Novel N-Heterocyclic Carbene Silver(I) Complexes: Synthesis, Structural Characterization, Antimicrobial and Cytotoxicity Potential Studies

Abstract

In this study, a series of unsymmetrical N,N-disubstituted benzimidazolium salts were synthesized as N-heterocyclic carbene (NHC) (2a-2j) and their Ag^I complexes. Their structures were characterized by elemental analyses, ¹H and ¹³C nuclear magnetic resonance (NMR) and infrared (IR) spectroscopy techniques. Further, both the salts and their silver(I)-NHC complexes have been evaluated for their potential antibacterial properties against a panel of bacterial strains namely, Micrococcus luteus, Listeria monocytogenes, Salmonella Typhimurium, Staphylococcus aureus and Pseudomonas aeruginosa. The results show that silver complexes are effective against SalmonellaTyphimurium, Listeria monocytogenes, and Micrococcus luteus with moderate to high activity, and their minimum inhibitory concentrations ranging from 0.0034 to 1.26 mg mL⁻¹. In addition, the benzimidazoles salts 2a-2j and silver-NHC complexes 3a-3j were screened for their antitumor activity. Complex 3e exhibited the highest antitumor effect with half-maximal inhibitory concentration (IC₅₀) value of 4.2 µg mL^-1 against MCF-7.

Keywords:
N-heterocyclic carbene; benzimidazolium salts; silver(I)-NHC complexes; antimicrobial and antitumor activities

Introduction

Organometallic complexes have an important role as design of therapeutic agents in biomedical applications. Organometallic compounds with N-heterocyclic carbene (NHC) ligands have been used in organic transformations and metal-based drugs lately.^1,2 The performance of organometallic compound in medical applications may closely be related to the choice of convenient ligands, and is crucially dependent on the respective trace metal binding forms.³ Heterocyclic azoles, such as imidazole, triazole, and especially benzimidazole have significant biological activities.^4-7 Especially, NHC derived from the imidazole or benzimidazole core have considerable antibacterial or anticancer activities.^8-13 NHCs are also a well-known class of organometallic ligands which are a strong s-donating and weak π-accepting when connected with metal ions. Metal-NHC complexes have exhibited an interesting propensity to bind deoxyribonucleic acid (DNA) by means of non-covalent interactions, which is believed to partially mediate their anti-proliferative activity, and to cleave DNA strands.^14-17 In addition to their antimicrobial and anticancer activity, metal-NHC complexes have also been intensely investigated due to their noteworthy applications in catalysis.^18-21 Silver has lower toxicity as compared to other transition metals. One reason for this could be the slower rate of release of silver ions, from the organometallic complexes, which also enhanced their biological activity. Among the many developed anticancer agents consisting of organometallic compounds, those based on silver-NHC complexes have attracted much attention because of their biological properties such as antimicrobial, anticancer, and antibacterial activities.^22-27 Ag-NHC complexes have been suggested as antimicrobial agents to prevent infections, in wound care antiseptics and in water purification.^28,29 Metallic silver was used as a treatment for gonorrhea before the development of antibiotics, while silver nitrate was commonly used as an antimicrobial compound before the discovery of penicillin in ancient times.³⁰ Silver nitrate is still being used as a preventive drug against the development of neonatal conjunctivitis in infants. The aim of this study was to design novel NHC salts and their Ag^I complexes and investigate their antimicrobial and cytotoxic activities. All salts and complexes structures were characterized by elemental analysis, Fourier transform infrared (FTIR), ¹H and ¹³C nuclear magnetic resonance (NMR) spectroscopies.

Experimental

Materials and methods

All procedures were carried out under an inert atmosphere using standard Schlenk line techniques. Chemicals and solvents were purchased from Sigma-Aldrich (Istanbul, Turkey). The solvents used were purified and dried by MBraun SPS 800 solvent purification system. Column chromatography was performed by using silica gel 60 (70-230 mesh). ¹H and ¹³C NMR spectra were recorded at 400 MHz (¹H), 100 MHz (¹³C) in CDCl₃ with tetramethylsilane as an internal reference. The NMR studies were carried out in high-quality 5 mm NMR tubes. Signals are quoted in parts per million as δ downfield from tetramethylsilane (δ = 0.00) as an internal standard. NMR multiplicities are abbreviated as follows: s = singlet, d = doublet, t = triplet, m = multiplet. Gas chromatography (GC) analyses of reactions mixtures were done using a Shimadzu GC 2010-Plus GCFID system. Column: TeknokromaTRB-5 capillary column, 30 m × 0.32 mm × 0.25 µm. Initial temperature at 50 °C, held for 1 min, ramp 2 °C min⁻¹ until 90 °C, held for 3 min, ramp 40 °C min⁻¹ until 240 °C, held for 10 min. The temperature of the injector and detector were held at 240 °C. Melting points (mp) were determined with Kofler bench at Isste of Borj Cedria (Hammam-Lif, University of Carthage, Borj Cedria, Tunisia).

General procedure for the preparation of benzimidazolium salts

The benzimidazolium salts 2a-2j were prepared by reacting N-(isobutyl)-benzimidazole (1 mmol) (1) with various alkyl chloride (1.1 mmol) in dimethylformamide (DMF; 5 mL) at 80 °C and the resulting mixture was stirred for 24 h. Diethyl ether (15 mL) was added to obtain a white crystalline solid, which was subsequently filtered off. The solid was washed with diethyl ether (3 × 10 mL), dried under vacuum, and the crude product was recrystallized from dichloromethane/diethyl ether (1:3 ratio).

1-(Isobutyl)-3-(3,4,5-trimethoxylbenzyl)benzimidazolium chloride (2a)

Yield: 93%; mp 136.2 °C; IR (KBr) ν / cm⁻¹ 1437; ¹H NMR (400 MHz, CDCl₃) δ 1.01 (d, 6H, CH_3a,b), 2.39 (hept, 1H, H_2’, J4 Hz), 3.78 (s, 6H, CH_3c,e), 3.84 (s, 3H, CH_3d), 4.40 (d, 2H, H_1’, J4 Hz), 5.81 (s, 2H, H_1”), 6.88 (s, 2H, H_3”,7”), 7.57-7.74 (m, 4H, H_4,5,6,7), 12.02 (s, 1H, H₂); ¹³C NMR (100 MHz, CDCl₃) δ 19.9 (C_a,b), 28.9 (C_2’), 51.6 (C_1”), 54.5 (C_1’), 56.7 (C_c,e), 60.9 (C_d), 106.1 (C_3”,7”), 113.2 (C₄), 113.8 (C₇), 127.2 (C₅), 128.6 (C₆), 131.2 (C₈), 131.7 (C₉), 138.6 (C_5”), 144.1 (C_2,2”), 153.9 (C_4”,6”).

