, THERMAL,

. The condensation of thiosemicarbazide with oxazolidinone produced 5-benzylidene-3-(4-chloro-phenyl)-6-oxo-5,6-dihydro-1H-[1,2,4]triazine-2-carbothioic acid amide (HL 1 ). Co(II) and Ni(II) ions were reacted with the HL 1 ligand. Elemental analysis and molar conductivity, along with mass, 1 H-NMR, IR, UV-Vis, and X-ray powder diffraction spectral examinations, were used to reveal the chemical structure of the synthesized complexes. The stoichiometry of the complexes was determined to be 1:2 (metallic: moiety) using analytical, spectroscopic, and thermal data. The thermal properties of the complexes were explored using thermogravimetric (TG-DTG/DTA) methods, as well as the decomposition stages. Several kinetic thermodynamic parameters such as free activation energy (ΔG*), activation enthalpy (ΔH*), activation entropy (ΔS*), pre-exponential factor (A), and the activation energy (E*) were estimated. Inhibitory effects towards colon carcinoma cells (HCT cell line), hepatocellular carcinoma (Hep G2 cell line), and breast carcinoma (MCF-7 cell line) were verified using various concentrations of the samples. A colorimetric method was used to determine the cell viability percent in comparison to doxorubicin drug as a control.


Materials
All the materials employed in the current study are of pure grade chemicals. CoSO4 (≥99%) and NiSO4 (99%) hydrate were obtained from "Fluka Chemical Co."

Synthesis of HL1 ligand
The HL1 ligand was produced by combining thiosemicarbazide (0.01 mol) with 3-bromo-4methoxy-benzylidene derivatives (0.01 mol) and oxazolinone in acetic acid (25 mL). The mixture was kept at a high temperature under reflux for 2 hours. After cooling, the resulting solid was crystallized in acetic acid, filtered off, subjected to ethanolic wash, and dried. The HL1 ligand was obtained as amber crystals with a yield of 78% and a melting point of 160 °C. 1

Synthesis of HL1 complexes
Free HL1 ligand (2 mmol) dissolved in hot DMF (20 mL) was mixed with an aqueous solution of the metal salt [Co(II) or Ni(II)] (1.0 mmol, 10 mL). The mixture was heated for 3 hours at temperature 60-70 °C. After allowing the solutions to sit for a night, a pink and light green colorful mixture of Co(II) and Ni(II) complexes was gathered. Filtered solid powder complexes are washed in ethanol and dried over CaCl2. TLC was used to evaluate the purity of the complexes. The melting point of all complexes is above 300 °C.

Physical measurements
Infrared spectra were collected in the 4000-400 cm −1 mid-range using a Perkin-Elmer 1420 Spectrometer. The UV-Vis. spectra were obtained in the DMSO solvent with a concentration of (1x10 −3 M) using a Jenway 6405 Spectrophotometer in the range 200-800 nm. For the freshly produced solutions, molar conductance was measured using a Jenway 4010 conductivity meter at 1x10 −3 mole in DMSO solvent. On General Electric QE 300 equipment, the NMR spectra were acquired, and chemical shifts were calculated in relation to TMS. On Gc/MS with CI (chemical ionization) and a Hewlett-Packard MS-Engine Thermosprary, the mass spectra were obtained. A Shimadzu TGA-50H thermal analyzer was used to perform the thermogravimetric and differential analysis (TGA-DTG/DTA) in an active nitrogen environment.

Assessment of cytotoxic activities of chemical compound
The cytotoxic effects of the tested compounds were evaluated at the Regional Center for Mycology and Biotechnology, Egypt. Trypan blue dyes, crystal violet, and dimethyl sulfoxide (DMSO) were obtained from Sigma Company. Trypsin-EDTA (0.25%), gentamycin, Lglutamine, HEPES buffer solution, RPMI-1640, DMEM, and Fetal Bovine serum were obtained from Lonza Company. All cells were subcultured twice a week and kept in a humidified setting with 5% CO2 at 37 C. The cells were planted in 100 µL of growth media, in a 96-well plate at a cell concentration of 1x10 4 cells per well. The microtiter plates were incubated in a humidified atmosphere with 5% CO2 at 37 °C for 48 hours. Different quantities of samples (50, 25, 12.5, 6.25, 3.125, and 1.56 µg) were introduced, and the incubation was maintained for two days, with viable cell yield evaluated utilizing a colorimetric method. The absorbance of the plates was dtermined on a Microplate reader (TECAN, Inc.) at 490 nm [14].

