Discovery of potent nucleotide pyrophosphatase/phosphodiesterase3 (NPP3) inhibitors with ancillary carbonic anhydrase inhibition for cancer (immuno)therapy.

Nucleotide pyrophosphatase/phosphodiesterase3 (NPP3) catalyzes the hydrolysis of extracellular nucleotides. It is expressed by immune cells and some carcinomas, e.g. of kidney and colon. Together with ecto-5'-nucleotidase (CD73), NPP3 produces immunosuppressive, cancer-promoting adenosine, and has therefore been proposed as a target for cancer therapy. Here we report on the discovery of 4-[(4-methylphthalazin-1-yl)amino]benzenesulfonamide (1) as an inhibitor of human NPP3 identified by compound library screening. Subsequent structure-activity relationship (SAR) studies led to the potent competitive NPP3 inhibitor 2-methyl-5-{4-[(4-sulfamoylphenyl)amino]phthalazin-1-yl}benzenesulfonamide (23, K i 53.7 nM versus the natural substrate ATP). Docking studies predicted its binding pose and interactions. While 23 displayed high selectivity versus other ecto-nucleotidases, it showed ancillary inhibition of two proposed anti-cancer targets, the carbonic anhydrases CA-II (Ki 74.7 nM) and CA-IX (Ki 20.3 nM). Thus, 23 may act as multi-target anti-cancer drug. SARs for NPP3 were steeper than for CAs leading to the identification of potent dual CA-II/CA-IX (e.g. 34) as well as selective CA-IX inhibitors (e.g. 31). This journal is © The Royal Society of Chemistry.