
==== Front
J Med Chem
J Med Chem
jm
jmcmar
Journal of Medicinal Chemistry
0022-2623
1520-4804
American Chemical Society

39196554
10.1021/acs.jmedchem.4c01117
Article
A New Class of Gold(I) NHC Complexes with Proapoptotic and Resensitizing Properties towards Multidrug Resistant Leukemia Cells Overexpressing BCL-2
https://orcid.org/0009-0007-3344-1602
Bannwart Franziska *†‡
Richter Leon F. §
Stifel Simon §
Rueter Johanna †‡
Lode Holger N. ∥
https://orcid.org/0000-0002-7847-4906
Correia João D. G. ⊥
https://orcid.org/0000-0002-4156-780X
Kühn Fritz E. *§
Prokop Aram *†‡#
† Department of Human Medicine, MSH Medical School Hamburg, Am Kaiserkai 1, 20457 Hamburg, Germany
‡ Department of Pediatric Hematology/Oncology, Helios Kliniken Schwerin, Wismarsche Str. 393-397, 19055 Schwerin, Germany
§ Department of Chemistry and Catalysis Research Center, Molecular Catalysis, Technical University of Munich, TUM School of Natural Sciences, Lichtenbergstr. 4, 85748 Garching bei München, Germany
∥ Department of Pediatric Hematology/Oncology, University Medicine Greifswald, Ferdinand-Sauerbruch-Str. 1, 17475 Greifswald, Germany
⊥ Centro de Ciências e Tecnologias Nucleares and Departamento de Engenharia e Ciências Nucleares, Instituto Superior Técnico, Universidade de Lisboa, Bobadela, Lisbon, LRS 2695-066, Portugal
# Experimental Oncology, Municipal Hospitals of Cologne, Ostmerheimer Str. 200, 51109 Cologne, Germany
* F.B.: phone, (+49) 385 520 6397; email, franziska.bannwart@medicalschool-hamburg.de.
* A.P.: phone, (+49) 385 520 6396; fax, (+49) 385 520 2704; email, aram.prokop@helios-gesundheit.de.
* F.E.K.: phone, (+49) 89 289 13096; email, fritz.kuehn@ch.tum.de.
28 08 2024
12 09 2024
67 17 1549415508
13 05 2024
15 08 2024
01 08 2024
© 2024 The Authors. Published by American Chemical Society
2024
The Authors
https://creativecommons.org/licenses/by-nc-nd/4.0/ Permits non-commercial access and re-use, provided that author attribution and integrity are maintained; but does not permit creation of adaptations or other derivative works (https://creativecommons.org/licenses/by-nc-nd/4.0/).

From previous studies, it is evident that metal–organic gold(I) complexes have antiproliferative activities. The aim of this study is not only to find new anticancer agents but also to overcome existing cytostatic resistance in cancer cells. The synthesis and medicinal evaluation of two cationic 1,3-disubstituted gold(I) bis-tetrazolylidene complexes 1 and 2 are reported. To determine apoptosis-inducing properties of the complexes, DNA fragmentation was measured using propidium iodide staining followed by flow cytometry. Gold(I) complex 1 targets explicitly malignant cells, effectively inhibiting their growth and selectively inducing apoptosis without signs of necrosis. Even in cells resistant to common treatments such as doxorubicin, it overcomes multidrug resistance and sensitizes existing drug-resistant cells to common cytostatic drugs. It is assumed that gold(I) complex 1 involves the mitochondrial pathway in apoptosis and targets members of the BCL-2 family, enhancing its potential as a therapeutic agent in cancer treatment.

FundaÃ§Ã£o para a CiÃªncia e a Tecnologia 10.13039/501100001871 PTDC/QUI-OUT/3854/2021 Koch-Foundation NA NA Kleist Foundation NA NA Foundation David NA NA Foundation Blankenheimerdorf NA NA FundaÃ§Ã£o para a CiÃªncia e a Tecnologia 10.13039/501100001871 UIDP/04349/2020 document-id-old-9jm4c01117
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pmcIntroduction

Following the fortuitous discovery of cisplatin in the 1960s by Rosenberg et al., the popularity of metallotherapeutic drugs and metal-based diagnostic agents grew rapidly.1,2 This was primarily due to their efficacy as anticancer chemotherapeutics.3 However, in some types of cancer, cells may develop resistance after initial treatment or persist despite treatment.4,5 Such resistance is either intrinsic or can be acquired during chemotherapy.6 Acquired cells may be resistant not only to the drug being treated but also cross-resistant to multiple cytostatic drugs. Mechanisms for the development of resistance include increased drug efflux, mutated drug targets, repair of DNA damage caused by cytostatic drugs, inactivation and metabolism of drugs and evasion of apoptosis.7,8 Overcoming drug resistance would positively impact patients’ chances of recovery.6 The goal is either to find other effective treatments or to resensitize the resistant cells. The latter is the subject of current research and could significantly expand patient treatment options.9 For example, transport systems such as P-glycoprotein (PGP) or other ATP-binding cassette (ABC) transport systems can be inhibited to minimize or eliminate drug efflux pathways.10,11 Besides resistance formation, a significant drawback of platinum-based medications is their lack of selectivity, which can result in severe negative effects due to their nonspecific mechanism of action and interaction with healthy cells, particularly those with high metabolism.12 In recent years, researchers have shifted their focus to other metal-based drugs in order to discover more cancer-cell-specific mechanisms of action and reduce side effects. Various gold compounds have since been characterized and found to exhibit intriguing antiproliferative properties.13−16 Gold(I) complexes are especially being considered potential substitutes for the usual platinum-based agents due to their distinct and more targeted mode of action.17−19 Unlike platinum compounds, multiple biological studies have shown that most gold compounds make considerably weaker bonds with cellular DNA, which suggests a distinct, DNA-independent mode of action.17 Auranofin (AF) is an organogold complex already approved for the treatment of rheumatoid arthritis that has shown apoptotic effects on neuroblastoma cells in previous studies.20 However, due to its significant side effects and low stability in vivo, it is now only considered a second choice. AF has been shown to selectively target the thioredoxin reductase (TrxR) system in cancer cells, affecting the formation of reactive oxygen species (ROS) in the cell.21,22 Unfortunately, gold complexes with labile ligands (such as AF) may irreversibly bind to transporter proteins such as human serum albumin (HSA) or antioxidants such as glutathione (GSH), preventing them from reaching the malignant cells in vivo.23 Therefore, designing the ligand is crucial to balance the reactivity and stability of the resulting gold complexes.24 Among the various types of ligands used to stabilize metal complexes, N-heterocyclic carbenes (NHCs) are particularly noteworthy. Generally, NHCs are defined as heterocyclic species that comprise a carbene carbon atom and one or more (usually 2) nitrogen atoms in the ring structure.25 NHCs exhibit exceptional versatility as they can be effortlessly customized considering the steric arrangement of the substituents and the substituents themselves. This renders them an ideal ligand system for cancer-fighting metal complexes in medicinal chemistry.26 Common variants of the NHC ligand type include imidazolylidenes (NHCim) and triazolylidenes (NHCtrz), whereas other classes such as tetrazolylidenes (NHCtetr) are barely investigated. The group of Berners-Price conducted pioneering research on organometallic gold-NHCim systems as potential anticancer agents.27,28 They reported a series of gold(I) bis-NHCim complexes and correlated their antiproliferative properties with the permeabilization of the mitochondrial membrane, a key property in inducing cell apoptosis.29,30 Modica-Napolitano et al. additionally suggested that the cationic charge and relatively large lipophilic ligands found in these complexes are responsible for a phenomenon referred to as delocalized lipophilic cations (DLCs).31 DLCs offer a way to selectively target cancer cells by accumulating toxic substances in the mitochondria due to negative membrane potentials. However, not all gold(I) NHC complexes described in the literature specifically target mitochondria. An interesting exception is a cationic caffeine-based gold(I) bis-NHC complex that was found to be an efficient and selective DNA quadruplex-interacting agent.32,33 Recently, we published an extensive study on gold(I) bis-NHCtrz complexes that have been investigated for their antiproliferative effects on cancer cells and demonstrated the possibility of straightforward backbone functionalization.34,35 As mentioned above, NHCs containing numerous (>2) nitrogen atoms within the cycle are still under-investigated,34,36−38 especially those based on tetrazolylidenes and their corresponding metal complexes. Raubenheimer and co-workers were among the pioneers to report gold(I) carbene complexes based on 1,4-disubstituted tetrazolylidene species in a synthetic and structural study and to investigate the antiproliferative activity of a gold(I) NHC/Phosphine complex in a follow-up study.39,40 Unlike normal imidazole and abnormal triazole-based bis-NHC complexes, the abnormal tetrazolium-based ligands (1,3-substituted) have a different substitution pattern. This might benefit their medicinal application because the gold center is more accessible for enzyme interactions. Within this framework, aimed at expanding the knowledge of these types of complexes and exploring their pharmacological properties, we describe herein two novel gold(I) bis-tetrazolylidene complexes and their ligand precursors and provide an extensive medicinal and mechanistic investigation.

Results and Discussion

Synthesis and Characterization of gold(I) Complexes 1 and 2

First, we prepared the tetrazolium salts L1 and L2, following literature procedures with slight modification (Scheme 1). The diaryl-substituted tetrazolium salt is accessible via a 5-step reaction pathway starting from mesityl hydrazine hydrochloride and mesityl isothiocyanate in a total yield of 15%.

Scheme 1 Chemical Synthesis Pathways to the Ligand Precursors L1 and L2 from the Respective Starting Materials

The dialkyl-substituted tetrazolium salt is obtained from a 3-step reaction pathway starting from 5-amino tetrazole in a total yield of 23%. For the sake of comparison, we synthesized the complexes and ligand precursors in the form of the corresponding hexafluorophosphate salts. The synthesis of the gold(I) bis-tetrazolylidene complexes 1 and 2 was achieved via the weak base route using K2CO3 as the base in acetone (Scheme 2). This simple approach contrasts the previously reported synthetic pathways to 1,4-disubstituted gold tetrazolylidene complexes, allowing us to work under ambient noninert conditions and eliminating the need for strong bases and highly toxic alkylating agents.39,41 The complexes 1 and 2, the ligand precursors L1 and L2 and all synthetic intermediates were characterized by common chemical analytical techniques, including elemental analysis, 1H-/13C NMR spectroscopy and thin layer chromatography. Furthermore, the ligand precursors and complexes were additionally characterized by electrospray-ionization mass spectrometry (ESI-MS). The most characteristic feature of the ligand precursors in the 1H NMR spectrum is the highly deshielded tetrazolium proton at a chemical shift of 10.12 ppm (L1) and 10.54 ppm (L2), respectively. For the gold(I) complexes 1 and 2, the absence of this proton indicates a successful synthesis. Additionally, in the 13C NMR spectrum of the gold(I) complexes, the typical Au-carbene peak appears quite far downfield shifted at 191.9 ppm (1) and 186.5 ppm (2), respectively. ESI-MS gives the expected m/z values for the cationic fragments of the ligand precursors and the gold(I) complexes. Furthermore, the purity of 1 and 2 was analyzed by high-pressure liquid chromatography (HPLC). The spectra and chromatograms can be found in the Supporting Information.

Scheme 2 Synthesis of the Gold(I) Bis-tetrazolylidene Complexes 1 and 2 from the Respective Ligand Precursors via the Weak Base Route Using K2CO3 in Acetone

Gold(I) Complex 1 Inhibits Cell Proliferation in Leukemia Cells

To investigate the extent to which this new class of complexes inhibits the growth of Nalm-6 cells, leukemia cells were incubated with gold(I) complex 1. The cell number of the treated cells was then measured (Figure 1A) and showed a significant difference even at 0.01 μM compared to the DMSO control. At the time of measurement after 24 h, a reduction in the number of cells can already be seen at 0.08 μM, indicating the onset of apoptosis, but in any case, an inhibition of proliferation.

