Recent Trends in the Design of Ruthenium Homometallic Polynuclear Complexes with Bioactive Ligands for Cancer Treatment

Abstract

Significant efforts have been devoted to discovering novel metal-based complexes with better cytotoxicity and specificity to tumor cells. Within the range of complexes studied for cytotoxic activity, Ru complexes have gained significant attention as one of the most promising classes of compounds offering advantages such as good scaffolds for the construction of new bioactive molecules with a variety of ligands. Ruthenium-based compounds demonstrate efficient penetration into cancer cells and show affinity for DNA binding with antitumor mechanisms, other than those of cisplatin. They were identified as perfect chemotherapeutics for cancer treatment due to their good tolerance by normal cells, negligible toxic effects and stronger activity towards Pt-drug-resistant tumor cell lines. Ru-based complexes may interact with multiple targets and show selective accumulation in cancer cells, which enhances their therapeutic potential. In recent years, the design of polynuclear complexes has aroused considerable interest in drug discovery research. The strategy to incorporate two or more metal centers into one precise molecular structure may result in better cytotoxic activity compared to the mononuclear precursors. That is why ruthenium-based multinuclear anticancer organometallic and complex compounds have attracted lots of attention. The objective of the current review is to highlight the key results obtained in research on ruthenium complexes, presenting the up-to-date advances of multinuclear homometallic ruthenium complexes as promising anticancer candidates. The reported outcomes shed new light on the fundamental biological interactions and antineoplastic modes of action of ruthenium-based complexes and organometallic compounds as well as significant information for the prediction of novel anticancer drugs.

1. Introduction

Over the last years, metal complexes have been widely utilized in the field of medicinal chemistry [1,2]. The best-known platinum-based anticancer agents have shown many limitations, such as low selectivity, chemoresistance, and toxic side effects to normal cells, such as neurotoxicity, nephrotoxicity, ototoxicity, hepatotoxicity, skin toxicity, etc. Consequently, there is a necessity for new compounds containing metals other than platinum as anticancer candidates. In this regard, Ru-based complexes have gained consideration in many aspects and emerged as the most prominent candidates [3,4].

The anticancer profiles of ruthenium compounds include improved selectivity, prominent bioactivity and bioavailability, as well as lower toxicity against healthy cells. Various less toxic and more selective Ru-based compounds have been investigated [5,6]. Research into the medical applications of these compounds has been facilitated by the wide variety of ruthenium coordination and its organometallic chemical properties, as well as in connection with the usage of Ru-based complexes in catalysis, photodynamic therapy (PDT), and photoactivated chemotherapy (PACT) [7]. The promising activity of Ru complexes is supposed to be connected with the capability of ruthenium to mimic Fe in interactions with biomolecules, specifically serum proteins, such as albumin and transferrin, which are overexpressed in the tumor cells [8,9]. Thereby, Ru complexes can target cancer cells selectively.

It has been proven that Ru complexes showed synergistic action when combined with conventional chemotherapeutic drugs [10]. Moreover, these complexes have been extensively used as phototherapeutic and bioimaging agents [11]. Additionally, Ru complexes can be used as tumor theranostic candidates for diagnosis and therapy of different cancers with reduced toxic effects [12,13,14].

In a physiological environment, the ruthenium cation is stable in Ru(II) and Ru(III) oxidation states, the former being more reactive [15]. Ruthenium(III) complexes have stable thermodynamical and kinetical behaviors. They are effective as prodrugs in hypoxic and acidic environments [16]. The Ru(III) ion is more passive than Ru(II) because of its higher nuclear charge. That is why Ru(II) compounds have higher chemical reactivity than Ru(III) ones [17]. The reduction of Ru(III) complexes yields the more active Ru(II) form by the so-called “activation by reduction mechanism”, which depends on reducing compounds, such as glutathione, ascorbate, and hypoxic cancer environments [18,19]. Nevertheless, the “activation by reduction” theory is still controversial [20]. The complexes in both the oxidation states have octahedral geometry with six-coordinated configuration, which can easily take part in various redox reactions [21]. Being in the central location in the second period of d-block metals, ruthenium shows early and late properties of these elements equally. Its partially filled 4d electronic subshell allows different valencies which enable the metal to form a variety of complexes. Additionally, the ligands’ variation modulates their steric and electronic properties, and can allow large platforms of prominent structural Ru-based moieties and different ligand-exchange reactions with biotargets. Thus, Ru-based compounds (octahedral coordination geometry) with lower nonspecific toxicity and higher bioactivity against platinum-resistant tumor cells are considered to be a good alternative that may broaden the spectrum of activity and offer different modes of action and chemoreactivity of Pt-based drugs with typical square-planar geometry [22,23,24]. Furthermore, in comparison with typical platinum drugs, a number of ruthenium compounds possess better water solubility in physiological environments, leading to improved efficacy against Pt-resistant cells [25] and balancing the hydrophilicity and hydrophobicity of Ru-based complexes and their absorption in tumor cells [26].

Many mononuclear Ru coordination compounds have received consideration due to their binding capability to DNA and induction of cytotoxicity and their unique photochemical, photophysical, and electrochemical properties. Successful clinical trial entries of the Ru-based complexes NAMI-A, NKP1339, KP1019, and TLD1443 along with the information on in vitro and/or in vivo activity of other ruthenium-based chemotherapeutic candidates provide great, promising prospects in anticancer drug development. The cytotoxic activity of cisplatin is principally associated with binding to DNA, while the biotargets of clinically approved Ru complexes are not completely clarified [27,28,29,30].

Most Ru-based complexes presently being studied are mononuclear [15,16,23,24,25,29]. The design of polynuclear analogous compounds could lead to the development of new complexes with improved bioactivity [4,9,29,30,31]. An attractive approach to overcome the drawbacks, such as toxicity, selectivity, uptake, and biocompatibility problems, associated with Ru-based complexes could be the design of polynuclear complexes. Recent studies on multinuclear complexes cover structurally novel scaffolds, and many of these compounds have shown encouraging results.

2. Homodinuclear Ru Complexes

Ruthenium medicinal chemistry has generated many promising results. Considerable research attention has been devoted to the interaction of Ru complexes with different biologically active ligands with DNA, and numerous new structures were synthesized and investigated, where di- or polynuclear ruthenium complexes have attracted serious attention. Because Ru has a higher coordination number than Pt, it offers additional coordination sites that can be utilized to modulate the properties of the obtained complexes and the interactions with DNA [31]. The varied redox behavior of Ru is also important for both the transport of drug candidates in the body and their reactions with biorelevant proteins [32]. Other significant features are the variances in the kinetics of ligand substitution and their water solubilities. Many dinuclear Ru compounds have displayed a cytotoxicity better or analogous to that of platinum drugs [33]. A wide variation of potent Ru-based drugs has been obtained, incorporating different ligands (DMSO, arenes, amines, imines, and polypyridyl compounds) [34]. The structural diversity of these complexes suggests that different types of ruthenium compounds may operate through distinct mechanisms of action [35].

Mendoza-Ferri et al. have reported newly synthesized polynuclear ruthenium(II)–arene complexes holding bis(pyridinone)alkane linking ligands that contained 3, 6, and 12 methylene groups (Figure 1) [36]. The Ru–arene complexes displayed potent activity in various human ovarian cancer and colon cancer cells. A noticeable effect of their spacer length on the in vitro antitumor action was found, which increased with the dimension of the alkane linking ligand, correlating to the complexes’ lipophilicity. The studied dinuclear compounds were more active than the similar mononuclear analogues. IC₅₀ in the same range as that recognized for Pt-based drugs was observed for the tested tumor cells. Based on water–octanol partition coefficients and hydrostability, the detailed SAR analysis revealed that the most lipophilic complex, containing a long alkyl chain (n = 12), exhibited high activity. The most active complex exhibited no cross-resistance to the cisplatin prodrug oxoplatin across the three resistant cell lines. Bioanalytical characterization of the compounds has shown that the studied ruthenium complexes hydrolyzed quickly, forming mainly diaqua species that exhibited affinity to transferrin and DNA, demonstrating that nucleobases and proteins could be possible targets for these ruthenium–arene polynuclear complex compounds.

The dinuclear Ru(II) complexes of great length with ammine, amine, and imine ligands, which are able to participate in long-range DNA interactions, have attracted considerable interest. The observed excellent cytotoxic activity of these compounds is thought to result from the formation of intra- and inter-strand long-range DNA adducts. Dinuclear Ru(II) complexes are believed to be capable of overcoming both acquired and intrinsic resistance to the classical anticancer drug cisplatin. The lengths and flexibility of the chains, the hydrogen bonds and charges of the linking ligands, as well as the labile ligand geometry are the main aspects for the synthesis of dinuclear Ru(II) antineoplastic drug candidates. Additionally, the chlorido ions, as leaving ligands, can be replaced with an easily coordinating solvent, for instance, DMSO. The solvolysis reactions and the in vitro cytotoxic activity evaluation with murine L1210 leukemia cells of Ru(II) dinuclear complexes with several bridged ligands in different conditions (H₂O, DMSO, H₂O-DMSO) have been investigated and reported [37]. It was found that ruthenium(II) complexes with hydrophobic DMSO were more efficiently permeable into the tumor cells. Their IC₅₀ values revealed that these complexes exhibited strong cytotoxic activity, signifying that the hydrophobic DMSO plays a significant role in their biological activity.

