Preparation, characterization of some transition metal complexes of hydrazone derivatives and their antibacterial and antioxidant activities

2.2.1 [(H₂L)]_, (M.F.: C₁₃H₁₃N₃OS and M.W.: 259)

Elemental analysis of (H₂L) gave, C = 60.0% (60.2), H = 4.70% (5.0) and N = 16.0% (16.2), (where the numbers in parenthesis represent the calculated value from the chemical formula). Electronic absorption spectra of (H₂L) in DMF solution, λ_max/nm: 250, 325. Selected IR frequencies (KBr disk, cm⁻¹): 3381, 3244, 1628 and 690.

2.3.1 [Pd(H₂L)(OH)Cl]·1/2H₂O] C_1, (M.F.: C₁₃H₁₅N₃O_1½S Pd and M.W.: 426.5)

Elemental analysis of C₁ gave; C = 36.5% (36.57), H = 3.20% (3.50), N = 9.80% (9.85), Cl = 8.30% (8.30) and Pd = 24.58% (24.85) (where the numbers in parenthesis represent the calculated value from the chemical formula). Electronic absorption spectra of C₁ in DMF solution, λ_max/nm: 323, 399, 520. Selected IR frequencies (KBr disk, cm⁻¹): 3431, 3194, 3135, 1602, 438 and 512.

2.3.2 [Cd(H₂L)Cl₂], C₂(M.F.: C₁₃H₁₃N₃OSCl₂Cd and M.W.: 442.5)

Elemental analysis of C₂ gave; C = 35.37% (35.25), H = 3.08% (2.94), N = 9.6% (9.5), Cl = 16.20% (16.00) and Cd = 25.4% (24.5) (where the numbers in parenthesis represent the calculated value from the chemical formula). Electronic absorption spectra of C₂ in DMF solution, λ_max/nm: 316. Selected IR frequencies (KBr disk, cm⁻¹):3413, 3171, 3069, 1707, 427 and 503.

2.3.3 [Cu(H₂L)(HL)·OH] C₃ (M.F.: C₂₆H₂₆N₆O₃S₂Cu and M.W.: 597.5]

Elemental analysis of C₃ gave; C = 52.60% (52.21), H = 4.50% (4.35), N = 14.30% (14.06) and Cu = 10.56% (10.63) (where the numbers in parenthesis represent the calculated value from the chemical formula). Electronic absorption spectra of C₃ in DMF solution, λmax/nm: 325, 379, 420 and 615. Selected IR frequencies (KBr disk, cm⁻¹): 3336, 3042, 3019, 1608, 1534, 452, and 575.

2.3.4 [Cu(H₂L)₂Cl]·1/2H₂O] C₄ (M.F.: C₂₆H₂₇N₆ O_2½ S₂ ClCu and M.W.: 626.0]

Elemental analysis of C₄ gave; C = 49.7% (49.84), H = 4.20% (4.31), N = 13.40% (13.42), Cl = 5.46% (5.67) and Cu = 10.05% (10.14) (where the numbers in parenthesis represent the calculated value from the chemical formula). Electronic absorption spectra of C₄ in DMF solution, λmax/nm: 338, 389, 440 and 607. Selected IR frequencies (KBr disk, cm⁻¹): 3432, 2923, 1674, 1604, 455, and 511.

2.3.5 [Cu₂(H₂L)₂(HL)(OH)] C₅ (M.F.: [C₃₉H₄₁N₉O₄S₃Cu₂] and M.W.: 938.1]

Elemental analysis of C₅ gave; C = 50.1% (49.9), H = 4.50% (4.3), N = 13.5% (13.4) and Cu = 14.32% (13.5) (where the numbers in parenthesis represent the calculated value from the chemical formula). Electronic absorption spectra of C₅ in DMF solution, λmax/nm: 340, 379 and 600. Selected IR frequencies (KBr disk, cm⁻¹): 3444, 3336, 3091, 1606, 1534, 485, and 571.

