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Modulators of the G protein-coupled A2A adenosine receptor (A2AAR) have been considered promising agents to treat Parkinson's disease, inflammation, cancer, and central nervous system disorders. Herein, we demonstrate that a thiophene modification at the C8 position in the common adenine scaffold converted an A2AAR agonist into an antagonist. We synthesized and characterized a novel A2AAR antagonist, 2 (LJ-4517), with Ki = 18.3 nM. X-ray crystallographic structures of 2 in complex with two thermostabilized A2AAR constructs were solved at 2.05 and 2.80 Å resolutions. In contrast to A2AAR agonists, which simultaneously interact with both Ser2777.42 and His2787.43, 2 only transiently contacts His2787.43, which can be direct or water-mediated. The n-hexynyl group of 2 extends into an A2AAR exosite. Structural analysis revealed that the introduced thiophene modification restricted receptor conformational rearrangements required for subsequent activation. This approach can expand the repertoire of adenosine receptor antagonists that can be designed based on available agonist scaffolds.

Based on high binding affinity of truncated 2-hexynyl-4'-thioadenosine (3a) at both A2A adenosine receptor (AR) and A3 AR, we explored structure-activity relationship (SAR) of the C2-substitution by altering chain length of the 2-hexynyl moiety, thereby evaluating the hydrophobic pocket size. A series of truncated N6-substituted 4'-thioadenosine derivatives with C2-alkynyl substitution were successfully synthesized from D-mannose, using a palladium-catalyzed Sonogashira coupling reaction as the key step, whose structures were confirmed by the X-ray crystal structure of 4h. As the size of the alkynyl group at the C2-position increased, the binding affinity improved; however, when the substituted group was larger than hexynyl, the binding affinity decreased. The introduction of a bulky hydrophobic group such as 3-halobenzyl group at the free N6-amino group decreased the binding affinity at hA2AAR. These results confirm our previous findings that a free amino group at N6-position and longer hydrophobic chain at C2-position are essential for hA2A AR binding affinity. The introduction of a bulky hydrophobic group at free N6-amino group maintained the binding affinity at hA3 AR. The binding mode of truncated 2-substituted-4'-thioadenosine derivatives to hA2A and hA3 AR were predicted by a molecular docking study., (© 2024 Wiley‐VCH GmbH.)

Building on the preceding structural analysis and a structure-activity relationship (SAR) of 8-aryl-2-hexynyl nucleoside hA 2A AR antagonist 2a , we strategically inverted C2/C8 substituents and eliminated the ribose moiety. These modifications aimed to mitigate potential steric interactions between ribose and adenosine receptors. The SAR findings indicated that such inversions significantly modulated hA 3 AR binding affinities depending on the type of ribose, whereas removal of ribose altered the functional efficacy via hA 2A AR. Among the synthesized derivatives, 2-aryl-8-hexynyl adenine 4a demonstrated the highest selectivity for hA 2A AR ( K i ,hA2A = 5.0 ± 0.5 nM, K i ,hA3 / K i ,hA2A = 86) and effectively blocked cAMP production and restored IL-2 secretion in PBMCs. Favorable pharmacokinetic properties and a notable enhancement of anticancer effects in combination with an mAb immune checkpoint blockade were observed upon oral administration of 4a . These findings establish 4a as a viable immune-oncology therapeutic candidate.

Based on hA 2A AR structures, a hydrophobic C8-heteroaromatic ring in 5'-truncated adenosine analogues occupies the subpocket tightly, converting hA 2A AR agonists into antagonists while maintaining affinity toward hA 3 AR. The final compounds of 2,8-disubstituted- N 6 -substituted 4'-thionucleosides, or 4'-oxo, were synthesized from d-mannose and d-erythrono-1,4-lactone, respectively, using a Pd-catalyst-controlled regioselective cross-coupling reaction. All tested compounds completely antagonized hA 2A AR, including 5d with the highest affinity ( K i,A 2A = 7.7 ± 0.5 nM). The hA 2A AR- 5d X-ray structure revealed that C8-heteroaromatic rings prevented receptor activation-associated conformational changes. However, the C8-substituted compounds still antagonized hA 3 AR. Structural SAR features and docking studies supported different binding modes at A 2A AR and A 3 AR, elucidating pharmacophores for receptor activation and selectivity. Favorable pharmacokinetics were demonstrated, in which 5d displayed high oral absorption, moderate half-life, and bioavailability. Also, 5d significantly improved the antitumor effect of anti-PD-L1 in vivo . Overall, this study suggests that the novel dual A 2A AR/A 3 AR nucleoside antagonists would be promising drug candidates for immune-oncology.

Modulators of the G protein-coupled A 2A adenosine receptor (A 2A AR) have been considered promising agents to treat Parkinson's disease, inflammation, cancer, and central nervous system disorders. Herein, we demonstrate that a thiophene modification at the C8 position in the common adenine scaffold converted an A 2A AR agonist into an antagonist. We synthesized and characterized a novel A 2A AR antagonist, 2 (LJ-4517), with K i = 18.3 nM. X-ray crystallographic structures of 2 in complex with two thermostabilized A 2A AR constructs were solved at 2.05 and 2.80 Å resolutions. In contrast to A 2A AR agonists, which simultaneously interact with both Ser277 7.42 and His278 7.43 , 2 only transiently contacts His278 7.43 , which can be direct or water-mediated. The n -hexynyl group of 2 extends into an A 2A AR exosite. Structural analysis revealed that the introduced thiophene modification restricted receptor conformational rearrangements required for subsequent activation. This approach can expand the repertoire of adenosine receptor antagonists that can be designed based on available agonist scaffolds.