Lipocalin 2 as a Potential Diagnostic and/or Prognostic Biomarker in Prostate...
thesis_14200651_submit
1. 1
Towards a Novel Glucose-Conjugated Ruthenium
RAPTA Anti-Cancer Drug
by
Atreyi Bhattacharya
(14200651)
Supervised
Directed by
Dr. Andrew Phillips
I hereby declare that all the work presented in this thesis is my own, unless clearly
indicated by citation.
Student Signature:
Submission Date:
2. 2
Abstract
The aim of this project was to synthesize a ruthenium triazapentadiene RAPTA complex
conjugated to a protected glucose molecule. A major problem associated with currently used
chemotherapeutic drugs is the unpleasant and unavoidable side effects caused by the death of
healthy cells such as in the bone marrow and hair follicles. Hence there is a need for a drug
that will show selectivity for cancer cells over healthy cells. This project aims to take
advantage of the Warburg effect in cancer cells to achieve selective uptake. The Warburg
effect is the observation that cancer cells have an excess of glucose receptors on their surface
and that these cells utilize up to 200 times the glucose that normal cells do. Previously the
Phillips group has prepared and tested several ruthenium RAPTA complexes with the
bidentate triazapentadiene moiety that have shown promising cytotoxic activity on certain
cell lines. The purpose of this project was to further explore the activity of this class of
ruthenium compounds, but with the added benefit of a glucose molecule that will, in theory,
aid the preferential uptake of the drug by cancer cells.
While the precursors for the complex were prepared in a facile manner, standard reaction
conditions were found unsuitable for the conjugation of the sugar derivative onto the
ruthenium complex. In several cases the complex was found to degrade. Moreover, the
glucose derivative was seen to be immiscible in the solvents used. These results indicate that
further investigation is required to find the optimum reaction conditions for the preparation of
the desired compound.
Keywords
Organometallic, drug-targeting, β-diketiminate, triazapentadiene, trifluoroamidine, PTA,
glucose derivative, selective uptake, cytotoxic activity, air-sensitive
3. 3
TABLE OF CONTENTS
ABSTRACT……………………………………………………………………………….............2
KEYWORDS……………………………………………………………………………………...2
ABBREVIATIONS………………………….…………………………………………...……….4
1. INTRODUCTION
1.1 Transition Metal Complexes as Therapeutic Agents ……………………………..……7
1.2 Organometallic Platinum Drugs ………………………………………………………..8
1.3 Organometallic RutheniuM Drugs …………………………………………………….9
1.4 RAPTA Complexes ………………………………………………………………...…12
1.5 The Phillips Group …………………………………………………………………… 15
1.6 The Warburg Effect …………………………………………………………….……. 17
1.7 A Novel Glucose-Conjugated RAPTA Compound ………………..………………… 19
2. MATERIALS AND METHODS
2.1 General Procedures ………………………….………………………..……………….20
2.2 Synthesis Of Cyclopentadienyl Bis-(Triphenylphosphine)Ruthenium Chloride, (C5H5)
Ru(PPh3)2Cl………………………………………………………………………………..21
2.3 Synthesis Of (η5
-C5H5)Ru(PPh3)((N(H)C(CF3))2N) …………………………………..21
2.4 Synthesis Of (η5
-C5H5)Ru(PTA)((N(H)C(CF3))2N)………………………...…………22
2.5 Synthesis Of 1,2,3,4,6-Penta-O-Acetyl-B-D-Glucopyranoside………………..………23
2.6 Synthesis Of 2-Bromoethyl-2,3,4,6-Penta-o-Acetyl-β-D-Glucopyranoside……..……24
2.7 Synthesis Of 1-(2-((3,4,5-triacetoxymethyl)tetrahydro-2h-pyran-2-yl)oxy)ethyl)-1,3,5-
Triaza-7-Phosphaadamantane-1-ium Bromide ……………………………………………25
2.8 Synthesis Of 1,3,5-Triaza-7-Phosphatricyclo[3.3.1.1]Decane…………...……………26
3. RESULTS AND DISCUSSION
3.1 Synthesis Of Cyclopentadienyl Bis-(Triphenylphosphine) Ruthenium Chloride, (C5H5)
Ru(PPh3)2Cl ………………………………………………………………………...……..27
3.2 Synthesis Of (η5
-C5H5)Ru(PPh3)((N(H)C(CF3))2N) ……………………..……………28
3.3 Synthesis Of (η5
-C5H5)Ru(PTA)((N(H)C(CF3))2N)…………………….……………..31
3.4. Synthesis Of 2-Bromoethyl-2,3,4,6-Penta-O-Acetyl-β-D-Glucopyranoside……...….33
3.5 Synthesis Of 1-(2-((3,4,5-triacetoxymethyl)tetrahydro-2h-pyran-2-yl)oxy)ethyl)-1,3,5-
Triaza-7-Phosphaadamantane-1-ium Bromide…………………………………………….34
4. 4
3.6 Preparation of Compound 1: Route A…………………………………………………36
3.7 Preparation of Compound 1 : Route B…………………………………………………37
4. CONCLUSIONS AND FUTURE WORK………………………………...………………40
5. ACKNOWLEDGEMENT…………………………………………………………...…….41
6. REFERENCE LIST………………………………………………………………………..42
Abbreviations
˚ Angle degree
˚C Degree Celsius
Å Angstrom
δ NMR chemical shift
η Ligand hapticity
λ Wavelength
μM Micromolar
μW Microwave
atm Atmospheres
CHCl3 Chloroform
13
C NMR Carbon NMR
Cp Cyclopentadienyl
5. 5
CpH Cyclopentadiene
d Doublet
dd Doublet of doublets
Et Ethyl
Etc. Etcetera
19
F NMR Fluorine NMR
1
H NMR Hydrogen NMR
L Ligand
m Multiplet
Me Methyl
mM Millimolar
MS Mass Spectrometry
NMR Nuclear Magnetic Resonance
31
P NMR Phosphorus NMR
PPh3 Triphenylphosphine
PTA 1,3,5-triaza-7-phosphatricyclo[3.3.1.1]decane
7. 7
1. Introduction
1.1 Transition Metal Complexes As Therapeutic Agents.
Drug discovery in today's world has been transformed by rapid advances in the fields of cellular
molecular biology and information science.[1] Cancer treatment has evolved towards agents
targeted toward specific pathways, notably those involved in cell division and signaling.[2] The
medicinal uses and applications of metals and metal complexes are of increasing clinical and
commercial importance and new chelating agents continue to be sought.[3,4] The field of
inorganic chemistry in medicine may usefully be divided into two main categories: firstly,
ligands as drugs which target metal ions in some form, whether free or protein-bound; and
secondly, metal-based drugs and imaging agents where the central metal ion is usually the key
feature of the mechanism of action. [5]
Knowledge of the crystal structure of both proteins and the complex, combined with physical
and biophysical studies on the complex, allows visualization of likely binding sites.[6] Human
serum albumin is of fundamental importance in the transport of drugs, metabolites, endogenous
ligands, and metal ions.[7] A further source of metal ion/protein interactions is provided by
transferrin, whose natural iron-binding site is accessible to many metal ions of similar radius and
charge including Ru2+,
Ga3+,
and Al3+.