1-(Isobutyl)-3-(3,4,5-trimethoxylbenzyl)-5,6-dimethyl benzimidazolium chloride (2b)

Yield: 92%; mp 198.2 °C; IR (KBr) ν / cm⁻¹ 1438; ¹H NMR (400 MHz, CDCl₃) δ 0.98 (d, 6H, CH_3a,b, J8 Hz), 2.34 (hept, 1H, H_2’, J8 Hz), 2.37 (d, 6H, CH_3c,d, J4 Hz), 3.75 (s, 3H, CH_3f), 3.81 (s, 6H, CH_3e,g), 4.30 (d, 2H, H_1’, J8 Hz), 5.71 (s, 2H, H_1”), 6.82 (s, 2H, H_3”,7”), 7.35 (s, 1H, H₄), 7.42 (s, 1H, H₇), 11.80 (s, 1H, H₂); ¹³C NMR (100 MHz, CDCl₃) δ 19.7 (C_c,d), 20.7 (C_a,b), 28.7 (C_2’), 51.2 (C_1”), 54.3 (C_1’), 56.6 (C_e,g), 60.8 (C_f), 105.9 (C_3”,7”), 112.8 (C₄), 113.4 (C₇), 128.8, (C_2”), 129.65 (C₈), 130.1 (C₉), 137.3 (C_5,6), 138.4 (C_5”), 142.7 (C₂), 153.7 (C_4”,6”).

1-(Isobutyl)-3-(4-tert-butylbenzyl)benzimidazolium chloride (2c)

Yield: 97%; mp 217.2 °C; IR (KBr) ν / cm⁻¹ 1542; ¹H NMR (400 MHz, CDCl₃) δ 1.03 (d, 6H, CH_3a,b), 1.23 (s, 9H, CH_3c,d,e), 2.44 (hept, 1H, H_2’, J8 Hz), 4.40 (d, 2H, H_1’, J8 Hz), 5.84 (s, 2H, H_1”), 7.33-7.67 (m, 8H, H_{4,5,6,7,3”,4”,6”,7”}), 12.0 (s, 1H, H₂); ¹³C NMR (100 MHz, CDCl₃) δ 19.3 (C_a,b), 28.6 (C_2’), 30.9 (C_c,d,e), 34.4 (C_8”), 51.8 (C_1”), 54.1 (C_1’), 112.8 (C₄), 113.6 (C₇), 126.0 (C_4”,6”), 126.7 (C₅), 126.8 (C₆), 127.7 (C_3”,7”), 129.6 (C_2”), 130.9 (C₈), 131.5 (C₉), 143.7 (C₂), 152.0 (C_5”).

1-(Isobutyl)-3-(4-tert-butylbenzyl)-5,6-dimethyl benzimidazolium chloride (2d)

Yield: 98%; mp 243.2 °C; IR (KBr) ν / cm⁻¹ 1558; ¹H NMR (400 MHz, CDCl₃) δ 1.01 (d, 6H, CH_3a,b, J8 Hz), 1.23 (s, 9H, CH_3c,d,e), 2.36 (s, 3H, CH_3c), 2.39 (s, 3H, CH_3d), 2.52 (hept, 1H, H_2’, J8 Hz), 4.32 (d, 2H, H_1’, J8 Hz), 5.76 (s, 2H, H_1”), 7.32-7.39 (m, 6H, H_{4,7,3”,4”,6”,7”}), 11.77 (s, 1H, H₂); ¹³C NMR (100 MHz, CDCl₃) δ 19.9 (C_a,b), 20.7 (C_c,d), 28.8 (C_2’), 31.2 (C_e,f,g), 34.7 (C_8”), 50.8 (C_1”), 54.3 (C_1’), 112.8 (C₄), 113.4 (C₇), 126.2 (C_4”,6”), 127.9 (C_3”,7”), 129.8 (C_5,6), 130.2 (C₈), 130.3 (C₉), 137.2 (C_2”), 127.87 (C_8,9), 142.80 (C₂), 152.30 (C_5”).

1-(Isobutyl)-3-(2,4,6-trimethylbenzyl)benzimidazolium chloride (2e)

Yield: 94%; mp 218.3 °C; IR (KBr) ν / cm⁻¹ 1469; ¹H NMR (400 MHz, CDCl₃) δ 1.00 (d, 6H, CH_3a,b, J8 Hz), 2.25 (s, 3H, CH_3d), 2.28 (s, 6H, CH_3c,e), 2.37 (hept, 1H, H_2’, J8 Hz), 4.42 (d, 2H, H_1’, J8 Hz), 5.90 (s, 2H, H_1”), 6.88 (s, 2H, H_4”,6”), 7.10 (d, 1H, H₄, J8 Hz), 7.37 (t, 1H, H₅, J8 Hz), 7.51 (t, 1H, H₆, J8 Hz), 7.64 (d, 1H, H₇, J8 Hz), 11.61 (s, 1H, H₂); ¹³C NMR (100 MHz, CDCl₃) δ 19.8 (C_c,e), 20.3 (C_a,b), 21.1 (C_d), 28.9 (C_2’), 47.5 (C_1”), 54.3 (C_1’), 113.1 (C₄), 114.0 (C₇), 125.2 (C_5,6), 126.9 (C_4”), 127.1 (C_6”), 130.2 (C₈), 131.3 (C₉), 131.8 (C_3”)_, 137.9 (C_5”,7”), 139.6 (C_2”), 144.2 (C₂).

1-(Isobutyl)-3-(4-methylbenzyl)-5,6-dimethyl benzimidazolium chloride (2f)

Yield: 93%; mp 223.2 °C; IR (KBr) ν / cm⁻¹ 1561; ¹H NMR (400 MHz, CDCl₃) δ 1.02 (d, 6H, CH_3a,b, J8 Hz), 2.27 (s, 3H, CH_3e), 2.34 (s, 3H, CH_3c), 2.35 (hept, 1H, H_2’, J4 Hz), 2.38 (s, 3H, CH_3d), 4.31 (d, 2H, H_1’, J8 Hz), 5.70 (s, 2H, H_1”), 7.11 (d, 2H, H_5,6, J8 Hz), 7.30-7.35 (m, 2H, H_{3”,4”,6”,7”}), 11.79 (s, 1H, H₂); ¹³C NMR (100 MHz, CDCl₃) δ 19.8 (C_a,b), 20.7 (C_c,d), 21.2 (C_e), 28.8 (C_2’), 50.9 (C_1”), 54.2 (C_1’), 112.8 (C₄), 113.4 (C₇), 128.18 (C_5,6), 129.7 (C_5”), 129.9 (C_3”,7”), 130.2 (C_4”,6”), 130.2 (C₈), 137.2 (C₉), 139.0 (C_2”), 142.8 (C₂).

