Synthesis, Spectroscopic, and Biological Studies of Mixed Ligand Complexes of Gemifloxacin and Glycine with Zn(II), Sn(II), and Ce(III)

Three novel mixed ligand metal complexes have been synthesized by the reaction of Zn(II), Sn(II), and Ce(III) with gemifloxacin (GMFX) in the presence of glycine (Gly) (1:1:1 molar ratio). The coordination possibility of the two ligands toward metal ions has been proposed in the light of elemental analysis, molar conductance, spectral infrared (IR), ultraviolet-visible (UV-Vis) and proton-nuclear magnetic resonance (1H NMR), and magnetic studies. Results suggest that GMFX and Gly interact with the metal ions as bidentate ligands. Electronic and magnetic data proposed the octahedral structure for all complexes under investigation. Antibacterial screening of the compounds was carried out in vitro against two Gram-positive bacteria, Clavibacter michiganensis and Bacillus megaterium, and two Gram-negative bacteria, Escherichia coli and Xanthomonas campestris. Antifungal activity was performed in vitro against Rhizoctonia solani, Sclerotinia sclerotiorum, Aspergillus niger, Botrytis cinerea, and Penicillium digitatum. The ligands and their complexes were also screened for their antioxidant activity. Results showed that some metal complexes showed more biological efficiency than the parent GMFX drug.


Introduction
Quinolones are a group of synthetic antibacterial agents used in treatment of many infections [1][2][3]. Fluoroquinolone gemifloxacin (GMFX) (Scheme 1A) is a synthetic broad-spectrum antibacterial agent [4] used in the treatment of acute bacterial exacerbation of chronic bronchitis and mild-to-moderate pneumonia. GMFX has excellent in vitro activity against both Gram-positive (G+ve) and Gram-negative (G−ve) organisms, including potent antibacterial activity against respiratory tract infection pathogens [4,5]. GMFX acts by inhibiting DNA synthesis through the inhibition of both DNA gyrase and topoisomerase IV, which are essential for bacterial growth. Fluoroquinolone antibiotics have also been found to have anti-inflammatory and anticancer effects. Recently, some experiments were carried out and revealed that GMFX has an anticancer effect on colon cancer via inhibition of metastasis [6]. The interaction of metal ions with diverse deprotonated fluoroquinolones has been thoroughly studied [7,8]. In many cases, the metal complexes of drugs are more active than the parent compound [8,9].
From this point of view, diverse metal complexes with fluoroquinolones have been synthesized in an attempt to investigate the physicochemical properties and evaluate their biological properties [10,11] The biological characterization of GMFX and its synthesized metal complexes was carried out by an antifungal activity test activity against some phytopathogenic fungi such as; Rhizoctonia solani J.G. Kühn., Sclerotinia sclerotiorum (Lib.) de Bary., Aspergillus niger van Tieghem., Botrytis cinerea Pers., and Penicillium digitatum Sacc. On the other hand, the bactericidal effect of the above mentioned substances was performed against two G+ve bacteria Xanthomonas campestris Pammel and Bacillus megaterium de Bary and two G-ve bacteria Escherichia coli Migula and Clavibacter michiganensis Smith. In addition, antioxidant activity test has been also carried out for GMFX ligand and its synthesized metal complexes by using two different methods 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2′azinobis (3-ethylbenzthiazoline-6-acid) (ABTS) for determine the radical scavenging activity (RSA%).

