Synthesis and anti-lung cancer evaluation of fused pyrazolo[3,4-b]pyridine linked isoxazoles and 1,2,3-triazoles: PEG-400 mediated one-pot reaction under microwave irradiation

Lung Cancer remains one of the leading causes of cancer-related mortality worldwide, and dysregulation of the epidermal growth factor receptor (EGFR) signaling pathway plays a central role in its progression. In the present study, a novel series of fused isoxazole and fused 1,2,3-triazole derivatives (4a-4 h and 5a-5 h) were rationally designed, synthesized and evaluated for their Anticancer potential against human lung Cancer cells. These compounds were synthesized via a single-step [3 + 2] cycloaddition reaction using (Z)-1-(4-fluorophenyl)-N-(4-iodobut-3-yn-2-ylidene)-3-methyl-1H-pyrazol-5-amine as the key precursor. In silico molecular docking studies revealed strong and stable interactions of the synthesized compounds with the ATP-binding pocket of the EGFR kinase domain, comparable to that of the reference drug, erlotinib. In vitro EGFR kinase inhibition assays confirmed the ability of the lead compounds to suppress kinase activity. Cytotoxicity studies on non-cancerous cell lines (HBEC30KT and HEK293) demonstrated minimal toxicity and a favorable therapeutic window.In contrast, selected compounds, particularly 4 h, 5d and 5 h, exhibited potent antiproliferative activity in lung Cancer cell lines H1975 and A549, with a marked reduction in cell viability and growth. Flow cytometric analysis revealed significant cell cycle arrest in the Sub-G1 phase, accompanied by induction of Apoptosis as confirmed by Annexin V-FITC staining. Furthermore, Transwell migration and soft agar colony formation assays demonstrated a strong inhibitory effect on Cancer cell migration and anchorage-independent growth, indicating effective suppression of metastatic and tumorigenic potential. In silico Absorption, Distribution, Metabolism, Excretion, and Toxicity (ADMET) analysis predicted favorable pharmacokinetic and safety profiles for the lead compounds.Collectively, the results suggest that fused isoxazole and fused 1,2,3-triazole derivatives, especially compounds 4 h, 5d and 5 h, represent promising lead molecules for the development of novel EGFR-targeted therapies for lung Cancer. Further in vivo studies and detailed pharmacological investigations are warranted to validate their therapeutic potential.

1,2,3-triazole; Anti-metastatic activity; Apoptosis; EGFR inhibition; Isoxazole; Lung cancer; Molecular docking.