Ruthenium coordination compounds of biological and biomedical significance. DNA binding agents

Coordination Chemistry Reviews 376 (2018) 75–94

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Coordination Chemistry Reviews journal homepage: www.elsevier.com/locate/ccr

Review

Ruthenium coordination compounds of biological and biomedical significance. DNA binding agents Viktor Brabec ⇑, Jana Kasparkova Institute of Biophysics, Czech Academy of Sciences, Kralovopolska 135, CZ-61265 Brno, Czech Republic

a r t i c l e

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Article history: Received 26 May 2018 Received in revised form 6 July 2018 Accepted 15 July 2018 Available online 14 August 2018 Keywords: Ruthenium DNA binding Photoactivation Radiosensitization Cytotoxicity

a b s t r a c t Ruthenium complexes exhibit a broad variety of biological and biomedical activities including anticancer efficiency. The reason is that the octahedral bonding of both Ru(II) and Ru(III) complexes affords an extensive repertoire of three-dimensional architectures, giving the potential for a high degree of site selectivity for binding to their biological targets. The mechanism of biological and biomedical action of ruthenium compounds is connected with their interactions with biomacromolecules. A lot of mechanistic studies revealed disposition of many ruthenium complexes to operate via mechanisms of action involving interactions with DNA, but distinctly different from those of the approved platinum anticancer drugs. In this Review, we discuss major DNA binding modes hitherto identified for ruthenium complexes of biological or biomedical significance and provide some typical examples. The introduction provides the reader with a brief overview of the approaches in the search for the new, transition metal-based agents of biological or biomedical significance to provide the context in which more recent research of DNA binding of ruthenium complexes has been evolved. We then describe main categories of binding modes between DNA and ruthenium compounds, such as coordinative, intercalative, minor groove binding, sequence specificity of DNA binding, the ability of ruthenium compounds to condense and cleave DNA, binding to A- and Z-DNA, DNA quadruplexes and other unusual DNA structures. A number of complexes based on ruthenium(II) centers have been reported to have excellent photophysical and radiosensitizing properties so that DNA photocleavage and synergistic enhancement of DNA damage due to a combined action of ruthenium complexes and ionizing radiation is also discussed. Finally, inhibition of DNA processing enzymes is reviewed as well. We believe that such a synthesis of disparate DNA binding modes of ruthenium complexes will help to generate new ruthenium complexes of improved biological and biomedical significance. Ó 2018 Elsevier B.V. All rights reserved.

Contents 1. 2.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Types of DNA binding modes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Coordinative binding. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.1. Dimethyl sulfoxide complexes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abbreviations: 2,3-dpp, 2,3-bis(2-pyridyl)pyrazine; 2-appt, 2-amino-4-phenylamino-6-(2-pyridyl)-1,3,5-triazine; apip, 2-(2-aminophenyl)imidazo[4,5-f][1,10]phenanthroline; bnbp, 2,6-bis-(6-nitrobenzimidazol-2-yl)pyridine; bpm, 2,20 -bipyrimidine; bpy, 2,20 -bipyridine; dppz, dipyrido[3,2-a:20 ,30 -c]phenazine; dppb, 1,4-bis(diphenylpho sphino)butane; dpq, dipyrido[3,2-d:20 ,3-f]quinoxaline; en, ethylenediamine; EtBr, ethidium bromide; iip, isoindolylimidazo[4,5,f][1,10]phenanthroline; Im, imidazole; hpip, 2-(2-hydroxyl-5-aminophenyl)imidazo[4,5-f][1,10]phenanthroline; Ind, indazole; ip, imidazo[4,5-f][1,10]phenanthroline; Me2bpy, 4,40 -dimethyl-2,20 -bipyridine; NAMI, Na [trans-Ru(III)((DMSO)Cl4(Im)]; NAMI-A, (H2Im)[trans-Ru(DMSO)Cl4(Im)]; nip, benzo[f]isoindole[4,5,f][1,10]phenanthroline; PARP, poly ADP ribose polymerase; phen, 1,10phenanthroline; pip, 2-phenylimidazo[4,5-f][1,10]phenanthroline; pipsh, 2-(4-benzothiazolyl)phenylimidazo[4,5-f][1,10]phenanthroline; p-terp, paraterphenyl; RAPTA-C, [(g6-p-cymene)Ru(1,3,5-triaza-7-phosphaadamantane)Cl2]; ROS, reactive oxygen species; Ru-CYM, [(g6-arene)Ru(II)(en)(Cl)]+ (arene = p-cymene); Ru-THA, [(g6-arene)Ru(II) (en)(Cl)]+ (arene = tetrahydroanthracene); SpymMe2, 4,6-dimethyl-2-mercaptopyrimidine anion; tatpp, 9,11,20,22-tetraazatetrapyrido[3,2-a:20 ,30 -c:300 ,200 -1:200 ,3000 0 -n]pentacene; tBu2bpy, 4,40 -di-tert-butyl-2,20 -bipyridine; tpphz, tetrapyridophenazine. ⇑ Corresponding author. E-mail address: [email protected] (V. Brabec). https://doi.org/10.1016/j.ccr.2018.07.012 0010-8545/Ó 2018 Elsevier B.V. All rights reserved.

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3. 4. 5.

6. 7.

V. Brabec, J. Kasparkova / Coordination Chemistry Reviews 376 (2018) 75–94

2.1.2. Heterocyclic complexes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.3. Arene complexes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.4. Dinuclear complexes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Intercalative binding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.1. Polypyridyl complexes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Minor groove binding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. Sequence specificity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5. DNA condensation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.6. Binding to A- and Z-DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.7. Binding to DNA quadruplexes and telomers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.8. Binding to other unusual DNA structures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.8.1. Binding to DNA bulges . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.8.2. Binding to DNA mismatches and abasic sites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.9. DNA cleavage. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Photoactivation and DNA photocleavage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Radiosensitization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Inhibition of DNA processing enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. Inhibition of topoisomerases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2. Inhibition of DNA transcription and DNA synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Summary and outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Medicinal coordination chemistry is now established as an acknowledged area of the interdisciplinary research. A key theme of this research has been to develop the fundamental principles that enable the design of effective anticancer metal-containing chemotherapeutic agents. Early research work on anticancer coordination compounds was concerned with the most widely used anticancer chemotherapeutics and successful platinum drugs, cisplatin, carboplatin, and oxaliplatin, approved worldwide for treating human tumors and additional three, heptaplatin, lobaplatin and nedaplatin approved for clinical use to treat cancer in humans in specific countries. However, there are several clinical problems relating to current platinum anticancer drugs which demand improvements in design. The first is drug resistance which can develop after repeated treatment, second, poor activity against some types of cancer (e.g., colon, lung and pancreatic cancer) and thirdly the occurrence of side-effects. These drawbacks have been the impetus for the development of improved anticancer drugs derived from not only platinum complexes but also other transition metal-based complexes. Notably, despite the intense research activity into new metal-based antitumor compounds, a new transition-metal based compound has not been hitherto approved. Broadening the spectrum of antitumor metallodrugs depends on understanding the mechanism of action of existing and new candidate antitumor agents with a view toward developing new modes of attack. It is therefore of great interest to understand details of molecular and biochemical mechanisms underlying the biological efficacy of the platinum compounds and other transition metal-based complexes. There is good evidence [1] that the preferential pharmacological target for platinum diammine drugs such as cisplatin and its clinically used derivatives in cancer cells is nuclear DNA, which is a molecule that contains the instructions an organism needs to develop, live and reproduce. The nitrogen N7 atom of the guanine residue is the most electron-dense site in DNA and is accessible in its major groove. Cross-linking of two adjacent purines leads to kinking of the DNA, recognition by high mobility group and other proteins, triggering apoptosis and cell death. In addition, it has been shown that: (i) there is a correlation between the antitumor activity of platinum compounds and their capability to induce in DNA a certain sort of conformational and other alter-

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ations; (ii) this correlation may be exploited for simple screening of new platinum complexes for antitumor activity in the search for new antitumor platinum drugs; (iii) this concept has already led to the synthesis of several new platinum antitumor compounds that violate the original structure–activity relationships. One approach in the search for the new, metal-based anticancer agent which would exhibit antitumor activity markedly different from that of cisplatin and its direct analogs is to examine complexes that would contain another transition metal. Possible advantages in using transition-metal ions other than platinum may involve additional coordination sites, alterations in ligand affinity and substitution kinetics, changes in oxidation state and photodynamic approaches to therapy [2]. In the design of these new drugs, ruthenium complexes have raised great interest [2–4]. The octahedral bonding of both Ru(II) and Ru(III) complexes affords an extensive repertoire of three-dimensional architectures, giving the potential for a high degree of site selectivity and implementation of favorable pharmacological attributes [5]. The pharmacological target for many antitumor ruthenium compounds has not been univocally identified although a number of antitumor ruthenium compounds have been found to inhibit DNA replication, exhibit mutagenic activity, induce SOS repair mechanism, bind to nuclear DNA including unusual DNA structures and reduce RNA synthesis, which is consistent with DNA binding of these compounds in vivo. Moreover, several ruthenium complexes have unequivocally been shown to have genomic DNA as their pharmacological target [6–9], and many ruthenium compounds are known to have high selectivity for binding to DNA [10–13]. Thus, also by analogy with platinum antitumor drugs DNA interactions of antitumor ruthenium agents are of great interest as well because understanding the cellular DNA damage responses to lesions generated by ruthenium complexes may considerably contribute to design and development of DNA-binding ruthenium agents. Since several review articles appeared recently [9,14–19] in which pharmacological properties of antitumor ruthenium compounds have been discussed, the present article is mainly focused on variety of DNA interactions of ruthenium complexes of biological or biomedical significance which result in novel lesions in DNA so that their origin may be connected with their biological (antitumor) effects or their use as new probes, and imaging or diagnostic agents. In this Review, we discuss major DNA binding modes

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hitherto identified for ruthenium complexes of biological or biomedical significance and provide some typical examples demonstrating each DNA binding mode of ruthenium complexes highlighted in this communication. If the sufficient experimental data is available, the relationship of the DNA interactions of the discussed ruthenium complexes to their biological activity has also been shortly mentioned. 2. Types of DNA binding modes Binding mode of the metallodrugs including ruthenium complexes with DNA may illustrate insight into the mechanism of action and effectiveness of these drugs [20]. Variety of physicochemical properties of ruthenium coordination compounds and the chemical and conformational polymorphism of nucleic acids offer a considerable amount of the modes by which ruthenium complexes can bind DNA or RNA (Fig. 1). There are two main categories of binding modes between DNA and ruthenium compounds: coordinative and noncoordinative binding. The coordinative binding is connected with the fact that the electrondeficient metal atoms in these complexes might act as electron acceptors for electron-rich DNA nucleophiles by the hydrolysis of ligands. The coordinative binding is irreversible and forms adducts consisting of DNA and Ru(II) complexes. Organometallic ruthenium complexes can bind to DNA via interaction with aromatic ligands via intercalation between DNA base pairs, groovebinding, molecular recognition based on shape and size, H-bonds, electrostatic interactions and hydrophobic forces [21,22]. Thus, ruthenium complexes of biological significance may contain more functional groups and can act and interact with DNA in a manner which combines two or more DNA binding modes [23]. As a result, they can distort DNA conformation in a different manner and extent providing a number of alternatives for diverse downstream processes leading to various mechanisms of tumor cell death. The aim of the following parts of this article is to provide an overall summary of the DNA binding modes of ruthenium complexes hitherto investigated as biologically significant agents. Rather than review all ruthenium complexes that bind DNA, we have chosen to focus on some typical examples of ruthenium complexes that display a particular type of DNA binding mode or their combinations. 2.1. Coordinative binding Transition metal-containing compounds are coordination complexes. Coordination refers to the ‘‘coordinate covalent bonds” between the ligands and the central atom. Antitumor ruthenium complexes can interact irreversibly with DNA via coordination

Fig. 1. Schematic representation of DNA binding modes of ruthenium complexes.

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bonds formed between ruthenium centers and base (Fig. 1), sugar residues and also phosphodiester backbone. 2.1.1. Dimethyl sulfoxide complexes Dimethyl sulfoxide complexes of both ruthenium(II) and ruthenium(III) exhibit cytotoxic activity comparable to cisplatin at an equitoxic dosage in animal models of metastasizing tumors, but with less severe side effects and prolonged host survival times [4]. Cis- and trans-[Ru(II)((CH3)2SO)4Cl2] (Fig. 2a,b) bind to DNA in cell-free media [24–26]. The DNA-binding mode of trans-[Ru (II)((CH3)2SO)4Cl2] includes the formation of 1,2-intrastrand cross-links and a small amount (1%) of interstrand cross-links. Cis isomer forms mainly monofunctional lesions on natural DNA. Both ruthenium isomers induce conformational alterations of nondenaturational character in DNA, with the trans compound being more effective. In addition, DNA adducts of trans-[Ru(II) ((CH3)2SO)4Cl2] are capable of inhibiting RNA synthesis by DNAdependent RNA polymerases, while the adducts of the cis isomer are not. Thus, several features of the DNA-binding mode of trans[Ru(II)((CH3)2SO)4Cl2] are similar to those of antitumor cisplatin [26], which may be relevant to biological effects of this antitumor ruthenium drug. On the other hand, the different DNA-binding mode of cis isomer is consistent with its less pronounced biological effects. In addition, another ruthenium complex of this series, Ru (II)(C6H6)(DMSO)Cl2 (Fig. 2c), also binds irreversibly to DNA, but without affecting its conformation. 2.1.2. Heterocyclic complexes Heterocyclic complexes of ruthenium(III), the general formula of which is (HB)[Ru(III)B2Cl4], where B stands for a heterocyclic base, such as imidazole (Im) or indazole (Ind) constitute another group of potential anticancer compounds [2,3]. Ruthenium(III) complexes are more inert toward ligand exchange reactions than ruthenium (II) compounds so that they need to be activated by reduction to ruthenium(II) [27]. Thus, anticancer ruthenium(III) complexes belong to pro-drugs which are reduced to the more reactive ruthenium(II) derivatives capable of coordinating with DNA and other biologically significant molecules [28]. The representative of this class of ruthenium cytostatics, (HInd)[Ru(III)Cl4(Ind)2] (KP1019, Fig. 2d) is highly active against colorectal tumor cells and is completely devoid of side effects and drug-induced lethality at therapeutically relevant doses [3]. (HInd)[Ru(III)Cl4(Ind)2] interacts with DNA forming cross-links or inducing strand breaks [29]. Interesting heterocyclic complexes of ruthenium(III) are also Na [trans-Ru(III)((DMSO)Cl4(Im)] (NAMI, Fig. 2e) and the corresponding imidazolium salt, (H2Im)[trans-Ru(DMSO)Cl4(Im)] (NAMI-A, Fig. 2f). The discovery of antimetastatic properties of NAMI-A in animal models at nontoxic dosages is unprecedented, and notably, NAMI-A is the only Ru compound to have reached clinical trials [27]. DNA binding of the complexes (HIm)[Ru(III)Cl4(Im)2], (HInd)[Ru(III)Cl4(Ind)2] and NAMI were investigated in detail in a cell-free medium [30]. They bind irreversibly to DNA, NAMI binds to DNA considerably faster than other two ruthenium compounds and cisplatin. In addition, when NAMI binds to DNA, it exhibits an enhanced base sequence specificity in comparison with other two ruthenium complexes. NAMI also forms on double-helical DNA bifunctional intrastrand adducts capable of terminating RNA synthesis in vitro while the capability of other two ruthenium compounds to form such adducts is markedly lower. This observation suggests that the bifunctional adducts of (HInd)[Ru(III)Cl4(Ind)2] and (HIm)[Ru(III)Cl4(Im)2] formed on rigid double-helical DNA are sterically more crowded by their octahedral geometry than those of NAMI. In addition, the adducts of all three heterocyclic ruthenium compounds affect the conformation of DNA, NAMI being most effective [30].

