METAL COMPLEXES HAVING THERAPEUTIC APPLICATIONS Field of the Invention

The present invention relates to metal complexes having histone deacetylase inhibitory activity. Also contemplated are methods of treatment of cancers comprising administering a metal complex of the invention to a patient.

Background to the Invention

Nearly 50% of all anti-cancer therapies are platinum (Pt)-based, yet surprisingly to date only three Pt drugs have been approved for worldwide clinical use, namely cisplatin, carboplatin and oxaliplatin. The cytotoxicity of Pt drugs is attributed to their ability to bind DNA and induce apoptosis. Despite their success, the widespread application and efficacy of classical Pt drugs is hindered by toxic side effects, their limited activity against many common human cancers and their susceptibility to intrinsic/acquired drug resistance. Some of these drawbacks, which undermine their curative potential against many malignancies, may be due to their lack of selectivity for the cell nucleus resulting in reduced cellular accumulation because of increased detoxification by cytoplasmic glutathione and/or metallothioneins or their reactions with other biomolecules such as proteins and

phospholipids. Once DNA binding has occurred, resistance mechanisms include increased DNA repair of adducts and an ability to tolerate greater levels of DNA damage. Alternative strategies are therefore required in an attempt to overcome these drawbacks such as developing a new class of chemotherapeutic with greater selectivity for cancer cells over normal cells and with a different mechanism of action to commercially available Pt drugs. The cytotoxicity of Pt-drugs such as cisplatin, c s-[Pt"(NH3)2Cl2], is attributed to their ability to undergo hydrolysis upon cell entry, forming hydrated species such as [Pt"(NH3)2(H20)2] ²⁺ which bind to DNA nucleobases (of which 60-65% consists of 1 ,2-intrastrand GpG crosslinks between two adjacent guanines), distorting the DNA helix and interfering with DNA processes such as transcription and replication. These distortions are thought to trigger apoptosis. However, Pt drugs react indiscriminately in the body giving rise to many of the toxic side effects associated with their use.

There is therefore an urgent need to develop novel therapeutics that overcome these toxic side effects and the resistance issues that have plagued conventional therapeutics. As such, the search for new molecular targets beyond DNA which may present unique opportunities for therapeutic exploitation, is currently the subject of intense investigation. Chromatin, a complex structure that plays a key role as an epigenetic regulator of gene expression, is one such target. Its fundamental repeating unit, the nucleosome, consists of core histones around which DNA coils. Some histone residues protrude the nucleosome and are subject to many enzyme-catalysed post-translational modifications including methylation and acetylation. Acetylation is controlled by two enzymes, histone

acetyltransferases (HATs) and histone deacetylases (HDACs), which work in harmony to acetylate and deacetylate core histone lysine residues respectively. Acetylation leads to an open chromatin structure upregulating transcription whereas deacetylation leads to a condensed structure and transcriptional repression. Inhibition of HATs or HDACs can therefore dramatically affect chromatin structure and reprogram transcription and in fact a range of structurally diverse HDAC inhibitors (HDACi) have already been shown to cause cell cycle arrest, differentiation and/or apoptosis of tumour cells. Several of these are now undergoing clinical trials. Suberoylanilide hydroxamic acid (SAHA), is the first FDA- approved HDACi to enter the clinic as an orally active treatment for advanced cutaneous T- cell lymphoma. It is well documented that HDAC inhibitors are subject to in-vivo metabolic degradation resulting in shorter half-lives, a limitation often associated with clinical development of hydroxamate-based drugs.

Metal complexes having dual histone deacetylase inhibitory and DNA binding activity are described in PCT/EP2010/060089. These complexes comprise a compound having a terminal HDAC inhibitory active site, coordinated to DNA-binding heavy metal ions by means of a linker comprising a monodentate, bidentate, or chelating oxygen donor group. The linker may be labile whereby when it is coordinated to a DNA binding metal ion the corresponding metal complex is susceptible to activation e.g. by hydrolysis in-vivo, releasing the HDAC inhibitor and leaving the metal ion free to bind DNA.

The chemical nuclease activity of copper-phenanthroline scaffolds is described in the literature. DNA strand scission by [Cu(Phen)2] ²⁺ arises from its ability to abstract hydrogen from DNA pentose rings in the presence of exogenous reductant (Cu ²⁺ to Cu ⁺ ) and oxidant

Gumienna-Kontecka (Fritsky et al., Inorg. Chem., 2013, 52, 13, 7633-7644) describe mono and oligonuclear Cu(ll) complexes of pyridine-2-hydroxamic acid and pyridine-2,6- dihydroxamic acids, neither of which are HDAC inhibitors. In the complexes reported, 'the ligands show the tendency to form bi- and trinuclear species with copper(ll) ions due to the {(Ν,Ν'); (Ο,Ο')} bis-(bidentate) chelating-and-bridging mode involving (0,0')-hydroxamate chelate formation combined with (Ν,Ν') chelating with participation of the pyridine and hydroxamic nitrogen atoms, so that the hydroxamate groups play a μ2-(Ν,0)^η¾^ role.

Golenya, Fritsky et al. (Cryst. Eng. Chem., 2014, 16, 10, 1904-1918) describes

mononuclear and polymeric Cu(ll) complexes of 3- and 4-pyridinehydroxamic acids, neither of which exhibit HDAC inhibitory activity. In the mononuclear complex [Cu(3- HPicHA)2(CI04)2] (1 ) reported, the hydroxamate is coordinated in a bidentate Ο,Ο'-fashion and the coordination sphere (elongated octrahedral) is completed by perchorlates.

Database Chemical Abstracts (REF) describes mixed ligand complexes of copper(ll) with salicylhydroxamic acid as primary ligand and amino acids, peptides, DNA units or amines as secondary ligands. Salicylhydroxamic acid is not a HDAC inhibitor.

Kasparkova et al (Angewante Chemie Int. Ed. Eng., 2015, 54, 48, 14478-14482) describes a photoactivatable Pt(IV)-HDAC inhibitor conjugate targeting genomic DNA and HDACs. The HDACi in question is suberoyl-bis-hydroxamic acid and it is coordinated to the Pt(IV) in a monodentate fashion.

It is an object of the invention to overcome at least one of the above-referenced problems.

Summary of the Invention

The present invention exploits the unique chemical environment of tumour cells by generating bioreductively-activated redox active metal-phenanthroline-HDAC inhibitor prodrugs that, upon entry into the reducing environment of a tumour cell, are activated by metal ion reduction, facilitating both oxidative DNA damage with concomitant release of the HDAC inhibitor which is free to inhibit HDACs within the tumour cell. A number of exemplary chemotypes, including [Cu(DPQ)(SAHA)] ⁺ , [Cu(DPPZ)(SAHA)] ⁺ ,

[Cu(DPQ)(Belinostat)] ⁺ and [Cu(DPPZ)(Belinostat)] ⁺ , have been tested and found tobind DNA, exhibit potent chemical nuclease and HDAC inhibition activity, and are typically cytotoxic at low microM and even nanoM concentrations (Table 1 ). Surprisingly, the complexes exhibit excellent HDAC inhibitory activity after 24 hours within SKOV-3 ovarian cancer cells, whereas in the same study the clinically used HDAC inhibitor SAHA demonstrated activity only after 72 hours.

According to a first aspect of the invention, the metal complex has a general formula I

(I)

which:

R-Z is a HDAC inhibitor in which Z a terminal hydroxamate group;

M is a redox active metal;

n+ is the positive charge on the complex cation;

B ^n" is a balancing counterion;

to M via 0,0' bidendate coordination; and

is a nitrogen donor ligand selected from a bipyridine ligand or a phenanthroline ligand or phenanthroline-type ligand,

with the proviso that R-Z is not a dual HDAC/PARP inhibitor.

Compared with the complexes disclosed in Kasparkova et al, the complexes of the invention exhibit improved stability due to the metal binding the HDAC inhibitor with 0,0' bidentate coordination. In addition, in contrast to the neutral complexes of Kasparova which require photoactivation, the charged complex of the invention is bio-reductively activated, allowing targeting of the compounds to tumour cells. one embodiment, M is a first-row transition element. In one embodiment, M is selected from copper, iron, manganese or cobalt.

In one embodiment, M is copper.

In one embodiment,

in which:

Ri to Rs are each, independently, selected from oxygen, hydrogen, hydroxyl, a carboxyl, a substituted or unsubstituted, branched or unbranched alkyl, alkenyl, cycloalkyl, aryl, aryloxy, alkyloxy, arylalkyloxy, amino, alkylamino, hydroxylamino, dialkylamino, or alkoxy group, or pyridine, or are fused to form a monocyclic or polycyclic aromatic hydrocarbon or heterocycle.

HDAC Inhibitors

The metal complexes of the invention may include many classes of hydroxamate-type HDAC inhibitors, such as the hydroxamate inhibitors described in Cancer Lett., 2009, 280, 233-241 , and the HDAC inhibitors described in the Journal of Hematology & Oncology, 2009, 2, 22 and Bioorg. Chem., 2016, 67, 18-42. Examples of hydroxamate-type inhibitors are described in patents US6,087,367, US6,552,065, and US6,888,027, which have a terminal hydroxamate group which is the active site inhibiting group. Preferred

hydroxamate-type HDAC inhibitors are SAHA (Vorinostat), Panobinostat, or Belinostat. In one embodiment, the HDAC inhibitor R-Z has a general formula III either in its protonated or deprotonated form:

in which: Rg and R10 are each, independently, a hydrogen, hydroxyl, a substituted or unsubstituted, branched or unbranched alkyl, for example a C1-C6 alkyl, alkenyl, cycloalkyl, for example a C ₄ -Cg cycloalkyl, aryl, acyl, heteroaryl, arylalkyl, heteroarylalkyl, aryloxy, alkyloxy, arylalkyloxy, aromatic polycycles, non-aromatic polycycles, mixed aryl and non-aryl polycycles, polyheteroaryl, non-aromatic polyheterocycles, and mixed aryl and non-aryl polyheterocycles, or a pyridine group,

n is an integer from 5 to 8.

Suitably, R10 is a hydrogen atom.

In a preferred embodiment of the invention, Rg is a substituted or unsubstituted phenyl group. Suitably, the phenyl group is substituted with a halogen, for example, a chloro, bromo, fluoro, or iodo group, a methyl, cyano, nitro, trifluoromethyl, amino, methylcyano, sulphonate, or aminocarbonyl group. In another embodiment, Rg is selected from the group consisting of methoxy, cyclohexyl, hydroxyl, benzyloxy, and pyridine.

