Synthesis and physicochemical, DFT, thermal and DNA-binding analysis of a new pentadentate N3S2 Schiff base ligand and its [CuN3S2]2+ complexes

A new N3S2 pentadentate Schiff base ligand derived from 5-bromothiophene-2-carbaldehyde, (E)-N1-((5-bromothiophen-2-yl)methylene)-N2-(2-((E)-((5-bromothiophen-2-yl)-methylene amino) ethyl ethane-1,2-diamine, is prepared. The ligand and its complexes are subjected to extensive physical and theoretical analyses and the results are consistent with their predicted compositions. Dicationic Cu(ii) complexes ([CuN3S2]X2) with a coordination number of 5 are proposed on the basis of the spectral data with N3S2 serving as a pentadentate ligand. The prepared complexes display a square pyramidal geometry around the Cu(ii) center. TG shows different thermal behavior for the N3S2 ligand and its complexes. Solvatochromism of the complexes is promoted by the polarity of the solvent used. A one-electron transfer Cu(ii)/Cu(i) reversible redox reaction is promoted by CV. SEM and EDS of the free ligand and its complexes support the morphology and composition changes observed upon the complexation of Cu(ii). As an outstanding goal to develop anticancer new metal chemotherapy, preliminary studies of the binding of the desired complexes with DNA were carried out, as it is through judging the strength of interactions that a future drug can be designed and synthesized. The viscosity and absorption results obtained for complex 1 indicated its enhanced CT-DNA binding properties as compared to those of complex 2 with Kb values of 3.2 × 105 and 2.5 × 105 M−1, respectively.


Background
Multidentate ligands are frequently used in organometallic chemistry because of their poly-bonding ability to coordinate with several metal ions. 1-3 Consequently, systems containing multidentate ligands are critical towards the success of coordination studies. 3 One of the most famous multidentate ligands are Schiff bases, which have attracted the attention of researchers due to their ability to coordinate to metals via several sites, which can stabilize novel structures around the metal center. [3][4][5][6][7][8] In general, Schiff base-transition metal complexes that have been extensively investigated because the Schiff base ligand can coordinate with metal ion centers via one or more sites leading to the synthesis of several types of complex with different metal centers and stereochemistry, and a broad range of applications. [8][9][10] Recently, several reports have shown that these ligands and their complexes can be used toward the design of new drug candidates exhibiting anti-cancer, enzyme inhibition, anti-malarial, antifungal, antibacterial, and anti-inammatory activity. [8][9][10][11][12][13][14][15][16] Copper is essential in the human body and plays a critical role in biological processes involving electron transfer reactions. In fact, Cu(II) complexes bearing {S, O, N} donor chelating ligands are excellent anti-cancer agents because of their strong DNA binding ability. [4][5][6][7][8][9][10] Due to the highly selective permeability of copper(II) ions through the cell membrane of cancer cells, copper is considered to be one of the most effective anti-tumor agents with low cost and few side effects. [7][8][9][10][11][12][13][14][15][16] Thus, various complexes bearing several types of ligand have been prepared and evaluated against cancer cells. [12][13][14][15][16][17][18][19][20][21][22][23][24] Pentadentate Schiff base ligands have received less attention as compared to mono-, di-, tri-, and tetradentate ligands due to the difficulty in their synthesis and unexpected multimode coordination behavior. [15][16][17][18][19][20][21][22][23][24] In view of the several coordination modes exhibited by pentadentate N 3 S 2 ligands derived from thiophene, their interesting structures, and the CT-DNA binding affinity of their Cu(II) complexes, we herein report two mononuclear copper(II) complexes obtained using a new pentadentate N 3 S 2

Material and instrumentation
All chemicals and solvents were purchased from Sigma and used without any further purication. TLC was performed to evaluate the purity of the as-synthesized compounds when needed. Elemental analysis was carried out on an Elementar-Vario EL analyzer. TG/DTG curves were recorded on a Perkin-Elmer thermogravimetric analyzer. FT-IR spectroscopy was recorded on a Perkin-Elmer Spectrum 1000 FT-IR spectrometer. UV-Vis spectroscopy was recorded on a TU-1901double-beam UV-visible spectrophotometer. All electrochemical experiments were carried out at room temperature under an argon atmosphere using a three-electrode cell Voltalab 80 potentio-state PGZ402 equipped with a Pt-electrode (Metrohm, A ¼ 0.0064 cm 2 ) used as the working electrode and platinum wire spiral (£1 mm) with a diameter of 7 mm used as the counter electrode.

