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Solvo-thermal Assisted-Synthesis; Experimental and Theoretical Characterization; and Biological Evaluations of Azo-Chelator-Ligand Chelates of Fe(II) and Zn(II) ions

Background and Objectives: The resistance of microbes against anti-bacteriological drugs leading to countless deaths and terminal ailments remains a basis for concern. Hence, the main interest of this study was to design, synthesize and report unusual compounds with basic hydrophilic moieties plus hydrophobic functions for anti-bacteriological studies. Materials and Methods: Analytical (melting points, micro-analysis (C. H.N.S) magnetic susceptibility (μeff), molar conductance plus solubility test) methods; spectral (Fourier Transform Infrared(FTIR)), electrospray ionization mass spectrometry (ESI-MS), nuclear magnetic resonance (Hplus C-NMR), electronic(UV-Vis)) measurements; theoretical (DFT) evaluations were utilized for the characterization of the chelator and its chelates. All synthesized compounds were examined for antimicrobial and antioxidant potentials while the chelator was singly evaluated for solvent extractive capacity. Results: A nitrogenous based chelator-ligand, (E)-1-(((4,6-dimethylpyrimidin-2-yl)imino)methyl) naphthalen-2-ol(LH) synthesized through reflux-condensation reaction of 2-amino-4,6dimethylpyrimidine with 2-hydroxy-1-napthaldehyde was acquired. Further reflux of the chelatorOriginal Research Article Chioma; AJACR, 10(1): 40-56, 2021; Article no.AJACR.76868 41 ligand with bivalent ions of iron-sulphate and zinc-acetate salts plus 2,2’-bipyridine resulted into separate bivalent-heteroleptic metallic chelates. The deprotonated nitrogen of the amine moiety and carbon of the carbonyl gave rise to the chelator-ligand with N2O2 chromophore detected around the metallic atom in the chelates. The μeff data plus UV-Vis spectral values of the chelates conformed to 6-coordinate octahedral geometry. All the chelates were high spin and non-ionic in dimethylsulfoxide (DMSO). The antimicrobial and antioxidant screening of the compounds presented moderate to fantastic results, while the metallic extractive proficiency of the chelator showed outstanding extractability for Fe and Znions with an efficiency of 79.34% and 51.92% correspondingly. Conclusion: All the synthesized compounds are novel and demonstrated prospective biological, plus metallic ions’ extractive potentials required for designs plus isolation of products also for such actions.


INTRODUCTION
Research undertakings on the improvement of metallic based antimicrobial drugs have had reasonable breakthroughs over the years. However, drug resilient bacteriological organisms has remained of utmost scientific concerns [1]. Countless metallic-based medications [2]. have been prepared as well as evaluated against manifold drug resisting microbes [3] like methicillin resistant Staphylococcus aureus, vancomycin resistant E. faecium and penicillin resistant S. pneumoniae strains [4], however, with only few making it to preclinical trials. The major challenge lies with the risk of metal poisoning due to high level of toxicity among metal based drugs [5]. Hence, the focus in the growth of more operative anti-bacteriological as well as anti-fungiform drugs is to synthesize drugs with broad spectrum antimicrobial potentials; of low toxicity, that can easily diffuse across microbial intracellular membrane, improved selectivity and with novel mechanisms of action [6].
The workability of anti-bacterial activities for most anti-bacteriological drugs remains a basis for their attraction [7] to the transpeptidase target enzyme within the bacteria, potential to permeate the bacterial cell membrane in addition to the grade of openness to β-lactamases emitted by certain microbial strains like Klebsille pnemonie for detoxification drugs [8,9]. For enhancement of the aforementioned potentials, our interest remains to design unusual compounds with basic hydrophilic moieties [7]; OH, NH, NH 2 , CO 2 H that stimulates lipophilicity of drugs via lipid cell membrane of countless microorganisms, as well as drugs having hydrophobic functions; C=N, -CH 2 -, C=C which triggers sensitivity amid resistant Gram positive microbial strains [9].
Pyrimidine based compounds are acknowledged for excellent potentials in stabilizing biomolecules [10,11] that could be used as connectors in organic synthesis and good ligating ability in the formation of metal complexes [12]. Metallic chelates encompassing pyrimidinyl function group are known to demonstrate exceptional antimicrobial actions, a consequence of their interesting topologies, assemblage liability, selectivity to countless molecular environs, capability to display infrequent geometries unusual to resistant bacteria as well as their capacity to avert prompted oxidative stress damages [13]. This work reported the solvothermal assisted-synthesis new azo-chelatorligand with its Fe(II) and Zn(II) chelates. The experimental and theoretical characterization in addition to the efficiency of the compounds as broad-spectrum in vitro antimicrobial agents were separately appraised