Figure 1 (A) After 24 h, the cell count of the Nalm-6 cells incubated with gold(I) complex 1 was measured. The cell count was significantly lower after 24 h of incubation with gold(I) complex 1, compared to the DMSO control. * (p < 0.05) indicates a significant difference (t test) between the treated cells and the control cells (DMSO). (B) The extracellular LDH concentration was measured after 1 h of incubation with gold(I) complex 1 as a marker for necrosis and compared to the positive control treated with 2% v/v Triton-X 100 (Triton). There is no evidence for necrosis even at much higher concentrations than those used previously. (C) Annexin V FITC and PI double staining was conducted after 48 h of treatment with gold(I) complex 1 to distinguish between vital, early apoptotic, late apoptotic, and necrotic cells. Necrosis could be largely excluded. * (p < 0.05) indicates a significant difference (t test) between the cells incubated with gold(I) complex 1 and the Triton treated necrotic cells. (D) Representative images from the experiment in C. The dot plots show the dose-dependent change of the Annexin V FITC and PI signal in the cells treated with increasing concentrations of gold(I) complex 1. (E,F) Nalm-6 cells were treated with gold(I) complex 1 (E) and the associated ligand L1 (F) to investigate their apoptotic effects. After 72 h of treatment, DNA fragmentation was measured using flow cytometry. Complex 1 shows a significant induction of apoptosis compared to the DMSO control. * (p < 0.05) indicates a significant difference (t test) between treated Nalm-6 cells vs DMSO control. The IC50 value of the gold(I) complex 1 is 0.014 μM, respectively. However, after treatment with L1, no apoptosis induction could be observed. All experiments were carried out in Nalm-6 cells in triplicates and the mean values ± SD are shown.

Gold(I) Complex 1 Is Not a Trigger for Necrosis

The question of whether an agent triggers apoptosis or necrosis (early, uncontrolled cell death), can be answered by measuring the release of lactate dehydrogenase (LDH) in the cell culture media after treatment with the test substance. For this purpose, Nalm-6 cells were incubated with gold(I) complex 1 and after 1 h, LDH was measured using a photometric assay (Figure 1B). After treatment of the cells with gold(I) complex 1, 100% of the cells are still vital and no LDH has leaked out. The results were compared to cells treated with 2% Triton-X 100, which served as a positive control. The concentrations investigated were deliberately chosen to be higher in order to investigate a wider therapeutic range, and even in concentrations up to 0.5 μM (30–40 times higher than the IC50) there are no indications for necrosis. Another way to obtain information about potential necrosis is staining with Annexin V FITC (A) and Propidium Iodide (PI). The selective staining of the dyes can be used to differentiate between vital cells (A–/PI−), early apoptosis (A+/PI−) and late apoptosis (A+/PI+) or rather necrosis (A–/PI+). For this purpose, Nalm-6 cells were incubated with different concentrations of gold(I) complex 1. The results show pronounced early apoptosis, while only very few cells are already in the late apoptosis stage and hardly any cells show signs of necrosis (Figure 1C,D).

Gold(I) Complexes 1 and 2 Exhibit Proapoptotic Properties in the Low Nanomolar Range

Previously, we have noticed a strong difference in pharmacologic activity for different wingtip (R) substituents on bis-NHC gold(I) complexes.34 Therefore, we have again synthesized a ligand precursor with lipophilic aromatic mesityl groups (L1) and another precursor with more flexible and less lipophilic iPr and tBu substituents (L2). To evaluate the efficacy of apoptosis induction, gold(I) complexes 1 and 2 were incubated on B-lymphoblastic leukemia (B-ALL) cells (Nalm-6). The cells were stained with Propidium Iodide after 72 h of incubation and apoptosis was analyzed using a modified cell cycle analysis by flow cytometry (Figure 1E, and SI). Apoptosis was detected after treatment with both complexes (Figure 1E, and SI), gold(I) complex 1 with an IC50 of 0.014 μM and 2 with an IC50 of 0.017 μM, which indicates a very high efficacy at already low concentrations. The two complexes differ only in their ligands and both ligands were tested for their own apoptotic effect on Nalm-6 cells (Figure 1F and SI). No apoptosis induced by the treatment of the cells with L1 or L2 could be detected. Thus, gold appears to play an essential role in apoptosis induction by the examined metal complexes.

As the IC50 values of complex 1 and 2 are comparable, they appear to be effective at a similar concentration. However, in our recent article, we previously focused on the more lipophilic complex (with Mes wingtips).34 For better comparability, the focus for further experiments was placed on gold(I) complex 1.

Gold(I) Complex 1 Shows High Selectivity for Cancer Cells

The selective efficacy of gold(I) complex 1, was tested on healthy human leukocytes ex vivo (generated by a member of the research group) and compared to Nalm-6 cells and Burkitt lymphoma (BJAB) cells. DNA fragmentation as a marker for apoptosis was measured after treatment with gold(I) complex 1 by flow cytometry (Figure 2A,B). Gold(I) complex 1 shows effective potency on the leukemia and lymphoma cells (Nalm-6 and BJAB) whereas there is no or very little apoptosis induction on the healthy human leukocytes, even at relatively high concentrations, such as 0.1 μM (Figure 2A,B) which indicates a high selectivity of the compound for malignant cells. However, this is an ex vivo experiment that can only give an indication of how the compound will behave in vivo. Nevertheless, it is an important indication for a selective anticancer agent that these complexes, at least in vitro or ex vivo, induce apoptosis almost exclusively in tumor cells and not in nonproliferative healthy leukocytes.

Figure 2 (A) Gold(I) complex 1 was tested on Nalm-6 and BJAB cells in comparison to healthy human leukocytes (ex vivo). Gold(I) complex 1 is significantly selective and hardly induces apoptosis on healthy leukocytes. * and ◆ (p < 0.05) indicate significant differences (t test) between treated nonproliferative healthy human leukocytes vs treated Nalm-6 and BJAB, respectively. (B) From the experiment described in A, the viability of Nalm-6 and BJAB cells was calculated in comparison to human healthy leukocytes. (C) Gold(I) complex 1 was tested on Nalm-6 cells in comparison to the conventional cytostatic drug cisplatin. While treatment with gold(I) complex 1 results in high apoptosis rates, the same concentration of cisplatin shows almost no effect on the Nalm-6 cells. * (p < 0.05) indicates a significant difference (t test) between Nalm-6 cells treated with gold(I) complex 1 vs treatment with cisplatin. (D) Gold(I) complex 1 and the conventional gold-containing drug AF were tested on Nalm-6 cells. Gold(I) complex 1 shows a 2-fold higher apoptosis rate in the low concentration range (0.05 μM). * (p < 0.05) indicates a significant difference (t test) between Nalm-6 cells treated with gold(I) complex 1 vs treatment with AF. In all experiments three replicates each were examined and the mean values ± SD are shown.

Gold(I) Complex 1 Is More Effective than Common Metal Complexes on Leukemia Cells

To compare the efficacy of gold(I) complex 1 with cisplatin, Nalm-6 cells were treated with both agents and examined for apoptosis via DNA fragmentation using flow cytometry (Figure 2C). There is a significant difference between the two metal complexes, with gold(I) complex 1 showing significantly higher apoptosis rates than cisplatin at the same concentration. Cisplatin appears to have little or no response on the Nalm-6 leukemia cells in the concentration range studied. According to this experiment, gold(I) complex 1 could have clinical relevance, especially in comparison with conventional cytostatic drugs of similar substance classes. Cisplatin has been shown do interact with DNA, therefore circular dichroism (CD) spectroscopy with calf tymus DNA (CT-DNA) was contucted to analyze the interaction of gold(I) complex 1 with DNA. However, the results indicate that gold(I) complex 1 (and 2) do not interact with DNA in contrast to cisplatin (see SI). Treatment of Nalm-6 cells with gold(I) complex 1 and AF (Figure 2D) shows that the novel gold compound has an even higher apoptotic effect at low concentrations compared to AF. Side effects that occur with the clinical use of AF and other metal complexes, could be prevented, or at least minimized at low doses.

The Apoptosis Triggered by Gold(I) Complex 1 Proceeds via the Mitochondrial Pathway

To analyze whether apoptosis induced by gold(I) complex 1 is proceeds via the mitochondrial pathway, Nalm-6 cells were treated with the gold(I) complex 1 for 48 h and stained with the dye 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetra-ethylbenz-imidazoyl-carbocyanine iodide (JC-1). Depending on the mitochondrial outer membrane potential (MOMP), the emission maximum of the fluorescent signal changes and the depolarization of the mitochondria is characterized by a shift from red (JC-1 aggregates) to green (JC-1 monomers) fluorescence that can be measured by flow cytometry. The change in MOMP is an important event of the intrinsic apoptotic pathway and initiates pore formation and Cytochrome C efflux from the mitochondrial matrix into the cytosol. The mitochondrial apoptosis pathway appears to play an important role in the mechanism of action of gold(I) complex 1, because a dose-dependent decrease in the MOMP could be observed (Figure 3A). A reduced membrane potential can be seen in the treated cells by the change in the detected JC-1 signal from red toward green (Figure 3B). MOMP reduction can occur through the oligomerization of the proapoptotic B-cell lymphoma 2 (BCL-2) family members BCL-2 associated X protein (BAX) and BCL-2 Antagonist/Killer (BAK). However, the formation of reactive oxygen species (ROS) can also cause such a membrane potential change. Other metal-based cytostatic drugs, such as cisplatin, have been shown to induce ROS in the cell.4 To detect ROS and ROS-induced apoptosis via DNA fragmentation, Nalm-6 cells were incubated with gold(I) complex 1 combined with 1.5 h by pretreatment with N-acetylcysteine (NAC), a ROS inhibitor. It has been shown that cells treated with only gold(I) complex 1 had significantly higher apoptosis levels than those treated with 1 (Figure 3C). In addition to this indirect detection of apoptosis with ROS involvement, ROS positive cells can also be detected directly using CellRox Green staining. Nalm-6 cells were treated with gold(I) complex 1 and the corresponding controls were inhibited with NAC. The measurement revealed a significantly higher ROS level in the cells treated with gold(I) complex 1 alone compared to the cells additionally treated with NAC (Figure 3D), suggesting that gold(I) complex 1 affects the presence of ROS.

Figure 3 Apoptosis induction by the gold(I) complex 1 is mediated via the mitochondrial and reactive oxygen species (ROS) pathway. (A) A significant change in MOMP could be detected after 48 h of treatment with gold(I) complex 1 using the JC-1 dye and was interpreted in relation to the DMSO control. * (p < 0.05) indicates a significant difference (t test) between gold(I) complex 1 treatment vs DMSO control. (B) Representative images from the experiment in A. The density dot plots show the dose-dependent change of the JC-1 aggregate and JC-1 monomer signal in the cells treated with increasing concentrations of gold(I) complex 1. Color gradients show the density of events and go from light gray (low density) to dark gray (high density of events). (C) The cells treated with N-acetylcysteine (NAC) showed significantly lower apoptosis rates compared to the samples without NAC. Therefore, the induction of apoptosis by gold(I) complex 1 seems to be ROS-dependent. The DNA fragmentation was analyzed 72 h after incubation with gold(I) complex 1 and the control substances by flow cytometry. Hydrogen peroxide (H2O2), known to react ROS positive in cells, was used as a positive control. The values are expressed in relation to the DMSO control. * (p < 0.05) indicates a significant difference (t test) between cells treated with gold(I) complex 1 vs treatment with 1 and NAC. (D) ROS positive cells could be detected 5 h after treatment with gold(I) complex 1 using CellRox staining. The ROS levels could be significantly inhibited with the ROS inhibitor NAC. H2O2 was used as a positive control. * (p < 0.05) indicates a significant difference (t test) between cells treated with gold(I) complex 1 vs treatment with 1 and NAC. (E) Representative Western Blot images of Nalm-6 cells incubated with gold(I) complex 1 and harvested after 24 h for protein expression analysis. The antibodies for procaspase 9 and BCL-2 were tested, and β-actin (ACTB) was used as housekeeping protein to ensure equal protein loading. An increased cleavage of procaspase 9 in treated Nalm-6 cells could be detected. (F) Semiquantitative analysis of procaspase 9 and cleaved caspase 9 protein bands showing an increased cleaved caspase 9 protein level. * (p < 0.05) indicates a significant difference (t test) between the DMSO control and cells treated with gold(I) complex 1. (G) Semiquantitative analysis of BCL-2 showing a decreased protein level due to the treatment with gold(I) complex 1. The density of the target protein bands was quantified and normalized by the density of the corresponding ACTB protein band. The results are expressed relative to the DMSO control. All experiments were carried out in Nalm-6 cells in triplicate, and the mean values ± SD are shown.

