O
1: m = 2, n = 1, R = H
2: m = 1, n = 1, R = H
3: m = 1, n = 2, R = H
5
Fig. 1. Chemical structure of the initial model compounds 1–5
4: m = 1, n = 1, R = 2-OCH3
structural modifications of pyrimido- or diazepino-
[2,1-f]-purines on the 5-HT receptors affinity and
5-HT /5-HT selectivity; in consequence, pharma-
cological assays showed that compound 4 behaved as
presynaptic agonist and postsynaptic partial agonist of
5-HT receptors, while 5 as pre- and post-synaptic
agonist of 5-HT receptors, respectively [20]. It has
been shown that different functional activity of these
compounds can be attributed to the enlargement of the
p system in 5 vs. 4 and/or to the diminished flexibility
of the heterocyclic fragment [20].
Introduction
A diversity of psychiatric disorders have been associ-
ated with dysfunction of serotonin-containing neurons,
including depression, anxiety, schizophrenia, bipolar
disorder, and Parkinson’s disease. Modulation of these
serotonin (5-HT) pathways, ranging from activation to
blockade of 5-HT receptor subtypes, gave basis for de-
velopment of several classes of psychotropic drugs or
designing compounds being under clinical trials.
Among 5-HT receptor subtypes, 5-HT , 5-HT ,
and 5-HT receptors have focused our interest as suit-
able targets for treatment of depression and anxiety [3,
15, 21, 22]. For several years we have been interested
in developing agents generally classified as long-chain
arylpiperazines (LCAPs) containing a different am-
ide/imide terminal fragment, which were mainly evalu-
ated towards 5-HT and 5-HT receptors, and re-
cently 5-HT [5, 7, 8, 19, 20, 28, 29]. Some of our pre-
vious structure–affinity and structure–intrinsic activity
studies were concerned with chemical modifications in
a group of compounds containing tricyclic theophylli-
nes with an annelated heterocyclic ring of lactam or
non-lactam structure, i.e., pyrimido[2,1-f]purine and
diazepino[2,1-f]purine [8, 16, 20].
To evaluate the influence of the 1,3-diazepine or
pyrimido-purine rings on the binding affinity, we de-
veloped a series of 8-arylpiperazinylpropylamine de-
rivatives of 7-alkyl, 7-arylalkyl substituted theophyl-
line (1,3-dimethyl-3,7-dihydropurine-2,6-dione) [5, 7,
27]. The structural modification involved the opening
O
N
O
2
N
N
N
N
H
N
It was found that 1,3-dimethyl-10-[3-(4-phenyl-
piperazin-1-yl)-propyl]-2,4-dioxo-1,3,6,7,8,9-hexahydro-
10H-1,3-diazepino-[2,1-f]-purine (1) and its analog 2
behaved as 5-HT postsynaptic antagonists, whereas
1,3-dimethyl-9-[3-(4-phenylpiperazin-1-yl)-butyl]-2,4-
dioxo-1,3,6,7,8,9-hexahydropyrimido-[2,1-f]-purine (3)
behaved as partial agonist of 5-HT receptors [8]
(Fig. 1). We successively examined the influence of
N
R
6: R = H
7: R = 2-OCH3
8: R = 3-Cl-PP
Fig. 2. General structure of 8-arylpiperazinylpropylamino derivatives
of 7-substituted purine-2,6-diones
16

New 8-aminoalkyl derivatives of purine-2,6-dione
of the terminal 1,3-diazepine or pyrimido-purine a Varian BB 200 (300 MHz) spectrophotometer in
cores in compounds 1–4 to give 8-arylpipera- DMSO solution. Chemical shifts are expressed in d
zinylpropylamine derivatives. Among them, the most (ppm), and the coupling constants, J, are given in
interesting 7-arylalkyl-8-alkylaminopurine-2,6-dione hertz (Hz). The purity of the compounds were rou-
derivatives displayed high-to-moderate affinity for tinely checked by thin layer chromatography (TLC)
5-HT receptors and moderate-to-low affinity for using Kieselgel 60 F sheets and the following elu-
5-HT sites (Fig. 2, Tab. 1). Those compounds, ex- ents: S : benzene/acetone = 7:3, v/v, S : benzene/ace-
amined in functional in vivo models, behaved as post- tone/methanol = 1:1:1, v/v/v. Spots were detected un-
synaptic 5-HT receptor antagonists [5].
der UV light. Elemental analyses were determined
To continue our research with 7-arylalkyl-8- with an Elementar Vario EL III apparatus and were
alkylaminopurine-2,6-dione derivatives we designed within 0.4% of the theoretical values.
and synthesized some analogs of the previously evalu-
The 8-bromo-1,3-dimethyl-1H-purine-2,6(3H,7H)-
ated series. Since 7-arylpiperazinylbutyl derivatives of dione (8-BrTh) 9 [11] and its 7-benzyl, 7-phenylethyl,
the previously reported [7] 8-alkoxy-purine-2,6-diones 7-phenylpropyl, 7-allyl, and 7-propyn-2-yn-1-yl (pro-
behaved as highly active 5-HT receptor ligands pargyl) derivatives 10–14 were synthesized according
(K = 11–19 nM) with distinct affinity for 5-HT recep- to the procedure published in the literature [10] and
tors (K = 51–83 nM) the first planned structural modi- were used for the synthesis of new compounds 15–35
fications consisted in elongation of the linker length and 36–42. 7-Arylalkyl- and 7-allyl-purine-2,6-dione
between 8-aminopurine-2,6-dione core and arylpipera- derivatives 15–33 were synthesized according to the
zine fragment from three to four carbon unit.
published procedure by nucleophilic substitution of
Further, by replacing an arylalkyl group with allyl and 10–14 with 10% molar excess of the appropriate pri-
propynyl substituents, we obtained a series of 8-aryl- mary amines (arylpiperazinylalkylamine or tetrahy-
piperazinylalkyl amino analogs with preserved p electron droisoquinolinylalkylamine) in boiling n-butanol in
system and similar low conformational flexibility.
the presence of K CO , (Scheme 1, Route A; Tab. 1)
To extend structure-activity relationships, we replaced [5]. In the same conditions, 7-propargyl-8-bromo-
the arylpiperazinylalkylamino fragment in the 8 position 1,3-dimethyl-3,7-dihydropurine-2,6-dione (14) under-
of purine 2,6-dione with arylpiperazinylalkoxy moiety in went the sigmatropic rearrangement yielding respec-
the series of previously reported propylene derivatives tive propyn-1-yn-1-yl derivatives 34 and 35.
6–8. This modification aimed at investigating the influ-
ence of electron-donor properties of the nitrogen-to- alkyl-purine-2,6-diones (36–42) were obtained from
oxygen switch on the receptor binding profile. 10–12 by heating them with 10% molar excess of respec-
8-Arylpiperazin-1-ylpropoxy- derivatives of 7-aryl-
The influence of the structural modification on the tive 3-[(4-aryl)-piperazin-1-yl]-propan-1-ols [12] in tolu-
activity for serotonin receptors was investigated in ene in the presence of finely powdered KOH (Scheme 1,
classic arylpiperazine (R = H, 2-OCH , 3-Cl, 4-F) or Route B; Tab. 2). Compounds 15–42 were isolated as
1,2,3,4-tetrahydroisoquinoline (THIQ) derivatives.
water-soluble hydrochloride salts and were purified by re-
Herein, we report on the synthesis of the new de- crystallization from anhydrous ethanol (compounds
signed compounds 15–42, their biological evaluation 15–33) or methanol (compounds 34 and 35).
related with 5-HT , 5-HT , 5-HT , 5-HT receptors,
and determination of their in vivo properties in animal
7-Benzyl-1,3-dimethyl-8-((4-(4-phenylpiperazin-
1-yl)butyl)amino)-1H-purine-2,6(3H,7H)-dione
hydrochloride (15)
models of anxiety and depression.
