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Article

In Vitro Evaluation of Silver-NHC Complexes Against a Clinical Isolate of Acanthamoeba castellanii: Time- and Dose-Dependent Effects

by
Zübeyda Akın-Polat
1,
Neslihan Şahin
2,*,
Shaima Hkiri
3,
Bui Minh Thu Ly
3,
İsmail Özdemir
4 and
David Sémeril
3,*
1
Departments of Medical Parasitology, Cumhuriyet University School of Medicine, 58140 Sivas, Türkiye
2
Department of Science Education, Faculty of Education, Cumhuriyet University, 58040 Sivas, Türkiye
3
Synthèse Organométallique et Catalyse, UMR-CNRS 7177, Strasbourg University, 67008 Strasbourg, France
4
Drug Application and Research Center, İnönü University, 44280 Malatya, Turkey
*
Authors to whom correspondence should be addressed.
Inorganics 2025, 13(6), 204; https://doi.org/10.3390/inorganics13060204
Submission received: 27 May 2025 / Revised: 16 June 2025 / Accepted: 17 June 2025 / Published: 18 June 2025
(This article belongs to the Special Issue Metal Complexes Containing Bioactive Ligands: Structure and Biological Evaluation)
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Abstract

:
The synthesis of a series of six chloro[N-alkyl-N-cinnamyl-benzimidazol-2-yliden]silver(I) complexes was successfully achieved, wherein allyl (3a), methoxymethyl (3b), benzyl (3c), 3-fluorobenzyl (3d), 4-fluorobenzyl (3e) and 4-methyl-benzyl (3f) substituents were grafted on the benzimidazole ring. The isolated silver N-heterocyclic carbene (NHC) complexes were identified by microanalyses and mass spectrometry and characterized by FT-IR and NMR spectroscopic techniques. Conclusive evidence for the structures of complexes 3c and 3d was provided by single-crystal X-ray crystallography. The in vitro inhibitory activity of the six Ag-NHC complexes was tested against trophozoites and cysts of the pathogenic Acanthamoeba castellanii strain and the efficacy sequence is as follows: 3d > 3c > 3f > 3a > 3b > 3e. At a concentration of 100 µM in complexes 3c, 3d and 3f and after 72 h of incubation, 5.3, 3.2 and 6.3% A. castellanii trophozoite viabilities were observed, respectively. The utilization of elevated silver(I) drug concentrations, 1000 µM, resulted in the near-total eradication of pathogenic protozoa.

Graphical Abstract

1. Introduction

Acanthamoeba spp. are free-living amoebae that are widely distributed in soil, freshwater, and the air [1,2]. Some species have been identified as opportunistic human pathogens. These protozoa can cause severe infections, including Acanthamoeba keratitis (AK), which is a painful corneal disease and granulomatous amoebic encephalitis, which is a typically fatal infection of the central nervous system occurring in immunocompromised individuals [3,4,5]. Acanthamoeba species exhibit a biphasic life cycle, characterized by the alternating phase between a metabolically active trophozoite stage and a dormant cyst stage. The latter stage is distinguished by its resilience to environmental stress and therapeutic agents. Furthermore, it has been demonstrated that both stages are capable of initiating infection [2,4].
AK has been identified as the most prevalent disease caused by Acanthamoeba. It primarily affects contact lens wearers, particularly those with corneal microtrauma or poor lens care practices, such as inadequate cleaning or exposure to contaminated water during activities like swimming or showering. The clinical manifestations include severe ocular pain, conjunctival hyperaemia, blurred vision, photophobia, and a foreign body sensation in the eye. Given the non-specific nature of these symptoms, they frequently masquerade as other forms of keratitis. This underscores the critical importance of a prompt and accurate diagnosis to avert severe consequences. In the absence of intervention, AK has the potential to escalate to a state of corneal ulceration, which may consequently lead to irreversible visual impairment or even total blindness [6,7,8,9].
Presently, there is an absence of an FDA-approved standard treatment protocol for AK. Treatment regimens generally consist of prolonged therapeutic interventions that incorporate topical antiseptics, such as polyhexamethylene biguanide and chlorhexidine, in combination with antimicrobials, including diamidines and azoles. Notwithstanding these therapeutic regimens, the degree of therapeutic success remains limited, and recurrence is not uncommon. Moreover, the majority of commercial contact lens disinfectants demonstrate an absence of amoebicidal activity against Acanthamoeba, thereby increasing the risk of infection for lens wearers [10,11,12]. These challenges underscore the pressing need to develop more effective, targeted therapeutic strategies for AK [13,14].
The exploration of organometallic compounds as potential drug candidates has emerged as a promising field within the bioinorganic chemistry [15,16]. Among these compounds, metal-N-heterocyclic carbene (NHC) complexes have attracted considerable attention in synthetic chemistry [17,18,19]. The introduction of NHCs into organometallic chemistry is attributed to Fischer in 1964. However, it was not until 1991 that a free NHC was successfully isolated, despite the existence of early reports of NHC metal complexes dating back to 1968 [20,21,22,23,24]. NHCs are distinguished by their robust stability, which is attributed to the coordination of two nitrogen atoms in close proximity, enabling them to form strong bonds with transition metals [25]. The bonding interactions between NHC ligands and metal centers are shaped by a combination of steric and electronic effects [26]. Initially, it was hypothesized that NHC complexes function exclusively as σ-donors. However, subsequent research has revealed a more intricate nature of these interactions. The inherent stability of NHCs, in conjunction with their facile synthesis, has resulted in their extensive utilization in the domain of coordination chemistry. Among metal-NHC complexes, Ag-NHC complexes are notable for their biological properties [27,28,29,30].
While biguanides, diamidines, and azoles have demonstrated efficacy in the treatment of Acanthamoeba, limitations such as prolonged treatment durations, toxicity to human corneal cells, and incomplete cyst eradication have prompted the exploration of novel agents [10,12]. Silver-based compounds, including silver nitrate, silver sulfadiazine, and silver acetate, have been recognized for their antimicrobial properties for a considerable duration. These compounds have been employed as effective microbiocidal agents for millennia [31,32,33,34]. Furthermore, studies have demonstrated the efficacy of silver nanoparticles in combating Acanthamoeba [35,36,37]. Notably, silver compounds exhibit minimal toxicity toward human cells, particularly mammalian cell membranes.
To date, and to the best of our knowledge, there has been no report of research on the use of silver-N-heterocyclic carbene complexes against A. castellanii. In the present article, we report on the synthesis of six chloro[N-alkyl-N-cinnamyl-benzimidazol-2-yliden]silver(I) complexes, 3a–f (Figure 1), and their in vitro tests against A. castellanii.

