Loraine Youssef

Stephanie Roualdes

Joelle Bassil

Mirvat Zakhour

Vincent R Rouessac

Claude Lamy

Michel Nakhl

Stéphanie Roualdès

Joëlle Bassil

photo-electrochemical studies in a single compartment cell for hydrogen generation by solar water splitting

Keywords: Anatase, Nitrogen doping, PECVD, Plasma power, Photo-electrochemistry, Hydrogen Fig

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Effect of plasma power on the semiconducting behavior of low-frequency PECVD TiO2 and nitrogen-doped TiO2 anodic thin coatings

Introduction

The 'Solar-to-Hydrogen' approach has been widely dis * Stéphanie Roualdès stephanie.roualdes@umontpellier.fr cussed over the past twenty years, especially with the increase of the negative side effects derived from the excess Extended author information available on the last page of the article Vol.:(0123456789 usage of fossil fuels [START_REF] Ribeiro | Fossil fuel energy impacts on health[END_REF][START_REF] Princen | The fossil fuel problem[END_REF][START_REF] Holtsmark | A comparison of the global warming effects of wood fuels and fossil fuels taking albedo into account[END_REF][START_REF]The hidden costs of fossil fuels[END_REF]. In fact, the main reasons behind hydrogen investigation as an alternative green fuel are its availability and its exceptional energetically efficient combustion generating only water as a by-product [START_REF] Chauhan | Fundamentals and use of hydrogen as a fuel[END_REF]. The problem remains in the fact that such a compound could not be found naturally as H2 but is chemically bounded to other elements like carbon or oxygen. For that, energy supply is necessary in order to split H2 combining products, thus producing hydrogen as a fuel from a primary source.

Many works have been performed in order to find convenient hydrogen primary sources with good gas yields. As a first trial, thermochemical processes have been presented for hydrogen production from fossil fuels. J. Jechura presented the process of H 2 generation from natural gas by steam methane reforming (SMR) [START_REF] Jechura | Hydrogen from natural gas via steam methane reforming (SMR)[END_REF]. A range of 95-98% hydrogen purity was obtained but, even though the process is cheap, the SMR-integrated systems still require high maintenance. Coal gasification has also been reported in various studies [START_REF] Raggio | Coal gasification pilot plant for hydrogen production[END_REF][START_REF] Deiana | Coal gasification for hydrogen production using nuclear energy coal gasification for hydrogen production using nuclear energy[END_REF][9][START_REF] Lan | Hydrogen production by catalytic gasification of coal in supercritical water[END_REF]. Still such techniques require highenergy initial supplies, they generate carbonated compounds particularly carbon dioxide ( CO2) as by-products and the produced H 2 is obtained with low purity percentages. Biological processes, taking advantages of the microorganisms' capacities in biomass digestion for H2 release, have also been investigated in the field of hydrogen generation [START_REF] Van Niel | Biological processes for hydrogen production[END_REF][START_REF] Orozco-Pulido R ; Loubette | Hydrogen production from biomass by integrating thermo-chemical and biological processes 13[END_REF][13]. Despite the fact that these investigations are very new and present good gas yield perspectives, the processes preparation and manipulation conditions remain delicate and sensitive [14]. Further studies should be done in order to improve many aspects of such processes, including low-cost materials and highefficiency microbes.

The previously cited investigations have been based on compounds that initially do not include hydrogen but whose reaction generates this energetic gas as a by-product. Then, the concept of 'Hydrogen generation by water splitting' emerged from the fact that H2O, the most available compound on earth, could be dissociated into its elements, oxygen and hydrogen. For that, water electrolysis using external electrical supply has been studied and optimized under several experimental conditions in order to conclude on the best materials performances [14 -18].

In a water-splitting system, oxidation occurs on the anode surface (linked to the positive pole of the external power source) generating oxygen, and reduction occurs on the cathode surface (linked to the negative pole of the external source) generating hydrogen. The most well-known watersplitting experiment, using external light source on semiconductor surface, was performed in 1972 by Fujishima and Honda [START_REF] Fujishima | Electrochemical photolysis of water at a semiconductor electrode[END_REF]. A UV light photo-active anode was used to split water into oxygen-generating protons traveling through the solution to reach the cathode surface. There protonic reduction occurred and hydrogen was produced. This approach was a tremendous discovery that opened many perspectives on further studies about 'photoelectrochemistry' or 'synthetic photo-synthesis' [START_REF] Kennedy | Photo-oxidation of water at a-Fe2O3 electrodes[END_REF][START_REF] Butler | Photoelectrolysis and physical properties of the semiconducting electrode WO2[END_REF]. However, the photo-anode efficiency should still be optimized and enhanced to fill in the major part of the solar spectrum. The semiconductor used in this study was TiO2 whose behavior is well described in the following theoretical part.

In a previous work by our group aiming at developing anode materials for solar water-splitting systems [START_REF] Youssef | Optimization of N-doped TiO2 multifunctional thin layers by low frequency PECVD process[END_REF], TiO2 thin layers with competitive microstructural and photocatalytic properties have been prepared using a onestep low-frequency plasma-enhanced chemical vapor deposition (PECVD) process, operating at temperature as low as 350 °C. Single-oriented pure and N-doped anatase films have been synthesized using titanium isopropoxide (TTIP) as a precursor, and NH3 as a doping agent. Optimized PECVD conditions have enabled to obtain homogeneous micro-columnar porous thin films with thicknesses close to 500 nm. Depth-profiling XPS analyses have proved the nitrogen incorporation into T iO2 lattice after ammonia introduction in the deposition chamber. As another proof of N-doping, Raman and XRD peaks shifting have been observed. Such thin films have been demonstrated to be efficient photocatalytic materials whose activity region can be tailored from UV to visible region by adjusting the proportion of doping agent in the plasma phase.

The present work is a sequel to the optimized TiO2 (nondoped) and N-TiO2 (nitrogen doped) low-frequency PECVD thin films previously developed [START_REF] Youssef | Optimization of N-doped TiO2 multifunctional thin layers by low frequency PECVD process[END_REF]. The structural, optical, and photo-electrochemical properties of slightly doped (implementing 5 Pa as N H3 partial pressure in the PECVD chamber) layers are investigated in the dark, under UV, and under visible light and compared to the nondoped TiO2 synthesized at two different plasma discharge powers.

State-of-art on the photo-electrochemical behavior of semiconductors

The overall water-splitting reaction is summarized as follows: H2O → H2 + ½ O2. For ideal electrolysis systems, for example, those using highly conductive metals as electrodes, a half mole of O2 and a mole of H2 are produced per mole of water. However, for systems using semiconductors or organic-based compounds, yield limitations generally appear due to the use of a low conducting electrode [START_REF] Fountaine | Efficiency limits for electrochemical water splitting[END_REF][START_REF] Duan | Ultrathin metal-organic framework array for efficient electrocatalytic water splitting[END_REF][START_REF] Swierk | Metal-free organic sensitizers for use in water splitting dye sensitized photoelectrochemical cells[END_REF]. Still, water electrolysis requires an external electrical power source in order to drive the reactions. One interesting approach for water splitting, derived from electrolysis, is photo-electrolysis of water. In such a process, water dissociation into its own elements is induced by solar energy on a single photo-electrode surface. This device eliminates the need for a separate power generator, reducing overall costs and increasing efficiency. The usual suggested hypothesis was the possibility of 'electrodeless' water photo-splitting. Cohen explained that water is completely transparent to a certain light range and could never be directly dissociated under energy sources of wavelengths higher than 190 nm. This means that electrode materials, properly responding to common light sources, are required [START_REF] Cohen | Studien über photochemische Gleichgewichte IV Das Ligchtgleichgewicht Knallgas-Wasserdampf[END_REF]. Semiconductors are widely known for their reaction abilities after absorbing a light source of wavelength smaller than their band gap, i.e., the energy difference between the filled valence band (VB) and the empty conduction band (CB).

When the material absorbs radiations of wavelengths at least corresponding to their band gap energy (according to the formula: Eg = hc/λ, E g is the band gap energy in eV, h is the Planck constant (6.62•10 -34 J s), c is the light velocity (3•10 8 m s -1 ), and λ is the wavelength in nm; reductive electrons (e -) are promoted from the Highest Occupied Molecular Orbital (HOMO) to the lowest unoccupied molecular orbital (LUMO). This generates oxidizing holes ( h + ) in the valence band leading to electron/hole pairs. The limiting photoactivity factor is the lifetime of the electronhole pairs. Increasing this latter factor leads to a higher material efficiency under light because of enhancement of redox reactions rate. According to literature [START_REF] Chovet | Cours de physique des semiconducteurs 28[END_REF]28], semiconductors have two different behaviors (p-type or ntype) in contact with a solution depending on the energy edges depletion on the material surface (Fig. 1).

An n-type semiconductor (Fig. 1a) has an electron excess near the conduction band. The level depletion derivative force acts in such a way to transport electrons into the electronic back contact through the external circuit towards the facing counter-electrode (reduction), while holes are preferably emitted into the solution. These holes are able to oxidize species in the electrolyte and the semiconductor behaves as a 'photo-anode.' A p-type semiconductor (Fig. 1b) has a hole excess near the valence band that are transported into the back contact by the derivative force while electrons pass through the solution where they can reduce species in the electrolyte. Thus, such a semiconductor is qualified of a 'photo-cathode.' These behaviors are observed on both intrinsic (non-modified by external species) and extrinsic (modified by metals or chemically doped) semiconductors. The latter explanations are important to understand the electrochemical systems proposed and studied in the literature.

The present work is mainly focused on a configuration in which a light activated photo-anode oxidizes water into oxygen (H2O + 2h + → 2H + + ½ O2). The generated protons diffuse through the H 2SO4 electrolyte while the electrons flow through the external circuit. The protons reduction into hydrogen occurs at the glassy carbon electrode surface ( 2H + + 2e -→ H2). The present work is more a study in the field of photo-water splitting or photo-electrochemical water molecule dissociation.

The choice of semiconductors as electrodes is not random [29]. These materials should have enough large band gaps to split the water molecule into its own elements hydrogen and oxygen. Numerous studies on photo-water splitting have been developed on common semiconductors. The most important point is that the semiconductor band gap edges must fill in the water oxidation/reduction levels which are theoretically 1.23 eV per electron (oxidation) and 0 eV (reduction) (vs. RHE, reversible hydrogen electrode). This is the reason why silicon is limited for hydrogen generation from water even though its band gap could cover a major part of the solar spectrum (1.1 eV-2.4 eV).

Here, a remark should be done considering that confusions occur generally between three concepts in the 'photoactive materials' field. The three concepts are 'photovoltaic,' 'photo-catalysis,' and 'photoelectrochemistry.' In fact, 'photovoltaic' process is converting solar light into electricity. This concept is applied to Solar Cells (SC) manufacture and elaboration [START_REF] Würfel | From basic principles to advanced concepts 31[END_REF]31].

