Literature Review On The Malignant Nature Of Cancers Biology Essay

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Cancer is a class of diseases in which a group of cells display uncontrolled growth (division beyond the normal limits), invasion (destruction of adjacent tissues), and sometimes metastasis (spread to other locations in the body via lymph or blood).1 These three malignant properties of cancers differentiate them from benign tumors, which are self-limited, and do not invade or metastasize.

Metastases takes place in many ways: through the lymphatic system, through the bloodstream, by spreading through spaces within the body, such as the bronchi or abdominal cavity, or through implantation. The most common way for cancer to spread is through the lymphatic system. The lymph system has its own channels that circulate throughout the body. These channels are very small and carry a tissue fluid called lymph throughout the body, an ideally pathway for the spread and growth of cancer.1

Most cancers form a tumor but some, like leukemia, do not. The branch of medicine concerned with the study, diagnosis, treatment, and prevention of cancer is oncology.

Cancer is the leading cause of death, it caused about 13 % of all human deaths in 2004.2 According to the American Cancer Society, 7.6 million people died from cancer in the world during 2007.3 The main types of cancer leading to overall cancer mortality each year are:

lung (1.3 million deaths per year)

stomach (803 000 deaths per year)

colorectal (639 000 deaths per year)

liver (610 000 deaths per year)

breast (519 000 deaths per year)

The WHO also reported that by 2030, deaths related to cancer will rise to 12 million deaths per year.2 Cancer may affect people at all ages, even foetuses, but the risk for most varieties increases with age (Fig. 1.1).4

Figure 1.1: Number of new cases and rates, by age and sex, all malignant neoplasms, UK, 2006.4

The increase of the survival rates is due to better cancer treatment, in particular thanks to the introduction of efficient anticancer drugs which largely contributed to this improvement. Cancer treatment aims to cure, prolong life and improve quality of life for patients. Some of the most common cancer types, such as breast cancer, cervical cancer and colorectal cancer, have high cure rates when detected early and treated according to best practice. Principal treatment methods are surgery, radiotherapy and chemotherapy. Fundamental for adequate treatment is an accurate diagnosis through imaging technology (ultrasound, endoscopy or radiography) and laboratory (pathology) investigations.2 Such treatments are the use of metals in anticancer therapy.

1.2. Platinum based anticancer drugs

The earliest reports on the therapeutic use of metals or metal-containing drugs in cancer and leukemia treatment date back to the 16th century. Hundreds of years of experience with inorganic anticancer agents was nearly forgotten until 1964, when the anticancer properties of cis-[PtCl2(NH3)2] (cis-diamminedichloro-platinum(II)) was discovered by Rosenberg.5 The various activities of metal ions in biology have stimulated the development of metal-based therapeutics, and thus the field of metal-based compounds in medicine has become very appealing to inorganic researchers.

Complexes that fragment when introduced into the cell and retain their carrier ligands are important. Those non-leaving ligands can mediate the interaction with the target molecule (i.e. DNA) and provide selectivity and/or controlled activity. An example of such complexes are the platinum(II) anticancer complexes, which lose anionic ligands and form coordinate bonds with their targets.6

cis-[PtCl2(NH3)2] (Fig. 1.2) was first synthesized by Michele Peyrone in 1844 and was known as Peyrone's chloride. More than a century later it was about to become, the first metal-containing anticancer drug, cisplatin. Cisplatin is one of the leading metal-based drugs, used in treating a variety of cancers,7 in particular testicular8,9 and ovarian cancers.10,11 Cisplatin is especially effective against squamous cell carcinoma and small cell lung carcinoma.12

Analogues of cisplatin, carboplatin and oxaliplatin (Fig. 1.2), have shown effectiveness as other second-generation chemotherapeutic agents for cancer.12 More recently have been the successes of Farrell's group with the synthesis of multiplatinum drugs.13 BBR3464 (Fig. 1.3) is a multinuclear platinum complex with two binding sites separated by a linker of variable length and has been claimed as the first platinum based compound with a DNA binding mode different to that of cisplatin. BBR3464 is currently undergoing phase II of clinical trials.14

cisplatin

oxaliplatin

carboplatin

Figure 1.2: Selected platinum compounds that are currently in clinical use.

