New N-Heterocyclic Carbene Gold and Platinum Complexes with 1,3-Dialkyl-4-anisyl-5-(4-chlorophenyl)imidazol-2-ylidene Ligands for the Treatment of Esophageal Adenocarcinoma

TL;DR

Researchers developed new gold and platinum complexes with potential to treat esophageal adenocarcinoma by inhibiting cancer cell growth and inducing cell death.

Contribution

New gold(I), gold(III), and platinum(II) complexes with N-heterocyclic carbene ligands show strong anticancer activity against esophageal adenocarcinoma.

Findings

Cationic triphenylphosphino-NHC-gold(I) and bis-NHC-gold(I) complexes showed strong antiproliferative effects in EAC cell lines.

Compounds induced caspase 3/7 activity and downregulated anti-apoptotic proteins in EAC cells.

NHC-gold(I) complexes suppressed cyclin D1 and induced reactive oxygen species in EAC cells.

Abstract

Encouraged by the promising anticancer activity of a iodidogold(I)-N-heterocyclic carbene (NHC) complex with a 1,3-diethyl-4-anisyl-5-(4-chlorophenyl)imidazol-2-ylidene ligand system, a series of new gold(I), gold(III) and platinum(II) complexes coordinated to this ligand system were designed, prepared, and characterized using NMR spectroscopy and mass spectrometry methods. A preliminary anticancer screening of the complexes using four esophageal adenocarcinoma (EAC) cell lines showed promising activities for the cationic triphenylphosphino-NHC-gold(I) and bis-NHC-gold(I) complexes, accompanied by strong antiproliferative, colony-, and spheroid-forming inhibitory effects. The compounds were relatively less toxic to the normal esophageal cell line Het-1A and the monocyte cell line THP-1. Moreover, these compounds induced caspase 3/7 activity and downregulated anti-apoptotic proteins (Bcl-XL, Bcl-2, and Mcl-1) in EAC cells. Further, the cell cycle promoter cyclin D1 was suppressed by these NHC-gold(I) complexes. Finally, we observed strong reactive oxygen species (ROS) induction in EAC cells with NHC-gold(I) complexes 8 and 11.

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Full text

1. Introduction

Gold compounds, including the gold(I) complex auranofin, are clinically applied for the therapy, aka chrysotherapy, of chronic inflammatory diseases such as rheumatoid arthritis (Figure 1) [1]. In preclinical experiments, auranofin also exhibited anticancer activity, mainly attributed to its potent inhibition of thioredoxin reductase (TrxR), leading to the formation of toxic reactive oxygen species (ROS) and the induction of apoptosis [1]. The mitochondrial protein import machinery includes the protein Mia40, which was identified as a target of auranofin in fungi and which can play a vital role in proliferating cells [2,3]. A significant proteasomal deubiquitinase (DUB) inhibition was also observed in cancer cells treated with auranofin [4,5]. Moreover, several clinical trials were launched with auranofin for the therapy of various cancers, including CLL, glioblastoma, lung, and ovarian cancer [6]. Consequently, the sound anticancer activities of auranofin prompted the development of improved auranofin derivatives and the design of suitable combination therapies of auranofin with other anticancer drugs [7,8,9]. In addition, neutral and cationic N-heterocyclic carbene (NHC) gold complexes exhibited strong anticancer activity, which was attributed in part to TrxR inhibition and to other mechanisms such as DNA binding (e.g., interaction with G-quadruplexes), p53-dependent apoptosis induction, and immunogenic cell death [10,11,12,13,14]. Our groups focused on the effects of NHC-gold complexes with 4,5-diarylimidazole-2-ylidene ligands, inspired by the natural tubulin-binding cis-stilbene, combretastatin A-4, on vital cytoskeletal components such as microtubules and actin filaments [15,16]. Notably, considerable damage to the actin cytoskeleton in cancer cells was also observed upon treatment with auranofin [17].

Esophageal cancer (EC) is a therapeutically challenging cancer disease and is globally ranked sixth in mortality (544,076 deaths) and seventh in incidence (604,100 new cases) [18,19]. Case numbers of esophageal adenocarcinoma (EAC) are rising rapidly in high-income countries, associated with lifestyle factors such as obesity, gastroesophageal reflux disease (GERD), and Barrett’s esophagus (BE) [20]. Surgery and chemoradiotherapy with the well-established anticancer platinum(II) complex cisplatin are currently used to treat EC [21]. Promising data in EC patients underscored the relevance of platinum drugs in combination with immune checkpoint inhibitors [22]. Gold complexes might also become suitable drugs for the therapy of EAC, and auranofin was reported to be active against EC [23].

Our group has recently disclosed a new iodidogold-NHC (N-heterocyclic carbene) complex 1a with the 1,3-diethyl-4-anisyl-5-(4-chlorophenyl)imidazol-2-ylidene ligand, which exhibited superior activity against esophageal adenocarcinoma (EAC) cells (Figure 1) [24]. Moreover, we have reported platinum(II)-NHC complexes such as 1b and 1c with activity against various tumor cell lines (Figure 1) [25]. In the current work, we focused on the design of new analogous complexes of the gold(I)-NHC (5, 6, 8, 11, and 12), gold(III)-NHC (13), and platinum(II)-NHC type (15) coordinated with the promising 1,3-dialkyl-4-anisyl-5-(4-chlorophenyl)imidazol-2-ylidene ligand system (Scheme 1). Their anticancer potential was analyzed in EAC cell lines. The most promising complexes, 8 and 11, were investigated for suppression of EAC colony and spheroid growth, induction of apoptosis and ROS generation in EAC cells.

2. Results

2.1. Chemistry

Based on the previously disclosed gold complex iodido-(1,3-diethyl-4-anisyl-5-(4-chlorophenyl)imidazol-2-ylidene)gold(I) 1a, which exhibited high activity against esophageal adenocarcinoma (EAC) cells [24], further new gold(I)-, gold(III)-, and platinum(II)-NHC complexes bearing the 1,3-di(m)ethyl-4-anisyl-5-(4-chlorophenyl)imidazole-2-ylidene ligand system were prepared. Initially, the close 1,3-dimethylimidazol-2-ylidene analog iodido(1,3-dimethyl-4-anisyl-5-(4-chlorophenyl)imidazol-2-ylidene)gold(I) 6 was synthesized. Starting from the TosMIC compound 2 [26], the N-methylimidazole 3 was prepared. Methylation of 3 with iodomethane afforded the 1,3-dimethylimidazolium iodide 4. The chlorido-gold(I) complex 5 was obtained from the reaction of 3 with Ag_2_O, followed by treatment with AuCl(DMS). Reaction of the chlorido complex 5 with KI gave the analogous iodido complex 6.