'Photo-catalysis' is more like accelerating a reaction under light effect. Photocatalytic effect is actually applied for organic molecules degradation and water purification using economical activation sources such as natural light [START_REF] Ajmal | Principles and mechanisms of photocatalytic dye degradation on T iO2 based photocatalysts: a comparative overview[END_REF][START_REF] Tayade | Photocatalytic degradation of dyes and organic contaminants in water using nanocrystalline anatase and rutile TiO2[END_REF][START_REF] Khataee | Photocatalytic degradation of organic dyes in the presence of nanostructured titanium dioxide: Influence of the chemical structure of dyes[END_REF]. 'Photo-electrochemistry' or 'Photo-galvanic' process is the study of complex chemical reactions induced or enhanced by light irradiation [35]. This is the field that interests our study in this paper.

Figure 2 is a schematic representation of possible semiconducting materials with their band edges for watersplitting application (from reference [START_REF] Liu | Thermodynamic and kinetic analysis of heterogeneous photocatalysis for semiconductor systems[END_REF]). For hydrogen generation by water splitting, a compromise should be made between a gap energy corresponding to the required light wavelengths domain (ultraviolet, visible, and infrared) and band gap edges well positioned according to the water oxidation/proton reduction redox couples. Taking the example of ZrO2 band edges, it could easily oxidize water into oxygen and induce hydrogen generation only when irradiated with short wavelengths (for example, 5 eV: λ ≤ 250 nm) situated in the UV range. In the case of silicon (Si), the wavelength absorption domain is enlarged (λ ≤ 1130 nm) but this material could not be used to produce hydrogen by water splitting due to its valence band edge situated above the O 2/H2O oxidation energy level.

In the present work, T iO2 has been used as a photo-anode semiconductor. As it can be noticed in Fig. 2, titanium dioxide presents good valence band position relatively to water oxidation redox couple and it has an average gap value (λ ≤ 414 nm).

3 Experimental section

Pure and Nitrogen-doped anodic layers synthesis procedure

Both TiO2 and N-TiO2 layers were deposited by a lowfrequency PECVD process according to the procedure previously described and optimized in a paper by our group [START_REF] Youssef | Optimization of N-doped TiO2 multifunctional thin layers by low frequency PECVD process[END_REF]. The TiO2 chemical precursor is Titanium Tetra-isopropoxide TTIP (purity ≥ 97%) from Sigma Aldrich. The doping agent is anhydrous ammonia NH3 supplied by a gas cylinder from Gazechim®. The partial pressures of vapors and gases in the PECVD chamber during the deposition step are 22.5 Pa for Ar + TTIP (Ar being used as a carrier gas), 17 Pa for O 2 (O2 being used as an oxidizing agent), and 5 Pa for N H3 (added in the plasma reactor for doping purpose). Two sample series were prepared: the series at low plasma discharge power (64 W) and the series at high plasma discharge power (100 W). 64 W has been chosen as the same low input power as that optimized in a previous study by our group dedicated to T iO2 deposition by radio-frequency PECVD [START_REF] Zhou | Novel photocatalytic TiO2-based porous membranes prepared by plasma-enhanced chemical vapor deposition (PECVD) for organic pollutant degradation in water 38[END_REF] and 100 W has been selected as the highest input power delivered by the lowfrequency generator used in this study. The deposition time was 20 min and the deposition temperature was 350 °C for all the samples, in a one-step process.

In this paper, TiO2 and N-TiO2 films prepared at 64 W and 100 W will be named 64W-TiO2, 64W-N-TiO2, 100W-TiO2, and 100W-N-TiO2, respectively.

Three different kinds of supports were used: single crystalline silicon (100) (from the Monsanto Electronic Materials Company, Boron-doped type p-Si (100), resistivity: 1-50 Ω), nickel foil (purity ≥ 99.9%) for structural characterizations, and a porous carbon substrate (commercial E-Tek® conductive black cloth for electrochemical characterizations).

Morphological and structural characterization techniques

In the previous work by our group [START_REF] Youssef | Optimization of N-doped TiO2 multifunctional thin layers by low frequency PECVD process[END_REF], the morphology of plasma layers had been observed for materials deposited on Si substrates. Here, the surfaces and cross sections morphologies were investigated on layers deposited on Ni (for crystal Fig. 2 Band gap energy edge position according to water oxidation/ proton reduction redox couples for thirteen common semiconductors [START_REF] Liu | Thermodynamic and kinetic analysis of heterogeneous photocatalysis for semiconductor systems[END_REF] morphology observation) and E-Tek® (for electrochemical functional studies) supports by scanning electron microscopy (SEM) using a Hitachi S4800 microscope.

The density of plasma layers deposited on Si was determined by X-ray reflectometry (XRR) using a Siemens/Bruker D5000 with a CuKα source (λ = 1.54056 Å). Manipulating with the same Si support, ESCALAB 250 from Thermo Electron was used for XPS depth profile correlations and a GES5E spectroscopic ellipsometer from Semilab (wavelength from 250 nm to 1000 nm, incidence angle of 70°) was used to evaluate the layer refractive index and optical properties. The ellipsometry data were simulated and fitted using Winelli2® software.

Electrochemical characterizations in a single compartment photo-electrochemical cell

The electrochemical characterizations of plasma-layered electrodes were performed in a single compartment Teflon® cell equipped with a 27-mm diameter transparent quartz window (Fig. 3). A three electrode configuration was implemented involving: a plasma layer deposited on an E-Tek® carbon cloth (in the form of a disc of 10 mm diameter making a constant surface of 0.785 cm 2 ) as the photoanode (working electrode), a commercial glassy carbon material (0.7 cm x 2 cm) as the cathode (counter-electrode), and a commercial Ag/AgCl (3 M KCl) from SI Analytics® (0.197 V vs. RHE) as the reference electrode. The three electrodes were immersed in a 0.5 M H2SO4 liquid electrolyte solution (prepared from a 100% H2SO4 solution from Merck Millipore® in 18 MΩ pure MilliQ® water from Millipore®). In order to ensure good electron collecting through the external circuit, both photo-anode and cathode were tightly connected to gold wires (Goodfellow ≥ 99.9% from Sigma Aldrich®) that were not immersed in the electrolyte solution in order to avoid metallic interference response. Two different irradiation sources were used: a 75 W (λ = 355 nm) [START_REF] Zhou | Novel photocatalytic TiO2-based porous membranes prepared by plasma-enhanced chemical vapor deposition (PECVD) for organic pollutant degradation in water 38[END_REF] from Eurosolar® and a 150 W filtered (λ ≥ 400 nm) from Van Cliff lamps for UV and visible, respectively. It should be mentioned that since the carbon cloth is not transparent and by that it will absorb the light before reaching the active layer, the plasma film was facing the lamp irradiation which was focused and adjusted to the cell quartz window in order to maintain constant irradiation in UV and visible for all the samples.

The whole cell was covered by aluminum foil during the illumination experiments to concentrate the light and to avoid room light interference when performing experiments in the dark. Lastly, before each electrochemical test, the electrolyte solution was degassed by pure argon bubbling for 40 min in order to get rid of the dissolved oxygen.

Impedance measurements were performed in order to evaluate the TiO2 and the N-TiO2 intrinsic electronic resistance and the electrode/electrolyte interface properties. Electrochemical Impedance Spectroscopy (EIS) was performed with a Solartron® SI 1260 frequency analyzer (in a range of 0.02 Hz-10 5 Hz with 10 mV AC amplitude). Both ZPlot® and ZView® softwares were used to record and fit the experimental data.

In order to investigate the n-type semiconductor behavior and the Oxygen Evolution Reaction (OER), the Open Circuit Voltage (OCV), the Cyclic Voltammetry (CV) at 5 mV s -1 , and the Linear Voltammetry (LV) at 1 mV s -1 were, respectively, recorded using a Solartron® SI 1287 galvanostat-potentiostat. The softwares are CorrWare 2® for experimental data recording and CorrView® for text files collecting.

Results and discussions

Fig. 3 Schematic representation of the single compartment photoelectrochemical cell

Coatings morphology and thickness observations by SEM

In a previous paper by our group [START_REF] Youssef | Optimization of N-doped TiO2 multifunctional thin layers by low frequency PECVD process[END_REF], SEM characterization of films on Si substrate had enabled to observe micro-columnar porous thin films with thicknesses close to 500 nm. Nevertheless, the crystal morphology could not be precisely depicted. This is the reason why dense nickel foils were used as supports in the present study (Fig. 4).

Figure 4a shows the 64W-TiO2 sample on Ni, the (101) facet orientation is clearly observed. Moreover, a dense thin layer starts to grow at the bottom of the upper layer for such a plasma power, while the 100W-TiO2 layer (Fig. 4b) shows a homogeneous columnar growth throughout the thickness. The latter layers expose 'flower-like' (101) facets while sharper and defined ones are exposed for the 64W-TiO2 layers. In Fig. 4c,d, respectively, corresponding to the 64W-N-TiO2 and 100W-N-TiO2 doped samples, the (101) facets coverage with a nitride shell is obviously observed as cited in the works of Cho et al. [38].

The coverage is more observed in the case of layers prepared at 64 W since the exposed facets are dense unlike the 'flower-like' facets of the layers synthesized at 100 W where the nitrided shell could have covered the scattered surface. Table 1 presents the films thicknesses measured by SEM cross section on nickel foil and E-Tek® cloth for the same deposition time of 20 min.

The first observation is that increasing the plasma power from 64 W to 100 W increases the growth rate since higher thicknesses were measured on both substrates at 100 W. The second observation is that lower thicknesses values were obtained on E-Tek® cloth. This could be due either to a hidden thickness by crystals grown inside the cloth or to the fact that metallic nucleation sites on nickel foil are highly activated by plasma and accelerated by the layer surface growth. The third observation is that the growth rate is slowed by doping process, especially on Ni support. In fact, when adding N H3 gas into the plasma phase, reactive species reactions are more favored into the plasma gaseous volume more than on substrate surface. This leads to a competition between species-species (volume) collisions and species-solid (substrate) interactions [START_REF] Guimond | Influence of gas phase and surface reactions on plasma polymerization[END_REF]. This process leads to lower growth rate.

Coatings refractive index, growth simulation, and Nitrogen composition

The layers refractive index and growth simulation were analyzed by ellipsometry measurements on Si wafer. This support is the most suitable for this kind of analysis since it is dense and reflective so the layer-substrate interface could be easily detected by the incident light beam.

According to the literature, the theoretical refractive index for crystalline dense anatase is 2.5-2.6 [START_REF] Nurfani | Effect of Ta concentration on the refractive index of T iO2:Ta studied by spectroscopic ellipsometry[END_REF][START_REF] Liu | A comparative study of amorphous, anatase, rutile, and mixed phase T iO2 films by mist chemical vapor deposition and ultraviolet photodetectors applications[END_REF]. The RI of TiO2 thin films could be affected by the layer microstructure including lattice modification, porosity, and crystalline amount [START_REF] Woo | Optical anisotropy of microstructure-controlled TiO 2 films fabricated by glancingangle deposition (GLAD)[END_REF].

In our case, the layer was analyzed at atmospheric pressure and its microstructure was fitted on the wavelength range 380 nm-1 µm using the stack model presented in Fig. 6.

The Cauchy optical behavior of dielectric materials well fits the bulk TiO2 material on this range. A diffusion layer, based on the Bruggeman effective medium approximation of the underlying bulk T iO2 mixed with the upper air, was used to simulate the top surface roughness of the films which could play a significant role in the interface processes in contact with the electrolyte. Table 2 shows the layer microstructure composition with the corresponding refractive index given at 633 nm (RI).