To improve the activity of ruthenium mononuclear 2,2′:6′,2″-terpyridine complexes [Ru(tpy)(L)(Cl)]Cl, where L = a non-labile bidentate ligand, Mulyana et al. obtained the dinuclear complexes with a general formula [{Ru(tpy)Cl}₂{μ-bb_n}]Cl₂ (bb_{n =} bis [4(4′-methyl-2,2′-bipyridyl)]-1, n-alkane, n = 7, 10, 12, 14), and investigated their nucleic acid binding and cytotoxicity [38]. The dinuclear complexes exhibited potent activity against the very sensitive murine leukemia L1210 cells with IC₅₀ = 5–10 μM, and showed tenfold higher activity than the respective mononuclear compound [Ru(tpy)(Me₂bpy)Cl]Cl (Me₂bpy = 4,4′-dimethyl-2,2′-bipyridine). To expand the family of binuclear complexes, the authors synthesized new complexes with substituted bb_n ligands (Figure 2) and studied their antineoplastic activity, hydrolysis rates, and binding capacity to guanosine-5′-monophosphate (5′-GMP) [39].

The multinuclear Ru(II)-based complex [{Ru(tpy)Cl}₂{μ-bb_n}]Cl_2, where the tpy is 2,2′:6′,2″-terpyridine, and the bbn is bis [4(4′-methyl-2,2′-bipyridyl)]-1,n-alkane (n = 12)}, showed higher anti-proliferative potential against two breast adenocarcinoma cell lines when compared both to cisplatin and carboplatin. The observed antitumor activity likely arises from both covalent and reversible interactions with DNA. Notably, the dinuclear ruthenium complexes with n = 10, 12, and 14 demonstrated higher potency than cisplatin across the tested cell lines. The complex with n = 12 exhibited the highest activity, while those with the shortest (n = 7) and longest (n = 16) linking chains showed the lowest activity. This superior activity was attributed to an optimal balance between lipophilicity, which facilitates cellular uptake, and the cytotoxicity resulting from the formation of covalent adducts with DNA. Nitro substituents in the tpy rings of these complexes decreased their activity, mainly in the most active compound (n = 12). The substitution of two methylene groups by two amine groups in the bridge ligand for the complexes (n = 7 and 16) decreased the activity. The highly potent complex (n = 12) was approximately four times more effective than cisplatin and then the dinuclear complexes [{Ru(bpy)₂Cl}₂{μBL}]Cl₂, for instance, with bidentate ligands, such as, 2-phenylazopyridine, 2,2′-azobispyridine, and 2-phenylpyridinylmethylene amine with mechanisms of action quite diverse from that of the classical Pt anticancer agents [40].

Some 1,10-phenanthroline bridging ligands BL and their ruthenium(II) complexes [(bpy)₂Ru(BL)Ru(bpy)₂][PF₆]₄, where bpy = 2,2′-bipyridine; PF₆ = hexafluorophosphate; BL = polyaromatic ligands with two equivalent chelating sites on the phenanthroline rings, bridged by a flexible alcoxyphenyl spacer moiety (Figure 3a–c), have been obtained and investigated for their antineoplastic activity [41]. The symmetrical structure of the bridging ligands allowed the production of dinuclear ruthenium(II) complexes with equivalent metal centers. The photophysical and electrochemical behaviors of the compounds have also been studied. Their in vitro cytotoxic activity has been assessed against cervical cancer HeLa, gastric cancer SGC-7901, and gastric cancer BGC-823 cells by the MTT test. The data showed that the dinuclear Ru(II) complexes displayed substantial dose-dependent cytotoxic activity to the tested tumor cell lines. The complex (Figure 3c) was the most potent and exhibited low IC₅₀ values (9.21 μM for BGC-823 and 10.38 μM for SGC7901 cells). The cytotoxicity of the complex, shown in Figure 3c, against HeLa cells was higher than that of [(bpy)₂Ru(L)Ru(bpy)₂][Cl₄], where L = 1,6-bis(3-(1H-imidazo-[4,5-f][1,10]phenanthrolin-2-yl)-9H-carbazol-9-yl)hexane, with IC₅₀ > 100 μM, which showed strong intercalative interaction and possible cell imaging application owing to its low antiproliferative activity, reported by Liu et al. [42]. The differences in cytotoxic activity between these series of complexes against HeLa, SGC-823, and SGC-7901 cells might be due to the different amount of methylene groups in their flexible alkane chains. The cytotoxicity of the complexes, depicted in Figure 3a–c, increased with the rise of methylene groups in the bridging ligands. The DNA quenching constants were found to be 1.34 × 10⁵, 1.46 × 10⁵, and 2.98 × 10⁵ mol/L for the complexes shown in Figure 3a–c, demonstrating that there were different kinds of interactions between the complexes and DNA.

Similar findings were reported by Beckford et al. for a series of trinuclear ruthenium complexes incorporating 1,3,5-triazine ligands and diverse η⁶–arene moieties. A set of trinuclear complexes comprising organometallic Ru fragments (-(arene)RuCl-) coordinated to 2,4,6-tris(di-2-pyridylamino)-1,3,5-triazine cores, with arene  =  benzene, p-cymene and hexamethylbenzene, have been synthesized. Their DNA interactions were broadly studied by various biophysical probes and by docking analysis. These compounds showed strong binding with DNA, found to be non-specific covalent or groove types of interactions. The complexes showed very low cytotoxic activity against tumor cell lines only at high (>100 μM) concentrations [43,44].

Tetrapyrido [3,2-a:2′,3′-c:3″,2″-h:2‴,3‴-j]phenazine (tpphz) ligands are a class of derivatives identified to interact with DNA due to their highly extended planar structure, similar to dipyridophenazine (dppz). They have been used by Gill et al. to obtain DNA-binding ruthenium(II) dinuclear complexes, shown in Figure 4. The complex in Figure 4a could specifically accumulate in the cellular nucleus coordinating to phenanthroline ligands alongside tpphz [45]. The complex (Figure 4a) was well tolerated by eukaryotic and prokaryotic cells, demonstrating suitability for applications in luminescence and transmission electron microscopy. Interestingly, despite its hydrophilic nature, the complex is internalized by live cells via a non-endocytotic mechanism. It enables visualization of structural changes in nuclear DNA through the cell cycle and shows promising potential for quadruplex DNA imaging [46].

Ruthenium binuclear polypyridyl complexes have been widely investigated over the past few decades due to their remarkable ability to selectively bind DNA. Svensson et al. have reported binuclear Ru(II) polypyridyl complex compounds, investigated their cell localization, uptake, and biomolecular interactions, and compared the results with those of well-known dipyridophenazine (dppz) complexes [47]. The structural isomers of the dinuclear ruthenium polypyridyl complex [μ-bipb(phen)₄Ru₂]Cl₄, shown in Figure 5, are [μ-meta-bipb(phen)₄Ru₂]Cl₄, Figure 5a, where meta-bipb = 1,3-bis(imidazo [4,5-f]-1,10-phenanthrolin-2-yl)benzene; phen = 1,10-phenanthroline; and [μ-para-bipb(phen)₄Ru₂]Cl₄, Figure 5b, where para-bipb = 1,4-bis(imidazo [4,5-f]-1,10-phenanthrolin-2-yl)benzene. The interactions of the enantiomerically pure forms of these structural isomers of the dinuclear Ru(II) polypyridyl complex with the mammalian CHO-K1 cell line have been examined to elucidate the potential of Ru(II) polypyridyl complexes as possible cellular imaging probes. Interestingly, while the two structural isomers of [μ-bipb(phen)₄Ru₂]Cl₄, Figure 5, showed similar staining patterns, the respective enantiomers (Δ and Λ) demonstrated significant localization differences. All complexes demonstrated the ability to penetrate the nucleus; however, the ΔΔ enantiomers of both isomers exhibited preferential accumulation within the nucleoplasm, whereas the ΛΛ enantiomers predominantly localized in the cytoplasm, displaying pronounced nucleolar staining. Notably, this differential subcellular distribution did not appear to arise from enantiomeric differences in the strength of DNA binding interactions. Although a definitive cause could not be established, it is plausible that variations in the nature rather than the strength of the interactions account for the observed localization patterns. The nucleoli are structures primarily composed of ribosomal RNA (rRNA), which exhibits substantial geometrical differences compared to the B-form of DNA. Consequently, the distinct intracellular distributions may reflect enantiomer-specific selectivity toward B-DNA versus rRNA [48]. Live-cell uptake of the complexes at 5 μM was both efficient and non-toxic, and proceeded through an energy-dependent pathway.

