Pyrazolyl-functionalised Ag(I)–NHC complexes: synthesis, characterisation, antibacterial activity, and computational investigation

Abstract

In this study, five pyrazolyl-functionalised azolium salts with varying N-substituents (R-group) on the (benz)imidazolium rings, i.e. 3-(2-(3,5-dimethyl-1H-pyrazol-1-yl) ethyl)-1-R-1H-imidazole-3-ium chloride, where R = methyl (1a), ethyl (1b), benzyl (1c) and 3-(2-(3,5-dimethyl-1H-pyrazol-1-yl)ethyl)-1-R-1H-benzo[d]imidazole-3-ium chloride, where R = methyl (1d), ethyl (1e) were synthesised. Corresponding silver(I)–N-heterocyclic carbene complexes 2a–e were also synthesised from the deprotonation of the salts. All the compounds were characterised by spectroscopic and analytical techniques. In addition, all the salts and Ag(I) complexes were utilised as in vitro antibacterial agents against a broad spectrum of bacterial strains. Among the synthesised complexes, compound 2c bearing a benzyl N-substituent demonstrated the highest potency with IC₅₀ values and bacterial inhibition percentages comparable to the standard drug neomycin. Theoretical methods, including DFT calculations, were used to examine electronic effects and SWISSADME to predict enhanced activity for the benzimidazole-containing compounds as compared to those bearing simple imidazole. Also examined is the role of the N-benzyl moiety in enhancing the antibacterial activity of complex 2c.

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1 Introduction

Antimicrobial resistance has emerged as a global health threat, and recent estimates indicate a growing trend in antibiotic resistance, with thousands of deaths reported annually.¹ Alarmingly, antimicrobial resistance appears to be spreading faster than the rate at which new, potent antibiotics are being introduced into clinical use. Alternative methods of combating the rapid drug resistance to microbes include structural modification and optimisation of existing antibiotics to improve the spectrum of activity while maintaining the safety and bioavailability profiles.² The lack of effective antibiotics can substantially compromise numerous medical procedures, including organ transplant surgery, the care of premature infants and the treatment of critically ill patients.³ Since ancient times, metallodrugs have been widely used for therapy and diagnostics of a wide range of ailments.⁴ Silver was historically used for its antibacterial properties until the discovery of antibiotics such as penicillin, which essentially replaced it in therapeutic applications.^5–7

The use of silver has reemerged in recent years owing to the high bioavailability of the Ag(I) cation, which significantly contributes to its antimicrobial activity.⁸ Apart from silver, several other metal-based antimicrobial agents have been reported to show promising efficacy against several microbes. A recent review reported some complexes based on manganese, cobalt, zinc, ruthenium, silver, iridium and platinum that exhibit good to excellent antibacterial activity against selected bacterial strains.⁹ Recent studies reported cationic aggregation-induced emission active Ru(II) and Ir(III) complexes, with the most potent having antibacterial activity in the range of 1–9 µM against the selected Gram-positive strains.^10,11 Moreover, the emergence of antimicrobial resistance has led to a decline in the use of metallic silver and simple silver salts in a variety of medicinal applications.¹²

A key challenge is the loss of activity due to the uncontrolled loss/release of silver cations. Although the mechanism of action of silver cations is not completely established, researchers believe that the Ag(I) cation has a unique affinity for soft bases, such as thiol groups (R-SH), which are found in enzymes or proteins on the bacterial cell membrane. Reports suggest that Ag(I) cations disrupt the bacterial electron transport chain activity, potentially leading to the generation of reactive oxygen species and the depletion of antioxidants in bacterial cells.^13–16 Hence, reducing the uncontrolled release of Ag(I) ions through ligand design and the development of silver compounds with precisely controlled structural variations is necessary. Imidazole and benzimidazole derivatives have shown antimicrobial activity, and studies have shown that such compounds, when complexed to transition metal centres (metallodrugs), often demonstrate superior antimicrobial potency than the free organic molecules.^9,17

Recently, the N-heterocyclic carbene (NHC) ligands, which are five-membered heterocyclic azolium entities commonly based on imidazoles and triazoles, have garnered immense attention in various spheres of synthetic chemistry, including catalysis and biological applications.^2,13 This is due to their structural stability, variability and ease of synthesis in high yields, which is vital in controlling the stability and biological activity of their silver(I) complexes.¹⁸ Additionally, research has shown that fusion with an aromatic benzene ring, such as in benzimidazoles, helps to further enhance the stability of the metal–carbene (M–C) bond.¹⁹ Consequently, many benzimidazole-based Ag(I)–NHC complexes have been reported to be air- and water-stable, vital for applications in biological systems. Hence, they are more widely studied than Ag(I)–NHC complexes of simple imidazolylidene ligands.²⁰ The strong M–C bond allows for the slow release of the silver ions into the biological medium, increasing their residence time in the system and subsequent increase in efficacy.²¹ In addition to the silver metal centre, structural modifications such as incorporating lipophilic groups on the NHC, such as alkyl chains, increase the lipophilicity of the Ag(I) complex, thus resulting in enhanced cell wall penetration.^22,23

As an illustration of the range of activity of Ag(I)–NHC complexes, Bensalah et al. reported minimum inhibitory concentrations (MICs) as low as 10 mmol L⁻¹ against S. aureus and 11 mmol L⁻¹ against MRSA.²⁴ Meanwhile, Mnasri and coworkers reported complexes with antibacterial activities against a broad range of bacterial strains comparable to ampicillin.²⁵ Ghdhayeb et al. reported their complexes to show inhibition zones of 20 and 25 mm against E. coli and S. aureus at 100 and 200 µg mL⁻¹, respectively.²⁶ In addition, several pyrazole derivatives have also been reported to have antibacterial activity comparable to standard drugs.²⁷

To complement experimental approaches, density functional theory (DFT), a widely used quantum-mechanical computational method, provides valuable insights into molecular geometry, electronic distribution, frontier molecular orbitals, and energetics.²⁸ In recent years, DFT has become an indispensable predictive tool used prior to experimental investigations, allowing researchers to model and optimise structures, assess reaction pathways, and evaluate key molecular properties such as binding energies, HOMO–LUMO gaps, and charge distribution.²⁹ These theoretical predictions not only guide experimental design but also serve to rationalise and validate experimental observations, thereby enhancing the reliability and efficiency of structure–activity relationship studies.³⁰ Herein, we report the utilisation of previously unreported imidazolium and benzimidazolium salts and their Ag(I)–NHC complexes as antibacterial agents using a combination of experimental and computational studies.