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Fig. 2. Selected ruthenium complexes that bind DNA coordinatively.

NAMI-A is also capable of binding to DNA and RNA, both in vitro and in cellulo [31–33]. However, such interactions occur at physiologically irrelevant concentrations so that DNA binding of NAMI-A has not been considered as a decisive factor responsible for the antimetastatic properties of this ruthenium(III) complex [34–36]. On the other hand, some results demonstrate that cytotoxicity of NAMI-A is correlated with DNA binding in four different human tumor cell lines. 2.1.3. Arene complexes Monofunctional Ru(II) complexes of the type [(g6-arene)Ru(II) (en)X]+ (arene = benzene or substituted benzene, en = ethylenedia mine, X = halide) (Fig. 2g) were shown to be effective inhibitors of the growth of cancer cells and form strong monofunctional adducts with DNA [37]. One of these complexes, [(g6-p-cymene) Ru(en)Cl]PF6 (Fig. 2h) was shown to bind after hydrolysis strongly,

selectively and coordinatively to guanine bases on DNA oligonucleotides. Interestingly, these monofunctional ruthenium(II) complexes did not inhibit topoisomerases. Later studies demonstrated that the (g6-arene)Ru(II) p bonds in the monofunctional [(g6-arene)Ru(II)(en)(Cl)]+ complexes are inert toward hydrolysis, but the chloride ligand is readily lost, and the complex is transformed into the corresponding more reactive, aquated species [38]. It has also been shown that in cell-free media ethylenediamine Ru(II) arene compounds, in which arene = biphenyl, dihydroanthracene, tetrahydroanthracene, p-cymene, or benzene, coordinatively bind preferentially to guanine residues in natural mammalian, double-helical DNA. In addition, DNA binding of the complexes containing biphenyl, dihydroanthracene, or tetrahydroanthracene ligands can involve combined coordination to guanine N7 and noncovalent, hydrophobic interactions between the arene ligand and DNA, which may include arene intercalation

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and minor groove binding [39,40]. In contrast, the single hydrocarbon rings in the p-cymene and benzene ruthenium complexes cannot interact with double-helical DNA by intercalation [40]. It has been suggested that the different character of conformational alterations induced in DNA as a consequence of its global modification, and the resulting thermal destabilization, may affect differently further ‘‘downstream” effects of damaged DNA [40] and consequently may result in different biological effects of this class of metal-based antitumor compounds. The following work was focused on the activity of two Ru(II) arene complexes from the [(g6-arene)Ru(II)(en)(Cl)]+ family (arene = tetrahydroanthracene and p-cymene, Ru-THA, and RuCYM, respectively) in two tumor cell lines, conformational distortions induced by monofunctional adducts of these complexes, and their recognition by DNA binding proteins and repair, i.e., the most important factors that modulate the antitumor effects of related platinum drugs [41]. These two ruthenium complexes modify DNA differently: one that may interact with DNA by intercalation (tricyclic-ring Ru-THA), and the other (mono-ring RuCYM) that cannot. The presence of the arene ligand in this class of ruthenium complexes capable of noncovalent, hydrophobic interaction with DNA considerably enhanced cytotoxicity in several tumor cell lines. An analysis of DNA duplexes modified by Ru-THA and Ru-CYM revealed substantial differences in the impact of their monofunctional adducts on the conformation and thermodynamic stability of DNA and DNA polymerization in vitro. In addition, the adducts of Ru-CYM are removed from DNA more efficiently than those of Ru-THA. Interestingly, the adducts of Ru (II) arene compounds (in contrast to those of cisplatin and its derivatives) are preferentially removed from DNA by mechanisms other than nucleotide excision repair. This provided additional support for a mechanism underlying antitumor activity of Ru(II) arene compounds different from that of conventional cisplatin. Hence, the character of DNA distortion induced in DNA by the adducts of Ru(II) arene complexes and the resulting thermodynamic destabilization of DNA very likely control the biological effects of this class of ruthenium complexes. The tethered Ru(II) half-sandwich complex [(g6:g1-C6H5 (CH2)3NH2)Ru(NO3)2] was shown to bind to mammalian DNA coordinatively, forming mainly monofunctional DNA adducts incapable to inhibit DNA transcription [42].

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The molecular targeting characteristics of two prototypical and related ruthenium-arene agents – the cytotoxic antiprimary tumor compound Ru-CYM (Fig. 2h) and the relatively non-cytotoxic antimetastasis compound [(g6-p-cymene)Ru(1,3,5-triaza-7-phos phaadamantane)Cl2] (RAPTA-C) (Fig. 2i) were investigated [5]. Both complexes were found to form the chromatin-bound adducts in cancer cells treated with these ruthenium complexes. Notably, Ru-CYM preferentially targeted the DNA of chromatin forming coordinative bonds between ruthenium and N7 atoms of guanine residues. Ru-CYM formed these bonds at the most distorted regions within the nucleosome core producing monofunctional adducts that result in a much more subtle distortion of the DNA in comparison with that resulting from the formation of the major DNA adducts of conventional cisplatin. These differences in conformational alterations in the double helix have been proposed to underlie the lower nucleotide excision repair efficiency observed for Ru-CYM relative to cisplatin adducts in vitro [41] and are also likely to yield differential effects on transcription factor binding and polymerase activity. Thus, these substantial distinctions in DNA-targeting potential, as well as localization and structure of the DNA adducts that form in the genome, may account for differences in mode of cell death between Ru-CYM and cisplatin by influencing adduct recognition and repair. In contrast, RAPTA-C appears to be primarily associated with the protein component of chromatin forming coordinative bonds between ruthenium and a glutamate carboxylate oxygen. The further studies indicate that the steric bulk of the phosphaadamantane ligand is the primary factor that distinguishes the histone/DNA site selectivity behavior between RAPTA-C and Ru-CYM [5]. 2.1.4. Dinuclear complexes A successful platform that has been exploited in the design of ruthenium compounds that interact irreversibly with DNA via coordination bonds comprises dinuclear arene Ru(II) complexes [43,44]. Typical examples of this class od ruthenium compounds are water-soluble dinuclear Ru(II) arene complexes, in which two {(g6-p-isopropyltoluene)RuCl[3-(oxo-jO)-2-methyl-4-pyridin onato-jO4]} units are linked by flexible chains of different length [(CH2)n (n = 4, 6, 8, 12)] (Fig. 3a) [45]. These dinuclear ruthenium drugs exert promising cytotoxic effects in human cancer cells

Fig. 3. Examples of dinuclear ruthenium complexes that interact irreversibly with DNA via coordination bonds.

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and bind DNA forming intrastrand and interstrand cross-links in one DNA molecule. An intriguing aspect of the DNA-binding mode of these dinuclear Ru(II) compounds is that they can crosslink two DNA duplexes and also proteins to DNA - a feature not observed for other antitumor ruthenium complexes. Thus, these results support the view that the concept for the design of interhelical and DNAprotein cross-linking agents based on dinuclear Ru(II] arene complexes with sufficiently long linkers between two Ru centers may result in new compounds which exhibit a variety of biological effects and can also be useful in nucleic acids research. Another example of dinuclear Ru(II) complexes that bind DNA coordinatively involves p-cymene di-ruthenium(II) complexes ([(g6-p-cymene)2Cl2Ru2X], with X = naphthazarin or quinizarin bridging ligands) (Fig. 3b,c) [46]. These complexes undergo aquation, forming the aqua-derivatives [(g6-p-cymene)2(H2O)2Ru2X]2+. These can further bind coordinatively to DNA via interstrand crosslinking, through both Ru centers and two N7 sites of consecutive guanines, to give [(g6-p-cymene)2(DNA1,2)Ru2X] complexes, by a mechanism similar to that of cisplatin; the formation of these interstrand cross-links leads to bending of DNA. The dinuclear complexes reduce the resistance factor and increase the selectivity toward tumor cells of both bridging ligands. 2.2. Intercalative binding In molecular biology, intercalation is defined as a process of the insertion of planar molecules of an appropriate size and chemical nature between the adjacent base pairs of double-helical DNA (Fig. 1) with concomitant unwinding and lengthening of the DNA helix. DNA-intercalator complexes are stabilized via van der Waals forces, hydrogen bonding, hydrophobic charge transfer and electrostatic complementarity [47,48]. Intensively studied DNA intercalators also include metallointercalators because the covalent attachment of organic intercalators to transition metal coordination complexes, yielding metallointercalators, can lead to novel DNA interactions that influence biological activity [23]. The frequently used metal ion in these DNA intercalators is also ruthenium because its complexes are not prone to decomposition in the biological milieu. To probe DNA intercalative binding modes of small DNA binding molecules, a fluorescent compound ethidium bromide (EtBr) is often used. EtBr intercalates DNA, the displacement of EtBr (quantified by fluorescence) by the titration of a compound has been often used as the only evidence of an intercalative binding. However, EtBr also binds DNA through interactions with the minor groove so that the displacement of EtBr by the titration of a compound can also be indicative of minor groove binding without intercalation. Thus, standard fluorescence methods such as intensity measurements, polarization, and solute quenching studies can reliably detect an interaction of the investigated compound with DNA, but they are of no use in distinguishing the different modes of DNA binding [49]. Therefore, we will only focus in this subsection on such ruthenium complexes for which intercalative DNA binding mode has been inferred on the basis of highresolution structural data or indirectly from various solution studies employing reliable criteria making it possible to distinguish intercalation from groove binding. 2.2.1. Polypyridyl complexes Ruthenium(II) polypyridyl complexes have been shown to exert interesting biological properties based on their excellent reactivity, imaging capability, binding ability, and redox chemistry [17]. The Ru(II) polypyridyl complexes frequently contain chelating ligands such as polypyridine, 1,10-phenanthroline (phen), and their derivatives. The intercalation of the Ru(II) polypyridyl complexes into DNA has frequently been shown to be an important compo-

nent of the mechanism of biological action of this class of ruthenium complexes [50]. A typical ruthenium complex that interacts with DNA solely via intercalation is substitutionally inert ruthenium(II) polypyridyl complex, [Ru(bpy)2(dppz)]2+ (bpy = 2,20 -bipyridine, dppz = dipyri dophenazine) [51], which has been developed as site- and structure-specific luminescent DNA binding agents [51,52]. A complex of this class [Ru(phen)2(dppz)]2+ adopts multiple intercalative geometries [53] whereas Ru(dppz) semi-intercalation induces bending of duplex DNA [54]; which is a structural distortion similar to that observed following modification by cisplatin and its clinically used derivatives [1]. Another member of this series of ruthenium complexes, [Ru(dppz)2(pip)]2+ (pip = 2-phenylimidazo [4,5-f][1,10]phenanthroline) (Fig. 4c) intercalates DNA and acts to stall DNA replication fork progression in human cancer cells, activating DNA replicative stress signalling responses and preventing cell growth by cell-cycle deregulation [7]. DNA replication is the target also for substitutionally inert ruthenium(II) polypyridyl complex [Ru(phen)2(tpphz)]2+ (tpphz = tetrapyridophenazine) [55]. Further studies also have provided the experimental evidence that this ruthenium-based intercalation targets multiple genome integrity pathways in cancer cells, thereby achieving enhanced selectivity compared to existing DNA-damaging agents such as cisplatin. Two mixed-ligand ruthenium(II) polypyridyl complexes [Ru (tpy)(ptn)]2+ (Fig. 4e) and Ru(dmtpy)(ptn)]2+ (Fig. 4f) (ptn = 3-(1,1 0-phenanthrolin-2-yl)-as-triazino[5,6-f]naphthalene, tpy = 2,20 :60 , 200 -terpyridine, dmtpy = 5,50 -dimethyl-2,20 :60 ,200 -terpyridine) have been shown [56] to bind to DNA in an intercalative mode and induce B to Z conformational transition of calf thymus DNA. The driving force of the Z-DNA conformation induced by Ru(II) complexes was proved to be not only the electrostatic interactions between the divalent cations and the negatively charged phosphates in DNA but also associated with the high DNA binding affinities of complexes. The capability to stabilize telomeric DNA through the formation of G-quadruplexes which denies the telomerase access, leading to apoptosis induction has been also shown for two Ru(II) polypyridyl complexes with hydrophobic ancillary ligands, namely [Ru(bpy)2 (5-idip)]2+ and [Ru(phen)2(5-idip)]2+ (5-idip = 2-indole-[4,5-f] [1,10]phenanthroline) (Fig. 4g and h, respectively) which affect multiple targets simultaneously [57]. Further mechanistic studies revealed that [Ru(phen)2(5-idip)]2+ induces apoptosis in HeLa cancer cells through mitochondria-mediated pathway and inhibition of telomerase activity. It has been well known that interaction of DNA with chiral complexes is generally enantioselective. Moreover, it has been also shown that DNA interactions with metal complexes with enantiomeric carrier ligands can exhibit different conformational features, which are processed differently by the cellular machinery leading to different cytotoxicities. A pair of enantiomeric ruthenium(II) complexes D- and K-[Ru(bpy)2(pbip)]2+ {pbip = 2-(4-bro mophenyl)imidazo[4,5-f]1,10-phenanthroline} (Fig. 4i and j, respectively) have been synthesized [58]. DNA-binding studies showed that both the enantiomers bind to double-helical DNA via intercalative mode, and the D form binds to DNA more strongly than the K form. Molecular simulation further showed that both the two enantiomers intercalate between base pairs of DNA in the minor groove and that the D form intercalates into DNA more deeply than the K form. In addition, the cell proliferation assays show that the D form induces a greater cytotoxicity than the K form on human cervical cancer HeLa cells, which is positively correlated with the results in DNA binding studies and molecular docking, and implies that the DNA binding affinities of ruthenium (II) polypyridyl complexes might constitute the part of their anticancer mechanisms. This study has explored the stereoselective

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Fig. 4. Selected ruthenium complexes that interact with double-helical DNA by intercalation.

source of difference of antitumor activity between enantiomers of ruthenium(II) complexes by comparative studies on the interactions of a pair of enantiomers with DNA. Regioisomers of the functional group of the main ligand (L) on a series of [Ru(phen)2L]2+ and [Ru(bpy)2L]2+ complexes, where L = 4-(1H-Imidazo[4,5-f][1,10]phenanthrolin-2-yl)benzonitrile (cpip),2-(4-formylphenyl)imidazo[4,5-f][1,10]phenanthroline (fpip), 2-(4-bromophenyl)imidazo[4,5-f][1,10]phenanthroline (bpip) and 2-(4-nitrophenyl)imidazo[4,5-f][1,10]phenanthroline (npip) were synthesized as potential therapeutics and their DNA binding properties were investigated [59]. The biophysical studies have revealed that the [Ru(phen)2L]2+ complexes intercalate more efficiently and are attracted to DNA even more than [Ru(bpy)2L]2+ derivatives. Interestingly, a profound effect on both DNA binding affinity and mode has been observed to be affected not only by the chemical structure of the main polypyridyl ligand but also by the auxiliary ligand; the aldehyde and nitrile group end on the polypyridyl ligand exhibited most effective DNA binding via an intercalative mode.

