Development of pyrazolo[1,5-a]pyrimidine-grafted coumarins as selective carbonic anhydrase inhibitors and tubulin polymerization inhibitors with potent anticancer activity.

This study presents the design, synthesis, and evaluation of a novel series of coumarin-based compounds (9a-t) as potential anticancer agents. The compounds were strategically designed to inhibit cancer-related carbonic anhydrase (CA) isoforms IX and XII and tubulin polymerization. Two approaches were employed for CA inhibition: utilizing the coumarin motif to occlude the CA active site entrance and incorporating zinc-binding groups (sulfonamide, carboxylic acid, and thiol) to interact with the catalytic zinc ion. The target compounds were also designed to inhibit tubulin polymerization by combining the privileged coumarin and pyrazolo[1,5-a]pyrimidine scaffolds. Biological evaluation of the target compounds (9a-t) revealed that sulfonamide-containing derivatives 9h and 9r exhibited potent inhibitory activity in the low nanomolar range against CA IX (K = 23 and 14 nM, respectively) and CA XII (K = 6 and 17 nM, respectively). In NCI-60 human tumor cell line screening, compounds 9k, 9m, and 9q demonstrated broad-spectrum anti-proliferative activity in the five-dose assay with MG-MID values of 7.31 μM, 10.68 μM, and 5.92 μM, respectively. Compound 9m showed significant tubulin polymerization inhibition with an IC = 5.28 μM, surpassing the efficacy of colchicine. Cell cycle analysis in MDA-MB-231 breast cancer cells revealed G2/M phase arrest for 9m, which induced significant apoptosis and modulated apoptotic markers. Molecular docking studies provided insights into the binding modes of the compounds with CA IX, CA XII, and tubulin. ADMET and toxicity predictions were performed to assess the drug-like properties of the compounds. These findings pave the way for further optimization of the coumarin scaffold to develop dual inhibitors of carbonic anhydrase IX/XII and tubulin polymerization.