Antimicrobial Studies of N-Heterocyclic Carbene Silver Complexes Containing Benzimidazol-2-ylidene Ligand

Seven novel 4-vinylbenzyl substituted N-heterocyclic carbene (NHC) silver complexes were synthesized from different benzimidazolium salts and silver (I) oxide in dichloromethane at room temperature. These new 4-vinylbenzyl substituted NHC silver complexes were characterized by spectroscopic (NMR, IR) and elemental analysis techniques. Using the agar dilution procedure, the antimicrobial activities of these synthesized new compoundswere investigated againstGram (+)/(−) bacterial and fungal strains. These NHC silver complexes showed effective activities against Escherichia coli, Pseudomonas aeruginosa (Gram-negative bacterial strains), Enterococcus faecalis, Staphylococcus aureus (Gram-positive bacterial strains), and Candida tropicalis andCandida albicans (fungal strains).

Herein, we report the synthesis, characterization, and antibacterial and antifungal activity studies of seven novel symmetrically and unsymmetrically p-vinylbenzyl-substituted NHC silver complexes which were prepared starting from 1-(4-vinylbenzyl)-3-alkyl benzimidazolium salts and silver (I) oxide in dichloromethane. The characterization of the NHC silver complexes is consistent with the proposed formula. Using the agar dilution procedure, the antimicrobial activities of these compounds are investigated against Gram (+)/(−) bacterial and fungal strains. NHC silver complexes in the antimicrobial study were observed to have higher activity against fungal strains than against Gram-positive and Gramnegative bacterial strains.

Materials and Methods.
In this study, the synthesis of silver (I) complexes was pursued in a dark medium under an inert atmosphere. All solvents and reagents were commercially bought. The solvents used in the synthesis, diethyl ether over Na, and dichloromethane over P 4 O 10 were distilled before they were used. The 1 H and 13  and fungal strains were also subcultured on RPMI 1640 Broth (Sigma-Aldrich Chemie GmbH Taufkirchen, Germany).

General Method for the Synthesis of 1-(4-Vinylbenzyl)-
3-alkylbenzimidazol-2-ylidene Silver Complexes, 1a-g. 1-(4-Vinylbenzyl)-3-alkylbenzimidazolium salts containing symmetrical and unsymmetrical groups were synthesized by reaction of 1-(4-vinylbenzyl)benzimidazole with various aryl halides in DMF according to the literature [25,26]. Under an inert atmosphere, Ag 2 O (0.5 mmol), 1-(4-vinylbenzyl)-3-alkyl benzimidazolium salt (1.0 mmol) and activated 4Å molecular sieves were put in CH 2 Cl 2 which was distilled prior to use over P 4 O 10 (20 mL) and were stirred in darkness at ambient temperature for one day. The Schlenk-type flask used was covered with aluminum foil to avoid exposure to light. The resulting solution was filtered through celite and the solvent was removed under reduced pressure. The crude product was washed with diethyl ether which was distilled prior to use over Na (3 × 10 mL) and was crystallized from dichloromethane/diethyl ether at room temperature.

Antimicrobial Activity.
The antimicrobial activity studies of the new synthesized NHC silver complexes were investigated according to the recommended agar dilution procedure [27,28]. The minimal inhibitory concentrations (MICs) for each silver (I) complex were tested against standard bacterial strains (E. faecalis, S. aureus, P. aeruginosa, and E. coli) and fungal strains (C. tropicalis and C. albicans). Their turbidities matched that of a McFarland number 0.5 turbidity standard. The stock solutions of all silver complexes were prepared in dimethylsulfoxide. All dilutions were carried out using distilled water. The concentrations of the tested silver (I) complexes were 6.25, 12.5, 25, 50, 100, 200, 400, and 800 g/mL. Fluconazole, ciprofloxacin, and ampicillin were used as the antifungal and antibacterial standard drugs. A loopful (0.01 mL) of the standardized inocula of the yeasts and bacteria (10 6 CFUs/mL) was spread over the surface of agar plates. All the samples were inoculated after 16-20 h of incubation for bacteria and 48 h for yeasts. The lowest concentration of the NHC silver complexes that prevented visible growth was considered to be the MIC.

