Central adenosine A 2A receptor is a promising target for drugs to treat Parkinson's disease (PD), and the central blockade of adenosine A 1 receptor improves cognitive function. In the present study, we investigated the effect of a novel adenosine A 1 and A 2A dual antagonist, 5-[5-amino-3-(4-fluorophenyl) pyrazin-2-yl]-1-isopropylpyridine-2(1H)-one (ASP5854), in animal models of PD and cognition. The binding affinities of ASP5854 for human A 1 and A 2A receptors were 9.03 and 1.76 nM, respectively, with higher specificity and no species differences. ASP5854 also showed antagonistic action on A 1 and A 2A agonist-induced increases of intracellular Ca 2ϩ concentration. ASP5854 ameliorated A 2A agonist 2-[p-(2-carboxyethyl) phenethylamino]-5Ј-N-ethylcarboxamidoadenosine (CGS21680)-and haloperidol-induced catalepsy in mice, with the minimum effective doses of 0.32 and 0.1 mg/kg, respectively, and it also improved haloperidol-induced catalepsy in rats at doses higher than 0.1 mg/kg. In unilateral 6-hydroxydopamine-lesioned rats, ASP5854 significantly potentiated l-dihydroxyphenylalanine (L-DOPA)-induced rotational behavior at doses higher than 0.032 mg/kg. ASP5854 also significantly restored the striatal dopamine content reduced by 1-metyl-4-phenyl-1,2,3,6-tetrahydropyridine treatment in mice at doses higher than 0.1 mg/ kg. Furthermore, in the rat passive avoidance test, ASP5854 significantly reversed the scopolamine-induced memory deficits, whereas the specific adenosine A 2A antagonist 8-((E)-2-(3,4-dimethoxyphenyl)ethenyl)-1,3-diethyl-7-methyl-3,7-dihydro-1H-purine-2,6-dione (KW-6002; istradefylline) did not. Scopolamine-or 5H-dibenzo [a,d]cyclohepten-5,10-imine (dizocilpine maleate) (MK-801)-induced impairment of spontaneous alternation in the mouse Y-maze test was ameliorated by ASP5854, whereas KW-6002 did not exert improvement at therapeutically relevant dosages. These results demonstrate that the novel, selective, and orally active dual adenosine A 1 and A 2A receptors antagonist ASP5854 improves motor impairments, is neuroprotective via A 2A antagonism, and also enhances cognitive function through A 1 antagonism.Adenosine is a ubiquitous neuromodulator in the central nervous system. Its major role in the central nervous system is to modulate neurotransmitter release, the postsynaptic components, and also the nonsynaptic components such as glial cell signaling. Adenosine exerts these diverse physiological actions through activation of specific G protein-coupled receptors termed A 1 , A 2A , A 2B , and A 3 (Fredholm et al., 1994).Adenosine A 2A receptors are specifically localized in the striatum (Svenningsson et al., 1999), where they are coexpressed with dopamine D 2 receptors in the GABAergic striaArticle, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.107.121962. ABBREVIATIONS:CGS21680, 2-[p-(2-carboxyethyl)phenethylamino]-5Ј-N-ethylcarboxamidoadenosine; NECA, N-ethylcarboxamidoadenosine; KW-6002, 7-methyl-3,7-dihydro-1H-purine-2,6-dion...

Abstract. Adenosine A 1 receptors in the brain are believed to play an important role in brain functioning. We have discovered a novel adenosine A 1 receptor antagonist, FR194921 (2-(1-methyl-4-piperidinyl)-6-(2-phenylpyrazolo[1,5-a]pyridin-3-yl)-3(2H)-pyridazinone), and characterized the pharmacological activity in the present study. FR194921 showed potent and selective affinity for the adenosine A 1 receptor without affinity for A 2A and A 3 receptors and did not show any species differences in binding affinity profile among human, rat, and mouse. Pharmacokinetic study in rats revealed that FR194921 was orally active and highly brain penetrable. Oral administration of FR194921 dose-dependently ameliorated the hypolocomotion induced by the A 1 receptor agonist N 6 -cyclopentyladenosine in rats, indicating this compound exerts A 1 -antagonistic action in vivo. In the passive avoidance test, scopolamine (1 mg / kg)-induced memory deficits were significantly ameliorated by FR194921 (0.32, 1 mg / kg). In two animal models of anxiety, the social interaction test and elevated plus maze, FR194921 showed specific anxiolytic activity without significantly influencing general behavior. In contrast, FR194921 did not show antidepressant activity even at a dose of 32 mg / kg in the rat forced swimming test. These results indicate that the novel, potent, and selective adenosine A 1 receptor antagonist FR194921 exerts both cognitive-enhancing and anxiolytic activity, suggesting the therapeutic potential of this compound for dementia and anxiety disorders.

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1 The pharmacological pro®le of adenosine A 1 receptors in human, guinea-pig, rat and mouse brain membranes was characterized in a radioligand binding assay by use of the receptor selective antagonist, Binding site anity was higher in rat cortical membranes than in membranes prepared from guinea-pig cortex and hippocampus, mouse cortex and human cortex. pK D values (M) were 9. 55, 9.44, 8.85, 8.94, 8.67, 9.39 and 8.67, respectively. The binding site density (B max ) was lower in rat cortical membranes than in guinea-pig or human cortical membranes. 3 The rank order of potency of seven adenosine receptor agonists was identical in each species. With the exception of 5'-N-ethylcarboxamidoadenosine (NECA), agonist anity was 3.5 ± 26.2 fold higher in rat cortical membranes than in human and guinea-pig brain membranes; anity in rat and mouse brain membranes was similar. While NECA exhibited 9.3 fold higher anity in rat compared to human cortical membranes, anity in other species was comparable. The stable GTP analogue, Gpp(NH)p (100 mM) reduced 2-chloro-N 6 -cyclopentyladenosine (CCPA) anity 7 ± 13.9 fold, whereas the anity of DPCPX was unaected. 4 The anity of six xanthine-based adenosine receptor antagonists was 2.2 ± 15.9 fold higher in rat cortical membranes compared with human or guinea-pig membranes. The rank order of potency was species-independent. In contrast, three pyrazolopyridine derivatives,

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