Synthesis, Characterization and Biological Evaluation of Mononuclear Dichloro-bis[2-(2-chloro-6,7-substituted Quinolin-3-yl)-1H-benzo[d]imidazole]Co(II) Complexes

A series of Co(II) complexes 3a-g of 2-(2-chloro-6,7-substituted quinolin-3-yl)-1Hbenzo[d]imidazole ligands 3a-g were prepared and characterized by various spectroscopic and physicochemical methods viz. FT-IR, ESI mass, H NMR, C NMR and UV-Visible spectroscopy, Thermogravimetric analysis, Magnetic susceptibility, Molar conductance and Elemental analysis. The 2-(2chloro-6,7-substituted quinolin-3-yl)-1H-benzo[d]imidazole ligands 3a-g have been synthesized by cyclocondensation of benzene-1,2-diamine with 2-chloroquinoline-3-carbaldehydes by using ceric ammonium nitrate as a catalyst in presence of hydrogen peroxide as an oxidant. The structures of all ligands were confirmed by IR, Mass, UV-Visible, H NMR and C NMR spectroscopy. All ligands 3a-g and their Co(II) complexes 3a-g were screened for their in vitro antimicrobial activity using twofold serial dilution technique against standard MTCC strains of two Gram-positive Staphylococcus aureus and Streptococcus pyogenes, two Gram-negative Escherichia coli and Pseudomonas aeruginosa bacteria and three Candida albicans, Aspergillus niger and Aspergillus clavatus fungus in comparison with standard drugs. All ligands 3a-g and complexes 3a-g also evaluated for antimycobacterial activity against standard Mycobacterium tuberculosis H37Rv strain.


INTRODUCTION
Generally, most of the infections and diseases caused in the human body are by fungus and bacteria. Recently it has been the challenge to overcome the diseases and infections which origins by the pathogenic microorganisms. Many organic compounds containing the heteroatoms in their structures were found as the potential drugs to fight against this kind of infections in the human body. By revering all the matters we have synthesized some novel benzimidazole molecules and their Co(II) complexes, which were then screened for antiinfective activities (viz., antibacterial, antifungal and antimycobacterium activity) in this research article because benzimidazole compounds shows promising biological and pharmacological activities in organic and coordination chemistry. In this concern many benzimidazole compounds are reported as antibacterial and antifungal agents in past literature [1][2][3][4], recently Chetan et al. have reported the antituberculosis activity of some benzimidazole molecules [5]. Further, the quinoline motifs have also good biological properties viz., antitumor activity and DNA binding capability [6][7][8]. Because of these versatile biological properties of benzimidazole and quinoline motifs we have decided to prepare 2-(2chloro-6,7-substituted quinolin-3-yl)-1Hbenzo[d]imidazole molecules and their Co(II) complexes.
The cobalt ion and benzimidazole molecule both are the components of vitamin B12, therefore, cobalt complexes containing benzimidazole ligands have attracted our attention due to their various biological properties and more especially, their structural similarity with vitamin B12. It is well known that coordination compounds containing transition metal complexes have wide research area according to their spectroscopic and structural representation for the active site of some metalloenzymes [9]. The Co(II) complexes of monodentate 2-substituted benzimidazole ligands by taking 1:2 mole ratio of cobalt(II)chloride salt and benzimidazole ligand respectively, were infrequently discussed in past literature, that is why in the present article, we reported the synthesis and characterization of the series of dichloro-bis[2-substituted benzimidazole]cobalt(II) complexes. The series of 2chloro-6,7-substituted quinoline-3-carbaldehydes used for the synthesis of benzimidazole molecules were synthesized by previously published method [10]. The in vitro antimicrobial activity was determined by agar disc diffusion method against selected bacterial and fungal strains, also evaluated for in vitro antimycobactrial activity against Mycobacterium tuberculosis H37Rv strain which was determined by broth micro dilution assay to identify their biological spectrum and strength.

Pathogens
The pathogenic bacterial and fungal strains were procured from Institute of Microbial Technology, Chandigarh. The bacteria were subcultured on Nutrient agar whereas, fungi were subcultured on Sabouraud's dextrose agar (SDA) and were incubated aerobically at 37 °C.