2. Results and Discussion

2.1. Synthesis of Silver Complexes

Six silver(I) complexes (3a-f), namely chloro[1-cinnamyl-3-allyl-benzimidazol-2-yliden]silver(I) (3a), chloro[1-methoxymethyl-3-cinnamyl-benzimidazol-2-yliden]silver(I) (3b), chloro[1-benzyl-3-cinnamyl-benzimidazol-2-yliden]silver(I) (3c), chloro[1-(3-fluorobenzyl)-3-cinnamyl-benzimidazol-2-yliden]silver(I) (3d), chloro[1-(4-fluorobenzyl)-3-cinnamyl-benzimidazol-2-yliden]silver(I) (3e) and chloro[1-(4-methyl-benzyl)-3-cinnamyl-benzimidazol-2-ylidene]silver(I) (3f), were obtained in satisfactory yields (33–86%) from a reaction of Ag2O with the benzimidazolium salt (1a–f) in dichloromethane for 24 h at room temperature, with the reaction protected from light by aluminum foil (Scheme 1). The benzimidazolium salts 1a–f were previously prepared by alkylation of 1-cinnamyl-benzimidazole (2) and aryl chloride. Note that the alkyl substituents grafted on the benzimidazole only slightly modify the σ-donor properties of the N-heterocyclic carbenes. Indeed, the 1JCH constants demonstrate minimal variability within the range of 220–225 Hz (Table 1). This finding indicates that these ligands exhibit weak σ-donor properties, which are comparable to those of 1,3-dicyclohexyl-imidazolium salt [38].
The silver(I) complexes demonstrated stability in the presence of air and moisture; however, they exhibited sensitivity to light. In the dark, these complexes remained stable in solution of dimethyl sulfoxide (DMSO) for at least three days. The characterization of the inorganic samples was performed using Fourier transform infrared spectroscopy (FT-IR), nuclear magnetic resonance spectroscopy (1H, 13C{1H}, and 19F{1H} NMR) and mass spectrometry (see the experimental section, Supplementary Materials and Table 1).
The benzimidazolium salts 1a–f exhibited a distinctive ν(CN) band within the FT-IR spectra, with a frequency range of 1556–1562 cm−1. Following the formation of silver complexes, no peaks were detected in this region, and a downshift was observed for the ν(CN) bands (1379–1394 cm−1). These values are in agreement with those previously observed by Panda, Ghosh and co-workers in the chloro[1-benzyl-3-tert-butylimidazol-2-yliden]silver(I) complex (ν(CN) at 1375 cm−1) [40]. The observed change in stretching frequency can be attributed to a weakening of the CN bond, which becomes a partial double bond after coordination. This assertion is supported by the X-ray structures of two complexes (vide infra), in which the distances of carbon-nitrogen bonds are longer than that of a C=N bond (Table 1).
A thorough analysis of the 1H NMR spectra of the silver-NHC complexes (3a–f) revealed the absence of the downfield-shifted resonance (δ = 10.07–12.17 ppm) of the acid proton (NCHN) from the spectra of the N-heterocyclic carbene precursor salts, indicative of deprotonation of the benzimidazolium carbene. In the 13C{1H} NMR spectra of these salts, the signals from the carbon atom between the two nitrogen atoms (NCHN), present in the 142.71–144.26 ppm range, undergo significant deshielding after coordination to the silver atom. The analysis of the spectra of the complexes reveals the presence of NC(Ag)N carbon peaks in the 188.49–189.04 ppm range for 3a, 3d and 3e. As is the case with numerous silver(I) complexes, the signal in question could not be observed in the case of complexes 3b, 3c and 3f [41]. During the coordination step, the trans conformation of the double bond of the cinnamyl substituent remains unchanged, the coupling constant between the two protons of the double bond is approximately 16 Hz (Table 1).
A detailed analysis of the mass spectra of the six silver(I) complexes confirmed the successful formation of the desired complexes. Each organometallic compound displays a peak that corresponds to the cations [M − Cl]+ at m/z = 481.05, 385.05, 431.06 and 445.08 for complexes 3a, 3b, 3c and 3f, respectively and/or [M − Cl + CH3CN]+ at m/z = 422.07, 472.08, 490.08, 490.10 and 486.11 for complexes 3a, 3c, 3d, 3e and 3f, respectively, with the expected isotopic profiles.

2.2. Single-Crystal X-Ray Diffraction Studies

The formation of a silver(I) complex of the (NHC)Ag-Cl type was confirmed through two single-crystal X-ray diffraction studies of complexes 3c and 3d. The complexes exhibit centrosymmetric crystallization in the P 1 ¯ triclinic space group, with the center of inversion situated at the midpoint of the [Ag2Cl2] bridge core.
The formation of dimers is achieved through the establishment of an additional Ag–Cl bond (Ag1–Cl1a of 2.968(3) and 2.9320(6) Å for complexes 3c and 3d, respectively), which are substantially longer than the direct Ag1–Cl1 bond (Ag1–Cl1 of 2.3854(13) and 2.3972(5) Å for complexes 3c and 3d, respectively) (Figure 2 and Figure 3). Dimer formation is frequently observed during the crystallization of (NHC)Ag-Cl complexes, as evidenced in chloro[1-(9-acridinyl)-3-methylimidazol-2-yliden]silver(I) [42] or chloro[1-(N,N-diethylcarbamoylmethyl)-3-mesitylimidazol-2-ylidene]silver(I) [43] complexes.
The Ccarbene-Ag-Cl alignment is angled (C1-Ag1-Cl1 163.55(7) and 162.33(6)° for complexes 3c and 3d, respectively), and due to dimer formation, the Ag2Cl2 moiety is out of the plane of the benzimidazole ring, resulting in dihedral angles of 12.5 and 14.6° for complexes 3c and 3d, respectively. The Ag1-Cl1 (2.3854(13) and 2.3972(5) Å for complexes 3c and 3d, respectively) and Ag1-C1 distances (2.096(3) and 2.102(2) for complexes 3c and 3d, respectively) are within the usual range [44,45]. The two aromatic rings, which correspond to the cinnamyl and benzyl/3-fluorobenzyl substituents, are inclined toward the benzimidazole moiety, forming dihedral angles of 79.3/64.1° and 64.6/79.8°, respectively.