The known chlorido complex 7 [24] was used as a starting compound for the synthesis of the cationic PPh_3_ complex 8, which was obtained by reaction of 7 with PPh_3_ and NaBF_4_.

The imidazolium iodide salt 9 [24] was the starting compound for the synthesis of the cationic bis-NHC gold complex 11 via the corresponding BF_4_^−^ salt 10. Imidazolium 9 was also applied for the synthesis of the bromidogold(I) complex 12 upon reaction with Ag_2_O followed by treatment with AuCl(DMS) and LiBr [27]. The analogous trisbromido-gold(III) complex 13 was obtained from the reaction of 12 with Br_2_ [28]. The iodido-silver(I) complex 14 was prepared from 9 and Ag_2_O. The reaction of 14 with K_2_PtCl_4_ in DMSO furnished the cis-dichlorido(DMSO)platinum(II) complex 15. However, we were not able to obtain a bis-PPh_3_ Pt(II) complex with the 1,3-diethyl-4-anisyl-5-(4-chlorophenyl)imidazole-2-ylidene ligand system as a close analog of the known complex 1c [25].

The new complexes were analyzed using nuclear magnetic resonance (^1^H and ^13^C NMR for each complex and ^11^B, ^31^P, and ^195^Pt NMR for the respective complexes) and mass spectrometry (EI and ESI-HRMS, see Section 4.3, and Figures S1–S25 in the supplementary materials). Elemental analyses were performed for the halogenido complexes 5, 6, 12–15, for which HRMS experiments were not performed due to expected stability issues such as halogenido ligand loss and exchange under ESI conditions in aqueous solvents (e.g., water/acetonitrile mixtures). In terms of stability and degradation studies of analogous halogenidogold(I)-NHC complexes, we refer to our previous work [24].

The disappearance of the imidazole-NCHN proton signal in the ^1^H NMR spectra of the complexes is a clear indicator for the coordination of the imidazol-2-ylidene ligand to the corresponding gold(I), gold(III), or platinum(II) center, together with a characteristic chemical shift in the metal-bound imidazole-NCN carbon signal in the ^13^C NMR spectra of the NHC complexes. The coupling constants (J) of the imidazole-2-ylidene ligand phenyl proton doublet signals were between 8.5 and 8.9 Hz, as expected for para-substituted benzene rings [24].

2.2. Cytotoxic Activity and Colony Formation Assay

The antiproliferative activities of complexes 5, 6, 11–13, and 15 against four EAC cell lines (FLO-1, SK-GT-4, OE19, and OE33) were evaluated using the hexosaminidase assay (Table 1). The known complexes 1a–c and the approved drugs auranofin and cisplatin served as positive control complexes. The iodidogold(I) complex 6 was distinctly more active than its chloridogold(I) analog 5 against OE19 cells. In the other three EAC cell lines, complex 5 was slightly more active than 6. Notably, the 1,3-dimethylimidazolylidene 6 was less active against SK-GT-4 and FLO-1 cells than the 1,3-diethylimidazolylidene 1a. The bromidogold(I) complex 12 and the trisbromidogold(III) complex 13 were less active than the iodido analog 1a against two EAC cell lines (SK-GT-4 and FLO-1). Complex 12 was twice as active as 13 against FLO-1 and OE19 cells, while they showed comparable activities against SK-GT-4 and OE33 cells.

The cationic NHC-gold(I) complexes 8 and 11 (also referred to as G2 and G4, respectively, in the following mechanistic studies) were the most active complexes in the new series. The bis-carbene complex 11 exhibited high (nanomolar) activity, comparable to that of the previously reported iodidogold(I) complex 1a, and it was more active than auranofin and cisplatin [24]. The new cis-dichlorido-DMSO-Pt(II) complex 15 was more active than its N,N-bisbenzyl analog 1b against OE19 cells but distinctly less active than 1b against SK-GT-4 and FLO-1 cells. Actually, 15 was inactive against FLO-1 and OE33 cells. The known bis-PPh_3_ complex 1c was the most active Pt(II) complex in this study, surpassing the activity of cisplatin and the DMSO complexes and falling within the IC_50_ range of the highly active gold complexes 1a and 11. It is worth noting that cisplatin was more active against FLO-1 than against SK-GT-4 cells, whereas the NHC-Pt complexes 1b, 1c, and 15 were more active against SK-GT-4 than against FLO-1 cells. Notably, complexes 8 (G2) and 11 (G4) did not affect the proliferation of the non-malignant esophageal cell line Het-1A at concentrations below 5 μM after 72 h. We observed cytotoxicity in these cells at higher doses of 5–10 μM, which are approximately 3.5–22-fold higher than their IC_50_ values in EAC cells, suggesting a considerable selectivity of 8 and 11 for cancer cells while sparing normal cells (Figure S26 and Table S1). Similarly, in THP-1 monocyte cells, complexes 8 and 11 did not show cytotoxicity at concentrations up to 5 μM, whereas a slight cytotoxicity (below 50%) was observed at 10 μM after 72 h.

The dose- and time-dependent activity of the cationic NHC-gold(I) complexes 8 (G2) and 11 (G4) against SK-GT-4, OE33, and FLO-1 EAC cells was investigated and compared with the activity of auranofin (Figure 2). After 24 h, complexes 8 and 11 showed only weak activity even at higher concentrations, whereas both complexes exhibited higher activity already at low doses after 48 h and 72 h. In contrast, auranofin showed a quicker onset of activity at higher doses (already after 24 h) but remained less active than 8 and 11 at lower doses after longer incubation times (72 h). In comparison to the previously published data of 1a, complexes 8 and 11 appeared to be more active than 1a, which, in spite of its low IC_50_ values, still showed ca. 20% (FLO-1) and ca. 40% (SK-GT-4) proliferating EAC cells at a concentration of 5 µM after 72 h [25]. We used the clonogenic assay to evaluate the long-term effects of complexes 8 (1.7 and 1.9 µM, respectively) and 11 (0.9 and 0.7 µM, respectively) in EAC cell lines OE33 and FLO-1 for 48 h. Both complexes significantly reduced colony number and size in OE33 and FLO-1 cells (Figure 3A,B), suggesting that the effects of complexes 8 and 11 are irreversible.