Li et al. have described the antineoplastic activity of some non-symmetric binuclear polypyridylruthenium(II) complexes (Rubb_n-Cl), Figure 6. These complexes consisted of one inert metal center and one coordinatively labile metal center, bridged by a bis [4(4′-methyl-2,2′-bipyridyl)]-1,n-alkane ligand with varying alkyl chain lengths (n = 7, 12, 16). The cytotoxic activity of the complexes was assessed by MTT against three eukaryotic cell lines—two kidney lines (BHK and HEK-293) and a liver cell line (HepG2). The results showed that the complexes were approximately 30 to 80 times more cytotoxic toward cancer cells than toward bacteria. Furthermore, an increase in alkyl chain length correlated with improved cytotoxicity in the HEK-293 and BHK cells. With an increase in lipophilicity, the cellular uptake increased, and the complexes showed improved cytotoxicity. However, in the HepG2 cell line, the cytotoxicity of the least lipophilic complex (n = 7) was abnormally high with an IC₅₀ value of 3.7 μM, which was lower than the values shown by the lipophilic complexes (n = 12, 16). Therefore, even though lipophilicity is a key factor in determining cellular uptake and cytotoxic activity, other parameters must also be taken into account. Moreover, the anticancer activity of these complexes has been attributed to their strong interactions with DNA [49]. To elucidate the unexpectedly large differences in the cytotoxic activities of Rubb_n-Cl complexes against eukaryotic cells, their intracellular localization was investigated using confocal microscopy. The outcomes suggested that the complexes (n = 12, 16) had better selectivity for rRNA, compared to DNA, than does the complex (n = 7). The superior level of DNA localization in HepG2 by the complex (n = 7), compared to the complexes (n = 12, 16), could be associated with its higher cytotoxic activity against HepG2 cells.

Liu et al. have also investigated the antitumor activity of two binuclear polypyridyl-ruthenium complexes (Figure 7) with variable bridging alkyl chains holding 3 and 10 carbon atoms. Their calf-thymus DNA-binding and plasmid-DNA photocleavage behaviors have been studied and the complexes showed partial DNA intercalation and selective localization in lysosomes. Theoretical calculations based on the density functional theory (DFT) method were performed to further understand the experimentally observed DNA-binding properties of the complexes. The results indicated that both complexes partially intercalate between the DNA base pairs. Cellular uptake and colocalization studies revealed that the complexes efficiently entered HeLa cells and localized within lysosomes. The in vitro antineoplastic activities of the complexes against HeLa and MCF-7 cancer cells were evaluated using an MTT cytotoxicity assay. In addition, high-content analysis (HCA) was employed to assess cytotoxic activity, apoptosis, and cell cycle arrest induced by the complexes. The complex (n = 10) exhibited moderate cytotoxic activity showing higher potency than the complex (n = 3), and the cytotoxic effects were not in line with DNA binding but depended on their lipophilicity. These results have shown that the length of the alkyl linkers could efficiently modulate their activity and that HCA was appropriate to quickly identify antiproliferation and could replace MTT assays to test the cytotoxic activity of chemotherapeutics. The complexes induced apoptosis through cell cycle arrest at the G0/G1 and S phases. Comprehensive elucidation of their mechanisms of action and structure–activity relationships is warranted to optimize and advance the therapeutic potential of these compounds [50]. Therefore, it can be settled that in most cases, the alkyl chain length is a critical factor influencing cytotoxic activity of the studied dinuclear ruthenium complexes, as it governs the lipophilicity and cellular uptake of the complexes.

Singh et al. have studied the antineoplastic activity of new dinuclear Ru(II)–polypyridyl complexes [Ru₂(N^N)₄(BPIMBp)]Cl₄, where N^N = 2,2′-bipyridine (bpy), 1,10-phenanthroline (phen), dipyrido [3,2-d:2′,3-f] quinoxaline (dpq), and [Ru₂(N^N)₄(BPIMBp)](PF₆)₄, where N^N = dipyrido [3,2-a:2′,3′-c] phenanzine (dppz), and 1,4′-bis[(2-pyridin-2-yl)-1H-imidazol-1-yl)methyl]-1,1′-biphenyl (BPIMBp) was used as a bridging ligand, Figure 8. These complexes were positively charged (IV+) cations and exhibited flexibility due to −CH₂ groups of the bridging ligand. They also contained terminal ligands with variable intercalative capabilities. The interaction of these complexes with calf-thymus DNA (CT-DNA) was studied by different methods. The complexes exhibited DNA interaction through the spacer, consistent with a groove-binding mode. The complexes of dipyrido [3,2-d:2′,3-f] quinoxaline and dipyrido [3,2-a:2′,3′-c] phenanzine have also shown an intercalative interaction. These complexes demonstrated moderate cytotoxic effects on HeLa, MCF-7, and HL-60 cells with IC₅₀ values in the range of 5–25 μM. The observed antitumor activity is mainly attributed to their ability to interact with DNA [51]. Liu et al. synthesized a series of twelve dinuclear Ru-arene metallacycles with 24-, 26-membered structures having 1-(3-((1H-imidazol-1-yl)methyl)benzyl)-1H-imidazole and 1-(4-((1H-imidazol-1-yl)methyl)benzyl)-1H-imidazole ligands and investigated their antineoplastic activity. The complexes demonstrated low to moderate cytotoxic activity against HeLa, MCF-7, and A549 tumor cells as well as the normal LO2 cells, suggesting a lack of selectivity toward cancer cells. The antineoplastic activity of these complexes is likely attributable to their capacity to induce DNA condensation [52].

The binuclear Ru(II)–arene complex compounds [{RuCl(η⁶-arene)}₂(μ-2,3-dpp)](PF₆)₂, arene = indane, benzene, p-cymene, hexamethylbenzene, and 2,3-dpp = 2,3-bis(2-pyridyl)pyrazine have been reported [53,54]. The photoactivation of the binuclear Ru–arene complex [{RuCl(η⁶-indane)}₂(μ-2,3-dpp)](PF₆)₂ (2,3-dpp = 2,3-bis(2-pyridyl)pyrazine) (Figure 9) has been studied in detail. Irradiation of the complexes had a substantial impact on their DNA interactions. The DNA-binding affinity of the complexes was markedly enhanced following irradiation. In the non-irradiated state, the complexes predominantly formed DNA adducts that only weakly impeded RNA polymerase progression, while irradiation of [{RuCl(η⁶-indane)}₂(μ-2,3-dpp)](PF₆)₂, shown in Figure 9, promoted the formation of adducts with significantly greater transcription-blocking efficiency and transformed the adducts into stronger blocks for RNA polymerase. The thermal reactivity of the compounds was restricted to a stepwise double aquation process exhibiting biexponential kinetics. In contrast, irradiation of the samples induced photoactivation, leading to the loss of the coordinated arene and generating ruthenium species that were more reactive than their ruthenium–arene precursors. The results have shown that photoactivation of binuclear ruthenium(II)–arene complexes simultaneously generates a highly reactive ruthenium species capable of binding to DNA and a fluorescent marker (the released arene). Notably, this photoreactivity occurs independently of oxygen. Therefore, these complexes show great potential for combining photoinduced cell death with fluorescence imaging to monitor the location and efficiency of the photoactivation process. Upon photoactivation, the binuclear complex [{RuCl(η⁶-indane)}₂(μ-2,3-dpp)](PF₆)₂, shown in Figure 9, can generate highly reactive and potentially cytotoxic ruthenium species along with the arene indane, which may serve as fluorescent probes. The photochemical studies of [{RuCl(η⁶-indane)}₂(μ-2,3-dpp)](PF₆)₂ indicated that UV and visible light led to the separation of indane ligands, visualized by their fluorescence, and the formation of strong diruthenium DNA adducts. These complexes therefore have the potential for simultaneously inducing photoinduced cell death and facilitating fluorescence imaging to monitor the spatial distribution and effectiveness of photoactivation.

The stereochemistry of the binuclear complex [{RuCl(η⁶-biphenyl)}₂(en)₂-(CH₂)₆][PF₆]₂ (en = ethylenediamine), Figure 10, has been elucidated [55]. The anticancer potential of the mononuclear complex [(η⁶-arene)RuCl(en)], which exhibited in vitro and in vivo potency in a human ovarian cancer A2780 xenograft model (cisplatin-resistant), has also been described. The studied binuclear Ru–arene complex [{RuCl(η⁶-biphenyl)}₂(en)₂-(CH₂)₆][PF₆]₂ could bind quickly to CT-DNA, favorably to G bases. Complexes of this type exhibited remarkably higher selectivity for G compared to the other nucleobases in the potential target DNA. The cytotoxic activity tends to increase with larger η⁶-arenes. The inhibition of DNA-directed RNA synthesis was more effective than that observed with the corresponding mononuclear complex [56]. Further enhancement of DNA recognition could be achieved by modifying the chelating ethylenediamine moiety, the linkers, and the arene ring systems.

