Structural, Theoretical and Biological Studies of (Z)-3-Amino-N-(3-Amino Pyrazine-2-Carbonyl) Pyrazine-2-Carbohydrazonic Acid (APA; L) and Its Cu 2+ , Co 2+ , Pt 4+ and Pd 2+ Chelates

New chelates derived from the novel ligand, (Z)-3-amino-N-(3-amino pyrazine-2-carbonyl)pyrazine-2-carbohydrazonic acid (APA, L), with Cu 2+ , Co 2+ , Pt 4+ and Pd 2+ salts were investigated. The results suggest that APA acts as mononegative tridentate in the case of Cu 2+ , binegative tetradentate in the case of Co 2+ and as mononegative bidentate towards Pt 4+ and Pd 2+ chelates. The results of the corrected µ eff . and spectral suggest the structures of the isolated chelates. The results of the corrected µ eff . and spectral suggest the geometries of the isolated chelates. Molecular modeling is deduced and chemical reactivity, energy components for chelates and also MEP for APA is illustrated. In Vitro, the SOD and radical scavengers like activity of the synthesized compounds Hep G2 liver cancer cells and cytotoxic activity were checked. Metal chelates show potent anti-oxidative activity. The results of cytotoxic activity assay against hepatocellular carcinoma cell line Hep G2 confirmed that Pt 4+ complex has the highest value, while APA, Cu 2+ , Co 2+ and Pd 2+ chelates have no significant cytotoxic activity.

The pervasive incidence of simple pyrazine molecules in nature, especially in the flavors of many food systems, their effectiveness at very low concentrations as well as the still increasing applications of synthetic pyrazines in the flavor and fragrance industry are queried for the high interest in these compounds [13]. Specific pyrazines, especially dihydropyrazines, are essential for all forms of life due to their DNA strand breakage activity and/or by their influencing of apoptosis [14]. Synthetic pyrazine derivatives have also been advantageous as drugs (antiviral, anticancer, antimycobacterial, etc.) fungicides, and herbicides [15]. Furthermore, 3-amino-6-chloro-pyrazine-6-carboxylic acid, as an example of a simple pyrazine compound, has shown an anti-auxin behavior. The significance of the pyrazine (1,4-diazine) ring for the activity of the biological herbicides can be assessed primarily according to the size of the investigated molecules. In this work, the potent active antioxidant and cytotoxic activities of the separated chelates on HepG2 liver cancer cells have been examined. The structures of the resulting APA and its chelates have been achieved on the basis of elemental analyses, spectral (IR, UV-vis., 1 H-NMR, 13 C-NMR mass), magnetic and thermal measurements and supported by Density Functional Theory (DFT) method.
The goal of our work is to check in detail the chelation behavior of the novel (Z)-3-amino-N-(3-aminopyrazine-2-carbonyl) pyrazine-2-carbohydrazonic acid (APA) and its chelates derived from Cu 2+ , Co 2+ , Pt 4+ and Pd 2+ ions, embedding structural articulation and molecular modeling of both APA and its chelates. Moreover, our attention is directed to evaluate the antioxidant and anticancer activity of the ligand as well as the detached chelates. mol) in absolute ethanol to N 2 H 4 (8 mL). A pale yellow solid of APA was obtained by continuous stirring of the reaction mixture on a heater at 60˚C for 1.5 h. Doubtless the addition of methyl 3-aminopyrazine-2-carboxylate to excess hydrazine hydrate assists of the formation of APA and excludes the formation of 3-amino-pyrazin-2-carbohydrazide ( Figure 1(c)). The novel product was obtained and collected by filtration followed by continuous washing several times with hot EtOH and ether. The isolated product was kept in a drier over anhydrous phosphorous pentaoxide. APA was recrystallized from absolute EtOH (Yield: 60%). The purity of APA was checked by spectral, TLC and its melting point (159˚C -160˚C).

Superoxide Dismutase Scavenging Activity
Both the metal chelates and APA were assessed against antioxidant activity [20].
The italicize of the ligand or its chelates to damped phenazine methosulphatemediated reduction of nitro blue tetrazolium dye was obtained from the results of the assay. The determination of SOD was calculated as reported earlier [18].

