Metal Complexes of Phenyl Glycine-o-Carboxylic Acid: Preparation, Characterization, Electrochemical and Biological Properties

Metal complexes are the effective therapeutic compound and it became more emerging field in the drug discovery and delivery. A novel ligand phenyl-glycine-o-carboxylic acid was synthesized and further complexed with the metal(II) chlorides. The synthesized metal complexes was interpreted by FT-IR spectroscopy, Ultra Violet-visible, 1 H-NMR, molar conductance, magnetic susceptibility and thermogravimetric study. The electrochemical properties of the ligand and its complexes were inquired in DMF. Antibacterial and fungal activities of the phenyl-glycine-o-carboxylic acid (ligand) and metal complexes were analyzed by three fungal and four bacteria pathogens. The ligand has no activity against Aspergillus terreus , but nickel, copper and cobalt chloride complexes showed good activity against Aspergillus terreus . On anti-bacterial activity compare to ligands and other metal(II) complexes the cobalt (II) complex revealed greater inhibition effect on selected bacteria.


INTROdUCTION
The study of coordination complexes reported the more than cennturies till now it plays significant role in many fields especially in the pharmacy and pharmacology fields. It related to the combinations of organic and inorganic chemistry concerning the synthesis, characterization and their applications. It mostly concern their structure, complexes were considered those compounds which do not obey the classical theory of valence.
The biological importance rich metal ions reported in divalent metals ions were cobalt, zinc, copper, manganese, and nickel owing the medicinal value all the five metals were selected for the this research. Naturally, the manganese obtained in daily life is only in trace amounts at different all forms. The human can 2 to 5 mg of vitamin B 12 and its derivatives, predominantly in the liver. Nickel gives the extraordinary binding potential for bone and skin which has played urged role in the skin pigmentations. Copper is extensively distributed in all human resources. Zinc is a very predominant micro-nutrient. A number of human diseases are compatriotic with lowered blood zinc.
The metal and carboxylate ligand coordination behaviors has been explored by several researches. The neutral ligand was decided for the present investigation. Newly formulated neutral ligand phenyl glycine-o-carboxylic acid is derived from anthranilic acid. A literature survey reported that the characterization of complexes of anthranilate ligands manifest different modes of coordination in which carboxylate anion and nitrogen of amine group 1-4 and both the oxygen of carboxylic acid 5,6 The crystal structure of Lithium, Sodium, Thallium complexes of anthranoylanthranilic acid have meshed through the oxygen of carboxylate group with trihydrate water molecule was described 7 . An azomethine nitrogen and carboxylate oxygen of mannich base 8 and Schiff base 9-11 , obtained from anthranilic acid are documented. In this paper, we have given a detailed outline of the preparation, and interpretation of the synthesized metal complexes, furthermore, the electrochemical and biological behavior were studied. In addition to that, the antifungal and antibacterial efficacies of the ligand as well as synthesized five metal complexes were studied in comparative aspects.

Materials
All the metal(II) chlorides like cobalt, nickel, manganese, zinc, and copper are hydrated forms which was used in the complexation process and are Merck and Sigma Aldrich grades. Ethanol, diethyl ether, anthranilic acid, chloroacetic acid, anhydrous sodium carbonate, and animal charcoal are acquired from Sigma Aldrich products.

Preparation of ligand
15 g of anthranilic acid, 12 g of chloroacetic acid, 22 g of anhydrous sodium carbonate, and 200 mL of water are taken in one liter round bottom flask. Then reflux condenser is fitted and boil the mixture under reflux for about 3-4 hours. Then, cooled and acidified with the concentrated hydrochloric acid when a light brown precipitate of a ligand is obtained. After that, the mixture is allowed it to complexation overnight. Then it reaction mixture is filtered using the Buchner funnel and then wash with water. The collected solid compound is dissolved in hot water. To that the solution of a hot dilute acid is added followed by 1 g decolorizing animal charcoal is mixed then boil and again filtered. Finally, the crystals of the pure product are obtained.

Preparation of the metal complexes
Appropriate amount of each metal chlorides and the ligand phenyl-glycine-o-carboxylic acid are dissolved by the ethanol solvent individually and then proper ratio of both are mixed well in the 500 mL round bottom flask. After that, the whole contents in the RB flask highly refluxed with the aid of reflux condenser around six hours. Final mixture of solutions were again concentrated under water bath then, cooled followed by recrystallized the all five metal complexes. Thus, the complexes were prepared, and the excess ligand is separated by washing with ether and dried under vacuum and make into anhydrous with the help of calcium chloride environment.