4+

Figure 1.3: An example of Farrell's multinuclear cationic trans-platinum(II) compound: BBR3464.14

The clinical success of cisplatin therapy is limited by severe toxic side effects and drug resistance, in particular nausea and vomiting, neuropathy, ototoxicity and nephrotoxicity. To overcome limitations associated with platinum-based drugs, researchers have focussed their attentions on compounds containing other metals.

1.3. Ruthenium(III) compounds in cancer therapy

It has been shown recently that ruthenium possesses a number of favourable conditions, having the ability to replace platinum and form a basis for anticancer drug design.15 Ruthenium is also found to be less toxic than platinum and that its activity as an anticancer agent resides in its ability to mimic the behaviour of iron, and bind to several biomolecules, such as human serum albumin and transferrin.16 A variety of ruthenium complexes with 2+ or 3+ oxidation states have shown activity against metastatic cancers.17 Two ruthenium complexes, NAMI-A, imidazolium [trans-tetrachloro(dimethylsulfoxide)-imidazoleruthenate(III)]18 and, KP1019, indazolium [trans-tetrachlorobis(1H-indazole)ruthenate(III)]19 have successfully completed phase I clinical trials and are currently undergoing phase II clinical trials (Fig. 1.4).20

NAMI-A

KP1019

Figure 1.4: Ruthenium(III) drugs which have completed phase I clinical trials, KP1019 and NAMI-A.20

NAMI-A was the first ruthenium anticancer agent to enter clinical trials. The drug was developed by the Alessio group and is a negatively charged complex containing an octahedral ruthenium(III) centre bound to a single imidazole ligand, with a trans positioned dmso ligand and four chlorides.21 They showed that the drug was not very active against cancer cell lines, which is usually the initial screen for potential activity as antitumor agents. However, the drug showed enhanced activity against metastases and appears to inhibit cancer growth as a result of a delayed process of metastasis, but has little impact on primary tumours in animal models.22,23

KP1019 was the second ruthenium anticancer agent to enter clinical trials. The drug was synthesised by the Keppler group in 2006.19 Both cisplatin and KP1019 are administered intravenously and therefore, proteins are the first binding partners in the blood stream. It is thought that the binding of platinum complexes to serum proteins leads to the side effects, while KP1019 binds to transferrin, and seems to be an important step in the mode of action.24 Despite the similarities between NAMI-A and KP1019, the drug was found to be a cytotoxin, active against primary tumours and being investigated for activity against colorectal cancers.

In recent years interest has shifted from ruthenium(III) compounds in clinical trails to ruthenium(II) complexes, in particular ruthenium(II) arene anticancer agents, some of which show antimetastatic activity similar to that of NAMI-A. It has been suggested that ruthenium(III) complexes are 'activated' by undergoing reduction in vivo to ruthenium(II), which coordinates more rapidly to biomolecules.17 There is a lower oxygen content and more acidic pH within tumours than in normal tissue and so the production of ruthenium(II) relative to ruthenium(III) should be favoured in tumours.17

1.4 Ruthenium(II) anticancer complexes

The majority of ruthenium compounds evaluated for anticancer activity are coordination compounds with the ruthenium in the +3 oxidation state. It has been proposed that in this oxidation state ruthenium is less active and is reduced in vivo to more active ruthenium(II) complexes, a process favoured in the hypoxic environment of a tumour.25 However, it should be noted that ruthenium(II) compounds also exhibit a low general toxicity and since cancer cells can also become oxidized at certain stages of their growth cycle, oxidation of the ruthenium cannot be excluded.25b

1.4.1 Ruthenium(II) arenes, an alternative to classic ruthenium anticancer agents

Similar to that of ruthenium(III) complexes, organometallic ruthenium(II) complexes of the type [(η6-arene)Ru(XY)Z] (XY=bi-dentate ligand and or two mono-dentate ligands, Z typically a halide), where XY are nitrogen or oxygen ligands (NN, NO, OO, N) have also been studied extensively, as potential anticancer agents. The general structure of the half-sandwich ruthenium(II) arene complexes is shown below in Scheme 1.1.

Scheme 1.1

These complexes all have the η6-arene occupying three coordination sites, a chelating ligand occupying two sites and a mono-dentate ligand occupying the final site. Depending on the nature of the ligand the complexes are either neutral or cationic (and isolated as salts). The presence of the η6-arene ring stabilises and protects the metal centre, preventing rapid oxidation to ruthenium(III).