The title compound was obtained in 65% yield, start-
Materials and Methods
ing from 10; m.p. 228–230°C; R = 0.77 (S ).
H-NMR (DMSO-d , d, ppm): 1.56–1.66 (m, 2H),
1.70–1.71 (m, 2H), 2.98–3.18 (m, 9H), 3.35–3.40 (m,
Chemical methodology
5H), 3.46 (d, 2H, J = 5.8 Hz), 3.74 (d, 2H, J = 5.8 Hz),
Melting points (m.p.) were determined in open glass 5.34 (s, 2H), 6.81–6.99 (m, 3H), 7.21–7.35 (m, 7H),
capillaries with Büchi 353 melting point apparatus 7.39–7.43 (t, 1H, J = 5.5 Hz), 10.87 (s, 1H). Analysis:
and are uncorrected. H-NMR spectra were taken with for C H ClN O × ½ H O (547.06): C, H, N.
17

O
N
N
R1
O
Scheme 1. The synthesis of 8-ami-
noalkyl derivatives of 7-substituted-
purine-2,6-dione derivatives 15–35 and
8-arylpiperazinylalkoxy derivatives of
7-substituted-purine-2,6-dione deriva-
tives 36–42: (i) R -Cl or R -Br, K CO ,
TEBA, Me CO, 12 h; (ii) arylpiperazi-
nylalkylamine or tetrahydroisoquino-
linylalkylamine, K CO , n-butanol, 40 h;
(iii) conc. HCl in anh. ethanol; (iv) aryl-
piperazinylpropanol, KOH, n-butanol,
40 h
N
Route A
ii, iii
N
N
H
N
O
O
n
N
N
N
N
H
R1
Br
i
O
O
15 - 35
15 - 35
N
N
N
N
Br
O
Route B
iv, iii
9
9
10 - 14
10 - 14
N
N
R1
O
O
N
N
N
36 - 43
36 - 42
R1 = phenylmethyl, phenylethyl, phenylpropyl,
allyl, propynyl
n = 1, 2
Amine = PP, 2-MPP, 3-Cl-PP, 4-F-PP, THIQ,
1,3-Dimethyl-7-phenethyl-8-((4-(4-phenyl-
piperazin-1-yl)butyl)amino)-1H-purine-
2,6(3H,7H)-dione hydrochloride (16)
H-NMR (DMSO-d , d, ppm): 1.58–1.62 (m, 2H),
1.67–1.69 (m, 2H), 2.99–3.19 (m, 9H), 3.21–3.53 (m,
9H), 3.77 (s, 3H), 5.21 (s, 2H), 6.90–7.04 (m, 5H),
7.20–7.37 (m, 6H), 10.48 (s, 1H). Analysis: for
C H ClN O × 2 H O (603.10): C, H, N.
The title compound was obtained in 54% yield, start-
ing from 11; m.p. 240–242°C; R = 0.84 (S ).
H-NMR (DMSO-d , d, ppm): 1.50–1.60 (m, 2H),
1.70–1.71 (m, 2H), 2.85–3.10 (m, 9H), 3.35–3.40 (m,
5H), 3.07 (t, 2H, J = 6.7 Hz), 3.46 (d, 2H, J = 5.5 Hz),
3.75 (d, 2H, J = 5.5 Hz), 4.25 (t, 2H, J = 6.7 Hz),
6.81–6.99 (m, 3H), 7.21–7.35 (m, 7H), 7.39–7.43 (t,
1H, J = 5 Hz), 10.51 (s, 1H). Analysis: for
C H ClN O × ½ H O (561.10): C, H, N.
1,3-Dimethyl-7-phenethyl-8-((4-(4-(2-methoxy-
phenyl)piperazin-1-yl)butyl)amino)-1H-purine-
2,6(3H,7H)-dione hydrochloride (19)
The title compound was obtained in 32% yield, start-
ing from 11; m.p. 264–265°C; R = 0.65 (S ).
H-NMR (DMSO-d , d, ppm): 1.53–1.57 (m, 2H),
1.70–1.75 (m, 2H), 2.91 (t, 2H, J = 7.7 Hz), 2.98–3.19
(m, 9H), 3.31–3.39 (m, 5H), 3.41–3.52 (m, 4H), 3.77
(s, 3H), 4.23 (t, 2H, J = 7.6 Hz), 6.88–7.03 (m, 4H),
7.17–7.28 (m, 6H), 10.48 (s, 1H). Analysis: for
C H ClN O × 2½ H O (626.12): C, H, N.
1,3-Dimethyl-8-((4-(4-phenylpiperazin-1-yl)
butyl)amino)-7-(3-phenylpropyl)-1H-purine-
2,6(3H,7H)-dione hydrochloride (17)
The title compound was obtained in 31% yield, start-
ing from 12; m.p. 244–246°C; R = 0.95 (S ).
H-NMR (DMSO-d , d, ppm): 1.52–1.65 (m, 2H),
1.75–1.88 (m, 2H), 1.87–1.97 (m, 2H), 2.56 (t, 2H,
J = 8.1 Hz), 3.01–3.18 (m, 9H), 3.30–3.39 (m, 5H),
3.47 (d, 2H, J = 5.4 Hz), 3.71 (d, 2H, J = 5.5 Hz), 4.12
(t, 2H, J = 7.1 Hz), 6.85 (t, 1H, J = 7.2 Hz), 7.11–7.30
(m, 10H), 11.04 (s, 1H). Analysis: for C H
ClN O × 1½ H O (593.12): C, H, N.
1,3-Dimethyl-8-((4-(4-(2-methoxyphenyl)-
piperazin-1-yl)butyl)amino)-7-(3-phenylpropyl)-
1H-purine-2,6(3H,7H)-dione hydrochloride (20)
The title compound was obtained in 30% yield, starting
from 12; m.p. 267–269°C; R = 0.75 (S ). H-NMR
(DMSO-d , d, ppm): 1.52–1.65 (m, 2H), 1.75–1.88 (m,
2H), 1.87–1.97 (m, 2H), 2.55 (t, 2H, J = 8.0 Hz),
3.01–3.20 (m, 9H), 3.31–3.38 (m, 5H), 3.47–3.53 (m,
4H), 3.78 (s, 3H), 4.12 (t, 2H, J = 7.1 Hz), 6.82 (t, 1H,
J = 7.2 Hz), 7.15–7.30 (m, 9H), 11.04 (s, 1H). Analy-
7-Benzyl-8-((4-(4-(2-methoxyphenyl)piperazin-
1-yl)butyl)amino)-1,3-dimethyl-1H-purine-
2,6(3H,7H)-dione hydrochloride (18)
The title compound was obtained in 33% yield, start-
ing from 10; m.p. 240–242°C; R = 0.60 (S ). sis: for C H ClN O × H O (613.31): C, H, N.
18

New 8-aminoalkyl derivatives of purine-2,6-dione
7-Benzyl-8-((4-(4-(3-chlorophenyl)piperazin-1-
yl)butyl)amino)-1,3-dimethyl-1H-purine-
2,6(3H,7H)-dione hydrochloride (21)
8-((3-(3,4-Dihydroisoquinolin-2(1H)-yl)pro-
pyl)amino)-1,3-dimethyl-7-phenethyl-1H-purine-
2,6(3H,7H)-dione hydrochloride (25)
The title compound was obtained in 64% yield, starting
from 11; m.p. 213–217°C; R = 0.63 (S ). H-NMR
(DMSO-d , d, ppm): 2.02–2.09 (m, 2H), 2.87–2.91 (m,
3H), 3.10–3.24 (m, 6H), 3.30–3.42 (m, 6H), 3.58–3.65
(m, 1H), 4.05 (t, 2H, J = 7.5 Hz), 4.20–4.25 (m, 1H), 4.51
(d, 1H, J = 7.3 Hz), 7.19–7.29 (m, 10H), 10.99 (s, 1H).
