Synthesis and evaluation of 7-amino-2-(2(3)-furyl)-5-phenylethylamino-oxazolo[5,4-d]pyrimidines as potential A2A adenosine receptor antagonists for positron emission tomography (PET).

The brain A2A adenosine receptor (A2AAR) participates with the dopamine D2 receptor in the control of movement and also might influence behavior. Because PET is an important tool for studying the roles of receptors in disease, a ligand for imaging the brain A2AAR is desirable. This report describes the synthesis and A2AAR antagonist activities of a panel of phenyl-substituted 7-amino-2-(2-furyl)-5-phenylethylamino-oxazolo[5,4-d]pyrimidines, 11aa-af, and their 3-furyl congeners, 11ba-bd. In competitive binding studies all compounds displaced [3H]CGS21680 from the A2AAR with Ki values of 14-33 nM with selectivity for the A2AAR over the A1AR of 5- to 94-fold. Autoradiography of brain sections showed a high level of unspecific binding that obscured specific binding. Thus, these compounds are not promising PET ligands.

Derivatives of 4,6-diamino-1,2-dihydro-2-phenyl-1,2,4-triazolo[4,3-a]quinoxalin-2H-1-one: potential antagonist ligands for imaging the A2A adenosine receptor by positron emission tomography (PET).

The importance of the brain A2A adenosine receptor (A(2A)AR) in movement disorders urges the development of radiolabeled ligands for imaging those receptors by positron emission tomography (PET). This study evaluated one class of A(2A)AR antagonists, derivatives of 4-amino-6-benzylamino-1,2-dihydro-2-phenyl-1,2,4-triazolo[4,3-a]quinoxalin-2H-1-one, 10a, as agents for imaging brain A(2A)ARs by PET.. Modifications of a literature synthesis of 10a efficiently generated analogs 10b-s for pharmacological evaluation. Radioligand binding experiments showed affinities for the rat brain A(2A)AR in the low nanomolar range but similar affinities for the A1AR and substantial unspecific binding. Autoradiography employing [3H]10a, showing that high unspecific binding obscured specific binding to both the A1AR and A(2A)AR. Thus, compounds 10b-s are unsuitable as ligands for imaging brain A(2A)ARs by PET.

Synthesis and evaluation of no-carrier-added 8-cyclopentyl-3-(3-[(18)F]fluoropropyl)-1-propylxanthine ([(18)F]CPFPX): a potent and selective A(1)-adenosine receptor antagonist for in vivo imaging.

This report describes the precursor synthesis and the no-carrier-added (nca) radiosynthesis of the new A(1) adenosine receptor (A(1)AR) antagonist [(18)F]8-cyclopentyl-3-(3-fluoropropyl)-1-propylxanthine (CPFPX), 3, with fluorine-18 (half-life = 109.6 min). Nucleophilic radiofluorination of the precursor tosylate 8-cyclopentyl-3-(3-tosyloxypropyl)-7-pivaloyloxymethyl-1-propylxanthine, 2, with nca [(18)F]KF under aminopolyether-mediated conditions (Kryptofix 2.2.2/K(2)CO(3)) followed by deprotection was straightforward and, after formulation, gave the radioligand ready for injection with a radiochemical yield of 45 +/- 7%, a radiochemical purity of >98% and a specific radioactivity of >270 GBq/micromol (>7.2 Ci/micromol). Preparation time averaged 55 min. The synthesis proved reliable for high batch yields ( approximately 7.5 GBq) in routine production (n = 120 runs). The radiotracer was pharmacologically evaluated in vitro and in vivo and its pharmacokinetics in rodents determined in detail. After iv injection a high accumulation of radioactivity occurred in several regions of mouse brain including thalamus, striatum, cortex, and cerebellum. Antagonism by the specific A(1)AR antagonists 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) and N(6)-cyclopentyl-9-methyladenine (N-0840), but not with the A(2)AR antagonist 3,7-dimethyl-1-propargylxanthine (DMPX), indicated specific and reversible binding of the radioligand to A(1)AR in cortical and subcortical regions of interest. In mouse blood at least two polar metabolites formed rapidly (50% at 5 min after tracer application). However, chromatographic analyses of brain homogenate extracts taken 60 min pi showed that >98% of radioactivity was unchanged radioligand. Chromatographic isolation and reinjection of peripherally formed radioactive metabolites revealed no accumulation of radioactivity in mouse brain, probably due to the polarity of the metabolites. These preliminary results suggest that nca [(18)F]CPFPX is a useful radioligand for the noninvasive imaging of the brain A(1)AR.

