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Ru(II) Compounds: Next-generation anticancer metallotherapeutics? Sreekanth Thota, Daniel A Rodrigues, Debbie C Crans, and Eliezer J. Barreiro J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 15 Feb 2018 Downloaded from http://pubs.acs.org on February 15, 2018

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Journal of Medicinal Chemistry

Ru(II) Compounds: Next-generation anticancer metallotherapeutics? Sreekanth Thota,†,‡ * Daniel A. Rodrigues,‡ Debbie C. Crans,§ Eliezer J. Barreiro‡ †

National Institute for Science and Technology on Innovation on Neglected Diseases

(INCT/IDN), Center for Technological Development in Health (CDTS), Fundação Oswaldo Cruz - Ministério da Saúde, Av. Brazil 4036 - Prédio da Expansão, 8º Andar - Sala 814, Manguinhos, 21040-361 - Rio de Janeiro - RJ – Brazil ‡

Laboratório de Avaliação e Síntese de Substâncias Bioativas (LASSBio), Institute of

Biomedical Sciences, Federal University of Rio de Janeiro (UFRJ), PO Box 68023, 21941-902, Rio de Janeiro, RJ, Brazil. §

Colorado State University, Department of Chemistry, Fort Collins, Colorado 80523, USA.

GRAPHICAL ABSTRACT:

ACS Paragon Plus Environment

Journal of Medicinal Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT: Metal based therapeutics are a precious class of drugs in oncology research that include examples of theranostic drugs, which are active both in diagnostic, specifically imaging, and therapeutics applications. Ruthenium compounds have shown selective bioactivity and the ability to overcome the resistance that platinum-based therapeutics face, making them effective oncotherapeutic competitors in rational drug invention approaches. The development of antineoplastic ruthenium therapeutics is of particular interest because ruthenium containing complexes NAMI-A, KP1019 and KP1339 entered clinical trials and DW1/2 is in preclinical levels. The very robust, conformationally rigid organometallic Ru(II) compound DW1/2 is a protein kinase inhibitor and presents new Ru(II) compound designs as anti-cancer agents. Over the recent years, numerous strategies have been used to encapsulate Ru(II) derived compounds in a nanomaterial system, 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 also can be photosensitizers for photodynamic therapy, which has proven to be an effective new, alternative, and noninvasive oncotherapy modality.

Key words:

Ru(II) therapeutics, Ru(II) theranostics, Anti-cancer drug design, Pre-clinical

cancer studies, Clinical trials, Nanomaterial, Protein kinase inhibitors, Photodynamic therapy.


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1. INTRODUCTION Tumor is the uncontrolled growth of anomalous cells in the body.1 Cancers range among the leading causes of death worldwide, accounting for 8.2 million deaths within five years of diagnosis.2 According to a WHO report, it is expected that annual cancer cases will rise from 14 million in 2012 to 26 million within the next two decades.3 There is no current oncotherapy that is able to cure most forms of disseminated tumors, so that the discovery of novel active chemotherapeutics is predominantly needed.4-5 There have been tremendous efforts to conquer cancer with current chemotherapy. The next generation of molecularly targeted drugs have potential in personalized medicine, because these approaches promise more efficacious and less adverse anti-tumor therapies in patients who have suffer from resistant cancers. Metallotherapeutics act by preventing cancer cell division and trigger cancer cell apoptosis by inducing DNA damage and disrupting DNA repair process.6-8 Metal scaffolds currently play an important role in medicinal chemistry and drug development after the serendipitous discovery and development of platinum compounds.9-10 The platinum-based drug cisplatin (Figure 1) is one of the most progressive and commonly used drugs in the clinic in the treatment of numerous forms of human cancers, but its therapeutic value is accompanied by serious side effects, and thus its effectiveness decreases by the increasing observed drug resistance.11 For these reasons, many researchers are actively searching for other alternative transition metal compounds and new ruthenium compounds have been reported as antitumor metallotherapeutics.12 2. DEVELOPMENT OF Ru(II) COMPOUNDS: Ruthenium-based therapeutics are promising candidates that show acceptable biological properties for chemotherapy and have emerged as a favorable adjunctive to the platinum-derived



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therapeutics.13 For the past few decades ruthenium therapeutics have successfully been used in clinical research and their mechanisms of antitumor action have been reported.14-15 Several reviews on the anticancer ruthenium compounds have been reported in 2016.16-20 Rutheniumbased anticancer metallotherapeutics21-22 are very appealing alternatives because of their different modes of action, and they were found to have certain merits over platinum-based therapeutics. Ruthenium compounds have desired properties that make these ruthenium scaffolds 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 for cancer cells compared with normal cells, iii) The higher selectivity of these compounds for their targets may be linked to selective uptake by the tumor compared with healthy tissue,23 iv) Ruthenium can mimic iron in binding to some biological molecules.23 Recently Alessio described some myths in the field of ruthenium antitumor therapeutics including discussing ruthenium therapeutics low toxicity because ruthenium mimics iron. He suggested that ruthenium therapeutics have inherently low toxicity but that ruthenium's ability to mimic iron is often confused with toxicity.13. Ruthenium belongs to the same group in the periodic table as iron, which is reflected by its high affinity for transferrin and by the its reductive activation in cells.23-24 Some ruthenium compounds are excellent candidates for clinical development, due the low cyto- and genotoxicity, different ligand exchange kinetics, transport, activation mechanisms and high biological activity. 2.1. Clinical trials and Patented compounds of Ruthenium. There are currently four ruthenium therapeutics in various stages of clinical trials, with one possibly about to obtain marketing approval for use in the clinic. The journey of antineoplastic ruthenium scaffolds that have entered clinical trials include Ru(III) species, imidazolium-(imidazole)(dimethylsulfoxide)tetrachlororuthenate(III)


