A Schiff base of 2-((E)-(2-amino-5-methylphenylimino)methyl)- 5-(difluoromethoxy)phenol and its applications on fluorescent chemosensor for selection of Mg2+ ion, molecular docking, and anticancer activity

A novel Schiff base of 2-((E)-(2-amino-5-methylphenylimino)methyl)-5-(difluoromethoxy)phenol (R) was synthesized and characterized by FTIR, 1H&13C-NMR, and mass spectrometry. The receptor turned yellow and then green in the presence of Mg2+ molecules, with the intervention of different metal ions. The selectivity and sensitivity of Mg2+ ion caused the maximum fluorescence emission intensity at 524 nm, with an excitation wavelength at 378 nm. Further experiments confirmed that receptor R binds with Mg2+. Job’s plot conforms to a 1:1 stoichiometry complex formation. The strong fluorescence is owing to the photoinduced electron/energy transfer effect. The receptor was recovered by an ethylenediaminetetraacetic acid titration and the emission intensity also returned to a value equivalent to the unbound ligand. protein data bank: 4J96 was used for the molecular docking of receptor R. The cytotoxicity effect treatment was carried out by increasing the concentration of HeLa cells to predict IC50 value. The highest occupied molecular orbital/lowest unoccupied molecular orbita energy gap calculated and compared between R results as 3.48 eV and R-Mg2+ alpha and the beta value calculated a low energy value at 2.27 eV.


INTRODUCTION
The most important magnesium (Mg 2+ ) ion, which is extremely abundant, is essential for a number of cellular processes, such as cell death, cell proliferation, biochemical reactions, enzymedriven reactions, stabilization of DNA conformation channel regulation, and signal transduction (Liu et al., 2018). A human body absorbing excess magnesium ions has side effects like nausea and diarrhea. The excess Mg 2+ ions also results in age-related diseases and nervous disorders. It is with great interest that fluorescent chemical sensors are designed to detect Mg 2+ ions (Tamil Selvan et al., 2018). For instance, certain low levels of Mg 2+ ions are related to the improvement of Parkinson's disease. As a consequence, the significance of Mg 2+ in many physiological conditions necessitates a new chemical apparatus to investigate Mg 2+ ion in both health and disease (Treadwell et al., 2018). Recently, numerous families of Mg 2+ response with fluorescent chemosensors containing moietybased ligand groups, including diketone, calyx, arene, crown ether, porphyrin, and (C = N) imine-like aromatic, have been formed in the region (Zhao et al., 2014). The presence of magnesium ions in mineral water supply is relatively quantitative. This is usually carried out by the ethylenediaminetetraacetic acid (EDTA) complexometric titration due to the presence of Mg 2+ in drinking water. On the contrary, this process is laborious, sensitive, and time-consuming, as well as it contains errors due to interference from numerous metal ions. However, the needed improvement and sensitivity of analytical types for Mg 2+ ion is not so effective in this regard (Men et al., 2015). Notably, fluorescence-based chemosensors provide real-time answers to a variety of lists that allow for complex measurements and easy handling. Significant efforts have, in recent times, been made to find new suitable fluorescent samples with all the desired properties, such as high sensitivity, selectivity, low-cost synthesis, aqueous solubility, and simple preparation (Orrego-Hernández et al., 2016). The key advantage of fluorescent chemosensors, "turn-on" sensors compared to "turn-off" sensors, is that it clearly detects a low concentration than a "black" setting, which decreases the probability of falsepositive signals and improves sensitivity, depending on the number of studies carried out. Moreover, colorimetric chemosensors have attracted much attention because it allows "naked-eye" detection, which is not complicated and expensive. These behaviors contribute to quantitative and qualitative information (Li et al., 2014). A variety of signaling mechanisms targeting the optical detection of different organisms were developed and implemented. The photoinduced electron/energy transfer (PET) signaling pathways consist of excimers and exciplex formations (Kim et al., 2002), the excited state of intramolecular and intermolecular proton transfers (Beer et al., 1998), metal-ligand charge transfer (MLCT) Kim and Yoon, 2002), intramolecular charge transfer (Xu et al., 2005), and closed-queuing network isomerization (Wu et al., 2007). The fluorescence sensors designed for metal ions have a huge significance these days. PET has been broadly used as a tool in such aspects. Many compounds contain amine and amide moiety or result in fluorescence quenching via PET by electron donating and accepting sites within the molecule. These group categories are called PET process chemosensors for "on-off" signals. Fluorescence quenching via PET due to structural flexibility (there is an unregulated torsional rotation between carbon-carbon bond which is covalently linked to two units) in these sensors is responsible for intramolecular rotation wherein a charge transfer to an excited single state is swiftly disabled (Gupta et al., 2018). Schiff base ligands, also called as "privileged ligands", are obtained by the condensation of aldehydes and amines prepared by a well-known procedure that is effective for various metal ions in different oxidation states. The efficiency of the Schiff base-metal complex is realized by their use and application across a range of fields, from catalysis to medicine and fluorescent chemosensors (Gao et al., 2018). Schiff base methods of binding metal complexes are briefly studied in the literature (Abu-Dief and Mohamed, 2015) due to their antioxidant activities (Li and Yang, 2009;Liu and Yang, 2009;Tarafder et al., 2001), antitumoral properties (Adsule et al., 2006;Jana et al., 2016), antimicrobial activity (Parashar et al., 1988;Raman et al., 2001), catalytic activities (Jia and Li, 2015;Mouri et al., 2010), and photophysical properties (Cozzi et al., 2003;Ziółek et al., 2008).
In this article, Scheme 1 shows the synthesis of compound R of 2-((E)-(2-amino-5-methylphenylimino)methyl)-5-(difluoromethoxy)phenol and mentions its use as a chemosensor for high sensitivity and selectivity toward Mg 2+ ions in different metals envisaged in this experiment. Magnesium was proposed by titration with additional metal ions and theoretical calculations. Moreover, the ligand R complexation with Mg 2+ ion was confirmed by EDTA titration and the cytotoxicity activity is related to molecular docking studies.

