Coordination Behavior and Biological Activity of Some Transition Metal Complexes with 2-Acetyl and 2-Formyl-3-Amino-1,4-Naphthoquinone Ligands

The aim of this work is to synthesize, characterize and evaluate the biological activity of 2-acetyl and 2-formyl-3-amino-1,4-naphthoquinone (L 1 - L 2 ) and their metal-Co(II), Ni(II) and Cu(II) chelates. The newly chelates were characterized by elemental analysis, IR, mass and 1 HNMR spectra, thermogravimetric analysis (TGA) and biological activity. The antibacterial and antifungal activities of the ligands and its metal complexes were screened against bacterial species (Staphylococcus aureus, Bacillus subtilis and Escherichia coli) and fungi (Candida albicans). Ampicillin and amphotericin were used as references for antibacterial and antifungal studies. The activity data show that the metal complexes have a promising biological activity comparable with parent free ligand against bacterial and fungal species.


Introduction
Chemical synthesis is a hot topic because of a global crisis of drug resistance, in pathogens of both clinical and agriculture importance. Many of these pathogens are resistant to multiple classes of antibiotics and is increasingly common for them to be resistant to practically all available drugs, leaving few alternatives for the treatment of infections, especially in immunocompromised patients [1] [2].
Despite addition of new classes of antimicrobials, the number of currently available drugs for infections treatment remains limited. Therefore, there is a continuing need to develop new, simpler, more effective and less toxic antimicrobials agents; so naphthoquinones and derivatives are a group of great importance that has attracted interest of the scientific community. Naphthoquinones are natural aromatic compounds that can be found in several plant families, as well as isolated of fungi, algae and bacteria. Traditionally used for their dyeing properties, however, recently a variety of biological activities of these compounds has been reported [3] [4]. In most cases, these pharmacological activities are related to redox and acid-base properties, which can be modulated synthetically by modifying the substituents attached to the 1,4-naphthoquinone ring, in order to enhance their therapeutic actions. At the present time, the synthetic methods should be designed according the principles of green chemistry to promote process more sustainable with the environmental and human safe. Because of this, in this chapter is described the chemistry and green synthesis of natural and synthetic naphthoquinones as potential antibacterial, antifungal, anti-parasitic and antiviral agents, as well as its mechanism of action. Contributing in the area of synthesis and screening of novel chemical compounds for antimicrobial action. Naphthoquinones are structurally related to naphthalene, are characterized by the presence of two carbonyl groups in the 1,4 position and 1,2 position with lower incidence, which are named as 1,4-naphthoquinones and 1,2-naphthoquinone respectively. Naturally present hydroxyl and methyl groups as substituents, can be found in free form or condensed with oligosaccharides. Naphthoquinones are highly reactive organic compounds, traditionally used as natural or synthetic dyes whose colors range from yellow to red [5] [6]. The effect of metal complexation on the antimicrobial activity of 1,4-naphthoquinones was investigated. Nickel-, chromium-, iron-, copper-, and cobalt-containing metal chelates of 5-amino-8hydroxy-1,4-naphtoquinone (2) and its acyl-derivatives (3 -8) were synthesized and characterized, and their antimicrobial activity was evaluated. Data from infrared spectroscopy indicate that naphthoquinones coordinate through oxygen and nitrogen atoms for (2), and through oxygen atoms when ligands were acyl derivatives (3 -8). Susceptibility tests for antimicrobial activity showed that 2 and its acyl derivatives were effective on inhibiting the growth of pathogenic bacteria such as Staphylococcus aureus, Streptococcus uberis and Bacillus cereus, but not Gram-negative bacteria. The metal complexation often caused decrease of biological activity. Nickel complex of (2) was the most effective against Gram-positive bacteria, showing MIC values ranging from 375 to 1400 mg/ml. Metal chelates may be useful tools for the understanding of the antimicrobial mechanism of 1,4-naphthoquinones on these bacteria [5].

Materials and Reagents
Analytical grade chemical reagents were used. All chemicals used in this study    Table 1. The procedure cited in respective reference [7] [8].

Preparation of Metal Complexes
The metal complexes were prepared by dissolving (1075 g and 1.005 g, 0.005 mol) of ligand (L 1 -L 2 ) respectively in hot ethanol (50 ml) and added drop wisely with stirring to a stoichiometric amount of 1:1 (M:L) molar ratio to (1.189 g,