Cell Cultures and Viability against HepG2
The safety of HepG2 cells in culture was carried out in triplicate and calculated by the MTT assay as reported in our earlier work [21]. [22] in materials studio package [23] was acquired for the data which is resolved for large-scale DFT calculations. DFT semi core pseudopods calculations were elicited with double numerical basis sets in addition to polarization functional. DNP basis sets are of comparable quality to 6-31G Gaussian basis sets [24]. The data acquired by DNP basis sets are more accurate than Gaussian basis sets of the same size as reported by Delley et al. [22]. The RPBE functional [25] is considered the best exchange-correlation function [26] establish on the generalized descended approximation (GGA) and is used to take account of the exchange and correlation impacts of electrons. Hence the geometric optimization is officiated without any symmetry curb. Table 1 summarized the elemental analyses (C, H, M, and Cl) of the isolated metal chelates and some physical properties. The empirical formulae of the chelates are rimmed by elemental analyses (C, H, N, M, Cl) and confirmed by thermal gravimetric data. The low values of molar conductance (8 -13 ohm −1 ·cm 2 ·mol −1 range) of the chelates in DMSO (Table 1) suggest their non-electrolytic nature [27]. Doubtless, the lower pH of the solution during complex formation (pH = 2, 3, 2 and 5 for Cu 2+ , Co 2+ , Pt 4+ and Pd 2+ , respectively) causes the block of the NH 2 groups and consequently the enolization of the carbonyl group occurred with the liberation of a proton during complex formation. Hence, all the compounds were obtained by losing one or two protons as shown in Table  suggest that the comparatively high pH values of the Co 2+ and Pd 2+ (pH = 3 and 5) complexes up stay for the liberations of two protons while the decline pH of Cu 2+ and Pt 4+ complexes give the chance to lose only one electron during the formation of the reactants.

IR Spectra
Two tautomer shapes [keto (a) and enol (b)] as elucidated in Figure 1(a), Figure  1(b) are suggested for APA on the basis of the results of IR spectra. The infrared spectrum of APA (KBr) shows three bands at 3416, 3306 and 3200 cm −1 imputable to ν(OH), ν a (NH 2 ) and ν s (NH 2 ) [28] vibrations, respectively. The band at 3150 cm −1 is attributed to ν(NH 2 ) vibration [29].  [31]. All these basics were ascertained and supported by studding the modeling of the ligand as shown in Figure 2. The infrared spectra of APA and the isolated metal chelates (Table 2) show that the ligand binds to Cu 2+ and Co 2+ metal ions in 2:1 (M:L) ratio and 1:1 in case of Pt 4+ and Pd 2+ metal ions. APA behaves as mononegative tetradentate coordinating via the two nitrogen of NH 2 groups and two oxygen atoms with losing only one proton from the OH group ( Figure 3) in case of C 10 H 13 Cu 2 N 8 O 4 Cl 3 . The bonding sites are revealed by: 1) the NH 2 and C=O groups are relocated to lower wavenumbers indicating that these groups are participate in chelation; 2) the observation of new band at 1331 cm −1 attributed to (C-O) group; 3) new bands are traced at 532 and 467 cm −1 assigned to M-O and M-N, respectively [32]. The spectrum of copper(II) chelate exhibits a broad band at 3416 cm −1 attributable to the water of coordination and water of solvation [33]. APA coordinates in a binegative tetradentate ligand via two NH 2 groups and two oxygen atoms with losing two protons from the two OH groups in case of Co 2+ , with the general formulae, C 12 H 18 Co 2 N 8 O 5 Cl 2 ( Figure 4). This conduct revealed by: 1) The hiding of the (CO) group indicates this group is taking part in coordination after deprotonation.
2) The bands at 3285 -3262 cm −1 attributable to (NH 2 ) are observed at lower wave numbers indicating that this group participates in bonding. Table 2. The most important IR bands of APA and its metal chelates.   6) The chelates exhibit a broad band at 3401 cm −1 assignable to H 2 O and EtOH [33].
In the complexes, C 14  nated water as well as ethanol [33]. Also, the bands observed in the range 544 -494 and 473 -461 cm −1 are assigned to M-O and M-N, respectively [32].

