Synthesis, Spectral Characterization, and In Vitro Cytotoxicity of Some Fe(III) Complexes Bearing Unsymmetrical Salen-Type Ligands Derived from 2-Hydroxynaphthaldehyde and Substituted Salicylaldehydes

Six Fe(III) complexes bearing unsymmetrical salen-type ligands derived from 2-hydroxynaphthaldehyde and substituted salicylaldehydes were synthesized by coordinating the unsymmetrical salen-type ligands with FeCl3.6H2O. ,e synthetic complexes were characterized by electrospray ionization mass spectra (ESI-MS), effective magnetic moments (μeff), and infrared (IR) and ultraviolet-visible (UV-Vis) spectra. ,e spectroscopic data are in good agreement with the suggested molecular formulae of the complexes. ,eir cyclic voltammetric studies in acetonitrile solutions showed that the Fe(III)/Fe(II) reduction processes are electrochemically irreversible. ,e in vitro cytotoxicity of the obtained complexes was screened on human cancer cell lines KB (a subline of Hela tumor cell line) and HepG2 (a human liver cancer cell line) and a normal human cell line HEK-293 (Human Embryonic Kidney cell line). ,e results showed that the synthetic Fe(III) complexes are highly cytotoxic and quite selective. ,e synthetic complexes bearing unsymmetrical salen-type ligands with different substituted groups in the salicyl ring indicate different cytotoxicity.


Introduction
e developments in transition metal complexes have gained considerable attention about various structures and potential applications in catalysis, analysis, advanced materials science, and biochemistry especially [1][2][3][4][5][6][7][8][9]. Besides the meaningful efficiency of platinum complexes as anticancer agents [10][11][12], recent bioinorganic chemists have focused on the design and preparation of new transition metal complexes with Schiff base ligands [13][14][15]. Schiff bases with donors (N, O, etc.) have been widely investigated due to their diverse pharmacological applications [16], in which tetradentate Schiff bases are derived from salicylaldehydes and diamines, which form the Schiff bases known as "salen" with an N2O2 donor group being able to coordinate with different metal ions [17]. ese diamine Schiff bases with OH groups in ortho positions are of interest because of the presence of tautomerism between keto-amine and enolimine forms [18]. e transition metal complexes of tetradentate Schiff bases have received much attention about their structure, magnetic and electrochemical characterization, and their potential application in biological functions lately. ey predominantly show their antiproliferative, antimalarial, antifungal, antipyretic, and antidiabetic activities [19,20]. Besides, many symmetrical tetradentate Schiff bases and their transition metal complexes have been extensively studied on the preparation, spectral characterization, and biological activity [21][22][23]; recently, unsymmetrical tetradentate Schiff base ligands and their complexes have been paid attention [24][25][26]. It should be realized that the coordinated ligands around central metal ions in natural systems are unsymmetrical. erefore, in this work, we continue with the synthesis, spectral characterization, and in vitro anticancer behavior of Fe(III) complexes bearing unsymmetrical salen-type Schiff bases derived from 2-hydroxynaphthaldehyde and substituted salicylaldehydes.

Materials and Methods
Chemical reagents used in the present study, such as ophenylenediamine (98%), 2-hydroxy-1-naphthaldehyde (tech.), and salicylaldehydes, were obtained from Across Organics and used without further purification. All solvents were distilled following the laboratory procedures before use.
Ultrahigh-performance liquid chromatography combined with hydride quadrupole time-of-flight tandem mass spectra (HP-TOF-MS) of the synthetic unsymmetrical tetradentate Schiff base ligands was 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 a 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 (ESI-MS) (m/z) were recorded on Agilent 6310 Ion Trap spectrometer. Infrared spectra (IR, 4000-400 cm −1 ) were carried out on a PerkinElmer Spectrum Two spectrophotometer using a 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) recorded in ppm. UV-Visible absorption spectra of the synthetic compounds (200-600 nm) were estimated in methanol solution (3 × 10 −5 M) with PerkinElmer Lambda UV-35 spectrophotometer. Magnetic susceptibility measurements of synthetic Fe(III) complexes were determined at room temperature using a magnetic susceptibility balance (Mark 1, serial No. 25179) of Sherwood Scientific, Ltd.

