Ruthenium Anticancer Agents

A Dissertation Based On Literature

Kamalpreet Singh

BSc. Candidate

999-596-192

Supervised By Judith Poë

Second Reader: Ulrich Fekl

Faculty Of Science

University Of Toronto

December 14th, 2015

ii

Abstract

As the leading source of deaths in economically developed countries and the second

leading source of deaths in developing countries, cancer has become a formidable foe in the war

against disease [2]. The success of the platinum based metallodrug, cisplatin, in targeting a range

of solid tumours has stimulated a wave of research into other potential metals which may be

utilized in cancer treatment. Here, a discussion is provided on the potential of ruthenium based

anti-cancer agents with a particular focus on what makes these agents different from cisplatin and

why the world needs these agents.

Ruthenium with contiguous oxidation states (+3 and +2) and due to its ability to mimic

iron, in vivo, was found to be significantly more biocompatible compared to platinum [20]. It was

not surprising then, that Ruthenium based anti-cancer agents had much larger tolerable dosages

and minimal detrimental side effects as compared to cisplatin[22][24][30]. In addition, the biological

targets and therapeutic effects of the ruthenium based agents were found to quite versatile. In

particular, NAMI-A, a famous Ruthenium anti-cancer agent was found to exert anti-metastatic

effects with no effect on primary tumours. This activity was believed to stem from the ability of

the agent to interrupt the nitric oxide-dependent angiogenesis [29]. Another famous ruthenium drug,

KP1019, was found to exert no anti-metastatic effects but rather target primary tumours in various

cell lines including cisplatin resistant ones. The anti-cancer activity of KP1019 was understood to

originate from its ability to produce reactive oxygen species which result in depolarization of the

mitochondrial membrane potential resulting in release of apoptotic proteins and thereby inducing

apoptosis [30]. Both NAMI-A and KP1019 have successfully completed phase I clinical studies [24].

Another class of ruthenium drugs that showed anti-metastatic activity were the RAPTA family of

complexes. This anti-cancer activity was attributed to the interaction of the RAPTA complexes

with various proteins, including PARP-1, a zinc based metalloprotein known to play a key role in

cancer resistance in chemotherapy[35]. Other Ru(II) based drugs, namely the RAED and Ru(II)

polypyridyl complexes, were found to be more like cisplatin as these agents also appeared to

induce apoptosis via interaction with DNA.

Clearly, Ruthenium based anti-cancer agents have a crucial role to play in the war against

cancer, as they offer lower toxicity to healthy cells, versatility in action and therapeutic effect and

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ability to target cisplatin resistant cell lines. Active research should be continued in the area till a so

called magic bullet is obtained.

iv

Table Of Contents

Topic Page

1. List of Tables & Figures vi

2. Acknowledgements viii

3. Introduction 1

4. Cisplatin 4

a. Synthesis 4

b. Mechanism of Action 6

c. Limitations 9

5. Ruthenium Biochemistry 11

a. Oxidation State 11

b. Ligand Exchange Kinetics 13

c. Iron Mimicking 14

6. Ruthenium Anticancer agents 16

a. NAMI-A 16

i. Synthesis 16

ii. Drug Metabolism & Biological Activity 17

iii. Anticancer Mechanics 22

iv. Clinical Data 23

v

b. KP1019 24

i. Synthesis 24

ii. Clinical Data 25

iii. Biological Activity & Transport 25

iv. Anticancer Mechanics 27

c. Other Candidates 29

i. Arene Complexes 29

ii. Ruthenium Polypyridyls 32

7. Concluding Remarks: why does the world need ruthenium agents? 35

8. References 37

vi

List Of Tables & Figures

Figure Pg

1. Paul Ehrlich & Magic Bullet (Figure1) 2

2. Cisplatin (Figure2) 4

3. Michele Peyrone’s Cisplatin Synthesis (Figure3) 5

4. Dhara’s Synthesis Of Cisplatin (Figure4) 6

5. Metabolism of Cisplatin (Figure5) 7

6. DNA & Cisplatin Interaction Sites (Figure6) 8

7. Cisplatin’s Induction Of Cell Death (Figure7) 9

8. A Chiral Ruthenium Sulfoxide Catalyst (Figure 8) 11

9. Ruthenium(II) and (III) in Cancer and Healthy Tissue(Figure9) 12

10. Ligand Exchange Kinetics (Figure10) 14

11. Ruthenium transport in healthy and cancer cells (Figure 11) 15

12. NAMI-A (Figure 12) 16

13. Synthesis of NAMI-A (Figure 13) 17

14. 1H-NMR of NAMI-A Hydrolysis (Figure 14) 19

15. Absorption Spectra NAMI-A & Ascorbic Acid Reaction (Figure 15) 21

16. Reduction of NAMI-A & Solution Chlorine Concentration(Figure16) 21

17. The Nitrosylation of NAMI-A (Figure17) 23

vii

18. KP1019 (Figure18) 24

19. Synthesis of KP1019 (Figure19) 25

20. Sodium Analogue of KP1019, KP1339 (Figure20) 24

21. KP1019 & Apo-lactoferrin (Figure21) 26

22. RAPTA & RAED type complexes (Figure22) 29

23. RAED complexes investigated by Sadler group (Figure23) 30

24. Various RAPTA complexes (Figure24) 32

25. General Synthesis of Ru(II) Arene complexes (Figure25) 32

26. mer-[Ru(terpy)Cl3] A Ru(II) polypyridyl complex (Figure26) 33

27. [Ru(phen)2(dppz)]2+ A DNA switch (Figure27) 33

28. [Ru(phen)2(dppz)]2+ derivatives studied by Tan group (Figure28) 34

viii

ACKNOWLEDGEMENTS

First of all, I would like to express my deepest gratitude to my supervisor, Professor Judith

Poë. This project would not be possible without her valuable guidance, critique, insightful

questions and patience with a novice undergrad such as myself. I could have not asked for a better

mentor. Thank you.

I would also like to thank Professor Ulrich Fekl for taking time out of his busy schedule to

be the second reader for the project. The critique of the report and feedback in general was much

appreciated, thank you.

In addition, I would like to thank David Armstrong and Fioralba Taullaj for their consistent

support and advice and my parents for their moral support.

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Introduction

Historically, the diseases threatening human existence have been mainly infectious in

nature. In fact, tuberculosis and influenza were among the leading causes of deaths in Canada

between 1921 and 1924[1]. Fortunately, progression in medicinal science and technology has

allowed for the development of wondrous antibiotics that have helped curb the lethal status of

many such infectious diseases. However, the war on disease is far from over as humans now face

a different kind of epidemic: cancer. As the leading source of deaths in economically developed

countries and the second leading source of deaths in developing countries, cancer has become a

formidable foe in the war against disease [2]. One of the reasons cancer is a tough disease to

eradicate, is due to the fact that there are actually more than 100 different types of cancers, the

majority of which are not well known[3][4]. Currently, the treatment to tackle the rapid and

uncontrolled cell division that characterizes cancer is comprised of surgery, radiotherapy and

chemotherapy [4]. In the surgery approach, the primary focus is to remove the malignant tumour

and any related lymph nodes from the patient [4]. Similarly, radiotherapy is employed to destroy

localized tumours in the body which, may or may not be accessible via surgery [4]. The two

approaches, although successful in removal of primary tumours have one major drawback; they

cannot resolve the issue of metastases [4] [5]. . Metastases is a process which refers to the spreading

of cancer cells from the primary tumour to distant sites [6]. Since both surgical and radiotherapy

approaches target only localized regions, they can only truly tackle one part of the body at a time.

This essentially means if metastases has occurred, then the cure via surgery or radiotherapy is only

temporary. This limitation became quite evident in the 1960s when physicians noticed that cure

rates following treatment via surgery and radiotherapy plateaued at about 33% [5]. In order to

improve the success rate of cancer therapy, an approach capable of targeting the whole body rather

than localized regions was desired. This realization served as a cue for the introduction of

chemotherapy to the field of cancer treatment.

