Toward Multi-Targeted Platinum and Ruthenium DrugsâA New

Review Cite This: Chem. Rev. 2019, 119, 1058−1137

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Toward Multi-Targeted Platinum and Ruthenium DrugsA New Paradigm in Cancer Drug Treatment Regimens? Reece G. Kenny and Celine J. Marmion* Centre for Synthesis and Chemical Biology, Department of Chemistry, Royal College of Surgeons in Ireland, 123 St. Stephen’s Green, Dublin 2, Ireland

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ABSTRACT: While medicinal inorganic chemistry has been practised for over 5000 years, it was not until the late 1800s when Alfred Werner published his ground-breaking research on coordination chemistry that we began to truly understand the nature of the coordination bond and the structures and stereochemistries of metal complexes. We can now readily manipulate and fine-tune their properties. This had led to a multitude of complexes with wide-ranging biomedical applications. This review will focus on the use and potential of metal complexes as important therapeutic agents for the treatment of cancer. With major advances in technologies and a deeper understanding of the human genome, we are now in a strong position to more fully understand carcinogenesis at a molecular level. We can now also rationally design and develop drug molecules that can either selectively enhance or disrupt key biological processes and, in doing so, optimize their therapeutic potential. This has heralded a new era in drug design in which we are moving from a single- toward a multitargeted approach. This approach lies at the very heart of medicinal inorganic chemistry. In this review, we have endeavored to showcase how a “multitargeted” approach to drug design has led to new families of metallodrugs which may not only reduce systemic toxicities associated with modern day chemotherapeutics but also address resistance issues that are plaguing many chemotherapeutic regimens. We have focused our attention on metallodrugs incorporating platinum and ruthenium ions given that complexes containing these metal ions are already in clinical use or have advanced to clinical trials as anticancer agents. The “multitargeted” complexes described herein not only target DNA but also contain either vectors to enable them to target cancer cells selectively and/or moieties that target enzymes, peptides, and intracellular proteins. Multitargeted complexes which have been designed to target the mitochondria or complexes inspired by natural product activity are also described. A summary of advances in this field over the past decade or so will be provided.

CONTENTS 1. Introduction 2. Multi-Targeted Platinum(II) Drugs 2.1. Tumour Cell Targeting by Platinum(II) Drugs 2.1.1. Glucose Receptor Targeting 2.1.2. Hormone Receptor Targeting 2.1.3. Integrin Receptor Targeting 2.1.4. Biotin Receptor Targeting 2.1.5. Other Targets 2.2. Platinum(II) Drugs Targeting DNA and at Least One Other Cellular Entity 2.2.1. Enzymes as Targets 2.2.2. Proteins as Targets 2.2.3. Mitochondria as a Target 2.2.4. Targets Inspired by Natural Product Activity 2.2.5. Other Targets 3. Multi-Targeted Platinum(IV) Prodrugs 3.1. Tumour Cell Targeting by Platinum(IV) Prodrugs 3.1.1. Glucose Receptor Targeting 3.1.2. CD44 Glycoprotein Targeting 3.1.3. Hormone Receptor Targeting

© 2019 American Chemical Society

3.1.4. Epidermal Growth Factor Receptor Targeting 3.1.5. Biotin Receptor Targeting 3.2. Platinum(IV) Prodrugs Targeting DNA and at least One Other Cellular Entity 3.2.1. Enzymes as Targets 3.2.2. Peptide Receptors as Targets 3.2.3. Proteins as Targets 3.2.4. Mitochondria as a Target 3.2.5. Targets Inspired by Natural Product Activity 3.3. Platinum(IV) Prodrugs Targeting DNA and at Least Two Other Cellular Entities 4. Multi-Targeted Ruthenium(II) and Ruthenium(III) Complexes 4.1. Enzymes as Targets 4.1.1. Cycloxygenases 4.1.2. Glutathione-S-Transferases 4.1.3. Thioredoxin Reductases 4.1.4. Poly(ADP-ribose) Polymerases

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Special Issue: Metals in Medicine Received: April 27, 2018 Published: January 14, 2019 1058

DOI: 10.1021/acs.chemrev.8b00271 Chem. Rev. 2019, 119, 1058−1137

Chemical Reviews 4.1.5. Cytochrome P450 4.1.6. Topoisomerases 4.1.7. Cyclic-Dependent Kinases 4.1.8. Peptidases 4.1.9. Carbonic Anhydrases 4.1.10. Aldo-Keto Reductases 4.1.11. Aromatases 4.1.12. Ureases 4.2. Proteins as Targets 4.2.1. Epidermal Growth Factor Receptor 4.2.2. P-Glycoprotein and MDR Protein 4.3. Targets Inspired by Natural Product Activity 4.4. Other Targets 5. Conclusions and Outlook Associated Content Supporting Information Author Information Corresponding Author ORCID Notes Biographies Acknowledgments Abbreviations References

Review

antibiotics in the 1940s. The development of these arseniccontaining drugs undoubtedly represented a significant milestone in medicinal inorganic chemistry. Notwithstanding this, metals have been exploited for medicinal purposes for centuries. The Edwin Smith papyrus, an ancient Egypian medical text, for example, provides one of the earliest reports of the medicinal use of copper and how it was employed to sterilize chest wounds.9 Gold-based medicines were used in China as far back as 2500 BC.10 Arsenic was used by a number of predominant physicians, including Hippocrates (460−377 BC) as escharotics in the form of orpiment (AssS3) and realgar (As2S2).11 To the best of our knowledge, some of the earliest reports of metals being exploited to treat cancer date back to the 1500s.12 In the 1700s, a solution of arsenic trioxide in potassium bicarbonate (1% w/v), developed by Thomas Fowler, was among the first treatments reported for leukemia.13 It was, however, the landmark discovery of the anticancer properties of cisplatin by Barnett Rosenberg (1926−2009) in the 1960s,14,15 and its introduction into the clinic as a cancer treatment in 1978 led to the more widespread establishment of metallodrugs as therapeutic agents.15−17 In fact, by 1998, several thousand platinum complexes had undergone preclinical screening as potential anticancer agents and, of these, 28 had entered clinical development.15 Interestingly, the synthesis of cisplatin was first reported in 1844 by Michele Peyrone (1813−1873), a young chemist working in Turin, Italy. It was Alfred Werner who elucidated its sterical configuration in 1892 while working in Zurich.1 Lebwohl and Canetta provide an eloquent account of the clinical development of cisplatin, including a historical perspective.15 It is surprising that, despite the wide array of metals available for incorporation into drug molecules and the fact that metalbased therapies have been practiced for centuries, most drugs to date are of organic origin. The disciplines of organic and inorganic chemistry should not, and must not, be treated as distinct. For example, metal ions can act as key structural scaffolds in which to support organic molecules. This was nicely illustrated by Meggers et al. over 10 years ago when they employed a ruthenium ion as a scaffold to orientate organic ligands in such a way so as to optimize their binding to protein kinases.18,19 In addition, metal complexes, with varying coordination numbers and varying geometries, can offer greater structural diversity while the redox active nature of some metal complexes can also be exploited in some circumstances.20,21 There are also examples of metal complexes incorporating labile ligands which can undergo important ligand substitution reactions with biomolecular targets, for example, the interaction between platinum drug adducts and DNA nucleobases.17 The ligands themselves can also serve to enhance the pharmacokinetic properties of the metallodrug or can, in their own right, possess some desired biological property. The use of metal-based drugs as antimicrobial,4,22 antidiabetic,4 and antiarthritic agents,4 in addition to their applications as diagnostic imaging agents and radiosensitizers,4 is a testament to their chemical and structural diversity.4,23 It is their use and potential as anticancer agents that form the focus of this review. Cancer treatment modalities include surgery, radiation therapy, and chemotherapy. Immunotherapy, a form of treatment which stimulates the immune system to destroy cancer cells,24 and oncolytic virotherapies25 are also emerging as promising options to complement existing therapeutic

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1. INTRODUCTION In 1893, the Swiss chemist Alfred Werner (1866−1919) was the first to propose the correct geometry of transition metal complexes when he described the structure of hexamminecobalt(III) chloride.1−3 This seminal work led to the establishment of coordination chemistry as a distinct discipline in chemistry and earned him the nickname “Inorganic Kekulé” as well as the Nobel Prize in Chemistry in 1913.3 As we now mark the 125th anniversary since the publication of this seminal paper on coordination chemistry,1 it is timely to reflect and celebrate progress within this important field. While the breadth of applications of metal complexes is wide-ranging,4,5 we wish to highlight in particular their use and potential as important therapeutic agents for the treatment of cancer. With a deeper understanding of the human genome and major advances in genomics, transcriptomics, proteomics, metabolomics, and radiomics,6 we now live in an exciting era in which we can more fully understand carcinogenesis at a molecular level. Developments in this space are enabling us to truly rationally design and develop drug molecules that can either selectively enhance or disrupt key biological processes and, in doing so, optimize their therapeutic potential. These developments have heralded a new era in drug design in which we are moving from a single- toward a multitargeted approach. This approach lies at the very heart of medicinal inorganic chemistry. This rational approach to drug design is not a new phenomenon. The German Nobel laureate Paul Ehrlich (1854−1915) pioneered the scientific concept of the “magic bullet” in the early 1900s. He postulated that one could design a drug to specifically kill disease-causing microbes without harming the body.7,8 Ehrlich and his team developed arsphenamine (Salvarsan), the first modern chemotherapeutic agent to be used clinically in the early 1910s as an effective treatment for syphilis.7 Salvarsan, together with the more water-soluble and less toxic Neosalvarsan, became the drugs of choice for the treatment of syphilis until the advent of 1059


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biogenesis stress.33,34 Oxaliplatin received FDA approval in 2002 as a treatment for colorectal cancer [in combination with 5-fluorouracil/leucovorin (5-FU/LV)].35 Some 16 years later, it, together with cisplatin and carboplatin, are still under active investigation as illustrated by their involvement in numerous ongoing clinical trials. For example, a search of active (not recruiting) US-based clinical trials in phase III, involving cisplatin, spanning the time frame Jan ’08−Jan ’18 and including all age groups and sexes, resulted in 27 hits, 24 of which included cisplatin as a cancer drug treatment in combination with others, as outlined in Table S1.36 An analogous search replacing “cisplatin” for “carboplatin” or “oxaliplatin” yielded 38 and 9 hits, respectively; 37 of which were directly relevant to carboplatin as a drug treatment and all 9 to oxaliplatin (Table S1). While these drugs continue to play a pivotal role in cancer therapeutic regimens, their widespread use is limited due to severe dose-limiting toxic side effects arising from their lack of selectivity for cancer cells over healthy cells. Rapidly growing cells are typically targeted such as those in the gastrointestinal tract, hair follicles, and bone marrow. Cisplatin is also particularly nephrotoxic and ototoxic. For example, it has been reported that between 40 and 80% of adult patients and at least 50% of children being treated with cisplatin develop permanent hearing loss.37,38 It should be noted that the presence of the dicarboxylate chelating agent in carboplatin confers the complex with greater stability arising from the chelate effect and, as such, the side effects associated with carboplatin are significantly less than those of cisplatin. Drug resistance is also a major challenge. The molecular processes underlying the development of platinum drug resistance are undoubtedly complex and multifactorial. Mechanisms of resistance may however fall under two main categories; (i) those that limit the formation of platinum-DNA adducts or (ii) those that prevent cancer cell death following platinum-DNA adduct formation.39 New strategies to overcome these drawbacks are constantly evolving. These include but are not limited to the exploitation of nanotechnologies to selectively deliver platinum drugs to tumor cells, thus potentially overcoming toxicity concerns but such delivery vehicles will not fully address resistance issues.17,40−42 An alternative is to move from single- to multitargeted drugs not only to enhance selectivity and/or efficacy but also to target resistance pathways. Developing drugs incorporating metals other than platinum is another option being actively investigated: these may have a mechanism of action and toxicity profile distinct to platinum drugs and may thus offer advantages over existing therapeutic regimens. The progression of the ruthenium(III) complexes, NAMI-A (7), Figure 2,43,44 KP1019 (8), Figure 2,45−47 and NKP-1339, the sodium salt of KP1019,47 to clinical trials, validates this latter approach. The applications of metal-based complexes as anticancer agents have been comprehensively reviewed over the years, including recent reviews or book chapters on platinum,17 ruthenium,48,49 osmium,48 copper,50,51 gold,52,53 and titanium54 complexes. This review, in contrast, has a distinct focus on the rational design and development of “multitargeted” metallodrugs. In this context, we use the term “multitargeted” to describe complexes which contain more than one biofunctional moiety; with cytotoxic complexes which are capable of not only binding DNA but also incorporating either tumor targeting vectors and/or ligands with distinct biological functions in their own right. In many

regimens. Chemotherapeutics work either alone or in combination regimens by blocking the unwanted proliferation of cancer cells. Platinum drugs constitute a major class of chemotherapeutic agents. In fact, it has been reported that nearly half of all cancer patients who require chemotherapy are treated with a platinum drug.26 Despite the introduction of cisplatin into the clinical setting over 40 years ago, only three platinum drugs have been approved to date for worldwide clinical use, namely cisplatin (1), carboplatin (2), and oxaliplatin (3), while three others, nedaplatin (4), heptaplatin (5), and lobaplatin (6) are in use in Japan, South Korea, and China, respectively, Figure 1.

Figure 1. Platinum drugs in clinical use (with generic names in brackets), together with the years in which they received either global or limited regulatory approval.

Cisplatin and carboplatin elicit their antitumor effect by first entering the tumor cell; they then undergo intracellular activation leading to platinum drug metabolites which can subsequently and irreversibly bind to deoxyribonucleic acid (DNA) nucleobases, more specifically to the N-7 of either guanine or adenine forming inter- and intrastrand cross-links. The formation of these platinum-DNA adducts ultimately leads to tumor cell apoptosis. It is noteworthy that, in recent times, the mechanism of action of these platinum drugs has also been associated with their ability to modulate the immune system. A detailed account of the molecular pathways underlying the immunogenic effects of these platinum drugs is provided by Lesterhuis et al.27 The reader is also directed to an excellent review by Lippard et al. in which they provide a comprehensive description of the mode of action of these classical platinum drugs.17 Dasari and Tchounwou provide a further insight into numerous pharmacological responses induced by cisplatin.28 Interestingly, despite the structural similarities between oxaliplatin, cisplatin and carboplatin recent research suggests that the mechanism of action of oxaliplatin differs somewhat from that of cisplatin and carboplatin. Oxaliplatin has been shown to trigger “immunogenic cell death” (ICD) and is now considered a bona fide ICD inducer.29−32 Immunogenic cell death inducers faciliate, rather than suppress, a T cell-dependent adaptive immune response specifically against dead cell-derived antigens.29−32 Oxaliplatin has also been shown to induce tumor cell death via ribosomal 1060


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2. MULTI-TARGETED PLATINUM(II) DRUGS The discovery of the antitumor activity of cisplatin spurred a “renaissance in inorganic chemistry”.55 Early efforts in this field of research focused on generating complexes of a similar structure to cisplatin (complexes in the cis configuration containing a central platinum ion in the +2 oxidation state, with ammine or substituted amine nonleaving group(s) and anionic labile ligands which could be substituted by DNA nucleobases. Hundreds of analogues were developed and their anticancer activities assessed, but none were found to offer any major advantage over cisplatin. While the presence of cisconfigured exchangeable ligands was once considered a major prerequisite for the anticancer activity associated with platinum drugs, there are now numerous examples of platinum complexes which violate this structure−activity relationship and yet possess potent anticancer properties.56 For example, trans platinum(II) complexes,17,56−58 and other nonconventional platinum(II) complexes 59 including polynuclear platinum(II) drugs60,61 are notable exceptions with one trinuclear complex, BBR3464,62 advancing to clinical trials. In more recent times, research groups have also endeavored to incorporate distinct functionalities into the “platin” framework to either enhance uptake into tumor cells by adding targeting moieties or by introducing ligands that can interact with other biological targets, in addition to DNA. We have thus divided the following section into two main parts: (i) tumor cell targeting by platinum(II) drugs and (ii) platinum(II) drugs targeting DNA and at least one other cellular entity.

Figure 2. Ruthenium(III) complexes which have advanced to clinical trials (7−8).

cases, these ligands, as single agents, have exhibited potent antitumor properties and some are already in clinical use as anticancer agents. The multitargeted complexes, highlighted in this review, have been specifically designed to limit doselimiting systemic toxic side effects and/or to circumvent resistance while trying to enhance therapeutic efficacy. We chose to particularly focus on multitargeted platinum and ruthenium complexes, as complexes with these metals are already either in clinical use or in clinical trials as anticancer agents. Major advances in this chemical space over the past decade or so are presented.

Figure 3. Chemical structures of platinum(II)-glycoconjugates (9−24) developed by Gao et al. containing an oxaliplatin scaffold and different sugar moieties. 1061


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2.1. Tumour Cell Targeting by Platinum(II) Drugs

the activity of this combination with that of oxaliplatin in a fixed ratio three-component FOLFOX in vitro combination. The in vivo study demonstrated that Glu-Pt (11a) exhibited a more favorable safety profile relative to oxaliplatin. It was also found to be more efficacious over oxaliplatin in their leukemiabearing mouse model. Combining Glu-Pt (11a) and 5-FU led to an increase in antitumor activity over the oxaliplatin-based FOLFOX regimen in certain cancer cell lines. Taken collectively, this study suggests that the Glu-Pt (11a) may offer advantages over treatments with oxaliplatin.73 Of the transporter proteins, GLUT-1 is one of the most widely studied to date. It is overexpressed in many cancer cells, including ovarian, prostate, renal, head and neck, bladder, and breast, among others,65 and is thus of particular interest in this research field.75 The 2-deoxyglucose (2-DG) sugar has been shown to exhibit a much higher affinity for GLUT-1 binding as compared to glucose. Gao et al. sought to exploit this selectivity to faciliate GLUT-1 mediated platinum drug tumor cell uptake. They developed a new series of oxaliplatin-like derivatives but this time incorporating a 2-DG-functionalized malonate donor with varying hydrogen, chloride, or fluoride substituents at the linker site, (12−14), Figure 3.76 Taking advantage of the fact that the crystal structure of GLUT-1 had already been reported,77 and using molecular modeling studies, the group determined the key binding site for 2-DG to be the same as that for the GLUT-1 substrate itself. They also determined that the 2-DG conjugated platinum(II) complexes could be recognized by this same substrate binding site. The cytotoxicity of the complexes was tested against A549, SKOV3, MCF7, HT29, H460, DU145, and noncancerous HEK293 cells. All three complexes demonstrated lower IC50 values across all cancer cell lines and higher IC50 values for the HEK293 cells. Of note is the fact that the complexes exhibited enhanced cytotoxicities in the cancer cells that overexpressed GLUT-1, more so than oxaliplatin, thus demonstrating strong evidence that these conjugates were indeed exhibiting selectivity toward this receptor. HT29 cells have a particularly high level of GLUT-1 overexpression. A clear correlation between expression of this protein and improvement of HT29 cytotoxicity over oxaliplatin was seen. Against this cell line, the IC50 value for oxaliplatin was 5.83 μM, while the IC50 values for the complexes ranged from 0.97 to 1.44 μM, an almost 5fold increase. Against most other cancer cell lines, the increase in potency was less than 2-fold. Against the normal cell line (HEK293), oxaliplatin had an IC50 of 49.46 μM in contrast to the complexes which had IC50 values ranging from 79.46 to 102.19 μM. These HEK293 cells have lower endogenous transporter expression. This study shows strong evidence of 2DG acting as an effective and selective transporter ligand of metal complexes toward cancer cells overexpressed with GLUT proteins, which covers a wide array of cancers.76 Additional studies by the same group sought to investigate not only the impact of varying the natural sugar substrates of GLUT (D-glucose, D-mannose, or D-galactose) attached to an oxaliplatin scaffold but also the type and length of linker between the sugar and the platinum(II) moiety.78−83 In one study, Gao et al. generated five new oxaliplatin-derived glycoconjugates incorporating either glucose (Glu-Pt (11a) and 11b), mannose (15a and 15b), or galactose (16a and 16b), Figure 3. The complexes exhibited comparable cytotoxicity irrespective of the sugar (glucose, mannose, and galactose) tethered, via the linker, to the platinum base against A549, HT29, A357, A431, ECA109, and SKOV3 cancer

2.1.1. Glucose Receptor Targeting. Cancer cells require large amounts of glucose for survival. However, because of their hypoxic environment, they are highly dependent on anaerobic glycolysis for the generation of adenosine triphosphate (ATP). This route is energetically inefficient in that for every mole of glucose, only 4 moles of ATP are generated. In contrast, most noncancerous cells generate ATP from the aerobic oxidation of pyruvate. This oxidation is significantly more efficient, generating 36 moles of ATP per mole of glucose.63 The over-reliance on anaerobic glycolysis by cancer cells, which is commonly referred to as “the Warburg effect”, leads to an overexpression of glucose transporter (GLUT) membrane proteins on the surface of these cells.63,64 GLUT proteins therefore represent an attractive drug target. As such, the generation of glycoconjugates which have been specifically designed to selectively transport drug molecules to cancer cells that overexpress GLUT proteins has been an active area of research for some time.65 Glycoconjugation also offers the advantage of conferring the new drug molecule with greater aqueous solubility, an attractive property in the drug development process. To the best of our knowledge, glufosfamide, a glucose derivative of the DNA alkylating agent ifosfamide mustard, was the first example of a glycoconjugate which was specifically designed to act as a cancer-targeting cytotoxic agent.66 The development of metallo-glycoconjugates soon followed, and numerous platinum-glycoconjugates have since been developed for this purpose. These typically incorporate a glucose unit which has been modified in such a way to facilitate its binding to either the ammine/amine nonleaving ligand of classical platinum(II) drugs67,68 or via an ethylenediamine ligand69 or via an O,O-bidentate leaving ligand such as malonate,68,70 while still, importantly, retaining the key structural features of the sugar moiety. This earlier work is nicely summarized by Hartinger et al. in a review published in 2008 on the role of metal complexes bearing carbohydrate ligands and the impact of the carbohydrate carriers on the overall antineoplastic activity of these compounds.71 There have since been a number of studies which have investigated the tumor cell uptake of platinum(II) complexes via a GLUT-mediated mechanism. To the best of our knowledge, Gao et al. were the first to report GLUT-mediated uptake of platinum(II) glycoconjugates.72 Their oxaliplatin analogues, in which the dicarboxylate leaving group was replaced with a glucose-functionalized malonate moiety, (9− 11a), Figure 3, were not only found to be highly water-soluble derivatives (150 times more soluble in water compared to oxaliplatin), but also exhibited 10 times greater in vitro cytotoxicities against A549, H460, SKOV-3, MCF7, HT29, and DU145 tumor cells. Of note is the fact that when in vitro studies were conducted in the presence of phlorizin, a flavanoid known to inhibit glucose uptake/transport, the cytotoxicities of the complexes decreased, validating their rationale behind the development of these conjugates.72 Building on this study,72 the same team investigated the in vivo efficacy of the fluoro-substituted derivative, Glu-Pt (11a), Figure 3, in L1210 leukemia-bearing DBA/2 mice and compared its efficacy to that of oxaliplatin.73 Given the wellestablished FOLFOX combination74 used clinically for the treatment of colorectal cancer, they also investigated the combination or synergistic impact of combining Glu-Pt (11a) with folinic acid (FA) and 5-fluorouracil (5-FU) and compared 1062


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cells.78 Their cytotoxicities were reduced significantly, as expected, in the presence of the GLUT inhibitor quercetin. As before, all the complexes showed significantly improved solubility (by several orders of magnitude) over cisplatin, carboplatin, and oxaliplatin and exhibited enhanced cytotoxicity over oxaliplatin against GLUT-expressing cancer cells.78 An in vivo study on the galactose complex, Gal-Pt (16a), Figure 3, showed it to be superior to cisplatin against a lung cancer mouse model and more effective than oxaliplatin against a colon cancer mouse model.79 Of note is the fact that Gal-Pt (16a), Figure 3, was over 25 times more water-soluble compared to oxaliplatin, potentially making it easier to be excreted by the kidneys, thus reducing heavy metal accumulation and associated systemic toxicities. This bodes well for its future development. The generation of methyl 6-amino-6-deoxy-D-pyranosideconjugated platinum(II) derivatives incorporating glucose (17), mannose (18), and galactose (19), Figure 3, while retaining the oxaliplatin scaffold (i.e., replacing the simple carbon linker in the aforementioned complexes with an amide linker to enhance stability) did not significantly change the properties of these complexes; they were still highly watersoluble, still dependent on GLUT for uptake and transport, and were either more cytotoxic or comparable to oxaliplatin against HT29, H460, DU145, A549, SKOV3, and MCF7 cells with the glucose derivative (17) showing greatest efficacy.80 Analogues of complexes 11, 15, and 16 were also developed by Gao et al., but in these analogues, the halides had been replaced with a methyl substituent; there was also a shorter carbon linker between the sugar moiety and the platinum center (20−22), Figure 3.81 These new complexes retained the aqueous solubility associated with these types of glycoconjugates as expected and were typically slightly more cytotoxic over oxaliplatin against HT29, A549, SKOV3, A431, and A357 cells. This study also investigated, for the first time, the in vitro DNA adduct formation of these types of glycoconjugates using a kinetic study in which the binding of the complexes to 5′guanosine monophosphate (5′-GMP) was monitored. The order of reactivity toward 5′-GMP followed the trend Gal-MePt (22) > Glu-Me-Pt (20) > Man-Me-Pt (21) ≈ oxaliplatin. The Glu-Me-Pt complex (20) was selected for further in vivo studies given its significantly enhanced water solubility over the other glycoconjugates. This in vivo study revealed that this conjugate exhibited a marked enhancement both in the therapeutic index and antitumor efficacy in a L1210 leukemia bearing DBA/2 mice model over cisplatin.81 Gao et al. went on to develop the platinum(II)-mannose derivatives (23 and 24), Figure 3, to study the impact of extending the chain length between the sugar and oxaliplatin base. This study showed that the complex containing the two carbon linker was notably more cytotoxic than that containing the three carbon linker, showing that chain length can have an effect on the cytotoxic properties of these types of complexes. This is likely due to changing their transport and uptake properties by GLUT receptors.82 The fluoro-substituted series (11a, 15a, and 16a), Figure 3, underwent further evaluation given that these complexes had significantly enhanced aqueous solubility over their nonflouride and methyl counterparts.83 The cytotoxicities of 11a, 15a, and 16a against HT29, H460, DU145, A549, SKOV3, and MCF7 varied but were generally comparable or better than oxaliplatin. There was no correlation between hydrophilicity and cytotoxicity. The glucose complex, (11a), was particularly

potent against the A549 cell line, being twice as potent as oxaliplatin. In the cisplatin-resistant SKOV-3 cells, the mannose complex (15a) had a 70% lower IC50 than oxaliplatin, while, in contrast, all three complexes were twice as potent as oxaliplatin against the H460 cell line. The glucose (11a) and mannose (15a) complexes were also twice as cytotoxic compared to oxaliplatin against the MCF7 cells. Again, the HT29 cells were the most sensitive, with a 10-, 5-, and 2-fold increase in potency for the glucose (11a), mannose (15a), and galactose (16a) derivatives, respectively. The glucose complex (11a), when tested for its selectivity compared to oxaliplatin against EBAS-2B-CM cells, was found to be less toxic. The complexes were also able to overcome cisplatin resistance, showing strong activity when tested against the HT29cisR cell line, with IC50 values ranging from 4.78 to 6.02 μM compared to cisplatin (IC50 of 19.35 μM). Significantly, using GLUT inhibitor mediated cell viability analysis, cytotoxicity studies using GLUT-1 knockdown cell lines, and platinum drug accumulation studies, the group was able to confirm that the cellular uptake of the complexes was indeed mediated by GLUT-1. The complexes were also found to bind DNA via platinum-guanine-guanine (Pt-GG) intrastrand cross-links. A kinetic study to test their intrinsic DNA reactivity revealed that the complexes reacted faster with the nucleophilic base 5’GMP when compared to oxaliplatin. In addition to their in vitro studies, the complexes were also found to have a safety and cytotoxicity profile better than oxaliplatin in both the L1210 bearing ascitic leukemia and human colon cancer xenograft animal models.83 Lippard et al. generated three additional oxaliplatin-type glycoconjugates and conducted a comprehensive study to elucidate the GLUT-mediated drug uptake mechanism. They were able to exploit the then recently published crystal structure of the bacterial xylose transporter XylE, a GLUT-1 homologue, in the design of their conjugates.84 The complexes retained the diaminocyclohexane (DACH) nonleaving ligand of oxaliplatin, but the O,O-bidentate leaving group was replaced by a malonate attached to a glucose via a linker of varying length, (25−27), Figure 4.85 The complexes were all

Figure 4. Chemical structures of platinum(II)-glucose conjugates in which the malonate is attached to the glucose via a linker of varying length (25−27).

shown to target genomic DNA. All were shown to exploit GLUT proteins for cellular uptake with a reduction in uptake observed with increasing linker chain length, consistent with the finding of Guo et al., as outlined earlier. Of significance, 25, while exhibiting potent cytotoxicity against prostate (DU145) and kidney (A498) cancer cells, both of which have high levels of GLUT-1 expression, exhibited low toxicity in matched 1063


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Figure 5. Chemical structures of D-glucose and platinum(II)-glycoconjugates containing each of the six D-glucose isomers (C1α, C1β, C2, C3, C4, and C6) (28−33).

clearly inform the future direction of research in this field. Lippard et al. sought to fill this void. They developed positional isomers of platinum glycoconjugates using [(trans-1,2diaminocyclohexane)(2,2-dimethylmalonato)platinum(II)] as the cytotoxic agent and investigated the position of substitution, and its impact on the biological profile of the resulting complexes. In this pivotal study, they successfully synthesized and characterized all six isomers (C1α, C1β, C2, C3, C4, and C6) of their platinum-glycoconjugate, attaching each of the isomers of D-glucose to the platinum via a two carbon spacer using an ether glycosidic linkage, (28−33, respectively), Figure 5. Their multistep syntheses involved a series of selective protection and deprotection steps and while the synthesis of some of these was reasonably facile, others, such as for C2-Glc-Pt (30), required an arduous 12-step synthetic pathway. The synthetic methodologies employed here will no doubt serve as an important platform for the development of other desired glycoconjugate positional isomers. The substitution position was found to influence the biological activity of the resulting conjugates. For example, the site of substitution modulated the uptake of the complexes into tumor cells with Glc-Pt 1α (28) and 30 accumulating most efficiently in tumor cells relative to the other positional isomers. When uptake experiments were conducted in the absence and presence of cytochalasin B, a GLUT-1 inhibitor, 30 exhibited highest GLUT-1-specific internalization. The specificity of 30 for GLUT-1 was further validated when the potencies of all Glc-Pts (28−33) in DU145 cells and in its GLUT-1 knockdown clone were compared. Interestingly, no

normal prostate epithelial (RWPE2) and kidney epithelial (CCD1105 KIDTr) cells; these epithelial cells have reduced GLUT-1 expression relative to the corresponding cancer cells. Complex 25 was preferentially taken up by the cancer cells over the normal epithelial cells with uptake in the DU145 cells being 4-fold higher than RWPE2 cells. Uptake was inhibited when the cells were treated with cytochalasin B, a potent GLUT inhibitor. Interestingly, uptake by an energy-dependent organic cation transporter (OCT), OCT-2, another membrane protein overexpressed on the surface of tumor cells, was also investigated and was likewise found to mediate uptake of 25 into cancer cells.85 While not specifically designed to target OCTs, this study is a reminder that complexes, whether rationally designed to do so or not, have the capacity to hit multiple targets. Despite a significant body of research already available on the role of glycoconjugates as potential targeted drug agents, Lippard et al. highlighted a major gap in the literature in that, over the past two decades, not a single report has been published describing the synthesis of a complete suite of positional isomers of a given glycoconjugate (from C1−C6 while retaining the same linker length) nor has the influence of the position of substitution on the biological acitivity of the corresponding glycoconjugate been investigated.86 These hydroxyl groups have been shown, through crystallographic studies, to play an integral part in hydrogen-bonding interactions between the sugar and numerous GLUT amino acid residues.77,87 Being able to identify the optimal hydroxyl group of D-glucose in which to tether a cytotoxic agent could 1064


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Figure 6. Chemical structures of estrogen-linked platinum(II) conjugates (34−43).

platinum unit. The linker length between the sugar moiety and platinum center has also been shown to impact the activity of the complexes and should also be taken into account when designing new platinum-glycoconjugates. 2.1.2. Hormone Receptor Targeting. The involvement of sex hormones in cancer progression in hormone-sensitive tissues is well-established.88 For example, most breast cancers (60−70%) are considered as being hormone-dependent due to an overexpression of estrogen receptor (ER) proteins.89 While the classical ER (ERα) is well-characterized, another, ERβ, has more recently been discovered.90 Exploiting steroids such as estradiol as carriers or as agents able to disrupt the biological function of ERs represents an attractive strategy for the rational design and development of anticancer agents. Some of the earliest reports of platinum(II)-steroid conjugates date back to the early 1980s, but these early studies did not investigate their specific interactions with ERs. For example, Gandolfi et al. developed platinum(II)-estradiol conjugates, but despite the sound rationale behind their development, they were not found to be highly cytotoxic.91−93 Bérubé et al. have also conducted extensive research in trying to bring forward a lead platinum(II)-steroid conjugate. They proposed that the length of the spacer linking the steroid to the cytotoxic platinum(II) moiety played a crucial role in the biological activity of the complex with the optimal chain length consisting of either 11 or 12 carbons in length.94 They have since advanced numerous examples of platinum(II)estradiol conjugates with some exhibiting ER binding affinities similar to that of 17-β estradiol itself.95−98

differences in uptake were observed in noncancerous cells that do not express the GLUT-1 transporter, suggesting that uptake was indeed mediated via a GLUT mechanism. Cytotoxicity was also influenced with 30 possessing greatest potency and 31 demonstrating least potency, of the positional isomers tested in DU145 cells. The team concluded from this extensive methodical study that the C2 position on D -glucose represented the optimal position in which to tether a “warhead” for GLUT-1-mediated selective delivery of the therapeutic agent.86 Taken collectively, these elegant studies by Gao et al. and Lippard et al. provide clear evidence of the potential inherent in GLUT-1 targeting sugars as promising vectors for the selectively delivery of cytotoxic platinum drug payloads to tumor cells. Many of their complexes were also shown to overcome cisplatin resistance. Given that a reduction in tumor cell uptake is a prominent feature associated with cisplatin resistance,38 linking these targeting vectors to platinum offers a fresh approach to potentially overcoming resistance. Incorporating sugar derivatives has the added benefit of endowing the resulting complexes with greater aqueous solubility. A major drawback of existing therapies is dose-limiting toxic side effects association with drug accumulation. Enhancing the aqueous solubility of drugs containing heavy metals such as those containing platinum is attractive as they may be more easily excreted from the body, lessening overall systemic toxicities. Future endeavors in this research space should also be cognizant of the findings by Lippard et al. in which they demonstrated that the C-2 position on D-glucose represented the best position in which to link the sugar to the cytotoxic 1065


