Silver(i) NHC complexes with antimicrobial and anticancer potential: synthesis, characterization, and DFT-derived molecular insights

Abstract

Ag(i)-NHC complexes are well known as an important class of organometallic compounds widely used in organometallic chemistry and pharmaceutical chemistry. Herein, we report the synthesis, characterization, biological activity, and density functional theory (DFT) studies of a series of benzimidazolium salts and their Ag(i)-NHC complexes bearing 5,6-dimethylbenzimidazole cores. The antifungal activity of the synthesized complexes was investigated against Candida albicans and Candida glabrata, while their antibacterial potential was evaluated against Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa. In addition, the anticancer properties of the compounds were assessed using HCT116 (human colorectal carcinoma), A549 (human lung cancer), and BEAS-2B (human normal lung epithelial) cell lines to determine their cytotoxic and selective antiproliferative effects. Overall, among the synthesized Ag-NHC complexes, 3a and 3e appear to be the most promising candidates for further development. Furthermore, the stability of the complexes in the biological media and their protein binding properties were investigated and the results revealed that the complexes displayed satisfactory stability and albumin-binding properties. Finally, DFT calculations were employed to optimize the molecular geometries and to investigate the electronic, thermodynamic, and vibrational properties of the complexes. FMO, NBO, and MEP analyses provide insight into the distribution of electron density and the nature of metal–ligand interactions.

A series of Ag(i)-NHC complexes have been synthesized and tested for their anticancer and antimicrobial activities.

1. Introduction

Cancer remains one of the leading causes of mortality worldwide and represents a major global health burden. According to the World Health Organization (WHO), approximately 10 million deaths were attributed to cancer in 2020 alone.¹ Despite significant advances in early diagnosis and therapeutic strategies, cancer treatment continues to face substantial challenges, including systemic toxicity, limited selectivity, and the development of drug resistance.

Treatment strategies are largely determined by the type, location, and stage of the tumor. Among these strategies, chemotherapy remains one of the most widely employed approaches. Platinum-based drugs such as cisplatin, carboplatin, and oxaliplatin have played a central role in the treatment of various malignancies.^2,3 However, their clinical use is often associated with severe side effects, including nephrotoxicity, neurotoxicity, and myelosuppression.³ In addition, prolonged exposure frequently leads to acquired resistance, significantly reducing therapeutic efficacy. These limitations have motivated researchers to explore alternative metal-based compounds with improved pharmacological profiles.

The design and synthesis of metal complexes with tailored structural and electronic properties have attracted increasing attention. Among them, metal-N-heterocyclic carbene (NHC) complexes have emerged as particularly promising candidates for anticancer applications.^4,5 NHC ligands are commonly regarded as phosphine analogues; they are strong σ-donating, two-electron donor ligands that often exhibit greater electron-donating ability than conventional tertiary phosphines.⁶ Moreover, their modular structure allows for fine-tuning of steric and electronic parameters, providing substantial flexibility in ligand design and optimization.

Transition metal-NHC complexes incorporating silver and gold have shown especially encouraging biological activities.^7,8 Silver-NHC complexes, in particular, serve as versatile precursors for the synthesis of a wide range of transition metal-NHC species and display notable structural diversity.^9,10 The absence of additional auxiliary ligands completing the silver coordination sphere can significantly influence structural motifs, reactivity patterns, and optical or biological properties.^11,12 At the same time, this simplicity facilitates a clearer understanding of the intrinsic role of the carbene ligand in determining the overall behavior of the complex.

Overall, the development of metal-NHC complexes represents a promising and rapidly expanding field in medicinal inorganic chemistry. Ongoing research into structure–activity relationships can contribute to the design of more effective and less toxic cancer drugs in the future.

The anticancer potential of Ag(i)-NHC complexes has been extensively demonstrated in recent years, particularly for benzimidazole-derived systems. Youngs and co-workers¹³ reported some of the earliest Ag(i)-NHC complexes displaying potent cytotoxic activity against leukemia and colon cancer cell lines, thereby highlighting silver-carbene complexes as promising alternatives to platinum-based drugs. Subsequent studies by Habib et al. showed that 5-methylbenzimidazole-derived Ag(i)-NHC complexes exhibited enhanced antiproliferative activity toward MDA-MB-231 breast and HCT-116 colon cancer cells compared with their corresponding benzimidazolium salts, indicating that coordination to the silver center substantially improves biological efficacy.¹⁴ Similarly, Çevik-Yıldız and co-workers synthesized a series of benzimidazole-based Ag(i)-NHC complexes that demonstrated submicromolar cytotoxicity against MCF-7 and MDA-MB-231 cell lines while maintaining improved selectivity toward cancer cells.¹⁵ More recently, Ulu et al. reported a series of benzimidazole-derived Ag(i)-NHC complexes that exhibited remarkable biological activity against both bacterial strains and cancer cells.¹⁶ In particular, all Ag(i)-NHC complexes showed higher cytotoxicity toward MCF-7 breast adenocarcinoma cells than cisplatin after 24 h of treatment, while the most active complex displayed approximately 24-fold greater potency than the reference drug. The same complexes also demonstrated pronounced antibacterial activity against both Gram-positive and Gram-negative bacteria, highlighting the multifunctional therapeutic potential of Ag(i)-NHC systems. Collectively, these findings emphasize that both ligand architecture and metal-centered electronic effects are critical determinants of the biological activity of Ag(i)-NHC complexes. Therefore, in this study, five novel benzimidazolium salts (2a–e) and their corresponding Ag(i)-NHC complexes (3a–e) were synthesized. The structures of the synthesized compounds were determined by ¹H and ¹³C Nuclear Magnetic Resonance (NMR), Fourier transform infrared (FTIR) spectroscopy, and liquid chromatography-high-resolution mass spectrometry (LC-HRMS). The purity of the synthesized benzimidazolium salts (2a–e) was tested by high performance liquid chromatography (HPLC). Furthermore, the structure of one of the Ag(i)-NHC complexes (3c) was elucidated by single-crystal X-ray diffractometry. The antimicrobial and anticancer activities of all prepared compounds were tested. Furthermore, the stability of the complexes in the biological media and their protein binding properties were investigated. In order to complement the experimental findings, density functional theory (DFT) calculations were carried out to provide detailed insight into the structural, electronic, and thermodynamic properties of the synthesized Ag(i) complexes 3a–e. DFT analyses provide valuable information on optimized geometries, frontier molecular orbitals, charge distribution, and thermodynamic parameters, which are not always directly accessible from experimental techniques alone. In the present study, DFT computations were performed to provide insight into the structural and electronic characteristics of the synthesized complexes and to complement the experimental observations. Although the calculated electronic descriptors reveal only minor differences among the complexes, they contribute to understanding the intrinsic electronic nature of the Ag-NHC framework, thereby highlighting the strong synergy between experimental and theoretical approaches.

2. Experimental section

2.1. General information

The reagents and solvents utilized in the synthesis of their Ag(i)-NHC complexes were procured commercially, and no purification step was necessary. The synthesis of the complexes was accomplished in an inert atmosphere by means of the Schlenk technique. The melting points of the substances were determined by employing capillary tubes with an Electrothermal digital melting point apparatus (Model IA9200, Barnstead, UK). The FT-IR spectra were collected using a PerkinElmer Spectrum One spectrometer in the range of 4000–650 cm⁻¹. The ¹H and ¹³C NMR spectra of the compounds were recorded in deuterated chloroform (CDCl₃) with the internal reference (Me₄Si) using a Bruker As 400 Mercury spectrometer (Bruker Optics Ltd, Milton, ON, Canada). The NMR data are abbreviated as follows: singlet (s), doublet (d), triplet (t), quartet (q), and multiplet (m). To obtain further information about the structures of the synthesized complexes, X-ray powder diffraction studies were performed. Powder XRD (PXRD) analysis of the compounds was carried out using a diffractometer (Rigaku RadB-Dmax II model, USA) with CuKα (λ = 1.54 Å) radiation at 40 kV and 40 mA. The 2θ diffraction angle range was 5–80°, and the scan rate was set to 6° min⁻¹.

2.2. Synthesis and structural characterization of benzimidazolium salts (2a–e)

To synthesize benzimidazolium salts, 1-alkyl benzimidazole (1.0 mmol) was dissolved in DMF under an inert atmosphere using the Schlenk technique, and then 2-2-bromoethyl-1,3-dioxane (1.0 mmol) was added. The mixture was first stirred at room temperature and then gradually heated to 50 °C and 75 °C. Diethyl ether was added to the mixture that had come to room temperature. The resulting white solid was filtered, washed with diethyl ether, and dried under vacuum. The resulting solid was recrystallized from a mixture of ethyl alcohol and diethyl ether.

2.2.1. 1-(Benzyl)-3-(2-(1,3-dioxane-2-yl)ethyl)-5,6-dimethylbenzimidazolium bromide, 2a

To prepare the benzimidazole salt 2a, 2.35 g (1.0 mmol) of 1-(benzyl)-5,6-dimethylbenzimidazole and 1.95 g (1.0 mmol) of 2-(2-bromoethyl)-1,3-dioxane were used.

Yield: white solid, 3.65 g, 85%, m.p.: 165–166 °C, FT-IR ν_(CN): 1560 cm⁻¹, HPLC purity: 95%, LC-MS analysis calculated (m/z) for the cationic part of [NHC]⁺: 351.21; found (m/z): 351.2073. ¹H NMR (400 MHz, CDCl₃) δ (ppm): 1.26 and 1.92 (m, 2H, NCH₂CH₂-1,3-dioxane-5-CH₂), 2.28 (q, J = 4 Hz, 2H, NCH₂CH₂-1,3-dioxane); 3.73 and 3.92 (m, 4H, NCH₂CH₂-1,3-dioxane-4,6-CH₂); 2.38 and 2.42 (s, 6H, C₆H₂-5,6-CH₃); 3.75 and 3.92 (m, 4H, NCH₂CH₂-1,3-dioxane-4,6-CH₂); 4.71 (t, J = 4 Hz, 2H, NCH₂CH₂-1,3-dioxane); 4.83 (t, J = 4 Hz, 1H, NCH₂CH₂-1,3-dioxane-2-CH); 5.86 (s, 2H, CH₂C₆H₅); 7,30-7,48 (m, 7H, Ar-H); 11.16 (s, 1H, NCHN). ¹³C NMR (100 MHz, CDCl₃) δ (ppm): 20.7 (C₆H₂-5,6-CH₃); 25.4 (NCH₂CH₂-1,3-dioxane-5-CH₂); 33.8 (NCH₂CH₂-1,3-dioxane); 42.5 (NCH₂CH₂-1,3-dioxane); 50.8 (CH₂C₆H₄Cl-2); 66.8 (NCH₂CH₂-1,3-dioxane-4,6-CH₂); 99.2 (NCH₂CH₂-1,3-dioxane-2-CH); 112.9, 113.3, 128.1, 128.9, 129.2, 129.7, 129.9, 133.3, 137.2 (Ar-C); 142.2 (NCHN).

2.2.2. 1-(2-Methylbenzyl)-3-(2-(1,3-dioxane-2-yl)ethyl)-5,6-dimethylbenzimidazolium bromide, 2b

To prepare the benzimidazole salt 2b, 2.50 g (1.0 mmol) of 1-(2-methylbenzyl)-5,6-dimethylbenzimidazole and 1.95 g (1.0 mmol) of 2-(2-bromoethyl)-1,3-dioxane were used.