The term chemotherapy was devised by the German chemist, Paul Ehrlich, who was

attracted to the idea of treating diseased individuals with chemicals [5]. Building on his approach

of treatment, Ehrlich would define chemotherapy as the use of chemicals to treat disease [5]. In

addition to finding chemicals for the treatment of infectious diseases, Ehrlich was also interested

2 | P a g e

in finding chemicals for cancer treatment; investigating aniline dye and some primitive alkylating

agents [5]. However like many others around this time, he was not very optimistic about success

and thus reported no major breakthroughs [5]. Indeed, this cynical interpretation of the potential of

cancer chemotherapy meant little industrial interest and very limited funding for academic research

for many years [5]. As a matter of fact, chemotherapy did not arrive at the forefront of cancer

research until the early 1940s when two pharmacologist, Gilman and Goodman, discovered

anticancer activity in nitrogen mustard [7]. Unfortunately, the anticancer activity was found to be

temporary and drug resistance was a major concern. Nonetheless, nitrogen mustard demonstrated

the potential of chemicals in the war against cancer inspiring research of chemotherapeutics, which

eventually led to the development of the National Cancer Chemotherapy Service Center (NCCSC)

at the National Cancer Institute (NCI) in 1955[5][7]. It was under the funding of this organization

Figure 1. Paul Ehrlich, shown in his office above, is considered the father of modern chemotherapy [9]. His work on the interactions between dyes and cellular structures lead to the realization that chemicals can be utilized to cure diseases [9]. Ehrlich was responsible for curing syphilis with Salvarsan, an arsenic based synthetic compound [9]. Ehrlich was also the master mind behind the concept of magic bullet, referring to the selective targeting of organelles by drugs [9]. The concept of magic bullet inspired the new generation of scientist which included Goodman and Gilman, pharmacologist who discovered the anti-cancer activity of nitrogen mustard. Paul Ehrlich received a Nobel Prize in physiology & medicine for his scientific contributions[9]. This image was obtained from [9].

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that, Barnett Rosenberg at Michigan State University discovered Cisplatin[7]. This platinum based

compound was found to show remarkable success in targeting testicular and ovarian cancer,

receiving FDA (Food and Drug Administration) approval in 1978 and by doing so introducing

metal based agents to the world of cancer [7]. Since its approval, Cisplatin has been utilized in the

treatment of a broad range of solid tumours, however, the occurrence of several detrimental side

effects and the issue of drug resistance have served to limit the remedial ability of this potent

compound [8]. These imperfections in Cisplatin have stimulated investigation of the anticancer

activity of other metals. This dissertation aims to highlight the potential of ruthenium based

compounds as anticancer agents. In particular, a comparison of such compounds with Cisplatin is

provided in order to answer the question, “Why does the world need ruthenium anti-cancer

agents?”

To begin, a brief review of Cisplatin chemistry will be provided. This is followed by an

introduction to ruthenium biochemistry with a particular focus on, what makes ruthenium a good

candidate for fighting cancer. The last segment of this paper will focus on the various ruthenium

compounds which show promise as anticancer agents.

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Cisplatin

Cis-diamminedichloroplatinum(II) commonly

known as cisplatin, is a coordination compound with

square planar geometry (Figure2)[8]. It was first

synthesized in 1844 by the Italian chemist Michele

Peyrone[10]. The compound was also utilized by Alfred

Werner in 1893 to support his theory of coordination

chemistry, in the process of which he elucidated the

square planar structure[8]. However, cisplatin only truly

became recognized after 1965 when, the Barnett

Rosenberg group at Michigan state university found

that certain electrolysis products of platinum electrodes, later found to be cisplatin, had the ability

to inhibit cell division in Escherichia coli[8]. This observation led to the investigation of the

anticancer properties of cisplatin which showed remarkable success in battling testicular and

ovarian cancer. Cisplatin gained FDA approval in 1978 and by doing so introduced metal based

agents to the war against cancer [8]. In this section, a brief discussion of the synthesis, anticancer

mechanics, adverse effects and limitations of cisplatin is provided. Since cisplatin will serve as the

benchmark to which ruthenium based agents will be compared, this discussion serves to provide

the foundation on which the remaining dissertation will be built.

Synthesis

The first synthesis of cisplatin was accomplished by pure luck when, Michele Peyrone

attempted to synthesize Magnus salt by combining a solution of acidified PtCl2 with excess

ammonia[10]. The reaction resulted in two different products, one green (Magnus’ green salt) and

the other yellow [10]. The two products were separated via a liquid extraction exploiting the

insolubility of Magnus’ salt in hydrochloric acid [10]. The isolated yellow precipitate was given the

name Peyrone’s chloride (now known as cisplatin) and was thought to be an isomer of Magnus’

salt with totally different properties[10]. The synthesis and subsequent separation conducted by

Peyrone is summarized in Figure 3.

Figure 2.Cis-diamminedichloroplatinum(II), commonly referred to as cisplatin is a coordination compound with square planar geometry [10]. This image was reproduced based on [10].

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Since the original synthesis, the process has seen many improvements, mainly targeting

the purity of product obtained, reaction time and overall yield, elements which seemed to be

lacking in early approaches [11]. Many modern synthesis of cisplatin conducted today are based on

the scheme developed by Dhara in 1970 (Figure 4) [11]. In this approach, K2[PtCl4] is added to a

saturated solution of KI, followed by the addition of NH3[11]. The reaction results in the production

of a yellow compound, cis-[PtI2(NH3)2] which is subsequently separated and dried [11]. Next, the

addition of aqueous AgNO3 is utilized to precipitate AgI, effectively removing iodine from the

platinum adduct [11]. The AgI is simply filtered off, leaving behind [Pt(OH2)2(NH3)2]2+ which on

treatment with KCl produces the desired yellow powder, cis-[PtCl2(NH3)2][11]. For the precise

details on the high purity and yield synthesis of cisplatin refer to [12]. It is worth noting that the

success of the synthesis by Dhara rests in the exploitation of the Trans-effect [11]. Discovered by

Chernyaev in 1926, the trans effect refers to the concept that the rate of substitution of a given

ligand depends on the ligand located trans (opposite) to it much more than the ligand located cis

(adjacent) [11]. The ability of various ligands to exert the trans effect is quite closely approximated

by the Spectrochemical series [13]:

CO, CN-, C2H4 > PR3, H- > CH3-, SC(NH2)2 > C6H5

- , NO2-, I-, SCN- > Br-, Cl- > py, NH3, OH-, H2O

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Considering the series, one can now

understand why the particular steps of the

reaction are employed and what makes this

synthesis successful. In the second step,

the trans effect provides one with

selectivity, allowing for the addition of

NH3 in the designated positions as the

stronger director (Iodine) makes the group

trans to it more labile and hence enforces

production of cis-product which ultimately

results in the production of Cisplatin[11].

Mechanism of action

The anti-cancer action of Cisplatin occurs at the end of a two part pathway, composed of a

blood plasma segment and a cellular segment (Figure 5) [11]. The first segment of this pathway

begins immediately after the drug enters the bloodstream. The cisplatin molecules find themselves

surrounded by a high chloride concentration (about 100 mM), which prevents the process of

aquation (replacement of ligands by water)[11]. However, cisplatin is an easy target for plasma

proteins such as human serum albumin which upon binding, are hypothesized to produce

deactivation of the drug and induce nephrotoxicity [11]. Studies have actually shown that 65 to 98%

of the platinum in blood plasma is protein bound after one day of drug administration [11]. Clearly,

protein binding is a major concern in the therapeutic pathway of cisplatin. The free cisplatin that

is left intact can go on to the next segment of the pathway, the cellular stage. The cisplatin

molecules enter the cell mainly via diffusion through the cell membrane [11], although new studies

suggest active transport via copper transporting proteins may also be possible [11].

Once inside the cell, a chloride ligand of cisplatin is replaced by water, producing a

monoaqua cisplatin adduct [11]. This substitution of a chlorine ligand is brought on by the low

intracellular chlorine concentration (about 4 mM) & allows for the violation of the spectrochemical

series (i.e. the chlorine ligand, which exert a stronger trans-effect relative to the ammine ligands is

Figure 4. The synthesis of cisplatin, cis-[PtCl2(NH3)2],

developed by Dhara[12]. This image was obtained from

[11].

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substituted)[11]. The aquation of the cisplatin molecule converts the cisplatin into a reactive charged

species that cannot escape the cell membrane and promptly reacts with a nitrogenous base

(typically guanine) to make a monofunctional DNA adduct [11]. Potential sites of DNA and cisplatin

interaction are shown in Figure 6. Next, ring closure promotes the conversion of the

monofunctional DNA adduct into a bifunctional adduct [11]. It may occur directly from the

monofunctional DNA adduct (nucleophilic attack of the nitrogen from the nitrogenous base to

replace chloride ligand) or via a second aquation followed by rapid ring closure [11]. As pointed out

above, the majority of the bifunctional DNA cisplatin adducts are either guanine-guanine (65% of

the time) and adenine-guanine (25% of the time)[11]. Such bifunctional adducts force the DNA to

take on a bent shape, effectively damaging the DNA [11][14]. To deal with the damaged DNA, the

cell may either signal transduction pathways which initiate apoptosis, the process of programmed

cell death, or attempt to repair the damage [14]. The repairing process can be conducted via two

pathways.

