Multi-target pyrazolopyrimidine-coumarin derivatives as potent CA IX/XII and tubulin polymerization inhibitors: Design, synthesis, and biological evaluation

In the current medical era, developing multi-target anticancer agents that simultaneously engage complementary cellular vulnerabilities represents a promising strategy to overcome the inherent limitations of single-target therapeutics. Building on insights from our previous investigation, we synthesized a novel series of coumarin-pyrazolo [1,5-a]pyrimidine hybrids (13a-n) designed to target tumor-associated carbonic anhydrases (CA IX/XII) and tubulin polymerization through rational molecular hybridization. Structure-activity relationship analysis revealed that a zinc-binding sulfonamide group was essential for CA inhibitory activity; coumarin-only analogs failed (Ki > 100 μM). Compounds 13g and 13n, bearing sulfonamide functionality, demonstrated nanomolar potency against hCA IX and hCA XII. Compound 13n emerged as the superior lead, achieving balanced dual-target inhibition: hCA IX (Ki = 27.1 nM), hCA XII (Ki = 20.9 nM), and tubulin polymerization (IC50 = 6.35 μM). Broad-spectrum NCI-60 screening revealed 13n′s potent antiproliferative activity across nine cancer types (GI50: 2.48-31.00 μM). Treatment of MCF-7 breast cancer cells with 13n resulted in significant G2/M phase arrest (13.81% to 31.97%) and robust induction of apoptosis (37-fold relative to control), mediated by p53-dependent signaling. Molecular analysis revealed elevated p53 expression (3.43-fold), increased Bax level (12.34-fold), reduced Bcl-2 expression (4.37-fold), and enhanced caspase-7 activation (7.35-fold). Molecular docking studies confirmed zinc coordination within the CA active sites and optimal positioning within the tubulin colchicine-binding pocket. In summary, compound 13n validates the design strategies employed to develop an efficient multi-target anticancer candidate, positioning this lead compound for further preclinical development.