Metal complexes derived from purine nucleobases are valuable tools for cancer diagnosis and therapy, as well as for other biomedical uses. Orts-Arroyo and collaborators have recently reported the synthesis of a binuclear Ru(III-adenine protonated complex [57]. The new binuclear Ru(III) complex of the nucleobase adenine [{Ru(μ-Cl)(μ-Hade)}₂Cl₄]Cl₂⋅2H₂O (Figure 11), where Hade = protonated adenine, has been characterized using electrochemical and spectral methods. It has been tested against colon cancer HCT-116 and gastric cancer AGS cells by MTT and cell cytometry at 48 and 72 h treatments. The Ru(III) complex exhibited a specific cytotoxic activity against AGS tumor cells in a dose- and time-dependent manner with IC₅₀ = 23.25 µM demonstrating an apoptotic pathway in the maximum concentration. McKenzie et al. have described the synthesis, characterization, and in vitro assessment of Ru(II) complexes bearing different oligonucleotides [58,59].

3. Complexes with Arylphosphines as Ancillary Ligands

The ligands of the metal complexes play a substantial role in moderating bioactivity. Arylphosphines, as ancillary ligands for the construction of ruthenium half-sandwich complexes, were found effective in studies of anticancer activity. Partially oxidized phosphorus(III) ligands exhibit strong hydrogen-bond-accepting abilities. Among these, phosphine oxides are recognized as some of the most potent hydrogen-bond acceptors known to date. Nevertheless, their potential influence on the structural integrity of DNA remains largely unexplored.

Reddy et al. have reported Ru–maltol complexes of the types [Ru₃(CO)₇(2L-2H)]₂(dppm) and [Ru₃(CO)₇(2L-2H)]₂(dppe), Figure 12, where L = 3-hydroxy-2-methyl-4H-pyran-4-one [60]. The compounds were synthesized by an interaction of [Ru₃(CO)₈(2L–2H)] with the bridging ligands 1,1-bis(diphenylphosphinomethane) (dppm) or 1,2-bis(diphenylphosphinoethane) (dppe). Cytotoxic evaluation of the complexes, along with the positive control cisplatin, has been performed on different tumor cells: K562 (chronic myelogenous leukemia, CML), HT-29 (colon), HL-60 (acute myloid leukemia, AML), DU-145 (prostate), MCF-7 (breast), GRANTA-519 (mantle cell leukemia, MCL), H460 (non-small cell lung carcinoma). The diphenylphosphinomethane- and diphenylphosphinoethane-bridged dimers of ruthenium–maltol compounds [Ru₃(CO)₇(2L-2H)]₂(dppm), and [Ru₃(CO)₇(2L-2H)]₂(dppe) exhibited significantly lower activity compared to their precursor complexes. A comparison between the two dimers revealed that increasing the chain length of the diphosphine bridge generally enhanced cytotoxic activity across most cell lines. However, the opposite trend was observed for the K562 cells, while no clear conclusions could be drawn for the HL-60 and GRANTA-519 cells. It is also noteworthy that [Ru₃(CO)₇(2L–2H)]₂(dppe) could not achieve the reported IC₅₀ values, as the complex showed poor solubility at concentrations above 25 μM. The diphenylphosphinomethane- and diphenylphosphinoethane-bridged dimers of Ru–maltol complexes, depicted in Figure 12, showed lower activity compared to the monomeric Ru–maltol complex comprising triphenylphosphine [Ru₃(CO)₇(2L-2H)(PPh₃)]. The reduced activity may be attributed to limited cellular uptake caused by the intrinsic bulkiness of the hexanuclear complexes.

Noffke et al. have described the tetranuclear ruthenium–arene complex [(η⁶-p-cym)₄Ru₄(2-dimpe^NMe)Cl₆]Cl₂, Figure 13, where 2-dimpe^NMe = 1,2-bis(di-N-methylimidadol-2-ylphosphino)ethane [61]. Notably, this ligand exhibits higher water solubility compared to that of 1,2-bis(diphenylphosphinoethane), dppe [62]. The complex was synthesized by reacting 2-dimpe^NMe with [(η⁶-p-cym)RuCl₂]₂. The four p-cymene ruthenium moieties exhibited a characteristic piano-stool geometry. The cytotoxic activity against different cell lines was evaluated using an MTT assay. With an approximate IC₅₀ value greater than 100 μM, the ruthenium–arene complex can be considered non-toxic. The tested tumor cells were human colon carcinoma Hct116, human hepatoma Huh 7, rat hepatoma H4IIE, and human ovarian carcinoma A2780 (cisplatin sensitive).

Lenis-Rojas et al. have reported the synthesis and structural characterization of the salt-type complex [(Ru(bipy)₂Cl)₂(μ-dppb)](OTf)₂, Figure 14, where dppb = 1,4-bis(diphenylphosphino)butane and −OTf = trifluoromethanesulfonate anion CF₃SO₃⁻ (triflate). Additional derivative complexes incorporating various bridging motifs have likewise been synthesized [63]. The complex [(Ru(bipy)₂Cl)₂(μ-dppb)](OTf)₂ was synthesized by reacting the Ru(II) precursor complex [Ru(bipy)₂Cl₂] with AgOTf. The newly obtained complex and its derivatives have been tested in vitro for cytotoxicity on A2780, MCF7, and MDAMB231 human tumor cell lines. The complex [(Ru(bipy)₂Cl)₂(μ-dppb)](OTf)₂ exhibited a higher percentage of A2780 cell apoptosis compared to the other derivatives. Considering the non-linear correlation between the IC₅₀ values and the percentage of cell apoptosis, the authors proposed that these compounds might also trigger an alternative form of programmed cell death. This hypothesis was further supported by the observed increase in autophagy levels following treatment with these complexes. The newly obtained complexes interacted with CT-DNA via intercalation, which appeared to induce some DNA degradation extent. This effect appeared to be associated with the generation of ROS. In the in vivo toxicity evaluation, the complex [(Ru(bipy)₂Cl)₂(μ-dppb)](OTf)₂ exhibited the highest toxicity toward zebrafish embryos, with an LC₅₀ value of 5.397 mg/L, indicating it to be the least biocompatible among the tested compounds.

Das et al. have reported organoruthenium(II) complexes [(η⁶-p-cym)RuCl(X)(Y)], Figure 15 [64]. The compound [(η⁶-p-cym)RuCl₂]₂(μ-dppe), Figure 15a, where dppe = 1,2-bis(diphenylphosphino)ethane, was obtained using the dimer complex [(η⁶-p-cym)RuCl₂]₂ as a precursor, which interacted with dppe in benzene medium. The cytotoxicity was evaluated by MTT assays. The human lung non-small cell carcinoma H460 line treated with the complex [(η⁶-p-cym)RuCl₂]₂(μ-dppe) demonstrated limited cytotoxicity, exhibiting an IC₅₀ value of 61.3 μM. This value is markedly higher than those observed for dppe alone (0.08 μM), cisplatin (1.7 μM), and several mononuclear ruthenium complexes reported within the same study. Owing to its comparatively poor cytotoxic performance, the complex was not subjected to further biological investigation. Furthermore, the complex was observed to show less cytotoxicity to non-cancerous HEL 299 cell lines. Additional neutral and cationic Ru(II) complexes [(η⁶-p-cym)RuCl(X)(Y)] with piano-stool geometries have been obtained and characterized by the same authors [64]. In the cationic complexes, the X and Y ligands were identified as either η²-coordinated phosphorus donors, such as 1,1-bis(diphenylphosphino)methane (dppm) and 1,2-bis(diphenylphosphino)ethane (dppe), or as their partially oxidized analogues, 1,2-bis(diphenylphosphino)methane monooxide (dppmo) and 1,2-bis(diphenylphosphino)ethane monooxide (dppeo), which exhibit strong hydrogen-bond-accepting capabilities. It has been found that the coordination of ruthenium to dppm and dppmo improved the cytotoxic activity of the complexes. The structurally similar compounds with the ligands dppm and dppmo [(η⁶-p-cym)Ru(η¹-dppm)Cl₂] (Figure 15b) and [(η⁶-p-cym)Ru(η¹-dppmo)Cl₂] (Figure 15c) showed good cytotoxic activity. The growth-inhibitory effects of these complexes on various human tumor cell lines have been investigated. The cytotoxic efficiency was tested against cervical cancer SIHA, breast cancer MCF7, prostate cancer PC3, ovarian cancer A2780, and hepatocellular carcinoma HEPG2 cell lines. Mechanistic studies on these complexes have revealed that their inhibitory effect on cancer cell growth involves the cell cycle arrest and the induction of apoptosis. Moreover, these complexes have demonstrated potent antimetastatic activity by preventing cancer cell invasion. They have been shown to interact with DNA through covalent binding without promoting its cleavage and to induce unwinding of plasmid DNA in a cell-free system. It can be concluded that the selection of ligands plays a pivotal role in maximizing cytotoxicity, and within the series examined, dppm and dppmo were identified as the most effective ligands.

