Synthesis, Characterization, and In Vitro Cytotoxicity of Unsymmetrical Tetradentate Schiff Base Cu(II) and Fe(III) Complexes

Unsymmetrical tetradentate Schiff base Fe(III) and Cu(II) complexes were prepared by the coordination of some unsymmetrical tetradentate Schiff base ligands with CuCl2·2H2O or FeCl3·6H2O. The obtained complexes were characterized by ESI-MS, IR, and UV-Vis. The spectroscopic data with typical signals are in agreement with the suggested molecular formulae of the complexes. Their cyclic voltammetric studies in acetonitrile solutions showed that the Cu(II)/Cu(I) and Fe(III)/Fe(II) reduction processes are at (−)1.882–(−) 1.782 V and at (−) 1.317–(−) 1.164 V, respectively. The in vitro cytotoxicity of obtained complexes was screened for KB and Hep-G2 human cancer cell lines. The results showed that almost unsymmetrical tetradentate Schiff base complexes have good cytotoxicity. The synthetic complexes bearing the unsymmetrical tetradentate Schiff base ligands with different substituted groups in the salicyl ring indicate different cytotoxicity. The obtained Fe(III) complexes are more cytotoxic than Cu(II) complexes and relative unsymmetric Schiff base ligands.


Introduction
Tetradentate salen-type Schiff bases obtained by the condensation of ethylendiamine derivatives with salicylaldehydes are attracted by many researchers because of their synthetic availability and interesting applications including catalytic, biological chemistry [1,2] and coordination chemistry [3]. Transition metal complexes of tetradentate Schiff base ligands occupy a principal role in coordination chemistry for analysis, catalysis, materials science, and biochemistry [4][5][6][7]. Metallosalens are highly versatile coordination compounds with a wide range of bioinorganic and medicinal chemistry such as enzyme mimics, sensing, bioimaging, and medicinal applications [8,9]. Some metal salen complexes have good DNA binding and RNA cleavage activity [10][11][12].
ey have shown diverse structures and properties generating a variety of stereochemistry and bonding interactions [13,14]. Besides, the synthesis, characterization, and applications of symmetrical tetradentate Schiff base complexes have been thoroughly studied and reported in the literature [15][16][17] recently, transition metal complexes with unsymmetrical tetradentate Schiff base ligands were reported for various applications [18][19][20][21]. Particularly, some synthetic Cu(II) and Fe(III) complexes with unsymmetrical tetradentate Schiff base ligands show potential DNA binding ability and bioactivity [22][23][24][25][26]. In this study, we continue to describe the synthesis, characterization, and in vitro cytotoxicity of the copper(II) and iron(III) complexes with some unsymmetrical tetradentate Schiff bases.

Materials and Methods
Analytical reagent grade chemicals such as o-phenylenediamine (98%) and salicylaldehydes were obtained from Across Organics and used without any purification. All solvents were purified by following the appropriate purification procedures.
Ultra-high performance liquid chromatography combined with hybrid quadrupole time-of-flight tandem mass spectra (UPLC-Q-TOF-MS) of the prepared unsymmetrical tetradentate Schiff base ligands were conducted on an ExionLC AC Series HPLC system coupled with a hybrid quadrupole time-of-flight tandem mass spectrometer (X500R QTOF System) equipped with Turboionspray source. Chromatographic separation was performed on a Kinetex C 18 column (30 mm × 2.1 mm, 1.7 µm), and the column temperature was maintained at 30°C. e mobile phase consisted of methanol and water containing 0.1% formic acid in gradient mode of 50% methanol for 0-5 min and 100% methanol at 5 min with a flow rate of 0.3 mL·min −1 . Electrospray ionization mass spectra (m/z) of the synthetic complexes were estimated on Agilent 6310 Ion Trap spectrometer (ESI-MS). Infrared spectra (4000-400 cm −1 ) were recorded on a Perkin Elmer Spectrum Two spectrophotometer using KBr pellet. 1 H-NMR and 13 C-NMR spectra were determined in DMSO-d 6 solution using a Bruker Advance 500 MHz NMR spectrometer with TMS as the internal standard and chemical shifts (δ) were recorded in ppm. UV-Visible absorption spectra of the complexes (200-600 nm) were measured in methanol solution (2 × 10 −5 M) on Perkin Elmer Lambda UV-35 spectrophotometer at room temperature. e effective magnetic measurements of the obtained complexes (µ eff ) were carried out at room temperature using a magnetic susceptibility balance (Mark 1, serial No. 25179) of Sherwood Scientific Ltd.

