Ru(II) Compounds: Next-Generation Anticancer Metallotherapeutics?Sreekanth Thota,*,†,‡ Daniel A. Rodrigues,‡ Debbie C. Crans,§ and Eliezer J. Barreiro‡

†National Institute for Science and Technology on Innovation on Neglected Diseases (INCT/IDN), Center for TechnologicalDevelopment in Health (CDTS), Fundacao Oswaldo Cruz, Ministerio da Saude, Av. Brazil 4036, Predio da Expansao, 8° Andar, Sala814, Manguinhos, 21040-361 Rio de Janeiro, RJ, Brazil‡Laboratorio de Avaliacao e Síntese de Substancias Bioativas (LASSBio), Institute of Biomedical Sciences, Federal University of Riode Janeiro (UFRJ), P.O. Box 68023, 21941-902 Rio de Janeiro, RJ, Brazil§Department of Chemistry, Colorado State University, Fort Collins, Colorado 80523, United States

ABSTRACT: Metal based therapeutics are a precious class of drugs inoncology research that include examples of theranostic drugs, which areactive in both diagnostic, specifically imaging, and therapeuticsapplications. Ruthenium compounds have shown selective bioactivityand the ability to overcome the resistance that platinum-basedtherapeutics face, making them effective oncotherapeutic competitorsin rational drug invention approaches. The development of antineoplasticruthenium therapeutics is of particular interest because rutheniumcontaining complexes NAMI-A, KP1019, and KP1339 entered clinicaltrials and DW1/2 is in preclinical levels. The very robust, conforma-tionally rigid organometallic Ru(II) compound DW1/2 is a proteinkinase inhibitor and presents new Ru(II) compound designs asanticancer agents. Over the recent years, numerous strategies havebeen used to encapsulate Ru(II) derived compounds in a nanomaterialsystem, improving their targeting and delivery into neoplastic cells. A new photodynamic therapy based Ru(II) therapeutic, TLD-1433, has also entered clinical trials. Ru(II)-based compounds can also be photosensitizers for photodynamic therapy, which hasproven to be an effective new, alternative, and noninvasive oncotherapy modality.

1. INTRODUCTION

Tumor is the uncontrolled growth of anomalous cells in thebody.1 Cancers are among the leading causes of deathworldwide, accounting for 8.2 million deaths within 5 yearsof diagnosis.2 According to a WHO report, it is expected thatannual cancer cases will rise from 14 million in 2012 to 26million within the next 2 decades.3 There is no currentoncotherapy that is able to cure most forms of disseminatedtumors, so the discovery of novel active chemotherapeutics ispredominantly needed.4,5 There have been tremendous effortsto conquer cancer with current chemotherapy. The nextgeneration of molecularly targeted drugs have potential inpersonalized medicine because these approaches promise moreefficacious and less adverse antitumor therapies in patients whohave suffer from resistant cancers.Metallotherapeutics act by preventing cancer cell division

and trigger cancer cell apoptosis by inducing DNA damage anddisrupting DNA repair process.6−8 Metal scaffolds currentlyplay an important role in medicinal chemistry and drugdevelopment after the serendipitous discovery and develop-ment of platinum compounds.9,10 The platinum-based drugcisplatin (Figure 1) is one of the most progressive andcommonly used drugs in the clinic in the treatment ofnumerous forms of human cancers, but its therapeutic value isaccompanied by serious side effects, and thus its effectivenessdecreases by the increasing observed drug resistance.11 For

these reasons, many researchers are actively searching for otheralternative transition metal compounds, and new rutheniumcompounds have been reported as antitumor metallotherapeu-tics.12

2. DEVELOPMENT OF Ru(II) COMPOUNDS

Ruthenium-based therapeutics are promising candidates thatshow acceptable biological properties for chemotherapy andhave emerged as a favorable adjunctive to the platinum-derivedtherapeutics.13 For the past few decades ruthenium therapeuticshave successfully been used in clinical research and theirmechanisms of antitumor action have been reported.14,15

Several reviews on the anticancer ruthenium compounds havebeen reported in 2016.16−20 Ruthenium-based anticancermetallotherapeutics21,22 are very appealing alternatives becauseof their different modes of action, and they were found to havecertain merits over platinum-based therapeutics. Rutheniumcompounds have desired properties that make these rutheniumscaffolds attractive alternatives for medicinal application. (i)They are active against some cisplatin resistant cell lines. (ii)They have low side effects due to their higher selectivity forcancer cells compared with normal cells. (iii) The higher

Received: November 15, 2017Published: February 15, 2018

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selectivity of these compounds for their targets may be linkedto selective uptake by the tumor compared with healthytissue.23 (iv) Ruthenium can mimic iron in binding to somebiological molecules.23 Recently Alessio described some mythsin the field of ruthenium antitumor therapeutics includingdiscussing ruthenium therapeutics’ low toxicity becauseruthenium mimics iron. He suggested that rutheniumtherapeutics have inherently low toxicity but that ruthenium’sability to mimic iron is often confused with toxicity.13

Ruthenium belongs to the same group in the periodic tableas iron, which is reflected by its high affinity for transferrin andby the its reductive activation in cells.23,24 Some rutheniumcompounds are excellent candidates for clinical development,due the low cytotoxicity and genotoxicity, different ligandexchange kinetics, transport, activation mechanisms, and highbiological activity.2.1. Clinical Trials and Patented Compounds of