Materials
The reagents/solvents adopted for this research without further purification were acquired from Merck o, Sigma-Aldrich o., and ACE Co. All the experimental works of this study were carried out at the research laboratory of the Department of Chemistry, Ignatius Ajuru University of Education, Rivers State, Nigeria form February-July, 2021.

The Extraction Process
The extraction of the metallic species from the aqueous phase into organic phase (portion) using the recently synthesized chelator-ligand was examined via solvent extraction. 20 mL of aqueous solutions comprising 0.0017M of the metallic acetate salts remained equilibrated with equal volume of chloroform consisting of 0.00045M of the chelator-ligand by stirring forcefully at 27 o C on a motorized-shaker for 2hrs.This was followed by careful transfer of the solution into a separatory funnel allowed to stand for ½-hr to establish equilibrium. The aqueous portion was then separated from the organic portion with concentration of metallic ions determined in both phases via atomic adsorption spectrophotometer (AAS). The extraction efficiency, E (%) was acquired according to Eq. 1 [15].
The solvent extraction procedures could be described by Eq. 2 in which metallic ion (M n+ ) forms a chelate with the chelator-ligand (LH) to afford a neutral structural assemblage (ML x ).
M n+ (aq) + xHL(org) ⇌ ML x (org) + nH The mechanism of extraction correlates with a cation interchange where a complex of stoichiometric formula ML x is established in the organic phase producing n mol of H + in the aqueous phase. Hence, the extraction constant (K ext ) can be stated as; Since the metallic ion M n+ is predominant in the aqueous phase and ML x is the only extractable specie, the distribution ratio, D can be calculated following Eq. 4. The D is used as the most imperative index for determining the adeptness of a solvent extraction procedure.

Biological Studies
Anti-bactericidal evaluation of the Fe(II) and Zn(II) chelates alongside their chelator-ligand were obtained via an agar well diffusion technique [16] with DMSO as reaction medium. The bacteria strains, reference drugs and procedural steps reported in our previous study [17]. were adopted with the sole aim of finding the most active compound with the best antibacterial potentials.
A disc evaluative practice was adopted in-vitro to appraise the anti-fungoid actions of the chelatorligand and its metal-chelates against A. niger, A. flevus and R. Stolonifer. Potato dextrose agar (PDA) served as medium for the harvest of matured conidia of fungal isolates which were suspended in ringer solution. A 1 mL conidial suspension for a separate fungoid isolate was inoculated on a petri dish of 90 mm with 20 mL PDA. The chelates with the chelator in DMSO were dissolved (10 mg/mL, 5 mg/mL and 1 mg/mL). Bored germ-free 7mm diameter disc holes were filled (0.15 L) with individual test compound's solution and experimental data acquired in duplicate at three altered concentrations. Fluconazole as adopted as reference drug while DMSO stood as the negative control. The inhibition growth zones were evaluated after 24 h and expressed as the inhibition zone in mm.

Quantum Chemical Calculation (QCC)
The chelator for appraisal remained evaluated via DFT, adopting Becke's 3-parameter hybrid interchange function fused to Lee-Yang-Parr correlation functional (B3LYP) by means of 6-311G++(d,p) simple set. All QCCs stood completed adopting Gaussian-9-package. Optimized structural geometry as well as visualization into HOMO plus LUMO calculations remained acquired via gauss-View 5.0.8 package enabling the determination of other parameters.