1-(Isobutyl)-3-(naphthyl)benzimidazolium chloride (2g)

Yield: 89%; mp 146.4 °C; IR (KBr) ν / cm⁻¹ 1665; ¹H NMR (400 MHz, CDCl₃) δ 1.04 (d, 6H, CH_3a,b, J8 Hz), 2.42 (hept, 1H, H_2’, J8 Hz), 4.41 (d, 2H, H_1’, J8 Hz), 6.08 (s, 2H, H_1”), 7.45-7.99 (m, 11H, H_{4,5,6,7,3”,5”,6”,7”,8”,10”,11”}), 12.15 (s, 1H, H₂); ¹³C NMR (100 MHz, CDCl₃) d 19.9 (C_a,b), 28.9 (C_2’), 51.7 (C_1”), 54.5 (C_1’), 113.1 (C₄), 114.0 (C₇), 125.3 (C_7”), 126.8 (C₅), 127.0 (C₆), 127.1 (C_3”), 127.2 (C_10”), 127.8 (C_5”), 128.2 (C_8”), 129.6 (C_6”), 130.4 (C_11”), 131.2 (C_2”), 131.8 (C_4”), 131.15 (C_9”), 133.1 (C₈), 133.3 (C₉), 144.15 (C₂).

1-(Isobutyl)-3-(anthracen-9-ylmethyl)benzimidazolium chloride (2h)

Yield: 89%; mp 278.9 °C; IR (KBr) ν / cm⁻¹ 1550; ¹H NMR (400 MHz, CDCl₃) δ 1.02 (d, 6H, CH_3a,b, J8 Hz), 2.34 (hept, 1H, H_2’, J8 Hz), 4.37 (d, 2H, H_1’, J4 Hz), 6.95 (s, 2H, H_1”), 7.03-8.54 (m, 13H, H_{4,5,6,7,4”,5”,6”,7”,9”,11”,12”,13”,14”}), 12.27 (s, 1H, H₂); ¹³C NMR (100 MHz, CDCl₃) δ 19.8 (C_a,b), 28.8 (C_2’), 45.6 (C_1”), 54.4 (C_1’), 112.8 (C₄), 114.4 (C₇), 121.8 (C_9”), 123.0 (C_6”,12”), 125.5 (C_5”,13”), 126.6 (C₅), 126.8 (C₆), 128.3 (C_4”,14”), 129.8 (C_7”,11”), 130.7 (C_4”,15”), 131.0 (C_8,9), 131.3 (C_8”,10”), 131.4 (C_10”), 131.7 (C_2”), 144.5 (C₂).

1-(Isobutyl)-3-(methoxyethyl)benzimidazolium chloride (2i)

Yield: 77%; mp 108.5 °C; IR (KBr) ν / cm⁻¹ 1567; ¹H NMR (400 MHz, CDCl₃) δ 1.01 (d, 6H, CH_3a,b, J8 Hz), 2.38 (hept, 1H, H_2’, J8 Hz), 3.30 (s, 3H, CH_3c), 3.91 (t, 3H, H_2”, J4 Hz), 4.38 (d, 2H, H_1’, J8 Hz), 4.87 (t, 2H, H_1”, J8 Hz), 7.58-7.84 (m, 4H, H_4,5,6,7), 11.46 (s, 1H, H₂); ¹³C NMR (100 MHz, CDCl₃) δ 19.8 (C_a,b), 28.9 (C_2’), 47.7 (C_1”), 54.3 (C_c), 59.1 (C_1’), 70.5 (C_2”), 112.8 (C₄), 114.2 (C₇), 126.9 (C_5,6), 131.4 (C₈), 132.1 (C₉), 143.9 (C₂).

1-(Isobutyl)-3-(isobutyl)-5,6-dimethylbenzimidazolium chloride (2j)

Yield: 87%; mp 127.9 °C; IR (KBr) ν / cm⁻¹ 1666; ¹H NMR (400 MHz, CDCl₃) δ 1.01 (d, 12H, CH_3a,b,c,d, J4 Hz), 2.39 (hept, 2H, H_2’,2”, J8 Hz), 2.44 (s, 6H, CH_3e,f), 4.39 (d, 4H, H_1’,1”, J8 Hz), 7.41 (s, 2H, H_4,7), 11.26 (s, 1H, H₂); ¹³C NMR (100 MHz, CDCl₃) δ 19.8 (C_a,b,c,d), 20.7 (C_e,f), 28.9 (C_2’,2”), 54.1 (C_1’,1”), 112.9 (C_4,7), 130.0 (C_8,9), 137.4 (C_5,6), 142.1 (C₂).

General procedure for the preparation of silver(I)-NHC complexes

A solution of benzimidazolium salt (1.0 mmol) and Ag₂O (1.0 mmol) in dichloromethane (15 mL) was stirred at room temperature for 24 h. The reaction mixture was filtered through celite and the solvent removed under reduced pressure. The crude product was recrystallized from dichloromethane/diethyl ether (1:3).

Chloro[1-(isobutyl)-3-(3,4,5-trimethoxylbenzyl)benzimidazole-2-ylidene]silver(I) (3a)

Yield: 75%; mp 136.2 °C; IR (KBr) ν / cm⁻¹ 1567; ¹H NMR (400 MHz, CDCl₃) δ 1.00 (d, 6H, CH_3a,b, J4 Hz), 2.36 (hept, 1H, H_2’, J4 Hz), 3.78 (s, 6H, CH_3c,e), 3.80 (s, 3H, CH_3d), 4.25 (d, 2H, H_1’, J8 Hz), 5.52 (s, 2H, H_1”), 6.50 (s, 2H, H_3”,7”), 7.33-7.49 (m, 4H, H_4,5,6,7); ¹³C NMR (100 MHz, CDCl₃) δ 20.3 (C_a,b), 29.4 (C_2’), 53.5 (C_1”), 56.4 (C_c,e), 56.9 (C_1’), 61.0 (C_d), 104.5 (C_3”,7”), 111.9 (C₄), 112.1 (C₇), 124.4 (C₅), 124.5 (C₆), 130.6 (C_2”), 133.8 (C₈), 134.2 (C₉), 138.2 (C_5”), 153.8 (C_4”,6”); elemental analysis calcd. for C₂₁H₂₆AgClN₂O₃: C 50.67, H 5.26, N 5.62%, found: C 50.1, H 5.4, N 5.8%.