RESULTS AND DISCUSSION
The elemental analysis results of the free HL1 ligand and its Co(II) and Ni(II) complexes are listed in Table 1. Table 1 also indicates several physical and microanalytical characteristics, such as, milting point, color and molar conductance value of the free HL1 ligand and its Co(II) and Ni(II) complexes. The data shows equimolar stoichiometry (metal: ligand), and the general formula of the complexes based on these results is: [M(HL1)2(SO4)], where M= Co(II) or Ni(II)]. The DMSO solution of Co(II) and Ni(II) complexes (1×10 -3 M) showed low conductivity. The molar conductance values for these complexes are 49 and 68 cm 2 mol −1 , respectively. These values indicated the non-electrolytic character of the complexes [15]. The chemical analysis using BaCl2 solution indicated that SO4 2ion is coordinated to the metal ion. Infrared spectra Table 2 states the diagnostic IR spectral bands of the HL1 ligand and its complexes, as well as their preliminary designations. Table 2 shows the IR spectral data of the Co(II) and Ni(II) complexes with their corresponding vibrational bands. The IR spectrum of free HL1 ligand shows varied intensity from fade-to-moderate strength absorbance bands at 3405 and 3188 cm −1 (NH2). The band at 1356 cm −1 was attributed to thioamide vibrations [15]. The presence of distinct thioamide bands in the HL1 free binding moiety, on the other hand, implies the existence of freebinding moiety in the thione form. The (NH) vibrational band for the free ligand was classified into the fade-to-moderate strength bands at 3285 cm −1 . The vibration movements of (C=O) bands for the ligand are allocated to the refereed band at 1683 cm −1 . A strong band of about 1630 cm −1 was also ascribed to the triazine ring's (C=N) ring. Both phenyl rings had aromaticity bands of (C=C) in the range of 1520-1596 cm −1 . In the IR spectra of the synthesized complexes, the absence of thioamide bands, particularly the (C=S) (1350 cm −1 ) band, and the greater shift of the (NH) band suggested that they were involved in coordination.

Molar conductivity
The DMSO solution of Co(II) and Ni(II) complexes (1×10 -3 M) showed low conductivity. The molar conductance of the synthesized complexes (Table 1) was found to be between 49 and 68 cm 2 mol −1 . These values indicated the non-electrolytic character of the complexes [16]. The deprotonated character of the ligand in most complexes, as well as the covalent attachment of SO4 to the metallic cations, account for the complexes' neutrality. This confirmed that the anions of these complexes are coordinated with the metal ion. This result was strongly supported by the chemical analysis, where SO4 2ion is detected by the addition of BaCl2 solution.

Electronic spectra
When the electronic spectra of the free HL1 ligand are compared to those of their corresponding Co(II) and Ni(II) complexes, certain changes are visible, which can be taken as a sign for complex creation. These bands emerged at 285 and 415 nm in neutral moderate for free binding moieties. These bands are thought to be caused by π→π* and n→π* transitions inside the functional binding moieties. In addition, metal complexes' absorption spectra (in DMSO) contain additive bands at distinct wavelengths. The Ni II complexes absorbance spectral revealed two distinct bands at 680 and 500 nm, which were ascribed to the transitions 3 A2g(F) → 3 T1g(P), and 3 A2g(F) → 3 T1g(F), respectively. The octahedral geometry of the Ni II complex is suggested by its spectra. As a result of 4 T1g(F) → 4 T2g(P), 4 T1g(F) → 4 A2g(F), 4 T1g(F) → 4 T2g(F), the electronic spectrum of the octahedral Co II complex has three types of transitions at 790, 631, and 539 nm, respectively. Based on the preceding explanation, Figure 1 is proposed.