Yield: white solid, 3.60 g, 81%, m.p.: 199–200 °C, FT-IR ν_(CN): 1563 cm⁻¹, HPLC purity: 92%, LC-MS analysis calculated (m/z) for the cationic part of [NHC]⁺: 365.27; found (m/z): 365.2235. ¹H NMR (400 MHz, CDCl₃) δ (ppm): 1.26 and 1.89 (m, 2H, NCH₂CH₂-1,3-dioxane-5-CH₂); 2.29 (m, 2H, NCH₂CH₂-1,3-dioxane); 2.35 (s, 3H, CH₂C₆H₄CH₃-2); 2.43 (s, 6H, C₆H₂-5,6-CH₃), 3.76 and 3.93 (m, 4H, NCH₂CH₂-1,3-dioxane-4,6-CH₂); 4.76 (t, J = 4 Hz, 2H, NCH₂CH₂-1,3-dioxane); 4.87 (t, J = 4 Hz, 1H, NCH₂CH₂-1,3-dioxane-2-CH); 5.84 (s, 2H, CH₂C₆H₄CH₃-2); 7,17-7,47 (m, 6H, Ar-H); 11.10 (s, 1H, NCHN). ¹³C NMR (100 MHz, CDCl₃) δ (ppm): 19.6 (CH₂C₆H₄-CH₃-2); 20.7 (C₆H₂-5,6-CH₃); 25.4 (NCH₂CH₂-1,3-dioxane-5-CH₂); 33.8 (NCH₂CH₂-1,3-dioxane); 42.5 (NCH₂CH₂-1,3-dioxane); 49.8 (CH₂C₆H₄CH₃-2); 66.8 (NCH₂CH₂-1,3-dioxane-4,6-CH₂); 99.4 (NCH₂CH₂-1,3-dioxane-2-CH); 112.8, 113.4, 126.7, 127.8, 129.9, 130.7, 131.3, 136.4, 137.1, 137.5 (Ar-C); 142.8 (NCHN).

2.2.3. 1-(3-Methylbenzyl)-3-(2-(1,3-dioxane-2-yl)ethyl)-5,6-dimethylbenzimidazolium bromide, 2c

To prepare the benzimidazole salt 2c, 2.50 g (1.0 mmol) of 1-(3-methylbenzyl)-5,6-dimethylbenzimidazole and 1.95 g (1.0 mmol) of 2-(2-bromoethyl)-1,3-dioxane were used.

Yield: white solid, 3.91 g, 88%, m.p.: 131–132 °C, FT-IR ν_(CN): 1562 cm⁻¹, HPLC purity: 99%, LC-MS analysis calculated (m/z) for the cationic part of [NHC]⁺: 365.27; found (m/z): 365.2232. ¹H NMR (400 MHz, CDCl₃) δ (ppm): 1.27 and 1.93 (m, 2H, NCH₂CH₂-1,3-dioxane-5-CH₂); 2.32 (m, 2H, NCH₂CH₂-1,3-dioxane); 2.36 (s, 3H, CH₂C₆H₄CH₃-3), 2.43 (s, 6H, C₆H₂-5,6-CH₃); 3.76 and 3.94 (m, 4H, NCH₂CH₂-1,3-dioxane-4,6-CH₂); 4.72 (t, J = 8 Hz, 2H, NCH₂CH₂-1,3-dioxane); 4.84 (m, 1H, NCH₂CH₂-1,3-dioxane-2-CH); 5.80 (s, 2H, CH₂C₆H₄CH₃-3); 7,14-7,46 (m, 6H, Ar-H); 11.30 (s, 1H, NCHN). ¹³C NMR (100 MHz, CDCl₃) δ (ppm): 21.4 (CH₂C₆H₄-CH₃-3); 20.7 (C₆H₂-5,6-CH₃); 25.4 (NCH₂CH₂-1,3-dioxane-5-CH₂); 33.8 (NCH₂CH₂-1,3-dioxane); 42.5 (NCH₂CH₂-1,3-dioxane); 50.9 (CH₂C₆H₄CH₃-3); 66.8 (NCH₂CH₂-1,3-dioxane-4,6-CH₂); 99.2 (NCH₂CH₂-1,3-dioxane-2-CH); 112.8, 113.3, 125.0, 128.6, 129.0, 129.8, 129.9, 133.2, 137.1, 139.1 (Ar-C); 142.3 (NCHN).

2.2.4. 1-(4-Methylbenzyl)-3-(2-(1,3-dioxane-2-yl)ethyl)-5,6-dimethylbenzimidazolium bromide, 2d

To prepare the benzimidazole salt 2d, 2.50 g (1.0 mmol) of 1-(4-methylbenzyl)-5,6-dimethylbenzimidazole and 1.95 g (1.0 mmol) of 2-(2-bromoethyl)-1,3-dioxane were used.

Yield: white solid, 4.04 g, 91%, m.p.: 147–148 °C, FT-IR ν_(CN): 1560 cm⁻¹, HPLC purity: 99%, LC-MS analysis calculated (m/z) for the cationic part of [NHC]⁺: 365.27; found (m/z): 365.2237. ¹H NMR (400 MHz, CDCl₃) δ (ppm): 1.93 (m, 2H, NCH₂CH₂-1,3-dioxane-5-CH₂); 2.28 (m, 2H, NCH₂CH₂-1,3-dioxane); 2.38 (s, 3H, CH₂C₆H₄CH₃-4), 2.42 (s, 6H, C₆H₂-5,6-CH₃); 3.77 and 3.95 (m, 4H, NCH₂CH₂-1,3-dioxane-4,6-CH₂); 4.71 (t, J = 8 Hz, 2H, NCH₂CH₂-1,3-dioxane); 4.84 (m, 1H, NCH₂CH₂-1,3-dioxane-2-CH); 5.79 (s, 2H, CH₂C₆H₄CH₃-4); 7,14-7,44 (m, 6H, Ar-H); 11.24 (s, 1H, NCHN). ¹³C NMR (100 MHz, CDCl₃) δ (ppm): 21.2 (CH₂C₆H₄-CH₃-4); 20.7 (C₆H₂-5,6-CH₃); 25.4 (NCH₂CH₂-1,3-dioxane-5-CH₂); 33.9 (NCH₂CH₂-1,3-dioxane); 42.5 (NCH₂CH₂-1,3-dioxane); 50.7 (CH₂C₆H₄CH₃-4); 66.8 (NCH₂CH₂-1,3-dioxane-4,6-CH₂); 99.2 (NCH₂CH₂-1,3-dioxane-2-CH); 112.8, 113.3, 128.1, 129.6, 129.9, 130.3, 137.1, 138.9 (Ar-C); 142.2 (NCHN).

2.2.5. 1-(3,5-Dimethylbenzyl)-3-(2-(1,3-dioxane-2-yl)ethyl)-5,6-dimethylbenzimidazolium bromide, 2e

To prepare the benzimidazole salt 2e, 2.64 g (1.0 mmol) of 1-(3,5-dimethylbenzyl)-5,6-dimethylbenzimidazole and 1.95 g (1.0 mmol) of 2-(2-bromoethyl)-1,3-dioxane were used.

Yield: white solid, 3.81 g, 83%, m.p.: 209–210 °C, FT-IR ν_(CN): 1563 cm⁻¹, HPLC purity: 97%, LC-MS analysis calculated (m/z) for the cationic part of [NHC]⁺: 379.24; found (m/z): 379.2386. ¹H NMR (400 MHz, CDCl₃) δ (ppm): 1.24 and 1.89 (m, 2H, NCH₂CH₂-1,3-dioxane-5-CH₂); 2.30 (m, 2H, NCH₂CH₂-1,3-dioxane); 2.28 (s, 3H, CH₂C₆H₃CH₃-3,5), 2.39 and 2.43 (s, 6H, C₆H₂-5,6-CH₃); 3.78 and 3.94 (m, 4H, NCH₂CH₂-1,3-dioxane-4,6-CH₂); 4.73 (t, J = 4 Hz, 2H, NCH₂CH₂-1,3-dioxane); 4.87 (t, J = 4 Hz, 1H, NCH₂CH₂-1,3-dioxane-2-CH); 5.72 (s, 2H, CH₂C₆H₃CH₃-3,5); 6.96–7,44 (m, 5H, Ar-H); 11.26 (s, 1H, NCHN). ¹³C NMR (100 MHz, CDCl₃) δ (ppm): 21.2 (CH₂C₆H₄-CH₃-3,5); 20.7 (C₆H₂-5,6-CH₃); 25.4 (NCH₂CH₂-1,3-dioxane-5-CH₂); 33.8 (NCH₂CH₂-1,3-dioxane); 42.5 (NCH₂CH₂-1,3-dioxane); 50.9 (CH₂C₆H₃CH₃-3,5); 66.7 (NCH₂CH₂-1,3-dioxane-4,6-CH₂); 99.3 (NCH₂CH₂-1,3-dioxane-2-CH); 112.8, 113.2, 125.6, 125.7, 129.7, 129.9, 130.7, 133.0, 137.0, 138.9 (Ar-C); 142.4 (NCHN).

2.3. Synthesis and structural characterization of Ag(i)-NHC complexes (3a–e)

DCM was added to the benzimidazolium salt (0.8 mmol) and Ag₂O (0.4 mmol) was taken into the Schlenk and stirred overnight at room temperature and in the dark. After the mixture was filtered, diethyl ether was added. The obtained product was washed with diethyl and dried under vacuum. Fig. 1 illustrates the general synthesis of Ag(i)-NHC complexes (3a–e).

Fig. 1. General synthesis of benzimidazolium salts (2a–e) and Ag(i)-NHC complexes (3a–e).

2.3.1. Bromo-1-(benzyl)-3-[2-(1,3-dioxane-2-yl)ethyl]-5,6-dimethylbenzimidazole-2-ylidinesilver(i), 3a

Yield: 68%, m.p.: 191–192 °C, FT-IR ν(CN):1440 cm⁻¹, LC-MS analysis calculated (m/z) for the cationic part of [(NHC)₂Ag]⁺: 807.31; found (m/z): 807.3058. ¹H NMR (400 MHz, CDCl₃-d₆) δ (ppm): 1.35 and 2.06 (m, 2H, NCH₂CH₂-1,3-dioxane-5-CH₂); 2.10 (q, 2H, J = 8 Hz, NCH₂CH₂-1,3-dioxane); 2.31 and 2.37 (s, 6H, C₆H₂-CH₃-5,6); 3.73 and 4.10 (m, 4H, NCH₂CH₂-1,3-dioxane-4,6-CH₂); 4.58 (t, J = 8 Hz, 1H, NCH₂CH₂-1,3-dioxane-2-CH); 4.54 (t, J = 8 Hz, 2H, NCH₂CH₂-1,3-dioxane); 5.56 (s, 2H, CH₂C₆H₅); 7.23–7.32 (m, 7H, Ar-H). ¹³C NMR (100 MHz, CDCl₃-d₆) δ (ppm): 20,4 (C₆H₂-CH₃-5,6); 25.6 (NCH₂CH₂-1,3-dioxane-5-CH₂); 35.2 (NCH₂CH₂-1,3-dioxane); 44.6 (NCH₂CH₂-1,3-dioxane); 53.1 (CH₂C₆H₅); 66.9 (NCH₂CH₂-1,3-dioxane-4,6-CH₂); 99.1 (NCH₂CH₂-1,3-dioxane-2-CH); 111.8, 112.2, 127.0, 128.3, 129.0, 132.3, 133.6, 135.3 (Ar-C). Elemental analysis, calculated for C₄₄H₅₄Ag₂Br₂N₄O₄: C: 49.00, H: 5.05, N: 5.20 (%); found: C: 49.13, H: 5.01, N: 5.37 (%).

