Synthesis , spectroscopic characterization and antimicrobial activities of N-methyl-o-methoxybenzohydroxamic acid and its metal ( Fe , Co , Ni , Zn ) complexes

A reportedly synthesized ligand, N-methyl-omethoxybenzohydroxamic acid (Nmombha), and its freshly prepared transition metal (Iron, Cobalt, Nickel, and Zinc) complexes have been synthesized. For characterization of reportedly synthesized ligand and its freshly prepared coordination compounds, spectroscopic techniques like Fourier transform infrared and ultraviolet/visible spectroscopy have been used. Spectroscopic studies show that the ligand acts as a bidentate chelate which forms a ring-like structure. The coordination of synthesized ligand with transition metals is via O, O donor sites, and it forms a fivemembered chelating ring on complex formation. Antimicrobial activities such as antibacterial and antifungal activities have been done for the said ligand and its complexes. Antibacterial activity of the synthesized ligand and its metal complexes are examined against four different species of Gram-positive and Gram-negative bacterial strains including Staphylococcusaureus, Klebsieludla, Esherichia coli, and Pseudomonas. Comparing the antibacterial effectiveness, the metal complexes having greater lipophilicity character show much higher antibacterial efficiency when compared with the free ligand. Cytotoxicity evaluation of the compounds shows that some of the samples are highly cytotoxic.


Introduction
Hydroxamic acids are synthetic organic compounds or naturally occurring compounds and can produce ions very easily. They consist of two groups, oxime (-N-OH) and the carbonyl group and are generally called "N-hydroxy amides" [1,2]. Most of the biochemists are interested with hydroxamic acids due to their important biological activities. These activities are due to chelate formation with metal ions because they can form chelates, especially with transition metals. The ligands are in keto form in most of the metal-hydroxamic acid such as iron, cobalt, nickel, and zinc. These coordinate through O, O-donor sites [3]. Several synthetic procedures are reported in the literature for the ligand synthesis, [4-7] but mostly the methods are time-consuming, expensive, and tiresome. Therefore, they are generally prepared by the reaction of hydroxylamine with esters (RCOOR) or acid chlorides (RCOCl) [8][9][10]. These acids can act as anti-tumor, antituberculosis, anti-cancer, anti-malarial, anti-fungal, anti-allergic, and hypotensive agents [11,12]. For specific enzymes like matrix metalloproteinanses, histonedeacetylases, ureases [13], and peroxidases [14], these acids act as effective and selective enzyme inhibitors. Transition metals show variable oxidation numbers and can react with several electronegative elements [15]. These metals have been utilized in many drugs resulting in enhanced therapeutic and pharmacological properties, such as antiinflammation, anti-microbial [16], and anti-cancer [17]. The transition metals are the best catalysts and can act as important catalysts in photochemistry and material synthesis. They show different magnetic, chemical, and optical properties [18]. In this work, we have synthesized coordination compounds of following metals Co (II), Fe (III), Zn (II) and Ni (II) from a reportedly synthesized ligand, Nmethyl-o-methoxybenzohydroxamic acid, and the characterization of coordination compounds have been done by FT-IR and UV/VIS spectroscopy. Anti-bacterial, anti-tumor, and anti-fungal activities have been tested as well.

Materials and Methods
The materials were used without any purification because all materials were of analytical grade (Sigma-Aldrich). The purity of synthesized ligand and its TMCs were tested by TLC. KBr pellets were used to record the IR spectrum on the FTIR spectrophotometer. UV/VIS spectrum was recorded in dimethylsulfoxide (DMSO) with UV/VIS spectroscopy (Shimadzu UV/VIS, 1700).

Preparation/synthesis of N-methyl-omethoxybenzohydroxamic acid
For the preparation of N-methyl-omethoxybenzohydroxamic acid, the compound o-Methoxybenzoyl chloride (4.3 g, 25 mmol) was added dropwise in a mixture which was ice-cooled containing N-methylhydroxylamine hydrochloride (2.1 g, 25 mmol) in ethanol (100 mL) and sodium bicarbonate (4.2 g, 50 mmol) as well, and continuously stirred for 30 minutes. The filtration was done carefully, and at very minimum pressure evaporation was proceeded. The obtained mass was put in boiled EtOAc (ethyl acetate, 50 mL) and filtration was done for the removal of undesired solid mass. On cooling the precipitation of ligand starts and the whole night it was kept in the refrigerator to form more and more crystals. The pure crystals of ligand were dried over silica. Ligand preparation is shown in (Scheme 1).

Scheme 1. Synthesis of N-methyl-o-methoxybenzohydroxamic acid Synthesis of metal complexes
TMCs were synthesized by mixing an equivalent mixture of the ligand with potassium hydroxide and were stirred for 15 minutes at room temperature in methanol. A freshly prepared aqueous solution of metal salts of transition elements (Fe, Co, Ni, and Zn) was added and stirred for two hours. The ratio of ligand and its metal salts was maintained 2:1 respectively. Through filtration, the solid products were collected and washed with distilled water, methanol, petroleum ether, and finally dried. The synthetic method for complexes is given below in (Scheme 2).

