Synthesis, Characterization and Biological Studies of Cu2+, Ni2+, Cd2+, and Pt4+ Complexes Derived from 3-(3H-1,2,4-Triazole-4-(5H)- Ylimino)Butane-2-One-Oxime (L)

Novel Cu2+, Ni2+, Cd2+, and Pt4+ complexes derived from 3-(3H-1,2,4-triazole-4-(5H)-ylimino)butane-2-one-oxime (L) were synthesized and characterized. FTIR spectra propose that L acts in different methods (bitriand/or tetradentate) depending on the metal used. Electronic spectra and magnetic measurements of the complexes suggest a structure that is tetrahedral for Ni2+, octahedral for Cd2+, while square-planar for Cu2+ and Pt4+ complexes. Density functional theory (DFT) parameters were applied for Cu2+, Ni2+, and Pt4+ complexes which prove the geometry of L towards the metal ions. Coats-Redfern and Horowitz-Metzger methods were used to calculate the kinetic and thermodynamic parameters of the isolated complexes. The cyclic-voltammogram curve of the Cu2+ complex was reported. The biological activity of L and its complexes were tested against three types of cancer cell lines, and against different bacterial strains.


Synthesis of Ni 2+ complex
[NiLCl(H 2 O)]Cl · 1.5H 2 O was obtained by adding the ligand (0.50 g; 3 mmol) in ethanol to an ethanolic solution of NiCl 2 · 6 H 2 O (0.72 g; 3 mmol). The green gel mixture at pH = 5.0 was formed and then the reactants were refluxed for 7 h. Finally, the complex was separated from the filtrate, continuously washed with ether, and finally dried in a dryer (yield: 60%). Elemental analyses: Anal. Found: for C 6  In vitro cytotoxicity of L and four complexes (Cu 2+ , Cd 2+ , Pt 4+ , and Ni 2+ ) was applied against three cancer cell lines [prostate cancer (PC-3), breast cancer (MCF-7), and Hela cell lines] brought from a holding corporation for the products and injections (VACSERA) located in Egypt. The doxorubicin as a typical anticancer medication was used. MTT, DMSO, and RPMI-1640 medium were obtained from Sigma Aldrich (MO, USA) while the fetal bovine serum was obtained from GIBCO (UK). Colorimetric assay effects were recorded at 750 nm using a plate reader (EXL 800). The relative cell practically in rate was computed by using the equation (A570 of treated examples/A570 of uncured sample) × 100. The effect on mammary MCF-7 observed with ELISA and the percentage of safety was estimated using the equation [1-(ODT/ODC)] × 100%, where ODT is the average optical density of cells cured with the compounds and ODC is the average optical density of uncured cells [35,36].

Antibacterial and antifungal activity
In order to test the complexes against two classes of bacteria: Staphylococcus aurous (Gram-positive) and Escherichia coli (Gram-negative), and two kinds of fungi: Candida albicans and Aspergillus flavus, the disc diffusion method was utilized [37,38].

Antioxidant activity and screening assay for erythrocyte hemolysis
Samples of blood from the rats were withdrawn in heparinized tubes. Red blood cells (RBCs) were detached from plasma and the buffy coat was washed about three times with 10 volumes of 0.15 M saline solution. After the middle of the last launder, the erythrocytes were centrifuged at 2,500 rpm for 10 min to obtain a constantly packed cell preparation. In the measured coordination polymer system, erythrocyte hemolysis was permeated by peroxyl radicals [39,40]. The same volume of 10% suspension of erythrocytes in a phosphate-buffered saline (PBS; pH = 7.4) and 200 mM 2,2 -azobis(2-amidinopropane) dihydrochloride solution were added to each other in PBS containing samples to be examined at varied concentrations. The reactant was cradled gently and incubated at 37°C for 2 h. After that, the mixture was emptied, diluted with eight volumes of PBS, and centrifuged for 10 min at 1,500 g. The supernatant absorption was recorded at 540 nm. Also, for achieving a complete hemolysis, the reactant was treated with 8 volumes of distilled water, and the supernatant after centrifugation was measured at 540 nm. L-ascorbic acid was wielded as a positive control.