2.3.2. Bromo-1-(2-methylbenzyl)-3-[2-(1,3-dioxane-2-yl)ethyl]-5,6-dimethylbenzimidazole-2-ylidinesilver(i), 3b

Yield: 65%, m.p.: 177–178 °C, FT-IR ν(CN):1446 cm⁻¹, LC-MS analysis calculated (m/z) for the cationic part of [(NHC)₂Ag]⁺: 837.35; found (m/z): 837.3359. ¹H NMR (400 MHz, CDCl₃-d₆) δ (ppm): 1.34 and 2.06 (m, 2H, NCH₂CH₂-1,3-dioxane-5-CH₂); 2.18 (m, 2H, NCH₂CH₂-1,3-dioxane); 2.27 (s, 3H, C₆H₄-CH₃-2); 2.38 and 2.39 (s, 6H, C₆H₂-CH₃-5,6); 3.73 and 4.10 (m, 4H, NCH₂CH₂-1,3-dioxane-4,6-CH₂); 4.60 (t, J = 8 Hz, 1H, NCH₂CH₂-1,3-dioxane-2-CH); 4.55 (t, J = 8 Hz, 2H, NCH₂CH₂-1,3-dioxane); 5.53 (s, 2H, CH₂C₆H₄); 6.66–7.30 (m, 6H, Ar-H). ¹³C NMR (100 MHz, CDCl₃-d₆) δ (ppm): 19.6 (C₆H₄-CH₃-2); 20,4 (C₆H₂-CH₃-5,6); 25.6 (NCH₂CH₂-1,3-dioxane-5-CH₂); 35.2 (NCH₂CH₂-1,3-dioxane); 44.6 (NCH₂CH₂-1,3-dioxane); 53.1 (CH₂C₆H₄); 66.9 (NCH₂CH₂-1,3-dioxane-4,6-CH₂); 99.1 (NCH₂CH₂-1,3-dioxane-2-CH); 111.8, 112.2, 127.0, 128.3, 129.0, 132.3, 133.6, 135.3 (Ar-C). Elemental analysis, calculated for C₄₆H₅₈Ag₂Br₂N₄O₄: C: 49.93, H: 5.28, N: 5.06 (%); found: C: 49.73, H: 5.24, N: 4.92 (%).

2.3.3. Bromo-1-(3-methylbenzyl)-3-[2-(1,3-dioxane-2-yl)ethyl]-5,6-dimethylbenzimidazole-2-ylidinesilver(i), 3c

Yield: 61%, m.p.: 167–168 °C, FT-IR ν(CN):1439 cm⁻¹, LC-MS analysis calculated (m/z) for the cationic part of [(NHC)₂Ag]⁺: 837.35; found (m/z): 837.3344. ¹H NMR (400 MHz, CDCl₃-d₆) δ (ppm): 1.35 and 2.09 (m, 2H, NCH₂CH₂-1,3-dioxane-5-CH₂); 2.17 (m, 2H, NCH₂CH₂-1,3-dioxane); 2.32 (s, 3H, C₆H₄-CH₃-3); 2.37 (s, 6H, C₆H₂-CH₃-5,6); 3.73 and 4.12 (m, 4H, NCH₂CH₂-1,3-dioxane-4,6-CH₂); 4.57 (t, J = 8 Hz, 1H, NCH₂CH₂-1,3-dioxane-2-CH); 4.53 (t, J = 8 Hz, 2H, NCH₂CH₂-1,3-dioxane); 5.50 (s, 2H, CH₂C₆H₄); 7.00–7.26 (m, 6H, Ar-H). ¹³C NMR (100 MHz, CDCl₃-d₆) δ (ppm): 21.4 (C₆H₄-CH₃-3); 20,4 (C₆H₂-CH₃-5,6); 25.6 (NCH₂CH₂-1,3-dioxane-5-CH₂); 35.1 (NCH₂CH₂-1,3-dioxane); 44.6 (NCH₂CH₂-1,3-dioxane); 53.1 (CH₂C₆H₄); 66.9 (NCH₂CH₂-1,3-dioxane-4,6-CH₂); 99.1 (NCH₂CH₂-1,3-dioxane-2-CH); 111.8, 112.2, 124.1, 127.7, 128.9, 129.2, 132.3, 133.7, 135.2, 138.9 (Ar-C). Elemental analysis, calculated for C₄₆H₅₈Ag₂Br₂N₄O₄: C: 49.93, H: 5.28, N: 5.06 (%); found: C: 49.72, H: 5.13, N: 4.93 (%).

2.3.4. Bromo-1-(4-methylbenzyl)-3-[2-(1,3-dioxane-2-yl)ethyl]-5,6-dimethylbenzimidazole-2-ylidinesilver(i), 3d

Yield: 67%, m.p.: 208–209 °C, FT-IR ν(CN):1440 cm⁻¹, LC-MS analysis calculated (m/z) for the cationic part of [(NHC)₂Ag]⁺: 837.35; found (m/z): 837.3390. ¹H NMR (400 MHz, CDCl₃-d₆) δ (ppm): 1.35 and 2.14 (m, 2H, NCH₂CH₂-1,3-dioxane-5-CH₂); 2.17 (m, 2H, NCH₂CH₂-1,3-dioxane); 2.37 (s, 3H, C₆H₄-CH₃-3); 2.31 (s, 6H, C₆H₂-CH₃-5,6); 3.73 and 4.10 (m, 4H, NCH₂CH₂-1,3-dioxane-4,6-CH₂); 4.58 (t, J = 8 Hz, 1H, NCH₂CH₂-1,3-dioxane-2-CH); 4.52 (t, J = 8 Hz, 2H, NCH₂CH₂-1,3-dioxane); 5.51 (s, 2H, CH₂C₆H₄); 7.09–7.26 (m, 6H, Ar-H). ¹³C NMR (100 MHz, CDCl₃-d₆) δ (ppm): 21.1 (C₆H₄-CH₃-3); 20,4 (C₆H₂-CH₃-5,6); 25.6 (NCH₂CH₂-1,3-dioxane-5-CH₂); 35.2 (NCH₂CH₂-1,3-dioxane); 44.5 (NCH₂CH₂-1,3-dioxane); 52.9 (CH₂C₆H₄); 66.9 (NCH₂CH₂-1,3-dioxane-4,6-CH₂); 99.1 (NCH₂CH₂-1,3-dioxane-2-CH); 111.8, 112.2, 127.1, 129.7, 132.3, 132.4, 133.6, 138.2 (Ar-C). Elemental analysis, calculated for C₄₆H₅₈Ag₂Br₂N₄O₄: C: 49.93, H: 5.28, N: 5.06 (%); found: C: 49.92, H: 5.14, N: 4.94 (%).

2.3.5. Bromo-1-(3,5-dimethylbenzyl)-3-[2-(1,3-dioxane-2-yl)ethyl]-5,6-dimethylbenzimidazole-2-ylidinesilver(i), 3e

Yield: 63%, m.p.: 186-187 °C, FT-IR ν(CN):1444 cm⁻¹, LC-MS analysis calculated (m/z) for the cationic part of [(NHC)₂Ag]⁺: 865.38; found (m/z): 865.3705. ¹H NMR (400 MHz, CDCl₃-d₆) δ (ppm): 1.33 and 2.17 (m, 2H, NCH₂CH₂-1,3-dioxane-5-CH₂); 2.18 (m, 2H, NCH₂CH₂-1,3-dioxane); 2.26 (s, 6H, C₆H₃-CH₃-3,5); 2.38 and 2.41 (s, 6H, C₆H₂-CH₃-5,6); 3.72 and 4.10 (t, 4H, J = 8 Hz and 12 Hz, NCH₂CH₂-1,3-dioxane-4,6-CH₂); 4.56 (m, 1H, NCH₂CH₂-1,3-dioxane-2-CH); 4.53 (m, 2H, NCH₂CH₂-1,3-dioxane); 5.45 (s, 2H, CH₂C₆H₃); 6.98–7.26 (m, 4H, Ar-H). ¹³C NMR (100 MHz, CDCl₃-d₆) δ (ppm): 20.4 and 20.4 (C₆H₃-CH₃-3,5); 21.3 (C₆H₂-CH₃-5,6); 25.6 (NCH₂CH₂-1,3-dioxane-5-CH₂); 35.2 (NCH₂CH₂-1,3-dioxane); 44.5 (NCH₂CH₂-1,3-dioxane); 53.0 (CH₂C₆H₃); 66.9 (NCH₂CH₂-1,3-dioxane-4,6-CH₂); 99.1 (NCH₂CH₂-1,3-dioxane-2-CH); 111.8, 112.1, 124.7, 124.9, 130.0, 132.3, 132.6, 133.6, 135.2, 138.6 (Ar-C).; 188.8 (Ag-C_carbene). Elemental analysis, calculated for C₄₈H₆₂Ag₂Br₂N₄O₄: C: 50.81, H: 5.51, N: 4.94 (%); found: C: 50.70, H: 5.45, N: 4.81 (%).

2.4. X-ray analysis

X-ray data of 3c were collected on a Bruker D8 QUEST diffractometer at 296(2) K using graphite-monochromated Mo Kα radiation by applying the φ and ω scan methods. Data collection was carried out using APEX2,¹⁷ while cell refinement and data reduction were applied using SAINT.¹⁷ The structure was solved by a dual-space algorithm using SHELXT-2018 (ref. 18) and refined by means of the full-matrix least-squares calculations on F² using SHELXL-2019.¹⁹ All H atoms were inserted in idealized positions and treated using a riding model, fixing the bond lengths at 0.98, 0.93, 0.97, and 0.96 Å for methine CH, aromatic CH, CH_2, and CH₃ atoms, respectively. The displacement parameters of the H atoms were fixed at U_iso(H) = 1.2U_eq (1.5U_eq for CH₃). Table 1 gathers information on crystal data, data collection, and structure refinement. Molecular graphic was created by using OLEX2.²⁰

Crystal data and structure refinement parameters for 3c.

CCDC depository	2497835
Color/shape	Colorless/block
Chemical formula	[Ag2Br2(C23H28N2O2)2]
Formula weight	1104.50
Temperature (K)	296(2)
Wavelength (Å)	0.71073 Mo Kα
Crystal system	Triclinic
Space group	P1̄ (no. 2)

Unit cell parameters
a, b, c (Å)	8.6000(8), 12.6723(12), 21.452(2)
α, β, γ (°)	81.586(3), 89.700(3), 85.723(3)
Volume (Å3)	2306.2(4)
Z	2
D calc. (g cm−3)	1.591
µ (mm−1)	2.627
Absorption correction	Multi-scan
T min., Tmax.	0.6452, 0.7454
F 000	1112
Crystal size (mm3)	0.06 × 0.05 × 0.03
Diffractometer	Bruker D8 QUEST
Measurement method	φ and ω scan
Index ranges	−10 ≤ h ≤ 10, −15 ≤ k ≤ 15, −26 ≤ l ≤ 26
θ Range for data collection (°)	0.960 ≤ θ ≤ 26.399
Reflections collected	70637
Independent/observed reflections	9440/4743
R int	0.0859
Refinement method	Full-matrix least-squares on F2
Data/restraints/parameters	9440/0/529
Goodness-of-fit on F2	1.016
Final R indices [I > 2σ(I)]	R 1 = 0.0597, wR2 = 0.1128
R Indices (all data)	R 1 = 0.1542, wR2 = 0.1390
Δρmax, Δρmin (e Å−3)	1.22, −1.06

2.5. Antimicrobial studies

Antimicrobial activity experiments were carried out in the Laboratory of the Department of Medical Genetics in the School of Medicine of İnönü University. In this study, Denovix DS-11 FX + (UV, Blue, Red, Green) Spectrophotometer/Fluorometer, Allsheng AMR-100 Microplate Reader, Daihan WIS 20 Shaking Incubator, Nüve EN 120 Incubator, Nüve NF 800R Cooled Centrifuge and Sigma 1-14 Microcentrifuge devices were used.

