Abstact

Phosphocoumarins and a first-in-class unsubstituted phosphoquinolinone are disclosed as previously unrecognized carbonic anhydrase (CA) inhibitors, displaying multimodal inhibition within a tunable coumarin-like scaffold. Acidic phosphocoumarins display inhibition of physiologically relevant human CAs, particularly tumor-associated isoforms IX and XII (K(I)s: 0.08-0.28 muM) through a composite, two-step mechanism: the ligand first anchors the zinc-bound water molecule before displacing it to directly coordinate the catalytic zinc ion, without CA-mediated hydrolysis. Conversely, a methyl-ester phosphocoumarin functions as an isoform-selective prodrug, undergoing CA-mediated cyclic phosphoester hydrolysis to selectively generate a potent hCA IX/XII inhibitor (K(I)s: 54-62 nM), whereas the phosphoquinolinone acts as a direct binder (K(I)s: 0.18-0.29 muM vs hCA IX/XII). The complementary mechanisms are supported by QM/MM and long-time scale MD simulations, crystallographic studies, (31)P NMR, HRMS, and MS/MS. Selected derivatives exhibit low-micromolar antiproliferative activity and induce apoptosis in cancer cells, fostering phosphorus-heterocycles as a mechanistically rich platform for isoform-selective CA inhibition and targeted drug design.