2.3. In Vitro Evaluation of Acanthamoeba castellanii

2.3.1. Effect on A. castellanii Trophozoites

The antiamoebic activities of the six silver(I) complexes (3a–f), the six benzimidazolium salts (1a–f), AgNO3, and Ag2O were evaluated against A. castellanii trophozoites after 24, 48 or 72 h of incubation at concentrations of 10, 100 or 1000 µM (Figure 4 and Figure 5).
A significant decrease in trophozoite viability was observed to be time- and concentration-dependent. The silver(I) complexes 3a–f exhibited significantly higher antiamoebic activities than their corresponding benzimidazolium salts (1a–f). At the highest concentration (1000 µM), all complexes demonstrated strong trophozoite-killing effects within 24 h. Complexes 3a, 3c, 3d and 3f completely eliminated trophozoite viability within 72 h. The residual viability in the 3b and 3e treatments was 3.4 and 2.2%, respectively (p < 0.05). In contrast, the corresponding carbene precursor salts exhibited only minimal inhibition at this concentration, with no statistically significant reduction in viability compared with the control group.
Overall, efficacy was reduced at 100 µM, but a similar trend was observed. Complexes 3c, 3d and 3f exhibited significant activity, with 5.3, 3.2 and 6.3% trophozoite viability, respectively, recorded after 72 h (p < 0.05). Ligands at the same concentration did not demonstrate any inhibitory effects. At the lowest tested concentration (10 µM), the complexes displayed minimal activities. All ligands yielded viability rates comparable to those of the untreated control group.
As illustrated in Figure 4, the efficacy of silver(I) complexes 3a–f against A. castellanii exhibits variation in the following sequence: 3d > 3c > 3f > 3a > 3b > 3e. This sequence provides compelling evidence that the lipophilicity [46,47] of the silver(I) complexes does not significantly impact the efficacy of these drugs. In fact, the two complexes with the lowest calculated logP [48] (complexes 3a and 3b with logP of 3.99 and 3.42, respectively; Table 2) are the least effective against A. castellanii. This observation underscores the pivotal function of the arylmethyl substituent grafted on to the N-heterocyclic carbene. The nature (H, F or CH3) and position (meta or para) of the substituent carried on this aromatic cycle are also of crucial importance. Indeed, the 3d complex, which contains a fluorine atom in the meta position of the benzylic substituent, exhibits higher reactivity compared with its less polar 3e counterpart, which possesses the fluorine atom in para position on the aromatic ring.
A study was conducted to ascertain the impact of AgNO3 and Ag2O on trophozoite viability. The study encompassed all concentrations and exposure times to ensure a comprehensive investigation. The findings indicate that neither compound exhibited a statistically significant reduction in trophozoite viability compared with untreated controls. Morphological evaluations substantiated these findings, disclosing the absence of cytopathic effects or aberrations from the conventional structure and motility of viable trophozoites (see Figure 5). The findings suggest that the silver salts examined were ineffective against Acanthamoeba trophozoites under these conditions, thereby emphasizing the enhanced efficacy and selectivity of the silver(I) complexes described in the present study.

2.3.2. Effect on A. castellanii Cysts

Following a 72 h observation period, no excystation or trophozoite formation was detected in cysts treated with complexes 3a, 3c, 3d or 3f at the highest tested concentration (1000 µM). This finding suggests the complete cysticidal activity of the complexes. Conversely, exposure to complexes 3b and 3e resulted in limited and delayed growth in cysts. Furthermore, minimal excystation was observed after 72 h at a concentration of 100 µM, and after 48 h at a concentration of 1000 µM, in cysts treated with complexes 3a, 3c, 3d and 3f. These results are of particular significance in light of the well-documented resistance of A. castellanii cysts to conventional antimicrobials [13]. The absence of any detectable activity by a ligand alone further emphasizes the importance of complexing with silver to achieve anti-amoebic potency.
At the lowest tested concentration (10 µM), none of the silver(I) complexes exhibited detectable cysticidal activity. In a similar manner, the ligands (1a–f) did not affect cyst viability at any time point (24, 48 or 72 h), and trophozoite outgrowth occurred at levels comparable to the untreated control. These results indicate that the ligands, when administered individually, were not efficacious against A. castellanii cysts under the conditions of the study. The present findings suggest that the silver(I) 3a–f complexes may offer an effective alternative or adjunctive therapeutic strategy by targeting both the trophozoite and cyst stages.

3. Materials and Methods

3.1. General

The synthesis of silver(I) complexes was carried out under an inert atmosphere of argon with dried solvents. 1H, 13C{1H} and 19F{1H} NMR spectra were recorded with Bruker Avance III spectrometer (300 or 500 MHz). The spectra were calibrated according to the residual protonated solvent in CDCl3 (δ = 7.26 and 77.16 ppm for 1H and 13C, respectively) or in DMSO-d6 (δ = 2.50 and 39.52 ppm for 1H and 13C, respectively). 19F NMR spectra are given relative to external CCl3F. Chemical shifts and coupling constants are reported in ppm and Hz, respectively. ESI-TOF spectra were recorded on a Bruker MicroTOF spectrometer. Infrared spectra were recorded on a Bruker FT-IR Alpha-P spectrometer. 1-Cinnamyl-benzimidazole (2) [49], 1-benzyl-3-cinnamyl-benzimidazolium chloride (1c) [39], 1-(3-fluorobenzyl)-3-cinnamyl-benzimidazolium chloride (1d) [39], and 1-(4-fluorobenzyl)-3-cinnamyl-benzimidazolium chloride (1e) [39] were prepared according to literature procedures.