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In the first, transcription of the DNA is inhibited followed by a restoration procedure called

transcription coupled repair (TCR) [14]. Initiated by RNA polymerase, TCR is a subpathay of the

nucleotide excision repair (NER) process and is responsible for removing abrasions from the

template DNA strands of actively

transcribed genes [15]. In the second

repair pathway, the cell imposes a cell

cycle arrest (i.e. DNA replication is

inhibited), in order to provide the cell

with time to make the necessary

repairs[14]. If the repair is not

successfully accomplished, apoptosis is

initiated [14]. In cases of severe DNA

damage, poly(ADP-ribose)polymerase

(PARP), responsible for cleaving NAD+

and thereafter transferring ADP-ribose

moieties (ADPR) to carboxyl groups of

nuclear proteins is found to be

hyperactive[14]. This hyperactive state

leads to the depletion of cellular

NAD+/ATP, which upon achieving a

lethally low concentration result in

death via necrosis, an alternative mode

of cell death[14]. Compared to the cell shrinkage, chromatin condensation and DNA fragmentation

that characterizes apoptosis, necrosis is cell death brought on by cytosolic swelling and early loss

of plasma-membrane integrity [14]. The mode of cell death induced by cisplatin has been found to

be dependent on the drug concentration; at higher concentrations necrosis triumphs, while

apoptosis is dominant at lower concentrations [14]. This is should not come as a surprise, since

necrosis requires excessive DNA damage which is more likely as the concentration of cisplatin in

the body is increased.

Figure 6. Potential sites on the various nitrogenous bases of

the DNA strand where cisplatin may bind. The N7 position

of the guanine base is often preferred [12]. Such binding can

lead to kinks in the DNA, which if not repaired leads to

apoptosis and/or necrosis[17]. This image was obtained from

[11].

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To summarize, cisplatin targets the rapid proliferation of cancer cells via induction of cell

death. The cell death is a result of bifunctional DNA cisplatin adducts which lead to kinks in the

DNA. The damaged DNA results in direct initiation of apoptosis via transduction pathways or

indirect initiation of apoptosis and necrosis via DNA repair pathways.

Limitations

Cisplatin is employed in treatment

of a wide range of solid tumours and is

considered one of the most effective

chemical agents in cancer chemotherapy

[16]. That being said, cisplatin is not a

perfect drug as rampant drug resistance

and cytotoxicity severely limit the drug’s

curative abilities [16]. In fact, studies of

patients with ovarian cancer showed a

success rate of a measly 15 to 20% upon

treatment with cisplatin over the duration

of five years [16]. The dismal rate of

success has been mainly attributed to the

drug resistance developed by primary

tumours [16] . Such drug resistance is

brought on by a multitude of factors

including reduced drug uptake, increased

drug inactivation, and greater DNA

adduct repair [16]. To obtain a complete

understanding of the drug resistance at the

molecular level, [14][16] should be

consulted. It is also worth noting that the molecular machinery responsible for cisplatin resistance

varies from tumour to tumour, that is to say, different tumours have different strengths of drug

resistance [16]. In addition to the issue of drug resistance, cisplatin has also been found to be

extremely toxic [8][17]. Adverse effects of cisplatin include, severe kidney problems, allergic

Figure 7. Pathways of cell death induced by cisplatin.

Necrosis has been found to occur at high concentrations of

the drug while apoptosis occurs at low concentrations. This

image was obtained from [14].

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reactions, decreased immunity to infections, gastrointestinal disorders, hemorrhage, and hearing

loss [8]. For a complete treatise of the nephrotoxicity and cytotoxicity associated with cisplatin,

refer to [8] and [17]. Such limitations have of the drug have inspired exploration of other platinum

metals (groups 8, 9 and 10) in order to develop a new metal based agent that not only bypasses

the limitations of cisplatin but also its potency. Amongst the many candidates being actively

examined, Ruthenium has been found to be relatively promising [18].

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Ruthenium Biochemistry

First recognized as a distinct metal in 1844,

Ruthenium (“Ru”) is the second least abundant platinum

metal [19]. Its ability to adopt multiple oxidation states,

ranging from -2 to +8, has allowed for the development

of numerous complexes with various applications [18].

Examples include, Ruthenium sulfoxides which have

been actively utilized in catalysis and Ruthenium

polypyridyls, which have been employed as DNA

cleavage agents and photosensitizers for solar cells [18]. In

this section, the oxidation states, ligand exchange

kinetics and iron mimicking abilities of ruthenium are discussed as these properties are known to

contribute to the biocompatibility of ruthenium, a highly desired feature in synthetic drugs [20][22].

Oxidation State

Although ruthenium is known to adopt a range of oxidation states, under physiological

conditions Ru(II) (d6, diamagnetic soft acid) and Ru(III) (d5, paramagnetic intermediate acid)

predominate[23][24]. Both Ru2+ (t62g) and Ru3+ (t52g) prefer low spin hexacoordinate octahedral

complexes, offering two additional ligand binding sites relative to square planar Pt(II) complexes

such as that in cisplatin[20] [24]. In addition, Ru3+ complexes are generally more biologically inert

relative to Ru2+ [20][23]. To explain this difference in reactivity, a so called “activation-by-reduction”

hypothesis was proposed by the Clarke group [23]. This hypothesis is based on an observation in

which, inert Ru (III) chlorido-ammine compounds such as fac-[RuCl3(NH3)3] and cis-

[RuCl2(NH3)4]Cl were found to undergo an in vivo reduction to Ru(II) species prior to aquation; a

process which results in a labile active species that can coordinate to biological target(s)[23]. So

simply speaking, the hypothesis states that the activation (aquation) of Ru(III) occurs indirectly

via a reduction of Ru(III) to Ru(II) followed by the aquation/activation. The in vivo reduction of

Ru(III) complexes is conducted by a variety of proteins such as glutathione, ascorbate,

mitochondrial and microsomal single electron transfer proteins[20]. Amongst these proteins,

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mitochondrial single electron transfer proteins are especially of interest as apoptosis can be

initiated in mitochondria inducing death of tumour cell [20]. It is also worth noting that cancer

tissues generally offer a more reductive environment relative to healthy tissues as they are low in

oxygen concentration, contain higher levels of glutathione and have lower pH[20]. The relatively

reductive environment of the cancer cells and the redox chemistry of ruthenium provides an

effective technique for administration (Figure9). That is to say, one can utilize the Ru(III)

complexes as prodrugs, which upon interaction with the reductive environment of the cancer tissue

are reduced to Ru(II) producing the active agent[20]. It is also possible for the Ru(II) to be oxidized

by molecular oxygen and cytochrome oxidase[20]. As a consequence, Ruthenium species inside the

cancer cells exist mostly in the form of the labile Ru(II), while inert Ru(III) species dominate

healthy cells. This essentially means, ruthenium agents are not as cytotoxic as platinum based

agents such as cisplatin for which no such redox effect exists.

In addition to Ru2+and Ru3+, Ru4+ complexes are also known to exist under physiological

conditions but require several acido, oxo, or sulfide ligands for stabilization; unfortunately little is

known about the pharmaceutical abilities of such compounds[23][24].

Figure 9. A schematic diagram depicting the oxidation states of ruthenium in the contrasting redox environments

of cancer and healthy tissue. As shown, labile Ru(II) dominates the reductive environment of cancer tissue, while

inert Ru(III) dominates the relatively oxidative environment of healthy tissues. This image was obtained from [20].

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Ligand Exchange Kinetics

Another factor which often contributes to the biological activity of a given metal is its rate

of ligand exchange [20]. This is due to the fact that coordination compounds are often subjected to

modifications induced by interactions with biological molecules and/or water[20]. As previously

discussed in the cisplatin section, such interactions are relatively important as they can lead to drug

activation or deactivation. Based on this idea, one may argue that one of the reasons cisplatin is an

effective antineoplastic drug is probably related to the ligand exchange kinetics of platinum.