It can be noticed that all the correlation factors are above 0.986 which is an indication of a good layer microstructure fitting with the Cauchy behavior and the chosen model of Fig. 6. As a microstructure correlation with XPS atomic percentages, the atmospheric nitrogen adsorbed atomic percentages have been found to be 0.55 at% at 400.14 eV on the non-doped 64 W and 0.79 at% at 399.93 eV on the nondoped 100 W layers. This adsorbed nitrogen is generally located in the diffusion layer. For almost the same diffusion percentages at non-doped 64 W (5%) and 100 W (4%), the RI lowering from 1.9794 (64 W) to 1.5937 (100 W) is mainly due to the disorder induced by N2 surface adsorption. Meanwhile, the non-doped T iO2 bulk keeps almost a constant RI for both powers as an indication of lattice conservation. This RI is lower than the theoretical anatase one even though the layer has been proved to be well crystallized at 350 °C [START_REF] Youssef | Optimization of N-doped TiO2 multifunctional thin layers by low frequency PECVD process[END_REF]. This fact is probably due to the layer porosity (nair = 1 < nanatase, where n is the refractive index of the medium) so that a porous layer introduces more air since the analysis is performed under atmospheric surrounding. This will be discussed in the XRR analysis. For the N-doped T iO2 samples at 64 W and 100 W, the optical correlation with the XPS results is deduced from Table 3.

The percentage values in Table 3 represent the mean value of the N1s atomic contents along the thin film from the surface (0 s etching time) to 2110s etching time. Both Ndoped 64 W and 100 W layers exhibit mostly the same insertion and adsorbed nitrogen atomic percentages, so this is not the limiting factor in that case. The difference is observed in the substitutional (Ti-N) nitrogen amount. As a correlation with the RI values, it is obvious that increasing the substitutional N-doping induces a bulk RI decrease (1.9219 for 64 W and 2.4693 for 100 W).

Even though the insertion and surface adsorption percentages are similar in both plasma powers, the diffusion RI decrease from 1.8803 (64 W) to 1.6205 (100 W) is explained by the higher diffusion concentration at 100 W. These microstructure differences between two layers synthesized at two different plasma powers are responsible of the behavior difference at the electrode-electrolyte interface presented in the electrochemical results.

Coatings density

The theoretical density reported for anatase is 3.82-3.97 g. cm -3 [START_REF] Lazzarelli | Blue chart gem identification[END_REF]. Using XRR data, a thin layer mass density ρm is calculated according to the formula 1 [START_REF] Lee | Diffusion, diffraction, réflectométrie et fluorescence de rayons-X à la PAC[END_REF]: 𝜌m = (𝜌Θc 2 ∕𝜌 2 NAr0)(∑AiCi∕∑ZiCi).

(

) 1
Θc is the critical angle (converted from degrees to radians) deduced from the minimum of the first derivative of the intensity versus angle plot, λ = 1.54056 Å (of the incident beam), NA is the Avogadro number (6.022•10 23 e.mol -1 ), r0 = 0.0000282 Å is the standard electron radius, Ai is the element molar mass, Ci is the element atomicity, and Zi is the element atomic number.

∑AiCi/∑ZiCi = (1 ⋅ ATi + 2 ⋅ AO)/(1 ⋅ ZTi + 2 ⋅ ZO) = 2.105 for TiO2.

(2)

The first derivative plots as a function of the angle for the non-doped and the doped 64 W and 100 W layers on Si (100) are plotted in Fig. 7.

For the non-doped 64 W and 100 W layers, Θc were found to be 0.25579° and 0.2285° (the mean value of the two minima on the curve), respectively. Using the formula cited previously, the calculated 64W-TiO2 density is 3.27 g cm -3 and the calculated 100W-TiO2 density is 2.61 g cm -3 . The two values are lower than the theoretical 3.82-3.97 g cm -3 as an indication of porosity existence. The 100W-TiO2 exhibits more porosity than the 64W-TiO2 which is in good agreement with the RI values. This could also be linked to the SEM observations shown in Fig. 4a andb where the 100W-TiO2 surface presents a 'flower-like' porous morphology that has a low RI of 1.5937 due to atmosphere inclusion.

The N-doped TiO2 molecular formula is TiO2-xNx since some Oxygen atoms are substituted by Nitrogen. According to that, the multiplying factor ∑AiCi/∑ZiCi would be equal to the following:

(1 ⋅ ATi + (2 -x) ⋅ AO + x ⋅ AN)∕(1 ⋅ ZTi + (2 -x) ⋅ ZO + x ⋅ ZN) = (48 + 32(2 -x) + 14x)∕(48 + 16(2 -x) + 7x). (3)
The critical angles were found to be 0.24761° and 0.26156°, respectively, for 64W-N-TiO2 and 100W-N-TiO2. Not considering the multiplying factor, the calculated values of the coating density are 1.46 mol cm -3 and 1.62 mol cm -3 , respectively, for 64W-N-TiO2 and 100W-N-TiO2. Then, the multiplying factor converts the unit from mol cm -3 to g.cm -3 .

The multiplying factor development leads to the following simplified ratio: (112 -18x)/(80 -9x). According to XPS results, x64 > x100 (x is the number of substituted oxygen, 64 and 100 refer to the plasma powers of 64 W and 100 W) since more oxygen atoms are substituted at 64 W. So:

112 -18x64 < 112 -18x100 . 80 -9x64 < 80 -9x100
Following this mathematical procedure, the multiplying factor at 64 W is lower than the one at 100 W. This result leads to a higher density for the 100W-N-TiO2. This is also in good agreement with the optical RI values that are higher at 100 W (doped) than at 64 W (doped) as an experimental proof of the higher lattice modification at low plasma power.

In fact, according to some references [START_REF] Chen | Reaction of N H3 with Titania: N-doping of the oxide and TiN formation[END_REF][START_REF] Swihart | Process control, diagnostics, and modeling in semiconductor manufacturing IV[END_REF], the variation in the nitrogen bulk amount is plasma power dependent. This is due to the nitrogen form present in the gaseous phase and which is directly related to the electronic density (the plasma power). The N H3 dissociation reaction in plasma phase has been well reported in reference [START_REF] Swihart | Process control, diagnostics, and modeling in semiconductor manufacturing IV[END_REF]. It has been demonstrated that, at the same chamber total pressure (which is the case for both doped samples), NH3 requires little plasma power to dissociate. Thus, the N + species intensity is higher in the low plasma power ranges and then reaches an almost steady state where N + species intensity increases slightly up to 150 W. This explains why the substitutional N-doping is more pronounced at 64 W and leads to optical and microstructural modifications. At 100 W, the bulk microstructure is less affected by nitrogen substitution while the surface state modification seems to be more expressed.

n-Type semiconducting behavior by OCV investigation

The definition of OCV of a photo-electrochemical system is the equilibrium potential without applying any external current. According to reference [START_REF] Rahul | Photoelectrochemical (PEC) and electrical studies on tungsten and molybdenum heteropolyoxometalate thin films 48[END_REF], when the semiconductor surface is in contact with the liquid electrolyte, a junction is formed. The latter one, for a n-type semiconductor surface, is represented in Fig. 8.

In real photo-electrochemistry conditions (Fig. 8a), and in case of a n-type semiconductor (like reported for T iO2), the light irradiation induces electron-hole separation and the added external bias voltage plays the role of the derivative force that conducts the electrons through the external circuit towards the cathode (reduction site) and the holes to the anode surface (electrolyte molecules oxidation).

At OCV conditions (equilibrium state where no current is added in Fig. 8b), when the supposed n-type semiconductor is irradiated, electron-hole separation occurs. Since there is no derivative force to conduct the electrons through the external circuit, these species in the depletion region migrate to the electrode surface and from there to the electrolyte [48]. Thus, under irradiation, the n-type semiconductor Fermi level shifts to lower energy levels towards the electrolyte redox potential. According to reference [START_REF] Muhibbullah | An equation of the width of the depletion layer for a step heterojunction[END_REF], the OCV (defined as an electrical field) is given by the following equation:

eV oc = E i(Fermi) - E eq(vs.
electrolyte redox potential), (4) where e is the elementary charge, Voc is the OCV, Ei(Fermi) is the Fermi energy level, and Eeq is the maximum level lowering when the anode is in contact with the electrolyte.

When the light is switched on, EFermi gets closer to Eelectrolyte and Voc drops (since e, the elementary charge, is a constant). When the light is switched off, a relaxation phenomenon occurs and the Voc increases again.

The OCV of the pure and doped TiO2 layers were evaluated under UV and visible irradiation for 800 s. The results are presented in Fig. 9.

It is clear that the four layers exhibit a n-type behavior since the OCV increases when the light is switched off and decreases again under illumination.

For the pure 64 W and 100 W T iO2 layers (Fig. 9a,b), the Voc is lower in the UV light than in the visible light. This is an indication that the Fermi level of the materials is closer to the electrolyte redox potential in UV. This conclusion is logical since the non-doped TiO2 absorbs the radiations at least corresponding to its gap in the UV.

The initial V oc decrease during the first 100 s of illumination is another proof of UV light absorbance. However, the Voc remains mostly stable under visible light as an indication that the Fermi level is not affected under visible light illumination for pure samples.

The remarkable point here is that the Voc drop when switching on the UV light again at 500 s is fast and reaches a steady state at 700 s for the 100W-TiO2 while it is slower and is still decreasing at 800 s for the 64W-TiO2. This means that the electron-hole pairs are instantly generated under UV illumination in the case of 100 W-TiO2.

The Voc reversibility under visible light has to be discussed for the 64 W and 100 W pure T iO2 (Fig. 9a,b, light gray plots). When the lamp is switched on at 500 s, the Voc drops quickly to its initial value in the case of 64 W (reversible drop) while it decreases slowly and to a higher Voc value in the case of 100 W. This phenomenon is explained in Fig. 10.

Correlating the Voc variations to the surface morphology and optical properties as determined in Sect. 4.2, some surface electron trap seems to exist in the 100W-TiO2. In fact, several experimental observations prove this hypothesis. The atmospheric N2 adsorption is higher at 100 W (0.79%) than at 64 W (0.55%) as reported in XPS analysis. Moreover, the adsorbed N at 100 W is located at a lower binding energy (399.93 eV) than at 64 W (400.14 eV). In addition to that, the 'flower-like' surface observed in the case of 100W-TiO2 seems to play a significant role in the material behavior at the electrode-electrolyte interface. The conclusion from Fig. 10 for the visible light non-reversibility of the Voc for 100W-TiO2 is that, under the first 100 s irradiation, the value is mainly stable. Switching off the light induces some state relaxation but when the light is switched on again, some electrons, still trapped in the surface state from the first 100 s illumination, limit the Voc decrease to its initial value.

Similar explanations could be presented in the case of the nitrogen-doped TiO2 layers (Fig. 9c,d). The Voc initial values under visible light are lower than under UV as a proof of the doping role in the charge separation enhancement in the visible range. Nitrogen doping creates N1s trap levels which leads to a decrease of the gap energy. Thus, the initial Fermi level is closer to the electrolyte redox potential and the Voc shifts to lower values. More N1s levels are generated in the band gap of the doped 64 W which lowers the Voc in both UV and visible light compared to the same layer synthesized at 100 W. These levels also play the role of electron traps so that it could be seen that the Voc stabilizes better in the case of the 64 W layers.