Analysis: for C H ClN O × 2H O (546.55): C, H, N.
The title compound was obtained in 45% yield, start-
ing from 10; m.p. 245–247°C; R = 0.78 (S ).
H-NMR (DMSO-d , d, ppm): 1.55–1.60 (m, 2H),
1.66–1.71 (m, 2H), 2.99–3.20 (m, 9H), 3.33–3.41 (m,
5H), 3.44 (d, 2H, J = 7.1 Hz), 3.84 (d, 2H, J = 6.4 Hz),
5.33 (s, 2H), 6.84–7.39 (m, 9H), 10.48 (s, 1H). Analy-
sis: for C H Cl N O × ½ H O (580.52): C, H, N.
8-((3-(3,4-Dihydroisoquinolin-2(1H)-yl)propyl)-
amino)-1,3-dimethyl-7-(3-phenylpropyl)-1H-
purine-2,6(3H,7H)-dione hydrochloride (26)
1,3-Dimethyl-7-phenethyl-8-((4-(4-(3-chloro-
phenyl)piperazin-1-yl)butyl)amino)-1H-purine-
2,6(3H,7H)-dione hydrochloride (22)
The title compound was obtained in 47% yield, starting
from 12; m.p. 223–225°C; R = 0.79 (S ). H-NMR
(DMSO-d , d, ppm): 1.87–1.97 (m, 2H), 2.12–2.17 (m,
2H), 2.61 (t, 2H, J = 8.0 Hz), 2.92–3.10 (m, 2H),
3.10–3.22 (m, 5H), 3.34 (s, 3H), 3.30–3.35 (m, 2H),
4.12 (t, 2H, J = 7.2 Hz), 4.20–4.25 (m, 2H), 4.25–4.28
(m, 1H), 4.50 (d, 1H, J = 6.9 Hz), 7.09–7.27 (m, 9H),
7.58–7.60 (m, 1H), 10.74 (s, 1H). Analysis: for
C H ClN O × 2¼ H O (564.57): C, H, N.
The title compound was obtained in 39% yield, starting
from 11; m.p. 239–241°C; R = 0.79 (S ). H-NMR
(DMSO-d , d, ppm): 1.52–1.57 (m, 2H), 1.72–1.75 (m,
2H), 2.89 (t, 2H, J = 7.6 Hz), 3.11–3.19 (m, 9H), 3.29–3.39
(m, 5H), 3.41–3.48 (m, 2H), 3.84–3.94 (m, 2H), 3.98 (t,
2H, J = 7.6 Hz), 6.84–7.31 (m, 10H), 12.41 (s, 1H). Analy-
sis: for C H Cl N O × 1½ H O (612.55): C, H, N.
1,3-Dimethyl-8-((4-(4-(3-chlorophenyl)piperazin-
1-yl)butyl)amino)-7-(3-phenylpropyl)-1H-purine-
2,6(3H,7H)-dione hydrochloride (23)
7-Benzyl-8-((4-(3,4-dihydroisoquinolin- 2(1H)-
yl)butyl)amino)-1,3-dimethyl-1H-purine-
2,6(3H,7H)-dione hydrochloride (27)
The title compound was obtained in 37% yield, starting
from 12; m.p. 234–237°C; R = 0.82 (S ). H-NMR
(DMSO-d , d, ppm): 1.59–1.66 (m, 2H), 1.75–1.79 (m,
2H), 1.90–1.97 (m, 2H), 2.57 (t, 2H, J = 8.1 Hz),
3.02–3.22 (m, 9H), 3.32–3.40 (m, 5H), 3.43–3.50 (d,
2H, J = 5.0 Hz), 3.79–3.84 (d, 2H, J = 5.8 Hz), 4.12 (t,
2H, J = 7.1 Hz), 6.84–7.27 (m, 10H), 10.57 (s, 1H).
Analysis: for C H Cl N O × H O (617.57): C, H, N.
The title compound was obtained in 34% yield, starting
from 10; m.p. 250–252°C; R = 0.73 (S ). H-NMR
(DMSO-d , d, ppm): 1.59–1.66 (m, 2H), 1.73–1.78 (m,
2H), 2.95–3.01 (m, 2H), 3.11–3.22 (m, 6H), 3.30–3.38
(m, 4H), 3.40–3.43 (m, 2H), 4.26–4.29 (m, 1H), 4.41
(d, 1H, J = 7.8 Hz), 5.33 (s, 2H), 7.14–7.34 (m, 9H),
7.39 (t, 1H, J = 5.4 Hz), 10.74 (s, 1H). Analysis: for
C H ClN O × H O (527.04): C, H, N.
7-Benzyl-8-((3-(3,4-dihydroisoquinolin-
2(1H)-yl)propyl)amino)-1,3-dimethyl-1H-
purine-2,6(3H,7H)-dione hydrochloride (24)
8-((4-(3,4-Dihydroisoquinolin-2(1H)-yl)butyl)
amino)-1,3-dimethyl-7-phenethyl-1H-purine-
2,6(3H,7H)-dione hydrochloride (28)
The title compound was obtained in 72% yield, starting
from 10; m.p. 227–230°C; R = 0.71 (S ). H-NMR
(DMSO-d , d, ppm): 1.88–1.95 (m, 2H), 2.71 (t, 2H),
2.75–2.81 (m, 4H), 3.33 (s, 3H), 3.52 (s, 3H),
3.61–3.67 (m, 4H), 4.95 (s, 2H), 6.84 (t, 1H, J = 4.6
The title compound was obtained in 32% yield, starting
from 11; m.p. 235–236°C; R = 0.65 (S ). H-NMR
(DMSO-d , d, ppm): 1.57–1.59 (m, 2H), 1.80–1.84 (m,
2H), 2.89 (d, 2H, J = 7.8 Hz), 3.18–3.34 (m, 13H),
3.60–3.64 (m, 1H), 4.24–4.28 (m, 3H), 4.46–4.50 (d,
Hz), 6.91–7.17 (m, 4H), 7.18–7.21 (m, 5H), 10.82 (s, 1H, J = 6.5 Hz), 7.14–7.28 (m, 10H), 10.89 (s, 1H).
1H). Analysis: for C H ClN O (496.02): C, H, N.
Analysis: for C H ClN O × 2H O (559.06): C, H, N.
19

8-((4-(3,4-Dihydroisoquinolin-2(1H)-yl)butyl)-
amino)-1,3-dimethyl-7-(3-phenylpropyl)-
1H-purine-2,6(3H,7H)-dione hydrochloride (29)
4.93–5.10 (m, 1H), 5.12–5.13 (m, 1H), 5.76–5.92 (m,
1H), 6.86–7.01 (m, 4H), 7.20–7.28 (m, 1H), 10.63 (s,
1H). Analysis: for C H ClN O (518.10): C, H, N.
The title compound was obtained in 31% yield, starting
from 12; m.p. 150–152°C; R = 0.67 (S ). H-NMR
(DMSO-d , d, ppm): 1.63–1.67 (m, 2H), 1.87–1.95 (m,
4H), 2.58 (t, 2H, J = 8.1 Hz), 2.61–2.98 (m, 2H),
3.13–3.22 (m, 7H), 3.32–3.38 (m, 5H), 4.14 (t, 2H,
J = 7.1 Hz), 4.23–4.29 (m, 1H), 4.44 (d, 1H, J = 6.4
Hz), 7.10–7.25 (m, 10H), 10.89 (s, 1H). Analysis: for
C H ClN O × 3½H O (600.09): C, H, N.