Treatment with Gold(I) Complex 1 Leads to Activation of Procaspase 9

After initiation of the mitochondrial apoptotic pathway, apoptotic protease activating factor 1 (APAF-1) proteins build an apoptosome mediated by Cytochrome C release. procaspase 9 can bind to this structure, dimerize and then be activated to caspase 9. Activated (cleaved) caspase 9 can then activate effector caspases to initiate the final stage of apoptosis. Twenty-four h after treatment of Nalm-6 cells with gold(I) complex 1, a cleavage from procaspase 9 can be detected by Western Blot. A cleaved caspase 9 protein band can be detected at 37 kDa in cells incubated with gold(I) complex 1 (Figure 3E). Quantitative analysis of the protein bands indicates that the level of cleaved caspase 9 is significantly increased after treatment with gold(I) complex 1 compared to untreated cells (Figure 3F). In addition, a slightly downregulated BCL-2 protein level was detected in Nalm-6 cells treated with gold(I) complex 1 (Figure 3E,G). It is therefore thought that the apoptosis induced by this compound involves the mitochondrion via activation of caspases. Apoptosis induced by gold complexes via (indirect) activation of caspases has been the subject of previous research and may be related to the induction of ROS upstream.42-45 Previous knowledge is supported by the results presented here.

Overcoming Multidrug Resistant Cells by Gold(I) Complex 1, Despite BCL-2 Overexpression

Overcoming multidrug resistance in cancer (cells) is an important part of the search for novel agents. To investigate whether gold(I) complex 1 has resistance overcoming properties, various cytostatic-resistant cell lines were tested for apoptosis induction. Therefore, chronic myeloid leukemia (CML) cells (K562) and daunorubicin-resistant K562 cells (NiWi-Dau), Burkitt lymphoma cells (BJAB) and vincristine-resistant BJAB cells (BiBo) as well as B-lymphoblastic leukemia cells (Nalm-6) and daunorubicin-resistant Nalm-6 (LiKa) cells were treated with gold(I) complex 1 and examined for DNA fragmentation using flow cytometry. NiWi-Dau cells show increased expression of the antiapoptotic protein BCL-2 and significant downregulation of the pro-apoptotic protein BAX. This altered expression pattern of both anti- and pro-apoptotic proteins probably contributes significantly to resistance to daunorubicin and other anthracyclines. Because the gold(I) complex 1 is as effective in the BCL-2 overexpressing NiWi-Dau cells as in the wild-type cell line (Figure 4A,D), it can be assumed that the complex has an impact on the BCL-2 pathway and is able to overcome multidrug resistance in the examined cells. In addition, the pro-apoptotic protein BAX does not seem to play a major role in the induction of apoptosis by gold(I) complex 1 because the same rate of apoptosis can occur in cells that show hardly any BAX expression level (NiWi-Dau cells). Vincristine-resistant BJAB cells (BiBo cells) are also characterized by an overexpression of BCL-2. The rate of gold(I) complex 1-induced apoptosis in the resistant BiBo cell line is significantly higher than in the wild-type cells, also compared by their IC50 values (Figure 4B,D). At a concentration of 0.025 μM, more than 90% of the resistant cells are already in a determined stage of cell death, while less than 25% of the BJAB cells are in apoptosis. These almost 4-fold higher results suggest that gold(I) complex 1 exerts an influence on BCL-2-dependent apoptosis and has a BCL-2 regulatory function. Again, it can be seen that 1 induces apoptosis in cytostatic-resistant cells and thus overcomes multidrug resistance.

Figure 4 (A) Gold(I) complex 1 was tested on chronic myeloic leukemia (CML) K562 cells and on the daunorubicin-resistant K562 cell line NiWi-Dau. Daunorubicin (Dauno) was applied in the concentration of 2 μM. Gold(I) complex 1 overcomes the resistance and is equally effective on the resistant cells. (B) Gold(I) complex 1 was tested on BJAB cells and on vincristine (VCR)-resistant cells (BiBo). Vincristine was applied in the concentration of 6 μM. Significantly higher levels of apoptosis were achieved in the VCR-resistant cells by the treatment with gold(I) complex 1 than in the wild-type cells, indicating that resistance has been overcome. (C) Gold(I) complex 1 was tested on Nalm-6 cells and on the daunorubicin-resistant Nalm-6 cell line LiKa. Daunorubicin was applied in the concentration of 56 nM The wild-type Nalm-6 cells were significantly more responsive to treatment with gold(I) complex 1 than the daunorubicin-resistant LiKa cells. * (p < 0.05) indicates a significant difference (t test) between wild-type cells and the resistant cell line treated with gold(I) complex 1. Three replicates were examined and the mean values of three replicates ± SD are shown. (D) IC50 values of the cell lines (A–C) treated with the gold(I) complex 1.

Daunorubicin-resistant (and coresistant to doxorubicin) Nalm-6 cells (LiKa) exhibit an overexpression of P-Glycoprotein (PGP), which is one potential reason for their resistance behavior. PGP is part of an unspecific ejection mechanism of the cell through which, for example, cytostatic drugs can be ejected from the tumor cell. The level of apoptotic cells induced by treatment with gold(I) complex 1 is significantly lower in the LiKa cells compared to Nalm-6 (Figure 4C,D). This indicates that the increased expression of PGP in the resistant cells leads to increased elimination of gold(I) complex 1. The gold(I) complex 1 thus appears to be a substrate of PGP.

Gold(I) Complex 1 Sensitizes Doxorubicin-Resistant Cells to Doxorubicin

To enhance the efficacy of conventional cytostatic drugs, it can be tested whether novel agents have synergistic effects with them. A synergistic effect is defined as the apoptosis rate of cells treated with two agents simultaneously being higher than the sum of the apoptosis rates of the individually treated cells.46 For this purpose, cells were treated with 1 and the cytostatic drug doxorubicin and DNA fragmentation as a marker for apoptosis was measured by flow cytometry. No synergistic effect was observed in the simultaneous treatment of the wild-type cell lines Nalm-6 (Figure 5A) and K562 (Figure 5B) with gold(I) complex 1 and doxorubicin, as the effect of both substances at the same time does not exceed the sum of the effects of the two individual substances. The daunorubicin-resistant cell lines LiKa and NiWi-Dau show a coresistance against other anthracyclines like doxorubicin. However, if the doxorubicin-resistant associated cell lines (LiKa and NiWi-Dau) are treated with gold(I) complex 1 and doxorubicin, a distinct synergistic effect can be demonstrated (Figure 5A and B). Comparing the apoptosis rate of the synergy on the wild-type Nalm-6 cells with the rate on the resistant LiKa cells, it is apparent that the resistance cannot be completely overcome. Nevertheless, a closer look at the treated LiKa cells reveals a significant synergistic effect (Figure 5A). At a concentration of 0.05 μM, an enormous increase in efficiency of +991% can be observed due to the combination with doxorubicin (Figure 5A). Although the relative apoptosis rate does not exceed that of the wild-type cell line, the initially doxorubicin-resistant LiKa cells are significantly sensitized to doxorubicin (Figure 5A). The synergistic effect is even more apparent when considering the K562 cells and the doxorubicin-resistant NiWi-Dau cells (Figure 6B). The synergistic effect on NiWi-Dau cells is highly significant in the experiment with the anthracycline and gold(I) complex 1 at both concentrations tested. Not only the synergy and the sensitization of the resistant cells are remarkable (Figure 5B). The apoptosis rate of NiWi-Dau cells in the synergy experiment significantly exceeds the rate of apoptotic K562 cells at the same concentrations and moreover the initial doxorubicin-resistant cells are again sensitized to doxorubicin (Figure 5B).

Figure 5 Important mitochondrial apoptosis targets are addressed by gold(I) complex 1. (A) Potential synergistic effects of gold(I) complex 1 and doxorubicin were tested on Nalm-6 and LiKa cells, the latter are coresistant to doxorubicin. (B) Potential synergistic effects of gold(I) complex 1 and doxorubicin (Doxo) were tested on K562 and NiWi-Dau cells, the latter showing coresistance to doxorubicin. The resistant cells show significant synergistic effects compared to the cells treated with gold(I) complex 1 and with doxorubicin alone. 1 resensitizes NiWi-Dau cells to doxorubicin. In both cell lines (A,B), synergistic effects of 1 with doxorubicin were observed, especially in the doxorubicin coresistant cell lines NiWi-Dau (A) and LiKa (B). * (p < 0.05) indicates a significant difference (t test) between cells treated with gold(I) complex 1 and doxorubicin alone, each versus treatment with both. (C) K562 and doxorubicin-resistant NiWi-Dau cells were incubated with gold(I) complex 1, doxorubicin and the combination of both and harvested after 24 h for Western Blot analysis. The antibodies against BCL-2, BAX and MCL-1 were tested and ß-actin (ACTB) was used as housekeeping protein to ensure equal protein loading. (D–F,H,I) The semiquantitative evaluation of the relative protein level of BAX (D) in K562 cells, BCL-2 in K562 (E) and NiWi-Dau cells (F) and MCL-1 in K562 (H) and NiWi-Dau cells (I) treated with gold(I) complex 1 is shown. The protein levels of BCL-2 (F) and MCL-1 (I) are downregulated in NiWi-Dau cells by gold(I) complex 1 and doxorubicin, whereas the BAX level is upregulated in K562 cells (D). Three independent passages were tested, one of them is presented above. The density of the target protein bands was quantified and normalized by the density of the corresponding ACTB protein band and the results are expressed relative to the DMSO control. No significant differences could be detected, but a trend can be seen. In all experiments the mean values of three replicates ± SD are shown. (G) The BAX/BCL-2 ratio in K562 cells was determined and calculated from the previous Western blotting results. A change in the ratio in favor of BAX can be seen, suggesting a proapoptotic metabolic situation in these cells.

In summary, the combinations of the two compounds, namely the conventional drug doxorubicin and the novel gold(I) complex 1, result in high apoptosis rates in the low nanomolar range and sensitize previously resistant cells. Side effects caused by high concentrations of individual drugs can be reduced or even avoided through synergistic effects.

Gold(I) Complex 1 Influences the BAX/BCL-2 Ratio

As described above, gold(I) complex 1 is involved in apoptosis induction via the mitochondrion. The semiquantitative analysis of protein expression by Western Blotting can provide further information about the exact signaling pathways that are targeted. Of particular interest here is the mechanism of sensitization of the doxorubicin-resistant NiWi-Dau cells. An important aspect of the intrinsic apoptosis pathway is the interplay between BAX and its antiapoptotic counterpart BCL-2. Various gold complexes have been investigated for their proapoptotic effects in previous studies. These gold complexes were often directed against a target of the BCL-2 family.47,48 To analyze the expression of different key proteins in K562 and NiWi-Dau cells, these cells were incubated for 24 h with the gold(I) complex 1, doxorubicin and the combination of both, respectively. Examining the protein level of BAX in NiWi-Dau cells, no protein band for the BAX protein can barely be seen (Figure 5C). In addition, a significant increase in the BCL-2 protein level can be observed in NiWi-Dau cells compared to the wild-type cell line K562. Treatment with gold(I) complex 1 and the combination of 1 and doxorubicin leads to an increased expression of BAX in K562 cells (Figure 5D), compared to untreated K562 cells (DMSO control).