Chloro[1-(isobutyl)-3-(3,4,5-trimethoxylbenzyl)-5,6-dimethyl benzimidazole-2-ylidene]silver(I) (3b)

Yield: 92%; mp 289.5 °C; IR (KBr) ν / cm⁻¹ 1583; ¹H NMR (400 MHz, CDCl₃) δ 0.99 (d, 6H, CH_3a,b, J8 Hz), 2.34 (s, 6H, CH_3c), 2.35 (hept, 1H, H_2’), 2.38 (s, 6H, CH_3d), 3.78 (s, 6H, CH_3e,g), 3.81 (s, 3H, CH_3f), 4.18 (d, 2H, H_1’, J8 Hz), 5.45 (s, 2H, H_1”), 6.45 (s, 2H, H_3”,7”), 7.15 (s, 1H, H₄), 7.21 (s, 1H, H₇); ¹³C NMR (100 MHz, CDCl₃) δ 20.3 (C_c,d), 20.5 (C_a,b), 29.3 (C_2’), 53.3 (C_1”), 56.3 (C_e,g), 56.8 (C_1’), 61.0 (C_f), 104.4 (C_3”,7”), 112.0 (C₄), 112.2 (C₇), 130.9 (C_2”), 132.4 (C₈), 132.7 (C₉), 138.45 (C_5”), 133.8 (C₅), 133.9 (C₆), 138.1 (C₂), 153.8 (C_4”,6”), C₂-Ag: not observed; elemental analysis calcd. for C₂₃H₃₀AgClN₂O₃: C 52.53, H 5.75, N 5.32%, found: C 52.7, H 5.9, N 5.5%.

Chloro[1-(isobutyl)-3-(4-tert-butylbenzyl)benzimidazole-2-ylidene]silver (3c)

Yield: 78%; mp 108.6 °C; IR (KBr) ν / cm⁻¹ 1450; ¹H NMR (400 MHz, CDCl₃) δ 1.02 (d, 6H, CH_3a,b, J8 Hz), 1.27 (s, 9H, CH_3c,d,e), 2.36 (hept, 1H, H_2’, J8 Hz), 4.24 (d, 2H, H_1’, J8 Hz), 5.58 (s, 2H, H_1”), 7.17-7.48 (m, 8H, H_{4,5,6,7,3”,4”,6”,7”}); ¹³C NMR (100 MHz, CDCl₃) δ 20.4 (C_a,b), 29.4 (C_2’), 31.3 (C_c,d,e), 34.7 (C_8”), 53.1 (C_1”), 56.9 (C_1’), 111.7 (C₄), 112.3 (C₇), 124.2 (C_4”), 124.3 (C_6”), 126.1 (C_5,6), 126.9 (C_3”,7”), 131.9 (C_2”), 133.8 (C₈), 134.3 (C₉), 151.7 (C_5”), C₂-Ag: not observed; elemental analysis calcd. for C₂₂H₂₈AgClN₂: C 56.97, H 6.08, N 6.04%, found: C 57.1, H 6.1, N 6.3%.

Chloro[1-(isobutyl)-3-(4-tert-butylbenzyl)-5,6-dimethyl benzimidazole-2-ylidene]silver(I) (3d)

Yield: 79%; mp 118.2 °C; IR (KBr) ν / cm⁻¹ 1450; ¹H NMR (400 MHz, CDCl₃) δ 1.01 (d, 6H, CH_3a,b, J4 Hz), 1.28 (s, 9H, CH_3c,d,e), 2.32 (s, 3H, CH_3c), 2.35 (hept, 1H, H_2’, J8 Hz), 2.37 (s, 3H, CH_3d), 4.16 (d, 2H, H_1’, J8 Hz), 5.51 (s, 2H, H_1”), 7.12-7.34 (m, 6H, H_{4,7,3”,4”,6”,7”}); ¹³C NMR (100 MHz, CDCl₃) δ 20.4 (C_c,d), 20.5 (C_a), 20.6 (C_b), 29.3 (C_2’), 31.3 (C_e,f,g), 34.7 (C_8”), 52.9 (C_1”), 56.8 (C_1’), 111.9 (C₄), 112.4 (C₇), 126.1 (C_4”,6”), 126.7 (C_3”,7”), 132.2 (C_8,9), 132.4 (C₅), 132.8 (C₆), 133.7 (C_2”), 133.8 (C₂), 151.5 (C_5”), C₂-Ag: not observed; elemental analysis calcd. for C₂₄H₃₂AgClN₂: C 58.60, H 6.55, N 5.69%, found: C 58.8, H 6.7, N 5.8%.

Chloro[1-(isobutyl)-3-(2,4,6-trimethylbenzyl)benzimidazole-2-ylidene]silver (3e)

Yield: 74%; mp 194.2 °C; IR (KBr) ν / cm⁻¹ 1467; ¹H NMR (400 MHz, CDCl₃) δ 0.95 (d, 6H, CH_3a,b, J4 Hz), 2.23 (s, 6H, CH_3c,e), 2.30 (hept, 1H, H_2’), 2.34 (s, 3H, CH_3d), 4.19 (d, 2H, H_1’, J8 Hz), 5.49 (s, 2H, H_1”), 7.24-7.47 (m, 6H, H_{4,5,6,7,4”,6”}); ¹³C NMR (100 MHz, CDCl₃) δ 20.4 (C_a,b), 20.5 (C_c,e), 21.3 (C_d), 29.4 (C_2’), 48.0 (C_1”), 57.4 (C_1’), 111.7 (C₄), 111.8 (C₇), 124.1 (C₅), 124.3 (C₆), 126.7 (C_4”,6”), 130.4 (C_5”,2”), 134.2 (C₈), 134.3 (C₉), 137.5 (C_3”), 139.6 (C_7”), C₂-Ag: not observed; elemental analysis calcd. for C₂₁H₂₆AgClN₂: C 56.07, H 5.82, N 6.22%, found: C 56.1, H 5.9, N 6.3%.

Chloro[1-(isobutyl)-3-(4-methylbenzyl)-5,6-dimethyl benzimidazole-2-ylidene]silver (3f)

Yield: 93%; mp 161.2 °C; IR (KBr) ν / cm⁻¹ 1433; ¹H NMR (400 MHz, CDCl₃) δ 1.00 (d, 6H, CH_3a,b, J4 Hz), 2.31 (s, 3H, CH_3c,d), 2.35 (hept, 1H, H_2’, J8 Hz), 2.36 (s, 3H, CH_3e), 4.17 (d, 2H, H_1’, J8 Hz), 5.50 (s, 2H, H_1”), 7.09-7.19 (m, 6H, H_{4,7,3”,4”,6”,7”}); ¹³C NMR (100 MHz, CDCl₃) δ 20.4 (C_c,d), 20.5 (C_a), 20.6 (C_b), 21.2 (C_e), 29.3 (C_2’), 53.1 (C_1”), 56.8 (C_1’), 111.9 (C₄), 112.4 (C₇), 127.0 (C_3”,7”), 129.8 (C_4”,6”), 132.2 (C_2”,5”), 133.7 (C_8,9), 133.8 (C₅), 138.3 (C₆), C₂-Ag: not observed; elemental analysis calcd. for C₂₁H₂₆AgClN₂: C 56.07, H 5.82, N 6.22%, found: C 56.1, H 5.9, N 6.3%.