Ruthenium. There are currently four ruthenium therapeuticsin various stages of clinical trials, with one possibly about toobtain marketing approval for use in the clinic. Theantineoplastic ruthenium scaffolds that have entered clinicaltrials include Ru(III) species, imidazolium(imidazole)-(dimethylsulfoxide)tetrachlororuthenate(III) (NAMI-A)25,26

and indazolium trans-tetrachlorobis(1H-indazole)ruthenate-(III)] (KP1019)27,28 and KP133929 (Figure 1) and Ru(II)-based therapeutic TLD1433.30 Ruthenium therapeutic (NAMI-A) was the first ruthenium based complex to reach humanclinical investigations. In preclinical investigations in several

tumor animal models, ruthenium based therapeutic NAMI-Aexhibited inhibition of tumor metastases and appeared to lackcytotoxic actions. It succeeded in phase I clinical studies, but inphase II clinical studies showed only limited efficacy whichprevented further clinical development of NAMI-A. Anotherruthenium therapeutic KP1019 entered phase I clinical trials.Its low solubility limited its further development, but in itsplace a more soluble sodium salt, KP1339, is currentlyundergoing clinical trials.29 Another interesting Ru(II)therapeutic TLD1433 (Figure 2) entered phase 1 and phase2a clinical trials for nonmuscle invasive bladder cancertreatment with photodynamic therapy (PDT).30

From the past decade, some Ru(II) complexes bearing 1,10-phenanthroline and 2,2-bipyridine exerted potent activitiesagainst numerous tumor cells31−33 whereas other rutheniumcomplexes were reported as protein kinase inhibitors.34 Manymedicinal chemists have discovered new Ru(II) scaffolds thatare already being investigated in preclinical studies at variousstages of development. For example, the ruthenium complexRAPTA-C,35 combination with erlotinib, exhibited efficientanticancer action. In the past few decades several advancementson patents of antineoplastic ruthenium complexes with a rangeof different scaffolds have been reported.36−43

2.2. Ru(II) Lead Compounds (Figure 1). RAED.Ruthenium compound with diamine scaffolds are of consid-eration as prospective novel metal-based antitumor therapeu-tics. RAED44 compounds are ruthenium-arene compoundsbearing 1,2-ethylenediamine ligand. They have the ability of

Figure 1. Structures of cisplatin and ruthenium clinical trial, preclinical, and lead compounds.

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binding to DNA and form adducts with guanine. Thisruthenium therapeutic displayed potent cytotoxicity in invitro neoplastic cells with DNA interaction.45

RM175. Another ruthenium organometallic lead compoundis RM175,46 [(η6-C6H5C6H5)RuCl(H2NCH2CH2NH2-N,N′)]+PF6−. Test with this ruthenium lead complex theapoptosis was induced through Bax, p53, and loss ofclonogenicity.46

RDC11. A ruthenium derived lead compound RDC1147

exhibited in vitro cytotoxic activity for numerous tumor celllines, including those that are sensitive to and those that areresistant to cisplatin. RDC11 treatment diminished chronictoxicity compared with cisplatin and hindered the growth of thevarious xenograft model cancers in mice more efficiently thancisplatin.47

DW1/2. DW1/248 is the first ruthenium antitumor agenttargeting a signal transduction pathway. DW1/2 binds to aprotein and, as such, can be characterized as a glycogensynthase kinase (GSK)-3β inhibitor. It is also a potent activatorof p53.KP418. A lead structure was found in the imidazole-

containing complex ICR (KP418),49 imidazolium trans-[tetrachloridobis(1H-imidazole)ruthenate(III)], which provedtherapeutic activity against murine P388 leukemia and B16melanoma.ONCO4417. This ruthenium lead structure more resembles

another ruthenium lead compound RM175, whereas insertion

of chloride instead of hexafluorophosphate anion (PF6−) in

RM175 results in another ruthenium lead compoundONCO4417.50 In vitro results concluded that ONCO4417has tantamount efficacy to platinum in many tumor cell lines.This ruthenium lead compound displayed significant efficacy ininhibiting tumor metastasis.RAPTA-C. Ru(II) complexes reported in 2004 bearing

phosphoadamantane and arene ligands as a class were namedRAPTAs. The [Ru(eta(6)-p-cymene)Cl(2)(pta)], where pta is1,3,5-triaza-7-phosphaadamantane, was termed RAPTA-C51

and found to induce cell death in Ehrlich ascites carcinoma(EAC) cells via p53-JNK and mitochondrial pathways.RAPTA-T. Another effective ruthenium-containing RAPTA-

derivative is the ruthenium(II)-arene drug Ru(η6-C6H5Me)-(pta)Cl2, abbreviated RAPTA-T.52 RAPTA-T showed anti-invasive and antimetastatic effects against breast cancer cells.UNICAM-1. A water-soluble ruthenium(II) organometallic

compound [Ru(p-cymene)(bis(3,5-dimethylpyrazol-1-yl)-methane)Cl]Cl is termed UNICAM-1.53 In A17 triple negativebreast cancer cell, this ruthenium lead complex significantlyreduces the growth.

3. CELLULAR UPTAKE AND CYTOTOXICITY OF Ru(II)COMPOUNDS

The drugs need to permeate the cell membrane to act on theliving cells. Cell membranes secure and organize cells, whichcontain numerous proteins and lipids, and its action is tomonitor what substances penetrate the cells. Cellular uptake ofsmall scaffolds can occur via energy-dependent (endocytosis,active transport) and energy-independent (facilitated diffusion,passive diffusion) processes (Figure 3).29 However, the uptakeof ruthenium compounds by tumor cells or other cells must besubstantial for selective and decisive cancer treatment. Cellularuptake of the ruthenium therapeutics has been analyzed by flowcytometry.54,55 The complex [Ru(phen)2(mitatp)]

2+ exhibitedsignificant antitumor activity against several tumor cells, andflow cytometric experiments and imaging experimentssuggested that the ruthenium compound could cross cellmembrane and accumulate in the nucleus, leading to inductionof G0/G1 cell cycle arrest and apoptosis.54,55 The rutheniumcompound [Ru(DIP)2(dppz)]

2+ showed cellular uptakethrough an energy-independent process.57 For the past decade

Figure 2. Structure of Ru(II) clinical trial photodynamic therapycompound.

