Development of Mononuclear (Arene)Ruthenium Complexes as Anticancer Agents: A Review

(Arene)ruthenium complexes have displayed promising activity against cancer and showed fewer side effects compared to platinum-based drugs. Activity tuning of arene complexes have been explored by varying, amines, phosphines and other N^N, N^O, N^S, N^C, S^O chelating ligands. (Arene)Ru(II) complexes of the type [(η 6 - arene)Ru(X)(Y)(Z)], [(η 6 - arene)Ru(L)(X)(Y)], [(η 6 -arene)Ru(L^L)X]Y and [(η 6 -arene)Ru(L^X)(Y)], where arene = cymene ( C ), benzene ( B ), toluene ( T ), hexamethylbenzene ( H ); L = amine, phosphine; L^L = en, diamine, diphosphine, (X^Y) = oxalate, (L^X) = acylacetonate; and (X), (Y) = halides, triflates etc. exhibit a high structural variety, and offer much potential in drug design. In this review, an overview of the progress in the field of mononuclear ruthenium complexes containing arenes and other co-ligands such as, PTA (1,3,5-triaza-7-phosphaadamantane), ethylenediamine (en), phosphines, thiosemicarbazone, acylthiourea and their bioactivity is presented.


Introduction
Cancer is one of the leading causes of death around the world (Acharya et al., 2021), ranking second for economically developed countries, and the third for developing countries (Süss-Fink, 2010). As a consequence of the development of anticancer drugs, rates of survival for cancer in females and males have shown a steady decrease since 1970 and 1985, respectively (Haberland et al., 2010). All credits go to the scientists for introducing efficient anticancer drugs, which made a visible contribution towards treating cancers. The discovery of cisplatin (1) which inhibits antitumoral properties marked the beginning of a new era of metal-based anticancer research. Platinum-based complexes like, carboplatin (2), oxaliplatin (3) (Figure 1) were also discovered later, which had significant influence on the field of metal based anticancer drugs.

Evolution of metal-based anticancer drugs
In the investigation on anticancer drugs, in 1931, Collier and Krauss studied the effect of inorganic metal salts and complexes on anticancer activity (Collier & Krauss, 1931 (Rosenberg, 1973). In 1978, cisplatin was clinically approved as an anticancer drug after Pascoe and Roberts confirmed that the cellular target of cisplatin is DNA (Pascoe & Roberts, 1974). Cisplatin presented an immense potential as an anticancer drug by demonstrating its antitumor activity for several tumour types. But it showed severe side effects such as neurotoxicity (nerve damage), alopecia (hair loss), vomiting, nephrotoxicity (kidney damage), drug resistance, bone marrow suppression, ototoxicity (loss of high frequency hearing) and vomiting when it was combined with chemotherapy (Prestayko, 1979;Lazarević et al., 2017;Allardyce et al., 2016;Zhao et al., 2019).

Generations of platinum anticancer drugs
Cisplatin, [PtCl 2 (en)], cis-[PtCl 4 (NH 3 ) 2 ] and [PtCl 4 (en)] belong to the first generation of anticancer drugs, which are platinum complexes containing chloride ligands; the binding of these platinum complexes takes place replacing chloride ligands. In the second generation of anticancer drugs, the chloride ligand was replaced by different ligands as in carboplatin (2) and [Pt(C 2 O 4 )(NH 3 ) 2 ]. In 1992, carboplatin (2) was introduced to the market and it showed less side effects than cisplatin due to its reduced neurotoxicity and nephrotoxicity (Lokich & Anderson, 1998). Though cisplatin showed low cytotoxicity in colorectal and breast tumors (Keppler et al., 1990), the third generation Pt(II) complex, oxaliplatin (3), was able to treat colon cancer liver metastases (Ono et al., 2012).