2 Experimental

2.1 Materials and methods

Reagent-grade acetylacetone, hydrazine hydrate, benzimidazole, 1-ethylimidazole and 1-methylimidazole were purchased from Merck. All reactions were carried out in a dry nitrogen atmosphere using standard Schlenk techniques. Thin-layer chromatography was done using TLC plates coated with silica gel. All solvents were distilled under nitrogen using the appropriate drying agent, stored in solvent reservoirs (containing 4 Å molecular sieves), and purged with nitrogen. ¹H- and ¹³C-NMR were recorded using the 600 MHz Bruker Ultra Shield spectrometer. Chemical shifts for ¹H and ¹³C spectra were recorded in ppm relative to the residual proton of CDCl₃ (¹H: δ 7.24; ¹³C: δ 77.0) and DMSO-d₆ (¹H: δ 2.50; ¹³C: δ 39.5) with tetramethylsilane as the internal standard. The elemental composition of the compounds was analysed using a CHN Thermo Scientific Flash 2000 Elemental Analyser. The FT-IR spectra were recorded using a PerkinElmer Attenuated Total Reflectance (ATR) spectrometer. UV-vis spectra were recorded with a PerkinElmer Lambda 365+ UV/vis spectrophotometer, and the compounds were dissolved in DMSO. Melting point analysis was performed on an MP-200D-HR Stuart high-resolution digital melting point apparatus.

2.2 Synthesis of ligand precursors

2.2.1 Synthesis of 3,5-dimethyl-1H-pyrazole. Hydrazine monohydrate (1.1 equiv.) in THF (2 mL mmol⁻¹) was added to a stirred solution of 2,4-pentanedione in THF (2 mL mmol⁻¹) over a period of 30 min via a syringe, and the reaction mixture was stirred overnight. The reaction mixture was concentrated in vacuo prior to adding water (50 mL). The desired product was extracted by DCM (3 × 50 mL), the combined organic extracts dried over anhydrous MgSO₄ and concentrated in vacuo to give 3,5-dimethyl-1H-pyrazole.³¹

2.2.2 Synthesis of 1-(2-chloroethyl)-3,5-dimethyl-1H-pyrazole. A 50 mL 1,2-dichloroethane solution of KOH (1.5 mol. equiv.), K₂CO₃ (1.5 mol. equiv.), tetrabutylammonium chloride (0.5 mol. equiv.), and 3,5-dimethylpyrazole (4.00 g, 0.04 mmol) was heated at 45 °C for 3.5 h. The solution was filtered, washed with water, and dried with anhydrous MgSO₄. The removal of the solvent under vacuum gave a yellow liquid as the pure product.³²

2.2.3 Synthesis of 1-methylbenzimidazole and 1-ethylbenzimidazole. Benzimidazole (1 mol equiv.) and excess KOH in DMF for 15 minutes, after which 1.5 mol equiv. of the respective alkyl source (iodomethane and bromoethane for 1-methylbenzimidazole and 1-ethylbenzimidazole, respectively) was added and left to stir at room temperature for 3 hours. The work-up of 1-ethylimidazole, 1-methylbenzimidazole, and 1-ethylbenzimidazole involved diluting the reaction mixture with water to break the DMSO and DMF and neutralising the residual base. All organics were then extracted with DCM (30 mL × 5). The combined organic extracts were then washed with more water till neutral to litmus, then dried with anhydrous magnesium sulphate. All volatiles were then removed under reduced pressure to yield the required product.^33–36

2.3 General procedure for the synthesis of salts 1a–e

A solvent-free mixture of 1-(2-chloroethyl)-3,5-dimethyl-1H-pyrazole and an appropriate N-substituted imidazole with a mole ratio 1 : 1 was heated for approximately 18 hours. The temperature of the reaction depended on the type of N-substituted imidazole. After the reaction time, the crude product was washed several times with ethyl acetate to remove any residual starting materials.³²

2.3.1 3-(2-(3,5-Dimethyl-1H-pyrazol-1-yl) ethyl)-1-methyl-1H-imidazole-3-ium chloride, (1a). Hygroscopic light brown oil. Yield 1.48 g, 90%. FT-IR: ν_max/cm⁻¹ 1555 (CN). ¹H-NMR (600 MHz, DMSO-d₆) δ 8.86 (s, 1H), 7.65 (d, J = 1.8 Hz, 1H), 7.51 (d, J = 1.8 Hz, 1H), 5.80 (s, 1H), 4.56–4.51 (m, 2H), 4.36 (t, J = 5.9 Hz, 2H), 3.82 (s, 3H), 2.07–2.06 (d, 6H). ¹³C-NMR (151 MHz, DMSO-d₆) δ 147.28, 139.91, 137.56, 123.95, 123.06, 105.48, 48.91, 47.68, 36.18, 13.75, 10.72. LRMS (ESI): m/z calcd for [C₁₁H₁₇N₄]⁺: 205.1448; found: 205.1455. Anal. calcd for C₁₁H₁₇ClN₄ C, 54.88; H, 7.12; N, 23.27; found: C, 54.79; H, 7.03; N, 23.15.

2.3.2 3-(2-(3,5-Dimethyl-1H-pyrazol-1-yl)ethyl)-1-ethyl-1H-imidazole-3-ium chloride, (1b). Hygroscopic light brown oil. Yield 1.73 g, 93%. FT-IR: ν_max/cm⁻¹ 1555 (CN). ¹H-NMR (600 MHz, DMSO-d₆) δ: 9.13 (s, 1H), 7.83 (d, J = 1.8 Hz, 1H), 7.64 (d, J = 1.8 Hz, 1H), 5.78 (s, 1H), 4.57 (d, J = 5.9 Hz, 2H), 4.39 (d, J = 5.9 Hz, 2H), 4.18 (q, J = 7.3 Hz, 2H), 2.05 (d, J = 12.5 Hz, 6H), 1.36 (t, J = 7.3 Hz, 3H). ¹³C-NMR (151 MHz, DMSO-d₆) δ: 147.27, 139.88, 136.74, 123.16, 122.54, 105.42, 49.02, 47.70, 44.60, 15.81, 13.74, 10.67. LRMS (ESI): m/z calcd for [C₁₂H₁₉N₄]⁺: 219.1605; found: 219.1755. Anal. calcd for C₁₁H₁₇ClN₄ C, 56.57; H, 7.52; N, 21.99, found: C, 56.48; H, 7.46; N, 21.85.

2.3.3 3-(2-(3,5-Dimethyl-1H-pyrazol-1-yl)ethyl)-1-benzyl-1H-imidazole-3-ium chloride, (1c). Compound 1c was synthesised the same way as 1a, except that the reaction temperature was raised between 100 and 110 °C. Hygroscopic off-white solid. Yield 1.64 g, 91%, mp 146–148 °C. FT-IR: ν_max/cm⁻¹ 1457 (CN). ¹H-NMR (600 MHz, DMSO-d₆) δ: 9.01 (s, 1H), 7.86 (d, J = 1.8 Hz, 1H), 7.71 (d, J = 1.8 Hz, 2H), 7.44–7.33 (m, 5H), 5.73 (s, 1H), 5.44 (s, 2H), 4.57 (t, J = 5.7 Hz, 2H), 4.38 (t, J = 5.7 Hz, 2H), 1.91–2.04 (d, 6H). ¹³C-NMR (151 MHz, DMSO-d₆) δ: 147.34, 139.81, 137.16, 135.40, 129.37, 129.13, 128.59, 123.57, 122.94, 105.41, 52.17, 49.23, 47.62, 13.75, 10.62. LRMS (ESI): m/z calcd for [C₁₇H₂₁N₄]⁺: 281.1761, found: 281.1762. Anal. calcd for C₁₇H₂₄ClN₄: C, 64.45; H, 6.68; N, 17.68, found: C, 64.37; H, 6.53; N, 17.62.