(NAMI-A)25-26

and

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indazolium transtetrachlorobis(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 human clinical investigations. Preclinical investigations in several tumor animal models, ruthenium based therapeutic NAMI-A exhibited inhibition of tumor metastases and appeared to lack cytotoxic actions. It succeeded phase I clinical studies, but in phase II clinical studies showed only limited efficacy which prevented further clinical development of NAMI-A. Another ruthenium therapeutic KP1019 entered phase I clinical trials. Its low solubility limited its further development, but in its place a more soluble sodium salt, KP1339, is currently undergoing clinical trials.29 Another interesting Ru(II)therapeutic TLD1433 (Figure 2) entered phase1 and 2a clinical trials for non-muscle invasive bladder cancer treatment with photodynamic therapy (PDT).30 From the last decade, some Ru(II) complexes bearing 1,10-phenanthroline and 2,2bipyridine exerted potent activities against numerous tumor cells31-33 whereas other ruthenium complexes were reported as protein kinase inhibitors.34 Many medicinal chemists have discovered new Ru(II) scaffolds which are already being investigated in pre-clinical studies at various stages of development. For example, the ruthenium complex RAPTA-C35, combination with erlotinib exhibited efficient anticancer action. In the past few decades several advancements on patents of antineoplastic ruthenium complexes with a range of different scaffolds have been reported.36-43 2.2. Ru(II) Lead compounds (Figure 1). RAED: Ruthenium compound with diamine scaffolds are of consideration as prospective novel metal-based antitumor therapeutics. RAED44 compounds are ruthenium-arene compounds bearing 1,2-ethylenediamine ligand. They have the ability of binding to DNA and form adducts



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with guanine. This ruthenium therapeutic displayed potent cytotoxicity in in vitro neoplastic cells with DNA interaction.45 RM175:

Another

ruthenium

organometallic

lead

compound

is

RM175,46

[(ɳ6-

C6H5C6H5)RuCl(H2NCH2CH2NH2-N,N¢)]+ PF6-. Test with this ruthenium lead complex the apoptosis was induced through Bax, p53 and loss of clonogenicity.46 RDC11: A ruthenium derived lead compound RDC1147 exhibited in vitro cytotoxic activity for numerous tumor cell lines, including those that are sensitive to and those that are resistant to cisplatin. RDC11 treatment diminished chronic toxicity compared with cisplatin and hindered the growth of the various xenograft model cancers in mice more efficiently than cisplatin.47 DW1/2: DW1/248 is the first ruthenium antitumor agent targeting a signal transduction pathway. DW1/2 binds to a protein, and as such can be characterized as a glycogen synthase kinase (GSK)-3β inhibitor. It is also a potent activator of p53. KP418: A lead structure was found in the imidazole-containing complex ICR (KP418),49 imidazolium trans-[tetrachloridobis(1H-imidazole)ruthenate(III)], which proved therapeutic activity against murine P388 leukaemia and B16 melanoma. ONCO4417: This ruthenium lead structure more resembling like another ruthenium lead compound RM175, whereas insertion of chloride instead of hexafluorophosphate anion (PF6-) in RM175 results in another ruthenium lead compound ONCO4417.50 In vitro results concluded that ONCO4417 has tantamount efficacy to platinum in many tumor cell lines. This ruthenium lead compound displayed significant efficacy in inhibiting tumor metastasis. RAPTA-C: Ru(II) complexes reported in 2004 bearing phosphoadamantane and arene ligands as a class were named RAPTAs. The [Ru(eta(6)-p-cymene)Cl(2)(pta)] where pta is 1,3,5-triaza-7-


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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 anti-metastatic effects against breast cancer cells. UNICAM-1: A water soluble ruthenium(II) organometallic compound [Ru(p-cymene)(bis(3,5dimethylpyrazol-1-yl)methane)Cl]Cl, termed UNICAM-1.53 In A17 triple negative breast cancer cell this ruthenium lead complex significantly reduces the growth.



Figure 1. Structures of Cisplatin, Ruthenium clinical trial, preclinical and lead compounds.


