Serotonin transporter activity of imidazolidine-2,4-dione and imidazo[2,1-f]purine-2,4-dione derivatives in aspect of their acid-base properties

Article abstract of DOI:10.1007/s00044-011-9883-y

Affinities of arylpiperazinylalkyl derivatives of imidazo[2,1-f]purine-2,4- dione and imidazolidine-2,4-dione for serotonin transporter and their acid-base properties were evaluated. The dissociation constant (pKa) of compounds 1-22 were determinated by p

Full text of DOI:10.1007/s00044-011-9883-y

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2012) 21:3455–3459
DOI 10.1007/s00044-011-9883-y
ORIGINAL RESEARCH
Serotonin transporter activity of imidazolidine-2,4-dione
and imidazo[2,1-f]purine-2,4-dione derivatives in aspect
of their acid–base properties
•
Agnieszka Zagorska Anna Czopek Maciej Pawłowski
•
•
´
Małgorzata Dybała Agata Siwek Gabriel Nowak
•
•
Received: 14 September 2011 / Accepted: 4 November 2011 / Published online: 19 November 2011
Ó The Author(s) 2011. This article is published with open access at Springerlink.com
Abstract Affinities of arylpiperazinylalkyl derivatives of
imidazo[2,1-f]purine-2,4-dione and imidazolidine-2,4-
dione for serotonin transporter and their acid–base prop-
erties were evaluated. The dissociation constant (pKa) of
compounds 1–22 were determinated by potentiometric
titration and calculated using pKalc 3.1 module of the
Pallas system. The data from experimental methods and
computational calculations were compared and suitable
conclusions were reached.
connecting with serotonin transporter (SERT). This trans-
porter is mediated extracellular uptake of serotonin from
the synaptic clefts. The SERT protein belongs to the large
family of transporters that are dependent on Na? ions.
Serotonin, Na? and Cl- form a quaternary complex with
the transporter before being co-transported across the
plasma membrane, followed by counter transport of K?. At
physiological pH = 7.4, serotonin is protonated and in the
case of the SERT 5-HT accumulation was not affected by
transmembrane pH differences (Rudnick et al., 1989;
Forrest et al., 2007). Many drug molecules contain ioniz-
able groups and hence penetrate across cell membranes,
through pores and via active transport mechanism in a pKa
dependent fashion, therefore pKa is an important factor on
estimating the pharmacological behavior of drugs and their
pharmacokinetic. This is particularly important in physio-
logical systems, where ionization state will affect the rate
at which the compound is able to diffuse across membranes
and obstacles such as the blood–brain barrier (BBB) (Luan
et al., 2005; Manallack, 2007).
Keywords Dissociation constant ꢀ Imidazolidine-2,
4-dione ꢀ Imidazo[2,1-f]purine-2,4-dione ꢀ
Serotonin receptor 5-HT1A ꢀ Serotonin transporter
Introduction
Studies on major depression, anxiety, schizophrenia,
mania, autism, obesity, and drug addiction have implicated
the involvement of serotonergic (5-HT) abnormalities in
these diseases. Serotonin acts via receptors which were
classified into seven families (5-HT1–7) and at least 14
different subtypes (Barnes and Sharp, 1999; Filip et al.,
2005; Hannon and Hoyer, 2008; Hoyer et al., 2002;
Pauwels, 2003). The level of 5-HT in central nervous
system (CNS) and regulation of its neurotransmission are
Since the early seventies until today, a large number of
selective SERT inhibitors (SSRIs) have been described.
However, only 5 products are currently marketed: fluoxetine
(ProzacÒ), escitalopram (LexaproÒ, CipralexÒ), sertraline
(ZoloftÒ), paroxetine (PaxilÒ), and fluvoxamine (Feva-
rinÒ). A major limitation of SSRIs and that extends to all
other classes of antidepressants as well, is the 2–6 weeks
delay in onset of therapeutic activity. This lengthy time to
achieve remission is suspected to result from indirect acti-
vation of somatodendric 5-HT1A autoreceptors (Chaput
et al., 1986; Invernizzi et al., 1992; Invernizzi et al., 1996).
The latest direction taken in antidepressant drug discovery
has been to design ligands with multiple targets. Preclinical
data obtained by co-administrating a SSRI with selective
5-HT1A antagonist, suggest that a single compound
´
A. Zagorska (&) ꢀ A. Czopek ꢀ M. Pawłowski
Department of Pharmaceutical Chemistry,
Jagiellonian University Medical College, Medyczna 9 St,
´
30-688 Krakow, Poland
e-mail: azagorsk@cm-uj.krakow.pl
M. Dybała ꢀ A. Siwek ꢀ G. Nowak
Department of Pharmacobiology, Jagiellonian University
´
Medical College, Medyczna 9, 30-688 Krakow, Poland
123