2.3. Complexes 8 and 11 Inhibit Spheroid Formation in EAC Cells

Complexes 8 (1.7 and 1.9 µM, respectively) and 11 (0.9 and 0.7 µM, respectively) inhibited spheroid formation by SK-GT-4 and FLO-1 EAC cells (Figure 4). The inhibitory effects of both complexes were more pronounced on FLO-1 spheroids, with a nearly complete inhibition at the applied concentrations. The inhibition of spheroid formation by 8 and 11 was also more pronounced in comparison with previous results for complex 1a in these EAC cell lines [24].

2.4. Complexes 8 and 11 Induce Apoptosis in EAC Cells

To investigate the cell death mechanisms induced by complexes 8 and 11, their potential to induce apoptosis in SK-GT-4 and OE33 EAC cells was assessed using the AnnexinV/PI assay and flow cytometry. Notably, cells treated with 8 and 11 (IC_50_ concentrations) for 72 h showed a considerable induction of both early and late apoptotic cells but only marginal numbers of necrotic cells, compared with untreated control populations (Figure 5). In contrast, complex 1a had not induced early apoptosis in SK-GT-4 cells in our previously published study [24].

As another indicator of apoptosis induction, the activation of caspases 3 and 7 by complexes 8 and 11 was studied (Figure 6). Both gold complexes 8 (1.7 and 1.9 µM, respectively) and 11 (0.9 and 0.7 µM, respectively) exhibited a 3–4-fold increase in caspase 3 and 7 activity in SK-GT-4 and FLO-1 EAC cells. The cells were then treated with auranofin, as previously reported, as it induces caspase 3/7 activity in a dose-dependent manner [29]. However, when the cells were treated with 8 (G2) or auranofin, an equivalent dose of the most potent compound, 11 (G4), did not result in significant caspase 3/7 activity in SK-GT-4 and FLO-1 cells, suggesting the superiority of complex 11 (G4) in inducing apoptosis.

To investigate the effects of complexes 8 (1.7 and 1.9 µM, respectively) and 11 (0.9 and 0.7 µM, respectively) on anti-apoptotic proteins Bcl-XL, Bcl-2, and Mcl-1 in SK-GT-4 and FLO1 cells, their expression was assessed and quantified by Western blot (Figure 7 and Figures S27 and S28). Both compounds 8 and 11 downregulated the expression of Bcl-XL, Bcl-2, and Mcl-1 in EAC cells after 48 h. Moreover, the cell cycle protein cyclin D1 was strongly suppressed by 8 and 11.

Auranofin has been reported to inhibit cytosolic and mitochondrial TrxR, thereby increasing reactive oxygen species (ROS) levels, which are associated with DNA damage, mitochondrial permeability transition, and an increased percentage of annexin V-positive cells [30]. To investigate the effects of our new complexes, we performed a fluorometric ROS assay on OE33 EAC cells treated with auranofin, complex 8 (G2), and complex 11 (G4). The results demonstrated that treatment with all compounds significantly increased ROS levels in OE33 cells in a dose-dependent manner, indicating the induction of oxidative stress and triggering apoptosis (Figure 8A). Additionally, a TrxR activity assay revealed a significant decrease in activity when cells were treated with auranofin, 8 (G2), and 11 (G4) at their respective IC_50_ concentrations after 48 h of incubation (Figure 8B). Conversely, we did not observe any ROS generation in Het-1A cells (Figure S29), suggesting the selectivity of these compounds towards EAC cells. Although further experiments are needed to establish a direct link between TrxR inhibition and ROS-driven apoptosis in EAC cells, we believe that this decrease in TrxR activity correlates with increased oxidative stress and apoptosis induction.

3. Discussion

New metal complex derivatives of the recently disclosed neutral 4-anisyl-5-(4-chlorophenyl)imidazol-2-ylidene-iodidogold(I) complex 1a were prepared by changing the N-ethyl substituents, the iodido ligand, and/or the metal ion and its oxidation state. The synthesis of the new gold-NHC complexes 5, 6, 8, 11–13 and platinum(II) complex 15 was straightforward using published procedures [15,16,24,25,27,28]. In this way, two antitumoral cationic gold(I)-NHC complexes, 8 and 11, were discovered. Complex 11 in particular exhibited high activities in the range of those of the iodidogold(I) complex 1a. Cationic NHC-gold(I) complexes were often found distinctly more active against cancer cells than their neutral chloridogold(I)-NHC analogs, while iodidogold(I) complexes reached excellently low IC_50_ values in cancer cells, similar to those of known cationic triphenylphosphinogold(I)-NHC complexes and biscarbene-gold(I) complexes [2,31,32]. It is also noteworthy that marginal modifications of the N-alkyl substituent of the imidazole ring (N-methyl instead of N-ethyl) of the 4-anisyl-5-(4-chlorophenyl)imidazole-based NHC ligand system applied in this study led to a substantial decrease in anticancer activity. This finding is in line with our previous reports that N-ethyl imidazole ring substituents are superior to N-methyl substituents in neutral chloridogold(I) complexes [31]. The replacement of the iodido ligand of 1a by a bromido ligand, as in complex 12, also reduced the anticancer activity as reported previously by Bian and coworkers for diarylimidazolylidene gold(I) complexes [32]. In a similar vein, the trisbromidogold(III) complex 13 showed only moderate activities and lower activities than the analogous bromidogold(I) complex 12 in two cell lines, indicating also an important role of the gold(I) oxidation state in the manifestation of anticancer activity against specific EAC cells. The cis-dichlorido(DMSO)platinum(II) complex 15 was only moderately active against SK-GT-4 and OE19 cells. It was inactive against FLO-1 cells, which is in stark contrast to the activity of 1b and cisplatin against FLO-1 cells. Only in the OE19 cells, complex 15 was superior to 1b. Notably, the known bis-PPh_3_-platinum complex 1c exhibited high activity against all tested EAC cell lines, which complements its previously reported anticancer activities against various other solid tumors [25]. Further on, complexes 8 and 11 demonstrated specificity towards cancer cells without inducing cytotoxicity in normal cell lines. Even at concentrations five times higher than those required to exhibit antiproliferative effects on EAC cells, these complexes did not prove themselves cytotoxic in the normal esophageal cell line Het-1A nor in the monocyte cell line THP-1. This suggests that complexes 8 and 11 are selectively toxic to cancer cells while sparing non-malignant and immune cells, which commends them for evaluation of their anticancer potential in future clinical trials.

