DOI: 10.26650/istanbuljpharm.2026.1845135 ISSN: 2587-2087

Strategic Hybridisation of Benzo[b][1,4]oxazin, Chalcone, and Phenyl-4-Piperidine Scaffolds for Enhanced Breast Cancer Cell Suppression via Gasdermin-Mediated Pyroptosis

Burak Kuzu, Mustafa Çakır, Eda Açıkgöz
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Background and Aims: Breast cancer persists as a leading cause of mortality due to cancer, and therapeutic strategies involving multifunctional molecules that simultaneously interfere with proliferative and metastatic mechanisms are increasingly being explored. We designed novel benzo[b][1,4]oxazin-chalcone-phenyl-4-piperidine hybrid molecules and evaluated their antiproliferative potential against MDA-MB-231 breast cancer cells, focusing on their ability to induce pyroptosis.Methods: The hybrids (BS1-5) were synthesised and structurally confirmed using spectroscopic techniques, including proton nuclear magnetic resonance (1H-NMR), carbon-13 nuclear magnetic resonance (13C-NMR), and high-resolution mass spectrometry (HRMS). Their antiproliferative activities were assessed using in vitro assays, including cell proliferation, colony formation, cell adhesion, and cellular imaging. Morphological changes were analysed via bright-field microscopy and hematoxylin and eosin (H&E) staining, while immunofluorescence was employed to detect gasdermin (GSDM) expression, a pyroptosis marker.Results: Treatment with BS-2 ((Z)-6-methyl-3-(2-oxo-2-(4-(piperidin-1-yl)phenyl)ethylidene)-3,4-dihydro-2H-benzo[b][1,4]oxazin-2-one) and BS-3 ((Z)-6-chloro-3-(2-oxo-2-(4-(piperidin-1-yl)phenyl)ethylidene)-3,4-dihydro-2H-benzo[b][1,4]oxazin-2-one) resulted in significant, concentration- and time-dependent suppression of cellular proliferation; notably, BS-2 showed IC50 values of 5.2 μM after 24 h and 3.5 μM after 48 h. Colony formation assays showed that BS-2 markedly reduced clonogenic growth, outperforming BS-3. Adhesion assays demonstrated that both compounds disrupted cell-surface interactions, indicating antimetastatic potential. Morphological analyses revealed hallmark pyroptotic features in BS-2–treated cells, and immunofluorescence confirmed a marked increase in GSDM expression, validating the induction of gasdermin-mediated pyroptosis.Conclusion: Benzo[b][1,4]oxazin-chalcone-phenyl-4-piperidine hybrids, particularly BS-2, exhibit potent antiproliferative and anti-adhesive effects on MDA-MB-231 cells through the induction of pyroptotic cell death. These findings highlight the potential of pharmacophore hybridisation for developing multifunctional anticancer agents, warranting further structural optimisation and in vivo evaluation.

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