Electrode-electrolyte interface by EIS and correlation with I-V curves by CV

The electrode-electrolyte interface has been studied in the dark, in UV and in visible light by EIS in the conditions previously mentioned in Sect. 3.3. The results are presented in Fig. 11.

The decrease of the charge transfer resistance (Rct) by the doping process is obvious by the scale lowering from the non-doped to the doped samples.

It could be noticed that no Rct modification occurred under visible light for the non-doped samples at both 64 W and 100 W. This is an indication that the charge transfer at the electrode-electrolyte is not affected by this wavelength range (> 400 nm) which is logical due to the band gap of both materials. In addition, the resistances of the layers deposited at 100 W are slightly lower than those of the films prepared at 64 W revealing a more intrinsic conductive microstructure at higher plasma power. Under UV light, the resistances decrease remarkably in the case of both plasma powers. This result is in good agreement with both materials band gap and is also coherent with the previous observation since the Rct at 100 W decreases more than at 64 W revealing another time the 100 W layer more conductive intrinsic properties.

For the 64W-N-TiO2, it could be observed that Rct decreases remarkably from the dark to UV. A good decrease is also observed under visible light as an indication of charge separation and mobility under this wavelength range (> 400 nm). In parallel, for 100 W-N-TiO2, the Rct decrease under UV light is almost not observed while it decreases under visible light. This means that the charge separation and mobility are better under visible light than under UV light.

The explanation of such differences is reported in the anodic CV curves (at slow scan rate of 5 mV s -1 ) and presented in Fig. 12. In the dark, no current was measured for these two layers. In the case of the 64 W-TiO2 layer (Fig. 12a), under UV light, the photo-current remarkably increased and the redox reaction peaks (anodic Ipa and cathodic Ipc) are displayed. The positive peak is attributed to the oxidation of T i 3+ appearing under UV illumination (Ti 3+ →Ti 4+ +1e -) while the negative peak corresponds to the reduction of the generated Ti 4+ species (Ti 4+ +1e -→Ti 3+ ) [START_REF] Jie | Photoelectrochemical characteristics of nano-titanium dioxide thin films prepared by RF magnetron sputtering[END_REF]. Moreover, both peaks (anodic E pa and cathodic Epc around 0.35 V-0.4 V) are close enough to consider a fast charge transfer which has been observed in EIS spectra under UV for this layer (Fig. 11). Under visible light, no photo-current was detected and no charge transfer occurred which is in good agreement with EIS results shown in Fig. 11. In the case of the 64W-N-TiO2 layer (Fig. 12d), the semiconducting behavior is also obvious under both UV and visible light in agreement with the EIS observations. Moreover, the generated photo-current is greater than the one in the non-doped 64 W TiO2. This shows the relevant advantage of nitrogen doping in T iO2. Epc slightly shifts to the left indicating a faster charge transfer under visible light. The conclusion is that the layers prepared at 64 W show a typical n-type semiconducting behavior and could be qualified of 'typical photo-materials' which activity is completely light-dependent.

In the case of the 100W-TiO2 layers (Fig. 12b), the Ti 3+ [START_REF] Ji | Surface Ti3+/ Ti4+ redox shuttle enhancing photocatalytic H2 production in ultrathin TiO2 nanosheets/CdSe quantum dots[END_REF][START_REF] Qiu | Synthesis of T i 3+ selfdoped TiO2 nanocrystals based on Le Chatelier's principle and their application in solar light photocatalysis[END_REF] species already exist in the material without illumination. The proof is the oxidation and reduction peaks existing in the dark. Under UV light, the photo-current increases especially in the 0-0.2 V vs. Ag/AgCl range and the redox peaks intensity increases slightly. This explains the Rct decrease in the UV displayed in Fig. 11. It could also be observed that Rct decreases remarkably from 64W-TiO2 to 100W-TiO2. This fact is due to the greater photo-current generated in the case of 100W-TiO2 due to the species mobility. This mobility results from a combination of the initial Ti 3+ and the Ti 3+ generated by UV irradiation. Under visible light, the material behavior is almost the same as in the dark proving that the initial T i 3+ does not tailor the layer band gap. The redox peaks intensities are slightly lower than the ones in the dark revealing the surface state role in trapping some mobile Ti 3+ decreasing by that the photocurrent and increasing the Voc values as reported in Fig. 9 and explained in Fig. 10. For the 100W-N-TiO2 (Fig. 12d), the Ti 3+ species still exist intrinsically into the material as the redox peaks exist in the dark. Their concentration increases slightly under UV and visible light as the photocurrent increases under irradiation. These results are in good agreement with the material EIS spectra shown in Fig. 11. Still, this increase is not really noticeable which proves the existence of an initial conductive phase for the layers deposited at 100 W. At the interface, in the electrodeelectrolyte equilibrium conditions (0 V vs. Ag/AgCl), it could be noticed that a higher photo-current is measured under visible light than in the dark or UV for the 100W-N-TiO2 (see the starting point of the CV curves in Fig. 12d). This reveals the nitrogen role in the band gap tailoring and in the Rct decrease under visible light. Thus, at a higher plasma power, the electrode contains an initial conductive phase including Ti 3+ . The electrode activity exists even in the dark and is barely enhanced under light (in the UV for the pure TiO2 and in the visible for the N-TiO2). The layers could be qualified of 'partial photomaterials' in which carriers are created by light and easily transferred by an initially existing conductive phase.

LV curves

LV has been performed for the four layers in the dark, UV, and visible light from 0 to 2 V vs. Ag/AgCl at a scan rate of 1 mV s -1 . The results are presented in Fig. 13.

Linear Voltammetry is a useful technique to study the oxygen evolution reaction (OER) current and the starting potential.

The photo-current is enhanced under UV light for the 64W-TiO2 as observed in Fig. 13a. This result is in good agreement with the previous electrochemical observations. In the case of the 100W-TiO2 (Fig. 13b), no current variation is observed between dark and visible light. The currents are higher than in the case of 64W-TiO2 as an evidence of initial conductive carriers in the material. These carriers are highly activated under UV light, their concentration increases under this light range and since the layer is initially conductive, they migrate to the electrodeelectrolyte surface causing instant electrolyte oxidation (at 1.4 V vs. Ag/AgCl). At the same time, an amount of Ti 3+ could be oxidized to Ti 4+ which causes current decrease and then lowering at 2 V vs. Ag/AgCl [START_REF] Ji | Surface Ti3+/ Ti4+ redox shuttle enhancing photocatalytic H2 production in ultrathin TiO2 nanosheets/CdSe quantum dots[END_REF][START_REF] Qiu | Synthesis of T i 3+ selfdoped TiO2 nanocrystals based on Le Chatelier's principle and their application in solar light photocatalysis[END_REF]. Similarly, the photo-current enhancement under visible light for the 64W-N-TiO2 (Fig. 13c) and 100W-N-TiO2 (Fig. 13d) is clear.

The higher current observed in the case of the 100W-N-TiO2 reveals the more intrinsic conductive properties due to the initial Ti 3+ species.

Conclusions

Anatase and Nitrogen-doped anatase thin layers with thicknesses of some hundred nanometers have been grown by low-frequency PECVD process at 64 W and 100 W according to previously optimized conditions. Three different substrates have been used for the deposition of layers: silicon for XRR, XPS, and ellipsometry measurements, Nickel foil for crystal growth morphology and nitride shell observations by SEM, and conductive porous carbon (E-Tek®) for electrochemical studies. Due to its intrinsic porosity, lower layer thicknesses have been obtained on the porous carbon when compared to dense substrates. XPS has revealed a higher substitutional Ti-N incorporation into the doped layers prepared at 64 W due to the higher amount of N + species in the lower plasma power ranges as previously reported in the literature. Ellipsometry microstructure simulation has shown that the films are rough, constituted of a bulk TiO2 on top of which lies a diffusion layer. In parallel, the layer refractive index variation from the theoretical value for anatase (2.5) was more noticed at 64 W than at 100 W. This is an indication of a higher lattice modification at lower plasma power as shown by XPS. These structural differences directly related to the plasma power in the preparation of films have a direct impact on the material photo-electrochemical behavior where the layers prepared at 64 W exhibit a 'fully semiconducting' behavior under UV and visible light, whereas the films deposited at 100 W already contain a conductive phase (Ti 3+ species) which activity can be enhanced under light.

As perspectives, layers with higher nitrogen amount should be characterized under the same conditions in order to evaluate the effect of doping incorporation on the structural, optical, and photo-electrochemical properties of anatase PECVD materials. Then optimized N-doped films should be integrated as anodes in photo-electrochemical systems for hydrogen generation by water splitting under solar light.

Fig. 1

 1 Fig. 1 Schematic representation of intrinsic energy band behaviors of a n-type semiconductor and b ptype semiconductor

Fig. 4

 4 Fig. 4 Surface and cross section SEM images of a 64W-TiO2, b 100W-TiO2, c 64W-N-TiO2, and d 100W-N-TiO2 deposited on nickel foil The TiO2 and N-TiO2 low-frequency PECVD thin layers deposited on porous carbon (E-Tek®) are presented in Fig. 5. The 'flower-like' morphology of the crystallized coatings depicted on Ni support could be noticed on the carbon cloth surface. The nitrogen doping seems to induce local agglomeration on some surface points. The crystallite edges become a little thicker after nitration.Table1presents the films thicknesses measured by SEM cross section on nickel foil and E-Tek® cloth for the same deposition time of 20 min.The first observation is that increasing the plasma power from 64 W to 100 W increases the growth rate since higher thicknesses were measured on both substrates at 100 W. The second observation is that lower thicknesses values were obtained on E-Tek® cloth. This could be due either to a hidden thickness by crystals grown inside the cloth or to the fact that metallic nucleation sites on nickel foil are highly activated by plasma and accelerated by the layer surface growth. The third observation is that the growth rate is slowed by doping process, especially on Ni support. In fact, when adding N H3 gas into the plasma phase, reactive species reactions are more favored into the plasma gaseous

Fig. 5 Table 1

 51 Fig. 5 Surface and cross section SEM images of a 64W-TiO2, b 100W-TiO2, c 64W-N-TiO2, and d 100W-N-TiO2 deposited on an E-Tek®

Fig. 6

 6 Fig. 6 TiO2 and N-TiO2 layer Cauchy simulation by ellipsometry on Si (100) wafer

Fig. 8

 8 Fig. 8 Electrode surface-electrolyte junction a real photo-electrochemistry and b simulated at Open Circuit Potential for a n-type semiconductor schematic representations

Fig. 10

 10 Fig. 10 OCV schematic variation under illumination for 100W-TiO2

Fig. 12

 12 Fig. 12 CV curves at 5 mV.s -1 in the dark, in UV, and in visible light for a 64W-TiO2, b 100W-TiO2, c 64W-N-TiO2, and d 100W-N-TiO2

Fig. 13

 13 Fig. 13 LV curves at 1 mV.s -1 in the dark, UV, and visible for a 64W-TiO2, b 100W-TiO2, c 64W-N-TiO2, and d 100W-N-TiO2