The BCL-2 protein level, on the other hand, is almost constant or increases slightly when treated with gold(I) complex 1 (Figure 5E). However, BCL-2 expression is relatively low in K562 cells, compared to NiWi-Dau. By treating the doxorubicin-resistant cells with gold(I) complex 1 and with 1 and doxorubicin, a reduced expression of BCL-2 can be detected (Figure 5F), which presumably shifts the ratio back in favor of the pro-apoptotic protein. However, due to high standard deviation, the proteins levels differ not statistically significant from each other, but nevertheless a clear trend can be seen. The ratio of BAX/BCL-2 in K562 cells was found to change in favor of BAX when treated with 1 and with 1 and doxorubicin (Figure 5G). The shift in this ratio could lead to an increased presence of pro-apoptotic proteins in the cell and facilitate apoptotic events. In NiWi-Dau cells, no ratio can be formed due to the barely visible BAX protein band, but it can be assumed that the BAX/BCL-2 ratio in these cells in general is clearly shifted in favor of the antiapoptotic protein due to the strongly increased BCL-2 protein level. Furthermore, from the results (Figure 5C and D) it can be concluded that treatment with gold(I) complex 1 as well as with 1 and doxorubicin in K562 cells seems to favor apoptosis induction possibly through an increased BAX protein level. In NiWi-Dau cells, on the other hand, no or barely any BAX protein band can be detected even after treatment, so it can be assumed that apoptosis occurs in these cells almost independently of BAX. The significantly increased BCL-2 level in NiWi-Dau cells compared to K562 (Figure 5E and F) cells is reduced by treatment with the gold(I) complex 1 and the combination of 1 and doxorubicin (Figure 5C,F). The formation of the BAX/BCL-2 ratio is not possible in these cells due to the absence of BAX. However, the relative BCL-2 protein level decreases by about 32% in the NiWi-Dau cells treated with doxorubicin compared to treatment with 1 and doxorubicin. Due to high standard deviation the difference between the doxorubicin and the doxorubicin + 1 treated cells not statistically significant. The gold(I) complex 1 attacks exactly where the cell has developed mechanisms to escape apoptosis: it leads to a reduced amount of antiapoptotic proteins such as BCL-2, which is probably partly the cause of resistance. Therefore, the reduced BCL-2 protein levels may possibly play a crucial role in overcoming resistance in the daunorubicin-resistant K562 (NiWi-Dau) cells. BCL-2 as an important antiapoptotic protein can therefore be downregulated by the novel gold(I) complex 1 and is eligible for a potential target of 1. Gold(I) complex 1 thus joins the family of gold complexes that have a pro-apoptotic effect on the BCL-2 family.

BCL-2 Family as Possible Targets of Gold(I) Complex 1

Another important antiapoptotic protein of the BCL-2 superfamily is Myeloid-cell-leukemia-1 protein (MCL-1), which is also a promising anticancer target. Targeted therapies can target cancer cells more specifically and reduce the potential side effects of chemotherapeutic drugs.6 It is already known that gold complexes target MCL-1.49 In NiWi-Dau cells, an increased expression level of MCL-1 can be observed compared to K562 cells (Figure 5C). In K562 cells, no effect of gold(I) complex 1 on the expression of MCL-1 protein levels could be demonstrated (Figure 5H). Due to the treatment with gold(I) complex 1, doxorubicin or the combination, a reduction in MCL-1 protein levels compared to DMSO control can be observed in NiWi-Dau cells (Figure 5I). However, the quantification revealed no significant reduction, but a trend.

In summary, treatment with gold(I) complex 1 reduced the protein levels of mitochondrial antiapoptotic proteins such as BCL-2 and MCL-1, particularly in the anthracycline-resistant NiWi-Dau cells. These results give a clear indication of the mechanism of action of gold(I) complex 1 and confirm the suspicion of apoptosis with mitochondrial involvement. Nevertheless, the significant synergistic effect of gold(I) complex 1 and doxorubicin could not be explained at the protein level.

As cancer cells develop resistance to conventional cytostatic drugs after relapse, the search for drugs to overcome this resistance has expanded. The approach of resensitizing resistant cells to the respective cytostatic drug has already been taken up in previous studies. The discovery that gold(I) complex 1 resensitizes doxorubicin-resistant cells, allowing conventional cytostatic drugs to induce apoptosis in the cells again, is an important discovery in the treatment of cancer relapse. Further research is underway to investigate the mechanisms of resensitization. This includes sensitization with epigenetic modifiers.50 Another approach is to identify the resistance mechanism of the cell and to combat the modified target with a specific targeted therapy.51 In the NiWi-Dau cells tested here, the reduction of BCL-2 protein levels was found to be a particularly interesting target for resensitization with gold(I) complex 1. Drug efflux systems such as PGP also play a role in the development of resistance of cancer cells to cytostatic drugs. For example, Huang et al. have described the influence of the ABCB1 transporter on cytostatic resistance and how it can be inhibited to resensitize cells.52 It is therefore conceivable that gold(I) complex 1 blocks drug efflux transporters and thus prevents the elimination of doxorubicin.

Conclusion

The novel gold(I) complex 1 is a highly potent complex that induces high apoptosis rates in various cancer cell lines even in the low nanomolar range. The complex investigated here has a much more potent effect on leukemia cells than previously investigated gold complexes.34,53-58 Gold(I) complex 1 is already effective at about one fiftieth of the IC50 compared to other potent gold complexes (with an IC50 of approximately 0.5 μM).34 Selective, controlled induction of cell death in tumor cells in vitro was demonstrated without detectable signs of necrosis. In particular, selective induction of apoptosis is of high clinical relevance, as it is essential to find potent anticancer agents that do not affect healthy cells. In comparison to conventional metal complexes such as cisplatin, which is clinically used as a cytostatic agent, and AF, which has also shown antiapoptotic properties, the gold(I) complex has been shown to be more effective. Mechanistically, it can be assumed that the versatile gold(I) complex 1 induces apoptosis via the mitochondrial pathway, which is in line with other gold complexes already investigated.59 A long-discussed target of gold complexes in general is TrxR, which has a direct influence on the ROS level of the cell.60 As gold(I) complex 1 increases the ROS level of the investigated cells, TrxR might be a potential target. Gold(I) complex 1 is not only highly effective in inducing apoptosis in cancer cells, but it is also particularly promising in overcoming multidrug resistance. Treatment with the gold(I) complex 1 can overcome existing multidrug resistance and moreover sensitize the previously resistant cells to the cytostatic drug. Again, evidence for a mitochondrial mechanism of action was found at the protein level and the BCL-2 family, in particular BCL-2 and MCL-1, may be important targets for 1.

However, it must be considered that these experiments are in vitro tests that would have to be tested in vivo to further clarify their efficacy. Based on the experiences with metal complexes that are already used clinically in anticancer therapy, such as cisplatin, the application of gold(I) complex 1 could also lead to some side effects. The extent to which the possible lower application dose could lead to a reduction in these potential side effects has to be evaluated in future studies. Furthermore, gold(I) complex 1 was tested exclusively on leukemia and lymphoma cells in the experiments performed here. However, previous studies with gold complexes have shown high efficacy on solid tumors.61,62 It may therefore be interesting to test gold(I) complex 1 on solid tumor cells as well.

Experimental Section

General Remarks and Synthesis

All reagents were purchased from commercial suppliers and used without further purification. NMR spectra were recorded on a Bruker AVANCE DPX 400 (1H NMR, 400.13 MHz; 13C NMR, 100.53 MHz). Chemical shifts are reported in parts per million and referenced to the residual signal of the deuterated solvent (acetonitrile-d3; 1.94 ppm, (CD3)2SO; 2.50 ppm). Elemental analyses (C/H/N) were performed by the microanalytical laboratory at Technische Universität München. ESI-MS data were acquired on a Thermo Fisher UltiMate 3000. Analytical reversed-phase HPLC-HESI-MS (heated ESI-MS) was performed on an UltiMate 3000 UHPLC focused chromatographic system (Dionex) connected to an LCQ Fleet mass spectrometer (Thermo Scientific) equipped with a C18 column (Hypersil GOLD aQ, 150 mm × 2.1 mm, 3 μm). 1-tert-Butyl,3-iso-propyl-tetrazolium perchlorate was prepared according to a literature procedure from commercially available 5-aminotetrazole.63-65 The identity and purity (>95%) of all biologically studied compounds were confirmed by analytical HPLC in conjunction with elemental analysis and NMR spectroscopy.

N-2-Dimesitylhydrazine-1-carbothioamide (1a)

In a 100 mL round-bottom flask, 1.45 g of 2,4,6-trimethylphenylhydrazine (7.77 mmol, 1.00 equiv) are suspended in 40 mL of diethyl ether. Subsequently, a mixture of 1.38 g of 2-isothiocyanato-1,3,5-trimethylbenzene (7.77 mmol, 1.00 equiv) and 2.20 mL of TEA (15.9 mmol, 2.04 equiv) in 15 mL of diethyl ether are added slowly. The reaction mixture is stirred for 1 h at 40 °C reflux. After the reaction time, the mixture is cooled to 0 °C and the precipitate is filtered and washed sequentially with cold diethyl ether (2 × 15 mL) and ethanol (1 × 20 mL). The crude residue is recrystallized with ethanol (56 mL) to obtain 1.52 g 1a (4.64 mmol, 60%) as a white crystalline solid. 1H NMR (400 MHz, (CD3)2SO, 294 K): δ [ppm] = 9.33 (s, 1H, HSCNHC), 9.22 (s, 1H, HNHNHCS), 6.85 (s, 2H, CH), 6.77 (s, 2H, CH), 6.73 (s, 1H, HNHNHCS), 2.30 (s, 6H, CH3o-mes), 2.23 (s, 3H, CH3,p-mes), 2.18 (m, 9H, CH3,op-mes).13C NMR (101 MHz, (CD3)2SO, 303 K): δ [ppm] = 179.8 (s, CNCSN), 140.6 (s, Car), 136.2 (s, Car), 135.3 (s, Car), 134.9 (s, Car), 131.4 (s, Car), 129.3 (s, Car), 128.1 (s, Car), 20.6 (s, CH3), 20.2 (s, CH3), 18.6 (s, CH3), 18.4 (s, CH3). EA (%): calcd C 69.68, H 7.69, N 12.83, S 9.79; found C 69.65, H 7.73, N 12.70, S 9.26. Rf = 0.11 (DCM) [UV].

Mesityl-(3-mesityl-1,2,3,4-oxatriazolium-5-yl)-amide (1b)

In a 50 mL round-bottom flask, 1.00 g 1a (3.05 mmol, 1.00 equiv) is suspended in 18 mL ethanol and the suspension is cooled to −10 °C. The cold suspension is acidified with conc HCl(aq) (1 mL) to pH 2 and 3.60 mL of an aqueous sodium nitrite solution (1M, 1.50 equiv) are added slowly. After stirring for 1 h at 25 °C, the reaction mixture is filtered, and the filtrate is subsequently alkalized with solid sodium carbonate to pH 11. After cooling overnight at 4 °C, the precipitate is filtered and washed with an excess amount of water (3 × 15 mL). After drying in vacuo, 808 mg of mesityl-(3-mesityl-1,2,3,4-oxatriazol-3-ium-5-yl)-amide 1b (2.50 mmol, 82%) is obtained as a light orange solid. 1H NMR (400 MHz, (CD3)2SO, 295 K): δ [ppm] = 7.21 (s, 2H, CH), 6.81 (s, 2H, CH), 2.34 (s, 3H, CH3,p-mes), 2.18 (s, 9H, CH3,o,p-mes), 2.07 (s, 6H, CH3,p-mes). 13C NMR (101 MHz, (CD3)2SO, 294 K): δ [ppm] = 142.7 (s, CNCON), 133.3 (s, Car), 131.2 (s, Car), 130.7 (s, Car), 130.3 (s, Car), 129.6 (s, Car), 128.4 (s, Car), 128.3 (s, Car), 20.8 (s, CH3), 20.4 (s, CH3), 17.9 (s, CH3), 16.1 (s, CH3). EA (%): calcd C 70.78, H 6.88, N 17.38, O 4.96; found C 69.39, H 6.97, N 16.63, S 1.97. Rf = 0.36 (DCM) [UV].

