New adenosine A2A receptor antagonists: Actions on Parkinson's disease models

Article abstract of DOI:10.1016/j.ejphar.2005.01.057

The 8-substituted 9-ethyladenine derivatives: 8-bromo-9-ethyladenine (ANR 82), 8-ethoxy- 9-ethyladenine (ANR 94), and 8-furyl-9-ethyladenine (ANR 152) have been characterized in vitro as adenosine receptor antagonists. Adenosine is deeply involved in the control of motor behaviour and substantial evidences indicate that adenosine A_2A receptor antagonists improve motor deficits in animal models of Parkinson's disease. On this basis, the efficacy of ANR 82, ANR 94, and ANR 152 in rat models of Parkinson's disease was evaluated. All compounds tested reversed the catalepsy induced by haloperidol. However, in unilaterally 6-hydroxydopamine-lesioned rats, only ANR 94 and ANR 152 potentiated l-dihydroxy-phenylalanine (l-DOPA) effect on turning behaviour and induced contralateral turning behaviour in rats sensitised to l-DOPA. Taken together the results of this study indicate that some 8-substituted 9-ethyladenine derivatives ameliorate motor deficits in rat models of Parkinson's disease, suggesting a potential therapeutic role of these compounds.

Full text of DOI:10.1016/j.ejphar.2005.01.057

European Journal of Pharmacology 512 (2005) 157–164
www.elsevier.com/locate/ejphar
New adenosine A2A receptor antagonists:
Actions on Parkinson’s disease models
Annalisa Pinnaa,b, Rosaria Volpinic, Gloria Cristallic, Micaela Morellib,
T
aCNR Institute for Neuroscience—Section Cagliari, Cagliari, Italy
bDepartment of Toxicology, University of Cagliari, Via Ospedale, 72 09124 Cagliari, Italy
cDepartment Chemical Sciences, University of Camerino, Camerino, Italy
Received 19 August 2004; received in revised form 25 January 2005; accepted 31 January 2005
Available online 29 March 2005
Abstract
The 8-substituted 9-ethyladenine derivatives: 8-bromo-9-ethyladenine (ANR 82), 8-ethoxy- 9-ethyladenine (ANR 94), and 8-furyl-9-
ethyladenine (ANR 152) have been characterized in vitro as adenosine receptor antagonists. Adenosine is deeply involved in the control of
motor behaviour and substantial evidences indicate that adenosine A2A receptor antagonists improve motor deficits in animal models of
Parkinson’s disease. On this basis, the efficacy of ANR 82, ANR 94, and ANR 152 in rat models of Parkinson’s disease was evaluated. All
compounds tested reversed the catalepsy induced by haloperidol. However, in unilaterally 6-hydroxydopamine-lesioned rats, only ANR 94
and ANR 152 potentiated l-dihydroxy-phenylalanine (l-DOPA) effect on turning behaviour and induced contralateral turning behaviour in
rats sensitised to l-DOPA. Taken together the results of this study indicate that some 8-substituted 9-ethyladenine derivatives ameliorate
motor deficits in rat models of Parkinson’s disease, suggesting a potential therapeutic role of these compounds.
D 2005 Elsevier B.V. All rights reserved.
Keywords: Adenosine A2A antagonist; Parkinson’s disease; Dopamine; 6-hydroxydopamine; l-DOPA (l-dihydroxy-phenylalanine)
1. Introduction
pathway (Fink et al., 1992; Schiffmann et al., 1991).
Stimulation of adenosine A2A receptors decreases the
binding affinity of dopamine for dopamine D2 receptors
(Dasgupta et al., 1996; Ferre´ et al., 1991) and elicits effects
opposite to dopamine D2 receptor activation at the level of
second messenger systems and early-gene expression (Le
Moine et al., 1997; Morelli et al., 1995). However,
stimulation, as well as blockade of adenosine A2A receptors,
induces behavioral and biochemical responses in dopamine
D2 receptor knockout mice, suggesting that adenosine A2A
receptor actions can occur independently from dopamine
(Aoyama et al., 2000; Chen et al., 2001; Zahniser et al.,
2000).
