Blockade of A2A adenosine receptors (A2AARs) and inhibition of monoamine oxidase B (MAO-B) in the brain are considered attractive strategies for the treatment of neurodegenerative diseases such as Parkinson's disease (PD). In the present study, benzothiazinones, e.g., 2-(3-chlorophenoxy)-N-(4-oxo-4H-3,1-benzothiazin-2-yl)acetamide (13), were identified as a novel class of potent MAO-B inhibitors (IC50 human MAO-B: 1.63 nM). Benzothiazinones with large substituents in the 2-position, e.g., methoxycinnamoylamino, phenylbutyrylamino, or chlorobenzylpiperazinylbenzamido residues (14, 17, 27, and 28), showed high affinity and selectivity for A2AARs (Ki human A2AAR: 39.5-69.5 nM). By optimizing benzothiazinones for both targets, the first potent, dual-acting A2AAR/MAO-B inhibitors with a nonxanthine structure were developed. The best derivative was N-(4-oxo-4H-3,1-benzothiazin-2-yl)-4-phenylbutanamide (17, Ki human A2A, 39.5 nM; IC50 human MAO-B, 34.9 nM; selective versus other AR subtypes and MAO-A), which inhibited A2AAR-induced cAMP accumulation and showed competitive, reversible MAO-B inhibition. The new compounds may be useful tools for validating the A2AAR/MAO-B dual target approach in PD.

Indazole- and indole-carboxamides were discovered as highly potent, selective, competitive, and reversible inhibitors of monoamine oxidase B (MAO-B). The compounds are easily accessible by standard synthetic procedures with high overall yields. The most potent derivatives were N-(3,4-dichlorophenyl)-1-methyl-1H-indazole-5-carboxamide (38a, PSB-1491, IC50 human MAO-B 0.386 nM, >25000-fold selective versus MAO-A) and N-(3,4-dichlorophenyl)-1H-indole-5-carboxamide (53, PSB-1410, IC50 human MAO-B 0.227 nM, >5700-fold selective versus MAO-A). Replacement of the carboxamide linker with a methanimine spacer leading to (E)-N-(3,4-dichlorophenyl)-1-(1H-indazol-5-yl)methanimine (58) represents a further novel class of highly potent and selective MAO-B inhibitors (IC50 human MAO-B 0.612 nM, >16000-fold selective versus MAO-A). In N-(3,4-difluorophenyl-1H-indazole-5-carboxamide (30, PSB-1434, IC50 human MAO-B 1.59 nM, selectivity versus MAO-A>6000-fold), high potency and selectivity are optimally combined with superior physicochemical properties. Computational docking studies provided insights into the inhibitors' interaction with the enzyme binding site and a rationale for their high potency despite their small molecular size.

2-Amino[1,2,4]triazolo[1,5-c]quinazolines were identified as potent adenosine receptor (AR) antagonists. Synthetic strategies were devised to gain access to a broad range of derivatives including novel polyheterocyclic compounds. Potent and selective A AR antagonists were discovered, including 3,5-diphenyl[1,2,4]triazolo[4,3-c]quinazoline (17, K human A AR 1.16 nm) and 5'-phenyl-1,2-dihydro-3'H-spiro[indole-3,2'-[1,2,4]triazolo[1,5-c]quinazolin]-2-one (20, K human A AR 6.94 nm). In addition, multitarget antagonists were obtained, such as the dual A /A antagonist 2,5-diphenyl[1,2,4]triazolo[1,5-c]quinazoline (13 b, K human A AR 51.6 nm, human A AR 11.1 nm), and the balanced pan-AR antagonists 5-(2-thienyl)[1,2,4]triazolo[1,5-c]quinazolin-2-amine (11 c, K human A AR 131 nm, A AR 32.7 nm, A AR 150 nm, A AR 47.5 nm) and 9-bromo-5-phenyl[1,2,4]triazolo[1,5-c]quinazolin-2-amine (11 q, K human A AR 67.7 nm, A AR 13.6 nm, A AR 75.0 nm, A AR 703 nm). In many cases, significantly different affinities for human and rat receptors were observed, which emphasizes the need for caution in extrapolating conclusions between different species.

8-Benzyl-substituted tetrahydropyrazino[2,1-f]purinediones were designed as tricyclic xanthine derivatives containing a basic nitrogen atom in the tetrahydropyrazine ring to improve water solubility. A library of 69 derivatives was prepared and evaluated in radioligand binding studies at adenosine receptor (AR) subtypes and for their ability to inhibit monoamine oxidases (MAO). Potent dual-target-directed A1 /A2A adenosine receptor antagonists were identified. Several compounds showed triple-target inhibition; one of the best compounds was 8-(2,4-dichloro-5-fluorobenzyl)-1,3-dimethyl-6,7,8,9-tetrahydropyrazino[2,1-f]purine-2,4(1H,3H)-dione (72) (human AR: Ki  A1 217 nM, A2A 233 nM; IC50 MAO-B: 508 nM). Dichlorinated compound 36 [8-(3,4-dichlorobenzyl)-1,3-dimethyl-6,7,8,9-tetrahydropyrazino[2,1-f]purine-2,4(1H,3H)-dione] was found to be the best triple-target drug in rat (Ki  A1 351 nM, A2A 322 nm; IC50 MAO-B: 260 nM), and may serve as a useful tool for preclinical proof-of-principle studies. Compounds that act at multiple targets relevant for symptomatic as well as disease-modifying treatment of neurodegenerative diseases are expected to show advantages over single-target therapeutics.

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