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

3. CELLULAR UPTAKE AND CYTOTOXICITY OF Ru(II) COMPOUNDS The drugs need to permeate the cell membrane to act on the living cells. Cell membranes secure and organize cells, which contain numerous proteins and lipids, and its action is to monitor what substances penetrate the cells. Cellular uptake of small scaffolds can occur via energy-dependent (endocytosis, active transport) and energy-independent (facilitated diffusion, passive diffusion) processes (Figure 3).29 However, the uptake of ruthenium compounds by tumor cells or other cells must be substantial for selective and decisive cancer treatment. Cellular uptake of the ruthenium therapeutics has been analyzed by flow cytometry.54-55 The complex [Ru(phen)2(mitatp)]2+ exhibited significant antitumor activity against several tumor cells and flow cytometric experiments and imaging experiments suggested that the ruthenium compound could cross cell membrane and accumulate in the nucleus, leading to induction of G0/G1 cell cycle arrest and apoptosis.54-55 The ruthenium compound [Ru(DIP)2(dppz)]2+ showed cellular uptake through an energy-independent process.57 For the last decade several reports on cellular uptake of ruthenium compounds has been published.54-57 Schobert et al., reported cellular uptake of Ru(II)-compounds as measured by ICP-OES spectrometry. They also discussed the effect of steroids and steroid binding proteins on hormone conjugates of ruthenium.58 One very interesting feature of anticancer Ru(II) therapeutics is the ability to inhibit the efflux pump P-glycoprotein (Pgp).59 Ru(II) compounds bearing natural antitumor naphthoquinone plumbagin exhibited augmented efficacy against resistant tumor cell lines and inhibit the drug efflux pump Pgp.60



Figure 3. General cellular uptake mechanisms of drugs. Reproduced with permission from ref. 29. Copyright 2017, Royal Society of Chemistry.

The crucial point in identifying substances is the selectivity index with acceptable biological action and negligible cytotoxicity. During the last two decades, several reports have been published on in vitro cytotoxicity of Ru(II) compounds on numerous human cancer cell lines.61-66 Several analyses have shown that numerous factors such as cell membrane changes and cell adhesion characters, apoptosis through mitochondrial pathway or inhibition of topoisomerase I and II could at least in part explain the observed cytotoxicity. Bergamo et al., reported the selectivity and cytotoxicity of ruthenium compounds in preclinical and clinical trials. In mammary carcinoma model differentiation of selectivity of the anti-neoplastic action of KP1019, RM175, RAPTA-T and NAMI-A. are shown in Figure 4.12 Non-selective cytotoxicity was observed when the compound inhibits the primary tumor and lung metastasis growth in a similar way. The in vivo results on a mouse metastasizing tumor demonstrated a moderate inhibition of primary tumor growth for KP1019, RM175, RAPTA-T and NAMI-A. However,


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the selectively varies. No selectivity is observed for KP1019 in contrast to the other three compounds. More than a 50% reduction of spontaneous lung metastasis formation was observed for RM175, RAPTA-T and NAMI-A.12

Figure 4. In mammary carcinoma model differentiation of the percent inhibition and selectivity of the anti-neoplastic 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 of primary tumor growth. Reproduced with permission from ref. 12 Copyright 2011, Elsevier Ltd.

4. DNA BINDING, PROTEIN BINDING AND APOPTOSIS OF RUTHENIUM COMPOUNDS 4.1. DNA Binding.



The nucleus DNA is one of the vital targets for many oncology drugs.67-68 DNA is one of the primary pharmacological targets for numerous FDA-approved metallotherapeutics (e.g. cisplatin, carboplatin, oxaliplatin) and organic oncology drugs (doxorubicin, gemcitabine, 5fluorouracil etc.).69 Significant emphases have been given to the design of compounds with ligand scaffolds that bind to DNA with site selectivity. Interaction of transition complexes with DNA has been a popular subject for researchers in the field of bioinorganic chemistry ever since the discovery of platinum therapeutics 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 forms adducts at guanine bases of oligonucleotide DNA and that RAPTA-C also binds to DNA.45 Ru(II) polypyridyl compounds afford favorable platforms for DNA-binding and delivering bioactive drugs to the cell. The compound is activated through the photoirradiation of the photolabile bond that utilizes the metal to ligand charge transfer (MLCT) band and these compounds have also been assessed as cellular probes. The binding mode of the metallotherapeutics with DNA provide insight into mechanism of action and effectiveness of these therapeutics.69 Ruthenium-based oncotherapeutics have displayed differences with respect to their DNA interactions depending on their structure. For example, organometallic piano-stool ruthenium(II) complexes containing biphenyl rings (RM175) interact strongly with DNA binding to guanines.76 Oncotherapeutics that target DNA are one of the most effective agents in clinical use and have produced significant improvement in the survival of cancer patients when used in combination with chemotherapeutics that have different mechanisms of action. Therefore, a detailed understanding of the interaction of ruthenium compounds with models of binding sites present in DNA is of paramount significance to unravel the mechanism of action of ruthenium-


