Novel benzyl-substituted N-heterocyclic carbene–silver acetate complexes: synthesis, cytotoxicity and antibacterial studies

Abstract

From the reaction of 1-methylimidazole (1a), 4,5-dichloro-1H-imidazole (1b^II) and 1-methylbenzimidazole (1c) with p-cyanobenzyl bromide (2a), non-symmetrically substituted N-heterocyclic carbene (NHC) [(3a–c)] precursors, 5,6-dimethyl-1H-benzimidazole (1d) and 4,5-diphenyl-1H-imidazole (1e) with p-cyanobenzyl bromide (2a) and benzyl bromide (2b), symmetrically substituted N-heterocyclic carbene (NHC) [(3d–f)] precursors were synthesised. These NHC-precursors were then reacted with silver(I) acetate to yield the NHC–silver complexes (1-methyl-3-(4-cyanobenzyl)imidazole-2-ylidene)silver(I)acetate (4a), (4,5-dichloro-1-(4-cyanobenzyl)-3-methyl)imidazole-2-ylidene)silver(I)acetate (4b), (1-methyl-3-(4-cyanobenzyl)benzimidazole-2-ylidene)silver(I)acetate (4c), (1,3-bis(4-cyanobenzyl)5,6-dimethylbenzimidazole-2-ylidene) silver(I) acetate (4d), (1,3-dibenzyl-5,6-dimethylbenzimidazole-2-ylidene) silver(I) acetate (4e) and (1,3-dibenzyl-4,5-diphenylimidazol-2-ylidene) silver(I) acetate (4f) respectively. Three NHC-precursors 3c–e and four NHC–silver complexes 4b and 4d–f were characterised by single crystal X-ray diffraction. Preliminary in vitro antibacterial activity of the NHC-precursors and NHC–silver complexes was investigated against Gram-positive bacteria Staphylococcus aureus, and Gram-negative bacteria Escherichia coli using the qualitative Kirby–Bauer disk-diffusion method. NHC–silver complexes have shown very high antibacterial activity compared to the NHC-precursors. All six NHC–silver complexes were tested for their cytotoxicity through MTT based in vitro tests on the human renal-cancer cell line Caki-1 in order to determine their IC₅₀ values. NHC–silver complexes 4a–f were found to have IC₅₀ values of 6.2 (±1.0), 7.7 (±1.6), 1.2 (±0.6), 10.8 (±1.9), 24.2 (±1.8) and 13.6 (±1.0) μM, respectively. These values represent improved cytotoxicity against Caki-1, most notably for 4c, which is a three times more cytotoxic than cisplatin (IC₅₀ value = 3.3 μM) itself.

---

1. Introduction

N-Heterocyclic carbenes (NHCs) are stable singlet carbenes that can act as excellent two electron donor ligands towards almost any element in the periodic table. They coordinate strongly to late transition metals and heavy main group elements, but are also known to bind to early transition metals and the lanthanoids. Öfele¹ and Wanzlick² successfully synthesised the first N-heterocyclic carbene transition metal complexes of chromium and mercury in 1968. The isolation of the first free carbene by Arduengo in 1991 set the scene for an ever-growing interest and advancement in the field of N-heterocyclic carbene chemistry.³ Shortly thereafter, the use of these ligands in organometallic chemistry, particularly in catalysis dramatically increased.^4,5 Very recently, NHCs found an application in NHC–silver complexes exhibiting antimicrobial activity, in particular for the possible treatment of cystic fibrosis (CF) and chronic lung infections^6–8 and maybe in the treatment of cancer.⁹

Silver salts have enjoyed a long history as antimicrobial agents and have proved to exhibit low toxicity for humans. Thus most of the biomedical studies on NHC–silver complexes have been conducted on their antimicrobial properties. After a thorough search of the literature, it was evident that only a handful of research groups have investigated the antimicrobial and antifungal properties of NHC–silver complexes. Young's 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.^6,8,10 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.¹¹ More recently Young's and colleagues have also shown that in vitro and murine efficacy and toxicity studies of nebulized methylated caffeine–silver(I) complex (SCC1), for treatment of pulmonary infections.¹²

Nowadays, compounds of wide structural diversity are used as therapeutic agents for cancer treatment. The discovery of the antitumour properties of cisplatin by Rosenberg¹³ has proven that not only drugs composed of the basic elements of the periodic table like carbon, oxygen, hydrogen and nitrogen but also those containing heavy elements may be toxic for tumour cells. Cisplatin, along with its second generation analogues, represent the most widely used chemotherapeutic agents.¹⁴ Clinical trials are not only restricted to the platinum element but also extended to ruthenium, iron, and titanium compounds.^15–18

Recently, 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.¹⁹ Zhu has reported that silver carboxylate dimers possess anticancer activity against human carcinoma cells.²⁰ McKeage has shown phosphine complexes of silver to be active anticancer agents, even against cisplatin resistant cell lines.²¹ 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).⁹ These silver complexes have been shown to be very stable and can be synthesized efficiently. We have recently reported the anticancer and antibacterial activity of symmetrically p-methoxybenzyl-substituted and benzyl-substituted N-heterocyclic carbene–silver complexes. All the reported NHC–silver complexes have shown medium to high anticancer and antibacterial activity.²²

Within this paper we present a new series of non-symmetrically and symmetrically p-cyanobenzyl- or benzyl-substituted N-heterocyclic carbene–silver acetate complexes, their synthesis, cytotoxicity and antibacterial studies.

2. Experimental

2.1 General considerations

All the solvents used were of analytical grade and were used without further purification. 4,5-Dichloro-1H-imidazole, 1-methylimidazole, 1-methylbenzimidazole, 5,6-dimethyl-1H-benzimidazole, 4,5-diphenyl-1H-imidazole, p-cyanobenzyl bromide, benzyl bromide, silver acetate, methyl iodide and K₂CO₃ were purchased from Sigma-Aldrich Chemical Company and were used without further purification. NMR spectra were measured on a Varian 400 MHz spectrometer. Chemical shifts are reported in ppm and are referenced to TMS. IR spectra were recorded on a Perkin Elmer Paragon 1000 FT-IR Spectrometer employing a KBr disc. UV–vis spectra were recorded on a Unicam UV4 Spectrometer. Electron spray mass spectrometry (MS) was performed on a quadrupole tandem mass spectrometer (Quattro Micro, Micromass/Water's Corp., USA), using solutions made up in 50% dichloromethane and 50% methanol. MS spectra were obtained in the ES+ (electron spray positive ionisation) mode for compounds 1b, 3a–f and 4a–f. CHN analysis was done with an Exeter Analytical CE-440 Elemental Analyser. Ag was estimated by spectrophotometric method (Atomic absorption spectra 55B Varian), while Cl, Br and I were determined in mercurimetric titrations. Crystal data were collected using an Oxford Diffraction SuperNova diffractometer fitted with an Atlas detector. 3c was measured with Mo-Kα (0.71073 Å), all others with Cu-Kα (1.54184 Å). 3c and 3e were collected at room temperature, all others at 100 K. An at least twice redundant dataset was collected, assuming that the Friedel pairs are not equivalent. For 3c a pseudo-empirical absorption correction based on redundant reflections was performed by the program SADABS.²³ For all others an analytical absorption correction based on the shape of the crystal was performed.²⁴ The structures were solved by direct methods using SHELXS-97²⁵ and refined by full matrix least-squares on F² for all data using SHELXL-97.²⁵ The hydrogen atoms of the water molecules in 3c and 3e were located in the difference fourier map. Their thermal displacement parameters were fixed to 1.5 times the equivalent one of the parent oxygen atom. O–H bond lengths were restrained to be 0.82 Å using DFIX. In 3d the hydrogen attached to C(9) was located in the difference fourier map and allowed to refine freely due to its involvement in hydrogen bonding. All other hydrogen atoms were added at calculated positions and refined using a riding model. Their isotropic temperature factors were fixed to 1.2 times (1.5 times for methyl groups) the equivalent isotropic displacement parameters of the carbon atom the H-atom is attached to. Anisotropic thermal displacement parameters were used for all non-hydrogen atoms. Suitable crystals of 3c–e were formed from the slow evaporation of a saturated methanol solution, while crystals of 4b and 4d–f were grown in a saturated dichloromethane solution with slow infusion of pentane. Further details about the data collection are listed in Table 1, as well as reliability factors.