Figure 3. General cellular uptake mechanisms of drugs. Reproduced from ref 29 (http://dx.doi.org/10.1039/C7CS00195A), Copyright 2017, withpermission of The Royal Society of Chemistry.

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several reports on cellular uptake of ruthenium compounds hasbeen published.54−57 Schobert et al. reported cellular uptake ofRu(II) compounds as measured by ICP-OES spectrometry.They also discussed the effect of steroids and steroid bindingproteins on hormone conjugates of ruthenium.58 One veryinteresting feature of anticancer Ru(II) therapeutics is theability to inhibit the efflux pump P-glycoprotein (Pgp).59

Ru(II) compounds bearing natural antitumor naphthoquinoneplumbagin exhibited augmented efficacy against resistant tumorcell lines and inhibit the drug efflux pump Pgp.60

The crucial point in identifying substances is the selectivityindex with acceptable biological action and negligiblecytotoxicity. During the past 2 decades, several reports havebeen published on in vitro cytotoxicity of Ru(II) compoundson numerous human cancer cell lines.61−66 Several analyseshave shown that numerous factors such as cell membranechanges and cell adhesion characters, apoptosis throughmitochondrial pathway, or inhibition of topoisomerase I andII could at least in part explain the observed cytotoxicity.Bergamo et al. reported the selectivity and cytotoxicity ofruthenium compounds in preclinical and clinical trials. Inmammary carcinoma model differentiation of selectivity of theantineoplastic action of KP1019, RM175, RAPTA-T, andNAMI-A is shown in Figure 4.12 Nonselective cytotoxicity wasobserved when the compound inhibits the primary tumor andlung metastasis growth in a similar way. The in vivo results on amouse metastasizing tumor demonstrated a moderate inhib-ition of primary tumor growth for KP1019, RM175, RAPTA-T, and NAMI-A. However, the selectively varies. No selectivity

is observed for KP1019 in contrast to the other threecompounds. More than a 50% reduction of spontaneous lungmetastasis formation was observed for RM175, RAPTA-T, andNAMI-A.12

4. DNA BINDING, PROTEIN BINDING, ANDAPOPTOSIS OF RUTHENIUM COMPOUNDS

4.1. DNA Binding. The nucleus DNA is one of the vitaltargets for many oncology drugs.67,68 DNA is one of theprimary pharmacological targets for numerous FDA-approvedmetallotherapeutics (e.g., cisplatin, carboplatin, oxaliplatin) andorganic oncology drugs (doxorubicin, gemcitabine, 5-fluorour-acil, etc.).69 Significant emphases have been given to the designof compounds with ligand scaffolds that bind to DNA with siteselectivity. Interaction of transition complexes with DNA hasbeen a popular subject for researchers in the field ofbioinorganic chemistry ever since the discovery of platinumtherapeutics as an anticancer agent.70,71 Several Ru(II)complexes have shown significant DNA binding affinity.72−75

Past literature studies have indicated that RAED-C formsadducts at guanine bases of oligonucleotide DNA and thatRAPTA-C also binds to DNA.45 Ru(II) polypyridyl com-pounds afford favorable platforms for DNA-binding anddelivering bioactive drugs to the cell. The compound isactivated through the photoirradiation of the photolabile bondthat utilizes the metal to ligand charge transfer (MLCT) band,and these compounds have also been assessed as cellularprobes. The binding mode of the metallotherapeutics with

Figure 4. In mammary carcinoma model differentiation of the percent inhibition and selectivity of the antineoplastic action of KP1019, RM175,RAPTA-T, and NAMI-A. The right red bar indicates the percent inhibition of lung metastases, and left green bar indicates the percent inhibition ofprimary tumor growth. Reproduced with permission from Journal of Inorganic Biochemistry (https://www.sciencedirect.com/journal/journal-of-inorganic-biochemistry), 2012, Vol. 106; Bergamo, A.; Gaiddon, C.; Schellens, J. H.; Beijnen, J. H.; Sava, G. Approaching tumour therapy beyondplatinum drugs: status of the art and perspectives of ruthenium drug candidates, Pages 90−99,12 Copyright 2011, with permission from Elsevier.

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DNA provides insight into mechanism of action andeffectiveness of these therapeutics.69 Ruthenium-based onco-therapeutics have displayed differences with respect to theirDNA interactions depending on their structure. For example,organometallic piano-stool ruthenium(II) complexes contain-ing biphenyl rings (RM175) interact strongly with DNAbinding to guanines.76

Oncotherapeutics that target DNA are one of the mosteffective agents in clinical use and have produced significantimprovement in the survival of cancer patients when used incombination with chemotherapeutics that have differentmechanisms of action. Therefore, a detailed understanding ofthe interaction of ruthenium compounds with models ofbinding sites present in DNA is of paramount significance tounravel the mechanism of action of ruthenium-derivedcomplexes.77 During the past decade, a huge amount of workhas been published on the synthesis, cytotoxicity, and DNA-binding ability of Ru(II) compounds.78−82

4.2. Protein Binding. Over the past few decades, theanalysis of plasma concentrations of oncotherapeutics hasdemonstrated its value to clinical studies for numerous vitaldrugs. Although protein binding is a major determinant of drugaction, with very few exceptions, it is clearly only one of manyfactors that influence the disposition of oncology drugs.Normally the protein-binding was analyzed by electronicabsorption and fluorescence quenching. High concentrationof proteins in plasma and the tendency of various drugs to bindthem have led drug discovery groups to identify the significanceof plasma protein binding (PPB) in modulating the effectivedrug concentration at pharmacological target sites. Overall, paststudies implied that proteins are biological targets of the Ru(II)polypyridyl scaffolds.83 Ru(II) compounds bind to major metal-transporting proteins from human blood such as human serumalbumin (HSA) and serum transferrin (Tf). The bindingaffinity of ruthenium compounds against these two proteinsshowed that HSA appears to be a more favorable bindingpartner.84