Parkinson’s disease is characterized by a motor impair-
ment caused by the degeneration of dopaminergic neurons
located in the substantia nigra pars compacta and by the
reduction of dopamine levels in the striatum. Replacement
therapy with the dopamine precursor l-dihydroxy-phenyl-
alanine (l-DOPA) is successful in most parkinsonian
patients. After several years of l-DOPA therapy, however,
many motor complications, including fluctuations in motor
responses and dyskinesia occur (Kopin, 1993). Therefore,
alternative therapeutic approaches are the target of active
research in Parkinson’s disease.
Adenosine A2A receptors are predominantly located in
the striatum (Jarvis and Williams, 1989; Rosin et al., 1998;
Svenningsson et al., 1999), where they are co-expressed
with dopamine D2 receptors on the indirect striatopallidal
Blockade of adenosine A2A receptors produces motor
stimulant effects (El Yacoubi et al., 2000; Griebel et al.,
1991; Hauber et al., 1998; Holtzman, 1991) and reverses
catalepsy induced by dopamine receptor blockade or by
dopamine depletion (Kanda et al., 1994; Mandhane et al.,
1997; Shiozaki et al., 1999; Wardas et al., 2001). Moreover,
studies in experimental 1-methyl-4-phenyl-1, 2, 3, 6-
T Corresponding author. Tel.: +39 70 6758663; fax: +39 70 6758612.
E-mail address: morelli@unica.it (M. Morelli).
0014-2999/$ - see front matter D 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.ejphar.2005.01.057

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A. Pinna et al. / European Journal of Pharmacology 512 (2005) 157–164
tetrahydropyridine (MPTP) and 6-hydroxydopamine (6-
OHDA) models of Parkinson’s disease have shown that
adenosine A2A receptor antagonists have positive effects on
motor impairments and potentiate dopamine agonist-stimu-
lated motor behaviour (Fenu et al., 1997; Grondin et al.,
1999; Jiang et al., 1993; Kanda et al., 1998; Pinna et al.,
1996; Pollack and Fink, 1996). Interestingly, recent clinical
evidences showed that adenosine A2A receptor antagonists
are effective in reducing motor impairment in parkinsonian
patients with low risk of dyskinesias (Chase et al., 2003;
Kase et al., 2003; LeWitt, 2004).
Test was terminated when the rat moved one paw or when
90 s had elapsed from the placement of the rat on the grid.
Catalepsy assessments were repeated every 10- to 15-min
intervals. At each test time, rats which did not assume the
given position on the grid after three attempts were
classified 0 s latency. Adenosine A2A receptor antagonists
were injected 90 min after haloperidol administration.
2.3. 6-hydroxydopamine lesion
Rats were anaesthetised with chloral hydrate (400 mg/kg
i.p.), placed in a David Kopf stereotaxic apparatus and
injected, through a stainless steel cannula in the left medial
forebrain bundle with 6-OHDA–HCl (8 Ag/4 Al of saline
containing 0.05% ascorbic acid), at coordinates AP=À2.2,
ML=+1.5, DV=À7.8, according to the atlas of Pellegrino
et al. (1979). Rats were pretreated with desipramine (10 mg/
kg i.p.) in order to prevent 6-OHDA-induced neurotoxicity
to noradrenergic neurons.
Recently, a class of 9-ethyladenine derivatives has been
characterized as adenosine receptor ligands (Camaioni et al.,
1998). Among them, the 8-bromo-9-ethyladenine (ANR
82), showed high affinity and moderate selectivity for the
human adenosine A2A receptor subtype (Volpini et al.,
2003). In order to improve the A2A binding affinity and
selectivity of ANR 82, the 8-bromine atom was replaced
with an ethoxy or a furyl substituent, to form the compound
8-ethoxy-9-ethyladenine (ANR 94) (Klotz et al., 2003) and
8-furyl-9-ethyladenine (ANR 152). These two compounds
showed higher selectivity and affinity, respectively, than the
origin compound.
