Synthesis, Characterization, Antimicrobial Properties, and Antioxidant Activities of Silver-N-Heterocyclic Carbene Complexes

The emergence of antimicrobial resistance has become a major handicap in the fight against bacterial infections, prompting researchers to develop new, more effective, and multimodal alternatives. Silver and its complexes have long been used as antimicrobial agents in medicine because of their lack of resistance to silver, their low potency at low concentrations, and their low toxicity compared to most commonly used antibiotics. N-Heterocyclic carbenes (NHCs) are widely used for coordination of transition metals, mainly in catalytic chemistry. In this study, several N-alkylated benzimidazolium salts 2a–j were synthesized. Then, the N-heterocyclic carbene (NHC) precursor was treated with Ag2O to give silver (I) NHC complexes (3a–j) at room temperature in dichloromethane for 48 h. Ten new silver-NHC complexes were fully characterized by nuclear magnetic resonance (NMR), Fourier transform infrared spectroscopy (FT-IR), elemental analysis, and LC-MSMS (for complexes) techniques. The antibacterial and antioxidant activities of salt 2 and its silver complex 3 were evaluated. All of these complexes were more effective against bacterial strains than comparable ligands. With MIC values ranging from 6.25 to 100 g/ml, the Ag-NHC complex effectively showed strong antibacterial activity. Antioxidant activity was also tested using conventional techniques, such as 2, 2-diphenyl-1-picrylhydrazine (DPPH) and hydrogen peroxide scavenging assays. In DPPH and ABTS experiments, compounds 3a, 3b, 3c, 3e, 3g, and 3i showed significant clearance.


Introduction
Te world faces a perilous challenge known as antimicrobial resistance (AMR), the ability of microorganisms to withstand commonly used antibiotics [1]. Tis surge in AMR presents a signifcant risk to public health, leading to higher death rates, increased medical expenses, and reduced efectiveness of antimicrobial therapy [2]. Te rise in AMR can be traced back to the misuse of antibiotics and the scarcity of new drugs to replace the currently compromised antimicrobial drugs [3]. In response to the issue of multiple treatment-resistant germs, the pharmaceutical and medical industries are actively seeking new, powerful, and less toxic antimicrobial drugs. Te term antimicrobial resistance (AMR) refers to the capacity of microorganisms to withstand commonly-used antibiotics [1]. If left unchecked, this phenomenon can escalate morbidity, mortality, and healthcare expenditures [2,3]. Currently, the heterocyclic compounds are the most sought-after components of efective anti-AMR medications [4][5][6][7]. Te role of these compounds in the creation of antibacterial agents is highlighted through several key examples. Furthermore, metal-NHC complexes have been primarily utilized in catalytic chemistry [8,9]. In recent times, NHC ligands have exhibited potential as carrier molecules for anticancer drugs [10]. Researchers continue to explore the vast potential of NHC-metal complexes, which is evident from the increasing number of published reviews [11,12]. Combatting bacterial and cancerous infections could be aided with the application of Silver (I)-N-Heterocyclic Carbene (Ag(I)-NHC) complexes. Recent years have witnessed a surge in research regarding these compounds' potential for antimicrobial and anticancer applications [13,14]. Creating efective antimicrobial Ag − NHC requires limiting the Ag + dissociation rate in afected regions. Te structure of the NHC ligand holds sway over the activity of NHC-silver complex [15], with factors such as hydrophobic substitution and steric bulk on the imidazole ring causing a delay in silver ion release [16]. Tacke's group showed that the minimum inhibitory concentration (MIC) of the silver complex against various Gram-positive, Gram-negative, and mycobacteria ranged from 20 to 3.13 μg/mL (35.3 to 5.52 μM) [17]. Haque et al.published a comparative study in which a series of mononuclear and binuclear silver (I) complexes were synthesized [18][19][20]. Furthermore, silver carbene complexes show biological efects. [21] Gave an overview of this achievement, including the structural features and synthetic routes and uses of silver NHC complexes [22][23][24]. Te presence of bulky electron-donating substituents attached to carbene ligands enhanced the antibacterial activity of silver complexes [23][24][25][26][27][28].
Here, we report the synthesis, characterization, and study of antibacterial and antifungal activities of ten novel asymmetric benzimidazole salts and their substituted NHC silver complexes. Te characterization of these NHC-silver complexes is consistent with the proposed formula. Using the agar dilution method, the antimicrobial activity of these compounds was examined against Gram (+)/(−) bacterial and fungal strains. In antibacterial studies it was observed that the NHC-silver complex was more active against fungal strains than against Gram-positive and Gram-negative bacterial strains. In addition, the antioxidant properties of these compounds were also investigated.