2.6.1 Antibacterial activity

The in vitro antibacterial activity studies were carried out in the department of Microbial Biotechnology, Genetic Engineering and Biotechnology Research Institute, Sadat City University, Egypt, by using Broth Dilution Method with some alterations (Wikler et al., 2009) to investigate the inhibitory effect of ligand and synthesized complexes (C₁-C₅) on the sensitive organisms Streptococcus pyogenes as Gram- positive bacterium and Escherichia coli as Gram–negative bacterium. Nutrient broth medium was prepared by using Brain Heart Infusion (BHI) broth and distilled water. The test compounds in measured quantities were dissolved in DMSO which has no inhibition activity to get two different concentrations (1 mg/mL, 5 mg/mL) of compounds. The strains selected for the study were prepared in (BHI) broth medium with shaking and autoclaved for 20 min 15 lb of pressure and at 121 °C before inoculation. The bacteria were then cultured for 24 h at 37 °C in an incubator. One ml of the standard bacterial culture was used as inoculation in a nutrient broth. For growth studies, culture of microbial cells were inoculated and grown aerobically in BHI broth for control and along with various concentrations of the test compounds in individual flasks. Growth was calculated turbidometrically at 650 nm using conventional Spectrophotometer, in which turbidity produced is measured by taking absorbance and compared with turbidity produced by control. The growth rate of different bacteria in absence as well as in presence of test compounds was performed for each concentration. Absorption measurements were accomplished by spectrophotometer after 24 and 48 h of incubation to determine the number of viable organisms per milliliter of sample and were used to the calculated the % inhibition (Umit et al., 2018; Abdülmelik et al., 2019; Nuraniye et al., 2019).

2.6.2 Assay for DPPH free-radical scavenging potential

The nitrogen centered stable free radical 2,2′-diphenyl-1-picrylhydrazyl (DPPH) has often been used to characterize antioxidants (Gülçin et al., 2010; Ercan and Ilhami, 2011; Leyla et al., 2015). It is reversibly reduced and the odd electron in the DPPH free radical gives a strong absorption maximum at λ 517 nm, which is purple in colour (Blois, 1958). Each sample at different concentrations (100, 200 and 300 µg) in HPLC methanol was added to the solution [3.9 mL, 0.004% (w/v)] of DPPH⁰ in methanol. The tubes were kept at an ambient temperature for 30 min and the absorbance was measured at λ 517 nm. All tests were performed in triplicate and mean were calculated. The scavenging activity (SCA) was expressed as a percentage of scavenging activity on DPPH: $$\text{SCA}\%=\left[({\text{A}}_{\text{control}}-{\text{A}}_{\text{test}})/{\text{A}}_{\text{control}})\right]\times 100$$ where A _control is the absorbance of the control (DPPH solution without test sample) and A _test is the absorbance of the test sample (DPPH solution plus scavenger).

Butylated hydroxyl anisole (BHA) and tert-Butylated hydroxyl qunione (TBHQ) were used as positive controls. All tests were run in triplicate and an average was used (Gülçin, 2006a,b; Ak and Gülçin, 2008).

3.1.1 ¹H NMR spectrum of the ligand

The ¹H NMR spectrum of the ligand was verified in DMSO-d₆ solution (Fig. 1). The spectrum shows that the different non-equivalent protons resonate at different values of applied field. The ¹H NMR spectrum exhibits one signal at δ 4.14 ppm assigned to —NH of amine protons and the multiple peaks observed in the region of (NH—CH₂) and (OC—NH—CH₂) signals indicate chemically non-equivalent methylene protons. Furthermore, the spectrum displays a multiple signal at δ (6.58–7.63 ppm) assigned to aromatic ring protons. Also, the spectrum depicts signals at δ (8.48, 11.46 and 7.63 ppm) to (2H, —CH⚌N—), (2H, —CO—NH—) and thiophene ring protons, respectively. Moreover, the spectrum exhibits signals at δ 3.77 ppm due to nonequivalent methylene protons. Thus, the ¹H NMR result supports the assigned of geometry.

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Fig. 1

Proton nuclear magnetic resonance spectrum of (Z)-2-(phenylamino)-N′-(thiophen-2-ylmethylene) acetohydrazide (H₂L).