Table 1 Crystal data and structure refinement for 3c–e, 4b and 4d–f

Identification code	3c	3d	3e	4b	4d	4e	4f
Empirical formula	C16 H16 N3 O Br	C25 H21 N4Br	C23 H25 N2 O Br	C14 H12 N3 O2 Cl2Ag	C27 H23 N4 O2Ag	C25 H25Ag N2 O2	C62 H54 N4 O4Ag2
Molecular formula	[C16 H14 N3]^+ [Br]^− × H2 O	[C25 H21 N4]^+[Br]^−	[C23 H25 N2]^+[Br]^− × H2 O	C14 H12 N3 O2 Cl2Ag	C27 H23 N4 O2Ag	C25 H25Ag N2 O2	C62 H54 N4 O4Ag2
Formula weight	346.23	457.37	425.36	433.04	543.36	493.34	1134.83
Crystal system	Monoclinic	Monoclinic	Triclinic	Monoclinic	Monoclinic	Monoclinic	Monoclinic
Space group	P21/n (#14)	P21/n (#14)	P1̄ (#2)	P21/c (#14)	P2/n (#13)	P 21/c (#14)	C2/c (#15)
Unit cell dimensions/Å (°)	a = 7.0820(2)	a = 7.26900(4)	a = 9.9811(3)	a = 4.40268(4)	a = 15.3823(1)	a = 11.7885(3)	a = 23.9368(7)
	b = 15.9788(3)	b = 12.62118(6)	b = 10.0794(4)	b = 16.7370(2)	b = 7.83949(7)	b = 47.3901(8)	b = 9.5997(2)
	c = 13.7653(3)	c = 23.4502(1)	c = 11.7361(3)	c = 21.1545(2)	c = 19.8044(2)	c = 7.8129(2)	c = 25.2845(7)
	α = 90	α = 90	α = 67.423(3)	α = 90	α = 90	α = 90	α = 90
	β = 90.326(2)	β = 97.0346(5)	β = 82.718(3)	β = 91.612(1)	β = 96.7996(7)	β = 99.281(2)	β = 119.297(4)
	γ = 90	γ = 90	γ = 76.645(3)	γ = 90	γ = 90	γ = 90	γ = 90
Volume/Å^3	1557.68(6)	2135.205(18)	1059.78(6)	1558.21(3)	2371.40(4)	4307.60(17)	5066.9(2)
Z	4	4	2	4	4	8	4
Density/Mg m^−3 (calculated)	1.476	1.423	1.333	1.846	1.522	1.521	1.488
Absorption coefficient/mm^−1	2.641	2.764	2.743	13.624	7.075	7.692	6.625
F(000)	704	936	440	856	1104	2016	2320
Crystal size/mm	0.3 × 0.2 × 0.2	0.1924 × 0.1119 × 0.0597	0.2009 × 0.1821 × 0.1689	0.3880 × 0.0694 × 0.0443	0.1983 × 0.0912 × 0.0700	0.2133 × 0.1538 × 0.1219	0.1491 × 0.0309 × 0.0169
Theta range for data collection	3.47 to 24.73°	3.80 to 76.88°	4.08 to 76.49°	3.37 to 76.32°	3.45 to 70.99°	3.73 to 76.95°	4.01 to 76.89°
Index ranges	−8 ≤ h ≤ 8,	−9 ≤ h ≤ 9,	−12 ≤ h ≤ 12,	−5 ≤ h ≤ 5,	−18 ≤ h ≤ 18,	−14 ≤ h ≤ 14,	−27 ≤ h ≤ 30,
	−18 ≤ k ≤ 18,	−15 ≤ k ≤ 15,	−12 ≤ k ≤ 12,	−20 ≤ k ≤ 20,	−8 ≤ k ≤ 9,	−59 ≤ k ≤ 59,	−12 ≤ k ≤ 12,
	−16 ≤ l ≤ 16	−29 ≤ l ≤ 29	−14 ≤ l ≤ 14	−25 ≤ l ≤ 26	−24 ≤ l ≤ 22	−9 ≤ l ≤ 9	−31 ≤ l ≤ 31
Reflections collected	22628	43690	23699	13661	31155	47192	24441
Independent reflections	2655 [R(int) = 0.0345]	4494 [R(int) = 0.0220]	4420 [R(int) = 0.0617]	3244 [R(int) = 0.0346]	4589 [R(int) = 0.0582]	9032 [R(int) = 0.0288]	5254 [R(int) = 0.0327]
Completeness to θmax	99.60%	99.80%	99.00%	99.00%	100.00%	99.40%	98.20%
Max. and min. Transmission	0.6202 and 0.4613	0.882 and 0.703	0.747 and 0.705	0.607 and 0.129	0.744 and 0.521	0.563 and 0.367	0.905 and 0.608
Data/restraints/parameters	2655/2/197	4494/0/277	4420/2/252	3244/0/201	4589/0/310	9032/0/547	5254/0/326
Goodness-of-fit on F2	1.04	1.061	1.062	1.167	1.085	1.155	1.069
Final R indices [I > 2sigma(I)]	R 1 = 0.0271,	R 1 = 0.0220,	R 1 = 0.0349,	R 1 = 0.0384,	R 1 = 0.0377,	R 1 = 0.0518,	R 1 = 0.0233,
	wR2 = 0.0664	wR2 = 0.0593	wR2 = 0.1016	wR2 = 0.0893	wR2 = 0.1022	wR2 = 0.1010	wR2 = 0.0551
R indices (all data)	R 1 = 0.0317,	R 1 = 0.0235,	R 1 = 0.0366,	R 1 = 0.0393,	R 1 = 0.0406,	R 1 = 0.0533,	R 1 = 0.0273,
	wR2 = 0.0688	wR2 = 0.0601	wR2 = 0.1032	wR2 = 0.0897	wR2 = 0.1048	wR2 = 0.1015	wR2 = 0.0564
Largest diff. peak and hole	0.425 and −0.615 e.Å^−3	0.311 and −0.331 e.Å^−3	0.605 and −0.519 e.Å^−3	2.310 and −0.888 e.Å^−3	1.171 and −0.602 e.Å^−3	3.974 and −2.735 e.Å^−3	0.348 and −0.581 e.Å^−3

---

CCDC 777915 (for 3c), 777917 (for 3d), 777916 (for 3e), 777918 (for 4b), 777919 (for 4d), 777921 (for 4e) and 777920 (for 4f) respectively, contain the supplementary crystallographic data for this paper. These data can be obtained free of charge from the Cambridge Crystallographic Data Centre viawww.ccdc.cam.ac.uk/data_request/cif.

2.2 Synthesis

Synthesis of 1-methyl-3-(4-cyanobenzyl)imidazolium bromide (3a). 4-Cyanobenzyl bromide (0.392 g, 2.00 mmol) was added in one portion to a stirred suspension of 1-methylimidazole (0.158 g, 2.00 mmol) in 30 mL of toluene. The mixture was stirred at room temperature for 3 d. Afterwards the solvent was removed under reduced pressure. The resultant residue was first washed with pentane and then with diethyl ether. Compound 3a (0.450 g, 1.61 mmol, 80.8% yield) was obtained as a colourless solid after drying under reduced pressure.

¹H NMR (δ ppm DMSO-d₆, 400 MHz): 9.27 (s, 1H, NCHN), 7.90 (d, J = 8.2 Hz, 2H, CH_Cyanobenzyl), 7.80 (s, 1H, CH_Imid), 7.74 (s, 1H, CH_Imid), 7.58 (d, J = 8.2 Hz, 2H, CH_Cyanobenzyl), 5.55 (s, 2H, CH₂), 3.85 (s, 3H, N–CH₃).

¹³C NMR (δ ppm DMSO-d₆, 100 MHz, proton decoupled): 140.6, 137.5, 133.3, 129.5, 124.6, 122.8, 118.8, 111.8 (NCN + CN + C_Imid + C_Cyanobenzyl), 51.6 (CH₂), 36.4(CH₃).

IR absorptions (KBr, cm⁻¹): 3368 (s), 3135 (w), 3067 (w), 3018 (s), 2975 (w), 2857 (w), 2230 (s), 1610 (w), 1578 (s), 1535 (s), 1420 (m), 1164 (s), 1020 (w), 824 (w), 761 (m), 652 (w), 622 (m), 550 (s).

UV-Vis (CH₃OH, nm): λ 225 (ε 10512), λ 268 (ε 3846), λ 366 (ε 1191).

MS (m/z, QMS-MS/MS): 198.32 [M⁺–Br].

Micro Analysis Calculated for C₁₂H₁₂N₃Br (278.15): Calcd.: C, 51.87%; H, 4.34%; N, 15.10%; Br, 28.72%; Found: C, 51.76%; H, 4.11%; N, 15.12%; Br, 28.70%.

Synthesis of 4,5-dichloro-1-(4-cyanobenzyl)imidazole (1b^II). 4,5-Dichloro-1H-imidazole (0.273 g, 2.00 mmol) and K₂CO₃ (0.414 g, 3.00 mmol) were stirred for 15 min in 30 mL of acetonitrile. 4-Cyanobenzyl bromide (0.392 g, 2.00 mmol) was added in one portion and stirring was continued at room temperature for further 2 d. After the solvent was removed under reduced pressure, 60 mL of water was added. The aqueous phase was extracted with CH₂Cl₂ (4 × 20 mL). Organic phases were combined and dried over magnesium sulfate. 1b^II was obtained as white solid after solvent removal under reduced pressure (0.407 g, 1.61 mmol, 80.7% yield).

¹H NMR (δ ppm CDCl₃, 400 MHz): 7.68 (d, J = 8.0 Hz, 2H, CH_Cyanobenzyl), 7.46 (s, 1H, NCHN), 7.25 (d, J = 8.0 Hz, 2H, CH_Cyanobenzyl), 5.17 (s, 2H, CH₂).

¹³C NMR (δ ppm CDCl₃, 100 MHz, proton decoupled): 139.6, 134.5, 132.9, 127.6, 127.0, 118.0, 113.6, 112.7 (NCN + CN + CCl + C_Cyanobenzyl), 49.0 (CH₂).

IR absorptions (KBr, cm⁻¹): 3450 (w), 3114 (m), 2230 (s), 1522 (m), 1476 (s), 1388 (m), 1342 (w), 1251 (s), 1174 (w), 1111 (w), 1022 (w), 984 (m), 863 (w), 821 (w), 770 (s), 689 (w), 664 (w), 628 (w), 563 (m).

UV-Vis (CH₃OH, nm): λ 225 (ε 8846), λ 274 (ε 3056), λ 366 (ε 430).