Herry et al. have recently described a related ruthenium complex featuring a distinct arene substituent and a short diphosphine bridging ligand, 1,1-bis(diphenylphosphino)methane (dppm) [65]. The mononuclear complex, [(η⁶-C₆H₆)RuCl₂(κ¹-dppm)], was initially prepared via the reaction of the dimeric precursor complex [(η⁶-C₆H₆)RuCl₂]₂ with dppm. Subsequent treatment of [(η⁶-C₆H₆)RuCl₂(κ¹-dppm)] with [(η⁶-C₆H₆)RuCl₂]₂ afforded the homodinuclear Ru,Ru complex [(η⁶-C₆H₆)RuCl₂(μ-dppm)RuCl₂(η⁶-C₆H₆)] (Figure 16), in a straightforward manner. Both monosubstituted and bimetallic complexes have been evaluated for cytotoxic activity against human ovarian cancer A2780 and the cisplatin resistant A2780cisR cancer cell lines along with the clinically approved cisplatin and RAPTA-C as positive and negative controls, respectively. The non-cancerogenic human embryonic kidney (HEK293) cell line was included to evaluate the interaction of the compounds with normal cells and to assess their selectivity. Cisplatin served as the positive control. The binuclear complex (Figure 16) exhibited substantially lower cytotoxic activity relative to both the mononuclear complex and cisplatin, with an IC₅₀ value of 30 μM in A2780 cells, compared to 16 μM and 2.3 μM for the mononuclear complex and cisplatin, correspondingly. In the cisplatin-resistant A2780cisR cell line, the dinuclear complex demonstrated moderately enhanced activity (IC₅₀ = 16 μM); however, this improvement was not significant when compared with cisplatin (IC₅₀ = 17.6 μM), and the compound remained less potent than the mononuclear complex (IC₅₀ = 13.8 μM). The observed reduction in cytotoxic activity of the dinuclear complex is likely attributable to diminished cellular uptake, possibly caused by the complex’s relatively large size, which may impede its ability to traverse the cellular membrane. The complex compounds showed somewhat lower activity against healthy cell line HEK293 than the malignant cells; however, they quite clearly were less toxic than cisplatin, which is attributed to the absence of Pt.

Arene-based organometallic ruthenium(II) complexes have garnered considerable pharmacological attention for their potent antitumor activities. Klaimanee et al. have recently reported similar complexes (Figure 17), with methyl-substituted ligands, 1,2-bis(diphenylphosphino)propane, and 2,4- bis(diphenylphosphino)pentane, producing the complex compounds [(η⁶-p-cym)RuCl₂]₂(μ-1,2-dppp), Figure 17a, and [(η⁶-p-cym)RuCl₂]₂(μ-2,4-dpppent), Figure 17b, correspondingly [66]. The complexes were synthesized by the interaction of the dimeric precursor [(η⁶-p-cym)RuCl₂]₂ with the corresponding ligand in THF medium under stirring. The obtained homobinuclear metallic compounds (Figure 17a,b) contained two distorted tetrahedral ruthenium(II) centers with η⁶-p-cymene, two chloride ligands, and the corresponding P-donor ligand. MTT assays against breast cancer cells, e.g., MCF-7, HCC1937, and MDA-MB-231, revealed that both complexes exhibited markedly higher anticancer activity than cisplatin. Complex [(η⁶-p-cym)RuCl₂]₂(μ-1,2-dppp), Figure 17a, was particularly potent against the tested tumor cell lines with IC₅₀ values of 0.35, 0.54, and 0.89 μM, respectively. The authors observed that increasing the concentration of the Ru complex resulted in a decrease in cell viability. Among the tested cell lines, MCF-7 cells were notably more sensitive to the complexes than HCC1937 and MDA-MB-7 cells. Remarkably, these complexes exhibited greater effectiveness compared to previously studied analogues. This enhanced activity may be attributed to the increased lipophilicity conferred by the methyl groups on the diphosphine backbone or to a higher propensity for M–P bond dissociation due to their electron-donating properties.

4. Ruthenium-Based Metalloassemblies

Supramolecular coordination complexes (SCCs), constructed via the self-assemblies of bioorganic ligands and metal cations, have been widely utilized across diverse applications. Their ability to interact with biomolecules through numerous non-covalent interactions is important for intense applicability in the biological fields, particularly in cancer therapy and cellular imaging. On the other hand, SCCs are attractive because of their solution capability due to their inherently charged assemblies and counter-ion effect. With their large, well-defined internal cavities, SCCs can encapsulate and deliver drugs at the anticipated locations, such as platinum complexes [67], porphin [68,69], pyrenyl-functionalized units [70,71,72], etc., to form the complex-in-a-complex systems with better cytotoxicity. Supramolecular coordination complexes can be principally separated into two classes: metallacycles with rhomboidal, triangular, rectangular, and hexagonal structures and metallacages with tetrahedral, hexahedral, and dodecahedral geometries.

The remarkable efficiency of bioactive Ru-based organometallic and coordination complexes has played a significant role in advancing the research on ruthenium supramolecular coordination complexes as prosperous antitumor systems. The ruthenium-based metalloassemblies can be obtained by integrating conventional polypyridine-coordinated Ru vertices or by introducing organoruthenium fragments containing Ru–C bonds. Working with Ru complexes in the +2 oxidation state offers an advantage. Furthermore, studies have revealed that kinetically inert ruthenium(II) ions are often favored in Ru-based species exhibiting bioactivity in vivo [73]. Rationalized synthetic processes for the formation of antineoplastic Ru-based metalloassemblies can also include reorganization of polypyridine–ruthenium fragments accompanied by coordination with organic linker ligands or additional metal cations as alternative strategies [74].

Arene–ruthenium organometallic complexes are undoubtedly an additional alternative for the construction of ruthenium metalloassemblies [75,76]. Arene–ruthenium complexes belong to the family of organometallic complexes with a half-sandwich piano-stool geometry, such as p-cymene (arene)–ruthenium compounds. Their octahedral geometry can be considered as pseudo-tetrahedral because of the presence of η⁶-arene or η⁵-Cp* ligands, described as monodentate. The organometallic Ru(II) half-sandwiched complexes are isostructural with their Os(II) [77], Rh(III) [78], and Ir(III) [79,80] analogues with anticancer activity [75]. The simplest arene–Ru complex is [(η⁶-benzene)RuCl₂]₂ with a dimeric structure consisting of two bridging and two terminal chloride ligands on the Ru(II) ions, together with two η⁶-coordinating benzene ligands [81]. The arenes coordinate in a facial arrangement, occupying three of the six coordination sites, while the remaining sites are occupied by labile ligands (H₂O or Cl⁻). The arenes occupy three of the six coordination sites in a facial arrangement, while the other three are taken by labile ligands such as water or chloride. Thus, the octahedral arene–ruthenium complexes have three accessible coordination sites. Consequently, this coordination arrangement enables the design of biologically relevant ruthenium supramolecular coordination complexes [82].