2.3.4 3-(2-(3,5-Dimethyl-1H-pyrazol-1-yl)ethyl)-1-methyl-1H-benzo[d]imidazole-3-ium chloride, (1d). White hygroscopic solid. Yield 1.85 g, 93%, mp 156–158 °C. FT-IR: ν_max/cm⁻¹ 1562 (CN). ¹H-NMR (600 MHz, DMSO-d₆) δ: ¹H-NMR (600 MHz, DMSO-d₆) δ: 9.74 (s, 1H), 8.01 (d, J = 8.3 Hz, 1H), 7.75 (d, J = 8.3 Hz, 1H), 7.67 (t, J = 7.7 Hz, 1H), 7.62 (t, J = 7.7 Hz, 1H), 5.72 (s, 1H), 4.90 (t, J = 5.7 Hz, 2H), 4.46 (t, J = 5.8 Hz, 2H), 4.09 (s, 3H), 2.02 (s, 3H), 1.94 (s, 3H). ¹³C-NMR (151 MHz, DMSO-d₆) δ: 147.26, 143.71, 139.79, 131.96, 131.49, 126.92, 126.88, 113.91, 113.66, 105.62, 47.20, 46.93, 33.69, 13.56, 10.67. LRMS (ESI): m/z calcd for [C₁₅H₁₉N₄]⁺: 255.1605; found: 255.1605. Anal. calcd for C₁₅H₁₉ClN₄: C, 61.96; H, 6.59; N, 19.27, found: C, 61.86; H, 6.44; N, 19.09.

2.3.5 3-(2-(3,5-Dimethyl-1H-pyrazol-1-yl)ethyl)-1-ethyl-1H-benzo[d]imidazole-3-ium chloride, (1e). White hygroscopic solid. Yield 1.77 g, 92%, mp 123–125 °C. FT-IR: ν_max/cm⁻¹ 1563 (CN). ¹H-NMR (600 MHz, DMSO-d₆) δ ¹H NMR (600 MHz, DMSO-d₆) δ 9.41 (s, 1H), 8.02 (d, J = 8.2 Hz, 1H), 7.76 (d, J = 8.1 Hz, 1H), 7.66 (t, J = 7.7 Hz, 1H), 7.62 (t, J = 7.7 Hz, 1H), 5.71 (s, 1H), 4.84 (t, J = 5.7 Hz, 2H), 4.50–4.46 (m, 2H), 4.46–4.42 (m, 2H), 1.94 (s, 3H), 1.92 (s, 3H), 1.47 (t, J = 7.3 Hz, 3H). ¹³C-NMR (151 MHz, DMSO-d₆) δ: 147.25, 142.97, 139.79, 131.59, 131.03, 126.95, 126.90, 113.96, 113.84, 105.56, 47.09, 46.97, 42.42, 14.85, 13.56, 10.64. LRMS (ESI): m/z calcd for [C₁₆H₂₁N₄]⁺: 269.1761, found: 269.1758. Anal. calcd For C₁₆H₂₁ClN₄: C, 63.04; H, 6.94; N, 18.38, found: C, 62.94; H, 6.81; N, 18.24.

2.4 General procedure for the synthesis of the Ag(I)–NHC complexes

A solution of an azolium salt (1.0 mmol) (1a–e), Ag₂O (1.0 mmol) in dry dichloromethane (15 mL) was stirred for 48 hours at room temperature in the dark. After the reaction time, the reaction mixture was filtered through Celite, and the solvent was removed under vacuum. The crude products (2a–e) were recrystallised from dichloromethane/diethyl ether (1 : 3).³⁷ All the Ag(I)–NHC complexes were obtained as white solids.

2.4.1 Chloro[3-(2-(3,5-dimethyl-1H-pyrazol-1-yl)ethyl)-1-methyl-1H-imidazole-2-ylidene]silver(I) [C₁₁H₁₆AgClN₄], (2a). Yield 0.258 g, 94%, mp 213–215 °C. FT-IR: ν_max/cm⁻¹ 1232 (CN). ¹H-NMR (600 MHz, CDCl₃) δ: 6.85 (d, J = 1.8 Hz, 1H), 6.41 (d, J = 1.8 Hz, 1H), 5.73 (s, 1H), 4.53 (t, J = 5.6 Hz, 2H), 4.27 (t, J = 5.6 Hz, 2H), 3.81 (s, 3H), 1.88 (s, 3H), 1.69 (s, 3H). ¹³C-NMR (151 MHz, CDCl₃) δ: 180.65, 148.67, 140.12, 122.02, 121.87, 105.33, 104.82, 49.18, 38.86, 13.51, 10.62. LRMS (ESI): m/z calcd for cationic fragment of [M–AgCl]⁺: 205.1453; found: 205.1535. Anal. calcd for C₁₁H₁₆AgClN₄: C, 38.01; H, 4.64; N, 16.12, found: C, 37.89; H, 4.58; N, 16.07.

2.4.2 Chloro[3-(2-(3,5-dimethyl-1H-pyrazol-1-yl)ethyl)-1-ethyl-1H-imidazole-2-ylidene]silver(I) [C₁₂H₁₈AgClN₄], (2b). Yield 0.35 g, 91%, mp 183–185 °C. FT-IR: ν_max/cm⁻¹ 1383 (CN). ¹H-NMR (600 MHz, CDCl₃) δ: 6.91 (d, J = 1.7 Hz, 1H), 6.50 (d, J = 1.7 Hz, 1H), 5.72 (s, 1H), 4.55 (t, J = 7.3 Hz, 2H), 4.30 (t, J = 7.3 Hz, 2H), 4.13 (q, J = 7.9 Hz, 2H), 2.20 (s, 3H), 1.85 (s, 3H), 1.43 (t, J = 7.3 Hz, 3H). ¹³C-NMR (151 MHz, CDCl₃) δ 179.29, 148.77, 140.11, 121.94, 120.24, 105.38, 51.65, 49.22, 47.10, 17.07, 13.53, 10.54. LRMS (ESI): m/z calcd for cationic fragment of [M–AgCl]⁺: calculated: 219.1610, found: 219.1755. Anal. calcd for C₁₂H₁₈AgClN₄ C, 39.86; H, 5.02; N, 15.49, found: C, 39.81; H, 4.90; N, 15.38.