2.4. Evaluation of turning behaviour
2.4.1. Potentiation of l-DOPA-induced turning behaviour
Two weeks after the unilateral 6-OHDA lesion, rats were
screened on the basis of their contralateral rotation in
response to l-DOPA (50 mg/kg i.p.)+benserazide (30 mg/
kg i.p.). Rats not showing at least 300 contralateral rotations
during the 2-h testing period were eliminated from the study.
Three days later, rats were administered with l-DOPA (3
mg/kg i.p.)+benserazide (6 mg/kg i.p.) in combination with
vehicle or with a dose of 5 mg/kg i.p of ANR 82, ANR 94,
or ANR 152. ANR compounds were administered at the
same time of l-DOPA. Benserazide was always injected 30
min before l-DOPA.
In this study, the in vivo activity of these three
derivatives was investigated by assessing the ability of
reversing locomotor deficits induced by haloperidol or
inducing contralateral turning behaviour in rats bearing an
unilateral 6-OHDA lesion of the nigrostriatal pathway
(Ungerstedt, 1971), two rodent models largely used to
evaluate the efficacy of antiparkinson drugs. In 6-OHDA-
lesioned rats, both potentiation of contralateral rotation
induced by a subthreshold dose of l-DOPA and induction of
contralateral turning, in rats previously sensitized to the
motor effect of l-DOPA, were evaluated.
In order to measure turning behaviour, rats were placed in
plexiglas hemispherical bowls (50 cm of diameter) 30 min
before the administration of benserazide to acclimatize and
the number of both contralateral and homolateral rotations
were counted by automated rotameters, every 10 min, for 2 h.
2. Materials and methods
2.1. Animals
Male Sprague–Dawley rats (Charles River, Italy) weigh-
ing 275–300 g were used. Rats were housed in groups of
five with free access to food and water and maintained in a
temperature-controlled (22F1 C) and light-controlled room
(12-h light/dark cycle). Testing was conducted during the
light phase. The guidelines of the European Community for
animal experiments were followed (86/609/EEC; D.L.,
27.01.1992, number 116).
2.4.2. Turning behaviour induced by ANR 82, ANR 94, and
ANR 152
Two weeks after the 6-OHDA infusion, rats were primed
one or four times with l-DOPA. Rats were, therefore,
injected twice a week (3-day interval) for 2 weeks with
vehicle or l-DOPA (6 mg/kg i.p.)+benserazide (6 mg/kg
i.p.). As shown by Fenu and Morelli (1998), this drug
treatment sensitised rats to adenosine antagonist-induced
turning behaviour. Rats not showing at least 150 contrala-
teral rotations during the first 2-h testing period in the first
administration were eliminated from the study. Three days
after the end of the single or four times priming procedure,
rats were injected with a dose of 5 mg/kg i.p. of ANR 82,
ANR 94, or ANR 152. The number of both contralateral and
homolateral rotations were counted by automated rota-
meters, every 10 min, for 2 h.
2.2. Catalepsy testing
Catalepsy was estimated through the vertical grid test.
The test was carried out by placing the rat with the four paws
Â
on a wire grid (43 25 cm) at an angle of about 708 respect
to the bench surface. Catalepsy was determined by measur-
ing the time in which the rat maintained the given position.

A. Pinna et al. / European Journal of Pharmacology 512 (2005) 157–164
159
NH
N
NH
2
2
NH
N
2
vortexing vigorously; the clear solution was injected in a
volume of 0.3 ml i.p. per 100 g body weight.
