A series of tricyclic imidazo[2,1-i]purinones and ring-enlarged analogues derived from xanthine derivatives have been prepared as adenosine receptor (AR) antagonists. In comparison with xanthines, the tricyclic compounds exhibit increased water solubility due to a basic nitrogen atom, which can be protonated under physiological conditions. Substituents were introduced that confer high affinity for A(2A) or A(3) ARs, respectively. A new capillary electrophoresis method was developed for the determination of the enantiomeric purity of selected chiral products using native and modified beta-cyclodextrins as chiral discriminators. The compounds were investigated in radioligand binding assays at rat brain A(1) and A(2A) ARs. Selected compounds were additionally investigated in radioligand binding assays at human recombinant A(3) ARs and in functional studies (adenylate cyclase assays) at A(1) ARs of rat fat cell membranes, A(2A) ARs of rat PC 12 cell membranes, and mouse A(2B) ARs of NIH 3T3 cell membranes. Structure-activity relationships were similar to those of corresponding xanthine derivatives. The 2-styrylimidazopurinones were less potent at A(2A) ARs as compared to 8-styrylxanthine derivatives. The most potent compound at A(2A) ARs was (S)-1,4-dimethyl-8-ethyl-2-styryl-imidazo[2,1-i]purinone (S-25) exhibiting a K(i) value of 424 nM at rat A(2A) ARs. The compound was highly selective for A(2A) receptors vs A(1) and A(3) ARs. Selectivity vs A(2B) ARs, however, was low. Among the 1-unsubstituted 2-phenyl-imidazo[2,1-i]purin-5-one derivatives, very potent and highly selective antagonists for human A(3) ARs were identified. The most potent A(3) antagonist of the present series was (R)-4-methyl-8-ethyl-2-phenyl-imidazo[2,1-i]purin-5-one (R-24) exhibiting a K(i) value of 2.3 nM and high selectivity for A(3) receptors vs all other AR subtypes.

Structure-activity relationships of 2-phenyl-imidazo[2,1-i]purin-5-ones as ligands for human A(3) adenosine receptors (ARs) were investigated. An ethyl group in the 8-position of the imidazoline ring of 4-methyl-2-phenyl-imidazopurinone leading to chiral compounds was found to increase affinity for human A(3) ARs by several thousand-fold. Propyl substitution instead of methyl at N4 decreased A(3) affinity but increased A(1) affinity leading to potent A(1)-selective AR antagonists. The most potent A(1) antagonist of the present series was (S)-8-ethyl-2-phenyl-4-propyl-4,5,7,8-tetrahydro-1H-imidazo[2,1-i]purin-5-one (S-3) exhibiting a K(i) value of 7.4 nM at rat A(1) ARs and greater than 100-fold selectivity versus rat A(2A) and human A(3) ARs. At human A(1) ARs 2-phenylimidazo[2,1-i]purin-5-ones were generally less potent and therefore less A(1)-selective (S-3: K(i)=98 nM). 2-, 3-, or 4-Mono-chlorination of the 2-phenyl ring reduced A(3) affinity but led to an increase in affinity for A(1) ARs, whereas di- (3,4-dichloro) or polychlorination (2,3,5-trichloro) increased A(3) affinity. The most potent and selective A(3) antagonist of the present series was the trichlorophenyl derivative (R)-8-ethyl-4-methyl-2-(2,3,5-trichlorophenyl)-4,5,7,8-tetrahydro-1H-imidazo[2,1-i]purin-5-one (R-8) exhibiting a subnanomolar K(i) value at human A(3) ARs and greater than 800-fold selectivity versus the other AR subtypes. Methylation of 4-alkyl-2-phenyl-substituted imidazo[2,1-i]purin-5-ones led exclusively to the N9-methyl derivatives, which exhibited largely reduced AR affinities as compared to the unmethylated compounds. [35S]GTP gamma S binding studies of the most potent 2-phenyl-imidazo[2,1-i]purin-5-ones at membranes of Chinese hamster ovary cells expressing the human A(3) AR revealed that the compounds were inverse agonists at A(3) receptors under standard test conditions. Due to their high A(3) affinity, selectivity, and relatively high water-solubility, 2-phenyl-imidazo[2,1-i]purin-5-ones may become useful research tools.

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The compound L-valine-3-{8-[(E)-2-[3-methoxyphenyl)ethenyl]-7-methyl-1-propargyl­xan­thine-3-yl}propyl ester hydrochloride (MSX-4) was synthesized as an amino acid ester prodrug of the adenosine A2A receptor antagonist MSX-2. It was found to be stable in artificial gastric acid, but readily cleaved by pig liver esterase.