Synthesis and Characterization of N-Heterocyclic Carbene
Silver Complexes, 1a-g. Different 1-(4-vinylbenzyl)-3-alkyl benzimidazolium salts as carbene precursors were synthesized [25,26]. The intended silver (I) complexes 1a-g were obtained from the interaction of Ag 2 O with benzimidazolium salts in CH 2 Cl 2 at room temperature after 24 h as white solid crystals in 73-86% yields according to known methods (Scheme 1) [17,24]. Their structures were characterized using spectroscopic and analytical techniques. The 1 H and 13 C NMR spectra of these new complexes are consistent with the proposed formula. The spectra of these products in dimethylsulfoxide (DMSO-d 6 ) supported the formation of the silver complexes because of the loss of proton (NCHN) signal of the benzimidazolium salts. Compound 1a exhibited characteristic a carbenic carbon peak in the 13 C NMR spectra as singlet at 189.0 ppm. The resonance for carbene carbon was not determined in the 1b-g complexes. This condition has been mentioned in the literature and has been given as a reason for the variable action of the NHC complexes [29][30][31]. The FT-IR data for NHC silver complexes exhibit a characteristic ](C=N) band at 1445. 13, 1443.45, 1444.25, 1439.17, 1403.38, 1449.43, and 1440.26 for 1a-g, respectively.

Antimicrobial Activity of N-(4-Vinylbenzyl) Substituted
Silver Complexes, 1a-g. Using an agar dilution procedure, the antimicrobial activities of the synthesized silver (I) complexes were determined. Antimicrobial activities were found to be effective in the tested complexes (1a-g) against both fungi and bacteria strains with MIC values between 200 and 25 gmL −1 . It was seen that these complexes are more effective against fungi strains than against bacteria strains. The obtained results are summarized in the Table 1. The silver carbene complexes showed effective activities against Escherichia coli, Pseudomonas aeruginosa (gram-negative bacterial strains), Enterococcus faecalis, Staphylococcus aureus (gram-positive bacterial strains), and Candida tropicalis and Candida albicans (fungal strains). Most of benzimidazolium salts containing both electron withdrawing and electron donating groups demonstrated antimicrobial activity. The 1b, 1e, and 1f silver carbene complexes which were containing benzyl, 2,4,6-trimethylbenzyl and 2,3,5,6-tetramethylbenzyl groups showed better antibacterial activity than the 1a, 1c, 1d, and 1g complexes which were including methyl, 2-methylbenzyl, 4-methylbenzyl, and 4-vinylbenzyl groups. Generally,results of this study showed that compound containing sterich effect on the nitrogen atom were more effective on antimicrobial activity. The NHC silver complexes including both symmetrical (1g) and unsymmetrical (1c, 1d) groups indicated the same activities against all bacteria and fungus. Also, these compounds (1c, 1d, and 1g) exhibited low activity for all bacteria and fungi strains. Among the NHC silver complexes, the 1a, 1b, 1e, and 1f complexes showed high activity against C. tropicalis at 25 gmL −1 . As positive control, ampicillin, ciprofloxacin, and fluconazole were used. It is seen from the obtained data in this work that the substituents on the Natoms play an important role in antimicrobial activity.

Conclusions
The seven new 4-vinylbenzyl substituted benzimidazol-2ylidene silver complexes were prepared by the reaction of different benzimidazolium salts with silver (I) oxide in dichloromethane and characterized using elemental analysis and spectroscopic techniques. Some of these complexes showed very good antibacterial activity, especially against Candida tropicalis and Candida albicans as the fungal strains.
The obtained results imply that the tested silver complexes displayed a different effect against Escherichia coli, Pseudomonas aeruginosa (Gram-negative bacterial strains), Enterococcus faecalis, Staphylococcus aureus (Gram-positive bacterial strains), and Candida tropicalis and Candida albicans (fungal strains). 1b, 1e, and 1f compounds exhibited very good activity against all bacteria and fungi strains. Obtained results are helpful for the synthesis of NHC silver complexes possessing high antimicrobial activity.