Highly potent and selective ectonucleotide pyrophosphatase/ phosphodiesterase i inhibitors based on an adenosine 5′-(α or γ)-Thio-(α,β- or β,γ)-methylenetriphosphate scaffold

Aberrant nucleotide pyrophosphatase/phosphodiesterase-1 (NPP1) activity is associated with chondrocalcinosis, osteoarthritis, and type 2 diabetes. The potential of NPP1 inhibitors as therapeutic agents, and the scarceness of their structure-activity relationship, encouraged us to develop new NPP1 inhibitors. Specifically, we synthesized ATP-α-thio-β,γ-CH₂ (1), ATP-α-thio-β,γ-CCl₂ (2), ATP-α-CH ₂-γ-thio (3), and 8-SH-ATP (4) and established their resistance to hydrolysis by NPP1,3 and NTPDase1,2,3,8 (<5% hydrolysis) (NTPDase = ectonucleoside triphosphate diphosphohydrolase). Analogues 1-3 at 100 μM inhibited thymidine 5′-monophosphate p-nitrophenyl ester hydrolysis by NPP1 and NPP3 by >90% and 23-43%, respectively, and only slightly affected (0-40%) hydrolysis of ATP by NTPDase1,2,3,8. Analogue 3 is the most potent NPP1 inhibitor currently known, K_i = 20 nM and IC₅₀ = 0.39 μM. Analogue 2a is a selective NPP1 inhibitor with K_i = 685 nM and IC₅₀ = 0.57 μM. Analogues 1-3 were found mostly to be nonagonists of P2Y₁/P2Y₂/P2Y₁₁ receptors. Docking analogues 1-3 into the NPP1 model suggested that activity correlates with the number of H-bonds with binding site residues. In conclusion, we propose analogues 2a and 3 as highly promising NPP1 inhibitors.