In fact, a number of ruthenium organometallic and coordination supramolecular assemblies have been effectively designed in the last years [83,84,85,86,87]. Many of them have displayed various structure-inherent anticancer properties and substantial noncovalent targeting abilities to DNA, leading to superior antitumor activity [88,89,90,91,92]. Additionally, their capability to photosensitize oxygen and generate ROS makes them highly efficient PDT agents against numerous types of malignances [93]. Supramolecular chemistry offers better design capability for creating complex assemblies compared to traditional synthetic coordination chemistry. This is typically a controlled formation of structures that closely resemble the dimensions of DNA or protein recognition motifs [94]. For instance, metallohelices, such as helicates and their achiral counterparts, mesocates, are commonly assembled from rigid bioorganic ligands that coordinate around two or more metallic centers, resulting in the formation of two- or three-stranded helices [95,96]. Due to their defined 3D stereochemical structures, tunable configurations, charges, and diameters, they have been accepted as potent mimics of protein helices and efficient agents to cause DNA-–photoactive cleavage [97,98,99,100,101,102,103]. Recently, new binuclear ruthenium(II) double-stranded metallohelices for anticancer therapy that were controllably synthesized by changing the steric interactions between ligand strands have been reported [104]. Therefore, studying the metallohelicate systems might be an optimistic avenue to discover renewed antitumor therapeutics in the future. Therrien et al. have described water-soluble chloride or tetrafluoroborate salt-type compounds of tri-and tetranuclear Ru–arene clusters of the type [(η⁶-C₆H₆)(η⁶-C₆Me₆)₂Ru₃(μ-H)₃(μ₃-O)][BF₄], [(η⁶-C₆H₆)(η⁶-1,4-ⁱPrC₆H₄Me)(η⁶-C₆Me₆)Ru₃(μ-H)₃(μ₃-O)][BF₄], [(η⁶-C₆H₆)₄Ru₄(μ-H)₄][BF₄]₂, [(η⁶-C₆H₅Me)₄Ru₄(μ-H)₄][BF₄]₂, and [(η⁶-C₆H₆)₄Ru₄(μ-H)₃(μ-OH)][Cl]₂. The series of ruthenium clusters has been tested against ovarian cancer A2780 and A2780cisR cells. Both triruthenium clusters were very potent (IC₅₀ < 10 µM) compared to cisplatin and Ru-based compounds on the whole, while the tetraruthenium clusters did not demonstrate substantial cytotoxicity. The pronounced difference observed between the Ru(III) and Ru(IV) cluster cations can be rationalized by their distinct supramolecular interactions with biomolecular environments. The triruthenium clusters exhibit an open hydrophobic cavity delineated by the three arene ligands, in conjunction with a hydrophilic oxo-cap that is predisposed to participate in hydrogen bonding. These complementary structural motifs facilitate specific supramolecular associations with aromatic systems and other functional groups within biomolecules. The same is not possible in the case of tetranuclear clusters. It is likely that these interactions are important in terms of their mode of action [105].

An alternative strategy has been confirmed to be relatively effective in formulating Ru–arene assemblies. This strategy includes stable binuclear clips, for instance, (p-cymene)₂Ru₂(oxalato)Cl₂, and bidentate linear ligands, such as 4,4′-bipyridine, 4,4′-bipyridylethylene, etc. The binuclear Ru(II) clips may be obtained by utilizing another tetradentate bridging ligand, for instance, N/O or O/O derivatives, allowing the development of ruthenium supramolecular assemblies, such as metallocycles or metallocages. In the last decades, this approach has been widely applied to produce numerous 2D and 3D biologically active metal-based assemblies.

Among the varied range of metallomacrocycles that incorporate diverse metallic centers, geometries, sizes, and functionalities for biomedical applications, ruthenium(II) metallacycles have attracted considerable attention due to their unique structural versatility and promising therapeutic potential. Owing to the favorable photophysical and redox properties of the Ru(II) center, as well as its ability to form stable coordination assemblies, these metallacycles have been extensively explored for both chemotherapeutic and phototherapeutic interventions in malignant diseases. A particularly effective synthetic approach involves the use of binuclear Ru–arene clip modules, which can be linked through appropriate pyridine-containing bidentate linear ligands to afford discrete, well-defined tetranuclear Ru(II) metallacycles with square geometries, as reported by Yan et al. [106]. The oxalato-based complexes [Ru₂(µ-η⁴-C₂O₄)Cl₂(η⁶-p-PrⁱC₆H₄Me)₂] and [Ru(η²-C₂O₄)(NH₃)(η⁶-p-PrⁱC₆H₄Me)] reacted with triphenylphosphine to give [Ru₂(µ-η⁴-C₂O₄)(PPh₃)₂(η⁶-p-PrⁱC₆H₄Me)₂][O₃SCF₃]₂ and [Ru(η²-C₂O₄)(PPh₃)(η⁶-p-PrⁱC₆H₄Me)]. The dichloro complex Ru₂(µ-η⁴-C₂O₄)Cl₂(η⁶-p-PrⁱC₆H₄Me)₂] was transformed to the cationic di-methanol compound [Ru₂(µ-η⁴-C₂O₄)(PPh₃)₂(η⁶-p-PrⁱC₆H₄Me)₂][O₃SCF₃]₂ which interacted with 4,4′-bipyridine to form the new tetranuclear metallomacrocycle [Ru₄(µ-η⁴-C₂O₄)₂(µ-η¹∶η¹-bipy)₂(η⁶-p-PrⁱC₆H₄Me)₄][O₃SCF₃]₄ with alternating oxalato- and 4,4′-bipyridine bridge ligands [96].

Han et al. have synthesized a series of metallarectangles, Figure 18a–d, through the coordination-driven self-assembly of an unsaturated binuclear Ru–arene clip with a range of linear bidentate pyridyl linkers. Binuclear complexes [(p-cymene)₂Ru₂(μ-CA)Cl₂] (CA = chloranilate) have been synthesized by the reaction of [(p-cymene)RuCl(μ-Cl)]₂ with H₂CA in a basic medium. Interaction of [(p-cymene)₂Ru₂(μ-CA)Cl₂] with bidentate ligands L such as pyrazine (prz), 4,4′-dipyridine (bpy), E-1,2-bis(4-pyridyl)ethene (bpe), and 2,5-bis(4-pyridyl)-1,3,5-oxadiazole (bpo), in the presence of AgOTf (OTf = CF₃SO₃) and CH₃OH, afforded the respective tetranuclear complexes [(p-cymene)₄Ru₄(μ-CA)₂(μ-L)₂](OTf)₄ (Figure 18a–d), correspondingly [107,108,109]. The single-crystal X-ray analysis of [(p-cymene)₄Ru₄(μ-CA)₂(μ-L)₂](OTf)₄ revealed that the ruthenium centers were connected by pyridyl ligands and bis–bidentate chloranilate (CA) ligands to construct the rectangular cavity with different dimensions and strong π-interactions between independent molecules to produce rectangle channels in solid state. The metallocycles exhibited excellent aqueous solubility and possessed positive charges, facilitating their noncovalent binding with DNA and contributing to their antineoplastic activity. The tetracationic bowl-shaped rectangle, illustrated in Figure 18c, has been described to display strong non-covalent interactions with calf-thymus DNA, attributed to the dimensional complementarity between the metallarectangle and the major grooves of DNA [110]. The binding between the positively charged metallarectangle and the negatively charged DNA backbone is predominantly governed by electrostatic interactions.

Building upon these preliminary studies, subsequent investigations into tetranuclear Ru-arene metallocycles have proliferated, focusing on their potential bioactive and antineoplastic properties. A series of such metallocyclic complexes were synthesized and systematically evaluated for their cytotoxic efficacy, as well as their interactions with DNA strands and protein targets. A set of ruthenium SCCs via [3 + 2] and [2 + 2] assembly have been reported (Figure 19) [111]. The set of [3 + 2] assembly with different biruthenium molecular clips, such as 2,5-dihydroxy-1,4-benzoquinone (dhbq, Figure 19a), 5,8-dioxydo-1,4-naphtaquinonato (donq, Figure 19b), and 6,11-dioxydo-5,12-naphtacenedionato (dotq, Figure 19c) and the ligand 1,3,5-tris(pyridine-4-ylethynyl) benzene, depicted in Figure 19d, have been synthesized [111]. The scheme for Ru-based SCCs via [3 + 2] assembly is presented in Figure 19e. The in vivo cytotoxic activity of the self-assemblies was assessed against liver SK-hep-1, cervix HeLa, colon HCT-15, lung A-549, and breast MDA-MB-231 cancer cell lines. The IC₅₀ values of the obtained SCCs were comparatively low for some complexes, particularly those containing 2,5-dihydroxy-1,4-benzoquinone and 6,11-dioxydo-5,12-naphtacenedionato ligands. The same authors have reported a set of [2 + 2] ruthenium SCCs based on the 4-pyridyl ligands (Figure 19f). The produced [2 + 2] Ru-based SCCs had rectangular geometry. The schematic presentation for Ru-based SCCs via [2 + 2] assembly is shown in Figure 19g. The cytotoxicity of these compounds has been evaluated against SK-hep-1 (liver), HeLa (cervical), HCT-15 (colon), and AGS (gastric) cancer cells. The mixed ruthenium assemblies exhibited high antineoplastic activity. It has been found that the presence of longer dipyridyl ligands promotes the formation of complexes with enhanced anticancer activity. Furthermore, both the geometries and sizes of these complexes significantly affect their biological performance [111].

Mishra et al. have synthesized ruthenium-based metallarectangles (Figure 20) by the interaction of various Ru–arene acceptors such as [Ru₂(p-cymene)₂(µ-η⁴-C₂O₄)Cl₂] (Figure 20a) and [Ru₂(p-cymene)₂(L)(Cl)₂] (Figure 20b–d), where L= 2,5-dihydroxy-1,4-benzoquinone (dhbq, Figure 20b), 5,8-dioxydo-1,4-naphtaquinonato (donq, Figure 20c), and 6,11-dioxydo-5,12-naphtacenedionato (dotq, Figure 20d) with a symmetrical donor ligand N,N′-bis(4-(pyridin-4-ylethynyl)phenyl)- terephthalamide [112]. The cytotoxicity of these metallarectangles was evaluated in human liver cancer SK-hep-1, gastric cancer AGS, and colorectal cancer HCT-15 cell lines. The studied metallarectangles exhibited greater cytotoxicity against all tested tumor cells compared to the recognized antitumor drugs doxorubicin and cisplatin. Moreover, exposure to the most active metallarectangle significantly upregulated the expression of the colorectal cancer suppressor genes APC and p53.