3.2. General Procedure for the Synthesis of Benzimidazolium Salts

The 1-cinnamyl-benzimidazole (2) (234 mg, 1 mmol) was first dissolved in DMF (5 mL) before the addition of aryl chloride (1 mmol). The resulting solution was then subjected to a heating process at a temperature of 80 °C for a duration of 24 h. Subsequent to a period of cooling to room temperature, the salt underwent precipitation by the addition of Et2O (100 mL). The white solid was filtered and washed twice with Et2O (2 × 10 mL) prior to being dried under vacuum.
1-Cinnamyl-3-allyl-benzimidazolium chloride (1a): Yield: 36%; FT-IR: ν(CN) 1556 cm−1; 1H NMR (300 MHz, CDCl3): δ = 11.78 (s, 1H, NCHN), 7.79–7.74 (m, 1H, arom CH), 7.72–7.67 (m, 1H, arom CH), 7.64–7.58 (m, 2H, arom CH), 7.41–7.37 (m, 2H, arom CH), 7.34–7.28 (m, 3H, arom CH), 6.92 (d, 1H, CH=CHPh, 3JHH = 15.9 Hz), 6.39 (dt, 1H, CH=CHPh, 3JHH = 15.9 Hz, 3JHH = 6.7 Hz), 6.18–6.05 (m, 1H, CH=CH2), 5.52–5.46 (m, 4H, CH2CH=CH and CH=CH2), 5.31 (d, 2H, CH2CH=CH2, 3JHH = 6.0 Hz); 13C{1H} NMR (126 MHz, CDCl3): δ = 143.88 (s, NCHN), 137.18 (s, CH=CHPh), 135.12, 131.67, 131.64, 128.99, 128.88, 127.30, 127.24, 127.08, 113.81, 113.78 (10 s, arom Cs), 129.71 (s, CH=CH2), 122.02 (s, CH=CH2), 120.32 (s, CH=CHPh), 50.37 (s, CH2CH=CHPh), 50.21 (s, CH2C=CH2) ppm. Elemental analysis (%): calcd for C19H19N2Cl (310.82): C: 73.42; H: 6.16; N 9.01; found C: 73.34; H: 6.21; N: 8.97.
1-Methoxymethyl-3-cinnamyl-benzimidazolium chloride (1b): Yield: 38%; FT-IR: ν(CN) 1556 cm−1; 1H NMR (500 MHz, CDCl3): δ = 11.94 (s, 1H, NCHN), 7.81 (d, 1H, arom CH, 3JHH = 9.0 Hz), 7.72 (d, 1H, arom CH, 3JHH = 9.0 Hz), 7.60–7.55 (m, 2H, arom CH), 7.34 (d, 2H, arom CH, 3JHH = 8.5 Hz), 7.26–7.19 (m, 3H, arom CH), 6.86 (d, 1H, CH=CHPh, 3JHH = 15.5 Hz), 6.39 (d br, 1H, CH=CHPh, 3JHH = 15.5 Hz), 6.03 (s, 2H, CH2OCH3), 5.42 (d, 2H, CH2CH=CH, 3JHH = 2.0 Hz), 3.46 (s, 3H, CH2OCH3); 13C{1H} NMR (126 MHz, CDCl3): δ = 144.14 (s, NCHN), 137.35 (s, CH=CHPh), 135.03, 131.73, 131.16, 129.03, 128.89, 127.66, 127.59, 127.09, 114.42, 113.73 (10 s, arom Cs), 120.07 (s, CH=CHPh), 79.15 (s, CH2OCH3), 57.96 (s, CH2OCH3), 50.43 (s, CH2CH=CHPh) ppm. Elemental analysis (%): calcd for C18H19N2OCl (314.81): C: 68.67; H: 6.08; N 8.90; found C: 68.64; H: 6.01; N: 8.86.
1-(4-Methyl-benzyl)-3-cinnamyl-benzimidazolium chloride (1f): Yield: 90%; FT-IR: ν(CN) 1556 cm−1; 1H NMR (300 MHz, CDCl3): δ = 12.17 (s, 1H, NCHN), 7.73 (d, 1H, arom CH, 3JHH = 9.9 Hz), 7.60–7.50 (m, 3H, arom CH), 7.42–7.39 (m, 4H, arom CH), 7.36–7.28 (m, 3H, arom CH), 7.18 (d, 2H, arom CH, 3JHH = 8.1 Hz), 6.92 (d, 1H, CH=CHPh, 3JHH = 15.6 Hz), 6.44 (dt, 1H, CH=CHPh, 3JHH = 15.6 Hz, 3JHH = 6.6 Hz), 5.80 (s, 2H, CH2C6H4CH3), 5.50 (d, 2H, CH2CH=CH, 3JHH = 6.6 Hz), 2.32 (s, 3H, C6H4CH3); 13C{1H} NMR (126 MHz, CDCl3): δ = 144.09 (s, NCHN), 139.55, 137.25, 131.73, 131.54, 130.23, 129.70, 129.06, 128.93, 128.50, 127.21, 127.11, 120.27, 113.92, 113.75 (14 s, arom Cs), 135.10 (s, CH=CHPh), 127.23 (s, CH=CHPh), 51.61 (s, CH2C6H4CH3), 50.30 (s, CH2CH=CHPh), 21.34 (s, C6H4CH3) ppm. Elemental analysis (%): calcd for C24H23N2Cl (374.90): C: 76.89; H: 6.18; N 7.47; found C: 77.02 H: 6.25, N: 7.39.