Results and Discussion
GMFX reacts with Zn(II), Sn(II), and Ce(III) in the presence of Gly as secondary ligand at 100 °C in ethanol to form solid complexes. The complexes were obtained as colored powdered materials. Table 1 summarizes the carbon, hydrogen, and nitrogen elemental analysis as well as melting point, magnetic measurements, and molar conductance. Moreover, the synthesized complexes were characterized using elemental analysis, IR, UV-Vis and 1 H NMR spectra analyses. The molar ratio for all prepared complexes is GMFX:M:Gly = 1:1:1 which was established from the results of the chemical analyses and all the prepared complexes were air stable solids at room temperature. The IR spectroscopic data confirm the presence of water in the composition of the complexes [18] with the number of bound water molecules in these complexes being different. The structures of the complexes suggested from the elemental analysis agree quite well with their proposed formula. The metal ions are coordinated to a deprotonated carboxylate oxygen and pyridone oxygen of GMFX and Gly through the carboxylate oxygen and the amino nitrogen. Here, metal ions react with GMFX and The biological characterization of GMFX and its synthesized metal complexes was carried out by an antifungal activity test activity against some phytopathogenic fungi such as; Rhizoctonia solani J.G. Kühn., Sclerotinia sclerotiorum (Lib.) de Bary., Aspergillus niger van Tieghem., Botrytis cinerea Pers., and Penicillium digitatum Sacc. On the other hand, the bactericidal effect of the above mentioned substances was performed against two G+ve bacteria Xanthomonas campestris Pammel and Bacillus megaterium de Bary and two G−ve bacteria Escherichia coli Migula and Clavibacter michiganensis Smith. In addition, antioxidant activity test has been also carried out for GMFX ligand and its synthesized metal complexes by using two different methods 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2 -azinobis (3-ethylbenzthiazoline-6-acid) (ABTS) for determine the radical scavenging activity (RSA%).

Results and Discussion
GMFX reacts with Zn(II), Sn(II), and Ce(III) in the presence of Gly as secondary ligand at 100 • C in ethanol to form solid complexes. The complexes were obtained as colored powdered materials. Table 1 summarizes the carbon, hydrogen, and nitrogen elemental analysis as well as melting point, magnetic measurements, and molar conductance. Moreover, the synthesized complexes were characterized using elemental analysis, IR, UV-Vis and 1 H NMR spectra analyses. The molar ratio for all prepared complexes is GMFX:M:Gly = 1:1:1 which was established from the results of the chemical analyses and all the prepared complexes were air stable solids at room temperature. The IR spectroscopic data confirm the presence of water in the composition of the complexes [18] with the number of bound water molecules in these complexes being different. The structures of the complexes suggested from the elemental analysis agree quite well with their proposed formula. The metal ions are coordinated to a deprotonated carboxylate oxygen and pyridone oxygen of GMFX and Gly through the carboxylate oxygen and the amino nitrogen. Here, metal ions react with GMFX and Gly, forming complexes of monomeric structure where the metal ions are six coordinate and complete the coordination number with two water molecules (Scheme 2). The molar conductance values of the GMFX and their metal complexes in dimethyl formamide (DMF) with standard reference using 1 × 10 −3 mol −1 solutions at room temperature were found to be in the range from 47.50 to 150.11 S cm 2 mol −1 . The data indicated that the complexes are electrolytes and the chloride ions were found as counter ions at all three complexes [19,20]. For our complexes, in order to verify that the chloride is ionic, their solutions were tested with aqueous solutions of AgNO 3 . A white precipitate was formed which indicated the presence of Cl − as counter ions. The magnetic moments (as B. M.) of the complexes were measured at room temperature. The Zn(II) and Sn(II) complexes were found to be of diamagnetic character with octahedral molecular geometry but the complex of Ce(III) is susceptible and their magnetic moments were 2.34 µ eff . Keys: s = strong, w = weak, v = very, m = medium, br = broad, sh = shoulder, ν = stretching, δb = bending.   The chloride content in the complexes was determined by using two methods; Mohr and Volhard. The lattice water for the three complexes was determined by heating an accurate weight of each complex from the ambient temperature up to~120 • C, the weight loss for each chelate were calculated. The weight loss of Sn(II) and Ce(III) complexes are 2.74%, and 2.46%, respectively. These data support the proposed complexes' chemical formulae and indicate the presence of one hydration water molecule for Sn(II) and Ce(III) complexes.
On the other hand, the measurement of the molar conductivity showed clear differences between the three prepared complexes (divalent and trivalent salt ions) as we have already specified in the manuscript (Table 1). Based on the above explanation, there is a steady relationship between increasing the conductivity of the compound and its solubility, as has been discussed by Uivarosi [21] who reported that the quinolone chelates of trivalent cations have shown an improved solubility compared to that of the free ligand and this behavior could be advantageous for pharmaceutical formulation.