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Figure 7. Chemical structures of testosterone-linked platinum(II) conjugates (44−49).

maintaining ERα binding affinity and had signficantly lower IC50 values compared to cisplatin for two of the three oxaliplatin analogues tested.100 This study demonstrates the influence of the labile leaving ligands on not only the antiproliferative activity of the resulting conjugates but also their ERα binding affinities.100 The presence of the bulkier cyclobutane-1,1-dicarboxylate leaving ligand was clearly impacting on the biological profile of these complexes. Given the prevalence of prostate cancer, Bérubé et al. went on to develop a series of platinum(II) complexes conjugated to a testosterone derivative via its 7α position. Testosterone was chosen on the basis that it could promote androgen targeting. Six new complexes were synthesized, with 17β-acetyltestosterone linked to a cisplatin core via amino acid N-donor bridges (L- and D-2-pyridylalanine, L- and D-methionine, L-histidine and 101 L-4-thiazolylalanine) (44−49 respectively), Figure 7. The team found that the amino acid stereochemistry had little impact on cytotoxicity. The pyridylalanine and thiazolylalanine complexes proved to be the most cytotoxic (44, 45, and 49) against LNCaP, PC3, and DU145 androgen receptor positive and negative (AR+ and AR−) cell lines, having IC50 values lower than cisplatin across all cell lines. These complexes were further tested against additional AR+ and AR− cell lines: HT1080 HT-29, M21, MCF-7, MDA-MB-231, MDA-MB-468, CEM, CEM-VLBb, and CEM-VLB/CEM. They were again found to be significantly more cytotoxic than cisplatin against these cell lines with the exception of the multidrug resistant leukemia CEM-VLB and wild-type leukemia CEM-VLB/CEM cells. Compounds 44 and 49 were tested on HT-1080 tumors grafted onto chick chorioallantoic membrane and showed good antitumor activity while showing low toxicity toward chick embryos.101 Overall the work by Bérubé et al. is detailed and promising, demonstrating that hormone receptor targeting is dependent not only on the cytotoxic platinum moiety but also on the nature of the linkers separating the platinum core from the steroid. Taken collectively, their studies form the basis of a design model for new hormone-targeting cytotoxic metallodrugs. Gust et al. provided a comprehensive review published in 2009 on how steroid ligands can be exploited to optimize the efficacy of cisplatin for the treatment of hormone-related cancers.102 Lippard et al. also cover work in this domain, including a description of liver-targeted platinum(II) complexes conjugated to bile acids, which are steroid acids taken up by hepatic epithelial cells by transporter proteins.17 2.1.3. Integrin Receptor Targeting. It has been welldocumented that cancer cells overexpress certain receptors

Over the past decade or so and within the scope of this review, Bérubé et al. have continued to build on their library of conjugates, including the development of a series of platinum(II)-estradiol complexes in which the estradiol was linked to the platinum center via a N-substituted 2-aminoalkylpyridyl chelating derivative with a carbon chain spacer of varying length (34−37), Figure 6.99 They found unsurprisingly that, with increasing chain length, solubility decreased. Incorporation of a poly(ethylene glycol) linker enhanced solubility while also allowing the team to alter the spacer length. The complexes, bearing hexyl, octyl, undecyl, and tetradecyl spacers which separate the cytotoxic platinum(II) core from the steroid nucleus (34−37), Figure 6, were tested against MCF-7 (ER+), MDA-MB-231 (ER−), MDA-MB-468 (ER−), and MDA-MB-436 (ER−) cell lines. Complexes 34−36 demonstrated good ER binding affinity, similar to estradiol 17β. All were also significantly more cytotoxic than cisplatin (between 4 and 9 times more potent) except against the MDA-MB-468 (ER−) cell line. There did not appear to be a direct correlation between alkyl chain length and cytotoxicity, though potency did vary slightly, with the complexes containing the 8 and 11 carbon chains (36 and 37) being the most potent. Eight synthetic steps were required to generate these conjugates with yields in the region of 20% which, although still low, represented a significant improvement on the synthesis of some of their earlier related derivatives. This optimization was faciliated by their judicious selection of a tetrahydropyran (THP) in place of a benzyl protecting group at the beginning of the syntheses which allowed for three chemical transformations in one step: decarboxylation and bis-deprotection.99 A later study by Bérubé et al. tested the efficacy of a series of estradiol-linked platinum(II) derivatives incorporating either the O,O-labile leaving ligand of carboplatin (38−40), Figure 6, or that of oxaliplatin (41−43), Figure 6.100 Six new complexes were developed, with varying carbon chain linkers between the cytotoxic platinum moiety and the estradiol for both “carboplatin-” and “oxaliplatin”-based structures. Their cytotoxicities were tested against the MCF-7 (ER+) and MDA-MB231 (ER−) cell lines and compared with cisplatin, carboplatin, and oxaliplatin. Interestingly, the team found that the carboplatin base had less of an effect on the inhibition of cancer cell proliferation, with IC50 values increasing from between 1.8 and 33 μM for the cisplatin analogues to between 21 and 74 μM for those of the carboplatin derivatives. Notably, the carboplatin analogues also resulted in a loss of ERα binding affinity. The oxaliplatin base proved more promising, 1066


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2.1.4. Biotin Receptor Targeting. Biotin, also known as vitamin H, has been shown to be rapidly taken up into tumor cells, which often overexpress receptors specific to biotin on their cell surface. As such, biotin has been identified and used as a selective drug transporter, with studies reporting the positive effects of conjugating biotin with classical chemotherapeutics such as doxorubicin and gemcitabine.110 Chakravarty and Kondaiah et al. sought to develop platinum(II)-biotin complexes which could exploit not only photoactivation but also the tumor-targeting properties of biotin to achieve highly targeted tumor-cell specificity.111 While photoinduced activation of platinum(IV) prodrugs has been well explored, there are fewer examples in which photoactivation has been investigated for platinum(II) drugs. Chakravarty and Kondaiah et al. had previously developed platinum(II) ferrocenylterpyridine (Fc-tpy) complexes which were found to be highly photocytotoxic toward cancer cells with low dark toxicity.112 They demonstrated that the Fc-tpy moiety, upon binding to platinum(II), behaves as an excellent photoinitiator within the photodynamic therapy spectral window (600−800 nm). The resulting complexes were shown to form reactive oxygen species (ROS), which ultimately led to cellular damage.112 This same group generated a series of platinum(II)-Fc-tpy and platinum(II)phenylterpyridine (Ph-tpy) complexes incorporating biotinconjugated acetylide ligands. They compared the effects of a metal-bound photoactive center versus one containing a phenyl photoinitiator. An acetylide biotin unit was bound to the platinum(II)-Fc-tpy moiety in complex 51, Figure 9, while it was separated with a 6-aminocaproic acid linker in complex 52, Figure 9. Complexes 53 and 56, Figure 9, represent nonbiotinylated complexes which were used as reference standards to investigate the role of the biotin unit in complexes 51 and 52, Figure 9. Complexes 54−56, Figure 9, were generated with a view to establishing the role of the Fc-tpy and Ph-tpy ligands as photosensitizers in 51−56, Figure 9. The biotinylated photoactive Fc-tpy complexes exhibited significant photoinduced cytotoxicity in visible light (400−700 nm) against human breast cancer cells (BT474) but were significantly less toxic in the dark (IC50 value: 7 μM in light vs >50 μM in the dark). They also exhibited selective uptake in these BT474 cancer cells over HBL-100 normal cells. The complexes did not show cytotoxicity in the dark, and, of note, their Fc-tpy ligand was not photocytotoxic by itself. Complexes

such as folate receptors, somatostatin receptors, epidermal growth factor receptors (EGFR), and integrins. Targeting these receptors is an attractive option in trying to selectively deliver a cytotoxic agent to tumor cells.103−107 Integrins, which are heterodimeric transmembrane cell adhesion glycoproteins, are a particularly attractive target. They play a key role in enhancing migration, invasion, and proliferation of cancer cells. They have also been linked to tumor angiogenesis.108 The latest development in this field was reported in 2017 when Sarli et al. described the synthesis and biological profile of a platinum(II)-peptide conjugate for integrin-targeted photodynamic therapy. Their cyclometalated complex, (50), Figure 8, bearing a N̂ Ĉ N 1,3-di(2-pyridyl)-benzene ligand

Figure 8. Chemical structure of a platinum(II)-peptide conjugate for integrin-targeted photodynamic therapy (50).

linked to a c(RGDyK) peptide was found to be mildly cytostatic toward six cancer cell lines with different levels of integrin expression. The platinum-peptide conjugate 50 was taken up rapidly into tumor cells by receptor-mediated endocytosis. It produced 1O2 upon irradiation. When rat bladder cancer cells (AY27) were incubated with the conjugate prior to blue light exposure (5 min), there was a significant reduction (50%) in cell survival compared to control cells, paving the way for the use of these types of conjugates in targeted photodynamic therapy.109

Figure 9. Chemical structures of biotinylated conjugated platinum(II) complexes (51, 52, 54, and 55) and nonbiotinylated derivatives (53 and 56). 1067


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51−53 were also shown to release their biotinylated ligands upon exposure to red light (647 nm, 50 mW), possibly leading to the generation of active platinum species, which were shown to bind to calf thymus DNA. Such controlled release of an active platinum species is desirable given the propensity of platinum drugs to undergo side reactions with biomolecules resulting in unwanted toxic side effects. The team postulated that the cytotoxicity observed for these complexes could be attributed to synergistic effects of the active platinum species, upon photorelease, and light-induced ROS-mediated apoptosis. Complexes 54 and 55 (i.e., the phenyl analogue of 51 and 52) had IC50 values of ∼4 μM, similar to that found for cisplatin. They were shown to act as DNA intercalators and exhibited selective cytotoxicity toward the cancer cells. The nonbiotinylated analogues, 53 and 56, exhibited reduced activities. Furthermore, they accumulated less in tumor cells even in the presence of externally added biotin. These findings validate the importance of the coordinated biotin in enhancing selective uptake of the biotin-conjugated complexes into tumor cells. This study also demonstrates that strategies for improving drug selectivity, such as the exploitation of targeted ligands like biotin and photoactivation, can be combined to make smarter chemotherapeutics.111 2.1.5. Other Targets. We described in a previous section (section 2.1.3) how a peptide could be exploited to target integrins on the surface of tumor cells. It should be pointed out that the conjugation of platinum to peptides is however not without its challenges. For example, conjugation of peptides to platinum drugs can be difficult given that the synthetic scales of these two classes of molecules differ by at least one order of magnitude. In addition, many of the amino acids making up the backbone of the peptide can also carry platinum reactive nitrogen or sulfur-containing nucleophilic groups which can compete and irreversibly bind to platinum, preventing the platinum from subsequently interacting with its ultimate target, DNA.113 The first platinum-peptide conjugate was reported by Kelland et al. in 1996.114 They tethered the minor groove binding agents netropsin and distamycin to platinum(II). Advances in solid phase peptide synthesis (SPPS) have undoubtedly facilitated the further progression of this class of drug molecules. The first SPPS of a peptide tethered to platinum(II) was reported in 2000.115 It should be pointed out that the use of SPPS is however not always straightforward. For example, often strongly acidic conditions and a mix of highly nucleophilic scavengers are required to cleave the peptide-resin anchor from the resin, conditions not usually compatible with platinum chemistry. Of the platinum(II)-peptide conjugates since reported, the majority focus on using the peptide as a “Trojan horse” to selectively deliver its platinum drug cargo to cancer cells. For example, Marchán et al., in 2009, developed dichloroplatinum(II) conjugates bearing dicarba analogues of octreotide (57), Figure 10. These conjugates retained their ability to bind to DNA.116 That same year, Hammer et al. developed platinum(II)-cyclic peptide conjugates in which the peptides were conjugated to the platinum center via an O,Omalonate linker system (58), Figure 10. These complexes were shown to not only target the CD13 receptor overexpressed on cancer cells but also were highly cytotoxic toward PC-3 cells, much more so than carboplatin.117 Kelley, Lippard, and team successfully conjugated a mitochondrial targeting decapeptide via an O,O-linker to a platinum(II)-ammine framework (59), Figure 10. Although less cytotoxic compared to cisplatin, the complex was shown to be delivered to the mitochondria and to

Figure 10. Chemical structures of dichloroplatinum(II) conjugates bearing dicarba analogues of octreotide (57), platinum(II)-cyclic peptide conjugates (58), and a platinum(II)-mitochondrial targeting decapeptide conjugate (59).

bind to mitochondrial DNA. Interestingly, the complex did not follow the mechanism of action of cisplatin in that it did not induce nuclear DNA damage nor did it induce cell cycle arrest.118 Our group also tried exploit peptides but, in our case, chose cationic antimicrobial peptides (CAPs). Many CAPs are known not only for their propensity to selectively target tumor cells but also many have been shown to possess intrinsic antitumor properties in their own right. These CAPs typically consist of between 5 and 50 amino acid residues and carry a net positive charge of between +2 to +9.119 In our first example, we derivatized the cationic antimicrobial P18 peptide and were successful in developing the first reported platinum(II)-CAP conjugate, (60), Figure 11, with the potential to be selectively cytotoxic toward tumor cells.113 The peptide was

Figure 11. Chemical structures of platinum(II) conjugates bearing derivatives of the cationic antimicrobial P18 (60) and buforin IIb (61) peptides. 1068


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Figure 12. Chemical structures of SAHA (62), Pt-malSAHA (63), Belinostat (64), Pt-malBel (65), valproic acid (66), and trans-Pt-valproate (67).

linked to the platinum core via a N,N-linker. While its anticancer activity was not reported, we highlight this work here for two reasons which may be of interest to the reader. First, we specifically chose to work with the D-stereoisomer of this peptide; D-peptides have been shown to evade proteolytic degradation and thus possess longer half-lives over their Lcounterparts in biological systems.120 Second, the use of the Disomer of P18 does not adversely affect its cytotoxic potential. In fact the D-P18 peptide had been shown to exhibit greater antiproliferative activity against a range of tumor cell lines relative to the L-form while also maintaining its selective cytotoxicity against tumor cells.121 In our second example, we attempted to harness the cell targeting, cell-penetrating, and antiproliferative effects of buforin IIb, another CAP, to target a cytotoxic platinum(II)DNA binding species to tumor cells while also delivering the peptide with potent antitumor properties in its own right.122 The tumor-cell selectivity associated with this 21 amino acid CAP is based on its capacity to electrostatically interact with cell surface negatively charged gangliosides.123 Unlike some CAPs, buforin IIb has also been shown to transverse the cell membrane without affecting its overall integrity. In cells, it induces apoptosis either via a mitochondria-dependent pathway124 or via interactions with the endoplasmic reticulum.125 The cytotoxic peptide had first to be derivatized so as to include an O,O-malonate terminal linker. The buforin IIb was generated using SPPS. A protected malonate linker was then manually added to the resin-bound buforin IIB using coupling chemistry. Cleavage from the resin and purification using HPLC followed. It was then successfully tethered to a cisplatin Pt(NH3)2 core to generate a dual-functional platinum(II)ammine-CAP conjugate (61), Figure 11.122 This conjugate was found to possess an order of magnitude greater cytotoxicity against the cisplatin resistance A2780 cells compared to a platinum-ammine-malonate standard, although its selectivity for tumor cells over a representative healthy cell line was not pronounced. The approach taken in this study to generate platinum(II) complexes bearing a tumor targeting and cytotoxic CAP could be extended to other peptides of this class.122 While this was a preliminary study, it does highlight the potential of employing CAPs as promising ligands. A number of CAPs have been shown to not only trigger

apoptosis in tumor cells via mitochondrial membrane disruption but also act as potent inhibitors of angiogenesis (blood vessel development) often associated with tumor progression.126 The folate receptor, a glycoprotein overexpressed on tumor cells, has also been exploited for the targeted delivery of platinum payloads to tumor cells. For example, Osella et al. developed a small series of platinum-folate conjugates but, due to their lack of water solubility and poor solubility in DMSO, their biological activities could not be investigated.127 Folate receptor-targeted liposomal systems have also been exploited to selectively deliver carboplatin to tumors,128 as have folatetargeted liposomes encapsulating cisplatin129 and folatedecorated nanogels encapsulating cisplatin,130 but these are beyond the scope of this review. The reader is directed to an eloquent recent review by Devereux et al.,107 in which they detail how folate-targeting can be exploited to improve efficacy in inorganic medicinal chemistry. Included in this review are examples of platinum(II)-folate conjugates and nanotechnologies encapsulating platinum drugs such as cisplatin.107 2.2. Platinum(II) Drugs Targeting DNA and at Least One Other Cellular Entity

The quest to enhance the anticancer activity of classical platinum drugs while overcoming their drawbacks has led to extensive research into identifying new cellular targets beyond DNA for therapeutic exploitation. Enzymes, which play a crucial role in almost every physiological and pathophysiological process, have long been considered as exploitable drug targets.131 In fact, it has been estimated that over 47% of approved drugs target the inhibition of enzymes.132,133 2.2.1. Enzymes as Targets. 2.2.1.1. Histone Deacetylases. Chromatin organization plays an instrumental role as an epigenetic regulator of gene expression. Chromatin packages DNA into the microscopic space of the eukaryotic nucleus by wrapping DNA around core histone proteins leading to a highly compact, tense structure.134 Acetylation of these core histones, catalyzed by histone acetyltransferases (HATs), leads to an open chromatin structure, and this allows transcription to take place. Histone deacetylases (HDACs), in contrast, deacetylate core histone proteins returning chromatin to its condensed state. The latter process down regulates tran1069


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scription. Inhibiting these enzymes impacts chromatin organization and ultimately transcription.135 These enzymes therefore represent an attractive target for therapeutic exploitation, and in fact, a number of HDAC inhibitors are now in clinical use as anticancer agents and others are in various stages of clinical trials. The first pan HDAC inhibitor to enter the clinic was suberoylanilide hydroxamic acid (Vorinostat; SAHA) (62), Figure 12. It received FDA in 2009 as a treatment for cutaneous T-cell lymphomas in patients with progressive, persistent, or recurrent disease on or following two systemic therapeutic regimes.136−138 Crystal structures of human HDACs with SAHA bound provide evidence that the hydroxamic acid moiety plays a key role in enzyme inhibition by binding directly to the active site zinc ion.139 The aliphatic linker chain fits neatly into the enzyme’s narrow channel, and the phenyl ring acts as a protein surface recognition domain.139 In the year preceding the clinical introduction of the hydroxamate-based HDAC inhibitor SAHA as a treatment for lymphoma, an extensive amount of research was reported on HDAC inhibitors and their potential use as anticancer agents. For example, according to a Pubmed search for the year 2008 and using the terms “histone deacetylase inhibitors” and “cancer”, there were a total of 552 hits. During this period, we had been working on hydroxamic acids as important bioligands and on their metal complexes as potential therapeutic agents.140−145 When SAHA entered the clinic, we instantly recognized the opportunity to combine into one drug molecule the DNA binding properties of platinum with the HDAC inhibition properties of SAHA. The preferential selectivity of HDAC inhibitors such as SAHA for tumor cells over healthy cells,135,146 and their ability to synergistically enhance the anticancer efficacy of existing drugs, including cisplatin,147 provided additional motivation behind our approach. Informed by a molecular modeling study, we derivatized SAHA in such a way to facilitate its binding to platinum while not compromising its HDAC inhibition potential. This was done by incorporating a malonate substituent on the phenyl ring of SAHA while keeping the hydroxamate moiety free to interact with the zinc ion at the HDAC active site. We subsequently developed cis-[PtII(NH3)2(malSAHA−2H)] (Pt-malSAHA), (63), Figure 12, the first dual-functioning platinum complex of its kind to be reported and confirmed its DNA binding, HDAC inhibitory activity and significantly reduced toxicity as compared to cisplatin against cisplatin sensitive and cisplatin resistance A2780 ovarian cancer cell lines. Interestingly, our complex was an order of magnitude less toxic compared to cisplatin against a representative healthy normal human dermal fibroblast (NHDF) cell line.148 A more in-depth study revealed that this complex retained its potent cytotoxicity against a larger panel of tumor cell lines (CH1, SW480, A549, and SKBR-3) having comparable cytotoxicity to that of cisplatin or SAHA. The complex was found to accumulate better in tumor cells, much more so compared to cisplatin or SAHA, but it bound to DNA less readily when compared to cisplatin. This is hardly surprising given that the chlorido ligands in cisplatin are considerably more labile compared to the bidentate malonato ligand of malSAHA. When investigated in cellulo, Pt-malSAHA bound DNA more effectively. This led us to investigate their mode of activation under cellular conditions. DNA binding was enhanced in the presence of thiol-containing molecules such as glutathione and thiourea, and activation occurred in cytosolic but not nuclear extracts of human cancer cells.149

Belinostat (Bel) (64), Figure 12, a second generation analogue of SAHA, entered the clinic in 2014 for the treatment of patients with relapsed or refractory peripheral T-cell lymphoma.150−152 It has also advanced to various stages of clinical trials for a number of solid malignancies, including ovarian cancer.153 As a follow up study to that described above, we developed [PtII(NH3)2(malBel−2H)] (Pt-malBel), a Bel analogue of Pt-malSAHA (63), Figure 12.154 This study revealed that Pt-malBel (65), Figure 12, had comparable cytotoxicity when compared to Pt-malSAHA (63) against the cisplatin sensitive A2780 ovarian cells. It was however found to be considerably more potent than Pt-malSAHA against the cisplatin-resistant A2780cisR ovarian tumor cells. Interestingly, and in contrast to Pt-malSAHA, Pt-malBel was not as cytotoxic when compared to both cisplatin and Bel alone. Exchanging malBel for malSAHA undoubtedly alters the pharmacokinetic profile of the resulting complex which may have resulted in, for example, a reduction in cellular accumulation, which may account for this finding. Valproic acid (VPA, 2-propylpentanoic acid) (66), Figure 12, is an established drug treatment for epilepsy and bipolar disorder, but it has also been shown to possess HDAC inhibition activity and, in this regard, antimetastatic and anticancer properties.155 With this in mind and inspired by the work of Farrell and others who reinvigorated research into the potential use of planar trans platinum amine (TPA) complexes as anticancer agents,57,156 and the fact that Lin et al. had previously described how VPA was capable of resensitizing cisplatin-resistant ovarian cancer cells in vitro,157 we developed two novel trans-platinum(II) complexes incorporating the HDAC inhibitor VPA, namely trans-[Pt(VPA−1H)2(NH3)(py)] (67), Figure 12, and trans-[Pt(VPA−1H)2(py)2] (py is pyridine).158 Farrell et al. had previously demonstrated that replacing the chlorido ligands in TPA complexes with carboxylato moieties could markedly enhance the cytotoxicity profile of the resulting complexes.156,159 Sodium valproate, transplatin, and the TPA complexes, trans-[PtCl2(py)2] and trans-[PtCl2(NH3)(py)], were shown to be inactive in both A2780 and A2780cisR ovarian cell lines at the maximum concentration used (100 μM). A previous study had indicated that the cytotoxicity profile of TPA complexes could be significantly enhanced when the ammine ligands of transplatin were substituted with N-donor heterocyclic ligands, such as py, with cytotoxicities on par with those of cisplatin. We, in contrast, found that replacement of the chlorido ligands in trans-[PtCl2(py)2] and trans-[PtCl2(NH3)(py)] by VPA ligands which afforded trans-[Pt(VPA−1H)2(py)2] and trans[Pt(VPA−1H)2(NH3)(py)], respectively, exhibited only marginally enhanced cytotoxicity against A2780 and A2780cisR cells when compared to cisplatin. Cytotoxicity was marginally enhanced in the cisplatin-resistant (A2780cisR) phenotype over the parental one (A2780). Interestingly, as you will see in section 3.2.1.2, changing the oxidation state of the platinum ion from +2 to +4, while retaining the VPA as a HDAC inhibitor ligand, had a marked effect on the efficacy of the resulting complexes. 2.2.1.2. Pyruvate Dehydrogenase Kinases. Dichloroacetate (DCA), a structural analogue of pyruvate, is an orally available small molecule inhibitor of pyruvate dehydrogenase kinase (PDK).160 Inhibition of PDK leads to an influx of pyruvate into the mitochondria which promotes glucose oxidation over glycolysis leading to suppression of in vitro and in vivo tumor cell growth.160,161 Furthermore, DCA appears to be well1070


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Figure 13. Chemical structures of platinum(II) complexes bearing DCA derivatives (68−73).

Figure 14. Chemical structures of platinum(II) complexes bearing CDK inhibitor ligands in the form of bohemine (74) or bohemine derivatives (75 and 76).

tolerated by patients.162 As such, it represents a potential metabolic-targeting therapy for the treatment of cancer. Li et al. developed two mixed-ammine/amine platinum(II) complexes incorporating 3-dichoroacetoxylcyclobutane-1,1dicarboxylate in which DCA was tethered to the cyclobutane ring via an ester bond, (68 and 69), Figure 13. While the compounds showed marked cytotoxic selectivity toward cancer cells (including cisplatin resistant SK-OV-3 cells) over BEAS2B normal cells, they were found to be poorly water-soluble.163 In an attempt to improve the physicochemical properties of the aforementioned complexes, the same group developed four new diam(m)ine platinum(II) complexes in which they retained the carboplatin-like dicarboxylate DCA-containing leaving group but altered the N-containing nonleaving ligand(s) (70−73), Figure 13. Their cytotoxicities were tested against A549, SK-OV-3, and SK-OV-3/DDP (cisplatin resistant) cell lines. Of the complexes, 71, an oxaliplatin-like analogue, was found to be most potent, being almost 60 times more potent than carboplatin against the A549 cells, 6 times more potent against SK-OV-3 cells, and over 15 times more potent against the cisplatin-resistant SK-OV-3/DDP cells. The same complex, 71, was found to efficiently release the DCA moiety via hydrolysis of the ester bond under physiological conditions. Complex 71 was also found to be approximately 10 times more soluble than the complexes reported previously.

This study demonstrates DCA to be a promising ligand for the creation of dual-action platinum(II) complexes that can overcome cisplatin resistance.164 2.2.1.3. Cyclin-Dependent Kinases. Cyclin-dependent kinases (CDKs) are sugar kinases that play a pivotal regulatory role in the cell cycle and additional roles in transcription, mRNA processing, and cell differentiation.165,166 These CDK enzymes are often found to be overactive in cancer cells. In addition, certain cancer cells downregulate the natural CDK inhibitor proteins found in normal cells, which regulate their activity. As such, CDKs have been identified as promising anticancer targets, and numerous CDK inhibitors have been developed to prevent tumor cell proliferation by blocking or inhibiting CDK-mediated roles. For example, palbociclib, a CDK inhibitor, in combination with an aromatase inhibitor, received accelated FDA approval in 2015 and later general FDA approval in 2016167 as a treatment for breast cancer that is estrogen receptor positive (ER+) and HER2 negative in postmenopausal women,168 while other CDK inhibitors, including abemaciclib, are in clinical trials.169 Previous studies have shown that complexing the CDK inhibitors bohemine or olomoucine to a platinum(II) framework significantly increased the cytotoxicity of the resulting complexes against several tumor cell lines.170 Brabec et al. followed up this study by developing three new complexes of 1071


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formula cis-[Pt(X)2Cl2] where X represented either bohemine (74), Figure 14, or bohemine derivatives (75 and 76), Figure 14.171 In vitro cytotoxicity studies against A2780 and A2780cisR ovarian cells, as well as against noncancerous cell lines of human origin (i.e., human fibroblast cells from embryonal lung tissue IMR-90 and Chinese hamster ovarian cells CHO-K1 and its mutant cell line MMC-2), revealed that the complexes exhibited either comparable or enhanced cytotoxicity to cisplatin against the cisplatin sensitive A2780 cell lines. They were, however, found to be considerably more cytotoxic than cisplatin against the cisplatin resistant variant. Complex 74, which was the least cytotoxic of the three against the A2780cisR cells but still significantly more cytotoxic than cisplatin (IC50 of 8.7 μM vs 24.7 μM, respectively) proved to be the only complex with selectivity toward the cancer cells over the normal ones. Complexes 75 and 76, while more cytotoxic against the tumor cells, had similar or lower IC50 values against the IMR-90 cells. The team concluded that 74, incorporating the nonderivatized form of bohemine, possessed a unique cytotoxic mechanism which was distinct from that of cisplatin and had the capacity to overcome cisplatin resistance. Interestingly, despite the rationale behind the development of these complexes, the team could not attribute this conclusion to its CDK inhibitory ability, as testing for this showed CDK inhibition was lost upon complexation of the bohemine to the platinum(II) framework. The complex did induce significant DNA platination, suggesting that its mechanism of action is still targeted toward DNA binding.171 It is also noteworthy that these complexes break the structure−activity relationship criteria originally associated with classical platinum(II) drugs in that the platinum(II) center is coordinated to the CDK inhibitors via tertiary N-donor atoms. A follow-up study by Brabec et al. sought to elucidate the different DNA-damaging mechanisms of 74 as compared to cisplatin.172 They did this through a series of biochemical and molecular biology studies focusing on the type of DNA damage occurring and the ability of the complex to interact with DNA binding proteins, DNA processing enzymes, and glutathione. The team identified six key differences between the mechanism of action of 74 to that of cisplatin which may account for its ability to overcome cisplatin resistance: (i) 74 had a slower rate of DNA binding; (ii) it was less able to form bifunctional DNA adducts; (iii) it reduced bending of DNA by the major adduct of 74; (iv) the platinum-DNA adduct formed, following complexation of the 74 metabolite to DNA, had a reduced affinity for DNA repair HMG proteins; (v) 74 had a greater ability to prevent DNA polymerization and transcription by RNA, and (vi) it reacted more slowly with glutathione as compared to cisplatin.172 Despite the fact that these complexes, which were orginally designed to target CDK enzymes, had no CDK inhibitory properties, they did possess promising in vitro cytotoxicities. They also had a DNA damaging profile significantly different to that of cisplatin. 2.2.1.4. Glutathione Peroxidase and Thioredoxin Reductases. Bednarski et al. synthesized a library of twenty eight monochlorido platinum(II) complexes coordinated to different heterocycles.173 A general structural formula for these complexes (77) is provided in Figure 15. Of the complexes developed, 50% were derived from a cisplatin base with one of the chlorido ligands substituted with the heterocycle, while the other 50% were trans-platinum(II) analogues. The ligands consisted of imidazoles and pyrazoles linked to various acylhydrazones, which the team had previously shown to be

Figure 15. General chemical structure (77) of cis- and transplatinum(II) complexes bearing imidazoles and pyrazoles linked to various acylhydrazones, previously shown to be weak GPx inhibitors.

weak inhibitors of the glutathione peroxidase (GPx) enzyme.174,175 The GPx enzyme plays a significant role as an antioxidant176 and is often linked to tumor resistance to chemotherapeutics. The team sought to develop cisplatin and transplatin analogues bearing these GPx inhibitor ligands with a view to advancing a new class of therapeutic with DNA binding and GPx inhibition properties. They also theorized that binding these ligands to platinum(II) could potentially improve their normally weak GPx inhibitory activity, resulting in irreversible inhibition. This, however, was found not to be the case. The complexes failed to inhibit GPx. They also investigated the capacity of their complexes to inhibit thioredoxin reductases (TrxR). Thioredoxin reductases (TrxR), together with thioredoxin (Trx), play central roles in the mitochondrial thioredoxin system. This system regulates multiple redox signaling pathways and, in recent times, has been recognized as a key modulator of tumor development.177 Inhibition of TrxR has also been shown to induce tumor cell suppression.177,178 Of the complexes tested, only some weakly inhibited TrxR. Despite these findings, the complexes did demonstrate good cytotoxicity results when tested against A2780, A2780cisR, SISO, and LCLC-103H cancer cell lines. In general, the cis complexes were more active than their trans analogues, and both demonstrated the ability to overcome cisplatin cross-resistance, with the trans complexes being more effective at this. The complexes were generally all less cytotoxic when compared to cisplatin with the exception of a couple of cases in the A2780cisR cell line. The complexes were shown to bind DNA, but there was no correlation between DNA binding and cytotoxicities, suggesting a different mechanism of action to that of cisplatin.173 Again, despite the fact that these complexes, which were orginally designed to target GPx enxymes while retaining their DNA binding capacity, had no GPx inhibitory properties, they did possess promising in vitro cytotoxicities with a mechanim of action distinct to classical platinum drugs. 2.2.1.5. Farnesyl Pyrophosphate Synthases. The mevalonate pathway is an essential eukaryotic metabolic pathway which produces the carbon building blocks of over 30000 biomolecules such as cholesterol, heme, and steroid hormones.179,180 Bisphosphonates are compounds with a strong affinity for calcified tissue such as bone mineral and, as such, have been used in the treatment of bone-related diseases such as osteoporosis, tumor-induced hypercalcemia, and bone metastases. They have been used as drugs in their own right or as drug-targeting and/or drug delivery vehicles. Lin, Luo, et al. set out to combine into one drug the bonetargeting capabilities of bisphosphonates with the anticancer properties of platinum with a view to advancing a new and effective bone-related cancer chemotherapeutic.181 They took zoledronic acid (ZL), an imidazolyl-containing bisphosphonate widely used in the clinical treatment of several bone diseases,182 and complexed it with two platinum(II) centers, 1072


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Figure 16. Chemical structures of platinum(II)-bisphosphonate complexes (78−81).