MS (m/z, QMS-MS/MS): 252.10 [M⁺].

Micro Analysis Calculated for C₁₁H₇N₃Cl₂ (252.10): Calcd.: C, 52.41%; H, 2.80%; N, 16.67%; Cl, 28.13%; Found: C, 52.31%; H, 2.79%; N, 16.57%; Cl, 28.10%.

Synthesis of 4,5-dichloro-1-(4-cyanobenzyl)-3-methylimidazolium iodide (3b). Methyl iodide (0.560 mL, 9.00 mmol) was added in one portion to a stirred suspension of 4,5-dichloro-1-(4-cyanobenzyl)imidazole (0.252 g, 1.00 mmol) in 30 mL of acetonitrile. The reaction mixture was heated under reflux for 24 h. Afterwards the solvent was removed under reduced pressure. The resultant residue was first washed with pentane and then with diethyl ether and the title compound 3b was isolated as a light yellow solid (0.296 g, 0.751 mmol, 75.1% yield).

¹H NMR (δ ppm DMSO-d₆, 400 MHz): 9.46 (s, 1H, NCHN), 7.93 (d, J = 8.0 Hz, 2H, CH_Cyanobenzyl), 7.57 (d, J = 8.0 Hz, 2H, CH_Cyanobenzyl), 5.63 (s, 2H, CH₂), 3.84 (s, 3H, N–CH₃).

¹³C NMR (δ ppm DMSO-d₆, 100 MHz, proton decoupled): 138.7, 137.5, 133.3, 129.2, 120.5, 118.8, 118.6, 112.0 (NCN + CN + CCl + C_Cyanobenzyl), 51.0 (CH₂), 35.6 (CH₃).

IR absorptions (KBr, cm⁻¹): 3430 (m), 3125 (w), 3046 (s), 2926 (m), 2855 (w), 2233 (s), 1580 (s), 1560 (s), 1509 (m), 1454 (m), 1417 (w), 1346 (s), 1197 (s), 1153 (s), 1029 (w), 877 (m), 820 (m), 603 (m), 553 (s).

UV-Vis (CH₃OH, nm): λ 224 (ε 14989), λ 269 (ε 5345), λ 361 (ε 1665).

MS (m/z, QMS-MS/MS): 267.96 [M⁺–I].

Micro Analysis Calculated for C₁₂H₁₀N₃Cl₂ I (394.04): Calcd.: C, 36.58%; H, 2.56%; N, 10.66%; Cl, 17.99%; I, 32.21%; Found: C, 36.47%; H, 2.48%; N, 10.57%; Cl, 17.98%; I, 32.18%.

Synthesis of 1-methyl-3-(4-cyanobenzyl)benzimidazolium bromide (3c). 4-Cyanobenzyl bromide (1.96 g, 10.0 mmol) was added in one portion to a stirred suspension of 1-methylbenzimidazole (1.32 g, 10.0 mmol) in 50 mL of toluene. The mixture was stirred at room temperature for 4 d. Afterwards the solvent was removed under reduced pressure. The resultant residue was first washed with pentane and then with diethyl ether. Compound 3c (2.50 g, 7.61 mmol, 76.1% yield) was obtained as a colourless solid after drying under reduced pressure.

¹H NMR (δ ppm DMSO-d₆, 400 MHz): 9.88 (s, 1H, NCHN), 8.03 (d, J = 8.2 Hz, 1H, CH_Benzimid), 7.88 (dd, J = 8.1, 3.8 Hz, 3H, CH_Benzimid), 7.70–7.59 (m, 4H, CH_Cyanobenzyl), 5.89 (s, 2H, CH₂), 4.09 (s, 3H, N–CH₃).

¹³C NMR (δ ppm DMSO-d₆, 100 MHz, proton decoupled): 143.7, 139.9, 133.2, 132.4, 131.0, 129.4, 127.1, 127.0, 118.8, 114.2, 113.9, 111.8 (NCN + CN + C_Benzimid + C_Cyanobenzyl), 49.5 (CH₂), 33.9 (CH₃).

R absorptions (KBr, cm⁻¹): 3575 (s), 3358 (s), 3077 (m), 3023 (m), 2233 (s), 1608 (m), 1568 (s), 1460 (m), 1346 (w), 1280 (w), 1226 (w), 1020 (m), 863 (m), 783 (m), 757 (s), 657 (m), 598 (w), 554 (w), 478 (w), 424 (m).

UV-Vis (CH₃OH, nm): λ 229 (ε 9655), λ 269 (ε 6138), λ 373 (ε 1472).

MS (m/z, QMS-MS/MS): 248.39 [M⁺–H₂O–Br].

Micro Analysis Calculated for C₁₆H₁₆N₃OBr (346.23): Calcd.: C, 55.51%; H, 4.66%; N, 12.14%; Br, 23.08%; Found: C, 55.23%; H, 4.51%; N, 12.12%; Br, 23.11%.

Synthesis of 1,3-bis(4-cyanobenzyl)-5,6-dimethylbenzimidazolium bromide (3d). 5,6-Dimethyl-1H-benzimidazole (0.146 g, 1.00 mmol) and K₂CO₃ (0.207 g, 1.50 mmol) were stirred for 15 min in 30 mL of acetonitrile. 4-Cyanobenzylbromide (0.392 g, 2.00 mmol) was added in one portion and stirring was continued at room temperature for further 4 d. After the solvent was removed under reduced pressure 30 mL of water were added. The precipitate was filtered, washed with diethyl ether and dried in suction at room temperature for 4 h to yield (0.350 g, 0.765 mmol, 76.5% yield) 3d as yellow solid.

¹H NMR (δ ppm DMSO-d₆, 400 MHz): 9.93 (s, 1H, NCHN), 7.90 (d, J = 8.0 Hz, 4H, CH_Cyanobenzyl), 7.72 (s, 2H, CH_Benzimid), 7.66 (d, J = 8.0 Hz, 4H, CH_Cyanobenzyl), 5.84 (s, 4H, CH₂), 2.32 (s, 6H, CH₃).

¹³C NMR (δ ppm DMSO-d₆, 100 MHz, proton decoupled): 142.6, 139.9, 137.3, 133.3, 129.9, 129.3, 118.8, 113.7, 111.8 (NCN + CN + C_Benzimid + C_Cyanobenzyl), 49.8 (CH₂), 20.4 (CH₃).

IR absorptions (KBr, cm⁻¹): 3430 (w), 3031 (w), 2951 (s), 2791 (w), 1608 (m), 1567 (s), 1488 (w), 1435 (s), 1413 (m), 1346 (w), 1309 (w), 1233 (w), 1186 (m), 1017 (w), 972 (w), 812 (s), 610 (w), 548 (s), 429 (w).

UV-Vis (CH₃OH, nm): λ 225 (ε 14115), λ 268 (ε 7126), λ 290(ε 5625), λ 362 (ε 1992).

MS (m/z, QMS-MS/MS): 377.14 [M⁺–Br].

Micro Analysis Calculated for C₂₅H₂₁N₄Br (457.37): Calcd.: C, 65.65%; H, 4.63%; N, 12.25%; Br, 17.47%; Found: C, 65.65%; H, 4.62%; N, 12.11%; Br, 17.77%.

Synthesis of 1,3-dibenzyl-5,6-dimethylbenzimidazolium bromide (3e). 5,6-Dimethyl-1H-benzimidazole (0.146 g, 1.00 mmol) and K₂CO₃ (0.207 g, 1.50 mmol) were stirred for 15 min in 30 mL of acetonitrile. Benzyl bromide (0.237 mL, 2.00 mmol) was added in one portion and stirring was continued at room temperature for further 4 d. After the solvent was removed under reduced pressure 30 mL of water were added. The precipitate was filtered, washed with diethyl ether and dried in suction at room temperature for 4 h to yield (0.335 g, 0.822 mmol, 82.2% yield) 3e as light yellow solid.

¹H NMR (δ ppm DMSO-d₆, 400 MHz): 10.09 (s, 1H, NCHN), 7.79 (s, 2H, CH_Benzimid), 7.51 (d, J = 7.1 Hz, 4H, CH_Benzyl), 7.44–7.32 (m, 6H, CH_Benzyl), 5.76 (s, 4H, CH₂), 2.32 (s, 6H, CH₃).

¹³C NMR (δ ppm DMSO-d₆, 100 MHz, proton decoupled): 141.8, 137.0, 134.6, 129.9, 129.4, 129.1, 128.6, 113.8 (NCN + C_Benzimid + C_benzyl), 50.2 (CH₂), 20.4 (CH₃).

IR absorptions (KBr, cm⁻¹): 3435 (s), 3115 (w), 3028 (w), 2965 (s), 1558 (s), 1497 (w), 1454 (s), 1427 (w), 1354 (w), 1225 (w), 1082 (w), 853 (w), 765 (m), 705 (s), 669 (m), 616 (w), 430 (m).

UV-Vis (CH₃OH, nm): λ 229 (ε 4622), λ 280 (ε 3341), λ 352 (ε 904).

MS (m/z, QMS-MS/MS): 327.17 [M⁺–H₂O–Br].

Micro Analysis Calculated for C₂₃H₂₅N₂OBr (425.36): Calcd.: C, 64.94%; H, 5.92%; N, 6.59%; Br, 18.78%; Found: C, 64.82%; H, 5.73%; N, 6.48%; Br, 18.67%.