2.3. Minor groove binding The minor groove binders (Fig. 1) constitute a group of DNA interactive agents which bind specific regions of the genome. The minor groove is narrow and shallow, only about 10 Å in width, whereas the major groove is deeper and wider, approximately 24 Å in width. Most DNA interactive proteins do bind in the major groove, but most small molecules of less than 1000 Da bind in the minor groove. The minor groove binders which have been characterized so far possess numerous biological activities such as antitumor, antiprotozoal, antiviral, and antibacterial properties. Minor groove binding has been demonstrated in the studies of Eriksson et al. [60,61] for D and K enantiomeric forms of [Ru(phen)3]2+ (Fig. 5a). The two-dimensional NMR study on the interaction of [Ru(phen)3]2+ with the self-complementary decanucleotide duplex [d(CGCGATCGCG)]2 has revealed minor groove binding for both enantiomers with the highest affinity for the adenine thymine (AT) regions. The observed AT specificity is more pronounced with the D as compared to the K enantiomer.

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Fig. 5. Selected ruthenium complexes that bind DNA through interactions with the minor groove.

Furthermore, neither of the enantiomers binds by classical intercalation. The results have been found compatible with binding of D[Ru(phen)3]2+ by insertion of two phenanthroline ligands into the minor groove, causing minor distortions of the DNA structure, whereas the K enantiomer binds in a mode that leaves the DNA structure unaffected. In contrast, other articles of Barton et al. [62–64] have described results of 1H NMR experiments on D-Ru(phen)2(dppz)2+ bound to a short duplex d(GTCGAC)2, which were interpreted to mean that Ru (phen)2(dppz)2+ intercalated into this duplex from the major groove side with a family of stacking orientations. This controversy has been resolved by irrefutable NMR evidence demonstrating that D-[Ru(phen)2dpq]2+ (dpq = dipyrido[3,2-d:20 ,3-f]quinoxaline), D[Ru(dmphen)2dpq]2+ (dmphen = 2,9-dimethyl-1,10-phenanthro line), D-[Ru(Me2phen)2dppz]2+ (Me2phen = 2,9-dimethyl-1,10-phe nanthroline), which are closely related to D-[Ru(phen)2(dppz)]2+, bind in the minor groove of the duplex d(GTCGAC)2 by intercalation [65–68]. The intercalation of the dppz ligand in [Ru(phen)2 (dppz)]2+ from the minor groove of the d(CCGGTACCGG)2 duplex has been also confirmed by X-ray structure analysis [53]. A ruthenium complex for which it has been shown that the minor groove is the most favored DNA binding site is also [Ru(tBu2bpy)2(2-appt)](PF6)2 [tBu2bpy = 4,40 -di-tert-butyl-2,20 bipyridine, 2-appt = 2-amino-4-phenylamino-6-(2-pyridyl)-1,3,5-t riazine] (Fig. 5b) [69]. UV–vis absorption titration, DNA melting studies, and competition dialysis using synthetic oligonucleotides [poly(dA-dT)2 and poly-(dG-dC)2] have shownd that [Ru(tBu2bpy)2(2-appt)](PF6)2 exhibits a binding preference for AT

sequences. As determined by cell viability assays, the ruthenium complex exhibits moderate cytotoxicities toward several human cancer cell lines. A nonintercalating ruthenium(II)-polypyridyl complex that has a catechol unit held at the end of an extended ligand, (E)-4-[2-(40 methyl-2,20 -bipyridine-4-yl)vinyl]benzene-1,2-diol, displays sequence selectivity and high-affinity binding to double-helical DNA through minor groove binding [70]. In addition, it displays preferential binding to alternating purine-pyrimidine sequences, particularly those that contain ATTA steps, with binding constants equivalent to those of high-affinity metallointercalators. A study contributing to better understanding the interactions between DNA and polypyridyl Ru(II) complexes has described the oxygen-sensing behavior of [Ru(ip)2(hnaip)]2+ (ip = imidazo[4,5-f] [1,10]phenanthroline and hnaip = 2-(2-hydroxy-1-naphthyl)imi dazo[4,5-f][1,10]phenanthroline) (Fig. 5c) and Ru(ip)2(dhpip)]2+ (dhpip = 2-(2,4-dihydroxyphenyl)imidazo[4,5-f][1,10]phenanthroline) (Fig. 5d) [71]. [Ru(IP)2(hnaip)]2+ binds DNA via a minor groove interaction with an intrinsic binding constant (Kb) of 7.9  104 M1 inducing also the B-to-Z DNA conformational transition. In contrast, Ru(ip)2(dhpip)]2+ which intercalates into DNA (Kb = 3.3  105 M1) is unable to induce the B-to-Z DNA transition. This study offers a new approach to evaluate the oxygen-sensing properties of DNA binders. A series of dinuclear ruthenium(II)–polypyridyl complexes of the type [Ru2(N-N)4(BPIMBp)]4+ (Fig. 5e), in which N-N is bpy, phen, dpq, dppz, and 1,40 -bis[(2-pyridine-2-yl)-(1H-imidazole-1-y l)methyl]-1,10 -biphenyl (BPIMBp) is a bridging ligand, have been

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synthesized and characterized [72]. These complexes are charged (4+) cations and flexible due to the –CH2 group of the bridging ligand and possess terminal ligands with variable intercalative abilities. Biophysical studies focused on the interaction of complexes with calf thymus DNA have revealed a groove-binding mode for all the complexes through a spacer and an intercalative mode for complexes containing dpq and dppz and bending and coiling of DNA suggesting its aggregation. Yellol et al. synthesized some neutral benzimidazole C,N-cyclometalated arene Ru(II) complexes (Fig. 5f–h), and these complexes are efficacious in several cancer cell lines [73]. Further studies indicated that one of these complexes (Fig. 5h) induces a high rate of apoptosis, good accumulation, S-phase cell cycle arrest, and weak binding to DNA in the minor groove. With the aim to develop some arene ruthenium(II) complexes based on fluorinated dipyrrin core which could be potential anticancer agents, half-sandwich complexes of the type [Ru(g6-arene)-(L)Cl], where arene is p-cymene and hexamethylbenzene and L is 4-fluorophenyldipyrrins (MFPdpmH) and 1,2,3,4,5-pentafluorophenyldipyrrins (PFPdpmH) were designed and synthesized [74]. The complexes exhibit significant cytotoxicity toward human lung cancer cell line (A549). Their binding through the minor groove of DNA has been established by molecular docking studies. External electrostatic interactions with DNA and/or DNA groove binding have been also demonstrated for water-soluble, enantiopure arene ruthenium compounds SRuSN-(1R,4S)-[(g6-p-cymene)Ru{jNH(bn),jNOH}Cl]Cl (bn = benzyl) (Fig. 5i) [75] and SRuSN-(1S,4R)-[(g6-p-cymene)Ru{jNH(pipsh),jN OH}Cl]Cl (pipsh = 2-(4-benzothiazolyl)phenylimidazo[4,5-f][1,10] phenanthroline) (Fig. 5j) [76]. Both enantiomers are active and versatile cytotoxic agents for which classical intercalation into the double-stranded DNA has been ruled out. 2.4. Sequence specificity It has become evident that DNA sequence specificity is an important component contributing to the cytotoxic potency of several antitumor agents [77]. Thus, tailoring the binding preference of DNA binding agents to particular sequences, such as critical genomic sequences, can be a useful strategy to create a tailormade antitumor agent. Early studies were focused on examinations of binding of tris(phenanthroline)ruthenium(II) ([Ru(phen)3]2+) enantiomers (Fig. 5a) to DNAs of different base compositions and structure [78]. Measurement of enantioselectivity made it possible the structural characterization of two noncovalent binding modes of the ruthenium(II) complexes to the DNA helix, one intercalatively bound mode showing a strong chiral preference for D-Ru(phen)2+ 3 and the other, a surface-bound mode along the DNA major groove, showing a weak preference for K-Ru(phen)2+ 3 . Notably, chiral discrimination for D-[Ru(phen)3]2+ increase with both the percent GC and increasing Na+ concentration. It has been suggested that the stereoselectivity is sensitive to the gradual changes in local DNA structure that are associated with variations in ionic strength. The ruthenium arene complexes [(g6-arene)Ru(en)Cl]+ with arene = benzene, p-cymene, biphenyl, dihydroanthracene, or tetrahydroanthracene (Fig. 2g,h) bind preferentially to N7 of guanosine, and reaction of complex [(g6-biphenyl)Ru(en)Cl]+ with 50 GMP and either 50 -AMP or 50 -CMP or 50 -TMP affords only the thermodynamically stable adduct [(g6-biphenyl)Ru(en)(N7-GMP)] [79]. Formation of H bonds between en-NH2 groups of the ruthenium complex and C6O of guanine enhances this selective coordination. Such high selectivity of Ru to guanine has also been observed for reactions of the (arene)RuII(en) complexes with natural DNA [40,41] and is not affected by the presence of excess cytochrome c, l-histidine, or glutathione [80,81], which may account

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for the low toxic side effects of such complexes compared with cisplatin [12]. The guanine base has been identified as the thermodynamically favored DNA binding site for organometallic ruthenium(II) anticancer complexes [(g6-arene)Ru(en)Cl][PF6] (arene = biphenyl, indane), and interestingly guanine-bound Ru(arene) units can migrate to the other guanine bases in the complementary strand [82]. Using a combination of MS-based ladder-sequencing and top-down MS approaches, the binding sites for the cytotoxic ruthenium arene anticancer complex [(g6-biphenyl)Ru(en)Cl]+ on a series of 15-mer single-stranded oligodeoxyribonucleotides with random nucleotide sequences were fully characterized [83]. The results identified besides guanine residues also unexpectedly thymine residues in single-stranded oligonucleotides as coordinative binding sites for this [(g6-biphenyl)Ru(en)Cl]+ complex. Thymines are competitive kinetically with guanines for binding to the organoruthenium anticancer complex also in G-quadruplex DNA [84]. 2.5. DNA condensation DNA condensation is essential for biological processes including DNA transcription and replication and receives additional impetus from an interest in gene therapy. DNA condensation refers to the process of compacting DNA molecules, and mechanistic details of DNA packing are essential for its functioning in the process of gene regulation in living systems. Two major interactions are proposed to drive the condensation of DNA [85]. (i) Electrostatic neutralization of negatively charged DNA backbones with multivalent cations decreases sufficiently repulsive energies to allow the tight packing of DNA. (ii) High binding affinity such as intercalation of metal complexes and organic aromatic cations into DNA reinforces the condensation of DNA. Several DNA-intercalating Ru(II) complexes with a pip derivative ligand [Ru(bpy)2(L)]2+ {L = pip, pipsh (Fig. 6a), 2-(4-benzoimidazolyl)phenylimidazo[4,5-f][1,10]phenanthroline] (pipnh) (Fig. 6b), 2-(4-benzoimidazolyl)phenylimidazo[4,5-f] [1,10]phenanthroline (pipsn), 2-(2-aminophenyl)imidazo[4,5-f] [1,10]phenanthroline) (apip) (Fig. 6c) or 2-(2-hydroxyl-5-amino phenyl)imidazo[4,5-f][1,10]phenanthroline (hpip)} (Fig. 6d) have been testified to possess high affinities for DNA and involve the condensation of DNA under neutral conditions [86,87], whereas [Ru(bpy)2(pip)]2+ was found to have no obvious effect on the condensation of DNA [88]. An effective approach to control the condensation and decondensation of DNA upon incorporation of luminescent of V-shaped di-ruthenium(II) complex [Ru2(bpy)4(mbpibH2)]Cl4 (mbpibH2 = 1, 3-bis([1,10]phenanthroline[5,6-d]imidazol-2-yl)benzene) (Fig. 6e) has been described by Gan et al. [85]. This dinuclear complex, compared with mononuclear Ru(II) complexes, has a greater charge and more variable molecular shape. It binds to the groove of herring sperm DNA with the binding constant (Kb) of 2.0  107 M1 (0.05 M NaCl, pH 7.2). Moreover, the di-Ru(II) complex induces the condensation of both herring sperm DNA to long chain-like particle clusters and circular plasmid pBR322 DNA to the particulate structure under neutral conditions. Potential nonviral vectors for DNA delivery based on two new luminescent ruthenium(II) polypyridyl complexes, [Ru(bpy)2(tptphen)]Cl2 (tpt-phen = triptycenyl-1,10-phenanthroline) (Fig. 6f) and [Ru(phen)2(tpt-phen)]Cl2 (Fig. 6g), have been developed [89]. A number of biophysical experiments have revealed that these complexes interact with DNA and efficiently condense DNA into globular nanoparticles. The observation that these complexes are taken up efficiently by tumor HeLa cells, do not cleave DNA and are biocompatible predisposes these complexes to have good gene transfection abilities.

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Fig. 6. Selected ruthenium complexes able to induce condensation of DNA.

Two Ru(II) arene complexes with imidazole (dichlorido complex) or bipyridyl (chlorido complex) ligands conjugated to 18b-glycyrrhetinic acid, an active triterpenoid metabolite of Glycyrrhiza glabra (Fig. 6h,i) have been explored as antitumor or antimicrobial agents [90]. In vitro assays have revealed moderate activity in several cancer cell lines, in some cases, the activity approached that of the conventional drug cisplatin. On the other hand, the bipyridyl (chlorido complex) has exhibited significant activity towards both Gram-positive S. aureus and Gramnegative E. coli bacteria. The experiments performed in a cellfree medium with plasmid DNA revealed that this ruthenium complex containing N,N-chelating ligand alters, upon hydrolysis, the conformation of DNA and induces condensation of DNA. The in cellulo experiments have further demonstrated the generation of intracellular reactive oxygen species (ROS). These findings have been proposed to explain promising toxicity towards human cancer cells. A series of 24, 26-membered Ru(II) dinuclear metal macrocycles containing 1-{3-[(1H-imidazole-1-yl)methyl]benzyl}-1Himidazole and 1-{4-[(1H-imidazole-1-yl)methyl]benzyl}-1H-imidazole ligands (Fig. 6j,k) have been synthesized and characterized with the aim to offer new anticancer agents with novel modes of binding to DNA [91]. In general, the complexes exhibited little or moderate anti-proliferative activity towards human cancer cell lines. Gel electrophoresis studies have revealed that the ruthenium(II) macrocycles affect the structure of supercoiled DNA significantly, and induce DNA condensation.