Platinum has a rate of aqua ligand exchange that falls in the order of minutes to days, a range

which is quite low compared to other metal based compounds which have aqua ligand exchange

kinetics in the order of microseconds to seconds (Figure10) [22]. This essentially means that

platinum based agents have great kinetic stability which prevents the occurrence of rapid

equilibrium reactions that may agitate the DNA-Drug adduct which is vital to drug action [22].

However, platinum is not the only metal with ligand exchange kinetics suitable for biological

targets. Ru (II) and Ru(III) also exhibit aqua ligand exchange kinetics that are on a time scale

comparable to Pt(II) and Pt(IV)[22]. In other words, ruthenium based agents also carry the ability

to form kinetically stable complexes which are not easily perturbed via rapid equilibrium reactions.

The first-order water exchange rate constants are roughly 10-2 per second (t1/2 ~1 minute) and 10-

6 per second (t1/2 ~19 hours) at 25 °C for Ru(II) and Ru(III) respectively[24]. This makes sense, as

Ru3+ with its greater ∆ has a higher free energy of activation (∆�‡) and as such is expected to have

a lower rate constant [24]. This information may make it appear as though, Ru3+ is not very well

fitted for biological use as the ligand exchange rates are simply too slow, however, this view can

be misleading as the ligand exchange for a given ion tends to vary with the nature of the

coordinated ligands[24]. So even though the rate of aqua ligand exchange is slow, this does not

necessarily mean Ru3+ cannot be utilized as a potent agent in chemotherapy.

Another reason, the ligand exchange kinetics of Ru2+ and Pt2+ are especially interesting is

due to the fact that they fall in a range that is similar to the rate of cellular division [22][25]. That is

to say, once these complexes are bound they stay bound for the duration of the cell’s life [25].

Furthermore, Ru2+ actually has the ability to bind to a range of biomolecules and not just DNA

(target of cisplatin) [25]. This feature may play a role in the anticancer mechanics of potential drugs,

allowing for the targeting of cisplatin resistant cell lines. This ability appears to stem from the

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capability of Ruthenium to form strong chemical bonds with a variety of ligands of differing

chemical hardness and electronegativities [25].

Figure10. A schematic of the water exchange constants of various metal ions at 25 ℃. Note, both Ruthenium and Platinum have ligand exchange rates that fall in the range of the rate of cellular division and are significantly lower than the rates exhibited by many other mono/divalent cations. This image was obtained from [24].

Iron Mimicking

In addition to the reduced cytotoxicity produced by the “activation by reduction” effect,

another reason for lower cytotoxicity of ruthenium is believed to be a result of its ability to mimic

iron. As a member of the Iron family, ruthenium is believed to bind many iron based biomolecules

including serum transferrin and albumin[20]. These proteins are involved in the solubilisation and

transportation of iron in mammals, processes which effectively lower the cytotoxicity of iron [20].

By potentially binding such proteins, ruthenium too can obtain greater solubility and efficient

transport, lowering its cytotoxicity.

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Furthermore, the greater iron requirement of rapidly dividing cancer cells, means these

cells often have a greater number of transferrin

receptors on their surfaces (Figure11). [20]. As

a result, such cells exhibit greater interactions

with ruthenium loaded transferrin proteins and

accumulate more Ruthenium relative to

healthy cells[20]. In fact, in vivo studies of

radio-labelled compounds in cancer cells have

shown 2-12 times (depending on cell type) the

amount of ruthenium uptake by cancer cells

versus healthy cells [20]. In comparison, protein

binding is actually quite problematic in the

action of cisplatin. As discussed earlier, the

binding of the drug to plasma proteins results

in drug deactivation and nephrotoxicity [11].

Clearly, this issue may be resolved by switching to Ru2+ ion based agents.

To conclude, ruthenium is a very strong candidate for employment in the synthesis of

antineoplastic agents as it offers valuable biocompatibility. This feature appears to be a product of

its ability to mimic iron, its oxidation states and its ligand exchange kinetics. In the following

sections, a survey of the key ruthenium based anticancer agents is provided.

Figure 11. A schematic diagram comparing the

transferrin mediated Ruthenium uptake in cancer and

healthy cells. Cancer cells have shown, 2-12 times as

much ruthenium compared to healthy cells. This image

was obtained from [20].

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Ruthenium Anticancer Agents

The biological activity of ruthenium complexes was first documented in work by Dwyer

in the 1950s [26]. Unfortunately, the work received little attention and was largely forgotten until

the discovery of cisplatin, which stimulated interest in other metals including ruthenium [26].

Today, a number of ruthenium based anticancer agents are known to exist. These agents include

traditional coordination compounds which bind biological targets directly and organometallic

compounds, which in addition to binding biological targets also produce reactive oxygen species

(ROS) that are able to further chemically modify molecules in the cell [24]. Some ruthenium

complexes can even act as competitive inhibitors, shutting down enzymes vital for cell survival

[24]. In the subsequent discussion, a survey of the key ruthenium based anticancer agents is

provided.

NAMI-A

Imidazolium trans-tetrachloro (dimethylsulfoxide)

imidazoleruthenate(III) commonly known as NAMI-A

(New Anti-tumour Metastasis Inhibitor A) (Figure12) was

the first ruthenium based anticancer agent to reach phase I

clinical studies [22] [24]. The drug has now successfully

completed phase I clinical studies for treatment of

metastatic cancer [24]. Here a discussion of the synthesis,

metabolism, anticancer mechanics and clinical data of

NAMI-A is provided.

Synthesis

The synthesis of NAMI-A begins with reaction of

the octahedral Ru3+ complex, RuCl3∙ 3H2O, with a mixture

of hydrochloric acid (HCl) and dimethylsulfoxide (DMSO) in ethanol [24]. The reaction results in

the addition of a chlorine and two DMSO ligands to the Ru3+ metal center, producing a 6 coordinate

anionic trans DMSO complex [24]. Next, addition of excess imidazole to the anionic compound in

Figure 12. NAMI-A abbreviated

[ImH][trans-RuCl4(DMSO)(Im)] (Im

=imidazole, DMSO = dimethylsulfoxide)

is a Ru3+ based octahedral complex that

has completed Phase I clinical studies in

the treatment of metastatic cancer. This

image was obtained from [24].

17 | P a g e

acetone results in replacement of one DMSO ligand by imidazole leading to the formation of a brick

red salt, NAMI-A, with imidazolium acting as the cationic counter-ion [24]. Much like Dhara’s

synthesis of cisplatin (Figure4), the synthesis of NAMI-A also appears to exploit the trans-directing

effect of ligands. In the case of NAMI-A, the trans-direction is conducted by the sulfur ligand of the

DMSO, which is able to utilize its unoccupied 3d orbitals to overlap with the occupied 4d orbitals

of Ru3+ ion, acting as a strong � acceptor and hence a strong trans-director[24]. The synthesis of

NAMI-A is summarized in Figure 13 below.

Figure 13. A summary of the synthesis of NAMI-A. This image was obtained from [24].

Drug Metabolism & Biological Activity

In order to understand how a particular compound functions in a biological setting, it is

often very important to learn about how the compound is broken down (i.e. metabolised). So, if one

is to learn about the anti-cancer mechanics of NAMI-A, one must first understand how the drug is

metabolised. To aid with this, a study was performed by the Reedijk group in which the hydrolysis

of NAMI-A in conditions closely resembling those of blood were examined via 1H-NMR [24]. To be

more specific, the hydrolysis of 5mM NAMI-A in phosphate buffer (0.05M phosphate, 0.15 M

NaCl, D2O), at pH 7.4 and 37℃ was examined [24]. This analysis is based upon the premise that the

protons of imidazole and DMSO coordinated to the paramagnetic Ru3+ (s =1/2) can be easily

identified in the NMR spectra [24]. However, due to the presence of the paramagnetic metal center

the signals are shifted upfield and broadened [24]. The observed 1H-NMR spectrum at various times

after initial addition of NAMI-A to phosphate buffer is presented in Figure 14.