2.4.3 Chloro[3-(2-(3,5-dimethyl-1H-pyrazol-1-yl)ethyl)-1-benzyl-1H-imidazole-2-ylidene]silver(I) [C₁₇H₂₀AgClN₄], (2c). Yield 0.27 g, 88%, mp 201–203 °C. FT-IR: ν_max/cm⁻¹ 1381 (CN). ¹H-NMR (600 MHz, CDCl₃) δ: 7.51–7.27 (m, 5H)), 6.85 (d, J = 7.4 Hz, 1H), 6.53 (d, J = 7.4 Hz, 1H), 5.69 (s, 1H), 5.25 (s, 2H), 4.59 (t, J = 5.4 Hz, 2H), 4.31 (t, J = 5.5 Hz, 2H), 2.19 (s, 3H), 1.78 (s, 3H). ¹³C-NMR (151 MHz, CDCl₃) δ 180.59, 162.57, 148.77, 140.16, 135.51, 129.09, 128.67, 127.71, 122.28, 121.01, 105.32, 55.74, 51.67, 49.16, 13.55, 10.57. LRMS (ESI): m/z calcd for cationic fragment of [M–AgCl]⁺: 281.1766, found: 281.1888. Anal. calcd for C₁₇H₂₀AgClN₄ C, 48.19; H, 4.76; N, 13.22, found: C, 48.10; H, 4.68; N, 13.13.

2.4.4 Chloro[3-(2-(3,5-dimethyl-1H-pyrazol-1-yl)ethyl)-1-methyl-1H-benzo[d]imidazole-2-ylidene]silver(I) [C₁₅H₁₈AgClN₄], (2d). Yield: 0.95 g, 92%, mp 225–228 °C. FT-IR: ν_max/cm⁻¹ 1388 (CN). ¹H-NMR (600 MHz, CDCl₃) δ: 7.4 (d, J = 8.3 Hz, 1H), 7.35 (d, J = 8.3 Hz, 1H), 7.28 (t, J = 7.7 Hz, 1H), 7.05 (t, J = 7.7 Hz, 1H)., 5.65 (s, 1H), 4.82 (t, J = 5.7 Hz, 2H), 4.46 (t, J = 5.8 Hz, 2H), 4.09 (s, 3H), 1.20 (s, 3H), 0.84 (s, 3H). ¹³C-NMR (151 MHz, CDCl₃) δ: 188.35, 147.45, 139.74, 133.51, 133.17, 124.54, 124.50, 112.44, 112.32, 105.36, 49.38, 47.57, 44.17, 16.50, 13.87, 10.13. LRMS (ESI): m/z calcd for cationic fragment of [M–AgCl]⁺: 255.1610, found: 255.1730. Anal. calcd for C₁₅H₁₈AgClN₄ C, 45.31; H, 4.56; N, 14.09, found: C, 45.23; H, 4.47; N,13.89.

2.4.5 Chloro[3-(2-(3,5-dimethyl-1H-pyrazol-1-yl)ethyl)-1-ethyl-1H-benzo[d]imidazole-2-ylidene]silver(I) [C₁₆H₂₀AgClN₄], (2e). Yield: 0.34 g, 89%, mp = 199–201 °C. FT-IR: ν_max/cm⁻¹ 1398 (CN). ¹H-NMR (600 MHz, CDCl₃) δ: ¹H NMR (600 MHz, chloroform-d) δ 7.46 (d, J = 8.2 Hz, 1H), 7.43 (t, J = 8.3 Hz, 1H), 7.32 (t, J = 8.3, Hz, 1H), 7.20 (d, J = 8.2 Hz, 1H), 5.60 (s, 1H), 4.90 (t, J = 7.2 Hz, 2H), 4.44 (dt, J = 23.5, 6.2 Hz, 4H), 2.02 (s, 3H), 1.54 (s, 3H), 1.40 (t, J = 7.2 Hz, 3H). ¹³C NMR (151 MHz, CDCl₃) δ 188.35, 147.45, 139.74, 133.51, 133.17, 124.54, 124.50, 112.44, 112.32, 105.36, 49.38, 47.57, 44.17, 16.50, 13.87, 10.13. LRMS (ESI): m/z calcd for cationic fragment of [M–AgCl]⁺: 269.1766, found: 269.1878. Anal. calcd for C₁₆H₂₀AgClN₄ C, 46.68; H, 4.90; N, 13.61, found: C, 46.79; H, 5.03; N, 13.51.

2.5 In vitro antibacterial study of the compounds

Ten compounds (five salts and their Ag(I)–NHC complexes) were tested for antibacterial activity against four bacterial strains, two Gram-positive (Staphylococcus aureus and Enterococcus faecalis) and two Gram-negative (Klebsiella pneumoniae and Pseudomonas aeruginosa). The broth microdilution method, as described by Moodley et al. (2018), was employed to determine the percentage inhibition and IC₅₀ values.³⁸ Briefly, the samples were serially diluted to obtain a concentration range of 6.25 to 200 µg mL⁻¹. Compounds 1a–e, 2c, and 2d were diluted in 5% DMSO. 2a, 2b, and 2e were diluted in 10% DMSO. All bacterial strains were grown in Mueller-Hinton Broth at pH 7.4 in a 30 °C incubator for 24 hours and adjusted to an OD equivalent to 0.5 McFarland's standard before aliquots of bacterial culture were added to the test samples. Samples with bacterial culture were incubated for 20 hours at 30 °C, followed by the addition of freshly prepared iodonitrotetratzolium chloride (2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl-2H-tetrazolium chloride). This was followed by an additional 4 hours of incubation at 30 °C and absorbance reading at 490 nm using a multimodal plate reader (Biotek Synergy HT, USA) with Gen 5 software (Biotek Synergy HT, USA Ver 2.01.14). All compounds were tested in triplicate on two separate occasions. The negative control was treated as per above, but contained bacterial cells exposed to 5% or 10% DMSO only, and this was used to represent 100% of live, metabolically active microbes. Statistical analysis was conducted using One-way ANOVA and the Tukey–Kramer post-test on GraphPad InStat software version 3.10 for Windows (GraphPad Software, San Diego, California, USA). Differences were considered significant when p < 0.05.

