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

## TL;DR

This paper introduces new benzoxazine–purine compounds that can kill cancer cells through different mechanisms, showing promise for targeted cancer therapy.

## Contribution

The study presents a novel class of benzoxazine–purine hybrids with divergent mechanisms of antiproliferative action.

## Key findings

- Compound 12 inhibits HER2/JNK1 kinases and induces pyroptosis-like cell death.
- Compound 9 promotes S-phase arrest and apoptotic-like cell death without kinase inhibition.
- Both compounds have favorable ADMET and physicochemical profiles suitable for drug development.

## Abstract

Background/Objectives: 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. Methods: 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. Results: Compounds 9 and 12 exhibited the most potent antiproliferative activity, with low micromolar IC50 values. Compound 12 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 8 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. Conclusions: The rational design of benzoxazine–purine hybrids resulted in the discovery of compounds with distinct mechanisms of action. Compound 12 induces inflammatory cell death by modulating kinases, while compound 9 acts through a kinase-independent apoptotic pathway. These results underscore the therapeutic potential of scaffold-based diversification for developing targeted anticancer agents.