Candida albicans (SC5314/ATCC MYA-2876) and Candida glabrata (ATCC 2001), which are pathogenic yeast species, were used in antifungal tests, and for antimicrobial tests Escherichia coli (ATCC 25922), Staphylococcus aureus (ATCC 29213), and Pseudomonas aeruginosa (ATCC 27853) bacterial species were used.

C. albicans and C. glabrata, which are pathogenic yeast species, were used in antifungal tests, and for antimicrobial tests E. coli, S. aureus and P. aeruginosa bacteria species were used. All types of bacteria and fungi used in the study were provided in the Laboratory of the Department of Medical Biology and Genetics in School of Medicine of İnönü University (Battalgazi, Malatya, Turkey).

C. albicans and C. glabrata, which are pathogenic yeast species, were used in antifungal tests, and for antimicrobial tests E. coli, S. aureus and P. aeruginosa bacteria species were used. All types of bacteria and fungi used in the study were provided in the Laboratory of the Department of Medical Biology and Genetics in School of Medicine of İnönü University (Battalgazi, Malatya, Turkey). A total of 8 mg of each compound was dissolved in dimethyl sulfoxide (DMSO) to prepare 80 µg per µL solution, and inhibitory activity was performed with 800 µg of compound per disk. In this method, test bacteria (∼1 × 10⁸ cells) inoculated into sterilized LB medium and yeasts (∼1 × 10⁷ cells) inoculated into YPD medium were gently mixed and transferred to a Petri dish under aseptic conditions. A disc containing the compound was placed in a Petri dish (90 mm diameter) and incubated at 37 °C for 24 h. A disc containing only DMSO was used as a negative control. Ampicillin (800 µg per disk) was used as the standard antibiotic, and caspofungin (800 µg per disk) was used as the standard antifungal. A clear zone of inhibition, measured in millimeters, was determined as an indicator of antibacterial and antifungal activity.

The BMD (Broth Microdilution) test, as described in EUCAST EDef 7.3.2 for yeasts and CLSI M07 for bacteria inside various media mentioned in these papers, was used to conduct the minimum inhibitory concentration (MIC) analyses. In a summary, the stock solution of chemically synthesized powdered compounds (NHCs) used in antifungal and antimicrobial tests was made in DMSO, and serial dilutions were made in flat-bottom 96-well plates, in YPD (Yeast Peptone Dextrose) medium (2% peptone, 2% glucose, 1% yeast extract) at pH 6.5 for yeasts, and LB (Luria–Bertani). To achieve the requisite cell density and quantities of chemical compounds to be evaluated, yeast (1–5 × 10⁵ CFU mL⁻¹) and bacteria (1 × 10⁶ CFU mL⁻¹) cell solutions (inoculums) were produced in sterile water and introduced in equal volumes to 96-well plates containing various concentrations of the chemicals. The final concentrations of the chemicals were between 0.8 and 800 µM after the cell solutions had been added, and the cell concentrations needed for the test were 0.5–2.5 × 10⁵ CFU mL⁻¹ for yeasts and 5 × 10⁵ CFU mL⁻¹ for bacteria in the last phase. After incubation in yeasts for 24 h at 37 °C and bacteria for 16 to 18 h at 37 °C, plates were examined visually for bacteria and spectrophotometrically for the MIC. The lowest drug concentration that results in a growth reduction of at least 50% or more in yeasts when compared to the control (no drug) cell group, as well as the lowest drug concentration that results in no discernible growth in bacteria, is known as the MIC value.

2.6. In vitro anticancer activity assay

Anticancer properties of the samples were reported by Sharma et al.,²¹ it was evaluated against HCT116 (human colon cancer), A549 (human lung cancer), and BEAS-2B (human healthy lung cell) cell lines. All stock solutions of the complexes were prepared in DMSO. All cells were cultured in DMEM medium supplemented with 10% fetal bovine serum and 1% penicillin and streptomycin at 37 °C in a 5% CO₂ atmosphere. After the cells covered the surface of the flask by 70–80%, the old medium was discarded and washed several times with sterile PBS (pH 7.4). Trypsin was then added and distributed evenly on the cell surfaces. After incubation with trypsin for 5 min at 37 °C, trypsin activity was inhibited by adding a 2 fold volume of fresh medium. The resulting solution was centrifuged at 1000 rpm for 7 min, and then the old medium was replaced with 5 mL of fresh medium. Cells were counted and diluted to obtain a final concentration of 1 × 10⁵ cells per mL, then the cell solution was added to 96-well cell plate wells (1 × 10⁴ cells per well). Plates containing cells were incubated at 37 °C in a 5% CO₂ atmosphere for 24 h for cell attachment, and anticancer activity analyzes of the compounds were performed using the MTT method. For analysis, the test substance was diluted with fresh broth medium to obtain the desired concentration (0.8–800 µM) from the stock while the cells were incubated. The old medium was aspirated from the wells containing the cells and 100 µL of broth containing the test substance was added to the wells. The plates were then incubated at 37 °C in 5% CO₂ for 24 h. After this period, the medium containing the test substance was aspirated from the wells and 10 µL of MTT solution (5 mg mL⁻¹) and 90 µL of fresh broth medium were added to each well to obtain a final concentration of 0.5 mg per mL MTT and then incubated for 4 h at 37 °C. Optical density was read at 570 nm and 630 nm in the ELISA reader. Cell viability percentages were determined using the formula [(570–630 nm) ((test compound applied) cell group)/(570–630 nm) (control (no compound applied) cell group)] × 100. IC₅₀ was calculated based on logarithmic cell viability percentages.

2.7. Solution stabilite studies

Stock solutions (1 mM) of complexes 3a–e were prepared in DMSO, and the final solutions were diluted with the corresponding diluent (PBS, 50 mM, pH 7.4) to 20 µM. The final concentration of DMSO was ≤2% (v/v). UV-Vis spectra of all solutions were recorded at intervals (0, 24, 48, 72, and 144 h) for six days using a microplate reader spectrophotometer (BioTek Eon, Winooski, VT, USA). The solutions were stored at 37 °C under dark conditions throughout the experiment.

2.8. Bovine serum albumin (BSA) binding analysis

The BSA binding properties of the complexes were also analyzed using the Benesi–Hildebrand method with recorded UV-Vis spectra, as previously reported by Şahin et al.²² Briefly, a 15 µM BSA (≥98%, lyophilized powder) stock solution was prepared in PBS (50 mM, pH 7.4). The 15 µM of BSA solution was incubated with different concentrations of each complex (0, 2, 4, 6, 8, 10, 15, and 20 µM) at 37 °C in the dark for 30 min. The complexes were dissolved in DMSO and the solutions were diluted with the corresponding diluent (PBS, 50 mM, pH 7.4). The final concentration of DMSO was ≤2% (v/v). To analyze the binding process, the absorbances of the BSA solution at the same wavelengths were recorded as a control sample. Changes in the spectra were recorded between 250–350 nm using a microplate reader spectrophotometer (BioTek Eon, Winooski, VT, USA).

2.9. Computational details

All geometry optimizations and harmonic vibrational frequency calculations for newly synthesized Ag(i) complexes (3a–e) were performed using the GAUSSIAN 16 software package.²³ The electronic structure calculations were performed with the B3LYP hybrid exchange–correlation functional.^24–26 For the non-metal atoms (C, H, N, O, and Br), the 6-311++G(d,p) basis set was employed, while the silver center was treated using the LANL2DZ effective core potential.²⁷ Optimized molecular structures, frontier molecular orbitals (FMOs), and molecular electrostatic potential (MEP) surfaces were visualized with GaussView 6.²⁸

Thermodynamic parameters, enthalpy (ΔH), entropy (S), Gibbs free energy (ΔG), and heat capacity (C_v), play a key role in describing the energetic and thermal behavior of molecular systems.²⁹ These quantities originate from translational, rotational, vibrational, and electronic contributions to the molecular partition function, with vibrational motion providing the dominant contribution. Accordingly, the vibrational energy (E_vib), vibrational entropy (S_vib), and vibrational heat capacity (C_{v vib}) for 3a–e were evaluated using eqn (1)–(5), derived from quantum-mechanical formulations.^30–33 The relevant terms are defined as follows: Θ_ν,j = hν_j/k → vibrational temperature, k → Boltzmann constant, h → Planck constant, and ν_j → jth fundamental frequency.

Q = Q_trans. × Q_rot. × Q_vib. × Q_elec. 1

2

3

4

5

FMO analyses were carried out to obtain deeper insights into the reactive regions and overall reactivity characteristics of 3a–e. The ionization potential (I = −E_HOMO) and electron affinity (A = −E_LUMO) values were estimated on the basis of Koopmans' theorem.³⁴ Using these fundamental parameters, a series of quantum-chemical reactivity descriptors namely chemical hardness (η), chemical potential (µ), electronegativity (χ), electrophilicity index (ω), maximum charge-transfer capacity (ΔN_max), back-donation energy (ΔE_{back-donation}), electron-accepting power (ω⁺), and electron-donating power (ω⁻) were subsequently derived according to eqn (6)–(13), following established theoretical formulations reported in the literature.^35–41

6

7

8

9

ω⁺ ≈ (I + 3A)²/(16(I − A)) 10

ω⁻ ≈ (3I + A)²/(16(I − A)) 11

12

13

Within the chosen computational framework, Natural Bond Orbital (NBO) analyses were carried out for 3a–e using second-order perturbation theory based on the Fock matrix formalism.^42,43 This methodology allows for a detailed evaluation of intramolecular donor–acceptor interactions and provides quantitative insight into their associated stabilization effects. The corresponding stabilization energies, E⁽²⁾, were computed using eqn (14), where q_i denotes the donor orbital occupancy, F_ij represents the off-diagonal Fock matrix element, and ε_i and ε_j are the energies of the donor and acceptor orbitals, respectively.

14

3. Results and discussion

3.1. Synthesis and characterization

As the NHC core structure, a 5,6-dimethylbenzimidazole-based ligand bearing aryl substituents was selected, since stable coordination with silver was anticipated. In the first step of the synthesis procedure, benzimidazolium salts (2a–e) were obtained in high yields (81–91%) through the reaction of 1-substituted 5,6-dimethylbenzimidazoles with 2-2-bromoethyl-1,3-dioxane. The structures of the synthesized compounds were fully characterized by ¹H and ¹³C NMR spectroscopies, FT-IR, and LC-HRMS mass spectrometry. In addition, their purity (92–99%) was confirmed by HPLC analysis.

Examination of the ¹H NMR spectra revealed the characteristic acidic NCHN proton signals as singlets at 11.16, 11.10, 11.30, 11.24 and 11.26 ppm, respectively. The methyl groups bearing to the benzimidazole ring appeared as singlets in the 2,38–2.43 ppm range. In the ¹³C NMR spectra, the NCHN carbon signals were observed at 142.2, 142.8, 142.3, 142.2, and 142.4 ppm, respectively. The aromatic carbon resonances appeared in the range of 112–139 ppm, while the methyl carbons attached to the benzimidazole ring were detected at 20.7 ppm.