The structural and electronic features of metal-arene bonding have been thoroughly reviewed.26a, 26b The η6-arene is considered as a -acid/-acceptor ligand towards ruthenium. Evidence comes from the 1H-NMR spectrum, upon arene coordination to the ruthenium center, the proton-resonance shifts to a lower frequency due to increased electron density and further evidence can be seen in the UV-vis spectrum.27, 28

Generally ruthenium(II) bonds are stable towards hydrolysis, although recently there have been reports that the photochemical displacement of the arene can occur in aqueous solution for dinuclear complexes such as [{(η6-indan)RuCl}2(µ-2,3-dpp)](PF6)2,29 and arene lability can be induced by the presence of strong -acceptor ligands bond elsewhere in the complex.

Presently there are two types of ruthenium(II) arene anticancer classes, one class developed by Sadler and the other by Dyson.30, 31 (Fig. 1.5).

Figure 1.5: Two classes of ruthenium(II) arene anticancer agents, ruthenium(II) agents of Sadler (left)30 and RAPTA agents of Dyson (right)31

The first class was synthesised by Sadler's group, a three-legged 'piano-stool' type conformation, consisting of an aryl group coordinated to the ruthenium metal, which is coordinated to a bi-dentate ethylenediamine and a chloride ligand. Sadler's group showed that this type of arene ruthenium complex is as potent as cisplatin and carboplatin in primary cell lines, and was also active against some cell lines which have formed a resistance against cisplatin.30 The authors also showed by changing the aryl group with more extended aryls (e.g. biphenyls, tetrahydroanthracene) the level of anticancer activity was increased. On the other hand changing the N-donor ligands with a more bulky ligand (e.g. N, N, N', N'-tetramethylethlenediamine) the anticancer activity would reduce.32

The second class of ruthenium(II) arene anticancer agents was synthesised by Dyson's group. These were termed RAPTA (1,3,5-triaza-7-phosphaadamantane) anticancer agents, similar in structure to the first class synthesised by Sadler's group, but instead the three remaining coordination sites are occupied with two chloride ligands and a mono-dentate 1,3,5-triaza-7-phosphatricyclo[3.3.1.1]decane ligand. Similar to that of NAMI-A, these agents were found to be inactive against primary tumours, but were found to be very active in vivo, against lung metastases in CBA mice.32b RAPTA compounds were found to be less toxic (in mice) than NAMI-A and thus could be administrated in high doses.31

1.4.2 Proposed mechanism of cytotoxic action (check article by Lang and Suss Fink Revie-check refs)

The mechanism of action of ruthenium(II) arenes is generally thought to involve hydrolysis of the Ru-X bond resulting in an active Ru-OH2 species (aquation), while the arene-Ru bond is robust. This species can exist over a range of pH values, but above the pH=pKa value (the pH at which 50 % of the species exists as Ru-OH2 and Ru-OH through deprotonation of the H2O ligand) the hydroxo Ru-OH species formed by deprotonation will be predominant (Scheme 1.1). This complex is usually considered to be a less reactive species. As hydroxide is a less labile ligand than water, it will not be so easily displaced by biomolecule targets. Thus ideally pKa values of ca. pH > 7 for aqua adducts should ensure that the active species predominates at physiological pH (7.2-7.4). The rate of hydrolysis is important; if the complexes hydrolyse too fast they may not reach the target site.33

Hydrolysis can be suppressed extracellularly due to high chloride concentration (ca. 0.1 M) but becomes impossible after the complex enters the cells due to lowerchloride concentraions (ca. 4-25 mM) found intracellularly. We thus obtain selective attraction inside the cell. The primary cellular target for ruthenium(II) arenes, as for many metal-based drugs is thought to be DNA34 and so factors affecting DNA binding such as rate and extent of binding and non-covalent interactions such as hydrogen bonding and DNA intercalation becomes important.33

1.4.3 Ruthenium(II) arene anticancer complexes: Structure versus activity

One of the earliest examples of a ruthenium(II) arene complex investigated as an anticancer drug candidate was [(η6-C6H6)Ru(dmso)Cl2].35 It has been suggested by the authors that the dmso derivative strongly inhibit topoisomerase II activity by cleavage complex formation via interaction with DNA and crosslink formation with topoisomerase II.