Materials and Methods.
All manipulations were carried out in air. All chemicals and solvents were purchased from Sigma-Aldrich and Merck. Te solvents such as dimethylformamide (DMF), dichloromethane, and diethyl ether were purifed by distillation over the drying agents. Melting points were determined with an Electrothermal-9200 melting points apparatus. Te elemental analysis measurements were determined by LECO CHNS-932 elemental analyser. Fourier transform infrared spectra were obtained in the range 450-4000 cm −1 on a Perkin Elmer Spectrum 100 Spectrophotometer. Te mass analysis was determined by using a Termo Scientifc Exactive Plus Benchtop Full-Scan Orbitrap Mass Spectrometer LC-MS/MS analyzer. 1 H NMR and 13 C NMR spectra were recorded at 400 MHz (1H), 100 MHz ( 13 C) in CDCl 3 with tetramethylsilane as an internal reference (Malaty, Turkey). Te NMR studies were carried out in high-quality 5 mm NMR tubes. Signals are quoted in parts per million as δ downfeld from tetramethylsilane (δ � 0.00) as an internal standard. NMR multiplicities are abbreviated as follows: s � singlet, d � doublet, t � triplet, and m � multiplet. In the investigation of antimicrobial properties of silver-NHCs, some microorganisms defned in the American Type Culture Collection (ATCC) were preferred. Mueller-Hinton Broth was purchased from HiMedia Laboratories Pvt. Ltd. (Mumbai, India), and RPMI 1640 broth was purchased from Sigma-Aldrich (Chemie GmbH, Taufkirchen, Germany). Te spectroscopic data of the new silver-NHCs are presented as follows.  13

General Procedure for the Preparation of Silver(I)-
NHC Complexes 3a-j. Benzimidazolium salt (1.0 mmol) (2a-j) and Ag 2 O (1.5 mmol) were dissolved in 15 mL of dichloromethane and stirred at room temperature in the dark for 48 hours. Under reduced pressure, the solvent was extracted from the reaction mixture after fltration through celite.

Determination of Minimum Inhibitory Concentration of the Silver-Complexes 3a-j.
Te antimicrobial activity of silver-NHCs complexes 3 was reported in terms of the minimum inhibitory concentration (MIC), according to previous work [29][30][31][32].

(ABTS) Radical Scavenging Activity.
Tis manipulation was carried out according to the protocol proposed by Re et al. [35] with some modifcations.

Synthesis and Spectral
Characterization. NHC precursors and silver (I)-NHC complexes were successfully synthesized using a modifed procedure [36]. After deprotonation in a basic medium, the frst step is by equimolar amounts of 5,6-dimethylbenzimidazole and 4-(2-chloroethyl) morpholine in DMSO in the presence of KOH at room temperature the N-alkylation was carried out under reaction for 2 hours. To generate a single product upon formation of the NHC ligand, a second alkylation was performed at 80°C using 1 equivalent of some alkyl halide in refuxing toluene for 48 h. Following thin layer chromatography, the reactions were observed to form salts (2a-j) for each of the target chemicals. Te reaction was monitored by thin layer chromatography, after which salt formation (2a-j) was observed for each target compound. Te benzimidazolium salts (2a-j) are stable to air and moisture in both solid and solution states. Te following protocol can be used to prepare silver-NHC complexes. Treat the free NHC with the correct silver source; (ii) treat the azole salt with an alkaline silver source such as Ag 2 O, AgCO 3 and Ag(OAc); and (iii) treat the azole salt with a silver salt in the presence of an external base. Today, a popular and readily available method for the synthesis of silver-NHC complexes is the in situ deprotonation of azole salts using AgO as the main silver source. Terefore, in this work, silver NHC complexes (3a-3j) were prepared by in situ deprotonation method. Te reaction was carried out at room temperature for 24 hours under dark conditions in the presence of dichloromethane as solvent. Te target complex was obtained as an air-stable white solid in 70% to 80% yield. Silver complexes 3a-3j are soluble in most organic solvents such as CH 2 Cl 2 , CHCl 3 , EtOAc, and DMSO, with the exception of nonpolar solvents such as n-pentane, n-hexane, and Et 2 O Scheme 1.