Nuclear Magnetic Resonance Spectra ( 1 H-NMR and 13 C-NMR)
Three signals at 9.8, 7.5 and 4.5 ppm, relative to TMS, are observed in the 1 H-NMR spectrum of APA in d 6 -DMSO ( Figure S1) and attributed to the protons of OH [34], NH and NH 2 [28] groups and obscured on adding D 2 O ( Figure S2). The protons of pyrazine ring are noticed in the (7.7 -8.3) region ppm. The observation of the OH signal suggests that APA is existed in the keto/enol forms as illustrated in Figure 7. Additional backing for the composition of the ligand is attested from the arising of ten signals in the 13     trend of π-electron densities [36], which is taken as a strong guide that APA is existed in the keto/enol forms. The 1 H-NMR spectrum of Pt 4+ chelate ( Figure S3) shows signals at 9.01, 3.93 and 3.56 ppm attributed to NH, NH 2 and OH (EtOH and H 2 O) protons, respectively. The protons of pyrazine ring are noticed in the 8.55 -7.96 ppm range. The protons signal of ethanol (CH 3 and CH 2 ) are observed at 1.35 and 4.38 ppm indicating the existence of EtOH outside the coordination sphere [37]. The Pd 2+ chelate spectrum ( Figure S4) exhibits three signals at 8.27, 3.85 and 3.89 ppm assigned to NH, NH 2 and OH (EtOH and H 2 O) protons, respectively. The protons of the pyrazine ring are traced in the 7.98 -7.59 ppm range. The monitor of the proton signals of CH 3 and CH 2 (EtOH) outside the coordination sphere at 1.05 and 3.45 ppm verify the proposed formula [37].

Mass Spectra
Molecular ion peak noticed at m/z = 274 (10%) corresponds to (C 10 H 10 N 8 O 2 ), M. wt. = 274.246, as shown in the mass spectrum of APA ( Figure 8). Elemental analyses and spectral are taken as strong brochure for the suggested geometry.

Spectral and Magnetic Results
All the electronic spectra of APA and its chelates were arises in Nujol mull. The chelates are distinctive by intense charge-transfer bands. The absorption band at 300 -344 nm (33,333 -28,901 cm −1 ) is raised to π → π * transition of pyrazine rings are shown in the spectrum of APA. The absorption band observed at 402 nm (24,815 cm −1 ) is assigned to n → π * of the NH 2 group, which shifts in chelates toward lower frequencies, supporting the chelation of the NH 2 group in bonding. Another two bands appear at 452 and 548 nm (22,123 and 18,284 cm −1 ) are assigned to π → π * of (C=N) azom and n → π * transitions of the carbonyl group, respectively [38] [39]. The 2 B 2 → 2 E and 2 B 2 → 2 B 1 transitions at 728 and 574 nm (13,736 and 17,421 cm −1 ) in the electronic spectrum of the Cu 2+ chelate (Nujol) confirms the presence of tetrahedral structure around the Cu 2+ ion [40]. The band at 525 nm (19,047 cm −1 ) is attributed to type L → M transition. The corrected magnetic moment value (μ eff = 1.6 BM) is calculated for each Cu 2+ ion [41]. Also, the Co 2+ chelate shows three bands at 691, 562 and 418 nm (14,471,17,793 and 23,923 cm −1 ). The first two bands are assigned to 4 A 2 → 4 T 1 (F) and 4 A 2 → 4 T 1 (P) transitions in a tetrahedral geometry around the Co 2+ ion. The band at 23,923 cm −1 is raised to charge-transfer (L → M) [42]. The corrected magnetic moment (μ eff = 4.4 BM) of Co 2+ ion supports a tetrahedral geometry around the Co 2+ [43]. The values of to be 10 Dq, B and β (519.9 cm −1 , 736.1 and 0.76), were calculated. Pt 4+ chelate exhibits two bands at 486 and 440 nm (20,576 and 22,727 cm −1 ) refers to the d-d transition bands 1 A 1g → 1 T 1g and 1 A 1g → 1 T 2g , respectively [17]. Two bands at 388 and 592 nm (25,773 and 16,891 cm −1 ) assigned to 1 A 1g → 1 B 1g and 1 A 1g → 1 E g transitions in a square-planar configuration for the diamagnetic C 11 H 14

DFT Calculations for Optimization of the Geometry
The optimization of APA and its metal chelates are displayed in Figures 2-6. The bond lengths and angles calculated by optimized molecular geometry are given in Tables S1-S10. The important points are summarized as follow:  (10), which are affected (decrease or increase) on chelation due to chemical bonding [45].
2) Tetrahedral geometry of the Cu(II) and Co(II) chelates, C 10 H 13 Cl 2 , is proposed on the basis of the values of bond angles, while octahedral geometry is propounded for C 14 H 25 PtN 8 O 6 Cl 3 , 2EtOH and a square-planar for C 11 H 14

Chemical Reactivity 1) Methods of Describing the Global Reactivity
The most important parameters in quantum calculations depend mainly on both the energies of the HOMO (π-donor) and LUMO (π-acceptor). These parameters are essentially behaved as an electron donor and electron acceptor, respectively, and these orbitals are known as Frontier Molecular Orbitals (FMOs). (Table 3) anticipate the stability of the synthesized molecules [18].