HDAC inhibitors of general formula II are described in US 6,087,367, especially the specific structures described in Table 1 spanning Columns 29 to 36 of US 6,087,367. The complete contents of US 6,087,367 are incorporated herein by reference. In one embodiment, the hydroxamate-type HDAC inhibitor R-Z of general formula lii preferably has the following structure (either in its protonated or deprotonated form):

SAHA

In another embodiment, the HDAC inhibitor R-Z is a hydroxamate-type HDAC inhibitor having a general formula IV either in its protonated or deprotonated form:

Z— Q ₂ — J— Q-|— A

(IV)

in which:

A is an aryl group; Qi is a covalent bond or an aryl leader group;

J is a sulfonamide linkage selected from where Rn is a sulfonamide substituent;

Q2 is an acid leader group; and

Z is a terminal hydroxamate group, with the proviso that then Qi is an aryl leader group.

The hydroxamate-type HDAC inhibitor R-Z of general formula IV preferably has the following structure either in its protonated or deprotonated form:

Belinostat: PXD-101

Compounds of general formula IV are described in WO02/30879, especially the generic compound number 4, 12, 15 on pages 17, 56 and 57, respectively, and the specific structure numbers 1 to 125 described on pages 58-72. The complete contents of WO02/30879 are incorporated herein by reference.

In another embodiment, the HDAC inhibitor R-Z is a hydroxamate-type HDAC inhibitor having a general formula V either in its protonated or deprotonated form:

(V)

in which:

Ri2 is H, halo, or a straight chain C1-C6 alkyl;

Ri3 and R14 are the same or different and independently selected from H, halo, Ci to C ₄ alkyl, such as CH ₃ and CF ₃ , N0 ₂ , C(0)Ri ₂ , OR12, SR19, CN, NR20R21 ;

Ri5 is selected from H, Ci to C10 alkyl, C ₄ to C9 cycloalkyl, C ₄ to Cg heterocycloalkyl,

C ₄ to Cg heterocycloalkylalkyl, cycloalkylalkyl, aryl, heteroaryl, arylalkyl,

heteroarylalkyl, -(CH ₂ ) _n C(0)Ri ₃ , -(CH ₂ ) _n O(0)Ri ₃ , amino acyl, and HON-C(O)-

CH=C(Ri)-aryl-alkyl;

R16 and Ri7 are the same or different and independently H , C1-C6 alkyl, acyl or acylamino, or R16 and R17 together with the carbon to which they are bound represent C=0, C=S, or R15 together with the nitrogen to which it is bound and R16 together with the carbon to which it is bound can form a C4-C9 heterocycloalkyl, a heteroaryl, a polyheteroaryl, a non-aromatic polyheterocycle, or a mixed aryl and non-aryl polyheterocycle ring;

R18 is selected from H, C1-C6 alkyl, C4-C9 cycloalkyl, C4-C9 heterocycloalkyl, acyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl, aromatic polycycles, non-aromatic polycycles, mixed aryl and non-aryl polycycles, polyheteroaryl, non-aromatic polyheterocycles, and mixed aryl and non-aryl polyheterocycles;

Rig is selected from C1-C4 alkyl, for example CH ₃ and CF ₃ , C(0)-alkyl, for example C(0)CH ₃ and C(0)CF ₃ ;

R20 and R21 are the same or different and independently selected from H, C1-C4 alkyl, and -C(0)-alkyl; and

η-ι, n2 and n ₃ are the same or different and independently selected from 0-6, when ni is 1 -6, each carbon atom can be optionally and independently substituted with Rg

Compounds of general formula V are described in US 6,552,056, especially the generic structures described on column 3 to 14, and the specific structure numbers 1 to 265 described on columns 23 to 142. In one embodiment, the HDAC inhibitor R-Z comprises the compounds falling within the scope of Claim 1 of US6,552,056. The complete contents of US 6,552,056 are incorporated herein by reference.

The hydroxamate-type HDAC inhibitor R-Z of general formula V preferably has the following structures either in their protonated or deprotonated forms selected from the group:

Panobinostat: LBH ₅₈ g H

Redox Active Metals

The redox active metal is typically a transition metal, and in one embodiment a first row transition element. Examples include copper, iron, manganese and cobalt. In one embodiment, the redox metal is selected from copper and iron. In one embodiment, where the metal is copper, the copper ion is in the +2 oxidation state with the overall complex being neutral. In one embodiment, the redox active metal excludes platinum. When the HDAC inhibitor-copper-phen complex enters a tumour cell, the reducing environment of the tumour cell activates the complex causing a Cu ²⁺ to Cu ⁺ reduction, resulting in the release of the HDAC inhibitor.

Nitorgen Donor Ligands

The nitrogen donor ligand is selected from bipyridine or phenanthroline (Phen) or a derivative thereof that is capable of forming a coordination complex with a redox active metal ion, whereby the coordination complex is typically capable of exhibiting chemical nuclease activity. In one embodiment, the nitrogen donor ligand has a structure of general formula II:

in which:

Ri to Rs are each, independently, selected from oxygen, hydrogen, hydroxyl, a carboxyl, a substituted or unsubstituted, branched or unbranched alkyl, alkenyl, cycloalkyl, aryl, aryloxy, alkyloxy, arylalkyloxy, amino, alkylamino, hydroxylamino, dialkylamino, or alkoxy group, or pyridine, or are fused to form a monocyclic or polycyclic aromatic hydrocarbon or heterocycle.

Examples of phenanthroline ligands are described in Kellett et al., Chem. Commun.

(Camb), 2012, 48, 6906-6908; (b) Kellett et al., J. Med. Chem., 2013, 56, 8599-8615; (c) M. McCann et al., Chem. Commun. (Camb), 2013, 49, 2341 -2343; (Kellett et al., Inorg.

Chem., 2014, 53, 5392-5404 and Kellett et al., ACS Chem. Biol., 2016, 1 1 , 159-171 . In one embodiment, the nitrogen donor-ligand is selected from bipyridine or 1 ,10- phenanthroline or phenanthroline derivatives selected from:

Bipy Phen DPQ DPPZ DPPN Phendio

Other examples of phenanthroline derivatives are described in Bencini et al., Coord. Chem. Rev., 2010, 254. 2096-2180; Santini et al., Chem. Rev., 2014, 1 14, 815-862; and Liu and Sadler, Acc. Chem. Res., 2011 , 44, 349-359. Counter ions and salts

Examples of suitable counter ions will be well known to the person skilled in the art, and include: acid addition salts such as the hydrochlorides, hydrobromides, perchlorates, phosphates, sulphates, hydrogen sulphates, alkylsulphates, arylsulphonates, acetates, benzoates, citrates, gluconates, maleates, fumarates, succinates, lactates, and tartrates; alkali metal cations such as Na, K, and Li; alkali earth metal salts such as Mg or Ca; or organic amine salts. The counterion may be one anion, or a plurality of anions, where the composite charge of the plurality of anions balances the charge on the complex cation. For ease of reference, the Metal Complex formulae provided hereafter do not illustrate a counter ion.

Metal Complexes

The invention also relates to a metal complex comprising a redox active metal ion such as Cu ²⁺ coordinated to a hydroxamic acid group of a HDAC inhibitor and a nitrogen donor ligand, especially a phenanthroline ligand, and typically capable of being reduced in a tumour cell to provide a free HDAC inhibitor and a redox active metal-nitrogen donor ligand complex ion that in one embodiment exhibits DNA binding and/or chemical nuclease activity (hereafter "Metal Complex"). The HDAC inhibitor may be in a protonated or deprotonated form. In one embodiment, the complex binds DNA, and exhibits potent chemical nuclease and HDAC inhibition activity. In one embodiment, the complex is cytotoxic at low microM or nanoM concentrations in the in-vitro cytotoxicity assay described below. Exemplary Metal Complexes of the invention have one of the following general formula VI, VII and VIII:

(VIM) in which N N , ₉ to Ris, n, n ^i , n2, n3, Q2, J, Q1 and A are as defined above.

In one embodiment, the Metal Complex of the invention has a structure selected from:

In one embodiment, the Metal Complex of the invention has a structure selected from: 

In another aspect, the invention provides a method of treating a proliferative disorder, for example a cancer, in an individual, comprising a step of administering to the individual a therapeutically effective amount of the Metal Complex of the invention.

In another aspect, the invention relates to a method of treating a cell to inhibit proliferation of the cell comprising a step of treating the cell with a therapeutically effective amount of the Metal Complex of the invention.

In another aspect, the invention relates to a method of treating a cancer comprising a step of treating an individual with a therapeutically effective amount of the Metal Complex of the invention, wherein the Metal Complex is typically capable of exhibiting DNA binding and HDAC inhibitory activity in-vivo. In another aspect, the invention relates to a method of treating a cancer comprising a step of treating an individual with a therapeutically effective amount of the Metal Complex of the invention, wherein the Metal Complex is typically capable of being activated in-vivo to provide an active HDAC inhibitor and an active DNA-binding heavy metal ion.

In another aspect, the invention provides a pharmaceutical composition comprising a therapeutically effective amount of the Metal Complex of the invention and a

pharmaceutically acceptable carrier.

In another aspect, the invention relates to the use of the Metal Complex of the invention as a medicament.

In another aspect, the invention relates to the use of the Metal Complex of the invention in the manufacture of a medicament for the treatment of cancer.

Methods for synthesising complexes of the invention are also provided. In one

embodiment, in which the counterion is a perchlorate ion, the method is a one-pot method, where the redox active metal perchlorate salt is first added, a non-aqueous suspension of HDAC inhibitor is then added, before a non-aqueous suspension of the nitrogen donor ligand is then added. The mixture is then agitated and a resultant precipitated is separated.

This, the invention relates to a one-pot method of synthesising a complex of the invention comprising the steps of:

providing an aqueous solution of a redox active metal perchlorate salt;

adding a non-aqueous solution of HDAC inhibitor to the aqueous solution of the redox active perchlorate salt to provide a first mixture, wherein the HDAC inhibitor has a terminal hydroxamate group;

adding a non-aqueous solution of nitrogen donor ligand to the first mixture to provide a second mixture, in which the nitrogen donor ligand is selected from a bipyridine ligand and a phenanthroline ligand or phenanthroline-type ligand;

agitating the second mixture to allow precipitating of the complex; and

separating the complex from the filtrate. In one embodiment, the second mixture comprises substantially equimolar amounts of redox active metal perchlorate salt, HDAC inhibitor, and nitrogen donor ligand.

In one embodiment, the non-aqueous solution of HDAC inhibitor and non-aqueous solution of nitrogen donor ligand are each, independently, warmed prior to addition to the aqueous solution of the redox active perchlorate salt.