Synthesis of 1,3-dibenzyl-4,5-diphenylimidazolium bromide (3f). 4,5-Diphenyl-1H-imidazole (0.220 g, 1.00 mmol) and K₂CO₃ (0.207 g, 1.50 mmol) were stirred for 15 min in 30 mL of acetonitrile. Benzyl bromide (0.237 mL, 2.00 mmol) was added in one portion and stirring was continued at room temperature for further 4 d. After the solvent was removed under reduced pressure 30 mL of water were added. The white precipitate was filtered, washed with diethyl ether to remove unreacted benzyl bromide and dried in suction at room temperature for 4 h to afforded the colourless crystalline product (0.327 g, 0.679 mmol, 67.9% yield) 3f.

¹H NMR (δ ppm CDCl₃, 400 MHz): 11.05 (s, 1H, NCHN), 7.56–6.89 (m, 20H, CH_Imid + CH_Benzyl), 5.44 (s, 4H, CH₂).

¹³C NMR (δ ppm CDCl₃, 100 MHz, proton decoupled): 137.2, 133.2, 132.1, 130.7, 130.4, 129.0, 129.0, 128.8, 128.4, 124.5 (NCN + C_Imid + C_Benzyl), 51.4 (CH₂).

IR absorptions (KBr, cm⁻¹): 3421 (m), 3030 (m), 2926 (m), 2855 (w), 1558 (m), 1497 (w), 1452 (s), 1352 (w), 1211 (w), 1178 (m), 1071 (w), 1024 (m), 755 (m), 698 (s), 645 (w), 514 (w), 462 (w).

UV-Vis (CH₃OH, nm): λ 237 (ε 17199), λ 255 (ε 11660), λ 299 (ε 3678).

MS (m/z, QMS-MS/MS): 401.53 [M⁺–Br].

Micro Analysis Calculated for C₂₉H₂₅N₂Br (481.43): Calcd.: C, 72.35%; H, 5.23%; N, 5.82%; Br, 16.60%; Found: C, 72.26%; H, 5.29%; N, 5.79%; Br, 16.58%.

Synthesis of (1-methyl-3-(4-cyanobenzyl)imidazole-2-ylidene) silver(I) acetate (4a). 1-Methyl-3-(4-cyanobenzyl)imidazolium bromide (0.278 g, 1.00 mmol) was dissolved in dichloromethane (30 mL) and silver acetate (0.333 g, 2.00 mmol) was added. The mixture was stirred at room temperature for 2 d. The pale yellow precipitate, presumably AgBr, was filtered and a clear solution was obtained. The volatile components were removed in vacuo to produce an off-white sticky solid. The solid was washed with diethyl ether and dried under reduced pressure to yield a white solid (0.260 g, 0.713 mmol, 71.4% yield) 4a.

¹H NMR (δ ppm DMSO-d₆, 400 MHz): 7.81 (d, J = 8.4 Hz, 2H, CH_Cyanobenzyl), 7.54–7.44 (m, 4H, CH_Imid + CH_Cyanobenzyl), 5.40 (s, 2H, CH₂), 3.75 (s, 3H, N–CH₃), 1.81 (s, 3H, COCH₃).

¹³C NMR (δ ppm DMSO-d₆, 100 MHz, proton decoupled): 179.2 (NCN), 174.7 (C=O), 143.2, 133.1, 128.9, 123.9, 122.6, 119.0, 111.1 (CN + C_Imid + C_Cyanobenzyl), 53.9 (CH₂), 38.6 (N–CH₃), 23.0 (COCH₃).

IR absorptions (KBr, cm⁻¹): 3401 (w), 3094 (w), 2926 (w), 2228 (s), 1579 (s), 1406 (s), 1232 (m), 1161 (w), 1019 (w), 920 (w), 824 (w), 771 (m), 739 (m), 647 (m), 550 (m).

UV-Vis (CH₃OH, nm): λ 230 (ε 8032), λ 273 (ε 4699), λ 368 (ε 1689).

MS (m/z, QMS-MS/MS): 305.90 [M⁺–O₂CCH₃].

Micro Analysis Calculated for C₁₄H₁₄N₃O₂Ag (364.15): Calcd.: C, 46.17%; H, 3.87%; N, 11.53%; Ag, 29.62%; Found: C, 46.37%; H, 3.92%; N, 11.41%; Ag, 29.59%.

Synthesis of (4,5-dichloro-1-(4-cyanobenzyl)-3-methyl)imidazole-2-ylidene) silver(I) acetate (4b). 4,5-Dichloro-1-(4-cyanobenzyl)-3-methylimidazolium iodide (0.394 g, 1.00 mmol) was dissolved in methanol (30 mL) and silver acetate (0.333 g, 2.00 mmol) was added. The mixture was stirred at room temperature for 2 d. The light pale yellow precipitate, presumably silver iodide, was filtered and a clear solution was obtained. The volatile components were removed in vacuo to produce an off-white sticky solid. The solid was washed with diethyl ether and dried under reduced pressure for 1 h to yield a white crystalline solid (0.385 g, 0.889 mmol, 88.9% yield) 4b.

¹H NMR (δ ppm DMSO-d₆, 400 MHz): 7.80 (d, J = 8.0 Hz, 2H, CH_Cyanobenzyl), 7.41 (d, J = 8.0 Hz, 2H, CH_Cyanobenzyl), 5.55 (s, 2H, CH₂), 3.80 (s, 3H, N–CH₃), 1.71 (s, 3H, COCH₃).

¹³C NMR (δ ppm DMSO-d₆, 100 MHz, proton decoupled): 181.4 (NCN), 175.0 (C=O), 141.4, 133.1, 128.3, 118.8, 118.5, 116.9, 111.3 (CCl + CN + C_Cyanobenzyl), 53.1 (CH₂), 38.2 (N–CH₃), 24.1(COCH₃).

IR absorptions (KBr, cm⁻¹): 3435 (w), 2925 (s), 2854 (w), 2229 (s), 1579 (s), 1435 (w), 1415 (w), 1385 (m), 1202 (w), 1134 (w), 1083 (w), 1020 (w), 876 (w), 814 (m), 466 (w).

UV-Vis (CH₃OH, nm): λ 234 (ε 10753), λ 271 (ε 5756), λ 366 (ε 2078).

MS (m/z, QMS-MS/MS): 374.89 [M⁺–O₂CCH₃].

Micro Analysis Calculated for C₁₄H₁₂N₃O₂Cl₂Ag (433.04): Calcd.: C, 38.83%; H, 2.79%; N, 9.70%; Cl, 16.37%; Ag, 24.90%; Found: Calcd.: C, 38.37%; H, 2.91%; N, 9.69%; Cl, 16.53%; Ag, 24.61%.

Synthesis of (1-methyl-3-(4-cyanobenzyl)benzimidazole-2-ylidene) silver(I) acetate (4c). 1-Methyl-3-(4-cyanobenzyl)benzimidazolium bromide (0.328 g, 1.00 mmol) was dissolved in methanol (30 mL) and silver acetate (0.333 g, 2.00 mmol) was added. The mixture was stirred at room temperature for 2 d. The pale yellow silver bromide suspension was filtered to give a colourless solution. The volume of the reaction was concentrated in vacuo. Addition of diethyl ether gave an off-white precipitate which was isolated by filtration, washed with diethyl ether and dried in vacuo to yield 4c (0.310 g, 0.739 mmol, 73.9% yield).

¹H NMR (δ ppm DMSO-d₆, 400 MHz): 7.77 (dd, J = 14.9, 8.1 Hz, 3H, CH_Benzimid), 7.67 (d, J = 7.8 Hz, 1H, CH_Benzimid), 7.50–7.37 (m, 4H, CH_Cyanobenzyl), 5.84 (s, 2H, CH₂), 4.07 (s, 3H, N–CH₃), 1.74 (s, 3H, COCH₃).

¹³C NMR (δ ppm DMSO-d₆, 100 MHz, proton decoupled): 181.4(NCN), 174.4(C=O), 142.5, 134.6, 133.5, 133.0, 128.5, 124.6, 124.6, 118.8, 112.7, 112.4, 111.1(CN + C_Benzimid + C_Cyanobenzyl), 51.4(CH₂), 36.1(N–CH₃), 23.8(COCH₃).

IR absorptions (KBr, cm⁻¹): 3435 (w), 3060 (w), 2229 (s), 1570 (s), 1410 (s), 1346 (w), 1194 (w), 1093 (w), 1020 (w), 797 (w), 750 (s), 654 (w), 552 (w), 464 (w).

UV-Vis (CH₃OH, nm): λ 223 (ε 14835), λ 237 (ε 11269), λ 275 (ε 7612), λ 285 (ε 6123).

MS (m/z, QMS-MS/MS): 355.93 [M⁺–O₂CCH₃].

Micro Analysis Calculated for C₁₈H₁₆N₃O₂Ag (414.21): Calcd.: C, 52.19%; H, 3.89%; N, 10.14%; Ag, 26.04%; Found: Calcd.: C, 52.27%; H, 4.01%; N, 10.29%; Ag, 25.98%.

Synthesis of (1,3-bis(4-cyanobenzyl)-5,6-dimethylbenzimidazole-2-ylidene) silver(I) acetate (4d). 1,3-Bis(4-cyanobenzyl)-5,6-dimethylbenzimidazolium bromide (0.456 g, 1.00 mmol) was dissolved in dichloromethane (40 mL) and silver acetate (0.333 g, 2.00 mmol) was added. The mixture was stirred at room temperature for 2 d. The pale yellow precipitate, presumably AgBr, was filtered and a clear solution was obtained. The volatile components were removed in vacuo to produce a light yellow sticky solid. The sticky solid was washed with diethyl ether and dried under reduced pressure for 1 h to yield a light yellow crystalline solid (0.440 g, 0.809 mmol, 80.9% yield) 4d.