Chloro[1-(isobutyl)-3-(naphthyl)benzimidazole-2-ylidene]silver(I) (3g)

Yield: 76%; mp 195.4 °C; IR (KBr) ν / cm⁻¹ 1437; ¹H NMR (400 MHz, CDCl₃) δ 1.02 (d, 6H, CH_3a,b, J8 Hz), 2.39 (hept, 1H, H_2’, J8 Hz), 4.27 (d, 2H, H_1’, J8 Hz), 5.77 (s, 2H, H_1”), 7.29-7.80 (m, 11H, H_{4,5,6,7,3”,5”,6”,7”,8”,10”,11”}); ¹³C NMR (100 MHz, CDCl₃) δ 20.4 (C_a,b), 29.5 (C_2’), 53.9 (C_1”), 56.9 (C_1’), 111.8 (C₄), 112.4 (C₇), 124.3 (C₅), 124.4 (C₆), 124.5 (C_7”), 126.5 (C_6”), 126.7 (C_3”), 126.8 (C_10”), 127.9 (C_5”), 128.0 (C_8”), 129.3 (C_11”), 132.3 (C_4”), 133.3 (C_9”), 133.3 (C₈), 133.7 (C₉), 134.4 (C_2”), C₂-Ag: not observed; elemental analysis calcd. for C₂₂H₂₂AgClN₂: C 57.72, H 4.84, N 6.12%, found: C 57.9, H 4.9, N 6.3%.

Chloro[1-(isobutyl)-3-(anthracen-9-ylmethyl)benzimidazole-2-ylidene]silver(I) (3h)

Yield: 74%; mp 252.3 °C; IR (KBr) ν / cm⁻¹ 1450; ¹H NMR (400 MHz, CDCl₃) δ 0.95 (d, 6H, CH_3a,b, J8 Hz), 2.29 (hept, 1H, H_2’, J8 Hz), 4.16 (d, 2H, H_1’, J8 Hz), 6.50 (s, 2H, H_1”), 7.01-7.12 (m, 2H, H_5”,6”), 7.29 (d, 1H, H_4”, J8 Hz), 7.40 (d, 1H, H_7”, J8 Hz), 7.49-7.57 (m, 4H, H_4,5,6,7), 8.10 (d, 2H, H_11”,12”), 8.23 (d, 2H, H_13”,14”, J8 Hz), 8.64 (s, 1H, H_9”); ¹³C NMR (100 MHz, CDCl₃) δ 20.4 (C_a,b), 29.4 (C_2’), 46.9 (C_1”), 57.3 (C_1’), 111.7 (C₄), 112.3 (C₇), 123.0 (C_5,6), 123.3 (C_9”), 124.2 (C_6”,12”), 125.4 (C_5”,13”), 127.8 (C_{4”,14”,7”,11”}), 130.1 (C_3”,15”), 130.5 (C_8”,10”), 131.2 (C_8,9), 131.6 (C_2”), C₂-Ag: not observed; elemental analysis calcd. for C₂₆H₂₄AgClN₂: C 61.49, H 4.76, N 5.51%, found: C 61.6, H 4.9, N 5.8%.

Chloro[1-(isobutyl)-3-(methoxyethyl)benzimidazole-2-ylidene]silver(I) (3i)

Yield: 77%; mp 188.5 °C; IR (KBr) ν / cm⁻¹ 1433; ¹H NMR (400 MHz, CDCl₃) δ 0.99 (d, 6H, CH_3a,b, J4 Hz), 2.33 (hept, 1H, H_2’, J4 Hz), 3.29 (s, 3H, CH_3c), 3.79 (t, 3H, H_2”, J4 Hz), 4.20 (d, 2H, H_1’, J8 Hz), 4.56 (t, 2H, H_1”, J8 Hz), 7.37-7.59 (m, 4H, H_4,5,6,7); ¹³C NMR (100 MHz, CDCl₃) δ 20.4 (C_a,b), 29.4 (C_2’), 49.5 (C_1”), 56.9 (C_c), 59.2 (C_1’), 72.0 (C_2”), 111.5 (C₄), 112.4 (C₇), 124.1 (C_5,6), 133.9 (C₈), 134.5 (C₉), C₂-Ag: not observed; elemental analysis calcd. for C₁₄H₂₀AgClN₂O: C 44.76, H 5.36, N 7.45%, found: C 44.8, H 5.5, N 7.6%.

Chloro[1-(isobutyl)-3-(isobutyl)-5,6-dimethylbenzimidazole-2-ylidene]silver(I) (3j)

Yield: 72%; mp 168.9 °C; IR (KBr) ν / cm⁻¹ 1600; ¹H NMR (400 MHz, CDCl₃) δ 0.97 (d, 12H, CH_3a,b,c,d, J8 Hz), 2.33 (hept, 2H, H_2’,2”, J8 Hz), 2.38 (s, 6H, CH_3e,f), 4.16 (d, 4H, H_1’,1”, J8 Hz), 7.20 (s, 2H, H_4,7); ¹³C NMR (100 MHz, CDCl₃) δ 20.4 (C_a,b,c,d), 20.5 (C_e,f), 29.3 (C_2’,2”), 56.5 (C_1’,1”), 111.9 (C_4,7), 132.6 (C_8,9), 133.6 (C_5,6), 168.7 (C₂); elemental analysis calcd. for C₁₇H₂₆AgClN₂: C 50.82, H 6.52, N 6.97%, found: C 50.9, H 6.9, N 7.2%.

Antibacterial activity

Bacterial strains, media and growth conditions

Bacteria strains used as microorganisms indicator for the antibacterial activity assays were: Micrococcus luteus (M. luteus) LB 14110, Staphylococcus aureus (S. aureus) American Type Culture Collection (ATCC) 6538, Listeria monocytogenes (L. monocytogenes) ATCC 19117, Salmonella Typhimurium (S. Typhimurium) ATCC 14028, Pseudomonas aeruginosa (P. aeruginosa) ATCC 49189 and E. coli were obtained from International Culture Collections (ATCC) and local culture collection of the Laboratory of Microorganisms and Biomolecules of the Centre of Biotechnology of Sfax, Tunisia.

These bacterial strains were grown overnight in Luria-Bertani (LB) agar medium (g L⁻¹): peptone 10; yeast extract 5 and NaCl 5 at pH 7.2 under aerobic conditions and constant agitation (200 rpm) at 30 °C for M. luteus LB14110 and L. monocytogenes ATCC 19117 and at 37 °C for S. aureus ATCC 6538, S. Typhimurium ATCC 14028 and P. aeruginosa ATCC 49189, and then diluted 1:100 in LB media and incubated for 5 h under constant agitation (200 rpm) at the appropriate temperature.

