Synthesis, Structural Investigations, DNA/BSA Interactions, Molecular Docking Studies, and Anticancer Activity of a New 1,4-Disubstituted 1,2,3-Triazole Derivative

We report herein a new 1,2,3-triazole derivative, namely, 4-((1-(3,4-dichlorophenyl)-1H-1,2,3-triazol-4-yl)methoxy)-2-hydroxybenzaldehyde, which was synthesized by copper(I)-catalyzed azide–alkyne cycloaddition (CuAAC). The structure of the compound was analyzed using Fourier transform infrared spectroscopy (FTIR), 1H NMR, 13C NMR, UV–vis, and elemental analyses. Moreover, X-ray crystallography studies demonstrated that the compound adapted a monoclinic crystal system with the P21/c space group. The dominant interactions formed in the crystal packing were found to be hydrogen bonding and van der Waals interactions according to Hirshfeld surface (HS) analysis. The volume of the crystal voids and the percentage of free spaces in the unit cell were calculated as 152.10 Å3 and 9.80%, respectively. The evaluation of energy frameworks showed that stabilization of the compound was dominated by dispersion energy contributions. Both in vitro and in silico investigations on the DNA/bovine serum albumin (BSA) binding activity of the compound showed that the CT-DNA binding activity of the compound was mediated via intercalation and BSA binding activity was mediated via both polar and hydrophobic interactions. The anticancer activity of the compound was also tested by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay using human cell lines including MDA-MB-231, LNCaP, Caco-2, and HEK-293. The compound exhibited more cytotoxic activity than cisplatin and etoposide on Caco-2 cancer cell lines with an IC50 value of 16.63 ± 0.27 μM after 48 h. Annexin V suggests the induction of cell death by apoptosis. Compound 3 significantly increased the loss of mitochondrial membrane potential (MMP) levels in Caco-2 cells, and the reactive oxygen species (ROS) assay proved that compound 3 could induce apoptosis by ROS generation.


Synthesis of 2-hydroxy-4-(prop-2-yn-1-yloxy)benzaldehyde
2,4-dihydroxybenzaldeyhde and potassium bicarbonate (10 mmol) were suspended in acetonitrile (30 mL) and refluxed for 45 min.Propargyl bromide (15 mmol) was added and the reaction mixture was refluxed for 30 h.When the completion of the reaction was confirmed by TLC, the reaction mixture was filtered while hot and the solvent was removed by rotary evaporation.The remaining black solid in the flask was extracted three times with chloroform and 3M HCl solution.After extraction, the combined organic phases were dried with anhydrous NaSO 4 , filtered and a brown solid was obtained after removing the solvent.The product was purified by silica gel column chromatography by eluting with a mixture of 10% ethyl acetate in n-hexane.Evaporation of the purified extract yielded pure product.The absorption titrations were carried out by adding increasing amounts of DNA (0-100 μM) to a solution of the compound at a fixed concentration (50 μM) contained in a quartz cell.The absorption spectra were obtained by adding the equal amount of CT-DNA to both reference and sample solutions to eliminate the absorbance of CT-DNA.After each addition, the reaction mixture was allowed to incubate for 5 minutes before the absorption spectrum was recorded.
The intrinsic binding constant K b of compound was calculated by using Wolfe-Shimmer equation 5 , Where [DNA] is the concentration of DNA in base pairs, ε a , ε f , and ε b correspond to A obs /[compound], the extinction coefficient for the free compounds, and the extinction coefficient for the compounds fully bound with DNA.In plot of the intrinsic binding constant K b is given by the ratio of the slope to y-intercept.The standard Gibbs free (ΔG) of compound bound to CT-DNA was obtained using the Van't Hoff equation.

Fluorescence spectral studies
The fluorescence quenching experiments were performed for additional support for binding of compound to CT-DNA.ethidium bromide (EB) is a well-known fluorescent probe for DNA S8 moiety and used in evaluation of the interaction mode between a compound and CT-DNA. 6In buffer solution, EB shows low fluorescence intensity, whereas the fluorescence intensity dramatically enhanced by treatment of EB (5 μM) with CT-DNA (25 μM) for 2 h at 24 °C.
Further, the compound in different amounts of concentrations (0-150 μM) were added to the EB+CT-DNA solution.The fluorescence spectra of 200 μL/well solutions in a 96-well plate were recorded with 510 nm excitation and the emission spectra of constant [EB+CT-DNA] with varying the compound concentration were recorded (λ em =500-700).Before recording the spectra, the solutions were thoroughly mixed and incubated for 10 minutes at room temperature.
The quenching efficiency of the compound was analysed using the Stern-Volmer equation.
where I o and I are the emission intensities of CT-DNA+EB complex in the absence and each addition of compound, respectively and [Q] is the concentration of quencher.The Stern-Volmer (quenching) constant, K sv was determined from the slope of the linear plot of To have an insight into the kinetics of the competitive binding process, the bimolecular quenching rate constant, k q values were also computed using the Stern-Volmer equation.K SV = k q τ 0 where τ 0 is the average fluorescence lifetime of the CT-DNA+EB complex in the absence of the quencher and its value is 23 nanoseconds at room temperature. 7Scatchard plots also gave the binding constant K bin as determined from the fluorescence titration using Scatchard equation.

log(I o -I) / I = logK bin + n log[Q]
where n is the number of binding sites per nucleotide. 8

Fluorescence Spectral Studies
Quenching of fluorescence emission of BSA by compound was performed in order to determine its binding constant.Stock concentration of BSA (5 μM) was prepared in 10 mM Tris-HCl buffer at pH = 7.4.The emission spectra of 5 μM BSA solutions in the absence and presence of compound of concentration ranging from 0 to 8 μM were acquired at room temperature.The

Figure S8 .S7 4 .
Figure S8.Annexin V and PI staining of untreated Caco2 cells and cells treated for 48 h with compound 3 at different drug doses (7.5, 15 and 30 μM).Viable cells are Annexin-and PI-.Necrotic cells are Annexin-and PI+.Early apoptotic cells are Annexin + and PI-.Late apoptotic cells are Annexin + and PI +.