Benzoxazine–Purine Hybrids as Antiproliferative Agents: Rational Design and Divergent Mechanisms of Action

<b>Background/Objectives:</b> Targeted cancer therapies increasingly rely on modulating specific cell death pathways and kinase signaling. Due to their structural versatility and potential to induce mechanistically distinct cytotoxic responses, benzoxazine-purine hybrids represent a promising scaffold for anticancer drug development. The objective of this study was to design and evaluate novel benzoxazine-purine derivatives for their antiproliferative activity and elucidate their underlying mechanisms of action. <b>Methods:</b> A series of benzoxazine-purine compounds was synthesized via a modular and efficient approach. The synthetic route involved a one-pot cyclization of substituted 2-aminophenols with epichlorohydrin, followed by tosylation and subsequent Mitsunobu coupling with halogenated purines. Their antiproliferative activity was assessed in MCF-7 (breast) and HCT-116 (colon) cancer cell lines using MTT assays. Selected compounds were evaluated further for kinase inhibition, effects on the cell cycle, membrane integrity (Annexin V/PI staining), ultrastructural changes (SEM), and caspase activation (Western blot). In silico ADMET profiling was also performed. <b>Results:</b> Compounds <b>9</b> and <b>12</b> exhibited the most potent antiproliferative activity, with low micromolar IC₅₀ values. Compound <b>12</b> showed dual HER2/JNK1 kinase inhibition and induced caspase-8-dependent pyroptosis-like cell death, characterized by membrane rupture and inflammatory features. In contrast, compound <b>8</b> lacked kinase inhibition and promoted S-phase arrest with apoptotic-like morphology. Both compounds demonstrated favorable physicochemical and ADMET profiles, including high intestinal absorption and an absence of mutagenicity. <b>Conclusions:</b> The rational design of benzoxazine-purine hybrids resulted in the discovery of compounds with distinct mechanisms of action. Compound <b>12</b> induces inflammatory cell death by modulating kinases, while compound <b>9</b> acts through a kinase-independent apoptotic pathway. These results underscore the therapeutic potential of scaffold-based diversification for developing targeted anticancer agents.