Ruthenium complexes with anticancer properties
In the search of anticancer agents containing metals other than platinum, the most promising one was found to be ruthenium. Ruthenium and osmium metallodrugs were originally designed to mimic the action of platinum in which, DNA was considered to be the primary target (Pragti et al., 2021). However, this perspective changed considerably during the last decade, because it was experimentally proven that ruthenium based anticancer drugs showed high potential as cytotoxic and cytostatic drugs which followed novel action mechanisms. One of the crucial factors that affects the anticancer activity of metal complexes is the ligand exchange kinetics in aqueous solutions. It varies in different metal cations (rates in the range of 10 -6 to 10 8 s -1 ). It was found that the rates of ligand exchange in ruthenium and platinum complexes are in the same range of 10 -3 to 10 -2 s -1 (Ranade, 2007). Therefore, ruthenium is considered to be a desirable alternative to platinum, as it mimics iron, which plays an active role in the physiological functions of the human body.
Remarkable features of ruthenium(II) centers are: (i) ruthenium 20and iron are biologically similar, (ii) they are in the Group 8, (iii) they have similar characteristics and many ruthenium complexes are not toxic, and (iv) they are quite selective of cancer cells, which is due to the ability of ruthenium to imitate iron in binding with biomolecules .

Figure 2. Timeline of discoveries of Ru-based anticancer drugs
Because of this ability, ruthenium complexes have been able to take the advantage of a human body's power to efficiently uptake and transport iron. Therefore, by imitating iron, ruthenium also has the ability to bind to transferrin and serum albumin proteins and transport within the body. Overexpression of transferrin receptors around cancer cells leads to an increased demand of iron. Hence, ruthenium based drugs can be delivered with high efficiency to the target cancer cells .
The ruthenium-arene unit has amphiphilic properties, because the arene unit is relatively hydrophobic, and that effect is counterbalanced by the metal centre which is relatively hydrophilic. The synthetic diversity provided by the arene ligand makes it an excellent scaffold for targeted chemotherapy (Dyson, 2007).

Evolution of ruthenium based anticancer drugs
Chloro-ammine complexes of ruthenium were the first to be studied as anticancer agents in a similar manner to well-known platinum complexes (1) -(3) (Figure 1).
In 1965, the ruthenium complex [Ru(NH 3 ) 4 Cl(OH)]Cl (4) (Figure 2) was used in initial investigations owing to its structural resemblance to Pt(IV) analogues. fac-[RuCl 3 (NH 3 ) 3 ] (5) showed induced-filamentous growth in E.coli which resembled cisplatin (Durig et al., 1976). Inert Ru(III) complexes were activated by reduction into the labile Ru(II) species inside the cellular surrounding (Kelman, Clarke, & Edmonds, 1976). The reduction of ruthenium diminished the π-acceptor property of the metal, which lead to the increased labiality of the chloride ligand, by facilitating hydrolysis, leading to activation. After that, they reacted with DNA, which paved the path for the anticancer activity.

Cytotoxicity of Ruthenium Complexes
Among the most investigated and advanced non-platinum anticancer metallodrugs are, ruthenium-based anticancer drugs, which have undergone considerable advances over the past two decades. Two representatives of ruthenium-based anticancer drugs are currently undergoing clinical trials, due to its high cytotoxic activity.

Indazole-based KP1019, KP1339 and IT-139 as anticancer agents
Ruthenium(III) complex, trans-[RuCl 4 (Ind) 2 [IndH] KP1019 (6) ( Figure 2) was investigated for its activity using freshly explanted human tumour cells in-vitro (Depenbrock et al., 1997). Based on experimental data, the tumour specific activity and its mode of action were found to be controlled by the mechanism of substantial binding to serum proteins in blood (Clarke, 1989). When appropriate plasma levels were reached, (6) was able to achieve clinical activity against various tumour types. The sodium salt of (6), which is KP1339 is also under clinical trials . The ruthenium(III) complex, IT-139 (8) ( Figure 2) has been used to treat cancer cells by targeting metastatic development in cancer patients (Lizardo et al., 2016), which was very effective in combination treatments and also as a single agent for carcinoid neuroendocrine tumours and colorectal cancer (Trondl et al., 2014).