Intracellular protein binding patterns of the clinical trialruthenium therapeutics KP1019 and KP1339 have beenreported. KP1019 is known to powerfully bind to serumproteins and hamper P-glycoprotein-mediated efflux, makingthis ruthenium therapeutic attractive for multidrug-resistanttumor therapy.85 Preclinical RAPTA-C compound has shown

significant binding affinity with two emerging protein targets,thioredoxin reductase and cathepsin B, for demonstrating theirantitumor action.86 Over the recent years several groupsreported significant protein binding affinity of Ru(II) basedscaffolds.83−86

4.3. Apoptosis. Facilitating apoptosis is a very effectiveapproach in the discovery of oncotherapeutics.87 Thecontribution of apoptosis on tumor size decrease has beenextensively investigated since many oncology drugs displaytheir action by promoting apoptosis.88 Apoptosis is a process ofprogrammed cell death and controls the development andhomeostasis in multicellular organisms and is often charac-terized by energy-dependent biochemical mechanisms anddistinct morphological characteristics. Activation of intrinsicand extrinsic pathways in cells leads to primarily induction ofapoptosis. The intrinsic pathway, also known as themitochondria-mediated pathway, is promoted by DNA damage,oxidative stress, and endoplasmic reticulum (ER) stress.29,89

Ruthenium derived clinical trial compound KP1019 binds totransferrin, which is more cytotoxic after reduction, and itcauses apoptosis via the mitochondrial pathway, also promotingthe formation of reactive oxygen species (ROS). Ru(II)compounds have been reported to induce apoptosis via themitochondrial pathway,90−92 autophagy pathway,93,94 andinduction of ROS-mediated apoptosis in tumor cells bytargeting thioredoxin reductase.95 DNA-intercalating Ru(II)compound [Ru(bpy)(phpy)(dppz)]+ has been reported as anantitumor agent and was found to be extremely cytotoxicagainst cancer cell lines. The high affinity for DNA binding ofthis ruthenium complex damages the transcription factor NF-κB on relevant DNA sequences. Any damage of thetranscription factor sequence leads to the inhibition of cellulartranscription and irreversible cancer cell apoptosis (Figure 5).82

Other reported antitumor actions of ruthenium complexes arethrough the inhibition of telomerase activity and stabilization ofG-quadruplex DNA intermediates.96

The preclinical ruthenium compound DW1/2 inhibits PI3Kand GSK3-β, which leads to apoptosis mediated by themitochondrial and p53 pathways and is the reportedmechanism of action of DW1/2 as an antitumor drug (Figure6).12

Another complex [Ru(MeIm)(npip)]2+ (npip 1/4 2-(4-nitrophenyl)imidazo[4,5-f ][1,10]phenanthroline) promotes

Figure 5. Representation of the antitumor action of the nucleus-targeting complex [Ru(bpy)(phpy)dppz]+. Reproduced with permission from ref 82.Copyright 2014, American Chemical Society.

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A549 human lung carcinoma cell apoptosis by both damagingmitochondrial homeostasis and death receptor signal pathways.This complex induces apoptosis of BEL-7402 cells through themitochondrial signal transduction pathway. The apoptoticpathways of several other ruthenium therapeutics have beenreported as well.97−100 Apoptotic and autophagy mechanismswere both induced by the [(η6-p-cymene)Ru(N,N′-hydrazinyl-thiazolo)Cl]Cl compound in the A2780 human ovarian cancercell line (Figure 7).65

5. Ru(II) COMPOUNDS AS PROTEIN KINASEINHIBITORS

The discovery of selective enzyme inhibitors is an importantactivity at the heart of medicinal chemistry and chemicalbiology. Protein kinases play a vital role in cell biology, regulatemost aspects of cellular processes, and are one of the mainoncology targets for drug discovery. Kinase inhibitors are oneof the most progressive and competently used inhibitors in thetreatment of numerous forms of human cancers. There aremore than 518 protein kinases encoded by the human genome,and many of them are associated with human cancers. Thedesign and discovery of compound scaffolds that perturbspecific protein functions are of significance for probingbiological processes and ultimately for the discovery of potent

Figure 6. Ru(II) therapeutic DW1/2 proposed mechanism of the invitro cytotoxicity in a model of human melanoma in vitro. Reproducedwith permission from Journal of Inorganic Biochemistry (https://www.sciencedirect.com/journal/journal-of-inorganic-biochemistry), 2012,Vol. 106; Bergamo, A.; Gaiddon, C.; Schellens, J. H.; Beijnen, J. H.;Sava, G. Approaching tumour therapy beyond platinum drugs: statusof the art and perspectives of ruthenium drug candidates, Pages 90−99,12 Copyright 2011, with permission from Elsevier.

Figure 7. Schematic portrayal of Ru(II)therapeutic inducing the activation of apoptotic and autophagy mechanisms in the A2780 cell line. The redcolor denotes mRNAs overexpression of specific genes in A2780 human ovarian cancer cells treated with the Ru(II) therapeutic compared withuntreated A2780 human ovarian cancer cells. Red intensities are proportional to mRNAs transcripts. Reproduced with permission from ref 65.Copyright 2015, American Chemical Society.