18 | P a g e

As shown in Figure 14, the two methyl groups of the coordinated DMSO are equivalent

owing to the Cs symmetry of the species and result in a broad singlet at roughly -14.8 ppm [24]. This

chemical shift is significantly upfield compared to free DMSO and DMSO coordinated to a

diamagnetic center [24]. The proton at the C5 position (“Im H5” in Figure14) on the coordinated

imidazole ring, manifests as a narrow singlet at roughly -3.2 ppm [24]. On the other hand, the non-

equivalent protons at the C2 and C4 position express as significantly broadened peaks at roughly -

5.6 and -7.8 ppm respectively [24]. The difference in the shift of the proton at the C5 position versus

the C2 and C4 position can be attributed to differences in the fermi contact shift term and the pseudo-

contact shift term. To put it simply, the C2 and C4 positions have greater spatial proximity to the

paramagnetic center and are also closer in terms of the number of bonds relative to C5 position and

thus have a greater shift (i.e. more upfield and broad) [27]. These somewhat questionable peak

assignments for the imidazole ring protons are based on previous characterization of NAMI.

Additional signals are obtained from the cationic counter ion, imidazolium. The counter ion having

a pKa of 6.95 exists largely in a deprotonated neutral form, contributing two singlet peaks at 7.49

and 8.96 ppm (Figure14)[24]. The singlet at 7.49 ppm is a result of the protons at the C4 and C5

position, which are equivalent due to resonance and tautomerization, both of which are fast relative

to the NMR time scale [24]. The singlet at 8.96 ppm is due to the proton at the C2 position. Refer to

Figure 13 for the numbering scheme of the imidazole ring.

As observed in Figure 14, the hydrolysis of NAMI-A is actually quite rapid and occurs on

the timescale of minutes. In fact, new broad peaks are observed a mere 10 minutes after the

dissolution of the drug accompanied by a decrease in the intensity of coordinated DMSO peak and

a shift in the coordinated imidazole peak [24]. The fact that the new peaks are broad indicates that the

metal retains its oxidation state, in other words exists as paramagnetic Ru3+ [24]. To understand the

species responsible for the emerging signals, a plot of area under signal peak (intensity) as a function

of time was created [24]. Based on the plot, it was proposed that one of the chloride ligands

coordinated to NAMI-A was substituted by water forming a mer-[RuCl3(dmso-S)(Im)(H2O)]

(NAMI-AH2O in Figure 14)[24]. Unfortunately, the aqua species was found to be short lived as the

coordinated DMSO is swiftly lost resulting in polynuclear species which could not be further

analyzed [24]. The half-life (t1/2) of the hydrolysis of NAMI-A was found to be roughly 20 minutes

at pH 7.4 with the stability increasing at lower pH values [24]. These experimental findings are

supported by a computational study which reports that the hydrolysis of a chlorine ligand is initially

19 | P a g e

more thermodynamically favored relative to DMSO and Imidazole [28]. However, as the hydrolysis

proceeds the dissociation of DMSO starts to compete with the dissociation of chlorine ligands, a

competition attributed to the similar thermodynamics of both pathways [28]. In addition the study

also noted that the hydrolysis of the coordinated imidazole ring is unlikely due to the high activation

barrier involved [28]. These findings based on the density functional theory (DFT) calculations

essentially mimic the experimental findings, providing validity for the proposed hydrolysis. Clearly,

NAMI-A is capable of forming active species (“aquated” complexes) that may react with substances

in the blood and/or inside the cell [24]. That being said, Ru3+ NAMI-A may not be directly

responsible for the biological action [24].

Figure 14. The hydrolysis of 5 mM NAMI-A in phosphate buffer at pH 7.4 and 37 ℃ and various after initial

addition of NAMI-A to phosphate buffer. Acetone, was utilized as an internal standard. This image was obtained

from [24].

Recall, the “activation-by-reduction” postulate by the Clarke group which states that the

biological action of Ru3+ proceeds via a bio-reduction to Ru2+ resulting in a labile species[23][24]. In

fact, DFT calculations have found that the Ru2+ based NAMI-A is much more readily hydrolyzed

compared to Ru3+ NAMI-A, with the entire process being exothermic for Ru2+ NAMI-A and slightly

endothermic for Ru3+ NAMI-A [28]. So it is entirely possible that the Ru3+ based NAMI-A, functions

20 | P a g e

as a prodrug which is converted into a labile species by bio-reductants, it is then the new labile

species which interacts with biomolecules and exerts the anticancer effect [24]. To investigate this

possibility, Brindell and group examined the reduction of NAMI-A by ascorbic acid (“H2A”), a bio-

reductant found in abundance (11-79 µM) in blood serum, using stopped-flow kinetics [24]. The UV-

visible absorption spectra before and at various times after the addition of H2A is provided in Figure

15 [24]. As shown by the dashed line in Figure 15, the absorption spectrum prior to the addition of

ascorbic acid consists of one strong absorbance band at roughly 390 nm with a molar extinction

coefficient of about 3240 M-1 cm-1 [24]. Based on the values of the molar extinction coefficient, it is

clear that the absorbance band is likely a result of a charge transfer interaction [24]. Immediately after

the introduction of H2A to the NAMI-A solution, a pronounced decreased in the intensity of the

charge transfer band at 390 nm was observed accompanied by emergence of two new absorption

peaks characteristic of d-d transitions (based on the much lower intensities compared to the earlier

charge transfer band) at 350 and 430 nm, this change is depicted by the solid line in Figure 15[24].

As time progressed, the new d-d transition peaks were found to gradually shift to lower wavelengths

indicating an increase in the crystal field energy of the Ru2+ metal center [24].

The second-order rate constant for the reduction of Ru3+ in NAMI-A to the corresponding

Ru2+ species by H2A was found to be 47.0 ± 1.8 M-1s-1 (pH 5 and 35℃)[24]. To achieve the reduction,

the H2A molecules first transfer an electron to one of the ligands coordinated to NAMI-A, the ligand

then transfers the electron to the metal center resulting in the reduction of Ru3+ to Ru2+ [24]. In other

words the electron transfer process between NAMI-A and ascorbic acid occurs via an outer sphere

mechanism [24]. In addition, Brindell also found that the presence of excess chloride ions in the

solvent can influence the kinetics of the electron transfer (i.e. reduction of NAMI-A)[24]. The excess

chloride ions were believed to hinder the aquation of the Ru2+ based NAMI-A, preventing the

formation of labile species which may react with Ru3+ NAMI-A to form a binuclear species [24]. In

this way, the Ru2+ form of NAMI-A was proposed to be playing a catalytic role in the reduction [24].

The impact of Chloride ions on the kinetics of the electron transfer is depicted in Figure 16 below.

21 | P a g e

Figure15. The UV-visible absorption spectra of 5 mM NAMI-A before the introduction of ascorbic acid, 12 seconds

after the introduction of ascorbic acid and during hydrolysis in the presence of NaCl at time intervals of 1,2, 4, 8 and 10

minutes[24]. This image was adapted from [24].

Figure 16. The absorption of NAMI-A at 402 nm after reduction with ascorbic acid in the presence of 0.24 M

NaClO4 and 0.24 M NaCl[24]. Note, perchlorate ion (ClO4-) being low on the spectrochemical series is not expected to

bind Ru3+ or Ru2+ and hence acts as a spectator ion[24]. This image was adapted from [24].

22 | P a g e

Anticancer Mechanics

NAMI-A is a unique anticancer agent in that it has essentially zero potency in targeting and

eliminating primary tumours like cisplatin [24] [26] [29]. That being said, the drug is far from useless as

it has shown remarkable selectivity and effectiveness in the inhibition of the formation and growth

of lung, mammary and B16F10 melanoma metastasis [29]. So even though the drug is not able to

fight tumours directly like cisplatin, by suppressing the spread of cancer cells it provides valuable

time which may be utilized to surgically remove the primary tumour and cure the patient [24]. The

anti-metastatic activity of NAMI-A is thought to be a result of a range of pathways including, cell

cycle arrest at the G2M premitotic phase, apoptosis in endothelial transformed cells, and interactions

with actin-type proteins on the cell surface and/or extracellular matrix collagen [29]. Although many

different pathways are proposed, the exact biological target of the drug remains elusive [24] [29].

However, it is generally agreed that NAMI-A has a weak affinity for DNA and its bases and hence

does not exert its anti-metastatic effect via interactions with DNA like cisplatin [24].

Recent work has led to the speculation that the target of ruthenium responsible for anti-

metastatic activity may be nitric oxide (NO) [29]. This view is based on the increased expression and

activity of Nitric oxide synthase (NOS) in various human tumours which suggests that a key role is

played by NO in cancer development [29]. Ruthenium complexes such as NAMI-A have the ability

to act as NO acceptors and donors in vivo and by doing so are able to interrupt the NO-dependent

angiogenesis, inhibiting the process of metastasis without having to target the DNA [29]. The

nitrosylation of NAMI-A under conditions similar to the human physiology (pH = 7.4, [NaCl] = 0.1

M, T = 37℃) is depicted in Figure 17.