Colorimetric assay for compounds that bind DNA
DNA methyl green (20 mg) was suspended in 100 mL of 0.05 M Tris-HCl buffers (pH 7.5) containing 7.5 mM MgSO 4 . The mixture was then stirred at 37°C for one day. Test samples (10 mg, 100 mg, 1,000 mg) were dissolved in EtOH; after that the solvent was separated under vacuum and to each tube 200 μL of the DNA/methyl green solution    was added. Samples were incubated in the dark at ambient temperature. After one day, the final absorbance of the samples was determined at 642.5-645 nm. Results were corrected for premier absorbance and normalized as the percentage of the untreated standard. The absorbance of the pure DNA (A 260/280 ) is ∼ 1.8.
decomposition steps for the complexes are tabulated in Table 3.

Results of kinetic data together with the thermal degradation steps of complexes
The results of kinetic data of the isolated complexes (Ni 2+ , Pt 4+ , and Cu 2+ ) as well as the thermodynamic parameters of activation were calculated by Eyring equation [43]. All the data are recorded in Table 4 by two isothermal methods such as Coats-Redfern (CR) [44] (Figures 9, 11, and 13) and Horowitz-Metzger (HM) [45] methods (Figures 10, 12, and  14). The results in Table 4 pointed out to the following remarks: (i) The stages of decomposition illustrate that the best fit is when n = 1, while the other values for n did not fit the best correlation. suggesting that the disordered structure of the activated fragments is less than that of the undecomposed complex and consequently the decomposition reactions are slow [43].

Cyclic voltammetry (CV)
The redox properties of Cu 2+ complex was studied by CV technique as shown in Figure 15.      angles are decreased or increased on bonding as a result of bonding. (iii) The bond angles in Pt 4+ and Cu 2+ complexes afforded square-planar geometry, while Ni 2+ complex shows tetrahedral geometry with sp 3 geometry. Finally, Cd 2+ complex illustrates octahedral geometry. (iv) We can predict an increase in the molecular weight and a decrease in the gas phase energy on the basis of the data in Table 5. (v) The energy of both HOMO (π-donor) and LUMO (π-acceptor) is important in quantum chemical monographs, since the orbital behaves as an electron donor and known as a HOMO while the orbital acts as an electron acceptor and nominated as a LUMO. These molecular orbitals are known as frontier molecular orbitals (FMOs). Table 5 depicted the chemical reactivity and site selectivity of the molecular systems by the DFT method concept. The energies of (E HOMO +E LUMO ) and energy band gap (E HOMO −E LUMO ) which feigned the end charge-transfer interaction within the molecule, electronegativity (χ), chemical potential (μ), global hardness (η), global softness (S) and global electrophilicity index (ω) are recorded in Table 5 [48,49,50]. The characterizations of kinetic stability and chemical reactivity of compounds depend on the energy gap (E HOMO −E LUMO ) which is considered the main stability index that facilities the description. Upon the group that enters in the conjugation, the low value of energy gap suggests the easier of charge-transfer which influences the biological activity of the compounds. It is well known that the soft ligand with small gap is more reactive and more polarized than the hard one due to the ease in donating electron to the acceptor. Table 6 illustrates some of energies the isolated complexes. This proposes that the suggested formula of this compound is approved with the experimental data. Scheme 2 explored the fragments of the Pt(IV) complex (1:1). The data of elemental and thermal analysis are also taken as an evidence for that assumption. The mass spectrum indicates the dissociation of Pt(IV) complex.