Result and Discussion Synthesis
The preparation of bidentate ligand Nmethyl-o-methoxybenzohydroxamic acid was done via nucleophilic substitution of o-methoxy benzoyl chloride with Nmethylhydroxylamine hydrochloride in the presence of a weak base such as sodium bicarbonate. Coordination compounds were prepared by the reaction of reportedly synthesized ligand with ML 2 Fe (II), Co (II), Zn (II), and Ni (II) in the presence of KOH in methanol. TLC was used to assure the purity of synthesized ligand and its coordination compounds.
The physical characteristics of the reportedly synthesized ligand and its TMCs are given below in (Table 1).

Infrared spectroscopy
The range maintained for IR spectra of reportedly synthesized ligand and its coordination compounds were in between 4000-400 cm -1 in the solid-state. The value of absorption bands is given below in ( Table 2)  Colorless crystals 85% Fe(Nmombha) 2 Reddish-brown 61% Co(Nmombha) 2 Pink solid 75% Ni(Nmombha) 2 Light green 72% Zn(Nmombha) 2 Colorless 77% Table 2. Infrared data for ligand and its transition metal complexes In the IR spectra of complexes, the most important characteristic is the absence of a

Ultraviolet spectroscopy
Visible ultraviolet spectroscopy (UV-Vis or UV/Vis) or ultraviolet spectrophotometer mainly refers to absorption or emission (reflection) spectroscopy in some of the ultraviolet and nearby spectral regions. It is revealed from the above statement that this spectrophotometer uses light in the visible and nearby ranges. In the visible region absorption or reflection directly affects the apparent color of the chemicals which is involved. In this range, excitation occurs in atoms and molecules. In absorption spectroscopy, the transition or excitation is from the ground state to the excited state, while it is reverse of fluorescence spectroscopy where the transition occurs from an excited state to the ground state. Therefore, these two spectroscopic techniques are complementary to each other [23]. The Ultraviolet absorbance of synthesized ligand and its freshly prepared complexes is studied for the first time. UV-Vis spectroscopy is carried out to observe the change in the electronic transition of ligands and its metal complexes. Dimethylsulfoxide (DMSO) was used to make the solutions of ligands and their complexes. The concentration for the complexes was kept 0.0001 M and 0.001 M. The wavelength was in the range of 200-400 nm. In the electronic spectrum of synthesized ligand (Nmombha) a band with is obtained at 357.5 nm wavelength. This is ascribed to the π-π * transition of ligand electrons whereas, in the spectra of complexes, a shift of 77-101 nm wavelength is observed in spectra of complexes that confirm the formation of complexes. In the spectrum of complex Fe (II), an absorption band at 431 nm is observed which may be ascribed to the d-d transition of electrons in metal complexes.

Antifungal activity
The in vitro antifungal activity of prepared/synthesized ligand and its freshly prepared coordination compounds have been performed by Agar Tube Dilution Protocol. Synthesized compound was tested against microorganisms such as Candida glabarata, Candida albicans, Trichophyton, Aspergillus niger, Microsporum canis, and Fusarium lini. In Petri plates using nutrient agar medium test organisms were grown. The synthesized ligand and its transition metal complexes were dissolved in DMSO. The concentration of compounds was 200 g/ml. The incubation of all plates was done at 27 degrees Centigrade for 7-10 days. The antifungal assay of synthesized ligand and its complexes were measured by MIC (minimum inhibitory concentration) [24]. The in vitro antifungal bioassay of synthesized ligand and its freshly prepared transition metal complexes have shown negligible and insignificant activity. The percent inhibition of ligand and its complexes is found 0%.

Brine shrimp lethality test (Cytotoxicity evaluation)
Brine shrimp lethality assay [25-27] is an easy, simplest one, low cost, a fast, and comprehensive assay that is commonly used for natural and synthetic origin of bioactive compounds. The pure compounds, metal complexes product extracts, and fractions can be tested for their bioactivity. It has advantages of being inexpensive, simple, and rapid. Commonly, this test is done for analysis of the cytotoxic effect of bioactive chemicals. It is a basic test for heavy metals [28] plant extracts, fungal toxins, cyanobacteria toxins, and cytotoxicity of dental material [29]. Brine shrimp lethality was first developed by Michael et al, in 1956. Further work was done by others. This assay has successfully used as a bioassay guide for antitumor agents and cytotoxic activity in 1982. Cytotoxicity of already prepared/synthesized ligand and its freshly prepared metal complexes were assessed by using cytotoxic evaluation tests according to the Michael et al, method [30]. Thirty shrimps were used to each of all test tubes of hydroxamic acid and its complexes. After 24 hours, the mortality of brine shrimps was observed. Some of the samples were highly cytotoxic and some of them show cytotoxicity at higher concentrations.
The results that were obtained from brine shrimp lethality are represented below in (Table 3).  Fig. 1-4). The enhanced antimicrobial activity of ligand and TMCs are related to their chelation property that enhances the lipophilicity of complexes which results in growth inhibition of the bacterial strains [32-34]. Among all the metal complexes, the higher antibacterial activity was shown by Zn (II) complex that may be due to its higher stability.

Conclusion
The reportedly synthesized ligand and its freshly prepared transition metal complexes were carefully and successfully synthesized and characterized by FT-IR and UV/Vis spectroscopic techniques. The IR spectroscopic data indicates the complexation of ligands with the oxygen atom of the hydroxamic acid. The ligand and its complexes did not show any significant activity against fungal strains. The compounds can be used as antibacterial agents because of their higher activity and can be used for synthesizing new drugs for different illnesses because they showed positive activity against both gram-negative and gram-positive strains of bacteria.