New 4,5-Diarylimidazol-2-ylidene–iodidogold(I) Complexes with High Activity against Esophageal Adenocarcinoma Cells

Inspired by the vascular-disrupting agent combretastatin A-4 and recently published anticancer active N-heterocyclic carbene (NHC) complexes of Au(I), a series of new iodidogold(I)–NHC complexes was synthesized and characterized. The iodidogold(I) complexes were synthesized by a route involving van Leusen imidazole formation and N-alkylation, followed by complexation with Ag2O, transmetalation with chloro(dimethylsulfide)gold(I) [Au(DMS)Cl], and anion exchange with KI. The target complexes were characterized by IR spectroscopy, 1H and 13C NMR spectroscopy, and mass spectrometry. The structure of 6c was validated via single-crystal X-ray diffraction. A preliminary anticancer screening of the complexes using two esophageal adenocarcinoma cell lines showed promising nanomolar activities for certain iodidogold(I) complexes accompanied with apoptosis induction, as well as c-Myc and cyclin D1 suppression in esophageal adenocarcinoma cells treated with the most promising derivative 6b.


Introduction
Esophageal cancer (EC) ranks seventh worldwide in terms of incidence (604,100 new cases) and sixth in overall mortality (544,076 deaths) [1]. Especially high morbidity and mortality rates for esophageal cancer are observed in East, Central, and South Asia, South and East Africa, and Northwest Europe. Moreover, these alarming numbers are expected to rise by 35-37% until 2030 [2]. Considering the poor prognosis of EC patients, frequently resulting from late diagnoses, this tumor is a considerable health issue for developed and developing countries [2,3]. EC is a rapidly growing cancer with a poor five-year survival rate of <20% [4]. The biology of EC is hardly understood compared to other cancers and typically shows extremely aggressive clinical features upon diagnosis [5]. Hence, it is one of the most challenging cancers to treat [6]. EC is divided into two histological subtypes [squamous cell carcinoma (ESCC) and adenocarcinoma (EAC)], which have diverse etiologies. ESCC is the most common EC worldwide, while EAC accounts for roughly two-thirds of EC cases in western countries [7]. The incidence of EAC is rapidly increasing across highincome countries due to obesity, an increase in gastroesophageal reflux disease (GERD), and Barrett's esophagus (BE) [8]. Current treatment options for esophageal cancer include endoscopic and surgical treatments and chemoradiotherapy based on platinum complexes such as cisplatin [9, 10]. Recently, the combination of platinum-based chemo(radio)therapy with immune checkpoint inhibitors revealed promising results in patients suffering from EC [11]. Thus, platinum complexes continue to be highly relevant for the management of EC, and metal-based drugs with other metals than platinum may have great potential for the treatment of this disease in the future. While platinum complexes are clinically applied for the treatment of various solid tumors, gold complexes such as auranofin (1a) are being used for the treatment of rheumatoid arthritis ("chrysotherapy") ( Figure 1) [12]. However, as early as 1986, auranofin was investigated as an active compound in a study with esophageal carcinoma [13]. Auranofin revealed anticancer properties in preclinical studies based on its considerable inhibition of thioredoxin reductase (TrxR) associated with oxidative stress by the formation of reactive oxygen species (ROS), leading to increased apoptosis induction [12]. In addition, auranofin was described as an inhibitor of proteasomal deubiquitinase in cancer cells [13,14]. The mitochondrial Mia40/CHCHD4 pathway playing a crucial role in the oxidation of freshly imported cysteine-rich proteins in mitochondria was identified as a target of auranofin in fungi, which might be relevant in vigorously proliferating cancer cells too [15,16]. Furthermore, derivatives of auranofin with improved anticancer properties were described [17,18]. Recent developments in the field of auranofin-derived gold compounds led to highly active thiolatopurine complexes showing strong DNA damaging effects aside from TrxR inhibition, apoptosis induction, ROS formation, and antiangiogenic effects [19]. Advances in the research of N-heterocyclic carbene (NHC) gold complexes established a prospering class of metal-based drugs, which