MYC, and hTERT, which suppress cancer cell growth. The p53 suppressor protein has also been shown to induce cell death by caspase-dependent signaling. Matrix metalloproteinases (MMPs), which are a family of zinc-containing proteolytic enzymes, in contrast, play an integral role in healthy tissue remodeling and degradation of proteinaceous components of the extracellular matrix.185 Their abnormal expression can result in excessive breakdown of the extracellullar matrix, and this degradation has been implicated in a variety of disease states, including tumor metastases.185 These enzymes therefore represent an attractive target for chemotherapeutic agents, in particular, antimetastatic agents. Decreased expression of MMP-1, MMP-2, MMP3, MMP-7, and MMP-9, in particular, in cancerous cells has been shown to result in suppression of cancer metastasis. Bisphosphonates are known to inhibit MMPs.185 With this in mind, Collucia et al. developed two novel platinum(II) complexes incorporating a diethyl[(methylsulfinyl)methyl]phosphonate (SMP) ligand, (82 and 83), Figure 17.186 The

with the ZL acting as a bridging linker. Zoledronic acid is a potent farnesyl pyrophosphonate synthase (FPPS) inhibitor.183 The coordination sphere around each platinum center was completed by ethylenediamine ligands, (78−81), Figure 16. They deliberately chose ZL on the basis that its structure could be fine-tuned by varying the R substituent and the length of the carbon chain linker which could potentially enhance their binding and biological properties. Cytotoxicity studies against U2OS, A549, HCT116, MDAMB-231, and HepG2 cell lines showed the complexes to be significantly more potent than the free ZL, though less potent than cisplatin. Complex 78, with the unmodified ZL as a bridging ligand and with the shortest carbon chain on the imidazole, was the most potent, demonstrating that chain length was important for activity. The complexes proved to be nonselective against most cell lines when compared to the free ligand. All induced more damage to normal human liver LO2 cells compared to the free ZL ligands. However, they were more selective than their ligands against HepG2 cells. Although the complexes were found to have a high affinity for bone mineral hydroxyapatite, this did not improve their selectivity for bone cancer cell lines. DNA binding studies revealed that the complexes did indeed bind DNA, albeit only moderately although, from biophysical studies, the interactions were identical with those of cisplatin. They were also shown to induce cell cycle arrest at the G2/M phase, again similar to that of cisplatin.181 A follow-up study by the same team sought to ascertain if the most potent complex in the previous study, 78, Figure 16, was able to inhibit FPPS and geranylgeranyl pyrophosphate (GGPP), crucial enzymes involved in the mevalonate pathway.184 These enzymes are believed to be key targets for the therapeutic effects of ZL as a drug. They first screened the complex against several tumor cells (SGC7901, HepG2, MCF7, MDA-MB-231, HCT116, and U2OS) and a normal gastric mucosal epithelial cell line (GES-1) and normal liver cell line (LO2). They found the complex to be most effective at inhibiting the cell profileration of GC7901 gastric cells. This cell line was next treated simultaneously with both the platinum complex 78 and either farnesol (FOH) or geranylgeraniol (GGOH), precursors of FPPS and GGPP, respectively, with a view to gaining a deeper insight into its mechanism of action. It was found that the IC50 value of 78 against this cell line (25.15 μM) was significantly raised by the inclusion of these enzyme precursors (to 83.72 μM for FOH and 65.69 μM for GGOH), demonstrating that the complex is, as seen in its free ligand, involved in the inhibition of the mevalonate pathway via inhibition of these enzymes. It is interesting that, while the complex appears to be carrying out its dual-functional role as hypothesized, the team did not obtain the selectivity they had hoped this would afford toward its original target of bone. 2.2.1.6. Matrix Metalloproteinases. The p53 protein is a known suppressor of tumor metastasis. When activated, it triggers the expression of numerous genes such as p21, PUMA,

Figure 17. Chemical structures of platinum(II)-phsophonate complexes (82−83).

SMP ligand was chosen to not only enhance bone specificity but also to improve the pharmacodynamic properties of the resulting complexes. Both 82 and 83 exhibited marked MMP inhibitory activity against MMP-3, -9, and -12 enzymes but only moderate inhibition of MMP-2. The observed inhibition was found to be via a noncompetitive mechanism. In contrast, both cisplatin and carboplatin did not possess any MMP inhibitory activity even when exposed to these enzymes after longer experimental reaction times.186 Despite the fact that other platinum phosphonate complexes, including a series of diam(m)ineplatinum(II) complexes bearing a bone-targeting N,N-bis(phosphonomethyl)glycine ligand,187 had been reported, these complexes represented the first examples of platinum(II) complexes possessing MMP inhibition activity. Natile and co-workers, in a follow up mechanistic study,188 developed a series of platinum(II) complexes with a view to establishing whether the presence of three potential coordination sites were necessary for MMP inhibition activity. They developed complexes with three chlorido or one chlorido and a dimethylmalonato as leaving groups, and ammonia (NH3), dimethyl sulfoxide (DMSO), or diethyl(aminomethyl)-phosphonate (AMP) as nonleaving ligands. Of the platinum complexes investigated, those that had three labile ligands exhibited MMP-3 inhibitory activity, whereas, significantly, those with only one or two labile ligands were inactive.188 An ESI-MS study revealed that reaction of 82 with the catalytic domain of MMP-3 resulted in the formation of a key type 1073


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cell cycle, cell survival, and immune response linked with cancer progression. STAT3, once activated, forms a homodimer which enters the nucleus where it can bind DNA. This promotes the translation of target genes which are associated with antiapoptosis, angiogenesis, and invasion/migration. STAT3 has been shown to be constituitively active in around 70% of human solid tumors, while in normal cells, it remains transient.193 STAT3 inhibition thus represents an attractive drug target. Oxadiazoles have been identified as effective STAT3 inhibitors, with the oxadiazole ring identified as being a key feature in known STAT3 inhibitors. With this in mind, Rimoldi, Ferri, and Gelain et al. developed three novel platinum(II) complexes bearing 1,2,5-oxadiazole ligands, (85− 87), Figure 19.194

MMP-3-[PtCl(OH)(SMP)] adduct where the platinum had lost one chlorido ligand and acquired a hydroxo ligand, most likely at the expense of the SMP phosphonic functionality.188 An NMR study further revealed that His 96 and His 224 can act as platinum(II) binding sites with binding of the platinum ion to His224, which is located in the specificity loop on MMP-3, being the most likely binding site. Such binding could thus induce some conformational change impacting the proximally located zinc(II) binding site.188 While the DNA binding properties of these platinum complexes were not investigated, we decided to include these studies in this review to highlight the potential in developing “multitargeted” platinum drugs with MMP inhibition properties. Zhang, Wang et al., in an unrelated study, tested the anticancer properties of a dinuclear mixed ligand complex consisting of two square planar platinum(II) centers connected by a phenylenediamine bridge, (84), Figure 18.189

Figure 19. Chemical structures of platinum(II) bearing oxadiazoles with STAT3 inhibitory properties (85−87). Figure 18. Chemical structure of a dinuclear mixed ligand platinum(II) complex with MMP inhibition properties (84).

Their cytotoxicities were tested againt HCT116 cells, as well as their ability to interupt STAT1 and STAT3 signaling. The ligands showed little or no cytotoxic effect. Of the three complexes, only 87 exhibited significant cytotoxicity. All ligands disrupted STAT3 activity with the ligand in 87 and 87 itself selectively interfering with STAT3 over STAT1. Compound 87 was also found to significantly inhibit STAT3 promotion. DNA binding studies involving 87 were performed using the model nucleobase 9-ethylguanine (9-EtG). This study confirmed that this complex had the potential to interact with DNA, which was validated by Western blot analysis studies. In vivo studies comparing the activity of the ligand in 87, 87 itself and cisplatin on a lewis lung carcinoma model revealed that the ligand, 87 and cisplatin could reduce tumor mass by approximately the same extent (with a reduction between 82.6 and 87.5%).194 2.2.2.2. Tubulin. Schobert et al. conducted a structure− activity relationship study on a series of novel platinum(II) complexes incorporating two different, cis-oriented, N-heterocyclic carbene (NHC) ligands.195 Of relevance to this review is the fact that during the course of their investigation the team tested the DNA binding and cytotoxicity of a platinum(II) complex incorporating a ligand with the same structure as combrestatin A-4, a clinically used tubulin-inhibiting anticancer agent (88), Figure 20. While tubulin inhibition was not covered in this study, it was noted that 88 had DNA binding

The complex had comparable cytotoxicity against MCF-7, HepG2, and A549 cell lines to cisplatin. The complex was found to upregulate p53, leading to interaction with its target genes: PUMA was upregulated, which typically leads to apoptosis. p21 was also activated, leading to the blocking of cell cycle progression, and MYC and hTERT were downregulated, which represents blocking of cancer progression by decreasing DNA synthesis. Of particular note was the fact that 84 was able to suppress the invasion and migration of human lung and luminal-like breast cancer cells. While the complex was not designed to specifically target MMPs, it was found to suppress the expression of a number of MMPs, including MMP-1, MMP-2, MMP-3, MMP-7, and MMP-9. Gel electrophoresis studies confirmed that the complex was also able to target and bind DNA.189 Developing complexes that target enzymes involved in cancer progression and metastases is clearly an attractive goal provided the inhibition is selective and the mechanistic basis underpinning this inhibition has been elucidated. 2.2.2. Proteins as Targets. Developments in proteomics will no doubt lead the way in identifying new protein targets for therapeutic exploitation.6 For example, anaplastic lymphoma kinase was identified as a potential target in ovarian cancer following a proteomic approach,190 as was cyclophilin as a potential prognostic factor and therapeutic target in endometrial carcinoma.191 Proteins, as potential targets of metallodrugs, have recently been highlighted in a review by Hartinger et al.192 Here, we highlight platinum(II) complexes which have been rationally designed to interact with key protein targets which have been implicated with cancer progression and/or metastases. 2.2.2.1. STAT3. Signal transducer and activator of transcription 3 (STAT3) is a transcription factor protein and member of the STAT3 protein family. This STAT3 family plays a central regulatory role in gene expression related to the

Figure 20. Chemical structure of a platinum(II)-combrestatin A-4 derivative (88). 1074


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would not only intercalate DNA given its planar cationic structure but also might possess interesting photophysical properties. Its DNA binding constant was found, however, to be several orders of magnitude lower than typical DNA intercalators (which have binding constants in the range 105− 106 M−1).51 Self-aggregation, which could compete directly with DNA binding, was proposed as a possible explanation. The complex was found to exhibit comparable cytotoxicity in HeLA cells to cisplatin and was approximately 4 times more cytotoxic compared to cisplatin in A549 lung cells. In a follow-up study by the same group, the cytotoxicity of 89 was screened against U2OS, MCF-7, HT-29, A2780, and A2780CP70 cancer cells as well as the normal fibroblast cell line MRC-5.197 The complex showed excellent cytotoxicity, being better or comparable to cisplatin across all cell lines except A2780 cells. It exhibited enhanced cytotoxicity against the cisplatin-resistant A2780CP70 cells, significantly more so than cisplatin, demonstrating an ability to overcome cisplatin resistance. The ligand alone was shown to have poor cytotoxicity across all cell lines, showing that the platinum center was important for the cytotoxicity observed. The complex readily accumulated in cells most likely due to its lipophilic properties and was distributed evenly between the cytoplasm and nucleus. A significant amount of complex was found to be taken up by the mitochondria. The complex was shown to damage genomic and mitochondrial DNA, enrich p53 and BAX levels, and induce mitochondrial-mediated apoptosis. Its mechanism was also shown to be p53independent, a promising observation as p53 is often downregulated in cancerous cells.197 Again, this is another example of a complex that breaks the structure activity relationship associated with cisplatin and yet demonstrates a promising cytotoxicity profile. It, and other platinum complexes containing planar π-conjugated ligands as DNA intercalators, could well serve as an interesting platform for the design of multitargeted platinum(IV) drugs.

ability and that it was also specifically cytotoxic to cancer cell lines (in the low nanomolar range), which are also sensitive to combrestatin A-4 (i.e., DLD-1 colon carcinoma and Panc-1 pancreatic cancer cells). The platinum(II)-combrestatin A-4 derivative, 88, was also highly cytotoxic toward the other cancerous cell lines tested but in the micromolar range. This shows that the ligand may have retained its inherent tubulininhibitory activity after complexation with platinum(II). This, combined with the evidence to suggest that incorporation of the combrestatin A-4 derivative into the platinum scaffold appears to provide cytoselectively, warrants further studies on its potential to act as a dual targeting agent. 2.2.3. Mitochondria as a Target. Noncovalent DNA binders have shown themselves to be promising alternatives to the typical irreversible DNA binding associated with classical platinum drugs. Planar π-conjugated ligands such as bipyridines (bpy) and phenanthrolines (phen), when coordinated to metal ions such as platinum(II)59 and copper(II),51 can intercalate DNA, causing it to unwind. Aldrich-Wright et al. have also developed a number of platinum(II) complexes incorporating planar aromatic ligands which not only intercalate DNA but also, following intercalation, bind directly to DNA nucleobases. The reader is directed to an eloquent recent review by Aldrich-Wright et al. in which these are comprehensively described.59 Lippard et al. conjugated the monoanionic tetradentate β-diketiminate ligand BDIQQ to platinum(II), resulting in the formation of [Pt(BDIQQ)]Cl, (89), Figure 21.196 The team postulated that the complex

Figure 21. Chemical structure of a platinum(II) complex bearing a monoanionic tetradentate β-diketiminate ligand BDIQQ (89).

Figure 22. Chemical structures of a photoactive platinum(II) complex Platicur (90), a platinum-acetylacetone derivative (91), and photoactivatable organometallic platinum(II) complexes of β-diketonates (92−94). 1075


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Figure 23. Chemical structures of a heteronuclear platinum(II)/ruthenium(II) complex (95) and their homonuclear analogues (96−97) as well as the structure of a platinum(II)/gold(I) complex (98).

2.2.4. Targets Inspired by Natural Product Activity. Chakravarty, Kondaiah, et al. developed a photoactivatable platinum(II) complex with curcumin coordinating the metal center via O,O-chelation.198 Curcumin, derived from yellow tumeric, is widely used in traditional Indian and Chinese medicine.199 It has remarkable antiflammatory properties, is able to inhibit proinflammatory transcription factors (NF-kB), which also endows it with anticancer capabilities. Despite this, its low aqueous solubility and poor bioavailability hinders its performance as a single agent. Curcumin has demonstrated significant photosensitizing properties, however, and has been shown to react with molecular oxygen to generate ROS in its triplet state. The team hoped to improve the cellular delivery of this photoactive curcumin ligand by complexing it with platinum(II) to create a photoactivatable dual-action anticancer agent. This complex was named Platicur (90), Figure 22, and was found to release the curcumin ligand and a cisplatin adduct upon irradiation with visible light (400 to 700 nm), thus acting as a prodrug. Its cytotoxicity against HaCaT, BT474, Hep3B, and T47D cell lines showed that the complex, following photoactivation, was indeed cytotoxic (IC50 values ranging from 12 to 18 μM at 400−700 nm) compared to its toxicity in the dark with IC50 values greater than 200 μM. An analogue of the complex with an acetylacetone ligand showed no photoactivated cytotoxicity (91), Figure 22, strongly suggesting that it was indeed the curcumin ligand that rendered the complex with its photoactivatable properties. The compound was also found to generate platinum-GMP cross-links with DNA upon light-induced activation. This study highlights the potential to generate photoactivatable platinum(II) prodrugs by attaching stable photoactive ligands which detach from the platinum center upon irradiation with light.198 Chakravarty, Kondaiah, et al., in a follow up study, developed three new organometallic platinum(II) complexes (92−94), Figure 22, in which they replaced curcumin with photoactivatable β-diketonates.200 They focused their attention on organometallic systems on the basis that organometallic complexes have shown promise as anticancer agents.201,202 Photoactivation has also been successfully employed in the development of organometallic agents capable of releasing, for example, inhibitors of enzymes.203 They compared the properties of these complexes with those of Platicur (90). All three complexes exhibited marked photoinduced cytotoxicities with IC50 values of ∼10 μM when subjected to visible light

(400−700 nm), while being considerably less toxic in the dark (IC50 value of ∼60 μM). Complexes 93 and 94, with their extended planar aromatic pyrenyl and anthracenyl pendant moieties, in particular, were found to be avid DNA intercalators. The complexes also demonstrated efficient DNA photocleavage with hydroxyl radicals being identified as the ROS inducing this effect. The complexes were also highly cytotoxic when activated with visible light (with IC50 values ranging from ∼8−14 μM) against HaCaT cells.200 This work highlights a novel approach toward designing photoactivable “multitargeted” platinum(II) prodrugs. 2.2.5. Other Targets. Smythe, Das, Thomas, et al. developed a novel platinum(II)−ruthenium(II) hybrid complex in which the metal centers were linked by an extended terpyridine ligand, namely 4-([2,2’:6′,2’’-terpyridine]-4′-yl)-N(pyridin-2-ylmethyl)aniline or tpypma) (95), Figure 23.204 In vitro studies revealed that, while this heteronuclear complex (95) was cytotoxic against A2780 tumor cells, it exhibited no cross resistance against the A2780cisR cell line and was ∼10 times less cytotoxic compared to cisplatin. In contrast, their mononuclear ruthenium(II) (96) and platinum(II) analogues (97) exhibited no cytotoxicity against these cell lines, suggesting that both metal centers were important for the antiproliferative activity observed. Of key interest however was that the complex did not seem to induce any significant signs of cell stress or death despite its antiproliferative activity. In other words, its mechanism of action could not be linked to an induction of apoptosis or necrosis in the cells, but instead cytosis (i.e., preventing cell growth). Further testing with and without the complex in A2780cisR cells showed that the complex was able to impede cell cycle progress, as seen in the significant increase in G1 phase cells (from 42.7% to 65.3%) and corresponding decrease in G2M phase cells (42.1% to 25.5%) in cells treated with and without 95, respectively). The team concluded that this complex blocks entry into the S Phase by up-regulating p27KIP1. The compound had no DNA binding ability nor did it induce any DNA damage. This interesting study shows a dramatically different mode of anticancer action observed when two DNA-binding, apoptosis-inducing metals are combined, producing a complex that carries none of these expected traits, instead preventing cancer growth in a completely unique manner. While platinum anticancer drugs typically target DNA, research on gold complexes has shown them to be cytotoxic via 1076


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distinctly different mechanisms, typically reacting with protein targets.52 Bodio, Casini, et al. set out to investigate the effects of combining into one drug complex a platinum(II) core and a gold(I) core with a view to generating a complex with a novel mechanism of action.205 A bridge consisting of a dipyridylamine unit coupled to a diphenylphosphine was used to link the two metal centers (98), Figure 23. Its cytotoxicity was tested against A2780, A2780cisR, A549, and noncancerous HEK-293T cells. Interestingly, despite the structural differences between the heteronuclear complex and its mononuclear analogues, there was no significant difference in cytotoxicities across all the cell lines tested. In other words, there was no additive or synergistic advantage observed when combining both metal centers into one drug molecule. While the complexes were able to overcome cisplatin resistance in the A2780cisR cell line, none showed selectivity toward cancerous cells. Their IC50 values against the tumor cells were comparable to those obtained when the HEK-293T cell lines were treated with these complexes. The complexes did have comparable IC50 values to cisplatin and auranofin, showing them to be at least potent if not selective. Studies on the DNA binding of the heteronuclear complex 98 indicated that, while there was some minor interaction with DNA, it was not sufficient to suggest that DNA was its primary target and the cause of its cytotoxic activity. The team suggested that the presence of the gold-phosphine moiety interfered with the ability of the platinum(II) center to interact freely with the DNA helix.205 Podophyllotoxin (PPT) is a compound with cathartic, antirheumatic, antiviral, and antimicrobial as well as anticancer properties.206 While it is a potent cytotoxic agent, it is highly toxic to healthy cells as well as cancerous cells. Several semisynthetic derivatives of PPT, however, have proved successful in the clinic, such as etoposide, teniposide, and etopophos, which are used in the treatment of various cancers such as non small cell lung cancer, testicular carcinoma, lymphoma, and Kaposi’s sarcoma.207 Etoposide is used as a combination therapy with cisplatin for non small cell lung carcinoma and malignant lymphoma.207 Its mechanism of action is thought to be mediated via the formation of a ternary chelate comprising etoposide, DNA, and the topoisomerase-II enzyme, an enzyme involved in the formation of microtubules and often overactive in cancers.207 Hui, Zhang, and Chen et al. developed three platinum(II) complexes incorporating a PPT moiety into a cisplatin-like scaffold (99−101), Figure 24, and assessed their cytotoxicities against A-549, HeLa, HCT-8, HepG2, K562, and ADM/K562 tumor cells.208

Complex 99 demonstrated remarkable potency across all cell lines, being significantly more potent than cisplatin in all cases, and was 50 times more effective against the multidrug resistant ADM/K562 cell line. Complex 99 was able to induce DNA damage in a dose-dependent manner similar to cisplatin. It also prevented microtubule formation and induced signs of apoptosis in HeLa cells. While the presence of the PPT ligand appeared to enhance the cytotoxicity of the complex, selectivity studies were not carried out. These would have been interesting given the high toxicity associated with the PPT ligand. In summary, there is now good evidence to validate the use of targeting vectors such as glucose derivatives, steroids, and peptides for the selective delivery of platinum cytotoxic agents to tumor cells. Attaching bioactive ligands to the platinum scaffold with a view to targeting more than one cellular entity appears to give less consistent results. While the rationale behind their development is sound, we have highlighted examples whereby a synergistic advantage has not been observed. This may well be due to differences in potencies between the cytotoxic platinum core and the bioactive ligand. Notwithstanding this, there does appear to be an advantage in combining two or more drug entities into one drug molecule in that most of the “multitargeted” complexes reported herein are highly cytotoxic toward cancer-resistant cell lines. Representative examples of “multitargeted” platinum(II) complexes incorporating ligands which are used clinically as drugs in their own right and/or are in clinical trials are provided in Table 1.

3. MULTI-TARGETED PLATINUM(IV) PRODRUGS Platinum(IV) prodrugs offer therapeutic advantages over their platinum(II) congeners. Unlike square planar platinum(II) complexes, the low spin d6 platinum(IV) center adopts an octahedral geometry and is therefore coordinatively saturated. As a result, they are more resistant to ligand substitution compared to their platinum(II) counterparts. This lower reactivity is attractive as it reduces unwanted side reactions with biomolecules, thus potentially lowering toxic side effects. Furthermore, the two additional axial ligands can be functionalized to either (i) enhance tumor cell targeting with a view to reducing systemic toxicity issues and/or (ii) improve overall efficacy. By adding additional functionalities, it may also be possible to generate complexes with a mechanism of action distinct from classical platinum(II) drugs and, in so doing, potentially circumvent resistance.17,220,221 The axial ligands may also be modified to facilitate their conjugation to carrier systems. Strategies employed for enhancing both the delivery and efficacy of platinum(IV) prodrugs using different carrier systems have previously been reviewed.42,222 Three notable platinum(IV) prodrugs have undergone clinical trials; namely ormaplatin (also called tetraplatin) (102), the more water-soluble iproplatin (103) and the orally active satraplatin (104), Figure 25, but, despite promising results, none to date have advanced into the clinic. More comprehensive details related to these clinical studies are provided in a review recently published by Lippard et al.17 In terms of their mode of action, these platinum(IV) prodrugs, upon tumor cell entry, undergo a 2-electron reduction, forming cytotoxic platinum(II) adducts with concomitant loss of their two “innocent” axial ligands.16 This “activation by reduction” is dependent both on the nature of

Figure 24. Chemical structures of platinum(II) complexes bearing podophyllotoxin moieties (99−101). 1077


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Table 1. Representative Examples of “Multi-Targeted” Platinum(II) Complexes Incorporating Ligands Which Are Used Clinically as Drugs and/or Are in Clinical Trials metallodrug conjugate number and figure no.

drug ligand

63 (Figure 12)148,149 65 (Figure 12)154

vorinostat (SAHA) belinostat

67 (Figure 12)158

valproic acid

68−73 (Figure 13)163,164

dichloroacetate

78−81 (Figure 16)181

zoledronic acid

88 (Figure 20)195 99−101 (Figure 24)208

combrestatin A-4 podophyllotoxin

drug ligand target

clinical use/preclinical properties of drugs

histone deacetylases histone deacetylases histone deacetylases pyruvate dehydrogenase kinases farnesyl diphosphonate synthase tubulin microtubules

in clinical use as a treatment for cutaneous T cell lymphoma;138 in clinical trials against a range of cancer types.209 in clinical use as a treatment for Peripheral T-cell lymphoma;150 in clinical trials against a range of cancer types210 in clinical use as a treatment for epilepsy and bipolar disorder;211 sensitizes cancer cells to ionisizing radiation212 and DNA-targeting anti-cancer drugs;213 also in clinical trials against a range of cancer types214 in clinical use as an orphan drug treatment for various acquired and congenital disorders of mitochondrial intermediary metabolism;215 has undergone or is undergoing clinical trials as a treatment of cancers including breast, head and neck, lung and brain cancers216 in clinical use as a treatment for bone diseases including osteoporosis182 and cancer treatment related-induced bone loss217 in multiple clinical trials including as a treatment for solid tumors218 in clinical use as a plant-derived anti-cancer agent;219 its derivatives (e.g. etoposide and teniposide) are used as treatments for a wide variety of cancers206

being actively pursued in which the “innocent” axial ligands are now being replaced with moieties that can either confer selectivity for tumor cells and/or enhance potency. One strategy involves the incorporation of axial ligands with tumor cell receptor targeting properties. 3.1. Tumour Cell Targeting by Platinum(IV) Prodrugs

3.1.1. Glucose Receptor Targeting. We described earlier, in section 2.1.1, why cancer cells over-rely on anaerobic glycolysis for the production of ATP. This reliance had led to an overexpression of GLUT membrane proteins on the surface of these cells. Overexpression of GLUT1, for example, has been shown to be a prognostic indicator for cancer.225 While we have already highlighted a number of platinum(II)carbohydrate conjugates which were rationally designed to

Figure 25. Chemical structures of early platinum(IV) prodrugs: ormaplatin (102), iproplatin (103), and satraplatin (104).

the ligands coordinated to the platinum(IV) core223 as well as the tumor cell type.224 There has been a significant shift in focus in this research space in relatively recent times. A more targeted approach is

Figure 26. Chemical structures of a series of glycosylated platinum(IV) complexes (105−117). 1078


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Figure 27. Chemical structures of glycosylated platinum(IV) complexes (118−122)

develop five additional glycosylated platinum(IV) prodrugs, again incorporating acetylated derivatives of glucose, mannose, and rhamnose, with a cisplatin scaffold and a varied axial linker chain (118−122), Figure 27.228 They wanted to establish whether or not the length of the linkers separating the sugar from the platinum(IV) center or steric effects had any impact on the overall cytotoxicity of the resulting complexes. The conjugation of these sugars to platinum was facilitated, this time, via an amide coupling reaction between peracetyl glucose, rhamnose, or mannose and either a propyl amino or ethyl amino linker at the reducing end and a carboxylic acid function on the platinum(IV) framework. Cytotoxicity results revealed that the mannose derivatives 121 and 122 exhibited high potency toward cancer cells, especially toward LNCaP while exhibiting low toxicities toward a normal cell line (3T3). Chain length did appear to impact cytotoxicity findings in that 122 with a shorter chain was more cytotoxic toward LNCaP compared with 121. The team also found a correlation between cytotoxicity and DNA targeting efficiency following reduction of the complexes in the presence of ascorbic acid.228 In vivo toxicity studies in Kunming mice further revealed that 122 was better tolerated than 121 and had a therapeutic index over 16-fold higher than oxaliplatin. A more recent study by the same group229 sought to investigate the impact of conjugating only one axial acetylated sugar ligand (glucose, rhamnose, and mannose with propyl amino or ethyl amino linkers) to either cisplatin and oxaliplatin cores and compared them to the bis complexes previously reported.228 The sixth coordination site was occupied, in all cases, by a hydroxido axial ligand. The team found that the monofunctionalized complexes were generally more cytotoxic than the bis analogues against Hela, MCF7, LnCAP, PC3, HepG2, and A549 cells, with those containing a cisplatin core generally more efficacious than their oxaliplatin analogues. This was further supported by in vivo studies where three of the monofunctionalized mannose derivatives (two of which contain an oxaliplatin base and the other a cisplatin base) were shown to inhibit tumor growth, while also demonstrating a 5− 11-fold increase in their therapeutic index over oxaliplatin and two previously reported bis complexes (121 and 122, Figure 27). Cellular drug update and DNA platination were also superior for the monofunctionalized complexes relative to

target these GLUT proteins; platinum(IV)-carbohydrate derivatives have also been reported. To the best of our knowledge, the first report of platinum(IV)-glycoconjugates dates back to 2000. Their anticancer properties, however, were not evaluated.226 Wang, Wang, et al. developed a series of 13 platinum(IV) glycoconjugates containing either a cisplatin or oxaliplatin core and incorporating different glycuronic acid derivatives of acetylated glucose (105 and 108 respectively, Figure 26), galactose (106 and 109 respectively, Figure 26), or mannose (107 and 110 respectively, Figure 26) as axial ligands or acetyl protected glucose (111, 115, 116, and 117, Figure 26), mannose, galactose (112, Figure 26), or rhamnose (114, Figure 26) sugars tethered to the platinum(IV) center via their carboxylate moieties.227 Of these complexes, those with a cisplatin core and galactose or mannose sugars showed the most potent cytotoxic effects; their cytotoxicity being equal to or greater than cisplatin and oxaliplatin in many cases against HeLa, HepG2, LCCap, MCF7, and A549 cancer cells. The complexes showed improved cellular uptake and DNA platination over cisplatin and oxaliplatin. However, the presence of a GLUT inhibitor phlorazin did not affect the cytotoxicity of these acetyl protected complexes, suggesting that their mode of action was not connected to the use of GLUTs.227 The presence of the acetylated hydroxyl groups may well have impacted GLUT-mediated uptake. Furthermore, we described in section 2.1.1 the findings of a comprehensive study by Lippard et al. in which they highlighted the importance of selecting the optimal position on the sugar backbone in which to bind the platinum so as not to negatively impact GLUT-mediated uptake into tumor cells. While these complexes reported by Wang, Wang, et al. are cytotoxic, the position and steric nature of the carboxylate moiety, in the case of these complexes, may well be impacting on GLUT-mediated uptake or lack thereof. Two complexes from the series (106 and 113), which demonstrated potent in vitro activities, were selected for further in vivo evaluation. These in vivo studies on a HepG2 tumor mouse model revealed that both complexes were able to inhibit tumor growth with relatively low toxicity.227 In a later study, the team, having identified the benefit of retaining a cisplatin core over an oxaliplatin one, proceeded to 1079


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Figure 28. Chemical structures of glycosylated platinum(IV) prodrugs bearing derivatives of glucose, galactose, and mannose appended to an oxaliplatin framework (123−130).

cisplatin, oxaliplatin, and the bis analogues. Furthermore, the monofunctionalized complexes demonstrated faster reduction, in the presence of ascorbic acid, compared to the bis complexes.229 This is in keeping with the findings of Gibson et al., who had previously demonstrated that the presence of a hydroxido axial ligand in the coordination sphere of platinum(IV) complexes can greatly facilitate electron transfer from ascorbate to the platinum(IV) center and thus increase this reduction rate.223 Later in 2017, the same team reported another series of monofunctionalized glycosylated platinum(IV) prodrugs. This time, derivatives of the nonacetylated forms of glucose, galactose, and mannose as axial ligands were appended to an oxaliplatin framework via linkers of varying length (123−130), Figure 28.230 Of the series, complexes 127−130 had superior cytotoxicities (in the low micromolar and even nanomolar range) against a range of tumor cell lines which were nearly 166-fold more efficacious than cisplatin, oxaliplatin, and satraplatin. There appeared to be a correlation between linker chain length and human serum albumin (HSA) binding with the hexadecanoic chain facilitating enhanced binding. There was also evidence to suggest cellular accumulation was mediated via the GLUT1 and OCT2 transporters, both of which are known to be overexpressed on tumor cell surfaces as stated previously. This was in contrast to the complexes (111− 117, Figure 26) they had previously reported. The fact that the sugar hydroxyl moieties in these complexes were all unprotected may well be a contributory factor in terms of

their capacity to interact with their GLUT1 and OCT2 targets. These complexes also appeared to preferentially kill tumor cells (293T and 3T3 cells) over noncancerous cells in vitro. Interestingly, while cytotoxicities of 127, 128, and 130 were comparable, the accumulation of 128 in tumor tissue was significantly higher. Complex 128 also demonstrated enhanced cancer-targeting abilities and decreased toxicity toward a mouse model of GLUT1-expressing breast cancer (MCF-7) in Balb/c mice over complexes 127 and 130.230 Developing the cisplatin analogues of these would be interesting given the earlier findings of the team in which they demonstrated that glycosylated platinum(IV) derivatives incorporating a cisplatin core were generally more cytotoxic than those containing an oxaliplatin one. Developing analogues of these also in which the sugar is tethered to the platinum(IV) core via a linker at the C-2 position of the sugar may also be worth investigating, given the findings by Lippard et al. in which this position was deemed the substitution position of choice for GLUT1mediated selective delivery of their platinum(IV) cytotoxic agents. All in all, the studies by Wang, Wang, et al. bring forth some interesting generalizations. Glycosylated platinum(IV) complexes incorporating a cisplatin core are generally more cytotoxic than those containing an oxaliplatin core.227,229 The monofunctionalized derivatives tend to be more cytotoxic relative to their bis derivatives, most likely due to an increase in their reduction rates.229 The GLUT-targeting ability of their glycosylated platinum(IV) derivatives appears to be lost when 1080