Morris and co-workers synthesized a series of ruthenium(II) arene complexes with three mono-dentate ligands [(p-cymene)Ru(X)(Y)(Z)], where X, Y or Z = halide, acetonitrile or isonicotinamide.36 The ruthenium(II) arene complexes showed that they were inactive (IC50, the dose which inhibits the growth of 50 % of the cells, > 50 µM) towards the human ovarian cells (A2780) in vitro. The authors suggested that the complexes may be too reactive with components of the cell culture medium and/or the cells and be deactivated by biomolecules before they reach their target sites.

To avoid this problem the authors decided to keep the ligand constant and vary the arene ring.37 They obtained reproducible cytotoxicity against A2780 human ovarian cancer cells for chelated diamine complexes of the type [(η6-arene)Ru(N,N)(X)]+ where N,N is typically ethylenediamine, and X is chloride (Fig. 1.6).

Figure 1.6: Five [(η6-arene)Ru(en)Cl]+ complexes (arenes shown separately) for which activity against A2780 human ovarian cancer cells has been investigated.37

Cells were incubated with the particular ruthenium(II) arene complex for 24 h, washed, and then the cell numbers were determined after growth on fresh medium or a further three days. Activity appeared to increase with size of the coordinated arene:

Arene/Pt complex

X

Y

IC50 (µM)

p-cymene

CH3CN

CH3CN

> 100

p-cymene

Cl

isonicotinamide

> 100

C6H5CO2CH3

H2NCH2CH2NH2

56

benzene

H2NCH2CH2NH2

17

p-cymene

H2NCH2CH2NH2

10

carboplatin

6

C6H5C6H­5

H2NCH2CH2NH(Et)

6

C6H5C6H­5

H2NCH2CH2NH2

5

dihydroanthracene

H2NCH2CH2NH2

2

cisplatin

0.6

tetrahydroanthracene

H2NCH2CH2NH2

0.5benzene < p-cymene < biphenyl < dihydroanthracene < tetrahydroanthracene, such that, the biphenyl complex has similar cytotoxicity to the anticancer drug carboplatin (IC50 6 μM) and the tetrahydroanthracene complex is as active as cisplatin (IC50 0.6µM) (Table 1.1).

Table 1.1: IC50 values for ruthenium(II) arene complexes [(η6-arene)Ru(X)(Y)(Cl)]A (A= PF6- for positively-charged complexes) in A2780 human ovarian cancer cells after 24 h drug exposure, and comparison with carboplatin and cisplatin.37 It appears that extended ligand groups, such as biphenyl and tetrahydroanthracene, improve the cytotoxicity of the drug, and while the introduction of an electron withdrawing group at the arene moiety such as COOCH3 results in complexes with poor cytotoxicity. The cationic complex [(η6-C6H5COOCH3)RuCl(en)]+ (Fig. 1.7), isolated as its hexafluorophosphate salt, showed a moderate activity on A2780 ovarian cancer cells (IC50 55 µM), because the presence of an electron-withdrawing group at the arene ligand reduces the activity of the complex, as compared to the p-cymene analogue [(p-cymene)RuCl(en)]PF6 (IC50 9 μM).36

Figure 1.7: Two [(η6-arene)Ru(en)Cl]+ complexes for which activity against A2780 human ovarian cancer cells vary with particular arene moiety.36

Recently, a new series of organometallic ruthenium(II) arene complexes with potential hydrogen-bonding groups attached to the pendant arm of the arene ligand have been prepared and studied for their activity as potential anticancer agents.38 The pta and dapta ligands (dapta = 3,7-diacetyl-1,3,5-triaza-5-phosphabicyclo[3.3.1]nonane) were used to obtain the neutral and cationic mononuclear ruthenium(II) arene complexes (Fig. 1.8). The cytotoxicity of these functionalised ruthenium(II) arene complexes showed no enhancement of the cytotoxicity toward the cancer cell lines screened, as compared to the analogous ruthenium(II) arene complexes without hydrogen-bonding substituents, namely toluene, p-cymene, hexamethylbenzene.38