¹H NMR (δ ppm DMSO-d₆, 400 MHz): 7.71 (d, J = 8.0 Hz, 4H, CH_Cyanobenzyl), 7.40 (d, J = 8.0 Hz, 4H, CH_Cyanobenzyl), 7.38 (s, 2H, CH_Benzimid), 5.72 (s, 4H, CH₂), 2.21 (s, 6H, CH₃), 2.05 (s, 3H, COCH₃).

¹³C NMR (δ ppm DMSO-d₆, 100 MHz, proton decoupled): 181.7(NCN), 176.3(C=O), 142.2, 134.3, 133.1, 132.3, 128.3, 118.9, 112.5, 111.1(CN + C_Benzimid + C_Cyanobenzyl), 51.6(CH₂), 31.0(CH₃), 20.1(COCH₃).

IR absorptions (KBr, cm⁻¹): 3436 (s), 3056 (w), 2924 (m), 2855 (m), 2230 (s), 1609 (m), 1577 (s), 1402 (s), 1184 (w), 1019 (m), 848 (w), 814 (w), 678 (w), 548 (m).

UV-Vis (CH₃OH, nm): λ 229 (ε 9120), λ 288 (ε 4110), λ 362 (ε 1546).

MS (m/z, QMS-MS/MS): 484.03 [M⁺–O₂CCH₃].

Micro Analysis Calculated for C₂₇H₂₃N₄O₂Ag (543.37): Calcd.: C, 59.68%; H, 4.26%; N, 10.31%; Ag, 19.85%; Found: C, 59.44%; H, 4.18%; N, 10.09%; Ag, 20.08%.

Synthesis of (1,3-dibenzyl-5,6-dimethylbenzimidazole-2-ylidene) silver(I) acetate (4e). 1,3-Dibenzyl-5,6-dimethylbenzimidazolium bromide (0.407 g, 1.0 mmol) was dissolved in CH₂Cl₂ (60 mL) and silver acetate (0.333 g, 2.0 mmol) was added. The mixture was stirred at room temperature for 2 d. The yellow silver bromide suspension was filtered to give a colorless solution. The volume of the reaction was concentrated in vacuo. Addition of diethyl ether gave an off-white precipitate which was isolated by filtration, washed with diethyl ether and dried in vacuo to yield 4e (0.390 g, 0.790 mmol, 79.0% yield).

¹H NMR (δ ppm DMSO-d₆, 400 MHz): 7.45 (s, 2H, CH_Benzimid), 7.39–7.24 (m, 10H, CH_Benzyl), 5.65 (s, 4H, CH₂), 2.25 (s, 6H, CH₃), 1.77 (s, 3H, COCH₃).

¹³C NMR (δ ppm DMSO-d₆, 100 MHz, proton decoupled): 185.9 (NCN), 176.1 (C=O), 136.8, 133.6, 132.4, 129.2, 128.3, 127.7, 112.7 (C_Benzimid + C_benzyl), 52.2 (CH₂), 23.8(COCH₃), 20.2 (CH₃).

IR absorptions (KBr, cm⁻¹): 3437 (s), 2923 (m), 1627 (w), 1577 (s), 1486 (w), 1448 (w), 1403 (s), 1337 (w), 1184 (w), 1024 (w), 845 (w), 707 (s).

UV-Vis (CH₃OH, nm): λ 284 (ε 7734), λ 360 (ε 2778), λ 438 (ε 2190).

MS (m/z, QMS-MS/MS): 434.72 [M⁺–O₂CCH₃].

Micro Analysis Calculated for C₂₅H₂₅N₂O₂Ag (493.35): Calcd.: C, 60.86%; H, 5.11%; N, 5.68%; Ag, 21.86%; Found: C, 60.72%; H, 5.15%; N, 5.77%; Ag, 21.73%.

Synthesis of (1,3-dibenzyl-4,5-diphenylimidazol-2-ylidene) silver(I) acetate (4f). 1,3-Dibenzyl-4,5-diphenylimidazolium bromide (0.481 g, 1.00 mmol) was dissolved in dichloromethane (50 mL) and silver acetate (0.333 g, 2.00 mmol) was added. The mixture was stirred at room temperature for 3 d. The pale yellow precipitate, presumably AgBr, was filtered and a clear solution was obtained. The volatile components were removed in vacuo to produce an off-white sticky solid. The sticky solid was washed with diethyl ether and dried under reduced pressure for 2 h to yield a white crystalline solid (0.461 g, 0.812 mmol, 81.2% yield) 4f.

¹H NMR (δ ppm CDCl₃, 400 MHz): 7.34–7.17 (m, 12H, CH_Imid + CH_Benzyl), 7.04–6.93 (m, 8H, CH_Benzyl), 5.33 (s, 4H, CH₂), 2.08 (s, 3H, COCH₃).

¹³C NMR (δ ppm CDCl₃, 100 MHz, proton decoupled): 178.9(NCN), 176.3(C=O), 136.1, 132.6, 130.6, 129.1, 128.6, 128.5, 127.9, 127.7, 127.3(C_Imid + C_Benzyl), 53.7(CH₂), 22.8 (COCH₃).

IR absorptions (KBr, cm⁻¹): 3437 (s), 2925 (m), 1630 (w), 1570 (s), 1497 (w), 1445 (m), 1405 (m), 1178 (w), 1021 (m), 923 (w), 875 (w), 759 (m), 732 (m), 700 (s).

UV-Vis (CH₃OH, nm): λ 214 (ε 16958), λ 263 (ε 9939), λ 364 (ε 2919).

MS (m/z, QMS-MS/MS): 508.73 [M⁺–O₂CCH₃].

Micro Analysis Calculated for C₃₁H₂₇N₂O₂Ag (567.43): Calcd.: C, 65.62%; H, 4.80%; N, 4.94%; Ag, 19.01%; Found: C, 65.54%; H, 4.75%; N, 4.63%; Ag, 18.91%.

2.3 Antibacterial studies

Preliminary in vitro antibacterial activity of non-symmetrically substituted and symmetrically substituted N-heterocyclic carbenes and their corresponding silver complexes were screened 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 3a–f and 4a–f, the qualitative Kirby–Bauer disk-diffusion method was applied.²⁶ All bacteria were individually cultured from a single colony in sterile LB medium²⁷ overnight at 37 °C (orbital shaker incubator). All the work carried out was performed under sterile conditions.

For each strain, 70 μL of culture were spread evenly on agar-LB medium. Four 5 mm diameter Whatman paper discs were placed evenly separated on each plate. Two stock solutions (90 : 10 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 (defined as the distance between the edge of the filter paper disc and the beginning of the bacterial growth) for each sample was measured in millimetres.

2.4 Cytotoxicity studies

Preliminary in vitrocell 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. These cell lines were chosen based on their regular and long-lasting growth behaviour, which is similar to the one shown in kidney carcinoma cells. They 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 5000-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 a 200 μL of a solution of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) in medium. The solution consisted of 30 mg of MTT in 30 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. 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

3.1 Synthesis

The synthetic route for non-symmetrically substituted and symmetrically substituted N-heterocyclic carbenes as ligand precursors and their corresponding silver complexes described in this work is given in Schemes 1, 2, 3 and 4. The non-symmetrically substituted NHC precursors 1-methyl-3-(4-cyanobenzyl)imidazolium bromide (3a) and 1-methyl-3-(4-cyanobenzyl)benzimidazolium bromide (3c) were prepared by stirring 1-methylimidazole (1a) and 1-methylbenzimidazole (1c) with p-cyanobenzyl bromide (2a) in toluene at room temperature for 3-4 d with 81% and 76% yields respectively. 4,5-Dichloro-1-(4-cyanobenzyl)imidazole (1b^II) is formed in 81% yield from the deprotonation of 4,5-dichloro-1H-imidazole (1b) with K₂CO₃ and subsequent alkylation with p-cyanobenzyl bromide (2a) in acetonitrile. 4,5-Dichloro-1-(4-cyanobenzyl)-3-methylimidazolium iodide (3b) was prepared by heating of 4,5-dichloro-1-(4-cyanobenzyl)imidazole (1b^II) with iodomethane in acetonitrile for 1d with a yield of 75%. The symmetrically substituted NHCs precursors 1,3-bis(4-cyanobenzyl)-5,6-dimethylbenzimidazolium bromide (3d), 1,3-dibenzyl-5,6-dimethylbenzimidazolium bromide (3e) and 1,3-dibenzyl-4,5-diphenylimidazolium bromide (3f) were prepared by stirring 5,6-dimethyl-1H-benzimidazole (1d) and 4,5-diphenyl-1H-imidazole (1e) with 2 equivalents of p-cyanobenzyl bromide (2a) and benzyl bromide (2b) in the presence of K₂CO₃ as a base in CH₃CN at room temperature for 4 d with 76%, 82% and 68% yields respectively.

Scheme 1 General reaction scheme for the synthesis of non-symmetrically substituted N-heterocyclic carbenes (3a and 3c) and their corresponding N-heterocyclic carbene–silver complexes (4a and 4c).

Scheme 2 General reaction scheme for the synthesis of non-symmetrically substituted N-heterocyclic carbene (3b) and its N-heterocyclic carbene–silver complex (4b).