[8] Abundant cysteine-rich small peptides, especially
glutathione (GSH), and proteins such as metallothionein represent detoxifying but also
deactivating pathways for inorganic drugs.[9]
An important distinction to be made is between drugs as chemotherapeutic agents, whose
function is to kill cells, and drugs acting by a pharmacodynamic mechanism—whose action must
be essentially reversible and/or short-lived.[10] Cytotoxic agents reduce the proliferation of a
tumor but lack of selectivity between normal and malignant tissue may render many agents of
little clinical utility. [11]
The nature of the target to be attacked by any drug obviously depends on the specific
application. Many cytotoxic metal complexes target DNA because of its importance in
replication and cell viability. Coordination compounds offer many binding modes to
8. 8
polynucleotides, including outer-sphere noncovalent binding, metal coordination to nucleobase
and phosphate backbonesites, as well as strand cleavage induced by oxidation using redox-active
metal centers. [13]The later transition metals such as platinum and ruthenium favor binding to
electron-rich nitrogens on the bases, especially guanine N7. Titanium and early metals may
display a mixture of nucleobase and phosphate backbone binding. The accessibility of different
oxidation states of metals such as Fe, Cu, Co, Ru, Mn, etc. may allow for redox chemistry
resulting in strand breakage. [12,13]
Transition metal complexes have been seen to have certain advantages over organic molecules as
therapeutic agents. Firstly, small organic molecules are usually tetrahedral, planar, or linear, and
often cannot be modified too adventurously to suit one's specific needs, whereas transition
metals ions offer a way to build complex, specially designed molecular scaffolds by exploiting
their variable oxidation states and the range of geometries exhibited by their coordination
spheres. [14,15]Secondly, one can achieve the desired biological function for a complex simply
by adjusting the auxiliary ligands since these greatly influence the thermodynamic and kinetic
properties of metal complexes. Thirdly, the synthesis of these complexes is much simpler and
more flexible process than that of organic molecules of similar complexity, which would
normally involve a linear and lengthy process requiring several protecting groups. [16]
1.2 Organometallic Platinum Drugs
Figure 1.2.1. Organometallic platinum complexes that have successfully completed clinical trials.
(1) Cisplatin (2) Carboplatin (3) Oxaliplatin
To date cis platin (1) is the most widely used organometallic drug for cancer but it leaves much
to be desired because of its side effects of nephrotoxicity, neurotoxicity, nausea, vomiting etc
9. 9
along with its ineffectiveness against resistant tumours. It’s mode of action is rapid hydrolysis in
the cytoplasm followed by coordination of the metal centre to guanine residues in DNA leading
to intra and inter strand DNA cross-linking, leading to apoptosis.[17] Only two other platinum
drugs have been approved for clinical use worldwide: carboplatin (2) and oxaliplatin (3). [18]
Both carboplatin and oxaliplatin are on the World Health Organization's List of Essential
Medicines. [19]
Carboplatin was introduced in the late 1980s and has since gained popularity in clinical treatment
due to its vastly reduced side effects compared to its parent compound cisplatin. It has been
found effective against some forms of cancer, mainly ovarian carcinoma, lung, head and neck
cancers as well as endometrial, esophageal, bladder, breast and cervical; central nervous system
or germ cell tumors; osteogenic sarcoma, and as preparation for a stem cell or bone marrow
transplant. [20]
Oxaliplatin is marketed as Eloxatin by Sanof as an advanced colorectal cancer treatment.[21]
According to in vivo studies, oxaliplatin fights carcinoma of the colon through non-targeted
cytotoxic effects. Like other platinum compounds, its cytotoxicity is thought to result from
inhibition of DNA synthesis in cells. In particular, oxaliplatin forms both inter- and intra-strand
cross links in DNA,which prevent DNA replication and transcription, causing cell death. [22]
1.3 Organometallic Ruthenium Drugs
While platinum based anti cancer drugs have shown a certain degree of success, there are two
main drawbacks: the negative side effects of the drug and the resistance that cells develop to it
over time calls for the exploration of other options such as ruthenium, whose numerous
properties qualify it as an antineoplastic drug contender. [23]
In the early development of analogs of the platinum compounds, complexes with ‘‘windows of
reactivity’’ similar to the platinum complexes were examined extensively. Whereas direct Ni and
Pd analogs of Pt complexes are too kinetically reactive to be of use as drugs, Ir and Os ammine
compounds are in general too inert. Ruthenium and rhodium compounds have shown greater
promise. [24]
10. 10
Ruthenium complexes can adopt numerous oxidation states, most commonly +2,+3 and +4 and
transition between these states occurs quite easily in the physiological environments found
within the body due to relatively low transition energies. The hexacoordinate, octahedral
geometry of Ru complexes presents one with an additional two ligands over the square planar
complexes of Pt allowing for more intensive tuning of the electronic and steric properties of the
complexes. The octahedral geometry of these molecules also aid in the stabilization of various
oxidation states, and ligand tuning can allow for controlled changes in the redox properties of the
ruthenium center.[25]
The relatively slow rate of ligand exchange for ruthenium complexes, in the range of around 10−2
to 10−4
s−1
, is on the scale of an average cell’s lifetime, which allows the ruthenium complex to
remain intact as it approaches the target as well as remain viable throughout its interaction with
the cells. [26]
It is understood that Ru(II) complexes are generally more reactive that Ru(III) complexes.
Cancer cells are generally growing and multiplying much more rapidly than normal healthy cells,
which creates an oxygen-deficient environment due to the raised metabolic rate. When this is
paired with the tendency of cancerous cells to contain higher levels of glutathione and a lower
pH, a chemically reducing environment is created. This allows for ruthenium complexes to be
administered as much less active, non-toxic Ru(III) compounds (as a prodrug), which can be
activated solely at the site of the cancerous cells. [27]
It is thought that the reduction occurs by mitochondrial proteins or microsomal single electron
transfer proteins, though it may also occur by trans-membrane electron transport systems which
reside outside the cell – implying that entry to the cancerous cells may not be required for the
drug to be effective. In theory it is also possible for the ruthenium compounds to be oxidized
back to its inactive form if it leaves the cancerous environment. [25]
Being in the same group as iron, ruthenium shares many characteristics with the element. It
bonds the same way as iron with sulfur and nitrogen donors which are found abundantly in
proteins within the body. Ruthenium complexes are thus able to take advantage of the body’s
ability to efficiently transport and uptake iron. They can be transported by binding to serum
11. 11
albumin and transferrin proteins, which allow for the efficient uptake of soluble iron for
metabolic purposes. Moreover, since rapidly dividing cells have an increased demand for iron,
the levels of transferrin receptors found on these cancerous cells are greatly increased (two to
twelve times that of healthy cells). This greatly increases the selectivity of the drug as the
majority of the dose is sequestered in cancerous tissues, bypassing most healthy cells,
contributing to the lower toxicity that is associated to the ruthenium drugs in comparison to
platinum. [23]
Figure 1.3.1. NAMI-A (left) and KP 1019 (right) are the only two ruthenium drugs that have made
it to clinical trials.
Two ruthenium drugs NAMI-A and KP-1019 have made it to clinical trials as antimetastatic and
antineoplastic agents. NAMI-A is thought to control angiogenesis (the process by which new
blood vessels are formed), possibly by interfering with nitric oxide metabolism and have anti-
invasive properties towards tumour cells and blood vessels, although it has been observed to
interact with DNA in vitro. DNA is thought to be the main target for KP-1019, which triggers
apoptosis.[28]
12. 12
1.4. RAPTA Complexes
Figure 1.4.1. Some examples of complexes with the Piano-Stool geometry.