Spectral methods
The FT-IR of all five metal complexes were measured with pellets of KBr using Shimadzu 24 FT-IR 8400S spectrometer between the ranges of 4000-400 cm -1 . Then, the electronic spectra of five metal complexes and their ligands were read by using Hitachi U-3400 spectrophotometer in the range of 1100-320 nm using cuvettes. The 1 HNMR was measured by BRUKER 400MHz spectrometer using TMS as an internal standard, CDCl 3 , and DMSO-D 6 as the solvent. The magnetic susceptibility measurements of the complexes were calculated using Gouy's balance at room temperature, and mercury tetrathiocynato cobaltate(II) was used as a calibrant. The molar conductance was recorded on Toshniwall's conductivity meter in acetonitrile. All melting points in degrees Celsius were measured with a capillary device by using the Stuart melting point apparatus. The antimicrobial activity of ligand and the metal complexes cobalt, copper, and nickel have been examined inimical to three fungal and four bacteria by using the Agar diffusion method.
The 100 µL test solution in DMSO was added to the nutrient Agar plates and potato dextrose agar plates. The plates were incubated at 37°C for 24 h for bacteria and 28 o C ± 2 o C for fungi. After 24 h the diameter (mm) of the incubation zone was recorded.

Outcomes & Validation Electrical conductance
The electrical conductance is weighed and the values of the molar conductance are tabulated in Table 1. The conductance of cobalt(II) complex is 1:1 electrolytic in nature 12 . From the all the spectral analysis and conductance results, cobalt(II) chloride complex is assigned as [CoL 3 ] 2+ [CoCl 4 ] 2 .

Magnetic susceptibility
The magnetic moment of the Manganese(II) chloride complex exhibits 5.93 B.M suggests that it exists in an octahedral geometry. The cobalt(II) chloride complex which is assigned to [CoL 3 ] 2+ [CoCl 4 ] 2with an octahedral geometry has a µ eff of 4.46 B.M which is supported by conductance data and the remaining values of five metal complexes are given in Table 1.

Melting behaviour
On heating, the complexes decompose at a temperature above 200 o C. It is revealed that all the complexes are stable. Melting behavior is tabulated in Table 1.

Infra-Red spectra of complexes
The infra-Red spectrum of ligand has a band at 3373 cm -1 is attributed to the OH group of carboxylic acids. The NH stretching vibrations is merged with the previous band. The band (1726 cm -1 ) C=O of aliphatic acid. The band (1705 cm -1 ) C=O of aromatic carboxylic acid. The band (1398 cm -1 ) C-N stretching 13 . The strong band appears (1662 cm -1 ) NH bending vibrations. The infra-red spectra of ligand are compared with the complexes confirming the complex formation. The shifting of bands from 3373 cm -1 to 3341 cm -1 , decreases the C-N stretching frequency to 1373 cm -1 . The asymmetric and symmetric stretch of the COOis not present proposes that the COOH is present in the complex and the shift of C=O stretch (1673 cm -1 ) proposes that oxygen of C=O is bonded with the M-O bond. The sharp band (1726 cm -1 ) is shifted to a higher frequency proposing that the aliphatic acid group is not combined with the metal ion. The bands at 3783 cm -1 indicate that both the hydroxyl group are present in the carboxylic acid. The new bands (605-575 cm -1 ) Metal-Oxygen stretch and the band (557-527 cm -1 ) are assigned to the M-N stretch of the complexes 14-16 .

Electronic spectra of five metal complexes
From the electronic spectral data t h e f o l l o w i n g a s s i g n m e n t , s t r u c t u r e a n d coordination of nickel, copper, and cobalt complexes were proposed which was listed in the Table 3 17 .

Fig. 5. Electronic Spectra of (i) Cobalt(II) chloride, (ii) Nickel(II) chloride, (iii) Copper(II) Chloride complexes 1 H NMR spectral studies
The spectrum of the complex is taken in DMSO-D 6 . The spectrum of ligand reveals different peaks at 6.48-7.82ppm corresponding to aromatic proton 13 and 3.94ppm corresponding to aliphatic CH 2 group 1.96ppm due to NH group. The peak at 8.15ppm assigns (2H) due to two carboxylic acid protons. In Zn(II) chloride complex shows different peaks at 6.53-7.84ppm corresponding to an aromatic proton, 3.98ppm corresponding to the aliphatic CH 2 group, and 2.50ppm assigned to the NH group of secondary amine. The peak at 8.25ppm responsible for -COOH (proton of carboxylic acid) attached to the aliphatic CH 2 group 12.6ppm responsible for -COOH (proton of carboxylic acid) bonded to an aromatic ring. The movement in the position of that proton in the spectrum of the complex suggests that the electron density around the proton is changed because of coordination bond of -C=O (carbonyl oxygen) in the acid functionality.