While the presence of substituents that can potentially hydrogen bond to DNA at the aromatic rings in titanocene-type drugs markedly increased their cytotoxicity,39 note that in the case of these ruthenium(II) arene complexes the effect of the hydrogen-bonding function is actually the opposite. The origins of this unexpected effect were not clearly identified.

d

e

f

g

c

b

a

Figure 1.8: Seven [(η6-arene)Ru(X)Cl2]+ complexes, where X= pta or dapta, with potential hydrogen bonding groups.38

Ruthenium(II) arene complexes appear to have a wide spectrum of cytotoxicity towards cancer cells. For example, the complexes [(η6­­-biphenyl)Ru(en)Cl]PF6 and [(η6­­-dihydroantracene)Ru(en)Cl]PF6 are active against not only A2780 human ovarian cancer cells, but also HT29 colon, Panc-1 pancreatic and NX02 lung cancer cells with IC50 ­values in the range 1-13 μM.40

1.4.4 Multinuclear ruthenium(II) arene complexes as anticancer agents

In contrast to multinuclear platinum complexes, analogous multinuclear organometallic ruthenium compounds have been rarely studied for their anticancer properties, with just a few examples to be found in the literature.15,41-45

Keppler's group have reported on the development of dinuclear ruthenium arene compounds (Scheme 1.2)46,47 with high in vitro anticancer activity (Table 1.2), whereas the mononuclear maltolato compound was found to be inactive.48,49

Scheme 1.2

Synthesis of the dinuclear ruthenium complexes

(a, n=3; b, n=6; c, n=12).46

Table 1.2: IC50 values of the complexes a-c in A2780 and SW480 cells, in comparison to the Pt complexes cisplatin, oxaliplatin, carboplatin, and BBR3464 and the ruthenium(III) compounds KP1019 and the mononuclear MALTOL.46

IC50 (µM)

Compound

A2780

SW480

a

25 ± 2

62 ± 14

b

30 ± 6

26 ± 8

c

1.5 ± 0.3

0.29 ± 0.05

Mononuclear MALTOL

>100

>100

Cisplatin

0.33 ± 0.04

4.5 ± 1.7

Carboplatin

61 ± 10

Oxaliplatin

0.40 ± 0.12

0.30 ± 0.08

BBR3464

0.01

KP1019

49 ± 11

More recently, Keppler's group have synthesized a series of mono-, di- and trinuclear ruthenium(II) arene complexes, and investigated their anticancer activity.50 The in vitro anticancer activity of the dinuclear, its closest mononuclear analogue, and the trinuclear complex (Fig. 1.9) was compared.

Figure 1.9: Dinuclear, mononuclear and trinuclear ruthenium(II) arene anticancer compounds.50

In both SW480 and A2780 cells, the dinuclear complex was identified as the most active species, while there is no meaningful difference between the mono- and the trinuclear complex (Table 1.3). When comparing the hydrophilicity of the three complexes (i.e. their solubility in water), the mononuclear complex is the most soluble, followed by the tri- and dinuclear derivatives (Table 1.3).

Table 1.3: Water solubility and IC50 values of ruthenium(II) arene complexes in human SW480 and A2780 cells.50

IC50 (μM)

Compound

Solubility (mM)

SW480

A2780

Dinuclear complex

3.9

26 ± 8

30 ± 6

Mononuclear complex

10.3

42 ± 1

88 ± 12

Trinuclear complex

7.4

59 ± 18

80 ± 7

Keppler's group also showed that the dinuclear compound represents a good compromise between solubility and hydrophilicity necessary for cellular uptake. They concluded by stating that the modification of the compound to link a higher number of ruthenium moieties improved the water solubility but showed decrease in biological activity.