Agar well diffusion method

According to Tagg et al.³¹ with certain adjustments, agar well dissemination technique was employed for the assurance of the antimicrobials activity of synthesized compounds. Growth medium (25 mL) was poured into Petri dishes at 50-70 °C and it was left to solidify under ultraviolet (UV) light (265 nm wavelength) for 15 min. Subsequently, a sterile cotton swab was dipped into overnight bacterial or yeast suspensions of indicator strains (adjusted to turbidity of 0.5 McFarland standard). An agar plate was inoculated by evenly streaking cotton swab over the agar medium. Then wells with a diameter of 8 mm were cut in the medium with a sterile cork borer. Stock solutions of the samples were diluted in sterile distilled water to get 500 µg mL⁻¹ concentrations. The tested samples and controls (100 µL) were dispensed into the wells. The plates were incubated at 37 °C for 24 h. Then the diameters of growth inhibition zones around the wells were measured.

Minimum inhibitory concentration (MIC)

The minimum inhibitory concentration (MIC) of the synthesized compounds was resolved as per rules M7-A6³² and M38-P³³ of National Committee for Clinical Laboratory Standard (NCCLS).³⁴ The test was conducted in clean 96-well microplates with a final volume in each microplate well of 100 µL. The synthesized compounds (20 mg mL⁻¹) were appropriately arranged in solution of dimethyl sulfoxide (DMSO)/water (1/9; v/v). The inhibitory activity of each synthesized compound was transferred to each well to get a two-fold sequential dilution of the original sample and visible growth after incubation. 25 µL of thiazolyl blue tetrazolium bromide (MTT), indicator solution (0.5 mg mL^(-1)) dissolved in clean water were added to the wells and incubated at room temperature for 30 min. This determination was done in triplicate and the results obtained were fundamentally the same. The detailed value is the average of the three tests.

Antioxidant activity: DPPH radical scavenging activity

Scavenging of 2,2-diphenyl-1-picrylhydrazyl radical (DPPH• examine) is the most generally announced technique for screening antioxidant activity. The methodology includes measurement of decrease in absorbance of DPPH at its assimilation maxima of 517 nm. This test determines the scavenging of stable radical species as indicated by the method of Kirby and Schmidt³⁵ with slight alterations. Briefly, synthesized compounds were dissolved in DMSO/water (1/9; v/v) and diluted with ultrapure water at different concentrations (1, 0.5, 0.250, 0.125, 0.0625, 0.03125 mg mL⁻¹). Then, 500 µL of a 4% (m/v) solution of DPPH radical in ethanol was mixed with 500 µL of samples. The mixture was incubated for 30 min in the dark at room temperature. The scavenging capacity was determined spectrophotometrically by monitoring the decrease in absorbance at 517 nm against a blank. The percentage of antiradical activity (ArA) had been calculated as follows:

(1) $\mathrm{ArA}\left(\%\right)=\left[\left(\mathrm{absorbance}\mathrm{of}\mathrm{control}-\mathrm{absorbance}\mathrm{of}\mathrm{test}\mathrm{sample}\right)/\mathrm{absorbance}\mathrm{of}\mathrm{control}\right]\times 100$

Cytotoxicity assay

Cytotoxicity of compounds was evaluated through MTT test on MDA-MB-231 and MCF7 cells as recently did by Mosmann et al.³⁶ To be concise, cells were refined into 96-well plates (Corning, USA) at a thickness of 5 × 10⁵cells for every well in 200 µL medium. After 24 h, cells were treated with various concentrations (10, 5, 2.5, 1 µg mL⁻¹) of compounds and further incubated for 48 h to assess the cytotoxicity of compounds. Toward the finish of the brooding period, 20 µL of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) solution was added to each well. An additional 4 h incubation at 37 °C was done, and then the medium was discarded. MeOH treated cells were utilized as controls. The relative cell viability was determined by reading absorbance at 450 nm with a microplate reader. The mean optical density (OD, absorbance) of treated and control wells was used to calculate the percentage of cell viability as follows:

(2) $\mathrm{Percentage}\mathrm{of}\mathrm{cell}\mathrm{viability}\left(\%\right)=\left(\mathrm{OD}\mathrm{treated}/\mathrm{OD}\mathrm{control}\right)$

Standard plate count

Bacterial number in the samples was determined using the standard plate count method. For each dilution series, the sample assumed to have 102 colony-forming unit (CFU) mL⁻¹ was used to inoculate Difco tryptic soy agar (TSA) plates (Fort Richard, Auckland, New Zealand). This assumption was based on the optical density of the most concentrated bacterial solution. For each plate count, a 100-µL aliquot of the sample was spread evenly onto a TSA plate and incubated at 37 °C overnight. For each tested sample, duplicate plate counts were made.

Colony-forming unit (CFU) is a measure of viable bacterial or fungal cells. CFU can be calculated using Miles method³⁷ and it is useful to determine the microbiological load and magnitude of infection in blood and other samples.

Results and Discussion

Preparation of benzimidazolium salts (2a-2j)

Nitrogen-containing heterocyclic salts have attracted attention in the fields of coordination chemistry due to their metal complexes showing high reactivity as catalyst and biologically agents. In this regards benzimidazolium salts (2a-2j) were synthesized via the two step N-alkylation process as depicted in Scheme 1. Compound 1 was obtained by N-alkylation of benzimidazole with isobutyl bromide (1-bromo-2-methylpropane) in the presence of KOH in DMSO. The benzimidazolium salts (2a-2j) were prepared by reacting N-(isobutyl)-benzimidazole (1) with various aryl halides in DMF at 80 °C for 24 h (Scheme 1). The reaction has been monitored following thin layer chromatography, and after this time the formation of salts (2a-2j) has been observed for every target compound. The benzimidazolium salts (2a-2j) were air- and moisture-stable both in the solid state and in solution.

Scheme 1
Synthesis of benzimidazoles salts 2a-2j.

The FTIR, ¹H and ¹³C NMR spectroscopies, and elemental analysis data of the title compound confirm the proposed structure. NMR spectra of all the compounds were analyzed in d-CDCl₃. In the ¹H NMR spectra, acidic proton (NCHN) for benzimidazolium salts (2a-2j) were seen at 12.02, 11.80, 12.02, 11.77, 11.61, 11.79, 12.15, 12.27, 11.46 and 11.26 ppm, respectively, as a characteristic sharp singlet. In the ¹³C NMR spectra of 2a-2j, the characteristic signals of the imino carbon (NCHN) were detected as typical singlets at 144.1, 142.7, 143.7, 142.8, 144.2, 142.8, 144.1, 144.5, 143.9 and 142.1 ppm, respectively. These values are consistent with related literature.³⁸ The formation of the benzimidazolium salts were also evidenced by their IR spectra, which showed CN bond absorption at 1437, 1438, 1542, 1558, 1469, 1561, 1665, 1550, 1567 and 1666 cm⁻¹ for the respective CN bond vibration of 2a-2j.