2.6. Binding to A- and Z-DNA Three different canonical forms of double-helical nucleic acid have been described (Fig. 7). The most common form, present in most DNA at neutral pH and physiological salt concentrations, is Bform. That is the classic, right-handed double helical structure. A thicker right-handed duplex with a shorter distance between the base pairs has been described for DNA under dehydrating conditions, RNA-DNA duplexes and double-helical RNA. This is called A-form nucleic acid. The third form of duplex DNA has a strikingly different, left-handed helical structure. This Z DNA is formed by stretches of alternating purines and pyrimidines, especially in negatively supercoiled DNA. The major and minor grooves, unlike A- and B-DNA, show little difference in width. Formation of this structure is generally unfavorable, although certain conditions can promote it. [Ru(tmp)3]2+ (tmp = tris(3,4,7,8-tetramethylphenanthroline)), [Ru(tmp)3]2+ (Fig. 7a) binds to A-DNA under conditions where little binding to B DNA is detected [92]. This ruthenium complex displays enantiomeric discrimination in its DNA binding, K-[Ru (tmp)3]2+ binds selectively to A-form polynucleotides and in the presence of light to cleave A-form polymers. K-[Ru(tmp)3]2+ has been proposed for use as a chiral probe useful to investigate DNA conformational heterogeneity in mapping sites in the A conformation along the double-helical molecule of nucleic acids. Z-DNA formation is primarily linked to the rate of transcription and may also affect chromatin recombination and nucleosome positioning, Z-DNA inducers and stabilizers are considered

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Fig. 7. Examples of ruthenium complexes that selectively bind A-DNA (a) or Z-DNA (b).

important regulators of several genes [93]. It has been demonstrated that the ruthenium complex [Ru(dip)2(dppz)]2+ (dip = 4,7diphenyl-1,10-phenanthroline) (Fig. 7b) efficiently induces the transition from B to Z DNA of various DNA sequences both in vitro and in vivo, which include non-alternating pyrimidinepurine and AT-rich segments, in a low-salt solution [94]. Because alternating AT-rich segments are common and often found in the upstream direction of genes, it has been suggested that the transformation of these segments from the B to the Z conformation induced by [Ru(dip)2(dppz]2+ can regulate gene transcription. A series of ruthenium(II) complexes - DNA intercalators – with imidazophenanthroline ligands (chiral polypyridyl Ru(II) complexes with expanding planar ligands ([Ru(bpy)2(ip)](ClO4)2, [Ru(bpy)2(iip)](ClO4)2, and [Ru(bpy)2(nip)](ClO4)2 (iip = isoindolyli midazo[4,5,f][1,10]phenanthroline, nip = benzo[f]isoindole[4,5,f] [1,10]phenanthroline)), which vary in size and planarity, have been also designed [95] to demonstrate the structural effect of DNA intercalators on their interference with DNA high-order structures, and to determine the link between the B–Z DNA conformational transformation and DNA condensation. Notably, no evident conformational change has been observed for [Ru(bpy)2(ip)](ClO4)2 complexes containing smallest planar ligand. With an increase in the planar sizes of the main ligands, [Ru(bpy)2(iip)](ClO4)2 complexes transform DNA secondary structure from B- to Z-form, whereas a higher-order structural change to complete DNA condensation is caused by [Ru(bpy)2(nip)](ClO4)2 complexes. These results contribute to the further understanding of the relation between the structures of Ru(II) polypyridyl complexes and the interaction with DNA, as well as offer the promising potential to control the B-Z transition. 2.7. Binding to DNA quadruplexes and telomers G-quadruplexes (Fig. 8a) are non-classical, higher-order DNA and RNA structures formed from G-rich sequences that are built around stacked tetrads of hydrogen-bonded guanine bases. Poten-

Fig. 8. Examples of ruthenium complexes that selectively bind DNA-quadruplexes (a, b), DNA-bulges (c), or DNA basic sites (d).

tial quadruplex sequences have been identified in G-rich eukaryotic telomeres, and in non-telomeric genomic DNA, e.g., in nucleasehypersensitive promoter regions or DNA in the upstream promoter of protooncogenes [96,97]. There is also particular interest in Gquadruplexes as targets for therapeutic intervention, and notably, an increasing number of metal complexes including Ru(II) complexes [98] have been reported as G-quadruplex DNA binders. Two ruthenium(II) polypyridyl complexes (Fig. 8a) have been reported with the aim of targeting bcl-2 gene which is overexpressed in various human tumor cells and is widely believed to be an apoptosis suppressor gene. The biophysical studies have revealed that these complexes induce and stabilize the formation of G-quadruplex in bcl-2 gene [98]. The results also show that these polypyridyl ruthenium (II) complexes also have the most significant effect on inhibiting Hela tumor cells proliferation and migration of ECV-304 cells. Similarly, the selectivity and ability to interact with bcl-2 DNA has been also described for [Ru(ip)3] (ClO4)22H2O [99]. This complex also induces cell apoptosis in in vitro assays. The ruthenium(II) polypyridyl complexes have also been shown to selectively bind to and stabilize c-myc G-quadruplex DNA ] (the c-myc proto-oncogene encodes a short-lived transcription factor that plays an important role in cell cycle regulation, differentiation,

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and apoptosis). Two polypyridyl ruthenium(II) complexes with alkynes, [Ru(bpy)2L](ClO4)2 (L = p-tepip and p-bepip; p-tepip = 2-(4-trimethylsilylpropargyl)-1H-imidazo[4,5f][1,10]phenanthroline; p-bepip = 2-(4-phenyacetylenephenyl)-1H-imidazo[4,5f][1,10] phenanthroline) selectively bind to and stabilize c-myc G-quadruplex DNA [100]. Notably, the replication of c-myc DNA is blocked by the latter two complexes so that these ruthenium (II) complexes have been proposed as candidates of inhibitors in chemotherapy through their binding and stabilization of c-myc G-quadruplex DNA. Recently, another study [101] demonstrated a novel class of ruthenium(II) terpyridine complexes that target the c-myc G-quadruplex DNA and are potent c-myc G4 DNA signaling telomerase inhibitors. As already mentioned in the Section 2.2.1., chiral recognition of DNA molecules is important because much evidence has indicated that transformations of chirality and diverse conformations of DNA are involved in a series of key biological events. Among these, enrichment of G-quadruplexes in the genome, and the exploration of their multiple structures has aroused great interest. It has been shown, on the basis of comparison of nearly one hundred different sequences capable of forming stable G-quadruplexes [102], that the enantioselectivity of G-quadruplexes is regulated by multiple factors, such as different link-sequences and tail-sequences. Several reports have shown that especially dinuclear ruthenium (II) complexes can induce the formation of and stabilize G-quadruplexes [103,104] offering a substantial advantage over mononuclear ruthenium(II) complexes. Dinuclear ruthenium(II) complexes exhibit greater variations in shape, charge, and size. By connecting two metal centers into a dinuclear complex, selectivity for G-quadruplex structures can be achieved, and the drug tolerance of tumor cells to conventional platinum antitumor agents can be reduced. In order to demonstrate the remarkable efficiency of dinuclear ruthenium(II) complexes to stabilize G-quadruplexes, dinuclear ruthenium(II) complexes have been designed [105]. For instance, the complex shown in Fig. 8b induces the formation and stabilization of antiparallel G-quadruplex structures of the human telomeric sequence (AG3(T2AG3)3, and can even lead to the conversion of a hybrid G-quadruplex to an antiparallel G-quadruplex. These complexes can also selectively bind and stabilize G-quadruplexes in the presence of excessive duplex DNA. Importantly, the complexes can significantly inhibit telomerase activity and HeLa cell proliferation. 2.8. Binding to other unusual DNA structures DNA does not always take the form of a double helix. It can often be found creating structures considered abnormal when compared to what is commonly considered DNA. Normally, DNA contains a B-form helix, but the improper formation of base pairs can lead to the formation of various unusual structures that greatly affect DNA’s conformation and flexibility. Moreover, RNA, namely viral and bacterial RNA, contains a larger proportion of nonduplex type unusual structures so that RNA may be a better target for ruthenium complexes capable of preferential binding to unusual structures of DNA. 2.8.1. Binding to DNA bulges An extra, unpaired base in one strand of a nucleic acid duplex is termed a ‘‘bulged” base. Nucleic acid bulges (Fig. 8c) have been involved in a number of biological processes such as frameshift mutagenesis, RNA splicing, and RNA-protein interactions. The structure and conformation of nucleic acid bulges have been under intensive investigation. It is believed that the conformation of the bulge is important for protein recognition. The binding of the stereoisomers of [{Ru(Me2bpy)2}2(m-bpm)]4+ (Fig. 8c) and the structurally related complexes [{Ru-(phen)2}2-

(m-bpm)]4+ and [{Ru(Me2phen)2}2(m-bpm)]4+ (Me2bpy = 4,40 -dime thyl-2,20 -bipyridine; bpm = 2,20 -bipyrimidine; Me2phen = 4,7-dimethyl-1,10-phenanthroline) to a tridecanucleotide d(CCGAGAATTCCGG)2 that contains a single adenine bulge site has been investigated [106]. The results demonstrate that meso[{Ru-(phen)2}2(m-bpm)]4+, in contrast to any of the stereoisomers of the corresponding Me2bpy complex, has a high affinity and selectivity for a single adenine bulge site. It binds the single adenine bulge site strongly and with greater affinity than any of the stereoisomers of the corresponding Me2bpy complex. The results of this study indicate that the dinuclear ruthenium complexes have excellent potential as DNA bulge probes. The latter conclusion has been corroborated by the results of the study in which DNA binding of the stereoisomers of a series of dinuclear ruthenium(II) complexes [{Ru(phen)2}2(m-BL)]4+ with flexible bridging ligands (BL) bb2 {1,2-bis[4(40 -methyl-2,20 -bipyri dyl)]ethane}, bb5 {1,5-bis[4(40 -methyl-2,20 -bipyridyl)]pentane}, bb7 {1,7-bis[4(40 -methyl-2,20 -bipyridyl)]heptane}, and bb10 {1,10-bis[4(40 -methyl-2,20 -bipyridyl)]decane} has been investigated [107]. Interestingly, an 1H NMR study of the binding of the DD-enantiomer of [{Ru(phen)2}2(m-bb7)]4+ to synthetic doublestranded oligodeoxyribonuceleotides confirmed the selectivity of the metal complex for the single adenine bulge. 2.8.2. Binding to DNA mismatches and abasic sites DNA mismatch is another type of DNA unusual structure. It represents a DNA defect occurring when two or more noncomplementary bases are aligned in the same base-pair step(s) of a duplex DNA. Mismatches can appear during replication of DNA, heteroduplex formation, action of mutagenic chemicals, ionizing radiation, or spontaneous deamination [108]. One of the early reports demonstrating binding of a ruthenium complex to mismatched DNA is that demonstrating binding of Ru (bpy)2(eilatin)2+ to a mismatched site in the 36 base pair oligonucleotide [109]. The heptacyclic aromatic alkaloid eilatin coordinated to an octahedral metal complex offered the possibility of high-affinity metalloinsertion. The reason is that while the intercalating ligands of complexes that bind well-matched DNA can slide easily between the regular base pairs, the more sterically expansive ligands of mismatch-specific complexes are simply too wide to fit into well-matched regular DNA [109]. Thus, the complexes bearing bulkier ligands can be able to bind mismatched sites, which contain the mismatched bases with the impaired hydrogen bonding and stacking. Unexpectedly, the selectivity of Ru(bpy)2(eilatin)2+ (Fig. 8d) in binding mismatched DNA was not high despite it contained the extremely expansive eilatin ligand very likely because the size and shape of the eilatin ligand allow stacking with both well-matched and mismatched DNA. Further studies with ruthenium complexes which bind preferentially to DNA mismatches investigated [Ru(bpy)2(bniq)]2+ (bniq = benzo[c][1,7]naphthyridine-1-isoquinoline), which incorporates the sterically expansive bniq ligand [110]. This complex is a highly selective luminescent probe for DNA mismatches and also abasic sites (Fig. 8d), possessing a 500-fold higher binding affinity toward these destabilized regions relative to well-matched base pairs. The results also implicate binding of the complex to a mismatch from the minor groove via metalloinsertion with extrusion of the destabilized mismatched bases. A similar binding preference for DNA mismatches and abasic sites in DNA have been also observed for complex [Ru(bpy)2(dppz)]2+ [111]. 2.9. DNA cleavage The DNA-binding properties of ruthenium complexes have aroused great interest during the past decades in relation to not

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Fig. 9. Examples of ruthenium complexes that cleave DNA in the dark (a) or under irradiation (b, c).

only anticancer drugs but also to sequence-specific DNA cleaving agents [112]. In 2007, MacDonnell and co-workers found out [113] that the ruthenium dimer [(phen)2Ru(tatpp)Ru(phen)2]Cl4 (tatpp = 9,11, 20,22-tetraazatetrapyrido[3,2-a:20 ,30 -c:300 ,200 -1:200 ,30000 -n]-pentacene) (Fig. 9a) could induce DNA cleavage in vitro and that this cleavage is enhanced under anaerobic conditions. This complex is known to intercalate and to bind DNA tightly, but DNA damage is caused by in situ reduction of the complex with glutathione. Moreover, exposure to air, even in a large excess of the reductive agent, attenuates the cleavage activity. The active species is the doubly protonated complex. The ruthenium(II) polypyridyl complexes, [(phen)2Ru(tatpp)]2+ and [(phen)2Ru(tatpp)Ru(phen)2]4+ cleave single strand of DNA in cell-free media in the presence of a mild reducing agent, such as glutathione, in a manner that is enhanced upon lowering the [O2] [8]. ROS are involved in the cleavage process as hydroxy radical scavengers attenuate the cleavage activity. Cleavage experiments in the presence of superoxide dismutase and catalase reveal a central role for H2O2 as the immediate precursor for hydroxy radicals. A mechanism has been proposed which explains the inverse [O2] dependence and ROS data and involves redox cycling between three DNA-bound redox isomers of [(phen)2Ru(tatpp)]2+ and [(phen)2Ru(tatpp) Ru(phen)2]4+. Further studies with half sandwich arene ruthenium(II) complexes of the type [Ru(arene)Cl(L)] (where arene = benzene or pcymene, L = thiophene benzhydrazone ligands) have investigated the possibility that these complexes could cleave DNA in breast MDA-MB-231 tumor cells forming single-strand breaks [114]. The results demonstrate that the complexes are capable of eliciting DNA cleavage effects, as evidenced by the comet assay. 3. Photoactivation and DNA photocleavage Recently, a variety of complexes based on ruthenium(II) centers have been reported to have excellent photophysical properties, and

consequently, attention has turned to the use of these complexes as biologically significant agents. Certain compounds of this class of ruthenium complexes exhibit enhanced cytotoxicity following light irradiation, providing a platform for relatively selective and improved tumor therapy [9,115]. The dinuclear Ru(II) arene complexes [{(g6-arene)RuCl}2 (m-2,3-dpp)](PF6)2, arene = indan, benzene, p-cymene, or hexamethylbenzene, 2,3-dpp = 2,3-bis(2-pyridyl)pyrazine, have been synthesized and characterized [116]. Upon irradiation with UVA light, complexes containing indan and benzene readily undergo arene loss, while complexes containing p-cymene or hexamethylbenzene not. UV or visible irradiation of [{(g6-indan)RuCl}2(m-2,3dpp)](PF6)2 in aqueous or methanolic solution leads to arene loss. The DNA binding of this complex is increased after irradiation; its non-irradiated form preferentially forms DNA adducts that only weakly block RNA polymerase, while its irradiation transforms the adducts into stronger blocks for RNA polymerase. The efficiency of irradiated [{(g6-indan)RuCl}2(m-2,3-dpp)](PF6)2 to form DNA interstrand cross-links is slightly greater than that of cisplatin. In contrast, the interstrand cross-linking efficiency of non-irradiated [{(g6-indan)RuCl}2(m-2,3-dpp)](PF6)2 is relatively low. DNA unwinding measurements supported the conclusion that both mono- and bifunctional adducts of [{(g6-indan)RuCl}2(m-2,3-dpp)] (PF6)2 with DNA can form. These results are consistent with the view that photoactivation of dinuclear Ru(II) arene complexes can simultaneously produce a highly reactive ruthenium species that can bind to DNA and a fluorescent marker (the free arene). Importantly, the mechanism of photoreactivity is also independent of oxygen. The dinuclear Ru(II) arene complexes investigated in this study, therefore, have the potential to combine both photoinduced cell death and fluorescence imaging of the location and efficiency of the photoactivation process. The work of Betanzos-Lara et al. [117] provides the example of a family of piano-stool Ru(II) arene complexes of the type [(g6-arene) Ru(N,N0 )(L)]2+ (where N,N0 is a chelating ligand and L is a pyridine or a pyridine derivative) that can selectively photodissociate the