Complexes of the general formula [CpMLn] (n = 2, 3, or 4) are often referred to as half sandwich
or “two-, three-, or four legged piano stool” complexes, with the Cp being regarded as the “seat”
and the other ligands as the “legs” of the stool. The chemistry of these complexes is diverse
stemming from the wide range of metals and co-auxiliary ligands. The term piano stool complex
may also refer to complexes bearing other face capping ligands such as η6
-arenes and thus is not
exclusively Cp based.[29]
To date, the most important water soluble cyclopentadienyl ruthenium anticancer complexes are
compounds of the type, (C5R5)Ru(PTA) Cl 2 (3.23), where R = H or CH3, and PTA is 1,3,5-
triazaphosphaadamantane. [30]
Severin et al. demonstrated that the cytotoxicity of these compounds is directly correlated with
the number and types of alkyl substituents attached to the cyclopentadienyl ring and this is
reflected in a study using TS/A murine adenocarcinoma cells which revealed that the
pentamethylcyclopentadienyl complex is over 100 times more effective than Cp complex at
inhibiting growth of tumour cells.[31] It is evident that substitution of the cyclopentadienyl
ligand gives rise to metal complexes with different properties than their unsubstituted
counterparts, resulting in improved activity in many instances.
13. 13
Figure 1.4.2. Left: Ru II arene ethylene diamine derivative. Right: RAPTA Derivative
Sadler et al. established that the mechanism of action of their compounds [(η6
-
arene)Ru(en)(Cl)]+
(en = ethylenediamine) as follows: first the hydrolysis of the Ru−Cl bond of
the prodrug occurs within the cell to generate an active [(η6
-arene)Ru(en)(H2O)]2+
species. This
step is suppressed in the blood because of the high chloride concentrations enabling [(η6
-
arene)Ru(en)(Cl)]+
to cross the cell and nuclear membranes. The much lower chloride
concentration (∼25 times lower) within the cell causes the hydrolysis of the chloride anion. The
aqua complex [(η6
-arene)Ru(en)(H2O)]2+
is then assumed to bind to nuclear DNA with a high
affinity for the N7 position of guanine bases as shown by NMR and X-ray crystallographic
studies and transcription mapping experiments.[32-34]
The Ru arene compounds can only form monofunctional adducts compared to cisplatin which is
known to form bifunctional adducts and DNA cross-links. Moreover, the fact that [(η6
-
arene)Ru(en)(Cl)]+
derivatives were found to be active against cisplatin-resistant cell lines,
indicates that the detoxification mechanism is different from the one of cisplatin.[35]
Figure 1.2.3. RAPTA Derivatives
Ru-RAPTA derivatives were originally designed to improve the aqueous solubility (pta = 1,3,4-
triaza-7-phosphatricyclo[3.3.1.1]decane). As for Ru(II) arene ethylenediamine compounds,
14. 14
RAPTA derivatives [36] containing two chloride ligands were also found to be susceptible to
hydrolysis and it was first anticipated that DNA was a primary target. [37]
Dyson et al. recently prepared RAPTA carboxylato derivatives (oxalo-RAPTA-C and carbo-
RAPTA-C, inspired by the structures of carboplatin and oxaliplatin, and these were seen to
operate by a different mode of action compared to cisplatin, Ru(II) arene ethylenediamine
compounds, and most of the known anticancer compounds in general.
They were found much less cytotoxic than cisplatin.Many of the RAPTA compounds could not
even be classified as cytotoxic and were also nontoxic to healthy cells.The extent of this
nontoxicity was proven in an in vivo study when healthy mice were treated at quite high doses
with RAPTA compounds without triggering toxic side effects.[38]
The most striking result observed was that both RAPTA-C and RAPTA-T inhibited lung
metastasis in CBA mice bearing the MCa mammary carcinoma (the number and weight of the
metastases were reduced) while having only mild effects on the primary tumor.[39]
The real target of these RAPTA complexes has not yet been determined, but at this stage of the
research, enzyme binding is the most probable explanation. It was shown by mass spectrometry
that RAPTA compounds form adducts with proteins [40] and that the reactivity of RAPTA-C
and cisplatin in the presence of proteins was much different.
Messori et al. studied the inhibition activity of a series of RAPTA compounds to two proteins,
i.e., cathepsin B (Cat B) and thioredoxin reductase (TrxR), possible targets for anticancer
metallodrugs, and found that all tested Ru compounds were inhibitors of Cat B while none of
them, with the exception of RAPTA-C, inhibited TrxR. [41] This finding was validated by
computer docking experiments. The Ru(II) center was found to coordinate to the active site
cysteine residue. Furthermore, other atoms of RAPTA (chloride, nitrogen of PTA, etc.) bind
other amino acids of Cat B, thereby stabilizing the metallodrug−enzyme complex. Interestingly,
a good correlation was observed between the inhibiting potency of the RAPTA derivatives and
the calculated stability of the corresponding Cat B/RAPTA adducts.
Other proteins have been proposed as the target for Ru organometallics such as P-Glycoprotein
(Pgp) is a plasma membrane protein that is responsible for drug efflux from cells and is involved
in multidrug resistance (MDR).[42,43]
15. 15
Dyson et al. recently described the preparation of a series of RAPTA-type complexes with
fluoro-substituted η6
-arene ligands. [44] Electron-withdrawing fluoro or CF3 units were added to
the arene to modulate the pKa values of the complexes. The activity of these organometallics was
found to be strongly influenced by the presence of the substituents. IC50 values of the fluoro
compounds were in general much lower than those of the nonfluorinated analogues.
1.5 The Phillips Group
Control over the rate of hydrolysis critical for the development of a new generation of prospective
drug candidates. [29] The labile halogens (such as in RAPTA-C, figure 1. 2.3.) do not truly allow for
direct hydrolytic control without changing the complex entirely. The Phillips group planned to
synthesize a Ru(II) complex that enabled direct control over hydrolysis.
Previously, chelating O,O-, N,O-, and N,N-chelating ligand systems have been employed which
allow for more controllable rates of complex hydrolysis without having a detrimental effect on
cytotoxcity (see oxalo RAPTA-C and carbo RAPTA-C, figure 1.2.3.).[45]
Controlling the steric and electronic environment can strongly influence the metal centre, effecting
ligand lability and consequently the rate of hydrolytic decomposition.
Figure 1.3.1. R= alkyl,fluoroalkyl, aryl, etc. R1 can be a variety of substituting patterns.
This can be achieved using N,N-chelating ligands, since trivalent nitrogen can accommodate an extra
functional position, compared to divalent oxygen. The β-diketiminate series of ligands (figure 1.3.1)
is a class of N,N-chelating ligand which has been highly adaptable in respect to controlling the
hydrolytic parameters.
16. 16
These mono-anionic chelating ligands bind a wide variety of metal centres, where the metal-
coordinated β-diketiminate adopts a relatively unconstrained planar six-membered ring, resulting in a
relatively small bite angle of less than 90˚.[46] The substitution pattern of the flanking N-aryl
substituents directly influences the steric environment around the metal centre, forming a partially
enclosed protective metal pocket such as in the active sites found in metallo-enzymes.
Figure 1.5.2. Aryl substituted β-diketiminate ligand coordiated to Ruthenium atom. R= H, CH3,
CF3 etc.
Studies by Phillips et al. on η6
-Ru-RAPTA complexes utilising the acid sensitive β-diketiminate
ligands found that these complexes (Figure 1.5.2.) were highly active on a number of cancer cell
lines, even exceeding the standard set for metal-based drugs. [47]
These complexes demonstrated selectivity for cancer cells over normal cells, which was proposed to
be due to the lower physiological pH associated with the former. However, their extremely low water
solubility proved problematic. One of the reasons for such poor aqueous solubility is that the β-
diketiminates have bulky hydrophobic aryl groups attached to the nitrogen position.