Table 2

 2 Layer microstructure composition and optical properties as determined by ellipsometry on Si (100)

	Sample	% of the full thick-		Bulk	Top	RI
		Correlation ness	RI	factor (fit on
		Bulk	Diffusion			380-1000 nm)
	64W-TiO2	95.0	5.0	2.390 1.979	0.986
	64W-N-TiO2 99.9	0.1	1.922 1.880	0.995
	100W-TiO2	96.0	4.0	2.318 1.594	0.990
	100W-N-	97.0	3.0	2.469 1.621	0.993
	TiO2

Table 3

 3 N1s atomic percentages deduced by depth profiles XPS for the nitrogen-doped 64 W and 100 W TiO2

	Sample	Substitutional N	Insertion and surface
		(396 eV)	adsorption N (398-
			402 eV)
	64W-N-TiO2	1.58%	0.98%
	100W-N-TiO2	0.59%	1.02%
			Fig. 7 XRR first derivative plot of the non-doped and N-doped 64 W
			and 100 W layers deposited on reflective Si (100)

Acknowledgements This project has been funded with support from Lebanese University (UL) in collaboration with AZM&SAADÉ foundation. We also thank Arie VAN DER LEE (IEM, Montpellier) for XRR analysis, Didier COT (IEM, Montpellier) for SEM observations, and Valérie FLAUD (ICGM, Montpellier) for thin films profile investigation by XPS.


domain: chim sdv

text:
Farukh Arjmand
email: farukh_arjmand@yahoo.co.in

Salman Khursheed

Thierry Roisnel

Hifzur R Siddique

Copper (II)-based halogen -substituted chromone antitumor drug entities: Studying biomolecular interactions with ct-DNA mediated by sigma hole formation and cytotoxicity activity

Copper-based antitumor drug entities 1-3 derived from substituted (F -, Br -, -CH 3 ) 3formylchromone pharmacophore were synthesized and thoroughly characterized by spectroscopic and single X-ray crystallographic studies. These complexes show structural novelty due to presence of the X-bonds in chromone scaffold which could facilitate higher propensity for nucleic acids via sigma σ-hole interactions. Therefore, structure-activity relationship of 1-3 was studied by performing ct-DNA binding, pBR322 cleavage and cytotoxicity activity to validate their potential to act as chemotherapeutic drug entities. The binding studies of 1-3 with ct-DNA were carried out employing many biophysical techniques and the corroborative results of these experiments showed intercalation mode of binding and the order of binding was found to be 2 > 1 >3. The structure of drug entities could facilitated strong halogen bonding interaction(in case of 1 &2) and stability of X bond was rationalized by sigma hole region of positive electrostatic potential on the surface of C-X covalent bond, as determined by gas phase B3LYP computational DFT studies. Interestingly, 2 exhibited most avid binding affinity due to presence of Br -electron withdrawing and polarizable group. Further, cleavage studies of 1-3 with pBR322 plasmid DNA were performed which demonstrated significant cleavage activity , the supercoiled form (Form I) of plasmid DNA was converted to nicked form (Form II) with the appearance of linearized form(Form III) in between two, implicating lethal double strand breaks of DNA. 2 showed predominantly higher cleavage activity following the similar trend as observed for binding studies.

The cytotoxicity of the complexes 1-3 was evaluated by MTT assay against the human liver carcinoma (Huh-7) and prostate cancer (DU-145) cell lines; complex 2 exhibited specific and selective cytotoxicity for the DU-145 cancer cell line with LC 50 value of 1.6μM.

Introduction

Copper is an endogenously biocompatible trace element which plays a key role in many vital enzymes in biological system [START_REF] Santini | Advances in Copper Complexes as Anticancer Agents[END_REF]. Copper complexes exhibit interesting chemical and biological properties viz., strong Lewis acid nature, redox active behavior with one electron Cu(II)/Cu(I) shuttle, high binding propensity for DNA, DNA cleaving ability via an oxidative pathway, antitumor and apoptosis inducing character [START_REF] Muhammad | Metal-based anticancer chemotherapeutic agents[END_REF][3][START_REF]Recent Advances in Anticancer Copper Compounds. Metal-based Anticancer Agents[END_REF][START_REF] Uma | Oxidative DNA cleavage mediated by a new copper(II) terpyridine complex: Crystal structure and DNA binding studies[END_REF]. Copper complexes have emerged as potent class of future generation anticancer agents, primarily due to their differential response towards cancerous and normal cells. Copper was found to be a co-factor for several angiogenesis mediators including vascular endothelial growth factor(VEGF) [START_REF] Sen | Copper-induced vascular endothelial growth factor expression and wound healing[END_REF], basic fibroblast growth factor (bFGF) [START_REF] Nasulewicz | Role of copper in tumour angiogenesisclinical implications[END_REF], interleukin-1 (IL-1) and IL-8 [START_REF] Moriguchi | The copper chelator trientine has an antiangiogenic effect against hepatocellular carcinoma, possibly through inhibition of interleukin-8 production[END_REF]. Tumors are dependent on angiogenesis for their growth, invasion and metastasis [9], since tumor cells are in a state of hypoxia due to altered metabolism and rapid growth. Furthermore, copper content is usually much higher in cancerous cells as compared to normal cells. Many copper complexes are also known to induce oxidative stress by the production of ROS or the depletion of GSH due to cellular accumulation of the redox active copper ions [START_REF] Gupte | Elevated copper and oxidative stress in cancer cells as a target for cancer treatment[END_REF].

Chromones-benzoannelated γ-pyrone heterocycles are considered as versatile privileged scaffolds in medicinal chemistry owing to their diverse pharmacological applications [START_REF] Gaspar | Chromone: a valid scaffold in medicinal chemistry[END_REF][START_REF] Keri | Chromones as a privileged scaffold in drug discovery: A review[END_REF]. They are excellent templates for the design of novel pharmacologically important compounds due to ease of substitution and structure modification. Therefore, many substituted derivatives of 3-formyl chromones have been synthesized and explored for medicinal properties for treating chronic neurodegenerative, inflammatory, and infectious diseases as well as diabetes and cancer [START_REF] Zwergel | Novel benzofuran-chromone and-coumarin derivatives: synthesis and biological activity in K562 human leukemia cells[END_REF][START_REF] Gaspar | Oxidative stress and neurodegenerative diseases: looking for a therapeutic solution inspired on benzopyran chemistry[END_REF][START_REF] Philip | Metal based biologically active compounds: Design, synthesis, DNA binding and antidiabetic activity of 6methyl-3-formyl chromone derived hydrazones and their metal (II) complexes[END_REF].

Chromone based compounds have been incorporated in cancer-targeted therapeutic drug molecules which were capable of inhibiting different types of cancer targets, viz., TNF αsignaling and protein kinases [START_REF] Talhi | Bis(4-hydroxy-2H-chromen-2-one): synthesis and effects on leukemic cell lines proliferation and NF-kappa B regulation[END_REF]. Copper (II) chromone complexes have shown cytotoxicity against different phenotypes of cancer cell lines with varied potencies [START_REF] Tang | Synthesis and biological studies of 4′, 7, 8-trihydroxyisoflavone metal complexes[END_REF][START_REF] Kalaiarasi | Synthesis, spectral characterization and biological evaluation of some copper(II) complexes containing 4-oxo-4H-chromene-3-carbaldehyde-4(N)-substituted thiosemicarbazones[END_REF][START_REF] Barve | Synthesis, molecular characterization, and biological activity of novel synthetic derivatives of chromen-4-one in human cancer cells[END_REF]. The cytotoxic activity of Cu(II) chromone Schiff base complexes on cell proliferation of chemoresistant lung cancer (A549) and breast (MCF-7) cancer cell line was evaluated and the complexes were capable of effectively overcoming cisplatin resistance in these cell lines which was attributed to chelation of the chromone ligand with metal ion [START_REF] Kalaiarasi | Synthesis, spectral characterization and biological evaluation of some copper(II) complexes containing 4-oxo-4H-chromene-3-carbaldehyde-4(N)-substituted thiosemicarbazones[END_REF]. In our laboratory, the cytotoxic potential of chromone-Cu(II) drug candidates was evaluated on human breast (MCF-7) and liver cancer (HepG2) cell lines which revealed good cytotoxic response at significantly lower IC 50 values of (5-10 μg mL -1 ) [START_REF] Arjmand | Design, synthesis and characterization of novel chromone based-copper(II) antitumor agents with N,N-donor ligands: comparative DNA/RNA binding profile and cytotoxicity[END_REF].

Recently, halogen bonding has fueled much interest in the subdomain area of small moleculenucleic acid interactions in medicinal inorganic chemistry [22]. Halogen bonding is a type of charge transfer interaction which is a qualitative analogue of hydrogen bonding [START_REF] Parker | Halogen bonding in DNA base pairs[END_REF][START_REF] Murray | σ-Holes, π-Holes and Electrostatically-Driven Interactions[END_REF]. The halogen bonds take place between a halide (X) in one molecule with neighboring electron-rich Lewis base site, similar to a hydrogen bonds. DNA is most amenable therapeutic drug target for many antitumor drugs in clinical use which possesses several easily accessible electron rich reactive sites such as nucleobase pairs and ribose sugar-phosphate backbone of double helix.

Interaction of small molecules with X-bond with DNA double helix could be interpreted on the basis of halogen bonding ,as formation of the halogen bond is thermodynamically more favorable and can be thus, be used to predict non-covalent interactions such as electrostatic interactions, groove binding and intercalation. Halogens in organic and inorganic compounds are known to polarize their covalent bonds to generate electropositive crown where the halogen acts as a Lewis acid to pair with Lewis bases, including oxygen and nitrogen atoms of biomacromolecules. These electrostatic pairs known as halogen bonds or X-bonds could contribute immensely to the understanding of drug-nucleic acid interactions [START_REF] Shields | Directional tendencies of halogen and hydrogen bonds[END_REF] We describe herein, new copper (II) based antitumor drug candidates 1-3 derived from substituted chromone scaffold where X= F -, Br -, and -CH 3 . The substitution of halogens and alkyls in chromone scaffold drastically alters the physiochemical and pharmacological properties of drug entities in following ways: i) They can affect the polarity and lipophilicity which enable the facile transportation/permeation via the cell membrane into the cells thus preventing off-target toxicity ii) prolong the lifetime of active metabolite iii) show cytotoxic response at low submicromolar dosage iv) inhibit cell proliferation by targeting TNF α-signaling and protein kinases. Besides, copper(II) embedded in bioactive chromone pharmacophore also plays a significant role by inducing ROS oxidative stress and mediating angiogenesis. We have evaluated the DNA binding affinity of Cu(II) chromone complexes 1-3 by a battery of biophysical methods. Since these complexes possess the X-bonds which could facilitate sigma σ-hole interactions, it was considered worthwhile to study their in vitro differential response with ct-DNA and cytotoxicity in cancerous cells. The present Cu(II) complexes-DNA model system demonstrated that among all X-bonds, bromine substituted chromone Cu(II) complex 2 exhibited avid binding propensity with DNA and exhibited selective cytotoxicity for the DU-145 cancer cell line at a low intracellular concentration.