Biological Activity
Modified Kirby-Bauer disc diffusion method [9], has been used to determine the antimicrobial activity of the tested samples [10]. Examined 100 μl of the tested bacteria or fungi and found it was developed in 10 ml of fresh media until they reached a count of approximately 10 8 cells/ml for bacteria and 105 cells/ml for fungi. 100 μl of microbial suspension was spread onto agar plates corresponding to the broth in which they were maintained. Isolated colonies of each organism that might be playing a pathogenic role should be selected from primary agar plates and tested for susceptibility by disc diffusion method of the many media available, NCCLS recommends Mueller-Hinton agar due to it results in good batch-to-batch reproducibility. Disc diffusion method for filamentous fungi tested by using approved standard method (M38-A) developed. For evaluating the susceptibilities of filamentous fungi to antifungal agent. Disc diffusion method for yeast developed by National Committee for Clinical Laboratory Standards using approved standard method (M44-P). Plates inoculated with filamentous fungi as Asprgillus flavus at 25˚C for 48 hours; Gram (+) bacteria as Staphylococcus aureus; Gram (−) bacteria as Escherichia coli, they were incubated at 35˚C -37˚C for 24 -28 hours and yeast as Candida albicans incubated at 30˚C for 24 -28 hours, then the diameters of the inhibition zones were measured in millimeters with slipping calipers of the National Committee for clinical Laboratory Standards [11], have been used standard discs of tetracycline (antibacterial agent), and amphotericin B (antifungal agent) served as positive controls for antimicrobial activity but filter discs impregnated with 10 μl of solvent (distilled water, chloroform, DMSO) were used as a negative control. The agar used is Mueller-Hinton agar that is rigorously tested for composition and pH. Further the depth of the agar in the plate is a factor to be considered in the disc diffusion method. This method is well documented and standard zones of inhibition have been determined for susceptible and resistant values. Blank paper discs (Schleicher and Schuell, Spain) with a diameter of 8.0 mm were impregnated with 10 μl of tested concentration of the stock solutions. When a filter paper disc impregnated with a tested chemical is placed on agar, the chemical will diffuse from the disc into the agar. This diffusion will place the chemical in the agar only around the disc. The solubility of the chemical and its molecular size will determine the size of the area of chemical infiltration around the disc. If an organism is placed on the agar, it will not grow in the area around the disc if it is susceptible to the chemical. This area of no growth around the disc is known as zone of inhibition or clear zone. For the disc diffusion, the zone diameters were measured [12], found that, agar based methods such test and disc diffusion can be good alternatives because they are simpler and faster than broth-based methods.

Elemental Analysis and Physical Properties
The results of elemental analyses and physical properties of the free ligands and its metal chelates shown in Table 1 are in good agreement with those required by proposed formulae. The isolated solid complexes are stable at room temperature, partly soluble in organic solvents (L 1 -L 2 ), but completely soluble in DMF and DMSO. Based on the above mentioned results, it can propose the general structural formulae of the complexes (M:L) ratio 1:1 is represented in Figure 1 and Figure 2.

Molar Conductivity Measurements
The metal chelates were dissolved in DMF at 25˚C ± 2˚C and the molar conductivities of 5 × 10 −4 M of their solutions were measured by recommended procedure [13]. The obtained molar conductance values are listed in Table 1. The molar conductivity value of Co(II), Ni(II) and Cu(II) chelates of free ligands (L 1 -L 2 ) are found to be 5.20 up to 7.76 Ω −1 •mol −1 •cm 2 . The chelates are nonionic in nature and they are considered as non-electrolytes.
The 1 H NMR spectrum (L 1 ) shows a singlet signal at δ 2.5 ppm of relative intensity (s, 3H) may be attributed to CH 3 protons, a multiplet at δ 7.2 -7.5 ppm of relative intensity 4H, may be assigned to four protons (m, 4H) in quinone, H arm . The multiplet signal observed at δ 8 ppm may be assigned to two protons of amino group (m, 2H, NH 2 ) [14].
On comparing the investigated 1 H-NMR signals of the cobalt chelate [CoC 12 H 9 NO 3 Cl 2 •(H 2 O) 2 ]•4H 2 O with those of the L 1 ligand protons signals, multiplets and the chemical shifts. It has been found that methyl protons of the free ligand are slightly shifted to δ 2.49 ppm. Also the NH 2 protons of the free ligands are slightly shifted to δ 7.7 ppm this suggests that the metal ion coordination takes place through the nitrogen atom of NH 2 group. The proton signal observed at δ 3.52 ppm, which may be assigned to the presence of water molecules is in agreement with the suggested formulae of metal chelates.
The 1 H NMR spectrum (L 2 ) shows a singlet signal at δ 9.6 ppm of relative intensity (s, 1H) may be attributed to CHO protons, a multiplet at δ 7.13 -7.5 ppm of relative intensity 4H, which may be assigned of four protons (m, 4 H) in quinone, H arm . investigated. It has been found that the aldehyde CHO protons of the free ligand are slightly shifted to δ 9.4 ppm. Also the NH 2 protons of the free ligands are slightly shifted to δ 7.8 ppm this suggests that the metal ion coordination takes place through the nitrogen atom of NH 2 group. The proton signal is observed at δ 3.18 ppm, which may be assigned to the presence of water molecules is in agreement with the suggested formulae of metal chelates. The 1 H-NMR spectrum of 2-formayl-3-amino-1,4-naphthoqunone nickel chelate shows several characteristic signals.  of a signal at m/z = 75.90 (mole mass = 79.54, RI = 40%) as rupture of C 5 H 5 NO (4-Hydroxypyridine) from the fragment. Pathway II shows a fragment at m/z = 371.79 (mole mass = 370.65, RI = 100%); which may be refer to the loss of CH 3 , followed by the appearance of a signal at m/z = 353.86 (mole mass = 352.64, RI = 52%); which may be refer to the loss of one molecules of water, Cl 2 and C 11 H 6 NO 2 (furo[2,3-b]quinolin-8-olate) leaving a fragment give a signal at m/z = 75.90 (mole mass = 79.54, RI = 40%).