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cells, in this case those expressing ER+ like some breast and ovarian cancers. 3.1.4. Epidermal Growth Factor Receptor Targeting. The epidermal growth factor receptors (EGFR) are transmembrane proteins which have been shown to regulate various cancer-related effectors such as Src Kinase, PI3 kinsase, and Ras proteins. They are also known to upregulate DNA repair mechanisms.236 Small molecule inhibitors of EGFR have been shown to be effective combinatorial drugs when used with metal complexes to treat cancer.236 Kowol, Heffeter, et al. developed a small family of platinum(IV) prodrugs bearing an EGFR-targeting peptide (LARLLT) as an axial ligand tethered to a platinum base containing the nonleaving DACH ligand of oxaliplatin (132−134), Figure 30.237 Despite the fact that many targeting peptides have been used to improve the selectivity of highly toxic anticancer agents, some of which have been described earlier in this review, the presence of the peptides in these complexes did not show any evidence of EGFR expression-dependent uptake of conjugates in four different cell lines with different verified EGFR expression levels, nor did they endow the complexes with an improved efficacy profile over their precursors. A fluorophore-coupled peptide was subsequently developed to help track the platinum conjugate, but again, there was no correlation between platinum drug uptake and EGFR expression levels. Despite the sound rationale behind this work, the results indicated that the LARLLT peptide, despite its known propensity for targeting EGFR proteins, was not the most suitable candidate in this case for facilitating the specific update of the platinum(IV) prodrug into tumor cells that overexpress EGFR.237 The presence of the linker used to faciliate the binding of the peptide to the platinum center or indeed the platinum center may well be negatively impacting on the targeting ability of the complexes. Interestingly, however, as we will highlight later in this review, targeting tumor cells which overexpress EGFR is a viable strategy, with some ruthenium(II)-EGFR targeting complexes showing promise as potential anticancer agents. Many platinum drugs have been shown to induce cancer cell death via an apoptosis pathway. Some cancer cells have developed a means of avoiding this cellular response by disabling apoptotic pathways using various mechanisms including forced expression of antiapoptotic Bcl-2 family proteins or via suppression of p53, a transcription factor known to modulate the expression of a number of genes known to trigger apoptosis.238 Targeting necrosis over apoptosis as a means of inducing cancer cell death has emerged as an alternative strategy to circumvent this resistance issue. Ang et al. developed platinum(IV)-peptide conjugates with a cisplatin or oxaliplatin core bearing an anti-HER2/neu peptide (NH2-Tyr-Cys-Asp-Gly-Phe-Tyr-Ala-Cys-Tyr-Met-AspVal-Gly-Gly-Lys-Lys(aminooxy)-CONH2 or ANHP) in one axial position and an acetate ligand in the other, (135 and 136), Figure 31.239 They chose an anti-HER2/nue peptide on the premise that the human EGFR 2 (HER2) cell surface protein is overexpressed in many cancers, notably in 15−30% of human breast cancers and is associated with poor disease prognosis.240,241 This receptor protein has also been linked with tumor progression and survival.239 The complexes were cytotoxic, comparable to those of cisplatin and oxaliplatin. They were selective for HER2 overexpressed NCI-N87 gastric cancer cells and BT-474 breast ductal carcinoma, both cell lines of which are apoptosis-

the sugars tethered to the platinum(IV) are in their acetylated forms.230 3.1.2. CD44 Glycoprotein Targeting. The cell surface glycoprotein receptor, CD44, represents another drug target of interest. It is highly expressed in many different types of cancers231 and has been shown to play a regulatory role in metastasis. It can interact with numerous extracellular matrix ligands including hyaluronic acid or hyaluronan (HA), promoting the migration and invasion processes associated with metastases.232,233 Sun, Tu, and co-workers sought to exploit the CD44 tumor targeting nature of HA. They did this by first modifying HA with ethylenediamine (EDA) to facilitate its binding to a platinum(IV) core via a succinate linker generating a HA-EDA-platinum(IV) nanoconjugate.234 The nanoconjugate was found to recognize the HA receptor, penetrate the cancer cell membrane, and had comparable cytotoxicity to cells known to express CD44 (melanoma B16− F10, liver carcinoma Hep G2, and kidney HEK-293) with significantly reduced toxicity toward normal cells as anticipated. On the basis of an in vivo study on melanoma-bearing mice, the nanoconjugate was shown to selectively target the tumor with minimal organ toxicity, validated by tissue distribution studies and imaging analysis, while also prolonging the life of the animals.234 3.1.3. Hormone Receptor Targeting. As stated earlier, the involvement of sex hormones in cancer progression in hormone-sensitive tissues is well-established.88 Exploiting steroids as carriers or as agents able to disrupt the biological function of hormone membrane receptors represents an attractive strategy for the rational design and development of anticancer agents. We earlier described the work of Bérubé et al. in which they rationally designed and developed a series of platinum(II)hormone receptor targeting conjugates but, to the best of our knowledge, did not develop any platinum(IV) derivatives. Lippard et al. were the first to develop a series of estrogentethered platinum(IV) complexes, following the rational that ER+ cells exposed to the hormone are sensitized to cisplatin.235 The estrogen ligands had first to be modified to facilitate their binding to the platinum(IV) ion, for example, (131), Figure 29. The complexes were found to induce an overexpression of HMGB1 in ER+ MCF-7 cells. While all complexes were cytotoxic, only one favored ER+ MCF-7 cells over ER− HCC1397 cells, being nearly 2-fold more cytotoxic in ER+ MCF-7 cells. This does indicate the potential inherent in these types of hormone-targeting prodrugs in selectively targeting tumor

Figure 29. Chemical structure of an estrogen-tethered platinum(IV) complex (131). 1081


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Figure 30. Chemical structures of platinum(IV) prodrugs bearing an EGFR-targeting peptide (LARLLT) (132−134).

into HER2 cancer cells was mediated by the presence of the HER2 targeting peptide ligand in their complexes.239 3.1.5. Biotin Receptor Targeting. As stated in section 2.1.4, biotin, also known as vitamin H, has been shown to be rapidly taken up into tumor cells, which often overexpress receptors specific to biotin on their cell surface. As such, biotin has been identified and used as a selective drug transporter, with studies reporting the positive effects of conjugating biotin with classical chemotherapeutics such as doxorubicin and gemcitabine.110 Wang, Gou, et al. sought to investigate whether biotin could likewise be used to selectively deliver a platinum(IV) prodrug to tumor cells. They complexed biotin to a cisplatin scaffold, generating two complexes with either one (137, Pt-Bio-I) or two (138, Pt-Bio-II) biotin moieties as axial ligands, Figure 32.242 Tethering these biotin moieties to the platinum(IV) core was found to significantly enhance uptake into breast cancer cells while lowering the accumulation of these complexes in breast epithelial cells. The monobiotinylated complex demonstrated superior cytotoxic properties over its bis-biotinylated analogue against the breast cancer cell lines (MCF-7 and MDA-MB-231). This may potentially be attributed to the presence of the hydroxido ligand in Pt-Bio-I

Figure 31. Chemical structures of platinum(IV)-HER2 receptor targeting peptide conjugates (135 and 136).

resistant. They were also found to have enhanced selectivity for cancerous cells over normal cells. They exhibited a biphasic mode of cytotoxicity, the induced necrosis in the first phase followed by a gradual phase of extended cell death. The team concluded that the accumulation of their platinum complexes

Figure 32. Chemical structures of platinum(IV) prodrugs targeting biotin receptors (137−141). 1082


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Figure 33. Chemical structure of ethacraplatin (142) and its monofunctionalized analogue (143).

recognized group of detoxification enzymes that play a key role in phase II biotransformations in the human body, some of which involve carcinogens, environmental pollutants, numerous secondary metabolites of oxidative stress, and in our context, cancer chemotherapeutic agents.244 They are classified as being the single-most important enzymes responsible for the metabolic breakdown of alkylating compounds.245,246 They work by catalyzing the nucleophilic S-conjugation between the GSH thiol group and electrophilic compounds; the metabolites which are generated can enter the mercapturic acid pathway which leads to their eventual elimination from the body.246 Of note is the fact that some cancer cell lines and tumors, including cisplatin-resistant cell lines, overexpress certain GST isoenzymes.246 The propensity of platinum complexes to react with soft nucleophilic groups, including thiol-containing groups, makes them classic targets for GST binding and thus detoxification.247 Armed with this insight, Dyson et al. chose to incorporate ethacrynic acid (EA), a small organic molecule with known GST inhibitory properties, into the cisplatin-like platinum(IV) scaffold. They generated ethacraplatin, (142) Figure, 33.248 They postulated that, upon tumor cell entry, it would be reduced to cisplatin which would then be free to bind DNA with concomitant release of the two EA ligands free to inhibit the GST enzymes (thus potentially evading detoxification). Cellular uptake should also be enhanced on the basis that the complex would be expected to be more lipophilic relative to cisplatin.248 Ethacraplatin was found to more cytotoxic against cisplatin-resistant breast MCF7 and T47D, lung A549, and colon HT29 carcinomas compared to cisplatin.248 In fact, the team went on to provide evidence that ethacraplatin was able to reverse cisplatin resistance in MCF-7 cancer cells. Using different biochemical and structural studies, and supported by molecular modeling analysis, the group was able to gain a deeper insight into the nature of the interaction between GST and ethacraplatin.248 They particularly focused on GST P1-1 as their protein target because of its importance in the mercapturic acid detoxification pathway. Interestingly and unexpectedly, while ethacraplatin was shown to bind to GST P1-1, the cisplatin scaffold remained bound to the enzyme while the EA ligands interacted with both active sites. These results account for the strong and irreversible GST inhibition observed for this complex.248 The cytotoxicity of ethacraplatin was further assessed, this time targeting a rare and aggressive form of an asbestos-related cancer, malignant pleural mesothelioma (MPM); this mesothelioma exhibits strong chemoresistance which has been linked to high levels of GST overexpression. In this study by Osella et al., neither ethacraplatin alone, nor a combination of cisplatin with EA, offered any advantage over cisplatin alone against MPM cells. Surprisingly GST activity was unaltered while levels of glutathione increased following treatment.249 In a more recent study, Ang, Montagner, Nowak-Sliwinska, Dyson, et al. developed a monofunctionalized ethacraplatin

impacting on the overall reduction rate of the complex or on its accumulation into the tumor cells. Gibson et al. previously demonstrated that the presence of a hydroxido axial ligand in the coordination sphere of platinum(IV) complexes can greatly facilitate electron transfer from ascorbate to the platinum(IV) center and thus increase this reduction rate.223 Pt-Bio-I had comparable cytotoxicity to cisplatin against MCF-7 cells at 72 h (10 vs 9 μM for cisplatin) but significantly improved cytotoxicity against the cisplatin resistant MDA-MB-231 cells at both 48 and 72 h (18 and 12 vs 45 μM and 40 μM for cisplatin). Cell cycle arrest studies on both complexes in MCF7 cells suggested that the active form and DNA damage mode of the complexes were similar to cisplatin, and this would compare favorably with the similar cytotoxicity profiles of both complexes and cisplatin against this cell line.242 Gou et al. developed this work further by complexing biotin as a single axial ligand to three platinum(IV) complexes, using cisplatin, DN603, and DN604 scaffolds (139−141) and a chlorido ligand in the other axial position, Figure 32.243 Chlorido ligands, like hydroxido ligands, are thought to facilitate reduction of the platinum(IV) to platinum(II) by forming bridges between the reducing agent and platinum center, thus facilitating electron transfer.223 They tested the cytotoxicity of the complexes against HepG2a, MCF7b, SGC7901c (cisplatin sensitive), and SGC-7901/Cisd (cisplatin resistant) cell lines. Complex 139, with the cisplatin base, was found to be more cytotoxic than cisplatin alone across all cell lines, between 2 and 3 times more so for the nonresistant cell lines, and an almost 10-fold increase in potency toward the cisplatin resistant cell line. The cellular accumulation studies of 139 revealed it did not have significant cellular uptake against the nonresistant cell lines but uptake increased by 4.5 times in the resistant cell line, offering insight into its ability to overcome cisplatin resistance through significantly higher cellular accumulation. Reduction studies with ascorbic acid showed that the prodrug was reduced to its platinum(II) form with the loss of its biotin axial ligand, which was then shown to be able to bind to streptavidin, a bacteria protein with an extremely high affinity for biotin. These promising results highlight how biotin may be exploited as a tumor cell targeting ligand for platinum(IV) prodrugs, with the added apparent benefit of being able to overcome cisplatin resistance. The team noted that further studies would need to be undertaken to understand how the nonreduced complexes interacted with the cell-surface biotin receptors.243 Please also refer to section 3.3 in which a “triple action” platinum(IV) complex derived from cisplatin and bearing a biotin targeting moiety as well as a cyclooxygenase (COX) inhibitor drug is described. 3.2. Platinum(IV) Prodrugs Targeting DNA and at least One Other Cellular Entity

3.2.1. Enzymes as Targets. 3.2.1.1. Glutathione-STransferases. Glutathione-S-transferases (GST) are a well1083


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Figure 34. Chemical structure of platinum(IV) complexes bearing HDAC inhibitor valproate (144−148) or phenylbutyrate (149−156) ligands or “innocent” carboxylate ligands (157−158).

analogue incorporating a hydroxido ligand in place of one of the axial EA ligands, (143), Figure, 33.250 The earlier study

had shown that the cytotoxicity of ethacraplatin could not be attributed to DNA binding as originally expected given that the 1084


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platinum complex remained tightly bound to the enzyme.248 We mentioned earlier the advantage of having a hydroxido ligand within the coordination sphere of platinum(IV) complexes to promote reduction of the platinum(IV) ion to platinum(II).223 The monofunctionalized ethacraplatin analogue (143), Figure, 33, generated by Ang, Montagner, Nowak-Sliwinska, Dyson, et al. was shown, as anticipated, to undergo in vitro reduction with release of the EA ligand as well as the cytotoxic platinum(II) DNA binding agent. Reduction of the monofuctionalised complex, in the presence of ascorbic acid, was almost 35 times faster than that of the bis analogue. The monofunctionalized complex retained its cytotoxicity against both cisplatin-sensitive and cisplatin-resistant tumor cells and was also shown to inhibit GST in a noncompetitive way. This complex also exhibited potent in vivo antitumor activity, with ∼80% inhibition of tumor growth in a human ovarian carcinoma tumor model.250 The early work of Gibson et al. in which they highlighted the impact of the axial ligands on the reduction potential of the corresponding platinum(IV) complexes223 has been nicely validated by these recent studies on glycosylated platinum(IV) derivatives developed by Wang et al.,229 the biotinylated platinum(IV) complexes developed by Guo et al.,242,243 and now this study by Ang, Montagner, Nowak-Sliwinska, Dyson, et al. on the monofunctionalized ethacraplatin derivative.250 3.2.1.2. Histone Deacetylases. We described, in section 2.2.1.1, the rationale behind the development of platinum drug conjugates incorporating ligands with HDAC inhibition properties. We also highlighted a number of FDA-approved hydroxamate-based HDAC inhibitors which have already been added to the chemotherapeutic arsenal of anticancer agents in clinical use, including suberoylanilide hydroxamic acid (SAHA, Vorinostat, Merck),251 belinostat (Onxeo),252,253 and panobinostat (Novartis).254 While our group generated platinum(II)-HDAC inhibitor conjugates incorporating derivatives of SAHA,148,149 and belinostat,154 there are no examples to date of platinum(IV) analogues incorporating these clinically used HDAC inhibitors. Some interesting results have however emerged related to platinum(IV)-valproate derivatives. Valproic acid (VPA) is also known to possess HDAC inhibition activity albeit to a much lesser extent than the hydroxamatebased inhibitors. Three teams independently reported the rational design and development of platinum(IV)-VPA complexes. Tang, Shen, et al. developed cis,cis,trans-diamminedichlorobisvalproatoplatinum(IV) or VAAP, (144), Figure 34, containing two valproate axial ligands.255 They then packaged this conjugate into PEG−PCL nanoparticles or dispersed it into a Tween 80 surfactant to promote tumor cell uptake. Both the nonPEGylated VAAP and PEG-PCL/VAAP were significantly more cytotoxic compared to cisplatin across multiple cancer cell lines. The VAAP complex 144, for example, was nearly 12 times more cytotoxic, as compared to cisplatin, in lung A549 cells. The team, however, did not assess its HDAC inhibition potential. In a parallel study, Osella et al. also developed VAAP (144), Figure 34, and they too checked its cytotoxicity against numerous tumor cell lines.256 It was found to be cytotoxic against four highly malignant and highly chemoresistant plural mesothelioma (MPM) cell lines, more so than cisplatin. Interestingly, this team noted that the isomer of VAAP (144), namely cis,cis,trans-diamminedichloridobis(n-octanoato)platinum(IV) (157), Figure 34, in which the HDAC inhibitor ligands had been replaced with “innocent” octanoate ligands,

exhibited similar and even some times higher cytotoxicity than the platinum(IV)-HDAC inhibitor conjugate (144). The team questioned whether the stoichiometric ratio of VAAP:platinum of 2:1 in the platinum(IV) complex was sufficient for a satisfactory synergistic effect. They ultimately attributed the improved cytotoxicity of the complexes, not to any synergy associated with the presence of the valproates serving as HDAC inhibitors but rather an increase in lipophilicity resulting from the presence of the axial monocarboxylate ligands. Gibson, Brabec, et al. took a slightly different approach in that they added either one or two VPA axial ligands to a platinum(IV) oxaliplatin framework (146 and 147), Figure 34, in contrast to VAAP which contained two VPA ligands and a cisplatin core (144), Figure 34.257 Oxaliplatin, when given in combination with the HDAC inhibitor trichostatin A, results in an additive cytotoxic effect when tested against gastric tumor cells. Their conjugates exhibited improved cytotoxicity over their platinum(IV) analogues without biologically active axial ligands against both cisplatin-sensitive and cisplatin-resistant cell lines. Cells treated with their oxaliplatin-type platinum(IV)-VPA complexes appeared to cause a marked-down regulation of HDACs which resulted in a decrease in the level of HDACs in cells, rather than any direct inhibition of the enzyme. The team concluded that this downregulation of HDACs was a major contributory factor accounting for the improved cytotoxicity observed for these complexes. This was in contrast with Osella’s study in which they concluded that lipophilicity, and not HDAC inhibition, was the primary factor for the cytotoxicity of VAAP (144). To address these competing findings, Gibson et al. undertook a further study to compare platinum(IV)-VPA complexes bearing a cisplatin equatorial core (144 and 145), Figure 34, against platinum(IV) analogues with biologically inert axial acetate ligands (158), Figure 34.258 Again, the VPA complexes proved to be more cytotoxic relative to the complexes bearing the “innocent” carboxylate-containing axial ligands. Again, their complexes were found to inhibit the expression of the HDAC protein, rather than inhibit the HDAC enzyme itself. The study also went on to demonstrate that the presence of the VPA ligands impacted on other cellular processes, including interfering with enzymes such as GST. The team concluded that the improved cytotoxicity observed for the VPA complexes was most likely due not only to the enhanced lipophilicity bestowed on the complexes by the presence of the VPA ligands but also to a reduction in HDAC expression and not HDAC inhibition, and that other biological interactions may also be playing a role.258 In a follow up study, Gibson et al. developed a library of platinum(IV) derivatives of cisplatin (144, 145, 149, and 150), Figure 34, oxaliplatin (146, 147, and 151), Figure 34, and trans-[Pt(n-butylamine)(piperidino-piperidine)Cl2]+ (148), Figure 34, incorporating either VPA or another HDAC inhibitor 4-phenylbutyrate (PhB), Figure 34, in one or both axial positions and compared their activities to platinum(IV) complexes bearing “innocent” carboxylate ligands.259 They sought to identify not only (i) the optimal platinum core framework but also (ii) the optimal axial HDAC inhibitor ligands for maximum cytotoxic activity. In all cases, the platinum(IV)-HDAC inhibitor conjugates that were derivatives of cisplatin were more cytotoxic that those that were derivatives of oxaliplatin. This is also consistent with the findings of Wang, Wang, et al. as described earlier; their 1085


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Figure 35. Chemical structures of platinum(IV) analogues (160−165) of Pt56MeSS (159) where TFA is trifluoroacetate.

platinum(IV)-glycoconjugates bearing a cisplatin core were generally more cytotoxic compared to those containing an oxaliplatin core.227 Of the complexes tested, cis,trans,cis[Pt(NH3)2(PhB)2Cl2] (150) was not only highly cytotoxic against several human cancer cell lines but was up to 100-fold more cytotoxic than cisplatin and even more so when compared to platinum(IV) derivatives of cisplatin with either two hydroxido, two acetato, or two VPA ligands. Again, a link between lipophilicity and cytotoxicity was established for the platinum(IV)-HDAC inhibitor derivatives. The platinum(IV)HDAC inhibitor complexes were more effective at binding to intracellular DNA compared to cisplatin and oxaliplatin. Interestingly, these complexes inhibited HDACs, more so than PhB and VPA alone. The team concluded that the enhanced cytotoxicity observed for these platinum(IV)-HDAC inhibitor conjugates was most likely due to a number of factors, including overall lipophilicity with a direct correlation between cellular uptake and cytotoxicities, a synergistic effect between the platinum and HDAC inhibitor moieties mediating various actions within the cell, including HDAC inhibition/down regulation. They go on to conclude that a “dual-functional” view of these conjugates is overly simplistic and that the complexes have multiple functions within the cell which combine to give an overall increase in their anticancer effects.259 Gandin, Gibson, et al. also investigated the effects of adding VPA axial ligands to the Pt(IV) base of Pt56MeSS (159), Figure 35. Pt56MeSS is a doubly charged platinum(II) oxaliplatin analogue in which the dicarboxylate leaving group has been replaced with a 5,6-dimethyl-1,10-phenanthroline as a N,N-bidentate donor ligand. This complex, which is incapable of covalently binding DNA, had shown promising in vitro cytotoxicity, particularly toward cisplatin-resistant and oxaliplatin-resistant cell lines.260 While its mechanism of action has yet to be fully elucidated, it is thought to act on the mitochondria.261 Nephrotoxicity has been cited as a drawback of this complex. Gandin, Gibson, et al. developed a series of platinum(IV) derivatives of Pt56MeSS bearing nonbioactive, lipophilic (160−162), and bioactive (VPA and PhB) axial ligands (163−165), Figure 35. The platinum(IV) complexes had on average improved cytotoxicity over cisplatin but were generally comparable or less effective when compared against Pt56MeSS itself. The presence or absence of the axial HDAC inhibitor ligands appeared to have little or no significant effect on the cytotoxicity of the resulting complexes. This contrasts with previous reports where the presence of the PhB axial ligands on the platinum(IV) form of cisplatin had a marked increase on cytotoxicities (up to 100 times). The synergistic properties previously observed for platinum(IV) complexes bearing HDAC inhibitor ligands were not present here, most likely related to the fact that these complexes cannot covalently bind to DNA.260

A collaborative study between Gibson, Brabec, Osella, et al. sought to investigate why the cytotoxicity of platinum(IV) complexes was greatly increased by the presence of octanoato (OA) axial ligands, more so than branched isomers like VPA.262 While both VPA and OA are known to possess HDAC inhibition ability, the HDAC inhibition properties of OA are significantly less than those of VPA. Earlier reports, as described above, theorized that the HDAC inhibitory activity of VPA was significant, having a synergistic effect with the platinum DNA-binding activity. As discussed earlier in this section, they had previously reported a correlation between complex lipophilicity arising from the presence of the axial ligands, cellular accumulation, and cytotoxicity. However, further studies revealed that the complexes bearing the OA ligands, with weaker HDAC inhibition properties, were significantly more cytotoxic across many cancer cell lines, an observation that could not be explained simply by OA being more effective at increasing cellular accumulation. This study sought to investigate this anomaly. It was again confirmed that the complex bearing two OA axial ligands, (157), Figure 34, was the most potent across a number of cell lines (A2780, A2780cisR, CHO-K1, MCF-7, LNCaP, PNT1a, HSF, MRC-5 (PD30), and HCT-116) with remarkably low IC50 values ranging from 0.014 to 0.14 μM, compared to cisplatin (with IC50 values ranging from 2.5 to 68.98 μM). The complex, as before, exhibited low HDAC inhibition activity, comparable to that of cisplatin. The team studied the amount of DNA-bound platinum, the effects on the mitochondria, and whether methylation of DNA was taking place to try to address why this ligand was having such a dramatic effect. Cellular accumulation was increased by the presence of the ligands in the complex as expected. The team found that the OA ligands were indeed released upon reduction of the platinum(IV) complex to its platinum(II) adduct, and while this platinum(II) adduct bound to DNA, the free OA ligands were shown to hypermethylate DNA, as well as to induce a reduction in mitochondrial membrane potential. The significance of DNA methylation has been reported in many studies, with both hypo- (less) and hyper- (more) methylation of DNA being linked to either promotion or suppression of tumor progression, with the general observation that DNA methylation regulation in cancer cells is drastically different to that in healthy cells.263 This methodical approach and collaboration between different groups has yielded intriguing results and highlights the benefits which result when teams come together to follow through on initial research findings. It also serves as an important reminder to remain open-minded when evaluating the mode of action of these types of “multitargeted” complexes. Kasparkova et al. explored a different approach in that they rationally designed and developed a “photoactivatable” platinum(IV)-diazido-suberoyl-bis-hydroxamate conjugate 1086


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results in translocation of pro-apoptotic mediators, ultimately leading to apoptosis.161 Tumour cells, with an over reliance on anaerobic glycolysis, can therefore be targeted while leaving normal cells unharmed. With this in mind, Dhar and Lippard developed mitaplatin (167), Figure 37, which contains a

(166), Figure 36. Suberoyl-bis-hydroxamic acid (SubH), a precursor to SAHA, is a potent HDAC inhibitor. It is also

Figure 36. Chemical structure of a photoactive platinum(IV)-HDAC inhibitor complex (166). Figure 37. Chemical structure of mitaplatin (167).

highly cytotoxic at micromolar concentrations.264,265 In addition, SubH has also been found to work synergistically with oxaliplatin in colorectal cancer cell lines.266 The platinum(IV)-diazido-suberoyl-bis-hydroxamate complex was found to be inactive in the dark, even in the presence of common biological reducing agents such as glutathione and ascorbic acid. Upon uptake into tumor cells, it can be activated using UVA irradiation (365 nm), which results in the simultaneous release of the cytotoxic platinum(II) DNAbinding adduct and the HDAC inhibitor ligands. The complex, upon photoactivation, was found to be highly cytotoxic against human ovarian carcinoma A2780 cells and cisplatin-resistant phenotype, much more so when compared to cisplatin or its photoactivatable platinum(IV) derivatives containing biologically inactive axial ligands. While 166 was shown to inhibit HDACs, it could also bind DNA with a marked increase in interstrand cross-links. Unlike conventional platinum(II)-DNA binding agents which typically contain primary or secondary amine donor ligands in which the hydrogens are thought to be important for hydrogen-bonding interactions with DNA,267 the nitrogen donor atoms of the bpy ligand in 166 clearly do not contain any hydrogens. Intriguingly, the DNA cross-links induced by this complex were found to block transcription and replication more effectively than DNA lesions induced by classical platinum(II) drugs.268 Erxleben, Montagner, et al. more recently joined this field of study. They too developed platinum(IV)-HDAC inhibitor conjugates, but in their case, they chose an oxaliplatin base but bearing the carboplatin dicarboxylate leaving ligand, 1,1cyclobutanedicarboxylate, with varying axial ligands. Complexes contained either two PhB axial ligands (152), or one PhB and either a hydroxide (153), an acetate (154), a succinate (155), or a benzoate (156), Figure 34, as the other axial ligand. The cytotoxicity of the complexes was tested against A375, BxPC3, LoVo, and A431 cell lines. As expected of carboplatin, their IC50 values were higher than those of cisplatin. The most potent complex, with a single PhB and benzoate ligand (156), Figure 35, had lower IC50 values than carboplatin against all cell lines and also showed the highest cellular accumulation and HDAC inhibition ability.269 3.2.1.3. Pyruvate Dehydrogenase Kinases. As previously mentioned, dichloroacetate (DCA), a structural analogue of pyruvate, is an orally available small molecule inhibitor of pyruvate dehydrogenase kinase (PDK).160 Inhibition of PDK leads to an influx of pyruvate into the mitochondria which shifts cellular metabolism from glycolysis to glucose, leading to a change in the mitochondrial membrane potential. This

cisplatin equatorial core and two DCA ligands in the axial positions. When tested against eight cancerous cell lines, including cervical HeLa, breast MCF-7, and ovarian A2780, it exhibited equal or improved cytotoxicity relative to many of the platinum(IV) complexes that had been reported at that time. It had comparable cytotoxicity to cisplatin and was more cytotoxic than DCA alone.270 Mitaplatin (167) had low toxicity against human fibroblast cells, validating their design hypothesis that cells dependent on glycolysis would be targeted. Mitaplatin induced DNA damage analogous to the signature platinum-DNA intrastrand cross-links associated with cisplatin, providing evidence that mitaplatin was indeed being reduced intracellularly to cisplatin. Follow up studies by Xue et al. supported these earlier findings: mitaplatin (167), Figure 37, exhibited strong cytotoxic selectivity toward cisplatin resistant cell lines over normal cells.271 Although platinum(IV) complexes are supposed to be “inert” on the basis that the metal center is coordinatively saturated, this was not found to be the case for mitaplatin. Hydrolysis studies revealed that mitaplatin is in fact not very stable under biological conditions including cell culture media, and yet, mitaplatin had been shown to be highly cytotoxic.272−274 They found that 50% of mitaplatin had undergone hydrolysis after only 2 h.272 This led the team to conclude that the rate of mitaplatin uptake into tumor cells must be faster than its rate of hydrolysis and that the cytotoxicity attributed to mitaplatin may well be due to a combination of mitaplatin and its hydrolysis products. Lippard et al. went on to show in an in vivo mouse xenograft model of triple-negative breast cancer that the efficacy of mitaplatin could be further improved by nanoparticle encapsulation.275 3.2.1.4. Cyclooxygenases. Cyclooxygenases (COX) are an important family of enzymes used by the body to catalyze the conversion of arachidonic acid into prostaglandins, mediators of inflammatory, and anaphylactic reactions. Three isoforms of COX are known: COX-1, -2, and -3.276 Of these, COX-2 has been found to be overexpressed in many tumor cells, including those of the skin, breast, esophagus, stomach, colorectal, pancreas, and bladder.277 This same isoform has been shown to play an influential role in tumor apoptosis and angiogenesis.278 Nonsteroidal anti-inflammatory drugs (NSAIDs) such as aspirin target COX enzymes. COX inhibition by NSAIDS reduces the formation of pro-inflammatory prostaglandins.279 A number of COX inhibitors have also been shown to act synergistically with established cancer drugs, including cisplatin, paclitaxel, and doxorubicin when used in combina1087


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Figure 38. Chemical structures of platinum(IV)-NSAID conjugates (168−172).

tion therapy.278 Neumann et al. set out to exploit this prodrug “activation by reduction” approach by generating platinum(IV) conjugates in which they incorporated the NSAIDs indomethacin and ibuprofen as axial ligands into either a cisplatin (168 and 170, respectively) or oxaliplatin core scaffold (169 and 171, respectively), Figure 38.280,281 Of the series, the indomethacin (168) and ibuprofen (170) complexes incorporating a cisplatin core were found to be significantly more cytotoxic compared to cisplatin and were also highly cytotoxic against cisplatin-resistant MDA-MB-231 breast cancer cells. Interestingly, despite being screened against two tumor cells with different levels of COX-2 expression, cisplatin sensitive colorectal HCT 116 carcinoma cells which do not express COX-2 and cisplatin resistant breast MDA-MB231 adenocarcinoma cells which exhibit high constitutive COX-2 expression, the complexes exhibited comparable cytotoxicities against both cell lines. When assessed for their COX inhibitory activity, the indomethacin conjugates exhibited potent COX inhibitory activity in contrast to the ibuprofen analogues which showed poor COX inhibition. The team concluded that while the complexes were all highly cytotoxic, their cytotoxicities could not be attributed to their COX inhibitory activity, to their potency as NSAIDs or to levels of cellular COX expression. They proposed that the presence of the NSAIDS enhanced the overall lipophilicity of the complexes leading to improved cellular uptake and that it was this change in their physicochemical properties that

accounted for the cytotoxicities of the complexes.280 It would be interesting to generate the asymmetrical analogues of these complexes in which one of the axial ligands is replaced with, for example, a hydroxido ligand. In so doing, the reduction potential of the resulting complexes would be altered, which may well impact the biological activities and possibly COX inhibition activity of the resulting complexes. A follow-up study by the same team supported their hypothesis. Employing the same NSAID ligands, they developed both platinum(II)- and platinum(IV)-NSAID conjugates with an oxaliplatin base (169 and 171), Figure 38. The platinum(IV)-NSAID conjugates, in all cases, were more cytotoxic compared to their platinum(II)-NSAID analogues. The platinum(IV)-NSAIDs were also able to overcome cisplatin resistance. Again, they proposed that COX-mediated pathways were not involved in the mechanism of action of these complexes.281 Aspirin, probably the most widely known NSAID, has been shown to prevent or induce remission of cancers.282 For example, it has been identified as a protective factor against colorectal cancer and several studies have been reported illustrating a decrease in the incidence of adenomas and colorectal cancers following long-term treatments with lowdoses of aspirin.283 An asymmetric platinum(IV) prodrug incorporating one aspirin axial ligand (172), Figure 38, was developed by Dhar et al.284 This prodrug, named Platin-A, was shown to be activated by reduction with release of the cisplatin 1088


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Figure 39. Chemical structures of platinum(II)-wogogin complexes (173−177) and platinum(II) precursors (178−179).

