New Arylpiperazinylalkyl Derivatives of 8-Alkoxy-purine-2,6-dione and Dihydro[1,3]oxazolo[2,3-f]purinedione Targeting the Serotonin 5-HT1A/5-HT2A/5-HT7 and Dopamine D2 Receptors

Article abstract of DOI:10.1002/ardp.201500015

To obtain potential antidepressants and/or antipsychotics, a series of new long-chain arylpiperazine derivatives of 8-alkoxy-purine-2,6-dione (10-24) and dihydro[1,3]oxazolo[2,3-f]purinedione (30-34) were synthesized and their serotonin (5-HT_1A, 5-HT_2A, 5-HT₆, 5-HT₇) and dopamine (D₂) receptor affinities were determined. The study allowed the identification of some potent 5-HT_1A/5-HT₇/D₂ ligands with moderate affinity for 5-HT_2A sites. The binding mode of representative compounds from both chemical classes (11 and 31) in the site of 5-HT_1A receptor was analyzed in computational studies. In functional in vitro studies, the selected compounds 15 and 16 showed antagonistic properties for the evaluated receptors. 8-Methoxy-7-{4-[4-(2-methoxyphenyl)-piperazin-1-yl]-butyl}-1,3-dimethyl-purine-2,6-dione (15) showed a lack of activity in terms and under the conditions of the forced swim, four plate and amphetamine-induced hyperactivity tests in mice, probably as a result of its high first pass effect in the liver.

Full text of DOI:10.1002/ardp.201500015

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Arch. Pharm. Chem. Life Sci. 2015, 348, 242–253
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Full Paper
New Arylpiperazinylalkyl Derivatives of 8-Alkoxy-purine-2,6-dione
and Dihydro[1,3]oxazolo[2,3-f]purinedione Targeting the
Serotonin 5-HT1A/5-HT2A/5-HT7 and Dopamine D2 Receptors
1
1
1
1
1
_
ꢀ
ꢀ
Grazyna Chłon-Rzepa , Agnieszka Zagorska , Adam Bucki , Marcin Kołaczkowski , Maciej Pawłowski ,
2
2
3
3
Grzegorz Satała , Andrzej J. Bojarski , Anna Partyka , Anna Wesołowska , Elzbieta Pe
and Karolina Słoczynska
kala4,
_
4
ꢀ
1
ꢀ
Department of Medicinal Chemistry, Jagiellonian University Medical College, Krakow, Poland
2
ꢀ
Department of Medicinal Chemistry, Institute of Pharmacology, Polish Academy of Sciences, Krakow,
Poland
Department of Clinical Pharmacy, Jagiellonian University Medical College, Krakow, Poland
3
4
ꢀ
ꢀ
Department of Pharmaceutical Biochemistry, Jagiellonian University Medical College, Krakow, Poland
To obtain potential antidepressants and/or antipsychotics, a series of new long-chain arylpiperazine
derivatives of 8-alkoxy-purine-2,6-dione (10–24) and dihydro[1,3]oxazolo[2,3-f]purinedione (30–34) were
synthesized and their serotonin (5-HT1A, 5-HT2A, 5-HT6, 5-HT7) and dopamine (D2) receptor affinities were
determined. The study allowed the identification of some potent 5-HT1A/5-HT7/D2 ligands with moderate
affinity for 5-HT2A sites. The binding mode of representative compounds from both chemical classes (11
and 31) in the site of 5-HT1A receptor was analyzed in computational studies. In functional in vitro studies,
the selected compounds 15 and 16 showed antagonistic properties for the evaluated receptors.
8-Methoxy-7-{4-[4-(2-methoxyphenyl)-piperazin-1-yl]-butyl}-1,3-dimethyl-purine-2,6-dione (15) showed
a lack of activity in terms and under the conditions of the forced swim, four plate and amphetamine-
induced hyperactivity tests in mice, probably as a result of its high first pass effect in the liver.
Keywords: Arylpiperazines / Depression / Serotonin receptor ligands
Received: January 7, 2015; Revised: February 18, 2015; Accepted: February 19, 2015
DOI 10.1002/ardp.201500015
Additional supporting information may be found in the online version of this article at the publisher's web-site.
:
agents, especially for affective disorders (major depression,
bipolar disorder) and schizophrenia, has declined [3, 4].
Introduction
A comprehensive study of mental disorders, based upon the
international diagnostic classification system [1], which covers
19 major groups [2] revealed that neuropsychiatric illnesses
represent a larger disease burden on society than either
cardiovascular disease or cancer. Nowadays, the investment in
research and development to create better psychotropic
Over the past 50 years, enormous progress has been made
in the clinical understanding of psychiatric disorders and
biochemical brain mechanisms, leading to the development
of effective drug treatments, for example, tricyclic antide-
pressants, serotonin reuptake inhibitors, benzodiazepines,
and atypical antipsychotic drug. Since the introduction of
aripiprazole in 2002 and then paliperidone, iloperidone,
asenapine, and lurasidone, there has been a relative hiatus in
the development of new atypical antipsychotic drugs. From
the chemical point of view, the new antidepressants and
atypical antipsychotics are based around arylpiperazine or
arylpiperidine core structures, which may explain their
_
ꢀ
Correspondence: Dr. Grazyna Chłon-Rzepa, Department of
Medicinal Chemistry, Jagiellonian University Medical College, 9
ꢀ
Medyczna Street, 30-688 Krakow, Poland.
interaction with
a large number of G-protein coupled
E-mail: mfchlon@cyf-kr.edu.pl
Fax: þ48-12-6205458
receptors (Rs), including 5-HT1A, 5-HT2A, 5-HT6, 5-HT7 as well
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as D2, D3, and D4 receptors. Clues from the clinic that atypical
antipsychotics agents can be used as augmentation therapy in
patients with poor responses to antidepressants led to the
creation of the multitarget strategy for development of CNS
active drugs.
them (compounds I–VI, Fig. 2) produced antidepressant-like
and/or specific anxiolytic effects in forced swim and four plate
tests in mice [15–20].
Continuing our investigation in a group of purine-2,6-dione
derivatives of LCAPs, and to extend the studies aimed at
verifying the impact of the alkoxy moiety in an 8 position of
purine-2,6-dione system on the selected serotoninergic and
dopaminergic Rs affinity, we designed and synthesized a
novel series of 8-methoxy analogs with the most thoroughly
studied arylpiperazines. In comparison to the previously
evaluated 8-alkoxy derivatives [15], we extended the length
of the linker between purine-2,6-dione core and arylpiper-
azine fragment from four to five carbon units to check the
influence of this modification on the receptor activity.
Moreover, a series of LCAPs derivatives of dihydro[1,3]-
oxazolo[2,3-f]purinediones, which may be regarded as 8-
methoxy-purine-2,6-dione analogs with methoxy group built
into the cyclic oxygen ring system, were obtained. Herein, we
report on their synthesis and evaluation for the selected
serotoninergic (5-HT1A, 5-HT2A, 5-HT6, 5-HT7) and dopaminer-
gic (D2) Rs as well as determination of their intrinsic activity for
validation of the multireceptor approach. Additionally,
computational studies were carried out to rationalize the
obtained binding data for representative compounds from
both chemical classes. Moreover, the potential antidepres-
Extending the concept of mixed 5-HT/D Rs targeting agents
as novel antipsychotics, a series of 1-aryl-4-(biarylmethylene)-
piperazines were discovered [5], which resulted in the
identification of bifeprunox (Fig. 1), a potential atypical
antipsychotic with reduced extrapyramidal side effects [6].
The structure–activity relationships in the group of 1-aryl-4-
(phenylarylmethyl)piperazines [7] led to obtaining SLV 313
(adoprazine) (Fig. 1) [8]. The multireceptor strategy has also
been explored in a series of isoquinoline- and quinoline-
amide- and sulfonamide-derivatives of long chain arylpiper-
azines (LCAPs) exemplified by structures A and B (Fig. 1) with
potential antipsychotic, antidepressant, and anxiolytic prop-
erties [9–11].