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Med Chem Res (2012) 21:3455–3459
combining SSRIs with 5-HT1A antagonism should have a
favorable therapeutic utility in the treatment (Artigas et al.,
1996; Ballesteros and Callodo, 2004; Adell et al., 2005;
Morphy and Rankovic, 2005; Millan, 2006). The most
important class of 5-HT1A receptor ligands are derivatives of
arylpiperazine. Simple arylpiperazines are non-selective
ligands for 5-HT receptor. The good selectivity and affinity
for 5-HT1A receptors show the majority of 4-substituted
arylpiperazines. These derivatives contain a flexible
aliphatic chain of different length (long-chain arylpipera-
zines, LCAPs), which connects the arylpiperazine
fragment with second terminal pharmacophoric group
(Lopez-Rodriguez et al., 2002; Paluchowska et al., 2007;
Paluchowska et al., 2005). For several years our attention has
been focused on developing LCAPs containing a different
amide/imide terminal fragment. In our earlier study a series
of arylpiperazinylalkyl derivatives with a complex terminal
part based on the purine moiety had been synthesized.
Compounds with pyrimido- and imidazo-[2,1-f]purine-2,
4-dione fragment have been tested in vitro for their 5-HT1A
and 5-HT2A receptor affinities and were potent 5-HT1A
receptor ligands with Ki within the range of 5.6–278 nM and
7-acetonyl-8-bromotheophylline, and 7-phenacyl-8-bro-
motheophylline, with double amount of appropriate aryl-
piperazinylpropylamine, in boiling 2-methoxyethanol
´
(Zagorska et al., 2009). Whereas initial spirohydantoins
were prepared from appropriate ketone by the Buchere–
Berg reaction with modification described by Goodson
et al. (1960). Compounds 13–22 were obtained by a two-
step procedure involving alkylation of hydantoin at N3
position and condensation of intermediates with the
substituted arylpiperazine or tetrahydroizoquinoline. The
synthesis and physicochemical properties are described
´
elsewhere (Zagorska et al., 2009; Czopek et al., 2010). The
synthetic procedures and physicochemical data of com-
pounds 16, 17, 21, and 22 have not been published yet.
Pharmacology in vitro
The assay was performed according to the method of
Owens et al. (1997) with slight modifications. [3H]-
Citalopram (spec. act. 50 Ci/mmol, NEN Chemicals) was
used for labeling 5-HT-transporter. Rat cerebral cortex was
homogenized in 30 volumes of ice-cold 50 mM Tris–HCl
containing 150 mM NaCl and 5 mM KCl, pH = 7.7 at
25°C and centrifuged at 20,0009g for 20 min. The
supernatant was decanted and pellet was resuspended in 30
volumes of buffer and centrifuged again. The resulting
pellet was resuspended in the same quantity of the buffer
and centrifuged third time in the same conditions. 240 ll
of the tissue suspension, 30 ll of 1 nM [3H]-citalopram,
and 30 ll of the analyzed compound or 30 ll of 1 lM
imipramine (displacer) were incubated at 22°C for 1 h. The
concentrations of analyzed compounds ranged from 10-10
to 10-5 M. Incubations were terminated by vacuum fil-
tration over Whatman GF/B filters and washed 5 times with
200 ll of ice-cold buffer. Radioactivity was measured in a
MicroBeta TriLux– liquid scintillation counter (Perkin
Elmer). All assays were done in duplicates.
´
demonstrated lack of affinity for 5-HT2A subtype (Zagorska
et al., 2009). The majority of imidazolidine-2,4-dione
derivatives displayed high affinity for 5-HT1A receptors
(23–350 nM), and some of them exhibited significant
affinity for 5-HT2A receptors (Czopek et al., 2010).
Continuing our research, we have been interested in
affinities of arylpiperazinylalkyl derivatives of imidazo
[2,1-f]purine-2,4-dione and imidazolidine-2,4-dione (hydan-
toin) for SERT and their acid-based properties presented as
dissociation constant (pKa) values. The aqueous ionization
constant of a molecule is denoted by its pKa values, where this
constant is equivalent to the pH at which a given ionizable
group on the molecule is half-ionized. In search of the struc-
ture activity relationship, the correlation with biological
activity data with received pKa values, was done.
Radioligand binding data were analyzed using iterative
curve fitting routines (GraphPAD/Prism, Version 3.0 – San
Diego, CA, USA). Ki values were calculated from the
Cheng–Prusoff equation (Cheng and Prusoff, 1973). The
results of in vitro binding studies (pKi) of the compounds
(1–22) are shown in Table 1.
Methods and materials
Chemistry
The chemical structures of derivatives of the imidazo
Measurement of pKa
[2,1-f]purine-2,4-dione
and
imidazolidine-2,4-diones
investigated are listed in the Table 1. The hydrochloride