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derived complexes.77 During the past decade, a huge amount of work has 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, the analysis of plasma concentrations of oncotherapeutics has demonstrated its value to clinical studies for numerous vital drugs. Although protein binding is a major determinant of drug action, with very few exceptions, it is clearly only one of many factors that influence the disposition of oncology drugs. Normally the protein-binding was analyzed by electronic absorption and fluorescence quenching. High concentration of proteins in plasma and the tendency of various drugs to bind them have led drug discovery groups to identify the significance of plasma protein binding (PPB) in modulating the effective drug concentration at pharmacological target sites. Overall, past studies implied that proteins are biological targets of the Ru(II) polypyridyl scaffolds.83 Ru(II) compounds bind to major metaltransporting proteins from human blood such as human serum albumin (HSA) and serum transferrin (Tf). The binding affinity of ruthenium compounds against these two proteins showed that HSA appears to be a more favorable binding partner.84 Intracellular protein binding patterns of the clinical trial ruthenium therapeutics KP1019 and KP1339 have been reported. KP1019 is known to powerfully bind to serum proteins, and hamper P-glycoprotein-mediated efflux, making this ruthenium therapeutic attractive for multidrug-resistant tumor therapy.85 Preclinical RAPTA-C compound has shown significant binding affinity with two emerging protein targets, thioredoxin reductase and cathepsin B, for demonstrating their antitumor action.86 Over the recent years several groups reported significant protein binding affinity of Ru(II) based scaffolds.83-86



4.3. Apoptosis. Facilitating apoptosis is a very effective approach in the discovery of oncotherapeutics.87 The contribution of apoptosis on tumor size decrease has been extensively investigated since many oncology drugs display their action by promoting apoptosis.88 Apoptosis is a process of programmed cell death and controls the development and homeostasis in multicellular organisms, and is often characterized by energy-dependent biochemical mechanisms and distinct morphological characteristics. Activation of intrinsic and extrinsic pathways in cells leads to primarily induction of apoptosis. The intrinsic pathway, also known as the mitochondriamediated pathway, is promoted by DNA damage, oxidative stress and endoplasmic reticulum (ER) stress.29, 89 Ruthenium derived clinical trial compound KP1019, binds to transferrin, which is more cytotoxic after reduction, and it causes apoptosis via the mitochondrial pathway, also promoting the formation of reactive oxygen species (ROS). Ru(II) compounds have been reported to induce apoptosis via the mitochondrial pathway,90-92 autophagy pathway,93-94 and induction of ROS-mediated apoptosis in tumor cells by targeting thioredoxin reductase.95 DNAintercalating Ru(II) compound [Ru(bpy)(phpy)(dppz)]+ has been reported as an antitumor agent and was found to be extremely cytotoxic against cancer cell lines. The high affinity for DNA binding of this ruthenium complex damages the transcription factor NF-κB on relevant DNA sequences. Any damage of the transcription factor sequence leads to the inhibition of cellular transcription and irreversible cancer cell apoptosis (Figure 5).82 Other reported antitumor actions of ruthenium complexes is through the inhibition of telomerase activity and stabilization of Gquadruplex DNA intermediates.96


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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.

The preclinical ruthenium compound DW1/2 inhibits either PI3K and GSK3-β, which leads to apoptosis mediated by the mitochondrial- and p53–pathway and is the reported mechanism of action of DW1/2 as an antitumor drug (Figure 6).12



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Figure 6. Ru(II) therapeutic DW1/2 proposed mechanism of the in vitro cytotoxicity in a model of human melanoma in vitro. Reproduced with permission from ref. 12 Copyright 2011, Elsevier Ltd.

Another

complex

[Ru(MeIm)(npip)]2þ

(npip

¼

2-(4-nitrophenyl)imidazo[4,5-f]

[1,10]phenanthroline) promotes A549 human lung carcinoma cell apoptosis by both damaging mitochondrial homeostasis and death receptor signal pathways. This complex induces apoptosis of BEL-7402 cells through the mitochondrial signal transduction pathway. The apoptotic pathways of several other ruthenium therapeutics have been reported as well.97-100 Apoptotic and autophagy mechanisms were both induced by the [(η6-p-cymene)Ru(N,N′-hydrazinylthiazolo)Cl]Cl compound in the A2780 human ovarian cancer cell line (Figure 7).65


