Antagonists of the A_2B adenosine receptor have recently emerged as targeted anticancer agents and immune checkpoint inhibitors within the realm of cancer immunotherapy. This study presents a comprehensive evaluation of novel Biginelli-assembled pyrimidine chemotypes, including mono-, bi-, and tricyclic derivatives, as A_2BAR antagonists. We conducted a comprehensive examination of the adenosinergic profile (both binding and functional) of a large compound library consisting of 168 compounds. This approach unveiled original lead compounds and enabled the identification of novel structure-activity relationship (SAR) trends, which were supported by extensive computational studies, including quantum mechanical calculations and free energy perturbation (FEP) analysis. In total, 25 molecules showed attractive affinity (K_i < 100 nM) and outstanding selectivity for A_2BAR. From these, five molecules corresponding to the new benzothiazole scaffold were below the K_i < 10 nM threshold, in addition to a novel dual A_2A/A_2B antagonist. The most potent compounds, and the dual antagonist, showed enantiospecific recognition in the A_2BAR. Two A_2BAR selective antagonists and the dual A_2AAR/A_2BAR antagonist reported in this study were assessed for their impact on colorectal cancer cell lines. The results revealed a significant and dose-dependent reduction in cell proliferation. Notably, the A_2BAR antagonists exhibited remarkable specificity, as they did not impede the proliferation of non-tumoral cell lines. These findings support the efficacy and potential that A_2BAR antagonists as valuable candidates for cancer therapy, but also that they can effectively complement strategies involving A_2AAR antagonism in the context of immune checkpoint inhibition.