inflammatory activity has been linked to its ability to downregulate COX enzymes as well as other inflammationassociated genes. Fang, Gou, et al. complexed a wogonin unit as a single axial ligand via a spacer to a platinum(IV) core resembling either cisplatin (173), oxaliplatin but with chlorido ligands instead of its dicarboxylate as leaving ligands (174), or oxaliplatin (175), Figure 39, to generate the first examples of platinum(IV)-wogonin conjugates.288 The team found that all three complexes had strong antiproliferative properties when compared to cisplatin and oxaliplatin against SGC7901, MCF7, HepG-2, HCT-116, and PC-3 cell lines. All three were also significantly more cytotoxic than wogonin across all cell lines. Of the series, 173 was the most potent, being comparable to cisplatin in most cases. Interestingly, 173 also demonstrated the strongest COX inhibition, DNA platination, and the fastest reduction rate relative to its platinum(II) adduct when in the presence of ascorbic acid. Compound 173 was also shown to generate ROS and to decrease the mitochondrial membrane potential.288 Incorporating wogonin into a platinum unit significantly enhanced its anticancer activity, validating not only the value in combining two drug entities into one drug molecule but also the important role that metals can play in enhancing the therapeutic efficacy of small organic drug molecules. The same team, in a follow-up study, attempted to improve the hydrophilicity of the aforementioned platinum(IV)wogonin complexes (173−175), Figure 39, by replacing the chlorido equatorial leaving ligands with a carboplatin-like cyclobutanedicarboxylate ligand which incorporated a carbonyl group in the cyclobutane ring.289 Two new platinum(IV)wogonin complexes were generated, (176 and 177), Figure 39. While the cytotoxicity profile of these varied when tested against MCF-7, HepG2, HCT-116, PC-33, SGC-7901, SGC7901/cDDP (cisplatin resistant), and noncancerous HUVEC cells, the complexes did demonstrate improved cytotoxicity and selectivity when compared to the parent wogonin molecule and the platinum(II) precursors 178 and 179. Given that 176 was more potent than 177, further studies focused on this derivative. Compound 176 showed stronger COX 1 and COX 2 inhibition properties compared to wogonin. It was also able to overcome the cisplatin resistance shown in SGC-7901/cDDP cells, which the team attributed to

adduct and liberation of the aspirin as expected. While aspirin alone did not exhibit cytotoxic effects against androgenunresponsive prostate PC3 and DU145 cells, Platin-A was found to be highly cytotoxic with a cytotoxicity profile comparable to that of cisplatin. It was later shown to possess anti-inflammatory properties via inhibition of COX-2.285 In the same year that Dhar et al. reported their Platin-A prodrug, Liu et al. reported this same complex, referring to it as Asplatin (172), Figure 38.286 They screened Asplatin against several cell lines (cervical HeLa, breast MCF-7, liver HepG2, lung A549, A549R, and normal human fibroblasts) and found Asplatin to be highly cytotoxic against all cell lines and more cytotoxic when compared to cisplatin. Asplatin was also shown to circumvent resistance, being highly cytotoxic against cisplatin-resistant tumor cells. When tested in vivo against mice-bearing lung A549 tumors, Asplatin (172) retained its higher cytotoxicity profile over cisplatin while also showing reduced toxic side effects. It was also shown to be fully reduced in the presence of equimolar ascorbic acid, releasing its cisplatin core and aspirin as a free ligand. While Asplatin was shown to bind DNA, the team did not assess its COX inhibition activity.286 They did, however, in a follow up study, conduct apoptosis analysis and gene expression studies, to more fully elucidate the mechanism of action of Asplatin. They found evidence to suggest that aspirin, upon its release from Asplatin, modulates the cellular response to the platinum cytotoxic agent. The complex promoted apoptosis via the Bcl-2 associated mitochondrial pathway. The team were able to show that Bcl-2 was downregulated, in contrast to BAX and BAK, which were upregulated, resulting in an increase in the permeability of the mitochondrial outer membrane. This permeability change allows cytochrome C to be released into the cytosol, ultimately promoting apoptosis via activation of caspase processing.287 This is one of few studies reported for platinum(IV) prodrugs in which gene expression analysis was exploited to more fully understand the mechanisms underpinning the anticancer properties of these types of complexes. Wogonin is a chinese herbal remedy, a natural O-methylated flavone currently in phase I clinical trials as both a single agent and in combination as a potential treatment of cancer in China.288 It has also demonstrated anti-inflammatory and antioxidant properties both in vitro and in vivo. Its anti1089


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antitumor agent in China. 291 It works by inhibiting phosphatase 2A (PP2A), a protein involved in cell regulation. Endothall (181), Figure 40, is a derivative of cantharadin which has been found to be less toxic compared to cantharadin itself, while retaining the ability to inhibit PP2A. Keppler et al. hypothesized that their platinum(IV)-endothall derivatives, upon activation inside tumor cells, would concomitantly release the DNA binding cisplatin moiety and two endothall axial ligands free to inhibit PP2A.290 Despite the sound rationale behind their development, cytotoxicity studies revealed that the new platinum(IV)-endothall conjugates were not as cytotoxic compared to cisplatin or cisplatin in combination with endothall, against a range of tumor cell lines with IC50 values in the 10−5 molar range. Inhibition of PP2A induced by the platinum(IV)-endothall derivatives or the released endothall were not however assessed. The rate of reduction could also be impacting on the level of cytotoxicity of these complexes. 3.2.2. Peptide Receptors as Targets. There is much evidence in the literature to suggest that peptide receptor targeting is an attractive strategy in drug design given that many tumor cells overexpress these receptors.103,292,293 Conjugation of peptides to platinum(IV)-DNA binding moieties can serve two main functions. They can selectively deliver their platinum(IV) payload to tumor cells. For example, platinum(IV) analogues of oxaliplatin incorporating the TAT (YGRKKRRQRRR) peptide have been reported.42,294 However, more relevant to this section are peptides that specifically target cell surface proteins which have been implicated in tumor growth and survival. 3.2.2.1. Integrins and Aminopeptidase N as Targets. Malignant tumor cells can stimulate the production of angiogenesis signaling molecules, leading to the formation of new blood vessels which can “feed” growing tumors with oxygen and nutrients, ultimately facilitating their growth and ability to metastasize. Several tumor cell surface proteins are

its ability to decrease intracellular GSH levels. Compound 176 also demonstrated strong cancer cell specificity with IC50 values ranging from 43.75 μM against HUVEC, to 3.29 μM against SGC-7901/cDDP, and 23.57 μM against HCT-116. The team also found 176 to be an inhibitor of NF-kB, a major chemoresistance-related antiapoptotic factor, which they also attributed to its remarkable performance against the cisplatinresistant cell line. The ability to overcome cisplatin resistance, high selectivity in vitro, and the dramatic improvement in cytotoxicity and enzyme inhibition ability over the wogonin and platinum parent compounds demonstrates nicely why the dual-functional drug design approach is so attractive in anticancer metal drug design. This study also highlights that for these complexes, the platinum(IV) complexes incorporating a cisplatin core outperform those containing an oxaliplatin core in terms of their overall cytotoxic effects. This is to be expected given the greater lability associated with the chlorido ligands in the cisplatin derivatives. 3.2.1.5. Phosphatase 2A. The development of two platinum(IV)-endothall prodrugs (182 and 183), Figure 40,

Figure 40. Chemical structures of cantharadin (180), endothall (181), and the platinum(IV)-endothall complexes (182 and 183) where en is ethylenediamine.

by Keppler et al. represents another example of how a derivative of a traditional Chinese medicine, cantharidin (180), Figure 40, can be exploited in the rational design of a new class of drug.290 Cantharidin (180), Figure 40, is used as an

Figure 41. Examples of platinum(IV)-NGR and -RGD conjugates (symmetrical and asymmetrical) (184−186) and platinum(IV)-c(RGDfk) peptide conjugates (187 and 188). 1090


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Figure 42. Chemical structure of an integrin-targeted patinum(IV) prodrug containing either a built-in AIG light-up apoptosis sensor (189) or an AIG luminogen (190).

upon light activation, demonstrated selectively cytotoxicity toward αVβ3 integrin expressing melanoma cancer cells.298 The RGD peptide as an integrin targeting moiety was also exploited by Marchán, Massaguer, et al.299 They developed two platinum(IV)-RGD peptide conjugates, but in their case, they incorporated a picoplatin scaffold. One conjugate contained a monomeric RGD peptide sequence, while the other contained a tetrameric RAFT-RGD peptide bearing four copies of the RGD motif, (187 and 188, respectively), Figure 41, both chosen for their affinity toward αVβ3 and αVβ5 cellsurface integrins. The team was particularly interested in comparing the impact of the presence of a monomeric versus tetrameric peptide for targeted drug delivery.299 Both the monomeric and tetrameric complexes were tested against the SK-MEL-28 malignant melanoma cells which were chosen on the basis of high expression levels of both αVβ3 and αVβ5 integrins. The antitumor efficacy of picoplatin was enhanced by 2.6-fold when incorporated into the platinum(IV)c(RGDfK) conjugate but was siginificantly more so (20-fold increase in efficacy) when in the platinum(IV)-RAFT{c(RGDfK)}4 form. The tetrameric complex was also significantly more potent than cisplatin. Against CAPAN-1 pancreatic cancer cells (chosen as a negative control given its low integrin expression) and noncancerous 1BR3G fibroblast cells, both conjugates showed negligible cytotoxicity, further highlighting the selective, targeted nature of RGD-coupled platinum complexes.299 Liu, Tang, et al. pioneered an integrin-targeted platinum(IV) prodrug in which one axial position was occupied by an integrin-targeted RGD peptide derivative while the other contained an apoptosis sensor made up of a tetraphenylsilole (TPS) fluorophore with aggregation-induced emission (AIE) characteristics attached to a caspase-3 enzyme specific Asp-

upregulated during this tumor angiogenesis process, including the αVβ3 and αVβ5 integrins and aminopeptidase N (APN). As such, these proteins represent a viable molecular target for drug therapies. For example, the Arg-Gly-Asp peptide motif (RGD) has a high binding affinity for these integrins, while the Asn-Gly-Arg (NGR) has a propensity to bind strongly to APN and weakly to αVβ3. Binding of these peptides to these cell surface proteins disrupts the angiogenesis pathway, ultimately leading to apoptosis. Using a succinate linker, Lippard et al. successfully conjugated these tripeptides to a cisplatin framework generating a small series of platinum(IV)-NGR and -RGD derivatives. The cytotoxicities of these mono- and disubstituted platinum(IV) complexes were assessed and their activities compared to nontargeting mono- and disubstituted platinum(IV) complexes with pentapeptides consisting of a cyclic disulfide-bridged RGD, (CRGDC)c, and the cyclic pentapeptide, (RGDfK)c. The platinum(IV)-RGD and -NGR complexes (184 and 185, respectively), Figure 41, exhibited enhanced inhibition of bovine capillary endothelial (BCE) tumor cell proliferation over their nontargeting counterparts and the unconjugated RGD and NGR peptides alone, with the platinum(IV)-RGD complexes exhibiting greater potency over the NGR complexes.295 The use of photodynamic therapy (PDT) is well-established for the treatment of many cancers.296 There are numerous examples of photoactivatable platinum(IV) prodrugs which, upon UV irradiation, release their cytotoxic platinum(II) adducts, with promising anticancer activities.42,297 Sadler and his group, who are leaders in this field, have taken this concept a step further in that they conjugated the RGD peptide sequence to a photoactivatable trans-platinum(IV) center, resulting in the formation of trans,trans,trans-[Pt(N3)2(OH)(pyridine)2(RGD derivative)] (186), Figure 41. This complex, 1091


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Glu-Val-Asp (DEVD) peptide (189), Figure 42.300 They hypothesized that the affinity of the platinum(IV) prodrug for αVβ3 integrins which are overexpressed on cancer cells would facilitate its selective uptake into tumor cells. Once inside, the platinum(IV) would be reduced, releasing both the cytotoxic platinum(II) derivative and the TPS-DEVD apoptosis sensor simultaneously. The platinum(II) moiety would then be free to induce apoptosis which activates caspase-3, which would go on to cleave the TPS-DEVD sequence. This TPS-DEVD is nonemissive in aqueous media due to free rotation of the phenylene rings. However, cleavage of the TPS-DEVD sequence results in the formation of a hydrophobic TPS residue, which tends to aggregate, leading to fluorescence enhancement as the phenyl rings are no longer free to rotate. This complex thus attempted to not only selectively target tumor cells and enhance its cellular uptake but also was designed to provide visual evidence of apoptosis through fluorescence monitoring. HPLC monitoring with ascorbic acid provided evidence of the formation of the reduced platinum(II) species. Fluorescence studies provided clear evidence that the TPS-DEVD ligand and the complex were almost nonfluorescent in DMSO/PIPES, while the TPS free ligand showed intense fluorescence in the same media. LC−MS of TPS-DEVD before and after treatment with caspase-3 was able to confirm its cleavage of this ligand. Several proteins were tested to see if the complex was indeed caspase-3 selective, including lysozyme, pepsin, BSA, and trypsin. Again only caspase-3 was shown to successfully cleave the ligand. Within the caspase protein family, only caspase 3 and 7 demonstrated cleavage, showing evidence that the complex could be considered selective to at least these two enzymes. Cytotoxicity of the complexes was tested against U87-MG (αVβ3 overexpressed) and MCF-7 (low αVβ3 expression) cells. Cell viability of the U87-MG cells clearly decreased as the prodrug concentration increased, while no trend of viability versus concentration was observed against the MCF-7 cells. This provides compelling evidence of a drug that is indeed integrin selective while also facilitating noninvasive and real-time imaging of drug-induced apoptosis. It has the added potential of being used as an indicator for the early detection of any therapeutic responses to the drug in situ.300 The same team designed a similar integrin-targeted platinum(IV) prodrug, again incorporating the RGD peptide as their targeting moiety but replacing their TPS-DEVD apoptosis sensor with an AIE luminogen.301 They also included a hydrophilic region separating the RGD peptide from the platinum(IV) center to enhance the aqueous solubility of the complex (190), Figure 42. Luminogens possessing AIG charecteristics have been shown to be versatile tools for biosensing and bioimaging purposes.302 The presence of the RGD peptide again facilitated tumor cell targeting with the complex exhibiting selective cytotoxicity toward MDA-MB231 cells (which overexpress αvβ3 integrins) over MCF-7 cells (negative control). Again, the prodrug did not fluoresce in aqueous media but became highly emissive upon its reduction to platinum(II) inside tumor cells. This study again provides sound evidence for the specific targeting nature of the RGD peptide and the usefulness of an AIE probe to detect and monitor drug activation.301 3.2.2.2. Neurotensin Receptor Targeting. Neurotensin (NT), a peptide made up of 13 amino acids, is well-recognized as an important neurotransmitter or neuromodulator303 and as an endocrine agent.304 It plays a role in numerous signaling

pathways via the actions of three NT receptors: NTR1, NTR2, and NTR3.292,305 Neurotensin has also been linked to the etiology of a number of disease states including cancer.304 Significant overexpression of NT receptors has been identified in many cancer types, including colon, pancreatic, prostate, and lung.305,306 Somatostatin is an example of another peptide which can act as a neurotransmitter or an endocrine hormone. Again, like NT, its actions are mediated via G protein-coupled somatastin receptors. These receptors have also been found to be overexpressed on the surface of many neuroendocrine tumors.292,307 Peptide analogues of NT and somatostatin have been investigated for diagnostic as well as therapeutic purposes.292 For example, peptide receptor radionuclide therapies have exploited radiolabeled somatostatin analogues like octreotate as a treatment for inoperable or metastasized neuroendocrine tumors.308 Ravera et al. developed a family of mono- and disubstituted platinum(IV)-peptide conjugates incorporating either a NT analogue (pseudoneurotensin = Lys-Lys-Pro-Tyr-Ile-Leu) or a somatostatin analogue [octreotate (D-Phe-Cys-Phe-D-Trp-LysThr-Cys-Thr-OH)]. Again, a succinato linker was employed to facilitate binding of the peptide to the platinum center (191− 195), Figure 43.306 The complexes were more cytotoxic

Figure 43. Chemical structures of platinum(IV)-NT/somatostatinanalogue peptide conjugates (191−195).

compared to the platinum(IV) succinate precursor against a range of cancer cell lines, including NT receptor positive cancer cells although cellular uptake was not found to be specific, most likely because the receptors could no longer recognize the peptide once bound to the platinum(IV) center. 3.2.2.3. N-Formyl Peptide Receptor Targeting. Our innate immune system has been shown to play a key role in the growth or regression of tumors and, as such, there has been a surge of interest in trying to develop immunotherapies for the treatment of cancer.309 While platinum drugs have been shown to act as potential immunomodulators,27 there are few examples in which this property has been exploited to rationally design and develop platinum(IV) complexes. NFormyl peptide receptors (FPRs), of which three have been identified in humans (FPR1-3), play an instrumental role in regulating innate immune responses including tissue repair and angiogenesis. Uncontrolled angiogenesis is associated with tumor growth, progression, and metastases.310 In addition, FPRs have been shown to be overexpressed in many malignant tumors. Exploiting an immine ligation strategy, Ang et al. conjugated peptides which not only behave as FPR1/2 targeting moieties but also as immune adjuvants [Annexin1 2-12 (ANXA1 2-12), Annexin12-26 (ANXA1 2-26), WKYMVm (m: D-Met), or fMLFK (f: formyl)] in one of the axial positions to a cisplatin pharmacophore, generating four potential platinum(IV)-immunochemotherapeutic agents, (general structure shown as 196) Figure 44.311 These peptides induce an immunostimulatory response by activating innate effectors including FPR1/2 expressing monocytes, dendritic 1092


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Figure 45. Chemical structures of the platinum(IV)-tubulin inhibitor conjugates (197−199).

Figure 44. General structure of platinum(IV)-FPR targeting conjugates (196).

They also exhibited lower toxicity toward LO2 human liver cells compared to their platinum(II) analogues. They were shown to induce apoptosis by arresting the cell cycle at the G2/M phase with strong evidence of inhibition of tubulin assembly.313 3.2.3.2. Anti-Apoptotic Proteins. α-Tocopheryl succinate (α-TOS), a vitamin E derivative, can inhibit antiapoptotic proteins, Bcl-2 and Bcl-xL, giving rise to mitochondriamediated apoptotic cell death. Armed with this knowledge, Lippard et al. developed two platinum(IV)-α-TOS complexes, namely cis,cis,trans-[Pt(NH3)2Cl2(α-TOS)2] and cis,cis,trans[Pt(NH3)2Cl2(α-TOS)(OEt)], (200), Figure 46.314 The

cells, and NK cells. The cytotoxicities of the complexes were evaluated against U-87MG, a highly malignant human glioblastoma linked to FPR1 overexpression and MCF-7 and MDA-MB-231, two human breast cancer lines which overexpress both FPR1 and FPR2 receptors. All complexes, with the exception of the fMLFK conjugate which was essentially nontoxic, exhibited comparable or slightly enhanced cytotoxicity compared to cisplatin. Pretreating the U-87MG cells with a free competitive WKYMVm peptide (to presaturate the FPR1/2 receptors) prior to treatment with the platinumWKYMVm complex led to a significant reduction in platinum drug uptake, providing evidence of selective drug uptake mediated by FPR1/2 receptors. The team went on to investigate the tumoricidal activity of platinum drug-activated peripheral blood mononuclear cells (PBMCs), to validate their hypothesis of immune-cell-mediated cytotoxicity. Briefly, the PBMCs were first incubated for 24 h with the platinum(IV)peptide conjugates and then cocultured with preseeded tumor cells in a 10:1 ratio for 72 h. The cell-mediated cytotoxicity in the presence of PBMCs, preincubated with the platinumpeptide complexes, was significantly enhanced against the MCF-7 and MDA-MB-231 cell lines but not against U-87MG with two platinum(IV)-WHYMVm conjugates being more potent compared to cisplatin. The peptide alone, although it had previously been shown to act as a potent activator of monocytes and NK cells, did not exert any significant cell cytotoxicity. The team went on to investigate the impact of their platinum(IV)-WHYMVm-acetate conjugate on secretion levels of pro-inflammatory cytokines TNF-α and IFN-γ, two key mediators of the innate immune system, and both of which in their own right have also been shown to be highly cytotoxic. Treatment of PBMCs with this platinum(IV)-WHYMVm conjugate dramatically enhanced TNF-α and IFN-γ secretion over untreated cells or cells treated with cisplatin. This comprehensive study has clearly shown the potential in trying to develop targeted cytotoxic platinum drugs which also promote tumor cell killing via an immune response.311 3.2.3. Proteins as Targets. 3.2.3.1. Tubulin. Tubulin inhibitors such as paclitaxel and docetaxel are well-established anticancer agents. They work as antimitotic agents by binding to the protein tubulin, thereby disrupting the mitosis pathway.312 Wang et al. developed platinum(IV) derivatives incorporating combretastatin A4-analogue (CA-4) as a single axial ligand anchored to a cisplatin or oxaliplatin equatorial core (197−199), Figure 45. The conjugates exhibited enhanced cytotoxicity in all cases against a range of cisplatinsensitive (MCF-7, HepG-2, HCT-116, Bel-7404, and NCIH460) and cisplatin-resistant (SGC-7901, A549) cell lines when compared to cisplatin, carboplatin, or oxaliplatin alone.

Figure 46. Chemical structure of cis,cis,trans-[Pt(NH3)2Cl2(α-TOS)(OEt)] (200).

symmetrical complex containing two α-TOS axial ligands was found to be only moderately cytotoxic. In contrast, the asymmetrical complex 200 exhibited potent cytotoxicity having between 7−220 times greater cytotoxicity compared to that of cisplatin, α-TOS alone, or combinations of cisplatin and αTOS when screened against several tumor cell lines, including lung A549, prostate PC-3, ovarian A2780, colorectal HCT-116, and breast MCF-7 cells. It was also 6 times more cytotoxic than cisplatin against the cisplatin-resistant A2780/CP70 cell line. In addition, it exhibited lower toxicity toward healthy cells. Not dissimilar to the COX and HDAC inhibition studies, the significantly enhanced efficacy associated with the asymmetrical complex was attributed to its enhanced lipophilicity and consequently its enhanced bioavailability. It was shown to accumulate in cells 15−20 times better than cisplatin.314 A mechanistic study revealed that cis,cis,trans[Pt(NH3)2Cl2(α-TOS)(OEt)] (200), Figure 46, induced apoptosis, caused S-phase cell cycle arrest, and disrupted mitochondrial function via Bcl-xL-Bax interruption. It was also shown to be capable of inducing DNA damage as indicated by its ability to upregulate γH2AX, p53, and pro-apoptotic proteins. 3.2.3.3. Double Minute 2 Homologue (MDM2) Protein. The tumor suppressor protein p53 prevents cancer cell proliferation. It is downregulated in many cancers. The 1093


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mouse double minute 2 homologue (MDM2) protein can bind to the transactivation domain of p53, inhibiting the action of p53. Chalcones have been shown to bind to the p53 binding site of MDM2. They can therefore act as MDM2 antagonists, and, in so doing, allow p53 to remain active.315 Several natural and (semi-) synthetic chalcones have shown promising anticancer activity.315 Many are also less toxic toward the body compared to other non-naturally occurring anticancer agents.316 Zhu et al. developed a platinum(IV)-chalcone complex, named calcoplatin (201), Figure 47.317 Chalcoplatin Figure 48. Chemical structure of the platinum(IV)-NER inhibitor conjugate (202).

ovarian and breast carcinomas.321 It is capable of forming both mono- and bifunctional adducts with DNA. Monofunctional adducts interfere with gene expression and promote mismatched base-pairing while bifunctional adducts leads to DNA intra- and interstrand cross-linking, inducing DNA double strand breaks (DSBs). Both eventually lead to cellular apoptosis due to extensive DNA damage.322 A drawback associated with chlorambucil is the development of tumor cell resistance, which can often be explained by upregulated DSB repair mechanisms in many cancer tumors. Double-strand break repair is promoted by the MRE11-RAD50-NBS1(MRN) complex. Gou et al. sought to generate platinum(IV)-DNA binding conjugates which could also suppress the DSB repair response. They developed two platinum(IV) conjugates bearing the same dicarboxylate leaving ligand and either DACH or ammines and with one chlorambucil and one chlorido ligand present in the axial positions (203 and 204 respectively), Figure 49.322,323 The team found that when tested against SGC-7901 (cisplatin-sensitive gastric cancer) SGC-7901/CDDP (cisplatin-resistant gastric cancer), A549 (cisplatin-sensitive lung cancer), and A549/CDDP (cisplatinresistant lung cancer), and human normal cell line HUVEC, 203 was more potent than chlorambucil, its parent platinum(II) complex without chlorambucil and cisplatin against the SGC-7901/CDDP and A549/CDDP cell lines. It had comparable cytotoxicity to cisplatin against the SGC-7901 and A549 nonresistant cell lines. The cytotoxicity of 204 was also comparable to that of its parent platinum(II) complex and oxaliplatin in all cases. The new complexes were both less toxic to the normal HUVEC cells compared to their platinum(II) precursors. Compound 203 was found to suppress MRN complex protein expression dramatically, which was attributed to its ability to overcome cisplatin resistance. 3.2.3.6. Hypoxia Inducible Factor 1 (HIF-1) Protein. Hypoxia inducible factor 1 (HIF-1) is a transcription factor protein which mediates the cellular response to tumor hypoxia. It has been identified as playing a significant role in the metabolism, angiogenesis, metastasis, and proliferation of hypoxic tumor cells. Inhibition of HIF-1α, a subunit of HIF1, has been identified as an attractive cancer drug target given that HIF-1α has been shown to be overexpressed in over 70% of human cancers.324,325 It has also been linked with poor patient prognosis.326 Among the limited number of HIF-1α inhibitors which have been developed, 1-benzyl-3-(5-hydroxymethyl-2-furyl)indazole has stood out as a promising candidate for this purpose given its potent HIF-1α inhibition activity. Gou et al. tethered this as a single axial ligand via a succinate linker to a platinum(IV) cisplatin scaffold, generating

Figure 47. Chemical structure of chalcoplatin (201).

was more cytotoxic compared to chalcone alone, cisplatin alone, or a combination of cisplatin and chalcone when tested against cervical HeLa, colorectal HCT-116, breast MCF-7, and lung A549 which are p53 wild-type cell lines. It was particularly cytotoxic against the cisplatin-resistant A549/cDDP cell line, demonstrating an ability to reverse cisplatin resistance. It was also found to induce a pattern of cell cycle arrest similar to chalcone and distinct from cisplatin. There was no significant increase in cytotoxicity however when chalcoplatin was tested against the p53 null HL-60 leukemia cell line, suggesting that the cytotoxicity of chalcoplatin is mediated via a p53dependent pathway. Chalcoplatin was also shown to be cancer-cell selective in that it was significantly less toxic when compared to cisplatin against normal human lung fibroblast cells. 3.2.3.4. DNA Excision Repair Protein ERCC-1. DNA is constantly being bombarded by factors derived from both endogenous and exogenous sources that can ultimately lead to the formation of DNA lesions, if left unchecked. Cells utilize various complex pathways to recognize and repair such DNA lesions, a phenomenon often referred to as the DNA damage response (DDR).318 Nucleotide excision repair (NER) is one such pathway. This pathway is of particular relevance to this review in that it has been shown to be exploited by cells to repair DNA lesions caused by exposure to alkylating agents and platinum drugs.319 Zhu et al. developed a platinum(IV) conjugate, (202), Figure 48 incorporating an axial ligand, (E)-2-(((8a,9a-dihydro-9Hfluoren-9-ylidene)hydrazono)methyl)benzoic acid, which had been shown to bind to ERCC1, a protein involved in the NER pathway. The platinum(IV)-NER inhibitor conjugate was significantly more cytotoxic compared to cisplatin and cisplatin in combination with the free NER inhibitor against both cisplatin-resistant and cisplatin-sensitive ovarian and lung cancer cell lines and was significantly more so against the resistant cells, suggesting a reversal of cisplatin resistance. Evidence of its ability to disrupt the NER pathway and to prevent DNA repair was also demonstrated.320 3.2.3.5. MRE11-RAD50-NBS1(MRN) Complex. Nitrogen mustards represent an important family of alkylating anticancer agents.321 Chlorambucil, a member from this class, is used clinically to treat chronic leukemia, lymphomas, and advanced 1094


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Figure 49. Chemical structures of platinum(IV) complexes incorporating the DNA alkylating nitrogen mustard, chlorambucil (203 and 204).

Figure 50. Chemical structures of hypoxia-targeted platinum(IV)-HIF-1α inhibitor conjugates (205−206).

Figure 51. Chemical structures of platinum(IV) prodrugs designed to target heat shock proteins (207−210).

two complexes (205 and 206), Figure 50, where the opposing axial ligand was either a chlorido or hydroxido ligand. The resulting complexes showed significantly improved cytotoxicity over cisplatin against both cisplatin-sensitive HCT-116, A549, MCF-7, and SGC7901 and cisplatin-resistant SGC7901/ CDDP cells. Complex 206, in particular, was shown to significantly downregulate the expression of HIF-1α and enhance cisplatin sensitivity of hypoxic HCT-116 cells, with the cytotoxicity of 206 increasing as cellular oxygen concentration of HCT-116 cells decreased. Complex 206 also demonstrated inhibition of tumor growth in a HCT-116 xenograft mouse model with low in vivo toxicity observed.327 3.2.3.7. Heat Shock Proteins. HSP70 is a stress-induced chaperone protein which is upregulated during a stress response in cells from either extrinsic (physiological, viral, or environmental) or instrinsic (replicative or oncogenic) stimuli.328 It has been shown to be overexpressed in many cancers including colorectal cancer and typically indicates poor prognosis. 328 In particular, membrane-bound HSP70 (memHSP70) has been shown to be selectively expressed in cancer cells. For example, from a study involving a population

of 1000 cancer patients, memHSP70 was shown to be present in 50% of the tumors and yet was not found to be expressed in healthy tissues. Griffith et al. developed symmetrical and asymmetrical oxaliplatin-based platinum(IV) prodrugs incorporating the 14-mer tumor penetrating peptide TKDNNLLGRFELSG or TPP (207 and 208), Figure 51, and the corresponding scrambled peptide, LNLETRLGFGDNKS (ScP), in one or both of the axial positions (209 and 210), Figure 51, with a view to targeting a memHSP70+ phenotype in colorectal cancer cells.329 The TPP peptide had previously been shown to be rapidly taken up in cancer cells overexpressing memHSP70. The complexes were tested against two colorectal cancer cell lines, HCT116 and HT29. The former (HCT116) cell line was selected on the basis that it exhibited low memHSP70 expression at 17%, in contrast to the HT29 cells which exhibited higher memHSP70 expression at 36%. The complexes presented significantly improved cell killing ability over oxaliplatin against the HT29 cell line, with the symmetrical complex, (208), Figure 51, being more effective than the monosubstituted derivative. TPP by itself showed little to no cytotoxicity. Oxaliplatin induced up to 53% 1095


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apoptotic pathway in LNCaP cells, and this was related to a disruption in mitochondrial function and accumulation of ROS. The mechanism of cell cycle arrest induced by 211 was akin to that of cisplatin and LND and occurred mainly at the S phase.337 While the rationale behind the development of these conjugates is very sound and the complexes possessed promising biological activities, it should be noted that LND is nonspecific. It targets numerous other metabolic enzymes and pathways. It is therefore challenging, like all of the complexes reported herein, to truly understand the mechanism of action of these intriguing complexes. 3.2.5. Targets Inspired by Natural Product Activity. Many blockbuster drugs are derived directly or indirectly from natural products.338,339 Camphor, found in the Cinnamomum camphora tree, has been exploited for centuries as a local anesthetic and antimicrobial agent. Its anticancer potential was first highlighted when it was shown to make cancer cells more susceptible to radiation.340 Jing, Zhang, et al. developed a platinum(IV) complex bearing a derivative of camphoric acid in one of its axial positions. The resulting prodrug, named camplatin (216), Figure 53, was linked to the MPEG-b-PCL-bPLL polymer which then self-assembled into a micellar structure. 341 Upon cellular accumulation mediated by endocytosis, the micelle released camplatin which, upon reduction, released cisplatin and camphoric acid. Camplatin was ∼7 and ∼13.5 times more cytotoxic when compared to cisplatin against ovarian A2780 and A2780cisR cell lines, respectively. Camplatin was shown to induce cancer cell death via an apoptotic pathway; it downregulated Bcl-2 while upregulated pro-apoptotic Bax gene expression. Chlorotoxin (CTX), a peptide consisting of 36 amino acids and derived from the venom of the giant Israeli yellow scorpion, Leiurus quinquestriatus hebraeus,342 has a high affinity for a number of proteins including matrix metalloproteases (MMP)343 as well as chloride ion channels.344 Chlorotoxin binding to these proteins leads to an inhibition of cell invasion and migration. Targeting these proteins represents a viable drug design strategy given that MMP-2 receptors343 and chloride ion channels344 are overexpressed on many tumor cell surfaces including those of the highly invasive human glioma cells. Exploiting this strategy, Lippard et al. developed a platinum(IV)-CTX conjugate, (217), Figure 53.345 Synthetically, because of the size of the peptide and the composition of the amino acids making up the peptide, many of which could potentially be platinated, standard synthetic protocols could not be employed to tether the peptide to the platinum scaffold. Instead, through careful adjustment of pH to avoid any unwanted platination reactions, together with selective amide coupling via EDC/NHS chemistry (where EDC is 1-ethyl-3[3-(dimethylamino)propyl]carbodiimide hydrochloride and NHS is N-hydroxysuccinimide), they were able to successfully generate 217, Figure 53, in which the CTX was bound to the platinum center via its N-terminus. While the complex was more cytotoxic than the peptide alone and the platinum(IV) precursor against cervical (HeLa), breast (MCF7), and lung (A549) cancer cells, it was not as cytotoxic when compared to cisplatin. The cancer cells were specifically chosen as they express varying levels of the CTX-targeted receptor. The team concluded that the presence of CTX in the conjugate was most likely acting as a targeting agent rather than a toxin in its own right.345

cell death against the HT29 cell line, in contrast to the mono and disubsituted TPP complexes which caused 70% and 80% cell death, respectively. When the TPP ligands were replaced with a scrambled peptide as a control, the complexes essentially lost all cytotoxicity against both cell lines, despite the fact that both contained the oxaliplatin moiety, suggesting that this nontargeting peptide prevents uptake of the complex into the cell.329 3.2.4. Mitochondria as a Target. Lonidamine (LND), as a single agent, has only limited antitumor activity. However, it has been shown to have great potential in modulating the activities of numerous therapeutic agents, including N-mustard alkylating agents,330 anthracyclines such as doxorubicin,331 and, of particular relevance to this review, cisplatin.332 It can also sensitize tumors to radiotherapy333,334 and photodynamictherapy334,335 regimens. It is particularly selective against a varied range of tumors while being nontoxic toward normal tissues at doses required to induce its therapeutic effect.336 It induces its therapeutic effects by multiple mechanisms, including targeting glycolytic pathways specific to tumor cells. It has been shown to retard aerobic glycolysis by reducing the activities of mitochondrial complex II and hexokinase (HK-II), both of which play a role in ATP production. It does not appear to inhibit aerobic glycolysis in noncancerous cells. Gou et al. sought to combine into one drug entity either a cisplatin, carboplatin, or oxaliplatin pharmacophore as their DNA binding moiety tethered to a LND ligand. Five platinum(IV)-LND conjugates were developed (211− 215) Figure 52, and their cytotoxicity profiles against MCF-7,

Figure 52. Chemical structures of platinum(IV) complexes incorporating lonidamine (211−215).

A549, LNCaP, U87 HCT-116, and noncancerous LO2 cells were assessed.337 The platinum(IV)-LND-carboplatin complex 212 was more effective than carboplatin across all cell lines: the platinum(IV)-LND-oxaliplatin complex 213 was an improvement over oxaliplatin in most cases, and the platinum(IV)LND-cisplatin complex 211 had an almost 2-fold increase in cytotoxicity over cisplatin against LNCaP cells and a significant increase over the combination of cisplatin and LND against this cell line. Reduction studies with ascorbic acid revealed that the complexes readily released their LND ligands upon reduction. The complexes induced cancer cell death via an 1096


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Figure 53. Chemical structure of camplatin (216) and a platinum(IV)-CTX conjugate (217).