Infrared Absorption Spectra
The infrared spectra of Zn(II), Sn(II), and Ce(III) complexes and free GMFX, Gly were measured as potassium bromide (KBr) discs. These spectra are shown in Figure 1 and the assignments are given in Table 2. The infrared spectra of the three complexes were compared with those of the free ligands in order to determine the site of coordination that may be involved in the chelation process. These peaks were expected to be involved in chelation. The position or the intensities of these peaks were expected to be changed upon complexation. The IR spectra of the complexes were very similar due to the same atoms of GMFX and Gly involved in the bonding to the metal. In most cases, the proposed mechanism of the interaction between GMFX and the metal cations was chelation between the metal ions through pyridone and carboxylic groups; therefore, it was important to give more attention to these group vibrations. The infrared spectra of the metal complexes exhibit a broad band between 3455 and 3411 cm −1 which corresponds to the ν(O-H) vibration and confirms the presence of water molecules in all complexes [18,22]. The two bands observed at 1720 and 1632 cm −1 in the spectrum of the free GMFX have been assigned to the stretching vibration of carboxylic ν(COOH) and the pyridone group ν(C=O), respectively [23], and at 1703 cm −1 for ν(C=O) carboxyl of Gly [24][25][26][27].
In the IR spectra of the complexes, the band corresponding to the carboxylic group appears with ∆ν = ν as (COO − ) − ν s (COO − ) > 200 cm −1 , indicating carboxylic group involvement in monodentate coordination [28,29]. These changes in the IR spectra suggest that GMFX is bound to metal ions via the carboxylate oxygen atom [23]. Also, the second important band at 1634 cm −1 , being attributed to the pyridone group, shifted to lower values at 1575 cm −1 for Sn(II), 1557 cm −1 for Zn(II), and 1559 cm −1 for Ce(III) being a good indication that this group is coordinated to the metal ions [30,31].
Regarding chelation through amino acids, the IR spectra exhibit significant features in the νNH 2 and νCOO − regions. It is worthwhile to mention here that free amino acids exist as zwitterions and the IR spectra of these cannot be compared entirely with those of metal complexes as amino acids in metal complexes do not exist as zwitterions. Free amino acids with NH 3 functions in particular show νNH 3 in the range of 3130-3030 cm −1 . In the complexes, NH 3 gets deprotonated and binds to metal through the neutral NH 2 group. The transformation of NH 3 to NH 2 must result in an upward shift in the νNH 2 of free amino acids. At the isoelectric point, they must show νNH 2 in the region 3500-3300 cm −1 [26]. In the present complexes, the IR spectra showed characteristic bands in the region of 3459-3181 cm −1 , which are lower compared to those of free νNH 2 . Hence, it can be concluded that the nitrogen of the amino group is involved in coordination. The IR spectra show strong evidence in support of the involvement of the carboxylate group in coordination.
In comparison with free amino acids, the νCOO − (asymmetric) and νCOO − (symmetric) record different shifts, which confirms the monodenticity [32] of the carboxylate group. The values of band shift ∆ν = ν as (COO − ) − ν s (COO − ) have indicated that the carboxylate group in Gly was chelated in a uni-negatively manner to the metal ions.