Physical methods
Elemental analysis was done for C, H, N and Co on a Euro EA Elemental Analyzer, EA-3000, RS-232. Infrared spectra were recorded on a Shimadzu FT-IR 8400 spectrophotometer using KBr pellets in the range 4000-400cm -1 . The 1 H NMR (400 MHz) and 13

Synthesis of Co(II) complexes 3a-g:
imidazole ligands 3a-g (1.0 mmol) was dissolved in hot ethanol (20 mL) and the resulting solution was treated with hydrated cobalt chloride salt (0.5 mmol) dissolved in ethanol (10 mL) with continuous stirring at room temperature. Then, the reaction mixture was stirred at 40 C to separate out the product 3a-g from the reaction mixture within 7-15 days. The obtained solid product then collected through whatmann 42 filter paper and wash with ethanol.

Scheme 2.
Reaction scheme for the synthesis of complexes 3a-g.

Assay for in vitro antimicrobial activity
Each of the test compounds and standards ciprofloxacin and ampicillin for antibacterial activity and griseofulvin and nystatin for antifungal activity were dissolved in DMSO initially at 2000 gmL -1 and then were serially diluted in two series for culture medium as: 1000, 500, 250 gmL -1 and 100, 50, 25, 12.5, 6.25 gmL -1 concentrations. The bacteria were subcultured on Nutrient agar and fungi were subcultured on Sabouraud's dextrose agar (SDA) and incubated aerobically at 37 °C. The minimum inhibitory concentrations (MICs) were defined as the lowest concentrations of the compounds that prevented visible growth. The used solvent was tested against all microbial strains and confirmed that it had no any kind of activity against microorganisms.

Agar disc diffusion method
This test was evaluated for each compound was performed in the petri plates. In subsequent studies, the selected standard drugs for antibacterial and antifungal activity were also tested for the comparative study between new compounds and standards. The culture of bacterial and fungal strains was prepared in 4 mL of Muller Hinton broth at 37 C for 24 hours in incubator. The turbidity of culture suspension was adjusted with sterile Muller Hinton broth in order to obtain turbidity comparable to a No. 1 McFarland turbidity standard. One mL of this suspension was pipetted into the Muller Hinton agar plate and distributed evenly over the surface of the medium by gently stirring the plate. The surface of the medium was allowed to dry for 15 minutes at room temperature and was pierced with 7 mm diameter holes. The serial twofold diluted series of synthesized compounds from 2000-6.25 gmL -1 concentrations were impregnated in the pierced holes (7 mm) of the discs (88 mm) and applied to the surface of inoculated plates. The Petri plates were placed in an incubator at 37 C. After 24 hours of incubation the Petri plates was examined by considering 22 mm ZOI as the susceptible [11].

Determination of MIC
The minimum inhibitory concentration (MIC) of the compounds was determined by the micro broth dilution technique using Muller Hinton broth. Serial twofold dilutions ranged from 1000, 500, 250 gmL -1 and 100, 50, 25, 12.5, 6.25 gmL -1 concentrations for compounds. The inoculums was prepared in broth which had been kept overnight at 37 C and which had been diluted with Muller Hinton broth to give a final concentration of 10 8 cfu mL -1 (where cfu = Colony forming unit) in the test tray. The micro plates were covered and placed in plastic bags to prevent drying. After incubation at 37 C for 24 hours, the MIC value was defined as the lowest concentration of the compound giving complete inhibition of visible growth and the zone of inhibition (ZOI) was measured in millimeter diameter [12].

Antitubercular activity
The ligands 3a-g and their Co(II) complexes 3a-g have been tested in the primary test against the standard strain of Mycobacterium tuberculosis H37Rv (ATCC 27294) in BACTEC 12B medium using a broth micro dilution assay, the Micro plate Alamar Blue Assay (MABA) susceptibility test [13] by using rifampin and isoniazid as a reference drugs. Compounds exhibiting fluorescence were tested in the BACTEC 460 radiometric system. The final concentration (>6.25 g/mL) of ligands 3a-g and their Co(II) complexes 3a-g were added in LJ medium after inspissation and the chemical containing medium was distributed in 7 mL amounts in screw capped tubes. After that the tubes were inspissated in the sloping position at 80 C for 45 minutes. The medium was inoculated according to the recommendation of W.H.O. [14]. The inoculums for the susceptibility test was prepared by adding approximately 2 mg of growth from the primary culture on a loop to a 5 mL sterile distilled water in 7 mL screw capped tubes together with six 3 mm glass leads. The tubes were shaken mechanically for 1 minute and a full 3 mm loopful of the suspension inoculated to each slope. Duplicate slopes for each ligands 3a-g and their Co(II) complexes 3a-g were inoculated. A drug free control slopes were set up with each test and tubes incubated at 37 C. The results of the tests were read after 4 weeks incubation.