7-Allyl-1,3-dimethyl-8-((4-(4-(4-fluorophenyl)-
piperazin-1-yl)butyl)amino)-1H-purine-
2,6(3H,7H)-dione hydrochloride (33)
The title compound was obtained in 26% yield, start-
ing from 13; m.p. 221–223°C; R = 0.25 (S ).
H-NMR (DMSO-d , d, ppm): 1.57–1.62 (m, 2H),
1.71–1.73 (m, 2H), 3.02–3.02 (m, 6H), 3.15 (s, 3H);
3.35 (s, 3H), 3.46–3.58 (m, 2H), 3.66–3.76 (m, 2H),
4.68–4.70 (m, 2H), 4.88–4,98 (m, 2H), 5.08–5.15 (m,
1H), 5.80–5.94 (m, 2H), 6.96–7.14 (m, 5H), 10.21 (s,
1H). Analysis: for C H ClFN O (506.02): C, H, N.
7-Allyl-1,3-dimethyl-8-((4-(4-phenylpiperazin-1-
yl)butyl)amino)-1H-purine-2,6(3H,7H)-dione
hydrochloride (30)
The title compound was obtained in 28% yield, start-
ing from 13; m.p. 245–247°C; R = 0.26 (S ).
H-NMR (DMSO-d , d, ppm): 1.58–1.62 (m, 2H),
1.75–1.79 (m, 2H), 3.05–3.11 (m, 6H), 3.14 (s, 3H);
3.35 (s, 3H), 3.35–3.36 (m, 2H), 3.47–3.53 (m, 4H),
4.69–4.70 (m, 2H), 4.96–5.09 (m, 1H), 5.12–5.13 (m,
1H), 5.84–5.92 (m, 1H), 6.72–6.76 (m, 1H),
6.82–6.87 (m, 1H), 6.96–6.99 (m, 4H), 10.84 (s, 1H).
Analysis: for C H ClN O (488.03): C, H, N.
1,3-Dimethyl-8-((3-(4-phenylpiperazin-1-yl)pro-
pyl)amino)-7-(prop-1-yn-1-yl)-1H-purine-
2,6(3H,7H)-dione hydrochloride (34)
The title compound was obtained in 40% yield, starting
from 14; m.p. 224–225°C; R = 0.36 (S ), R = 0.74
(S ). H-NMR (DMSO-d , d, ppm): 2.39 (s, 3H),
2.64–2.69 (m, 2H), 2.99–3.02 (m, 2H), 3.09–3.15 (m,
2H), 3.42 (s, 3H), 3.43–3.56 (m, 2H), 3.56 (s, 3H),
3.57–3.65 (m, 4H), 4.19–4.23 (m, 2H), 6.84–6.96 (m,
1H), 6.97–7.00 (m, 2H), 7.22–7.26 (m, 3H), 10.23 (s,
1H) . Analysis: for C H ClN O (471.21): C, H, N.
7-Allyl-1,3-dimethyl-8-((3-(4-(2-methoxyphenyl)-
piperazin-1-yl)propyl)amino)-1H-purine-
2,6(3H,7H)-dione hydrochloride (31)
8-((3-(4-(2-Methoxyphenyl)piperazin-1-yl)pro-
pyl)amino)-1,3-dimethyl-7-(prop-1-yn-1-yl)-
1H-purine-2,6(3H,7H)-dione hydrochloride (35)
The title compound was obtained in 21% yield, start-
ing from 13; m.p. 207–209°C; R = 0.25 (S ), R =
0.73 (S ). H-NMR (DMSO-d , d, ppm): 2.02–2.09
(m, 2H), 3.09–3.14 (m, 6H), 3.15 (s, 3H), 3.36 (s,
3H), 3.38–3.53 (m, 4H), 3.77 (s, 3H), 3.81–3.84 (m,
2H), 4.71–4.73 (m, 2H), 5.02–5.09 (m, 1H),
5.12–5.13 (m, 1H), 5.77–5.93 (m, 1H), 6.88–7.01 (m,
4H), 7.35–7.39 (m, 1H), 10.52 (s, 1H). Analysis: for
C H ClN O (504.08): C, H, N.
The title compound was obtained in 25% yield, starting
from 14; m.p. 237–239°C; R = 0.22 (S ), R = 0.72 (S ).
H-NMR (DMSO-d , d, ppm): 2.40 (s, 3H), 2.62–2.67
(m, 2H), 3.18–3.38 (m, 2H), 3.17–3.63 (m, 8H), 3.43 (s,
3H), 3.59 (s, 3H), 3.90 (s, 3H), 4.21–4.26 (m, 2H),
6.92–6.96 (m, 2H), 7.15–7.25 (m, 3H), 10.35 (s, 1H) .
Analysis: for C H ClN O (501.23): C, H, N.
7-Allyl-1,3-dimethyl-8-((4-(4-(2-methoxyphenyl)-
piperazin-1-yl)butyl)amino)-1H-purine-
2,6(3H,7H)-dione hydrochloride (32)
7-Benzyl-1,3-dimethyl-8-(3-(4-phenylpiperazin-
1-yl)propoxy)-1H-purine-2,6(3H,7H)-dione
hydrochloride (36)
The title compound was obtained in 27% yield, start-
ing from 13; m.p. 240–242°C; R = 0.22 (S ), R = The title compound was obtained in 57% yield, start-
0.70 (S ). H-NMR (DMSO-d , d, ppm): 1.59–1.62 ing from 10; m.p. 158–161°C; R = 0.16 (S ),
(m, 2H), 1.75–1.82 (m, 2H), 3.15 (s, 3H), 3.16–3.27 R = 0.77 (S ). H-NMR (DMSO-d , d, ppm): 2.07–
(m, 6H), 3.35 (s, 3H), 3.43–3.47 (m, 4H), 3.77 (s, 2.25 (m, 2H), 3.08–3.11 (m, 6H), 3.20 (s, 3H), 3.37 (s,
3H), 3.77–4.15 (m, 2H), 4.71–4.73 (m, 2H), 3H), 3.55 (d, 2H, J = 6.2 Hz), 3.81 (d, 2H, J = 8.5 Hz),
20

New 8-aminoalkyl derivatives of purine-2,6-dione
1,3-Dimethyl-8-(3-(4-(3-chlorophenyl)piperazin-
1-yl)propoxy)-7-(3-phenylpropyl)-1H-purine-
2,6(3H,7H)-dione hydrochloride (40)
4.53 (t, 2H, J = 5.6 Hz), 5.26 (s, 2H), 6.87 (t, 3H,
J = 7.2Hz) , 6.97 (d, 2H, J = 9.3 Hz), 7.22–7.37 (m,
5H), 10.71 (s, 1H). Analysis: for C H ClN O × H O
(543.05): C, H, N.
The title compound was obtained in 50% yield, start-
ing from 12; m.p. 214–218°C; R = 0.37 (S ),
R
= 0.88 (S ). H-NMR (DMSO-d , d, ppm):
1,3-Dimethyl-8-(3-(4-phenylpiperazin-1-
yl)propoxy)-7-(3-phenylpropyl)-1H-purine-
2,6(3H,7H)-dione hydrochloride (37)
1.87–1.97 (m, 4H), 2.02–2.30 (m, 2H), 2.55 (t, 2H,
J = 7.2 Hz), 3.04–3.11 (m, 4H), 3.15 (s, 3H), 3.28 (s,
3H), 3.42–3.58 (m, 4H), 3.74 (t, 2H, J = 6.4 Hz), 3.82
(t, 2H, J = 2.3 Hz), 6.85 (d, 1H, J = 1.3 Hz), 6.94 (d,
2H, J = 2.6 Hz), 7.02 (s, 1H), 7.09–7.25 (m, 5H),
11.54 (s, 1H). Analysis: for C H Cl N O × 3½H O
(650.55): C, H, N.