showed eminent anticancer activities based on TrxR inhibition and DNA interaction, adding well to the currently available arsenal of anticancer platinum complexes and the leading gold(I) complex auranofin [20][21][22][23][24]. Another emphasis was laid on the investigation of the effects on cytoskeletal structures, especially actin filaments and microtubules, by gold(I)-NHC complexes with 4,5-diarylimidazole-2-ylidene ligands derived from the tubulin-binding natural product combretastatin A-4 [25][26][27]. Meanwhile, the disruption of the actin cytoskeleton was described for cancer cells treated with auranofin too [28]. Recently, the relevance of the iodido ligand of neutral 4,5-dianisylimidazole-2-ylidene-iodidogold(I) complexes was proved, which revealed promising in vitro and in vivo antihepatoma activities of the iodido complex 1b when compared with analogous gold(I)-NHC complexes bearing halo ligands (bromido), pseudohalo ligands (isocyanato), or acetato ligands ( Figure 1) [29].
for the management of EC, and metal-based drugs with other metals than platinum may have great potential for the treatment of this disease in the future.
While platinum complexes are clinically applied for the treatment of various solid tumors, gold complexes such as auranofin (1a) are being used for the treatment of rheumatoid arthritis ("chrysotherapy") ( Figure 1) [12]. However, as early as 1986, auranofin was investigated as an active compound in a study with esophageal carcinoma [13]. Auranofin revealed anticancer properties in preclinical studies based on its considerable inhibition of thioredoxin reductase (TrxR) associated with oxidative stress by the formation of reactive oxygen species (ROS), leading to increased apoptosis induction [12]. In addition, auranofin was described as an inhibitor of proteasomal deubiquitinase in cancer cells [13,14]. The mitochondrial Mia40/CHCHD4 pathway playing a crucial role in the oxidation of freshly imported cysteine-rich proteins in mitochondria was identified as a target of auranofin in fungi, which might be relevant in vigorously proliferating cancer cells too [15,16]. Furthermore, derivatives of auranofin with improved anticancer properties were described [17,18]. Recent developments in the field of auranofin-derived gold compounds led to highly active thiolatopurine complexes showing strong DNA damaging effects aside from TrxR inhibition, apoptosis induction, ROS formation, and antiangiogenic effects [19]. Advances in the research of N-heterocyclic carbene (NHC) gold complexes established a prospering class of metal-based drugs, which showed eminent anticancer activities based on TrxR inhibition and DNA interaction, adding well to the currently available arsenal of anticancer platinum complexes and the leading gold(I) complex auranofin [20][21][22][23][24]. Another emphasis was laid on the investigation of the effects on cytoskeletal structures, especially actin filaments and microtubules, by gold(I)-NHC complexes with 4,5-diarylimidazole-2-ylidene ligands derived from the tubulin-binding natural product combretastatin A-4 [25][26][27]. Meanwhile, the disruption of the actin cytoskeleton was described for cancer cells treated with auranofin too [28]. Recently, the relevance of the iodido ligand of neutral 4,5-dianisylimidazole-2-ylidene-iodidogold(I) complexes was proved, which revealed promising in vitro and in vivo antihepatoma activities of the iodido complex 1b when compared with analogous gold(I)-NHC complexes bearing halo ligands (bromido), pseudohalo ligands (isocyanato), or acetato ligands ( Figure 1) [29].
With iodido being the optimal ligand for neutral 4,5-dianisylimidazol-2-ylidenegold(I) complexes in hepatoma, subtle changes in the 4,5-dianisylimidazole-based NHC ligand might lead to iodidogold(I) complexes with improved activities against various tumor entities. This work studied the impact of various alterations of the NHC ligand of 1b on the activity against invasive EAC cells.  With iodido being the optimal ligand for neutral 4,5-dianisylimidazol-2-ylidene-gold(I) complexes in hepatoma, subtle changes in the 4,5-dianisylimidazole-based NHC ligand might lead to iodidogold(I) complexes with improved activities against various tumor entities. This work studied the impact of various alterations of the NHC ligand of 1b on the activity against invasive EAC cells.