Synthesis of Unsymmetrical Tetradentate Schiff Base
Ligands. Unsymmetrical tetradentate Schiff base ligands were prepared by one-pot method including two-step reactions similarly according to the known procedures [27,28]. In the first step, monocondensed half-units were prepared by the condensation of o-phenylenediamine with 5-t-butylsalicylaldehyde or 5-methoxysalicylaldehyde. In the second step, the monocondensed half-unit was mixed with a methanol solution of relative salicylaldehydes. O-phenylenediamine (15.5 mmol) dissolved in methylene chloride (25 mL) was added in a 100 ml flask containing 5-t-butylsalicylaldehyde or 5-methoxysalicylaldehyde (15.5 mmol) and was stirred for 3 h. After the monocondensed half-units were obtained completely by TLC checking, relative salicylaldehyde (15.5 mmol) in methanol (25 mL) was added and the new mixture was stirred under ultrasound for 1 h more and then the productive precipitates were collected after filtered and washed by cold ethanol. e products were recrystallized from ethyl acetate and dried in vacuo.

Preparation of Unsymmetrical Schiff Base Complexes.
Unsymmetrical Schiff base complexes were prepared from the coordination between the obtained unsymmetrical Schiff base ligands and CuCl 2 ·2H 2 O or FeCl 3 ·6H 2 O in a molecular ratio 1 : 1. 1.0 mmol CuCl 2 ·2H 2 O or FeCl 3 ·6H 2 O dissolved in ethanol was added to an ethanol solution of 1.0 mmol ligand. e reaction mixtures were refluxed at the presence of 1.0 mmol Na 2 CO 3 for 3 hrs; then, the reaction mixtures were cooled to room temperature. e productive precipitates were collected after filtered and washed by cold ethanol and then dried in vacuo.

Electrochemical Studies.
e electrochemical studies of all complexes were performed using Zahner IM6 instrument. e cyclic voltammograms of Cu(II) complexes and Fe(III) complexes were recorded using 1.0 × 10 −3 M concentration in acetonitrile solution and 0.1 M LiClO 4 as supporting electrolyte. e working electrode was platinum Bioinorganic Chemistry and Applications wire which was polished, washed, and dried. e reference electrode was Ag/AgCl/KCl and platinum wire was the counter electrode. All experiments were performed in standard electrochemical cells at room temperature at a scan rate of 100 mV·s −1 with the potential window −3 V to +3 V vs Ag/AgCl/KCl reference electrode.

In Vitro
Cytotoxicity. MTT (3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium) method was used to estimate in vitro cytotoxicity of obtained ligands and synthetic complexes. Human cancer cells KB and Hep-G2 were cultured in DMEM with 10% fetal bovine serum, 100 µg/mL streptomycin, 100 units/mL penicillin, and 2 mmol/L L-glutamine at 37°C in a humidified atmosphere with 5% CO 2 and 95% air. Cancer cells were cultivated in 96-well plates for 24 hrs followed by treating with different concentrations of complexes in DMSO and incubated continuously for 48 hrs more. en, testing cells were exposed to 10 µL of freshly prepared MTT (5 mg/ml) solution and incubated for 4 h at 37°C in an atmosphere of 5% CO 2 . e formazan crystals obtained during MTT incubation were dissolved in 100 µL of DMSO. e absorbance was recorded at 540 nm on Genios TECAN spectrophotometer. e experiments were carried out in triplicate for every concentration of the complexes. e percent viable cells were plotted as a function of concentration to determine the IC 50 values presented in Table 1.
In the high resolution mass spectra, Q-TOF-MS, the pseudo-molecular ion signals of the obtained unsymmetrical ligands are observed as [ In  (Table 3).