The title compound was obtained in 53% yield, start-
ing from 12; m.p. 200–202°C; R = 0.35 (S ),
R
= 0.88 (S ). H-NMR (DMSO-d , d, ppm):
1.98–2.08 (m, 2H), 2.24–2.47 (m, 2H), 2.58 (t, 2H,
J = 8.2 Hz), 3.03–3.15 (m, 4H), 3.10–21 (m, 4H), 3.31
(s, 3H), 3.35 (s, 3H), 3.81 (d, 2H, J = 10.5 Hz), 4.07
(t, 2H, J = 6.9 Hz), 4.52 (t, 2H, J = 5.9 Hz), 6.85 (t,
1H, J = 7.2 Hz), 7.11 (d, 2H, J = 9.2 Hz), 7.14–7.21
(m, 2H), 7.22–7.27 (m, 5H), 10.50 (s, 1H). Analysis:
for C H ClN O × H O (553.11): C, H, N.
8-(3-(4-(4-Fluorophenyl)piperazin-1-yl)propoxy)-
1,3-dimethyl-7-phenethyl-1H-purine-2,6(3H,7H)-
dione hydrochloride (41)
The title compound was obtained in 20% yield, start-
ing from 11; m.p. 178–179°C; R = 0.42 (S ).
H-NMR (DMSO-d , d, ppm): 2.06–2.12 (m, 2H),
2.96–2.99 (m, 2H), 3.01–3.16 (m, 6H), 3.23 (s, 3H),
3.36 (s, 3H), 3.40–3.50 (m, 2H), 3.54–3.58 (m, 2H),
3.72–3.76 (m, 2H), 4.21–4.26 (m, 2H), 6.99–7.20 (m
5H), 7.22–7.30 (m, 4H), 10.65 (s, 1H). Analysis: for
C H ClFN O (557.06): C, H, N.
7-Benzyl-8-(3-(4-(2-methoxyphenyl)piperazin-1-
yl)propoxy)-1,3-dimethyl-1H-purine-2,6(3H,7H)-
dione hydrochloride (38)
The title compound was obtained in 22% yield, start-
ing from 10; m.p. 145–147°C; R = 0.30 (S ),
R = 0.86 (S ). H-NMR (DMSO-d , d, ppm): 2.18–
2.28 (m, 2H), 2.99 (t, 2H, J = 11.5 Hz), 3.09–3.12 (m,
4H), 3.20 (s, 3H), 3.37 (s, 3H), 3.46–3.57 (m, 4H), 3.78
(s, 3H), 4.53 (t, 2H, J = 5.4 Hz), 5.26 (s, 2H),
6.89–7.02 (m, 4H), 7.28–7.38 (m, 5H), 10.55 (s, 1H).
Analysis: for C H ClN O × 2H O (590.26): C, H, N.
8-(3-(4-(4-Fluorophenyl)piperazin-1-yl)pro-
poxy)-1,3-dimethyl-7-(3-phenylpropyl)-
1H-purine-2,6(3H,7H)-dione hydrochloride (42)
The title compound was obtained in 17% yield, start-
ing from 11; m.p. 151–152°C; R = 0.46 (S ).
H-NMR (DMSO-d , d, ppm): 1.99–2.10 (m, 4H),
2.95–2.97 (m, 2H), 3.00–3.13 (m, 6H), 3.23 (s, 3H),
3.35 (s, 3H), 3.43–3.50 (m, 2H), 3.55–3.60 (m, 2H),
3.71–3.74 (m, 2H), 4.21–4.26 (m, 2H), 7.05–7.20 (m
5H), 7.22–7.30 (m, 4H), 10.42 (s, 1H). Analysis: for
C H ClFN O (571.09): C, H, N.
7-Benzyl-8-(3-(4-(3-chlorophenyl)piperazin-1-
yl)propoxy)-1,3-dimethyl-1H-purine-2,6(3H,7H)-
dione hydrochloride (39)
PHARMACOLOGY
The title compound was obtained in 57% yield, starting
from 10; m.p. 129–132°C; R = 0.27 (S ), R = 0.88
(S ). H-NMR (DMSO-d , d, ppm): 2.22–2.78 (m, 2H),
3.03–3.14 (m, 6H), 3.19 (s, 3H), 3.37 (s, 3H),
3.78–3.98 (m, 4H), 4.53 (t, 2H, J = 5.9 Hz), 5.25 (s,
2H), 6.84 (d, 1H, J = 1.3 Hz), 6.96 (d, 2H, J = 1.8 Hz),
7.04 (t, 1H, J = 2.3 Hz), 7.22–7.37 (m, 5H), 11.22 (s,
1H). Analysis: for C H Cl N O (559.50): C, H, N.
In vitro evaluation
Investigated compounds (7–9 concentrations) were
tested in competition binding experiments for native
5-HT , 5-HT receptors, as well as for cloned hu-
man 5-HT and 5-HT receptors, according to the pre-
viously published procedures [2, 18, 28].
21

For 5-HT and 5-HT receptors, experiments were 8-OH-DPAT which were administered 60 and 15 min,
carried out using membranes from rat hippocampus and respectively, before testing and ( )DOI that was given
[ H]-8-OH-DPAT or rat cortex and [ H]-ketanserin, re- immediately before scoring. The experimental proce-
spectively. Following incubation, the receptor prepara- dures used were approved by the I Local Ethics Com-
tions were rapidly filtered under vacuum through GF/B mission at the Jagiellonian University in Kraków.
glass fiber filters which were washed extensively with
an ice cold 50 mM Tris buffer (pH 7.4) using a Brandel
Lower lip retraction (LLR) in rats
harvester. Radioactivity was determined by liquid scin-
tillation counting in a Beckman LS 6500 apparatus [2].
Binding assays on membranes from HEK 293 cells
stably expressing human 5-HT or 5-HT receptors
[28] were performed with the use of [ H]-LSD, and
[ H]-5-CT as radioligands, respectively. The experi-
ments were performed in a 96-well plate using a Mul-
tiPROBE II Liquid Handling System. After 1-h incu-
bation at 37°C, the assay samples were rapidly
filtered using a Unifilter harvester and plates were
subsequently washed with ice-cold 50 mM Tris buffer
(pH 7.4). Radioactivity retained on the filters was
quantified on a Microbeta plate reader.
LLR was assessed according to the method described
by Berendsen et al. [1]. The rats were individually
placed in cages (30 × 25 × 25 cm) and were scored
three times (at 15, 30 and 45 min after administration
of the tested compounds) as follows: 0 = lower incisors
not visible, 0.5 = partly visible, 1 = completely visible.
The total maximum score amounted to 3/rat. In a sepa-
rate experiment, the effect of the studied compounds
on LLR induced by 8-OH-DPAT (1 mg/kg) was tested.
Head-twitch responses in mice
The inhibition constants (K ) were calculated from
the Cheng-Prusoff equation [4]. Results are expressed
as the means of at least three separate experiments.
In order to habituate mice to the experimental envi-
ronment, each animal was randomly transferred to
a 12 cm (diameter) and 20 cm (height) glass cage,
lined with sawdust 20 min before the treatment. Head
twitches in mice were induced by ( )DOI (2.5 mg/kg)
[9]. Immediately after treatment, the number of head
twitches was counted during 20 min. ID (the dose
inhibiting the head twitches in mice by 50%) was cal-
culated using Graph Pad Prism 5 software.
Animals and drugs used
The experiments were performed on male Albino
Swiss mice (24–28 g) obtained from a licensed
breeder (Staniszewska, Ilkowice, Poland) and male
Wistar rats (accredited facility at Faculty of Phar-
macy, Jagiellonian University, Kraków, Poland). The
animals were housed in groups of 15 (mice) or 4 (rats)
for 3–4 days in polycarbonate Makrolon type 3 cages,
in an environmentally controlled experimental room
Forced swim test in mice
The experiment was carried out according to the
method of Porsolt et al. [23]. Briefly, mice were indi-
vidually 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.