In one embodiment, the method comprises a step of adding a strong base to the second reaction mixture prior to or during the agitation step.

In one embodiment, the HDAC inhibitor is selected from SAHA, Panabinostat and

Belinostat.

In one embodiment, the nitrogen donor ligand is selected from BIPY, PHEN, DPQ, DPPZ, DPPN and Phendio.

In one embodiment, the redox active metal is selected from a first row transition element.

In one embodiment, the redox active metal is copper.

In another embodiment in which the redox active metal salt is not a perchlorate, for example is a nitrate, sulphate or chloride, the method comprises the steps of:

providing a non-aqueous solution of a redox active metal salt;

adding a non-aqueous suspension of nitrogen donor ligand to the non-aqueous solution of a redox active metal salt, in which the nitrogen donor ligand is selected from a bipyridine ligand and a phenanthroline ligand or phenanthroline-type ligand; agitating the first mixture to precipitate an intermediate complex;

separating the intermediate complex from the filtrate;

resuspending the intermediate complex in a non-aqueous solvent;

adding a non-aqueous solution of HDAC inhibitor to the resuspended intermediate complex to provide a second mixture, wherein the HDAC inhibitor has a terminal hydroxamate group;

agitating the second mixture to precipitate the final complex; and

separating the final complex from the filtrate. In one embodiment, the salt is selected from a nitrate, sulphate, or chloride.

In one embodiment, the redox active metal is a first row transition metal. In one embodiment, the redox active metal is copper.

In one embodiment, the redox active metal salt is suspended in ethanol. In one embodiment, the nitrogen donor ligand is suspended in ethanol.

In one embodiment, the HDAC inhibitor is suspended in methanol.

In one embodiment, the second mixture comprises base, for example potassium hydroxide. In one embodiment, the method employs substantially equimolar amounts of redox active metal salt, HDAC inhibitor, and nitrogen donor ligand.

In one embodiment, the agitation steps are carried out under reflux.

Other aspects and preferred embodiments of the invention are defined and described in the other claims set out below.

Brief Description of the Figures

Figure 1. Multifunctional Cu ²⁺ complexes reduce HDAC activity: SKOV-3 Cells were seeded in a 75 cm ² tissue culture flask then grown to 70-80% confluency. Cells were incubated with Cu(ll) complexes and SAHA corresponding to the IC50 value for 24, 48 or 72 Hrs. Nuclear isolates were isolated according to the protocol recommended by the manufacturer. SKOV-3 nuclear extracts were tested for their HDAC activity according to the protocol reccomended by the manufacturer (Epigentek, USA).

Figure 2. DNA cleavage reactions in the presence or absence of ROS specific scavengers: 400 ng of SC pUC19 was incubated in for 30 minutes at 37 °C with concentrations of 1 , 2.5, 3.75 & 5 μΜ Cu-SAHA-Phen / Cu-SAHA-DPQ or Cu-SAHA-Phendio; 2.5, 5, 10 & 15 μΜ Cu-SAHA-Bipy or SAHA; 1 , 2.5, 5 & 10 μΜ Cu-SAHA-DPPZ; in the presence of 25 mM NaCI, 0.5 mM Na-L-ascorbate, 80 mM HEPES. Lanes 1 - 4: DNA + Complex; Lanes 5 - 8: + 10mM Kl; Lanes 9 - 12: + 10mM NaN ₃ ; Lanes 13 - 16: + 10 mM Tiron; Lanes 17 - 20: + 10% DMSO.

Figure 3. Radical scavenger interactions: Panel A: ROS scavengers utilized within this study, Panel B: Molecular structure of Tiron (TH2), Panel C: Reaction equations of radical species and their respective scavengers.

Figure 4. Cu ²⁺ induce oxidative DNA damage: EdU incorporation assay data (SK-OV-3 treated cells).

Detailed Description of the Invention

All publications, patents, patent applications and other references mentioned herein are hereby incorporated by reference in their entireties for all purposes as if each individual publication, patent or patent application were specifically and individually indicated to be incorporated by reference and the content thereof recited in full.

Definitions and general preferences

Where used herein and unless specifically indicated otherwise, the following terms are intended to have the following meanings in addition to any broader (or narrower) meanings the terms might enjoy in the art:

Unless otherwise required by context, the use herein of the singular is to be read to include the plural and vice versa. The term "a" or "an" used in relation to an entity is to be read to refer to one or more of that entity. As such, the terms "a" (or "an"), "one or more," and "at least one" are used interchangeably herein.

As used herein, the term "comprise," or variations thereof such as "comprises" or

"comprising," are to be read to indicate the inclusion of any recited integer (e.g. a feature, element, characteristic, property, method/process step or limitation) or group of integers (e.g. features, element, characteristics, properties, method/process steps or limitations) but not the exclusion of any other integer or group of integers. Thus, as used herein the term "comprising" is inclusive or open-ended and does not exclude additional, unrecited integers or method/process steps.

As used herein, the term "disease" is used to define any abnormal condition that impairs physiological function and is associated with specific symptoms. The term is used broadly to encompass any disorder, illness, abnormality, pathology, sickness, condition or syndrome in which physiological function is impaired irrespective of the nature of the aetiology (or indeed whether the aetiological basis for the disease is established). It therefore encompasses conditions arising from infection, trauma, injury, surgery, radiological ablation, poisoning or nutritional deficiencies.

As used herein, the term "treatment" or "treating" refers to an intervention (e.g. the administration of an agent to a subject) which cures, ameliorates or lessens the symptoms of a disease or removes (or lessens the impact of) its cause(s) (for example, the reduction in accumulation of pathological levels of lysosomal enzymes). In this case, the term is used synonymously with the term "therapy". Additionally, the terms "treatment" or "treating" refers to an intervention (e.g. the administration of an agent to a subject) which prevents or delays the onset or progression of a disease or reduces (or eradicates) its incidence within a treated population. In this case, the term treatment is used synonymously with the term "prophylaxis". As used herein, an effective amount or a therapeutically effective amount of an agent defines an amount that can be administered to a subject without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio, but one that is sufficient to provide the desired effect, e.g. the treatment or prophylaxis manifested by a permanent or temporary improvement in the subject's condition. The amount will vary from subject to subject, depending on the age and general condition of the individual, mode of administration and other factors. Thus, while it is not possible to specify an exact effective amount, those skilled in the art will be able to determine an appropriate "effective" amount in any individual case using routine experimentation and background general knowledge. A therapeutic result in this context includes eradication or lessening of symptoms, reduced pain or discomfort, prolonged survival, improved mobility and other markers of clinical improvement. A therapeutic result need not be a complete cure.

In the context of treatment and effective amounts as defined above, the term subject (which is to be read to include "individual", "animal", "patient" or "mammal" where context permits) defines any subject, particularly a mammalian subject, for whom treatment is indicated. Mammalian subjects include, but are not limited to, humans, domestic animals, farm animals, zoo animals, sport animals, pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows; primates such as apes, monkeys, orangutans, and chimpanzees; canids such as dogs and wolves; felids such as cats, lions, and tigers;

equids such as horses, donkeys, and zebras; food animals such as cows, pigs, and sheep; ungulates such as deer and giraffes; and rodents such as mice, rats, hamsters and guinea pigs. In preferred embodiments, the subject is a human. In this specification, the term "cancer" should be taken to mean a cancer selected from the group consisting of: fibrosarcoma; myxosarcoma; liposarcoma; chondrosarcom; osteogenic sarcoma; chordoma; angiosarcoma; endotheliosarcoma; lymphangiosarcoma;

lymphangioendotheliosarcoma; synovioma; mesothelioma; Ewing's tumor;

leiomyosarcoma; rhabdomyosarcoma; colon carcinoma; pancreatic cancer; breast cancer; ovarian cancer; prostate cancer; squamous cell carcinoma; basal cell carcinoma;

adenocarcinoma; sweat gland carcinoma; sebaceous gland carcinoma; papillary carcinoma; papillary adenocarcinomas; cystadenocarcinoma; medullary carcinoma;

bronchogenic carcinoma; renal cell carcinoma; hepatoma; bile duct carcinoma;

choriocarcinoma; seminoma; embryonal carcinoma; Wilms' tumor; cervical cancer; uterine cancer; testicular tumor; lung carcinoma; small cell lung carcinoma; bladder carcinoma; epithelial carcinoma; glioma; astrocytoma; medulloblastoma; craniopharyngioma;

ependymoma; pinealoma; hemangioblastoma; acoustic neuroma; oligodendroglioma; meningioma; melanoma; retinoblastoma; and leukemias. In a preferred embodiment, the cancer is selected from the group comprising: breast; cervical; prostate; ovarian, colorectal, lung, lymphoma, and leukemias, and/or their metastases.

"Lower alkyl" means an alkyl group, as defined below, but having from one to ten carbons, more preferable from one to six carbon atoms (eg. "C - C - alkyl") in its backbone structure. "Alkyi" refers to a group containing from 1 to 8 carbon atoms and may be straight chained or branched. An alkyi group is an optionally substituted straight, branched or cyclic saturated hydrocarbon group. When substituted, alkyi groups may be substituted with up to four substituent groups, at any available point of attachment. When the alkyi group is said to be substituted with an alkyi group, this is used interchangeably with "branched alkyi group". Exemplary unsubstituted such groups include methyl, ethyl, propyl, isopropyl, a- butyl, isobutyl, pentyl, hexyl, isohexyl, 4, 4-dimethylpentyl, octyl, 2,2,4-trimethylpentyl, nonyl, decyl, undecyl, dodecyl, and the like. Exemplary substituents may include but are not limited to one or more of the following groups: halo (such as F, CI, Br, I), Haloalkyl (such as CCI 3 or CF3), alkoxy, alkylthio, hydroxyl, carboxy (-COOH), alkyloxycarbonyl (- C(O)R), alkylcarbonyloxy (-OCOR), amino (-IMH2), carbamoyl (-NHCOOR-or-OCONHR), urea (-NHCONHR-) or thiol (-SH). Alkyi groups as defined may also comprise one or more carbon double bonds or one or more carbon to carbon triple bonds. "Lower alkoxy" refers to O-alkyl groups, wherein alkyi is as defined hereinabove. The alkoxy group is bonded to the core compound through the oxygen bridge. The alkoxy group may be straight-chained or branched; although the straight-chain is preferred.

Examples include methoxy, ethyloxy, propoxy, butyloxy, t-butyloxy, i-propoxy, and the like. Preferred alkoxy groups contain 1 -4 carbon atoms, especially preferred alkoxy groups contain 1 -3 carbon atoms. The most preferred alkoxy group is methoxy.