salts of investigated compound 1–22 of the analytical
purity (mp, TLC, elemental analysis) were used for the
potentiometric titration.
The pKa measurements were determined by potentiometric
titration (alkalimetric), using a Compact Titrator Mettler
Toledo G21 equipped with an integrated burette drive, and
combined glass electrode DGi115-SC, compact rod stirrer,
and 20 ml burette. Titrator was pre-programmed with
standard tried-and-tested methods and calculations. The pH
Compounds 1–12 were obtained in the cyclocondensa-
tion reaction of 7-acetic-8-bromotheophylline aldehyde,
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Med Chem Res (2012) 21:3455–3459
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Table 1 The structures of compounds 1–22, their SERT activity (pKi), experimental and theoretical pKa values
O
X
N
N
N
O
O
N
HN
O
HN
O
R
N
II:
III:
I:
N
N
O
N
R
R
Z
HN
B:
A: HN
Compd
Core
X
R
Z
pKi [SERT]
Exp pKa
pKa Pallas
1
I
H
H
H
A
A
A
A
A
A
A
B
A
A
A
B
A
A
A
A
B
A
A
A
A
B
H
6.35
6.95
7.53
4.95
5.09
7.52
7.25
4.52
6.65
4.69
6.72
5.61
5.96
5.96
6.07
6.19
Nd
8.09
8.19
9.29
9.29
9.20
9.29
9.29
9.20
9.20
8.72
9.29
9.29
9.20
8.72
8.95
8.95
8.85
8.95
8.38
8.95
8.95
8.85
8.95
8.38
2
I
2–OCH3
3–Cl
H
3
I
8.35
4
I
CH3
CH3
CH3
CH3
CH3
C6H5
C6H5
C6H5
C6H5
–
7.55
5
I
2–OCH3
3–Cl
2,3–diCl
–
7.61
6
I
8.12
7
I
7.61
8
I
8.66
9
I
H
8.79
10
11
12
13
14
15
16
17
18
19
20
21
22
I
2–OCH3
3–Cl
H
8.44
I
10.61
10.41
10.48
9.60
I
II
II
II
II
II
III
III
III
III
III
H
–
2–OCH3
3–Cl
3–CF3
–
–
10.31
9.96
–
–
10.93
10.55
10.32
10.80
11.08
10.90
–
H
6.00
6.01
6.04
5.62
5.40
–
2–OCH3
3–Cl
3–CF3
–
–
–
–
where Ct is a titrant concentration, Ca is a concentration of
sample at each measured point.
electrode was first calibrated with buffers (pH = 7.00 and
pH = 9.00). Sample (5 9 10-5 M) were prepared in water
solutions (between 10–20 ml). Typically, more than 120
pH readings were collected for each titration. The deion-
ized water used for the aqueous solution was twice dis-
tilled, degassed, and filtered with a Hydrolab Polska HLP5s
System. The 0.0512 M sodium hydroxide solution were
prepared from substances delivered by POCH. The buffers
pH = 7.00 and pH = 9.00 used for calibration were
obtained from Beckman Coulter. The pKa were expressed
as the mean of values of results from three titrations and are
listed in Table 1.
Calculations
Calculations of pKa were performed using Pallas 3.1
(CompuDrug Chemistry Ltd, 1995). Program applied
logarithm, adapted after Hammett and Taft takes into
account all necessary electronic, steric, and other effects
and relies on an extended database of almost a thousand
equations.
Regression analysis was performed using the Statistica
for Windows program (Statistica for Windows, version 9,
Statsoft Inc.2009). The significance level of the performed
calculations was above 95%.
The following equation was used for the calculation of
the pKa values:
2Ct ꢁ Ca
pKa ¼ pH þ log
ð1Þ
Ca ꢁ Ct
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Med Chem Res (2012) 21:3455–3459
Fig. 1 Correlation between pKa
values and pKi SERT of
compounds 1–7 (left) and 13–22
(right)
Results and discussion
confirm that the applied potentiometric method is useful in
characterization of the acid–base properties of closely
related compounds contrary to values of pKa predicted by
Pallas program. There is no correlation between values of
pKa predicted by Pallas program and experimental. The
moderate correlation between activity for SERT and pKa,
indicating that acid–base properties are one of the impor-
tant factors, which could influent and modify the activity
for SERT.
The library consisting of twenty two compounds was
investigated. Based on their structural features, this library
could be divided into two sublibraries: the first contained
various arylpiperazinylpropyl derivatives of imidazo[2,1-
f]theophylline, and the second derived from imidazolidine-
2,4-dione. Comparing the affinity for SERT obtained for
imidazo[2,1-f]purine-2,4-dione and respective imidazoli-
dine-2,4-dione analogues revealed higher activity in the
first mentioned series. The most potent SERT ligands
were compounds 3, 6, and 7 with pKi within the range of
7.25–7.53, which were containing 2,3-dichloro or 3-chlor-
ophenylpiperazine fragment in their structures. Compounds
1, 2, 9, 11, 12, 15, 16, 19, and 20 displayed moderate to very
low affinity for the SERT (5.61–6.95), whereas other were
practically devoid of any affinity.
Open Access This article is distributed under the terms of the
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