Apoptosis is strongly induced by the most promising complexes 8 and 11. This is consistent with previous reports on the induction of apoptosis by auranofin and complex 1a in tumor cells [1,24]. However, complex 1a did not induce early apoptosis in SK-GT-4 cells in our previously published study [24]. The pro-apoptotic activities of 8 and 11 were associated with a suppression of various anti-apoptotic proteins. In particular, the observed gold complex-mediated downregulation of Mcl-1 has been reported previously in association with apoptosis induction in FLO-1 and SK-GT-4 cells treated with other drugs [33]. The induction of apoptotic cell death by 8 and 11 is supported by the suppression of cyclin D1 and the inhibition of cell-cycle progression, including mitosis, in treated cells. Cyclin D1 is relevant to the progression of FLO-1 cells, and its suppression is also relevant to the treatment of other cancers, thereby broadening the therapeutic potential of the new cationic NHC-gold(I) complexes 8 and 11 [34,35].

The suppression of the formation of new EAC cell colonies and spheroids by complexes 8 and 11 is another promising effect, likely due to a consequent inhibition of cancer stem-like cells (CSCs). The effect of 8 and 11 on EAC spheroids appears to be more pronounced than that of 1a [24]. The inhibition of tumor growth by CSCs and the reversal of the mesenchymal properties of EAC cells are crucial for blocking EAC metastasis [36]. In FLO-1 and SK-GT-4 EAC cells, EMT reversal can also reduce cancer cell motility and invasion [37].

Our study demonstrates that auranofin and gold complexes 8 and 11 significantly induce ROS generation in the EAC cell line OE33. Auranofin, known for its anti-rheumatic effects, inhibits TrxR, disrupts the cell’s redox balance, and increases ROS formation, leading to oxidative stress and apoptosis of cancer cells. By extending this mechanism to EAC cells, auranofin effectively induced ROS and cytotoxicity. Gold complexes 8 and 11 similarly inhibited TrxR and increased ROS levels in EAC cells. This ROS-mediated cytotoxicity is specific to cancer cells, as these complexes showed minimal toxicity and no ROS generation in normal esophageal Het-1A and monocyte THP-1 cells, even at higher concentrations, underscoring their potential for targeted therapy. The probable mechanism behind the inhibition of TrxR activity may involve binding to crucial cysteine and selenocysteine residues by the newly described gold(I) complexes, but further research is needed to confirm this. The combination of gold complexes with HDAC inhibitors might be promising given the known effects of HDAC inhibition on thioredoxin and thioredoxin-interacting proteins in EAC [38,39].

Moreover, increased ROS formation can modulate the tumor immune response. An auranofin-type (triethylphosphine)gold complex with the clinically approved EGFR inhibitor erlotinib upregulated ROS formation in acute myeloid leukemia (AML) models, which was accompanied by a differentiation of AML cells to dendritic cells and a modulation of the immune microenvironment [40]. Analogously, NHC-gold(I) complex conjugates with glabridin and triterpene molecules enhanced ROS formation and induced dendritic cell maturation and immunogenic cell death in liver cancers [41,42]. Notably, a dinuclear NHC-gold(I) complex based on a dimeric bis-4,5-di(4-fluorophenyl)imidazol-2-ylidene ligand system also exhibited promising in vitro and in vivo activities against liver cancer models based on upregulated ROS levels leading to endoplasmic reticulum stress and ferroptosis associated with enhanced immunogenic cell death [43]. Thus, a beneficial immunomodulatory mechanism of the gold(I) complexes 8 and 11 in the tumor microenvironment can be expected based on their proven ROS-forming properties in EAC cells, albeit these immune effects remain to be evaluated in future studies.

Our findings indicate that the gold complexes 8 and 11 are promising anticancer agents, as they induce ROS formation and apoptosis in EAC cells. The current manuscript did not establish the mechanistic link between 8- and 11-induced inhibition of TrxR activity, increased oxidative stress, and apoptosis induction in EAC cells. This is a limitation of the study, and further research is needed to clarify the exact molecular pathways, conduct drug-protein target binding assays, evaluate in vivo activity, and investigate suitable combination therapies to improve treatment effectiveness.

4. Materials and Methods

4.1. General Procedures

Column chromatography: silica gel 60 (230–400 mesh, Merck, Darmstadt, Germany). Melting points (uncorrected), Electrothermal 9100 (Thermo Fisher Scientific, Geel, Belgium); IR spectra, Perkin-Elmer Spectrum One FT-IR spectrophotometer with an ATR sampling unit (Perkin Elmer, Rodgau, Germany); NMR spectra, Bruker Avance 300/500 spectrometer (Bruker, Billerica, MA, USA); chemical shifts (δ) are given in parts per million (ppm) downfield from tetramethylsilane as an internal standard; mass spectra, Thermo Finnigan MAT 8500 (EI), UPLC/Orbitrap (ESI-HRMS, using water/acetonitrile mixtures as solvent, Thermo Fisher Scientific, Geel, Belgium).

4.2. Materials

Starting compounds and reagents were obtained from abcr (Karlsruhe, Germany), Sigma-Aldrich (Darmstadt, Germany), and TCI (Zwijndrecht, Belgium). The complexes 1a–c and 7, as well as the imidazolium salt 9 used in this study, were prepared and published before [24,25]. The anisyl-TosMIC compound 2 was synthesized according to a reported procedure [26].

4.3. Synthesis

** 1-Methyl-4-anisyl-5-(4-chlorophenyl)imidazole (3) **

4-Chlorobenzaldehyde (59 mg, 0.42 mmol) was dissolved in ethanol (15 mL), treated with 33% MeNH_2_/EtOH (260 mL, 2.10 mmol) and acetic acid (150 mL, 2.63 mmol), and heated for 2 h under reflux. Anisyl-TosMIC 2 (127 mg, 0.44 mmol) and K_2_CO_3_ (500 mg, 3.62 mmol) were added and the reaction mixture was stirred for 4 h under reflux. The volatiles were evaporated and the residue was suspended in ethyl acetate, washed with water, dried over Na_2_SO_4_, filtered, and the filtrate was concentrated in vacuum. The residue was purified by column chromatography (ethyl acetate, silica gel 60). Yield: 83 mg (0.28 mmol, 67%); colorless amorphous solid; υmax(ATR)/cm^−1^ 2989, 2938, 2836, 1613, 1578, 1514, 1496, 1483, 1453, 1416, 1371, 1294, 1247, 1194, 1171, 1120, 1091, 1029, 1013, 955, 833, 808, 798, 744, 735, 704, 654; ^1^H NMR (300 MHz, CDCl_3_) δ 3.45 (3 H, s), 3.74 (3 H, s), 6.7–6.8 (2 H, m), 7.2–7.3 (2 H, m), 7.3–7.5 (4 H m), 7.52 (1 H, s); ^13^C NMR (75.5 MHz, CDCl_3_) δ 32.2, 55.2, 113.6, 113.8, 114.4, 126.5, 128.1, 127.6, 127.9, 129.2, 131.9, 134.5, 137.6, 137.0, 137.6, 138.6, 158.4; m/z (EI, %) 298 (100) [M^+^], 283 (33), 220 (17), 152 (13).