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



5. Ru(II) COMPOUNDS AS PROTEIN KINASE INHIBITORS The discovery of selective enzyme inhibitors is an important activity at the heart of medicinal chemistry and chemical biology. Protein kinases play a vital role in cell biology, regulate most aspects of cellular processes and are one of the main oncology targets for drug discovery. Kinase inhibitors are one of the most progressive and competently used inhibitors in the treatment of numerous forms of human cancers. There are more than 518 protein kinases encoded by the human genome, and many of them are associated with human cancers. The design and discovery of compound scaffolds that perturb specific protein functions is of significance for probing biological processes and ultimately for the discovery of potent and safe drugs. Many medicinal chemists are already targeting these kinases and have discovered new compound scaffolds, some which are already approved by FDA, with others in clinical trials and others still in developing stage.101-103 Some literature results reported that Ru(II) based therapeutics DW1/2 and NP309 are protein kinase inhibitors, which suggest that these Ru(II) compounds act as inhibitors of the GSK3 and Pim1 half-sandwich inhibitors.48 The first reports of very stable and conformationally rigid ruthenium(II) compounds containing the natural product staurosporine have been reported as protein kinase inhibitors.104-105 The Ru(II) lead compound bearing staurosporine (indocarbazole alkaloid) is reported as a subnanomolar ATP competitive protein kinase inhibitor (Figure 8(a)).103 These reports suggested that Ru(II) bearing a staurosporine ligand in a Ru(II) complex binds to the ATP binding site of Pim-1 (Figure-8(b)). The co-crystallized ruthenium compound bearing staurosporine (PDB code 1YHS) with Pim-1 describes a Ru(II) compound mimicking the binding mode of staurosporine (Figure 8(c) & 8d).103 Biersack et al. reported another interesting Ru(II) complex ((arene)Ru(II)compounds)


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containing the tyrphostin type epidermal growth factor receptor (EGFR) inhibitors.106 These targeted Ru(II) compounds as protein kinase inhibitors represent a major advance in oncotherapy.

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-1protein kinase (yellow color, PDB code 2BZI) (d) The superimposed cocrystal structure of Pim-1 (PDB code 2BZI) with staurosporine (blue color, PDB code 1YHS) reveals



the close match in binding mode between the organometallic compound and the natural product. Reproduced with permission from ref. 103 Copyright 2007, Elsevier Ltd.

6. MECHANISM OF ACTION OF Ru(II) COMPOUNDS Many researchers are now seeking to develop new mechanisms of cell death by affecting the general molecular framework by these ruthenium therapeutics.29,107-111 The ruthenium therapeutics are most potent when they induce apoptosis is by blocking transcription. After the early discovery of ruthenium compounds Clarke has reported a mechanism of the ruthenium compounds by “activation by reduction”. Many research laboratories worldwide were conducting experiments to determine the mechanisms of ruthenium(II) compounds, and to elucidate how Ru(II) compounds carry out their antitumor effects. From the literature reports so far, ruthenium compounds exert their anticancer actions by impacting the mitochondrial pathway, autophagy pathway & ROS mediated apoptosis. Recently Zheng et al. reported common representation of main targets and predicted several mechanisms of action of Ru(II) therapeutics as antitumor candidates (Figure 9).29


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Figure 9. General portrayal of the principal targets and projected mechanisms of action of ruthenium complexes as oncotherapeutics. Reproduced with permission from ref. 29 (Copyright 2017, Royal Society of Chemistry)

7. RECENT ADVANCES IN NANOMATERIALS The emergence of bionanomaterials gives scientists a new tool to solve oncotherapy problems.112 Nanotechnology has the potential to provide novel, paradigm-shifting solutions to medical emergencies and is currently used as a drug carrier for oncotherapy.113 Cancer



nanotechnology is being diligently examined and executed in cancer therapy, signifying a major advance in detection, diagnosis, and therapy. Many oncology laboratories are using advanced techniques to discover more precise acting nanotechnology based cancer therapy, that diminishes the adverse effects of the conventional ones. Novel drug delivery system (NDDS) are more and more applied in oncotherapy and diagnosis. In oncotherapy, diagnosis, imaging and drug delivery, nanomaterials including metallic and non-metallic nanoparticles, polymeric nanoparticles, nanowires, carbon nanotubes and quantum dots are currently being developed further with appropriate strategies. Nanoparticles can be designed through numerous modifications such as altering their shape, size, physical and chemical properties to program them for targeting the desired cells. Metal nanoparticles target the neoplastic cells either through active or passive targeting. Previously safe and efficacious functionalized nanomaterials were reported for Ru(II)–selenium nanoparticles,114 Ru(II)–gold nanomaterials,121 Ru(II)–silica composites,29 Ru(II)–carbon nanotubes,120 organic and biomaterial containing Ru(II) nanomaterials. Over the recent years, numerous strategies have been used to encapsulate Ru(II)-derived compounds in a nanomaterial system and ameliorate their targeting and delivery into neoplastic cells. Ruthenium-encapsulated silica nanoparticles were developed with increased cellular uptake and photoactivation (Figure 10A). Development of a Ru(II) photoactive compound to be grafted on the top of the UCNPs to produce DOX-UCNP@mSiO2-Ru has been accomplished (Figure 10B). Porous silicon nanoparticles (PSiNPs) to deliver photosensitized ruthenium compounds for photodynamic therapy have been introduced (Figure 10C). Another ruthenium nanoparticle system, RuPOP@MSNs, induced cancer cell apoptosis by the AKT & MAPK signaling pathways (Figure 10D).


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Figure 10. (A) Mechanized MSNPs graphical portrayal. (B) Schematic representation and UCNP@mSiO2 nanoparticles TEM image (1), Drug release from DOX-UCNP@mSiO2-Ru nanoparticles (2). (C) Synthetic route for pSiNP-Ru-PEG-Man. (D) RuPOP@MSNs construction reaction pathways. Reproduced with permission from ref. 29 (Copyright 2017, Royal Society of Chemistry).