Scheme 3 General reaction scheme for the synthesis of symmetrically substituted N-heterocyclic carbenes (3d and 3e) and their corresponding N-heterocyclic carbene–silver complexes (4d and 4e).

Scheme 4 General reaction scheme for the synthesis of symmetrically substituted N-heterocyclic carbene (3f) and its N-heterocyclic carbene–silver complex (4f).

The NHC precursors were characterized by spectral (¹H, ¹³C NMR, IR, UV-visible and mass) and elemental analysis studies. Additionally, the solid state structure of the NHC precursors 3c–e were determined by single crystal X-ray diffraction. The ¹H NMR spectra of all NHC precursors 3a–f show a characteristic downfield shift in the range δ = 9.27–11.05 ppm for the NCHN proton attributable to the positive charge of the molecule.^28–30 In addition, their identities have also been confirmed by a base peak for the [M⁺–Br] fragments in their positive mode ESI mass spectra.

The NHC–silver complexes (1-methyl-3-(4-cyanobenzyl)imidazole-2-ylidene) silver(I) acetate (4a), (4,5-dichloro-1-(4-cyanobenzyl)-3-methyl)imidazole-2 ylidene) silver(I) acetate (4b), (1-methyl-3-(4-cyanobenzyl)benzimidazole-2-ylidene) silver(I) acetate (4c), (1,3-bis(4-cyanobenzyl)5,6-dimethylbenzimidazole-2-ylidene) silver(I) acetate (4d), (1,3-dibenzyl-5,6-dimethylbenzimidazole-2-ylidene) silver(I) acetate (4e) and (1,3-dibenzyl-4,5-diphenylimidazol-2-ylidene) silver(I) acetate (4f) were synthesized by the reaction of 3a–f with 2 equivalents of silver acetate in dichloromethane. The reaction mixture was stirred for 1–3 d at room temperature or refluxed for 2–4 d to afford the NHC–silver acetate complexes as off white solid in 23% to 79% yield. The complexes were characterized by spectral (¹H, ¹³C NMR, IR, UV-visible and mass) and elemental analysis studies. Furthermore, the solid state structures of 4b and 4d–f were analysed by single crystal X-ray diffraction. The absence of a downfield NCHN signal and presence of new signals at 2.08–1.70 ppm for the acetate protons in all the ¹H NMR spectra for 4a–f however, indicates a successful complex formation. The ¹³C NMR resonances of the carbene carbon atoms in complexes 4a–f occur in the range δ 181.7–178.9 ppm respectively. These signals are shifted downfield compared to the corresponding precursors of 3a–fcarbene carbons resonance at the range 141.8–137.2 ppm respectively which further demonstrates the formation of expected NHC–silver acetate complexes. Also the appearance of the ¹³C NMR resonances for the carbonyl and methyl carbons of the acetate group of complexes 4a–f in the range 176.3–173.6 and 24.1–20.1 ppm respectively showed the formation of the NHC–silver complexes.^6,8 Furthermore, positive mode ESI mass spectra of all six NHC–silver complexes (4a–f) are dominated by [M⁺–O₂CCH₃] fragment peaks arising from the loss of one acetate ligand.

3.2 Structural discussion

Suitable crystals for X-ray crystallography to determine the molecular structure of 3c–e were grown from the slow evaporation of a saturated methanol solution, while crystals of 4b and 4d–f were formed in a saturated dichloromethane solution with slow infusion of pentane. Compounds 3c, 3d, 4b and 4d–f crystallised in the monoclinic space groupP2₁/n (#14), P2₁/c (#14), P2/n (#13), P2₁/c (#14) and C2/c (#15) respectively while compound 3e crystallised in the triclinic space groupP1̄(#2). Compounds 3e and 4e crystallised with 2 and 8 molecules in the unit cell, whilst 3c, 3d, 4b, 4d and 4f crystallised with 4 molecules in the unit cell. Compounds 3c and 3e have a lattice held water molecules in the unit cell of the determined structure. In compounds 3d, 4b and 4d–f there is an absence of any lattice held water molecules or organic solvent molecules in the unit cells of the determined structures. The molecular structures of the compounds 3c–e, 4b and 4d–f are shown in Fig. 1–8. The crystal data and refinement details for all seven compounds are tabulated in Table 1, whereas selected bond lengths and bond angles are compiled in Table 2.

Fig. 1 X-Ray diffraction structure of 3c; molecule; thermal ellipsoids are drawn on the 15% probability level.

Fig. 2 X-Ray diffraction structure of 3d; molecule; thermal ellipsoids are drawn on the 15% probability level.

Fig. 3 X-Ray diffraction structure of 3e; molecule; thermal ellipsoids are drawn on the 15% probability level.

Fig. 4 X-Ray diffraction structure of 4b; molecule; thermal ellipsoids are drawn on the 50% probability level.

Fig. 5 X-Ray diffraction structure of 4d; molecule; thermal ellipsoids are drawn on the 50% probability level.

Fig. 6 X-Ray diffraction structure of 4e; molecule; thermal ellipsoids are drawn on the 50% probability level.

Fig. 7 X-Ray diffraction structure of 4f; molecule; thermal ellipsoids are drawn on the 50% probability level.

Fig. 8 Dimer held together by Ag⋯Ag interaction of 4f; thermal ellipsoids are drawn on the 50% probability level, phenyl groups are represented by their ipso carbons only.

Table 2 Selected bond lengths (Å) and angles (°) for compounds 3c–e, 4b and 4d–f

Bond lengths/Å	3c	3d	3e	Bond lengths/Å	4b	4d	4e	4f
N(2)–C(9)	1.330(3)	1.3311(18)		N(2)–C(9)	1.362(5)	1.352(4)		1.398(2)
N(2)–C(10)	1.392(2)	1.3935(17)		N(2)–C(10)	1.383(5)	1.385(4)
C(9)–N(3)	1.323(3)	1.3325(18)		C(9)–N(3)	1.356(5)	1.359(4)
C(11)–N(3)	1.389(3)	1.3950(18)		C(11)–N(3)	1.382(5)
C(10)–C(11)	1.388(3)	1.3923(18)		C(10)–C(11)	1.352(6)
N(1)–C(8)			1.330(2)	C(15)–N(3)		1.393(4)
N(1)–C(9)			1.391(2)	C(10)–C(15)		1.387(4)
C(8)–N(2)			1.330(2)	C(10)–Cl(1)	1.700(4)
C(14)–N(2)			1.394(2)	C(11)–Cl(2)	1.693(4)
C(9)–C(14)			1.388(2)	Ag–C(9)	2.069(4)	2.052(3)
				Ag–O(1)	2.140(3)	2.113(2)		2.1585(12)
				O(1)–C(13)	1.255(5)
				O(2)–C(13)	1.241(5)
				C(13)–C(14)	1.530(6)
				O(1)–C(26)		1.276(4)
				O(2)–C(26)		1.239(4)
				C(26)–C(27)		1.508(5)
				N(1)–C(8)			1.358(5)	1.351(2)
				N(3)–C(33)			1.359(5)
				N(1)–C(10)				1.394(2)
				C(8)–N(2)			1.354(5)	1.348(2)
				C(33)–N(4)			1.361(5)
				C(9)–C(10)				1.363(2)
				Ag–C(8)				2.0896(17)
				O(1)–C(30)				1.285(2)
				O(2)–C(30)				1.242(2)
				C(30)–C(31)				1.514(3)
				N(1)–C(9)			1.395(5)
				C(14)–N(2)			1.394(5)
				C(9)–C(14)			1.391(6)
				Ag(1)–C(8)			2.073(4)
				Ag(1)–O(1)			2.171(3)
				O(1)–C(24)			1.271(5)
				O(2)–C(24)			1.235(6)
				C(24)–C(25)			1.497(6)
				N(3)–C(34)			1.398(5)
				C(39)–N(4)			1.391(5)
				C(34)–C(39)			1.390(6)
				Ag(2)–C(33)			2.073(4)
				Ag(2)–O(3)			2.137(3)
				O(3)–C(49)			1.281(5)
				O(4)–C(49)			1.242(6)
				C(49)–C(50)			1.511(6)

---

Bond angles [°]	3c	3d	3e	Bond angles [°]	4b	4d	4e	4f
N(2)–C(9)–N(3)	110.33(17)	109.81(13)		N(2)–C(9)–N(3)	104.5(3)	105.3(2)
C(9)–N(2)–C(10)	108.24(16)	108.78(11)		C(9)–N(2)–C(10)	110.8(3)	111.6(2)
C(9)–N(3)–C(11)	108.46(16)	108.62(11)		C(9)–N(3)–C(11)	111.2(3)
C(10)–C(11)–N(3)	106.60(16)	106.43(12)		C(10)–C(11)–N(3)	106.6(4)
C(11)–C(10)–N(2)	106.36(17)	106.36(11)		C(11)–C(10)–N(2)	106.9(3)
N(1)–C(8)–N(2)			110.39(15)	C(9)–Ag–O(1)	173.86(14)	170.49(10)
C(8)–N(1)–C(9)			108.20(14)	C(13)–O(1)–Ag	105.5(3)
C(8)–N(2)–C(14)			108.20(14)	C(26)–O(1)–Ag		107.55(19)
C(9)–C(14)–N(2)			106.48(14)	C(9)–N(3)–C(15)		111.1(2)
C(14)–C(9)–N(1)			106.71(13)	N(2)–C(10)–C(15)		106.1(2)
				C(10)–C(15)–N(3)		106.0(2)
				N(2)–C(8)–N(1)			106.0(3)	104.88(14)
				C(8)–N(1)–C(10)				111.51(14)
				C(10)–C(9)–N(2)				106.27(15)
				C(9)–C(10)–N(1)				106.06(15)
				C(8)–N(2)–C(9)				111.28(14)
				C(8)–Ag–O(1)				163.40(6)
				C(30)–O(1)–Ag				107.04(11)
				C(8)–N(2)–C(14)			111.0(3)
				C(9)–C(14)–N(2)			106.1(3)
				C(14)–C(9)–N(1)			106.1(3)
				C(8)–N(1)–C(9)			110.8(3)
				C(8)–Ag(1)–O(1)			165.38(15)
				O(2)–C(24)–O(1)			122.7(4)
				C(33)–N(4)–C(39)			110.8(3)
				C(34)–C(39)–N(4)			106.4(3)
				C(39)–C(34)–N(3)			106.2(3)
				C(33)–N(3)–C(34)			110.7(3)
				C(33)–Ag(2)–O(3)			166.60(14)
				O(4)–C(49)–O(3)			123.8(4)