Ag(i)-NHC complexes (3a–e) were synthesized according to the procedure reported by Wang and Lin.⁴⁴ The reaction was carried out in dichloromethane and in the dark. The complexes were obtained with moderate yields (61–68%). The structures of the synthesized complexes were characterized using ¹H and ¹³C NMR spectroscopies, LC-HRMS, and FTIR techniques. Furthermore, the molecular structure of the complex 3c was unambiguously determined by single-crystal X-ray diffraction analysis.

Analysis of the ¹H and ¹³C NMR spectra revealed the disappearance of the characteristic acidic proton and carbon signals of the benzimidazolium salts, providing clear evidence of complex formation. Signals corresponding to the aromatic region were observed at 6.66–7.32 ppm in the ¹H NMR spectra and 111–139 ppm in the ¹³C NMR spectra. Furthermore, the peak associated with the Ag–C_carbene bond was not observed in complexes 3a–d, but was observed at 188.8 ppm in complex 3e. FT-IR, LC-MS, HPLC, ¹H NMR, and ¹³C NMR spectra of the synthesized complexes are given in the SI (Fig. S1–S25).

To verify the phase purity of the crystal structure, PXRD patterns of 3a, 3b, 3c, 3d, and 3e complexes recorded in the range (2θ = 0–80°) were shown in Fig. 2. The PXRD patterns of the metal complexes displayed sharp crystal peaks indicating their crystalline phases.⁴⁵ Therefore, these findings revealed the synthesized complexes was pure enough. In addition, the mean crystallite size of the complexes was calculated using the Scherer formula.⁴⁶ The mean crystallite sizes of the 3a, 3b, 3c, 3d, and 3e complexes were 34.4, 81, 61.5, 55.3, and 46.9 nm, respectively.

Fig. 2. PXRD patterns of 3a, 3b, 3c, 3d, and 3e complexes.

3.2. Antimicrobial evaluation of Ag(i)-NHC complexes

Ag(i)-NHC complexes (3a–e) were evaluated for their antibacterial and antifungal potentials using the disk diffusion method against three bacterial species including E. coli (Gram-negative bacterium), S. aureus (Gram-positive bacterium), and P. aeruginosa (Gram-negative bacterium), as well as two yeast species C. albicans and C. glabrata. Ampicillin was used as the positive control group for bacteria, and caspofungin was used as the positive control group for yeasts (Table 2). Moreover, Fig. S26 presents photographic images of the inhibition zones of Ag(i)-NHC complexes.

Table 2. The inhibition diameter zone values (mm) of Ag(i)-NHC complexes (3a–e), ampicillin and caspofungin.

Zones of inhibition (mm)
Sample code	C. albicans	C. glabrata	E.coli	P. aeruginosa	S. aureus
3a	7.8 ± 0.12	8.6 ± 0.14	12.5 ± 0.41	9.8 ± 0.22	15.5 ± 0.5
3b	8.3 ± 0.51	8.8 ± 0.16	11.1 ± 0.33	11.0 ± 0.05	18.3 ± 2.03
3c	7.1 ± 0.08	8.4 ± 0.09	11.5 ± 0.41	10.9 ± 0.09	12.2 ± 0.15
3d	8.0 ± 0.12	7.5 ± 0.08	11.3 ± 0.21	11.1 ± 0.17	20.3 ± 0.61
3e	9.9 ± 0.08	8.5 ± 0.05	11.3 ± 0.22	11.4 ± 0.05	18.3 ± 0.3
Ampicillin	—	—	17.6 ± 1.84	12.5 ± 0.41	15.3 ± 0.25
Caspofungin	23.5 ± 0.36	26.5 ± 0.39	—	—	—

When the antimicrobial activities of Ag(i)-NHC complexes against E. coli bacteria were compared with ampicillin (17.6 ± 1.84 mm), 3a complex showed the highest inhibition zone diameter of 12.5 ± 0.41 mm with the highest inhibition area. The complex 3c with 11.5 ± 0.41 mm, 3e and 3d complexes with 11.3 ± 0.21 mm and 11.3 ± 0.22 mm, 3b complex with 11.1 ± 0.33 mm zone diameters showed moderate activity. All Ag(i)-NHC complexes had lower zone diameters than ampicillin and showed moderate inhibition against E. coli bacteria.

When the antimicrobial activities of Ag(i)-NHC complexes against S. aureus bacteria were compared with ampicillin (15.3 ± 0.25 mm), 3d complex showed the highest inhibition zone diameter of 20.3 ± 0.61 mm with the highest inhibition area and had higher zone area than ampicillin. 3e and 3b complexes with 18.3 ± 0.3 mm and 18.3 ± 2.03 mm zone diameters had higher zone areas than ampicillin. 3a complex had 15.5 ± 0.5 mm zone diameter. The complex with the lowest inhibition zone diameter was 3c with 12.2 ± 0.15 mm having the lowest zone area.

When the antimicrobial activities of Ag(i)-NHC complexes against P. aeruginosa bacteria were compared with ampicillin (12.5 ± 0.41 mm), 3e complex showed the highest inhibition zone diameter of 11.4 ± 0.05 mm with the highest inhibition area. 3d complex with 11.1 ± 0.17 mm, 3b complex with 11 ± 0.05 mm, and 3c complex with 10.9 ± 0.09 mm zone diameters showed moderate activity. The complex with the lowest inhibition zone diameter was 3a with 9.8 ± 0.22 mm having the lowest zone area. All Ag(i)-NHC complexes had lower zone diameters than ampicillin.

When the antimicrobial activities of Ag(i)-NHC complexes against C. albicans yeast were compared with caspofungin (23.5 ± 0.36 mm), 3e complex showed the highest inhibition zone diameter of 9.9 ± 0.08 mm with the highest inhibition area. 3b complex with 8.3 ± 0.51 mm, 3d complex with 8 ± 0.12 mm, and 3a complex with 7.8 ± 0.12 mm zone diameters showed moderate activity. The complex with the lowest inhibition zone diameter was 3c complex with 7.1 ± 0.08 mm having the lowest zone area. All Ag(i)-NHC complexes had lower zone diameters than caspofungin.

When the antimicrobial activities of Ag(i)-NHC complexes against C. glabrata yeast were compared with caspofungin (26.5 ± 0.39 mm), 3b complex showed the highest inhibition zone diameter of 8.8 ± 0.16 mm with the highest inhibition area. 3a complex with 8.6 ± 0.14 mm, 3e complex with 8.5 ± 0.05 mm, and 3c complex with 8.4 ± 0.09 mm, zone diameters showed moderate activity. The complex with the lowest inhibition zone diameter was 3d complex with 7.5 ± 0.08 mm having the lowest zone area. All Ag(i)-NHC complexes had lower zone diameters than caspofungin.

In spectrum evaluation, Ag(i)-NHC complexes demonstrated broad-spectrum antimicrobial activity against both bacteria and yeasts. 3d complex showed particularly high activity against S. aureus bacteria, while 3e complex exhibited the highest antifungal activity against C. albicans yeast. 3a complex was the most effective against E. coli bacteria. 3e complex showed the highest activity against P. aeruginosa bacteria.

Taken together, according to the results presented in the Table 2, all complexes exhibited antibacterial activity against Gram-positive bacteria compared to Gram-negative bacteria. These results may be due to the fact that the cell wall of Gram-positive bacteria is single-layered, while the cell wall of Gram-negative bacteria is multi-layered. As expected, this multi-layered structure makes it difficult for the complexes to penetrate the cell wall.⁴⁷ This phenomenon is supported by similar studies reported previously. For instance, Doğan Ulu et al. evaluated the antibacterial activity of Ag(i)-NHC complexes against E. coli as and B. subtilis (Gram-positive) bacteria and some complexes exhibited better antibacterial activity against Gram-positive bacteria when compared to Gram-negative counterpart.⁴⁷ In another study, Yiğit et al. synthesized p-nitrobenzyl-substituted Ag(i)-NHC complexes and assessed antibacterial properties against four Gram-positive (S. aureus, E. faecalis, S. aureus MRSA, and B. cereus), four Gram-negative (E. coli, K. pneumonia, P. aeruginosa, and A. baumannii). The authors reported that overall antibacterial efficacy of compounds was higher against Gram-positive strains than Gram-negative strains.⁴⁸ Similarly, Boubakri et al. evaluated the antibacterial activity of N-alkylbenzimidazole-stabilized Ag(i) complexes against S. aureus, P. aeruginosa, and E. coli and some complexes displayed higher antibacterial activity against Gram-positive bacteria when compared to Gram-negative.⁴⁹

In order to more comprehensively evaluate and quantitatively determine the antimicrobial efficacy of the compounds, MIC values were determined (Table 3). Ligand 1a exhibited narrow-spectrum activity (MIC: 400 µM vs. C. albicans/C. glabrata; 800 µM vs. E. coli; inactive vs. P. aeruginosa). Benzimidazolium salt 2a showed a complete collapse of antifungal activity against both Candida species, suggesting that quaternization of the nitrogen center abolishes the antifungal pharmacophore of the scaffold. Crucially, complex 3a restored a uniform MIC of 200 µM across all five organisms, including P. aeruginosa-an activity absent in both precursors. This restoration constitutes direct evidence that the antimicrobial profile of the Ag-NHC complexes is an intrinsic property of the coordination framework and cannot be attributed to the organic components alone.

Table 3. MIC (µM) values of 1a–e, 2a–e, and 3a–e against to tested microorganisms^a.

Sample code	MIC (µM)
	C. albicans	C. glabrata	E. coli	P. aeruginosa	S. aureus
1a	400	400	800	Ineffective	800
1b	200	200	800	Ineffective	400
1c	400	50	Ineffective	Ineffective	200
1d	200	50	25	Ineffective	25
1e	200	200	12.5	Ineffective	12.5
2a	Ineffective	Ineffective	800	Ineffective	200
2b	800	Ineffective	400	Ineffective	100
2c	400	400	400	Ineffective	100
2d	800	Ineffective	400	Ineffective	100
2e	400	400	200	Ineffective	12.5
AgNO3	200	25	12.5	6.25	50
3a	200	200	200	200	200
3b	100	200	100	200	100
3c	200	100	100	200	100
3d	200	200	100	200	100
3e	200	200	100	200	100
Ampicillin	—	—	50	Ineffective	12.5
Caspofungin	25	6.25	—	—	—

^aIneffective: MIC > 800 µM.

Ligand 1b (MIC 200 µM vs. both Candida spp.) displayed superior antifungal potency relative to 1a. Benzimidazolium salt 2b largely abolished antifungal activity, consistent with the pattern observed across the series. Complex 3b achieved MIC values of 100 µM for C. albicans, E. coli, and S. aureus, and 200 µM for C. glabrata and P. aeruginosa, representing the broadest spectrum of activity in this subseries.

Ligand 1c was notable for a selective antifungal MIC of 50 µM against C. glabrata-the lowest value recorded across the entire free ligand series. Benzimidazolium salt 2c resulted in an 8-fold loss of this selective potency (50–400 µM). Complex 3c reestablished balanced broad-spectrum activity (100 µM vs. C. glabrata, E. coli, and S. aureus; 200 µM vs. C. albicans and P. aeruginosa), again with activation of P. aeruginosa efficacy from a baseline of complete inactivity in both precursors.

