Synthesis, Characterization of New Metal Complexes of Ligand [3-(4-hydroxyphenyl)-2-(3-(4-methoxybenzoyl) thio ureido)propanoicacid] (HMP ) and Study of the Biological Activity

A new ligand [3-(4-hydroxyphenyl)-2-(3-(4-methoxybenzoyl)thioureido)propanoic acid] (HMP) was synthesized by reaction of Tyrosine and, 4-methoxy benzoylisothiocyanate , and used for the synthesis of a series of new Metal complexes Cobalt(II) Nickel (II), Copper (II), Zinc(II) , Palladium(II), Cadmium (II), mercury (II). The mineral complexes were characterized by molar conductivity, elemental analyzes (C H N), and spectroscopic techniques Such as : electronic spectral UV-Vis , Infrared FT-IR , 1 H-NMR, 13 C-NMR and magnetic properties . The results confirmed that the ligand (HMP) behaves as a Bidentate and coordinates with the metal ion via the oxygen atom of the carboxyl group and a nitrogen atom of the amine group. The antimicrobial properties of all newly synthesized compounds were also demonstrated against the bacterial pathogenic organisms Escherichia coli (G) and Staphylococcus aureus (G+) by the agar-well diffusion technique. These complexes are more effective against bacteria compared to the Amoxicillin antibiotic. The aim of this study describes the synthesis and characterization of the new ligand (HMP) derived from Tyrosine and its metal complexes with knowing their effect on selected bacteria


Introduction
The major role in structure blocks of proteins and intermediates in metabolism is played by amino acids.One of the 20 non-essential amino acids that are used by cells to synthesized proteins is tyrosine (1) , Chelating ligands based on N, O and S donor atoms show the immense extent organic performance (2) .The body produces it naturally from another amino acid called phenylalanine, which is found in meat, dairy products, eggs, oats, wheat and beans, and is used continuously within the body (3) .It is worth noting that the body's inability to manufacture tyrosine from phenylalanine is a condition called phenylketonuria .The brain uses it to make neurotransmitters such as dopamine and adrenaline (4) .Some compounds of metal ions with amino acid derivatives are very important in the biological framework and in the study of the effects of pharmaceutical drugs used as therapeutics or to increase functional similarity and reduce the effect of some toxic metals (5) .In previous studies, new ligand compounds (HNP) were synthesized from tyrosine with (4-Nitrobenzoyl isothiocyanate) and metal ions (manganese, cobalt, nickel, zinc, cadmium and mercury) as a result of electronic spectra studies (6) , and also has been prepared ligand derived of Vanillaldehyde and (L-tyrosine and L-phenylalanine) and its complexes with iron(III) were and determined using various physicochemical such as FT-IR and UV-Vis (7) .The aim of this work to prepare new ligand [3-(4-

Material and reagent :
The chemicals used in this work were all reagent grade by from known sources BDH, Fluka and Sigma-Aldrich Instruments: Nuclear magnetic resonance was recorded using Bruker 500 MHz USA at Tahran university / Central Laboratory of the College of Science and using DMSO-d 6 AndTMS as aggregates to determine the zero point.Electronic spectra were obtained using ( Spectraphotometer Shimadzu UV-Vis -1700 ) at 25 ºC in (1x10 -3 ) M DMSO.FT-IR Spectra were recorded with a Shimadzu 8000Sspectrophotometer using KBr discs in the (400-4000) cm -1 range.Elemental analysis (C H N) have been determined for prepared ligand, also a few of their complexes at Tehran University / Iran utilizing Elementar Analysensysteme GMBH،device.Metal contents of the complexes were determined by atomic absorption technique by using Shimadzu(A620).Magnetic susceptibility measurements were obtained by balance magnetic susceptibility by model MSB .

Synthesis of ligand (HMP)
The ligand was prepared by two steps : 1-Preparation of the(4-methoxy benzoyl isothiocyanate) Mixture of 4-methoxy benzoyl chloride (4.446g ، 26mmol) and Ammonium thiocyanate (1.979g, 26mmol) in 25mL of acetone was stirred under refluxed for 3 hrs , and then filtered, the filtrate was used for further reaction.(0.177 g ,1mmol) in10mL ethanol , were added drop wise to the solution of the ligand (HMP -K + ) .The precipitate formed immediately after stirring the mixture at room temperature for 3hr.The resulting precipitates were filtered off, washed with ethanol, and water recrystallized from ethanol and dried.Physical properties were given in Table (1).