For several years, we have been interested in developing
LCAPs derivatives of 1,3-dimethyl-purine-2,6-dione, which
were mainly evaluated toward 5-HT1A, 5-HT2A, 5-HT7, and
recently also for 5-HT6 Rs [12–20]. The 8-unsubstituted or 8-
alkoxy- and 8-morpholinyl-purine-2,6-dione derivatives
showed the features of dual 5HT1A/5HT2A or 5-HT1A/5HT7 Rs
ligands with a moderate affinity toward D2 sites. Some of
Figure 1. Structures of mixed 5-HT/D receptor agents: SLV313 (adoprazine) [8], bifeprunox [6], and isoquinoline-amide (A) [9] and
quinoline-sulfonamide (B) [10] LCAPs derivatives.
Figure 2. The structures of the pharmacologically active compounds I–VI from earlier studies.
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sant-, anxiolytic-, and antipsychotic-like properties of the
most interesting compound 15 were evaluated in the
behavioral tests in mice.
converted into water-soluble salts using conc. hydrochloric
acid in acetone.
The synthesis of arylpiperazinylmethyl-dihydro[1,3]oxazolo
[2,3-f]purinediones was accomplished as shown in Scheme 2.
8-Bromo-1,3-dimethyl-purine-2,6-dione reacted with the re-
spective substituted oxiranes in the presence of a catalytic
amount of pyridine in anhydrous 1-propanol.
Results and discussion
Chemistry
The elemental analysis data and some physical properties of
these compounds are reported in the Experimental section.
The structures of the investigated compounds and their
syntheses are presented in Schemes 1 and 2.
In vitro evaluation
The starting 8-bromo-7-(3-chloropropyl)-1,3-dimethyl-pu-
rine-2,6-dione (2) [21], 8-bromo-7-(4-chlorobutyl)-1,3-dimeth-
yl-purine-2,6-dione (3) [15], and the new 8-bromo-7-(5-
chloropentyl)-1,3-dimethyl-purine-2,6-dione (4) were pre-
pared in a reaction of 8-bromo-1,3-dimethyl-purine-2,6-dione
(1) [22] with the appropriate bromochloroalkane according to
the previously described method [15]. The 7-v-chloroalkyl-8-
alkoxy-1,3-dimethyl-purine-2,6-diones 5–9 were obtained in a
reaction of 2, 3, or 4 with the appropriate sodium alcoholate
in a corresponding alcohol medium [15]. The designed
derivatives 10–24 were synthesized by nucleophilic substitu-
tion of 5–9 with the appropriate arylpiperazines in the
presence of K2CO3 in toluene (10–18) and in 1-propanol (19–
24). Compounds 10–24 were separated by column chroma-
tography as free bases. Compounds 15, 19–24 were then
Radioligand binding assays were employed to determine the
affinity and the selectivity profiles of the new synthesized
compounds 10–24 and 30–34 for serotonin 5-HT1A, 5-HT2A, 5-
HT6, 5-HT7, and dopamine D2 Rs. For compounds 10–24 as
close analogs of previously reported 8-alkoxypurine-2,6-
diones, the inhibition constant (Ki) values were determined
in 7–9 compound concentrations, according to the previously
published procedures [11, 23, 24]. The affinity data of the
compounds 10–24 are presented in Table 1. For compounds
30–34 as derivatives of the dihydro[1,3]oxazolo[2,3-f]purine-
dione system, never before evaluated for these targets,
preliminary in vitro evaluation in two concentrations (10ꢀ6
M
and 10ꢀ7 M) was provided to establish percent of specific
binding according to described procedure [25].
Scheme 1. The synthesis of 7-arylpiperazinylalkyl-8-alkoxy-purine-2,6-diones 10–24. Reagents and conditions: (a) RONa, ROH, reflux;
(b) arylpiperazine derivatives, toluene, K2CO3, reflux; (c) arylpiperazine derivatives, 1-propanol, K2CO3, reflux.
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Scheme 2. The synthesis of arylpiperazinylmethyl oxiranes 25–29 and arylpiperazinylmethyl derivatives of dihydro[1,3]oxazolo[2,3-
f]purinediones 30–34. Reagents and conditions: (a) epichlorohydrin; (b) pyridine, 1-propanol.
Table 1. The binding data of the 10–24 for 5-HT and D2 Rs.
Ki (nM) ꢁ SEM
Comp.
R
R1
n
5-HT1A
5-HT2A
5-HT6
5-HT7
D2
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
C2H5
C2H5
C2H5
C3H7
C3H7
C3H7
H
2-OCH3
3-Cl
4-F
H
2-OCH3
3-Cl
4-F
H
1
688 ꢁ 55
106 ꢁ 9
72 ꢁ 6
3643 ꢁ 428
20 ꢁ 2
4 ꢁ 1
154 ꢁ 14
498 ꢁ 61
116 ꢁ 9
22 ꢁ 3
>30,000
>20,000
>20,000
>10,000
>20,000
>20,000
2927 ꢁ 264
2330 ꢁ 2715
>20,000
>10,000
6979 ꢁ 835
719 ꢁ 73
4796 ꢁ 571
>10,000
262 ꢁ 17
NT
747 ꢁ 69
283 ꢁ 33
147 ꢁ 9
447 ꢁ 51
69 ꢁ 8
12 ꢁ 1
23 ꢁ 2
283 ꢁ 23
204 ꢁ 11
190 ꢁ 22
90 ꢁ 7
71 ꢁ 6
106 ꢁ 9
53 ꢁ 4
59 ꢁ 7
54 ꢁ 2
125 ꢁ 9
267 ꢁ 21
112 ꢁ 10
–
1303 ꢁ 106
216 ꢁ 25
432 ꢁ 41
4012 ꢁ 473
196 ꢁ 23
24 ꢁ 3
60 ꢁ 7
404 ꢁ 31
87 ꢁ 8
35 ꢁ 4
8 ꢁ 1
1
1
1
2
2
2
2
3
3
3
3
3
3
3
245 ꢁ 15
695 ꢁ 75
60 ꢁ 7
10 ꢁ 2
222 ꢁ 17
66 ꢁ 7
57 ꢁ 6
6 ꢁ 1
157 ꢁ 20
289 ꢁ 37
331 ꢁ 39
412 ꢁ 27
178 ꢁ 23
429 ꢁ 41
333 ꢁ 29
72 ꢁ 8
H
2-OCH3
3,4-diCl
H
2-OCH3
3,4-diCl
91 ꢁ 8
36 ꢁ 4
7 ꢁ 1
34 ꢁ 5
13 ꢁ 2
4 ꢁ 1
79 ꢁ 10
11 ꢁ 1
15 ꢁ 1
288 ꢁ 18
22 ꢁ 2
20 ꢁ 2
–
17 ꢁ 3
NT
NT
Compound I
Compound II
Compound III
Compound IV
Buspirone
Olanzapine
Clozapine
253 ꢁ 14
28 ꢁ 2
NT
NT
25 ꢁ 2
NT
21 ꢁ 2
207 ꢁ 26
155 ꢁ 19
–
–
–
4 ꢁ 0.9
–
7 ꢁ 0.8
–
7 ꢁ 0.6
–
–
–
18 ꢁ 2
NT, not tested.
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Generally the synthesized derivatives 10–24 displayed
high-to-moderate affinity for selected serotonin: 5-HT1A
(5ꢀ688 nM), 5-HT2A (22ꢀ695 nM), 5-HT7 (12ꢀ747 nM), and
D2 (4ꢀ432 nM) Rs and, except 21 and 24, a lack of activity for
5-HT6 sites (Table 1). The binding results have showed a
positive influence of the methoxy moiety in comparison with
the previously reported 8-alkoxy analogs [15]. Compounds
14–16 were 2- to 4-fold and 5- to 13-fold more active for 5-HT7
sites, respectively, than 8-ethoxy analogs and 5- to 13-fold
more active than 8-propoxy analogs [15]. At the same time,
this modification only slightly increased 5-HT1A and decreased
5-HT2A Rs affinity.