Synthesis of
5-Bromothiophene-2-carbaldehyde (0.026 mol) was added to diethylenetriamine (0.013 mol) in the absence of solvent and the resulting mixture was stirred for 30 min at RT until a viscous oil was formed. The temperature increased and the viscosity of the reaction mixture ensured the condensation reaction occurred. Dichloromethane (10 mL) was added to the reaction mixture and the resulting solution was sealed and stirred for 1 h. A colorless oily product was obtained aer evaporation of the solvent.
Yield: 85%. The product was a colorless oil at RT.

Synthesis of complexes 1-2
A solution of N 3 S 2 (0.18 mmol) in EtOH (5 mL) was added to the Cu(II) salt (0.17 mmol) dissolved in freshly distilled EtOH (20 mL). The color of the reaction mixture changed from brown to blue and the temperature increased upon the addition of the ligand solution. The product complex is poorly soluble in EtOH and precipitates from the reaction mixture. A reduction in the volume of the reaction mixture under vacuo led to most of the blue Cu(II) complex being precipitated, which was then ltered and washed with cooled EtOH and ethyl acetate. 2.3.

DNA binding experiments
The experimental titration absorption spectra were recorded in Tris-HCl buffer (5 mM Tris-HCl/50 mM NaCl buffer at pH 7.2) using a Cu(II) complex concentration of 5.0 Â 10 À5 M (complex 1) and 1.0 Â 10 À5 M (complex 2) throughout the experiment. The CT-DNA concentrations were varied between 0 and 5.0 Â 10 À5 M (complex 1) and 0 and 1.0 Â10 À3 M (complex 2), maintaining the total mixture volume constant at 10.0 mL. The resulting mixed solution of Cu(II)/CT-DNA was allowed to equilibrate for 10 min at RT for each experiment prior to carrying out the absorption measurement.
The desired Cu(II) complexes were prepared by mixing an equivalent amount of N 3 S 2 with the hydrated CuX 2 salt in EtOH at RT under an atmosphere. The preparation of complexes 1 and 2 was conrmed by the color and temperature changes observed in the reaction. The addition of the colorless ligand solution to the copper salt solution was accompanied by a distinct color change from brown to blue and the isolated complexes were characterized using spectroscopic, electrochemical, and thermal analysis.
The complexes are very soluble in coordinating-solvents such as water, DMSO, and DMF, and poorly soluble in alcohols including ethanol, which strongly indicates the complexes are ionic. The molar conductivity of the aqueous complex solutions was 185-210 mS cm À1 , which is within the range observed for a [1 : 1] electrolyte.
The structures of the N 3 S 2 ligand and its complexes were analyzed using EA, MS, FT-IR, CV, NMR, UV-Vis, SEM, EDS, and TG/DTG. DFT calculations on the free ligand were performed using Gaussian 09 soware. The analysis of the complexes revealed the construction of [Cu:N 3 S 2 ]X 2 -type square pyramid complexes. The mass spectra, conductance, and water solubility of the as-prepared complexes supported their dicationic mononuclear salt nature, as illustrated in Scheme 1.

Optimized structure of N 3 S 2
Since the ligand is an oil at RT, it was not possible to collect a stable and suitable crystal for X-ray analysis. Subsequently, the structure of the N 2 S 2 ligand was optimized at the DFT-B3LYP level of theory, as depicted in Fig. 1. The optimized molecular structure of the N 2 S 2 ligand revealed the (E,E)-isomer of both C]N groups was the kinetically favored isomer with the least internal strict repulsion effect, which forces the S-heterocyclic rings to be in the same plane creating a semi-vacant site center suitable to be occupied by metal ions. The presence of two aromatic rings conjugated to the two C]N groups results in the increased acidity of the N-H group in the ligand.