This problem was solved by using a ligand with certain properties related to the β-diketiminate
scaffold, i.e., an N,N-chelating, negatively charged species with acid sensing ability, whilst
eliminating the bulky aryl substituents.
17. 17
Figure 1.5.3. Conformations adopted by metal-triazapentadiene complexes. R1 = H, alkyl, aromatic.
R2 = H, alkyl, aromatic.
The 1,3,5-triaza-2,4-pentadiene is a ligand ideal in this case for several reasons. It is closely related
to the β-diketiminate family and can form a number of coordination binding modes with metals
(Figure 1.5.3.) [48] The U-shaped binding mode is desired for the triazapentadienyl ligand-metal
complex since, the chelating ligand adds stability (chelate effect) to the metal compound.
Additionally , in the U-shaped conformation, the triaza-ligand contributes a negative charge which
when combined with the negative charge afforded by the anionic Cp-ligand provides the +2
oxidation state for the ruthenium metal, creating a charge balance negates the need for synthesising
salt-like ruthenium complexes to obtain water solubility, already provided by the hydrophilic PTA
ligand.
Unique to the triazapentadienyl system and which is absent in a number of other Ru(II) and Ru(III)
anticancer complexes is the direct control of hydrolysis achievable by alteration of the α-C
substituents (designated R1, figure 1.5.3). The central nitrogen atom of the triazapentadienyl system
is known to participate in facile acid-base equilibria,[49] thus providing pH sensing properties to the
complexes they are incorporated into.[50]
1.6 The Warburg Effect in Cancer Cells
Glucose is an extremely important cellular resource taken up by a cell using receptors on its
surface and broken down in a process called Glycolysis, which is followed by the aerobic
oxidation of pyruvate in the mitochondria. A common phenotype of many types of rapidly
growing cancer cells is a high rate of glycolysis that is up to 200 times higher than those of their
normal tissues of origin, followed by lactic acid fermentation in the cytosol. Cancer cells often
18. 18
display dysregulated metabolism and freely take advantage of the abundant resources available
within the body. This observation is known as the Warburg Effect.[51]
This effect may be a consequence of damage to the mitochondria in cancer, or an adaptation to
low-oxygen environments within tumors, or a result of cancer genes shutting down the
mitochondria because they are involved in the cell's apoptosis program .
It may also be an effect associated with cell proliferation. Since glycolysis provides most of the
building blocks required for cell proliferation, cancer cells (and normal proliferating cells) have
been proposed to need to activate glycolysis, despite the presence of oxygen, to proliferate.
Lewis C. Cantley and colleagues at the Harvard Medical School are believed to have identified
the enzyme that gives rise to the Warburg effect, a form of the pyruvate kinase enzyme, called
tumor M2-PK which is produced in all rapidly dividing cells, and is responsible for enabling
cancer cells to consume glucose at an accelerated rate. This enzyme form is not usually found in
healthy tissue, though it is apparently necessary when cells need to multiply quickly, e.g. in
healing wounds or hematopoiesis.[52,53]
The researchers were able to curb the growth of cancer cells by forcing the cells to switch to
pyruvate kinase's alternative form by inhibiting the production of tumor M2-PK.While the exact
chemistry of glucose metabolism is likely to vary across different forms of cancer; the
researchers identified PKM2 in all of the cancer cells they had tested.
Figure 1.6.1. 2-18F-2-deoxyglucose used to detect and image tumours.
This characteristic enhanced glucose uptake observed in cancer cells has been utilized to detect
and image cancers via (Positron Emission Tomography) PET detection of 2-18F-2-
19. 19
deoxyglucose, which preferentially concentrates within tumors as a result of their rapid uptake of
glucose. [54]This metabolic shift releases cells from the typical restraints on growth, and
provides a potential way to distinguish them from healthy cells – allowing for treatments that
may be selective for cancerous cells.
1.7 A Novel Glucose-Conjugated RAPTA Compound
Figure 1.7.1. Proposed Compound 1.
Glycol-conjugation has recently emerged as a promising strategy for targeting cancer cells. The strategy
of using the Warburg effect to an advantage was inspired by the widespread clinical use of 2-deoxy-2-
(18
F)fluoro-D-glucose (figure 1.6.1), a radio-labeled glucose analog. The field of glucose-conjugated anti
cancer drugs has been growing rapidly since 1995 with several such drugs in development and the first-
in-class conjugate glufosfamide in advanced clinical trials.[55]
This project draws inspiration from the above and aims to synthesis the above Compound 1. In theory the
glucose part of the compound should allow easy uptake into cancer cells and the ruthenium part could
then inhibit the proliferation of the cell, i.e. a selective, cytotoxic agent.
20. 20
2. Materials and Methods
2.1 General Procedures
Synthesis of starting materials, products and reagents were carried out under purified N2
atmosphere using standard Schlenk techniques, unless stated otherwise. All glassware was pre-
dried in an oven at 130˚C and the flasks underwent several purge/refill cycles before the
introduction of solvents or reagents. All solvents were degassed with N2 prior to use and/or
degassed using standard literature procedures. [56] All chemicals (reagent grade) were
purchased from Sigma Aldrich or Acros Organics unless stated otherwise. Trifluoroacetamidine
(Apollo Scientific, UK) was used as received and stored in a refrigerator at -20˚C.
NMR spectra were recorded using a Varian VNMR 300 MHz instrument. Chemical shifts for 1H
and 13C spectra were referenced to Me4Si (TMS, Trimethylsilane), and the 19F and 31P NMR
referenced to residual solvent peaks. Column chromatography purifications were performed
using glass columns (10-50 mm wide) and silica (230-400 mesh particle size). Mass spectra were
recorded using either a solution or nano-electrospray ionisation (ESI) technique on a Waters
Alliance HT Micromass Quattro LCT (MeOH/H2O, 60/40) TOF instrument with a cone voltage
of 35 volts and a capillary voltage of 2800V (+) and 2500 V (-) and are reported in the form of
m/z (intensity relative to base = 100). Microwave reactions were carried out using a CEM
Discover Microwave which has a maximum operating wattage of 300W.
21. 21
2.2 Synthesis Of Cyclopentadienyl Bis-(Triphenylphosphine)Ruthenium Chloride, (C5H5)
Ru(PPh3)2Cl
Scheme 2.2
RuCl3(H2O)3 (1.04 g, 4 mmol) was dissolved in 20 mL of absolute ethanol under nitrogen and
the resulting dark brown solution filtered. The filtrate was added directly into a refluxing
suspension containing ethanol (200 mL) and triphenylphosphine (4.20g, 16.0 mmol)
immediately. A second ethanolic solution (20 mL) of freshly cracked cyclopentadiene (4 ml) was
added to the reaction mixture and this dark solution was refluxed for 3 hours. The completion of
the reaction is indicated by the dark brown solution changing to bright orange. This mixture
was then allowed to cool in a refrigerator overnight, before orange crystals of the product were
collected under vacuum filtration, washed with cold ethanol (2 x 20 mL) and diethyl ether ( 2 x
20 mL).
Yield: 2.67 g, 92%.