Chemistry
The synthesis of the new iodidogold(I) complexes 6a-n was carried out following procedures from the literature (Scheme 1) [25][26][27]29]. The TosMIC reagent 2 was prepared as described before [30]. Reagent 2 was treated with EtNH 2 and the corresponding aryl aldehyde to form the 1-ethyl-4,5-diarylimidazoles 3. N-Alkylation with ethyl iodide led to the imidazolium iodides 4. The reaction of 4 with Ag 2 O followed by transmetalation of the NHC-silver(I) intermediates with chloro(dimethylsulfide)gold(I) [Au(DMS)Cl] resulted in In addition to the diethylimidazol-2-ylidene complexes 1b and 6a-o, the new 1banalogous dimethylimidazol-2-ylidene complex 6o was prepared from the known chloridogold(I) precursor 5o for comparison purposes (Scheme 2) [24]. In addition to the diethylimidazol-2-ylidene complexes 1b and 6a-o, the new 1banalogous dimethylimidazol-2-ylidene complex 6o was prepared from the known chloridogold(I) precursor 5o for comparison purposes (Scheme 2) [24]. as described before [30]. Reagent 2 was treated with EtNH2 and the corresponding ary aldehyde to form the 1-ethyl-4,5-diarylimidazoles 3. N-Alkylation with ethyl iodide led to the imidazolium iodides 4. The reaction of 4 with Ag2O followed by transmetalation of the NHC-silver(I) intermediates with chloro(dimethylsulfide)gold(I) [Au(DMS)Cl] resulted in the chloridogold(I) complexes 5. Finally, the chlorido ligands of 5 were replaced by iodido ligands upon reaction with KI, thus, generating the target complexes 6a-n. Scheme  In addition to the diethylimidazol-2-ylidene complexes 1b and 6a-o, the new 1banalogous dimethylimidazol-2-ylidene complex 6o was prepared from the known chloridogold(I) precursor 5o for comparison purposes (Scheme 2) [24]. The stability of complexes 5e and 6e was studied by 1 H NMR spectroscopy in DMSOd 6 upon addition of 5% D 2 O (Figure 2). After 24 h, new signals of the N-ethyl groups appeared, which grew with prolonged incubation. These new signals can be assigned to Single crystals suitable for X-ray diffraction structure elucidation were obtained via slow diffusion of n-hexane into a saturated solution of 6c in CHCl3 at 4 °C. The 6c crystallizes in the monoclinic space group P21/n with Z = 4. A plot of the molecular structure of 6c is given in Figure 3A. The linearity of the I-Au-C vector and the bond distance between Au and the carbene carbon are well in agreement with values from the literature [31]. Selected bond lengths and angles are given in the caption of Figure 3; crystallographic details Single crystals suitable for X-ray diffraction structure elucidation were obtained via slow diffusion of n-hexane into a saturated solution of 6c in CHCl 3 at 4 • C. The 6c crystallizes in the monoclinic space group P2 1 /n with Z = 4. A plot of the molecular structure of 6c is given in Figure 3A. The linearity of the I-Au-C vector and the bond distance between Au and the carbene carbon are well in agreement with values from the literature [31]. Selected bond lengths and angles are given in the caption of Figure 3; crystallographic details are assembled in Table 1. A view along crystallographic axis a reveals the formation of pseudo-dimers in a head-to-tail arrangement with a short Au-Au non-bonded contact of d(Au-Au) = 3.806 Å ( Figure 3B).