Aromatic protons of benzimidazolium salts (2a-2j) were detected in the range of 6.30-8.02 ppm. −CH− protons of isobutyl group on all benzimidazolium salts where seen as heptets in the range of 2.25-2.44 ppm. Methylic protons of isobutyl group on benzimidazolium salts (2a-2j) were resonated between 0.98 and 1.04 as doublets. In the ¹H NMR spectra of 2a-2j, NCH₂CH protons appeared as doublet between 4.30 and 4.42 while NCH₂C protons were detected as typical singlets between 4.87 and 6.95 (Supplementary Information section).

The ¹³C NMR spectra showed aromatic carbons of benzimidazolium salts (2a-2j) in the range of 105.8-153.8 ppm. Terminal carbons of the isobutyl group of all benzimidazolium salts (2a-2j) gave peaks at 19.9, 19.7, 19.3, 19.9, 19.8, 19.8, 19.9, 19.8, 19.8 and 19.8, respectively, while -CH carbons of the isobutyl group were observed at 28.9, 28.7, 28.6, 28.8, 28.9, 28.8, 28.9, 28.8, 28.9 and 28.9, respectively (Supplementary Information section).

The reaction of the benzimidazolium salt with 1 equiv. Ag₂O in dichloromethane resulted in the silver-NHC complex as a crystalline solid. Monitoring of the reaction by ¹H NMR spectroscopy in d-CDCl₃ showed that benzimidazolium salts were completely transformed into silver complexes with moderate yields (72-93%) under argon atmosphere. Synthesis of the Ag^I complexes were conducted in the absence of light and all complexes were stored in the dark (Scheme 2).

Scheme 2
Protocol synthesis of Ag−NHC 3a-3j.

The silver complexes are stable in the air and toward the moisture with good solubility in polar solvents. The successful formation of silver carbene complexes was indicated by the absence of a peak of NCHN proton in their ¹H NMR spectra (Figure 1), further suggested full conversion to silver(I) NHC complexes (3a-3j). In the ¹³C NMR spectra of 2a-2j, the characteristic signals of carbon (NCHN) were seen as singlets between 142.1-144.2 ppm for benzimidazolium salts while for complexes 3a-3j (Figure 2) a signal of carbon (NCN) have shifted greatly downfield region compared to the corresponding benzimidazolium salt (2a-2j) and were observed at 186.7 ppm for complex 3j, however, the rest of carbon signal for the rest of complexes were not observed.

Figure 1
¹H NMR spectra (400 MHz, CDCl₃) of silver complex 3c.

Figure 2
¹³C NMR spectra (100 MHz, CDCl₃) of silver complex 3c.

At the same time, formation of the Ag^I-NHC complexes (3a-3j) was proven by IR spectra, which showed CN bond vibrations at 1567, 1583, 1450, 1450, 1467, 1433, 1437, 1450, 1433, 1600 cm⁻¹, respectively. The analytical data are in good agreement with the compositions proposed for all the new compounds we prepared and are summarized in Table 1.

Table 1
Physical and spectroscopic properties of new benzimidazolium salts 2a-2j and Ag^I complexes 3a-3j

Compound	Molecular formula	Isolated yield / %	mp / ºC	ν(CN) / cm-1	H(2) 1H NMR / ppm	C(2) 13C NMR / ppm
2a	C21H27ClN2O3	93	136.2	1437	12.02	144.1
2b	C23H31ClN2O3	92	198.2	1438	11.80	142.7
2c	C22H29ClN2	97	217.2	1542	12.02	143.7
2d	C24H33ClN2	98	243.2	1558	11.77	142.8
2e	C21H27ClN2	94	218.3	1469	11.61	144.2
2f	C21H27ClN2	93	223.3	1561	11.79	142.8
2g	C22H23ClN2	89	146.4	1665	12.15	144.1
2h	C26H25ClN2	89	278.9	1550	12.27	144.5
2i	C14H21ClN2O	77	108.5	1567	11.46	143.9
2j	C17H27ClN2	87	127.9	1666	11.26	142.1
3a	C21H26AgClN2O3	75	136.2	1567	-	not observed
3b	C23H30AgClN2O3	92	289.5	1583	-	not observed
3c	C22H28AgClN2	78	108.6	1450	-	not observed
3d	C24H32AgClN2	79	118.2	1450	-	not observed
3e	C21H26AgClN2	74	194.2	1467	-	not observed
3f	C21H26AgClN2	93	161.2	1433	-	not observed
3g	C22H22AgClN2	76	195.4	1437	-	not observed
3h	C26H24AgClN2	74	252.3	1450	-	not observed
3i	C14H20AgClN2O	77	188.5	1433	-	not observed
3j	C17H26AgClN2	72	168.9	1600	-	168.7

Asekunowo et al.³⁹ have recently reported the synthesis of a series of mono-carbene silver halides [R₂NHC]-AgCl and have demonstrated the influence of halide ions and the solvent on the structural formulas of Ag^I-NHCs. Similar results have been communicated by Fremontet al.⁴⁰ Ion-pair complexes have been obtained in the reaction of N,N-dimethylimidazoliumiodide with Ag₂O in dichloromethane (DCM).⁴¹ Fluxional behavior between [R₂NHC] AgX and [(R₂NHC)₂ − Ag] + [AgX₂]− species was observed in solution for most of the complexes.⁴² Mechanism of formation of silver N-heterocyclic carbenes was reported by Lin et al.⁴³ in 2007. According to these results, the monomeric compound NHC−Ag−X, in which the two ligands adopt a linear disposition, seems to be the most favorable species in CH₂Cl₂ solution.

Biological activities

All the synthesized benzimidazolium salts (2a-2j) and their corresponding silver(I) complexes (3a-3j) were investigated for antibacterial against the both gram (+)/(−) bacteria. The DMSO did not exhibit any antimicrobial activity as reported earlier.^44,45 The antimicrobial activities of the NHC precursors (2a-2j) and their corresponding silver complexes (3a-3j) are summarized in Table 2.

