New Organometallic Ru(II) Compounds with Lonidamine Motif as Antitumor Agents

The combination of one molecule of organic and metal-based fragments that exhibit antitumor activity is a modern approach in the search for new promising drugs. In this work, biologically active ligands based on lonidamine (a selective inhibitor of aerobic glycolysis used in clinical practice) were introduced into the structure of an antitumor organometallic ruthenium scaffold. Resistant to ligand exchange reactions, compounds were prepared by replacing labile ligands with stable ones. Moreover, cationic complexes containing two lonidamine-based ligands were obtained. Antiproliferative activity was studied in vitro by MTT assays. It was shown that the increase in the stability in ligand exchange reactions does not influence cytotoxicity. At the same time, the introduction of the second lonidamine fragment approximately doubles the cytotoxicity of studied complexes. The ability to induce apoptosis and caspase activation in tumour cell MCF7 was studied by employing flow cytometry.

It is known that the introduction of bioactive organic moieties into the structure of metal-based agents can increase anticancer activity and selectivity due to the interaction with several molecular targets [33][34][35]. One of the most important metabolic features of malignant cells is their increased glycolytic activity known as the Warburg Effect [36,37]. Lonidamine ( Figure 2) stimulates lactate production in noncancer cells and reduced glycolysis in their malignant counterparts by inhibiting mitochondrial-associated hexokinase glycolysis in their malignant counterparts by inhibiting mitochondrial-associated hexokinase or reprogramming cellular metabolism and mitochondrial function [38][39][40][41]. Lonidamine is widely studied for the treatment of different types of cancer [42][43][44] and is of special interest in the development of dual-acting anticancer compounds.  Previously, lonidamine was introduced into the structures of Pt(IV) complexes [35,45,46] and Ru(II/III) compounds [47][48][49]. The obtained platinum prodrugs and ruthenium twin drugs showed a significantly improved cytotoxicity, superiority to cisplatin and lonidamine, and also some degree of selectivity [35]. The Ru(III) complexes were also shown to be non-competitive thioredoxin reductase inhibitors that effectively induce apoptosis via caspase activation incubation for 24 h. The cytotoxicity of the Ru(III) complexes as well as cellular uptake, apoptosis induction, and thioredoxin reductase inhibition positively correlate with the length of the linker between the ruthenium center and lonidamine moiety [49]. Two organometallic Ru(II) lonidamine conjugates showed promising cytotoxicity on human glioblastoma cell lines and also exhibited a degree of selectivity towards these cells [47].
This work aims to introduce lonidamine-containing ligands into the structure of organometallic Ru(II) compounds to study the antitumor activity and its dependence on the distance between the lonidamine moiety and ruthenium centre, ligand exchange reactions, and the number of lonidamine moieties in the molecule as well as a possible mode of action of cell death via apoptosis induction and caspase activation.

Materials and Methods
All solvents were purified and degassed before use [50]. Ligands 1-6 were prepared following the published procedure [47,49]. NMR spectra were recorded on a Bruker Avance II 400 spectrometer at room temperature at 400.13 ( 1 H) and 100.61 ( 13 C{ 1 H}) MHz. 2D NMR measurements were carried out using standard pulse programs. Chemical shifts were referenced relative to the solvent signal for 1 H and 13 C spectra. Elemental analysis was performed with MicroCube Elementar analyzer. Electrospray ionization (ESI) mass spectra were recorded using a TSQ Endura (Thermo Fisher Scientific, Waltham, MA, USA) instrument. Each analysed compound was dissolved in methanol (HPLC grade) and injected directly into the ionization source through a syringe pump. The spectra were glycolysis in their malignant counterparts by inhibiting mitochondrial-associated hexokinase or reprogramming cellular metabolism and mitochondrial function [38][39][40][41]. Lonidamine is widely studied for the treatment of different types of cancer [42][43][44] and is of special interest in the development of dual-acting anticancer compounds.  Previously, lonidamine was introduced into the structures of Pt(IV) complexes [35,45,46] and Ru(II/III) compounds [47][48][49]. The obtained platinum prodrugs and ruthenium twin drugs showed a significantly improved cytotoxicity, superiority to cisplatin and lonidamine, and also some degree of selectivity [35]. The Ru(III) complexes were also shown to be non-competitive thioredoxin reductase inhibitors that effectively induce apoptosis via caspase activation incubation for 24 h. The cytotoxicity of the Ru(III) complexes as well as cellular uptake, apoptosis induction, and thioredoxin reductase inhibition positively correlate with the length of the linker between the ruthenium center and lonidamine moiety [49]. Two organometallic Ru(II) lonidamine conjugates showed promising cytotoxicity on human glioblastoma cell lines and also exhibited a degree of selectivity towards these cells [47].
This work aims to introduce lonidamine-containing ligands into the structure of organometallic Ru(II) compounds to study the antitumor activity and its dependence on the distance between the lonidamine moiety and ruthenium centre, ligand exchange reactions, and the number of lonidamine moieties in the molecule as well as a possible mode of action of cell death via apoptosis induction and caspase activation.