N
N
N
N
N
C H I
N
N
2
5
N
+
K CO
2
3
6-OHDA–HCl, desipramine, benserazide, and l-DOPA
were purchased from Sigma-Aldrich Co. (St. Louis, MO,
USA). Haloperidol was purchased from commercial source
(Serenase, Lusofarmaco, Italy), diluted in distilled water,
and administered s.c. The drugs administered parenterally
were dissolved in saline and injected in a volume of 0.3 ml
i.p. per 100 g body weight or in a volume of 0.1 ml s.c. per
100 g body weight.
N
H
N
Adenine
7-ethyladenine
9-ethyladenine
NBS
NH
2
N
N
Br
N
N
2.6. Data analysis and statistic
8-bromo-9-ethyladenine (ANR 82)
C H OH But Sn
2
5
3
O
For catalepsy evaluation, the mean of total time spent by
rats in immobility during each test section and S.E.M. were
calculated. Significance between groups was evaluated by
one way analysis of variance (ANOVA) followed by a
Newman–Keuls post hoc test.
NaOH
(PPh) PdCl
3
2
NH
NH
2
2
N
N
N
N
N
N
O
O
N
N
In the turning behaviour experiments, mean and S.E.M.
of the total number of rotations were calculated. Signifi-
cance between groups was evaluated by one-way ANOVA
followed by a Newman–Keuls post hoc test.
8-ethoxy-9-ethyladenine (ANR 94)
8-furyl-9-ethyladenine (ANR 152)
Fig. 1. Chemical structures of ANR 82, ANR 94, and ANR 152. NBS=N-
bromosuccinimide.
2.5. Drugs
3. Results
The 8-substituted 9-ethyladenine derivatives, ANR 82,
ANR 94, or ANR 152, were synthesized starting from
commercially available adenine, which was alkylated using
ethyl iodide in the presence of potassium carbonate. Both
the 9-ethyladenine and its N-7 isomer were obtained and the
two isomers were separated through chromatography and
the structure was assigned to each compound using
spectrophotometric techniques (Fig. 1).
As shown by previous studies (Klotz et al., 1998,
2003) ANR 82, ANR 94, and ANR 152 have been
evaluated in human recombinant adenosine receptors,
stably transfected into Chinese hamster ovary (CHO)
cells, utilizing radioligand binding studies (A1, A2A, A3),
or adenylyl cyclase activity assay (A2B). Receptor binding
affinity was determined using [3H]CCPA (2-chloro-N6-
cyclopentyladenosine) as radioligand for adenosine A1
receptors, whereas [3H]NECA (5V-N-ethylcarboxamidoade-
nosine) was used for binding adenosine A2A and
adenosine A3 receptor subtypes. The results of those
studies are shown in Table 1.
The 9-ethyladenine was treated with N-bromosuccini-
mide (NBS) to obtain the first final product: the 8-bromo-
9-ethyladenine (ANR 82; Camaioni et al., 1998). Reaction
of ANR 82 with ethanol in the presence of sodium
hydroxide produced the 8-ethoxy-9-ethyladenine (ANR
94), while the 8-furyl-9-ethyladenine (ANR 152) was
obtained by treating ANR 82 with tributylstannylfuran.
The synthesis of these two compounds will be reported
elsewhere (In publication).
