4-Substituted-1,2,3-triazolo nucleotide analogues as CD73 inhibitors, their synthesis, in vitro screening, kinetic and in silico studies

• Three series of nucleotide analogues were designed as AMP or ADP mimics. • Potent competitive inhibitors of CD73 were identified. • One derivative exhibited higher efficacy than AOPCP in the blockade of T cells proliferation. Three series of nucleotide analogues were synthesized and evaluated as potential CD73 inhibitors. Nucleobase replacement consisted in connecting the appropriate aromatic or purine residues through a triazole moiety that is generated from 1,3-dipolar cycloaddition. The first series is related to 4-substituted-1,2,3-triazolo-β-hydroxyphosphonate ribonucleosides. Additional analogues were also obtained, in which the phosphonate group was replaced by a bisphosphonate pattern (P–C–P–C, series 2) or the ribose moiety was removed leading to acyclic derivatives (series 3). The β-hydroxyphosphonylphosphonate ribonucleosides (series 2) were found to be potent inhibitors of CD73 using both purified recombinant protein and cell-based assays. Two compounds ( 2a and 2b ) that contained a bis(trifluoromethyl)phenyl or a naphthyl substituents proved to be the most potent inhibitors, with IC 50 values of 4.8 ± 0.8 µM and 0.86 ± 0.2 µM, compared to the standard AOPCP (IC 50 value of 3.8 ± 0.9 µM), and were able to reverse the adenosine-mediated immune suppression on human T cells. This series of compounds illustrates a new type of CD73 inhibitors.