Ligand 1d was the most potent free ligand for antibacterial activity (MIC 25 µM vs. E. coli and S. aureus). Benzimidazolium salt 2d produced a 16-fold loss of E. coli potency (25–400 µM), representing the most severe attenuation upon salt formation in the series. Complex 3d fully reconstituted broad-spectrum activity, with MIC 100 µM vs. E. coli and S. aureus, and 200 µM for all remaining organisms including P. aeruginosa.

Ligand 1e achieved the lowest MIC values among all free ligands: 12.5 µM vs. both E. coli and S. aureus, directly comparable to the reference antibiotic ampicillin (12.5 µM). Benzimidazolium salt 2e was the only salt in the series to fully preserve S. aureus potency (12.5 µM), although E. coli activity suffered a 16-fold loss (12.5–200 µM). Complex 3e restored balanced activity (100 µM vs. E. coli and S. aureus; 200 µM for the remaining organisms), confirming the cross-series pattern.

MIC values of AgNO₃ (C. albicans: 200; C. glabrata: 25; E. coli: 12.5; P. aeruginosa: 6.25; S. aureus: 50 µM) were generally lower than those of the Ag-NHC complexes. This finding is mechanistically significant: rather than reflecting inferior activity, the higher MIC values of the Ag-NHC complexes are consistent with a controlled-release mechanism in which the NHC ligand modulates the bioavailability of Ag⁺. Importantly, the consistent emergence of P. aeruginosa activity (200 µM) across all five Ag-NHC complexes, in the complete absence of activity in all 10 ligand and salt precursors, constitutes definitive evidence that the biological effect originates from the coordination scaffold itself-not from uncoordinated silver ions or the free organic components.

Although the antimicrobial mechanism of Ag(i)-NHC complexes is not yet fully elucidated, various possible mechanisms have been proposed in the literature. It is emphasized that the mechanisms proposed in this study remain hypothetical, as no direct mechanistic investigations were conducted. Therefore, the observed antimicrobial activities are discussed only in light of previously reported literature findings. For example, positively charged Ag⁺ ions may have bound to negatively charged cell membranes, causing cell membrane/wall leakage and/or rupture.⁴⁰ However, the limited duration of action of Ag-containing antimicrobial agents is a significant disadvantage. Therefore, due to their stability, Ag(i)-NHC complexes may have released active Ag⁺ cations over a long period of time. This could be another reason underlying the improvement of antimicrobial activity.⁵⁰ On the other hand, the presence of certain anions such as sulfides, phosphates, and chlorides, as well as certain cations such as calcium and magnesium, can enhance the biological activity of Ag(i).⁵⁰ Therefore, the presence of bromide ions in the complexes may have contributed to the increase in antimicrobial activity. Similarly, Ronga et al. reported that the complexes must meet two requirements to exhibit antimicrobial activity.⁵¹ Firstly, they need to retain their ligands by releasing silver cations for an extended period, and secondly, they need to guarantee sufficient lipophilicity to allow the complex to penetrate and advance across the membrane, thereby deactivating the active enzyme sites of microorganisms. Another possible mechanism is that Ag⁺ ions interact with the bacterial membrane, binding to oxygen, nitrogen, and sulfur biomolecules present in the bacterial cell and disrupting cell growth, leading to cell death.⁵² The relationship in the variable antimicrobial activity exhibited by different structures is related to the substituents attached to the nitrogen atom in the benzimidazole rings of the complexes. These findings are consistent with literature studies reporting that Ag(i)-NHC complexes have significant antibacterial effects.^50,53,54

3.3. Cytotoxicity studies

In this study, the anticancer potential of the synthesized Ag(i)-NHC complexes (3a–e) was evaluated against BEAS-2B healthy lung epithelial cells, HCT116 colorectal carcinoma cells, and A549 lung cancer cells, using cisplatin as a reference drug (Table 4). Additionally, the anticancer activity of the complexes was statistically evaluated by one-way ANOVA using Tukey's multiple comparison (Table S1). The biological activity profiles clearly demonstrate that structural variations among the complexes significantly influence cytotoxicity and selectivity.

Table 4. IC₅₀ (µM) values Ag(i)-NHC complexes (3a–e) and cisplatin against BEAS-2B healthy lung epithelial cells, HCT116 colorectal carcinoma cells, and A549 lung cancer cells. Different letters indicate statistically significant difference at p < 0.05. The differences between the experimental groups were analyzed by one-way ANOVA using Tukey's multiple comparison.

Sample code	BEAS-2B	HCT116	A549
3a	121.73 ± 9.19a	17.47 ± 2.31f	51.37 ± 4.98c
3b	18.43 ± 0.22d	132.69 ± 14.14c	24.56 ± 2.50d
3c	10.74 ± 0.83e	165.13 ± 13.03b	46.67 ± 1.52c
3d	2.42 ± 0.17f	99.48 ± 0.05d	95.34 ± 8.24b
3e	73.57 ± 2.33c	45.46 ± 7.19e	14.71 ± 1.48e
Cisplatin	103.26 ± 3.26b	254.78 ± 6.11a	189.44 ± 5.13a

When the toxicity toward healthy BEAS-2B cells was considered, complex 3a exhibited the lowest cytotoxicity (IC₅₀ = 121.73 µM), even higher than cisplatin (103.26 µM), indicating a relatively favorable safety profile, and the difference was considered statistically significant for a p < 0.05. Complex 3e also showed comparatively low toxicity in healthy cells. In contrast, complex 3d displayed extremely high cytotoxicity toward BEAS-2B cells (IC₅₀ = 2.42 µM), suggesting a lack of selectivity and limiting its therapeutic applicability. Besides, the difference was considered statistically significant for a p < 0.05. Complexes 3b and 3c also demonstrated considerable toxicity in healthy cells, which may restrict their potential as drug candidates without further structural optimization.

In HCT116 colorectal cancer cells, complex 3a showed the most pronounced anticancer activity (IC₅₀ = 17.47 µM), markedly outperforming cisplatin (254.78 µM). This substantial difference (p < 0.05) indicates a strong antiproliferative effect of 3a against colorectal carcinoma cells. Complexes 3e and 3d also exhibited enhanced cytotoxicity compared to cisplatin, whereas 3b showed relatively weaker activity in this cell line (p < 0.05).

In A549 lung cancer cells, complex 3e emerged as the most potent derivative (IC₅₀ = 14.71 µM), demonstrating significantly stronger activity than cisplatin (189.44 µM). Besides, the difference was considered statistically significant for a p < 0.05. The remaining complexes (3a–d) also exhibited improved cytotoxic effects compared to the reference drug (p < 0.05), although their selectivity varied considerably.

The high anticancer activity of the synthesized Ag(i)-NHC complexes (3a–e) has been attributed to the lipophilic character of the compounds and the controlled release of Ag⁺ ions. Possible mechanisms of action include: (i) degradation of cell wall proteins by Ag⁺ ions,⁵⁵ (ii) increased bioavailability due to the stability provided by NHC ligands,⁵⁶ (iii) facilitated membrane permeability due to high lipophilicity,⁵⁷ and (iv) inhibition of cancer cell metabolism (replication and transcription) by bromide ions.⁴⁷ The present findings are in agreement with previous studies reported in the literature, which demonstrate the activity of Ag(i)-NHC complexes against various cancer cell lines.^58–60 It is emphasized that the mechanisms proposed remain hypothetical, as no direct mechanistic investigations were conducted. Therefore, the observed anticancer activities are discussed only in light of previously reported literature findings.

On the other hand, selectivity is a critical parameter in anticancer drug development. Notably, complex 3d, despite its strong cytotoxic profile, showed higher toxicity toward healthy cells than toward cancer cells. This lack of discrimination between normal and malignant cells suggests that 3d may induce nonspecific cytotoxic mechanisms. In contrast, complex 3a demonstrated a markedly higher IC₅₀ value in BEAS-2B cells than in HCT116 cells, indicating selective cytotoxicity toward colorectal cancer cells. Similarly, complex 3e showed lower toxicity in healthy cells compared to its strong activity in A549 lung cancer cells, suggesting selective anticancer potential.

The calculated selectivity indices (SI) further support these observations. Complex 3a (SI = 6.97) and complex 3e (SI = 5.00) both exceeded the commonly accepted selectivity threshold (SI > 2), highlighting their therapeutic promise. These findings suggest that subtle structural differences in the NHC framework and substituent patterns play a decisive role in modulating both potency and selectivity.

Overall, among the synthesized Ag(i)-NHC complexes, 3a and 3e appear to be the most promising candidates for further development. Their superior cytotoxicity compared to cisplatin in cancer cell lines, combined with relatively lower toxicity toward healthy cells, indicates a favorable therapeutic window. Future studies should focus on detailed structure–activity relationship (SAR) analysis, mechanistic investigations to elucidate their mode of action, and in vivo preclinical evaluations to validate their anticancer potential. Structural refinement aimed at enhancing selectivity while minimizing toxicity may further improve their pharmacological profile.

3.4. Solution stabilite studies

To determine the stability of metal complexes in biological applications, solution stability was investigated at 37 °C (PBS, pH 7.4) at specific time intervals (0, 24, 48, 72, and 144 h) as shown in Fig. 3. As shown in Fig. 3, no significant shift was detected except for minor fluctuations for the complexes.⁶¹ These results demonstrate the stability of these complexes in PBS-DMSO solution under physiological conditions.

Fig. 3. UV-Vis absorption spectra of complexes series, 3a, 3b, 3c, 3d, and 3e in PBS (50 mM, pH 7.4).

3.5. BSA binding analysis

Serum albumin is the most abundant protein in blood plasma and plays a major role in the transport and delivery of many drugs to disease sites. Therefore, studying the interactions between biologically active compounds and proteins can provide useful information about structural features that determine the therapeutic efficacy, pharmacological response, and dosage forms of drugs.⁶² BSA is widely used for protein binding studies due to its structural similarity to human serum albumin. Therefore, the BSA binding properties of the complexes were evaluated (Fig. 4 and S27–S30). A gradual increase in the absorbance intensity of BSA at 280 nm was observed with increasing concentration of complexes in the solution. This result suggested an apparent interaction between the complexes and BSA, leading to conformational changes in the protein structure and increased exposure of aromatic amino acid residues. Furthermore, the recorded absorbance values were assessed using the Benesi–Hildebrand equation.²² The results revealed that complex 3b showed the strongest binding affinity with a value of 7.9 × 10⁴ M⁻¹ (Fig. 4). In contrast, the binding constants of complexes 3a, 3c, 3d, and 3e were recorded as 1.7 × 10⁴ M⁻¹, 1.3 × 10⁴ M⁻¹, 6.3 × 10⁴ M⁻¹, and 1.1 × 10⁴ M⁻¹, respectively (Fig. S27–S30). The results of this study demonstrated that methyl-substituted complexes play a positive role in the interaction between BSA and complexes. Indeed, complexes substituted with 2-methyl and 4-methyl groups (complexes 3b and 3d) exhibited higher binding affinity compared to both unsubstituted complexes and complexes substituted with 3-methyl and 3,5-dimethyl groups (complexes 3c and 3e).

Fig. 4. UV-Vis spectra of BSA solution with the addition of complex 3b (0–20 µM); inset: the plot of 1/[µM] vs. 1/(A − A₀).