---

In the five-membered ring (NCNCC) of compounds 3c–e, the bond distances and angles are in good agreement with each other and the bond distances found in the similar compound 1,3-diisopropylbenzimidazolium bromide reported in literature (see Table 2).³¹ The NHC–silver complexes 4b and 4d–f are mononuclear complexes. In the NHC–silver complexes 4b and 4d–f reported here, the bond lengths and angles in and directly around the NHC core agree very well among each other and with literature data.^32–36 Comparing the precursors 3d and 3e with the corresponding complexes 4d and 4e (see Table 2), one finds a slight increase of both the C(9)–N and C(8)–N distances. In the NHC–silver complexes 4b, 4d, 4e and 4fsilver is two-coordinate in an approximately linear fashion. In 4f there is a significant argentophilic interaction with an Ag⋯Ag distance of 2.9107(3) Å (Fig. 7). This is accompanied by a slight increase of the silver–carbene bond length (Ag–C(8) = 2.0896(17) Å), compared to those in the complexes 4b, 4d and 4e (see Table 2).

3.3 Biological evaluation

The in vitro antibacterial activities of NHC-precursors and NHC–silver complexes were tested using the qualitative Kirby-Bauer disk diffusion method. The antibacterial activities of the NHC-precursors and their corresponding NHC–silver complexes are summarized in Fig. 9–12. The results are tabulated in Tables 3 and 4, respectively. The metal salt (silver 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.^22,37 With respect to our previously tested compounds,²² the concentration of the stock solutions is reduced by 4-folds, hence the amount of compound used in each test is significantly less, indicating a remarkably stronger biological activity. Almost no antibacterial activity was observed for compounds 3a–c against the both Gram-positive bacteria Staphylococcus aureus and Gram-negative bacteria Escherichia coli. Medium to high antibacterial activity was observed for compounds 3d–f against Gram-positive bacteria Staphylococcus aureus but no antibacterial activity was observed against Gram-negative bacteria Escherichia coli. The compounds 4a–d have shown medium antibacterial activity against the both Gram-positive bacteria Staphylococcus aureus and Gram-negative bacteria Escherichia coli. The best antibacterial activity was observed for the compounds 4e and 4f against Gram-positive bacteria Staphylococcus aureus with areas of clearance 12 to 15 mm while medium activity was observed towards Gram-negative bacteria Escherichia coli with areas of clearance 6 to 7 mm at the highest amount (0.44 μmol) used (see Tables 3 and 4).

Fig. 9 Area of clearance on Staphylococcus aureus (Gram +ve) by 3a–c and 4a–c.

Fig. 10 Area of clearance on Escherichia coli (Gram −ve) by 3a–c and 4a–c.

Fig. 11 Area of clearance on Staphylococcus aureus (Gram +ve) by 3d–f and 4d–f.

Fig. 12 Area of clearance on Escherichia coli (Gram −ve) by 3d–f and 4d–f.

Table 3 Area of clearance for compounds 3a–f in mm

Compounds		Staphylococcus aureus (Gram +ve)	Escherichia coli (Gram −ve)
3a/mm	0.11 μmol (30.5 μg)	1	1
	0.15 μmol (42.8 μg)	1	1
	0.22 μmol (61.1 μg)	1	1
	0.44 μmol (122.3 μg)	2	1
3b/mm	0.11 μmol (43.3 μg)	1	1
	0.15 μmol (60.6 μg)	1	1
	0.22 μmol (86.6 μg)	1	2
	0.44 μmol (173.3 μg)	1	2
3c/mm	0.11 μmol (36.1 μg)	1	1
	0.15 μmol (50.5 μg)	1	1
	0.22 μmol (72.2 μg)	1	1
	0.44 μmol (144.4 μg)	1	2
3d/mm	0.11 μmol (50.3 μg)	5	1
	0.15 μmol (70.4 μg)	5	1
	0.22 μmol (100.6 μg)	6	2
	0.44 μmol (201.2 μg)	7	2
3e/mm	0.11 μmol (44.8 μg)	6	1
	0.15 μmol (62.7 μg)	6	1
	0.22 μmol (89.6 μg)	10	1
	0.44 μmol (179.2 μg)	10	1
3f/mm	0.11 μmol (52.9 μg)	9	1
	0.15 μmol (74.1 μg)	10	1
	0.22 μmol (105.9 μg)	11	2
	0.44 μmol (211.8 μg)	13	2

---

Table 4 Area of clearance for compounds 4a–f in mm

Compounds		Staphylococcus aureus (Gram +ve)	Escherichia coli (Gram −ve)
4a/mm	0.11 μmol (40.0 μg)	3	5
	0.15 μmol (56.0 μg)	4	5
	0.22 μmol (80.1 μg)	5	6
	0.44 μmol (160.2 μg)	6	7
4b/mm	0.11 μmol (47.6 μg)	4	5
	0.15 μmol (66.6 μg)	5	6
	0.22 μmol (95.2 μg)	5	7
	0.44 μmol (190.5 μg)	6	8
4c/mm	0.11 μmol (45.5 μg)	4	5
	0.15 μmol (63.7 μg)	4	6
	0.22 μmol (91.1 μg)	5	6
	0.44 μmol (182.2 μg)	7	7
4d/mm	0.11 μmol (59.7 μg)	4	5
	0.15 μmol (83.6 μg)	4	6
	0.22 μmol (119.5 μg)	5	6
	0.44 μmol (239.0 μg)	5	7
4e/mm	0.11 μmol (54.2 μg)	6	5
	0.15 μmol (75.9 μg)	9	6
	0.22 μmol (108.5 μg)	10	6
	0.44 μmol (217.0 μg)	12	7
4f/mm	0.11 μmol (62.5 μg)	10	5
	0.15 μmol (87.5 μg)	11	6
	0.22 μmol (125.0 μg)	13	6
	0.44 μmol (250.0 μg)	15	7

---

Based on the above discussions it can be stated that (i) the NHC–silver complexes 4a–f are significantly more active against both Gram-positive bacteria Staphylococcus aureus and Gram-negative bacteria Escherichia coli than the NHC-precursors 3a–f. (ii) It was concluded that, as the NHC-precursors and NHC–silver complexes concentration increases, the antimicrobial activity becomes higher and (iii) It was also observed that, compared to the NHC-precursors the NHC–silver complexes exhibited enhanced antibacterial activity, which is due to the synergistic effect of the increased lipophilicity of the complexes. Chelation decreases the polarity of the metal ion, which further leads to the enhanced lipophilicity of the complex. Since the bacterial cell wall is surrounded by a lipid membrane which favours the passage of lipid soluble materials, increased lipophilicity allows the penetration of complex into and through the membrane and deactivates the active enzyme sites of the microorganisms.³⁸

In comparison with the known reported NHC-precursors and NHC–silver complexes from the literature^6,8,22 the NHC-precursors (3a–f) and their corresponding NHC–silver complexes (4a–f) have remarkably higher antibacterial activity.

3.4 Cytotoxicity studies

We are interested in the different types of NHC–silver complexes as possible anti-cancer drugs. Therefore the in vitro cytotoxicity of non-symmetrically substituted NHC–silver acetate complexes 4a–c and symmetrically substituted NHC–silver acetate complexes 4d–f was evaluated by MTT-based assays³⁹ on the human cancerous renal-cell line Caki-1. This test involves a 48 h drug exposure period, followed by a 24 h recovery time. The log dose response curve for NHC–silver acetate complexes 4a–f can be viewed in Fig. 13 and 14, respectively. Non-symmetrically substituted NHC–silver acetate complexes 4a–c, which contains 1-methylimidazole, 4,5-dichloro-1-(4-cyanobenzyl)imidazole and 1-methylbenzimidazole groups, has an IC₅₀ values 6.2 (±1.0), 7.7 (±1.6) and 1.2 (±0.6) μM, respectively. Compound 4c is the most promising candidate in this paper because of lowest IC₅₀ value and has an approximately five-fold and six-fold increase in magnitude when compared with compounds 4a and 4b respectively. In comparison with cisplatin (IC₅₀ value = 3.3 μM), 4c has an approximately three-fold increase in magnitude. Symmetrically substituted NHC–silver acetate complexes 4d–f, which also contains 5,6-dimethyl-1H-benzimidazole and 4,5-diphenyl-1H-imidazole groups, has an IC₅₀ values 10.8 (±1.9), 24.2 (±1.8) and 13.6 (±1.0) μM, respectively. Compounds 4d and 4f show a very similar IC₅₀ value and two-fold increase in magnitude when compared with 4e and in comparison with cisplatin the IC₅₀ values for the three compounds are not impressive.