2.6 Computational studies

Geometry optimisation of the imidazolium salts was done using the Gaussian16 program.³⁹ Gauss View version 5 by Semichem, Inc.⁴⁰ was used to generate the input files and visualise the output. Density functional theory (DFT) calculations were performed using the B3LYP functional and the 6-31+G(d,p) basis set.⁴¹ The LanL2DZ basis set was used for the Ag atom in the Ag(I)–NHC complexes.⁴² A frequency calculation yielded the IR spectra and confirmed the structures were minima with no imaginary frequencies. The UV-visible spectra were obtained using the TDDFT keyword.⁴³ The TDDFT calculation also gave the frontier molecular orbital (FMO) energies, that is, the highest occupied molecular orbital (E_HOMO) and lowest unoccupied molecular orbital (E_LUMO).⁴⁴ These energies were used to determine the emission properties of the salts and the complexes and to estimate the quantum chemical descriptors. The descriptors included bandgap energy (ΔE) (eqn (1)), ionisation potential (IP) (eqn (2)),⁴⁵ global softness (S) (eqn (3)), chemical hardness (η) (eqn (4)),⁴⁵ electrochemical potential (µ) (eqn (5)),⁴⁵ electronegativity (χ) (eqn (6)),⁴⁵ and electrophilicity index (eqn (7)).⁴⁵ The XYZ coordinates of the optimised structures, including the simulated IR and UV-visible spectra, are available in the (SI) file.

ΔE = E_HOMO − E_LUMO (1)

IP = −E_HOMO (2)

χ = −µ (6)

Drug-likeness scores and bioactivity prediction were carried out using the online SwissDrugDesign program. SWISSADME predicted the following parameters: lipophilicity, water solubility, pharmacokinetics, and drug-likeness of the azolium salts that were used as ligand precursors for the synthesis of Ag(I)–NHC complexes.^46–48

3. Results and discussion

3.1 Synthesis and characterisation of the azolium salts and Ag(I)–NHC complexes

The first reaction involved the formation of 3,5-dimethyl-1H-pyrazole from 2,4-pentanedione and hydrazine hydrate. The reaction proceeded through the nucleophilic attack of hydrazine on the two carbonyl groups of 2,4-pentanedione. The intermediate is a hydrazone, which undergoes ring closure to form a pyrazole ring.⁴⁹ N-Alkylation that involved the deprotonation of 3,5-dimethyl-1H-pyrazole, with the pyrazole nitrogen acting as a nucleophile, that attacked one of the carbon atoms of 1,2-dichloroethane, produced 1-(2-chloroethyl)-3,5-dimethyl-1H-pyrazole. In the reaction, tetrabutylammonium chloride was the phase transfer catalyst. The nitrogen displaced the chlorine atom, forming the product 1-(2-chloroethyl)-3,5-dimethyl-1H-pyrazole. All the azolium salts were synthesised by the solvent-free method of reacting 1-(2-chloroethyl)-3,5-dimethyl-1H-pyrazole and the desired imidazole source under mild temperatures. The reaction proceeded via the S_N2 reaction route, where the halide on the 1-(2-chloroethyl)-3,5-dimethyl-1H-pyrazole was displaced by the lone pair of electrons on the sp² nitrogen in the substituted imidazole.⁴⁹ The wingtips N-substituents (R group, Scheme 1) were chosen as methyl, ethyl and benzyl.

Scheme 1 Synthesis route for the azolium salts (1a–e) and the Ag(I)–NHC complexes (2a–e).

The pyrazolyl functionalised imidazolium and benzimidazolium salts (1a–e) (Scheme 1) were obtained in high yields. Preliminary evidence for the successful synthesis of the azolium salts was seen as the appearance of an acidic imidazolium proton between 9 and 10 ppm in their ¹H-NMR spectra. The other two protons on the imidazolium moieties appeared as two doublets of one proton, each between 7 and 8 ppm for compounds 1a–c. The presence of a substituted phenyl ring for compounds 1d and 1e gave rise to two doublets and two triplets for the four phenyl protons of the benzimidazolium salts.³² The key characteristic signals on the ¹³C-NMR were due to the imidazolium carbon atoms observed between 137 and 139 ppm (see SI Fig. S2–S11).

The Ag(I)–NHC complexes were obtained by reacting the imidazolium and benzimidazolium salts with Ag₂O in dichloromethane in the dark at room temperature (Scheme 1). Ag₂O is a weak (metal oxide) base often used as a Lewis acid to donate empty orbitals to electron-rich species.⁵⁰ The basic nature allows it to deprotonate the imidazolium salt for in situ carbene formation, where the silver(I) ion subsequently donates an empty orbital to the electron-rich carbene.⁵¹ The complexes of the simple imidazolylidene ligands (2a–c) are unstable in air and moisture as they turn from white to brown hygroscopic solids. In contrast, the benzimidazolylidene-based 2d and 2e are stable in air and moisture due to the added stabilisation of the delocalised electrons of the fused phenyl ring.

Successful formation of the Ag(I)–NHC complexes was characterised by the loss of the acidic proton on the salt and the formation of the Ag–carbene bond, which is observed as a generally weak signal around 180 ppm for complexes 2a–c and around 190 ppm in the ¹³C NMR spectra of complexes 2d–e.⁵² The downfield shift of the imidazolium C2-carbon signals from 137–139 ppm to 180 and further down to 190 ppm also indicated the successful formation of the Ag(I)–C bond. However, in most cases, the Ag(I)–C signal appears weak in the ¹³C NMR spectrum, and in some reported instances, it was not observable due to its lability.^26,53,54

The experimental FT-IR spectra show the ν_(CN) bands for azolium salts 1a–e at 1555, 1558, 1457, 1562 and 1563 cm⁻¹, respectively (see SI, Fig. S32–S41). All the salts are highly hygroscopic and imbibed varying quantities of water, as indicated by the broad peaks at around 3300 cm⁻¹, a commonly reported observation for the handling and storage of such salts.⁵⁵ The hygroscopic nature of the azolium salts also affected their elemental analysis data, which showed variable numbers of water molecules.⁵⁶ After complexion, the CN vibrations of the Ag(I)–NHC complexes, 2a–e, shifted to lower energy regions, such as 1232, 1383, 1381, 1388 and 1398 cm⁻¹, respectively, due to the presence of the electropositive Ag(I) centre, which draws the electron density towards itself.³² The simulated IR spectra of the azolium salts and their Ag(I)–NHC complexes showed bands in similar regions comparable to those of the experimental spectra (see SI, Fig. S32–S41). The simulated UV-vis spectra (see SI, Fig. S42–S51) show the absorption bands in the same regions (200–350 nm) as reported in the literature.⁵⁷ Elemental analysis showed that the synthesised Ag(I) complexes were coordinated to only one NHC ligand through the carbene (C2) carbon.

The calculated mass-to-charge ratios of the azolium salts gave comparable values to the experimental, indicating the successful synthesis of the proposed structures and the purity of the compounds. The most intense fragments belonged to the molecular ion peaks, representing the molecular weight of the cationic fragments of the salts. The mass spectrometry results of the complexes showed that the Ag(I) complexes were ionised on the Ag(I)–carbene bond, forming the positive ion (which is the imidazolium salt) and the negative counterion comprising the silver and chloride ions (–Ag–Cl).⁵⁴ Thus, the spectroscopic and analytical data confirm the successful synthesis and isolation of the proposed compounds in high bulk purity.

