Trifluorinated Pyrimidine-Based A_2B Antagonists: Optimization and Evidence of Stereospecific Recognition

Abstract

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.

Identification of V6.51L as a selectivity hotspot in stereoselective A2B adenosine receptor antagonist recognition

“…To experimentally validate this hypothesis, the ISAM-140 enantiomers were separated and their absolute configuration unequivocally assigned. Besides this goal, the enantiomeric separation and pharmacological characterization of this reference A 2B AR antagonist allowed to confirm the expected higher affinity of the S enantiomer, in line with the original modeling hypothesis 11 and recent similar results obtained with derivatives of this scaffold 10 , 13 , 14 .…”

“…Single crystals suitable for X-ray analysis were grown by slow evaporation of each enantiomer in ethanol. The structures were solved and the data extracted from X-ray crystallography of both monocrystals presented in the Supporting Information (Supplementary Table S1 ) 13 . The crystal structures of ( S )-ISAM-140 and ( R )-ISAM-140 (monoclinic, Fig.…”

“…The QresFEP protocol has been broadly applied to investigate the A 2A AR mutational landscape 26 – 28 , showing exceptional sensitivity to capture the correct affinity shifts for different chemotypes. The binding model of (S) -ISAM-140 to the WT and L249V 6.51 mutant versions of A 2A AR was here assumed to be the same as our docking model of this compound to the WT A 2B AR 13 . That model suggested that the high A 2B AR affinity of ( ±) ISAM-140 was due to the stereoselective optimal fitting of the ( S ) isomer to the A 2B AR binding site, facilitated by the Val sidechain in position 6.51 of this receptor 13 .…”

“…The binding model of (S) -ISAM-140 to the WT and L249V 6.51 mutant versions of A 2A AR was here assumed to be the same as our docking model of this compound to the WT A 2B AR 13 . That model suggested that the high A 2B AR affinity of ( ±) ISAM-140 was due to the stereoselective optimal fitting of the ( S ) isomer to the A 2B AR binding site, facilitated by the Val sidechain in position 6.51 of this receptor 13 . The calculated recovery of the binding affinity of (S) -ISAM-140 upon the L249V 6.51 mutation in the A 2A AR, which is in line with the experimental design of this A 2B -like mutation on the A 2A AR, further confirms the validity of the binding model for this chemotype on the A 2B AR.…”

“…1 ) 11 , 12 . The prediction of the active stereoisomer for this chemotype was later confirmed indirectly by experimental characterization of the active stereoisomers for representative compounds of a series of cyanopyrimidines 10 , fluorinated tricyclic derivatives 13 and aza-bioisosteres of the pentagonal heterocycle 14 . This binding model proposed that the stereospecific complementarity to the A 2B AR cavity was due to the optimal accommodation of the thiophene/furan ring around the chiral center of the core scaffold (Fig.…”