1,3-Dimesityltetrazolium-5-olate (1c)

In a pressure tube, 781 mg 1b (2.42 mmol, 1.00 equiv) are dissolved in 18 mL ethanol and alkalized with NaOH(aq) (10 m, 3 mL) to pH 9. After stirring for 2 days at 90 °C reflux, the reaction mixture is allowed to reach 25 °C and is subsequently diluted with 30 mL of water. The formed precipitate is filtered, washed with water (2 × 10 mL), and dried in vacuo to obtain 742 mg 1c (2.30 mmol, 95%) as an orange solid. 1H NMR (400 MHz, (CD3)2SO, 294 K): δ [ppm] = 7.18 (s, 2H, CH), 7.14 (s, 2H, CH), 2.37 (s, 3H, CH3,p-mes), 2.33 (s, 3H, CH3,p-mes), 2.14 (s, 6H, CH3,o-mes), 2.11 (s, 6H, CH3,o-mes).13C NMR (101 MHz, (CD3)2SO, 294 K): δ [ppm] = 159.8 (s, CNCON), 141.7 (s, Car), 140.6 (s, Car), 135.5 (s, Car), 133.9 (s, Car), 133.6 (s, Car), 129.4 (s, Car), 129.2 (s, Car), 127.9 (s, Car), 20.8 (s, CH3), 20.7 (s, CH3), 17.1 (s, CH3), 16.3 (s, CH3). EA (%): calcd C 70.78, H 6.88, N 17.38, O 4.96; found C 70.66, H 6.85, N 17.21. Rf = 0.05 (DCM); 0.79 (EtOAc) [UV].

1,3-Dimesityltetrazolium-5-thiolate (1d)

In a 50 mL round-bottom flask, 896 mg 1c (2.78 mmol, 1.00 equiv) are suspended in 10 mL toluene. After the addition of 1.18 g Lawesson’s reagent (2.92 mmol, 1.05 equiv), the reaction mixture is stirred for 3 days at 120 °C reflux. After the reaction time, the reaction mixture is transferred to a separation funnel and washed with a saturated sodium hydrogen carbonate solution (3 × 20 mL). The combined organic phases are dried over MgSO4, and the solvent is removed in vacuo. The crude residue is further purified by recrystallization with ethyl acetate (16 mL) to obtain 520 mg 1d (1.54 mmol, 55%) as yellow solid. 1H NMR (400 MHz, (CD3)2SO, 294 K): δ [ppm] = 7.24 (s, 2H, CH), 7.17 (s, 2H, CH), 2.37 (s, 3H, CH3,p-mes), 2.35 (s, 3H, CH3,p-mes), 2.14 (s, 6H, CH3,o-mes), 2.07 (s, 6H, CH3,o-mes). 13C NMR (101 MHz, (CD3)2SO, 294 K): δ [ppm] = 175.3 (s, CNCON), 142.4 (s, Car), 141.3 (s, Car), 135.3 (s, Car), 134.0 (s, Car), 132.3 (s, Car), 129.4 (s, Car), 129.0 (s, Car), 20.8 (s, CH3), 20.8 (s, CH3), 17.1 (s, CH3), 16.3 (s, CH3). EA (%): calcd C 67.42, H 6.55, N 16.55, S 9.47; found C 67.15, H 6.68, N 16.46, S 9.21. Rf = 0.94 (EtOAc) [UV].

1,3-Dimesityltetrazolium Hexafluorophosphate (L1)

In a 25 mL round-bottom flask, 457 mg 1d (1.35 mmol, 1.00 equiv) are dissolved in 2 mL of conc HNO3(aq) and the reaction mixture is stirred for 2 h at 100 °C reflux. After the reaction time, the solution is filtered over Celite into a solution of 330 mg ammonium hexafluorophosphate (2.03 mmol, 1.50 equiv) in water (2.5 mL). After cooling to 4 °C, the precipitate is filtered and washed with water (3 × 2 mL). For further purification the product is dissolved in a small amount of acetonitrile and precipitated by addition of diethyl ether to obtain 370 mg L1 (0.82 mmol, 61%) as a white solid. 1H NMR (400 MHz, CD3CN, 294 K): δ [ppm] = 10.12 (s, 1H, Htetr), 7.28 (s, 4H, CH), 2.43 (s, 3H, CH3, p-mes), 2.42 (s, 3H, CH3, p-mes), 2.17 (s, 6H, CH3,o-mes), 2.16 (s, 6H, CH3,o-mes). 13C NMR (101 MHz, (CD3)2SO, 294 K): δ [ppm] = 152.1 (s, CNCON), 143.8 (s, Car), 143.3 (s, Car), 134.7 (s, Car), 134.5 (s, Car), 131.4 (s, Car), 130.2 (s, Car), 128.8 (s, Car), 128.0 (s, Car), 124.0 (s, Car), 20.9 (s, CH3), 20.9 (s, CH3), 17.3 (s, CH3), 17.0 (s, CH3). EA (%): calcd C 50.45, H 5.12, N 12.39, F 25.20, P 6.85; found C 50.61, H 4.84, N 12.59. Rf = 0.10 (DCM) [UV].

General Procedure for the Synthesis of 1,3-Substituted Tetrazol-5-ylidene gold(I) Bis–NHC Complexes

The respective proligand (2.00 equiv), the gold precursor chloro(tetrahydrothiophene)gold(I) (1.00 equiv) and the weak base potassium carbonate (6.00 equiv) are weighed in a glass vial and suspended in 4 mL of acetone. After stirring for 16 h, the solvent is removed in vacuo. The crude residue is suspended in a small amount of dichloromethane and filtered over Celite. Subsequently, the filtrate is concentrated in vacuo (1 mL).

Bis-(1,3-dimesityltetrazol-5-ylidene)gold(I) Hexafluorophosphate (1)

The general procedure for 1,3-substituted tetrazolylidene gold(I) bis–NHC complexes is applied, using 70 mg of the proligand L1 (155 μmol, 2.00 equiv). The crude product is further dissolved in a small amount of DCM and purified by column chromatography (20 × 200 mm, 6.7 g Silica, DCM → DCM/MeOH 97:3) to obtain 35.4 mg of the product 1 (37.1 μmol, 48%) as a white solid. 1H NMR (400 MHz, CD3CN, 294 K): δ [ppm] = 7.21 (s, 4H, CH), 7.14 (s, 4H, CH), 2.48 (s, 6H, CH3,p-mes), 2.40 (s, 6H, CH3,p-mes), 2.04 (s, 12H, CH3,o-mes), 1.83 (s, 12H, CH3,o-mes). 13C NMR (101 MHz, CD3CN, 295 K): 191.9 (s, Ctetr), 144.5 (s, Car), 143.7 (s, Car), 138.7 (s, Car), 135.7 (s, Car), 135.5 (s, Car), 133.0 (s, Car), 131.6 (s, Car), 130.8 (s, Car), 130.7 (s, Car), 118.3 (s, Car), 21.5 (s, CH3), 21.3 (s, CH3), 17.6 (s, CH3), 17.3 (s, CH3). EA (%): calcd, C 47.80, H 4.65, N 11.74, Au 20.63, P 3.24, F 11.94; found, C 48.13, H 4.76, N 11.35. Rf = 0.17 (DCM/MeOH = 97:3) [UV]. ESI-MS (m/z): [1–PF6]+ calcd, 809.33; found, 809.27.

Bis-(1-tert-Butyl,3-iso-propyl-tetrazol-5-ylidene)gold(I) Hexafluorophosphate (2)

The general procedure for 1,3-substituted tetrazolylidene gold(I) Bis–NHC complexes is applied, using 70 mg of the proligand L2 (223 μmol). The crude perchlorate salt is further purified by filtration over Celite into a solution of ammonium hexafluorophosphate (27.2 mg, 167 μmol, 1.50 equiv) in water and acetone (4 mL, H2O/acetone = 1:1). After removing the acetone under reduced pressure, the product is extracted with dichloromethane (3 × 6 mL) and the combined organic phases are dried over MgSO4. The solvent is removed in vacuo to obtain the product 2 is obtained as a slightly yellow solid with a yield of 61.2 mg (90.2 μmol, 81%). Crystals suitable for SC–XRD were grown by slow diffusion of Et2O into a solution of 2 in MeCN at 20 °C.

1H NMR (400 MHz, CD3CN, 294 K): δ [ppm] = 5.15 (hept, 2J = 6.7 Hz, 2H, CH), 1.91 (s, 18H, CH3, t-butyl), 1.69 (s, 6H, CH3, o-isopropyl), 1.68 (s, 6H, CH3, o-isopropyl). 13C NMR (101 MHz, CD3CN, 295 K): 186.5 (s, Ctetr), 65.0 (s, Ct-butyl), 60.9 (s, CH), 30.7 (s, Ct-butyl), 21.7 (s, Cisopropyl). EA (%): calcd C 28.33, H 4.75, N 16.52, Au 29.03, P 4.57, F 16.80; found C 28.60, H 4.80, N 16,29. Rf = 0.66 (DCM/MeOH = 10:2) [UV]. ESI-MS (m/z): [2-PF6]+ calcd, 533.24; found, 533.17.

Used Cell Lines and Cultivation of Cells

The cell lines used were grown in RPMI 1640 medium (Gibco, Invitrogen, Karlsruhe, Germany) and cultivated at 37 °C with 5% CO2. The medium was supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS; Merck KGaA, Darmstadt, Germany) and 1% (v/v) penicillin streptomycin (ThermoFisher Scientific Inc., Waltham, USA). The cells are splitted twice a week and transferred to 75 cm2 cell culture flasks at a concentration of 0.5 × 105 cells/mL. In preparation for experiments, the suspension cells are prepared the day before in a concentration of 3 × 105 cells/mL and standardized growth conditions are ensured. On the day of performance, the suspension cells are seeded at a concentration of 1 × 105 cells/mL in 12-well plates in 2 mL of RPMI 1640 medium. Different concentrations of gold complexes and cytostatic drugs are added to the cells depending on the test being performed and incubated for different lengths of time, as described in the following sections. The gold complexes are dissolved in dimethyl sulfoxide (DMSO; Serva Electrophoresis GmbH, Heidelberg, Germany) in stock solutions with a concentration of 40 mM. A DMSO control with the highest concentration of the corresponding test substance, not exceeding 0.5%, is added to each test preparation.

The suspension cells B-lymphocyte leukemia cells (Nalm-6), B-lymphoma cells (BJAB) and chronic myeloid leukemia cells (K562) are used, as well as associated, specially generated, multidrug-resistant cell lines.

Measurement of Cell Concentration and Viability

The CASYCell Counter and Analyzer System (OMNI Life Science GmbH, Bremen, Germany) is used to measure the cell concentration, inhibition of proliferation and viability induced by the compounds under investigation. There is a specific setting for each cell line used. 100 μL of the cell suspension are resuspended after 24 h in 10 mL CASYton (ready to use isotonic saline solution) and measured. The cell count of the treated cells with DMSO is taken as the maximum.

Determination of DNA Fragmentation

In the late phase of apoptosis, DNA fragmentation can be observed as a final process. Therefore, the cells incubated with gold compound are centrifuged after 72 h (5900g, 5 min, 4 °C) and then resuspended in 200 μL 2% (v/v) formaldehyde (Carl Roth GmbH, Karlsruhe, Germany). After 30 min incubation on ice, the cells are centrifuged (370g, 5 min, 4 °C), resuspended in 180 μL 2:1 (v/v) ethanol/1× PBS (Merck KGaA, Darmstadt, Germany; ThermoFisher Scientific Inc., Waltham, USA) and incubated again for 15 min on ice. After washing, 50 μL RNase A Solution (10 mg/mL in 1× PBS; VWR International GmbH, Darmstadt, Germany) is added to eliminate the RNA and incubated at 37 °C for 30 min. The cells are then centrifuged again (370g, 5 min, 4 °C) and stained with 200 μL 1× PBS containing 50 μg/μL propidium iodide (PI). PI is a DNA intercalating dye and allows the identification of cells with hypodiploid DNA. The experiment is quantified via flow cytometry (FACS Lyric, Becton Dickinson GmbH, Heidelberg, Germany), the data evaluation was performed by Cell Quest software. The IC50 values determined to compare potency on different cell lines were obtained using the QuestGraph IC50 Calculator (AAT Bioquest, Inc., https://www.aatbio.com/tools/ic50-calculator).