1 H and 13 C-NMR spectra of the N 3 S 2 ligand
The experimental 1 H-NMR spectrum of the N 3 S 2 ligand was recorded in CDCl 3 and shown in Fig. 3a; the theoretical spectrum is depicted in Fig. 3b and a comparison between the theoretical and experimental 1 H NMR spectra is shown in Fig. 3c. The 1 H NMR spectrum shows two sharp triplet signals at d ¼ 2.4 and 2.9 ppm corresponding to ]NCH 2 CH 2 NHCH 2 -CH 2 N] and ]NCH 2 CH 2 NHCH 2 CH 2 N], respectively. No signal for the NH proton was detected due to the rapid D/H exchange parallel to the CDCl 3 /CHCl 3 singlet observed at d ¼ 7.2 ppm (the NH proton appears as a broad singlet peak at d ¼ 4.3 ppm in the freshly prepared NMR solution of the ligand), which is in consistent with the calculated acidity of the NH group. The thiophene protons were observed as two multiplet peaks at d ¼ 6.7 and 7.6 ppm, and the azomethine proton (N] CH) was detected as a singlet at d ¼ 8.2 ppm.
The theoretical 1 H-NMR spectrum shows several peaks belonging to the aliphatic, thiophene, and azomethine protons in the N 3 S 2 ligand, which are consistent with the experimental spectrum. For comparison, the theoretical 1 H-NMR spectrum was plotted against the experimental one, as depicted in Fig. 3c. Fig. 3c shows the matched linear relationship between the theoretical and experimental 1 H-NMR spectra, which reects the excellent degree of agreement.

FT-IR and DFT-IR spectroscopy
FT-IR spectroscopy was utilized to monitor the condensation reaction during the ligand formation step. The FT-IR spectra of the starting materials, 5-bromothiophene-2-carbaldehyde and N1-(2-aminoethyl)ethane-1,2-diamine, before and aer the condensation reaction were recorded (Fig. 5). The formation of This journal is © The Royal Society of Chemistry 2020 the ligand was conrmed by two major changes: (1) the primary N-H stretching vibration in diethylenetriamine observed at 3340 and 3270 cm À1 (Fig. 5a) is reduced to one single peak at 3240 cm À1 due to the formation of the secondary amine in the ligand (Fig. 5b); and (2) the stretching vibration belonging to the C]O group in carbaldehyde observed at 1742 cm À1 is shied by $60 cm À1 due to the formation of the C]N-group (1688 cm À1 ) in the ligand (Fig. 5c).
DFT-IR calculations were also carried out for the free ligand, as shown in Fig. 5d. The experimental result was expected to be lower than the theoretically calculated value because the DFT-IR calculations were performed for a free molecule in the gaseous state, while the experimental spectrum was recorded in the solid state. [25][26][27] The theoretical and experimental FT-IR spectra are illustrated in Fig. 5c and d for comparison, in which the vibrational frequencies and intensities were in agreement with each other.
The FT-IR spectra of the as-synthesized complexes are similar to that observed for the free ligand with slight shis in the peak positions (Fig. 6). Fig. 6 illustrates the differences observed in the FT-IR spectra recorded for the ligand and complex 2. In complex 2, the water peak vibrations are observed at $3425 (n (O-H) ) and 1422 (n (bend) ) cm À1 indicating the presence of uncoordinated water molecules in the lattice of the complex and not in the ligand, since the complex is water soluble, but the ligand is not. The n (N-H) band observed at 3250 cm À1 in the complex was shied to a lower wavenumber with high intensity compared to that of the N 3 S 2 ligand (3320 cm À1 ), which may be attributed to the coordination of the NH group to the copper metal center. In addition, the n (C]N) vibration peak of the complex was shied by $23 cm À1 (from 1688 to 1665 cm À1 ) due to the formation of the C]N / Cu(II) bond. The most important observation in this FT-IR study was the presence of a sharp peak at 510 cm À1 in the complex spectrum due to the n (Cu-N)