"Halogen" means the non-metal elements of Group 17 of the periodic table, namely bromine, chlorine, fluorine, iodine and astatine.

The terms "alkyi", "cycloalkyl", "heterocycloalkyl", "cycloalkylalkyl", "aryl", "acyl", "aromatic polycycle", "heteroaryl", "arylalkyl", "heteroarylalkyl", "amino acyl", "non-aromatic polycycle", "mixed aryl and non-aryl polycycle", "polyheteroaryl", "non-aromatic polyheterocyclic", "mixed aryl and non-aryl polyheterocycles", "amino", and "sulphonyl" are defined in

US6, 552,065, Column 4, line 52 to Column 7, line 39. "Halogen" means the non-metal elements of Group 17 of the periodic table, namely bromine, chlorine, fluorine, iodine and astatine.

The terms "aryl leader group" and "acid leader group" are defined in pages 25-54 of WO02/30879. "Substituted" unless otherwise defined means substituted with a substituent selected from halogen, oxygen, hydrogen, hydroxyl, a carboxyl, a substituted or unsubstituted, branched or unbranched alkyl, alkenyl, cycloalkyl, aryl, aryloxy, alkyloxy, arylalkyloxy, amino, alkylamino, hydroxylamino, dialkylamino, or alkoxy group, or pyridine, or are fused to form a monocyclic or polycyclic aromatic hydrocarbon or heterocycle.

The terms "salt" and "counter ion" designate a pharmaceutically acceptable salts/counter ions and can include acid addition salts such as the hydrochlorides, hydrobromides, phosphates, nitrates, sulphates, hydrogen sulphates, alkylsulphates, arylsulphonates, acetates, benzoates, citrates, gluconates, maleates, fumarates, succinates, lactates, and tartrates; alkali metal cations such as Na, K, Li; alkali earth metal salts such as Mg or Ca; or organic amine salts. Exemplary organic amine salts are tromethamine (TRIS) salts and amino acid salts (e.g. histidine salts) of the compounds of the invention.

As used herein, the term "Histone deacetylase inhibitor" or "HDAC inhibitor" (R-Z) refers to a hydroxamate-type HDAC inhibitor, i.e. a HDAC inhibitor having a terminal hydroxamate group, that typically exhibits HDAC inhibitory activity in a tumour cell. The HDAC inhibitor preferably has at least micromolar and preferably nanomolar inhibitory activity (IC50) against any HDAC isoform as determined using the HDAC inhibition assay described in Yuan et al (Bioorganic and Medicinal Chemistry, 2017 - http://dx.doiorg/10.1016/j.bmcx.2017.05.058). In one embodiment, the HDAC inhibitor is selected from the group consisting of: SAHA or SAHA-like HDAC inhibitors (general formula III above); Panabinostat or Panabinostat-like inhibitors (general formula V); and Belinostat or Belinostat-like inhibitors (general formula IV). In one embodiment, the HDAC inhibitor is selected from the group consisting of: SAHA or SAHA-like HDAC inhibitors (general formula III above); Panabinostat or Panabinostat-like inhibitors (general formula V); and Belinostat or Belinostat-like inhibitors (general formula IV). In one embodiment, the HDAC inhibitor is selected from the group consisting of: SAHA, Panabinostat and

Belinostat.

As used herein the term "dual HDAC/PARP inhibitor" refers to a hybrid compound that targets PARP and HDAC concurrently. Dual HDAC/PARP inhibitors are described in Yuan et al (Bioorganic and Medicinal Chemistry, 2017). The dual PARP/HDAC inhibitor has inhibitory activity against any PARP isoform, for example PARP1 or 2 and any HDAC isoform, for example HDAC 1 or 6, with I C50- values in at least the micromolar, and preferably nanomolar range, as determined by the HDAC inhibitory assay described in Yuan et al. In one embodiment, the dual PARP/HDAC inhibitor is a hybrid of the PARP inhibitor Olaparib and a hydroxamate-type HDAC inhibitor.

Therapeutic Compositions and Methods of Administration

The invention provides methods of, and compositions for, treatment and prevention by administration to a subject in need of such treatment of a therapeutically or prophylactically effective amount of a metal complex of the invention.

Various delivery systems are known and can be used to administer a compound or composition of the invention, e.g., encapsulation in liposomes, microparticles,

microcapsules. Methods of introduction include but are not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compounds or compositions may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local. In addition, it may be desirable to introduce the compounds or compositions of the invention into the circulation system by any suitable route. Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent. It may be desirable to administer the compounds or compositions of the invention locally to the area in need of treatment; this may be achieved, for example and not by way of limitation, by topical application, by injection, by means of a catheter, by means of a suppository, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers.

Alternatively, the compounds can be delivered in a vesicle, in particular a liposome (see Langer, Science, 1990, 249, 1527-1533; Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327). In yet another embodiment, the compounds or compositions of the invention can be delivered in a controlled release system. In one embodiment, a pump may be used (see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng., 1987, 14, 201 ; Buchwald et al., Surgery, 1980, 88, 75; Saudek et al, N. Engl. J. Med., 1989, 321 , 574). In another embodiment, polymeric materials can be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Press., Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, J. Macromol. Sci. Rev. Macromol. Chem., 1983, 23, 61 ; see also Levy et al., Science, 1985, 228, 190; During et al., Ann. Neurol., 1989, 25, 351 ; Howard et al., J. Neurosurg., 1989, 71 , 105). In yet another embodiment, a controlled release system can be placed in proximity of the therapeutic target, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 1 15-138 (1984)). Other controlled release systems are discussed in the review by Langer (Science, 1990, 249, 1527-1533).

The present invention also provides pharmaceutical compositions. Such compositions comprise a therapeutically effective amount of an Active Compound of the invention, and a pharmaceutically acceptable carrier. In a specific embodiment, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals and more particularly in humans.

The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the compound or pro-drug of the invention is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol and water.

The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like.

The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulation can include standard carriers such as

pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E. W. Martin. Such compositions will contain a therapeutically effective amount of the compound or pro-drug of the invention, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration.

In a preferred embodiment, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anaesthetic such as lignocaine to, ease pain at the, site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. In the case of cancer, the amount of the therapeutic of the invention which will be effective in the treatment or prevention of cancer will depend on the type, stage and locus of the cancer, and, in cases where the subject does not have an established cancer, will depend on various other factors including the age, sex, weight, and clinical history of the subject. The amount of therapeutic may be determined by standard clinical techniques. In addition, in vivo and/or in vitro assays may optionally be employed to help predict optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the cancer, and should be decided according to the judgment of the practitioner and each patient's circumstances. Routes of administration of a therapeutic include, but are not limited to, intramuscularly, subcutaneously or intravenously. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.

The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the compositions of the invention.

Exemplification

The invention will now be described with reference to specific Examples. These are merely exemplary and for illustrative purposes only: they are not intended to be limiting in any way to the scope of the monopoly claimed or to the invention described. These examples constitute the best mode currently contemplated for practicing the invention.

Unless otherwise stated, all chemicals were purchased from Sigma-Aldrich (Arklow, Ireland) as reagent grade and used without further purification. SAHA (Gediya et a/., J. Med. Chem., 2005, 48, 15, 5047-51 ),Phendione (Kellett et al., Inorg. Chem., 2014, 53, 5392-5404), DPQ (Kellett et al., Inorg. Chem., 2014, 53, 5392-5404) and DPPZ (Kellett et al., Inorg. Chem., 2014, 53, 5392-5404) were prepared by previously reported methods. Infra-Red spectra were recorded on a Perkin Elmer Spectrum GX Bruker spectrometer. Electrospray lonisation mass spectrometry (ESI-MS) experiments were carried out on an Advion Expression Compact Mass Spectrometer. Elemental analysis (C, H, N) were carried out on an Exeter Analytical CE440 elemental analyser. Cu analysis was carried out on a Varian 55B atomic absorption spectrometer. CI analysis was determined via combustion in an oxygen flask followed by titration with mercuric nitrate.

Example 1

[Cu(SAHA. _{1 H} )(DPPZ)]CI0 ₄

Cu-SAHA-DPPZ

Scheme 1 Synthesis of [CU(SAHA-IH)(DPPZ)]CI0 ₄ (CU-SAHA-DPPZ)

Cu(CIC>4)2-6H20 (106.4 mg, 0.287 mmol) was dissolved in 1 ml of deionised water . To the Cu solution, warm solutions of SAHA (75.9 mg, 0.287 mmol) and DPPZ (81.04 mg, 0.287 mmol) in methanol (3ml) were added sequentially. The resulting suspension was stirred at room temperature for 30 min. The solid green product (Cu-SAHA-DPPZ) was filtered, washed with cold deionised water, methanol and dried under vacuum, Scheme 1 . Yield 127 mg, 60.5%. C32H31CICUN6O7 requires C - 54.09 %; H - 4.40 %; N - 1 1 .83 %; Cu - 8.94 %; CI - 4.99 % ; found C - 53.70 %; H - 3.83 %; N - 1 1.65 %; Cu - 9.05 %; CI 4.80 %. ESI- MS ([M-H] ⁺ ) (MeOH) m/z: 609 ([Cu(SAHA.i _H )(DPPZ)] ⁺ ) 265.2 (SAHA). IR (KBr) cm ^"1 : 3346.37, 3071.09, 1652.96, 1592.59, 1073.17.

Example 2

[Cu(SAHA_ _{1 H} )(Phendio)]CIO

Cu-SAHA-Phendio

Scheme 2 Synthesis of [Cu(SAHA-iH)(Phendio)]CI0 ₄ (Cu-SAHA-Phendio) Cu(CIC>4)2-6H20 (108.6 mg, 0.293 mmol) was dissolved in 1 ml of deionised water. Warm solutions of SAHA (77.7 mg, 0.293 mmol) in 1 ml of methanol and 1 ,10-phenanthroline- 5,6-dione (Phendio) (61 .7 mg, 0.293 mmol) in 4 ml of ethanol were added sequentially. A solution of potassium hydroxide (16.4 mg, 0.293 mmol) in 1 ml of deionised water was added and the solution left to stir at room temperature for 30 minutes. The filtrate was left to slowly evaporate at room temperature for -5 days. A green solid by-product was filtered and the solvent from the filtrate was concentrated in vacuo. The waxy solid was suspended in deionised water and agitated. A brown solid (Cu-SAHA-Phendio) was filtered, washed with cold deionised water, methanol and dried under vacuum, Scheme 2. Yield 105 mg, 67%. [Cu(SAHA.i _H )(Phendio)]CIO ₄ 0.5 H ₂ 0 (Cu-SAHA-Phendio):

C26H26CICU N4O10 requires C - 47.79 %; H - 4.01 %; N - 8.57 %; Cu - 9.72 %; CI - 5.43 %; found C - 47.55 %; H - 3.99 %; N - 8.37 %; Cu - 9.51 %; CI - 5.21 %. IR (ATR) cm ^"1 :

3377.67, 3222.67, 1693.41 , 1598.73, 1070.18.