3.6. Description of the structure

Molecular structure of 3c was demonstrated by X-ray analysis, and its molecular structure, along with the designated atom-numbering scheme, was depicted in Fig. 2 as selected bond lengths and bond angles are provided in Table 4. The asymmetric unit of 3c consists of two half-molecules, labeled as A and B, with each molecule being completed by inversion symmetry, which is −x + 1, −y + 1, −z + 2 for molecule A and −x, −y + 2, −z + 1 for molecule B. In the following discussion, parameters for molecule B are quoted in square brackets, and only one (molecule A) of the two molecules is shown in Fig. 5 for the sake of simplicity.

Fig. 5. An inversion-symmetric dimer in the structure of 3c. Symmetry transformation used to generate equivalent atoms is −x + 1, −y + 1, −z + 2.

As shown in Fig. 5, the structure of NHC dimer contains a dimetallocyclic Ag₂Br₂ core with a crystallographic center of inversion at the mid-point of the Ag⋯Ag^a line, which is frequently found for silver complexes. Each silver atom is tri-coordinated with one carbene carbon atom and two bromide ions, and the two silver atoms are linked through two bromide bridges to afford an Ag₂Br₂ quadrangular arrangement. The silver atoms exhibit a pseudo-T-shape with the largest obtuse angle of 141.8(2)° [144.5(2)°] as a result of coordination from an additional bridging bromine atom. The Ag1/Br1/Ag1^a/Br1^a ring is strictly planar because of the inversion center. The Ag1–Br1^a distance of 2.7660(10) Å [2.7905(11) Å] is longer than the Ag1–Br1 bond of 2.5871(10) Å [2.5667(11) Å]. The absence of argentophilic interaction within the Ag₂Br₂ core is reflected by the Ag1⋯Ag1^a separation of 3.5759(3) Å [3.6179(3) Å], which is longer than the sum of two van der Waals radii for Ag (3.44 Å).⁶³ The angles of C1–Ag1–Br1^a, Br1–Ag1–Br1^a and Ag1–Br1–Ag1^a are 121.9(2)° [120.35(19)°], 96.24(3)° [95.15(3)°] and 83.76(3)° [84.85(3)°], respectively. The Ag1–C1 distance is 2.126(6) Å [2.113(7) Å], and the internal ring angle (N1–C1–N2) at the carbene center is 105.7(5)° [105.2(5)°]. The fact that N1–C1 and N2–C1 bonds are significantly shorter than N1–C2 and N2–C9 bonds demonstrates delocalization within the imidazole ring. The dihedral angle between the carbene and methylbenzene rings is 69.8(3)° [68.8(3)°], and they form the dihedral angles of 7.81(13)° [8.54(14)°] and 65.5(2)° [66.5(2)°] with the centric Ag₂Br₂ core, respectively. Furthermore, the substituents bound to the nitrogen atoms of the central NHC ring are placed in trans positions. The dioxane ring adopts the usual chair conformation and the puckering parameters⁶⁴ are Q = 0.555(10) Å [0.557(8) Å], q₂ = 0.022(10) Å [0.006(8) Å], q₃ = 0.555(10) Å [0.557(8) Å], θ = 2.7(10)° [0.0(8)°] and φ₂ = 144(26)° [19(74)°] for the atom sequence C20/O1/C21/C22/C23/O2. All the above-mentioned data are comparable to those of some known NHC-Ag–Br complexes.^58,65–69

3.7. Molecular geometry and thermodynamic parameters

Fig. 6 presents the DFT-optimized molecular structures of the newly synthesized silver(i) complexes 3a–e, and the corresponding selected experimental and calculated geometric parameters are compiled in Table 5. The DFT-optimized structures reproduce the experimentally determined bond lengths and bond angles with good agreement, with deviations typically within 0.05–0.20 Å for Ag–Br bonds and less than 0.06 Å for Ag–C(NHC) bonds, while larger deviations are observed for certain bond angles, particularly those involving the bridging bromide ligands which can be attributed to the absence of crystal packing effects in the gas-phase calculations. Single-crystal X-ray diffraction analysis shows that the complexes form centrosymmetric dimers featuring a characteristic µ₂-bromide-bridged Ag₂Br₂ core. Similar µ₂-halide bridged dinuclear silver(i) N-heterocyclic carbene complexes, in which each Ag(i) center coordinates one carbene carbon and two bridging halide ligands to form distorted Ag₂X₂ quadrangular units, have been observed in the literature for Ag-NHC bromide and chloride systems.⁷⁰ These structural motifs have been structurally characterized and discussed as common coordination modes for silver(i) NHC halide complexes. The Ag–Br interactions are markedly asymmetric, with shorter Ag–Br bond lengths in the range of 2.57–2.59 Å and longer Ag⋯Br contacts of 2.77–2.79 Å observed experimentally, a feature that is well reproduced by DFT calculations (2.64–2.98 Å). The Br–Ag–Br bond angles lie between 94.65° and 95.65°, while the Ag–Br–Ag angles fall in the range of 85.35–85.35°, consistent with a rhomboidal Ag₂Br₂ core.⁷⁰ Notably, the Ag–C(NHC) bond distances remain nearly invariant across the entire series (2.113–2.126 Å experimentally and ≈2.180 Å from DFT), indicating that variations in the benzyl substitution pattern exert minimal influence on the primary Ag–carbene interaction. Furthermore, key structural parameters within the benzimidazole ring, including the N1–C1 and N2–C1 bond lengths (≈1.35–1.36 Å) and the N1–C1–N2 angle (≈105–106°), are essentially unchanged upon coordination, highlighting the structural rigidity of the benzimidazol-2-ylidene framework and the preservation of its electronic characteristics in these Ag(i) complexes.

Fig. 6. The optimized structures of 3a–e.

Experimental and DFT-optimized bond parameters of 3a–e.

Parameters	Exp.		DFT
Bond lengths (Å)	Molecule A	Molecule B	3a	3b	3c	3d	3e
Ag1–Br1	2.5871(10)	2.5667(11)	2.6408	2.6437	2.6393	2.6387	2.6364
Ag1–Bra	2.7660(10)	2.7905(11)	2.9615	2.9596	2.9681	2.9690	2.9762
Ag1–C1	2.126(6)	2.113(7)	2.179	2.180	2.179	2.179	2.178
N1–C1	1.361(8)	1.355(8)	1.362	1.363	1.363	1.363	1.363
N1–C2	1.388(7)	1.399(7)	1.396	1.395	1.396	1.396	1.396
N2–C1	1.355(8)	1.350(8)	1.356	1.356	1.356	1.356	1.357
N2–C9	1.399(7)	1.402(8)	1.395	1.396	1.395	1.395	1.395

^aA is the symmetry code being −x + 1, −y + 1, −z + 2 for molecule A and −x, −y + 2, −z + 1 for molecule B.

Parameters	Exp.		DFT
Bond angles (Å)	Molecule A	Molecule B	3a	3b	3c	3d	3e
Br1–Ag1–C1	141.8(2)	144.5(2)	148.9	148.2	149.2	149.4	149.8
Br1a–Ag1–C1	121.9(2)	120.35(19)	116.1	116.1	115.9	115.9	115.5
Br1–Ag1–Br1a	96.24(3)	95.15(3)	94.95	95.65	94.86	94.75	94.65
Ag1–Br1–Ag1a	83.76(3)	84.85(3)	85.04	84.35	85.13	85.25	85.35
Ag1–C1–N1	127.4(5)	127.7(5)	126.2	125.9	126.2	126.2	126.1
Ag1–C1–N2	126.9(5)	127.1(5)	127.7	128.1	127.8	127.8	127.8
N1–C1–N2	105.7(5)	105.2(5)	106.0	106.0	106.0	106.0	106.0
C1–N1–C2	110.8(5)	111.2(5)	110.8	110.8	110.8	110.8	110.8
C1–N2–C9	110.8(5)	111.7(5)	110.9	110.9	110.9	110.9	110.9

Thermodynamic parameters obtained from DFT frequency calculations (Table S2) indicate that all complexes possess extremely small dipole moments (DM = 0.0001–0.02 D), confirming their highly symmetric nature and supporting the centrosymmetric dinuclear Ag₂Br₂ motifs observed in the solid state. The calculated Gibbs free energies show a systematic stabilization across the series, with ΔG values ranging from −7667.747902 a.u. for 3a to −7824.959045 a.u. for the more substituted complex 3e. Among the series, complex 3c exhibits a particularly balanced thermodynamic profile, combining a relatively high entropy value (S = 390.785 cal mol⁻¹ K⁻¹) with moderate vibrational and thermal contributions (ΔE_vib = 627.401 kcal mol⁻¹; ΔE_thermal = 629.179 kcal mol⁻¹), which may contribute to its favorable crystallization behavior. A gradual increase in both vibrational and thermal energies from 3a to 3e correlates with the increasing steric demand of the benzyl substituents, while the Ag₂Br₂ core remains intact and energetically robust. Consistently high polarizability values (α = 689.275–734.527 a.u.) across the series further reflect the presence of soft, µ₂-bromide-bridged silver(i) centers, in agreement with previously reported dinuclear Ag-NHC halide systems.^71,72 Taken together, these results demonstrate that while substituent variation subtly modulates molecular flexibility and entropy, the Ag₂Br₂ framework retains its thermodynamic stability throughout the series.

3.8. Vibrational analysis

The experimental and scaled vibrational frequencies of complexes 3a–e, together with the corresponding vibrational assignments, are compiled in Table S2. Accordingly, the calculated frequencies show good agreement with the experimental FT-IR data after application of the scaling factor, with most characteristic bands reproduced within an acceptable deviation range, confirming the reliability of the DFT model for these systems. In the high-frequency region (3200–2800 cm⁻¹), all complexes display intense bands associated with aromatic and aliphatic C–H stretching vibrations. The aromatic ν(C–H) modes are predicted around 3139–3111 cm⁻¹, while the asymmetric stretching modes of CH₂ and CH₃ groups appear in the 3073–3009 cm⁻¹ region. The experimental data indicate bands at 3047, 3055, and 3061 cm⁻¹. The consistent presence of these bands across the series indicates that substitution on the benzyl moiety does not significantly perturb the local C–H bonding environment. The mid-frequency region (1600–1400 cm⁻¹) is dominated by ν(C C) and ν(C–N) stretching vibrations of the benzimidazole framework, often coupled with in-plane bending modes of CH₂ and CH₃ groups. Characteristic ν(C C) stretching vibrations in aromatic and conjugated systems typically appear in the mid-IR region around 1650–1430 cm⁻¹, and such assignments have been reported in both experimental and theoretical studies of related organic and coordination compounds.^73,74 These values are closely matched by the calculated ones. The persistence of these bands at similar frequencies for all complexes highlights the structural rigidity of the NHC backbone and suggests that coordination to silver does not disrupt the conjugated heteroaromatic system. Bands appearing in the 1400–1200 cm⁻¹ region arise mainly from mixed ν(CC)/ν(CN) stretching modes combined with CH₂ wagging, twisting, and CH₃ umbrella motions. Notably, vibrations associated with the C–N bond of the carbene framework are consistently identified around 1331–1317 cm⁻¹, providing spectroscopic support for the integrity of the NHC coordination environment across the series. In the lower-frequency region (below 1200 cm⁻¹), the spectra are characterized by various bending, rocking, wagging, and twisting modes of CH₂ and CH₃ groups, as well as ring-breathing vibrations of the benzimidazole and dioxane units. The calculated frequencies successfully reproduce these complexes coupled motions, although slightly larger deviations are observed in this region, which is typical for low-energy vibrational modes that are sensitive to intermolecular effects and solid-state packing. As a result, the close correspondence between experimental and scaled theoretical frequencies for complexes 3a–e confirms the validity of the vibrational assignments and demonstrates that substitution on the benzyl group mainly affects band intensities and minor shifts, rather than introducing new vibrational features. These findings further support the conclusion that the Ag₂Br₂-NHC core remains structurally and electronically conserved throughout the series.