Figure 54. Chemical structures of a “quadruple action” platinum(IV) prodrug (218) and dinuclear (219) and mononuclear (220 and 221) adducts.

3.3. Platinum(IV) Prodrugs Targeting DNA and at Least Two Other Cellular Entities

(218), Figure 54, was compared to that of the dimer (219), the monomeric dual action complexes (220−221), Figure 54, and cisplatin, oxaliplatin, and Pt56MeSS (159), Figure 35, against nine human cancer cell lines [(2008 ovarian, A431 cervical, H157 lung, HCT-15 colon, A375 melanoma, and BxPC3 pancreas as well as KRAS mutated, PSN1 pancreas, MIAPaCa-2 pancreas, and LoVo colon cancer cells]. The quadruple action complex 218 was found to have remarkable anticancer activity, significantly more so than each of the complexes as dual functioning agents or the single agents. Its average IC50 value across the nine cell lines was also found to be ∼15-fold lower than that of 219, suggesting that the presence of the bioactive axial ligands was contributing to the enhanced cytotoxicity observed. Of particular note is the fact that the “quadruple action” complex 218 demonstrated a 200−450-fold greater cytotoxicity profile relative to cisplatin against KRAS-mutated pancreatic and colon cancers and was 40-fold more selective toward KRAS-mutated cells compared to noncancerous cells. KRAS are genes that encode a family of

Most if not all of the “multitargeted” or “multifunctional” or “multimodal” platinum complexes reported to date involve “dual” targeting, by either employing targeting agents to selectively deliver platinum DNA-binding payloads to tumor cells or endowing the platinum DNA binding drug with some additional functionality. Gibson et al. developed an interesting dinuclear “quadruple action” platinum(IV) prodrug with two platinum centers designed to release simultaneously four different bioactive moieties upon entering the cancer cell.346 The framework was made up of one cisplatin DNA binding core, the previously discussed non-DNA-binding Pt56MeSS core (159), Figure 35, which is believed to act on the mitochondria, a DCA ligand previously shown to be a PDK inhibitor which also acts on the mitochondria, and a PhB HDAC inhibitor ligand. The cytotoxicity profile of this novel quadruple action complex 1097


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Figure 55. Chemical structure of platinum(IV) complex bearing a biotin receptor targeting moiety (222) and a “triple action” complex containing both a biotin receptor targeting moiety and a COX inhibitor ligand (223).

HepG2, and PC3 and had comparable cytotoxicities to cisplatin against SGC7901 cells. Both complexes were shown to have significantly improved cytotoxicities over cisplatin against the cisplatin-resistant SGC7901 cells with 223 being 9fold more cytotoxic than cisplatin. Of note also is the fact that 223 had lower toxicity compared to both cisplatin and 222 against the human normal LO2 cells, suggesting selectivity for tumor cells over normal ones. As stated earlier, tumor progression has been associated with increased COX expression in tumor cells. Both complexes exhibited concentration-independent COX-1 and COX-2 inhibition in vitro with both exhibiting different activities and selectivities for COX-1 and COX-2 compared to indomethacin. Angiogenesis is often assessed by monitoring the ability of endothethial cells to sprout, migrate, and form vascular tubes on a matrigel matrix. The antiangiogenic properties of both 222 and 223 were assessed and both were found to disrupt capillary-like tube formation in EA.hy926 endothelial cells. Furthermore, both were shown to reduce not only tumor-associated inflammation but also the invasiveness of the highly agressive PC-3 cells.348 It would certainly be interesting to further exploit this prototype, retaining, for example, the biotin targeting moiety and substituting the COX inhibitor with other bioactive ligands. Conjugating GLUT-targeting sugars or peptides with known receptor targeting properties to a platinum(II) or a platinum(IV) framework have already shown promising targeting and cytotoxic properties. Replacing biotin with these targeting vectors, while retaining the DNA binding platinum center and another bioactive ligand also warrants further investigation. All in all, these studies on the rational design and development of multitargeted platinum(IV) prodrugs clearly demonstrate the enormous potential inherent in combining multiple drug entities into one drug molecule. Most of the platinum(IV) prodrugs described had the capacity to overcome classical platinum(II) drug resistance, and many of those that were tested in vivo were also found to be less toxic than platinum drugs in clinical use. This bodes well for their future development. One does need to be continuously mindful of the fact that the concentration or dose required of one of the drug entities to induce its biological effect may fall outside the range of the other. This should be considered when designing drugs of this type if one is to hope for a true synergistic effect. One also needs to be mindful of the complex milieu found with a given cell and within biological systems. While drugs may be designed to hit specific targets, we have highlighted plenty of evidence in this review in which a number of prodrugs described herein are actually hitting targets over and above those that they were initially intended to hit.

proteins that bind GTP and play a role in cell differentiation, proliferation, and survival. KRAS mutations in cancer cells typically have poor prognosis and no effective chemotherapeutic treatment. The team also tested the complex in 3D cell cultures, which aim to mimic the in vivo environment of tumor cells more closely. Here, against HCT-15, BxPC3, and KRAS-mutated PSN1 spheroid models, the quadruple action complex 218 was more potent than cisplatin in all cases and comparable to oxaliplatin in the non-KRAS mutated cells. The team also demonstrated that, upon cellular activation, the quadruple action drug was capable of covalently modifying DNA following release of the cisplatin core, inhibited HDACs via PhB, and interfered with mitochondrial function due to the combined action of both DCA and Pt53MeSS. This study also demonstrated that complexation of the bioactive ligands to the platinum framework does not adversely impact on their biological activities. The quadruple action complex 218 was also found to be particularly potent against the highly aggressive and extremely difficult to treat KRAS-mutated cell lines: KRAS-mutated cancers have been referred to as “undruggable”.347 The development of this quadruple action platinum(IV) prodrug certainly highlights that this multitargeted approach to drug design can lead to drug candidates which appear to offer significant advantages over the sum of their parts. This work has led to the development of a new prototype capable of targeting difficult-to-treat cancers. This may well pave the way toward the generation of a new class of therapeutic that may overcome resistance issues, a major bugbear associated with many current cancer therapeutic regimens. Gou et al. developed a triple action platinum(IV) complex derived from cisplatin and bearing a biotin targeting ligand in one axial position and a NSAID indomethacin ligand in the other, (223), Figure 55.348 The complex was designed to not only bind DNA but also to target biotin receptors for preferential uptake of the complex into cancer cells that overexpress biotin receptors on their surfaces, as well as to inhibit COX activity, given that COX enzymes are overexpressed in numerous tumor cells. The cytotoxicity of conjugate 223 against HCT-116, HepG2, PC3, SGC7901, cisplatinresistant SGC7901, and human normal liver LO2 cells was determined and compared to cisplatin and a complex without the NSAID ligand (222), Figure 55. Both 222 and 223 were highly cytotoxic against all cell lines, with IC50 values ranging from 0.81 to 19.27 μM with complex 222 being more cytotoxic compared to the biotin-targeting 223. This difference may well be due to different reduction rates. Chlorido or hydroxido axial ligands in asymmetrical platinum(IV) complexes have been shown to generally promote reduction.223 Complex 222 was also more cytotoxic compared to cisplatin against HCT-116, 1098


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Table 2. Representative Examples of ‘Multi-Targeted‘ Platinum(IV) Complexes Incorporating Ligands Which Are Used Clinically as Drugs and/or Are in Clinical Trials metallodrug conjugate number and figure no.

drug ligand

142−143 (Figure 33)248,250 144−148 (Figure 34)255−258,260 149−156 (Figure 34)259,260,269 218(Figure 54)346 167 (Figure 37)270 218(Figure 54)346

ethacrynic acid

168−172 (Figure 38)280,281,285,286

indomethacin, ibuprofen, and aspirin endothall

182−183 (Figure 40)290 313

valproic acid 4-phenyl butyrate dichloroacete

197−199 (Figure 45) 200 (Figure 46)314

combrestatin A4 α-tocopheryl succinate

203−204 (Figure 49)322,323 211−215 (Figure 52)337

chlorambucil lonidamine

drug ligand target glutathione-Stransferases histone deacetylases histone deacetylases pyruvate dehydrogenase kinases cyclooxygenases

phosphatase 2A protein tubulin antiapoptotic proteins (Bcl-2 and Bcl-xL) MRN protein complex mitochondria

clinical use/preclinical properties of drugs in clinical use as a diuretic treatment for edema349 in clinical use as a treatment for epilepsy and bipolar disorder;211 Sensitizes cancer cells to ionisizing radiation212 and DNA-targeting anti-cancer drugs;213 Also in clinical trials for various cancer types214 sodium salt in clinical use as a treatment for urea cycle disorders; in vitro anti-tumor activities350

in clinical use as an orphan drug treatment for various acquired and congenital disorders of mitochondrial intermediary metabolism;215 has undergone or is undergoing clinical trials as a treatment for cancers including breast, head and neck, lung and brain cancers216 in clinical use as NSAIDs; NSAIDs such as aspirin have been shown to reduce the risk of developing colorectal cancer and reduce colorectal cancer mortality rates;351 NSAIDs included in combination therapies with cisplatin, paclitaxel and doxorubicin352 derivatives used in traditional chinese medicine for the treatment of liver, lung and digestive tract cancers353 in multiple clinical trials including as a treatment for solid tumors218 has undergone numerous in vitro and clinical testing against various cancers354

in clinical use as a treatment for chronic lymphocytic leukemia (CLL), Hodgkin lymphoma, and nonHodgkin‘s lymphoma355 originally introduced into the clinic as an anti-spermatogenic agent, now known to possess anti-cancer activity by sensitizing tumors to chemotherapy, radiotherapy, and photodynamic therapy334

Representative examples of “multitargeted” platinum(IV) complexes incorporating ligands which are used clinically as drugs in their own right and/or are in clinical trials are provided in Table 2.

section 4.1.7. Meggers et al., in contrast, generated a series of organometallic ruthenium(II) complexes which mimicked the 3D structure of the highly potent protein kinase inhibitor, staurosporine. Interestingly, in these complexes, the ruthenium(II) center remained inert, only serving as a structural scaffold to optimize the three-dimensional (3D) structure of the bioactive ligand rather than having any biological properties in its own right. The resulting complexes were however more potent inhibitors of protein kinases such as Pim-1 and glycogen synthase kinase 3b compared to staurosporine alone.18,19,358,361 This work nicely demostrates how the presence of metal centers can compliment and enhance the molecular diversity of organic-based bioactive molecules in the quest to advance new therapeutics with superior biological properties. The reader is directed to pertinent reviews by Meggers in which he describes how biologically relevant chemical space can be explored using metal complexes358 and how the unique shapes of metal complexes can be exploited for targeting enzymes361 and proteins.362 While a “multitargeted” approach has been well-exploited for the generation of platinum complexes as described in the previous sections of this review, this field is also rapidly developing relative to ruthenium complexes. Despite the fact that the only ruthenium complexes to enter clinical trials to date are in the +3 oxidation state, there appears to be particular interest in organometallic “piano stool” pseudo octahedral ruthenium(II) complexes in which an η6-π-bonded arene ligand forms the “seat” while the three remaining ligands that complete the coordination sphere form the “legs”. The arene coordinated to the ruthenium center endows the complex with hydrophobic properties, facilitating its passage though cell membranes while the remaining ancillary ligands can be rationally chosen to produce new complexes with distinct mechanisms of action. Structure activity relationship studies have also shown that the nature of the arene ligand and the mono or chelating ligands can play distinct yet synergistic roles in terms of fine-tuning the biological properties of the resulting

4. MULTI-TARGETED RUTHENIUM(II) AND RUTHENIUM(III) COMPLEXES Ruthenium complexes represent an attractive alternative to platinum drugs. While many of their aqua species display similar ligand exchange kinetics to platinum drugs, they can also exploit the iron-transporter protein transferrin to facilitate their uptake into tumor cells. They can also act as prodrugs in that ruthenium(III) complexes are kinetically inert. They can however be reduced to their ruthenium(II) analogues in the hypoxic environment found in tumor cells and this property can be exploited as a strategy for drug delivery.356 As stated in the introduction, the ruthenium(III) complexes, NAMI-A (imidazolium trans-[tetrachloro(dimethyl sulfoxide)(1Himidazole)ruthenate(III)]) (7), Figure 2,43,44 KP1019 (indazolium trans-[tetrachlorobis(1H-indazole)-ruthenate(III)]) (8), Figure 2,45−47 and NKP-1339, the sodium salt analogue of KP101947 have already advanced to clinical trials. The reader is directed to an interesting chapter by Alessio and Messori in which the different actions of “the deceptively similar” ruthenium(III) drug candidates KP1019 and NAMI-A are compared.357 The earliest reports of “multitargeted” ruthenium complexes containing bioactive ligands date back to pioneering work published in the mid-2000s when Meggers et al. developed staurosporin mimics containing the (η6-arene)ruthenium(II) framework,18,19,358 and Keppler et al. developed (η6-arene)ruthenium(II) complexes bearing cyclin-dependent kinase (CDK)-inhibiting paullone ligands.359 The Keppler team demonstrated that they could overcome the poor aqueous solubility associated with paullones by coordinating paullone derivatives to a central ruthenium(II) scaffold and, in so doing, enhance their bioavailability and thus their therapeutic potential.359,360 This work is described in more detail in 1099


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Figure 56. Chemical structures of an organoruthenium(II) complex bearing an ethylenediamine ligand (224) and organoruthenium complexes bearing a PTA ligand (225 and 226).

Figure 57. Chemical structures of (arene)ruthenium(II) complexes bearing the NSAID ligands naproxen (227), diclofenac (228), ibuprofen (229), and aspirin (230).

complexes.363−365 For example, of the (η6-arene)ruthenium(II) complexes reported to date, probably two of the most important of this complex type are those containing either a bidentate ethylenediamine ligand (224 or RM175), Figure 56, or the RAPTA-type complexes incorporating the monodentate 1,3,5-triaza-7-phosphatricyclo[3.3.1.1]decane (PTA) ligand, (225 or RAPTA-C and 226 or RAPTA-T), Figure 56. While structurally quite similar, they behave in very different ways under biological conditions. Compound 224 can either covalently bind DNA via the N7 of guanine or bind noncovalently via intercalation of the arene; these interactions lead to cancer cell death via modulation of the p53-p21-Bax pathway.366,367 In contrast, RAPTA-type complexes behave differently, both chemically and biologically. RAPTA-T, (226), Figure 56, for example, has been shown to be activated selectively under the hypoxic environment of solid tumors. It has been also shown to inhibit metastasis in experimental in vitro and in vivo models.46,368−370 Hartinger et al. employed a proteomics approach to gain a deeper insight into the mechanism of action and protein targets of RAPTA-type complexes.371 They were able to identify 15 cancer-related proteins which could be associated with the observed antimetastatic, antiangiogenic, and antiproliferative properties of RAPTA-type complexes (e.g., MK, PTN, and FGF3), which are angiogenesis and metastasis related effectors. Additional targets associated with cell cycle regulation were also identified (i.e., GNL3, CGBP1, FAM32A, and VIR).371 While there is a drive to design and develop multitargeted complexes, this study is an important reminder that metallodrugs can interact with more than their intended targets and highlights the importance of keeping an open mind when assessing their therapeutic potential.

4.1. Enzymes as Targets

4.1.1. Cycloxygenases. As outlined in section 3.2.1.4, COX inhibition represents an attractive molecular target with a number of platinum(II)- and platinum(IV)-COX inhibitor conjugates having already been reported. The COX enzymes (particularly the COX-2 isoform) are upregulated in many malignant tumors. Nonsteroidal anti-inflammatory drugs (NSAIDs), in addition to targeting COX, have also been shown to target lipoxygenase (LOX), another enzyme that plays a key role in angiogenic activity via the formation of 12(S)-HETE, which is also upregulated in tumor cells. Mukhopadhyay et al. reported the first examples of ruthenium(II)-COX/LOX inhibitor conjugates with some intriguing findings. They developed four (η6-arene)ruthenium(II) complexes bearing the NSAID ligands naproxen (227), diclofenac (228), ibuprofen (229), and aspirin (230), Figure 57.372 Interestingly, while all four complexes were found to inhibit COX and LOX more effectively than their noncomplexed NSAID counterparts, only 229 was found to have any significant DNA binding activity. When tested against A549, MCF7, and HeLa cells for their growth inhibition properties, the results varied widely. Complexes 227, 228, and 229 showed marked antiproliferative activity toward A549 and MCF7 cells. Complexes 228 and 229 were effective against HeLa while 227 was not, and complex 230 did not show any marked antiproliferative activity toward any cell line. It is worth noting that the free NSAIDs did not demonstrate any antiproliferative activity. Also of interest is the fact that the diclofenac compound, 228, with the highest antiproliferative activity, was also the most potent COX inhibitor and the only DNA binder of the group. Cellular accumulation studies would have been interesting here given the findings from the platinum work and the influence of the coordinated NSAID 1100


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ligands on the overall hydrophobicity of the complexes.372 It would also be interesting to explore the stability of these complexes in solution given the strain on the chelate ring formed between the central ruthenium ion and the carboxylate oxygens. There are numerous examples of multinuclear organometallic ruthenium-arene complexes which have been developed as potential anticancer agents but not necessarily as multitargeted agents.373 de Oliveira Silva et al. have been working in this space for some time but with a distinct focus on generating mixed-valence dinuclear ruthenium(II,III) complexes incorporating carboxylate ligands derived from drugs of clinical importance including NSAIDs and γ-linolenic acid.374−380 The team particularly focused on malignant gliomas (brain tumors) due to their current poor prognosis which has been attributed to their invasive nature. This type of cancer is also resistant to most conventional chemotherapies. Of their dinuclear ruthenium NSAID series, they identified one lead candidate, namely chloridotetrakis(ibuprofenato)diruthenium(II,III) or [Ru2(Ibp)4Cl] (where Ibf is ibuprofen) (231), Figure 58.381 This lead complex (231) was found to be

ingly, the three complexes were shown to inhibit the cell migration process for the U87MG and the A172 human glioma cell lines.381 4.1.2. Glutathione-S-Transferases. Building on their previous work in which they developed novel platinum(IV)GST inhibitor conjugates in the form of a platinum(IV)-EA complex (where EA is ethacrynic acid),248 (see section 3.2.1.1), Dyson, Lo Bello, Ang, et al. tethered EA to a ruthenium(II)-arene scaffold, via an imidazole linker, to create three new complexes, similar in structure to RAPTA-C, (225), Figure 56, incorporating either chlorido ligands (234 or ethaRAPTA), Figure 59, or dicarboxylate ligands (235−236), Figure 59.382 Given that the pharmacophore responsible for the GST inhibition activity of EA is the phenolic group comprising the α,β-unsaturated conjugated carbonyl system and the 2,3-dichlorinated aromatic ring, the group were confident that they could introduce the imidazole linker via modification of the α-acetic acid group without comprising the GST-inhibition activity of the resulting ligand. Like EA, the complexes were good GST P1-1 inhibitors although their interactions with GST P1-1 were different to those of EA. The ruthenium complexes were shown to rapidly inactivate the enzyme. To elucidate the mode of inactivation, investigations using the cysteine-modified GST mutants revealed that covalent binding occurs at Cys 47 and, to a lesser extent, at the Cys 101 residue. The complexes were cytotoxic against both cisplatin-sensitive and cisplatin-resistant human ovarian carcinoma cell lines; they were 10-fold more potent than RAPTA-C and a reference standard (η6-pcym)ruthenium−imidazole complex (where cym is η6-pcymeme) and 3−5 fold more potent than EA, suggesting that GST P1-1 may be a potential target of these complexes in vitro.382 Bhattacharyya, Dyson, et al. went on to demonstrate that ethaRAPTA (234), Figure 59, was also highly cytotoxic against the cisplatin-resistant MCF-7 breast cancer cells, triggering multiple pathways associated with apoptosis, including those involving endonuclease G, caspases, and c-Jun N-terminal kinases.383 The same group went on to generate RAPTA-C analogues but this time tethering the EA moiety to its arene ring. Two complexes were developed (237 and 238), Figure 59, both of which were shown not only to bind the enzyme at the H-site but also to interact with the reactive cysteine residues of GST P1-1.384 In a related study, Marchetti, Dyson, et al. further derivatized EA, but this time by incorporating either phosphine (239) or pyridine linkers (240 and 241), Figure 59, to facilitate their binding to the ruthenium center. They also generated a ruthenium(III)-NAMI-A type complex (242), Figure 59.385 Interestingly, despite the Ru−N bond in the N-donor-based complexes being more labile in aqueous-DMSO solutions relative to the Ru−P bond in the P-donor complex, there was little difference in their cytotoxicities against human ovarian A2780 and cisplatin-resistant A2780cisR cancer cells and human embryonic kidney (HEK-293) cells. This is surprising, given that one would expect the Ru−N bond in the complex to partially dissociate in advance of being taken up by cells. The complexes were, however, significantly more cytotoxic compared to RAPTA-C, NAMI-A, and EA alone. Furthermore, the complexes, irrespective of the stability of the Ru-ligand bond, exhibited similar levels of GST activity in the different cell lines.385

Figure 58. Chemical structures of multinuclear organometallic ruthenium-arene complexes (231−233).

cytotoxic against C6 rat glioma cells,375 and its method of action was suggested to involve several proteins including p21, p27, p53, Bax, and COX-1.376 The complex was also effective in vivo against a C6 rat orthotopic glioma tumor model, decreasing tumor size by both intraperitoneal or alzet osmotic pump infusion.377 Toxicology studies in normal Wistar rats suggested that the complex was well-tolerated.377 The complex was also able to inhibit cell proliferation of p53 wild-type U87MG and A172 cells, as well as p53 mutant type U138MG and U251MG cells. Inhibition was shown to be clearly both dose- and time-dependent. The studies suggested that p53 and COX-1 played a key role in the anticancer properties of the complex due to the U138MG cell line being the most resistant, which has a mutant form of p53 and low COX-1 mRNA expression.377 Given that cisplatin, NAMI-A, KP1019, and NKP1339 are examples of chlorido complexes exhibiting potent anticancer activities, and the fact that the chlorido ligands are thought to play a key role as leaving ligands capable of promoting ligand substitution reactions, the team looked at the effect of replacing the chlorido ligand in 231 with either a triflate or hexafluorophosphate. They generated two new analogues, [Ru2(Ibp)4(CF3SO3)] (232) and [Ru2(Ibp)4(EtOH)2]PF6 (233), Figure 58. While the three complexes accumulated in human glioma cell lines, the triflate and hexafluorophosphate analogues demonstrated less ability to inhibit cell proliferation in U87MG and A172 cell lines compared to the original chlorido complex (231). Interest1101


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Figure 59. Chemical structures of organoruthenium(II) complexes incorporating an EA derivative either linked to the ruthenium center via an imidazole linker (234−236) or tethered to the arene ring (237−238), or linked via phosphine or pyridine moieties (239−242), a ruthenium(III)NAMI-A type complex (242), a RAPTC-C-EA analogue (243), and organoruthenium(II) complexes with different ratios of ruthenium to EA (244−246). 1102


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Figure 60. Chemical structures of RAPTA complexes bearing derivatives of picolinamide as ancillary ligands where PTA is 1,3,5-triaza-7-phosphatricyclo-[3.3.1.1]decane (247−250), organoruthenium(II) derivatives incorporating lapachol (251), plumbagin (252), lawsone (253), and juglone (254), and organoruthenium(II) lawsone (255) and nitroso-naphthalene derivatives (256).

cytotoxic, especially against the chemo-resistant A2780cisR ovarian cells with the complex being ∼5 times more potent compared to cisplatin. Moreover, 245, unlike 234, which contains the EA-modified imidazole ligand, was endowed with some degree of cancer cell selectivity being less toxic to human embryonic kidney HEK-293 cells.388 These comprehensive studies demonstrate that these complexes are not only targeting GST enzymes but also are triggering multiple pathways associated with apoptosis. Again, these studies highlight the need to keep an open mind when evaluating the mode of action of these “multitargeted” prototypes. They also further serve to reinforce the enormous potential inherent in this “multitargeted” approach to drug design. 4.1.3. Thioredoxin Reductases. Thioredoxin reductase (TrxR), as stated previously, has been identified as an enzyme responsible for suppressing apoptosis in cancer cells. It has also been identified as a potential target for anticancer active metallodrugs, including ruthenium(II)-based derivatives.389 Mukhopadhyay et al. generated a small series of ruthenium(II)-arene-PTA or RAPTA complexes containing derivatives of picolinamide as ancillary ligands, (247−250), Figure 60.390 The amphiphilic PTA ligand was chosen to overcome the poor aqueous solubility often associated with these organometallic complexes. They selected picolinamides as metal complexes incorporating picolinamides, including those of iridium, rhodium, and ruthenium, had previously been shown to

More recently, Ratanaphan, Dyson, et al. generated another RAPTA-C-EA analogue (243), Figure 59, this time linking the EA ligand to the ruthenium(II)-arene scaffold via its phenyl ring.386 They compared the activity of this complex against BRCA1-defective HCC1937 breast cancer cells versus BRCA1competent MCF-7 breast cancer cells. The team found that the complex caused significantly more damage to the BRAC1 gene in the BRCA1-defective HCC1937 cells than to the BRCA1competent MCF-7 cells. Expression of BRCA1 mRNA in the BRCA1-competent cells was reduced; while in contrast, its expression was shown to have increased in the BRCA1defective cells. The expression of the BRCA1 protein, however, was significantly reduced in both breast cancer cell types.386 Exploiting the emerging technique of functional identification of target by expression proteomics (FITExP), Dyson et al. were able to identify key protein targets of RAPTA-EA (243), Figure 59, in breast cancer cells with RAPTA-EA causing upregulation of mainly oxidative stress-related proteins. This was in contrast to RAPTA-T (224), Figure 56, which upregulated multiple proteins suggesting a broader mechanism of action involving suppression of both metastasis and tumorigenicity.387 Dyson et al. also went on to develop a new generation of ruthenium(II)-cym EA conjugates (the study also included osmium(II) analogues), this time varying the metal to EA ratios from 1:1, 1:2, and 2:1 (244−246), Figure 59. Complex 245, Figure 59, containing two EA derivatives, was most 1103


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exhibit promising anticancer activities.390 The complexes did not possess any significant DNA binding ability, which would be typical of RAPTA complexes. Of the four complexes developed, only one 249 showed significant antiproliferative effects against the MCF-7, A2780, and A549 cancer cell lines, which was on par with the known anticancer drug adriamycin. Again, of the complexes developed, only 249 demonstrated significant Trx-R inhibition, indicating this as the possible reason for its antiproliferative effects. It was not clear why only 249 exhibited TrxR inhibition and antiproliferative effects, given the structural similarities between all four complexes (247−250), Figure 60. While the compound was not clearly identified as being multitarget, it is interesting that RAPTA compounds do not have the same DNA-binding propensity as other ruthenium(II) anticancer complexes, where it is considered their main target. Instead it appears they work on different cellular targets entirely, such as Trx-R as demonstrated by this work.390 Tabrizi and Chiniforoshan likewise sought to generate ruthenium(II)-arene complexes with the capacity to target TrxR enzymes. They synthesized four ruthenium(II)-arene naphthoquinone complexes [incorporating lapachol (251), plumbagin (252), lawsone (253) and juglone (254)], Figure 60, on the premise that naphthoquinones, both naturally occurring and synthetic analogues, have been extensively studied due to their numerous pharmacological applications.391 Among these, they have been shown to possess anticancer properties.392 The well-known anticancer drug, Doxorubicin, for example, contains a naphthoquinone core.392 The cytotoxic effects of some naphthoquinones, including plubmagin, have been shown to be mediated via ROS generation, ultimately leading to apoptosis via the extrinsic and intrinsic mitochondrial pathways.393,394 Some drugs, including Doxorubicin, which incorporate a naphthoquinone core, have also been shown to inhibit topoisomerase II, via intercalation, and this has also been proposed as another possible mode of action of naphthoquinones.395,396 The cytotoxicity of 251−254, Figure 60, was tested against a range of tumor cell lines (A375, HepG2, MCF7, LoVo, A2780, and HCT8) and followed the general trend of 251 > 252 > 253 > 254 [with the lapachol analogue (251) being the most potent and the juglone analogue (254) being the least potent]. The complexes were also shown to be active under hypoxic conditions. The free ligands showed negligible cytotoxicity across all cell lines. Compound 251 was found to be slightly more potent than cisplatin against all cell lines with the exception of A375 cells. The complexes were also shown to induce cell death via an apoptotic pathway. All four complexes exhibited chemoselectivity properties with all having lower toxicities when compared to cisplatin against noncancerous HEK293 fibroblasts. When tested for their TrxR inhibition, all four complexes were found to effectively inhibit TrxR in the nanomolar range, with compounds 251 and 252 being particularly potent, more so than compounds 253 and 254.391 It would appear that substituting the N,N-donor ligands in 247−250 with O,O-donor ligands in 251−254 may well be impacting on the stability of the complexes and their capacity to induce TrxR inhibition. Hartinger, Kandioller, et al. also exploited lapachol as an O,O-bidentate ligand for complexation to a ruthenium(II)arene core. They generated an analogue of 251 whereby they replaced the PTA ligand with a chlorido ligand. They also included osmium(II)- and rhodium(II)-derivatives in their

study for comparative purposes. The resulting complex was more cytotoxic compared to the osmium and rhodium analogues, exhibiting low micromolar in vitro cytotoxity against a range of tumor cell lines (CH1, SW480, A549, HCT-116, and HL60). It was shown to be activated by fast hydrolysis to the corresponding aqua species under aqueous conditions. A preliminary mechanistic study revealed that the complex led to ROS-induced apoptosis and cell cycle arrest, although its capacity to inhibit TrxR was not investigated.397 In a follow-up study, Hartinger et al. sought to investigate the influence of the substituent in position 3 of lapachol and the impact of O,O- versus N,O-coordination on its druglike properties. They developed a small library of ruthenium(II)arene naphthoquinone complexes in which the naphthoquinone was based on a lawsone core, (255), Figure 60. Their respective oximes were generated to investigate the influence of the first coordination sphere on the anticancer activity of the resulting complexes.396 Interestingly, complexation of the oxime ligands to the ruthenium(II) precursor resulted in the formation of a nitroso-naphthalene ligand coordinated to the ruthenium(II) center (256), Figure 60, rather than the expected oximes. They mostly focused on the reduction and stability of their complexes while also assessing their cytotoxicity against SW480 and CH1/PA-1 cells (IC50 values ranging from 12− 78 μM). While they did not perform any studies on the TrxR inhibitory activity or dual-activity nature of their complexes, the study did reveal that the complexes were not reduced in the presence of the two-electron reductants, glutathione and ascorbic acid. They, instead, underwent reactions with these reducing agents, with decomposition occurring when reacted with glutathione and the formation of a unique product when reacted with ascorbic acid. This study showcases how ruthenium(II)-arene complexes, such as these, may be susceptible to reactions, other than reduction, with reducing agents found within the reducing environment of many tumor cells.396 4.1.4. Poly(ADP-ribose) Polymerases. Poly(ADP-ribose) polymerases (PARPs) are a family of nuclear proteins which play an instrumental role in genomic stability.398,399 The PARP-1 isoform, which is one of the most abundant members of this family, plays a particularly important role in DNA replication, transcriptional regulation, and DNA damage repair. PARP-1 catalytic activity is low under physiological conditions. However, upon DNA damage, it is upregulated, leading to the recruitment of DNA-damage response proteins to the sites of DNA damage. These response proteins trigger the base excision repair (BER) pathway. Of particular note here is the fact that PARP-1 binds to the DNA lesions induced by platinum anticancer drugs.400 The small molecule PARP inhibitor Olaparib is already in clinical use for the treatment of ovarian cancer399,401 and is in advanced stages of clinical trials for the treatment of breast cancer.402 Given the promising anticancer activity associated with ruthenium complexes, Zhu et al. sought to develop ruthenium complexes bearing a PARP inhibitor ligand.403 Their rationale was based on the fact that any DNA damage induced by the ruthenium complex would not be repaired, thus potentially increasing efficacy and overcoming resistance. They synthesized three half sandwich (η6-arene)ruthenium(II) complexes incorporating either nicotinamide (257), Figure 61, quinazolin-4(3H)-one (258), Figure 61, or 3-aza-5[H]-phenanthridin-6-one (259), Figure 61 (first, second, and third generation PARP inhibitors, respectively), and compared their cytotoxicity, DNA binding, 1104


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metallodrugs capable of inhibiting these enzymes, thus restoring drug efficacy. Furthermore, there are a number of P450s that are overexpressed in some tumor cells where they have been shown to play a key role in cancer initiation, progression, and drug resistance.405 Inhibition of P450s is therefore considered to be an attractive drug target.405 A major consideration, though, is that these enzymes are widely expressed throughout the body. They also play crucial roles in numerous biosynthetic pathways. Inhibitors tend not to be isoform selective. Therefore, long-term systemic inhibition of these enzymes can result in unwanted drug interactions and may also lead to altered hormone levels. To overcome these challenges, a possible strategy is to fine-tune the properties of the inhibitors such that they can be activated to selectively inhibit some desired P450 isoforms in a regulated way. This is what Glazer et al. set out to achieve. They hypothesized that if they could inhibit P450s while delivering a highly cytotoxic DNA damaging agent to tumor cells, they may be able to sensitize the cells to the cytotoxic payload. Furthermore, they could potentially avoid deactivation of their cytotoxic agent by hepatic P450s.406 They developed three photoactive ruthenium(II)-bpy complexes (260−262), Figure 62, incorporating the P450 inhibitors metyrapone (260), etomidate (261), and a third novel P450 inhibitor (262) as ligands. These complexes were designed as prodrugs which would release the P450 inhibitor ligands and a DNA-damaging ruthenium(II)-bpy species only upon activation by light. All three complexes were shown to induce potent DNA damage and cytochrome P450 inhibition, following light activation at 425 nm. Compound 262, for example, exhibited a 136-fold difference in protein inhibition following photoinduction as compared to in the dark. Of note is the fact that the ruthenium center was able to induce DNA damage even in the presence of this protein, suggesting that DNA was the preferred target. The complexes were not tested for their cytotoxicity against cancer cell lines. This proof-of-concept study does show a clear ability for complexes, such as these, to damage more than one target under selective photoactivated conditions, although their