Activity against Esophageal Cancer
Initially, the antiproliferative activities of complexes 6a-o against cell lines (FLO-1 and SK-GT-4) were evaluated using the hexosaminidase assay ( Table 2 and Figure 4A). The known iodido complex 1b and the chlorido complexes 5a-g were analyzed for comparison. Except for complex 6l, all iodidogold(I) complexes 6 exhibited moderate to high activities against FLO-1 cells, while the SK-GT-4 cells were less sensitive in most cases. Complexes 6b, 6i, and 6m were the most active compounds that inhibited the growth of EAC cells in a dose-and time-dependent manner, with IC 50 values in the nanomolar concentration range of 0.26-0.4 µM and 0.12-0.45 µM in the cases of SK-GT-4 and FLO-1, respectively. These complexes were also distinctly more active than the known complex 1b. In analogy to the high activity of the 4-chlorophenyl derivative 6b, which was the most active compound of this series, the 3,5-dichlorophenyl derivative 6f was distinctly more active against the SK-GT-4 cells than its close congeners 6e and 6g. In contrast, the 3-chloro-4,5-dimethoxyphenyl analog 6l was inactive. The chloridogold(I) complexes 5 were generally less active than their iodidogold(I) analogs, except for complexes 5a and 5g, which were more active against SK-GT-4 cells than the iodido complexes 6a and 6g. In addition, complex 6o, the N,N-dimethyl analog of 6k, was more active than 6k. showed the strongest colony formation suppression (i.e., reduced colony size and number) at IC50 and semi-IC50 concentrations. It completely inhibited colony formation in terms of size and number (p < 0.01) at its IC50 dose after treatment for 72 h ( Figure 4B,C).
In addition, at IC50 doses, the colony formation was almost completely inhibited by complex 6i, while 6d was less inhibitory. These data suggested that the anticancer effects of the tested gold complexes are irreversible.  Compounds 6b, 6d, and 6i were selected for further evaluation of their inhibitory activity against EAC cell lines (SK-GT-4 and FLO-1). The colony formation was performed to understand the long-term effect of these gold complexes on EAC cells. Complex 6b showed the strongest colony formation suppression (i.e., reduced colony size and number) at IC 50 and semi-IC 50 concentrations. It completely inhibited colony formation in terms of size and number (p < 0.01) at its IC 50 dose after treatment for 72 h ( Figure 4B,C). In addition, at IC 50 doses, the colony formation was almost completely inhibited by complex 6i, while 6d was less inhibitory. These data suggested that the anticancer effects of the tested gold complexes are irreversible.
Several studies showed cancer stem cells (CSCs) are involved in tumor initiation, aggressiveness, and drug resistance in EAC. Hence, targeting CSCs is an attractive strategy to treat [32]. It was observed that CSCs form spheroids in ultra-low attachment plates. Hence, we used a spheroid formation assay to assess the effects of gold complexes on EAC CSCs. The complexes 6b, 6d, and 6i inhibited spheroid formation (both size and numbers) by EAC cells (Figure 5A,B). Complexes 6b and 6d showed the strongest inhibitory effects on spheroids. Next, the highly antiproliferative complexes 6b, 6d, and 6i (IC 50  To understand the mechanistic changes in proteins due to cell cycle arrest, we performed a Western blot to study the levels of cyclin D1 and cMyc. Cyclin D1 is known to drive cell cycle progression, while c-Myc regulates cyclin D1 to induce proliferation and tumor growth [33]. Moreover, these complexes suppressed the expression of c-Myc and cyclin D1, which is in line with their strong cell death/apoptosis induction ( Figure 6E). Hence, we further studied the ability of 6b, 6d, and 6i (IC 50 concentration) to induce apoptosis in EAC cell lines using the Annexin V/PI assay and flow cytometry. Cell populations treated with the gold complexes (IC 50 concentration) for 72 h showed increased percentages of apoptotic (especially late apoptotic) and necrotic cells when compared with untreated control populations ( Figure 7A-D, p < 0.01). In line with this finding, the number of viable cells was reduced in the treated populations. The Western blot analysis showed that all three gold(I) complexes (IC 50 concentration) increased cleaved PARP as a sign of apoptosis after 72 h. The expression of the anti-apoptotic factors Bcl-XL, Bcl-2, and Mcl-1 was suppressed in cells treated with the gold complexes. No significant differences in pro-apoptotic Bax expression were observed compared to untreated cells ( Figure 7E). These data suggest that gold complexes induced apoptosis by inhibiting the apoptotic proteins involved in cancer cell survival and, hence, can be used in the combination with current chemotherapeutic agents.

