Table of Contents
International Journal of Inorganic Chemistry
Volume 2012, Article ID 121540, 13 pages
http://dx.doi.org/10.1155/2012/121540
Research Article

Novel Nonsymmetrically p-Benzyl-Substituted (Benz)imidazole N-Heterocyclic Carbene-Silver(I) Acetate Complexes: Synthesis and Biological Evaluation

Conway Institute of Biomolecular and Biomedical Research, Centre for Synthesis and Chemical Biology (CSCB), UCD School of Chemistry and Chemical Biology, University College Dublin, Belfield, Dublin 4, Ireland

Received 24 November 2011; Accepted 11 January 2012

Academic Editor: Alfonso Castiñeiras

Copyright © 2012 Frauke Hackenberg et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Nonsymmetrically substituted N-heterocyclic carbene (NHC) precursors 1ad and 3ad were synthesised by first reacting 1H-(benz)imidazole with p-cyanobenzyl bromide to give 4-(1H-imidazole-1-ylmethyl)benzonitrile (1) and 4-(1H-benzimidazole-1-ylmethyl)benzonitrile (3) and afterwards introducing benzyl bromide, 1-(bromomethyl)-4-methylbenzene, 1-(bromomethyl)-4-methoxybenzene, and methyl 4-(bromomethyl)benzoate. The NHC-silver(I) acetate complexes (1-benzyl-3-(4-cyanobenzyl)-2,3-dihydro-1H-imidazole-2-ylidene) silver(I) acetate (2a), (1-(4-cyanobenzyl)-3-(4-methylbenzyl)-2,3-dihydro-1H-imidazole-2-ylidene) silver(I) acetate (2b), (1-(4-cyanobenzyl)-3-[4-(methoxycarbonyl)benzyl]-2,3-dihydro-1H-imidazole-2-ylidene) silver(I) acetate (2c), (1-benzyl-3-(4-cyanobenzyl)-2,3-dihydro-1H-benzimidazole-2-ylidene) silver(I) acetate (4a), (1-(4-cyanobenzyl)-3-(4-methylbenzyl)-2,3-dihydro-1H-benzimidazole-2-ylidene) silver(I) acetate (4b), (1-(4-cyanobenzyl)-3-(4-methoxybenzyl)-2,3-dihydro-1H-benzimidazole-2-ylidene) silver(I) acetate (4c), and (1-(4-cyanobenzyl)-3-[4-(methoxycarbonyl)benzyl]-2,3-dihydro-1H-benzimidazole-2-ylidene) silver(I) acetate (4d) were yielded by reacting these NHC precursors with silver(I) acetate. The silver(I) acetate complex 4b was characterised by single crystal X-ray diffraction. Preliminary in vitro antibacterial studies against the Gram-positive bacteria Staphylococcus aureus and the Gram-negative bacteria Escherichia coli, using the Kirby-Bauer disc diffusion method, were carried out on the seven NHC-silver(I) acetate complexes 2ac and 4ad. Also the IC50 values of these seven complexes were determined by an MTT-based assay against the human renal cancer cell line Caki-1. The complexes 2ac and 4ac revealed the following IC50 values, respectively, 25 (±1), 15 (±2), 5.4 (±0.8), 16 (±2), 7.1 (±1), 20 (±4), and 14 (±1) μM.

1. Introduction

N-Heterocyclic carbenes (NHCs) are versatile ligands in silver complexes exhibiting antimicrobial activity, in particular for the possible treatment of pulmonary infections accompanying cystic fibrosis (CF) and chronic lung infections [13] and maybe in the treatment of cancer [4]. Youngs’ research group have reported antimicrobial activity of NHC-silver complexes derived from 1H-imidazole, 4,5-dichloro-1H-imidazole and xanthines against a panel of highly resistant pathogens recovered from the respiratory tract of cystic fibrosis (CF) patients [1, 3, 5]. Another important contribution by the Ghosh research group led to the synthesis and antimicrobial evaluation of NHC-silver complexes derived from 1-benzyl-3-tert-butylimidazole [6]. In addition, in vitro and murine in vivo efficacy and toxicity studies of nebulised methylated caffeine-silver(I) complex (SCC1), for treatment of pulmonary infections [7]. Recently, a larger number of known compounds were evaluated as potential antibiotics by Roland et al. [8].

In addition, silver complexes have been reported to have anticancer activity in vitro. Egan has reported that silver complexes of coumarin derivatives possess anticancer activity against certain types of cancer [9]. Zhu et al. has reported that silver carboxylate dimers possess anticancer activity against human carcinoma cells [10]. Liu et al. has shown phosphine complexes of silver to be active anticancer agents, even against cisplatin-resistant cell lines [11]. Youngs and coworkers have reported anticancer activity of NHC-silver complexes derived from 4,5-dichloro-1H-imidazole against the human cancer cell lines OVCAR-3 (ovarian), MB157 (breast), and HeLa (cervical) [4]. We have reported the anticancer (CAKI-1, renal) and antibacterial (E. coli, S. aureus) activity of benzyl-substituted N-heterocyclic carbene-silver [1217] and carbene-gold complexes [18, 19].

Within this paper we present a new series of nonsymmetrically p-benzyl-substituted N-heterocyclic carbene-silver acetate complexes derived from imidazole and benzimidazole, their synthesis, cytotoxicity, and antibacterial studies.

2. Experimental

2.1. General

All reactions were carried out under aerobic conditions. All silver(I) acetate reactions were carried out under exclusion of light. 1H-Imidazole, 1H-benzimidazole, benzyl bromide, 4-methylbenzyl bromide, methyl 4-(bromomethyl)benzoate, 4-cyanobenzyl bromide, silver(I) acetate, and K2CO3 were procured commercially from Sigma-Aldrich Chemical Company and were used without further purification. IR spectra were recorded on a Perkin-Elmer Paragon 1000 FT-IR spectrometer employing a KBr disc. NMR spectra were measured either on a Varian 300 MHz or 400 MHz spectrometer. All chemical shifts are reported in ppm and referenced to TMS. ESI MS was performed on a quadrupole tandem mass spectrometer (Quattro Micro, Micromass/Water’s Corp., USA), using solutions in 100% MeOH. MS spectra were obtained in the ES+ (electron spray positive ionisation) mode for all compounds. CHN Analysis was carried out in an Exeter Analytical CE-440 elemental analyzer. Crystal Data was collected using an Agilent Technologies (former Oxford Diffraction) SuperNova diffractometer fitted with an Atlas detector. 4b was measured with Mo-Kα (0.71073 Å) at 100 K. A four times redundant dataset was collected, assuming that the Friedel pairs are not equivalent. An analytical absorption correction based on the shape of the crystal was performed [20]. The structure was solved by direct methods using SHELXS-97 [21] and refined by full matrix least-squares on F2 for all data using SHELXL-97 [21]. Hydrogen atoms were added at calculated positions and refined using a riding model. Their isotropic thermal displacement parameters were fixed to 1.2 (1.5 for methyl groups) times the equivalent ones of the parent atom. Anisotropic thermal displacement parameters were used for all nonhydrogen atoms. A suitable crystal of 4b was grown in a saturated solution of chloroform with slow infusion of pentane. Further details about the data collection are listed in Table 1.

tab1
Table 1: Crystal data and structure refinement for 4b.

CCDC 853680 (for 4b) contains the supplementary crystallographic data for this paper. These data can be obtained free of charge from the Cambridge Crystallographic Data Centre via http://www.ccdc.cam.ac.uk/cgi-bin/catreq.cgi/.

2.2. Synthesis

4-(1H-Imidazole-1-ylmethyl)benzonitrile (1)
1H-Imidazole (1.478 g, 22.03 mmol), 4-(bromomethyl)benzonitrile (4.319 g, 22.03 mmol), and K2CO3 (4.560 g, 33.00 mmol) were stirred in CH3CN at room temperature for 2 d. After the solvent was removed under reduced pressure, water (30 mL) was added. The aqueous phase was extracted with CH2Cl2(3x, 20 mL), and the combined organic phases were dried with magnesium sulfate. After removing the solvent under reduced pressure, the product (1) was yielded (3.511 g, 19.14 mmol, 87% yield) as a white powder.
1H NMR (δ ppm, CDCl3, 300 MHz): 7.65 ( ), 7.56 (s, 1H, CHimid), 7.21(d, , 2H, CHcyanobenzyl), 7.12 (d, 1H, CHimid), 6.89 (s, 1H, NCHN), 5.20 (s, 2H, CH2).
13C NMR (δ ppm, CDCl3, 100 MHz, proton decoupled): 139.6, 134.4, 132.9, 128.4, 128.2, 118.7, 113.9, 111.9 (NCHN, CN, CHimid), 50.2 (CH2).
IR absorptions (KBr, cm−1): 3427 (m), 3132 (s), 3063 (m), 2981 (s), 2852 (s), 2226 (s), 1608 (m), 1567 (s), 1411 (s), 1355 (m), 1209 (s), 1161 (s), 769 (m), 634 (m), 550 (m).
MS (m/z, QMS-MS/MS): 182.07 [M+-H].
Microanalysis calculated for C11H9N3 (183.21): calcd.: C, 72.11%; H, 4.95%; N, 22.94%; found: C, 72.03%; H, 5.05%; N, 22.92%.