The 8-ethoxy derivatives ANR 94 showed affinity versus
the adenosine A2A receptor comparable to that of ANR 82,
while it resulted more selective than ANR 82 at the
adenosine A2A receptor (ANR 94 A1/A2A ratio=52, A2B
/
A
A
2A=652, and A3/A2A=456; ANR 82 A1/A2A ratio=5,
2B/A2A=16, and A3/A2A=534; Table 1). The substitution
ANR 82, ANR 94 and ANR 152 were dissolved by
adding dimethylsulfoxide (DMSO), polyethylene glycol
(PEG 400) and water of the ratio (50:350:600), and
of the bromine atom with a furyl ring, led to a compound,
ANR 152, endowed with affinity higher than ANR 82 at all
Table 1
Affinity of 8-substituted 9-ethyladenines at human adenosine receptor subtypes
Compound
PM
A1
A2A
A2B
A3
ANR 82
ANR 94
ANR 152
242.1
207.2
229.2
280 (250–320)
52 (24–113)
46 (24–91)
840 (630–1120)
N30,000
380 (250–560)
27,800 (22,300–34,700)
21,000 (11,100–41,000)
4700 (2900–7600)
2400 (2100–2600)
24 (16–34)
3.7 (3.0–4.6)
Ki values for adenosine A1 receptors are from competition experiments with [3H]CCPA, for adenosine A2A and adenosine A3 receptors [3H]NECA was used as
a radioligand. In the case of adenosine A2B receptors Ki-values were calculated from IC50-values determined by inhibition of NECA-stimulated adenylyl
cyclase activity. Ki values are in nM with 95% confidence intervals in parentheses.

160
A. Pinna et al. / European Journal of Pharmacology 512 (2005) 157–164
Antagonism of haloperidol-induced catalepsy
Halop. + vehicle
Halop. + ANR 82
Halop. + ANR 94
Halop. + ANR 152
180
150
120
90
*
60
**
**
**
30
**
**
**
**
**
**
0
60 min
90 min
10 min
30 min
Fig. 2. Effect of ANR 82 (5 mg/kg i.p.; n =8), ANR 94 (5 mg/kg i.p.; n =5), or ANR 152 (5 mg/kg i.p.; n =9) on catalepsy induced by haloperidol (0.2 mg/kg
s.c.; n =7) in rats, 10, 30, 60, and 90 min after drug administration. Results are meanFS.E.M. of intensity of catalepsy measured as time spent on cataleptic
posture by each rat in test section. **p b0.0001, *p b0.05 versus haloperidol+vehicle. Statistical significance was determined by one-way ANOVA followed
by Newman–Keuls post hoc test.
adenosine receptors, making this compound the most active
of the series (Table 1).
the basis of preliminary studies showing that 1 mg/kg of
ANR 82, ANR 94, or ANR 152 had low efficacy on
catalepsy, whereas 5 mg/kg was fully effective.
3.1. Catalepsy
Haloperidol (0.2 mg/kg s.c.) produced significant cata-
lepsy in rats at 40 min after drug administration and reached
the maximum at 60–70 min (data not shown). Therefore, the
administration of the new adenosine A2A receptor antago-
nists was made 90 min after haloperidol, in order to evaluate
their effects on deeply cataleptic rats.
At the dose of 5 mg/kg i.p. the adenosine A2A receptor
antagonists ANR 82, ANR 94 and ANR 152 did not modify
spontaneous motility in rats, whereas at higher doses (10, 15
mg/kg) they induced hypermotility (data not shown).
Therefore, in order to prevent non specific interference with
catalepsy the dose of 5 mg/kg i.p. of the adenosine A2A
receptor antagonists was used in this study.
At a dose of 5 mg/kg i.p. ANR 82, ANR 94, or ANR 152
significantly reversed the catalepsy induced by 0.2 mg/kg of
haloperidol during the 90-min testing period (Fig. 2). The
effect of ANR 82 and ANR 152 was maximal at 10–30 min,
whereas the effect of ANR 94 was maximal at 30–60 min.