(ambient temperature 22
1°C; relative humidity
50–60%; 12:12 light:dark cycle, lights on at 8 a.m.).
Standard laboratory pellets and filtered water were
freely available. Each experimental group consisted
of 5–10 animals/dose, and all the animals were used
only once. Compounds 21, 42 and diazepam (Polfa,
Poznañ) were suspended in a 1% aqueous solution of
Tween 80 while imipramine (ADAMED Ltd,,
Pieñków, Poland), 8-hydroxy-2-(di-n-propylamino)-
tetralin (8-OH-DPAT hydrobromide, RBI), ( )-1-
Four-plate test in mice
(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (( )DOI The four-plate test apparatus (BIOSEB, France) con-
hydrochloride, RBI) were dissolved in distilled water. sists of a cage (25 × 18 × 16 cm) floored by four iden-
All compounds, except 8-OH-DPAT that was given tical rectangular metal plates (8 × 11 cm) separated
subscutaneously (sc), were injected intraperitoneally from one another by a gap of 4 mm. The top of the
(ip) in a volume of 10 ml/kg (mice) or 2 ml/kg (rats) cage is covered by a transparent Perspex lid that pre-
30 min before the test, excluding diazepam and vents escape behavior. The plates are connected to
22

New 8-aminoalkyl derivatives of purine-2,6-dione
Spontaneous locomotor activity
a device that can generate electric shocks. Following
a 15-s habituation period, the animal’s motivation to
explore a novel environment was suppressed by an
electric foot shock (0.8 mA, 0.5 s) every time it
moved from one plate to another during a 1-min test
session. This action was referred to as a ‘punished
crossing’, and was followed by a 3 s shock interval,
during which the animal could move across plates
without receiving a shock.
The locomotor activity was recorded with an Opto M3
multi-channel activity monitor (MultiDevice Software
v.1.3, Columbus Instruments). Mice were individually
placed in plastic cages (22 × 12 × 13 cm) for 30 min
habituation period, and then the crossings of each
channel (ambulation) were counted during the first
6 min with data recording every 1 min. Data recorded
Tab. 1. Binding affinities of the 8-aminoalkylamino derivatives of 7-substituted-purine-2,6-dione derivatives 6–8 and 15–29 for 5-HT receptors
O
N
O
m
N
N
N
N
H
N
n
K
SEM [nM]
i
Compound
m
n
Aminea
5-HT1A
5-HT2A
5-HT6
5-HT7
6b
7b
2
2
2
0
1
2
0
1
2
0
1
2
0
1
2
0
1
2
1
1
1
2
2
2
2
2
2
2
2
2
1
1
1
2
2
2
PP
50
2
445 15
> 1 μM
> 1 μM
> 1 μM
> 1 μM
> 1 μM
94
25
3
1
2-MPP
3-ClPP
PP
8
0.3
304
346
6
3
8b
10 0.4
130
90
9
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
39
68
20
2
4
1
259 13
298 21
116 14
162 18
400 25
6
2
2
4
1
3
5
PP
25
PP
958 17
> 10 μM
> 10 μM
> 10 μM
> 1 μM
911 22
98 22
> 1 μM
> 1 μM
> 1 μM
> 1 μM
> 10 μM
> 1 μM
36
2-MPP
2-MPP
2-MPP
3-ClPP
3-ClPP
3-ClPP
THIQ
THIQ
THIQ
THIQ
THIQ
THIQ
4.3 0.5
4.5 0.3
4.5 0.4
40
10
179
9
20
25
66
18
3
9
2
68 13
115 17
114 21
428 58
65
113
85
8
6
204 14
146
454 32
3590 220
> 10 μM
8
3517 654
1789 233
574 44
488 22
354 39
302 19
56
7
2335 308
269 23
99
7
104 15
239 13
PP – phenylpiperazine; 2-MPP – 2-methoxyphenylpiperazine; 3-ClPP – 3-chlorophenylpiperazine; THIQ – 1,2,3,4-tetrahydroisoquinoline;
compounds reported in ref. [5]; affinities for 5-HT and 5-HT receptors are presented herein for the first time
23

Lower lip retraction in rats
after 1 min (i.e., the time equal to the observation
period in the four-plate test) and recorded between
2–6 min (i.e., the time equal to the observation period
in the forced swim test) were calculated. The cages
were cleaned up with 70% ethanol after each mouse.
8-OH-DPAT induced LLR with the maximum possible
score being 90%. Compound 21 evoked LLR with score
being 23% and simultaneously it had no effect on the
8-OH-DPAT-induced LLR. Derivative 42 dose-depen-
dently attenuated LLR evoked by 8-OH- DPAT in rats
(Tab. 4); calculated ID dose is 19.92 (18.80-21.05)
mg/kg.
Statistical analysis
The obtained data are presented as the mean SEM
and were analyzed by a one-way ANOVA followed
by Bonferroni’s test.
Head twitch responses in mice
Both compounds tested dose-dependently antago-
nized the effect of ( )DOI in mice. Accordingly to
differences in their 5-HT receptor affinities, com-
pound 21 produced stronger effect than 42 as com-
pared their ID doses (Tab. 4).
Results
Forced swim test
Table 5 shows that 21 at doses of 20 and 30 mg/kg
(but not 10 mg/kg) significantly reduced (by 33%) the
immobility time of mice in the forced swim test.
Compound 42 administered at a dose of 20 mg/kg,
slightly but non-significantly, attenuated the immobil-
ity time. Imipramine, given as a reference drug, was
The newly synthesized derivatives of purine-2,6-dione
15–42 showed a diversified level of affinity for 5-HT
receptors, ranging from 4.3 nM to 899 nM, and displayed
high-to-low affinity for 5-HT R (68–17330 nM) and for
5-HT R (10–26000 nM). The compounds practically did
not bind to 5-HT R, except compound 23 possessing ineffective at a dose of 5 mg/kg, but at higher ones
a K = 98 nM. The receptor binding properties of the (10 and 20 mg/kg), it significantly shortened (by 52%
new compounds are presented in Tables 1, 2 and 3. and 27%, respectively) the immobility time of mice.