Material and measurements
Most of the chemicals were bought from Sigma-Aldrich and were kept in the laboratory without further purification. Selective study of specific metal ions, including Ni 2+ , Fe 3+ , Cu 2+ , Ca 2+ , Co 2+ , Mg 2+ , Sn 2+ , Ti 2+ , Pb 2+ , Zn 2+ , Cd 2+ , Hg 2+ , Mn 2+ , Al 3+ , and Ag + , as well 1 H& 13 C-NMR quantification were carried out on a Bruker spectrometer ( 1 H& 13 C-NMR for 400 and 100 MHz), and the solvent used was dimethyl sulfoxide (DMSO). The UV-vis spectra were achieved using a UV-2450-Shimadzu spectrometer at room temperature by using the liquid compound mixing solution in distilled water and ethanol. The fluorescence emission spectra were determined on a spectrometer from Perkin Elmer LS45.

Cell culture and cytotoxicity assays
HeLa cells were grown with 10% fasting blood sugar in Dulbecco's Modified Eagle's Medium. All the cells were treated with an antibiotic solution (0.1 mg ml −1 streptomycin, 0.25 mg ml −1 amphotericin B, and 100 units/ml penicillin) and grown under normal conditions at 37°C in cell culture (95% humidity, 5% CO 2 , respectively). In the culture media, the cells were seeded into 96-well plates at a density of 4 × 10 3 cells per well, and then compounds of 0, 7.8, 15.6, 31.2, 62.5, 125, 250, 500, 1,000, and 2,000 μM (final concentration) were added. The cells were then incubated in an atmosphere of 5% CO 2 and 95% air for 24 hours, at 37°C. Enzyme-linked immunosorbent assay was used to measure the absorbance of the cells.

Molecular docking
The protein was first prepared using the protein preparation wizard and the docking experiments within Maestro 9.5 were conducted using Schrodinger's Glide program. Ligand preparation was carried out using LigPrep, followed by the minimization and optimization of the force field for liquid simulations (Dodda et al., 2017). The G score was measured in kcal/mol, which included hydrogen bonds, internal strength, hydrophobic interactions, stacking interactions, ligand-protein interaction energies, and square deviation and desolvation of the center. The extra-precision (XP)'s glide module visualizes the specific interactions between ligands and proteins. Grids were developed using version 9.5 of Schrodinger's Glide following a standard procedure.

Theoretical calculation
The geometric optimization of receptor R and complex formation of Mg 2+ provided lowest unoccupied molecular orbita (LUMO) and highest occupied molecular orbital (HOMO) energy gap values calculated by the density functional theory (DFT) using the B3LYP/6-31G level of the Gaussian 09 program. The receptor R and Mg 2+ bind the complexes of chemosensor activity of alpha and beta value calculations, while the complexes are formed using the B3LYP/6-31G basis set of Mg 2+ ions.
The UV-vis spectra of probe R showed major absorption peaks at 378 nm that can be attributed to π-π* transition. Although various metal ions of 0.5 equiv. concentration were used, the interference of Mg 2+ ions with probe R and the absorption intensity at 378 nm increased immediately and moved to a new absorption band at 415 nm. As shown in Fig. 2, solution R is colorless to light yellow, when seen by the naked eye. This is due to the presence of Mg 2+ , which moves the absorption band in an upward direction at 378 nm. As shown in the UV-vis absorption spectrum (Fig. 3), the addition of Mg 2+ ions (0.5 equiv.) increased the absorption strength from 378 nm to another peak at 415 nm, with two isosbestic points at 337 and 396 nm.