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The ¹H NMR spectrum of the ligand was verified in DMSO-d₆ solution δ ppm: (Fig. 1). The ¹H NMR spectrum exhibits signal at 2.36 (s, 2H, CH₂), 4.14 (br, 1H, NH), 6.58–7.63 (m, 8H, aromatic system), 5.94 (s,1H, CH), 11.36 (s,1H, NH) (amide α to hydrazone linkage).

3.1.2 ¹³C NMR spectrum of the ligand

The ¹³C NMR spectrum of the ligand (DMSO-d₆) features a signal at 57.5 ppm corresponding to the methylene group. The aromatic carbons of the phenyl ring are observed at 113.5, 120.8, 129.5 and 147.6 ppm (El-Saied et al., 2017) whereas the thiophene ring carbons appear at 127.2, 128.5 and 130 ppm (Nursen and Perihan, 2004; Chandrasekaran et al., 2014). The signal of the azomethine carbon is observed at 125 ppm (Juvansinh et al., 2015). The chemical shift appeared at 171 ppm can be assigned to the carbonyl group (see Fig. 2).

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Fig. 2

¹³C NMR of (Z)-2-(phenylamino)-N′-(thiophen-2-ylmethylene) acetohydrazide.

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3.1.3 Mass spectrum of the ligand

The electron impact (EI) mass spectrum of the ligand (Fig. 3) confirms the proposed formula, the observed peak at m/z = 259amu (M) corresponding to the ligand moiety (C₁₃H₁₃N₃OS, atomic mass m/z = 259 molecular ion peak). The series of peaks in the range m/z = 51 [C₄H₄-H]⁺, 77 [C₆H₅]⁺, 106 [C₇H₈N]⁺ base peak, 132 [C₈H₈NO-2H]⁺, 149 [C₈H₉N₂O]⁺, may be assigned to different fragments.

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Fig. 3

Mass spectrum of (Z)-2-(phenylamino)-N′-(thiophen-2-ylmethylene)acetohydrazide(H₂L).

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3.1.4 Infrared spectra of the ligand and its metal complexes

The IR spectrum of free ligand is compared with IR spectra of its metal complexes, to investigate the mode of bonding Schiff base ligand to metal ion. The characteristic bands of the IR spectrum of the Schiff base ligand are listed in Table 2 and Fig. 4. The broad band at 1628 cm⁻¹ is assigned to the (—C⚌O, —CH⚌N) stretching frequency. The IR spectrum of the ligand additionally shows a broad band at 3381, 3244 cm⁻¹ due to the stretching vibration of υ(NH) group (Table 2). On complexation, the IR spectra of all complexes display the bands of the imine groups has shifted to lower wave number as compared of free Schiff base ligand. Also the bands of carbonyl group was shifted to lower wavenumber accompanied by a decrease in their intensity, indicating that they are involved in complex formation (Morsy et al., 2011; Samar, 2017). Complexes (C₃, C₅) showed that the ligand in keto-enolform and enolic oxygen is added to the coordination of complexes (Cescon, 1962; Kumar and Ramesh, 2005; Balasubramanian et al., 2006; Yin and Chen, 2006). The appearance of two new bands at 427–485 and 503–575 cm⁻¹ ranges corresponds to ν(M−N) and ν(M−O), respectively (Fernandez et al., 2006; Majumder et al., 2006). Complexes (C₁ and C₄) under investigation included water molecules. The broad bands in the 3336–3444 cm⁻¹ region are due to water of crystallization or coordinated of hydroxo group (Fathy and Samar, 2004). From the IR spectral data and the results mentioned above, it is concluded that, the ligand behaved as a neutral bidentate ligand coordinating through the imine nitrogens and the carbonyl groups oxygen in complexes (C₁, C₂). While in complex C₅ the ligand produces binuclear complex.

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Fig. 4

Infrared spectra of ligand and its metal complexes (C₁, C₂, C₄).