The elongation of the linker length from three- to four-
carbon units significantly increased the affinity for 5-HT1A (6-
to 34-fold), 5-HT7 (6- to 23-fold), and D2 (6- to 9-fold) sites. The
five-member alkyl spacer did not affect the binding to
evaluated Rs (14 vs. 18). In comparison to the previously
reported analogs [15], the elongation of the linker length
from four- to five-carbon units increased 5-HT1A but
decreased 5-HT2A affinity. The influence on the affinity for
5-HT7 Rs is not so decisive; however, 22 and 23 are more active
for these sites than their previously reported analogs with a
four-methylene spacer [15].
receptor, which simulates the conformational flexibility of the
protein. This procedure allowed us to capture important
ligand–receptor interactions, differentiating their receptor
affinity. The arylpiperazine fragment, common for both
compounds, provided significant interactions with Asp3.32
(charge-reinforced hydrogen bond), as well as with Phe6.52
(p–p stacking) and Lys191 (p-cation). The 8-alkoxy-purine-2,6-
dione moiety of compound 11 reached additional stabilizing
interactions with Tyr2.64 (p–p stacking) and Gln2.65 (H-bond)
(Fig. 3A). Such beneficial interactions were inaccessible for
any enantiomer of compound 31 (Fig. 3B). Moreover, the
unfavorable conformation of the molecule determined by the
oxazole ring fused to the purine-2,6-dione scaffold, caused
steric hindrance in the binding sites of some homology
models, mirroring the lack of in vitro affinity for the target
protein.
Compounds with unsubstituted phenyl ring display a lower
affinity for 5-HT1A, 5-HT7, and D2 Rs than their 2-OCH3 and 3-Cl
analogs (e.g. 10 vs. 11, 12 or 14 vs. 15 and 16). The
introduction of 4-F into the phenyl ring generally did not
improve the affinity for evaluated Rs, except 12 which
appeared to be a selective 5-HT2A ligand (Table 1). On the
other hand, the introduction of 3,4-diCl moiety (21 and 24)
significantly increased 5-HT6 affinity (Table 1). It is noteworthy
that all of the tested LCAPs derivatives of dihydro[1,3]oxazolo-
[2,3-f]purinedione 30–34 were inactive in the screening
binding assays. The percentages of specific binding for most
of these compounds were up to 90% at both concentrations
studied (Supporting Information). This resulted in exclusion of
these compounds from the subsequent phases of the study.
The above results have conclusively proven that a change in
the chemical character of the cyclic amide fragment affects
the receptor affinity. Summing up, 15 containing 2-methoxy-
phenylpiperazine moiety could be regarded as a 5-HT1A/5-HT7
Rs ligand with D2 dopaminergic Rs activity. In turn, 16 with 3-
chlorophenylpiperazinyl fragment showed features of a
5HT1A/5HT2A/5-HT7 Rs ligand. Hence, in the series of the
tested 8-methoxy derivatives, the four-methylene group alkyl
spacer was preferable to obtain potent 5-HT1A/5-HT2A/5-HT7
ligands with a high-to-moderate affinity for D2 Rs.
Figure 3. Binding modes of compounds 11 (A) and 31 (B) in the
site of 5-HT1A receptor. Compound 31 is represented by R-
(orange) and S- (green) enantiomers. Amino acid residues
engaged in ligand binding (within 4 Å from the ligand atoms)
are displayed as sticks, whereas those forming H-bonds (dotted
yellow lines) or p–p stacking/p-cation interactions (dotted green
lines) are represented as thick sticks. For the sake of clarity, a
part of ECL2 and its residues were hidden. TMH, transmem-
brane helix; ECL, extracellular loop.
Molecular modeling
Computational studies have been engaged to predict binding
mode of the presented compounds, as well as to explain
pronounced differences in the receptor binding affinity
between compounds representative for both chemical classes.
Compound 11 from the 8-alkoxy-purine-2,6-dione class and its
closest analog 31 (the derivative of oxazolo[2,3-f]purine-
dione) were docked to a set of homology models of 5-HT1A
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to this type of Rs. This compound revealed weak antagonistic
properties for 5-HT2A and 5-HT7 Rs (Table 2).
Functional in vitro evaluation
The preliminary functional activity of the selected compounds
15 and 16 on evaluated Rs, on intracellular cAMP levels, was
determined at Cerep (Le Bois l'Eveque, 86600 Celle L'Eves-
cault, France) according to the previously published methods
[26–30]. The assays were carried out in HEK-293 and CHO cells
which stably express human 5-HT1A, 5-HT2A, 5-HT7, and D2 Rs,
respectively, with WAY 10063, ketanserin, methiothepin, and
butaclamol (antagonistic effect) and 8-OH-DPAT, and 5-HT
(agonistic effect) as reference compounds.
The compound 15 showed a strong 5-HT1A and D2 Rs
antagonistic effect and a weak 5-HT7 antagonistic activity
(Table 2). In turn, 16 exerted strong 5-HT1A antagonistic
activity as well as displayed 73% of control agonistic response
Behavioral evaluation
Both the receptor profile and functional activity of 15 (Tables
1
and 2) have prompted us to evaluate its potential
antidepressant- and anxiolytic-like properties, using forced
swim and four-plate test in mice, respectively [31, 32].
Serotonin 5-HT1A R is well known to mediate antidepres-
sant- and anxiolytic-like activity in rodents and humans [33]. In
the tests conducted, 15 in comparison with diazepam and
imipramine, used as reference drugs, showed neither
antidepressant- nor anxiolytic-like activity (Table 3). The
previously reported 8-ethoxy analog I (Fig. 1) with a similar
receptor 5-HT1A affinity but a different agonistic functional
Table 2. The functional in vitro evaluation of 15 and 16.
% inhibition of control agonistic response
(antagonistic effect)
% of control agonistic response
(agonistic effect)
Comp.
5-HT1A
5-HT2A
5-HT7
D2
5-HT1A
5-HT7
15a
16
105
110
NT
42
44
47
94
NT
NT
73
NT
5.0
NT, not tested.
aTest concentration: 10ꢀ6 M.
Table 3. Effects of 15 in the forced swim test (A) and four-plate test (B) in mice.
Treatment
Dose [mg/kg]
Mean ꢁ SEM
Immobility time [s]
(A)
Vehicle
15
–
179.4 ꢁ 5.0
164.4 ꢁ 13.5
168.0 ꢁ 4.2
167.2 ꢁ 7.8
173.5 ꢁ 8.4
0.625
1.25
2.5
5
10
–
184.3 ꢁ 13.4 F(5,49) ¼ 0.54752, ns
162.7 ꢁ 6.8
Vehicle
Imipramine
5
10
20
170.4 ꢁ 10.9
119.6 ꢁ 13.0a
77.8 ꢁ 12.2b, F(3,36) ¼ 16.757, p < 0.0001
(B)
Vehicle
15
Number of punished crossings/60 s
–
2.6 ꢁ 0.3
2.5
5
10
–
1.3 ꢁ 0.2
2.7 ꢁ 0.4
2.3 ꢁ 0.4, F(3,34) ¼ 3.0414, p < 0.05
Vehicle
Diazepam
4.2 ꢁ 0.4
1.25
2.5
5
5.8 ꢁ 0.3a
6.4 ꢁ 0.5b
6.6 ꢁ 0.4b, F(3,36) ¼ 6.455 p < 0.01
Compound 15 and diazepam were administered i.p. 60 min, while imipramine 30 min before the test; n ¼ 8–10 mice per group.
ap < 0.05.
bp < 0.01 vs. vehicle.
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profile at 5-HT1A sites produced antidepressant- and anxio-
lytic-like effects in mice [15]. The above information indicates
that the impact of an alkoxy moiety on the affinity to the 5-HT
Rs studied, the functional profile, and psychotropic activity is
not decisive.