Materials and Methods
All solvents were purified and degassed before use [50]. Ligands 1-6 were prepared following the published procedure [47,49]. NMR spectra were recorded on a Bruker Avance II 400 spectrometer at room temperature at 400.13 ( 1 H) and 100.61 ( 13 C{ 1 H}) MHz. 2D NMR measurements were carried out using standard pulse programs. Chemical shifts were referenced relative to the solvent signal for 1 H and 13 C spectra. Elemental analysis was performed with MicroCube Elementar analyzer. Electrospray ionization (ESI) mass spectra were recorded using a TSQ Endura (Thermo Fisher Scientific, Waltham, MA, USA) instrument. Each analysed compound was dissolved in methanol (HPLC grade) and injected directly into the ionization source through a syringe pump. The spectra were Previously, lonidamine was introduced into the structures of Pt(IV) complexes [35,45,46] and Ru(II/III) compounds [47][48][49]. The obtained platinum prodrugs and ruthenium twin drugs showed a significantly improved cytotoxicity, superiority to cisplatin and lonidamine, and also some degree of selectivity [35]. The Ru(III) complexes were also shown to be noncompetitive thioredoxin reductase inhibitors that effectively induce apoptosis via caspase activation incubation for 24 h. The cytotoxicity of the Ru(III) complexes as well as cellular uptake, apoptosis induction, and thioredoxin reductase inhibition positively correlate with the length of the linker between the ruthenium center and lonidamine moiety [49]. Two organometallic Ru(II) lonidamine conjugates showed promising cytotoxicity on human glioblastoma cell lines and also exhibited a degree of selectivity towards these cells [47].
This work aims to introduce lonidamine-containing ligands into the structure of organometallic Ru(II) compounds to study the antitumor activity and its dependence on the distance between the lonidamine moiety and ruthenium centre, ligand exchange reactions, and the number of lonidamine moieties in the molecule as well as a possible mode of action of cell death via apoptosis induction and caspase activation.

Materials and Methods
All solvents were purified and degassed before use [50]. Ligands 1-6 were prepared following the published procedure [47,49]. NMR spectra were recorded on a Bruker Avance II 400 spectrometer at room temperature at 400.13 ( 1 H) and 100.61 ( 13 C{ 1 H}) MHz. 2D NMR measurements were carried out using standard pulse programs. Chemical shifts were referenced relative to the solvent signal for 1 H and 13 C spectra. Elemental analysis was performed with MicroCube Elementar analyzer. Electrospray ionization (ESI) mass spectra were recorded using a TSQ Endura (Thermo Fisher Scientific, Waltham, MA, USA) instrument. Each analysed compound was dissolved in methanol (HPLC grade) and injected directly into the ionization source through a syringe pump. The spectra were recorded during 30 s in the m/z range 150-1400 in both positive and negative ionization modes with spray voltage 3.4 and 2.5 kV, correspondingly. The human HCT116 colorectal carcinoma, A549 non-small cell lung carcinoma, MCF7 breast adenocarcinoma (80 mg, 0.193 mmol) in 5 mL of CH2Cl2 was added to the dimer (η 6 -p-cymene-RuCl2)2 solution (59 mg, 0.096 mmol) in 5 mL CH2Cl2. The reaction was stirred for 10 h at room temperature. The reaction mixture was evaporated to 1 mL, and 10 mL of ether and 15 mL of hexane were added. The resulting orange solid was filtered off, washed with hexane, and dried in a vacuum. Yield 70 mg (50%), Tdec. = 103-105 °C. 1
(80 mg, 0.193 mmol) in 5 mL of CH 2 Cl 2 was added to the dimer (η 6 -p-cymene-RuCl 2 ) 2 solution (59 mg, 0.096 mmol) in 5 mL CH 2 Cl 2 . The reaction was stirred for 10 h at room temperature. The reaction mixture was evaporated to 1 mL, and 10 mL of ether and 15 mL of hexane were added. The resulting orange solid was filtered off, washed with hexane, and dried in a vacuum. Yield 70 mg (50%), T dec . = 103-105 • C. 1  recorded during 30 s in the m/z range 150-1400 in both positive and negative ionization modes with spray voltage 3.4 and 2.5 kV, correspondingly. The human HCT116 colorectal carcinoma, A549 non-small cell lung carcinoma, MCF7 breast adenocarcinoma and SW480 colon adenocarcinoma cell lines were obtained from the European collection of authenticated cell cultures (ECACC; Salisbury, UK).