Recently reported Ru(II) polypyridyl/thiols protected SeNPs114-115 were found to be both extremely sensitive cellular imaging agents and which induced the cell death. They are thus referred to as theranostics, that is, carrying out both diagnostic as well as therapeutic purposes at the same time. Luminescent, multi-functionalized Ru(II) polypyridyl-encapsulated selenium nanoparticles can inhibit bFGF-induced angiogenesis by suppressing the AKT and Erk signaling pathways which induce apoptosis (Figure 11).



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 ref. 115 Copyright 2012, Elsevier Ltd.

Another ruthenium compound (Ru-MUA@Se) exerts multiple functions by having dual-target inhibitors that directly suppress the tumor growth by inducing apoptosis and exerting dependence on ROS generation.116 Nanoparticle/Ru(II) polypyridyl complex assembled for NIR-activated release of a DNA covalent-binding agent.117 Ru(II)polypyridine compound encapsulated within


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liposomes reduces the TNBC tumor growth and is a favorable theranostic approach for breast cancer 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 radio resistance.119 A Ru(II) compound deposited on single-walled carbon nanotube composites was used for bimodal photodynamic and photothermal therapy.120 Consequently, effective nano-approaches for oncotherapy in the past few years have witnessed much progress involving nanotechnology strategies.121-122

Figure 12. Schematic portrayal of Ruthenium polypyridyl complex [Ru(phen)2dppz](ClO4)2(Ru) encapsulated with in bilayer of Liposomes (Lipo-Ru) reduces the triple-negative breast cancer (TNBC) and is a favorable theranostic approach for cancer therapy. Reproduced with permission from ref. 118. Copyright 2017, American Chemical Society. 8. NOVEL THERAPEUTIC APPROACHES FOR Ru(II) COMPOUNDS. 8.1. Photodynamic therapy.



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Over the recent years, scientists are searching for advanced innovative approaches in cancer treatment. Photodynamic therapy has proven to be a new, impressive, attractive, and noninvasive oncotherapy modality. Photodynamic therapy123 was developed for the management of neoplastic and non-malignant diseases and to treat specific types of tumors (i.e. bladder, lung and urinary tumors). Photodynamic therapy (PDT) has the capability to meet many currently unmet medical emergencies. The working principle of PDT is the light activation of a photosensitizer (PS), which produces ROS that are toxic and thus must be managed appropriately. These ROS then lead to apoptosis, with only minimal damage to normal tissues.124 Recent literature evidences suggesting that many Ru(II) derived compounds act as photosensitizers

for

both

one-

and

two-photon

photodynamic

therapy.125-130

These

metallotherapeutics are potential replacements to the current photosensitizers (PSs). Zhang and her coworkers reported on development of Ru(II) complex-functionalized single-walled carbon nanotubes (Ru@SWCNTs) as nanotemplates for bimodal two-photon photodynamic therapy (TPPDT) and photothermal therapy (PTT) with irradiation (808 nm) are illustrated in Figure13.120 These Upon treated with light, the Ru(II) compounds are liberated, and then which produce 1

O2 upon the two-photon laser irradiation (808 nm). These results suggest that bimodal therapy

potentially exerts a greater significant antitumor effect compared with one-modal therapy.120 Ru(II) therapeutics for two-photon photodynamic therapy have been reported.131-132 Ru(II)-based therapeutics are reported as radiosensitizers when used in combination with clinically–relevant doses of radiation therapy.133 From the clinical examination, they reported that these ruthenium scaffolds should be equally efficient, with no indication of cross-resistance to platinum therapeutics. These results also suggest impressive radiosensitization with clinically– relevant doses of RT compared with p53-mutated or p53-null, higher radiosensitizing activity in


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p53-wildtype cells and a biological mechanism of action that has been shown to affect increasing the destruction of DNA by these ruthenium scaffolds.133 These significant outcomes highlight the immense potential of ruthenium compounds in PDT.

Figure 13. Schematic portrayal of Ru(II) complex-functionalized single-walled carbon nanotubes (Ru@SWCNTs) for bimodal two-photon photodynamic therapy (TPPDT) and photo thermal therapy (PTT) with irradiation (808 nm). Reproduced with permission from ref. 120. Copyright 2017, American Chemical Society.