Furthermore, the dose of 5 mg/kg i.p. of the adenosine
A2A receptor antagonists used in this study was chosen on
Potentiation of L-DOPA-induced contralateral turning
1200
1000
800
600
400
200
0
**
Homolateral turning
Contralateral turning
*
-200
-400
Fig. 3. Effect of administration of l-DOPA (3 mg/kg i.p.)+vehicle (n =5), l-DOPA (3 mg/kg i.p.)+ANR 82 (5 mg/kg i.p.; n =4), l-DOPA (3 mg/kg i.p.)+
ANR 94 (5 mg/kg i.p.; n =6), or l-DOPA (3 mg/kg i.p.)+ANR 152 (5 mg/kg i.p.; n =6). Ordinate indicates the total number of turns measured in 2 h; positive
and negative values represent contralateral and homolateral rotations, respectively. Results are mean+S.E.M. of total turns. **p b0.0001, *p b0.05 versus
l-DOPA alone. Statistical significance was determined by one-way ANOVA followed by Newman–Keuls post hoc test.

A. Pinna et al. / European Journal of Pharmacology 512 (2005) 157–164
161
The duration of the anticataleptic effect of ANR 82 and
ANR 152 was similar lasting about 80 min (Fig. 2). In
contrast, the anticataleptic effect of ANR 94 had a longer
duration, over 150 min (data not shown). As shown in Fig.
2, rats did not return cataleptic within the 90-min testing.
one or four administrations of l-DOPA (6 mg/kg i.p.) and
3 days later they were treated with ANR 82, ANR 94 and
ANR 152.
Administration of ANR 94 (5 mg/kg) or ANR 152 (5 mg/
kg) produced significant contralateral rotation in rats that
received four l-DOPA (6 mg/kg) primings (Fig. 4). On the
other hand, rats with a single l-DOPA priming, did not
rotate in response to 5 mg/kg of ANR 94 or ANR 152 (Fig.
4). In contrast, ANR 82 (5 mg/kg) failed to induce
significant contralateral turning in either four or single
l-DOPA-primed rats (Fig. 4).
3.2. Potentiation of l-DOPA-induced turning behaviour
Administration of ANR 82 failed to increase the number
of contralateral rotations induced in 6-OHDA-lesioned rats
by a subthreshold dose of l-DOPA (3 mg/kg i.p.; Fig. 3). In
contrast, both ANR 94 and ANR 152, significantly
increased the number of contralateral rotations induced by
l-DOPA (3 mg/kg) in 6-OHDA-lesioned rats (Fig. 3). The
increase in the number of rotations produced by ANR 152
was less marked than that observed with ANR 94 (Fig. 3).
Similarly to the anticataleptic effect, the potentiation of
ANR 152 on l-DOPA-induced turning behaviour lasted
about 80 min, whereas the effect of ANR 94 effect lasted up
to 120–130 min. Moreover, after ANR 94+l-DOPA
administration the contralateral turns increased to a max-
imum of 148F20 turns at 40 min, whereas contralateral
turns produced by ANR 152+l-DOPA reached a maximum
of 85F20 at 30 min. Therefore, the most pronounced effect
of ANR 94 on l-DOPA-induced turning behaviour appears
to be due to both stronger efficacy and longer duration.
Administration of ANR 94 and ANR 152 resulted in few
homolateral turns (Fig. 3).
The effect of ANR 152 on turning behaviour was
extinguished 80 min after administration, whereas the effect
of ANR 94 lasted up to 150 min.
Both ANR 94 and ANR 152 induced a low intensity
homolateral turning behaviour during the 2-h testing (Fig. 4).
4. Discussion
The results of this study indicate that the new adenosine
A2A receptor ligands 8-substituted 9-ethyladenine derivates
are effective in two rat models of Parkinson’s disease: the
haloperidol catalepsy reversal and the 6-OHDA model of
contralateral turning behaviour.