Synthesis of Unsymmetrical Salen-Type Schiff Base
Ligands. Unsymmetrical salen-type Schiff base ligands were prepared following a two-step procedure similar to the known procedure [27,28]. In the first step, monocondensed half-units were prepared by the condensation of o-phenylenediamine with 2-hydroxy-1-naphthaldehyde. In the second step, the monocondensed half-unit was mixed with an ethanol solution of relative salicylaldehydes. O-Phenylenediamine (15.4 mmol) dissolved in ethanol (20 mL) was added in a 100 ml flask containing 2-hydroxy-1-naphthaldehyde (15.5 mmol) in ethanol (15 mL) and was stirred for 3 h. After the monocondensed half-units were obtained completely by TLC checking, relative salicylaldehyde (15.5 mmol) in ethanol (15 mL) was added and the new mixture was put in an ultrasonic case, Hwashin Power Sonic 405, for 1 h. en, the productive precipitates were collected after being filtered and washed by cold ethanol. e obtained products are soluble in DMSO, methanol, dichloromethane, and ethyl acetate. e products were recrystallized from ethyl acetate and dried in vacuo. (

Preparation of Unsymmetrical Salen-Type Schiff Base
Complexes. Unsymmetrical salen-type Schiff base complexes were prepared from the obtained ligands and FeCl 3 .6H 2 O in 1 : 1 molecular ratio following the published procedure similarly [28][29][30]. 1.0 mmol FeCl 3 .6H 2 O dissolved in ethanol was added to an ethanol solution of 1.0 mmol ligand. e reaction mixture was refluxed in the presence of 1.0 mmol Na 2 CO 3 for 3 hrs; then, the reaction mixture was cooled to room temperature. e productive precipitate was collected after being filtered and washed by cold ethanol and then dried in vacuo. [

Electrochemical Studies.
e electrochemical studies of all complexes were carried out using the Zahner-elektrik IM6 instrument. e cyclic voltammograms of Fe(III) complexes were recorded using 1.0 × 10 −3 M concentration in acetonitrile solution and LiClO 4 0.1 M as supporting electrolyte. e working electrode was a platinum electrode. e reference electrode was Ag/AgCl/KCl and platinum wire was the counterelectrode. 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.
e different concentrations of each tested complexes were 128.0, 32.0, 8.0, 2.0, and 0.5 μg/ml. en, tested human cells were exposed to 20 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 optical density (OD) was determined at 550 nm on Genios TECAN spectrophotometer.
e experiments were done in triplicate for every concentration of the tested complexes. e cell viability was calculated with regard to the untreated cell control (positive control), which was set to 100% viability. e dead cell control (negative control) was set to 0% viability. (1) e percent cell inhibitions were plotted as a function of concentration to estimate the IC 50 (the concentration at which a substance exerts half of its maximal inhibitory effect) values presented in Table 1.
e Fe(III) complexes were prepared following the coordination of FeCl 3 .6H 2 O with each obtained ligand in good yields (85-94%) in ethanol at pH 9.0 (Scheme 1). e synthetic unsymmetrical tetradentate Schiff base complexes are soluble in DMSO, acetonitrile, methanol, and dichloromethane. ese complexes were characterized by ESI-MS, IR, UV-Vis spectroscopies, and magnetic moments (μ eff ).
From the high-performance mass spectra, the pseudomolecular ion signals of the obtained ligands are found as [M + H] + , clearly indicating molecular masses suitable for the suggested formulae. On ESI-MS spectra of synthetic unsymmetrical complexes, pseudomolecular ion peaks are assigned to [M-Cl] − for Fe(III) complexes. ey are in excellent agreement with the suggested formulae ( Table 2).
On 1 H-NMR spectra, usually, a typical signal at about 15.63 ppm was found for a proton of OH of naphthyl group as a singlet [35], but typical signals at 15.63-15.81 ppm as doublets, so there must be tautomerism between keto-amine and enol-imine forms here (Scheme 1) and protons of NH groups were found.
ere are typical signals at 10.81-11.93 ppm as singlets were assigned to protons of OH of salicyl groups [36]. e typical signals at 9.56-9.60 ppm as doublets were probably observed for protons of HC−N groups and at 8.98-9.03 ppm as singlets are for protons of HC � N groups. e proton signals of aromatic groups were found at about 6.80-8.44 ppm. When the salicyl group containing fluor (H 2 L2) proton signals were observed as multilets as usual, there are proton signals at 1.34 ppm as singlet for 9 protons of t-butyl and at 3.88 ppm as singlet for 3 protons of methoxy group.
On 13 C-NMR spectra, there are typical signals at 174.31-176.65 ppm for C 16     ere are few differences in UV-Vis spectra of the ligands with different substituted groups.
UV-Vis spectra of synthetic unsymmetrical salen-type Fe(III) complexes are obtained in Figure 2. Upon complexation, the maximum absorption bands of obtained ligands were shifted to different wavelengths, indicating the coordination of the ligands to metal [29].
e absorption bands with wavelengths of maximum absorption at 223-234 nm were assigned to π⟶π * electronic transition of aromatic rings; 295-300 nm and 334-342 nm could present for n⟶π * electronic transitions of free electrons on N and O of C�N and C-O. A band is observed at 431-440 nm, which can be assigned to the electronic transitions of C�O. A weak band is observed at about 500 nm, which can be assigned to LMCT transitions [24,37]. e d-d bands were not observed due to the low concentration (3.0 × 10 −5 M) of the solutions. ese bands should be of low intensity in the region of 550-650 nm. ere are also small differences in UV-Vis spectra of Fe(III) complexes bearing ligands with different substituted groups.
Fe(III) complexes exhibit effective magnetic moment values of 5.78 − 6.00 BM due to the presence of high-spin five unpaired electrons, which indicate an octahedral geometry around Fe(III) ions [38,39].

