Synthesis of Novel Indazole- and Theophylline-Substituted 1,3,4-Thiadiazoles Based EGFR Inhibitors Targeting Human Breast Cancer Cells

Epidermal growth factor receptor (EGFR) overexpression plays a critical role in breast cancer progression, metastasis, and resistance to chemotherapy, making EGFR an important therapeutic target for the development of novel anticancer agents. This study aims to design, synthesize, and biologically evaluate a new series of indazole- and theophylline-substituted 1,3,4-thiadiazole derivatives as the potential EGFR inhibitors against human breast cancer cells. A library of novel thiadiazole derivatives was synthesized through multistep synthetic protocols and structurally characterized using standard spectroscopic techniques. The anticancer activity of the synthesized compounds was evaluated against MCF-7 human breast cancer cells using cytotoxicity assays. Molecular docking studies were performed to investigate the binding interactions of the compounds within the EGFR active site. Furthermore, the most active compound was subjected to mechanistic studies including EGFR phosphorylation inhibition, apoptosis induction, AKT signaling analysis, and cell cycle distribution assays. Among the synthesized derivatives, the compound 4n demonstrated the most potent cytotoxic activity against MCF-7 cells with an IC50 value of 22.92 µM. Molecular docking analysis revealed a strong binding affinity toward EGFR (−8.29 kcal/mol), which was superior to that of the reference ligand. Biological investigations confirmed that compound 4n effectively inhibited EGFR phosphorylation and suppressed downstream AKT signaling. In addition, the compound significantly induced apoptosis (15.32%) and caused cell cycle arrest at the sub-G2/M phase, indicating its ability to inhibit breast cancer cell proliferation through EGFR-mediated pathways. The findings demonstrate that indazole- and theophylline-substituted 1,3,4-thiadiazole derivatives, particularly compound 4n, possess promising EGFR inhibitory and anticancer activities. The combined synthetic, computational, and biological results suggest that compound 4n may serve as a potential lead candidate for the development of targeted therapeutics against EGFR-positive breast cancer.