All chemicals were purchased from Sigma-Aldrich, Fluka and Agar Powder, reagent grade and were used as received. For bacteria test strains both Gram-negative (Klebsiella spp. and Pseudomonas aeruginosa) and Gram-positive (Bacillus cereus and Staphylococcus aureus) were obtained from Department of Microbiology, Khon Kaen University.

All ligands were prepared by condensation of syringaldehyde with one of the five hydrazides indicated (Scheme 1) in the molar ratio 1:1 in MeOH (5 ml) with constant stirring at 40˚C for 6 hrs and concentrating the reaction mixture before allowing it to cool at room temperature. The products were obtained with high percentages of the yield as shown in Table 1.

Copper (II) chloride dihydrate was added to the methanolic solution of syringaldehyde hydrazones in the molar ratio of 1:1 over a period of 3 hrs with constant stirring in dark. The products were separated by centrifugation and dried in vacuum.

The assay was performed as described Sharma et al. [15] , the DPPH radical scavenging activity of the compounds was measured. Briefly, 0.2 mM 2,2-Diphynyl-1-picrylhydrazyl (DPPH) solution was prepared with methanol. 100 µL sample was added to 3.9 cm³ DPPH solution and was incubated in dark for 30 min at a temperature of 37˚C then the absorbance was measured at λ_max = 517 nm, calculate the percentage of anti-free radical activities.

Prepare FRAP (ferric reducing/antioxidant power) by preparing acetate buffer pH 3.6, 10 mM TPTZ (2, 4, 6-tripyridly-s-trizine) in solution 40 mM of HCL, and 20 mM of ferric chloride and then mix at the proportion of 10:1:1 (v/v). This will get FRAP solution, then prepare sample solution in purified water with the required concentrations [15] .

All the newly synthesized compounds were screened for antibacterial activity against both gram-negative (Pseudomonas aeruginosa and Klebsiella spp.) and gram-positive bacteria (Staphylococcus aureus and Bacillus cereus) by determining minimal inhitory concentration (MIC) and minimal bactericidal concentration (MBC) using the broth dilution method. Bacterial colonies were taken from fresh cultures and incubated in the tubes containing 5 mL of trypticase soy broth at 37˚C for 6 hours until achieving visible growth. Turbidity was adjusted to 0.5 McFarland standard by adding 0.05 mL of 1% w/v BaCl₂·2H₂O in phosphate buffered saline (PBS) to 9.95 mL of 1% v/v H₂SO₄ in PBS. Finally, 10 mg of each compound was dissolved in 10 mL of dimethyl sulfoxide (DMSO) to obtain concentration if 1 mg/mL [16] .

By using MCF-7 cells during growth stage (9 × 10⁴ cells/mL) 45 μL. Add sample element 5 μL diluted by DMSO 5% mixed with 384-well plated and incubate at a temperature of 37˚C with 5% of CO₂ for 3 days, then add solution resazurin 62.5 μg/mL 12.5 μL, incubate 4 hours at a temperature of 37˚C. Then draw a graph for the relation between the concentration and the value of response of cells to find IC₅₀ [17] [18] [19] .

All experiments were carried out in triplicates. Data obtained were analyzed by using one-way analysis of variance (ANOVA) and Pearson’s correlation coefficient was performed. Significant differences between the means at 95% (p < 0.05) level were considered statistically significant. Data was recorded as mean ± standard deviation.

Physical chemistry feature of compound from synthesis, both ligands; syringaldehyde semicarbazone (L1), syringaldehyde thiosemicarbazone (L2), syringaldehyde isonicotinic acid hydrazone (L3), syringaldehyde nicotinic hydrazone (L4), syringaldehyde salicylic hydrazone (L5) and complex between ligands and copper (II) ion are C1, C2, C3, C4 and C5. The synthesized substances will have color, molecular formula, melting point, percentage of elemental substances, and percentage of products in different figures as in Table 1 and Table 2 respectively.

*calc., #found.

All synthesized substances are polar compounds. But polarity is predominant in ligands (L1-L5). These ligands are reacted with copper (II) ions to form complexes (C1-C5) with reduced polarity due to is a reduced charge. This is caused by the neutralization of negatively charged ligands with positively charged copper (II) ion.

The hydrazonate Schiff base ligands employed in the present work were synthesized in good yields (80% - 91%) by the condensation reaction of corresponding syringaldehyde with selected hydrazide in methanolic solvent 1:1 stoichiometry. They yield green colored copper complexes when conjugated with CuCl₂·2H₂O. Their compositional analyses indicate a general molecular formula as [M(ligand)(H₂O)Cl] and absence of conductivity in DMSO solvent suggesting a nonelectrolyte nature for them.

The significant peaks in ir spectra of parent compounds and their probable assignments useful for determining the coordination mode of the present ligands are summarized in Table 3.