1-Benzyl-3-(4-cyanobenzyl)-2,3-dihydro-1H-imidazole-2-ylium Bromide (1a)
4-(1H-Imidazole-1-ylmethyl)benzonitrile (1) (0.500 g, 2.73 mmol) and 1.5 equivalents of benzyl bromide (0.701 g, 4.10 mmol) were dissolved in toluene (30 mL) and heated under reflux for 6 h. After removing the solvent under reduced pressure, the obtained sticky white product was washed with pentane (2x, 10 mL), diethylether (3x, 5 mL), and THF (2x, 10 mL) to yield a white powder (0.512 g, 1.44 mmol, 53% yield).
1H NMR (δ ppm, CDCl3, 300 MHz): 11.36 (s, 1H, NCHN), 7.71 (bs, 4H, CHcyanobenzyl), 7.44 (s, 5H, CHbenzyl), 7.08 (s, 1H, CHimidazole), 7.04 (s, 1H, CHimidazole), 5.78 (s, 2H, CH2), 5.48 (s, 2H, CH2).
13C NMR (δ ppm, CDCl3, 100 MHz, proton decoupled): 137.9 (NCHN), 137.8, 133.2, 132.1, 129.9, 129.8, 129.7, 129.0, 121.8, 121.7, 117.9, 113.6 (CN + Cimid + Ccyanobenzyl + Cbenzyl), 53.9, 52.6 (CH2).
IR absorptions (KBr, cm−1): 3461 (m), 3130 (s), 3058 (s), 2980 (s), 2920 (s), 2226 (s), 1608 (m), 1567 (s), 1411 (m), 1355 (m), 1209 (s), 1160 (s), 770 (s), 634 (m), 549 (m).
MS (m/z, QMS-MS/MS): 274.12 [M+-Br].
Microanalysis calculated for C18H16BrN3 (354.24): calcd.: C, 61.03%; H, 4.55%; N, 11.86%; Found: C, 60.96%; H, 4.52%, N, 11.68%.

1-(4-Cyanobenzyl)-3-(4-methylbenzyl)-2,3-dihydro-1H-imidazole-2-ylium Bromide (1b)
4-(1H-Imidazole-1-ylmethyl)benzonitrile (1) (0.500 g, 2.73 mmol) and 1.5 equivalents of 1-(bromomethyl)-4-methylbenzene (0.759 g, 4.10 mmol) were dissolved in toluene (30 mL) and heated under reflux for 6 h. After removing the solvent under reduced pressure, the obtained sticky white product was washed with pentane (2x, 10 mL), diethylether (3x, 5 mL), and THF (2x, 10 mL) to yield a white powder (0.784 g, 2.13 mmol, 78% yield).
1H NMR (δ ppm, CDCl3, 300 MHz): 10.74 (s, 1H, NCHN), 7.76 (d, J = 8.2 Hz, 2H, CHcyanobenzyl), 7.64 (d, J = 8.2 Hz, 2H, CHcyanobenzyl), 7.52 (s, 1H, CHimidazole), 7.35–7.26 (m, 2H, CHimid, CHmethylbenzyl), 7.18 (d, J = 7.6 Hz, 3H, CHimid, CHmethylbenzyl), 5.82 (s, 2H, CH2), 5.45 (s, 2H, CH2), 2.33 (s, 3H, CH3).
13C NMR (δ ppm, CDCl3, 100 MHz, proton decoupled): 139.8(NCHN), 138.3, 136.9, 133.0, 130.2, 129.9, 129.4, 128.9, 122.5, 121.9, 118.1, 113.2 (CN + Cimid + Ccyanobenzyl + Cmethylbenzyl), 53.4, 52.3 (CH2), 21.2 (CH3).
IR absorptions (KBr, cm−1): 3455 (m), 3130 (s), 3052 (s), 2981 (s), 2852 (s), 2227 (s), 1609 (m), 1566 (s), 1411 (m), 1209 (m), 1160 (s), 769 (s), 635 (m), 551 (s).
MS (m/z, QMS-MS/MS): 288.11 [M+-Br].
Microanalysis calculated for C19H18BrN3 (368.27): calcd.: C, 61.97%; H, 4.93%; N, 11.41%. Found: C, 62.34%; H, 5.01%; N, 11.21%.

1-(4-Cyanobenzyl)-3-(4-methoxybenzyl)-2,3-dihydro-1H-imidazole-2-ylium Bromide (1c)
4-(1H-Imidazole-1-ylmethyl)benzonitrile (1) (0.500 g, 2.73 mmol) and 1.5 equivalents of 1-(bromomethyl)-4-methoxybenzene (0.59 mL, 4.1 mmol) were dissolved in toluene (30 mL) and heated under reflux for 6 h. After removing the solvent under reduced pressure, the obtained sticky white product was washed with pentane (2x, 10 mL), diethylether (3x, 5 mL), and THF (2x, 10 mL) to yield a white powder (1.038 g, 2.70 mmol, 99% yield).
1H NMR (δ ppm, CDCl3, 300 MHz): 11.24 (s, 1H, NCHN), 7.69 (d, J = 9.2 Hz, 4H, CHcyanobenzyl), 7.50–7.17 (m, 4H, CHmethoxybenzyl), 6.92 (s, 2H, CHimidazole), 5.81 (s, 2H, CH2), 5.42 (s, 2H, CH2), 3.80 (s, 3H, CH3).
13C NMR (δ ppm, CDCl3, 100 MHz, proton decoupled): 138.3(NCHN), 137.0, 133.0, 130.7, 129.8, 129.3, 124.3, 122.2, 121.8, 121.5, 118.1, 114.9, 113.2 (CN + Cimid + Ccyanobenzyl + Cmethylbenzyl), 55.4 (OCH3), 53.3, 52.4 (CH2).
IR absorptions (KBr, cm−1): 3414 (m), 3059 (s), 2986 (s), 2964 (s), 2223 (s), 1610 (m), 1553 (s), 1516 (s), 1257 (m), 1146 (s), 1032 (m), 846 (m), 629 (s), 562 (m).
MS (m/z, QMS-MS/MS): 304.06 [M+-Br].
Microanalysis  calculated for C19H18BrN3O (384.27): calcd.: C, 59.39%; H, 4.72%; N, 10.94%. Found: C, 59.01%; H, 4.80%; N, 10.47%.

1-(4-Cyanobenzyl)-3-[4-(methoxycarbonyl)benzyl]-2,3-dihydro-1H-imidazole-2-ylium Bromide (1d)
4-(1H-Imidazole-1-ylmethyl)benzonitrile (1) (0.500 g, 2.73 mmol) and 1.5 equivalents of methyl 4-(bromomethyl)benzoate (0.939 g, 4.10 mmol) were dissolved in toluene (30 mL) and heated under reflux for 6 h. After removing the solvent under reduced pressure, the obtained sticky white product was washed with pentane (2x, 10 mL), diethylether (3x, 5 mL), and THF (2x, 10 mL) to yield a white powder (0.461 g, 1.12 mmol, 41% yield).
1H NMR (δ ppm, CDCl3, 300 MHz):
13C NMR (δ ppm, CDCl3, 100 MHz, proton decoupled): 160.1 (C=O), 142.6 (NCHN), 137.9, 137.2, 133.1, 131.3, 130.6, 130.0, 129.9, 129.0, 122.4, 122.2, 117.9, 113.4 (Cimid + Ccyanobenzyl + Cmethoxycarbonylbenzyl), 55.1, 52.2 (CH2), 32.2 (CH3).
IR absorptions (KBr, cm−1): 3403 (m), 3132 (s), 3062 (s), 2983 (s), 2851 (s), 2226 (s), 1718 (m), 1609 (m), 1566 (s), 1412 (s), 1286 (s), 1161 (s), 769 (s), 635 (m), 556 (s).
MS (m/z, QMS-MS/MS): 332.09 [M+-Br].
Microanalysis calculated for C20H18BrN3O2 (412.28): calcd.: C, 58.26%; H, 4.40%; N, 10.19%; found: C, 59.01%; H, 4.68%; N, 10.89%.

