Styryl dyes with N-Methylpiperazine and N-Phenylpiperazine Functionality: AT-DNA and G-quadruplex binding ligands and theranostic agents

• Synthesis of new monomethine, unsymmetrical styryl dyes based on benzothiazole and piperazine scaffolds. • High binding affinities of dyes toward ds-DNA, ds-RNA and G-quadruplexes. • New dyes with N -methylpiperazine function showed significant preference for AT-DNA polynucleotide sequence and demonstrated minor groove binding. • New dyes with N -phenylpiperazine function showed high affinities for G-quadruplexes and micromolar and submicromolar antiproliferative activity toward human tumour cell lines. • Accumulation of dye in the mitochondrial space. New monomethine, unsymmetrical styryl dyes consisting of benzothiazole and N -methylpiperazine or N -phenylpiperazine scaffolds were synthesized, and their binding affinities for different ds-polynucleotides and G-quadruplex were studied. Substitution of piperazine unit with methyl or phenyl group strongly influenced their binding modes, binding affinities, spectroscopic responses and antiproliferative activities. Compounds with N -methylpiperazine substituents showed a significant preference for AT-DNA polynucleotides and demonstrated AT-minor groove binding, which manifested in strong fluorescence increase, significant double helix stabilization, and positive induced circular dichroism spectra. These compounds formed complexes with G-quadruplex by π-π stacking interactions of dye with the top or bottom G-tetrad. Bulkier compounds with N -phenylpiperazine function are probably bound to ds-polynucleotide by partial intercalation between base pairs. On the other hand, they showed stronger stabilization of G-quadruplex compared to methyl-substituted compounds. Fluorimetric titrations pointed to possible mixed stoichiometry’s: 1:1 complex with π-π stacking interactions of dye on the top or bottom G-tetrad and 1:2 complex with dye positioned between two G-quadruplex molecules. Bulkier dyes with N -phenylpiperazine fragments demonstrated micromolar and submicromolar antiproliferative activity that was especially pronounced for leukaemia and lymphoma. Flow cytometric assay shows dose- and time-dependent increase in SubG0/G1 phase. Furthermore, the compounds enter the cells readily and accumulate in the mitochondrial space, co-localize with the standard mitochondrial markers.