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monodentate ligand (L) when excited with UVA or white light. Such a unique feature allows control of the hydrolysis reaction of the complexes and, therefore, the formation of a reactive aqua species that otherwise would not form in the dark. The possibility of phototriggering the binding to DNA of the Ru(II) arene pyridine or pyridine-derivative complexes has been investigated as well. Studies performed with mammalian DNA in cell-free media suggest combined weak monofunctional coordinative and intercalative DNA binding modes. Two cytotoxic DNA-intercalating ruthenium(II) complexes, [Ru(bpy)2(apip)]2+ and [Ru(bpy)2(hpip)]2+ (see Section 2.5.), cleave plasmid DNA upon irradiation at 365 nm [87]. The studies on the mechanism of photocleavage demonstrate that superoxide anion 1 radical (O 2 ) and singlet oxygen ( O2) play an important role. Similarly, upon irradiation at 365 nm, effective cleavage of plasmid DNA has been observed for ruthenium(II) polypyridyl complexes [Ru(bpy)2(L)](ClO4)2 and [Ru(phen)2(L)](ClO4)2 (L = 2-(3-nitro-4-hy droxylphenyl)imidazo[4,5-f][1,10]phenanthroline or 2-(3-amino-4hydroxylphenyl)imidazo[4,5-f][1,10]phenanthroline) [118]. The photoinduced plasmid DNA cleavage (by UV light, 360 nm) by dinuclear Ru(II) polypyridyl complexes containing three, six and ten methylene chains have also been investigated [112,119]. It has been shown that the length of the alkyl linker has a dominant effect on the DNA binding mode of these dinuclear Ru(II) complexes. While the complexes containing three and ten methylene chains intercalate DNA only partially, the complex containing six methylene chain binds DNA most strongly by classical intercalation and exhibit the highest DNA photocleaving activity. These results have indicated that the partial or nonclassical intercalative mode might be less favorable for the DNA cleavage than the classical intercalative mode. The ruthenium(II) complexes [Ru(tpy)(dpoq)Cl]+ (Fig. 9b) and [Ru(tpy)(dpoq)CH3CN]2+ (dpoq = dipyrido[1,2,5]oxadiazolo[3,4-b] quinoxaline) represent another group of DNA-intercalating agents which, when irradiated under light (365 nm), efficiently photocleave DNA both under aerobic and anaerobic condition [120]. The mechanistic studies have revealed that the photocleavage reaction functions through both oxygen-independent (photoinduced electron transfer) and oxygen-dependent (singlet oxygen generation) pathways. These complexes have been proposed as promising photodynamic therapy (PDT) candidates used for treating hypoxic tumors. Ruthenium(II) arene anticancer complex [(g6-p-terp)RuII(en) Cl]+ (p-terp = paraterphenyl) (Fig. 9c) has been designed to investigate whether it could be considered a potential candidate agent for improved photodynamic anticancer chemotherapy. It was shown [121] that this complex exhibits promising toxic effects in several human tumor cell lines and concomitantly its DNA binding mode involves combined intercalative and monofunctional (coordination) binding modes. In vitro studies have confirmed that when photoactivated by UVA or visible light, [(g6-p-terp)RuII(en)Cl]+ efficiently photocleaves DNA, also in hypoxic media. Studies of the mechanism underlying DNA cleavage by photoactivated [(g6-pterp)RuII(en)Cl]+ have revealed that the photocleavage reaction does not involve generation of ROS, although the contribution of singlet oxygen (1O2) to the DNA photocleavage process cannot be entirely excluded [122]. Notably, the mechanism of DNA photocleavage by [(g6-p-terp)RuII(en)Cl]+ appeared to involve direct modification of mainly those guanine residues to which this complex is coordinatively bound. As some tumors are oxygen-deficient and cytotoxic effects of photoactivated ruthenium compounds containing [Ru(g6-arene)]2+ do not require the presence of oxygen, this class of ruthenium complexes may be also considered potential candidate agents for improved photodynamic therapy. Wang et al. have reported [123] that the ferrocenyl pyridinebased arene Ru(II) complex, [(p-cymene)Ru(bpy)(py-Fc)]2+, where

py-Fc = 4-pyridyl ferrocene, is an efficacious photosensitizer that kills cancer cells. This complex produces both hydroxyl radicals and 1O2 as well as photoinduced monodentate ligand dissociation upon visible light irradiation (440 nm). The complex produces DNA photodamage under light irradiation and has significant photoactivated anticancer efficacy. In addition, the same research group also determined the effect of substituents on the photoactivity of [(g6-p-cymene)Ru(dpb)(py-R)]2+ (dpb = 2,3-bis(2-pyridyl)be nzoquinoxaline, py-R = 4-substituted pyridine, R = N(CH3)2, NH2, OCH3, H, COOCH3 and NO2) [124]. The complexes induce DNA photocleavage and DNA photobinding by photoinduced ligand dissociation and 1O2 production. Ruthenium(II) complexes trans-[Ru(sac)2(dpq)2], trans-[Ru (sac)2(dppz)2] and its cis isomer, where sac is artificial sweetener saccharin (o-sulfobenzimide; 1,2-benzothiazole-3(2H)-one1,1-dio xide (Hsac)), represent another important class of photoactivatable agents capable of DNA cleavage [20]. Saccharin is known to act as carbonic anhydrase IX inhibitor which is a biomarker for highly aggressive and proliferative tumor in hypoxic stress, so inhibition of carbonic anhydrase IX is a potential strategy for anticancer chemotherapy. The photo-induced dissociation of monodentate saccharin ligand is observed when irradiated at UVA light of 365 nm. The complexes show significant binding affinity to doublehelical DNA through significant intercalation through planar dpq and dppz ligands. The complexes show appreciable photoinduced plasmid DNA cleavage activity upon irradiation of low power UVA light of 365 nm from supercoiled to its nicked circular form at micromolar complex concentrations. Photocleavage mechanistic studies in the presence of O2 have revealed the involvement of ROS generated upon photoactivation leading to nicking of supercoiled DNA to nicked circular form. In the absence of O2, photocleavage of DNA through the formation of photoinduced ligand dissociated Ru-DNA complex is also observed. Two ruthenium nitrosyl complexes containing a thiocarbonyl ligand, with the formula cis-[Ru(phen)2(L)(NO)](PF6)3 (L = thiourea or thiobenzamide) have been examined for DNA photocleavage activity as well [125]. An efficient release of nitric oxide by blue light has been demonstrated. Interestingly, the complex containing thiourea cleaves DNA even in the dark, while both complexes show great DNA photocleavage activity in blue light. This process has been proposed to work mainly through NO and hydroxyl radical production. Additionally, these complexes have shown promising vasodilator activity. Metallosupramolecular chemistry was used to design a new class of synthetic agents, namely, tetracationic supramolecular cylinders, that bind strongly and noncovalently in the major groove of DNA. To gain additional information on interactions of the cylinders with DNA, photonuclease activity of ruthenium(II) metallosupramolecular cylinder [Ru2L3]4+, where L is a bispyridyl-imine ligand, has also been explored [126]. The molecular biophysics analyzes have revealed that [Ru2L3]4+ interact with BDNA predominantly through major groove binding and unwound negatively supercoiled plasmid DNA preferentially in regular alternating purine-pyrimidine sequences. Photocleavage studies have shown that [Ru2L3]4+ induces single-strand breaks on irradiation by visible and UVA light and cleaves DNA mainly at guanine residues contained preferentially in regularly alternating purinepyrimidine nucleotides.

4. Radiosensitization Chemotherapeutics are often used in combination with external ionizing radiation in the treatment of cancers and several metallodrugs that inhibit DNA replication are potent radiosensitizers [127]. Combination therapy employing a DNA-damaging agent

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with external ionizing radiation offers significant benefits compared to either treatment in isolation and is an established treatment regimen for a range of cancers. To date, metal complexes that have been examined as radiosensitizers include ruthenium complexes as well [128,129]. In an effort to develop a ruthenium-based compound that functions as a radiosensitizer, the nuclear-targeting complex [Ru(dppz)2(pip)]2+ was synthesized. It has been shown [7] that this complex functions as a radiosensitizer in human cervical cancer cells. It generates a substantial increase in cell sensitivity to ionizing radiation (up to 6 Gy for 20 h) while possessing mild cytotoxicity, which appears to be a substantial advantage in a therapeutic situation as off-target tissue toxicity by the sensitizer may be minimized. It has been shown that complex [Ru(dppz)2(pip)]2+ intercalates DNA and acts to stall replication fork progression in human cancer cells, activating DNA replicative stress signaling responses and preventing cell growth by cell-cycle deregulation [7]. A later study has demonstrated that a related complex [Ru(phen)2 (tpphz)]2+ functions as a DNA replication inhibitor and a radiosensitizer with efficiency comparable to cisplatin, which occurs through a synergistic enhancement of DNA damage [55]. The other example of a ruthenium compound which has received attention as a radiosensitizer was the class of ruthenium-based DNA binding complexes comprising those containing bis-benzimidazole derivatives: [Ru(bbp)]Cl3, [Ru(bbp)2]Cl2 (in which bbp = 2,6-bis(benzimidazol-1-yl)pyridine), and [Ru (bnbp)2]Cl2 (where bnbp = 2,6-bis-(6-nitrobenzimidazol-2-yl)pyri dine). Evaluation of their radiosensitization capacities in vitro has revealed [130] that [Ru(dppb)2]Cl2 is particularly effective in sensitizing human melanoma A375 cells toward radiation. Along with this potency, [Ru(bnbp)2]Cl2 exhibits a high degree of selectivity between human cancer and normal cells. Mechanistic studies have revealed that [Ru(bnbp)2]Cl2 promotes radiation-induced accumulation of intracellular ROS by reacting with cellular glutathione and then causing DNA strand breaks.

somerase IB. Compound [Ru(SpymMe2)(bipy)(dppb)]PF6 is the most efficient topoisomerase inhibitor. Further analysis indicated that the ruthenium complex inhibits the cleavage reaction impeding the binding of the enzyme to DNA and slows down the religation reaction. Molecular docking showed that [Ru(SpymMe2)(bipy) (dppb)]PF6 preferentially binds closer to the residues of the active site of the topoisomerase and lies on the DNA groove downstream of the cleavage site in the topoisomerase–DNA complex. Notable ruthenium(II) compounds to have undergone tests for their ability to act as potential inhibitors acting on topoisomerase IIa are chiral Ru(II) complexes bearing furan ligands, D/K-[Ru (bpy)2(pocl)]2+ and D/K-[Ru(bpy)2(poi)]2+ (pocl = 2-(5-chloro furan-2-yl)imidazo[4,5-f][1,10]phenanthroline, poi = 2-(5–5-iodo furan-2-yl)imidazo[4,5-f][1,10]phenanthroline) [136]. These Ru (II) complexes show antitumor activities against several tumor cell lines. These Ru(II) complexes gradually penetrate into the interior of the nucleus suggesting that DNA is the cellular target of these enantiomers. Notably, D-[Ru(bpy)2(poi)]2+ acts as efficient poisons of topoisomerase IIa in vitro and in living cells. These results demonstrate that complex D-[Ru(bpy)2(poi)]2+ binds to DNA and stabilizes the topoisomerase IIa-DNA cleavable complex thus acting as a topoisomerase IIa poison. The ability to explore a much wider range of ruthenium(II) complexes capable of inhibiting topoisomerases arose when ruthenium (II) polypyridyl complexes with 1,8-naphthalimide were designed and synthesized [137]. DNA-binding studies of [Ru(phen)2(pnip)]2+ (Fig. 10b) and [Ru(bpy)2(pnip)]2+ {pnip = 12-[N-(p-phenyl)-1,8-nap thalimide]imidazo[40 ,50 -f][1,10]phenanthroline} demonstrate that the two complexes interact with double-helical DNA via an intercalative mode. Topoisomerase inhibition assays indicated that the two Ru(II) complexes are efficient dual topoisomerase I and IIa poisons. Moreover, these complexes exhibit plasmid DNA photocleavage under irradiation at 365 nm.

5. Inhibition of DNA processing enzymes

Transcription is the first step of gene expression by which the information in a strand of DNA is copied into a new molecule of messenger RNA by the enzyme RNA polymerase. Many anticancer and antimicrobial drugs inhibit transcription, and most transcription inhibitors have useful pharmacological properties. To explore the transcription inhibition activity of ruthenium compounds, ruthenium(II) polypyridyl complexes with potential high DNA-binding ability have been designed by extending the conjugated plane of the intercalative ligand and introducing electropositive pendants to the ancillary ligand (Fig. 10c) [138]. These complexes intercalate into DNA base pairs with very high affinity. Owing to their high DNA-binding ability, the complexes effectively inhibit the DNA transcription activity by blocking the binding of T7 RNA polymerase to the template DNA. As efficient transcription inhibitors, the complexes demonstrate high in vitro antitumor activity against several tumor cell lines. Similarly, two complexes [(g6-p-cymene)Ru(bpm)Cl][PF6] (bpm = 2,20 -bipyrimidine) (Fig. 10d) and [(g6-p-cymene)Ru(phen) Cl][PF6] were selected for further studies of the transcription inhibition activity ruthenium compounds [139]. The results have revealed the presence of combined covalent (coordinative), noncovalent intercalative, and monofunctional coordination binding modes of DNA binding for these investigated Ru(II) complexes upon hydrolysis. The inhibition of RNA synthesis by T7 RNA polymerase on the template plasmid DNA pSP73KB containing adducts of the investigated Ru(II) arene complexes was examined by gel electrophoresis. The results indicate that RNA synthesis on these templates is prematurely terminated. The major stop sites occur at similar positions and are solely at guanine residues, for both Ru(II) arene complexes.

5.1. Inhibition of topoisomerases A mechanism proposed for the mechanism of action of Ru(II) complexes is also the inhibition of topoisomerases I and II, which are essential enzymes that control and modify the topological states of DNA [131,132]. Thus, ruthenium compounds have also received attention as possible topoisomerase inhibitors. Topoisomerases are a class of ubiquitous enzymes that modulate the topological problems associated with DNA replication, transcription, and other nuclear processes by introducing transient single- or double-strand breaks in the DNA (topoisomerase I or II, respectively). Ruthenium compounds which have received attention as possible topoisomerase inhibitors consisting of ruthenium(II) compounds are the Ru(II)(g6-p-cymene) compounds carrying bioactive flavonol ligands (Fig. 10a) [133]. These compounds are potent antiproliferative agents forming covalent bonds to DNA and inhibit topoisomerase IIa in vitro [133,134]. The ruthenium(II) compounds [Ru(pySH)(bpy)(dppb)]PF6, [Ru (HSpym)(bpy)(dppb)]PF6 and [Ru(SpymMe2)(bpy)(dppb)]PF6, where dppb = 1,4-bis(diphenylphosphino)butane; Spy = 2-mercap topyridine anion; Spym = 2-mercaptopyrimidine anion and SpymMe2 = 4,6-dimethyl-2-mercaptopyrimidine anion, represent further examples of ruthenium-based topoisomerase inhibitors [135]. In vitro cell culture experiments have revealed significant antiproliferative activity for these complexes. These ruthenium compounds bind to DNA through electrostatic interactions and inhibit the DNA supercoiled relaxation mediated by human topoi-

5.2. Inhibition of DNA transcription and DNA synthesis

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Fig. 10. Examples of ruthenium complexes that inhibit DNA topoisomerases (a, b) or RNA-polymerase (c, d).