3.2 In vitro antibacterial studies of the compounds 1a–e and complexes 2a–e

The antibacterial efficacy of the synthesised salts (1a–e) and their corresponding Ag(I)–NHC complexes (2a–e) was tested by studying their inhibition of two Gram-negative (K. pneumoniae and P. aeruginosa) and two Gram-positive bacterial strains (S. aureus and E. faecalis) at concentrations of 200, 100, 50, 25 and 12.5 µg mL⁻¹. Statistical analysis was carried out, and it was concluded that almost all the compounds had a significant inhibition at the highest concentration of 200 µg mL⁻¹. Neomycin, an antibacterial drug used as the standard, belongs to a class of antibiotics known as aminoglycosides, which inhibit bacterial protein synthesis, resulting in cell death. The antibiotic is most effective at inhibiting Gram-negative bacteria (K. pneumoniae and P. aeruginosa) due to its ability to penetrate their thinner outer membrane compared to the thicker one of Gram-positive strains.⁵⁸ Neomycin also exhibits notable activity against the Gram-positive bacterial strains (S. aureus and E. faecalis), but its clinical utility is limited by resistance mechanisms.⁵⁹

A comparative evaluation of the antibacterial activity of the imidazolium salts (1a–e) and the corresponding Ag(I)–NHC complexes (2a–e) (Table 1) revealed that the complexes consistently exhibited superior bacterial inhibition. Enhancement of activity upon complexation is similar to previous studies, which have reported that Ag(I)–NHC complexes demonstrated significantly greater antibacterial efficacy than their precursor salts.^60–62 This is supported by the well-established antibacterial activity of silver, which is known to be a good agent for disrupting the bacterial cell wall and inhibiting DNA replication, resulting in reduced enzymatic activity and cell death.^63–65 It is noteworthy that all the Ag(I) compounds showed significantly better or comparable antimicrobial activity than neomycin against all the tested bacteria strains. Also, all the compounds statistically exhibited comparable activities showing that the presence of a benzimidazolium moiety in 2d and 2e did not increase antibacterial activity.

Table 1 Highest percentage inhibition of the azolium salts (1a–e) and their Ag(I)–NHC complexes (2a–e)^a

Compound	Gram-positive		Gram-negative
	S. aureus	E. faecalis	K. pneumoniae	P. aeruginosa
a All data reported at 200 µg mL^−1 concentration (unless noted otherwise).
1a	15.11 (±7.55)	19.55 (±7.14)	13.56 (±4.07)	2.52 (±2.18)
1b	17.90 (±12.81)	0.50 (±6.27)	3.70 (±6.80)	4.50 (±1.36)
1c	10.67 (±3.92)	3.06 (±8.54)	28.56 (±3.67)	4.46 (±8.68)
1d	24.29 (±9.03)	1.67 (±1.87)	23.26 (±4.55)	3.38 (±3.63)
1e	18.06 (±5.54)	29.88 (±5.85)	5.70 (±2.68)	7.04 (±3.67)
2a	61.04 (±5.46)	81.32 (±8.55)	84.67 (±8.67)	85.46 (±5.49)
2b	59.01 (50) (±2.23)	91.02 (50) (±0.57)	85.60 (±3.61)	81.34 (±5.37)
2c	60.77 (±4.85)	86.71 (±2.12)	93.02 (±1.08)	83.97 (±5.49)
2d	59.53 (±3.87)	73.69 (±3.92)	90.00 (±1.87)	80.16 (±6.57)
2e	51.61 (±5.56)	90.72 (±0.97)	88.79 (±1.48)	96.53 (±7.51)
Neomycin	44.19 (±3.84)	63.07 (±7.32)	89.02 (±1.79)	44.41 (±9.37)

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Compound 2b is especially worth mentioning, because it showed a significantly high percentage inhibition of 59% at a lower concentration of 50 µg mL⁻¹ compared to neomycin with 44% at 200 µg mL⁻¹ against S. aureus.⁶⁶ It also did better against E. faecalis, a common cause of hospital-acquired infections, accounting for 60% of reported cases.⁶⁷ Neomycin showed 63% inhibition, lower than the least active complex, 2d (74%), against the deadly microbe. The better activity of the complexes than neomycin, a broad-spectrum standard antibiotic,⁶⁸ against both Gram-positive strains showed the potential efficacy of these Ag(I)–NHC as antibacterial agents.

The good activity of the complexes against K. pneumoniae was indicated by the fact that all had inhibitions ranging between 84–93%, comparable to the 89% of neomycin. When tested against P. aeruginosa, the complexes showed better bacterial inhibition (80–87%) than the 44% for the control drug, neomycin. Cumulatively, these results show the promising antibacterial efficacy of the five Ag(I)–NHC complexes presented in this report. In context, the antibacterial activity of the five Ag(I)–NHC complexes was comparable to that reported by Atli and Aksu Their Ag(I)–NHC complexes with MIC values against P. aeruginosa in the range 0.61–1.25 mg mL⁻¹, but were more sensitive against S. aureus with an MIC of 0.61 µg mL⁻¹.⁶⁹ Mnasri et al. reported complexes with better activity than ours, and had MIC values between 0.24 and 125 µg mL⁻¹ against S. aureus.²⁶ One of the best reported Ag(I)–NHC complexes by Tutar and Celik is based on benzimidazole with an MIC of 15.6 µg mL⁻¹ against S. aureus and E. faecalis.⁷⁰

Furthermore, the antibacterial activity of the Ag(I)–NHC complexes was evaluated using the half-maximal inhibitory concentration (IC₅₀) method based on the molar concentrations of the compounds (Table 2) to study some potential structure–activity relationships. Overall, the complexes exhibited greater efficacy against the Gram-negative strains than the Gram-positive strains, due to the thinner peptidoglycan layer in the Gram-negative cell wall, allowing easy penetration by antibacterial agents.⁷¹ The activity of complexes 2a–e shows a distinct structure–activity relationship (SAR) influenced by either the nature of the N-substituent (R group) or the backbone of the azolium moiety. The increase in activity is based on changes in steric bulk and lipophilicity of the complexes due to the alkyl N-substituents. This is exhibited by 2c, which is the most potent with low IC₅₀ values against both Gram-positive (S. aureus = 197 µM; E. faecalis = 20 µM) and Gram-negative strains (K. pneumoniae = 26 µM; P. aeruginosa = 62 µM). The enhanced activity of 2c can be attributed to increased lipophilicity facilitated by the aromatic substituent. Increasing the chain of the alkyl group from methyl (2a) to ethyl (2b) decreased the activity of the complex against the Gram-positive strains; however, it did improve the potency of the benzimidazolium-based counterpart (2e vs. 2d). The two complexes with an ethyl N-substituent group (2b and 2e) showed better activity against the Gram-negative strains than related ones (2a and 2d) bearing the smaller methyl group.