As shown, the coordination of the NO to Ru3+ results in the reduction of Ru3+ metal center,

producing a ruthenium nitrosyl complex [Ru2+-NO+][29]. The change in the oxidation state upon NO

binding is verified by the presence of a very weak, molar extinction coefficient of about 50-60 M-1

cm-1, absorption band at roughly 500 nm [29]. As previously mentioned, this is significantly different

from the parent ruthenium(III) metal center the spectrum of which features a strong ligand to metal

charge transfer band [24][29]. The ruthenium nitrosyl complex features a linear Ru-N-O arrangement

with the electrophilic NO having a stretching frequency greater than 1870 cm-1 [29].

23 | P a g e

Figure 17. The Nitrosylation of NAMI-A under physiological conditions. This image was obtained from [29].

Clinical Trial Data

Phase I clinical studies of NAMI-A began in 1999, making NAMI-A the first ruthenium

based anticancer agent to reach human clinical development [22]. The study investigated the effect

of varying doses on 24 patients with varying metastatic solid tumours such as colorectal, lung,

melanoma, ovarian and pancreatic cancers, administrating NAMI-A via 3 hour intravenous infusion

daily for 5 days every 3 weeks [22]. NAMI-A was found to be relatively nontoxic up to doses of 500

mgm-2 day-1, after which point patients developed painful blisters on hands and feet [22][24]. This

dosage is significantly greater than the 20-140 mgm-2day-1 range considered tolerable for cisplatin

[24]. Clearly, cisplatin is much more toxic than NAMI-A. The half-life of ruthenium clearance from

plasma was found to be 50 ±19 hours and no distinct relationship was found between the amount of

drug bound to white blood cells and the dose administrated [24]. In addition, analysis of genomic

DNA from white blood cells showed no significant binding of ruthenium to GG and AG DNA

sequences [24].

24 | P a g e

KP1019

Synthesized by the Keppler group, Indazolium-trans-bis(1H-

indazole)-tetrachlororuthenate(III), often referred to as

KP1019 (or FFC14A), is another popular Ru3+ based

anticancer agent[22][24][30]. Showing significant potency against

colon carcinomas and cisplatin resistant cell lines, it is the

second ruthenium based anticancer agent to reach clinical

studies [22] [30]. Here a discussion of the drug synthesis, clinical

data, biological activity, and anticancer mechanics is offered.

Synthesis

Much like the synthesis of NAMI-A, the synthesis of

KP1019 also utilizes the commercially available RuCl3∙

3H2O as the starting material [24] [30]. Addition of this

octahedral precursor to a refluxing mixture of HCl and

ethanol followed by addition of excess indazole, results

in the red-brown powder known as KP1019[30]. The

resultant complex much like NAMI-A was also found to

be a salt, with an indazolium serving as the counter ion

to the trans-indazole Ru3+ anionic complex. This

synthesis is summarized in Figure 19.

Unfortunately, the synthesized KP1019 exhibited poor solubility in water and as a result

could not be directly employed for examination in clinical studies [30]. To resolve this issue, an

alternative approach was devised in which the 35 times more soluble sodium analogue, KP1339

(Figure 20), was employed as the precursor [30]. To be more precise, KP1019 was prepared in situ

by dissolving KP1339 in an isotonic solution of NaCl at room temperature [30]. The isotonic mixture

was then transferred into a clean container where upon addition of excess indazolium chloride, the

desired complex, KP1019, was obtained [30]. This in situ synthesis is performed just before infusion

and administration of the drug [30]. The sodium based precursor, KP1339, can be prepared from

25 | P a g e

KP1019 via a simple salt metathesis reaction with ammonium salts [30]. The results from the clinical

trials of the drug are summarized below.

Clinical Data

Phase I clinical studies of KP1019 investigated the pharmacokinetics and dosage of the drug

via intravenous administration to patients with various solid tumours [30]. The drug was administrated

in doses ranging from 25 to 600 mg twice, weekly, over a 3 week period and found to cause up to

10 weeks of disease stabilization, independent of the drug dosage with no significant dose limiting

toxicity [24][30]. The drug was found to be mainly (80-90%) protein bound in the blood plasma and

exhibited a terminal half-life in the range of 69-284 hours [30]. Much like NAMI-A, the tolerable

dosage range of KP1019 is also considerably greater than that of cisplatin [24].

Biological Activity & Transport

KP1019 has exhibited great stability in its solid and solution forms, making it easy to store and

administrate [30]. That being said, the occurrence of gradual hydrolysis, as observed by the

replacement of chloro ligands by hydroxyl and/or aqua groups, has been found by a study based on

electrospray ionization mass spectrometry (ESI-MS) [30]. Such hydrolysis was found to be both

temperature and pH sensitive, with greater drug stability being observed at lower pH and

26 | P a g e

temperature values [30]. The half-life of the hydrolysis at physiological conditions (pH 7.4 and 37℃)

was found to be less than half an hour [30]. This suggests that the drug is capable of achieving rapid

metabolism, forming labile active

derivatives which can go on to form adducts

with numerous biological molecules. In

particular, the drug is believed to bind with

two plasma proteins, serum albumin and

transferrin [30]. Investigation of the drug-

protein binding kinetics have suggested that

KP1019 thermodynamically prefers serum

albumin, but kinetically favors transferrin

[30]. This finding advocates that transferrin

acts as a shuttling system for the drug while

serum albumin acts as a storage, regulating

drug accumulation [24] [30].

As previously discussed, the

transport of ruthenium by transferrin is

particularly attractive as it provides a facile, non-toxic, and somewhat selective method of

transportation [20]. The binding of the drug to transferrin has been elucidated by a crystal structure

of apo-lactoferrin (a close homolog of human transferrin) [24][30]. As shown in Figure 21, the binding

of KP1019 occurs via His253 contained in the active site [24] [30]. Further examination of the transferrin

based drug transport shows that at least one iron(III) must be bound to the protein in order for it to

properly function, as the sole binding of ruthenium(III) appears to cause conformational changes

which hinder protein recognition [30]. The release of the ruthenium drug typically requires acidic

conditions and as a result are crucial to the activity of the complex [30]. This protein mediated

transportation of the drug may be one of the major factors contributing to the relatively low toxicity

of the drug compared to cisplatin for which no such interaction exist.

Similar to NAMI-A, the Ru3+ metal center of KP1019 is also susceptible to reduction into

the more labile Ru2+ by bio reductants such as ascorbic acid and glutathione[24][30]. In other words,

KP1019 may also be a prodrug, which upon reduction is converted into a reactive species

27 | P a g e

responsible for anticancer activity. This activation-by-reduction hypothesis is supported studies of

colon carcinoma cell line SW480, which have showed a correlation between greater antineoplastic

activity and greater reduction potential of the Ru(III) metal center [30]. The mechanics for the

anticancer activity of the drug are provided below.

Potential Anticancer mechanics

In vitro studies of KP1019 have demonstrated the potency of the agent in successfully

inducing apoptosis in two colorectal tumor cell lines, SW480 and HT29[30]. Apoptosis induced by

KP1019 is characterized by the downregulation of bcl2 (an anti-apoptotic protein), the activation of

caspase 3 (a pro-apoptotic protein) and depolarization of the mitochondrial membrane [31]. This

anticancer activity is believed to stem from the agent’s ability to produce hydrogen peroxide (H2O2),

a reactive oxygen species (ROS) [31]. The formation of reactive oxygen species by the ruthenium

based agent is not very surprising, as it is well known that iron can react with unsaturated fatty acids

and their hydroperoxides in a Fenton type reaction to produce H2O2 [31]. As a member of the iron

group, ruthenium can mimic iron in biological settings and therefore has the ability to partake in the

mentioned interaction as a substitute for iron. The produced H2O2 can go on to react with membrane

lipids to produce even more H2O2 [31]. These labile species induce cytotoxic and genotoxic effects

via interacting with macromolecules in the cell such as DNA, where such species cause oxidation

of bases, promutagentic adducts and strand breaks [31]. In fact, KP1019 can actually react directly

with DNA like cisplatin, however, such direct interactions occur at a much lower intensity compared

to cisplatin [31]. In addition, the DNA damage produced by KP1019 is believed to be partially due to

the reactive oxygen species and not a sole result of the drug-DNA adducts (like in the case of

cisplatin) [31]. That being said, the low intensity of the DNA strand-breaks produced by KP1019 may

not actually be enough to kill cells, indicating that unlike in the case of cisplatin, DNA damage is

not a key site for initiation of apoptosis [31]. This difference in site of action may be the reason

KP1019 can target cisplatin resistant cell lines [30].