Results and discussion

Synthesis and characterization

The copper (II) complexes 1-3 derived from substituted (F-, Br-,-CH 3 ) 3-formylchromone were

Spectroscopic characterization

In the IR spectra of complexes 1-3, the characteristic bands assigned to υ(HC=O) and υ(-C=O) of 3-formylchromone derivatives observed at 1694 and 1671 cm -1 ,respectively were shifted to lower wave number by 15-25 cm -1 indicating the complexation of these groups with metal ions (Fig. S1) [START_REF] Shebl | Structural variety in copper (II) complexes of 3-formylchromone: Synthesis, spectral, thermal, molecular modeling and biological studies[END_REF]. The bands which appeared at 592 cm -1 and 1263 cm -1 were assigned to υ(C-Br) and υ(C-F)

stretching vibrations, respectively. In complex 3, two characteristic bands at 2907 cm -1 and 3069 cm -1 corresponded to sp 2 and sp 3 hybridized carbon atoms, respectively. The band at 1060 cm -1 was attributed to υ(C-O) stretch, indicating the ethoxylation of the 3-formylchromone derivative at 2-position. In the far-IR of complexes 1-3, the metal coordination was substantiated by the bands at 426-500 cm -1 .

The solid state X-band EPR spectra of complexes 1-3 were recorded at room temperature on 9.1 GHz frequency under the magnetic field strength of 3000 G (Fig. S2). The EPR spectra of 1-3 displayed the anisotropic band centered at g values as shown in Table S4. These values are in agreement with tetragonally elongated octahedral geometry of Cu(II) ion with d 9 configuration [START_REF] Yousuf | Mechanistic insights into a novel chromone-appended Cu(ii) anticancer drug entity: in vitro binding profile with DNA/RNA substrates and cytotoxic activity against MCF-7 and HepG2 cancer cells[END_REF] i.e. (eg) 4 •(a 1 g) 2 •(b 2 g) 2 •(b 1 g) 1 . The trend of g-tensor values is g ║ >g ┴ >2.0023 suggested that unpaired electron mainly resides in dx 2 -y 2 orbital. Furthermore, the complexes possess g ║ < 2.3, revealing an appreciable metal-ligand covalent character of the complexes [START_REF] Morgan | Supramolecular assembly of hydrogen bonding, ESR studies and theoretical calculations of Cu (II) complexes[END_REF][START_REF] El-Sonbati | Synthesis, characterization of Schiff base metal complexes and their biological investigation[END_REF].

Electronic spectra of complexes 1-3 were recorded in DMSO at room temperature which revealed two well resolved absorption maxima in the range 260 to 290 nm and 300-315 nm attributed to ππ* and n-π* electronic transitions, respectively. Broad d-d bands observed in the visible part of spectra in the range 400-642 nm are typical for tetragonally distorted octahedral environment around the Cu (II) center [START_REF] Sharma | Octahedral copper (ii)-diimine complexes of triethylenetetramine: effect of stereochemical fluxionality and ligand hydrophobicity on Cu II/Cu I redox, DNA binding and cleavage, cytotoxicity and apoptosis-inducing ability[END_REF].

Description of the X-ray crystal structures

The complex 1 crystallizes in the monoclinic space group P21/n possessing the lattice parameters a = 9.9291(4) A 0 , b = 5.1435(2) A 0 , c = 27.7256(10) A 0 and α = γ = 90 0 , β = 98.1990 [START_REF] Gupte | Elevated copper and oxidative stress in cancer cells as a target for cancer treatment[END_REF] for other Cu (II) octahedral complexes [START_REF] Jiang | Synthesis, crystal structure, cytotoxic activities and DNA-binding properties of new binuclear copper(II) complexes bridged by N,N′-bis(N-hydroxyethylaminoethyl)oxamide[END_REF].

Other crystallographic data viz., selected bond lengths and bond angles for 1-3 are summarized in Table S1-S3.

DNA binding studies

Metal-based drug candidates can uncoil the helical structure of ct-DNA, undrape the embedded electron rich nucleobases to the exterior helix leading to an efficacious binding with the drug entities either in covalent or non-covalent binding mode viz., electrostatic, groove binding and intercalation [START_REF] El-Sonbati | Synthesis, characterization of Schiff base metal complexes and their biological investigation[END_REF][START_REF] Pages | Metal complex interactions with DNA[END_REF]. The binding of copper(II)-based halogen substituted-chromone derivatives 1-3 with ct-DNA was followed by a battery of different complementary techniques such as UVvis absorption, florescence spectroscopy, EPR spectroscopy, circular dichorism and cyclic voltammetry.

Electronic absorption titration of complexes 1-3 was carried out in presence and in the absence of ct-DNA at room temperature (Fig. 2). Upon addition of increasing concentration (0.00-3 × 10 -5 M) of ct-DNA to a fixed concentration ( 2×10 -4 M ) of 1-3 , a sequential decrease of the absorption intensity was observed at the intra-ligand band at 250-300 nm exhibiting " hypochromism" of 43% in case of 1 , 54% for 2 and 41% for 3 , respectively. There was a pronounced red shift of 7-8 nm in complexes 1 and 2, suggesting intercalative mode of binding while a moderate shift of 1-2 nm was observed in case of 3 which implicated groove binding mode [START_REF] Yang | Copper (II) complexes with NNO ligands: synthesis, crystal structures, DNA cleavage, and anticancer activities[END_REF]. The rationale for intercalation mode of binding in complexes 1 & 2 is presence of aromatic π-π stacking chromophore which could easily "slip in" or insert between the nucleobases while 3 articulates groove binding due to the non-planarity of the methyl groups, therefore prefers to bind by electrostatic interaction to the negatively charged phosphate oxygen of DNA helix. The binding strengths of 1-3 with ct-DNA was quantified by the intrinsic binding constant K b , using the Wolfe-Shimer equation (1) [START_REF] Wolfe | Polycyclic aromatic hydrocarbons physically intercalate into duplex regions of denatured DNA[END_REF].

[DNA]/ϵ a -ϵ f = [DNA]/ϵ b -ϵ f + 1/K b (ϵ b -ϵ f ) (1)
Where [DNA] is the ct-DNA concentration and ϵ a , ϵ f, and ϵ b are apparent (A abs / [Cu (II) complex]), free and bound complex molar absorptivity coefficients, respectively. From the plot of [DNA/ϵ a -

ϵ f ] vs [DNA] with a slope of 1/ (ϵ b -ϵ f ) and intercept of 1/ [K b (ϵ a -ϵ f )]
, the values of K b were derived by the ratio of slope to intercept. Table 2 The binding constant values for complexes 1-3 obtained upon interaction with ct-DNA.

Emission spectra The intrinsic binding constant, K b values of the complexes 1-3 with ct-DNA are depicted in Table 2 and were found to be in the order of magnitude 2 >1 > 3. Interestingly, the K b values of 1 & 2 were found to be two-fold higher in magnitude as compared to complex 3 which has been attributed to the presence of halogens on their ligand scaffolds as halogen substituents provide hydrophobic, polar and planar environment suitable for intercalation with ct-DNA [35]. Notably, bromine substituted complex 2 revealed highest binding affinity towards DNA , which can be envisaged by a model that shows that the electron density from DNA nucleobases could be directly transferred to polarizable C-Br bond of ligand scaffold via the sigma σ-hole (Fig. 3) with further "proof of concept" obtained by docking and computational B3LYP /DFT studies [START_REF] Scholfield | Halogen bonding (X-bonding): A biological perspective[END_REF]. Albeit no such observation was seen in case of fluorine-substituted moiety of 1 while the complex 3 exhibited lowest K b value with ct-DNA which can be explained by the lack of planarity and steric hindrance due to methyl group present on ligand moiety. Similarly, binding constant (K) values of 1-3 were obtained from the Scatchard equation [START_REF] Healy | Quantitative determination of DNA-ligand binding using fluorescence spectroscopy[END_REF] with ct-DNA and the binding propensity further substantiated that complex 2 exhibited remarkable binding with ct-DNA as compared to complex 1 and 3 (Table 2).

Complex Absorption spectra, K b (× 10 4 M -1 ) K (× 10 4 M -1 ) K sv (× 10 4 M -1 )
The validation of binding mode of 1-3 was evaluated by performing competitive binding studies with ethidium bromide, EB. Ethidium bromide is a typical intercalator of ct-DNA possessing planar phenantridine ring by which it intercalates in between adjacent base pairs of the ct-DNA double helix, thus emitting intense florescence at ca.600 nm [START_REF] Alagesan | Potentially cytotoxic new copper (II) hydrazone complexes: synthesis, crystal structure and biological properties[END_REF]. The enhanced florescence emission has been attributed to the formation EB-ct-DNA complex and may be quenched with the co-existence of motifs that intercalates ct-DNA equally or strongly than ethidium bromide. Upon addition of 1-3 to EB-ct-DNA system such that [complex]/[ct-DNA]=1:1 to 5:1, the significant quenching of the band at 600 nm was observed (Fig. 4), resulting florescence emission was 32% -33% of the initial EB-ct-DNA florescence intensity in case of complex 1 & 2 while it was 50% for 3. These experimental results demonstrate that 1 & 2 displaced EB from EB-ct-DNA system more efficiently than 3, thus implicating their non-covalent intercalative mode of binding with ct-DNA and groove binding interaction in case of 3. In an another florescence quenching experiment, Stern -Volmer constant, K sv values for 1-3 were calculated (equation 2) which depicted quenching of florescence, in the order 2 >1 >3 well corroborated with other binding studies ascertaining strong affinity of complexes 1 and 2 for ct-DNA as compared to 3. The calculated Stern-Volmer constant, K sv values for 1, 2 and 3 were found to be 5.04, 6.13 and 4.19, respectively. The liquid state X-band EPR spectra of complexes 1-3 were recorded in DMSO at LNT in the absence and presence of ct-DNA which revealed two g ║ and two g ┴ signals (Fig. 5). Upon addition ct-DNA, EPR spectra of complex 2 exhibited weak signatures with minor shift in g-values,

I 0 /I =1+K sv. r (2)
indicating the binding of complex 2 with ct-DNA [START_REF] Draksharapu | Binding of copper(ii) polypyridyl complexes to DNA and consequences for DNA-based asymmetric catalysis[END_REF][START_REF] Raja | Copper (II) complexes of tridentate pyridylmethylethylenediamines: role of ligand steric hindrance on DNA binding and cleavage[END_REF]. However, the spectra of complex-ct-DNA showed that the coordination number of the metal in 1-3 was not perturbed. Similar intensity pattern was observed in the complex-ct-DNA spectra of 1 and 3 (Fig. S3).

Morphological studies

Due to the presence of various potential amenable binding sites in DNA viz., the phosphate group and electron donor atoms on nucleobases, metal ions carry out the dramatic condensation of DNA to a compact, rod like and toriodal structure, most commonly observed by electron microscopy.