FTIR Spectroscopy.
Bands in N-alkylated benzimidazoles (1) and salts (2) are attributable to Caliph-Nbenzimi stretching vibrations [37,38] and sometimes appear broad due to signal overlap with residual water molecules. Te signal at 2875 cm −1 is caused by the CH stretching vibration of the aromatic ring. Although the Khalifa H stretching vibration of the alkyl chain is thought to be responsible for the appearance of signals in the 2875-2963 cm −1 range in the spectrum of salt 2, these signals in the silver 3 complex were found to be due to the coordination of the Ag(I) ion weakening and electrondonating properties of the alkyl group. For complex 3, the CN stretching vibration occurs in a specifc four-fnger mode (f. fs) [39,40]. Te CN stretching vibration of the benzimidazole ring of salt 2 appears at 1356-1539 cm −1 , while complex 3 does not appear in this mode. Te CH vibrations of aromatic compounds occur at 729-901 cm −1 .
Te synthesis of salt 2 was confrmed by observing specifc signals for alkyl protons with chemical shifts of 0-6 ppm and aromatic protons in the range of 6-8 ppm. In addition, new signals of the most deshielded protons and carbons (NCHN) appear between 9 and 12 ppm between 140 and 145 ppm in 13C NMR and facilitate the synthesis of NHC ligands 2 confrmed [41]. During silver metallization, the NCHN proton resonance of [42] NHC salts disappeared at 9-12 ppm, which may indicate Ag-NHC bonding. Te aliphatic-CH 2 protons of the benzyl substituents of the benzimidazolium salts were observed to be between δ � 5.43-5.50 ppm. In addition, CH 2 proton singlets of morpholine were found at δ � 2.40 ppm and 3.60 ppm.
In the 13 C NMR spectrum of complex 3 of all silver NHCs, the characteristic signal of the C(2) carbon of benzimidazolium salt has completely disappeared, and the characteristic AgC(carbene) bond resonance of complex 3 was not observed, which is also the case in the literature mentioned and given as the reason for the fuctuating behavior of silver NHC [43]. Tis can be attributed to the      Bioinorganic Chemistry and Applications dynamic behavior of the silver complex in solution and the poor relaxation of the carbine quarter carbon. For complexes 3a-3j, aliphatic-CH 2 carbon resonances of benzyl substituents were detected between δ � 47.08-52.6 ppm. In addition, the CH 2 carbon resonance of morpholine was detected between δ � 52.6-67.01 ppm. Te elemental analysis data of the silver complex are also consistent with the expected structure. Figures 1 and 2. Table 1 summarizes some physical and spectroscopic data for the novel carbene-silver complex 3.
Silver NHCs exhibit multiple structural modes both in the solid state and in solution. Te properties of NHC ligands, temperature, solvent, and counterions are also afected by the silver NHC structure [43,45]. However, it is often not possible to determine the structure of silver-NHC complexes in solution due to the presence of diferent species that participate in rapid equilibration at ambient temperature. However, it is often impossible to determine the structure of silver-NHC complexes in solution due to the presence of diferent species that participate in rapid equilibration at room temperature.

Ligand exchange equilibria between neutral monocarbene complexes [AgX(NHC)] or ion pairs [Ag(NHC) 2 ] + [AgX 2 ]
were demonstrated by variable temperature NMR studies using the association mechanism. To better understand the structure of our complexes, many attempts have been made to generate suitable silver complex crystals using solvent diffusion methods using diferent solvent systems including CH 2 Cl 2 /Et 2 O and EtOH/Et 2 O. Despite all eforts, it was not possible to isolate single crystals suitable for X-ray examination from the silver complexes. However, in the absence of crystallographic data, mass spectroscopy can be used to elucidate the structure of silver-NHC complexes. To understand the behavior of these complexes in solvent, only one of the complexes 3, was studied by mass spectrometer LC/MS/ MS. For this reason, complex 3a was chosen as the model complex for mass analysis. Figure 3.
Te fragmentation leading to the m/z � 406.28 can occur via the mechanism of fragmentation given in Figure 4.