-p-cymene){N-(6-(1H-imidazol-1-yl)hexyl)-1-(2,4-dichlorobenzyl)-1H-indazole-3-carboxamide}ruthenium(II)-N oxalate (13)
Silver oxalate Ag2C2O4 (67 mg, 0.22 mmol) was added to the dimer (η 6 -p-cymene-RuCl2)2 solution (69 mg; 0.11 mmol) in 40.0 mL H2O. The reaction mixture was stirred for 12 h. Precipitated AgCl was filtered off, and the solvent was evaporated under a vacuum. The resulting ruthenium complex was dissolved in 18.0 mL of MeOH, and a solution of compound 4 (100 mg, 0.22 mmol) in 2.0 mL of MeOH was added. The reaction mixture was stirred for 8 h, the solvent was evaporated under a vacuum, and the product was precipitated with hexane and filtered. The resulting orange precipitate was dried in a vacuum. Yield 78 mg (72%). 1  RuCl2)2 solution (69 mg; 0.11 mmol) in 40.0 mL of H2O. The reaction mixture was stirred for 12 h. Precipitated AgCl was filtered off, and the solvent was evaporated under a vacuum. The resulting ruthenium complex was dissolved in 18.0 mL of MeOH, and a solution of compound 6 (122 mg, 0.22 mmol) in 2.0 mL of MeOH was added. The reaction mixture was stirred for 8 h, the solvent was evaporated under a vacuum, and the product was precipitated with hexane and filtered. The resulting orange precipitate was dried in a vacuum. Yield 98 mg (65%), Tmelt. = 84-86 °C. 1

Log P Determination
Log P values of the new compounds were determined by the HPLC method [42,43] using a Phenomenex Kinetex 5 µ XB-C18 100 Å column 150 × 4.6 mm using two mobile phases: phase A was 20 mM MOPS, 0.15% decylamine, pH = 7.4; phase B was 0.25% 1-octanol in methanol. Briefly, samples dissolved in methanol with uracil as an internal standard were injected into the column and eluted with mobile phase B between 70%, 80%, and 90%. The log P values were calculated as previously described [43] using benzaldehyde, methyl benzoate, ethoxybenzene, naphthalene, and 1-chloronaphthalene as standards. These experiments were repeated three times for each of the compounds.

Cell Death Studies
The antiproliferative activity was studied by MTT assays as published previously [45].

TrxR1 Assay
The activity of rat TrxR1 in the presence of target compounds was determined in vitro using hepatocyte homogenate as we described previously [49].