However Therrien and co-workers showed that an increase in nuclearity results in an increase in biological activity. They reported the synthesis of water-soluble metallaprisms that are able to encapsulate planar aromatic molecules (e.g. pyrene, coronene),51 or complexes (e.g. [Pd(acac)2], [Pt(acac)2]).45 These "complex-in-a-complex" systems showed high cytotoxicity toward human ovarian cancer cell line, A2780.45 ­­­More recently the group have synthesized tetranuclear metallarectangles (Fig. 1.10)52 of the general formula [Ru4(arene)4(N∩N)2(OO∩OO)2]4+ (arene = p-cymene, hexamethylbenzene; OO∩OO=2,5-dihydroxy-1,4-benzoquinonato, 2,5-dichloro-1,4-benzoquinonato; N∩N = pyrazine, 4,4'-bipyridine, 1,2-bis(4-pyridyl)ethylene), prepared from a particular dinuclear ruthenium(II) arene complexes (Fig. 1.11).45,51

Figure 1.10: ORTEP representation of ruthenium(II) hexamethylbenzene tetranuclear metallarectangle.52

Figure 1.11: Capped stick representations of ruthenium(II) hexamethylbenzene tetranuclear metallarectangle (left) and diethyl ether encapsulated by ruthenium(II) hexamethylbenzene tetranuclear metallarectangle (right).52

The authors evaluated the activity of the water-soluble complexes against the A2780 ovarian cancer cell line. All the complexes showed moderate to excellent activity with IC50 values in the range 4-66 µM. It is likely that these large rectangular complexes would be taken up more efficiently by tumor cells,53 which are permeable to large, non-natural molecules, whereas healthy cells are less able to take up such structures, which should provide a degree of selectivity and ultimately lead to reduced drug side effects.

1.5. Metallodendrimers

Dendrimers are complex molecules, built around a central core, having well defined molecular structures.54 The term dendrimers is built from two Greek words "dendros" meaning "tree" or "branch" and "meros" meaning "part". These compounds can be obtained by a series of reaction steps and both the regular and highly branched types exist (Fig. 1.12).55 They are represented in a symmetrical fashion with all tiers pointing outwards. These macromolecules can have a wide range of functionalities located on the periphery. Functionalization of the periphery with transition metals, gives the complex several advantages in the field of catalysis such as, enhanced catalytic activity when compared to other mononuclear analogues.55b,55c These types of branched macromolecules contains metals and are known as metallodendrimers.

Figure 1.12: Regular dendrimer (left) and a highly branched dendrimer (right).55

1.5.1 Synthesis, characterization and properties

The divergent route and the convergent route are two synthetic strategies for the formation of various dendrimers. Both approaches involve a repetition of steps, with each repetition yielding an additional generation. Each route has its own characteristics and so to obtain the desired dendritic product, care has to be taken when choosing the synthetic approach.

The divergent route implies that the synthesis is started with a multifunctional core molecule and is extended to the periphery in a stepwise manner.55 In such a manner, starting at the polyalkylamine core, the poly(propyleneimine) dendrimers are synthesised (Fig. 1.13). The nitrogen atoms serve as branch points that are then reacted with acrylonitrile via a "Michael addition" to give a branched alkyl chain structure. The end-group is reduced yielding a new set of primary amines and thus the process can be repeated for further branching.56 This approach is sometimes troublesome because as the generations increase, so do the number of structural defects. As the generations increase, it also becomes more difficult to purify the dendrimer.

Figure 1.13: Poly(propylene imine) dendrimer synthesis via the divergent approach.56

Thus a second approach is required: the convergent approach (sometimes known as the "defect-free" method) entails making dendrons (a dendritic wedge without a core) and reacting them to a core molecule in the last step of a synthesis. Having such a large "molecular difference" between the reactant and the product, facilitates ease of purification.

Early dendritic structures were synthesised using the divergent route, which have been thoroughly investigated by Tomalia57 and Newkome,58 for poly(amidoamine) (PAMAM) dendrimers and arboral systems respectively. In 1978, Buhleier reported the first synthesis of the poly(propylene imine) dendrimers, which were also prepared by the divergent route.59 The convergent approach was introduced by Hawker and Fréchet, where synthesis started at the periphery and extended towards the core (Fig. 1.14).60 The last type of dendrimer is a convergently produced phenylacetylene dendrimer synthesised by Xu and Moore (Fig. 1.15).61

Figure 1.14: Convergent approach, making use of wedges that can be connected to a core in the last step of a reaction by Hawker and Fréchet.60

Figure 1.15: Convergently synthesised phenylacytylene dendrimer by Xu and Moore. 61