D-Amphetamine produces locomotor hyperactivity in
animals as a result of increased dopaminergic activity in
mesolimbic system [34, 35] and its effect is blocked by
antipsychotics having antagonist properties toward dopa-
mine Rs [36]. Taking into account the above information, the
influence of 15 on the amphetamine-induced hyperactivity
in mice was studied. The obtained results indicate that
15, given at doses of 1.25–10 mg/kg i.p., did not attenuate
D-amphetamine-induced hyperactivity in mice (Supporting
Information) and, therefore, showed no antipsychotic-like
activity.
which suggests that a high hepatic first pass effect may occur
in mice following intraperitoneal administration of 15.
Noteworthy, the value of CLint of 15 is significantly higher
than that exhibited by an antidepressant imipramine (CLint
¼ 0.02 mL/mg/min with t0.5 ¼ 66 min) [37]. It, therefore, seems
probable that the lack of activity in vivo of 15 may be related
to its high first pass effect. Another explanation might be a
poor distribution to the site of action. Further pharmacologi-
cal and pharmacokinetic studies are warranted to explain this
phenomenon.
Experimental
General
Reagents and organic solvents were from Sigma–Aldrich, Alfa
Aesar, and Chempur. Monitoring of the reaction was carried
out by TLC performed on silica gel 60 F254 aluminum sheets
(Merck) with the following solvents: dichloromethane/meth-
anol: A: 9.5:0.5, B: 9:1, C: dichloromethane/triethylamine 9:1,
D-ethyl acetate/methanol 9:1. Spots were detected by their
absorption under UV light (l ¼ 254 nm). 1H-NMR spectra were
taken on a Varian Mercury-VX (300 MHz) spectrometer in
CDCl3 (4, 7–18, and 25–34) or DMSO-d6 (19–24) solutions,
using signals of solvents residual 1H atoms as internal
standard (d ¼ 7.26 ppm and 2.48 ppm, respectively). Chemical
shifts were expressed in d (ppm) and the coupling constants J
in Hertz (Hz). The splitting patterns were designated as
follows: s (singlet), d (doublet), t (triplet), q (quartet), m
(multiplet). The UPLC-MS/MS system consisted of a Waters
Acquity UPLC (BEH, C18 column; 2.1 mm ꢂ 100 mm, and 1.7 mm
particle size) coupled to a Waters TQD mass spectrometer
(electrospray ionization mode ESI-tandem quadrupole). The
column was maintained at 40°C, and eluted under gradient
conditions from 95% to 0% of eluent A over 10 min, at a flow
The metabolic studies of 15
The lack of activity in vivo of 15 prompted as to examine its
metabolism using mouse liver microsomes. The samples for
analysis were taken at three different time points, that is,
after 5, 15, and 30 min of incubation. At the first time point
five major metabolites (M1–M5) were observed, M6 was
found after 15 and M7 after 30 min from the start of
experiment. On the basis of ions fragmentation (MS/MS) the
metabolic fate, the chemical structures, and percentage
content of metabolites M1–M7 were proposed (Fig. 4). The
exemplary liquid chromatography results and the ions present
in LC/MS fragmentation spectra of 15 were also presented
(Supporting Information).
From the time course of disappearance of the parent
compound during incubation with microsomes, the half-life
(t0.5) of 15 was determined and then its intrinsic clearance
(CLint) was estimated. The evaluated compound demonstrat-
ed a high CLint (122.0 mL/mg/min, and a short t0.5 ¼ 7.1 min),
Figure 4. Proposed metabolic pathways of 15 using mouse liver microsomes.
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248

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Arch. Pharm. Chem. Life Sci. 2015, 348, 242–253
Arylpiperazinylalkyl Derivatives of Purine-2,6-dione
Archiv der Pharmazie
rate of 0.3 mL/min. The eluent A consisted of water/formic
acid (0.1%). A total of 10 mL of each sample were injected. The
UPLC/MS purity of all the investigated compounds was
determined to be over 98%. Elemental analyses were taken
with Elementar Vario EL III apparatus and were found within
ꢁ0.4% of the theoretical values. Melting points (mp) were
1.92 (m, 2H, O–CH2CH2CH3), 3.37 (s, 3H, N1–CH3), 3.51 (s, 3H,
N3–CH3), 3.55 (t, J ¼ 6.9 Hz, 2H, CH2Cl), 4.42–4.48 (t, J ¼ 6.8 Hz,
2H, O–CH2CH2CH3). LC/MS: m/z calc. 343.15, found 343.47.
Anal. (C15H23ClN4O3) C, H, N.
General procedure for the synthesis of 7-
arylpiperazinylalkyl-8-alkoxy-purine-2,6-dione derivatives
10–24
€
determined with a Buchi apparatus and were uncorrected.
Column chromatography separations were carried out on
column with Merck Kieselgel 60 using solvents A–D.
The appropriate 8-alkoxy-purine-2,6-diones 5–9 (50 mmol),
respective arylpiperazines (50 mmol), and anhydrous K2CO3
(100 mmol) were refluxed in (10 mL) toluene (10–18) or 1-
propanol (19–24) for 30 h .The mixture was filtered off and the
solvent was evaporated under reduced pressure. The final
products were purified by column chromatography (A). In
case of 15 and 19–24, free bases were converted into
hydrochloride by treatment with an excess of conc. HCl to
pH 3 in an acetone solution. The precipitated salts were
washed with acetone and filtered off.
Synthesis of compounds 1–4
The starting 8-bromo-1,3-dimethyl-purine-2,6-dione (1) was
synthesized by a procedure published elsewhere [22]. 8-Bromo-
7-(3-chloropropyl)-1,3-dimethyl-purine-2,6-dione (2) [21], 8-bro-
mo-7-(4-chlorobutyl)-1,3-dimethyl-purine-2,6-dione (3) [15], and
new 8-bromo-7-(5-chloropentyl)-1,3-dimethyl-purine-2,6-dione
(4) were obtained from 1 according to the previously described
method [13].
8-Bromo-7-(5-chloropentyl)-1,3-dimethyl-purine-2,6-
dione 4
8-Methoxy-1,3-dimethyl-7-[3-(4-phenylpiperazin-1-yl)-
propyl]-purine-2,6-dione 10
Yield 58%, mp 151–153°C, Rf ¼ 0.60 (A), H-NMR d: 2.23–2.31
1
Yield81%,mp111–112°C,Rf ¼ 0.82(A),1H-NMRd:1.38–1.58(m,2H,
N7–(CH2)2CH2(CH2)2), 1.72–2.02 (m, 4H, N7–CH2CH2CH2CH2CH2),
3.40 (s, 3H, N1–CH3), 3.56 (s, 3H, N3–CH3), 3.51–3.62 (m, 2H,
CH2CH2–Cl), 4.34 (t, J¼ 7.2 Hz, 2H, N7–CH2CH2). LC/MS: m/z calc.
363.02, found 363.26. Anal. (C12H16BrClN4O2) C, H, N.
(m, 2H, N7–CH2CH2CH2), 2.48–2.56 (m, 2H, N7–CH2CH2CH2),
3.02–3.13 (m, 4H, N–(CH2)2), 3.38 (s, 3H, N1–CH3), 3.53 (s, 3H,
N3–CH3), 3.55–3.76 (m, 4H, (CH2)2N–Ph), 4.14 (s, 3H, O–CH3),
4.21–4.26 (m, 2H, N7–CH2CH2), 6.85–6.99 (m, 3H, Ph), 7.22–
7.31 (m, 2H, Ph). LC/MS: m/z calc. 413.22, found 413.39. Anal.
(C21H28N6O3) C, H, N.
Synthesis of compounds 5–8
The 7-(3-chloropropyl)-8-methoxy-1,3-dimethyl-purine-2,6-dione
(5) [15], 7-(4-chlorobutyl)-8-methoxy-1,3-dimethyl-purine-
2,6-dione (6) [15], and new 7-(5-chloropentyl)-8-alkoxy-
1,3-dimethyl-purine-2,6-diones (7–9) were prepared by a
published procedure [15].