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3.1.5 Electronic spectra and magnetic moments

The electronic spectra of hydrazone ligand and M²⁺, M¹⁺ complexes [where M = Pd(II), Cd (II) and Cu(I, II)] were recorded in 10⁻³ DMF solution in the range 190–800 nm. Within the UV spectrum of the ligand (Table 3) was observed the existence of two absorption bands assigned to the transition π–π* at 250, 325 nm (Table 3). While the electronic spectrum of Pd(II) complex (C₁) displayed bands at 323, 399 and 520 nm, this spin-allowed transition may correspond to ¹A_1g → ¹B_1g transition indicating a square planar geometry (Shankarwar et al., 2015; Hanaa and Ocala, 2015). The electronic spectrum of Cd(II) complex exhibited non-ligand band at 316 nm, referring to L → M charge transfer transition in tetrahedral geometry (Mohamed et al., 2018). Pd(II) and Cd(II) complexes exhibit diamagnetic character (Azam et al., 2012).

Also, The electronic spectra of the copper complexes (C₃-C₅), showed strong absorption bands in the spectra at 325–340 nm attributed to the transition of the ligand between different energy levels. The other band at 379–440 nm is probably assigned to ligand to metal charge transfer (LMCT) transition. One more spectral band appeared at 600–615 for Cu complexes which is due to d-d transitions, which are consistent with the distorted square-pyramidal structure (Li and Xu, 1999; Hathaway et al., 1987). The room temperature magnetic moment value of the Cu complex (C₃) is found to be 1.75B.M. which are close to the spin only value of one unpaired electron. However, the complexes (C₄ and C₅) showed diamagnetic character. The proposed structures are shown in Scheme 2.

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Scheme 2

Suggested chemical structure of Pd(ll), Cd(ll), Cu(l, ll) complexes.

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3.1.6 Thermal studies of the ligand and its complexes

The thermogravimetric (TG/DTG) analysis is very important technique used to assess the stability of compounds and support their geometry structure. TG/DTG results of ligand and its complexes (C₁-C₅) are listed in Table 4 and Figs. 5a–5c. The thermal studies of the reported compounds show good agreement with the suggested theoretical formulae obtained from analytical and spectral data (Ahamed et al., 2003).

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Fig. 5a

TG/ DTG curves of the ligand.

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Fig. 5b

TG/ DTG curve of the Pd(ll) complex (C₁).

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Fig. 5c

TG/ DTG curve of the Cu(l) complex (C₄).

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3.1.7 Kinetic and thermodynamic parameters

The kinetic parameters of activation energy (E), reaction order (n) and the pre-exponential factor (A) were calculated using the Coats–Redfern equation (Coats and Redfern, 1964). As well as the thermodynamic activation parameters for the thermal decomposition steps in complexes; enthalpy (ΔH*), entropy (ΔS*) and free energy of decomposition (ΔG*) were evaluated using the following relationships: $$\mathrm{\Delta }{H}^{\ast }=E^{\ast }-RT$$ $$\mathrm{\Delta }{S}^{\ast }=R[\mathrm{l}\mathrm{n}(Ah/kT)-1]$$ $$\mathrm{\Delta }{G}^{\ast }=\mathrm{\Delta }{H}^{\ast }-T\mathrm{\Delta }{S}^{\ast }$$ where k is Boltzmann constant and h is Plank’s constant and R is the gas constant. The kinetic parameters obtained from the previously mentioned methods are listed in Table 5 and Figs. 6a–6d.

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Fig. 6a

Coats-Redfern plot for complex C₁.

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Fig. 6b

Coats-Redfern plot for complex C₂.

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Fig. 6c

Coats-Redfern plot for complex C₃.

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Fig. 6d

Coats-Redfern plot for complex C₄.

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Fig. 7

X-ray diffraction pattern of C₄ complex.

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Fig. 8a

Optimized molecular structure of ligand.

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Fig. 8b

Optimized molecular structure of Pd(II) complex (C₁).

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Fig. 8c

Optimized molecular structure of Cd(II)complex (C₂).

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Fig. 9

Antibacterial activity for the ligand and its metal complexes against E. coli.

