We report the identification of two subsets of fluorinated nonxanthine A2B adenosine receptor antagonists. The novel derivatives explore the effect of fluorination at different positions of two pyrimidine-based scaffolds. The most interesting ligands combine excellent hA2B affinity (K i < 15 nM) and remarkable subtype selectivity. The results of functional cAMP experiments confirmed the antagonistic behavior of representative ligands. The compounds were designed on the basis of previous molecular models of the stereoselective binding of the parent scaffolds to the hA2B receptor, and we herein provide refinement of such models with the fluorinated compounds, which allows the explanation of the spurious effects of the fluorination at the different positions explored. These models are importantly confirmed by a synergistic study combining chiral HPLC, circular dichroism, diastereoselective synthesis, molecular modeling, and X-ray crystallography, providing experimental evidence toward the stereospecific interaction between optimized trifluorinated stereoisomers and the hA2B receptor.

A systematic exploration of bioisosteric replacements for furan and thiophene cores in a series of potent A 2B AR antagonists has been carried out using the nitrogen-walk approach. A collection of 42 novel alkyl 4-substituted-2-methyl-1,4dihydrobenzo [4,5]imidazo[1,2-a]pyrimidine-3-carboxylates, which contain 18 different pentagonal heterocyclic frameworks at position 4, was synthesized and evaluated. This study enabled the identification of new ligands that combine remarkable affinity (K i < 30 nM) and exquisite selectivity. The structure−activity relationship (SAR) trends identified were substantiated by a molecular modeling study, based on a receptor-driven docking model and including a systematic free energy perturbation (FEP) study. Preliminary evaluation of the CYP3A4 and CYP2D6 inhibitory activity in optimized ligands evidenced weak and negligible activity, respectively. The stereospecific interaction between hA 2B AR and the eutomer of the most attractive novel antagonist (S)-18g (K i = 3.66 nM) was validated.

We present and thoroughly characterize a large collection of 3,4-dihydropyrimidin-2­(1H)-ones as A2BAR antagonists, an emerging strategy in cancer (immuno) therapy. Most compounds selectively bind A2BAR, with a number of potent and selective antagonists further confirmed by functional cyclic adenosine monophosphate experiments. The series was analyzed with one of the most exhaustive free energy perturbation studies on a GPCR, obtaining an accurate model of the structure–activity relationship of this chemotype. The stereospecific binding modeled for this scaffold was confirmed by resolving the two most potent ligands [(±)-47, and (±)-38 K i = 10.20 and 23.6 nM, respectively] into their two enantiomers, isolating the affinity on the corresponding (S)-eutomers (K i = 6.30 and 11.10 nM, respectively). The assessment of the effect in representative cytochromes (CYP3A4 and CYP2D6) demonstrated insignificant inhibitory activity, while in vitro experiments in three prostate cancer cells demonstrated that this pair of compounds exhibits a pronounced antimetastatic effect.

Using a previously unexplored, efficient, and versatile multicomponent method, we herein report the rapid generation of novel potent and subtype-selective DRD 2 biased partial agonists. This strategy exemplifies the search for diverse and previously unexplored moieties for the secondary/allosteric pharmacophore of the common phenyl-piperazine scaffold. The pharmacological characterization of the new compound series led to the identification of several ligands with excellent DRD 2 affinity and subtype selectivity and remarkable functional selectivity for either the cAMP (22a and 24d) or the β-arrestin (27a and 29c) signaling pathways. These results were further interpreted on the basis of molecular models of these ligands in complex with the recent DRD 2 crystal structures, highlighting the critical role of the secondary/allosteric pharmacophore in modulating the functional selectivity profile.