8.2. Photo Activated Chemo Therapy. Although the PDT strategy has been applied in oncotherapy, the major limitation of 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-mediated toxicity is independent of oxygen levels must be developed and this technique is called photo-activated chemotherapy (PACT). The chemical compounds for this technique exhibit light-activated characteristics and are referred to as photo-activated chemotherapy agents.127, 135 The mechanism of action of these agents is based on the ligand exchange to create metal centers able to form DNA adducts or photo-release of a bioactive compound.135-136 Ligand exchange plays a vital part in PACT. Glazer et al. denoted antitumor Ru(II) compounds bearing methylated bipyridyl, pyridylbenzazole ligands and their photochemical and photobiological action.137-138 Very recent work has been reported about interaction between Ru(II)trans tetrapyridyl ruthenium therapeutic and an oligonucleotide controlled by light irradiation.139 The strategy of using Ru(II)-photocaged therapeutics has been denoted in several recent studies in the literature that associate a drug with a complex to the superior restraint of the biological action and release of the drug in selected tissues using light.140-142 Abiraterone is an inhibitor of cytochrome P450 17A1 inhibitor (CYP17A1), useful in metastatic prostate cancer therapy. The aid of abiraterone Ru(II)-photocaged complexes led to the selective release of abiraterone after exposure to visible light and potent CYP inhibition.141 These results suggest that the use of this strategy can avert the adverse effects of the parent drugs. This combined mechanism can also be achieved since after activation by light the ligand is released (drug), which has biological activity. In addition, the resulting complex may still be able to cause damage to the DNA, resulting in a synergistic activity.143 A last approach is to use


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the combination of mechanisms for developing more efficient Ru(II) therapeutics with dual potential PDT and PACT.144-145

9. SUCCESSES, FUTURE CHALLENGES & SCOPES FOR DRUG DISCOVERY Many pharmaceutical industries as well as non-profit government and non-government organizations all over the world are eagerly expecting the development of novel oncotherapeutics. However, the drug invention and development is proceeding slowly with low success rates, specifically in last steps of the process, the clinical trials. This is augmented by the increasing development of drug resistance, which remains a formidable challenge. Metallo therapeutics are a unique class of drugs in oncology research, also contributing to many possibilities in imaging, cancer therapy and theranostics at the same time. Currently almost 50% of patients undergoing chemotherapy receive some type of a platinum medication, however, drug resistance to platinum drugs limits its applications and tracing for adjunctive metal therapeutics. During the last three decades ruthenium compounds with varying scaffolds have shown tremendously selective bioactivity, as well as the capacity to overcome the resistance of platinum-based therapeutics, making them successful oncotherapeutic competitors in a rational drug invention approach. It is gratifying that four of the ruthenium-derived therapeutics entered human clinical trials but unfortunately, the results of phase-1 and phase-2 clinical studies did not support the continuation of two of these drugs (NAMI-A, and KP1019) to Phase III clinical trials.29 Two compounds remain under consideration in clinical trials KP1339,29 and the theranostic compound TLD1433. Some ruthenium based chemotherapeutics have been proven to be mitochondria-targeting oncotherapeutic candidates. Most of the developed Ru(II) scaffolds are lipophilic and carry a positive charge, which expedite their dispersion across the cell



membrane. These evidences suggest that Ru(II) therapeutics are significant candidates for future anticancer drug discovery. Comprehensive investigations have been undertaken to alter the structure of Ru(II) compound scaffolds to ameliorate their drug efficacy, while so far minimal effort has been conducted towards determining drug combinations.35,146-147 Drug combination treatments in tumor models indicate effective induced apoptosis without added toxicity (Figure 14).35 Further development of drug combinations in the future would allow for analysis of their synergistic antineoplastic action in preclinical drug-resistant tumor models, with the objective of ameliorating the therapeutic potential and protract life expectancy of the patients. These potential outcomes afford valuable, authentic knowledge on ruthenium-derived candidate medicaments and modern insights for future optimized oncotherapy protocols. Novel oncotherapeutics with effective molecular mechanisms of action are imperative in chemotherapy to kill specific tumor types, and to conquer toxic adverse effects. The detailed mechanisms of action of some of the ruthenium based therapeutics is still under some investigation and present a big challenge for inorganic medicinal chemists.


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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 and therefore which induce apoptosis. In A2780 and A2780cisR cells, genesis of DNA bridges and micronuclei by mitotic defection. Due to the genesis of DNA bridges, cytokinesis failure occur which leads to senescence. Reproduced with permission from ref. 35. Copyright 2017, Nature Publishing group. In a recent investigation, 95% of potential oncotherapeutics entering clinical development failed, correlating with an average of 90% for compounds in all therapeutic areas, which is a great challenge in the oncology drug discovery. We have summarized some of the issues above relating to the complexities of translation.

Indeed, in addition to the developmental and

mechanistic aspects of drug development, increasing attention to speciation and formulation may also assist this translational aspect of drug development.148, 149 Complex clinical trials with an



effective targeted translational research constituent enable major advances in the understanding of specific cancer types and directly contribute to defining new tailored standards of patient care. From all this evidence, it is the hope that Ru(II) derived therapeutics will enter into the market as a supportive to platinum based chemotherapeutics. We present an overview of the comprehensive development of Ru(II) compounds as anticancer metallotherapeutics, which should inspire young budding researchers and senior researchers to enter the interesting fields of metallotherapeutics.