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Fig. 10

Antibacterial activity for the ligand and its metal complexes against S. pyogenes.

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Fig. 11

Antioxidant activity of ligand and its complexes (C₁-C₄) as determined by DPPH.

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From the results, the following remarks can be pointed out:

• –

  The positive sign of ΔG* for the complexes under investigation revealed that the free energy of the final residue is higher than that of the initial compound and all the decomposition steps are non-spontaneous processes.

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  The negative values of ΔS*for the degradation process indicated a more order activated complex than reactant and/or the reaction was slow (Frost and Pearson, 1961).

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  All the complexes have negative entropy, which indicates that the complexes are formed spontaneously.

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  The positive values of ΔH* means that the decomposition processes were endothermic (El-Ayaan et al., 2007).

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  Kinetics of the thermal decomposition stages of the complexes under study obeys, in most cases, the first order kinetics.

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  The values of ΔG* increased for the subsequent decomposition stage of complex C₁. This increase in the values of ΔG* reflected that the rate of removal of the subsequent ligand was lower than that of the precedent ligand, this is due to increasing in the values of TΔS* from one step to another which overrides the values of ΔH* (Maravalli and Goudar, 1978; Yusuff and Sreekala, 1990; Vinodkumar et al., 2000).

• –

  It was found that the order of the activation energy values of the first decomposition step for the complexes is: C₃ > C₅ > C₄ > C₁ > C₂.

3.1.8 X-ray diffraction

XRD pattern of copper complex (C₄) is revealed in Table 6 and Fig. 7. The X-ray powder diffraction analysis of (C₄) complex was carried out to give information about atomic or molecular arrangement as shown in Fig. 7. The spectra showed sharp peaks indicating their crystalline nature. The X-ray powder diffraction patterns were attained in the range of 5–90° and in steps of 0.050°. The full width at the half- maximum (FWHM) of diffraction peaks observed from the refinement was used to evaluate the particle size. Also, to investigate the crystalline information about complex, the average crystallite sizes (D) were calculated from the most sharp peaks using Sherrer’s formula (Harikumaran and Appukuttan, 2012). $$\text{D}=\left(\text{K}\lambda \text{/}\beta Cos\theta \right)$$

The diffraction peaks indicate that synthesized of copper complex (C₄) is in the nanometer range. Also, the average crystallite size of C₄ complex is 4.107 nm. From the value of (D), we can obtain the dislocation density (δ) value of complex which indicates the number of dislocation lines per unit area of the crystal. The value of (δ) is related to the average crystallite size (D) from the equation: $$\delta =1/{\left(\text{D}\right)}^2$$

The calculated value of (δ) found to be 0.0593.

3.1.9 Molecular modeling

Many attempts to prepare crystals for X-ray crystallography were unsuccessful. The computational strategy in this manuscript is to determine the geometry of the ligand and its complex. Thus, molecular modeling calculations were considered. Thus, the geometries of the complexes were optimized by hyper chem. 8.03 Molecular Modeling and Analysis Program using the molecular mechanics calculations (PM3) (HyperChem, 2007).

The optimized geometries of the ligand and its complexes are shown in Figs. 8a–8c. The optimized lengths of bonds in addition to bond angles (Supplementary materials) are listed in Tables (S₁-S₃). The energetic properties of these compounds are listed in Table 8.

From selected bond lengths collected in Table 7 one can conclude the following remarks:

• In all complexes the metal ions are coordinated to the ligand via N9 and O14 atoms.

• A large variation in C6⚌N7 and N7—N9 bonds length on coordination via the N7 atom.

• The C6⚌N7 bond length increases in all complexes than ligand.

• In Pd complex the bond distance N7—N9 is enlarged due to their participation in coordination which becomes weaker due to the formation of Pd—N bonds (El-Gammal et al., 2014) M—N bond of Cd complex is larger than Pd complex.

• In general, the M—Cl bond length is longer than that of M—N and M—O bonds.

• The bond angles of the ligand moiety are altered somewhat upon coordination (Mohamed et al., 2014).

• The other bond lengths and bond angles also suffer some changes, but not significantly.