With
Molinspiration cheminformatic, all pharmacokinetic factors stood acquired for prediction of bioactivities of the chelator though the property explorer appraisal afforded relevant data on the relative cytotoxicity of the chelator.

Synthesis and Preliminary Investigational Data
The synthesized chelates with their chelatorligand exhibited various shades of colour ( Table 1) (Table 1). Excellent correlations were observed amid acquired experimental and calculated values for percentage metallic ratios determined through complexometric titration method as presented in table 1. The molar conductance values (Table 1) of the chelates obtained in dimethylsulphoxide (DMSO) were between 9.7-12.0 Ohm -1 mol -1 cm 2 indicative of non-ionic status for the soluble chelates. The latter arises from literature reports which affirms that values of 45-90 Ohm -1 mol -1 cm 2 and 90-120 Ohm -1 mol -1 cm 2 are generally for 1:1 and 1:2 ionic chelates. The molar conductance data remained intensely validated by the micro (CHNS) plus quantitative analyses data in which no anions were noticed. The solubility tests of the chelator and its chelates obtained in polar and non-polar organic solvents gave varied solubilities ( Table 2). The compounds were majorly or slightly insoluble in H 2 O, CH 2 Cl 2 and CH 3 NO 2 . Moreover, the compounds had good solubilities in chloroform, dimethylsulfoxide and dimethylformamide but were sparingly soluble in ethanol, methanol and dichloromethane. The chelator and chelates had moderate-good (46-59%) yields (Table 1). The micro analysis, C, H, N, S data for the chelator and its chelates shown in table 1 have been obtained and the values remains consistent with the theoretical values corroborating 1:1:1 stiochiometries for the heteroleptic chelates and conforms with the empirical formula suggested for each compound

Extraction of Metallic Ions Using the Synthesized Chelator-Ligand
The extraction proficiencies of the chelator-ligand were evaluated by means of solvent extraction on d 6 and d  The table 3 below illustrated the number of metallic ions in the aqueous as well as organic phases after the extraction in addition to the extractability of the chelator-ligand.
From the extraction data presented in table 3, it was noticed that the chelator displayed high extractability for Fe 2+ with extraction efficiency of 79.34%, and showed a fair extractability potential for Zn 2+ with extraction efficiencies of 51.92%. The chelator-ligand exhibited moderately good extractability for the metallic ions.

NMR Studies
The 1 H-NMR spectrum of the chelator-ligand was shown Fig. 4 and its signal positions presented in table 4a. The methyl protons on the pyrimidine ring gave sharp singlet peak around 3.34 ppm while the C 5 proton resonated around 6.65 ppm. Equally, the protons (H 15

FT-IR Data
The FT-IR spectrum of the chelator-ligand presented no bands arising from amino groups as noticed within the spectrum of the precursor amine adopted for synthesis indicative of condensation via the amino group with the aldehyde [18]. Acquired spectral bands necessary for structural estimations for the synthesized compounds are presented in Table  5. The broad to medium band centered at 3440 cm -1 remained apportioned to intra-protonbonding vibrations (νO-H….N) of an enol assemblage frequently detected in chelators consisting of terminal OH functions. The band remained absent in the spectra of the chelates validating chelation via the naphthol oxygen atom of the chelator. Though, observed medium-broad bands from 3348 cm -1 -3447 cm -1 in the spectra of metallic chelates remained attributed to νOH of hydrated H 2 O molecules [11]. While the sharpmedium H-stretching bands of the aromatic rings, ѵ(Ar-H), within the chelator appeared amid 3013-3001 cm −1 , the chelates displayed medium asymmetric plus symmetric stretching vibrations for the alkyl groups within 2929-2913 cm -1 . The absorption band at 1628 cm -1 due to C=N moiety moved to lower/higher frequencies in the chelates to the range 1614-1667 cm -1 , substantiating participation of the azomethine N in chelation with the M(II) ions [12]. The new band, C=N in addition to the cyclic C=C vibrations almost appeared at equal strength in the chelator from 1628 cm -1 to 1593 cm -1 but were observed at lower/higher frequencies by ±30cm −1 in the chelates. The C=N function was apportioned to participation of the imine N in interaction with the M(II) ions, while the C=C was a consequence of aromatic conjugations plus the effect of chelation [19,20]. The status of ν(C=N) remained same (single) in the spectra of all compounds suggestive of Fermi resonance [12,21]. On the other hand, the band around 1290 cm -1 within the chelator's spectrum was accredited to v(C-O) which displayed significant shifts to greater/lesser frequencies in the spectra of the chelates owing to chelation. The sharp band in the spectrum of the chelator observed at 981 cm