1) The negative values of E HOMO and E LUMO and their neighboring orbitals
2) The coordination sites (electrophilic attack) on aromatic compounds are easily anticipated using FMOs theory. The uttermost overlap between the HOMO on one molecule and the LUMO on the other illustrates that the reaction takes place. The most important factor in any reaction depends essentially on the interaction between HOMO and LUMO. The goal of the computations is to find out the highest values of molecular orbital coefficients. Consequently, the elevated value of molecular orbital coefficients for the ligand orbitals indicates its activity for chelation. All these keynotes are deduced from the data obtained from calculations indicating that the nitrogen of NH 2 groups, oxygen of deprotonated C-OH and carbonyl groups with largest values of molecular orbital coefficients.
3) Linert et al. [45] showed that the bond strength increases as the adjacent bonds become weaker according to Guttmann's variation rules. This view agrees with the results of increasing of the value of E HOMO accompanying by elongation followed by the weakness of the metal-ligand bonds and the narrowness of the positions abutted to the metal-ligand centers and thus becomes quite strong. 4) The level of HOMO is essentially domestic on the N (20), N (19) and O(10) and O(12) atoms ( Figure 9) suggesting that these atoms are the most active nucleophile sites on the central metal ion. This indicates that these centers have high values of HOMO density coefficients and in face of the metal ions.
5) The energy gap (E HOMO -E LUMO ) [45] is used to calculate the kinetic stability and chemical reactivity of the APA. The theoretical model for illustrating the geometry and approval barriers in different modulate, which governs the biological bustle of the molecule, depends mainly on the energy gap. The softness of the molecule (more polarized) is known from the small gap. Accordingly, the reactivity of soft molecules than hard ones is mainly due to the easily offer electrons to an acceptor. The charge-transfer is easily occurred due to the small energy gap in case of APA suggests which effectuating the biological activity of the molecule. The groups that enter into conjugation causes the low value of energy gap. 6) The donating electron capacity is weaker due to the lowering of the HOMO energy values. Contrarily, the molecule becomes good electron donor when the HOMO energy becomes higher. The ability of a molecule to receive electron depends on the LUMO energy [45].
Both the site selectivity and chemical reactivity of the molecular systems is figured out from DFT. The energies of both (E HOMO + E LUMO ) and (E HOMO − E LUMO ) construe the charge-transfer interaction, electronegativity (χ), chemical potential (µ), global hardness (η), global softness (S) and global electrophilicity index (ω) within the molecule [46] [47]. The results are depicted in Table 3.
The inverse value of the global hardness bestowed the softness (σ) as follow: The electrophilicity index is considered the most important quantum chemical factor which describes the toxicity of different pollutants in relation to their reactivity and site selectivity [48]. The biological activity of drug receptor interaction is illustrated from the value of electrophilicity. The new reactivity index is utilized to measure the stabilization energy when additional electronic charge from the environment from the system is needed. The measurements of the molecular stability and reactivity depend on the values of η and σ. The metal ion acts as a Lewis acid while APA behaves as a Lewis base in chelate formation.

Molecular Electrostatic Potential (MEP)
The electrostatic potential mapped onto the constant electron density surface is obtained from the plot of MEP. Also, it is important in disquisition of the molecular structure with its physiochemical property nexus and hydrogen bonding interplay [49] [50] [51]. The values of V(r) at r (x, y, and z) is mooted in terms of the interaction energy between the electrical charge formed from the electrons of molecule, nuclei and proton located at r [52] [53]. The plot of 3D of MEP for APA is labeled in Figure 10. The results showed that the maximum negative region suggests the most suitable site for electrophilic attack which symbolized by a red color. The blue color represents the maximum positive region prefers site for nucleophile attack. Equivocally the increase of potential is in the order red < green < blue, where blue depicts the strongest attraction and red shows the strongest repulsion. The negative potential regions are existed over the more electronegative atoms meanwhile the regions with positive potential are found over the hydrogen atoms.

Other Molecular Properties
Binding energy estimations detect that the increase of the value of calculated binding energy of complexes in comparison to that of APA indicating that the stability of the formed metal chelate is higher than that of APA. DFT calculations conjecture this energy as shown in Table 4.