8-Methoxy-7-{3-[4-(2-methoxyphenyl)-piperazin-1-yl]-
propyl}-1,3-dimethyl-purine-2,6-dione 11
1
Yield 49%, mp 127–129°C, Rf ¼ 0.50 (A), H-NMR d: 2.26–2.33
(m, 2H, N7–CH2CH2CH2), 2.50–2.58 (m, 2H, N7–CH2CH2CH2),
3.02–3.13 (m, 4H, N–(CH2)2), 3.36 (s, 3H, N1–CH3), 3.53 (s, 3H,
N3–CH3), 3.55–3.76 (m, 4H, (CH2)2N–Ph), 3.98 (s. 3H,
Ph–O–CH3), 4.15 (s, 3H, O–CH3), 4.23–4.27 (m, 2H, N7–CH2CH2),
6.94–7.80 (m, 4H, Ph). LC/MS: m/z calc. 443.24, found 443.35.
Anal. (C22H30N6O4) C, H, N.
7-(5-Chloropentyl)-8-methoxy-1,3-dimethyl-purine-2,6-
dione 7
Yield 80%, mp 79–81°C, Rf ¼ 0.49 (A), 1H-NMR d: 1,44–1.49 (m,
2H, N7–(CH2)2CH2CH2), 1.75–1.85 (m, 4H, N7–CH2CH2CH2CH2),
3.38 (s, 3H, N1–CH3), 3.52 (s, 3H, N3–CH3), 3.55 (t, 2H,
CH2CH2Cl), 4.11 (t, 2H, N7–CH2CH2), 4.12 (s, 3H, OCH3). LC/MS:
m/z calc. 315.12, found 314.88. Anal. (C13H19ClN4O3) C, H, N.
7-{3-[4-(3-Chlorophenyl)-piperazin-1-yl]-propyl}-8-
methoxy-1,3-dimethyl-purine-2,6-dione 12
1
Yield 67%, mp 137–140°C, Rf ¼ 0.27 (A), H-NMR d: 2.23–2.31
7-(5-Chloropentyl)-8-ethoxy-1,3-dimethyl-purine-2,6-
dione 8
(m, 2H, N7–CH2CH2CH2), 2.48–2.56 (m, 2H, N7–CH2CH2CH2),
3.02–3.13 (m, 4H, N–(CH2)2), 3.36 (s, 3H, N1–CH3), 3.53 (s, 3H,
N3–CH3), 3.55–3.76 (m, 4H, (CH2)2N–Ph), 4.15 (s, 3H, O–CH3),
4.21–4.26 (m, 2H, N7–CH2CH2), 7.10–7.43 (m, 4H, Ph). LC/MS:
m/z calc. 447.19, found 447.43. Anal. (C21H27ClN6O3) C, H, N.
Yield 74%, mp 84–86°C, Rf ¼ 0.75 (A), 1H-NMR d: 1.44–1.49 (m,
5H, N7–(CH2)2CH2CH2 and O–CH2CH3), 1.75–1.83 (m, 4H,
N7–CH2CH2CH2CH2CH2), 3.38 (s, 3H, N1–CH3); 3.53 (s, 3H,
N3–CH3), 3.49–3.52 (m, 2H, CH2Cl), 4.05–4.10 (t, J ¼ 6.9 Hz, 2H,
N7–CH2), 4.50–4.53 (q, J ¼ 7.2 Hz, O–CH2CH3). LC/MS: m/z calc.
329.13, found 328.92. Anal. (C14H21ClN4O3) C, H, N.
7-{3-[4-(4-Fluorophenyl)-piperazin-1-yl]-propyl}-8-
methoxy-1,3-dimethyl-purine-2,6-dione 13
1
Yield 48%, mp 119–121°C, Rf ¼ 0.26 (A), H-NMR d: 1.73–1.88
7-(5-Chloropentyl)-1,3-dimethyl-8-propoxy-purine-2,6-
dione 9
(m, 2H, N7–CH2CH2CH2), 2.37–2.49 (m, 2H, N7–CH2CH2CH2),
2.56–2.63 (m, 4H, N–(CH2)2), 3.07–3.14 (m, 4H, (CH2)2N–Ph),
3.38 (s, 3H, N1–CH3), 3.53 (s, 3H, N3–CH3), 4.12 (s, 3H, O–CH3),
4.05–4.17 (m, 2H, N7–CH2CH2), 6.81–7.01 (m, 4H, Ph). LC/MS:
m/z calc. 431.22, found 431.33. Anal. (C21H27FN6O3) C, H, N.
Yield 84%, mp 48–50°C, Rf ¼ 0.79 (A), 1H-NMR d: 1.03 (t,
J ¼ 7.2 Hz, 3H, O–CH2CH2CH3), 1.41–1.46 (m, 2H, N7
(CH2)2CH2CH2), 1.77–1.82 (m, 4H, N7–CH2CH2CH2CH2), 1.85–
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249

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Arch. Pharm. Chem. Life Sci. 2015, 348, 242–253
G. Chłon-Rzepa et al.
ꢀ
Archiv der Pharmazie
8-Methoxy-1,3-dimethyl-7-[4-(4-phenylpiperazin-1-yl)-
O–CH2CH3), 1.60–1.80 (m, 4H, N7–CH2CH2CH2CH2CH2), 3.00–
3.18 (m, 6H, (CH2)2N-Ph and N7–(CH2)3CH2CH2), 3.20 (s, 3H,
N1–CH3), 3.38 (s, 3H, N3–CH3), 3.40–3.56 (m, 2H, N(CH2)2 axial),
3.75–3.85 (m, 2H, N(CH2)2 equat.), 4.10 (t, J ¼ 6.5 Hz 2H,
N7–CH2CH2), 4.50 (q, J ¼ 7.1 Hz, 2H, O–CH2CH3), 6.80–7.00 (m,
3H, Ph), 7.24–7.32 (m, 2H, Ph), 10.37 (s, 1H, Hþ) LC/MS: m/z calc.
455.27, found 454.99. Anal. (C24H34N6O3 ꢃ HCl) C, H, N.
butyl]-purine-2,6-dione 14
1
Yield 45%, mp 117–119°C, Rf ¼ 0.26 (A), H-NMR d: 1.43–1.61
(m,
2H,
N7–(CH2)2CH2CH2),
1.75–1.88
(m,
2H,
N7–CH2CH2(CH2)2), 2.38–2.49 (m, 2H, N7–(CH2)3CH2), 2.56–
2.63 (m, 4H, CH2N(CH2)2), 3.07–3.14 (m 4H, (CH2)2N–Ph), 3.38
(s, 3H, N1–CH3), 3.53 (s, 3H, N3–CH3), 4.05–4.17 (m, 5H, OCH3
and N7–CH2), 6.85–7.25 (m, 5H, Ph). LC/MS: m/z calc. 427.24,
found 427.41. Anal. (C22H30N6O3) C, H, N.
8-Ethoxy-7-{5-[4-(2-methoxyphenyl)-piperazin-1-yl]-
pentyl}-1,3-dimethyl-purine-2,6-dione hydrochloride 20
1
8-Methoxy-7-{4-[4-(2-methoxyphenyl)-piperazin-1-yl]-
butyl}-1,3-dimethyl-purine-2,6-dione 15
Yield 62%, mp 157–159°C, Rf ¼ 0.49 (A), H-NMR d: 1.21–1.26
(m, 2H, N7–(CH2)2CH2(CH2)2), 1.38 (t, J ¼ 7.1 Hz, 3H,
O–CH2CH3), 1.60–1.80 (m, 4H, N7–CH2CH2CH2CH2CH2), 2.82–
3.18 (m, 6H, (CH2)2N–Ph and N7-(CH2)3CH2CH2), 3.20 (s, 3H,
N1–CH3), 3.37 (s, 3H, N3–CH3), 3.40–3.58 (m, 2H, N(CH2)2), 3.76
(s, 3H, Ph–OCH3), 4.00 (t, J ¼ 6.5 Hz 2H, N7–CH2CH2), 4.50 (q,
J ¼ 7.0 Hz, 2H, O–CH2CH3), 6.82–7.02 (m, 4H, Ph), 10.77 (s, 1H,
Hþ). LC/MS: m/z calc. 485.28, found 484.97. Anal. (C25H36N6O4
ꢃ HCl) C, H, N.