The parent syringaldehyde compound (precursor) exhibits the carbonyl stretching frequency at 1671 cm⁻¹ which is replaced after the condensation reaction by the imino stretching frequency at 1600 - 1699 cm⁻¹ and additional stretches at 957 - 976 cm⁻¹ due to hydrazinic N-N and C=O linkages respectively. The appearance of the ν (N-H) and ν (C=O) stretching vibrations indicate that in the solid state these ligands essentially exist in the keto form [20] . The broad band at 1600 - 1615 cm⁻¹ arises out of the vibrations of the delocalized (>C=N-N=C<) linkage [21] . The complexes also display a new band in the far-infrared region at 355 - 330 cm⁻¹ which can be assigned to stretching frequencies. Following metal complexation, the imino stretch is shifted to lower wave numbers whereas the hydrazinic N-N band shifts to higher wave numbers indicating participation of the azomethine group in copper complexation. The additional broad absorption observed in the visible 750 - 940 nm for copper complexes correspond to ²B_1g ²B_2g transition in the square planar environment in these compounds [22] .

When syringaldehyde hydrazonate copper complexes are calculated for ESR, it shows the results as in Table 4.

It well-known that x-band ESR spectra of copper (II) complexes can provide valuable information about the coordination environment around copper (II) ions. The spectra of four coordinate complexes in frozen DMSO glass show four resolved copper hyperfine lines which is indicative of monomeric copper (II) complexes having axial geometry. The calculations of ESR parameters yielding following relationship in the g values g_// > g_^ > g_e (2.0023) is consistent with a d_x²-_y² ground state in the square-planar environment similar to many analogous compounds reported in literature [23] . For copper (II) complexes the g_// is a moderately sensitive function for indicating covalency. For the covalent complexes g_// is less than 2.3 and for ionic environments it is normally 2.3 or larger. Present compounds showing g_// is between 2.25 - 2.29 obviously have a significant covalent character in their metal-ligand bonds in agreement with the observation of Kivelson and Neiman [24] . The degree of distortion f (=g_///A_//) appears to be an empirical index of deviation from idealized geometry. Values of 110 - 120 cm⁻¹ are typical for planar complexes, whereas the range 130 - 150 cm⁻¹ is characteristic of slight to moderate distortion and 180 - 250 cm⁻¹ indicate considerable distortion [25] . The present compounds found to be ranking from 142 - 177 cm⁻¹ suggestive of slight to moderate distortion in geometry.

vs = very strong, s = strong, sh = shoulder, w = weak.

ESR data recorded at 77 K in DMSO solvent.

The ability of anti-free radical activities by using the technique DPPH technique illustrate the results by the percentage of free radical caused from the reaction made between the substance used in experiment and solution DPPH, calculating the spectral absorbance value at λ_max 517 nm, the wave length value that DPPH can highly absorb. It is found that the value of spectral absorbance is changed. Hence, the sample substance has anti-free radical effect by making reaction with solution DPPH [26] . The DPPH free radical receive electron or hydrogen free radical from the sample substance used in experiment, resulting consistency and changed to be independent atom which can be observed from the spectral absorbance at the λ_max 517 nm. The calculation of inhibitory concentration 50% is illustrated in Table 5

It is found that the novel Schiff base ligands have anti-free radical effect DPPH at a high percentage. All complexes showed anti-free radical DPPH and has concentration dependence. as the ligands but have lower proficiency than the ligands because of the bigger structure resulting the steric effect that makes it difficult to provide hydrogen free radical. For the results of anti-free radical activity by reducing Fe³⁺ to be Fe²⁺ (FRAP technique), it is revealed that both ligands and complexes can reduce Fe³⁺ [27] [28] .

Prepare solution of samples in different concentrations and test antibacterial growth ability for the type Escherichia coli, Klebsiella spp., Staphylococcus aureus and Bacillus cereus for 24 hours. Then measure clear zone which find the results as in Table 6 for their copper complexes.

From Table 6, it is found that all 5 complexes have antibacterial effect for all tested strains, especially C3 and C5, good antibacterial effects against Staphylococcus aureus. Moreover, it is also found that all complexes have effects on the dose dependence in nature.

Note: Values are expressed as Mean ± S.D (n = 3).

The researcher used the complexes to test the effect of anti-breast cancer cell in metastasis stage (breast adenocarcinoma MCF-7 cell line). In addition, the researcher also studies whether the selected solution has poisonous effect to normal Vero cell line, as in Table 7.

From Table 7, it is revealed that the complex substances C1, C2, C3 and C4 are not poisonous to the normal cell except C5 which has poisonous effect in high concentrations. The effect of anti-breast cancer growth MCF-7 cell line is found that all sub stances have anti-cancer cell growth at a good effect with the value of IC₅₀, low value, especially the C5 (IC₅₀ 9.75 μM). The efficiency of anti-cancer cell effect of sample substances ranking from much to less is as follows: C5 > C2 > C1 > C4 > C3.