Synthesis of the main metabolite in human blood of the A1 adenosine receptor ligand [18F]CPFPX.

In human blood, the PET radiotracer [(18)F]CPFPX (1) is metabolized to numerous metabolites, one (M1) being the most prominent in plasma 30 min p.i. Because the mass of injected tracer is < or = 5 nmol, concentrations in plasma are too low to analyze. Human liver microsomes generate main metabolites having HPLC retention times identical to those in plasma. HPLC-MS tentatively identified M1 as 2. Synthesis of 2 and identical HPLC-MS spectra of 2 and M1 confirmed that assignment.

Autoradiographic comparison of in vitro binding characteristics of various tritiated adenosine A2A receptor ligands in rat, mouse and pig brain and first ex vivo results.

The adenosine A(2A) receptor in the basal ganglia is involved in the control of movement and plays a role in movement disorders such as Parkinsonism. Developing ligands to evaluate that receptor by noninvasive methods such as positron emission tomography has a high priority. In vitro radioligand binding guides the selection of ligands for in vivo application. This study measured the binding of the adenosine A(2A) receptor antagonist [(3)H]MSX-2 (3-(3-hydroxypropyl)-8-m-methoxystyryl)-7-methyl-1-propargylxanthine) to rat, mouse and pig brain by autoradiography. Other studies measured binding to membranes from PC12 pheochromocytoma cells. Those binding parameters were compared to those of the adenosine A(2A) receptor antagonist [(3)H]ZM241385 (4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino)ethyl)phenol), the adenosine A(2A) receptor agonist [(3)H]CGS21680 (2-[p-(2-carboxyethyl)-phenethylamino]-5'-N-ethylcarboxamidoadenosine) and the unselective adenosine receptor agonist [(3)H]NECA (5'N-ethylcarboxamido)adenosine). The potency order (K(d)) in the three species was [(3)H]ZM241385<[(3)H]MSX-2<[(3)H]NECA<[(3)H]CGS21680. The density of [(3)H]MSX-2 binding sites was greater in the striatum than in the cortex. Preliminary ex vivo experiments showed that by 10min after iv injection, [(3)H]MSX-2 and [(3)H]CGS21680 crossed the blood-brain barrier to the extent of almost 1% ID/g brain tissue, but [(3)H]NECA and [(3)H]ZM241385 to only 0.2% ID/g. The prior administration of unlabeled ZM241385 significantly lowered brain uptake of [(3)H]MSX-2. In conclusion, [(3)H]MSX-2 has a high affinity and sufficient selectivity for the adenosine A(2A) receptor. It penetrates the blood-brain barrier. Sensitivity to photoisomerization is a limitation. Further investigations assess its suitability as a ligand for imaging the brain adenosine A(2A) receptor.

Metabolism of the A(1)1 adenosine receptor positron emission tomography ligand [18F]8-cyclopentyl-3-(3-fluoropropyl)-1-propylxanthine ([18F]CPFPX) in rodents and humans.

Studies of plasma from mice, rats, and human volunteers evaluated methods for the extraction and quantification of the positron emission tomography ligand [(18)F]8-cyclopentyl-3-(3-fluoropropyl)-1-propylxanthine ([(18)F]CPFPX) and identification of its metabolites in plasma by thin-layer chromatography and high-performance liquid chromatography (HPLC). Analysis of human, mouse, and rat plasma extracts by HPLC identified four identical radioactive metabolites in each species. The low mass of radioligand administered to humans (0.5 - 5 nmol) prevented direct identification of metabolites. However, incubating liver microsomes with CPFPX and analysis by means of liquid chromatography-mass spectrometry (LC-MS) identified seven compounds, four having the same retention times as the metabolites in human plasma. Analysis of microsomal metabolites by LC-MS identified five [M + H](+) ions of m/z equivalent to hydroxy derivatives, 339, one of m/z equivalent to an oxo derivative, m/z 337, and one of m/z equivalent to a difunctionalized oxo-desaturation species, m/z 335, which is prominent in rat and mouse plasma and is the main metabolite in human plasma. An [M + H](+) ion corresponding to a N-dealkylated derivative was not detected. Thus, like the natural methylxanthines, CPFPX seems to undergo oxidation by liver microsomes but, unlike those methylxanthines, dealkylation did not occur. LC-MS experiments with "in source" fragmentation identified the cyclopentyl moiety to be the most functionalized part of the molecule by liver microsomes and in vivo oxidations. Except for two metabolites, hydroxylated at the N1 propyl chain, all oxidative modifications found took place at the cyclopentyl ring.