Click-modifiable isatin hydrazones as COX-2, VEGFR-2, and carbonic anhydrase inhibitors: A multi-target approach to cancer therapy

In pursuit of novel therapeutic strategies for complex multifactorial diseases such as cancer, a set of isatin hydrazone/1,2,3-triazole hybrids was rationally designed and synthesized to target multiple cancer-related pathways, including COX-2, VEGFR-2, and tumor-associated carbonic anhydrases (CA IX and XII). In vitro COX-2 inhibition assays revealed eight hybrids with nanomolar potency (IC50 = 50–90 nM), comparable to celecoxib, with compounds 5h and 5j exhibiting comparable selectivity indices to celecoxib. For VEGFR-2 inhibition, compound 5f surpassed sunitinib (IC50 = 64 vs. 92 nM), while 5j retained ∼75 % of its activity. Compounds 5f, 5j, and 5l were potent and selective inhibitors of hCA IX (Kᵢ = 27.3–37.2 nM). Cytotoxicity screening identified 5h and 5j as the most active against MCF7, A549, Caco-2, and MDA-MB-231 cell lines (IC50 = 1.3–12.49 μM) with good safety in normal WI-38 cells. Mechanistic studies on 5j indicated cell cycle arrest and apoptosis induction via upregulation of Caspase-9 and Bax and downregulation of Bcl-2. Compound 5j was also subjected to additional cell-based evaluations, including wound-healing, cell viability assays under hypoxic conditions, and PGE2 quantification, which further supported its multi-target profile. The in vitro human plasma stability of compound 5j was confirmed, exhibiting a half-life (t1/2) of approximately 7 h. In vivo, 5j significantly reduced tumor growth in an MDA-MB-231 xenograft model, comparable to 5-FU. Docking simulations confirmed favorable interactions with COX-2, VEGFR-2, and hCA IX, supporting the observed biological activities.