Mass Spectra of 2-Formyl-3-Amino-1,4-Naphthoqunone (L 2 ) and
Its Copper Complex The electron impact mass spectrum of [CuC 11 H 7 NO 3 Cl 2 •(H 2 O) 2 ] shows many fragment ions which consists of two principle pathways as shows in Scheme 2.
The signal that appears at m/z = 371.29 (mole mass = 371.66) may be referred to the appearance of main general molecular weight of metal chelate which undergo two pathways of fragmentation. Pathway I stated that; the main metal chelate lose two molecules of water, hydroxyl ion and Cl 2 leaving a fragment at m/z = 247.94 (mole mass = 247.71, RI = 11%) followed by the appearance of a signal at m/z = 77.23 (mole mass = 79.54, RI = 35%); which may be refer to the loss of C 11 H 6 NO (furo[2,3-b]quinolin-8-olate). Pathway II shows a fragment at m/z = 333.94 (mole mass = 335.63, RI = 18.38%); which may be refer to the loss of two molecules of water, followed by the appearance of a signal at m/z = 263.73 (mole mass = 264.72, RI = 15.34%); which may be refer to the loss of Cl 2 , followed by the appearance of a signal at m/z = 77.23 (mole mass = 69.54, RI = 35%) as rupture of C 11 H 7 NO (1,4-dioxo-2,3-dihydrona-phthalene-2-carbonitrile) from the fragment.

Thermogravimetric Analyses (TGA)
The TGA thermal analyses data of the synthesized metal chelates are tabulated in Table 4. Open Journal of Inorganic Non-metallic Materials   Table 4.  (Table 4). The final residue is quantitatively proved to be copper(II) oxide. The total experimental mass loss 74.37% agrees well with the calculated mass loss of 78.60%.

Biological Activity
The comparison of biological activity of the free ligands (L 1 up to L 2 ) and its complexes with the standard disc of Ampicillin (antibacterial G + agent and antibacterial G − agent), Amphotericin B (antifungal agent), towards the different organisms was carried out. The data are listed in Table 5 and shown in Figure 4 and Figure 5. The free ligands and its metal chelates were screened against Staphylococcus aureus and Bacillis subtilis (G+) and Escherichia coli (G-) bacteria and candida albicans (fungi) to assess their potential antimicrobial agents. Open Journal of Inorganic Non-metallic Materials

Biological Activity of 2-Acetyl-3-Amino-1,4-Naphthoqunone (L 1 )
The biological activity of Ligand (2-Acetyl-3-Amino-1,4-Naphthoquinone) L 1 and its metal complexes Figure 4 shows higher results than that of the free ligand. But all of them are lower than standard. Therefore, the biological activity of the complexes follow the order Co(II) > Cu(II) > Ni(II) against Staphylococcus aureus, Bacillus subtilis and Escherichia coli organisms. But with Candida albicans the biological activity follows the order Co(II) > Ni(II) > Cu(II). Open Journal of Inorganic Non-metallic Materials
But all of them are lower than standard. Therefore, the biological activity of the complexes follow the order Co(II) > Cu(II) > Ni(II) against Bacillus subtilis and Escherichia coli organisms for (L 2 ) and complexes Meanwhile, the biological activity of the complexes follow the order Cu(II) > Co(II) > Ni(II) against Staphylococcus aureus. But with candida albicans the biological activity follows the order Ni(II) > Co(II) > Cu(II).
The importance of this, lies in the fact that these complexes could be applied fairly in the treatment of some common diseases caused by E. coli e.g. Septicemia, Gastroenteritis, Urinary tract infections and hospital acquired infections according to [16]. However, the complexes were specialized in inhibiting Gram-positive and Gram-negative bacterial strains. The importance of this unique property of the investigated complexes lies in the fact that, it could be applied safely in the treatment of infections caused by any of these particular strains. Generally, the activity of the free ligand was increased upon complexation with metal ions; the enhancement in activity can be explained on the basis of chelation theory, reported by [17] [18]. Chelation reduces the polarity of the metal ion considerably, mainly because of the partial sharing of its positive charge with donor groups and the possible p electron delocalization over the whole chelate ring. Chelation not only reduces the polarity of metal ion, but also increases the lipophilic character of the chelate. As a result of this, the interaction between the metal ion and the cell walls is favored, resulting in interference with normal cell processes.

Conclusion
In the present study, the free ligands (L1, L2) and its metal complexes Co(II), Ni(II) and Cu(II) were prepared and structurally identified. The structures of free ligands and its metal chelates are proved by elemental analyses and applying spectroscopic measurements (FT-IR, H-NMR, and mass spectra) and confirmed by thermal analyses. The synthesized free ligands are found to be biologically active and their metal complexes showed significantly enhanced antibacterial and antifungal activities against microbial strains in comparison to the free ligand.

Conflicts of Interest
The authors declare that they have no conflict of interest.