Harnessing dual CA/EGFR inhibition via pyrazolo[3,4-d]pyrimidine-nucleobase/Sulfonamide hybrids: Synthesis, antiproliferative, pro-apoptotic and in silico investigations

Abstract

A novel series of pyrazolo[3,4-d]pyrimidine derivatives conjugated with uracil, cytosine, and benzene sulfonamide moieties (5a–11b) were designed as dual inhibitors of tumor-associated carbonic anhydrase (CA) isoforms and epidermal growth factor receptor (EGFR). Compounds 11a and 11b emerged as the most potent candidates, exhibiting nanomolar inhibition against CA II, IX, and XII. Notably, 11a inhibited CA IX with a Ki of 2.6 nM, while 11b showed potent inhibition of CA II (Ki= 7.1 nM) and CA IX (Ki = 2.8 nM). Furthermore, both compounds surpassed erlotinib in EGFR inhibition, with IC50 values of 69 nM (11a) and 64 nM (11b). Biological evaluation revealed significant antiproliferative activity against MCF-7, HePG-2, HCT-116, and A549 cancer cell lines under normoxic and hypoxic conditions. Under hypoxia, 11a demonstrated superior potency (IC50 range: 4.04–9.58 µM). Flow cytometry indicated that 11a induced G1 phase arrest, while 11b triggered G2/M arrest and pronounced apoptosis, reducing cell viability to 12.4%. Molecular analysis confirmed the activation of the intrinsic apoptotic pathway through the upregulation of BAX, caspase-9, and p53, alongside Bcl-2 downregulation. Molecular docking and 100 ns molecular dynamics simulations confirmed stable binding within CA-II, CA-IX, and EGFR active sites. These findings identify 11b as a highly promising multi-target lead for further anticancer development.