Multifaceted Anticancer Activity of Flavanone/Chromanone Intermediates for Five-Membered Heterocyclic Derivatives: Targeting Oxidative Stress, Apoptosis, and MAPK Signaling in Colorectal Cancer

This study explores the multifaceted anticancer mechanisms of flavanone analogues and spiropyrazoline condensed with flavanone ring against colorectal cancer (CRC) cell lines. Five-membered heteroaromatic scaffolds, in particular, have gained prominence in medicinal chemistry as they offer enhanced metabolic stability, solubility and bioavailability, crucial factors in developing effective drugs. Building upon previous findings, we investigated three lead derivatives (<b>1</b>, <b>3</b>, and <b>5</b>) with potent antiproliferative activity (IC₅₀ < 35 μM). The compounds induced pronounced oxidative stress, evidenced by increased lipid peroxidation and reduced membrane fluidity, primarily within the hydrophobic layers of cell membranes. Preincubation with the antioxidant N-acetylcysteine (NAC) significantly attenuated these effects, confirming the pivotal role of reactive oxygen species (ROS) in their cytotoxicity. Mechanistic studies revealed that the derivatives triggered intrinsic apoptosis, characterized by the cleavage of PARP and the activation of caspase-9 and caspase-3. Furthermore, the compounds modulated key signaling pathways involved in cell survival and proliferation. Specifically, they inhibited the pro-oncogenic ERK1/2 MAPK pathway while inducing cell line-dependent alterations in p38 and JNK activity. Concurrently, all derivatives reduced the level of the transcription factor Nrf2, a master regulator of antioxidant defense and a mediator of chemoresistance in CRC. Collectively, these findings indicate that flavanone/chromanone derivatives exert their anticancer activity through a synergistic mechanism involving ROS generation, disruption of redox homeostasis, inhibition of Nrf2 signaling, and modulation of MAPK-dependent apoptotic pathways. These results highlight the therapeutic potential of flavanone-based compounds and their spiropyrazoline analogues as multifunctional anticancer agents targeting oxidative stress and survival signaling in colorectal cancer.