Exclusion of Necrosis via the Lactate Dehydrogenase (LDH) Outflow

The exclusion of necrosis is measured via the lactate dehydrogenase (LDH) release after one hour. For this purpose, Nalm-6 cells are seeded at a concentration of 1 × 10 cells/mL in RPMI 1640 medium without FBS and treated for one hour with the gold compound at different concentrations. The LDH assay (Roche Molecular Systems Inc., Rotkreuz, Switzerland) was performed according to the manual. As a positive control, Nalm-6 cells were treated with 2% v/v Triton X-100 (Merck KGaA, Darmstadt, Germany) and defined as 100% cytotoxicity. DMSO control results are subtracted from all measured results to avoid possible background signals.

Differentiation between Late and Early Apoptosis and Necrosis via Annexin V/Propidium Iodide Staining

The Annexin-V-Fluos staining kit (Roche, Mannheim, Germany) can be used to determine the different stages of apoptosis and necrosis. Annexin-V dye binds to phosphatidylserine, which is normally localized on the inner membrane of the cell and migrates to the outside of the membrane during early apoptosis. Only then can the dye bind; it cannot pass through the membrane. PI cannot pass through the intact membrane either. Late apoptosis leads to permeabilization of the membrane and thus to the entry of PI dye, which can now intercalate with the DNA. If an isolated PI signal is found, this indicates necrosis. Nalm-6 cells are centrifuged 48 h after treatment with the gold compounds (8000 rpm, 5 min, 4 °C) and resuspended in 100 μL Annexin/PI staining solution. After incubation for 10–15 min, 200 μL incubation buffer is added and the cells are quantified using flow cytometry and Cell Quest software.

Interaction with CT-DNA by Circular Dichroism (CD) Spectroscopy

One mg of CT-DNA (Sigma-Aldrich) was dissolved in 7.58 mL of PBS buffer (10 mM phosphate buffer, 140 mM NaCl, 3 mM KCl, pH 7.4 at 25 °C) to obtain a 200 μM (in base pairs) solution. The solution was then diluted 1:2 and quantified by UV–vis spectroscopy by ε (260 nm) = 13,200 mol–1 dm3 cm–1 to give the actual concentration in DNA base pairs. The respective stock solution of the complexes was prepared by dissolving an adequate amount of the complexes (to achieve double the target concentration) in 1 mL DMSO and diluting it with 49 mL PBS buffer. For a blank measurement, 500 μL of the stock DNA solution was mixed with 500 μL of a mixture of DMSO and PBS (1:50). For the experiments with the complexes, 500 μL of the DNA stock solution was mixed with 500 μL of the complex stock solution. All CD spectra were collected on a Jasco J-810 spectrometer in the range of 230 to 320 nm with a measuring velocity of 100 nm/min, and a data point interval of 0.1 nm. Final DMSO concentrations remained below 1%.

CellRox

Reactive oxygen species (ROS) are formed in the cell during oxidative cell stress and can thus initiate cell death pathways. These ROS can be stained and detected using CellROX Green Reagent (ThermoFisher Scientific Inc., Waltham, USA). The procedure was according to the manual with slight modifications. 30% H2O2 was used as a positive control instead of TBHP. The fluorescence signal from stained ROS is measured by flow cytometry.

Measurement of the Mitochondrial Outer Membrane Potential (MOMP)

The permeabilization of the mitochondrial outer membrane leads to a change in MOMP and initiates the apoptosis cascade.

5,5′,6,6′-Tetrachloro-1,1′,3,3′-tetra-ethylbenzimidazoyl-carbocyanine iodide (JC-1) is a dye used to detect the reduced MOMP. The cells are incubated for 48 h with the compound to be analyzed. The cells are then centrifuged (800g, 5 min, 4 °C) and the pellet is resuspended in phenol free RPMI medium. After addition of 6.25 μL JC-1 (ThermoFisher Scientific Inc., Waltham, USA), the cells are incubated (30 min, 37 °C) and then centrifuged (1500g, 5 min, 4 °C). The pellet is resuspended in 1x PBS and the percentage of cells with reduced MOMP is measured using flow cytometry and Cell Quest software.

Protein Extraction and Western Blotting

The analysis of protein expression was performed with cells treated for 24 h with the gold compound. Control cells were incubated with DMSO. Proteins were extracted from the cells by lysis in RPIA lysis buffer (50 mM Tris HCL, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 1 mM EDTA, pH 7.4) supplemented with protease inhibitors (Roche, Mannheim, Germany). The lysates were then incubated on ice (60 min) and centrifuged (12,000g, 30 min, 4 °C). The total protein in the supernatant was stored at −80 °C for Western blot analysis. The Bicinchoninic acid assay (BCA; Thermo Scientific, Rockford, USA) was used to determine the protein concentration of the samples in order to determine the loading volume required with 40 μg protein. The concentration was calculated and quantified using a standard curve of bovine serum albumin (BSA). Forty μg protein were diluted with 4x Laemmli sample buffer (12% SDS, 25% glycerol, 150 mM Tris HCl, 0.03% bromophenol blue, 20% ß-mercaptoethanol) and denatured (5 min, 95 °C). The single proteins were separated on 12% acrylamide gels by gel electrophoresis using the Laemmli running buffer (Serva, Heidelberg, Germany). The proteins were then transferred to nitrocellulose membranes using Towbin transfer buffer (Serva, Heidelberg, Germany). The membranes were stained with Ponceau red to check the uniform protein loading. After blocking in 5% (m/v) skim milk in PBST (1x PBS, 0.05% Tween-20), primary antibodies were diluted in 5% BSA in PBST and incubated on the membrane for two hours at room temperature. The following primary antibodies were used: anticaspase 9 (R&D Systems, Minneapolis, USA), anti-MCL-1, anti-BCL2, anti-BAX (Proteintech Europe, Manchester, UK), and anti-β-actin (Sigma, St. Louis, USA). After washing three times in 1× PBST, the corresponding secondary antibody was incubated for two hours at room temperature. As secondary antibodies, antimouse IgG, antirabbit IgG and antigoat IgG (Promega, Madison, USA) were diluted in 5% skim milk in PBST. After washing again three times in PBST, the membranes were detected using the ECL Western Blotting Detection Reagent (GE Healthcare, Chicago, USA) and the Super Signal West Pico Chemiluminescent Substrate (Thermo Scientific, Rockford, USA). The images were captured using the Imager Chemi Premium System (VWR, Pennsylvania, USA). The densitometric analysis was performed with the VWR Image Capture Software (software version; VWR, Pennsylvania, USA) and the signal intensity of the target protein bands was normalized to the signal intensity of the housekeeping protein ACTB.

Isolation of Healthy Human Leukocytes

Human healthy leukocytes were isolated to investigate the selectivity of the compounds in proliferative cells. The blood from which the white blood cells were taken was made available to us by the coauthor Aram Prokop. 40 mL human blood was diluted in 14 mL RPMI 1640 medium (containing 20% (v/v) heat inactivated FBS and 1% (v/v) penicillin streptomycin). To each 5 mL of blood suspension, 4 mL of Lymphosep (sucrose–epichlorohydrin copolymer) lymphocytes separation solution (Biowest, Nuaillé, France) was added to create a separation with buffy coat using a concentration gradient. The suspension is centrifuged (2000 rpm, 18 min, 18 °C). Leukocytes are then transferred from the buffy coat into a 50 mL tube and dissolved 1:1 in RPMI medium and centrifuged again (2000 rpm, 5 min, 18 °C). The cell pellet is resuspended in 10 mL RPMI medium (20% FBS) and the cells are seeded in 12-well plates at a concentration of 3 × 10 cells/mL.

Statistical Analysis

The data collected was run in triplicate. The standard deviation (SD) was calculated and is shown in the figures as error bars. To assess the significance of the results, a two-tailed t test was performed with an accepted significance at p ≤ 0.05. The mean values of the results ± SD are shown in the figures and the significance is marked with an asterisk (*). The graphs and statistics were generated using Microsoft Office Excel.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jmedchem.4c01117.NMR spectra, MS spectra ,and HPLC chromatograms, as well as biological assays (PDF)

Molecular formula strings file containing the SMILE structures of 1 and 2 (CSV)

Supplementary Material

jm4c01117_si_001.csv

jm4c01117_si_002.pdf

Author Contributions

Franziska Bannwart and Leon F. Richter contributed equally; Franziska Bannwart: Conceptualization, Data Curation, Formal analysis, Investigation, Project administration, Validation, Visualization, Writing – Original Draft, Writing – Review & Editing; Leon F. Richter: Conceptualization, Data Curation, Formal analysis, Investigation, Project administration, Validation, Visualization, Writing – Original Draft, Writing – Review & Editing; Simon Stifel: Investigation; Johanna Rueter: Investigation, Supervision, Writing – Review & Editing; Holger N. Lode: Supervision, Writing – Review & Editing; João D. G. Correia: Funding acquisition, Resources, Supervision, Writing – Review & Editing; Fritz E. Kühn: Funding acquisition, Resources, Supervision, Writing – Review & Editing; Aram Prokop: Funding acquisition, Resources, Supervision, Writing – Review & Editing; All authors have read and agreed to the published version of this article.

The authors declare no competing financial interest.

Acknowledgments

F.B., J.R., and A.P. gratefully thank the Foundation Blankenheimerdorf ev (Blankenheim, Germany), the Foundation David (Cologne, Germany), the Dr. Kleist Foundation (Berlin, Germany), and the Koch-Foundation (Berlin, Germany) for financial support. Also, they thank the pharmacy of the Helios Clinics Schwerin, Germany, for providing the cytostatic drugs for their experiments. J.D.G.C. thanks Fundação para a Ciência e Tecnologia” (FCT, Portugal) for funding through projects UIDP/04349/2020 and PTDC/QUI-OUT/3854/2021. L.F.R. thanks Melanie Hoffmann and Dr. Aras Kartouzian for their valuable input and experimental support.

Abbreviations

A Annexin V FITC

ABC ATP-binding cassette

ACTB β-actin

AF auranofin

APAF-1 apoptotic protease activating factor-1

BAK BCL-2 antagonist/killer

BAX BCL-2 associated X protein

BCL-2 B-cell-lymphoma 2

BSA bovine serum albumin

B-ALL B-lymphoblastic leukemia

CD circular dichroism

CML chronic myeloic leukemia

CT-DNA calf thymus DNA

Dauno daunorubicin

DLCs delocalized lipophilic cations

DMSO dimethyl sulfoxide

Doxo doxorubicin

ESI-MS electronspray ionization mass spectrometry

FBS fetal bovine serum

GSH glutathion

HPLC high pressure liquid chromatography

HSA human serum albumin

H2O2 hydrogen peroxide

JC-1 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetra-ethylbenzimidazoyl-carbocyanine iodide