Ultraviolet-Visible Spectroscopy Analysis
Application of ultraviolet spectroscopy can be useful for structural determination of chelates since they all absorb in this region [24,33,34]. Formation of mixed ligand complexes was confirmed by the electronic absorption spectra. The electronic absorption spectra of the ligand along with the Zn(II), Sn(II), and Ce(III) complexes from 200 to 800 nm are shown in (Figure 2). Free GMFX showed bands at 213-245 nm and 276-343 nm which may be assigned to π-π* and n-π* transitions. Respectively. The shift of the absorption bands to higher or lower values and the appearance of new bands for the complexes are attributed to the mixed ligand complexation.
by the electronic absorption spectra. The electronic absorption spectra of the ligand along with the Zn(II), Sn(II), and Ce(III) complexes from 200 to 800 nm are shown in (Figure 2). Free GMFX showed bands at 213-245 nm and 276-343 nm which may be assigned to π-π* and n-π* transitions. Respectively. The shift of the absorption bands to higher or lower values and the appearance of new bands for the complexes are attributed to the mixed ligand complexation.
The decrease or increase of the absorption bands and the appearance of new bands upon coordination could be caused by the following effects: (1) the expected increase of the metal ion mass upon coordination; (2) the increase of the electron density on the metal ions by ligand, and (3) the decrease of the electron density on oxygen and nitrogen donor atoms. The complexes of Zn(II), Sn(II), and Ce(III) showed bands in the range of 402-468 nm, which may be assigned to ligand-metal charge transfer (Table 3).

Proton-Nuclear Magnetic Resonance Spectra Analysis
The suggested molecular structures of the complexes were also proved by nuclear magnetic resonance (NMR) spectroscopy. The 1 H NMR spectra of the ligands and their metal complexes in CD3OD displayed distinct signals with appropriate multiplets; the signal assignments are shown in (Figure 3). The 1 H NMR spectrum obtained for GMFX shows peaks in the range δ = 8.02-8.72 ppm The decrease or increase of the absorption bands and the appearance of new bands upon coordination could be caused by the following effects: (1) the expected increase of the metal ion mass upon coordination; (2) the increase of the electron density on the metal ions by ligand, and (3) the decrease of the electron density on oxygen and nitrogen donor atoms. The complexes of Zn(II), Sn(II), and Ce(III) showed bands in the range of 402-468 nm, which may be assigned to ligand-metal charge transfer (Table 3).