Tab. 2. Binding affinities of the 8-arylpiperazinylalkylamino derivatives of 7-allyl/propynyl substituted-purine-2,6-diones 30–35 for 5-HT recep-
tors
O
N
R1
O
N
N
N
N
H
N
n
N
R2
K
SEM [nM]
i
R1
R2
Compound
n
5-HT1A
5-HT2A
5-HT6
5-HT7
30
31
32
33
34
35
allyl
allyl
2
1
2
2
1
1
H
70
15
20
9
302 15
5706 663
> 10 μM
> 10 μM
> 10 μM
> 10 μM
> 10 μM
476 38
274 15
622 33
269 18
1200 6 5
740 86
2-OCH3
2-OCH3
4-F
2
3
1411 158
475 69
allyl
allyl
133 17
899 112
230 34
72 9
prop-1-yn-1-yl
prop-1-yn-1-yl
H
902 54
> 10 μM
2-OCH3
24

New 8-aminoalkyl derivatives of purine-2,6-dione
Tab. 3. Binding affinities of the 8-arylpiperazinylpropoxy derivatives of 7-substituted purine-2,6-diones 36–42 for 5-HT receptors
O
N
O
m
N
N
N
O
N
N
R
K
i
SEM [nM]
Compound
m
R
5-HT1A
5-HT2A
5-HT6
5-HT7
36
37
38
39
40
41
42
0
H
H
7.6 1.0
348 41
154 12
480 62
388 21
398 53
80 11
> 1 μM
> 1 μM
> 1 μM
> 1 μM
> 1 μM
> 1 μM
89
165 15
75
5
2
0
0
2
1
2
64
48
7
6
2-OCH3
3-Cl
3-Cl
4-F
6
477 66
151 20
631 47
295 17
936 54
105 12
4-F
34
2
210 31
717 15
63
4
Tab. 4. Induction of lower lip retraction (LLR) (A) by the tested com-
pounds, and their effect on the 8-OH-DPAT-induced LLR (B) in rats
and ( )DOI-induced head twitches (C) in mice
Tab. 5. Effects of compounds 21, 42 and imipramine in the forced
swim test in mice
Dose
(mg/kg)
Immobility time (s)
mean SEM
Treatment
Vehicle
LLR mean SEM behavioral
score
ID50
Dose
(mg/kg)
–
185.6 8.1
169.3 9.5
Treatment
10
20
30
A
B
C
123.9 11.3a
21
Vehicle
–
0.1 0.1
0.2 0.1
0.7 0.3
0.7 0.2a
0.0 0.0
0.3 0.2
0.0 0.0
2.7 0.2
2.5 0.2
2.5 0.2
2.4 0.3
2.3 0.4
2.0 0.2
1.6 0.1a
124.0 17.6b
F (3, 28) = 6.657 p < 0.01
184.7 9.7
21
10
20
30
10
20
30
19.79
(18.24-21.35)
10
20
30
155.0 14.1
42
210.3 9.1
F (3, 31) = 4.649 p < 0.01
162.7 6.8
42
30.22
(29.29-31.16)
Vehicle
Imipramine
–
5
170.4 10.9
10
20
77.8 12.2b
119.6 13.0a
All compounds were administrated ip 30 min before the test, except
8-OH-DPAT (sc) and ( )DOI that were given 15 min and immediately,
respectively, before scoring; n = 5-6 animals per group. p < 0.05 vs.
the respective vehicle group (one way ANOVA followed by Bonfer-
roni’s post-hoc test); ID (the dose inhibiting the head twitches in
mice by 50% calculated using Graph Pad Prism 5 software), confi-
dence limits (95%) given in parentheses
F (3, 36) = 16.757 p < 0.0001
All compounds were administrated ip 30 min before the test; n = 8–9
mice per group. p < 0.05, p < 0.001 vs. the respective vehicle
group (one way ANOVA followed by Bonferroni’s post-hoc test)
25

Tab. 6. Effects of compounds 21, 42 and diazepam in the four plate
test in mice
Tab. 7. Effects of compounds 21, 42, imipramine and diazepam on
the spontaneous locomotor activity in mice
Dose
(mg/kg)
Number of punished crossings
mean SEM
Number of crossings
mean SEM during
Treatment
Dose
(mg/kg)
Treatment
Vehicle
Vehicle
–
2.2 0.2
3.1 0.4
1 min
2–6 min
21
10
20
–
69.0 17.8
15.0 8.9a
17.7 6.3a
211.33 20.2
34.3 18.4b
84.2 33.7b
3.5 0.3a
20
30
F (2, 23) = 5.709 p < 0.01
2.2 0.4
21
42
10
20
30
F (2, 26) = 6.247
p < 0.05
F (2, 26) = 12.530
p < 0.001
2.1 0.3
30
NT
NT
2.44 1.5b
1.8 0.3
42
F (1, 16) = 106.550
p < 0.001
F (3, 36) = 0.342 ns
Vehicle
–
1.25
2.5
5
4.2 0.4
Vehicle
–
NT
NT
NT
116.1 41.9
28.7 17.4
18.4 9.4a
5.8 0.3b
10
20
6.4 0.5b
Diazepam
6.6 0.4a
Imipramine
Vehicle
F (2, 27) = 4.031
p < 0.05
F (3, 36) = 6.455 p < 0.01
–
1.25
2.5
5
47.6 21.3
34.0 12.6
30.3 16.3b
23.9 12.2b
NT
NT
NT
NT
All compounds were administrated ip 30 min, excluding diazepam
which was administered 60 min, before the test; n = 9–10 mice per
group. p < 0.05, p < 0.001 vs. the respective vehicle group (one way
ANOVA followed by Bonferroni’s post-hoc test); ns – non significant
Diazepam
F (3, 34) = 1.314
ns
Four-plate test
All compounds were administrated ip 30 min, excluding diazepam
which was administered 60 min, before the test; n = 9–10 mice per
group. p < 0.05, p < 0.001 vs. the respective vehicle group (one
way ANOVA followed by Bonferroni’s post-hoc test); ns – non signifi-
cant. NT – not tested
Table 6 shows that 21 at a dose of 20 mg/kg (but not
10 mg/kg) significantly increased (by 59%) the number
of punished crossings recorded in the four-plate test in
mice. Compound 42 given at doses of 10, 20 and
30 mg/kg was ineffective in that test. Administered as
a reference drug, diazepam, at doses of 1.25, 2.5 and
5 mg/kg significantly increased (by 28%, 52% and 57%,
respectively) the number of punished crossings in mice.
Discussion
First of all, we analyzed the impact of elongation of
the linker length between purine-2,6-dione core and
arylpiperazine fragment from three- to four-carbon
units on the selected 5-HT receptors affinity. Gener-
Spontaneous locomotor activity
Derivative 21 administered at doses of 20 and 30 mg/ ally this modification confirmed the rules describing
kg significantly decreased spontaneous locomotor ac- relation between the length of a spacer and the affinity
tivity of mice during 1-min and 4-min observation ses- of LCAP derivatives for 5-HT R. All the 2-meth-
sions (Tab. 7). Compound 42 (30 mg/kg) produced oxyphenylpiperazine (2-MPP) derivatives containing
a significant sedative effect during a 4-min observation four-methylene group spacer (18–20) displayed high
period (i.e., the period of time identical to the observa- affinity for 5-HT R and 5-HT R, 4.3–4.5 nM and
tion period in the forced swim test). Both imipramine 10–40 nM, respectively, and behaved as dual 5-HT /
(20 mg/kg) and diazepam (2.5 and 5 mg/kg) signifi- 5-HT receptor ligands. Their unsubstituted phenyl-
cantly decreased locomotor activity of mice during ob- piperazine counterparts (15–17) displayed slightly
servation period of 4- and 1-min, respectively.
lower affinity with K ranging from 20 to 68 nM for
26

New 8-aminoalkyl derivatives of purine-2,6-dione
5-HT and from 25 to 36 nM for 5-HT receptors. In- for 5-HT sites and even more dramatically, from 5-
terestingly, one of the unsubstituted phenylpiperazi- to 80-folds, it decreased affinity for 5-HT R (e.g., 17
nes (16) was classified as dual 5-HT / 5-HT ligand vs, 32). Thus 31 and 32 behaved as 5-HT ligands
that preferentially bind to 5-HT R (S
= 2.6). with 5-HT /5-HT selectivity ratio 18 and 31, respec-
Introduction of 3-chloro substituent decreased affinity tively. Propynyl substituent in 7-position revealed to
for both 5-HT and 5-HT receptors, and 3-chloro- be unfavorable for binding of compounds 34 and 35
phenylpiperazine (3-Cl-PP) derivatives (21–23) dis- for 5-HTRs tested. Interestingly, introduction of a halo-
played high-to-moderate affinity for 5-HT , 5-HT
gen atom in 4-position of phenylpiperazine yielded
and 5-HT receptors. At the same time this modifica- compound that preferentially binds to 5-HT Rs (32
tion shifted affinity for 5-HT R from μM to nM vs. 33).