LDH lactate dehydrogenase

MCL-1 myeloid cell leukemia-1

MOMP mitochondrial outer membrane potential

NAC N-acetylcysteine

NHCim imidazol-2-ylidene

NHCtetr tetrazolylidene

NHCtrz 1,2,3-triazol-5-ylidene

NHCs N-heterocyclic carbenes

PGP P-glycoprotein

PI propidium iodide

ROS reactive oxygen species

Triton Triton-X-100

TrxR thioredoxin reductase

tht tetrahydrothiophene

VCR vincristine
==== Refs
References

Muggia F. M. ; Bonetti A. ; Hoeschele J. D. ; Rozencweig M. ; Howell S. B. Platinum Antitumor Complexes: 50 Years since Barnett Rosenberg’s Discovery. J. Clin. Oncol 2015, 33 (35 ), 4219–4226. 10.1200/JCO.2015.60.7481.26503202
Rosenberg B. ; Van Camp L. ; Krigas T. Inhibition of Cell Division in Escherichia Coli by Electrolysis Products from a Platinum Electrode. Nature 1965, 205 (4972 ), 698–699. 10.1038/205698a0.14287410
Turel I. Special Issue: Practical Applications of Metal Complexes. Molecules 2015, 20 (5 ), 7951–7956. 10.3390/molecules20057951.26007166
Hour T.-C. ; Lai Y.-L. ; Kuan C.-I ; Chou C.-K. ; Wang J.-M. ; Tu H.-Y. ; Hu H.-T. ; Lin C.-S. ; Wu W.-J. ; Pu Y.-S. ; Sterneck E. ; Huang A-M. Transcriptional Up-Regulation of SOD1 by CEBPD: A Potential Target for Cisplatin Resistant Human Urothelial Carcinoma Cells. Biochem. Pharmacol. 2010, 80 (3 ), 325–334. 10.1016/j.bcp.2010.04.007.20385105
Hsin I.-L. ; Wang S.-C. ; Li J.-R. ; Ciou T.-C. ; Wu C.-H. ; Wu H.-M. ; Ko J.-L. Immunomodulatory Proteins FIP-Gts and Chloroquine Induce Caspase-Independent Cell Death via Autophagy for Resensitizing Cisplatin-Resistant Urothelial Cancer Cells. Phytomedicine 2016, 23 (13 ), 1566–1573. 10.1016/j.phymed.2016.09.003.27823620
Hunger S. P. ; Raetz E. A. How I Treat Relapsed Acute Lymphoblastic Leukemia in the Pediatric Population. Blood, J. Am. Soc. Hematol. 2020, 136 (16 ), 1803–1812. 10.1182/blood.2019004043.
Longley D. B. ; Johnston P. G. Molecular Mechanisms of Drug Resistance. J. Pathol. 2005, 205 (2 ), 275–292. 10.1002/path.1706.15641020
Holohan C. ; Van Schaeybroeck S. ; Longley D. B. ; Johnston P. G. Cancer Drug Resistance: An Evolving Paradigm. Nat. Rev. Cancer 2013, 13 (10 ), 714–726. 10.1038/nrc3599.24060863
Alves R. ; Gonçalves A. C. ; Jorge J. ; Almeida A. M. ; Sarmento-ribeiro A. B. Combination of Elacridar with Imatinib Modulates Resistance Associated with Drug Efflux Transporters in Chronic Myeloid Leukemia. Biomedicines 2022, 10 (5 ), 1158 10.3390/biomedicines10051158.35625893
Kostka L. ; Sivák L. ; Šubr V. ; Kovářová J. ; Šírová M. ; Říhová B. ; Sedlacek R. ; Etrych T. ; Kovář M. Simultaneous Delivery of Doxorubicin and Protease Inhibitor Derivative to Solid Tumors via Star-Shaped Polymer Nanomedicines Overcomes P-Gp- and STAT3-Mediated Chemoresistance. Biomacromolecules 2022, 23 (6 ), 2522–2535. 10.1021/acs.biomac.2c00256.35584053
Chen H. ; Shien K. ; Suzawa K. ; Tsukuda K. ; Tomida S. ; Sato H. ; Torigoe H. ; Watanabe M. ; Namba K. ; Yamamoto H. ; Soh J. ; Asano H. ; Miyoshi S. ; Toyooka S. Elacridar, a Third-Generation ABCB1 Inhibitor, Overcomes Resistance to Docetaxel in Non-Small Cell Lung Cancer. Oncol. Lett. 2017, 14 (4 ), 4349–4354. 10.3892/ol.2017.6678.28959367
Oun R. ; Moussa Y. E. ; Wheate N. J. The Side Effects of Platinum-Based Chemotherapy Drugs: A Review for Chemists. Dalt. Trans. 2018, 47 (19 ), 6645–6653. 10.1039/C8DT00838H.
Olelewe C. ; Awuah S. G. Mitochondria as a Target of Third Row Transition Metal-Based Anticancer Complexes. Curr. Opin. Chem. Biol. 2023, 72 , 102235 10.1016/j.cbpa.2022.102235.36516614
Tialiou A. ; Chin J. ; Keppler B. K. ; Reithofer M. R. Current Developments of N-Heterocyclic Carbene Au(I)/Au(III) Complexes toward Cancer Treatment. Biomedicines 2022, 10 (6 ), 1417 10.3390/biomedicines10061417.35740438
Borutzki Y. ; Skos L. ; Gerner C. ; Meier-Menches S. M. Exploring the Potential of Metal-Based Candidate Drugs as Modulators of the Cytoskeleton. ChemBioChem. 2023, 10.1002/cbic.202300178.
Van Der Westhuizen D. ; Bezuidenhout D. I. ; Munro O. Q. Cancer Molecular Biology and Strategies for the Design of Cytotoxic Gold(i) and Gold(Iii) Complexes: A Tutorial Review. Dalt. Trans. 2021, 50 (47 ), 17413–17437. 10.1039/D1DT02783B.
Yeo C. I. ; Ooi K. K. ; Tiekink E. R. T. Gold-Based Medicine: A Paradigm Shift in Anti-Cancer Therapy?. Molecules 2018, 23 , 1410 10.3390/molecules23061410.29891764
Porchia M. ; Pellei M. ; Marinelli M. ; Tisato F. ; Del Bello F. ; Santini C. New Insights in Au-NHCs Complexes as Anticancer Agents. Eur. J. Med. Chem. 2018, 146 , 709–746. 10.1016/j.ejmech.2018.01.065.29407992
Liu X. ; Li H. ; Yin X. NaOH-Promoted One-Pot Aryl Isothiocyanate Synthesis under Mild Benchtop Conditions. Phosphorus, Sulfur Silicon Relat. Elem. 2021, 196 (9 ), 839–844. 10.1080/10426507.2021.1927031.
Krabbendam I. E. ; Honrath B. ; Bothof L. ; Silva-Pavez E. ; Huerta H. ; Peñaranda Fajardo N. M. ; Dekker F. ; Schmidt M. ; Culmsee C. ; César Cárdenas J. ; Kruyt F. ; Dolga A. M. SK Channel Activation Potentiates Auranofin-Induced Cell Death in Glio- and Neuroblastoma Cells. Biochem. Pharmacol. 2020, 171 , 113714 10.1016/j.bcp.2019.113714.31738894
Freire Boullosa L. ; Van Loenhout J. ; Flieswasser T. ; De Waele J. ; Hermans C. ; Lambrechts H. ; Cuypers B. ; Laukens K. ; Bartholomeus E. ; Siozopoulou V. ; De Vos W. H. ; Peeters M. ; Smits E. L. J. ; Deben C. Auranofin Reveals Therapeutic Anticancer Potential by Triggering Distinct Molecular Cell Death Mechanisms and Innate Immunity in Mutant P53 Non-Small Cell Lung Cancer. Redox Biol. 2021, 42 , 101949 10.1016/j.redox.2021.101949.33812801
Chen L. ; Ren A. ; Zhao Y. ; Chen H. ; Wu Q. ; Zheng M. ; Zhang Z. ; Zhang T. ; Zhong W. ; Lin J. ; Zhu H. Direct Inhibition of Dioxygenases TET1 by the Rheumatoid Arthritis Drug Auranofin Selectively Induces Cancer Cell Death in T-ALL. J. Hematol. Oncol. 2023, 16 (1 ), 4–9. 10.1186/s13045-023-01513-6.36658588
Mirabelli C. K. ; Johnson R. K. ; Sung C. M. ; Faucette L. ; Muirhead K. ; Crooke S. T. Evaluation of the in Vivo Antitumor Activity and in Vitro Cytotoxic Properties of Auranofin, a Coordinated Gold Compound, in Murine Tumor Models. Cancer Res. 1985, 45 , 32–39.3917372
Mirabell C. K. ; Johnson R. K. ; Hill D. T. ; Faucette L. F. ; Girard G. R. ; Kuo G. Y. ; Sung C. M. ; Crooke S. T. Correlation of the in Vitro Cytotoxic and in Vivo Antitumor Activities of Gold(I) Coordination Complexes. J. Med. Chem. 1986, 29 , 218–223. 10.1021/jm00152a009.3081721
Hopkinson M. N. ; Richter C. ; Schedler M. ; Glorius F. An Overview of N-Heterocyclic Carbenes. Nature 2014, 510 (7506 ), 485–496. 10.1038/nature13384.24965649
Ott I. Metal N-Heterocyclic Carbene Complexes in Medicinal Chemistry. Advances in Inorganic Chemistry 2020, 75 , 121–148.
Hickey J. L. ; Ruhayel R. A. ; Barnard P. J. ; Baker M. V. ; Berners-Price S. J. ; Filipovska A. Mitochondria-Targeted Chemotherapeutics: The Rational Design of Gold(I) N-Heterocyclic Carbene Complexes That Are Selectively Toxic to Cancer Cells and Target Protein Selenols in Preference to Thiols. J. Am. Chem. Soc. 2008, 130 (38 ), 12570–12571. 10.1021/ja804027j.18729360
Berners-Price S. J. ; Filipovska A. Gold Compounds as Therapeutic Agents for Human Diseases. Metallomics 2011, 3 (9 ), 863–873. 10.1039/c1mt00062d.21755088
Lazreg F. ; Cazin C. S. J. Medical Applications of NHC-Gold and -Copper Complexes. N-Heterocyclic Carbenes: Effective Tools for Organometallic Synthesis 2014, 173–198. 10.1002/9783527671229.ch07.
Baker M. V. ; Barnard P. J. ; Berners-Price S. J. ; Brayshaw S. K. ; Hickey J. L. ; Skelton B. W. ; White A. H. Cationic, Linear Au (I) N-Heterocyclic Carbene Complexes: Synthesis, Structure and Anti-Mitochondrial Activity. Dalt. Trans. 2006, (30 ), 3708–3715. 10.1039/b602560a.
Modica-Napolitano J. S. ; Aprille J. R. Delocalized Lipophilic Cations Selectively Target the Mitochondria of Carcinoma Cells. Adv. Drug Delivery Rev. 2001, 49 (1–2 ), 63–70. 10.1016/S0169-409X(01)00125-9.
Stefan L. ; Bertrand B. ; Richard P. ; Le Gendre P. ; Denat F. ; Picquet M. ; Monchaud D. Assessing the Differential Affinity of Small Molecules for Noncanonical DNA Structures. ChemBioChem. 2012, 13 (13 ), 1905–1912. 10.1002/cbic.201200396.22930447
Bertrand B. ; Stefan L. ; Pirrotta M. ; Monchaud D. ; Bodio E. ; Richard P. ; Le Gendre P. ; Warmerdam E. ; De Jager M. H. ; Groothuis G. M. M. ; Picquet M. ; Casini A. Caffeine-Based Gold(I) N-Heterocyclic Carbenes as Possible Anticancer Agents: Synthesis and Biological Properties. Inorg. Chem. 2014, 53 (4 ), 2296–2303. 10.1021/ic403011h.24499428
Schlagintweit J. F. ; Jakob C. H. G. ; Wilke N. L. ; Ahrweiler M. ; Frias C. ; Frias J. ; König M. ; Esslinger E. M. H. J. ; Marques F. ; MacHado J. F. ; Reich R. M. ; Morais T. S. ; Correia J. D. G. ; Prokop A. ; Kühn F. E. Gold(I) Bis(1,2,3-Triazol-5-Ylidene) Complexes as Promising Selective Anticancer Compounds. J. Med. Chem. 2021, 64 (21 ), 15747–15757. 10.1021/acs.jmedchem.1c01021.34670090