Yield43%,mp120–123°C,Rf ¼ 0.28(A),1H-NMRd:1.45–1.60(m,
2H, N7–(CH2)2CH2–CH2), 1.74–1.87 (m, 2H, N7–CH2CH2(CH2)2),
2.37–2.46(m,2H,N7–(CH2)3CH2),2.57–2.67(m,4H,CH2N(CH2)2),
3.00–3.25 (m, 4H, (CH2)2N–Ph), 3.38 (s, 3H, N1–CH3), 3.53
(s, 3H, N3–CH3), 4.03–4.16 (m, 5H, OCH3 and N7–CH2), 6.83–
7.01 (m, 4H, Ph). LC/MS:, m/z calc. 457.25, found 457.53. Anal.
(C23H32N6O4) C, H, N. 15 ꢃ HCl: mp 169–171°C, Anal. (C23H32N6O4
ꢃ HCl) C, H, N.
7-{5-[4-(3,4-Dichlorophenyl)-piperazin-1-yl]-pentyl}-8-
7-{4-[4-(3-Chlorophenyl)-piperazin-1-yl]-butyl}-8-
methoxy-1,3-dimethyl-purine-2,6-dione 16
ethoxy-1,3-dimethyl-purine-2,6-dione hydrochloride 21
Yield 64%, mp 169–171°C, Rf ¼ 0.41 (A), H-NMR d: 1.18–1.28
1
Yield 46%, mp 82–85°C, Rf ¼ 0.26 (A), 1H-NMR d: 1.48–1.63 (m,
2H, N7–(CH2)2CH2CH2), 1.73–1.88 (m, 2H, N7–CH2CH2(CH2)2),
2.35–2.49 (m, 2H, N7–(CH2)3CH2), 2.56–2.63 (m, 4H, CH2N
(CH2)2), 3.08–3.17 (m 4H, (CH2)2N–Ph), 3.38 (s, 3H, N1–CH3),
3.53 (s, 3H, N3–CH3), 4.05–4.17 (m, 5H, OCH3 and N7–CH2),
6.85–7.03 (m, 4H, Ph). LC/MS: m/z calc. 461.21, found 461.32.
Anal. (C22H29ClN6O3) C, H, N.
(m, 2H, N7–(CH2)2CH2(CH2)2), 1.38 (t, J ¼ 7.1 Hz, 3H,
O–CH2CH3), 1.60–1.80 (m, 4H, N7–CH2CH2CH2CH2CH2), 3.00–
3.17 (m, 6H, (CH2)2N–Ph and N7–(CH2)3CH2CH2), 3.20 (s, 3H,
N1–CH3), 3.37 (s, 3H, N3–CH3), 3.40–3.57 (m, 2H, N(CH2)2 axial),
3.80–3.85 (m, 2H, N(CH2)2 equat.), 4.00 (t, J ¼ 6.5 Hz, 2H,
N7–CH2CH2), 4.50 (q, J ¼ 7.1 Hz, 2H, O–CH2CH3), 7.00 d, 1H,
Ph), 7.22 (s, 1H, Ph), 7.43 (d, 1H, Ph), 10.30 (s, 1H, Hþ). LC/MS:
m/z calc. 523.19, found 523.44. Anal. (C24H32Cl2N6O3 ꢃ HCl) C,
H, N.
7-{4-[4-(4-Fluorophenyl)-piperazin-1-yl]-butyl}-8-
methoxy-1,3-dimethyl-purine-2,6-dione 17
1
Yield 41%, mp 113–115°C, Rf ¼ 0.26 (A), H-NMR d: 1.46–1.62
1,3-Dimethyl-7-[5-(4-phenylpiperazin-1-yl)-pentyl]-8-
propoxy-purine-2,6-dione hydrochloride 22
(m,
2H,
N7–(CH2)2CH2–CH2),
1.73–1.88
(m,
2H,
N7–CH2CH2(CH2)2), 2.37–2.49 (m, 2H, N7–(CH2)3CH2), 2.56–
2.63 (m, 4H, CH2N(CH2)2), 3.07–3.14 (m 4H, (CH2)2N–Ph), 3.38
(s, 3H, N1–CH3), 3.53 (s, 3H, N3–CH3), 4.05–4.17 (m, 5H, OCH3
and N7–CH2), 6.81–7.01 (m, 4H, Ph). LC/MS: m/z calc. 445.23,
found 445.36. Anal. (C22H29FN6O3) C, H, N.
Yield 56%, mp 179–180°C, Rf ¼ 0.42 (A), 1H-NMR d: 0.97 (t,
J¼ 7.4 Hz,3H,O–CH2CH3),1.20–1.38(m,2H,N7–(CH2)2CH2(CH2)2),
1.60–1.80 (m, 6H, N7–CH2CH2CH2CH2CH2 and O–CH2CH2CH3),
2.95–3.18 (m, 6H, (CH2)2N–Ph and N7–(CH2)3CH2CH2), 3.20 (s, 3H,
N1–CH3), 3.37 (s, 3H, N3–CH3), 3.40–3.60 (m, 2H, N(CH2)2 axial),
3.72–3.85 (m, 2H, N(CH2)2 equat.), 4.00 (t, J¼ 6.3 Hz, 2H,
N7–CH2CH2), 4.40 (t, J¼ 7.1 Hz, 2H, O–CH2CH2CH3), 6.84 (t,
J¼ 7.2 Hz, 1H, Ph), 7.00 (d, J ¼ 8.0 Hz, 2H, Ph), 7.20–7.30 (m, 2H,
Ph), 10.18 (s, 1H, Hþ). LC/MS: m/z calc. 469.29, found 468.96. Anal.
(C25H36N6O3 ꢃ HCl) C, H, N.
8-Methoxy-1,3-dimethyl-7-[5-(4-phenylpiperazin-1-yl)-
pentyl]-purine-2,6-dione 18
1
Yield 39%, mp 110–112°C, Rf ¼ 0.56 (A), H-NMR d: 1.27–1.38
(m,
2H,
N7–(CH2)2CH2(CH2)2),
1.58–1.60
(m,
2H,
N7–(CH2)3CH2CH2), 2.32–2.40 (m, 2H, N7–(CH2)3CH2CH2),
2.56–2.60 (m, 4H, N(CH2)2), 3.15–3.20 (m, 4H, (CH2)2N–Ph),
3.38 (s, 3H, N1–CH3), 3.53 (s, 3H, N3–CH3), 4.05 (t, J ¼ 6.5 Hz 2H,
N7–CH2CH2), 4.10 (s, 3H, O–CH3), 6.80–6.95 (m, 3H, Ph), 7.22–
7.35 (m, 2H, Ph). LC/MS: m/z calc. 441.26, found 44.89. Anal.
(C23H32N6O3) C, H, N.
7-{5-[4-(2-Methoxyphenyl)-piperazin-1-yl]-pentyl}-1,3-
dimethyl-8-propoxy-purine-2,6-dione hydrochloride 23
1
Yield 59%, mp 190–192°C, Rf ¼ 0.45 (A), H-NMR d: 0.97 (t, J¼
7.4 Hz, 3H, O–(CH2)2CH3), 1.18–1.26 (m, 2H, N7–(CH2)2CH2(CH2)2),
1.59–1.84(m,6H,N7–CH2CH2CH2CH2CH2andO–CH2CH2CH3),2.95–
3.18 (m, 6H, (CH2)2N–Ph and N7–(CH2)3CH2CH2), 3.20 (s, 3H,
N1–CH3),3.37(s,3H,N3–CH3),3.41–3.55(m,4H,N(CH2)2),3.76(s,3H,
Ph–OCH3), 4.05 (t, J¼ 6.0 Hz, 2H, N7–CH2(CH2)4), 4.42 (t, J¼ 6.5 Hz,
2H, O-CH2C2H5), 6.86–7.02 (m, 4H, Ph), 10.42 (s, 1H, Hþ). LC/MS: m/z
calc. 499.30, found 499.13. Anal. (C26H38N6O4 ꢃ HCl) C, H, N.