Thermal Studies (TGA/DTG)
The degradable stages, temperature ranges degradable products as well as the weight loss percentages of Cu 2+ chelate are shown in Table S11. Also, Figure S8 illustrates the TGA/DTG curves of the chelate. The results suggest that the experimental weight loss agrees with the calculated values. The residue was confirmed by chemical analysis. The first stage at 32˚C -102˚C with weight loss of 3.3 (Calcd. 3.3%) matches to the loss of H 2 O (lattice molecules) was shown from the TG thermogram of C 10 H 13 Cu 2 N 8 O 4 Cl 3 . The weight loss of 27.1 (Calcd. 27.2%) at 102˚C -246˚C is assigned to the loss of the coordination water molecules and (C 4 H 4 N 3 + Cl) portions as shown from the second step. The removal of (C 4 H 3 N 3 ) portions with weight loss of 17.1 (Calcd. 17.2%) at 247˚C -336˚C is obtained from the third step. The fourth step at 336˚C -416˚C with weight wastage of 14.1 (Calcd. 13.4%) indicates the removal of 2HCl molecules. Finally, the fifth step is noticed in the 417˚C -569˚C range with gradual mass loss corresponds to (N 2 + C) fragments, 7.3 (Calcd. 7.4%). The final remainder is [Cu 2 (O) 2 ] + C (Found 31.1, Calcd. 31.5%).

The Antioxidant Activity
Antioxidant activity utilizing superoxide dismutase like activity assay was used Table 4. Some of energetic properties of APA and its complexes calculated by DMOL 3 using DFT-method. to check out all the chelates. In contrarily, the Pd 2+ chelate shows no antioxidant activity while APA showed from low to mediate anti-oxidative activity as shown in Figure 11. The Cu 2+ , Co 2+ and Pt 4+ chelates have the highest activity of quenching phenazin methosulphate radicals at 76.3%, 83.0% and 88.1% inhibition, respectively. The high potency of antioxidant is due to the fared of chelation. This is due to structure-activity relationship: they differ in the coordination geometries and stability and distortion. Moreover, the difference in the dipole moment of the complexes affects the stability, the distortion and the activity of the complexes. The compounds under investigation are considered as important group of promising antioxidants and also help to attenuate oxidative stress and used in protection against the harmful action of reactive oxygen species, mainly oxygen free radicals. Figure 11. Superoxide dismutase scavenging radicals like activity of APA and its chelates.

Cytotoxic and Antitumor Activity
APA and its metal chelates were tried against hepatocellular carcinoma cell line HepG2 for their antitumor activity ( Figure 12). After few days' incubation of the HepG2 liver cancer cells using different concentrations of APA and its chelates, Pt 4+ proved to have the highest cytotoxic activity with IC 50 of 79.8 µM.
The viability of HepG2 tumor cells after incubation with Pt 4+ chelate is highly affected. The cell layer partially condensed forming cell-free areas and finally detached from the culture plate. APA and its chelates (Cu 2+ ,Co 2+ and Pd 2+ ) show no significant cytotoxic activity. The data indicate that there is no significant difference in cell death in comparison to the untreated control (negative control).

Conclusion
Metal chelates (Cu 2+ , Co 2+ , Pt 4+ and Pd 2+ ) derived from the novel ligand, (Z)-3amino-N-(3-aminopyrazine-2-carbonyl) pyrazine-2-carbohydrazonic acid (APA, L) were synthesized and achieved by conventional physical and chemical, spectroscopic, magnetic and TGA studies. APA behaves in tridentate and tetradentate manners as in the case of Cu 2+ and Co 2+ , respectively. Also, the ligand acts in a bidentate manner towards Pt 4+ and Pd 2+ chelates. Both the corrected µ eff . and spectral data propose the geometries of the chelates and the molecular modeling.
The chemical activity, energy components and also MEP for APA are figured out.
In Vitro, cytotoxic activity and superoxide dismutase radical scavenger-like activity of the synthesized compounds HepG2 liver cancer cells were examined.
Metal chelates show the potent anti-oxidative activity. The results of cytotoxic activity assay against hepatocellular carcinoma cell line HepG2 confirmed that Pt 4+ complex has the highest value, while APA, Cu 2+ , Co 2+ and Pd 2+ chelates have no significant cytotoxic activity.
Angle Degree (˚) Angle Degree (˚) Angle Degree (˚) Open Journal of Inorganic Chemistry