The ESI-MS Studies
The ESI-mass spectrum (Fig. 6) of the chelatorligand presented dual pathways of disintegration with a base peak m/e+ at 278.12 conforming to the estimated molecular weight (278.85) for the chelator-ligand (Table 7). This substantiates the formation of form HL chelator from 2-hydroxy-1napthaldehyde plus 2-amino-4,6dimethylpyrimdine. The signals at m/z 250. 17,193.66 plus 142.92 were arising from loss of COH , NC 2 H 2 O and C 2 N 2 H components while the peak at m/z 124.81 remained undoubtedly due to OH lost separately. Additionally, the spectrum had L+1 peak at m/z 279.24, a small strength peak at m/z 280.11 which can be ascribed to additional mass units, a result of C-13 existence as well as extra average peak around 276.12 owing to lose of protons

Biological Evaluations
Acquired antibacterial studies data are accessible in Table 8 below. From the table, it is obvious that the heteroleptic Fe(II) and Zn(II) chelates were largely sensitive with varying degrees of inhibitory impacts on the growth of the screened organisms compared to the ligands ('L and B'); a consequence of chelation [13,14]. Also as observed from the data, the enhanced sensitivity of the chelates with their chelator against the gram positive stains than the gram negative microbes is explainable on the basis of differences in the cell-wall compositions of the microbes as well as the alteration in the penetrability plus permeation of the compounds through the microbial cell membranes [22]. The over-all inhibition of the microbes by the chelator with inhibitory zones of 12.0-19.0 mm could be attributable to the presence of π-electrons within the cyclic moieties as well as the C=N functions frequently acknowledged to improve antibacterial actions [29,30] heterocyclic assemblages. The Fe(II) chelate demonstrated inhibitory growth effect better than that of the chelators ('L' and 'B') against only B. cereus (19.5 mm) and S.aureus (21.0 mm) indicating its potential significance in new antibacterial drug designs. Though the Zn(II) chelate remained moderate in its actions against the tested microbes. The latter may be assigned to creation of persuasive protein poisons within the cell-surfaces of the microbes averting further penetration of the Zn(II) chelate into the inner portions of the microbial cells as well as imposing lessened lipophilicity of same chelate which automatically reduces permeation into the lipid cell membrane of the microbes [20,31]. The antifungal activities of HL chelator-ligand and its asymmetrical metallic chelates against A. niger, A. flevus and R. Stolonifer are contained in Table  9. The chelator-ligand had activity against all the tested species except R. Stolonifer which showed resistance. The Fe(II) and Zn(II) chelates were suggestively active against A. flevus and A. niger only with inhibitory zones of 15.0 -21.0 mm.

Antioxidant Studies
The chelator-ligand with its M 2+ chelates were appraised for radical scavenging actions using DPPH (1,1-diphenyl-2-picryl-hydrazyl) radical at 200, 100 plus 50 g/mL concentrations in 1mL DMSO. Acquired data on the basis of percentage inhibition are obtainable in Table 10. A good analysis of the data designates that the chelator plus its chelates largely demonstrated radical scavenging actions in DPPH assay. Inhibitory values commonly reflect degree of radical scavenging actions. The chelator-ligand substantively exhibited percentage inhibitory values smaller or comparable to that of standard suggestive of its anti-oxidant abilities. The potentials of the chelator-ligand got enhanced noticeably on chelation with M(II) ions. Largely, the metallic chelates presented superior DPPH radical scavenging actions.
Subsequently, the data of the DPPH antioxidant actions attested that the compounds could be adopted for design as well as syntheses of drugs for the management of pathological illnesses rising from oxidative stress.