Antimicrobial Properties of Carbene-Based Silver-
Complexes 3a-j. Te minimum inhibition concentration (MIC), which is the lowest concentration of test sample that completely inhibits the growth of microorganisms, was determined for the antibacterial study by the broth dilution method and the disc difusion method, respectively [47]. Zones of inhibition against Escherichia coli (ATCC 25988), Pseudomonas aeruginosa (ATCC 27853), Klebsilla pneumonia (ATCC 700603), Staphylococcus aureus (ATCC 29213), and Methicillin-resistant Staphylococcus aureus (ATCC 43300) were measured, and the minimum inhibitory concentrations of test samples were determine. Figure 5 Te MIC values of compounds 2-3 against all bacterial strains are tabulated in Table 2. (Figure 6) As shown in Table 2, all silver NHCs inhibited the growth of bacterial and fungal strains. First, when we evaluated the antibacterial activity of silver NHC against Gram-negative bacterial strains, we could say that the 3a, 3c, and 3e complexes showed the same activity against Escherichia coli (ATCC 25988) bacteria. Te 3e, 3h, and 3i complexes showed the least activity against Pseudomonas aeruginosa (ATCC 27853) bacteria. Te 3c complex showed lower activity against Klebsiella pneumoniae (ATCC 700603) bacteria. Of all the silver complexes, complexes 3b and 3a are the most sterically demanding and most active against Gram-negative bacterial strains. Likewise, all silver-NHC complexes were less active than ampicillin against Staphylococcus aureus (ATCC 29213). Promising results can be said to have been obtained when the antibacterial activity of all silver-NHC complexes was evaluated compared with standard drugs such as ampicillin and fucarbazole.
If we evaluate the antibacterial activity of silver NHCs against fungal strains, it can be said that some silver NHCs are less active than fuconazole, an antifungal drug used for many fungal infections. For example, all complexes except 2e and 2g showed the same anti-C. albicans (ATCC 14053) Ag-C(carbene) bond resonance was not observed as a reason for the fuxional behavior of the Ag-NHCs [44]. Bold values represent the number of the synthesized compounds. 8 Bioinorganic Chemistry and Applications activity as fuconazole. Finally, silver NHC showed less or equal inhibitory activity against Gram-negative bacterial strains compared to Gram-positive bacterial strains and fungal strains. Tese results can be attributed to the outer membrane of Gram-negative bacteria making them more resistant to antimicrobial agents. Tese results suggest that N atom substitution of benzimidazol-2-ylidene ligands plays an important role in the antibacterial activity.   10 Bioinorganic Chemistry and Applications Te antibacterial activity of NHC salts 2 showed lower minimal inhibitory concentration when compared to that of silver (I)-NHC complexes 3. Interestingly, the silver-ligand bonding of complexes 3 resulted in promising activity against all bacterial strains, similar to that of ciprofoxacin. Tis could be due to the synergistic efects of silver-ligand bonding, which enhances the lipophilicity of complexes. It has been previously demonstrated that the silver-ligand bond is the critical factor for antibacterial activity, over other factors like degree of polymerization, chirality, or solubility of these complexes. In addition, the Ag(I)-NHC complexes displayed a broad antibacterial spectrum, potentially caused by ligand exchange phenomenon with S-(thiols).
Direct interaction occurs between silver ions and biological ligands, such as membranes, proteins, DNA and enzymes, while N-or O-donors at potential target points, such as bacterial sulfur containing proteins and enzymes, play a signifcant role [48]. Te antimicrobial potential of silver complexes depends on the ease of the ligand exchange process that enables release of Ag + ions, with the NHC ligands in silver (I)-NHC complexes only acting as carriers of silver ions to target sites in biological systems [49,50]. Antimicrobial activity is infuenced not only by the nature of silver complexes but also by the type of bacterial strains present, making it difcult to draw clear conclusions about the structure activity relationship that displays antimicrobial  potential [51]. However, Table 2 indicates that the sensitivity of B. cereus to silver compounds is apparent from the larger zones of inhibition and smaller MIC values [52]. In addition, the presence of the benzimidazole aromatic ring increases lipophilicity and activity, which helps silver ions to pass through the cell membrane and enter the cell, destroying the function of organelles, resulting in obstruction of the respiratory system and respiratory system. Metabolic mechanisms of microorganisms [26,53]. Te cellular functions of microorganisms are afected by the interaction of silver ions with cellular proteins and DNA, which interact with thiol groups of various enzymes, causing them to denature. Te activity of silver complexes on bacteria and fungi is closely related to their solubility, stability, lipophilicity and release rate of Ag + ions. In previous studies, the authors reported increased antibacterial activity due to the lipophilicity of the silver-NHC complex and the increased release rate of Ag + ions [27,54]. For this reason, we here attempted to use diferent N-substituents on the NHC backbone to compare the efect of N-substituents on stability and lipophilicity. In addition, we believe that ether functional substituents can increase water solubility, so silver NHC may be more efective in antibacterial detection.

Conclusion
In conclusion, a series of silver-NHC complexes have been synthesized and studied using various spectroscopic and analytical techniques. Te antibacterial properties of each silver-NHC complex were tested against four Gram-negative, three Gram-positive, and one fungal strains. Tese silver-NHC complexes showed antibacterial activity against bacteria and fungi with MIC values ranging from 6.25 to 100 g/ml. Diferent substituents on the NHC ligands were found to alter the biological activity of the complex against bacteria. Diferent nitrogen atom substituents have diferent efects on antibacterial activity. A bulkier and lipophilic substituent directly attached to the nitrogen atom of the benzmidazol- 2-ylidene   compound   3a  3b  3c  3d  3e  3f  3g  3h  3i  3j  2a  2b  2c  2d  2e  2f  2g  2h  2i  ligand positively afects antibacterial activity. Terefore, this study will help researchers develop new antibacterial and antifungal drugs with higher potency. [28,43,[55][56][57].

Data Availability
Te data used to support the fndings of this study are available from the corresponding author upon request.

Conflicts of Interest
Te authors declare that there are no conficts of interest.