Synthesis and evaluation of cyclobut-3-ene-1,2-dione-3-hydrazones with benzothiazole moiety as novel anticancer agents inducing nonapoptotic oncosis-like cell death

Abstract

We report the design, synthesis, and biological evaluation of novel hybrid anticancer agents bearing three pharmacophores in one molecule: benzothiazole, cyclobut-2-ene-1,2-dione and hydrazone moieties. Several derivatives have demonstrated potent anticancer activity and high selectivity towards T-lymphoblastic leukaemia (CCRF-CEM) and colorectal cancer (HCT116) cell lines. The effects of in situ formed metal complexes on anticancer activity were investigated, revealing that the Fe(III) complexes of some derivatives were more active than the parental ligands, whereas the Cu(II) and Zn(II) complexes did not enhance cytotoxicity. However, compound 6 exhibited a decrease in cytotoxic activity upon the addition of iron, suggesting that the balance between extracellular and intracellular metal chelation is crucial for the anticancer activity of these compounds. Fluorescence microscopy using U2OS cells revealed the colocalization of specific derivatives with cellular components, including mitochondria (Pearson's coefficient for colocalization yielded positive correlation values of 0.85 and 0.83 for compound 6 at two concentrations), accompanied by a reduced mitochondrial membrane potential, endoplasmic reticulum vacuolization and reduction in lysosomal integrity. Moreover, cell cycle analysis revealed that compound 6 induced concentration-dependent G2/M cell cycle arrest, further contributing to its anticancer activity. These results imply the potential of these compounds to induce nonapoptotic, oncosis-like cell death. Lipinski's rule of five analysis, along with calculated drug-likeness and drug-score factors, indicates that most of these compounds have properties compatible with oral bioavailability and potential for further development. This study presents a promising new class of hybrid anticancer agents with a unique mechanism of action and favourable drug-like properties.

Keywords: Anticancer; Fluorescence microscopy; Mitochondrial membrane potential; Molecular hybridization; Nonapoptotic cell death; Oncosis; Vacuolization.