[Ru(bpy)(phpy)(dppz)]+ (phpy = 2-phenylpyridine) is a DNAintercalating Ru(II) complex that selectively accumulates in cell nuclei and is toxic in several cancer cell lines [6]. The high DNA binding affinity of this complex results in effective inhibition of DNA transcription investigated by comparing the amount of mRNA produced by T7 RNA polymerase and disruption of the binding of transcription factor NF-jB to its consensus DNA sequences (rB sites), thereby leading to the inhibition of cellular transcription and irreversible cancer cell apoptosis. Thus, the cytotoxic effects of [Ru(bpy)(phpy)(dppz)]+ have been related to the inhibition of DNA transcription [6], which results in the down-regulation of genes regulated by transcription factors. As ruthenium-based anticancer agents also induce DNA damage [6–9], conjugation of ruthenium complexes with an inhibitor of DNA damage repair may have synergistic effects to enhance the antitumor activity of the ruthenium complex. The well-known compounds that prevent the repair of DNA damage are the inhibitors of the enzyme poly (ADP-ribose) polymerase (PARP) [140]. Three organometallic ruthenium(II)-arene anticancer complexes

based on PARP-1 inhibitors have been designed, namely [RuCl2(cymene)L] {L = nicotinamide, quinazolin-4(3H)-one or 3-aza-5[H]-phenanthridin-6-one} and their in vitro cytotoxicity, as well as mechanism of action, were investigated [141]. The complexes show significantly enhanced cytotoxicity in human cancer cells compared with the free ligands of PARP-1 inhibitors and have multi-targeting properties. They exhibit the characteristics of DNA-binding metallic drugs, such as binding to DNA and effectively inhibiting transcription. On the other hand, the complexes also efficiently inhibit the catalytic activity of PARP-1 in vitro. DNA synthesis is a process by which copies of nucleic acid strands are made. DNA synthesis occurs just prior to cell division through a process called replication. When replication begins the two strands of DNA are separated by a variety of enzymes. Thus opened, each strand serves as a template for producing new strands of DNA. This whole process is catalyzed by enzymes called DNA polymerase. A number of DNA synthesis inhibitors are efficient anticancer and antimicrobial drugs.

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The monofunctional Ru(II)-arene complex [(g6-arene)Ru(II)(en) Cl] , where the arene is para-terphenyl, exhibits promising cytotoxic effects in human tumor cells including those resistant to conventional cisplatin [121]. The effect of this Ru(II)-arene complex on DNA synthesis was determined using the [3H]-thymidine incorporation assay in ovarian tumor A2780 cells. The results indicate that the Ru(II) terphenyl complex is capable of inhibiting DNA synthesis in living cells. +

6. Summary and outlook In this Review, we have highlighted various DNA binding modes of ruthenium complexes of biological or biomedical significance, how these complexes target various DNA conformations and affect some DNA processing enzymes. We show that ruthenium complexes upon their binding to DNA may produce in this biomacromolecule a broad variety of structural motifs recognized by various damaged-DNA binding-proteins or enzymes, which may affect numerous cellular downstream processes participating in biological or biomedical effects. The reason is that the physicochemical properties and activity of ruthenium complexes can be changed by a variety of organic ligands around the ruthenium metal center. Thus, ruthenium complexes have become highly desirable across numerous fields of molecular biology and pharmacology in which DNA plays a role. It is, therefore, unsurprising that significant efforts continue in the area of preparing new ruthenium compounds that bind to DNA basically in ways different from those observed for hitherto investigated biologically active ruthenium or platinum complexes. The efforts we highlight here also include the ability of ruthenium complexes to photocleave DNA and induce synergistic enhancement of DNA damage due to a combined action with ionizing radiation. DNA interactions have been up to recently the important focus within the field of ruthenium complexes of biological or biomedical significance very likely also because the established pharmacological target of so far most successful anticancer metallodrugs used in the clinic, i.e., platinum cytostatics, is DNA. Hence, numerous mechanistic studies trying to clarify the mechanism of action of new ruthenium complexes have been typically focused mainly on DNA binding. On the other hand, cells contain the immense quantities of other biomolecules, such as proteins, polysaccharides, lipids, etc. and the structural properties of ruthenium complexes also provide a basis for targeting these biomolecules. Thus, undoubtedly, theoretical background needed for design and development of new ruthenium complexes useful for biological or biomedical applications would also benefit from studies affording information about interactions of ruthenium complexes with other cellular components than DNA. Nevertheless, an important step in studies aimed at understanding of the molecular basis of the mechanism of action of metal-based compounds of biological significance remains to determine their biological target(s) and how interactions with the target affect their structure, function, and recognition by other cellular components. Biological effects of metal-based compounds involve complex processes (for instance biodistribution, cellular accumulation, toxicity, resistance) and each of these processes may have different targets and mechanisms. Thus, for understanding mechanisms underlying biological effects of metal-based compounds and their use in other biomedical applications, it is of fundamental importance to identify their cellular (pharmacological) target related to their biological effects and applications. On this occasion, it is important to admonish that the mere fact that the investigated agent modifies properties of a certain biomacromolecule does not automatically mean that this biomacromolecule is the cellular target related to the biological effect. Only those modifications which directly lead to this effect should be consid-

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ered the target. Several tests have been already described in the literature supporting the view that nuclear DNA is the important functional target of the investigated agents and should be applied to substantiate extensive studies of their DNA interactions in relation to their biological effects. We anticipate that summarization of the typical results on DNA binding of biologically active ruthenium compounds may help to shed light on their potency to affect processes in living cells and will make it possible to design a wide range of new classes of probes, luminescent biosensors and imaging or diagnostic agents, biocatalysts, nanomaterial complexes, therapeutics (including photosensitizers for PDT, radiosensitizers and antimicrobials) and theranostics. 7. Conflict of interest None. Acknowledgements This work was supported by the Czech Science Foundation – Czech Republic [grant numbers 17-09436S and 17-05302S]. References [1] V. Brabec, O. Hrabina, J. Kasparkova, Cytotoxic platinum coordination compounds. DNA binding agents, Coord. Chem. Rev. 351 (2017) 2–31. [2] M.J. Clarke, F. Zhu, D.R. Frasca, Non-platinum chemotherapeutic metallopharmaceuticals, Chem. Rev. 99 (1999) 2511–2533. [3] B.K. Keppler, K.-G. Lipponer, B. Stenzel, F. Kratz, New tumor-inhibiting ruthenium complexes, in: B. Keppler (Ed.), Metal complexes in cancer chemotherapy, VCH Verlagsgesellschaft, VCH Publishers, Weinheim, New York, 1993, pp. 187–220. [4] G. Sava, E. Alessio, A. Bergano, G. Mestroni, Sulfoxide ruthenium complexes: non toxic tools for the selective treatment of solid tumour metastases, in: M.J. Clarke, P.J. Sadler (Eds.), Topics in Biological Inorganic Chemistry. Metallopharmaceuticals, Springer, Berlin, 1999, pp. 143–169. [5] Z. Adhireksan, G.E. Davey, P. Campomanes, M. Groessl, C.M. Clavel, H. Yu, A.A. Nazarov, C.H.F. Yeo, W.H. Ang, P. Dröge, U. Rothlisberger, P.J. Dyson, C.A. Davey, Ligand substitutions between ruthenium–cymene compounds can control protein versus DNA targeting and anticancer activity, Nat. Commun. 5 (2014). [6] H. Huang, P. Zhang, B. Yu, Y. Chen, J. Wang, L. Ji, H. Chao, Targeting nucleus DNA with a cyclometalated dipyridophenazineruthenium(II) complex, J. Med. Chem. 57 (2014) 8971–8983. [7] M.R. Gill, S.N. Harun, S. Halder, R.A. Boghozian, K. Ramadan, H. Ahmad, K.A. Vallis, A ruthenium polypyridyl intercalator stalls DNA replication forks, radiosensitizes human cancer cells and is enhanced by Chk1 inhibition, Sci. Rep. 6 (2016). [8] C. Griffith, A.S. Dayoub, T. Jaranatne, N. Alatrash, A. Mohamedi, K. Abayan, Z.S. Breitbach, D.W. Armstrong, F.M. MacDonnell, Cellular and cell-free studies of catalytic DNA cleavage by ruthenium polypyridyl complexes containing redox-active intercalating ligands, Chem. Sci. 8 (2017) 3726–3740. [9] L. Zeng, P. Gupta, Y. Chen, E. Wang, L. Ji, H. Chao, Z.-S. Chen, The development of anticancer ruthenium(II) complexes: from single molecule compounds to nanomaterials, Chem. Soc. Rev. 46 (2017) 5771–5804. [10] R. Zhao, R. Hammitt, R.P. Thummel, Y. Liu, C. Turro, R.M. Snapka, Nuclear targets of photodynamic tridentate ruthenium complexes, Dalton Trans. 10926–10931 (2009). [11] L.L. Zeng, Y. Xiao, J. Liu, L.F. Tan, Synthesis, characterization, DNA-binding and cytotoxic properties of Ru(II) complexes: Ru(MeIm)4L2+ (MeIm=1methylimidazole, L = phen, ip and pip), J. Mol. Struct. 1019 (2012) 183–190. [12] R. Aird, J. Cummings, A. Ritchie, M. Muir, R. Morris, H. Chen, P. Sadler, D. Jodrell, In vitro and in vivo activity and cross resistance profiles of novel ruthenium (II) organometallic arene complexes in human ovarian cancer, British J. Cancer 86 (2002) 1652–1657. [13] G.I. Pascu, A.C.G. Hotze, C. Sanchez-Cano, B.M. Kariuki, M.J. Hannon, Dinuclear ruthenium(II) triple-stranded helicates: luminescent supramolecular cylinders that bind and coil DNA and exhibit activity against cancer cell lines, Angew. Chem., Intl. Ed. 46 (2007) 4374–4378. [14] M. Abid, F. Shamsi, A. Azam, Ruthenium complexes: an emerging ground to the development of metallopharmaceuticals for cancer therapy, Mini-Rev. Med. Chem. 16 (2016) 772–786. [15] F.E. Poynton, S.A. Bright, S. Blasco, D.C. Williams, J.M. Kelly, T. Gunnlaugsson, The development of ruthenium(II) polypyridyl complexes and conjugates for in vitro cellular and in vivo applications, Chem. Soc. Rev. 46 (2017) 7706– 7756.

92

V. Brabec, J. Kasparkova / Coordination Chemistry Reviews 376 (2018) 75–94

[16] K.D. Zheng, Q. Wu, Y. Ding, W.J. Mei, Arene ruthenium(II) complexes: the promising chemotherapeutic agent in inhibiting the proliferation, migration and invasion, Mini-Rev. Med. Chem. 16 (2016) 796–803. [17] A. Notaro, G. Gasser, Monomeric and dimeric coordinatively saturated and substitutionally inert Ru(II) polypyridyl complexes as anticancer drug candidates, Chem. Soc. Rev. 46 (2017) 7317–7337. [18] A. Bergamo, P.J. Dyson, G. Sava, The mechanism of tumour cell death by metal-based anticancer drugs is not only a matter of DNA interactions, Coord. Chem. Rev. 360 (2018) 17–33. [19] S.M. Meier-Menches, C. Gerner, W. Berger, C.G. Hartinger, B.K. Keppler, Structure-activity relationships for ruthenium and osmium anticancer agents – towards clinical development, Chem. Soc. Rev. 47 (2018) 909–928. [20] P. Kumar, S. Dasari, A.K. Patra, Ruthenium(II) complexes of saccharin with dipyridoquinoxaline and dipyridophenazine: structures, biological interactions and photoinduced DNA damage activity, Eur. J. Med. Chem. 136 (2017) 52–62. [21] A.F.A. Peacock, P.J. Sadler, Medicinal organometallic chemistry: designing metal arene complexes as anticancer agents, Chem. Asian J. 3 (2008) 1890– 1899. [22] A.M. Pizarro, P.J. Sadler, Unusual DNA binding modes for metal anticancer complexes, Biochimie 91 (2009) 1198–1211. [23] H.-K. Liu, P.J. Sadler, Metal complexes as DNA intercalators, Acc. Chem. Res. 44 (2011) 349–359. [24] G. Mestroni, E. Alessio, M. Calligaris, W.M. Attia, F. Quadrifoglio, S. Cauci, G. Sava, S. Zorzet, S. Pacor, C. Monti-Bragadin, M. Tamaro, L. Dolzani, Chemical, biological and antitumor properties of ruthenium(II) complexes with dimethylsulfoxide, Prog. Clin. Biochem. Med. 10 (1989) 71–87. [25] F. Loseto, E. Alessio, G. Mestroni, G. Lacidogna, A. Nassi, D. Giordano, M. Coluccia, Interaction of RuCl2 (dimethylsulphoxide)4 isomers with DNA, Anticancer Res. 11 (1991) 1549–1553. [26] O. Novakova, C. Hofr, V. Brabec, Modification of natural, double-helical DNA by antitumor cis- and trans-[Cl2(Me2SO)4Ru] in cell-free media, Biochem. Pharmacol. 60 (2000) 1761–1771. [27] E. Alessio, Thirty years of the drug candidate NAMI-A and the myths in the field of ruthenium anticancer compounds: a personal perspective, Eur. J. Inorg. Chem. 2017 (2017) 1549–1560. [28] G. Sava, S. Pacor, F. Bregant, V. Ceschia, Metal-complexes of ruthenium – a potential class of selective anticancer drugs, Anticancer Res. 11 (1991) 1103– 1107. [29] S. Kapitza, M.A. Jakupec, M. Uhl, B.K. Keppler, B. Marian, The heterocyclic ruthenium(III) complex KP1019 (FFC14A) causes DNA damage and oxidative stress in colorectal tumor cells, Cancer Lett. 226 (2005) 115–121. [30] J. Malina, O. Novakova, B.K. Keppler, E. Alessio, V. Brabec, Biophysical analysis of natural, double-helical DNA modified by anticancer heterocyclic complexes of ruthenium(Ill) in cell-free media, J. Biol. Inorg. Chem. 6 (2001) 435–445. [31] D. Pluim, R.C.A.M. van Waardenburg, J.H. Beijnen, J.H.M. Schellens, Cytotoxicity of the organic ruthenium anticancer drug Nami-A is correlated with DNA binding in four different human tumor cell lines, Cancer Chemother. Pharmacol. 54 (2004) 71–78. [32] A.A. Hostetter, M.L. Miranda, V.J. DeRose, K.L.M. Holman, Ru binding to RNA following treatment with the antimetastatic prodrug NAMI-A in Saccharomyces cerevisiae and in vitro, J. Biol. Inorg. Chem. 16 (2011) 1177– 1185. [33] D. Musumeci, L. Rozza, A. Merlino, L. Paduano, T. Marzo, L. Massai, L. Messori, D. Montesarchio, Interaction of anticancer Ru(III) complexes with single stranded and duplex DNA model systems, Dalton Trans. 44 (2015) 13914–13925. [34] A. Levina, A. Mitra, P.A. Lay, Recent developments in ruthenium anticancer drugs, Metallomics 1 (2009) 458–470. [35] M.I. Webb, C.J. Walsby, EPR as a probe of the intracellular speciation of ruthenium(III) anticancer compounds, Metallomics 5 (2013) 1624–1633. [36] A. Bergamo, G. Sava, Linking the future of anticancer metal-complexes to the therapy of tumour metastases, Chem. Soc. Rev. 44 (2015) 8818–8835. [37] R.E. Morris, R.E. Aird, P.D. Murdoch, H.M. Chen, J. Cummings, N.D. Hughes, S. Parsons, A. Parkin, G. Boyd, D.I. Jodrell, P.J. Sadler, Inhibition of cancer cell growth by ruthenium(II) arene complexes, J. Med. Chem. 44 (2001) 3616– 3621. [38] F. Wang, H. Chen, S. Parsons, I.D.H. Oswald, J.E. Davidson, P.J. Sadler, Kinetics of aquation and anation of ruthenium(II) arene anticancer complexes, acidity and X-ray structures of aqua adducts, Chem. Eur. J. 9 (2003) 5810–5820. [39] H.M. Chen, J.A. Parkinson, S. Parsons, R.A. Coxall, R.O. Gould, P.J. Sadler, Organometallic ruthenium(II) diamine anticancer complexes: arenenucleobase stacking and stereospecific hydrogen-bonding in guanine adducts, J. Am. Chem. Soc. 124 (2002) 3064–3082. [40] O. Novakova, H. Chen, O. Vrana, A. Rodger, P.J. Sadler, V. Brabec, DNA interactions of monofunctional organometallic ruthenium(II) antitumor complexes in cell-free media, Biochemistry 42 (2003) 11544–11554. [41] O. Novakova, J. Kasparkova, V. Bursova, C. Hofr, M. Vojtiskova, H. Chen, P.J. Sadler, V. Brabec, Conformation of DNA modified by monofunctional Ru(II) arene complexes: recognition by DNA-binding proteins and repair. Relationship to cytotoxicity, Chem. Biol. 12 (2005) 121–129. [42] M. Melchart, A. Habtemariam, O. Novakova, S.A. Moggach, F.P.A. Fabbiani, S. Parsons, V. Brabec, P.J. Sadler, Bifunctional amine-tethered ruthenium(II) arene complexes form monofunctional adducts on DNA, Inorg. Chem. 46 (2007) 8950–8962.