Figure 61. Chemical structures of (η6-arene)ruthenium(II) complexes incorporating either nicotinamide (257), quinazolin-4(3H)one (258), or 3-aza-5[H]-phenanthridin-6-one (259) (first, second, and third generation PARP inhibitors, respectively)

and PARP inhibition capabilities against RAPTA-C (225), Figure 56, a ruthenium(II)-arene complex which has shown significant antitumor and antimetastatic potential both in in vitro and in vivo studies.404 The complexes showed varying degrees of cytotoxicity against A549, A2780, HCT116, and Hcc1937 cells (with IC50 values ranging from ∼38−500 μM), while the free PARP inhibitor ligands were not cytotoxic. The complexes were nontoxic toward normal human lung MRC-5 cells. Complexes 258 and 259 were however more potent than RAPTA-C with complex 259 being the most potent in the series. When tested for PARP inhibition, complex 259, the most cytotoxic, was also the most potent PARP-1 inhibitor and was a slighly more potent inhibitor compared to the ligand alone. While PARP inhibition is not normally associated with cisplatin, its PARP inhibition was found to be on par with 258 (with IC50 values of ∼12 vs 13 μM, respectively) which was not accounted for in this study. The team also presented evidence that complex 259 could bind DNA, but to a much lesser extent than RAPTA-C (225), Figure 56, most probably due to steric hindrance as a result of the presence of the PARP inhibitor ligand.403 4.1.5. Cytochrome P450. Cytochrome P450s are a group of enzymes which play a key role in drug metabolism. Hepatic P450s, in particular, are responsible for the degradation of xenobiotics including anticancer agents. One strategy to reduce or prevent these unwanted degradation reactions is to generate

Figure 62. Chemical structures of ruthenium(II) bipyridyl complexes incorporating cytochrome P450 inhibitors (260−262). 1105


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Figure 63. Chemical structures of organoruthenium(II) complexes incorprorating topoisomerase inhibitor ligands (263−267).

anticancer agents.408 Flavonoids are complexes which have been shown to inhibit topoisomerase IIα, while demonstrating anticancer activity. In fact members from this class have undergone clinical trials as anticancer agents.409 Hartinger et al. synthesized a family of ruthenium(II)-arene complexes with derivatized flavonols as ligands. Their initial study with four complexes (general structure shown as 263), Figure 63, showed that chelation of the 3hydroxy-4-keto structural motif of 3-hydroxyflavones to the ruthenium center did not show a significant change or improvement in cytotoxicity against CH1, SW48, and A549 carcinoma cells when compared to the free ligands.410 The complexes and their flavonoid ligands were all fluorescent with

impact on biological pathways, mediated by cytochrome P450s, would need to be further explored.406 The reader is also directed to a recent review by Dutkiewicz and Mikstacka in which they summarize the current state of knowledge regarding computational approaches related to ligand-enzyme interactions for cytochrome P450s with a particular focus on structure-based drug design for cytochrome P450 family 1 inhibitors.407 This family plays an important role in the metabolism of drugs and chemical procarcinogens. 4.1.6. Topoisomerases. Topoisomerase IIα is an enzyme which is overexpressed in many human cancers, and a number of inhibitors of this enzyme, including doxorubicin, etopside, and mitoxantrone, are now routinely used in the clinic as 1106


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an emission maximum at ca. 520 nm. Using fluorescence confocal laser scanning microscopy, the team was able to establish that the complexes localized in the endoplasmic reticulum (ER). They concluded that the ER was most likely the preferred target of these complexes and that it acted as a reservoir for these agents. The complexes were shown to readily bind DNA and also inhibit topoisomerase IIα. The study revealed that the nature of the substituent on the phenyl ring of the flavone ligand had a significant effect on the cytotoxicity of the complexes, with a chloro substituent affording the most potent cytotoxicity and topoisomerase IIα inhibition.410 A later study by the same group reported ten similar complexes in which the phenyl substituent on the hydroxyflavone ligand had been further modified (general structure shown as 264, Figure 63).409 The complexes, in this study, were found to be considerably more water-soluble (10-fold) compared to the parent ligands. The complexes also showed low micromolar cytotoxicities against CH1, SW480, A549, 5637, LCLC103H, and DAN-G cell lines. Their IC50 values were, in general, considerably lower than other reported ruthenium(II)-arene complexes reported at that time, suggesting that the flavonoid ligands were key determinants in the cytotoxicity profile of these complexes. The complexes were found to bind DNA, although they had only a minor impact on the cell cycle. They did however possess cyclin-dependent kinase (CDK2) and topoisomerase IIα inhibition activity in vitro. Following a structure−activity relationship approach, the team was able to ascertain a direct correlation between the cytotoxicity and topoisomerase inhibition activity of the complexes with the substitution pattern of the coordinated flavonol ligand. Those complexes containing para- and metasubstituted ligands demonstrated highest cytotoxicity and topoisomerase inhibition activities, while those incorporating ortho or nonsubstituted ligands had lower activities. In constrast, while most of the complexes exhibited CDK2 inhibition activity, similar to the well-known CDK2 inhibitor roscovitine, there was no obvious correlation between the CDK2 inhibition pattern observed for these complexes and their corresponding in vitro cytotoxicities.409 The team went on to develop a series of 3-hydroxyflavoneand 3-hydroxyquinolinone-derived ruthenium(II)-arene complexes (general structure shown as 265, Figure 63), with a view to investigating the impact not only of lipophilicity of the arene ligand and different halogenido ligands on overall complex stability but also of these and complex hydrolysis rates on overall cytotoxicities. The quinolinone complexes were included in this study to determine the impact of the heteroatom (nitrogen in this case) in the bioactive ligand backbone on the overall cytotoxicity of the resulting complexes.411 The behavior of these complexes in aqueous solution was investigated with stability constants and pKa values for ligands and corresponding complexes reported. A total of thirteen complexes were prepared (265a−265m), Figure 63. When tested against CH1, SW480, and A549 cell lines, all complexes exhibited potent cytotoxicities in the low micromolar range. The complexes bearing different arene ligands [toluene (265h and 265i) versus cym (265a and 265c) versus biphenyl (265j and 265k)], and hence different lipophilic properties, exhibited similar cytotoxicities so the influence of lipophilicity of the arene ligands on cytotoxicity was not deemed to be of significance. This was in contrast to other

studies, including those involving ruthenium(II)-arene-ethylenediamine complexes, which demonstated a strong correlation between the lipophilicity of their arene ligands and cytotoxicities.412 The team concluded that the contribution of the flavonoid ligands toward the overall lipophilicity of the complexes was greater in their systems and that any influence related to the slight modification of the arene ligands was only marginal. Furthermore, changing the ligands from 3-hydroxyflavones to 3-hydroxyquinolinones and changing the halide ligands from chlorido to bromido to iodo appeared to have little or no influence on cytotoxicities. The quinolinone complexes 265l and 265m, for example, exhibited cytotoxicities in the same range as 265a. While all complexes were shown to interact with the DNA model compound 5′-GMP, their dual-targeting nature was however not investigated.411 Most ruthenium(II)-arene complexes reported to date contain cym or related structures. Sadler et al. have shown that incorporation of an expanded π-system can lead to complexes with higher cytotoxicities most likely due to their enhanced ability to intercalate between DNA base pairs. The biological properties of RAPTA complexes appear not to depend on the nature of the arene ligand. More recently, it has been shown that functionalization of the arene ligand coordinated to the ruthenium(II) center can lead to complexes with additional features.384,388,413−416 To further explore the pharmacological properties of the complexes previously described (265), Figure 63, and to enhance their aqueous solubility profiles, Hartinger et al. focused on their 8-oxyquinolinato complexes and substituted the apolar η6-p-cym with an an L-phenylalanine (Phe)-derived arene ligand and varied the halogen substituents on the 8oxyquinolinato ligand (general structure shown as 266, Figure 63).417 Interestingly, Phe, in its own right, is a stereospecific inhibitor of human intestinal alkaline phosphatase, another enzyme which is known to be overexpressed in many tumors.418 The incorporation of the Phe ligand was found to have little impact on the overall lipophilicity of their complexes. In fact, they were less lipophilic when compared to their cym analogues. Substituting the cym ligand with Phe had little impact also on the in vitro antiproliferative properties of the resulting complexes. Of note, the most potent complex was 266a, which contains the unsubstituted 8-oxyquinolinato ligand, the same ligand found in the clinically tested gallium complex KP46.419 Building on this aforementioned study, the same team went on to study the impact of substituting η6-p-cym with the Phederived arene in 3-hydroxyflavonol organoruthenium(II) complexes on their biological activity and chemical properties and compared their findings to analogous cym derivatives.417 The compounds (general structure shown as 267, Figure 63) were cytotoxic against HCT116, NCI-H460, SiHa, and SW480 cells, with IC50 values as low as 1 μM in NCI-H460 nonsmall cell lung cancer cells. Despite a representative Phe-derived complex accumulating better in tumor cells (and this was independent of concentration used and incubation times), the IC50 values of the Phe-derived compounds did not follow the same trends as those observed for their cym analogues. The complexes did bind DNA, with similar binding to that of cisplatin, albeit to a lesser extent.420 Through many structure−activity relationship studies, as outlined above, Hartinger et al. have undoubtedly established that the major contributory factor accounting for the antiproliferative activity of their organoruthenium complexes 1107


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remain intact in order to retain its CDK inhibition activity.426 Incorporation of the six-membered flat ring in indolo[3,2c]quinolines as compared to the indolo[3,2-d]benzazepine derivatives, which contained the seven-membered nonplanar azepine ring, resulted in a pronounced enhancement in cytotoxicity against CH1, SW480, and A549 tumor cell lines. This increase in antiproliferative activity was attributed to the greater capacity of the flat indolo[3,2-c]-quinoline backbone to intercalate into DNA. Binding of the indolo[3,2-c]-quinolines to the ruthenium(II)-arene framework resulted in a slight increase in antiproliferative activity. In contrast, ruthenium(II)arene-indolobenzazepine complexes resulted in a 6.8−16-fold increase in antiproliferative activity depending on the cell line. The difference in the coordination sphere around the metal center may account for differences in the kinetic lability of the ruthenium(II)-arene-indolo[3,2-c]quinolines, which may result in the difference observed in terms of the cytotoxicity of these complexes. While CDK inhibition studies were conducted on the osmium analogues, no data was presented related to the ruthenium complexes.425 While cytotoxic, the complexes exhibited low stability in both organic and aqueous media, resulting in the rapid dissociation of the ligands from the metal-arene scaffold.425 In order to increase the hydrolytic stability of the complexes, the team went on to develop a new family of ruthenium(II)arene (and osmium(II)-arene) complexes with 2-substituted indoloquinolines.427 They employed iminopyridines as chelating moieties in place of ethylenediamine ones. The resulting complexes (general structure shown as 272, Figure 65) demonstrated potent cytotoxicity against CH1, SW480, and A549 tumor cells, although the team was unable to define any clear-cut structure−activity relationship due to overlapping ranges of variation. The CDK inhibition activity of the complexes was not investigated.427 3-(1H-Benzimidazol-2-yl)-1H-pyrazolo[3,4-b]pyridines have also been shown to possess potent CDK1 inhibition activity as well as antiproliferative activity.428 Building on the promising results related to the ruthenium(II)-arene-indolobenzazepine complexes previously described, Keppler et al. sought to exploit the ruthenium(II)-arene scaffold again, but this time incorporating these pyridines with CDK1 inhibition properties (general structure shown as 273), Figure 65. Complexation of these ligands to the organoruthenium(II) center, while enhancing the solubility for the pyridines in biological medium, lowered their cytotoxicity against CH1, SW480, and A549 cell lines. CDK inhibition studies also revealed that the ligands had lost their CDK inhibition ability upon complexation to the ruthenium ion.429 Returning to the indolobenzazepine series, the same team went on to develop two further complexes but this time incorporating two different indolobenzazepines derivatives (274−275), Figure 65.430 Complexation of these ligands to the ruthenium ion had a beneficial effect on their solubility as well as increasing their cytotoxic potency. The complexes exhibited low micromolar cytotoxicity against A549, SW480, CH1, LNCaP, N87, and T47D cell lines and were found to induce tumor cell death via an apoptotic pathway. While CDK inhibition activity was observed for both complexes in cell free media, there did not appear to be any correlation between cytotoxicity and CDK inhibition activity. The team concluded that mechanisms, other than CDK inhibition, were more likely the reasons for the observed antiproliferative activities observed.430

was driven not by the nature of the arene ligand nor the halogen ligands but rather by the cytotoxicities of the coordinated flavonol ligands. They also established that the complexes were more potent inhibitors of topoisomerase IIα compared to the free flavonols which was also dependent on the phenyl ring substituents. Chalcones are a class of naturally occurring plant metabolite compounds which have demonstrated a wide variety of biological activities including antifungal, antiviral, antibacterial, anti-inflammatory, and anticancer properties. In particular, their ability to inhibit topoisomerase has been linked to their cytotoxicity. Radulovic et al. developed a series ruthenium(II)DMSO-chalcone complexes. In a previous study these complexes were shown to possess potent DNA binding, DNA cleavage, and topoisomerase inhibitory activity.421 Two of these were selected for further testing, 268 and 269, Figure 64.422 Against HeLa, FemX, MDAMB231, K562, and A549

Figure 64. Chemical structure of ruthenium(II)-DMSO-chalcone complexes.

cell lines, they demonstrated moderate IC50 values, ranging from 27−76 μM. Complex 269, which demonstrated more potent DNA cleavage than complex 268, was, or nearly was, twice as potent as complex 268 in all cases, providing support that DNA binding ability is an important factor in the cytotoxicity of these complexes. Along with their previously reported potent topoisomerase-II inhibitory ability, these complexes demonstrate the dual-targeting properties of a ruthenium complex with natural products as ligands.422 4.1.7. Cyclic-Dependent Kinases. Protein kinases play key roles in cellular processes such as metabolism, proliferation, and apoptosis. Kinase inhibitors seek to provide a therapeutic option by interupting kinase-associated pathways in cancers. Inspired by the success of the cyclin-dependent kinase (CDK) inhibitor flavopiridol423 which, at the time, had advanced to phase II clinical trials as a treatment for chronic lymphocytic leukemia, Keppler et al. set out to build on their previous work in which they had developed the first examples of ruthenium-arene-paullone derivatives.359,360 Paullones are known to possess potent CDK inhibition properties.424 In one study, the team chose indolo[3,2-d]benzazepine (paullone) derivatives as ligands for complexation to their ruthenium(II)arene framework (general structure shown in 271, Figure 65).425 They included in their study the structurally related indolo[3,2-c]quinolines to generate an alternative class of compounds (general structure shown as 270, Figure 65) given that quinolones form the basis of many biologically active compounds, and the indole backbone is a basic structural feature in anticancer agents including vinblastine.425 Previous work had shown that paullones themselves are not suitable for complexation to metal ions. Rather peripherary metal ion binding moieties embedded within the paullone scaffold are necessary in order to facilitate such binding. Previous studies had also indicated that the paullone lactam unit needed to 1108


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Figure 65. General chemical structures of ruthenium complexes designed to inhibit cyclin-dependent kinases (270−275).

Figure 66. Chemical structures of organoruthenium(II)-indirubin conjugates (276).

myelocytic leukemia. Of the mixture of medicines found in Danggui Longhui Wan, Indigo naturalis was suggested to be responsible for the antileukemic properties associated with this

Another source of inspiration for the Keppler group came from the properties of a traditional Chinese herbal remedy, Danggui Longhui Wan, which is used in China to treat chronic 1109


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concentrations for the inhibition of Cathepsin B exopeptidase and endopeptidase activities in MCF-10A neoT whole cell lysates comparable to those obtained for nitroxoline, a potent and selective Cathepsin B inhibitor.439 While the complex was not cytotoxic against MCF-10A neoT and U87MG cancer cell lines, both of which express high levels of proteolytically active Cathepsin B, it was found to significantly inhibit extracellular matrix degradation at a noncytotoxic concentration of 1.25 μM.438 The degradation of the ECM is considered one of the key processes associated with tumor invasion and metastasis.438 A follow-up structure activity relationship study was undertaken to elucidate the effect of adding various substituents on the clioquinol ligand framework and how these changes impacted on the physicochemical and biological properties of the resulting complexes (general structure shown as 278), Figure 67.440 The complexes were assessed for their anticancer as well as antibacterial properties to ascertain the impact of the halogen substitution on positions 5 and 7 of the quinoline ring. Human lung and bone cancer cells and E. coli, B. cereus, and S. aureus bacterial strains were used in this study. The cytotoxicity results indicated that the substitution pattern on the ligand itself does indeed result in significant changes in anticancer activity, with the bromo substituents in positions 5 and 7 of the quinolone ring appearing to play an important role in the antiproliferative activity of the corresponding ruthenium complexes. In contrast, the presence of the methyl group in position 2 appeared to diminish the anticancer activity. Taking the complexes which contained one and two bromo substituents, respectively, as lead candidates, the team went on to demonstrate that the bromo-substituted ligands impaired cell viability in a concentration-dependent manner with more pronounced cytotoxicity against the human osteosarcoma cells compared to the lung adenocarcinoma cells. The disubstituted derivative was more cytotoxic than the monosubstituted one in both cell lines. Moreover, both complexes exhibited antibacterial activity against all strains with the monosubstituted derivative being more potent than the disubstituted one. 4.1.9. Carbonic Anhydrases. Hypoxia and acidosis are common features associated with many tumors. These features result in these tumors having a different metabolism to normal cells. Metabolism leads to the production of many products, including protons and bicarbonate which are generated by the catalytic activity of zinc-containing carbonic anhydrase (CA) enzymes. Two isoforms of CA, namely CA IX and CA XII, are associated with hypoxia tumors.441 Inhibition of these tumorassociated CA isoforms is therefore yet another attractive cancer drug target, and, already, a number of drugs targeting these isoforms have been developed as either antiproliferative or antimetastatic agents.441,442 Classical CA inhibitors contain an aromatic or heteroaromatic sulfonamide motif (ArSO2NH2) which can bind directly to the CA active site zinc ion, thus inhibiting its catalytic action.443 Several CA inhibitors containing this moiety are in clinical use, such as acetazolamide, methazolamide, and ethoxazolamide.444 To the best of our knowledge, Poulsen, Supuran, et al. were the first to report benzenesulfonamides bearing triazole units tethered to an organoruthenium framework (279 and 280), Figure 68.443 They also developed the ferrocenyl analogues, inspired by the encouraging results at that time of ferrocifens (incorporating a ferrocenyl unit linked to the breast cancer drug tamoxifen) and ferroquine (containing a ferrocenyl unit linked to the antimalarial agent chloroquine). The rutheno-

remedy and, more specifically, the red-colored isomer of indigo, indirubin. This latter derivative was found to inhibit CDKs, most likely by binding to the ATP binding site of the protein through the lactam moiety and the N1′-H group via three hydrogen bonds.431 Keppler et al. sought to exploit the pharmacophore of indirubin, 276, Figure 66, and combined into one ligand the benzimidazolylquinolinone and indolylquinoxalinone structures to generate 3-(1H-benzimidazol-2-yl)1H-quinoxalin-2-one, which is well-suited for complexation to metal ions via the diimine moiety.432 A series of ruthenium(II) (and osmium(II)) arene complexes were generated, and their cytotoxicity assessed against CH1, SW480, and A549 cancer cell lines. Differences in structural features did not appear to impact significantly on the cytotoxicity of the complexes. The osmium complexes were generally more cytotoxic compared to their ruthenium analogues, which the team attributed to the higher inertness associated with osmium over ruthenium. They suggested that either coordination was not playing a significant role in the mechanism of action or the higher reactivity associated with the ruthenium derivatives could be leading to off target deactivating side reactions. Despite the cytotoxicity of the complexes being in the low micromolar range, the effects of the ligands on cell cycle appeared to be diminished when complexed to the metal ions, which would suggest that effects beyond the cell cycle were contributing to the cytotoxicities observed. The impact on CDK inhibition was however not reported. 4.1.8. Peptidases. Cathepsin B is a ubiquitously expressed lysosomal cysteine peptidase enzyme. It represents yet another attractive cancer drug target due to its involvement in numerous pathologies and oncogenic processes.433 Its upregulation, for example, is associated with tumor growth, signaling, and the degradation of the extracellular matrix, the latter of which promotes tumor metastasis and invasion of other cells. In an effort “to teach an old drug new tricks”, Turel, Gobec, et al. sought to combine into one drug entity the apoptotic agent, clioquinol, with an organoruthenium moiety.434 The complex, 277, Figure 67, was found to possess antiproliferative

Figure 67. Chemical structure of the organoruthenium(II)-clioquinol complex and analogues (general structure shown as 278).

activity in leukemia cell lines, which was found to be mediated via caspase activation. Mechanistic studies revealed that the complex did intercalate DNA, exhibited proteasome-independent inhibition of the NFκB signaling pathway and had no effects on cell-cycle distribution, suggesting a mechanism of action different to the clioquinol ligand.434 Given that ruthenium-quinolone complexes had already shown promising Cathespsin inhibition activity,389,435−437 Kos, Turel, et al. went on to explore whether their organoruthenium-clioquinol complex could likewise inhibit Cathepsin B.438 The complex did indeed inhibit Cathepsin B with half-maximal inhibitory 1110


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Figure 68. Chemical structure of the organoruthenium(II) complexes containing carbonic anhydrase inhibitors (279−283).

correlation was found between the antiproliferative activity of their complexes and both the nature of the arene ligand as well as the anionic counterion which informed a future study as outlined in the next paragraph.445 In this study, the two teams came together to develop a series of eight ionic ruthenium(II)-pentamethylcyclopentadienyl benzenesulfonamide sandwich complexes (general structure shown as 281 and 282), Figure 68. The CA inhibition activity of this series was then screened against physiologically dominant human CA I and II, mitochondrial isozymes VA and VB, and the cancer-associated CA IX isozyme. Their cytotoxic properties against MCF7, MDA-MB231, and MM96L tumor cells and the normal human neonatal foreskin fibroblasts NFF, were also assessed.444 In this study, the organoruthenium framework was linked to the CA inhibitors via the benzene ring of the sulphonamide pharmacophore. Unlike their earlier study described above in which the pharmacophore was linked via triazole units, the presence of the organoruthenium moiety resulted in a decrease in binding affinity toward the isozymes tested, most likely due to its steric bulk. Of the complexes developed, complex 282

cenyl-benzenesulfonamide derivatives generally exhibited superior CA inhibition activity over the ferrocenyl analogues across the three CA isozymes (human CA I, II, and IX) investigated (with the exception only of compound 280 at hCA IX). The CA inhibition potency and isozyme selectivity were also found to be dependent on the orientation of the metallocene unit with the complexes containing the 1,4triazole regioisomer (279), typically enhancing hCA IX selectivity over those containing the 1,5-triazole regioisomer (280). The ruthenocenyl complexes were also found to be more potent CA inhibitors compared to clinically used CA inhibitors. The anticancer properties of the complexes were, however, not reported.443 In the meantime, the cytotoxic properties of a series of ionic ruthenium-arene-cyclopendadienyl complexes were reported by Williams, Parsons, et al. These complexes exhibited potent antiproliferative activity against a range of tumorigenic cell lines while retaining moderate-to-good selectivity toward the normal human normal human neonatal foreskin fibroblasts (NFF). While these complexes did not contain any CA inhibitor ligands, this work is highlighted here because a 1111


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Figure 69. Chemical structure of nonorganoruthenium(II) derivatives incorporating CA inhibitors.

complexes developed (general structure shown as 285), Figure 68, 285d exhibited highest CA binding affinity. To further probe for host−guest interactions, the team were able to cocrystallize the human CA II protein with 285d. The complex interacted with this protein in a similar fashion to previously reported sulphonamide inhibitors. The sulphonamide group was shown to bind the zinc ion at the active site; the aryl spacer was found to fit into the hydrophobic funnel-shaped cavity, and the ruthenium-arene motif served as an achor at the entrance to the cavity. The ruthenium coordination sphere appears to stay intact despite the presence of the labile chlorido ligand.447 It is precisely this type of structural insight that is required in order to more fully inform the future development of these types of artificial metal-based CA inhibitor conjugates. Moving away from the ruthenocene framework, Tsukahara, Takashima, et al. developed a ruthenium(II)-bipy complex tethered to a benzenesulfonamide moiety, (286), Figure 69.448 The team investigated the CA inhibition activity of this complex and found it to have comparable activity relative to commercially available CA inhibitors. While the complex was not designed to be “multitargeted”, extensive photophysical and kinetic studies associated with this complex were described. These included a detailed discussion on the electron transfer mechanisms associated with the regulation of the catalytic activity of CA.448 Patra et al. generated two additional nonorganoruthenium(II) derivatives designed not only to bind DNA but also to target CAs (287 and 288), Figure 69. In these examples, the ruthenium(II) center contained two different planar N-donor ring systems as well as the artificial sweetener saccharin (osulfobenzimide), which is known to act as a CA IX inhibitor.449 The saccharin ligands were found to dissociate following light activation using UV-A light at 365 nm. As anticipated, the complexes were able to bind DNA with binding constants on the order of 105 M−1. Photoinduced DNA cleavage at

exhibited highest levels of CA inhibition which was attributed to the greater distance separating the ruthenium-cyclopentadienyl group and the sulphonamide. The complexes were, however, found to be cytotoxic in the low micromolar concentrations in the cancer cell lines tested but to a lesser extent than cisplatin. They also demonstrated lower levels of toxicity toward the NFF cells.444 The team went on to generate twenty structurally diverse metallocene complexes as potential CA inhibitors; of these two were ruthenocene derivatives (283 and 284), Figure 68, and the remainder were ferrocene derivatives.446 They compared their activities to the ruthenocenes (279 and 280), Figure 68, and ferrocenes that they had previously reported.443 The series exhibited moderate-to-good CA inhibition properties in vitro, and several displayed selectivity toward cancer-associated CAs relative to off-target CAs. Compounds 279 and 280 were more potent CA inhibitors relative to their ferrocene analogues against all isozymes tested. Of the meta-substituted derivatives, 283 and 284 exhibited similar inhibition activity compared to the ferrocene analogue of 280 and, in all cases, was more potent relative to the ferrocene analogue of 279. Although only a small number of ruthenocene complexes were investigated in this study, the findings do suggest that the ruthenium center can offer advantages over the iron center in these complexes in terms of CA inhibition activities.446 In the meantime, Ward et al. provided key structural insights into the interactions between piano stool ruthenium(II)-arene complexes containing sulphonamide-based CA inhibitors and CA inhibition.447 They selected human CA II as a model enzyme in which to host their piano stool organoruthenium(II)-CA inhibitor “guests” for a number of reasons. The fact that this enzyme is overexpressed in many tumors makes it an attractive drug target as stated earlier. It also contains a large binding cavity (15 A° deep and 15 A° diameter at its mouth), which can accommodate metal complexes. Furthermore, the protein itself is monomeric and stable. Of the piano stool 1112


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Figure 70. Chemical structures of organoruthenium complexes incorporating acetazolamide

Figure 71. Chemical structures of ruthenium(III) and organoruthenium(II) complexes investigated for their AKR inhibition activity (292−298).

micromolar complex concentrations was also observed for both complexes. Mechanistic studies, in the presence of O2, revealed that DNA cleavage involved ROS. Photoinduced DNA cleavage was also found to occur, under anaerobic conditions, which was attributed to the formation of a ruthenium-DNA complex following light activated loss of the saccharin ligands. The complexes were, however, not assessed for their cytotoxic properties nor their CA inhibition activities.449 This preliminary study certainly paves the way for complexes of this type to be further explored as potential CA targeting and DNA cleavage agents. We mentioned previously that acetazolamide is a known sulphonamide-based CA inhibitor. It has been shown, as a single agent, to reduce tumor growth and, when given in combination with other therapeutics, can retard tumor development.450 There are only a small number of ruthenium complexes reported that incorporate this CA inhibitor.

Ruthenium(II)-bipy and ruthenium-phen complexes containing acetazolamide were previously shown to possess in vitro antibacterial and in vivo anticancer properties.451 Marchetti et al. generated some novel (η6-p-cym)ruthenium and RAPTA analogues incorporating acetazolamide (289−292), Figure 70.452 With dependence on the reaction conditions, the complexes contained acetazolamide either as a neutral Nmonodentate ligand (290), or a monoanionic N,N-bidentate ligand (290), or as a dianionic N,N-bidentate ligand (291). The cytotoxicity of 290 and 291, both of which had demonstrated reasonable stability under aqueous conditions and both strucures of which had been characterized by X-ray crystallography, were assessed against cisplatin-sensitive and cisplatin-resistant A2780 ovarian tumor cell lines but were not cytotoxic. They were, likewise, not toxic toward nontumorogenic HEK3 cells. Their CA inhibition activities were not assessed.452 1113


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Figure 72. Chemical structures of organoruthenium(II)-letrozole complexes (299−302).

4.1.10. Aldo-Keto Reductases. The 1C family of aldoketo reductase (AKR) enzymes play crucial roles in the biosynthesis of hormones, neurosteroids, and prostaglandins. Overexpression of these enzymes is associated with the progression of hormone-dependent cancers, including breast, prostate, and endometrium cancers. Expression of AKR1Cs, and especially of AKR 1C1 and AKR 1C3, has been linked to resistance to a number of different anticancer drugs, including cisplatin and carboplatin.453,454 Rizner, Turel, et al. were the first to explore the impact of ruthenium complexes on the inhibition of this class of enzymes. They investigated the AKR inhibition activity of ten compounds: two ruthenium(III) pyridyl complexes (292 and 293), Figure 71, three organoruthenium(II)-pyridyl complexes (294−296), Figure 71, the ruthenium(III) and ruthenium(II) precursors without the N,N-pyridyl ligands, and the three parent N,N-ligands, the latter possessing water-solubilizing methoxycarbonyl groups.455 Of these, the two organoruthenium(II) complexes (292 and 293) and both ruthenium precursors demonstrated the ability to inhibit AKR, with 294 inhibiting only AKR1C3 and 295 inhibiting AKR 1C1, AKR 1C2, and AKR 1C3, which are potential drug targets for breast cancer.455 Retaining the same ruthenium(III) and organoruthenium(II) frameworks, the same group went on to develop four new complexes but, this time, completing the coordination spheres with the zinc ionophores pyrithione and its oxygen-containing analogue 2-hydroxypyridine N-oxide with the capacity to inhibit ADKIC enzymes (general structures shown 297 and 298 respectively), Figure 71.456 The compounds were assessed for their inhibition activity against three enzymes of the ADK subfamily IC, chosen because of their involvement in the development or progression of a number of cancers, including breast cancer. Employing a kinetic study and complimenting this with docking simulations, the team revealed differences in the mechanism of actions of the complexes. Both organoruthenium(II) complexes and the ruthenium(III) complex with the N,O-bidentate ligand showed combined reversible and irreversible enzyme inhibition by binding to the active and peripheral sites of the enzymes, respectively. The O,S-ligand and its ruthenium(III) complex showed irreversible inhibition by binding to the peripheral site only. Of the six

agents evaluated, the organoruthenium(II) complexes (general structure shown as 298, Figure 71) were the most potent AKR1C inhibitors. These latter two complexes also exhibited a relatively high degree of selectivity toward AKR 1C1 (AKR 1C1 > AKR 1C3 ≫ AKR 1C2). This is an interesting result given that there are only seven amino acid differences between AKR 1C1 and AKR 1C2 enzymes. The team went on to assess the cytotoxicity of the agents on a model cell line of hormonedependent MCF-7 breast cancer. The complexes bearing the O,S-donor ligand as well as the ligand itself were cytotoxic with the ligand and its organoruthenium(II) complex being 50-fold more cytotoxic than the corresponding ruthenium(III) complex. The O,O-ligand and complexes bearing this ligand, in contrast, were not cytotoxic. This difference in cytotoxicity was potentially attributed to the increased stability of the O,Schelating ligands over the O,O-analogues.456 4.1.11. Aromatases. Maysinger et al. developed a complex designed to combine the anticancer properties of ruthenium(II)-arene complexes and letrozole, an aromatase inhibitor, which is administered after surgery to postmenopausal women with hormonally responsive breast cancer.457 Aromatase is an enzyme responsible for the conversion of androgens to estrogens. In estrogen receptor positive (ER+) breast cancer, increased levels of the estrogen estradiol have been linked to tumor progression. Inhibition of aromatase can therefore reduce estradiol levels and may therefore play a role in preventing tumor progression. The complexes consisted of an organoruthenium(II)-arene structure with a single letrozole ligand coordinated to the ruthenium center in a monodentate fashion with the coordination sphere completed by either chlorido ligands only (300−302) or, in one case, a chlorido ligand and a triphenylphosphene ligand (299), Figure 72. Their cytotoxicities were assessed against two tumor cell lines: human breast cancer cells (MCF-7) and human glioblastoma cells (U251N). Of the complexes tested under estrogendeprived conditions, the one containing the triphenylphosphine ligand, 299, was found to be significantly more cytotoxic against MCF-7 breast cancer cells compared to its chlorido analogues, suggesting the importance of this ligand in the mode of action of these complexes. Against U251N brain cancer cells, the complex was only mildly cytotoxic. The 1114


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tyrphostin molecule (between 2−17 times lower IC50 values). Complex 308 was found to be particularly effective against multidrug-resistant and EGFR positive MCF-7/Topo cells. The toluene complex, 310, in contrast, displayed melanomaspecific activity attributed to mTOR signaling interference and independent of EGFR inhibition. The complexes were all shown to bind strongly to DNA without altering its topology, which is a marked difference to the DNA topology changes induced by platinum drugs following DNA binding.459 These initial findings present EGFR-targeting as a promising design strategy for ruthenium(II) anticancer complexes and have since been followed up with additional studies as outlined below. We have seen many examples in which the presence of a metal center can enhance the biological activity of bioactive ligands. Wang et al. present yet another example. They demonstrated that EGFR inhibition by 4-anilinoquinazolines (4-AQs), analogues of the EGFR inhibitor gefitinib, which is used clinically to treat non small cell lung cancer,460 can be enhanced when complexed to an organoruthenium(II) base.461 Their complexes incorporating 4-AQ (311−316), Figure 75, were shown to have high inhibitory activity toward EGFR while also retaining the DNA binding capacity associated with the ruthenium(II) center. Complexation to the ruthenium(II) center enhanced the ability of the 4-AQ EGFR inhibitor ligands to induce early stage apoptosis. Some of the complexes also demonstrated improved anticancer efficacy over a known cytotoxic ruthenium complex ([(η6-p-cym)Ru(EDA)Cl](PF6) and the clinically used gefitinib toward MCF-7 breast cancer cells.461 Wang, Luo, et al., in a follow up study, developed new derivatives of the EGFR inhibitor, 4-AQ. They retained the 4AQ pharmacophore but modified it in such a way so as to incorporate either chelating ethylenediamine or monodentate imidazole groups at the 6-position and, in so doing, facilitated their complexation to either runthenium(II) or ruthenium(III) centers. Five new octahedral ruthenium-DMSO complexes were generated, 317−321, Figure 75, one of which contained ruthenium in the +2 oxidation state, 317. Like gefitinib, the target of these complexes appears to be the EGFR protein. When tested for EGFR inhibition, as well as their cytotoxicity against EGFR negative and EGFR positive MCF-7 cancer cells, only complexes 318 and 319 were shown to possess significant EGFR inhibiting ability, while only complex 318 showed significant and selective cytotoxicity against the EGFR positive MCF-7 cell line and only negligible cytotoxicity toward the EGFR negative cell line. Complex 318 was also shown to induce early stage apoptosis, more actively than gefitinib. This complex therefore represents another example of a multitargeted agent capable of blocking EGFR signaling as well as having the capacity to induce early stage apoptosis cascades.462 In a follow-up study, the team generated an expanded library of organometallic ruthenium(II) complexes incorporating pcym, benzene, 2-phenylethanol, or indane as arene ligands and 4-AQ derivatives as ancillary ligands and tested their EGFR inhibition, DNA binding ability, and antiproliferative activity (322−329), Figure 75.463 They also employed molecular modeling studies to help elucidate their mode of action. Two of the fourteen complexes synthesized were chosen as representative structures to establish mode of DNA binding on calf-thymus DNA and were found to be strong, competitive minor groove DNA binders. When tested for their EGFR inhibiting abilities, ten and fourteen complexes were found to

aromatase inhibition ability of the complexes was, however, not evaluated.457 4.1.12. Ureases. An interesting study by Hartinger, Hanif, et al. led to the development of five ruthenium(II)-arene complexes incorporating thiourea derivatives, chosen because of their urease inhibition properties, (303−307), Figure 73.458

Figure 73. Chemical structures of (η6-arene)ruthenium(II) complexes incoporating thiourea derivatives, chosen because of their urease inhibition properties, (303−307).