Fig. 13 Cytotoxicity curves from typical MTT assays showing the effect of compounds 4a–c on the viability of Caki-1 cells.

Fig. 14 Cytotoxicity curves from typical MTT assays showing the effect of compounds 4d–f on the viability of Caki-1 cells.

Non-symmetrically and symmetrically substituted NHC–silver acetate complexes show almost similar IC₅₀ values; both compound classes are easily soluble in DMSO and all compounds are stable in saline solution with respect to silver chloride precipitation. It was also observed that, compared to known reported NHC–silver complexes from the literature,^9,22 the NHC–silver complexes (4a–f) have shown slightly high cytotoxic activity.

4. Conclusions and outlook

Six new non-symmetrically substituted and symmetrically substituted N-heterocyclic carbene–silver acetate derivatives (4a–f) were synthesised through the reaction of appropriately non-symmetrically substituted and symmetrically substituted N-heterocyclic carbenes (3a–f) with silver acetate. Almost all the complexes have shown high antibacterial activity compared to the precursors against both Gram-positive bacteria Staphylococcus aureus and Gram-negative bacteria Escherichia coli. While screening this novel series we have noted a marked improvement in antibacterial activity with respect to the compounds previously reported.²² We were able to significantly reduce the substrate concentration in the stock solution (from 18.4 μM previously utilised down to 4.4 μM, four-fold) and achieve a greater area of clearance in many cases (from 7 mm with complexes (4,5-dichloro-1,3-bis(4-methoxybenzyl)imidazole-2-ylidene)silver(I)acetate and (1,3-dibenzylimidazole-2-ylidene)silver(I)acetate²² to 15 mm with compound 4f when tested against Staphylococcus aureus for example). We are currently looking at the possibility of determining MIC₅₀ values for the best compounds. NHC–silver complexes 4a–f yielded antitumor IC₅₀ values of 6.2 (±1.0), 7.7 (±1.6), 1.2 (±0.6) and 10.8 (±1.9), 24.2 (±1.8) and 13.6 (±1.0) μM respectively on the Caki-1 cell line. One of the compounds contained in this paper (1-methyl-3-(4-cyanobenzyl)benzimidazole-2-ylidene)silver(I) acetate (4c) shows the highest cytotoxicity [IC₅₀ value of 1.2 (±0.6) μM] up to now for the N-heterocyclic carbene–silver complexes synthesised in our research group, indicating the high potential as an anti-cancer drug. Further work is currently underway in order to improve these values by performing formulation experiments to improve solubility of these NHC-silver acetate complexes, which should allow for in vivo testing of 4c in the nearby future.

Acknowledgements

The authors thank the Irish Research Council for Science Engineering and Technology (IRCSET) for funding through a postdoctoral fellowship for Dr Siddappa Patil.

References

1. K. Öfele, J. Organomet. Chem., 1968, 12, 42.
2. H. W. Wanzlick and H. J. Schönberr, Angew. Chem., Int. Ed. Engl., 1968, 7, 141.
3. A. J. Arduengo, R. L. Harlow and M. Kline, J. Am. Chem. Soc., 1991, 113, 361.
4. W. A. Herrmann, Angew. Chem., Int. Ed., 2002, 41, 1290.
5. D. Bourissou, O. Guerret, F. P. Gabbai and G. Bertrand, Chem. Rev., 2000, 100, 39.
6. K. M. Hindi, T. J. Siciliano, S. Durmus, M. J. Panzner, D. A. Medvetz, D. V. Reddy, L. A. Hogue, C. E. Hovis, J. K. Hilliard, R. J. Mallet, C. A. Tessier, C. L. Cannon and W. J. Youngs, J. Med. Chem., 2008, 51, 1577.
7. A. Kascatan-Nebioglu, M. J. Panzner, C. A. Tessier, C. L. Cannon and W. J. Youngs, Coord. Chem. Rev., 2007, 251, 884.
8. A. Kascatan-Nebioglu, A. Melaiye, K. M. Hindi, S. Durmus, M. J. Panzner, L. A. Hogue, R. J. Mallett, C. E. Hovis, M. Coughenour, S. D. Crosby, A. Milsted, D. L. Ely, C. A. Tessier, C. L. Cannon and W. J. Youngs, J. Med. Chem., 2006, 49, 6811.
9. D. A. Medvetz, K. M. Hindi, M. J. Panzner, A. J. Ditto, Y. H. Yun and W. J. Youngs, Metal-Based Drugs, 2008 , Article ID 384010, 7 pages, http://dx.doi.org/10.1155/2008/384010.
10. A. Melaiye, R. S. Simons, A. Milsted, F. Pingitore, C. Wesdemiotis, C. A. Tessier and W. J. Youngs, J. Med. Chem., 2004, 47, 973.
11. S. Ray, R. Mohan, J. K. Singh, M. K. Samantaray, M. M. Shaikh, D. Panda and P. Ghosh, J. Am. Chem. Soc., 2007, 129, 15042.
12. C. L. Cannon, L. A. Hogue, R. K. Vajravelu, G. H. Capps, A. Ibricevic, K. M. Hindi, A. Kascatan-Nebioglu, M. J. Walter, S. L. Brody and W. J. Youngs, Antimicrob. Agents Chemother., 2009, 53, 3285.
13. B Rosenberg, L. V. Van Camp and T. Krigas, Nature, 1965, 205, 698.
14. E. R. Jamieson and S. J. Lippard, Chem. Rev., 1999, 99, 2467.
15. J. M. Rademaker-Lakhai, D. V. Bongard, D. Pluim, J. H. Beijnen and J. H. M. Schellens, Clin. Cancer Res., 2004, 10, 3717.
16. C. G. Hartinger, M. A. Jakupec, S. Zorbas-Seifried, M. Groessl, A. Egger, W. Berger, H. Zorbas, P. J. Dyson and B. K. Keppler, Chem. Biodiversity, 2008, 5, 2140.
17. A. Vessières, S. Top, W. Beck, E. Hillard and G. Jaouen, Dalton Trans., 2006, 529.
18. P. M. Abeysinghe and M. M. Harding, Dalton Trans., 2007, 3474.
19. B. Thati, A. Noble, B. Creaven, M. Walsh, M. McCann, K. Kavanagh, M. Devereux and D. Egan, Cancer Lett., 2007, 248, 321.
20. H. Zhu, X. Zhang, X. Liu, X. Wang, G. Liu, A. Usman and H. Fun, Inorg. Chem. Commun., 2003, 6, 1113.
21. J. Liu, P. Galetis, A. Farr, L. Maharaj, H. Samarasinha, A. McGechan, B. Baguley, R. Bowen, S. Berners-Price and M. McKeage, J. Inorg. Biochem., 2008, 102, 303.
22. S. Patil, J. Claffey, A. Deally, M. Hogan, B. Gleeson, L. M. M. Méndez, H. Müller- Bunz, F. Paradisi and M. Tacke, Eur. J. Inorg. Chem., 2010, 1020.
23. G. M. Sheldrick, SADABS Version 2.03, University of Göttingen, Germany, 2002.
24. Program CrysalisPro Version 1.171.33.55, Oxford Diffraction Limited, 2010.
25. G. M. Sheldrick, SHELXS-97 and SHELXL-97, University of Göttingen, Germany, 1997.
26. A. Bondi, H. E. Spaulding, E. D. Smith and C. C. Dietz, Am. J. Med. Sci., 1947, 213, 221.
27. S. E. Luria, Bacteriol Rev., 1947, 11, 1.
28. W. A. Herrmann and C. Kocher, Angew. Chem., Int. Ed. Engl., 1997, 36, 2162.
29. A. J. Arduengo, H. V. Rasika-Dias, J. C. Calabrese and F. Davidson, Organometallics, 1993, 12, 3405.
30. J. C. Garrison, C. A. Tessier and W. J. Youngs, J. Organomet. Chem., 2005, 690, 6008.
31. H. V. Huynh, Y. Han, J. H. H. Ho and G. K. Tan, Organometallics, 2006, 25, 3267.
32. P. De Fremont, N. M. Scott, E. D. Stevens, T. Ramnial, O. C. Lightbody, C. L. B. Macdonald, J. A. C. Clyburne, C. D. Abernethy and S. P. Nolan, Organometallics, 2005, 24, 6301.
33. Y. Han, Y. T. Hong and H. V. Huynh, J. Organomet. Chem., 2008, 693, 3159.
34. C. P. Newman, G. J. Clarkson and J. P. Rourke, J. Organomet. Chem., 2007, 692, 4962.
35. V. Lillo, J. Mata, J. Ramirez, E. Peris and E. Fernandez, Organometallics, 2006, 25, 5829.
36. M. Viciano, E. Mas-Marza, M. Sanau and E. Peris, Organometallics, 2006, 25, 3063.
37. B. Gleeson, J. Claffey, D. Ertler, M. Hogan, H. Müller-Bunz, F. Paradisi, D. Wallis and M. Tacke, Polyhedron, 2008, 27, 3619.
38. R. Karvembu, C. Jayabalakrishnan and K. Natarajan, Transition Met. Chem., 2002, 27, 574.
39. T. Mosmann, J. Immunol. Methods, 1983, 65, 55.

---

Footnote

† CCDC reference numbers 777915–777921. For crystallographic data in CIF or other electronic format see DOI: 10.1039/c0mt00034e

---