Mass spectra
The mass spectral of HL1 indicated a powerful ion peak at m/z 356, which corresponds to the chemical formula C17H13N4ClOS. By removing the NHCS group, the ion of HL1 (Figure 2) fragmented to create a peak at m/z 297. The ion with m/z 297 lost its NH group, resulting in an ion with m/z 282, which lost its C=O to provide a peak at m/z 254. The ion at m/z 254 was fragmented, resulting in a stable peak at m/z 117. The hydrogen cyanide (HCN)    The TG results are supported by a single medium peak for DTG at 104 °C, which was assigned to liberate one NH2 molecule and one chlorine atom. The residual complex begins to decompose around 120 °C, and the partial structure is destroyed at 800 °C. Many tiny gas molecules are liberated at this moment. Two DTG peaks (329, 784 °C) and three DTA peaks (60 °C (endo), 337 °C (endo), 520 °C (endo)) may be seen for this phase, indicating that at least three intermediary steps are created during this degradation stage. This stage is responsible for weight loss (reported to be 55.23%). CoO contaminated with carbon atoms is recognized as the final stable residue after 800 °C. At 30 o C, the Ni(II) complex begins to lose mass and shows four breakdown phases. Five DTG (64, 288, 364, 498, and 757 o C) and two obvious DTA (67 (endo), and 757 o C (endo)) are used in these phases. It's worth noting that the ligand decomposes in two steps when complexed with Ni(II), whereas it pyrolyzes in four steps when decomposed alone. The loss of terminal groups for the organic moiety of the HL1 ligand, which accounts for 14.12% weight loss, is attributable to the first stage, which leaves the other organic portion connected with nickel ions behind. The ligand from labor separates in an unexpected way from the second to fourth breakdown phases, losing 48.95% of its mass. Nickel oxide (NiO) polluted with a few carbon atoms, is the ultimate remaining product of the Ni(II) complex.

Kinetic studies
The kinetic characteristics of the decomposition processes were elucidated using different kinetic approaches such as Horowitz-Metzger (HM) and Coats-Redfern (CR) [17,18]. The foregoing approaches were used to compute parameters such as Gibbs free energy change; ΔG*, enthalpy change; ΔH*, entropy change; ΔS*, frequency factor; A, and activation energy; E (Figure 4). Each method's activation energy values were in good agreement with one another. The thermogravimetric study was also used to find an appropriate mechanism for each material's thermal degradation process. Table 3 lists the computed thermodynamic parameters from TG and DTG. The order parameter for the decomposition stage of interest was chosen as the n value that offered the greatest fit (r ≈ 1). The A and E values were calculated using the intercept and linear slope of such a stage. The following points should be noted: (i) All phases of complex decomposition have a best fit for (n = 1), suggesting that they are all first-order decompositions.
(ii) Negative activation entropies ΔS suggest that the activated complexes are more ordered than the reactants and that the reactions are sluggish. (iii) ΔH values are positive indicate that the breakdown processes are endothermic.

XRD study
Powder X-ray diffraction (XRD) data prpoased a monoclinic structure for the HL1 ligand and its Ni(II) and Co(II) complexes ( Figure 5). The mean crystallite sizes of the free ligand and its complexes (D) were calculated using the Scherrer equation [D = 0.9λ/(β cosθ)], where β is the full width at half maximum of the diffraction peak), θ is Bragg diffraction angle, and λ is X-ray wavelength (1.5406 Å). The average crystallite sizes of all compounds were found to be ∼ 23-37 nm.

Cytotoxicity studies
The cytotoxic and anticancer effects of the HL1 ligand and its Co(II) and Ni(II) complexes against cell lines MCF-7, Hep G2, and HCT were tested according to MTT assay [19]. Different concentrations of the tested compound were used and cell viability (percent) was evaluated using a colorimetric method. The cytotoxicity results were listed in Table 4. Table 5 summarizes the results of the 50% inhibitory concentration (IC50) data. The HL1 ligand was found to be active against HCT, HePG-2, and MCF-7 cell lines when compared to the standard anticancer medication doxorubicin. MCF-7, HePG-2, and HCT cell lines were also reported to be active against Co(II) and Ni(II) complexes. The cytotoxic activity results reveal that the Co(II) and Ni(II) complexes show fairly less activity against all the tested MCF-7, HCT, and HePG-2 cell lines, and in general, the activity order of the compounds can be represented as HL1 > Ni(II) > Co(II). The higher activity of the free HL1 ligand in comparison with the metal complexes may be owing to the effect of metal ions on the normal cell membrane [20]. The synthesized complexes display marked cytotoxic activity against the tested cancer cell lines (MCF-7, HCT, and HePG-2), and the IC50 values for the complexes were decreased compared with that of the free HL1.