The same authors have described large metallarectangles via self-assembly of an unsymmetrical amide donor (N-(4-(pyridin-4-ylethynyl)phenyl)isonicotinamide) ligand and two different lengths of Ru–arene acceptors [Ru₂(μ-η⁴-C₂O₄)(MeOH)₂(η⁶-p-PrⁱC₆H₄Me)₂][O₃SCF₃]₂ and [Ru₂(p-cymene)₂(donq)(OH₂)₂ O₃SCF₃]₂, where donq is 5,8-dioxydo-1,4-naphthaquinonato. They tested the new complexes in vitro for anticancer potency against human colorectal Colo320, lung A549, breast MCF-7, and lung H1299 cancer cell lines. The anticancer efficacy of the studied metallarectangles was observed to be significantly better against all the tested cell lines than that of the reference cisplatin [113].

Moreover, by choosing linear or V-shaped dipyridines to bind the different binuclear Ru(II)–arene clips, a series of tetranuclear metallocycles have been fabricated and their corresponding antineoplastic activity has been explored [114,115]. Metallacycles of dicarboxylate-bridged Ru–arene precursors [Ru₂(μ-η⁴-OO∩OO)(η⁶-p-iPrC₆H₄Me)₂][CF₃SO₃]₂ (OO∩OO = oxalate, 2,5-dihydroxy-1,4-benzoquinonato (dobq), and 5,8-dihydroxy-1,4-naphthoquinonato (donq), reported in [114], effectively inhibited the growth in human colon HCT-15 and human gastric AGS cancer cell lines. The tetranuclear cationic metalla-bowls of bispyridine amide donor ligands, reported in [115], have the general formula [Ru₄(p-cymene)₄(N∩N)₂(OO∩OO)₂][CF₃SO₃]₄ (N∩N = 2,6-bis(N-(4-pyridyl-carbamoyl)pyridine, where OO∩OO = 2,5-dihydroxy-1,4-benzoquinonato, 5,8-dioxydo-1,4-naphthaquinonato, or hoxonato ligands. Consistent with prior investigations of Ru–arene complexes, the obtained metalla-bowls exhibited pronounced antiproliferative activity against human liver (SK-hep-1), gastric (AGS), and colorectal (HCT-15) cancer cells. The extent of growth inhibition observed surpassed that of the reference chemotherapeutic agents doxorubicin and cisplatin. In colorectal tumor cells, exposure to the most active metalla-bowl led to the upregulation of the tumor suppressor genes APC and p53, indicating a potential mechanistic basis for the observed cytotoxic effects. Furthermore, Co–Ru bimetallic rectangles have been demonstrated to act as new possible agents for the induction of apoptosis and autophagy in human cancer cell lines [116]. Collectively, these investigations have deepened the understanding of the bioactivity of tetranuclear Ru–arene metallocycles, highlighting their promising antineoplastic potential.

Recently, the use of ruthenium-based complexes as PDT agents has further enriched their therapeutic effectiveness. Tetranuclear arene–ruthenium(II) complex compounds comprising the porphyrin scaffold have also been considered for PDT as promising photosensitizing chemotherapeutics. The bioactivity of these tetranuclear arene–ruthenium(II) complexes was evaluated on human melanoma tumor cells, and their intracellular localization and cellular uptake were studied [117]. With 5,10,15,20-tetra(4-pyridyl)porphyrin (4-tpp) as a central unit, the derivatives of para-cymene and toluene [(η⁶-p-MeC₆H₄Prⁱ)RuCl₂]₄(4-tpp) and [(η⁶-C₆H₅Me)RuCl₂]₄(4-tpp) have been found to be very active against melanoma cell lines [117]. The tetranuclear ruthenium complexes exhibited excellent phototoxicity. Cellular localization and uptake studies of porphyrin arene–ruthenium(II) complexes [(η⁶-p-MeC₆H₄Prⁱ)RuCl₂]₄(4-tpp) and [(η⁶-C₆H₅Me)RuCl₂]₄(4-tpp) showed that the studied compounds accumulated in the melanoma cellular cytoplasm differently than lysosomes, representing a new class of promising photosensitizers capable of associating chemotherapeutic potency with phototherapeutic anticancer therapy. The same authors have demonstrated that the isomeric 5,10,15,20- tetra(3-pyridyl)porphyrin (3-tpp) derivatives of para-cymene and toluene [(η⁶-p-MeC₆H₄Prⁱ)RuCl₂]₄(3-tpp) and [(η⁶-C₆H₅Me)RuCl₂]₄(3-tpp) were very efficient at very low doses (5 µM) to induce antiproliferation of human Me300 melanoma cells [118]. The complexes displayed dual synergistic effects, integrating the favorable features of arene–ruthenium chemotherapeutic agents with those of porphyrin photosensitizers.

Mattsson et al. have described tetranuclear rectangular arene–ruthenium cationic complexes, Figure 21, combining 2,5-dihydroxy-1,4-benzoquinato (dbq) derivatives and a dipyridyl linker [119]. Cationic arene–ruthenium-based tetranuclear rectangulars have been obtained from the dinuclear arene–ruthenium complexes [Ru₂(arene)₂(OO∩OO)₂Cl₂] (arene = p-cymene, hexamethylbenzene; OO∩OO = 2,5-dihydroxy-1,4-benzoquinonato, 2,5-dichloro-1,4-benzoquinonato) by reaction with a bipyridine linker N∩N = E-1,2-bis(4-pyridyl)ethene in the presence of AgO₃SCF₃ in methanol, producing tetranuclear cations [Ru₄(arene)₄(N∩N)₂(OO∩OO)₂][CF₃SO₃]₄. The cytotoxicity of these water-soluble complexes has been evaluated on the human ovarian cancer A2780 cell line. The studied compounds were found to be potent against the tested cancer cells [119].

Motivated by the remarkable photophysical and photochemical characteristics of BODIPY, Gupta et al. have synthesized Ru metallorectangles containing a BODIPY-based dipyridine linker and evaluated their antineoplastic potential [120]. Some of the compounds displayed very selective antineoplastic potency and interacted strongly with DNA and proteins. Among them, the ruthenium metallorectangle, depicted in Figure 22, showed improved cytotoxicity against several tumor cell lines compared to that of cisplatin, e.g., lung A549 (IC₅₀ = 3.09 ± 0.17 μM), breast MCF-7 (IC₅₀ = 2.72 ± 0.21 μM), cervical HeLa (IC₅₀ = 4.31 ± 0.22 μM), and brain U87 (IC₅₀ = 0.24 ± 0.06 μM) cancer cell lines. The characteristic green fluorescence of the BODIPY ligand and associated aggregation-induced emission (AIE) allowed imagining of the compounds inside the cells using confocal microscopy.

To further enhance the biological performance of Ru-based supramolecular rectangles, the same research group recently reported a new series of Ru (2 + 2) assemblies constructed from thiophene-functionalized dipyridyl BODIPY ligands [121]. Confocal laser scanning microscopy analyses indicated that the obtained rectangles mainly localized in the cellular cytoplasm. Interestingly, the incorporation of the thiophene moiety into the BODIPY framework resulted in a pronounced enhancement of both cytotoxic activity and selectivity toward cancer cells, underscoring the critical role of ligand functionalization in modulating biological behavior. The complexes displayed dose-dependent cytotoxic activity against various tumor cell lines. The cytotoxicity of the studied rectangles showed selective activity against MCF-7 breast cancer cells while showing reduced toxic effects toward human non-malignant fibroblast WI38 cell lines. Moreover, the investigation revealed that cervical tumor cells also responded favorably to these compounds. The net fluorescence attributable to BODIPY permitted the visualization of their position inside tumor cells. Furthermore, the metallarectangles exhibited a considerable tendency to bind with biological macromolecules. Although previous binding studies have revealed substantial interactions between these metallorectangles and biomacromolecules such as DNA and proteins, additional investigations were warranted to elucidate their potential as photosensitizing agents in photodynamic therapy (PDT). Recently, Xu et al. have obtained a new Ru(II) metallacycle with a similar structure as that shown in Figure 22, by the inclusion of aza-BODIPY as a NIR-II fluorescence emitter. The constructed Ru(II) metalacyclic compound was able to emit over 1000 nm and to achieve precise NIR-II fluorescence for chemo- and phototherapy [122]. Its antineoplastic capability, assessed against lung (A549), cervix (Hela), and liver (HepG2) tumor cells, was notable in cisplatin-resistant cells with low toxic effects on healthy cells. In addition to its inhibitory effect on cell proliferation, the antimetastatic properties of the Ru(II) metallacycle were also investigated, and significant inhibition of the migration and invasion of tumor cells has been observed [122].