Proton-Nuclear Magnetic Resonance Spectra Analysis
The suggested molecular structures of the complexes were also proved by nuclear magnetic resonance (NMR) spectroscopy. The 1 H NMR spectra of the ligands and their metal complexes in CD 3 OD displayed distinct signals with appropriate multiplets; the signal assignments are shown in (Figure 3). The 1 H NMR spectrum obtained for GMFX shows peaks in the range δ = 8.02-8.72 ppm for the protons of an aromatic ring, a multiplet at δ = 1.14-1.33 ppm corresponding to -CH 2 and -CH cyclopropane group, between 3.32 and 3.36 ppm for a -CH 2 aliphatic group, and a singlet peak at δ = 11.0 ppm for the proton of carboxylic acid. The 1 H NMR spectrum of glycine shows a peak at δ = 11.0 ppm for the proton of carboxylic acid and a peak at δ = 2.0 ppm for the amine (Table 4).
Comparing the main signals of the complexes with those of GMFX and Gly, the 1 H NMR spectra of the complexes showed chemical shift values that were only slightly changed compared with the free ligands, except for the carboxylic acid proton signal: the resonance of the carboxylic proton (COOH) was not detected in the spectra of the three complexes, suggesting coordination of GMFX and Gly through its carboxylation to oxygen atoms [24,25]. Also, the 1 H NMR spectra for the complexes exhibit a new peak in the range of 4.50-4.89 ppm, due to the presence of water molecules in the complexes [35]. for the protons of an aromatic ring, a multiplet at δ = 1.14-1.33 ppm corresponding to -CH2 and -CH cyclopropane group, between 3.32 and 3.36 ppm for a -CH2 aliphatic group, and a singlet peak at δ = 11.0 ppm for the proton of carboxylic acid. The 1 H NMR spectrum of glycine shows a peak at δ = 11.0 ppm for the proton of carboxylic acid and a peak at δ = 2.0 ppm for the amine (Table 4).
Comparing the main signals of the complexes with those of GMFX and Gly, the 1 H NMR spectra of the complexes showed chemical shift values that were only slightly changed compared with the free ligands, except for the carboxylic acid proton signal: the resonance of the carboxylic proton (COOH) was not detected in the spectra of the three complexes, suggesting coordination of GMFX and Gly through its carboxylation to oxygen atoms [24,25]. Also, the 1 H NMR spectra for the complexes exhibit a new peak in the range of 4.50-4.89 ppm, due to the presence of water molecules in the complexes [35].    The results of antibacterial test showed that the tested ligands and its metal complexes were able to inhibit the growth of all studied bacterial strains in a dose dependent manner (Table 5). In particular, all GMFX-metal complexes demonstrated the highest significant activity against E. coli at 10,000 ppm compared to all other treatments. Whereas, GMFX 10,000 ppm and its Sn and Zn complexes at 1000 ppm showed moderate activity. In contrast. GMFX at 1000 and 100 ppm and its metals complexes at 100 ppm showed the lowest significant activity against E. coli. In case of the antibacterial activity against C. michiganensis, the obtained results showed that GMFX and all its metal complexes inhibited its growth completely at all tested concentrations. In case of X. campestris and B. megaterium, the GMFX and all its metal complexes at 10,000 ppm showed the highest significant activity compared to all other treatments. The Ce(III)-GMFX complex at 100 ppm showed the lowest significant activity. The potential antibacterial efficacy of the tested ligand and its metal complexes is thought to depend on the specific toxicity of the studied metals. The low activity observed against E. coli, especially at low concentrations, could be due to the presence of lipopolysaccharides in the outer membrane of the G−ve bacteria, which make them inherently resistant to external agents. Cole et al. [36] have reported that the differences among G−ve and G+ve bacteria regarding the bactericidal effect are due to the structure of their cell walls, where the G−ve bacteria have a more complex cell wall composed of a thin peptidoglycan layer and an outer membrane containing lipopolysaccharides, whereas G+ve bacteria are protected by a wall predominantly composed by one type of macromolecule (pepidoglycan). The obtained results are in agreement with Sadeek et al. [37] who concluded that there is a significant increase in antibacterial activity of metal complexes compared to uncomplexed ligand.

Antifungal Activity
Results of the in vitro antifungal activity testing of the studied ligand GMFX and its metal complexes against R. solani and B. cinerea showed that GMFX at 500 and 1000 ppm demonstrated the highest significant activity compared either to all other treatments or the two tested synthetic fungicides azoxystrobin and cycloheximide ( Table 6). On the other hand, when the fungicidal activity against S. sclerotiorum was tested, the obtained results showed that azoxystrobin has significantly higher activity than GMFX. There were no significant differences between the Zn(II) and Ce(III) complexes. In the case of antifungal activity against A. niger, GMFX exhibited the highest significant activity at all tested concentrations and was similar to the Zn(II) complex and azoxystrobin. Regarding the activity against P. digitatum, GMFX at 1000 ppm and its Zn(II) complex had the highest significant activity, which was similar to both tested synthetic fungicides, and were followed by all other metal complexes. The increased biological activity of metal chelates was explained by the concept of cell permeability and chelation theory. Upon chelation, the polarity of a metal ion is reduced due to the partial sharing of the positive charge with the donor groups of the ligand and as a consequence of overlap with the ligand orbitals. Chelation increases the delocalization of electrons over the whole chelate ring and thus increases the lipophilic nature of the central ion. This increase in lipophilicity enhances the passage of the complex through lipid membranes, facilitating the penetration of cells [38][39][40].