Thermal analysis
In Manganese(II) chloride complex, the curve depicts that the range between 100-120°C was due to the elimination of two lattice water molecules and loss of chloride around 230°C 16.1% (Cal 16.6%) assisted by an endothermic peak of about 136.1°C and 195.9°C in the DTA curve revealed that the complex melts before decomposition. Further, it decomposes from 300-570°C with a weight loss of 67.9% (calc 70%) corresponding to the decomposition of the ligand combined with an exothermic peak of 446°C on the DTA curve was observed. In Cobalt(II) chloride complex, it is stable up to 150°C 19,20 . Above this temperature decomposes the cobalt(II) anion accompanied by an endothermic peak of 151.8°C on DTA showing melting of the complex. In the temperature range between 270-350°C, the anionic complex gets slowly decomposed, furthermore, the second stage of degradation occurs between the range of temperature from 350-580°C with 44.4% (cal 44.6%) mass loss and an exothermic peak with 441.7°C and 556.7°C on DTA are thereby, response to the ligand decomposition. The weight of cobalt oxide is estimated as 19% (cal 17.6%) are stable. The curve of the Nickel(II) chloride complex depicts an initial weight loss of 14.2% (calc 14.8%) at 50-200°C leads to the separation of one lattice water and non-coordinated ligand, an endothermic peak with 76°C on DTA depicts that there is an occurrence of melting of the complex. The second stage from 250-550°C with the gradual decrease of mass loss of 73.8% (calc 79.7%) corresponds to the removal of two chlorine atoms and the remaining part and an exothermic peak with 384.5°C and decomposition occurs at 512.9°C. The weight of the final residue is about 12% (calc 10.5%) corresponding to stable nickel oxide.

Electrochemical behaviour
The electron transfer property of ligand and all the complexes were studied in DMF solution except cobalt(II) chloride complex. It was studied in methanol. All were recorded in 0.001M electrolytes in the range of -2 to +2 volt potential in the 50 mV/s scan rate except manganese(II) complexes. The manganese(II) chloride complex was scanned at about 20 mV/s. The free ligand exhibits quasi reversible one-electron reduction at E 1/2 value is -0.907V (DE p = 211 mV) and two irreversible oxidation potentials at E pa 0.566V and 1.221V. Manganese(II) chloride complex reveals the quasi reversible reduction 21 with respect to Mn(II) Mn(I) at E 1/2 value of -0.765V (DE p = 189mV). Cobalt(II) chloride complex reveals an irreversible reduction and two irreversible oxidation. The first irreversible reduction is allotted to metal-based Co(II) Co(I), two oxidative responses at (E pa )=0.738V and 1.536V were assigned to 2Co(II) 2Co(III) oxidation. Nickel(II) chloride complex exhibits irreversible reduction related to Ni(II) Ni(I), quasi reversible one-electron oxidation corresponding to Ni(II) Ni(III) and it exhibits an irreversible oxidative response at (E pa ) = 1.349V assigned to Ni(III) Ni(IV) oxidation. Copper(II) chloride complex displays quasi reversible oneelectron reduction 22 with respect to Cu(II) Cu(I) at E 1/2 value of -0.842V (DE p = 200 mV) and it exhibits an irreversible oxidative reduction at Epc = 0.0108V.The Zinc(II) chloride exhibits quasi reversible one-electron reduction concerning Zn(II) Zn(I) at an E 1/2 value of -0.781V (DEp = 161 mV). From the results, it is conclude that, the metal complexex zinc, copper and manganes are quasi reversible reduction, the reduction waves with E 1/2 in the range of 0.765V to 0.842V is assigned to M(II) M(I) reduction, Ni(II) complex exhibits quasi reversible oxidation and Cobalt (II) complex exhibits irreversible reduction and oxidation. The potentials are summarized in Table 5.

BIOLOGICAL ACTIVITY Antifungal activity
The antifungal activities of three metal complexes and their ligand has been examined was inimical to 3 fungal pathogens-Aspergillus niger, Aspergillus flavus, and Aspergillus terreus by the Agar well diffusion method, and the outcomes are validated and tabulated in Table 6 Antifungal activity of ligand compared with their metal(II) chloride complexes. The ligand has no activity against Aspergillus terreus, but copper, nickel and cobalt complexes showed good activity against Aspergillus terreus. Cobalt(II) complex reveals a better activity inimical to Aspergillus niger and Aspergillus flavus.

Antibacterial activity
Phenyl glycine-o-carboxylic acid and its complexes have been examined by the growth of bacteria such as Klebsiella pneumonia, E s c h e r i c h i a c o l i , B a c i l l u s a n t h r a x , a n d Staphylococcus aureus. The ligand has no activity against Klebsiella pneumonia. The ligand phenyl-glycine-o-carboxylic acid and their selected metal complexes depict a moderate activity inimical to B. anthrax. The metal complexes show fair activity against E. coli. The copper complex shows less activity against S. aureus. From the above outcomes, it is concluded that the phenyl-glycine-o-carboxylic acid and its metal complexes manifest a good activity inimical to the growth of various microorganisms.

CONCLUSION
The Phenyl-glycine-o-carboxylic acid and its complexes has been concocted and signalized that the ligand is employed as a bidentate, where the carbonyl oxygen and nitrogen of the secondary amine are bound to the metal ion. The electrical conductance analysis illustrates, that the cobalt complex is in a 1:1 electrolyte. 1 H NMR spectral study elucidates that; the hydroxyl group of the carboxylic acid is not ionized. Remarkably, CV studies manifest that manganese(II), Copper(II) and Zinc(II) complexes flourish in a quasi reversible one-electron transfer reduction process whereas, the Cobalt(II) and Nickel(II) complexes flaunt an irreversible reduction and oxidation process. The ligand and the complexes make evident that it has a good antimicrobial activity inimical to the fungal and bacterial strains.