Electrochemical Studies.
e electrochemical behaviors of the synthetic unsymmetrical salen-type Fe(III) complexes were studied using cyclic voltammetry (CV). Cyclic voltammograms were recorded using a Zahnerelektrik IM6 instrument with a standard three-electrode   setup, a carbon graphite as the working electrode, a platinum wire as the counterelectrode, and Ag/AgCl as the reference electrode, at room temperature with 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 voltammograms of synthetic Fe(III) complexes are shown in Figure 3. Synthetic Fe(III) complexes possess well-defined cathodic peaks at (−) 0.603-(−) 0.693 V for irreversible reduction of Fe(III) ⟶ Fe(II) probably. A similar type of cathodic signal was observed in the reported Fe(III) complexes [40]. Some differences in the reduction potentials of the Fe(III) complexes must be expected from the electronic effects of the electron-withdrawing and electron-donating substituted groups (Table 4). showed the worse cytotoxic activity in several times for the normal human cell line HEK-293 with IC 50 being 9.33 and 6.34 μM, respectively.

In Vitro
is selectivity is quite good and similar to the selectivity of ellipticine with IC 50 � 6.69 μM for HEK-293.

Conclusions
In this study, we have reported the synthesis and characterization of novel Fe(III) complexes bearing various unsymmetrical salen-type ligands.
e ligands with electron-donating and electron-withdrawing substituted groups have some effects on their spectral properties. e UV-Vis absorption bands for LMCT of the Fe(III) complexes were observed at 430-440 nm. Interestingly, the cyclic voltammograms of the Fe(III) complexes show cathodic peaks for irreversible reduction of Fe(III)⟶ Fe(II) at (−) 0.603-(−) 0.693 V. e obtained Fe(III) complexes were all estimated on the cytotoxicity in vitro for KB and HepG2 human cancer cells.
e results showed that the synthetic Fe(III) complexes have excellent cytotoxicity for KB and HepG2 human cancer cells (IC 50 < 15 and 65 μM, respectively). Among them, [Fe(III)L1Cl] and [Fe(III)L6Cl] showed the best cytotoxic activity for KB and HepG2 with IC 50 � 0.81 and 1.87 μM, respectively, even better than the standard compound, ellipticine, with IC 50 � 1.14 and 2.11 μM for KB and HepG2 respectively.

Data Availability
All data used to support this report's findings are included within the article and in supplementary materials.