Synthesis and Characterization
Previously, we have reported the synthetic route and antiproliferative activity data for the lonidamine-modified imidazole ligands 1-6 [47,49] and utilized them for the preparation of various Ru(III) and Ru(II) compounds, including complex 8 [47]. In this work, new complexes 7, 9-11 were obtained by coordination of imidazole ligands 1, 3-5 with the ruthenium dimer ((η 6 -p-cymene)RuCl 2 ) 2 in CH 2 Cl 2 in the ratio 2:1 (Scheme 1). It has been found that Ru(II) organometallic compounds with chloride ligands easily entered into ligand exchange reactions with several solvent molecules, such as water or DMSO [52]. DMSO is widely used in in vitro tests, while the transformation of organometallic compounds in DMSO-containing solutions can hinder the study of biological activity. To overcome the mentioned problem, we have proposed an approach to obtaining Scheme 1. Synthesis of Ru(II) complexes.
Complexes 12-17 with the oxalate or malonate moiety were prepared in two steps procedure: first the formation in situ ruthenium aqua complexes from the ruthenium dimer with silver oxalate or malonate, correspondingly, were carried out and later, coordination of aqua complex with ligands 2, 4, and 6. Complexes 18-19 were prepared by coordination ligands 2, 6, and ((η 6 -p-cymene)RuCl 2 ) 2 in the ratio 4:1 (Scheme 1). All obtained complexes, 7-19, were fully characterized with 1 H and 13 C{ 1 H} NMR spectroscopy, ESI mass-spectrometry, and elemental analysis which have fully confirmed the structure of expected products (see Supplementary Figures S1-S6).
It has been found that Ru(II) organometallic compounds with chloride ligands easily entered into ligand exchange reactions with several solvent molecules, such as water or DMSO [52]. DMSO is widely used in in vitro tests, while the transformation of organometallic compounds in DMSO-containing solutions can hinder the study of biological activity. To overcome the mentioned problem, we have proposed an approach to obtaining analogues resistant to the ligand exchange reactions. This was achieved by replacing the chloride ligands with the dicarboxylic acid moiety or introducing a second imidazole ligand into the coordination sphere.
The stability of complexes 7-19 in DMSO-containing solutions has been studied by NMR spectroscopy. 1 H NMR spectra of compounds 7-11 bearing two chloride ligands include additional signals corresponding to ligand exchange products when a DMSOcontaining solvent is used; whereas, compounds with an oxalate or malonate fragment as well as complexes 18-19 with two imidazole ligands do not show any additional signals, hence demonstrating no transformation of the complex in the solution (Figure 3). Complexes 12-19 were also found to be stable in pure DMSO. It has been found that Ru(II) organometallic compounds with chloride ligands easily entered into ligand exchange reactions with several solvent molecules, such as water or DMSO [52]. DMSO is widely used in in vitro tests, while the transformation of organometallic compounds in DMSO-containing solutions can hinder the study of biological activity. To overcome the mentioned problem, we have proposed an approach to obtaining analogues resistant to the ligand exchange reactions. This was achieved by replacing the chloride ligands with the dicarboxylic acid moiety or introducing a second imidazole ligand into the coordination sphere.
The stability of complexes 7-19 in DMSO-containing solutions has been studied by NMR spectroscopy. 1 H NMR spectra of compounds 7-11 bearing two chloride ligands include additional signals corresponding to ligand exchange products when a DMSOcontaining solvent is used; whereas, compounds with an oxalate or malonate fragment as well as complexes 18-19 with two imidazole ligands do not show any additional signals, hence demonstrating no transformation of the complex in the solution (Figure 3). Complexes 12-19 were also found to be stable in pure DMSO.  The lipophilicity of complexes 12-14 with oxalate moiety was determined by HPLC (Table 1). For complexes 7-11 and 18, 19 we observed irreversible absorption on the column. Complexes showed high lipophilicity, as was expected, and an increase in Log P values with an increase in linker length. To confirm the key role of lonidamine in the cytotoxicity of the obtained complexes, analogue 21 without lonidamine moiety was obtained (Scheme 2). The complex was synthesized by coordination of ligand 20 to ruthenium aqua complex with an oxalate group obtained in situ.
umn. Complexes showed high lipophilicity, as was expected, and an increase in Log P values with an increase in linker length. To confirm the key role of lonidamine in the cytotoxicity of the obtained complexes, analogue 21 without lonidamine moiety was obtained (Scheme 2). The complex was synthesized by coordination of ligand 20 to ruthenium aqua complex with an oxalate group obtained in situ. Scheme 2. Synthesis of the Ru(II) analogue without lonidamine.
An increase in the linker length about twice increases activity, however, it significantly raised lipophilicity. Unfortunately, we did not observe any selectivity toward the cancer cells in the experiments with the non-tumorigenic WI38 cell line (IC50 25.05 ± 0.03 for 15). Moreover, the antiproliferative study confirmed the significance of lonidamine moiety in the compound, as ligand 20 and complex 21 exhibited no activity. For further cell death studies by flow cytometry, complexes 12, 14, 18, and 19 were chosen, and cisplatin was used as a reference drug (Figure 4). Cytometric studies of apoptosis induction and caspase activation on the HCT116 cell line revealed that Ru(II) organometallic compounds with lonidamine-containing ligands at an early stage (after 24 h of incubation) do not lead to significant apoptosis induction (~15%), and there are no cells with activated caspases (0%). This is probably due to the slow transformation to the active form of the Ru-prodrug. However, after 48h, and especially after 72h organometallic derivatives start to show significant apoptosis induction which is accompanied by caspase activation. Cytometric studies of apoptosis induction and caspase activation on the HCT116 cell line revealed that Ru(II) organometallic compounds with lonidamine-containing ligands at an early stage (after 24 h of incubation) do not lead to significant apoptosis induction (~15%), and there are no cells with activated caspases (0%). This is probably due to the slow transformation to the active form of the Ru-prodrug. However, after 48 h, and especially after 72 h organometallic derivatives start to show significant apoptosis induction which is accompanied by caspase activation.
Thioredoxin reductases belong to the thioredoxin system and play a crucial role in regulating redox processes, transcription, and protection from reactive oxygen species. TrxR1 is one of the cytosolic isoforms of this enzyme, which is overexpressed in cancer cells, making it a target for developing new anticancer therapies [53]. Due to the presence of the selenocysteine enzyme in the active centre, the majority of known TrxR inhibitors are electrophilic compounds [54], making it necessary to study the inhibitory effect of new compounds on TrxR1 in vitro.
To assess whether the engagement of thioredoxin reductase 1 contributes to the cytotoxic action of novel Ru(II) complexes, we evaluated selected compounds as TrxR1 inhibitors in a functional in vitro assay. Complexes 12, 15, and 18, which comprise lonidamine/oxalate, lonidamine/malonate, and bis-lonidamine moieties, respectively, were tested at a final concentration of 100 µM ( Figure 5). We have found that these Ru(II) complexes lack significant TrxR1 inhibitory properties regardless of the ligand's nature, unlike previously reported Ru(III) complexes [40]. Thus, it appears that the ruthenium oxidation state and the presence of the Ru-C bond play a definitive role in the compounds' mechanism of action.
To assess whether the engagement of thioredoxin reductase 1 contributes to the cytotoxic action of novel Ru(II) complexes, we evaluated selected compounds as TrxR1 inhibitors in a functional in vitro assay. Complexes 12, 15, and 18, which comprise lonidamine/oxalate, lonidamine/malonate, and bis-lonidamine moieties, respectively, were tested at a final concentration of 100 μM ( Figure 5). We have found that these Ru(II) complexes lack significant TrxR1 inhibitory properties regardless of the ligand's nature, unlike previously reported Ru(III) complexes [40]. Thus, it appears that the ruthenium oxidation state and the presence of the Ru-C bond play a definitive role in the compounds' mechanism of action.