** 1,3-Dimethyl-4-anisyl-5-(4-chlorophenyl)imidazolium iodide (4) **

1-Methyl-4-anisyl-5-(4-chlorophenyl)imidazole 3 (78 mg, 0.26 mmol) was dissolved in acetonitrile (25 mL) and iodomethane (2 mL) was added. The reaction mixture was stirred at 85 °C for 24 h. The solvent was evaporated and the residue was dried in vacuum. Yield: 110 mg (0.25 mmol, 96%); yellow gum; υmax(ATR)/cm^−1^ 3429, 3007, 2964, 2837, 1628, 1607, 1577, 1517, 1495, 1418, 1398, 1294, 1251, 1178, 1111, 1090, 1051, 1013, 919, 861, 837, 817, 792, 725, 668; ^1^H NMR (300 MHz, CDCl_3_) δ 3.75 (3 H, s), 3.83 (3 H, s), 3.86 (3 H, s), 6.8–6.9 (2 H, m), 7.1–7.3 (2 H, m), 7.2–7.4 (4 H m), 10.15 (1 H, s); ^13^C NMR (75.5 MHz, CDCl_3_) δ 34.9, 35.2, 55.3, 114.6, 116.1, 116.5, 123.3, 129.4, 130.8, 131.9, 132.0, 132.5, 136.5, 136.8, 160.9; m/z (EI, %) 298 (100) [M^+^-CH_3_], 283 (13), 220 (12), 142 (61), 127 (23).

** Chlorido-[1,3-dimethyl-4-anisyl-5-(4-chlorophenyl)imidazole-2-ylidene]gold(I) (5) **

A mixture of imidazolium salt 4 (116 mg, 0.26 mmol), CH_2_Cl_2_ (15 mL), and silver(I) oxide (60 mg, 0.26 mmol) was stirred at room temperature in the dark for 1 d. The suspension was filtered through Celite and the filtrate was concentrated in vacuum. The residue was dissolved in CH_2_Cl_2_ and the silver(I) compound iodido-[1,3-dimethyl-4-anisyl-5-(4-chlorophenyl)imidazole-2-ylidene]silver(I) (82 mg, 0.15 mmol, 58%) was precipitated with n-hexane. The silver(I) intermediate was dissolved in CH_2_Cl_2_ (15 mL) and treated with chloro(dimethylsulfide)gold(I) (55 mg, 0.18 mmol). The reaction mixture was stirred at room temperature for 18 h. The suspension was filtered, the filtrate was concentrated in vacuum, and the residue recrystallized from CH_2_Cl_2_/n-hexane. Yield: 66 mg (0.12 mmol, 81%); amber solid of m.p. 210–211 °C; ^1^H NMR (300 MHz, CDCl_3_) δ 3.70 (3 H, s), 3.72 (3 H, s), 3.80 (3 H, s), 6.8–6.9 (2 H, m), 7.0–7.2 (4 H, m), 7.33 (2 H, d, J = 8.6 Hz); ^13^C NMR (75.5 MHz, CDCl_3_) δ 36.7, 36.8, 55.3, 114.6, 114.5, 119.1, 19.7, 126.2, 129.3, 130.2, 131.6, 131.7, 131.9, 135.6, 160.4, 171.2; m/z (EI, %) 544 (96) [M^+^], 509 (100), 347 (17), 298 (16), 255 (17), 50 (64). Anal. Calcd. For C_18_H_17_AuCl_2_N_2_O: C, 39.65%; H, 3.14%; Found: C, 39.79%; H, 3.22%.

** Iodido-[1,3-dimethyl-4-anisyl-5-(4-chlorophenyl)imidazole-2-ylidene]gold(I) (6) **

Chlorido-[1,3-dimethyl-4-anisyl-5-(4-chlorophenyl)imidazole-2-ylidene]gold(I) 5 (60 mg, 0.11 mmol) and KI (73 mg, 0.439 mmol) were dissolved in acetone (2 mL) and the reaction mixture was stirred at room temperature for 24 h. The solvent was evaporated, the residue was dissolved in CH_2_Cl_2_ and filtered over celite followed by filtration over a plug of silicate. The residue was recrystallized from CH_2_Cl_2_/n-hexane. Yield: 56 mg (0.088 mmol, 80%); off-white solid of mp 190–191 °C; ^1^H NMR (300 MHz, CDCl_3_) δ 3.70 (3 H, s), 3.73 (3 H, s), 3.79 (3 H, s), 6.8–6.9 (2 H, m), 7.1–7.3 (4 H, m), 7.34 (2 H, d, J = 8.7 Hz); ^13^C NMR (75.5 MHz, CDCl_3_) δ 36.3, 36.4, 55.3, 114.3, 114.5, 119.1, 119.7, 126.1, 129.3, 130.1, 131.6, 131.7, 135.6, 160.4, 181.5; m/z (EI, %) 636 (51) [M^+^], 509 (100), 465 (8), 255 (10), 215 (7). Anal. Calcd. For C_18_H_17_ClIN_2_O: C, 33.96%; H, 2.69%; Found: C, 34.06%; H, 2.72%.

Chlorido-[1,3-diethyl-4-anisyl-5-(4-chlorophenyl)imidazole-2-ylidene]gold(I) (7) [24]

Iodido-[1,3-diethyl-4-anisyl-5-(4-chlorophenyl)imidazole-2-ylidene]silver(I) (77 mg, 0.134 mmol) was dissolved in CH_2_Cl_2_ (15 mL) and treated with chloro(dimethylsulfide)gold(I) (49 mg, 0.16 mmol). The reaction mixture was stirred at room temperature for 18 h. The suspension was filtered, the filtrate was concentrated in vacuum, and the residue recrystallized from CH_2_Cl_2_/n-hexane. Yield: 62 mg (0.11 mmol, 82%) [24].