3.3. General Procedure for the Preparation of Silver Complexes

In a Schlenk tube, a solution of benzimidazolium salt (1.0 mmol) in CH2Cl2 (50 mL) was prepared under argon atmosphere. Subsequently, Ag2O (2.2 mmol) was added to the mixture, which was stirred in a dark environment at room temperature for a period of 24 h. Following this, the reaction mixture was filtered through a celite bed, and the volume of the resulting solution was reduced to approximately 5 mL. The silver complex was precipitated with the addition of Et2O (60 mL). The mixture was subsequently filtered, washed with Et2O, and then dried under vacuum.
Chloro[1-cinnamyl-3-allyl-benzimidazol-2-yliden]silver(I) (3a): Yield: 79%; FT-IR: ν(CN) 1387 cm−1; 1H NMR 300 MHz, DMSO-d6): δ = 7.86–7.83 (m, 1H, arom CH), 7.78–7.74 (m, 1H, arom CH), 7.46–7.40 (m, 4H, arom CH), 7.30–7.21 (m, 3H, arom CH), 6.74 (d, 1H, CH=CHPh, 3JHH = 15.9 Hz), 6.55 (dt, 1H, CH=CHPh, 3JHH = 15.9 Hz, 3JHH = 6.1 Hz), 6.14–6.02 (m, CH=CH2), 5.28 (d, 2H, CH2CH=CH, 3JHH = 6.1 Hz), 5.25 (dd, 1H, CH=CH2, 3JHH = 9.9 Hz, 3JHH = 1.2 Hz), 5.20 (dd, 1H, CH=CH2, 3JHH = 17.1 Hz, 3JHH = 1.2 Hz), 5.13 (d, 2H, CH2CH=CH2, 3JHH = 5,4 Hz), 13C{1H} NMR (126 MHz, DMSO-d6): δ = 188.49 (s, NCN), 135.66, 133.37, 133.34, 128.57, 128.01, 126.52, 124.58, 112.28, 112.25 (9 s, arom Cs), 133.36 (s, CH=CHPh), 124.04 (s, CH=CHPh), 124.02 (s, CH=CH2), 118.29 (s, CH=CH2), 50.87 (s, CH2CH=CHPh), 50.59 (s, CH2C=CH2) ppm. MS (ESI-TOF): m/z = 381.05 [M − Cl]+ and 422.07 [M − Cl + CH3CN]+ (expected isotopic profiles). Elemental analysis (%): calcd for C19H18N2AgCl (417.68): C: 54.64; H: 4.34; N 6.71; found C: 54.72; H: 4.39; N: 6.62.
Chloro[1-methoxymethyl-3-cinnamyl-benzimidazol-2-yliden]silver(I) (3b): Yield: 56%; FT-IR: ν(CN) 1379 cm−1; 1H NMR (500 MHz, DMSO-d6): δ = 7.88–7.83 (m, 2H, arom CH), 7.49–7.47 (m, 2H, arom CH), 7.42 (d, 2H, arom CH, 3JHH = 7.0 Hz), 7.28 (t, 2H, arom CH, 3JHH = 7.2 Hz), 7.22 (t, 1H, arom CH, 3JHH = 7.2 Hz), 6.75 (d, 1H, CH=CHPh, 3JHH = 16.0 Hz), 6.55 (dt, 1H, CH=CHPh, 3JHH = 16.0 Hz, 3JHH = 6.0 Hz), 5.83 (s, 2H, CH2OCH3), 5.31 (d, 2H, CH2CH=CH, 3JHH = 6.0 Hz), 3.31 (s, 3H, CH2OCH3); 13C{1H} NMR (126 MHz, DMSO-d6): δ = 135.57, 133.71, 133.12, 128.62, 128.08, 126.57, 124.49, 112.50, 112.48 (9 s, arom Cs), 133.40 (s, CH=CHPh), 124.46 (s, CH=CHPh), 80.01 (s, CH2OCH3), 56.19 (s, CH2OCH3), 50.73 (s, CH2CH=CHPh) ppm. MS (ESI-TOF): m/z = 385.05 [M − Cl]+ (expected isotopic profiles). Elemental analysis (%): calcd for C18H18N2OAgCl (421.67): C: 51.27; H: 4.30; N 6.64; found C: 51.18; H: 4.26; N: 6.57.
Chloro[1-benzyl-3-cinnamyl-benzimidazol-2-yliden]silver(I) (3c). Yield: 86%; FT-IR: ν(CN) 1394 cm−1; 1H NMR (300 MHz, DMSO-d6): δ = 6.82 (d, 1H, arom CH, 3JHH = 9.9 Hz), 6.70 (d, 1H, arom CH, 3JHH = 9.9 Hz), 6.43–6.23 (m, 12H, arom CH), 5.71 (d, 1H, CH=CHPh, 3JHH = 15.9 Hz), 5.52 (dt, 1H, CH=CHPh, 3JHH = 15.9 Hz, 3JHH = 6.0 Hz), 4.70 (s, 2H, CH2Ph), 4.26 (d, 2H, CH2CH=CH, 3JHH = 6.0 Hz); 13C{1H} NMR (126 MHz, DMSO-d6): δ = 136.43, 135.78, 133.69, 129.00, 128.79, 128.26, 128.24, 127.54, 126.71, 124.36, 124.32, 112.52, 112.51 (13 s, arom Cs), 133.48 (s, CH=CHPh), 124.57 (s, CH=CHPh), 52.17 (s, CH2Ph), 50.78 (s, CH2CH=CHPh) ppm. MS (ESI-TOF): m/z = 431.06 [M − Cl]+ and 472.08 [M − Cl + CH3CN]+ (expected isotopic profiles). Elemental analysis (%): calcd for C23H20N2AgCl (467.74): C: 59.06; H: 4.31; N 5.99; found C: 58.98; H: 4.35; N: 5.87.
Chloro[1-(3-fluorobenzyl)-3-cinnamyl-benzimidazol-2-yliden]silver(I) (3d): Yield: 33%; FT-IR: ν(CN) 1385 cm−1; 1H NMR (500 MHz, DMSO-d6): δ = 7.85 (dd, 1H, arom CH, 3JHH = 7.5 Hz, 4JHH = 1.5 Hz), 7.72 (dd, 1H, arom CH, 3JHH = 7.0 Hz, 4JHH = 1.5 Hz), 7.45–7.35 (m, 5H, arom CH), 7.29–7.19 (m, 5H, arom CH), 7.14 (td, 1H, arom CH, 3JHH = 8.7 Hz, 4JHH = 2.5 Hz), 6.74 (d, 1H, CH=CHPh, 3JHH = 16.0 Hz), 6.55 (dt, 1H, CH=CHPh, 3JHH = 16.0 Hz, 3JHH = 6.0 Hz), 5.76 (s, 2H, CH2C6H4F), 5.30 (d, 2H, CH2CH=CH, 3JHH = 6.0 Hz); 13C{1H} NMR (126 MHz, DMSO-d6): δ = 189.04 (s, NCN), 162.19 (d, CF, 1JCF = 244.9 Hz), 135.66, 133.54, 133.29, 128.58, 128.02, 126.53, 124.21, 124.17, 112.41, 112.28 (10 s, arom Cs), 139.11 (d, arom Cquat, 3JCF = 7.3 Hz), 133.29 (s, CH=CHPh), 130.87 (d, arom CH, 3JCF = 8.3 Hz), 124.57 (s, CH=CHPh), 123.36 (d, arom CH, 4JCF = 2.9 Hz), 114.94 (d, arom CH, 2JCF = 20.8 Hz), 114.30 (d, arom CH, 2JCF = 21.9 Hz), 51.32 (s, CH2C6H4F), 50.68 (s, CH2CH=CHPh); 19F{1H} NMR (282 MHz, DMSO-d6): δ = -112.42 (s, C6H4F) ppm. MS (ESI-TOF): m/z = 490.08 [M − Cl + CH3CN]+ (expected isotopic profiles). Elemental analysis (%): calcd for C23H19N2FAgCl (485.73): C: 56.87; H: 3.94; N 5.77; found C: 56.85; H: 3.88; N: 5.74.
Chloro[1-(4-fluorobenzyl)-3-cinnamyl-benzimidazol-2-yliden]silver(I) (3e): Yield: 51%; FT-IR: ν(CN) 1391 cm−1; 1H NMR (500 MHz, DMSO-d6): δ = 7.84 (d, 1H, arom CH, 3JHH = 9.0 Hz), 7.73 (d, 1H, arom CH, 3JHH = 9.0 Hz), 7.46–7.39 (m, 6H, arom CH), 7.28 (t, 2H, arom CH, 3JHH = 7.5 Hz), 7.22 (t, 1H, arom CH, 3JHH = 7.2 Hz), 7.17 (t, 2H, arom CH, 3JHH = 9.0 Hz), 6.74 (d, 1H, CH=CHPh, 3JHH = 16.0 Hz), 6.54 (dt, 1H, CH=CHPh, 3JHH = 16.0 Hz, 3JHH = 6.0 Hz), 5.72 (s, 2H, CH2C6H4F), 5.29 (d, 2H, CH2CH=CH, 3JHH = 6.0 Hz); 13C{1H} NMR (126 MHz, DMSO-d6): δ = 188.64 (s, NCN), 161.76 (d, CF, 1JCF = 244.8 Hz), 135.67, 133.55, 133.25, 128.59, 128.04, 126.55, 124.16, 124.13, 112.40, 112.34 (10 s, arom Cs), 133.31 (s, CH=CHPh), 132.54 (d, arom Cquat, 4JCF = 3.1 Hz), 129.59 (d, arom CH, 3JCF = 8.4 Hz), 124.57 (s, CH=CHPh), 115.64 (d, arom CH, 2JCF = 21.7 Hz), 51.20 (s, CH2C6H4F), 50.68 (s, CH2CH=CHPh); 19F{1H} NMR (282 MHz, DMSO-d6): δ = −114.15 (s, C6H4F) ppm. MS (ESI-TOF): m/z = 490.10 [M − Cl + CH3CN]+ (expected isotopic profiles). Elemental analysis (%): calcd for C23H19N2FAgCl (485.73): C: 56.87; H: 3.94; N 5.77; found C: 56.92; H: 4.11; N: 5.72.
Chloro[1-(4-methyl-benzyl)-3-cinnamyl-benzimidazol-2-ylidene]silver(I) (3f): Yield: 81%; FT-IR: ν(CN) 1387 cm−1; 1H NMR (300 MHz, DMSO-d6): δ = 7.83 (d, 1H, arom CH, 3JHH = 9.0 Hz), 7.73 (d, 1H, arom CH, 3JHH = 8.7 Hz), 7.45–7.39 (m, 4H, arom CH), 7.30–7.20 (m, 5H, arom CH), 7.12 (d, 2H, arom CH, 3JHH = 7.8 Hz), 6.72 (d, 1H, CH=CHPh, 3JHH = 15.9 Hz), 6.53 (dt, 1H, CH=CHPh, 3JHH = 15.9 Hz, 3JHH = 6.0 Hz), 5.67 (S, 2H, CH2C6H4CH3), 5.27 (d, 2H, CH2CH=CH, 3JHH = 6.0 Hz), 2.23 (s, 3H, C6H4CH3); 13C{1H} NMR (126 MHz, DMSO-d6): δ = 137.35, 135.65 133.31, 133.29, 129.33, 128.58, 128.02, 127.43, 126.54, 124.09, 124.06, 112.41, 112.33 (13 s, arom Cs), 133.52 (s, CH=CHPh), 124.57 (s, CH=CHPh), 51.79 (s, CH2C6H4CH3), 50.61 (s, CH2CH=CHPh), 20.64 (s, C6H4CH3) ppm. MS (ESI-TOF): m/z = 445.08 [M − Cl]+ and 486.11 [M − Cl + CH3CN]+ (expected isotopic profiles). Elemental analysis (%): calcd for C24H22N2ClAg (481.77): C: 59.83; H: 4.60; N 5.81; found C: 59.97 H: 4.49; N: 5.74.