Table 2 IC₅₀ (µM) of the Ag(I)–NHC complexes against the tested bacterial strains

Compound	Gram-positive		Gram-negative
	S. aureus	E. faecalis	K. pneumoniae	P. aeruginosa
2a	296	103	161	136
2b	323	133	88	107
2c	197	20	26	62
2d	443	272	125	155
2e	419	108	60	98
Neomycin	21	8	276	77

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Thus, it can be suggested that the longer carbon chain enhanced lipophilicity, and reports have shown that complexes with long alkyl chain N-substituents have a slower release rate of metal ions than those with shorter carbon chains, which enhanced the antibacterial activity of complexes 2b and 2e against the Gram-negative strains.⁷² These results are consistent with reports showing that antibacterial activity depends on a combination of factors and is generally strain dependent, and different bacteria respond differently to the same antibacterial agents.⁷³ Thus, the activity of the compounds should be analysed separately against each strain. However, fine-tuning the NHC ligand structure can lead to enhanced antimicrobial properties for Ag(I)–NHC complexes, particularly against resistant Gram-negative strains.

3.3 Computational study

3.3.1 Chemical reactivity descriptors. In this study, DFT calculations were carried out to get insight into the reported compounds' structure-chemical reactivity properties (Table 3). The energy difference between the lowest unoccupied molecular orbital (LUMO) and the highest occupied molecular orbital (HOMO), ΔE (E_LUMO − E_HOMO), was calculated according to eqn (1). The HOMO and LUMO levels are critical for electron mobility, which is related to the compound's biological activity.⁷⁴ The HOMO is the preferred location for oxidation, whereas the LUMO is where the molecule is most likely to be reduced; hence, the compound has superior biological activity when the HOMO is located primarily on the donor atoms, and the LUMO is located on the acceptor atoms.⁷⁵ The smaller the HOMO–LUMO energy gap, the greater the compound's potential for biological activity, as it allows for better electron mobility.⁷⁶ In contrast, a larger ΔE value indicates high stability and low chemical reactivity.⁷⁷

Table 3 Chemical reactivity descriptors for the reported compounds^a

	HOMO	LUMO	ΔE	IP	S	η	µ	χ	ω
a ΔE is the HOMO–LUMO energy gap, S is global softness, I is ionisation potential, η is global hardness, µ is chemical potential, χ is electronegativity, and ω is electrophilicity index.
1a	−9.130	−5.093	4.037	9.130	0.495	2.022	−7.112	7.112	12.529
1b	−9.097	−4.991	4.105	9.097	0.487	2.053	−7.044	7.044	12.087
1c	−9.032	−4.850	4.182	9.032	0.478	2.091	−6.941	6.941	11.523
1d	−9.129	−5.293	3.836	9.129	0.521	1.918	−7.211	7.211	13.555
1e	−9.099	−5.224	3.875	9.098	0.516	1.938	−7.161	7.161	13.233
2a	−6.050	−0.993	5.057	6.050	0.396	2.528	−3.521	3.521	2.452
2b	−6.034	−0.972	5.062	6.034	0.395	2.531	−3.502	3.502	2.424
2c	−6.052	−1.225	4.827	6.052	0.414	2.414	−3.638	3.638	2.742
2d	−6.347	−1.880	4.467	6.347	0.448	2.234	−4.113	4.113	3.787
2e	−6.093	−1.875	4.218	6.093	0.474	2.109	−3.984	3.984	3.763

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The HUMO–LUMO analysis shows that the benzimidazolium salts have a smaller energy gap and should have better activity than the imidazolium salts. The antibacterial activity of 2d and 2e (Tables 1 and 2) can be ascribed to the presence of the benzene ring, which stabilises the entire benzimidazolium moiety and controls the steady release of the Ag(I) ions.²¹ Based on the HOMO–LUMO energy gap, the order of activity is 1d > 1e > 1b > 1a > 1c. From Table 3, the imidazolium and benzimidazolium salts have smaller HOMO–LUMO energy gaps than the corresponding Ag(I) complexes. Thus, the order of activity for the complexes based on the HOMO–LUMO energy gap is 2e > 2d > 2c > 2a > 2b.

The order of activity against the different strains based on their IC₅₀ values for S. aureus is 2c > 2b > 2a > 2e > 2d; for E. faecalis is 2c > 2a > 2e > 2b > 2d; for K. pneumoniae is 2c > 2e > 2b > 2d > 2a and for P. aeruginosa is 2c > 2b > 2e > 2a > 2d. Complexes 2d and 2e have the smallest HOMO–LUMO energy gap, indicating that the benzimidazole is more favourable than the simple imidazole. With the exception of 2c, the benzimidazolium-based complexes were generally more active than the imidazolium-based ones, confirming the HOMO–LUMO prediction that 2d and 2e have smaller energy gaps than 2a and 2b. This is despite the fact that the in vitro antibacterial activity is strain-sensitive. Serdaroğlu and coworkers also reported that their NHC precursor had a smaller HOMO–LUMO energy gap (4.3522 eV) than its Ag(I)–NHC complex (5.1704 eV). However, the Ag(I)–NHC complex exhibited better antibacterial activity than the salt.³⁷

Global softness, hardness, and ΔE can be used to predict compound stability. A big energy gap and high global hardness value indicate a more stable compound with reduced biological activity.⁷⁸ Global hardness is defined as the resistance to charge transfer, while global softness demonstrates the compound's susceptibility to charge transfer. The order of stability of the five salts is 1c > 1b > 1a > 1d > 1e, while the order for the Ag(I) complexes is 2b > 2a > 2c > 2d > 2e. Based on the global hardness values for the complexes, the most active complex is 2e, followed by 2d, with 2b and 2a being the least active, while compound 2c lies in between. However, the IC₅₀ values showed compound 2c as the most active against all the bacterial strains.

Since the activity of the complexes is strain-sensitive, it is not easy to link the DFT predictions to the in vitro evaluations, and numerous investigations have examined frontier molecular orbital maps to ascertain the possible reactivity of molecules.⁷⁹ None of the salts exhibited in vitro antibacterial activity similar to that of the Ag(I)–NHC complexes (2a–e), despite the salts' global softness being on par with, and occasionally even better than, that of the complexes. This implies that although DFT is a useful tool for predicting a compound's reactivity, experimental results must always be utilised to confirm the predictions.⁸⁰

The lower ionisation energies and electronegativity values for the Ag(I)–NHC complexes compared to the salts indicate that less energy is required to remove electrons, implying improved electron-donating ability and activity.⁸¹ The electrophilicity index measures the ability of a compound to accept electrons. The higher the electrophilicity index, the greater the electron acceptance. Thus, compounds 1a–e are better electron acceptors, while 2a–e are good electron donors. It may be concluded that while compounds 2a–e have larger energy gaps than 1a–e, their softness and ability to readily donate electrons give the Ag(I)–NHC complexes superior in vitro antibacterial activity over the azolium salts.⁸²

The FMO analysis of the salts showed that the LUMO orbitals are situated on the imidazolium moiety, while the HOMO orbitals are on the pyrazole moiety (Fig. 1). Thus, the imidazolium moiety acts as the electron acceptor, while the pyrazole donates the electrons.⁷⁵ For the Ag(I)–NHC complexes, the LUMO is primarily on the imidazolium moiety and slightly on the silver and chlorine atoms, while the HOMOs are on the silver and chlorine atoms. Thus, in the Ag(I)–NHC complexes, the chlorine and silver atoms can both donate and accept electrons. The high HOMO and LUMO energies of the Ag(I)–NHC complexes compared to their respective salts showed that the metal complexes are better electron donors and thus have better in vitro activity than the salts.⁸¹

Fig. 1 FMOs of selected salts (1a, 1c and 1d) and their Ag(I)–NHC complexes (2a, 2c and 2d).