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and safe drugs. Many medicinal chemists are already targetingthese kinases and have discovered new compound scaffolds,some of which are already approved by FDA, with others inclinical trials and others still in the developing stage.101−103

Some literature results reported that Ru(II) based therapeuticsDW1/2 and NP309 are protein kinase inhibitors, whichsuggest that these Ru(II) compounds act as inhibitors of theGSK3 and Pim1 half-sandwich inhibitors.48 The first reports ofvery stable and conformationally rigid ruthenium(II) com-pounds containing the natural product staurosporine have beenreported as protein kinase inhibitors.104,105 The Ru(II) leadcompound bearing staurosporine (indocarbazole alkaloid) isreported as a subnanomolar ATP competitive protein kinaseinhibitor (Figure 8a).103 These reports suggested that Ru(II)bearing a staurosporine ligand in a Ru(II) complex binds to theATP binding site of Pim-1 (Figure 8b). The cocrystallizedruthenium compound bearing staurosporine (PDB code 1YHS)with Pim-1 describes a Ru(II) compound mimicking thebinding mode of staurosporine (Figure 8c and Figure 8d).103

Biersack et al. reported another interesting Ru(II) complex((arene)Ru(II)compounds) containing the tyrphostin typeepidermal growth factor receptor (EGFR) inhibitors.106

These targeted Ru(II) compounds as protein kinase inhibitorsrepresent a major advance in oncotherapy.

6. MECHANISM OF ACTION OF Ru(II) COMPOUNDSMany researchers are now seeking to develop new mechanismsof cell death by affecting the general molecular framework bythese ruthenium therapeutics.29,107−111 The ruthenium ther-apeutics are most potent when they induce apoptosis byblocking transcription. After the early discovery of rutheniumcompounds Clarke has reported a mechanism of the rutheniumcompounds by “activation by reduction”. Many researchlaboratories worldwide were conducting experiments todetermine the mechanisms of ruthenium(II) compounds andto elucidate how Ru(II) compounds carry out their antitumoreffects. From the literature reports so far, rutheniumcompounds exert their anticancer actions by impacting themitochondrial pathway, autophagy pathway, and ROS mediatedapoptosis. Recently Zeng et al. reported common representa-tion of main targets and predicted several mechanisms of actionof Ru(II) therapeutics as antitumor candidates (Figure 9).29

7. RECENT ADVANCES IN NANOMATERIALSThe emergence of bionanomaterials gives scientists a new toolto solve oncotherapy problems.112 Nanotechnology has thepotential to provide novel, paradigm-shifting solutions tomedical emergencies and is currently used as a drug carrierfor oncotherapy.113 Cancer nanotechnology is being diligentlyexamined and executed in cancer therapy, signifying a majoradvance in detection, diagnosis, and therapy. Many oncologylaboratories are using advanced techniques to discover more

Figure 8. Ruthenium(II) therapeutics that are ATP-competitive protein kinase inhibitors. (a) Shape mimicry: structure of lead staurosporine. (b)Cocrystal structure of pseudo-octahedral ruthenium half-sandwich complex with Pim-1 protein kinase (yellow color, PDB code 2BZI). (c)Enlargement of the cocrystal structure of pseudo-octahedral ruthenium half-sandwich complex with Pim-1 protein kinase (yellow color, PDB code2BZI). (d) The superimposed cocrystal structure of Pim-1 (PDB code 2BZI) with staurosporine (blue color, PDB code 1YHS) reveals the closematch in binding mode between the organometallic compound and the natural product. Reproduced with permission from Current Opinion inChemical Biology (https://www.sciencedirect.com/journal/current-opinion-in-chemical-biology) 2007, Vol. 11; Meggers, E. Exploring biologicallyrelevant chemical space with metal complexes, Pages 287−292,103 Copyright 2007, with permission from Elsevier.

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precise acting nanotechnology based cancer therapy, whichdiminishes the adverse effects of the conventional ones. Noveldrug delivery system (NDDS) are more and more applied inoncotherapy and diagnosis. In oncotherapy, diagnosis, imagingand drug delivery, nanomaterials including metallic andnonmetallic nanoparticles, polymeric nanoparticles, nanowires,carbon nanotubes, and quantum dots are currently beingdeveloped further with appropriate strategies.Nanoparticles can be designed through numerous mod-

ifications such as altering their shape, size, and physical andchemical properties to program them for targeting the desiredcells. Metal nanoparticles target the neoplastic cells througheither active or passive targeting. Previously safe and efficaciousfunctionalized nanomaterials were reported for Ru(II)−selenium nanoparticles,114 Ru(II)−gold nanomaterials,121 Ru-(II)−silica composites,29 Ru(II)−carbon nanotubes,120 andorganic and biomaterial containing Ru(II) nanomaterials. Overthe recent years, numerous strategies have been used toencapsulate Ru(II)-derived compounds in a nanomaterialsystem and ameliorate their targeting and delivery intoneoplastic cells.

Ruthenium-encapsulated silica nanoparticles were developedwith increased cellular uptake and photoactivation (Figure10A). Development of a Ru(II) photoactive compound to begrafted on the top of the UCNPs to produce DOX-UCNP@mSiO2-Ru has been accomplished (Figure 10B). Porous siliconnanoparticles (PSiNPs) to deliver photosensitized rutheniumcompounds for photodynamic therapy have been introduced(Figure 10C). Another ruthenium nanoparticle system,RuPOP@MSNs, induced cancer cell apoptosis by the AKTand MAPK signaling pathways (Figure 10D).Recently reported Ru(II) polypyridyl/thiols protected

SeNPs114,115 were found to be extremely sensitive cellularimaging agents and to induce cell death. They are thus referredto as theranostics, that is, carrying out both diagnostic andtherapeutic purposes at the same time. Luminescent, multi-functionalized Ru(II) polypyridyl-encapsulated selenium nano-particles can inhibit bFGF-induced angiogenesis by suppressingthe AKT and Erk signaling pathways which induce apoptosis(Figure 11).Another ruthenium compound (Ru-MUA@Se) exerts multi-

ple functions by having dual-target inhibitors that directlysuppress the tumor growth by inducing apoptosis and exerting

Figure 9. General portrayal of the principal targets and projected mechanisms of action of ruthenium complexes as oncotherapeutics. Reproducedfrom ref 29 (http://dx.doi.org/10.1039/C7CS00195A), Copyright 2017, with permission of The Royal Society of Chemistry.