3.4. X-Ray Crystal Structure Analysis

Single crystals of silver(I) complexes 3c and 3d, suitable for X-ray analysis, were obtained through the slow diffusion of Et2O into a CH2Cl2 solution of the complex. The crystal structures of complexes 3c and 3d were determined using, respectively, a Bruker APEX-II CCD and a Bruker PHOTON-III CPAD with Mo-Kα radiation (λ = 0.71073 Å). The final structure was solved with SHELXT-2018/2 [50], which revealed the non-hydrogen atoms of the molecule. Following anisotropic refinement, the location of all hydrogen atoms was ascertained through the use of a Fourier difference map. The structure was refined with SHELXL-2019/3 [51] using the full-matrix least-square technique (use of F square magnitude; x, y, z, and βij for C, Cl, N, F and Ag atoms; x, y, and z in riding mode for H atoms) (Table 3). The Cambridge Crystallographic Data Centre (CCDC) contains the supplementary crystallographic data for the structures. The data can be obtained free of charge via www.ccdc.cam.ac.uk/structures.

3.5. In Vitro Effects on A. castellanii

3.5.1. A. castellanii Strain and Culture

The A. castellanii PAT06 strain (sequence type T4, GenBank accession no. EF429131), which was previously isolated from a patient with severe AK, was utilized in the study. The strain was cultivated axenically in proteose peptone-yeast extract-glucose (PYG) medium. The cultures were maintained in 25 cm2 tissue culture flasks (Corning, USA) and incubated at 25 °C [52].
Trophozoites in the exponential growth phase (72–96 h) were harvested by centrifugation at 500× g for 10 min. Subsequently, the cell pellets were washed twice with a sterile Neff’s saline solution (1.2 g NaCl, 0.4 g MgSO4·H2O, 0.4 g CaCl2·2H2O, 1.42 g Na2HPO4, and 1.36 g KH2PO4 dissolved in 100 mL of distilled water). The cell concentrations were determined using a Neubauer hemocytometer, and the suspension was adjusted to a final concentration of 1 × 104 trophozoites/mL in PYG medium with ≥95% viability. The prepared suspension was utilized immediately for viability assays.
Mature cysts were obtained by culturing trophozoites on non-nutrient agar (NNA) plates presmeared with Escherichia coli, followed by incubation for six weeks at 25 °C. The resulting cysts were harvested by washing with a sterile Neff’s saline solution, with the concentration subsequently adjusted to 1 × 104 cysts/mL for the ensuing assays.

3.5.2. A. castellanii Trophozoites

Stock solutions of the silver(I) complexes and their respective ligands were prepared in 100% dimethyl sulfoxide (DMSO). Working solutions at final concentrations of 10, 100, and 1000 µM were prepared by diluting the stock solutions with PYG medium. The final DMSO concentration in all experimental groups, including the highest tested concentration, was standardized to 10% (v/v). AgNO3 and Ag2O were prepared in PYG medium at final concentrations of 10, 100, and 1000 µM.
A. castellanii trophozoites were seeded into sterile 24-well tissue culture plates at a density of 104 trophozoites per mL per well. Subsequently, the cultures were subjected to incubation with the test compounds at the designated concentrations for a duration of 24, 48 and 72 h at a temperature of 25 °C. Following the incubation period, the adherent trophozoites were meticulously detached using a sterile cell scraper and resuspended by gentle pipetting. The resulting cell suspensions were stained with 0.3% methylene blue for a period of 10 min. The viability of the trophozoites was determined by staining them with methylene blue, a method that distinguishes between viable (unstained) and non-viable (blue-stained) cells. The cell count was performed using a Neubauer hemocytometer under a light microscope. The percentage of viable trophozoites was subsequently calculated relative to the untreated control.
The control group consisted of trophozoites cultured in PYG medium alone. The solvent control group was maintained in PYG medium containing 10% (v/v) DMSO. All experiments were performed in quadruplicate.

3.5.3. A. castellanii Cysts

In order to assess the impact of silver(I) complexes and ligands on A. castellanii cysts, the cysts were prepared at a final concentration of 1 × 104 cysts/mL. Subsequently, the samples were subjected to incubation with the test compounds at the indicated concentrations at 25 °C for 24, 48 and 72 h.
Following the incubation period, the cyst suspensions were centrifuged and washed three times with sterile Neff’s saline. The treated cysts were then inoculated onto non-nutrient agar (NNA) plates that had been smeared with Escherichia coli and incubated at 25 °C for 14 days.
The viability of the cyst was monitored daily using phase-contrast microscopy. Cysts were considered viable if excystation occurred and motile trophozoites were observed. In the absence of trophozoite emergence, the cysts were considered non-viable.

3.6. Statistical Analysis

The experiments described herein were conducted in four independent biological replicates, with each replicate comprising technical triplicates. The results are expressed as the mean ± standard deviation (SD). The statistical analysis was carried out using GraphPad Prism version 9.0. To assess the statistical significance of the observed variations among the multiple treatment groups, we employed a one-way analysis of variance (ANOVA), which was then followed by a Tukey’s multiple comparison test. For the purpose of performing for-a-value comparisons, a two-tailed Student’s t-test was utilized. In the context of statistical analysis, a p-value of less than 0.05 was determined to be statistically significant.