Characterisation was identical to that reported in literature. [57]
2.3 Synthesis Of (η5-C5H5)Ru(PPh3)((N(H)C(CF3))2N)
Scheme 2.3
22. 22
A solution of (η5
-C5H5)Ru(PPh3)2Cl (250 mg, 0.34mmol) and trifluoroacetamidine (80 mg, 0.7
mmol) C(NH)(NH2)(CF3) in degassed toluene was heated to 130˚C for 12 hours or 45 minutes in
a 5 mL microwave reactor vial. The resulting orange solutions were filtered to remove
ammonium chloride and the solvent removed under reduced pressure to furnish dark orange
crude residues. Purification was accomplished using column chromatography (silica gel) with n-
pentane/diethyl ether mixtures as eluents (4:1). The products were collected as the first orange
band. These elutes were combined, reduced to dryness and the orange solids collected in a vial.
1
H NMR: (30˚C 299.88 MHz, CDCl3), δ(ppm): 7.40-7.25 (m, 15H, aryl, PPh3), 4.24 (s, 5H, Cp)
19
F NMR (25˚C, 282.14 MHz, CDCl3), δ (ppm): -74.34 (s, CF3)
31
P NMR (25˚C, 161.82 MHz, CDCl3), δ (ppm): 58.55 (s, PPh3)
ESI-MS (25˚C, CH2Cl2), (m/z) positive mode: 636.1 [ M+ H, 100 %], 374.1 [M- PPh3, 20 %]
Yield: 90 mg, 42 %
2.4 Synthesis Of (η5-C5H5)Ru(PTA)((N(H)C(CF3))2N)
Scheme 2.4
The precursor complex (100 mg, 0.158 mmol) was combined with 1,3,5-triaza-7-
phosphaadamantane (25 mg , 0.158 mmol) in degassed toluene and refluxed for 1 hour or
alternatively, irradiated in a microwave reactor for 20 minutes at 130˚C. The resulting red
solution was reduced to dryness in air and the crude solid was loaded onto a column of silica
using a minimum amount of CH2Cl2. Using a mixture of n-pentane/diethyl ether as eluents, any
unreacted starting material and PPh3 were removed. The product was extracted from the column
using methanol, concentrated to dryness and taken up in a minimum of CH2Cl2. Precipitation of
23. 23
the pure product was achieved after the addition of n-pentane and cooling in a refrigerator. The
red solid was filtered, washed with n-pentane and dried in vacuo.
1
H NMR: (30˚C 299.88 MHz, CDCl3), δ(ppm): 4.61-4.45 (AB spin quartet, 6H, 2
JHH = 13 Hz,
NCH2N ), 4.33 (s, 5H, Cp), 3.97 (s, 6H, PCH2N)
19
F NMR (25˚C, 282.17 MHz, CDCl3), δ (ppm): -73.95 (s, CF3)
31
P NMR (25˚C, 121.39 MHz, CDCl3), δ (ppm): -23.85 (s, PTA)
ESI-MS (25˚C, CH2Cl2), (m/z) positive mode: 531 [ M+ H, 100 %], 374 [M- PTA, 70 %]
Yield: 56%
2.5 Synthesis Of 1,2,3,4,6-Penta-O-Acetyl-β-D-Glucopyranoside
Scheme 2.5
Sodium acetate (4 g, 48.7 mmol) and acetic anhydride (70 mL, 0.74 mol) were added to a 250
mL double necked round bottom flask. The reaction was stirred until a suspension formed. A
condenser was then fitted to the flask and heated to boiling point (140 C) in an oil bath with slow
stirring. 2 g of glucose was added to the suspension via a wide-mouthed funnel and the reaction
was removed from heat. When the reaction had subsided, it was heated back up to boiling point,
and then removed from heat whilst 6 g of glucose was added slowly in portions to the reaction,
ensuring that the boiling temperature of the reaction was maintained. Once the reaction had
subsided, the reaction was then brought to full boil for ten minutes. After the reaction had cooled
down, it was poured onto 300mL of ice and left to stand for an hour. The solid was filtered and
washed with ice water. The solid was then recrystallised using methanol.
24. 24
Yield: 10.52 g, 61 %
1
H NMR: (CDCl3, 400 MHz): δ 5.70 ( d, J = 8.3 Hz, 1H, H-1), 5.23 (t, J = 9.4 Hz, 1H, H-3),
5.16- 5.07 ( m, 2H, H-2/H-4), 4.27 (dd, J = 12.5, 4.5 Hz, 1H, HA
-6), 4.09 (dd, J = 12.5, 2.1 Hz,
1H, HB
-6), 3.82 (ddd, J = 10.0, 4.5, 2.2 Hz, 1H, H-5), 2.10 (s, 3H, CH3-1), 2.07 (s, 3H, CH3-3),
2.01 (s, 6H, CH3-2/4), 1.99 (s, 3H, CH3-6)
2.6 Synthesis Of 2-Bromoethyl-2,3,4,6-Penta-o-Acetyl-β-D-Glucopyranoside
Scheme 2.6
Under moisture free conditions, 1,2,3,4,6-penta-o-acetyl-b-D-glucopyranoside (3 g, 7.7 mmol),
bromoethanol (2 mL, 28 mmol) and dry DCM (30 mL) were added to a 3-necked round bottom
flask. At 0˚C, boron trifluoride diethyl etherate (5 mL, 40 mM) was added to the reaction flask
via a dropping funnel slowly over a period of 15 minutes. The reaction was then stirred overnight
at room temperature, resulting in a dark orange solution. TLC (EtOAc/toluene 1:1 ) was used to
show complete reaction. The reaction was diluted with 25 mL of dichloromethane and then
poured onto 50 mL ice water. The organic layer was then separated, dried using sodium sulfate,
filtered and concentrated down. The crude product was then purified by silica gel
chromatography (EtOAc/toluene 1:2).
Yield: 2.74 g, 60 %
Rf (EtOAc/Toluene 1:2): 0.60
1
H NMR: (CDCl3, 300 MHz): δ 5.22 ( t, J = 9.5 Hz, 1H, H-3), 5.08 (t, J = 9.6 Hz, 1H, H-4), 5.01
( dd, J = 9.6, 8.0 Hz, 1H, H-2), 4.58 (d, J = 7.9 Hz, 1H, H-1), 4.26 (dd, J = 12.3, 4.8 Hz, 1H,H A
-
26. 26
2.8 Synthesis Of 1,3,5-Triaza-7-Phosphatricyclo[3.3.1.1]Decane
Scheme 2.8
5.4 g of NaOH and 20 g of tetrakis(hydroxymethyl)phosphonium chloride and 37.8 g of
formaldehyde were cycled using N2 and vacuum and 110 mL of distilled water was degassed
using N2. A 50% w/w solution of NaOH was prepared via cannula and added to the
tetrakis(hydroxymethyl)phosphonium chloride with stirring. After the clear colourless solution
warmed to room temperature, 37.8 g of the formaldehyde was added. 11.8 g of
hexamethylenetetraamine was dissolved in the solution and it is allowed to stand overnight. The
solution was then transferred to an evaporating dish and placed under a hood for evaporation and
allowed to stand until it was 90 % solid. The solid was then filtered via a Buchner funnel,
washed with several aliquots of cold ethanol, and allowed to dry in air. The solid was then
recrystallized by dissolving in hot ethanol followed by a slow cooling to room temperature which
resulted in the formation of a pure, white crystalline solid.
Yield: 6 g, 70 %
1
H NMR (D2O), δ(ppm): 4.07 (d, 6H, PCH2N), 4.62 (s, 6H, NCH2N)
31
P NMR (D2O), δ(ppm): -102.