Antibacterial assay (in vitro) :
The biological activity of the ligand (HMP) and their complexes has been tested against two types of Gram-negative bacteria (E-coli) and Gram-positive bacteria (Staphylococcus aureus) were studied Each of these bacteria is one of the most common types of pathogenic bacteria.biological effectiveness solutions were prepared at a concentration of 10 -3 mg/L each of the metal complexes and its ligand dissolved in dimethyl sulfoxide (DMSO).The medium was prepared according to the instructions provided by Muller-Hinton Agar Company by dissolving (37 g) of agar in 1liter of distilled water .Bacteria is spread in plates and on the surface of the food medium (Muller-Hinton agar) using (Loop full), likewise formation several holes with a diameter of 6 mm in these plates using a sterile alcohol drill.The prepared solutions were added to these bores in an amount of 0.1mL using (Micropipette) and placed in the incubator for 24 hours at a temperature of 37°C, after which the diameter of the inhibition zone was measured in millimeters (8) .In addition, the antibiotic Amoxicillin was tested for comparison with prepared compounds .During the experiments, bacteria and Amoxicillin were placed in nutrient agar medium respectively.Different test microorganisms were inoculated into agar medium .The Gram method was also used to diagnose bacteria.Microbial growth was determined by measuring the area of the inhibition diameter with the help of a scale .The diameter of the inhibitory region was measured after 24h in solvent (DMSO) for bacteria and Amoxicillin respectively.

Characterization of ligand (HMP) and its metal complexes
The prepared compounds were stable in air and moisture at room temperature.It was insoluble in water, diethyl ether and carbon tetrachloride , but soluble in dimethylformamide (DMF), dimethyl sulfide (DMSO) , absolute ethanol (ETOH) also acetone.

Elemental analysis data for the Ligand and Complexes
Synthesized ligand (HMP) and its metal complexes are shown in Table (1).They are consistent with the suggested stoichiometries.The melting points and color of the synthesized ligand and its metal complexes are as well as shown in Table (1).

Molar conductivities of all synthesized
The molar conductivity of the metal compounds was studied in a solution of dimethyl sulfoxide (10 -3 M) and at ambient temperature using Cond 3110 SET1 model, and the test showed that all the complexes are non-electrolytes

Biological activity study
The biological activity of the ligand and their complexes has been tested against two types of bacteria by using the inhibition Zone method .
Escherichia Coli: when we comparing the results with the percentage of Amoxicillin we find the Ligand (HMP) ,Cd ,Co and Hg complexes Showed a greater inhibition than Amoxicillin while the Ni , Cu ,Zn and Pd Complex showed the lower inhibition than Amoxicillin antibiotic.
Staphylococcuse aurous: when we comparing the results from the results in table (4) and Figures (5,6) ,with the percentage of Amoxicillin antibiotic inhibition we find the Hg complex showed a greater inhibition than Amoxicillin , As for the ligand and the Cd complex they showed an inhibition rate similar to Amoxicillin, while the Ni, Co ,Cu , Zn and Pd complexes showed smaller than Amoxicillin.Electronic and magnetic measurements suggested tetrahedral Structure for all complexes except Cu (II) and Pd (II) complex has a square planar geometry .The biological efficiency of the ligand(HMP) and its complexes were performed and compare it the antibiotic Amoxicillin and the experimentation showed that the ligand and all the complexes have good activity against bacteria.

Fig ( 1 )
Fig (1): FTIR spectra of ligand(HMP) and Co complex ) ppm belonging to (1H,NH amine ) , And the appearance of a single signal at δ (2.50) ppm which is attributed to the solvent( d 6 -DMSO ) , And the emergence of multiple signals at δ (2.73-3.39)ppm due to (2H,CH 2 and (1H,CH) , And the appearance of a single signal at δ (3.93) ppm belonging to (3H, OCH 3 ) as well as the emergence of a single signal at δ (9.71) ppm belonging to (1H, OH phenolic).It was also observed that multiple signals appeared at δ (6.69-8.41)ppm, which belong to aromatic protons, and a single signal appeared at δ ((7.79) ppm , due to (1H,NH sec.CO ) and a mono signal at δ (11.68) ppm which is attributed to (1H,COOH)