8-Ethoxy-1,3-dimethyl-7-[5-(4-phenylpiperazin-1-yl)-
pentyl]-purine-2,6-dione hydrochloride 19
1
Yield 67%, mp 128–130°C, Rf ¼ 0.45 (A), H-NMR d: 1.22–1.26
(m, 2H, N7–(CH2)2CH2(CH2)2), 1.40 (t, J ¼ 7.1 Hz, 3H,
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Arch. Pharm. Chem. Life Sci. 2015, 348, 242–253
Arylpiperazinylalkyl Derivatives of Purine-2,6-dione
Archiv der Pharmazie
7-{5-[4-(3,4-Dichlorophenyl)-piperazin-1-yl]-pentyl}-8-
ethoxy-1,3-dimethyl-8-propoxy-purine-2,6-dione
hydrochloride 24
(CH2)2þCH2CH), 3.44–3.69 (m, 2H, CH2CHþNCH2), 6.68–6.78
(m, 1H, Ph), 6.89–6.98 (m, 1H, Ph), 7.23–7.28 (m, 1H, Ph). LC/
MS: m/z calc. 286.06, found 288.19 (MþH)þ. Anal.
(C13H16Cl2N2O) C, H, N.
Yield 65%, mp 201–203°C, Rf ¼ 0.43 (A), 1H-NMR d: 0.98 (t, J¼
7.4 Hz, 3H, O–(CH2)2CH3), 1.20–1.28 (m, 2H, N7–(CH2)2CH2(CH2)2),
1.60–1.80 (m, 6H, N7–CH2CH2CH2CH2CH2 and O–CH2CH2CH3),
2.96–3.20 (m, 6H, (CH2)2N–Ph and N7–(CH2)3CH2CH2), 3.20 (s, 3H,
N1–CH3),3.37(s,3H,N3–CH3),3.46(d,J ¼ 9.7 Hz,2H,N(CH2)2axial),
3.85 (d, J¼ 10.5 Hz, 2H, N(CH2)2 equat.), 4.04 (t, J ¼ 6.0 Hz, 2H,
N7–CH2(CH2)4),4.41(t,J ¼ 6.3 Hz,2H,O–CH2C2H5),6.97(d,1H,Ph),
7.21 (s, 1H, Ph), 7.42 (d, 1H, Ph), 10.65 (s, 1H, Hþ). LC/MS: m/z calc.
537.48, found 537.03. Anal. (C25H34Cl2N6O3 ꢃ HCl) C, H, N.
General procedures for the preparation of derivatives of 7-
arylpiperazinylmethyldihydro[1,3]oxazolo[2,3-f]-
purinediones 30–34
A
mixture
of
8-bromo-1,3-dimethyl-purine-2,6-dione
(10 mmol) and 20 mmol of the corresponding substituted
oxiranes was heated under reflux in anhydrous 1-propanol in
the presence of catalytic amount of pyridine (0.3 mL) for 10 h.
After cooling, the solvent was evaporated, the residue was
mixed with 10% NaOH, and washed with water. The final
compounds were purified by column chromatography using C
(30, 31, 34), B (32) or D (33) as solvent.
General procedures for the preparation of derivatives of
phenylpiperazinylmethyloxiranes 25–29 [38]
Epichlorohydrin (1.9 mmol) was added all at once and with
vigorous stirring to corresponding phenylpiperazines
(1.9 mmol) containing 0.6 mL of water. The reaction mixtures
weremaintainedbetween18and35°C(1 h), andnextheatedto
75°C and treated during 15 min with 2.31g of aqueous NaOH
(38.4%). The cooled solutions were filtered and the final
compounds were purified by column chromatography (C).
7-[(4-Phenyl)-piperazin-1-yl-methylene]-1,3-dimethyl-6,7-
dihydro-1,3-oxazolo[2,3-f]-purine-2,4(1H,3H)-dione 30
Yield 89%; mp 123–124°C, Rf ¼ 0.4 (C); 1H-NMR d: 3.21–3.40
(m, 8H, N(CH2)4N, 3.45 (s, 3H, N3–CH3), 3.55 (s, 3H, N1–CH3),
4.20 (t, J ¼ 8.02 Hz, 1H, CH2), 4.40–4.52 (m, 1H, CH2), 4.55–4.68
(m, 2H, CH2), 5.92–6.05 (m, 1H, CH), 6.84–6.98 (m, 3H, Ph),
7.25–7.34 (m, 2H, Ph). LC/MS: m/z calc. 396.19, found 397.45
(MþH)þ. Anal. (C20H24N6O3) C, H, N.
3-(4-Phenyl-piperazinyl)-1,2-epoxypropane 25
Yield83%;mp82–84°C; Rf ¼ 0.71 (C);1H-NMR d:2.29–2.35 (m, 1H,
NCH2), 2.50–2.53 (m, 1H, CH2CH), 2.63–2.70 (m, 2H,
CH2CHþNCH2), 2.74–2.85 (m, 4H, N(CH2)2), 3.11–3.27 (m, 5H, N
(CH2)2þCH), 6.80–6.94 (m, 3H, Ph), 7.24–7.29 (m, 2H, Ph). LC/MS:
m/z calc. 218.14, found 219.30(MþH)þ.Anal.(C13H18N2O) C, H, N.
7-[(4-(-20-Methoxyphenyl)-piperazin-1-yl-methylene]-1,3-
dimethyl-6,7-dihydro-1,3-oxazolo[2,3-f]-purine-2,4-
(1H,3H)-dione 31
1
Yield 90%; mp 132–133°C, Rf ¼ 0.34 (C); H-NMR d: 3.25–3.48
3[4-(20-Methoxyphenyl)-piperazinyl]-1,2-epoxypropane 26
Yield 90%; mp 76–77°C; Rf ¼ 0.71 (C); 1H-NMR d: 2.34–2.39 (m,
1H, NCH2), 2.51–2.53 (m, 1H, CH2CH), 2.60–2.89 (m, 6H,
CH2CHþNCH2þN(CH2)2), 3.12–3.24 (m, 5H, N(CH2)2þCH), 3.86
(s, 3H, OCH3), 6.85–7.00 (m, 4H, Ph). LC/MS: m/z calc. 248.15,
found 249.33 (MþH)þ. Anal. (C14H20N2O2) C, H, N.
(m, 8H, N(CH2)4N, 3.42 (s, 3H, N3–CH3), 3.58 (s, 3H, N1–CH3),
3.80 (s, 3H, OCH3), 4.17 (t, J ¼ 8.02 Hz, 1H, CH2), 4.46–4.54 (m,
1H, CH2), 4.58–4.67 (m, 2H, CH2), 5.87–6.05 (m, 1H, CH), 6.85–
7.04 (m, 4H, Ph). LC/MS: m/z calc. 426.20, found 427.45
(MþH)þ. Anal. (C21H26N6O4) C, H, N.
7-[(4-(-30-Chlorophenyl)-piperazin-1-yl-methylene]-1,3-
dimethyl-6,7-dihydro-1,3-oxazolo[2,3-f]-purine-2,4-
(1H,3H)-dione 32
3[4-(30-Chlorophenyl)-piperazinyl]-1,2-epoxypropane 27
1
Yield 90%; mp 139–141°C; Rf ¼ 0.71 (C); H-NMR d: 2.48–2.59
1
(m, 2H, CH2CHþNCH2), 2.62–2.85 (m, 4H, N(CH2)2), 3.18–3.29
(m, 6H, N(CH2)2þCH2CHþNCH2), 3.59–3.61 (m, 1H, CH), 6.76–
6.90 (m, 3H, Ph), 7.14–7.19 (m, 1H, Ph). LC/MS: m/z calc. 252.10,
found 253.73 (MþH)þ. Anal. (C13H17ClN2O) C, H, N.