Frontier molecular orbital calculations
An evaluation of the HOMO/LUMO energies is benecial toward estimating the chemical behavior of the N 3 S 2 ligand. The HOMO/LUMO energy gap controls many chemical reactivity descriptors, such as hardness, electrophilicity, quantum chemistry terms, chemical potential, electronegativity, and local reactivity. [25][26][27][28] For example, the nucleophilicity of the ligand can be evaluated by its ability to donate electrons, which is associated with the HOMO energy level, while the electron affinity is characterized by the LUMO. Fig. 7 shows a schematic representation of the HOMO/LUMO orbitals in the gaseous phase. The HOMO is located at À5.2311 eV, while the LUMO is located   at À1.5252 eV with an energy gap of 3.7059 eV. The calculated energy gap value reveals the ease of HOMO to LUMO electron excitation, which is reected in the HOMO being the predominant molecular orbital and consistent with overall nature of the pentadentate ligand as a strong electron donor with a high degree of nucleophilicity. It is very easy for electrons to be excited from the ground to excited state with such a small energy gap. The HOMO/LUMO gap is related to the chemical reactivity or kinetic stability, since both have negative values. Consequently, the HOMO and LUMO decide the chemical stability of the ligand. [22][23][24][25][26][27][28][29][30][31] Several parameters related to the HOMO/LUMO energy gap value have been theoretically calculated and are illustrated Table 1.

UV-Vis spectroscopy
The electronic absorption behavior of the N 3 S 2 ligand and its complexes were measured at RT in ethanol and water, respectively. The absorption bands observed for the ligand were also assigned using theoretical calculations at the TD-DFT/B3LYP/3-21 level of theory. The UV-Vis spectrum of the N 3 S 2 ligand shows highly intense transitions at l max ¼ 240 (sharp) and 280 nm (3.0 Â 10 4 M À1 L À1 ), which correspond to the p-p* transitions, as shown in Fig. 8a. The absorption maxima in the timedependent DFT UV-Vis spectra were observed at 275 (sharp) and $1100 nm (broad, out of range >800 nm), as shown in Fig. 8b. The theoretical UV-Vis calculations of the molecular orbital geometry revealed the visible absorption maximum of the N 3 S 2 ligand corresponds to the electron transition between the HOMO and LUMO. A good agreement between the theoretical-TD-DFT results and experimental UV-Vis spectra was observed and the slight $5 nm shi was attributed to the solvent effect. [26][27][28][29][30] In water, complex 1 and 2 exhibit similar electronic behavior. The signals corresponding to the p-p* electron transition were shied from 280 (free ligand) to $250 nm in the complexes due to the coordination of the ligand to the Cu(II) center. In addition, broad bands in the visible region of 600-640 nm were observed upon complexation with the Cu(II) center, which were not exhibited by the free ligand or CuX 2 starting material. These bands were attributed to the blue color of the resulting N-Cu(II) complexes. The blue color absorption was assigned to the d-d electron transition in the square pyramid geometry around the Cu(II) complex center, as shown in Fig. 9 for complex 1.    Water, EtOH, DMF, and DMSO were used to evaluate the solvatochromism phenomena observed due to the solubility of the dicationic complexes. The UV-Vis spectra of complex 2 recorded in the selected solvents exhibit a broad band at 600-800 nm. The complexes exhibited a signicant positive l max shi upon increasing the polarity of the solvent due to the expected Jahn-Teller effect observed at the Cu(II) center with a d 9 electronic conguration and coordination number of 5.
The UV-Vis spectra of complex 2 shi depending on the solvent's donor number polarity, as shown in Fig. 10a.
Bathochromic shis were recorded due to the direct coordination of the polar solvent molecules to the vacant sites of the ve coordination Cu(II) center with different strengths, which is in accordance with the mechanism of solvatochromism reported for this type of complex. [32][33][34] Accordingly, the l max values observed for complex 2 in the different solvents studied increases linearly when Gutmann's donor number (DN) of the selected solvent increased. The linear trend in l max observed for complex 2 verses DN is shown in Fig. 10b.