Ruthenium hexanuclear prismatic and octanuclear cubic metallocages are also of great interest. Water-soluble Ru–arene prismatic metallocages have demonstrated exceptional potential as drug delivery systems for encapsulating lipophilic antineoplastic agents in anticancer treatment. Therrien et al. have obtained a hexanuclear cation prismatic metallocage [Ru₆(p-iPrC₆H₄Me)₆(tpt)₂-(dhbq)₃](O₃SCF₃)₆, which incorporated ruthenium p-cymene building blocks, bridged by 2,5-dihydroxy-1,4-benzoquinonato (dhbq) ligands with triangle-type pyridyl linkers, such as 2,4,6-tris (pyridin-4-yl)-1,3,5-triazine (tpt) [123]. The obtained metallocage [Ru₆(p-iPrC₆H₄Me)₆(tpt)₂-(dhbq)₃](O₃SCF₃)₆ acted as a carrier, and its high charge may have promoted enhanced uptake in tumor cells. Many isostructural metalloprisms with slight modifications in the bridge ligands have been obtained in a similar approach, for instance, by using 5,8-dioxido-1,4-naphthoquinonato (donq) as a bridge ligand of ruthenium clips, which led to more spacious metalloprisms [124]. Cationic Ru–arene metalloprisms [Ru₆(p-cymene)₆(tpt)₂(OO∩OO)₃](O₃SCF₃)₆, where OO∩OO represents 9,10-dioxo-9,10-dihydroanthracene-1,4-diolato and 6,11-dioxo-6,11-dihydronaphthacene-5,12-diolato, have been synthesized from the corresponding binuclear Ru–arene complexes [Ru₂(p-cymene)₂(OO∩OO)Cl₂] via reaction with 2,4,6-tris (pyridin-4-yl)-1,3,5-triazine (tpt) [124].

On the other hand, with the substitution of the tridentate tpt derivatives with larger tridentate ligands, e.g., 1,3,5-tris{2-(pyridin-4-yl)vinyl}benzene (pvb), larger hexa- and octanuclear cages can be successfully produced [125]. Cationic Ru–arene assemblies [Ru₆(p-cymene)₆(tris-pvb)₂(μ₂-Cl)₆][CF₃SO₃]₆, [Ru₆(p-cymene)₆(tris-pvb)₂(OO∩OO)₃][CF₃SO₃]₆ (tris-pvb = 1,3,5-tris{2-(pyridin-4-yl)vinyl}benzene), and [Ru₈(p-cymene)₈(NN∩NN)₂(OO∩OO)₄][CF₃SO₃]₈ were successfully synthesized through coordination-driven self-assembly. The tritopic ligand 1,3,5-tris{2-(pyridin-4-yl)vinyl}benzene (tris-pvb) and the tetratopic ligands 1,2,4,5-tetrakis{2-(pyridin-4-yl)vinyl}benzene and 1,2,4,5-tetrakis{2-(pyridin-4-yl)ethynyl}benzene (NN∩NN) acted as bridging linkers to promote the formation of discrete, multinuclear architectures. These assemblies were obtained via the reaction of the corresponding binuclear Ru–arene precursors, [Ru₂(p-cymene)₂(μ-Cl)₂Cl₂] and [Ru₂(p-cymene)₂(OO∩OO)Cl₂], with the appropriate multidentate ligands under mild conditions [125]. The OO∩OO bridging ligands employed include oxalato, 2,5-dioxido-1,4-benzoquinonato, 2,5-dichloro-1,4-benzoquinonato, 5,8-dioxido-1,4-naphthoquinonato, 5,8-dioxido-1,4-anthraquinonato, and 6,11-dioxido-5,12-naphthacenedionato, which impart distinct electronic and steric characteristics to the resulting assemblies. Variation of these bridging units allows fine-tuning of the redox properties and overall topology of the supramolecular frameworks. The resulting cationic species, stabilized by the η⁶-bound p-cymene ligands, display well-defined geometries characteristic of arene–ruthenium coordination-driven assemblies [125]. The large cationic metalla-assemblies allow encapsulation of various guest molecules [67,68,69,70,71,72]. The exceptional structural tunability of structures and molecular encapsulation characteristics toward aromatic planar guests, combined with the promising bioactivity of Ru–arene octanuclear and hexanuclear prismatic metallocages, highlight them as potential drug delivery vectors with remarkable selectivity for tumor cells [126,127,128].

Octanuclear cubic metallocages represent a class of attractive organometallic chemotherapeutics. Ru–arene octanuclear cubic metallocages can be conveniently assembled through the combination of appropriate Ru–arene clips and tetrapodal panel donors, e.g., the photoactive ligand tetrapyridyl porphyrin (tpp). Therrien et al. have synthesized a series of porphyrin-based organometallic cubes [129,130]. Some octanuclear ruthenium p-cymene and rhodium and iridium pentamethylcyclopentadienyl metalla-assemblies have been synthesized via coordination-driven self-assembly of tetrapyridyl porphyrins (tpp) with binuclear clips, (η⁶-MeC₆H₄Prⁱ)₂Ru₂(μ₄-C₆HRO₄)Cl₂ (R = C₁₁H₂₃). All complexes displayed potent antiproliferative activity against MCF-7, B16, and A549 cancer cell lines, with IC₅₀ values of about 0.1 μM, while showing similar effects in the non-tumorigenic NIH 3T3 cell line [129]. Ruthenium-based assemblies containing tetrapyridylporphyrins have been evaluated as photosensitizers (in photodynamic therapy) [130]. To further expand the development of new discrete ruthenium(II) octanuclear cubic cages with significant antineoplastic activity, Adeyemo et al. have recently obtained Ru(II) cubic cages constructed from tetradentate pyridyl ligands linked to various binuclear Ru(II) acceptor clips [131,132]. These novel octanuclear Ru(II) cages were prepared through coordination-driven self-assembly between a tetradentate pyridyl ligand, N,N,N′,N′-tetra(pyridin-4-yl)benzene-1,4-diamine, and different binuclear p-cymene Ru(II) acceptors: [Ru₂(μ–η⁴-C₂O₄)(CH₃OH)₂(η⁶-p-cymene)₂](O₃SCF₃)₂, [Ru₂(μ–η⁴-C₆H₂O₄)(CH₃OH)₂(η⁶-p-cymene)₂](O₃SCF₃)₂, [Ru₂(dhnq)(H₂O)₂(η⁶-p-cymene)₂](O₃SCF₃)₂, and [Ru₂(dhtq)(H₂O)₂(η⁶-p-cymene)₂](O₃SCF₃)₂. The octanuclear self-assembled cages have demonstrated potent in vitro antineoplastic activity against human lung adenocarcinoma A549 and human cervical cancer HeLa cells, compared to cisplatin. Subsequent studies exploring the mechanism of action indicated that the observed cell death resulted from the generation of reactive oxygen species [132].

5. Conclusions

Ruthenium complex compounds have demonstrated substantial promise as antineoplastic agents with potentially reduced toxic side effects owing to their high selectivity against tumor cells. Ru-based complexes have demonstrated the capacity to associate with a broad spectrum of biological macromolecules, notably proteins and DNA.

While the predominant studies have focused on mononuclear ruthenium complexes, the potential of polynuclear Ru-based compounds remains rather underexplored. Despite the achieved promising prospects of these multifunctional drug candidates, some key issues of the currently known Ru polynuclear complexes still restrict their prevalent application. Some important aspects are still understudied: (1) detailed mechanistic studies on the possible mechanisms of action of homometallic ruthenium complexes are still scarce; (2) the SAR of these compounds remains basically unclear and necessitates further exploration; (3) their interactions with different targeting biomolecules are at their preliminary stage; (4) the in vivo experiments should be enhanced and systematically studied. Solving these issues is important and would lead to better design strategies for the development of new metallodrugs.

Significant approaches to increase the anticancer profile of structurally different polynuclear ruthenium complexes and organometallics are the incorporation of appropriate biologically active ligands with therapeutic values and the improvement of specific host–guest characteristics of Ru-based metalla-assemblies to enable better activity and selectivity, novel mechanisms of action, low toxicity, and modulated pharmacokinetic profiles. Through the functionalization of the obtained polynuclear ruthenium candidates, new targeting organelles could be revealed, contributing to the development of agents with a wide range of biological and medicinal applications. The reviewed findings and results clearly indicate that this field of drug discovery definitely needs further active exploration in the future in order to obtain alternative classes of effective anticancer chemotherapeutic and theranostic agents.