Table 2
Zone of bacterial inhibition of the synthesized salts 2 and silver complexes 3

Compound	Zone of bacterial inhibition / mm
	Micrococcus luteus (LB 14110)	Listeria monocytogenes (ATCC 19117)	Salmonella Typhimurium (ATCC 14028)	Staphylococcus aureus (ATCC 6538)	Pseudomonas aeruginosa
3a	21 ± 0.6	15 ± 0.5	19 ± 0.54	17 ± 0.25	17 ± 0.13
3b	23 ± 0.6	16 ± 0.6	19 ± 0.5	18 ± 0.3	18 ± 0.14
3c	36 ± 0.5	17 ± 0.2	19 ± 0.5	19 ± 0.5	23 ± 0.2
3d	31 ± 0.5	15 ± 0.5	17 ± 0.10	19 ± 0.11	17 ± 0.19
3e	26 ± 0.33	23 ± 0.5	19 ± 0.5	19 ± 0.18	21 ± 0.45
3f	37 ± 0.2	17 ± 0.3	19 ± 0.5	21 ± 0.1	21 ± 0.4
3g	29 ± 0.32	17 ± 0.5	23 ± 0.44	19 ± 0.15	23 ± 0.5
3h	31 ± 0.4	17 ± 0.2	17 ± 0.2	21 ± 0.2	19 ± 0.2
3i	39 ± 0.2	23 ± 0.2	23 ± 0.3	23 ± 0.2	21 ± 0.4
3j	35 ± 0.44	23 ± 0.5	23 ± 0.15	23 ± 0.3	21 ± 0.25
2a	21 ± 0.22	-	22 ± 0.22	18 ± 0.05	18 ± 0.22
2b	19 ± 0.2	20 ± 0.2	16 ± 0.3	20 ± 0.2	18 ± 0.2
2c	17 ± 0.2	18 ± 0.3	18 ± 0.22	16 ± 0.0	16 ± 0.5
2d	23 ± 0.2	17 ± 0.2	15 ± 0.2	21 ± 0.2	17 ± 0.2
2e	19 ± 0.2	19 ± 0.22	19 ± 0.33	19 ± 0.23	19 ± 0.22
2f	31 ± 0.4	23 ± 0.7	31 ± 0.4	26 ± 0.2	19 ± 0.22
2g	23 ± 0.3	17 ± 0.4	23 ± 0.4	19 ± 0.2	19 ± 0.2
2h	11 ± 0.4	15 ± 0.5	14 ± 0.10	15 ± 0.15	17 ± 0.10
2i	33 ± 0.32	-	17 ± 0.15	19 ± 0.1	19 ± 0.15
2j	21 ± 0.4	19 ± 0.5	19 ± 0.24	19 ± 0.5	20 ± 0.16
Fluconazole	24

Compound 3i is more active than compounds 3h and 3c against the Micrococcus luteus LB 14110. The complexes have shown antibacterial activity to different extents, depending on the nature of the ligand. On comparison with the benzimidazolium salts, the silver complexes have shown enhanced activity. The complexes exhibited enhanced antibacterial activity, which is due to the synergistic effect that increases the lipophilicity of the complexes. Observed antibacterial activity of these complexes is comparable to that of previous silver complexes.⁴⁶

MIC determination

The MIC values of tested silver complexes and their starting material against Listeria monocytogenes ATCC 19117, Salmonella Typhimurium ATCC 14028 and Micrococcus luteus are presented in Table 3.

Table 3
Minimal bacterial inhibitory concentration (MIC) of benzimidazoles salts and silver-NHC complexes

Microorganism indicator	Compound	MIC / (mg mL-1)
Listeria monocytogenes ATCC 19117	2h	1.25
	2j	0.635
	3f	0.0058
	ampicillin	0.049
Salmonella Typhimurium ATCC 14028	2h	1.26
	2j	0.041
	3f	0.0034
	ampicillin	0.635
Micrococcus luteus	2h	0.3225
	2j	0.3125
	3f	0.0034
	ampicillin	0.0195

Silver complex 3f showed good activity than that of ampicillin against Micrococcus luteus with an MIC of 0.0034 mg mL⁻¹, while an MIC of 0.0034 mg mL⁻¹ was observed in the case of Salmonella Typhimurium for the silver complex 3f. MICs of other compounds remained within the tested range.

Cytotoxic activities

The benzimidazolium salts and their corresponding Ag^I−NHC complexes were screened colorimetrically using MTT cell assay in two human cancer cell lines, namely MCF7 and MDA-MB-231, to assess their cytotoxicity. The results are presented in Table 4.

Table 4
Anticancer activity of synthesized of benzimidazoles salts 2a-2j and silver-NHC complexes 3a-3j

Compound	Anticancer activity IC50 / (µg mL-1)
	MCF7	MDA-MB-231
3a	4.2 ± 3.5	4.3 ± 3.3
3b	4.1 ± 3.6	2.6 ± 4.3
3c	3.2 ± 3.1	2.7 ± 5.9
3d	1.8 ± 3.1	15 ± 2.8
3e	4.2 ± 1.8	0.0 ± 00
3f	0.69 ± 3.2	1.94 ± 2.6
3g	1.4 ± 4.1	3.4 ± 2.9
3h	2.1 ± 3.2	2.7 ± 2.9
3i	0.63 ± 3.1	1.96 ± 2.5
3j	1.4 ± 4.1	3.5 ± 2.9
2a	2.1 ± 3.2	2.8 ± 2.8
2b	NA	NA
2c	3.2 ± 5.9	6.2 ± 3.2
2d	NA	5.2 ± 3.1
2e	0.6 ± 2.9	3.1 ± 5.9
2f	> 100	> 100
2g	> 100	> 100
2h	> 100	> 100
2i	> 100	> 100
2j	> 100	> 100
Tetracyclinea	NT	NT

• ^a
  The concentration was 30 µg. NA: not active; IC₅₀: half maximal inhibitory concentration; MCF7 and MDA-MB-231: human cancer cell lines; NT: not tested. Values are mean value ± standard deviation of three different replicates.

As shown in Table 4, the cytotoxicity of 3i and 3f was much stronger in MCF7 with half-maximal inhibitory concentration (IC₅₀) values of 0.63 and 0.69 µg mL⁻¹, respectively, as compared to their activity on MDA-MB-231 cells. The cytotoxicity of compound 3j in MCF7 and MDA-MB-231 cells was 1.4 and 3.5 µg mL⁻¹, while the IC₅₀ values of compound 2a were 2.1 and 2.8 µg mL⁻¹ against MCF7 and MDA-MB-231, respectively. The compound2d was inactive against MCF7.

Conclusions

In conclusion, a series of novel unsymmetrically substituted N-heterocyclic carbene precursors and their silver complexes were synthesized and characterized by ¹H NMR, ¹³C NMR, IR and elemental analyses. New silver complexes showed high antibacterial activity compared with the precursors against gram (+)/(−) bacteria and fungi strains. In addition, the silver(I)-NHC complexes 3i and 3f showed good antitumor activity against MDA-MB-231, and MCF-7 cell lines. More studies are now in progress including a larger collection of bacteria of different species in order to determine if the antimicrobial activity is species dependent and its possible application in different fields. New biological active Pt or Au-NHC complexes will be prepared with the aim to develop robust cancer chemotherapeutic agents. Furthermore, we will test the properties of both transmetallation reaction and catalysis of silver complexes described in this work.

Acknowledgments

This work was supported by the Research Supporting Project (RSP2019/75), King Saud University, Riyadh, Saudi Arabia.

Supplementary Information

Supplementary information is available free of charge at http://jbcs.sbq.org.br as PDF file.

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Publication Dates

• Publication in this collection
  09 Oct 2020
• Date of issue
  Oct 2020

History

• Received
  05 Mar 2020
• Accepted
  03 June 2020