Biological evaluation, synthesis and characterization of transition metal complexes of N-Methyl o and p- substituted benzohydroxamic acids

The four new transition metal complexes as Copper (II), Nickel (II), Cobalt (II) and Cadmium (II) of N-Methyl benzohydroxamate compounds have been prepared by refluxing 2:1 molar ratio of the oand p-substituted hydroxamic acid ligands along with transition metal salts in hot toluene, using a Dean Stark water separator. The elemental analysis and FT-IR Spectroscopic technique were used to characterizing the hydroxamic acid ligands and its transition metal complexes. Spectroscopic analysis exhibits that the hydroxamic acid ligand behaves as a bidentate chelator and coordinating with metal atom through carbonyl oxygen and deprotonated hydroxyl group. The antibacterial and antifungal activity of hydroxamic acid ligands and their transition metal complexes were evaluated. Transition metal complexes display stronger antibacterial activity than their ligands because of the chelating property of ligands to metal ions and as a result chelation increases the lipophilicity of the complexes. However, ligands and their transition metals complexes did not display any antifungal activity against the Aspergillus Niger (fungus). This study shows that several transition metal complexes are successfully synthesized and these complexes exhibit high antibacterial activity with less toxicity. This research encourages further synthesis of transition metal complexes and the invention of future metal based drugs.


Introduction
Hydroxamic acids having a general formula RCONHOH derived from oxoacids by substituting the hydroxyl (OH) by NHOH.
These compounds are the derivates of both hydroxylamines and carboxylic acids [1]. Hydroxamic acids play an essential role in many biochemicals, industrial, analytical pharmaceutical fields. The antioxidant and antiradical properties of hydroxamic acids have also been reported [2]. It has also been observed that the hydroxamic acids are act as effective corrosion inhibitors for copper [3]. The hydroxamic acids are utilized in the production of therapeutics targeting cancer, Alzheimer's, cardiovascular diseases, allergic diseases and malaria [4, 5]. Furthermore, hydroxamate compounds have been employed as insecticides and antimicrobials [6]. Hydroxamic acid moieties has recently been imparted for their potential use as, tumor growth, inhibitors of hypertension, asthma, inflammation and other pharmaceutical application [7, 8]. These bidentate hydroxamic acid ligands are behave as a chelated ligand toward metals atoms as copper, nickel, cadmium, iron [1]. The transition metal complexes with various ligands play a major role in the form of numerous chemical compounds [9]. These diverse transition metals as, zinc copper, nickel and iron display important role in many biological systems [10] and show strong antibacterial activity against the different pathogenic microorganisms. The strong interaction of transition metal ion along with antibiotic drugs has shown the antifungal, antioxidant, antimalarial activities [10, 11]. Transition metal complexes display variable oxidation states and interact with various negatively charged molecules. Hence, the transition metal complexes have started the production of new metal-based drugs having pharmaceutical application and offers therapeutic opportunities [12,13]. Transition metal compounds also used as antiinflammatory and antidiabetic and antiinfective agents. These complexes have many side effects but most widely used anticancer agent due to its great efficacy [12]. Some of the transition metals such as cupper, vanadium, titanium and gold metal complexes show significant anticancer activity [14]. In this analysis, four new transition metal complexes as Copper (II), Nickel (II), Cobalt (II) and Cadmium (II) of N-Methyl benzohydroxamate were synthesized and characterized by elemental analysis and FT-IR Spectroscopic technique. Antibacterial and antifungal activity of these compounds has been determined.

Materials and methods Reagents
All the chemicals were obtained from sigma Aldrich (99%) and utilized without any purification. All the chemicals and transition metal salts were of analytical grade. In this research work the chemicals which are utilized; N-Methylhydroxylamine Hydrochloride, o-Mithoxybenzoyl chloride p-Nitrobenzoyl chloride, Sodium Hydrogen carbonate (NaHCO3), Potassium hydroxide (KOH), Toluene, Methanol, Ethyl ether, Ethyl alcohol, Dimethyl sulfoxide (DMSO), cobalt chloride, nickel chloride, copper sulphate and cadmium acetate.

Synthesis of o-and p-substituted benzohydroxamic acid
The o-and p-substituted N-methyl benzohydroxamic acids, N-methyl omethoxybenzohydroxamic acid (L1) and Nmethyl p-nitrobenzohydroxamic acid (L2) have been prepared by in a similar fashion according to the method described earlier by Ulrich and Sayigh [15]. The Nmethylhydroxylamine hydrochloride (25 mmol) was added to the dissolving mixture of sodium hydrogen carbonate (50mmol) and was poured o-and p-substituted benzoyl chloride (25mmol) dropwise. The ratio between these compounds was 2:1 molar ratio respectively. Sodium hydrogen carbonate (NaHCO3) act as a catalyst. The resulting mixture was further stirred for 35 minutes. After the filtration, the solvent was evaporated. The solid mass was obtained on removing solvent at low pressure are dissolved in hot acetic ether and again filtered. On cooling the hydroxamic acids have started to precipitate and the filtrate were placed in refrigerator overnight to obtain the desire crystals. The general synthetic method is carried out shown in (Scheme 1).