[Cu(SAHA_ _{1 H} )(Bipy)]CI0 ₄ [Cu(SAHA. _{1 H} )(Phen)]CI0 ₄ [Cu(SAHA _{.1 H} )(DPQ)]CI0 ₄ (Cu-SAHA-Bipy) (Cu-SAHA-Phen) (Cu-SAHA-DPQ)

Scheme 3 Synthesis of [CU(SAHA-IH)(W,W^]CI0 ₄ (where N,N' = Bipy, Phen & DPQ)

Copper(ll) perchlorate hexahydrate (Cu(CI04)2-6H20) (100 mg, 0.270 mmol) was dissolved in a minimum volume of deionised water (1 ml). Warm solutions of SAHA (71 .4 mg, 0.270 mmol) and nitrogen donor ligand of either Bipy (42.5 mg, 0.270 mmol), Phen (48.7 mg, 0.270 mmol) or DPQ (46.7 mg, 0.270 mmol) in 1 ml of methanol were added sequentially. A solution of potassium hydroxide (15.1 mg, 0.270 mmol) in 1 ml of deionised water was immediately added and the resulting suspension stirred at room temperature for 30 min. The solid product was filtered, washed with cold deionised water , methanol and dried under vacuum. [Cu(SAHA.i _H )(Bipy)]CIO ₄ 0.5 H ₂ 0 (Cu-SAHA-Bipy): Green solid, Yield 107.3 mg, 67.7 %. C24H28CICUN4O8 requires C - 48.08 %; H - 4.71 %; N - 9.35 %; Cu - 10.60 %; CI - 5.91 %; found C - 48.39 %; H - 4.38 %; N - 9.30 %; Cu - 10.67 %; CI - 5.73 %. ESI-MS ([M-H] ⁺ ) (MeOH) mass-to-charge ratio (m/z): 483 ([Cu(SAHA.i _H )(BIPY)] ⁺ ) 265.2 (SAHA) 219.8 ([Cu(BIPY)] ⁺ ]). IR (ATR) cm ^"1 : 3567.39 (s,s), 2931 .19, 1660.60 (s,s) 1595.55. (s,s) 1071.57 (s,br). [Cu(SAHA.i _H )(Phen)]CIO ₄ 0.5 H ₂ 0 (Cu-SAHA-Phen): Blue solid, Yield 90 mg, 80 %. C26H28CICUN4O9 requires C - 48.83 %; H - 4.41 %; N - 8.76 %; Cu - 9.94 %; CI - 5.54 %; found C - 49.25 %; H - 4.56 %; N - 8.62 %; Cu - 10.36 %; CI - 5.44 %. IR (ATR) cm ^"1 : 3495.44, 3065.47, 1650.33 1535.02 1070.18. ESI-MS ([M-H] ⁺ ) (MeOH) m/z: 506.1 ([Cu(SAHA-i _H )(Phen)] ⁺ ) 265.1 (SAHA) 243.9 ([Cu(Phen)] ⁺ ). [Cu(SAHA.

IH)(DPQ)]CI0 ₄ (CU-SAHA-DPQ): Grey solid, Yield 96.8 mg, 53.01 %. C28H27CICUN6O7 requires C - 51.07 %; H - 4.13 %; N - 12.76 %; Cu - 9.65 %; CI - 5.38 %; found C - 50.65 %; H - 3.81 %; N - 12.57 %; Cu - 9.63 %; CI - 4.97 %. ESI-MS ([M-H] ⁺ ) (MeOH) m/z: 558.1 ([CU(SAHA.IH)(DPQ)] ⁺ ) 294.8 ([Cu(DPQ)] ⁺ ) 265.1 (SAHA). IR (ATR) cm ^"1 : 3350.21 ,

3050.31 , 1658.57, 1595.07, 1087.07.

Scheme 4 Synthesis of [Cu(Belinostat-i _H )(DPPZ)]CIC>4

Copper(ll) perchlorate hexahydrate (0.1090g; 0.294 mmol; 1 eq; limiting reagent) was dissolved in the minimum volume (1 ml) of Dl H2O. Hot methanolic solutions of Belinostat (0.0934g; 0.294 mmol; 1 eq; 4ml) and DPPZ (0.0828g; 0.294 mmol; 1 eq; 1 ml) were added sequentially and the resulting suspension was left to stir at room temperature for 30 minutes. A solid precipitate was filtered and dried under vacuum. [Cu(Belinostat-iH)(DPPZ)]CIC>4, green solid, 178.8 mg, 79.8 %. [Cu(Belinostat-i _H )(DPPZ)]CI0 ₄ ^■ MeOH requires C - 51.39%; H - 3.42%; N - 10.58%; Cu - 8.00%; CI - 4.46%. Found C - 51.35%; H - 2.96%; N - 10.75%; Cu - 7.93%; CI - 4.70%. ESI-MS ([M-H] ⁺ ; MeOH) 661 .9 ([Cu(Belionstat IH)(DPPZ)] ⁺ ) 345 ([Cu(DPPZ)] ⁺ ). ATR-IR (cm ^-1 ) 1510, 1019 & 1 130.

Example 4

Scheme 5 Synthesis of [Cu(Belinostat-iH)(DPQ)]CI0 ₄

Copper(ll) perchlorate hexahydrate (0.1051 g; 0.284mmol; 1 eq; limiting reagent) was dissolved in the minimum volume (1 ml) of Dl H2O. Hot methanolic solutions of Belinostat (0.0906g; 0.284mmol; 1 eq; 4ml), DPQ (0.0653g; 0.284mmol; 1 eq; 1 ml) were added sequentially. KOH (0.0159g; 0.284mmol; 1 eq) in 1 ml de-ionised water was added and the resulting suspension was left to stir at room temperature for 30 minutes. A solid precipitate was filtered and dried under vacuum. [Cu(Belinostat.i H)(DPQ)]CI0 ₄ , grey solid, 174 mg, 86.1 %. [Cu(Belinostat.i _H )(DPQ)]CI0 ₄ ^■ H ₂ 0 requires C - 47.68%; H - 3.17%; N - 1 1.50%; Cu - 8.70%. Found C - 47.48%; H - 2.84%; N - 1 1.29%; Cu - 8.73%. ESI-MS ([M-H] ⁺ ; MeOH) 61 1 .8 ([Cu(Belionstat.i _H )(DPQ)] ⁺ ) 327 ([Cu(DPQ)] ⁺ ). ATR-IR (cm ^"1 ) 1521 , 952 & 1074.

Example 5

[Cu(DPQ)[(S0 ₄ ) [Cu(DPQ)(N0 ₃ ) ₂ ]

Scheme 6 Synthesis of [Cu(DPQ)(NOs)2] and [Cu(DPQ)]S0 ₄

Copper(ll) nitrate trihydrate (0.1042g; 0.431 mmol; 1 eq; limiting reagent) or copper(ll) sulphate (0.1 1 12g; 0.697mmol; 1 eq; limiting reagent) were suspended in 5ml of EtOH at reflux. Suspensions of DPQ or DPPZ (1 eq) in 5ml of hot EtOH were added and the resulting suspension was left to stir at reflux for 30 minutes. The solution was allowed to cool and a solid precipitate was filtered and dried under vacuum.

SO

rCu((DPQ)(N0 ₃ R Blue solid, 164mg, 90.7%. ATR-IR (cm ^"1 ) 1472.35; 1260.98; 720.54 [Cu(DPQ)(N0 ₃ ) ₂ ] ^■ MeOH ^■ H ₂ 0 requires C - 38.34%; H - 3.00%; N - 17.89%; found C - 38.57%; H - 3.35; N - 17.45%.

rCu((DPQ)lS0 ₄ . Green solid, 273mg, 99.5%. ATR-IR (cm ^"1 ) 1400.44; 1 140.88; 809.57 [Cu(DPQ)](S0 ₄ ) ^■ H ₂ 0 requires C - 41.03%; H - 2.46%; N - 13.67%; found C - 39.87%; H - 2.28; N - 13.82%.

Example 6

Scheme 7 Synthesis of [Cu(DPPZ)(N0 ₃ ) ₂ ]; [Cu(DPPZ)](S0 ₄ ); [Cu(DPQ)(CI) ₂ ]; [Cu(DPPZ)(CI) ₂ ].

Copper(ll) nitrate trihydrate (0.1068g; 0.442mmol; 1 eq; limiting reagent) copper (II) chloride (0.1038g; 0.772mmol; 1 eq limiting reagent) or copper(ll) sulphate (0.1029g; 0.645mmol; 1 eq; limiting reagent) were suspended in 5ml of EtOH at reflux. Suspensions of DPQ or DPPZ (1 eq) in 5ml of hot EtOH were added and the resulting suspension was left to stir at reflux for 30 minutes. The solution was allowed to cool and a solid precipitate was filtered and dried under vacuum. rCu((DPPZ)(N0 ₃ R Blue solid, 135mg, 64.9%. ATR-IR (cm ^"1 ) 1487.05, 1269.69, 813.21 [Cu(DPPZ)(N0 ₃ ) ₂ ] ^■ H ₂ 0 requires C - 44.31 %; H - 2.48%; N - 17.23%; found C - 44.65%; H - 2.28; N - 17.71 %.

rCu((DPPZ)lSQ ₄ , Green solid, 252mg, 72.7%. ATR-IR (cm ^"1 ) 1485.54; 1274.41 ; 812.59 [Cu(DPPZ)](S0 ₄ ) ^■ H ₂ 0 requires C - 47.01 %; H - 2.63%; N - 12.18%; found C - 47.32%; H - 2.46; N - 12.52%.

rCu((DPQ)(CI)?l Green solid, 217mg, 77.6%. ATR-IR (cm ^"1 ) 1564.49; 1395.41 ; 815.30 [Cu(DPQ)(CI) ₂ ] ^■ H ₂ 0 requires C - 43.71 %; H - 2.62%; N - 14.56%; found C - 43.63%; H - 2.33; N - 14.28%.

rCu((DPPZ)(CI?)l Green solid, 272mg, 84.5%. ATR-IR (cm ^"1 ) 1483.91 ; 1061.82; 798.35 [Cu(DPPZ)(CI) ₂ ] ^■ H ₂ 0 requires C - 49.73%; H - 2.78%; N - 12.89%; found C - 49.52%; H - 2.64; N - 12.45%. Example 7

Belinostat, KOH

[Cu(W,/V'ligand)](X) ₂ eOH, 80°C, 30 min

[Cu(Belinostat. _{1 H} )(W,W'ligand)](X)

X = N0 ₃ or CI

W.W'ligand =

Scheme 8 Synthesis of [Cu(W,W ligand)](X) ₂ where X = NOs or CI; Ν,Ν' ligand = DPQ or DPPZ.