Quantum Chemical Studies
The Frontier Molecular orbital allows for the prediction of reactivity of the chelator whose active site could be established by the distribution of the orbital frontiers. The HOMO is seen as a nucleophile, while LUMO. The LUMO is seen as an electrophile which accepts electrons from nucleophile. Several chemical reactivity descriptors, such as the chemical potential, global hardness and electrophilicity, have been calculated and is presented in Table 11.  The global reactivity and local descriptors remained evaluated via Koopman's theorem equations expressed as: Ionization potential (I) = -E-homo Electron affinity (A) = -E-Lumo Electronegativity (χ) = (I +A)/2 Hardness (η) = (I -A)/2 Softness (S) = 1/2 η Electrophipilicity (ω) = μ2/2η where μ which is chemical potential is -χ Low energy gap value denotes high chemical reactivity [32], while high energy gap signifies low chemical reactivity. The energy gap of the chelator was observed to be low which suggested high chemical reactivity. The electrophilicity is a measure in stabilization of energy when a compound accepts an extra electronic charge within its environ. Often perceived an improved descriptor of general chemical reactivity consisting of together the capacity of an electron-loving specie to obtain an extra electronic charge as well as the nonacceptance of a compound to undergo an electronic charge interchange in its environ. It offers basic facts on electron transfer (chemical potential) and stability (hardness). The 'χ' ascertains the attraction of electrons to an atom within a covalent bond. When two dissimilar atoms are bonded covalently, the shared electrons will be more strongly pulled towards the more electronegative atom [33]. The optimized, HOMO in addition to LUMO orbitals of our synthesized chelator are given below as Figs. 1-3 separately.

. LUMO of HL Chelator
The result as presented in Table 12 showed that the chelator's pharmokinetic parameters where within the Lipinski rule of five. This entails that the chelator will be of good oral bioavailability. A bioactivity score of more than 0 signifies a chelator's activeness. Between -0.5-0, it is moderately active and a value less than -0.5 suggests inactiveness. Table 13 shows the bioactivity scores of the chelator. Except for its enzyme inhibitor and Kinase inhibitor probability scores which fell within the moderately active zone. The chelator showed values that suggests inactivity for the remaining drug-likeness parameters. The risks of toxicity (mutagenicity, tumorigenicity, irritation, reproduction) as well as the physicochemical features (miLogP, solubility, drug likeness plus drug score) remained evaluated via a procedure established by property explorers and illustrated in Table 14.
The cLogp and the solubility were observed to be within the acceptable criteria. , which suggested good absorption and distribution [33].

CONCLUSION
The N 2 O 2 chelator-ligand with its bivalent heteroleptic Fe and Zn chelates were obtained through reflux-condensation reaction. Spectral plus analytical techniques were utilized for the characterization of the synthesized compounds. The deprotonated nitrogen of the amine moiety and carbon of the carbonyl reagent gave rise to the chelator-ligand with N 2 O 2 chromophore detected around the metallic atom of the chelates. The µ eff data plus UV-Vis spectral values of the chelates conformed to 6-coordinate octahedral geometry and assigned same. The Fe 2+ chelate displayed high spin whereas the Zn 2+ chelate assumed spin-free configuration, however both chelates were non-ionic in dimethylsulfoxide (DMSO). The antimicrobial screening of the chelates presented moderate to excellent results but with respect to standard drug used, the tested compounds were found to be moderately active. The bivalent heteroleptic chelates demonstrated good scavenging actions like the standard drug adopted. The chelatorligand was further evaluated for metallic extractive proficiency and an outstanding extractability for Fe 2+ and Zn 2+ ions with an efficiency of 79.34% and 51.92% was acquired correspondingly.

SIGNIFICANT STATEMENT
Reflux-chelative process was unraveled in this study as a synthetic method for an N 2 O 2 chelator-ligand with an enol tautomeric assemblage. Principally, the study established that the chelates demonstrated outstanding biological actions over their chelator against tested microbial organisms. However, the metallic ions' extractive potentials of the chelatorligand as observed remained prospective