Richter L. F. ; Marques F. ; Correia J. D. G. ; Pöthig A. ; Kühn F. E. Exploiting Click-Chemistry: Backbone Post-Functionalisation of Homoleptic Gold(i)-1,2,3-triazole-5-ylidene complexes. Dalton. Trans. 2023, 52 , 17185 10.1039/D3DT03052K.37942578
Aucamp D. ; Kumar S. V. ; Liles D. C. ; Fernandes M. A. ; Harmse L. ; Bezuidenhout D. I. Synthesis of Heterobimetallic Gold(i) Ferrocenyl-Substituted 1,2,3-Triazol-5-Ylidene Complexes as Potential Anticancer Agents. Dalt. Trans. 2018, 47 (45 ), 16072–16081. 10.1039/C8DT03116A.
Hoyer C. ; Schwerk P. ; Suntrup L. ; Beerhues J. ; Nössler M. ; Albold U. ; Dernedde J. ; Tedin K. ; Sarkar B. Synthesis, Characterization, and Evaluation of Antibacterial Activity of Ferrocenyl-1,2,3-Triazoles, Triazolium Salts, and Triazolylidene Complexes of Gold(i) and Silver(I). Eur. J. Inorg. Chem. 2021, 2021 (14 ), 1373–1382. 10.1002/ejic.202100024.
Schaper L. A. ; Wei X. ; Altmann P. J. ; Öfele K. ; Pöthig A. ; Drees M. ; Mink J. ; Herdtweck E. ; Bechlars B. ; Herrmann W. A. ; Kühn F. E. Synthesis and Comparison of Transition Metal Complexes of Abnormal and Normal Tetrazolylidenes: A Neglected Ligand Species. Inorg. Chem. 2013, 52 (12 ), 7031–7044. 10.1021/ic4005449.23706110
Gabrielli W. F. ; Nogai S. D. ; McKenzie J. M. ; Cronje S. ; Raubenheimer H. G. Tetrazolyl and Tetrazolylidene Complexes of Gold: A Synthetic and Structural Study. New J. Chem. 2009, 33 (11 ), 2208–2218. 10.1039/b907022b.
Gabrielli W. F. ; Nogai S. D. ; Nell M. ; Cronje S. ; Raubenheimer H. G. Neutral Mononuclear and Dinuclear Complexes of Gold(I) Featuring Azole Ligands: Synthesis, Structure and Cytotoxicity. Polyhedron 2012, 34 (1 ), 188–197. 10.1016/j.poly.2011.12.026.
Kinzhalov M. A. ; Legkodukh A. S. ; Anisimova T. B. ; Novikov A. S. ; Suslonov V. V. ; Luzyanin K. V. ; Kukushkin V. Y. Tetrazol-5-Ylidene Gold(III) Complexes from Sequential [2 + 3] Cycloaddition of Azide to Metal-Bound Isocyanides and N4 Alkylation. Organometallics 2017, 36 (20 ), 3974–3980. 10.1021/acs.organomet.7b00591.
Soto K. M. ; Luzardo-Ocampo I. ; López-Romero J. M. ; Mendoza S. ; Loarca-Piña G. ; Rivera-Muñoz E. M. ; Manzano-Ramírez A. Gold Nanoparticles Synthesized with Common Mullein (Verbascum Thapsus) and Castor Bean (Ricinus Communis) Ethanolic Extracts Displayed Antiproliferative Effects and Induced Caspase 3 Activity in Human HT29 and SW480 Cancer Cells. Pharmaceutics 2022, 14 (10 ), 2069 10.3390/pharmaceutics14102069.36297503
Quero J. ; Royo J. C. ; Fodor B. ; Gimeno M. C. ; Osada J. ; Rodríguez-Yoldi M. J. ; Cerrada E. Sulfonamide-Derived Dithiocarbamate Gold (I) Complexes Induce the Apoptosis of Colon Cancer Cells by the Activation of Caspase 3 and Redox Imbalance. Biomedicines 2022, 10 (6 ), 1437 10.3390/biomedicines10061437.35740458
Liu B. ; Li J. ; Zhou P. ; Pan W. ; Li N. ; Tang B. Real-Time in Situ Sequential Fluorescence Activation Imaging of Cyt c and Caspase-9 with a Gold-Selenium-Bonded Nanoprobe. Anal. Chem. 2021, 93 (50 ), 16880–16886. 10.1021/acs.analchem.1c03872.34886667
Rouco L. ; Sanchez-Gonzalez A. ; Alvariño R. ; Alfonso A. ; Vazquez-Lopez E. M. ; García-Martínez E. ; Maneiro M. Combined Effect of Caspase-Dependent and Caspase-Independent Apoptosis in the Anticancer Activity of Gold Complexes with Phosphine and Benzimidazole Derivatives. Pharmaceuticals 2021, 14 (1 ), 10 10.3390/ph14010010.
Foucquier J. ; Guedj M. Analysis of Drug Combinations: Current Methodological Landscape. Pharmacol. Res. Perspect. 2015, 3 (3 ), e00149 10.1002/prp2.149.26171228
Choroba K. ; Machura B. ; Szlapa-Kula A. ; Malecki J. G. ; Raposo L. ; Roma-Rodrigues C. ; Cordeiro S. ; Baptista P. V. ; Fernandes A. R. Square Planar Au (III), Pt (II) and Cu (II) Complexes with Quinoline-Substituted 2, 2′: 6′, 2 ″-Terpyridine Ligands: From in Vitro to in Vivo Biological Properties. Eur. J. Med. Chem. 2021, 218 , 113404 10.1016/j.ejmech.2021.113404.33823390
Nandy A. ; Dey S. K. ; Das S. ; Munda R. N. ; Dinda J. ; Saha K. D. Gold (I) N-Heterocyclic Carbene Complex Inhibits Mouse Melanoma Growth by P53 Upregulation. Mol. Cancer 2014, 13 , 57 10.1186/1476-4598-13-57.24625085
Zhai J. ; Jia Y. ; Zhao L. ; Yuan Q. ; Gao F. ; Zhang X. ; Cai P. ; Gao L. ; Guo J. ; Yi S. ; Chai Z. ; Zhao Y. ; Gao X. Turning On/Off the Anti-Tumor Effect of the Au Cluster via Atomically Controlling Its Molecular Size. ACS Nano 2018, 12 (5 ), 4378–4386. 10.1021/acsnano.8b00027.29667812
Rauscher S. ; Greil R. ; Geisberger R. Re-Sensitizing Tumor Cells to Cancer Drugs with Epigenetic Regulators. Curr. Cancer Drug Targets 2021, 21 (4 ), 353–359. 10.2174/1568009620666210108102723.33423645
Li Z. ; Chen C. ; Chen L. ; Hu D. ; Yang X. ; Zhuo W. ; Chen Y. ; Yang J. ; Zhou Y. ; Mao M. ; Zhang X. ; Xu L. ; Ju S. ; Shen J. ; Wang Q. ; Dong M. ; Xie S. ; Wei Q. ; Jia Y. ; Zhou J. ; Wang L. STAT5a Confers Doxorubicin Resistance to Breast Cancer by Regulating ABCB1. Front. Oncol. 2021, 11 , 697950 10.3389/fonc.2021.697950.34336684
Huang W. ; Yang S. ; Cheng Y.-S. ; Sima N. ; Sun W. ; Shen M. ; Braisted J. C. ; Lu W. ; Zheng W. Terfenadine Resensitizes Doxorubicin Activity in Drug-Resistant Ovarian Cancer Cells via an Inhibition of CaMKII/CREB1Mediated ABCB1 Expression. Front. Oncol. 2022, 12 , 1068443 10.3389/fonc.2022.1068443.36439493
Schmidt C. ; Albrecht L. ; Balasupramaniam S. ; Misgeld R. ; Karge B. ; Brönstrup M. ; Prokop A. ; Baumann K. ; Reichl S. ; Ott I. A Gold (I) Biscarbene Complex with Improved Activity as a TrxR Inhibitor and Cytotoxic Drug: Comparative Studies with Different Gold Metallodrugs. Metallomics 2019, 11 (3 ), 533–545. 10.1039/c8mt00306h.30516775
Ott I. ; Qian X. ; Xu Y. ; Vlecken D. H. W. ; Marques I. J. ; Kubutat D. ; Will J. ; Sheldrick W. S. ; Jesse P. ; Prokop A. ; Bagowski C. P. A Gold (I) Phosphine Complex Containing a Naphthalimide Ligand Functions as a TrxR Inhibiting Antiproliferative Agent and Angiogenesis Inhibitor. J. Med. Chem. 2009, 52 , 763–770. 10.1021/jm8012135.19123857
Rubbiani R. ; Kitanovic I. ; Alborzinia H. ; Can S. ; Kitanovic A. ; Onambele L. A. ; Stefanopoulou M. ; Geldmacher Y. ; Sheldrick W. S. ; Wolber G. ; Prokop A. ; Wolfl S. ; Ott I. Benzimidazol-2-Ylidene Gold (I) Complexes Are Thioredoxin Reductase Inhibitors with Multiple Antitumor Properties. J. Med. Chem. 2010, 53 (24 ), 8608–8618. 10.1021/jm100801e.21082862
Serebryanskaya T. V. ; Lyakhov A. S. ; Ivashkevich L. S. ; Schur J. ; Frias C. ; Prokop A. ; Ott I. Gold (I) Thiotetrazolates as Thioredoxin Reductase Inhibitors and Antiproliferative Agents. Dalt. Trans. 2015, 44 (3 ), 1161–1169. 10.1039/C4DT03105A.
Schmidt C. ; Karge B. ; Misgeld R. ; Prokop A. ; Franke R. ; Brönstrup M. ; Ott I. Gold (I) NHC Complexes: Antiproliferative Activity, Cellular Uptake, Inhibition of Mammalian and Bacterial Thioredoxin Reductases, and Gram-Positive Directed Antibacterial Effects. Chem.—Eur. J. 2017, 23 (8 ), 1869–1880. 10.1002/chem.201604512.27865002
Schmidt C. ; Karge B. ; Misgeld R. ; Prokop A. ; Brönstrup M. ; Ott I. Biscarbene Gold (I) Complexes: Structure-Activity-Relationships Regarding Antibacterial Effects, Cytotoxicity, TrxR Inhibition and Cellular Bioavailability. Medchemcomm 2017, 8 (8 ), 1681–1689. 10.1039/C7MD00269F.30108879
Ahrweiler-Sawaryn M.-C. ; Biswas A. ; Frias C. ; Frias J. ; Wilke N. L. ; Wilke N. ; Berkessel A. ; Prokop A. Novel Gold (I) Complexes Induce Apoptosis in Leukemia Cells via the ROS-Induced Mitochondrial Pathway with an Upregulation of Harakiri and Overcome Multi Drug Resistances in Leukemia and Lymphoma Cells and Sensitize Drug Resistant Tumor Cells to Apoptosis. Biomed. Pharmacother. 2023, 161 , 114507 10.1016/j.biopha.2023.114507.36958194
Abdalbari F. H. ; Telleria C. M. The Gold Complex Auranofin: New Perspectives for Cancer Therapy. Discovery Oncol. 2021, 12 , 42 10.1007/s12672-021-00439-0.
Xiao Q. ; Liu Y. ; Jiang G. ; Liu Y. ; Huang Y. ; Liu W. ; Zhang Z. Heteroleptic Gold (I)-BisNHC Complex with Excellent Activity In Vitro, Ex Vivo and In Vivo against Endometrial Cancer. Eur. J. Med. Chem. 2022, 236 , 114302 10.1016/j.ejmech.2022.114302.35395440
Gurba A. ; Taciak P. ; Sacharczuk M. ; Młynarczuk-Biały I. ; Bujalska-Zadrożny M. ; Fichna J. Gold (III) Derivatives in Colon Cancer Treatment. Int. J. Mol. Sci. 2022, 23 (2 ), 724 10.3390/ijms23020724.35054907
Koren’ A. O. ; Gaponik P. N. Regioselective of Tetrazole and 5-Substituted Tetrazoles N2 Alkylation by Alcohols. Chem. Heterocycl. Compd. 1990, 26 , 1366–1370. 10.1007/BF00473965.
Henry R. A. ; Finnegan W. G. An Improved Procedure for the Deamination of 5-Aminotetrazole. J. Am. Chem. Soc. 1954, 76 (1 ), 290–291. 10.1021/ja01630a086.
Voitekhovich S. V. ; Gaponik P. N. ; Lyakhov A. S. ; Ivashkevich O. A. Synthesis and Structure of 1-Tert-Butyl-3-R-Tetrazolium Salts. Chem. Heterocycl. Compd. 2001, 37 (8 ), 949–959. 10.1023/A:1012779315893.