Minimum Inhibitory Concentration of Fungicidal Activity
Minimum inhibitory concentration (MIC) tests were carried out for the most inhibited tested fungi (B. cinerea and P. digitatum). The effects of the different concentrations of GMFX and its metal complexes against the above-mentioned fungi were variable. In particular, MIC values in the case of B. cinerea were evaluated by 2000 ppm in case of the Ce(III)-GMFX complex and 2100 ppm in the case of the [Zn(II) or Sn(II)]-GMFX complexes and uncomplexed GMFX. Regarding P. digitatum, the MIC values were ordered in the following manner: Sn(II)-GMFX < Ce(III)-GMFX < free GMFX < Zn(II)-GMFX where the values were measured as 1400, 2100, 2200, and 2600 ppm, respectively.

Antioxidant Activity
We have screened the antioxidant activity of GMFX and its metal complexes to evaluate their electron-donating activity. Results showed the highest significant antioxidant activity for the GMFX-Sn complex (p < 0.05) (Figure 4), compared to all other treatments. In particular, the DPPH and ABTS scavenging activity evidenced that Stannum presented the best percentage of antiradical activity. These results are in accordance with Bukhari et al. [41] and Chen et al. [42] who reported that the antioxidant activity of some ligands was increased by the presence of metal, such as Sn(IV).
recorded as the mean of fungal mycelium growth inhibition (%) from three replicates ± SDs. Values followed by the different letter in each vertical column for each fungus are significantly different according to Tukey post hoc test at p < 0.05.

Antioxidant Activity
We have screened the antioxidant activity of GMFX and its metal complexes to evaluate their electron-donating activity. Results showed the highest significant antioxidant activity for the GMFX-Sn complex (p < 0.05) (Figure 4), compared to all other treatments. In particular, the DPPH and ABTS scavenging activity evidenced that Stannum presented the best percentage of antiradical activity. These results are in accordance with Bukhari et al. [41] and Chen et al. [42] who reported that the antioxidant activity of some ligands was increased by the presence of metal, such as Sn(IV).
In any case, the antiradical activity of the GMFX-Sn complex could be due to its hydrogen donating ability, as reported by Corona-Bustamante et al. [43]. A possible exchange mechanism is that the proton from the GMFX ligand is donated and the charge is stabilized inside the entire complex, changing its charge delocalization capacity. The high scavenging activity of the Sn complex may explain its low antimicrobial activity, since the oxygenated compounds may contribute to the attachment to and destruction of the microorganism's cell wall, hence increasing the permeability of cytoplasm fluid and finally leading to cell death.

Materials
All chemicals used for preparation of the complexes were of analytical reagent grade, being commercially available from different sources and used without further purification. Gemifloxacin was purchased from Obour Pharmaceutical Industrial Company (Cairo, Egypt). Glycine, AgNO3, K2CrO4, and ethanol (99.8%) were purchased from Fluka, Sigma Aldrich Company (Seelze, Germany). SnCl2•2H2O (98%) and ZnCl2 (98%) were purchased from Carlo Erba company (Milan, Italy). CeCl3•7H2O was purchased from Sigma Aldrich company (Steinheim, Germany).  In any case, the antiradical activity of the GMFX-Sn complex could be due to its hydrogen donating ability, as reported by Corona-Bustamante et al. [43]. A possible exchange mechanism is that the proton from the GMFX ligand is donated and the charge is stabilized inside the entire complex, changing its charge delocalization capacity. The high scavenging activity of the Sn complex may explain its low antimicrobial activity, since the oxygenated compounds may contribute to the attachment to and destruction of the microorganism's cell wall, hence increasing the permeability of cytoplasm fluid and finally leading to cell death.