Yield 90%; mp 178–179°C, Rf ¼ 0.68 (B); H-NMR d: 3.29–3.44
(m, 8H, N(CH2)4N, 3.46 (s, 3H, N3–CH3), 3.58 (s, 3H, N1–CH3),
4.21 (t, J ¼ 8.02 Hz, 1H, CH2), 4.47–4.54 (m, 1H, CH2), 4.59–4.70
(m, 2H, CH2), 5.94–6.08 (m, 1H, CH), 6.76–6.91 (m, 3H, Ph),
7.15–7.23 (m, 1H, Ph). LC/MS: m/z calc. 430.15, found 431.90
(MþH)þ. Anal. (C20H23ClN6O3) C, H, N.
3[4-(20,30-Dichlorophenyl)-piperazinyl]-1,2-epoxypropane
28
1
Yield 87%; mp 145–146°C, Rf ¼ 0.68 (C); H-NMR d: 2.34–2.88
7-[(4-(-20,30-Dichlorophenyl)-piperazin-1-yl-methylene]-
1,3-dimethyl-6,7-dihydro-1,3-oxazolo[2,3-f]-purine-2,4-
(1H,3H)-dione 33
(m, 6H, N(CH2)2þCH2CHþNCH2), 3.14–3.16 (m, 4H, N(CH2)2),
3.44–3.62 (m, 3H, CH2CHþNCH2þCH), 6.93–6.98 (m, 1H, Ph),
7.14–7.16 (m, 2H, Ph). LC/MS: m/z calc. 286.06, found 288.19
(MþH)þ. Anal. (C13H16Cl2N2O) C, H, N.
Yield 90%; mp 166–167°C, Rf ¼ 0.28 (D); 1H-NMR d: 3.28–
3.46 (m, 8H, N(CH2)4N, 3.51 (s, 3H, N3–CH3), 3.61 (s, 3H,
N1–CH3), 4.20–4.32 (t, J ¼ 5.5Hz, 1H, CH2), 4.48–4.56 (m, 1H,
CH2), 4.57–4.67 (m, 2H, CH2), 5.95–6.04 (m, 1H, CH),
6.93–6.98 (m, 1H, Ph), 7.16–7.18 (m, 2H, Ph). LC/MS: m/z
calc. 464.11, found 466.34 (MþH)þ. Anal. (C20H22Cl2N6O3) C,
H, N.
3[4-(30,40-Dichlorophenyl)-piperazinyl]-1,2-epoxypropane
29
1
Yield 82%; mp 156–157°C, Rf ¼ 0.68 (C); H-NMR d: 2.42–2.86
(m, 6H, N(CH2)2þCH2CHþNCH2), 3.10–3.22 (m, 5H,
N
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Arch. Pharm. Chem. Life Sci. 2015, 348, 242–253
G. Chłon-Rzepa et al.
ꢀ
Archiv der Pharmazie
7-[(4-(-30,40-Dichlorophenyl)-piperazin-1-yl-methylene]-
1,3-dimethyl-6,7-dihydro-1,3-oxazolo[2,3-f]-purine-2,4
(1H,3H)-dione 34
above it. The total duration of immobility was recorded
during the last 4 min of a 6 min test session.
Yield 90%; mp 155–156°C, Rf ¼ 0.7 (C); 1H-NMR d: 3.32–3.49
(m, 8H, N(CH2)4N, 3.56(s, 3H, N3–CH3), 3.64(s, 3H, N1–CH3), 4.23–
4.31 (t, J ¼ 5.5Hz, 1H, CH2), 4.54–4.61 (m, 1H, CH2), 4.66–4.75 (m,
2H, CH2), 6.03–6.11 (m, 1H, CH), 6.71–6.80 (m, 1H, Ph), 6.89–7.01
(m, 1H, Ph), 7.22–7.28(m, 1H, Ph). LC/MS:m/zcalc. 464.11, found
466.34 (MþH)þ. Anal. (C20H22Cl2N6O3) C, H, N.
Four-plate test in Swiss Albino mice
The experiment was assessed according to the method of Aron
et al. [32]. The method and conditions of experiment were
previously reported [20]. The number of punished crossings
received by an animal was recorded during the 60 s period.
D-Amphetamine-induced hyperactivity in CD-1 mice
Locomotor activity was recorded with an Opto M3 multichan-
nel activity monitor (MultiDevice Software v.1.3, Columbus
Instruments). The mice were individually placed in plastic
cages (22 cm ꢂ 12 cm ꢂ 13 cm) for 30 min habituation period,
and then the crossings of each channel (ambulation) were
counted during 1 h with data recording every 5 min.
In vitro radioligand binding assays
Compounds 10–24 were tested in competition binding
experiments by displacement of respective radioligands from
cloned human Rs, all stably expressed in HEK-293 cells: [3H]-8-
OH-DPAT, [3H]-ketanserin), [3H]-LSD, [3H]-5-CT, and [3H]-
raclopride for 5-HT1A, 5-HT2A, 5-HT6, 5-HT7, and dopamine
D2 Rs, respectively, according to the previously published
procedures [11, 23, 24]. Each compound was tested in
triplicate at 7–8 concentrations (10ꢀ11–10ꢀ4 M). The inhibition
constants (Ki) were calculated based on the method described
in literature [39]. Results were expressed as means of at least
two separate experiments.
The metabolic studies in mouse liver microsomes
The incubation systems were composed of the tested
compound (20 mM in 100 mM potassium phosphate buffer,
pH 7.4), mouse liver microsomes (Sigma–Aldrich) (0.8 mg/mL),
and NADPH-regenerating system [44]. The resulting mixture
was incubated for 5, 15, or 30 min at 37°C. Then, an internal
standard (levallorphan, 20 mM) was added. The incubation
was terminated at different time points by the addition of
perchloric acid (69–72%, by volume). Proteins were sedi-
mented by centrifugation. The resulting supernatant was
analyzed using UPLC/MS in order to determine the quantity of
starting material left in solution and possible metabolites. The
tests without NADPH-regenerating system were conducted in
parallel. All probes were done in duplicate [45, 46].
Compounds 30–34 were tested in a screening assay at two
finalconcentrations:10ꢀ6 M and10ꢀ7 M. Detailed conditionsof
the assays for the respective Rs were previously reported [25].
Results were expressed as a percentage of specific binding.
Molecular modeling
The homology models of human 5-HT1A serotonin receptor
used for docking studies were described in previously
published papers [20, 40, 41].
The homology models were built and optimized according
to a validated method [42] on the basis of b2 adrenergic
receptor crystal structure (PDB ID: 2RH1) [43]. Ligand
structures were optimized using LigPrep and Jaguar tools.
Glide XP flexible docking was carried out using default
parameters, setting H-bond constraints on Asp3.32.
Glide, Jaguar, and LigPrep were implemented in Small-
Molecule Drug Discovery Suite (Schroödinger, Inc.), which was
licensed for Jagiellonian University Medical College.
Statistical analysis
All the data are presented as the mean ꢁ SEM. The statistical
significance of results was evaluated by a one-way analysis of
variance (ANOVA) followed by Bonferroni's comparison test.
A p < 0.05 was considered statistically significant.
This study was supported by the NCN funded grants no. UMO-
2011/01/B/NZ4/00695 and no. 2012/07B/NZ7/01173.
In vivo experiments
The authors have declared no conflict of interest.
The experiments were performed on male Albino Swiss or CD-
1 mice (22–28 g). Detailed conditions of the procedures and
reagents were previously reported [20]. D-Amphetamine
(Sigma–Aldrich) was administered subcutaneously (sc),
30 min before the test; D-amphetamine and 15 were dissolved
in distilled water. All the experimental procedures were
approved by the Local Ethics Committee for Animal Experi-
ments of Jagiellonian University in Cracow.
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