• The bond angles in Pd(II) complex are quite near to a square planer, and tetrahedral geometry in Cd(II) predicting dsp² hybridization. The bond angle values show a large distortion around the metal centers.

• The HOMO and LUMO namely the Frontier Molecular Orbitals (FMOs) are very popular quantum chemical parameters. The chemical reactivity is a function of interaction between HOMO and LUMO levels of the reacting species (Kenichi, 1982). The higher E_HOMO implies that the molecule is a good electron donor and has great ability to complex formation. LUMO energy presents the ability of a molecule receiving electron (Geerlings et al., 2003; Seda et al., 2009). The lower E_LUMO value, the more ability of the molecule is to accept electrons (Guo and Chengha, 2007).

3.2.1 In vitro antibacterial activity

The antibacterial activities of ligand H₂L as well as its metal complexes (C₁- C₅) were tested against S. pyogenes as Gram- positive bacteria and E. coli as Gram–negative bacteria. According to the data depicted in Figs. 9 and 10, the following results are summarize as follow:

1-All of the tested compounds are found to have remarkable antibacterial activity. The active sequence of the ligand and its tested complexes against S. pyogenes in the concentration of 1 μg/mL, 5 μg/mL follows the trend: C₁ > C₂ > C₃ > C₅ > C₄ > H₂L.

2- Also all the tested complexes except C_4, have higher activity than their metal free ligand against E. coli for concentration of 1 μg/mL as the follow trend: C₂ > C₃ > C₅ > C₁ > H₂L > C₄, and for the concentration of 5 μg/mL, the activity follows the trend: C₂ > C₃ > C₅ > C₁ > H₂L, C₄.

3- It is found that, C₁ complex has the highest toxicity against Gram-positive bacteria in both two concentrations; C₂ has the highest toxicity against gram-negative bacteria in both concentrations. Moreover, it is found that all tested complexes are more toxic against both Gram-positive and Gram-negative bacteria than that of the free ligand in the both concentrations, except C₄ in gram-negative bacteria, having higher antibacterial activities than that of the free ligand. This would suggest that, chelation/complexation could enhance the lipophilic character of the central metal atom, which subsequently favors its permeation through the lipid layers of the cell membrane and blocking the metal binding sites on enzymes of microorganism which as explained by Tweedy’s chelation theory (Tweedy, 1964). It is suspected that other factors such as liposolubility, dipole moment, conductivity influenced by metal ion, geometry, number and type of metal ions and counter ions, and moieties as azomethine linkage present in these compounds may be possible reasons for their remarkable antibacterial activities (Mehmet et al., 2010; Dharmpal et al., 2017; Saeed et al., 2009; Najma et al., 2010).

3.2.2 Antioxidant activity

The process of scavenging DPPH-free radicals has been used to assess the antioxidant activity of the present compounds (Bilgicli et al., 2015). The data of the suppression ratio for DPPH are shown in Fig. 11. The results were compared with those of standard antioxidants. The percentage radical scavenging activity of the synthesized compounds has been carried out at different concentrations (100, 200 and 300 µg mL⁻¹). The DPPH scavenging activity was expressed as IC₅₀, the effective concentration at which 50% of the radicals were scavenged, have been calculated to assess the antioxidant activities. A lower IC₅₀ value indicates greater antioxidant activity. IC₅₀ values lower than 10 mg/mL usually imply effective activities in antioxidant properties. Fig. 11 shows that, all of the tested compounds exhibited very good radical scavenging activities at different concentrations. From the obtained results, C₃,C₄ complexes (IC₅₀ = 0.019, 0.211 µg mL⁻¹) showed good activity better all tested compounds and the positive standard. The ligand (IC₅₀ = 0.514 µg mL⁻¹) possesses the lowest scavenging ability as compared to the positive standard and all tested complexes. The order of IC₅₀ values of ligand and its metal(II,I) complexes was as follows: C₃ > C₄ > C₁ > C₂ > Ligand. The free radical scavenging activity of the compounds depends on the structural factors and other structural features as type and geometry of metal ions.