Electronic Spectra and Magnetic Moments.
On the UV-Vis spectra of the obtained ligands there were three main absorption bands with maximum absorption wavelengths (λ abs ) at about 235 nm (42,553 cm −1 ) assigned to the π ⟶ π * electronic transitions of the aromatic rings, at about 275 nm (36,364 cm −1 ) and 335 nm (29,851 cm −1 ) attributed to n ⟶ π * electronic transitions associated with the transfer of lone pair situated at N and O of C�N and C−O groups, respectively [25]. ere is a little difference between UV-Vis spectra of these unsymmetrical tetradentate ligands H 2 L1-H 2 L5 (Figure 1). Upon complexation, n ⟶ π * transition of ligand shifts to a longer wavelength; this indicates the coordination of ligand to metal [26].
In UV-Vis spectra of unsymmetrical tetradentate Schiff base Cu(II) complexes, besides the main absorption bands of interligand charge transfer transitions with λ abs at about 245-250 nm and 300-360 nm (n ⟶ π * ), a new broad lowenergy absorption band with λ abs is observed at 380-500 nm which can be assigned to ligand-to-metal charge transfer (LMCT) and metal-to-ligand (MLCT) transitions [29,30]. Magnetic measurements and electronic spectra were conducted in order to obtain information about geometry of the complexes. Copper(II) complexes, in the present study, show µ eff values 1.81-2.00 BM which were consistent with presence of one unpaired electron. is behavior suggests square-planar geometry for the copper(II) complexes [31,32].
In UV-Vis spectra of unsymmetrical tetradentate Schiff base Fe(III) complexes, besides the main absorption bands with wavelength maximum at 242-247 nm, 297-305 nm and a shoulder at 372-390 nm which may be assigned to interligand charge transfer transitions (n ⟶ π * ), there is a new broad low-energy absorption band with λ abs at 375-485 nm which can belong to LMCT and MLCT transitions. e d-d bands were also not observed due to the low concentration

Electrochemical Studies.
e electrochemical behaviors of the synthetic unsymmetrical tetradentate Schiff base Cu(II) and Fe(III) complexes were investigated using cyclic voltammetry (CV). Cyclic voltammograms were recorded using a Zahner-elektrik IM6 instrument with a standard three-electrode setup, a platinum working electrode, a platinum wire as the counter electrode, and Ag/AgCl/KCl as the reference electrode, at room temperature with voltage scan rate � 100 mV·s −1 . e concentration of complexes in acetonitrile was 1.0 × 10 −3 M and 0.1 M LiClO 4 was used as supporting electrolyte. e cyclic voltammetric profile of synthetic Cu(II) complexes is given in Figure 4. Interestingly, the CVs of synthetic Cu(II) complexes show cathodic peaks at (−)1.882-(−) 1.782 V for the reduction of Cu(II) ⟶ Cu(I). A similar type of cathodic response was found in reported Cu(II) complexes [33]. Some slight differences in the reduction potentials of these Cu(II) complexes should be attributed to the effects of the electrondonating methoxy and electron-withdrawing halogen substituted groups (Table 4).
Similarly, the cyclic voltammograms of synthetic Fe(III) complexes are given in Figure 5. Synthetic Fe(III) complexes possess well-defined cathodic peaks at (−) 1.317-(−) 1.164 V for the reduction of Fe(III) ⟶ Fe(II) probably. A similar type of cathodic signals was observed in the reported Fe(III) complexes [34]. e reduction progresses of these Fe(III) complexes are seemingly taken easier than the ones of the Cu(II) complexes. Some difference in the reduction   Compound

Conclusions
Series of Cu(II) and Fe(III) complexes with unsymmetrical tetradentate Schiff base ligands were synthesized in good yields and characterized by ESI-MS, IR, UV-Vis, and CV spectroscopies. e characteristic spectra of ligands have changed when the coordination of the ligands to metals was carried out. e electron-donating and electron-withdrawing substituted groups of ligands have some effects on their spectral properties. e strong UV-Vis absorption bands for MLCT of the Cu(II) complexes were observed at 422-440 nm, while the weak UV-Vis absorption bands for

Data Availability
e data used to support the findings of this study are included within the article and the supplementary information file.

Conflicts of Interest
e authors declare that there are no conflicts of interest regarding the publication of this paper.