Instruments
Elemental C, H, N analysis was carried out on a PerkinElmer CHN 2400 (Waltham, MA, USA). The percentage of metal ions was determined by using the atomic absorption method with a PYE-UNICAM SP 1900 spectrometer (Cambridge, UK) fit with the corresponding lamp. IR spectra were recorded from KBr discs using an FT/IR-460 Plus model Jasco-32 spectrophotometer (Easton, PA, USA) in the range from 4000 to 400 cm −1 . 1 H NMR spectra were recorded on a Varian 400 NMR spectrometer (Urbana, IL, USA) using CD 3 OD as solvent. Electronic spectra were obtained using T80 UV/Vis spectrometer (Taylors, SC, USA) PG Instruments Ltd. Magnetic susceptibilities of the powdered samples were measured on a Sherwood scientific magnetic balance (Cambridge, UK) using the Gouy method with Hg[Co(SCN) 4 ] as calibrant. Molar conductance of 1 × 10 −3 M solutions of ligand and metal complexes in DMF was measured at room temperature using a CONSORT K410 (Turnhout, Belgium). All measurements were carried out at ambient temperature with freshly prepared solutions. Melting points were recorded on a Buchi apparatus.

Bactericidal Activity Test
The antibacterial test of the studied ligand and its metal complexes was investigated by the disc diffusion method of Bhunia et al. [20,44,45] against X. campestris, B. megaterium, E. coli, and C. michiganensis with some minor modifications. All strains were listed in National Collection of Plant Pathogenic Bacteria Catalogue (NCPPB) and have been cultivated, lyophilized, and freeze-conserving as pure cultures in the collection of the School of Agricultural, Forestry, Food and Environmental Sciences, University of Basilicata, Potenza, Italy. The bacterial suspension of each strain was prepared in sterile distilled water plus soft agar (0.7%) at concentration 10 8 colony form unit (CFU) mL −1 (optical density, OD ≈ 0.2 nm). A mixture of 0.7% soft agar and bacterial suspension (9:1 v/v) was prepared and 4 mL of this suspension was poured into each 10 mL KB Petri dish 90 mm in diameter. Blank Discs (6 mm)-OXOID (Milan, Italy) were placed over KB-plate surfaces after complete solidification and 20 µL aliquots from each ligand and metal complexes at the following concentrations (1:5) 5000, 250, and 62.5 ppm.

Fungicidal Activity Test
The antifungal screening was evaluated against five phytopathogenic fungi: R. solani, S. sclerotiorum, A. niger, B. cinerea, and P. digitatum. The tested fungi were previously identified by classical and molecular methods and stored in the mycotheca of School of Agricultural, Forestry, Food and Environmental Sciences, University of Basilicata, Potenza, Italy with the following collection numbers: R. solani 1312; S. sclerotiorum 593; A. niger 1008; B. cinerea 479, and P. digitatum 1395. Different concentrations of each treatment at 1000, 500, and 250 ppm was prepared in potato dextrose agar (PDA). Fourteen mL aliquots of previous prepared media supplemented with each treatment was poured into Petri dishes. After the complete dry off of the agar surface under laminar flow, 0.5 cm diameter of fungal disks square cut off from 96 h fresh cultures were singularly inoculated in the center of each Petri dish prepared as above. All plates were incubated at 24 ± 2 • C for 4 days under dark conditions. The diameter of fungal mycelium growth measured in mm. Petri dishes containing only PDA were also inoculated with fungal disks of the same fungi and used as a control. Each treatment was carried out in triplicate. The fungitoxicity was expressed as growth inhibition percentage (GIP) and calculated according to the formula of Zygadlo et al. [46] (Equation (1)), herein reported. Compared to synthetic fungicide azoxystrobin 1 µL mL −1 and cycloheximide 0.1 µg mL −1 to PDA nutrient medium: where: GC = average diameter of fungus colony grown on PDA alone (control); GT = average diameter of fungus colony grown on PDA containing each treatment.