27. 27
3.Results and Discussion
3.1 Synthesis Of Cyclopentadienyl Bis-(Triphenylphosphine) Ruthenium Chloride, (C5H5)
Ru(PPh3)2Cl
Scheme 2.2
(η5
-C5H5)Ru(PPh3)2Cl is a stable and easily obtainable precursor. Both the triphenylphosphine
ligands are readily displaced under mild conditions.[58] Reactions involving replacement of
chloride with other anions, or by neutral ligands have also been reported.[59] (η5
-
C5H5)Ru(PPh3)2Cl was synthesised by reaction of hydrated ruthenium trichloride, freshly
‘cracked’ cyclopentadiene and triphenylphosphine in the polar protic ethanol. These conditions
facilitated reduction of ruthenium trichloride to the desired η5
-CpRu(II) species. Precipitation of
an orange microcrystalline solid upon reaction completion provides the desired complex in
excellent yields (>90%).[60]
Figure 3.1.1. 1
H NMR of (η5-C5H5)Ru(PPh3)2Cl showing the peaks for Cp and PPh3, with some solvent and grease peaks.
28. 28
Figure 3.1.2 31
P NMR of (η5-C5H5)Ru(PPh3)2Cl showing the singlet peaks for PPh3 ligand at -38
ppm.
This complex was then characterised by 1-dimensional (1D) NMR (nuclear magnetic resonance)
spectroscopy and ESI (electrospray ionisation) mass spectrometry (MS).
In the positive ion mode, the molecular ion peak, [M – Cl-
]+
, was observed at 691 m/z displaying the
characteristic Ru-isotope pattern. [60]
The formation of the product can be clearly observed from the 1
H NMR: the Cp signal is
identifiable as a single peak in the 1
H-NMR spectrum at 4.12 ppm while the peaks for
triphenylphosphine lie in the 7.12-7.39 ppm region.
The 31
P NMR shows one peak at -38.82, for the two triphenyl phosphine ligands, as expected.
3.2 Synthesis Of (η5-C5H5)Ru(PPh3)((N(H)C(CF3))2N)
29. 29
Scheme 2.3
The synthesis of (η5
-C5H5)Ru(PPh3)((N(H)C(CF3))2N) was reported by Woodward et al.,who
synthesised a variety of low valent first and second row metal complexes bearing fluorinated
triazapentadienyl ligands by condensation of fluorinated nitriles, such as trifluoroacetonitrile, in
the presence of a metal precursor.[61] Hursthouse and co-workers have also described the in situ
synthesis of the same fluorinated triazapentadienyl ligand, ((N(H)C(CF3))2NH) bound to a
variety of phosphine exchange reactions are also amenable to microwave irradiation with total
reaction times as low as 10 min with quantitative yields.[62] Hence this complex was prepared at
first by refluxing overnight and later by microwaving in a sealed tube. This complex was then
characterised by 1-dimensional (1D) NMR (nuclear magnetic resonance) spectroscopy and ESI
(electrospray ionisation) mass spectrometry (MS). In the positive ion mode, the molecular ion peak,
[M + H, 100 %] was observed at 636.1, and [M- PPh3, 20 %] at 374.1.
1
H NMR showed the multiplet arising from the aryl protons on the PPh3 ligand in the 7.43-7.26
ppm region and the cyclopentadiene signal at 4.15 ppm.
The product shows up in 31
P NMR as a singlet at 58.38 ppm due to the PPh3 ligand bound to
Ruthenium atom, while the free triphenyl phosphine ligands is seen at -5.39 ppm.
The singlet from the CF3 group on the amidine ligand was clearly visible at -74.34 ppm.
30. 30
Figure 3.2.1. 1
H NMR of (η5
-C5H5)Ru(PPh3)((N(H)C(CF3))2N) showing the peaks for Cp (4.15 ppm)
and PPh3 (7.43-7.26) , with some solvent and grease peaks.
Figure3.1.2. 31
P NMR of (η5-C5H5)Ru(PPh3)2Cl showing the singlet peaks for ruthenium-bound
PPh3 ligand (58 ppm) and free PPh3 (-5 ppm).
31. 31
Figure 3.2.3. 19
F NMR showing the singlet of CF3 group (-74 ppm).
3.3 Synthesis Of (η5-C5H5)Ru(PTA)((N(H)C(CF3))2N)
Scheme 2.4
In the solution 1
H NMR spectra of the final products, all complexes displayed the characteristic
AB spin quartet associated with the PTA ligand with 2J HH = 13Hz typical for an AB spin
system and of the N-CH2N bridge of the PTA system. Ideally no PPh3 aromatic proton signals
should be present, if complete conversion to product were to occur. Here it is evident that some
starting material may be present in the resulting product which was removed via silica column
chromatography.
32. 32
Figure 3.3.1. 1
H NMR of (η5-C5H5)Ru(PTA)((N(H)C(CF3))2N) showing the peaks for PTA and Cp
(3.9 ppm) as well as PPh3 (7.3 ppm) , with some solvent and grease peaks.
Figure 3.3.2. 31
P NMR of (η5-C5H5)Ru(PPh3)2Cl showing the singlet peaks for ruthenium-bound
PTA (-23.8 ppm) and free PPh3 (-5 ppm).
33. 33
Figure 3.3.3. 19
F NMR showing the singlet of CF3 group (-74 ppm).
Solution 31P NMR spectroscopy was highly informative for differentiating the conversion of
complexes (η5
-C5H5)Ru(PPh3)((N(H)C(CF3))2N) to (η5
-C5H5)Ru(PTA)((N(H)C(CF3))2N) with a
signal for the P-donor atom of the PTA ligand located in the upfield region at approximately -
23.88 ppm, which is in contrast to its precursor complex which is characterised by a downfield
singlet at +58 ppm (PPh3). The single peak at -102 ppm seems to be a degradation product or
possibly P(Ph)5.
The expected singlet from the CF3 on the amidine ligand is seen at -73 ppm, indicating that the
ligand is still intact and attached to the Ru-atom.
3.4. Synthesis Of 2-Bromoethyl-2,3,4,6-Penta-O-Acetyl-β-D-Glucopyranoside
34. 34
Scheme 2.7
Figure 3.4.1. 1
H NMR of 2-Bromoethyl-2,3,4,6-Penta-O-Acetyl-β-D-Glucopyranoside with some
solvent and grease peaks.
In this reaction the boron trifluoride diethyl etherate acts as an activator for the acyl protected
glucose molecule and the completion of the reaction is indicated most characteristically in proton
NMR by the ethyl protons in the bromoethanol that have successfully replaced the acyl ion at the
anomeric position. The ethyl proton peaks are important indicators of this product in proton
NMR.
3.5 Synthesis Of 1-(2-((3,4,5-triacetoxymethyl)tetrahydro-2h-pyran-2-yl)oxy)ethyl)-1,3,5-
Triaza-7-Phosphaadamantane-1-ium Bromide.
Scheme 2.5
35. 35
Figure 3.5.1. 1
H NMR of 1-(2-((3,4,5-triacetoxymethyl)tetrahydro-2h-pyran-2-yl)oxy)ethyl)-1,3,5-
Triaza-7-Phosphaadamantane-1-ium Bromide with some solvent and grease peaks.
Figure 3.5.2. 31
P NMR of 1-(2-((3,4,5-triacetoxymethyl)tetrahydro-2h-pyran-2-yl)oxy)ethyl)-1,3,5-
Triaza-7-Phosphaadamantane-1-ium Bromide showing the singlet peaks for PTA (-84.8 ppm).
36. 36
The success of this reaction was judged by the presence of the characteristic peaks for PTA at
around 4 ppm as well as the ethyl proton peaks. The reaction I extremely air sensitive and the
product was kept under nitrogen at all times.