[43] M.G. Mendoza-Ferri, C.G. Hartinger, M.A. Mendoza, M. Groessl, A.E. Egger, R.E. Eichinger, J.B. Mangrum, N.P. Farrell, M. Maruszak, P.J. Bednarski, F. Klein, M. A. Jakupec, A.A. Nazarov, K. Severin, B.K. Keppler, Transferring the concept of multinuclearity to ruthenium complexes for improvement of anticancer activity, J. Med. Chem. 52 (2009) 916–925. [44] M.-G. Mendoza-Ferri, C.G. Hartinger, R.E. Eichinger, N. Stolyarova, K. Severin, M.A. Jakupec, A.A. Nazarov, B.K. Keppler, Influence of the spacer length on the in vitro anticancer activity of dinuclear ruthenium-arene compounds, Organometallics 27 (2008) 2405–2407. [45] O. Novakova, A.A. Nazarov, C.G. Hartinger, B.K. Keppler, V. Brabec, DNA interactions of dinuclear RuII arene antitumor complexes in cell-free media, Biochem. Pharmacol. 77 (2009) 364–374. [46] H.J. Lozano, N. Busto, G. Espino, A. Carbayo, J.M. Leal, J.A. Platts, B. Garcia, Interstrand DNA covalent binding of two dinuclear Ru(II) complexes. Influence of the extra ring of the bridging ligand on the DNA interaction and cytotoxic activity, Dalton Trans. 46 (2017) 3611–3622. [47] R.K. Gupta, G. Sharma, R. Pandey, A. Kumar, B. Koch, P.Z. Li, Q. Xu, D.S. Pandey, DNA/protein binding, molecular docking, and in vitro anticancer activity of some thioether-dipyrrinato complexes, Inorg. Chem. 52 (2013) 13984– 13996. [48] S. Tabassum, M. Zaki, M. Afzal, F. Arjmand, New modulated design and synthesis of quercetin-Cu-II/Zn-II-Sn-2(IV) scaffold as anticancer agents: in vitro DNA binding profile, DNA cleavage pathway and Topo-I activity, Dalton Trans. 42 (2013) 10029–10041. [49] D. Suh, J.B. Chaires, Criteria for the mode of binding of DNA binding agents, Bioorg. Med. Chem. 3 (1995) 723–728. [50] C.P. Tan, S.H. Wu, S.S. Lai, M.X. Wang, Y. Chen, L.J. Zhou, Y.P. Zhu, W. Lian, W.L. Peng, L.N. Ji, A.L. Xu, Synthesis, structures, cellular uptake and apoptosisinducing properties of highly cytotoxic ruthenium-Norharman complexes, Dalton Trans. 40 (2011) 8611–8621. [51] A.E. Friedman, J.C. Chambron, J.P. Sauvage, N.J. Turro, J.K. Barton, Molecular light switch for DNA – Ru(bpy)2(dppz)2+, J. Am. Chem. Soc. 112 (1990) 4960– 4962. [52] B.M. Zeglis, V.C. Pierre, J.K. Barton, Metallo-intercalators and metalloinsertors, Chem. Commun. 4565–4579 (2007). [53] H. Niyazi, J.P. Hall, K. O’Sullivan, G. Winter, T. Sorensen, J.M. Kelly, C.J. Cardin, Crystal structures of K-[Ru(phen)2d ppz]2+ with oligonucleotides containing TA/TA and AT/AT steps show two intercalation modes, Nat. Chem. 4 (2012) 621. [54] J.P. Hall, K. O’Sullivan, A. Naseer, J.A. Smith, J.M. Kelly, C.J. Cardin, Structure determination of an intercalating ruthenium dipyridophenazine complex which kinks DNA by semiintercalation of a tetraazaphenanthrene ligand, Proc. Natl. Acad. Sci. USA 108 (2011) 17610–17614. [55] M.R. Gill, P.J. Jarman, S. Halder, M.G. Walker, H.K. Saeed, J.A. Thomas, C. Smythe, K. Ramadan, K.A. Vallis, A three-in-one-bullet for oesophageal cancer: replication fork collapse, spindle attachment failure and enhanced radiosensitivity generated by a ruthenium(II) metallo-intercalator, Chem. Sci. 9 (2018) 841–849. [56] L.-Y. Li, H.-N. Jia, H.-J. Yu, K.-J. Du, Q.-T. Lin, K.-Q. Qiu, H. Chao, L.-N. Ji, Synthesis, characterization, and DNA-binding studies of ruthenium complexes [Ru(tpy)(ptn)]2+ and Ru(dmtpy)(ptn)]2+, J. Inorg. Biochem. 113 (2012) 31–39. [57] Q.Q. Yu, Y.A. Liu, L. Xu, C.P. Zheng, F.L. Le, X.Y. Qin, Y.Y. Liu, J. Liu, Ruthenium (II) polypyridyl complexes: cellular uptake, cell image and apoptosis of HeLa cancer cells induced by double targets, Eur. J. Med. Chem. 82 (2014) 82–95. [58] W.-X. Hong, F. Huang, T. Huan, X. Xu, Q. Han, G. Wang, H. Xu, S. Duan, Y. Duan, X. Long, Y. Liu, Z. Hu, Comparative studies on DNA-binding and in vitro antitumor activity of enantiomeric ruthenium(II) complexes, J. Inorg. Biochem. 180 (2018) 54–60. [59] L. Perdisatt, S. Moqadasi, L. O’Neill, G. Hessman, A. Ghion, M.Q.M. Warraich, A. Casey, C. O’Connor, Synthesis, characterisation and DNA intercalation studies of regioisomers of ruthenium(II) polypyridyl complexes, J. Inorg. Biochem. 182 (2018) 71–82. [60] M. Eriksson, M. Leijon, C. Hiort, B. Norden, A. Graslund, Minor groove binding of [Ru(phen)3]2+ to d(CGCGATCGCG)2 evidenced by two-dimensional nuclear magnetic resonance spectroscopy, J. Am. Chem. Soc. 114 (1992) 4933–4934. [61] M. Eriksson, M. Leijon, C. Hiort, B. Nordén, A. Gräslund, Binding of D- and K[Ru(phen)3]2+ to [d(CGCGATCGCG)]2 studied by NMR, Biochemistry 33 (1994) 5031–5040. [62] C.M. Dupureur, J.K. Barton, Use of selective deuteration and 1H NMR in demonstrating major groove binding of D-[Ru(phen)2d ppz]2+ to d (GTCGAC)2, J. Am. Chem. Soc. 116 (1994) 10286–10287. [63] C.M. Dupureur, J.K. Barton, Structural studies of K- and D-Ru(phen)2d ppz2+ bound to d(GTCGAC)2: Characterization of enantioselective intercalation, Inorg. Chem. 36 (1997) 33–43. [64] R.E. Holmlin, E.D.A. Stemp, J.K. Barton, Ru(phen)2d ppz2+ luminescence: dependence on DNA sequences and groove-binding agents, Inorg. Chem. 37 (1998) 29–34. [65] I. Greguric, J.R. AldrichWright, J.G. Collins, A 1H NMR study of the binding of D-[Ru(phen)2DPQ]2+ to the hexanucleotide d(GTCGAC)2. Evidence for intercalation from the minor groove, J. Am. Chem. Soc. 119 (1997) 3621– 3622. [66] J.G. Collins, A.D. Sleeman, J.R. Aldrich-Wright, I. Greguric, T.W. Hambley, A 1H NMR study of the DNA binding of ruthenium(II) polypyridyl complexes, Inorg. Chem. 37 (1998) 3133–3141.

V. Brabec, J. Kasparkova / Coordination Chemistry Reviews 376 (2018) 75–94 [67] J.G. Collins, J.R. Aldrich-Wright, I.D. Greguric, P.A. Pellegrini, Binding of the Dand K-enantiomers of [Ru(dmphen)2dpq]2+ to the Hexanucleotide d (GTCGAC)2, Inorg. Chem. 38 (1999) 5502–5509. [68] A. Greguric, I.D. Greguric, T.W. Hambley, J.R. Aldrich-Wright, J.G. Collins, Minor groove intercalation of D-[Ru(Me2phen)2d ppz]2+ to the hexanucleotide d(GTCGAC)2, Dalton Trans. 849–855 (2002). [69] D.-L. Ma, C.-M. Che, F.-M. Siu, M. Yang, K.-Y. Wong, DNA binding and cytotoxicity of ruthenium(II) and rhenium(I) complexes of 2-amino-4phenylamino-6-(2-pyridyl)-1,3,5-triazine, Inorg. Chem. 46 (2007) 740–749. [70] A. Ghosh, P. Das, M.R. Gill, P. Kar, M.G. Walker, J.A. Thomas, A. Das, Photoactive RuII-polypyridyl complexes that display sequence selectivity and high-affinity binding to duplex DNA through groove binding, Chem. Eur. J. 17 (2011) 2089–2098. [71] L. Chen, H. Chao, Q. Zhao, H. Li, Unique optical oxygen-sensing performance of [Ru(IP)2(HNAIP)]2+ during the groove-binding-induced B-to-Z DNA conformational transition, Inorg. Chem. 54 (2015) 8281–8287. [72] S.B. Singh, A.S. Kumbhar, A. Khan, Honeycomb-like ordered assembly of DNA induced by flexible binuclear ruthenium(II)–polypyridyl complexes, Chem. Eur. J. 22 (2016) 15760–15771. [73] G.S. Yellol, A. Donaire, J.G. Yellol, V. Vasylyeva, C. Janiak, J. Ruiz, On the antitumor properties of novel cyclometalated benzimidazole Ru(II), Ir(III) and Rh(III) complexes, Chem. Commun. 49 (2013) 11533–11535. [74] R.P. Paitandi, R.S. Singh, S. Mukhopadhyay, G. Sharma, B. Koch, P. Vishnoi, D.S. Pandey, Synthesis, characterization, DNA binding and cytotoxicity of fluorodipyrrin based arene ruthenium(II) complexes, Inorg. Chim. Acta 454 (2017) 117–127. [75] I. de la Cueva-Alique, S. Sierra, L. Muñoz-Moreno, A. Pérez-Redondo, A.M. Bajo, I. Marzo, L. Gude, T. Cuenca, E. Royo, Biological evaluation of water soluble arene Ru(II) enantiomers with amino-oxime ligands, J. Inorg. Biochem. 183 (2018) 32–42. [76] Y. Benabdelouahab, L. Muñoz-Moreno, M. Frik, I.d.l. Cueva-Alique, M.A.E. Amrani, M. Contel, A.M. Bajo, T. Cuenca, E. Royo, Hydrogen bonding and anticancer properties of water-soluble chiral p-cymene RuII compounds with amino-oxime ligands, Eur. J. Inorg. Chem. (2015) 2295–2307. [77] M. Palumbo, Introduction to DNA sequence-specific agents, in: G.B. Jones, M. Palumbo (Eds.), Advances in DNA Sequence-Specific Agents, Elsevier, 1998, pp. 1–6. [78] J.K. Barton, J.M. Goldberg, C.V. Kumar, N.J. Turro, Binding modes and base specificity of tris(phenanthroline)ruthenium(II) enantiomers with nucleic acids: tuning the stereoselectivity, J. Am. Chem. Soc. 108 (1986) 2081–2088. [79] H.M. Chen, J.A. Parkinson, R.E. Morris, P.J. Sadler, Highly selective binding of organometallic ruthenium ethylenediamine complexes to nucleic acids: novel recognition mechanisms, J. Am. Chem. Soc. 125 (2003) 173–186. [80] F. Wang, J. Bella, J.A. Parkinson, P.J. Sadler, Competitive reactions of a ruthenium arene anticancer complex with histidine, cytochrome c and an oligonucleotide, J. Biol. Inorg. Chem. 10 (2005) 147–155. [81] F. Wang, J. Xu, A. Habtemariam, J. Bella, P.J. Sadler, Competition between glutathione and guanine for a ruthenium(II) arene anticancer complex: detection of a sulfenato intermediate, J. Am. Chem. Soc. 127 (2005) 17734– 17743. [82] K. Wu, Q. Luo, W.B. Hu, X.C. Li, F.Y. Wang, S.X. Xiong, P.J. Sadler, Mechanism of interstrand migration of organoruthenium anticancer complexes within a DNA duplex, Metallomics 4 (2012) 139–148. [83] K. Wu, W.B. Hu, Q. Luo, X.C. Li, S.X. Xiong, P.J. Sadler, F.Y. Wang, Competitive binding sites of a ruthenium arene anticancer complex on oligonucleotides studied by mass spectrometry: ladder-sequencing versus top-down, J. Am. Soc. Mass Spectrom. 24 (2013) 410–420. [84] K. Wu, S.Y. Liu, Q. Luo, W.B. Hu, X.C. Li, F.Y. Wang, R.H. Zheng, J. Cui, P.J. Sadler, J.F. Xiang, Q. Shi, S.X. Xiong, Thymines in single-stranded oligonucleotides and G-quadruplex DNA are competitive with guanines for binding to an organoruthenium anticancer complex, Inorg. Chem. 52 (2013) 11332–11342. [85] G.-L. Gan, H. Chao, X.-P. Cai, Z.-S. Jiang, H. Li, Condensation and salt-induced decondensation of DNA upon incorporation of a V-shaped luminescent [Ru2(bpy)4(mbpibH2)]4+, J. Inorg. Biochem. 129 (2013) 9–14. [86] B. Sun, J.X. Guan, L. Xu, B.L. Yu, L. Jiang, J.F. Kou, L. Wang, X.D. Ding, H. Chao, L.N. Ji, DNA condensation induced by ruthenium(II) polypyridyl complexes Ru(bpy)2(PIPSH)2+ and Ru(bpy)2(PIPNH)2+, Inorg. Chem. 48 (2009) 4637– 4639. [87] Y.-J. Liu, Z.-H. Liang, X.-L. Hong, Z.-Z. Li, J.-H. Yao, H.-L. Huang, Synthesis, characterization, cytotoxicity, apoptotic inducing activity, cellular uptake, interaction of DNA binding and antioxidant activity studies of ruthenium(II) complexes, Inorg. Chim. Acta 387 (2012) 117–124. [88] J.-Z. Wu, L. Li, T.-X. Zeng, L.-N. Ji, J.-Y. Zhou, T. Luo, R.-H. Li, Synthesis, characterization and luminiscent DNA-binding study of a series of ruthenium complexes containing 2-arylimidazo[f]1,10-phenanthroline, Polyhedron 16 (1997) 103–107. [89] S.S. Bhat, V.K. Revankar, A. Khan, R.V. Pinjari, M. Necas, Luminescent ruthenium(II) polypyridyl complexes as nonviral carriers for DNA delivery, Chem. Asian J. 12 (2017) 254–264. [90] Y. Kong, F. Chen, Z. Su, Y. Qian, F.-X. Wang, X. Wang, J. Zhao, Z.-W. Mao, H.-K. Liu, Bioactive ruthenium(II)-arene complexes containing modified 18bglycyrrhetinic acid ligands, J. Inorg. Biochem. 182 (2018) 194–199. [91] H.Y. Wang, Y. Qian, F.X. Wang, A. Habtemariam, Z.W. Mao, P.J. Sadler, H.K. Liu, Ruthenium(II)-arene metallacycles: crystal structures, interaction with DNA, and cytotoxicity, Eur. J. Inorg. Chem. 1792–1799 (2017).