3.3.2 Molecular electrostatic potential analysis. The 3-D molecular electrostatic potential (MEP) maps are useful for locating the sites where nucleophilic and electrophilic attacks may occur. Electronegative groups usually have negative potentials due to their ability to attract bonding electrons.^83,84 The positive potential in blue is the region where electrophilic attacks occur. The red region designates a negative potential and area for nucleophilic attack.⁸⁵ In Fig. 2, compounds 1a–e are more electrophilic as denoted by the blue regions that lie over the imidazolium moieties. The LUMO (Fig. 1) on the imidazolium moieties of compounds 1a–e further supports this, demonstrating that these areas are electron acceptors.⁸⁶ MEP surfaces for the Ag(I)–NHC complexes indicated a negative potential centred on the chlorine atom, the most electronegative in the compounds. Thus, the MEP analysis is consistent with the FMO study, which revealed that the HOMO of the complexes is exclusively located on the silver and chlorine atoms. The slight prevalence of a positive potential in compounds 2a–e indicates that they are more vulnerable to a nucleophilic rather than an electrophilic attack.⁸⁷

Fig. 2 MEP surfaces of compounds 1a–e and 2a–e.

3.3.3 SWISSADME prediction. SwissDrugDesign was used to estimate the ligand precursors' lipophilicity, water solubility, pharmacokinetics, and drug-likeness. Log P_o/w is a measure of a compound's lipophilicity, or how easily it dissolves in a fatty environment like a cell membrane. According to Lipinski's rule of five, a drug-like molecule should have a log P value smaller than five in order to ensure adequate aqueous solubility and absorption.⁴⁶ It has been demonstrated that a compound's antibacterial activity varies with the length of its carbon chain, with longer chains showing greater activity than shorter ones. This happens because the longer carbon chains increase the compound's lipophilicity, which makes it easier to cross the bacterial cell membrane.^68,88

Among the investigated log P_o/w values (Table 4) of the complexes, the best lipophilicity was for 2e, which consists of an ethyl substituent on the benzimidazolium moiety. The good lipophilicity of compound 2c can be linked to its good antibacterial activity, which is exhibited by the best IC₅₀ values across all the tested strains. Complex 2e had the second-best IC₅₀ values against E. faecalis, K. pneumoniae, and P. aeruginosa (Table 2), thus also showing the role played by its good lipophilicity.

Table 4 Computational SWISS ADME prediction of compounds^a 2a–e

Compound	Consensus log Po/w	Water solubility (mg mL^−1)	P-gp substrate	Lipinski
a Log P: lipophilicity. Water solubility was calculated using the ESOL method a quantitative structure–property relationship (QSPR)-based model.
2a	0.83	0.179	No	Yes, 0 violations
2b	1.11	0.108	No	Yes, 0 violations
2c	1.96	0.0084	Yes	Yes, 0 violations
2d	1.90	0.0087	Yes	Yes, 0 violations
2e	2.17	0.0053	Yes	Yes, 0 violations

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When compounds 2a and 2d are compared to compounds 2b against 2e, it is clear that the benzimidazolium moiety increased the compounds' lipophilicity. The log P_o/w value of 2c indicated that the benzyl moiety is more lipophilic than methyl and ethyl groups. Compounds 2c–e were identified as potential P-gp substrates that use the P-glycoprotein transporter to cross the cell membrane.⁸⁹ As a result, due to their high lipophilicity, these compounds can easily permeate the bacterial cell membrane.

4. Conclusion

In this work, imidazolium and benzimidazolium-based salts and their Ag(I)–NHC complexes were synthesised, and their in vitro antibacterial and computational studies were conducted. The Ag(I)–NHC complexes showed better antibacterial activity than their respective salts and the control drug, neomycin. The antibacterial studies showed that the percentage inhibition of the compounds depended, to a large extent, on the concentration of the compound. DFT predicted the benzimidazolium-based Ag(I)–NHC complexes to be better than the imidazolium-based ones. The Ag(I)–NHC complexes were predicted to be better electron donors than their respective salts due to the presence of the silver and chlorine atoms. However, the IC₅₀ results showed that the imidazolium-based Ag(I)–NHC complex with a benzyl N-substituent exhibited the best activity, and this was supported by the SWISSADME prediction, which showed the ligand precursor of the same complex to have good lipophilicity. The results showed that the benzimidazolium moiety and a longer carbon chain increased antibacterial activity. Fine-tuning the NHC ligand structure can enhance the antimicrobial properties of Ag(I)–NHC complexes, particularly against strains that have developed resistance to currently available drugs. Thus, future considerations should prioritise altering the chain length of the carbon N-substituent of especially benzimidazolium-based Ag(I) complexes.

Conflicts of interest

There are no conflicts of interest to declare.

Abbreviations

ADME	Absorption, distribution, metabolism and excretion
DFT	Density functional theory
DMSO	Dimethyl sulfoxide
FMO	Frontier molecular orbital
HOMO	Highest occupied molecular orbital
LUMO	Lowest unoccupied molecular orbital
IC50	Half-maximal inhibitory concentration
MEP	Molecular electrostatic potential
MIC	Minimum inhibitory concentration
MRSA	Methicillin-resistant Staphylococcus Aureus
NHC	N-Heterocyclic Carbene
NMR	Nuclear magnetic resonance
SAR	Structure–activity relationship

Data availability

The data supporting this article have been included as part of the supplementary information (SI). Supplementary information: experimental and simulated spectra as well as the XYZ coordinates. See DOI: https://doi.org/10.1039/d5ra05570a.

Acknowledgements

The authors thank the National Research Foundation of South Africa and the University of KwaZulu-Natal for financial support. TS thanks the National Research Foundation of South Africa for a Competitive Programme for Rated Researchers grant [CPRR230527110705]. TS and NR acknowledge the Centre for High Performance Computing (CHPC), Cape Town, South Africa, for the computational resources.

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