3.6 Preparation of Compound 1: Route A
Scheme 3.6
2-Bromoethyl-2,3,4,6-Penta-O-Acetyl-B-D-Glucopyranoside and (η5
-C5H5) Ru(PTA)
((N(H)C(CF3))2N) were taking in a 1:1 equivalent ratio and cycled before adding degassed
toluene. The solution was then refluxed at 80˚C and 130˚C for 1hr, 2hr and overnight. The
solution was also microwaved at 150W, 175W and 200W for 10min, 20min, 45min and 1hr at
80˚C and 130˚C. The red solutions were filtered and the filtrate was dried under vacuum and
thereafter analyzed using 1
H NMR and 31
P NMR.
Figure 3.6.1. 31
P NMR showing singlet peaks for the red solid at -58 ppm and -5 ppm..
37. 37
In all cases, The reacting solution did turn red indicating that the Ru complex was still intact but
from a visual inspection the glucose derivative seemed to not have dissolved much in toluene.
The 1H NMR did not show the characteristic doublet and singlet in the 4.07-4.62 ppm region
indicating the absence of PTA in the reaction product.
Figure 3.6.2. 1
H NMR of the red solid with some solvent and grease peaks.
While 31
P NMR did show a peak for P atom on PTA at -58.37, it is most probably that of the
starting material.
3.7 Preparation of Compound 1 : Route B
Scheme 3.7
38. 38
1-(2-((3,4,5-triacetoxymethyl)tetrahydro-2h-pyran-2-yl)oxy)ethyl)-1,3,5-Triaza-7-
Phosphaadamantane-1-ium Bromide and (C5H5) Ru(PPh3)2Cl were taking in a 1:1 equivalent
ratio and cycled before adding degassed toluene. The solution was then refluxed at 80˚C and
130˚C for 1hr, 2hr and overnight. The solution was also microwaved at 150W, 175W and 200W
for 10min, 20min, 45min and 1hr at 80˚C and 130˚C. The red solutions were filtered and the
filtrate was dried under vacuum and thereafter analyzed using 1
H NMR and 31
P NMR.
Figure 3.7.1. 1
H NMR of the yellow-black solution.
On reaction, in all cases, the reacting solution turned yellow-black, indicating that the Ru
complex had decomposed, and as before the sugar seemed unreacted. The proton NMR showed
no trace of the PTA ligand and the 31
P NMR showed several peaks that are probably from
degradation products. It is possible that the bromide ion is too harsh a nucleophile and is causing
the ruthenium complex to decompose. Alternatively it is possible that the reaction conditions
used above are too harsh and more research needs to be done on finding the optimum conditions.
Lastly the problem of the sugar not dissolving in the solvent used (toluene) needs to be addressed
and alternatives found.
39. 39
Figure 3.7.2. 31
P NMR of the yellow-black solution showing degradation products.
40. 40
4. Conclusions and Way Forward
The reaction conditions employed so far for the synthesis of Compound 1 from various
precursors have been found unsuitable. Route B above may hold some advantages over route A,
mainly that the bromide anion may be substituted by a milder nucleophile such as PF6
-
. A major
reason for the failure of both routes could be the insufficient solubility of the glucose derivative
in the solvent use; hence alternatives need to be explored. Some reactions were carried out in
Chloroform which has the inertness of toluene towards the reactions but lacks the high boiling
point, but sufficiently high temperatures for reaction to occur could not be achieved.
The future possibilities of this work is vast since parts of the compound can be varied, such as
the cyclopentadienyl ring, the phosphine ligands used in the precursors, the use of alkyl
substituted PTA ligands, the counter-ion, etc. Reaction conditions need to be optimized too, and
the most suitable solvent for the final reaction determined.
In conclusion, this novel glycol-RAPTA compound could potentially be a highly selective
cytotoxic drug, hence it is worthwhile to persevere in preparing Compound 1 and investigate
further.
41. 41
Acknowledgement
I would like to thank Dr. Andrew Phillips for giving me the opportunity to work in his laboratory
and to learn under his guidance. I would also like to thank Declan Armstrong for his patience and
help at every step of the way. I have learnt a lot over the summer thanks to Brian Gaynor, Darren
Lawlor and Crystal Connor who have been extremely accommodating. Lastly I would like to
thank University College Dublin for the V.V. Giri Scholarship without which none of this would
have been possible.
42. 42
Reference List
[1] Weinstein, J. N.; Myers, T. G.; O’Connor, P. M.; Friend, S. H.; Fornace, A. J., Jr.; Kohn, K.
W.; Fojo, T.; Bates, S. E.; Rubinstein, L. V.; Anderson, N. L.; Buolamwini, J. K.; van Osdol, W.
W.; Monks, A. P.; Scudiero, D. A.; Sausville, E. A.; Zaharevitz, D. W.; Bunow, B.;
Viswanadhan, V. N.; Johnson, G. S.; Wittes, R. E.; Paull, K. D. (1997), Science, 275, 343–349.
[2] Boyle, F. T.; Costello, G. F. (1998), Chem. Soc. Rev., 27, 251–261.
[3] Andersen, O. (1999), Chem. Rev., 99, 2683–2710.
[4] Liu, Z. D.; Hider, R.C. (2002), Coord. Chem. Rev., 232, 151–172.
[5] Reynolds, J. E. F. Ed., (1996), Martindale The Extra Pharmacopoeia, 31sted.; The Royal
Pharmaceutical Society; London.
[6] He, X. M.; Carter, D. C. (1992), Nature, 358, 209–215.
[7] Bertucci, C.; Domenici, E. (2002), Curr. Med. Chem., 9, 1463–1481.
[8] Sun, H.; Li, H.; Sadler, P. (1999), J. Chem. Rev., 99, 2817–2842.
[9] Chan, J.; Huang, Z.; Merrifield, M. E.; Salgado, M. T.; Stillman, M. J. (2002), Coord. Chem.
Rev., 233, 319–339.
[10] Albert, A. (1979), Selective Toxicity, 6th ed.; Wiley: New York.
[11] Weinstein, J. N.; Myers, T. G.; O’Connor, P. M.; Friend, S. H.; Fornace, A. J., Jr.; Kohn, K.
W.; Fojo, T.; Bates, S. E.; Rubinstein, L. V.; Anderson, N. L.; Buolamwini, J. K.; van Osdol, W.
W.; Monks, A. P.; Scudiero, D. A.; Sausville, E. A.; Zaharevitz, D. W.; Bunow, B.;
Viswanadhan, V. N.; Johnson, G. S.; Wittes, R. E.; Paull, K. D., (1997), Science, 275, 343–349.
[12] Petering, D.; Xiao, J.; Nyayapati, S.; Fulmer, P.; Antholine, W. (1999), The Royal Society of
Chemistry: Cambridge, page 135–157.
43. 43
[13] Claussen, C. A.; Long, E. C. (1999), Chem. Rev., 99, 2797–2816.
[14] E. Meggers, Angew., (2011), Chem. Int. Ed., 50, 2442–2448.
[15] E. Meggers, (2009), Chem. Commun.,1001–1010.
[16] Bioactive iridium and rhodium complexes as therapeutic agents Chung-Hang Leunga,∗,
Hai-Jing Zhonga, Daniel Shiu-Hin Chanb, Dik-Lung Mab.
[17] R.C. Todd, S.J. Lippard (2009), Metallomics, 1 , 280–291
[18] N.J. Wheate, S. Walker, G.E. Craig, R. Oun (2010), Dalton Trans., 39, 8113–8127.