Elemental analysis and conductance
The ligands 3a-g were micro analyzed for C, H and N whereas, complexes 3a-g were micro analyzed for C, H, N and Co elements satisfactorily and results are comparatively in good agreement with their structures ( Table 1). The molar conductivity values of all complexes 3a-g observed in the range of 6.98-18.33 cm 2  -1 mol -1 indicating non-electrolyte nature of all complexes. 1

H NMR
The 1 H NMR spectral study of all newly synthesized ligands 3a-g shows the characteristic broad singlet of imidazolic hydrogen of secondary amine (-NH group) at 11.69-12.69 ppm, which is the confirmation about the formation of benzimidazole ring [15]. The aromatic ring protons for all ligands were observed in the range of 6.91-8.78 ppm. The proton of methoxy and methyl substitution on quinoline ring gives single line at 3.79 ppm and 2.84 ppm for ligand 3b and 3c respectively.
The signals consequent to the -NH group of benzimidazole ring in the 1 H NMR spectra of Co(II) complexes 3a-g, are remain unchanged with a variation of 0.05-0.09 ppm. The aromatic region of the metal complexes is considerably distorted therefore, the signals obtained cannot be identified, and this distortion is probably due to the metal ion present in the compounds. The signals corresponding to hydrogens of methoxy group (-OCH3) and methyl group (-CH3) are not changed or little bit shifted to down field or up field from the free ligands.

C NMR
The number of carbons of the all benzimidazole ligands can be count from the number of signals obtained, which represent most of chemically non-equivalent carbons in the compounds.

IR spectra
Hofmann showed that benzimidazole molecules display the strong intermolecular hydrogen bond [16] for secondary amine in the region of 3500- The  (C=N) stretching bands were observed get shifted towards the lower frequency region in the IR spectra of Co(II) complexes, which is indicating the complex formation of benzimidazole ligands with Co(II) ions through the tertiary nitrogen of imidazole ring.

GC-MS spectra of ligands
The mass spectra of all ligands show total molecular ion peaks and fragmented molecular ion peaks with respective to their molecular weight in form of cations or anions. The fragmentation in all ligands at 116-118 m/z is predictable to benzimidazole ring ion. The representative mass fragmentation prototype of ligand 3b is portrayed in Scheme 3.

Electronic spectra and magnetic susceptibility
The electronic spectra of benzimidazole ligands 3a-g and their complexes 3a-g shows two specific bands according to   * and n  * at 230-257 nm (43478-38911 cm -1 ) and 272-310 nm (36765-32258 cm -1 ) with the variation of 15-20 nm in each Co(II) complexes than their corresponding ligands. The ligand to metal charge transfer transition in all Co(II) complexes demonstrated as a broad shoulder at 400-453 nm (25000-22075 cm -1 ).
The electronic spectrums of all complexes 3a-g shows bands at 565-672 nm (17699-14881 cm -1 ) and 690-785 nm (14493-12739 cm -1 ) which are assigned to the d-d transitions due to the 4 A2  4 T1(F) and 4 A2  4 T1(P), which are related with the proposed tetrahedral geometry to the all Co(II) complexes (18) except 3c complex, which have 4 B2g  4 A1g, 4 B2g  4 Eg, 4 B2g  4 B1g transitions suggesting the square pyramidal geometry around the Co(II) metal ion [19]. The transition 4 A2  4 T2 is may be merge with the ligand to metal charge transfer transition in all complexes except 3g complex, which, shows three bands in their UV-Visible spectrum (Figure 1). All bands observed for all complexes 3a-g are summarized in Table 2. Table 2. Electronic spectral and magnetic susceptibility data of complexes 3a-g.

Complex
Electronic spectral bands (cm -1 ) Assignment  Continuation Figure 1. UV-Visible spectrums of ligands 3a-g and their complexes 3a-g.

TG analysis
The thermogravimetric analysis of complexes was taken at heating rate of 20 C per minute in nitrogen atmosphere from 30-1000 C temperature.
The complexes 3a and 3e shows 3 molecules of water, whereas, 3b and 3d complexes shows 4 molecules of water. The complexes 3c and 3g shows 1.5H2O and 2.5H2O molecules of water respectively in their outer sphere except 3f complex. All complexes decomposed in two to three steps ( Figure  2) and the thermogravimetric data of all complexes are summarized in following Table 3.