4. Conclusions

In summary, the present study described the synthesis of six new chloro[N-alkyl-N-cinnamyl-benzimidazol-2-yliden]silver(I) complexes, with isolated yields ranging from 33 to 86%. These complexes were thoroughly characterized by a combination of analytical techniques, including FT-IR and NMR spectroscopic analyses, as well as microanalysis and mass spectrometry. Subsequently, a substantial in vitro inhibitory activity against trophozoites and cysts of the A. castellanii strain was exhibited, signifying a superior efficacy in comparison to the ligands and commercially available silver complexes as Ag2O and AgNO3. At a concentration of 100 µM, complexes bearing benzyl, 3-fluorobenzyl and 4-methyl-benzyl substituents on the benzimidazole ring exhibited significant activity, with 5.3, 3.2 and 6.3% trophozoite viability, respectively, recorded after 72 h. These findings provide substantial support for the preclinical development of silver-based NHC compounds as novel anti-Acanthamoeba agents, particularly for the treatment of AK. Subsequent studies will concentrate on the optimization of the substituents of the benzimidazole ligand in silver complexes and the investigation of their mode of action.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/inorganics13060204/s1, characterizing data of 1-cinnamyl-3-allyl-benzimidazolium chloride (1a) with Figure S1. FT-IR spectrum, Figure S2. 1H NMR spectrum and Figure S3. 13C{1H} NMR spectrum; characterizing data of 1-methoxymethyl-3-cinnamyl-benzimidazolium chloride (1b) with Figure S4. FT-IR spectrum, Figure S5. 1H NMR spectrum and Figure S6. 13C{1H} NMR spectrum; characterizing data of 1-(4-methyl-benzyl)-3-cinnamyl-benzimidazolium chloride (1f) with Figure S7. FT-IR spectrum, Figure S8. 1H NMR spectrum and Figure S9. 13C{1H} NMR spectrum; characterizing data of chloro[1-cinnamyl-3-allyl-benzimidazol-2-yliden]silver(I) (3a) with Figure S10. FT-IR spectrum, Figure S11. 1H NMR spectrum, Figure S12. 13C{1H} NMR spectrum and Figures S13 and S14. Mass spectrum (ESI-TOF); characterizing data of chloro[1-methoxymethyl-3-cinnamyl-benzimidazol-2-yliden]silver(I) (3b) with Figure S15. FT-IR spectrum, Figure S16. 1H NMR spectrum, Figure S17. 13C{1H} NMR spectrum and Figure S18. Mass spectrum (ESI-TOF); characterizing data of chloro[1-benzyl-3-cinnamyl-benzimidazol-2-yliden]silver(I) (3c) with Figure S19. FT-IR spectrum, Figure S20. 1H NMR spectrum, Figure S21. 13C{1H} NMR spectrum and Figure S22. Mass spectrum (ESI-TOF), characterizing data of chloro[1-(3-fluorobenzyl)-3-cinnamyl-benzimidazol-2-yliden]silver(I) (3d) with Figure S23. FT-IR spectrum, Figure S24. 1H NMR spectrum, Figure S25. 13C{1H} NMR spectrum, Figure S26. 19F{1H} NMR spectrum and Figure S27. Mass spectrum (ESI-TOF); characterizing data of chloro[1-(4-fluorobenzyl)-3-cinnamyl-benzimidazol-2-yliden]silver(I) (3e) with Figure S28. FT-IR spectrum, Figure S29. 1H NMR spectrum, Figure S30. 13C{1H} NMR spectrum, Figure S31. 19F{1H} NMR spectrum and Figure S32. Mass spectrum (ESI-TOF); characterizing data of chloro[1-(4-methyl-benzyl)-3-cinnamyl-benzimidazol-2-ylidene]silver(I) (3f) with Figure S33. FT-IR spectrum, Figure S34. 1H NMR spectrum, Figure S35. 13C{1H} NMR spectrum and Figures S36 and S37. Mass spectrum (ESI-TOF).

Author Contributions

Conceptualization, N.Ş. and D.S.; methodology, Z.A.-P., N.Ş. and D.S.; validation, Z.A.-P., N.Ş. and D.S.; formal analysis, Z.A.-P., N.Ş., S.H. and D.S.; investigation, Z.A.-P., N.Ş., S.H. and B.M.T.L.; resources, Z.A.-P. and D.S.; data curation, Z.A.-P., N.Ş. and D.S.; writing—original draft preparation, Z.A.-P., N.Ş. and S.H.; writing—review and editing, İ.Ö. and D.S.; visualization, Z.A.-P.; supervision, İ.Ö. and D.S. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding authors.

Acknowledgments

We would like to thank Julia Walochnik from the Department of Medical Parasitology at the Clinical Institute of Hygiene at the University of Vienna, Vienna, Austria, for providing the Acanthamoeba strain.

Conflicts of Interest

The authors declare no conflicts of interest.