Thermal analysis
The thermal behavior of the N 3 S 2 ligand and complex 2 were investigated using TG/DTG under an open air atmosphere over a temperature range of 0-900 C at a heating rate of 10 C min À1 (Fig. 11). Fig. 11a shows the TG curve obtained for the N 3 S 2 ligand, which displays noticeable thermal stability up to 140 C. Decomposition begins aer 140 C and ends at $260 C. The ligand was totally decomposed into light gases including SO 2 , NO 2 , and CO 2 in a broad step with $100% weight loss. No intermediate degradation steps, physical phonemes, or residues were recorded and the compound exhibited a simple onestep thermal decomposition mechanism.
Complexes 1 and 2 exhibit similar thermogravimetric behavior. The TG/DTG curve obtained for complex 2 shows three main steps (Fig. 11b). The rst step (<100 C) corresponds to the loss of uncoordinated water molecules in accordance with the FT-IR results. The second decomposition step at 280-450 C (40% weight loss) was attributed to the decomposition of the ligand from the backbone of complex 2 to give CuBr 2 as the nal product. The third step starts from 460 C and ends at 750 C, which was attributed to the reaction between CuBr 2 and O 2 with the elimination of bromide ions in one broad step to form copper oxide (Cu]O, 18%) as the nal product.

Electrochemistry of complex 2
As a representative example, the electron-transfer conductance of complex 2 in acetonitrile was investigated using cyclic voltammetry, as shown in Fig. 12. The N 3 S 2 ligand is electroinactive  This journal is © The Royal Society of Chemistry 2020 RSC Adv., 2020, 10, 21806-21821 | 21815 from 0 to À1.5 V, while complex 2 exhibits a one electron redox transfer process in this range. The electrochemical behavior observed at the Pt working electrode was E 1/2 ¼ À0.760 V, i pa /i pc ¼ 0.92, and DE p ¼130 mV, and the plot of i pc vs. v 1/2 was linear with slope ¼ 0.991. All these parameters suggest that the Cu(I)/ Cu(II) redox process becomes quasi-reversible with responses at À650 and À780 mV (vs. Ag/AgNO 3 ).