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dependence on ROS generation.116 Nanoparticle/Ru(II)polypyridyl complex assembled for NIR-activated release of aDNA covalent-binding agent.117 Ru(II)polypyridine compoundencapsulated within liposomes reduces the TNBC tumorgrowth and is a favorable theranostic approach for breastcancer therapy (Figure 12). These results indicate that the Ru-liposome system has greater significance on nanooncology.118

Ruthenium compounds carrying functionalized multiwalled

carbon nanotubes are able to antagonize tumor multidrug

resistance and radioresistance.119 A Ru(II) compound

deposited on single-walled carbon nanotube composites was

used for bimodal photodynamic and photothermal therapy.120

Consequently, effective nanoapproaches for oncotherapy in the

Figure 10. (A) Mechanized MSNPs graphical portrayal. (B) Schematic representation and UCNP@mSiO2 nanoparticles TEM image (1) and drugrelease from DOX-UCNP@mSiO2-Ru nanoparticles (2). (C) Synthetic route for pSiNP-Ru-PEG-Man. (D) RuPOP@MSNs construction reactionpathways. Reproduced from ref 29 (http://dx.doi.org/10.1039/C7CS00195A), Copyright 2017, with permission of The Royal Society of Chemistry.

Figure 11. Schematic representation of inhibition of bFGF-induced angiogenesis and apoptosis (suppressing the AKT and ERK signaling pathways)by luminescent Ru(II) polypyridyl encapsulated selenium nanoparticles. Reproduced with permission from Biomaterials (https://www.sciencedirect.com/journal/biomaterials), 2013, Vol. 34; Sun, D.; Liu, Y.; Yu, Q.; Zhou, Y.; Zhang, R.; Chen, X.; Hong, A.; Liu, J. The effects of luminescentruthenium(II) polypyridyl functionalized selenium nanoparticles on bFGF-induced angiogenesis and AKT/ERK signaling, Pages 171−180,115Copyright 2012, with permission from Elsevier.

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past few years have witnessed much progress involvingnanotechnology strategies.121,122

8. NOVEL THERAPEUTIC APPROACHES FOR Ru(II)COMPOUNDS8.1. Photodynamic Therapy. Over the recent years,

scientists are searching for advanced innovative approaches in

cancer treatment. Photodynamic therapy has proven to be anew, impressive, attractive, and noninvasive oncotherapymodality. Photodynamic therapy123 was developed for themanagement of neoplastic and nonmalignant diseases and totreat specific types of tumors (i.e., bladder, lung, and urinarytumors). Photodynamic therapy (PDT) has the capability tomeet many currently unmet medical emergencies. The working

Figure 12. Schematic portrayal of ruthenium polypyridyl complex [Ru(phen)2dppz](ClO4)2(Ru) encapsulated within bilayer of liposomes (Lipo-

Ru), which reduces the triple-negative breast cancer (TNBC) and is a favorable theranostic approach for cancer therapy. Reproduced withpermission from ref 118. Copyright 2017, American Chemical Society.

Figure 13. Schematic portrayal of Ru(II) complex-functionalized single-walled carbon nanotubes (Ru@SWCNTs) for bimodal two-photonphotodynamic therapy (TPPDT) and photothermal therapy (PTT) with irradiation (808 nm). Reproduced with permission from ref 120. Copyright2017, American Chemical Society.

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principle of PDT is the light activation of a photosensitizer(PS), which produces ROS that are toxic and thus must bemanaged appropriately. These ROS then lead to apoptosis, withonly minimal damage to normal tissues.124

Recent literature evidence suggests that many Ru(II) derivedcompounds act as photosensitizers for both one- and two-photon photodynamic therapy.125−130 These metallotherapeu-tics are potential replacements to the current photosensitizers(PSs). Zhang and her co-workers reported on development ofRu(II) complex-functionalized single-walled carbon nanotubes(Ru@SWCNTs) as nanotemplates for bimodal two-photonphotodynamic therapy (TPPDT) and photothermal therapy(PTT) with irradiation (808 nm), illustrated in Figure 13.120

Upon treatment with light, the Ru(II) compounds are liberatedand then produce 1O2 upon the two-photon laser irradiation(808 nm). These results suggest that bimodal therapypotentially exerts a greater significant antitumor effectcompared with one-modal therapy.120 Ru(II) therapeutics fortwo-photon photodynamic therapy have been reported.131,132

Ru(II)-based therapeutics are reported as radiosensitizerswhen used in combination with clinically relevant doses ofradiation therapy.133 From the clinical examination, theyreported that these ruthenium scaffolds should be equallyefficient, with no indication of cross-resistance to platinumtherapeutics. These results also suggest impressive radio-sensitization with clinically relevant doses of RT comparedwith p53-mutated or p53-null, higher radiosensitizing activity inp53-wild-type cells and a biological mechanism of action thathas been shown to affect increasing the destruction of DNA bythese ruthenium scaffolds.133 These significant outcomeshighlight the immense potential of ruthenium compounds inPDT.8.2. Photoactivated Chemotherapy. Although the PDT

strategy has been applied in oncotherapy, the major limitationof this approach is related to the low levels of oxygen (hypoxia),commonly found in solid tumors.134 Therefore, for these types

of tumors an innovative approach in which light-mediatedtoxicity is independent of oxygen levels must be developed, andthis technique is called photoactivated chemotherapy (PACT).The chemical compounds for this technique exhibit light-activated characteristics and are referred to as photoactivatedchemotherapy agents.127,135 The mechanism of action of theseagents is based on the ligand exchange to create metal centersable to form DNA adducts or photorelease of a bioactivecompound.135,136 Ligand exchange plays a vital part in PACT.Glazer et al. denoted antitumor Ru(II) compounds bearingmethylated bipyridyl, pyridylbenzazole ligands and theirphotochemical and photobiological action.137,138 Very recentwork has been reported about interaction between Ru(II)trans-tetrapyridyl ruthenium therapeutic and an oligonucleotidecontrolled by light irradiation.139

