Development of dual A2AR/EGFR targeting pyrazoline-thiazole hybrids for restored apoptotic balance in colorectal cancer

Colorectal Cancer (CRC) is the third most common cause of cancer-related mortality globally, claiming approximately 800,000 lives annually. Despite standard treatments like 5-fluorouracil, therapeutic success is frequently challenged by systemic toxicity, drug resistance, and high recurrence rates. This study aimed to overcome these limitations by developing novel thiazolyl-pyrazoline hybrids as dual inhibitors of the Adenosine 2 A Receptor (A2AR) and Epidermal Growth Factor Receptor (EGFR) that mechanistic convergence on the PI3K/Akt signaling axis regulating cell survival and apoptotic resistance. Using a molecular hybridization strategy based on lead pharmacophores Gefitinib and Tozadenant, three chemical series were developed and validated through a multi-disciplinary pipeline including molecular docking and iterated biological testing. Lead compounds, 6aii and 10b, have been identified as the most potent analogs of the series, exhibiting exceptional cytotoxicity against Caco2 cells at IC₅₀ 0.037 ± 0.10 μM and 0.6125 ± 0.13 μM, respectively. Mechanistic investigations confirmed these hybrids induce S and G2/M phase cell cycle arrest and stimulate restore Apoptosis balance by upregulating pro-apoptotic genes (Caspase-3, TP53, and Bax) and downregulating the anti-apoptotic one (Bcl-2). Enzyme inhibition activity on EGFR and A2AR target depicted low-to-sub micromolar activity concentrations on respective targets (IC₅₀ 1.53 ± 0.12-8.37 ± 0.03 μM and 0.037 ± 0.21-0.6125 ± 0.15 μM, respectively). Compound 6ii further demonstrated the lowest cAMP expression biolevels confirming significant hampering of the A2AR's downstream signaling pathway. Moreover, the in vivo validation using AOM-induced CRC mouse models illustrated the effective restoration of the colonic crypt architecture, suppression of EGFR overexpression, and exhibition of favorable safety profiles regarding liver serum enzyme levels. This research provides a robust therapeutic framework for multi-target drug discovery in advanced colorectal Cancer management.