Synthesis and antimicrobial properties of cycloheptyl substituted benzimidazolium salts and their silver(I) carbene complexes

Chemistry

The ligand precursors, cycloheptyl substituted benzimidazolium chlorides 2, were synthesized by quaternization of N-(2-cycloheptylethyl)benzimidazole (1) with various substituted benzyl chlorides. Compound 1 was synthesized by treatment of benzimidazole with 2-cycloheptylethyl chloride in tetrahydrofuran (THF) in the presence of sodium hydride (NaH). The reaction conditions for the synthesis of compound 1 and benzimidazolium chlorides 2a–d are given in Scheme 1. The structures of benzimidazolium salts were confirmed by the ¹H NMR, ¹³C NMR, IR spectroscopic methods and elemental analysis [20].

Scheme 1

Synthesis of benzimidazolium salts and silver(I)-NHC complexes.

Various procedures have been reported for the synthesis of silver-NHC complexes. Among them, reaction of an azolium salt with an alkaline silver compound is the most commonly used method [21], [22]. In this study, Ag₂O was used as the source of alkaline silver (I). Silver-NHC complexes 3a-d were synthesized in 16%–34% yields by reaction of the benzimidazolium chlorides 2a–d with 0.5 equivalent Ag₂O in dichloromethane. All reactions were carried out in the dark and complexes were also stored in the dark. The synthetic route to the silver-NHC complexes 3a–d is given in Scheme 1. The structures of complexes were established by spectroscopic data and elemental analysis. In the ¹H NMR spectra of 3a–d, the disappearance of a downfield signal of acidic benzimidazolium proton (NCHN) is consistent with the formation of silver-NHC complexes. In the ¹³C NMR spectra of 3a–d, the lack of the signal for the imino carbon (NCHN) also supports the proposed structures. Unfortunately, the signal of the carbene carbon could not be detected because of the fluxional behavior of silver-NHC complexes. This feature has been discussed in the literature [22]. Fluxional behavior between (NHC)AgX and [(NHC)₂Ag]⁺[AgX₂]^- species in solution has been shown for many complexes [22]. However, it has been demonstrated that monomeric (NHC)AgX species are the most favorable form in dichloromethane [23]. All attempts to obtain a single crystal of 3 suitable for X-ray crystallographic analysis failed.

Antimicrobial evaluation

Minimal inhibitory concentrations (MICs) were determined against Staphylococcus aureus, Enterococcus faecalis (Gram-positive), Escherichia coli and Pseudomonas aeruginosa (Gram-negative) bacterial strains and fungal strains C. albicans and C. tropicalis. The MIC values of synthesized compounds are summarized in Table 1. Ampicillin, ciprofloxacin and fluconazole were used as standard drugs for comparison. As shown in Table 1, MICs against bacteria and fungi are in the range of 800–6.25 μg/mL. All compounds inhibit the growth of the selected bacteria and fungi strains. The silver-NHC complexes are more active than benzimidazolium chlorides.

Benzimidazolium salts and complexes are more active against Gram-positive bacteria compared to Gram-negative bacteria strains. The activities of the newly synthesized silver complexes are comparable with the activities of standard drugs ampicillin and fluconazole for S. aureus and fungi strains. As already mentioned, the lipophilicity of complexes is the primary factor for enhanced antimicrobial activities. The lipophilicity enhances the passage of the drug through a lipid that surrounds the microorganism’s cell wall [18], [24]. We previously reported the synthesis and antimicrobial properties of coumarin functionalized silver-NHC complexes [19] and when antimicrobial activities of complexes 3a–d are compared with those of the complexes of the coumarin derivatives, it is obvious that complexes 3a–d have better antimicrobial properties.

Synthesis of 1-(2-cycloheptylethyl)benzimidazole (1)

A mixture of benzimidazole (3 g, 25 mmol) (50 mL) and NaH (600 mg, 25 mmol) in anhydrous THF (50 mL) was stirred for 1 h at ambient temperature, then treated with 1-chloro-2-cycloheptylethane (4 g, 25 mmol) and heated at 70°C for 48 h with continuous stirring. After cooling, the mixture was filtered through celite and the filtrate was concentrated. Compound 1 was obtained in 68% yield as a yellow oil; ¹H NMR (CDCl₃): δ 8.14 (s, 1H, -NCHN-), 7.07–7.59 (m, 4H, ArH), 4.13 (t, 2H, J=7 Hz, -NCH₂CH₂C₇H₁₃), 0.85–1.70 (m, 15H, -NCH₂CH₂C₇H₁₃); ¹³C NMR (CDCl₃): δ 143.9, 143.4, 133.7, 122.1, 121.3, 119.4, 110.3, 44.0, 34.9, 32.1, 29.5, 25.2, 24.6.

General procedure for preparation of benzimidazolium salts 2a–d

A solution of 1 (3 mmol, 0.73 g) in dried dimethylformamide (DMF) (5 mL) was treated with the corresponding benzyl chloride (3 mmol) and the mixture was heated for 24 h at 80°C, after which it was cooled and concentrated under reduced pressure. The residue was crystallized from ethanol/ether (1/1).

General procedure for preparation of silver complexes 3a–d

A mixture of Ag₂O (120 mg, 0.5 mmol), a benzimidazolium salt 2a–d (1 mmol) and activated 4 Å molecular sieves in dichloromethane (25 mL) was stirred at room temperature for 48 h. Then the mixture was filtered through celite and the filtrate was concentrated under reduced pressure. The residue was crystallized from dichloromethane/n-hexane at ambient temperature and washed with n-hexane (3×5 mL). All manipulations for the synthesis of silver-NHC complexes were carried out in the dark.

Antimicrobial activities of benzimidazolium salts and silver complexes

Antimicrobial performances of the benzimidazolium salts 2a–d and silver-NHC complexes 3a–d were determined by using the agar dilution procedure recommended by the Clinical and Laboratory Standards Institute [25], [26]. MICs were obtained against standard bacterial strains S. aureus ATCC 29213, E. faecalis ATCC 29212, E. coli ATCC 25922 and P. aeruginosa ATCC 27853 and the fungal strains C. albicans ATCC 10231 and C. tropicalis ATCC 13803 purchased from American Type Culture Collection (Rockville, MD). Bacterial strains were subcultured on a Muller Hinton broth (HiMedia Laboratories Pvt. Ltd. Mumbai, India). The fungal strains were subcultured on an RPMI 1640 broth (Sigma-Aldrich Chemie GmbH Taufkirchen, Germany). Their turbidities matched that of a McFarland no. 0.5 turbidity standard [27]. The stock solutions were prepared in dimethyl sulfoxide (DMSO). All dilutions were done with distilled water. The concentrations of the tested compounds were 800, 400, 200, 100, 50, 25, 12.5 and 6.25 μg/mL. Ampicillin and ciprofloxacin were used as antibacterial standard drugs, while fluconazole was used as an antifungal standard drug. A loopful (0.01 mL) of the standardized inoculum of the bacteria and yeasts (10⁶ CFUs/mL) was spread over the surface of agar plates. All inoculated plates were incubated at 35°C and the results were evaluated after 16–20 h of incubation for bacteria and 48 h for yeasts. The lowest concentration of the compounds that prevented visible growth was considered to be the MIC.