(1-Benzyl-3-(4-cyanobenzyl)-2,3-dihydro-1H-imidazole-2-ylidene) Silver(I) Acetate (2a)
1-Benzyl-3-(4-cyanobenzyl)-2,3-dihydro-1H-imidazole-2-ylium bromide (1a) (0.499 g, 1.41 mmol) and 2 equivalents of silver(I) acetate (0.471 g, 2.82 mmol) were dissolved in 30 mL of methanol/dichloromethane (1 : 1) and stirred in darkness at room temperature for 4 d. After filtering off the AgBr byproduct, the solvent was reduced to 3 mL and by adding dropwise 10 mL of pentane a white powder precipitated. The solvent was decanted and the white precipitate was washed with pentane (2x, 10 mL) and diethylether (3x, 10 mL) to yield a white powder (0.422 g, 0.96 mmol, 68% yield).
1H NMR (δ ppm, CDCl3, 300 MHz): 7.70 (m, 5H, CHcyanobenzyl, CHbenzyl), 7.39 (m, 4H, CHcyanobenzyl, CHbenzyl), 7.01 (d, 2H, CHimid), 5.40 (s, 4H, CH2), 2.05 (s, 3H, ).
13C NMR (δ ppm, CDCl3, 100 MHz, proton decoupled): 188.1 (NCN), 166.1 (C=O), 144.1, 137.5, 136.9, 133.1, 131.2, 130.6, 129.1, 128.2, 127.5, 117.9 (Ccyanobenzyl + Cimid + Cbenzyl), 52.4, 51.2 (CH2), 21.7 ( ).
IR absorptions (KBr, cm−1): 3410 (s), 3154 (s), 2230 (s), 1564 (s), 1411 (s), 1237 (m), 777 (m), 672 (w), 557 (m).
MS (m/z, QMS-MS/MS): 382.23 [M+-OCOCH3].
Microanalysis calculated  for C20H18AgN3O2 (440.24): calcd.: C, 54.56%; H, 4.12%; N, 9.54%; found: C, 55.01%; H, 4.24%; N, 9.07%.

(1-(4-Cyanobenzyl)-3-(4-methylbenzyl)-2,3-dihydro-1H-imidazole-2-ylidene) Silver(I) Acetate (2b)
1-(4-Cyanobenzyl)-3-(4-methylbenzyl)-2,3-dihydro-1H-imidazole-2-ylium bromide (1b) (0.501 g, 1.36 mmol) and 2 equivalents of silver(I) acetate (0.451 g, 2.72 mmol) were dissolved in 30 mL of methanol/dichloromethane (1 : 1) and stirred in darkness at room temperature for 4 d. After filtering off the AgBr byproduct, the solvent was reduced to 3 mL, and by adding dropwise 10 mL of pentane a white powder precipitated. The solvent was decanted, and the white precipitate was washed with pentane (2x, 10 mL) and diethylether (3x, 10 mL) to yield a white powder (0.296 g, 0.650 mmol, 48% yield).
1H NMR (δ ppm, CDCl3, 300 MHz): 7.70 (m, 4H, CHcyanobenzyl, CHmethylbenzyl), 7.40 (m, 4H, CHcyanobenzyl, CHmethylbenzyl), 7.01 (d, 2H, CHimid), 5.40 (d, 4H, CH2), 1.91 (d, 6H, , ).
13C NMR (δ ppm, CDCl3, 100 MHz, proton decoupled): 178.4 (NCN), 176.5 (C=O), 140.4, 138.0, 133.2, 132.9, 129.5, 128.4, 121.9, 121.3, 118.0, 112.8, 110.0 (CN + Cimid + Ccyanobenzyl + Cmethylbenzyl), 55.2, 52.7 (CH2), 30.9 ( ), 23.3 ( ).
IR absorptions (KBr, cm−1): 3391 (s), 3053 (m), 2227 (s), 1567 (s), 1410 (s), 1238 (m), 1158 (m), 771 (s), 690 (s), 624 (m).
MS (m/z, QMS-MS/MS): 392.23 [M+-OCOCH3].
Microanalysis calculated for C21H20AgN3O2 (454.27): calcd.: C, 55.52%; H, 4.44%; N, 9.25%; found: C, 55.48%; H, 4.62%; N, 9.09%.

(1-(4-Cyanobenzyl)-3-[4-(methoxycarbonyl)benzyl]-2,3-dihydro-1H-imidazole-2-ylidene) Silver(I) Acetate (2c)
1-(4-Cyanobenzyl)-3-[4-(methoxycarbonyl)benzyl]-2,3-dihydro-1H-imidazole-2-ylium bromide (1d) (0.449 g, 1.09 mmol) and 2 equivalents of silver(I) acetate (0.363 g, 2.18 mmol) were dissolved in 30 mL of methanol/dichloromethane (1 : 1) and stirred in darkness at room temperature for 3d. After filtering off the AgBr byproduct, the solvent was reduced to 3 mL, and by adding dropwise 10 mL of pentane a white powder precipitated. The solvent was decanted, and the white precipitate was washed with pentane (2x, 10 mL) and diethylether (3x, 10 mL) to yield a white powder (0.309 g, 0.620 mmol, 57% yield).
1H NMR (δ ppm, CDCl3, 300 MHz): 7.68 (d, 4H, CHcyanobenzyl, CHmethoxycarbonylbenzyl), 7.40 (d, 4H, CHcyanobenzyl, CHmethoxycarbonylbenzyl), 7.01 (s, 2H, CHimid), 5.75 (d, 2H, CH2), 5.72 (d, 2H, CH2), 3.91 (s, 3H, ), 2.07 (s, 3H, ).
13C NMR (δ ppm, CDCl3, 100 MHz, proton decoupled): 178.3 (NCN), 156.2 (C=O), 140.2, 133.0, 130.5, 128.5, 122.0, 118.0, 112.9, 110.0, 55.4, 55.3 (CH2), 30.9 ( ), 22.4 ( ).
IR absorptions (KBr, cm−1): 3417 (s), 3080 (s), 2229 (s), 1720 (m), 1566 (s), 1412 (s), 1281 (w), 656 (s).
MS (m/z, QMS-MS/MS): 440.23 [M+-OCOCH3].
Microanalysis calculated for C22H20AgN3O4 (498.28): calcd.: C, 53.03%; H, 4.05%; N, 8.43%; found: C, 52.66%; H, 4.12%, N, 8.01%.

4-(1H-Benzimidazole-1-ylmethyl)benzonitrile (3)
1H-Benzimidazole (1.488 g, 12.70 mmol), 4-(bromomethyl)benzonitrile (2.489 g, 12.70 mmol), and K2CO3 (2.633 g, 19.05 mmol) were stirred in CH3CN at room temperature for 2 d. After the solvent was removed under reduced pressure, water (30 mL) was added. The aqueous phase was extracted with CH2Cl2(3x, 20 mL), and the combined organic phases were dried with magnesium sulfate. After removing the solvent under reduced pressure, the product (3) was yielded as a white powder (2.637 g, 11.81 mmol, 93% yield).
1H NMR (δ ppm, CDCl3, 300 MHz): 7.98 (s, 1H, NCHN), 7.86 (d, 1H, CHbenzimid), 7.64 (d, 2H, CHcyanobenzyl), 7.24 (m, 5H, CHcyanobenzyl, CHbenzimid), 5.45 (s, 2H, CH2).
13C NMR (δ ppm, CDCl3, 100 MHz, proton decoupled): 138.2, 137.9, 134.6, 134.2, 128.9, 128.1, 120.3, 119.1, 118.4, 113.7 (NCHN, CN, Cbenzimid), 47.3 (CH2).
IR absorptions (KBr, cm−1): 3427 (m), 3060 (m), 2222 (s), 1615 (m), 1490 (s), 1457 (s), 1364 (m), 1284 (s), 1259 (s), 1167 (s), 761 (m), 745 (m), 556 (s).
MS (m/z, QMS-MS/MS): 232.09 [M+-H].
Micro analysis calculated for C15H11N3 (223.27): calcd.: C, 77.23%, H, 4.75%; N, 18.01%; found: C, 76.47%; H, 4.73%; N, 17.77%.