Considering that stimulation, or blockade of adenosine
receptors potentiates and reverses haloperidol-induced
catalepsy, respectively (Kanda et al., 1994; Mandhane et
al., 1997; Shiozaki et al., 1999), the effects of the 8-
substituted 9-ethyladenine derivatives were at first inves-
tigated in this experimental paradigm in order to confirm
that binding to adenosine A2A receptors resulted in func-
tional antagonistic actions on this receptor. All three
compounds investigated, reversed haloperidol-induced cata-
3.3. Turning behaviour in l-DOPA-sensitised rats
In order to evaluate the ability of the new adenosine
A2A receptor antagonists to induce contralateral turning
behaviour by themselves, different groups of rats received
Contralateral turning induced
by A2A antagonists
Contralateral turning
1000
800
600
400
200
0
4-L-DOPA primings
1-L-DOPA priming
*
*
-200
-400
ANR 82
ANR 94
ANR 152
Homolateral turning
Fig. 4. Total rotations after ANR 82 (5 mg/kg i.p.; n =7), ANR 94 (5 mg/kg i.p.; n =5), or ANR 152 (5 mg/kg i.p.; n =5) in rats primed with one (squared
columns) or four (dashed columns) administrations of benserazide (6 mg/kg i.p.)+l-DOPA (6 mg/kg i.p.) 2 weeks after 6-OHDA lesioning. Compounds were
administered 3 days after priming. Ordinate indicates the total number of turns measured in 2 h; positive and negative values represent contralateral and
homolateral rotations, respectively. Results are mean+S.E.M. of total turns. *p b0.05 versus single priming. Statistical significance was determined by one-
way ANOVA followed by Newman–Keuls post hoc test.

162
A. Pinna et al. / European Journal of Pharmacology 512 (2005) 157–164
lepsy, thus showing a pharmacological profile similar to that
of proved adenosine A2A receptor antagonists (Kanda et al.,
1994; Mandhane et al., 1997; Shiozaki et al., 1999). The
assessment of the anticataleptic effect revealed some differ-
ences among the derivatives examined. ANR 82 and ANR
152 were maximally effective right after their administration
and their action lasted for about 80 min. In contrast, the
effect of ANR 94 showed a slower onset but a longer
duration as compared to ANR 82 and ANR 152.
might contrast the potentiation of l-DOPA and produce no
increase of l-DOPA contralateral turning or contralateral
turning in l-DOPA-sensitised rats (Bishop and Walker,
2003).
Influences of adenosine A2A receptors on dopamine D1-
mediated behaviours have been explained by functional
antagonistic influences of striatopallidal and striatonigral
pathways on output structures (Ferre´ et al., 1997; Hauber et
al., 2001; Pinna et al., 1996). Further studies on binding
activity of these compounds might clarify this issue;
however, the effectiveness of ANR 82 in the catalepsy
paradigm deserves interest since ANR 82 is the precursor of
ANR 94 and ANR 152.
On the basis of the ability in reversing catalepsy showed
by the 8-substituted 9-ethyladenine derivatives, accounting
for a functional antagonism at adenosine A2A receptors, the
effects of these compounds were further investigated using
unilaterally 6-OHDA-lesioned rats.
Interestingly, our results, show that the adenosine A2A
receptor antagonists ANR 94 and ANR 152, differently
from other selective adenosine A2A receptor antagonists like
SCH 58261 or KW 6002, produce contralateral turning in l-
DOPA-primed rats even when are administered alone,
without concomitant administration of l-DOPA. This result
suggests that this class of adenosine A2A receptor antago-
nists, in the presence of dopamine receptor supersensitivity,
like after repeated l-DOPA treatment, might be effective as
monotherapy.
Numerous experimental evidences indicate that adeno-
sine A2A receptor antagonists potentiate the contralateral
turning induced by a subthreshold dose of l-DOPA (Fenu et
al., 1997; Koga et al., 2000; Pinna et al., 2001). Admin-
istration of ANR 94, as well as ANR 152, significantly
potentiated l-DOPA-induced rotational behaviour, whereas
ANR 82 was not effective in this test. Furthermore, ANR 94
and ANR 152 were able to induce contralateral turning in l-
DOPA-sensitised rats even without concomitant adminis-
tration of l-DOPA. The positive response on turning
behaviour predicts that ANR 94 and ANR 152, similarly
to other selective adenosine A2A receptor antagonists, might
have antiparkinsonian effects. The higher efficacy of ANR
94 as compared to ANR 152 might be due to its higher
selectivity toward adenosine A2A receptors, whereas the
longer duration might be related to its longer half-life,
although pharmacokinetic studies on these compounds have
not been performed yet.
