Multi-Targeted Azacoumarin–Cyanocinnamate Hybrids Induce G2/M Arrest and Apoptosis via Tubulin, and COX-2/VEGFR Modulation: insights from in vitro mechanistic basis and in vivo validation

Abstract

Cancer remains a significant global health concern, with breast cancer ranking among the leading causes of cancer-related mortality in women. In pursuit of multi-targeted anticancer agents, we designed and synthesized a novel series of 7-hydroxy azacoumarin–α-cyanocinnamate hybrids and evaluated their therapeutic potential through comprehensive in vitro and in vivo studies. Structural characterization was confirmed using NMR, IR, and elemental analysis. Among the synthesized compounds, compound 7 exhibited the most potent cytotoxic activity against MCF-7 cells (IC₅₀ = 7.65 µM) and MDA MB 231 (IC₅₀ = 9.7 ± 1.15 µM), with notable selectivity over non-tumorigenic MCF-10A cells (IC₅₀ = 52.02 µM), as compared to the reference drug doxorubicin. Mechanistic in vitro investigations revealed that compound 7 induced G₂/M phase arrest and apoptosis, accompanied by upregulation of pro-apoptotic markers (Bax, p53) and suppression of Bcl-2. Additionally, compound 7 significantly inhibited tubulin polymerization and demonstrated marked antioxidant activity in the FRAP assay (IC₅₀ = 144.71 µM), as well as selective COX-2 inhibition (IC₅₀ = 1.264 µM, SI = 5.93). In vivo evaluation using the Ehrlich ascites carcinoma (EAC) model confirmed its anticancer efficacy, with 85.92% reduction in viable EAC cells and substantial tumor volume suppression at 10 mg/kg. Notably, compound 7 mitigated EAC-induced hepatorenal toxicity by restoring liver and kidney biomarkers and reducing oxidative stress and lipid peroxidation. Furthermore, it significantly downregulated pro-inflammatory (TNF-α) and angiogenic (VEGFR-II) markers while preserving normal tissue histoarchitecture. Collectively, these findings highlight compound 7 as a promising multi-functional lead candidate with cytotoxic, antioxidant, anti-inflammatory, and anti-angiogenic activities, meriting further development in cancer therapeutics.