Discussion
New derivatives of the published 4,5-dianisylimidazol-2-ylidene-iodidogold(I) complex 1b were prepared by changing the modification of one of the ligand's anisyl residues. The synthesis of the new iodidogold(I)-NHC complexes 6a-o was straightforward and based on previously published works by our groups and by Bian and coworkers [25][26][27]29]. In this way, many highly antitumoral iodidogold(I)-NHC complexes were identified. The antiproliferative activities of several new iodidogold(I) complexes against two invasive EAC cell lines were superior to the activity of the known complex 1b. They were also much more active than their chloridogold(I) precursors. Neutral chloridogold(I)-NHC complexes were often reported to be less anticancer active than analogous cationic NHC-gold(I) complexes [34,35]. Chloridogold-NHC complexes were casually developed as selective antiparasitic agents due to their relatively low toxicity to human cells [36]. However, with the exchange of the chlorido ligand for an iodido ligand, the cytotoxicity of neutral iodidogold(I) complexes reached excellent IC 50 values in cancer cells, being in the active concentration range of known cationic triphenylphosphinogold(I)-NHC complexes and biscarbene-gold(I) complexes. Future studies will reveal how far neutral iodidogold(I)-NHC complexes and cationic NHC-gold(I) complexes differ in their performance in cancers in terms of activity, cellular localization/accumulation, and mechanisms of action. Nevertheless, it is noteworthy that, already, slight modifications of one of the phenyl rings of the 4-anisyl-5-arylimidazole-based NHC ligand system applied in this study can lead to strong changes in the anticancer activity of the tested iodidogold(I) complexes. For instance, while the 4-bromophenyl derivative 6c was virtually inactive against the SK-GT-4 cells, its close 4-chlorophenyl analog 6b is the most active complex identified in this study. In addition, while complexes 1b, 6b, 6d, 6i, and 6m showed high antiproliferative activities against FLO-1 cells and SK-GT-4 cells, the FLO-1 cells were much more sensitive to certain complexes such as 6a, 6c, 6e and 6g than the SK-GT-4 cells. FLO-1 cells are p53-mutant cells, and the gold complexes may take advantage of the absence of p53, the "guardian of the genome", to kill the FLO-1 cells. Analogously, higher sensitivities of p53-knockout HCT-116 colon carcinoma cells, when compared with p53-wildtype HCT-116 cells, were observed only recently for various NHC-gold(I) complexes and phosphinogold(I) complexes [19,35].
Apoptosis is strongly induced by the most promising iodidogold(I) complex 6b. This is in line with previous reports about the induction of apoptosis by auranofin (1a) and complex 1b in tumor cells [12,29]. The downregulation of Mcl-1 was described before in association with apoptosis induction in FLO-1 and SK-GT-4 cells [37]. In contrast to the described G2/M arrest of HepG2 hepatoma cells caused by 1b, the new complexes 6b, 6d, and 6i exhibited no cell cycle arrest in EAC cells. Instead, high sub-G1 levels generated by these complexes indicate a strong preference to induce cell death. The observed suppression of cyclin D1 and c-Myc by the complexes 6b, 6d, and 6i might prohibit the ability of treated cells to enter the proper cell cycle, including mitosis, and directly paves the way to the induction of cell death instead. Both factors are relevant for EAC progression in FLO-1 cells [38,39]. In addition, the suppression of c-Myc and/or cyclin D1 is important for the treatment of other cancer diseases, which broadens the therapeutic scope of the newly discovered gold complexes [40,41].
The suppression of the formation of colonies and spheroids by EAC cells by complexes 6b, 6d, and 6i is another positive attribute. The downregulation of the stem cell marker CD44 is a hint at an efficient targeting of esophageal cancer stem-like cells (CSCs) and the inhibition of mesenchymal features of EAC cells associated with metastasis formation [42]. EMT reversal in FLO-1 and SK-GT-4 EAC cells can also suppress paracrine effects and the production of exosomes [43].
Since platinum complexes are crucial components of currently applied clinical therapies of EAC, future studies with gold(I)-NHC complexes will probably shed more light on common and distinctive modes of action of platinum and gold complexes. The binding to cysteine and selenocysteine proteins (e.g., thioredoxin reductase) may play a role in the mode of action of the new iodidogold(I) complexes. The relevance of selenium was also highlighted by the suppression of selenium-binding protein 1 as a sign of EAC formation [44]. The combination of gold complexes with HDAC inhibitors might be promising when considering the known effects of HDAC inhibition on thioredoxin and thioredoxininteracting protein in EAC [45,46]. In addition, aurora kinase inhibitors revealed promising anticancer effects in combination with docetaxel or cisplatin in p53-mutant FLO-1 cells. Thus, they might also be suitable combination partners for the treatment of EAC together with active iodidogold(I)-NHC complexes [47,48]. c-Myc inhibitors might also be suitable combination partners [40]. Moreover, our groups have recently identified highly active c-Myb inhibitors [49][50][51]. This transcription factor was found to be upregulated in EAC and crucial for the immune escape of EAC cells via the miR-145-5p/SPOP/PD-L1 axis [52][53][54]. Hence, a combination of 6b with a potent c-Myb inhibitor seems worth being investigated in EAC cells.