Fluorescence sensing of Mg 2+
The selectivity of receptor (sensor) R (10 µM) for various metal ions, such as Ni 2+ , Mn 2+ , Fe 3+ , Co 2+ , Ca 2+ , Mg 2+ , Sn 2+ , Cu 2+ , Ti 2+ , Pb 2+ , Zn 2+ , Cd 2+ , Hg 2+ , Al 3+ , and Ag + (50 µM) was investigated with CH 3 CH 2 OH:H 2 O solution (1:4, v/v, 20-mM HEPES buffer, pH 7.0) at room temperature. As shown in Figure 4, the fluorescence spectrum from 490 to 560 nm was obtained with an exciting value of 378 nm, and probe R (10 µM) exhibited an enhancement from weak emission intensity of fluorescence around at 524 nm due selectivity and sensitivity of Mg 2+ ions in the titration. Concomitantly, all the remaining metal ions showed no response for any emission changes. Probe R (10 µM) solution was induced by Mg 2+ to change from blue to green, which determined the existence of Mg 2+ as shown in Figure 5, under fluorescence light.
Furthermore, with the addition of Mg 2+ ions of 5 equiv., the fluorescence emission intensity at 524 nm gradually increased as shown in Figure 6. The weak fluorescence intensity at 524 nm is assumed to be the intramolecular charge transfer of PET phenomenon caused by the intramolecular H bonding atom between C=N and a hydroxyl group. As shown in Scheme 2, the fluorescence emission of the free R was feeble due to the lone pair electron of the N atom of the PET mechanism. The binding of R-Mg 2+ complex resulted in the inhibition of PET and -CH=N isomerization at the excited state, which remarkably increased the fluorescence emission intensity.
The stoichiometry between R and Mg 2+ was determined using Job's plot with CH 3 CH 2 OH/H 2 O solution (1:4, v/v, 0.01 M HEPES buffer, pH 7.0) as shown in Figure 7. The fluorescence intensity at 524 nm calculated by a gradual increase in the maximum emission intensity of tested Mg 2+ ions with 0.5 µM mole fraction indicated the 1:1 complex of the binding mode of R and Mg 2+ . The result, further confirmed by the molecular peak ion at m/z 292.00, was assumed as [R-H + +Mg 2+ ] + (calculated m/z 289.00) in the ESI-Mass spectrum.

Effect of pH and binding reversibility
The pH titration implied as the optimal pH to sense metal ions and the spectrum is shown in Figure 9. The fluorescence emission of R was examined in the overall pH range, and from the figure it is evident that there is no change in the intensity and the existence of Mg 2+ ion enhancement intensity of fluorescence at 524 nm in maximum at pH 7.0. In this experiment, pH 7.0 was used for all measurements. EDTA (10 μM) solution was used to test the reversibility of sensor R (10 μM) binding with Mg 2+ (50 μM) to validate novel chemosensors for practical use. Due to the binding receptor R and Mg 2+ complexation, as shown in Figure 10, the turn-on fluorescence intensity was observed at 524 nm. The test revealed a strong R-Mg 2+ complex fluorescence strength due   to the presence of Mg 2+ ion. Although R-Mg 2+ −EDTA complex did not promote high intensity, it showed the reversibility of receptor R to Mg 2+ ions.

The cytotoxicity activity
With the HeLa cell line, the cytotoxicity effects of R can be estimated. The IC 50 values of this assay for the HeLa cells were obtained by conducting the cell viability experiment by treatment with compound-varying concentrations. Figure 11 shows the cytotoxicity analyses of the HeLa cell line with specific ligand preparation concentrations. HeLa cells were treated with ligand R for about 36 hours (Figure 11b and c), and the empty cells can be seen in Figure 11a. The ligand R was used to treat HeLa cells with respect to different concentrations to analyze the cytotoxic activity.
The cell viability (%) of the ligand and its various concentrations (Dhineshkumar et al., 2019) with its respective IC 50 values are given in Table 1. Plots of the HeLa cell line viability and the concentration of the ligand treatment are shown in Figure 12.
The IC 50 values of the inhibited cells take up much time. From this experiment, the key recovery mechanism for unaccepted aspects effects the rationalization of the IC 50 values with short time intervals.