Suspensions of [Cu(DPPZ)(CI) ₂ ] (0.1040 g; 0.250 mmol; 1 eq; limiting reagent), [Cu((DPQ)(CI) ₂ ] (0.1036 g; 0.283 mmol; 1 eq; limiting reagent) were stirred at reflux for 2 minutes in 5ml of MeOH. Belinostat (1 eq) and KOH (1 eq) in 5 ml or 1 ml of MeOH or de- ionised water respectively were added. The resulting suspension was left to stir at reflux for 30 minutes. The solution was allowed to cool and a solid precipitate was filtered and dried under vacuum.

rCu(Belinostat-i _H )(DPQ)(CI)l. Green solid, 123mg, 67.1 %. [Cu(Belinostat-i _H )(DPQ)](CI) 1 H ₂ 0 requires C - 52.25%; H - 3.48%; N - 12.61 %; Cu - 9.53%. Found C - 51.77%; H - 3.06%; N - 12.32%; Cu - 9.43%. ESI-MS ([M-H] ⁺ ; MeOH) 661.8 ATR-IR (cm ^"1 ). rCu(Belinostat-i _H )(DPPZ)(CI)l. Green solid, 151 mg, 86.5%. [Cu(Belinostat.i _H )(DPPZ)](CI) 1.5 H ₂ 0 requires C - 52.94%; H - 3.37%; N - 1 1.23%; Cu - 8.49%. Found C - 53.21 %; H - 3.06%; N - 1 1.06%; Cu - 8.89%. ESI-MS ([M-H] ⁺ ; MeOH) 661 .9 ([Cu(Belionstat.i _H )(DPPZ)] ⁺ ). ATR-IR (cm ^"1 ). Example 8

Scheme 9: General procedure for synthesis of [Cu(DPQ)(Belinostat-iH)]N03; [Cu(DPPZ)(Belinostat-iH)]N03 and [Cu(DPQ)(Belinostat-iH)]HS0 ₄ .

Suspensions of [Cu((DPQ)](N0 ₃ )2 (0.101 1 g; 0.265mmol; 1 eq; limiting reagent), [Cu((DPPZ)]NO ₃ (0.1019g; 0.217mmol; 1 eq; limiting reagent) or [Cu((DPPZ)]S0 ₄ (0.1040g; 0.250mmol; 1 eq; limiting reagent) were stirred at reflux for 2 minutes in 5ml of EtOH. Belinostat (1 eq) and KOH (1 eq) in 5 ml or 1 ml of EtOH or de-ionised water respectively were added. The resulting suspension was left to stir at reflux for 30 minutes. The solution was allowed to cool and a solid precipitate was filtered and dried under vacuum. rCu(Belinostat-i _H )(DPQ)lNQ3. Green solid, 150mg, 92%. [Cu(Belinostat-i _H )(DPQ)](HS0 ₄ ) requires C - 50.25%; H - 3.34%; N - 14.15%; Cu - 9.17%. Found C - 50.35%; H - 2.95%; N - 14.07%; Cu - 9.31 %. ESI-MS ([M-H] ⁺ ; MeOH) 61 1 .8 ([Cu(Belionstat.i _H )(DPQ)] ⁺ ). ATR- IR (cm- ¹ ). rCu(Belinostat-i _H )(DPPZ)lNQ3. Green solid, 136.4mg, 86.7%. [Cu(Belinostati _H )(DPQ)](CI) : 0.5 H ₂ 0 requires C - 52.28%; H - 3.19%; N - 12.93%; Cu - 8.38%. Found C - 52.48%; H - 2.97%; N - 12.45%; Cu - 8.82%. ESI-MS ([M-H] ⁺ ; MeOH) 661.8 ATR-IR (cm ^"1 ). rCu(Belinostat-i H)(DPPZ)lHS0 ₄ . Green solid, 142.8mg, 97%. [Cu(Belinostati _H )(DPPZ)](CI) 1 .5 H ₂ 0 requires C - 52.07%; H - 3.31 %; N - 1 1.04%; Cu - 8.35%. Found C - 51.94%; H - 3.13%; N - 10.92%; Cu - 8.74%. ESI-MS ([M-H] ⁺ ; MeOH) 661 .9 ([Cu(Belionstat IH)(DPPZ)] ⁺ ). ATR-IR (cm ^-1 ).

Example 9

÷ Γ

Belinostat,

[Cu(DPQ)](S0 ₄ ) MeOH, Reflux, 30

[Cu(Bel. _{1 H} )(DPQ)](HS0 ₄ )

Scheme 10:Synthesis of [Cu(DPQ)(Belinostat-i _H )]HS04.

A suspension of [Cu((DPQ)](S0 ₄ )2 (0.1040g; 0.265mmol; 1 eq; limiting reagent), was stirred at reflux for 2 minutes in 5ml of MeOH. A suspension of belinostat (0.0846 g; 0.265 mmol; 1 eq) in 5ml of MeOH was added. The resulting suspension was left to stir at reflux for 30 minutes. The solution was allowed to cool and a solid precipitate was filtered and dried under vacuum. rCu(Belinostat-i H)(DPQ)lHS0 ₄ . Green solid, 159mg, 84.5%. [Cu(Belinostat-i _H )(DPQ)](HS0 ₄ ) : 0.5 H ₂ 0 requires C - 47.90%; H - 3.19%; N - 1 1 .56%; Cu - 8.74%. Found C - 48.29%; H - 2.93%; N - 1 1.28%; Cu - 8.87%. ESI-MS ([M-H] ⁺ ; MeOH) 61 1 .8 ([Cu(Belionstat IH)(DPQ)] ⁺ ). ATR-IR (cm ^"1 ).

Biological evaluation

Cell Culture

Two cancerous cell lines, outlined in Table 1 , were used in this study. These cell lines are routinely used for measuring in vitro cytotoxicity of novel test compounds. Cells were cultured in a standard 75 cm ³ flask (Corning®, Austria) containing RPMI 1640 medium (Sigma-Aldrich, Ireland) supplemented with 10% Fetal Bovine Serum (Gibco®, Ireland) at 37 °C in a humidified atmosphere at 5% CO2. Every 3-4 days cells reached 70-80% confluency after which they were harvested with trypsin-EDTA (ATCC, LGC, United Kingdom) and re-suspended in media. Table 1 Cell line panel employed in this study

Cell line Tissue, disease type

SK-OV-3 Ovarian, Adenocarcinoma

DU145 Prostate, carcinoma

Guava ViaCount® Assay

Prior to complex addition, SK-OV-3 and DU 145 cells were seeded overnight in 96 well tissue culture plates (Costar) at an initial density as outlined in Table 2. Table 2 In vitro cytotoxicity: Seeding densities (24-72 Hrs) for tested cell lines

Cell Density (cells ml ¹ )

Cell Line 24 Hrs 72 Hrs

SK-OV-3 4 x 10 ⁴ 1 x 10 ⁴

DU145 2 x 10 ⁵ 1 x 10 ⁵

DMSO stocks of Doxorubicin (Sigma-Aldrich, Ireland) and complexes were prepared at ~ 10 mM. Stock solutions were diluted in supplemented RPMI 1640 medium to give the following final concentrations in 200 μΙ wells: 10, 7.5, 5, 2.5 and 1 .25 μΜ, while Doxorubicin stocks were diluted giving the following final concentrations: 1 , 0.75, 0.5, 0.25 and 0.125 μΜ. A DMSO control of the highest drug incubation was also included. Cells were incubated for 24, 48 and 72 hr at 37 °C in a humidified atmosphere with 5% CO2. After drug incubation, cells were washed once with 200 μΙ_ of phosphate buffered saline (PBS) then harvested with 100 μΙ 1 X trypsin. After additio of 100 μΙ_ media, the cells were transferred to round bottom 96 well plates (Greiner, Austria) containing 10 μΙ ViaCount® reagent and incubated in the dark at RT for 10 mins prior to analysis. Viability was assessed on a Guava EasyCyte HT flow cytometer using Guava ViaCount® software.

Tabulated IC50 values are included in Table 3. Table 3A: In vitro cytotoxicitY: Tabulated IC50 values for test compounds. TJata points representative of average of triplicate measurements, IC50 ± SD, N=3.

SK-OV-3 IC50 (μΜ)" DU145 ICso UiM)" Compound 24 Hrs 72 Hrs 24 Hrs 72 Hrs

SAHA >10 μΜ 1.46 ± 0.20 >10 μΜ 1.72 ± 0.14

Cu-SAHA-DPQ 2.31 ± 0.35 1.40 ± 0.21 5.50 ± 1.08 1.10 ± 0.12

Cu-SAHA-DPPZ 1.54 ± 0.14 0.40 ± 0.54 1.06 ± 0.61 2.75 ± 0.36

To investigate the in vitro cytotoxic activity of the Cu(ll) complexes, their IC50 values (concentration which inhibits 50% cellular proliferation) were assessed using the Guava ViaCount® assay. A panel of 4 human cancers namely: MDA-MB-231 (breast), HepG2 (liver), A549 (lung) and HT29 (colon) were employed. Cytotoxic experiments were conducted at 24 and 72 hour time intervals and the IC50 values were calculated by fitting data to sigmoidal dose-response curves (72 hour results shown).

Table 3B: In vitro cytotoxicity

* Data points represent an average of triplicate measurements. Vehicle 5%DMSO:30%PEG-

300

IC50 (μΜ) ± SD; N=3. Vehicles were found to have no effects

on cell viability. Preparation of nuclear isolates

SK-OV-3 cells were seeded in a 75 cm ³ flask and grown until 80-90% confluency. The cells were treated at isotoxic concentrations corresponding to the I C50 value (Error! Reference source not found.) for 24 hr (Cu-SAHA-Phen, Cu-SAHA-Phendio, Cu-SAHA-DPQ, Cu- SAHA-DPPZ, 48 hr (Cu-SAHA-Bipy) or 72 hr (SAHA). Cells were washed twice with ice cold PBS, scraped and collected into a 1 .5 ml microcentrifuge tube (Eppendorf, Germany). Nuclear extracts were prepared following the protocol recommended by the manufacturer of the EpiQuik® Nuclear Extraction Kit (Epigentek, USA). Protein concentration of the nuclear isolates was determined by following the protocol of the manufacturer of the Quick Start™ Bradford protein assay (Bio-Rad, CA, USA).