Characterisation of dendrimers is complex due to the shear size and symmetry of the macromolecules. A number of techniques can be used to fully characterise the dendritic complexes, such as NMR spectroscopy (1H, 13C, 15N, 31P), elemental analysis and chromatography techniques (HPLC and SEC).62 There exists softer analytical methods such as ESI (electrospray ionization) and MALDI-TOF (matrix-assisted laser desorption ionization-time of flight) mass spectrometry which allow for in-depth analysis of macromolecules, by giving the molecular ion of the dendrimer.63

1.5.2 General applications of metallodendrimers

Early work of Tomalia and Newkome mainly focused on the synthesis and characterisation of dendrimers.57,58 More recently, the focus has shifted, with research now in areas of functionality and applications of dendrimers. Such areas include medicinal chemistry,64 host-guest chemistry65 and catalysis.66

A high density of functional groups on the periphery attracted researchers to the area of medicinal chemistry. Functionalization of the periphery with identical biologically active groups showed enhanced activities.67 Roy and co-workers investigated the activity of glycodendrimers, having an L-lysine core with various carbohydrates substituted on the periphery, compared to the monofunctional residue (Fig. 1.16).68 As a biological catalyst, the glycodendrimer showed enhanced binding properties compared to the monofunctional residue.68

Figure 1.16: A glycodendrimer with an L-lysine core and various carbohydrates on the periphery, synthesized by Roy and co-workers.68

It has been shown that dendrimers can possess cavities within their macromolecular structure that can be used to accommodate guest molecules.69 In light of these findings; research is focusing on this property, for the development of sophisticated drug-delivery systems (Fig. 1.17).

Figure 1.17: Dendritic box synthesised by Jansen and co-wokers, used as a drug delivery agent.69

Functionalization of the dendrimer arms with various transition-metals showed promise in medicine and catalysis as mentioned before. Metallodendrimers used in medicine and catalysis can have the active metal centre located at the core or on the periphery (Fig. 1.18).

Figure 1.18: Catalytically active transition-metal complexes can be attached to the periphery (a), the core (b), at the focal point of a wedge (c), and at the periphery of a wedge (d).54

= Transition Metal

An example of a multinuclear metallodendrimer synthesised by Smith and co-workers is the poly(propylene imine)pyridyliminepalladium dendrimer (Fig. 1.19), which was used in ethylene polymerisation studies.70 The dendritic palladium complex also shows high activity and high efficiency in Heck cross-coupling reactions in the coupling of an aryl halide with electron-deficient or electron-rich olefins.70

Figure 1.19: Poly(propylene imine)pyridyliminepalladium metallodendrimer.70

1.5.3 Enhanced permeability and retention (EPR) effect

The EPR effect is a phenomenon in which macromolecules (such as polymers and dendrimers) can accumulate at the tumour site due to an increase in blood vessel permeability within the cancerous tissues compared to normal tissues (Fig. 1.20).71 The normal endothelial layer surrounding the blood vessels feeding healthy tissues restricts the size of molecules that can diffuse from the blood stream. In contrast, the endothelial layer of blood vessels in cancerous tissues is more porous providing access to the surrounding tissue. Furthermore, diseased tissue do not usually have a lymphatic drainage system. Once macromolecules have entered the tissue they are retained and show increased bio-availability. A tetraruthenium cluster was found to be highly active against the polio virus without damaging the host cells, thereby offering the potential of developing highly selective drugs.72

Figure 1.20: Diagram representing the enhanced permeability and retention (EPR) effect.71

1.5.4 Metallodendrimers used as anticancer agents

More recently it has been shown that polynuclear platinum complexes have shown to be a very important group of antitumor active compounds, by showing a different toxicity profile and slight different mode of action than cisplatin. They cross-link the DNA differently, instead they cross link the DNA in a 1→ 4 compared to the 1→2 GG pattern of cisplatin.73

It was shown that synthesis of polymeric platinates is another means to increase platinum solubility, reduce toxicity and localise more drug in the tumour via the enhanced permeability and retention effect, to partially overcome mechanisms of resistance.74,75

The study done by Kapp and co-workers focused their attention to the design of drugs with increased selectivity for breast tumors.76 They coupled the DAB(PA)4 (N, N, N', N',-tetrakis(3-amino-propyl)butane-1,4-diamine) polyimine dendrimer with the well-known [1,2-bis(4-flurophenyl)ethylenediamine]platinum(II) complex (Fig. 1.21).77,78 They concluded that the platinum functionalised dendrimer operates as a carrier for the shuttling of platinum into the cell nuclei of the cancerous cell, with no cytotoxic effects seen.