Materials
Starting compounds and reagents were obtained from abcr (Karlsruhe, Germany), Sigma-Aldrich (Darmstadt, Germany) and TCI (Zwijndrecht, Belgium). Compound 1b was prepared according to a literature procedure, and analytical data of the newly prepared compound were in line with published data [24]. The known intermediates 3k-m and 4k-m, 5k, and 5o were also prepared following procedures from the literature [25][26][27].

Synthesis of Diethylimidazolium Iodides 4-Typical Procedure
Imidazoles 3 (1.00 equiv.) were dissolved in acetonitrile (60.0 mL/mmol) and treated with ethyl iodide (55.0 equiv.). The reaction mixture was stirred at 85 • C for 24-70 h. The reaction was concentrated in vacuum, and the residue was dissolved in a small amount of CH 2 Cl 2 and dropped into Et 2 O, leading to the precipitation of the product. The solvent was decanted, and the residue was dried in vacuum. The products were obtained as brown or off-white solids or oils in yields of 74-91%.

Synthesis of Chloridogold(I) Complexes 5-Typical Procedure
Imidazolium salts 4 (1.00 equiv.) were dissolved in CH 2 Cl 2 (30 mL/mmol) and Ag 2 O (0.60 equiv.) was added. The reaction mixture was stirred at room temperature for 6 h at r.t. in the dark. Thereupon, AuCl(SMe2) (1.10 equiv.) was added, and the reaction mixture was stirred at room temperature for 24 h in the dark. The mixture was filtered through Celite ® , the filtrate was concentrated in vacuum, and the product was recrystallized from CH 2 Cl 2 /

Proliferation Assay
A total of 5000 EAC cells/well (SK-GT-4 and FLO-1) were plated in a 96-well plate using complete DMEM. After 24 h of plating, EAC cells were treated with gold complexes at different concentrations. After 72 h, the medium was removed, and cell viability was measured using the hexosaminidase enzymatic assay [59]. The percentage of inhibition was calculated by comparing cell viability after compound treatment with controls.

Colony Formation Assay
A total of 500 cells/well of EAC were plated in 6-well plates. After 24 h, the EAC cells were treated with IC 50 and semi-IC 50 concentrations of 6b, 6d, and 6i. Compounds containing media were replaced after 72 h with complete DMEM to remove the test compounds. The cells were grown for 10-12 days to form colonies. The resulting colonies were washed and fixed using a 10% formalin solution. After 20 min, the formalin was removed, and the fixed cells were washed and stained with 1% crystal violet solution in 10% ethanol. After staining, colonies were washed to remove crystal violet, dried, counted, and compared to controls [60]. We scanned the stained and dried 6-well plates using a Canon Image RUNNER Advance scanner to make figures.