Imidazole-based NAMI, NAMI-A and KP418 as anticancer agents
In 1975, [RuCl 2 (DMSO) 4 ] was explored for induced filamentous growth in E.coli and was found to possess similar properties as cisplatin (Monti Bragadin et al., 1975). However, trans-[Ru II Cl 4 (DMSO) 2 ] displayed only a slight effect on primary tumors, but a significant reduction was shown in the volume of lung metastases (Pacor et al., 1991). The trans-[Ru III Cl 4 (DMSO) 2 ] complex was unstable in aqueous solution during hydrolysis, immediately liberating DMSO. Investigation of Ru(III) complexes with imidazole ligands lead to the discovery of Na[RuCl 4 (DMSO)(HIm)] NAMI, (7) (Figure 2). The complex (7) containing both N and S donor ligands was found to be an antitumor metastasis inhibitor ( Mestroni et al., 1993) due to : (i) the effective inhibition of spontaneous metastasis formation (ii) good solubility and (iii) higher stability. Additionally, the S donor displayed a strong kinetic trans-effect, resulting in an increment of lability of the ligands of (7) in biological media. The reduction of the metal occurred before the hydrolysis of (7), which could be catalysed by biological reductants ( Mestroni et al., 1993). NAMI (7) was also investigated for its antitumor activity (Meier-Menches et al., 2018). trans-[RuCl 4 (DMSO)(Im)][HIm] (NAMI-A (9) ( Figure  2) displayed increased stability in air compared to (7), but both showed similar pharmacological effects (Pillozzi et al., 2014). Phase I studies of complex (9) was completed in 2004, and it was the first ruthenium based anticancer agent to enter clinical trials (Rademaker-Lakhai et al., 2004). KP418, (Im) 2 [ImH], and (6) were shown to induce apoptosis through the mitochondrial pathway in the cell lines of SW480 (colorectal carcinoma) (Kapitza et al., 2005). KP418, showed therapeutic activity against B16 melanoma and P388 leukaemia (Trondl et al., 2014).

Cytotoxicity of (arene)ruthenium(II) complexes
The activity of arene ruthenium complexes can be identified based on many properties, but the main focus regarding the activity would be the cytotoxicity of these complexes, involving their antitumoral and antimetastatic properties. Cytotoxicity of ruthenium complexes with phosphines, amines, and other N^N, N^O, N^S, N^C, S^O chelating ligands are presented below.

PTA as a P donor ligand
Among the monodentate ligands used in (arene)ruthenium complexes, the PTA ligand (L 1 ) is one of the most widely employed. The anticancer activity of [(η 6 -arene)Ru(PTA)(X)(Y)] was studied by   (11C) displayed anti-angiogenic properties as well (Portoghese, 1990). Variation of the anionic ligands of (11C) by replacing the chloro ligand with iodo, bromo and isocyanato ligands paved the way to new complexes which were able to show antimicrobial activity, but in contrast, antiviral activity was not seen ( Allardyce et al., 2003).  (Figure 8) showed an increase of cytotoxicity due to the replacement of chloride ligands by the oxalate ligand (Wee et al., 2006).
The aquation of (11C) in water has been studied using NMR and UV-Vis spectroscopy (Scolaro et al., 2008), and it was determined that the activation step in the cytotoxicity is aquation. Suppression of hydrolysis was observed in the blood plasma, because of the high chloride concentration in blood plasma, approximately around 100 mM, but once the compound penetrated the cell cytoplasm, aquation was activated due to the sudden drop of chloride concentrations in the cell cytoplasm, allowing the labile aqua ligand to undergo substitution by biomolecules (Dyson, 2007).
The complexes (19 and 20) were resistant to hydrolysis, but also cytotoxic, presumably activated using a different mechanism which involved the slippage of the arene ring (Vock et al., 2008). RNA or DNA are generally considered as the drug targets inside the cancer cells, but serum proteins can also act as targets. RAPTA compounds are strong inhibitors of cathepsin B, and can slightly inhibit thioredoxin reductase as well (Ang et al., 2011). The complex (11C) has the ability to slow down cell division in cancer cells, and also to induce apoptosis. The primary target of (11C) is presumed to be proteins instead DNA as proposed in platinum metallodrugs (Wu et al., 2008).