3.9. FMO & MEP analyses

To further elucidate the electronic properties of the complexes, frontier molecular orbital analysis and global reactivity descriptors were evaluated. The calculated HOMO–LUMO energies and associated global reactivity parameters for complexes 3a–e are summarized in Table S4. The calculated FMO energies for complexes 3a–e reveal only minor variations across the series, indicating that substitution on the benzyl moiety has a limited influence on the overall electronic structure of the Ag-NHC core. The HOMO energies span a narrow range from −5.394 eV (3a) to −5.348 eV (3e), while the LUMO levels vary between −1.120 eV (3b) and −1.069 eV (3e). As a result, the HOMO–LUMO energy gaps remain nearly constant (ΔE = 4.269–4.287 eV), suggesting comparable kinetic stability and electronic robustness for all complexes. The chemical hardness (η) values are similarly uniform (2.135–2.144 eV), further supporting the notion that the µ₂-bromide-bridged Ag₂Br₂ framework dominates the electronic behavior of the system. Consistent with these findings, the chemical potential (µ) and electronegativity (χ) parameters exhibit only slight changes across the series, with µ ranging from −3.255 eV (3b) to −3.209 eV (3e). The electrophilicity index (ω) shows a modest decrease from 2.481 eV for 3b to 2.406 eV for 3e, indicating a subtle reduction in electrophilic character as the steric bulk of the N-aryl substituent increases. This trend is mirrored in the maximum charge transfer values (ΔN_max = 1.500–1.525), which remain relatively high and suggest that all complexes can accommodate significant electron density without destabilization. The nearly invariant back-donation energy (ΔE_back ≈ −0.535 eV) across the series highlights the consistent Ag-carbene σ-donation/π-back-donation balance, reinforcing the electronic resilience of the Ag-NHC interaction regardless of benzyl substitution pattern. Consequently, these electronic descriptors indicate that structural modification at the benzyl substituent primarily introduces steric effects, while the electronic properties of the dinuclear Ag₂Br₂-NHC core remain largely preserved.

The HOMO and LUMO distributions depicted in Fig. 7 clearly indicate a pronounced separation of the frontier molecular orbitals between the metal-halide core and the ligand framework in complexes 3a–e. For all complexes, the HOMO is predominantly localized on the Ag₂Br₂ core, with major contributions from the Ag(i) centers and the µ₂-bromide bridges. This localization highlights the metal/halide-centered nature of the HOMO and underscores the decisive role of Ag–Br interactions in shaping the electronic structure. In contrast, the LUMO is mainly concentrated on the π systems of the benzimidazole-based NHC ligands, while contributions from the metal centers remain minor. The ligand-centered character of the LUMO suggests that electron-accepting behavior is primarily governed by the benzimidazole framework and that the lowest-energy electronic transitions may involve charge transfer from the metal-halide core toward the ligand scaffold, consistent with a metal-to-ligand charge transfer (MLCT) character. Across the series, the HOMO–LUMO energy gaps remain within a narrow range of approximately 4.27–4.29 eV, indicating that the overall electronic stability of the complexes is largely preserved despite variations in the benzyl substituents. Nevertheless, the localization of the LUMO on the ligand implies that substituent effects can subtly modulate the electron-accepting ability of the ligands, potentially influencing the biological interaction profiles of the complexes.

Fig. 7. HOMO–LUMO (isoval: 0.02), and MEP (isoval: 0.0004) plots of 3a–e.

Furthermore, the molecular electrostatic potential (MEP) maps provide further insight into their charge distribution and possible reactive sites.^75,76 Accordingly, MEP surfaces of complexes 3a–e (Fig. 4) reveal a relatively balanced charge distribution rather than sharply separated highly positive and negative regions. The most negative electrostatic potential is mainly located around the µ₂-bromide ligands and, to a lesser extent, on the heteroatoms of the dioxane framework, confirming their electron-rich character and their dominant contribution to the electrostatic profile of the Ag₂Br₂ core. In contrast, strongly positive regions are not prominently observed. Instead, areas of moderately positive to near-neutral potential (displayed in light green tones) are largely distributed over the benzimidazole backbone and the benzyl substituents. This indicates that these ligand fragments do not behave as strongly electron-deficient sites, but rather provide a polarizable surface capable of engaging in weak electrostatic and noncovalent interactions. The Ag(i) centers do not appear as strongly positive regions on the MEP surfaces, indicating that the positive charge is effectively distributed through Ag–Br and Ag–C(NHC) bonding interactions rather than being localized on the metal center. Across the series, the overall MEP patterns remain highly similar, suggesting that substitution at the N-position primarily modulates the spatial extent of the electrostatic surface rather than inducing drastic changes in charge localization. Such a distribution, combining electron-rich bromide regions with relatively neutral ligand surfaces, may be favorable for biological interactions, where subtle electrostatic complementarity rather than strong localized charges often governs binding to biomolecular targets.

3.10. NBO analysis

Natural Bond Orbital (NBO) analysis was performed to gain deeper insight into the intra- and intermolecular electronic interactions governing the stability of complexes 3a–e. An overview of the most relevant resonance interactions is presented in Table S5. In all cases, the electronic structure is dominated by extensive π → π* delocalization within the benzimidazole and aryl fragments, with stabilization energies typically ranging from ∼17 to 52 kcal mol⁻¹. These interactions reflect efficient conjugation across the heterocyclic backbone and remain largely unaffected by variations in benzyl substitution, underscoring the electronic rigidity of the NHC framework. A key feature common to all complexes is the presence of significant donor–acceptor interactions involving the bromide ligands and the silver centers. Lone-pair donation from Br to vacant Ag orbitals (LP (Br) → LP* (Ag)) consistently contributes stabilization energies of approximately 8-19 kcal mol⁻¹, confirming the µ₂-bromide bridging mode and highlighting the partially covalent character of the Ag–Br interactions. In several cases, additional Br lone-pair donation into σ* orbitals of neighboring C–C or C–O bonds is observed. The Ag(i) centers themselves also participate actively in donor–acceptor interactions. Lone pairs on silver donate electron density into σ orbitals of adjacent ligand bonds (LP(Ag) → σ*(C–C/C–H)), with stabilization energies typically in the range of 6.29–24.32 kcal mol⁻¹. These interactions point to a non-negligible back-donation component that complements the strong σ-donation from the carbene carbon, reinforcing the Ag-NHC bond without significantly altering its strength across the series. Notably, nitrogen lone pairs on the benzimidazole moiety engage in pronounced LP(N) → π* interactions with adjacent aromatic systems, yielding stabilization energies of ∼30–60 kcal mol⁻¹. This consistent behavior across complexes 3a–e further emphasizes the preservation of electronic conjugation within the heterocycle, even upon coordination to silver and incorporation into a dinuclear framework. Among the series, complex 3c exhibits a particularly well-distributed network of donor–acceptor interactions involving Br, Ag(i), and the ligand framework, without extreme localization of stabilization energy in any single interaction. This balanced electronic delocalization may contribute to its favorable thermodynamic profile and enhanced crystallization behavior, as suggested by both the DFT thermodynamic data and the solid-state structural features. Consequently, the NBO findings confirm that the electronic stabilization of complexes 3a–e arises from a synergistic combination of π-delocalization within the ligand scaffold, strong σ-donation from the NHC carbon, and substantial Br → Ag charge transfer within the µ₂-bromide-bridged Ag₂Br₂ core. Substituent effects are therefore largely steric in nature, while the fundamental electronic architecture of the dinuclear silver framework remains conserved.

3.11. Structure–activity relationship (SAR) analysis

Although complexes 3a–e share the same Ag(i)-NHC coordination framework and differ only in the position and number of methyl substituents on the benzyl moiety, they exhibited distinct antimicrobial selectivity and antiproliferative activities. The DFT calculations revealed nearly identical frontier orbital energies and HOMO–LUMO energy gaps for all complexes, indicating that methyl substitution exerts only a minor influence on the intrinsic electronic structure of the Ag-NHC core. Therefore, the observed biological differences cannot be explained solely on the basis of electronic descriptors. Instead, the biological behavior of the complexes is likely governed by the combined influence of several physicochemical factors.¹³ Although methyl substitution is not expected to markedly alter the total lipophilicity of the complexes, changing the position or number of methyl groups modifies the three-dimensional presentation of the benzyl substituent and the spatial distribution of the hydrophobic surface around the Ag(i)-NHC scaffold.⁷⁷ Such subtle structural differences may influence how the complexes interact with biological membranes, serum proteins, and intracellular biomolecular targets, ultimately affecting cellular uptake and biological selectivity. In addition, the different substitution patterns may slightly alter the steric accessibility of the Ag(i) center, which could further contribute to differences in biomolecular recognition. Although the MEP maps display similar electrostatic characteristics, slight differences in the spatial orientation of the benzyl substituents may influence how the complexes are presented to biological interfaces. The experimental stability studies demonstrated that all complexes remain sufficiently stable under physiological conditions, while the BSA-binding experiments confirmed their ability to interact with serum proteins, suggesting that differences in protein transport and bioavailability may also contribute to the observed biological responses.⁷⁸ Collectively, these findings indicate that the distinct antimicrobial and antiproliferative activities of complexes 3a–e arise from the interplay of steric effects, hydrophobic surface distribution, solution behavior, protein-binding characteristics, and the intrinsic properties of the Ag(i)-NHC framework rather than from substantial changes in the electronic structure alone.

4. Conclusion

In summary, a series of µ₂-bromide-bridged dinuclear silver(i)-NHC complexes (3a–e) bearing benzimidazole-based ligands were successfully synthesized and comprehensively characterized by spectroscopic methods, single-crystal X-ray diffraction, and DFT calculations. The synthesized complexes were tested for both antimicrobial and anticancer activities. Overall, the synthesized Ag(i)-NHC complexes exhibit good antimicrobial activity, with complexes 3a and 3e appearing to be the most promising candidates for further development in cancer studies. Their superior cytotoxicity compared to cisplatin in cancer cell lines and their relatively lower toxicity against healthy cells indicate a favorable therapeutic window. Furthermore, the stability of the complexes in the biological media and their protein binding properties were investigated. The results revealed that the complexes displayed satisfactory stability and BSA-binding properties. X-ray analyses reveal centrosymmetric Ag₂Br₂ cores with rhomboidal geometries, while DFT-optimized structures closely reproduce the experimental metrics, confirming the robustness of the dinuclear framework. FMO analysis demonstrates a clear separation between a metal/halogen-centered HOMO and a ligand-centered LUMO, suggesting ligand-directed electron-accepting behavior and potential metal-to-ligand charge-transfer character. Thermodynamic and NBO analyses further indicate that substituent variations subtly modulate molecular flexibility without disrupting the electronic integrity of the Ag₂Br₂ core. MEP maps highlight predominantly neutral to mildly positive regions over the ligand scaffold, consistent with delocalized charge distribution and favorable interaction profiles.

Conflicts of interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability

CCDC 2497835 contains the supplementary crystallographic data for this paper.⁷⁹

The data supporting this article have been included as part of the supplementary information (SI). Supplementary information is available. See DOI: https://doi.org/10.1039/d6ra03398a.