The condensation process has been attributed to diverse ionic interactions such as electrostatic and other forces viz., free energy of bending, mixing entropy, coulombic etc. [START_REF] Bloomfield | DNA condensation by multivalent cations[END_REF]. The condensation or aggregation phenomena plays a critical role in many antitumor drugs. DNA condensation has become a dynamic useful technique as it can be used to transfer DNA containing genes of therapeutic interest from solution to target cells involving gene therapy [START_REF] Hormann | Multiply intercalator-substituted Cu (II) cyclen complexes as DNA condensers and DNA/RNA synthesis inhibitors[END_REF][START_REF] Teif | Condensed DNA: condensing the concepts[END_REF]. Since DNA possess negative charge density due to nucleobases present in the major and minor grooves, positively charged metal ions can easily intercalate within these nucleobases, thus showing molecular recognition and in vivo modification of DNA in the condensed state. In this study, condensation of ct-DNA was achieved to examine the morphological changes under scanning electron microscope (SEM) by evaporating an equimolar mixture of 1-3 in Tris-HCl buffer/ 50mM NaCl at pH=7.3. The SEM micrographs of 1-3 showed single crystalline motifs in rod shapes (Fig. 8). On the contrary, the SEM micrographs of condensates of 1-3 with ct-DNA revealed the randomization and formation of globular structures, thus indicating substantial interaction of ct-DNA with complexes similar to earlier reports on copper-DNA condensates [START_REF] Zehra | Enantiomeric Amino Acid Schiff Base Copper(II) Complexes as a New Class of RNA-Targeted Metallo-Intercalators: Single X-ray Crystal Structural Details, Comparative in Vitro DNA/RNA Binding Profile, Cleavage, and Cytotoxicity[END_REF][START_REF] Rajalakshmi | DNA condensation by copper (II) complexes and their anti-proliferative effect on cancerous and normal fibroblast cells[END_REF].

Molecular docking studies

The molecular docking study of 1-3 was carried out with the d(CGCGAATTCGCG)2 (PDF ID :

1BNA) dodecamer sequence of DNA helix [START_REF] Gilad | Docking studies on DNA intercalators[END_REF] to estimate the most probable binding affinity along with the sterically stable conformations (Fig. 9). The binding energies for the best docked pose of complexes were quantified as -333.98 kJ/mole for 1, -347.07 kJ/mole and -294.72 KJ/mole for 2 and 3, respectively which revealed efficient DNA-drug interaction with the notably higher binding energy of complex 2 than the rest of complexes. Moreover, the results of docking implicated that complexes 1-3 fit snugly in the intercalative sites of DNA helix and moderately deep in adenine-cytosine rich region stabilized by hydrogen bonding, van der Waals interactions and hydrophobic contacts of aromatic chromone ring motif with the minor groove of ct-DNA; as the minor groove of DNA is considered to be lucrative target for many small molecules [START_REF] Fu | Water-soluble DNA minor groove binders as potential chemotherapeutic agents: synthesis, characterization, DNA binding and cleavage, antioxidation, cytotoxicity and HSA interactions[END_REF].

These interactions are considered to be the main driving force of the intercalative binding mode in addition to electrostatic interactions between the polarized bromine and the lone pair electrons of the nitrogen and oxygen atom of a neighboring nucleobases of DNA helix.

The greater binding energy of complex 2 was particularly supported by halogen donation via σhole of C-Br covalent bond. This highly directional attraction between halogen atom (bromine in our case) and an electron donor nucleobase (adenine) helps in modulating the physicochemical properties of the molecular scaffolds; they affect the polarity and lipophilicity, which in turn could change membrane and blood brain barrier permeation of the molecule. Furthermore, the difficulty in metabolization of carbon-halogen, specifically C-Br bond of 2 could prolong the lifetime of active motif [START_REF] Hernandes | Halogen atoms in the modern medicinal chemistry: hints for the drug design[END_REF]. The electron donor nucleobase to halogen interaction in complex 2 was found to be nitrogen atom of adenine wherein nitrogen atoms are seen to transfer its electron density directly into the σ-hole of C-Br covalent bond, thus stabilizing DNA-drug interactions. Also the increasing stabilization was attributed to the aromatic character of nucleobase which allows them to act as electron donors via their ᴨ-electrons above and below their rings.

B3LYP/DFT Computational studies

The electrostatic potential mapped structure was obtained to model the σ-hole region on the surface of C-Br covalent bond which was shown by the emergence of electropositive region (Fig. 3a; blue) along the C-Br axis in complex 2, consistent with the presence of more polarizable Br̄ atom. This region is surrounded by an electroneutral ring (Fig. 3a; white) and farther out, an electronegative region (Fig. 3a; red). The presence of this σ-hole region indicates that complex is 2 more likely to serve as halogen bond donor to nucleobases via., its Br̄ atom, hence giving an indication of the specific and enhanced binding affinity of 2 as compared to 1 and 3 with ct-DNA.

Moreover, B3LYP/DFT calculations were performed on 1-3 to get an insight onto the frontier molecular orbitals and their energy gap values which help to shed light on the chemical reactivity, kinetic stability and electrical transport properties of a molecule. The HUMO-LUMO energy gap is also used to check bioactivity from intramolecular charge transfer and correlated with various biological aspects like antibacterial, antioxidant and DNA binding aspects [START_REF] Mabkhot | Novel enaminone derived from thieno [2,3-b] thiene: Synthesis, x-ray crystal structure, HOMO, LUMO, NBO analyses and biological activity[END_REF][START_REF] Lian | Mixed-ligand copper (II) Schiff base complexes: the role of the co-ligand in DNA binding, DNA cleavage, protein binding and cytotoxicity[END_REF]. The negative values of HOMO and LUMO indicates the stability of the complexes (Fig. 10) .The higher value

In vitro cytotoxic activity

The in vitro cytotoxic activity of complexes 1-3 was evaluated in terms of LC 50 (the lethal concentration of the drug required to kill 50% of the cells) values by colorimetric 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay [START_REF] Siddique | Lupeol, a novel androgen receptor inhibitor acts as a double-edged sword: Competitive binding as well as transcriptional inhibition[END_REF] on the panel of two cancer lines viz., human liver carcinoma (Huh-7), prostate cancer (DU-145) and one normal prostate epithelial (PNT1) cell lines. Complexes 1-3 (2.5-10 μM) were treated for 48 hours which resulted in potential dose-dependent growth inhibition of both Huh-7 and DU-145 cancer cell lines irrespective of origin of the cells (Fig. 11), but complex 2 showed higher inhibition than 1 and 2

suggesting that the presence of Br -is lucrative in complex 2. Interestingly, no significant cell death was observed in the normal PNT1 cell lines upon treatment with complexes 1-3.

Cleavage activity

The cleavage reaction of complexes 1-3 was monitored by agarose gel electrophoresis employing supercoiled pBR322 DNA as a substrate in a medium of 5 mM Tris-HCl/50 mM NaCl buffer at pH 7.2 under the influence of applied electric field . Upon gel electrophoresis of pBR322 DNA, the fastest migration results in the conversion of a supercoiled form (Form I) to a relaxed nicked form (Form II) and appearance of linearized form of DNA (Form III) in between the two, implicating efficient double stranded cleavage of the DNA [START_REF] Siddique | Lupeol, a novel androgen receptor inhibitor acts as a double-edged sword: Competitive binding as well as transcriptional inhibition[END_REF]. Concentration-dependent (5-25 μΜ) plasmid DNA cleavage experiments of 1-3 depicted the conversion of SC DNA (Form I) to NC DNA (Form II) and upon increasing the concentration of 1-3, there was the appearance of linearized form (Form III) which suggested double-stranded cleavage of plasmid DNA induced by 1-3, with the significantly higher cleavage exhibited by complex 2 at 20 μΜ (Fig. 12 a and S4).

Furthermore, the plasmid DNA cleavage activity of 2 was investigated to examine mechanistic pathway in the presence of additives viz., DMSO and EtOH as hydroxyl radical scavengers, NaN 3 as singlet oxygen scavenger ( 1 O 2 ) and SOD as superoxide anion radical (Fig. 12 b) . During the reaction, it was ascertained that there was no significant quenching of plasmid DNA cleavage efficiency in presence of DMSO and EtOH, hence excluding the possibility of hydrolytic cleavage pathway, involving diffusible hydroxyl radicals in the cleavage reaction. However, in the presence of SOD and NaN 3 significant cleavage was observed, suggesting the oxidative cleavage pathway of complex 2 and demonstrated that ROS, singlet oxygen and superoxide anion radical could be responsible for cleavage activity [START_REF] An | Double-strand DNA cleavage by copper complexes of 2, 2′-dipyridyl with electropositive pendants[END_REF] The further prediction of cleavage activity of complex 2 was discerned in presence of activators such as H 2 O 2 , glutathione (GSH), 3-mercaptopropionic acid (MPA) and methyl green (MG) which revealed the significant enhancement of DNA cleavage activity. The activity efficacy for complex 2 followed the order as H 2 O 2 > GSH > MPA > MG.

Synthesis of complex 2

Complex 2 was synthesized by stirring ethanolic solutions of copper (II) nitrate trihydrate (0.241 g, 1 mmol) with 6-bromo-3-formylchromone (.506 g, 2 mmol) in the molar ratio 2:1 under reflux conditions for 4h. The resulting solution was filtered and allowed to evaporate slowly at room temperature. Very fine green coloured crystals suitable for single X-ray crystallography were obtained after 3-4 days of slow evaporation of the reaction mixture which were found to be quite

Molecular docking studies

The molecular docking studies were performed by employing HEX 8.0 software. [START_REF] Gonzalez-Alvarez | Oxidative Cleavage of DNA by a New Ferromagnetic Linear Trinuclear Copper(II) Complex in the Presence of H 2 O 2 /Sodium Ascorbate[END_REF][START_REF] Mustard | Docking essential dynamics eigenstructures[END_REF] The B-DNA dodecamer d(CGCGAATTCGCG) 2 (PDB ID : 1BNA) crystal structure was downloaded from the http://www.rcsb.org./pdb (protein data bank). Before employing docking experiments, the structure of complexes 1-3 were converted into PDB format and the visualization was done by using Discovery Studio molecular graphics program.

Computational studies

Single-point density functional theory (DFT) calculations and geometry optimization were carried out by the using hybrid B3LYP functional. The optimized structure was further calculated using def2-TZVP basis set for all atoms to calculate the HOMO and LUMO energies.

The def2-TZV/J main, and def2-SVP/J auxiliary, Coulomb fitting basis set were used as implemented in the ORCA, to accelerate the calculations.

In vitro binding and cleavage experiments

The DNA binding experiments were performed in Tris-HCl/NaCl (5:50 mM) buffer at pH 7.3 by employing absorption spectroscopy, emission spectroscopy, cyclic voltammetry and circular dichroism following previously used standard methods adopted by our laboratory [START_REF] Macindoe | HexServer: an FFT-based protein docking server powered by graphics processors[END_REF][START_REF] Arjmand | Design, synthesis and crystal structure determination of dinuclear copper-based potential chemotherapeutic drug entities; in vitro DNA binding, cleavage studies and an evaluation of genotoxicity by micronucleus test and comet assay[END_REF] The cleavage experiments of complexes 1-3 were carried out using agarose gel electrophoresis in Tris-HCl/NaCl (5:50 mM) buffer at pH 7.3 using supercoiled pBR322 DNA (300 ng).

Cytotoxic studies

In vitro antitumor screening of complexes 1-3 was performed on human cancer lines of two different histological origin viz., Du145 (prostate) and Huh7 (Liver). The effect of complexes 1-3 on cell viability was determined by MTT assay. Briefly, cells were seeded in 96-well dishes at a density of 3.5 × 10 3 cells per well. Cells were allowed to attach for 24h before incubation with fresh medium containing either complex 1/2/3 (2.5-10.0 mM) for 48h. After incubation for 48h at 37 0 C, MTT assay was performed as described previously [START_REF] Chauhan | DNA binding studies of novel copper (II) complexes containing l-tryptophan as chiral auxiliary: in vitro antitumor activity of Cu-Sn 2 complex in human neuroblastoma cells[END_REF].