** Triphenylphosphino-[1,3-diethyl-4-anisyl-5-(4-chlorophenyl)imidazole-2-ylidene]gold(I) tetrafluoroborate (8) **

Chlorido-[1,3-diethyl-4-anisyl-5-(4-chlorophenyl)imidazole-2-ylidene]gold(I) 7 (62 mg, 0.11 mmol) was dissolved in acetone (10 mL) and treated with NaBF_4_ (23 mg, 0.205 mmol) and PPh_3_ (38 mg, 0.143 mmol). The reaction mixture was stirred at room temperature for 24 h. The suspension was filtered, the filtrate was concentrated in vacuum, and the residue recrystallized from CH_2_Cl_2_/n-hexane. Yield: 90 mg (0.10 mmol, 91%); colorless solid of m.p. 110 °C (dec.); ^1^H NMR (300 MHz, CDCl_3_/acetone-d_6_) δ 1.3–1.5 (6 H, m), 3.81 (3 H, s), 4.3–4.4 (4 H, m), 6.99 (2 H, d, J = 8.9 Hz), 7.36 (2 H, d, J = 8.8 Hz), 7.4–7.7 (19 H, m); ^13^C NMR (75.5 MHz, acetone-d_6_) δ 17.1, 17.2, 45.0, 45.2, 55.8, 115.1, 115.2, 120.2, 120.7, 127.7, 129.9, 130.0, 131.5, 132.7, 133.1, 133.2, 133.5, 133.6, 135.7, 136.1, 161.5, 161.7, 183.4; ^31^P NMR (121.5 MHz, acetone-d_6_) δ 40.8; ^11^B NMR (96.3 MHz, acetone-d_6_) δ −0.89; HR-MS (ESI) for C_38_H_36_ON_2_AuClP [M^+^-BF_4_], calcd. 799.1914, found 799.1923.

** 1,3-Diethyl-4-anisyl-5-(4-chlorophenyl)imidazolium tetrafluoroborate (10) **

A mixture of 1,3-diethyl-4-anisyl-5-(4-chlorophenyl)imidazolium iodide 9 (119 mg, 0.348 mmol), acetone (30 mL), and NaBF_4_ (59 mg, 0.54 mmol) was stirred at room temperature for 24 h. The mixture was filtered over MgSO_4_, the filtrate was concentrated in vacuum, and the residue was dried. Yield: 128 mg (0.348 mmol, 100%); yellow gum; υmax(ATR)/cm^−1^ 3425, 3129, 2980, 2938, 2837, 1607, 1559, 1517, 1493, 1460, 1398, 1352, 1294, 1251, 1177, 1068, 1015, 839, 806, 783, 733, 709, 655; ^1^H NMR (300 MHz, acetone-d_6_) δ 1.4–1.5 (6 H, m), 3.81 (3 H, s), 4.2–4.3 (4 H, m), 6.9–7.0 (2 H, m), 7.4–7.5 (4 H, m), 7.6–7.7 (2 H m), 9.80 (1 H, s); ^13^C NMR (75.5 MHz, acetone-d_6_) δ 15.4, 15.5, 43.7, 44.0, 55.8, 115.2, 118.2, 125.8, 133.1, 133.3, 133.4, 133.8, 135.9, 136.5, 161.9; ^11^B NMR (96.3 MHz, acetone-d_6_) δ −0.46. m/z (ESI, %) 341.1405 (100) [M^+^-BF_4_], 337.1907 (40).

** Bis-[1,3-diethyl-4-anisyl-5-(4-chlorophenyl)imidazole-2-ylidene]gold(I) tetrafluoroborate (11) **

Imidazolium salt 10 (120 mg, 0.32 mmol) was dissolved in CH_2_Cl_2_/methanol (1:1, 30 mL) and treated with Ag_2_O (84 mg, 0.34 mmol). The resulting mixture was stirred in the dark at room temperature for 5 h. Chloro(dimethylsulfide)gold(I) (56 mg, 0.184 mmol) was added and stirring was continued for a further 20 h. The suspension was filtered, the filtrate was concentrated in vacuum, and the residue was redissolved in CH_2_Cl_2_, and filtered over MgSO_4_/Celite. The filtrate was concentrated and the remainder was dried in vacuum. Yield: 113 mg (0.12 mmol, 65%); colorless solid of m.p. 136–137 °C (dec.); ^1^H NMR (300 MHz, CDCl_3_/acetone-d_6_) δ 1.2–1.3 (6 H, m), 1.4–1.5 (3 H, m), 3.80 (3 H, s), 3.82 (3 H, s), 4.1–4.2 (4 H, m), 4.3–4.4 (4 H, m), 6.9–7.0 (4 H, m), 7.2–7.4 (8 H, m), 7.4–7.5 (4 H, m); ^13^C NMR (75.5 MHz, acetone-d_6_) δ 17.8, 17.9, 45.2, 45.4, 55.8, 115.2, 115.3, 119.8, 120.4, 127.5, 129.5, 130.0, 130.3, 130.6, 130.7, 131.8, 132.7, 132.8, 132.9, 133.2, 133.5, 133.6, 134.1, 135.0, 135.2, 136.0, 161.7, 185.4; ^31^P NMR (121.5 MHz, acetone-d_6_) δ 40.8; ^11^B NMR (96.3 MHz, acetone-d_6_) δ −0.96; HR-MS (ESI) for C_40_H_42_O_2_N_4_AuCl_2_ [M^+^-BF_4_], calcd. 877.2345, found 877.2356.

** Bromido[1,3-diethyl-4-anisyl-5-(4-chlorophenyl)imidazole-2-ylidene]gold(I) (12) **

Imidazolium salt 9 (103 mg, 0.22 mmol) in a mixture of CH_2_Cl_2_ (6 mL) and methanol (2 mL) was combined with silver(I) oxide (29 mg, 0.12 mmol) and stirred overnight under protection from light. Then Me_2_SauCl (65 mg, 0.22 mmol) and LiBr (191 mg 2.2 mmol) were added and the suspension was stirred for an additional 6 h. The gray precipitate was separated by filtration over a bed of Celite and silica gel, and the filtrate was evaporated to dryness under reduced pressure to yield a colorless oil. The residue was recrystallized from CH_2_Cl_2_/n-hexane. Yield: 130 mg (0.21 mmol, 96%); off-white solid of m.p. 182–183 °C (dec.); ^1^H NMR (300 MHz, CDCl_3_) δ 1.2–1.3 (6 H, m), 3.79 (3 H, s), 4.1–4.2 (4 H, m), 6.8–6.9 (2 H, m), 7.0–7.2 (4 H, m), 7.32 (2 H, d, J = 8.7 Hz); ^13^C NMR (75.5 MHz, CDCl_3_) δ 16.9, 44.2, 44.3, 55.3, 114.3, 114.5, 119.3, 119.9, 126.4, 129.3, 130.7, 131.3, 131.7, 131.8, 135.6, 160.4, 173.0; m/z (EI, %) 618 (32) [M^+^], 537 (100), 465 (8), 255 (10), 215 (7). Anal. Calcd. For C_20_H_21_AuBrClN_2_O: C, 38.89%; H, 3.43%; Found: C, 38.95%; H, 3.48%.