The strategy of using Ru(II)-photocaged therapeutics hasbeen denoted in several recent studies in the literature thatassociate a drug with a complex to the superior restraint of thebiological action and release of the drug in selected tissuesusing light.140−142 Abiraterone is an inhibitor of cytochromeP450 17A1 inhibitor (CYP17A1), useful in metastatic prostatecancer therapy. The aid of abiraterone Ru(II)-photocagedcomplexes led to the selective release of abiraterone afterexposure to visible light and potent CYP inhibition.141 Theseresults suggest that the use of this strategy can avert the adverseeffects of the parent drugs.This combined mechanism can also be achieved since after

activation by light the ligand is released (drug), which hasbiological activity. In addition, the resulting complex may stillbe able to cause damage to the DNA, resulting in a synergisticactivity.143 A last approach is to use the combination ofmechanisms for developing more efficient Ru(II) therapeuticswith dual potential PDT and PACT.144,145

Figure 14. Schematic illustration of cellular mechanism involved in the activity of RAPTA-C and erlotinib combinations. In ECRF24 cells,destruction of DNA may lead to failure to progress through G1/S checkpoint, which therefore induces apoptosis. In A2780 and A2780cisR cells,genesis of DNA bridges and micronuclei is by mitotic defection. Due to the genesis of DNA bridges, cytokinesis failure occurs, which leads tosenescence. Reproduced with permission from ref 35 (http://dx.doi.org/10.1038/srep43005), licensed under a Creative Commons Attribution 4.0International License (https://creativecommons.org/licenses/by/4.0/legalcode), Nature Publishing Group.

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9. SUCCESSES, FUTURE CHALLENGES, AND SCOPESFOR DRUG DISCOVERY

Many pharmaceutical industries as well as nonprofit govern-ment and nongovernment organizations all over the world areeagerly expecting the development of novel oncotherapeutics.However, drug invention and development are proceedingslowly with low success rates, specifically in last steps of theprocess, the clinical trials. This is augmented by the increasingdevelopment of drug resistance, which remains a formidablechallenge. Metallotherapeutics are a unique class of drugs inoncology research, also contributing to many possibilities inimaging, cancer therapy, and theranostics at the same time.Currently almost 50% of patients undergoing chemotherapyreceive some type of a platinum medication; however, drugresistance to platinum drugs limits its applications and tracingfor adjunctive metal therapeutics.During the past 3 decades ruthenium compounds with

varying scaffolds have shown tremendous selective bioactivity,as well as the capacity to overcome the resistance of platinum-based therapeutics, making them successful oncotherapeuticcompetitors in a rational drug invention approach. It isgratifying that four of the ruthenium-derived therapeuticsentered human clinical trials, but unfortunately, the results ofphase 1 and phase 2 clinical studies did not support thecontinuation of two of these drugs (NAMI-A, and KP1019) tophase III clinical trials.29 Two compounds remain underconsideration in clinical trials: KP133929 and the theranosticcompound TLD1433. Some ruthenium based chemotherapeu-tics have been proven to be mitochondria-targeting oncother-apeutic candidates. Most of the developed Ru(II) scaffolds arelipophilic and carry a positive charge, which expedite theirdispersion across the cell membrane. The evidence suggeststhat Ru(II) therapeutics are significant candidates for futureanticancer drug discovery.Comprehensive investigations have been undertaken to alter

the structure of Ru(II) compound scaffolds to ameliorate theirdrug efficacy, while so far minimal effort has been conductedtoward determining drug combinations.35,146,147 Drug combi-nation treatments in tumor models indicate effective inducedapoptosis without added toxicity (Figure 14).35 Furtherdevelopment of drug combinations in the future would allowfor analysis of their synergistic antineoplastic action inpreclinical drug-resistant tumor models, with the objective ofameliorating the therapeutic potential and protracting lifeexpectancy of the patients. These potential outcomes affordvaluable, authentic knowledge on ruthenium-derived candidatemedicaments and modern insights for future optimizedoncotherapy protocols. Novel oncotherapeutics with effectivemolecular mechanisms of action are imperative in chemo-therapy to kill specific tumor types and to conquer toxicadverse effects. The detailed mechanisms of action of some ofthe ruthenium based therapeutics are still under someinvestigation and present a big challenge for inorganicmedicinal chemists.In a recent investigation, 95% of potential oncotherapeutics

entering clinical development failed, correlating with an averageof 90% for compounds in all therapeutic areas, which is a greatchallenge in the oncology drug discovery. We have summarizedsome of the issues above relating to the complexities oftranslation. Indeed, in addition to the developmental andmechanistic aspects of drug development, increasing attentionto speciation and formulation may also assist this translational

aspect of drug development.148,149 Complex clinical trials withan effective targeted translational research constituent enablemajor advances in the understanding of specific cancer typesand directly contribute to defining new tailored standards ofpatient care.From all this evidence, it is the hope that Ru(II) derived

therapeutics will enter into the market as a supportive toplatinum based chemotherapeutics. We present an overview ofthe comprehensive development of Ru(II) compounds asanticancer metallotherapeutics, which should inspire youngbudding researchers and senior researchers to enter theinteresting field of metallotherapeutics. It is important toconclude that the pharmacokinetic profile of ruthenium-basedtherapeutics is yet to be ascertained in humans, but emergentresults remain positive, keeping our hope alive that designingeffective ruthenium-derived compounds to selectively targettumor cells is an achievable goal resulting in a Ru compoundthat will progress to clinical use.

