Various series of 4,6-disubstituted-2-thiopyridine derivatives were synthesized and evaluated as potential ecto-5'-nucleotidase (CD73) inhibitors. Altogether, about ninety compounds were prepared using a general synthetic pathway involving one or two steps (eventually one-pot) procedures. Variation of the nature of the substituents in positions 4 and 6 (methyl, trifluoromethyl or phenyl) of the thiopurine ring, as well as on the thiol function, was examined and led to marked differences both in term of reactivity and ability to interfere with the putative target protein. Using a functional assay on immune cells, few compounds belonging to series 4 were shown to be able to antagonize the inhibition of the T-cell proliferation at both 100 μM and 10 μM (completely for 4 ab and partially for 4 ai), that is as potent as AOPCP which entirely reversed the inhibitory impact of exogenous ATP on T cell proliferation until 62.5 μM. In addition, we have shown that both compounds (4 ab and 4 ai) were also capable of moderately inhibiting the hA