Synthesis of Metal (II) N-Methyl benzohydroxamate complex
The Metal (II) N-methyl benzohydroxamate compounds have been prepared by refluxing 2:1 molar ratio of the ligand Nmethylbenzohydroxamic acid with transition metal (II) salts in hot toluene about 6-7h with refluxing and Dean-Stark apparatus was used to remove the water. After cooling and filtration, the solution was evaporated at low pressure. Solid residue was precipitated by boiling acetic ether and recrystallized in ethyl alcohol (Scheme 2).
Where X is NO 2

Scheme 1. Reaction between hydroxylamine and p-substituted benzoyl chloride
Where R is NO2 and R 1 is OCH3

Scheme 2. Reaction between the o-and p-substituted hydroxamic acids and transition metal ions
Anti-bacterial assay Agar well method was utilized to determine the antibacterial activity of the synthesized compounds against different types of bacteria including Escherichia coli, Klebsiella pneumoniae, Salmonella typhi and Staphylococcus aureus. The Mueller Hinton agar plates were made by following instructions. The wells of 6 milimeters (mm) width were made in to the petri dishes seeded with bacterial culture by the help of sterilized cork borers. Transition metal hydroxamate complexes were added in 2mL of DMSO. The 100 mL of stock solution (2mg/mL in dimethyl sulfoxide) of complexes were poured in each well. The petri dishes were then incubated overnight at 37ᵒC. Pure DMSO and Amoxicillin (30 µg/mL) were utilized as positive and negative controls. The antibacterial activity of the complexes was investigated by measuring the diameters of inhibition zone in millimeters [16,17]. Ant-fungal assay Anti-fungal assay of synthesized compound has been determined by the agar well diffusion method. Aspergillus Niger (fungi) was utilized for antifungal activity of synthesized compound and this fungus was isolated from environment. The solution of hydroxamic acids and their TMCs (0.02mg) used for antifungal activity were made in 1mL of DMSO assayed against Aspergillus niger (fungus). After this, the solution of potato dextrose agar (PDA) was prepared and autoclaved then this potato dextrose agar (PDA) solution was poured in petri dishes under. The walls (6mm) were prepared in petri dishes by sterilized borers. The antifungal activity of the compounds was noted after 3, 7, and 14 days. Moreover, the diameter of inhibition zone was determined at the end of the incubation period [13,18].

Results and discussion Synthesis of o-and p-substituted benzohydroxamic acid and their complexes
These ligands, N-methyl omethoxybenzohydroxamic acid (L1) and Nmethyl p-nitrobenzohydroxamic acid (L2) were prepared by reacting Nmethylhydroxylamine hydrochloride with acid chloride. Sodium hydrogen carbonate (NaHCO3) was utilized as catalyst. Transition metal hydroxamate complexes were obtained by the reaction between some transition metal ions such as, Copper (II), Nickel (II), Cobalt (II) and Cadmium (II) and the relevant hydroxamic acids ligands (L1 and L2) in 1:2 molar ratios in hot toluene. The calculated values of ligands and complexes are in good yield with according to the observed (Table 1 & 2).

Infra-red spectroscopy of hydroxamic acid ligands
The IR spectra of hydroxamic acid ligands are reported in solid state in the region 4000-400cm -1 . The significant stretching absorptions are given in (Table 3)

Table 3. Infrared spectral data for the ligands (L1 and L2) and their transition metal complexes
Compounds

Infra-red spectroscopy of transition metal complexes
To compare the vibrational spectra of hydroxamic acid ligands with the derived transition metal complexes. The significant characteristic is the absence of hydroxyl v(OH) peak in the IR spectra of complexes, due to the replacement of OH of free ligands by transition metal ions, indicating the existence of ligand as deprotonated form in complexes. The significant peak in the free hydroxamic acid ligands is the carbonyl groups which have stretching frequencies occur in the range of 1609-1718cm -1 . The shifting of stretching frequency of C=O group to lower value range 1584-1603cm -1 in the IR spectra of transition metal compounds, which confirm the synthesis of metal complexes [19,20]. Thus, suggested on the transition metal ion a five membered chelating ring is formed which furthermore supported the existence of hydroxamic acid as a bidentate chelating ligand in complexes [21]. The C-N bands are reported in the region 1459-1499cm -1 on complexation. More interestingly, the carbonyl oxygen acts as a donor atom with electron withdrawal from the carbonyl group enhance the electron density on C-N group. Hence, increase of the C-N frequency and decrease of the carbonyl frequency are expected as outcome in the IR spectra of complexes Amoxicillin (30µg/mL) were utilized as positive and negative controls. The inhibition zone values are reported in mm given in the (Table 4). When Inhibition zone values are less than 10mm reported as weak, from 10-16mm are moderate and above than 16mm are shown as active [20]. It was reported that all transition metal complexes show inhibition against all tested Gram-negative and Gram-positive bacterial strains at various rates. The newly synthesized transition metal complexes display effective bactericidal activity as compare with its hydroxamic acid ligands, which is due to the chelation property of hydroxamic acid ligand to the metal ions. Thus, chelation will increase the lipophilicity of the complexes and an effective penetration of complexes through the cell wall of bacteria to inhibit the growth of various strains of bacteria [11,13,18]. Furthermore, the hydroxamic acids and its transition metal complexes such as, Coppe (II), Nickel (II), Cobalt (II) and Cadmium (II) show moderate action against the four strains of bacteria.