Cell Cycle Analysis
A total of 200,000 EAC cells (SK-GT-4 and FLO-1) per well were plated in 6-well plates. After 24 h, EAC cells were treated with IC 50 and semi-IC 50 concentrations of compounds 6b, 6d, and 6i. After 72 h, EAC cells were washed, resuspended in PBS, and fixed using an ice-cold fixing solution (70% ethanol in PBS), followed by storage overnight at 4 • C. The next day, EAC cells were centrifuged, washed with PBS, resuspended, permeabilized, and stained with FxCycleTM PI/RNase staining solution (Invitrogen). The cell cycle was studied by flow cytometry using an FACS Calibur analyzer (Becton Dickinson, Mountain View, CA, USA). The experimental datasets were plotted using ModFit LT™ software (Verity Software House, Topsham, ME, USA).

Apoptosis Assay
A total of 200,000 EAC cells/well were plated in a 6-well plate in complete DMEM, and, after 24 h, the EAC cells were treated with IC 50 concentrations of compounds 6b, 6d, and 6i. After 72 h, cells were trypsinized, washed and stained using the Annexin V-FITC Early Apoptosis Detection Kit (Cell Signaling Technology#6592) following the manufacturer's instructions, and studied by flow cytometry.

Western Blot Analysis
A total of 500,000 cells of EAC cell lines were plated in a 10 cm cell culture Petri dish, and, after 24 h, cells were incubated with IC 50 concentrations of 6b, 6d, and 6i for 72 h. Cells were washed and lysed in lysis buffer with phosphatase and protease inhibitor (Roche), followed by sonification. The protein lysate was centrifuged at 6000 rpm for 10 min at 4 • C. Protein determination was performed using the Pierce BCA protein assay kit to estimate protein contents. A total of 50 µg of protein from each group was subjected to gel electrophoresis and further transferred onto polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA) at 90 V for 2 h under cold conditions. These PVDF membranes were then blocked for 1 h by using 5% milk in TBST, washed with TBST, and incubated with primary antibodies at 4 • C overnight. The next day, membranes were washed using TBST to remove primary antibodies and incubated with respective secondary anti-mouse and antirabbit antibodies (Cell Signaling Technology, anti-mouse#7076, anti-rabbit#7074) for 1 h. The proteins were identified by using the GE Health Care chemiluminescence system (Piscataway, NJ, USA), imaged using the Bio-Rad ChemiDoc-XRS+ instrument, and processed by image lab. Antibodies for detecting cyclin D1 (CST#2922), Bcl-XL (CST#2762), Bcl-2 (CST#4223), c-Myc (CST#9402), Bax (CST#2772), Mcl-1 (CST#4572), and PARP (CST#9542) were bought from Cell Signaling Technology (Beverly, MA, USA), and GAPDH (G-9) was purchased from Santa Cruz Biotech Inc. (Santa Cruz, CA, USA).

Statistical Analysis
All values are shown as the mean ± SD. Experimental data were examined using an unpaired two-tailed t-test by comparing to the corresponding control group. A probability value of less than 0.05 was considered as statistically significant (* p < 0.05, ** p < 0.01).

Conclusions
The NHC ligand system of a previously published anticancer active iodidogold(I)-NHC complex was successfully optimized in terms of anticancer properties, and high activities against EAC cells were achieved for several new gold complexes. These compounds induced programmed cell death and suppressed colony and spheroid formation by EAC cells at low doses. Together with their promising suppressive effects on cyclin D1 and c-Myc expression, there exists a considerable potential of iodidogold(I)-NHC complexes as new candidates for the treatment of problematic tumor diseases such as EAC. Deeper investigations of the mechanisms of action of these gold compounds will provide more information about their prospects as new anticancer drugs. An extension of compound testing to other tumor entities than EAC will also be of great interest given the described anticancer properties of the newly discovered gold complexes.