Figure 1.21: A platinum functionalised metallodendrimer synthesised by Kapp and co-workers.76In 2009 Zhao and co-workers synthesized a multinuclear chloropyridyliminecopper(II) complex. The dendritic complex contained seven copper(II) centers (Fig. 1.22).79

Figure 1.22: A multinuclear (top) and mononuclear copper complex (bottom) synthesized by Zhao and co-workers.79

Following synthesis of the complexes, they investigated the complexes as potential anticancer agents, which to their surprise showed enhanced in vitro cytotoxicity. The copper(II) complexes were studied against leukemia cells (MOLT-4), breast cancer cells (MCF-7), and Chang Liver cells (Table 1.4). In comparison with the mononuclear copper derivative, the authors showed an enhanced improvement in cytotoxicity. Furthermore the multinuclear copper complex demonstrated enhanced cytotoxicity compared to cisplatin against MOLT-4 and cisplatin-resistance MCF-7.

Table 1.4: IC50 values of multinuclear and mononuclear Cu-complexes versus cisplatin.79

IC50 (μM)

Compound

MOLT-4

MCF-7

Chang liver

Cisplatin

15.5 ± 4.2

-

73.5 ± 3.7

Multinuclear Cu-complex

11.1 ± 0.6

10.2 ± 1.5

8.7 ± 0.7

Mononuclear Cu-complex

24.7 ± 2.4

73.1 ± 4.9

-

1.5.5 Ruthenium arene metallodendrimers

Functionalised metallodendrimers with ruthenium arene moieties on the periphery are few and rare in the literature. A ruthenium arene functionalised metallodendrimer was recently synthesized by Pettirossi and co-workers.80 They synthesized the multicationic ruthenium arene metallodendrimer (Fig. 1.23) by coupling of [(η6-p-cymene)Ru(κ3-dpk-OCH2CH2OH)]X (dpk=2,2'-dipyridyl ketone, X=PF6) with polypropylenimine dendrimer DAB-dendr-(NH2)n {n = 4, 8, 16} mediated by 1,1'-carbonyldiimidazole (CDI). The metallodendrimers were characterised with multidimensional and multinuclear NMR techniques (1H, 13C, 1H-COSY, 1H-NOESY, 1H,13C-HMQC NMR, and 1H,13C-HMBC NMR spectroscopy). The authors showed that the attachment of an organometallic moiety to a dendritic structure neither alters the relative anion-cation orientation nor does it cause a significant spatial proximity of two metal centers. The solvophobicity of the metallodendrimers is much higher than that of neutral dendrimers and increases with the generation.

Figure 1.23: First ruthenium arene metallodendrimer synthesized by Pettirossi and co-workers.80

To the best of our knowledge, there are no ruthenium arene metallodendrimers used as anticancer agents. This further gives motivation for our present study.

1.6. Aims and Objectives

Aims

The general aims of this project are to synthesize and characterize a range of highly functionalized multinuclear ruthenium(II) metallodendrimers. Their biological activity as potential anticancer agents will be evaluated.

Objectives

The specific objectives of the research project are:

Synthesis and characterisation of Schiff-Base end-group modified poly(propyleneimine) dendrimers (1st and 2nd generations) (Fig. 1.24).

Figure 1.24: 1st and 2nd generation Schiff-Base end group modified poly(propyleneimine) dendrimers.

Subsequent complexation with ruthenium, using arene-type derivatives. Such as [(p-cymene)RuCl2]2,81 [(hexamethylbenzene)RuCl2]2,82 and

[(η6-C6H5OCH2CH2OH)RuCl2]2 (Fig. 1.25).83

Figure 1.25: Various ruthenium arene precursors, [(p-cymene)RuCl2]2 (top left),81 [(hexamethylbenzene)RuCl2]2 (top right),82 and

[(η6-C6H5OCH2CH2OH)RuCl2]2 (bottom).83

Synthesis of mononuclear analogues to compare biological activity.

Evaluation of the dendritic complexes as potential anticancer agents

All compounds will be characterized by a variety of analytical and spectroscopic techniques, which include NMR and IR spectroscopy, elemental analysis and mass spectrometry.

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