HDAC activity

The SK-OV-3 nuclear extracts were tested for HDAC activity following the protocol recommended by the manufacturer of the EpiQuik® Colorimetric HDAC Activity/Inhibition assay kit (Epigentek, USA). The standard curve was constructed using the standard included in the kit; the absolute amount of deacetylated product was calculated from the standard curve. HDAC activity was calculated by:

OD(Control— blank)— OD (Sample— blank)

HDAC activity =

Standard curve slope

Values in the bar chart were normalised to the control (untreated) sample. 4 μg of protein was added to each sample.

Copper-SAHA-A/,A/'-Chemotypes and Their Interactions with Nucleic Acids

General chemicals

poly[d(G-C) ₂ ] (P9389, £ ₂₆₀ = 16,800 M(bp) ^"1 cm ^"1 ), poly[d(A-T) ₂ ] (P0833, £ ₂₆₀ = 16,800 M(bp) ^"1 cm ^"1 ), ethidium bromide (EtBr), netropsin (N9653), Actinomycin D (A-1410), Ν,Ν'- Dimethylformamide (DMF, 22705-6) and salmon testes DNA (stDNA, D1626- 1 G) were purchased from Sigma-Aldrich and used without further purification. pUC19 vector (New England Bio-Labs, N3041 ), UltraPure calf thymus DNA (ctDNA, Invitrogen, 15633-019, £ ₂₆₀

= 12.824 M(bp)- ¹ cm ^"1 ), sodium chloride (NaCI, Ambion, AM9760G) and 2-[4-(2- hydroxyethyl)piperazin-1 -yl]ethanesulfonic acid (HEPES) buffer (Fisher, 10041703) were purchased from respective suppliers and used without further purification. Competitive EtBr displacement

Competitive EtBr displacement assays were conducted using a method previously reported by Mc Cann, M; Kellett, A. et al. Chem. Comm. 2013, 49, 2341 -2343. Briefly, a working solution of 20 μΜ ctDNA, poly[d(G-C) ₂ ] or poly[d(A-T) ₂ ]; 25.2 μΜ EtBr; 40 mM NaCI in HEPES buffer (80 mM, pH 7.2) was prepared. Stock solutions of metal complexes, SAHA and groove binding drugs were prepared in Λ/,Λ/'-Dimethyl formamide (DMF) at -10 mM and further diluted with 80 mM HEPES. 50 μΙ of DNA-Et working solution were placed into each well of a 96 well microplate, with the exception of blanks which contained 100 μΙ of HEPES buffer. Serial aliquots of the metal complexes, SAHA and groove binding drugs were added to the working solution and the final volume was adjusted to 100 μΙ in each well such that the final concentrations of ctDNA and EtBr were 10 and 12.6 μΜ, respectively. The plate was incubated at room temperature for 1 hour, protected from light. Microplates were analysed using a Bio-Tek synergy HT multi-mode microplate reader with excitation and emission wavelengths set to 530 and 590 nm respectively. Each drug concentration was measured in triplicate and the apparent binding constants were calculated using K _app = K _e * 12.6/C ₅ o, where K _e = 9.5 10 ⁶ M(bp) ^"1 .

Viscosity measurements

Viscosity measurements were conducted using a method previously reported by Mc Cann, M; Kellett, A. et al., Chem. Comm., 2013, 49, 2341 -2343. Briefly, a 15 ml solution of stDNA was prepared at 1 10 ^"3 M in 80 mM HEPES buffer for each working sample. Stock solutions prepared in DMF were added according to the gradual increasing [drug]/[DNA] (r) ratios of 0.02, 0.04, 0.06, 0.08, 0.10, 0.12, 0.14, 0.16, 0.18 and 0.2. Viscosity values, n, (unit: cP) were directly obtained by running 0# spindle in working samples at 60 rpm via DV-ll-Programmable Digital Viscometer equipped with Enhanced Brookfield UL Adapter at room temperature. Data were presented as η / η ₀ versus [compound]/[DNA] ratio, in which Ho and η refers to viscosity of each DNA working sample in the absence and presence of complex' ¹¹ .

Fluorescence quenching

Fluorescence quenching assays were conducted using a method previously reported by Mc Cann, M; Kellett, A. et al., Chem. Comm., 2013, 49, 2341 -2343. Briefly, a working solution of 50 μΜ UltraPure ctDNA (£ ₂₆ o = 12,824 M (bp) ^"1 cm ^"1 ) along with either 10 μΜ EtBr or Hoechst 33258 (Sigma) in HEPES buffer (80 mM, pH = 7.2) and NaCI (40 mM) was prepared. Stock solutions of metal complexes, metal salts, free ligands and groove binding drugs were prepared at -4.0 mM in DMSO and diluted further with 80 mM HEPES. 50 μΙ_ of DNA-EtBr or DNA-Hoechst working solutions were placed each well of a 96 well microplate with the exception of the blanks which contained 100 μΙ_ HEPES buffer and 5 μΜ of either Hoechst or EtBr. Serial aliquots of the tested compound were added to the working solutions and the volume was adjusted to 100 μΙ_ in each well such that the final concentration of ctDNA and EtBr/Hoechst were 25 μΜ and 5 μΜ, respectively. The plate was allowed to incubate at room temperature for 5 minutes before being analysed using a Bio-Tek synergy HT multi-mode microplate reader with excitation and emission

wavelengths being set to 530 and 590 nm for EtBr detection or 360 nm and 460 nm for Hoechst 33258 detection. Concentrations of the tested compounds were optimized such that fluorescence was 30-40% of the initial control at their highest reading. Each drug concentration was measured in triplicate, on at least two separate occasions. From a plot of fluorescence versus added drug concentration, the Q value is given by the concentration required to effect 50% removal of the initial fluorescence of bound dye. ^[1]

Thermal melting

Thermal melting analysis were conducted using a method previously reported by Molphy, Z; Kellett, A. et al., Inorg. Chem., 2014, 53, 5392-5404. Analysis was carried out on an Agilent Cary 100 dual beam spectrophotometer equipped with a 6 x 6 Peltier multicell system with temperature controller. For poly[d(G-C)2] ; in a final volume of 1 ml using Starna black-walled quartz cuvettes with tight-fitting seals, 2 mM NaOAc buffer (pH = 5.0), 1 mM NaCI and poly[d(G-C)2] (Sigma, P9389) were added to give a final absorbance of between 0.18 and 0.20 absorbance units at 260 nm (£max = 8400 IVr ¹ cnr ¹ ). For poly[d(A-T) ₂ ]; in a final volume of 1 ml using Starna black-walled quartz cuvettes with tight-fitting seals, 50 mM NaOAc buffer (pH = 5.0), 250 mM NaCI and poly[d(A-T) ₂ ] (Sigma, P0883) were added to give a final absorbance of between 0.18 and 0.20 absorbance units at 260 nm (£max = 6600 M ^"1 cm ^"1 ). Stock solutions of metal complexes, netropsin, and actinomycin D, prepared beforehand in DMF, were further diluted in 80 mM HEPES (pH 7.2). An aliquot of test reagent was then added to each cuvette such that an r value of 0.1 was achieved (r =

[compound]/[nucleotide]). The test reagent and respective alternating copolymer were then incubated for 10 min at 20 °C prior to commencing the temperature ramp. Thermal melting measurements were recorded at 260 nm at 0.25 s intervals. Temperature was ramped at 3 °C/min over the range of 20.0-97.0 °C. The spectral bandwidth (SBW) was set to 1.

Temperature was calibrated, for each measurement, using a temperature probe placed in an identical black-walled cuvette containing equivalent buffer and NaCI concentrations. Samples were run in triplicate, and the melting temperature TM (°C) was calculated using the built-in derivative method on the instrument ¹²¹ .

Nuclease activity in the presence or absence of ROS scavengers

The presence or absence of ROS specific scavengers were used to determine the effect on the DNA cleavage abilities of each Cu complex. The procedure was adapted from a previously reported method (Molphy et al Front. Chem. 2015, 3(18), 1 -9). Briefly, in a final volume of 20 μΙ, 80 mM HEPES (pH = 7.2), 25 mM NaCI, 1 mM Na-L-ascorbate, and 400 ng of pUC19 DNA treated with drug concentrations of 1 , 2.5, 3.75 and 5 μΜ (Cu-SAHA- Phen, Cu-SAHA-DPQ and Cu-SAHA-Phendio), 2.5, 5, 10, 15 μΜ (Cu-SAHA-Bipy), 1 , 2.5, 5, 10 μΜ (Cu-SAHA-DPPZ) and 250 nM, 500 nM 1 μΜ and 2.5 μΜ (Cu-Phen) in the presence or absence of ROS scavengers/stabilisers: Kl (10 mM), NaN3 (10 mM), DMSO (10%) and Tiron (10 mM). Reactions were incubated for 30 minutes at 37 °C and quenched with 6X loading dye (Fermentas) containing 10mM Tris-HCI, 0.03% bromophenol blue, 0.03% xylene cyanole FF, 60% glycerol and 60 mM EDTA. Samples were then loaded onto an agarose gel (1 .2%) containing 4 μΙ of EtBr. Electrophoresis was completed at 70 V for 2 hrs in 1X TAE buffer.

EdU incorporation assay

SK-OV-3 cells were seeded at an intial density of 6 x 10 ⁴ cells ml ^"1 overnight. Cu(ll) complexes were added to SK-OV-3 cells at isotoxic concentrations corresponding to the IC50 value and incubated for 24 hours. EdU incorporation was measured according to the protocol recommended by the manufacturer of the EdU cell proliferation kit (Baseclick GmBH). Analysis was performed on a Guava EasyCyte HT flow cytometer. DNA metallodrug interactions

In order to investigate the apparent binding constants {K _app ) or binding affinity, of the complex series towards canonical double stranded (dsDNA) - ctDNA, we utilised a high throughput competitive EtBr displacement assay. Actinomycin D (Goodisman, J;

Dabrowiak, J.C et al. Biochemistry 1992, 31 , 1046-1058) and netropsin (Zimmer, C;

Guschlbauer, W. et al., Nuc. Acid. Res., 1979, 6, 2831 -2837) were used throughout the study due to their known interchelative and minor groove binding properties, respectively. The presence of the phenazine ligands DPQ and DPPZ in the coordination environment around the Cu ²⁺ metal centre serve to significantly enhance DNA binding with calculated Kapp values (table )exhibiting a -10 fold increase compared to the Phen derivative (7.13 x 10 ⁶ M(bp ^"1 ) (Cu-SAHA-DPQ) versus 6.67 x 10 ⁶ M(bp ^"1 ) (Cu-SAHA-Phen)) (Table 4).