As previously mentioned, in addition to inducing DNA damage, the H2O2 also reacts with

unsaturated fatty acids in the cell including those within the mitochondrial membrane [31]. This

interaction of the H2O2 with the unsaturated fatty acids in the mitochondrial membrane results in a

loss of mitochondrial membrane potential (MMP), which is believed to be the main factor behind

the anticancer effect of the drug [31]. The reduction in MMP can be observed 2-6 hours after the

28 | P a g e

introduction of the drug, depending on the drug concentration [30]. MMP is a function of the charge

difference between mitochondrial matrix and the cytosol [32]. So a decrease in MMP essentially

means a reduced difference between the mitochondrial matrix and the cytosol (i.e. greater

mitochondrial matrix permeability). The increased permeability of the mitochondrial matrix allows

for the release of multiple pro-apoptotic proteins contained within the inner mitochondrial matrix,

resulting in apoptosis [32]. This is quite a potent mechanism as it is able to bypass the need to induce

DNA damage in order to initiate apoptotic signals, like in the case of cisplatin. This is beneficial as

it eliminates the issue of drug resistance brought on by DNA repair. That being said, KP1019 is not

free of drug resistance.

The potency of KP1019 appears to be unhindered by the activity of many different multidrug

resistance-associated proteins such as MRP1, BCRP, and LRP [30]. However, the drug was found to

be sensitive to high concentrations of the drug effluxing protein, P-glycoprotein [24] [30]. The protein

results in a reduced intracellular accumulation of the drug, inducing a type of drug resistance [30].

However, it should be noted that even though drug resistance is an issue, the resistance to KP1019

over time is a mere 10% compared to the acquired resistance against cisplatin [24].

Evidently, KP1019 is a potent anticancer agent, capable of inducing apoptosis via production

of cellular oxidative stress [31]. Although the drug can bind DNA, much like cisplatin, DNA-damage

does not appear to be the main pathway with which KP1019 induces apoptosis; the main site of

action of KP1019 is the mitochondrial matrix [31]. This difference in site of action makes KP1019 a

valuable candidate in cancer chemotherapy as cisplatin resistant cell lines may be targeted [30]. It

should be noted that the drug does face an issue similar to cisplatin, acquired drug resistance.

However, the resistance to KP1019 has been found to be significantly lower compared to cisplatin

drug resistance [24].

Next, a brief introduction is provided to other potential ruthenium anticancer agents. In

particular, the Ru(II) based agents, ruthenium arene compounds and ruthenium polypyridyls are

discussed.

29 | P a g e

Other Ruthenium Candidates

The success of ruthenium as an anticancer agent has been demonstrated by the ruthenium

(III) based agents, NAMI-A and KP1019. Although these drugs are quite potent in what they do,

they are far from being what one would call a magic bullet. The potency of NAMI-A is lacking in

the sense that it is not able to target primary tumors, combating only the issue of metastasis[29] [33].

Meanwhile, KP1019 is flawed by the issue of acquired drug resistance [24] [30]. That being said, it is

quite clear that ruthenium has immense potential as a metal base for a chemotherapeutic agent and

is therefore being further investigated. In particular, recent work on the ruthenium based anticancer

agents is focused on employment of ruthenium in the +2 oxidation state, as this is believed to be the

active form of ruthenium in Ru(III) based drugs[20][33]. In this section, a brief introduction is

provided to the two prominent types of Ru(II) based anticancer agents, ruthenium arene and

ruthenium polypyridyls complexes.

Ruthenium(II) Arene Complexes

Commonly referred to as “piano stool”

complexes, Ru(II) arene complexes can be sub-

classified into two main families(Figure22); RAPTA

([Ru(η6-arene)(PTA)X6], where PTA is 1,3,5-triaza-7-

phosphoadamantane) type complexes and RAED

([Ru(η6-arene)(en)Cl]+ where en stands for

ethylenediamine) type complexes[33][34]. The

anticancer activity of such complexes was first

reported in 2001 by Sadler, who was investigating the

RAED family of complexes[33] [34]. In particular, Sadler

and his group explored the ability of the RAED type

complexes (Figure23) in targeting a human ovarian

cancer cell line called A2780 [33]. All RAED type

complexes were found to be active against the A2780,

with the potency increasing as a function of the size of

the Arene ligand [33]. In other words, the increased

lipophilicity of the compound appeared to contribute

Figure 22. The structures of RAPTA and

RAED type complexes. This image was

obtained from [34].

30 | P a g e

to its anticancer activity [33]. The observed trend for increasing activity against A2780 as a function

of arene ligand was found to be [33]:

Benzene < p-cymene < Biphenyl < Dihydroanthracene < Tetrahydroanthracene

In addition to targeting A2780, the RAED series of agents were also found to be cytotoxic against

cisplatin-resistant cell lines and MCa mammary carcinoma targeting both primary and metastatic

tumours [34]. The anticancer abilities of the RAED group of agents originate from the DNA

coordination of the species via the N7 on the Guanine base (like cisplatin) and the occurrence of

DNA intercalation upon use of large arene ligands (Biphenyl and up)[33][34].

RAPTA complexes much like RAED complexes also have a piano-stool shape, coordinated

halogen groups and an eta six arene group. The only real distinction between the structures of the

two families is the presence of PTA ligand, a sterically easygoing amphiphilic phosphine ligand[34].

The first RAPTA complex (called RAPTA-C) was reported in 2001 by Allardyce and Dyson[34].

RAPTA-C was found to induce DNA damage at pH values lower than 7, demonstrating potential in

selectively targeting tumour cells which are known to be hypoxic compared to healthy cells [34]. In

addition, studies of the drug hydrolysis showed that much like cisplatin, RAPTA-C also experienced

hydrolysis in the presence of low chlorine concentration resulting in the replacement of the chlorine

ligands by aqua ligands [34]. Computational studies have actually supported this finding, reporting

that under acidic conditions, RAPTA agents tend to form mono-aqua complexes[34]. Subsequent

studies of RAPTA agents-DNA adducts involving ESI-MS and 1H-NMR spectroscopy have

Figure23. The various RAED type complexes examined by the Sadler group. This image was obtained from [33].

31 | P a g e

showed that most signals corresponded to mono-chlorido complexes with purine bases or

nucleosides coordinated via N7[34]. The N7 site of Guanine (just like RAED and cisplatin) appeared

to be favored, however adducts with adenine and thymine were also observed [34]. Even though DNA

and RAPTA adducts appear to form quite easily, study of DNA models under physiological

conditions have shown that there is not a strong relationship between the DNA-drug adducts and

the induced cytotoxicity [34][35]. This finding led to the investigation of the interaction of RAPTA

agents with proteins. Investigation of such interactions showed that RAPTA complexes actually had

quite a high affinity for protein molecules [35]. In fact, the RAPTA-protein interactions were found

to be favored over RAPTA-DNA interactions, a preference attributed to the presence of hydrophobic

arene groups on the proteins [34]. Initial studies of the RAPTA-protein interactions were focused on

the model proteins, cytochrome c and ubiquitin [34][35]. RAPTA agents appeared to bind these

proteins via aromatic side groups and showed preferential binding to these proteins over other model

proteins such as superoxide dismutase [35]. This selectivity in protein binding, unlike cisplatin,

appeared to be brought on by the greater steric demand of the agent and its hydrophobic interactions

rather than electronic effects [34]. Further investigation of the protein interactions involved an

examination of enzymes known to play a part in cancer, cysteine protease cathepsins B (cat B) and

soleno-enzyme thioredoxin reductase [35]. Such studies found that the RAPTA agents were not active

against thioredoxin reductase, but RAPTA-C and RAPTA-T were good inhibitors of cat B [34].

Recent studies have started to investigate the interaction of the RAPTA agents with poly-(adenosine

diphosphate(ADP)-ribose) polymerase (PARP-1), a zinc based metalloprotein known to play a key

role in cancer resistance in chemotherapy[35]. Early results have showed that RAPTA-T is able to

inhibit the protein with about the same potency as the benchmark inhibitor, 2-aminobenzamide [35].