SEM and EDS
The surface morphologies of both the free N 3 S 2 ligand and complex 2 were subjected to SEM and EDS. The SEM image of the free ligand displays a semi-square single phase with a block over block morphology with unequal boundaries and various micrometer volumes (Fig. 13a). The SEM image of complex 2 shows a different morphology with a smooth, homogeneous, and uniform rod-like morphology with various pore sizes (Fig. 13b).
Since the SEM image of the surface of the free N 3 S 2 ligand was different than that of its corresponding complex, the change in the morphology of the ligand before and aer its coordination to Cu(II) conrmed the formation of the L-M complex and allowed us to differentiate the chemical composition of the N 3 S 2 ligand and its complex. Therefore, the composition of the N 3 S 2 ligand and its complex were determined using EDS, as shown in Fig. 13c and d, respectively. Comparison of the spectra indicated that the N 3 S 2 ligand only contains C, N, S, and Br, while its complex contains C, N, S, Br, and Cu, which again conrmed the formation of the copper complex. The absence of O atoms in the ligand and complex indicates the stability of the compounds toward atmospheric O 2 . The absence of unknown peaks reects the high purity of both the ligand and its complex.
3.12. CT-DNA binding affinity of complexes 3.12.1. Absorption spectroscopy. Absorption spectroscopy is considered to be one of the most useful methods to evaluate the DNA binding affinity of a molecule. [35][36][37][38][39][40] The affinity of complex 1 and 2 toward CT-DNA was investigated using the UV-Vis titration spectra recorded in Tris-HCl buffer solution. The UV-Vis spectra of the target compounds were expected to  This journal is © The Royal Society of Chemistry 2020 RSC Adv., 2020, 10, 21806-21821 | 21817 change upon drug-DNA binding. Bathochromic red shi hypochromism interactions are commonly observed due to strong p-p stacking (aromatic-DNA base pairs) and indicate intercalative binding interactions. [38][39][40] To estimate the binding ability of the Cu(II) complexes, the intrinsic binding constant (K b ) was evaluated by monitoring the changes in the absorbance spectra verses the CT-DNA concentration using the following equation. [35][36][37][38] [DNA]/(3 a À 3 f ) where [DNA] is the concentration of DNA in the base pairs and 3 f , 3 a , and 3 b are the free-, apparent-, and metal-bound complex extinction coefficients, respectively. K b is the equilibrium binding constant (M À1 ) for complex 1 bound to DNA. When plotting [DNA]/(3 a À 3 f ) vs.
[DNA], K b was obtained using the ratio of the slope to the intercept. The plot of [DNA]/(3 a À 3 f ) vs.
[DNA] was used to calculate and compare the K b values obtained for complex 1 (Fig. 14) and complex 2 (Fig. 15). Fig. 14 shows the UV-Vis spectra of CT-DNA-1. A high concentration of complex 1 (5 Â 10 À5 M) was used in this experiment in order to monitor the complex absorption behavior in both the UV and visible light regions during the DNA titration experiment. The two characteristic absorption peaks observed at 250 and 625 nm decrease in intensity upon the addition of CT-DNA at different concentrations, as shown in Fig. 14. Fig. 15 shows the UV-Vis spectra recorded during the complex 2-CT-DNA binding titration experiment at 255 nm using a lower concentration of complex 2 (1 Â 10 À5 M).
The UV-abs. spectral titrations behavior of DNA with the Cu(II) complexes were used to make the comparison between the desired complexes with the recent synthesized Schiff bases/ Cu(II) complexes. The small shi in Abs. wavelength and the intercalating binding constant (K b ) values were used as a criterion in judging the [Cu(II):DNA] binding strength as seen in Table 2.
Depending on the results illustrated in Table 1, one can say that complexes 1 and 2 prepared in this study are classied as a good binder among their peers' complexes, as their K b values are higher than all the complexes except complexes in entries 5 and 7. Such high activities can be attributed to the pentadentate coordination mode of N 3 S 2 Schiff base ligand, which gave their Cu(II) desired complexes an additional stability and opportunities to bind the DNA strings in different types and more binding modes, these features are equivalent to those previously observed for Cu(II) complexes. [38][39][40] 3.12.2. Viscosity. To clarify the nature of the Cu(II) complexes upon interaction with CT-DNA and determine which of the complexes is the better binder, the binding modes were investigated using viscosity measurements. The values of the relative specic viscosity were (h/h o ) 1/3 plotted against [complex]/[DNA] (Fig. 16). The viscosity of DNA was increased upon interaction with the complexes because they make the DNA longer. [37][38][39][40] In this study using identical conditions, it was observed that increasing the complex concentration leads to an increase in the DNA viscosity (complex 1 > complex 2). Thus, complex 1 is a slightly better DNA binder than complex 2, which was in agreement with the DNA binding results.

Conclusions
A new Schiff base, (E)-N1-((5-bromothiophen-2-yl)methylene)-N2-(2-((E)-((5-bromothiophen-2-yl)methylene)amino)ethyl)ethane-1,2diamine, was synthesized via the condensation of 5bromothiophene-2-carbaldehyde and diethylenetriamine. The N 3 S 2 ligand was characterized using spectroscopic and theoretical analyses, and the condensation reaction used in its synthesis was  monitored through FT-IR spectroscopy. Water soluble square pyramid dicationic complexes with the general formula [Cu(N 3 S 2 )] X 2 were formed because the N 3 S 2 ligand acts as a pentadentate ligand. The TG results demonstrated the different thermal behavior observed between the free ligand and its complexes. The polarity of the solvent used plays a critical role in controlling the solvatochromatic behavior of the complexes. SEM and EDS supported the complexation of the N 3 S 2 ligand to the Cu(II) metal center. The complexes exhibited a one electron redox transfer process with negative voltages. Both the viscosity and absorption spectra showed that complex 1 is a better CT-DNA binder than complex 2 with K b values of 3.20 Â 10 5 and 2.51 Â 10 5 M À1 , respectively. The hypochromism interactions indicated and intercalative binding between the aromatic-DNA base pairs and the complexes theophine aromatic ring via p-p stacking interactions. The preliminary complex:DNA binding results observed in this study reected the desired complexes as very good binder, which means that such compounds can be used as new chemotherapy.

Conflicts of interest
The authors declare no conict of interest.