range. Among 3-Cl-PP derivatives the most potent
Last but not least, through the replacement of a ni-
compounds 21 and 23 behaved as mixed 5-HT / trogen atom with an oxygen in the spacer connecting
5-HT /5-HT and 5-HT /5-HT /5-HT ligands, respec- purine-2,6-dione core with phenylpiperazine we ob-
tively.
tained a series of arylpiperazinylalkoxy derivatives
In contrast to data reported by other authors, [17, (36–42). Direct comparison of the selected analogs re-
24] replacement of 2-MPP moiety with 1,2,3,4-tetra- vealed that this modification has not dramatically
hydroisoquinoline (THIQ) revealed to be unfavorable modified the receptor affinity; it only slightly de-
for affinity for 5-HT , 5-HT , and 5-HT receptors. creased affinity for 5-HT R (Tab. 3). Interestingly
Among THIQ derivatives 27–29 only compound 28, enough, a shift of halogen atom from 3-position of
containing phenylethyl substituent in 7-position, dis- compound 41 to 4-position yielded compound 42 with
played affinity for 5-HT and 5-HT Rs lower than significantly increased affinity for both 5-HT and
100 nM. According to the general rule of the influ- 5-HT sites. However, in opposite to other literature
ence of the linker length, propylene analogs (24–26) data, this modification did not increase 5-HT /5-HT
were even less active.
selectivity.
Regarding different classes of 5-HT receptors de-
Within the evaluated series, we could not found
a decisive dependence between the length of alkylene scribed in the central nervous system, much attention
fragment connecting purine-2,6-dione core with addi- has been devoted to the role of the 5-HT , 5-HT ,
tional aryl moiety in 7-position. With respect to the and 5-HT receptor subtypes in such psychiatric dis-
linker length in 7-position all the 2-MPP displayed al- orders as anxiety and depression. In fact, several
most the same affinity for 5-HT R (5 nM). The high- authors have shown that agonists/partial agonists/an-
est affinity for 5-HT R within the series of unsubsti- tagonists of 5-HT receptors, antagonists of 5-HT
tuted, 2-MPP, and THIQ derivatives was displayed by receptors, as well as 5-HT receptor antagonists exert
compounds with an ethylene spacer, while, within anxiolytic- and/or antidepressant-like effect [3, 14,
compounds containing 3-Cl-PP moiety benzyl group 26]. Moreover, Volk et al. has recently reported on
revealed to be the best. Interestingly enough, a three- a series of oxindole derivatives of LCAP substituted
methylene spacer in 7-position separating purine-2,6- in position 3 and 4 of phenylpiperazine with halogen
dione and phenyl ring was preferential for binding of atom with significant antidepressant and anxiolytic
PP and 3-Cl-PP derivatives, e.g., 17 and 23, for properties [25]. Taking into account 5-HT receptor
5-HT Rs.
profile of the investigated derivatives, we selected 3-
In the following step, we studied the replacement of and 4-halogen substituted phenylpiperazine deriva-
an arylalkyl group in 7-position of purine-2,6-dione tives – compound 21 (mixed 5-HT /5-HT /5-HT
with allyl and propynyl substituents. The 8-aryl- ligand) and 42 (mixed 5-HT /5-HT ligand), for in
piperazinylalkylamino analogs obtained (30–35), pre- vivo functional studies.
served p electron system and similar low conforma-
It is generally accepted that the 8-OH-DPAT-
tional flexibility, but diverse electronic sp and sp induced LLR in rats is mediated by postsynaptic
configuration. For direct comparison, we have synthe- 5-HT receptors, whereas the hypothermia evoked
sized allyl analogs of benzyl counterparts containing by 8-OH-DPAT in mice is connected with activation
phenylpiperazine (PP) (6, 15) and 2-MPP (7, 18) – com- of presynaptic 5-HT ones [1, 13]. The results of
pounds 30, and 31, 32, respectively (Tab. 2). This LLR model indicate that compound 42 behaves as
modification slightly (2–3 folds) decreased affinity a postsynaptic 5-HT receptor antagonist. Unexpect-
27

edly, derivative 21 with high 5-HT affinity poorly
In conclusion, we synthesized a series of aryl-
imitated 8-OH-DPAT-induced LLR, producing activ- piperazines and their THIQ analogs containing new
ity that did not reach significant level (Tab. 4). Again 8-alkylamino- (15–35) and 8-alkoxy- purine-2,6-
in the hypothermia model, compound 42 attenuated diones (36–42). The structural modifications allowed
temperature of mice body; however, its effect was not us to select some mixed 5-HT /5-HT /5-HT (21)
changed by WAY 100635, a 5-HT antagonist. Com- and 5-HT /5-HT (16, 17, 19, 20, 42) receptor
pound 21 produced a weak and non-significant hypo- ligands or compounds preferentially binding with
thermic effect (data not showed). Both studied com- 5-HT R (31, 32). Replacement of an arylalkyl group
pounds demonstrated 5-HT antagonistic properties in 7-position of purine-2,4-dione with allyl substitu-
towards the ( )DOI-induced head twitches in mice; ent opens the chance to design new 5-HT ligands with
their ID doses are consistent with their 5-HT affin- preserved p electron system and lower molecular
ity data. The above results show that compound 21 weight. Hence compounds displaying mixed 5-HT re-
can be classified as a very weak postsynaptic 5-HT
ceptors’ profile are interesting with regard to their po-
agonist and 5-HT antagonist while analog 42 is tential antidepressant/anxiolytic activity, the most
a postsynaptic 5-HT /5-HT antagonist. At the suc- promising ones 21 and 42 were pharmacologically
cessive stage, screening experiments were performed evaluated in animal preclinical models of depression
in order to establish potential antidepressant and/or and anxiety. Behavioral studies revealed that com-
anxiolytic activity of both compounds. Our results in- pound 21 evoked specific antidepressant/anxiolytic
dicate that compound 21 produced an antidepressant- effects in mice, while derivative 42 is devoid such ac-
like effect in the forced swim test in mice. The typical tivity presumably due to their different intrinsic activ-
antidepressant imipramine (used as a reference drug) ity toward 5-HT receptors.
was more active than derivative 21. Furthermore,
compound 21 exerted anxiolytic-like activity detected
in the four-plate test in mice. Interestingly, its effect
was weaker in terms of potency and active doses than
that produced by diazepam (used as a reference anxio-
lytic drug). At the same time, 21 at doses active in the
four-plate and the forced swim tests potently attenu-
ated the spontaneous locomotor activity of mice dur-
ing the initial 1- and 4-min (i.e., at the time identical
to the observation period in the four-plate and the
forced swim tests, respectively). In comparison, imi-
pramine and diazepam at doses producing a distinct
antidepressant- and anxiolytic-like effect, respec-
tively, slightly reduced the locomotor activity of mice.
The sedation induced by compound 21 indicates that
its potential antidepressant/anxiolytic activity seems
to be specific; however, such strong sedative effect
practically excluded this ligand from being regarded
as a potential drug. Compound 42 with narrower re-
ceptor profile produced no effect in both screening
models used in mice. On the basis of obtained results
we may hypothesize that the lack of antidepres-
sant/anxiolytic activity for analog 42 can be due to
different intrinsic activity of the compound toward
5-HT receptors as compared with 21, since both
compounds are classified as 5-HT antagonists. As
concluded by Carr and Lucki [3], postsynaptic 5-HT
receptors are essential for producing the anti-
depressant-like effect of 5-HT receptor agonists.
Acknowledgments:
This study was partly supported by the Polish Ministry of Science
and Higher Education (MNiSW), grant no. N N405 378437.
Radioligand binding experiments were financially supported
by the Norwegian Financial Mechanism as part of the
Polish-Norwegian Research Fund, grant no. PNRF–103–AI-1/07.
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Received: December 20, 2011; in the revised form: July 31, 2012;
accepted: September 27, 2012.
29