With Carbohydrate phosphine ligands
In order to tune and enhance the biological behaviour of drugs, a bioactive molecule can be incorporated to a metal complex. To perform that, various bioactive fragments within phosphine ligands have been used in the arene ruthenium frame (Biancalana et al., 2017). The cancer cell selectivity is provided by the carbohydrate fragment in the arene ruthenium complex against several cancer cell lines (Patra et al., 2016). The complexes (21) containing a carbohydrate-based ligand (Figure 8) showed intermediate anticancer activities, but they demonstrated lower cytotoxicity against nontumorigenic cells, which showed that it has cancer cell selectivity (Berger et al., 2008;Pelletier et al., 2010). By enhancing the lipophilicity of the carbohydrate moiety, the performance of this complex could be improved (Berger et al., 2008).

With monodentate phosphine ligands
Phosphine Complexes containing silicon-side groups with the general formula [Ru(cymene)Cl 2 (phosphine)] showed IC 50 values which were in the same range as of cisplatin in human leukemia cancer cells (HL-60) (Aznar et al., 2013). Similarly, complexes with aminomethylphosphanes (22) [Ru(ɳ 6 -p-cymene )Cl 2 (PPh 2 R)] ( Figure 9) showed cytotoxic activities close to cisplatin against the MCF7 (human breast adenocarcinoma) and A549 (human lung adenocarcinoma) cell lines. Ruthenium arene complexes with triphenylphosphine ligands showed enhanced ability to bind with DNA and changed its secondary and tertiary structures, in contrast to neutral complexes in which the PPh 3 ligand was absent, that could bind to DNA solely in a covalent manner (Sáez et al., 2014). The R group of PPh 2 R ligands has a variety of roles which includes its employment as a scaffold for tethering specific functionalities to the ruthenium center. The (arene)ruthenium complex tethered to BODIPY to the phosphino moiety via an amide bond (26) is highly florescent (Bertrand et al., 2018).

With monodentate N donor ligands
(Arene)ruthenium complexes of the type (27) had the ability to induce cell death through inhibition of DNA synthesis. In comparison to the free anthracene-based ligand, the uptake and the accumulation of the complex in the cells was accelerated (Vock et al., 2007).  (28) showed reasonable cytotoxicity towards cancer cells and in contrast, their cytotoxicity towards model healthy cells was less (Kilpin et al., 2012). Naphthalimide-based complexes (28) showed higher antitumor activity than the prototype complex (11C), which illustrated that the naphthalimide moiety induced higher cytotoxicity than the prototype complex (11C). Ruthenium complex of mebendazole (29), which was a widely known anthelmintic drug showed activity against HeLa cancer cell (Akhtar et al., 2017)

Cationic (arene)Ru(II) complexes containing ethylenediamine (en) displayed elevated cytotoxicity both in vivo and in vitro.
Complexes (30A and 30B) containing the cymene ligand and the complex (31) containing the biphenyl (bph) ligand retarded the growth of the human ovarian cancer cell line (A2780), which had IC 50 values similar to carboplatin. In contrast, the complex (32) with the tetrahydroanthracene (tha) ligand was hydrophobic, and showed similar antiproliferative ability as cisplatin (Aird et al., 2002).