Conclusions
The ruthenium organometallic compounds with lonidamine ligand connected by an imidazole linker were prepared. The presence of oxalate, malonate moiety, or second lonidamine ligand leads to high stability in the ligand exchange reaction. These complexes showed good antiproliferative activity and high lipophilicity but also some increase in activity with an increase in the length of the linker. The study on the mechanism of cell death revealed slow induction of apoptosis without activation of caspases. In contrast to previously studied Ru(III) complexes with the same ligand, the TrxR1 is not inhibited by Ru(II) organometallic analogues. The new compounds described herein represent an interesting and promising class of antiproliferative ruthenium complexes that will be studied further, including in vivo evaluation.

Conclusions
The ruthenium organometallic compounds with lonidamine ligand connected by an imidazole linker were prepared. The presence of oxalate, malonate moiety, or second lonidamine ligand leads to high stability in the ligand exchange reaction. These complexes showed good antiproliferative activity and high lipophilicity but also some increase in activity with an increase in the length of the linker. The study on the mechanism of cell death revealed slow induction of apoptosis without activation of caspases. In contrast to previously studied Ru(III) complexes with the same ligand, the TrxR1 is not inhibited by Ru(II) organometallic analogues. The new compounds described herein represent an interesting and promising class of antiproliferative ruthenium complexes that will be studied further, including in vivo evaluation.  Data Availability Statement: Data will be made available on request.

Conflicts of Interest:
The authors declare no conflict of interest.