It is important to conclude that the pharmacokinetic profile of ruthenium-

based therapeutics is yet to be ascertained in humans, but emergent results remain positive, keeping our hope alive that designing effective ruthenium-derived compounds to selectively target tumor cells is an achievable goal resulting in a Ru-compound that will progress to clinical use. Corresponding Author: *For S.T: phone, +55-21-38829234; fax, +55-21-22900494; ∗E-mail: [email protected], [email protected] ORCID Sreekanth Thota: 0000-0002-7501-3987 Daniel A Rodrigues: 0000-0001-7898-7093 Debbie C. Crans: 0000-0001-7792-3450 Eliezer J Barreiro: 0000-0003-1759-0038

Author Contributions: The manuscript was written with contributions from all authors. All authors have given approval to the final manuscript. Notes: The authors declare no competing financial interest.


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Biographies: Sreekanth Thota, Ph.D., is a Visiting Researcher at the Center for Technological Development in Health, Fiocruz & LASSBio, UFRJ in Rio de Janeiro, Brazil. He studied Pharmaceutical Chemistry at Kakatiya University (India) and Rajiv Gandhi University of Health Sciences (Bangalore, India) and obtained his Ph.D from Jawaharlal Nehru Technological University Hyderabad (India) in 2011. He then did postdoctoral work at Colorado State University, USA under the supervision of Prof. Debbie C Crans. He received the CAPES-Fiocruz Visiting Researcher award in 2014. His research interest is focused on synthesis, drug design, drug development and medicinal chemistry and has published over 40 articles in peer-reviewed journals and book chapters and a book concerning the field of medicinal chemistry. He is the inventor of many bioactive scaffolds. Daniel A. Rodrigues, M. S., obtained his M.S degree in Chemistry from Chemistry Institute, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil. Currently, his Ph.D. research is being carried out at Laboratório de Avaliação e Síntese de Substâncias Bioativas (LASSBio) at the Institute of Biomedical Sciences of Federal University of Rio de Janeiro, and is related to the medicinal chemistry field, particularly the design, synthesis and biological evaluation of multitarget ligands acting as anticancer agents. Debbie C. Crans, Ph.D., is a Professor in Chemistry in Organic, Inorganic Chemistry, Chemical Biology and Cell and Molecular Biology at Colorado State University. She was an undergraduate at the H. C. Ørsted Institute in Denmark, a graduate student at Harvard University and a postdoctoral fellow at the University of California, Los Angeles. Crans’s work has systematically explored speciation and solution chemistry of transition metal complexes, polyoxometalates and



their activities in more complex biological systems. She has been recognized with the first Vanadis Award in 2004 and an Arthur Cope Scholar award in 2015 for her fundamental work on vanadium complexes in diabetes. She is currently investigating the structural and functional involvement of menaquinone in the membrane associated electron transfer complex in pathogens. Eliezer J. Barreiro, Ph.D., concluded his scientific education (Docteur-Ès-Sciences d′État) in Medicinal Chemistry at the University of Grenoble, France, in 1978. He spent four years as Associate Professor of Organic Chemistry at Federal University of São Carlos, S.P. from 1979 to 1983 before he joined the Federal University of Rio de Janeiro where he got a permanent position. He works in medicinal chemistry field and founded the Laboratório de Avaliação e Síntese de Substâncias Bioativas (LASSBio) at the Institute of Biomedical Sciences of Federal University of Rio de Janeiro. Professor Barreiro has published over 330 journal articles and book chapters, a book concerning in medicinal chemistry and he is the inventor of 25 patents of bioactive compounds. Acknowledgments: S.T, D.A.R, and E.J.B. acknowledge the support of the Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq-BR,) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES-BR) & Oswaldo Cruz Foundation (Fiocruz-BR). S.T is thankful to Dr. Carlos M. Morel, Director, CDTS-Fiocruz for his support for carrying out this research. The author is thankful to Prof. Carlos Alberto Manssour Fraga and Prof. Lídia M. Lima for their insights and critical review of the manuscript. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The authors are grateful to the reviewers for their careful comments and precious suggestions.


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ABBREVATIONS USED: ATP, Adenosine Triphosphate; BSA, Bovine Serum Albumin; CNS, Central Nervous System; DNA, Deoxyribonucleic acid; EGFR, Epidermal growth factor receptor; ER, Endoplasmic Reticulum; HAS, Human Serum Albumin; ICP-OES, Inductively Coupled Plasma Optical Emission Spectrometry; MLCT, Metal to Ligand Charge Transfer; m-RNA, Messenger RNA; NDDS, Novel Drug Delivery System; NIR, Near Infrared; NPs, Nanoparticles; PACT, Photo Activated Chemo Therapy; PDT, Photo Dynamic Therapy; PEG, Polyethylene glycol; Pgp, Pglycoprotein; PPB, Plasma Protein Binding; PS, Photosensitizer; PTT, Photo Thermal Therapy; RDC, Ruthenium Derived Compounds; ROS, Reactive Oxygen Species; RT, Radiation Therapy; SeNPs, Selenium Nanoparticles; TNBC, Triple-Negative Breast Cancer; TPPDT, Two-Photon Photodynmaic Therapy; WHO, World Health Organization; References: 1.

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