** Trisbromido[1,3-diethyl-4-anisyl-5-(4-chlorophenyl)imidazole-2-ylidene]gold(III) (13) **

To a solution of bromido[1,3-diethyl-4-anisyl-5-(4-chlorophenyl)imidazole-2-ylidene]gold(I) 12 (72 mg, 0.12 mmol) in CH_2_Cl_2_ (2 mL) was added bromine (28 mg, 0.18 mmol). The mixture was stirred for 3 h in the dark, and the solvent was removed under reduced pressure, as well as the excess of bromine. The residue was recrystallized from CH_2_Cl_2_/n-hexane. Yield: 81 mg (0.10 mmol, 87%); orange solid of m.p. 105 °C (dec.); ^1^H NMR (300 MHz, CDCl_3_) δ 1.3–1.4 (6 H, m), 3.80 (3 H, s), 4.2–4.3 (4 H, m), 6.8–7.0 (2 H, m), 7.1–7.2 (4 H, m), 7.37 (2 H, d, J = 8.5 Hz); ^13^C NMR (75.5 MHz, CDCl_3_) δ 15.3, 44.2, 44.3, 55.4, 114.5, 114.7, 117.6, 118.2, 124.8, 129.5, 131.8, 131.9, 132.0, 132.7, 133.8, 134.3, 136.5, 138.6, 160.9; m/z (EI, %) 618 (33) [M^+^-2Br], 537 (100), 269 (15), 160 (20), 96 (36). Anal. Calcd. For C_20_H_21_AuBr_3_ClN_2_O: C, 30.90%; H, 2.72%; Found: C, 30.96%; H, 2.78%.

** Iodido-[1,3-diethyl-4-anisyl-5-(4-chlorophenyl)imidazole-2-ylidene]silver(I) (14) **

A mixture of imidazolium salt 9 (105 mg, 0.22 mmol), CH_2_Cl_2_ (15 mL), and silver(I) oxide (51 mg, 0.22 mmol) was stirred at room temperature in the dark for 1 d. The suspension was filtered through Celite and the filtrate was concentrated in vacuum. The residue was dissolved in CH_2_Cl_2_ and the product precipitated with n-hexane. Yield: 93 mg (0.16 mmol, 73%); brown gum; ^1^H NMR (300 MHz, CDCl_3_) δ 1.2–1.3 (6 H, m), 3.79 (3 H, s), 4.0–4.2 (4 H, m), 6.8–6.9 (2 H, m), 7.0–7.2 (4 H, m), 7.31 (2 H, d, J = 8.5 Hz); ^13^C NMR (75.5 MHz, CDCl_3_) δ 17.4, 44.8, 44.9, 55.2, 114.2, 114.4, 119.6, 120.2, 126.7, 129.2, 130.2, 131.6, 131.7, 131.8, 135.4, 160.2, 183.2; m/z (EI, %) 576 (3) [M^+^], 449 (48), 339 (100), 312 (49).

** cis-Dichlorido-[1,3-diethyl-4-anisyl-5-(4-chlorophenyl)imidazole-2-ylidene]-dimethylsulfoxidoplatinum(II) (15) **

The silver complex 14 (72 mg, 0.125 mmol) was dissolved in DMSO (6 mL), treated with K_2_PtCl_4_ (52 mg, 0.125 mmol), and stirred at 60 °C for 24 h. CH_2_Cl_2_ was added, the reaction mixture was filtered, and the filtrate was washed with water and dried over Na_2_SO_4_. The volatiles were removed in vacuum and the remainder was recrystallized from CH_2_Cl_2_/n-hexane. Yield: 58 mg (0.085 mmol, 68%); amorphous solid; ^1^H NMR (300 MHz, CDCl_3_) δ 1.2–1.3 (6 H, m), 3.55 (6 H, s), 3.78 (3 H, s), 4.3–4.6 (4 H, m), 6.8–6.9 (2 H, m), 7.1–7.2 (4 H, m), 7.29 (2 H, d, J = 8.7 Hz); ^13^C NMR (75.5 MHz, CDCl_3_) δ 15.6, 41.0, 43.9, 44.0, 46.2, 55.2, 114.1, 114.3, 119.3, 119.9, 126.4, 129.1, 130.1, 131.4, 131.8, 131.9, 135.4, 142.1, 160.3; ^195^Pt NMR (64.3 MHz, CDCl_3_) δ 959.8; m/z (EI, %) 535 (12) [M^+^-2Cl—DMSO], 506 (5), 340 (22), 313 (44), 120 (19), 44 (28), 36 (100). Anal. Calcd. For C_22_H_27_Cl_3_N_2_O_2_PtS: C, 38.58%; H, 3.97%; Found: C, 38.66%; H, 4.00%.

4.4. Anticancer Activity

4.4.1. Cell Line and Culture Conditions

OE19, OE33 (Sigma-Aldrich), Het-1A (ATCC), FLO-1, THP-1, and SK-GT-4 cells (a gift from Dr. Anant’s lab, University of Kansas Medical Center, KS) were cultured in complete RPMI (L-glutamine and HEPES, Corning, Lowell, MA, USA, and Cytiva, Marlborough, MA, USA). Complete RPMI was prepared by adding 10% fetal bovine serum (heat-inactivated, Biowest Bradenton, FL, USA) and 1% antibiotic-antimycotic solution (Corning, MA). Het-1A cells were cultured in complete BEBM media prepared as per instructions provided by the BEGM kit (Lonza/Clonetics Corporation, Walkersville, MD, USA, Catalog No. CC-3170). The cells were seeded in a coated flask with a mixture of 0.01 mg/mL fibronectin, 0.03 mg/mL bovine collagen type I, and 0.01 mg/mL bovine serum albumin dissolved in culture medium. EAC cells were cultured in a 5% CO_2_ atmosphere at 37 °C. The stock solutions of the gold complexes were prepared in DMSO and diluted in the media to achieve the required concentration. The maximum DMSO concentration was 0.2%. All procedures were performed in accordance with standard guidelines and regulations, as well as with the manufacturer’s instructions.