1-Benzyl-3-(4-cyanobenzyl)-2,3-dihydro-1H-benzimidazole-2-ylium Bromide (3a)
4-(1H-Benzimidazole-1-ylmethyl)benzonitrile (3) (0.500 g, 2.24 mmol) and 1.5 equivalents of benzyl bromide (0.575 g, 3.36 mmol) were dissolved in toluene (30 mL) and heated under reflux for 6 h. After removing the solvent under reduced pressure, the obtained sticky white product was washed with pentane (2x, 10 mL), diethylether (3x, 5 mL), and THF (2x, 10 mL) to yield a white powder (0.624 g, 1.54 mmol, 69% yield).
1H NMR (δ ppm, CDCl3, 300 MHz): 12.02 (s, 1H, NCHN), 7.73 (d, J = 8.0 Hz, 2H, CHcyanobenzyl), 7.65 (d, J = 8.0 Hz, 2H, CHcyanobenzyl), 7.53 (d, J = 10.9 Hz, 6H, CHbenzimidazole, CHbenzyl), 7.39 (d, J = 5.1 Hz, 3H, CHbenzimid, CHbenzyl), 6.10 (s, 2H), 5.83 (s, 2H).
13C NMR (δ ppm, CDCl3, 100 MHz, proton decoupled): 143.7 (NCHN), 137.7, 133.1, 132.1, 131.2, 129.5, 129.1, 128.3, 127.4, 117.9, 113.9, 113.3 (CN + Cbenzimid + Ccyanobenzyl + Cbenzyl), 51.9, 50.7 (CH2).
IR absorptions (KBr, cm−1): 3451 (s), 3384 (s), 3121 (s), 3035 (s), 2877 (s), 2228 (s), 1607 (m), 1554 (s), 1454 (s), 1184 (s), 1020 (m), 770 (s), 622 (m).
MS (m/z, QMS-MS/MS): 324.14 [M+-Br].
Microanalysis calculated for C22H18BrN3 (404.30): calcd.: C, 65.36%; H, 4.49%; N, 10.39%; found: C, 65.40%; H, 4.40%, N, 10.21%.

1-(4-Cyanobenzyl)-3-(4-methylbenzyl)-2,3-dihydro-1H-benzimidazole-2-ylium bromide (3b)
4-(1H-Benzimidazole-1-ylmethyl)benzonitrile (3) (0.500 g, 2.24 mmol) and 1.5 equivalents of 1-(bromomethyl)-4-methylbenzene (0.622 g, 3.36 mmol) were dissolved in toluene (30 mL) and heated under reflux for 6 h. After removing the solvent under reduced pressure, the obtained sticky white product was washed with pentane (2x, 10 mL), diethylether (3x, 5 mL), and THF (2x, 10 mL) to yield a white powder (0.787 g, 1.88 mmol, 84% yield).
1H NMR (δ ppm, CDCl3, 300 MHz): 11.94 (s, 1H, NCHN), 7.72 (d, J = 8.2 Hz, 2H, CHcyanobenzyl), 7.64 (d, J = 8.2 Hz, 2H, CHcyanobenzyl), 7.53 (d, J = 16.2 Hz, 4H, CHbenzimidazole), 7.39 (d, J = 8.0 Hz, 2H, CHmethylbenzyl), 7.18 (d, J = 8.0 Hz, 2H, CHmethylbenzyl), 6.10 (s, 2H, CH2), 5.77 (s, 2H, CH2), 2.33 (s, 3H, CH3).
13C NMR (δ ppm, CDCl3, 100 MHz, proton decoupled): 143.5 (NCHN), 139.5, 137.8, 133.0, 131.2, 130.1, 129.1, 128.3, 127.3, 118.0, 114.0, 113.4, 113.1 143.7, 137.7, 133.1, 132.1, 131.2, 129.5, 129.1, 128.3, 127.4, 117.9, 113.9, 113.3 (CN + Cbenzimid + Ccyanobenzyl + Cmethylbenzyl), 51.8, 50.7 (CH2), 21.2 (CH3).
IR absorptions (KBr, cm−1): 3465 (s), 3397 (s), 3127 (s), 3032 (s), 2968 (s), 2885 (s), 2230 (s), 1608 (m), 1561 (s), 1416 (m), 1372 (s), 1191 (s), 1019 (m), 761 (s), 613 (m), 554 (m).
MS (m/z, QMS-MS/MS): 338.13 [M+-Br].
Microanalysis calculated for C23H20BrN3 (418.33): calcd.: C, 66.04%; H, 4.82%; N, 10.04%; found: C, 65.99%; H, 4.79%, N, 10.04%.

1-(4-Cyanobenzyl)-3-(4-methoxybenzyl)-2,3-dihydro-1H-benzimidazole-2-ylium Bromide (3c)
4-(1H-Benzimidazole-1-ylmethyl)benzonitrile (3) (0.500 g, 2.24 mmol) and 1.5 equivalents of 1-(bromomethyl)-4-methoxybenzene (0.484 mL, 3.36 mmol) were dissolved in toluene (30 mL) and heated under reflux for 6 h. After removing the solvent under reduced pressure, the obtained sticky white product was washed with pentane (2x, 10 mL), diethylether (3x, 5 mL), and THF (2x, 10 mL) to yield a white powder (0.651 g, 1.50 mmol, 67% yield).
1H NMR (δ ppm, CDCl3, 300 MHz): 11.91 (s, 1H, NCHN), 7.72 (d, J = 8.2 Hz, 2H, CHcyanobenzyl), 7.63 (d, J = 8.2 Hz, 2H, CHcyanobenzyl), 7.60–7.41 (m, 6H, CHbenzimid, CHmethoxybenzyl), 6.89 (d, J = 8.6 Hz, 2H, CHmethoxybenzyl), 6.09 (s, 2H, CH2), 5.75 (s, 2H, CH2), 3.77 (s, 3H, CH3).
13C NMR (δ ppm, CDCl3, 100 MHz, proton decoupled): 144.3 (NCHN), 137.9, 133.1, 131.2, 129.9, 129.1, 127.3, 124.2, 117.9, 114.8, 113.9, 113.2 (CN + Cbenzimid + Ccyanobenzyl + Cmethoxybenzyl), 55.34 (OCH3), 51.5, 50.6 (CH2).
IR absorptions (KBr, cm−1): 3420 (s), 3122 (s), 3035 (s), 2968 (s), 2840 (s), 2229 (s), 1792 (w), 1609 (s), 1555 (s), 1514 (s), 1459 (s), 1370 (s), 1254 (s), 1178 (s), 1112 (s), 1013 (s), 763 (s), 613 (s).
MS (m/z, QMS-MS/MS): 355.08 [M+-Br].
Microanalysis calculated for C23H20BrN3O (434.33): calcd.: C, 63.60%; H: 4.64%; N, 9.67%; found: C, 63.41%; H, 4.70%; N, 9.69%.

1-(4-Cyanobenzyl)-3-[4-(methoxycarbonyl)benzyl]-2,3-dihydro-1H-benzimidazole-2-ylium Bromide (3d)
4-(1H-Benzimidazole-1-ylmethyl)benzonitrile (3) (0.500 g, 2.24 mmol) and 1.5 equivalents of methyl 4-(bromomethyl)benzoate (0.770 g, 3.36 mmol) were dissolved in toluene (30 mL) and heated under reflux for 6 h. After removing the solvent under reduced pressure, the obtained sticky white product was washed with pentane (2x, 10 mL), diethylether (3x, 5 mL), and THF (2x, 10 mL) to yield a white powder (0.735 g, 1.59 mmol, 71% yield).
1H NMR (δ ppm, CDCl3, 300 MHz): 12.09 (s, 1H, NCHN), 8.03 (d, J = 8.2 Hz, 2H, CHcyanobenzyl), 7.72 (d, J = 8.2 Hz, 2H, CHcyanobenzyl), 7.66 (d, J = 8.2 Hz, 2H, CHbenzimidazole), 7.58 (d, J = 8.2 Hz, 2H, CHbenzimidazole), 7.51 (d, J = 5.5 Hz, 4H, CHmethoxycarbonylbenzyl), 6.07 (s, 2H, CH2), 5.96 (s, 2H, CH2), 3.91 (s, 3H, CH3).
13C NMR (δ ppm, CDCl3, 100 MHz, proton decoupled): 166.20 (C=O), 143.88 (NCHN), 139.76, 139.48, 133.32, 131.50, 130.17, 129.56, 129.03, 127.42, 118.86, 114.45, 114.39, 111.93 (CN + Cbenzimid + Ccyanobenzyl + Cmethoxycarbonylbenzyl), 52.75 (OCH3), 50.14, 49.96 ( + ).
IR absorptions (KBr, cm−1): 3433 (s), 2963 (s), 2228 (s), 1717 (s), 1560 (m), 1435 (m), 1416 (m), 1283 (s), 1189 (m), 1107 (m), 748 (m), 601 (m).
MS (m/z, QMS-MS/MS): 382.05 [M+-Br].
Microanalysis calculated for C24H20BrN3O2 (462.34): calcd.: C, 62.35%; H, 4.36%; N, 9.09%; found: C, 61.90%; H, 4.14%; N, 9.10%.