Previous studies have suggested that dopamine deficits
produce modification in adenosine transmission (Ekonomou
et al., 2004; Pinna et al., 2002) facilitating adenosine A2A
receptors activity (Morelli et al., 1995). Since adenosine
A
2A receptor stimulation plays a negative role in the control
of motor behaviour (Hauber and Munkle, 1997; Morelli et
al., 1994; Rimondini et al., 1997), blockade of adenosine
A
2A receptors has been suggested to be a useful therapeutic
strategy for increasing the efficacy of dopamine receptor
agonists like l-DOPA (Kase et al., 2003; Morelli, 2003;
Richardson et al., 1997). Complications in chronic l-
DOPA-treated parkinsonian patients appear to be due to
the advanced dopamine neuron degeneration as well as the
chronic intermittent l-DOPA administration; therefore, a
reduction of l-DOPA dosage might effectively contrast the
appearance of side effects (Fredduzzi et al., 2002; Grondin
et al., 1999; Kanda et al., 1998; Pinna et al., 2001).
The results obtained with these new synthesis adenosine
A2A receptor antagonists, show that ANR 82 although
antagonised catalepsy induced by haloperidol was not
effective in the turning behaviour tests. The origin for this
difference are not clear, however, since haloperidol cata-
lepsy is due to a selective acute dopamine D2 receptor
blockade, whereas contralateral turning induced by l-DOPA
is due to stimulation of both dopamine D1 and D2 receptors,
one might envision in these different mechanisms the basis
of this discrepancy. ANR 82 might, in fact, induce reversal
of dopamine D2-mediated catalepsy through the functional
antagonistic interaction with dopamine D2 receptors, which
are coexpressed on striatopallidal neurons with adenosine
A2A receptors (Fink et al., 1992; Schiffmann et al., 1991).
This antagonistic effect involves the interaction between the
adenosine A2A and dopamine D2 receptors at both receptors
and second messenger level (Dasgupta et al., 1996; Ferre´ et
al., 1991); although, an interaction at the level of cholinergic
or g-aminobutyric acid (GABA)ergic neurons, whose
release is controlled by adenosine A2A receptors, cannot
be excluded (Kurokawa et al., 1996; Mori and Shindou,
2003; Ochi et al., 2000). At the same time, ANR 82 by
binding non-adenosine receptors, which interact with
dopamine D1 receptors (e.g. serotonin 5-HT2 receptors),
In line with these suggestions, recent preliminary clinical
trials have reported that adenosine A2A receptor antagonists
improved l-DOPA efficacy without inducing dyskinesia
(Chase et al., 2003; Kase et al., 2003; LeWitt, 2004).
Adenosine A2A receptor antagonists are among the most
promising class of drugs for the treatment of Parkinson’s
disease, therefore, the synthesis of new adenosine A2A
receptor antagonists and their testing in models of Parkin-
son’s disease are of great importance for the development of
new, more effective treatments, devoid of side effects.
Acknowledgments
The authors wish to thank Dr. Liliana Del Zompo for
revising the English version of this manuscript.

A. Pinna et al. / European Journal of Pharmacology 512 (2005) 157–164
163
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Grondin, R., Be´dard, P.J., Hadj Tahar, A., Gre´goire, L., Mori, A.,
Kase, H., 1999. Antiparkinsonian effect of a new selective ade-
nosine A2A receptor antagonist in MPTP-treated monkeys. Neuro-
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This research was supported by grants from CNR
(recipient: Annalisa Pinna). This study was funded by
MURST project FIRB 2005.
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