Determination of Minimum Inhibitory Concentration (96-Well Microplate Method)
The MIC was considered as the fungicidal effect of each treatment, defined as the lowest concentration of each ligand and metal complexes that definitely inhibits the fungal growth. MIC was determined on 96-well culture plates by a micro-dilution method following the technical procedures of Lehtinen et al. [47]. Spore formation was ascertained after nine days incubation under a light microscope (Axioskop, ZEISS, West Germany). The stock solution of each treatment was prepared in Potato Dextrose Broth (PDB) at 1000, 1200, 1400, 1600, 1800, 2000, 2200, 2400, 2600, 2800, and 3000 µg mL −1 . The absorbance of fungal growth was read at λ = 450 nm using the Elisa Microplate reader instrument (DAS s.r.l., Rome, Italy). All samples were tested in triplicate.

Antioxidant Activity
In the present study, two tests were performed (ABTS and DPPH) following the methodological procedures of the basic principles of Martysiak-Żurowska and Wenta [48], as follows. In these trials, the above mentioned two assays were carried out to determine the radical scavenging activity (RSA%) of the studied ligand and its metal complexes at 100% utilizing the following formula: Radical Scavenging Activity (RSA%) = (1 − A t /A c ) × 100%; where A t is the absorbance of sample and A c is the absorbance of colorimetric radical substance without sample.

DPPH: 2,2-diphenyl-1-picrylhydrazyl Assay
The DPPH method is considered one of the most useful techniques for measuring the antiradical activity of a compound and it depends on the use of the stable free radical DPPH. The electronic delocalization of this radical is responsible for its characteristic deep violet color and hence the compounds which will be able to donate a hydrogen atom will convert the DPPH radical into its reduced form (neutral stable molecule) and then the color switch from deep violet to pale yellow [49,50].
Stock radical solution (DPPH solution) was prepared by dissolving 20 mg of DPPH in 15 mL of ethanol. Thereafter, 1 mL of DPPH solution was diluted with 29 mL of ethanol.
Test procedures: 50 µL was diluted at (1:20) using 950 µL of DPPH solution and was incubated at darkness for 30 min at room temperature. Later on, all samples were centrifuged at 8000 rpm for 5 min and the absorbance was measured at 515 nm in triplicate using a UV-Vis spectrophotometer (LKB Biochrom 4050 Ultrospec II, Arizona, USA) considering the value of the reference sample (ethanol). The stock solution of DPPH was prepared fresh and all determinations were carried out in triplicate.
ABTS: 2,2 -azinobis (3-ethylbenzthiazoline-6-acid) Assay The ABTS method is considered the most sensitive method and is characterized by higher repeatability for observing the kinetics of specific enzymes. The detectability and sensitivity of ABTS are higher than the DPPH assay.
Stock radical solution. ABTS solution was prepared by dissolving 38 mg of ABTS in 10 mL of a aqueous sodium persulphate solution (2.45 mM). and the solution was conserved in darkness for 16 h at room temperature. Then, 1 mL of stock ABTS •+ solution was diluted with 29 mL of ethanol.
Test procedures: 20 µL of each studied ligand and the metal complexes was diluted at (1:50) using 980 µL of radical ABTS •+ solution and was incubated after that in darkness at room temperature for 2 h. All samples were centrifuged at 8000 rpm for 5 min and the absorbance was measured at 734 nm by using UV-Vis spectrophotometer (LKB Biochrom 4050 Ultrospec II) considering the value of reference sample (ethanol). The stock solution of ABTS was prepared fresh and all determinations were carried out in triplicate.

Statistical Analysis
The outcomes of the biological tests were statistically analyzed using statistical package for the social sciences (SPSS) (version 13.0. Prentice Hall, Chicago, IL, USA, 2004). Experimental data were expressed as mean ± SD and comparisons were employed by Tukey post-hoc test for detecting any significance differences among different treatments at p < 0.05.