(1-Benzyl-3-(4-cyanobenzyl)-2,3-dihydro-1H-benzimidazole-2-ylidene) Silver(I) Acetate (4a)
1-Benzyl-3-(4-cyanobenzyl)-2,3-dihydro-1H-benzimidazole-2-ylium bromide (3a) (0.150 g, 0.370 mmol) and 2 equivalents of silver(I) acetate (0.124 g, 0.740 mmol) were dissolved in 30 mL of dichloromethane and stirred in darkness at room temperature for 3 d. After filtering off the AgBr byproduct, the solvent was reduced to 3 mL, and by adding dropwise 10 mL of pentane a white powder precipitated. The solvent was decanted, and the white precipitate was washed with pentane (2x, 10 mL) and diethylether (3x, 10 mL) to yield a white powder (0.094 g, 0.192 mmol, 52% yield).
1H NMR (δ ppm, CDCl3, 300 MHz): 7.64 (d, 2H, CHcyanobenzyl), 7.30 (m, 11H, CHcyanobenzyl, CHbenzimid, CHbenzyl), 5.74 (s, 2H, CH2), 5.66 (s, 2H, CH2), 2.09 (s, 3H, ).
13C NMR (δ ppm, CDCl3, 100 MHz, proton decoupled): 189.21 (NCN), 165.20 (C=O), 142.29, 136.61, 133.19, 129.23, 128.74, 128.50, 128.00, 124.63, 118.95, 113.02, 112.72, 111.28, 109.99 (Ccyanobenzyl, Cbenzimid, Cbenzyl), 52.56, 51.85 (CH2), 23.82 ( ).
IR absorptions (KBr, cm−1): 3476–3364 (m), 3037-2922 (s), 2229 (m), 1607 (s), 1562 (s), 1390 (s), 1335 (m), 740 (s), 657 (s), 614 (s), 552 (m), 454 (m).
MS (m/z, QMS-MS/MS): 430.05 [M+-OCOCH3].
Microanalysis calculated for C24H20AgN3O2 (490.30): calcd.: C, 58.79%; H, 4.11%; N, 8.57%; found: C, 58.71%; H, 4.03%; N, 8.69%.

(1-(4-Cyanobenzyl)-3-(4-methylbenzyl)-2,3-dihydro-1H-benzimidazole-2-ylidene) Silver(I) Acetate (4b)
1-(4-Cyanobenzyl)-3-(4-methylbenzyl)-2,3-dihydro-1H-benzimidazole-2-ylium bromide (3b) (0.498 g, 1.19 mmol) and 2 equivalents of silver(I) acetate (0.399 g, 2.39 mmol) were dissolved in 30 mL of dichloromethane and stirred in darkness at room temperature for 3d. After filtering off the AgBr byproduct, the solvent was reduced to 3 mL, and by adding dropwise 10 mL of pentane a white powder precipitated. The solvent was decanted, and the white precipitate was washed with pentane (2x, 10 mL) and diethylether (3x, 10 mL) to yield a white powder (0.462 g, 0.916 mmol, 77% yield).
1H NMR (δ ppm, CDCl3, 300 MHz): 7.25 (m, 12H, CHcyanobenzyl, CHmethylbenzyl, CHbenzimi), 5.73 (d, 2H, CH2), 5.61 (d, 2H, CH2), 2.32 (s, 3H, ), 2.08 (s, 3H, ).
13C NMR (δ ppm, CDCl3, 100 MHz, proton decoupled): 179.1 (NCN), 138.5, 133.9, 133.7, 132.9, 131.7, 131.7, 129.8, 127.9, 127.3, 124.5, 118.2, 112.4, 111.6 (Ccyanobenzyl, Cbenzimid, Cmethylbenzyl), 55.5, 52.8 (CH2), 22.8, 21.1 ( , ).
IR absorptions (KBr, cm−1): 3417 (m), 3030 (s), 2229 (m), 1571 (s), 1391 (s), 796 (m), 743 (m), 668 (m), 612 (m)
MS (m/z, QMS-MS/MS): 445.95 [M+-OCOCH3].
Microanalysis calculated for C25H22AgN3O2 (504.33): calcd.: C, 59.54%; H, 4.40%; N, 8.33%; found: C, 60.04%; H, 4.38%, N, 8.37%.

(1-(4-Cyanobenzyl)-3-(4-methoxybenzyl)-2,3-dihydro-1H-benzimidazole-2-ylidene) Silver(I) Acetate (4c)
1-(4-Cyanobenzyl)-3-(4-methoxybenzyl)-2,3-dihydro-1H-benzimidazole-2-ylium bromide (3c) (0.499 g, 1.15 mmol) and 2 equivalents of silver(I) acetate (0.383 g, 2.30 mmol) were dissolved in 30 mL of dichloromethane and stirred in darkness at room temperature for 2 d. After filtering off the AgBr byproduct, the solvent was reduced to 3 mL, and by adding dropwise 10 mL of pentane a white powder precipitated. The solvent was decanted, and the white precipitate was washed with pentane (2x, 10 mL) and diethylether (3x, 10 mL) to yield a white powder (0.418 g, 0.805 mmol, 70% yield).
1H NMR (δ ppm, CDCl3, 300 MHz): 7.25 (m, 12H, CHcyanobenzyl, CHmethoxybenzyl, CHbenzimi), 5.74 (d, 2H, CH2), 5.58 (d, 2H, CH2), 3.78 (s, 3H, CH3), 2.07 (s, 3H, ).
13C NMR (δ ppm, CDCl3, 100 MHz, proton decoupled): 178.8 (NCN), 159.8 (C=O), 133.9, 133.7, 132.9, 128.8, 127.9, 124.4, 118.1, 114.5, 112.4, 111.5 (Ccyanobenzyl, Cbenzimid, Cmethoxybenzyl), 55.3, 53.2 (CH2), 30.9 ( ), 22.9 ( ).
IR absorptions (KBr, cm−1): 3390 (m), 2929 (s) [C–H], 2228 (s), 1608 (s), 1571 (s), 1513 (s), 1393 (s), 1249 (s), 1176 (s), 1116 (w), 1024 (s), 799 (s), 745 (s), 538 (s).
MS (m/z, QMS-MS/MS): 461.97 [M+-OCOCH3].
Microanalysis calculated for C25H22AgN3O3 (520.33): calcd.: C, 57.71%; H, 4.26%; N, 8.08%; found: C, 57.72%; H, 4.18%; N, 8.27%.

(1-(4-Cyanobenzyl)-3-[4-(methoxycarbonyl)benzyl]-2,3-dihydro-1H-benzimidazole-2-ylidene) Silver(I) Acetate (4d)
1-(4-Cyanobenzyl)-3-[4-(methoxycarbonyl)benzyl]-2,3-dihydro-1H-benzimidazole-2-ylium bromide (3d) (0.499 g, 1.08 mmol) and 2 equivalents of silver(I) acetate (0.360 g, 2.16 mmol) were dissolved in 30 mL of dichloromethane and stirred in darkness at room temperature for 2 d. After filtering off the AgBr byproduct, the solvent was reduced to 3 mL, and by adding dropwise 10 mL of pentane a white powder precipitated. The solvent was decanted, and the white precipitate was washed with pentane (2x, 10 mL) and diethylether (3x, 10 mL) to yield a white powder (0.361 g, 0.658 mmol, 61% yield).
1H NMR (δ ppm, CDCl3, 300 MHz): 7.39 (m, 12H, CHcyanobenzyl, CHmethoxycarbonylbenzyl, CHbenzimi), 5.74 (d, 2H, CH2), 5.72 (d, 2H, CH2), 3.90 (s, 3H, CH3), 2.08 (s, 3H, ).
13C NMR (δ ppm, CDCl3, 100 MHz, proton decoupled: 179.3 (NCN), 166.3 (C=O), 140.0, 139.4, 133.8, 133.7, 133.0, 130.4, 127.9, 127.2, 124.8, 118.1, 112.7, 112.2, 111.8 (Ccyanobenzyl, Cbenzimid, Cmethoxycarbonylbenzyl), 53.4, 53.0 (CH2), 52.3 ( ), 22.6 ( ).
IR absorptions (KBr, cm−1): 3422 (m), 2952 (s), 2229 (m), 1715 (s), 1573 (s), 1394 (s), 1283 (s), 1106 (m), 1018 (w), 739 (m).
MS (m/z, QMS-MS/MS): 490.07 [M+-OCOCH3].
Microanalysis calculated for C26H22AgN3O4 (548.34): calcd.: C, 56.95%; H, 4.04%; N, 7.66%; found: C, 56.77%; H, 4.03%; N, 7.48%.