93

[92] H.Y. Mei, J.K. Barton, Chiral probe for A-form helices of DNA and RNA: tris (tetramethylphenanthroline)ruthenium(II), J. Am. Chem. Soc. 108 (1986) 7414–7416. [93] L. Yang, S. Wang, T. Tian, X. Zhou, Advancements in Z-DNA: development of inducers and stabilizers for B to Z transition, Curr. Med. Chem. 19 (2012) 557– 568. [94] Z.G. Wu, T. Tian, J.P. Yu, X.C. Weng, Y. Liu, X. Zhou, Formation of sequenceindependent Z-DNA induced by a ruthenium complex at low salt concentrations, Angew. Chem. Int. Ed. 50 (2011) 11962–11967. [95] S. Zou, G. Li, T.W. Rees, C. Jin, J. Huang, Y. Chen, L. Ji, H. Chao, Interfering with DNA high-order structures using chiral ruthenium(II) complexes, Chem. Eur. J. 24 (2018) 690–698. [96] D. Rhodes, H.J. Lipps, G-quadruplexes and their regulatory roles in biology, Nucleic Acids Res. 43 (2015) 8627–8637. [97] L.H. Hurley, Secondary DNA structures as molecular targets for cancer therapeutics, Biochem. Soc. Trans. 29 (2001) 692–696. [98] C. Wang, Q.Q. Yu, L.C. Yang, Y.Y. Liu, D.D. Sun, Y.C. Huang, Y.H. Zhou, Q.L. Zhang, J. Liu, Ruthenium (II) polypyridyl complexes stabilize the bcl-2 promoter quadruplex and induce apoptosis of Hela tumor cells, BioMetals 26 (2013) 387–402. [99] J. Zhang, Q. Yu, Q. Li, L. Yang, L. Chen, Y. Zhou, J. Liu, A ruthenium(II) complex capable of inducing and stabilizing bcl-2 G-quadruplex formation as a potential cancer inhibitor, J. Inorg. Biochem. 134 (2014) 1–11. [100] S. Zhang, Q. Wu, H. Zhang, Q. Wang, X. Wang, W. Mei, X. Wu, W. Zheng, Microwave-assisted synthesis of ruthenium(II) complexes with alkynes as potential inhibitor by selectively recognizing c-myc G-quadruplex DNA, J. Inorg. Biochem. 176 (2017) 113–122. [101] Q.-P. Qin, T. Meng, M.-X. Tan, Y.-C. Liu, S.-L. Wang, B.-Q. Zou, H. Liang, Synthesis, characterization and biological evaluation of six highly cytotoxic ruthenium(II) complexes with 4’-substituted-2,2’:6’,2’’-terpyridine, MedChemComm 9 (2018) 525–533. [102] X. Hu, D. Yang, T. Yao, R. Gao, M. Wumaier, S. Shi, Regulation of multi-factors (tail/loop/link/ions) for G-quadruplex enantioselectivity of D- and K-[Ru (bpy)2(dppz-idzo)]2+, Dalton Trans. 47 (2018) 5422–5430. [103] C. Rajput, R. Rutkaite, L. Swanson, I. Haq, J.A. Thomas, Dinuclear monointercalating RuII complexes that display high affinity binding to duplex and quadruplex DNA, Chem. Eur. J. 12 (2006) 4611–4619. [104] S. Shi, J. Liu, T.M. Yao, X.T. Geng, L.F. Jiang, Q.Y. Yang, L. Cheng, L.N. Ji, Promoting the formation and stabilization of G-quadruplex by dinuclear RuII complex Ru2(obip)L4, Inorg. Chem. 47 (2008) 2910–2912. [105] L. Xu, X. Chen, J. Wu, J. Wang, L. Ji, H. Chao, Dinuclear ruthenium(II) complexes that induce and stabilise G-quadruplex DNA, Chem. Eur. J. 21 (2015) 4008–4020. [106] J.L. Morgan, D.P. Buck, A.G. Turley, J.G. Collins, F.R. Keene, Meso-[{Ru (phen)2}2(l-bpm)]4+: a high-affinity DNA bulge probe {bpm=2,2’bipyrimidine; phen=1,10-phenanthroline}, Inorg. Chim. Acta 359 (2006) 888–898. [107] J.L. Morgan, C.B. Spillane, J.A. Smith, D.P. Buck, J.G. Collins, F.R. Keene, Dinuclear ruthenium(II) complexes with flexible bridges as non-duplex DNA binding agents, Dalton Trans. 4333–4342 (2007). [108] G. Rossetti, P.D. Dans, I. Gomez-Pinto, I. Ivani, C. Gonzalez, M. Orozco, The structural impact of DNA mismatches, Nucleic Acids Res. 43 (2015) 4309– 4321. [109] B.M. Zeglis, J.K. Barton, Binding of Ru(bpy) 2(eilatin)2+ to matched and mismatched DNA, Inorg. Chem. 47 (2008) 6452–6457. [110] A.N. Boynton, L. Marcélis, A.J. McConnell, J.K. Barton, A Ruthenium(II) complex as a luminescent probe for DNA mismatches and abasic sites, Inorg. Chem. 56 (2017) 8381–8389. [111] M.H. Lim, H. Song, E.D. Olmon, E.E. Dervan, J.K. Barton, Sensitivity of Ru (bpy)2d ppz2+ luminescence to DNA defects, Inorg. Chem. 48 (2009) 5392– 5397. [112] P. Liu, B.-Y. Wu, J. Liu, Y.-C. Dai, Y.-J. Wang, K.-Z. Wang, DNA binding and photocleavage properties, cellular uptake and localization, and in-vitro cytotoxicity of dinuclear ruthenium(II) complexes with varying lengths in bridging alkyl linkers, Inorg. Chem. 55 (2016) 1412–1422. [113] T.K. Janaratne, A. Yadav, F. Ongeri, F.M. MacDonnell, Preferential DNA cleavage under anaerobic conditions by a DNA-binding ruthenium dimer, Inorg. Chem. 46 (2007) 3420–3422. [114] N. Mohan, M.K.M. Subarkhan, R. Ramesh, Synthesis, antiproliferative activity and apoptosis-promoting effects of arene ruthenium(II) complexes with N, O chelating ligands, J. Organomet. Chem. 859 (2018) 124–131. [115] M.R. Gill, J.A. Thomas, Ruthenium(II) polypyridyl complexes and DNA-from structural probes to cellular imaging and therapeutics, Chem. Soc. Rev. 41 (2012) 3179–3192. [116] S.W. Magennis, A. Habtemariam, O. Novakova, J.B. Henry, S. Meier, S. Parsons, I.D.H. Oswald, V. Brabec, P.J. Sadler, Dual triggering of DNA binding and fluorescence via photoactivation of a dinuclear ruthenium(II) arene complex, Inorg. Chem. 46 (2007) 5059–5068. [117] S. Betanzos-Lara, L. Salassa, A. Habtemariam, O. Novakova, A.M. Pizarro, G.J. Clarkson, B. Liskova, V. Brabec, P.J. Sadler, Photoactivatable organometallic pyridyl ruthenium(II) arene complexes, Organometallics 31 (2012) 3466– 3479. [118] G.-J. Lin, Z.-Z. Li, J.-H. Yao, H.-L. Huang, Y.-Y. Xie, Y.-J. Liu, Cytotoxicity in vitro, apoptosis, cellular uptake, cell cycle distribution, mitochondrial membrane potential detection, DNA binding, and photocleavage of ruthenium(II) complexes, Aust. J. Chem. 66 (2013) 555–563.

94

V. Brabec, J. Kasparkova / Coordination Chemistry Reviews 376 (2018) 75–94

[119] P. Liu, J. Liu, Y.Q. Zhang, B.Y. Wu, K.Z. Wang, Synthesis, DNA binding and photocleavage, and cellular uptake of an alkyl chain-linked dinuclear ruthenium(II) complex, J. Photochem. Photobiol. B-Biol. 143 (2015) 89–99. [120] H.-J. Yu, S.-M. Huang, H. Chao, L.-N. Ji, Synthesis, crystal structure and anaerobic DNA photocleavage of ruthenium complexes [Ru(tpy)(dpoq)Cl]+ and [Ru(tpy)(dpoq)CH3CN]2+, J. Inorg. Biochem. 149 (2015) 80–87. [121] T. Bugarcic, O. Novakova, A. Halamikova, L. Zerzankova, O. Vrana, J. Kasparkova, A. Habtemariam, S. Parsons, P.J. Sadler, V. Brabec, Cytotoxicity, cellular uptake, and DNA interactions of new monodentate ruthenium(II) complexes containing terphenyl arenes, J. Med. Chem. 51 (2008) 5310–5319. [122] V. Brabec, J. Pracharova, J. Stepankova, P.J. Sadler, J. Kasparkova, Photoinduced DNA cleavage and cytotoxicity of a ruthenium(II) arene anticancer complex, J. Inorg. Biochem. 160 (2016) 149–155. [123] T.J. Wang, Q.X. Zhou, Y.Y. Zhang, Y. Zheng, W.B. Wang, Y.J. Hou, G.Y. Jiang, X.X. Cheng, X.S. Wang, A ferrocenyl pyridine-based Ru(II) arene complex capable of generating center.OH and 1O2 along with photoinduced ligand dissociation, RSC Adv. 6 (2016) 45652–45659. [124] Y. Chen, W. Lei, Y. Hou, C. Li, G. Jiang, B. Zhang, Q. Zhou, X. Wang, Fine control on the photochemical and photobiological properties of Ru(II) arene complexes, Dalton Trans. 44 (2015) 7347–7354. [125] C.D.S. Silva, I.A. Paz, F.D. Abreu, A.P. de Sousa, C.P. Veríssimo, N.R.F. Nascimento, T.F. Paulo, D. Zampieri, M.N. Eberlin, A.C.S. Gondim, L.C. Andrade, I.M.M. Carvalho, E.H.S. Sousa, L.G.F. Lopes, Thiocarbonyl-bound metallonitrosyl complexes with visible-light induced DNA cleavage and promising vasodilation activity, J. Inorg. Biochem. 182 (2018) 83–91. [126] J. Malina, M.J. Hannon, V. Brabec, Interaction of dinuclear ruthenium(II) supramolecular cylinders with DNA: sequence-specific binding, unwinding, and photocleavage, Chem. Eur. J. 14 (2008) 10408–10414. [127] H. Ali, J.E. van Lier, Metal complexes as photo- and radiosensitizers, Chem. Rev. 99 (1999) 2379–2450. [128] S.H. Liu, J.H. Zhao, K.K. Deng, Y. Wu, J.W. Zhu, Q.H. Liu, H.H. Xu, H.F. Wu, X.Y. Li, J.W. Wang, Q.F. Guo, Effect of radiation on cytotoxicity, apoptosis and cell cycle arrest of human osteosarcoma MG-63 induced by a ruthenium(II) complex, Spectrochim. Acta, Part A 140 (2015) 202–209. [129] R. Carter, A. Westhorpe, M.J. Romero, A. Habtemariam, C.R. Gallevo, Y. Bark, N. Menezes, P.J. Sadler, R.A. Sharma, Radiosensitisation of human colorectal cancer cells by ruthenium(II) arene anticancer complexes, Sci. Rep. 6 (2016). [130] Z.Q. Deng, L.L. Yu, W.Q. Cao, W.J. Zheng, T.F. Chen, Rational design of ruthenium complexes containing 2,6-bis(benzimidazolyl)pyridine

[131] [132] [133]

[134]

[135]

[136]

[137]

[138]

[139]

[140] [141]

derivatives with radiosensitization activity by enhancing p53 activation, ChemMedChem 10 (2015) 991–998. J.C. Wang, DNA topoisomerases, Annu. Rev. Biochem. 65 (1996) 635–692. J.C. Wang, Cellular roles of DNA topoisomerases: a molecular perspective, Nat. Rev. Mol. Cell Biol. 3 (2002) 430–440. A. Kurzwernhart, W. Kandioller, S. Bächler, C. Bartel, S. Martic, M. Buczkowska, G. Mahlgassner, M.A. Jakupec, H.-B. Kraatz, P.J. Bednarski, V.B. Arion, D. Marko, B.K. Keppler, C.G. Hartinger, Structure-activity relationships of targeted RuII(g6-p-cymene) anticancer complexes with flavonol-derived ligands, J. Med. Chem. 55 (2012) 10512–10522. A. Kurzwernhart, W. Kandioller, C. Bartel, S. Bachler, R. Trondl, G. Muhlgassner, M.A. Jakupec, V.B. Arion, D. Marko, B.K. Keppler, C.G. Hartinger, Targeting the DNA-topoisomerase complex in a double-strike approach with a topoisomerase inhibiting moiety and covalent DNA binder, Chem. Commun. 48 (2012) 4839–4841. M.S. de Camargo, M.M. da Silva, R.S. Correa, S.D. Vieira, S. Castelli, I. D’Anessa, R. De Grandis, E. Varanda, V.M. Deflon, A. Desideri, A.A. Batista, Inhibition of human DNA topoisomerase IB by nonmutagenic ruthenium(II)-based compounds with antitumoral activity, Metallomics 8 (2016) 179–192. C. Qian, J. Wu, L. Ji, H. Chao, Topoisomerase IIa poisoning and DNA doublestrand breaking by chiral ruthenium(II) complexes containing 2-furanylimidazo[4,5-f][1,10]phenanthroline derivatives, Dalton Trans. 45 (2016) 10546–10555. Y. Sun, J. Li, H. Zhao, L. Tan, Ruthenium(II) polypyridyl complexes with 1,8naphthalimide group as DNA binder, photonuclease, and dual inhibitors of topoisomerases I and IIa, J. Inorg. Biochem. 163 (2016) 88–94. F. Gao, X. Chen, J.-Q. Wang, Y. Chen, H. Chao, L.-N. Ji, In vitro transcription inhibition by ruthenium(II) polypyridyl complexes with electropositive ancillary ligands, Inorg. Chem. 48 (2009) 5599–5601. S. Betanzos-Lara, O. Novakova, R. Deeth, A. Pizarro, G. Clarkson, B. Liskova, V. Brabec, P. Sadler, A. Habtemariam, Bipyrimidine ruthenium(II) arene complexes: structure, reactivity and cytotoxicity, J. Biol. Inorg. Chem. 17 (2012) 1033–1051. Z. Herceg, Z.-Q. Wang, Functions of poly(ADP-ribose) polymerase (PARP) in DNA repair, genomic integrity and cell death, Mutation Res. 477 (2001) 97–110. Z. Wang, H. Qian, S.-M. Yiu, J. Sun, G. Zhu, Multi-targeted organometallic ruthenium(II)-arene anticancer complexes bearing inhibitors of poly(ADPribose) polymerase-1: a strategy to improve cytotoxicity, J. Inorg. Biochem. 131 (2014) 47–55.