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Inorganics 13 00204 g001
Figure 1. Silver complexes 3a–f tested against Acanthamoeba castellanii in the present study.
Figure 1. Silver complexes 3a–f tested against Acanthamoeba castellanii in the present study.
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Inorganics 13 00204 sch001
Scheme 1. Synthesis of silver complexes 3a–f.
Scheme 1. Synthesis of silver complexes 3a–f.
Inorganics 13 00204 sch001
Inorganics 13 00204 g002
Figure 2. Molecular structure of silver(I) complex 3c. The ORTEP drawing, with 50% probability thermal ellipsoid, shows the atom labelling. Important bond lengths (Å) and angles (°): Ag1-Cl1 2.3854(13), Ag1-Cl1a 2.968(3), Ag1-C1 2.096(3), N1-C1 1.350(4), N1-C2, 1.396(3), C1-Ag1-Cl1 163.55(7), Cl1-Ag1-Cl1a 86.49(6), Cl1a-Ag1-C1 109.24(9), N1-C1-Ag1 128.69(19), Ag1-C1-N2 125.1(2) and N2-C1-N1 105.7(2).
Figure 2. Molecular structure of silver(I) complex 3c. The ORTEP drawing, with 50% probability thermal ellipsoid, shows the atom labelling. Important bond lengths (Å) and angles (°): Ag1-Cl1 2.3854(13), Ag1-Cl1a 2.968(3), Ag1-C1 2.096(3), N1-C1 1.350(4), N1-C2, 1.396(3), C1-Ag1-Cl1 163.55(7), Cl1-Ag1-Cl1a 86.49(6), Cl1a-Ag1-C1 109.24(9), N1-C1-Ag1 128.69(19), Ag1-C1-N2 125.1(2) and N2-C1-N1 105.7(2).
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Inorganics 13 00204 g003
Figure 3. Molecular structure of silver(I) complex 3d. The ORTEP drawing, with 50% probability thermal ellipsoid, shows the atom labelling. Important bond lengths (Å) and angles (°): Ag1-Cl1 2.3972(5), Ag1-Cl1a 2.9320(6), Ag1-C1 2.102(2), N1-C1 1.364(3), N1-C2, 1.355(3), C1-Ag1-Cl1 162.33(6), Cl1-Ag1-Cl1a 86.06(2), Cl1a-Ag1-C1 110.52(6), N1-C1-Ag1 125.43(15), Ag1-C1-N2 128.48(15) and N2-C1-N1 105.58(18).
Figure 3. Molecular structure of silver(I) complex 3d. The ORTEP drawing, with 50% probability thermal ellipsoid, shows the atom labelling. Important bond lengths (Å) and angles (°): Ag1-Cl1 2.3972(5), Ag1-Cl1a 2.9320(6), Ag1-C1 2.102(2), N1-C1 1.364(3), N1-C2, 1.355(3), C1-Ag1-Cl1 162.33(6), Cl1-Ag1-Cl1a 86.06(2), Cl1a-Ag1-C1 110.52(6), N1-C1-Ag1 125.43(15), Ag1-C1-N2 128.48(15) and N2-C1-N1 105.58(18).
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Inorganics 13 00204 g004
Figure 4. The viability of trophozoites at concentrations of 1000, 100 and 10 µM against the silver(I) complex (3a–f) and the benzimidazolium salts (1a–f) was assessed after 24, 48 and 72 h against the pathogenic A. castellanii. Data are expressed as the mean ± SD of four independent experiments (* p < 0.05 vs. control).
Figure 4. The viability of trophozoites at concentrations of 1000, 100 and 10 µM against the silver(I) complex (3a–f) and the benzimidazolium salts (1a–f) was assessed after 24, 48 and 72 h against the pathogenic A. castellanii. Data are expressed as the mean ± SD of four independent experiments (* p < 0.05 vs. control).
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Figure 5. Inverted microscope images showing the viability of A. castellanii trophozoites treated with 1000 µM concentrations of silver(I) complexes (3a–f), AgNO3 and Ag2O after 24, 48 and 72 h (magnification: 200×).
Figure 5. Inverted microscope images showing the viability of A. castellanii trophozoites treated with 1000 µM concentrations of silver(I) complexes (3a–f), AgNO3 and Ag2O after 24, 48 and 72 h (magnification: 200×).
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Table 1. Spectroscopic data of silver(I) complexes 3a–f and their corresponding benzimidazolium salts 1a–f.
Table 1. Spectroscopic data of silver(I) complexes 3a–f and their corresponding benzimidazolium salts 1a–f.
Benzimidazolium SaltsSilver Complexes
FT-IR
ν(CN) (cm−1)		1H NMR
NCHN (CDCl3; ppm)		13C{1H} NMR
NCHN (CDCl3; ppm)		 FT-IR
ν(CN) (cm−1)		13C{1H} NMR
NC(Ag)N (DMSO-d6; ppm)
1a 155611.78 (1JCH = 220 Hz)143.883a1387188.49
1b 155611.94 (1JCH = 223 Hz)144.143b1379Not observed
1c[39]156012.02 (1JCH = 221 Hz)144.033c1394Not observed
1d[39]156212.15 (1JCH = 220 Hz)144.263d1385189.04
1e[39]156210.07 (1JCH = 220 Hz)142.713e1391188.64
1f 155612.17 (1JCH = 225 Hz)144.093f1387Not observed
Table 2. Calculated logP [48] for silver(I) complexes 3a–f.
Table 2. Calculated logP [48] for silver(I) complexes 3a–f.
Silver ComplexCalculated logP
3a3.99
3b3.42
3c4.60
3d4.82
3e4.86
3f5.01
Table 3. Selected crystallographic data for complexes 3c and 3d.
Table 3. Selected crystallographic data for complexes 3c and 3d.
3c3d
CCDC depository24536832451735
Chemical formulaAg2Cl2C46H40N4Ag2Cl2C46H38F2N4
Molar mass (g.mol−1)935.46971.44
Temperature (K)173(2)120(2)
Crystal systemTriclinicTriclinic
Space group P 1 ¯ P 1 ¯
a (Å)8.725(6)8.6142(3)
b (Å)10.626(9)10.6406(4)
c (Å)10.982(9)11.1741(4)
α (°)106.338(10)107.7810(10)
β (°)91.58(2)91.7150(10)
γ (°)100.33(2)101.2310(10)
Volume (Å3)957.9(13)952.22(6)
Z11
ρcalc. (g·cm−3)1.6221.694
μ (mm−1)1.2011.219
F000472488
Crystral size (mm)0.180 × 0.140 × 0.1200.140 × 0.120 × 0.080
Radiation (λ/Å)0.71073 Mo Kα0.71073 Mo Kα
θ range for data collection (°)1.939 ≤ θ ≤ 27.9472.42 ≤ θ ≤ 27.88
Reflections collected3715535981
Rint0.05080.0468
Goodness-of-fit on F21.0211.008
R1, wR2 [I > 2σ(I)]R1 = 0.0334, wR2 = 0.0738R1 = 0.0264, wR2 = 0.0559
R1, wR2 (all data)R1 = 0.0487, wR2 = 0.0816R1 = 0.0340, wR2 = 0.0594
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Akın-Polat, Z.; Şahin, N.; Hkiri, S.; Ly, B.M.T.; Özdemir, İ.; Sémeril, D. In Vitro Evaluation of Silver-NHC Complexes Against a Clinical Isolate of Acanthamoeba castellanii: Time- and Dose-Dependent Effects. Inorganics 2025, 13, 204. https://doi.org/10.3390/inorganics13060204

AMA Style

Akın-Polat Z, Şahin N, Hkiri S, Ly BMT, Özdemir İ, Sémeril D. In Vitro Evaluation of Silver-NHC Complexes Against a Clinical Isolate of Acanthamoeba castellanii: Time- and Dose-Dependent Effects. Inorganics. 2025; 13(6):204. https://doi.org/10.3390/inorganics13060204

Chicago/Turabian Style

Akın-Polat, Zübeyda, Neslihan Şahin, Shaima Hkiri, Bui Minh Thu Ly, İsmail Özdemir, and David Sémeril. 2025. "In Vitro Evaluation of Silver-NHC Complexes Against a Clinical Isolate of Acanthamoeba castellanii: Time- and Dose-Dependent Effects" Inorganics 13, no. 6: 204. https://doi.org/10.3390/inorganics13060204

APA Style

Akın-Polat, Z., Şahin, N., Hkiri, S., Ly, B. M. T., Özdemir, İ., & Sémeril, D. (2025). In Vitro Evaluation of Silver-NHC Complexes Against a Clinical Isolate of Acanthamoeba castellanii: Time- and Dose-Dependent Effects. Inorganics, 13(6), 204. https://doi.org/10.3390/inorganics13060204

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