2.3. Antibacterial Studies

The silver(I) acetate complexes were screened in preliminary in vitro antibacterial tests against two bacterial strains. The test organisms included Staphylococcus aureus (SA) (NCTC 7447) as a Gram-positive bacteria and Escherichia coli (E. coli) as Gram-negative bacteria.

To assess the biological activity of compounds 2a–c and 4a–d, the qualitative Kirby-Bauer disk-diffusion method was applied [22]. All bacteria were individually cultured from a single colony in sterile LB medium [23] overnight at 37°C in an orbital shaker incubator. All the work carried out was performed under sterile conditions.

For each strain, 70 μL of culture was spread evenly on agar-LB medium. Four 5 mm diameter Whatman paper discs were placed evenly separated on each plate. Two stock solutions (9 : 1 DMSO : H2O) of every compound were prepared at 2.2 μM and 4.4 μM to be able to test the effect of different concentrations. Each plate was then tested with 5 μL and 7 μL of 2.2 μM solution and 5 μL and 10 μL for the 4.4 μM solution. The plates were covered and placed in an incubator at 37°C for 24 h. The plates were then removed and the area of clearance, which is defined as the distance between the edge of the filter paper disc and the beginning of the bacterial growth, was measured for each sample in mm.

2.4. Cytotoxicity Studies

Preliminary in vitro cell tests were performed on the human cancerous renal cell line Caki-1 in order to compare the cytotoxicity of the compounds presented in this paper. This cell line was chosen based on its regular and long-lasting growth behaviour, which is similar to the one shown in kidney carcinoma cells. The cells were obtained from the ATCC (American Tissue Cell Culture Collection) and maintained in Dulbecco’s Modified Eagle medium containing 10% (v/v) FCS (fetal calf serum), 1% (v/v) penicillin streptomycin, and 1% (v/v) L-glutamine. Cells were seeded in 96-well plates containing 200 μL microtitre wells at a density of 5,000 cells/200 μL of medium and were incubated at 37°C for 24 h to allow for exponential growth. Then the compounds used for the testing were dissolved in the minimal amount of DMSO (dimethylsulfoxide) possible and diluted with medium to obtain stock solutions of 5 × 10−4 M in concentration and less than 0.7% of DMSO. The cells were then treated with varying concentrations of the compounds and incubated for 48 h at 37°C. Then, the solutions were removed from the wells, and the cells were washed with PBS (phosphate buffer solution) and fresh medium was added to the wells. Following a recovery period of 24 h incubation at 37°C, individual wells were treated with 200 μL of a solution of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) in medium. The solution consisted of 22 mg of MTT in 40 mL of medium. The cells were incubated for 3 h at 37°C. The medium was then removed, and the purple formazan crystals were dissolved in 200 μL DMSO per well. For all tests, cells with low passage numbers were used. A Wallac Victor (Multilabel HTS Counter) Plate Reader was used to measure absorbance at 540 nm. Cell viability was expressed as a percentage of the absorbance recorded for control wells. The values used for the dose response curves represent the values obtained from four consistent MTT-based assays for each compound tested.

3. Results and Discussion

In Schemes 1 and 2, the synthetic routes for the asymmetric substituted N-heterocyclic carbene ligand precursors as well as their corresponding silver(I) acetate complexes are given. The initial precursors 4-(1H-imidazole-1-ylmethyl)benzonitrile (1) and 4-(1H-benzimidazole-1-ylmethyl)benzonitrile (3) were prepared by stirring 1H-imidazole and 1H-benzimidazole with 4-(bromomethyl)benzonitrile in acetonitrile and K2CO3 at room temperature for 2d with 69 and 93% yields, respectively. 1-Benzyl-3-(4-cyanobenzyl)-2,3-dihydro-1H-imidazole-2-ylium bromide (1a), 1-(4-cyanobenzyl)-3-(4-methylbenzyl)-2,3-dihydro-1H-imidazole-2-ylium bromide (1b), 1-(4-cyanobenzyl)-3-(4-methoxybenzyl)-2,3-dihydro-1H-imidazole-2-ylium bromide (1c), and 1-(4-cyanobenzyl)-3-[4-(methoxycarbonyl)benzyl]-2,3-dihydro-1H-imidazole-2-ylium bromide (1d) were synthesised by stirring (1) with benzyl bromide, 1-(bromomethyl)-4-methylbenzene, 1-(bromomethyl)-4-methoxybenzene, and 4-(bromomethyl)benzoate in toluene under reflux for 6 h to yield the N-heterocyclic carbene ligand precursors in 53, 78, 99, and 41%, respectively. The benzimidazole containing precursors 1-benzyl-3-(4-cyanobenzyl)-2,3-dihydro-1H-benzimidazole-2-ylium bromide (3a), 1-(4-cyanobenzyl)-3-(4-methylbenzyl)-2,3-dihydro-1H-benzimidazole-2-ylium bromide (3b), 1-(4-cyanobenzyl)-3-(4-methoxybenzyl)-2,3-dihydro-1H-benzimidazole-2-ylium bromide (3c), and 1-(4-cyanobenzyl)-3-[4-(methoxycarbonyl)benzyl]-2,3-dihydro-1H-benzimidazole-2-ylium bromide (3d) followed the same reaction route to give the respective yields of 69, 84, 67, and 71%.

121540.sch.001
Scheme 1: General reaction scheme for the synthesis of imidazole containing asymmetric substituted NHC precursors 1a–d and their corresponding NHC-silver(I) acetate complexes 2a–c.
121540.sch.002
Scheme 2: General reaction scheme for the synthesis of benzimidazole containing asymmetric substituted NHC precursors 3a–d and their corresponding NHC-silver(I) acetate complexes 4a–d.

In the absence of light, the silver(I) acetate complexes (1-benzyl-3-(4-cyanobenzyl)-2,3-dihydro-1H-imidazole-2-ylidene) silver(I) acetate (2a), (1-(4-cyanobenzyl)-3-(4-methylbenzyl)-2,3-dihydro-1H-imidazole-2-ylidene) silver(I) acetate (2b), and (1-(4-cyanobenzyl)-3-[4-(methoxycarbonyl)benzyl]-2,3-dihydro-1H-imidazole-2-ylidene) silver(I) acetate (2c) were prepared by stirring the respective precursor with 2 equivalents of silver(I) acetate in a mixture of dichloromethane and methanol (1 : 1) at room temperature for 3-4 d. The silver complexes were obtained in 68, 48, and 57% yield respectively. The synthesis of the asymmetric benzimidazole silver(I) acetate complexes was carried out under the same conditions but in dichloromethane and with shorter reaction times of 2-3d to give the silver(I) acetate complexes (1-benzyl-3-(4-cyanobenzyl)-2,3-dihydro-1H-benzimidazole-2-ylidene) silver(I) acetate (4a), (1-(4-cyanobenzyl)-3-(4-methylbenzyl)-2,3-dihydro-1H-benzimidazole-2-ylidene) silver(I) acetate (4b), (1-(4-cyanobenzyl)-3-(4-methoxybenzyl)-2,3-dihydro-1H-benzimidazole-2-ylidene) silver(I) acetate (4c), and (1-(4-cyanobenzyl)-3-[4-(methoxycarbonyl)benzyl]-2,3-dihydro-1H-benzimidazole-2-ylidene) silver(I) acetate (4d) in 52, 77, 70, and 61% yields.

The asymmetric substituted N-heterocyclic carbene ligand precursors as well as the silver(I) acetate complexes were fully characterized by spectral (1H NMR, 13C NMR, IR, mass) and elemental analysis studies.

Furthermore, the solid state structure of the silver(I) acetate complex 4b was determined by single crystal X-ray diffraction.