So it appears that the anticancer activity of the RAPTA based agents stems from their ability to

interfere with the expression and activity of some key proteins that are involved in the regulation of

the cell cycle, this interruption in protein functioning ultimately induced apoptosis[35].

Today a range of RAPTA derivatives are known (Figure24), unfortunately, only RAPTA-C

and RAPTA-T exert noteworthy anticancer effects [34]. RAPTA-T is active against invasive cancer

cells and metastatic tumours [34]. This ability is attributed to the induced changes in the cytoskeleton

leading to loss in the cell’s flexibility which is crucial in the detachment and reattachment processes

[34]. RAPTA-C on the other hand, has been shown to inhibit tumour growth of A2780 transplanted

onto the CAM (chicken chlorioallantoic membrane) model by about 75% at a dose of 0.2 mgkg-1

32 | P a g e

per day for 5 days [34]. In addition, RAPTA-C has also been found to be active against mice with

LS174T, a colorectal adenocarcinoma cell line, inhibiting tumor growth by 50% at 100 mgkg-1 per

day for 11 days [34].

Figure24. Some RAPTA derivatives. This image was obtained from [34].

The synthesis of Ru(II) arene complexes proceeds via a dinuclear ruthenium complex[24]. To

synthesize this dinuclear species, the commercially RuCl3∙ 3H2O is reacted with a non-aromatic

precursor such as cyclohexa-1, 4-diene in ethanol [24]. This reaction results in the production of H2

which acts as a reductant, converting the Ru3+ metal center to Ru2+ [24]. The resultant dinuclear

complex can be reacted with the desired ancillary ligand in subsequent steps, allowing for a facile

production of the desired Ru(II) arene compound [24]. The general synthesis of Ru(II) arene

complexes is summarized in Figure 25.

Figure 25. The general synthesis of Ru(II) arene complexes. This image was obtained from [24].

Ruthenium(II) Polypyridyl Complexes

Another group of Ru(II) complexes that have shown potential for application in cancer

chemotherapy are the Ru(II) Polypyridyl complexes. This class of ruthenium agents is particularly

attractive due to its facile synthesis, stability in aqueous solutions and luminescence [36].

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Investigation of the biological activity of such complexes

was initiated in the 1950s by the Dwyer group, who

reported the antiviral and bacteriostatic activity of a

family of tris(polypyridyl) complexes [33] [36]. At the time

of this discovery, no biological target was known,

however, recent studies of coordinatively saturated metal

complexes have suggested that the bio-target of such

complexes is most likely DNA [33]. This is exciting news

as DNA is the main target of the famous anti-cancer

agent, cisplatin. It is not surprising then, that many

studies of the anticancer activity of Ru(II) polypyridyl

complexes are based on the employment of Ru(II)

complexes that are somewhat structurally similar to

cisplatin[33]. This point is demonstrated in the study

performed by the Novakova group. The study investigated the anticancer activity of Ru(II)

polypyridyl complexes with labile chlorine ligands, just like cisplatin, against various murine and

human tumour cell lines [33]. The group reported significant anti-cancer activity by one of complexes,

mer-[Ru(terpy)Cl3] (Figure 26), which was found to crosslink DNA just like cisplatin[33]

. In more recent approaches, the focus has

shifted away from the Ru(II) chloro-polypyridyls

towards the exploration of relatively inert

ruthenium (II) polypyridyls[33]. The potential of

such complexes was demonstrated by the Barton

group which exploited the luminescence abilities

of [Ru(phen)2(dppz)]2+ (Figure27) to detect DNA

defects[37]. Although, the complex was capable of

binding DNA, it was found to be inactive against

cancer [33]. This finding supports the observation

made by the Novakova group, that a mere binding interaction with the DNA is not enough to induce

anti-cancer activity and that an accompanying abrasion is crucial[33]. Further studies of the anti-

cancer activity of inert ruthenium(II) polypyridyls were performed by the Tan group which

Figure26. A Ru(II) polypyridyl complex found to be active against murine and human tumour cell lines by the Novakova group. This image was obtained from [33].

34 | P a g e

examined the anti-cancer activity of [Ru(phen)2(dppz)]2+ analogues[33]. In particular, the group

found that the replacement of the dppz ligand by a �-carboline resulted in complexes (Figure28)

which were able to induce simultaneous autophagy and ROS dependent apoptosis in tumour cells

[33][38]. This cytotoxicity of the agent was attributed to DNA affinity, lipophilicity and membrane

penetration abilities [38].

Clearly, ruthenium is a versatile metal capable of forming complexes with various

geometries and biological activity which can exploited to selectively and effectively combat cancer

cells. In the next section, the discussion conducted till this point will be utilized in order to answer

the key question, “Why does the world need ruthenium anti-cancer agents?”

35 | P a g e

Concluding Remarks:

Why does the world need ruthenium anti-cancer agents?

In dealing with a disease as powerful and widespread as cancer, time is a precious

commodity, that is to say, there is little room for research into therapeutic approaches that are

essentially a dead end. In this last section, a brief discussion is provided to convince the reader that

ruthenium anti-cancer agents are not a dead end, a discussion which also answers the key question

posed by this dissertation; why does the world need ruthenium anti-cancer agents?

As previously discussed, Ruthenium biochemistry offers some unique features which are

simply not available for other metals such as platinum, the metal base of cisplatin. To be more

specific, Ruthenium has the ability to adopt two contiguous oxidation states, +3 and +2, in vivo

which allows for the possibility of a so called activation by reduction effect[20]. The greater lability

of the +2 form, coupled with the reducing environment of cancer cells compared to healthy cells,

allows for the administration of the ruthenium agents in a selective manner[20]. In simpler terms,

ruthenium agents exert lower cytotoxicity to healthy cells. No such effect is available for a drug like

cisplatin. It should also be noted that Ruthenium adopts octahedral geometry in both these oxidation

states and therefore offers two additional ligand sites compared to square planar Pt2+ complexes[24].

In addition, ruthenium being a member of the iron family can also mimic iron in vivo[20]. This

mimicking ability means ruthenium can be transported around the biological body by iron carrier

proteins such as transferrin[20]. This feature combined with the increased transferrin receptors on

cancer cell surfaces, further adds to the selective accumulation of the ruthenium based drugs in

vivo[20]. Once, again platinum based drugs do not offer such a feature. Both these unique biochemical

features of ruthenium mean the ruthenium based drugs are highly biocompatible, offering a greater

selectivity in the targeting of cancer cells and a lower toxicity to healthy cells compared to their

platinum based counter parts.

If the biochemical features were not quite convincing, one can further look at the various

ruthenium drugs as compared to cisplatin. Although a number of ruthenium based drugs can bind

DNA much like cisplatin, DNA does not seem to be the main biological target of these drugs in

many cases[24][29][30][31][35]. Instead, one ruthenium drug, KP1019, is found to target mitochondrial

pathways, the housing unit of apoptotic proteins, effectively bypassing the need to damage DNA to

induce apoptotic signals[31]. This unique mechanism of action makes this new drug quite potent in

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combating a range of cancer cell lines, including those that are resistant to cisplatin. Other ruthenium

drugs such as RAPTA complexes and NAMI-A are unique in that they can target one of the daunting

issues in cancer treatment, metastasis[29][35]. This anti-metastatic action of these drugs makes them

significantly different from cisplatin which is incompetent in the matter. Clearly, ruthenium based

anti-cancer agents are quite versatile not only in their site of action but also in the induced

therapeutic effect.

From an economic point of view, Ruthenium is also a much better choice compared to other

precious metals such as platinum and gold. In fact, ruthenium is actually about 20 times cheaper

than platinum [39]. This should mean the produced ruthenium drugs should be significantly cheaper

as compared to cisplatin. The affordability of the drugs is quite an important factor as this allows

for the treatment of individuals of various socioeconomic classes. Based on this discussion, it is

quite clear that Ruthenium based anti-cancer agents are the future of cancer chemotherapy and not

a dead end. These agents have a crucial role to play in the war against cancer, as they offer lower

toxicity to healthy cells, versatility in action and therapeutic effect and ability to target cisplatin

resistant cell lines. In fact, in my humble opinion Ruthenium may one day be responsible for the

anti-cancer agent that one may classify as a magic bullet. In order to achieve this, active research in

the field should continue with a particular focus on determining with confidence, what the true

mechanism of anti-cancer action is NAMI-A, KP1019 and other potential Ru(II) agents.

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