Figure 12. (Arene)Ru(II) complexes with ethylenediamine
Arene-ruthenium-ethylenediamine units showed favorable binding towards N7 of guanine in DNA (Chen et al., 2003). When two monodentate N-donor ligands were replaced by a chelating diamine ligand, the (arene)Ru(II) complexes were found to be inactive towards the A2780 cell line. In terms of structure-activity relationship, the complexes with a more hydrophobic arene ligand and a stable bidentate N^N-donor ligand along with exchangeable halide ligand showed a higher cytotoxicity (Iida et al., 2016).
The complex with the biphenyl ligand (31) acts as a potential DNA intercalator (Liu et al., 2006). The complex (31) showed powerful stereospecific hydrogen bonding between its NH group and the C6 carbonyl group in guanine in DNA, suggesting that simultaneous stereospecific hydrogen bonding, intercalation, and covalent coordination are involved in the recognition behaviour of DNA in arene-ruthenium-diamine complexes .
It was suggested that DNA binding of complexes containing bph (31), tha (32) and dihydroanthracene (dha) (33) are due to a combination of (i) non-covalent hydrophobic interactions, (ii) covalent Ru-N (guanine N7) coordination between DNA and the arene ligand, (iii) minor groove binding, and (iv) arene intercalation. In contrast to complexes containing multiple arene rings (e.g., bph, tha, dha), complexes with single arene rings such as benzene and cymene ligands were unable to interact with DNA using intercalation (Kostrhunova et al., 2008).

With guanidine as N^N and N^O donor ligands
Guanidine plays an important role in both inorganic and organic chemistry, which is found in many natural compounds. Guanidines were tested for its nuclease activity and it showed cytotoxic properties (Jeyalakshmi et al., 2019). Due to the Yshaped CN 3 unit present) in the guanidine ligand (34 and 35), it is an electronically and sterically flexible ligand and can be used in a wide range of biological applications, such as, antitumor, anti-inflammatory, antimalarial and urease inhibition (Murtaza et al., 2011). The donor atoms of guanidine differ as N^N and N^O, in the complexes (34 and 35) respectively, and it has a direct influence on the rate of hydrolysis, and thereby on cytotoxicity (Habtemariam et al., 2006).

With β-ketoamine as a N^O donor ligand
Electronic and steric properties around the ruthenium ion can be fine-tuned using the β-ketoamine ligand in a (arene)Ru(II) complex (36) (Figure14) by varying the arene and the properties of the R group. These complexes showed significant anticancer properties in vitro, and cytotoxicity against human ovarian cancer cells (Pettinari et al., 2013).

With picolinate as a N^O donor ligand
The (arene)Ru(II) complex [(cymene)RuCl(picolinate)] (37) bound efficiently to DNA and showed antimetastatic activity and antiproliferative activity, despite its low level of genotoxicity and cytotoxicity .

With NHC as a N^C donor ligand
Cationic (arene)Ru(II) complexes (38a-f) ( Figure 15) with benzothiazole-functionalized NHC ligand (NHC = nitrogenheterocarbene) were studied for their cytotoxic activity. Their invitro cytotoxicity was evaluated using six cancer cell lines, A549, HT-29, HeLa, A2780, LoVo and HCT-116 (colon cancer) ( Chen et al., 2020). The complexes (38a) and (38b) were found to be inactive against these cell lines, and (38d) demonstrated significant cytotoxicity against the cell lines A22780 and HT-29, which occurred due to the increase in the length of the alkyl substituent.

With acylthiourea as a S^O donor ligand
(Arene)Ru(II) complexes (39) with the formula [Ru(cymene)(PPh 3 ) (S^O)]PF 6 having acylthiourea were evaluated for the cytotoxic activity on five cell lines, MCF-10A, DU145, A549, MRC-5 and MDA-MB-231 (Cunha et al., 2020). These complexes showed high selectivity towards breast cancer cells compared to cisplatin, and they were cytotoxic against the A549 and DU145 cell lines. The complex (39a) with thienyl as R 1 was cytotoxic to all the above cell lines. The cytotoxicity was enhanced by the increase of the chain length of R 2 , because the increase of chain length amplified the lipophilicity of the complexes, thereby increasing the cellular uptake of these agents.