Molecular docking
In an attempt to understand our compound's binding mode, the ligand was docked by the Protein Data Bank with the crystal structure of the FGF receptor two kinases [protein data bank (PDB):4J96]. The protein was first prepared using the protein preparation wizard, and the docking experiments were conducted using the XP mode of the Schrodinger Glide program (Maestro 9.5). The 2D diagram interactions of active compounds are shown in Figure 13. From the docking studies, we find that the compound showed the highest G scores comparatively with others. Figure 14 shows a composite 3D model in the complex.

Theoretical studies
In order to gain an insight into the binding mode of receptor R fluorescence-sensing mechanism against Mg 2+ , DFT calculations at the B3LYP/6-31G level using Gaussian 09 software were carried out to recognize the optimized structures of R and its Mg 2+ complex.The optimized molecular structure of probe R and R-Mg 2+ exhibited in the ground state was computed by the B3LYP calculation with a 6-31G (d,p) basis set as shown in Figure 15. The electron density distributions and orbital energy transfer from the HOMO and LUMO investigated the probe R and R-Mg 2+ complex.
Although sensor R and the HOMO electron densities were primarily located on the hydroxyl group and replaced the   amine group, the transfer of 4-(2,2-difluoroethyl)-2-hydorxy benzaldehyde group occurred through LUMO orbital energies. It was an electron transfer that contributed to the energy level from HOMO to LUMO. However, the imine group of C=N isomerization and PET of probe R may also be responsible for fluorescent nature in the coordinate complex 1:1 ratio (Dhineshkumar et al., 2018).
Optimized theoretical calculations were conducted to calculate the coordinated sensor R to R-Mg 2+ complex and the energy levels of both the LUMO and HOMO energy gap values ( Figure 16).
The sensor R showed that HOMO (5.40 eV) and LUMO (3.12 eV) orbital energies were completely involved in working together in ligand-to-ligand charge transfer with a bandgap of 3.48 eV. The mechanism of 1:1 stoichiometry of equal binding with R-Mg 2+ predicts that HOMO and LUMO can be assumed as alpha and beta, HOMO Alpha = 3.12 eV and LUMO Alpha = 1.69 eV with calculated energy gap at 1.43 eV, while HOMO Beta = 5.40 eV and LUMO Beta = 1.68 eV with calculated energy gap at 3.72 eV. The metal complex formed with R-Mg 2+ exhibited an alpha value with a low energy gap and the beta value exhibited a high energy gap. The R-Mg 2+ complex bound by the imine group (-C=N) and hydroxyl group had a strong bond formation due to elimination of the PET process on turn-on fluorescence. As a result, sensor R exhibited a electronegativity attractive value of 3.101 eV. While electrophilicity      Table 2.

Charge distribution
Gaussian program of Mulliken method using a B3LYP/6-31G level calculation was used to calculate the charge distribution of the molecule. In this molecule, every atom charge distribution calculation was delineated. The bond length between atoms are shown by increasing and decreasing the distribution of negative and positive charges, and Mulliken atomic charge values and plots are shown in Figure 17. The Mulliken value with the negative charge for the high and low carbon groups, such as C16, C15, C13, C11, C12, C10, C9, C7, C6, C5, C4, C3, C2, and C1 atoms, and the N8, O14, F15, and O16 atoms in compound R, are also shown. The hydrogen atoms were split with fluorine atoms and linked to oxygen atoms, while all the remaining hydrogen atom were split with their positions. Mulliken charge was based on estimating calculation partial atomic charges from the computational chemistry in working Mulliken population analysis (Arockia doss et al., 2020; Dhineshkumar et al., 2020).  Figure 16. Optimized structural geometry of chemosensors and its complexes R and R-Mg 2+ from left to right.

CONCLUSION
In summary, the Schiff base of 2-((E)-(2-amino-5methylphenylimino)methyl)-5 (difluoromethoxy)phenol was synthesized and investigated by analytical techniques. The sensitivity and selectivity of Mg 2+ ions due to fluorescence emission intensity at 524 nm was ascertained by other metal ions, with an excitation wavelength at 378 nm. The stoichiometry of 1:1 complex was recognized for mole fraction of 0.5. The receptor R and the existence of Mg 2+ ions in the R-Mg 2+ complex was confirmed by the turn-on fluorescence mass spectra and EDTA titration. Furthermore, different pH's used confirm the optimum pH. The cytotoxicity effect of inhibition strong force to HeLa cells was analyzed with the compound. Molecular docking of PDB: 4J96 protein was also studied for this compound.

AUTHOR CONTRIBUTIONS
CAS and EDK conceptualed, designed, and finalized Schemes 1 and 2. CAS, EDK, and MY carried out the research work and wrote the article. All the authors read and agreed to published this version of the manuscript.

CONFLICT OF INTEREST
All the authors declare that there are no conflicts of interest.

FUNDING
This research received no external funding.