Synthesis, Characterization, Thermal Properties, and Antimicrobial Activities of 5-(Diethylamino)-2-(5-nitro-1H-benzimidazol-2-yl)phenol and Its Transition Metal Complexes

Synthesis and antimicrobial activities of new metal [Co(II), Cu(II), Ni(II), and Fe(II)] complexes from 5-(diethylamino)-2-(5-nitro-1H-benzimidazol-2-yl)phenol are described. The newly synthesized ligands were characterized by IR, 1H NMR, and LC-MS analysis, and metal-ligand complex formations were confirmed by using atomic absorption spectroscopy and elemental analysis. All complexes show significant in vitro antibacterial activities against E. coli and S. aureus strains and in vitro antifungal activity against C. albicans and A. niger strains by using serial dilution method. The antibacterial activities were expressed as the minimum inhibitory concentration (MIC) in μg/mL. Thermal properties and electrochemical behavior of novel transition metal complexes have been studied.

Several derivatives are reported in literature for the synthesis of benzimidazole-metal complexes for fluorescent probes and bioorganic application, but antimicrobial activities of these classes of compounds have received little attention. In the literature, there are no reports 2 ISRN Organic Chemistry available describing synthesis and antimicrobial activities of metal complexes of 5-(diethylamino)-2-(5-nitro-1H-benzimidazol-2-yl)phenol. In this paper, we have synthesized novel ligand 5-(diethylamino)-2-(5-nitro-1H-benzimidazol-2-yl)phenol and its metal complexes and studied their antimicrobial activities.

Biological Activity.
All compounds were evaluated for in vitro antibacterial activities against E. coil and S. aureus strains and in vitro antifungal activity tested against C. albicans and A. niger strains by using serial dilution method.

Medium.
Isosensitest medium was used throughout the assay, as it is pH buffered. Although NCCLS recommends the use of Mueller Hinton medium for susceptibility testing [28], the isosensitest medium had comparable results for most of the tested bacterial strains [29].

Preparation of the Plates.
Plates were prepared under aseptic conditions. A sterile 96 well plate was labelled. A volume of 100 μL of test material in 10% (v/v) DMSO (usually a stock concentration of 4 mg/mL) was pipetted into the first row of the plate. To all other wells, 50 μL of nutrient broth was added. Serial dilutions were performed using a multichannel pipette. Tips were discarded after use such that each well had 50 μL of the test material in serially descending concentrations. To each well, 10 μL of resazurin indicator solution was added. Using a pipette, 30 μL of 3.3x strength isosensitised broth added to each well to ensure that the final volume was single strength of the nutrient broth. Finally, 10 μL of bacterial suspension (5 × 10 6 cfu/mL) was added to each well to achieve a concentration of 5 × 10 5 cfu/mL. Each plate was wrapped loosely with cling film to ensure that bacteria did not become dehydrated. Each plate had a set of controls: a column with a broad-spectrum antibiotic as positive control, a column with all solutions with the exception of the test compound, and a column with all solutions with the exception of the bacterial solution, adding 10 μL of nutrient broth instead. The plates were prepared in triplicate and placed in an incubator set at 37 • C for 18-24 h. The colour change was then assessed visually. Any colour changes from purple to pink or colourless were recorded as positive. The lowest concentration at which colour change occurred was taken as the MIC value. The average of three values was calculated and that was the MIC for the test material and bacterial or fungal strain [30].

Antimicrobial Activity.
The new ligand and their metal complexes were evaluated for their in vitro antibacterial activity against E. coli and S. aureus strains and in vitro antifungal activity against C. albicans and A. niger strains by using serial dilution method. The minimum inhibitory concentration (MIC) measurement determined for compounds showed significant growth inhibition zones using serial dilution method. The MIC (μg/mL) values are recorded in Figure 1. The results mentioned in Figure 1 indicate that most of the tested compounds displayed variable inhibitory effects on growth of tested against bacterial strain and antifungal strain.
The metal complex 4d showed excellent antibacterial activity against E. coli and S. aureus strains but metalcomplexes 4a-4c showed moderate activity against tested antibacterial strains. The inhibitory growth of metal-complexes 4a-4d are almost double than novel synthesized ligand 5-(diethylamino)-2-(5-nitro-1H-benzimidazol-2-yl)phenol (3), and the compound 4b is less active against S. aureus antibacterial strain. Regarding the structure-activity relationship of the novel compound 5-(diethylamino)-2-(5nitro-1H-benzimidazol-2-yl)phenol complex with cobalt(II) metal showed better activity than Fe, Ni, and Cu metals. The results mentioned in Figure 1 showed that ligand as well as metal complexes show good inhibitory growth in case C. albicans as well as A. niger strains. All metal complexes 4a-4d showed better activity than ligand (3). These results indicate that after coordination of biologically important transition, metal with 5-(diethylamino)-2-(5nitro-1H-benzimidazol-2-yl)phenol has substantial effect on the antimicrobial activity against tested microorganism. In general, most of the tested compounds revealed better activity against the antibacterial strain (E. coli, S. aureus) and antifungal strain (C. albicans, A. niger). It was also noticed that ligand (3) and metal complexes 4a-4d showed stronger antibacterial activity than antifungal one.

Thermal Stability.
In order to examine the thermal stability of these complexes, thermal gravimetric (TG) and differential scanning colorimeter (DSC) analysis were carried out between 40 and 600 • C under a nitrogen atmosphere. The TG curves of the complexes are shown in Figure 2

Conclusion
In conclusion, we have synthesized new ligand 5-(diethylamino)-2-(5-nitro-1H-benzimidazol-2-yl)phenol and their metal complexes. These novel compounds were evaluated for in vitro antibacterial activity against E. coli and S. aureus strains as well as for antifungal activity against C. albicans and A. niger strains using serial dilution technique.

Experimental Section
4.1. General. All commercial reagents and solvents were procured from s.d. fine chemicals (India) and were used without purification. The reaction was monitored by TLC using on 0.25 mm E-Merck silica gel 60 F 254 precoated plates, which were visualized with UV light. Melting points were measured on standard melting point apparatus from Sunder industrial product Mumbai and are uncorrected. The FT-IR spectra were recorded on Perkins-Elmer 257 spectrometer using KBr discs. 1 H-NMR spectra were recorded on VXR 300-MHz instrument using TMS as an internal standard.  (2 mL). The reaction mixture was stirred for 24 h at room temperature. The product, thus, separated was filtered, washed with water followed by methanol, and dried to give 4a-4d. Metal-ligand complexation was confirmed by using atomic absorption spectroscopy. Atomic absorption spectra were recorded using atomic absorption spectrometer model GBC 932 (GBC Scientific Equipment, Australia). Exactly weighed dye samples were dissolved in 20 mL of dimethyl sulphoxide and diluted to 100 mL with distilled water and analyzed by GBC 932 plus atomic absorption spectrometer (AAS). Acetylene was used as fuel, and air was used as carrier gas. Certified 1000 mg/L standard solution of iron (Merck, Mumbai) was used to perform calibration using hallow cathode lamp for iron at 248.3 nm wavelength. The samples were prepared in such a manner that they will result in 2 mg/L solution containing 1 : 2 complexes. Samples were analyzed form different metals using atomic absorption spectrometer analysis. Table 1 compares the experimental results of AAS analysis and with one calculated on the theoretical basis. The results of AAS analysis are in well agreement with the predicted results within the limitations of the experimental error, which confirms the proposed 1 : 2 metal complex stoichiometric between metal and ligand.