Rationally designed Pyrazolo[1,5-a]pyrimidines as dual inhibitors of CA IX/XII and CDK6: A novel approach for NSCLC treatment

Developing novel anticancer agents that target critical pathways in non-small cell lung cancer (NSCLC) presents a considerable challenge. This study synthesized 16 pyrazolo[1,5-a]pyrimidine derivatives with zinc-binding groups through molecular hybridization to achieve dual-target inhibition of tumor-associated carbonic anhydrase (CA) isoforms IX/XII and cyclin-dependent kinase 6 (CDK6). In-vitro assays indicated that sulfonamide-bearing compounds displayed enhanced CA inhibition, with compounds 7d, 11b, and 11d presenting K_i values of 11.2, 18.4, and 19.7 nM for CA IX, while compounds 11a and 11c exhibited K_i values of 14.8 and 8.7 nM for CA XII. Cytotoxicity assays conducted on NSCLC cell lines A549 and NCI–H1734 demonstrated that compounds 7c, 7d, 7i, and 11d exhibited superior activity relative to Roscovitine in both cell lines. While these compounds demonstrated limited inhibition of cyclin-dependent kinase 4 (CDK4), 7d and 11d effectively inhibited CDK6, with IC₅₀ values of 0.054 and 0.069 μM, respectively, which are comparable to Palbociclib. Analyses of the cell cycle and apoptosis demonstrated a strong G1 arrest and a notable induction of apoptosis. Molecular docking confirmed essential binding interactions with CA IX/XII and CDK6, while in-silico ADMET predictions suggested favorable pharmacokinetics, despite potential toxicity concerns. Compounds 7d and 11d represent potential dual-target inhibitors for the treatment of NSCLC.