Furthermore we sought to investigate the intercalative effects of the complex series on salmon testes DNA (stDNA) using viscosity measurements. Introduction of an intercalative agent such as actinomycin D result in an increase in relative viscosity due to

conformational changes induced after intercalation between the DNA base pairs

accordingly, while introduction of DNA surface-binding species such as netropsin and

[Co(NH ₃ )6]CI ₃ induce a moderate or diminished effect (Table 4). Cu-SAHA-DPPZ and Cu- SAHA-DPQ produce an enhanced viscosity profile with Cu-SAHA-DPPZ showing on par intercalative activity to the known DNA intercalator actinomycin D. Interestingly, the Cu- SAHA-Phendio and Cu-SAHA-Bipy complexes show only moderate intercalative effects which are in agreement with observations for the K _app measurements. Overall, a trend in intercalative activity was observed in the Cu(ll) complexes where: Cu-SAHA-DPPZ>Cu- SAHA-DPQ»Cu-SAHA-Phen»Cu-SAHA-Phendio>Cu-SAHA-Bipy. Fluorescence quenching experiments of limited bound ctDNA solutions of EtBr

(intercalator) and Hoechst 33258 (minor groove binder) were employed (Table 4).

Compared to the minor groove binding agent netropsin or intercalator actinomycin D, the complex series did not exhibit a degree of discrimination for quenching either EtBr or Hoechst with the Cu-SAHA-DPPZ and Cu-SAHA-DPQ complexes having the highest Q values of the series and also being higher than Cu-Phen. However, both of these complexes displace Hoescht with slight preference over EtBr wheras with Cu-Phen, this effect is essentially reversed. As expected, no significant was observed for both the Cu- SAHA-Bipy and Cu-SAHA-Phendio complexes at concentrations >150μΜ.

Table 4: Summarised DNA binding data (A) competitive ethidium bromide displacement, (B) fluorescence quenching and (C) viscosity analysis.

(A) (B) (C)

Complex Cso"(nM) Ka _PP ^b M(bp)-' Q (EtBr, ff (Hoescht, η/ηο ^Λ μΜ) μΜ)

Actinomycin D 04.10 2.92 x 10 ⁷ 04.78 26.34 1.14

Netropsin 46.27 2.50 x 10 ⁶ 20.04 02.40 1.00

[Co(NH ]Ch >300 NC NC NC 0.82

Cu-Phen ¹ 179.21 6.67 x 10 ⁵ 20.38 34.96 1.17

Cu-SAHA-Phen 124.00 9.65 x 10 ⁵ 83.10 78.10 1.05

Cu-SAHA-DPQ 16.80 7.13 x lO ⁶ 14.60 13.40 1.14

Cu-SAHA-DPPZ 13.30 9.00 x 10 ⁶ 15.30 14.20 1.07

Cu-SAHA-Phendio > 150 NC >150 >150 1.01

Cu-SAHA-Bipy > 150 NC >150 >150 1.03

SAHA > 150 NC >150 >150 0.98

Concentration required to reduce fluorescence by 50%. ^b Apparent binding constant (Ka _PP = Ke x 12.6/Cso, where Ke = 9.5 x 10 ⁶ M(bp ^" '). - concentration required to effect 50% removal of the initial fluorescence of bound dye.

dRelative viscosity at drug load of 0.18%. NC = Not Calculated

In order to gain insight into base-specific nucleotide binding, thermal melting analysis and competitive EtBr displacement assays on two synthetic alternating co-polymers of adenine- thymine, poly[d(A-T)2] and guanine-cytosine, poly[d(G-C)2] (Table 5). Actinomycin D substantially stabilised the thermal denaturation of poly[d(G-C)2], netropsin stabilised the thermal denaturation of poly[d(A-T)2] with both compounds exhibiting almost equal amounts of stabilisation (-12.60 °C) within these respective polymers. TM analysis of both agents highlights binding specificity with neglible stabilisation effects observed for the disfavoured polynucleotide. These findings are in agreement with previous studies in which Actinomycin D exhibits interchelative preference towards G C rich copolymers

(Goodisman, J; Dabrowiak, J.C et al, Biochemistry 1992, 31 , 1046-1058) and Netropsin forms preferable interactions with a compressed minor groove in T-tracts of A T rich DNA (Zimmer, C; Guschlbauer, W et al., Nuc. Acid. Res., 1979, 6, 2831 -2837. All of the Cu ²⁺ complexes stabilize the thermal melting temperature of poly[d(G-C)2] to varying extents (~2 - 12.6 °C). Cu-SAHA-DPQ had the strongest stabilisation effect (Δ7 _Μ 12.57 ± 0.73), Cu- SAHA-DPPZ followed closely {AT _M 9.23 ± 1.56). Cu-Phen enhanced the thermal temperature of poly[d(G-C) ₂ ] (Δ7 _Μ 6.64 ± 1 .58) thereafter followed by Cu-SAHA-Phen (Δ7 _Μ 4.30 ± 0.51 ). Compared to ploy[d(G-C)2] all of the complexes had neglible or negative effects on thermal stabilization. The Cu ²⁺ complexes bind both G C and A co-polymers where K _app values being similar for both nucleotides tested. Table 5: Effect of Actinomycin D, Netropsin, SAHA and Cu ²⁺ complexes on the thermal denaturation of poly[d(G- C) ₂ ] and poly[d(A-T) ₂ ]

Poly [d(A-T) ₂ ] Poly [d(G-C) ₂ ]

Complex Cso' faM) Kap _P ^b (M(bp ^l ) Cso' faM) Kapp ^b (M(bp ^l )

(°C)

Netropsin 2.08 5.75 x 10 ⁷ 12.61 ± 0.61 >500 NC 2.51 ±

0.51

Actinomycin D >500 NC -0.31 ± 0.48 2.28 5.25 x 10 ⁷ 12.10 ±

0.95

Cu-Phen -0.02 ± 0.29 6.64 ±

1.58

Cu-SAHA-Phen 121.40 9.86 x 10 ⁵ -1.98 ± 0.15 1 15.80 1.03 x lO ⁶ 4.30 ±

0.51

Cu-SAHA-DPQ 23.30 5.13 x lO ⁶ -1.07 ± 0.14 16.60 6.80 x 10 ⁶ 12.57 ±

0.73

Cu-SAHA-DPPZ 13.20 9.10 x 10 ⁶ -1.03 ± 0.48 16.40 7.32 x 10 ⁶ 9.23 ±

1.56

Cu-SAHA-Phendio >150 NC -0.39 ± 0.71 >150 NC -2.52 ±

0.66

Cu-SAHA-Bipy >150 NC -1.72 ± 0.73 >150 NC -3.85 ±

0.51

SAHA >150 NC -0.73 ± 0.48 >150 NC 2.51 ±

0.51

Cso - Concentration required to reduce fluorescence by 50%. ^b Kap _P - Apparent binding constant (Ka _PP = Ke x 12.6/Cso, where Ke = 9.5 x 10 ⁶ M(bp ^" '). ^C A TM - Difference in thermal melting (TM) of drug treated nucleotide at r = 0.1 compared with drug-untreated nucleotide. NC - Not Calculated.

The chemical nuclease activity of the complex series in the presence or absence of ROS specific scavengers were employed to identify both single and double stranded DNA damage and possible ROS species involved in DNA damage. Treatment of supercoiled Fl pUC19 with Cu-Phen lanes 1 -4) results in FN and FIN bands at 1 μΜ & 2.5 μΜ respectively with complete degradation of the DNA strand occurring at 5 μΜ (data not shown). In order to identify the predominant ROS species involved in strand scission ROS-specific scavengers such as Kl, NalS , Tiron and DMSO were added to the reaction mixture during the chemical nuclease experiments. Incubation of Cu-Phen with tiron (4, 5-di hydroxy- 1 ,3- benzenedisulfonic acid disodium salt or Thb) (Figure 2, Lanes 13-16) a superoxide (O2 ^' ~) scavenger (Kiss, T, Martin, R.B ei a/., J. Am. Chem. Soc, 1989, 1 1 1 , 361 1 -3614) significantly hinders the cleavage of Fl to FN & FIN at 1 & 2.5μΜ respectively. Similiarly, DMSO, a hydroxyl radical (ΌΗ) scavenger (Mazzer, P.A; Bose, R.N et al., J. Inorg.

Biochem., 2007, 101 , 44-55 & Franco, R; Cidlowski, J.A et al., J. Biol. Chem., 2007, 282, 30452-30465) had nearly an identical effect (Figure 2, Lanes 17-20) to tiron where cleavage activity from to FIN was also hindered at the same concentration. Incubation with Kl (H2O2 scavenger (Dunand, C; Penel, C et al., New Phytologist, 2007, 174, 332-341 & Steffens, B; Sauter, M et al., Plant Cell, 2012, 24, 3296-3306), Figure 2, Lanes 5-8) and NaN3 (singlet oxygen CO2) scavenger (Franco, R; Cidlowski, J.A et al., New Phytol., 2007, 174, 332-341 , Figure 2, Lanes 9-12) had marginal effects on the cleavage activity. This suggests two predominant ROS species formed during the reaction, mainly 02 ^° ~ and ΌΗ.

In vitro DNA damage

The capability of the Cu ²⁺ complexes to induce oxidative DNA damage in vitro was assessed using a 5-ethynyl-2'-deoxyuridine (EdU) incorporation assay. Exogenous nucleoside analogues of thymidine such as EdU can be incorporated into cellular DNA following oxidative DNA damage. The complexes Cu-SAHA-DPQ and Cu-SAHA-DPPZ were found to have -30% reduction in EdU incorporation compared to control (non drug treated cells). Moreover, the complex Cu-SAHA-Phendio was found to have on par to the clinical agent, doxorubicin (Figure 4).

Equivalents

The foregoing description details presently preferred embodiments of the present invention. Numerous modifications and variations in practice thereof are expected to occur to those skilled in the art upon consideration of these descriptions. Those modifications and variations are intended to be encompassed within the claims appended hereto.