Activity tuning of (arene)ruthenium complexes
The cytotoxicity of these (arene)ruthenium complexes were determined using various assays such as, tube formation assay (Yamamoto et al., 2003), adhesion assay (Gurgul et al., 2020), migration and invasion assay (Chambers et al., 2002), wound healing assay (Zamora et al., 2015), colony formation assay (Chen et al., 2021), RT-PCR, and western blotting. (i) Fine tuning of the bidentate ligand (L^L, L^X and X^Y) is used as one such method. Chelate ligands generally exhibit higher resistance towards substitution, and as a result the aquation is controlled by the suitable choice of the other ligands in the molecule. The toxicity of these complexes can be changed by the appropriate choice of the X ligand (Aird et al., 2002). One such example is, the change of the bidentate ligand from en to acac. Apart from increasing the pK a of the aqua complex significantly (Fernández et al., 2004), acac influenced the recognition of the complex by DNA and other targets. This selective recognition is critical for the activity of the drugs that mainly targets DNA. (Arene)Ru(II) complexes with indoloquinolines as N^N ligands have been used, because they can act as kinase inhibitors.
(ii) The nature of the exchangeable ligand (X/Y) is another factor that can be varied in order to tune the cytotoxicity of arene ruthenium complexes, because it affects the extent of hydrolysis of the Ru-X bond. For an example, though the hydrolysis difference between chloride and bromide is negligible, the hydrolysis of iodide as a halide is up to seven-fold slower than the chloride and bromide ligands. Ruthenium-pyridine bond is even more inert than iodide, and it completely blocks the hydrolysis. These inert halides are not cytotoxic and these inert species can be triggered to undergo hydrolysis using various strategies. [(cym)Ru(bpm)(py)](PF 6 ) 2 (bpm = 2,20-bipyrimidine) in which pyridine is inert, is activated using visible light to dissociate the pyridine ligand (Barragán et al., 2011). By using controlled irradiation, reactive aqua species can be cleanly generated, and it gains ability to bind with DNA, through phototriggered binding of anticancer pro-drugs.
(iii) Activation by ligand oxidation is another mechanism for fine tuning of ruthenium arene complexes. Redox mechanisms are involved in ruthenium arene thiolato-complex activation (Jaouen & Dyson, 2007). For an example, the tripeptide glutathione (GSH) is involved in the activation by oxidation of RM175 (10) in buffered solutions (Wang, Xu ei al., 2005).
(iv) Another main factor is the nature of the arene ligand. The arene complexes are not static, where benzene or hexamethylbenzene in (arene)Ru(II) complexes, can rotate around the perpendicular axis compared to biphenyl, which allows the optimization of arene interactions with DNA (Palermo et al., 2016). Thermodynamic properties and DNA recognition can be modified site-specifically in ruthenium arene complexes by varying the type of arene as, para-, meta-and ortho-isomers (Palermo et al., 2016). It is shown that the para complex displays higher cytotoxicity towards cancer cells, compared to the metaand ortho-isomers (Bugarcic et al., 2008). para-Arene complexes can bind to DNA bases through both intercalation and coordination, whereas the other less toxic isomers are able to bind only through monofunctional coordination.

Conclusions
(Arene)ruthenium complexes are an emerging class of anticancer drugs, owing to their fewer side effects compared to platinum anticancer agents. The relationship between the structure and cytotoxicity of (arene)Ru (  Monti Bragadin, C., Ramani, L., & Samer, L. (1975). Effects of cis dichlorodiammineplatinum (II) and related transition metal complexes on Escherichia coli. Antimicrob.Agents Chemother., 7 (6)