4.4.2. Proliferation Assay

5000 cells per well (SK-GT-4, FLO-1, OE19, Het-1A, and OE33) were seeded in a 96-well plate with complete media. After 24 h, cells were exposed to various concentrations of gold complexes. After 24–72 h, the medium was discarded, and cell viability was assessed using the hexosaminidase enzymatic assay [44]. A CCK-8 assay (Abcam#ab228554, Cambridge, MA, USA) was performed on THP-1 cells. Briefly, 5000 cells were seeded in each well of a 96-well plate. After 24 h, the cells were treated with the compounds. At the designated time points, an equal volume of CCK-8 reagent was added to the wells, and the plates were incubated for 1 h. Absorbance was then measured at 450 nm [45]. The percentage of inhibition was calculated by comparing cell viability after compound treatment with the viability of control cells.

4.4.3. Colony Formation Assay

500 EAC cells (FLO1 and OE33) per well were seeded in 6-well plates. After 24 h, the cells were treated with complexes 8 (1.7 and 1.9 µM, respectively) and 11 (0.9 and 0.7 µM, respectively). The media containing the compounds was replaced with complete RPMI after 48 h to eliminate the test substances. The cells were then cultured for 10–12 days to develop colonies. These colonies were washed with PBS, fixed with 10% formalin for 20 min, then washed again and stained with 1% crystal violet solution in 10% ethanol. Following staining, the colonies were rinsed to remove excess dye, dried, counted, and compared to control groups [46].

4.4.4. Spheroid Formation Assay

500 EAC cells were seeded in an ultra-low attachment 96-well plate (Corning, Lowell, MA, USA) using spheroid medium made from serum-free DMEM supplemented with heparin salt (4 µg/mL), EGF (20 ng/mL), FGF (20 ng/mL), 1% antibiotic-antimycotic, and B27 supplement. After 2 days, spheroids were treated with 8 (1.7 and 1.9 µM, respectively) and 11 (0.9 and 0.7 µM, respectively). After 7 days, spheroids were counted and imaged [46].

4.4.5. Apoptosis Assay

200,000 EAC cells per well were seeded in a six-well plate with complete RPMI medium. After 24 h, the cells were treated with IC_50_ concentrations of compounds 8 (1.4 and 1.7 µM, respectively) and 11 (0.23 and 0.14 µM, respectively). After 72 h of incubation, the cells were trypsinized, washed, and stained with the Annexin V-FITC Early Apoptosis Detection Kit (Cell Signaling Technology #6592, Danvers, MA, USA) according to the manufacturer’s instructions, and finally analyzed by flow cytometry.

4.4.6. Caspase 3/7 Activation Assay

10,000 EAC cells (SK-GT-4 and FLO-1) per well were plated in an opaque, black, clear-bottom plate using complete RPMI media. After 24 h, the SK-GT-4 and FLO-1 cells were treated with complexes 8 (1.7 and 1.9 µM, respectively) and 11 (0.9 and 0.7 µM, respectively), and with an equivalent dose of complex 11 for auranofin and 8. Following 48 h of incubation, the cells with media were exposed to Caspase 3/7 reagent (Caspase-Glo^®^ 3/7 Assay System, Promega, G8090, Madison, WI, USA) at a 1:1 ratio to the media. After an additional hour of incubation, luminescent readings were taken using a plate reader [46].

4.4.7. Western Blot Analysis

500,000 EAC cells (SK-GT-4 and FLO-1) were seeded in a 10 cm Petri dish. After 24 h, they were treated with complexes 8 (1.7 and 1.9 µM, respectively) and 11 (0.9 and 0.7 µM, respectively) for 48 h. Cells were then washed, lysed in buffer containing phosphatase and protease inhibitors (Roche, Basel, Switzerland), and sonicated. The lysate was centrifuged at 6000 rpm for 10 min at 4 °C. Protein content was measured using the Pierce BCA assay. Equal amounts (50 µg) of protein from each sample were run on gel electrophoresis and transferred onto PVDF membranes (Millipore, Burlington, MA, USA) at 90 V for 2 h under cold conditions. The membranes were blocked with 5% non-fat skimmed milk in TBST for 1 h, washed, and then incubated with primary antibodies at 4 °C overnight. The next day, membranes were washed and incubated with secondary anti-mouse and anti-rabbit antibodies (Cell Signaling Technology, Danvers, MA, USA) for 1 h. Proteins were detected using the GE Healthcare chemiluminescence system and imaged with the Bio-Rad ChemiDoc-XRS+ system, with images processed in Image Lab. Antibodies for cyclin D1 (CST#2922), Bcl-XL (CST#2762), Bcl-2 (CST#4223), and Mcl-1 (CST#4572) were obtained from Cell Signaling Technology (Danvers, MA, USA), while GAPDH (G-9) was purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

4.4.8. Reactive Oxygen Species Assay

The ROS assay was performed with 10,000 OE33 cells per well, grown in complete RPMI medium in black, clear-bottom 96-well plates, and treated with auranofin, complex 8, and complex 11 after 24 h. After 72 h, the wells were treated with the ROS staining kit from Abcam (Cellular ROS Assay Kit (Red), ab186027, Cambridge, MA, USA). After 37 min, readings were taken with a fluorometric plate reader (Ex/Em 520/605 nm) [47].

4.4.9. Thioredoxin Reductase Activity Assay

The TrxR activity assay was conducted in SK-GT-4 and OE33 cells using the Thioredoxin Reductase Colorimetric Assay Kit (Cayman Chemical, Item No. 10007892, Ann Arbor, MI, USA). Cells were seeded at a density of 600,000 cells per 10 cm tissue culture dish and incubated for 24 h. Following this incubation period, the cells were treated with Auranofin, complex 8 (G2), and complex 11 (G4). After 48 h of treatment, the cells were collected and lysed. Then, the TrxR activity was measured colorimetrically. The lysates were incubated with DTNB and NADPH substrates, and absorbance was measured at one-minute intervals after a 30 s shaking incubation. A colorimeter set to 405 nm was used to measure absorbance, enabling determination of TrxR activity.

4.4.10. Statistical Analysis

All values are shown as the mean  ±  SD. Experimental data were examined using an unpaired two-tailed t-test or one-way ANOVA by comparing with the corresponding control group. A probability value of less than 0.05 was considered statistically significant.