Luminescent bis(benzo[d]thiazolyl)quinoxaline: facile synthesis, nucleic acid and protein BSA interaction, live-cell imaging, biopharmaceutical research and cancer theranostic application

A series of quinoxaline-2-hydroxyphenylbenzothiazole scaffolds were synthesized and characterized using NMR, UV, fluorescence spectroscopy and LCMS. These newly synthesized compounds were found to be cytotoxic in human epithelioid cervix carcinoma (HeLa) and human colon cancer cell lines (Caco-2). Selectivity of the compounds 7e and 7g are more than 9 fold higher in Caco-2 cells with respect to the normal cell line HEK-293. The most fluorescent compound 7e has displayed high cytoselectivity, significant cellular uptake in HeLa cells and strong binding efficacy with DNA and BSA. The most potent compound 7g has primarily classified as BCS class 4 and BDDCS class 4.


Synthesis and characterization of 2-benzothiazolyl phenol (BTP) (3)
The equal molar of 2-aminothiophenol (1) and 2-hydroxybenzaldehyde (2) was dissolved in ethanol and an adequate amount of silica gel was added to make the slurry. The prepared slurry was further air dried in vacuum followed by microwave irradiation at 490 watt for 15 min. The progress of reaction was monitored by TLC using hexane/ethyl acetate (3:1) solvent system. After completion of the reaction, ethyl acetate was added to the solid support and the product was recovered by filtering the solution from the resin by whattman filter paper. The solution is then transferred to the beaker and air dried. Subsequently, the solvent was reduced gradually and white needle-like crystals of benzothiazolylphenol (BTP) (3) was obtained with high yield. 1

General procedure for the synthesis of BTP-Quinoxaline series (7a-7g)
2,3-bis (bromomethyl quinioxaline) series (6a-6g) and BTP (1:2 ratio) were dissolved in acetone followed by the slurry preparation using adequate amount basic alumina. It is noteworthy to mention that 1.1 equivalents of K 2 CO 3 were added to the reaction mixture for speed up the reaction. It is then air dried and subjected to microwave at 490 watts (120° C) for 30 min. The progress of the reaction was monitored eventually by TLC using hexane/ethyl acetate (3:1) solvent system. After completion of the reaction, the compound was recovered by adding ethyl acetate to it and the alumina is filtered off using whattman filter paper. The fine needle like crystals of compound 7a-7g was obtained by slow evaporation of ethylacetate with high yield.

DNA binding experiment
Interaction of complex (7e) with DNA was recorded using UV-Visible absorption spectroscopy and ethidium bromide displacement assay by fluorescence spectroscopy.

UV-visible spectral studies
The DNA binding assay were carried out using Tris-HCl as a buffer (5 mM L -1 Tris-HCl /50mM L -1 NaCl in water, pH 7.4) using an aqueous solution of the compound 7e. The purity of the CT-DNA was checked using nanodrop instrument and it was found to be 1.8:1 which indicated that DNA are free from proteins. The CT-DNA concentration in buffer were calculated by using the UV absorbance value at 260 nm and from the known molar absorption coefficient value and it was found to be 6600 M -1 cm -1 [1]. DNA binding experiments were performed by incubating constant complex concentration and increasing CT-DNA concentration [for 5 minute at room temperature] to give sufficient time to interact with DNA. The absorbance of the complex was measured after each succeeding addition of CT-DNA.
The intrinsic DNA binding constant (K b ) was obtained using the equation (1) [

Fluorescence study:
To know the emission property of all the synthesized complexes (7a-g), an initial investigation was performed using spectrofluorometric method in water by using absorption maximum as their excitation energy. DNA binding titration was also carried out in Tris-HCl buffer (5 mM Tris-HCl in water, pH 7.4) using aqueous solution of complex 7e by fluorescence spectroscopic method. Following successive addition of CT-DNA, the sample was kept for 5 min. After completion of this titration, the graph was plotted between Intensity and Wavelength.

Ethidium bromide displacement assay
EtBr is also known as a trypanocidal dye which has numerous biological applications [2,3].
EtBr displacement is periodically used as a diagnostic approach to determine the capability of compounds to bind with DNA [4].

BSA binding study
BSA is a carrier protein that increases solubility of the drug in the blood by binding to it [6].
Fluorescence spectroscopy is an efficient method to study the interaction of the BSA with the compound by quenching mechanism [7].