Synthesis, biological evaluation and molecular docking studies of N-propylsulfonyl indole-linked...

Ectonucleotidases, including NTPDases and ecto-5'-nucleotidase (e-5'NT/CD73), regulate extracellular purinergic signaling by converting ATP to adenosine, a pathway critically involved in immune response, inflammation, and cancer progression. In this study, a novel library of 22 N-propylsulfonyl-substituted indole-based hydrazinecarbothioamides (5a-5v) was synthesized and structurally characterized. Biological evaluation against human e-5'NT and NTPDase1, -2, -3, and - 8 revealed that several compounds exhibited low micromolar inhibitory activity, with 5n (IC₅₀ = 1.7 µM), 5o (IC₅₀ = 1.7 µM), 5f (IC₅₀ = 1.0 µM), and 5i (IC₅₀ = 1.6 µM) emerging as the most promising derivatives, showing strong potency and isoform selectivity. Structure-activity relationship analysis indicated that both electronic and steric features of substituents significantly influence activity and enzyme preference. Molecular docking studies performed on e-5'NT demonstrated that active compounds adopt consistent binding modes within the catalytic pocket, stabilized by key residues such as Asp-506, Phe-500, Phe-417 and Arg-395. Binding free energy calculations (MM-GBSA) supported strong ligand-protein interactions ( ~ - 70 kcal/mol). The docking protocol was validated by redocking, yielding an RMSD value well below the accepted threshold. Molecular dynamics simulations (500 ns) confirmed stable complex formation, with low RMSD values (~ 1-3 Å), limited residue fluctuations, and persistent interactions with catalytic residues. Surface and compactness parameters (rGyr, SASA) remained stable, indicating consistent ligand accommodation. In silico ADME analysis suggested favorable drug-like properties for most compounds, particularly for the lead candidates. Overall, these findings identify 5n and 5o as the most promising lead compounds, supported by both experimental and computational results, and highlight this scaffold as a valuable platform for the development of selective ectonucleotidase inhibitors.