Due to the positive charge of the molecule, the 1H NMR spectra of all NHC precursors 1a–d and 3a–d show a characteristic downfield shift in the range δ = 10.74–12.09 ppm for the NCHN proton [2325]. In addition, their identities have also been confirmed by a base peak for the [M+-Br] fragments in their positive mode ESI mass spectra. A successful formation of the complexes 2a–c and 4a–d is indicated by the absence of a downfield NCHN signal and presence of new signals at 2.09–1.91 ppm for the acetate protons in all the 1H NMR spectra. The 13C NMR resonances of the carbene carbon atoms in complexes 2a–c and 4a–d occur in the range δ 188.1–178.3 ppm respectively. These signals are shifted downfield compared to the corresponding precursors of 1a–d and 3a–d carbene carbons resonance at the range 144.3–137.9 ppm, respectively, which further demonstrates the formation of expected NHC-silver(I) acetate complexes. Also the appearance of the 13C NMR resonances for the carbonyl and methyl carbons of the acetate group of complexes 2a–c and 4a–d in the range 176.5–156.2 and 23.3–21.1 ppm, respectively, showed the formation of the NHC-silver(I) acetate complexes [1, 3]. Furthermore, positive-mode ESI mass spectra of all six NHC-silver(I) acetate complexes (2a–c, 4a–d) are dominated by [M+-O2CCH3] fragment peaks arising from the loss of one acetate ligand.

3.1. Structural Discussion

The crystal of 4b was formed in a saturated solution of chloroform with slow infusion of pentane. It crystallized in the monoclinic space group P21/n (no. 14) in absence of any solvent molecules. The crystal data and refinements are found in Table 1, whereas selected bond lengths and bond angles are compiled in Table 2. The X-ray structure shows that the benzimidazole ring is planar and the bond lengths and angles in the five-membered imidazole ring (NCNCC) are in good agreement with those in similar compounds reported earlier by our group [1316]. Complex 4b crystallises in two different species, which are shown in Figures 1 and 2.

tab2
Table 2: Selected bond lengths [Å] and angles [°] for 4b.
121540.fig.001
Figure 1: X-ray diffraction structure of 4b showing the major occupied Ag position; thermal ellipsoids are drawn on the 50% probability level.
121540.fig.002
Figure 2: X-ray diffraction structure of 4b showing the minor occupied Ag position; thermal ellipsoids are drawn on the 50% probability level, Ag2 with fixed radius.

The major species (95%, Figure 1) shows a nearly linear bond angle of 170.76 (7)° at the two-coordinated silver atom. The bond distance Ag(1)–C(9) of 2.060 (2) Å agrees very well with previously reported examples of this compound class [1317] and shows that the silver is strongly bonded to the carbene. The Ag(1)–O(1) bond length of 2.1088 (14) Å is quite short compared to the ones our group previously reported but is still within range of the corresponding ones in similar molecules (see e.g., [26, 27]). This short distance suggests a predominantly covalent character of the Ag(1)–O(1) bond.

In comparison to this, the minor species (5%, Figure 2), where the silver atom is bonded to both oxygen atoms of the acetate group, the bond lengths to the oxygen atoms are much longer (Ag(2)–O(1): 2.408(6) Å, and Ag(2)–O(2): 2.554(5) Å). Furthermore the bond angle C(9)–Ag(2)–O(1) is no longer linear with 135.9(4)° and the C(9)–Ag(2)–O(2) angle is found to be 164.9(3)°. This bonding pattern is characteristic for a predominantly ionic coordination of the acetate to the silver. Both of these modes have been observed earlier (see e.g., [17]). Having both of them in the same structure suggests a rather small energy difference between the two forms.

3.2. Biological Evaluation
3.2.1. Antibacterial Testing

Using the Kirby-Bauer disk diffusion method, the in vitro antibacterial activity of the NHC-silver(I) acetate complexes was tested and summarised in Figures 3 and 4. The metal salt (silver(I) acetate) used to prepare the complexes and the solvent (DMSO) used to prepare the stock solutions played no role in growth inhibition on the same bacteria as previously reported [17, 28].

121540.fig.003
Figure 3: Area of clearance on Staphylococcus aureus (Gram +ve) by 2a–c and 4a–d.
121540.fig.004
Figure 4: Area of clearance on Escherichia coli (Gram −ve) by 2a–c and 4a–d.

Compounds 2b and 2c showed almost no antibacterial activity against the Gram-positive bacteria Staphylococcus aureus, and compound 4d showed the best activity against this bacterial strain, but, in comparison to previous reported NHC-silver(I) acetate complexes, the activity is more in a medium range. Hereby, an area of clearance of 0 mm is considered as no activity, areas of 1–4 mm as low, 5–9 as medium, and areas of clearance ≥10 mm as high activity. Low antibacterial activity was observed for complexes 2c and 4b against the Gram-negative bacteria Escherichia coli and all other complexes (2a, 2b, 4a, 4c, and 4d) exhibited only medium activity against this bacteria strain.

3.2.2. Cytotoxicity Studies

Our interest focuses on the probable difference in activity due to the influence of different lipophilicity in the two compound classes. The in vitro anticancer activity of the asymmetric substituted NHC-silver(I) acetate complexes was tested in an MTT-based assay against the human renal cancer cell line Caki-1. In this test a 48 h drug exposure period was followed by a 24 h recovery period, and the log dose response curves for complexes 2a–c and 4a–d are shown in Figures 5 and 6, respectively. The IC50 values of the imidazole containing complexes are : 25 (±1), : 15 (±2), : 5.4 (±0.8) μM. Slightly less activity was observed for the more lipophilic NHC-silver(I) acetate complexes 4a–d, which gave IC50 values of 16 (±2), 7.1 (±1), 20 (±4), 14 (±1) μM, respectively. Compounds 2c and 4b show the highest cytotoxic activities with single-digit micromolar IC50 values.

121540.fig.005
Figure 5: Cytotoxicity curves from typical MTT assays showing the effect of compounds 2a–c on the viability of Caki-1 cells.
121540.fig.006
Figure 6: Cytotoxicity curves from typical MTT assays showing the effect of compounds 4ad on the viability of Caki-1 cells.

It has been shown that there is no difference between the cytotoxic activities of the two compound classes, since all compounds show a good level of activity. The solubility of all compounds in DMSO was good, and they are stable in saline solution with respect to silver chloride precipitation.

4. Conclusion and Outlook

In summary, a series of seven nonsymmetrically p-cyanobenzyl-substituted NHC-silver(I) acetate complexes 2a–c and 4a–d were synthesised by reacting appropriate nonsymmetrically p-cyanobenzyl-substituted N-heterocyclic carbenes with silver(I) acetate. The preliminary antibacterial activity of the NHC-silver(I) acetate complexes was tested in vitro against two bacterial strains, where complex 4d showed superior activity against Staphylococcus aureus and complex 2b against Escherichia coli. Against the renal cancer cell line Caki-1, the NHC-silver(I) acetate complexes 2ac and 4ad yielded IC50 values of 25 (±1), 15 (±2), 5.4 (±0.8), 16 (±2), 7.1 (±1), 20 (±4), and 14 (±1) μM, respectively. The complex 2c, however, gave a superior IC50 value of 5.4 (±0.8) μM. Further work is currently underway to improve these results by varying the substituents on the imidazole ring in order to enhance stability and on the benzimidazole nitrogen atoms in order to improve biological activity. Overall, this should lead to enhanced solubility, stability, and activity in biological media and enable in vivo testing of a NHC-silver(I) acetate in the nearby future.

Acknowledgment

This project was funded by the UCD School of Chemistry and Chemical Biology.