Their cytotoxicity against tumor cells was however not evaluated. While the thiourea-derived ligands exhibited moderate urease inhibition activity, their correponding complexes were inactive. We have described numerous examples in which the addition of enzyme inhibitors to a metal ion scaffold can enhance the inhibition properties of the enzyme inhibitor itself. In contrast, this study showcases how the conjugation of enzyme inhibitor ligands to metal centers does not always lead to an enhancement in enzyme inhibition activities. 4.2. Proteins as Targets

4.2.1. Epidermal Growth Factor Receptor. As described earlier, the epidermal growth factor receptors (EGFR) are an important class of transmembrane proteins that have already been identified as promising targets for the generation of cancer cell selective platinum(II) and platinum(IV) drugs. These proteins have been shown to regulate various cancerrelated effectors such as Src Kinase, PI3 kinase, and Ras protein. They are also known to upregulate DNA repair mechanisms. Small molecule inhibitors of EGFR have also shown promise, when given in combination with metal complexes, for the treatment of various cancers.236 Inspired by these findings, Schobert et al. developed ruthenium(II)arene complexes incorporating EGFR inhibitor tyrphostin derivatives and tested them for their anticancer activity (308− 310), Figure 74.459 The complexes were found to have enhanced cytotoxicity and selectivity against human 518A2 melanoma, HL-60 leukemia, Kb-V1/Vbl cervix carcinoma, and MCF7/Topo breast carcinoma compared to the free

Figure 74. Chemical structures of (η6-arene)ruthenium(II) complexes incorporating EGFR inhibitor tyrphostin derivatives (308− 310). 1115


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Figure 75. Chemical structures of organoruthenium(II) complexes incorporating 4-anilinoquinazolines (4-AQs) (311−316; 322−334), analogues of the EGFR inhibitor gefitinib, and nonorganoruthenium(II)-4-AQ derivatives (317−321; 335−339).

be stronger EGFR inhibitors than gefitinib. It would certainly be interesting to more fully explore the precise mode of

inhibition and to identify the role of the ruthenium(II) center. The complexes were also tested for their antiproliferative 1116


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Figure 76. Chemical structures of ruthenium(II) complexes targeting efflux proteins (340−344).

The complexes demonstrated the greatest potency against the non small cell lung A549 cell line, with 332 having a comparable IC50 value to that of cisplatin and being greater than gefitinib, [(η6-p-cym)Ru(en)Cl](PF6), and the two in combination, thus verifying the synergistic potential in combining both pharmacophores into one drug molecule. The complex also induced much more early stage apoptosis of cancer cells relative to its ruthenium(II) precursor or the EGFR-inhibiting 4-AQ ligands, again demonstrating the potential inherent in this dual-targeting approach.467 The same group, a year later, reported a series of nonorganometallic ruthenium(II)-4-AQ derivatives.468 They complexed the ruthenium(II)-polypyridyl substrates, cis-[Ru(bpy)2Cl2], and cis-[Ru(phen)2Cl2], which are nontoxic to tumor cells, to 4-AQ ligands to test if the ligands endowed the complexes with cytotoxic characteristics. Five complexes were prepared in total (335−339), Figure 75. This study revealed that the presence of the EGFR-targeting ligands in these previously benign cis-[Ru(bpy)2Cl2] and cis-[Ru(phen)2Cl2] complexes endowed the resulting complexes with antiproliferative properties. Hydrolysis studies showed rapid hydrolysis of chlorido ligands but showed that the 4-AQ ligands remained stable and intact. When tested against a series of EGRF overexpressing cancer cells, 338 was found to be the most active, with an IC50 value comparable to cisplatin and gefitinib in most cases. Interestingly, while 339 showed the strongest EGFR inhibiting ability, its cytotoxicity was lower than that of 338. Complexes 338 and 339 demonstrated high DNA minor groove binding capability. These studies demonstrated quite remarkably that co-ordinating EGFR inhibiting moieties to a nonorganoruthenium(II) center can not only improve the EGFR targeting properties of the ligand but also the anticancer properties of the metal complexes, thus providing further validation of the advantages inherent in this dual-targeting approach.468 4.2.2. P-Glycoprotein and MDR Protein. Many multidrug resistance (MDR) cells overexpress certain efflux proteins. Such overexpression can lower intracellular drug concentrations either by blocking uptake or increasing efflux.469 Of these efflux proteins, P-glycoprotein (Pgp) and MDR protein (MDR 1) represent important members of this family.470 Dyson, Juillerat-Jeanneret, et al. sought to exploit the overexpression of these proteins on tumor cells. They also sought to take adavantage of the lower toxicity of rutheniumarene complexes owing to their higher selectivity toward

activity toward HeLa human cervical cancer cells, both with overexpressed and normal levels of EGFR. They were compared to gefitinib, cisplatin, and [(η6-p-cym)Ru(EDA)Cl](PF6) as an “(arene)ruthenium-moiety-only” control. Complexes 311, 314−316, 322−325, and 327 exhibited selective inhibitory acitivity toward the EGFR positive cell line, and, of these, 314, 316, 323, 324, and 325 were more effective than gefitinib. Complex 314 was the most cytotoxic, even more so than cisplatin. Cancer cell selectivity was also verified in that 322−329 were found to be less than half as toxic toward noncancerous human bronchial epithelial cells when compared to their cytotoxicity against the cancerous cell lines. Molecular modeling studies were employed to further validate the dual targeting nature of these complexes. The complexes were docked to the EGFR kinase domain and to DNA via either GN7 guanine coordination or minor groove binding. Of note is the fact that each complex contained two chiral centers (Ru* and N*) resulting in four possible stereoisomers. The docking scores of each of these stereoisomers for one of these complexes (323) into the ATP-binding pocket of EGFR and into the minor groove of a β-form DNA duplex were first compared, and no pronounced difference was observed. Only one stereoisomer of each of the fourteen complexes was therefore chosen for further docking studies. Hydrolysis of ruthenium(II)-arene complexes containing chlorido ligands is thought to be a key step in their activation toward biomolecules.364,464−466 Docking studies therefore also included the respective hydrolyic adducts of each of the fourteen complexes. These studies revealed that the hydrolysis of these complexes enhanced their inhibitory activity against EGFR as well as their reactivity toward DNA, and, in so doing, promotes their anticancer potential.463 Wang, Zhao, et al., the following year, described five additional ruthenium(II)-arene-4AQ complexes (330−334), Figure 76, but unlike the previous study which contained a library of complexes with different arene ligands, here the arene is p-cym and four new 4-AQ derivatives coordinated as monodentate ligands with chlorido ligands completing the coordination sphere.467 Again, the complexes were found to act as DNA minor groove binders. They were also all capable of inhibiting EGFR. Their cytotoxicity against A549, HeLa, PC-3, MCF-7, and A431 cell lines (both under and overexpressed in EGFR) was also assessed and compared to gefitinib, cisplatin, and [(η6-p-cym)Ru(EDA)Cl](PF6), as well as [(η6-p-cym)Ru(en)Cl](PF6) and gefitinib in combination. 1117


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malignant cells.471 They developed five ruthenium(II)-arene complexes, incorporating modified phenoxazine- and anthracene-based MDR modulator ligands (340−344), Figure 76, and compared their cytotoxicities as well as their MDR reversal properties with the parent MDR modulator ligands.471 Three of these complexes (340−342), Figure 76, were chosen for biological evaluation against A549, HT29, T47D, and TS/A cell lines. Complexes 343 and 344 were not included, owing to their poor aqueous solubility even in the presence of DMSO. Despite all three complexes (340−342) containing ligands designed to inhibit MDR proteins, complexation of these ligands to the ruthenium-arene center did not appear to significantly impact the cytotoxicity profile of the resulting complexes, with the ligands and complexes exhibiting cytotoxicities within the same order of magnitude. Of the complexes investigated, complex 342 with the anthracene-based ligand was the most potent, being 2−5 times more potent than both complexes 340 and 341 and being significantly more potent than the free anthracene-based ligand. This complex was shown to inhibit not only Pgp but also DNA synthesis, with its ligand likely being a DNA intercalator. Furthermore, because of the fluorescent nature of the ligand and complex, their uptake into tumor cells could be investigated. This study revealed that, relative to the free ligand, 342 is taken up faster into tumor cells and appears to accumulate in the cell nucleus. This most likely accounts for the differential in cytotoxicities between the ligand and complex. This study nicely demonstrates that while MDR inhibition may have played some role in the efficacy associated with these complexes as initially hypothesized, it was another structural process which contributed to the potency of the most active compound. This again highlights the need for broad analysis when testing these “multifunctional” complexes, as it cannot be assumed they will only act on their primary targets of interest.471

ruthenium complexes are generally less toxic to normal cells and tissues as illustrated previously by the toxicity profile of NAMI-A and KP1019. Ruthenium complexes also tend to evoke less drug resistance relative to platinum drugs.475 The team found that complexing plumbagin to the (η6-arene)ruthenium(II) scaffold did indeed significantly improve the selectivity of the resulting complex toward multidrug-resistant KB-V1/Vbl cervix carcinoma cells, while having significantly less toxic effects toward nonmalignant chicken heart fibroblasts (CHF). The cytotoxicity of 345 was attributed to DNA binding, as typically expected from both plumbagin and ruthenium(II) complexes, and its ability to inhibit the Pgp drug efflux pumps, which are overexpressed in KB-V1/Vbl cells, possibly explaining its ability to overcome drug resistance. The complex did not however show improved efficacy over plumbagin against 518A2 melanoma and HCT-116 colon carcinoma cells. The complex was however able to alter the shape of linear DNA more extensively than plumbagin, which was attributed to the presence of the large ruthenium fragment alongside plumbagin’s typical DNA-intercalating naphthoquinone unit.474 4.4. Other Targets

We have already highlighted numerous examples of “multitargeted” ruthenium(II)-arene complexes incorporating bioactive ligands in which the activity of the bioactive ligand has been enhanced upon complexation to the metal center. Here we present yet another such class. Dyson, Nazarov, et al. generated two hybrid complexes comprising an antimetastatic ruthenium-arene fragment tethered to an indazole-3-carboxylic acid derivative of lonidamine.476 They selected lonidamine as their ligand of choice owing to the fact that it is used clinically in combination with radiotherapy and temolomide to treat brain tumors. Its mode of action is attributed to its ability to inhibit aerobic glycolysis. In order to complex it to the ruthenium(II)-arene scaffold, they had to first modify it. This they did by incorporating an imidazole linker. Interestingly, incorporation of this linker appeared to enhance the cytotoxic profile of the parent lonidamine molecule. Two complexes were generated with different arene units; one containing a cym ring (347) and the other a toluene one (346), Figure 78. The complexes

4.3. Targets Inspired by Natural Product Activity

The natural product plumbagin and a close analogue juglone are naphthoquinone derivatives. Plumbagin possesses antioxidant, antimicrobial, anti-inflammatory, antimalarial, and anticancer properties,472 while juglone, as an Indian traditional medicine, has been used to treat inflammation and bacterial, viral, and fungal infections, as well as cancer.473 Both induce cancer cell death via an apoptotic mechanism. Plumbagin has shortcomings, however, not the least of which is its low activity against some cancers and its lack of selectivity. To address these drawbacks, Biersack et al. were successful in tethering plumbagin to an (η6-arene)ruthenium(II) framework, generating 345, Figure 77, but they were unable to isolate the corresponding juglone derivative in sufficient purity for further studies.474 Their motivation was fueled by the fact that

Figure 78. Chemical structures of organoruthenium(II)-lonidamine derivatives (346−347).

346 and 347 exhibited markedly enhanced cytotoxicity over the parent lonidamine and RAPTA-C complexes which would suggest that combining both the ruthenium(II)-arene center and the aerobic glycolysis inhibitor into one drug molecule is beneficial. The cym derivative 347 was more cytotoxic compared to the toluene analogue. Relative to cisplatin,

Figure 77. Chemical structures of (η6-p-cym)ruthenium(II)-plumbagin derivative (345). 1118


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Figure 79. Chemical structures organoruthenium(II)-chlorambucil derivatives (348−350).

Figure 80. Chemical structures of organoruthenium(II)-antiobiotic derivatives (351−353).

interfere with cellular energy processes was, however, not investigated.476 Dyson, Hartinger, Nazarov, et al. sought to exploit the DNA alkylating properties of the well-known nitrogen mustard chlorambucil (which is already in clinical use as an anticancer agent) by complexing it to their RAPTA scaffold, known for its propensity to bind proteins,477,478 with a view to generating a novel multitargeted agent.413 The chlorambucil unit was first modified to include an additional arene ring capable of binding to the ruthenium(II) center. Complexes 348−350, Figure 79, were generated with varying coligands. The impact of varying the distance between the ruthenium center and the

however, both complexes exhibited lower cytotoxicities. The cym derivative, which was also more stable than the toluene analogue, was selected for further studies. Its cytotoxicity against three human glioblastoma cell lines was evaluated and compared to lonidamine, given the fact that lonidamine, at that time, was being clinically evaluated as a treatment for brain cancers. Both the modified lonidamine and the cym complex 347 were highly cytotoxic against the glioblastoma cells investigated (LN18, LN229, and LNZ308), especially the complex, in contrast to the parent lonidamine drug, which essentially exhibited no activity. The ability of complex 347 to 1119


naproxen, diclofenac, ibuprofen, and aspirin

ethacrynic acid

metyrapone, etomidate

clioquinol

acetazolamide

letrozole

lonidamine

chlorambucil

ofloxacin, nalidixic acid, cinoxacin, levofloxacin, oxolinic acid, and ciprofloxacin

Ru(II)

Ru(II)

Ru(II)

Ru(II)

Ru(II)

Ru(II)

Ru(II)

Ru(II) and Ru(III)

234−246 (Figure 59)382,384,385 260−262 (Figure 62)406 277−278 (Figure 67)434 289−291 (Figure 70)452 299−302 (Figure 72)457 346−347 (Figure 78)476 348−350 (Figure 79)413 351−353 (Figure 80)436,479,483

drug ligand

Ru(II)

metal ion and oxidation state

227−230 (Figure 57)372

metallodrug conjugate number and figure no.

bacterial DNA gyrases and topoisomerases

DNA

hexokinase

aromatase

peptidases and proteosomes carbonic anhydrase

glutathione-Stransferases cytochrome P450

cyclooxygenases

drug ligand target

in clinical use as a treatment for chronic lymphocytic leukemia (CLL), Hodgkin lymphoma, and non-Hodgkin lymphoma488 bacterial DNA gyrases and topoisomerases in clinical use as antibiotic treatments for bacterial infections489,490

in clinical trials in combination with anticancer drugs487

in clinical use as a post-surgery treatment for hormonally-responsive breast cancer486

in clinical use as a treatment for glaucoma, epilepsy, altitude sickness, ulcers452

in clinical use as an anti-parasitic treatment485

in clinical use as a treatment for Cushing‘s syndrome484

in clinical use as NSAIDs; NSAIDs such as aspirin have been shown to reduce the risk of developing colorectal cancer and reduce colorectal cancer mortality rates;351 NSAIDs included in combination therapies with cisplatin, paclitaxel and doxorubicin352 in clinical use as a diuretic treatment for edema349

clinical use/preclinical properties or drugs

Table 3. Representative Examples of “Multi-Targeted” Ruthenium Conjugates Incorporating Ligands Which Are Used Clinically As Drugs and/or Are in Clinical Trials

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these ciprofloxacin derivatives exhibited in vitro cytotoxicity in the low micromolar range against a panel of tumor cell lines, including the fluoroqiunolone ligand shown in 353. Our rationale was to try and develop a new class of prophylactic metallo-antibiotic with anticancer activity given that cancer patients, particularly those who sustain neutropenia (where the white blood cell count is lower than normal) during the course of their disease, are often highly predisposed to infections. Furthermore, therapy-related myelosuppression and immune defects associated with the cancer disease process are also risk factors for infection.482 While we were successful in generating a platinum(II)-complex incorporating this ciprofloxacin derivative, due to poor aqueous solubilty, we were unable to progress biological testing (unpublished results). We thus turned our attention to the organoruthenium(II) framework. The resulting complex 353, Figure 80, was found to be highly cytotoxic, in the low micromolar range, against a range of tumor cell lines (A2780, A549, HCT116, and PC3), comparable to cisplatin. Of particular interest was the fact that 353 retained low micromolar cytotoxicity against the human colon cancer cell line HCT116p53 in which the tumor suppressor p53 had been knocked out. This suggested that the antitumor properties associated with this complex were independent of the status of p53 (in contrast to cisplatin). While we had hoped that the ciprofloxacin derivative would bestow the complex with potent antibacterial activity, in the bacterial strains tested (a laboratory Escherichia coli strain and a clinical isolate resistant to first, second, and third generation βlactam antibiotics), it only demonstrated moderate antimicrobial activity. Nevertheless, this study demonstrated the potential in designing metallo-antibiotics as a prophylactic treatment for cancer patients at particularly high risk of infection. Furthermore, the influence of the aliphatic tail present in the ciprofloxacin analogue in 353 and not in oflaxacin, nalidixic acid, cinoxacin, levofloxacin, or oxolinic acid may well be endowing the complex with the required physicochemical properties to facilitate its uptake into tumor cells and hence account for its potent cytotoxic properties.483 In summary, we have showcased a multitude of ruthenium complexes, the majority of which exhibited potent anticancer activity. Many of these were also found to be highly efficacious against resistant cancer cell lines. In some cases, the metal center endowed with the bioactive ligands had more potent activities compared to the free ligands themselves. For example, the ruthenium-EGFR inhibitor conjugates were more potent EGFR inhibitors compared to clinically used EGFR inhibitors. This certainly bodes well for the future development of ruthenium complexes both as anticancer agents and as alternatives to current organic-based chemotherapeutic regimens. Representative examples of “multitargeted” ruthenium complexes incorporating ligands which are used clinically as drugs in their own right and/or are in clinical trials are provided in Table 3.

chlorambucil moiety was also investigated. The resulting complexes exhibited low micromolar cytotoxicities against A2780, A2780cisR, and MCF-7 cells and were superior to chlorambucil, the parent RAPTA complex and a mixture of both in cisplatin-resistant tumor cells. Their stability in solution and their reactivity toward DNA substrates and proteins were also investigated. The study revealed that the η6arene bond in complex 350a was relatively stable with only slow cleavage in solution, and that this stability may account for the different cytotoxic effect of this complex when compared to the combination of chlorambucil and the parent RAPTA complex. The study also revealed that the complex does hydrolyze in solution allowing the complex coordinate EtG and that this hydrolysis proceeds more rapidly than alkylation of EtG by the chlorambucil unit.413 The team acknowledged that further refinement of the complex class may be necessary in order to enhance the stability of the ruthenium(II)-arene bond and, in so doing, enhance the simultaneous extent of both DNA alkylation and protein ruthenation.413 Turel et al. reported the crystal structure and DNA binding properties of the first example of a ruthenium(II)-arene complex incorporating a known antibiotic, namely oflaxacin as a bidentate ligand (351a), Figure 80.479 In a follow-up study, the same team generated two additional analogues, one containing nalidixic acid (351b) and the other containing cinoxacin (351c), Figure 80. The physicochemical properties and cytotoxicities of all three complexes were then compared.480 The complexes were found to undergo rapid hydrolysis to the aqua species following loss of the chlorido ligands, endowing them with the potential to interact with biomolecules including DNA. While all three complexes were shown to possess DNA binding capability in that they formed adducts with 5′-GMP via its nucleophilic N7, only one complex, that containing the ofloxacin ligand (351a), demonstrated any cytotoxicity and only in one of the three cell lines tested (CH1).480 Building on this work, the same team generated four additional complexes but this time moving away from an organometallic framework to complexes of general formula [Ru([9]aneS3)(DMSO-κS)(quinolonato-κ2O,O)](PF6). Here the complexes contained the antibiotic quinolones nalidixic acid (352b), cinoxacin (352c), levofloxacin (352d), and oxolinic acid (352e), Figure 80. The complexes were found to be relatively stable under aqueous conditions, in contrast to their organoruthenium analogues.480 Their enhanced stability was attributed primarily to the slow substitution of the DMSO ligand. The complexes had a high binding affinity for serum proteins. They were shown to bind to calf thymus-DNA via intercalation. Compounds 352b and 352c were also found to weakly inhibit Cathepsins B and S, enzymes associated with tumor cell progression. The complexes were however not cytotoxic against HeLa and A549 tumor cells (with IC50 values 227 μM to >600 μM). We too rationally designed and developed a (η6-p-cym)ruthenium(II)-antibiotic derivative (353), Figure 80, but this time incorporating a ciprofloxacin analogue which had previously been shown to not only possess potent antimicrobial but also potent anticancer properties. Azema et al. had previously reported a structure activity study of thirty one 7((4-substituted)piperazin-1-yl) derivatives of ciprofloxacin in which they endeavored to correlate the lipophilic properties of the ciprofloxacin analogues with antitumor activity.481 Two of

5. CONCLUSIONS AND OUTLOOK At a recent Faculty event in our home Institution, the RCSI Professor of Otolaryngology, Head and Neck Surgery, delivered an inspiring lecture which included a reference to cisplatin and how it is used, in combination with other drugs, to treat head and neck cancers. He specifically referred to cisplatin as being “king” in his world and that of his patients. The incredible boon that this drug has offered as a treatment 1121


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complexes to enter clinical trials to date are in the +3 oxidation state, it is interesting to note that the majority of the “multitargeted” ruthenium complexes developed so far are organometallic pseudo octahedral ruthenium complexes in the +2 oxidation state. It is envisaged that research in this chemical space will continue to thrive. There is no doubt that cancer is a multifactorial disease that is not yet fully understood. It is also truly difficult to fully elucidate the mechanism of action of drug molecules given the complex mix of biomolecules and pathways that they can interact with in the human body. This review highlights this well, showing that many new complexes either did not act upon their expected targets or interacted with other targets that were not considered in the original hypothesis. The stories of the platinum(IV)-HDAC inhibitor and platinum(IV)-COX inhibitor conjugates illustrates this well. These complexes, while originally designed to inhibit HDAC and COX enzymes, respectively, attained their improved cytotoxicites for reasons other than direct enzyme inhibition. The methodical approach employed to more fully elucidate the mechanism of action of the platinum(IV)-valproates and the collaborative efforts between different groups working on these complexes not only yielded intriguing results but also highlights the benefits which result when teams come together to follow through on initial research findings. This review also clearly illustrates that combining two or more entities with some desired functionality into one drug molecule offers advantages over single agents. These multitargeted drugs appear to be more than the sum of their parts. While the precise mode of action of the complexes may never fully be elucidated, a common trend shows that many of these possess the ability to overcome the issues of cancer drug resistance. This multitargeted approach may therefore ultimately lead to NMEs with the ability to act as a potential alternative treatment for cancers where cisplatin is not or is no longer effective. We have also highlighted numerous examples where vectors such as sugars and peptides have been successfully exploited to selectively deliver their platinum payloads to tumor cells. This tumor cell targeting approach may well lead to NMEs with improved selectivity for tumor cells, thus offering the possibility of reduced systemic toxicities. While we have highlighted many examples where the conjugation of known drugs to a metal center has resulted in a synergistic advantage, this was not always found to be the case. As this research field continues to grow, one should be mindful that the doses required to induce a potent biological effect for organic drugs or bioactive organic-based ligands may fall outside the range required to induce a cytotoxic effect, arising from the presence of the cytotoxic platinum or ruthenium moiety. Furthermore, one should also be aware of any side effects associated with known drugs being conjugated to metal centers. In conclusion, this is a rapidly developing field of research which has already demonstrated huge potential. The move toward a more rational multitargeted approach to drug design has yielded many innovative solutions from numerous international research groups as outlined in this review. We now need to engage proactively with the pharma sector if we are to truly translate these exciting metallochemotherapeutics to the next stage in the drug development process. Given the enduring success of platinum drugs and the current momentum in this field, it is only a matter of time before we see a multitargeted metallodrug reach the clinical stage.

for different types of cancers has driven research teams around the world to develop more and better metal-based chemotherapeutics. While significant progress has been made in this regard, we feel it is important to acknowledge the enduring clinical success of cisplatin, despite its drawbacks. Cisplatin has been in clinical use for some 40 years now. Despite major advances in the medical oncology field, it, along with its analogues carboplatin and oxaliplatin, continue to play a crucial role as part of treatment regimens for numerous cancers. Their success is also reflected in the number of ongoing clinical trials which involve these drugs, as outlined earlier. Despite the remarkable success of cisplatin, it does have significant drawbacks, which include is severe dose-limiting toxic side effects as well as major drug-resistance issues. While DNA represents the primary target of cisplatin, there is no doubt that there has been a paradigm shift in research focus in relatively recent times to try to identify drug targets beyond DNA for therapeutic exploitation. Harnessing our immune system to improve cancer therapies is also now becoming a reality.491 For example, carboplatin, in combination with paclitaxel or nab-paclitaxel and the immunotherapeutic Keytruda (pembrolizumab), received FDA approval in 2018 as a first-line treatment for patients with metastatic, squamous nonsmall cell lung cancer.492 These advances have undoubtedly heralded a new era in drug design in which we are moving from a single- toward a multitargeted approach. The success of combination chemotherapies for the treatment of a wide array of cancers has undoubtedly played a part in driving this change. Combination chemotherapies have limitations however in that each drug will have its own pharmacokinetic profile and so the overall treatment regimen is hard to control. A single drug designed to hit multiple biological targets may overcome this shortcoming. There is now concrete evidence in the literature to suggest that this multitargeted approach is fast becoming a reality. For example, an analysis of new molecular entities (NMEs) approved by the FDA during the period 2015−2017 was recently reported.493 This report highlights that of the NMEs approved within this time frame, 31% were biotech drugs such as proteins, peptides, and monoclonal antibodies, 34% were single target drugs, 10% represented therapeutic combinations while multitarget drugs accounted for 21% of these NMEs. It was noted that the percentage of “multitargeted” NMEs was on the increase. It is this multitargeted approach in metallodrug design and action that we have endeavored to showcase in this review. We have particularly focused on progress within this domain over the past decade or so. We can see that while platinum drug development continues to thrive, there is now a paradigm shift toward the rational design and development of “multitargeted” platinum drugs, both in the +2 and +4 oxidation states. These drugs not only target DNA but also contain either vectors to confer selectivity for tumor cells and/or moieties that target other cellular entities, including enzymes, peptides, intracellular proteins, and the mitochondria. Some research teams have also drawn inspiration for the design of their multitargeted drugs based on natural product activity. Complexes of other transition metals are also being actively pursued as alternative classes of chemotherapeutics. We have endeavored to highlight recent developments related to multitargeted ruthenium complexes given that complexes containing this metal have already advanced to clinical trials as anticancer agents. Despite the fact that the only ruthenium 1122


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ASSOCIATED CONTENT

(AR) (4-AQ) (bpy) (CA) (CAPs) (CDKs) (COX) (cym) (DNA) (DACH) (DCA) (AMP) (SMP) BDIQQ (DMSO) (DDR) (DSB) (ER) (EGFR) (ER-) (ER+) (EA) (EDC)

androgen receptor 4-anilinequinazolines bipyridines carbonic anhydrase cationic antimicrobial peptides cyclin-dependent kinases cyclooxygenase η6-p-cymeme deoxyribonucleic acid diaminocyclohexane dichloroacetate diethyl(aminomethyl)-phosphonate diethyl[(methylsulfinyl)methyl]phosphonate β-diketiminate ligand dimethyl sulfoxide DNA damage response double strand break endoplasmic reticulum epidermal growth factor receptors estrogen receptor negative estrogen receptor positive ethacrynic acid 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride (EDA) ethylenediamine (9-EtG) 9-ethylguanine (ECM) extracellular matrix (FOH) farnesol (FPPS) farnesyl pyrophosphate synthase (Fc-tpy) ferrocenylterpyridine (5-FU) 5-fluorouracil (5-FU/LV)) 5-fluorouracil/leucovorin (FA) folinic acid (FPRs) N-formyl peptide receptors (GGOH) geranylgeraniol (GGPP) geranylgeranyl pyrophosphate (GLUT) glucose transporter (GPx) glutathione peroxidase (5‘-GMP) 5′-guanosine monophosphate (HATs) histone acetyltransferases (HDACs) histone deacetylases (HA) hyaluronan (NHS) N-hydroxysuccinimide (Ibf) ibuprofen (ICD) immunogenic cell death (LDH) lactic dehydrogenase (LOX) lipoxygenase (LND) lonidamine (MMPs) matrix metalloproteinases (MDR) multi-drug resistance (NMEs) new molecular entities (NHC) N-heterocyclic carbene (NSAIDs) nonsteroidal anti-inflammatory drugs (9-EtG) nucleobase 9-ethylguanine (NER) nucleotide excision repair (OA) octanoato (OCT) organic cation transporter (Pgp) P-glycoprotein (phen) phenanthrolines (Phe) L-phenylalanine (PhB) 4-phenylbutyrate (Phe-tpy) phenylterpyridine (Pt-GG) platinum-guanine-guanine

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.chemrev.8b00271. Table of ongoing clinical trials involving cisplatin, carboplatin, and/or oxaliplatin in the U.S. (PDF)

AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected]. ORCID

Celine J. Marmion: 0000-0003-0637-6693 Notes

The authors declare no competing financial interest. Biographies Reece Kenny graduated with a first class honours degree in Chemical Sciences with Medicinal Chemistry from the Dublin Institute of Technology, Ireland, in 2014. He then joined the Royal College of Surgeons in Ireland, where he is an Apjohn Scholar carrying out a Ph.D. under the supervision of Professor Marmion. His research is focused on developing new metal-based chemotherapeutic agents with targeted bioactive ligands. Celine J. Marmion received her Ph.D. from the University of Surrey, UK, in 1994. She held a lecturing position in chemistry (1994−1995) at St. Mary’s University College, Strawberry Hill, UK, and then graduated with a Postgraduate Certificate in Education from the University of Kingston in 1996. She took up a lecturing position in Chemistry at the Royal College of Surgeons in Ireland (RCSI) in 1997 and was promoted to senior lecturer in 2003, to Associate Professor in 2013 and to Full Professor in 2018. She is a Fellow of the Royal Society of Chemistry and of the Institute of Chemistry of Ireland (ICI) and is currently Vice-President of the ICI. Professor Marmion has a passion for and a track record in trying to bridge the interface between medicinal inorganic chemistry and biology. In this regard, she helped establish the Irish Biological Inorganic Chemistry Society and is currently its President. In education, she has received a number of teaching awards, most notably, a national ‘Teaching Hero’ award in 2016. She has also received two innovation awards for her research commercialization achievements. Her current research interests revolve around the rational design and development of innovative, multitargeted metallodrugs.

ACKNOWLEDGMENTS This material is based upon works supported by the Science Foundation Ireland under Grants [11/RFP.1/CHS/3095], [12/TIDA/B2384], and [17/TIDA/5009]. This work has also been funded by the RCSI under the Apjohn Scholarship programme. Funding under the Programme for Research in Third-Level Institutions and cofunding under the European Regional Development fund (BioAT programme) is also acknowledged. The authors would also like to acknowledge COST CM1105 and CA13135 for providing a platform to progress fruitful collaborations. ABBREVIATIONS (ATP) adenosine triphosphate (AKR) aldo-keto reductase (NH3) ammonia 1123


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Review

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phosphatase 2A photodynamic therapy podophyllotoxin proinflammatory transcription factors 2-propylpentanoic acid pyridine pyruvate dehydrogenase kinase reactive oxygen species ruthenium-arene-PTA signal transducer and activator of transcription 3 solid phase peptide synthesis suberoylanilide hydroxamic acid suberoyl-bis-hydroxamic acid 4-([2,2′:6′,2″-terpyridine]-4′-yl)-N-(pyridin-2ylmethyl)aniline tetrahydropyran thioredoxin thioredoxin reductase topoisomerase trans platinum amine trifluoroacetate 1,3,5-triaza-7-phospha-tricyclo-[3.3.1.1]decane valproic acid zoledronic acid

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