[19] "WHO Model List of EssentialMedicines" (PDF). World Health Organization. October
2013. Retrieved 08 August 2015
[20] Wheate NJ, Walker S, Craig GE, Oun R (2010), Dalton Transactions 39 (35): 8113–27.
[21] Ehrsson H, Wallin I, Yachnin J (2002), Medical Oncology, 19 (4): 261 –265.
[22] Graham, Joanne; Mushin, Mohamed; Kirkpatrick, Peter (2004), Nature Reviews Drug
Discovery 3 (1): 11–2.
[23] Bergamo, Alberta; Sava, Gianni (2011), Dalton Transactions, 40 (31): 7817–23.
[24] N. Farrell (2004), Comprehensive Coordination Chemistry II, 9, 809–840.
[25] Page, Simon (2012), "Ruthenium compounds as anticancer agents". Education in
Chemistry.
[26] Bruijnincx, Pieter C.A.; Sadler, Peter J. (2009), Advances in Inorganic Chemistry, 61. p. 1.
[27] Antonarakis, Emmanuel S.; Emadi, Ashkan (2010). Cancer Chemotherapy and
Pharmacology 66 (1): 1–9.
44. 44
[28] Amin, Amr; Buratovich, Michael (2009), Mini-Reviews in Medicinal Chemistry, 9 (13):
1489–503.
[29] Kavanagh J., Rational design and synthesis of a new generation of water-soluble, organo-
ruthenium complexes with antiproliferative properties, (2012), Chapter 3, page 166.
[30] Tan, Yu Qian; Dyson, Paul J.; Ang, Wee Han (2011). Organometallics 30 (21): 5965–71.
[31] Dutta, B.; Scolaro, C.; Scopelliti, R.; Dyson, P.J.; Severin, K. (2008), Organometallics., 27,
1355.
[32] Novakova O.; Chen H.; Vrana O.; Rodger A.; Sadler P. J.; Brabec V. (2003),
Biochemistry, 42, 11544–11554.
[33] Liu H.-K.; Wang F.; Parkinson J. A.; Bella J.; Sadler P. J., (2006), Chem.—Eur. J., 12,
6151–6165.
[34] Wang F.; Xu J.; Habtemariam A.; Bella J.; Sadler P. J. (2005), J. Am. Chem. Soc. , 127,
17734–17743.
[35] Ang, Wee Han; Casini, Angela; Sava, Gianni; Dyson, Paul J. (2011), Journal of
Organometallic Chemistry, 696 (5), 989–98.
[36] Allardyce C. S.; Dyson P. J.; Ellis D. J.; Heath S. L. (2001), Chem. Commun., 1396–1397.
[37] Gossens C.; Dorcier A.; Dyson P. J.; Rothlisberger U.(2007), Organometallics , 26, 3969–
3975.
[38] Scolaro C.; Bergamo A.; Brescacin L.; Delfino R.; Cocchietto M.; Laurenczy G.; Geldbach
T. J.; Sava G.; Dyson P. J. (2005), J. Med. Chem., 48, 4161–4171.
[39] Dyson P. J.; Sava G. (2006), Dalton Trans., 1929–1933 and references therein.
[40] Casini A.; Mastrobuoni G.; Ang W. H.; Gabbiani C.; Pieraccini G.; Moneti G.; Dyson P. J.;
Messori L. (2007), ChemMedChem, 2, 631–635.
45. 45
[41] Casini A.; Gabbiani C.; Sorrentino F.; Rigobello M. P.; Bindoli A.; Geldbach T. J.;
Marrone A.; Re N.; Hartinger C. G.; Dyson P. J.; Messori L. (2008), J. Med. Chem., 51, 6773–
6781.
[42] Melchart M.; Sadler P. J. (2006) Wiley-VCH: Weinheim, Germany, 39−64.
[43] Vock C. A.; Ang W. H.; Scolaro C.; Phillips A. D.; Lagopoulos L.; Juillerat-Jeanneret L.;
Sava G.; Scopelliti R.; Dyson P. J. (2007), J. Med. Chem., 50, 2166–2175.
[44] Renfrew A. K.; Phillips A. D.; Tapavicza E.; Scopelliti R.; Rothlisberger U.; Dyson P. J.
(2009), Organometallics , 28, 5061–5071.
[45] Ang, W. H.; Daldini, E.; Scolaro, C.; Scopelliti, R.; Juillerat-Jeannerat, L.; Dyson, P. J.
(2006), Inorg. Chem., 45, 9006.
[46] (a) Schreiber, D. F.; Ortin, Y.; Muller-Bunz, H.; Phillips, A. D. (2011), Organometallics.,
30, 5381.
(b) Phillips, A. D.; Laurenczy, G.; Scopelliti, R.; Dyson, P. J. (2007), Organometallics., 26,
1120.
[47] (a) Bourget-Merle, L.; Lappert, M. F.; Severn, J. R. (2002), Chem. Rev., 102, 3031.
(b) Budzelaar, P. H. M.; van Oort, A. B.; Orpen, A. G. (1998), Eur. J. Inorg. Chem. 1998, 10,
1485.
[48] Phillips, A. D.; Zava, O.; Scopelliti, R.; Nazarov, A. A.; Dyson, P. J. (2010),
Organometallics., 29, 417.
[49] Dias, R.; Flores, J. A.; Pellei, M.; Morresi, B.; Lobbia, G. G.; Singh, S.; Kobayashi, Y.;
Yousufuddin, M.; Santini, C. (2011), Dalton Trans., 40, 8569.
[50] (a) Kopylovich, M. N.; Pombeiro, A. J. L.; Fischer, A; Kloo, L.; Kukushkin, V. Y. (2003),
Inorg. Chem., 42, 7239.
46. 46
(b) Kopylovich, M. N.; Haukka, M.; Kirillov, A. M.; Kukushkin, V. Y.; Pombeiro, A. J. L.
(2007). Chem. Eur. J., 13, 786.
[51] Gatenby RA; Gillies RJ (2004), "Why do cancers have high aerobic glycolysis?". Nature
Reviews Cancer 4 (11): 891–9.
[52] Newberg A, Alavi A, Reivich M (2002), Semin Nucl Med 32 (1): 13–14.
[53] Christofk HR, Vander Heiden MG, Harris MH, Ramanathan A, Gerszten RE, Wei R,
Fleming MD, Schreiber SL, Cantley LC (2008), Nature 452 (7184): 230–233.
[54] Pedersen PL (2007), J. Bioenerg. Biomembr. 39 (3): 211–222.
[55] Calvaresi E. C., Hergenrother P. J., (2013), Chem Sci, 4(6), 2319–2333
[56] Amarego, W. L. F.; Chai, C. L. L. (2003), Purification of Laboratory Chemicals, 5th Ed,
Elsevier.
[57] Bruce, M. I., (1977), Australian. J. Chem., 30, 1601.
[58] Ashby, G. S.; Bruce, M. I.; Tomkins, I. B.; Wallis, R. C. (1979), Australian. J. Chem., 32,
1003.
[59] Blackmore, T.; Bruce, M. I.; Stone, F. G. A. (1971), J. Chem. Soc. A., 2376.
[60] Bruce, M. I. (1977), Australian. J. Chem. , 30, 1601.
[61] Robinson, V.; Taylor G. E.; Woodward, P. (1981), J. Chem. Soc. Dalton., 1169.
[62] Hursthouse, M. B.; Mazid, M. A.; Robinson, S. D.; Sahajpal, A. (1994), J. Chem. Soc. Dalton
Trans., 3615.