References

  1. K. M. Hindi, T. J. Siciliano, S. Durmus et al., “Synthesis, stability, and antimicrobial studies of electronically tuned silver acetate N-heterocyclic carbenes,” Journal of Medicinal Chemistry, vol. 51, no. 6, pp. 1577–1583, 2008. View at Publisher · View at Google Scholar · View at Scopus
  2. A. Kascatan-Nebioglu, M. J. Panzner, C. A. Tessier, C. L. Cannon, and W. J. Youngs, “N-Heterocyclic carbene-silver complexes: A new class of antibiotics,” Coordination Chemistry Reviews, vol. 251, no. 5-6, pp. 884–895, 2007. View at Publisher · View at Google Scholar · View at Scopus
  3. A. Kascatan-Nebioglu, A. Melaiye, K. Hindi et al., “Synthesis from caffeine of a mixed N-heterocyclic carbene-silver acetate complex active against resistant respiratory pathogens,” Journal of Medicinal Chemistry, vol. 49, no. 23, pp. 6811–6818, 2006. View at Publisher · View at Google Scholar · View at Scopus
  4. W. J. Youngs, D. A. Medvetz, K. M. Hindi, M. J. Panzner, A. J. Ditto, and Y. H. Yun, “Anticancer activity of Ag(I) N-heterocyclic carbene complexes derived from 4,5-dichloro-1H-imidazole,” Metal-Based Drugs, vol. 2008, Article ID 384010, 2008. View at Publisher · View at Google Scholar
  5. A. Melaiye, R. S. Simons, A. Milsted et al., “Formation of water-soluble pincer silver(I)-carbene complexes: a novel antimicrobial agent,” Journal of Medicinal Chemistry, vol. 47, no. 4, pp. 973–977, 2004. View at Publisher · View at Google Scholar · View at Scopus
  6. S. Ray, R. Mohan, J. K. Singh et al., “Anticancer and antimicrobial metallopharmaceutical agents based on palladium, gold, and silver N-heterocyclic carbene complexes,” Journal of the American Chemical Society, vol. 129, no. 48, pp. 15042–15053, 2007. View at Publisher · View at Google Scholar · View at Scopus
  7. C. L. Cannon, L. A. Hogue, R. K. Vajravelu et al., “In vitro and murine efficacy and toxicity studies of nebulized SCC1, a methylated caffeine-silver(I) complex, for treatment of pulmonary infections,” Antimicrobial Agents and Chemotherapy, vol. 53, no. 8, pp. 3285–3293, 2009. View at Publisher · View at Google Scholar · View at Scopus
  8. S. Roland, C. Jolivalt, T. Cresteil et al., “Investigation of a series of silver-N-Heterocyclic carbenes as antibacterial agents: Activity, synergistic effects, and cytotoxicity,” Chemistry—A European Journal, vol. 17, no. 5, pp. 1442–1446, 2011. View at Publisher · View at Google Scholar
  9. B. Thati, A. Noble, B. S. Creaven et al., “In vitro anti-tumour and cyto-selective effects of coumarin-3-carboxylic acid and three of its hydroxylated derivatives, along with their silver-based complexes, using human epithelial carcinoma cell lines,” Cancer Letters, vol. 248, no. 2, pp. 321–331, 2007. View at Publisher · View at Google Scholar · View at Scopus
  10. H. L. Zhu, X. M. Zhang, X. Y. Liu et al., “Clear Ag-Ag bonds in three silver(I) carboxylate complexes with high cytotoxicity properties,” Inorganic Chemistry Communications, vol. 6, no. 8, pp. 1113–1116, 2003. View at Publisher · View at Google Scholar · View at Scopus
  11. J. J. Liu, P. Galettis, A. Farr et al., “In vitro antitumour and hepatotoxicity profiles of Au(I) and Ag(I) bidentate pyridyl phosphine complexes and relationships to cellular uptake,” Journal of Inorganic Biochemistry, vol. 102, no. 2, pp. 303–310, 2008. View at Publisher · View at Google Scholar · View at Scopus
  12. S. Patil and M. Tacke, “NHC-silver(I) acetates as bioorganometallic anticancer and antibacterial drugs,” in Insights into Coordination, Bioinorganic and Applied Inorganic Chemistry, M. Melník, P. Segľa, and M. Tatarko, Eds., pp. 555–566, Press of Slovak University of Technology, Bratislava, Slovakia, 2011. View at Google Scholar
  13. S. Patil, A. Deally, B. Gleeson et al., “Synthesis, cytotoxicity and antibacterial studies of novel symmetrically and non-symmetrically p-nitrobenzyl-substituted N-heterocyclic carbene-silver(i) acetate complexes,” Zeitschrift fur Anorganische und Allgemeine Chemie, vol. 637, no. 3-4, pp. 386–396, 2011. View at Publisher · View at Google Scholar
  14. S. Patil, A. Deally, B. Gleeson, H. Müller-Bunz, F. Paradisi, and M. Tacke, “Novel benzyl-substituted N-heterocyclic carbene–silver acetate complexes: synthesis, cytotoxicity and antibacterial studies,” Metallomics, vol. 3, no. 1, pp. 74–88, 2011. View at Publisher · View at Google Scholar · View at Scopus
  15. S. Patil, K. Dietrich, A. Deally et al., “Synthesis, cytotoxicity and antibacterial studies of novel symmetrically and nonsymmetrically 4-(methoxycarbonyl)benzyl-substituted n-heterocyclic carbene-silver acetate complexes,” Helvetica Chimica Acta, vol. 93, no. 12, pp. 2347–2364, 2010. View at Publisher · View at Google Scholar · View at Scopus
  16. S. Patil, A. Deally, B. Gleeson, H. Müller-Bunz, F. Paradisi, and M. Tacke, “Synthesis, cytotoxicity and antibacterial studies of symmetrically and non-symmetrically benzyl- or p-cyanobenzyl-substituted N-Heterocyclic carbene—silver complexes,” Applied Organometallic Chemistry, vol. 24, no. 11, pp. 781–793, 2010. View at Publisher · View at Google Scholar · View at Scopus
  17. S. Patil, J. Claffey, A. Deally et al., “Synthesis, cytotoxicity and antibacterial studies of p-methoxybenzyl- substituted and benzyl-substituted n-heterocyclic carbene-silver complexes,” European Journal of Inorganic Chemistry, no. 7, pp. 1020–1031, 2010. View at Publisher · View at Google Scholar · View at Scopus
  18. L. Kaps, B. Biersack, H. Müller-Bunz et al., “Gold(I)-NHC complexes of antitumoral diarylimidazoles: Structures, cellular uptake routes and anticancer activities,” Journal of Inorganic Biochemistry, vol. 106, no. 1, pp. 52–58, 2012. View at Publisher · View at Google Scholar
  19. S. Patil, A. Deally, F. Hackenberg et al., “Novel benzyl- or 4-cyanobenzyl-substituted N-heterocyclic (Bromo)(carbene)silver(I) and (Carbene)(chloro)gold(I) complexes: Synthesis and preliminary cytotoxicity studies,” Helvetica Chimica Acta, vol. 94, no. 9, pp. 1551–1562, 2011. View at Publisher · View at Google Scholar
  20. Agilent Technologies, “Program CrysalisPro Version 1.171.34.49,” 2011.
  21. G. M. Sheldrick, “A short history of SHELX,” Acta Crystallographica Section A, vol. 64, no. 1, pp. 112–122, 2007. View at Publisher · View at Google Scholar · View at Scopus
  22. A. Bondi, H. E. Spaulding, E. D. Smith, and C. C. Dietz, “A routine method for the rapid determination of susceptibility to penicillin and other antibiotics,” The American Journal of the Medical Science, vol. 213, no. 2, pp. 221–225, 1947. View at Google Scholar
  23. S. E. Luria, “Recent advances in bacterial genetics,” Bacteriological Reviews, vol. 11, no. 1, pp. 1–40, 1947. View at Google Scholar · View at Scopus
  24. W. A. Herrmann and C. Kocher, “N-heterocyclic carbenes,” Angewandte Chemie International Edition, vol. 36, no. 20, pp. 2162–2187, 1997. View at Google Scholar
  25. A. J. Arduengo, H. V. Rasika Dias, J. C. Calabrese, and F. Davidson, “Homoleptic carbene-silver(I) and carbene-copper(I) complexes,” Organometallics, vol. 12, no. 9, pp. 3405–3409, 1993. View at Google Scholar · View at Scopus
  26. C. C. Scarborough, B. V. Popp, I. A. Guzei, and S. S. Stahl, “Development of 7-membered N-heterocyclic carbene ligands for transition metals,” Journal of Organometallic Chemistry, vol. 690, no. 24-25, pp. 6143–6155, 2005. View at Publisher · View at Google Scholar · View at Scopus
  27. M. J. Panzner, A. Deeraksa, A. Smith et al., “Synthesis and in vitro efficacy studies of silver carbene complexes on biosafety level 3 bacteria,” European Journal of Inorganic Chemistry, no. 13, pp. 1739–1745, 2009. View at Publisher · View at Google Scholar · View at Scopus
  28. B. Gleeson, J. Claffey, D. Ertler et al., “Novel organotin antibacterial and anticancer drugs,” Polyhedron, vol. 27, no. 18, pp. 3619–3624, 2008. View at Publisher · View at Google Scholar · View at Scopus