■ AUTHOR INFORMATIONCorresponding Author*Phone: +55-21-38829234. Fax: +55-21-22900494. E-mail:stsreekanththota@gmail.com, sthota@cdts.fiocruz.br.ORCIDSreekanth Thota: 0000-0002-7501-3987Eliezer J. Barreiro: 0000-0003-1759-0038Author ContributionsThe manuscript was written with contributions from allauthors. All authors have given approval to the final manuscript.NotesThe authors declare no competing financial interest.BiographiesSreekanth Thota, Ph.D., is a Visiting Researcher at the Center forTechnological Development in Health, Fiocruz & LASSBio, UFRJ inRio de Janeiro, Brazil. He studied Pharmaceutical Chemistry atKakatiya University (India) and Rajiv Gandhi University of HealthSciences (Bangalore, India) and obtained his Ph.D. from JawaharlalNehru Technological University Hyderabad (India) in 2011. He thendid postdoctoral work at Colorado State University, U.S., under thesupervision of Prof. Debbie C. Crans. He received the CAPES-FiocruzVisiting Researcher Award in 2014. His research interest is focused onsynthesis, drug design, drug development, and medicinal chemistryand has published over 40 articles in peer-reviewed journals and bookchapters and a book concerning the field of medicinal chemistry. He isthe inventor of many bioactive scaffolds.

Daniel A. Rodrigues obtained his M.S. degree in Chemistry fromChemistry Institute, Federal University of Rio de Janeiro, Rio deJaneiro, Brazil. Currently, his Ph.D. research is being carried out atLaborato rio de Avaliaca o e Sıntese de Substancias Bioativas(LASSBio) at the Institute of Biomedical Sciences of FederalUniversity of Rio de Janeiro, and is related to the medicinal chemistryfield, particularly the design, synthesis, and biological evaluation ofmultitarget ligands acting as anticancer agents.

Debbie C. Crans, Ph.D., is a Professor of Chemistry in Organic,Inorganic Chemistry, Chemical Biology, and Cell and MolecularBiology at Colorado State University. She was an undergraduate at theH. C. Ørsted Institute in Denmark, a graduate student at HarvardUniversity, and a postdoctoral fellow at the University of California,Los Angeles. Crans’s work has systematically explored speciation andsolution chemistry of transition metal complexes, polyoxometalates,and their activities in more complex biological systems. She has been

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recognized with the first Vanadis Award in 2004 and an Arthur CopeScholar award in 2015 for her fundamental work on vanadiumcomplexes in diabetes. She is currently investigating the structural andfunctional involvement of menaquinone in the membrane associatedelectron transfer complex in pathogens.

Eliezer J. Barreiro, Ph.D., concluded his scientific education(Docteur-Es-Sciences d’Etat) in Medicinal Chemistry at the Universityof Grenoble, France, in 1978. He spent 4 years as Associate Professorof Organic Chemistry at Federal University of Sao Carlos, S.P., from1979 to 1983 before he joined the Federal University of Rio de Janeirowhere he got a permanent position. He works in the medicinalchemistry field and founded the Laboratorio de Avaliacao e Sıntese deSubstancias Bioativas (LASSBio) at the Institute of BiomedicalSciences of Federal University of Rio de Janeiro. Professor Barreirohas published over 330 journal articles and book chapters and a bookconcerning medicinal chemistry, and he is the inventor of 25 patents ofbioactive compounds.

■ ACKNOWLEDGMENTS

S.T, D.A.R, and E.J.B. acknowledge the support of theConselho Nacional de Desenvolvimento Cientifico e Tecnolo-gico (CNPq-BR), Coordenacao de Aperfeicoamento de Pessoalde Nıvel Superior (CAPES-BR), and Oswaldo Cruz Founda-tion (Fiocruz-BR). S.T. is thankful to Dr. Carlos M. Morel,Director, CDTS-Fiocruz, for his support for carrying out thisresearch. The author is thankful to Prof. Carlos AlbertoManssour Fraga and Prof. Lıdia M. Lima for their insights andcritical review of the manuscript. The funders had no role instudy design, data collection and analysis, decision to publish,or preparation of the manuscript. The authors are grateful tothe reviewers for their careful comments and precioussuggestions. The authors also acknowledge support from theInstituto Nacional de Ciencia e Tecnologia de Inovacao emDoencas de Populacoes Negligenciadas (INCT-IDPN).

■ ABBREVATIONS USED

ATP, adenosine triphosphate; BSA, bovine serum albumin;CNS, central nervous system; DNA, deoxyribonucleic acid;EGFR, epidermal growth factor receptor; ER, endoplasmicreticulum; HAS, human serum albumin; ICP-OES, inductivelycoupled plasma optical emission spectrometry; MLCT, metalto ligand charge transfer; m-RNA, messenger RNA; NDDS,novel drug delivery system; NIR, near-infrared; NP, nano-particle; PACT, photoactivated chemotherapy; PDT, photo-dynamic therapy; PEG, polyethylene glycol; Pgp, P-glycopro-tein; PPB, plasma protein binding; PS, photosensitizer; PTT,photothermal therapy; RDC, ruthenium derived compound;ROS, reactive oxygen species; RT, radiation therapy; SeNP,selenium nanoparticle; TNBC, triple-negative breast cancer;TPPDT, two-photon photodynmaic therapy; WHO, WorldHealth Organization

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