Biological activity
The antimicrobial activity data of all ligands 3a-g and their Co(II) complexes 3a-g are summarized in Table 4.
Antibacterial data collected for the above synthesized complexes 3a-g display good activity comparable with reference drugs in some of the cases, whereas moderate antibacterial activity was found for others against all bacterial strains by means of MIC values ranging from 6.25 to 1000 gmL -1 . A few of these 3a-g complexes are more active than their parent ligand as well as the Reference drugs used for the study. The compounds, microorganism and minimum inhibition concentration (MIC) are as follows.
The complexes 3a and 3g exhibits similar activity to standard drug ampicillin (250 gmL -1 MIC and 15 mm ZOI) against Staphylococcus aureus. The 3a complex demonstrates less activity than that of the free ligand, whereas, 3g complex shows comparatively similar activity to its free ligand 3g.
Contrarily the complexes 3c (12.5 gmL -1 MIC and 25 mm ZOI) and 3e (6.25 gmL -1 MIC and 30 mm ZOI) show two times and four times greater activity than reference drug ciprofloxacin and also shows more activity than those of their free ligands against Staphylococcus aureus. 3c complex shows comparatively similar activity to ampicillin reference drug and it's free ligand (100 gmL -1 MIC and 17 mm ZOI), whereas, 3d complex shows similar MIC value with ampicillin reference drug and four times less activity than that of free ligand.
In this context 3c and 3e complexes can be considered as potentially biological active compounds better than those of free ligands as well as used reference drugs too.
It is obvious from antifungal screening data that ligands 3a,b,d,e,f were more active against Candida albicans with 12.5, 500, 100, 500 and 250 gmL -1 MIC values having 27 mm, 6 mm, 14 mm, 8 mm and 19 mm ZOI respectively. The activity compared with reference drug nystatin shows ligand 3a is eight fold more active and ligand 3d is activity wise similar to it. In this direction ligands 3b,e and 3f shows similar and two times greater activity respectively compared to reference drug griseofulvin.
Only ligands 3d and 3e shows similar MIC values (100 gmL -1 and 17 mm ZOI) against Aspergillus niger and Aspergillus clavatus respectively to both of the used reference drugs nystatin and griseofulvin.
The complexes 3b, 3c and 3d have 250 gmL -1 MIC value and 13  3 mm ZOI, two times greater than the used reference drug griseofulvin, out of them, complexes 3b and 3c are more active than those of free ligands, whereas, 3d complex have less activity than that of free ligand. The complex 3f shows comparatively similar activity with 250 gmL -1 MIC value and 14 mm ZOI to griseofulvin reference drug and less than that of free ligand. The complexes 3a, 3e and 3g shows similar activity to both used reference drug nystatin and griseofulvin with 100 gmL -1 MIC value and 19 mm ZOI, and potentially more active than those of their respective free ligands. The complexes 3c and 3d displays similar activity with 100 gmL -1 MIC value having 20 mm ZOI to the both reference drugs nystatin and griseofulvin and they are more active than those of free ligands too.
Antimycobacterial activity is carried out by Alamar assay protocol and the obtained data is designated in Table 5. The result expressed as Minimum Inhibition Concentration in gmL -1 . All ligands 3a-g and their Co(II) complexes 3a-g effected <90% inhibition in primary screening (i.e., MIC <12.5 gmL -1 ). The ligands 3a-g and their complexes 3a-g were not subjected to the further evaluation due to lake of inhibition in primary screening. Table 5. Antimycobacterial activity of ligands 3a-g and their complexes 3a-g (Minimum Inhibition Concentration in gmL -1 ).
complexes. The antimicrobial data exhibited that most of Co(II) complexes 3a-g are more active than those of corresponding free ligands against bacterial and fungal strains. The 3c, 3e and 3f complexes and 3a,d,e ligands display comparatively good activity more than used reference drugs against bacterial and fungal strains, which show that they are the promising leads for antimicrobial drug development. None of the compounds showed any specific inhibition towards Mycobacterium tuberculosis strain H37Rv in primary screening

ACKNOWLEDMENTS
Authors are thankful to Rajiv Gandhi National Fellowship Scheme, UGC, New Delhi for the financial support and Tuberculosis Antimicrobial Acquisition and Coordinating Facility (TAACF), U.S.A. for anti-mycobacterial activity.