Description of X-ray crystal structures

Single crystal X-ray structural studies of complexes 1-3 were performed on a D8 VENTURE Bruker AXS diffractometer, employing graphite-mono-chromated Mo-Kα radiation generated from a fine-focus sealed tube (λ = 0.71073 °A) at 150 K. The structure was solved by dual-space algorithm using the SHELXT program and further refined with full-matrix least-square methods based on F2 (SHELXL). All non-hydrogen atoms were refined with anisotropic atomic displacement parameters. H atoms were finally included in their calculated position. The drawing of the complex was realized with PLATON [START_REF] Spek | PLATON Procedure, A Multipurpose Crystallographic Tool[END_REF] A summary of selected crystallographic information for complexes 1-3 is given in Table 2.

Scheme 1

 1 Scheme 1 Synthetic route for complexes 1, 2 and 3

  0 . The complex shows slight distorted octahedral geometry CuO 6 coordination environment with ligation from four carbonyl oxygen [Cu-O=1.9325(12)] of fluoro substituted chromone ligand scaffold and two oxygen [Cu-O=1.958 (13)] from solvate ethanol moiety. The bond lengths of Cu-O at axial positions were slightly larger due to static Jahn-Teller distortion. Further extension of the crystal structure occurs through π-π stacking interactions, resulting in the 3D supramolecular architecture as shown in Fig. 1. The ORTEP view of the complex 2 is shown in Fig. 1. The O-O donor bromine substituted formylchromone ligand scaffold binds to the Cu (II) ion to form 6-membered chelate rings. Due to static Jahn-Teller distortion, there was considerable variations in equatorial and axial bond lengths. The complex crystallized in the triclinic system in P-1 space group. The optimized lattice parameters are a = 5.1134 (5) Å, b = 9.7980 (9) Å, c = 14.9650 (14) Å and α = 85.415 (3) °, β = 83.430(3) °, γ = 89.988(4) °. Single crystal X-ray study of complex 3 revealed that it crystallized in the triclinic P-1 space group possessing the lattice parameters, a = 5.1349 (4) A 0 , b = 11.6066 (9) A 0 , c = 12.9630 (11) A 0 and α = 105.733 (3) 0 , β = 97.741 (3) 0 , γ = 90.384 (3) 0 per unit cell. An ellipsoid view of complex 3 along with atom numbering scheme is depicted in fig. 1. The structure of complex 3 represent a centrosymmetric Cu(II) ion coordinated to a methyl substituted chromone ligand scaffold through O,O-atoms with a solvate ethoxy group at 2-position along with two ethanol moieties at the axial position. All the Cu-O bond distances in complexes 1-3 were observed within the range reported

Fig. 1 .

 1 Fig. 1. Single crystal X -ray structure of complexes 1 -3 (50 % probability ellipsoids).

Fig. 2 .

 2 Fig. 2. Absorption spectra of complexes 1 (a), 2 (b) and 3 (c) in absence and presence of increasing concentrations of ct-DNA in Tris -HCl buffer (pH=7.3). Inset: plots of [DNA]/(ϵ a -ϵ f) (M 2 cm) vs [DNA] for the absorption titration with 1-3. The arrows specify the change (increase/decrease) in intensity of absorbance with increasing ct-DNA.

Fig. 3 .

 3 Fig. 3. Electrostatic potential surface depicting σ-hole in blue colour (a) in complex 2. Selected halogen (Br) interactions of complex 2 with nucleobases, cytosine (a) and guanine (b). Hydrogen atoms have been omitted for clarity.

Fig. 4

 4 Fig. 4 Emission spectra of EB-ct-DNA in the absence and presence of complexes 1 (a), 2 (b) and 3 (c) in Tris-HCl buffer at pH 7.3. Arrow indicates change in intensity with increasing concentration of complexes 1-3.

Fig. 5 .Fig. 6 .

 56 Fig. 5. X-band EPR (LNT) spectra of complex 2 (red), complex 2 + ct-DNA (blue).

Fig. 7 .

 7 Fig. 7. CD spectra of complexes 1-3

Fig. 8 .

 8 Fig. 8. SEM images of 1 (a), 2 (c), 3 (e) showing surface morphology upon condensation with ct-DNA (b, d & f, respectively).

Fig. 9

 9 Fig. 9 Molecular docked model of complex 1 (a) complex 2 (b) and complex 3 (c) fitted into the G-C region of the minor groove of the ct-DNA dodecamer duplex of sequence d(CGCGAATTCGCG) 2 , PDB ID: 1BNA.

Fig. 10 .

 10 Fig. 10. Frontier molecular orbitals of complexes 1-3 using the B3LYP/DFT method.

Fig. 11 .

 11 Fig. 11. Survival of tumor cells as a function of different concentrations of 1, 2 & 3 determined by MTT test, after 48 h incubation.

Fig. 12 .Experimental Materials and instrumentation 6 - 3 Synthesis of complex 1 Complex 1

 126311 Fig. 12. Agarose gel electrophoresis demonstrating the cleavage of plasmid pBR322 DNA (300 ng) by complex 2 at 37 °C in the presence of different exogenous agents. (a) Lane 1, DNA control; lane 2, DNA + 2 (5 μM); lane 3, DNA + 2 (10 μM); lane 4, DNA + 2 (15 μM); lane 5, DNA + 2 (20 μM); (b) lane 1, DNA control, lane 2, DNA + 2 (20 μM) + H 2 O 2 (0.4 μM), lane 3, DNA + 2 (20 μM) + NaN 3 (0.4 μM), lane 4, DNA + 2 (20 μM) + SOD (0.4 μM), lane 5, DNA + 2 (20 μM) + MPA (0.4 μM), lane 6, DNA + 2 (20 μM) + EtOH (5 μL); lane 7, DNA + 2 (20 μM) + DMSO (5 μL); lane 8, DNA + 2 (20 mM) + GSH (20 μM); lane 9, DNA + 2 (20 μM) + Methyl green ( 0.4 μΜ).

a

  GoF is defined as {Σ [ w (F 0 2 -F c 2 )]/(n-P)}1/2 where n is the number of data and p is the number of parameters.b R = {Σ||F 0 |-|F c ||/Σ|F 0 |, wR 2 = {Σ w(F 0 2 -F c 2 ) 2 /Σ w(F 0 2 ) 2 } 1/2 .ConclusionsNew Cu (II) drug entities 1-3 derived from substituted (F -, Br -, -CH 3 ) bioactive chromone pharmacophore were synthesized as antitumor chemotherapeutic agents. The complexes 1-3 were thoroughly characterized by various spectroscopic techniques (IR, UV-vis, EPR and ESI-MS) and single X-ray crystallography. Interaction of complexes 1-3 with ct-DNA was carried out employing UV-vis, fluorescence, cyclic voltammetry, EPR and circular dichroism techniques. The corroborative results of these experiments with ct-DNA showed higher binding propensity of complex 2 as compared to complexes 1 & 3. It was observed that complex 2 facilitates strong halogen bonding interaction via, Br -and stability of halogen bond was rationalized by the presence of sigma hole region as observed by gas phase B3LYP computational DFT studies. Moreover, the cleavage activity of complexes 1-3 with pBR322 DNA suggested double strand cleaving ability of the complexes and the mechanism was mediated by oxidative cleavage pathway involving SOD and NaN 3 . In vitro cytotoxic activity of complexes 1-3 was carried out on a panel of two human cancer cell lines by MTT assay and the growth curve data revealed greater potency of complex 2 towards human liver carcinoma (Huh-7) and prostate cancer (DU-145) cell lines than the other two complexes 1 & 3.

  stable towards air and soluble in organic solvents such as EtOH, THF, DMF and DMSO. Yield: 76%, melting point: 209 o C. CCDC no. 1987454. Elemental analysis calculated for C 28 H 32 Br 2 CuO 10 : C, 44.73; H, 4.29. Found: C, 44.78; H, 4.35. FT-IR (KBr, υ max /cm -1 ): 3089 υ(-C-

	H), 1656 υ(-C=O), 1045 υ(-C-O), 535 υ(-C-Br). UV-vis (1×10 -3 M, DMSO, λ max , nm): 296, 315,
	380 and 660.
	Synthesis of complex 3
	Complex 3 was synthesized by stirring ethanolic solutions of copper (II) nitrate trihydrate (0.241
	g, 1 mmol) with 6-methyl-3-formylchromone (.376 g, 2 mmol) in the molar ratio 2:1 under reflux
	conditions for 6h. The resulting solution was filtered and allowed to evaporate slowly at room
	temperature. Green crystals suitable for single X-ray crystallography were obtained after 5-6 days
	of slow evaporation of the reaction mixture which were found to be quite stable towards air and
	soluble in organic solvents such as EtOH, THF, DMF and DMSO. Yield: 83 %, melting point: 223
	o C. CCDC no. 1969139. Elemental analysis calculated for C 30 H 38 CuO 10 : C, 57.91; H, 6.16. Found:
	C, 57.96; H 6.23. FT-IR (KBr, υ max /cm -1 ): 2974 υ(-C-H), 1606 υ(-C=O), 1060 υ(-C-O). UV-vis
	(1×10 -3 M, DMSO, λ max , nm): 298, 335, 357 and 698.

Table 2

 2 Crystallographic data for complexes 1-3. Formula C 28 H 32 CuF 2 O 10 C 28 H 32 Br 2 CuO 10 C 30 H 38 CuO 10

	Parameters	1	2	3
	CCDC	1970411	1987454	1969139
	Fw (g mol -1 )	630.07	751.89	622.14
	Crystal system	Monoclinic	Triclinic	Triclinic
	Space group	P21/n	P-1	P-1
	a (Å)	9.9291(4) Å	5.1134(5)	5.1349(4)
	b (Å)	5.1435(2) Å	9.7980(9)	11.6066(9)
	c (Å)	27.7256(10) Å	14.9650(14)	12.9630(11)
	α (deg)	90 °	85.415(3) °	105.733(3) °β
	(deg)	98.1990(10) °	83.430(3) °	97.741(3) °γ
	(deg)	90 °	89.988(4) °	90.384(3) °U
	(Å 3 )	1401.48(9)	742.43(12)	736.10(10)
	ρ calc (g/cm 3 )	1.493	1.682	1.403

Acknowledgements

The authors are thankful to SAIF, CIL, Panjab University, and Chandigarh for providing elemental analysis facility. The authors also acknowledge the University Sophisticated Instrumentation Facility (USIF), Aligarh Muslim University, for carrying out the SEM studies. The author (Salman Khursheed) sincerely expresses his gratitude to University Grants Commission (UGC), New Delhi, for providing the fellowship through UGC-assisted DRS-SAP, DST-FIST and DST-PURSE Programme.

Conflicts of interest

There are no conflicts to declare.

Highlights

 Substituted chromone Copper (II) complexes were synthesized and characterized.  DFT computational studies were also performed.  Electrophoretic studies revealed an oxidative cleavage pathway.  Cytotoxicity was evaluated against the human liver carcinoma (Huh-7) and prostate cancer (DU-145) cell lines.  Copper (II) complex 2 exhibited selective cytotoxicity against DU-145 cell line at GI 50 =1.6μM.