Antitumor activity
The in vitro cytotoxicity of L and its complexes was scanted by MTT-based assays [35,36,51]. Through colorimetric technique, MTT assay is considered an adequate method to decide the cytotoxicity and for measuring cell growth. It is known that the yellow color of [MTT; 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] changes to purple due to the formation of formazan via mitochondrial dehydrogenases of the cells [52]. The purple formazan which is insoluble becomes soluble on the addition of suitable solvent to form the colored solution. And hence the value of the absorbance at specific wavelength of the colored solution was purposed. In comparing the cured cells with the compounds and those obtained by unreacted control cells of the amount of purple formazan formed, we get the efficacy of the complexes in triggering death of cells concluded by the formation of a potion response curve and the results are shown in Table 7. The data obtained show that the estimating of L has availed chemotherapy since it exhibits a very strong activity towards mammary gland breast (MCF-7) and human cervical epithelioid carcinoma (Hela) with IC 50 values of 7.91 μg/mL and 6.83 μg/mL, respectively. Also, the ligand has a strong activity against prostate cell line (PC-3) with IC 50 value of 12.13 μg/mL. Almost the same result is with the Ni(II) complex as it has a very strong activity towards (MCF-7) and (Hela) with IC 50 values of 9.98 μg/mL and   water to the dye was allowed, which led to the development of the pale carbinol, then the absorbance declines as observed from the colorimetric curves. The offset was calculated by a spectrophotometric assay as a decrease in the absorbance at 630 nm. The results were announced as inhibition concentration 50% value (IC 50 ) calculated by linear regression of data plotted on a semi-log scale and represented in Table 8. In this assay, doxorubicin was used as a control. Doxorubicin or Adriamycin, this drug belongs to a group of antitumor medications or the socalled antimetabolic drugs. It works to stop the growth of tumor cells by interfering with the DNA, which is the genetic material present in the cell. L displayed a very strong inhibitory activity and powerfully intercalate DNA Approximately, the activity of tested chelates is arranged in the order Ni(II) > Cu(II) > Cd(II) > Pt(IV) comparing to Doxorubicin with IC 50 values as shown in Table 8.

Antioxidant activities
The ligand (L) and its metal complexes were examined for their antioxidant activity by the erythrocyte hemolysis. The results of the antioxidant activity with vitamin C are depicted in Table 9 and represented in Figure 22. The ligand showed a high antioxidant activity with respect to erythrocyte hemolysis. The activity of the tested complexes is arranged in the order Co(II) > Pd(II) > Fe(III) in comparison to vitamin C with respect to erythrocyte hemolysis.

Antibacterial and antifungal activity
In the antimicrobial activities, the ampicillin (a broad-field antibiotic) acts as a positive control for the antibacterial test while DMSO was utilized as a negative control and solvent. The diameter of inhibition zone of ampicillin towards E. coli is 25 mm and towards S. aurous is 21 mm. Amphotericin B (antifungal agent) recorded the antifungal activities towards the fungal strains A. flavus and C. albicans of 17 mm and 21 mm, respectively. Only Cd(II) and Cu(II) compounds exhibit an antifungal effectiveness towards fungal strain C. albicans with 27 mm and 11 mm inhibition zone diameter, respectively. While Ni(II) and Pt(IV) chelates do not display any antifungal reactivity  towards fungal strain C. albicans. Cd(II) complex shows a potent activity against fungal strain A. flavus and Ni(II) and Cu(II) compounds show moderate antifungal activities against fungal strain A. flavus. While Pt(II) complex does not show any antifungal activities against fungal strain A. flavus. It was perceived that the ligand has no effect against bacteria or fungi as represented in Table 10. The antimicrobial activities of L and its chelates are shown in Figure 23. The efficient and effective propagation of compounds via cell membrane or interaction with microorganism cell wall give rise to a high antibacterial activity, which is known as lipophilic characteristics (i.e., penetrating the lipid cell wall linked to the polarity of central metal ion and raised with the formation of chelation ring, sharing the positive charge on metal ion with the N -donor atom of L causing π-electron delocalization on the chelation ring, which is also corroborated by the works in [53,54,55]).

=
Zone of inhibition by test compound (diameter) Zone of inhibition by standard (diameter) × 100.

Conclusions
A novel ligand, 3-(3H-1,2,4-triazole-4-(5H)-ylimino)butane-2-one-oxime (L), and its complexes were synthesized and characterized using chemical, spectral (FTIR, UV-Vis, mass), cyclic voltammetry, and magnetic measurements. Electronic spectra and magnetic measurements of the complexes suggest a structure that is tetrahedral for Ni 2+ , octahedral for Cd 2+ , while square-planar for Cu 2+ and Pt 4+ complexes. Also, DFT parameters were applied for Cu 2+ , Ni 2+ , and Pt 4+ complexes which prove the geometry of L towards the metal ions. Kinetic and thermodynamic parameters of the isolated complexes were calculated by CR and HM methods. The biological activity of L and its complexes was tested against three types of cancer cell lines, and against different bacterial strains.